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+{"text": "【兽医文献】\n标题: High seroprevalence of SARS-CoV-2 in cats linked to human infection in a Latin American country with elevated COVID-19 transmission and mortality\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nUsing social media listening to\nidentify the real-world challenges\nfaced by dog owners globally\nwhen administering oral\nmedications\nGeorgina Tarrant 1*, Taranpreet Rai 1,2, Andrea Wright 3,\nTravis Street 1 and Kevin Wells 1,2\n1 Surrey DataHub, vHive, School of Veterinary Medicine, University of Surrey, Guildford,\nUnited Kingdom, 2 Centre for Vision, Speech and Signal Processing, University of Surrey, Guildford,\nUnited Kingdom, 3 Outcomes Research, Zoetis Inc., Parsippany, NJ, United States\nIntroduction: Pet owner compliance with oral medication administration\nrepresents a significant challenge in veterinary medicine, yet limited real-world\ndata exists on the experiences and barriers faced by dog owners during at-\nhome “pilling” processes.\nMethods: This study employed social media listening (SML) to analyze 4,787\nrelevant posts from X, Reddit, Facebook, blogs, and forums discussing oral\nmedication administration to dogs. Large language models (LLMs) were used\nfor data cleaning, relevancy filtering and analysis. Topic modeling, sentiment\nand thematic analyses were conducted to identify key themes and challenges.\nResults: Analysis revealed significant anxiety and fear associated with medication\nadministration, with 12% of posts mentioning anxiety and additional fear-related\nterms appearing in 1-3% of posts. Only 7.6% of posts discussed soft/chewable\nmedications, which showed positive sentiment and preference. Geographic\nanalysis showed posts predominantly from English-speaking countries (US\n70.3%, UK 17.8%). Five major themes emerged from X, Reddit, blogs and forums:\ngeneral medication/veterinary experiences, pill types and practices, pill delivery\nmethods, specific medications/preventatives, product reviews/natural remedies.\nFinancial concerns were prominent, with pet owners describing medication\ncosts as barriers to optimal care. Successful pilling strategies included hiding\npills in peanut butter, cheese or meat products; crushing pills and mixing with\nfood, and using distraction techniques.\n\nSuccessful pilling strategies included hiding\npills in peanut butter, cheese or meat products; crushing pills and mixing with\nfood, and using distraction techniques.\nDiscussion: The study identified key barriers to compliance including financial\nconstraints, fear and anxiety, mistrust of veterinary advice and practical\nadministration challenges. Pet owners showed higher adherence when\ntreatments visibly improved quality of life or addressed chronic conditions.\nChewable formulations were preferred but raised concerns about accidental\noverdosing. The methodology demonstrated that SML combined with AI\nanalysis effectively captures real-world pet owner experiences.\nConclusions: This novel approach revealed that dog owners face significant\npsychological and practical barriers when administering oral medications.\nChewable formulations may improve compliance, though proper storage and\neducation are essential. The study provides veterinarians with evidence-based\nsuccessful pilling strategies reported by pet owners and highlights the need for\nbetter communication about treatment benefits, financial planning options and\nalternative delivery methods to improve medication adherence.\nOPEN ACCESS\nEDITED BY\nMaurice Pitesky,\nUniversity of California, Davis, United States\nREVIEWED BY\nBlagica Sekovska,\nSaints Cyril and Methodius University of\nSkopje, North Macedonia\nChristine Pye,\nUniversity of Liverpool, United Kingdom\n*CORRESPONDENCE\nGeorgina Tarrant\ngeorgina.tarrant@surrey.ac.uk\nRECEIVED 26 September 2024\nACCEPTED 21 May 2025\nPUBLISHED 30 June 2025\nCITATION\nTarrant G, Rai T, Wright A, Street T and\nWells K (2025) Using social media listening to\nidentify the real-world challenges faced by\ndog owners globally when administering oral\nmedications.\nFront. Vet. Sci. 12:1502236.\ndoi: 10.3389/fvets.2025.1502236\nCOPYRIGHT\n© 2025 Tarrant, Rai, Wright, Street and Wells.\nThis is an open-access article distributed\nunder the terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other forums is\npermitted, provided the original author(s) and\nthe copyright owner(s) are credited and that\n\nThis is an open-access article distributed\nunder the terms of the Creative Commons\nAttribution License (CC BY).The use,\ndistribution or reproduction in other forums is\npermitted, provided the original author(s) and\nthe copyright owner(s) are credited and that\nthe original publication in this journal is cited,\nin accordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Original Research\nPUBLISHED  30 June 2025\nDOI  10.3389/fvets.2025.1502236\n\nTarrant et al.\b\n10.3389/fvets.2025.1502236\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nKEYWORDS\nsocial media, large language models, pulsar platform, topic modeling, palatability, oral\nformulations, canines, dogs\n1 Introduction\nMedication adherence in dogs, particularly for oral dosing, is crucial\nfor the effective management of various health conditions. Common\nailments requiring oral medications include infections, skin disease,\nshort-term pain (e.g., post-operative), chronic pain from diseases like\narthritis, allergic dermatitis, and controlling parasitic infestations (1, 2).\nHowever, few studies have explored factors affecting compliance with\nmedication regimens (3, 4). Such studies have typically shown that pet\nowner adherence to veterinary recommendations for oral medications\nfalls short of ideal coverage (5). For example, Lavan et al. found that while\nalmost all veterinary hospitals in their study recommended 12 months\nof flea and tick prevention, the estimated actual coverage was only\n6.1 months based on owner medication purchases from their veterinary\nhospital over a 12-month period (6). This gap between recommendation\nand practice is further highlighted in a separate study by Lavan et al.,\nwhich revealed that a considerable proportion of dog owners purchased\nonly one or two doses of oral flea and tick medication per year.\nSpecifically, 30 to 42% of dog owners obtained only one dose of oral flea\nand tick medication per 12-month period, depending on the product (7).\nA study by Mwacalimba et al. showed a compliance rate of 7.1 months\nfor monthly heartworm preventatives in Australia, while a similar study\nby Mwacalimba et  al. showed compliance of 7.3 months in the\nUnited States (8, 9). A further study by Mwacalimba et al. found that 25%\nof pet owners in the United States purchased a heartworm preventative\nat least once in a 2-year period (10).\nPurchase compliance for chronic conditions and regular treatments\namong dog owners is generally thought to be  low (11), which\nconcomitantly impacts outcomes. Pet owners’ compliance with\n\nPurchase compliance for chronic conditions and regular treatments\namong dog owners is generally thought to be  low (11), which\nconcomitantly impacts outcomes.Pet owners’ compliance with\npreventative medicines is particularly challenging as owners may\nquestion antiparasitic treatment necessity, perceive veterinarians as\n“upselling” products, or be concerned about side effects (12). Similarly,\nsuboptimal compliance with other medications may lead to suboptimal\ntreatment outcomes, prolonged recovery times, and drug resistance\ndevelopment. Veterinary practice management strategies such as\nmonthly payment plans and creating tailored annual care plans for pets\ncan help to address financial barriers to treatment (13, 14).\nPet owners’ dosing adherence to routine daily, weekly, or monthly\nlong-term veterinary prescription recommendations is crucial for\ntreatment efficacy. Maintaining consistent medication schedules for\ndogs can be challenging, particularly for long-term treatments (e.g.,\nparasite prevention and arthritis pain medication) and chronic\nconditions requiring daily administration (e.g., allergic dermatitis).\nOther challenges include treatment duration, inability to give\nmedicine with food, inconvenient dosing schedules, administering\nmultiple prescribed medications, perceived adverse effects causing pet\nowners to stop administering medication, and complex treatment\nplans (3, 15–17). Some owners discontinued medication after\nobserving possible side effects such as diarrhea, while many modified\nmedications without veterinary advice by opening capsules, crushing\ntablets, or changing dosing schedules to facilitate administration (3).\nCompliance improves when veterinarians explain medications,\nsimplify the treatment regime, and demonstrate how to administer the\nmedication (3). Communication is vital to determine an owner’s\npriorities, and treatment should be customized to both patient and\nowner. Recent studies indicate that longer-acting medications may\nimprove compliance in areas such as ectoparasite protection (6, 11,\n18–20). However, prolonged drug use can reduce efficacy. For\n\nRecent studies indicate that longer-acting medications may\nimprove compliance in areas such as ectoparasite protection (6, 11,\n18–20).However, prolonged drug use can reduce efficacy.For\nexample, extended, repeated, or poorly managed antibiotic use in dogs\ncan contribute to antimicrobial resistance (AMR), especially for\nchronic conditions requiring extended therapy, such as recurrent skin\ninfections, urinary tract infections, or osteomyelitis (21). It has been\nfound that long-term exposure to monthly fipronil treatments has\nselected for resistant flea strains (22), and resistant Dirofilaria immitis\nstrains that cause heartworm disease have emerged (23).\nSuccessfully administering regular oral medications to dogs is\nthought to represent a momentous challenge for many pet owners (1,\n3, 5, 17, 24), with approximately one-third of pet owners in a\nNew Zealand study reporting difficulties administering prescribed\nmedications to their pets. Pet owners reported several strategies that\nhelped with medication administration, including using food,\nmodifying their behavior or their pets, using positive reinforcement,\nor combining multiple approaches (3). Canine resistance to\nadministration of oral medications is thought to be the most common\nchallenge (3). This often stems from factors such as a perceived\nunpleasant taste, smell, or associated anxiety. Aleo et al. found that\nsophisticated flavor enhancers that improve taste, aroma, and texture\ncan increase a dog’s willingness to consume tablets (25). As a result,\nvarious formulations have been developed, including film-coated\ntablets, capsules, powders, liquid solutions, and chewable and\npalatable tablets designed to improve treatment adherence. However,\neven with flavor enhancers, excessively chewy tablets may still\nbe refused. Adding an enticing scent can encourage dogs to pick up a\ntablet (1, 5). Chewable formulations are considered a palatable\nalternative to film-coated tablets and have been shown to increase\ncompliance through ease of administration and increased acceptance\nby dogs (26). Various strategies can be employed by pet owners to\n\nChewable formulations are considered a palatable\nalternative to film-coated tablets and have been shown to increase\ncompliance through ease of administration and increased acceptance\nby dogs (26).Various strategies can be employed by pet owners to\nencourage ingestion, although these approaches may not always\nbe successful. Soft gelatin capsules can improve dosing compliance,\nparticularly if the capsule size is decreased to make it easier for the dog\nto ingest. This suggests that optimizing drug delivery systems to make\nthe pills more palatable is likely to enhance dosing compliance (27).\nThis study investigates the practical challenges pet owners face\nwhen administering oral medications to dogs, comparing different\ntypes, such as coated pills, soft chewables, and flavored or flavorless\npills. Prior work has relied on surveys and/or focus groups and may\nexhibit sensitivity to participant selection bias and response bias to\nquestions. A complementary alternative is to employ social media\nlistening (SML) data with AI-driven (artificial intelligence-driven)\ntopic modeling and regional demographic analysis. Using this\napproach, we examine ‘the collective wisdom of the [online] crowd’\nby extracting unsolicited real-world pet owner experiences publicly\nshared on social media (SM) platforms. Key themes are identified\nusing AI tools and correlated with demographic data to provide an\nenhanced understanding of canine medication administration issues\nin pets across the globe.\n\nTarrant et al.\b\n10.3389/fvets.2025.1502236\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nSocial media listening is a powerful tool for extracting insights\nquickly and efficiently from text content (28). SML involves actively\ntracking and analyzing unsolicited online content related to specific\ntopics across SM media to understand sentiment, trends, and behavior\n(29). This technique can be used to explore a variety of topics from pet\nowner perceptions of feline and canine pruritus (30–32) to the role of\nwomen in livestock farming in Sub-Saharan Africa (28). In this study,\nwe harness SML and AI topic modeling to understand the challenges\nexperienced by pet owners around the world when administering oral\nmedications to their dogs.1\nThe aims of this study were to (i) better understand practical and\ncommonly encountered challenges experienced by pet owners when\nadministering oral medications to dogs of all sizes, (ii) to examine the\nunique challenges of medicating large dogs, and (iii) to examine the\nexperience of different pilling types and administration methods.\n2 Methodology\nAn overview of the data collection and analysis workflow is\nprovided in Figure 1. In brief, we use a commercial SML platform\n(designed for marketing applications such as tracking brand\nawareness) to ‘scrape’ SM content. This process is initiated by creating\ntailored search expressions to collect SML data. Data are then cleaned\nand pre-processed (to remove advertisements/promotions, irrelevant\ncontent, retweets/reposts, etc.) and filtered to extract key SM posts for\ndownstream analysis.\n2.1 Data collection\nTo extract (commonly referred to as ‘scrape’) SM data, either\nbespoke software can be  employed (that may have legal data\nimplications) or a variety of subscription-based commercial SML\nplatforms may be used, e.g., Sprout, Hootsuite, Meltwater, etc. These\nhave large-scale scraping agreements and licenses with major SM\noutlets such as X (formerly Twitter), Reddit, Instagram, etc. We have\nused Pulsar Platform™, one of the largest and best-known SML\nplatforms, to conduct topic searches using bespoke keywords and key\n\nWe have\nused Pulsar Platform™, one of the largest and best-known SML\nplatforms, to conduct topic searches using bespoke keywords and key\nphrases. A tailored set of search expressions was developed to\nmaximize sensitivity for retrieving SM posts relevant to administering\noral medications to dogs of all sizes, particularly daily pilling with\nchewable, flavored, or non-flavored products, in conjunction with\nexpert veterinary information. The structure of the Boolean logic\nsearch expression is shown in Figure 2. The search was limited to\nanglophone posts worldwide. American Kennel Club (AKC) dog\nbreeds were used to expand the search beyond mentions of dogs.\nRetweets were excluded as these are a form of duplicate in this context.\nOther exclusions were added as necessary. Further details on Facebook\nscrapes are available in Supplementary Table S1.\nPulsar Platform™ was used to scrape SM posts from X, Reddit,\nblogs, and forums. A time window of 30 days was selected for the data\n1  A large language model (LLM) is a machine learning model that has been\npre-trained on vast volumes of data. LLMs can recognize and generate text\nand perform other natural language processing tasks such as classification (28).\nscraping from all sources. X, Reddit, blogs, and forums were scraped\nfrom 4th May to 10th June 2024. A maximum of 30 days of historical\ndata are available from Facebook; therefore, this platform was scraped\nfrom 7th May to 6th June 2024.\nPreliminary raw data were manually reviewed after 24 h, and\nrevisions were made to the search exclusions to improve the quality\nof the resulting data. Bulk scraping of SM frequently produces\nirrelevant data despite careful optimization of search terms. As\nsuch, the analysis of raw SM data from any platform needs to\nbe treated with caution. This is often due to the coincidental use of\nkeywords on irrelevant topics or posts that are impossible to\nanticipate. In this study, we inadvertently scraped a large number of\nderogatory, racist, and/or misogynist posts, as this coincided with\nthe derogatory/emotive use of words pertinent to this topic. Adult/\n\nIn this study, we inadvertently scraped a large number of\nderogatory, racist, and/or misogynist posts, as this coincided with\nthe derogatory/emotive use of words pertinent to this topic.Adult/\npornographic content was similarly problematic. This and related\nterms were excluded in so far as possible via exploratory pilot work\nbefore the initial scraping of the raw SML data, and further\nexclusion processes were needed at the pre-processing stage to\navoid contaminating the SML data. This kind of contamination can\nroutinely account for a substantial amount of SML data and\nhighlights the importance of cleaning and filtering SML data before\nconducting any substantial analyses.\nResults (SM posts) from all 33 Pulsar searches were downloaded\nwhen the 30-day search was complete, resulting in ~27 k SM posts.\n2.2 Data cleaning and pre-processing\nIt was necessary to clean and pre-process the data before filtering\nfor relevancy and conducting analyses.\nSML data often contains duplicates across different scrapes. These\nduplicates arise for various reasons. One cause was overlaps between\nsearches created manually. For example, a broad and potentially noisy\nsearch for “medications” might be followed by a narrower search for\n“oral medications.” This is done to examine whether the narrower\nsearch has sufficient coverage. Another source of duplicates was posts\nthat are retweets but miss the retweet exclusion. This can happen due\nto tagging issues on X. Duplicates can also occur when posts are\npublished by the same user on multiple SM platforms. Reposts by the\nsame user on the same forum can also create duplicates. This might\nhappen if the original response was not as the user desired.\nThe X, Reddit, blogs, and forums data were cleaned to remove\nduplicates using Python, and an LLM (GPT-4) was used to repeat this\nprocess for the concatenated Facebook data. We  decided not to\nremove special characters (e.g., emojis) from the data at this stage as\nthese might provide additional context to the posts. The title and post\ncontent were concatenated to optimize zero-shot relevancy filtering\n\nWe  decided not to\nremove special characters (e.g., emojis) from the data at this stage as\nthese might provide additional context to the posts.The title and post\ncontent were concatenated to optimize zero-shot relevancy filtering\nby providing context relating to the data source.\nAfter filtering, 75% of the posts were retained, ensuring cost-\neffective and efficient analysis. Posts exceeding 640 words were then\nremoved to minimize the complexity and costs associated with\nprocessing through LLMs, as longer posts often contribute less\nrelevant content. Manual spot checks of posts longer than 640 words\nwere implemented, confirming this observation. Additionally, posts\nwith fewer than 15 words were excluded to enhance the accuracy of\nzero-shot classification, as these shorter entries typically lack sufficient\ncontext for reliable classification. See Supplementary Figure S1 for a\nhistogram justifying this word limit.\n\nTarrant et al.\b\n10.3389/fvets.2025.1502236\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n2.3 Zero-shot relevancy filtering\nIn one of our previous SML studies (33), data scraped from\npublicly available SM was manually labeled as relevant or irrelevant to\nthe topic under investigation by a human reviewer: a tedious and\npotentially error-prone task. By contrast, in this latest work,\nwe  leveraged an LLM (OpenAI’s GPT-4 model) for zero-shot\nclassification through “prompt engineering,” an iterative process\nwhere users provide conversational instructions and examples to the\nLLM to optimize performance (28, 34). Thus, an LLM categorized\nSML posts, with a representative sample of classified posts verified by\na human. Our test runs demonstrated that GPT-4 was more accurate\nin classifying the relevancy of the post than ChatGPT. A sample of the\nposts labeled as ambivalent was manually spot-checked, and it was\nfound that most of these were irrelevant (advertisements or\nsimilar promotions).\n2.4 Audience profiling, demographic,\ngeographical, and sentiment analysis\nOnce filtered for relevancy, a total of 3,629 relevant posts from X,\nReddit, blogs, and forums and a further 1,158 relevant posts from\nFacebook were used for subsequent analysis out of a total of 26,846\nposts contained in the original raw data.\nSentiment analysis was applied to these filtered posts using the\nPulsar Platform™ sentiment AI model (35). Posts were automatically\nassigned sentiment scores of −1, 0, or 1, indicating negative, neutral, or\npositive attitudes. The sentiment model recognizes explicitly positive/\nnegative words and their context, understanding negations such as “not\ngood” and modifiers such as “really good.” Geographical analysis of the\nindividuals posting by country and city was undertaken in some cases.\nX platform has sophisticated geolocation capabilities for many users,\nwhile Facebook is more limited, as posts within a group are assigned to\nthe location of the group or the page owner. The authors of posts on\nReddit, blogs, and forums can be geolocated using the country code of\n\nThe authors of posts on\nReddit, blogs, and forums can be geolocated using the country code of\nthe internet domain or the author’s location where available.\nA key aim of the study was to investigate the challenges faced by\npet owners when administering pills to large dogs specifically. To this\nend, we specifically further segmented data for large dog breeds by\nconsidering explicit mentions of large dogs or synonyms for large\ndogs, mentions of breeds with a “standard” size, and 26 AKC large\nbreeds along with their synonyms. A further four filters were\ndeveloped to detect mentions of different oral medication types,\nspecifically pills that are (i) shiny or coated, (ii) flavored, (iii)\nnon-flavored, and (iv) soft chewables.\nFIGURE 2\nBoolean expression developed to scrape social media content from X, Reddit, blogs, and forums using Pulsar Platform™.\nFIGURE 1\nOverview of the data collection and analysis process.\n\nTarrant et al.\b\n10.3389/fvets.2025.1502236\nFrontiers in Veterinary Science\n05\nfrontiersin.org\n2.5 Topic modeling\nData scraped from X, Reddit, blogs, and forums were analyzed\nfirst. The topic modeling themes were generated by combining\nNon-Negative Matrix Factorization (NMF) and an LLM, in this case,\nGPT-4o, to identify key topics within the data. NMF is a well-known\ntopic modeling method (36) and the goal is to identify word/topic\npatterns or structures within the data (28, 37). The LLM was prompted\nto interrogate the data and extract topics. From the top five topics, the\nLLM was prompted to output the top 20 keywords associated with\neach topic for manual checking.\nThe next stage of the topic modeling analysis involved making\nsense of the ‘word salads’ that arose from the above step.\nConventionally, this can be undertaken manually; however, we chose\nto explore an automated approach that used prompting with an LLM\nto make sense of the data-driven topics and generate plausible themes\nthat could be understood by a human observer instead. The LLM was\nprompted to generate a description of each topic and assign individual\nSM posts to the topics. For data quality checks, the LLM was also\nrequired to provide example posts matching each theme, and these\nwere easily manually checked against the input data. All extracted\nexemplar posts from each theme existed within the raw data, although\nsome of the examples were slightly re-worded by the LLM. This\nprocess was repeated for Facebook data.\nFollowing the initial topic modeling exercise, the data were further\ninterrogated with specific probes to retrieve any posts discussing\nspecific issues associated with pilling.\n3 Results\n3.1 Data collection, processing, and\nfiltering\nThe initial data extraction from Reddit, X, blogs, and forums using\nPulsar Platform™ resulted in a raw dataset containing 21,340 posts,\nas shown in the data funnels illustrated in Figure 3. Following data\npre-processing to remove blank posts (e.g., Reddit posts that had been\ndeleted after publication, leaving the title available but devoid of\n\nFollowing data\npre-processing to remove blank posts (e.g., Reddit posts that had been\ndeleted after publication, leaving the title available but devoid of\ncontent), duplicates, and posts <15 words or >640 words, 17,603 posts\nwere remaining. These posts were filtered using a zero-shot approach\nutilizing the GPT-4 LLM as described previously. This left a total of\n3,629 posts for analysis.\nA total of 5,506 Facebook posts were scraped using the 32\nFacebook searches on Pulsar Platform™. Only 22 of the 32 Facebook\nsearches contained relevant data; the remaining 10 Facebook searches\nfound zero posts in the selected period. Zero duplicates were found,\nand ~90% of posts remained after removing posts <15 words and >640\nwords. The pre-processing step removed 503 posts to leave 5,003 posts.\nThe processed posts were filtered using the GPT-4 zero-shot relevancy\nfilter, leaving 1,158 posts remaining for analysis, as shown in Figure 3.\n3.2 Audience profiling and demographic\nanalysis\nOnly ~16% of posts could be confidently predicted as male or\nfemale, with most posts by users of unknown gender. There were 3,201\nunique authors in the dataset, of which 8.4% (n = 272) were female\nand 8.1% (n = 262) were male. The remaining authors were of\nunknown gender (n = 2,667).\nHowever, keywords in user biographies on X platform, the only\nplatform scraped that provides this information, appeared to show a\nslant toward female posters, with “mom,” “children,” and “family”\nbeing more closely associated with female posters. Slightly more male\nthan female posters were observed for the large dog breeds, but the\nsample size was considered to be too small to draw meaningful\nconclusions. Studies have shown that men often own larger, sporty\ndog breeds, as this reflects the owner’s self-image (38).\nAccording to the available demographic information from\nauthor’s user biographies on the X platform, the most frequently\nmentioned interests were “dogs” (n = 34, 29.1%) and “mom”\n(n = 32, 27.4%), the pronouns “she/her” (n = 31, 26.5%) and\n“animal” (n = 20, 17.1%). Not all X users have an author\n\n1%) and “mom”\n(n = 32, 27.4%), the pronouns “she/her” (n = 31, 26.5%) and\n“animal” (n = 20, 17.1%).Not all X users have an author\nbiography; therefore, these data are not available for some authors.\nThe majority of posts (see Table 1 for the distribution of posts\nbetween the top four countries) were authored by people from the\nUnited States (n = 445, 70.3%), with other English-speaking countries\nrepresented: United Kingdom (n = 144, 17.8%), Australia (n = 81,\n7%), and Canada (n = 44, 5%). Location data are deduced from the\nauthors’ profile or geolocation data. The posts collected were largely\nfrom anglophone nations, and the search expressions were written in\nFIGURE 3\nPipelines showing total social media posts extracted from X, Reddit,\nblogs, forums, and Facebook (raw data) through the data cleaning\nand pre-processing stage and zero-shot relevancy filtering. The raw\ndata from Facebook were exported from 22 separate Pulsar\nPlatform™ searches and concatenated.\nTABLE 1  Geolocation of social media posts discussing the administration\nof oral medications to dogs by top four countries with sentiment\n(positive, negative, and neutral).\nCountries\nResults\n(posts)\nPositive\nNegative\nNeutral\nUnited States\n445\n258\n176\n11\nUnited Kingdom\n144\n64\n39\n41\nAustralia\n81\n42\n13\n26\nCanada\n44\n17\n15\n12\n\nTarrant et al.\b\n10.3389/fvets.2025.1502236\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nthe English language. Posts were collected from numerous countries\nin addition to the top four countries included in Table 1. Geolocation\nof social media posts discussing the administration of oral\nmedications to dogs by the top four countries with a sentiment\n(positive, negative, and neutral). Figure  4 demonstrates the full\ngeographical spread of post locations. This shows that the SML\nnarrative around dog pilling had global reach despite the dataset\nbeing limited to posts in English rather than other languages. The\ngeolocations of the posts are focused on cities, although this could\nsimply reflect the larger human and dog populations in these areas.\n3.3 Sentiment analysis\nThe relevant SM posts were analyzed by sentiment and labeled\npositive, negative, or neutral within the platform. The wordclouds\nin Figure 5 show the keywords from the posts by relative word\nfrequency and sentiment for (A) oral medication administration\nand (B) chewable medication administration. A wordcloud is a\nvisual representation of text data, where the size of each word\ncorresponds to its frequency within the corpus of text, and the color\nof the keyword indicates the sentiment attributed to it. The majority\nof keywords had a positive sentiment, particularly in relation to\n“medication,” “home,” and “help.” It is interesting to note that\nmentions of “pain,” “pill,” and “tablets” had neutral sentiments,\nwhile mentions of “pills,” “bad,” “eat,” “prescribed,” “trying,” “gave,”\nand “giving” were negative.\n3.4 Pill-type content within social media\ndata\nThe relevant SM posts were filtered to find posts that mention\ncoated pills, flavored (palatable) pills, unflavored pills (including\nflavorless, odorless, and plain), soft or chewy medications, and posts\ndiscussing the challenges of administering pills to large dogs based on\nmentions of large dogs and AKC large dog breeds. Additional oral\nmedications and supplements such as treats, vitamins, painkillers,\nantibiotics, supplements, probiotics, and drug(s) were considered, but\n\nAdditional oral\nmedications and supplements such as treats, vitamins, painkillers,\nantibiotics, supplements, probiotics, and drug(s) were considered, but\nthese were found to bring in irrelevant mentions such as police dogs\nand other working dogs, as well as advertisements for nutribiotics for\npets. None of the five detailed filters on pill types returned a large\nvolume of posts, making it more challenging to draw meaningful\nconclusions from the data.\nOnly 0.2% (n = 10) of the SM posts met the coated pills filter.\nThere were nine unique authors, of which two were male, one female,\nand seven were of unknown gender. Topic modeling found that the\nmost frequently found topic based on post content was “pain” with\nfour mentions.\nNo posts met the flavored pills filter, while only one post matched\nthe unflavored pills filter.\nA total of 7.6% (n = 311) of posts met the filter for soft or chewy\npills, and those posts had 266 unique authors from the United States.\nFemale authors accounted for 8.3% (n = 27) posts, male authors 7.9%\n(n = 23), and unknown gender 83.8% (n = 261). Only keywords with\nover 24 mentions were included in the wordcloud (see Figure 5B), and\nnone of those keywords had a negative sentiment. The keywords “soft”\nand “medication” had positive sentiment, while “chewable” and “food”\nhad neutral sentiment. This suggests there may be a preference for soft\nand chewable pill formats.\nLooking at the 2.5% of posts (n = 101) that mention “chewable”\nspecifically, it is apparent that pet owners often face challenges when\nadministering pills to their dogs, with many turning to chewable\nmedications as a solution due to their ease of use and appeal to pets.\nChewable medications are perceived as convenient, as one user noted,\n“One chewable a month is easy to remember!” and effective, with\nanother mentioning that “Ivermectin is a super safe drug and used all\nthe time for dogs […] They get it as a monthly chewable.” The appeal\nFIGURE 4\nCoordinates map showing where the majority of social media posts discussing the administration of oral medications to dogs originate from, broken\ndown by geo-coordinates.\n\nTarrant et al.\b\n10.3389/fvets.2025.1502236\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nalso lies in the treat-like nature of these chewables, making dogs more\nlikely to consume them willingly, as one owner shared, “Our girl dog\nhas to take a chewable medicine an hour before breakfast, so we have\nto give the boy dog a treat so he  gets something too.” However,\nchewables are not always universally accepted, with some dogs refusing\nthem despite attempts to hide them in favorite foods, such as “Put it in\nham, cheese, and all their other favorites. They ate around it, but the\nmed itself.” Additionally, there are concerns about chewables being too\nappealing, as another owner expressed worry that their dog could\naccidentally ingest a large quantity if left within reach, describing how\nthe “tablets [are] yummy on purpose, which is great for ease of\nadministration, but it can obviously cause problems.” The slightly\nmixed reception highlights the balance between making medications\npalatable and ensuring safe administration practices among pet owners.\nThe large dogs’ filter matched 7% (n = 292) of posts. These posts\nwere authored by 249 unique authors from the United States. Male\nauthors accounted for 7.6% (n = 34) of posts and female authors 5.6%\n(n = 14) of posts. Trending topics were health (n = 117 posts), dogs\n(n = 115 posts), and clinical medicine (n = 113 posts), while the most\nfrequently mentioned keywords in the dataset were “medication,”\n“dogs,” and “home.” The majority of keywords in these posts had a\npositive sentiment, but it was not possible to draw any firm conclusions\non discussions around larger dogs and issues around pilling.\n3.5 Topic modeling outputs\nTo better understand the wider online narrative surrounding\npilling in dogs, we undertook topic modeling and produced topic\nsummaries on original posts surrounding the issues of administering\nnon-flavored, flavored, or chewable pills by pet owners.\nExtracted posts were pre-processed as described in the previous\nsection before conducting data-driven topic modeling using NMF on\n\nExtracted posts were pre-processed as described in the previous\nsection before conducting data-driven topic modeling using NMF on\nthe filtered and pre-processed data. There were 3,630 posts labeled as\n“relevant” by the classifier for topic modeling. An additional class\ncalled “ambivalent” was produced by the classifier (n = 160 posts), but\nthese were manually spot-checked, and the majority were found to\nbe  irrelevant to the task at hand, such as posts containing\nadvertisements or similar promotions.\nTopic modeling generated five topics (themes) along with the\ntop 20 keywords associated with each topic. An LLM approach was\nadopted to make sense of each of the five ‘word salads’ generated from\nthe topic modeling. These are presented below.\n3.5.1 Topic modeling outputs for X, Reddit, blogs,\nand forums\n3.5.1.1 Theme 1: General medication experience and\nveterinary visits\nThis theme captures posts concerned with the overall experience\nof medicating dogs, involving visits to the vet and the general process\nof administering medication. Common issues discussed include\nanxiety management, pain relief, and the challenges faced by dog\nowners when giving medication to their pets. The posts often share\npersonal anecdotes, advice, and experiences about managing their\ndog’s health, including specific incidents of medicating older dogs or\ndealing with new medication regimes. Anonymized examples include\nthose shown in Figure 6.\n3.5.1.2 Theme 2: Types of pills and general medication\npractices\nPosts under this theme (Figure 7) focus on specific types of pills,\nsuch as tablets and worm medications, and the general practices\ninvolved in giving these pills to dogs. There is mention of different\nFIGURE 6\nExample social media posts matching topic modeling Theme 1:\nGeneral medication and veterinary visits.\nFIGURE 5\nWordcloud (A) shows the most frequently mentioned keywords in\nsocial media posts relating to pet owner experiences administering\noral medications to dogs by sentiment, and (B) shows the most\nfrequently mentioned keywords in social media posts relating to pet\n\nowner experiences administering chewable oral medications to dogs\nby sentiment. Green is a positive sentiment, red is negative, and gray\nis neutral. Keyword size is proportional to the frequency of mentions\nof the keyword.\n\nTarrant et al.\b\n10.3389/fvets.2025.1502236\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nmedications, including heartworm prevention and daily medications.\nDiscussions often include the effectiveness of these medications, how\nwell dogs accept them, and tips on making the process easier, such as\nmixing pills with food to ensure these are consumed.\n3.5.1.3 Theme 3: Pill delivery methods\nThis theme is centered around the various methods and tricks\nused to successfully administer pilling medication. Posts (Figure 8)\nfrequently mention using pill pockets, treats, cheese, chicken, peanut\nbutter, and other creative ways to hide the medication. There is a focus\non the practical side of pill delivery, sharing tips and hacks among pet\nowners to ensure successful dose administration. Table 2 summarizes\nthe successful pilling strategies posted on SM.\n3.5.1.4 Theme 4: Specific medications and preventative\ntreatments\nPosts in this theme (examples in Figure  9) discuss specific\nmedications and preventative treatments, such as those for ticks, fleas,\nand heartworm. There is a particular emphasis on chewable tablets\nand oral medications. The theme covers not only the types of\nmedications but also the routines (e.g., monthly treatments) and safety\naspects of using these products.\n3.5.1.5 Theme 5: Product reviews and natural remedies\nThis theme (Figure  10) includes posts that review specific\nproducts, share links to resources, and discuss natural remedies. There\nis mention of products such as Apoquel (a film-coated or chewable\nformulation of oclacitinib maleate indicated for the treatment of\npruritus) (39). Posts mention various formats of images or links to\nthese products and discussions on natural alternatives such as garlic.\nThe focus is on sharing information about different medication\noptions, including pharmaceutical and natural treatments, and\nproviding opinions on their effectiveness and ease of use.\n3.5.2 Topic modeling outputs for Facebook\nThe same aforementioned topic modeling approach was\nundertaken using Facebook data. The resulting topic themes are\ndescribed below:\n\n3.5.2 Topic modeling outputs for Facebook\nThe same aforementioned topic modeling approach was\nundertaken using Facebook data.The resulting topic themes are\ndescribed below:\n3.5.2.1 Theme 1: Veterinary and animal care resources\nThis theme focuses on technical and informational content related\nto animal care, featuring links to resources (e.g., websites) and\ndiscussions about care techniques, adoption information, and\nveterinary advice.\n3.5.2.2 Theme 2: Foster care and adoption stories\nThis theme indicates a strong focus on the personal and emotional\naspects of fostering and adopting dogs. Descriptors such as “home,”\n“love,” “family,” and “adoption” highlight narratives or testimonials\nrelated to the experiences of fostering pets, adopting them into new\nhomes, and the affectionate bonds that develop between dogs\nand humans.\n3.5.2.3 Theme 3: Specific animal care events and logistics\nThis theme revolves around a specific animal care event where\npets can be  chipped, vaccinated for rabies and other infectious\ndiseases, and supplied with routine antiparasitics. This is an organized\nevent like a vaccination drive or informational session.\n3.5.2.4 Theme 4: Individual pet stories with a health focus\nThis theme revolves around individual stories of pets with specific\nhealth issues or care needs, particularly chronic conditions. Words like\n“diagnosed,” “cushings,” “I love my medicine wrapped […] cheese,”\nand “I love […] puzzles” suggest detailed accounts of pets’ health\njourneys and their daily care routines, which may include unique\nactivities or therapies designed for their comfort and stimulation.\n3.5.2.5 Theme 5: Special needs and high-care pets\nThis theme centers on pets with serious medical needs, as\nindicated by quotes such as “He was dying from a liver shunt when\nwe rescued him,” “He needs an echocardiogram,” and “Today was a\nvery expensive rescue day with 4 dogs.” These focus on the challenges\nand processes involved in caring for pets with serious health\nconditions, including fundraising for medical expenses and the\ncommunity support required to manage such cases.\nFIGURE 7\n\n” These focus on the challenges\nand processes involved in caring for pets with serious health\nconditions, including fundraising for medical expenses and the\ncommunity support required to manage such cases.FIGURE 7\nExamples of social media posts matching topic modeling Theme 2:\nTypes of pills and general medication practices.\nFIGURE 8\nExamples of social media posts matching topic modeling Theme 3:\nPill delivery methods and tricks.\n\nTarrant et al.\b\n10.3389/fvets.2025.1502236\nFrontiers in Veterinary Science\n09\nfrontiersin.org\n3.5.3 Thematic analysis for fear and anxiety\nWe also undertook thematic analysis using the curated data\n(n = 4,787 posts from Reddit, X, blogs, forums, and Facebook) and AI\ntools to automate our analysis. The analysis of SM posts reveals the\nfollowing frequencies of fear and anxiety-related terms:\n•\t Anxiety: 592 (12%) mentions\n•\t Scared: 128 (3%) mentions\n•\t Fear: 96 (2%) mentions\n•\t Stress: 116 (2%) mentions\n•\t Worry: 83 (2%) mentions\n•\t Nervous: 82 (2%) mentions\n•\t Afraid: 55 (1%) mentions\n•\t Panic: 33 (1%) mentions\nThe thematic analysis of SM posts reveals the following\ncommon themes related to fear and anxiety among dogs and\npet owners:\nTABLE 2  Summary of successful pilling techniques used domestically and posted on social media.\nPilling approach\nLLM interpreted strategy\nExample post(s)\nPeanut butter\nPeanut butter is a popular choice for hiding pills due to its strong flavor\nand sticky texture, which makes it difficult for dogs to spit out the pill.\n“…I dip my dogs’ pills in peanut butter. We can try that here.” /\n“Without even thinking I gave my pup a pill with peanut\nbutter…”\nCheese\nAmerican (i.e., processed) cheese is frequently used to wrap pills. The\ntexture helps envelop the pill completely, making it hard for dogs to\ndetect.\n“The art of crushing up my dogs pills and melting them into\ncheese will always amaze me.” / “Do not feel bad, my dogs will\nnot eat edible heartworm medicine and they had to have sharp\ncheddar not American cheese to take their pills”\nMeat or meat products\n(chicken, lamb, beef etc.)\nWrapping pills in slices of ham, sausage, or other types of meat is\nanother common approach. The strong flavor and scent of the meat can\neffectively mask the pill.\n“…For cats I use a pill pusher. Dogs, wrap it in meat / cheese /\npeanut butter.” / “gave my dog his pills wrapped in some\nmortadella and he gobbled it so fast he did not even have time\nto chew lmao…”\nCrushing pills and mixing with\nfood\nFor dogs who are adept at detecting whole pills, owners often crush the\npills and mix them into wet food, cheese, or other strong-smelling\nfoods.\n\n“The art of crushing up my dogs pills and melting them into\ncheese will always amaze me.” / “My little dog does not want\nmedicines. I crush a pill, mix with water and give him in his\nmouth with a syringe….”\nDistraction and deception\nAs an example, giving the dog a regular treat first and then quickly\nfollowing it with the treat containing the pill can sometimes distract the\ndog enough that they do not notice the pill.\n“If you have another dog, give that dog a slice of cheese\nwithout medicine, and then give the cheese with meds in it.” /\n“…Sometimes pills or tablets taste foul when wet, so the poor\ndogs need something that completely covers the pill without\ngetting wet….”\nFIGURE 9\nExamples of social media posts matching topic modeling Theme 4:\nSpecific medications and preventative treatments.\nFIGURE 10\nExamples of social media posts matching topic modeling Theme 5:\nProduct reviews and natural remedies.\n\nTarrant et al.\b\n10.3389/fvets.2025.1502236\nFrontiers in Veterinary Science\n10\nfrontiersin.org\n3.5.3.1 Theme 1: Vet visits and medication\nDiscussions often revolved around visits to the vet, medications,\nand concerns about how dogs react to these situations. Common\nwords include “vet,” “medication,” and expressions of uncertainty or\ndistress such as “just” and “do not know,” indicating anxiety related\nto medical care.\n3.5.3.2 Theme 2: Home and family environment\nThis theme highlights the emotional bonds between pets and\ntheir families, fostering environments, and the needs of dogs within\nthese settings. Words such as “home,” “love,” “family,” and “foster”\npoint to anxiety related to adjusting to new environments or changes\nin the household.\n3.5.3.3 Theme 3: Feeding and medication administration\nThis theme focuses on the challenges of administering medication\nto dogs, particularly pills. Common terms include “pills,” “treats,”\n“cheese,” and “peanut butter,” suggesting strategies pet owners use to\nreduce anxiety when giving medication.\n3.5.3.4 Theme 4: Tick, flea, and parasite prevention\nThis theme focuses on flea, tick, and heartworm prevention and\nassociated anxiety about infestations or treatments. Words such as\n“tick,” “flea,” “heartworm,” and “prevention” indicate worries related\nto parasites and their impact on pets.\n3.5.3.5 Theme 5: Adoption and care\nMany posts discuss adoption, care for rescue animals, and the\nneed for assistance. Terms such as “adopted,” “help,” “care,” “rescue,”\nand “need” suggest anxiety around the adoption process and providing\nadequate care for pets, especially rescues.\n3.5.4 Thematic analysis for financial costs\nTo further our research, we conducted AI-enhanced thematic\nanalysis using the methodology and datasets (n = 4,787 posts)\ndescribed in Section 3.5.3. When examining these SM posts,\nwe identified the following dominant themes related to the financial\ncosts associated with veterinary care:\n3.5.4.1 Theme 1: Veterinary and emergency costs\nA number of posts highlight that vet visits and emergency\ntreatments are a major financial burden. Users mention struggles\n\n5.4.1 Theme 1: Veterinary and emergency costs\nA number of posts highlight that vet visits and emergency\ntreatments are a major financial burden.Users mention struggles\nwith paying for unexpected surgeries or emergency vet care,\ndifficulty affording routine vaccinations and checkups, and financial\nstrain leading to delays in seeking care or choosing fewer\noptimal treatments.\n3.5.4.2 Theme 2: Medication and long-term treatment\nOngoing medication for chronic conditions such as seizures,\nallergies, or anxiety was noted as a recurring expense. This includes\nspecialized prescriptions and behavioral medications for anxiety-\nrelated issues.\n3.5.4.3 Theme 3: Pet insurance and budgeting\nSome users discussed pet insurance as a way to mitigate costs but\nalso expressed frustration over the high premiums, exclusions in\ncoverage, or limited reimbursements and expressed uncertainty about\nwhether insurance is worth the cost for aging pets.\n3.5.4.4 Theme 4: Pet adoption and initial set-up costs\nThere were mentions of people considering or delaying adoption\ndue to the adoption fees and costs such as vaccinations.\n3.5.4.5 Theme 5: Daily and lifestyle adjustments\nPet owners shared stories of having to cut back on personal\nexpenses to care for their dogs, including skipping travel, delaying\npersonal purchases, and making lifestyle adjustments to account for\npet-related expenses.\n3.5.4.6 Theme 6: Donation and support appeals\nA few posts linked to donation platforms, such as PayPal or\nGoFundMe, show that some owners (and pet shelters) rely on public\nsupport to afford care for their dogs.\nWhile themes 1–6 relate to veterinary medical prescriptions, they\nlack focus on oral medications. For this reason, the analysis was\nrepeated using a filtered dataset with n = 359 posts from Reddit, X,\nblogs, and forums that mentioned “financial,” “finance,” “cost,” “costs,”\nor “expensive.” The dominant themes from this analysis were:\n3.5.4.6.1 Financial theme 1: Recurring monthly costs\nPet owners struggle with ongoing expenses for basic dog care,\nsharing specific estimates like “I pay about $30/month for a 22 lb. dog.\n\n” The dominant themes from this analysis were:\n3.5.4.6.1 Financial theme 1: Recurring monthly costs\nPet owners struggle with ongoing expenses for basic dog care,\nsharing specific estimates like “I pay about $30/month for a 22 lb.dog.\n$10 for heartworm medicine and $8 for flea and tick prevention…,”\nwhile comparing premium versus budget options. Essential\npreventative medications add to the financial burden, along with\ninsurance premiums. These posts highlight the challenge of budgeting\nfor and sustaining these long-term costs.\n3.5.4.6.2 Financial theme 2: High medical and treatment costs\nThe expense of veterinary care represents a major concern,\nparticularly specialist visits, advanced diagnostics (blood work,\nimaging), and daily medications for chronic health or behavioral\nissues. Many express shock at how quickly vet bills accumulate, even\nfor basic care, creating significant financial stress from unexpected or\nongoing medical needs.\n3.5.4.6.3 Financial theme 3: Cost of supplements and health\nproducts\nOwners frequently mention supplements and alternative health\naids for senior dogs or those with special needs. While these products\nenhance the quality of life (QoL), they are framed as financially\nburdensome. Posts demonstrate how owners are willing to spend\nmore on their pet’s wellbeing despite the financial strain this causes.\n3.5.4.6.4 Financial theme 4: Perception of expense as a barrier to\nownership\nComments questioning whether certain products or treatments\nare “too expensive” or necessary reveal deeper concerns about\naffordability. Many owners’ resort to seeking cheaper alternatives or\nmaking compromises due to financial constraints, suggesting that\nthese barriers may ultimately deter optimal pet care or even pet\nownership altogether.\n3.5.5 Thematic analysis for perceived benefits of\ntreatment\nAdditional thematic analysis was conducted to investigate\nwhether the pet owner’s perceived benefit of treatment was a\n\nTarrant et al.\b\n10.3389/fvets.2025.1502236\nFrontiers in Veterinary Science\n11\nfrontiersin.org\ncontributing factor in veterinary medicines compliance. As before,\nwe used AI tools to analyze n = 4,787 posts from Reddit, X, blogs,\nforums, and Facebook. The dominant themes found in the data that\nwere most relevant to our study (see section 1.1. of the\nSupplementary materials for all themes and theme descriptions) were:\n•\t Anxiety and behavioral treatments\n•\t Home life and emotional support\n•\t Medical issues and specialized diets\nThe extracted themes using the full curated dataset were related\nto veterinary medicines but with a broader scope than oral\nmedications alone. Therefore, further analysis was conducted on a\nfiltered data sample containing posts mentioning keywords relating to\nthe positive benefits and the negative side of treatment. There were\n1,417 posts (40.5% of the full dataset) in the positive benefits sample\nand 807 (23%) in the negative sample. The following themes were\nextracted for the positive benefits of treatments as perceived by\npet owners:\n3.5.5.1 Effective parasite control (fleas, ticks, and heartworm)\nPet owners frequently praised medications for their efficacy in\ncontrolling external and internal parasites such as fleas, ticks, and\nheartworm. Products such as monthly chewable tablets are highlighted\nfor maintaining the health of both dogs and cats, offering convenience\nand reliable protection.\n3.5.5.2 Emotional bonding\nMedications are viewed as lifesaving and enabling deeper human–\nanimal bonds. Owners shared emotionally rich stories of adopting\npets with health conditions and successfully managing those\nconditions, reinforcing their connection and mutual trust.\n3.5.5.3 Chronic condition management and improved\nquality of life\nLong-term treatments for conditions such as arthritis, Cushing’s\ndisease, and allergies are seen as dramatically improving pets’ comfort\nand behavior. Many users report that proper medication restored their\npets’ personalities, reduced pain, and reversed weight loss.\n3.5.5.4 Daily management and owner resilience\n\nMany users report that proper medication restored their\npets’ personalities, reduced pain, and reversed weight loss.3.5.5.4 Daily management and owner resilience\nOwners describe the mental and physical effort of daily care for\nmedicated pets: administering doses, monitoring symptoms, and\nadjusting routines. Despite challenges, they express deep satisfaction\nin seeing their pets recover or maintain a good QoL.\nThe following dominant themes were extracted for the negative\naspects of treatments as perceived by pet owners:\n3.5.5.5 Ineffectiveness and trial-and-error of treatments\nMany users report frustration with the unpredictability and\ninconsistency of pet treatments. Owners often mention trying multiple\nmedications or methods without reliable results.\n3.5.5.6 Anxiety, pain, and emotional distress in pets\nA recurring theme involves treatments leading to or failing to\nalleviate anxiety, pain, or distress, especially in dogs. Owners describe\nhow treatments do not address root causes and sometimes even\nexacerbate the discomfort.\n3.5.5.7 Concerns over over-medication and side effects\nSeveral posts mention the concerns about the overuse of\nmedications, harsh side effects, and a preference for more natural or\nholistic approaches.\n3.5.5.8 Financial strain and accessibility issues\nTreatment costs are a major burden. Pet owners frequently\nreference the high expense of vet visits, medications, and emergency\ncare, making them question the value or accessibility of\nsome treatments.\n3.5.5.9 Mistrust toward vets and pharmaceutical advice\nThere’s a strong undercurrent of mistrust toward veterinary\nadvice, particularly when treatment seems profit-driven or when\nmultiple vet opinions contradict each other.\n3.5.5.10 Emotional toll on pet owners\nOwners often describe the emotional burnout and sadness\ninvolved in managing chronic illness in their pets, especially when\ntreatments are not effective. This is more prominent among those with\nlong-term caregiving roles.\n4 Discussion\n4.1 Effectiveness of social media listening\nand large language models in capturing\nreal-world pet owner experiences\n\nThis is more prominent among those with\nlong-term caregiving roles.4 Discussion\n4.1 Effectiveness of social media listening\nand large language models in capturing\nreal-world pet owner experiences\nThis study demonstrated the utility of using SML and AI-led\nanalysis using LLMs in capturing and analyzing real-world\ndescriptions of the experiences of pet owners when administering oral\nmedications to their dogs. By leveraging a commercial social listening\nplatform for data scraping with targeted search terms, the study\nsuccessfully gathered a substantial dataset from diverse SM sources,\nincluding X, Reddit, Facebook, blogs, and forums. These searches were\nintended to be broad enough to bring in sufficient posts for valid\nstatistical analysis while being sufficiently narrow to minimize noise\nfrom irrelevant posts.\nThe subsequent use of AI tools, including LLMs for data cleaning\nand filtering, ensured that the dataset was relevant and manageable,\nenabling a focused analysis of pet owner challenges in administering\noral medications to dogs.\nThe application of LLMs for zero-shot relevancy filtering proved\nto be a compelling advancement over manual classification methods.\nThis approach not only increased efficiency but also enhanced the\naccuracy of the analysis, as evidenced by the successful identification\nof relevant posts despite the initial presence of noise in the dataset,\nsuch as duplicate content and unrelated posts. This study’s\nmethodology highlights the potential of LLMs in large-scale SM\nresearch, particularly in fields where manual data handling would\nbe impractical due to the volume of data.\nWe found that the data scraped across different platforms had\nvarying levels of utility. This may be reflective of platform-specific\nbehaviors/formats. Posts from Facebook were of limited value in\nproviding insights into veterinary issues. This largely consisted of\npositive dog-related stories rather than individuals seeking help\nor solutions provided. Such information may exist on this\n\nTarrant et al.\b\n10.3389/fvets.2025.1502236\nFrontiers in Veterinary Science\n12\nfrontiersin.org\nplatform, but if so, it is likely hidden in private groups that are\ninaccessible to SML platforms. X appears to provide important\ncontent and, by the nature of the platform, maybe more closely\nassociated with the posting of problems and issues. Similar blogs\nand forums, such as Reddit, often contain long stories but with\nproblems and solutions posted alongside, which, along with X,\nappear to be a useful source of information on pet owners’ real-\nworld experiences.\n4.2 Comparison of oral medication types\nThe findings from this study reveal distinct challenges associated\nwith different types of oral medications for dogs. A prominent challenge\nis that despite a number of different pilling types/offerings, flavored\ntreatments are often preferred (1, 25), but this alone may not be a\npanacea for medication adherence issues. The topic modeling outputs for\nX, Reddit, blogs, and forums revealed several pill delivery techniques\n(Theme 3: Pill delivery methods), including the use of pill pockets and/\nor treats to hide pills, chicken or strong-smelling foods such as cheese\nand peanut butter to entice reluctant dogs to eat crushed pills. However,\nit should be noted that oral products are carefully formulated to ensure\nreliable and predictable drug dissolution, absorption, and performance,\nand the impact of an owner crushing, powdering, mixing, or hiding the\nmedication with foods/liquids, etc. (as in Table 2. Summary of successful\npilling techniques used domestically and posted on social media.) may\nalter drug performance. The impact of this is largely unknown; therefore,\nincorporating easy-to-give characteristics into the product at the\ndevelopment phase removes such uncertainty of outcome. The findings\nunderscore the need for continued innovation in medication\nformulation, potentially incorporating advanced flavor enhancers or\nnovel delivery systems to improve compliance.\n4.3 Geographical and demographic\nvariations\nThe geographic analysis of SM posts discussing the administration\n\n4.3 Geographical and demographic\nvariations\nThe geographic analysis of SM posts discussing the administration\nof oral medications to dogs reveals an international presence across a\nnumber of countries on these topics, unsurprisingly dominated by the\nUnited States contributing the majority (n = 445, 70.3%), followed by\nthe United Kingdom (n = 144, 17.8%), Australia (n = 81, 7%), and\nCanada (n = 44, 5%). This distribution reflects the language of the\nsearch terms, which were in English, and possibly the higher\nprevalence of dog ownership in these regions. Posts are predominantly\nlocated in urban areas, likely due to the larger human and pet\npopulations in cities, which could skew the geographic distribution\ntoward these areas. There was no particularly noticeable difference in\nthe ratios of men and women posting on these issues.\nSentiment analysis of these posts indicates a generally positive\noutlook among pet owners when discussing medication administration\nto dogs. Positive sentiments were particularly associated with keywords\nsuch as “medication,” “home,” and “help,” suggesting a supportive and\nsolution-oriented discourse. However, negative sentiments emerged in\ndiscussions involving difficulties with pill administration, highlighted\nby words such as “pills,” “bad,” “eat,” and “trying.” Interestingly,\nmentions of “pain” and “tablets” remained neutral, reflecting a more\nfactual and less emotionally charged tone compared with other topics\non SM. Overall, while the sentiment leans positive, there is a\nmeaningful portion of posts reflecting the challenges and frustrations\nof medicating dogs, especially regarding pill forms and large dog\nbreeds. A marginal predominance of male dog owners posting about\nlarge dog breeds was observed, though the limited sample size\nprecludes definitive conclusions. Previous research studies have shown\nthat men frequently select larger, more athletic dog breeds, as these\nchoices often reflect the owner’s self-image and identity (38).\n4.4 Non-compliance with preventative,\nshort, and long-term medications\n\nPrevious research studies have shown\nthat men frequently select larger, more athletic dog breeds, as these\nchoices often reflect the owner’s self-image and identity (38).4.4 Non-compliance with preventative,\nshort, and long-term medications\nAdenot et al. and Lavan et al. both identified pet owner-related\nfactors as major causes of treatment non-compliance, including\nadministering doses at incorrect times, outside optimal efficacy\nintervals, or creating gaps in medication purchase (1, 2). Posts in\nTheme 4: Specific medications and preventative treatments (section\n3.5.1) captured the online discourse on the topic of chewable and\nother oral medications, the safety of these products, and dosing\nroutines, suggesting that pet owners are seeking support from other\npet owners. Pet owners may demonstrate higher compliance when\ntreating discernible and/or chronic conditions that visibly impact the\npet’s QoL, when treatments are of short duration (such as antimicrobial\ncourses), or when they fully comprehend the prescribed treatment\nprotocol (3, 4, 15). Communication and education are, therefore,\nfundamental to improving compliance across all veterinary care areas.\nA study by Odom et al. reported that 95% of pet owners surveyed\nclaimed to understand the prescribed treatments either “very well” or\n“extremely well,” suggesting effective vet-client communication.\nHowever, Adams et al. discovered that while initial antimicrobial\ncompliance rates were high (as measured by container openings), they\ndeclined sharply when daily dosing exceeded twice daily, a finding\nlater corroborated by Odom et al. (3, 4). Both studies recommended\nless frequent dosing regimens to improve compliance, with Odom\net al. demonstrating the effectiveness of combining this approach with\na long-acting ectoparasiticide (3). The latter study also concluded that\nadequate veterinary medication instructions are crucial to enhancing\npet owner compliance (3). A review by Manasa et al. found long-\nacting formulations to be effective in treating numerous animal health\nconditions across species, including chronic conditions, pain\n\nA review by Manasa et al.found long-\nacting formulations to be effective in treating numerous animal health\nconditions across species, including chronic conditions, pain\nmanagement, novel antiparasitics, and the reduction of anxiety and\nstress. The authors also postulated that long-acting medications have\nthe potential to transform veterinary care by improving treatment\ncompliance and reducing the challenges pet owners face when\nadministering medications to their pets (40).\nPreventative treatments, particularly antiparasitics, typically\nexhibit lower compliance rates because pet owners often fail to\nrecognize their necessity (12, 30, 31). Although our data did not\nexplicitly reveal pet owner reluctance toward antiparasitics,\nwe identified “a strong undercurrent of mistrust toward veterinary\nadvice, particularly when treatment seems profit-driven” that may\nbe related. Conversely, some pet owners discussed “concerns about\nflea, tick and heartworm prevention and associated anxiety about\ninfestations or treatments” on Facebook (in Section 3.5.4, Theme 4:\nTick, flea, and parasite prevention), using “words [that] indicate\nworries related to parasites and their impact on pets.” Veterinarians\ncan improve pet owner compliance with flea and tick prevention\n\nTarrant et al.\b\n10.3389/fvets.2025.1502236\nFrontiers in Veterinary Science\n13\nfrontiersin.org\nthrough several methods: offering financial incentives such as\nvouchers and discounts, implementing smartphone reminder systems,\nenhancing parasite education, and recommending extended-duration\nparasite control products (2). According to a recent study,\napproximately 50% of pet owners stated they received no\ndemonstration on medication administration, while one-third\nencountered difficulties, primarily due to resistant pets (3).\nProlonged medication regimens can significantly diminish\ntreatment efficacy over time. Research has demonstrated that\nextended pharmaceutical use often leads to the development of drug\nresistance (21, 40). While antimicrobial resistance is widely\nrecognized, resistance to antiparasitic treatments poses serious\nconcerns. For instance, fipronil-resistant flea populations have\nemerged, drastically reducing the efficacy of these once-reliable\ntreatments (22). This reality is reflected in pet owner experiences:\n“We adopted a stray kitten and have treated her twice with Kitten\nFrontline. On the skin not the fur. I have two other cats and a dog.\nNever have I had a pet resistant to treatment! We have bathed her\ntwice, applied medication twice and the fleas persist.” This pattern\nextends to veterinary medicine, where extended or improperly\nmanaged antibiotic courses in dogs treating chronic conditions such\nas recurrent skin infections contribute to antimicrobial resistance\n(21). Similarly, long-term monthly fipronil administration has been\nselected for resistant flea strains, and resistant heartworm-causing\nDirofilaria immitis populations have developed, undermining\npreviously effective therapeutic approaches (23).\nFor pets with chronic conditions, the positive response to\ntreatment, as evidenced by the pet owner’s perceived benefits analyzed\nin Section 4.6, suggests that demonstrable improvements to a pet’s QoL\nare more likely to encourage treatment adherence. Odom et al. found\nthat senior dogs had significantly higher medication compliance rates.\n\n6, suggests that demonstrable improvements to a pet’s QoL\nare more likely to encourage treatment adherence.Odom et al.found\nthat senior dogs had significantly higher medication compliance rates.\nThis could be  due to older pets having more medical conditions\nrequiring treatment and older pets being more used to receiving\ntreatment. Owners will likely have gained more experience\nadministering medications and may have higher awareness and\nunderstanding of the importance of medication compliance for their\npet (3), although older pets visit their veterinarian for preventatives less\nfrequently than their younger counterparts (13). Chronic conditions\nlead to more frequent veterinary interactions, thereby creating more\nopportunities for vet–client communication and enhancing compliance\n(13). Social pressures may also apply with pet owners being compelled\nto care properly for visibly unwell older pets. Conditions such as\nosteoarthritis may require major changes in daily routine and introduce\nstrong motivation for compliance (3).\n4.5 Financial factors affecting compliance\nFinance is a barrier for many pet owners that “may ultimately\ndeter optimal pet care or even pet ownership altogether” (Financial\ntheme 4: Perception of expense as a barrier to ownership). Purchase\ncompliance is thought to be low (11) as fewer doses are sold compared\nto veterinarian recommendations (5, 6, 18), “many [pet] owners’\nresort to seeking cheaper alternatives” (financial theme 4), and some\npet owners are unable to afford treatment. Financial theme 1:\nRecurring monthly costs (in Section 3.5.4) are concerned with pet\nowners’ financial challenges paying for essential dog care, including\nantiparasitics. Although these are an essential part of responsible pet\nownership (12), they “add to the financial burden.” Fortunately, “posts\ndemonstrate how owners are willing to spend more on their pet’s\nwellbeing; despite the financial strain” (Financial theme 3: Costs of\nsupplements and health products).\nEmergency care (Section 3.5.4, Theme 1: Veterinary and\nemergency costs) is another “financial strain leading to delays in\n\nEmergency care (Section 3.5.4, Theme 1: Veterinary and\nemergency costs) is another “financial strain leading to delays in\nseeking care or having to choose less optimal treatments.” The\nrecurring expenses incurred when caring for a pet with a chronic\ncondition (Theme 2: Medication and long-term treatment in section\n3.5.4) also cause financial pain. Pet owners “express shock at how\nquickly vet bills accumulate, even for basic care, creating significant\nfinancial stress from unexpected or ongoing medical needs” (Financial\ntheme 2: High medical and treatment costs). “Pet insurance [can]\nmitigate costs,” but pet owners “expressed frustration over the high\npremiums” and question “whether insurance is worth the cost for\naging pets (Theme 3: Pet insurance and budgeting in Section 3.5.4).\nA sick pet leads to “making lifestyle adjustments” including\ncutting back on other expenses to pay veterinary fees (Theme 5: Daily\nand lifestyle adjustments, section 3.5.4). Crowd funding is also\nmentioned (Theme 6: Donation and support appeals, section 3.5.4)\nwith “posts linked to donation platforms like PayPal or GoFundMe.”\nImplementing strategies like monthly payment options and\npersonalized yearly care plans for pets can help overcome financial\nobstacles to veterinary treatment (13, 14), particularly with routine\npreventative treatments such as antiparasitics and annual vaccinations.\n4.6 Pet owner perceived benefits of\ntreatment\nPet owners are more likely to adhere to treatment if that treatment\nvisibly improves the pet’s QoL (14). Improvements to a pet’s overall\nQoL are just one of the pet owner’s perceived benefits of treatment as\nfound in our “Perceived benefits” dataset. Demonstrable improvements,\nsuch as “medications are viewed as lifesaving” (Emotional bonding\ntheme), indicated that a positive response to treatment is more likely\nto encourage treatment adherence for pets with chronic conditions.\nThis is reflected in the “Daily management and owner resilience” theme\nwhere pet owners said that “Despite challenges, they express deep\nsatisfaction in seeing their pets recover or maintain a good [QoL].”\n\nThis is reflected in the “Daily management and owner resilience” theme\nwhere pet owners said that “Despite challenges, they express deep\nsatisfaction in seeing their pets recover or maintain a good [QoL].”\n“Home life and emotional support” and “Medical issues and\nspecialized diets” were dominant themes in the full dataset (Reddit, X,\nblogs, forums, and Facebook, as described in Section 3.5.5). These\nthemes are likely to be  related to the management of chronic\nconditions rather than short-term or preventative treatments.\nOngoing treatments for conditions such as arthritis, Cushing’s disease,\nand allergies are viewed as “dramatically improving pets’ comfort”\nwith “Many users report[ing] that proper medication restored their\npets’ personalities, reduced pain and reversed weight loss.” (Chronic\ncondition management and improved quality of life theme, Section\n3.5.5). Caring for a pet requiring daily medications is a “mental and\nphysical effort […] administering doses, monitoring symptoms, and\nadjusting routines,” meaning that pet owners must develop resilience\n(Daily management and owner resilience, Section 3.5.5).\nPreventative measures such as antiparasitics are also seen as\nbeneficial, with “Pet owners frequently praised medications for their\nefficacy in controlling external and internal parasites” and “monthly\nchewable tablets are highlighted for maintaining the health of both\ndogs and cats, offering convenience and reliable protection” [3.5.5\nEffective parasite control (fleas, ticks, and heartworm)].\n\nTarrant et al.\b\n10.3389/fvets.2025.1502236\nFrontiers in Veterinary Science\n14\nfrontiersin.org\n4.7 Negative aspects of treatment as\nperceived by pet owners\nNegative perceptions of treatment included “frustration with the\nunpredictability and inconsistency of pet treatments” where\nveterinarians try “multiple medications or methods without reliable\nresults.” (Ineffectiveness and trial-and-error of treatments theme,\nSection 3.5.5). “Mistrust toward vets and pharmaceutical advice” was\nof particular note, especially “when multiple vet opinions contradict\neach other” (Mistrust toward vets and pharmaceutical advice theme,\nSection 3.5.5).\nOur data illustrated pet owner concerns about “overuse of\nmedications, harsh side effects…” (Concerns over over-medication\nand side effects theme, section 3.5.5). This is consistent with Odom\net al. (3), who found that some pet owners stopped treatment after\ntheir pet showed signs of potential side effects such as diarrhea (3).\nThe “Emotional toll on pet owners” theme (section 3.5.5) was\npoignant as pet owners caring for chronically ill pets “often describe\nthe emotional burnout and sadness involved in managing chronic\nillness in their pets, especially when treatments are not effective.”\n4.8 Implications for veterinary\nprofessionals and pharmaceutical\ncompanies\nStudies by Lavan et al. and Mwacalimba et al. reveal a considerable\ngap between recommended parasiticide treatment and actual oral\nprevention coverage (7–9, 19). To improve adherence, veterinary\nprofessionals should emphasize year-round protection to pet owners\nand recommend longer-acting medications, which have improved\ncompliance (18, 19). However, recommendations by country vary, and\na risk-based approach may be preferred over continuous dosing where\nthe parasite is not endemic (20). Additionally, addressing the tendency\nof owners to purchase only one dose annually can enhance treatment\noutcomes and overall canine health.\nOne alternative to pilling is via some form of injection, which may\nbe potentially long-lasting, thereby overcoming the issues of regular\npill dosing needed and associated issues with prescription compliance\n\nOne alternative to pilling is via some form of injection, which may\nbe potentially long-lasting, thereby overcoming the issues of regular\npill dosing needed and associated issues with prescription compliance\n(41). However, not all dogs are a good fit for injectable treatments such\nas heartworm preventatives (8–10). Although we did not explicitly use\nwords associated with injections as part of our search strategy,\nwe naively proposed that this may arise as part of the discussions\naround pilling and pilling alternatives. However, in the dataset used\nin this analysis, there were few mentions of injections or injectables in\nthe posts (e.g., ProHeart 6, a 6-monthly vet-administered injectable to\nprevent Heartworm disease in dogs, manufactured by Zoetis Inc., was\nmentioned, Figure 9). This suggests either little understanding of\nalternative drug administration methods or no particular desire to\nseek alternatives to domestically administrated pilling.\n4.9 Limitations\nAs with any study that uses a sample population to understand the\nwider population, there are potential limitations associated with\nsampling methodology. Reliance on SM data collection introduces\npotential biases as the personality types that choose to post on SM\nmay not represent the broader pet owner population. Nonetheless,\ngiven the popularity and propensity of SM in modern daily lives and\nthe non-controversial nature of the topics discussed here, such risks\nare thought to be minimal. While our comprehensive multi-platform\napproach captured a rich diversity of experiences, future research\ncould enhance this methodology by developing sophisticated\ntechniques to cluster posts from the same author that would allow for\neven more precise analysis by accounting for multiple submissions\npotentially originating from the same individuals across different\nplatforms, potentially reducing overrepresentation of certain\nexperiences or incidents. The development of search expressions\nfocused on the topic of dog pilling may have limited the dataset’s\ncomprehensiveness. To mitigate this, we specifically tried to optimize\n\nThe development of search expressions\nfocused on the topic of dog pilling may have limited the dataset’s\ncomprehensiveness.To mitigate this, we specifically tried to optimize\nsensitivity over specificity in the initial data scrapes and then filtered\nthe resulting data off-platform for relevancy, etc. Additionally,\nconstraints on Facebook’s search feature, specifically the search syntax\nand the limit to 20 keyword combinations per search, plus the need to\nfilter out unwanted content, may potentially have led to the exclusion\nof some relevant posts. The Facebook restriction meant that to\nbe practicable, and multiple searches had to be run that focused on the\nmost popular large dog breeds and mentions of dogs in general as\nopposed to searching for all large dog breeds, mentions of large dogs,\nand breeds that come in large (standard) size. This will not have\nunduly affected the results, as irrelevant posts should have been\nfiltered out by the zero-shot filter.\n4.10 Future work\nFuture research should consider broadening the demographic\nscope to include non-English language posts, providing an expanded\nglobal perspective on pet owner challenges in administering\nmedications. Expanding the scope could also aid in creating a more\ncomprehensive dataset. Conducting longitudinal studies to track\nchanges in attitudes and experiences over time will offer deeper\ninsights into evolving trends in the landscape of veterinary care and\npet medication administration. Incorporating image analysis\nalongside text analysis, which is now feasible with the latest (in 2024)\ngeneration of LLMs, could enhance the understanding of SM content\nwhile comparing findings with traditional survey methods could\nvalidate the results.\nExploring real-time SM monitoring could aid in the early\ndetection of medication-related issues or trends, and applying\nsimilar methodologies to study medication administration in\nother companion animals, such as cats, will expand the research’s\napplicability. Developing targeted interventions based on these\nfindings can improve medication adherence, ultimately leading to\n\nDeveloping targeted interventions based on these\nfindings can improve medication adherence, ultimately leading to\nbetter health outcomes for pets. Additionally, further refining\nrelevancy filters and addressing issues such as unwanted content\nin SML studies will enhance data quality and reproducibility.\nDeveloping and testing targeted interventions based on the\nfindings to improve medication adherence would be a logical next\nstep in this research.\nUnforeseen content matches that are irrelevant to the present\nstudy are almost inevitable in many SML studies. Future work to aid\nresearchers in removing this kind of content would be of great utility.\n\nTarrant et al.\b\n10.3389/fvets.2025.1502236\nFrontiers in Veterinary Science\n15\nfrontiersin.org\n5 Conclusion\nThis study provides key insights into the challenges pet \nowners face in administering oral medications to their dogs. \nWe  demonstrated that of those encountering problems, many \nreport issues of fear and anxiety about the pilling process. We have \nfound that chewables can be more popular and easier-to-use in \nsome cases, but there is a fear of over-dosing if the pills are seen \nas too appetizing. This may, in part, be  mitigated by proper \nstorage of these medications including the use of a suitable, safe \ndelivery point for home delivery of medications.\nOur SML data has also been used to compile a list of the most \nsuccessful strategies used to successfully administer oral medication. \nThis may be  useful for vets and owners alike. Successful pilling \napproaches adopted by pet owners included hiding pills in peanut \nbutter, cheese, meat, or meat products such as chicken, lamb, or beef; \ncrushing pills and mixing them with food; or distraction and deception \ntechniques like feeding the dog a regular treat before feeding a treat \ncontaining a pill (see Figure 11). Veterinarians may wish to default to \nchewable or palatable formulations when they are available or consider \nalternatives to oral medications when longer-lasting injectables are \nindicated and available to reduce pet owner anxiety and stress.\nFIGURE 11\nInfographic summarizing the overall methodology, social media data collection, sentiment analysis, major challenges for pet owners when \nadministering oral medications, and common strategies for success, as well as key takeaways from the study.\n\nTarrant et al.\b\n10.3389/fvets.2025.1502236\nFrontiers in Veterinary Science\n16\nfrontiersin.org\nThe novel methodology has proved valuable in uncovering real-\nworld data, complementing traditional research methods such as\nsurveys and focus groups. Such work might also inform and shape\nquestionnaires on specific veterinary topics.\nData availability statement\nThe data supporting the conclusions of this article will be made\navailable by the authors, without undue reservation.\nEthics statement\nThe studies involving humans were approved by the University of\nSurrey Ethics Committee and a favorable ethical opinion was granted,\nreference FHMS 21–22,026 EGA—Amend 3. The studies were conducted\nin accordance with the local legislation and institutional requirements.\nWritten informed consent for participation was not required from the\nparticipants or the participants’ legal guardians/next of kin in accordance\nwith the national legislation and institutional requirements.\nAuthor contributions\nGT: Data curation, Formal analysis, Investigation, Methodology,\nProject administration, Visualization, Writing  – original draft,\nWriting – review & editing. TR: Formal analysis, Investigation,\nMethodology, Software, Visualization, Writing – review & editing.\nAW: Conceptualization, Investigation, Writing – review & editing.\nTS: Investigation, Methodology, Project administration, Software,\nVisualization, Writing – review & editing. KW: Conceptualization,\nInvestigation, Methodology, Supervision, Writing  – review &\nediting.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article.\nAcknowledgments\nThe authors would like to acknowledge the support of Zoetis in\nthe ideation of this study.\nConflict of interest\nAW was employed by Zoetis Inc.\nThe authors declare that this study received funding from Zoetis\nunder the auspices of a wider collaboration: Surrey DataHub within\nthe Veterinary Health Innovation Engine (vHive).\nThe funder had the following involvement in the study:\nconceptualization, decision to publish, approval of the manuscript.\n\nThe funder had the following involvement in the study:\nconceptualization, decision to publish, approval of the manuscript.\nThe author(s) declared that they were an editorial board member of\nFrontiers, at the time of submission. This had no impact on the peer\nreview process and the final decision.\nGenerative AI statement\nThe author(s) declare that Gen AI was used in the creation of this\nmanuscript. Generative AI was used for intermediate stage text\nediting. This is in addition to Generative AI being used to process and\nextract topics from the data as described in the manuscript.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. Any product\nthat may be evaluated in this article, or claim that may be made by its\nmanufacturer, is not guaranteed or endorsed by the publisher.\nSupplementary material\nThe Supplementary material for this article can be found online\nat: https://www.frontiersin.org/articles/10.3389/fvets.2025.1502236/\nfull#supplementary-material\nReferences\n1.\tAdenot CC, Abdelhakim HE. 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+{"text": "【兽医文献】\n标题: Temporal transcriptomic profiling of channel catfish (Ictalurus punctatus) gills after acute copper exposure reveals suppressed immune gene responsiveness to Flavobacterium covae infection\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nTYPE  Case Report\nPUBLISHED  03 June 2026\nDOI  10.3389/fvets.2026.1854874\nOPEN ACCESS\nEDITED BY\nDavid Levine,\nUniversity of Tennessee at Chattanooga,\nUnited States\nREVIEWED BY\nAjay Vikram Singh,\nFederal Institute for Risk Assessment\n(BfR), Germany\nJohnny Altwal,\nCornell University College of Veterinary\nMedicine, United States\n*CORRESPONDENCE\nLucas Rannier Ribeiro Antonino\nCarvalho\nlucas.carvalho@ki.se\nRECEIVED 14 April 2026\nREVISED 16 May 2026\nACCEPTED 20 May 2026\nPUBLISHED 03 June 2026\nCITATION\nde Souza MMN, de Lima RR, de\nAraújo DV, Dantas AFM and Carvalho\nLRRA (2026) Customized 3D orthopedic\nexoprostheses for dogs with\namputations and congenital\nmalformations: a case series study.\nFront. Vet. Sci. 13:1854874.\ndoi: 10.3389/fvets.2026.1854874\nCOPYRIGHT\n© 2026 de Souza, de Lima, de Araújo,\nDantas and Carvalho. This is an\nopen-access article distributed under\nthe terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other\nforums is permitted, provided the\noriginal author(s) and the copyright\nowner(s) are credited and that the\noriginal publication in this journal is\ncited, in accordance with accepted\nacademic practice. No use, distribution\nor reproduction is permitted which does\nnot comply with these terms.\nCustomized 3D orthopedic\nexoprostheses for dogs with\namputations and congenital\nmalformations: a case series\nstudy\nMiriã Mamede Noronha de Souza 1,2, Ramon Rodrigues de Lima 2,\nDannielly Virgínia de Araújo 3, Antonio Flávio Medeiros Dantas 1\nand Lucas Rannier Ribeiro Antonino Carvalho 2,4*\n1Postgraduate Program in Animal Science and Health, Center for Rural Health and Technology, Federal\nUniversity of Campina Grande, Patos, Brazil, 23D Medicine Vet, João Pessoa, Brazil, 3Veterinary\nMedicine Residency Program, Federal Rural University of Pernambuco (UFRPE), Recife, Brazil,\n4Department of Physiology and Pharmacology – Fyfa, Karolinska Institutet, Stockholm, Sweden\nLimb loss, particularly in cases of partial amputation, and congenital limb mal­\nformations in dogs may affect mobility, biomechanics, and quality of life. In this\n\nIn this\ncontext, customized orthopedic exoprostheses emerge as a promising therapeu­\ntic alternative, capable of partially restoring locomotor function, redistributing\nloads, and improving postural stability. This study aimed to describe the develop­\nment and clinical application of customized three-dimensional (3D) orthopedic\nexoprostheses in dogs with amputations and congenital malformations, using a\ncase series design. Six canine patients were included, three with involvement of\nthoracic limbs and three with involvement of pelvic limbs. Regarding etiology,\nfour cases were associated with partial limb amputations and two with congenital\nmalformations. All patients underwent clinical evaluation, anatomical measure­\nments, and individualized prosthetic planning. The three-dimensional models\nwere developed in software and printed in polylactic acid (PLA) using fused depo­\nsition modeling (FDM) technology. After hand-finishing, padding, and securing\nwith compressive bandages, the devices were adapted to the patients’ residual\nlimbs. Clinical monitoring was conducted, with emphasis on assessing func­\ntionality, skin integrity, gait stability, and response to rehabilitation. The results\ndemonstrated good acceptance by the animals, with no skin lesions, mechani­\ncal failures, or definitive refusal to use the devices. In all cases, improvements in\nmobility, stability, and quality of life were observed, with progressive adaptation\nand functional return to locomotion. Therefore, this study highlights the clinical\npotential of producing veterinary orthopedic exoprostheses using 3D printing,\nemphasizing its potential as an accessible, personalized, and functional strategy\nfor the rehabilitation of dogs with missing or malformed limbs.\nKEYWORDS\ncanine, prosthetics, rehabilitation, socket, three-dimensional printing\n\nde Souza et al.\b\n10.3389/fvets.2026.1854874\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nIntroduction\nExoprostheses are devices that function as partial or total substi­\ntutes for lost limbs, contributing to body weight redistribution and\nassisting in postural stability and locomotor functionality during\nstance and ambulation (1, 2). In animals, limb loss and the resulting\nbiomechanical alterations directly impact locomotor performance and\noverall well-being and are associated with reduced social activ­\nity (3, 4).\nOrthopedic disorders affect a relevant proportion of the animal\npopulation and are frequently associated with traumatic injuries (5).\nComplete limb amputation is widely performed in veterinary medi­\ncine and has been associated with generally favorable outcomes, with\nmost dogs achieving good functional adaptation and satisfactorily to\ngood quality of life following surgery (6, 7). Similar outcomes have\nalso been reported in feline populations (8). However, variations in\nactivity levels, exercise tolerance, and behavioral patterns have been\nreported in a subset of cases, as perceived by owners (6, 7).\nIn contrast, partial limb amputation remains less frequently\nreported in the literature, with limited evidence regarding long-term\nfunctional outcomes and owner-reported satisfaction. Although func­\ntional adaptation may occur, partial limb loss has been associated with\na higher rate of complications and less well-defined long-term out­\ncomes, highlighting a gap in current knowledge (9).\nThese conditions, although etiologically distinct, share a common\noutcome characterized by limb deficiency and consequent biome­\nchanical imbalance. Another cause of biomechanical alteration is con­\ngenital abnormalities which represent one of the main causes of\nneonatal disorders and are characterized by structural and/or func­\ntional deformities that develop during gestation or are present at birth,\npotentially compromising the animal’s viability (10).\nSimilar to amputations, which may result from both conditions,\npartial or total limb loss can affect the animal’s quality of life, hinder­\n\nSimilar to amputations, which may result from both conditions,\npartial or total limb loss can affect the animal’s quality of life, hinder­\ning participation in rehabilitation therapies and increasing the risk of\nsecondary deformities and joint degeneration, in addition to reducing\nphysical activity levels and functional autonomy (11).\nIn this context, additive manufacturing technology has emerged\nas a transformative tool in veterinary medicine, with applications\nacross different fields, including orthopedics, dentistry, and, more\nrecently, surgical planning. According to Lima and Carvalho (12), this\ntechnology enables the creation of anatomical models from imaging\nexaminations, facilitating structural visualization, and supporting\nclinical decision-making. Furthermore, the availability of affordable\nprinters and software has increased the feasibility of this approach.\nIn veterinary medicine, orthotic and prosthetic devices are used\nto support locomotor function in patients with limb loss or impair­\nment. Prostheses can be classified as endoprostheses, when surgically\nanchored to bone structures, or exoprostheses, when externally fitted\nto the residual limb, with the primary aim of restoring stability and\nreducing the biomechanical alterations associated with segmental\nabsence (11). Orthoses, in turn, are external devices designed to sup­\nport, protect, or control the movement of body segments, allowing\nrestriction or guidance of joint mobility according to clinical\nneeds (13).\nIn addition, prostheses and orthoses produced through 3D print­\ning have become increasingly important for the functional reintegra­\ntion of patients, promoting restoration of well-being and segmental\nkinetic recovery, as well as reducing pain associated with prolonged\nbiomechanical overload (4, 14). Within this framework, different\nthermoplastic materials can be employed, including polylactic acid\n(PLA), thermoplastic polyurethane (TPU), and nylon, each with dis­\ntinct mechanical properties that directly influence device performance\nunder weight-bearing conditions. While PLA is widely used due to its\n\nWhile PLA is widely used due to its\nease of processing and biocompatibility, it presents limited impact\nresistance (15), TPU provides superior flexibility and energy absorp­\ntion, which are advantageous in dynamic loading environments (16),\nwhereas polyamides, such as nylon, offer higher mechanical strength\nand durability, making them more suitable for high-stress applications\nin locomotor devices (17).\nThe effective design and fabrication of orthopedic exoprostheses\nrequire a comprehensive understanding of anatomical, kinetic, and\nbiomechanical principles to ensure optimization of the interaction\nbetween the device and the patient (18). The suitability of animals for\nexoprosthetic rehabilitation is influenced by multiple factors, includ­\ning species, patient temperament and behavior, neurological and\northopedic status, as well as the caregiver’s ability to maintain appro­\npriate follow-up and daily management of the device (19). In addition,\nthe use of these devices may be discouraged in aggressive animals\nbecause of the difficulties associated with handling, adaptation, and\nlong-term safe use (13). Regarding residual limb characteristics, pres­\nervation of approximately 50% of the radius/ulna or tibia/fibula has\nbeen associated with improved prosthetic attachment and may facili­\ntate the use of socket-based prostheses when clinically indicated for\nrehabilitation (20).\nDespite technological advances, there remains a scarcity of litera­\nture addressing the clinical application of 3D-printed orthopedic exo­\nprostheses in dogs, particularly considering the anatomical, adaptive,\nand biomechanical challenges associated with these devices.\nConsidering this scenario, the present study aimed to describe the\ndevelopment and application of customized 3D-printed orthopedic\nexoprostheses in dogs with partial limb amputations and congenital\nmalformations affecting limb development, through a case series\nusing three-dimensional modeling and 3D printing techniques as a\nfunctional and accessible therapeutic alternative.\nMethods\nThis study was conducted in collaboration with the private com­\n\nMethods\nThis study was conducted in collaboration with the private com­\npany 3D Medicine—3D Solutions for Health, located in João Pessoa,\nBrazil. The study was performed in accordance with ethical principles\nand approved by the Animal Use Ethics Committee of the Federal\nUniversity of Campina Grande (CEUA/CSTR) under protocol\nnumber 44.2024.\nThe development of digital models for the fabrication of custom­\nized exoprostheses was performed using the three-dimensional com­\nputer-aided design (CAD) software Blender® (Blender Foundation,\nAmsterdam, Netherlands), based on direct anatomical measurements\nof each patient obtained using measuring tape, digital calipers, and\nrulers. Advanced imaging techniques, such as computed tomography\nor three-dimensional rendering, were not used. The main collected\nparameters included residual limb height relative to the ground with\nproximal joints anatomically aligned, residual limb circumference at\nthree different levels, limb diameter, and the distance between the\ndistal end of the residual limb and the nearest proximal joint. These\nmeasurements were used to create a simple cylindrical three-dimen­\nsional digital phantom of the residual limb, intended primarily to\n\nde Souza et al.\b\n10.3389/fvets.2026.1854874\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nprovide a spatial reference for the anatomical dimensions and propor­\ntions of each patient. This phantom served as the anatomical basis for\nindividualized prosthetic modeling, allowing adaptation of socket\ndimensions, limb alignment, and prosthetic height according to the\nmorphology and biomechanical requirements of each animal.\nThe three-dimensional files were exported to the slicing software\nBambuStudio® (v.1.10.1.50, BambuLab, Shenzhen, China), which gen­\nerated layer-by-layer printing instructions for Fused Deposition\nModeling (FDM) technology. The devices were manufactured using\nan X1 Carbon Combo 3D printer (BambuLab, Shenzhen, China), with\npolylactic acid (PLA) selected as the printing material due to its bio­\ncompatibility, compressive strength, low cost, and renewable origin\n(derived from corn starch and sugarcane) (21).\nThe printing protocol was adapted from Carvalho (16) and\nincluded the following technical parameters: 0.4-mm nozzle diameter,\n0.2-mm layer height, 1.6-mm wall thickness, 50% gyroid infill pattern,\nprinting speed ranging from 200 to 250 mm/s, and tree-type support\nstructures when required. All prostheses were developed using stan­\ndardized technical parameters, including material selection and print­\ning configurations, to ensure adequate mechanical resistance, reduced\nweight, and patient comfort.\nAfter printing, support structures were manually removed, and\neach component underwent fine sanding for surface finishing.\nSubsequently, the exoprostheses were internally lined with acrylic\npadding and cushioned fabric to enhance comfort, prevent abrasions,\nand improve adaptation to the residual limb. Fixation was achieved\nusing compressive bandages individually adjusted according to the\nmorphology of the residual limb. Additionally, the bases of the pros­\ntheses were fitted with anti-slip rubber soles to improve ground trac­\ntion and prevent slipping during ambulation.\nImmediately after prosthesis fitting, device acceptance was quali­\n\nAdditionally, the bases of the pros­\ntheses were fitted with anti-slip rubber soles to improve ground trac­\ntion and prevent slipping during ambulation.Immediately after prosthesis fitting, device acceptance was quali­\ntatively evaluated based on weight-bearing capacity, gait performance,\nand behavioral response to the device. Acceptance was classified as:\n(1) poor, characterized by hesitation or resistance to prosthesis use,\ninconsistent or absent weight-bearing, and difficulty initiating ambu­\nlation; (2) satisfactory, characterized by partial functional adaptation,\nprogressive weight-bearing, and gradual gait improvement during the\nadaptation period; and (3) good to excellent, characterized by imme­\ndiate spontaneous use of the prosthesis, consistent weight-bearing\nfrom the first fitting, stable ambulation, and absence of evident dis­\ncomfort or resistance to the device.\nClinical follow-up included periodic assessments of functional\nadaptation, skin integrity, gait stability, and overall tolerance to the\nprosthetic device. Professional physiotherapeutic follow-up was rec­\nommended for all patients; however, owner adherence to specialized\nrehabilitation programs was limited. Therefore, only general home-\nbased rehabilitation guidance was provided to the owners, consisting\nof controlled leash-guided walking, assisted weight-shifting exercises,\ngradual stimulation of supported stance, and proprioceptive stimula­\ntion on different floor surfaces. Exercise intensity and progression\nwere empirically adjusted according to each patient’s tolerance and\nclinical adaptation, following general principles of veterinary rehabili­\ntation (22).\nCase reports\nAll dogs included in this study presented with amputated or mal­\nformed residual limbs affecting either thoracic or pelvic limbs and\nunderwent adaptation to customized exoprostheses manufactured\nthrough 3D printing. Table 1 summarizes the clinical characteristics\nand the underlying conditions that indicated the use of these devices,\nwhile Table 2 presents data related to residual limb measurements,\n\nTable 1 summarizes the clinical characteristics\nand the underlying conditions that indicated the use of these devices,\nwhile Table 2 presents data related to residual limb measurements,\nnumber of prosthetic adjustments, immediate device acceptance, and\nfollow-up duration.\nThe clinical reports of dogs undergoing adaptation to customized\n3D-printed exoprostheses are presented below, with emphasis on the\ncharacteristics of the affected limb, fabrication process, adaptation\nperiod, and response to the device.\nCase 1. A 3-year-old, 17.5-kg male mixed-breed dog presented\nwith a history of congenital malformation affecting the right pelvic\nlimb, involving the tarsal joint. The condition resulted in significant\nlocomotor impairment, absence of proprioception, lameness, and skin\nlesions caused by direct contact of the malformed structure with the\nground (Figure 1A). Given these limitations, the fabrication of a cus­\ntomized 3D-printed exoprosthesis was indicated as an alternative to\nimprove functionality and overall quality of life. Detailed measure­\nments of the affected limb were collected and used to develop a three-\ndimensional model, which was subsequently manufactured using 3D\nprinting (Figure 1B). The measurements collected were stump dis­\ntance to the ground of 8 cm, residual limb length of 11.5 cm, and cir­\ncumference of 15.7 cm. From the first exposure to the exoprosthesis,\nthe dog demonstrated satisfactory adaptation, with partial weight-\nbearing on the affected limb and more balanced body weight redistri­\nbution. The owner actively participated in the rehabilitation process,\ncarefully following the instructions provided by the veterinarian and\ntechnical team (Figure 1C). Short-term clinical follow-up confirmed\ncomplete adaptation to the prosthetic device. No skin lesions, discom­\nfort, or complications related to prosthesis use were observed. The\nTABLE 1  Clinical characterization of dogs fitted with customized exoprostheses manufactured using 3D printing.\nCase\nBreed\nSex\nAge\nWeight (kg)\nAffected limb\nLevel of articular\ninvolvement\nClinical history\n1\nMixed-breed\nM\n3 years\n17.5\nRight pelvic limb\nTarsal joint\n\nCase\nBreed\nSex\nAge\nWeight (kg)\nAffected limb\nLevel of articular\ninvolvement\nClinical history\n1\nMixed-breed\nM\n3 years\n17.5\nRight pelvic limb\nTarsal joint\nCongenital malformation\n2\nMixed-breed\nM\n8 years\n13.3\nLeft pelvic limb\nTarsal joint\nTraumatic amputation\n3\nLabrador Retriever\nM\n10 years\n40\nLeft thoracic limb\nCarpal joint\nSurgical amputation\n4\nMixed-breed\nM\n13 years\n14\nRight thoracic limb\nCarpal joint\nTraumatic amputation\n5\nMixed-breed\nM\n3 years\n15.3\nLeft thoracic limb\nProximal radioulnar third\nTraumatic amputation\n6\nMixed-breed\nF\n9 months\n11.6\nLeft pelvic limb\nInterphalangeal joint\nCongenital malformation\nThe table summarizes the main clinical characteristics of dogs fitted with customized 3D-printed exoprostheses, including breed, sex, age, body weight, affected limb, level of articular\ninvolvement, and the underlying etiology of amputation or malformation.\n\nde Souza et al.\b\n10.3389/fvets.2026.1854874\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nsuccessful adaptation highlighted the effectiveness of the device and\nthe improvement in the animal’s quality of life, allowing stable and\nfunctional locomotion over time.\nCase 2. An 8-year-old male mixed-breed dog weighing 13.3 kg was\npresented with a history of traumatic amputation of the left pelvic limb\nat the distal tarsal level. The amputation resulted from a stab wound,\nconstituting an act of animal abuse that occurred outdoors, severely\ncompromising the animal’s tarsal joint. After the initial period of com­\nplete healing of the residual stump, the animal was referred to for spe­\ncialized evaluation with the aim of creating a customized prosthesis that\ncould restore locomotor function and improve quality of life\n(Figure 2A). During the initial consultation, the patient showed evident\ndiscomfort during movement, as well as increased sensitivity to palpa­\ntion in the amputated region, supporting the need for an adaptive inter­\nvention. In this context, an exoprosthetic approach was considered as a\ntherapeutic option and selected following discussion with the owner,\nconsidering the clinical condition and the goal of limb preservation. For\nthe development of the prosthesis, detailed anatomical measurements\nof the stump were taken, including the distance from the stump to the\nground (15 cm), the length of the limb to the stump (13 cm), and the\ncircumference of the region (12.6 cm). This information was fundamen­\ntal for the elaboration of the personalized three-dimensional model,\nwhich would allow the manufacture of an anatomical and functional\ndevice, compatible with the patient’s specificities (Figure 2B).\nAfter the manufacture and initial adjustment of the prosthesis, the\nanimal was fitted with the device and subjected to a progressive adap­\ntation protocol (Figure 2C). Although it showed some hesitation and\ncaution in the first moments of use, the dog demonstrated significant\nimprovement in locomotion over time, demonstrating the ability to\n\nAlthough it showed some hesitation and\ncaution in the first moments of use, the dog demonstrated significant\nimprovement in locomotion over time, demonstrating the ability to\nadapt to the new artificial limb. Clinical follow-up was rigorous, with\nconstant monitoring of the functional response and the performance\nof necessary technical adjustments to ensure proper alignment, device\nstability, and comfort during use.\nCase 3. A 10-year-old neutered male Labrador Retriever was pre­\nsented with a history of low amputation of the left thoracic limb, per­\nformed at the carpal level as part of treatment for squamous cell\ncarcinoma (SCC) (Figure 3A). After initial recovery, the animal began\nto show signs of apathy, muscle atrophy in the amputated limb, lame­\nness, and limited movement, factors that motivated the indication for\nthe use of a customized exoprosthesis. During the physical evaluation,\nprecise measurements of the stump were collected, including a length\nof 15 cm, a circumference of 12 cm, and a distance from the stump to\nthe ground of 6 cm, which served for the development of the three-\ndimensional digital model. The model had subsequently materialized\nthrough 3D printing. The prosthesis was carefully adjusted to the limb\nto provide comfort and stability (Figure 3B). From the first contact\nwith the prosthesis (Figure 3C), the dog was able to support the limb\non the ground, distributing body weight evenly and significantly\nTABLE 2  Residual limb characteristics, prosthetic adaptation, and follow-up information of dogs fitted with customized 3D-printed exoprostheses.\nCase\nStump\nlength\n(cm)\nStump\ncircumference\n(cm)\nDistance from\nstump to\nground (cm)\nNumber of\nprosthetic\nadjustments\nImmediate\ndevice\nacceptance\nFollow-up\nduration\n(months)\n1\n11.5\n15.7\n8\n0\nSatisfactory\n4\n2\n13\n12.6\n15\n0\nPoor\n4\n3\n15\n12\n6\n0\nGood/excellent\n4\n4\n13\n12.6\n10\n0\nGood/excellent\n3\n5\n15\n11\n20\n2\nPoor\n8\n6\n6\n9\n8\n1\nSatisfactory\n9\nFIGURE 1\n(A) Lateral view photographic record of the congenital malformation in the right pelvic limb of a male dog. (B) Customized virtual model of the 3D\nsocket-style exoprosthesis. (C) Immediate record after the first fixation of the 3D prosthesis, showing the support of the amputated limb on the\n\n(B) Customized virtual model of the 3D\nsocket-style exoprosthesis.(C) Immediate record after the first fixation of the 3D prosthesis, showing the support of the amputated limb on the\nstructure, with balanced distribution of body weight on two bases, offering greater stability to the animal.\n\nde Souza et al.\b\n10.3389/fvets.2026.1854874\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nreducing impacts on the contralateral limb. These improvements were\nmaintained during short-term follow-up, with benefits in mobility and\ncomfort during locomotion.\nCase 4. A 13-year-old, 14 kg, male mixed-breed dog was presented\nwith a history of traumatic amputation of the right thoracic limb at\nthe carpal joint. The amputation was caused by a domestic accident in\nwhich the gate of the residence trapped the limb, leading to its trau­\nmatic removal. During the initial assessment, the animal presented\nwith a lack of proprioception and muscle atrophy in the affected limb\nbut did not present with weight-bearing wounds or crepitus\n(Figure 4A). It was observed that the dog supported the stump on the\nground as a compensatory mechanism during locomotion. Given\nthese conditions, a customized exoprosthesis was developed to\nimprove its mobility and quality of life. The measurements collected\nwere stump distance to the ground of 10 cm, residual limb length of\n13 cm, and circumference of 12.6 cm. The prosthesis was internally\nlined with padded material to ensure comfort and fitted to the stump\nwith a compressive bandage, providing greater stability (Figure 4B).\nFrom the first fitting, the animal was able to successfully support the\nprosthetic limb on the ground, demonstrating improved functionality\nduring gait (Figure 4C). In addition, rehabilitation guidance was pro­\nvided to facilitate adaptation to the prosthetic device, including con­\ntrolled leash-guided walking, assisted weight-shifting exercises,\nstimulation of supported stance, and proprioceptive exercises per­\nformed on different floor surfaces. Initially, the dog performed super­\nvised walking sessions of approximately 5 min, with gradual\nprogression according to its tolerance and adaptation. During follow-\nup, the owner reported improved acceptance of physical activity,\nreduced fatigue during ambulation, and a significant improvement in\nposture while feeding. Periodic short-term follow-up assessments\n\nDuring follow-\nup, the owner reported improved acceptance of physical activity,\nreduced fatigue during ambulation, and a significant improvement in\nposture while feeding.Periodic short-term follow-up assessments\nwere conducted to monitor comfort, prosthesis adaptation, locomotor\nperformance, and overall quality of life.\nCase 5. A 3-year-old, 15.3 kg, male mixed-breed dog was pre­\nsented with a history of traumatic amputation of the left thoracic limb\nin the proximal radioulnar third (Figure 5A). The accident occurred\nwhen the animal was only 2 months old, after a vehicle ran over the\nlimb, resulting in its partial loss of the limb. At the time, the dog was\ntaken to the veterinary hospital, where it received emergency care and\ntreatment for the open wound until complete healing. During the pro­\ncess, an infection occurred at the site, but the condition was success­\nfully controlled. However, the lack of physiotherapy compromised the\nanimal’s mobility, leading to lateral deviation of the elbow joint,\nmarked muscle atrophy of the limb, and inability to bear weight. In\naddition, the animal exhibited pain-related hypersensitivity during\npalpation of the affected limb, without evidence of active skin lesions\nat the time of evaluation. A custom-made exoprosthesis was devel­\noped with the aim of improving the patient’s functional capacity and\nquality of life, with a length of 15 cm, a circumference of 11 cm, and a\ndistance from the stump to the ground of 20 cm (Figure 5B). The exo­\nprosthesis was covered with acrylic batting to ensure comfort and\nprevent injuries or abrasions. Fixation was performed with crepe ban­\ndage, well-adjusted with the aid of adhesive tape, providing stability\nand safety in the use of the device.\nInitially, the dog had difficulty adapting to the prosthesis,\nshowing sensitivity in the affected region, marked limb lateraliza­\ntion, reduced weight-bearing capacity, and instability during\nstance and ambulation. Due to the greater prosthetic height and\nthe severe biomechanical alterations observed in this case, the ini­\n\nDue to the greater prosthetic height and\nthe severe biomechanical alterations observed in this case, the ini­\ntial rehabilitation approach focused primarily on supported stance,\ngradual weight redistribution, and tolerance to prosthesis use.\nEarly adaptation exercises included short periods of assisted stand­\ning, controlled static weight-bearing, postural support exercises,\nand brief leash-guided walking sessions over short distances to\nprogressively stimulate limb loading while minimizing discomfort\nand fatigue. This case represented one of the most biomechanically\ncomplex scenario in the series due to marked articular lateraliza­\ntion, muscle atrophy, and prolonged absence of prior physiother­\napy, requiring specific design adaptations to ensure stability and\ncomfort. During follow-up, the animal demonstrated progressive\nimprovement in locomotion and functional adaptation to the pros­\nthesis (Figure 5C). Simultaneously, the dog was also undergoing\ntreatment for a transmissible venereal tumor (TVT), achieving\ncomplete remission without alterations in physiological\nparameters.\nCase 6. A nine-month-old, mixed-breed female dog weighing\n11.6 kg was presented with a congenital malformation of the phalan­\nges of the left pelvic limb, distal to the tarsal joint, while maintaining\nlimb functionality (Figure 6A). The structural anomaly involved an\natypical bone conformation, characterized by the presence of two pha­\nlanges in the affected limb, preventing adequate locomotion and\nseverely compromising limb functionality. The initial clinical evalua­\ntion included detailed visual inspection, orthopedic and neurological\nexamination, and precise measurement of the stump for planning a\ncustomized prosthesis. The measurements collected were stump dis­\ntance to the ground of 8 cm, residual limb length of 6 cm, and circum­\nference of 9 cm. These data were fundamental for the development of\nFIGURE 2\n(A) Lateral view photographic record of a dog with a history of traumatic amputation of the left pelvic limb, resulting from a stab wound. (B) Virtual\n\nThese data were fundamental for the development of\nFIGURE 2\n(A) Lateral view photographic record of a dog with a history of traumatic amputation of the left pelvic limb, resulting from a stab wound.(B) Virtual\nmodel of the angled 3D exoprosthesis, made from precise measurements of the residual stump, designed with a wider base to promote stability.\n(C) First fixation of the device to the stump, showing support of the prosthetic limb and the beginning of functional adaptation to gait.\n\nde Souza et al.\b\n10.3389/fvets.2026.1854874\nFrontiers in Veterinary Science\n06\nfrontiersin.org\na precise and individualized three-dimensional model that considered \nthe patient’s anatomical particularities (Figure 6B). After manufactur­\ning, the device underwent finishing, internal padding, and individual­\nized adaptation procedures to provide stability and prevent discomfort \nor skin lesions at the stump interface. During the adaptation period, \nthe patient demonstrated a progressive return to locomotion with \nexcellent acceptance of the exoprosthesis. Initial rehabilitation guid­\nance included supervised walking sessions lasting approximately 20 \nFIGURE 3\n(A) Photographic record of the left thoracic limb before amputation due to squamous cell carcinoma. (B) Three-dimensional model of the 3D \nexoprosthesis, manufactured with reduced height and anatomical design for close adjustment to the carpus. (C) Dog in sternal recumbency position \nduring the initial adaptation phase, already using the prosthesis on the left thoracic limb and demonstrating contact of the prosthetic stump with the \nground.\nFIGURE 4\n(A) Photographic record of a dog showing healed traumatic amputation of the right thoracic limb caused by a domestic accident. (B) Customized 3D \nexoprosthesis covered with acrylic blanket. (C) Record of the use of the device after adaptation, showing firm support on the ground and improvement \nin locomotor functionality.\nFIGURE 5\n(A) Lateral view photograph of a dog with traumatic amputation of the left thoracic limb due to being run over. (B) 3D digital model of the \nexoprosthesis, made with an elongated height to compensate for the articular lateralization observed in the patient. (C) First adaptation of the \nprosthesis, fixed to the stump with a compressive bandage and soft inner lining, demonstrating the beginning of gait with functional support.\n\nde Souza et al.\b\n10.3389/fvets.2026.1854874\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nto 30 min, emphasizing gradual gait retraining, supported weight-\nbearing, and functional muscle conditioning. Over the course of\nshort-term follow-up, marked improvement in mobility, balance, and\nlocomotor confidence was observed, allowing the animal to ambulate\nwith greater autonomy and stability. According to owner reports and\nclinical observations, the patient eventually regained the ability to per­\nform more dynamic movements, including running while using the\nprosthetic device, resulting in a substantial positive impact on quality\nof life (Figure 6C).\nDiscussion and conclusions\nThe use of three-dimensional modeling and FDM printing tech­\nnologies has proven to be effective and accessible, allowing the pro­\nduction of customized devices according to the morphology of each\npatient. The anatomical variability among canine patients, although a\nstrong point for 3D printing that allows customization, also means\nthat each prosthesis is unique and requires a meticulous and individu­\nalized design and manufacturing process (23). In addition to bone\nparticularities, the condition of the residual musculature proved to be\na determining factor since different degrees of atrophy and muscle\nasymmetry influence load distribution and stability throughout the\nadaptation process (13, 24).\nThe customization enabled by 3D printing is a determining factor\nfor increasing the functional effectiveness of prostheses, directly\nreflecting on the adaptation process, and improving the quality of life\nof animals. This degree of individualization favors faster rehabilitation\nprocesses and enhances the return of patients to their usual behavioral\nand locomotor functions (2).\nSimilarly, previous studies have demonstrated that dogs fitted with\nsocket prostheses can achieve good functional outcomes and maintain\na satisfactory quality of life, although successful adaptation is closely\nrelated to proper fit and careful management of potential complica­\ntions such as pressure sores (25). In the present study, in addition to\n\nIn the present study, in addition to\nstructural customization, adaptation was favored by the padded inner\nlining and the protection of the stump with a cotton sock, which\ncontributed to adequate tolerance to the device, without the occur­\nrence of clinically relevant skin complications.\nThe fabrication and adaptation of custom-printed orthopedic exo­\nprostheses using 3D printing demonstrated promising results during\nthe short-term follow-up period, with observed improvements in\nmobility, stability, and quality of life of the dogs treated. These findings\nare in line with the reports of Marcellin-Little (1) and Cruz et al. (14),\nwho highlight the importance of prostheses in restoring locomotor\nfunctionality and preventing compensatory overload in healthy limbs.\nStudies with exoprostheses reveal that, despite the animal’s initial\ntolerance, it requires physiotherapy follow-up and adjustments to help\nwith adaptation over time. Models with external fixation and absence\nof bone fixation have less control of movements, greater mechanical\nwear, and worse impact absorption, especially in running activities or\nabrupt changes of direction, which compromises functionality and\nacceptance by the animal (11, 23).\nDue to their snap-fit nature, they predominantly rely on external\nfixation methods, such as bandages, which can compromise the stabil­\nity and safety of the device during use. The effectiveness of fixation is\neven more critical in amputations of higher limbs, where the contact\narea for external fixation is reduced, making it difficult to obtain a firm\nand lasting fit (4). In Case 5, characterized by amputation in the proxi­\nmal radioulnar third, slower adaptation and initial challenges in sta­\nbilizing the device were observed, mainly due to marked muscle\natrophy, articular lateralization, and pain-related hypersensitivity\nduring limb manipulation, highlighting how more proximal levels of\namputation can compromise prosthetic stability and prolong the time\nrequired for functional consolidation.\nSolutions such as adaptable straps and silicone coatings have been\n\nSolutions such as adaptable straps and silicone coatings have been\ntested, but they still do not offer ideal adhesion in dynamic move­\nments. Thus, external fixation appears as one of the main limitations\nrelated to this type of prosthesis for animals, unlike humans where\nmodels with external fixation are widely used (26–28).\nAdditional engineering strategies achievable through additive\nmanufacturing have been described to improve prosthetic stability\nand biomechanical performance in dogs, including anatomically con­\ntoured socket interfaces, customized load-distribution structures, and\ngeometric modifications designed to better withstand gait forces and\nFIGURE 6\n(A) Lateral view photograph of a female dog with a congenital malformation in the left pelvic limb, characterized by alterations in the tarsal joint.\n(B) Three-dimensional model of the 3D exoprosthesis, developed at an intermediate height and with structural reinforcement for stability. (C) Use of the\ndevice during the adaptation phase, showing active support of the prosthetic limb on the ground and gradual resumption of locomotion.\n\nde Souza et al.\b\n10.3389/fvets.2026.1854874\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nimprove weight redistribution during locomotion (29, 30).\nFurthermore, finite element analysis (FEA) has been reported as a\nuseful tool for evaluating stress distribution and mechanical resistance\nin 3D-printed prosthetic components subjected to gait-related loading\nconditions (29). Although these approaches were not evaluated in the\npresent study, they represent relevant engineering strategies that may\ncontribute to the optimization of veterinary exoprosthetic devices.\nRegarding the rehabilitation and adaptation process, the dogs\nincluded in the present study demonstrated gradual and individual­\nized functional responses to prosthesis use, although most owners\nreported noticeable improvements in mobility and overall quality of\nlife, consistent with previous reports in dogs fitted with orthopedic\nprosthetic devices (13). The adaptation period varied among patients\nand appeared to be influenced by factors such as stump morphology,\ndegree of muscle atrophy, biomechanical alterations, and previous\nlocomotor compensation patterns. Similar challenges have been\nreported in the literature. In a prospective study involving 43 dogs\nfitted with orthoses or prostheses, Rosen et al. (31) observed that 91%\nof the animals developed at least one complication within a 12-month\nperiod, with skin-related lesions, including abrasions, hair loss, and\nwounds, representing the most common adverse findings.\nMechanical problems with the device (e.g., need for repair) were\nalso described, occurring in approximately 7–40% of dogs, depending\non the type of orthosis/prosthesis; non-acceptance of the device (resis­\ntance or refusal to use) in approximately 10–55%; abandonment or\npremature discontinuation of use before veterinary accommodation\nin 10–27%; and another category, such as “major” skin complications\n(wounds requiring veterinary treatment) versus minor ones, present\nin about 17% of cases among those who had skin complications (31).\nComparable challenges have also been described in dogs undergoing\n\nComparable challenges have also been described in dogs undergoing\npartial limb amputation managed with socket prostheses, including\nvariability in adaptation and the occurrence of complications (9, 25).\nIn all evaluated cases, favorable clinical evolution was observed,\nwith no records of skin lesions or major complications associated with\nprosthesis use, suggesting that the internal coating and padding strate­\ngies adopted in the exoprostheses were effective in protecting the\nresidual limb and minimizing discomfort during ambulation, similar\nto that described by Carr et al. (32), who emphasized that adjustments\nin internal padding thickness represent a key strategy for correcting\nprosthetic misalignment or limb length discrepancies and maintaining\nappropriate gait patterns.\nDespite the favorable outcomes observed, some limitations of the\npresent study should be considered. Radiographic evaluation was not\nincorporated into the prosthetic planning process, although imaging\ntechniques could provide additional anatomical and biomechanical\ninformation useful for individualized prosthetic design, as reported\nby Wendland et al. (33). In addition, standardized professional phys­\niotherapeutic follow-up was not achieved due to limited owner adher­\nence, resulting in reliance on non-standardized home-based\nrehabilitation guidance, which may have influenced functional adap­\ntation among patients, as rehabilitation is considered essential for\noptimizing the functional use of orthotic and prosthetic devices (18).\nAnother limitation was the absence of long-term follow-up,\nrestricting the assessment of chronic biomechanical adaptations, pros­\nthesis durability, and potential late orthopedic complications.\nFurthermore, objective functional assessment methods, such as\nkinetic or kinematic gait analysis and force platform evaluation, were\nnot performed. Consequently, functional outcomes were based on\nqualitative clinical assessment of gait, weight-bearing, and prosthesis\nadaptation. Future studies involving larger populations, prolonged\nfollow-up, imaging support, standardized rehabilitation protocols, and\n\nConsequently, functional outcomes were based on\nqualitative clinical assessment of gait, weight-bearing, and prosthesis\nadaptation.Future studies involving larger populations, prolonged\nfollow-up, imaging support, standardized rehabilitation protocols, and\nobjective gait analysis are necessary to further validate the clinical\napplicability of customized veterinary exoprostheses.\nEven considering these limitations, customized three-dimensional\nexoprostheses demonstrated important clinical applicability as func­\ntional and relatively low-cost rehabilitation alternatives, particularly\nin cases where preservation of the residual limb allowed individual­\nized prosthetic adaptation. The accessibility of additive manufacturing\ntechnologies, combined with individualized digital modeling, may\nsubstantially expand therapeutic possibilities for veterinary locomotor\nrehabilitation.\nIn summary, three-dimensional modeling and 3D printing proved\nto be effective tools for the production of customized orthopedic exo­\nprostheses in dogs, promoting improvements in mobility, postural\nstability, and overall quality of life. The application of additive manu­\nfacturing in the development of veterinary orthopedic exoprostheses\nrepresents an important advancement in Veterinary Medicine and\nhighlights the potential of personalized rehabilitation strategies for\nanimals with amputations or congenital limb deformities.\nData availability statement\nThe raw data supporting the conclusions of this article will be\nmade available by the authors, without undue reservation.\nEthics statement\nThe animal studies were approved by Ethics Committee on\nAnimal Use (CEUA), Center for Rural Health and Technology\n(CSTR), Federal University of Campina Grande (UFCG), Patos, Brazil\n(protocol no. 44/2024). The studies were conducted in accordance\nwith the local legislation and institutional requirements. Written\ninformed consent was obtained from the owners for the participation\nof their animals in this study. Written informed consent was obtained\nfrom the participants for the publication of this case report.\nAuthor contributions\nMS:\n\nWritten\ninformed consent was obtained from the owners for the participation\nof their animals in this study.Written informed consent was obtained\nfrom the participants for the publication of this case report.Author contributions\nMS:\nConceptualization,\nData\ncuration,\nInvestigation,\nMethodology, Visualization, Writing – original draft, Writing – review\n& editing. RL: Conceptualization, Methodology, Visualization,\nWriting – review & editing. DA: Methodology, Writing – review &\nediting, Visualization. AD: Visualization, Writing – review & editing,\nInvestigation,\nResources,\nSupervision,\nValidation.\nLC:\nConceptualization, Methodology, Supervision, Validation, Writing –\noriginal draft, Writing  – review & editing, Funding acquisition,\nInvestigation, Project administration, Resources.\nFunding\nThe author(s) declared that financial support was received for this\nwork and/or its publication. This work was supported by Karolinska\n\nde Souza et al.\b\n10.3389/fvets.2026.1854874\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nInstitute through an institutional publication agreement. The funding\ncovered the open access publication fees.\nAcknowledgments\nThe authors thank the Federal University of Campina Grande\n(UFCG) for its institutional support, the Coordination for the\nImprovement of Higher Education Personnel (CAPES) for the schol­\narship, and Professor Dr. Danilo Ayres (in memoriam) for his contri­\nbution and assistance in this work.\nConflict of interest\nAuthors MS, RL and LC were employed by company 3D\nMedicine Vet.\nThe remaining author(s) declared that this work was conducted\nin the absence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declared that Generative AI was not used in the\ncreation of this manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the review­\ners. Any product that may be evaluated in this article, or claim that\nmay be made by its manufacturer, is not guaranteed or endorsed\nby the publisher.\nReferences\n1.\tMarcellin-Little DJ. \"Bandaging, external coaptation, and external devices for compan­\nion animals\". In: Johnston SA and Tobias KM, editors. Textbook of Small Animal Surgery.\nSt. Louis, MO: Elsevier (2018). p. 737–51.\n2.\tSouza MMN, Lima JNG, Menezes DJA, Carvalho LRRA. Impacts of exoprosthesis use\nin dogs with partial amputation and limb malformations: a systematic review. Front Vet\nSci. 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+{"text": "【兽医文献】\n标题: Evaluation of the surface thermal response of peripheral nerve blocks in dogs undergoing trauma or orthopedic surgery\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nFlow cytometric features of\nlymphoid subsets in healthy and\ndiseased cats\nCesar Guillermo Meneses-Nava 1, Alessandra Ubiali 1,\nGreta Dossi 1, Sabine E. Hammer 2, Barbara C. Rütgen 3,\nChiara Locatelli 1, Angelica Stranieri 1 and Valeria Martini 1*\n1 Department of Veterinary Medicine and Animal Sciences, University of Milan, Lodi, Italy,\n2 Immunology, Department of Biological Sciences and Pathobiology, University of Veterinary\nMedicine, Wien, Austria, 3 Clinical Pathology, Department of Biological Sciences and Pathobiology,\nUniversity of Veterinary Medicine, Wien, Austria\nIntroduction: Flow cytometry (FC) is widely used in humans and dogs to\ndiagnose and characterize hematopoietic neoplasms. Conversely, its use in\nfeline patients is still limited, leading to a lack of standardized protocols and\nsubjective data interpretation.\nMethods: Herein, we describe FC features of circulating lymphoid subsets in a\ntotal of 20 cats: 9 healthy cats, 6 diseased cats without hematopoietic neoplasm,\nand 5 cats with probable chronic lymphocytic leukemia (CLL), using a panel of\n10 antibodies and a multicolor approach, in terms of both cell size (nFSC) and\ndegree of antigen expression (MFI).\nResults: Three main subsets were identified in healthy cats and diseased cats\nwithout hematopoietic neoplasm (namely, CD5 + CD45R-, CD21 + CD45R +\nand CD5 + CD45R+). CD4 + CD8- cells outnumbered CD4- CD8 + cells. Low\npercentages of CD4 + CD8 + and CD134 + cells were also present. MHCII had\nhigher fluorescence intensity in B- than in T-cells. CD9 was not expressed on\nleukocytes surface, but on small events possibly referable to platelet clumps. In\ndiseased cats without hematopoietic neoplasm, each T-cell subset was larger in\nsize than in healthy cats. Finally, in cats with probable CLL the leading phenotype\nwas CD5 + CD45R-CD4 + CD8-CD134 + MHCII+ and cell size overlapped with\nthe one of the other diseased cats.\nDiscussion: Our results are expected to lay the ground for a more standardized\napproach to feline samples for FC, and a more objective data interpretation,\n\nDiscussion: Our results are expected to lay the ground for a more standardized\napproach to feline samples for FC, and a more objective data interpretation,\nultimately leading to improved diagnostic accuracy. Further studies are needed\nto assess the biological, diagnostic and prognostic value of specific FC patterns\nin feline medicine.\nKEYWORDS\nflow cytometry, peripheral blood, lymphocytes, FSC, MFI\n1 Introduction\nFlow cytometry (FC) is widely used to diagnose and characterize lymphoma and leukemia\nin dogs. It allows the differentiation of reactive from neoplastic conditions by identifying a\npredominant homogeneous population or immunophenotypic aberrancies (1). Conversely, its\nuse is still limited in cats because of the low availability of species-specific monoclonal\nantibodies and the high frequency of samples unsuitable for processing (2).\nOne of the most important aspects for differentiating a reactive versus a neoplastic process\nis the identification of cell clonality. During their development, the genes encoding the T-cell\nOPEN ACCESS\nEDITED BY\nGabriele Rossi,\nMurdoch University, Australia\nREVIEWED BY\nTim Williams,\nUniversity of Cambridge, United Kingdom\nIlona Holcomb,\nImpriMed Inc., United States\n*CORRESPONDENCE\nValeria Martini\nvaleria.martini@unimi.it\nRECEIVED 03 June 2025\nACCEPTED 22 July 2025\nPUBLISHED 01 August 2025\nCITATION\n​Meneses-Nava CG, Ubiali A, Dossi G,\nHammer SE, Rütgen BC, Locatelli C,\nStranieri A and Martini V (2025) Flow\ncytometric features of lymphoid subsets in\nhealthy and diseased cats.\nFront. Vet. Sci. 12:1640229.\ndoi: 10.3389/fvets.2025.1640229\nCOPYRIGHT\n© 2025 Meneses-Nava, Ubiali, Dossi,\nHammer, Rütgen, Locatelli, Stranieri and\nMartini. This is an open-access article\ndistributed under the terms of the Creative\nCommons Attribution License (CC BY). The\nuse, distribution or reproduction in other\nforums is permitted, provided the original\nauthor(s) and the copyright owner(s) are\ncredited and that the original publication in\nthis journal is cited, in accordance with\naccepted academic practice. No use,\ndistribution or reproduction is permitted\nwhich does not comply with these terms.\nTYPE  Original Research\n\nNo use,\ndistribution or reproduction is permitted\nwhich does not comply with these terms.TYPE  Original Research\nPUBLISHED  01 August 2025\nDOI  10.3389/fvets.2025.1640229\n\nMeneses-Nava et al.\b\n10.3389/fvets.2025.1640229\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nreceptor gamma (TCRγ) and Immunoglobulin heavy chain receptor\n(IgH) undergo rearrangement. Thus, virtually each rearrangement is\nunique to individual lymphocytes. In a reactive population, a\npolyclonal expansion would be  observed, while a single or\npredominant rearrangement indicates expansion of a population with\nthe same antigen receptor gene. In cats, identification of clonality of\nTCRγ or IgH is currently performed by PCR for Antigen Receptor\nRearrangement (PARR) (3–5). However, while all neoplasms are\nclonal in origin, not all clonal lymphocyte expansions are neoplastic\nand can be secondary to infectious or inflammatory diseases.\nIn this scenario, FC analysis of lymphoid population could be a\nsupport in differentiating truly neoplastic samples. Unfortunately,\ninformation about immunophenotypic characteristics of lymphoid subsets\nusing FC in cats is limited, with few reports carried out in peripheral blood\n(PB) (6–8) and in lymph nodes (LN) (9, 10). In particular, currently there\nis no reference information on lymphoid subsets in PB of healthy cats,\nother than the percentage of CD4 + and CD8 + cells (6, 7). A deep\nknowledge of the composition and FC features of lymphoid circulating\ncells in normal and pathological conditions other than lymphoid\nneoplasms is mandatory, for a better interpretation of the data obtained on\nroutine diagnostic samples, ultimately leading to a more accurate diagnosis.\nThe aim of this study is to describe FC features of lymphoid\nsubsets in PB from healthy and diseased cats, in terms of percentage\nof different populations, cell size and degree of antigen expression, in\norder to provide reference information possibly useful for\ndifferentiating between reactive and neoplastic processes.\n2 Materials and methods\n2.1 Sample collection\nAll samples included in the present study had been delivered to the\nlaboratory of clinical pathology of the Veterinary Teaching Hospital (VTH,\nUniversity of Milan) after being sampled for diagnostic purposes or routine\n\n1 Sample collection\nAll samples included in the present study had been delivered to the\nlaboratory of clinical pathology of the Veterinary Teaching Hospital (VTH,\nUniversity of Milan) after being sampled for diagnostic purposes or routine\nhealth check, with a written informed consent of the owner. Thus, specific\napproval of the Ethical Committee to use leftover specimens for research\npurposes was not required (EC decision dated 29 October 2012, renewed\nunder protocol 02–2016, University of Milan).\nFeline PB samples collected into EDTA tubes and delivered to the\nlaboratory between January and March 2025 were considered eligible\nif fulfilled the following criteria: (1) adequate blood-to-EDTA ratio and\nlack of visible clots; (2) complete blood count (CBC) performed with\nan automated laser-based hematology analyzer (Sysmex XN-1000 V,\nSysmex Corporation, Kobe, Japan) and blood smear evaluation; (3)\navailability of full signalment data and clinical records at the time of\nsampling; (4) negative result of a snap-test for FIV and FeLV infection\nat the time of sampling or within the last month. Different snap-test had\nbeen used, based on the referring veterinarian preferences. Still, all of\nthem detected FIV antibodies and FeLV antigens.\nThereafter, samples were subdivided into healthy and diseased cats\naccording to the criteria listed below.\nHealthy cats fulfilled all the following additional criteria: (1) were\nsampled for routine health check or within a pre-anesthetic protocol\nfor elective orchiectomy or ovariectomy; (2) were in good nutritional\ncondition (Body Condition Score 4–6/9); (3) were asymptomatic and\nwere not receiving treatments at the time of sampling; (4) more than\n2 months had passed before last vaccine administration; (5) CBC and\nbiochemistry results were within normal limits.\nDiseased cats were further subdivided into two groups, based on\nthe underlying disease.\nGroup 1 - diseased cats had any disease except hematopoietic\nneoplasia. So, they were retained in the study if: (1) had no solid lesion\ncompatible with lymphoid neoplasia and (2a) a final diagnosis other\n\nGroup 1 - diseased cats had any disease except hematopoietic\nneoplasia.So, they were retained in the study if: (1) had no solid lesion\ncompatible with lymphoid neoplasia and (2a) a final diagnosis other\nthan hematopoietic neoplasia could be reached within few days from\nsampling or (2b) PARR gave polyclonal results for both TCRγ and\nIgH. PARR was performed on WBC pellets according to already\npublished protocols (5, 10, 11).\nGroup  2  - diseased cats had PB infiltration from lymphoid\nneoplasia, based on: (1) a single lymphoid subset prevalent at FC\nanalysis; and (2) monoclonal result for at least one gene at PARR analysis.\n2.2 Flow cytometry\nFC was performed on EDTA PB samples within 24 h from\nsampling, according to already published protocols for cats (12). The\nantibody panel (Table 1) had been designed prior to study initiation,\nTABLE 1  Details of antibodies used to characterize circulating lymphoid subsets in 20 cats via flow cytometry.\nAntigen\nExpected specificity\nClone\nConjugation\nSource\nCD5\nT-cells\nf43\nPE\nSouthernBiotech, Birmingham,\nAL, USA\nCD21\nB-cells\nCA2.1D6\nAlexaFluor647\nBio-Rad, Oxford, UK\nCD45R\nB- and T-cells subsets\nRA3-6B2\nFITC\nBio-Rad\nCD4\nT-helper cells\n3-4F4\nFITC\nSouthernBiotech\nCD8\nT-cytotoxic cells\nfCD8\nPE\nSouthernBiotech\nCD134\nActivated lymphocytes\n7D6\nAlexaFluor647\nBio-Rad\nCD9\nActivated lymphocytes, platelets\nMM2/57\nPE\nBio-Techne, Milan, Italy\nMHCII\nMonocytes and lymphocytes\n169-1B5.2\nAlexaFluor488\nBio-Techne\nCD18\nAll leukocytes\nCA1.4E9\nAlexaFluor647\nBio-Rad\nCD44\nAll leukocytes\nIM7\nFITC\nBD Pharmingen, San Diego, CA,\nUSA\n\nMeneses-Nava et al.\b\n10.3389/fvets.2025.1640229\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nselecting antibodies commercially available that had already been used\non feline samples in the literature or were reported to be cross-reactive\nwith feline antigens from the manufacturer. In this latter case, cross-\nreactivity was preliminarily tested on random feline PB samples from\nthe laboratory routine activity, and antibodies were included in the\npanel only if positive signals were obtained on the expected\npopulation, and not on the other ones. Otherwise, the antibody clone\nwas not included in the panel, and therefore not used in the present\nstudy. All antibodies and cocktails had been titered to select the best\nworking dilution before study initiation.\nFive different tubes were prepared, and an adequate volume of PB\nwas put into each of them, in order to have 500,000 cells/tube, based\non instrumental WBC count. In all tubes, 25 μL of a blocking solution\ncontaining fetal bovine serum (FBS) were added, to prevent aspecific\nantibody binding. Thereafter, antibody cocktails were added to the five\ntubes, as detailed in Table 2. 7-Amino-Actinomycin D (7-AAD, BD\nPharmingen, San Diego, CA, USA) was included in each sample to\ncheck cells viability. After 10 min of incubation at room temperature,\n1 mL of a red blood cells lysis buffer was added to each tube. Once the\nsample had become transparent, a washing step was performed by\ncentrifugation at 277 g for 8 min, the supernatant discarded, and the\ncell pellet resuspended in 500 μL of PBS for final acquisition.\nUnfortunately, Tube D could not be tested in a subset of cats due to\ntemporary lack of the anti-MHCII antibody.\nAll samples were acquired with a BriCyte E6 flow cytometer\n(Mindray, Shenzhen, China) equipped with 2 lasers (blue and red) and\n4 fluorescence channels and analyzed with the specific software\nMRFlow (Mindray). For each tube, 10,000 nucleated cells were\nacquired. For each sample from healthy cats and for samples from\nGroup1 diseased cats, the percentage of the following lymphoid\nsubsets\nwas\nrecorded:\nCD5 + CD45R+,\nCD21 + CD45R+,\n\nFor each tube, 10,000 nucleated cells were\nacquired.For each sample from healthy cats and for samples from\nGroup1 diseased cats, the percentage of the following lymphoid\nsubsets\nwas\nrecorded:\nCD5 + CD45R+,\nCD21 + CD45R+,\nCD5 + CD45R-, CD4 + CD8-, CD4-CD8+, CD4 + CD8+, CD134+,\nMHCII+CD21+, MHCII+CD21- (Supplementary material 1). The\npercentages were then coupled with the absolute lymphocyte count\nobtained with instrumental WBC count and blood smear evaluation,\nto calculate the absolute concentration of each subset. In addition, for\neach subset, the Median Fluorescence Index (MFI) of antibody-\npositive cells was recorded, as well as the median forward-scatter\n(FSC-H) value. These parameters were not recorded for\nCD4 + CD8 + subset, due to the low number of cells with this\nphenotype. In order to allow comparison with data obtained on other\ninstruments, for each antigen an MFIratio was calculated, by dividing\nthe MFI of antibody-positive cells to the one of unstained lymphoid\ncells in the same fluorescence channel and regarded as an indicator of\nthe degree of antigen expression (12). Pairwise, the FSC-H value of\neach subset was normalized with a ratio with the FSC-H value of\ngranulocytes in the same sample (nFSC), to quantify cell size (13).\nFinally, CD18-MFIratio and CD44-MFIratio were calculated for the\nlymphoid population as a whole. Concerning Group2 samples, the\naforementioned parameters were recorded setting a gate to include\nonly neoplastic cells.\n2.3 Statistical analyses\nAll data were inserted in an electronic datasheet, and descriptive\nstatistics were calculated. A Friedman test for paired samples was\nperformed to compare nFSC values among subsets within the group\nof healthy cats and within Group1 diseased cats, using Bonferroni\ncorrection for post-hoc comparisons. Wilcoxon test was used to assess\npossible differences in CD5-MFIratio between CD5 + CD45R + and\nCD5 + CD45R- cells, CD45R-MFI ratio between CD5 + CD45R + and\nCD21 + CD45R + cells,\nand\nMHCII-MFIratio\nbetween\nMHCII+CD21 + and MHCII+CD21- cells within healthy cats and\nwithin Group1 diseased cats.\n\nMann–Whitney test was used to assess possible differences in\nnFSC and MFIratio values of each lymphoid subset between healthy\nand Group 1 diseased cats.\nOnly descriptive data are presented for Group 2 diseased cats, due\nto low caseload and variable phenotype among cases.\nAll analyses were performed with SPSS Statistical software for\nWindows, v29.0. Significance was set at p ≤ 0.050.\n3 Results\nOverall, 20 feline PB samples were included in the present study,\nincluding 9 (45%) healthy cats, and 11 (55%) diseased cats (6 in\nGroup1 and 5 in Group2).\nBased on 7-AAD staining, all samples had ≥97.0% vital cells.\n3.1 Healthy cats\nHealthy cats included 8 (88.9%) Domestic Short Hair (DSH) and\n1 (11.1%) Maine coon. Five (55.6%) were female (1 spayed) and 4\n(44.4%) were males (1 neutered). Median age was 1 year (range:\n6 months to 8 years).\nDescriptive statistics of FC data are presented in Table 3. Overall,\nCD5 + CD45R- was the most numerous subset, followed by\nCD21 + CD45R+,\nwhereas\nCD5 + CD45R + cells\nwere\nless\nrepresented. Considering CD4 and CD8 expression, CD4 + CD8- cells\noutnumbered CD4-CD8+, whereas CD4 + CD8 + cells were not\ndetected in 1 (11.1%) case and accounted for <1% in the other 8\n(88.9%) cases. CD134 + cells were poorly represented, never exceeding\n1,000 cells/μL. Finally, MHCII+ cells were more commonly CD21-\nthan CD21+. No MHCII-CD21 + cell was detected in any sample.\nTube D (Table 2) was not tested in 1 case.\nAll lymphoid cells were smaller in size than granulocytes, with a\nmedian nFSC ranging from 0.60 to 0.67. Significant differences among\nsubsets were found (p = 0.002), with CD134 + cells being larger than\nboth CD4 + CD8- and CD4-CD8 + cells (p = 0.037 for both\npost-hoc analyses).\nTABLE 2  Antibody cocktails used to characterize circulating lymphoid\nsubsets in 20 cats via flow cytometry.\nTube\nFluorescence channel\nFL1 (530/30)\nFL2 (585/40)\nFL4\n(660/20)\nUnstained\nNone\nNone\nNone\nA\nCD45R\nCD5PE\nCD21\nB\nCD4\nCD8\nCD134\nC\nCD44\nCD9\nCD18\nD\nMHCII\nnone\nCD21\n\nMeneses-Nava et al.\b\n10.3389/fvets.2025.1640229\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nConcerning MFI analyses, CD5-MFIratio was higher in\nCD5 + CD45R- than in CD5 + CD45R + cells (p = 0.008), CD45R-\nMFIratio was significantly higher in CD21 + CD45R + than in\nCD5 + CD45R + cells (p = 0.008), and MHCII-MFIratio was higher\nin MHCII+CD21 + than in MHCII+CD21- cells (p = 0.012). These\ndata are represented in Figure 1.\nNo CD44 + CD9 + CD18 + cell was detected in any sample.\nHowever, variable amounts of CD9 + events were present among\nsamples. When back-gating that population of cells, it appeared to\nbe mostly distributed in the low-left angle and along the diagonal of\nthe morphological scattergram (Supplementary material 2). Those\nevents were thus considered possible platelet clumps and were not\nfurther analyzed.\n3.2 Diseased cats without hematopoietic\nneoplasia (Group 1)\nThe 6 cats in Group 1 included 5 (83.3%) DSH and 1 (16.7%)\nChartreux. There were 5 (83.3%) male (2 neutered) and 1 (16.7%)\nTABLE 3  Flow cytometric features of lymphoid subsets in 9 healthy cats.\nCell subset\nParameter\nMedian\nRange\nAll lymphocytes\nCount (cells/μL)\n4,564\n2,697–5,384\nnFSC\n0.60\n0.57–0.98\nCD18-MFIratio\n37.65\n24.17–157.11\nCD44-MFIratio\n134.80\n107.34–\n179.32\nCD5 + CD45R-\nCount (cells/μL)\n1,586\n648–3,136\nnFSC\n0.62\n0.57–0.69\nCD5-MFIratio\n545.63\n391.76–\n709.68\nCD21 + CD45R+\nCount (cells/μL)\n1,080\n421–2,405\nnFSC\n0.63\n0.59–0.66\nCD21-MFIratio\n202.23\n98.37–698.11\nCD45R-MFIratio\n66.38\n32.16–145.41\nCD5 + CD45R+\nCount (cells/μL)\n134\n65–439\nnFSC\n0.64\n0.61–0.69\nCD5-MFIratio\n312.86\n83.42–388.46\nCD45R-MFIratio\n28.56\n13.51–66.83\nCD4 + CD8-\nCount (cells/μL)\n1,260\n393–1783\nnFSC\n0.60\n0.56–0.67\nCD4-MFIratio\n61.67\n34.31–82.44\nCD4-CD8+\nCount (cells/μL)\n429\n143–548\nnFSC\n0.61\n0.56–0.66\nCD8-MFIratio\n219.24\n141.51–\n430.81\nCD4 + CD8+\nCount (cells/μL)\n7\n0–29\nCD134+\nCount (cells/μL)\n198\n54–569\nnFSC\n0.67\n0.59–0.74\nCD134-MFIratio\n61.20\n21.92–134.59\nMHCII+CD21+\nCount (cells/μL)\n1,078\n314–2061\nnFSC\n0.63\n0.59–0.67\nMHCII-MFIratio\n294.20\n164.30–\n514.78\nMHCII+CD21-\nCount (cells/μL)\n1,574\n960–2,788\nnFSC\n0.63\n0.58–0.68\nMHCII-MFIratio\n27.55\n18.95–76.01\nnFSC = ratio between median forward scatter (FSC-H) value of the population of interest\nand granulocytes in the same sample; MFIratio = ratio between median fluorescence index\nof stained and unstained cells.\nFIGURE 1\n\n63\n0.58–0.68\nMHCII-MFIratio\n27.55\n18.95–76.01\nnFSC = ratio between median forward scatter (FSC-H) value of the population of interest\nand granulocytes in the same sample; MFIratio = ratio between median fluorescence index\nof stained and unstained cells.FIGURE 1\nHistogram overlays showing differences in fluorescence intensity\nbetween lymphoid subsets in 9 healthy cats. All differences were\nstatistically significant (p < 0.05) (A) Fluorescence intensity of CD5\nwas higher in CD5 + CD45R- (green line) than in\nCD5 + CD45R + (red line) cells. (B) Fluorescence intensity of CD45R\nwas higher in CD21 + CD45R + (red line) than in\nCD5 + CD45R + (green line) cells. (C) Fluorescence intensity of\nMHCII was higher in MHCII+CD21 + (red line) than in MHCII+CD21-\n(green line) cells.\n\nMeneses-Nava et al.\b\n10.3389/fvets.2025.1640229\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nfemale. Median age was 1.5 years (range: 8 months to 13 years). They\nwere diagnosed with: hypertrophic cardiomyopathy, chronic kidney\ndisease, lung carcinoma, dermatitis, trauma from car accident, and\ndental abscess (1 each). No PARR analysis was needed, since none of\nthem had a clinical suspicion of lymphoid neoplasia, nor a prevalent\nlymphoid subset at FC.\nDescriptive\nstatistics\nof\nFC\ndata\nare\npresented\nin\nSupplementary material 3.\nOverall, the relative prevalence of cell subsets overlapped the one\nencountered in healthy cats. CD4 + CD8 + cells were not detected in\n1 (16.7%) case, accounted for <1% in 4 (66.7%) cases, and for 1.28%\nin 1 (16.7%) case. CD134 + cells never exceeded 1,000 cells/μL. Tube\nD (Table 2) was tested only in 3 cats. Among them, MHCII+ cells were\nmore commonly CD21- than CD21+. No MHCII-CD21 + or\nCD44 + CD9 + CD18 + cell was detected in any sample.\nAll lymphoid cells were smaller in size than granulocytes.\nSignificant differences in nFSC were found among subsets (p = 0.008).\nAt post-hoc analyses, only CD134 + cells were significantly larger in\nsize than CD21 + CD45R + cells (p = 0.002).\nConcerning MFI analyses, CD5-MFIratio was significantly higher\nin CD5 + CD45R- than in CD5 + CD45R + cells, and CD45R-\nMFIratio was significantly higher in CD21 + CD45R + cells than in\nCD5 + CD45R + cells (p = 0.028 for both analyses).\nThe parameters for which a significant difference between healthy\ncats and Group1 diseased cats was found, are shown in Figure 2. In\nparticular, CD5 + CD45R + and CD5 + CD45R- cells were larger in\nsize in reactive samples (p = 0.002 for both analyses), as well as\nCD4 + CD8- (p = 0.003), CD4-CD8 + (p = 0.013) and CD134 + cells\n(p = 0.018). Conversely, no difference in cell counts or MFIratios\nwas found.\n3.3 Diseased cats with lymphoid neoplasia\n(Group 2)\nAll 5 cats diagnosed with lymphoid neoplasia were DSH. Three\n(60%) were female (2 spayed) and 2 (40%) were male (1 neutered).\nMedian age was 11 years (range: 9 to 17 years). All of them presented\nwith mature lymphocytosis as the sole abnormality. Median\n\nThree\n(60%) were female (2 spayed) and 2 (40%) were male (1 neutered).Median age was 11 years (range: 9 to 17 years).All of them presented\nwith mature lymphocytosis as the sole abnormality.Median\nlymphocytes count was 67,525 cells/μL (range: 17314 to 691,564 cells/\nμL). Diagnostic workup and staging procedures varied among cases,\nbut solid lesions were never detected. A prevalent T-cell population\nwas detected in FC and clonal TCRγ rearrangement was confirmed by\nPARR, thus leading to a probable diagnosis of T-cell chronic\nlymphocytic leukemia (T-CLL). The phenotype of neoplastic cells\nwas  CD5 + CD45R-CD4 + CD8-CD134 + MHCII+ in 3 cases,\nCD5 + CD45R-CD4 + CD8-CD134-MHCII+ in 1 case, and\nCD5-CD45R-CD4 + CD8-CD134-MHCII+ in 1 case. In all cases,\nneoplastic cells stained positive for CD44 and CD18, and negative for\nCD21 and CD9. Raw data of nFSC and MFIratios of antibody-positive\nneoplastic cells are represented in Figure 3, together with those of the\nrespective cellular subsets in healthy cats and Group1 diseased cats.\n4 Discussion\nTo the authors knowledge, this is the first study describing FC\nfeatures of circulating lymphoid subsets in healthy cats using a 10\nantibody panel. Data on diseased cats with and without lymphoid\nneoplasia are also presented.\nFlow cytometry is only at its debut in feline oncology. In the past,\nindeed, it was mentioned in the diagnostic workup of occasional case\nFIGURE 2\nBox-plot showing flow cytometric features of lymphoid subsets that significantly differed between 9 healthy (light blue boxes) and 6 diseased without\nhematopoietic neoplasia (orange boxes) cats (p < 0.05). nFSC = ratio between median forward scatter (FSC-H) value of the population of interest and\ngranulocytes in the same sample. For each box, the main horizontal line represents the median value, the lower and upper limits of the box represent\nthe first and third quartile, the box represents the interquartile range, and the upper and lower whiskers represent the upper and lower value that is not\n\nan outlier. (A) nFSC of CD5 + CD45R- cells. (B) nFSC of CD5 + CD45R + cells. (C) nFSC of CD4 + CD8- cells. (D) nFSC of CD4-CD8 + cells. (E) nFSC of\nCD134 + cells.\n\nMeneses-Nava et al.\b\n10.3389/fvets.2025.1640229\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nreports (14, 15), while studies specifically focused on FC analysis of\noncological samples date back to less than one decade ago (2, 8, 9, 16,\n17). Importantly, data on non-neoplastic cells in cats are limited to two\nrecent studies (10, 12). Herein, we applied a large antibody panel and\nreport a number of FC features for each lymphoid cell subset in\nhealthy cats, including absolute count, cell size (expressed as nFSC)\nand degree of antigen expression (expressed as MFIratio).\nWhile designing the antibody panel, we  encountered some\nunexpected findings with CD9 antibody, which prevented us from\nconfirming the cross-reactivity with feline antigen reported by the\nmanufacturer. Still, we  retained the antibody in the panel since\nwe believe that our findings could help improve FC analysis of feline\nsamples, irrespective of the precise antigen detected by the antibody.\nAccording to the published literature, CD9 should be expressed on all\nleukocytes and platelets (18–20). However, we  detected no\nCD9 + leukocyte. Unfortunately, CD9 expression has never been\ntested on feline fresh PB samples. Thus, we could not define whether\nour findings are linked to the antibody clone or to minimal to null\nCD9 expression on circulating feline WBC. However, we found a\nvariable amount of CD9 + events among samples and among tubes\nwithin single samples. Better separation of WBC subclasses was\nobtained in the morphological scattergram when excluding those\nCD9 + events (Supplementary Figure 2). This fact, together with the\npeculiar distribution of CD9 + events in the morphological\nscattergram, and concomitant negative staining for the panleukocyte\nmarkers CD44 and CD18, prompted us to speculate that those events\ncould represent platelet clumps. Further studies are needed to confirm\nthis hypothesis.\nOverall, three main populations were identifiable in healthy\nsubjects and Group1 diseased cats, namely CD5 + CD45R-,\nCD21 + CD45R + and CD5 + CD45R+, in decreasing order of\nprevalence. CD45R has been used in the literature to identify B-cells\n\nOverall, three main populations were identifiable in healthy\nsubjects and Group1 diseased cats, namely CD5 + CD45R-,\nCD21 + CD45R + and CD5 + CD45R+, in decreasing order of\nprevalence.CD45R has been used in the literature to identify B-cells\nin cats (21, 22). However, its expression on effector T-cytotoxic cells\nand neoplastic T-cell has also been reported (23–25). Our results\nsuggest that CD45R expression is not limited to B-cells even in normal\nconditions. Concomitant use of additional B- and T-cell markers is\nnecessary to accurately identify lymphoid subsets. Additionally,\nanalysis of CD45R- and MHCII-MFIratios could be  of aid in\ndiscriminating among the 3 subclasses, based on the significant\ndifferences we reported, in both healthy and Group1 diseased cats.\nUnfortunately, we did not couple CD45R with further T-cell markers,\nsuch as CD4 and CD8. Thus, the full antigenic pattern of\nCD5 + CD45R + cells and their the biological role still have to\nbe defined.\nWhen comparing healthy and Group1 diseased cats, an increase\nin the cell size of all T-cell subsets was noted. This could be attributed\nto their activation, although not all cats had inflammatory/infectious\ndiseases. Irrespective of the underlying mechanisms, this difference is\nrelevant from a diagnostic point of view, since it remarks that\nmoderate increase in T-cell size alone should not be considered a\ncriterium for diagnosing lymphoid neoplasia in cats.\nWhen considering CD4 and CD8 expression, herein we confirmed\nthe higher prevalence of CD4 + CD8- cells already reported in healthy\ncats in literature (6, 26). More interestingly, we  reported a small\namount of CD4 + CD8 + cells in >80% samples, without differences\nbetween healthy and Group1 diseased cats. It seems reasonable to\nFIGURE 3\nDot graphs showing flow cytometric features of T-cell subsets in 9 healthy (light blue dots), 6 diseased without hemaopoietic neoplasia (orange dots)\nand 5 diseased with chronic lymphocytic leukemia (green dots) cats. nFSC = ratio between median forward scatter (FSC-H) value of the population of\n\nnFSC = ratio between median forward scatter (FSC-H) value of the population of\ninterest and granulocytes in the same sample. Since all neoplastic samples had a MHCII+CD21- phenotype, nFSC values were taken from this\npopulation also in healthy and diseased cats without hematopoietic neoplasia, in order to allow comparison. MFIratio = ratio between antibody-stained\nand unstained cells in the same fluorescence channel.\n\nMeneses-Nava et al.\b\n10.3389/fvets.2025.1640229\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nargue that in the two remaining cats (1 healthy and 1 diseased cat)\nCD4 + CD8 + cells count was lower than the detection limit of the\ntechnique, rather than being truly absent. In the literature, low\npercentages of CD4 + CD8 + cells have been found in normal or\nreactive lymph nodes in cats (10), whereas higher proportions can\nbe  found in mediastinal masses. This is due to the fact that\nCD4 + CD8 + cells represent a transient stage during T-cell\nmaturation (27). The percentage of CD4 + CD8 + cells in mediastinal\nmasses is a leading criterium for FC differential between mediastinal\nlymphoma and thymoma in dogs, but the same is not valid in cats (16,\n28). Altogether, our results and the data already published suggest that\nCD4 + CD8 + phenotype should not be considered a hallmark of\nneoplasia in cats.\nWe also found a low number of CD134 + cells in circulation\nwithout differences between healthy and Group1 diseased cats.\nCD134, also named OX40, is a member of the tumor necrosis factor\n(TNF) receptor superfamily, and works as a co-stimulator of\nlymphocyte activation, preventing apoptosis and enhancing\nproliferation (29, 30). In cats, this molecule is considered a key-driver\nof the progression of FIV infection, although almost all studies on this\ntopic report in-vitro results (31–33). All cats included in the present\nstudy tested negative for FIV antibodies within the last month prior\nto sampling. Thus, in the future, it could be interesting to test the\nconcentration of CD134 + cells in FIV-infected cats with different\nstage of disease, in order to assess its possible clinical relevance in the\nmanagement of such patients. In addition, 3 out of 5 neoplastic\nsamples stained positive for CD134. To the authors’ knowledge, this is\nthe first report of CD134 expression in neoplastic cells in cats. Thus,\nit is still unknown whether it reflects a different stage of maturation of\nthe cells, or has any prognostic implication.\nConcerning the data from 5 cats diagnosed with probable T-CLL\n\nThus,\nit is still unknown whether it reflects a different stage of maturation of\nthe cells, or has any prognostic implication.Concerning the data from 5 cats diagnosed with probable T-CLL\nwe present herein, T-cell lineage was confirmed in all cases by PARR\nTCRγ monoclonal results and FC expression of CD4. Our results\nagree with a recent study, which reported a high prevalence of\nCD4 + neoplastic lymphocytosis in cats, whereas B-cell lymphocytosis\nwas generally not clonal, and indeed not represented in our caseload\n(8). Of notice, Rout and colleagues also identified a less common\n“CD5low” subgroup, bearing a worse prognosis. In contrast, the\nmajority of the cases in the present study (3 out of 4 CD5+) had lower\nCD5-MFIratio than normal T-cells, and one case was even CD5-.\nUnfortunately, we lack follow-up data for our cohort, thus preventing\ncomparison with the survival data for the “CD5low” disease.\nNeoplastic cells were similar to those of the non-neoplastic\ncounterparts, either in healthy or Group1 diseased cats, with the only\nexception of CD5-MFIratio (Figure 3). In particular, nFSC values\noverlapped in Group1 and Group2 diseased cats (Figure 3). Once\nmore, this fact suggests that cell size alone should not be used to\ndifferentiate between neoplastic and non-neoplastic cells in cats.\nAnother interesting finding is the level of expression of CD18.\nWe already reported that this integrin is expressed at low levels in\ncirculating lymphocytes in healthy cats (12). Based on our present\nresults, CD18 seems to be overexpressed on neoplastic cells. On one\nside, this could play a role in the spread of the disease, since this\nmolecule is involved in leukocyte rolling and extravasation (34). On\nthe other hand, the differential degree of expression could serve as a\nlaboratory tool to be included in the diagnostic process for suspected\nfeline leukemias. Further studies are needed to support\nboth hypotheses.\nThe low number of samples included represent the main limitation\nof the present study. This is particularly true for diseased cats, where\n\nFurther studies are needed to support\nboth hypotheses.The low number of samples included represent the main limitation\nof the present study.This is particularly true for diseased cats, where\nsubjects with different diseases were included in Group1, and all\nGroup2 cats had a T-cell neoplasia. Also, the cats included had\ndifferent breed, sex and age possibly biasing our results. Because of\nthis, they should not be interpreted as reference interval for healthy\ncats or laboratory parameters to differentiate among clinical\nconditions. Rather, they represent a basis to improve FC analysis of\nfeline samples in a diagnostic setting. Finally, the lack of follow-up\ndata for cats with probable CLL prevented us from making any\nhypothesis about possible prognostic role of the different phenotypes\nwe recorded.\nIn conclusion, this is the first study reporting FC features of\nlymphocyte subsets in cats, applying a large antibody panel, and\nhighlighting some differences between healthy and diseased cats,\neither with neoplastic or non-neoplastic conditions. The antibody\npanel we present is composed of fluorochrome-conjugated antibodies\navailable in the market, and as such can be easily reproduced in any\nveterinary FC facility. However, it is important to note that some of\nthe antibody clones we used are reported not to work on preserved\nsamples (35). Thus, the panel is suitable only to analyze fresh samples,\nwhich is overall generally recommended in FC practice. Future studies\nshould address the biological and diagnostic relevance of FC data\nin cats.\nData availability statement\nThe raw data supporting the conclusions of this article will\nbe made available by the authors, without undue reservation.\nEthics statement\nThe requirement of ethical approval was waived by Ethical\nCommittee, University of Milan for the studies involving animals\nbecause all samples included in the present study had been delivered\nto the laboratory of clinical pathology of the Veterinary Teaching\nHospital (VTH, University of Milan) after being sampled for\n\ndiagnostic purposes or routine health check, with a written informed\nconsent of the owner. Thus, specific approval of the Ethical Committee\nto use leftover specimens for research purposes was not required (EC\ndecision dated 29 October 2012, renewed under protocol 02–2016,\nUniversity of Milan). The studies were conducted in accordance with\nthe local legislation and institutional requirements. Written informed\nconsent was obtained from the owners for the participation of their\nanimals in this study.\nAuthor contributions\nCM-N: Investigation, Methodology, Writing – original draft. AU:\nInvestigation, Writing – review & editing. GD: Investigation, Writing –\nreview & editing. SH: Investigation, Writing – review & editing. BR:\nInvestigation, Writing – review & editing. CL: Investigation, Writing –\nreview & editing. AS: Investigation, Writing – review & editing. VM:\nConceptualization, Formal analysis, Investigation, Methodology,\nResources, Validation, Writing – original draft.\n\nMeneses-Nava et al.\b\n10.3389/fvets.2025.1640229\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. The present study was\nfunded by MyLav srl, Alessano, Lecce, Italy. The funder was not\ninvolved in the study design, collection, analysis, interpretation of\ndata, the writing of this article, or the decision to submit it\nfor publication.\nAcknowledgments\nThe authors wish to thank the VTH veterinary staff and the\nprivate vets who sent their samples to our clinical pathology laboratory\nand flow cytometry facility.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the reviewers.\nAny product that may be evaluated in this article, or claim that may\nbe made by its manufacturer, is not guaranteed or endorsed by\nthe publisher.\nSupplementary material\nThe Supplementary material for this article can be found online\nat: https://www.frontiersin.org/articles/10.3389/fvets.2025.1640229/\nfull#supplementary-material\nReferences\n1.\tComazzi S, Gelain ME. 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+{"text": "【兽医文献】\n标题: Methionine and its derivatives in dairy cow nutrition: implications for intestinal barrier function, periparturient performance, and metabolic health\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nAssociation between subclinical\nmastitis pathogens and passive\ntransfer of immunity in calves\nGokcenur Sanioglu Golen  *\nDepartment of Microbiology, Faculty of Veterinary Medicine, Aksaray University, Aksaray, Türkiye\nIntroduction: Colostrum plays a critical role in providing passive immunity in\nnewborn calves, and its immunological effectiveness is closely linked to the\nhealth status of the producing cow. Subclinical mastitis, an inflammation of the\nmammary gland without visible clinical signs, frequently caused by pathogens\nsuch as Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae, and\nCorynebacterium bovis, can impair colostrum quality. Haptoglobin (Hp) and\nserum amyloid A (SAA) are acute phase proteins that increase during infection\nand inflammation and may serve as biomarkers for detecting subclinical mastitis\nand assessing its impact on calf health.\nMethods: Colostrum and serum samples were collected from 20 healthy cows\nand 20 cows diagnosed with subclinical mastitis, along with their newborn calves.\nBacteriological cultures were performed to identify mastitis pathogens. Hp, SAA,\nand total immunoglobulin (Ig) concentrations were measured using ELISA kits.\nCorrelations between colostrum and calf serum biomarker levels were analyzed.\nCalves were monitored for diarrhea in the first week of life, and fecal samples\nfrom diarrheic calves were tested for common enteric pathogens.\nResults: All cows in the subclinical mastitis group were positive for at least one mastitis-\ncausing pathogen, most frequently S. aureus (35%), followed by E. coli (30%), S. agalactiae\n(20%), and C. bovis (15%). Cows with subclinical mastitis had significantly higher Hp\nand SAA levels and lower Ig concentrations in colostrum compared with healthy cows\n(p < 0.05). Calves from the subclinical mastitis group also exhibited higher serum Hp\nand SAA concentrations and lower serum Ig levels (p < 0.05). Positive correlations\nwere found between colostrum and calf serum Hp and SAA concentrations, while\n\n05).Calves from the subclinical mastitis group also exhibited higher serum Hp\nand SAA concentrations and lower serum Ig levels (p < 0.05).Positive correlations\nwere found between colostrum and calf serum Hp and SAA concentrations, while\ncolostrum Ig showed negative correlations with both biomarkers. Four calves from the\nsubclinical mastitis group developed diarrhea, all with low serum Ig concentrations,\nand fecal analysis revealed rotavirus, coronavirus, or E. coli K99.\nDiscussion: Subclinical mastitis, particularly infections caused by S. aureus\nand E. coli, is associated with increased inflammatory markers and reduced\nimmunoglobulin content in colostrum, leading to impaired passive immune\ntransfer in calves. Elevated Hp and SAA in calves may indicate both passive\ntransfer from colostrum and early activation of the neonatal immune system.\nThese findings highlight the importance of pathogen-driven alterations in\ncolostrum composition and support the potential of Hp and SAA as biomarkers\nfor monitoring subclinical mastitis and predicting calf health outcomes.\nKEYWORDS\nbiomarker, Escheria coli, immunoglobulin, pathogen, Staphylococcus aureus\n1 Introduction\nCalf immunity, development, and growth are critical factors affecting cow productivity.\nThe first 4 week period is of critical importance in minimizing calf morbidity and mortality.\nHowever, in the neonatal period, their neonatal innate responses may not be fully developed\nand they are unable to form an active immune response. Neonatal calves rely primarily on\nOPEN ACCESS\nEDITED BY\nCarol Geralyn Chitko-McKown,\nAgricultural Research Service (USDA),\nUnited States\nREVIEWED BY\nMarina Erm,\nEstonian University of Life Sciences, Estonia\nAdil Karakus,\nIVC Evidensia, United Kingdom\n*CORRESPONDENCE\nGokcenur Sanioglu Golen\ngokcenursanioglu@aksaray.edu.tr\nRECEIVED 12 July 2025\nACCEPTED 12 August 2025\nPUBLISHED 02 September 2025\nCITATION\nSanioglu Golen G (2025) Association between\nsubclinical mastitis pathogens and passive\ntransfer of immunity in calves.\nFront. Vet. Sci. 12:1664685.\ndoi: 10.3389/fvets.2025.1664685\nCOPYRIGHT\n© 2025 Sanioglu Golen. This is an\n\nFront.Vet.Sci.12:1664685.doi: 10.3389/fvets.2025.1664685\nCOPYRIGHT\n© 2025 Sanioglu Golen.This is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY). The use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\naccordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Original Research\nPUBLISHED  02 September 2025\nDOI  10.3389/fvets.2025.1664685\n\nSanioglu Golen\b\n10.3389/fvets.2025.1664685\nFrontiers in Veterinary Science\n02\nfrontiersin.org\npassive immunity transferred from cows for protection against\ndisease. Colostrum plays a central role in immune transfer; it contains\nmany immune components critical for immunity such as\nimmunoglobulins, cytokines and acute phase proteins (APPs), lipids,\ncarbohydrates, and micronutrients, and it is also affected by subclinical\nmastitis pathogens (1).\nIn dairy farming, the health of a calf after birth is related to the\nquality of the colostrum it receives from its cows milk. The amount\nand biological activity of these components are closely related to the\ncow’s health status and in particular the inflammatory condition of the\nmammary gland (1, 2). Subclinical mastitis, a condition characterized\nby inflammation of the mammary tissue without obvious clinical\nsigns, may cause alteration in colostrum content and negatively affect\nthe transfer of passive immunity (2).\nSubclinical mastitis, which is an inflammation of the mammary gland\nthat occurs without obvious clinical symptoms, is commonly caused by\npathogens such as Staphylococcus aureus (S. aureus), Escherichia coli\n(E. coli),  Streptococcus agalactiae (S. agalactiae), and Corynebacterium\nbovis (C. bovis), and can negatively affect colostrum quality. Acute phase\nproteins like haptoglobin (Hp) and serum amyloid A (SAA) increase in\nresponse to infection and inflammation, making them potential\nbiomarkers for identifying subclinical mastitis and evaluating its effects\non calf health. Haptoglobin (Hp) and serum amyloid A (SAA) are acute\nphase proteins (APP) that play an important role in cattle health and can\nrise rapidly during infection. The increase in APP levels in milk during\nmastitis makes these proteins useful as markers of mammary infections\nor inflammations. Therefore, Hp and SAA are considered important\nbiomarkers for both the diagnosis of infection and the early detection of\nsubclinical mastitis (2–4). The synthesis of Hp in both the liver and\nmammary tissue enables the use of milk Hp concentration as an indicator\n\nThe synthesis of Hp in both the liver and\nmammary tissue enables the use of milk Hp concentration as an indicator\nof local inflammation. Recent studies have shown that Hp is not only\npresent in systemic circulation but can also be locally produced in the\nmammary gland and transferred to milk (5, 6). Especially in cases of\nsubclinical mastitis, increases in milk Hp and SAA levels may be observed,\nwhile colostrum quality and immunoglobulin (Ig) levels may decrease,\nwhich could negatively affect immune transfer to calves (7). Recent\nstudies have reported that milk Hp levels are significantly elevated in cows\nwith subclinical mastitis, which increases (8).\nAs stated by Honkala et al. (1), colostrum plays a decisive role in\nthe immunity of newborns. Cytokines and APPs found in colostrum,\nespecially Hp, can contribute directly to the immune development of\ncalves beyond passive immunity. On the other hand, some studies\nemphasize that the M-SAA3 isoform of SAA found in colostrum has\nlocal effects, particularly in the intestinal system, and that its passage\ninto the systemic circulation is limited. High levels of inflammatory\nmarkers such as Hp and SAA in calves with insufficient total Ig levels\nmay indicate inadequate passive immunity transfer (9). In this context,\ncomparing Hp, SAA, and total Ig levels measured in colostrum and\ncalf blood may help understand the effects of subclinical mastitis on\ncalf health.\n2 Materials and methods\nThis study was conducted on a commercial dairy farm located in\nAksaray, Türkiye, which maintains a large herd of lactating Jersey\ncows. The animals, all in their second to fourth lactation, were housed\nin a free-stall system under the same management and environmental\nconditions, milked twice daily, and clinically healthy at the time of\ncalving. They were fed a total mixed ration formulated to meet the\nnutritional requirements of lactating dairy cows and had ad libitum\naccess to clean water.\nA total of 40 multiparous cows were selected from a larger\npopulation of calving animals over a two-month period. Selection\n\nA total of 40 multiparous cows were selected from a larger\npopulation of calving animals over a two-month period.Selection\ncriteria included the availability of complete health records and the\nabsence of systemic illness or clinical mastitis. Within 48 h\npostpartum, the cows were evaluated using the California Mastitis\nTest (CMT), somatic cell count (SCC), and bacteriological culture of\ncolostrum samples.\nSubclinical mastitis was diagnosed based on the presence of all three\nof the following criteria: (1) SCC > 200,000 cells/mL, (2) a positive CMT\nscore (≥trace), and (3) isolation of at least one mastitis-causing bacterium\n(S. aureus, E. coli, S. agalactiae, or C. bovis) from colostrum culture. Cows\nmeeting these criteria were assigned to the subclinical mastitis (SCM)\ngroup (n = 20). The healthy control group (n = 20) consisted of cows with\nSCC < 200,000 cells/mL, negative CMT results, and no bacterial growth\nin culture. Group selection ensured parity and calving dates were\ncomparable between groups.\nEach calf born to a selected cow was included in the study,\nresulting in 40 cow–calf pairs. Colostrum samples were aseptically\ncollected within 48 h after calving. Ten milliliters of each sample were\ntransferred into sterile tubes and stored for microbiological analysis.\nCultures were performed on 5% sheep blood agar and MacConkey\nagar, and incubated aerobically at 37 °C for 24 h. Bacterial colonies\nwere identified using standard microbiological techniques, including\nmorphology, Gram staining, and biochemical testing.\nBlood samples were obtained from the jugular vein of each calf\nbetween 24 and 36 h of life using sterile vacutainer tubes. Calves were fed\ncolostrum within the first 2 h after birth and again within the first 12 h,\nreceiving a total of approximately 4–6 liters during the first day of life.\nColostrum samples were centrifuged at 3,000 rpm for 15 min to\nseparate the supernatant. Calf serum samples were stored at −20 °C\nuntil analysis.\nThe sample size (n = 20 per group) was determined based on\nprevious studies examining colostrum quality and passive immunity\n\nCalf serum samples were stored at −20 °C\nuntil analysis.The sample size (n = 20 per group) was determined based on\nprevious studies examining colostrum quality and passive immunity\nin calves, with the goal of detecting significant differences in\nimmunoglobulin and acute phase protein levels. The design achieved\n80% statistical power at a significance level of 0.05, while also\naccounting for practical constraints such as the availability of cows\nmeeting the inclusion criteria during the calving period.\nHp concentrations in colostrum and calf serum were measured\nusing a commercial ELISA kit [Bioassay Technology Laboratory, Cat.\nNo: E1035Bo; (10)]. SAA levels were determined using an ELISA kit\nfrom MyBioSource (Cat. No: MBS036405). Total immunoglobulin\n(Ig) concentrations in calf serum were assessed using an ELISA kit\nfrom Elabscience (Cat. No: BOFI00152). Immunoglobulin\nconcentrations in colostrum were directly measured using a\ncommercial bovine Ig ELISA kit (Elabscience, Cat. No: BOFI00152)\naccording to the manufacturer’s instructions. Colostrum samples were\ndiluted 1:10 prior to analysis. Optical density was read at 450 nm using\na microplate reader, and concentrations were calculated from a\nstandard curve. In addition to calf serum Ig measurements, these\ncolostrum Ig values were used to assess colostrum quality and their\ncorrelation with passive transfer efficiency. Sample dilutions were\nprepared according to the manufacturer’s instructions: 1:10 for\ncolostrum and 1:1000 for serum. Optical density was read at 450 nm\n\nSanioglu Golen\b\n10.3389/fvets.2025.1664685\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nusing a microplate reader, and concentrations were calculated based\non standard curves.\nAll statistical analyses were performed using IBM SPSS Statistics\nversion 26.0. Data distribution was assessed using the Shapiro–Wilk\ntest to determine the appropriate statistical method. Normally\ndistributed variables were analyzed using independent samples t-tests,\nwhile Pearson correlation analysis was used to assess relationships\nbetween continuous variables. Statistical significance was defined as\np < 0.05. Bonferroni correction was applied for multiple comparisons.\nFresh fecal samples were collected from calves exhibiting diarrhea\nwithin the first week of life. These samples were analyzed for the\npresence of rotavirus, coronavirus, and enterotoxigenic E. coli (K99\nantigen) using the IDEXX Rota-Corona-K99 ELISA test kit, following\nthe manufacturer’s instructions. The results were analyzed using the\nsame statistical parameters as described above.\n3 Results\nBacteriological analysis of colostrum samples obtained from cows\nin the subclinical mastitis (SCM) group showed that all 20 milk\nsamples (100%) were positive for at least one mastitis-causing bacterial\npathogen. The most frequently isolated microorganism was S. aureus,\ndetected in 35% of the samples, followed by E. coli (30%), S. agalactiae\n(20%), and C. bovis (15%), as shown in Table 1. No bacterial growth\nwas identified in any of the samples from the healthy control group.\nAnalysis of inflammatory markers revealed significant\ndifferences between groups. Hp and SAA levels in the milk of cows\ndiagnosed with subclinical mastitis were significantly higher than\nthose in the healthy group. Specifically, the mean milk Hp\nconcentration in the SCM group was 85.3 ± 12.5 μg/mL, compared\nto 49.6 ± 10.8 μg/mL in the healthy group (p = 0.004). Similarly,\nmilk SAA concentrations were 63.8 ± 9.7 μg/mL in the SCM group\nand 35.2 ± 7.3 μg/mL in the control group (p = 0.012). In the\ncorresponding calves, serum Hp and SAA levels were also\nsignificantly elevated in the SCM group. Calf serum Hp levels\n\n004).Similarly,\nmilk SAA concentrations were 63.8 ± 9.7 μg/mL in the SCM group\nand 35.2 ± 7.3 μg/mL in the control group (p = 0.012).In the\ncorresponding calves, serum Hp and SAA levels were also\nsignificantly elevated in the SCM group.Calf serum Hp levels\naveraged 93.1 ± 11.2 μg/mL in the SCM group and 60.7 ± 9.4 μg/\nmL in the healthy group (p = 0.001), while SAA levels were\n71.4 ± 10.1 μg/mL and 41.5 ± 8.2 μg/mL, respectively (p = 0.008).\nIn terms of immune status, immunoglobulin (Ig) concentrations\nin colostrum were significantly lower in the SCM group (mean ± SD:\n45.8 ± 7.6 mg/mL) compared to the healthy group (63.4 ± 8.2 mg/mL;\np = 0.002). Similarly, total Ig concentrations in calf serum were\nsignificantly lower in the SCM group (19.7 ± 3.8 mg/mL) compared\nto the healthy group (27.6 ± 4.2 mg/mL; p = 0.023). A strong positive\ncorrelation was found between colostrum Ig and calf serum Ig levels\n(r = 0.71, p < 0.05). Colostrum Ig also showed moderate negative\ncorrelations with colostrum Hp (r = −0.52, p < 0.05) and SAA\n(r = −0.49, p < 0.05), indicating that mammary inflammation may\nbe associated with reduced immunoglobulin content.\nDuring the first week of life, diarrhea was observed in four of the\nforty calves (10%) included in the study. All affected calves were\nfrom the subclinical mastitis group, representing an incidence of\n20% within that group, while no cases of diarrhea were reported in\nthe healthy control group. Although this difference was not\nstatistically significant (Fisher’s exact test, p = 0.10), the finding may\nbe biologically relevant. Each of the diarrheic calves had serum\nimmunoglobulin levels below 20 mg/mL, consistent with failure of\npassive transfer. Pathogen screening via IDEXX Rota-Corona-K99\nELISA tests identified rotavirus in two calves, coronavirus in one\ncalf, and K99-positive E. coli in one calf. These findings suggest a\npossible link between maternal subclinical mastitis, impaired passive\nimmunity, and increased neonatal susceptibility to enteric pathogens\n(Figure 1).\n4 Discussion\nThis study aimed to investigate the relationship between Hp\n\nThese findings suggest a\npossible link between maternal subclinical mastitis, impaired passive\nimmunity, and increased neonatal susceptibility to enteric pathogens\n(Figure 1).4 Discussion\nThis study aimed to investigate the relationship between Hp\nand SAA levels in the colostrum of cows with subclinical mastitis\n(SCM) and systemic concentrations of Hp, SAA, and total\nimmunoglobulin (Ig) in their neonatal calves. The findings\ndemonstrated that both Hp and SAA levels were significantly\nelevated in the colostrum and serum of the SCM group, whereas\ntotal Ig concentrations in the calves were significantly lower\ncompared to the healthy group. These results suggest that\nsubclinical mastitis affects colostrum composition and may impair\npassive immune transfer in calves (Table 2).\nMastitis is characterized by local inflammation and increased\npermeability of the mammary epithelium, which alters the\nimmunological profile of colostrum. Acute phase proteins (APPs),\nincluding Hp and SAA, are known to increase in colostrum during\ninflammatory responses. Previous studies have identified Hp as a\npromising diagnostic biomarker in subclinical mastitis. Carinelli\net al. (2) demonstrated its diagnostic utility, and in a subsequent\nstudy, the same group developed a biosensor-based method for\nrapid, field-based Hp detection in milk. Carinelli et al. (2) also\nshowed that milk Hp levels were both sensitive and specific for\ndistinguishing subclinical from clinical mastitis, and that Hp\nresponses varied by pathogen.\nIn our study, S. aureus and E. coli were identified as the\npredominant pathogens in subclinical mastitis (SCM) milk\nsamples, a finding that is in line with the well-established role of\nthese bacteria as major causative agents of intramammary\nTABLE 1  Bacterial pathogens isolated from milk samples in the subclinical mastitis group.\nPathogen\nNumber of isolated samples\n(n = 20)\nPercentage (%)\nS. aureus\n7\n35%\nE. coli\n6\n30%\nS. agalactiae\n4\n20%\nC. bovis\n3\n15%\nTotal\n20\n100%\n\nSanioglu Golen\b\n10.3389/fvets.2025.1664685\nFrontiers in Veterinary Science\n04\nfrontiersin.org\ninfections in dairy cows. The frequent isolation of these pathogens\nnot only reflects their widespread prevalence in bovine mastitis\ncases but also underscores their significant impact on udder health\nand milk quality. Importantly, the association of these\nmicroorganisms with acute phase responses highlights the intricate\ninterplay between pathogen type and host immune activation.\nSpecifically, the markedly elevated levels of haptoglobin (Hp)\nobserved in samples positive for S. aureus infection are consistent\nwith previous reports (11), suggesting that this pathogen induces\na more pronounced acute phase response compared to other\nbacteria. This may be attributable to the chronic and often\npersistent nature of S. aureus infections, which are known to\ntrigger sustained inflammatory signaling and tissue damage. In\ncontrast, while E. coli is also capable of stimulating acute phase\nprotein synthesis, its effects may be more transient due to the\ntypically acute but shorter duration of coliform infections.\nOur findings also align with the results of Åkerstedt et al. (12),\nwho assessed Hp and SAA in the milk of clinically healthy cows\nand reported a correlation between elevated APPs and changes in\nmilk composition, including protein and lactose levels. In their\nmultivariate analysis, high somatic cell counts were associated with\nincreased APP concentrations and reduced casein and lactose\ncontent. These changes may negatively affect both milk processing\ncharacteristics and the ability of colostrum to support passive\nimmunity in calves.\nThe synthesis of Hp locally in the mammary gland, in addition\nto hepatic production, supports the utility of colostrum as a matrix\nfor evaluating local inflammation (4). However, differences\nbetween serum and plasma Hp measurements have been noted (5),\nand should be  considered when comparing biological fluids.\nDespite this, we observed a moderate positive correlation between\nHp levels in colostrum and those in calf serum, supporting a\npotential immunological link between maternal colostrum and\n\nDespite this, we observed a moderate positive correlation between\nHp levels in colostrum and those in calf serum, supporting a\npotential immunological link between maternal colostrum and\nneonatal systemic responses.\nThe elevated Hp and SAA levels in calf serum suggest activation of\nthe neonatal inflammatory response. Whether these proteins are\ntransferred passively or reflect endogenous synthesis is a matter of\ndebate. Honkala et  al. (1) proposed that some APPs can cross the\nintestinal barrier shortly after birth, contributing to early immune\nmodulation. In particular, IL-6-mediated stimulation of Hp synthesis\nmay shift the neonatal immune response from an innate Th2-dominant\nprofile toward a more adaptive Th1-dominant state. Hendriks et al. (8)\nalso demonstrated that milk Hp concentrations are highest within the\nfirst 24 h postpartum, consistent with our sampling timeline and findings.\nFIGURE 1\nComparison of haptoglobin (Hp), serum amyloid A (SAA), and total immunoglobulin (Ig) concentrations between diarrheic (n = 4) and non-diarrheic\n(n = 36) calves. Bars represent mean ± standard deviation (SD). All diarrheic calves were born to cows with subclinical mastitis.\nTABLE 2  Mean ± standard deviation (SD) concentrations of haptoglobin (Hp), serum amyloid A (SAA), and total immunoglobulin (Ig) in colostrum and\ncalf serum according to maternal health status.\nParameter\nMilk (subclinical)\nMilk (healthy)\nCalf serum\n(subclinical)\nCalf serum\n(healthy)\np-value\nHaptoglobin (μg/mL)\n85.3 ± 12.5\n49.6 ± 10.8\n93.1 ± 11.2\n60.7 ± 9.4\n0.004\nSAA (μg/mL)\n63.8 ± 9.7\n35.2 ± 7.3\n71.4 ± 10.1\n41.5 ± 8.2\n0.012\nTotal Ig (mg/mL)\n45.8 ± 7.6\n63.4 ± 8.2\n19.7 ± 3.8\n27.6 ± 4.2\n0.002* (milk)/0.023\n(serum)\nValues are compared between cows diagnosed with subclinical mastitis and healthy controls. Ig levels were assessed in both colostrum and calf serum. p-values are shown for between-group\ncomparisons.\n\nSanioglu Golen\b\n10.3389/fvets.2025.1664685\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nIn contrast, the extent to which SAA is absorbed systemically\nremains uncertain. Honkala et al. (1) noted that SAA isoforms in\ncolostrum primarily exert localized effects on the gastrointestinal\ntract and are minimally present in systemic circulation. This could\nexplain the weaker correlation between colostral and serum SAA\nlevels in our study. Orro et al. (13) also observed that systemic SAA\nlevels in calves increase in response to microbial colonization and\nvary with age, suggesting that early postnatal inflammation may\nreflect environmental exposures rather than passive SAA transfer.\nIn this study, colostrum Ig concentrations were directly measured\nand found to be significantly reduced in the SCM group compared to\nhealthy cows, confirming that subclinical mastitis compromises\ncolostrum quality. Total Ig concentrations are a well-established\nindicator of passive transfer effectiveness. The significantly lower Ig\nlevels observed both in colostrum and in the serum of calves from the\nSCM group provide direct evidence that subclinical mastitis\ncompromises colostrum quality. The strong positive correlation\nbetween colostrum Ig and calf serum Ig levels in our study aligns with\nprevious findings by Furman-Fratczak et al. (9) and Chigerwe et al.\n(14), who reported that colostrum Ig concentration is the primary\ndeterminant of passive transfer success. The observed negative\ncorrelation between colostrum Ig and colostral Hp/SAA\nconcentrations supports the concept that inflammation in the\nmammary gland can impair immunoglobulin synthesis, secretion, or\nstability, as suggested by Baumrucker and Bruckmaier (15). This\nrelationship underscores the importance of controlling subclinical\nmastitis not only to reduce local inflammation but also to preserve the\nimmunological value of colostrum. According to Furman-Fratczak\net al. (9), serum IgG concentrations below 10 g/L are indicative of\nfailure of passive transfer (FPT), which increases susceptibility to\ninfectious diseases and mortality in neonatal calves. In our study, the\n\nAccording to Furman-Fratczak\net al.(9), serum IgG concentrations below 10 g/L are indicative of\nfailure of passive transfer (FPT), which increases susceptibility to\ninfectious diseases and mortality in neonatal calves.In our study, the\nnegative correlation between total Ig and APPs (Hp and SAA) suggests\nthat inflammatory colostrum may impair immunoglobulin absorption\nor reflect concurrent infection risks in the calf.\nDžermeikaitė et al. (16) reported that high SAA levels in milk\nare associated with low lactose concentrations, implicating\nmammary epithelial dysfunction in subclinical inflammation. This\nsupports the idea that inflammation-induced alterations in\ncolostrum composition may impact neonatal health. A limitation\nof this study is that potential confounding factors such as parity\nand environmental conditions, although standardized in our herd,\ncould still influence colostrum composition and calf immunity.\nThe occurrence of diarrhea in four calves from the SCM group,\nall of which had serum Ig levels below 20 mg/mL, further highlights\nthe clinical implications of impaired passive immunity. The\nidentification of enteric pathogens—rotavirus, coronavirus, and\nK99 + E. coli—in these calves underscores their increased\nvulnerability to infections. Although the sample size limits statistical\nsignificance (Fisher’s exact test, p = 0.10), the findings are consistent\nwith prior research linking FPT with higher morbidity. However, the\nsmall number of diarrheic calves (n = 4) limits the statistical power\nof the subgroup analysis. Although Fisher’s exact test was applied, the\nsmall sample size means these results should be interpreted with\ncaution, and larger-scale studies are needed before drawing firm\ncausal conclusions. Honkala et  al. (1) emphasized the role of\ncolostrum-derived APPs in shaping neonatal immune development,\nwhile Furman-Fratczak et al. (9) stressed the importance of achieving\nadequate IgG levels to reduce disease incidence.\nOur findings also corroborate the study by Kabu et al. (17), which\nreported significantly elevated Hp and SAA levels in diarrheic calves\n\n(9) stressed the importance of achieving\nadequate IgG levels to reduce disease incidence.Our findings also corroborate the study by Kabu et al.(17), which\nreported significantly elevated Hp and SAA levels in diarrheic calves\ncompared to healthy controls. Similarly, Hajimohammadi et al. (18)\nfound that APP concentrations rose in parallel with diarrhea severity\nand were correlated with clinical signs, suggesting their potential as\ndiagnostic and prognostic biomarkers in neonatal disease. Şeliman\net al. (19) reported significantly elevated Hp and SAA levels in cattle\nwith dermatophytosis before treatment, which decreased following\ntherapy. Similarly, increased levels of Hp and SAA in the colostrum\nof cows with subclinical mastitis were mirrored in their calves, along\nwith reduced passive immunity. Both studies highlight that Hp and\nSAA are reliable biomarkers of infection and useful indicators for\nmonitoring disease progression and treatment response.\n5 Conclusion\nIn conclusion, this study demonstrated that subclinical mastitis,\nmost frequently associated with S. aureus and E.coli, increases\nhaptoglobin (Hp) and serum amyloid A (SAA) concentrations in\ncolostrum while reducing its immunoglobulin (Ig) content. Calves\nborn to SCM-affected cows exhibited elevated serum Hp and SAA,\nreduced Ig levels, and a higher incidence of diarrhea, indicating\nimpaired passive transfer of immunity and increased susceptibility to\nenteric pathogens. These findings highlight the pathogen-driven\nalterations in colostrum composition as a critical factor influencing\nneonatal health. Early detection and control of SCM are therefore\nessential not only to protect colostrum immunological quality but\nalso to prevent passive transfer failure and reduce the risk of\ncalf diarrhea.\nData availability statement\nThe original contributions presented in the study are included in\nthe article/supplementary material, further inquiries can be directed\nto the corresponding author.\nEthics statement\nThe animal study was approved by We  confirm that written\ninformed consent was obtained from the owners for the participation\n\nEthics statement\nThe animal study was approved by We  confirm that written\ninformed consent was obtained from the owners for the participation\nof their animals in this study. This has been confirmed and approved\nby the Local Ethics Committee for Animal Experiments at our\ninstitution. Accordingly, we  have uploaded the original ethics\ncommittee approval letter along with its English translation as a\n“Review Only” file to support this statement (Animal Experiments\nLocal Ethics Committee Approval No: 2025/05/39). The study was\nconducted in accordance with the local legislation and\ninstitutional requirements.\nAuthor contributions\nGS:\nConceptualization,\nFormal\nanalysis,\nInvestigation,\nMethodology, Resources, Validation, Writing  – original draft,\nWriting – review & editing.\n\nSanioglu Golen\b\n10.3389/fvets.2025.1664685\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nFunding\nThe author(s) declare that no financial support was received for\nthe research and/or publication of this article.\nAcknowledgments\nThe author thanks the entire staff of the Department of\nMicrobiology, Aksaray University, for their invaluable support\nthroughout the study.\nConflict of interest\nThe author declares that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe author declares that no Gen AI was used in the creation of\nthis manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. Any product\nthat may be evaluated in this article, or claim that may be made by its\nmanufacturer, is not guaranteed or endorsed by the publisher.\nReferences\n1.\tHonkala MA Cow colostrum acute phase proteins (SAA, Hp) and cytokines\n(IL-1β, IL-6): Associations with inflammatory response in neonatal calves. Eesti\nMaaülikool. (2021)\n2.\tCarinelli S, Fernández I, González-Mora JL, Salazar-Carballo PA. Haptoglobin\nelectrochemical diagnostic method for subclinical mastitis detection in bovine milk.\nChemosensors. (2023) 11:378.\n3.\tAras Z, Yavuz O. Evaluation of fetal heart serum amyloid A concentrations in\ninfectious cattle abortion cases. Heliyon. 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+{"text": "【兽医文献】\n标题: Intersectionality, equity-mindedness, and access to care\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nSeroprevalence study for selected\nzoonotic vector-borne pathogens\nin sheep from endemic areas of\nCroatia\nLjubo Barbic 1*, Vladimir Stevanovic 1, Maja Mauric Maljkovic 2,\nGorana Miletic 1, Ivona Coric 1, Vladimir Savic 3, Viktor Masovic 3,\nMaja Bogdanic 4,5, Alan Medic 6 and Tatjana Vilibic-Cavlek 4,5*\n1 Department of Microbiology and Infectious Diseases with Clinic, Faculty of Veterinary Medicine,\nUniversity of Zagreb, Zagreb, Croatia, 2 Department of Animal Breeding and Livestock Production,\nFaculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia, 3 Poultry Center, Croatian\nVeterinary Institute, Zagreb, Croatia, 4 Department of Virology, Croatian Institute of Public Health,\nZagreb, Croatia, 5 School of Medicine, University of Zagreb, Zagreb, Croatia, 6 Department of\nEpidemiology, Zadar County Institute of Public Health, Zadar, Croatia\nSurveillance is crucial in controlling and preventing vector-borne zoonotic diseases\n(VBDs). We  analyzed the seroprevalence of selected vector-borne zoonotic\npathogens in sheep from endemic areas and their role as possible sentinels for\nVBDs. A total of 300 sheep from seven farms at three micro-locations were\ntested for the presence of IgG antibodies against tick-borne encephalitis virus\n(TBEV), West Nile virus (WNV), Usutu virus (USUV), Borrelia burgdorferi s.l., and\nCrimean-Congo hemorrhagic fever virus (CCHFV) using ELISA with confirmation\nof borderline/positive results by VNT. Seropositivity for at least one pathogen was\nobserved in 18.0% (54/300) of sheep. The highest seroprevalence was confirmed\nfor TBEV (9.7%; 29/300), followed by WNV (3.0%; 9/300) and B. burgdorferi s.l.\n(2.7%; 8/300), while USUV and inconclusive flavivirus (TBEV/WNV/USUV) infections\nhad the same seroprevalence of 1.3% (4/300). None of the serum samples tested\npositive for CCHFV. Geographic micro-location was a significant risk factor for\nUSUV (p = 0.045), TBEV (p = 0.03), and B. burgdorferi s.l. (p = 0.015) infections,\nbut not for WNV. The farm distance from the household (TBEV p < 0.001, B.\n\n3% (4/300).None of the serum samples tested\npositive for CCHFV.Geographic micro-location was a significant risk factor for\nUSUV (p = 0.045), TBEV (p = 0.03), and B.burgdorferi s.l.(p = 0.015) infections,\nbut not for WNV.The farm distance from the household (TBEV p < 0.001, B.\nburgdorferi s.l. p = 0.005) and sheep breed (TBEV p < 0.001, B. burgdorferi s.l.\np < 0.001) were found as risk factors for seropositivity to tick-borne (TBEV, B.\nburgdorferi s.l.), but not to mosquito-borne diseases (WNV, USUV). Of the other risk\nfactors, sheep shearing was statistically significant, with unshared sheep showing\na higher probability of tick-borne diseases (p = 0.048). Sex, age, herd size, and the\npresence of clinical signs were not associated with the seroprevalence. Serologic\nevidence of VBDs suggests their sentinel potential for mapping micro-foci of\nzoonotic pathogens’ activity and identifying high-risk areas for public health.\nFurther studies are needed to confirm this observation.\nKEYWORDS\ntick-borne encephalitis virus, Borrelia burgdorferi s.l., West Nile virus, Usutu virus,\nCrimean-Congo hemorrhagic fever virus, sentinels\nOPEN ACCESS\nEDITED BY\nVesna Milicevic,\nUniversity of Belgrade, Serbia\nREVIEWED BY\nEva Bártová,\nUniversity of Veterinary and Pharmaceutical\nSciences Brno, Czechia\nTeufik Goletic,\nUniversity of Sarajevo,\nBosnia and Herzegovina\nDimitrije Glisic,\nUniversity of Belgrade, Serbia\n*CORRESPONDENCE\nLjubo Barbic\nljbarbic@gmail.com\nTatjana Vilibic-Cavlek\ntatjana.vilibic-cavlek@hzjz.hr\nRECEIVED 30 March 2025\nACCEPTED 07 May 2025\nPUBLISHED 02 June 2025\nCITATION\nBarbic L, Stevanovic V, Mauric Maljkovic M,\nMiletic G, Coric I, Savic V, Masovic V,\nBogdanic M, Medic A and ​\nVilibic-Cavlek T (2025) Seroprevalence study\nfor selected zoonotic vector-borne\npathogens in sheep from endemic areas of\nCroatia.\nFront. Vet. Sci. 12:1602706.\ndoi: 10.3389/fvets.2025.1602706\nCOPYRIGHT\n© 2025 Barbic, Stevanovic, Mauric Maljkovic,\nMiletic, Coric, Savic, Masovic, Bogdanic,\nMedic and Vilibic-Cavlek. This is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY). The use, distribution or\n\nThis is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY).The use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\naccordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Brief Research Report\nPUBLISHED  02 June 2025\nDOI  10.3389/fvets.2025.1602706\n\nBarbic et al.\b\n10.3389/fvets.2025.1602706\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n1 Introduction\nZoonotic vector-borne diseases (VBDs) are becoming an\nincreasing public health problem in many regions of the world (1).\nVBDs represent more than 17% of all infectious diseases, causing\nmore than 700,000 human deaths annually, with the highest disease\nburden in tropical and subtropical areas. The spread of VBDs has been\nfacilitated by different factors, including global travel and trade,\nunplanned urbanization, climate change, and the vectors’ adaptation\nand spread (2).\nAmong vector-borne pathogens, Borrelia burgdorferi s.l. (Lyme\ndisease; LD) and tick-borne encephalitis virus (TBEV) are most\nwidely distributed in Europe (1). Other flaviviruses, such as West Nile\nvirus (WNV) and Usutu virus (USUV), are also endemic in many\nEuropean countries, causing outbreaks (WNV) or sporadic infections\n(USUV) in humans (3). In addition, several epidemics of Crimean-\nCongo hemorrhagic fever (CCHF) have occurred in EU/EEA\nneighboring countries since 2013, including the Balkan region, Russia,\nand Turkey (4).\nSince zoonotic VBDs are spreading rapidly, surveillance is crucial\nin their prevention and control. To define priorities for developing\nintegrated surveillance systems that accurately model and predict the\nhuman risk of VBDs, it is important to understand the practical\noptions of connecting the surveillance data of both animals and\nhumans (5). As clinical cases of emerging diseases in humans usually\nindicate a widespread of zoonotic pathogens in an area, various animal\nsurveillance models are used as an early warning system, yielding\nvaluable results. Animal surveillance can address the significant public\nhealth challenge of gathering information on the introduction and\nemergence of new pathogens in a given area, a prerequisite for an\neffective response to protect human health (6–8).\nDifferent animal species have been tested as sentinels for VBDs,\nincluding wild birds (WNV and USUV) (9), horses (TBEV, WNV, and\nUSUV) (10), poultry (WNV and USUV) (11), and dogs (TBEV and\n\nDifferent animal species have been tested as sentinels for VBDs,\nincluding wild birds (WNV and USUV) (9), horses (TBEV, WNV, and\nUSUV) (10), poultry (WNV and USUV) (11), and dogs (TBEV and\nWNV) (12). Captive and free-ranging birds have been used for WNV\nsurveillance for decades. As primary WNV reservoirs, infections in\nbirds occurred more frequently than in humans and horses (13). In\naddition, chickens (Gallus gallus domesticus) have been routinely used\nfor arbovirus surveillance and monitoring in different settings and on\ndifferent continents. After infection, chickens do not exhibit any\nclinical signs but produce neutralizing antibodies (11). The use of\nsentinel chickens seems to be a more sensitive indicator of virus\nactivity when compared with the detection of seroconversion in wild\nbirds (14). While clinically apparent WNV infections in horses are\nrarely observed, seroprevalence studies in horses may allow the\ntracing of flavivirus transmission and help to estimate the risk for\nhuman infections (10). Whereas some studies provided evidence that\ndogs could be useful sentinels for WNV monitoring (15), others\nindicated that the role of dogs and horses in the early detection of\nhuman cases is debatable (16).\nHowever, studies that use sheep as sentinels to predict human risk\nare limited. A study conducted in Germany has shown that\nseroprevalence in free-ranging animals, particularly in sheep and\ngoats, can be a useful additional tool to identify TBEV foci in both\nendemic and non-endemic areas (17). Very few studies have analyzed\nWNV infection in sheep, with no data on USUV (18). Sheep and cattle\nbecame infected with the CCHFV in experimental inoculations but\ndeveloped only mild and transient fever. The viremia duration and\nlevel are usually low, with detectable antibodies shortly after cessation\nof viremia (19). Although CCHF in domestic ruminants is typically\nsubclinical, there is some evidence that they become reservoirs and\ncan be used as sentinels for the circulation of CCHFV, especially in\nnon-endemic areas (20).\nIn Croatia, B. burgdorferi s.l., TBEV, WNV, and USUV are the\n\nIn Croatia, B.burgdorferi s.l., TBEV, WNV, and USUV are the\nmost commonly detected vector-borne zoonotic pathogens, all\nendemic in continental regions (21). The reported number of LD\nvaries from 400 to 800. In addition, TBE is continuously recorded with\na bimodal seasonality (April–August and October–November). The\nLD and TBE endemicity is highest in northwestern and eastern\ncounties between the Sava and Drava rivers (21, 22). WNV infections\nin humans, horses, and poultry have been continuously reported in\nCroatia since 2012 (21), while USUV infections were detected\nsporadically in humans (2013, 2018, 2024) and birds (2018, 2022)\n(23–25). CCHFV has not been detected in Croatia so far.\nThe multidisciplinary approach enables the early detection of an\nincrease in pathogen activity of VBDs or confirmation of the\nemergence of a new pathogen in a given area, as has also been\nobserved in Croatia in recent years (6, 7). Chickens and horses were\nused as sentinels to detect seasonal WNV incursions in Croatia,\nrevealing a significant correlation between the geographical\ndistribution of high WNV seroprevalence in tested animals and\nhuman WNV infections (26). However, data on the sheep are limited.\nThis study aimed to analyze the seroprevalence of selected vector-\nborne zoonotic pathogens in sheep from endemic areas in eastern\nCroatia and assess the potential role of sheep as sentinels of VBDs.\n2 Materials and methods\nIn this study, 300 sheep from Vukovar-Srijem County, the\neasternmost region of Croatia, were tested for the presence of IgG\nantibodies against TBEV, WNV, USUV, B. burgdorferi s.l., and\nCCHFV. Blood samples were collected from animals from seven farms\nat three micro-locations: four in Borovo, one in Vukovar, and two in\nTrpinja (Figure 1).\nSample size calculations were performed using the RiBESS+ tool\ndeveloped by the European Food Safety Authority (27). The calculation\nincorporated the official sheep population data for Vukovar-Srijem\nCounty, the reported sensitivity of the enzyme-linked immunoassay\n(ELISA) used, and an assumed seroprevalence of 1%, reflecting the\n\nThe calculation\nincorporated the official sheep population data for Vukovar-Srijem\nCounty, the reported sensitivity of the enzyme-linked immunoassay\n(ELISA) used, and an assumed seroprevalence of 1%, reflecting the\nabsence of previously documented USUV infections in sheep.\nSerum samples were collected in the second half of April to obtain\nreliable data of the epidemiological status before the onset of peak\nvector activity.\nEpidemiological and clinical data of the tested sheep are presented\nin Table 1. Clinical signs were assessed both at the time of sampling\nand retrospectively based on owner-reported observations over the\npreceding 12 months. The following categories were evaluated:\nneurological,\nrespiratory,\nreproductive,\ngastrointestinal,\ndermatological signs, and lameness. Information regarding any\nadditional observed clinical signs was also collected. Clinical\nexaminations performed during sampling across all seven farms\nrevealed no detectable clinical abnormalities. However, according to\nthe owner of Farm 1, located in Borovo, episodes of dermatitis\naffecting all animals were reported during the winter (approximately\n3 months prior), characterized by skin erythema and partial wool loss.\n\nBarbic et al.\b\n10.3389/fvets.2025.1602706\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nThese lesions resolved spontaneously without the administration of\nany treatment.\nAll serum samples were screened using an ELISA for the detection\nof flavivirus antibodies (WNV, USUV, and TBEV) with the ID Screen®\nFlavivirus Competition (Innovative Diagnostics, Grabels, France).\nSamples with borderline or positive ELISA results were additionally\ntested by a virus neutralization test (VNT), as described by Ilic et al.\n(28), to exclude cross-reactivity and confirm infection with a specific\nflavivirus. A VNT titer of ≥10 was considered a positive result. In the\ncase of positive VNT results with more than one flavivirus, the\npathogen with an antibody titer at least fourfold higher than the others\nwas considered to be the causative agent. Samples with less than\nfourfold titer differences using different pathogens in VNT were\nconsidered inconclusive, classified as flavivirus-positive, and excluded\nfrom further risk factor analysis for a particular disease or group\nof diseases.\nThe ID Screen® Borreliosis Double Antigen Multi-species ELISA\n(Innovative Diagnostics, Grabels, France) was used for the detection\nof antibodies against B. burgdorferi s.l., and the ID Screen® CCHF\nDouble Antigen Multi-species ELISA (Innovative Diagnostics,\nGrabels, France) for CCHFV antibodies.\nAll commercial ELISA assays were performed according to the\nmanufacturer’s instructions.\nEpidemiological data were systematically collected for each\nanimal to assess possible risk factors associated with arboviral\ninfections. The analyzed parameters included farm location, flock size,\nbreed, sex, age, clinical signs, type of water bodies, and distance to\nwater bodies and households. Risk factor analyses were conducted for\nFIGURE 1\nSheep sampling area in Croatia (Vukovar-Srijem County) with three micro-locations.\nTABLE 1  Epidemiological data on sheep farms from Vukovar-Srijem County included in the study.\nCharacteristic\nVukovar\nTrpinja\nBorovo\nFarm 1\nFarm 1\nFarm 2\nFarm 1\nFarm 2\nFarm 3\nFarm 4\nFlock size\n100\n286\n50\n50\n10\n150\n70\n% of flock tested\n73.0\n28.0\n40.0\n64.0\n40.0\n33.3\n57.1\nTested females/males\n73/0\n79/1\n20/0\n32/1\n4/0\n47/3\n38/2\nBreed\n\nCharacteristic\nVukovar\nTrpinja\nBorovo\nFarm 1\nFarm 1\nFarm 2\nFarm 1\nFarm 2\nFarm 3\nFarm 4\nFlock size\n100\n286\n50\n50\n10\n150\n70\n% of flock tested\n73.0\n28.0\n40.0\n64.0\n40.0\n33.3\n57.1\nTested females/males\n73/0\n79/1\n20/0\n32/1\n4/0\n47/3\n38/2\nBreed\nRomanov\nMerino\nMerino\nTsigai\nMerino\nTsigai\nRomanov\nAverage age and range\n(years)\n3.8 (1.1–4.8)\n4.4 (1.4–12.3)\n6.2 (2.1–9.2)\n4.1 (1.1–8.2)\n6.2 (5.6–6.6)\n4.0 (2.2–4.9)\n3.5 (2.3–5.0)\nObserved clinical signs\nNo\nNo\nNo\nYes\nNo\nNo\nNo\nWater body type\nStream\nRiver\nStream\nRiver\nRiver\nRiver\nRiver\nDistance to water (km)\n0.02\n10.0\n0.2\n0.1\n0.2\n0.1\n0.3\nDistance to households\n(km)\n0.5\n0.05\n0.05\n2.0\n0.05\n1.5\n0.02\nShearing performed\nNo\nYes\nYes\nNo\nNo\nNo\nNo\n\nBarbic et al.\b\n10.3389/fvets.2025.1602706\nFrontiers in Veterinary Science\n04\nfrontiersin.org\neach VBD, with additional analyses for tick-borne infections (TBEV,\nCCHFV, and B. burgdorferi s.l.) and mosquito-borne infections (WNV\nand USUV) due to epidemiological differences.\nStatistical analyses were performed using Statistica v.14 (TIBCO\nSoftware Inc., 2020), Medcalc Odds Ratio Calculator v.23 (MedCalc\nSoftware Ltd., 2025), and R 4.4.0 (R Core Team, Vienna, Austria,\n2024). Descriptive statistics are presented as numbers and percentages.\nOdds ratios (OR) with 95% confidence intervals (95% CI) were used\nfor single bivariate risk factors. Logistic regression analysis was used\nto calculate ORs for numerical and multivariate risk factors, while\nANOVA type II was used to assess the overall influence of the whole\nvariable. When complete separation occurred for multivariate\nvariables (cells with 0), Firth correction was applied using the logistf\npackage. The relation between variables was analyzed using Pearson’s\ncorrelation coefficient (r; numerical variables) and Cramér’s V (V;\nnominal variables). Differences were considered significant at p < 0.05.\n3 Results\nOut of 300 tested sheep serum samples, 54 (18.0%) were positive\nfor at least one vector-borne zoonotic pathogen (Table 2). The highest\nseroprevalence was confirmed for TBEV (9.7%). The seroprevalence\nof WNV was 3.0% B. burgdorferi s.l. 2.7%, while USUV and\ninconclusive flavivirus (TBEV/WNV/USUV) infections had the same\nseroprevalence of 1.3%. None of the serum samples tested positive\nfor CCHFV.\nTBEV seropositivity was confirmed at all micro-locations in six\nout of seven farms, with seroprevalence rates from 5.0 to 15.0%.\nWNV-positive animals were confirmed at all micro-locations on\nfive of the seven farms, with seroprevalence ranging from 1.4 to\n3.9%. The lowest seroprevalence was found for USUV, with only\nfour positive animals confirmed on two farms at two micro-\nlocations, with a seroprevalence of 4.1 and 1.0%, respectively\n(Table\n2).\nFour\nsamples\n(1.3%)\nwere\nconsidered\nas\nflavivirus-positive due to cross-reactivity. Cross-reactivity between\n\n1 and 1.0%, respectively\n(Table\n2).Four\nsamples\n(1.3%)\nwere\nconsidered\nas\nflavivirus-positive due to cross-reactivity.Cross-reactivity between\nWNV and TBEV was confirmed in three samples and between\nUSUV and TBEV in one sample (Table 2). Serological evidence of\nB. burgdorferi s.l. infection was confirmed in 8 animals (2.7%) at\ntwo micro-locations with seroprevalence rates of 1.0 and 5.5%,\nrespectively.\nLocation as a risk factor was significant for USUV, TBEV and\nB. burgdorferi s.l. infections, but not for WNV. For mosquito-borne\ndiseases, location had no significant influence, while it was found as a\nrisk factor for tick-borne diseases (ANOVA p = 0.006) (Table 3).\nWhen analyzing the risk between micro-locations, Borovo was\nselected as a reference category, as the highest number of animals was\ntested there. No significant association between micro-location and\nWNV or USUV seropositivity was found at the location Trpinja, as\nwas the case for a group of tick-borne diseases. In contrast, on the\nsame location, the probability of TBEV infection (p = 0.02) and tick-\nborne diseases overall (p = 0.01) was significantly lower and a\nborderline reduction in the risk of B. burgdorferi s.l. infection was also\nobserved (p = 0.07). In contrast, in Vukovar, we observed a significant\nincrease in the risk of USUV infections (p = 0.03), a decrease in\nB. burgdorferi s.l. seropositivity (p = 0.04) as well as lower risk of tick-\nborne diseases (p = 0.03) (Table 3).\nThe type of water body and the distance of the farm from it as risk\nfactors had no significant influence on the seroprevalence of any\nVBDs or a group of diseases (Table 3).\nThe distance of the farm from the household was found as an\nimportant risk factor for seropositivity to TBEV (p < 0.001),\nB. burgdorferi s.l. (p = 0.005) and accordingly also for tick-borne\ndiseases (p < 0.001), but not for WNV, USUV, and mosquito-borne\ninfections (Table 3). To exclude the influence of location on these\nresults because of just one farm analyzed at Vukovar micro-location\nand two at Trpinja, the same findings were analyzed for the four farms\n\n001), but not for WNV, USUV, and mosquito-borne\ninfections (Table 3).To exclude the influence of location on these\nresults because of just one farm analyzed at Vukovar micro-location\nand two at Trpinja, the same findings were analyzed for the four farms\nin Borovo micro-location, and distance to households was confirmed\nas a significant risk factor also on the same location at the farm level\n(p = 0.01).\nTABLE 2  Seroprevalence of sheep to vector-borne zoonoses by micro-location and farm in Croatia (N, %).\nLocation\nFarm\nNumber\nof tested\nanimals\nWNV\nUSUV\nTBEV\nFlavivirus\nB. burgdorferi\ns.l.\nMosquito-\nborne\npathogens\nTick-\nborne\npathogens\nPositive\nto at least\none\npathogen\nVukovar\nTotal\n73\n1 (1.4)\n3 (4.1)\n5 (6.9)\n0 (0.0)\n0 (0.0)\n4 (5.5)\n5 (6.8)\n9 (12.3)\nTrpinja\nFarm 1\n80\n3 (3.8)\n1 (1.3)\n4 (5.0)\n1 (1.3)\n1 (1.3)\n4 (5.0)*\n5 (6.3)*\n10 (12.5)\nFarm 2\n20\n0 (0.0)\n0 (0.0)\n1 (5.0)\n1 (5.0)\n0 (0.0)\n0 (0.0)*\n1 (5.0)*\n2 (10.0)\nTotal\n100\n3 (3.0)\n1 (1.0)\n5 (5.0)\n2 (2.0)\n1 (1.0)\n4 (4.0)\n6 (6.0)\n12 (12.0)\nBorovo\nFarm 1\n33\n1 (3.0)\n0 (0.0)\n6 (18.2)\n1 (3.0)\n2 (6.1)\n1 (3.0)*\n8 (24.2)*\n10 (30.3)\nFarm 2\n4\n2 (NA)\n0 (0.0)\n0 (0.0)\n0 (0.0)\n0 (0.0)\n2 (NA)\n0 (0.0)\n2 (NA)\nFarm 3\n50\n2 (4.0)\n0 (0.0)\n11 (22.0)\n1 (2.0)\n5 (10.0)\n2 (4.0)*\n16 (32.0)*\n19 (38.0)\nFarm 4\n40\n0 (0.0)\n0 (0.0)\n2 (5.0)\n0 (0.0)\n0 (0.0)\n0 (0.0)\n2 (5.0)\n2 (5.0)\nTotal\n127\n5 (3.9)\n0 (0.0)\n19 (15.0)\n2 (1.6)\n7 (5.5)\n5 (3.9)\n26 (20.5)\n33 (26.0)\nTotal\n300\n9 (3.0)\n4 (1.3)\n29 (9.7)\n4 (1.3)\n8 (2.7)\n13 (4.3)\n37 (12.3)\n54 (18.0)\nAll samples tested negative to Crimean-Congo hemorrhagic fever virus. *Numbers of positive animals without four flavivirus (TBEV/WNV/USUV)-positive animals, in which it was not\npossible to determine the pathogen; NA—insufficient sample size of tested animals for farm-level analysis; WNV, West Nile virus; USUV, Usutu virus; TBEV, tick-borne encephalitis virus;\nmosquito-borne pathogens: WNV, USUV; tick-borne pathogens: TBEV, B. burgdorferi s.l.\n\nBarbic et al.\b\n10.3389/fvets.2025.1602706\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nWhen analyzing differences in VBDs seroprevalence at the farm\nlevel, a significant impact on seroprevalence was confirmed\n(p = 0.002), but as only animals from one farm at the Vukovar micro-\nlocation were tested, a close association between farm and location\n(V = 1) was confirmed, so we do not present the results.\nThe sheep breed was confirmed as an important risk factor with\na significantly higher probability of infections with TBEV (p = 0.003),\nB. burgdorferi s.l. (p = 0.002) and tick-borne pathogens (p < 0.001) in\nTsigai sheep than in Romanov sheep, which were used as the\nreference category. The seroprevalence rates in Merinolandschaf\nsheep were not significantly different (Table 4). When analyzing the\ncorrelation between the variables, sheep breed and location, the\ncorrelation was high (V = 0.83, 95% CI 0.79–0.86), with only\nMerinolandschaf present in Trpinja and only Romanov sheep\nin Vukovar.\nOf the other risk factors analyzed, only sheep shearing was\nstatistically significant, with unshared sheep showing a higher\nprobability of tick-borne infections (p = 0.048) (Table 4).\n4 Discussion\nInfection of sheep with various zoonotic VBD pathogens has been\ndocumented in several studies, but their use as sentinel animals needs\nto be evaluated. In this study, we analyzed the seroprevalence of\nzoonotic VBDs in sheep from WNV and TBEV endemic areas in\nCroatia, as well as areas with sporadic evidence of USUV infection. In\naddition, we tested sheep for B. burgdorferi s.l., the most common\nvector-borne bacteria in Croatia, and the possible introduction of\nCCHFV. Sheep in all three sampling micro-locations and on all tested\nfarms were positive for TBEV, WNV, USUV, and B. burgdorferi s.l.\nantibodies, suggesting their possible sentinel role for selected\nzoonotic pathogens.\nThe highest seroprevalence was recorded for TBEV (9.7%). It was\nhigher than the 0.53 and 0.42% seroprevalence in Germany and\nBelgium, tested by VNT and plaque reduction neutralization test,\nrespectively (29, 30). The observed difference could be attributed to\n\nThe highest seroprevalence was recorded for TBEV (9.7%).It was\nhigher than the 0.53 and 0.42% seroprevalence in Germany and\nBelgium, tested by VNT and plaque reduction neutralization test,\nrespectively (29, 30).The observed difference could be attributed to\nthe endemic occurrence and high risk of TBEV infection in this part\nof Croatia (22).\nVery few studies analyzing WNV seroprevalence in sheep are\navailable for European countries. In a study conducted in Turkey, 1%\nof sheep tested positive for WNV neutralizing antibodies (31). A\nseropositivity of 2.2% was observed by VNT in the Astrakhan region\nof Russia (32). The WNV seroprevalence of 3.0% in Croatia is similar\nto the seroprevalence results in sheep from Egypt, northeast Ethiopia,\nand Tunisia (3.5, 3.5, and 3.2%, respectively), which are also enzootic\nregions for WNV (33–35). With extremely high seroprevalence in\nhorses (36), the surveillance system in this area could be compromised,\nmaking sheep a possible alternative species for WNV surveillance in\nregions with high virus activity.\nTo the best of our knowledge, this study gives the first serological\nevidence of USUV infections in sheep globally. The detection of\nUSUV-seropositive sheep in Vukovar-Srijem County is not surprising,\nas the first serologic evidence of human USUV infection in Croatia\nwas confirmed in 2012 in a resident of this region (37).\nAthanosiu et al. (38) confirmed a B. burgdorferi s.l. seroprevalence\nof 23.58% in sheep in Greece. Similarly, a seroprevalence study in\nSlovakia found the seropositivity to B. burgdorferi s.l. of 15.8% (1999)\nand 17.5% (2000) (39). In the Alto Adige-South Tyrol, Italy, the\nseropositivity in sheep was 14.1% (1990) (40). In the present study,\nseropositivity was much lower (2.7%), which is consistent with the low\nincidence of human borreliosis in this Croatian region (21).\nAll sheep were tested negative for CCHFV, which was expected\nsince this arbovirus had not yet been confirmed in Croatia. Further\ninvestigation is necessary due to the high risk of CCHF emergence,\ngiven the recent seropositive sheep confirmation in neighbouring\n\nAll sheep were tested negative for CCHFV, which was expected\nsince this arbovirus had not yet been confirmed in Croatia.Further\ninvestigation is necessary due to the high risk of CCHF emergence,\ngiven the recent seropositive sheep confirmation in neighbouring\ncountries (41, 42).\nThe antibody detection in sheep suggests their sentinel potential\nfor VBD pathogens. For highly prevalent pathogens such as WNV, the\npossible advantage of sheep in endemic areas is noteworthy. In\nVukovar-Srijem County, the testing area in the present study, a high\nIgG seroprevalence of WNV exceeding 50% was recorded in horses\nduring 2024 (data of the Faculty of Veterinary Medicine, University of\nZagreb). This challenges the continued use of horses as sentinel\nTABLE 3  Seroprevalence of vector-borne zoonoses in sheep according to geographic and environmental risk factors in Croatia (OR, 95% CI, p).\nRisk factor\nWNV\nUSUV\nTBEV\nB. burgdorferi\ns.l.\nMosquito-\nborne\npathogens\nTick-borne\npathogens\nLocation\nBorovo*\np = 0.54\np = 0.045\np = 0.03\np = 0.015\np = 0.878\np = 0.006\nTrpinja\n0.76 (0.15–\n3.17) p = 0.71\n3.86 (0.2–566.03)\np = 0.37\n0.3 (0.1–0.78)\np = 0.02\n0.24 (0.03–1.13)\np = 0.07\n1.02 (0.25–3.96)\np = 0.98\n0.29 (0.1–0.7) p = 0.01\nVukovar\n0.33 (0.02–\n2.12) p = 0.32\n12.46 (1.18–\n1684.65)\np = 0.03\n0.41 (0.13–1.08)\np = 0.09\n0.11 (0.001–0.92)\np = 0.04\n1.39 (0.33–5.43)\np = 0.63\n0.33 (0.11–0.84)\np = 0.03\nWater bodies\n0.27 (0.03–\n2.18) p = 0.22\n6.84 (0.7–66.69)\np = 0.098\n0.55 (0.22–1.4)\np = 0.21\n0.13 (0.01–2.2) p = 0.16\n0.98 (0.3–3.28) p = 0.98\n0.44 (0.18–1.1) p = 0.08\nDistance from water bodies\n(km)\n1.034 (0.88–\n1.19) p = 0.65\n0.99 (0.72–1.22)\np = 0.91\n0.91 (0.80–1.01)\np = 0.104\n0.91 (0.67–1.08)\np = 0.37\n1.02 (0.89–1.15)\np = 0.75\n0.92 (0.82–1.01)\np = 0.097\nDistance from household (km)\n1.07 (0.38–\n2.55) p = 0.89\n0.58 (0.05–2.46)\np = 0.54\n2.50 (1.52–4.18)\np < 0.001\n4.50 (1.73–15.22)\np = 0.005\n0.90 (0.36–1.94)\np = 0.81\n2.74 (1.71–4.45)\np < 0.001\n*Reference category in the logistic regression; the ANOVA p-value represents the influence of location; WNV, West Nile virus; USUV, Usutu virus; TBEV, tick-borne encephalitis virus;\n\n52–4.18)\np < 0.001\n4.50 (1.73–15.22)\np = 0.005\n0.90 (0.36–1.94)\np = 0.81\n2.74 (1.71–4.45)\np < 0.001\n*Reference category in the logistic regression; the ANOVA p-value represents the influence of location; WNV, West Nile virus; USUV, Usutu virus; TBEV, tick-borne encephalitis virus;\nmosquito-borne pathogens: WNV/USUV; tick-borne pathogens: TBEV/B. burgdorferi s.l. Statistically significant differences are bold.\n\nBarbic et al.\b\n10.3389/fvets.2025.1602706\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nanimals in the upcoming transmission season and underscores the\nneed for integrating additional species into the surveillance\nframework. The easy access to serum samples collected for veterinary\nimportant infectious disease surveillance, such as brucellosis, and the\nlarge population of these animals offer the advantage of including\nsheep in the surveillance programme for VBD pathogens.\nIn addition to confirming the activity of zoonotic pathogens in\nspecific areas, surveillance with sentinel animals could provide\ninformation on epidemiological risk factors in a particular location. In\nthis study, we confirmed that location had a significant influence on the\nrisk of infection with VBD pathogens. This was not confirmed for WNV\nand mosquito-borne pathogens overall, likely due to the vector\npopulation density and the high viral activity of WNV observed in this\narea over the last decade. The impact of location in a small geographic\narea, as investigated in this study, on mosquito-borne infections could\nbe minimized compared to tick-borne diseases, given that mosquitoes\ntravel long distances, with a mean distance of 1.33 km (43). Even though\nthe risk of infection from less prevalent mosquito-borne pathogens, such\nas USUV, in this region is location-dependent in some instances. This\nindicates a general and highly prevalent spread of WNV throughout the\nstudy area, as well as the circulation of USUV in some micro-foci.\nLocation was a significant risk factor for TBEV, B. burgdorferi s.l.,\nas well as tick-borne diseases overall. The observed differences\nbetween mosquito-borne and tick-borne VBDs can be explained by\nthe significantly different movement distances of ticks compared to\nmosquitoes. Slovák et al. (44) confirmed that nymphs have the highest\ninfection rates with TBEV among tick stages. At the same time, the\nindependent movement of nymphs is very short, and they were\npredominantly recaptured 2–3 meters from their release points, while\nalmost 50% of adults were found to be more than 5 meters away, with\n\nAt the same time, the\nindependent movement of nymphs is very short, and they were\npredominantly recaptured 2–3 meters from their release points, while\nalmost 50% of adults were found to be more than 5 meters away, with\nsome dispersing up to 7–8 meters. These findings confirm that all tick\nstages exhibit minimal autonomous movement (without involvement\nof the host’s movement), resulting in microlocalization of\nTBEV circulation.\nFrom the other geographic and environmental risk factors for\nVBDs in this study, we found that the distance of the flock from the\nhousehold is a significant risk factor for TBEV, B. burgdorferi s.l., and\ntick-borne diseases overall. This finding could be the result of the\nabove-explained differences in the movement of the vectors, as well as\nconfirmation that the dispersal of nymphs into pastures was minimal\ncompared to the woodland (45).\nIn an analysis of host- and management-related risk factors\nfor infection with VBD pathogens in sheep, we confirmed breed\nas an important risk factor. Due to the high correlation between\nbreed and location, this finding requires further investigation.\nSex, age, flock size, and clinical signs did not influence\nseroprevalence. An unexpected result was that the risk of tick-\nborne infection was statistically higher in flocks without sheep\nshearing practices. This could be  a consequence of the easier\nattachment of tick vectors to a host when sheep were not sheared,\nbut may also reflect broader farming practices. Regularly shared\nflocks are handled more frequently, increasing the likelihood of\nearly tick infestation detection. Differences in exposure risk may\nalso be influenced by other factors not addressed in our study,\nsuch as the level of flock management and the degree of system\nextensiveness, which is a limitation of the study. The role of\nshearing is, therefore, a risk factor likely multifactorial in origin\nand should be interpreted within the broader context of flock\nmanagement and environmental exposure.\nIn conclusion, the results of this study suggested the potential\n\nThe role of\nshearing is, therefore, a risk factor likely multifactorial in origin\nand should be interpreted within the broader context of flock\nmanagement and environmental exposure.In conclusion, the results of this study suggested the potential\nof sheep as sentinels for mosquito-borne and tick-borne zoonotic\ndiseases. The lower WNV seroprevalence in sheep compared to\nhorses, the most commonly used sentinel species for WNV,\nsuggests that even sheep may have advantages in endemic regions\nwith high viral activity. In this study, to the best of our knowledge,\nwe confirmed for the first time USUV infection in sheep, which\nexpands the possibility of collecting epidemiological data using\nthis species as sentinel animals. However, it should be noted that\nage was not identified as a significant risk factor for VBDs\nseropositivity. Due to cumulative exposure, older animals are\noften more likely to be  seropositive, and their usefulness as\nsentinels for identifying recent pathogen circulation may\nTABLE 4  Seroprevalence of vector-borne zoonoses in sheep according to host- and management-related risk factors in Croatia (OR, 95% CI, p).\nRisk factor\nWNV\nUSUV\nTBEV\nB. burgdorferi s.l.\nMosquito-borne\npathogens\nTick-borne\npathogens\nRomanov breed*\np = 0.17\np = 0.18\np < 0.001\np < 0.001\np = 0.672\np < 0.001\nMerino breed\n5.77 (0.91–\n111.62) p = 0.11\n0.47 (0.04–2.89)\np = 0.42\n0.78 (0.22–2.53)\np = 0.68\n3.29 (0.17–482.26)\np = 0.43\n1.70 (0.47–6.83) p = 0.42\n0.95 (0.3–2.94) p = 0.92\nTsigai breed\n4.31 (0.54–87.96)\np = 0.21\n0.19 (0.001–2.04)\np = 0.19\n4.02 (1.64–10.89)\np = 0.003\n22.25 (2.65–2904.02)\np = 0.002\n1.05 (0.20–4.88) p = 0.95\n5.3 (2.22–14.12)\np < 0.001\nSex\n1.97 (0.1–37.04)\np = 0.65\n4.23 (0.21–85.84)\np = 0.35\n1.55 (0.18–13.37)\np = 0.69\n2.24 (0.12–42.48)\np = 0.59\n1.37 (0.07–25.18) p = 0.83\n1.29 (0.15–11.08) p = 0.81\nAge\n1.06 0.75–1.35\np = 0.69\n1.00 (0.55–1.43)\np = 0.99\n1.07 (0.89–1.24)\np = 0.44\n1.17 (0.88–1.46) p = 0.2\n1.04 (0.78–1.29) p = 0.74\n1.11 (0.95–1.28) p = 0.17\nHerd size\n1.00 (0.99–1.01)\np = 0.91\n1.00 (0.99–1.01)\np = 0.96\n0.99 (0.99–1.002)\np = 0.4\n0.99 (0.99–1.007)\np = 0.95\n1.00 (0.99–1.01) p = 0.9\n0.99 (0.99–1.003) p = 0.52\nShearing\n1.01 (0.25–4.13)\np = 0.99\n0.67 (0.07–6.53)\np = 0.73\n\n99\n1.07 (0.89–1.24)\np = 0.44\n1.17 (0.88–1.46) p = 0.2\n1.04 (0.78–1.29) p = 0.74\n1.11 (0.95–1.28) p = 0.17\nHerd size\n1.00 (0.99–1.01)\np = 0.91\n1.00 (0.99–1.01)\np = 0.96\n0.99 (0.99–1.002)\np = 0.4\n0.99 (0.99–1.007)\np = 0.95\n1.00 (0.99–1.01) p = 0.9\n0.99 (0.99–1.003) p = 0.52\nShearing\n1.01 (0.25–4.13)\np = 0.99\n0.67 (0.07–6.53)\np = 0.73\n0.39 (0.14–1.06)\np = 0.06\n0.28 (0.03–2.3) p = 0.23\n0.89 (0.27–2.98) p = 0.85\n0.4 (0.16–0.99) p = 0.048\nClinical signs\n1.06 (0.13–8.8)\np = 0.95\n0.89 (0.05–16.92)\np = 0.94\n2.41 (0.9–6.48)\np = 0.08\n2.81 (0.54–14.51)\np = 0.22\n0.68 (0.09–5.39) p = 0.71\n2.46 (0.97–6.22) p = 0.06\n*Reference category in the logistic regression; the ANOVA p-value represents the influence of breed; WNV, West Nile virus; USUV, Usutu virus; TBEV, tick-borne encephalitis virus; mosquito-\nborne pathogens: WNV/USUV; tick-borne pathogens: TBEV/B. burgdorferi s.l. Statistically significant differences are bold.\n\nBarbic et al.\b\n10.3389/fvets.2025.1602706\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nbe questionable. Therefore, further studies are needed to evaluate\nthe role of sheep as sentinels for mapping microfoci of zoonotic\npathogens’ activity and determining high-risk areas for\npublic health.\nThe findings of this study are relevant within the “One Health”\nframework, which emphasizes the interdisciplinary collaboration\nbetween human, animal, and environmental health. The detected\nseropositivity to multiple zoonotic VBD pathogens (TBEV, WNV,\nUSUV, B. burgdorferi s.l.) in sheep in various micro-locations supports\nthe inclusion of livestock surveillance in integrated early-warning\nsystems. By highlighting geographic and environmental risk factors\nsuch as farm location, breed susceptibility, and proximity to\nhouseholds, the study provides insight into the micro-epidemiology\nof zoonotic VBDs. These data can be used to improve risk mapping at\nthe local level, allowing for more focused public health interventions,\nsuch as tick and mosquito control measures and public education\ncampaigns in high-risk zones.\nData availability statement\nThe raw data supporting the conclusions of this article will\nbe made available by the authors, without undue reservation.\nEthics statement\nEthical approval was not required for the study involving animals\nin accordance with the local legislation and institutional requirements\nbecause samples tested in the study are residual samples obtained\nfrom a mandatory surveillance program on sheep infectious diseases\napproved by the Ministry of Agriculture, Veterinary Directorate.\nHowever, part of the research was conducted as a component of the\ngraduation thesis of one of the co-authors, DVM. In accordance with\nthe internal procedures of the Faculty of Veterinary Medicine,\nUniversity of Zagreb, all graduate theses must receive prior ethical\napproval regardless of the source or nature of the samples. Therefore,\nethical approval was formally requested to comply with the\ninstitutional requirements, and was granted by the Ethics Committee\nof the Faculty (Decision: Class: 640-01/22-02/07, Reg. No.:\n\nTherefore,\nethical approval was formally requested to comply with the\ninstitutional requirements, and was granted by the Ethics Committee\nof the Faculty (Decision: Class: 640-01/22-02/07, Reg.No.:\n251-61-01/139-22-42).\nAuthor contributions\nLB: Conceptualization, Supervision, Writing – original draft. VSt:\nMethodology, Writing – review & editing. MMM: Formal analysis,\nMethodology, Writing  – original draft. GM: Investigation,\nWriting – review & editing. IC: Investigation, Writing – review &\nediting. VSa: Funding acquisition, Writing – review & editing. VM:\nInvestigation, Resources, Writing  – review & editing. MB:\nVisualization, Writing – review & editing. AM: Writing – review &\nediting. TV-C: Conceptualization, Visualization, Writing  –\noriginal draft.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. This research was funded\nby the European Union Next Generation EU Project, supported by the\nMinistry of Science and Education of the Republic of Croatia, Grant\nNumber NPOO1, of the Croatian Veterinary Institute: Epidemiology\nof avian flaviviruses in the “One Health” context; FLAVIR (to VSa).\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nThe author(s) declared that they were an editorial board member\nof Frontiers, at the time of submission. This had no impact on the peer\nreview process and the final decision.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. Any product\nthat may be evaluated in this article, or claim that may be made by its\nmanufacturer, is not guaranteed or endorsed by the publisher.\nSupplementary material\nThe Supplementary material for this article can be found online\n\nAny product\nthat may be evaluated in this article, or claim that may be made by its\nmanufacturer, is not guaranteed or endorsed by the publisher.Supplementary material\nThe Supplementary material for this article can be found online\nat: https://www.frontiersin.org/articles/10.3389/fvets.2025.1602706/\nfull#supplementary-material\nReferences\n1.\tVilibić-Čavlek T, Bogdanić M, Savić V, Barbić L, Stevanović V, Kaić B. Tick-borne\nhuman diseases around the globe. Chapter 1 In: G Dobler, W Erber, M Bröker, L Chitimia-\nDobler and HJ Schmitt, editors. The TBE book. 7th ed. Singapore: Global Health Press (2024)\n2.\tWorld Health Organization. (2024). Vector-borne diseases. 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+{"text": "【兽医文献】\n标题: The detrimental impact of seminal plasma on the quality of cryopreserved sperm in goat bucks: freezing–thawing as the most harmful step\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nImpacts of Bacillus-based biotics\nand an enzyme cocktail on\ngrowth performance, immunity,\nand gut pathogenic\nmicroorganisms of nursery pigs\nunder commercial conditions\nSeong-Min Koo , Jae-Hyeok Lee , Sang-Hyon Oh  and\nJae-Cheol Jang *\nDivision of Animal Science and Institute of Agricultural and Life Science, Gyeongsang National\nUniversity, Jinju, Republic of Korea\nIntroduction: The prohibition of antibiotics in animal feed has increased interest\nin alternatives, such as phytogenic compounds, pro- and prebiotics, organic acids,\nand exogenous enzymes. Among these, Bacillus-based biotics and enzyme cocktail\nare the most commonly used feed additives. However, their effects on growth\nperformance, immunity, and gut health in nursery pigs, as well as their interactions\nwith pathogens under commercial conditions, remain unclear. This study investigated\nthe impact of these additives on growth performance, immunity, and pathogenic\nmicroorganisms in the gut under commercial conditions.\nMaterial and methods: Two hundred nursery pigs were assigned to one of\nfive dietary treatments: (1) CON: a basal corn-soybean meal diet, (2) A: basal\ndiet with 0.05% probiotics, (3) B: basal diet with 0.1% synbiotics containing one\nstrain, (4) C: basal diet with 0.1% synbiotics containing two strains, and (5) D:\nbasal diet with 0.1% enzyme cocktail.\nResults: The growth performance did not show significant differences according\nto the feed additives. In terms of immunity, B treatment increased immunoglobulin\nM levels, while D treatment increased immunoglobulin A levels during weeks 0–2\n(p < 0.01). Additionally, both B and D treatments decreased Mycoplasma spp. in the\ngut, as indicated by log fold change (LFC) values of −1.571 and −1.529, respectively.\nConclusion and implications: Therefore, this study highlights the potential of\nBacillus-based biotics and enzyme cocktail as practical alternatives for reducing\npathogenic microorganisms such as Mycoplasma spp. and improving immunity\nin nursery pigs under commercial conditions.\nKEYWORDS\n\nand improving immunity\nin nursery pigs under commercial conditions.KEYWORDS\nalternative feed additives, Bacillus-based biotics, commercial farm, Mycoplasma,\nnursery pig\n1 Introduction\nThe weaning period is one of the most stressful phases for nursery pigs, characterized by\nvarious stressors such as environmental, social, and dietary changes, which can lead to negative\neffects such as reduced feed intake and weight loss (1, 2). These changes can adversely affect\nthe gastrointestinal tract and can lead to diseases such as diarrhea in vulnerable nursery pigs.\nOPEN ACCESS\nEDITED BY\nElena De Felice,\nUniversity of Camerino, Italy\nREVIEWED BY\nUgonna Henry Uzoka,\nMichael Okpara University of Agriculture,\nNigeria\nRuili Li,\nYantai Academy of Agricultural Sciences,\nChina\n*CORRESPONDENCE\nJae-Cheol Jang\njaejang1278@gnu.ac.kr\nRECEIVED 13 May 2025\nACCEPTED 18 June 2025\nPUBLISHED 25 July 2025\nCITATION\nKoo S-M, Lee J-H, Oh S-H and Jang J-C\n(2025) Impacts of Bacillus-based biotics and\nan enzyme cocktail on growth performance,\nimmunity, and gut pathogenic\nmicroorganisms of nursery pigs under\ncommercial conditions.\nFront. Vet. Sci. 12:1627739.\ndoi: 10.3389/fvets.2025.1627739\nCOPYRIGHT\n© 2025 Koo, Lee, Oh and Jang. This is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY). The use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\naccordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Original Research\nPUBLISHED  25 July 2025\nDOI  10.3389/fvets.2025.1627739\n\nKoo et al.\b\n10.3389/fvets.2025.1627739\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nHowever, the prohibition of antibiotics and some minerals due to\nissues such as antibiotics resistance and residues in the body has\nprompted research into alternative feed additives (3, 4). Among these\nadditives, probiotics, synbiotics, and enzyme cocktail have been\ncommonly used in swine diet.\nProbiotics are defined as live microorganisms that when given in\nadequate amounts, and they help establish a balanced gut microbial\ncommunity in the intestine (5, 6). The major effects of orally administered\nprobiotics on the gut include: (1) regulation of intestinal microbial\ncommunities; (2) inhibition of pathogenic bacteria; (3) immune\nregulation; (4) enhancement of epithelial cell proliferation and\ndifferentiation and strengthening of the intestinal barrier (7). These key\neffects enhance the responsiveness of intestinal epithelial and immune\ncells to the gut microbiota, improve the overall metabolic function of the\ngut microbial community, and suppress pathogens, thereby shifting the\ngut microbiota towards a more beneficial state (8). Synbiotics, which\ncombine probiotics and prebiotics, offer many of the same benefits as\nprobiotics alone. For instance, Girard et al., (9) showed that synbiotics\nsupplementation promotes the growth of beneficial gut bacteria while\nreducing harmful metabolites. Notably, many probiotics and synbiotics\nformulations utilize Bacillus spp. as their primary bacterial component,\nwith Bacillus subtilis and Bacillus licheniformis being the most commonly\nused species (10). Bacillus subtilis is known for secreting numerous\nantimicrobial peptides, including bacteriocins and lipopeptides, which\ncollectively inhibit a broad range of gut pathogens (11), and Bacillus\nlicheniformis produces lichenysin, a lipopeptide antibiotic that disrupts\npathogen cell membranes and biofilms and provides strong antibacterial\neffects in the intestinal environment (12). Both species help maintain gut\nhealth and are often used alone or in combination to achieve\ncomplementary benefits (13).\n\nBoth species help maintain gut\nhealth and are often used alone or in combination to achieve\ncomplementary benefits (13).\nEnzyme cocktails are a mixture of multiple enzymes designed to\nmaximize efficiency by utilizing the synergy between each enzyme. These\nblended enzymes hydrolyze cell-wall polysaccharides and phytic acids to\nmaximize the utilization of phosphorus, proteins, and minerals, thereby\nimproving the digestibility of energy and amino acids (14). Concurrently,\nenzyme cocktails modulate the gut microbiota by generating fermentable\noligosaccharides through exogenous carbohydrases (15). These\noligosaccharides promote the growth of beneficial bacteria, increase\nmicrobial diversity, and improve fermentation profiles, thus supporting\nintestinal health and function (16). Several studies have also shown that\nsupplementing pig diets with enzyme cocktails improves growth\nperformance, enhances nutrient digestibility, and beneficially modulates\nthe gut bacterial community (17, 18).\nPig farms are complex environments with diverse microorganisms,\nsome of which can impact the productivity and health of pigs. Notably,\nporcine reproductive and respiratory syndrome virus (PRRSV),\nporcine circovirus-2 (PCV-2), Mycoplasma hyopneumonia, (MH),\nPasteurella multicide A (PMA), Haemophilus parasuis (HP),\nActinobacillus pleuropneumonia type 2, (APP2), and Actinobacillus\npleuropneumonia type 5 (APP5) are major pathogens in the porcine\nrespiratory disease complex (PRDC), responsible for enzootic\npneumonia (19, 20). Among these pathogens, MH is the primary\ncausative agent of enzootic pneumonia in swine, leading to chronic\nrespiratory disease and economic losses (21). In response, attempts\nhave been made to regulate the pathogenic gut microbiota using\nprobiotics and prebiotics as alternative approaches (22, 23). For\ninstance, Pahumunto et al. (24) evaluated potential probiotics strains\nfor their effects on porcine pathogens and immune modulation.\nHowever, the mechanisms of interaction between probiotics,\nsynbiotics, enzyme cocktail, and the gut microbiota are not yet clearly\n\n(24) evaluated potential probiotics strains\nfor their effects on porcine pathogens and immune modulation.However, the mechanisms of interaction between probiotics,\nsynbiotics, enzyme cocktail, and the gut microbiota are not yet clearly\nunderstood, and the effects of these feed additives in nursery diets have\nshown inconsistent results.\nTherefore, this study is notable for being conducted directly under\ncommercial farm conditions, with the aim of evaluating the effects of\nBacillus-based biotics and enzyme cocktail supplementation in nursery\npig diets on growth performance, immune status, gut health, and the\nreduction of pathogenic microorganisms. By conducting the research\nunder commercial farm conditions, this study provides practical\ninsights into how these additives perform in real-world settings, offering\napplicable nutritional strategies beyond controlled laboratory conditions.\n2 Materials and methods\nAll experimental protocols involving animals in the present study\nwere approved by the Institutional Animal Care and Use Committee\n(IACUC) of the Gyeongsang National University (GNU-231030-P0204).\nThis experiment was conducted in a commercial farm to investigate the\nantibody titers of PRRSV, PCV-2, MH, APP2, APP5, PMA, and HP,\nwhich are considered major causative agents of porcine respiratory\ndisease complex, using a serum ELISA test. In this experimental farm,\nseropositive reactions were observed for PRRSV, PCV-2, and MH.\n2.1 Obtained feed additives\nFeed additive used in all experiments were composed as follows:\nProbiotics (PRO) Bacillus subtilis 1.0 × 108 cfu/g, Bacillus coagulans\n1.0 × 108 cfu/g, and Clostridium butyricum 1.0 × 106 cfu/g; synbiotics\n(SYO) Bacillus licheniformis 1.0 × 108 cfu/g and more than 2%\nfructooligosaccharides; synbiotics + (SYO+) Bacillus licheniformis\n1.0 × 108 cfu/g, Bacillus subtilis 1.0 × 108 cfu/g, and more than 2%\nfructooligosaccharides; enzyme cocktail (EC) At least 500,000 unit/kg\nof phytase, at least 1,500,000 unit/kg of α-amylase, at least 40,000 unit/\nkg of cellulase, at least 50,000 unit/kg of xylanase, at least 300,000 unit/\n\n0 × 108 cfu/g, and more than 2%\nfructooligosaccharides; enzyme cocktail (EC) At least 500,000 unit/kg\nof phytase, at least 1,500,000 unit/kg of α-amylase, at least 40,000 unit/\nkg of cellulase, at least 50,000 unit/kg of xylanase, at least 300,000 unit/\nkg of neutral protease, at least 500 unit/kg of lipase, and at least\n1,500,000 unit/kg of α-amylase. All feed additives were provided by\nWoogene B&G (WOOGENE B&G CO. LTD, Seoul, Republic of Korea).\n2.2 Experimental procedure and diet\nA total of 200 crossbred ([Landrace × Yorkshire]) × Duroc)\nnursery pigs (initial body weight of 5.60 ± 0.05 kg) were used in this\nstudy. The pigs were allocated to five dietary treatments based on sex\nand initial body weight (BW), using a randomized complete block\ndesign (RCBD), with four replicates of 10 pigs per pen. The\nexperimental treatments included: (1) CON: a corn-soybean meal-\nbasal diet (basal diet), (2) Treatment A: basal diet supplemented with\n0.05% probiotics (PRO), (3) Treatment B: basal diet with 0.1%\nsynbiotics (SYO) containing one strain, (4) Treatment C: basal diet\nwith 0.1% SYO containing two strains, and (5) Treatment D: basal diet\nwith 0.1% enzyme cocktail (EC). The study lasted for a total of 35 days.\nThe experimental diets (Table 1) were formulated according to the\nnutrient requirements outlined by the National Research Council\n(NRC) 2012 and were divided into two phases following the phase\nfeeding program for nursery pigs: Phase 1 (P1) and Phase 2 (P2).\nPhase 1 lasted for 14 days, and Phase 2 for 20 days. The chemical\n\nKoo et al.\b\n10.3389/fvets.2025.1627739\nFrontiers in Veterinary Science\n03\nfrontiersin.org\ncomposition of the diets included 22.2% crude protein (CP) and\n3,400 kcal/kg metabolizable energy (ME) for Phase 1, and 20.1% CP\nwith the same ME level for Phase 2.\n2.3 Housing and sampling\nEach replicate consists of 10 pigs housed in pens with a slotted floor\nstructure (3.0 × 1.8 m2). Each pen was equipped with a feeder and an\nautomatic waterer, allowing pigs ad libitum access to feed and water\nthroughout the experimental period. The room’s temperature was\ninitially maintained at 30°C and progressively lowered to 25°C until the\ncompletion of the trial with humidity between 60 and 70%. Body weight\n(BW) was recorded at the start of the experiment, d 14 (P1), and d 35\n(P2). Feed intake was recorded daily, and residual feed was measured at\nd 14 and d 35 to calculate average daily gain (ADG), average daily feed\ndisappearance (ADFD), and gain to feed ratio (G: F ratio).\nBlood samples were collected from the jugular vein of two\nrandomly selected male pigs from each pen at d 14 and d 35 to analyze\nthe immune indicators. Additionally, fecal samples were collected\nfrom one pig per pen, selected based on proximity to the average body\nweight, at the end of the experiment for gut microbiota analysis.\n2.4 Sample analysis\nBlood samples were collected in serum separator tubes (SST) and\ncentrifuged at 3,500 RPM for 15 min to separate the serum. The\nseparated serum was transferred to a 1.5 mL microtubes and stored at\n−25°C. To assess immune responses, levels of immune globulin G (IgG),\nimmune globulin A (IgA), and immune globulin M (IgM) were analyzed\nusing enzyme-linked immunosorbent assay (ELISA) kits, following the\nmanufacturer’s protocol [IgG (ab291065, Abcam, USA), IgA (ab190536,\nAbcam Singapore Pte Ltd., USA) IgM (ab190537, Abcam, USA)].\nFecal samples were collected in fecal tubes, and processed\naccording to the 16S sequencing standard workflow (Illumina, San\nDiego, USA). The sequence data generated were analyzed using\nQIIME2 (ver. 2023.9, QIIME2 development team, Boulder, CO,\n\nFecal samples were collected in fecal tubes, and processed\naccording to the 16S sequencing standard workflow (Illumina, San\nDiego, USA).The sequence data generated were analyzed using\nQIIME2 (ver.2023.9, QIIME2 development team, Boulder, CO,\nUSA). Raw reads were demultiplexed and quality-filtered using the\nDADA2 plugin in QIIME2, truncating reads with Phred scores below\n20 and removing chimeric sequences. Taxonomic assignment was\nperformed against the SILVA 138 reference database (25). Then,\namplicon sequence variants (ASVs) were obtained through the\nfeature-table generated by the QIIME2 pipeline. Alpha-diversity\nmetrics, including Chao1 richness, Shannon diversity, and Simpson\ndiversity, were analyzed to compare the control and treatment groups\nusing QIIME2. For beta-diversity, Jaccard and Bray-Curtis indices\nwere computed, and permutational multivariate analysis of variance\n(PERMANOVA) was conducted to evaluate the statistical significance\nof microbial distribution difference. Additionally, the log fold change\n(LFC) of microbial communities between the control and treatment\ngroups was obtained at both the family and genus levels through\nANCOM-BC analysis. The LFC obtained from ANCOM-BC analysis\nwere adjusted using the false discovery rate (FDR) procedure (26).\n2.5 Statistical analysis\nStatistical analyses were performed using the general linear model\n(GLM) of SAS (SAS 9.4, SAS Institute Inc., Cary, NC, U. S) to evaluate\nthe significance of the collected data, with dietary treatment as the\nfixed effect. Outliers were identified and removed by applying the\nadjusted quartile method to each pen and cage. For PERMANOVA\nanalysis, distribution-free permutation techniques were employed to\nobtain p-values via QIIME2 (27). Statistical differences were\nconsidered highly significant at p < 0.01, significant at p < 0.05, and\ntendencies were noted if 0.05 < p ≤0.1.\n3 Results\n3.1 Growth performance\nThe effects of adding PRO, SYO, SYO+, and EC to the nursery pig\ndiet on the growth performance of nursery pigs are shown in Table 2.\nBW showed no significant differences at the initial, P1, and P2, and\n\n05 < p ≤0.1.3 Results\n3.1 Growth performance\nThe effects of adding PRO, SYO, SYO+, and EC to the nursery pig\ndiet on the growth performance of nursery pigs are shown in Table 2.BW showed no significant differences at the initial, P1, and P2, and\nthe ADG, ADFD, and G: F ratio were also not significantly different\namong treatments.\nTABLE 1  Composition of the basal nursery pig diet (Phase 1, Phase 2)a.\nItem\nPhase 1\nPhase 2\nMetabolizable energy (kcal/kg)\n3,400\n3,400\nCP (%)\n22.2\n20.1\nADF (%)\n3.2\n3.7\nNDF (%)\n7.4\n9.1\nCrude Fat (%)\n5.1\n5.7\nChemical compositiona\nSID Lys (%)b\n1.50\n1.35\nSID Thr (%)\n0.88\n0.78\nSID Met (%)\n0.45\n0.57\nSID Met+Cys (%)\n0.70\n0.82\nSID Trp (%)\n0.21\n0.19\nSID Ile (%)\n0.80\n0.71\nSID Val (%)\n0.90\n0.81\nSID Arg (%)\n1.23\n1.10\nSID His (%)\n0.49\n0.46\nSID Leu (%)\n1.57\n1.53\nSID Phe (%)\n0.90\n0.82\nSID Phe + Tyr (%)\n1.57\n1.43\nBCAA\nSID Lys (%)\n100.0\n100.0\nSID Ile (%)\n53.6\n52.9\nSID Val (%)\n59.8\n60.2\nSID Leu (%)\n104.6\n113.3\nCa: P (Total)\n1.22\n1.18\nCa: P (Available)\n1.61\n1.64\nCa: P (Digestible)\n1.64\n1.69\naCalculated value.\nbStandard ileal digestibility.\n\nKoo et al.\b\n10.3389/fvets.2025.1627739\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n3.2 Immune status\nTable  3 showed the immune status of nursery pigs fed diets\nsupplemented with PRO, SYO, SYO+, and EC. IgG concentration\nshowed no significant differences during P1 but displayed tendencies\nduring P2 (p = 0.06). IgA concentrations were highly significant in\nTreatment D during P1 (p < 0.01), with no significant differences\nobserved during P2 (p > 0.1). IgM concentrations showed highly\nsignificant differences during P1 (p < 0.01) and P2 (p < 0.01) in\nTreatment B.\n3.3 Fecal microbiota diversity analysis\nAlpha-diversity at the family and genus levels, estimated using\nChao1 richness, Shannon diversity, and Simpson diversity (Table 4),\nwas not affected by the addition of PRO, SYO, SYO+, and EC to\nnursery pig diets. Beta-diversity analysis using PERMANOVA\nindicated no significant differences between the control (CON) and\nother treatments, as shown by both Jaccard and Bray-Curtis indices\n(Table 5).\n3.4 Fecal microbiota LFC analysis\nFigure  1 illustrates the log fold change (LFC) in microbial\ncommunities between the control and Treatment A (PRO). At the family\nlevel, Anaerovoracaceae increased (lfc = 0.178) while Deferribacteraceae\ndecreased (lfc = −1.836). At the genus level, Pectinophilus decreased\n(lfc = −3.735). Figure  2 shows LFC comparing the control and\nTreatment B. At the family level, increases were observed in Pirellulaceae\n(lfc = 1.597), Corynebacteriaceae (lfc = 1.461), Christensenellaceae\n(lfc = 1.204), RF39 (lfc = 1.043), Muribaculaceae (lfc = 0.980), and\nAnaerovoracaceae (lfc = 0.707). At the genus level, Mycoplasma\n(lfc = −1.571) and Pectinophilus (lfc = −3.904) decreased. Figure  3\npresents the LFC between the control and Treatment C. At the family\nlevel, Anaerovoracaceae decreased (lfc = −0.081). At the genus level,\nEubacterium_nodatum (lfc = −2.406) and Pectinophilus (lfc = −3.904)\ndecreased, while UCG-002 increased (lfc = 1.003). Figure 4 illustrates\nthe LFC at the family level comparing the control and Treatment D, with\nincreases in Pirellulaceae (lfc = 1.897) and Anaerovoracaceae\n\n406) and Pectinophilus (lfc = −3.904)\ndecreased, while UCG-002 increased (lfc = 1.003).Figure 4 illustrates\nthe LFC at the family level comparing the control and Treatment D, with\nincreases in Pirellulaceae (lfc = 1.897) and Anaerovoracaceae\n(lfc = 0.752), while decreases were observed in Mycoplasmataceae\n(lfc = −1.700) and Selenomonadaceae (lfc = −3.374). At the genus level,\nMycoplasma (lfc = −1.529) and Pectinophilus (lfc = −3.374) decreased.\n4 Discussion\n4.1 Effects of various feed additives on pig\ngrowth performance\nFeed additives are commonly used in animal nutrition to enhance\nthe health and productivity of livestock, with probiotics, synbiotics, and\nenzyme cocktails being the most frequently applied. Probiotics help\nmaintain intestinal microbial balance and promote overall health by\nimproving gut epithelial barrier function, activating the immune\nsystem, degrading toxin receptors, and modulating the gut microbiota\n(28, 29). Synbiotics complement probiotics by providing specific\nsubstrates for fermentation, enhancing the availability of beneficial\nbacteria and offering additional health benefits to the host (30). In this\nTABLE 2  Effects of adding PRO, SYO, SYO+, and EC on growth performance in nursery pigsa.\nItem\nTreatmentb\nSEMc\np-values\nControl\nA\nB\nC\nD\nBody weight (kg)\nInitial\n5.58\n5.62\n5.65\n5.59\n5.58\n0.06\n0.99\n2 week\n7.70\n7.62\n7.83\n7.89\n8.09\n0.10\n0.59\n5 week\n14.55\n14.41\n14.28\n14.20\n15.13\n0.20\n0.62\nAverage daily gain (g)\n0–2 week\n150.76\n132.90\n152.63\n158.01\n174.26\n5.18\n0.16\n2–5 week\n341.41\n339.62\n322.43\n315.39\n351.44\n6.78\n0.44\nOverall\n263.60\n254.50\n252.51\n250.69\n279.77\n5.31\n0.39\nAverage daily feed disappearance (g)\n0–2 week\n229.85\n210.56\n224.83\n223.22\n235.69\n3.03\n0.10\n2–5 week\n516.87\n495.27\n508.14\n486.59\n533.85\n6.10\n0.22\nOverall\n394.74\n378.04\n392.74\n378.14\n405.68\n4.50\n0.33\nG: F ratio\n0–2 week\n0.65\n0.64\n0.67\n0.74\n0.75\n0.02\n0.47\n2–5 week\n0.66\n0.69\n0.63\n0.66\n0.67\n0.01\n0.75\nOverall\n0.66\n0.68\n0.64\n0.67\n0.69\n0.01\n0.85\naA total of 200 crossbred pigs was fed from average initial body weight 5.60 ± 0.05 kg and the average of final weight was 14.51 kg. Experimental pigs were randomly allocated to 5 treatments\nwith 4 replicates (10 pigs per pen).\n\n75\nOverall\n0.66\n0.68\n0.64\n0.67\n0.69\n0.01\n0.85\naA total of 200 crossbred pigs was fed from average initial body weight 5.60 ± 0.05 kg and the average of final weight was 14.51 kg.Experimental pigs were randomly allocated to 5 treatments\nwith 4 replicates (10 pigs per pen).\nbControl = basal diet; A = basal diet + PRO 0.05% (t/500 g); B = basal diet + SYO 0.1% (t/1 kg); C = basal diet + SYO+ 0.1% (t/1 kg); D = basal diet + EC 0.1%(t/1 kg).\ncStandard error of mean.\n\nKoo et al.\b\n10.3389/fvets.2025.1627739\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nstudy, the PRO, SYO, and SYO + used Bacillus spp. or Clostridium spp.,\nwhich are representative strains in probiotics and synbiotics known for\nproducing various enzymes and short-chain fatty acids (SCFA) (31).\nSCFA, including acetate, propionate, and butyrate, interact with SCFA-\nactivated receptors free fatty acid receptor 2 (FFA2) and free fatty acid\nreceptor 3 (FFA3) in L-cells, influencing appetite regulation (32).\nL-cells secrete glucagon-like peptide-1 (GLP-1) and peptide YY (PYY),\nwhich act via the vagus-brainstem-hypothalamic pathway (33). Within\nthe\nhypothalamus,\nthe\narcuate\nnucleus\n(ARC)\ninvolves\npro-opiomelanocortin (POMC) and cocaine and amphetamine\nregulated transcript (CART), where GLP-1 and PYY interact with Y2\nreceptors to increase POMC activity and suppress appetite (34). This\nmechanism may explain the lack of significant differences in overall\nADFD between the PRO, SYO, and SYO + treatment groups and the\nControl group. Batterham et al. (35) found that chronic peripheral\nadministration of PYY3-36  in mice increased POMC activity,\ndecreased appetite, and reduced body weight. However, these results\nwere not inconsistent. According to a study by Cheng and Kim et al.\nTABLE 3  Effects of adding PRO, SYO, SYO+, and EC on immune indicators in nursery pigs.\nItem\nTreatmenta\nSEMb\np-values\nControl\nA\nB\nC\nD\nIg G\n0–2 week\n205.43\n225.50\n153.81\n173.14\n265.61\n15.48\n0.16\n2–5 week\n179.55\n145.55\n148.51\n104.55\n133.39\n8.29\n0.06\nIg A\n0–2 week\n47.75B\n27.85B\n25.62B\n42.47B\n60.07A\n4.66\n< 0.01\n2–5 week\n74.06\n43.11\n45.20\n39.60\n51.75\n6.07\n0.41\nIg M\n0–2 week\n28.10B\n22.50B\n49.73A\n34.00B\n24.96B\n2.53\n< 0.01\n2–5 week\n38.71BC\n39.18BC\n59.68A\n30.20C\n46.78B\n2.50\n< 0.01\naControl = basal diet; A = basal diet + PRO 0.05% (t/500 g); B = basal diet + SYO 0.1% (t/1 kg); C = basal diet + SYO+ 0.1% (t/1 kg); D = basal diet + EC 0.1%(t/1 kg).\nbStandard error of mean.\nTABLE 4  Effects of adding PRO, SYO, SYO+, and EC on alpha-diversity in nursery pigs.\nItem\nTreatment\nSEMa\np-values\nControl\nA\nB\nC\nD\nFamily\nChao1\n57.50\n55.50\n57.00\n61.50\n55.75\n1.434\n0.73\nShannon\n3.89\n3.94\n4.16\n4.12\n3.92\n0.074\n0.72\nSimpson\n0.88\n0.89\n0.91\n0.91\n0.88\n0.009\n0.81\nGenus\nChao1\n143.83\n140.75\n145.50\n152.75\n130.25\n3.335\n0.32\nShannon\n5.19\n5.21\n5.49\n5.40\n4.85\n0.092\n0.21\nSimpson\n0.93\n0.94\n0.96\n0.94\n0.91\n0.009\n0.45\n\n50\n55.50\n57.00\n61.50\n55.75\n1.434\n0.73\nShannon\n3.89\n3.94\n4.16\n4.12\n3.92\n0.074\n0.72\nSimpson\n0.88\n0.89\n0.91\n0.91\n0.88\n0.009\n0.81\nGenus\nChao1\n143.83\n140.75\n145.50\n152.75\n130.25\n3.335\n0.32\nShannon\n5.19\n5.21\n5.49\n5.40\n4.85\n0.092\n0.21\nSimpson\n0.93\n0.94\n0.96\n0.94\n0.91\n0.009\n0.45\naStandard error of mean.\nTABLE 5  Effects of adding PRO, SYO, SYO+, and EC on beta-diversity PERMANOVA results in nursery pigs.\nBeta-diversity\nDietary\ntreatmenta\nSample size\nPermutations\npseudo-F\np-value\nJaccard\nCON\nTreatment A\n8\n999\n0.74\n0.61\nTreatment B\n8\n999\n0.98\n0.49\nTreatment C\n8\n999\n0.86\n0.69\nTreatment D\n8\n999\n1.10\n0.33\nBray-Curit\nTreatment A\n8\n999\n0.90\n0.52\nTreatment B\n8\n999\n1.72\n0.15\nTreatment C\n8\n999\n0.73\n0.64\nTreatment D\n8\n999\n0.88\n0.55\naControl = basal diet; A = basal diet + PRO 0.05% (t/500 g); B = basal diet + SYO 0.1% (t/1 kg); C = basal diet + SYO+ 0.1% (t/1 kg); D = basal diet + EC 0.1%(t/1 kg).\n\nKoo et al.\b\n10.3389/fvets.2025.1627739\nFrontiers in Veterinary Science\n06\nfrontiersin.org\n(36) found that supplementing microorganisms as feedstuffs and feed \nadditives improved the ADG by 1.83%, and the daily feed intake by \n0.24% in pigs and broilers. Also, Duarte et al. (37) highlighted that \nadding synbiotics consisting of xylanases and Bacillus spp. to nursery \npig diets improved ADG and the G: F ratio, demonstrating their \npotential to enhance the intestinal health of nursery pigs challenged \nwith enterotoxigenic Escherichia coli. Therefore, the complexity of the \neffects of probiotics and synbiotics on nursery pig growth and health \nsuggests that their efficacy depends on specific microbial compositions, \nstrain types, specific condition, and physiological contexts. In addition, \nthe limitations of the additive effects seem to be influenced by the \nfollowing socio-environmental factors. First, the experiment was \nconducted in a commercial setting with 10 animals per pen, making it \ndifficult to control for social factors among individuals. Second, the \nlimited number of repetitions made it challenging to achieve statistical \nsignificance between treatment groups. Third, due to the nature of \ncommercial farms, there were inherent limitations in controlling \nenvironmental factors such as various diseases. However, this study did \nnot measure SCFAs, so these findings remain hypothetical. Therefore, \nwe suggest that additional gut metabolites are needed.\nFIGURE 1\nThe log fold change (Control vs. A group/Family & Genus), where ‘f__’ represent family and ‘g__’ represent genus.\nFIGURE 2\nThe log fold change (Control vs. B group/Family & Genus), where the left side represents family (f__) and the right side represent genus (g__).\n\nA group/Family & Genus), where ‘f__’ represent family and ‘g__’ represent genus.FIGURE 2\nThe log fold change (Control vs.B group/Family & Genus), where the left side represents family (f__) and the right side represent genus (g__).\n\nKoo et al.\b\n10.3389/fvets.2025.1627739\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nEnzyme cocktail can enhance nutrient digestibility, as seen \nwith phytase activity increasing phosphorus bioavailability (38). \nAdditionally, unlike probiotics and synbiotics, enzyme cocktails \ndirectly degrade specific nutrients, improving their availability \nand potentially enhancing the growth performance of nursery \npigs (39). However, in this study, EC treatment did not \nsignificantly affect nursery pig growth performance. These \ninconsistencies highlight the complexity of enzyme cocktail \nefficacy, which may be  influenced by factors such as host \nresponse, and environmental conditions. Also, several studies \nhave identified varying factors affecting enzyme cocktail \nperformance, including enzyme activity, enzyme activity level, \nsubstrate availability, fiber composition, enzyme matrix, and \nexperimental conditions (40–43). Nevertheless, Trindade Neto \net al. (44) reported that adding enzyme cocktail on nursery pig \ndiet improved growth performance. Zhang et al. (45) found that \nsupplementing corn-soybean meal diets for nursery pigs with \nexogenous multi-enzyme has the potential to enhance gut \nhealth, improve nutrient digestion, and boost growth \nperformance. Although similar results were not observed, the \nFIGURE 3\nThe log fold change (Control vs. C group/Family & Genus), where ‘f__’ represent family and ‘g__’ represent genus.\nFIGURE 4\nThe log fold change (Control vs. D group/Family & Genus), where the left side represents family (f__) and the right side represent genus (g__).\n\nKoo et al.\b\n10.3389/fvets.2025.1627739\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nenzyme cocktail suggests potential for improving growth\nperformance in nursery pigs. Therefore, future research should\nevaluate various combination and supplementation levels of\nenzymes to optimize enzyme cocktail efficacy for digestibility\nand growth performance.\n4.2 Effects of various feed additives on pig\nimmune status\nThe representative indicators of serum immunoglobulins, IgG,\nIgA, and IgM are produced by B cell, which primarily manage humoral\nimmunity. IgM provides a rapid immune response to maintain tissue\nbalance, IgA forms the immune system in the gastrointestinal mucosa,\nand IgG is the most abundant antibody and plays the largest role in\ndefense against antigens (46, 47).\nIn the study, the enzyme cocktail used contained phytase,\nwhich breaks down phytic acid [myo-inositol 1,2,3,4,5,6-hexakis\n(dihydrogen phosphate); InsP6], a form of phosphorus commonly\nfound in plants. Phytic acid is classified as an anti-nutritional\nfactor due to its inability to be digested by pigs, which limits the\navailability of phosphorus. The phosphates fraction for animal\navailability depend on the breakdown of InsP6, which can\nbe degraded by phytase into less-phosphorylated InsP5 to InsP1,\nmyo-inositol, and orthophosphates (48). The inositol phosphates\nisomers (InsPs) produced in this process are recognized by\nmacrophages and toll-like receptor 2 (TLR2) in the intestinal\nmucosa, leading to the activation of dendritic cells (DCs). The\nactivated DCs induce regulatory T cells that modulate immune\nresponses and increase IgA levels (49, 50).\nDuring the 0–2 week and 0–5 week periods, the addition of SYO\nto the diets of nursery pigs improved IgM concentration. The\nincrease in IgM may be related to Bacillus licheniformis present in\nSYO. Bacillus licheniformis is known to exert antiviral and\nimmunoregulatory effects (45). In a study by Wang et  al. (51),\nBacillus licheniformis was found to reduce the expression of\npro-inflammatory cytokines (TNF-α and IL-1β) and increase levels\nof anti-inflammatory cytokines (IL-10 and IL-4). IL-10 and IL-4 are\n\nIn a study by Wang et  al.(51),\nBacillus licheniformis was found to reduce the expression of\npro-inflammatory cytokines (TNF-α and IL-1β) and increase levels\nof anti-inflammatory cytokines (IL-10 and IL-4).IL-10 and IL-4 are\nkey co-factors for B cell proliferation and promote differentiation\ninto plasmablasts, which secrete IgM or IgG (52, 53). Therefore, this\nis thought that Bacillus licheniformis in SYO stimulates the immune\nsystem, inducing anti-inflammatory cytokines, which in turn\nincrease IgM. However, in the case of SYO+, the increase in IgM was\nnot observed, which is suggested to be  due to the interaction\nbetween Bacillus licheniformis and Bacillus subtilis within SYO+.\nThese findings suggest that interaction between Bacillus licheniformis\nand Bacillus subtilis in SYO + may have reduced the ability of\nBacillus licheniformis to raise IgM levels observed with the single-\nstrain SYO supplement.\nConsequently, the addition of SYO and enzyme cocktail in\nnursery pig diet can activate the immune system. These findings\nsuggest that the inclusion of SYO + and enzyme cocktail in\nnursery pig diets can stimulate the immune system, highlighting\nthe potential for improved health management in commercial pig\nfarming. However, this study did not measure anti-inflammatory\nmarkers, and these findings remain hypothetical. Therefore,\nwe  suggest that additional immunological or metabolomic\nanalyses are needed.\n4.3 Effects of various feed additives on pig\nfecal microbiota\nFeed additives play a significant role in modulating gut microbiota\ncomposition. Probiotics, prebiotics, and synbiotics often referred to as\nbioactive components, can promote gut health by inhibiting the growth of\npathogen and supporting beneficial gut microbiota. Previous studies have\nshown that these bioactive components influence gut microbial diversity\n(Alpha-diversity and Beta-diversity) and alter gut microbiota composition\n(54, 55). We investigated the effects of various feed additives on microbial\nalpha and beta diversity, with beta diversity analyzed by PERMANOVA\n\nWe investigated the effects of various feed additives on microbial\nalpha and beta diversity, with beta diversity analyzed by PERMANOVA\nusing a multivariate distance matrix (27). However, in our study, the\naddition of feed additives in nursery pig feed did not significantly affect\nalpha-diversity and beta-diversity. The gut microbiota of nursery pigs is\nsignificantly influenced by various internal and external factors, and as it\nstabilizes over time, the diversity of the gut microbiota may decrease (56,\n57). In this study, the result of fecal analysis did not show significant\ndifferences in microbial diversity among treatments. The microbiota is a\ndynamic and complex structure that can be influenced by various factors\nsuch as age, dietary composition, time, and breeding environment (58–60).\nAccording to Fonseca et al. (61) study, the alpha-diversity tended to increase\nduring the first 4 days but no significant difference was observed at over\ntime. Also, the study by Da Sliva et al. (62) reported that beta-diversity was\nunchanged over time despite the addition of various probiotics to broiler\ndiets, which in line with our result, suggesting that microbial diversity\ntemporarily increased during the initial period of the experiment, but the\neffect did not remain over time. However, in this study, we did not perform\nlongitudinal sampling, which prevented us from analyzing microbial\ntrajectories over time. Moreover, we did not measure gut metabolites that\ncould support the proposed mechanisms. Future studies integrating\nmetabolomic analyses with microbial profiling would strengthen\ncausal inferences.\nAmong the major findings, the significant reduction of\nMycoplasma\nspp.\nby\nsynbiotics\nand\nenzyme\ncocktail\nsupplementation is noteworthy, given the pathogen’s importance\nin swine respiratory disease. Log Fold Change (LFC) analysis\ndoes not represent the absolute or relative abundance within a\nsingle sample but rather indicates the fold change between two\nsamples (63). In our study, LFC analysis of feed additive\nsupplementation showed a reduction in pathogenic Mycoplasma\n\nIn our study, LFC analysis of feed additive\nsupplementation showed a reduction in pathogenic Mycoplasma\nspp. in the SYO treatment group. Mycoplasma spp., lacking a cell\nwall, can evade immune system recognition, attach to cell\nreceptors, penetrate cell membranes, and cause inflammatory\nresponses (64). However, the antibiotic compounds produced by\ncertain strains stimulate the immune system by promoting anti-\ninflammatory cytokine release and B-cell activation, thereby\nenhancing immunoglobulin secretion (65, 66). Consequently, the\nreduction of Mycoplasma spp. in the SYO group may be attributed\nto lichenysin, an antibiotic produced by Bacillus licheniformis.\nLichenysin, a modified form of surfactin, is a lipopeptide\nbiosurfactant used against Mycoplasma spp. (67, 68). It penetrates\nthe cell membrane of Mycoplasma spp., inducing micelle\nformation, destabilizing the membrane, and leading to growth\ninhibition or cell death (69).\nInterestingly, the reduction in Mycoplasma spp. was not observed in\nthe PRO and SYO + groups, possibly due to interactions between Bacillus\nsubtilis and Bacillus licheniformis. These strains primarily utilize carbon\nsources as nutrients (70), and competition may reduce their inhibitory\n\nKoo et al.\b\n10.3389/fvets.2025.1627739\nFrontiers in Veterinary Science\n09\nfrontiersin.org\neffects on Mycoplasma spp. Vijayalakshmi et al. (70) also reported that\nBacillus subtilis exhibits higher stability under various conditions compared\nto Bacillus licheniformis. Additionally, differences in antibiotics produced\nby these strains may explain the lack of reduction in Mycoplasma spp. The\nMycoplasma spp., lacking a cell wall, have a higher exposure and content of\nmembrane lipids (71). Surfactin, produced by Bacillus subtilis, has lower\nlipid affinity and surface activity than lichenysin (72), which may account\nfor the observed results. However, these effects have not been consistent.\nWhile some studies reported that co-culturing Bacillus subtilis and Bacillus\nlicheniformis was effectively inhibits pathogens (73, 74). Nevertheless,\nstudies on competitive and symbiotic interactions between the two strains\nin pigs were limited, we cannot rule out the possibility that they vie for the\nsame resources. Therefore, these results suggest that in vitro co-culture and\nin vivo validation studies are needed to clarify their compatibility and\nmechanisms of interaction.\nIn the enzyme cocktail treatment group, a reduction in Mycoplasma\nspp. was observed, though the mechanism remains unclear. The EC used\nin this study contained phytase and lipase. Phosphorus released during\nphytic acid degradation by phytase can stimulate SCFA production,\nenhance mineral absorption, and lower pH, inhibiting pathogenic bacteria\ngrowth (75, 76). Galié et al. (77) reported that various enzymes degrade\nbiofilm structures, thereby inhibiting pathogen adhesion and survival.\nHowever, lipid-hydrolyzing enzymes like lipase can release long-chain fatty\nacids, potentially promoting the growth of Mycoplasma spp. (78, 79). Thus,\nthe reduction in Mycoplasma spp. in the EC group likely results from the\ninteraction between phytase and lipase, balancing phytase’s inhibitory\neffects on pathogenic bacteria with lipase’s potential growth-promoting\neffects on Mycoplasma spp.\n5 Conclusion\nThe incorporation of probiotics, synbiotics, and an enzyme cocktail\n\n5 Conclusion\nThe incorporation of probiotics, synbiotics, and an enzyme cocktail\ninto the diets of nursery pigs, under commercial farming conditions,\ndid not significantly impact growth performance directly. However,\npositive effects were observed in immune status and gut microbiota.\nNotably, nursery pigs exhibited increased feed intake, which is\nanticipated to contribute positively to future growth. Therefore, this\nstudy suggests that Bacillus-based biotics and enzyme cocktails have\npotential as alternatives to antibiotics for controlling infections caused\nby pathogenic microorganisms in nursery pigs under commercial\nconditions. However, we suggest that additional studies with increased\nreplicates and pen numbers are necessary for application in commercial\nlarge-scale farms.\nData availability statement\nThe datasets presented in this study can be found in online\nrepositories. This data can be found here: NCBI repository, accession\nnumber PRJNA1290052.\nEthics statement\nThe animal study was approved by Institutional Animal Care and\nUse Committee (IACUC) of the Gyeongsang National University\n(GNU-231030-P0204). The study was conducted in accordance with\nthe local legislation and institutional requirements.\nAuthor contributions\nS-MK: Formal analysis, Investigation, Software, Visualization,\nWriting – original draft, Writing – review & editing. J-HL: Investigation,\nWriting – original draft. S-HO: Formal analysis, Software, Writing – original\ndraft. J-CJ: Conceptualization, Data curation, Formal analysis, Funding\nacquisition, Methodology, Project administration, Resources, Supervision,\nValidation, Writing – original draft, Writing – review & editing.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. This study was supported\nby a contract research project funded by Woogene B&G. The funder\nwas not involved in the study design, collection, analysis, interpretation\nof data, the writing of this article, or the decision to submit it\nfor publication.\nAcknowledgments\nThe author(s) would like to acknowledge Woogene B&G for\nproviding the feed additives.\n\nAcknowledgments\nThe author(s) would like to acknowledge Woogene B&G for\nproviding the feed additives.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. 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+{"text": "【兽医文献】\n标题: Effect of bluetongue serotype 3 vaccines on probability of viremia and NSAID usage in Dutch dairy cattle herds\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nA behavioral study of live\ncaptured coypu (Myocastor\ncoypus) and raccoons (Procyon\nlotor) with respect to animal\nwelfare\nFranziska M. Schöttes 1†, Friederike Gethöffer 1†, Daniel Tost 1,\nNicole Kemper 2 and Ursula Siebert 1*\n1 Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover,\nHannover, Germany, 2 Institute for Animal Hygiene, Animal Welfare and Farm Animal Behavior,\nUniversity of Veterinary Medicine Hannover, Hannover, Germany\nSince 2016, coypu (Myocastor coypus) and raccoons (Procyon lotor) have been\nlisted as invasive alien species (IAS) of European Union concern (The Implementing\nRegulation (EU) 1141/2016). The three-stage management plan stipulates the\nprevention of the further spread of species already established in Germany (Regulation\n(EU) 1143/2014 of the European Parliament and of the Council). Live trapping is\na commonly used instrument to catch animals in hunting practice, but also in\nspecies conservation and pet protection. As part of a comprehensive study to\nimprove animal welfare in live trapping, this paper focuses on a behavioral study\nwith the aim of assessing the behavior of trapped animals in relation to stress. Video\nrecordings were analyzed using a species adjusted ethogram and a quantitative\nobservation method with focus on the animal in the trap over a maximum six-hour\nperiod. Blood and hair samples were taken for endocrinological examinations. The\nresults showed large species-specific and individual differences in the expression\nof a wide range of behavior and coping strategies. As part of the stress assessment,\nit was concluded that external factors, among others the type of trap, have an\ninfluence on the behavior of coypu and raccoon. The raccoons showed different\nbehaviors depending on the individual data. The endocrinological examinations\nof the stress parameters cortisol and dehydroepiandrosteron (DHEA) measured\nin serum and hair revealed differences between the species, indicating differing\nbasal values. For coypu, the measurements indicated differences in serum and\n\nFor coypu, the measurements indicated differences in serum and\nhair cortisol levels between juvenile and adult coypu. The study shows substantial\nindicators, such as the design of the trap type and the duration of capture, can\nbe used to contribute to improve practices in live capture of (wild) animals.\nKEYWORDS\nlive trap, distress, behavior, ethology, capture impact, invasive species, wildlife\nmanagement, cortisol\n1 Introduction\nThe Coypu (Myocastor coypus) and the raccoon (Procyon lotor) are established Invasive\nAlien Species (IAS) in all federal states of Germany (1–3), their population having increased\nconsiderably over the last decades (4). Both animals are classified as IAS of European Union\nconcern under European regulation 1143/2014 (2). According to this regulation, an\nimplementation of management following the guidelines of prevention, early detection and\nOPEN ACCESS\nEDITED BY\nEdward Narayan,\nSouthern Cross University, Australia\nREVIEWED BY\nJames Edward Brereton,\nSparsholt College, United Kingdom\nDenise Pereira Leme,\nSanta Catarina State University, Brazil\n*CORRESPONDENCE\nUrsula Siebert\nUrsula.Siebert@tiho-hannover.de\n†These authors have contributed equally to\nthis work and share first authorship\nRECEIVED 28 April 2025\nACCEPTED 27 June 2025\nPUBLISHED 30 July 2025\nCITATION\nSchöttes FM, Gethöffer F, Tost D,\nKemper N and Siebert U (2025) A behavioral\nstudy of live captured coypu (Myocastor\ncoypus) and raccoons (Procyon lotor) with\nrespect to animal welfare.\nFront. Vet. Sci. 12:1619604.\ndoi: 10.3389/fvets.2025.1619604\nCOPYRIGHT\n© 2025 Schöttes, Gethöffer, Tost,\nKemper and Siebert. This is an open-access\narticle distributed under the terms of the\nCreative Commons Attribution License\n(CC BY). The use, distribution or reproduction\nin other forums is permitted, provided the\noriginal author(s) and the copyright owner(s)\nare credited and that the original publication\nin this journal is cited, in accordance with\naccepted academic practice. No use,\ndistribution or reproduction is permitted\nwhich does not comply with these terms.\nTYPE  Original Research\nPUBLISHED  30 July 2025\nDOI  10.3389/fvets.2025.1619604\n\nSchöttes et al.\b\n10.3389/fvets.2025.1619604\nFrontiers in Veterinary Science\n02\nfrontiersin.org\neradication is required (5). In Germany, coypu and raccoon are\nsubjected to hunting law in most federal states (6). In Lower Saxony,\nhunting seasons for coypu and juvenile raccoons are open all year;\nclosed seasons only exist for adult raccoons (7). A recently published\nsurvey among hunters from Lower Saxony shows percentages of live\ntrappings ranging from 44–74% (8, 9).\nThe international standard reflecting animal welfare in trapping\nis represented by the Agreement of Human Trapping Standards\n(AIHTS) for the trapping of mammals in restraining and killing traps\n(10). The AIHTS is controversially discussed, as it does not take into\naccount all welfare aspects nor does it consider the current\nunderstanding of animal welfare sufficiently (11, 12). Among others,\nthe AIHTS disregards many physiological indicators (e.g., vital\nparameters, enzyme and hormone levels) and keeps the behavioral\nindicators for poor animal welfare to a minimum. So far, research has\nfocused on the practicability and efficiency of trapping (13–16), but\nincreasingly, researchers emphasize the importance of more\ncomprehensive knowledge about welfare aspects for several species,\ne.g., ethical approaches like the humaneness of methods in trapping\n(17–21). Animal welfare has gained more attention in public\nawareness in many countries over the last decades, not only in\nlivestock and laboratories, but also in wildlife conservation and\nhunting practice (22–25).\nResearch on animal welfare has been significantly influenced by\nthe popular “model of five freedoms,” which focuses on livestock and\nits avoidance of negative experiences (26, 27). Its further development\nwent towards a “Five Domains” model, which includes the internal\nand external conditions and focuses on the associated psychological\nexperiences (28, 29). The updated five domains model includes\nnutrition, the physical environment, health, and behavioral\ninteractions, which form the basis of the mental state of animals (30).\nThis model also serves as a guideline for improved welfare in\n\nThe updated five domains model includes\nnutrition, the physical environment, health, and behavioral\ninteractions, which form the basis of the mental state of animals (30).This model also serves as a guideline for improved welfare in\nzoological facilities and the wildlife conservation sector (31, 32).\nAlmost one hundred years ago in 1936, Selye started to define\nand explore the meaning of stress (33, 34), after discovering the\nrelationship between certain pathological findings and the activation\nof the hypothalamic–pituitary–adrenal-axis (HPA). Even then,\nhe differentiated between positive (eustress) and negative (distress)\nexpressions of stress (35). Ever since, researchers have successfully\ndeduced the physiological factors and signal cascades involved,\ndiscovering the complex interplay of nervous, endocrine and immune\nmechanisms. Within this interplay, the sympathetic-adreno-medullar\naxis and the hypothalamus-pituitary–adrenal axis are defined (36).\nSo far, the sympathetic nervous system influences numerous organs,\ngenerally by mobilizing energy. The transmitters (catecholamines) are\nresponsible for, for example, the increase in the heart rate and blood\npressure and a redirection of blood flow (targeting on skeletal muscles\nand reducing gastrointestinal organs) in order to ensure a quick\nflight, while pain perception is temporarily decreased. Meanwhile, by\nthe release of glucocorticoids, the metabolism is regulated,\nsuppressing the immune system and generating energy. While the\nsympathetic nervous system acts within seconds, the rise in, for\nexample, cortisol in the blood occurs within minutes (37). Modern\nresearch tries to measure transmitters, changes of immune cell\nnumbers, or mediated chemicals as well as of hormonal blood\nconcentrations (38–41). Behavioral approaches to assess stress in\nanimals are common in the laboratory as well as farm animals (42–\n44). Due to missing empirical data, behavioral stress evaluation in\nwild animals is rare (45, 46). Nevertheless, especially in (wild)\nanimals, an evaluation and interpretation of these findings\nare challenging.\n\nDue to missing empirical data, behavioral stress evaluation in\nwild animals is rare (45, 46).Nevertheless, especially in (wild)\nanimals, an evaluation and interpretation of these findings\nare challenging.\nBy definition, according to Fraser, “an animal is said to be in a\nstate of stress if it is required to make abnormal or extreme adjustments\nin its physiology or behavior in order to cope with adverse aspects of\nits environment and management.” (47). The way of dealing with an\nunpleasant situation, also referred to as coping, is divided into\ndifferent approaches: escape, remove, search, wait (48). Coping\nstrategies are not only perceivable in connection with stress-related\nbehavior, but also part of the concept of behavioral adaptation (49).\nTherefore, coping is seen as the animal’s ability to compensate for a\nchange of situation (50). When a state is reached that exceeds an\nanimal’s coping behavior in response to a stressor, this is referred to as\n“strain” in stress research. Over the decades, stress research in animals\nhas paid more attention to the various aspects of the meaning of the\nword stress and the manifestation of stress in physiological and\nbehavioral expressions (33, 47, 51). The question how to quantify\nstress, the measurability and the classification of stress levels was the\nfocus of earlier research (52), but even then it became clear that there\nwas no single method that could be  used in all situations (53).\nNowadays, there is a tendency to include several parameters in order\nto be able to assess the stress factor in many different ways (54–57).\nBoth from a hunting perspective and for ethical reasons, it is\nconsidered important to analyze the behavior of the animals caught,\ntaking into account animal welfare aspects in order to ensure the best\npossible practice in live capture. To the authors�� knowledge, there are\nno behavioral studies of live trapped coypu. Only a few behavioral\nstudies exist that describe species-specific behavioral sequences of free\nranging or captive coypu from different countries (58–62), most of\n\nTo the authors’ knowledge, there are\nno behavioral studies of live trapped coypu.Only a few behavioral\nstudies exist that describe species-specific behavioral sequences of free\nranging or captive coypu from different countries (58–62), most of\nwhich describe activity patterns or movement behavior in terms of\nhome range sizes. For raccoons, the behavior during a catch in\nfoothold traps is briefly described in terms of activity (63). Related\nstudies were conducted for different species or trap designs (20,\n46, 64–66).\nA comprehensive individual animal observation based on a\nbehavior catalog has not yet been compiled for coypu and raccoons.\nFor wild animal species, often only parts of ethograms are created for\ndifferent study reasons (67). A standardized ethogram for various\nspecies, primarily mammals and birds, was already designed in the\npast to be able to assess visual behavior patterns of wildlife (68), and\nthe idea of using shared ethograms uniformly is still relevant today\n(69, 70). Standardized behavior recording is also indicated as a way to\nregularly monitor well-being for captive animals (71).\nThere are different approaches for the assessment of stress. In the\ncontext of laboratory animal research, pain, suffering, distress, or\nlasting harm are used as indicators and the stress load is classified on\nthis basis and established in an EU Directive (2010/63, Annex VI).\nComparatively, a confinement of laboratory animals for less than 24 h\nis classified as low severity, but inducing escape and avoidance\nreactions is expected to cause moderate distress. Due to the differences\nbetween laboratory and wild animals as well as environmental factors,\nthe applicability of this guideline on live capture in the field is not\ngiven, but could serve as a point of reference. Furthermore, scoring\nsheets, used in behavioral studies for animal experiments to assess\nstress load assessment (72, 73), are not defined for coypu and raccoons.\nOpen field tests are another widely used experimental strategy for the\nquantitative study of behavior, e.g., exploratory or avoidance behavior,\n\nSchöttes et al.\b\n10.3389/fvets.2025.1619604\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nas they test reactions to a new, pre-defined environment, divided into\ncategories (74–76), but these are also difficult to transfer to fieldwork.\nThis study considers animal welfare aspects for coypu in three\nmost frequently used live trap types in Germany. Since the capture of\nnon-target species must be considered, the raccoon was chosen as a\nrepresentative bycatch species in order to illustrate species-specific\ndifferences. The study was designed to implement the basic conditions\nof the AIHTS, including additional parameters. We focus on the\nbehavior and hormonal response to evaluate the stress of live captured\ncoypu and raccoons in two of five restraining trap designs known in\nthe literature (77, 78). As there is no standardized observation\nprocedure for the species examined in our study, we decided to use the\ngenerally valid continuous focus method (79), referring to two-level\nhierarchy in the ethogram, which enabled us, on the one hand, to\nbe certain that no behavior was overlooked and, on the other hand, to\nrecord both the behavioral class and the behavioral pattern in detail.\nThe underlying ethogram was developed according to substantial\nresearch in zoo and wildlife animals (80). Our hypothesis assumed\nthat the behavior of the captured animals is influenced by external\nfactors and strain-related parameters, but not by the animal’s\nindividual data.\n2 Materials and methods\n2.1 Study area and setup\nAll animals were captured from 2019 to 2022 at different locations\nin Lower Saxony with permission granted by 33.8-42502-04-19/3190\nfrom the Lower Saxony State Office for Consumer Protection and Food\nSafety (LAVES). All trapping sites were characterized by access to\nrunning or stagnating water and surrounded by varying habitat features\nsuch as meadows, reed beds, forests, and cultivated areas. Three\ndifferent live trap types were tested for animal welfare aspects: first, a\nwire grid trap (WG trap; common design used in waterboard\nassociations) with a rocker trigger under the food basket at the head\n\nThree\ndifferent live trap types were tested for animal welfare aspects: first, a\nwire grid trap (WG trap; common design used in waterboard\nassociations) with a rocker trigger under the food basket at the head\nend of the trap. This type of trap was set up without cover. Second, the\nsheet metal trap (SM trap; “Trapper-Neozoen®,” Raiffeisen\nWarengenossenschaft Osnabrücker Land eG, Melle, Germany), a\nclosed galvanized steel tube that is released by shifting weight beyond\nthe center of the axle. Third, a square wooden box trap (WB trap;\nFuchsfalle.de, Horb am Neckar, Germany), with a rocker trigger. Each\ntrap was combined with a self-developed technical system. This\ncomprised a video system, consisting of a 130° wide-angle camera with\n940nm lens (KEYESTUDIO Raspberry Pi Camera, Shenzhen KEYES\nRobot Co. Ltd., Shenzhen, China) and LED ring (INSTAR IR LED\nRing, INSTAR Deutschland GmbH, Hünstetten Bechtheim, Germany),\nconnected to a Raspberry Pi 3® computer (Raspberry Pi 3 Model B+,\nRaspberry Pi Foundation, Cambridge, United Kingdom), to which a\ntemperature sensor and a surf stick (Huawei E3372, Huawei\nTechnologies Deutschland GmbH, Düsseldorf, Germany) were\nconnected. Images of the catch could be retrieved at any time via an\ninternet connection and received by smartphone. A Python script was\nset up to operate the system. When an animal was caught by triggering\nthe trap mechanism, the camera started continuous recording via a reed\ncontact sensor. The data were stored on the Raspberry Pi 3® computer.\nAdditionally, a trap alarm (TRAPMASTER professional® and\nTRAPMASTER professional Neo® EPV Electronics GmbH,\nLüdenscheid, Germany) trap detector was installed. An audio recorder\n(Wildlife acoustics songmeter 4®, Wildlife Acoustics, Inc., Maynard,\nMA, USA) was placed next to the trap to continuously collect audio\ndata. A common camera trap was set up to monitor the surrounding\narea, referred to as “additional cameras” in the following text. Only\nvegetarian baits, like fruit and vegetables, were used to attract coypu\nand raccoons. The traps were set up all year and during the daytime,\n\nOnly\nvegetarian baits, like fruit and vegetables, were used to attract coypu\nand raccoons.The traps were set up all year and during the daytime,\nexcept for closed hunting seasons.\n2.2 Trapping and sampling\nWhen the trap closed, each animal’s behavior was video-recorded\nof 31 coypus and 8 raccoons for up to 6 h. This corresponds to the\nmaximum duration time in the trap permitted by the authorities. After\nthese 6 h the animals were transferred in a capture box with a locking\nslide and anesthetized with medetomidine hydrochloride (Domitor®,\nVetoquinol GmbH, Ismaning, Germany) and ketamine (Ketamine 10%,\nEcuphar GmbH, Greifswald, Germany). Blood and hair samples were\ntaken, and vital parameters such as rectal body temperature, oxygen\nsaturation, heart rate, and respiratory rate were monitored during\nanaesthesia. For euthanasia, T61® (T61®, MSD Animal Health, Intervet\nDeutschland GmbH, Unterschleißheim, Germany) was administered\nintracardially after blood sampling, and a general examination was\nperformed according to a standardized protocol. The individual\nanimals were weighed and body measurements were documented. For\ncomparison, five intracardially obtained blood samples per species from\nanimals shot by hunters that had not previously been in a trap were\ncollected immediately after death as a control group. Serum levels of\nCortisol and DHEA were determined at the Clinical Endocrinology\nLaboratory of the Clinic for Cattle, University of Veterinary Medicine\nHannover, Foundation, Hannover, Germany (81, 82). For this purpose,\nall blood samples were prepared using a Z307® universal centrifuge\n(HERMLE Labortechnik GmbH, Accel 3, 4500 rpm, 12:00 min). Serum\nsamples were stored at −20°C and analyzed with a radioimmunoassay\n(RIA) for cortisol (Cortisol RIA IM1841, Backman Coulter Inc., Brea,\nCA, USA) and Dehydroepiandrosteron (DHEA, DHEA RIA IM1138\nBackman Coulter Inc.) after validation (83) within 3 months of storage.\nFor the analysis of the hair Cortisol and DHEA levels, hair samples were\nsent to the Institute for Doping Analysis and Sports Biochemistry\n\n) after validation (83) within 3 months of storage.For the analysis of the hair Cortisol and DHEA levels, hair samples were\nsent to the Institute for Doping Analysis and Sports Biochemistry\nKreischa (IDAS, Kreischa, Germany). The samples were treated\naccording to the internal protocol of IDAS Kreischa dated 12/29/2021,\nusing a deuterated internal standard for the quantification of each\nanalyte, determining hormone levels with gas chromatography and\nmass spectrometry coupling (Agilent 1290 Infinity-HPLC with Sciex\nTripleQuad 6500+, e.g., Agilent GC 7890/ 7000 GC-QQQ). For further\nexamination, the carcasses were x-rayed (GIERTH X-Ray international\nGmbH, Riesa, Germany) and subjected to pathological dissection to\ndetect capture-related injuries, which will be  discussed in a\nfollow-up paper.\n2.3 Data preparation and analysis\nThe dataset was prepared using Excel (Microsoft Corporation\n2016, Microsoft Excel). Based on the clinical examinations and X-ray,\nthe age of the animals was estimated and classified as juvenile (<1year)\nor adult (>1year) (see Supplementary material), by body measurements,\n\nSchöttes et al.\b\n10.3389/fvets.2025.1619604\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nweight, sexual status (mature or immature), and growth plates \n(presence or absence) in comparison with literature (84–87). \nWe referred time and season according to Central European Time.\nThe video recordings were analyzed with the software Mangold \nINTERACT version 14.3 and 18.7 (Mangold INTERACT, Mangold \nInternational GmbH, Arnstorf, Germany). We developed a species-\nspecific ethogram that formed the basis for the behavioral analysis. \nThereafter, we  categorized the behavior classes into movement \nbehavior, exploratory behavior, foraging behavior, resting behavior, \ncomfort behavior, automutilative behavior, and visualized \nvocalization in the video (in the following referred to as movement, \nexploration, \nforaging, \nresting, \ncomfort, \nautomutilation, \nvocalization; see ethogram, Supplementary material Tables S2-5). \nHowever, the classes automutilation and vocalization were only \nincluded in the descriptive analysis. Each behavior class contained \nthe corresponding individual behavior pattern, such as walking in \nthe movement class, shown in the detailed ethogram (see Figure 1). \nIndividual behavior was defined in terms of frequency and \nduration, with the start and end times of each behavior pattern \ndocumented using a quantitative observation method with focus \non the animal in the trap over a maximum six-hour period. The \nvideo recordings of six animals caught in pairs in an SM trap were \ntreated separately.\nFrom each class, selected behavior patterns were considered \nseparately. These included nasalizing, gnawing, being startled, \ntrembling, pausing, coprophagia, sleeping, and grooming. These either \nstood out in relation to stress behavior during the behavioral analysis \nor had a species-specific reference.\nFIGURE 1\nEthogram with seven behavior classes and associated behavior patterns as basis for video analysis of coypu and raccoon trapped in live traps.\n\nSchöttes et al.\b\n10.3389/fvets.2025.1619604\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nIndependent variables were assigned to three groups: individual\ndata, external factors, and strain-related parameters (see\nSupplementary material Table S1). For the variables weight, rectal body\ntemperature, external temperature, and internal trap temperature,\nwe formed subgroups for better comparability. In our study, there was\na data pool of 55 coypu and 48 raccoons, from which only the animals\nwhose capture was recorded in a video were considered in this paper.\nIn order to avoid bias by narrowing down, the entire data pool,\nincluding individuals not included in the behavioral analysis, was used\nas the basis for grouping individual parameters.\nIn a separate analysis of the acoustic recorder data including all\naudible vocalizations of the trapped animals, we created a detailed\nethogram, and call types of coypu were described for the first time\n(88). For this reason, the audible vocalizations were not included in\nthe statistical behavioral analysis.\n2.4 Statistical analysis\nTo check for observer reliability, we used the Cohen-Kappa (κ) test\n(89), which showed substantial agreement (κ = 0.72) (90).\nThe behavior classes served as dependent variables for the\nhypothesis, which was tested in three different models. In these, the\nindividual, external, and strain-related factors were used as independent\nvariables with interactions. Behavioral data were not normally\ndistributed for both species (Shapiro–Wilk >0.05, see Table 1).\nSAS Enterprise Guide 7.1 ® (SAS Enterprise Guide, SAS Institute\nInc., Cary, NC, USA) (91) was used for all descriptive and R ® (R, R\nCore Team, Vienna, Austria) (92) for statistical calculations. For\nstatistical modeling of behavioral data, we used the non-parametric\nsimple analysis of variance (ANOVA) and generalized linear models\n(GLM) with the applied regression data set carData (93). Figures were\nplotted using ggplot2 (94). The model design was selected based on\nthe good explanation of the effects, the Akaike Information Criterion\n(AIC), in combination with the Mc Fadden’s R-squared parameter\n\nFigures were\nplotted using ggplot2 (94).The model design was selected based on\nthe good explanation of the effects, the Akaike Information Criterion\n(AIC), in combination with the Mc Fadden’s R-squared parameter\n(R2), where values >0.60 were accepted. An ANOVA Model was\ncalculated to verify the significance of the variables in the model\nselection. The level of significance was specified at 0.05. To analyze\nwhether hormonal values of captured animals differed between trap\ntypes, we  used the non-parametric simple analysis of variance\n(ANOVA) and Wilcoxon signed-rank test.\n3 Results\n3.1 Coypu\n3.1.1 Capture data\nA total of 31 videos of coypu trapped in the WG trap (n = 17), the\nSM trap (n = 11), and the WB trap (n = 3) were analyzed. The sex ratio\nwas balanced (16 female/15 male) and the adults in the video (21)\noutnumbered the juveniles (10). All further recorded individual,\nexternal, and strain-related data are listed below (see Tables 2–4).\n3.1.2 Behavioral data\nIn total, 179 h of video footage were recorded, with a mean video\nlength of 5.77 h per catch. Broken down by trap type, the sample size\nnumber for the WB trap was very small and therefore assessed to a\nlimited extent.\nThere were large differences depending on whether duration or\nfrequency of each behavior class was assessed (see Figure 2).\n3.1.2.1 Individual data\nFor all behavior classes, an influence of the individual factors (age,\nsex, weight class) was rejected due to AIC and R2 value below the limit\nrange of the individual data model. The strain-related variables had no\ninfluence on the duration or frequency of the expression of the\nbehavior classes.\n3.1.2.2 External factors\nThe external factors, including the type of trap as one of the most\nimportant factors, influenced the expression of the five main\nbehavioral classes in coypu (see Figure  3). The differences were\nsignificant for the duration and frequency of exploration and resting\nbehavior and frequency of movement (see Table 5). Significantly high\nduration of movement was detected in WG or SM traps. A high\nfrequency of movement, exploration, and resting was observed in the\nWG trap.\n\nSignificantly high\nduration of movement was detected in WG or SM traps.A high\nfrequency of movement, exploration, and resting was observed in the\nWG trap.\nImpact of external factors on shown behavior was evaluated using\nGLMs (see S6-S9). Catches in a WG trap resulted in significantly\nhigher frequencies of movement (p-value [f] = 0.002), exploration (p-\nvalue [f] = 0.000), and resting (p-value [f] = 0.007). Low durations of\nexploration were detected in spring trapping in the WG trap (p-value\n[d] = 0.024). The duration of comfort was lower when trapping in the\nWG trap in combination with low outside temperatures (p-value\n[d] = 0.037).\nThe seasons only had a minor influence on the behavioral classes\nof coypu. The duration of the resting period was shorter when the\nanimals were caught in winter, but not significantly (p-value\n[d] = 0.100). Higher Frequencies in exploration (p-value [f] = 0.006)\nand resting (p-value [f] = 0.014) were recorded during catches\nin winter.\nThe influence of daytimes, like trapping at midday (p-value\n[d] = 0.099) or afternoon (p-value [d] = 0.002), resulted in an increase\nin duration in movement. Midday catches also led to an increase in\nfrequency in movement (p-value [f] = 0.005), exploration (p-value\n[f] = 0.049), and resting (p-value [f] = 0.005). Catching at night led\nto a significant increase in duration of exploration (p-value\n[d] = 0.017). Catches during the morning showed a significantly\nincreased frequency of comfort (p-value [f] = 0.043). The duration of\nTABLE 1  Normal distribution of classes calculated with the Shapiro–Wilk test (>0.05) for coypu and raccoon in duration (d) and frequency (f).\nSpecies\nMovement\nExploration\nResting\nComfort\nForaging\nCoypu\nd: 0.0078\nf: 0.0242\nd: 0.0506\nf: 0.0030\nd: 0.0072\nf: 0.0989\nd: <0.0001\nf: <0.0001\nd: <0.0001\nf: <0.0001\nRaccoon\nd: 0.0001\nf: 0.0061\nd: 0.4487\nf: 0.5746\nd: 0.5434\nf: 0.8336\nd: 0.1043\nf: 0.2107\nd: 0.0021\nf: 0.0007\n\nSchöttes et al.\b\n10.3389/fvets.2025.1619604\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nresting was significantly higher during dry weather (p-value\n[d] = 0.040).\nNo self-inflicted injuries were detected in coypu. There were 14\nvocalizations, which were visible in the video of coypu trapped in the\nWG trap, with a total duration of 0.86 min, 69 in the SM trap with a\ntotal duration of 63.78 min and none in the WB trap.\nAll animals expressed a wide variety of different behavioral\npatterns. The values for duration and frequency showed that the\npatterns were generally expressed very briefly and repeatedly, which\nindicates continual switching between each.\nAs expected, exploration was expressed differently depending on\nthe species. As coypu primarily expressed exploration by use of its\nnose, we named this behavior “nasalizing.” It was expressed very\nfrequently (p-value [f] = 0.000) and with significantly high duration\n(p-value [d] = 0.001), particularly in the WG trap. As a more intensive\nexpression of exploration, gnawing was expressed less frequently, but\nwith significantly high duration (p-value [d] = 0.017) and frequency\n(p-value [f] = 0.005) in the WG trap and, in contrast, only rarely or\nnot at all in the other trap types. All other behavioral patterns\nconsidered here were expressed much less frequently. Although not\nreaching significance or high numbers, “being startled” was mainly\nand longest shown by coypu caught in the SM trap, “trembling” was\nonly expressed twice in SM trap.\nSeventeen of 31 coypu showed coprophagia in the trap. It was\nshown in the WB trap with significantly high frequency (p-value\n[f] = 0.012) but occurred in all trap types. Sleeping animals were only\ndiscovered in the closed trap types (SM, WB), but in few cases.\nGrooming was frequently expressed in all traps.\nThe behavioral patterns “nasalizing,” “gnawing,” and “coprophagia”\ndiffered significantly in duration and frequency between trap types\n(see Table 6 below).\n3.1.3 Paired catches\nOn three occasions, two juveniles were jointly trapped in the SM\ntrap. These could be assigned to the same litter and were all caught\n\n3.1.3 Paired catches\nOn three occasions, two juveniles were jointly trapped in the SM\ntrap.These could be assigned to the same litter and were all caught\nTABLE 2  Animal-related data of live captured coypu are depicted in this\ntable.\nAnimal-related data\nTotal*\nDataset (n)\nSex\n31\nMale\n16\nFemale\n15\nAge [yy]\n31\nAdult\n21\nJuvenile\n10\nWeight [kg]\nWeight class\n31\nLight\n6\nMedium\n13\nHeavy\n12\n*: p < 0.05, else not significant.\nTABLE 3  External factors of live captured coypu are depicted in this table.\nExternal factors\nTotal*\nDataset (n)\nTemperature outside\n[°C] Tout\n30\nT0\n7\nT1\n11\nT2\n12\nNA\n1\nWeather conditions\n31\nDry\n15\nCloudy\n5\nRainy\n6\nFoggy\n3\nWindy\n2\nSeason [meteorological]\n31\nSpring\n7\nSummer\n0\nFall\n12\nWinter\n12\nDaytime [Central\nEuropean Time]\n31\nMorning\n2\nMidday\n1\nAfternoon\n4\nEvening\n11\nNight\n13\n*: p < 0.05, else not significant.\nTABLE 4  Strain-related data of live captured coypu are depicted in this\ntable.\nStrain-related\nparameters\nTotal*\nDataset (n)\nRectal body\ntemperature [C°]\nTrec\n30\nLow\n7\nMedium\n10\nHigh\n13\nNA\n1\nTemperature trap\ninside [C°]\nTtrap\n11\nTt0\n0\nTt1\n5\nTt2\n6\nNA\n20\nSerum Cortisol\n[nmol/l]\nSCortisol\n30\nSC1\n12\nSC2\n10\nSC3\n8\nNA\n1\nFull body index\nGS\n31\nGS0\n12\nGS1\n11\nGS2\n7\nGS3\n1\n*Of individuals considered in behavioral analysis of this paper; NA = Not available.\n\nSchöttes et al.\b\n10.3389/fvets.2025.1619604\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nat the same location during winter. Females predominated, with a\nratio of 6:1 (f/m). All individuals belonged to the light weight class.\nThree showed low rectal body temperatures (32.9°C, 33.0°C,\n32.9°C), while three exhibited medium values (34.1°C, 34.4°C,\n34.7°C).\nMovement of all six animals was expressed with little duration (d\nmean 45.28 min) but high frequency (f mean 441.83). Exploration (d\nmean 27.73 min; f mean 112.83) and resting (d mean 180.93 min; f\nmean 322.17) were both detected in high duration and frequency, little\ncomfort (d mean 3.20 min; f mean 14.33) and foraging (d mean 11.25\nmin; f mean 26.67) was displayed. Among the interactive behavior of\nthe animals caught in pairs, exploration and resting occurred most\nfrequently and long lasting, resting representing the highest\nproportion (Figure 4).\nApart from interactive behavior in comparison with single catches\nof juveniles, the animals trapped in pairs showed significantly higher\nresting and exploration in terms of duration and frequency of\nbehavioral classes. Movement was expressed with longer durations,\nbut less frequently than in single-caught animals. Comfort behavior\nwas shown with less duration and frequency. The foraging behavior\nwas recorded with the same frequency but shorter duration for paired\ncaught coypu.\n3.1.4 Occurrence of animals outside the trap\nThere were 42 sightings of animals, which occurred outside the\ntraps, during 12 catches of coypu. In order to avoid a bias between the\nopen and closed trap types, only observations of animals made by\nadditional camera devices were included.\nThe average dwelling time of these animals amounted to 7.66 min\nper presence. In total, the animals were present for an average of\n26.83  min per trapping period (maximum 6 h). There was no\nprominent time of occurrence during the catch (see Table 7). The\nspecies recorded included 24 coypu (Myocastor coypus), 13 rats\n(Rattus norvegicus), three mallard ducks (Anas platyrhynchos), and\ntwo songbirds (Passeri spp.), which were not determined further.\n\nThe\nspecies recorded included 24 coypu (Myocastor coypus), 13 rats\n(Rattus norvegicus), three mallard ducks (Anas platyrhynchos), and\ntwo songbirds (Passeri spp.), which were not determined further.\nMost sightings of coypu were documented during a catch in the\nWG trap (17/5), fewer in SM trap (7/4), and none during a catch in\nthe WB trap (0/3) (see Supplementary material).\nDuring the animals’ occurrence outside the trap, the trapped\nanimals displayed exploration and resting (see Figure 5). Among\nthese, “observing” and “sitting” were prominently detected.\n3.2 Raccoon\n3.2.1 Capture data\nOf eight raccoons that were trapped, five were caught in the WG\ntrap and three in the WB trap. The sex was distributed in favor of\nmales 1/7 (f/m). Of these, seven animals were estimated to\nbe  juvenile and one adult (see Supplementary material for\nindividual data).\n3.2.2 Behavioral data\nA total of 34.71 h of video material was analyzed. Each video\nlasted 4.34 h on average. Due to the low sample size, these results\ncould be used only to a limited extent for behavior assessment of the\nFIGURE 2\nDistribution of observed duration [min.] (above) and frequency [number of events] (below) of the five behavior classes per catch, per trap type for\ncoypu. The dot indicates the median, the line indicates the 25–75% quantile range and the violin shows the distribution of observed durations or\nfrequencies. Movement was expressed in high frequencies with a low mean duration. Exploration was designated with a lower frequency, but lasted\nlonger on average. Resting was shown less frequently, but with long duration times. Comfort and foraging had both a shorter duration and frequency.\n\nSchöttes et al.\b\n10.3389/fvets.2025.1619604\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nraccoons and therefore serve as an approximation of species-specific\ndifferences. The differences in the durations and frequencies of the\nbehavioral classes can be seen in Figure 6.\nThe models indicated that individual data as well as external\nvariables had influence on raccoons behavior in the traps. The model\nincluding strain-related influences was rejected for all classes.\n3.2.2.1 Individual predictors\nThe individual data had great influence on the behavior classes for\nthe raccoon. Adult raccoons showed significantly more frequent\nmovement, resting, foraging and comfort. Juveniles expressed foraging\nsignificantly longer and frequenter.\nFemales showed a significantly higher frequency of movement (p-\nvalue [f] = 0.003), resting (p-value [f] = 0.064), and foraging (p-value\n[f] = 0.020). Duration of movement (p-value [d] = 8.300) and foraging\n(p-value [f] = 0.008) was also higher in females. In contrast, males\ndisplayed significantly shorter duration and less frequency of\nmovement (p-value [d] = 7.360; [f] = 0.001) and foraging (p-value\n[d] = 0.004; [f] = 0.012).\nAnimals of high weight class showed significantly high foraging\nin terms of duration (p-value [d] = 0.008) and frequency (p-value\n[f] = 0.020). Resting (p-value [f] = 0.064) and comfort (p-value\n[f] = 0.009) were detected with significantly higher frequency in heavy\nweight classes. Increased duration of movement could be seen in\nmedium (p-value [d] = 0.045) and heavy weight animals (p-value\n[d] = 8.300). In addition, medium weight animals displayed shorter\nduration times of movement (p-value [d] = 0.045). In light (p-value\n[d] = 0.002; [f] = 0.000) or medium weight animals (p-value\n[d] = 0.002; [f] = 0.000), foraging occurred in shorter duration and\nless frequently.\n3.2.2.2 External predictors\nThe trap type was one of the main influencing variables of the\nexternal factors. Duration and frequency of exploration, resting, and\ncomfort significantly differed between trap types (see Table  8;\nFigure 7).\n\n3.2.2.2 External predictors\nThe trap type was one of the main influencing variables of the\nexternal factors.Duration and frequency of exploration, resting, and\ncomfort significantly differed between trap types (see Table  8;\nFigure 7).\nAnimals caught in the WG trap showed significantly more frequent\ncomfort (p-value [f] = 0.000) and resting (p-value [f] = 0.010) and higher\ndurations of exploration (p-value [d] = 0.040) and foraging (p-value\n[d] = 0.004). In the WB trap, resting (p-value [f] = 0.045) and comfort\n(p-value [f] = 0.001) occurred more frequently. In addition, higher\nduration could be seen in comfort behavior (p-value [d] = 0.002).\nThe trap type was one of the main influencing variables of the\nexternal factors. Duration and frequency of exploration, resting, and\ncomfort significantly differed between trap types (see Table 8).\nAnimals caught in the WG trap showed significantly more frequent\ncomfort (p-value [f] = 0.000) and resting (p-value [f] = 0.009) and higher\ndurations of exploration (p-value [d] = 0.040) and foraging (p-value\n[d] = 0.004). In the WB trap, resting (p-value [f] = 0.045) and comfort\nTABLE 5  Results of the ANOVA model showing the expression of behavior classes classified by trap type for coypu.\nBehavior class\nDuration\nSignificance\nFrequency\nSignificance\nPr>ChiSq\nDF\nPr>ChiSq\nDF\nMovement\n0.0847\n2\n< 0.0001\n2\n***\nExploration\n< 0.0001\n2\n***\n< 0.0001\n2\n***\nComfort\n0.5169\n2\n0.5008\n2\nForaging\n0.7085\n2\n0.3911\n2\nResting\n< 0.0001\n2\n***\n0.0008\n2\n***\n***: p < 0.001.\nFIGURE 3\nBehavior classes in duration [min.] per catch period [maximum 6 h]\nof coypu, differentiated by trap type.\n\nSchöttes et al.\b\n10.3389/fvets.2025.1619604\nFrontiers in Veterinary Science\n09\nfrontiersin.org\n(p-value [f] = 0.001) occurred more frequently. In addition, higher\nduration could be seen in comfort behavior (p-value [d] = 0.002).\nSimilar to the coypu, the behavioral patterns of the trapped\nraccoons showed short duration times and high frequencies (see\nTable 9).\nExploration was primarily expressed by raccoons by their use of\npaws to explore the surroundings, which was why “palpating” was\ndetected mostly with significant frequencies in the WG trap (p-value\n[f] = 0.045). More intensive exploration was carried out by “scratching\nthe object.” In the WG trap, the raccoon often drew grass material into\nthe trap. During the catch, the raccoons “groomed” themselves\nsignificantly more frequently and for longer times in the WB trap (p-\nvalue [d] = 0.000; [f] = 0.005). “Shaking” was also one of the behaviors\nshown, but with less duration and frequency, while “sleeping” was shown\nwith high duration, but only very rarely. In the comparison of traps,\nsignificantly higher values for the duration and frequency of “sleeping”\nwere detected in the WB trap (p-value [d] = 0.039; [f] = 0.022).\nThe behavior “sleeping” and “grooming” differed significantly in\nduration and frequency between trap types. “Palpating” showed\nsignificant differences in frequency only (see Table 10 below).\n3.3 Endocrinological data\n3.3.1 Differences between species\nEndocrine examinations of cortisol and DHEA for 24 raccoons\n(12 males, 12 females) and 58 coypus (27 males, 31 females) revealed\ndistinct higher levels of serum and hair cortisol and lower serum\nDHEA levels in coypus than in raccoons (see Figure 8), whereas for\nhair DHEA, no significant difference could be detected. The serum\nDHEA concentrations negatively correlated with the cortisol values\n(see Supplementary material). The serum/hair DHEA quotient was\nalso not comparable between the species for coypus and raccoons,\nrespectively (Wilcoxon p-value = 4.341e-05, see Supplements).\n3.3.2 Differences within species\nIn order to assess a possible stress reaction, concentrations of\n\nThe serum/hair DHEA quotient was\nalso not comparable between the species for coypus and raccoons,\nrespectively (Wilcoxon p-value = 4.341e-05, see Supplements).3.3.2 Differences within species\nIn order to assess a possible stress reaction, concentrations of\ncortisol and the steroid hormone DHEA were measured in the serum\nand in the hair of the examined coypu and raccoons (see Figure 9).\nThe differences in stress parameters measured in serum and hair\nfound between sexes were not statistically significant within the\nspecies (see Supplementary material), but age (differentiated between\njuvenile and adult) did bring significant differences for serum cortisol\nlevels (Wilcoxon p = 0.082 raccoons, 0.0369 coypu) as well as for hair\ncortisol levels (Wilcoxon p = 0.0004929) in coypu.\nDifferences in juvenile (n = 26 blood samples, 27 hair samples) vs.\nadult (30 blood samples, 31 hair samples) serum and hair cortisol\nlevels were statistically significant in coypu (Wilcoxon p = 0.03697;\nTABLE 6  Differences between the behavioral patterns calculated in one way analysis, classified by trap type for coypu.\nBehavioral\npattern\nDuration\nSignificance\nFrequency\nSignificance\np-value\nDF\np-value\nDF\nNasalizing\n0.0015\n2\n**\n0.0003\n2\n***\nGnawing\n0.0168\n2\n*\n0.0050\n2\n**\nCoprophagia\n0.0110\n2\n*\n0.0121\n2\n*\n***: p < 0.001, **: p < 0.01, *: p < 0.05, else not significant.\nFIGURE 4\nBehavior classes in duration [min.] (above) and frequency [number of\nevents] (below) per catch period of paired trapped juvenile coypu.\nThe white boxplots contain the overall behavior (including the\ninteractive behavior), the gray boxplots contain only the interactive\nbehavior in respect to exploratory and resting behavior.\nTABLE 7  Frequencies of sightings of coypu outside the traps, by trap\ntype, and hours of the trapping per catch period.\nInterval (hours)\nWire grid trap\nSheet metal trap\n0\n5\n1\n1\n0\n2\n2\n1\n3\n3\n4\n0\n4\n6\n1\n5\n0\n0\n6\n1\n0\n\nSchöttes et al.\b\n10.3389/fvets.2025.1619604\nFrontiers in Veterinary Science\n10\nfrontiersin.org\n0.0004929). Differences in juvenile (n = 13 blood samples, 9 hair \nsamples) vs. adult (n = 10 blood samples, 7 hair samples) raccoons \nserum and hair cortisol levels were not statistically significant \n(Wilcoxon p = 0.1475, 0.351).\nThere were no differences in hormonal findings between the \ntested trapped animals and the control group either for coypu or \nraccoons (see Supplementary material).\n4 Discussion\nAs a part of a holistic assessment of animal welfare in trapped \ncoypu and raccoons, this study focused on behavior of these animals \nduring their capture. This paper evaluated the performance of the \ntrapped animals in combination with hormonal and somatically \nfindings, aiming to determine the best practice for stress evaluation. \nFIGURE 5\nExpressed behavior in classes of the trapped animals during the presence of another animal in the environment, displayed duration in minutes. \nEspecially exploration/movement or resting behavior, in this case sitting, was presented. Comfort behavior was not observed during presence of other \nanimals.\nFIGURE 6\nDistribution of observed duration [min.] (above) and frequency [number of events] (below) of the five behavior classes per catch, per trap type for \nraccoon. The dot indicates the median, the line indicates the 25–75% quantile range, and the violin shows the distribution of observed durations or \nfrequencies. The time duration of exploration was long and of high frequency, followed by resting behavior with a long duration and slightly lower \nfrequency. Movement, comfort, and foraging were shown with a low duration. Movement was detected in high frequency, comfort and foraging with \nvery low occurrence.\n\nSchöttes et al.\b\n10.3389/fvets.2025.1619604\nFrontiers in Veterinary Science\n11\nfrontiersin.org\nThe present study comprises captures of 31 coypu and eight raccoons,\nshowing that both coypu and raccoons exhibit diverse, species-\nspecific, and individual behaviors during capture, which provide\nevidence of different coping strategies and stress reactions. None of\nthe AIHTS indicators of poor animal welfare occurred consistently,\nbut automutilative and frustration-driven behavior patterns were\noccasionally observed in raccoons. The combination of behavioral\nanalysis and physiological parameters highlights the need for a\ndifferentiated and expanded assessment of stress and animal welfare\nduring live capture.\nIn our study, we opted for a neutral observational recording of\nbehavior, which ensures a high degree of comparability, by\nrecording behaviors based on a predefined definition, without any\ninterpretation by the observer. However, it is a longstanding\nrealization that the same behavioral expressions can be motivated\ndifferently (95). Therefore, the assessment of behavioral patterns\nmay lead to varying main classifications. For example, according\nto the ethogram, the behavior of “sitting” was categorized into the\nclass of resting behavior, although it should not be  used\nsynonymously with resting, but could also express “waiting” or\n“persevering.” In this way, the allocated meaning of various\nrecorded behaviors can be discussed, especially in relation to stress\nbehavior. We can state that the captured animals expressed all main\nclasses of expected behavior according to the underlying ethogram.\nFor coypu, the categories were comparable to those described in\nother studies (60, 96). According to the AIHTS on which our study\nis based, two indicators that serve as signs of poor welfare are\nstipulated in the assessment of trapped mammals (10). The first\nindicator, excessive immobility and lack of reaction, was not\nexpressed by coypu or raccoons in this study.\nMeanwhile, we  found species-specific differences in the\nbehavioral recordings that might hint at coping strategies for both\n\nThe first\nindicator, excessive immobility and lack of reaction, was not\nexpressed by coypu or raccoons in this study.Meanwhile, we  found species-specific differences in the\nbehavioral recordings that might hint at coping strategies for both\nspecies. Raccoons primarily showed palpating and scratching at\ntrap parts, whereas coypu merely displayed nasalizing and\ngnawing. In behavioral research, animals` reactions to an aversive\nsituation are classified within the coping concept (97). Recurring\nstriking individual differences in coping style were described for\nvarious species as proactive or reactive tendencies with\ncorresponding characteristics (98). Efforts have been made in\nmouse models (Mus musculus domesticus) to genetically breed less\naggressive animals or more stress-tolerant animals, which has\nprovided further insights into the link between genes and behavior\n(99). Nowadays, it is assumed that animals have personalities with\ndifferences in behavioral expression between individuals of a\nspecies, which are shaped by their genetics, environment, and\nexperiences (100–102). Among all our data, clear individual\ndifferences in behavioral patterns were detected. Against the\nbackground of individual personalities with individual coping\nbehavior, a stress assessment must therefore be viewed in a more\ndifferentiated light.\nTABLE 8  Results of the ANOVA model showing the expression of behavior classes classified by trap type for raccoon.\nBehavior class\nDuration\nSignificance\nFrequency\nSignificance\nPr>ChiSq\nDF\nPr>ChiSq\nDF\nMovement\n0.5018\n1\n0.3889\n1\nExploration\n< 0.0001\n1\n***\n< 0.0001\n1\n***\nComfort\n< 0.0001\n1\n***\n< 0.0001\n1\n***\nForaging\n0.5211\n1\n0.1104\n1\nResting\n< 0.0001\n1\n***\n0.0001\n1\n***\n***: p < 0.001.\nFIGURE 7\nBehavior classes in duration [min.] per catch period [maximum 6 h]\nof raccoon, differentiated by trap-type.\n\nSchöttes et al.\b\n10.3389/fvets.2025.1619604\nFrontiers in Veterinary Science\n12\nfrontiersin.org\nAs expected, species-specific differences were found, particularly\nwith regard to exploration behavior. The raccoons showed behavior\nalready known from other studies (45), in which it in the first place\nexplored with use of its paws and climbed nimbly through the traps.\nIts mouth and teeth, especially canines, were also used in the\nprocessing of the trap materials. On the contrary, the coypu primarily\nused its nose for exploration, which is why the term nasalizing was\nadded to the ethogram. Its paws were used to explore the trap walls or\nto dig. Animals that perform a greater number of different motor\nactions are more successful at solving a new task (103). Raccoons are\nknown for their ability to solve innovative problems, developing\nmultiple solutions to a novel problem (45, 104). This and the ability to\nalter its behavior in response to environmental stimuli are\ncharacteristics of a behaviorally flexible species, which appears to have\nan influential role on the type and manifestation of coping\nbehavior (105).\nTABLE 9  Extracted behavior patterns of the total data set with minimum, median, and maximum values of duration time and frequency, shown in\nminutes per catch period (maximum 6 h) for raccoon.\nBehavior\npattern\nDuration per catch period\nFrequency per catch period\nMinimum\nMedian\nMaximum\nMinimum\nMedian\nMaximum\nSleeping\n0.00\n25.90\n89.53\n0.00\n1.50\n7.00\nShaking\n0.04\n0.33\n0.85\n2.00\n13.00\n28.00\nGrooming\n1.75\n15.51\n54.74\n8.00\n29.50\n53.00\nPalpating\n5.73\n14.85\n38.01\n35.00\n82.50\n231.00\nPulling material\ntowards itself\n0.00\n2.88\n31.10\n0.00\n11.00\n57.00\nScratching the object\n15.33\n63.17\n104.88\n25.00\n219.00\n377.00\nTABLE 10  Differences between the behavioral patterns calculated in one-way analysis, classified by trap type for raccoon.\nBehavioral\npattern\nDuration\nSignificance\nFrequency\nSignificance\np-value\nDF\np-value\nDF\nSleeping\n0.0389\n1\n*\n0.0219\n1\n*\nShaking\n0.1319\n1\n0.2937\n1\nGrooming\n0.0003\n1\n***\n0.0053\n1\n**\nPalpating\n0.1393\n1\n0.0447\n1\n*\nScratching the object\n0.0986\n1\n0.0699\n1\n***: p < 0.001, **: p < 0.01, *: p < 0.05, else not significant.\nFIGURE 8\nSpecies differences in endocrine examination of serum and hair cortisol, serum and hair DHEA and quotients.\n\nSchöttes et al.\b\n10.3389/fvets.2025.1619604\nFrontiers in Veterinary Science\n13\nfrontiersin.org\nIn this study, frequency and duration were used to evaluate\nquantitative and qualitative dimensions of behavior. Remarkably, in\nmost cases, a short duration and high frequency of individual behavior\nresults in a pattern of constantly changing behaviors (20). If the coping\nstrategy of the animal fails, an increase in duration, frequency, and\nintensity of coping behavior patterns might occur (48). It is also\ndescribed that animals stuck in a certain situation will try out a variety\nof coping strategies (48), which in this evaluation becomes particularly\nevident in the frequent occurrence of exploratory behavior in raccoons\nand coypu. The short duration and frequent changes of behavioral\npatterns suggest that an individual behavior is started, but not\ncompleted. In addition, it was observed that a behavior that had just\nbeen started was interrupted, followed by another behavior that could\nnot be assigned to a trigger and often seemed out of context. This\nbehavior is described in ethology as a displacement activity (106–108),\narising from nervousness or a stressful situation and was already\ndescribed for other animal species (109). A further expression of\ncoping as a response to an aversive situation or due to the influence of\nexternal stressors is the expression of abnormal behavior (48). One\nform, the behavioral stereotypies, are also considered successful\ncoping behavior and are associated with reduced stress (50) although\nthey have not yet been interpreted consistently. Stereotypies could not\nbe observed in this study, but it should be kept in mind that the\ntrapping period was limited.\nThe second indicator that serves as a sign of poor welfare in the\ncontext of AIHTS is automutilative behavior, which is defined as self-\ndirected biting leading to severe injury (self-mutilation). We identified\nautomutilation in raccoons, which manifested as biting or scratching,\nalthough occurring in very low numbers. It is apparent that this was\ndone out of frustration at not being able to escape the trap.\n\nWe identified\nautomutilation in raccoons, which manifested as biting or scratching,\nalthough occurring in very low numbers.It is apparent that this was\ndone out of frustration at not being able to escape the trap.\nAdditionally, aggressive behaviors that occurred in raccoons, such as\ntugging at the object, indicate frustration (110–112) and were also\nconsidered as an expression of coping strategy (48). Similar behavior,\nlike bar biting or digging, were also documented for other captured\nspecies in the literature (64). For coypu, no aggression or\nautomutilative behavior was detected.\nIt was found that the individual neurophysiological reactions of\nanimals to a stressful situation are also reflected in the activity level of\nthe animals (74, 98). This finding is associated with behavioral\ndifferences in a variety of contexts (113). Different levels of “active” or\n“passive” behavior were observed over the entire trapping period. A\ndistinct trend towards temporal divisions could only be  derived\ndescriptively for movement behavior, which was recorded more at the\nbeginning than at the end of the six-hour trapping period. A difference\nin movement behavior, which may indicate an activity level, could\nbe detected in the case of raccoons. A generally higher level of activity\nwas detected in female raccoons, particularly in frequency and\nduration of movement compared with male raccoons that showed\nlower frequencies and duration times of movement behavior. The\nparticularly high activity and resilience in the first moments after trap\nclosure described in other studies could only be observed in few cases\n(64, 114). Nevertheless, over the course of the maximum six-hour\ncatch we could not confirm any fatigue of the animals with a decrease\nin exploration activity over a longer trapping period, as described in\nthe literature (63). Also, other behavioral signs that would have\nindicated habituation to the trap were not observed, so that the\nassumption that habituation does not take place in such situations can\nbe emphasized (20). It is also assumed that influencing the amount of\n\nAlso, other behavioral signs that would have\nindicated habituation to the trap were not observed, so that the\nassumption that habituation does not take place in such situations can\nbe emphasized (20).It is also assumed that influencing the amount of\ntime the animal is exposed to the stressor has no effect on the\nmeasured behavior (115).\nIn addition to the direct influencing factors, there are many\nexternal factors to which the trapped animal is indirectly exposed and\nwhich could influence the animal’s behavior. Due to its construction,\nenvironmental stimuli (climatic factors like precipitation, temperature,\nor sunlight as well as acoustic or visual attractions) are best perceivable\nfor trapped animals in the WG trap. Correspondingly, we detected\nhigh values especially for movement and exploration. In contrast, the\nSM trap was isolated from its surroundings, and high resting and\ncomfort was expressed by raccoons in the WB and WG trap. For\nraccoons, the duration of comfort was higher when caught in the WB\ntrap, supporting the use of a closed trap type. In addition, the wood\nseemed to have a slightly (sound) insulating effect, which could\nbe beneficial. Evidently, the special feature of the SM trap, a movable\npipe, startled the animals when the trap tipped over.\nSeasonal variations appeared in frequency of exploratory and\nresting behavior in coypu and comfort behavior in raccoons. The open\ntrap type exposes the trapped animals to the weather. Coypu caught\nin the WG trap in windy weather showed higher exploration, slightly\nmissing the significance. They also displayed lower durations of\ncomfort and resting during low outside temperatures or in winter\nFIGURE 9\nDifferences within species in endocrine examination of serum and hair cortisol, serum, and hair DHEA in terms of age.\n\nSchöttes et al.\b\n10.3389/fvets.2025.1619604\nFrontiers in Veterinary Science\n14\nfrontiersin.org\ncatches. However, a low temperature is associated with a reduced\nactivity phase (60). In contrast, a high movement behavior of the\ncoypu was recorded at low outside temperatures, which could\nbe justified with lack of isolation and thermoregulation.\nThe daily activity of non-native species may differ in new\nenvironments (116), which is why the comparability of extra-\nEuropean studies are not always given. The coypu and raccoons are\nconsidered to be crepuscular and nocturnal species (58, 60, 61, 117,\n118). Contrary to what we expected, a diurnal rhythm in form of\ncrepuscular and nocturnal activity of coypu could not be observed in\nthe trapped animals. The duration of movement behavior of coypu\nwas higher when caught at noon or in the afternoon. Longer\nmovement is usually only documented during periods of cold weather,\nwhich we  could not confirm (119). Longer-lasting exploratory\nbehavior at night in coypu could be associated with physiologically\nincreased activity in the daytime (60). During midday, the activity of\ncoypu increased in frequency of movement and exploration behavior,\nwhile comfort behavior showed higher frequencies in the morning.\nAlthough trapped raccoons showed more persistent exploratory\nbehavior and foraging in the evening, resting frequencies were also\nsignificantly higher, while at night lower resting times were\ndocumented, which corresponds to the nocturnal rhythm and the\nresting times of animals in the wild (120). Interestingly, we found that\ntrapped coypu switched to either exploration or resting behavior if\nanimals outside the trap were present. Here, mostly sitting was\nencoded (see Supplementary material), which might be interpreted as\npausing and vigilance. Sitting is usually expressed in the active period\nof day (60), which is why an assignment to the behavior class resting\ncan be  discussed. Additionally, observing was recorded almost\nexclusively if other animals were present.\nHowever, the expression of physiological behavior of the targeted\n\nAdditionally, observing was recorded almost\nexclusively if other animals were present.However, the expression of physiological behavior of the targeted\nspecies is an important indicator for animal welfare (121). Few\nbehaviors, such as coprophagia (122, 123) could be interpreted as\nphysiological for coypu. Coprophagia in captive coypu is usually\nconducted daily after returning to the nest in the morning hours or\nat midday (60), but was rarely expressed during live capture. Sleeping\nand lying are reported very rarely, probably because these behaviors\nare mainly expressed in the nest (60). Additional behaviors of social\nrodents, such as grooming each other (84), cannot be practiced in a\nsingle capture. Without doubts, the expression of physiological\nbehavior is limited during the trapping period of 6 h, due to\ntemporally and spatial limitation. The diurnal rhythm cannot\nbe exhibited, and resources like water and nesting material are not\naccessible. This could also be  a reason for the low frequency of\nencoded grooming, as the animals usually clean themselves after\nswimming or in the nest (60).\nSince both coypu and raccoons are species living in social\ngroups, isolation during live trapping restricts their social behavior.\nDuring captures of juvenile coypu pairs, interactive behavior was\nexpressed repeatedly over the capture period. The assumption that\ntwo animals might calm each other down could be confirmed, as\nmore resting behavior was shown compared to individually captured\njuveniles. It also was remarkable that less frequent movement\nbehavior was shown in the paired captures. In addition, social\nbehavior could be observed, mainly expressed by resting behavior as\ncontact lying (60). In the five domains model, the expression of\nphysiological behavior is one of three basic pillars of mental status\n(121, 124).\nChanges in physiological parameters can provide a further\nmeasurability of stress. In addition to increased heart rates and body\ntemperatures, an increase in the traditional stress hormone cortisol in\nserum samples is often described in trapped animals (12, 64, 114, 125).\n\nIn addition to increased heart rates and body\ntemperatures, an increase in the traditional stress hormone cortisol in\nserum samples is often described in trapped animals (12, 64, 114, 125).\nOther biomarkers like DHEA are being established as a method in the\nlatest research, as the interaction of many measuring points allows\nmore complete conclusions to be drawn about the neurological stress\nreactions (126). In this study, cortisol and DHEA parameters were\nmeasured in hair and blood that provide information about both acute\nand chronic stress reactions. Our results showed no significant\ndifferences between the different trap types or sex, although there were\nsignificant age-specific differences. However, significantly different\nserum cortisol and DHEA levels were measured between the two\nspecies, indicating different basal levels. The correlations between\nbehavioral expression and neurophysiological reaction described in\nthe literature (98, 99) could not be reflected in our results.\nTaking into account European and national legislation, such as the\nAIHTS, we believe that live trapping does not do sufficient justice to\nanimal welfare. In this study, essential points of reference in the behavior\nof the animals during live trapping were shown and the complexity of the\ndifferent parameters are clarified, which is why an all-encompassing\nconsideration of all methods is always necessary. Extended guidelines\nadapted to the modern understanding of animal welfare are necessary in\norder to be  able to carry out responsible live capture in the future,\nespecially in the area of conflict with invasive species.\n5 Limitations of the study\nThis study is limited due to restrictions of the animal experiment to\na six-hour trapping time, although a realistic trapping time in practice\nmay last considerably longer. The predefined time limitation in the\nexperimental setup resulted in significantly higher animal welfare\nstandards than those commonly applied in practice, creating a gap that is\nethically questionable and must be taken into account when transferring\n\nThe predefined time limitation in the\nexperimental setup resulted in significantly higher animal welfare\nstandards than those commonly applied in practice, creating a gap that is\nethically questionable and must be taken into account when transferring\nthe study’s findings to real-world applications. It was neither possible to\ncarry out uniform behavioral tests nor to use a comparable ethogram nor\nto generate a large sample size due to field study conditions. A comparison\nstudy to access “normal behavior” would be  required especially for\nraccoons, where behavioral studies are rare. The results of this study\ncannot be generalized, as there are too many differences between the trap\ntypes and species, which is why an assessment of all conditions is essential.\nFor these reasons, an assessment of behavior in relation to animal welfare\nis only possible to a limited extent, which should be taken into account in\nfuture studies. Our findings are intended as indications of an improved\nlive trapping of coypu and bycatch and does not fulfill the claim to\ngeneral validity.\n6 Conclusion\nWe conclude that both species-specific and individual behavior\npatterns, as suggested in this study, can serve as indicators of animal\nwelfare. The design, including insulation, and placement of the trap as\nwell as the handling of the trapped animals also contribute significantly\nto animal welfare. Therefore, adapting live traps to the specific needs,\nbehaviors, and physiological characteristics of different animal species is\nessential to ensure animal welfare. Species-specific activity patterns, stress\n\nSchöttes et al.\b\n10.3389/fvets.2025.1619604\nFrontiers in Veterinary Science\n15\nfrontiersin.org\nresponses and coping strategies should be considered. Only by aligning\ntrapping conditions with the physiological requirements of the animals,\nstress, injury, or suffering during capture can be minimized. It has been\nconfirmed that trapping (related) species as pairs can improve animal\nwelfare, and therefore it is suggested to support further research on multi-\nspecies trapping. We emphasize the consideration of the length of time\nthe animals remain in the trap as relevant to regulation and recommend\nshort intervals. Every practitioner of live trapping is responsible for\nconscientious and animal-friendly work, which is why we appeal to those\nwho use live traps to consider this during handling.\nData availability statement\nThe original contributions presented in the study are included in\nthe article/Supplementary material, further inquiries can be directed\nto the corresponding author.\nEthics statement\nThe animal study was approved by Lower Saxony State Office for\nConsumer Protection and Food Safety (LAVES). The study was\nconducted in accordance with the local legislation and\ninstitutional requirements.\nAuthor contributions\nFS: Investigation, Writing – review & editing, Software, Methodology,\nFormal analysis, Validation, Writing – original draft, Data curation,\nVisualization. FG: Validation, Writing – review & editing, Methodology,\nData curation, Investigation, Writing – original draft, Software, Formal\nanalysis, Visualization. DT: Visualization, Data curation, Validation,\nSoftware, Writing – original draft, Formal analysis, Writing – review &\nediting. NK: Supervision, Writing – original draft, Writing – review &\nediting. US: Validation, Supervision, Methodology, Writing – review &\nediting, Funding acquisition, Writing  – original draft, Resources,\nConceptualization.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. This research received\nexternal funding with grants from the Lower Saxony Ministry of Food,\n\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article.This research received\nexternal funding with grants from the Lower Saxony Ministry of Food,\nAgriculture and Consumer Protection, Germany. We acknowledge\nfinancial support by the Open Access Publication Fund of the\nUniversity of Veterinary Medicine Hannover, Foundation, Germany.\nAcknowledgments\nWe would like to thank the Lower Saxony Ministry of Food,\nAgriculture and Consumer Protection for commissioning us to carry\nout the study and for their trust in our scientific work. We would also\nlike to thank the staff of the Lower Saxony Chamber of Agriculture for\ntheir boundless support and excellent assistance in trapping coypu\nand raccoons. We are grateful to Frances Sherwood-Brock for the\nproofreading work and to Fritjof Freise for his support with statistical\nquestions. Last but not least, our thanks go to our colleagues who\nenthusiastically helped in every situation and any time of day in the\nfield with data analysis or other matters.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declare that no Gen AI was used in the creation of\nthis manuscript.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. 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+{"text": "【兽医文献】\n标题: Seasonal and age effects on in vitro embryo production in domestic cats under a standardized protocol\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nRoad transportation is associated\nwith decreased intestinal motility\nin horses\nSharanne L. Raidal 1,2*, Francesca Freccero 3, Ann Carstens 2,\nSarah Weaver 4 and Barbara Padalino 5,6\n1 Melbourne Veterinary School, The University of Melbourne, Werribee, VIC, Australia, 2 School of\nAgricultural, Environmental and Veterinary Sciences, Faculty of Science, Charles Sturt University,\nWagga Wagga, NSW, Australia, 3 Department of Veterinary Medical Sciences, University of Bologna,\nBologna, Italy, 4 School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy, SA,\nAustralia, 5 Faculty of Science and Engineering, Southern Cross University, Lismore, NSW, Australia,\n6 Department of Agricultural and Food Sciences, University of Bologna, Bologna, Italy\nBackground: Transportation of horses has been associated with colic and changes\nto the gastrointestinal microbiome. Percutaneous abdominal ultrasonography\nusing wireless, point-of-care transducers can be used to assess gastrointestinal\nmotility in field settings.\nObjectives: Characterization of intestinal motility and salivary cortisol responses\nof horses completing commercial transportation of 10-12h.\nStudy design: Prospective observational study of 30 horses with diverse signalment\nand transport histories. Clinical parameters, sonographic assessment of intestinal\nmotility and saliva samples, were collected before departure (Tpre or T0), after\noff-loading (T1) and 2h after arrival (T2).\nResults: After transportation, intestinal motility grades were reduced [Tpre median\n(IQR) composite motility grade: 8 (7-10), T1: 6 (4-7), T2: 7 (6-7); p < 0.001] and\nqualitative changes were observed in small intestinal sonographic appearance.\nHigh ambient temperatures during transport were associated with reduced\nintestinal motility at T1 (composite motility grade vs arrival temperature rs −0.45,\np = 0.017) and T2 (cecal motility grade vs arrival temperature rs −0.74, p < 0.001).\nHorses with high heart rates, high sweat scores or abnormal demeanour on\n\n45,\np = 0.017) and T2 (cecal motility grade vs arrival temperature rs −0.74, p < 0.001).Horses with high heart rates, high sweat scores or abnormal demeanour on\narrival demonstrated decreased intestinal motility. Salivary cortisol concentrations\nincreased after transportation (mean difference, 95% CI, for T0 vs T1 was 1.66,\n1.09−2.53 nmol/L) and were inversely associated with intestinal motility. Signalment\nand past travel history were not predictive of intestinal motility, but horses with\nunknown or no prior travel history (mean 12.8, 95%CI 8.2-17.4 nmol/L) had higher\ncortisol concentrations prior to departure than horses known to have travelled\npreviously (7.9, 5.8−9.9 nmol/L, p = 0.023).\nConclusions: These findings suggest that transportation is associated with transient\nreductions in intestinal motility, particularly during hot ambient conditions and\nin horses with increased cortisol response. Possible effects of provision of water\nand food during transport warrant further investigation.\nKEYWORDS\ntransport, equine, gastrointestinal physiology, health, welfare, cortisol, stress,\nultrasonography\nOPEN ACCESS\nEDITED BY\nNikola Čobanović,\nUniversity of Belgrade, Serbia\nREVIEWED BY\nAna Kaić,\nUniversity of Zagreb, Croatia\nMuhammad Thohawi Elziyad Purnama,\nAirlangga University, Indonesia\n*CORRESPONDENCE\nSharanne L. Raidal\nsharanne.raidal@unimelb.edu.au\nRECEIVED 15 June 2025\nACCEPTED 22 July 2025\nPUBLISHED 18 August 2025\nCITATION\nRaidal SL, Freccero F, Carstens A,\nWeaver S and Padalino B (2025) Road\ntransportation is associated with decreased\nintestinal motility in horses.\nFront. Vet. Sci. 12:1647236.\ndoi: 10.3389/fvets.2025.1647236\nCOPYRIGHT\n© 2025 Raidal, Freccero, Carstens, Weaver\nand Padalino. This is an open-access article\ndistributed under the terms of the Creative\nCommons Attribution License (CC BY). The\nuse, distribution or reproduction in other\nforums is permitted, provided the original\nauthor(s) and the copyright owner(s) are\ncredited and that the original publication in\nthis journal is cited, in accordance with\naccepted academic practice. No use,\ndistribution or reproduction is permitted\nwhich does not comply with these terms.\n\nNo use,\ndistribution or reproduction is permitted\nwhich does not comply with these terms.\nTYPE  Original Research\nPUBLISHED  18 August 2025\nDOI  10.3389/fvets.2025.1647236\n\nRaidal et al.\b\n10.3389/fvets.2025.1647236\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n1 Introduction\nTransportation has been associated with the development of colic\nin horses associated with gastrointestinal impaction, displacement,\nobstruction or colitis in epidemiologic (1–3) and in survey (4, 5)\nstudies. Altered intestinal motility has been suggested as contributing\nto colic (6), and might be related to changes in mucosal transport of\nwater and nutrients (7), or to changes in the gastrointestinal\nmicrobiome (8, 9). Decreased gastric emptying and alkaline gastric\nfluid content, consistent with reflux of duodenal content into the\nstomach, has been demonstrated in horses transported 12 h overnight\n(10), suggesting abnormal gastrointestinal motility.\nFeeding prior to and during transportation has been\nrecommended to reduce the severity of gastric ulceration subsequent\nto transportation (10), and makes intuitive sense to many owners.\nTransport regulations relating to road transport of horses in Europe\nmandate provision of liquid and, if necessary, feed every 8 h during\nroad transportation.1 However, the provision of feed to horses with\nreduced gastrointestinal motility might predispose to impactions or\nother abnormalities and hence contribute to adverse outcomes\nassociated with travel. To date, few studies have prospectively\nevaluated the effect of feeding on gastrointestinal function in\ntransported horses. Percutaneous, transabdominal ultrasonography is\na non-invasive technique that has been used to demonstrate alterations\nin gastrointestinal motility associated with pasture and stable\nmanagement (11), medications (12–15), anesthesia (16) and colic\nsurgery (16, 17), but has not been previously applied to the evaluation\nof possible transport effects on intestinal motility in horses.\nPhysiological alterations, such as increased heart and respiratory\nrate, increased rectal temperature, and endocrine changes, including\nalterations to the hypothalamic, pituitary adrenal (HPA) axis,\nβ-endorphin (18–20) and serotonergic responses (21), have been\nassociated with transportation and may be influenced by past travel\n\nhistory, temperament, signalment factors and management strategies\nprior to travel, and with conditions during the journey, including\norientation within the transport vehicle, travel distance and duration,\nambient conditions such as temperature and humidity. Cortisol\nincreases after road transportation in horses (18, 22–26) and has been\nused to characterize the hypothalamic–pituitary stress response in this\nspecies. The current study aimed to characterize intestinal motility by\nnon-invasive, ultrasonographic assessment of client-owned horses\nassociated with commercial land transportation of 10 – 12 h duration,\nand to relate gastrointestinal motility to factors evident prior to or\nduring transportation and to the cortisol response observed with travel.\nWe hypothesized that 1. Commercial road transportation without\nprovision of food or water is associated with transiently decreased\nintestinal motility, 2. Factors evident prior to or during transportation\nmight predict horses at risk for decreased motility, and 3. Horses with\n1  Council Regulation (EC) No. 1/2005 on the protection of animals during\ntransport and related operations and amending Directives 64/432/EEC and\n93/119/EC and Regulation (EC) No. 1255/97. Chapter V Watering and\nfeeding interval, journal times and resting periods, regulation 1.4(c), page\n25. https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32005R0001,\naccessed 25/06/15.\nhigher salivary cortisol concentrations prior to or following\ntransportation will have greater reduction in intestinal motility.\n2 Materials and methods\n2.1 Study design\nThis study was conducted as a prospective study using horses\ncompleting commercial transportation of 10 to 12 h duration (Figure 1)\nand was approved by the Charles Sturt University Animal Ethics\nCommittee (approval number A21415). Horses were deemed suitable\nfor inclusion if they were >12 months of age, well-handled, free of\nsubstantive injury or illness on veterinary examination, completing a full\njourney (depot to depot), amenable to and available for veterinary and\nsonographic evaluation at least once prior to transportation and on\n\narrival. A detailed and standardized data sheet (Supplementary File 1)\nwas completed for each horse enrolled in the study. Veterinary\nexaminations and ultrasonographic assessment (detailed below) were\ncompleted, where possible, at the transport depot on the evening prior\nto transportation (T-1), within 60 min prior to transportation (T0),\nwithin 60 min of arrival (T1), and approximately 2 h after arrival (T2).\nAs these were commercial horse movements, researchers were not able\nto standardize veterinary examination at T-1 or T0 relative to arrival,\ndeparture or feeding practices for all horses. As such, times of\nexamination, arrival, feeding and departure were all recorded.\nInformation on horse use, management prior to transportation, reason\nfor transportation (racing, competition, breeding, relocation or other),\ntravel history of each horse, and pre-transport management were\nobtained from the owner, agent or transport company. Horse age was\nprovided by owner or agent, and confirmed by assessing microchip,\nbrands and/or dentition, as available. Availability and consumption of\nfood and water at T-1, T0 and T2 were recorded; at T1 (off-loading),\nultrasonographic and veterinary examinations were completed prior to\nfeeding. Connections (owners, trainers or agents) were asked to\ndocument food intake, clinical observations (demeanour, appetite,\ncolicky signs, performance) following transportation using a\nstandardized template (Supplementary File 1) and without knowledge of\nultrasonographic findings, to document any adverse effects subsequent\nto the study and possibly associated with transportation.\n2.2 Transport details\nThe study was completed with the support of Ultimate Horse\nTransport,2 departing from and arriving into company depots. Trucks\ndeparted at 0500 h for southbound journeys, or at 0400 h for\nnorthbound journeys, and arrived at their destination 10 – 12 h later,\ndepending on the number of animals to be dropped off or picked up\nen route, and on local traffic conditions. Horses were offered ad-lib\nwater at all times at the depot, and were fed at least twice overnight or,\n\nHorses were offered ad-lib\nwater at all times at the depot, and were fed at least twice overnight or,\nif resident for longer periods, were additionally fed twice daily. Feeds\ncomprised lucerne hay with additional commercial pelleted feed as\nneeded, or according to owner preferences.\n2  1,236 Tooma Rd., Burra, NSW; ultimatehorsetransport.net.au.\n\nRaidal et al.\b\n10.3389/fvets.2025.1647236\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nResearchers shadowed the transporter on a preliminary\njourney (Trip 1, October 2022) to familiarize themselves with\ndepots, and with operations around receival and departure of\nhorses; no data were collected on this initial trip. Journeys were\nroutine (weekly) routes and were included in the current study\nwhen two or more horses suitable for inclusion were scheduled to\ntravel, when researchers were available subject to other\ncommitments, and to encompass a range of weather conditions.\nDuring transportation horses were managed at the discretion of\nUltimate staff, and were not fed or watered in the transport\nvehicle. Horses were restrained in single bays facing forwards or\nsideways. Route details and meteorologic information were\nrecorded by researchers using available weather data3 and the\nenvironment within the transport vehicle was monitored\ncontinuously using a weather tracker (Kestrel: 4000 Pocket\nWeather Tracker, Nielsen-Kellerman, Boothwyn, Pennsylvania).\nTransport route, distance, duration and driving speed were\nrecorded using a Garmin Edge 540 Cycling Computer (Garmin\nAustralasia Pty Ltd., Marsden Park, NSW) placed next to the\nweather meter. Loading was videoed and information on loading\nevents was recorded based on researcher observation. Events\nduring transportation were recorded based on driver anamnesis.\nHorses were unloaded and information on horse demeanour and\na sweat score [adapted from Zeyner et al. (27)] were recorded on\narrival. Water was available on arrival, and feed was provided\nimmediately following completion of initial sonographic\nassessment and veterinary examination at T1.\n3  http://www.bom.gov.au/\n2.3 Veterinary examination\nVeterinary examinations were performed by researchers (all\nveterinarians with equine clinical experience and advanced\nqualifications), assisted by veterinary and equine science students, and\nwere recorded on each horse’s data collection template\n(Supplementary File 1). Body condition score (BCS) and size (height,\n\nBody condition score (BCS) and size (height,\ngirth and shoulder width) were recorded using a measuring stick and\nmeasuring tape. Body weight was estimated from girth measurements,\nand size measurements were converted into a single ordinal size\ncategory (extra large, large, medium, small).\nPhysical examination included inspection of each horse for ocular\nor nasal discharge or injury, assessment of demeanour, rate and\ncharacter of respiration, bilateral cardiothoracic and abdominal\nauscultation, evaluation of circulatory integrity (mucous membrane\ncolor and refill, jugular fill, peripheral pulses), and rectal temperature.\nAbdominal auscultation was performed at the upper  and lower\nquadrants bilaterally, with a subjective grading assigned to each\nquadrant based on the intensity of borborygmi (0 = silent, no motility\nheard during 30 s, 1 = less than normal motility, 2 = normal motility,\n3 = increased motility) and, in the upper right quadrant, the frequency\nof ileocecal sounds was recorded as previously described (14, 28).\n2.4 Characterization of gastrointestinal\nmotility\nUltrasonographic assessment was completed as previously\ndescribed (15), using a portable, handheld ultrasound probe (GE\nVScan Air CL, GE HealthCare, Mascot, NSW). Briefly, images were\nobtained from four acoustic windows to evaluate the duodenum\n(DUOD, 16th or 17th intercostal space, adjacent to the cranial pole of\nFIGURE 1\nOverview of study design and data collection. Trip 1 was completed by researchers in an observational capacity only (no horses were included in the\nstudy); data were collected from horses undergoing seven subsequent trips. The number of horses available for inclusion and sample timing are shown\n(T-1 and T0 are shown as time of day, T1 and T2 are shown as hh:mm after arrival). Further detail is available in Supplementary information.\n\nRaidal et al.\b\n10.3389/fvets.2025.1647236\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nthe right kidney), caecum (CAEC, along the lateral cecal band, ventral\nto the costochondral junction), colon (COLN, ventral midline or\nparamedian longitudinal orientation) and jejunum (JEJM, right (or\nleft) inguinal region). Once optimized, 60s video clips of the region of\ninterest were saved using unique, randomly generated 4-digit codes,\npre-assigned for each examination. Random identification codes were\ngenerated using a standard computer spreadsheet software (Microsoft\nExcel, Version 2,560, 2025). De-identified clips were independently\nassessed as previously described (15) by three researchers with\nspecialist qualifications in diagnostic imaging (AC) or equine internal\nmedicine (FF, SLR). For DUOD recordings, researchers counted the\nnumber of concentric duodenal contractions. Motility was subjectively\ngraded in each acoustic window, as described previously (15). For\neach clip, results from the three independent evaluations were\ncombined by taking the mode response. Where discordant results\nwere recorded (defined as the number of DUOD contractions\ndiffering by >2 or grade assigned differing by >1), clips and comments\nwere reviewed to achieve a consensus result prior to decoding based\non consideration of the character of intestinal movement observed\nand careful differentiation of patient or probe movement from\nintestinal contraction.\n2.5 Cortisol determination\nSaliva samples were collected for cortisol determination the\nevening prior to departure (T-1), immediately prior to loading on the\nmorning of transport (T0) and within 2 h of arrival (between T1 and\nT2 examinations, subsequently deemed to be  T1). Sampling was\nperformed as previously described (29). Briefly, the horse’s head was\ncradled in one arm and a cotton gauze swab held in Doyen forceps was\npassed through the diastema between the incisors and premolar teeth.\nThe gauze was placed on the horse’s tongue, and the horse allowed to\n‘chew’ for 1 min. On removal from the mouth, the swab was placed in\na 20 mL syringe, and the plunger was used to compress the gauze and\n\nThe gauze was placed on the horse’s tongue, and the horse allowed to\n‘chew’ for 1 min.On removal from the mouth, the swab was placed in\na 20 mL syringe, and the plunger was used to compress the gauze and\nexpress saliva (typically ~1 mL) into duplicate Eppendorf containers.\nSamples were placed on ice and stored at -12°C for up to 24 h prior to\nstorage at -80°C. Saliva cortisol concentration was determined in\nduplicate using a commercial ELISA kit (Arbor Assay, MI). The limit\nof detection was 0.12 nmol/L, and intra-assay coefficient of variation\nwas 12%, calculated as SD/mean x 100 from all duplicate samples, as\npreviously described (30).\n2.6 Statistical methods\nThe 30 horses included in the current study were a convenience\nsample based on commercial movements. Power analysis based on\nprevious findings (15) suggested eight horses were sufficient to\ndiscriminate a mean difference of two duodenal contractions in the\n60s observation window with α = 0.05 and power of 0.9, assuming a\nstandard deviation of 1. Additional horses (n = 30) were recruited, as\navailable, to enable exploratory assessment of multiple explanatory\nvariables. An overview of explanatory and outcome variables is\nprovided in Supplementary Table S1.\nAll results were explored initially with descriptive statistics, and\ncontinuous data were tested for normality by the Shapiro-Wilks test.\nDescriptive data relating to subjective observations were grouped and\nanalyzed as categorical or ordinal variables (per data sheet,\nSupplementary Table S1) using non-parametric methods. The effect\nof transport on the number of duodenal contractions, the number of\nileocecal sounds, and salivary cortisol concentrations was assessed by\nlinear effects model and post-hoc differences were determined by\nTukey test. Categorical and non-normal continuous data resistant to\ntransformation were analyzed by Friedman’s test and subsequently by\nDunn’s test for pairwise multiple comparisons. In order to provide a\ncomplete data set for pre-transportation US assessment, motility\nresults from T-1 and T0 were combined such that ‘pre-transport’\n\nIn order to provide a\ncomplete data set for pre-transportation US assessment, motility\nresults from T-1 and T0 were combined such that ‘pre-transport’\nvalues (Tpre) were derived from the single available result, or from the\ngreater value obtained at T-1 or T0. Associations between outcome\nvariables (measures of intestinal motility and salivary cortisol\nconcentrations) and explanatory variables were explored by\nunivariable analyses: relationships between continuous explanatory\nand outcome variables were assessed by Pearson correlation if\nnormally distributed or by Spearman correlation; combinations of\ncontinuous and categorical variables were explored by Spearman\ncorrelation, with effect sizes characterized by unpaired t-test or\nanalysis of variance, or equivalent non-parametric test, as appropriate;\neffects of categorical variables on categorical outcome data were\nassessed by Spearman correlation.\n3 Results\n3.1 Transport details\nThe study was completed following horses on seven trips, four\nsouthbound and three northbound (Supplementary File 2). Travel\nduration ranged from 9:47 to 13:15 h, with distances covered\nbetween 703 and 1,108 km. Temperatures ranged from a minimum\nof 10.5°C prior to trip 7, to a maximum of 37.7°C enroute during\ntrip 8. Horses were not offered feed or water during transport, and\nwere restrained in single bays facing forwards or sideways. The\nmajority (n = 23) of horses loaded well, six required moderate\nencouragement to load, and one loaded poorly. Drivers reported at\nall horses traveled well, and each trip was completed without\nunexpected incident or delay.\n3.2 Animals\nThirty horses of mean age 8.4 years (range 18 months to 20 years)\nand mean weight 493 kg (range 230 to 814 kg) were recruited for the\ncurrent study. The study population comprised 17 geldings, one entire\nmale, and 12 females, ranging in body condition score from 2 to 5.\nThere were 13 Quarter Horses, six Thoroughbreds, two Standardbreds\nand nine other breeds (four ponies, three Warmbloods, one Australian\nStock Horse, and one Shire-cross).\n3.3 Veterinary examination\n\nThere were 13 Quarter Horses, six Thoroughbreds, two Standardbreds\nand nine other breeds (four ponies, three Warmbloods, one Australian\nStock Horse, and one Shire-cross).3.3 Veterinary examination\nPre-departure findings are summarized in Supplementary File 3.\nHorse and researcher availability were managed such that each horse\nwas examined on at least one occasion (T-1 and/or T0) prior to\ntransport. Consistent with inclusion criteria, no horse demonstrated\n\nRaidal et al.\b\n10.3389/fvets.2025.1647236\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nsubstantive abnormalities on clinical examination prior to\ntransportation at either timepoint.\nSweat scores were recorded immediately on off-loading; only\nhorses on trips 6 and 8 were visibly sweating (Figure 2), with four (of\nsix) and seven (of eight) horses scored at 1 or 2 on arrival. Veterinary\nexamination on arrival (T1) was completed for all 30 horses between\n10 min and 1:55 h after arrival. Minor increases in heart rate were\nnoted at T1 and persisted at T2 (Supplementary File 3). There were no\nabnormalities noted on cardiac auscultation for any horse. Jugular\nrefill and peripheral perfusion were considered normal in all horses.\nRectal temperature and respiratory rate increased after travel, but were\nnot\nsignificantly\ndifferent\nto\npre-transport\nvalues\n(Supplementary File 3). Gastrointestinal borborygmi included gas\nsounds on auscultation and/or percussion in four horses, and was\nsignificantly reduced in all four quadrants relative to pre-transport\nfindings (Supplementary File 3). Other minor abnormalities were\nnoted on examination of 12 horses (Supplementary File 3).\nVeterinary assessment or partial assessment was recorded for 23\nhorses at T2. Abnormal findings evident at T1 were largely resolved or\nunchanged (ie. none had progressed). Heart rate was still mildly elevated\nrelative to pre-transport values, and all but two horses had normal\nmucous membranes. Abnormalities were not appreciated on thoracic\nauscultation of any horse. Borborygmi remained decreased at T2 in the\nright\nventral\nquadrant\nand\nbased\non\ncomposite\nscore\n(Supplementary File 3). One horse still had gas on abdominal auscultation.\nThe majority (22 of 28 horses) were observed to eat readily on arrival and\nmost (22 of 28) drank readily after off-loading; observations were not\nrecorded for two horses. No adverse health outcomes were recorded by\nowners or reported to the transport company subsequent to horse\nmovements included in the current study.\n3.4 Sonographic assessment of intestinal\nmotility\nUltrasonographic assessment was possible for all 30 horses at T-1\n\n3.4 Sonographic assessment of intestinal\nmotility\nUltrasonographic assessment was possible for all 30 horses at T-1\nand / or at T0. Results are provided in greater detail in\nSupplementary File 4. Briefly, examinations were performed between\n3:40 and 10:45 pm (T-1), depending on arrival of horses into the\ndeparture depot, and prior to transportation (T0) between 1:28 and\n6:10 am. Where paired evaluations were completed, there was no\ndifference between results obtained at T-1 in comparison with results\nobtained at T0 for DUOD (p = 0.913, n = 23), CAEC (p = 0.965,\nn = 23), JEJM (p = 0.518, n = 22), or when all motility grades were\nsummed to give a composite score (p = 0.051 n = 22). Motility at T-1\nwas greater than that at T0 in COLN window (median difference 0.5,\n98.3%CI 1.0 to 0, p = 0.022). The time interval between feeding and\nsonographic assessment at T-1 or T0 had no effect on the number of\nduodenal contractions (p = 0.697) or DUOD grade (p = 0.748), CAEC\n(p = 0.973), COLN (p = 0.900), JEJM (p = 0.808), or on the composite\nmotility grade (p = 0.977).\nSonographic assessment was repeated between 2 min and 1:29 h\nafter off-loading horses at the arrival depot (T1), prior to horses being\nfed, and again at T2, 1:43 to 4:12 h after arrival. When compared to Tpre\nvalues, transportation was associated with decreased motility grades in\nDUOD (p < 0.001), CAEC (p = 0.024), COLN (p < 0.001) and JEJM\n(p = 0.004) windows, and in the composite motility score (p < 0.001), as\nshown in Figure 3. Results at T2 were equivalent to pre-transport values\nfor all windows except COLN and composite grades.\nQualitative differences were recognized in images obtained after\ntransportation, particularly in the DUOD and JEJM windows (Figure 4;\ncineloops are provided as Supplementary Files). Qualitative changes in\nduodenal function were recognized in one horse at T-1, six horses at T1,\nand one horse at T2. In each instance, evaluators recognized decreased\nor abnormal contractility and increased, typically anechoic, fluid content\nin the duodenal lumen. In one case gas shadowing was observed.\n\nIn each instance, evaluators recognized decreased\nor abnormal contractility and increased, typically anechoic, fluid content\nin the duodenal lumen.In one case gas shadowing was observed.\nQualitative changes in small intestinal function in the JEJM window\nwere recognized in 12 horses at T1, and were characterized as moderately\ndistended, fluid-filled loops with, variably, decreased motility,\npartitioning or sedimentation of content. Less pronounced changes\npersisted in two horses in T2 recordings.\n3.5 Factors associated with intestinal\nmotility after transport\nAmbient conditions on the day of transport were significantly\nassociated with sonographic measures of intestinal motility following\ntransportation (Table 1), with reduced cecal and composite motility\ngrades at T1 observed in association with high ambient temperatures\nduring the journey and on arrival. This effect was still present, in fact was\nmore pronounced, at T2. Heart rate on arrival (T1) was inversely\nFIGURE 2\nH29 on arrival (left) showing sweat score 2; she is also splinting her abdominal musculature (‘tucked up’), evidenced by muscular line extending from\nher flank toward her thorax (arrow). Sweat scores on arrival are shown (right) for all horses.\n\nRaidal et al.\b\n10.3389/fvets.2025.1647236\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nassociated with duodenal motility at this time, and changes identified on\narrival (T1) including heart rate, sweat score and demeanour (abnormally\nquiet or abnormally agitated), were associated with reduced duodenal\nand cecal motility. There was no direct and consistent association\nbetween auscultatory findings and sonographic motility grades but, at\nT1, auscultation of the left ventral abdomen was positively associated\nwith JEJM motility grade, and auscultation of the right ventral abdomen\nwas positively associated with COLN, JEJM and COMP grades. The\ncomposite auscultation grade was positively associated with JEJM and\nCOMP sonographic motility grades at this time. Horses that had spent\n>1 day at the transport depot were more likely to have decreased\nsonographic motility on arrival, and the amount of food consumed the\nnight prior to departure was positively associated with the.\n3.6 COMP motility grade on arrival\nSonographic findings at T1 or T2 were not readily associated with\nappetite nor fecal output on arrival. Sonographic measures of motility\nprior to transportation (T-1 or T0) were not associated with results\nfollowing transportation, nor was motility at T1 associated with\nmotility at T2. Signalment factors including use, level of exercise and\npast travel history were not associated with sonographic motility at\nany time during the current study.\n3.7 Cortisol response to transportation\nTransportation was associated with increased median salivary\ncortisol concentrations in samples collected between 40 min and\n2:48 h after transportation (T1, Figure 5), relative to values obtained\nprior to transportation (T0). Factors associated with salivary cortisol\nconcentrations at T-1 and T1 are shown in Table 2. None of the\nfactors evaluated was associated with salivary cortisol concentrations\nat T0. Horses with no previous experience of travel, or with unknown\ntravel histories, had significantly higher cortisol concentrations at T-1\nthan was observed for horses that had traveled occasionally or\n\nHorses with no previous experience of travel, or with unknown\ntravel histories, had significantly higher cortisol concentrations at T-1\nthan was observed for horses that had traveled occasionally or\nfrequently. Higher temperatures during travel (waypoint temperature)\nFIGURE 3\nEffects of transportation on sonographic assessments of intestinal motility. The number of duodenal contractions (count) is shown as median\n(horizontal line), mean (+), quartiles (box), range (whiskers), with all data points shown; motility grades (ordinal data) are shown as violin plots with\nmedian (red line) and quartiles (dotted lines) indicated. As graded data were ordinal variables and DUOD count data were nonparametric, all have been\nanalyzed by Friedman test with post-hoc comparisons determined using Dunn’s method. Significant effects were observed for DUOD (p = 0.001),\nCAEC (p = 0.026), COLN (p < 0.001), JEJM (p = 0.005) and the composite motility score (p < 0.001), but not for duodenal contractions (p = 0.257);\nsignificant pairwise comparisons are shown. Results prior to transportation at T-1 and T0 are shown, but not included in analyses. DUOD = nephro-\nduodenal window, CAEC = caecal window, COLN = ventral colon window, JEJM = jejunal (inguinal) window; # = number.\n\nRaidal et al.\b\n10.3389/fvets.2025.1647236\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nand on arrival, and lower humidity at waypoint and on arrival, were\nassociated with higher cortisol concentrations at T1. Cortisol at T1\nwas significantly and inversely associated with distance, duration of\ntravel and arrival time. Salivary cortisol concentrations increased as\nthe interval between arrival and sampling increased, and a similar\neffect was observed for time of sampling. Cortisol responses varied\nwith trip, with horses on trip 8 showing greater cortisol responses at\nall sampling times.\n4 Discussion\nThis study demonstrated decreased intestinal motility associated\nwith road transportation, and prospectively evaluated environmental\nand horse factors that might be associated with the magnitude of\nobserved change. Using four acoustic windows on the right side of the\nhorse, sonographic measures of intestinal motility were reduced after\ntransportation in the duodenal (DUOD), jejunal (JEJM), and colon\n(COLN) windows, and when all scores were combined (COMP).\nHorses in the current study demonstrated no clinically apparent\nadverse effects and recovered quickly after transportation, but\nsonographic assessment also identified qualitative changes consistent\nwith small intestinal dysmotility. As part of veterinary assessment of\ntransported horses, such changes might be monitored on arrival and\ninform management or husbandry decisions, such as ongoing\nmonitoring, feeding, or performance expectations. These changes\nwere easily recognized and were quite different to normal motility\npatterns. Taken together with previous studies that reported decreased\ngastric emptying and reflux of duodenal content into the stomach in\ntransported horses (10), these findings suggest that transportation\nmight be  associated with loss of normal aboral movement of\nintestinal contents.\nIn the current study, ambient temperature likely exacerbated the\nobserved effect on motility. Heat stress during transportation has\nbeen recognized as a welfare issue for horses (31), and heat stroke\nhas been identified in owner surveys of health problems associated\n\nHeat stress during transportation has\nbeen recognized as a welfare issue for horses (31), and heat stroke\nhas been identified in owner surveys of health problems associated\nwith transportation in this species (32). Variations in hormonal and\nautonomic regulation during road transport of horses in tropical\nconditions have been linked to ambient conditions (33) but, to the\nauthors’ knowledge, this is the first study to demonstrate an\nassociation between ambient temperature and intestinal function in\ntransported horses. On arrival, an association was evident between\nreduced motility and increased sweat score, abnormal demeanour\nand increased heart rate, suggesting that these factors, evident on\nphysical examination, might predict horses at increased risk of\nadverse gastrointestinal sequelae to transportation. The inverse\nrelationship observed between heart rate and motility is consistent\nwith a switch to sympathetic dominance as part of the sympathetic-\nadrenal medullary stress response in anticipation of or response to\ntransportation and off-loading to a novel environment. Sweat score\nand demeanour on arrival were also associated with reduced motility\nat T2, suggesting that visibly affected horses might fail to recover as\nwell as other horses following transport. Sonographic evaluation of\nintestinal motility might provide complementary information to that\nobtained from auscultation. The grade of intestinal sounds\n(borborygmi) on abdominal auscultation was positively associated\nwith some sonographic measures of intestinal motility, suggesting\nthat the reciprocal finding (decreased gastrointestinal sounds) might\npredict decreased motility, although sonographic and audible\nmeasures of motility were not strongly associated at any time point\nFIGURE 4\nRepresentative jejunal (top row) and duodenal (bottom) images showing qualitative abnormalities recognized after transportation; all images were\nobtained at T1. For jejunal images, ventral is to the top of the image; for duodenal images, cranial is to the left of the image. (a) H16 – jejunal loops are\n\nFor jejunal images, ventral is to the top of the image; for duodenal images, cranial is to the left of the image.(a) H16 – jejunal loops are\nmore prominent and there is increased anechoic content with partitioning of content (arrow), there is normal to increased motility evident in cineloop\n(Supplementary material); (b) H12 – as for recording 4725, sedimentation is more obvious (arrow); (c) H23 – mildly distended jejunal loops are evident\n(arrows) with anechoic content; motility is normal to slightly decreased (Supplementary material); (d) H2 – duodenal motility is reduced, duodenum\n(arrow) remains slightly distended, with anechoic content; (e) H12 – there is fluid content in a dilated duodenum and a second viscus noted more\nsuperficially and ventrally (arrowhead) containing more echogenic fluid; the right kidney is more evident to the left (dorsal) of frame in this image; (f)\nH16 – there is anechoic fluid in a moderately distended duodenum (arrow).\n\nRaidal et al.\b\n10.3389/fvets.2025.1647236\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nor, anecdotally, when researchers were able to perform both\nassessments concurrently. Similar observations have been made in\nhuman studies, where sonographic evaluation has been interpreted\nas superior to auscultation for assessment of intestinal function in\npeople (34, 35).\nHorses that had been at the transport depot for a protracted\ntime, or that ate little overnight prior to transport, were more likely\nto have decreased intestinal motility on sonographic evaluation at\noff-loading. However, to the extent evaluated in the current study,\nfactors identified prior to transportation, including prior transport\nTABLE 1  Factors significantly associated with sonographic motility after off-loading from transport (T1) and on recovery (T2, 2 h after arrival).\nOutcome variable\nExplanatory factor(s)\nSignificance\nP=\nEffect size\nT-1 Intestinal motility\nDUOD count\nT-1 [cortisol]\n0.018\nrs = −0.469\nDUOD grade\nT-1 [cortisol]\n0.008\nrs = −0.336\nCOLN grade\nT-1 [cortisol]\n0.046\nrs = −0.402\nCOMP grade\nT-1 [cortisol]\n0.018\nrs = −0.468\nT1 Intestinal motility\nDUOD count\nHR at T1\n0.021\nr = −0.419\nDUOD grade\nHR at T1\n0.004\nrs = −0.506\nCAEC grade\nWaypt temp\n0.018\nrs = −0.435\nArrival temp\n0.048\nrs = −0.371\nTime at depot prior to travel\n0.016\nDepot ≤1d 2 (1–3),\ndepot >1d 1 (1–1)*\nCOLN grade\nRV ausc at T1\n0.012\nrs = 0.261\nJEJM grade\nLV ausc at T1\n0.030\nrs = 0.226\nRV ausc at T1\n0.019\nrs = 0.245\nCOMP ausc at T1\n0.011\nrs = 0.265\nCOMP grade\nArrival temp\n0.017\nrs = −0.448\nRV ausc at T1\n0.001\nrs = 0.329\nCOMP ausc at T1\n0.014\nrs = 0.256\nTime at depot prior to travel\n0.008\nDepot ≤1d 7 (6–9),\ndepot >1d 4 (4–6.75)\nFeed overnight prior to travel\n0.050\nAte all 7 (5–9), ate ~½ 6 (4–11), ate <½ 4 (3–4)*\nT2 Intestinal motility\nDUOD count\nSweat score at T1\n0.068\nrs = −0.350\nDemeanour at T1\n0.020\nNormal 3.88 (3.11–4.65), abnormal 2.54 (1.73–\n3.36)**\nCAEC grade\nWaypt temp\n<0.001\nrs = −0.782\nArrival temp\n<0.001\nrs = −0.740\nWaypt humidity\n0.002\nrs = 0.630\nArrival humidity\n<0.001\nrs = 0.566\nSweat score at T1\n0.001\nrs = −0.579\nDemeanour at T1\n0.004\nNormal 2 (1.5–2),\nabnormal 1 (1–1)*\nT1 [cortisol]\n0.007\nrs = −0.497\nJEJM grade\nT0 [cortisol]\n0.041\nrs = −0.439\nCOMP grade\nSweat score at T1\n0.013\nrs = −0.462\n\n002\nrs = 0.630\nArrival humidity\n<0.001\nrs = 0.566\nSweat score at T1\n0.001\nrs = −0.579\nDemeanour at T1\n0.004\nNormal 2 (1.5–2),\nabnormal 1 (1–1)*\nT1 [cortisol]\n0.007\nrs = −0.497\nJEJM grade\nT0 [cortisol]\n0.041\nrs = −0.439\nCOMP grade\nSweat score at T1\n0.013\nrs = −0.462\n*Median (IQR); **Mean (95% CI). DUOD, duodenum; CAEC, caecum; COLN, ventral colon; JEJM, jejunum; COMP grade, composite (summed) motility score; waypt temp, waypoint\ntemperature (weather data from mid-point of trip); arrival temp, ambient temperate on arrival; ausc, auscultation; HR, heart rate; RV, right ventral abdomen; LV, left ventral abdomen; COMP,\ncomposite auscultation score; composite (sum of motility scores); IQR, interquartile range (non-parametric data); 95% CI, 95% confidence interval (parametric data). Interactions between\ncontinuous explanatory data with continuous outcomes data were explored by Pearson (parametric) or Spearman (non-parametric) correlation; effects of categorical explanatory factors on\ncontinuous data were explored by analysis of variance or t-test (or non-parametric equivalent, as appropriate), and effects of categorical explanatory data on categorical outcomes were\nevaluated using Spearman correlation.\n\nRaidal et al.\b\n10.3389/fvets.2025.1647236\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nhistory and signalment factors such as breed, size or intended use,\nwere not predictive of alterations in intestinal motility, before or\nafter the journey. Prior to travel (T-1, T0), there was no clear\nassociation between intestinal motility and other observations, such\nas feed consumption, water intake, fecal output or physical exam\nfindings. Despite the absence of external evidence of physiologic\n‘stress’ response, sonographic measures of intestinal motility at T-1\nwere inversely associated with salivary cortisol concentrations,\nsuggesting that intestinal function was adversely affected by the\nhypothalamic–pituitary stress response. In the time frame available\nfor assessment in the current study, sonographic results at off-loading\nor 2 h after arrival were not predictive of food intake or fecal output\nfollowing transport, and were only weakly associated with\ncortisol responses.\nCortisol concentrations in saliva samples obtained at all time\npoints in the current study were well above those reported in resting\nhorses (29), and consistent with previous studies on transported\nhorses. Values obtained at T-1 and T1 were increased relative to data\nfrom T0, demonstrating reversal of the expected diurnal variation\n[values should be highest in the morning (29)]. Horses with prior\ntravel experience had slightly lower cortisol values at T-1 than was\nobserved for horses that had not traveled before or where travel history\nwas unknown, suggesting that prior experience mitigated the stress\nresponse associated with travel. Hot weather was associated with the\nhighest cortisol values, but no consistent association was identified\nwith signalment, body size, or factors evident on clinical examination.\nSurprisingly, cortisol concentrations increased as the time interval\nbetween arrival and sampling increased, and with samples derived later\nin the day. While these findings might be confounded by other factors,\nin the current study, horses were off-loaded into an environment with\n\nWhile these findings might be confounded by other factors,\nin the current study, horses were off-loaded into an environment with\nwhich they had no prior experience. By following horses in a\ncommercial transport environment, the current study differs from\nprevious results evaluating horses transported from and returning to\nfamiliar environments (29, 36, 37), and might suggest that, rather than\nameliorating endocrine responses to transportation, arrival into a\nnovel environment may be an additional stressor for horses.\nSonographic assessment of intestinal motility was readily\ncompleted in a field setting with portable hand-held ultrasound\ntransducers. Horses in the current study spanned a range of handling\nexperience and temperaments but readily accepted the procedure with\nminimal restraint. Sonographic evaluation proved effective to further\ncharacterize horse health beyond what is possible with veterinary\nexamination alone, demonstrating health conditions that might have\ninfluenced wellbeing during transport (pregnancy, abnormal\nabdominal fluid, as documented in Supplementary File 4). Although\nnot pursued in the current study, ultrasonographic evaluation might\ninclude assessment of gastric size and content (15, 17) or pulmonary\nchanges (38).\nFIGURE 5\nSalivary cortisol concentrations the evening prior to travel (T-1),\nimmediately prior to loading (T0) and on arrival (T1). Data are shown\nas median (horizontal line), quartiles (boxes) and range (whiskers),\nwith all data points shown. Data were non-normal and transformed\nfor analysis by linear effects model. A significant time effect is evident\n(p < 0.001), with results of post-hoc pairwise comparisons (Tukey\ntest) shown.\nTABLE 2  Factors significantly associated with saliva cortisol\nconcentrations the night prior to transportation (T-1) and after transport\n(T1).\nOutcome\nvariable\nExplanatory\nfactor(s)\nSignificance\nP=\nEffect size\nT-1 Cortisol concentration\nTravel history\n0.029\nNil/unknown\n12.8 (8.2–17.4),\nocc/freq 7.9\n(5.8–9.9)\nnmol/L**\nT1 Cortisol concentration\nWaypoint\ntemperature\n<0.001\nrs = 0.572\nArrival temperature\n0.002\nrs = 0.535\nWaypoint humidity\n<0.001\nrs = −0.585\nArrival humidity\n0.001\nrs = −0.569\n\n029\nNil/unknown\n12.8 (8.2–17.4),\nocc/freq 7.9\n(5.8–9.9)\nnmol/L**\nT1 Cortisol concentration\nWaypoint\ntemperature\n<0.001\nrs = 0.572\nArrival temperature\n0.002\nrs = 0.535\nWaypoint humidity\n<0.001\nrs = −0.585\nArrival humidity\n0.001\nrs = −0.569\nDistance traveled\n0.002\nrs = −0.533\nDuration of travel\n0.004\nrs = −0.514\nTime of arrival\n0.011\nrs = −0.457\nTime of sampling\n0.018\nrs = −0.428\nInterval btw arrival\n& sampling\n0.002\nrs = 0.535\nTrip\n<0.001\nTrip 8 16.2\n(13.0–19.0),\nother trips 5.8\n(4.5–9.5)\nnmol/L*\nSweat score\n<0.001\nrs = 0.653\nAbnormal\ndemeanour\n0.005\nrs = 0.497\n*Median (IQR); **Mean (95% CI). occ, occasional; freq, frequent; btw, between; IQR,\ninterquartile range (non-parametric data); 95% CI, 95% confidence interval (parametric\ndata). Interactions between continuous explanatory data with continuous outcomes data\nwere explored by Pearson (parametric) or Spearman (non-parametric) correlation; effects of\ncategorical explanatory factors on continuous data were explored by analysis of variance or\nt-test (or non-parametric equivalent, as appropriate), and effects of categorical explanatory\ndata on categorical outcomes were evaluated using Spearman correlation. No significant\ninteractions were observed for salivary cortisol concentrations immediately prior to\ndeparture (T0).\n\nRaidal et al.\b\n10.3389/fvets.2025.1647236\nFrontiers in Veterinary Science\n10\nfrontiersin.org\n4.1 Limitations\nAs might be expected in commercial settings, trips included in the\ncurrent study were opportunistic, of slightly different durations, and\ndifficult to schedule. In some weeks an unseasonably low number of\nhorses was booked for travel, meaning that some journeys were canceled\ndue to insufficient numbers of horses meeting inclusion criteria and with\nowner permission for participation in the study. As these were\ncommercial horse movements, researchers did not influence\nmanagement practices of horses prior to departure, during transportation\nor on arrival. To facilitate rapid acquisition of ultrasonographic images\nand veterinary examination data immediately on arrival, sonographic\nassessment was restricted to the right side of the horse. Data analysis\nincluded available results at T-1 and T0 but, in some circumstances,\nhorses were not available for assessment at both time points. To enable\nrepeated measures analyses without missing data points, results at T-1\nand T0 were pooled. Although the validity of data handling in this way\nmight be  contested, the combined pre-transport (Tpre) result was\nconsidered to reflect the best available measure of predictive variables,\nand direct comparison of available results suggested there was no\nsignificant difference between values obtained at T-1 and T0. However,\ndiurnal variation and the effect of management practices, particularly the\ntime since feeding, might be expected to influence values of interest,\nincluding intestinal motility. The failure to identify any such effect might\nsuggest that factors in the transport environment influenced normal\nhomeostatic mechanisms.\nVeterinary examination at T1 was completed between 10 min and\n1:55 h after arrival, and sonographic assessment was completed between\n2 min and 1:29 h after arrival. Due to logistic constraints in a commercial\nsetting, these were much wider windows than was planned and this\nallowed variable recovery prior to examination for horses. Continuous\n\nDue to logistic constraints in a commercial\nsetting, these were much wider windows than was planned and this\nallowed variable recovery prior to examination for horses.Continuous\ndata recoding with ‘smart textiles’, telemetry ECG or electronic sensors\n(14) would be a pragmatic response to this challenge in future studies.\nHowever, evaluation of horses at T2 yielded more significant differences\nand greater effect size than was apparent for T1 data, suggesting that\nabnormalities persisting in horses after transportation and off-loading\nmight be of more value than their immediate responses.\nThe apparent lack of effect on intestinal motility of explanatory\nfactors such as travel history, signalment, management and physical\nexamination findings prior to transportation should be  interpreted\ncautiously given incomplete data collection, the relatively small number\nand the heterogenous population of horses included in the current study.\nBehavior during transport was based on driver anamnesis rather than\ndirect observation. Given these limitations, multivariate analysis was\ndeemed inappropriate and some apparent associations might reflect\ninteractions between factors that could not be reliably evaluated. For\nexample, counter-intuitive associations between relative humidity and\njourney duration might be confounded by the strong observed effect\nassociated with ambient temperature. Horses in the current population\nwere well handled, loaded easily and traveled well. Our findings,\ntherefore, may not be transferrable to less well-handled horses. Finally,\nas an observational study of commercial horse movements, the current\nstudy could not evaluate the effects of provision of feed or water during\nthe journey, and we were unable to include a control group of horses\nfasted for an equivalent period of time without transportation. In a\ncommercial transport setting, we were unable to collect reliable measures\nof urine volume or fecal output and consistency. Future studies might\ninclude more rigorous evaluation of fecal output and urine production\n\nIn a\ncommercial transport setting, we were unable to collect reliable measures\nof urine volume or fecal output and consistency.Future studies might\ninclude more rigorous evaluation of fecal output and urine production\nas measures of physiologic response to transport conditions.\n5 Conclusion\nIn the current study, transport without provision of food or water\nwas associated with quantitative and qualitative adverse effects on\nintestinal motility, confirming previous observations relating to gastric\nemptying (10). High ambient temperatures potentiated this effect and\nwere also associated with increased salivary cortisol concentrations.\nUntil we have further evidence upon which to base feeding practices\nduring and subsequent to transportation, horses should be provided\nwith food and water on arrival and be  evaluated carefully after\ntransport, as some characteristics readily identifiable on arrival (sweat\nscore, demeanour and heart rate) might identify horses at increased\nrisk for decreased intestinal motility or delayed recovery. Other\nabnormalities evident on physical examination in the current study\nwere relatively uncommon, of minor severity and variably involved the\ngastrointestinal tract, respiratory tract or minor injury to limbs or body\nsurfaces. Future studies should ascertain whether the provision of food\nand/or water during travel might ameliorate the detrimental effects on\nintestinal motility observed in the current study or, conversely, whether\nfasting during transportation might protect horses from colic\nsubsequent to the observed motility disorders. Conversely, ingestion of\nfeed, particularly in the absence of adequate water intake during travel,\nmight predispose to impaction or colic if the observed changes are a\nresponse to the transport environment. The experimental methodology\nused in the current study is well suited to addressing research questions\ndesigned to further characterize gastrointestinal responses\nto transportation.\nData availability statement\nThe original contributions presented in the study are included in\n\nData availability statement\nThe original contributions presented in the study are included in\nthe article/Supplementary material, further inquiries can be directed\nto the corresponding author/s.\nEthics statement\nThis study was reviewed and approved by the Animal Care and\nEthics Committee at Charles Sturt University (A21415). The studies\nwere conducted in accordance with the local legislation and\ninstitutional requirements. Written informed consent was obtained\nfrom the owners or their agent for the participation of their animals\nin this study. Written informed consent was obtained from the\nindividual(s) for the publication of any potentially identifiable images\nor data included in this article.\nAuthor contributions\nSR: Conceptualization, Data curation, Formal analysis, Funding\nacquisition, Investigation, Methodology, Project administration,\nResources, Software, Validation, Visualization, Writing – original draft,\nWriting – review & editing. FF: Funding acquisition, Investigation,\nMethodology, Resources, Validation, Visualization, Writing – original\ndraft, Writing  – review & editing. AC: Conceptualization, Data\ncuration, Formal analysis, Funding acquisition, Investigation,\nMethodology, Resources, Validation, Visualization, Writing – original\ndraft, Writing – review & editing. SW: Data curation, Formal analysis,\n\nRaidal et al.\b\n10.3389/fvets.2025.1647236\nFrontiers in Veterinary Science\n11\nfrontiersin.org\nInvestigation, Methodology, Resources, Validation, Writing – original\ndraft, Writing  – review & editing. BP: Conceptualization, Data\ncuration, Formal analysis, Funding acquisition, Investigation,\nMethodology, Resources, Validation, Visualization, Writing – original\ndraft, Writing – review & editing.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. This study was funded by\nthe Morris Animal Foundation (grant no. D22EQ-025).\nAcknowledgments\nThe support of Katrina O’Brien, Jack Holstein and all the drivers\nand horse carers from Ultimate Horse Transport is gratefully\nacknowledged. The assistance and interest of CSU students Matthew\nDuke, Natalie Jefferson, Emma McTavish, Jakob Motha, and Tayla\nPrice are gratefully acknowledged.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nThe author(s) declared that they were an editorial board member\nof Frontiers, at the time of submission. This had no impact on the peer\nreview process and the final decision.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. 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(2023) 13:9858. doi: 10.1038/s41598-023-37069-1\n\nRaidal et al.\b\n10.3389/fvets.2025.1647236\nFrontiers in Veterinary Science\n12\nfrontiersin.org\n23.\tFazio E, Ferlazzo A. Evaluation of stress during transport. Vet Res Commun. (2003)\n27:519–24. doi: 10.1023/b:verc.0000014211.87613.d9\n24.\tSmith BL, Jones JH, Hornof WJ, Miles JA, Longworth KE, Willits NH. Effects of\nroad transport on indices of stress in horses. Equine Vet J. (1996) 28:446–54. doi:\n10.1111/j.2042-3306.1996.tb01616.x\n25.\tStull CL, Morrow J, Aldridge BA, Stott JL, McGlone JJ. Immunophysiological\nresponses of horses to a 12-hour rest during 24 hours of road transport. Vet Rec. (2008)\n162:609–14. doi: 10.1136/vr.162.19.609\n26.\tStull CL, Rodiek AV. Physiological responses of horses to 24 hours of transportation\nusing a commercial van during summer conditions. J Anim Sci. (2000) 78:1458–66. doi:\n10.2527/2000.7861458x\n27.\tZeyner A, Romanowski K, Vernunft A, Harris P, Kienzle E. Scoring of sweat losses\nin exercised horses--a pilot study. J Anim Physiol Anim Nutr. (2014) 98:246–50. doi:\n10.1111/jpn.12073\n28.\tSanchez LC. Disorders of the gastrointestinal system In: SM Reed, WM Bayly and\nDC Sellon, editors. Equine Internal Medicine. 4th ed. Philadelphia, PA: W.B. Saunders\n(2018). 709–842.\n29.\tBohák Z, Szabó F, Beckers JF, Melo de Sousa N, Kutasi O, Nagy K, et al. Monitoring\nthe circadian rhythm of serum and salivary cortisol concentrations in the horse. Domest\nAnim Endocrinol. (2013) 45:38–42. doi: 10.1016/j.domaniend.2013.04.001\n30.\tGardela J, Carbajal A, Tallo-Parra O, Olvera-Maneu S, Álvarez-Rodríguez M, Jose-\nCunilleras E, et al. Temporary relocation during rest periods: relocation stress and other\nfactors influence hair cortisol concentrations in horses. Animals. (2020) 10:642. doi:\n10.3390/ani10040642\n31.\tNielsen SS, Alvarez J, Bicout DJ, Calistri P, Canali E, Drewe JA, et al. Welfare of\nequidae during transport. EFSA J. (2022) 20:e07444. doi: 10.2903/j.efsa.2022.7444\n32.\tPadalino B, Raidal SL, Hall E, Knight P, Celi P, Jeffcott L, et al. Risk factors in\nequine transport-related health problems: a survey of the Australian equine industry.\nEquine Vet J. (2017) 49:507–11. doi: 10.1111/evj.12631\n\n(2022) 20:e07444.doi: 10.2903/j.efsa.2022.7444\n32.Padalino B, Raidal SL, Hall E, Knight P, Celi P, Jeffcott L, et al.Risk factors in\nequine transport-related health problems: a survey of the Australian equine industry.Equine Vet J.(2017) 49:507–11.doi: 10.1111/evj.12631\n33.\tLertratanachai S, Poochipakorn C, Sanigavatee K, Huangsaksri O, Wonghanchao\nT, Charoenchanikran P, et al. Cortisol levels, heart rate, and autonomic responses in\nhorses during repeated road transport with differently conditioned trucks in a tropical\nenvironment. PLoS One. (2024) 19:e0301885. doi: 10.1371/journal.pone.0301885\n34.\tDrake A, Franklin N, Schrock JW, Jones RA. Auscultation of bowel sounds and\nultrasound of peristalsis are neither compartmentalized nor correlated. Cureus. (2021)\n13:e14982. doi: 10.7759/cureus.14982\n35.\tFelder S, Margel D, Murrell Z, Fleshner P. Usefulness of bowel sound\nauscultation: a prospective evaluation. J Surg Educ. (2014) 71:768–73. doi:\n10.1016/j.jsurg.2014.02.003\n36.\tPadalino B, Raidal SL, Knight P, Celi P, Jeffcott L, Muscatello G. Behaviour during\ntransportation predicts stress response and lower airway contamination in horses. PLoS\nOne. (2018) 13:e0194272. doi: 10.1371/journal.pone.0194272\n37.\tTateo A, Padalino B, Boccaccio M, Maggiolino A, Centoducati P. Transport stress\nin horses: effects of two different distances. J Vet Behav. (2012) 7:33–42. doi:\n10.1016/j.jveb.2011.04.007\n38.\tRibonnet C, Palmers K, Saegerman C, Vanderperren K, van Galen G. Perioperative\nlung ultrasonography in healthy horses undergoing general anesthesia for elective\nsurgery. J Vet Intern Med. 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+{"text": "【兽医文献】\n标题: Editorial: Case reports in veterinary neurology and neurosurgery\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nEpidemiological analysis of\nporcine reproductive and\nrespiratory syndrome viruses in\n2020–2023 in China and the\nimpact of serum acclimatization\non production performance of\nsows farm\nShuangxi Li 1,2 and Jinlian Hua 1,2*\n1 College of Veterinary Medicine, Northwest A&F University, Yangling, China, 2 Shaanxi Centre of Stem\nCells Engineering & Technology, Yangling, China\nPorcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus\n(PRRSV), leads to severe economic losses in the pig farming industry. Currently,\nthere is no specific treatment for PRRSV. This study investigated the epidemiological\ncharacteristics of PRRSV in large-scale pig farms across 24 provinces and municipalities\nin China from 2020 to 2023. Subsequently, serum acclimatization of gilts was\nimplemented in large-scale pig farms to evaluate its impact on the reproductive\nperformance of sows and the growth performance of piglets. The results showed\nthat 14 provinces had PRRSV-positive rates exceeding 10%, with Yunnan Province\nreporting the highest rate at 23.5%, whereas Zhejiang had the lowest at 3.5%.\nThe PRRSV-positive rate exhibited clear seasonality, with markedly higher rates\nin winter and spring compared to summer and autumn. The introduction of gilts\nwas identified as the most important cause of PRRS outbreaks, with an incidence\nrate as high as 49%. Following serum acclimatization, the average total number of\npiglets per litter, the average number of live piglets per litter, the number of piglets\nweaned per litter, and the total weaning weight per litter were all significantly\nhigher than those before acclimatization and in unacclimated sows (p < 0.05).\nThe results indicated that serum acclimatization could effectively improve the\nreproductive performance of sows and the growth performance of piglets. This\nstudy provides valuable data for understanding the epidemiology of PRRSV and\nsupports serum acclimatization as a potential strategy for PRRSV prevention and\ncontrol in China.\nKEYWORDS\nPRRSV, prevalence, time of infection, serum acclimatization, reproductive\n\nThis\nstudy provides valuable data for understanding the epidemiology of PRRSV and\nsupports serum acclimatization as a potential strategy for PRRSV prevention and\ncontrol in China.KEYWORDS\nPRRSV, prevalence, time of infection, serum acclimatization, reproductive\nperformance, production performance\n1 Introduction\nPorcine reproductive and respiratory syndrome (PRRS), commonly referred to as blue ear\ndisease in pigs, is a highly contagious and lethal disease caused by the PRRS virus (PRRSV),\nposing a serious threat to the swine industry (1, 2). It is also a typical immunosuppressive\ndisease, often leading to severe immune suppression, including the inhibition of antibody\nproduction (3, 4). Additionally, PRRSV frequently co-infects with bacteria, parasites, and other\nOPEN ACCESS\nEDITED BY\nMengmeng Zhao,\nFoshan University, China\nREVIEWED BY\nZhendong Zhang,\nYangzhou University, China\nHongliang Zhang,\nChinese Academy of Agricultural Sciences,\nChina\n*CORRESPONDENCE\nJinlian Hua\njinlianhua@nwsuaf.edu.cn\nRECEIVED 18 April 2025\nACCEPTED 02 June 2025\nPUBLISHED 23 June 2025\nCITATION\nLi S and Hua J (2025) Epidemiological analysis\nof porcine reproductive and respiratory\nsyndrome viruses in 2020–2023 in China and\nthe impact of serum acclimatization on\nproduction performance of sows farm.\nFront. Vet. Sci. 12:1614039.\ndoi: 10.3389/fvets.2025.1614039\nCOPYRIGHT\n© 2025 Li and Hua. This is an open-access\narticle distributed under the terms of the\nCreative Commons Attribution License\n(CC BY). The use, distribution or reproduction\nin other forums is permitted, provided the\noriginal author(s) and the copyright owner(s)\nare credited and that the original publication\nin this journal is cited, in accordance with\naccepted academic practice. No use,\ndistribution or reproduction is permitted\nwhich does not comply with these terms.\nTYPE  Original Research\nPUBLISHED  23 June 2025\nDOI  10.3389/fvets.2025.1614039\n\nLi and Hua\b\n10.3389/fvets.2025.1614039\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nviruses, further complicating prevention and control efforts. Currently,\neffective control of this disease remains elusive. PRRS causes\nreproductive disorders in pregnant sows and respiratory symptoms in\npigs of all ages (5, 6). Infected pregnant sows may experience\ndecreased farrowing rates, with abortion, stillbirth, mummification,\nand weak piglets affecting 30 to 100% of cases. Infected fattening pigs\noften exhibit poor appetite, dyspnea, multiple secondary infections,\nslow growth, and reduced feed efficiency (7).\nPRRSV, a member of the genus Betaarterivirus, has spherical or\novoid, enveloped virions. These virions measure 45–80 nm in\ndiameter and contain an icosahedral nucleocapsid structure. The\nPRRSV genome is approximately 15.4 kb in length (8). PRRSV is\ndivided into two gene types: the European type (PRRSV-1),\nrepresented by the Lelystad virus (LV strain), and the North American\ntype (PRRSV-2), represented by the VR-2332 strain (9, 10).\nThe primary strategy for preventing PRRS currently relies on\nvaccination, with a vaccination rate of 70% in pig farms across China.\nThe effectiveness and safety of vaccines are crucial criteria in their\ndevelopment and deployment. Presently, PRRS attenuated live\nvaccines are highly effective (11); however, they carry a potential risk\nof pathogenicity. In contrast, PRRS inactivated vaccines offer sufficient\nsafety but exhibit poor immunogenicity. They are effective primarily\nagainst homologous strains, offering inadequate protection against\nheterologous strains and lacking cross-protection capabilities.\nConsequently, their overall effectiveness remains unsatisfactory (4,\n7, 12–14).\nSerum acclimatization is a technique used to protect pigs by\ndeliberately controlling the timing of herd infections and\nadministering serum or tissues containing low-dose viruses. In recent\nyears, PRRS serum acclimatization has become a widely adopted and\neffective method for preventing and controlling PRRS in large-scale\npig farms. This approach has gained increasing support from pig farm\n\nIn recent\nyears, PRRS serum acclimatization has become a widely adopted and\neffective method for preventing and controlling PRRS in large-scale\npig farms.This approach has gained increasing support from pig farm\nmanagers and veterinarians (15). The process involves collecting\npositive serum from specific serotypes of PRRSV strains, diluting it in\nappropriate proportions, and inoculating the entire pig herd with the\nserum. This induces a high level of uniform antibodies, thereby\nblocking virus excretion and protecting uninfected pigs, ultimately\ncontrolling PRRSV within the farm (16). The advantages of serum\nacclimatization include the ability to manage the infection timing,\nachieve uniform antibody levels, maintain herd consistency, and\npotentially enhance the reproductive and production performance of\nsows in affected farms.\nDuring this experiment, 28,054 samples were collected from\npig farms across 24 provinces and municipalities in China,\nspanning January 2020 to June 2023. The objective was to\ninvestigate the epidemiological characteristics of PRRSV in China.\nSubsequently, in large-scale pig farms located in Xuwen County,\nGuangdong Province, serum acclimatization was applied to gilts.\nThe reproductive performance of sows and the production\nperformance of piglets were then measured. The aim was to\ndetermine whether serum acclimatization could enhance the\nreproductive performance of sows and the production\nperformance of piglets. This study provides new data and\nexperiential references for the prevention and control of PRRSV\nin large-scale pig farms, offering new directions and ideas for\nPRRSV management.\n2 Materials and methods\n2.1 Main reagents and instruments\nThe magnetic bead method virus DNA/RNA extraction kit was\nobtained from Hangzhou Bioer Technology Co., Ltd. (Hangzhou,\nChina). The PRRSV RT-qPCR detection kit was acquired from\nThermo Fisher Scientific Co., Ltd. (Shanghai, China). Compound\namoxicillin powder (no. 110802092) was sourced from Zhejiang\nHisun Animal Healthcare Products Co., Ltd. (Hangzhou, China).\nThe fully automated nucleic acid extraction and purification\n\n, Ltd.(Shanghai, China).Compound\namoxicillin powder (no.110802092) was sourced from Zhejiang\nHisun Animal Healthcare Products Co., Ltd.(Hangzhou, China).The fully automated nucleic acid extraction and purification\ninstrument, NPA-32P, was obtained from Hangzhou Bioer Technology\nCo., Ltd. (Hangzhou, China). The nucleic acid concentration was\ndetermined using the NanoDrop One instrument from Thermo Fisher\nScientific Co., Ltd. (Shanghai, China). For real-time fluorescent\nquantitative PCR, the LightCycler 96 instrument, acquired from\nRoche Co., Ltd. (Basel, Switzerland), was used.\n2.2 Analysis of the epidemiological\ncharacteristics of PRRSV in large-scale pig\nfarms across different regions\n2.2.1 Analysis of the detection rate of PRRSV\nBetween January 2020 and June 2023, samples were collected\nfrom large-scale pig farms across 24 provinces and municipalities. On\naverage, 13,454 blood samples were taken per province, amounting to\na total of 322,896 samples for nucleic acid detection. The detailed\ninformation of pig farms was listed in Supplementary Table S1.\n2.2.2 Analysis of the time of PRRSV infection\nFrom July 2021 to June 2023, blood and throat swab samples were\ncollected monthly from 961 individual pig farms, representing 192\ncompanies across 24 provinces and municipalities. Nucleic acid tests\nwere then conducted on these samples. The detailed information of\npig farms and the positive number of samples were listed in\nSupplementary Tables S2, S3.\n2.2.3 Analysis of the number of pig farms with\nPRRS\nFrom January 2021 to December 2022, the large-scale pig farms\nacross the country was surveyed and analyzed each month. The\nnumber of farms surveyed is detailed in Table 1. The study statistically\nanalyzed the number of pig farms affected by PRRS and the incidence\nTABLE 1  The number of pig farms surveyed each month.\nYears\nJan\nFeb\nMar\nApr\nMay\nJun\nJul\nAug\nSep\nOct\nNov\nDec\n2021\n552\n563\n593\n618\n632\n638\n637\n609\n585\n534\n488\n481\n2022\n486\n471\n460\n441\n409\n391\n386\n388\n395\n405\n422\n430\n\nLi and Hua\b\n10.3389/fvets.2025.1614039\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nrate of this disease. The detailed information of pig farms was listed in\nSupplementary Table S4.\n2.3 Serum acclimatization on large-scale\npig farm\n2.3.1 The sample source of serum acclimatization\nA positive serum sample containing a strain of the NADC30-like\ngene type of PRRSV was obtained from a large-scale pig farm in\nGuangdong, where this strain is prevalent. Subsequently, piglets\ntesting positive for PRRSV were selected and euthanized. Serum\nsamples were collected and confirmed to be positive only for the\nPRRSV antigen using RT-qPCR. These serum samples were sent to\nShanghai Sangon Biotech Co., Ltd. for sequencing of the viral nucleic\nacids. The positive serum, containing PRRSV, was then quantified and\nstored at −80°C for future serum acclimatization.\n2.3.2 The procedure of serum acclimatization\nBased on quantitative results, the serum was diluted to ensure\neach milliliter contained 500 virions. Subsequently, 90-day-old gilts\nreceived an injection of 2 mL of serum containing 1,000 virions\nadministered into the neck muscle. Compound amoxicillin was\nprovided via water for three consecutive days, beginning on the day\nof injection. The serum injection was repeated on the third day.\nWithin 10 to 14 days following the initial serum injection, PRRSV\nantibodies began developing in the gilts. One month post-\nacclimatization, the PRRSV antibody positive rate in the gilts\nreached\n100%,\nconfirming\nthe\nsuccess\nof\nthe\nserum\nacclimatization process.\n2.3.3 The time and number of gilts for serum\nacclimatization\nThe serum acclimatization site was located on a pig farm in\nXuwen County, Guangdong Province. From February 2021 and July\n2022, the farm did not engage in serum acclimatization, and during\nthis period, there were 3,817 gilts. From August 2022 to December\n2023, a total of 11,363 gilts underwent acclimatization. Concurrently,\nthe number of non-acclimatization gilts totaled 18,251. Statistical\nanalyses were conducted on the reproductive performance of sows\nand the growth performance of piglets.\n2.4 RT-qPCR detection\n\nConcurrently,\nthe number of non-acclimatization gilts totaled 18,251.Statistical\nanalyses were conducted on the reproductive performance of sows\nand the growth performance of piglets.2.4 RT-qPCR detection\nViral RNA was extracted using the magnetic bead method,\nfollowed by RT-qPCR to detect PRRSV nucleic acids. The RT-qPCR\nprogram included reverse transcription at 45°C for 10 min and\npre-denaturation at 95°C for 10 min. This was followed by 40 cycles\nof denaturation at 95°C for 15 s and annealing/extension at 60°C for\n45 s. The judgment criteria were as follows, based on the amplification\ncurve and cycle threshold (Ct) value: a Ct value of ≤40 indicated a\nPRRSV-positive result. If the Ct value was between 40 and 45, the\nexperiment was repeated. Should the Ct value remain in this range or\nfall below 40 upon repetition, the sample was deemed PRRSV-\npositive. A Ct value >45 or the absence of an amplification curve\nindicated a negative result for PRRSV. The threshold of Ct value is set\n40 based on commercialized reagent kit (purchased from Thermo\nFisher Scientific Co., Ltd). The detection method used in this study\nwas also in accordance with the instructions provided in the kit.\n2.5 Statistical analysis\nStatistical analysis was performed using SPSS 27.0 software.\nIndependent-samples T test was used for comparison between groups.\np-value of less than 0.05 was considered statistically significant,\nP* < 0.05, P** < 0.01, P*** < 0.001.\nStatistical analysis was conducted using SPSS 27.0 software. An\nindependent-samples t-test was employed to compare the groups. A\np-value of less than 0.05 was considered statistically significant, with\n* indicating p < 0.05, ** indicating p < 0.01, and *** indicating\np < 0.001.\n3 Results\n3.1 Sequencing analysis of PRRSV strain\nPositive samples with Ct values below 30 were selected for\nsequencing of the ORF5 region. To obtain the sequences, PCR was\nperformed using the specific primer pair PRRSV-ORF5-F and\nPRRSV-ORF5-R. PCR products were then analyzed via electrophoresis\non 1% agarose gels. The resulting fragments were cloned into pEASY-\n\nTo obtain the sequences, PCR was\nperformed using the specific primer pair PRRSV-ORF5-F and\nPRRSV-ORF5-R.PCR products were then analyzed via electrophoresis\non 1% agarose gels.The resulting fragments were cloned into pEASY-\nblunt vectors (TransGen Biotech, Beijing), and the positive clones\nwere sequenced at Sangon Biotech (Qingdao, China). A phylogenetic\ntree was constructed using the neighbor-joining method in MEGA\nv.7.1.0 software with default settings, including 1,000 bootstrap\nreplicates, based on the gene sequences obtained in this study and\nreference sequences from GenBank. The initial tree was drawn to\nscale, with branch lengths representing the number of substitutions\nper site. The resulting tree was visualized using iTOL v.6 (Interactive\nTree of Life, http://itol.embl.de/).\nAll the sequenced strains cluster into a single large branch\n(Figure  1). Compared to the reference strain, these sequenced\nstrains exhibit the highest homology with the NADC30 strain.\nAdditionally, the homology among the sequenced virus strains\nthemselves is relatively high. Consequently, a representative strain,\nnamed\nXW1-WGS-XQXH-20220908-1,\nwas\nselected\nfor\nthe experiment.\n3.2 Epidemiological analysis of PRRSV\n3.2.1 Analyses of the detection rate of PRRSV in\ndifferent regions\nThe analysis results revealed that, among the 24 provinces and\nmunicipalities (Figure 2A), Yunnan Province had the highest positive\ndetection rate at 23.5%. In contrast, Zhejiang had the lowest rate at\n3.5%. Two provinces, Yunnan and Shanxi, exhibited a positive rate\nexceeding 20%, while 14 provinces had a positive rate exceeding 10%\n(Figure 2B).\n3.2.2 Analysis of the time of PRRSV infection\nThe results demonstrated that the positive rate of PRRSV\ndisplayed clear seasonality. Specifically, the positive rate was\n\nLi and Hua\b\n10.3389/fvets.2025.1614039\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nsignificantly higher during winter and spring (October to March)\ncompared to summer and autumn (April to September). Additionally,\nno significant difference was observed between the detection rates\nfrom abnormal pig blood samples and throat swab samples, with both\nexhibiting similar detection patterns. The average positive rate of\nblood samples was 11.4%, the average positive rate of throat swab\nsamples was 9.2% (Figure 3).\n3.2.3 Analysis of the number of pig farms with\nPRRSV infection\nThe results indicated that the number of infected pig farms and\nthe incidence rate were higher in 2021 than in 2022. The lowest\nnumber of infections occurred from May to July, while the number of\ninfections and the incidence rate peaked from October to February.\nAdditionally, the 2021 infection numbers surpassed those of 2022\n(Figure 4).\n3.3 The impact of serum acclimatization on\nthe reproductive performance of gilts and\nthe growth performance of piglets\n3.3.1 The impact of serum acclimatization on the\nreproductive performance of gilts\nFigure 5 illustrated the effects of serum acclimatization on the\nreproductive performance of gilts in pig farms. The findings indicate\nno significant differences in the abortion rate, pregnancy rate, or\nparturition rate of gilts before and after acclimatization\n(Figures 5A–C). However, the average total number of piglets per litter\npost-acclimatization is 13.17, significantly greater than the\npre-acclimatization number (p < 0.01) (Figure  5D). Similarly, the\naverage number of live piglets per litter post-acclimatization was\n11.66, a significant increase compared to pre-acclimatization levels\n(p < 0.01) (Figure 5E). Furthermore, the average stillbirth rate per\nlitter post-acclimatization decreased significantly to 8.25%, compared\nto pre-acclimatization figures (p < 0.05) (Figure 5F).\n3.3.2 The impact of serum acclimatization on the\ngrowth performance of piglets\nThe results indicated that the average number of weaned piglets\nper litter after serum acclimatization was 11.12, which is significantly\n\n05) (Figure 5F).3.3.2 The impact of serum acclimatization on the\ngrowth performance of piglets\nThe results indicated that the average number of weaned piglets\nper litter after serum acclimatization was 11.12, which is significantly\nhigher than the number before acclimatization (p < 0.05) (Figure 5G).\nThe average total weight of piglets per litter post-serum acclimation\nwas 77.82 kg, also significantly greater than the weight before\nacclimatization (p < 0.01) (Figure  5H). However, there was no\nsignificant difference in the individual weight of weaned piglets before\nand after serum acclimatization (p > 0.05) (Figure 5I). Furthermore,\nthe mortality rate of weaned piglets after serum acclimatization was\n4.63%, notably lower than the rate before acclimation (p < 0.01)\n(Figure 5J).\n3.4 The impact of serum acclimatization\nand non-acclimatization on the\nreproductive performance of gilts and\ngrowth performance of piglets\n3.4.1 The effect of acclimatization and\nnon-acclimatization on the abortion rate of gilts\nThe results showed that from the 1st batch of gilts to the 7th batch,\nthe abortion rates between acclimatization and non-acclimatization\ngilts were similar and did not differ significantly (p > 0.05). However,\nfrom the 8th batch of gilts to the 17th batch, the non-acclimatization\ngilts exhibited consistently higher abortion rates compared to the\nacclimatization gilts. Notably, the highest abortion rate for\nnon-acclimatization gilts reaching 19.63% in the 16th batch of gilts\n(Figure 6A). Throughout the entire period, the average abortion rate\nfor acclimatization gilts was 2.76%, which was lower than the 5.22%\nobserved for non-acclimatization gilts. This difference, however, was\nnot statistically significant (p > 0.05) (Figure 6B).\n3.4.2 The effect of acclimatization and\nnon-acclimatization on the pregnancy rate of\ngilts\nThe results indicated that from the 1st batch of gilts to the 5th\nbatch, the pregnancy rate of the acclimatization gilts consistently\nFIGURE 1\nPhylogenetic trees of reference strains of PRRSV and the\nrepresentative strains of PRRSV were constructed based on the ORF5\n\ngene sequences. Positive samples with Ct values below 30 were\nselected for sequencing of the ORF5 region. PCR was performed\nusing the specific primer pair PRRSV-ORF5-F and PRRSV-ORF5-R.\nPCR products were analyzed.\n\nLi and Hua\b\n10.3389/fvets.2025.1614039\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nexceeded that of non-acclimatization gilts. However, from the 6th \nbatch of gilts to the 17th batch, the pregnancy rates of acclimatization \nand non-acclimatization gilts were closely similar, with no significant \ndifference observed (Figure 6C). Over the entire period, the average \npregnancy rate of the acclimatization gilts was 91.59%, which was \nslightly higher than the 90.75% for non-acclimatization gilts, though \nthis difference was not statistically significant (p > 0.05) (Figure 6D).\n3.4.3 The effect of acclimatization and \nnon-acclimatization on the parturition rate of \ngilts\nThe results indicated that, from the 1st batch of gilts to the 17th \nbatch, the parturition rate of the acclimatization gilts consistently \nexceeded that of the non-acclimatization except the 7th batch. The \nmost pronounced difference in parturition rates occurred in 16th \nbatch (Figure 6E). Over the entire period, the average parturition rate \nfor acclimatization gilts was significantly higher than for \nnon-acclimatization gilts (p < 0.01) (Figure 6F).\n3.4.4 The effect of acclimatization and \nnon-acclimatization on the total number of \npiglets per litter\nThe results indicated that from 1st batch of gilts to the 17th batch, \nthe total number of piglets per litter was consistently higher in the \nserum acclimatization gilts compared to non-acclimatization \n(Figure 6G). Over the entire period, the average total number of\n\nOver the entire period, the average total number of\n\n0.0%\n5.0%\n10.0%\n15.0%\n20.0%\n25.0%\nYunnan\nShanxi\nHeilongjiang\nGansu\nHebei\nLiaoning\nHubei\nHenan\nShandong\nGuizhou\nShaanxi\nInner Mongolia\nJiangsu\nJiangxi\nHainan\nSichuan\nHunan\nTianjin\nBeijing\nGuangxi\nGuagndong\nAnhui\nChongqing\nZhejiang\n1\n2\n3\n4\n5\n6\n7\n8\n9\n10 11 12 13 14 15 16 17 18 19 20 21 22 23 24\nThe detction rate\n 24 provinces and municipalities in China\nB\nFIGURE 2\nThe PRRSV positive rate in 24 provinces and municipalities. (A) The samples from 24 provinces and municipalities in China. (B) The PRRSV positive \ndetection rate. Between January 2020 and June 2023, samples were collected from large-scale pig farms across 24 provinces and municipalities. \n①–㉔ represent different provinces and municipalities in China.\n\nLi and Hua\b\n10.3389/fvets.2025.1614039\nFrontiers in Veterinary Science\n06\nfrontiersin.org\npiglets per litter in serum acclimatization gilts was 13.17, which was\nsignificantly higher than that in non-acclimatization gilts (p < 0.001)\n(Figure 6H).\n3.4.5 The effect of acclimatization and\nnon-acclimatization on the number of live piglets\nper litter\nThe results showed that from 3rd batch of gilts to the 17th batch,\nthe acclimatization gilts consistently produced a higher number of live\npiglets per litter compared to the non-acclimatization gilts (Figure 6I).\nOver the entire period, the average number of live piglets per litter for\nthe serum acclimatization gilts was 11.66, significantly surpassing that\nof the non-acclimatization gilts (p < 0.001) (Figure 6J).\n3.4.6 The effect of acclimatization and\nnon-acclimatization on the stillbirth rate per litter\nThe results indicated that, in the 16th batch, the stillbirth rate per\nlitter for acclimatization gilts was significantly lower than that of\n0.0%\n2.0%\n4.0%\n6.0%\n8.0%\n10.0%\n12.0%\n14.0%\n16.0%\n18.0%\n2021-07\n2021-08\n2021-09\n2021-10\n2021-11\n2021-12\n2022-01\n2022-02\n2022-03\n2022-04\n2022-05\n2022-06\n2022-07\n2022-08\n2022-09\n2022-10\n2022-11\n2022-12\n2023-01\n2023-02\n2023-03\n2023-04\n2023-05\n2023-06\nThe detection rate\nThe time for collecting samples\nBlood\nThroat  swab\nFIGURE 3\nThe time of PRRSV infection. From July 2021 to June 2023, blood and throat swab samples were collected monthly from 961 individual pig farms\nacross 24 province sand municipalities. Nucleic acid in a total of 495,711 blood samples and 418,788 throat swab samples was measured by RT-qPCR.\n0.0%\n1.0%\n2.0%\n3.0%\n4.0%\n5.0%\n6.0%\n7.0%\n8.0%\n9.0%\n0\n5\n10\n15\n20\n25\n30\n35\n40\nJan\nFeb\nMar\nApr\nMay\nJun\nJul\nAug\nSep\nOct\nNov\nDec\nThe rate of pig farms infected PRRSV\nThe number of pig farms infected PRRSV\nThe time of pig farms infected PRRSV\nThe number of pig farms infected PRRSV in 2021\nThe number of pig farms infected PRRSV in 2022\nThe rate of pig farms infected PRRSV in 2021\nThe rate of pig farms infected PRRSV in 2022\nFIGURE 4\nThe number of pig farms and incidence rate with PRRSV infection. From January 2021 to December 2022, the large-scale pig farms across the country\n\nFrom January 2021 to December 2022, the large-scale pig farms across the country\nwas surveyed and analyzed each month. The pig farms with obvious clinical symptoms of PRRS and positive nucleic acid results are identified as PRRS-\npositive farms.\n\nLi and Hua\b\n10.3389/fvets.2025.1614039\nFrontiers in Veterinary Science\n07\nfrontiersin.org\n\n\u0003\n\n0.0 \n0.5 \n1.0 \n1.5 \n2.0 \n2.5 \n3.0 \n3.5 \nBefore serum acclimatization\nAfter serum acclimatization\nThe rate of abortion (%)\nA\n80.0 \n82.0 \n84.0 \n86.0 \n88.0 \n90.0 \n92.0 \n94.0 \nBefore serum acclimatization\nAfter serum acclimatization\nThe pregnancy rate (%)\nB\n80.0 \n81.0 \n82.0 \n83.0 \n84.0 \n85.0 \n86.0 \n87.0 \n88.0 \n89.0 \n90.0 \nBefore serum acclimatization\nAfter serum acclimatization\nThe parturition rate (%)\nC\n12.6 \n12.7 \n12.8 \n12.9 \n13.0 \n13.1 \n13.2 \n13.3 \nBefore serum acclimatization\nAfter serum acclimatization\nThe number of piglets per litter\n**\nD\n10.6 \n10.8 \n11.0 \n11.2 \n11.4 \n11.6 \n11.8 \nBefore serum acclimatization\nAfter serum acclimatization\nThe number of live piglets per litter\n  **\nE\n2.0 \n4.0 \n6.0 \n8.0 \n10.0 \n12.0 \n14.0 \nBefore serum acclimatization\nAfter serum acclimatization\nThe stillbirth rate per litter (%)\n *\nF\n5.0 \n6.0 \n7.0 \n8.0 \n9.0 \n10.0 \n11.0 \n12.0 \nBefore serum acclimatization\nAfter serum acclimatization\nThe number of weaned piglets per litter\n ***\nG\n60.0 \n65.0 \n70.0 \n75.0 \n80.0 \n85.0 \nBefore serum acclimatization\nAfter serum acclimatization\nTotal weight of piglets per litter˄kg)\n **\nH\n2.0 \n3.0 \n4.0 \n5.0 \n6.0 \n7.0 \n8.0 \nBefore serum acclimatization\nAfter serum acclimatization\nPiglet weight after weaning(kg)\nI\n1.0 \n2.0 \n3.0 \n4.0 \n5.0 \n6.0 \n7.0 \n8.0 \n9.0 \n10.0 \n11.0 \nBefore serum acclimatization\nAfter serum acclimatization\nMortality rate of piglets at weaning (%)\n***\nJ\nFIGURE 5\nThe impact of serum acclimatization on the reproductive performance of gilts and the growth performance of piglets. (A) The effects of serum \nacclimatization on the abortion rate. (B) The effects of serum acclimatization on the pregnancy rate. (C) The effects of serum acclimatization on the \n(Continued)\n\nLi and Hua\b\n10.3389/fvets.2025.1614039\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nnon-acclimatization gilts. During other periods, the stillbirth rates per\nlitter for the acclimatization and non-acclimatization gilts were similar\n(Figure 6K). Over the entire study period, no significant difference\nwas observed in the stillbirth rate per litter between the two groups of\ngilts (p > 0.05) (Figure 6L).\n3.4.7 The effect of acclimatization and\nnon-acclimatization on the number of weaned\npiglets per litter\nThe results indicated that, in the 8th and 9th batch, the number of\nweaned piglets per litter in the serum acclimatization group was slightly\nlower than in the non-acclimatization group. However, during other\nperiods, the acclimatization group exhibited a higher number of weaned\npiglets per litter compared to the non-acclimatization group (Figure 6M).\nOverall, the average number of weaned piglets per litter throughout the\nentire period was significantly higher in the acclimatization group than\nin the non-acclimatization group (p < 0.01) (Figure 6N).\n3.4.8 The effect of acclimatization and\nnon-acclimatization on the total weight of piglets\nper litter\nThe results indicated that from 1st batch of gilts to 11th batch, the\ntotal weight of piglets per litter in the acclimatization group followed\na trend similar to that of the non-acclimatization group. However,\nfrom 12th batch to 17th batch, the total weight per litter in the\nacclimatization group exceeded that of the non-acclimatization group,\nwith the difference increasing progressively (Figure 6O). Over the\nentire period, the average total weight of piglets per litter in the\nacclimatization group was significantly higher than that of the\nnon-acclimatization group (p < 0.01) (Figure 6P).\n3.4.9 The effect of acclimatization and\nnon-acclimatization on the weight of weaned\npiglets\nThe results indicated that, the weight trend of weaned piglets in\nacclimatization closely resembled that of the non-acclimatization\ngroup (Figure 6Q). No significant difference was observed in the\nweight of weaned piglets between the acclimatization and\n\nNo significant difference was observed in the\nweight of weaned piglets between the acclimatization and\nnon-acclimatization groups throughout the entire period (p > 0.05)\n(Figure 6R).\n3.4.10 The effect of acclimatization and\nnon-acclimatization on the mortality rate of\nweaned piglets\nThe results indicated that from 1st batch of gilts to 9th batch, the\nmortality rate of weaned piglets in the acclimatization group was\nsimilar to that of the non-acclimatization group. However, from 10th\nbatch of gilts to 17th batch, the mortality rate of weaned piglets in the\nacclimatization group was lower than that of the non-acclimatization\ngroup (Figure 6S). Across the entire period, the mortality rate of\nweaned piglets in the acclimatization group remained lower than that\nin the non-acclimatization group, although the difference was not\nstatistically significant (p > 0.05) (Figure 6T).\n4 Discussion\nPRRS is caused by PRRSV and is considered one of the most\nserious swine diseases affecting the pig industry in China and globally\n(5, 17, 18, 32). PRRSV is a highly variable RNA virus, and traditional\nvaccination methods increasingly fail to offer effective cross-\nprotection, particularly against NADC30-like PRRSV strains.\nConsequently, conducting an epidemiological investigation of large-\nscale pig farms is crucial for understanding the characteristics of\nPRRS. Recent studies have addressed the genetic diversity and\nprevalence of PRRSV in China, yet many have been restricted by\nregional scope, farm size, or small sample sizes (19–24). In response\nto these limitations, this study examines the incidence rate, timing of\ninfection, number of affected pig farms, and causes of PRRSV across\n244 large-scale pig farms in 24 provinces and municipalities in China.\nThe results revealed variability in the detection rate of PRRSV\nacross 24 provinces and municipalities. Yunnan Province recorded the\nhighest positive rate at 23.5%, while Zhejiang exhibited the lowest at\n3.5%. Notably, 14 provinces reported a positive rate exceeding 10%.\nThis indicates regional differences influenced by varying terrain,\n\nYunnan Province recorded the\nhighest positive rate at 23.5%, while Zhejiang exhibited the lowest at\n3.5%.Notably, 14 provinces reported a positive rate exceeding 10%.This indicates regional differences influenced by varying terrain,\naltitude, and climate. Arruda et al. highlighted the impact of these\nfactors on the transmission of airborne diseases, such as PRRS (25,\n26). Additionally, the results demonstrated clear seasonality in the\npositive rate of PRRSV, with significantly higher rates in winter and\nspring compared to summer and autumn. The introduction of gilts is\nidentified as the primary catalyst for PRRS outbreaks, with an\nincidence as high as 49%. Other studies have confirmed the\npronounced seasonality of PRRSV incidence (27), aligning with our\nsurvey results. In summary, by analyzing samples from 24 provinces\nand municipalities, we have investigated the incidence and diversity\nof PRRSV in China. These findings provide valuable epidemiological\ndata and insights that enhance the understanding of PRRS from an\nepidemiological standpoint and may contribute to the prevention and\ncontrol of PRRSV in China.\nSerum acclimatization of PRRS has emerged as an effective\nmethod for the clinical prevention and control of the disease in large-\nscale pig farms in recent years. This approach has gained support\nfrom an increasing number of pig farm managers and veterinarians.\nSerum acclimatization offers procedural advantages. First, the\nacclimatization timing of the pigs can be  artificially controlled,\nallowing for greater consistency. Second, the purity of the\nacclimatization PRRSV serum type can enhance the reproductive\nperformance of sows and the productive performance of piglets to\nsome extent (15, 33). In this study, the pathogen responsible for a\nPRRS outbreak at a large-scale pig farm in Xuwen County,\nparturition rate. (D) The effects of serum acclimatization on the average total number of piglets per litter. (E) The effects of serum acclimatization on\n\nIn this study, the pathogen responsible for a\nPRRS outbreak at a large-scale pig farm in Xuwen County,\nparturition rate.(D) The effects of serum acclimatization on the average total number of piglets per litter.(E) The effects of serum acclimatization on\nthe average number of live piglets per litter. (F) The effects of serum acclimatization on the average stillbirth rate per litter. (G) The effects of serum\nacclimatization on the average number of weaned piglets per litter. (H) The effects of serum acclimatization on the average total weight of piglets per\nlitter. (I) The effects of serum acclimatization on the weight of weaned piglets. (J) The effects of serum acclimatization on the mortality rate of weaned\npiglets. From February 2021 and July 2022, there were 3,817 gilts which did not engage in serum acclimatization. From August 2022 to December\n2023, the gilts were acclimated monthly, the total number of acclimatization gilts was 11,363. A p-value of less than 0.05 was considered statistically\nsignificant, with * indicating p < 0.05, ** indicating p < 0.01, and *** indicating p <���0.001.\nFIGURE 5 (Continued)\n\nLi and Hua\b\n10.3389/fvets.2025.1614039\nFrontiers in Veterinary Science\n09\nfrontiersin.org\n\n1.0 \n5.0 \n9.0 \n13.0 \n17.0 \n21.0 \n1\n2\n3\n4\n5\n6\n7\n8\n9\n10 11 12 13 14 15 16 17\nThe abortion rate  (%)\nThe batch of gilts\n Acclimatization\nNon-acclimatization\nA\n0.0 \n1.0 \n2.0 \n3.0 \n4.0 \n5.0 \n6.0 \n7.0 \n Acclimatization\nNon-acclimatization\nThe average abortion rate  (%)\nB\n80.0 \n85.0 \n90.0 \n95.0 \n100.0 \n1\n2\n3\n4\n5\n6\n7\n8\n9\n10 11 12 13 14 15 16 17\nThe pregnancy rate (%)\nThe batch of gilts\n Acclimatization\nNon-acclimatization\nC\n80.0 \n82.0 \n84.0 \n86.0 \n88.0 \n90.0 \n92.0 \n94.0 \n Acclimatization\nNon-acclimatization\nThe average pregnancy rate (%)\nD\n60.0 \n65.0 \n70.0 \n75.0 \n80.0 \n85.0 \n90.0 \n95.0 \n1\n2\n3\n4\n5\n6\n7\n8\n9\n10\n11\n12\n13\n14\n15\n16\n17\nThe parturition rate  (%)\nThe batch of gilts\n Acclimatization\nNon-acclimatization\nE\n76.0 \n78.0 \n80.0 \n82.0 \n84.0 \n86.0 \n88.0 \n90.0 \n Acclimatization\nNon-acclimatization\nThe average parturition rate  (%)\n **\nF\n12.0 \n12.5 \n13.0 \n13.5 \n14.0 \n1\n2\n3\n4\n5\n6\n7\n8\n9\n10 11 12 13 14 15 16 17\nThe number of piglets per litter\nThe batch of gilts\n Acclimatization\nNon-acclimatization\nG\n12.2 \n12.4 \n12.6 \n12.8 \n13.0 \n13.2 \n13.4 \n Acclimatization\nNon-acclimatization\nThe average number of piglets per litter\n***\nH\n9.5 \n10.0 \n10.5 \n11.0 \n11.5 \n12.0 \n12.5 \n1\n2\n3\n4\n5\n6\n7\n8\n9\n10 11 12 13 14 15 16 17\nThe number of live piglets per litter\nThe batch of gilts\n Acclimatization\nNon-acclimatization\nI\n10.9 \n11.0 \n11.1 \n11.2 \n11.3 \n11.4 \n11.5 \n11.6 \n11.7 \n11.8 \n Acclimatization\nNon-acclimatization\nThe average number of live piglets per litter\n***\nJ\nFIGURE 6 (Continued)\n\nLi and Hua\b\n10.3389/fvets.2025.1614039\nFrontiers in Veterinary Science\n10\nfrontiersin.org\n0.0\n2.0\n4.0\n6.0\n8.0\n10.0\n12.0\n14.0\n16.0\n1\n2\n3\n4\n5\n6\n7\n8\n9\n10 11 12 13 14 15 16 17\nThe stillbirth rate per litter (%)\nThe batch of gilts\nAcclimatization\nNon-acclimatization\nK\n2.0\n3.0\n4.0\n5.0\n6.0\n7.0\n8.0\n9.0\n10.0\nAcclimatization\nNon-acclimatization\nThe average stillbirth rate per litter (%)\nL\n8.5\n9.0\n9.5\n10.0\n10.5\n11.0\n11.5\n12.0\n1\n2\n3\n4\n5\n6\n7\n8\n9\n10 11 12 13 14 15 16 17\nThe number of weaned piglets per litter\nThe batch of gilts\nAcclimatization\nNon-acclimatization\nM\n5.0\n6.0\n7.0\n8.0\n9.0\n10.0\n11.0\n12.0\n13.0\nAcclimatization\nNon-acclimatization\nThe average number of weaned piglets per litter\n**\nN\n60.0\n65.0\n70.0\n75.0\n80.0\n85.0\n90.0\n95.0\n1\n2\n3\n4\n5\n6\n7\n8\n9\n10 11 12 13 14 15 16 17\nThe total weight of piglets per litter˄kg)\nThe batch of gilts\nAcclimatization\nNon-acclimatization\nO\n60.0\n65.0\n70.0\n75.0\n80.0\n85.0\nAcclimatization\nNon-acclimatization\nThe average total weight of piglets per litter˄kg)\n**\nP\n6.0\n6.5\n7.0\n7.5\n8.0\n8.5\n1\n2\n3\n4\n5\n6\n7\n8\n9\n10 11 12 13 14 15 16 17\nThe piglet weight after weaning (kg)\nThe batch of gilts\nAcclimatization\nNon-acclimatization\nQ\n2.0\n3.0\n4.0\n5.0\n6.0\n7.0\n8.0\nAcclimatization\nNon-acclimatization\nThe average piglet weight after weaning (kg)\nR\n0.0\n2.0\n4.0\n6.0\n8.0\n10.0\n12.0\n14.0\n1\n2\n3\n4\n5\n6\n7\n8\n9\n10 11 12 13 14 15 16 17\nThe mortality rate of piglets at weaning (%)\nThe batch of gilts\nAcclimatization\nNon-acclimatization\nS\n1.0\n2.0\n3.0\n4.0\n5.0\n6.0\n7.0\n8.0\nAcclimatization\nNon-acclimatization\nThe average mortality rate of piglets at weaning (%)\nT\nFIGURE 6\nThe effect of serum acclimatization and non-acclimatization on the reproductive performance of gilts and the growth performance of piglets from the\n1st batch of gilts to 17th batch. (A) The comparison of the abortion rate in different batch gilts between serum acclimatization and non-acclimatization.\n(B) The comparison of average abortion rates between acclimatization and non-acclimatization. (C) The comparison of pregnancy rate between\n(Continued)\n\nLi and Hua\b\n10.3389/fvets.2025.1614039\nFrontiers in Veterinary Science\n11\nfrontiersin.org\nGuangdong Province, was isolated and identified. An analysis of its\ngenomic characteristics confirmed that it belongs to the NADC30-\nPRRSV strain. Since this isolated strain represents the current\nprevalent strains and was obtained from this particular farm, it holds\na certain level of representativeness. Consequently, the positive serum\nfrom this isolated strain was used for serum acclimatization.\nRegarding serum acclimatization methods, some studies\nrecommend two injections within a short timeframe (28, 29), while\nothers advocate for a single injection (15, 16). Results consistently\ndemonstrate that single and double injection protocols yield\nsignificantly better outcomes than no serum acclimatization. However,\nno comparative studies between these two methods have been\npublished. Thus, in this experiment, the protocol of administering two\ninjections over three days was selected. Although this approach\nincreases labor costs, it addresses the large number of gilts involved\nand mitigates concerns about improper injections potentially affecting\nacclimatization outcomes. The experiment confirmed that serum\nacclimatization is effective for PRRS prevention and control. Future\nresearch will focus on comparing these methods to identify the most\neffective and cost-efficient strategy.\nThe results demonstrated no significant differences in abortion\nrate, pregnancy rate, parturition rate, and the average weight of\nweaned piglets before and after acclimatization. However, significant\ndifferences were found in the average total number of piglets per litter,\naverage number of live piglets per litter, average stillbirth rate per litter,\nthe number of piglets weaned per litter, total weight of piglets weaned\nper litter, and the mortality rate of weaned piglets before and after\nacclimatization. When compared to non-acclimatization, serum\nacclimatization resulted in significant improvements in parturition\nrate, average total number of piglets per litter, average number of live\n\nWhen compared to non-acclimatization, serum\nacclimatization resulted in significant improvements in parturition\nrate, average total number of piglets per litter, average number of live\npiglets per litter, number of piglets weaned per litter, and total weaning\nweight per litter. The above results indicate that serum acclimatization\ncan improve the reproductive performance of sows and the production\nperformance of piglets to a certain extent. Similar findings were\nreported by Wu et al. (15), who observed a significant increase in the\naverage number of live piglets per litter following serum\nacclimatization. However, in this study, there were no significant\ndifferences in abortion rate or pregnancy rate of gilts after\nacclimatization when compared with either pre-acclimatization or\nun-acclimatization sows. This suggests that serum acclimatization has\nno significant effect on abortion rate and pregnancy rate of sows.\nSerum acclimatization is an effective method for preventing and\ncontrolling PRRSV, but it must be performed following strict operational\nstandards to ensure safety and efficacy. Combining our experimental\nresults with data from previous studies, we find that the success of serum\nacclimatization primarily involves the following aspects: First, it is\nessential to rigorously test the serum for pathogens before acclimatization.\nThis testing ensures that all pathogens are negative except for the PRRSV\nantigen, which confirms the disease infection status of the pigs and\nprevents the spread of other diseases during subsequent adaptation\nphases. Second, sequencing of the PRRSV strain is necessary to\ndetermine whether the strain infecting the pig farm is the dominant\nepidemic strain in the farm or region in recent years. Third, determining\nthe optimal dilution ratio of the acclimatization serum is crucial. This\nstep ensures that pigs produce high levels of antibodies quickly post-\nacclimatization while maintaining relatively mild clinical symptoms of\nPRRS, avoiding severe stress, abortion, and other adverse effects (30).\n\nThis\nstep ensures that pigs produce high levels of antibodies quickly post-\nacclimatization while maintaining relatively mild clinical symptoms of\nPRRS, avoiding severe stress, abortion, and other adverse effects (30).\nSerum acclimatization is recognized as an effective strategy for\npreventing and controlling PRRS, but it faces certain challenges in\npractice. Notably, the purified serum used in acclimatization often\nfails to provide cross-protection against various PRRSV strains,\nparticularly those with significant genetic divergence. Moreover, as an\nRNA virus, PRRSV is prone to mutations, posing a risk of virulence\nreversion after several generations of transmission in pig farms.\nConsequently, there exists a potential for virulence reversion during\nthe serum acclimatization process, which could exacerbate PRRS-\nrelated damage (31). Therefore, it is crucial to continuously monitor\nthe clinical symptoms of pigs after acclimatization. A key issue in\nclinical production is how to reduce the virulence of the domesticated\nPRRSV strain while enhancing its infectivity, enabling a lower viral\ndose to convert the pig herd to a positive status without causing severe\nsymptoms. Additionally, serum acclimatization requires stringent\ndisease management, efficient production operations, and strong team\nconfidence. In large-scale pig farms, where multiple diseases are\nprevalent and management is suboptimal, inadequate technical\ninfrastructure can hinder the success of serum acclimatization (30).\nDespite the ongoing mutation of PRRSV, PRRS continues to pose\na significant threat as a major infectious disease within the global pig\nindustry. It remains a critical target for prevention and control efforts\non pig farms. In China, the pressure to manage PRRS in pig farms is\nparticularly substantial. However, with ongoing, in-depth research\ninto the epidemiology and fundamental aspects of PRRS, alongside\nacclimatization and non-acclimatization. (D) The comparison of average pregnancy rates between acclimatization and non-acclimatization. (E) The\n\nHowever, with ongoing, in-depth research\ninto the epidemiology and fundamental aspects of PRRS, alongside\nacclimatization and non-acclimatization.(D) The comparison of average pregnancy rates between acclimatization and non-acclimatization.(E) The\ncomparison of parturition rate between acclimatization and non-acclimatization. (F) The comparison of average parturition rates between\nacclimatization and non-acclimatization. (G) The comparison of the number of piglets per litter between acclimatization and non-acclimatization.\n(H) The comparison of the average number of piglets per litter between acclimatization and non-acclimatization. (I) The comparison of the number of\nlive piglets per litter between acclimatization and non-acclimatization. (J) The comparison of the average number of live piglets per litter between\nacclimatization and non-acclimatization. (K) The comparison of the stillbirth rate per litter between acclimatization and non-acclimatization. (L) The\ncomparison of the average stillbirth rate per litter between acclimatization and non-acclimatization. (M) The comparison of the number of weaned\npiglets per litter between acclimatization and non-acclimatization. (N) The comparison of the average number of weaned piglets per litter between\nacclimatization and non-acclimatization. (O) The comparison of the total weight of piglets per litter between acclimatization and non-acclimatization.\n(P) The comparison of the average total weight of piglets per litter between acclimatization and non-acclimatization. (Q) The comparison of the\nweight of weaned piglets between acclimatization and non-acclimatization. (R) The comparison of the average weight of weaned piglets between\nacclimatization and non-acclimatization. (S) The comparison of the mortality rate of weaned piglets between acclimatization and non-acclimatization.\n(T) The comparison of the average mortality rate of weaned piglets between acclimatization and non-acclimatization. From August 2022 to December\n\n(S) The comparison of the mortality rate of weaned piglets between acclimatization and non-acclimatization.(T) The comparison of the average mortality rate of weaned piglets between acclimatization and non-acclimatization.From August 2022 to December\n2023, a total of 11,363 gilts underwent acclimatization. Concurrently, the number of non-acclimatization gilts totaled 18,251. From April 2023, the gilts\nacclimatized began to give birth every month until August 2024. The reproductive performance and the growth performance were analyzed and\ncompared each month. These pigs were divided into 17 batches according to the duration of parturition (one batch/month).\nFIGURE 6 (Continued)\n\nLi and Hua\b\n10.3389/fvets.2025.1614039\nFrontiers in Veterinary Science\n12\nfrontiersin.org\nthe enhancement of prevention and control measures, comprehensive\nprevention and control technologies for PRRS are expected to improve\nsignificantly. Consequently, PRRS will increasingly become a\ncontrollable, preventable, and treatable disease.\n5 Conclusion\nThis study investigated the incidence of PRRSV, the timing of\ninfection, the number of infected farms, and the causes of the disease\nin large-scale pig farms across China. Following this, serum\nacclimatization was applied to gilts in these farms to assess its impact\non the reproductive performance of sows and the production\nperformance of piglets. The findings showed that the PRRSV-positive\nrate was highest in Yunnan Province and lowest in Zhejiang Province.\nThe prevalence of PRRSV exhibited significant seasonality, with\nhigher incidence during winter and spring. The introduction of gilts\nwas identified as the primary cause of PRRS outbreaks. Post-serum\nacclimatization, improvements were observed in the reproductive\nperformance of sows and production performance of piglets. These\nresults suggest that serum acclimatization is an effective strategy for\nthe prevention and control of PRRS in large-scale pig farms. These\nresults provide national-level epidemiological data, advancing the\nunderstanding of PRRS from an epidemiological perspective, and may\naid in the prevention and control of PRRSV in China. They also offer\ndata support and experiential references for large-scale pig farms to\nprevent and control PRRS through serum acclimatization.\nData availability statement\nThe original contributions presented in the study are included in\nthe article/supplementary material, further inquiries can be directed\nto the corresponding author.\nEthics statement\nThe animal studies were approved by Animal Experiment Ethics\nCommittee of Northwest A&F University. The studies were conducted\nin accordance with the local legislation and institutional requirements.\nWritten informed consent was obtained from the owners for the\nparticipation of their animals in this study.\n\nThe studies were conducted\nin accordance with the local legislation and institutional requirements.Written informed consent was obtained from the owners for the\nparticipation of their animals in this study.\nAuthor contributions\nSL: Investigation, Data curation, Writing  – original draft,\nWriting  – review & editing, Visualization, Formal analysis. JH:\nFunding acquisition, Project administration, Writing  – review &\nediting, Conceptualization, Supervision.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. The work was supported by\nNational Key Research and Development Program of China, grant/\naward number: 2023YFF1000904; National Natural Science\nFoundation of China, grant/award number: 32372970; Key\nTechnologies Demonstration of Animal Husbandry in Shaanxi\nProvince, grant/award number: 2024NYGG005.\nAcknowledgments\nWe are grateful to all the staff at Shaanxi Centre of Stem Cells\nEngineering & Technology for their assistance with these experiments.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declare that no Gen AI was used in the creation of\nthis manuscript.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. Any product that may be evaluated in this article, or claim\nthat may be made by its manufacturer, is not guaranteed or endorsed\nby the publisher.\nSupplementary material\nThe Supplementary material for this article can be found online\nat: https://www.frontiersin.org/articles/10.3389/fvets.2025.1614039/\nfull#supplementary-material\nReferences\n1.\tJeong J, Kim S, Park C, Kang I, Park KH, Ham HJ, et al. Effect of vaccination with\na porcine reproductive and respiratory syndrome subunit vaccine on sow reproductive\nperformance in endemic farms. Vet Res. 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+{"text": "【兽医文献】\n标题: Evaluating stillborn and litter size as indicators of PRRSV detection in live piglets and the use of stillborn tongue fluids as risk-based samples for PRRSV monitoring\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nInfluences on limited\nantimicrobial use in small-scale\nfreshwater aquaculture farms in\ncentral Thailand\nNour Alhusein 1*, Boonrat Chantong 2, Sarin Suwanpakdee 2,\nAnuwat Wiratsudakul 2, Virginia C. Gould 3†,\nKantima Wichuwaranan 4, Kornrawan Poonsawad 4,\nVarapon Montrivade 4, Nutcha Charoenboon 1,\nLuechai Sringernyuang 4, Matthew B. Avison 5, Helen Lambert 1,\nWalasinee Sakcamduang 2, Henry Buller 6‡ and\nKristen K. Reyher 3*‡\n1 Population Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom, 2 Faculty\nof Veterinary Science, Mahidol University, Nakhon Pathom, Thailand, 3 Bristol Veterinary School, University\nof Bristol, Bristol, United Kingdom, 4 Faculty of Social Science and Humanities, Mahidol University, Nakhon\nPathom, Thailand, 5 School of Cellular and Molecular Medicine, University of Bristol, Bristol, United Kingdom,\n6 College of Life and Environmental Sciences, University of Exeter, Exeter, United Kingdom\nIntroduction: Recent years have shown substantial growth both in the scale\nand the spread of freshwater aquaculture in Thailand, raising concerns about\npotential widespread unsustainable use of antimicrobials.\nMethods: This mixed-methods study used surveys and qualitative interviews to\nexamine conditions of freshwater aquaculture farming in central Thailand in relation\nto animal health, disease management and patterns of antimicrobial use.\nResults: Freshwater aquaculture in this area of Thailand was largely a domestic\nventure operated as a source of additional household income to increase financial\nsecurity. Aquaculture was often integrated with other types of farming; initial outlay\nwas reduced by repurposing unused crops, food, or animal manure (e.g. chicken\ndroppings and pig dung) to fertilise aquaculture ponds. Among farmers representing\ntwenty farms who were surveyed during 2019, only six farmers representing six\nfarms said they used antimicrobials. These included oxytetracycline, enrofloxacin,\nnorfloxacin, ciprofloxacin and sulphonamides. Farmers doubted the benefits of\n\nAmong farmers representing\ntwenty farms who were surveyed during 2019, only six farmers representing six\nfarms said they used antimicrobials.These included oxytetracycline, enrofloxacin,\nnorfloxacin, ciprofloxacin and sulphonamides.Farmers doubted the benefits of\nusing antimicrobials to treat aquatic animals; some believed antimicrobials stunted\ngrowth. The high cost of medicines and prohibitive regulations also discouraged\nantimicrobial use. Farmers linked disease occurrence to changes in the weather,\nthe emergence of new diseases and variable water quality. They relied on farm\nmanagement practices to maintain the health of their aquatic animals, using lime\nand salt to maintain and improve water quality and pH and to disinfect aquaculture\npools. Farmers also reported obtaining juvenile fish and shrimp selectively from\nfarms known to produce healthy stock. Specialised veterinary services for aquatic\nfarming were rare, so farmers relied on their own experimentation with medicines,\npeer advice and recommendations of shopkeepers who sold both aquatic feed and\nmedicines.\nConclusion: This study unexpectedly reveals limited use of antimicrobials linked to\nsocio-economic and ecological features of small-scale family aquaculture farms.\nKEYWORDS\nantimicrobial use, antibiotics use, aquaculture, Thailand, farms, qualitative methods\nOPEN ACCESS\nEDITED BY\nCharlotte Doidge,\nUniversity of Nottingham, United Kingdom\nREVIEWED BY\nDaniela Farias,\nMonterey Bay Aquarium, United States\nNiamh Mahon,\nJames Hutton Institute, United Kingdom\n*CORRESPONDENCE\nNour Alhusein\nnour.alhusein@bristol.ac.uk\nKristen K. Reyher\nKristen.Reyher@bristol.ac.uk\n†PRESENT ADDRESS\nVirginia C. Gould,\nSchool of Psychological Science, University of\nBristol, Bristol, United Kingdom\n‡These authors share senior authorship\nRECEIVED 25 March 2025\nACCEPTED 04 June 2025\nPUBLISHED 11 July 2025\nCITATION\nAlhusein N, Chantong B, Suwanpakdee S,\nWiratsudakul A, Gould VC, Wichuwaranan K,\nPoonsawad K, Montrivade V, Charoenboon N,\nSringernyuang L, Avison MB, Lambert H,\nSakcamduang W, Buller H and\nReyher KK (2025) Influences on limited\nantimicrobial use in small-scale freshwater\n\naquaculture farms in central Thailand.\nFront. Vet. Sci. 12:1600051.\ndoi: 10.3389/fvets.2025.1600051\nCOPYRIGHT\n© 2025 Alhusein, Chantong, Suwanpakdee,\nWiratsudakul, Gould, Wichuwaranan,\nPoonsawad, Montrivade, Charoenboon,\nSringernyuang, Avison, Lambert,\nSakcamduang, Buller and Reyher. This is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY). The use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\naccordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Original Research\nPUBLISHED  11 July 2025\nDOI  10.3389/fvets.2025.1600051\n\nAlhusein et al.\b\n10.3389/fvets.2025.1600051\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n1 Introduction\nFast-developing aquaculture sectors in several Southeast Asian\ncountries have been seen as contributors to the rapidly expanding and\ndiversifying antimicrobial resistance (AMR) documented in animal\nproduction systems over the last two decades (1–3). Analysis of point\nprevalence surveys reporting AMR from aquatic food animals in Asia\nhas found concerning levels of resistance to medically important\nantimicrobials in foodborne pathogens (4). Increased antimicrobial\nuse (AMU) is associated with the emergence of AMR in bacteria, with\nsignificant implications for both animal and human health (5). Schar\net al. (6) estimated that by 2030, AMU in aquaculture will constitute\n5.7% of global AMU from human, terrestrial and aquatic food\nproducing animal sectors, and will carry the highest use intensity per\nkilogram of biomass. While these projections are based on estimations\ndue to the paucity of surveys on AMU in aquaculture and the diversity\nof the aquaculture industry, it emphasizes the urgent need for effective\nsurveillance and antimicrobial stewardship policies in the aquaculture\nsector (6).\nThailand has been part of the Asian aquaculture surge, being\nranked 11th among aquaculture country producers with a production\nof 1,001 thousand tonnes of live weight of aquatic animals in 2022 (2).\nIts industries predominantly center on coastal production of shrimp,\nmolluscs and prawn in salt-water ponds. More recent years, however,\nhave seen the significant growth of inland, fresh-water aquaculture in\nThailand (7). Here the early emphasis has been rather more on the\nproduction of fish species, notably catfish and tilapia, though shrimp\nproduction has grown rapidly and displayed a similar dynamism to\nthat of the coastal regions (8). In 2022, inland aquaculture production\nin Thailand accounted for 46.6% of total aquaculture production\nvalue, with the remainder from marine/coastal aquaculture (2).\nThe growth of inland Thai aquaculture (both shrimp and fish) has\n\nIn 2022, inland aquaculture production\nin Thailand accounted for 46.6% of total aquaculture production\nvalue, with the remainder from marine/coastal aquaculture (2).The growth of inland Thai aquaculture (both shrimp and fish) has\ngiven rise to many debates, despite its very clear profitability for those\ninvolved (9, 10). There are concerns about the sustainability of the\nolder model of domestic rice production in many areas of central\nThailand - the widescale and highly profitable conversion of paddy\nfields to aquaculture ponds has accompanied this transition, changing\nthe nature and regulation of the rural communities that rice\ntraditionally sustained (8, 11, 12). A second concern has been the use,\nover-use and management of fresh-water resources, both river and\ncanals, as critical and replenishable growing media for aquatic species\nat a time when other uses of water are being employed, particularly as\na means of moving effluent and wastewater out of sites of production.\nLittle empirical evidence currently exists as to whether inland\naquaculture alone makes a major contribution to river and canal\npollution or whether, alternatively, aquaculture can place itself\nprincipally as the victim of pollution from urban and industrial\nsources (13).\nA third concern about Thai aquaculture, alongside most\nagriculture systems globally, has been expressed: that emergence of\nAMR in environmental or animal-borne pathogens is driven by AMU\non aquaculture farms, resulting in the increased potential for AMR\ninfections in humans (14–17). Indeed, the rapid growth and spread of\nthe Thai marine aquaculture industry in the early 2000s was\ncharacterized by high profit margins sustained by a wide range of\nchemical inputs (including the use of antimicrobials) to prevent and\ntreat diseases as well as improve water quality (18). These inputs took\nplace in an environment where many production-related diseases\nwere present and unchecked, where increasingly concentrated fish and\nshrimp species were vulnerable to disease outbreaks and where,\ninitially, a relatively unregulated legal framework governed AMU in\n\nanimal farming (19–21). The result was that much of the Thai\naquaculture sector developed with relatively high levels of prophylactic\nAMU being the norm, particularly in the coastal zone (22).\nRecent years have seen establishment of several significant\nregulatory and policy initiatives in Thailand to survey, monitor and\nreduce AMU in aquaculture (23, 24). Under these regulations, the list\nof antimicrobial drugs permitted for use in aquaculture (single drug\nformula) includes amoxicillin, enrofloxacin, oxytetracycline,\nsarafloxacin, oxolinic acid, toltrazuril, and sulfamonomethoxine\nsodium. Permitted combination drug formulas include sulfadiazine/\ntrimethoprim,\nsulfadimethoxine\nsodium/trimethoprim,\nsulfadimethoxine sodium/ormetoprim, sulfamonomethoxine sodium/\ntrimethoprim and sulfadimidine/Trimethoprim (23). Several market\ninitiatives have followed suit with major retailers and food processing\ngroups seeking to limit AMU within their Thai supply chains (e.g., the\nRaised Without Antibiotics initiative) (25). The first five-year National\nStrategic Plan on AMR in Thailand was published in 2016, covering\nthe period 2017 to 2021 (26). Since then, legislation has tightened the\navailability and accessibility of antimicrobials for aquaculture and\nreinforced the role of the veterinarian in antimicrobial prescribing.\nMoreover, AMU for growth promotion has been prohibited, and\ncertain antimicrobials considered critically important for human\nhealth have been made unavailable for use in aquaculture. These\nchanges were in parallel with those in many other major international\nfood-trading nations.\nFifty kilometres outside Bangkok, a wide river flows south\nthrough the agricultural district of ‘Plakat’ (a pseudonymised district\nname has been used to maintain anonymity), part of a larger\nadministrative province. This is a flat fertile landscape with an\nelaborate network of irrigation systems and canals, bearing witness to\nagricultural and industrial activities following a period of land\nreformation. Rice fields and other field crops, vegetable farms,\n\nThis is a flat fertile landscape with an\nelaborate network of irrigation systems and canals, bearing witness to\nagricultural and industrial activities following a period of land\nreformation.Rice fields and other field crops, vegetable farms,\nfishponds, shrimp ponds, chicken, duck and pig farms are numerous\nin the district. There are also industrial factories and solar energy\npower stations. In agricultural terms, over the last two decades there\nhas been a notable shift (as there has across much of central Thailand)\nfrom the more longstanding production of rice to more profitable, low\nsalinity freshwater fish (notably tilapia) and shrimp production. Local\nfish markets serving Bangkok and nearby provinces provide critical\ncommercial infrastructure, along with a range of aquaculture sector\nindustries which include food-processing factories as well as feed and\nanimal suppliers. With such animal production—and its associated\ncommercial and market exigencies - come new and different pressures\nnotably around health (both animal and public health), disease\nmanagement and resource control, which interlink with the growing\nnational agenda of antimicrobial surveillance and reduction\nmentioned above.\nIn an effort to identify the principal drivers of AMU in aquaculture\nand understand how inland, small-scale aquaculture farmers respond\nto shifts in animal health practice and regulation, our team spent a\nyear in Plakat district, conducting research with freshwater prawn and\nfish farmers as part of a wider project to build a holistic picture of\nAMR drivers in Thailand from the One Health (human-animal-\nenvironment) perspective (27). We investigated aquaculture farm\nmanagement practices and attitudes towards AMU as well as the\n\nAlhusein et al.\b\n10.3389/fvets.2025.1600051\nFrontiers in Veterinary Science\n03\nfrontiersin.org\ninformation and advisory networks they had for information about\nantimicrobials and aquatic animal health.\n2 Materials and methods\nA mixed methods approach was employed comprising a farmer\nsurvey aimed to investigate AMU in farms, along with semi-structured\nlocal community (household) interviews undertaken as part of this\nresearch programme. These interviews were oriented to human health\nand AMU but provided additional contextual information to life\naround the farms (Figure 1).\n2.1 Materials and participant recruitment\n2.1.1 Farmer survey\nThe purpose of the questionnaire survey was to explore the\naquaculture farms and animal health management practices as well as\nAMU. A questionnaire was developed by the Thai and UK research\nteams that combined closed, open-ended and multiple choice\nquestions covering three main areas: first, farm structure and farmer\ndemographics; second, animal production and health; and third,\nmedicine use including knowledge, advisory systems and networks\nused by farmers, with a particular emphasis on antimicrobials (Farmer\nsurvey in Appendix S1). Data collection was conducted between\nOctober 1, 2019, and ended on December 20, 2019. A purposive\nsampling was followed based on geographical area and farm type. The\nfarms were geographically centered around the principal settlement\nof ‘Plakat’ but stretched across the river basin and extensive local canal\nsystem. A total of 20 aquaculture farms were recruited: nine fish farms\nand 11 shrimp farms. The survey was conducted face-to-face (social\ndistancing in place due to COVID-19 pandemic) with the farm owner\nby researchers (Researchers’ initials: BC, SS and/or AW). On a few\noccasions, co-workers were present and contributed to the discussion.\nInformed consent was obtained from all participants. All participants\nneeded to sign a consent form before participating in the study. Only\nadults participated in the study. Open-ended questions were all in\nThai, were audio recorded and subsequently translated to English and\n\nAll participants\nneeded to sign a consent form before participating in the study.Only\nadults participated in the study.Open-ended questions were all in\nThai, were audio recorded and subsequently translated to English and\nanalysed. Responses to the closed and multiple-choice questions from\nthe questionnaire were analysed and will be  reported in future\nmanuscripts. Researchers (BC, SS, and AW) have a specialty and\ntraining in veterinary sciences.\n2.1.2 Additional interviews with households\nAdditional qualitative interviews were undertaken with local\nresidents, including aquaculture farmers, in the district as part of the\nwider project’s aim to understand local attitudes towards human\nhealth, AMU and AMR. These also provided additional information\non aquafarming and animal health practices. Nineteen households\nwere interviewed by researchers (KW, KP, and NC) and later analysed\nfor the aquafarming theme. Data collection took place between\nFebruary 2019 and August 2021. The sample was purposively selected\nbased on geographical area and socio-economic diversity. Initial\ncontact was made by the village health volunteers (community health\nworkers) who introduced the study on behalf of the research team\n(28). Households who were happy to participate were introduced to\nthe researchers. All visits were made on an invitation/permission basis\nfrom the key informant of the household. Social distancing rules were\napplied due to COVID-19 pandemic. To establish rapport with the\nparticipants, researchers conducted several visits to each household.\nOn average, at least 5 visits were made to each household. Time per\nvisit varied from the early (30–60 min) to the later (1–3 h) period of\nfieldwork. An informed consent was obtained from all the households.\nOnly adults participated in the study. Other adult family members\ncontributed to interviews if they were present. Interviewees were in\neffect self-selected from among family members according to\navailability, but efforts were made to ensure a representative spread of\noccupations and ages across the overall sample. The three main themes\n\nInterviewees were in\neffect self-selected from among family members according to\navailability, but efforts were made to ensure a representative spread of\noccupations and ages across the overall sample.The three main themes\nfor investigation included living conditions (livelihoods, food and\nwater sources), health and treatment-seeking (including medicine use)\n(Interview topic guide in Appendix S2). Interviews were conducted in\nThai. Researchers used both audio recorders and field notes to record\nthese visits. Out of 19 households, almost half (nine) were identified\nFIGURE 1\nA diagram showing the methods and the data sources involved in this study.\n\nAlhusein et al.\b\n10.3389/fvets.2025.1600051\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nas running aquafarming businesses. Researchers (KW, KP, and NC)\nare trained qualitative researchers with background in anthropology.\n2.2 Data management and analysis\nInterview transcripts and responses to open-ended questions were\nrecorded, transcribed and translated into English by local Thai\nprofessional transcription/translation service. These transcripts were\nthen coded using open coding and analysed thematically following the\nguidelines described by Braun and Clarke (29) using NVivo qualitative\ndata analysis software (Version 12, QSR International, Melbourne,\nAustralia). A thematic analysis approach enables exploring the data in\ndepth and identifying patterns of meaning across the data. The first\nstep involved researcher (NA) reviewing the transcripts through the\nstages of familiarization and assigning initial inductive codes to a\nsub-sample of transcripts. An initial coding framework was developed\nand discussed with the rest of the research team. The remaining\ntranscripts were then indexed by researcher (NA) using the coding\nframework and refinements were made as necessary. Researcher (NA)\nthen re-read, reviewed and clustered the identified codes to form\ninductive themes and sub-themes and assigned names to the themes.\nThe process was iterative and since the data were translated from Thai,\ncross-checking of accuracy of interpretation went on throughout the\nanalysis through continuous discussion with the Thai researchers and\nthe rest of the research team. Illustrative quotes from the farmer\nsurvey are presented with descriptors including the farm type (fish or\nshrimp) and the farm ID number (e.g., fish farmer F3 or shrimp\nfarmer S4). Illustrative quotations from household data are presented\nwith descriptors including the farm type (fish or shrimp) and\nhousehold ID number (e.g., fish farmer HH38). As the two datasets\nwere collected by two different teams, the IDs assigned for the\nparticipants were not linked.\n3 Results\n3.1 Farm characteristics\nMost farmers had members of their families living on the farm\n\ng., fish farmer HH38).As the two datasets\nwere collected by two different teams, the IDs assigned for the\nparticipants were not linked.3 Results\n3.1 Farm characteristics\nMost farmers had members of their families living on the farm\nand working with animals. All farms were run by the farmer; none\nwere contracted from a company. Fifteen farmers had established\nthe farms by themselves, three were inherited and two rented. Fish\nfarmers mainly raised tilapia (Oreochromis niloticus). Other\nspecies included giant gourami (Osphronemus goramy), Jullien’s\ngolden carp (Probarbus jullieni) and other carp species\n(Cyprinidae). Shrimp farmers raised Pacific white shrimp\n(Litopenaeus\nvannamei)\nand\ngiant\nfreshwater\nprawn\n(Macrobrachium\nrosenbergii)\n(Farm\ncharacteristics\nin\nSupplementary Table S1).\n3.2 Household characteristics\nIt was common for household members to have multiple\noccupations (Household characteristics in Supplementary Table S2,\nalso see section 3.5.2. Economic fragility). Of the nine households,\nseven had private ponds used to raise fish or shrimp. Others had a\ncage (or floating baskets) used for raising fish in shared ponds or in\nthe river (Figure 2).\nThree main themes and nine subthemes were generated from the\nanalysis (Figure 3).\n3.3 Health management practices\n3.3.1 Pond preparation and disinfection\nFarmers grew fish and shrimp in ponds dug out of the ground.\nTo prepare the ponds for new stock, farmers would leave emptied\nponds to dry out, clean the floor of the ponds and then add new\nwater. Drying periods varied; one farmer spoke of allowing the floor\nto dry for 40–60 days while others mentioned a few days to 2 weeks.\nOnce the soil was dry, most farmers scattered lime (calcium\nhydroxide or calcium carbonate) on the pond floor to disinfect the\nground. Farmers also reported using other chemicals including\nherbicides or quicklime (calcium oxide) with two interviewees\nspeaking of disinfecting their ponds with ‘germ-killing’ medicines (a\nlocal term generally used to refer to antibiotics or disinfectants).\nTreated ponds were left between one to 10 days, before water\nwas added.\n\nTreated ponds were left between one to 10 days, before water\nwas added.\n“We put in lime first to kill the germs.\nResearcher: For how many days, approximately?\n“Three days.” Shrimp farmer S4.\n“To prepare the ponds, we fill them with water. Fill it up to the\nheight that we think the fish can go in. Then disinfect the ponds\nusing ‘germ-killing’ medicines.”\nResearcher: Do you also use lime before (putting the water in) at all?\n“No, no need.” Fish farmer F8.\nAlmost all farms obtained water for the ponds from the canal\nsystem connected to the river. Water levels were monitored and usually\nadjusted to a level of around 1 m depth. Additives were often added to\nthe ponds. These included salts, micro-organisms, ‘germ-killing’\nmedicines and fertilizers. The pond water then was usually left for\naround a week before the fish or shrimp were introduced. One farmer\nmentioned waiting for the water to ‘turn green’ before adding juvenile\nshrimp. Some farmers also checked the pH of the water.\n“We scatter microbes (micro-organisms) and germ-killing\nmedicines into the ponds, then we fill in water. After 3 days or\na week, the shrimplets (baby shrimp) can go in.” Shrimp\nfarmer S5.\n“Sometimes we also add salt into some ponds to treat the soil and\nthe water, to not let the water be too acidic or basic.” Fish farmer F5.\n“(…) add water. Followed by tablet fertilizer to prime the floor.\nThat’s it.” Shrimp farmer S10.\n\nAlhusein et al.\b\n10.3389/fvets.2025.1600051\nFrontiers in Veterinary Science\n05\nfrontiersin.org\n“Interviewee: When the water goes in, we add microbes (micro-\norganisms) to treat it first.\nResearcher: That’s before you put the shrimps in.\nInterviewee: Yes. We use lime and microbes.\nResearcher: Do you buy the microbes or do you make that yourself?\nInterviewee: We buy. Sometimes we use (product name). We use \n(product name) to ferment first, then we use (another product) to \nadjust the water condition…\nResearcher: From then, do you leave it for many more days?\nInterviewee: We leave it for another week before we put the shrimp \nin.” Shrimp farmer S4.\n3.3.2 Stock management\nBuying the aquatic animals from trusted sources was vital to \nfarmers as such a practice meant fewer diseases later. New fish were \nusually kept together in a single, protective ‘nursery pond’ for an \ninitial two and a half to three months. When the fish had grown in size \nand weight, they were divided between different ponds. Just over half \nthe farmers reported co-housing different species. It was common for \nshrimp farmers to raise giant freshwater prawns and white shrimp \ntogether in the same pond. One farmer raised fish and shrimp \ntogether. Other animals such as poultry (in small-scale production) \nroamed freely around the ponds or walked on the nets covering \nthe ponds.\n“But if you want to raise the fish together with the shrimp, you have \nto put the shrimp in first. We take that water to test in a lab; there’s \na lab in (town). If the water passes (the standards), then we can \nrelease the shrimps. Fifteen days after the shrimps go in, the fish can \nFIGURE 2\nLeft: fishpond. Middle: drained fishpond with protective nets. Right: shared fishery in a pond or river.\nFIGURE 3\nThematic map of the themes and subthemes identified in the data.\n\nAlhusein et al.\b\n10.3389/fvets.2025.1600051\nFrontiers in Veterinary Science\n06\nfrontiersin.org\ngo in – tilapias and seven-striped carp and Chinese fish. Then\nwe raise them.” Fish farmer F7.\n3.3.3 Feeding and nutrition\nFarmers explained that the aquaculture farming rearing cycle was\ngenerally operated over a one-year period. This period could\nbe reduced to 6 to 8 months depending on the farmer’s feeding style,\nwhether they used organic feed or commercial supplements.\nCommercial supplements accelerated the growth of fish and shrimp,\nbut not everyone could afford them. Some farmers used animal\nmanure, sourced from their other livestock such as ducks, chicken,\nor pigs, to feed fish or shrimp in the ponds. This practice was seen as\na way to reduce cost as well as being wise use of resources. One\nfarmer mentioned having direct pipelines from their pig sheds\nthrough which dung was pumped into the ponds. Another farmer\nsold the extra dung they had to other farmers. Animal manure usage,\nhowever, was disapproved of by some other farmers due to its\nunpleasant smell. Farmers also reported using other types of organic\nor locally available sources of fertilizers such as vegetables from their\ngarden, lotus, hay or rice bran either alone or mixed with powdered\nfishmeal usually given to small fish. One farmer mentioned using the\nwastewater from a fertilizer factory and another fed fermented\npineapple peel to the shrimp. One participant described using unused\nnoodles from a nearby factory. Vitamins were also used, though one\nfarmer described them as a waste of money.\n“I took pig dung to feed the fish. When I wash the pig dung from this\ncoop, the water flows down two pipes; it flows into the fishpond.”\nFish farmer HH38.\n“Feed this fish only grain and rice bran. Mix rice bran with pellet\nfood. Chicken manure, (…) even the low cost, but the son does not\nlike it, he does not want it. Chicken manure is smelly too. Big\npond over there (different farm). The owner of the big pond raised\nit with chicken manure. Feed both pellet food and chicken\nmanure. That fishpond was delivered by a truck of chicken\n\nChicken manure is smelly too.Big\npond over there (different farm).The owner of the big pond raised\nit with chicken manure.Feed both pellet food and chicken\nmanure.That fishpond was delivered by a truck of chicken\nmanure. Feeding the fish like that makes the fish grow fast.” Fish\nfarmer HH03.\n“The noodles were brought to the fish to eat, but the noodles had to\nbe marinated. Other farms do this. It has to add molasses water to mix\nin the tank as well. They buy from sugar factories.” Fish farmer HH03.\n“I feed the fish with leftover food, vegetables that I bought to cook\nand then I throw the rest to the fish in the pond.” Fish farmer HH25.\n3.4 Disease control and medicine use\n3.4.1 Disease drivers\nMany farmers reported that diseases amongst their stock had\nincreased over the last decade. While some farmers offered no\nexplanation for this, others felt different factors were responsible\nincluding weather changes, the emergence of new diseases and\ndecreasing water quality due to pollution.\n“In the past they never got sick; if we found a few sick or dead ones\nwe will just remove them.” Shrimp farmer S11.\n“Lately, we do not know what’s wrong with the fish, but they keep\ndying.” Fish farmer F6.\n“If the water is good and the weather is good, we do not need to use\nany medicine.” Fish farmer F4.\nThere was no consensus whether it was the heat or the cold that\ncaused disease in the fish or shrimp, but it seemed that the extreme\nweather conditions led to disease spreading.\n“The disease outbreak is usually at the end of winter/beginning (of)\nsummer. Maybe, because of the heat, not sure, March–April. I give them\nmedication and add a lot of water. I do not know, maybe because it is\ngetting warm around that time of the year.” Fish farmer F6.\n“Researcher: You mentioned that the animals died a lot during the\npast year. How did it happen? What were the symptoms?\nInterviewee (1st male): It is the heat.\nInterviewee (2nd male): The weather is too hot, and they (the fish)\ngot sick.” Fish farmer and co-worker F7.\n“If the weather keeps getting colder, we will probably have to buy\nthem medication.” Fish farmer F9.\n\nWhat were the symptoms?Interviewee (1st male): It is the heat.Interviewee (2nd male): The weather is too hot, and they (the fish)\ngot sick.” Fish farmer and co-worker F7.“If the weather keeps getting colder, we will probably have to buy\nthem medication.” Fish farmer F9.\n“They are currently healthy, because right now the temperature is\ndropping. Once it got cooler, they are able to survive. But they would\nalso start showing symptoms if it is getting too cold.” Shrimp\nfarmer S5.\nWater condition was considered vital in raising healthy aquatic\nanimals and was monitored continuously, largely through attention to\nits color, appearance and pH. Poor-looking water was often treated\nusing additives such as salt, micro-organisms and ‘germ-killing’\nmedicines. Two farmers spoke of the impact of rain on the water\nconditions. Others spoke of the importance of oxygenating the water,\nsometimes artificially. Critically, these water quality and environmental\nfactors played a part in determining subsequent antimicrobial and\nother medicine use.\n“After 1 month, we add more water and salt. We have to take note\nof the water color. If the color starts to look bad – if it’s not green or\nclear – then we have to add salt or minerals.” Shrimp farmer S9.\n“It depends on the weather and the water. For example, when we left\nthem out during the rain, then they would start dying. They are\nunable to survive under that condition, because of the dropping pH,\nand alkaline, the shrimps are unable to adapt.” Shrimp farmer S5.\n“I think the rain is toxic. I think. My opinion, not sure how true it\nis.” Fish farmer F8.\n“It depends on the water. If the water quality is good, it would\nbe acceptable for us not to add any medicine.” Shrimp farmer S3.\nHalf the farmers reported having significant animal disease\noutbreaks or mass dying events recently, with six reporting\n\nAlhusein et al.\b\n10.3389/fvets.2025.1600051\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nestimated mortality rates of over 20% - in one case, rising to 80%\nof stock. One farmer described disease symptoms in fish as “red\nbelly, disease in the eye, swollen navel” (Fish farmer F9). Another\nlooked for specific symptoms such as bruises as signs of disease.\nFor most, shrimp or fish feeding rates and mortality levels served\nas the basic indicators of animal population health. One farmer\nmentioned using a lifting net to examine the fish closely or\nshining a light at them at night.\n“The amount of death is uncertain because we can see only\nwhen the dead fish are floating.” Shrimp farmer S2.\n“When they got sick, when they started to die. Once they died,\nthey will float, and we just need to determine what kills them.\nLike, if they have bruises, we would have a look and decide on\nbacterial or viral, and then we would treat them accordingly.”\nFish Farmer F6.\n“Researcher: From your experience, you lift Yo (a tool for trapping\nfishes) to see how they are?\n“Yes. We also can see when the fishes come up to the side of the bank.\nWe shine the light to see them during the nighttime… we check\nwhether they (shrimp) have tight texture or black gum…have foods\nin their intestines.” Shrimp farmer S7.\n3.4.2 “It does not work”: AMU on aquaculture\nfarms\nFarmers were generally divided as to whether AMU on\naquaculture farms had increased or decreased in the last decade.\nThose that mentioned AMU decreasing argued that medicine cost\nwas high and that aquaculture farming simply did not generate\nenough revenue to cover this cost. Some doubted the benefits of using\nantimicrobials in treating aquatic stock, believing antimicrobials\nactively stunted growth. For a few respondents, government\nregulations along with food chain and market imperatives mitigated\nagainst the use of antimicrobials.\n“(AMU) decreased, to decrease the running cost of the farm. Because\nantibiotics are expensive and sometimes ineffective.” Shrimp\nfarmer S9.\n“Mostly, they (aquaculture farmers in general) do not use\n(antimicrobials) anymore… It does not work.” Fish farmer F4.\n\n“(AMU) decreased, to decrease the running cost of the farm.Because\nantibiotics are expensive and sometimes ineffective.” Shrimp\nfarmer S9.“Mostly, they (aquaculture farmers in general) do not use\n(antimicrobials) anymore… It does not work.” Fish farmer F4.\n“However, even medicines did not really help… when we give them\nmedications it looks like they will not grow, it looks like the medicines\nstunted their growth.” Fish farmer F7.\n“I heard that people say it’s not good if we use too much. If we sell\nthe shrimps and the merchant/buyer checks and detects the drug\nuse, we will not be able to sell our shrimps anymore. That is the\nreason why I try to limit the usage of medicine with my shrimps.”\nShrimp farmer S3.\n“For medicine, we may not use it at all because the Department of\nFisheries already told that it will be problem if medicine is detected.\nWe used to face that problem.” Shrimp farmer S6.\nSuch farmers preferred to use other methods to control disease.\nThese included selecting reputed aquatic animal providers from which\nto purchase stock, using herbal medicines, e.g., Turmeric and adding\nvitamins to enhance growth. As stated above, water quality was seen\nas an important factor in keeping the stock healthy, often with\nadditions of salts and lime. In the case of major disease outbreaks,\nfarmers regularly removed any floating dead fish and shrimp and\nwould deploy emergency harvesting if needed. Emergency harvests\nwere either sold for sauce production or became landfill. Infected\nponds were then disinfected, and water was pumped out to prepare\nfor a new batch.\n“Mostly, I do not use (antimicrobials). Because we focus on the\nspecies (of aquatic animals) that we got from different farms…they\nare not similar.” Shrimp farmer S6.\n“Now people mostly use organic methods… we just will not use it\n(antimicrobials). We will concentrate on natural/organic ways.”\nShrimp farmer S4.\n“Yes. I use only herbs I bought from the food shop. I remember that\nit made from turmeric, which human also can eat … If vitamins,\nwe use sometimes … When I find that some ponds grow slowly,\n\nWe will concentrate on natural/organic ways.”\nShrimp farmer S4.“Yes.I use only herbs I bought from the food shop.I remember that\nit made from turmeric, which human also can eat … If vitamins,\nwe use sometimes … When I find that some ponds grow slowly,\nI make a decision by myself to use vitamin supplements.” Shrimp\nfarmer S6.\n“To avoid using antimicrobials, we prepare the pool and water by\nadding microorganisms; oxygenate the water, to help strengthen the\nanimals, so that they will not be bothered by any diseases.” Fish\nfarmer F3.\n“Researcher: What did you do with the dead ones, did you bury\nthem? Or sell them?\nInterviewee: We  sold them. We  sold them as “Phla-ra” (salt-\nfermented fish (not fish sauce) commonly used in many Thai dishes).\nThirteen tons of Phla-ra.” Fish farmer F8.\n“During (disease outbreak) we  catch them; if we  see they die,\nwe must hurry to catch them. If we let it be, they will all die.” Shrimp\nfarmer S8.\nOther farms surveyed reported that their use of antimicrobials had\nincreased in recent years in response to growing disease prevalence.\nProphylactic use of antimicrobials was often described, particularly when\nan outbreak on a neighbouring farm was discussed. As a treatment,\nantimicrobials were largely considered to be a last resort and were far\nfrom being seen as being effective in treating sick animals or populations.\nMost farmers were unable to name specific antimicrobial medicines,\nrecognising them rather by their appearance or characteristics.\n“I think the use (of antimicrobials) is increased. Nowadays, diseases\nof aquatic animals are more and more every day. We need to use\nantibiotics for curing their diseases.” Fish farmer F2.\n“However, I occasionally give them medicine for disease prevention\nif there is a disease outbreak and others say I should give them as a\nprevention measure.” Shrimp farmer S4.\n\nAlhusein et al.\b\n10.3389/fvets.2025.1600051\nFrontiers in Veterinary Science\n08\nfrontiersin.org\n“(…giving them antimicrobials) To stimulate them. (…) To\nprevent them from getting sick… we do not give them that\noften, sometimes we  would refrain from giving them the\nmedication. If they look responsive, we will not give them any.”\nShrimp farmer S3.\n“We do not know what else we could do. We already try changing\nthe water, and aerating; we already did everything, but it did not\nwork. So, we have to turn to using medications.” Fish farmer F6.\n“(…) Like when they ate (the medicine) for seven days in a row,\nthey stop (dying). However, if we stop giving them the meds, they\nwill start dying again. They are not completely cured. We do not\nknow how to solve this.” Fish farmer F8.\n“If we  see giant freshwater prawns die, then we  use yellow\nmedicine. That’s it. Nothing much here because we do not want\nto invest too much. The price of the shrimp (to sell) is low.”\nShrimp farmer S3.\nOut of the 20 farms, only six farms reported using antimicrobials\nincluding oxytetracycline, fluoroquinolones (enrofloxacin, norfloxacin\nand ciprofloxacin) and sulphonamides. Farmers reported purchasing\nantimicrobials from feed companies or stores and mixing these with feed\non the farms. Farmers did not mention obtaining antimicrobials from\nveterinarians. Other additives were also used including multivitamins,\namino acids and probiotics. The dosage and frequency were mostly\ndetermined by the farmer and varied significantly between farms,\nsuggesting the absence or lack of knowledge of common guidelines\nwithin this small-scale commercial aquaculture sector. Treatment plans\nwere pond- and population-based. When some fish or shrimp showed\nsigns of sickness, the entire pond would be treated with the medicines\nmixed with the feed or water and spread manually into the pond.\n“There is a powder medicine that we can mix in water and put in\nthe pond.”\nResearcher: That would be for the entire pond?\n“Yes. Some people would mix it in the food. It’s different for each\nfarm.” Shrimp farmer S11.\n\n“There is a powder medicine that we can mix in water and put in\nthe pond.”\nResearcher: That would be for the entire pond?“Yes.Some people would mix it in the food.It’s different for each\nfarm.” Shrimp farmer S11.\n“We cure them all in a pond. If this pond was cured, I can leave them\nand take care of the next pond.” Shrimp farmer S8.\n3.4.3 Farmers’ perspectives on AMU and AMR\nFarmers were asked what they thought about the long-term\nimpact of AMU on humans, animals or the environment.\nMisinformation or lack of awareness was evident as some farmers felt\nthere was no negative impact from AMU.\n“I have not seen any effects from the use of antimicrobial so far,\nneither has anyone come to clarify/educate us about it.” Fish\nfarmer F3.\n“It’s not harmful to humans… My opinion would be that our use\nof medicines is not harmful and does not affect the consumer; even\nthe biologist that gave us the medication said that there is\nno effect.”\nResearcher: What about these medicines – germ killer? Do you think\nthey have any effects on the consumer?\n“No.” Shrimp farmer S4.\n“Researcher: Do you know about the effects of using antibacterial\ndrugs on human, animals and environment? Is there any effect when\nwe use them for a long time?\n“Interviewee: No, I do not know. I do not really study about this, so\nI do not know much.” Shrimp farmer S7.\nOther farmers believed that AMU could affect consumers due\nto chemicals accumulating in the body and subsequently causing\nillnesses. However, these farmers explained that using\nantimicrobials was necessary, especially in large farms with\nmany animals.\n“I know that if we use a significant amount of medicines with the\nanimal, it will affect the consumer as well. The antibiotics we used\nwould accumulate in the human body, causing it to resist drug\naction afterwards. So, yes. I know that it would cause drug resistance\nin the human body when we use it to treat illness later on.” Fish\nfarmer F6.\n“If we use too much medicine for a long time, it would definitely\nbe harmful to humans because it would accumulate in our body. But\nit is necessary to use medication because we need to medicate the\n\n” Fish\nfarmer F6.“If we use too much medicine for a long time, it would definitely\nbe harmful to humans because it would accumulate in our body.But\nit is necessary to use medication because we need to medicate the\nanimal when they get sick, especially for those big farms that have a\nlot of animals.” Shrimp farmer S11.\n3.4.4 Advice and support networks for disease\nmanagement and AMU provision\nThe farmers interviewed stated that they had autonomy over the\nhealth management and treatment decisions taken on their farms.\nFarmers tended to learn what treatment to use through their own\nexperimentation with different products and their impact upon illness\nsymptoms and animal health.\n“I decide from the symptom whether to use Oxy (oxytetracycline),\nSulfa (sulfonamides), Enro (enrofloxacin), or others. There are 2 to\n3 primary medicines that I use nowadays.” Fish farmer F8.\n“If I use any product, and it’s good, then I continue using that\n(laugh)… I have no one to do research for me. So, I must do it\nmyself. If I give the fishes any medicines and see that during the\n3 days they get better, then I continue using that one. But if they\ndo not get better within 3 days, then I  have to change the\nmedicine.” Fish farmer F8.\n\nAlhusein et al.\b\n10.3389/fvets.2025.1600051\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nSome sought treatment recommendation and advice from more\nexperienced peers, animal husbandry personnel and from storekeepers\nwho sold aquatic food and medicines. Farmers occasionally referred\nto these sellers and personnel as ‘vets’; farmers would describe the\nsymptoms to the sellers and the sellers dispensed the medicines. Some\nstores also offered water testing services.\n“I asked the store owner too, and other people who used the same\nproducts or had the same experiences. So, they can give me advice\non what to do or what medicines to use.” Fish farmer F3.\n“I would consult with people at the drug store. They usually\nhave a veterinarian there. Sometimes I  would ask the\nveterinarians who can prescribe medicine that I know. I would\ntell them the symptoms��- such as having red scales, red gill and\ndroopy eyes - then ask which medicine I should use. They would\ngive suggestions based on that. We do not meet face-to-face for\nthe veterinarian to check the water condition and tell us exactly\nwhich medicine we need to use specifically for such a disease.\nI  would be  more sure like 100% if that is the case.” Fish\nfarmer F8.\nDedicated aquatic veterinary support was considered rare in the\narea and farmers took every opportunity to contact a veterinarian\nwhenever one came into the neighbourhood, though none of the\nfarmers interviewed reported a veterinarian visiting their farms for\nroutine checks or in case of an illness or outbreak. One farmer said\nhe had attended a training course on aquatic farming, and another\nsaid they had done their own reading.\n“At the beginning, I usually ask the agriculturist/academic. But after\na while, I started to study things myself. I tried different things while\nseeing what other people use. That’s how I  ended up with this\nproduct. Actually, others suggest me to use Amoxy (amoxycillin) as\nwell.” Fish farmer F6.\n“A fisheries scientist used to come to the district office, so\nI memorized the names of the medications. I would call and asked\n\nThat’s how I  ended up with this\nproduct.Actually, others suggest me to use Amoxy (amoxycillin) as\nwell.” Fish farmer F6.“A fisheries scientist used to come to the district office, so\nI memorized the names of the medications.I would call and asked\nhim, whether this meds would work or not, and he said that it can.\nSo, I made the decision.” Fish farmer F6.\n“I attended training about fish diseases before. The instructor\ninstructed us to use which medicine with what symptom. I had the\ninstructor’s phone number, so I used to call him and ask him for the\nsuggestion of medication relating to the fishes’ symptom, but I lost\nthe number.” Fish farmer F6.\n3.5 Socioecological influences in the\ndevelopment of aquaculture\n3.5.1 Shifting land use from traditional rice\nfarming to other agro-ventures including\naquafarming\nMany participants described how land was, in the past, mainly\nused for rice farming and that rice fields spread as far as the eye could\nsee. Participants mentioned that, during the 1990s, the use of land\nstarted to shift due to a fall in rice prices and subsequent economic\nloss. Additionally, participants explained that the rice farming\nworkforce has reduced over the years due to aging with younger\ngenerations not wanting to undertake low-income occupations.\nSubsequently, new business ventures started to appear. Factories were\nbuilt and other agricultural systems were promoted including\ngardening, plantations, aquaculture farming and livestock farming to\ngenerate more income.\n“Many people are doing the shrimp pond business because the\nincome is good. Those who cannot do the rice field will have shrimp\nponds. The shrimp pond business has been around for about\n10 years because people had left the area and when they came back,\nthey started a shrimp pond business.” Fish farmer HH25.\n“Our family switched to raising fish in the old fields, digging old\nfields into fishponds. At that time, farming was not good. Oh, now\nfarming requires a lot of investment, a lot of things. My parents are\nold, and I cannot farm myself. So, our family has switched to raising\nfish instead.” Fish farmer HH25.\n\nAt that time, farming was not good.Oh, now\nfarming requires a lot of investment, a lot of things.My parents are\nold, and I cannot farm myself.So, our family has switched to raising\nfish instead.” Fish farmer HH25.\n“My family used to farm rice. I do not plant rice anymore, now\nI raise shrimp. In the past, most of the rice fields were cultivated in\nthis area, 80–90% of the area was rice fields. Today it is transformed\ninto a pond and mixed farming. It has changed in the past 10 years\nfrom rice fields to shrimp ponds. However, when shrimp prices are\nnot good, there are farmers who turn back to rice farming. But my\nfamily has always been raising shrimp since we have been raising\nshrimp and never changed. I’ve raised white shrimp before. I planted\nManila tamarind on the edge of the shrimp pond, cultivated as an\nadditional income. One year it can sell for over a hundred thousand\nbaht.” Shrimp farmer HH19.\n3.5.2 Economic fragility\nAll the households involved in aquaculture farming also had\nother sources of income. Household members worked in various\nagriculture projects such as rice farming, growing vegetables, fruit\ngardens, lotus ponds and aquaculture farming as well as livestock\nfarming including poultry and pigs. Other occupations such as\nworking in factories and cookery were also common. Participants\nexplained that having different occupations provided more financial\nstability to support families. For example, one household had to\nclose their chicken farm in 2006 due to avian influenza. However,\nthey had other sources of income from raising shrimp and growing\nrice as well as working as a truck driver. Another household, which\nhad a chicken farm contracted to sell eggs to a larger farm, closed\nthe chicken egg business due to lower egg prices being imposed by\nthe larger farm. They had to sell all the chickens back to the larger\nfarm (from which they were originally purchased) at a financial loss.\nThis household, however, also had a tilapia pond they could rely on.\nOne participant explained that the concept of holding several\nfarming projects was encouraged by the government-promoted\n\nThis household, however, also had a tilapia pond they could rely on.One participant explained that the concept of holding several\nfarming projects was encouraged by the government-promoted\n“sufficient economy” ideology which advocated the wise use of one’s\nown resources and the redeployment rather than wasting of\nthese resources.\n“The people choose to do many kinds of agriculture because they\nmight face a lack of income if one of their businesses has some\ncomplications.” Shrimp farmer HH19.\n\nAlhusein et al.\b\n10.3389/fvets.2025.1600051\nFrontiers in Veterinary Science\n10\nfrontiersin.org\n“My job is to raise fish and plant lotuses. My husband (HH227(02))\nis the one who picked up the lotus from the pond. I also help my\nhusband. My other job is raising my grandchildren… my husband\nwill go out to feed the fish at 6 am. The pond has tilapia and yisok\n(Julien’s golden carp). Our ponds often sell fish to fishmongers who\nsell fish in the fish market. They will come and catch fish at the pond\nthemselves.” Fish farmer HH27.\n“I repaired the equipment myself and also repaired the machines for\nother people in the community.” Fish farmer HH27.\n“My house is sectioned according to the sufficiency economy’s\nprototype that is the two rai areas (about 3,800–6,400 square\nmeters) of the land is for the house, various kinds of vegetable\ngardens, a fishpond and a pig shed. The rest of the land, about 36\nrai, is paddy field which is shared with other types of agriculture.”\nFish farmer HH38.\nNotably, most households were multi-generation residencies\nwhere grandparents, siblings and grandchildren lived together in one\nhouse or adjacent houses on the same piece of land. This concentration\nof family members provided labor for these multiple occupations.\nParticipants described helping each other in running different projects\nwith stay-at-home members (elderly, unemployed) generally helping\nwith commercial cooking (restaurants, catering) and aquaculture\nfarming or livestock feeding.\n“This fishpond belongs to my son. I could not do it, I let the kids feed\nthe fish. When catching fish for sale, there will be many relatives who\nare selling fish in the fish market. We do not have to do anything.\nMy son works at the university. He  returned on Saturday and\nSunday. My daughter is a fish feeder… This pond is raising tilapia,\ncarp and yisok. It has been raised for 7–8 months.” Fish\nfarmer HH03.\n4 Discussion\nThis study aimed to investigate the socio-economic and ecological\nconditions of freshwater aquaculture farming and the drivers for\nAMU in a district in central Thailand. Our findings demonstrated that\n\nIt has been raised for 7–8 months.” Fish\nfarmer HH03.4 Discussion\nThis study aimed to investigate the socio-economic and ecological\nconditions of freshwater aquaculture farming and the drivers for\nAMU in a district in central Thailand.Our findings demonstrated that\nthere was limited use of antimicrobials across the farmers in this study.\nThose farmers expressed doubt about the benefits of antimicrobials,\nboth in relation to disease treatment and aquatic animal growth. High\ncost and prohibitive market regulations also discouraged farmers from\nusing antimicrobials. Disease drivers were often linked to weather\nchanges, the emergence of new diseases and decreasing water quality\ndue to pollution. Farmers depended on improved farm management\npractices to maintain the health of the aquatic animals; when that\nfailed, many farmers applied emergency harvesting techniques (i.e.,\nno treatment but catching animals that were still alive). Aquaculture\nveterinary support was limited in the area and many farmers relied on\ntheir social networks and on experimenting with medicines in relation\nto decision making about disease treatment. Aquaculture farms in this\narea took the shape of domestic ventures operated by family members.\nThese families were often found to invest in different juxtaposed small\nbusinesses in an attempt to maintain or improve the financial stability\nof the household. Aquaculture farming was considered a profitable\nbusiness that did not require high input and labor. Initial outlay was\noften reduced by repurposing unused crops, food or animal manure\nto fertilize the ponds and stimulate aquatic animal growth instead of,\nor in addition to, using commercial feed.\nIt is interesting that many farmers in this study stated that they did\nnot use antimicrobials in their farms. Farmers referred to antimicrobial\ntreatments as ineffective and expensive. Traditional antimicrobial\nadministration methods are often problematic and complex in\naquaculture; dispensing antimicrobials directly into water or adding\nantimicrobials to aquatic feed can be ineffective, possibly harmful and\n\nTraditional antimicrobial\nadministration methods are often problematic and complex in\naquaculture; dispensing antimicrobials directly into water or adding\nantimicrobials to aquatic feed can be ineffective, possibly harmful and\ncould lead to AMR (31–34). This is due to difficulties in adjusting a\ntherapeutic dose uniformly across the pond, the effects of the natural\nenvironment on the antimicrobial as well as associated toxicity (e.g.,\nto denitrifying bacteria leading to a build-up of toxic ammonia) (32).\nAdditionally, diseased fish often do not eat, which reduces the success\nrate of medicating the feed (32). Accordingly, specialist veterinary\nadvice is important to facilitate effective antimicrobial administration\nin aquaculture systems. It is possible, therefore, that, in our study area,\ndue to the lack of veterinary support, our participants’ own attempts\nat antimicrobial treatment failed. This may have resulted in their\nopinions about the lack of effectiveness of these drugs, which led them\nto stop using antimicrobials and look for alternative disease\nmanagement options. However, a few participants still reported using\nantimicrobials despite limited observed benefit. Other studies have\nnoted that antimicrobial use may fluctuate from year to year even in\nthe same area, subject to changing climate and disease incidence; one\nreview article noted that farmer surveys often described inconsistent\nreports of antimicrobial use (35). Another study in Vietnam that\nmonitored antimicrobial use found that although 45% of farmers\nbelieved antimicrobials had no effect on curing diseases, 86% of the\nfarmers who held that opinion still used antibacterials for either\ntreatment or prophylaxis (36). These reports suggest that our results\nindicating low use of antimicrobials should be  interpreted with\ncaution; although the findings indicate little use, it would be valuable\nin our study area to see whether abstinence from AMU continues into\nthe future.\nAnother relevant issue to AMR is the reported use of\nfluoroquinolones in this study. One attraction of using\n\nAnother relevant issue to AMR is the reported use of\nfluoroquinolones in this study.One attraction of using\nfluoroquinolones in aquaculture is that these molecules are stable in\naquatic environments, rendering them easy to manage for\naquaculture disease treatment (37). Indeed, enrofloxacin is widely\nused globally (24). However, the use of fluoroquinolones in\naquaculture has been banned in many countries due to their\nimportance of some fluoroquinolones to human medicine (especially\nciprofloxacin and norfloxacin). This is because when resistance to one\nfluoroquinolone (whether used only in farming, or used to treat\nanimals and humans) emerges, it usually confers resistance to all\nfluoroquinolones (15, 24).\nLimited AMU in aquaculture, as was observed in this study,\nwould broadly be considered positive in relation to AMR. However,\nto ensure its sustainability, improving farm management practices\nsuch as sourcing disease-free juveniles, administrating vaccinations,\ncoordinating spatial planning, assisting with disease identification\nthrough veterinary support, as well as enhancing farmers’ knowledge\nthough training and enforcing stricter regulations and monitoring\nis vital to compensate for the health needs of the aquatic animals and\nto ensure farm biosecurity (38). Farmers in this study adopted\nseveral management practices they considered important to protect\n\nAlhusein et al.\b\n10.3389/fvets.2025.1600051\nFrontiers in Veterinary Science\n11\nfrontiersin.org\ntheir stock from diseases: pond preparation and disinfection, healthy\nstock acquisition and good nutrition regimes, to name a few. These\nfarms resembled the description of the small-scale aquaculture\nfarms described by the work of Little et al. (10, 39) which describes\naquaculture as an activity practiced predominantly by farmers “for\nwhom aquaculture constitutes one element of a larger total livelihood\nportfolio.” In these systems, farmers often adopt lower-input and\nlower-risk practices similar to extensive farming systems. This is, for\ninstance, by crop diversification, improving pond preparation,\nchoosing better-quality stock and using improved quality feed\nregimes, subsequently reducing disease incidence and the need for\ntreatment (40, 41). Similar multi-culture agricultural systems such\nas home garden systems in Vietnam, Indonesia and Sri Lanka have\nbeen encouraged for their socio-economic and ecological benefits,\nand have been highlighted for their potential role in alleviating\npoverty (42–45). In Thailand, the effects of the extensification and\nthe diversification of crops, as seen in our study area, has been\nshown to lead to resilience in facing diseases (e.g., rotating rice and\nshrimp production showed reduced disease susceptibility when\ncompared to back-to-back shrimp farming) (13). Governmental\nsupport has also provided knowledge and production management\nplans which have, in turn, encouraged farmers to establish mixed\ncropping agriculture (18). Land has often been divided into an area\nfor the main farming activity, the secondary activity and a\nsupplementary activity, and has included plans for production of\ndifferent types of plants, livestock and fisheries (18). Repurposing\nlocal resources such as we saw in in our study area (e.g., using left-\nover crops and food to feed aquatic animals) has also\nbeen encouraged.\nAnother characteristic of Thai aquaculture farming was that\nfarmers were seen to be well connected to vertical and horizontal\nknowledge networks (46). Their vertical network went along the\n\nAnother characteristic of Thai aquaculture farming was that\nfarmers were seen to be well connected to vertical and horizontal\nknowledge networks (46).Their vertical network went along the\nsupply chain and also included government representatives,\npharmaceutical and feed companies. Farmers also belonged to social\nnetworks of interlinked farmer groups and small clubs known locally\nas “Chum Rom.” These networks created a rural seminar culture\nwhere farmers shared their knowledge and practices, and industry\nand government representatives attended and added to discussions.\nHowever, in our study area, a dedicated aquatic veterinary support\nwas considered rare and none of our participants reported a\nveterinarian visiting their farms for routine checks or in case of an\nillness or outbreak. Farmers also referred to medicines using generic\nterms such as yellow medicine or germs killing medicines and might\nnot be aware of products containing antibiotics, similar to what is\nreported elsewhere (47). This renders farmers relying on sellers’\nadvice who may have interests in providing particular type of advice\nor products. The need for specialized veterinary services independent\nof pharmaceutical and feed companies is vital to ensure an accurate\nand impartial advice especially around AMU. In particular, farmer –\nveterinarian relationship was found to be a barrier or facilitator in\nreducing AMU, depending in the dynamic of the relationship (48).\nDespite improved management practices, findings of our\nstudy indicated that farmers were still facing high disease and\nmortality rates among their aquatic animals. This could risk the\nsuccessful adaptation of extensive farming styles with low AMU,\nresulting in farmers switching to more intensified farming\npractices with higher AMU (37, 49). Reasons for high disease\nincidence were unclear, but farmers cited water pollution as one\nof the drivers of disease occurrence. The effects of water pollution\non aquaculture farming have previously been investigated in\nThailand costal aquaculture regions (50, 51). These studies found\n\nThe effects of water pollution\non aquaculture farming have previously been investigated in\nThailand costal aquaculture regions (50, 51).These studies found\nthat aquaculture water quality was indeed affected by urban\npollution in the canals and included faecal coliforms, human\nEscherichia coli, tetracycline resistance genes and nitrogen (50).\nThe aquaculture water was a source of salinity and herbicides.\nAdditionally, high AMR prevalence was found to be associated\nwith a high prevalence of faecal indicator bacteria which was\nhighest in peri-urban canal water feeding the aquaculture systems\n(51). Another study in Taiwan investigated the potential cross-\ncontamination problems between aquaculture systems and\nsurrounding waters (52). This work demonstrated that aquaculture\nactivities (i.e., usage of antimicrobials) impacted the surrounding\naquatic environments and, at the same time, the surrounding\nanthropogenic activities impacted aquaculture waters. These\nstudies show that aquaculture (even with low AMU) would still\nbe  in danger of transmission of AMR from the environment.\nAnother factor potentially contributing to environmental AMR\ntransmission and selection in aquaculture systems is the use of\nanimal manure in fertilizing the ponds (35, 37), a practice applied\nby some of the farmers in our study. A few of these even\nhad integrated systems where manure was pumped from other\nanimal sheds (e.g., pig or chicken) to the ponds. This practice was\nseen by farmers as economically sustainable. However, it is well\nestablished that manure is a reservoir of resistant bacteria and\nantimicrobial compounds (53). For example, in a study\ninvestigating the impact of integrated fish farming (a practice\ncombining livestock and fish farming, where animal manure is\nshed directly into fish ponds) on AMR, the level of resistance in\nAcinetobacter spp. was found to increase from 1 to 5% of bacterial\nisolates resistant prior to integration to 100% resistance to\noxytetracycline and sulfamethoxazole and to more than 80%\nresistance to ciprofloxacin after 2 months (54). Another study has\n\nwas found to increase from 1 to 5% of bacterial\nisolates resistant prior to integration to 100% resistance to\noxytetracycline and sulfamethoxazole and to more than 80%\nresistance to ciprofloxacin after 2 months (54).Another study has\nshown the correlation between diffusion of fluoroquinolone\nresistance genes and biofertilizers utilization in Chinese shrimp\naquaculture (55). There is certainly a need to raising awareness\namong aquaculture farmers of the risks of using contaminated\nmanure as well as the wider role of veterinary and\nhusbandry support.\nAnother relevant finding linked to drivers of AMU was that\nparticipants linked disease incidence to changes in climate, and\nparticularly the effect of heat on fish health. Prophylactic AMU\nwas seen more commonly when farmers anticipated weather\nchanges. Interestingly, increased local temperature has been\nassociated with increasing AMR in human infections and this\nassociation was consistent across most classes of antibacterials\nand pathogen (56). Another study has found that increased\ntemperature was associated with increased odds that faecal\nsamples from the environment were positive for resistant E. coli\n(57). If AMR were also to increase on fish farms as a function of\ntemperature, it would be expected that antimicrobials would then\nhave reduced ability to cure bacterial infections in treated\nanimals. Increased temperature also affects the chemical activity\nand uptake of medicines in the pond environment (31). All these\nfactors mean a possible increase in disease incidence on\naquaculture farms as the temperature rises, along with forward\npressure on AMU, further exacerbating AMR. There is a specific\n\nAlhusein et al.\b\n10.3389/fvets.2025.1600051\nFrontiers in Veterinary Science\n12\nfrontiersin.org\nneed, therefore, to support farmers in developing disease\nmanagement plans as the climate warms.\n4.1 Limitations\nThis study was a qualitative investigation, and the findings are\nspecific to the research geographical area and may not be generalizable\nto other geographical areas. Future studies could include several areas\nwith freshwater aquaculture. Farmers accounts of their use of\nantimicrobials, disease incidence and mortality rates are subject to\nrecall bias.\n5 Conclusion\nFarmers in this study reported limited AMU in small-scale\nfamily aquaculture farming due to intentional and unintentional\nsocio-economic and ecological factors. Governmental support to\nencourage reduced disease risk and crop diversification as well as\nmarket regulation concerning the residues of antimicrobials allowed\nin food may have encouraged farmers to reduce their use of\nantimicrobials. Past experience of ineffective treatment of farmed\naquatic animals with antimicrobials and the high cost of these\nmedicines have also played a part in the low AMU reported.\nIncreased disease rates were attributed to weather changes, the\nemergence of new diseases and decreasing water quality due to\npollution. The lack of specialist aquatic veterinary support might\nleave farmers subject to pressure from commercial drug sellers\nwhen they seek disease management advice and misuse may also\nmake treatment failure more likely, particularly as temperatures\nrise. There is a need to investigate the effects of climate change on\naquaculture farming across Southeast Asia as well as its associated\ndisease and treatments patterns. Further attention is needed to\nunderstand and raise awareness about the risks of using\ncontaminated animal manure in aquaculture farming. Future\npolicies should attempt to fill the gap in specialist veterinary\nprovision to freshwater aquaculture, address the need to provide\nevidence-based information and advice on farm management\npractices that reduce the need for prophylactic and therapeutic\n\nFuture\npolicies should attempt to fill the gap in specialist veterinary\nprovision to freshwater aquaculture, address the need to provide\nevidence-based information and advice on farm management\npractices that reduce the need for prophylactic and therapeutic\nAMU, and particularly address disease prevention in the face of a\nchanging climate.\nData availability statement\nThe datasets presented in this article are not readily available\nbecause of the risk of identifying participants. Requests to access the\ndatasets should be  directed to Nour Alhusein, nour.alhusein@\nbristol.ac.uk.\nEthics statement\nThe studies involving humans were approved by Mahidol\nUniversity Social Science Institutional Review Board [(Certificate of\nApproval No. 2019.026.0702), MUSSIRB No. 2019/024 (B2)], and\nFaculty of Veterinary Science, Mahidol University – Institute Animal\nCare and Use Committee (FVS-MU-IACUC), (license no: UI-01292-\n2558) and registered with University of Bristol, UK. The studies were\nconducted in accordance with the local legislation and institutional\nrequirements. The study used mixed methods including a survey of\nfarmers and semi-structured interviews with adult members of\nhouseholds in the local community. For the farmer survey, written\nconsent was obtained from all participants. For semi-structured\ninterviews, written consent was requested before the commencing of\ndata collection from all households. Some households initially\npreferred to give a verbal consent. In this case, the verbal consent was\nobtained and witnessed by another member of the research team,\nother household members and village health volunteers (Community\nhealth workers) who helped in the recruitment. Subsequent\nconfirmation of consent was ensured on an ongoing basis (e.g.,\nrepeated requests for permission for household visits), in line with the\nAssociation of Social Anthropologists of the UK (ASA) Ethical\nGuidelines 2021 for good research practice (30). Later, written consent\nwas obtained from all households for other data collection activities\n(e.g., household survey, stool samples, reported elsewhere). For each\n\nLater, written consent\nwas obtained from all households for other data collection activities\n(e.g., household survey, stool samples, reported elsewhere).For each\nactivity, the research team explained to participants the study\nobjectives and procedures, the voluntary nature of participation,\nprinciples of confidentiality and anonymization of data. The\nconsenting procedure (written or verbal) was approved by Mahidol\nUniversity Social Science Institutional Review Board.\nAuthor contributions\nNA: Investigation, Writing – review & editing, Formal analysis,\nWriting – original draft, Visualization, Software. BC: Writing – review\n& editing, Data curation, Methodology, Investigation. SS: Writing –\nreview & editing, Data curation, Investigation, Methodology. AW:\nWriting – review & editing, Investigation, Data curation, Methodology.\nVG: Project administration, Validation, Writing – review & editing,\nData curation, Resources. KW: Data curation, Writing – review &\nediting. KP: Writing – review & editing, Data curation. VM: Writing –\nreview & editing, Data curation. NC: Writing – review & editing, Data\ncuration. LS: Supervision, Methodology, Investigation, Writing  –\nreview & editing, Conceptualization, Funding acquisition. MA:\nResources, Funding acquisition, Writing  – review & editing,\nSupervision, Conceptualization, Investigation. HL: Funding\nacquisition, Writing  – review & editing, Conceptualization,\nMethodology,\nInvestigation,\nSupervision.\nWS:\nSupervision,\nMethodology, Investigation, Writing  – review & editing,\nConceptualization, Funding acquisition. HB: Formal analysis,\nSoftware, Conceptualization, Methodology, Funding acquisition,\nWriting – original draft, Investigation, Supervision. KR: Writing –\nreview & editing, Investigation, Funding acquisition, Supervision,\nConceptualization, Methodology.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. This work was funded by\ngrant MR/S004769/1 from the Antimicrobial Resistance Cross\n\nAlhusein et al.\b\n10.3389/fvets.2025.1600051\nFrontiers in Veterinary Science\n13\nfrontiersin.org\nCouncil Initiative supported by the seven United Kingdom research\ncouncils and the National Institute for Health Research.\nAcknowledgments\nWe thank all the community members and the farmers who\nparticipated in this study. There is a preprint associated to this manuscript\nin medRxiv DOI: https://doi.org/10.1101/2024.02.11.24302655.\nConflict of interest\nProfessor LS is an appointed member of the AMR-NAP (2023–\n2027) drafting committee in Thailand.\nThe remaining authors declare that the research was conducted in\nthe absence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. Any product that may be evaluated in this article, or\nclaim that may be made by its manufacturer, is not guaranteed or\nendorsed by the publisher.\nSupplementary material\nThe Supplementary material for this article can be found online\nat: https://www.frontiersin.org/articles/10.3389/fvets.2025.1600051/\nfull#supplementary-material\nSUPPLEMENTARY APPENDIX S1\nFarmer survey.\nSUPPLEMENTARY APPENDIX S2\nInterview topic guide.\nSUPPLEMENTARY TABLE S1\nFarm characteristics.\nSUPPLEMENTARY TABLE S2\nHousehold characteristics.\nReferences\n1.\tGarlock T, Asche F, Anderson J, Bjørndal T, Kumar G, Lorenzen K, et al. A global\nblue revolution: aquaculture growth across regions, species, and countries. Rev Fish Sci\nAquacult. (2020) 28:107–16. doi: 10.1080/23308249.2019.1678111\n2.\tFood and Agriculture Organization of the United Nations (FAO). The state of world\nfisheries and aquaculture 2024 – Blue transformation in action. 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+{"text": "【兽医文献】\n标题: article_476_Does occurrence of ventricular arrhythmia reduce the surviva\n\nIn dogs with osteoarthritis, is intra-articular allogenic \nmesenchymal stem cell therapy more effective than \nplacebo effect?\n\nA Knowledge Summary by\n\nMegan Moloney BVMSci 1*\n\n1 VSM Building, University of Surrey, Daphne Jackson Rd, Guildford GU2 7AL \n* Corresponding Author (moloneymegan@hotmail.co.uk)\n\nISSN: 2396-9776 \nPublished: 10 Aug 2022 \nin: Veterinary Evidence Vol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.473 \nReviewed by: Andy Morris (BSc[Hons] BVSc CertAVP[GSAS] \nMRCVS) and Lesca Monica Sofyan \n(BaAVBS[HonsII] MVS DVM) \nNext Review Date: 06 Jan 2024\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.473  \nNext review date: 06 Jan 2024 \np a g e | 2 of 13\n\nKNOWLEDGE SUMMARY\n\nPICO question \nIn dogs diagnosed with osteoarthritis in the hip, elbow, stifle or shoulder joint, is treatment with intra-\narticular allogenic mesenchymal stem cell therapy, in comparison with a placebo effect, more effective at \nreducing lameness and pain?\n\nClinical bottom line\nCategory of research question\nTreatment\nThe number and type of study designs reviewed\nAll three papers were randomised controlled trials\nStrength of evidence\nWeak\nOutcomes reported\nIntra-articular allogenic stem cell therapy is effective at reducing pain and lameness in dogs with\nosteoarthritis when compared to a placebo effect. Two studies indicated a statistically significant\nimprovement in both client and veterinary outcome measurements. Client outcome measurements utilised\nincluded: the canine brief pain inventory; a measure of any changes in pain and lameness based on owners\nperception, and the client-specific outcome measure; and an evaluation of the impact of osteoarthritis on\nthree client selected activities and how this changed with treatment. Veterinary outcome measurements\nincluded veterinary pain score based on manipulation of the limb, veterinary assessment of clinical\noutcomes and veterinary pre and post lameness examinations, all of which were subjective measures.\nThe final study identified a statistically significant improvement in both pain and lameness based on owner\nassessments utilising the canine brief pain inventory and the Hudson Visual Analogue Scale for lameness\nscoring. No statistically significant improvement was identified when considering subjective and objective\nveterinary measurements including force plate gait analysis and veterinary orthopaedic examination\nConclusion\nThere is moderate evidence from owner observation and veterinary assessment to suggest that intra-\narticular allogenic (adipose and umbilical derived) stem cell therapy has some efficacy for reducing pain and\nlameness compared to a placebo effect. However, it must be noted that these studies did not compare the\nuse of intra-articular allogenic stem cells with conventional treatments such as intra-articular corticosteroid\ninjections. Therefore, comparison trials are required.\nWhilst all three papers showed significant improvements in the subjective measurements, objective data\n\nTherefore, comparison trials are required.Whilst all three papers showed significant improvements in the subjective measurements, objective data\noutcomes and assessment by board certified veterinary surgeons failed to find a significant improvement in\npeak vertebral force or lameness with the use of intra-articular stem cell therapy in comparison to a\nplacebo effect. Furthermore, whilst no significant adverse reactions to intra-articular stem cell therapy\nwere recorded, information regarding the safety for multiple dosing is lacking and ambiguity remains as to\nthe most appropriate lineage and quantity of allogenic stem cells for the best clinical effect\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.473  \nNext review date: 06 Jan 2024 \np a g e | 3 of 13\n\nThe evidence \nTwo of the three papers (Harman et al., 2016; and Maki et al., 2020) investigated the use of allogenic adipose \nderived mesenchymal stem cells whilst the last paper (Kim et al., 2019) investigated the use of allogenic \numbilical derived mesenchymal stem cells.\n\nSummary of the evidence\n\nHarman et al. (2016)\nPopulation: 93 client owned dogs from nine different clinical study sites across\nthe US.\n52 female and 41 male dogs varying from large to small breeds.\nInclusion criteria:\n•\nAge: 9 months + (average age was 8.1 years).\n•\nWeight: 2.5 kg + (average weight was 29.63 kg).\n•\nSigns and diagnosis: Physical exam and radiographic\nconformation of osteoarthritis (OA) in one or two of the\nfollowing joints: hips, elbows, stifles or shoulders. Owner\nconfirmed pain / lameness for at least 3 months prior and a\nsubjective veterinary assessed pain on manipulation score of\n≥ 3 based on a scoring system of 1–5 (where 1 indicated no\nresponse to palpation and 5 indicated the dog did not allow\nmanipulation/palpation) for each arthritic joint.\nExclusion criteria:\n•\nParticipants could not be pregnant, lactating or in oestrus.\n•\nNo known malignant or benign interfering neoplasia.\nSample size: 93 dogs.\nIntervention details:\n•\nAdipose derived mesenchymal stem cells (MSCs) from a\nhealthy donor dog were manufactured in accordance with\nstandard operating procedures.\n•\nEach study site was expected to enrol 10 participants, five\nfor group A and five for group B. Overall 47 dogs were\nenrolled into group A and 46 into group B.\n•\nPhysical examination and radiographs were used to confirm\nthe presence of OA.\n•\nParticipants were separated into two groups according to a\nblinded randomisation chart – group A treated with stem\ncells and group B treated with placebo.\n•\nDogs were sedated and given an intra-articular injection of\neither 0.7 ml of saline or 0.7 ml of stem cell solution that\ncontained a target dose of 12 x 106 viable adipose stem cells.\nHow to apply this evidence in practice\nThe application of evidence into practice should take into account multiple factors, not limited to:\nindividual clinical expertise, patient’s circumstances and owners’ values, country, location or clinic where\nyou work, the individual case in front of you, the availability of therapies and resources.\nKnowledge Summaries are a resource to help reinforce or inform decision making. They do not override the\n\nKnowledge Summaries are a resource to help reinforce or inform decision making.They do not override the\nresponsibility or judgement of the practitioner to do what is best for the animal in their care.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.473  \nNext review date: 06 Jan 2024 \np a g e | 4 of 13\n\n•\nOwners were instructed to restrict exercise post injection.\n•\nDogs were assessed at day 0 and day 60.\nStudy design: Prospective, double-blinded, placebo controlled, randomised trial.\nOutcome studied: Subjective:\n•\nClient-specific outcome measurement (CSOM).\n•\nPain on manipulation – veterinary subjective pain on\nmanipulation score based on patient response to\nmanipulation of the limb.\n•\nVeterinary assessment of clinical outcomes – veterinary\nglobal score.\n•\nOwner assessment of clinical outcomes – owner global\nscore.\n•\nSafety – monitoring for adverse events.\nMain findings:\n(relevant to PICO question):\n•\nOwner CSOM showed a statistically significant improvement\nin the treated versus the placebo group (p = 0.02).\n•\nVeterinary pain score on manipulation showed a statistically\nsignificant improvement in the treated versus the placebo\ngroup (p = 0.01).\n•\nVeterinary global outcome score showed a statistically\nsignificant improvement in the treated versus the placebo\ngroup (p = 0.0085).\n•\nOwner global outcome showed an improvement in the\ntreated versus the placebo group however this\nimprovement was not statistically significant.\n•\n15 adverse events were reported throughout this trial: six in\nthe treated group and nine in the control group. In each\ngroup, two of these events were deemed to be serious\nhowever all were assessed to be not related nor caused by\nthe administered cell product.\n•\nIn total 74 of the original 93 enrolled dogs completed the\nstudy, 38 in group A (the stem cell treated group) and 36 in\ngroup B (the placebo group).\nLimitations:\n•\nUncontrolled environmental conditions – dogs were taken\nhome with discharge instructions however owner\ncompliance is uncontrollable.\n•\nLoss of 19 participants to follow-up due to non-compliance\nwith the protocol (11) and low enrollment at sites (eight).\n•\nDifference in severity of OA between dogs – dogs were\nallowed to have one or two joints affected.\n•\nSubjective outcomes.\n•\nMeasure of pain on manipulation of the limb by a\nveterinarian was subjective.\n•\nQuantity of viable adipose stem cells unmeasured –\npotential for discrepancies between the amount of viable\nstem cells injected into each patient.\n•\n\n•\nSubjective outcomes.•\nMeasure of pain on manipulation of the limb by a\nveterinarian was subjective.•\nQuantity of viable adipose stem cells unmeasured –\npotential for discrepancies between the amount of viable\nstem cells injected into each patient.•\nConflict of interest – not a truly independent study. The lead\nauthor is an employee and shareholder of the funding\ncompany, VetStem Biopharma.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.473  \nNext review date: 06 Jan 2024 \np a g e | 5 of 13\n\nKim et al. (2019)\nPopulation: 51 client owned dogs (28 male and 23 female) at the University of\nFlorida Small Animal Hospital.\nInclusion criteria:\n•\nAge: 12 months to 11 years.\n•\nWeight: 11.5 kg to 60 kg.\n•\nSigns and diagnosis; visible unilateral forelimb lameness for\nmore than 6 months attributable to elbow osteoarthritis\n(OA). Osteoarthritic changes to the elbow confirmed on\ncomputerised tomography (CT) imaging. Owner assessment\nof Canine Brief Pain Inventory score (CBPI) (Hudson et al.,\n2004) greater than 2 for baseline pain severity and pain\ninterference scores.\n•\nGeneral health; otherwise healthy. Any medication must\nhave been at a stable dose 4 weeks prior to and throughout\nthe trial.\nExclusion criteria:\n•\nLameness for any reason other than forelimb elbow OA.\nSample size: 51 dogs.\nIntervention details:\n•\n51 dogs were randomly assigned to umbilical cord derived\nmesenchymal stem cell (UMSC) therapy or placebo groups\nvia the method of minimisation.\n•\nStem cells were obtained and cell banks generated from\ncanine umbilical cords.\n•\nAll dogs were sedated and underwent arthrocentesis to\nconfirm the presence of osteoarthritis in the joint via\ncytological evaluation of the joint fluid.\n•\n28 dogs were injected with intra-articular UMSC (0.5 ml\ncontaining approximately 7 x 106 cells) and 23 with intra-\narticular saline (0.5 ml) via the preplaced needle used for\narthrocentesis.\n•\nOwners were advised to restrict their dog’s activity for 2–3\ndays post injection.\n•\nAll dogs were assessed at baseline, 1, 3 and 6 months post\ntreatment using force plate gait analysis, owner assessed\nHudson Visual Analog Scale (HVAS) and CBPI and veterinary\northopaedic exams.\nStudy design: Prospective, double-blinded, placebo-controlled randomised trial.\nOutcome studied: Objective:\n•\nForce plate gait analysis.\nSubjective:\n•\nOwner assessed CBPI level of pain – primary outcome\nmeasure.\n•\nOwner assessed degree of lameness using HVAS.\n•\nVeterinary orthopaedic exam.\n•\nSafety – monitoring for adverse events.\nMain findings:\n(relevant to PICO question):\n•\nNo significant difference in age, body weight, proportion of\ndogs using non-steroidal anti-inflammatory drugs (NSAIDs)\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.473  \nNext review date: 06 Jan 2024 \np a g e | 6 of 13\n\nand symmetry index between the treatment and placebo\ngroups.\n•\nOwner assessed CBPI scores – statistically significantly\nhigher treatment success rate in the UMSC treated group\ncompared with the control group at 1 and 6 months after\ntreatment. No significant difference (p = 0.056) observed at\n3 months.\n•\nMean HVAS mood and sum indexed HVAS scores both\nsignificantly improved following treatment in the UMSC\ngroup. No significant differences were observed for the\nplacebo group.\n•\nPlate vertical force (PVF) – no differences were seen in\neither the treatment or control group.\n•\nVeterinary orthopaedic exam – no differences seen in either\ngroup.\n•\nSeven serious adverse events occurred – five in the UMSC\ngroup, and two in the placebo group. All were classified as\nunlikely to be related to treatment.\nLimitations:\n•\nLack of representation of the wider clinical population – the\npaper assessed the efficacy of UMSC treatment on unilateral\nosteoarthritis cases. The majority of osteoarthritis cases\nseen in dogs are bilateral.\n•\nUnder-powered study – the group sizes for the UMSC and\nplacebo intervention were different and the total number of\nsubjects completing the study was below the original target\nset by the pre-study power analysis.\n•\nSubjective primary outcome variable CBPI - prone to bias.\nBias was limited by blinding and subjecting both groups to\nthe same confounding variables.\n•\nLess stringent definition of success based on CBPI – a\nreduction of pain interference score (PIS) >2 is usually used\nas a measure of success whereas this study utilised a\nreduction of PIS >1 to indicate success.\n•\nUncontrolled environmental conditions – dogs were taken\nhome with discharge instructions however owner\ncompliance was uncontrollable.\n•\nNo indication as to whether the same veterinarian\nconducted the orthopaedic exam on the dogs at each time\npoint.\n•\nOnly the subjective outcomes studied in this paper improved\nwith the UMSC treatment, objective outcomes measured did\nnot.\n•\nShort time period – in total the study only ran for 6 months.\n•\nConflict of interest – several authors were employed by the\nAnimal Cell Therapies company, though it is declared that\n\n•\nShort time period – in total the study only ran for 6 months.•\nConflict of interest – several authors were employed by the\nAnimal Cell Therapies company, though it is declared that\nthe research was conducted in the absence of commercial\nrelationships.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.473  \nNext review date: 06 Jan 2024 \np a g e | 7 of 13\n\nMaki et al. (2020)\nPopulation: 20 client owned dogs, 12 female and 8 male, from two private\nveterinary clinics in Hong Kong.\nInclusion criteria:\n•\nAge: 1 year + (median age 11.25 years).\n•\nWeight: 9 kg + (median weight 25.5 kg) and body condition\nscore of 7/9 or less.\n•\nSigns and diagnosis: clinical signs of osteoarthritis (OA) for at\nleast 1 month in one or both hip joints with radiographic\nevidence of arthritic changes. Noticeable lameness, limited\nrange of motion, and evident pain on palpation /\nmanipulation at the time of evaluation.\n•\nPatient must have undergone at least 1 month of medical\nand / or physical therapy / cage rest management with little\nor no improvement and all treatments had to be stopped at\nleast a week prior to start of trial.\nExclusion criteria:\n•\nPatients could have no additional significant illnesses nor\nhave had any surgery in the affected area within the\nprevious year.\nSample size: 20 dogs.\nIntervention details:\n•\nStem cells were obtained from fat tissue of a healthy 5\nmonth old female donor dog during an ovariohysterectomy.\n•\nAll patients were put under general anaesthesia for\ntreatment.\n•\n16 dogs were injected intra-articularly with mesenchymal\nstem cells (MSCs) at different concentrations – five received\n5x106 cells per joint, six received 25x106 cells per joint, and\nfive received 50x106 cells per joint. The other four dogs were\ninjected intra-articularly with saline.\n•\nBlood was taken and pain and lameness scores were\nrecorded before treatment, at day 0 (day of treatment), day\n5, day 30 and day 90 after the injection.\nStudy design: Prospective, double blinded, placebo controlled, randomised control\ntrial.\nOutcome studied: Objective:\n•\nBlood – anti / pro inflammatory and immunomodulatory\nbiomarkers.\nSubjective:\n•\nLameness score – owner canine brief pain inventory (CBPI)\nscore and veterinary pre and post assessment forms.\n•\nPain score – owner CBPI.\nMain findings:\n(relevant to PICO question):\n•\nLameness scores – 6/7 (86%) of the dogs with low to\nmoderate lameness scores showed improved lameness\nscores following MSC administration however, only 3/4\n(75%) of dogs with severe lameness scores showed\n\nMain findings:\n(relevant to PICO question):\n•\nLameness scores – 6/7 (86%) of the dogs with low to\nmoderate lameness scores showed improved lameness\nscores following MSC administration however, only 3/4\n(75%) of dogs with severe lameness scores showed\nimproved lameness scores (P <0.05). Dogs injected with MSC\nhad a statistically significant improvement of lameness\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.473  \nNext review date: 06 Jan 2024 \np a g e | 8 of 13\n\nwithin the first 30 days (P <0.05). Overall compared with the \nplacebo group, improvement in lameness with MSCs was \nextremely statistically significant (P <0.0001). \n• \nInterleukin receptor antagonist protein (IRAP) levels – all \ndogs that were seen to have improvement in lameness also \nhad increased IRAP levels indicating that increased IRAP \nlevels could be a good indicator for lameness improvement. \n(No power factor was given so to be taken with caution). \n• \nCBPI owner assessed pain scores – results mirrored the \nveterinary assessed lameness scores of the participants. All \nanimals receiving MSCs were seen to have improved pain \nscores. \n• \nNo statistical significance between age of dog and lameness \nresponse to MSC treatment or sex of the dog and lameness \nresponse to MSC treatment. \nLimitations: \n• \nSmall sample size and small placebo group containing only \nfour participants – very underpowered study. \n• \nVariable group sizes. \n• \nVolume of stem cell injection and saline injection not stated \n– unknown whether they were the same or not. \n• \nVarying degrees of OA in all participants. \n• \nSubjective primary outcomes. \n• \nInjection site ambiguity – entry into the joint was not always \nconfirmed by aspiration and was sometimes only judged on \n‘surgeon feel of a slight pop’. Increased chance for human \nerror. \n• \nUncontrolled environmental conditions – owner instructions \nfor at home care were broad and not monitored. \n• \nAmbiguity in the numbers of enrolled dogs at each stage \nwith attrition not clearly described. \n• \nConflict of interest – the study was funded by VetCell \nTherapeutics (VCT) USA and VCT Asia who are both stem cell \nmanufacturers. \n• \nTwo dogs were lost to follow-up due to receiving other \nmedications / treatment during the study period.\n\nAppraisal, application and reflection\n\n• \nTwo dogs were lost to follow-up due to receiving other \nmedications / treatment during the study period.Appraisal, application and reflection\n\nThe aim of osteoarthritis treatment is to decrease pain and increase limb function – enhancing the quality of \nlife of the patient. The current treatment of choice for canine osteoarthritis (OA) is non-steroidal anti-\ninflammatory drugs (NSAIDs) alongside other conventional therapies including the use of polysulphated \nglycosaminoglycans, nutritional modifications, physical therapy and weight management (Pettitt & German, \n2015). Although proven to be effective at combatting pain and lameness in dogs suffering with OA, owner \ncompliance to daily administration of NSAIDs can be poor (Harman et al., 2016). New interventions such as \nplatelet rich plasma (PRP) and mesenchymal stem cell injections are becoming increasingly popular for their \nreduced side effects and safety as long-term treatment (Singh, 2012). There are two forms of stem cell therapy \navailable, allogenic and autologous. Most commercially available stem cell therapies for use in dogs are \nautologous. This Knowledge Summary looked into the efficacy of the use of allogenic stem cells for treatment \nof OA when compared to a placebo effect. The papers reviewed provided some moderate subjective evidence. \nNo objective evidence was observed. One of the papers (Kim et al., 2019) showed no improvement in \nlameness based on both subjective and objective veterinary measurements thus indicating that allogenic stem \ncell treatment alone may have a limited effect on improvement of lameness.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.473  \nNext review date: 06 Jan 2024 \np a g e | 9 of 13\n\nComparisons between the use of autologous and allogenic stem cells for OA in dogs are yet to be investigated.\nBoth forms of stem cell therapy offer benefits and limitations. Allogenic stem cells offer the benefit of being\navailable ‘off the shelf’ and appear to have a relatively long duration of action (Kim et al., 2019) however\nquestions as to the safety of multiple dosages of allogenic stem cells should be considered. Studies of allogenic\nstem cell use in animals for other disease processes have identified complications due to graft-versus-host\ndisease (Michálek et al., 2003; and Wi et al., 2021). All three papers reviewed only assessed single dose\ninterventions and the longest time period studied in any of these papers was 6 months (Kim et al., 2019),\nmeaning that long-term adverse effects and the total duration of improvement of a single intra-articular\ninjection of stem cells is unknown. There is also consideration to be made as to the ethics of collecting\nallogenic stem cells. In two of the studies (Kim et al., 2019; and Maki et al., 2020) the stem cells were collected\nas a by-product from donor dogs that were already undergoing surgery for other procedures, however in the\nfinal study (Harman et al., 2016) it is not stated as to whether the donor dog was undergoing a general\nanaesthetic for any reason other than stem cell collection. Collection of allogenic stem cells carries all the risks\nof a general anaesthetic and does not benefit the donor in any way thus it poses a major ethical issue.\nAutologous stem cells pose less of a risk when considering the patients’ immune system (Khaddour et al.,\n2020) however they require harvesting from the patient themselves which poses a risk through the need for\nsurgical collection. Autologous stem cells also pose an ethical issue as their production warrants the need for\nat least two general anaesthetics for the patient (RVC Canine Stem Cell Treatments Owners' Frequently Asked\nQuestions, 2021). Further safety data is required for both forms of stem cell therapy to determine the risk,\nbenefit ratio.\n\nMesenchymal stem cells can be obtained from a number of different tissues in the body (Hass et al., 2011). \nBoth adipose and umbilical derived stem cells were used in these studies. One study (Kim et al., 2019) \nsuggested that umbilical derived stem cells may be better than adipose derived as they are a population of \nyounger stem cells with a greater ability to differentiate and proliferate. As well as stem cell lineage, the \nnumber of viable stem cells in the solution injected into the osteoarthritic joints differed amongst all three \npapers. One paper (Maki et al., 2020) compared intra-articular injection of different quantities of stem cells \nand did not identify any significant difference between lower and higher dosages in any of the measured \noutcomes. Increased volumes of stem cell did appear to have an increased level of IL-10 biomarker in the \npatients’ serum however, due to sample sizes this correlation was not significant. Age, lineage and quantity of \nthe stem cells may well impact their efficacy and therefore it may be beneficial for future studies to compare \nthese.\n\nThe sample size across all three studies was small. Only one of the papers (Kim et al., 2019) had done a pilot \nstudy to calculate a power analysis and even in this study the clinical trial sample size was smaller than that \ncalculated therefore indicating a lack of power in the study. Not having a large enough study group could \nmean that the results were not true of the wider population and statistical analysis done on the results would \nhave been invalid (Suresh & Chandrashekara, 2012).\n\nAll studies were blinded.  The necessity for blinding was obvious in all three studies as there was a significant \nplacebo effect noted in each. However, the effects of the stem cells went beyond that of the placebo.\n\nAll studies were blinded.The necessity for blinding was obvious in all three studies as there was a significant \nplacebo effect noted in each.However, the effects of the stem cells went beyond that of the placebo.\n\nThe papers reviewed all used subjective primary measure outcomes which have a high potential for bias. This \nbias was mitigated through use of two well regarded subjective measures – Canine Brief Pain Inventory and \nHudson Visual Analog Scale scores, both of which have been evaluated in multiple studies and are well \naccepted (Hudson et al., 2004; and Brown et al., 2008). Force plate gait analysis used in one paper (Kim et al., \n2019) is an objective measure however it is very difficult to take accurately and has lots of room for error \n(McLaughlin, 2001).\n\nIn all of the papers the animals were client owned and taken home throughout the duration of the study and \ntherefore there was a lack of environmental control. However, this provided a perspective on how the animal \ncoped within their usual environment.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.473  \nNext review date: 06 Jan 2024 \np a g e | 10 of 13\n\nThere was a potential limitation in the conflict of interest in two of the papers (Harman et al., 2016; and Kim et \nal., 2019). Both papers had input from employees of major stem cell research companies. This limitation was \nmitigated through the declaration of the remaining authors expressing that the research was conducted in the \nabsence of any commercial or financial relationship however further funding is required for true independent \nstudies to take place.\n\nOverall, there is weak evidence to suggest that allogenic stem cell use in dogs with osteoarthritis is more \nefficacious than a placebo effect. However, consideration must also be taken when providing allogenic stem \ncell use as a treatment to owners such as donor, recipient, dosing, cell therapy formulation, route of \nadministration and veterinary surgeon experience (Maki et al., 2020). Whilst these studies have suggested that \nallogenic stem cell use is effective as treatment for canine OA when compared to a placebo effect, it would be \nmore useful to compare and recognise its uses to the current known treatments for canine OA, such as NSAIDs \nor intra-articular corticosteroids.\n\nMethodology\n\nSearch strategy \nDatabases searched and dates \ncovered: \nCAB Abstracts via CAB Direct (2007–2021) \nScopus via Scopus.com (2005–2021) \nPubMed via the NCBI interface (2005–2022) \nSearch terms: CAB Abstracts: \n1. ((“stem cell*”) AND (OA OR osteoarthritis) AND (shoulder \nOR stifle OR hip OR elbow) AND (dog OR canine) AND \n(placebo OR control)) \n2. ((“stem cell*” OR mesenchymal OR MSC OR stem) AND \n(osteoarthritis OR OA OR DJD OR “degenerative joint \ndisease”) AND (shoulder OR stifle OR hip OR elbow) AND \n(canine OR dog) AND (placebo OR control))\n\nScopus: \n1. ((“stem cell*”) AND (OA OR osteoarthritis) AND (shoulder \nOR stifle OR hip OR elbow) AND (dog OR canine) AND \n(placebo OR control)) \n2. ((“stem cell*” OR mesenchymal OR MSC OR stem) AND \n(osteoarthritis OR OA OR DJD OR “degenerative joint \ndisease”) AND (shoulder OR stifle OR hip OR elbow) AND \n(canine OR dog) AND (placebo OR control))\n\nPubMed: \n1. ((“stem cell*”) AND (OA OR osteoarthritis) AND (shoulder \nOR stifle OR hip OR elbow) AND (dog OR canine) AND \n(placebo OR control)) \n2. ((“stem cell*” OR mesenchymal OR MSC OR stem) AND \n(osteoarthritis OR OA OR DJD OR “degenerative joint \ndisease”) AND (shoulder OR stifle OR hip OR elbow) AND \n(canine OR dog) AND (placebo OR control)) \nDates searches performed: 06 Jan 2022\n\n((“stem cell*” OR mesenchymal OR MSC OR stem) AND \n(osteoarthritis OR OA OR DJD OR “degenerative joint \ndisease”) AND (shoulder OR stifle OR hip OR elbow) AND \n(canine OR dog) AND (placebo OR control)) \nDates searches performed: 06 Jan 2022\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.473  \nNext review date: 06 Jan 2024 \np a g e | 11 of 13\n\nExclusion / Inclusion criteria \nExclusion: \n• \nTreatment group received additional therapies beyond \nallogenic stem cell therapy. \n• \nStem cells were obtained from a different species. \n• \nStem cells were not allogenic. \n• \nStem cells were autologous. \n• \nNo placebo / control group used in the study. \n• \nPaper did not answer the PICO. \n• \nLameness levels not measured. \n• \nReview papers. \nInclusion: \n• \nAllogenic stem cells compared to a placebo / control group. \n• \nAny allogenic stem cell type. \n• \nClinical signs of osteoarthritis visible for 1 month or greater. \n• \nAnswered the PICO. \n• \nMeasured lameness levels. \n• \nPaper written in the English language.\n\nSearch outcome \nDatabase \nNumber \nof \nresults \nExcluded – \nPaper did \nnot answer \nthe PICO \nExcluded – \nStudy group did \nnot compare to \nplacebo / \ncontrol \nExcluded –  \nDid not use \npurely \nallogenic \ncanine stem \ncells \nExcluded –  \nDid not \nmeasure \nlameness as \nan outcome \nTotal \nrelevant \npapers \nCAB \nAbstracts \n26 \n18 \n4 \n1 \n0 \n3 \nScopus \n33 \n24 \n2 \n3 \n1 \n3 \nPubMed \n28 \n21 \n3 \n1 \n0 \n3 \nTotal relevant papers when duplicates removed \n3\n\nCONFLICT OF INTEREST\n\nThe author declares no conflict of interest.\n\nREFERENCES \n1. Brown, D., Boston, R.C., Coyne, J.C. & Farrar, J.T. (2008). Ability of the Canine Brief Pain Inventory to \ndetect response to treatment in dogs with osteoarthritis. [online]. Available \nat: https://www.CanineBPI.com [Accessed 6 Jan 2022].\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.473  \nNext review date: 06 Jan 2024 \np a g e | 12 of 13\n\n2. Harman, R., Carlson, K., Gaynor, J., Gustafson, S., Dhupa, S., Clement, K., Hoelzler, M., McCarthy, T.,\nSchwartz, P. & Adams, C. (2016). A Prospective, Randomized, Masked, and Placebo-Controlled Efficacy\nStudy of Intraarticular Allogeneic Adipose Stem Cells for the Treatment of Osteoarthritis in\nDogs. Frontiers in Veterinary Science. 3, 16. DOI: https://doi.org/10.3389/fvets.2016.00081\n3. Hass, R., Kasper, C., Böhm, S. & Jacobs, R. (2011). Different populations and sources of human\nmesenchymal stem cells (MSC): A comparison of adult and neonatal tissue-derived MSC. Cell\nCommunication and Signalling - BioMed Central. 9(12). DOI: https://doi.org/10.1186/1478-811X-9-1\n4. Hudson, J.T., Slater, M.R., Taylor, L., Scott, H.M. & Kerwin, S.C. (2004). Assessing repeatability and\nvalidity of a visual analogue scale questionnaire for use in assessing pain and lameness in\ndogs. American Journal of Veterinary Research. 65(12), 1634–1643.\nDOI: https://doi.org/10.2460/ajvr.2004.65.1634\n5. Khaddour, K., Hana, C.K. & Mewawalla, P. (2020). Hematopoietic Stem Cell Transplantation - StatPearls\n- NCBI Bookshelf. StatPearls.[online]. Available\nat: https://www.ncbi.nlm.nih.gov/books/NBK536951/ [Accessed 10 Jan 22].\n6. Kim, S.E., Pozzi, A., Yeh, J.C., Lopez-Velazquez, M., Au Yong, J.A., Townsend, S., Dunlap, A.E.,\nChristopher, S.A., Lewis, D.D., Johnson, M.D. & Petrucci, K. (2019). Intra-Articular Umbilical Cord\nDerived Mesenchymal Stem Cell Therapy for Chronic Elbow Osteoarthritis in Dogs: A Double-Blinded,\nPlacebo-Controlled Clinical Trial. Frontiers in Veterinary Science. 6(December), 1–10.\nDOI: https://doi.org/10.3389/fvets.2019.00474\n7. Maki, C.B., Spaas, J.H., Camiel Alice Samoy, Y., Fahie, M., C-bcc, W., Beck, A., Cheuk Chi Wallis, C.-B.,\nChoo, J., Ramos, T., Tong, R., Borjesson, D.L. & Izadyar, F. (2020). Intra-articular Administration of\nAllogeneic Adipose Derived MSCs Reduces Pain and Lameness in Dogs With Hip Osteoarthritis: A\nDouble Blinded, Randomized, Placebo Controlled Pilot Study. Frontiers in Veterinary Science. 1, 570.\nDOI: https://doi.org/10.3389/fvets.2020.00570\n\nIntra-articular Administration of\nAllogeneic Adipose Derived MSCs Reduces Pain and Lameness in Dogs With Hip Osteoarthritis: A\nDouble Blinded, Randomized, Placebo Controlled Pilot Study.Frontiers in Veterinary Science.1, 570.DOI: https://doi.org/10.3389/fvets.2020.00570\n8. McLaughlin, R.M. (2001). Kinetic and Kinematic Gait Analysis in Dogs. The Veterinary Clinics of North\nAmerica : Small Animal Practice. 31(1), 193–201. DOI: https://doi.org/10.1016/S0195-5616(01)50045-\n5\n9. Michálek, J., Collins, R.H., Hill, B.J., Brenchley, J.M., Douek, D.C. (2003). Identification and monitoring\nof graft-versus-host specific T-cell clone in stem cell transplantation. The Lancet. 361 (9364), 1183–\n1185. DOI: https://doi.org/10.1016/S0140-6736(03)12917-0\n10. Pettitt, R.A. & German, A.J. (2015). Investigation and management of canine osteoarthritis. In\nPractice. DOI: https://doi.org/10.1136/inp.h5763\n11. (2021). RVC Canine Stem Cell Treatments Owners' Frequently Asked Questions. [online]. Available\nat: https://www.rvc.ac.uk/small-animal-vet/specialist-referrals/advanced-techniques/stem-cell-\nclinic/information-for-dog-owners#panel-will-my-dog-be-treated-with-stem-cells-from-other-\ndogs [Accessed 24 Jan 2022].\n12. Singh, J.A. (2012). Stem cells and other innovative intra-articular therapies for osteoarthritis: What\ndoes the future hold? BMC Medicine. 44. DOI: https://doi.org/10.1186/1741-7015-10-44\n13. Suresh, K., Chandrashekara, S. (2012). Sample size estimation and power analysis for clinical research\nstudies. Journal of Human Reproductive Sciences. 5(1), 7-13. DOI: https://doi.org/10.4103/0974-\n1208.97779\n14. Wi, H., Lee, S., Kim, Y., No, J.G., Lee, P., Lee, B.R., Oh, K.B., Hur, T.Y. & Ock, S.A. (2021).\nImmunosuppression-enhancing effect of the administration of allogeneic canine adipose-derived\nmesenchymal stem cells(cA-MSCs) compared with autologous cA-MSCs in vitro. Journal of Veterinary\nScience.  22(5), 1–14. DOI: https://doi.org/10.4142/JVS.2021.22.E63\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.473  \nNext review date: 06 Jan 2024 \np a g e | 13 of 13\n\nIntellectual Property Rights \nAuthors of Knowledge Summaries submitted to RCVS Knowledge for publication will retain \ncopyright in their work, and will be required to grant RCVS Knowledge a non-exclusive license \nof the rights of copyright in the materials including but not limited to the right to publish, re-\npublish, transmit, sell, distribute and otherwise use the materials in all languages and all \nmedia throughout the world, and to license or permit others to do so.\n\nDisclaimer \nKnowledge Summaries are a peer-reviewed article type which aims to answer a clinical \nquestion based on the best available current evidence. It does not override the responsibility \nof the practitioner. 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+{"text": "【兽医文献】\n标题: Ecologies of care: qualitative insights from a psychosocial study on veterinarians’ experiences in Northeastern Italy\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nThe effect of kinesiology taping\non gait variability in healthy dogs\nChing-Ming Liu 1, Syu-Lun Lin 1, Hsiao-Man Liu 2 and\nJanice L. Huntingford 3*\n1National Taiwan University Veterinary Hospital, Taipei City, Taiwan, 2Information School, University of\nWashington, Seattle, WA, United States, 3Essex Animal Hospital, Essex, ON, Canada\nIntroduction: Kinesiology taping is commonly utilized in sports and rehabilitation\nfor both humans and horses. Evaluating its effectiveness includes both subjective\nand objective kinesiology assessments. However, there is limited research on the\nuse of kinesiology taping in dogs. This study examines the effects of kinesiology\ntaping on gait variability in healthy dogs.\nMaterials and methods: A total of eight client-owned dogs varying in age,\nbreed, and body weight were recruited for the study. Three taping methods were\napplied to the biceps femoris muscle. Mobility, as well as static and dynamic\nparameters, were assessed using a scale, a stance analyzer, and a pressure-\nsensitive walkway for data collection and statistical evaluation.\nResults: Kinesiology taping on clinically healthy dogs had minimal impact on\nmobility and stance, and only a partial intrinsic effect on gait. Only 3 of 29 kinetic\nand temporospatial parameters showed a statistically significant difference\n(p < 0.05) between taping types. Over days with different taping methods,\nthe number of significantly affected kinetic and temporospatial variable items\nranged between 2 and 7 out of 29, with inconsistent distribution. The effect of\ntaping on gait also varied depending on whether hair was present or clipped.\nDiscussion: These findings suggest that kinesiology taping has minimal and\ninconsistent effects on gait variables in healthy dogs, with limited variation\nacross taping methods and a moderate influence due to the presence of hair.\nThis study provides preliminary data on intrinsic neuromuscular modulation\nin response to skin stimulation, enhancing our understanding of canine gait\n\nThis study provides preliminary data on intrinsic neuromuscular modulation\nin response to skin stimulation, enhancing our understanding of canine gait\nbiomechanics. The insights gained may help guide future research into dynamic\nstability, compensatory strategies, and neurosensory responses during healthy\nmovement in healthy dogs.\nKEYWORDS\nkinesiology taping, dog, pressure sensitive walkway, kinetic variable, temporospatial\nvariable\n1 Introduction\nKinesiology taping (KT) was developed by a Japanese chiropractor, Kenzo Kase, in the\n1970s. This technique involves the use of a pharmaceutical-free elastic woven cotton strip with\na heat-sensitive acrylic adhesive, which is applied to the skin. It is designed to treat various\nmusculoskeletal issues in clinical settings and to enhance muscle function in athletes in human\nmedicine (1, 2). Numerous effects of KT have been hypothesized, including pain reduction,\nwound edema reduction, normalization of muscle function, and improvement of\nproprioceptive feedback. Various clinical effects of KT have been reviewed in a diversity of\nconditions and populations (3). Unlike conventional sports tape, which limits movement, the\neffect of KT is to promote movement. It mimics the thickness and flexibility of the skin and\nOPEN ACCESS\nEDITED BY\nHeidi Reesink,\nUniversity of California, Davis, United States\nREVIEWED BY\nAndrew J. Spence,\nTemple University, United States\nSherman Canapp,\nCanapp Sports Medicine, United States\n*CORRESPONDENCE\nJanice L. Huntingford\ndrjanhuntingford@gmail.com\nRECEIVED 20 June 2025\nREVISED 18 November 2025\nACCEPTED 21 November 2025\nPUBLISHED 08 December 2025\nCITATION\nLiu C-M, Lin S-L, Liu H-M and\nHuntingford JL (2025) The effect of\nkinesiology taping on gait variability in healthy\ndogs.\nFront. Vet. Sci. 12:1650607.\ndoi: 10.3389/fvets.2025.1650607\nCOPYRIGHT\n© 2025 Liu, Lin, Liu and Huntingford. This is\nan open-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY). The use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\n\nThe use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\naccordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Original Research\nPUBLISHED  08 December 2025\nDOI  10.3389/fvets.2025.1650607\n\nLiu et al.\b\n10.3389/fvets.2025.1650607\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nprovides support to muscles without restricting the range of motion\nof limbs.\nThere are three main functions of KT. First, it decreases local\nedema by lifting the skin and promoting blood and lymph flow\nunderneath the dermis (4, 5). Second, it stimulates cutaneous\nmechanoreceptors, which increase afferent feedback to the central\nnervous system, thus reducing pain sensation (6). Finally, it\nstimulates the peripheral nerves, lowering the firing threshold of the\nmotor neurons, enhancing the recruitment of motor units, and\nincreasing the excitability of the motor cortex, thereby improving\nmuscle function (7). The recruitment of the muscle spindle fibers via\nthe sensorimotor pathway improves muscle tone (8). Two types of\nKT techniques are used to produce different effects on muscle\nfunction— facilitation and inhibition. Kase (54), who developed the\noriginal concept, proposed that applying the tape in the direction of\nmuscle contraction facilitated muscle function, whereas applying it\nin the opposite direction inhibited it. Tape recoil resembles muscle\ncontraction, moving toward the anchor. When applied in the same\ndirection as the muscle origin (fixed) to the insertion (movable), it\nenhances contraction. In contrast, applying tape from the insertion\nto the origin may inhibit contraction. In humans, for example,\nthigh-to-knee taping boosts quadriceps muscle activity. A neutral\napplication involves no tension and is not intended to affect muscle\nactivity. However, systematic reviews of KT research have shown\ninconsistent evidence regarding the effectiveness of tape direction\nin enhancing muscle strength (9).\nIn humans, KT has been used as an adjunctive therapy for\ndecreasing post-mastectomy lymphedema (10, 11) and for alleviating\npain in plantar fasciitis (12), knee osteoarthritis (6), shoulder\ndysfunction (13), and carpal tunnel syndrome (14). Other adjunct\nuses are for the treatment of cerebral palsy (8), muscular recovery\n(15), endurance and motor control performance enhancement for\nathletes, and the prevention of injuries (9).\n\nOther adjunct\nuses are for the treatment of cerebral palsy (8), muscular recovery\n(15), endurance and motor control performance enhancement for\nathletes, and the prevention of injuries (9).\nKT has been used in the equine athlete for both competition\nenhancement and rehabilitation (16). It can be used to assess and treat\nmuscular conditions, postural imbalances, and fascia restrictions. The\neffects on muscles, tendons, and ligament injuries are well addressed,\nand according to Molle (16), KT can be used in neurologic pathologies\nas well as lymphatic conditions.\nIn dogs, KT has been used in lymphatic conditions (17) for the\nassistance and treatment of muscular conditions, postural imbalances,\nfascia restrictions, and gait disorders, but there is a paucity of related\nresearch. Canine KT books have been published (18, 19), and KT\ntraining courses exist; however, there is a lack of evidence about the\neffect of KT on the gait performance of dogs or any of the other\nconditions that KT has been proposed to treat in the canine.\nSeveral quantitative gait analysis systems have been validated for\nthe evaluation of gaits in dogs (20). These systems include, but are not\nlimited to, image capture systems, force plates, weight distribution\nplatforms, pressure-sensitive walkways, and inertial measurement\nunits (IMUs) (20, 21). Stance analyzers were utilized to detect static\nweight distribution on limbs with orthopedic disease (22–24) and to\nevaluate the outcomes of orthopedic surgery (25, 26). The pressure-\nsensitive walkway detects both force and non-force information about\ngait, as well as specific gait patterns for different breeds and sizes of\ndogs (27–29), dogs with neurological or orthopedic disease (30–32),\nand rehabilitation outcomes (33).\nThe purpose of this study was to determine the effects of KT on\nthe gait of clinically healthy dogs, whether different types of KT\n(facilitation, inhibition or neutral) on biceps femoris muscle would\naffect the gait, and if the KT effect would be changed by shaving the\nhair over the muscle. We hypothesized that there would be differences\n\nWe hypothesized that there would be differences\nin gait variables with different KT methods on normal dogs and that\nthe hair would affect the effects of taping.\n2 Materials and methods\n2.1 Experimental design and data collection\nThis clinical research was a prospective, randomized, double-\nblinded, crossover study on the effect of KT in eight client-owned\ndogs, which were recruited at National Taiwan University\nVeterinary Hospital. The protocol was reviewed and approved by\nthe National Taiwan University Institutional Animal Care and Use\nCommittee, and the number was NTU-111-EL-00056. The\ninclusion criteria included healthy client-owned dogs, 1 to 15 years\nof age, and between 5 kg and 30 kg body weight of any breed,\ngender, age, and size. Dogs were excluded if they had abnormal\nmobility, were allergic to the taping, or were difficult to handle for\nthe stance or gait evaluation. The dog owners were informed and\nsigned the consent form before enrolling their animals in the\nstudy. After normal physical, orthopedic, and neurological\nexaminations, the dogs were enrolled in the trial. The trial\nconsisted of a baseline period folowed by three consecutive\nrandomized taping periods: K + (facilitation), K− (inhibition), and\nK = (neutral or no effect). Washout periods were included between\neach taping period.\nThe first 2 days of baseline data collection were without taping\nafter clipping the hair on the left thigh using a 2-mm clipper\n(C6-PetPro, ELEMENT, Huei You Trading Co., New Taipei City,\nTaiwan). For the first 2 days, the disability score Cincinnati Orthopedic\nDisability Index (CODI) was evaluated in each dog, and the data for\nstatic analysis using a stance analyzer (Companion Stance Analyzer;\nLiteCure LLC®, Newark, DE, USA) were collected. In the CODI\nquestionnaire, there were eight items—walking, running, jumping,\ngetting up, lying down, climbing stairs, descending stairs, and\nposturing to urinate or defecate—and the owner selected the five\nsituations their dog most frequently experienced. In the stance\nanalysis, dogs were guided by handlers to stand naturally on the\n\nIn the stance\nanalysis, dogs were guided by handlers to stand naturally on the\nweight distribution platform, placing one foot in each quadrant, with\na balanced center of gravity and head facing forward. At least 10\nreadings were taken per dog, and the 5 most consistent values were\nused. Limb weight distribution was expressed as body weight\npercentages. Following the static stance analysis, dynamic gait analysis\nwas performed, consisting of five valid passes of walk and five valid\npasses of trot with a velocity range of ±0.3 m/s and an acceleration of\n±0.5 m/s2. For this analysis, the dog was loosely leashed by a handler\nat the left side with their own comfortable speed of walking, and the\ndata were collected from the pressure sensitive walkway (PSW)\nchipboard and platform equipped with 15,360 sensors covering an\narea of 203 × 54.2 cm working with a measuring frequency of 100 Hz\n(CaniGait; FDM Type 2 from Zebris Medical GmbH, Allgäu,\nGermany). The walkway was covered with a 1.5-mm-thick yoga mat\n\nLiu et al.\b\n10.3389/fvets.2025.1650607\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nmade of natural rubber (TAIMAT, Taiwan) to prevent irritation\nand slipping.\nAfter the baseline training, the dogs were randomized into three\ngroups by online randomizing software (RANDOM. ORG) as to\nwhich type of taping would be received. Every dog eventually received\neach of the taping groups K+, K−, and K=. After measuring and\nrecording the length needed to cover the interested area, the taping\nwas performed on the left thigh with I-strip 5-cm wide kinesiotape\n(KINESIO CANINE; Kinesio Holding Corporation, New Mexican,\nUSA) covering the origin and insertion of the biceps femoris muscle.\nThe taping steps followed the standard taping procedure on\nCANINEEXERCISES website. Kinesio tape contains elastic fibers\naligned lengthwise. When the tape is stretched, its recoil creates a force\ndirected toward the end that was first anchored. If this force vector\nfollows the direction of muscle contraction, it facilitates the muscle\nfunction, which means helping the muscle contraction; if it goes in the\nopposite direction, it inhibits it, which means hindering muscle\ncontraction. The spray glue (Adherent; Mueller Sports Medicine, WI,\nUSA) was used on the anchor and the endpoint to make the tape stick\nfirmly at two ends. The direction of K + was from the origin to the\ninsertion of the biceps femoris muscle, that is, from the sacrotuberous\nligament to the medial tibial tuberosity. The direction of K− was\nopposite to K+. Both K + and K− used 50% recoil tension by\nstretching the tape to extend with 50% available length. The K = taping\nused 0% recoil tension taping from either end. The tape was left on for\n3 days, after which it was removed, and a 7-day washout period with\nno tape on the dog occurred before it received the next taping.\n(Figure 1).\nThe static stance and the gait of walk and trot, as well as the\nCODI, were measured before taping (D0) and were measured 15 min\nafter taping on Day 1(D1). On the second day (D2), the CODI, the\nstatic stance, and the gait of walk and trot were evaluated. On the third\n\nOn the second day (D2), the CODI, the\nstatic stance, and the gait of walk and trot were evaluated.On the third\nday (D3), the CODI and the static stance and the gait of walk and trot\nwere evaluated, and then the tape was removed and the same\nFIGURE 1\nExperimental design.\n\nLiu et al.\b\n10.3389/fvets.2025.1650607\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nmeasurements were retaken (D4). All the measurements were\nperformed in the morning between 9 a.m. to 11 a.m., the same time\nas D1. Between the trial groups, there was a wash-out period without\ntaping for at least 7 days. After all the dogs completed all three taping\ngroups, the hair was allowed to grow, and then the K + taping was\napplied using the same method as previously used for the K + taping.\nThe pressure-sensitive walkway recorded two groups of variables of\ngaits: kinetic gait variables and temporospatial variables. The kinetic\ngait variables, comprising 10 force-related items, included vertical\nimpulse and average maximal force for all four limbs, as well as\nbilateral symmetry. The temporospatial variables, consisting of 19\nmobility-related items, included velocity (1 item), stride length (1\nitem), cadence (1 item), step length (4 items), step width (2 items),\nhind limb reach on both sides, and stance and swing phases for all\nfour limbs.\nThe taping was performed by a veterinarian (CML) with both KT\nand canine rehabilitation training (CKTP-certified kinesio taping\npractitioner and CCRP-certified canine rehabilitation practitioner)\n(see Figure 2). The evaluator was another veterinarian (SLL) with\nCCRV\n(certified\ncanine\nrehabilitation\nveterinarian).\nThe\nrandomization was blinded from the owners and the evaluator. All the\nmeasured data were collected for statistics.\n2.2 Statistical analysis\nThis study contained repeated measurements with designed\nconditions. Comparative analysis of the three taping methods was\nconducted over time for all dogs, and gait performance was examined\nwith respect to clipped versus haired taping. For dogs using different\ntaping methods, comparisons were also made on the same day (Day\n1, Day 2, and Day 3), with one point assigned per item if a statistically\nsignificant difference was found (p < 0.05). The data were analyzed by\nIBM software SPSS version 25. The collected static stance data,\ndynamic kinetic, and temporospatial data were a number of unpaired\n\n05).The data were analyzed by\nIBM software SPSS version 25.The collected static stance data,\ndynamic kinetic, and temporospatial data were a number of unpaired\nsamples that originated from the same population, which were\nnon-normally distributed by the Kolmogorow–Smirnov test. They\nwere compared between three or more than three groups with the\nKruskal–Wallis test. The collected data were also compared in pairs\nusing the Mann–Whitney test within different groups, Wilcoxon test\nFIGURE 2\nThe taping methods.\n\nLiu et al.\b\n10.3389/fvets.2025.1650607\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nbetween the same group under different conditions. For all statistical\nanalyses, values of p < 0.05 were considered significant.\n3 Results\nEight dogs were included in the data analysis, including one\nHusky, two Labrador Retrievers, two Golden Retrievers, two Mixed\nbreeds, and one Yorkshire Terrier. The mean age was 7.7 years\n(7.7 ± 4.43 years, range from 1.5–12 years) while the mean body\nweight was 21.6 kg (21.6 ± 8.62 kg, range from 6.2–30.6 kg). Three\ndogs were castrated males, four were spayed females, and one dog was\nan intact female.\nThe CODI was performed on every visit (total 11 times), and all\nshowed a score of 0. The observed items were scored by clients, and\nthe results of 11 scores, which included baseline two evaluations and\nthree sets of three evaluations in each dog, did not show any changes\non baseline, during taping periods, and taping with hair (another three\nevaluations were after the hair grew).\nFor the static stance, there were 17 measurements, which were 2\nmeasurements on the baseline and 3 sets of 5 measurements in each\ndog, including the average stance of the left front limb, the average\nright front limb, the average left hind limb, and the average right hind\nlimb. The results of the comparison of four stance variables between\nthree different taping methods (n = 8) showed only one significant\ndifference (p < 0.05) on the right front limb, which was on the diagonal\nto the taped limb. The post hoc analysis showed that the significant\ndifference was between no effect taping and inhibition taping (Table 1).\nThere were 17 measurements for the PSW analysis when\ncomparing three different methods of taping for 48 hours at the walk.\nThe comparison of 29 variables showed 3 significant differences\n(p < 0.05), which were on the vertical impulse of the left hind, and the\naverage maximal force of left hind and right hind on the measurements\nbetween three different taping methods on the walk (n = 8). The value\nwas K = higher than K + , and K + higher than K−. Post hoc analysis\n\nThe value\nwas K = higher than K + , and K + higher than K−.Post hoc analysis\nshowed there was no significant difference between K + and K=.\nWhen comparing K + with K− and K = with K−, it showed differences\n(p < 0.05) in the vertical impulse of the left hind and the average\nmaximal force of the left hind, which were both on the taping limb.\nWhen comparing K = with K−, they showed differences (p < 0.05) on\nthe vertical impulse of left hind, and average maximal force of left hind\nand right hind. There were 3 significant differences (p < 0.05), which\nwere on hind step width, left hind reach, and symmetry of front on the\nmeasurements between three different taping methods on trot (n = 8).\nIn hind step width, the value of K− was higher than K+, and K + was\nhigher than K=. In the left hind reach, K + was higher than K−, and\nK− was higher than K=. In symmetry of front, the value of K = was\nhigher than K+, and K + was higher than K−. The post hoc analysis\nshowed there were differences (p < 0.05) in all these three variables\nwhen comparing K + with K=. There was no difference in all these\nthree variables when comparing K + with K−. There were differences\n(p < 0.05) in hind step width and symmetry of front when comparing\nK = with K−. (Table 2; for detailed data, see Supplementary Material).\nThere was no significant difference in the velocity, which is\nexpected due to the group comparison of dogs of varying sizes.\nSimilarly, no differences were observed in stride length and step length\ndue to the cross-over group comparison over time. There were no\ndifferences in stance and swing phase during both walk and trot with\ndifferent taping methods.\nRegarding the comparison between taping with hair (H) and with\nclipped (C), one dog experienced an idiopathic seizure during the hair\ngrowth period. The owner was unwilling to return for the taping with\nTABLE 1  The comparison of three different taping methods in stance.\nAll dogs (n = 8) clipped taping in stance\nStance (% of body weight)\nLt_FL\nRt_FL\nLt_HL\nRt_HL\nFacilitate taping period (K+)\nMean\n31.14\n31.24\n18.81\n18.87\nSD\n6.07\n6\n3.87\n4.96\nMedian\n32\n30\n19\n19\nCI\n19.24−43.04\n19.49−43\n11.23−26.39\n9.14−28.59\n\nAll dogs (n = 8) clipped taping in stance\nStance (% of body weight)\nLt_FL\nRt_FL\nLt_HL\nRt_HL\nFacilitate taping period (K+)\nMean\n31.14\n31.24\n18.81\n18.87\nSD\n6.07\n6\n3.87\n4.96\nMedian\n32\n30\n19\n19\nCI\n19.24−43.04\n19.49−43\n11.23−26.39\n9.14−28.59\nNo effect taping period (K=)\nMean\n31.61\n30.38\n19.33\n18.68\nSD\n6.78\n6.2\n4.16\n4.56\nMedian\n32\n30\n18.5\n18\nCI\n18.33−44.89\n18.22−42.53\n11.18−27.49\n9.75−27.62\nInhibit taping period (K−)\nMean\n29.83\n32.35\n19.61\n18.22\nSD\n7.03\n6.03\n4.45\n5.52\nMedian\n31\n32.5\n20\n18\nCI\n16.04−43.61\n20.53−44.17\n10.89−28.32\n7.41−29.03\nK-W test (p-value < 0.05)\n0.051\n0.03\n0.467\n0.418\nK + VS. K=\n0.294\nK + VS. K−\n0.113\nK = VS. K− (p-value < 0.05)\n0.009\nLt_FL, left front limb Rt_FL, right front limb Lt_HL, left hind limb Rt_HL, right hind limb.\nSD, Standard deviation; CI, Confidence intervals; K–W test, Kruskal–Wallis test.\n\nLiu et al.\b\n10.3389/fvets.2025.1650607\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nhair session, and the dog’s clinical condition was not stable enough to\ncontinue in the study. Seven dogs completed the study. The\ncomparison of 29 variables in the walking trials revealed 9 significant\ndifferences (p < 0.05) between haired and clipped taping conditions\n(n = 7). These differences were observed in vertical impulse of the left\nhind limb (C > H), stride length (C > H), step length of the left hind\nlimb (C < H) and right hind limb (C < H), step width of the hind limb\n(C > H), hind reach of the left (C < H) and right limbs (C < H), stance\nphase of the left hind limb (C > H), and swing phase of the left hind\nlimb (C < H). In the trotting trials (n = 7), two significant differences\n(p < 0.05) were found: vertical impulse of the right front limb (C > H)\nand step width of the hind limb (C > H). There were two significant\ndifferences (p < 0.05), which were on the vertical impulse of the right\nfront (C > H) and step width of the hind (C > H) on the measurements\nbetween with hair and clipped taping on trot (n = 7). (Table 3; for\ndetailed data, see Supplementary Material).\nComparison of taping methods on the same day among all\ndogs (n = 8) showed that the average number of significantly\ndifferent items during walking ranged from 4.5 to 6.01 on Day 1.\nThe greatest differences were observed between K + and K=,\nfollowed by K + versus K−, with K = versus K − showing the\nfewest differences. On Day 2, the range was 5.63 to 5.76, with\nK + versus K − showing the highest differences, followed by\nK + versus K=, and K = versus K − being the lowest. On Day 3, the\nrange was 3.5 to 6.63, again with K + vs. K = being the highest,\nfollowed by K + versus K−, and K = vs. K − being the lowest.\nWhen trotting, the number of items with significant differences\n(p < 0.05) across taping methods on Day 1 ranged from 2.875 to\n3.5, with K = versus K − showing the most differences, followed\nby K + versus K−, and K + versus K = showing the fewest. On Day\n2, the range was 2.375 to 5.375, with K + versus K = highest,\n\n05) across taping methods on Day 1 ranged from 2.875 to\n3.5, with K = versus K − showing the most differences, followed\nby K + versus K−, and K + versus K = showing the fewest.On Day\n2, the range was 2.375 to 5.375, with K + versus K = highest,\nfollowed by K + versus K−, and K = versus K − was the lowest. On\nDay 3, it ranged from 4 to 6.375, with K + versus K − highest,\nfollowed by K + versus K=, and K = versus K − was the lowest. The\npatterns of these comparisons with different taping methods were\ninconsistent. Likewise, the distributions of kinetic and\ntemporospatial variables also varied inconsistently. (Figures 3, 4;\nfor detailed data, see Supplementary Material).\nTABLE 2  The comparison of the measurements with significant differences in walk and trot with three different taping methods (n = 8).\nVariables\nTaping\nMean\nSD\nMedian\nConfidence\nintervals\nK-W\ntest\nPost hoc analysis\np\nvalue\nK + VS.\nK=\nK + VS.\nK−\nK = VS.\nK−\nWalk\nVertical\nimpulse\n(VI)\n[Newton-\nsecond]\nVI _LR\nK+\n3468.59\n358.9\n3485.94\n2765.14\n4172.04\n0.009\n0.891\n0.009\n0.006\nK=\n3523\n272.28\n3454.71\n2989.33\n4056.68\nK−\n3419.51\n266.21\n3424.18\n2897.74\n3941.29\nAverage\nmax force\n(AVGF)\n[%body\nweight]\nAVGF_\nLR\nK+\n41.67\n4.51\n41.59\n32.84\n50.51\n0.048\n0.902\n0.038\n0.028\nK=\n41.75\n4.34\n41.19\n33.25\n50.25\nK−\n40.66\n4.98\n39.68\n30.89\n50.43\nAverage\nmax force\n(AVGF)\n[%body\nweight]\nAVGF_\nRR\nK+\n40.01\n4.52\n39.62\n31.16\n48.86\n0.042\n0.487\n0.078\n0.014\nK=\n40.4\n4.25\n40.18\n32.07\n48.74\nK−\n39.23\n4.56\n38.21\n30.3\n48.17\nTrot\nStep width\n(SWD)\n[cm]\nSWD_\nHind\nK+\n5.17\n1.31\n4.98\n2.6\n7.75\n0.008\n0.008\n0.942\n0.006\nK=\n4.67\n1.63\n4.53\n1.48\n7.86\nK−\n5.21\n1.44\n5.06\n2.39\n8.04\nHind reach\n(Hind R)\n[cm]\nHind\nR_L\nK+\n5.4\n20.64\n−0.74\n−35.05\n45.86\n0.02\n0.005\n0.277\n0.089\nK=\n0.33\n14.08\n−3.39\n−27.27\n27.92\nK−\n2.51\n14.29\n−2.12\n−25.5\n30.53\nSymmetry\nindex (SI)\n[%]\nSI _Front\nK+\n4.32\n4.11\n2.97\n−3.73\n12.37\n0.022\n0.012\n0.821\n0.023\nK=\n5.1\n3.5\n4.88\n−1.76\n11.96\nK−\n4.17\n3.51\n3.23\n−2.71\n11.04\nThree different taping methods: facilitate taping period K+, no effect taping period K=, inhibit taping period K−; LR, left rear, RR, right rear; L, left; SD, Standard deviation; Green background\nmeans kinetic related; Yellow background means temporospatial related; K-W test, Kruskal–Wallis test.\n\nLiu et al.\b\n10.3389/fvets.2025.1650607\nFrontiers in Veterinary Science\n07\nfrontiersin.org\n4 Discussion\nKinesiology taping (KT) is widely used in human sports medicine\ndue to its perceived benefits in injury prevention and muscle\nperformance enhancement. This concept has recently been applied to\nhealthy dogs. Two key questions remain: (1) Does taping direction\n(facilitation vs. inhibition) affect dogs similarly to humans? (2) Is KT\neffective when applied over fur? While KT use is growing, scientific\nevidence in dogs is limited.\nA study by Noel et al. was among the first to investigate KT in\nhealthy canines. They applied tape with 25% stretch in a proximal-to-\ndistal direction across the tarsal joint, aligning with a facilitation\ntechnique, to assess its impact on pelvic limb movement. Gait analysis\nperformed within 2 h of application revealed no significant changes\nin kinetic or kinematic parameters during walking, trotting, or\nstepping over obstacles (34). Unlike their study, this research evaluated\nthe extended effects of different KT applications on canine gait,\nincorporating both kinetic and temporospatial measurements over\nmultiple days.\nTo our knowledge, this is the first study to investigate the sustained\neffects of different KT methods in healthy dogs over an extended\nperiod. The CODI results showed that the taping did not affect\nobservable mobility in healthy dogs. Given the subjective nature of\nCODI, static and dynamic objective analyses (stance analyzer and\npressure-sensing walkway) were also performed (35). These objective\nanalyses showed no significant differences in stance based on taping\nmethod in the taped leg. This aligns with human research where KT’s\nimmediate and delayed effects on femoral quadriceps performance,\nbalance, and lower limb function in healthy women found no change\nin static balance (36). While another study in humans demonstrated\nthat KT benefits dynamic activities in children with cerebral palsy, it\nhad no effect on static activities (37). The taping did not affect the\nstatic stance results regardless of the health status in human studies.\n\nWhile another study in humans demonstrated\nthat KT benefits dynamic activities in children with cerebral palsy, it\nhad no effect on static activities (37).The taping did not affect the\nstatic stance results regardless of the health status in human studies.\nInterestingly, we observed effects on non-taped legs (p < 0.05),\nsuggesting that dogs perceive the sensation of the tape while standing\nand adjust their other legs to maintain balance. Unlike humans, who\ncan verbalize these sensations, dogs’ responses are reflected in stance\nchanges, likely representing anticipatory postural adjustments where\nthe central nervous system reorganizes the activity of individual\npostural muscles to compensate, ensuring stance balance (38).\nIt has been proposed that KT may facilitate or inhibit muscle\nfunction depending on the taping direction. However, study findings\nare inconsistent; many report no significant facilitatory or inhibitory\neffects of KT on muscle strength or electromyographic activity\nTABLE 3  The comparison of the measurements showing significant difference with clipped/haired facilitate taping (n = 7).\nClipped\nHaired\nMean\nSD\nMedian\nConfidence\nintervals\nMean\nSD\nMedian\nConfidence\nintervals\np-value\nIn walk\nVertical\nimpulse (VI)\n[newton-\nsecond]\nVI _LR\n3456.28\n456.29\n3475.54\n2559.66\n4352.9\n3364.93\n451.31\n3366.71\n2478.09\n4251.76\n0.03\nStride length\n[cm]\nStride_\nlength\n65.49\n15.89\n70.78\n34.26\n96.73\n67.25\n15.1\n73.8\n37.58\n96.93\n0.02\nStep length\n(SL) [cm]\nSL_LR\n32.47\n8.4\n35.15\n15.95\n48.98\n33.59\n7.58\n36.42\n18.7\n48.49\n0.04\nSL_RR\n32.62\n7.9\n34.51\n17.1\n48.14\n34.36\n6.48\n36.42\n21.63\n47.09\n0.01\nStep width\n(SWD)[cm]\nSWD_\nHind\n6.33\n1.73\n6.49\n2.94\n9.73\n5.74\n1.74\n5.67\n2.33\n9.15\n0.04\nHind reach\n(Hind R)\n[cm]\nHind\nR_L\n11.53\n6.03\n12.42\n−0.32\n23.38\n14.06\n6\n13.97\n2.26\n25.85\n0.05\nHind\nR_R\n10.98\n6.44\n13.41\n−1.67\n23.63\n12.58\n7.28\n13.55\n−1.73\n26.88\n0.01\nStance phase\n(ST) [%]\nST _LR\n63.31\n2.36\n63.6\n58.67\n67.96\n60.73\n6.4\n61.73\n48.15\n73.31\n0.01\nSwing phase\n(SW) [%]\nSW _LR\n36.69\n2.36\n36.4\n32.04\n41.33\n39.27\n6.4\n38.27\n26.69\n51.85\n0.01\nIn trot\nVertical\nimpulse (VI)\n[newton-\nsecond]\nVI _RF\n6286.43\n1622.55\n5972.28\n3098.12\n9474.74\n5726.39\n921.24\n5807.85\n3916.14\n7536.63\n0.01\nStep width\n(SWD) [cm]\nSWD_\nHind\n5.38\n1.47\n5.19\n2.49\n8.27\n5.08\n1.38\n5.36\n2.37\n7.79\n0.04\nLR, left rear; RR, right rear; RF, right front; L, left; SD, Standard deviation; Green background means kinetic-related; Yellow background means temporospatial-related.\n\nLiu et al.\b\n10.3389/fvets.2025.1650607\nFrontiers in Veterinary Science\n08\nfrontiersin.org\n(39–42). In our study, the PSW analysis, our dynamic measurement\nshowed that taping with the inhibition technique (insertion to origin)\nproduced the weakest effects on kinetic variables, irrespective of\nwhether the dog was walking or trotting. While our study showed\nminimal overall effects, different taping methods may still influence\ngait patterns differently in dogs. Therefore, applying tape in the\nincorrect direction is unlikely to yield beneficial outcomes.\nWhen comparing different taping methods on the same day during\nwalking and trotting, the variable changes were not consistently\nassociated with specific days or taping types. The specific gait variables,\nwhether kinetic or temporospatial, showed that significant differences\nvaried independently. We assume that the dogs likely adjusted their\nneuromuscular activity and limb locomotion in response to the subtle\nstimulation from the thin elastic tape, helping maintain their gait\nstability in a healthy state. These findings indicate that taping effects\nvaried across days when the tape remained in place for 48 h.\nThis prompts a question that warrants further investigation: when\ntaping is needed for several consecutive days, is it more effective to\nreapply the tape daily or to leave it on to preserve consistent outcomes?\nA human study by Sheikhi et al. (43) investigated the immediate\neffects of various taping tensions (0, 50, and 75%) on tuck jump\nperformance in 75 active individuals. The tape was applied bilaterally\nto lower limb muscles, such as the gastrocnemius, biceps femoris,\nvastus lateralis, vastus medialis, rectus femoris, and gluteus medius,\nfor 72 h. No significant differences were observed between the KT\ngroups at the immediate, 24-h, and 72-h time points (43). In contrast,\nour study found that different taping methods in dogs affected gait\npatterns variably across days during both walking and trotting,\nalthough the overall taping effects remained limited.\nQuadrupedal dogs, unlike bipedal humans, possess a greater\n\nIn contrast,\nour study found that different taping methods in dogs affected gait\npatterns variably across days during both walking and trotting,\nalthough the overall taping effects remained limited.Quadrupedal dogs, unlike bipedal humans, possess a greater\ncompensatory ability. A study of 39 amputee dogs using pressure-\nsensitive walkways examined forelimb and hindlimb amputations at\ndifferent levels. High amputations involved hip or scapular\ndisarticulation, while low amputations were distal limb removals. The\nlevel of forelimb amputation did not affect weight distribution, but\nhigh hindlimb amputations led to increased loading on the opposite\nlimb (30). Dogs with permanent limb loss compensate consistently. In\ncontrast, our study showed that healthy dogs responded inconsistently\nto temporary unilateral taping on the biceps femoris.\nThe effect of KT on the enhancement of muscle activity has been\nobserved in healthy human athletes experiencing muscle soreness.\nKirmizigil et al. (44) published a study investigating the effects of KT\nFIGURE 3\nThe average number of items with significant differences compared to different taping methods with each other on the same day under walk (n = 8).\n\nLiu et al.\b\n10.3389/fvets.2025.1650607\nFrontiers in Veterinary Science\n09\nfrontiersin.org\non delayed onset muscle soreness after exercise. The tape was applied\nbilaterally to the rectus femoris of healthy amateur athletes. The results\nindicated some beneficial effects on performance and balance (44).\nFor this reason, KT is commonly used by competitive athletes to\nalleviate soreness and maintain performance, rather than to\nincrease strength.\nIn humans with musculoskeletal diseases, there is evidence\nsuggesting that kinesiology tape may positively affect muscle strength\nduring weight-bearing movements (45). During non-weight-bearing\nmovements, a human study by Cho et al. (46) demonstrated that KT\ntherapy applied to patients with chronic lateral epicondylitis resulted\nin improved pain-free grip strength. The impact of KT on strength\nevaluation seems to differ between humans and animals (46). In\nhumans, strength is typically assessed using open-chain exercises,\nwhereas in animals, it is usually evaluated during closed-chain\nactivities such as walking or trotting.\nVithoulka et al. (42) showed that the application of KT to the\nanterior surface of the thigh in the direction of vastus medialis,\nlateralis, and rectus femoris fascia could increase eccentric muscle\nstrength. However, a study in horses investigating the effects of KT on\nthe trajectory of the forelimb and the muscle activity of the\nM. brachiocephalicus and the M. extensor carpi radialis at the walk and\ntrot found no significant differences among no tape, with tape, and\npost tape conditions (47). To date, no research has demonstrated an\nenhancement of muscle activity in healthy dogs using KT. In our\nstudy, healthy dogs performed activities with taping at the walk and\ntrot, but no consistently significant results were obtained. The healthy\ndogs appear to maintain homeostasis as effectively as possible.\nOur results showed that some kinetic and temporospatial variables\nhad significant differences (p < 0.05) when comparing three different\ntaping methods across different days. However, the variable items\nchanged inconsistently. This could indicate that intrinsic body\n\nOur results showed that some kinetic and temporospatial variables\nhad significant differences (p < 0.05) when comparing three different\ntaping methods across different days.However, the variable items\nchanged inconsistently.This could indicate that intrinsic body\nmodulations were caused by the stimulation of the skin via taping.\nThis inner drive of movement generated by the elastic tape might be\nrelated to Ia afferent neurons and alpha motor neurons (48) and the\nrelated reflex. Bagheri et al. (49) examined the H-reflex recruitment\ncurve of the gastrosoleus muscle under several conditions: no\ntreatment (control), KT, KT applied over skin with topical anesthesia,\ntopical anesthesia alone, and sham taping without tension. Their\nresults showed that KT, whether applied to normal or anesthetized\nFIGURE 4\nThe average number of items with significant differences compared to different taping methods with each other on the same day under trot (n = 8).\n\nLiu et al.\b\n10.3389/fvets.2025.1650607\nFrontiers in Veterinary Science\n10\nfrontiersin.org\nskin, enhanced the H-reflex parameters. In contrast, topical anesthesia\nalone suppressed these parameters, while sham taping had no effect.\nThese findings indicate that kinesiology taping promotes muscle\nactivity, likely by stimulating cutaneous receptors that influence the\nmotor neuron pool of the gastrosoleus muscle (49). Further research\nis needed to explore its influence on sensory input and motor\nresponses in canine models.\nThe comparison between taping with hair and clipped is interesting.\nIn our analysis, the results suggest that the presence or absence of hair\ninfluences the effect of taping. To explore how taping over hair might still\nbe effective, we focused on kinetic parameters such as vertical impulse\nand peak vertical force on the applied limb, which directly reflect\nchanges in force. Gillette and associates (50) mentioned that both peak\nvertical force and vertical impulse are indicators of limb usage. In our\nstudy, we only assessed the effects of facilitative taping on the left hind\nlimb during both walk and trot, comparing conditions with hair and\nclipped. Since a dog’s fur is considered a physical barrier, it may diminish\nthe effectiveness of taping. This decision is supported by a previous study\nconducted on healthy young adults, which compared three groups: one\nreceiving kinesiology tape from origin to insertion (facilitation), another\nfrom insertion to origin (inhibition), and a control group with no taping.\nThe results showed no changes in the inhibition or control groups, while\nfacilitation taping led to increased muscle tone, elasticity, and stiffness\n(40). Our findings demonstrated a significant decrease (p < 0.05) in\nforce-related variables in walk and trot when hair was present, indicating\na diminished or minimal taping effect. Other variables indirectly related\nto the taped limb exhibited variable changes, potentially indicating\ncompensatory adaptations. Overall, the results suggest that hair reduces\nthe efficacy of kinesiology taping and that application on clipped areas\n\nOther variables indirectly related\nto the taped limb exhibited variable changes, potentially indicating\ncompensatory adaptations.Overall, the results suggest that hair reduces\nthe efficacy of kinesiology taping and that application on clipped areas\nis necessary to achieve the intended therapeutic effects.\nTheoretically, KT offers contraction on the skin mimicking a second\nlayer of skin, which produces tactile input. It is similar to the innocuous\nmechanical stimulation of low-threshold mechanoreceptors by which\nskin or hair react to perceive the stimuli—this is as opposed to the high-\nthreshold mechanoreceptors that respond to harmful mechanical\nstimulation (51). In hairy skin, tactile stimuli are transduced through\nthree types of hair follicles (51). It has been suggested that KT may\nstimulate hair follicles to trigger signal input in dogs, as noted by the\nKinesio Group (52). In our study, notable differences were found\nbetween taping on shaved versus unshaved skin, even though overall\nmobility was unaffected. It is still unclear whether the direct skin contact\nwith the tape does not arouse enough stimulation, or if the presence of\nhair itself interferes with the effectiveness of kinesiology taping—both\npossibilities need to be explored further. Additionally, the exact influence\nof hair follicle stimulation on how dogs respond to KT remains\nundefined and calls for more research.\nOur results offered partial support for the initial hypothesis. As\nthe first study to examine various kinesiology taping (KT) techniques\nin healthy dogs over time, it identified only subtle and inconsistent\nimpacts on gait kinetics and temporospatial parameters, indicating\nthat taping has a limited effect on canine movement. Interestingly,\nnon-taped limbs showed compensatory changes, likely due to\nproprioceptive feedback. The inconsistent variable changes across\ndifferent taping methods in healthy dogs did not align with the\nexpected facilitation or inhibition mechanisms. Furthermore, the\npresence of hair significantly affected taping effectiveness,\n\nThe inconsistent variable changes across\ndifferent taping methods in healthy dogs did not align with the\nexpected facilitation or inhibition mechanisms.Furthermore, the\npresence of hair significantly affected taping effectiveness,\nunderscoring the importance of skin stimulation in KT outcomes.\nThese results emphasize the adaptive capacity of healthy dogs and\npoint to the need for additional studies in clinical settings.\n4.1 Limitations and conclusion\nThere were notable limitations to our study. The main limitation\nof this study was the small number of dogs, which reduced the\nstatistical power. Recruiting a large sample for a prospective study\nrequiring multiple three consecutive days of visits was challenging, as\nmost owners could not commit to the protocol due to time constraints.\nAnother limitation was the diverse range of breeds and sizes,\nresulting in a heterogeneous sample population. The KT area was\napplied in proportion to the target muscle by length. However, no\nresearch exists that determines the ideal KT area in relation to dog size\nor taping area. The same tape width was used across different-sized\ndogs, and the adequacy of this dosage remains uncertain. In addition,\nsize and breed differences, hair length also posed a challenge in\nassessing KT on fur. This study included two short-coated dogs, two\ncombination-coated dogs, three double-coated dogs, and one silky-\ncoated dog, demonstrating diversity.\nAnother limitation of this study is the difficulty in evaluating\nmuscle strength or activity in dogs. KT research in humans has\nfocused on several outcomes, including muscle activity, neuromuscular\nefficiency, rate of force development, and movement performance\n(53). Among these, movement performance—such as ground reaction\nforce—is the most feasible and convenient to assess in dogs. At\npresent, gait analysis remains the main approach for evaluation;\nhowever, it does not fully reflect the functional performance of the\nbiceps femoris, a key muscle involved in stifle extension. Wireless\nelectromyography (EMG) offers a dependable means of assessing\n\nWireless\nelectromyography (EMG) offers a dependable means of assessing\nindividual muscle activity and can objectively detect kinematic\nchanges influenced by specific muscles. Nevertheless, since KT\nfunctions through tactile stimulation of the skin to activate nerves,\napplying any device to the skin under dynamic conditions, such as\nEMG sensors, may interfere with the taping effect, presenting\na dilemma.\nAnother limitation was that the PSW sensors measured the foot\nat a frequency of 100 Hz, which only recognizes walking and trotting,\nnot running. We are unsure if the KT effect differs during high-speed\nmotion, given the potential compensation of limbs at higher velocities\nwhen running.\nWe concluded that in healthy dogs, KT had no impact on overall\nmobility or stance of the affected leg, with only minor effects observed\nduring walking and trotting. Different KT application methods led to\nslight, inconsistent changes in gait pattern. Variability in kinetic and\ntemporospatial data was expected due to the adaptability of healthy\ndogs. These fluctuations likely reflect natural gait variability. While KT\nmay influence nerve input and trigger compensatory limb\nadjustments, its overall impact on performance appears limited.\nInterestingly, only a decreased vertical impulse on the taped limb\nwhen taping with hair was observed, warranting further research on\nKT’s effect on hair follicles.\n4.2 Future work\nThe results in this study lay an essential foundation for future\nstudies examining other muscle groups that significantly contribute to\nmuscle atrophy and functional decline in dogs. Consideration should\nbe given to applying double or triple taping on counteracting muscle\nsides that work in synchrony during extension or flexion. Several\n\nLiu et al.\b\n10.3389/fvets.2025.1650607\nFrontiers in Veterinary Science\n11\nfrontiersin.org\nparameters, both kinetic and kinematic, showed significant variability.\nThe relationship between these factors and the dog’s response to KT\nneeds further exploration. It remains unclear how these elements\ninfluence performance. To establish conclusive results, a more\ncomprehensive study design is required. Future assessments could\ninclude evaluating the effects of KT on fur, potentially by applying tape\nto smooth-coated dogs like French Bulldogs or Pointers to study its\nimpact on hair follicles.\nData availability statement\nThe original contributions presented in the study are included in\nthe article/Supplementary material, further inquiries can be directed\nto the corresponding author.\nEthics statement\nThe animal studies were approved by National Taiwan University\nInstitutional Animal Care and Use Committee. The studies were\nconducted in accordance with the local legislation and institutional\nrequirements. Written informed consent was obtained from the\nowners for the participation of their animals in this study.\nAuthor contributions\nC-ML: Methodology, Writing  – review & editing, Writing  –\noriginal draft. S-LL: Writing  – review & editing, Data curation,\nInvestigation. H-ML: Writing – review & editing, Formal analysis. JH:\nSupervision, Writing – review & editing.\nFunding\nThe author(s) declare that no financial support was received for\nthe research and/or publication of this article.\nAcknowledgments\nThe authors would like to thank NTU Statistical Consulting\nService of Statistical Education Center for assistance with data analysis.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\n\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. Any product\nthat may be evaluated in this article, or claim that may be made by its\nmanufacturer, is not guaranteed or endorsed by the publisher.\nSupplementary material\nThe Supplementary material for this article can be found online\nat: https://www.frontiersin.org/articles/10.3389/fvets.2025.1650607/\nfull#supplementary-material\nReferences\n1.\tKim, H, and Lee, B. The effects of kinesio tape on isokinetic muscular function of\nhorse racing jockeys. J Phys Ther Sci. (2013) 25:1273–7. doi: 10.1589/jpts.25.1273\n2.\tMostert-Wentzel, K, Swart, JJ, Masenyetse, LJ, Sihlali, BH, Cilliers, R, Clarke, L,\net al. Effect of kinesio taping on explosive muscle power of gluteus maximus of male\nathletes. South Afr J Sports Med. 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Kinesio taping improves pain and function in\n\nAuxetic structures used in kinesiology tapes can improve form-fitting and\npersonalization.Sci Rep.(2022) 12:13509.doi: 10.1038/s41598-022-17688-w\n6.Lu, Z, Li, X, Chen, R, and Guo, C.Kinesio taping improves pain and function in\npatients with knee osteoarthritis: a meta-analysis of randomized controlled trials. Int J\nSurg. (2018) 59:27–35. doi: 10.1016/j.ijsu.2018.09.015\n7.\tSimoneau, GG, Degner, RM, Kramper, CA, and Kittleson, KH. Changes in ankle\njoint proprioception resulting from strips of athletic tape applied over the skin. J Athl\nTrain. (1997) 32:141.\n8.\tPina, AKA, and Lima, AKP d. Effects of application of Kinesio taping on the Tibialis\nanterior muscle in cerebral palsy: case series. Res Soc Dev. (2022) 11:e44811225861. doi:\n10.33448/rsd-v11i2.25861\n9.\tWilliams, S, Whatman, C, Hume, PA, and Sheerin, K. Kinesio taping in treatment\nand prevention of sports injuries: a meta-analysis of the evidence for its effectiveness.\nSports Med. (2012) 42:153–64. doi: 10.2165/11594960-000000000-00000\n10.\tMarotta, N, Lippi, L, Ammendolia, V, Calafiore, D, Inzitari, MT, Pinto, M, et al.\nEfficacy of kinesio taping on upper limb volume reduction in patients with breast\ncancer-related lymphedema: a systematic review of randomized controlled trials. Eur J\nPhys Rehabil Med. (2023) 59:237–47. doi: 10.23736/S1973-9087.23.07752-3\n11.\tPajero Otero, V, Garcia Delgado, E, Martin Cortijo, C, Romay Barrero, HM, de\nCarlos Iriarte, E, and Avendano-Coy, J. Kinesio taping versus compression garments for\ntreating breast cancer-related lymphedema: a randomized, cross-over, controlled trial.\nClin Rehabil. (2019) 33:1887–97. doi: 10.1177/0269215519874107\n12.\tGarcia-Gomariz,\nC,\nGarcia-Martinez,\nMT,\nAlcahuz-Grinan,\nM,\nHernandez-Guillen, D, and Blasco, JM. Effects on pain of kinesiology tape in patients\nwith plantar fasciitis: a randomized controlled study. Disabil Rehabil. (2024) 46:5490–6.\ndoi: 10.1080/09638288.2024.2304645\n13.\tHanson, JH, Ostrem, JD, and Davies, BL. Effect of kinesiology taping on upper\ntorso mobility and shoulder pain and disability in US masters national championship\n\nLiu et al.\b\n10.3389/fvets.2025.1650607\nFrontiers in Veterinary Science\n12\nfrontiersin.org\nswimmers: an exploratory study. J Manip Physiol Ther. (2019) 42:247–53. doi: 10.1016/j.\njmpt.2018.11.009\n14.\tde Sire, A, Curci, C, Ferrara, M, Losco, L, Spalek, R, Cisari, C, et al. Efficacy of\nkinesio taping on hand functioning in patients with mild carpal tunnel syndrome. A\ndouble-blind randomized controlled trial. J Hand Ther. (2022) 35:605–12. doi: 10.1016/j.\njht.2021.04.011\n15.\tMohamed, HK, Yousef, AM, Kamel, H-EH, Oweda, KA, and El-samea, GAA.\nKinesio taping and strength recovery of postnatal abdominal muscles after cesarean\nsection. Egypt J Phys Ther. (2020) 4:12–9. doi: 10.21608/ejpt.2020.41427.1016\n16.\tMolle, S. Kinesio taping fundamentals for the equine athlete. Vet Clin North Am\nEquine Pract. (2016) 32:103–13. doi: 10.1016/j.cveq.2015.12.007\n17.\tBrunke, M. Kinesiology taping In: B Bockstahler, editor. Essential facts of physical\nmedicine, rehabilitation and sports medicine in companion animals. 1st ed. Barbara\nBockstahler, ed. Verden: VBS GmbH (2019). 263–7.\n18.\tBredlau-Morich, K. Kinesiology taping for dogs. North Pomfret: Trafalgar Square\nBooks (2020).\n19.\tMikail, S. Kinesiology taping for dogs. Josink Kolkweg: Thysol Group BV (2022).\n20.\tCarr, BJ, Levine, D, and Marcellin-Little, DJ. Gait changes resulting from\northopedic and neurologic problems in companion animals. Adv Small Anim Care.\n(2023) 4:1–20. doi: 10.1016/j.yasa.2023.05.001\n21.\tTorres, BT. Objective gait analysis In: FM Duerr, editor. Canine Lameness.\nHoboken, NJ: John Wiley & Sons (2020). 15–30.\n22.\tAlves, J, Santos, A, Jorge, P, Lavrador, C, and Carreira, L. Evaluation of four clinical\nmetrology instruments for the assessment of osteoarthritis in dogs. Animals. (2022)\n12:2808. doi: 10.3390/ani12202808\n23.\tAlves, JC, Santos, A, Jorge, P, Lavrador, C, and Carreira, LM. Characterization of\nweight-bearing compensation in dogs with bilateral hip osteoarthritis. Top Companion\nAnim Med. (2022) 49:100655. doi: 10.1016/j.tcam.2022.100655\n24.\tClough, WT, Canapp, SO Jr, Taboada, L, Dycus, DL, and Leasure, CS. Sensitivity\n\nCharacterization of\nweight-bearing compensation in dogs with bilateral hip osteoarthritis.Top Companion\nAnim Med.(2022) 49:100655.doi: 10.1016/j.tcam.2022.100655\n24.Clough, WT, Canapp, SO Jr, Taboada, L, Dycus, DL, and Leasure, CS.Sensitivity\nand specificity of a weight distribution platform for the detection of objective lameness\nand orthopaedic disease. Vet Comp Orthop Traumatol. (2018) 31:391–5. doi: 10.1055/\ns-0038-1667063\n25.\tDiGiovanni, LC, Roush, JK, and Berke, K. Preoperative and postoperative stance\nanalysis in dogs with patellar luxation confirms lameness improvement after surgery.\nAm J Vet Res. (2023) 84:1–7. doi: 10.2460/ajvr.22.10.0186\n26.\tWilson, ML, Roush, JK, and Renberg, WC. Comparison of the effect of dog,\nsurgeon and surgical procedure variables on improvement in eight-week static weight-\nbearing following tibial plateau levelling osteotomy. Vet Comp Orthop Traumatol. (2018)\n31:396–404. doi: 10.1055/s-0038-1667139\n27.\tDemircioglu, I, Gündemir, O, Demiraslan, Y, Güngören, G, Ince, N, and Yilmaz, B.\nTemporospatial and kinetic gait analysis in Aksaray Malakli shepherd dogs [article].\nTurk J Vet Anim Sci. (2022) 46:2:559–64. doi: 10.55730/1300-0128.4226\n28.\tFahie, M, Cortez, J, Ledesma, M, and Su, Y. Pressure mat analysis of walk and trot\ngait characteristics in 66 normal small, medium, large, and giant breed dogs [article].\nFront Vet Sci. (2018) 5:256. doi: 10.3389/fvets.2018.00256\n29.\tGundemir, O, Duro, S, Aydin Kaya, D, and Zenginler Yazgan, Y. Temporo-spatial\nand kinetic gait parameters in English setter dogs. Anat Histol Embryol. (2020) 49:763–9.\ndoi: 10.1111/ahe.12572\n30.\tFilho, TG, Rahal, SC, Kano, WT, Mesquista, LR, Mamprim, MJ, Silva, JP, et al. Gait\nanalysis of amputee dogs using a pressure-sensitive walkway. Vet Comp Orthop\nTraumatol. (2024) 37:189–95. doi: 10.1055/s-0044-1779727\n31.\tGuadalupi, M, Crovace, AM, Monopoli Forleo, D, Staffieri, F, and Lacitignola, L.\nPressure-sensitive walkway system for evaluation of lameness in dogs affected by\nunilateral cranial cruciate ligament rupture treated with porous Tibial tuberosity\n\nGuadalupi, M, Crovace, AM, Monopoli Forleo, D, Staffieri, F, and Lacitignola, L.Pressure-sensitive walkway system for evaluation of lameness in dogs affected by\nunilateral cranial cruciate ligament rupture treated with porous Tibial tuberosity\nadvancement. Vet Sci. (2023) 10:696. doi: 10.3390/vetsci10120696\n32.\tLima, CGD, da Costa, RC, Foss, KD, and Allen, MJ. Temporospatial and kinetic\ngait variables of Doberman pinschers with and without cervical spondylomyelopathy.\nAm J Vet Res. (2015) 76:848–52. doi: 10.2460/ajvr.76.10.848\n33.\tCharalambous, D, Strasser, T, Tichy, A, and Bockstahler, B. Ground reaction forces\nand Center of Pressure within the paws when stepping over obstacles in dogs. Animals\n(Basel). (2022) 12:1702. doi: 10.3390/ani12131702\n34.\tNoel, RC, Shaw, LM, Millis, NH, Janas, K, and Millis, DL. Kinesiology taping does\nnot affect tarsal joint motion during selected exercises in dogs. Vet Sci. (2025) 12:439.\ndoi: 10.3390/vetsci12050439\n35.\tKieves, NR. Objective gait analysis: review and clinical applications [review]. Vet\nClin North Am Small Anim Pract. (2022) 52:857–67. doi: 10.1016/j.cvsm.2022.03.009\n36.\tLins, CA, Borges, DT, Macedo, LB, Costa, KS, and Brasileiro, JS. Delayed effect of\nKinesio taping on neuromuscular performance, balance, and lower limb function in\nhealthy individuals: a randomized controlled trial. Braz J Phys Ther. (2016) 20:231–9.\ndoi: 10.1590/bjpt-rbf.2014.0161\n37.\tda Costa, CSN, Rodrigues, FS, Leal, FM, and Rocha, N. Pilot study: investigating\nthe effects of Kinesio taping® on functional activities in children with cerebral palsy.\nDev Neurorehabil. (2013) 16:121–8. doi: 10.3109/17518423.2012.727106\n38.\tAruin, AS. The effect of changes in the body configuration on anticipatory postural\nadjustments. Mot Control. (2003) 7:264–77. doi: 10.1123/mcj.7.3.264\n39.\tCai, C, Au, I, An, W, and Cheung, R. Facilitatory and inhibitory effects of Kinesio\ntape: fact or fad? J Sci Med Sport. (2016) 19:109–12. doi: 10.1016/j.jsams.2015.01.010\n40.\tLopes, M, Torres, R, Romão, D, Dias, M, Valério, S, Espejo-Antúnez, L, et al.\n\ndoi: 10.1123/mcj.7.3.264\n39.Cai, C, Au, I, An, W, and Cheung, R.Facilitatory and inhibitory effects of Kinesio\ntape: fact or fad?J Sci Med Sport.(2016) 19:109–12.doi: 10.1016/j.jsams.2015.01.010\n40.Lopes, M, Torres, R, Romão, D, Dias, M, Valério, S, Espejo-Antúnez, L, et al.\nKinesiology tape increases muscle tone, stiffness, and elasticity: effects of the direction\nof tape application. J Bodyw Mov Ther. (2022) 30:176–80. doi: 10.1016/j.jbmt.2022.01.003\n41.\tPoon, KY, Li, S, Roper, M, Wong, M, Wong, O, and Cheung, R. Kinesiology tape\ndoes not facilitate muscle performance: a deceptive controlled trial. Man Ther. (2015)\n20:130–3. doi: 10.1016/j.math.2014.07.013\n42.\tVithoulka, I, Beneka, A, Malliou, P, Aggelousis, N, Karatsolis, K, and\nDiamantopoulos, K. The effects of Kinesio-taping® on quadriceps strength during\nisokinetic exercise in healthy non athlete women. Isokinet Exerc Sci. (2010) 18:1–6. doi:\n10.3233/IES-2010-0352\n43.\tSheikhi, B, Letafatkar, A, Marchetti, PH, Eftekhari, F, Wallace, BJ, Maselli, F, et al.\nEffects of Kinesio taping on Tuck jump performance in competitive male athletes. Int J\nSports Med. (2023) 44:516–23. doi: 10.1055/a-2035-8005\n44.\tKirmizigil, B, Chauchat, JR, Yalciner, O, Iyigun, G, Angin, E, and\nBaltaci, G. The effectiveness of Kinesio taping in recovering from delayed onset muscle\nsoreness: a crossover study. J Sport Rehabil. (2020) 29:385–93. doi: 10.1123/jsr.2018-0389\n45.\tYam, ML, Yang, Z, Zee, BC-Y, and Chong, KC. Effects of Kinesio tape on\nlower limb muscle strength, hop test, and vertical jump performances: a meta-\nanalysis. BMC Musculoskelet Disord. (2019) 20:1–12. doi: 10.1186/s12891-019-2564-6\n46.\tCho, YT, Hsu, WY, Lin, LF, and Lin, YN. Kinesio taping reduces elbow pain during\nresisted wrist extension in patients with chronic lateral epicondylitis: a randomized,\ndouble-blinded, cross-over study. BMC Musculoskelet Disord. (2018) 19:193. doi:\n10.1186/s12891-018-2118-3\n47.\tZellner, A, Bockstahler, B, and Peham, C. The effects of Kinesio taping on the\ntrajectory of the forelimb and the muscle activity of the Musculus brachiocephalicus and\n\nBMC Musculoskelet Disord.(2018) 19:193.doi:\n10.1186/s12891-018-2118-3\n47.Zellner, A, Bockstahler, B, and Peham, C.The effects of Kinesio taping on the\ntrajectory of the forelimb and the muscle activity of the Musculus brachiocephalicus and\nthe Musculus extensor carpi radialis in horses. PLoS One. (2017) 12:e0186371. doi:\n10.1371/journal.pone.0186371\n48.\tKonishi, Y. Tactile stimulation with kinesiology tape alleviates muscle weakness\nattributable to attenuation of Ia afferents. J Sci Med Sport. (2013) 16:45–8. doi: 10.1016/j.\njsams.2012.04.007\n49.\tBagheri, R, Pourahmadi, MR, Sarmadi, AR, Takamjani, IE, Torkaman, G, and Fazeli, SH.\nWhat is the effect and mechanism of kinesiology tape on muscle activity? J Bodyw Mov Ther.\n(2018) 22:266–75. doi: 10.1016/j.jbmt.2017.06.018\n50.\tGillette, RL, and Angle, TC. Recent developments in canine locomotor analysis: a\nreview. Vet J. (2008) 178:165–76. doi: 10.1016/j.tvjl.2008.01.009\n51.\tAbraira, VE, and Ginty, DD. The sensory neurons of touch. Neuron. (2013)\n79:618–39. doi: 10.1016/j.neuron.2013.07.051\n52.\tKinesio-Group. Kinesio taping canine for dog lovers. Albuquerque: Kinesio IP\nLLC (2020).\n53.\tMagalhães, I, Bottaro, M, Mezzarane, RA, Neto, FR, Rodrigues, BA,\nFerreira-Júnior, JB, et al. Kinesiotaping enhances the rate of force development but not\nthe neuromuscular efficiency of physically active young men. J Electromyogr Kinesiol.\n(2016) 28:123–9. doi: 10.1016/j.jelekin.2016.04.003\n54.\tKase, K, Wallis, J, Kase, T. 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+{"text": "【兽医文献】\n标题: Beyond traditional biomarkers: do serum cystatin C and urinary cystatin B improve diagnostic precision in feline and canine renal disease? A systematic review and meta-analysis\n\nTYPE Editorial\nPUBLISHED 28 April 2026\nDOI 10.3389/fvets.2026.1843968\nOPEN ACCESS\nEDITED AND REVIEWED BY\nFrancisco Javier Salguero,\nUK Health Security Agency (UKHSA),\nUnited Kingdom\n*CORRESPONDENCE\nAgnieszka ˙Zak-Bochenek\nagnieszka.zak-bochenek@upwr.edu\nRECEIVED 31 March 2026\nACCEPTED 09 April 2026\nPUBLISHED 28 April 2026\nCITATION\n˙Zak-Bochenek A and Siwi´nska N (2026)\nEditorial: Biomarker identification and\nnovel therapies in equine medicine.\nFront. Vet. Sci. 13:1843968.\ndoi: 10.3389/fvets.2026.1843968\nCOPYRIGHT\n© 2026 ˙Zak-Bochenek and Siwi´nska.\nThis is an open-access article distributed\nunder the terms of the Creative\nCommons Attribution License (CC BY).\nThe use, distribution or reproduction in\nother forums is permitted, provided the\noriginal author(s) and the copyright\nowner(s) are credited and that the\noriginal publication in this journal is\ncited, in accordance with accepted\nacademic practice. No use, distribution\nor reproduction is permitted which does\nnot comply with these terms.\nEditorial: Biomarker identification\nand novel therapies in equine\nmedicine\nAgnieszka ˙Zak-Bochenek\n1* and Natalia Siwi´nska2\n1Department of Immunology, Pathophysiology and Veterinary Preventive Medicine, Wrocław\nUniversity of Environmental and Life Sciences, Wroc��aw, Poland, 2Department of Internal Medicine and\nClinic of Diseases of Horses, Dogs and Cats, Wrocław University of Environmental and Life Sciences,\nWrocław, Poland\nKEYWORDS\nbiomarkers, colic, horses, osteoarthritis, strangles\nEditorial on the Research Topic\nBiomarker identification and novel therapies in equine medicine\nWithin veterinary medicine, equine research has experienced substantial growth in\nrecent years. This trend is largely due to the evolving perception of horses from primarily\nbeing seen as livestock to being valued as companion animals. This shift underscores\nthe need to expand both traditional diagnostic methods and innovative approaches to\nenhance disease detection, treatment options, and overall equine health management.\nDespite significant advancements, there remain critical gaps in our understanding of equine\ndiseases, particularly in the integration of traditional and modern diagnostic techniques.\n\nDespite significant advancements, there remain critical gaps in our understanding of equine\ndiseases, particularly in the integration of traditional and modern diagnostic techniques.\nRecent studies have highlighted the importance of combining classical diagnostic practices\nwith advanced technologies to improve disease outcomes. However, there is still a need\nfor comprehensive research that bridges these methodologies, particularly in the areas of\nbiomarker identification and novel therapeutic strategies.\nOsteoarthritis (OA) is a prevalent and debilitating condition affecting horses across\nbreeds and disciplines. According to a 2013 survey conducted in the United Kingdom,\nOA was diagnosed in up to 13.9% of the horses examined (1). The proportion of affected\nanimals increases markedly with advancing age, and the disease typically assumes a\nchronic, progressive course. Numerous studies have therefore focused on elucidating the\npathomechanisms underlying intra-articular alterations, with the dual aims of improving\ndiagnostic approaches and developing novel therapeutic strategies (2). The present\nResearch Topic encompasses three articles addressing chronic lameness and OA in horses,\nunderscoring both the clinical importance and the scientific relevance of this condition.\nIn the study by Kwonet al., the effects of an eggshell membrane–based supplement on\nchronic lameness were investigated. The evaluation employed conventional assessment\ntools, including subjective gait analysis, rider assessment, joint-angle kinematics, and\nradiographic examination. The authors demonstrated the feasibility of enhancing these\nclassical methods with advanced counting techniques to standardize and systematize\noutcome measures. Chabronova et al. presented advanced research on the molecular\npathogenesis of equine OA, specifically focusing on the role of small nucleolar RNAs\n(snoRNAs) and the ribosomal alterations they may induce. Using an experimentally\ninduced model of OA, snoRNAs were identified by high-throughput sequencing. In total,\nFrontiers in Veterinary Science\n01\nfrontiersin.org\n\n˙Zak-Bochenek and Siwi´nska\n10.3389/fvets.2026.1843968\n44 snoRNAs exhibited altered expression across the various\ncomparisons, the majority of which were canonical snoRNAs.\nThese findings indicate that snoRNA expression is dynamically\nregulated in equine OA, highlighting their potential utility as early\nmolecular biomarkers and as targets for therapeutic intervention.\nAs these studies illustrate, investigation of the pathomechanisms\ndriving joint inflammation frequently relies on experimental\nmodels that induce controlled inflammatory responses within the\njoint. The study by Korac et al. is consistent with contemporary\nimplementation of the 3R (Replacement, Reduction, Refinement)\nprinciple in biomedical research, as it aimed to determine a safe\nand effective concentration of interleukin-1β (IL-1β) for inducing a\nmodel inflammatory state.\nAmong the disorders commonly affecting horses, colic warrants\nparticular attention, as it is most frequently an acute condition\ncharacterized by severe abdominal pain and associated with\nsubstantial mortality. Colic is estimated to occur in approximately\n4–10% of horses annually, most often requiring conservative\n(medical) management, but in a subset of life-threatening cases\nnecessitating surgical intervention (3), (4). A key objective of\ncontemporary research is the development of rapid diagnostic\napproaches, especially those capable of guiding the choice of\ntherapeutic strategy. In the study presented in this Research\nTopic, Crosby et al. evaluated the clinical utility of alactic base\nexcess (ABE) by analyzing data from 538 horses with colic at\ndifferent stages of disease progression. ABE is derived from\nblood gas analysis (base excess–extracellular fluid) combined with\nmeasurement of L-lactate concentration. While the measurement\nof L-lactate concentrations in serum and peritoneal fluid is\nalready in clinical use as a biomarker of intestinal ischemia in\nhorses with colic, the authors proposed an extension of this\nconcept (5). Although the investigation did not demonstrate\nstatistically significant differences in ABE values between horses\n\nAlthough the investigation did not demonstrate\nstatistically significant differences in ABE values between horses\nmanaged conservatively and those requiring surgical intervention,\nABE levels were significantly higher in non-surviving horses,\nsuggesting its potential prognostic relevance. Moreover, the study\nshowed significantly elevated L-lactate and glucose concentrations\nin horses that underwent surgery and in non-survivors, further\nunderscoring the diagnostic and prognostic value of these\nbiochemical parameters.\nStrangles, caused by Streptococcus equi subspecies equi, is a\nhighly contagious, globally distributed respiratory disease of horses,\npredominantly affecting young animals (6, 7). The disease is\nendemic worldwide and is characterized by high morbidity (up to\n100% in immunologically naive herds) but generally low mortality,\nand it frequently causes outbreaks in environments with high\nanimal density (7). Consequently, S. equi is a frequent focus of\nresearch on rapid diagnostic techniques and preventive strategies,\nincluding vaccination (7). Within this Research Topic, we highlight\nthe work of Zu et al., who, for the first time, proposed a method\nfor detecting S. equi in specimens such as nasal swabs using an\nassay that integrates recombinase-aided amplification (RAA) with\nCRISPR/Cas12a technology. This approach exhibits high analytical\nsensitivity and specificity. Despite the technological sophistication\nof the platform, the endpoint readout is straightforward: the\npresence of S. equi can be visually confirmed under ultraviolet light.\nThis feature facilitates potential implementation of the assay in\nroutine veterinary practice.\nIt should also be emphasized that, as in other animal species,\nhorses are susceptible to neoplastic diseases. Among ovarian\nneoplasms in mares, granulosa cell tumors (GCTs) are the most\nfrequently reported. The study by Derar et al. outlines current\ndiagnostic and therapeutic options and, through a retrospective\nanalysis, explores potential etiological factors associated with\nthe development of GCTs. The authors describe the role of\n\nThe study by Derar et al.outlines current\ndiagnostic and therapeutic options and, through a retrospective\nanalysis, explores potential etiological factors associated with\nthe development of GCTs.The authors describe the role of\nvarious imaging modalities and histopathological examination in\nestablishing a definitive diagnosis, and they report a high rate of\nreturn to fertility following ovariectomy.\nCollectively,\nthe\ncontributions\nin\nthis\nResearch\nTopic\nillustrate the broad spectrum of contemporary equine research,\nencompassing\nthe\napplication\nof\nadvanced\ndiagnostic\nand\ntherapeutic\ntechniques\nalongside\nrefinement\nof\nclassical\nmethodologies. The studies discussed herein directly address\nthe central questions posed in the scope of the Research\nTopic—namely, “How can traditional and modern diagnostic\nmethods be effectively integrated? What are the most promising\nbiomarkers\nfor\nequine\ndiseases?”—and\nthereby\nfulfill\nits\nintended aims.\nAuthor contributions\nA˙Z-B: Conceptualization, Writing – original draft. NS: Writing\n– review & editing.\nConflict of interest\nThe author(s) declared that this work was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declared that generative AI was used in\nthe creation of this manuscript. The Writefull tool was used\nfor proofreading.\nAny alternative text (alt text) provided alongside figures in\nthis article has been generated by Frontiers with the support of\nartificial intelligence and reasonable efforts have been made to\nensure accuracy, including review by the authors wherever possible.\nIf you identify any issues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. Any product that may be evaluated in this article, or\nclaim that may be made by its manufacturer, is not guaranteed or\nendorsed by the publisher.\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n\n˙Zak-Bochenek and Siwi´nska\n10.3389/fvets.2026.1843968\nReferences\n1. Ireland JL, Wylie CE, Collins SN, Verheyen KLP, Newton JR. 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+{"text": "【兽医文献】\n标题: Partial culling and HPAI control in poultry: insights from Lower Saxony, Germany\n\nTYPE Editorial\nPUBLISHED 03 February 2026\nDOI 10.3389/fvets.2026.1787676\nOPEN ACCESS\nEDITED AND REVIEWED BY\nChiara Palmieri,\nThe University of Queensland, Australia\n*CORRESPONDENCE\nFelisbina Queiroga\nfqueirog@gmail.com\nRECEIVED 14 January 2026\nACCEPTED 21 January 2026\nPUBLISHED 03 February 2026\nCITATION\nQueiroga F, Horta RdS, Giuliano A and\nDagli MLZ (2026) Editorial: Advancements and\nchallenges in veterinary oncology.\nFront. Vet. Sci. 13:1787676.\ndoi: 10.3389/fvets.2026.1787676\nCOPYRIGHT\n© 2026 Queiroga, Horta, Giuliano and Dagli.\nThis is an open-access article distributed\nunder the terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other forums is\npermitted, provided the original author(s) and\nthe copyright owner(s) are credited and that\nthe original publication in this journal is cited,\nin accordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nEditorial: Advancements and\nchallenges in veterinary oncology\nFelisbina Queiroga1*, Rodrigo dos Santos Horta2,\nAntonio Giuliano3 and Maria L´ucia Zaidan Dagli4\n1Centro de Ciência Animal e Veterinária (CECAV), University of Trás-os-Montes and Alto Douro, Vila\nReal, Portugal, 2Department of Veterinary Medicine and Surgery, Veterinary School, Universidade\nFederal de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil, 3Harvest Veterinary Oncology\nCenter (HVOC), Hong Kong, Hong Kong SAR, China, 4Department of Pathology, School of Veterinary\nMedicine and Animal Science, University of São Paulo (USP), São Paulo, Brazil\nKEYWORDS\ncancer, guideline, liquid biopsy, precision medicine, tumor biology\nEditorial on the Research Topic\nAdvancements and challenges in veterinary oncology\nVeterinary oncology has undergone remarkable transformation over the past decade,\ndriven by rapid advances in molecular biology, imaging, therapeutics, and translational\nresearch (1). These developments have substantially expanded diagnostic precision and\ntherapeutic possibilities for companion animals with cancer. Despite this progress,\n\nThese developments have substantially expanded diagnostic precision and\ntherapeutic possibilities for companion animals with cancer.Despite this progress,\nsignificant challenges remain, including the identification of reliable prevention and early\ndetection strategies, optimization of multimodal treatments and integration of molecular\nand genetic data into clinical decision-making (2, 3). This Research Topic, Advancements\nand Challenges in Veterinary Oncology, includes 12 contributions that reflect the breadth of\ncontemporary veterinary oncology, spanning clinical management, translational research,\nmolecular oncology, and evidence-based guidelines.\nA major thematic pillar across the published articles relates to diagnostics and tumor\ncharacterization, including the need to improve how tumors are detected, assessed, and\nclassified. Ko et al. combined circulating cell-free DNA profiling with machine learning for\nearly detection of canine hemangiosarcoma, highlighting both the potential and current\nlimitations of liquid biopsy in aggressive, heterogeneous tumors. Within the diagnostic\nand biomarker axis, Rösch et al. evaluated serum survivin, Ki-67, thymidine kinase 1, and\nthe survivin–lymphocyte ratio in dogs with chronic nasal disease. While not diagnostic\nindividually, survivin-related markers were increased in malignant cases, supporting their\npotential role in early suspicion and monitoring, while underscoring the overlap between\nneoplastic and inflammatory conditions. Vincenti et al. addressed a highly practical\nand widely relevant issue by evaluating combined cross-sectional and tangential surgical\nmargin assessment in dogs and cats with different tumor types. Their findings highlight\nthe risk of underestimating margin involvement when relying on a single trimming\napproach and support more standardized and informative pathology reporting, with\ndirect implications for surgical planning and post-operative management. Díaz-Santana\net al. reported a case of metastatic renal giant-cell sarcoma in an African pygmy\n\nDíaz-Santana\net al.reported a case of metastatic renal giant-cell sarcoma in an African pygmy\nhedgehog, providing detailed pathological and immunohistochemical characterization\nof an uncommon studied species. Although case reports are not designed to change\npractice on their own, they strengthen the collective knowledge base, support comparative\npathology, and remind us that oncological challenges extend beyond the classical canine\nand feline settings. The Research Topic also includes work directly aligned with the\nFrontiers in Veterinary Science\n01\nfrontiersin.org\n\nQueiroga et al.\n10.3389/fvets.2026.1787676\ndevelopment of immunology-enabling tools, which are essential\nfor both research and clinical translation. Li et al. reported the\npreparation and functional validation of a rabbit anti-canine\nCD3 monoclonal antibody, addressing a practical methodological\nbarrier in canine immunophenotyping and immune profiling. The\navailability of validated reagents of this nature is fundamental for\nconsistent immunohistochemical analyses and for advancing the\nstudy of tumor immune microenvironments in veterinary species.\nBeyond primary diagnostics, the Research Topic also addresses\ntumor characterization and disease-specific overviews that help\nclinicians interpret heterogeneous presentations and rationally\nselect interventions. Marcinowska et al. provided a descriptive\nreview of canine lung carcinoma, summarizing etiology, clinical\npresentation, staging, and current treatment options, while\nframing\npersistent\ncontroversies\naround\nadjuvant\ntherapies.\nImportantly, the review reinforces that surgery remains the\ncornerstone of treatment when feasible, while pointing to emerging\ntargeted and immunotherapeutic strategies as areas of active\ndevelopment in dogs, aligned with the broader movement toward\npersonalized oncology.\nAnother set of contributions focuses on therapeutic strategies\nand treatment optimization, particularly in settings where durable\nlocal control or meaningful palliation remains difficult. Norquest\net al. explored the combination of zoledronate and radiation\ntherapy in canine osteosarcoma, integrating in vitro assessment\nwith a prospective clinical cohort. Their work supports the\nfeasibility and tolerability of the combined approach and raises\nimportant questions regarding optimal timing and biological\nvariability in response. Also for canine osteosarcoma, Griffin et al.\ninvestigated clinical outcomes in dogs with axial and appendicular\nbone tumors treated with surgical stabilization combined with\nnon-stereotactic radiation therapy. Their bi-institutional study\ndemonstrates that this combined approach can achieve meaningful\nfunctional improvement, acceptable complication rates, and\n\nTheir bi-institutional study\ndemonstrates that this combined approach can achieve meaningful\nfunctional improvement, acceptable complication rates, and\nencouraging survival outcomes, even in advanced orthopedic\noncology cases, supporting its role as a viable limb-sparing\nand palliative strategy. Petrucci et al. reviewed metronomic\nchemotherapy,\nintegrating\nmechanistic\nconcepts,\nincluding\nantiangiogenic and immunomodulatory effects, with available\nclinical data and practical considerations. Importantly, the authors\nclearly acknowledge the current limitations of the evidence,\nreinforcing the need for well-designed prospective studies while\nproviding clinicians with a balanced overview of how metronomic\napproaches may be incorporated into practice.\nAmaral et al. addressed the interface between nutrition and\noncology in dogs and cats. By integrating evidence on cancer-\nassociated metabolic changes, cachexia, the role of obesity,\nand nutritional interventions and supplementation, this review\npositions nutrition as a core element of supportive oncology\ncare, contributing to functional preservation, quality of life, and\nimproved treatment tolerance.\nTherapeutic advancement is also dependent on establishing\nshared standards. In this context, Polton et al. provided a\ncomprehensive consensus and guideline document for melanoma\nin dogs and cats, addressing staging, prognostic features, treatment\nmodalities, and areas of uncertainty. By consolidating current\nknowledge and expert guidance, this contribution offers a\npragmatic framework for clinicians managing one of the most\nbiologically complex and clinically challenging tumor types\nin companion animals. Another key therapeutic challenge in\nveterinary oncology is the implementation of strategies that are\nbiologically rational, clinically safe, and realistically applicable.\nTaken together, these contributions illustrate how veterinary\noncology is advancing through multiple, interconnected fronts,\nincluding refinement of local and systemic therapies, consensus\nbuilding and clinical standardization, development of enabling\n\nTaken together, these contributions illustrate how veterinary\noncology is advancing through multiple, interconnected fronts,\nincluding refinement of local and systemic therapies, consensus\nbuilding and clinical standardization, development of enabling\nimmunology tools, and the steady expansion of minimally invasive\ndiagnostics and biomarker research. At the same time, the\nResearch Topic clearly reflects the field’s ongoing challenges,\nsuch as biological heterogeneity, limited high-level evidence for\nsome interventions, and the need for robust, clinically deployable\ndiagnostic strategies. By presenting both progress and limitations in\na balanced manner, this Research Topic aims to support clinicians\nand researchers working to deliver more precise, evidence-\ninformed, and patient-centered veterinary cancer care.\nAuthor contributions\nFQ: Writing – original draft, Writing – review & editing,\nInvestigation, Methodology, Conceptualization. RH: Writing –\nreview & editing, Writing – original draft, Conceptualization,\nMethodology, Investigation. AG: Writing – original draft, Writing\n– review & editing. MD: Writing – review & editing, Writing –\noriginal draft.\nConflict of interest\nThe author(s) declared that this work was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nThe author FQ declared that they were an editorial board\nmember of Frontiers at the time of submission. This had no impact\non the peer review process and the final decision.\nGenerative AI statement\nThe author(s) declared that generative AI was not used in the\ncreation of this manuscript.\nAny alternative text (alt text) provided alongside figures in\nthis article has been generated by Frontiers with the support of\nartificial intelligence and reasonable efforts have been made to\nensure accuracy, including review by the authors wherever possible.\nIf you identify any issues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\n\nIf you identify any issues, please contact us.Publisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. Any product that may be evaluated in this article, or\nclaim that may be made by its manufacturer, is not guaranteed or\nendorsed by the publisher.\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n\nQueiroga et al.\n10.3389/fvets.2026.1787676\nReferences\n1. Christensen J, Johnson K, Ettinger S, Garrett L, Gordon I, Ireifej S,\net al. AAHA oncology guidelines for dogs and cats. J Am Anim Hosp. (2026)\n63. doi: 10.5326/JAAHA-MS-7549\n2. Pimentel\nPAB,\nGiuliano\nA,\nBeczkowski\nPM,\nHorta\nRDS.\nMolecular\nprofile\nof\ncanine\nhemangiosarcoma\nand\npotential\nnovel\ntherapeutic\ntargets.\nVet\nSci.\n(2023)\n10:387.\ndoi:\n10.3390/vetsci1006\n0387\n3. Chon E, Hendricks W, White M, Rodrigues L, Haworth D, Post G. Precision\nmedicine in veterinary science. Vet Clin North Am Small Anim Pract. (2024) 54:501–\n21. doi: 10.1016/j.cvsm.2023.12.006\nFrontiers in Veterinary Science\n03\nfrontiersin.org\n\n---"}
+{"text": "【兽医文献】\n标题: Identifying veterinary surgeons&#x02019; barriers to, and potential solutions for, improving antimicrobial stewardship among sheep farmers in Northern Ireland\n\nReceived: 24 June 2023\nRevised: 11 September 2023\nAccepted: 10 October 2023\nDOI: 10.1002/vro2.75\nOR IG I NA L R E SE A RC H\nImproving analgesia provision for sheep: An analysis of farm\nmedicine records and attitudes towards pain relief on sheep\nfarms in Northern Ireland\nPaul E. Crawford\nKim Hamer\nFiona Lovatt,\nMalgorzata C. Behnke\nPhilip A. Robinson,,\n1Department of Animal Health, Behaviour and\nWelfare, Harper Adams University, Shropshire,\nUK\n2School of Biodiversity, One Health and Veterinary\nMedicine, College of Medical, Veterinary and Life\nSciences, University of Glasgow, Garscube\nCampus, Glasgow, UK\n3School of Veterinary Science, Sutton Bonington\nCampus, University of Nottingham, Nottingham,\nUK\n4Flock Health Ltd., Egglesburn Farm, Eggleston,\nBarnard Castle, Durham, UK\n5Harper & Keele Veterinary School, Harper Adams\nUniversity Campus, Shropshire, UK\n6Keele University, Staffordshire, UK\nCorrespondence\nPaul E. Crawford, Department of Animal Health,\nBehaviour and Welfare, Harper Adams University,\nNewport, Shropshire, TF10 8NB, UK.\nEmail: paul@paulmrcvs.com\nAbstract\nBackground: Management of pain is critical to improve the welfare of farmed livestock\nand meet consumer expectations. There is limited published information about the use\nof analgesic drugs in the sheep sector.\nMethods: A mixed-method approach was followed. The range of analgesic drugs used\non 52 Northern Irish sheep farms was determined through analysis of medicine purchase\nrecords. Through interview and discussion groups, with both farmer and veterinarian\nparticipants, attitudes towards the use and adoption of such medicines were explored.\nResults: The use of non-steroidal anti-inflammatory drugs (NSAIDs) was widespread\nand highly variable. One-third of farmers in the sample did not purchase any NSAID.\nMeloxicam was the most commonly purchased NSAID by mass (72%) and standardised\ndose (73%). During interviews and discussions, farmers outlined the benefits they saw\nin using NSAIDs and how veterinarians influenced their uptake of these medicines. Use\nof corticosteroid was evidenced on 50% of the farms that supplied medicine records for\nanalysis.\n\nDuring interviews and discussions, farmers outlined the benefits they saw\nin using NSAIDs and how veterinarians influenced their uptake of these medicines.Use\nof corticosteroid was evidenced on 50% of the farms that supplied medicine records for\nanalysis.\nConclusions: Veterinarians can influence farmers to adopt NSAIDs for the provision\nof analgesia in their sheep and farmers observed the benefits they delivered. However,\nmany farmers are still to be reached with this message, perhaps due to being largely\nself-sufficient and rarely engaging with veterinarians. Veterinarians have the opportu-\nnity to challenge farmers about the provision of analgesia, especially when farmers seek\nantibiotics for painful conditions such as lameness. Currently, the lack of an authorised\nproduct in the UK, with associated treatment guidance and industry promotion, may\nlimit veterinarians’ confidence in prescribing drugs for pain control in sheep.\nINTRODUCTION\nPain in sheep is a complex sensory and emotional experience\narising directly, following damage to tissue and secondar-\nily to inflammation.1 Pain is influenced by previous painful\nexperiences and social position within the flock.1,2 In sheep\nfarming, pain can arise from a wide range of disease and\nmanagement-related factors.3,4\nThe importance of adequate analgesia has been recog-\nnised and is enshrined in legislation in many countries.4\nThe management of pain in livestock continues to come\nunder increasing scrutiny from the public2,4,5; additionally,\npainful conditions, such as lameness, carry an economic\ncost to farmers.6 However, before pain can be effectively\nThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original\nwork is properly cited.\n© 2023 The Authors. Veterinary Record Open published by John Wiley & Sons Ltd on behalf of British Veterinary Association.\nmanaged, those caring for sheep need to recognise its pres-\nence and appreciate the need for pain to be treated promptly\n\n© 2023 The Authors.Veterinary Record Open published by John Wiley & Sons Ltd on behalf of British Veterinary Association.managed, those caring for sheep need to recognise its pres-\nence and appreciate the need for pain to be treated promptly\nand appropriately.3,4 Although no universal, validated pain\nscoring system yet exists for sheep, previous reviews have\nhighlighted subjective and objective tools that can aid in\npain assessment1,2,4; while many of these are more suited to\nresearch settings,7 research continues to develop systems for\non-farm use.8 Industry bodies have prepared guidance notes\nfor farmers9,10 to make information on pain assessment acces-\nsible to them. Previous studies have highlighted that while\nboth farmers and veterinarians are cognisant of and can iden-\ntify procedures that may cause pain and can identify signs of\npain, there is a variation in how participants in these studies\nrank the severity of the pain.1,11 Furthermore, sheep are known\nVet Rec Open. 2023;10:e75.\nwileyonlinelibrary.com/journal/vro2\nof \nhttps://doi.org/10.1002/vro2.75\n\nof \nVETERINARY RECORD OPEN\nto moderate their behavioural expression, which may mask\nsigns of pain following disturbance or handling, influenced by\nthe management system and prior experiences.1,12,13\nAll of the major classes of analgesic drugs have been shown\nto provide some level of analgesia in sheep.2,3 Currently, how-\never, in the UK, there are no products specifically authorised\nfor use in sheep to provide long-term analgesia.14 Under\nthe ‘Cascade’ system,15 products authorised for use in other\nfood-producing animals may be prescribed for sheep, by a\nveterinarian, on a case-by-case basis for the protection of ani-\nmal welfare. The lack of any non-steroidal anti-inflammatory\ndrugs (NSAIDs) authorised for use in sheep in the UK has\nbeen identified as having major animal welfare implications.\nCalls have been made for novel approaches to the genera-\ntion of the data necessary to prove the efficacy and safety of\nsuch products for use within the sheep sector.16 For cattle,\nNSAIDs have proven practical to administer and efficacious\nin providing long-term analgesia when administered on-farm\nby the farmer.5 However, there is evidence of underuse of these\nmedicines in cattle and calls for further education to reinforce\nthe benefits of timely analgesia.5\nCorticosteroids (CSs), such as dexamethasone, but not\nNSAIDs, have been used for their anti-inflammatory proper-\nties in a range of livestock species, including for provision of\nanalgesia, despite potential side effects and limited evidence\nof clinical pain-relieving properties.17,18 Historically, evidence\nfor any long-term therapeutic benefit of CS has been limited\nto specific circumstances, at least in cattle.19\nThe metric mg/population-corrected unit (PCU) was ini-\ntially developed for comparing antibiotic use where flocks,\nbreeds and production systems differed between nations.20\nThe PCU metric standardises antibiotic use based on stan-\ndardised, averaged body masses for the class(es) of livestock\nunder consideration. Subsequently, they have been utilised\nwidely to quantify, compare and benchmark antibiotic use\n\n20\nThe PCU metric standardises antibiotic use based on stan-\ndardised, averaged body masses for the class(es) of livestock\nunder consideration.Subsequently, they have been utilised\nwidely to quantify, compare and benchmark antibiotic use\nnationally, also as an indicator of antibiotic use on and\nbetween individual farms. To the best of the authors’ knowl-\nedge, no such work has been undertaken regarding analgesia\nprovision in sheep.\nThis report presents a preliminary study that aimed to\ndevelop an understanding of the attitudes and behaviours of\nNorthern Irish sheep farmers and their veterinarians towards\nNSAID and CS use in sheep. Additionally, it sought to iden-\ntify stimuli to achieve wider use of pain relief in sheep and any\nbarriers to increased analgesia provision in the sector.4,21\nMETHODS\nA mixed-method research approach was utilised. Data com-\nprised records of medicines purchased over a 12-month\nperiod, semi-structured interviews with farmers and veteri-\nnarians, and discussion groups with participants drawn from\nthe same sectors. Convenience sampling was used for selec-\ntion of participants throughout each phase of data collection,\nwith no restriction on how many elements a participant could\ncontribute to. These data were collected as part of a wider\nprogramme considering medicine use and stewardship in the\nNorthern Ireland sheep flock.\nMedicine records were obtained from on-farm records\n(eight farmers) or directly from the farmer’s veterinary\npractice’s sales records (50 farmers), with six farmers supply-\ning records from both sources. All farmers were asked about\ntheir livestock enterprises. There were 13 (25%) sheep-only\nlivestock enterprises. During discussion with the remaining\n39 (75%), their medicine purchases were allocated to each\nof their enterprises. Only data pertaining to the sheep flock\nwere analysed. Medicine purchase data were transcribed into\nMicrosoft Excel22 for analysis. Each flock’s mean antibiotic\nconsumption was calculated in mg/PCU using the University\nof Nottingham Antibiotic calculator.23 To contextualise the\nquantity of NSAID and CS purchased, flock size, total antibi-\n\nEach flock’s mean antibiotic\nconsumption was calculated in mg/PCU using the University\nof Nottingham Antibiotic calculator.23 To contextualise the\nquantity of NSAID and CS purchased, flock size, total antibi-\notic purchased and antibiotic consumption, in mg/PCU, were\nused as denominators. Spearman’s R was used to determine\nthe significance of the relationship between pairs of variables.\nStandardised, semi-structured interviews and discussion\ngroups were undertaken with farmers and veterinarians\n(Supporting Information S1). Electronic recordings or con-\ntemporaneous notes were taken from interviews and dis-\ncussion groups. Following transcription, NVivo24 software\nwas utilised during coding. The first author undertook all\nthe medicine record analysis, interviewing and facilitation of\ndiscussion groups, and transcription. During interviews and\ndiscussion groups, participants were assured that their knowl-\nedge of sheep medicine was not being tested. They were not\nasked for the rationale behind specific treatments or dose\nrates. Where these were offered, the information was recorded.\nOne follow-up question addressing CS was sent to veterinari-\nans by email (Supporting Information S1). After initial coding,\nsections relevant to pain management and the use of NSAIDs\nwere collated and exemplar quotes were identified.\nRESULTS\nMedicine records\nThree NSAIDs, meloxicam, flunixin and ketoprofen, and one\nCS, dexamethasone, were identified in the records. Meloxi-\ncam (72%) was, by mass of active component, the most\npurchased, followed by flunixin (14%), ketoprofen (12%) and\ndexamethasone (2%).\nTo account for differing potencies and dose rates, the num-\nber of standardised doses purchased was calculated utilising\ndose rates the authors considered represented best-practice\nclinical use in the UK: meloxicam 1 mg/kg, ketoprofen\n3.3 mg/kg, flunixin 2.2 mg/kg and dexamethasone 0.14 mg/kg.\nThis showed meloxicam was the most utilised product (73%),\nfollowed by dexamethasone (16%), flunixin (7%) and ketopro-\nfen (4%) (Figure 1).\nMedicine records revealed that 14 (33%) farmers had pur-\n\n3 mg/kg, flunixin 2.2 mg/kg and dexamethasone 0.14 mg/kg.This showed meloxicam was the most utilised product (73%),\nfollowed by dexamethasone (16%), flunixin (7%) and ketopro-\nfen (4%) (Figure 1).Medicine records revealed that 14 (33%) farmers had pur-\nchased no NSAID during the 12 months covered by the\nrecords supplied (Table 1). Of the 38 farmers who did pur-\nchase NSAIDs, 17 (30%) purchased 50 mL or more. The CS\ndexamethasone was purchased by 26 (50%) farmers, with half\nof these purchasing 50 mL or more. Both NSAID and CS were\npurchased by 23 (44%) farmers. Eleven (21%) farms purchased\nneither NSAID nor CS (Table S1).\nAll antibiotics purchased were identified from the records\nand analysed. Following calculation of the mean antibiotic\nconsumption for each flock, in mg/PCU using the University\nof Nottingham Antibiotic use calculator,23 this was compared\n\nVETERINARY RECORD OPEN\nof \nTA BL E \nFrequency distribution of quantities by volume of each non-steroidal anti-inflammatory drug (NSAID) and corticosteroid (CS) product\npurchased by farmers, as identified in the 12-month sample of medicine records supplied relating to 52 Northern Irish sheep farms.\nNone\nLess than mL\n–mL\n–mL\nmL and greater\nNSAID\nMeloxicam\n17 (33%)\n16 (31%)\n6 (12%)\n2 (4%)\n11 (21%)\nKetoprofen\n50 (96%)\n0 (0%)\n1 (2%)\n0 (0%)\n1 (2%)\nFlunixin\n46 (88%)\n3 (6%)\n0 (0%)\n1 (2%)\n2 (4%)\nCS\nDexamethasone\n26 (50%)\n8 (15%)\n5 (10%)\n10 (19%)\n3 (6%)\nNote: In 11 of the records, no NSAID or CS purchases were identified; 24 records indicated purchase of both NSAID and CS. The majority of farms (94%) that purchased NSAID,\npurchased only one NSAID. Two (4%) farms purchased two different NSAIDs and one (2%) purchased all three of the NSAIDs identified.\nF IG U R E \nRelative proportions in standardised dose (based on the\nauthors’ experience of clinical practice in the UK) for the single\ncorticosteroid and three non-steroidal anti-inflammatory products\nidentified in the 12-month sample of medicine records supplied relating to\n52 Northern Irish sheep farms.\nTA BL E \nSignificance of association between flock size and antibiotic\nuse on levels of non-steroidal anti-inflammatory (NSAID) use and\ncorticosteroid (CS) use and between the level of NSAID and CS use on\nNorthern Ireland farms supplying medicine records.\nRelationship\nSpearman’s\nR, rs\np-Value\n(two-tailed)\nFlock size versus NSAID (n = 52)\n−0.134\n0.35\nFlock size versus CS (n = 52)\n−0.058\n0.68\nMean antibiotic purchase versus NSAID\n(n = 52)\n0.264\n0.06\nMean antibiotic purchase versus CS\n(n = 52)\n0.153\n0.28\nTotal antibiotic purchase versus NSAID\n(n = 52)\n0.094\n0.51\nTotal antibiotic purchase versus CS\n0.0408\n0.77\nNSAID versus CS (n = 52)\n0.338\n0.014a\nNSAID versus CS (n = 41)\n0.077\n0.63\naOnly the relationship between NSAID and CS use appeared significant; however, when\nthe 11 non-users of both medications were excluded, there was no significant relationship.\nwith NSAID purchase calculated in mg/PCU (Figure 2) to\nillustrate the wide range of both antibiotic and NSAID pur-\nchase between farms, having accounted for flock size. The\n\nwith NSAID purchase calculated in mg/PCU (Figure 2) to\nillustrate the wide range of both antibiotic and NSAID pur-\nchase between farms, having accounted for flock size.The\nrelationship between NSAID and CS purchases, with each of\ntotal flock size, total flock antibiotic consumption and stan-\ndardised antibiotic consumption (mg/PCU) per sheep, were\ntested for significance using Spearman’s R.25 No significant\nrelationship between any pairs was observed (Table 2).\nInterviews and discussion groups\nThe mean farmer interview duration was 46 min (range 20–\n74 min) and the mean veterinarian interview duration was\n55 min (range 31–84 min) (Table 3). The interviews and dis-\ncussion groups covered a wide range of topics pertaining to\nmedicine use on the sheep flock—only information related to\npain perception and management, and the purchase and use\nof NSAID and CS is reported here. Additional quotations are\nprovided in the Supporting Information S2.\n1 Farmers demonstrated an understanding that some of their\nsheep suffered from painful conditions and that not only\ncould they benefit from some form of pain relief, but that\nprovision of an anti-inflammatory was an essential part of\nthe sheep’s treatment.\nSF65—To treat contagious digital dermatitis, everything\nhad to be given—meloxicam as well as an antibiotic. I\nalso used it to treat ewes with peri-orbital dermatitis.\nA simple antibiotic cured the infection but to stop the\nirritation, they needed meloxicam as well; otherwise,\nthey just kept rubbing and rubbing their face until one\ndid so much damage, and she had to be euthanased.\nDG02—A caesarean or something like that there I would\ngive it to them for a couple of days—it definitely helps.\nI think it is necessary that you do give them something\nfor the pain.\n2 Some farmers chose to pre-emptively purchase NSAID as\npart of their lambing preparations, so they would have the\nmedicine to hand if it was needed.\nSF30—Before lambing, I go to my veterinary practice\nwith a shopping list: bottles of calcium and antibi-\notic injection and some sort of anti-inflammatory and\nsome sort of painkiller. I like having them in the cup-\n\nSF30—Before lambing, I go to my veterinary practice\nwith a shopping list: bottles of calcium and antibi-\notic injection and some sort of anti-inflammatory and\nsome sort of painkiller.I like having them in the cup-\nboard. Then, if you have problems around lambing, an\newe with mastitis, for example, she is going to get pain\nrelief.\n3 This awareness or use of pain relief was not universal and\nsome farmers indicated that some sheep received analgesia,\nbut not all.\nSF33—Sheep with contagious ovine digital dermatitis\n(CODD) have been proving very difficult to get\ncured. You get to a certain level of improvement\n\nof \nVETERINARY RECORD OPEN\nF IG U R E \nQuantity of antibiotic and non-steroidal anti-inflammatory drugs (NSAID) identified in the medicine purchase records supplied by 52\nNorthern Irish sheep farmers and standardised to account for the varied flock sizes using the mg/population-corrected unit (mg/PCU) metric.\nTA BL E \nParticipants’ demographics for the dataset addressing medicine use in the Northern Ireland sheep flock.\nFarmers\nVeterinarians\nInterviews\nTwenty-seven farmers, farming in five of the six counties of Northern\nIreland. Farmers represented flocks of 40–730 breeding sheep.\nSixty-three percent had other livestock enterprises.\nTwenty-four recorded electronically.\nThree recorded by contemporaneous notes.\nFifteen veterinarians from 12 veterinary practices based in Northern\nIreland, who serve sheep-farming clients across all six counties of\nNorthern Ireland.\nAll recorded electronically.\nDiscussion\ngroups\nThirteen discussion groups with average 14 (range 8–25) participants.\nFarmers participating were predominantly male and over 50 years\nof age.\nFive electronically recorded.\nEight recorded by contemporaneous notes.\nTwo discussion groups of five and seven participants, drawing\nveterinarians from 10 veterinary practices based in Northern\nIreland, who serve sheep-farming clients.\nBoth electronically recorded.\nwith them. I have been using Alamycin LA (oxyte-\ntracycline 200 mg/mL, Norbrook Pharmaceuticals)\nfor that and a wee bit of Hexasol (oxytetracycline\n300 mg/mL combined with flunixin 20 mg/mL, Nor-\nbrook Pharmaceuticals) for pain, if you thought they\nwere needing an anti-inflammatory.\n4 Farmers generally indicated that their veterinarian had\ninitially recommended the use of NSAID.\nDG02—My veterinarian’s recommendations got me\nstarted using painkillers—you would notice a dif-\nference. You maybe did not have to use as much\nantibiotic actually. The painkiller was maybe actually\ndoing more good than the antibiotic was what you\nwere finding sometimes.\n5 Veterinarians were positive about the benefits of providing\npain relief for sheep following difficult lambings, surgery\n\nThe painkiller was maybe actually\ndoing more good than the antibiotic was what you\nwere finding sometimes.5 Veterinarians were positive about the benefits of providing\npain relief for sheep following difficult lambings, surgery\nand when asked to prescribe treatments for conditions they\nconsidered to be painful. They also relayed feedback from\nfarmers about the efficacy of the pain relief. Mastitis and\nlameness were the two disease conditions most frequently\nidentified by them as benefitting from analgesia.\nV11—I like everything that is assisted during lambing to\nget meloxicam. Everything. Also, anything with mas-\ntitis, anything that is lame. I am a big NSAID advocate.\nPainkillers are key.\n6 One veterinarian observed that farmers were surprised\nat how effective meloxicam was at reducing straining in\nsheep who had suffered a vaginal prolapse. Addition-\nally, veterinarians had also observed that their farming\nclients appreciated the benefits of the pain relief pro-\nvided and that farmers now came into the practice look-\ning for pain-relieving products with sales increasing over\ntime.\nV20—Farmers say ‘I want to come in and have a chat\nto you before lambing’. They come to get their lube,\ngloves, antibiotics, pain relief and their colostrum. The\nuse of NSAIDs is a massive improvement in the last\n10 years, and the number of sheep that get pain relief\ncompared to years ago—there are many more sheep\ngetting them.\n7 A wide range of factors were suggested for this increased\ndemand from clients for analgesic products, including\n\nVETERINARY RECORD OPEN\nof \nthe influence of social media, animal welfare campaigns,\ngreater education on the issue and veterinarians talking\nto their clients. Some suggested that younger farmers and\nfemale farmers would be the first ones to give pain relief and\nask about what options existed to improve pain manage-\nment in their flocks. However, one veterinarian noted that\nthey were only getting through to a few farmers and that\nthere were lots of sheep farmers that they never engaged\nwith, as they did not seek veterinary advice. Another, while\nspecifically mentioning cattle, discussed how he felt some\nfarmers administer some form of medication to a non-\nspecifically dull animal ‘just in case’ it was to deteriorate\novernight and they encouraged farmers to consider using\nan NSAID rather than an antibiotic. An unrelated sheep\nfarmer who had adopted such practice was positive about\nit.\nDG—You don’t have to use as much antibiotic actually. I\nhave found an anti-inflammatory was as good as the\nantibiotic, you know. The painkiller was maybe doing\nmore good than the antibiotic was, I was finding.\n8 Some veterinarians highlighted uncertainty in the mind of\ntheir farming clients as to which products contained a pain-\nrelieving component and which pain-relieving product\nwould be best for their sheep.\nV10—There are a lot, especially of sheep farmers, that\nthink there is a painkiller in Pen Strep (procaine peni-\ncillin 200 mg/mL, and dihydrostreptomycin sulfate\n250 mg/mL, Norbrook Pharmaceuticals), for example,\nso it is trying to get them to understand that an anti-\ninflammatory, pain relief drug is a separate product to\nan antibiotic.\n9 The lack of a product containing an NSAID autho-\nrised for use in sheep was identified by prescribers, with\nsome showing awareness of the need to apply a statutory\nwithdrawal.\nV15—Painkillers is a difficult one because there is nothing\nlicensed out there. We would use meloxicam.\nV20—I know maybe it is not licensed in sheep, (but)\nwe use away at the flunixin and meloxicam products.\nWhen we are prescribing it, we would put a 28-day\nmeat withdrawal on it.\n\nWe would use meloxicam.V20—I know maybe it is not licensed in sheep, (but)\nwe use away at the flunixin and meloxicam products.When we are prescribing it, we would put a 28-day\nmeat withdrawal on it.\nCorticosteroids were only mentioned during the interviews\nby one farmer who indicated he had previously used them,\nwith limited success, as part of a protocol to treat lambs with\njoint ill.\n10 No veterinarian advocated, during interview or discus-\nsion group, a role for the use of dexamethasone in the\nmanagement of painful conditions in sheep. Through\nthe interviews and follow-up email question veterinarians\nindicated that they may use it on a case-by-case basis where\nthe diagnosis was unknown. While some indicated that\nthere was a demand from their clients for access to the\nproduct, others could not identify a reason why a farmer\nwould require CS to be supplied by the bottle.\nV12—Invariably, farmers who come to the desk describ-\ning vague signs and reluctant to present the sheep\nfor clinical examination, will leave with some sort\nof cortisone if they are not in lamb, you check if\nthey are not in lamb, and a vitamin and probably an\nantibiotic—penicillin type or oxytetracycline based,\nare my favourites for those sheep.\nDISCUSSION\nWhile Northern Ireland sheep farmers participating in this\nstudy expressed positivity about the benefits of pain relief\nmedicines and the use of some form of anti-inflammatory was\nwidespread, it was not universal. This provides an opportunity\nfor the industry to consider the barriers to use of NSAIDs and\nto promote their use to unengaged farmers.\nTo manage pain effectively, first the pain must be recog-\nnised and then an efficacious medicine must be administered.4\nThe farmers already using NSAIDs and veterinarians in this\nstudy were clear about which conditions they considered\nto be painful. The range of conditions that both groups\nidentified (obstetrical, surgical, mastitis and lameness cases)\nreflected previous reports on ovine conditions benefitting\nfrom analgesia.11,26 The farmers recognised the benefits that\nanalgesia provided in aiding recovery and fed this informa-\n\n11,26 The farmers recognised the benefits that\nanalgesia provided in aiding recovery and fed this informa-\ntion back to their veterinarians. Observing this response to\ntreatment influenced farmers’ behaviour, resulting in them\nchoosing to maintain a supply of NSAIDs on farms so there\nwas some available immediately if needed, especially during\nthe lambing period. This change hopefully improved animal\nwelfare and will encourage more farmers to follow suit, given\nthe significance of peer influence across a range of farming\nsituations.27\nFarmers indicated that it was their veterinarian who had\ninitially suggested using an NSAID as part of treatment proto-\ncols, reinforcing the previously stated view that veterinarians\nare trusted advisors to farmers.21 One veterinarian had iden-\ntified a need within farmers to do something when faced\nwith a cow who was dull (and no specific diagnosis could\nbe reached). His approach was to encourage farmers to use\nNSAIDs rather than antibiotics as the first-line treatment.\nThis first-line use of NSAIDs was reflected in the comments\nfrom a farmer, who, when faced with a dull sheep, realised\nthat pain rather than infection may be the cause of the dull-\nness and inappetence and reported a positive outcome using\nNSAIDs. This is an example of how past behaviour may influ-\nence attitudes moving forward,28 with sheep farmers electing\nto use NSAIDs promptly in future cases and talking openly\nto their peers about the benefits they perceive during discus-\nsion group sessions. Encouraging more farmers to assess their\nsheep and consider the potential benefits of analgesia rather\nthan immediately reaching for antibiotics could improve the\nstewardship of antibiotics through the creation of a new\nbehavioural pattern. In time, the use of pain relief as a first-\nline treatment could come to define what constitutes a ‘good\nfarmer’.29\nOver a quarter (27%) of participating farmers were not\nusing NSAIDs. Insufficient data were collected to definitively\nsay whether this was due to a lack of clinical indication for\nanalgesics, although the quantity of antibiotic purchased by\n\nof \nVETERINARY RECORD OPEN\nsome would suggest clinical infectious disease was present.\nFew, if any, sheep suffering from an infectious condition will\nnot benefit from NSAID provision. Overall, while no sig-\nnificant relationship was identified between antibiotic and\nNSAID purchases, the results suggested a potential trend\nthat may have been statistically significant with a larger\ndataset. Further study is needed to characterise the conditions\nbeing treated with antibiotics and the barriers to those sheep\nconcurrently receiving analgesia.\nAreas where further advice on pain recognition and treat-\nment is potentially warranted and highlighted in these results\ninclude lameness, CODD specifically and neonatal lambs.\nWhen treating CODD, a farmer stated that ‘if you thought\nthey were needing an anti-inflammatory’ (SF33), they used a\ncombination antibiotic and anti-inflammatory product, while\nmost of their lame sheep were treated with an antibiotic only.\nThis, combined with the low level of use of NSAIDs on some\nfarms purchasing significant quantities of antibiotics, suggests\nthat there is a pain hierarchy, where some lame animals are\ndeemed worthy of analgesia and others are not. This finding\nreinforces the need for farmer and veterinarian education on\npain recognition and the development and dissemination of\npain-scoring tools validated for general application in an on-\nfarm environment.30 Given that only minimal reference was\nmade by farmers and veterinarians to the provision of analge-\nsia for routine husbandry procedures such as tail-docking and\ncastration, this is another area that may benefit from further\nengagement, given the calls for improvements in the welfare\nof lambs undergoing such procedures.4,30,31\nThree NSAIDs were supplied by veterinary practices. None\nare authorised in the UK and thus defined doses were not\navailable, complicating comparison between the relative pro-\nportions of each product supplied and in determining the\nnumber of sheep likely to have been treated based on the\nvolumes sold onto each farm. NSAIDs are recognised as pro-\n\nNSAIDs are recognised as pro-\nviding some analgesia in sheep.4,18,26 However, authoritative\nefficacy studies of NSAID use in clinical cases in the field are\nlacking.26 Veterinarians stated concerns about the lack of an\nauthorised product for pain management in sheep and may\nlack the confidence to determine the optimal product and\ndose, to use for providing analgesia in sheep. The lack of an\nauthorised product will inhibit promotion of NSAIDs through\navenues such as professional publications. In the human med-\nical field, advertising prescription-only medicines directly to\nthe public has been shown to significantly increase patients’\nconfidence in talking to doctors about their concerns and\nspecific solutions.32 If farmers fail to see promotion of named\nanalgesics, they may wrongly assume that, as there is no pro-\nmotion of pain-relieving medication, pain is not a welfare\nconcern in sheep.\nMeloxicam, the most commonly supplied NSAID, was\ndescribed by veterinarians as their standard treatment or a\npreference based on experience. This product is authorised in\nsome countries outside the UK, for use in sheep at 1 mg/kg.33\nThis is twice the authorised dose that UK veterinarians are\nfamiliar prescribing for cattle. They were not asked specifi-\ncally what dose of each product they prescribed in this study.\nThis avoided participants feeling that they were being tested\non clinical knowledge, which may have reduced their trust\nand thus openness in participation.34 There remains a knowl-\nedge gap surrounding the exact dosing strategy recommended\nby veterinarians and how they determine the dose, treat-\nment frequency, duration and withdrawal periods of these\nproducts.\nPressure should continue to be put on the pharmaceutical\nindustry, or state action taken, to provide safety and efficacy\ndata on efficacious analgesia products for sheep. Veterinari-\nans could then use this information to formulate prescribing\nprotocols for all sheep, specifically neonatal lambs, to facilitate\nprogress in analgesia provision. Additionally, an authorised\nproduct would have a specific sheep meat withdrawal period,\n\nVeterinari-\nans could then use this information to formulate prescribing\nprotocols for all sheep, specifically neonatal lambs, to facilitate\nprogress in analgesia provision.Additionally, an authorised\nproduct would have a specific sheep meat withdrawal period,\nremoving the need to apply the statutory sheep meat with-\ndrawal period, 28 days in this instance, required under the\ncascade.15\nComparative studies have failed to demonstrate any analge-\nsia provided by dexamethasone.35 It has been reported that CS\ncan have a positive effect on ameliorating clinical signs, such\nas inappetence and pyrexia, as well as improving the general\nwellbeing of a sick animal,19,36 without improving clinical out-\ncome except in limited, specific conditions.19 Responses from\nveterinarians and farmers in this study indicated that some\ncontinue to use CS for such purposes despite a lack of evidence\nof efficacy in sheep. However, where pain is the cause of dull-\nness or inappetence, a greater impact on animal welfare may\nbe gained through encouraging these farmers to administer\nan appropriate dose of an NSAID instead of the CS. Further\nwork is needed to understand why farmers are requesting\nveterinary practices to prescribe CS products, which were\nauthorised in cattle and only to be used in sheep under the\ncascade. It may be that farmers have followed some veteri-\nnarians’ use of these products where a diagnosis is unknown\nor other rational treatments have failed; alternatively, veteri-\nnarians still supply them, failing to appreciate the superior\nanalgesic benefits NSAIDs can provide. As CS are known to\ninduce parturition, they are contraindicated where the preg-\nnancy is to be maintained. This may mean sheep miss out\non analgesia for conditions such as lameness, where a farmer\nrelies on CS, but knows not to administer them to pregnant\newes. In New Zealand, where meloxicam is authorised for use\nin sheep, the product carries no contraindications for use dur-\ning pregnancy33 and may offer safe and efficacious analgesia\nfor sheep throughout pregnancy.\nQuality assurance schemes often require farmers to review\n\nQuality assurance schemes often require farmers to review\nantibiotic use.37 The disparity in NSAID use between farms\nsuggests that a review of analgesia provision is also important\nto improve animal welfare. The lack of authorised dosages, dif-\nfering potencies and concentrations of active ingredients in\nNSAIDs makes the analysis and benchmarking of these prod-\nucts complicated. If suitable metrics could be developed and\nvalidated, then an objective review of the farmers’ provision of\npain relief could be instigated.\nVeterinarians should ensure that when they attend to a\nsheep suffering from a painful condition and provide anal-\ngesia, they could discuss which product they have dispensed\nand why with the farmer.30 Veterinarians participating in this\nstudy highlighted that farmers are not always aware of the\npurpose of medications administered to their animals. This\nwill allow farmers to learn about and appreciate the benefits\nof pain relief in sheep, hopefully going on to adopt analgesia\ninto their normal routine practices. However, one veterinarian\nhere highlighted that such opportunities to interact with many\nsheep farming clients can be rare because these clients tend\n\nVETERINARY RECORD OPEN\nof \nto be self-sufficient, emulating previous observations about\nsheep farmers.38\nVeterinarians and farmers highlighted that veterinary con-\nsultation over the best method of treating animals was central\nto farmers adopting NSAIDs for pain relief in their flocks.\nClear efficacy and safety data should be developed to enable\nwider promotion of analgesia for sheep. To further promote\nanimal welfare, new approaches need to be considered to\nencourage more engagement between farmers and their vet-\nerinarians. Until suitable metrics have been developed for\nbenchmarking NSAID use, a subjective approach can be\nadopted. Veterinarians should be encouraged to challenge\nfarmers each time antibiotics are prescribed, both on whether\nthe antibiotic is necessary for the condition and whether\nanalgesia is also required.\nAU T HOR C ON T R I BU T ION S\nAll authors contributed to the design of the wider project from\nwhich these data are drawn. Paul E. Crawford conducted all\ndata collection and analysis. All authors contributed to the\nwriting and critical review of the manuscript.\nAC K NOW L E D G E M E N T S\nPaul E. Crawford was in receipt of a PhD scholarship from the\nDepartment of Agriculture, Environment and Rural Affairs,\nNorthern Ireland, when this work was undertaken. A subsec-\ntion of the data was presented in abstract form at the Inter-\nnational Sheep Veterinary Conference, March 2023, Seville,\nSpain. The authors thank Janet Roden for her assistance with\nthe statistical elements and all the participants who gave their\ntime and records to this study.\nC ON F L IC T S OF I N T E R E ST STAT E M E N T\nThe authors declare they have no conflicts of interest.\nDATA AVA I L A B I L I T Y STAT E M E N T\nDue to the confidentiality agreements entered between\nresearcher and participants, the raw data cannot be made\navailable.\nET H IC S STAT E M E N T\nThe authors confirm that the ethics policies of the journal,\nas noted on the journal’s author guidelines page, have been\nadhered to. Ethical approval was obtained from the Research\nEthics Committee of Harper Adams University (approval\nnumber: 0010-202101-PGMPHD).\n\nEthical approval was obtained from the Research\nEthics Committee of Harper Adams University (approval\nnumber: 0010-202101-PGMPHD).\nORC I D\nPaul E.Crawford\nhttps://orcid.org/0000-0003-3666-6050\nKimHamer\nhttps://orcid.org/0000-0002-6142-5366\nFionaLovatt\nhttps://orcid.org/0000-0003-2298-9174\nMalgorzataC.Behnke\nhttps://orcid.org/0000-0003-4352-\n6312\nPhilip A.Robinson\nhttps://orcid.org/0000-0002-0577-3147\nR E F E R E NC E S\n1. Fitzpatrick J, Scott M, Nolan A. Assessment of pain and welfare in sheep.\nSmall Rumin Res. 2006;62:55–61. https://doi.org/10.1016/j.smallrumres.\n2005.07.028\n2. Small A, Fisher AD, Lee C, Colditz I. Analgesia for sheep in commer-\ncial production: where to next? Animals. 2021;11:1127. https://doi.org/10.\n3390/ani11041127\n3. Lizarraga I, Chambers JP. Use of analgesic drugs for pain management in\nsheep. 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+{"text": "【兽医文献】\n标题: Case Report: Diagnosis and treatment of equine ascending placentitis: compilation of 17 case reports\n\nTYPE Original Research\nPUBLISHED 22 August 2025\nDOI 10.3389/fvets.2025.1591214\nOPEN ACCESS\nEDITED BY\nGeorgios A. Papadopoulos,\nAristotle University of Thessaloniki, Greece\nREVIEWED BY\nPanagiotis Tassis,\nAristotle University of Thessaloniki, Greece\nPanagiotis Sakkas,\nAristotle University of Thessaloniki, Greece\n*CORRESPONDENCE\nMaamer Jlali\nmaamer.jlali@adisseo.com\nRECEIVED 10 March 2025\nACCEPTED 23 July 2025\nPUBLISHED 22 August 2025\nCITATION\nJlali M, Hincelin C, Manceaux C and Ozbek S\n(2025) Efficacy of a new biosynthetic bacterial\n6-phytase on growth performance, bone\nmineralization, and nutrient digestibility in\nnursery and growing pigs.\nFront. Vet. Sci. 12:1591214.\ndoi: 10.3389/fvets.2025.1591214\nCOPYRIGHT\n© 2025 Jlali, Hincelin, Manceaux and Ozbek.\nThis is an open-access article distributed\nunder the terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other forums is\npermitted, provided the original author(s) and\nthe copyright owner(s) are credited and that\nthe original publication in this journal is cited,\nin accordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nEfficacy of a new biosynthetic\nbacterial 6-phytase on growth\nperformance, bone\nmineralization, and nutrient\ndigestibility in nursery and\ngrowing pigs\nMaamer Jlali1*, Cl´ementine Hincelin2, Celsa Manceaux3 and\nSarper Ozbek1\n1Adisseo France S.A.S., European Laboratory of Innovation, Science and Expertise, Saint-Fons, France,\n2Adisseo France S.A.S., Antony, France, 3Parc Technologique du Zoopôle, Zootests, Ploufragan, France\nTwo studies were carried out to investigate the effects of a novel bacterial\nbiosynthetic 6-phytase on growth performance, bone mineralization, and\napparent total digestibility (ATTD) of phosphorus (P) in weaned piglets and\ngrowing pigs. They were carried out on 192 weaned piglets with initial body\nweight (BW): 9.3 ± 1.2 kg (33 days of age) and 360 growing pigs with initial\nBW: 33.3 ± 4.9 kg (85 days) for 43 and 84 days, respectively, according to a\nrandomized complete block design with three treatments. The treatments were\n\n3 ± 1.2 kg (33 days of age) and 360 growing pigs with initial\nBW: 33.3 ± 4.9 kg (85 days) for 43 and 84 days, respectively, according to a\nrandomized complete block design with three treatments.The treatments were\na positive control (PC) diet formulated to meet nutrient requirements, a negative\ncontrol (NC) diet reduced similarly in calcium (Ca) and digestible P by 0.12%\npoints in piglets and by 0.14, 0.11, and 0.10% points, respectively, in phases 1, 2,\nand 3 in growing pigs, compared to the PC diet; and the NC diet supplemented\nwith the new 6–phytase at 500 phytase units (FTU) per kg of diet (PHY). The\nmineral depletion decreased final BW (−6.3%, p = 0.005; −3.0%, p < 0.05),\naverage daily gain (ADG: −8.8%, p = 0.003; −4.3%, p < 0.05), bone ash content\n(−24.7%, p < 0.001; −9.6%, p = 0.005), bone P content (−24.6%, p < 0.001;\n−6.5%, p = 0.11), and ATTD of P (−11.8% points, p < 0.001; −9.2% points,\np < 0.001) and increased the feed–to–gain (F:G) ratio (+2.1%, p < 0.001; +4.7%,\np < 0.05) in weaned piglets and growing pigs, respectively. Compared to animals\nfed the NC diet, phytase addition improved the final BW (+6.5%, p = 0.006,\n+2.3%, p < 0.05), ADG (+9.0%, p = 0.005, +3.4%, p < 0.05), F:G ratio (−3.1%,\np < 0.001, −2.4%, p < 0.05), metacarpal ash content (+27.6%, p < 0.001, +9.3%,\np = 0.004), and metacarpal P content (+29.3%, p < 0.001, +7.0%, p = 0.06) in\nweaned piglets and growing pigs, respectively. The final BW, ADG, and bone ash\ncontent in animals fed the NC diet supplemented with phytase were comparable\nto animals fed the PC diet. The supplementation of phytase to the NC diet\nimproved (P < 0.001) the ATTD of P by 13.5 and 24.6% points (p < 0.001) in\nweaned piglets and growing pigs, respectively. Compared to the NC diet, phytase\nsupplementation also improved the ATTD of Ca (+7.8% points, p) and N (+2.5%\npoints) in growing pigs. This finding indicates the ability of this novel biosynthetic\nbacterial 6–phytase to restore performance and bone mineralization in piglets\nand growing pigs fed P– and Ca–reduced diets.\nKEYWORDS\nefficacy, phytase, mineralization, piglets, pigs\n\nThis finding indicates the ability of this novel biosynthetic\nbacterial 6–phytase to restore performance and bone mineralization in piglets\nand growing pigs fed P– and Ca–reduced diets.KEYWORDS\nefficacy, phytase, mineralization, piglets, pigs\nFrontiers in Veterinary Science\n01\nfrontiersin.org\n\nJlali et al.\n10.3389/fvets.2025.1591214\n1 Introduction\nPhosphorus (P) present in plant-feed ingredients constitutes\nthe major part (50% to 75%) of the phosphorus supply in swine\ndiets. However, a significant proportion of the total P (up to\n80%) is present in the form of phytate-P, which is poorly digested\nby non-ruminant animals due to the low level of endogenous\nphytase activity present in the gastrointestinal tract (GIT) of these\nanimals (1–3).\nIt is well known that phytate can form complexes with\nprotein and minerals within the GIT, reducing the digestibility and\nabsorption of nutrients in monogastric animals (4, 5). Exogenous\nphytase is well-known to hydrolyze the phytate complex present\nin most oilseeds, protein meals, and cereals (6, 7). Hence,\nsupplementation of swine diets with microbial phytase has largely\nproven to be an effective way to enhance the bioavailability of\nP from phytate-P, improving P digestibility, performance, and\nbone mineralization of animals fed diets reduced in P and\ncalcium (Ca) and reducing the excretion of phosphorus in the\nenvironment (8–10). However, the effects of such exogenous\nphytase on these parameters could largely depend on its source,\ndose, and intrinsic characteristics of the evaluated phytase and\nfeed composition, especially in terms of dietary P and Ca levels. A\nnovel bacterial 6-phytase characterized by a high affinity for myo-\ninositol hexakisphosphate (IP6) and other myo-inositol phosphate\n(IP) esters, a high activity over a wide pH range, and a high intrinsic\nthermostability, which is important during feed processing, was\nrecently developed. In addition, its efficacy in broilers and laying\nhens was also investigated (10, 11). However, there are limited data\nto demonstrate the efficacy of this phytase in nursery and growing\npigs. Therefore, the aim of this study was to investigate the effects\nof this novel bacterial 6-phytase on growth performance, bone\nmineralization, and apparent total tract digestibility (ATTD) of\nnutrients in nursery and growing pigs fed diets reduced in digestible\nP and Ca.\n2 Materials and methods\n\n2 Materials and methods\nAll experimental procedures were carried out according\nto legislation governing the ethical treatment of animals, and\ninvestigators were certified by the French government to conduct\nanimal experiments. All experimental procedures were conducted\nunder the authorization number G-03-159-4 at the Center\nof Expertise in Research and Nutrition (Adisseo France SAS,\nMalicorne, France) and internal standard operating procedures at\nZootests (Ploufragan, France).\n2.1 Animals, diets, and experimental design\n2.1.1 Experiment 1—Weaned piglets\nThe experiment was conducted at the Center of Expertise in\nResearch and Nutrition (Adisseo France SAS, 03600 Malicorne).\nA total of 192 weanling male piglets (initial BW: 9.3 ±1.2 kg)\nwere blocked by initial body weight and randomly allotted to\nthree dietary treatments using a randomized complete block design.\nDietary treatments consisted of a positive control (PC) diet with\nadequate supply of all nutrients according to the National Research\nCouncil (NRC) 2012 including Ca (0.70 and 0.66% for phases 1\nand 2, respectively) and digestible P (0.33 and 0.31% for phases\n1 for 14 days and 2 for 28 days, respectively); a negative control\n(NC) reduced in Ca and digestible P similarly by 0.12% unit (as\nthe amount of digestible P which can be released with the present\nphytase at 500 FTU/kg diet) compared to the PC diet; and a NC\ndiet supplemented with this new 6-phytase at 500 FTU/kg diet\n(Rovabio PhyPlus, Adisseo France SAS, France). The composition\nand nutrient characteristics of the basal diets are presented in\nTable 1. Titanium dioxide was included in all diets of phase 2 (post-\nweaning 2) at 5 g/kg as an indigestible marker for apparent total\ntract digestibility (ATTD) of P assessment. Diets were offered as\npellets throughout the experimental period. Piglets had ad libitum\naccess to experimental diets and water for 42 d. The body weight\n(BW) and feed intake (FI) were recorded two times (at days\n14 and 42) to determine the average daily gain (ADG), average\ndaily feed intake (ADFI), and feed-to-gain (F:G) ratio. Digestibility\n\nThe body weight\n(BW) and feed intake (FI) were recorded two times (at days\n14 and 42) to determine the average daily gain (ADG), average\ndaily feed intake (ADFI), and feed-to-gain (F:G) ratio.Digestibility\nmeasurements were carried out on days 34 to 38 of the experiment,\nand fresh fecal samples were collected every day from 16 pens\nper treatment, then pooled by pen, and immediately stored at\n−20◦C until further analysis and determination of the total tract\ndigestibility (ATTD) of P. On day 43, all piglets (24 piglets per\ntreatment) from 12 pens per treatment used for fecal sample\ncollection were anesthetized by intramuscular injection of Zoletil\n100 (1 mL/10 kg BW) and then euthanized by T61 (1 mL/10 kg\nBW), and the right feet from the fore were excised to determine\nmetacarpal bones (III and IV) ash and P contents.\n2.1.2 Experiment 2—Growing pigs\nThe experiment was conducted at Zootests SAS (Ploufragan,\nFrance). A total of 360 growing pigs (180 barrows and 180 gilts)\nwith an average initial BW of 33.4 ± 4.9 kg (85 days of age) and\nindividually identified with radio-frequency identification (RFID)\ntags were divided into two runs and assigned to three treatments in\na randomized complete block design. Dietary treatments consisted\nof a positive control (PC) diet with adequate supply of all nutrients\nincluding Ca and digestible P; a negative control (NC) reduced\nin digestible P (0.17 vs. 0.31, 0.15 vs. 0.26, and 0.13 vs. 0.23%\nfor phases 1, 2, and 3, respectively) and Ca (0.55 vs. 0.69, 0.50\nvs. 0.61, and 0.46 vs. 0.56% for phases 1, 2, and 3, respectively)\ncompared to the PC diet; and a NC diet supplemented with the new\n6-phytase at 500 FTU/kg diet (Rovabio PhyPlus, Adisseo France\nSAS, France). Experimental diets were fed in three phases (grower\n1 for 29 days, grower 2 for 25 days, and finisher for 28 days).\nThe composition and characteristics of basal diets are presented\nin Table 2. Each treatment was presented equally in each block\n(n = 4 blocks with 15 pigs/treatment in each block). Each block\nwas equipped with two Self Automatic Feeders (SAF) that allow\n\nThe composition and characteristics of basal diets are presented\nin Table 2.Each treatment was presented equally in each block\n(n = 4 blocks with 15 pigs/treatment in each block).Each block\nwas equipped with two Self Automatic Feeders (SAF) that allow\nfor recording daily individual body weight and individual feed\nconsumption. Titanium dioxide was included in all diets of phase\n1 at 5 g/kg as an indigestible marker for ATTD of nutrients. From\ndays 109 to 113 (phase 1) of the experiment, fresh fecal samples\nwere collected by rectal stimulation from 16 pigs per treatment\nduring 5 consecutive days, then pooled by pig, and immediately\nstored at −20◦C until further analysis. At the end of phase 1\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n\nJlali et al.\n10.3389/fvets.2025.1591214\nTABLE 1 Composition and characteristics of basal diets for weanling\npiglets (Experiment 1).\nPhase 1\n(post-weaning 1)\nPhase 2\n(post-weaning 2)\nPC\nNC\nPC\nNC\nComposition, %\nCorn\n45.63\n45.45\n58.95\n60.31\nWheat\n12.28\n13.79\n15.00\n15.00\nSoybean meal 48%\n27.39\n27.09\n19.27\n19.04\nWhey powder\n10.00\n10.00\n-\n-\nRapeseed oil\n1.00\n0.60\n1.91\n1.47\nMonocalcium\nphosphate\n0.79\n0.12\n1.19\n0.52\nCalcium carbonate\n0.55\n0.53\n0.68\n0.66\nL-Lysine HCl 98%\n0.43\n0.44\n0.42\n0.42\nDL-methionine 99%\n0.13\n0.12\n0.07\n0.06\nL-Threonine 98.5%\n0.13\n0.13\n0.12\n0.12\nL-Tryptophan 98%\n0.02\n0.02\nSalt\n0.24\n0.24\n0.43\n0.43\nPremixa\n1.45\n1.45\n1.45\n1.45\nTitanium dioxide\n-\n-\n0.50\n0.50\nCalculated nutrients, %\nDry matter\n88.51\n88.38\n87.52\n87.37\nCrude protein\n19.09\n19.12\n15.75\n15.75\nCrude fat\n3.36\n2.97\n4.55\n4.16\nCrude fiber\n2.96\n2.97\n2.85\n2.87\nAsh\n5.39\n4.84\n4.62\n4.07\nNE, kcal/kg\n2,412\n2,412\n2,475\n2,475\nDig. Lysine\n1.23\n1.23\n0.98\n0.98\nDig. Methionine\n0.39\n0.39\n0.29\n0.29\nDig. Cystine\n0.29\n0.29\n0.26\n0.26\nDig. Met+Cys\n0.68\n0.68\n0.55\n0.55\nDig. Threonine\n0.73\n0.73\n0.59\n0.59\nDig. Tryptophan\n0.20\n0.20\n0.17\n0.17\nDig. Arginine\n1.10\n1.09\n0.88\n0.88\nDig. Valine\n0.78\n0.78\n0.64\n0.64\nCalcium\n0.70\n0.58\n0.66\n0.54\nTotal P\n0.60\n0.45\n0.59\n0.44\nDigestible P\n0.33\n0.21\n0.31\n0.19\nPhytate-P\n0.21\n0.22\n0.21\n0.22\nSodium\n0.17\n0.17\n0.17\n0.17\naPremix provides per kg feed: vitamin A, 9715 IU; vitamin D3, 1943 IU; vitamin E (α-\ntocopherol), 48.4 IU; vitamin B1, 1.9 mg; vitamin B2, 4.8 mg; vitamin B3, 19.5 mg; vitamin B5,\n14.5 mg; vitamin B6, 2.9 mg; vitamin B12, 29 µg; vitamin K3, 1.9 mg; Fe, 135 mg; I, 0.73 mg;\nCu, 87 mg; Mn, 58.4 mg; Zn, 103 mg; Se, 0.30 mg; choline, 290 mg. PC, positive control diet;\nNC, negative control diet similarly reduced by 0.12% unit of digestible P and Ca.\n(on day 113 of the experiment), 16 pigs were randomly chosen\nper treatment. They were anesthetized by intramuscular injection\nof Zoletil 100 (1 mL/10 kg BW) and then euthanized by T61 (1\nmL/10 kg BW), and the right feet from the fore were excised to\ndetermine metacarpal bones (III and IV) ash and P contents.\n2.2 Chemical analyses\nRepresentative samples of all experimental diets were analyzed\nfor dry matter, crude fat, ash, crude fiber, nitrogen (N), Ca, total\nP, phytate-P, and phytase activity. In addition, in Experiment 1,\ntotal P was analyzed in fecal samples, and in Experiment 2, total\n\nIn addition, in Experiment 1,\ntotal P was analyzed in fecal samples, and in Experiment 2, total\nP, Ca, and N were analyzed in fecal samples. Dry matter, crude fat,\nash, crude fiber, and N were determined using the method AOAC\nprocedure (method 934.01; AOAC, 2006). Total P and Ca were\nanalyzed using the inductively coupled plasma (ICP) spectroscopy\nmethod (method 985.01 A, B, and C, AOAC, 2006). Phytate-P\nwas analyzed using the colorimetric method. The concentration of\ntitanium dioxide in diets and ileal digesta was analyzed following\nthe procedure of Myers et al. (12). Ash content in metacarpal bones\nwas determined by removing the adhering tissue, drying the bone at\n110◦C for 12 h, and extracting fat with ether. The dry fat-free bones\nwere ashed in a muffle furnace at 550◦C for 3 h, according to AOAC\nInternational (2000, method 932.A6). P content in metacarpal\nbones was measured using dry-ashed bone samples. The activity\nof phytase in the experimental diets was analyzed according to\nthe ISO standard methodology (ISO Standard 300242, 2009). One\nunit of phytase is defined as the amount of enzyme that liberates\none micromole of inorganic orthophosphate from phytic acid per\nminute at pH 5.5 and 37◦C.\n2.3 Calculations and statistical analysis\nThe ATTD (%) of P, Ca, and N in the experimental diets was\ndetermined using the following equation:\nATTD = (1 −[(Tid/Tif) x (Nf/Nd)]) x 100\nwhere Tid and Tif are the concentration of titanium in diets and\nfeces, respectively; Nd and Nf are the concentration of nutrients (P,\nCa, or N) in diets and feces, respectively.\nData were analyzed using the MIXED procedure of SAS (SAS\nInst. Inc., Cary, NC). Residuals were checked for normality. In\nExperiment 1, block (n =32) and treatment (n = 3) were used as\nfixed variables. In Experiment 2, an individual pig was used as an\nexperimental unit. Treatment, gender, and animals’ batch, and their\ninteractions were considered as fixed variables. Initial body weights\nof pigs were used as covariates for growth performance parameters\n(BW, ADG, ADFI, and FCR). Comparisons of least square means\n\nTreatment, gender, and animals’ batch, and their\ninteractions were considered as fixed variables.Initial body weights\nof pigs were used as covariates for growth performance parameters\n(BW, ADG, ADFI, and FCR).Comparisons of least square means\nfor each significant effect were performed using the Tukey-Kramer\ntest. Statistical significance was set at a P < 0.05, and 0.05 < P <\n0.10 was considered a trend.\nFrontiers in Veterinary Science\n03\nfrontiersin.org\n\nJlali et al.\n10.3389/fvets.2025.1591214\nTABLE 2 Composition and characteristics of basal diets for growing-finishing pigs (Experiment 2).\nPhase 1 (from 85 to 115 d)\nPhase 2 (from 115 to 140 d)\nPhase 3 (from 140 to 182 d)\nPC\nNC\nPC\nNC\nPC\nNC\nComposition, %\nCorn\n73.74\n75.32\n81.49\n82.674\n87.42\n86.75\nWheat bran\n1.00\n1.00\n2.00\n2.00\n4.47\n5.92\nSoybean meal 48%\n19.75\n19.48\n12.80\n12.64\n5.19\n5.00\nSoy oil\n1.51\n1.00\n0.59\n0.19\n0.00\n0.00\nCalcium carbonate\n0.86\n0.84\n0.81\n0.79\n0.79\n0.78\nMonocalcium\nphosphate\n1.20\n0.41\n0.95\n0.34\n0.81\n0.24\nL-Lysine HCl 98%\n0.41\n0.42\n0.38\n0.38\n0.37\n0.37\nDL-Methionine 99%\n0.07\n0.07\n0.03\n0.03\n0.00\n0.00\nL-Threonine 98.5%\n0.11\n0.11\n0.10\n0.10\n0.10\n0.10\nL-Tryptophan 98%\n0.03\n0.03\n0.03\n0.03\n0.04\n0.04\nSalt\n0.43\n0.43\n0.43\n0.43\n0.41\n0.41\nPremixa\n0.40\n0.40\n0.40\n0.40\n0.40\n0.40\nTitanium dioxide\n0.50\n0.50\n-\n-\n-\n-\nNutrient characteristics, %\nDry matter\n87.33\n87.16\n87.04\n86.91\n86.82\n86.76\nCrude protein\n15.52\n15.52\n12.95\n12.97\n10.24\n10.32\nCrude fat\n4.49\n4.03\n3.76\n3.40\n3.35\n3.37\nCrude fiber\n2.94\n2.96\n2.78\n2.80\n2.69\n2.79\nAsh\n4.65\n4.00\n4.11\n3.60\n3.67\n3.24\nNet energy, kcal/kg\n2,475\n2,475\n2,475\n2,475\n2,475\n2,475\nDig. Lysine\n0.98\n0.98\n0.79\n0.79\n0.61\n0.61\nDig. Methionine\n0.30\n0.30\n0.23\n0.23\n0.17\n0.17\nDig. Met+Cys\n0.55\n0.55\n0.45\n0.45\n0.36\n0.36\nDig. Threonine\n0.59\n0.59\n0.49\n0.49\n0.40\n0.40\nDig. Tryptophan\n0.17\n0.17\n0.14\n0.14\n0.11\n0.11\nDig. Arginine\n0.88\n0.88\n0.69\n0.69\n0.49\n0.49\nDig. Valine\n0.64\n0.64\n0.53\n0.53\n0.42\n0.42\nCalcium\n0.69\n0.55\n0.61\n0.50\n0.56\n0.46\nTotal P\n0.59\n0.42\n0.52\n0.38\n0.48\n0.36\nDigestible P\n0.31\n0.17\n0.26\n0.15\n0.23\n0.13\nPhytate-P\n0.22\n0.22\n0.22\n0.22\n0.22\n0.23\nSodium\n0.17\n0.17\n0.17\n0.17\n0.16\n0.16\naPremix provides per Kg feed: vitamin A, 2,680 IU; vitamin D3, 536 IU; vitamin E (alfa-tocopherol), 13.3 IU; vitamin B1, 0.5 mg; vitamin B2, 1.3 mg; vitamin B3, 5.4 mg; vitamin B5, 4.0 mg;\nvitamin B6, 0.8 mg; vitamin B12, 8 µg; vitamin K3, 0.5 mg; Fe, 37.3 mg; I, 0.2 mg; Cu,24 mg; Mn, 16.1 mg; Zn, 28.4 mg; Se, 0.1 mg; choline, 80 mg. PC, positive control diet; NC, negative control\ndiet similarly reduced by 0.14, 0.11, and 0.10% units in digestible P and Ca for grower 1, grower 2, and finisher phase, respectively, compared to the PC diet.\n3 Results\nThe analyzed values of dietary nutrients and phytase activities\nin experimental diets are presented in Tables 3, 4, respectively.\nThe analyzed dietary Ca and total P contents were, in general, in\n\n3 Results\nThe analyzed values of dietary nutrients and phytase activities\nin experimental diets are presented in Tables 3, 4, respectively.The analyzed dietary Ca and total P contents were, in general, in\nagreement with the expected values. Analyzed values of phytate-P\nwere, on average, 0.21% and 0.22% in experiments 1 and 2,\nrespectively. Analyzed phytase activities in the experimental diets\nsupplemented with phytase were 415 (phase 1), 507 (phase 2),\nand 474 (phase 1), 511 (phase 2), and 501 (phase 3) FTU/kg diet\nin experiments 1 and 2, respectively. In the two experiments, no\nmortality was observed in the groups, and all the animals were in\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n\nJlali et al.\n10.3389/fvets.2025.1591214\ngood health and readily consumed diets throughout the duration\nof both experiments.\n3.1 Experiment 1—Weaned piglets\nThe effects of dietary treatments on growth performance, bone\nmineralization, and ATTD of P in piglets are presented in Tables 5,\n6. Piglets fed with the NC diet reduced in digestible P and Ca had\nlower final body weight (-6.3%, p = 0.005), ADG (-8.8%, p = 0.003),\nand ADFI (-6.9%, p = 0.02) and a higher F:G ratio (+2.1%, p =\n0.02) in comparison with piglets fed with the PC diet. In addition,\nTABLE 3 Analyzed nutrients of experimental diets (Experiment 1).\nItem\nPost-weaning 1\nPost-weaning 2\nPC\nNC\nPHY\nPC\nNC\nPHY\nDry matter, %\n90.50\n90.20\n90.00\n87.80\n87.50\n88.20\nCrude\nprotein, %\n18.80\n19.20\n19.00\n15.10\n15.20\n15.20\nCrude fat, %\n3.00\n2.30\n2.50\n4.10\n4.00\n3.60\nCrude fiber, %\n2.30\n2.30\n2.40\n2.00\n2.00\n2.20\nAsh, %\n5.10\n4.80\n4.70\n4.20\n4.30\n4.60\nCalcium, %\n0.72\n0.58\n0.61\n0.66\n0.55\n0.54\nTotal\nphosphorus,\n%\n0.63\n0.47\n0.46\n0.57\n0.43\n0.45\nPhytate-P, %\n0.25\n0.22\n0.23\n0.21\n0.20\n0.15\nPhytase\nactivity,\nFTU/kg diet\n47\n82\n415\n115\n112\n507\nPC, positive control diet; NC, negative control diet; similarly reduced by 0.12% unit of\ndigestible P and Ca; PHY, negative control diet supplemented with phytase (Rovabio PhyPlus)\nat 500 FTU/kg diet.\nmineral reduction decreased (p < 0.001) the ash and P contents\nin metacarpal bones by 24.7 and 24.6%, respectively. Compared\nto piglets fed with the NC diet, piglets fed with the NC diet\nTABLE 5 Growth performance of weaned piglets fed diets without or with\nphytase for 42 days (Experiment 1).\nItem\nTreatment\nAdjusted\nSEM\nP-\nvalue\nPC\nNC\nPHY\nPhase 1, 0–14d\nBW d0, kg\n9.25\n9.34\n9.32\n0.03\n0.13\nBW d14, kg\n13.66\n13.50\n13.99\n0.17\n0.13\nADG d0-14,\ng/piglet/d\n315.23\n297.15\n333.92\n11.59\n0.09\nADFI d0-14,\ng/piglet/d\n515.64\n509.05\n525.40\n11.96\n0.63\nFCR d0-14\n1.672ab\n1.761a\n1.590b\n0.034\n0.004\nPhase 2, 14-42d\nBW d42, kg\n36.14a\n33.86b\n36.05a\n0.53\n0.006\nADG d14-42,\ng/piglet/d\n802.82a\n727.10b\n787.73a\n15.28\n0.003\nADFI d14-42,\ng/piglet/d\n1,398.14a\n1,286.47b\n1,365.89ab\n27.98\n0.02\nFCR d14-42\n1.740ab\n1.768a\n1.731b\n0.01\n0.04\nGlobal period, 0-42d\nADG d0-42,\ng/piglet/d\n640.29a\n583.78b\n636.46a\n12.58\n0.004\nADFI d0-42,\ng/piglets/d\n1,103.97a\n1,027.33b\n1,085.73ab\n21.23\n0.04\nFCR d0-42\n1.724b\n1.760a\n1.705b\n0.01\n0.001\na,bWithin a row, least square means without a common superscript letter differ (p < 0.05).\nPC, positive control diet; NC, negative control diet; similarly reduced by 0.12% unit of\n\n97a\n1,027.33b\n1,085.73ab\n21.23\n0.04\nFCR d0-42\n1.724b\n1.760a\n1.705b\n0.01\n0.001\na,bWithin a row, least square means without a common superscript letter differ (p < 0.05).PC, positive control diet; NC, negative control diet; similarly reduced by 0.12% unit of\ndigestible P and Ca; PHY, negative control diet supplemented with phytase (Rovabio PhyPlus)\nat 500 FTU/kg diet; SEM, standard error of the mean.\nTABLE 4 Analyzed nutrients of experimental diets (Experiment 2).\nBatch 1\nBatch 2\nItem\nPhase 1\nPhase 2\nPhase 3\nPhase 1\nPhase 2\nPhase 3\nPC\nNC\nPHY\nPC\nNC\nPHY\nPC\nNC\nPHY\nPC\nNC\nPHY\nPC\nNC\nPHY\nPC\nNC\nPHY\nDry matter, %\n88.70\n88.20\n87.50\n88.00\n87.00\n87.40\n87.40\n86.90\n86.90\n87.60\n87.40\n87.10\n87.40\n87.00\n86.90\n88.60\n88.00\n88.20\nCrude protein, %\n14.90\n14.90\n14.80\n12.40\n12.40\n12.50\n10.60\n10.60\n10.60\n15.30\n15.10\n15.50\n12.80\n13.30\n12.50\n10.30\n10.50\n10.50\nCrude fat, %\n3.90\n4.10\n3.70\n3.30\n2.50\n2.60\n2.90\n2.50\n2.50\n3.80\n3.40\n3.10\n3.30\n2.70\n2.90\n3.30\n3.50\n3.30\nCrude fiber, %\n2.60\n2.70\n2.90\n2.20\n2.20\n2.30\n2.00\n2.30\n2.10\n2.50\n3.00\n2.80\n2.80\n2.60\n2.80\n2.30\n2.50\n2.50\nAsh, %\n4.70\n4.20\n4.20\n3.50\n3.00\n3.00\n3.20\n2.70\n2.70\n4.60\n4.20\n4.10\n3.50\n3.30\n3.10\n3.10\n2.90\n2.80\nCalcium, %\n0.68\n0.49\n0.51\n0.51\n0.48\n0.46\n0.47\n0.43\n0.42\n0.66\n0.54\n0.49\n0.54\n0.48\n0.42\n0.54\n0.38\n0.39\nTotal P, %\n0.58\n0.40\n0.39\n0.48\n0.36\n0.37\n0.46\n0.35\n0.36\n0.57\n0.39\n0.41\n0.49\n0.38\n0.38\n0.47\n0.34\n0.35\nPhytate-P, %\n0.23\n0.21\n0.22\n0.21\n0.20\n0.23\n0.18\n0.22\n0.22\n0.22\n0.25\n0.25\n0.22\n0.22\n0.23\n0.22\n0.22\n0.23\nPhytase activity,\nFTU/kg diet\n0\n118\n474\n88\n82\n511\n133\n93\n501\n29\n23\n485\n38\n64\n424\n67\n63\n424\nPC, positive control diet; NC, negative control diet, similarly reduced by 0.14, 0.11, and 0.10% units in digestible P and Ca for grower 1, grower 2, and finisher phase, respectively, compared\nwith PC diet; PHY, negative control diet supplemented with phytase (Rovabio PhyPlus) at 500 FTU/kg diet.\nFrontiers in Veterinary Science\n05\nfrontiersin.org\n\nJlali et al.\n10.3389/fvets.2025.1591214\nsupplemented with phytase at 500 FTU/kg diet had a greater final\nbody weight (+6.5%, p = 0.006) and ADG (+9.0%, p = 0.005) and\na lower F:G ratio (-3.1%, p < 0.001). ADFI tended (p = 0.06) to be\nTABLE 6 Bone traits of weaned piglets fed diets without or with phytase\nfor 42 days (Experiment 1).\nItem\nTreatment\nAdjusted\nSEM\nP-\nvalue\nPC\nNC\nPHY\nATTD of P\n58.01a\n46.19b\n59.67a\n0.89\n<0.001\nBone dry matter, %\n54.31a\n51.99b\n54.20a\n0.36\n<0.001\nBone ash, g\n5.13a\n3.87b\n4.93a\n0.11\n<0.001\nBone ash, %\n18.39a\n14.96c\n17.44b\n0.21\n<0.001\nBone ash, % DM\n33.86a\n28.78c\n31.92b\n0.30\n<0.001\nBone P, g\n0.896a\n0.676b\n0.874a\n0.02\n<0.001\nBone P, %\n3.21a\n2.62b\n3.09a\n0.04\n<0.001\nBone P, % DM\n5.84a\n5.03b\n5.66a\n0.07\n<0.001\na,b,cWithin a row, least square means without a common superscript letter differ (p < 0.05).\nPC, positive control diet; NC, negative control diet; similarly reduced by 0.12% unit of\ndigestible P and Ca; PHY, negative control diet supplemented with phytase (Rovabio PhyPlus)\nat 500 FTU/kg diet; SEM, standard error of the mean; DM, dry matter.\nhigher in piglets that received phytase-supplemented NC diet than\nthose fed with the NC diet. In addition, phytase supplementation on\ntop of the NC diet improved (p < 0.001) bone ash and P contents\nby 27.6% and 29.3%, respectively, in comparison with the NC diet.\nThere was no significant difference between piglets fed with the NC\ndiet supplemented with phytase and those fed with the PC on final\nBW (p = 0.91), ADG (p = 0.83), ADFI (p = 0.55), F:G ratio (p\n= 0.18), bone ash content (p = 0.19), and bone P content (p =\n0.13). Piglets fed with the NC diet had lower (p < 0.001) ATTD\nof P as compared to piglets fed with the PC diet. However, the\nsupplementation of phytase to the NC diet improved (p < 0.001)\nthe ATTD of P by 13.5% points.\n3.2 Experiment 2—Growing pigs\nThe effects of dietary treatments on growth performance,\nbone mineralization, and ATTD of nutrients in growing pigs are\npresented in Tables 7, 8. No significant interaction was observed\nbetween dietary treatment and sex on growth performance\nparameters, bone traits, and ATTD of nutrients. Growing pigs\nfed with the NC diet reduced in digestible P and Ca had lower\n(p < 0.05) final BW (−3.0%) and ADG (−4.3%) and a higher\n\nGrowing pigs\nfed with the NC diet reduced in digestible P and Ca had lower\n(p < 0.05) final BW (−3.0%) and ADG (−4.3%) and a higher\nF:G ratio (+4.7%) in comparison with piglets fed the PC diet. In\nTABLE 7 Growth performance of growing pigs fed diets without or with phytase for 84 days (Experiment 2).\nTreatment\nAdjusted\nP-value\nItem\nPC\nNC\nPHY\nSEM\nInitial\nBW\nTreatment\n(T)\nSex\n(S)\nBatch\n(B)\nT x S\nT x B\nPhase 1\nBW 85d, kg\n33.3\n33.4\n33.3\n0.260\n-\n0.99\n0.53\n0.003\n0.98\n1.00\nBW 114d, kg\n61.4a\n59.2b\n60.5a\n0.496\n<0.001\n<0.001\n<0.001\n<0.001\n0.32\n0.76\nADG 85-114d, kg/pig/d\n0.962a\n0.888b\n0.931a\n0.010\n<0.001\n<0.001\n<0.001\n<0.001\n0.32\n0.76\nADFI 85-114d, kg/pig/d\n2.14\n2.09\n2.14\n0.021\n<0.001\n0.408\n<0.001\n0.05\n0.08\n0.43\nFCR 85-114d\n2.25b\n2.37a\n2.32a\n0.018\n<0.001\n0.002\n0.21\n<0.001\n0.31\n0.34\nPhase 2\nBW 140d, kg\n86.6a\n84.0b\n85.3ab\n0.590\n<0.001\n0.004\n<0.001\n0.18\n0.92\n0.90\nADG 115-140d, kg/piglet/d\n0.983\n0.966\n0.974\n0.007\n<0.001\n0.501\n<0.001\n<0.001\n0.75\n0.85\nADFI 115-140d, kg/piglet/d\n2.74\n2.74\n2.74\n0.022\n<0.001\n0.970\n<0.001\n<0.001\n0.66\n0.41\nFCR d115-140\n2.79\n2.85\n2.82\n0.015\n<0.001\n0.261\n0.02\n0.13\n0.88\n0.63\nPhase 3\nBW d169, kg\n109.2a\n105.9b\n108.3a\n0.631\n<0.001\n0.002\n<0.001\n0.02\n0.83\n0.83\nADG d141-169, kg/piglet/d\n0.784\n0.785\n0.804\n0.007\n<0.001\n0.3696\n<0.001\n<0.001\n0.73\n0.51\nADFI d141-169, kg/piglet/d\n2.79b\n2.86ab\n2.90a\n0.021\n<0.001\n0.035\n<0.001\n<0.001\n0.76\n0.05\nFCR d141-169\n3.60\n3.69\n3.66\n0.025\n<0.001\n0.287\n0.21\n0.13\n0.29\n0.80\nGlobal period\nADG d85-169, kg/pig/d\n0.903a\n0.864b\n0.893a\n0.006\n<0.001\n0.002\n<0.001\n0.02\n0.83\n0.83\nADFI d85-169, kg/pig/d\n2.472\n2.471\n2.498\n0.017\n<0.001\n0.620\n<0.001\n<0.001\n0.87\n0.10\nFCR d85-169\n2.74c\n2.87a\n2.80b\n0.012\n<0.001\n<0.001\n0.09\n<0.001\n0.59\n0.10\na,b,cWithin a row, least square means without a common superscript letter differ (p < 0.05).\nPC, positive control diet; NC, negative control diet; similarly reduced by 0.12% unit of digestible P and Ca; PHY, negative control diet supplemented with phytase (Rovabio PhyPlus) at 500\nFTU/kg diet; SEM, standard error of the mean.\nFrontiers in Veterinary Science\n06\nfrontiersin.org\n\nJlali et al.\n10.3389/fvets.2025.1591214\nTABLE 8 Bone traits and apparent total digestibility of phosphorus, calcium, and nitrogen in growing pigs fed diets without or with phytase for 84 days\n(Experiment 2).\nTreatment\nAdjusted\nP-value\nItem\nPC\nNC\nPHY\nSEM\nTreatment (T)\nSex (S)\nBatch (B)\nT x S\nT x B\nBone traits\nDry matter, %\n62.5\n61.1\n62.2\n0.29\n0.10\n0.82\n0.04\n0.57\n0.50\nBone ash, g\n8.3a\n7.5b\n8.2a\n0.11\n0.00\n0.42\n<0.001\n0.48\n0.94\nBone ash, % DM\n35.2a\n33.7b\n35.2a\n0.31\n0.02\n0.05\n<0.001\n0.42\n0.95\nBone P, g\n1.38\n1.29\n1.38\n0.02\n0.07\n0.33\n0.11\n0.68\n0.73\nBone P, % DM\n5.90\n5.70\n5.90\n0.06\n0.43\n0.45\n0.53\n0.40\n0.63\nATTD\nPhosphorus\n43.63b\n34.42c\n59.06a\n1.76\n<0.001\n0.12\n0.87\n0.65\n0.26\nCalcium\n43.49c\n51.62b\n59.41a\n1.41\n<0.001\n0.10\n0.43\n0.62\n0.13\nNitrogen\n74.26b\n75.84b\n78.37a\n0.57\n<0.001\n<0.001\n<0.001\n0.37\n0.93\na,b,cWithin a row, least square means without a common superscript letter differ (p <0.05).\nPC, positive control diet; NC, negative control diet; similarly reduced by 0.12% unit of digestible P and Ca; PHY, negative control diet supplemented with phytase (Rovabio PhyPlus) at 500\nFTU/kg diet; SEM, standard error of the mean.\naddition, digestible P and Ca reduction decreased (p = 0.005) the\nash content in metacarpal bones by 9.6%. Bone P content tended\nto be lower (−6.5%, p = 0.11) in pigs that received the NC diet in\ncomparison with pigs fed with that received the PC diet. Compared\nto growing pigs fed with the NC diet, pigs fed with the NC diet\nsupplemented with phytase at 500 FTU/kg diet had a greater (p <\n0.05) final body weight (+2.3%) and ADG (+3.4%) and a lower\nF:G ratio (−2.4%). In addition, phytase supplementation on top of\nthe NC diet improved (p < 0.05) bone ash content by 9.3% and\ntended (p = 0.06) to improve P content by 7.0%, in comparison\nwith the NC diet. There was no significant difference between pigs\nfed with the NC diet supplemented with phytase and those fed\nwith the PC diet on final BW, ADG, ADFI, bone ash content,\nand bone P content. However, the F:G ratio remains higher (p <\n0.05) in pigs fed with the phytase-supplemented diet than those\nfed the PC diet. Regarding the digestibility of nutrients, digestible\nP and Ca reduction decreased (p < 0.001) the ATTD of P by\n9.2% points, increased (p = 0.01) ATTD of Ca by 8.3% points,\n\n05) in pigs fed with the phytase-supplemented diet than those\nfed the PC diet.Regarding the digestibility of nutrients, digestible\nP and Ca reduction decreased (p < 0.001) the ATTD of P by\n9.2% points, increased (p = 0.01) ATTD of Ca by 8.3% points,\nand maintained the ATTD of N. However, the supplementation\nof phytase improved (p < 0.05) the ATTD of P (+24.6% points),\nCa (+7.8% points), and N (+2.5% points) in comparison with the\nNC diet.\n4 Discussion\nThe first phytases were commercialized in 1990, and within a\ndecade, phytases were being added routinely to swine diets to break\ndown phytic acid, thereby increasing the availability of P, improving\ngrowth performance, and reducing the excretion of P into the\nenvironment (13). However, the benefits of adding exogenous\nphytases to livestock feeds, especially in pigs, can mainly depend on\ntheir source and dose as well as on the nutritional characteristics\nof diets. Growth performance is often used as an indicator of\nphytase efficacy on P utilization, particularly when animals are\nfed P-deficient diets (9, 14). As expected, the dietary reduction\nin digestible P and Ca reduced growth performance in piglets\nand growing pigs in comparison with animals fed a P- and Ca-\nadequate diet. These results are in agreement with many previous\nstudies (9, 15–17). Similarly, Lee et al. (18) reported that the growth\nperformance of pigs was reduced when dietary Ca and P were below\nthe requirement. These results indicate the essentiality of P and Ca\nto optimize the productivity of piglets and growing pigs. Indeed, P\nis the second most abundant bodily mineral next to calcium (19).\nIt is an essential nutrient required for normal muscle growth, and\nit plays vital roles in maintaining osmotic and acid–base balance,\nenergy metabolism, and protein synthesis (19, 20). It appears also\nfrom these results that reducing P and Ca levels by 0.12% points\nfrom the NRC (2012) recommended values in piglets and growing\npigs has a negative impact on growth performance. Otherwise,\nseveral studies have reported improvements in performance when\n\n12% points\nfrom the NRC (2012) recommended values in piglets and growing\npigs has a negative impact on growth performance.Otherwise,\nseveral studies have reported improvements in performance when\nphytase was used in piglets and growing pigs (21–24).\nIn the current study, the addition of phytase at 500 FTU/kg diet\nto a diet similarly reduced in digestible P and Ca by 0.12% units fed\nto weaned piglets improved the final BW, ADG, and F:G ratio by\n6.5%, 9.0%, and 3.1%, respectively. These results indicate the ability\nof phytase to release P and Ca and probably the other nutrients,\nwhich therefore improves the growth performance of nursery and\ngrowing pigs. In the current study, the improvements in growth\nperformance parameters observed with phytase supplementation\nwere lower in growing pigs compared to those in weaned piglets.\nIndeed, final BW, ADG, and FCR were improved by 2.3%, 3.4%,\nand 2.4%, respectively, in growing pigs fed a phytase-supplemented\ndiet in comparison with those fed an NC diet. These differences\nin terms of improvements may be because weaned piglets are\nmore sensitive to P and Ca reduction compared to growing\npigs, and they need these minerals for skeletal development and\nmineralization. As previously reported by Cambra-Lopez et al.\n(25) and recently by Babatunde and Adeola (9), the age of pigs\ncan influence the efficacy of phytase on growth performance. It\nis important to notice in the current study that the magnitude\nof the efficacy of phytase on growth performance seems to be\nreduced during the growing period. Several other factors, such\nas diet composition, dietary phytate-P level, and source and dose\nof phytase can also influence the magnitude of phytase response\nFrontiers in Veterinary Science\n07\nfrontiersin.org\n\nJlali et al.\n10.3389/fvets.2025.1591214\nin nursery and growing pigs (21, 22, 26). It is also important\nto highlight that in the experiment performed on growing pigs,\nphytase supplementation at 500 FTU/kg diet was able to partially\nrestore the F:G ratio compared to pigs fed the nutrient adequate-\nPC diet. This observation could be explained by the negative effect\nof a high reduction in digestible P and Ca, especially during the\nfirst grower phase (similarly by 0.14% points) on the F:G ratio,\nwhich was translated during the whole experimental period. The\nbone ash and P content results in piglets and growing pigs are\nconsistent with the growth performance results. Many studies have\ndemonstrated that dietary Ca and P play an indispensable role\nin the bone development and therefore on bone ash content of\npiglets and pigs (20, 27, 28). As observed on growth performance,\nthere was a negative effect of dietary reduction in P and Ca on\nash content in metacarpal bones of piglets and growing pigs.\nThis could be an indicator that reducing digestible P and Ca\nsimilarly by 0.12% points from NRC (2012) recommended level\nin weaned piglets and growing pigs was sufficient to induce\nnegative effects not only on growth performance but also on bone\nmineralization. In agreement with previous studies (23, 29, 30),\nphytase supplementation at 500 FTU/kg diet in P- and Ca-deficient\ndiets improved the metacarpal bones ash and P contents in weaned\npiglets and growing pigs. The levels of metacarpal bones ash and\nP contents in animals fed with the phytase-supplemented diets\nbecame equivalent to those fed with the PC-nutrient adequate\ndiets. This observation indicates the effectiveness of this new\nbiosynthetic 6-phytase to improve the utilization of P and Ca in\npigs fed the P- and Ca-deficient diets. Indeed, growing pigs fed\nwith the low P and Ca diet had higher ATTD of Ca than those\noffered adequate P and Ca diet. A higher Ca utilization at a lower\ndietary Ca concentration has already been shown by Varley et al.\n(31) and Dersjant-Li and Dusel (32) in growing pigs fed diets\n\nA higher Ca utilization at a lower\ndietary Ca concentration has already been shown by Varley et al.(31) and Dersjant-Li and Dusel (32) in growing pigs fed diets\nreduced in Ca and digestible P. It seems that the absorption and\nuse efficiency of dietary Ca increases when less Ca is provided.\nIn the present study, the result that 6-phytase supplementation\nincreased the ATTD of P in Experiment 1 and ATTD of P and\nCa in Experiment 2 is expected and it agrees with the results from\nprevious experiments (9, 33–36). These improvements of P and\nCa digestibility could be explained by the mechanism of action\nof phytase on phytate which is probably efficient to release or\nremove minerals, especially Ca, from the phytate bond as well as\nlimiting the interactions between nutrients and phytate. However,\nBournazel et al. (37) reported that addition of a microbial phytase\nat 500 FTU/kg diet did not influence the ATTD of Ca but increased\nthe retainable Ca in growing pigs. These different outcomes may\nbe due to phytase type and dose, Ca level and source, as well as\nthe Ca:P ratio which all these factors may influence the response\nof phytase on ATTD of Ca. In addition to P and Ca, phytase\naddition also improved the ATTD of N. These results confirm\nthat phytate may chelate not only dietary Ca but also protein\nand basic amino acids such as lysine, arginine, and histidine, but\nwhen phytate is hydrolyzed by exogenous phytase, the chelated\nprotein and amino acids will be released and absorbed, which\nwill increase the utilization of proteins next to minerals (38). As\nreported by several studies (21, 23, 39), the phytases from the new\ngeneration (from bacterial origin) are highly efficient at low pH\nespecially at the upper part of gastrointestinal tract (GIT) which\nmay lead to limit the nutrient–phytate complexes within the GIT\nand improving thereby the bioavailability of nutrients including\nminerals and amino acids for monogastric animals. However, some\nlimitations of these two studies including the efficacy of this novel\nphytase depending on its dose and nutritional conditions (different\n\nHowever, some\nlimitations of these two studies including the efficacy of this novel\nphytase depending on its dose and nutritional conditions (different\nenergy and nutrient density) can be highlighted. Therefore, further\nstudies are needed to elucidate the mode of action of this new\nbiosynthetic 6-phytase according to dietary phytate level and its\ndose on the bioavailability of nutrients beyond P, Ca, and N in\nmonogastric animals.\n5 Conclusion\nIn conclusion, this study confirmed that reducing digestible\nP and Ca similarly by 0.12% points in weaned piglets and\nby 0.14, 0.11, and 0.10% points, in phases 1, 2, and 3,\nrespectively, in growing pigs, from the NRC (2012) recommended\nlevel, was sufficient to induce negative effects on growth\nperformance, bone mineralization, and P digestibility in both\nweaned piglets and growing pigs. The addition of the novel\nbiosynthetic 6-phytase to P- and Ca-deficient diets fed to\nweaned piglets and growing pigs has the potential to improve\nnutrient digestibility and, therefore, enhance performance and\nbone mineralization.\nData availability statement\nThe raw data supporting the conclusions of this article will be\nmade available by the authors, without undue reservation.\nEthics statement\nThe animal study was approved by French government\nto\nconduct\nanimal\nexperiments.\nThe\nstudy\nwas\nconducted\nin\naccordance\nwith\nthe\nlocal\nlegislation\nand\ninstitutional requirements.\nAuthor contributions\nMJ:\nConceptualization,\nData\ncuration,\nFormal\nanalysis,\nInvestigation, Methodology, Software, Validation, Visualization,\nWriting – original draft, Writing – review & editing. CH:\nConceptualization, Investigation, Validation, Writing – review &\nediting. CM: Investigation, Validation, Writing – review & editing.\nSO: Investigation, Project administration, Validation, Writing –\nreview & editing.\nFunding\nThe author(s) declare that no financial support was received for\nthe research and/or publication of this article.\nFrontiers in Veterinary Science\n08\nfrontiersin.org\n\nJlali et al.\n10.3389/fvets.2025.1591214\nAcknowledgments\nThe authors thank the staffof the breeding facilities of\nthe Center of Expertise in Research and Nutrition (Malicorne,\nFrance) and of Zootests (Ploufragan, France) for valuable\ntechnical assistance.\nConflict of interest\nMJ, CH and SO were employed by Adisseo France S.A.S. CM\nwas employed by Zootest.\nGenerative AI statement\nThe author(s) declare that no Gen AI was used in the creation\nof this manuscript.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. Any product that may be evaluated in this article, or\nclaim that may be made by its manufacturer, is not guaranteed or\nendorsed by the publisher.\nReferences\n1. Kemme PA, Jongbloed AW, Mroz Z, Kogut J, Beynen AC. Digestibility of nutrients\nin growing-finishing pigs is affected by Aspergillus niger phytase, phytate and lactic\nacid levels: 1. Apparent ileal digestibility of amino acids. Livestock Prod Sci. (1999)\n58:107–17. doi: 10.1016/S0301-6226(98)00203-6\n2. Tahir M, Shim MY, Ward NE, Smith C, Foster E, Guney AC, et al. Phytate\nand other nutrient components of feed ingredients for poultry. Poult Sci. 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+{"text": "【兽医文献】\n标题: Interventions for reducing antimicrobial resistance in livestock in sub Saharan Africa: systematic review\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nCase Report: Congenital gingival\nangiomatosis in a newborn lamb\nD. Zapico 1,2*, V. Pérez 1,2, M. Criado 1,2, P. Mendívil 2, M. Silva 1,2,\nJ. Benavides 2, M. C. Ferreras 1,2 and J. Espinosa 1,2\n1 Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de León, León, Spain,\n2 Instituto de Ganadería de Montaña, CSIC-ULE, León, Spain\nProliferative vascular disorders are rare congenital tumor-like lesions that arise\nfrom anomalies in blood vessel development. A newborn Assaf lamb presented\nwith a diffuse, multinodular, dark-red overgrowth of the periodontal gingiva,\naccompanied by refusal to suckle. The animal developed diarrhea and died 2 days\nafter birth. Necropsy was performed, and histopathological examination of the oral\nlesions revealed a marked expansion of the gingival stroma by multiple, closely\npacked, small- to medium-sized vascular channels. These were lined by a single\nlayer of von Willebrand factor- and vimentin-positive endothelial cells and were\npartially surrounded by alpha smooth muscle actin (α-SMA) - and vimentin-positive\nspindle cells, consistent with well-differentiated capillaries and post-capillary\nvenules. Taken together, these findings were consistent with diffuse vascular\ngingival hamartomatosis/angiomatosis. The congenital nature and distribution\nof the lesions are supportive of a malformation of the periodontal vasculature.\nKEYWORDS\nlamb, gingiva, vascular malformation, hamartoma, angiomatosis\n1 Introduction\nProliferative gingival lesions in lambs and adult sheep are most commonly associated with\norf virus infection (contagious ecthyma) (1, 2). Other causes of gingival swelling include\nperiodontitis (3), trauma, dentigerous cysts (4, 5), and, less frequently, neoplasia. The most\ncommon oral tumors in sheep, although rare by themselves, are squamous cell carcinoma and,\nto a lesser extent, fibrosarcoma (6–9), whereas other neoplasms such as ossifying fibroma (10),\nodontoameloblastoma (11), or hemangioma (12) have been sporadically reported.\n\nNon-neoplastic proliferative vascular lesions (e.g., vascular hamartomas, arteriovenous\nmalformations, and angiomatosis) are infrequent in domestic animals (13, 14). Microscopic\nsimilarity and overlap in terminology often make the histopathological classification of these\ndisorders challenging. In practice, the term “vascular hamartoma” has been most widely used\n(15). Gingival vascular hamartomas have been reported mainly in calves and, less frequently,\nin cats. However, their presentation is usually focal, small in size, without extension to the\nteeth, and without interference with suckling or feeding (16–24). A case of vascular hamartoma\nhas also been described in the skin of a Polwarth lamb (25). In the present study, we report a\nneonatal Assaf lamb with diffuse vascular proliferation extensively involving the gingival\nregion. To the authors’ knowledge, this is the first report of a vascular malformation of this\nseverity and distribution in the gingiva of a lamb.\n2 Case description\nA male newborn Assaf lamb from an intensive dairy farm of this breed showed apathy,\nanorexia, and refusal to suckle immediately after birth. Within the first hours of life, the animal\nOPEN ACCESS\nEDITED BY\nIrina Amorim,\nUniversity of Porto, Portugal\nREVIEWED BY\nGabriela Fernandes Silva,\nUniversity of Porto, Portugal\nHossein Hamali,\nUniversity of Tabriz, Iran\n*CORRESPONDENCE\nD. Zapico\ndzaps@unileon.es\nRECEIVED 09 September 2025\nREVISED 28 October 2025\nACCEPTED 04 November 2025\nPUBLISHED 18 November 2025\nCITATION\nZapico D, Pérez V, Criado M, Mendívil P,\nSilva M, Benavides J, Ferreras MC and\nEspinosa J (2025) Case Report: Congenital\ngingival angiomatosis in a newborn lamb.\nFront. Vet. Sci. 12:1702299.\ndoi: 10.3389/fvets.2025.1702299\nCOPYRIGHT\n© 2025 Zapico, Pérez, Criado, Mendívil, Silva,\nBenavides, Ferreras and Espinosa. This is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY). The use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\n\nThe use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\naccordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Case Report\nPUBLISHED  18 November 2025\nDOI  10.3389/fvets.2025.1702299\n\nZapico et al.\b\n10.3389/fvets.2025.1702299\nFrontiers in Veterinary Science\n02\nfrontiersin.org\ndeveloped progressive weakness and diarrhea and ultimately died at\n2 days of age. The carcass was submitted to the Diagnostic Pathology\nService of the University of León for post-mortem examination.\n3 Diagnostic assessment\nAt necropsy, the animal presented with severe, diffuse, exophytic\novergrowth, and dark-red discoloration of the periodontal gingiva,\nwhich partially or completely covered the incisors, premolars, and\nmolars in both the maxilla and the mandible (Figures 1A,B). These\nchanges were absent from the dental pad (Figure 1A) and the diastema\n(Figure 1B). Another lesion observed during post-mortem examination\nwas segmental distension of the small intestine, containing mild to\nmoderate amounts of gas and liquid content, consistent with catarrhal\nenteritis. This condition was most likely secondary to insufficient\ncolostrum intake caused by impaired or painful suckling. Therefore,\nit can be concluded that catarrhal enteritis represented the final cause\nof death in this lamb.\nTissue samples of the entire mandible and maxilla (including\nteeth, gingiva, and alveolar bone), as well as from the other organs of\nthe animal, were fixed in 10% neutral phosphate-buffered formalin.\nSubsequently, the mandibular and maxillary samples showing\nproliferative lesions were treated with a demineralization solution\ncomposed of sodium citrate and formic acid (50/50%, v/v) for 7 days.\nAfterward, samples were carved, dehydrated through a graded alcohol\nseries and xylene treatment, and embedded in paraffin wax. Sections\n3-μm thick were obtained and stained with Harris’s hematoxylin and\neosin for histopathological examination.\nImmunohistochemical analysis of von Willebrand factor\n(A008229-2, Dako-Agilent Technologies, Santa Clara, CA, USA,\n1:200), alpha smooth muscle actin (α-SMA) (M0851, Dako-Agilent\nTechnologies, Santa Clara, CA, USA, 1:100 dilution), and vimentin\n(M0725, Dako-Agilent Technologies, Santa Clara, CA, USA, 1:1,000\ndilution) was further performed. The immunohistochemical\n\nThe immunohistochemical\nprocedure was carried out as described elsewhere (26). Briefly, heat-\nmediated antigen retrieval was performed with PT Link® system\n(Dako-Agilent Technologies, Santa Clara, CA, USA), using pH 6.0\ntarget retrieval solution for vimentin and pH 9.0 solution for\nα-SMA, for 20 min at 95 °C. Sections were then submerged for\n30 min in 3% H2O2 in methanol solution at room temperature to\nblock endogenous tissue peroxidase. For the von Willebrand factor,\nantigen retrieval was performed for 15 min at room temperature\nusing proteinase K (Dako-Agilent Technologies, Santa Clara, CA,\nUSA) after deparaffination, hydration, and peroxidase blockage.\nTissue slides were then incubated overnight with the primary\nantibody in a humidified chamber at 4 °C. Afterward, sections were\nexposed to appropriate anti-mouse or anti-rabbit secondary\nantibody for 40 min at room temperature using the EnVision\nSystem® (Dako-Agilent Technologies, Santa Clara, CA, USA).\nImmunostaining was developed using 3,3-diaminobenzidine (Dako-\nAgilent Technologies, Santa Clara, CA, USA) as a chromogenic\nsubstrate, and slides were counterstained with hematoxylin.\nAppropriate species- and isotype-matched immunoglobulins were\nused as a control. These included sections with an isotype control\nfor the primary antibody and the omission of the primary antibody.\nAs positive controls, sections from the jejunum of clinically healthy\nsheep were used.\nFIGURE 1\nMaxilla and mandible. Exophytic, multinodular enlargement with dark-red discoloration of the gingiva, partially to completely covering the teeth (A,B),\nwhile sparing the dental pad (A) and the diastema (B).\n\nZapico et al.\b\n10.3389/fvets.2025.1702299\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nHistopathological examination revealed a marked exophytic\nexpansion of the free gingiva resulting from the proliferation of\nnumerous disorganized, densely packed, small- to medium-sized\nvascular channels filled with blood in the stroma, partially or\ncompletely covering the teeth. Proliferating vascular channels were\nembedded in mild to moderate fibrous stroma and had irregular\nlumina lined by a single layer of flattened endothelial cells. These\nvascular structures extended from the alveolar crest toward the base\nof the overlying epithelium. The epithelium was markedly ulcerated\nand hyperplastic, with prominent anastomosing rete ridges\n(Figures 2A,B). Multifocal areas of epithelial swelling and hydropic\nchange (ballooning degeneration) were also observed (Figure 2A,\ninset). Additional findings included an atypical orientation of\nperiodontal ligament fibers and mesenchymal cells parallel to the long\naxis of the tooth (Figure 2C), as well as extensive areas of alveolar bone\nremodeling, characterized by increased osteoclastic activity and\nosteolysis (bone resorption) concurrent with immature bone\nformation by variable numbers of osteoblasts (Figure 2D). These\nchanges were interpreted as physiologic processes associated with\nnormal tooth eruption rather than true pathologic lesions.\nImmunohistochemical analysis revealed that the endothelium of\nthe proliferative vascular channels (Figure 3A) was strongly positive\nfor von Willebrand factor (Figure 3B) and vimentin in the cytoplasm,\nwith variable immunoreactivity for α-SMA (Figure 3C). Larger-caliber\nvascular structures were partially surrounded by elongated spindle-\nshaped cells exhibiting cytoplasmic α-SMA (Figure 3C) and vimentin\n(Figure 3D) immunolabeling, consistent with perivascular smooth\nmuscle cells and pericytes. Overall, these vascular channels were\nconsidered compatible with structurally normal capillaries and post-\ncapillary venules.\n4 Discussion\nCongenital hemangiomas have been described in several animal\n\nOverall, these vascular channels were\nconsidered compatible with structurally normal capillaries and post-\ncapillary venules.4 Discussion\nCongenital hemangiomas have been described in several animal\nspecies, including sheep (13, 14, 27). These benign vascular tumors are\ncomposed of variably sized, blood-filled vascular spaces lined by a\nsingle layer of flat endothelial cells, but they lack the smooth muscle\nand pericytic support characteristic of normal vessels (13, 14). In the\npresent case, the vascular channels were partially supported by\nα-SMA- and vimentin-positive perivascular cell elements, compatible\nwith smooth muscle cells or pericytes, suggesting a non-neoplastic\nprocess. However, some reports indicate that hemangiomas may\noccasionally display pericytic components, and therefore their absence\ncannot be  considered a definitive diagnostic criterion (15).\nFurthermore,\nhemangiomas\nare\ntypically\nsolitary\nand\nFIGURE 2\nTissue sections of the teeth, periodontium, gingiva, and alveolar bone. Hematoxylin and eosin. (A) Exophytic expansion of the free gingiva due to\nextensive vascular proliferation within the stroma, completely covering the tooth. The epithelium is markedly ulcerated and hyperplastic, with\nprominent rete ridges. Magnification: 40×. Inset: epithelial swelling and hydropic (ballooning) degeneration (top right). Magnification: 200×.\n(B) Decidual tooth with normal dental pulp, dentin, and an artefactual space resulting from enamel loss during tissue processing. The surrounding\ngingiva shows changes similar to those described in (A). Magnification: 40×. (C) Periodontal ligament fibers and fibroblasts are oriented parallel to the\nlong axis of the tooth. A focal hemorrhage is also present. Magnification: 200×. (D) Woven bone fragments with adjacent osteoclasts within Howship’s\nlacunae (center), consistent with osteolysis and alveolar bone resorption, accompanied by moderate numbers of osteoblasts lining thin, irregular,\nnewly formed bony trabeculae (bottom). Magnification: 200×.\n\nZapico et al.\b\n10.3389/fvets.2025.1702299\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nwell-circumscribed masses, which does not match the multifocal and\nperiodontal distribution of the gingival lesions observed here.\nMalignant vascular tumors, such as hemangiosarcoma, may occur as\nmultiple lesions, but they usually exhibit invasive growth and are\ncomposed of irregular vascular channels lined by one or more layers\nof spindle cells with varying degrees of atypia and mitotic activity (13,\n14). These histological features were not present in the current case.\nThe term hamartoma refers to a non-neoplastic overgrowth of\nmature tissue elements native to the site of origin (13). Vascular\nhamartomas appear as single, well-demarcated, expansile masses\ncomposed of different proportions of capillaries, arterioles, and\nvenules (13, 14, 28). They are congenital in nature and result from\ndevelopmental anomalies in blood vessels, leading to an architectural\ndisarray of the local vasculature (15). In this case, the disorganized\nproliferation of vascular channels lined by flat endothelial cells and\nvariably supported by perivascular cell elements, compatible with\nmature capillaries and post-capillary venules, was consistent with the\nmicroscopic features of a vascular hamartoma. However, the\nwidespread, multifocal distribution of the gingival lesions does not\nentirely conform to the classic definition of this condition. On the\nother hand, the concept of angiomatosis encompasses a heterogeneous\ngroup of non-neoplastic vascular proliferations, including specific\nsyndromes such as bovine cutaneous angiomatosis and progressive\nangiomatosis of dogs and cats (13, 14, 29). More broadly, the term can\nbe applied to describe multiple vascular hamartomas within the same\ntissue or anatomical region (30). Accordingly, the multifocal\ndistribution of the gingival lesions in this lamb provides a rational\nbasis for the diagnosis of gingival angiomatosis.\nThe exact etiology or pathogenesis of this vascular proliferation\nremains uncertain. Nevertheless, the topographic distribution of the lesions\n\nThe exact etiology or pathogenesis of this vascular proliferation\nremains uncertain.Nevertheless, the topographic distribution of the lesions\nstrongly suggests an origin related to periodontal structures. Although the\nvascular anatomy of the ovine periodontium has not been well\ncharacterized, studies in other species indicate that the free gingiva is\nsupplied by supraperiosteal arterioles, periodontal ligament arterioles, and\nvessels emerging from the alveolar crest. These anastomose with\ndescending venules beneath the sulcular epithelium form the dento-\ngingival or crevicular plexus (31–34). A congenital vascular malformation\nof this plexus could explain both the distribution of the gingival lesions and\nthe absence of proliferations in edentulous areas such as the dental pad.\nFinally, contagious ecthyma should also be considered among the\ndifferential diagnoses. Atypical proliferative lesions caused by orf virus\nhave been reported in the gingiva of sheep and lambs. These are\ncharacterized by marked papillomatous epithelial hyperplasia supported\nby a highly vascularized stroma, changes that have been associated with a\nviral VEGF-like protein (35). Other common microscopic features\ninclude epithelial cell swelling, vacuolization, ballooning degeneration,\nand eosinophilic, intracytoplasmic inclusion bodies (36). While the farm\nwhere this case occurred had a history of contagious ecthyma, molecular\nconfirmation of the virus was not possible. Nevertheless, the congenital\nnature of the vascular proliferations described here, together with their\nFIGURE 3\nHigh magnification microphotographs of the proliferating vascular structures. (A) Hematoxylin and eosin. Closely packed vascular channels with\nirregular lumen, lined by a single layer of flat endothelial cells. (B) Immunohistochemistry (IHC) for the von Willebrand factor. Endothelial cells show\nstrong immunoreactivity for von Willebrand factor. (C) IHC for α-SMA. Vascular endothelium displays variable α-SMA labeling, while larger vessels are\n\n(B) Immunohistochemistry (IHC) for the von Willebrand factor.Endothelial cells show\nstrong immunoreactivity for von Willebrand factor.(C) IHC for α-SMA.Vascular endothelium displays variable α-SMA labeling, while larger vessels are\npartially surrounded by α-SMA-positive spindle-shaped cells. (D) IHC for vimentin. Both perivascular walls and endothelium are positive for vimentin.\nMagnification: 200×.\n\nZapico et al.\b\n10.3389/fvets.2025.1702299\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nextensive distribution in a 48-h-old lamb, strongly argues against a viral\netiology, as lesions of this magnitude could not have developed within\nsuch a short postnatal period. Additionally, no poxviral inclusion bodies\nwere identified in the sections examined.\nIn conclusion, diffuse gingival angiomatosis in neonatal lambs\nappears to be an exceptionally rare condition, with no comparable\nreports currently available in the veterinary literature. The congenital\nnature and multifocal distribution of the lesions, together with their\nlocation restricted to periodontal structures, highlights a unique\nvascular malformation process rather than true neoplastic proliferation.\nThis case expands the spectrum of vascular anomalies described in\nruminants and underscores the need for further studies on the vascular\nanatomy and developmental disorders of the ovine periodontium.\nData availability statement\nThe original contributions presented in the study are included in\nthe article/supplementary material, further inquiries can be directed\nto the corresponding author.\nEthics statement\nEthical approval was not required for the studies involving animals in\naccordance with the local legislation and institutional requirements because\nthe case derived from a death animal submitted for necropsy. Written\ninformed consent was obtained from the owners for the participation of\ntheir animals in this study. Written informed consent was obtained from\nthe participant/patient(s) for the publication of this case report.\nAuthor contributions\nDZ: Investigation, Methodology, Writing – original draft. VP:\nInvestigation, Writing – original draft. MC: Investigation, Writing –\noriginal draft. PM: Investigation, Writing  – original draft. MS:\nInvestigation, Writing – original draft. JB: Investigation, Resources,\nWriting – original draft. MCF: Investigation, Writing – original draft.\nJE: Investigation, Methodology, Supervision, Writing – original draft,\nWriting – review & editing.\nFunding\nThe author(s) declare that no financial support was received for\n\nMCF: Investigation, Writing – original draft.JE: Investigation, Methodology, Supervision, Writing – original draft,\nWriting – review & editing.Funding\nThe author(s) declare that no financial support was received for\nthe research and/or publication of this article.\nAcknowledgments\nThe authors wish to acknowledge the collaboration of personnel\nfrom the Animal Health Department at the University of León and\nthank them for their assistance with the laboratory analyses,\nparticularly Eva Martín de Valmaseda. The authors are also grateful to\nSERSA, Veterinary Services, especially Ana Casas and Pío Méndez, for\nreferring the lamb to the Pathological Diagnostic Service of the\nFaculty of Veterinary Sciences of León.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. Any product\nthat may be evaluated in this article, or claim that may be made by its\nmanufacturer, is not guaranteed or endorsed by the publisher.\nReferences\n1.\tFitzgerald WG, Cassidy JP, Markey BK, Doherty ML. Profiling oral and digital\nlesions in sheep in Ireland. Ir Vet J. (2015) 68:30. doi: 10.1186/S13620-015-0055-0\n2.\tMcElroy MC, Bassett HF. The development of oral lesions in lambs naturally\ninfected with orf virus. Vet J. 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+{"text": "【兽医文献】\n标题: Longitudinal study of mammary microbiota dynamics and mastitis prevention in Holstein cows by dry-off strategy\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nTYPE  Case Report\nPUBLISHED  25 June 2026\nDOI  10.3389/fvets.2026.1751730\nOPEN ACCESS\nEDITED BY\nBruno André Lopes,\nSouthfields Veterinary Specialists,\nUnited Kingdom\nREVIEWED BY\nJerzy Pawel Gawor,\nUniversity of Veterinary Medicine\nHannover, Germany\nFaye Rawson,\nLangford Vets Small Animal Referral\nHospital, United Kingdom\n*CORRESPONDENCE\nJennifer A. Woolf\njennifer@wvfcinc.com\nRECEIVED 21 November 2025\nREVISED 03 May 2026\nACCEPTED 05 June 2026\nPUBLISHED 25 June 2026\nCITATION\nWoolf JA (2026) Case report: Injury\nplausibility for skull fractures in two\nkittens.\nFront. Vet. Sci. 13:1751730.\ndoi: 10.3389/fvets.2026.1751730\nCOPYRIGHT\n© 2026 Woolf. This is an open-access\narticle distributed under the terms of the\nCreative Commons Attribution License\n(CC BY). The use, distribution or\nreproduction in other forums is\npermitted, provided the original\nauthor(s) and the copyright owner(s) are\ncredited and that the original publication\nin this journal is cited, in accordance\nwith accepted academic practice. No\nuse, distribution or reproduction is\npermitted which does not comply with\nthese terms.\nCase report: Injury plausibility for\nskull fractures in two kittens\nJennifer A. Woolf *\nWoolf Veterinary Forensics Consulting, Inc., Dublin, CA, United States\nA forensic postmortem examination (FPME) was performed on two kittens. In\neach case, investigators were given conflicting stories describing how the kitten\ndied. The goal for each FPME was to determine the cause of death with the hope\nof also determining which story was more plausible. In both cases, animal control\nofficers responded to a call to retrieve a deceased kitten. In the first case, the kit­\nten’s owner reported the kitten had died when thrown to the floor by the owner’s\nboyfriend; the boyfriend reported he had accidentally dropped the kitten. In the\nsecond case, an ex-girlfriend of the owner reported the owner had crushed the\nkitten’s head against a wall; the owner stated the kitten had come between a wall\nand a piece of furniture being moved, causing its accidental death. Findings from\nthe radiographs, FPME, and histopathology showed the cause of death in the first\n\nFindings from\nthe radiographs, FPME, and histopathology showed the cause of death in the first\ncase to be blunt force trauma resulting in a non-pathological skull fracture and\ntraumatic brain injury (TBI). The distribution and type of injuries were consistent\nwith being thrown against the floor and were not consistent with an accidental\nfall. Findings from the radiographs and FPME of the second case also showed\nblunt force trauma to the head leading to TBI and death, but could not deter­\nmine which story was more plausible due to a limited investigation resulting in\na lack of information necessary to make the distinction. Together, the two cases\ndemonstrate the importance of a FPME in cases of suspicious death while also\ndemonstrating the limitations of FPME alone in determining whether a death is\naccidental or malicious. They also demonstrate the limitations of current litera­\nture on animal abuse and the need for more studies and case reports specific to\nanimal maltreatment.\nKEYWORDS\nanimal cruelty, blunt head injuries, skull fractures, veterinary forensic postmortem\nexamination, veterinary forensics\n1 Introduction\nTwo cases of kittens with fatal skull fractures due to potential animal cruelty are presented.\nIn the first, the owner’s boyfriend claimed that the kitten died accidentally when it was dropped,\nbut the owner said the boyfriend intentionally threw it to the floor. In the second case, the\nowner’s ex-girlfriend reported that the owner crushed the kitten’s head against a wall, but he\nsaid the kitten accidentally became caught between a piece of furniture that was being moved\nand the wall.\nOne of the challenges of investigating animal cruelty is determining the true cause of injury\nwhen there are conflicting stories between the alleged perpetrator and witness(es), such as the\ntwo cases here. Often, one person’s story indicates an accidental injury or death while another\nperson’s story indicates animal maltreatment. Those investigating the case must then use avail­\nable evidence —including, but not limited to, the forensic postmortem examination (FPME)\n\nThose investigating the case must then use avail­\nable evidence —including, but not limited to, the forensic postmortem examination (FPME)\n— to determine which story is the most plausible to explain the injury or death. By reasoning\n\nWoolf\b\n10.3389/fvets.2026.1751730\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nthrough what is known in the literature about injuries to animals,\nknowledge of normal animal behavior, and knowledge of the biome­\nchanics of injury, veterinarians may be able to determine whether a\ngiven story is consistent with the animal’s injuries.\nThere is more extensive information in the literature on child abuse\nthan on animal abuse. In a study of child abuse by Pierce et al. (1) it was\nnoted that different characteristics in cases of femoral fractures from\nstair falls in children could help determine if a case was more likely due\nto abuse or accident. This was termed injury plausibility. These charac­\nteristics included histories that were vague on details, fractures that did\nnot have biomechanical conditions agreeing with the history provided,\ndelays in seeking care despite the child being in obvious pain, and addi­\ntional injuries found during the initial examination.\nSimilar to what is seen in child abuse (1, 2), certain false histories\nsuch as an accidental fall are not uncommon in animal cruelty. A story\nthat is inconsistent with the injuries seen is one potential red flag for\nabuse, whether in animals or children. While the FPME is important\nto any given case, there are often multiple possible explanations for the\nfindings. Stories that are vague or incomplete may require further\ninvestigation from professionals in other fields such as animal control\nand law enforcement before a determination of intentional abuse or\naccident can be made.\nIn child abuse studies looking at falls of less than 3 feet in infants,\nsevere head injury is uncommon (3), despite the increased mass of an\ninfant’s head in relation to the body. No studies were found by the\nauthor that examined the rate and type of injury in cats that fall short\ndistances. Most of what is known about fall-related injuries in cats are\nfrom studies of falls from significant heights as seen in high-rise syn­\ndrome (HRS) where cats fall a distance of at least 2 stories (approxi­\nmately 7.3 m). In HRS, injuries to the head specifically tend to involve\n\n3 m).In HRS, injuries to the head specifically tend to involve\norofacial injuries, and both traumatic brain injury (TBI) and death are\nuncommon (4–6). These studies in children and cats are important\nbecause they suggest that accidental falls in the home such as the alleged\ndrop in the first case are unlikely to result in serious harm or fatal injury.\nBoth kittens in the cases presented had skull fractures. A study by\nKnight and Meeson (7) looked at skull fractures in cats that had trau­\nmatic head injuries. The trauma was classified as either falling from an\nunspecified height, road traffic accident (RTA), bite wound, or gun­\nshot wound. Of the 75 cats in the study, 89% were injured by RTA and\nonly 3% of the skull fractures were caused by falls. Approximately 70%\nof all of the cats had fractures of the upper and lower jaws and cranio­\nfacial areas. Only 7 (9%) of the cats had caudal skull fractures, defined\nas those involving the calvarium, parietal, occipital, presphenoid and\nbasisphenoid bones. Of those 7 cats, none of the fractures were associ­\nated with falls. Knowing the typical pattern of skull fractures due to\ntrauma can help discern inconsistencies in stories provided to explain\nan animal’s injuries such as those of the two kittens.\nIn all of these studies examining traumatic injuries in cats (4–7),\nthe indicated cause of injury is something other than abuse; it is\nunknown whether the history provided for the cause of injury or\ndeath was plausible because it is unknown if the possibility of animal\ncruelty was ever considered. For instance, in the Knight and Meeson\nstudy (7), RTA included cats witnessed as having been hit by a car and\nthose that were found by a road. It is possible some of those cats were\nmis-classified as RTA when they were actually due to another type of\ntrauma, including the possibility of abuse. Nevertheless, these studies\nprovide important information for comparison of the injuries sus­\ntained by the two kittens in this case report to what is known about\nfalls and traumatic skull fractures in cats.\nAnother inconsistency sometimes seen in cases of animal mal­\n\nAnother inconsistency sometimes seen in cases of animal mal­\ntreatment is a description of the animal’s behavior that does not\nseem likely given what is known of normal behavior for the species.\nFor instance, cats naturally have an instinct to right themselves\nwhen falling so they will land on their feet; this is called the right­\ning reflex. In a study by Sechzer et al. (8), kittens aged 0–70 days\nwere tested three times per week for their postural reflexes until\nthe reflex was fully developed. To test for the righting reflex, kittens\nwere held in a supine position then dropped a distance of about\none foot (approximately 61 cm). With this test, the researchers\nfound the righting reflex begins development at 3 weeks of age and\nis fully developed by 6 weeks of age. They also demonstrated that\na cat can right itself quickly, needing as little as a foot of distance\nto do so. This information about falls, skull fractures, and the right­\ning reflex can be used to determine the plausibility of the injuries\nto the two kittens.\n2 Case description\nIn case number one, animal control services were requested to\ndispose of a deceased kitten. The animal control officer (ACO) was\ntold by the owner that her boyfriend (height 1.9 m) had thrown the\nkitten against the hardwood floor from a standing position. Following\nimpact the kitten died less than a minute later. When the ACO inter­\nviewed the boyfriend, he said that he had dropped it accidentally. A\nFPME was performed to determine the cause of death and the plausi­\nbility of each story.\nPrior to the FPME, full body, postmortem radiographs were\nobtained [right lateral (Figure 1A) and ventrodorsal (Figure 1B)]. The\nimages showed soft tissue swelling dorsal to the cranium without for­\neign material or gas. No fractures were identified. There was also con­\ncern for generalized decreased bone density.\nOn FPME, the 6-week-old, intact female, domestic shorthair\nkitten was thin, had fleas, and a small amount of formed feces attached\nto the perianal fur. The dorsal aspect of the head was soft on palpation.\n\nOn FPME, the 6-week-old, intact female, domestic shorthair\nkitten was thin, had fleas, and a small amount of formed feces attached\nto the perianal fur.The dorsal aspect of the head was soft on palpation.\nDiffuse scleral hemorrhage was noted in the dorsolateral aspect of the\nright eye. There were no fractured teeth, no oral injuries, and no fluid\nin the ear canals or nostrils.\nReflection of the skin over the head revealed mottled hemor­\nrhagic, edematous subcutaneous tissues at the dorsal aspect extending\nfrom the level of the eyes caudally to the atlanto-occipital junction,\nright laterally to the ear, and left laterally to the level of the eye.\nMultifocal and coalescing petechiae and ecchymoses were noted on\nthe subcutis over the dorsal aspect of the neck and shoulder region\nextending laterally to both elbows, more extensive on the right than\nthe left (Figure 1C). Ecchymoses were also noted on the subcutis of\nthe right hip (Figure 2A).\nInternal FPME showed multifocal and coalescing petechiae and\necchymoses on the pleural aspect of the right, mid-dorsal thoracic wall\nat ribs 1–5 and on the diaphragmatic aspect of the right side of the liver.\nThe muscles of the head were removed revealing a single, com­\nplete, non-displaced fracture at the occipital bones extending from\none side of the skull to the other in an inverted, asymmetrical “V,”\nlonger on the right side. This fracture followed the normal suture lines\nbetween the occipital and parietal bones (Figure 2B). Within the cra­\nnial vault, hemorrhage was noted in the surrounding tissues of the\nbrain and meninges.\n\nWoolf\b\n10.3389/fvets.2026.1751730\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nOn histopathology, the occipital lobes and cerebellum had \nacute hemorrhage into the leptomeninges. Sections of the occipi­\ntal and parietal bones had acute hemorrhage next to the suture \nlines where the fracture was located. The bones had normal \narchitecture with no evidence of disease or a congenital or genetic \ndisorder. Sections from a femur were examined and also found to \nbe normal negating the radiographic concerns for decreased bone \ndensity.\nFIGURE 1\nCase 1 – (A) Postmortem radiograph in approximately right lateral positioning. Swelling can be seen in the soft tissues at the dorsocaudal aspect of the \nhead. (B) Postmortem radiograph in approximately ventrodorsal positioning. Swelling is seen at the dorsal aspect of the head. (C) Dorsal aspect of head, \nneck, shoulders, and thorax, and lateral aspects of forelimbs of the kitten as seen at the forensic postmortem examination. Note the petechiae and \necchymoses over the shoulders and humeri, more concentrated on the right than the left (white arrows) and the hemorrhage into the muscles and soft \ntissues over the caudal aspect of the head (black arrowheads).\nFIGURE 2\nCase 1 – (A) Right lateral aspect of pelvic region of the kitten as seen at the forensic postmortem examination. Note the ecchymoses over the right hip \n(arrows). (B) The caudal and right lateral aspect of the skull of the kitten as seen at the forensic postmortem examination. The fracture follows the \nnormal suture lines between the occipital and parietal bones (arrows).\n\nWoolf\b\n10.3389/fvets.2026.1751730\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nThe cause of death was TBI secondary to the skull fracture, con­\nsistent with being forcefully picked up over the shoulders and thrown\nto the ground; it was inconsistent with an accidental drop.\nIn the second case, the owner’s ex-girlfriend reported that the\nowner told her he had killed his kitten by crushing the kitten’s head\nagainst a wall; she had not witnessed this personally. The owner said\nit died after becoming caught between the wall and a piece of furni­\nture that was being moved.\nRight lateral and ventrodorsal postmortem radiographs were\ntaken of the entire body. Findings included a suspicion for one or\nmore skull fractures (Figures 3A,B). No other significant findings\nwere noted.\nA FPME was performed on an approximately 10-week-old, intact\nmale, domestic shorthair kitten. The kitten’s body was in good condi­\ntion with no indication of external parasites. All of the deciduous\nteeth and claws were intact. No hemorrhage was associated with the\nglobe of either eye, but the eyelids were bruised. Blood-tinged fluid\nwas on the face, nose, and front paws. Additionally blood was noted\nwithin the right ear canal and at both nares. Clotted blood was found\nwithin the mouth, nasal cavities, trachea, and esophagus.\nReflecting the skin over the head revealed moderate edema and\ncontusions on the left caudal aspect of the head with corresponding\nhemorrhage on the subcutis of the overlying skin. The contusions\nextended through the muscles and the soft tissues to the skull. No\nother contusions were noted anywhere else on the body or the sub­\ncutis. Multifocal petechiae and ecchymoses were present on approxi­\nmately 10% of the lungs with no other significant findings within the\nthoracic or abdominal cavities or viscera.\nAfter removal of soft tissue, multiple fractures were identified in\nthe skull. Most fractures followed suture lines and affected the nasal,\nfrontal, maxilla, temporal, parietal, basisphenoid, and occipital bones\n(Figure 3C). The mandibles were intact and without significant find­\n\nMost fractures followed suture lines and affected the nasal,\nfrontal, maxilla, temporal, parietal, basisphenoid, and occipital bones\n(Figure 3C).The mandibles were intact and without significant find­\nings. Hemorrhage was seen within the skull and on the brain, par­\nticularly around and between the caudal cerebrum and the\ncerebellum. Histopathology was not performed for financial reasons.\nThe cause of death for this second kitten was also determined to\nbe TBI secondary to multiple skull fractures. However, because the\nforces involved in the two stories were potentially quite similar (a\ncrushing force applied to both sides of the kitten’s head), the plausibil­\nity of accident versus abuse could not be made.\n3 Discussion\nIn both cases, the main goals of the FPME were to determine the\ncause of death and whose story better fit the injuries seen. Cause of\ndeath was clearly established in both to be TBI secondary to skull\nfractures. Determining the plausibility of each story, however, was\nonly possible in the first case.\nThe velocity of a thrown kitten can significantly exceed the velocity\nreached by a dropped kitten. This will increase the force of impact and\nsubsequently increase the amount of expected injury compared to an\naccidental fall (9). The impacted surface will also affect the severity of\ninjury. Greater injury is likely when a cat impacts hard surfaces such as\nhardwood floors compared to soft surfaces such as grass (10). Also, the\nincreased velocity from being thrown may not allow adequate time for\na kitten to right itself, which can change the location of expected injury\nrelative to a kitten falling accidentally. A kitten that rights itself may\nhave injury on the ventral aspects of the body if it sustains any injury\nat all. One that is unable to right itself before impact is more likely to\nsustain injury to the dorsal and/or lateral aspects of the body.\nTherefore, it was determined that the injuries to the first kitten\nwere consistent with being picked up forcefully over the shoulders and\nthen thrown to the ground. This conclusion was based on multiple\n\nTherefore, it was determined that the injuries to the first kitten\nwere consistent with being picked up forcefully over the shoulders and\nthen thrown to the ground.This conclusion was based on multiple\nfactors. One, had the kitten been dropped accidentally, it would have\nrighted itself to land on its feet, not its head. Two, the contusions over\nthe dorsal neck and shoulders were consistent with being grabbed\nforcefully before being hurled to the floor. Three, the contusions were\nslightly more prevalent on the right side of the body than the left, con­\nsistent with impacting the floor in a dorsally recumbent and slightly\nright-lateral position. Four, cats that suffer trauma do not typically have\nfractures at the caudal aspect of the skull (7). Five, had the injuries\noccurred due to an accidental drop, it is more likely they would have\nFIGURE 3\nCase 2 – Postmortem radiographs of the skull in (A) approximately right lateral and (B) approximately rostrocaudal positioning. The author was\nconcerned for one or more skull fractures based on these images but could not determine with certainty that fractures were present versus that\npositioning artifacts were being seen. (C) The craniodorsal aspect of the skull of the kitten as seen at the forensic postmortem examination. Note the\nmultiple fractures in the nasal, frontal, and maxilla bones, some of which follow the natural suture lines (arrows).\n\nWoolf\b\n10.3389/fvets.2026.1751730\nFrontiers in Veterinary Science\n05\nfrontiersin.org\ninvolved the ventral regions. Finally, due to the relatively minimal force\nthat would come from being dropped when compared to being thrown,\nserious injury from being dropped accidentally was simply unlikely\nwithout an underlying condition predisposing the kitten to injury.\nThe second kitten’s injuries were isolated to the head and could have\noccurred if the kitten had its head crushed against a solid surface by a\nsignificant force without having any other part of the body hit that sur­\nface with an equivalent force. This could be consistent with the story of\nthe owner intentionally crushing the kitten’s head against a wall.\nHowever, there was not enough information known about the story of\nalleged movement of furniture to determine it implausible. Other infor­\nmation that would have been useful would have included the weight,\ndimensions, and style of furniture (e.g., sofa vs. dresser, flat on the floor\nvs. on feet), the manner in which it was being moved (e.g., carried vs. slid\nacross the floor), the surface on which it was being moved (e.g., carpet\nvs. hardwood), and the speed at which the owner could move it. It would\nalso need to be known how much of the kitten the owner claimed was\ncaught between the furniture and the wall. Without this additional infor­\nmation, it could not be determined whether the ex-girlfriend’s story or\nthe owner’s story was more plausible nor could either story be dismissed.\nAn important limitation in many investigations of animal maltreat­\nment is the inability to know with absolute certainty what caused the\ninjury. There may never be a confession to the alleged crime and the\nevidence may not be adequate to remove all doubt about the cause.\nWhile the evidence was enough to determine which story was most plau­\nsible for the injuries in the first kitten, it was not sufficient to differentiate\nbetween the alleged stories explaining the injuries in the second kitten.\nPostmortem radiographs are an essential part of a FPME but they\n\nPostmortem radiographs are an essential part of a FPME but they\nalso have limitations. Because they are taken after death, the body may\nbe in rigor mortis or frozen making standard positioning impossible.\nThis issue was seen with both kittens. Additionally, the images are often\ntaken while the body is still bagged. This not only adds to difficulty in\npositioning but it can also add artifact. Decomposition may cause arti­\nfact as well, such as increased intestinal gas which may not have been\npresent antemortem. Nevertheless, postmortem radiographs allow the\nveterinarian to look for fractures – whether acute, healing, or healed –\nand foreign bodies such as projectiles. These can easily be missed on a\nFPME unless the veterinarian is expecting them in advance. Computed\ntomography (CT) can be more useful than radiographs, but often this\nis either inaccessible or unaffordable for an agency.\nAnother limitation in investigation of animal maltreatment is a lack\nof published studies on the injuries seen in these cases. It would be\nunethical to perform prospective studies in live animals to determine\nthe type of injuries seen with different types of abuse. Studies in\ndeceased animals may not be helpful because of the lack of a vital reac­\ntion normally seen with antemortem injuries. Additionally, deceased\nanimals will not stand, move, or react in the way live animals do making\nit difficult to replicate the dynamics involved in abuse situations.\nRetrospective studies depend on a case having been generated by a vet­\nerinarian, animal control, or law enforcement. Because many instances\nof animal abuse go unreported, they will not be available for study.\nFinally, studies already published involving trauma to animals\nmay unknowingly include false stories of accidental injury. Rarely\ndoes a study discussing trauma indicate whether or not the trauma\nclassification\nconsidered\nthe\npossibility\nof\nmaltreatment.\nUndiscovered, fabricated stories of accidents may skew the results\nmaking injuries seen from abuse appear to be consistent with those\nseen in accidents.\nTherefore, it is important for cases of animal maltreatment to be\n\nUndiscovered, fabricated stories of accidents may skew the results\nmaking injuries seen from abuse appear to be consistent with those\nseen in accidents.Therefore, it is important for cases of animal maltreatment to be\npublished. With time and enough cases, patterns may emerge to allow\nfor better assessment of when a story is consistent with the injuries\nseen and when it is implausible.\n4 Conclusion\nIn both cases, opposing stories were provided to explain the skull\nfractures seen in the two kittens. A FPME was needed in each case to\ndetermine which story was more plausible. In the first case, the injuries\nseen were consistent with being picked up and thrown to the floor; they\nwere inconsistent with being dropped. In the second case, both stories\ncould potentially be plausible to explain the crushing skull fractures\nseen without any other injuries elsewhere on the body. Without greater\ndetails regarding the story of furniture being moved, it cannot be ruled\nout, nor could it be determined that the ex-girlfriend was lying.\nData availability statement\nThe original contributions presented in the study are included in\nthe article/supplementary material, further inquiries can be directed\nto the corresponding author.\nEthics statement\nEthics review and approval and written informed consent were\nnot required as per local legislation and institutional requirements.\nWritten informed consent was obtained from the participants for the\npublication of this case report.\nAuthor contributions\nJW: Writing  – original draft, Writing  – review & editing,\nConceptualization, Investigation.\nFunding\nThe author(s) declared that financial support was not received for\nthis work and/or its publication.\nConflict of interest\nJW was employed by Woolf Veterinary Forensics Consulting, Inc.\nGenerative AI statement\nThe author(s) declared that Generative AI was not used in the\ncreation of this manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\n\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\n\nWoolf\b\n10.3389/fvets.2026.1751730\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the review­\ners. Any product that may be evaluated in this article, or claim that\nmay be made by its manufacturer, is not guaranteed or endorsed by\nthe publisher.\nReferences\n1.\tPierce MC, Bertocci GE, Janosky JE, Aguel F, Deemer E, Moreland M, et al. Femur\nfractures resulting from stair falls among children: an injury plausibility model. Pediatrics.\n(2005) 115:1712–22. doi: 10.1542/peds.2004-0614\n2.\tPennock AT, Gantsoudes GD, Forbes JL, Asaro AM, Mubarak SJ. Stair falls: caregiver's\n\"missed step\" as a source of childhood fractures. J Child Orthop. (2014) 8:77–81. doi:\n10.1007/s11832-014-0551-x\n3.\tPierce MC, Kaczor K, Lohr D, Richter K, Starling. A practical guide to differentiating\nabusive from accidental fractures: an injury plausibility approach. Clin Pediatr Emerg\nMed. (2012) 13:166–77. doi: 10.1016/j.cpem.2012.06.009\n4.\tVnuk D, Pirkić B, Maticić D, Radisić B, Stejskal M, Babić T, et al. Feline high-rise\nsyndrome: 119 cases (1998-2001). J Feline Med Surg. (2004) 6:305–12. doi: 10.1016/j.\njfms.2003.07.001\n5.\tBonner SE, Reiter AM, Lewis JR. Orofacial manifestations of high-rise syndrome in cats: a\nretrospective study of 84 cases. J Vet Dent. (2012) 29:10–8. doi: 10.1177/089875641202900103\n6.\tCandela Andrade M, Slunsky P, Nerlich A, Aguilera-Rojas M, Brunnberg L. High-rise\nsyndrome in cats (part 2): injury patterns and survival rate. J Feline Med Surg. (2025) 27.\ndoi: 10.1177/1098612X251334096\n7.\tKnight R, Meeson RL. Feline head trauma: a CT analysis of skull fractures and their\nmanagement\nin\n75\ncats.\nJ\nFeline\nMed\nSurg.\n(2019)\n21:1120–6.\ndoi:\n10.1177/1098612X18819183\n8.\tSechzer JA, Folstein SE, Geiger EH, Mervis RF, Meehan SM. Development and matura­\ntion of postural reflexes in normal kittens. Exp Neurol. (1984) 86:493–505. doi:\n10.1016/0014-4886(84)90084-0\n\nJ\nFeline\nMed\nSurg.(2019)\n21:1120–6.doi:\n10.1177/1098612X18819183\n8.Sechzer JA, Folstein SE, Geiger EH, Mervis RF, Meehan SM.Development and matura­\ntion of postural reflexes in normal kittens.Exp Neurol.(1984) 86:493–505.doi:\n10.1016/0014-4886(84)90084-0\n9.\tLefman S, Prittie JE. High-rise syndrome in cats and dogs. J Vet Emerg Crit Care. (2022)\n32:571–81. doi: 10.1111/vec.13206\n10.\tCandela Andrade M, Slunsky P, Nerlich A, Aguilera-Rojas M, Brunnberg L. High-rise\nsyndrome in cats (part 1): epidemiology and risk factors. J Feline Med Surg. (2025) 27.\ndoi: 10.1177/1098612X251334091\n\n---"}
+{"text": "【兽医文献】\n标题: Advancing evidence-informed practice in modern zoos: research priorities for animal welfare, conservation, and social legitimacy\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nTYPE  Original Research\nPUBLISHED  21 April 2026\nDOI  10.3389/fvets.2026.1792929\nOPEN ACCESS\nEDITED BY\nInkar Alejandro Castellanos-Huerta,\nUniversity of Arkansas, United States\nREVIEWED BY\nSoheil Sadr,\nFerdowsi University of Mashhad, Iran\nAlisher Safarov,\nTashkent State Agrarian University,\nUzbekistan\n*CORRESPONDENCE\nAbdulrahman A. Alkheraif\nalkheraif@qu.edu.sa\nRECEIVED 21 January 2026\nREVISED 06 March 2026\nACCEPTED 27 March 2026\nPUBLISHED 21 April 2026\nCITATION\nAL-Sobayil F, Sadan M, Alkheraif AA and\nHaridy M (2026) Surgical and\npathological interventions of\noropharyngeal and laryngeal disorders in\ncamels (Camelus dromedarius).\nFront. Vet. Sci. 13:1792929.\ndoi: 10.3389/fvets.2026.1792929\nCOPYRIGHT\n© 2026 AL-Sobayil, Sadan, Alkheraif and\nHaridy. This is an open-access article\ndistributed under the terms of the\nCreative Commons Attribution License\n(CC BY). The use, distribution or\nreproduction in other forums is\npermitted, provided the original\nauthor(s) and the copyright owner(s) are\ncredited and that the original publication\nin this journal is cited, in accordance\nwith accepted academic practice. No\nuse, distribution or reproduction is\npermitted which does not comply with\nthese terms.\nSurgical and pathological\ninterventions of oropharyngeal\nand laryngeal disorders in camels\n(Camelus dromedarius)\nFahd AL-Sobayil 1, Madeh Sadan 1, Abdulrahman A. Alkheraif 2*\nand Mohie Haridy 2\n1Department of Clinical Sciences, College of Veterinary Medicine, Qassim University, Buraydah, Saudi\nArabia, 2Department of Pathology and Laboratory Diagnosis, College of Veterinary Medicine, Qassim\nUniversity, Buraydah, Saudi Arabia\nAim: This study aimed to characterize the clinical, hematobiochemical, radio­\ngraphic, ultrasonographic, laryngoscopic, gross, and histopathological features\nof oropharyngeal and laryngeal disorders, epulis (osteolipoma and osseous meta­\nplasia), dulla entrapment (tonsillitis and sialadenitis), and obstructive laryngeal\nmass (mycotic pyogranulomatous laryngitis) and to describe the surgical proce­\ndures used for their management in dromedary camels.\n\nMethods: Oropharyngeal and laryngeal lesions in four camels were evaluated\nthrough comprehensive clinical, imaging, hematobiochemical, and pathological\nassessments.\nResults: One camel with epulis is presented with a large mandibular mass caus­\ning partial oral obstruction, impaired mastication, and swallowing. Radiographs\nshowed a soft tissue density mass on the right cranial mandible. Two camels are\npresented with dulla entrapment, dysphagia, neck stiffness, and mild respira­\ntory distress. The camel with tonsillitis exhibited dark pinpoint mucosal lesions\non the surface of the soft palate with spiny feed impaction; however, the camel\nwith sialadenitis had multiple purulent abscesses in the dulla and submandibu­\nlar swelling. The fourth camel with obstructive laryngeal mass had a marked\nrespiratory difficulty, and the radiographs revealed a large, irregular soft-tissue\nmass within the laryngeal lumen. Ultrasonography showed an echogenic, vari­\nably anechoic mass, and laryngoscopy confirmed luminal mass obstruction. After\nsurgical removal of these affections in the four camels, histopathology revealed\nosteolipoma and osseous metaplasia, velar suppurative tonsillitis, suppurative\nsialadenitis of the minor palatine salivary glands, and mycotic pyogranulomatous\nlaryngitis, respectively.\nConclusion: This study reports four oropharyngeal and laryngeal disorders in\ncamels with obstructive clinical signs. Imaging, laryngoscopy, and histopathology\nenabled accurate diagnosis, and timely-applied surgery is effective for managing\nthese conditions and rescuing the lives of affected camels.\nKEYWORDS\nanimals, diagnostic imaging, osteolipoma, pathology, sialadenitis\n\nAL-Sobayil et al.\b\n10.3389/fvets.2026.1792929\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nIntroduction\nThe importance of camels is obtained from their major roles in\nproducing milk, meat, wool, and leather, as well as their prominence\nin camel racing within Arab heritage (1–3). Today, they serve as a key\nsource of both meat and milk. Moreover, the general health status of\na camel directly influences the volume and quality of its milk and\nmeat, as well as overall production efficiency (1, 2, 4, 5). The gingiva\n(gum) of the camel is a specialized oral tissue adapted to browsing on\ncoarse, thorny desert vegetation; it exhibits distinctive anatomical and\nfunctional features compared with other domestic ruminants (6).\nGum disorders in camels (Camelus dromedarius) range from mild\ngingivitis to severe periodontal disease and tumor-like growths in the\noral cavity. Recent reports have described maxillary tumors, including\nameloblastoma, intraosseous squamous cell carcinoma, and central\nodontogenic fibroma, emphasizing the clinical significance of oral\nlesions in this species (7–9). If left untreated, gingival and periodontal\nlesions can impair mastication, reduce feed intake, cause weight loss,\nand decrease overall productivity, highlighting the need for early diag­\nnosis and appropriate management (10).\nThe oropharynx extends from the palatoglossal arches to the\nbase of the epiglottis. Its roof is formed by the soft palate and the\npalatine diverticulum (11, 12). In camels, the dulla is a downward\nand forward extension of the soft palate. It is remarkably long,\nstretching from the caudal margin of the hard palate to a point\ncaudal to the epiglottis, reaching the level of the arytenoid carti­\nlages. The structure of the dulla is more pronounced in adult\nmales than in females, and it commonly protrudes outside the\nmouth during the rutting season as a sign of sexual behavior (1).\nSeveral types of injuries in dulla have been reported in the adult\nmale camels during the rutting season, including protrusions, lac­\nerated wounds, abscesses, hematomas, impaction of feed materi­\n\nSeveral types of injuries in dulla have been reported in the adult\nmale camels during the rutting season, including protrusions, lac­\nerated wounds, abscesses, hematomas, impaction of feed materi­\nals, and gangrene (1, 13). Injuries of the dulla are often caused by\npointed or abrasive edges of the cheek teeth and canines, by she-\ncamels during copulation, or by other males during fights.\nFurthermore, blunt objects, feed materials, and straw can cause\nsuch injuries. As a result, an inflammation accompanied by edem­\natous, fluctuating swelling of the dulla may occur, hindering\nretraction of the dulla into the mouth cavity and progressively\nworsening the condition. Consequently, these injuries are\ncommon reasons for performing the soft palate surgeries in camels\n(1, 11, 13). Tonsillitis in camels refers to inflammation of the ton­\nsillar tissue. Tonsils serve as a primary immunological barrier in\nthe oropharynx. Anatomical investigations have demonstrated\nthat the lingual and palatine tonsils in camels are well-developed\nlymphoid structures. They are capable of reacting to ingested and\ninhaled antigens, making them susceptible to the inflammatory\nresponses when exposed to pathogens or traumatic insults (14).\nTonsillitis may occur as a primary condition or alongside other\noropharyngeal disorders. Affected animals commonly exhibit dys­\nphagia, excessive salivation, malodorous breath, and varying\ndegrees of respiratory compromise, particularly when tonsillar\nenlargement partially obstructs the upper airway (15). Tonsillitis\nhas been reported in association with salivary gland disorders and\ndulla entrapment, suggesting a contributory role in the mechani­\ncal and inflammatory airway obstruction. Persistent or untreated\ncases may adversely affect feed intake, body condition, and pro­\nductivity (16). Minor salivary glands in camels are widely distrib­\nuted in the oral mucosa and are essential for lubrication and oral\nhealth. These glands may be affected by inflammatory, obstruc­\ntive, cystic, or neoplastic lesions, including sialadenitis and muco­\n\nMinor salivary glands in camels are widely distrib­\nuted in the oral mucosa and are essential for lubrication and oral\nhealth.These glands may be affected by inflammatory, obstruc­\ntive, cystic, or neoplastic lesions, including sialadenitis and muco­\nceles, which can cause swelling, pain, and feeding difficulties if left\nuntreated (17, 18).\nThe larynx of the camel is a cartilaginous and muscular organ\nlocated at the cranial end of the trachea and plays a vital role in respi­\nration, airway protection, and phonation. Anatomical and histological\ninvestigations have shown that the camel larynx is composed of the\nepiglottic, thyroid, cricoid, and paired arytenoid cartilages, supported\nby intrinsic and extrinsic muscles, and lined by specialized mucosa\nadapted to respiratory function (19). Laryngeal affections in camels,\nalthough infrequently reported, include inflammatory conditions,\ntraumatic or obstructive lesions, and rare mass-forming abnormali­\nties, which may result in respiratory distress, dysphagia, abnormal\nvocalization, and reduced feeding efficiency. Early clinical evaluation\nand endoscopic examination are therefore essential for accurate diag­\nnosis and effective management of laryngeal disorders in this species\n(1, 12, 19–21).\nSeveral oropharyngeal and laryngeal disorders have been docu­\nmented in earlier studies in different animal species, including sialad­\nenitis in equine (22, 23), canine (24), feline (25), and small (26), and\nlarge ruminants (6); palatine tonsillitis in equine (27), canine (28), and\nsmall (29) and large ruminants (30); and laryngitis in horses (31),\ncanine (32), and small (33), and large ruminants (34). Osteolipoma\nhas been documented in equine (35), canine (36), and small (37) and\nlarge ruminants (38, 39) in various tissues, but it has not been reported\nin the gingival tissue. Clinical, hematobiochemical, radiographic,\nultrasonographic, laryngoscopic, and histopathological evaluations\nare essential for identifying causes, understanding the pathogenesis,\nand formulating the appropriate treatment strategies for oropharyn­\n\nClinical, hematobiochemical, radiographic,\nultrasonographic, laryngoscopic, and histopathological evaluations\nare essential for identifying causes, understanding the pathogenesis,\nand formulating the appropriate treatment strategies for oropharyn­\ngeal and laryngeal disorders. As a result, these conditions are fre­\nquently cited as key indications for surgical intervention to achieve a\ncurative outcome (1, 22, 36). Despite the widespread popularity of\ncamels, to the authors’ knowledge, there is no prior research on clini­\ncal, hematobiochemical, radiographic, ultrasonographic, laryngo­\nscopic, and histopathological findings or the treatment of\noropharyngeal and laryngeal disorders in camels. Therefore, the pres­\nent study is conducted to describe several oropharyngeal and laryn­\ngeal disorders in camels and to evaluate the efficacy of surgical\nresection as treatment.\nMaterials and methods\nAnimals and clinical cases\nFour adult camels (two males, two females) were admitted to the\nUniversity Veterinary Hospital, Qassim University, Saudi Arabia, in\n2025. The camel’s age ranged from 1 to 10 years (mean ± SD:\n7 ± 2 years) and weight from 300 to 700 kg (450 ± 120 kg). Breeds of\ncamels were Wadeh (three) and Asfar (one). The camels were selected\nbased on clinical signs, hematobiochemical results, imaging, laryngos­\ncopy, histopathology, and relevant medical history of the oropharyn­\ngeal and laryngeal disorders. The latter were documented and\ncategorized by etiology and lesion type (Figure 1). The Committee of\nAnimal Welfare and Ethics at Qassim University, in accordance with\nthe Laboratory Animal Control Guidelines, approved the study pro­\ntocol (No. 367; March 2025).\n\nAL-Sobayil et al.\b\n10.3389/fvets.2026.1792929\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nClinical examination\nClinical examinations were routinely performed on the\naffected camels. The oral, pharyngeal, and laryngeal cavities were\nexamined physically to describe the conditions: epulis (osteoli­\npoma and osseous metaplasia), tonsillitis, sialadenitis, and\nobstructive laryngeal mass (mycotic granulomatous laryngitis).\nThe physical characteristics of the injured gums, dulla, and larynx\nwere reported, and the cause, type, and duration of each lesion\nwere determined. The animals’ ages, breeds, and sexes were\nrecorded, and all assessed parameters were compared among the\ncamels examined.\nHematological and biochemical\nexamination\nThe hematological and biochemical parameters were examined\npreoperatively in all camels. EDTA blood samples were analyzed using\nan automated hematology analyzer (VetScan HM5, Abaxis, Union\nCity, CA, USA) for complete blood counts. Sera were collected, cen­\ntrifuged, and analyzed with an automated biochemical analyzer\n(VetScan VS2, Abaxis, Union City, CA, USA) to determine the total\nprotein (TP), albumin (ALB), globulin (GLOB), glucose (GLU), cre­\natinine (CR), creatine kinase (CK), and blood urea nitrogen (BUN);\nthe liver enzymes [aspartate aminotransferase (AST), alanine amino­\ntransferase (ALT), gamma-glutamyl transferase (GGT), and alkaline\nphosphatase (ALP)]; and the electrolytes and minerals [calcium (Ca),\nphosphorus (P), magnesium (Mg), sodium (Na), and potassium (K)].\nResults were compared with the published reference ranges (4, 39).\nRadiographic, laryngoscopic, and\nultrasonographic examinations\nTwo standard radiographic projections, dorsoventral and latero­\nmedial, were obtained for the affected camels while they were main­\ntained in a sitting position under light sedation (Intravenous/xylazine\nHCl (2%)/a dose of 0.1 mg/kg). Radiography was performed using a\nMinx Ray HF 100/30 unit (Toshiba, Tokyo, Japan), and the obtained\nimages were subjectively assessed before surgical intervention.\nUltrasonographic examination was subsequently carried out on the\n\n1 mg/kg).Radiography was performed using a\nMinx Ray HF 100/30 unit (Toshiba, Tokyo, Japan), and the obtained\nimages were subjectively assessed before surgical intervention.Ultrasonographic examination was subsequently carried out on the\nsedated camel while placed in a lateral recumbency, using a 3.5–\n7.5 MHz linear transducer (Aloka, Tokyo, Japan). The ventral cervical\nregion was adequately prepared to allow imaging of the larynx, follow­\ning the technique described by Sadan et al. (5). Laryngoscopic exami­\nnation was done after restraining the affected camel in the sternal\nrecumbency under light sedation, with the head elevated, following\nthe procedure described by Nadjmi et al. (40).\nSurgical technique\nPreoperative medications\nPreoperatively, camels received intravenous fluids (0.9%\nsaline\n1–3 L\nand\n5%\ndextrose\n1–2 L),\nintramuscular\nFIGURE 1\n(A) Cranial view of a gingival osteolipoma in a camel (arrow). (B) Lateral view of a gingival osteolipoma in a camel (arrow). (C) Close image of gingival\nosteolipoma in a camel (arrow). (D) Lateral radiograph revealed soft-tissue–density mass located on the lingual (inner) cranial aspect of the right\nmandible in a camel (arrow). (E) Close image of lateral radiograph revealed soft-tissue–density mass located on the lingual (inner) cranial aspect of the\nright mandible in a camel (arrow). (F) Surgically resected gingival osteolipoma in camel (arrow).\n\nAL-Sobayil et al.\b\n10.3389/fvets.2026.1792929\nFrontiers in Veterinary Science\n04\nfrontiersin.org\npenicillin–streptomycin (30,000  IU/kg and 10 mg/kg), flunixin\nmeglumine (1.1 mg/kg), and a systemic hemostatic agent (10 mL IM).\nSurgical management of epulis (osteolipoma)\nThe affected camel was fasted for 24 h. Under sedation with xyla­\nzine (0.3 mg/kg IV) and local infiltration of 2% lignocaine, the mass\nof epulis was surgically excised (Figure 1) following aseptic prepara­\ntion (7, 39). Hemorrhage was controlled by applying gauze soaked in\ntincture of benzoin for 2 minutes, after which the area was dressed in\npovidone-iodine–impregnated gauze.\nSurgical management of tonsillitis and sialadenitis\nSurgical resection of the affected dulla was performed as definitive\ntreatment to relieve the clinical signs, following comprehensive clini­\ncal and hematobiochemical evaluations. After sedation [intravenous\nxylazine HCl at a dose of 0.3 mg/kg (Norbrook Laboratories, Newry,\nUK)], the affected camels were secured in sternal recumbency, and the\ninjured dulla was carefully exteriorized using long sponge forceps,\ncombined with gentle digital pressure over the swollen pharyngeal\narea. The protruded dulla was grasped with a sterile surgical towel and\nextended to allow proper aseptic preparation, particularly at its base.\nLocal infiltration anesthesia was administered at the base of the dulla\nusing 2% lidocaine hydrochloride (Norbrook Laboratories, UK). Once\nadequate anesthesia was achieved, overlapping interrupted horizontal\nmattress sutures were applied to establish hemostasis using coated\npolyglactin 910 (No. 2) (United Medical Industries Co., Ltd., Riyadh).\nThe injured portion of the dulla was then amputated approximately\n3 cm distal to the suture line, and the surgical wound was closed rou­\ntinely using a simple continuous suture pattern.\nSurgical management of the laryngeal mass\nThe laryngeal mass was surgically excised under general anesthe­\nsia following a complete clinical and diagnostic evaluation. Anesthesia\nwas induced with xylazine (0.2 mg/kg IV) and ketamine (2–4 mg/kg\n\nSurgical management of the laryngeal mass\nThe laryngeal mass was surgically excised under general anesthe­\nsia following a complete clinical and diagnostic evaluation.Anesthesia\nwas induced with xylazine (0.2 mg/kg IV) and ketamine (2–4 mg/kg\nIV; Zoetis, NJ, USA), intubated, and maintained with 2% isoflurane\n(Piramal Critical Care Inc., Bethlehem, PA) delivered in 100% oxygen.\nVia a ventral midline cervical approach, the larynx was exposed, and\na longitudinal incision was made through the cricothyroid membrane\nto access the laryngeal lumen for mass removal (41). The mass was\nsurgically detached and resected at its point of attachment.\nPostoperative management and follow-up\nPostoperatively, antibiotics and anti-inflammatory drugs were\nadministered for 5 days, along with adjunct vitamin AD₃E and\nmeloxicam. Intravenous fluids were provided for 3 days. Camels were\nstall-confined and monitored daily for 1 month, then discharged after\nadequate healing. Long-term outcomes were assessed via owner fol­\nlow-up at 6 months. All animals recovered without complications.\nHistopathological examination\nThe excised epulis, dullas, and laryngeal masses were fixed in 10%\nneutral buffered formalin, paraffin-embedded, sectioned (5 μm), and\nstained with H&E. Slides were evaluated blindly by a board-certified\nveterinary pathologist. Special stains (Sudan Black and PAS) were\napplied to detect lipid cells and fungal hyphae, respectively.\nResults\nClinical findings\nThis study reported three types of oropharyngeal and one type of\nlaryngeal disorders, namely epulis (osteolipoma and osseous metapla­\nsia), tonsillitis, sialadenitis, and mycotic granulomatous laryngitis (see\nFigures 1–7; Table 1). A 1-year-old female camel with epulis presented\nwith a history of partial oral cavity obstruction due to a large mass\n(Figure 1), which was interfering with the normal mastication and\ncausing difficulty in swallowing. The animal appeared restless and had\na reduced appetite. Upon oral examination, a large, oblong prolifera­\ntive mass was observed affecting the lower right gingiva.\n\nThe animal appeared restless and had\na reduced appetite.Upon oral examination, a large, oblong prolifera­\ntive mass was observed affecting the lower right gingiva.\nTwo male camels (aged 8 and 10 years) with entrapped dullas due\nto tonsillitis and sialadenitis, respectively, were presented with dyspha­\ngia, neck stiffness, and mild respiratory distress. Tonsillitis appeared\nas pinpoint dark-brown raised foci on the mucosal surface of the soft\npalate, which was impacted with spiny food materials (Figure 2).\nConversely, the camel with sialadenitis (Figure 3) had an entrapped\ndulla with multiple abscesses, containing greenish-yellow pus that\nopened on the surface, and multifocal ulcerated mucosa. The camel\nhad a dry mouth, no salivation, and swelling of the submandibu­\nlar region.\nA 7-year-old female camel presented with a history of partial\nlaryngeal cavity obstruction with a large mass. The obstructive laryn­\ngeal mass interfered with normal respiration and caused stiffness,\nextension of the head and neck, and dyspnoea (Figure 4). The animal\nappeared restless and had a reduced appetite.\nHematological and biochemical findings\nThe camel with epulis had reduced red blood cell (RBC) count\n(6.75 × 106/μL; reference value: 11.3 ± 1.4 × 106/μL), along with\ndecreased hematocrit value (16.78%; normal: 28.9 ± 2.7%) and\nhemoglobin concentration (14.2 g/dL; normal: 16.0 ± 2.3 g/dL).\nConversely, mean corpuscular hemoglobin (MCH) and mean cor­\npuscular hemoglobin concentration (MCHC) were elevated to 21 pg.\n(normal: 14.7 ± 2.4 pg) and 84.5 g/dL (normal: 57.6 ± 9.0 g/dL),\nrespectively. Biochemical tests revealed an increment in the levels of\npotassium (5.6 mmol/L; normal: 3.8 ± 0.2 mmol/L), calcium\n(10.6 mg/dL; normal: 8.6 ± 0.7 mg/dL), and glucose (147 mg/dL;\nnormal: 61 ± 19 mg/dL) and reduction in the levels of sodium\n(149 mmol/L; normal: 163 ± 2.0 mmol/L), albumin (2.9 g/dL;\nnormal: 4.2 ± 0.4 g/dL), and total proteins (4.6 g/dL; normal:\n7.9 ± 0.4 g/dL).\nIn a camel affected by tonsillitis, the hematobiochemical profile\nshowed several alterations: elevated MCHC (52.5 g/dL) and white\n\n0 mmol/L), albumin (2.9 g/dL;\nnormal: 4.2 ± 0.4 g/dL), and total proteins (4.6 g/dL; normal:\n7.9 ± 0.4 g/dL).In a camel affected by tonsillitis, the hematobiochemical profile\nshowed several alterations: elevated MCHC (52.5 g/dL) and white\nblood cell (WBC) count (19.39 × 103/ell) with marked neutrophilia\n(76.2%), lymphocytopenia (14.5%), and monocytopenia (0.7%). Red\ncell distribution width (RDW) was elevated (28.6% and RDW 24.2\nfly). Glucose was also elevated at 217 mg/dL.\nThe hematobiochemical profile in camels affected with sialadenitis\ndisplayed macrocytosis and hypochromic anemia. The RBC count\n(8.97 × 106/μL), hemoglobin (15.6 g/dL), and hematocrit (26.63%)\n\nAL-Sobayil et al.\b\n10.3389/fvets.2026.1792929\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nwere decreased, indicating anemia. Indicators of red blood cell size \nand hemoglobin content; mean corpuscular volume (MCV) (30 fl), \nMCH (17.4 pg), and MCHC (58.7 g/dL) were elevated, indicating \nmacrocytosis. RDWc (26% and RDWs 26.6 fl) was also higher than \nnormal. Total WBC were increased (18.94 × 103/μL) with prominent \nneutrophilia (63.8%), eosinophilia (26.3%), lymphocytopenia (9.3%), \nand monocytopenia (0.6%). Alkaline phosphatase (ALP) activity was \nslightly below the reference range (45 U/L), but glucose was elevated \n(159 mg/dL).\nThe camel with the laryngeal mass had reduced RBC count \n(7.29 × 106/μL; reference value: 11.3 ± 1.4 × 106/μL) and hematocrit \nvalue (13.12%; normal: 28.9 ± 2.7%). Conversely, eosinophil count \n(3.96; normal: 2.05 ± 0.57 × 103cell/μL), MCH (18.1 pg.; normal: \n14.7 ± 2.4 pg), and MCHC (77 g/dL; normal: 57.6 ± 9.0 g/dL) were \nelevated. Biochemical analyses revealed increments in the levels of \npotassium (5 mmol/L; normal: 3.8 ± 0.2 mmol/L), calcium (9.7 mg/\ndL; normal: 8.6 ± 0.7 mg/dL), and glucose (127 mg/dL; normal: \n61 ± 19 mg/dL) and reduction in the levels of creatine kinase (CK) \n(35 mmol/L; normal: 138 ± 22 U/L), gamma-glutamyl transferase \n(GGT) (11 mmol/L; normal: 13 ± 5.0 U/L), sodium (147 mmol/L; \nnormal: 163 ± 2.0 mmol/L), and albumin (3 g/dL; normal: \n4.2 ± 0.4 g/dL).\nFIGURE 2\n(A) Tonsillitis in a camel. Please note the pinpoint dark brown raised foci on the surface of the mucosa of the soft palate (dulla) (arrow). (B) Impacted soft \npalate (dulla) with spiny food materials (arrow).\nFIGURE 3\n(A) Sialadenitis in a camel. Please note the entrapped soft palate filled with multiple abscesses (arrow). (B) Impacted soft palate (dulla) with food \nmaterials with greenish-yellow pus that opened on the surface, and areas covering the mucosa were ulcerated (arrow).\n\nAL-Sobayil et al.\b\n10.3389/fvets.2026.1792929\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nRadiographic, laryngoscopy, and\nultrasonographic examinations\nRadiographic assessment of the epulis showed a soft-tissue–den­\nsity mass located on the lingual (inner) cranial aspect of the right\nmandible (Figures 1D,E) and localized on the gingival soft tissue. The\nsoft tissue mass contained deep radiopaque granules corresponding\nto osseous tissue and had no continuation with the mandibular bony\ntissue. Radiographs of the laryngeal mass revealed a large, irregular\nsoft-tissue mass occupying the laryngeal lumen, outlined by the radio­\nlucent laryngeal air as a negative contrast background (Figure 5A).\nUltrasonography of the mass demonstrated a sizable, highly vascular\nechogenic lesion containing variable anechoic areas within the mass\n(Figure 5B). Also, laryngoscope examination confirmed the mass\nwithin the laryngeal lumen (Figures 6A,B). Surgical removal was suc­\ncessfully performed, and the excised tissue appeared as an oval pyo­\ngranulomatous mass, had a firm consistency, and was firmly adherent\nto the laryngeal tissue (Figure 7).\nGross and histopathological findings\nEpulis appeared as a large mass on the right cheek that origi­\nnated from the gum and had no osseous continuation with the man­\ndibular bone (Figure 1C). The mass measured 25× 5× 3.3 cm and\nwas covered with ulcerated and dirty yellowish diphtheritic mem­\nbrane that penetrated with foodstuffs. The mass was solid and greasy\nin texture, with a gritty sound in the cut section (Figures 1C,F).\nHistologically, the epulis mass revealed osteolipoma composed of\nlarge areas of mature fat cells, supported by fibrous connective tissue\nsepta (Figures 8A,B), and focal areas of woven bone. Fusiform- mes­\nenchymal cells circumscribed the lesion (Figure 8B). In addition to\nosteolipoma, focal areas of osteoid bony tissues, as ectopic bone\ntissue (osseous metaplasia) containing myeloid, were observed in the\ndense connective tissue layer of the gum (Figures 8C,D). The overly­\ning epithelium was necrotic and ulcerated, and the superficial layer\n\nThe overly­\ning epithelium was necrotic and ulcerated, and the superficial layer\nof the gum was severely infiltrated with neutrophils (Figure 9A).\nFocal gingival epithelial hyperplasia was associated with severe\ninflammatory cell infiltration (Figure 9B). Foodstuffs invaded the\nloose connective tissue layer, leaving a foreign body suppurative\nreaction (Figure 9C).\nThe soft palate tonsils (velar tonsils) are formed of disseminated\nnodules with crypts on the oral surface of the soft palate, almost near\nthe junction with the hard palate. Tonsillitis appeared as pinpoint,\ndark brown raised foci on the mucosa of the soft palate (Figure 2A).\nThe soft palate was swollen, edematous, bright red, and impacted with\nspiny food materials. Histologically, the normal tonsillar nodules are\nformed of primary and secondary lymphoid follicles, separated by\nparafollicular lymphoid tissue, and distributed within the connective\ntissue of the soft palate, which is covered by stratified squamous kera­\ntinized epithelium. The nodule had a crypt, which is lined by a non-\nkeratinized stratified squamous epithelium (Figure 10A). The soft\npalate tonsillar follicles sometimes appeared hyperplastic and had\nseveral germinal centers (Figure 10A). The tonsillar nodules also\nrevealed suppurative necrosis and depletion of the lymphoid follicles\nleaving thickened interfollicular dense connective tissue (Figure 10B).\nThe lymphoid follicles of the tonsils were also severely necrosed and\ndevoid of lymphocytes associated with necrosis of crypt’s epithelium\nleaving the tonsillar nodule without structural outlines (Figure 10C).\nThe epithelial ridge of the overlying mucosa of the soft palate was\nhyperplastic. The submucosa was diffusely infiltrated with neutrophils\n(Figure 10D). Massive necrosis of the tonsils induced a pressure atro­\nphy of the salivary glands due to surrounding fibrosis. Thorns were\nobserved in the area of massive necrosis, along with giant cells and\ncolonies of bacterial infection (Figure 10E).\nThe minor palatine salivary glands, branched tubule-alveolar\n\nThorns were\nobserved in the area of massive necrosis, along with giant cells and\ncolonies of bacterial infection (Figure 10E).The minor palatine salivary glands, branched tubule-alveolar\nmucous and seromucous glands, are located within the propria and\nsubmucosa in the hard palate and increase toward the apex of the soft\npalate. The camel with sialadenitis grossly revealed multiple abscesses\nof dulla, contained greenish-yellow pus that opened on the surface,\nand multifocal areas of the covering mucosa were ulcerated.\nHistologically, hemorrhages and multiple abscesses were observed in\nthe submucosa of the affected soft palate. The overlying mucosa was\nulcerated and healed with granulation tissue (Figure 11A).\nDestruction, necrosis, and fibrosis of the salivary glands (Figures 11A–\nC) and periductal fibrosis (Figure 11C) were observed in the tissue\nsurrounding the large abscesses. Ductal obstruction with exudate\nencircled with organized connective tissue, where the surrounding\nglandular tissues had a liquefactive necrosis (Figure 11D).\nGrossly, the laryngeal mass was large oval mass that had a firm\nconsistency, measured 20 cm x 6 cm x 4.4 cm in diameter and was\nfirmly adherent to the laryngeal tissue (Figure 7A). The mass com­\nposed of disseminated caseated abscesses that circumscribed with tan\nwhitish gray layer of connective (pyogranulomatous lesions)\n(Figure 7B). The histopathology of the mass revealed mycotic granu­\nlomatous laryngitis with a necrotic eosinophilic center, containing\ncellular debris rich of eosinophils and fungal hyphae (Figure 12D). It\nsurrounded by a cellular zone composed of macrophages, epithelioid,\ngiant cells, and rich eosinophils (Figures 12A,B). The cellular zones\nwere surrounded by a fibrous layer rich with eosinophilic infiltrate and\ngiant cells (Figure 12B). The interstitium between the pyogranuloatous\nlesions was rich in neutrophlic, eosinophilic, macrophages and giant\ncell infiltration (Figure 12C). To improve the visualization of the\nfungal elements, periodic acid-Schiff was applied, and the pyogranu­\n\nThe interstitium between the pyogranuloatous\nlesions was rich in neutrophlic, eosinophilic, macrophages and giant\ncell infiltration (Figure 12C).To improve the visualization of the\nfungal elements, periodic acid-Schiff was applied, and the pyogranu­\nlomatous lesions revealed plenty of hyphae that characterized by par­\nallel cell walls, distinct septa, and a dichotomous branching pattern\n(Figures 12D, 13A,B). These morphological characteristics are in\nFIGURE 4\nA 7-year-old she-camel presented with a history of partial\nobstruction of the laryngeal cavity by a large mass\n(pyogranulomatous laryngitis), which was interfering with normal\nrespiration, causing stiffness, extension of the head and neck, and\ndyspnea.\n\nAL-Sobayil et al.\b\n10.3389/fvets.2026.1792929\nFrontiers in Veterinary Science\n07\nfrontiersin.org\ncompromise with Aspergillus Spp. Fungal hyphae were present mainly\nin the pyogranulomatous lesions and also invaded adjacent structures.\nTreatment outcomes\nA strong correlation was reported between the duration of inter­\nvention for the oropharyngeal and laryngeal disorders and the general\nhealth conditions of the affected camels. Favorable outcomes were\nassociated with early diagnosis, prompt treatment, and diligent post-\noperative management. Follow-up assessments were conducted over\na period of 6 months through phone communication and field visits\nwith camel owners. The effect of surgical resection of osteolipoma,\nentrapped dullas, and mycotic laryngeal mass on the ability to eat,\nswallow, and breathe of the affected camels, and if any abnormal sur­\ngical-site discharge, and the overall functional recovery parameters\nwere evaluated. All camels showed full restoration of normal function\nwithin 6 months after surgery.\nDiscussion\nInjuries involving the oropharyngeal and laryngeal lumen are seri­\nous conditions in camels and often necessitate surgical intervention to\nmanage their associated complications. However, the literature pro­\nvides limited information regarding the clinical presentation, hema­\ntobiochemical alterations, imaging characteristics, histopathological\nfeatures, and the therapeutic approaches for these lesions. Therefore,\ndetailed clinical, laboratory, radiographic, ultrasonographic, laryngo­\nscopic, and histopathological findings of the oropharyngeal and laryn­\ngeal disorders were reported, and the surgical outcomes in treating\nthese conditions in camels were highlighted. Three types of oropha­\nryngeal and one type of laryngeal disorders, namely, epulis (osteoli­\npoma), tonsillitis, sialadenitis, and mycotic pyogranulomatous\nlaryngitis, respectively, were reported.\nClinical examination is routinely used to diagnose the oropha­\nryngeal and laryngeal disorders in camels. In the present study, a\nFIGURE 5\n\nClinical examination is routinely used to diagnose the oropha­\nryngeal and laryngeal disorders in camels.In the present study, a\nFIGURE 5\n(A) A lateral radiograph of pyogranulomatous mycotic laryngitis in a camel revealed a large, irregular soft-tissue mass occupying the laryngeal lumen,\noutlined by the radiolucent laryngeal air as a negative contrast background (arrow). (B) Ultrasonography revealed a sizable, highly vascular echogenic\n(white arrow) lesion containing variable anechoic areas within the mass (blue arrow).\nFIGURE 6\n(A) Laryngoscopic examination confirmed the pyogranulomatous mass within the laryngeal lumen (arrow). (B) Close image of the mass within the\nlaryngeal lumen (arrow).\n\nAL-Sobayil et al.\b\n10.3389/fvets.2026.1792929\nFrontiers in Veterinary Science\n08\nfrontiersin.org\ncamel with an osteolipoma had a history of partial obstruction of\nthe oral cavity due to a large mass that interfered with normal\nmastication and caused difficulty in swallowing. These observa­\ntions were consistent with those described in earlier reports\n(35–39). The camels with entrapped dulla, due to tonsillitis and\nsialadenitis, had dysphagia, neck stiffness, and mild respiratory\ndistress. These clinical findings are similar to the findings reported\nby Sadan and El-Shafaey (1). The camel affected with pyogranu­\nlomatous laryngitis had a partial obstruction of the laryngeal\nlumen, which impaired normal respiration and resulted in neck\nand head extension, stiffness, and noticeable dyspnea. The affected\ncamel showed signs of restlessness and a diminished appetite\n(33, 34).\nThe hematological examination of the camel with osteolipoma\nrevealed anemia, which could be due to the bleeding accompanying\nthe osteolipoma and recurrent traumatization. Elevated potassium\nlevels, along with decreased levels of sodium, albumin, and total pro­\ntein, indicated anorexia, dehydration, and inflammatory responses\nassociated with the osteolipoma. The increase in glucose level could\nbe related to the mobilization of fat stores as a glucose source, particu­\nlarly under fasting conditions or prolonged inflammation in camels\n(5, 42, 43). In a camel affected by tonsillitis, the hematobiochemical\nchanges are consistent with systemic inflammation and stress. The\nMCHC was elevated, reflecting altered erythrocyte hemoglobin con­\ntent compared with established reference ranges in healthy camels (5,\n42–44). The elevated WBC count with marked neutrophilia and con­\ncurrent reductions in lymphocyte and monocyte percentages indi­\ncated a pronounced inflammatory leukogram typical of the infectious\nconditions in camels. RDW was also elevated, suggesting increased\nvariability in erythrocyte size. Furthermore, the glucose level was\nelevated due to stress-related hyperglycemia, which is commonly\n\nRDW was also elevated, suggesting increased\nvariability in erythrocyte size.Furthermore, the glucose level was\nelevated due to stress-related hyperglycemia, which is commonly\nobserved during acute disease in camels. These alterations are aligned\nwith the pathological inflammatory findings (5, 42, 43).\nIn camels with sialadenitis, the hematobiochemical alterations are\nconsistent with systemic inflammation and stress, including mild\nanemia, macrocytosis and anisocytosis, leukocytosis, neutrophilia,\neosinophilia, lymphocytopenia, and monocytopenia, as they are com­\nmonly reported in inflammatory conditions in camels (5, 42, 43).\nFIGURE 7\n(A) The resected laryngeal pyogranulomatous mass. Please note that the large oval mass had a firm consistency, measured 20 cm × 6 cm × 4.4 cm in\ndiameter, and was firmly adherent to the laryngeal tissue. (B) The mass is composed of disseminated caseated abscesses that are circumscribed with a\ntan whitish-gray layer of connective tissue.\nTABLE 1  Clinical findings of oropharyngeal and laryngeal disorders in four camels.\nDisorder\nAge/sex\nMain clinical\nsigns\nLocal\nfindings\nOsteolipoma with\nosseous metaplasia\n(Epulis)\n1-year-old\nfemale\nPartial oral\nobstruction,\ndysphagia, reduced\nappetite,\nrestlessness\nLarge oblong\nproliferative\nmass on the\nlower right\ngingiva\nTonsillitis\n(Entrapped dulla)\n8-year-old\nmale\nDysphagia, neck\nstiffness, mild\nrespiratory distress\nPin-point dark\nbrown raised\nfoci on the soft\npalate mucosa,\nimpacted with\nspiny food\nmaterial\nSialadenitis\n(Entrapped dulla)\n10-year-old\nmale\nDysphagia, neck\nstiffness,\nsubmandibular\nswelling, dry\nmouth\nMultiple\nabscesses in the\nsoft palate\ncontaining\ngreenish-yellow\npus; ulcerated\nmucosa\nMycotic\ngranulomatous\nlaryngitis\n7-year-old\nfemale\nDyspnea, head and\nneck extension,\nreduced appetite,\nrestlessness\nLarge\nobstructive\nlaryngeal mass\n\nAL-Sobayil et al.\b\n10.3389/fvets.2026.1792929\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nThe camel affected by mycotic pyogranulomatous laryngitis dem­\nonstrated a reduction in red blood cell count and hematocrit values,\nbut an increase in eosinophil percentage, MCH, and MCHC. Increased\nlevels of potassium, calcium, and glucose and decreased levels of CK,\nGGT, sodium, and albumin were also reported. These alterations may\nreflect the reduced feed intake, respiratory compromise, and the pro­\nlonged inflammatory nature of the condition. Similar findings were\nreported in chronic granulomatous and mycotic disorders in camels\nand other large ruminants (6, 45–47).\nThe radiographic and ultrasonographic findings in the present study\nprovide valuable insights into the diagnostic characteristics of soft-tissue\nmasses affecting the oropharyngeal and laryngeal regions of camels. In\nosteolipoma, the identification of a well-defined soft-tissue mass con­\ntaining discrete radiopaque foci without continuity with the mandibular\nbone supports its origin from gingival soft tissues rather than the man­\ndibular framework. Similar radiographic features have been described\nin osteolipomatous and mineralizing soft-tissue lesions in large animals\n(6, 45). In pyogranulomatous mycotic laryngitis, the radiographs\nrevealed an irregular intraluminal soft-tissue mass outlined by the laryn­\ngeal air column, a negative contrast effect commonly reported in\nobstructive laryngeal lesions (6, 48). Ultrasonography demonstrated a\nvascular echogenic mass with mixed anechoic areas, which is consistent\nwith chronic granulomatous inflammatory lesions in ruminants and\ncamelids (45, 48). Laryngoscopic examination provided a definitive con­\nfirmation of the intraluminal obstruction and is considered a critical\ndiagnostic modality for upper airway diseases in large animals (6). The\nsuccessful surgical excision of these obstructive masses highlighted the\nimportance of integrating multimodal imaging with endoscopic evalu­\nation to achieve accurate diagnosis and to guide effective management,\n\nThe\nsuccessful surgical excision of these obstructive masses highlighted the\nimportance of integrating multimodal imaging with endoscopic evalu­\nation to achieve accurate diagnosis and to guide effective management,\nas recommended in earlier camelid and ruminant clinical reports (48).\nThe gum of camels is histologically composed of a covering\nmucosa, loose superficial, and dense deep connective tissue layers. Oral\ndisorders in camels at Maiduguri Abattoir, Borno State, Nigeria were\nreported in 313 heads of camels, comprising 114 (41.67%) males and\n199 (58.33%) females. The prevalence rate of the disorders observed\ninclude dental tartar and calculus (5.42%), inward rotation of incisors\n(2.87%), fractured teeth (7.66%), maleruption (0.31%), oligodontia\n(0.31%), gingivitis (4.15%), ulcerated cheek (0.63%), and presence of\nforeign body (0.31%) in the mouth. About 22.61% of the total heads\nhad one disorder or another; 7.98 and 14.06% of the disorders were\namong male and female heads, respectively (49). Three cases of tooth\nroot abscesses in camels (Old World Camelids) were reported (50).\nFour camels had maxillary masses that appeared as multicystic and\nexpanded the maxillary bone. The tumors were diagnosed by histo­\npathologic examination as conventional ameloblastoma, two cases as\nintraosseous squamous cell carcinoma, and central odontogenic\nFIGURE 8\nEpulis of camel revealed osteolipoma composed of: (A) Large areas of mature fat cells supported by fibrous connective tissue septa (H&E,\nBar = 400 μm). (B) Focal areas of woven bone surrounded by fusiform mesenchymal cells that circumscribed the lipomatous tissue (H&E,\nBar = 200 μm). (C,D) Focal areas of osteoid bony tissue (osseous metaplasia) were observed as ectopic osseous tissue containing myeloid tissue in the\ndense layer of the gum (H&E, C: Bar = 400 μm, D: Bar = 200 μm).\n\nAL-Sobayil et al.\b\n10.3389/fvets.2026.1792929\nFrontiers in Veterinary Science\n10\nfrontiersin.org\nfibroma with ossification (7). A fibrous epulis (1.2 cm in diameter) has\nbeen recorded in a one-humped camel on the labial part of the upper\ngum (51). Whorls and bundles of fibroblasts and fibrocytes were the\nmain components of the epulis, with two keratin foci centrally located.\nOdontogenic tumors have been recorded in camels, characterized by\nodontogenic epithelium and mesenchyme, and they originate from sites\nof teeth in the mandibular and maxillary bone (7). The present case was\nneither fibromatous epulis nor an odontogenic tumor, as the former\ncontained collagenous fibers and fibroblastic proliferation, whereas the\nlatter contained odontogenic epithelium, which was absent in osteoli­\npoma and osseous metaplasia. Osteolipoma has been observed very\nrarely in humans (less than 1% of mesenchymal lipomas). Only 20 cases\nof oral osteolipoma have been reported in humans based on 69 searches\nin the PubMed database (52). To the authors’ knowledge, oral or gingi­\nval osteolipoma has never been recorded in veterinary medicine. Oral\nfibrolipoma has been recorded in 112 dogs in a multi-institutional ret­\nrospective compilation of cases submitted to diagnostic pathology ser­\nvices, which are characterized histologically by a typical lipoma with\nvariable amounts of paucicellular and collagenous fibrous components\nbut devoid of osseous tissue (53). Osteolipoma, characterized by mature\nyellow adipocytes and residual bony trabeculae, has been recorded in\nthe ribs of a sheep (37) and a Leonberger (54) named intraosseous\nlipoma (37). A lipoma with marked calcified osseous tissue and some­\ntimes fat cell necrosis is diagnosed as osteolipoma, osseous lipoma,\nossifying lipoma, or lipoma with osseous metaplasia (55, 56). An intra­\ncranial ossifying lipoma was recorded in a juvenile pig (57) and a labo­\nratory rat (58), characterized by lobules of adipose tissue interrupted by\nfibrous connective tissue septa, an outer layer of well-differentiated\ncortical bone, and an inner core of adipose tissue and hematopoietic\n\ncells (57). Two cases of osteolipoma in canine skin were reported with\nfindings similar to those of the present study (56). Osseous metaplasia\nis the formation of bone in abnormal locations in which fibroblast-like\ncells differentiate into osteoblasts and osteoid formation. Abnormal\nlocations of bone formation in dogs commonly include mammary\ntumors and salivary mucocele (59), as well as other tissues such as eyes,\nskin, lateral abdominal wall, and the external auditory canal (60). In\ncamels, osseous and osteo-lipomatous metaplasia were recorded in the\nliver (61, 62) and metastatic gastric adenocarcinoma (63). Apart from\nosteolipoma in the present case, focal areas of osseous metaplasia of the\ndense fibrous layer of the gum were observed. This is the first record of\nosseous metaplasia in gingivitis in camel and veterinary medicine.\nA few papers studied the anatomy and histology of camel tonsils. A\ncamel has six types of tonsils. They are well-developed and visible, par­\nticularly the palatine one. The lingual, palatine, velar, and paraepiglottic\ntonsils are arranged into closely assembled lymphoid follicles and show\na multitude of crypt openings into the oropharyngeal tube. However, the\nnasopharyngeal tonsils (pharyngeal and tubal) include loosely con­\nnected follicles that extend into the overlaying epithelium (14, 64). There\nFIGURE 9\n(A) The overlying epithelium of the epulis was necrotic and ulcerated, associated with severe neutrophilic infiltration in the superficial layer of the gum\n(H&E, bar = 200 μm). (B) Gingival epithelial hyperplasia was associated with severe inflammatory cell infiltration (H&E, bar = 200 μm). (C) Foodstuffs\ninvaded the loose connective tissue layer, leaving a foreign body suppurative reaction (H&E, bar = 100 μm).\n\nAL-Sobayil et al.\b\n10.3389/fvets.2026.1792929\nFrontiers in Veterinary Science\n11\nfrontiersin.org\nare no previous reports on tonsillitis in camels, but it is common in dogs\nand cats (65). Suppurative nasopharyngeal tonsillitis and palatine tonsil­\nlar cryptitis were recorded in calves infected with Mycoplasma bovis, and\nthe histopathological picture revealed degenerative and necrotic changes\nof the cryptic epithelium and lymphoid follicles. The lumen of these ton­\nsillar crypts was occluded by necrotic cellular debris, resulting in micro­\nabscesses (66). The present case of camel also presented with suppurative\ntonsillitis in the velar tonsils. The foodstuff penetrating the soft palate\nwas observed grossly and histopathologically, which might incite inflam­\nmation. Foreign body giant cells, bacterial colonies, and thorns were\nobserved in the soft palate tissue. Actinomyces -associated plant thorns\nare implicated in the deep stomatitis in animals (67). The major salivary\nglands of camels are the parotids, mandibular, and sublingual salivary\nglands (68); however, the minor salivary glands are the labial, buccal,\npalatine, von Ebner’s glands, and lingual salivary glands (18). The pala­\ntine salivary glands were in the caudal part of the hard palate and in the\nentire length of the soft palate. The palatine glands were most numerous\nat the apex of the soft palate (18). Sialadenitis in camels has not been\nrecorded except as part of rabies infection. Moreover, sialadenitis might\nbe a sequel to sialolithiasis. Sialolithiasis has been recorded in camels and\nother farm animals (17, 69, 70). The present case of sialadenitis did not\nsuffer from sialolithiasis. The sialadenitis in the present case might be\ndue to a bacterial infection of the soft palate, leading to entrapment\nduring expulsion. Several abscesses, hemorrhage, and ulceration of the\nmucosa of the soft palate were detected, suggesting that palatine sialad­\nenitis is a primary condition concurrent with inflammation of the soft\npalate (dulla). Inflammation of minor salivary glands in dogs has been\nreported and may be of non-specific cause (71).\n\nInflammation of minor salivary glands in dogs has been\nreported and may be of non-specific cause (71).\nLaryngitis is not as common a primary lesion but usually an exten­\nsion from the upper or lower respiratory tract, except in cats (72). A\nsearch of the PubMed database revealed records of diphtheroid necrotic\ninflammation of the larynx in calves and cattle (73, 74). Necrotizing sto­\nmatitis, glossitis, pharyngitis, laryngitis, ruminitis, and liver abscesses\nand thrombosis of the vena cava, as well as interdigital foot rot in cattle,\nare commonly associated with Fusobacterium necrophorum subsp. nec­\nrophorum (75). Laryngitis in camelids was recorded only once, with\nlaryngeal abscessation (not involving the cartilage) in a 10-day-old\nalpaca, and Mannheimia hemolytica was isolated from the case (76). In\nthe present case, mycotic pyogranulomatous laryngitis with morphologi­\ncal features of fungal hyphae, in the context of aspergillosis, was diag­\nnosed. Mycotic laryngitis is common in immunocompromised human\npatients, with a higher prevalence of Aspergillus infection (77). The\nFIGURE 10\n(A) The tonsillar nodules were composed of primary and secondary lymphoid follicles separated by parafollicular lymphoid tissue and had crypts that\nwere lined by a non-keratinized stratified squamous epithelium. Some tonsillar follicles were hyperplastic and revealed several germinal centers (H&E,\nBar = 400 μm). (B) Tonsil nodules revealed suppurative necrosis of the lymphoid follicles, leaving thickened interfollicular dense connective tissue (H&E,\nBar = 200 μm). (C) The epithelial ridges of the overlying mucosa of the soft palate were hyperplastic, and the submucosa was diffusely infiltrated with\nneutrophils (H&E, Bar = 400 μm). (D) The follicles of the tonsils were also severely necrosed and devoid of lymphocytes associated with necrosis of\ncrypts, leaving an eosinophilic coagulum without structural outlines (H&E, Bar = 400 μm). (E) Thorns were observed in the content of massive necrosis\n\n(E) Thorns were observed in the content of massive necrosis\nand associated with giant cells (inset) and colonies of bacterial infection (H&E, Bar = 400 μm, inset; H&E, Bar = 100 μm).\n\nAL-Sobayil et al.\b\n10.3389/fvets.2026.1792929\nFrontiers in Veterinary Science\n12\nfrontiersin.org\ncamel in the present case was in good body condition and had no signs\nof starvation or immunosuppression. Aspergillus spp. are saprophytic\nfilamentous fungi commonly found in soil, decaying vegetation, and on\nseeds and grains, with occasional potential to infect living animal hosts\n(78). Fungal spores could gain entry into the deep tissue of the tongue,\npharyngeal or laryngeal mucosa, producing deep pyogranulomatous\nlesions such as wooden tongue disease (79). So, the fungus might enter\nthe deep layer of submucosa of the larynx through penetration by food­\nstuffs that proceed to the mycotic pyogranulomas. Bronchopulmonary\nand disseminated infections are major forms of Aspergillosis in dogs and\ncats (80). Aspergillus species causes mycotic pneumonia, gastroenteritis,\nmastitis, placentitis, and abortions in ruminants and guttural pouch\ninfections, keratomycosis, and pneumonia in horses (81). There are no\nlesions in the present case except mycotic laryngitis.\nThe present work has several limitations. The small number of cases\nlimits generalizability and precludes statistical analysis or determination\nof prevalence and risk factors. Furthermore, part of the follow-up evalu­\nation depended on the owner’s reports, introducing potential subjective\nbias. Practical challenges included the need for specialized diagnostic\ntools (radiography, ultrasonography, and laryngoscopy) and skilled per­\nsonnel for safe handling and surgical intervention in camels, which may\nnot be readily available in field settings. Future studies should include\nlarger, multicenter investigations to better define epidemiology and risk\nfactors. Incorporation of molecular diagnostics and fungal culture is\nrecommended to confirm etiological agents and guide targeted therapy.\nConclusion\nThis study documented four distinct upper respiratory and oro­\npharyngeal conditions in camels: epulis (osteolipoma with osseous\nmetaplasia), tonsillitis, and sialadenitis associated with entrapped dulla,\n\nConclusion\nThis study documented four distinct upper respiratory and oro­\npharyngeal conditions in camels: epulis (osteolipoma with osseous\nmetaplasia), tonsillitis, and sialadenitis associated with entrapped dulla,\nand mycotic granulomatous laryngitis. These disorders were character­\nized clinically by varying degrees of oral or laryngeal obstruction, dys­\nphagia, respiratory distress, neck stiffness, and reduced appetite.\nHematological and biochemical analyses consistently revealed anemia,\ninflammatory leukogram changes (particularly neutrophilia and/or\neosinophilia), and metabolic alterations, including electrolyte imbal­\nances, reflecting systemic inflammatory and stress responses.\nDiagnostic imaging (radiography and ultrasonography), together with\nlaryngoscopic examination, proved essential for identifying the loca­\ntion, extent, and nature of the masses. Histopathological examination\nconfirmed the definitive diagnoses, including osteolipoma with osseous\nmetaplasia and fungal pyogranulomatous inflammation consistent with\nAspergillus spp. infection. Surgical excision of the affected dulla or\nabnormal masses resulted in a complete functional recovery in all cases\nwithin 6 months of follow-up. Favorable outcomes were strongly asso­\nciated with early diagnosis, prompt surgical intervention, and appropri­\nate postoperative care. Therefore, a comprehensive diagnostic approach\ncombined with timely-applied surgical management represents an\neffective strategy for treating oropharyngeal and laryngeal disorders\nin camels.\nFIGURE 11\nSialadenitis of minor salivary glands of soft palate revealed (A) hemorrhages and multiple abscesses in the submucosa, and the overlying mucosa were\nulcerated and healed with granulation tissue (H&E, Bar = 400 μm). (B) Destruction, necrosis, and fibrosis of salivary glands (H&E, Bar = 200 μm), and\n(C) periductal fibrosis (H&E, Bar = 200 μm) were detected in the tissue surrounding the abscesses. (D) Ductal obstruction with exudate, organized\n\n(B) Destruction, necrosis, and fibrosis of salivary glands (H&E, Bar = 200 μm), and\n(C) periductal fibrosis (H&E, Bar = 200 μm) were detected in the tissue surrounding the abscesses.(D) Ductal obstruction with exudate, organized\nperiductal fibrosis, and the surrounding glandular tissues revealed liquefactive necrosis were noticed (H&E, Bar = 200 μm).\n\nAL-Sobayil et al.\b\n10.3389/fvets.2026.1792929\nFrontiers in Veterinary Science\n13\nfrontiersin.org\nData availability statement\nThe datasets presented in this study can be found in online \nrepositories. The names of the repository/repositories and acces­\nsion number(s) can be found in the article/supplementary \nmaterial.\nEthics statement\nAll experimental procedures were conducted in accordance \nwith the Laboratory Animal Control Guidelines of Qassim \nUniversity, which adhere to the principles outlined in the Guide \nfor the Care and Use of Laboratory Animals of the U.S. National \nFIGURE 12\n(A) Mycotic granulomatous laryngitis with a necrotic eosinophilic center containing cellular debris rich in eosinophils and fungal hyphae, surrounded by \nmacrophages, epithelioid cells, giant cells, and rich eosinophilic infiltration (H&E, Bar = 400 μm). (B) The necrotic centers were surrounded by a fibrous \nlayer rich with eosinophilic infiltrate and giant cells (H&E, Bar = 100 μm). (C) The tissues separated the pyogranulomatous lesions, which were rich in \nneutrophils, eosinophils, macrophages, and giant-cell infiltration (H&E, Bar = 100 μm). (D) Hyphae had parallel cell walls, distinct septa, and a \ndichotomous branching pattern (Aspergillus hyphae) (H&E, Bar = 200 μm).\nFIGURE 13\n(A) Fungal elements revealed hyphae that were characterized by parallel cell walls, distinct septa, and a dichotomous branching pattern (Aspergillus \nspp.) (PAS, bar = 400 μm). (B) Fungal hyphae were present in granulomatous lesions and invaded adjacent structures (PAS, bar = 200 μm).\n\nAL-Sobayil et al.\b\n10.3389/fvets.2026.1792929\nFrontiers in Veterinary Science\n14\nfrontiersin.org\nInstitutes of Health (NIH Publication No. 86–23, revised 1996).\nThe study protocol (Approval No. 370) was reviewed and\napproved by the Committee for Animal Welfare and Ethics,\nQassim University, Saudi Arabia. The studies were conducted in\naccordance with the local legislation and institutional require­\nments. Written informed consent was obtained from the owners\nfor the participation of their animals in this study.\nAuthor contributions\nFA-S: Methodology, Validation, Investigation, Conceptualization,\nVisualization, Writing  – original draft. MS: Investigation,\nVisualization,\nValidation,\nConceptualization,\nSupervision,\nMethodology, Writing  – original draft. AA: Methodology,\nVisualization, Validation, Writing – original draft. MH: Visualization,\nMethodology, Conceptualization, Validation, Supervision, Writing –\noriginal draft.\nFunding\nThe author(s) declared that financial support was received for this\nwork and/or its publication. The researchers would like to thank the\nDeanship of Graduate Studies and Scientific Research at Qassim\nUniversity for financial support (QU-APC 2026).\nConflict of interest\nThe author(s) declared that this work was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declared that Generative AI was not used in the\ncreation of this manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organiza­\ntions, or those of the publisher, the editors and the reviewers. Any\nproduct that may be evaluated in this article, or claim that may be\nmade by its manufacturer, is not guaranteed or endorsed by the\n\nAny\nproduct that may be evaluated in this article, or claim that may be\nmade by its manufacturer, is not guaranteed or endorsed by the\npublisher.\nReferences\n1.\tSadan M, El-Shafaey ES. Diagnosis and surgical repair of soft palate (dulla) injuries in\ncamel (Camelus dromedarius). J Camel Health. (2020) 1:38–42. Available at: https://www.\nresearchgate.net/publication/339443065_Diagnosis_and_surgical_repair_of_soft_palate_\nDulla_injuries_in_camel_Camelus_dromedaries#fullTextFileContent\n2.\tBornstein S. \"Evolution, distribution, and economic importance of the camels\". 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+{"text": "【兽医文献】\n标题: article_738_Does a metoclopramide CRI reduce the risk of regurgitation a\n\n__\nThis Open Access work is\ndistributed under a Creative\nCommons Attributions 4.0\nInternational License. You\nare free to share (copy and\nredistribute the material in\nany medium or format) or\nadapt (remix, transform,\nand build upon the material\nfor any purpose, even\ncommercially), provided you\nfully cite this original work.\n__\nVeterinary Evidence is an\nonline, open access, peer-\nreviewed journal owned\nand published by RCVS\nKnowledge.\nVeterinary Evidence (2026) Vol 11 Iss 2 | Page 1 of 6\nDOI: https://doi.org/10.18849/ve.v11i2.737\nKNOWLEDGE SUMMARY\nKeywords: EXTERNAL FIXATION; FELINE; FRACTURE; INTERNAL FIXATION; ORTHOPAEDIC SURGERY;\nPOSTOPERATIVE COMPLICATIONS\nComparing postoperative complications of\ninternal versus external fixation for closed\ndiaphyseal tibial fractures in cats\nStephen Keith John, BVMSci MRCVS1*\nJake Chitty, BVM&S DipECVSMRCVS1\n1 East Neuk Veterinary Clinic, Netherton Estate, Station Road, St. Monans, United Kingdom, KY10 2DW\n* Corresponding author email: sj00501@outlook.com\nCategory of research\nNumber and type of study\ndesigns reviewed\nStrength of evidence\nOutcomes reported\nConclusion\nTreatment.\nThere were no publications that directly answered the PICO\nquestion.\nZero.\nBoth internal and external fixation are published techniques\ndeployed in tibial fracture fixation in cats, however no study\nhas directly compared the postoperative complications in these\ngroups for closed diaphyseal tibial fractures.\nGiven the lack of evidence to answer the PICO question, the veteri­\nnarian should consider their choice of stabilisation technique on the\nmethods available and their personal experience. Both internal and\nexternal skeletal fixation (ESF) are published methods of feline\ntibial fracture fixation. A related study demonstrated that ESF has\na high risk of complication for feline tibial fracture repair but given\nthe absence of evidence for exclusively closed fracture types, this\nconclusion cannot be drawn for the PICO.\nSubmitted: 24 April 2025; published: 28 April 2026\nPICO question\nIn cats with closed diaphyseal tibial fractures, does internal fixation, when compared to external\nfixation, result in a lower postoperative complication rate?\n\nSubmitted: 24 April 2025; published: 28 April 2026\nPICO question\nIn cats with closed diaphyseal tibial fractures, does internal fixation, when compared to external\nfixation, result in a lower postoperative complication rate?\nClinical bottom line\nHow to apply this evidence in practice\nThe application of evidence into practice should take into account multiple factors, not limited\nto: individual clinical expertise, patient’s circumstances and owners’ values, country, location\nor clinic where you work, the individual case in front of you, the availability of therapies and\nresources.\nKnowledge Summaries are a resource to help reinforce or inform decision-making. They do not\noverride the responsibility or judgement of the practitioner to do what is best for the animal in\ntheir care.\nClinical scenario\nA 4-year-old domestic shorthair cat presents to your clinic with an acute non-weight bearing\nlameness of the right pelvic limb after falling from height. On clinical examination, you localise\n\nthe pain and feel crepitus and instability at the level of the mid tibia. Radiographs reveal a closed\nmid-diaphyseal tibial fracture. The contralateral limb and pelvis are unaffected. Your clinic has\nthe capability to perform external fixation and internal fixation for the repair, but you wish to\ndeploy a technique that results in the lowest risk of developing postoperative complications.\nThe evidence\nThe literature search found no evidence that addressed the PICO question from a literature\nsearch.\nVeterinary Evidence (2026) Vol 11 Iss 2 | Page 2 of 6\nAppraisal, application and reflection\nTibial fractures are relatively common in cats, accounting for approximately 10% of feline\nfractures (Hill, 1977), with 73% of these involving the diaphyseal region (Brunnberg et al,\n2003). Tibial fracture fixation is understood to encounter higher rates of complications than\nfractures of other bones; non-union, delayed union, implant failure, and osteomyelitis being\ncomplications outlined in the literature (El-shafey et al., 2022; Brunnberg, 2003). Nolte et\nal. (2005) suggested that the over-presentation of non-union seen in tibial fractures could\nbe associated with lack of soft tissue covering of the area. Other intrinsic factors such as a\nhigh cortical to cancellous bone ratio, lack of muscular attachments, and the biomechanical\nforces that act upon the tibia are other cited reasons for the higher rate of non-union seen\n(Glyde & Arentt, 2006). Extrinsic risk factors reported include age, weight, and degree of\ncomminution (Nolte et al, 2005).\nThere are many techniques for open reduction and internal fixation (ORIF) including interlocking\nnail, bone plating, intramedullary pinning, and clamp-rod (DeCamp, 2006). These techniques can be\napplied in differing approaches including full open, Open But Do Not Touch (OBDNT), or minimally\ninvasive plate osteosynthesis (MIPO) technique (Seaman & Simpson, 2004; Vannini, 2010; Schmierer\n& Pozzi, 2017; Butterworth, 2016; Duhautois, 2003). Advantages of MIPO included the minimal dis­\n\nAdvantages of MIPO included the minimal dis­\nruption to the biological environment, which may facilitate faster bone healing (Istim & Arican, 2022).\nInternal fracture fixation is considered by veterinary surgeons as it facilitates accurate reduction\nand increased stability of fracture fragments to allow for bone healing (Pozzi et al., 2021). Unlike\nexternal fixation, implant removal for internal fixation is not anticipated and durability of the\nimplants used is often increased, hence metalwork is less likely left in situ after explantation.\nHowever, disadvantages of internal fixation include the possibility of increased tissue damage\nfrom the surgical approach potentially delaying healing, and internal implants serving as a nidus\nfor infection (Johnston et al., 2018).  Therefore, the veterinary surgeon must consider the fracture\npatient assessment score and biological cost when planning fracture fixation methods, balanced\nagainst the mechanical and biological environment of the fracture.\nExternal skeletal fixation (ESF) can be used in multiple different configurations such as uniplanar,\nbiplanar, circular and circular-linear hybrid ESFs (Zurita & Craig, 2022) but generally consist of\nmultiple pins with clamps and a connecting bar. Free form fixators have been described in\nliterature, where epoxy or resin-acrylic bars can provide fixation, often at less expense than\nthe other configurations mentioned previously (Roberts & Meeson, 2022). External fixation\ncan be applied using closed reduction or via an OBDNT approach. External skeletal fixation\nstabilisation for fracture repair uses a biologic approach to osteosynthesis, allowing stability\nof the fractured bone and preservation of the haematoma and surrounding soft tissues (Palmer\net al., 1992). However, the percutaneous nature of ESF means that infection is an increased risk\ncompared to internal fixation (Chitty et al., 2025; Jaeger & Wosar, 2018).  A limitation to ESF is\nthe anatomic reconstruction and fracture gap compression is challenging, if perfect anatomical\n\n, 2025; Jaeger & Wosar, 2018).A limitation to ESF is\nthe anatomic reconstruction and fracture gap compression is challenging, if perfect anatomical\nreconstruction is not achieved a high strain environment can be created. This can be better\naddressed via anatomic reconstruction and rigid fixation, where more modern design plates\nsuch as Locking Compression Plates allow for compression of the fracture gap (Moreno et al.,\n2018; Haaland et al., 2009).\n\nJohn & Chitty | Page 3 of 6\nThere was no evidence that directly addressed the PICO question, although one published\nstudy (Perry & Bruce, 2015) compared complications between internal fixation via ORIF to\nESF in tibial fractures in 57 cats aged between 3 months and 200 months. Perry & Bruce\n(2015) defined complications as ‘any undesirable outcome associated with the surgical\nprocedure’ and further classified major complications where surgical intervention was\nrequired, and minor where the complication could be conservatively managed. Of the total\ncomplication rate of 23/59 (40.4%) in the study, only one of these was in the ORIF group.\nConversely, 50% (22/44) of all ESF fixations encountered at least a minor complication.\nComplications included pin tract infection/pin loosening, delayed union, non-union, pin\nloosening in absence of any infection, osteomyelitis, frame disruption leading to fragment\ndisplacement, and mild distal tibial valgus deformity. All nine major complications were\nencountered in the ESF group; four of these cases required limb amputation, another four\ncases required surgical revision, and one case was euthanised due to extreme lethargy of\nunknown aetiology. When specifically assessing infection, the ORIF group had no reported\npostoperative infections, whereas the ESF group had a postoperative infection rate of 27.2%\n(12/44). This study included 16/59 cats (27%) with open fractures within their evaluation.\nDirect comparison of this study to the PICO should be acknowledged cautiously as open fractures\nwere included which may have influenced the overall postoperative complication rate. This study\nwas a retrospective study with low case numbers which sits low on the hierarchy of evidence\n(Howick et al., 2011).\nA study by Beever et al. (2017) analysed postoperative complications when ESFs were deployed\nfor feline fracture stabilisation. Of the 140 cats included in the study, 24% (34/140) of these\nhad tibial fractures. This study found that only 3% (1/34) of tibial fractures stabilised by ESF\n\n(2017) analysed postoperative complications when ESFs were deployed\nfor feline fracture stabilisation.Of the 140 cats included in the study, 24% (34/140) of these\nhad tibial fractures.This study found that only 3% (1/34) of tibial fractures stabilised by ESF\nproduced fixator-associated complications, superficial pin tract infection being the only reported\npostoperative complication. Limitations of this study include its retrospective nature with a small\nnumber of cases, so it sits low on the hierarchy of evidence.\nConsidering the absence of evidence directly addressing the PICO question, veterinary\nsurgeons should deploy the techniques that they are competent in and are comfortable\nusing from personal experience of fracture stabilisation and repair, with consideration of\nthe patient and owners’ limitations/ability to manage postoperative care.\nA prospective or retrospective study comparing postoperative complications with internal\nversus external fixation for closed diaphyseal tibial fractures in cats is required for the PICO\nto be addressed.\nIn conclusion, there is no evidence that directly compared internal fixation to external fixation\nfor cats with closed tibial fractures. Related literature shows that internal fixation does carry a\nlower complication rate when compared to ESF when all types of fractures are considered, but\nno conclusion can be drawn from the available evidence on whether this would be applicable to\nexclusively closed fracture types.\nSearch strategy\nDatabases searched and dates\ncovered\nCAB Abstracts on OVID interface 1973 to 2025 Week 07\nPubMed Accessed via the NCBI website 1920 to February 2025\nSearch strategy\nPubMed:\n1.\ncat OR feline\n2.\n(tibia OR tibial) AND fracture\n3.\n(fixation OR fixator OR stabilise OR stabilize) AND (internal OR external)\n4.\n4. 1 AND 2 AND 3\nMethodology\n\nSearch strategy (cont.)\nSearch strategy\nPubMed:\n1.\t\ncat OR feline\n2.\t\n(tibia OR tibial) AND fracture\n3.\t\n(fixation OR fixator OR stabilise OR stabilize) AND (internal OR external)\n4.\t\n4. 1 AND 2 AND 3\nDates searches performed\n21 February 2025\nSearch outcome\nDatabase\nNumber of results\nExcluded — not specific to PICO question\nTotal relevant papers\nCAB Abstracts\n160\n160\n0\nPubMed\n71\n71\n0\nTotal relevant papers when duplicates removed\n0\nExclusion / Inclusion criteria\nExclusion\n•\t\nStudies including open fractures\n•\t\nOnly on technique deployed\n•\t\nArticles irrelevant to PICO\n•\t\nBook chapters\n•\t\nCase reports\nInclusion\n•\t\nProspective studies\n•\t\nRetrospective studies\nAuthor contributions\nStephen Keith John: Writing - Original Draft, Writing - Review & Editing. Jake Chitty: Writ­\ning - Review & Editing\nORCiD\nStephen Keith John: https://orcid.org/0009-0003-2581-043X\nJake Chitty: https://orcid.org/0000-0001-7011-006X\nReferences\n1.\t\nBeever, L., Giles, K. & Meeson, R. (2017). Postoperative complications associated with \nexternal skeletal fixators in cats. Journal of Feline Medicine and Surgery. 19(7), 727–736. \nDOI: https://doi.org/10.1177/1098612X17699466\n2.\t\nBrunnberg, L., Forterre, F., Kaiser, S., Burger, M. & Waibl, H. (2003). Tibia and fibular \nfractures in the cat. Kleintierpraxis. 48, 9–23.\n3.\t\nButterworth, S.J. (2016). The tibia and fibula, in: T.J Gemmill & D.N. Clements, ed., \nBSAVA Manual of Canine and Feline Fracture Repair and Management. 2nd ed. Gloucester: \nWiley, 301–318.\n4.\t\nChitty, J.W., Sharp, C.A.T., Low, D., Chapman, T., Singleton, D., Murdoch, A. & Aldridge, P. (2025). \nShort-term complications of internal versus external fixation of closed diaphyseal tibial fractures \nin 95 skeletally immature dogs. Veterinary Surgery. DOI: https://doi.org/10.1111/vsu.14221\n5.\t\nDeCamp, C.E, Flo, G.L & Piermattei, D.L. (2006). Fractures of the Tibia and Fibula. in: \nHandbook of Small Animal Orthopedics and Fracture repair. 4th ed. St Louis: Saunders \nElsevier, 633–660.\nVeterinary Evidence (2026) Vol 11 Iss 2 | Page 4 of 6\n\nJohn & Chitty | Page 5 of 6\n6.\nDuhautois, B. (2003). Use of Veterinary Interlocking Nails for Diaphyseal Fractures in\nDogs and Cats: 121 cases. Veterinary Surgery. 32(1), 8–20. DOI: https://doi.org/10.1053/\njvet.2003.50008\n7.\nEl-shafey, S., El-Mezyen, A., Behery, A. & Abd El Raouf, M. (2022). Tibial and Fibular\nFractures in Dogs and Cats: Retrospective Study. Zagazig Veterinary Journal. 50(1), 52–61.\n8.\nGlyde, M. & Arentt, R. (2006). Tibial fractures in the dog and cat: Options for management. Irish\nVeterinary Journal [online]. 59(5), 290–295. Available from: https://researchportal.murdoch.edu.\nau/esploro/outputs/journalArticle/Tibial-fractures-in-the-dog-and/991005544180707891\n[Accessed 15/07/2025].\n9.\nHaaland, P.J., Sjöström, L., Devor, M. & Haug, A. (2009). Appendicular fracture repair in dogs\nusing the locking compression plate system: 47 cases. Veterinary and Comparative Orthopaedics\nand Traumatology. 22(04), 309–315. DOI: https://doi.org/3415/vcot08-05-0044\n10.\t Hill, F.W.G. (1977). A survey of bone fractures in the cat. Journal of Small Animal Practice.\n18(7), 457–463. DOI: https://doi.org/10.1111/j.1748-5827.1977.tb05912.x\n11.\t Howick, J., Chalmers, I., Glasziou, P., Greenhalgh, T., Heneghan, C., Liberati, A., Moschetti,\nI., Phillips, B. & Thornton, H. (2011). The Oxford 2011 Levels of Evidence. Oxford Centre for\nEvidence-Based Medicine. Available at: http://www.cebm.net/index.aspx?o=5653 [Accessed\n14/04/2026].\n12.\t Istim, C. & Arican, M. (2022). Clinical and radiographic evaluation of minimally invasive\nplate osteosynthesis (MIPO) in dogs with tibial fractures. Veterinární medicína. 67(6), 316–\n322. DOI: https://doi.org/17221/42/2021-vetmed\n13.\t Jaeger, G. & Wosar, M. (2018). External Skeletal Fixation, in S.A., Johnston & K.M., Tobias,\nVeterinary Surgery: Small Animal, 2nd St Louis: Saunders, 691–702.\n14.\t Johnston, S. von Pfeil, D.J.F., Déjardin, L.M., Roe, S.C. & Weh, J.M. (2018). Internal Fracture\nFixation, in S.A., Johnston & K.M., Tobias, Veterinary Surgery: Small Animal, 2nd St Louis:\nSaunders, 654–687.\n\nM., Tobias,\nVeterinary Surgery: Small Animal, 2nd St Louis: Saunders, 691–702.14.Johnston, S.von Pfeil, D.J.F., Déjardin, L.M., Roe, S.C.& Weh, J.M.(2018).Internal Fracture\nFixation, in S.A., Johnston & K.M., Tobias, Veterinary Surgery: Small Animal, 2nd St Louis:\nSaunders, 654–687.\n15.\t Moreno, M. Zambrano, S., Déjardin, L.M. & Saunders, W.M. (2018). Bone Biomechanics and\nFracture Biology, in A., Johnston & K.M., Tobias, Veterinary Surgery: Small Animal, 2nd ed. St\nLouis: Saunders, 641–643.\n16.\t Nolte, D.M., Fusco, J.V. & Peterson, M.E. (2005). Incidence of and predisposing factors\nfor nonunion of fractures involving the appendicular skeleton in cats: 18 cases (1998–\n2002). Journal of the American Veterinary Medical Association. 226(1), 77–82. DOI: https://doi.\norg/10.2460/javma.2005.226.77\n17.\t Palmer, R.H., Hulse, D.A., Hyman, WA. & Palmer, D.R. (1992). Principles of Bone Healing and\nBiomechanics of External Skeletal Fixation. Veterinary Clinics of North America: Small Animal\nPractice. 22(1), 45–68. DOI:  https://doi.org/10.1016/S0195-5616(92)50004-3\n18.\t Perry, K.L. & Bruce, M. (2015). Impact of fixation method on postoperative complication\nrates following surgical stabilization of diaphyseal tibial fractures in cats. Veterinary and\nComparative Orthopaedics and Traumatology. 28(02), 109–115. DOI: https://doi.org/10.3415/\nvcot-14-08-0120\n19.\t Pozzi, A. Lewis, D.D. & Scheuermann, L.M. (2021). A review of minimally invasive\nfracture stabilization in dogs and cats. Veterinary Surgery. 50(S1), O5–O16. DOI:\nhttps://doi.org/10.1111/vsu.13685\n20.\t Roberts, V.J. & Meeson, R.L. (2022). Feline Femoral Fracture Fixation: What are\nthe options? Journal of Feline Medicine and Surgery. 24(5), 442–463. DOI: https://doi.\norg/1177/1098612x221090391\n21.\t Schmierer, P.A. & Pozzi, A. (2017). Guidelines for surgical approaches for minimally invasive\nplate osteosynthesis in cats. Veterinary and Comparative Orthopaedics and Traumatology. 30(04),\n272–278. DOI: https://doi.org/10.3415/vcot-16-07-0105\n22.\t Seaman, J.A. & Simpson, A.M. (2004). Tibial fractures. Clinical Techniques in Small Animal\n\nVeterinary and Comparative Orthopaedics and Traumatology.30(04),\n272–278.DOI: https://doi.org/10.3415/vcot-16-07-0105\n22.Seaman, J.A.& Simpson, A.M.(2004).Tibial fractures.Clinical Techniques in Small Animal\nPractice. 19(3),151–167. DOI: https://doi.org/10.1053/j.ctsap.2004.09.007\n23.\t Vannini, R. (2010). Fracture planning in cats, SCIVAC Congress. 15–18 September. Bologna:\nWVOC, 757–759.\n24.\t Zurita, M. & Craig, A. (2022). Feline Diaphyseal Fractures: Management and treat­\nment options. Journal of Feline Medicine and Surgery. 24(7), 662–674. DOI: https://doi.\norg/10.1177/1098612X221106354\n\nContribute to the evidence\nThere are two main ways you can contribute to the evidence base while also enhancing your \nCPD:\n•\t\nTell us your information need \n•\t\nWrite a Knowledge Summary\nEither way, you will be helping to add to the evidence base, and strengthen the decisions that \nveterinary professionals around the world make to give animals the best possible care. Learn \nmore here: https://veterinaryevidence.org/index.php/ve/author-hub\nLicence\nCopyright (c) 2026 Stephen Keith John & Jake Chitty\nIntellectual property rights\nAuthors of Knowledge Summaries submitted to RCVS Knowledge for publication will retain \ncopyright in their work, and will be required to grant to RCVS Knowledge a non-exclusive \nlicence to publish including but not limited to the right to publish, re-publish, transmit, sell, \ndistribute and otherwise use the materials in all languages and all media throughout the \nworld, and to licence or permit others to do so.\nDisclaimer\nKnowledge Summaries are a peer-reviewed article type which aims to answer a clinical \nquestion based on the best available current evidence. It does not override the responsibility of \nthe practitioner. Informed decisions should be made by considering such factors as individual \nclinical expertise and judgement along with patient’s circumstances and owners’ values. \nKnowledge Summaries are a resource to help inform and any opinions expressed within the \nKnowledge Summaries are the author’s own and do not necessarily reflect the view of the \nRCVS Knowledge. Authors are responsible for the accuracy of the content. While the Editor \nand Publisher believe that all content herein are in accord with current recommendations and \npractice at the time of publication, they accept no legal responsibility for any errors or omissions, \nand make no warranty, express or implied, with respect to material contained within. For further \ninformation please refer to our Terms of Use.\nVeterinary Evidence (2026) Vol 11 Iss 2 | Page 6 of 6\n\n---"}
+{"text": "【兽医文献】\n标题: article_754_Evidence-based veterinary medicine and contextualised care\n\n__\nThis Open Access work is\ndistributed under a Creative\nCommons Attributions 4.0\nInternational License. You\nare free to share (copy and\nredistribute the material in\nany medium or format) or\nadapt (remix, transform,\nand build upon the material\nfor any purpose, even\ncommercially), provided you\nfully cite this original work.\n__\nVeterinary Evidence is an\nonline, open access, peer-\nreviewed journal owned\nand published by RCVS\nKnowledge.\nVeterinary Evidence (2026) Vol 11 Iss 2 | Page 1 of 19\nDOI: https://doi.org/10.18849/ve.v11i2.752\nARTICLE\nKeywords: BARRIERS; CONTEXTUALISED CARE; PET OWNERSHIP; SMALL ANIMAL; VETERINARY\nPRACTICE\nBarriers to the delivery of contextualised care\nin UK small animal veterinary practice\nSally Everitt, BVSc MSc PhD1*\nAndréa Taylor, MA2\nAshley Doorly1\nKatherine Cropley, BSc2\nAllison Osterberg, MSc2\nKatie Mantell, BA1\n1 RCVS Knowledge, 1 Hardwick Street, London, United Kingdom, EC1R 4RB\n2 Kaleidoscope Health and Care, 604, Cannon Wharf, Pell Street, London, United Kingdom, SE8 5EN\n* Corresponding author email: sallymeveritt@btinternet.com\nSubmitted: 18 February 2026; published: 03 June 2026\nOne-sentence summary\nThis paper reports on the perceived barriers to the delivery of contextualised care in UK small\nanimal veterinary practice.\nAbstract\nObjective: To identify the main barriers to the delivery of contextualised care in UK small\nanimal veterinary practice, and to understand how these are experienced in practice.\nBackground: The term contextualised care was introduced in the veterinary context in a\npublished article by members of Veterinary Humanities UK. Although the term has been\nwidely adopted there has been no explicit research into contextualised care in practice.\nEvidentiary value: This paper presents the first empirical evidence using quantitative and\nqualitative data on the barriers to contextualised care as perceived by over 400 veterinary\nprofessionals and over 700 dog and cat owners. This evidence should help veterinary teams to\nidentify potential barriers to the delivery of contextualised care.\nMethods: The study used a mixed methods approach analysing data from questionnaires,\n\nThis evidence should help veterinary teams to\nidentify potential barriers to the delivery of contextualised care.Methods: The study used a mixed methods approach analysing data from questionnaires,\ninterviews, and focus groups.\nResults: The most frequently reported barriers related to lack of continuity of care and\nfinancial constraints, but the research also found that fear of regulatory scrutiny, missed\nor incorrect diagnoses, and client complaints could act as a barrier to adapting care to\nthe individual animal, owner, and context. The study found some variation in the barriers\nencountered based on experience, role, and practice type.\nConclusion: Overcoming the barriers to contextualised care will require support at all levels,\nincluding from professional bodies, educators, and those with leadership positions in veterinary\npractice.\nApplication: This research should help veterinary teams to overcome potential barriers to the\ndelivery of contextualised care.\nIntroduction\nThe term ‘contextualised care’ was introduced in the veterinary context to describe an approach\nin which ‘different treatment pathways are able to offer equally acceptable patient journeys in\ndifferent contexts’ and ‘that is intentionally shaped by the aims, knowledge, experiences and\ncircumstances of individual animal caregivers and veterinary professionals, acknowledging the\n\nwider contexts of each clinical encounter, to deliver the most appropriate welfare-focused care\nfor every animal’ (Skipper et al., 2021, 2024).\nThe idea of adapting care to the individual circumstances of patients and their owners, as well\nas the context in which the care is provided, may previously have been described as the ‘art of\nveterinary practice’ (May, 2015; Rollin, 2020). However, there are a number of reasons why it may\nnow be more difficult to adopt this approach, including: advances in the range of diagnostic tests\nand treatments available, with associated higher costs leading to concerns about affordability and\naccessibility; changes in the way that veterinary services are provided; and the fact that pets are\nbeing increasingly seen as family members, leading to altered expectations for veterinary care\n(Corr et al., 2024).\nThe discussion in the UK has contrasted contextualised care with gold standard care, that is\ncare associated with intensive and technologically advanced approaches, which is assumed to\nbe the preferred option, in the belief that it is most likely to result in the best outcomes (Skipper\net al., 2021; Englar, 2023). Concerns have also been raised that advances in veterinary medicine\nmay have led to a situation where some available diagnostic investigations and treatments are\nunaffordable to owners and not in the best interests of the animal (Grimm et al., 2018; Quain et\nal., 2021).\nDiscussions regarding contextualised care have also been brought into sharper focus because\nof the Competition and Markets Authority (CMA) market investigation into veterinary services\nfor household pets, with a number of organisations referencing contextualised care in their\nresponses (CMA, 2024).\nDespite the widespread use of the term, there is currently little evidence into how contextualised\ncare is understood and delivered in practice. To answer these questions, RCVS Knowledge partnered\nwith Kaleidoscope Health and Care to conduct a research project to explore how contextualised care\n\nTo answer these questions, RCVS Knowledge partnered\nwith Kaleidoscope Health and Care to conduct a research project to explore how contextualised care\nis understood, experienced, and applied within UK small animal veterinary practice, what barriers\nprevent its delivery, what it looks like when done well, and what changes might be needed to support\nthis approach. The research focused on veterinary care for dogs and cats as these make up the\nmajority of small animal consultations and have been most impacted by the changes in the delivery\nof veterinary services (Sánchez-Vizcaíno et al., 2017).\nThis paper reports specifically on the main barriers to the delivery of contextualised care identified\nby veterinary professionals working in small animal practice and dog and cat owners.\nMethods\nStudy design and data collection tools\nThis research employed a mixed-methods approach to capture both the breadth and depth\nof stakeholder experiences including both quantitative data (questionnaires) and qualitative\ndata (focus groups, interviews and free-text questionnaire responses) from those working in\nveterinary practice and from dog and cat owners.\nDevelopment of the research framework and data collection instruments was informed by\nfindings from a review of relevant literature as well as discussions at the ‘National Forum on\nContextualised Care: Shaping the Strategy’, held in London in February 2025. At this event\nmore than 70 participants, including veterinary surgeons, Registered Veterinary Nurses\n(RVNs), receptionists, educators, regulators, leaders of veterinary associations, leaders from\ncharity and rescue settings, and pet owners participated in discussions focused on the relevance\nof contextualised care, and the barriers and enablers to its delivery.\nThe data collection tools included two surveys, one for veterinary professionals (veterinary\nsurgeons and RVNs in clinical practice) and one for owners of dogs and cats. These composed\nmulti-select, multiple-choice, and Likert-type questions which collected quantitative data, and\n\nThese composed\nmulti-select, multiple-choice, and Likert-type questions which collected quantitative data, and\nopen-text fields which captured qualitative data. The draft surveys were piloted with a subset\nVeterinary Evidence (2026) Vol 11 Iss 2 | Page 2 of 19\n\nEveritt et al. | Page 3 of 19\nof the target populations, which informed refinements to question phrasing, response options,\nand survey logic prior to full launch.\nInterview guides were tailored to specific roles (e.g. veterinary surgeon, RVN, practice adminis­\ntrator, veterinary educator) but all followed a similar structure based on the research questions,\nwith flexibility for probing and exploration of emergent themes. The focus group guide was\nalso framed by the research questions and aimed to explore pet owners’ experiences and\nperspectives of obtaining appropriate veterinary care.\nParticipants and recruitment\nBoth surveys were hosted on the Alchemer online survey platform and were active for four\nweeks in April 2025. Response rates were monitored throughout the data collection period.\nThe inclusion criteria for the veterinary professional survey were qualified veterinary surgeons\nand RVNs, over 18 years of age, currently practising in the UK who provide clinical care to dogs\nand/or cats. The survey was promoted through multiple RCVS Knowledge channels including a\npress release, newsletters, and social media.\nThe target sample size of 381 was calculated based on an estimated population of 35,000 to\n37,000 veterinary surgeons and RVNs working in small animal or mixed practices in the UK, in\norder to achieve a 95% confidence level and a 5% margin of error. Sample size and composition\nwas monitored against a sampling framework based on RCVS data to ensure that a range of\npractice types, job roles, and experience were represented (RCVS, 2024; Rosolin et al., 2024).\nA heavier weighting was given to veterinary surgeons due to their central role in clinical\ndecision-making. Targeted recruitment helped address the initial underrepresentation of\nRVNs, professionals from charity settings, and recent graduates, though the final sample\nincluded a slight overrepresentation (3%) of early-career veterinary surgeons.\nThe inclusion criteria for the pet owner survey were current ownership of at least one\n\nThe inclusion criteria for the pet owner survey were current ownership of at least one\ndog and/or cat and having visited a veterinary practice in the UK within the past three\nyears. Exclusion criteria were being under 18 years of age and residing outside the UK.\nThe survey used convenience sampling, recruiting participants through Kaleidoscope’s\nprofessional networks and organic social media posts on Facebook. After two weeks of\ndata collection, response rates were below the target of 381. Recruitment was enhanced\nthrough paid Facebook advertising targeting UK pet owners, supplemented by addition of\nan incentive prize draw (£100 voucher randomly awarded to one participant who provided\ncontact information).\nFifteen semi-structured 30-minute online interviews were conducted in April and May 2025.\nParticipants were recruited through purposive sampling to ensure diversity in role, experience,\nand practice type. Participants included veterinary surgeons, RVNs, practice managers and\nreceptionists from various practice settings (corporate, independent, charity), and veterinary\neducators.\nTwo 1-hour focus groups were conducted online via Microsoft Teams in May 2025. Ten dog and\ncat owners were recruited from survey participants who expressed interest in further involvement.\nParticipants received £25 gift vouchers as compensation for their time. Interviews and focus groups\nwere audio-recorded and transcribed with participant consent.\nData analysis\nThe quantitative survey data were cleaned and analysed using PSPP version 2.0. Frequencies,\npercentages, means, and standard deviations were calculated as appropriate. Chi-square tests\nwere used to examine differences in nominal variables (e.g. barrier selection, professional role,\npractice ownership type). Mann–Whitney U and Kruskal–Wallis tests were used to examine\nordinal variables in Likert-scale responses, as appropriate to the number of comparison\ngroups. This non-parametric approach was used given the discrete, ordered response categories\nand distribution characteristics typical of attitudinal data.\n\nVeterinary Evidence (2026) Vol 11 Iss 2 | Page 4 of 19\nStatistical significance was set at α = 0.05. Bonferroni correction was applied for multiple\ncomparisons within each analysis family to control family-wise error rate. Where expected\ncell counts were insufficient for robust chi-square analysis (n < 5), subgroups were excluded\nfrom comparative testing to avoid violations of test assumptions.\nWhere statistically significant differences were detected using chi-square or Kruskal–Wallis\ntests, post-hoc pairwise comparisons were not conducted. Therefore, while we report significant\nvariation among subgroups, we describe patterns in the data rather than identifying specific\npairwise differences.\nThe qualitative data from interview and focus groups transcripts were coded by a team\nof three researchers using an initial coding framework developed deductively from the\nresearch framework and research questions. Additional inductive codes were developed\nduring analysis as new themes emerged from the data. Discrepancies were discussed and\nresolved through consensus.\nOpen-text responses from both surveys were initially coded using artificial intelligence platforms\n(Claude Sonnet 4 and Gemini 2.5 ProPreview). The results were cross-checked and all survey\nresponses reviewed in full twice by researchers, who revised the AI-suggested themes as\nneeded.\nFor analysis of open-text responses of the pet owner data, the research team employed\nquota sampling to select a qualitative analysis sample of 385 from the full quantitative\nsample of 718. The quota sampling strategy prioritised inclusion of underrepresented\ngroups (men, non-binary respondents, ethnic minorities, and participants under 55 years)\nto offset the overall sample skew toward women, white ethnicity, and older age groups.\nAll open-text responses from the 417 veterinary professional survey participants were\nincluded in qualitative analysis.\nResults were compared across data sources and participants to identify areas of convergence,\ncomplementarity, and discrepancy.\nResults\nDemographics\n\nResults were compared across data sources and participants to identify areas of convergence,\ncomplementarity, and discrepancy.Results\nDemographics\nThe survey of veterinary professionals achieved an initial sample of 418 participants. During\ndata cleaning, one participant was excluded due to straight-lining responses, resulting in a\nfinal sample of 417 participants, made up of 313 veterinary surgeons (75%) and 104 RVNs\n(25%). This sample was predominantly female (83%), with 15% male and 2% selecting other\noptions. Experience levels among veterinary surgeons were distributed across graduation\ncohorts: 28% qualified since 2020, 45% qualified between 2000–2019, and 27% qualified\nbefore 2000. Among RVNs, 67% had 10 or more years of experience. The sample consisted\nof 52% working in corporate practices, 33% in independent practices, 13% in charity prac­\ntices, and 8% in other practice types (percentages exceed 100% as respondents could select\nmultiple categories). The final sample achieved a 95% confidence level with a 5% margin of\nerror when compared to RCVS workforce data, with a 3% overrepresentation of early career\nveterinary surgeons. The majority (65%) described themselves as working in first opinion\npractice, 10% in Emergency/Out of Hours provision, 14% in Veterinary Hospitals, 3% in\nuniversity practice, 8% in referral practice, and 2% in charity practice (percentages exceed\n100% as respondents could select multiple categories).\nThe survey of pet owners resulted in 763 completed responses. Application of inclusion and\nexclusion criteria led to exclusion of 45 participants, resulting in a final sample of 718. The pet\nowner sample was majority female (71%), white ethnicity (76%), and older age groups (58%\naged 55+). The majority of the sample (87%) had owned a dog and/or cat for six years or more,\nwhile 5% had between three and six years of ownership experience, 3% had less than 3 years’\nexperience and 5% were breeders. Pet insurance coverage among the sample was split, with\n\nEveritt et al. | Page 5 of 19\n44% having no insurance, 44% having insurance for all their cats and/or dogs and 12% having\ninsurance for some, but not all, of their cats and/or dogs.\nPerceived barriers to contextualised care – veterinary professionals\nIn response to the question in which veterinary professionals were asked to select the three\nbiggest barriers to providing contextualised care, the most frequently selected barriers were\nlack of continuity of care (47%) and owner financial constraints limiting the care they could\nprovide (42%), while 45% of respondents selected that they experience very few barriers to pro­\nviding contextualised care. However, a significantly higher proportion of veterinary surgeons\nthan RVNs reported experiencing very few barriers. RVNs consistently reported higher rates of\nbarriers across most categories, with statistically significant differences for continuity of care\nchallenges, financial constraints, feeling most comfortable when providing ‘gold standard’ care,\nand feeling restricted by practice protocols and guidelines (Figure 1).\nFigure 1: The biggest barriers to providing contextualised care (up to three selected), shown by\nrole (RCVS Knowledge 2025b).\nDifferences also emerged among veterinary surgeons when analysing perceived barriers by years\nsince qualification. Those graduating since 2020 reported substantially higher rates across most\nbarriers and lower rates of experiencing very few barriers than all other cohorts. These differences\nwere most marked for continuity of care challenges, financial constraints, communicating about\ncosts, and feeling most comfortable providing ‘gold standard’ care (Figure 2).\nDifferences by time since graduation were also found in responses to the question ‘I tend to rely\non tests and diagnostics to get a clear answer because I am worried about client complaints or\ndisciplinary action if I make the “wrong” decision’, with just 9% among those qualified in the\n1980s agreeing or strongly agreeing with the statement compared to 50% among those qualified\nsince 2020.\n\nSimilarly, feeling relieved when pets are insured increased from 36% to 83% across these\nsame cohorts. Conversely, confidence in providing appropriate care without a definitive\ndiagnosis decreased from 92% among veterinary surgeons who qualified before 2010 to\n66% among those who qualified since 2020.\n\nVeterinary Evidence (2026) Vol 11 Iss 2 | Page 6 of 19\nFigure 2: The biggest barriers to providing contextualised care (up to three selected), shown for\nveterinary surgeons only, by year of qualification (RCVS Knowledge 2025b).\nThe qualitative data helps to illuminate the reasons for this.\n‘I think contextualised care is more difficult for younger vets because they are frightened\nthey are going to miss things. A younger vet will do a lot more blood testing than I would\nbecause they haven’t got that experience yet’ (Veterinary surgeon).\n‘New grads feel so overwhelmed, they fear the RCVS is going to look at them, or owners are,\nthey are going to get complaints’ (RVN).\n‘I feel that they [new graduates] are really good at knowing what the options are, and they’re\nreally good for treatment, knowing what the best one is for that condition in isolation but I think\nthey lack confidence in being able to make a call to say ‘I think this is the best for you’. Because I\nthink that’s a clinical confidence as well as a communication confidence’ (Veterinary educator).\nDifferences in perceived barriers were also identified by practice type, with continuity of\ncare, owner financial constraints, and feeling most comfortable when able to provide ‘gold\nstandard’ care selected as barriers to the delivery of contextualised care at significantly\nhigher rates by veterinary professionals working in corporate practice than those working\nin independent practice or charity settings (Figure 3).\nHigher percentages of veterinary surgeons in corporate practice also reported relief when pets are\ninsured (77% vs 55% independent, 27% charity), and fewer reported confidence in providing care\nwithout definitive diagnosis (72% vs 89% independent, 95% charity).\nPet owners expressed concerns that vets may be under pressure to follow standardised protocols\nwhich they perceive as a barrier to providing contextualised care.\n‘Newly trained vets seem like they’re going through a set protocol which will direct you into\ncertain treatments. This worries me. It’s difficult finding a vet with all the experience who can\n\n‘Newly trained vets seem like they’re going through a set protocol which will direct you into\ncertain treatments.This worries me.It’s difficult finding a vet with all the experience who can\nlook at an animal and think yes it doesn’t need surgery’ (Pet owner).\n‘When it comes to personalised care, it has a lot to do with the vet… the amount of pressure,\ntargets, facilities, if they are focusing on one species or multiple’ (Pet owner).\n\nEveritt et al. | Page 7 of 19\nFigure 3: The biggest barriers to providing contextualised care (up to three selected), shown\nby practice type (RCVS Knowledge 2025b).\nLack of continuity of care\nLack of continuity of care was perceived as a major barrier to the delivery of contextualised\ncare, with 47% of veterinary professionals selecting ‘It is difficult to provide contextualised\ncare when owners see different members of the practice team at each visit.’\nPet owners also strongly valued continuity of care, with 78% agreeing or strongly agreeing that\nit is important to see the same veterinary team members when visiting the practice.\nThe qualitative data provides a deeper understanding of why lack of continuity of care creates\na barrier to the provision of contextualised care, highlighting concerns about capturing and\nsharing information in the practice:\n‘The common complaint is “I always see a different person every time I go in.” They’re\nhaving to start the story from scratch, and sometimes you get duplication of testing\nbecause the next vet isn’t fully aware of what’s gone on before or even the thinking of\nthe previous day’ (Veterinary surgeon).\n‘Lack of continuity of care [is a barrier]. We still don’t have a good culture of the same vet\nseeing the same owner and we aren’t always good about recording details about the client\nbecause we are nervous about including personal details in the notes’ (RVN).\nThe challenge extended beyond simple information transfer to encompass the importance of\nbuilding relationships in order to provide continuity of care:\n‘I think it’s easiest to deliver [contextualised care] if you have a good relationship with the\nclient but unfortunately [clients] rarely see the same doctor because they are working part\ntime and it’s hard to get an appointment with them. It’s so much easier [when] you get to\nknow the client’s approach. You get to know the client’s willingness to do things... if you have\nthat relationship it’s much easier to discuss the case in depth then to find the appropriate\nroute’ (Veterinary surgeon).\n\nIt’s so much easier [when] you get to\nknow the client’s approach.You get to know the client’s willingness to do things...if you have\nthat relationship it’s much easier to discuss the case in depth then to find the appropriate\nroute’ (Veterinary surgeon).\n‘Seeing a different vet every time you visit [means the] vet has no personal relationship with\nme or with the dog, therefore carries no direct knowledge of me or of the dog or the dog’s\nhistory’ (Pet owner).\n\nVeterinary Evidence (2026) Vol 11 Iss 2 | Page 8 of 19\nThe consequences of lack of continuity of care can lead to pet owners losing confidence in the\nadvice provided and negatively impacting the relationship and trust between the pet owner and\nthe veterinary team:\n‘Different vets at the clinic might give different advice or options for treating my dog which\ncan be confusing and make it harder to choose the right option for my dog’ (Pet owner).\n‘At my current vet practice, there’s been a lot of changeover of vets. This means my pet is\nseen by a different vet each time, and that makes it challenging for anyone to develop a\nrelationship or trust. Often, different vets give different advice as well, which means we’re\ngetting conflicting information’ (Pet owner).\n‘I feel that the vet team often don’t agree with and therefore ignore/dismiss information I’ve\ngiven them’ (Pet owner).\n‘Doing my own research to understand conditions and medications along with side effects\nand bringing up this in consultations [is] met with hostility when I seem to know more or ask\ndeeper questions about things pertaining to my dogs’ illnesses’ (Pet owner).\nFinancial constraints\nFinancial constraints were perceived as a major barrier to the delivery of contextualised care,\nwith 42% of veterinary professionals selecting ‘I find it difficult when financial constraints of\nthe owner limit the care that I am able to provide.’ The percentage was higher for RVNs than\nveterinary surgeons (56% v 37%, Figure 1), veterinary surgeons that qualified since 2020 (71%,\nFigure 2), and veterinary professionals working in corporately owned practices (57%, Figure 3).\nWhen veterinary professionals were asked how often owner financial constraints influenced\ntheir care of individual patients 47% responded always, 43% often, 9% sometimes, and only 1%\nrarely.\nThe qualitative data indicates that veterinary professionals experience tensions between\nproviding optimal care and managing the financial concerns and limitations of pet owners:\n‘It is sometimes difficult for owners to understand/accept that financial constraints may\n\nlimit the care that is available to their pet’ (Veterinary surgeon).\n‘Owners not accepting their own financial limitations, i.e. offering cheaper options, but\nowners becoming aggressive and blaming staff for the fact that it’s not possible for the\npatient to receive “gold standard” treatment’ (Veterinary surgeon).\n‘Due to the current economic climate, we’ve seen more patients be euthanised or [owners]\nwait to bring their pet in for care due to financial concerns, so by the time we see someone\nthey may be much worse off than they could’ve been’ (RVN & practice manager).\nThere are also indications that some veterinary professionals feel commercial pressure to\ngenerate income for the practice.\n‘I am in the position of practising [contextualised care] in my full-time job and am very\ncomfy with it but find my locum work really hard as I work for corporates that want to push\neverything at a client even when I don’t feel it is needed’ (Veterinary surgeon).\n‘I feel some clinics may be incentivised to increase client spend in order to repay large\nequipment purchases such as MRI/CT’ (RVN).\n‘In the corporates, it is about bringing in the money. I know that there is pressure on my\nmanager to bring in money, what more could we be doing. We have a team meeting this\n\nEveritt et al. | Page 9 of 19\nafternoon before the shift starts and that will be an uncomfortable conversation for me\nbecause I will still not be recommending things that are unnecessary. So probably one\nbarrier is organisational financial goals and priorities. And they are businesses. I get that,\nthey have to run, they have to pay their staff, I understand that’ (Veterinary surgeon).\nOwners report that they consider the costs of veterinary care less often than concerns around\nquality of life and evidence of effectiveness (Figure 4). However, when asked about barriers to\ncontextualised care, 31% of pet owners selected feeling guilty when they cannot afford all the\noptions on offer (Figure 6).\nFigure 4: Factors considered by dog and cat owners when making choices about diagnostic and\ntreatment options (RCVS Knowledge 2025b).\nWhen asked about satisfaction with aspects of care received over the past three years, only\n42% of pet owners felt satisfied that veterinary teams considered their financial situation\nwhen making recommendations, with 24% expressing dissatisfaction.\nThe qualitative data also indicates that the costs of veterinary care can be a barrier.\n‘Treatments are often tailored to the dog but these treatments can be extremely expensive\nand not covered through insurance e.g. canine behaviourist’ (Pet owner).\n‘Just costs [are the barrier], especially since vets are now not allowed to advise human\nmedicines for pets, meaning the licenced alternative is hugely expensive’ (Pet owner).\n‘[The] biggest problem for me is the ridiculous cost of out of hours veterinary care. My\nown vets are great but not open at weekends. The out of hours service is run by a different\ncompany and the cost is extortionate’ (Pet owner).\nCommunication about costs emerged as a specific challenge for both veterinary professionals\nand pet owners. Whilst only 16% of veterinary professional respondents overall selected difficulty\ncommunicating effectively with owners about costs as one of their top three barriers, this figure\n\nWhilst only 16% of veterinary professional respondents overall selected difficulty\ncommunicating effectively with owners about costs as one of their top three barriers, this figure\nvaried substantially by experience, with 33% of recent graduates (2020–present) identifying this\nas a barrier compared to 0% of those qualified in the 1980s (Figure 2).\nThe qualitative data revealed the complexity of discussing costs sensitively.\n\n‘The vets that do [cost communication] well literally provide estimates for the client and it’s\nall done in the appointment. One of the vets is the best at it. And you do hear her saying “gold\nstandard would be this, but we can also try this and there is no harm in trying this first” so\nthe client doesn’t feel bad if they can’t afford gold standard. So, she’s brilliant but it’s about\ngetting that level taught out to all other vets’ (Practice manager).\n‘Sometimes owners also feel upset when you offer gold star care simply because it would\nfeel dishonest not to offer even if you are happy to work with them. I’ve been accused of\n“shaming” owners for not being able to afford the gold star option even when I’m very happy\nto adjust’ (RVN).\nFrom the pet owner perspective, lack of transparent cost communication from veterinary practices\nemerged as a significant concern.\n‘The vets always assume money is no object and I am not usually told the costs (e.g. for blood\ntests) in advance’ (Pet owner).\n‘The offers we received were mostly best for the practice, not for the pet. I strongly believe\nthat tailoring the best possible options for a certain pet is a very good approach. However,\nowners who cannot fully afford more expensive treatment should be informed of the [other\noptions]’ (Pet owner).\n‘When my vet went from being independent and sold out to a big company, suddenly they\ndidn’t want to talk money, rather it’s we need to do this test, or this test ... I’m currently with\nan independent vet who is open and happy to talk about money ... It needs to be not a taboo\ntopic, honest and open from the beginning so it’s not a shock at the end’ (Pet owner).\nHowever, when asked about how they would like their vet to inform them of the costs of treatment\nthe results show that very few pet owners want to be asked directly about how much they are\nwilling or able to spend (Figure 5). Instead, the top option (selected by 44% of respondents) was to\nbe given the full range of options to enable them to make a choice. The next most popular option,\n\nInstead, the top option (selected by 44% of respondents) was to\nbe given the full range of options to enable them to make a choice.The next most popular option,\nselected by 25% of the sample, was to be given a recommendation about what is best for the pet\nregardless of cost. These results did not differ significantly with the level of insurance cover.\nFigure 5: Dog and cat owners’ preferences for discussion of costs (RCVS Knowledge 2025b).\nVeterinary Evidence (2026) Vol 11 Iss 2 | Page 10 of 19\n\nDefensive medicine\nSurvey respondents articulated how fear may prevent veterinary professionals from using \nclinical judgement to tailor care appropriately. Veterinary surgeons responding to open-text \nsurvey questions said:\n‘I find that vets are often so scared of stepping out of a box that they don’t even consider \nan alternative approach’ (Veterinary surgeon).\n‘So many vets are terrified of disciplinary action that this informs how they practice’ \n(Veterinary surgeon).\n‘I think you would find there is a lot of anxiety out there. I try to be pragmatic but 100% there \nare nights when I come home obsessing over whether I should have done this or that, did I \nwrite that properly, should I have offered this? That is all emotional mental load’ (Veterinary \nsurgeon).\n‘It isn’t just a mental health approach but it’s building resilience within our veterinary \npopulation, to help them understand that change is okay and not having gold standard \nis okay. Also helping them to deal with unhappy clients and emotional clients. Because I \nthink there is a lot of defensiveness over that’ (Veterinary surgeon).\nPet owners also acknowledge the impact emotions can have. When asked to choose the three \nstatements that best reflect the barriers they experienced in receiving personalised care, 43% \nselected feeling very emotional when pets are unwell and 31% sometimes feeling guilty about \nnot being able to afford all the treatment options on offer (Figure 6).\nFigure 6: The biggest barriers to receiving contextualised care, identified by pet owners (up to \nthree selected) (RCVS Knowledge 2025b).\nDiscussion\nA positive finding of this study is that 45% of veterinary professionals and pet owners report that \nthey encounter very few barriers in providing or receiving contextualised care, confirming that \nthis is something that is achievable in practice. However, this research has identified a number \nof perceived barriers to contextualised care.\nEveritt et al. | Page 11 of 19\n\nLack of continuity of care was perceived as a significant barrier to the delivery of contextualised\ncare by both veterinary professionals and pet owners. In human medicine continuity of care\nhas been shown to be associated with both improved clinical outcomes and patient satisfaction,\nparticularly for those with chronic conditions (Cabana & Jee, 2004; Van Walraven et al., 2010).\nIn this study, pet owners associated continuity of care with building trusting relationships\nand ensuring consistent care. Inconsistency of care and lack of trust have been reported to\nbe associated with reduced perception of value and increased likelihood of switching practice\n(Brown, 2018; Hickey et al., 2024). Veterinary professionals also acknowledge these concerns.\nIn addition, they identified the ability to capture and share information within the practice\nteam as a barrier to the delivery of contextualised care, highlighting concerns about recording\ncontextual information about the owner in the patient notes.\nThere are a number of reasons why continuity of care may be more difficult to deliver now than\nit was in the past. Changes in the delivery of veterinary services over the last twenty years have\nseen significant increases in the use of out of hours service providers and referral services,\nmeaning that pet owners may visit several different practices with their animal, even during the\ncourse of a single condition.  There has also been a significant increase in flexible and part-time\nworking, meaning that it may be more difficult for pet owners to see the same members of the\nveterinary team, even at the same practice (Kinnison et al., 2014; Rosolin et al., 2024).\nIt may therefore be necessary for veterinary practices to consider whether continuity of care can\nbe delivered at a team level. This will require contextual information to be captured and shared\nbetween members of the practice team. Recording any information about the client will need to be\ndone with client consent and in a way that is compliant with data protection regulations, which may\n\nRecording any information about the client will need to be\ndone with client consent and in a way that is compliant with data protection regulations, which may\nrequire guidance from professional bodies. However, it has been noted that the delivery of care by\nmultidisciplinary teams and the expansion of digital and remote technologies have created both\nopportunities and challenges for continuity of care (Freeman & Hughes, 2010; Pyatt et al., 2020).\nThe costs of veterinary care have risen significantly in recent years with the costs of veterinary\nand other pet services in the UK reported to have increased by over 75% since 2015 (Office for\nNational Statistics, 2025), with significant rises also being reported in other countries (Egenvall\net al., 2024).\nThe reasons for these increases are complex, but include advances in diagnostic and treat­\nment options, changes in owner expectations, increased popularity of pets with exaggerated\nconformation, as well as changes in the delivery of veterinary services (O’Neill et al., 2023;\nCMA, 2024; Corr et al., 2024). However, whatever the reasons, the increase in the costs of\nveterinary care has led to issues of affordability for pet owners and potential adverse welfare\noutcomes for pets (Croney et al., 2025; O’Connor et al., 2025) .\nThe affordability of veterinary services also impacts the wellbeing of veterinary professionals\n(Kipperman et al., 2017; Williams, 2025), with financial constraints influencing the care of indi­\nvidual patients and a recent survey of UK veterinary professionals showing that the affordability\nof veterinary services was rated as the third greatest challenge facing the veterinary profession\n(46%) after client expectations/demands (54%) and stress levels (49%) (Rosolin et al., 2024).\nAlthough contextualised care, in terms of adapting care to the individual circumstances of the\nowner, may be seen as a potential way to mitigate financial constraints, this research shows that\nthere are still barriers relating to having open conversations about the costs of veterinary care.\n\nThe survey data showed that when owners are asked how they would like their vet to let them\nknow about the potential costs of treatment, the most frequently selected options were to be\ngiven the full range of options to enable them to make a choice or to be given a recommendation\nabout what is best for the pet regardless of cost. However, less than 10% wanted to be asked\ndirectly about how much they can afford and even fewer wanted to have an early conversation\nVeterinary Evidence (2026) Vol 11 Iss 2 | Page 12 of 19\n\nabout what their insurance covers. Despite veterinary surgeons reporting relief when animals\nare insured, owner preferences regarding the discussion of costs do not appear to vary in line\nwith level of insurance, suggesting that other contextual factors are important.\nThese findings provide some evidence as to why discussions of the cost in veterinary consulta­\ntions may perceived as challenging and the need for these discussions to move from discussion\nof tangibles, such as time and services, to outcomes related to the pet’s health and well-being\n(Coe et al., 2007, 2009; Groves et al., 2022).\nThe impact of owners not being able to afford veterinary care has an impact on the welfare of\nanimals and the wellbeing of pet owners and veterinary professionals. Many pet owners in this\nstudy report sometimes feeling very emotional when their animal is unwell and feeling guilty\nif they cannot afford all the treatment options on offer. These findings are supported by other\nresearch that suggests that decisions regarding an animal’s care are impacted by the owner’s\nemotional attachment to the animal rather purely financial concerns (Brown, 2018; Corr et al.,\n2024; Strand et al., 2024) which may lead to conflict between the veterinary care that they can\nafford and the care they would like their pet to receive.\nThere is also evidence that the high costs of veterinary care can impact the wellbeing of veterinary\nprofessionals, and that recently qualified vets in particular may feel unprepared to deal with financial\nconstraints that impact their ability to treat animals to their preferred standard or necessitate having\nto perform euthanasia when treatment is unaffordable (Kipperman et al., 2017; Williams, 2025).\nAlongside age and experience, there is evidence that professional identity can impact the re­\nsponse to contextual challenges. Two distinct variants of veterinary identity have been proposed:\n‘diagnosis-focused’ identity, which prioritises definitive diagnosis and best-evidence treatment;\n\nTwo distinct variants of veterinary identity have been proposed:\n‘diagnosis-focused’ identity, which prioritises definitive diagnosis and best-evidence treatment;\nand ‘challenge-focused’ identity, where priorities were also reported to include engaging with\nthe client, and contextual challenges including limited finances (Armitage-Chan & May, 2018). It\nmay be that those with a challenge-focused identity find it easier to practise contextualised care.\nThe anxiety around disciplinary action documented in responses suggests that there is a perception\nthat current professional standards discourage the clinical flexibility required for contextualised care,\neven though the Royal College of Veterinary Surgeons (RCVS) has recently updated their guidance to\nexplicitly make reference to providing contextualised care (RCVS, 2025).\nThe fear of regulatory scrutiny, missed or incorrect diagnoses, and client complaints may be\ncontributing to a defensive approach to practice in which additional tests or treatments that are\nof little or no benefit to the patient are carried out, raising the costs of veterinary care and acting\nas an additional barrier to the delivery of contextualised care (Summerton, 2000; Assing Hvidt\net al., 2017; Gibson et al., 2022),\nThis study detected several differences in the perceived importance of barriers to contextualised\ncare by experience, role, and type of practice. It is not surprising that those that have graduated\nmore recently generally perceive greater barriers to the delivery of contextualised care as they\nhave less experience to draw on. However, to ensure that they have the knowledge and skills to\ndeliver contextualised care it is important that they are not only exposed to a range of evidence-based\ntreatment options but have the people and communication skills to work with pet owners in poten­\ntially emotionally charged situations, to find the most appropriate option.\nWhile changes have already been made in the UK to ensure that the majority of clinical education\n\nWhile changes have already been made in the UK to ensure that the majority of clinical education\nmust focus upon casework in the ‘general practice’ context (RCVS, 2023), it is important to ensure\nthat ideas relating to professional identity, or a single most appropriate approach, are not instilled\nthrough the hidden curriculum (Roder & May, 2017; Kunze & Seals, 2020).\nThe American Association of Veterinary Medical Colleges have recently published an implementation\nstrategies guide to prepare graduates to deliver contextually appropriate, evidence-based care across\nEveritt et al. | Page 13 of 19\n\na range of clinical settings, indicating that these concerns are not limited to the UK (Fedesco et al.,\n2025).\nThis research also found that RVNs consistently perceived greater barriers to the delivery of\ncontextualised care across most categories. Since our sample of RVN respondents was skewed\ntowards those with greater experience (> 10 years), it is more likely that this finding relates to\ndifferences in role rather than experience. Going forward it will be essential to ensure that vet­\nerinary nurses also have the knowledge and skills to support and deliver contextualised care.\nWhile the quantitative data indicates that those working in corporate practices were more likely to\nreport a range of barriers to contextualised care, interpretation of these findings is complicated by\nthe fact that nearly half (48%) of corporate practice respondents in the sample qualified in the last\nfive years, compared with 15% of respondents working in independent practice, suggesting years\nof experience may be a confounding factor.\nHowever, it is also important to recognise that the qualitative data reveals a clear perception\namong both veterinary professionals and pet owners that corporate ownership is associated\nwith a range of barriers to contextualised care, and there is evidence to support this from other\nareas where private equity has become involved in the delivery of healthcare (Singh et al., 2022;\nBruch et al., 2023; Zhu et al., 2024).\nThis study has both strengths and weaknesses. This is the first study of contextualised care to\ncollect and analyse data from veterinary professionals and dog and cat owners. The involvement\nof stakeholders at two meetings, before and after data collection, enabled us to incorporate a\nwide range of perspectives both into the framing of the questions and providing feedback on the\nfindings to develop actionable recommendations (RCVS Knowledge, 2025b). One disadvantage\nof this approach is that we asked a wide range of questions, and with hindsight focusing on a\nnarrower range of topics in more depth might have given more robust results. However, this\n\nOne disadvantage\nof this approach is that we asked a wide range of questions, and with hindsight focusing on a\nnarrower range of topics in more depth might have given more robust results.However, this\nresearch now provides a broad foundation for future work. The questionnaires were completed\nby a self-selecting sample, and therefore the responses were subject to selection bias. While it\nwas possible to compare the representativeness of the veterinary professional data, at least in\nterms of certain criteria, against RCVS workforce data, any assessment of the representativeness\nof the pet owner data is limited by the absence of published demographic data for the UK\nveterinary-visiting pet owner population, although there is evidence from other veterinary\nquestionnaires that respondents to veterinary questionnaires are primarily female (Belshaw\net al., 2025; Glasspool et al., 2025).\nThe data for this study were collected over a concentrated period (April–July 2025), capturing a\nsnapshot of contextualised care during this timeframe. As this coincided with the CMA investi­\ngation in veterinary services in domestic pets, the discourse around and media coverage of this\ninvestigation may have influenced the responses of both veterinary professionals and pet owners.\nWhile this paper reports on the most frequently reported barriers to the delivery of contextualised care\nit should be remembered that for certain individuals, other factors such as access to transportation,\nlanguage, and cultural differences may be of great significance.\nThe questionnaires were limited to responses from veterinary surgeons and RVNs, and dog and\ncat owners respectively. However, it is reasonable to assume that the principles of contextualised\ncare, and adapting the care provided to the individual circumstances and context in which care is\ndelivered, will be relevant to all members of the veterinary team including those working in other\ncontexts including farm, equine, and exotic practice, as well as with wildlife.\nConclusion\n\nConclusion\nThis paper presents the first empirical evidence on the barriers to contextualised care as\nperceived by both veterinary professionals and dog and cat owners.\nVeterinary Evidence (2026) Vol 11 Iss 2 | Page 14 of 19\n\nThe main barriers to contextualised care identified were lack of continuity of care and financial\nconstraints. Continuity of care is important to the delivery of contextualised care as it enables a\ntrusting relationship to develop and for contextual information, including the owner’s personal\nvalues and circumstances, to be shared alongside clinical information. While financial constraints\nwere identified as a barrier to contextualised care, they are more accurately characterised as an\nimportant contextual factor. However, the problems identified around discussing the costs of care\ndo act as a barrier to the delivery of contextualised care.\nA strong theme that emerged in this study was the impact of emotions on the veterinary consultation,\nwith veterinary professionals identifying fear of regulatory scrutiny, complaints, and clinical failure as\nbarriers to implementing contextualised care, and with pet owners reporting that they feel emotional\nwhen pets are unwell and feel guilty about not being able to afford treatment.\nThis research identified a number of differences in the barriers veterinary professionals perceived to\ncontextualised care based on their level of experience, role in practice, and type of practice in which\nthey work. It is therefore important to recognise that the barriers to contextualised care may be felt\ndifferently by individuals for a range of reasons, and to ensure that all members of the practice team\nare supported to understand the context in which they are working to deliver contextualised care.\nThis research was limited to responses from veterinary professionals working in small animal\npractice, and dog and cat owners respectively. However, the principles of contextualised care\nwill be relevant to those working in other contexts including farm, equine, and exotic practice,\nas well as with wildlife.\nTo overcome the barriers to contextualised care it will be important to ensure that those working\nin veterinary practice receive support to develop the knowledge and skills to deliver contextualised\n\nTo overcome the barriers to contextualised care it will be important to ensure that those working\nin veterinary practice receive support to develop the knowledge and skills to deliver contextualised\ncare. This will require support at all levels, including from professional bodies, educators and those\nwith leadership positions in veterinary practice (RCVS Knowledge, 2025a).\nEthical approval\nRCVS Ethics review panel 2025-115.\nInformed consent\nInformed consent for use of anonymised data was obtained from all participants as part of the\nrecruitment process.\nAuthor contributions\nSally Everitt: Conceptualisation, Visualization, Writing – Original draft. Andréa Taylor:\nMethodology (lead), Formal analysis (lead), investigation, Writing – Review & Editing. Ashley\nDoorly: Project Administration, Visualization, Writing – Review & Editing. Katherine Cropley:\nMethodology (supporting), Formal Analysis (Supporting,) Investigation. Allison Osterberg:\nMethodology (Supporting), Formal Analysis (Supporting,) Investigation. Katie Mantell: Funding\nAcquisition, Writing – Review & Editing.\nAcknowledgements\nThanks to Naheem Bashir (Kaleidoscope Health and Care) for his expert advice and review in\nthe data set up and analysis phases.\nFunding\nBattersea supported this research through their grants programme but they were not involved\nin the research and did have any input into the findings or the recommendations.\nORCiD\nSally Everitt: https://orcid.org/0009-0009-7739-7134\nAndréa Taylor: https://orcid.org/0000-0002-3684-3312\nAshley Doorly: https://orcid.org/0000-0003-3336-1665\nEveritt et al. | Page 15 of 19\n\nVeterinary Evidence (2026) Vol 11 Iss 2 | Page 16 of 19\nKatherine Cropley: https://orcid.org/0009-0008-3168-8457\nAllison Osterberg: https://orcid.org/0009-0009-7945-8869\nKatie Mantell: https://orcid.org/0000-0002-1292-5014\nConflict of Interest\nThe authors declare no conflicts of interest.\nReferences\n1.\nArmitage-Chan, E. & May, S.A. (2018). 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Co-Produced Care in Veterinary\nServices: A Qualitative Study of UK Stakeholders’ Perspectives. Veterinary Sciences. 7(4), 149.\nDOI: https://doi.org/10.3390/vetsci7040149\n29.\t Quain, A., Ward, M.P. & Mullan, S. (2021). Ethical Challenges Posed by Advanced Veterinary Care\nin Companion Animal Veterinary Practice. Animals. 11(11), 3010. DOI: https://doi.org/10.3390/\nani11113010\n30.\t RCVS. (2023). RCVS Standards and Guidance for the Accreditation of Veterinary Degree Programmes\n2023, [online]. Available at: https://www.rcvs.org.uk/sites/default/files/2025-12/1rcvs-\nstandards-and-guidance-updated-dec2025.pdf [Accessed: January 23, 2026].\n\nRCVS.(2023).RCVS Standards and Guidance for the Accreditation of Veterinary Degree Programmes\n2023, [online].Available at: https://www.rcvs.org.uk/sites/default/files/2025-12/1rcvs-\nstandards-and-guidance-updated-dec2025.pdf [Accessed: January 23, 2026].\n31.\t RCVS. 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DOI: https://\ndoi.org/10.1001/jamainternmed.2024.0062\n\nEveritt et al. | Page 19 of 19\nLicence\nCopyright (c) 2026 Sally Everitt, Andréa Taylor, Ashley Doorly, Katherine Cropley, Allison \nOsterberg & Katie Mantell\nIntellectual property rights\nAuthors of articles submitted to RCVS Knowledge for publication will retain copyright in their \nwork, and will be required to grant to RCVS Knowledge a non-exclusive licence of the rights \nof copyright in the materials including but not limited to the right to publish, re-publish, trans­\nmit, sell, distribute and otherwise use the materials in all languages and all media through­\nout the world, and to licence or permit others to do so.\nDisclaimer\nAny opinions expressed in articles and other publication types published in Veterinary Evidence \nare the author’s own and do not necessarily reflect the view of the RCVS Knowledge. Veterinary \nEvidence is a resource to help inform, and the content herein should not override the responsibility \nof the practitioner. Practitioners should also consider factors such as individual clinical expertise \nand judgement along with patient’s circumstances and owners’ values. Authors are responsible \nfor the accuracy of the content. While the Editor and Publisher believe that all content herein are \nin accord with current recommendations and practice at the time of publication, they accept \nno legal responsibility for any errors or omissions, and make no warranty, express or implied, with \nrespect to material contained within. For further information please refer to our Terms of Use.\n\n---"}
+{"text": "【兽医文献】\n标题: Identification of antimicrobial resistant bacteria isolated from Hyalomma excavatum and Hyalomma dromedarii infesting camels in Aljouf region, Saudi Arabia\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nEstablishment of a TaqMan qPCR\nmethod with MGB probe for the\nspecific detection of BVDV field\nstrains circulating in China\nLele An 1,2, Xiaoting Ren 1, Yetao Zhang 2, Shubin Zhang 1 and\nYongqing Zhao 1,2*\n1 Key Laboratory of Biotechnology and Bioengineering of State Ethnic Affairs Commission, Biomedical\nResearch Center, Northwest Minzu University, Lanzhou, China, 2 College of Life Science and\nEngineering, Northwest Minzu University, Lanzhou, China\nBovine viral diarrhea virus (BVDV), a highly mutable pathogen, poses a significant\nthreat to the cattle industry in China. Therefore, the development of a rapid,\nsensitive, and specific diagnostic assay is essential for effective surveillance and\ncontrol. In this study, a TaqMan real-time quantitative PCR (qPCR) assay utilizing\na minor groove binder (MGB) probe was developed for the detection of BVDV,\nwith a focus on strains currently circulating in China. Universal primers and\nan MGB probe targeting the conserved 5′ untranslated region (5′UTR) of both\nBVDV-1 and BVDV-2 were designed based on complete genome sequences\navailable in GenBank. Following optimization of the reaction conditions, the assay\ndemonstrated a detection limit of 1.265 copies/μL using a plasmid standard.\nThe method exhibited high specificity for BVDV-1 and BVDV-2, with no cross\nreactivity observed with other common bovine pathogens. Intra- and inter-assay\ncoefficients of variation were below 1.5%, indicating excellent repeatability and\nreproducibility. When applied to field serum samples collected from free-range\ncattle in various regions of China, the assay achieved a 100% concordance rate\nwith a commercial reference kit (IDEXX RealPCR™ BVDV RNA Test). These results\nsuggest that the established TaqMan MGB qPCR assay is a reliable and efficient\ntool for the detection and epidemiological investigation of BVDV-1 and BVDV-2\ninfections in cattle herds across China.\nKEYWORDS\nBVDV-1, BVDV-2, TaqMan MGB qPCR, clinical diagnosis, epidemiological investigation\n1 Introduction\n\nKEYWORDS\nBVDV-1, BVDV-2, TaqMan MGB qPCR, clinical diagnosis, epidemiological investigation\n1 Introduction\nBovine viral diarrhea virus (BVDV), a member of the genus Pestivirus within the family\nFlaviviridae, is a significant pathogen affecting cattle worldwide. It poses substantial threats to\nthe cattle industry due to its economic and health impacts (1–3). BVDV is a single-stranded,\npositive-sense RNA virus with a genome of approximately 12.3 kilobases (kb). The genome\ncontains a single open reading frame (ORF), flanked by two untranslated regions (UTRs): the\n5′UTR and 3′UTR. Phylogenetic classification of BVDV isolates primarily relies on sequence\nanalysis of the 5′UTR and coding regions of viral proteins, including Npro (a viral protease),\nstructural protein E2, and non-structural proteins NS4B and NS5A (4–9). Among these, the\n5′UTR is the most widely used marker for genotyping due to its high conservation and\ndiscriminatory power. Based on the 5′UTR, different BVDV variants have been identified,\nleading to the current classification of BVDV into three genotypes: BVDV-1 (24 subgenotypes,\n1a–1x), BVDV-2 (5 subgenotypes, 2a–2e), and BVDV-3 (HoBi-like pestivirus; 4 subgenotypes,\nOPEN ACCESS\nEDITED BY\nRohana P. Dassanayake,\nAgricultural Research Service (USDA),\nUnited States\nREVIEWED BY\nSultan Ali,\nUniversity of Agriculture, Pakistan\nDavid Holthausen,\nUnited States Department of Agriculture\n(USDA), United States\n*CORRESPONDENCE\nYongqing Zhao\n179122111@xbmu.edu.cn\nRECEIVED 24 May 2025\nACCEPTED 11 July 2025\nPUBLISHED 25 July 2025\nCITATION\nAn L, Ren X, Zhang Y, Zhang S and\nZhao Y (2025) Establishment of a TaqMan\nqPCR method with MGB probe for the\nspecific detection of BVDV field strains\ncirculating in China.\nFront. Vet. Sci. 12:1634429.\ndoi: 10.3389/fvets.2025.1634429\nCOPYRIGHT\n© 2025 An, Ren, Zhang, Zhang and Zhao.\nThis is an open-access article distributed\nunder the terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other forums is\npermitted, provided the original author(s) and\nthe copyright owner(s) are credited and that\nthe original publication in this journal is cited,\n\nThe use,\ndistribution or reproduction in other forums is\npermitted, provided the original author(s) and\nthe copyright owner(s) are credited and that\nthe original publication in this journal is cited,\nin accordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Original Research\nPUBLISHED  25 July 2025\nDOI  10.3389/fvets.2025.1634429\n\nAn et al.\b\n10.3389/fvets.2025.1634429\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n3a–3d) (10–15). Of these, BVDV-1 is the predominant genotype\nglobally, accounting for approximately 88.2% of reported isolates (8).\nThe rapid expansion of the global cattle industry has increased\nawareness of the economic and clinical impacts of BVDV. Increasing\nevidence highlights that BVDV prevalence is associated with a range\nof economically important clinical diseases, including decreased\nreproductive performance and acute fatal hemorrhagic disease (16).\nNotably, BVDV can cross the placental barrier during early gestation,\nresulting in the birth of persistently infected (PI) calves, which serve\nas critical viral reservoirs (17). In China, BVDV-1 and BVDV-2 are\nconsidered the primary sources of infection in cattle herds, with their\npersistent circulation posing substantial risks to both animal health\nand the economic sustainability of the cattle industry (10, 18–22).\nCurrent diagnostic approaches for BVDV include real-time\nquantitative PCR (qPCR), enzyme linked immunosorbent assay\n(ELISA), immunofluorescence assay (IFA), and viral isolation (6, 23,\n24). Among these, qPCR demonstrates superior sensitivity for\ndetecting acute infections. However, challenges arise from the high\ngenetic variability of BVDV. Global genomic surveillance reveals\nextensive genetic divergence among BVDV variants, both between\ngenotypes (e.g., BVDV-1 vs. BVDV-2) and within subgenotypes. In\nChina, co-circulation of multiple BVDV-1 and BVDV-2 subgenotypes\nhas been documented (4, 10, 22, 25–28). As more BVDV variants\ncirculate within cattle herds, this increases the risks associated with\nsensitive detection, particularly in clinical cases involving\nserum samples.\nTo address these limitations, we developed a TaqMan MGB qPCR\nassay targeting the conserved 5′UTR region. This method was\nvalidated using serum samples collected from calves in diverse\nChinese cattle herds between 2022 and 2024, with the aim of\nenhancing the detection of wild-type BVDV strains amidst evolving\nviral diversity.\n2 Materials and methods\n2.1 Serum sample collection\n\n2 Materials and methods\n2.1 Serum sample collection\nThis study was approved by the Animal Ethics Committee of the\nCollege of Life Science and Engineering, Northwest Minzu\nUniversity (Approval No. CLSEAEC 2020–003). A total of 174 whole\nblood samples were collected from free-range, unimmunized cattle\nherds in five Chinese provinces between 2022 and 2024. The\ndistribution of samples across provinces was as follows: Henan\n(n = 28), Jilin (n = 36), Gansu (n = 54), Sichuan (n = 24), and\nQinghai (n = 32).\nWhole blood samples were transported on ice to the laboratory\nwithin 1 h of collection. The samples were allowed to clot at room\ntemperature (25 ± 1°C) for 2–3 h, followed by centrifugation at\n3,000 × g for 10 min at 4°C. The resulting serum was aliquoted into\nsterile cryovials and stored at −80°C until further analysis.\n2.2 RNA extraction and cDNA synthesis\nTotal RNA was extracted from the serum samples using the\nMagMAX\nViral/Pathogen\nNucleic\nAcid\nIsolation\nKit\n(ThermoFisher Scientific Inc.), following the manufacturer’s\nprotocol. The RNA was then reverse transcribed into\ncomplementary DNA (cDNA) using the SuperScript IV First-\nStrand Synthesis System (ThermoFisher Scientific Inc.). The\nresulting cDNAs were stored at −20°C for subsequent use in the\nqPCR assays.\n2.3 BVDV strain collection\nEight representative BVDV strains were isolated, identified, and\npreserved at the Laboratory of Biomedical Research Center, Northwest\nMinzu University. The strains used were as follows: 22Anhui-7\n(GenBank: OQ338174.1), 22Gansu-F2 (GenBank: OQ338175.1),\n22Sichuan-B8 (GenBank: OQ338177.1), 22NX-69 (GenBank:\nON165517.1), 22AH-1 (GenBank: ON624286.1), 21SD-16 (GenBank:\nON624287.1), BVDV1/GS (GenBank: PV806860.1), and 21NM-44\n(GenBank: ON411191.1).\n2.4 Other pathogens for specific testing\nIn consideration of the current epidemiological landscape of\nvarious viral pathogens in Chinese cattle herds, we focused on six\ncommon pathogens: foot-and-mouth disease virus (FMDV), bovine\ncoronavirus (BCoV), bovine respiratory syncytial virus (BRSV),\nbovine parvovirus (BPV), classical swine fever virus (CSFV), and\n\nbovine parainfluenza virus 3 (BPIV3). Nucleic acids were extracted\nfrom clinical serum samples that tested positive for these specific\npathogens. Viral DNA/RNA was isolated using the MagMAX Viral/\nPathogen Nucleic Acid Isolation Kit, and the extracted nucleic acids\nwere reverse transcribed into cDNA using the SuperScript IV kit. The\nresulting DNAs and cDNAs were stored at −20°C for\nsubsequent analysis.\n2.5 Designing a pair of universal primers\nand one MGB probe\nTo identify relatively conserved regions in the BVDV genome, full\ngenomic sequences of BVDV-1 and BVDV-2 strains, isolated from\nvarious countries and over different time periods, were retrieved from\nthe GenBank database (Supplementary Table S1, DOI: 10.6084/\nm9.figshare.29143592). Multi-sequence alignment analysis was\nconducted using the ClustalW program within the MegAlign software.\nBased on the identified conserved regions in the BVDV genome, a\npair of universal primers and an MGB probe were designed (Figure 1).\nAll oligonucleotides were commercially synthesized by Tsingke Co.,\nLtd. (Xi’an, China).\n2.6 Preparation of standard positive\nplasmids\nTwo representative BVDV strains, 21SD-16 (BVDV-1, GenBank:\nON624287.1) and 22Sichuan-B8 (BVDV-2, GenBank: OQ338177.1),\nwere maintained at −80°C in our laboratory. The target sequences\nderived from these two strains were amplified by PCR and cloned into\nthe pMD18-T vector (TaKaRa, China). After verification by gene\n\nAn et al.\b\n10.3389/fvets.2025.1634429\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nsequencing (Tsingke Co., Ltd., Xi’an, China), the two standard positive \nplasmids were designated as pMD18-T -BVDV1 and pMD18-T \n-BVDV2. The DNA copy number was calculated by the following \nformula: DNA copy number = (N × 6.02 × 1023 × 10−9)/(l × 660), \nwhere N is the amount of DNA in nanograms, and l is length of the \nplasmid in bp.\nThe concentrations of the pMD18-T-BVDV1 and pMD18-T-\nBVDV2 plasmids were determined to be 1.265 × 1011 copies/μL and \n8.016 × 1010 copies/μL, respectively. The standard positive plasmids \nwere serially diluted ten-fold, and the diluted plasmid standards with \ndifferent copy numbers were used as templates to evaluate the \nperformance of the BVDV TaqMan MGB qPCR assay.\n2.7 Development of a TaqMan MGB \nprobe-based qPCR assay for BVDV\nTo develop a robust detection assay, two plasmid standards were \nused as DNA templates in a TaqMan MGB probe-based qPCR assay. \nThe reaction conditions were systematically optimized using response \nsurface methodology (RSM) to establish the final qPCR protocol. \nInitial optimization involved fixing the MGB probe concentration at \n0.2 μmol/L and evaluating the optimal primer concentration (0.35, \n0.40, or 0.45 μmol/L). Based on the optimized primer concentration, \nwe then assessed the optimal MGB probe concentration (0.25, 0.30, \nor 0.35 μmol/L). Finally, the annealing temperature (58°C, 60°C, or \nFIGURE 1\nSequence alignment information for primers and probes. The nucleotide bases color-coded as follows: cytosine (C) in blue, thymine (T) in red, adenine \n(A) in green, and guanine (G) in purple.\n\nAn et al.\b\n10.3389/fvets.2025.1634429\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n62°C)\nwas\noptimized\nusing\nthe\nselected\nprimer\nand\nprobe concentrations.\n2.8 Construction of the standard curve\nTo establish the standard curve for the TaqMan qPCR assay with\nan MGB probe, a series of diluted vector solutions (ranging from 103\nto 108 copies/μL) were used as DNA templates and amplified under\nthe optimized detection conditions. To ensure reliability, each\nconcentration was analyzed in triplicate.\n2.9 Estimations for sensitivity and\nspecificity of the assay\nBased on the optimized detection system, we  evaluated the\nsensitivity of TaqMan qPCR with MGB probes using a series of nucleic\nacid concentrations ranging from 100 copies/μL to 104 copies/μL as\nDNA templates. Additionally, sensitivity tests were conducted on\nplasmid pMD18-T-BVDV1, with concentrations ranging from\n1.265 × 100 to 1.265 × 106 copies/μL, using a conventional PCR\ninstrument. Deionized water was used as the negative control. To\nfurther assess the specificity of the detection method, we used TaqMan\nqPCR with MGB probes to detect BVDV and tested the cross-\nreactivity with other non-BVDV pathogens previously mentioned.\n2.10 Inclusivity assessment of a TaqMan\nqPCR assay for BVDV detection\nEight representative epidemic BVDV strains—22NX-69, 22AH-1,\n21SD-16, 21NM-44, 22Anhui-7, 22Gansu-F2, BVDV1/GS, and\n22Sichuan-B8—were selected for inclusivity assessment. Viral RNA\nwas extracted from each strain, reverse transcribed into cDNA, and\nsubsequently analyzed using the established TaqMan MGB\nqPCR assay.\n2.11 Assessment of repeatability and\nreproducibility of TaqMan qPCR for BVDV\ndetection\nTo evaluate repeatability, the assay was tested using plasmid\nstandards pMD18-T-BVDV1 and pMD18-T-BVDV2 at three\nconcentrations (103, 105, and 107 copies/μL). Both intra-assay\n(within-run) and inter-assay (between-run) precision were assessed\nby performing triplicate measurements. The arithmetic mean (x̄),\nstandard deviation (SD), and coefficient of variation (CV) of Ct values\nwere calculated to assess method stability and repeatability.\n2.12 Detection of BVDV in clinical serum\n\nThe arithmetic mean (x̄),\nstandard deviation (SD), and coefficient of variation (CV) of Ct values\nwere calculated to assess method stability and repeatability.2.12 Detection of BVDV in clinical serum\nsamples from multiple regions of China\nTo evaluate the clinical applicability of the TaqMan-MGB qPCR\nassay for BVDV detection, bovine serum samples collected from\nmultiple provinces in China were analyzed. The performance of our\nassay was compared with a commercial BVDV qPCR kit (IDEXX\nRealPCR™, IDEXX Laboratories, Shanghai, China) using clinical\nsamples. The positive coincidence rate was calculated as follows:\n(number of concordant positive results)/ [number of concordant\npositives + (TaqMan MGB qPCR negatives that were IDEXX method\npositives)] × 100%.\n2.13 BVDV phylogeny based on TaqMan\nMGB qPCR detection\nTo determine the prevalent BVDV-1 and BVDV-2 genotypes\nacross five Chinese provinces, one TaqMan MGB qPCR-positive\namplicon from each province was randomly selected for direct\nsequencing (Tsingke Biotechnology Co., Ltd., Xi’an, China). Sequence\nalignment was performed using the ClustalW algorithm in MEGA 5.0\nsoftware, followed by phylogenetic tree construction using the\nneighbor-joining method with 1,000 bootstrap replicates.\n3 Results\n3.1 Development and validation of a\nTaqMan MGB probe-based qPCR assay\nUsing Design-Expert 13 software (State-Ease Inc., USA),\nwe optimized and analyzed the reaction conditions through response\nsurface methodology (RSM). The model identified significant\nparameter interactions (p < 0.05), with the optimal conditions shown\nin Figure 2. The optimal amplification conditions were determined\nwhen the fluorescence signal intensity reached a plateau. The final\nparameters included a primer concentration of 0.42 μmol/L, a probe\nconcentration of 0.30 μmol/L, and an annealing temperature\nof 60.2°C.\nTo validate the detection performance (sensitivity and specificity),\nwe further optimized the TaqMan MGB qPCR assay under practical\nconditions. The final reaction conditions were as follows: 0.4 μmol/L\nprimer, 0.3 μmol/L MGB probe, and an annealing temperature of\n\n2°C.To validate the detection performance (sensitivity and specificity),\nwe further optimized the TaqMan MGB qPCR assay under practical\nconditions.The final reaction conditions were as follows: 0.4 μmol/L\nprimer, 0.3 μmol/L MGB probe, and an annealing temperature of\n60°C. The 20 μL reaction mixture contained 10 μL of 2 × Pro Taq HS\nProbe Premix (Accurate Biotechnology, China), 1 μL template DNA,\n0.8 μL each of upstream/downstream primers (BVDV-F/R, 10 μM),\n0.6 μL MGB probe (BVDV-P, 10 μM), and ddH₂O to adjust the\nvolume. The thermal cycling protocol included an initial denaturation\nat 95°C for 2 min, followed by 45 cycles of 95°C for 5 s and 60°C\nfor 30 s.\n3.2 Standard curve generation for BVDV\nquantification\nStandard curves were generated using GraphPad Prism version\n8.0.1 (GraphPad Software, USA). The logarithm of plasmid copy\nnumbers (x-axis) was plotted against Ct values (y-axis), yielding\nlinear regression equations:y = −3.355x + 41.555 (R2 = 0.998) and\ny = −3.386x + 41.601 (R2 = 0.996) for the two standards, respectively\n(Figure  3). The amplification efficiencies were 98.6 and 97.4%,\nwhich\nmeet\nthe\nMIQE\nguidelines\ncriteria\n(R2 > 0.98,\n90% < E < 110%). These results demonstrate a strong linear\n\nAn et al.\b\n10.3389/fvets.2025.1634429\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nrelationship between Ct values and logarithmic plasmid\ncopy numbers.\n3.3 Evaluation of TaqMan qPCR specificity\nand sensitivity for BVDV detection\nDetection specificity of the TaqMan MGB qPCR assay\ndemonstrated\nthat\nonly\nBVDV-1(21SD-16)\nand\nBVDV-\n2(22Sichuan-B8) yielded characteristic sigmoidal amplification\ncurves. No cross-reactivity was observed with other bovine pathogens,\nincluding FMDV, BCoV, BRSV, BPIV3, CSFV, BPV, or with the\nnegative control (ddH₂O) (Figure 4).\nAssay sensitivity was evaluated using serial dilutions of the plasmid\nstandards. The minimum detectable copy numbers were 1.265 copies/\nμL for pMD18-T-BVDV1 and 8.016 copies/μL for pMD18-T-BVDV2\n(Figure 5). Both plasmid standards were tested in 20 replicates at their\nrespective detection limits, yielding a 100% detection rate in both cases\n(detection rate >95%). Thus, the overall limit of detection (LOD) for the\nFIGURE 2\nResponse Surface Methodology (RSM) Optimization of BVDV TaqMan qPCR. (A) Effect of primer-probe concentration interaction on Ct values.\n(B) Effect of primer-probe concentration interaction on ΔRn. (C) Effect of primer-annealing temperature interaction on Ct values. (D) Effect of primer-\nannealing temperature interaction on ΔRn. (E) Effect of probe-annealing temperature interaction on Ct values. (F) Effect of probe-annealing\ntemperature interaction on ΔRn.\nFIGURE 3\nTaqMan MGB qPCR Standard Curve for BVDV Quantification. (A) Plasmid standard pMD18-T-BVDV1 (1.265 × 103 ~ 1.265 × 108 copies/μL) was used as\nthe template. (B) Plasmid standard pMD18-T-BVDV2 (8.016 × 103 ~ 8.016 × 108 copies/μL) was used as the template.\nFIGURE 4\nSpecificity Test of the BVDV TaqMan MGB qPCR Detection Method.\nIn the presented amplification graph, the x-axis represents the\nnumber of cycles, while the y-axis represents the relative\nfluorescence units (RFU). The amplification conditions for the\nfollowing viral nucleic acids were tested: BVDV-1 (21SD-16), BVDV-2\n(22Sichuan-B8), FMDV, BCoV, BRSV, BPV, CSFV, BPIV3, and a\nnegative control (ddH₂O).\n\nAn et al.\b\n10.3389/fvets.2025.1634429\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nFIGURE 6\nSensitivity test for conventional PCR detection of BVDV. The target gene was amplified using plasmid standard pMD18-T-BVDV1 and analyzed by\nnucleic acid electrophoresis. In the electrophoresis pattern, Lane M: Molecular weight standard (DL 2000 DNA Marker); Lane 1: Negative control; Lane\n2 ~ 8: Plasmid standard concentrations ranging from 1.265 × 106 to 1.265 × 100 copies/μL.\nassay was established at 1.265 copies/μL. For comparison, conventional\nPCR using serially diluted pMD18-T-BVDV1 demonstrated a detection\nlimit of 1.265 × 103 copies/μL. This result confirms that the TaqMan\nMGB qPCR assay offers significantly enhanced sensitivity relative to\nconventional PCR (Figure 6).\n3.4 Inclusion analysis\nThe inclusive test (n = 3) demonstrated 100% detection of eight\nclassical genotypic BVDV strains by the TaqMan® MGB qPCR assay\nestablished in this study. The results are shown in Figure 7. This\nindicates that the newly developed assay is highly inclusive of the eight\nclassical epidemic strains.\n3.5 Repeatability analysis\nIntra-assay and inter-assay repeatability tests were conducted using\nvarying copy numbers of two plasmid standards as templates. The intra-\nassay coefficients of variation (CVs) ranged from 0.083 to 0.490%, while\nthe inter-assay CVs ranged from 0.873 to 1.167%. All CVs were below\n1.5% (Table 1), indicating high precision. These results demonstrate that\nthe established qPCR assay exhibits excellent repeatability and stability.\n3.6 Detection of clinical samples\nTo further evaluate the clinical applicability of the assay for the\ndifferential diagnosis of BVDV-associated viral infections, a total of 174\nFIGURE 5\nAnalytical sensitivity evaluation of TaqMan MGB qPCR for BVDV Detection. The amplification curves for BVDV-1 and BVDV-2 were generated using\nplasmid standards pMD18-T-BVDV1 and pMD18-T-BVDV2, respectively. In panels (A) and (B), the plasmid concentrations for curves 1 to 5 ranged from\n\nThe amplification curves for BVDV-1 and BVDV-2 were generated using\nplasmid standards pMD18-T-BVDV1 and pMD18-T-BVDV2, respectively.In panels (A) and (B), the plasmid concentrations for curves 1 to 5 ranged from\n1.265 × 104 to 1.265 × 100 copies/μL for BVDV-1 and from 8.016 × 104 to 8.016 × 100 copies/μL for BVDV-2. Curve 6 represents the negative control.\n\nAn et al.\b\n10.3389/fvets.2025.1634429\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nbovine serum samples were tested using the established TaqMan MGB\nqPCR method. As shown in the bar chart (Figure 8), the results were fully\nconsistent with those obtained using a commercial BVDV nucleic acid\ndetection kit, yielding a 100% positive agreement rate. The overall positive\ndetection rate among the tested serum samples was 33.9%. Region-\nspecific positive rates were as follows: 25.0% (7/28) in Henan Province,\n47.2% (17/36) in Jilin Province, 27.8% (15/54) in Gansu Province, 37.5%\n(9/24) in Sichuan Province, and 34.4% (11/32) in Qinghai Province. These\nresults demonstrate that the developed qPCR assay possesses excellent\nclinical applicability for BVDV detection in diverse field settings.\n3.7 Genetic evolution analysis of BVDV 5′-\nUTR gene\nIn the analysis of clinical samples, sequence homology and\nphylogenetic tree construction were performed on BVDV-\npositive samples collected from different regions. The results\nrevealed that the Henan strain shared 97.44% nucleotide identity\nwith the BVDV-1 HN1736 strain (GenBank accession no.\nMN442374.1). The Jilin strain exhibited 93.80% homology with\nthe BVDV-1 NM2312 isolate (GenBank accession no.\nPP992316.2). The Qinghai strain showed 96.58% similarity to the\nBVDV-1 MRI3543 strain (GenBank accession no. LR822280.1).\nThe Gansu strain demonstrated 98.32% homology with the\nBVDV-2 B9497 isolate (GenBank accession no. MH231134.1),\nwhile the Sichuan strain shared 97.48% identity with the same\nBVDV-2 isolate.\nPhylogenetic analysis further confirmed that the Henan, Jilin,\nand Qinghai strains clustered within the BVDV-1 genotype, while the\nGansu and Sichuan strains grouped within the BVDV-2 genotype.\nNotably, the Henan and Jilin strains were closely related, forming a\ndistinct phylogenetic branch, suggesting a potential shared lineage or\ntransmission source. Similarly, the Gansu and Sichuan strains\nclustered together, indicating a close genetic relationship and\nsuggesting possible interregional transmission between Aba\n\nSimilarly, the Gansu and Sichuan strains\nclustered together, indicating a close genetic relationship and\nsuggesting possible interregional transmission between Aba\nPrefecture in Sichuan Province and the Gannan region in Gansu\nProvince (see Figure 9).\n4 Discussion\nSince the first isolation and identification of BVDV in China in\nthe 1980s, numerous outbreaks have been reported across various\nFIGURE 7\nInclusivity analysis of TaqMan MGB qPCR for BVDV detection. In the presented amplification graph, the x-axis represents the number of cycles, while\nthe y-axis represents the relative fluorescence units (RFU). The amplification conditions for the following BVDV strains were tested: 21SD-16, 22NX-69,\n21NM-44, 22AH-1, 22Anhui-7, 22Sichuan-B8, 22Gansu-F2, BVDV1/GS, and a negative control (ddH₂O).\nTABLE 1  Intra-group and inter-group repeatability verification of TaqMan MGB qPCR methods.\nPlasmid\nstandards\nNumber of\ncopies\n(Copies/μL)\nIntra- group reproducibility tests\nInter-group reproducibility tests\nMean\nvalue x̄\nStandard\ndeviation SD\nCoefficient of\nvariation CV\n(%)\nMean\nvalue x̄\nStandard\ndeviation SD\nCoefficient of\nvariation CV\n(%)\npMD18-T-BVDV1\n1.265 × 107\n17.80\n0.040\n0.226\n17.99\n0.175\n0.971\n1.265 × 105\n24.15\n0.118\n0.490\n24.01\n0.257\n1.071\n1.265 × 103\n31.45\n0.026\n0.083\n31.49\n0.275\n0.873\npMD18-T-BVDV2\n8.016 × 107\n14.80\n0.062\n0.417\n14.91\n0.161\n1.077\n8.016 × 105\n21.62\n0.090\n0.415\n21.77\n0.192\n0.880\n8.016 × 103\n28.40\n0.065\n0.231\n28.81\n0.336\n1.167\n\nAn et al.\b\n10.3389/fvets.2025.1634429\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nprovinces (10). As a highly contagious disease that poses a significant\nthreat to cattle health, BVDV has become endemic in many regions of\nChina. The prevalent BVDV genotypes vary among countries and\nregions, and with the shift toward intensive, large-scale farming in\nChina, the increased cross-regional movement of cattle has created\nfavorable conditions for the spread of infectious diseases.\nBVDV-infected animals may develop persistent infections (PI)\nand immunosuppression, both of which exacerbate disease severity\nand predispose affected herds to co-infections. Notably, PI cattle\ncontinuously shed the virus throughout their lives, serving as major\nreservoirs for BVDV transmission (29). Furthermore, PI animals are\nat risk of progressing to fatal mucosal disease (BVD-MD), which\nrepresents a severe threat to the sustainable development of China’s\nlivestock industry (30, 31). Therefore, early detection and timely\nelimination of PI cattle are crucial for reducing the risk of BVDV\noutbreaks and ensuring effective BVDV control and eradication.\nDue to the virus’s widespread distribution, high mutation rate,\nand genetic adaptability, numerous BVDV subtypes have emerged,\nenabling the virus to evade host immune responses (4). This genetic\ndiversity necessitates continuous improvements in diagnostic\ntechniques to facilitate broad-spectrum detection across genotypes\nand the early identification of animals in the latent stage of infection.\nSuch advancements are vital for BVDV prevention and control, herd-\nlevel virus eradication, quality monitoring of veterinary biological\nproducts, and the reduction of economic losses in the cattle industry.\nIn this study, we developed a TaqMan MGB-based real-time PCR\nassay targeting the conserved regions of the 5′-UTR gene across\nBVDV-1 and BVDV-2 subtypes. Primers and MGB probes were\ndesigned based on sequence alignment of representative strains\ndownloaded from NCBI. The assay was optimized for specificity and\nvalidated against common bovine pathogens, including FMDV, BCoV,\n\nPrimers and MGB probes were\ndesigned based on sequence alignment of representative strains\ndownloaded from NCBI.The assay was optimized for specificity and\nvalidated against common bovine pathogens, including FMDV, BCoV,\nBRSV, CSFV, BPIV3, and BPV. Only BVDV-1 and BVDV-2 strains\nyielded positive amplification signals, confirming the assay’s high\nspecificity and lack of cross-reactivity. The limit of detection was\ndetermined to be 1.265 copies/μL, demonstrating superior sensitivity\ncompared to previously reported methods. For example, a SYBR\nGreen-based qPCR method developed by Hou et  al. (32) had a\ndetection limit of 5.2 copies/μL, and a TaqMan probe assay reported\nby Zhang et al. (33) showed a detection threshold of 10 copies/μL. The\nenhanced sensitivity of our assay is attributed to the use of MGB\nprobes, which utilize fluorescence resonance energy transfer (FRET)\nprinciples and can detect single-nucleotide mismatches with high\nprecision (34). Furthermore, MGB probes offer improved thermal\nstability, minimizing degradation under ambient conditions, a\nlimitation common in traditional molecular beacon systems (35, 36).\nRepeatability tests indicated a coefficient of variation below 1.5%\nfor both intra- and inter-assay experiments, reflecting the method’s\nrobust stability and reliability. Clinical validation with 174 field\nsamples demonstrated a 100% concordance rate with results from a\ncommercial BVDV nucleic acid detection kit, confirming the assay’s\nhigh accuracy and practical applicability. The ability to sensitively\ndetect low viral loads and differentiate BVDV from other pathogens,\ncombined with a rapid turnaround time (typically within 1–2 h),\nsupports the assay’s suitability for clinical diagnostics and large-scale\nepidemiological surveillance.\nDespite these advantages, certain limitations remain. Given the high\ngenetic variability of BVDV, future improvements should focus on\nexpanding primer and probe coverage to include a broader range of viral\nsubtypes and emerging variants. Additionally, the sample size used for\n\nGiven the high\ngenetic variability of BVDV, future improvements should focus on\nexpanding primer and probe coverage to include a broader range of viral\nsubtypes and emerging variants.Additionally, the sample size used for\nclinical validation was relatively limited. Subsequent studies involving\nlarger sample sets from diverse geographic regions and herd types are\nneeded to further assess the assay’s broad applicability and robustness.\n5 Conclusion\nIn summary, this study developed a TaqMan MGB-based real-\ntime PCR assay for the detection of BVDV genotypes 1 and 2. The\nassay demonstrated high specificity, excellent sensitivity, strong\nrepeatability, and good clinical applicability. Combined with sequence\nanalysis, it enabled precise differentiation between BVDV-1 and\nBVDV-2 without cross-reactivity with other bovine viruses. Clinical\nsample results showed strong agreement with those obtained from\ncommercial kits, and phylogenetic analysis confirmed the assay’s\nuniversal detection capabilities for both major BVDV genotypes. This\nmethod provides valuable technical support for BVDV control\nefforts, including herd-level purification, clinical diagnostics, vaccine\ndevelopment, and epidemiological monitoring.\nFIGURE 8\nDetection of bovine serum clinical samples. (A) Comparison of TaqMan qPCR assay with commercial nucleic acid detection kit. (B) Distribution of\nBVDV positive clinical samples in different regions of China.\n\nAn et al.\b\n10.3389/fvets.2025.1634429\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nData availability statement\nThe datasets presented in this study can be  found in online \nrepositories. The names of the repository/repositories and accession \nnumber(s) can be found in the article/supplementary material.\nEthics statement\nThe animal study was approved by Animal Ethics Committee of \nthe College of Life Science and Engineering, Northwest Minzu \nUniversity. The study was conducted in accordance with the local \nlegislation and institutional requirements.\nAuthor contributions\nLA: Data curation, Formal analysis, Investigation, Writing – original \ndraft. XR: Methodology, Writing – review & editing. YeZ: Methodology, \nWriting – review & editing. SZ: Methodology, Writing – review & editing. \nYoZ: Conceptualization, Funding acquisition, Investigation, Methodology, \nProject administration, Supervision, Writing – review & editing.\nFunding\nThe author(s) declare that financial support was received for the \nresearch and/or publication of this article. This research was funded \nFIGURE 9\nGenetic evolution of BVDV 5’-UTR gene in positive clinical samples from different regions of China.\n\nAn et al.\b\n10.3389/fvets.2025.1634429\nFrontiers in Veterinary Science\n10\nfrontiersin.org\nby the Lanzhou Science and Technology Bureau, with the research\nfunding number being 2023-1-31.\nAcknowledgments\nWe are grateful to cattle farmers from different provinces in China\nfor providing us with the samples needed for this research.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as potential conflicts of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. 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+{"text": "【兽医文献】\n标题: Dose-dependent modulation of hepatic cytochrome P450 enzymes by tenvermectin: implications for medication safety and combination therapy\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nGPS tracking of free-roaming\ndogs and human spillover risk of\nEchinococcus granulosus in\nhighly endemic Peru\nKatherine Morucci 1, Lizzie Ortiz Cam 2, Elvis W. Diaz 2,\nGuillermo Porras-Cotrina 2, Javier Bustos 3, Manuela Verastegui 4,\nCesar M. Gavidia 5 and Ricardo Castillo-Neyra 1,2,6*\n1 School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, United States, 2 One\nHealth Unit, School of Public Health and Administration, Universidad Peruana Cayetano Heredia,\nLima, Peru, 3 Center for Global Health, Universidad Peruana Cayetano Heredia, Lima, Peru, 4 Infectious\nDiseases Laboratory Research–LID, Faculty of Sciences and Philosophy, Universidad Peruana\nCayetano Heredia, Lima, Peru, 5 School of Veterinary Medicine, Universidad Nacional Mayor de San\nMarcos, Lima, Peru, 6 Department of Biostatistics, Epidemiology and Informatics, Perelman School of\nMedicine at University of Pennsylvania, Philadelphia, PA, United States\nIntroduction: Cystic echinococcosis (CE), a neglected disease that results from\ninfection with the larval stage of the Echinococcus granulosus sensu lato (s.l.)\ntapeworm, poses significant zoonotic risk to humans and is a persistent threat\nin developing agricultural communities around the world. While the prevalence\nof human CE in the central highlands of Peru has previously been estimated\naround 5–7%, true prevalence is likely higher given the protracted period of\nasymptomatic disease, reduced medical access of at-risk populations, increased\ncontact between herders, livestock, and herding dogs, and poor understanding\nof local disease epidemiology. To beIer understand CE epidemiology in a highly\nendemic region of Peru, we studied the movement of free-roaming dogs in the\ncommunity of Chanchayllo, Junin, Peru.\nMethods: We performed copro-ELISA to identify E. granulosus s.l. positive\ndogs, tracked the ranging behavior and calculated home ranges of 19 owned,\nfree-roaming dogs to understand the movement of the definitive host of\nE. granulosus s.l. on the landscape. Specifically, we investigated the spatial\n\ngranulosus s.l.positive\ndogs, tracked the ranging behavior and calculated home ranges of 19 owned,\nfree-roaming dogs to understand the movement of the definitive host of\nE.granulosus s.l.on the landscape.Specifically, we investigated the spatial\nassociation between E. granulosus-infected dog home ranges and proximity to\ntheir owners’ houses and a local slaughterhouse.\nResults: Echinococcus granulosus s.l. infection prevalence was alarmingly high\nin our canine population, with 85% positivity (binomial exact 95% CI: 62.1–\n96.8%). All dog home ranges overlapped with their owners’ households, and\nnotably, even negative dog households overlapped with nearby positive dog\nhome ranges.\nDiscussion: These data suggest that widespread environmental contamination\nof E. granulosus s.l. egg-containing feces may be a significant driver of locally\nelevated disease prevalence in human populations. We use our findings to\nunderstand the local disease ecology of CE in free roaming dogs, assess pillover\nrisk, and guide future intervention strategies aimed at reducing human cases.\nOur findings suggest that existing trategies delivering anthelmintic drugs to\nindividual households have the potential to reduce spillover of E. granulosus s.l.\nKEYWORDS\nanimal movement, dog, Echinococcus granulosus, GPS tracking, one health, spatial\nanalysis, tapeworm\nOPEN ACCESS\nEDITED BY\nVerónica Roxana Flores,\nNational University of Comahue, Argentina\nREVIEWED BY\nEdmundo Larrieu,\nNational University of La Pampa, Argentina\nMarina De La Reta,\nCONICET Patagonia Norte, Argentina\n*CORRESPONDENCE\nRicardo Castillo-Neyra\ncricardo@upenn.edu\nRECEIVED 15 June 2025\nACCEPTED 28 October 2025\nPUBLISHED 18 November 2025\nCITATION\nMorucci K, Ortiz Cam L, Diaz EW, ​\nPorras-Cotrina G, Bustos J, Verastegui M,\nGavidia CM and ​Castillo-Neyra R (2025) GPS\ntracking of free-roaming dogs and human\nspillover risk of Echinococcus granulosus in\nhighly endemic Peru.\nFront. Vet. Sci. 12:1647590.\ndoi: 10.3389/fvets.2025.1647590\nCOPYRIGHT\n© 2025 Morucci, Ortiz Cam, Diaz,\nPorras-Cotrina, Bustos, Verastegui, Gavidia\nand Castillo-Neyra. This is an open-access\n\nFront.Vet.Sci.12:1647590.doi: 10.3389/fvets.2025.1647590\nCOPYRIGHT\n© 2025 Morucci, Ortiz Cam, Diaz,\nPorras-Cotrina, Bustos, Verastegui, Gavidia\nand Castillo-Neyra.This is an open-access\narticle distributed under the terms of the\nCreative Commons Attribution License\n(CC BY). The use, distribution or reproduction\nin other forums is permitted, provided the\noriginal author(s) and the copyright owner(s)\nare credited and that the original publication\nin this journal is cited, in accordance with\naccepted academic practice. No use,\ndistribution or reproduction is permitted\nwhich does not comply with these terms.\nTYPE  Original Research\nPUBLISHED  18 November 2025\nDOI  10.3389/fvets.2025.1647590\n\nMorucci et al.\b\n10.3389/fvets.2025.1647590\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nIntroduction\nFree-roaming dogs play a critical role in the ecology of\nEchinococcus granulosus sensu lato (s.l.), the parasitic tapeworm that\ncauses cystic echinococcosis (CE) in humans and animals. CE is a\nzoonotic disease that poses a significant threat to human and animal\nhealth, with canines serving as definitive hosts and livestock as\nintermediate hosts (1, 2). Infection remains a significant public health\nconcern in regions that engage in livestock production, such as the\ncentral Andes in Peru (3). Previous studies have emphasized the\nprevalence of CE in regions like Junin due to the close proximity and\ninteraction between dogs and livestock, which are central to the\neconomy and agricultural practices of these areas (4, 5).\nEchinococcus granulosus has an indirect life cycle which requires\ninfection of multiple hosts to successfully survive from an egg to\nsexual maturity. The reproductive stage of E. granulosus parasitizes the\ngastrointestinal tract of its definitive host, wild and domestic canids\n[CDC-DPDx (6–8)]. Eggs shed in feces are highly environmentally\nstable and may persist outside of a host for extended periods of time\n(8, 9). Parasite eggs hatch upon consumption by an intermediate host,\nmigrate into the bloodstream, and become lodged in viscera where\nparasite larvae contribute to slow-growing cystic lesions [CDC-DPDx\n(6, 8, 10)]. To complete its life cycle, E. granulosus larva must\nbe consumed by scavenging canids feeding upon infected offal. While\nherbivorous species such as sheep tend to serve as the intermediate\nhost, unfortunately, humans that consume E. granulosus s.l. eggs may\nalso become infected and develop cysts in various organs, such as the\nlungs and liver (11–13). Without medical screening, CE may go\nundetected for years, or until cysts grow large enough to contribute to\nlife-threatening disease that warrants surgical intervention (4, 14–17).\nThat said, surgical interventions may not be readily available to the\nmost vulnerable populations due to reduced medical access in\n\nThat said, surgical interventions may not be readily available to the\nmost vulnerable populations due to reduced medical access in\ndeveloping agricultural economies (18–20). The persistence of CE in\nendemic regions poses a serious threat to human health and wellbeing.\nHalting transmission requires a clear understanding of animal\nreservoirs, environmental conditions that favor parasite persistence,\nand human behaviors that facilitate spillover. This challenge calls for\na truly One Health approach—where animal, human, and\nenvironmental health overlap and where integrated interventions are\nmost urgently needed (21).\nThe district of Canchayllo, in Junin, Peru, is a rural region located\nin the Central Andes. This region is known for its agricultural\neconomy, and specifically, many families in this district engage in\nlivestock management. The interdependence between sheep, herders,\nand herding dogs is central to this lifestyle. Unfortunately, these\nconditions provide a favorable environment for the transmission of\nE. granulosus s. l. Previous studies have estimated the prevalence of\nhuman CE in the central highlands of Peru at 5–7% based on\nultrasound-based population surveys (3, 19). However, we believe that\nprevalence is likely higher given the protracted period of asymptomatic\ndisease, reduced medical access of at-risk populations, high degree of\ncontact between herders, dogs, and their livestock, and poor\nunderstanding of local disease epidemiology (19, 22).\nLittle is known about the contribution of free-roaming dogs to the\nepidemiology of CE in the Central Andes in Peru due to the limited\nnumber of integrative research initiatives bridging human and\nveterinary health. This study sought to better understand E. granulosus\ns.l. spillover risk, defined here as the transmission of parasite eggs\nfrom infected dogs to humans as an accidental, dead-end host, by\nexamining parasite prevalence and ranging behavior in free-roaming\ndogs. While we refer to this phenomenon as spillover throughout the\nmanuscript, we acknowledge that this term is usually used to describe\n\nWhile we refer to this phenomenon as spillover throughout the\nmanuscript, we acknowledge that this term is usually used to describe\ncross-species transmission events involving biological barriers that\nprevent sustained transmission. In this case, humans serve as a\ndead-end host as they are susceptible to infection upon exposure, but\ndo not permit subsequent infection of other hosts. Our goal was to\nestablish an environmentally focused understanding of regional\nfeatures that may be contributing to local disease transmission. To this\nend, we tracked free-roaming dogs to understand the movement of\nthe definitive host of E. granulosus on the landscape and performed\ncopro-ELISA to identify E. granulosus s.l. positive animals.\nMost studies on echinococcosis in dogs have focused on working\ndogs. In contrast, our study aimed to better understand pet dogs,\nwhich may also play an important role in transmission to humans.\nTherefore, our first aim was to estimate the intensity of space use (e.g.,\nprobability of dog presence in specific areas) to inform spatially-\ntargeted dog interventions. Specifically, we investigated the spatial\nassociation between E. granulosus s.l.-infected dog home ranges and\nproximity to their owners’ houses and a local slaughterhouse. In\naddition, knowing that free-roaming dogs often defecate along the\nborders of their territory to establish a “property line” (23), our second\naim was to estimate the home range and core areas used by these dogs\nas a proxy for the spatial extent of spillover risk (e.g., transmission\nfrom dog feces to humans). We use our findings to increase our\nunderstanding of free-roaming dog ecology and spillover risk to\ninform interventions aimed at reducing human infection.\nMethods\nStudy area\nThis study was conducted from June to July of 2023 in the district\nof Canchayllo, Jauja, Junin, Peru. Chanchayllo is one of the 34 districts\nin the province of Jauja and is located in the Central Andes within the\nNor Yauyos Cochas Landscape Reserve. The district covers an area of\n974.69 km2 and is located at 3,800 masl. The climate is cold, with\n\nChanchayllo is one of the 34 districts\nin the province of Jauja and is located in the Central Andes within the\nNor Yauyos Cochas Landscape Reserve.The district covers an area of\n974.69 km2 and is located at 3,800 masl.The climate is cold, with\ntemperatures ranging between 6 and 20 degrees Celsius.\nThis region is home to 1,601 inhabitants, most of whom are engaged\nin livestock farming, and specifically, sheep herding (24). The\ninterdependence between humans, their dogs, and sheep herds\nassociated with agrarian lifestyles not only provides a favorable\nenvironment for the completion of the E. granulosus s.l. life cycle, but also\nfor the accidental spillover into human populations. Moreover, suspected\nand actual case data from nearby medical centers between 2019 and 2022\nsuggest that up to 31% of the total population within this district exhibits\nclinical signs compatible with CE symptomatology (unpublished data).\nThis suggests prevalence in this area may be much higher than previously\npublished findings in endemic regions (3, 19). We decided to conduct\nour study in two communities within this highly endemic district, given\nthe unique occupational and lifestyle hazards presented to herders and\ntheir family members and suspected case prevalence.\nTwo sites within the district of Canchayllo were selected for\ninclusion in this study: the communities of San Juan de Pachacayo and\nCanchayllo (Figure  1). The selection of each site was based on\nproximity to a regional slaughterhouse that does not have appropriate\n\nMorucci et al.\b\n10.3389/fvets.2025.1647590\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nbarriers to prevent canine access (Figure 2), and thus may serve as a\npotential source of contaminated offal for free-roaming dogs.\nMoreover, the presence of a local river served as a geographical barrier\nthat we predicted would permit dog movement between the study sites\nwhile limiting significant East–West displacements. We hypothesized\nthat (1) free-roaming owned dogs associated with households near the\nslaughterhouse in the community of Pachacayo would be more likely\nto be infected with E. granulosus s.l. than dogs associated with homes\nin the community of Canchayllo, and (2) that the home ranges of\nE. granulosus-infected dogs will overlap with a majority of households\nin the study communities, representing a high risk of parasite spillover.\nSelection and recruitment\nWe identified potential participants based on the vicinity of their\nhomes to the Pachacayo slaughterhouse and the presence of free-\nroaming dogs outside or near their household. Specifically,\nwe attempted to balance participation from households located in the\ncommunities of Pachacayo and Canchayllo to determine if the presence\nof the slaughterhouse influenced dog movement and infection.\nWe then conducted abbreviated door-to-door interviews to identify a\nsubset of animals that were owned but spent the majority of their time\nfree-roaming outside of the owner’s household. Owners were informed\nof study objectives, agreed to have feces collected from their pet, and\nto have the location of their dog tracked and mapped over the study\nperiod of 10 days. We recorded the sex, age, and physical characteristics\nof enrolled dogs and collected brief information about the degree of\ninteraction between the dog and any other animals or livestock kept in\nor near the house. We did not intend to identify individual-level factors\nassociated with positivity status or ranging behavior, and thus\nwe prioritized inclusion based on location of owner’s homes and dog\ndemographic features were unbalanced. Exclusion criteria included\n\nWe did not intend to identify individual-level factors\nassociated with positivity status or ranging behavior, and thus\nwe prioritized inclusion based on location of owner’s homes and dog\ndemographic features were unbalanced.Exclusion criteria included\ndogs that were 6-months of age or younger, small-bodied breeds/dogs,\nand animals with restricted access to the outdoors. After identifying a\nsubset of dogs to enroll, we asked owners to restrain their animals while\nwe placed a GPS-embedded collar around their necks.\nData collection\nWe identified 20 dogs to enroll in our study. Dogs were manually\nrestrained by their owners to be fitted with cloth collars containing a\nMobile Action i-gotU GT-600 GPS logger. Based on our previous\nwork (25), GPS loggers were programmed to record GPS coordinates\nof the animal at 3-min intervals. Dogs were visited daily to monitor\nthe GPS tracker battery levels and the quality of recorded data. One\ndog was immediately lost to follow up due to the removal of the collar\nand tracking device, but collars remained in place and devices were\nsuccessfully retrieved from the other 19 animals included in this study.\nWhile collar retrieval times varied between 7 to 10 days, GPS loggers\nwere occasionally found turned off and thus recording times varied\nfrom 3 to 10 days.\nFecal sample management and\nCopro-ELISA analysis\nApproximately 2 grams of fresh feces were collected from each of\nour study animals. After asking owners about their dogs’ elimination\nFIGURE 1\nMap of the study region demonstrating (A) the location of the District of Canchayllo, Jauja - Junin, within the central Andes of Peru, and a street view\nshowing the interconnectivity between the two study sites, the communities of San Juan de Pachacayo and Canchayllo, and their relation to the\nPachacayo slaughterhouse and participant households, and (B) a photograph of a street containing houses in the community of Canchayllo.\n\nMorucci et al.\b\n10.3389/fvets.2025.1647590\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nroutines, our team waited for each animal to defecate to collect their\nfeces. If a dog was not observed defecating during the initial visit,\nwe conducted multiple follow-up visits to avoid the use of canine\npurgative treatments. In the field, fecal samples were saved and\npreserved in PBS formalin and potassium dichromate and maintained\nat room temperature during transport to Laboratorio de Epidemiología\ny Economía Veterinaria, Facultad de Medicina Veterinaria,\nUniversidad Nacional Mayor de San Marcos, Lima, Peru. The time\nbetween collection and arrival at the lab varied between 2 to 10 days.\nTo identify positive samples in the lab, we implemented a validated\nsandwich copro-ELISA technique based on the capture and detection\nof somatic E. granulosus antigens (26). A cut-off point was selected as\nthe highest value for both sensitivity and specificity by using ROC\ncurves over the already known infected and non-infected dog\nstool samples.\nSpatial data analysis\nThe GPS data collected was downloaded as csv files from the\nbackend of the iGotU application and exported into the statistical\nsoftware R1 for further analysis and cleaning.\n1  https://www.R-project.org, version 3.4.0\nFor each dog, we used two distinct computational methods to\ncalculate dog home ranges (HRs) and core areas based on their\nabilities to address our aim of identifying areas that may present\nelevated spillover risk to owners based on dog movement and\ninfection status. First, we generated HRs and core areas based on the\nminimum convex polygon (MCP) approach using the adehabitatHR\npackage in R. To determine the most appropriate stable HR level for\nour analyses, we plotted 80–100% MCP HR levels for all dogs(S1).\nWe found that most dogs’ HRs were stable through the 95% level, and\nthat ranging behavior beyond this point was highly variable. As such,\nHRs were calculated by mapping the smallest polygon that fit 95% of\neach dog’s nearest ranging data points. The furthest 5% of ranging\npoints for all dogs were excluded as random visitations. The MCP\n\nAs such,\nHRs were calculated by mapping the smallest polygon that fit 95% of\neach dog’s nearest ranging data points.The furthest 5% of ranging\npoints for all dogs were excluded as random visitations.The MCP\npolygons were used to estimate HR areas and to calculate spatial\nvariables concerning HR geometry, such as HR centroids. HR\ncentroids were then used to measure distances between each animal’s\nHR and structures of interest, such as their owner’s household and the\nslaughterhouse. Core areas, which reveal where dogs spend the\nmajority of their time, were calculated using the 50% isopleth.\nWe also calculated 95 and 50% isopleths using the kernel\nutilization distribution (KUD) approach to determine areas where\ndogs spend the greatest proportion of their time within their HR. This\ntechnique assesses the density of ranging data points and can\ndistinguish between regions where a dog is most likely to be found at\nany given point in time, from areas that are more transiently occupied,\nsuch as those used to traverse a core area or HR. We  used this\nFIGURE 2\nImages of the Pachacayo slaughterhouse which permits access to scavenging free-roaming dogs. Despite the presence of an external barrier (A), dogs\nare often found on the inside of the wall (B) due to the gate being left open during operation hours, and can readily access the facility from entrances\nat the back of the property. Moreover, dogs are frequently found throughout the facility where all animal processing takes place (C), where we have\nobserved them consuming discarded offal.\n\nMorucci et al.\b\n10.3389/fvets.2025.1647590\nFrontiers in Veterinary Science\n05\nfrontiersin.org\napproach, and specially the 50% isopleth, to identify where a dog\nwould most likely be encountered for targeted interventions.\nStatistical analysis\nNon-parametric analyses were performed to identify associations\nbetween infection status, dog demographic data, HR size and distances\nbetween HRs and areas of interest. All further statistical analyses were\nconducted in R.\nEthics\nIACUC ethical approval was obtained from Universidad Peruana\nCayetano Heredia (approval number: 67258) and the University of\nPennsylvania (approval number: 806514).\nResults\nCopro-ELISA\nWe collected 20 total fecal samples. Of those samples, 18 were\ncollected from dogs included in our study, but we were unable to observe\none of the collared dogs defecate during our time in the field. Two\nadditional samples were taken from dogs that were found frequently\nranging alongside dogs in our study. Overall, 17 of 20 dogs (85%;\nbinomial exact 95% CI:62.1–96.8%) were copro-ELISA positive (Table 1).\nDog ranging analysis\nUsing the MCP approach, we estimated HRs areas for each dog\nin our study (Figure 3). We calculated the weighted centroid for\neach dog’s HR, which was then used to determine the distances\nbetween animals’ HRs, their owners’ household, and the Pachacayo\nslaughterhouse (Table 1). The average distance between a dog’s HR\nand household is 289.74 m, which is significantly less than the\naverage distance between HRs and the slaughterhouse, 1720.46 m\n(Mann–Whitney U test: p = 2.192e-05). There was only a single\ncase in which a dog’s MCP core area did not overlap or border their\nowner’s home, indicating that 18/19 dogs in our study spend the\nvast majority of their time near home. In addition, only seven dogs\nhad HRs that overlapped with the slaughterhouse. The association\nbetween HRs that overlap with the slaughterhouse and E. granulosus\ns.l. infection status was insignificant (Fisher’s test: p = 0.26). Finally,\nHR size did not influence infection status (Mann–Whitney U test:\np = 0.07332). No individual-level dog demographic factors were\n\ngranulosus\ns.l.infection status was insignificant (Fisher’s test: p = 0.26).Finally,\nHR size did not influence infection status (Mann–Whitney U test:\np = 0.07332).No individual-level dog demographic factors were\nstatistically associated with positivity status or ranging behavior.\nTABLE 1  Ranging behavior and home range sizes of dogs in Canchayllo, Junin, Peru.\nDog\nID\nSex\nAge\n(y)\nCommunity\nInfected\n95%\nMCP\nHR\nsize\n(km2)\n95%\nKUD\nHR\nsize\n(km2)\n50%\nMCP\nCore\nArea\nsize\n(km2)\n50%\nKUD\nCore\nArea\nsize\n(km2)\nDistance\n95% MCP\nHR\ncentroid\nand home\n(m)\nDistance\n95% MCP\nHR centroid\nand\nslaughter-\nhouse (m)\n1\nM\n7\nPachacayo\n1\n1.000\n1.092\n0.514\n0.045\n691.77\n338.17\n2\nF\n6\nCanchayllo\n1\n0.020\n0.029\n0.002\n0.002\n13.37\n2546.80\n3\nF\n4\nPachacayo\n0\n0.037\n0.036\n0.003\n0.003\n30.00\n461.48\n4\nM\n1\nPachacayo\n1\n1.644\n1.830\n0.307\n0.196\n639.25\n441.09\n5\nM\n2\nCanchayllo\n1\n0.329\n0.166\n0.010\n0.009\n74.44\n4105.04\n6\nM\n2\nCanchayllo\n0\n0.707\n1.385\n0.050\n0.217\n81.10\n4521.32\n7\nM\n1\nCanchayllo\n1\n4.810\n3.806\n0.660\n0.446\n1735.44\n5311.12\n8\nM\n1\nCanchayllo\n1\n0.258\n0.208\n0.009\n0.012\n155.58\n3200.84\n9\nM\n1\nPachacayo\n1\n0.401\n0.422\n0.035\n0.040\n136.76\n228.61\n10\nM\n2\nPachacayo\n1\n0.175\n0.099\n0.009\n0.007\n151.04\n186.50\n11\nM\n1\nPachacayo\n1\n0.048\n0.097\n0.004\n0.004\n36.80\n346.62\n12\nM\n3\nPachacayo\n1\n1.975\n1.637\n0.342\n0.138\n880.29\n629.44\n13\nF\n4\nPachacayo\n1\n0.097\n0.128\n0.011\n0.002\n80.97\n369.89\n14\nM\n2\nCanchayllo\n0\n0.048\n0.043\n0.003\n0.003\n12.05\n3738.54\n15\nM\n2\nPachacayo\n1\n0.259\n0.168\n0.009\n0.007\n68.40\n246.60\n16\nM\n2\nPachacayo\n1\n0.123\n0.076\n0.006\n0.007\n58.22\n149.49\n17\nM\n5\nPachacayo\n1\n0.024\n0.032\n0.002\n0.001\n1.58\n1001.82\n18\nM\n7\nCanchayllo\n1\n0.014\n0.016\n0.001\n0.001\n18.46\n3673.71\n19\nM\n1\nPachacayo\n1\n0.026\n0.042\n0.003\n0.005\n95.30\n1001.73\nMCP, Minimum convex polygon; KUD, Kernel utilization distribution; HR, Home range.\n\nMorucci et al.\b\n10.3389/fvets.2025.1647590\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nUsing the MCP approach, all dogs’ home ranges overlapped with\ntheir owners’ households as well as with many other houses in the\ncommunities. To explore the potential threat of free-roaming dogs on\nE. granulosus spillover to humans, we estimated the geospatial spread\nof KUD HRs across the study region. Home ranges generated using\nthe KUD function provide a more accurate model of spatial utilization\nbehavior in that they distinguish between areas where dogs commonly\nspend time from the paths that animals use to traverse between areas\nof interest. In other words, the KUD-generated HRs provide a better\nestimation of risk areas. We overlaid copro-ELISA positivity status on\nKUD HRs to create a visual risk map depicting E. granulosus\ns.l.-positive dogs HRs, or areas that are likely contaminated with feces\nfrom infected animals (Figure 4). Notably, the HRs of the 16 infected\nanimals overlap with all participant households in the study region.\nThis suggests that there is a significant risk of spillover to individuals\nliving in these communities based on environmental contamination.\nDiscussion\nEchinococcus granulosus s.l. infection prevalence was alarmingly\nhigh in our canine population, with 85% positivity. Importantly, the\nFIGURE 3\nHome ranges and core areas for each dog were estimated using two distinct methods. (A) The minimum convex polygon (MCP) approach was\nimplemented to calculate the smallest polygon that encapsulates the nearest 95% of ranging data points. Using the 95% MCP home ranges,\nwe calculated centroids (red cross-hatch) to measure distances from the weighted center of each dog’s home range to their owner’s home (dark gray\ncircles) and the slaughterhouse. We also estimated home ranges using the kernel utilization distribution (KUD) approach. The 95% KUD home ranges\nwere used to determine where a dog is most likely to be found throughout their home range, and as a proxy to determine risk of environmental\n\nWe also estimated home ranges using the kernel utilization distribution (KUD) approach.The 95% KUD home ranges\nwere used to determine where a dog is most likely to be found throughout their home range, and as a proxy to determine risk of environmental\ncontamination due to the probability of encountering egg-containing feces on the landscape (B). Core areas were calculated using the 50% MCP\n(C) and KUD (D) isopleths, respectively.\n\nMorucci et al.\b\n10.3389/fvets.2025.1647590\nFrontiers in Veterinary Science\n07\nfrontiersin.org\ntotal area covered by E. granulosus-infected dog home ranges overlap\nwith all households in the study communities. This is a remarkable\nfinding considering that we only sampled 20 dogs and suggests that\nwidespread environmental contamination of E. granulosus\negg-containing feces may be  a significant driver of local disease\npersistence and a high exposure risk for individuals living in this area.\nAlthough dogs tend to defecate repeatedly in specific locations within\ntheir territory (23), contamination is not necessarily limited to these\nareas. Several studies have shown that Echinococcus eggs are highly\nresistant in the environment and can be dispersed passively by biotic\nand abiotic vectors, such as wind and coprophagous insects (27–29).\nThese mechanisms allow egg dissemination in a radial pattern from\nthe original site of deposition, potentially expanding the\ncontamination zone beyond the dog’s most heavily used areas. This\nhighlights both the importance of considering both direct deposition\nsites and secondary dispersal routes when assessing environmental risk.\nOur observations of the slaughterhouse revealed that, despite a\nperimeter wall and gate, it remained accessible to free-roaming dogs\nthrough open entrances and rear access points. A few dogs were seen\nconsuming discarded viscera inside the premises, with no formal\nsystem for offal disposal or personnel restricting access. Thus, the\nfacility represents a potential source of infection. But we found no\nsignificant association between infection status, home range size, or\nproximity to the slaughterhouse. In fact, dogs in our study rarely\nvisited the slaughterhouse, and when they did, these visits did not\ncoincide with operational hours, suggesting that this facility is unlikely\nto be a major source of infection. However, many families in the study\nregion do not rely on the slaughterhouse, and instead slaughter\nlivestock near their homes, a practice often accompanied by feeding\ndiscarded offal deemed unfit for human consumption to dogs (30–32).\n\nHowever, many families in the study\nregion do not rely on the slaughterhouse, and instead slaughter\nlivestock near their homes, a practice often accompanied by feeding\ndiscarded offal deemed unfit for human consumption to dogs (30–32).\nThis pattern is consistent with our findings, as all dog home ranges\noverlapped with owners’ households, and even those with the smallest\nhome ranges that do not overlap with the slaughterhouse were\npositive. Although our questionnaire did not directly ask about\nat-home slaughter, informal conversations with participants and other\ncommunity members, as well as direct field observations, confirmed\nthat this is a widespread practice in both study communities. This\nrisky behavior likely plays an important role in the high observed\nprevalence, yet remains largely unaddressed by health programs\nin Peru.\nPrevious intervention strategies to control E. granulosus\ntransmission in the central highlands of Peru, including Junín, have\nprimarily stemmed from government-led mass treatment campaigns\nfocused on periodic deworming of dogs using praziquantel (33–36).\nThese efforts were often accompanied by educational messaging\ndiscouraging the feeding of offal to dogs and encouraging improved\nslaughterhouse hygiene (37, 38). However, such campaigns have faced\nsignificant limitations, including inconsistent implementation due to\nlimited resources, lack of follow-up, low community participation, and\nlimited integration of veterinary and public health services (39). In\nmany rural communities like Canchayllo, access barriers and limited\nengagement of local stakeholders have further weakened the\neffectiveness of these interventions (40, 41). Given our findings—\nparticularly the persistence of infection in dogs and their frequent\nmovement between households and communal areas—these past\nstrategies have been insufficient to disrupt the parasite’s life cycle. A\nmore sustainable and targeted approach is urgently needed, including\nenhanced surveillance and context-specific education campaigns\ngrounded in a One Health framework (42).\n\nA\nmore sustainable and targeted approach is urgently needed, including\nenhanced surveillance and context-specific education campaigns\ngrounded in a One Health framework (42).\nAn important finding of this study was that the HRs of all dogs\nand all but one dog’s core area overlap with their households,\nindicating that they spend most of their time near their owners’\nhomes. This information supports that existing intervention strategies\nbased on delivering anthelmintic drugs to individual households have\nthe potential to reduce the environmental presence of infectious\nE. granulosus eggs. Delivering deworming agents to dog owners would\noffer a simple solution to ensure that dogs receive the necessary\npreventative care to curtail the zoonotic cycle that places owners at\nincreased risk of infection. Given that dogs are often found near home,\nthere would be plenty of opportunities for public health inspectors\nand dog owners to find the dogs and to offer them consumable\npreventative treatment at necessary intervals to provide protection\nagainst reinfection. While this would not address pre-existing\nenvironmental contamination, it would be a critical step in reducing\nfurther spillover risk to humans.\nOur study has several strengths, but also some limitations. While\nwe distributed our sampling across the different study communities,\nwe did not have access to a household roster to conduct true random\nsampling. Additionally, the tracking period for animals varied and was\nlimited to less than a week. It is possible that seasonal events (e.g.,\nlivestock fairs) influence movement patterns in ways that could affect\nE. granulosus s.l. transmission. We did not use purgatives, which may\nFIGURE 4\nKUD dog home ranges and their households colored by E.\ngranulosus-s.l. infection status. Red home ranges indicate E.\ngranulosus s.l.-positive animals and blue home ranges correspond to\nnegative animals. All households are overlapped by positive dog\nhome ranges.\n\nMorucci et al.\b\n10.3389/fvets.2025.1647590\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nhave increased the chances of false negatives; however, given the high\nprevalence we observed, we believe this did not significantly affect our\nresults. Finally, we did not explore in detail the practice of home\nslaughtering by dog owners, which may help explain some of\nour findings.\nOur findings suggest that in areas where E. granulosus s.l. is highly\nendemic (43), humans may face a high risk of infection regardless of\nthe infection status of their own dog due to environmental\ncontamination and overlap by nearby positive, free-roaming dog\nhome ranges. Additionally, dogs experience high infection rates\nregardless of direct access to slaughterhouses, highlighting the\nimportance of informal and home-based slaughtering practices. The\nconsistent overlap between dogs’ core areas and their owners’\nhouseholds underscores the potential for household-targeted\ninterventions. Our findings support other studies that propose\nintegrated control strategies that go beyond slaughterhouse\nmanagement and include regular deworming of dogs, safe disposal of\nlivestock offal, community education on zoonotic transmission routes,\nand improved surveillance of canine infections. Targeting\ninterventions at the household and community level is likely to\nbe essential for reducing human risk and breaking the transmission\ncycle of this neglected but persistent zoonosis. Targeted interventions\ncould be informed by the spatial ecology of free-roaming dogs, which\nis closely intertwined with human behavior, the environment, and\nlivestock management systems. While our study contributes valuable\ndata to the understanding of free-roaming dog ecology, it represents\nonly one component of a broader, complex system. Additional\nresearch is essential to build a comprehensive evidence base capable\nof supporting integrated, One Health–oriented approaches to the\ncontrol of this persistent zoonotic disease.\nData availability statement\nThe original contributions presented in the study are publicly\navailable. This data can be found here: https://doi.org/10.5281/\n\nData availability statement\nThe original contributions presented in the study are publicly\navailable.This data can be found here: https://doi.org/10.5281/\nzenodo.17081023.\nEthics statement\nThe animal studies were approved by IACUC ethical approval was\nobtained from Universidad Peruana Cayetano Heredia (approval\nnumber: 67258) and University of Pennsylvania (approval number:\n806514). The studies were conducted in accordance with the local\nlegislation and institutional requirements. Written informed consent\nwas obtained from the owners for the participation of their animals in\nthis study.\nAuthor contributions\nKM: Conceptualization, Data curation, Formal analysis,\nInvestigation, Visualization, Writing – original draft, Writing – review\n& editing, Methodology. LO: Conceptualization, Investigation, Project\nadministration, Supervision, Writing – original draft, Writing – review\n& editing. ED: Conceptualization, Data curation, Formal analysis,\nMethodology, Visualization, Writing – original draft, Writing – review\n& editing. GP-C: Investigation, Writing – original draft, Writing –\nreview & editing. JB: Investigation, Supervision, Writing – original\ndraft, Writing – review & editing. MV: Formal analysis, Validation,\nWriting  – original draft, Writing  – review & editing. CG:\nConceptualization, Formal analysis, Investigation, Supervision,\nWriting  – original draft, Writing  – review & editing. RC-N:\nConceptualization, Funding acquisition, Supervision, Writing  –\noriginal draft, Writing – review & editing.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. This project was supported\nby NIH-NIAID (K01AI139284 to RCN) and the Fogarty International\nCenter (D43TW012741 to RCN, EWD, and LOC). The funders had\nno role in study design, data collection and analysis, decision to\npublish, or preparation of the manuscript.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\n\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.Generative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. Any product\nthat may be evaluated in this article, or claim that may be made by its\nmanufacturer, is not guaranteed or endorsed by the publisher.\nSupplementary material\nThe Supplementary material for this article can be found online\nat: https://www.frontiersin.org/articles/10.3389/fvets.2025.1647590/\nfull#supplementary-material\n\nMorucci et al.\b\n10.3389/fvets.2025.1647590\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nReferences\n1.\tCraig PS, McManus DP, Lightowlers MW, Chabalgoity JA, Garcia HH, Gavidia CM,\net al. Prevention and control of cystic echinococcosis. Lancet Infect Dis. (2007) 7:385–94.\ndoi: 10.1016/S1473-3099(07)70134-2\n2.\tCraig PS, Rogan MT, Campos-Ponce M. 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+{"text": "【兽医文献】\n标题: Editorial: Germplasm conservation and semen banking in livestock: strategies, challenges, and applications\n\nTYPE Original Research\nPUBLISHED 26 June 2026\nDOI 10.3389/fvets.2026.1878293\nOPEN ACCESS\nEDITED BY\nTugay Ayasan,\nOsmaniye Korkut Ata University, Türkiye\nREVIEWED BY\nYuanxiao Li,\nHenan University of Science and\nTechnology, China\nErcan Mevliyaogullari,\nBa¸skent University, Türkiye\n*CORRESPONDENCE\nRuiping Li\nliruipin@wzvcst.edu.cn\nSuzhen Liu\nliusuzhen@wzvcst.edu.cn\nRECEIVED 11 May 2026\nREVISED 04 June 2026\nACCEPTED 09 June 2026\nPUBLISHED 26 June 2026\nCITATION\nLi R, Liu S, Dong L, Liu Z, Xu S, Zeng T,\nLi C, Ma H and Chai Y (2026) Cordyceps\ncicadae polysaccharide and plant extract\nas a potential antibiotic-free feed\nadditive: effects on growth performance,\nantioxidant capacity, and cecal\nmicrobiota of Yandang chickens.\nFront. Vet. Sci. 13:1878293.\ndoi: 10.3389/fvets.2026.1878293\nCOPYRIGHT\n© 2026 Li, Liu, Dong, Liu, Xu, Zeng, Li,\nMa and Chai. This is an open-access\narticle distributed under the terms of the\nCreative Commons Attribution License\n(CC BY). The use, distribution or\nreproduction in other forums is\npermitted, provided the original author(s)\nand the copyright owner(s) are credited\nand that the original publication in this\njournal is cited, in accordance with\naccepted academic practice. No use,\ndistribution or reproduction is permitted\nwhich does not comply with these terms.\nCordyceps cicadae\npolysaccharide and plant extract\nas a potential antibiotic-free feed\nadditive: effects on growth\nperformance, antioxidant\ncapacity, and cecal microbiota of\nYandang chickens\nRuiping Li1*, Suzhen Liu1*, Liyan Dong1, Zhangguo Liu2,\nShixiang Xu1, Tao Zeng3, Chong Li1, He Ma1 and Yiqiu Chai4\n1Wenzhou Vocational College of Science and Technology, Wenzhou, China, 2Zhejiang A&F University,\nHangzhou, China, 3Zhejiang Academy of Agricultural Sciences, Hangzhou, China, 4Key Laboratory of\nEntomogenous Fungus Resources Research and Development of Wenzhou, Zhejiang Institute of\nSubtropical Crops, Wenzhou, China\nThis study was conducted to investigate the effects of a composite of\nCordyceps cicadae polysaccharide (CCP) and plant extract (PE) on growth\nperformance, serum antioxidant status, and cecal microbiota of Yandang\n\nchickens, a Chinese indigenous breed, with the aim of evaluating its potential\nas a natural feed additive candidate for antibiotic-free poultry production. A\ntotal of 315 1-day-old healthy Yandang chickens were randomly allocated\nto seven groups: blank control, vaccinated control, antibiotic control, CCP\ngroup, PE group, low-dose combination group, and high-dose combination\ngroup. Supplementation with CCP at 50 mg/kg BW significantly increased\naverage daily gain (ADG) compared with the vaccinated control (p < 0.05);\nthe high-dose combination did not differ significantly from the CCP group in\nADG. All additive-treated groups significantly elevated serum total protein and\nalbumin levels while reducing creatinine content (p < 0.05). The CCP group\nand high-dose combination group significantly increased serum total antioxidant\ncapacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and glutathione\nperoxidase (GSH-Px) activities, and markedly decreased malondialdehyde (MDA)\ncontent (p < 0.05), with the high-dose combination showing the strongest\nantioxidant activity among all treatment groups. Cecal microbiota analysis\nrevealed that the high-dose combination significantly increased the relative\nabundance of Bacteroidetes, a phylum known for its capacity to degrade\ncomplex polysaccharides. PERMANOVA based on Bray–Curtis distance revealed\nsignificant differences in overall microbial community structure among groups\n(p = 0.001). In conclusion, CCP supplementation was associated with improved\ngrowth performance, while the CCP-PE composite, especially the high-dose\ncombination, enhanced antioxidant capacity and modulated cecal microbiota in\nYandang chickens under the conditions of this study. The high-dose combination\nFrontiers in Veterinary Science\n01\nfrontiersin.org\n\nLi et al.\n10.3389/fvets.2026.1878293\nexerted the optimal comprehensive effects on antioxidant capacity and intestinal\nflora, and may be considered a promising natural feed additive candidate for\nantibiotic-free poultry production.\nKEYWORDS\nantibiotic-free production, antioxidant status, cordyceps cicadae polysaccharide,\ngrowth performance, gut microbiota, Yandang chicken\n1 Introduction\nThe global ban on in-feed antibiotics, driven by growing\nconcerns over bacterial resistance, drug residues, and intestinal\nmicrobiota disturbance, has created an urgent need for safe, green,\nand residue-free alternatives in sustainable poultry production.\nChinese herbal polysaccharides and plant extracts are considered\npromising natural feed additive candidates due to their antioxidant,\nimmunomodulatory, and gut microbiota-modulating properties\nand leave no harmful drug residues after application (1).\nCordyceps cicadae polysaccharide (CCP) is a major bioactive\ncomponent of the traditional entomopathogenic fungus Cordyceps\ncicadae. Our previous studies have demonstrated that CCP\nimproves antioxidant capacity (2), enhances immune function (3),\nand improves growth performance (4) in chickens. Recent evidence\nhas further shown that CCP can regulate signaling pathways such\nas NF-κB and MAPK and modulate gut microbiota metabolism (5).\nSimilarly, plant extracts (PE) have been widely reported\nto improve nutrient digestibility, intestinal morphology, and\nantioxidant status in poultry (6–8). However, most studies have\nfocused on the individual effects of CCP or plant extracts\nalone, while the combined effects and co-application potential of\nCCP and PE remain largely unexplored. Given their functional\ncomplementarity-polysaccharides acting as prebiotic substrates to\nmaintain gut microbial balance, and plant extracts providing potent\nantioxidant and anti-inflammatory activities-we hypothesize that\ntheir combined use may produce a more comprehensive protective\neffect than either component alone.\nTo test this hypothesis, the present study was conducted\nto investigate the effects of dietary supplementation with CCP,\n\nTo test this hypothesis, the present study was conducted\nto investigate the effects of dietary supplementation with CCP,\nPE, and their combinations on growth performance, serum\nbiochemical\nand\nantioxidant\nindices,\nand\ncecal\nmicrobiota\ncommunity structure in Yandang chickens, a Chinese indigenous\nbreed. This study aims to determine the optimal application\nscheme and provide a scientific basis for developing the CCP-PE\ncomposite as a novel green feed additive candidate for antibiotic-\nfree poultry production.\n2 Materials and methods\n2.1 Ethics statement\nThe animal trial was conducted at the Wenzhou Seed\nand Seedling Science Park. All experimental procedures were\napproved by the Institutional Animal Care and Use Committee\nof the Zhejiang Academy of Agricultural Sciences (Approval\nNo.: 2022ZAASLA59). All efforts were made to minimize\nanimal suffering, and humane treatment was implemented during\nall procedures, including sample collection and euthanasia by\ncervical dislocation.\n2.2 Experimental reagent\nCCP was provided by the Zhejiang Institute of Subtropical\nCrops (Zhejiang, China). It was prepared as a light-yellow powder\nfrom the fruiting bodies of Cordyceps cicadae through drying, hot-\nwater extraction, ethanol precipitation, deproteinization via the\nSevag method, macroporous resin purification, and final drying,\nfollowing established protocols.\nA compound essential oil product (Guangdong Reborn\nTechnology Group Co., Ltd., Guangdong, China) was used as\nthe PE. This additive was a flowable, microencapsulated powder.\nAccording to the manufacturer’s specification, its main active\ncomponents were cinnamaldehyde (≥6%), carvacrol (≥0.2%), and\neucalyptus oil (≥0.5%), formulated with mixed botanical extracts\nas the carrier (≥30%) derived from Coptis chinensis, Cinnamomum\ncassia, Syzygium aromaticum, and Pogostemon cablin.\nZinc bacitracin (Lukang Biochemistry Co., Ltd., batch no.\nP181013) was used as the antibiotic control. The live Newcastle\ndisease vaccine (La Sota strain) was obtained from Bomeilai\nBiological Products Co., Ltd.\n2.3 Experimental animals and management\nA total of 315 one-day-old healthy Yandang chickens (half male\n\n, batch no.P181013) was used as the antibiotic control.The live Newcastle\ndisease vaccine (La Sota strain) was obtained from Bomeilai\nBiological Products Co., Ltd.2.3 Experimental animals and management\nA total of 315 one-day-old healthy Yandang chickens (half male\nand half female) with similar initial body weights were obtained\nfrom Zhejiang Lüyan Co., Ltd. (Zhejiang, China). The experiment\nwas conducted at the Wenzhou Seed and Seedling Science Park.\nBirds were housed in a double-tiered cage system under natural\nlighting and ventilation. Routine sanitation procedures were strictly\nimplemented throughout the 42-day experimental period.\nChickens were randomly allocated to seven treatment groups\nwith three replicates per group and 15 birds per replicate. All\nbirds had ad libitum access to feed and clean drinking water.\nA basal diet formulated to meet the nutritional requirements of\nYandang chickens was provided by Wenzhou Zhengtai Agriculture\nand Animal Husbandry Co., Ltd.; its composition and nutritional\nlevels are presented in Table 1.\nExcept for the blank control group (Group I), all birds were\nvaccinated against Newcastle disease via eye drop with a live\nattenuated vaccine (La Sota strain) on days 14 and 28. Health\nstatus and general behavior were observed and recorded daily. The\nblank control was intentionally maintained without vaccination to\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n\nLi et al.\n10.3389/fvets.2026.1878293\nTABLE 1 Ingredients and nutrient levels of the antibiotic-free basal diet (%, air-dry basis).\nIngredient\nContent (1–3\nweeks)\nContent (4–6\nweeks)\nCorn\n58.60\n67.70\nWheat bran\n8.35\n5.10\nWheat middlings\n6.00\n6.00\nCorn gluten meal\n6.00\n7.00\nSoybean meal\n16.95\n10.1\nLimestone\n1.55\n1.55\nPremix∗\n2.55\n2.55\nTotal\n100.00\n100.00\nNutrient Levels#\nNutrient\nContent (1–3 weeks)\nContent (4–6 weeks)\nMetabolizable\nenergy (MJ/kg)\n12.60\n12.70\nCrude protein\n21.27\n19.94\nCalcium\n1.01\n0.85\nTotal phosphorus\n0.6\n0.54\nMethionine\n0.50\n0.40\nLysine\n1.15\n1.10\n∗The premix provided per kilogram of diet: Vitamin A 5,000 IU, Vitamin B1 9.8 mg, Vitamin\nB2 28.8 mg, Vitamin B6 19.6 mg, Vitamin B12 0.1 mg, Vitamin D 10,000 IU, Vitamin E 75.0 mg,\nVitamin K3 18.8 mg, nicotinic acid 196.5 mg, folic acid 4.8 mg, biotin 2.5 mg, Cu (as copper\nsulfate) 4.0 mg, Fe (as ferrous sulfate) 40.0 mg, Zn (as zinc sulfate) 37.6 mg, Mn (as manganese\nsulfate) 50.0 mg, Se (as sodium selenite) 0.2 mg, I (as potassium iodide) 0.2 mg.\n#Nutrient levels were calculated values.\nserve as a non-immunized baseline reference, to assess the potential\ngrowth depression associated with vaccination.\n2.4 Experimental design\nA single-factor completely randomized design was adopted.\nA total of 315 Yandang chickens were randomly assigned to\nseven treatment groups, with three replicates of 15 birds each.\nThe treatment groups were as follows: blank control (Group\nI), vaccinated control (Group II), antibiotic control (Group III),\nCCP alone at 50 mg/kg BW (Group IV), low-dose combination\nof CCP 25 mg/kg BW + PE 125 mg/kg BW (Group V),\nhigh-dose combination of CCP 50 mg/kg BW + PE 125\nmg/kg BW (Group VI), and PE alone at 250 mg/kg BW\n(Group VII). Detailed treatment information is presented in\nTable 2.\nAll additives were administered via oral gavage (0.5 mL per\nbird) once daily at 14:00 during two periods: days 11–17 and\ndays 25–31. This route was selected to ensure precise and\nuniform intake of the test substances by each individual bird.\nAll groups, including the blank and vaccinated controls, received\nthe same gavage and eye-drop procedures with physiological\nsaline to eliminate any potential confounding effects from\n\nAll groups, including the blank and vaccinated controls, received\nthe same gavage and eye-drop procedures with physiological\nsaline to eliminate any potential confounding effects from\nhandling stress.\n2.5 Measurements and analytical methods\n2.5.1 Determination of growth performance\nOn days 10 and 42 of the experiment, all chickens were fasted\nfor 12 h, and their individual fasting body weights were accurately\nrecorded. Feed intake per replicate was precisely determined by\nrecording the amount of feed offered and the amount refused\nthroughout the experimental period. ADG, ADFI, and F/G were\ncalculated for each treatment group.\n2.5.2 Determination of serum biochemical and\nantioxidant indices\nOn day 42, six birds were randomly selected from each replicate\n(18 birds per treatment group) for blood collection from the wing\nvein. Blood samples were left to clot at ambient temperature before\ncentrifugation at 3,000 r/min for 15 min at 4 ◦C to separate the\nserum. The serum samples were stored at −20 ◦C until analysis.\nSerum concentrations of TP, TC, ALB, TG, CREA, and\nUN were determined using colorimetric kits (Beijing Huaying\nBiotechnology Research Institute, Beijing, China) on a Mindray\nBS-420 automatic biochemical analyzer. Serum T-AOC, as well as\nthe activities of SOD, GSH-Px, CAT, and the content of MDA,\nwere measured using corresponding colorimetric kits from the\nsame manufacturer on an A6 semi-automatic biochemical analyzer\n(Beijing Songshang Technology Co., Ltd., Beijing, China), following\nthe manufacturer’s instructions.\n2.5.3 Analysis of cecal microbial diversity\nOn day 42, three birds were randomly selected from each\nreplicate (nine birds per treatment group) and euthanized by\ncervical dislocation. The cecal contents were aseptically collected\ninto sterile cryopreservation tubes, immediately frozen in liquid\nnitrogen, and stored at −80 ◦C until DNA extraction.\nTotal bacterial genomic DNA was extracted from the cecal\ncontent samples using the PowerSoil R⃝DNA Isolation Kit (MO BIO\nLaboratories, Carlsbad, CA, USA) according to the manufacturer’s\nprotocol. The concentration and purity of the extracted DNA were\n\nTotal bacterial genomic DNA was extracted from the cecal\ncontent samples using the PowerSoil R⃝DNA Isolation Kit (MO BIO\nLaboratories, Carlsbad, CA, USA) according to the manufacturer’s\nprotocol.The concentration and purity of the extracted DNA were\ndetermined by A260/A280 ratio, and DNA integrity was verified by\n1.8% agarose gel electrophoresis.\nThe V3-V4 hypervariable region of the bacterial 16S rRNA\ngene was amplified by PCR using the universal primer pair\n338F\n(5′-ACTCCTACGGGAGGCAGCA3′)\nand\n806R\n(5′-\nGGACTACHVGGGTWTCTAAT3′).\nPCR\namplification\nwas\nperformed using high-fidelity DNA polymerase (Q5, New England\nBiolabs). The PCR products were purified using VAHTSTM\nDNA Clean Beads, quantified using Quant-iTTM dsDNA HS\nreagent, and pooled in equal amounts. Sequencing libraries\nwere constructed using the TruSeq\nR⃝DNA PCR-Free Sample\nPreparation Kit (Illumina, San Diego, CA, USA), and library\nquality was assessed using a Qubit fluorometer (Thermo Fisher\nScientific, Waltham, MA, USA). Paired-end sequencing (PE250)\nwas performed on the Illumina HiSeq 2,500 platform by Beijing\nBiomarker Technologies Co., Ltd. (Beijing, China).\nFrontiers in Veterinary Science\n03\nfrontiersin.org\n\nLi et al.\n10.3389/fvets.2026.1878293\nTABLE 2 Experimental design and treatment details.\nGroup\nTreatment description\nAdditive and dosage (administered in\n0.5 mL solution per bird)\nVaccination status\nI\nBlank control\nPhysiological saline (0 mg/kg BW)\nNo\nII\nVaccinated control\nPhysiological saline (0 mg/kg BW)\nYes∗\nIII\nAntibiotic control\nZinc bacitracin (50 mg/kg BW)\nYes∗\nIV\nCordyceps cicadae polysaccharide\nCCP (50 mg/kg BW)\nYes∗\nV\nLow-dose combination\nCCP (25 mg/kg BW) + PE (125 mg/kg BW)\nYes∗\nVI\nHigh-dose combination\nCCP (50 mg/kg BW) + PE (125 mg/kg BW)\nYes∗\nVII\nPlant extract\nPE (250 mg/kg BW)\nYes∗\n∗Birds in Groups II to VII were vaccinated against Newcastle disease on days 14 and 28.\nBW, body weight; CCP, Cordyceps cicadae polysaccharide. The PE refers to the compound essential oil product described in Section 2.2.\nRaw sequencing reads were subjected to quality control,\nfiltering, and assembly to generate high-quality clean tags.\nOperational taxonomic units (OTUs) were clustered at 97%\nsequence similarity using USEARCH software (9). Taxonomic\nassignment of OTUs was performed against the Silva bacterial\ntaxonomic database (Release 132). Alpha diversity indices (Chao1,\nAce, Shannon, and Simpson) and beta diversity (PCoA) were\ncalculated and visualized using the QIIME (version 1.9.1) pipeline\nand R software v3.6.3 (The R Foundation for Statistical Computing,\nVienna, Austria).\n2.5.4 Statistical analysis\nAll experimental data were analyzed using SPSS version\n26.0 (IBM Corp., Armonk, NY, USA). For growth performance\nparameters (ADG, ADFI, and F/G), cage means served as the\nexperimental unit (n = 3 per group). For serum biochemical,\nantioxidant,\nand\ncecal\nmicrobiota\nindices,\nindividual\nbirds\nserved as the statistical unit. Differences among groups were\nexamined\nby\none-way\nANOVA\nfollowed\nby\nthe\nStudent–\nNewman–Keuls (S–N–K) test for multiple comparisons. Unless\notherwise stated, the vaccinated control (Group II) served as the\nprimary comparator for treatment effects. Statistical significance\nwas set at p < 0.05. All results are presented as mean\n± SD.\n3 Results\n3.1 Growth performance and serum\nbiochemical indices\nAs shown in Table 3, ADG was significantly higher in\n\nStatistical significance\nwas set at p < 0.05.All results are presented as mean\n± SD.3 Results\n3.1 Growth performance and serum\nbiochemical indices\nAs shown in Table 3, ADG was significantly higher in\nGroups I and IV compared with Group II (p < 0.05). ADFI\nwas significantly higher in Groups I, IV, and VI compared\nwith Group II (p < 0.05). Feed-to-gain ratio (F/G) in all\ntreatment groups (IV–VII) was numerically lower than in\nthe vaccinated control (Group II), although the differences\nwere not significant (p > 0.05). Group III also showed no\nsignificant difference in F/G compared with Group II (p\n> 0.05).\nTABLE 3 Growth performance of Yandang chickens.\nGroup\nADG (g)\nADFI (g/d)\nF/G\nI\n10.47 ± 1.58a\n26.10 ± 0.63a\n2.49 ± 0.18\nII\n9.21 ± 1.41c\n23.30 ± 0.54f\n2.53 ± 0.08\nIII\n9.56 ± 1.24abc\n23.90 ± 0.59d\n2.50 ± 0.24\nIV\n10.23 ± 1.44ab\n24.35 ± 0.41c\n2.38 ± 0.10\nV\n9.75 ± 1.63abc\n23.69 ± 0.32e\n2.43 ± 0.14\nVI\n10.06 ± 1.63abc\n24.85 ± 0.34b\n2.47 ± 0.14\nVII\n9.49 ± 1.76bc\n23.44 ± 0.71f\n2.47 ± 0.03\nIn each column, means marked with different lowercase letters differ significantly at p < 0.05;\nthis notation applies to all subsequent tables.\nSerum biochemical parameters are presented in Table 4.\nSerum TP and ALB levels in all treatment groups (IV–VII)\nwere significantly higher than those in Group II (p < 0.05).\nSerum CREA content in all treatment groups (IV–VII) was\nsignificantly lower than in the vaccinated Groups II (p <\n0.05). Serum TC was significantly higher in Groups III, IV,\nV, VI, and VII compared with Group II (p < 0.05). Serum\nUN in Group VI and TG in Group V were numerically\nlower\nthan\nin\ntheir\nrespective\ncontrol\ngroups;\nhowever,\nthese\ndifferences\ndid\nnot\nreach\nstatistical\nsignificance\n(p > 0.05).\n3.2 Serum antioxidant indices\nAs\nshown\nin\nTable 5,\nGroup\nVI\nexhibited\nthe\nhighest\nT-AOC\namong\nall\ngroups\n(p\n<\n0.05),\nfollowed\nby\nGroup\nIV,\nwhich\nwas\nsignificantly\nhigher\nthan\nall\ngroups except Group VI (p < 0.05). For SOD, CAT, and\nGSH-Px,\nboth\nGroups\nIV\nand\nVI\nexhibited\nsignificantly\nhigher activities compared with Groups I, II, and III (p\n< 0.05).\nNotably, Groups I and II also showed significantly higher T-\nAOC, SOD, CAT, and GSH-Px activities, and lower MDA content,\nthan Group III (p < 0.05).\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n\nLi et al.\n10.3389/fvets.2026.1878293\nTABLE 4 Serum biochemical parameters of Yandang chickens.\nGroup\nTP (g/L)\nTC (mmol/L)\nALB (g/L)\nCREA (μmol/L)\nUN (mg/dl)\nTG (mmol/L)\nI\n18.87 ± 7.17b\n1.89 ± 0.70bc\n9.05 ± 3.44b\n28.34 ± 8.25a\n1.26 ± 0.40\n0.37 ± 0.08\nII\n16.07 ± 4.32c\n1.57 ± 0.67c\n7.91 ± 2.47b\n29.89 ± 8.28a\n1.24 ± 0.19\n0.32 ± 0.11\nIII\n25.22 ± 2.30a\n2.30 ± 0.37ab\n11.56 ± 1.02a\n23.26 ± 2.87b\n1.26 ± 0.15\n0.38 ± 0.07\nIV\n23.28 ± 4.85a\n2.01 ± 0.51ab\n10.35 ± 1.60a\n22.54 ± 3.25b\n1.28 ± 0.18\n0.33 ± 0.08\nV\n23.66 ± 3.58a\n2.01 ± 0.31ab\n10.68 ± 1.45a\n22.49 ± 2.45b\n1.24 ± 0.18\n0.31 ± 0.08\nVI\n23.95 ± 0.15a\n2.17 ± 0.22ab\n11.08 ± 1.11a\n21.64 ± 2.59b\n1.13 ± 0.11\n0.33 ± 0.06\nVII\n25.47 ± 2.15a\n2.38 ± 0.31a\n11.58 ± 0.98a\n23.06 ± 2.25b\n1.21 ± 0.18\n0.35 ± 0.08\nDifferent lowercase superscript letters (a, b, c) within the same column represent significant differences between groups at p < 0.05; values marked with identical letters have no statistically\nsignificant difference.\nTABLE 5 Serum antioxidant indices of Yandang chickens.\nGroup\nT-AOC (U/mL)\nSOD (U/mL)\nCAT (U/mL)\nGSH-Px (U/mL)\nMDA (nmol/mL)\nI\n11.03 ± 0.82d\n71.42 ± 3.10d\n46.95 ± 4.10d\n833.07 ± 31.52d\n4.74 ± 0.55c\nII\n12.98 ± 1.20c\n79.14 ± 3.52c\n52.05 ± 5.24c\n875.88 ± 19.75c\n4.40 ± 0.35d\nIII\n7.04 ± 1.09f\n51.09 ± 1.72g\n29.50 ± 2.52g\n685.31 ± 10.53g\n6.04 ± 0.45a\nIV\n14.78 ± 1.34b\n85.71 ± 4.61b\n58.37 ± 3.81b\n919.78 ± 41.99b\n4.06 ± 0.20e\nV\n10.44 ± 0.76d\n65.90 ± 3.33e\n41.56 ± 9.96e\n811.45 ± 21.25e\n5.26 ± 0.49b\nVI\n17.11 ± 1.35a\n129.30 ± 5.19a\n65.08 ± 5.01a\n964.54 ± 22.87a\n3.12 ± 0.48f\nVII\n9.39 ± 1.82e\n60.25 ± 2.61f\n33.49 ± 4.84f\n755.64 ± 26.93f\n5.53 ± 0.31b\nLowercase letters a and b indicate significant inter-group differences at p < 0.05; means sharing the same letter are not significantly different.\n3.3 Effects on the gut microbiota of\nYandang chickens\n3.3.1 Sequencing results\nOne sample from Group IV was unavailable for sequencing;\ntherefore, a total of 62 cecal content samples were analyzed. From\nthese samples, 4,799,221 paired-end reads were obtained. After\nassembly and quality filtering, 4,561,941 clean tags were generated,\nwith a minimum of 47,773 and an average of 73,580 clean tags per\nsample. Rarefaction curves based on OTU numbers approached the\nsaturation plateau (Figure 1), indicating that the sequencing depth\nwas sufficient to capture the majority of microbial diversity present\n\nRarefaction curves based on OTU numbers approached the\nsaturation plateau (Figure 1), indicating that the sequencing depth\nwas sufficient to capture the majority of microbial diversity present\nin the samples (10).\n3.3.2 Alpha diversity of gut microbiota\nAs shown in Table 6, Group VII exhibited a numerically\nlower Simpson index and a numerically higher Shannon index\ncompared with Groups I–III, although the differences were not\nstatistically significant (p > 0.05). These results suggest that PE\nsupplementation may tend to increase the richness and evenness\nof the cecal microbiota.\n3.3.3 Beta diversity of gut microbiota\nPrincipal\ncoordinates\nanalysis\n(PCoA)\nshowed\nthat\nsamples from Groups II, IV, VI, and VII were relatively\nFIGURE 1\nRarefaction curves for cecal microbial samples of Yandang chickens.\nEach curve reflects the relationship between the number of sampled\nsequences and observed operational taxonomic units (OTUs)\nclustered at a 97% sequence similarity threshold. All curves gradually\nreach a plateau, confirming sufficient sequencing depth for\nmicrobiota analysis. Groups I–VII are defined in Table 2.\nclustered,\nindicating\nhigh\nwithin-group\nsimilarity,\nwhereas\nthose\nfrom\nGroup\nIII\nwere\nmore\ndispersed\n(Figure 2).\nPERMANOVA\nbased\non\nBray–Curtis\ndistance\nrevealed\nsignificant\ndifferences\nin\noverall\nmicrobial\ncommunity\nstructure\namong\ngroups\n(R2\n=\n0.158,\np\n=\n0.001;\nTable 7).\nFrontiers in Veterinary Science\n05\nfrontiersin.org\n\nLi et al.\n10.3389/fvets.2026.1878293\nTABLE 6 Gut microbial alpha diversity in Yandang chickens.\nGroup\nChao1\nACE\nSimpson\nShannon\nI\n296.67 ± 19.54\n294.73 ± 17.66\n0.09 ± 0.04\n3.34 ± 0.28ab\nII\n302.17 ± 12.37\n296.81 ± 9.37\n0.08 ± 0.04\n3.36 ± 0.33ab\nIII\n296.75 ± 19.21\n297.77 ± 11.02\n0.08 ± 0.04\n3.44 ± 0.32ab\nIV\n302.01 ± 9.23\n296.96 ± 8.33\n0.09 ± 0.04\n3.30 ± 0.24ab\nV\n294.76 ± 24.44\n288.85 ± 20.30\n0.11 ± 0.04\n3.18 ± 0.36b\nVI\n296.98 ± 24.35\n286.58 ± 15.06\n0.09 ± 0.03\n3.35 ± 0.25ab\nVII\n297.32 ± 16.57\n292.62 ± 18.73\n0.07 ± 0.04\n3.56 ± 0.46a\nLowercase letters a and b indicate significant inter-group differences at p < 0.05; means sharing the same letter are not significantly different.\nFIGURE 2\nPrincipal coordinates analysis (PCoA) of cecal bacterial communities\nfrom Yandang chickens based on the Bray–Curtis distance matrix.\nEach dot represents an individual sample (n = 62). The percentage of\ntotal community variation explained by PC1 and PC2 is marked on\nthe corresponding axes. Groups I–VII are defined in Table 2.\n3.3.4 Gut microbiota composition at the phylum\nlevel\nAt the phylum level, Firmicutes and Bacteroidetes were the\ndominant phyla in the cecum, accounting for 39%−49% and\n30%−45% of all sequences, respectively (Figure 3). Compared with\nGroups I–III, Group VII showed a 20.0%, 15.8%, and 1.1% increase\nin the relative abundance of Firmicutes, respectively. Group V also\nexhibited higher Firmicutes abundance than Groups I and II.\nRegarding Bacteroidetes, Group VI showed a 7.4%, 5.8%, and\n32.8% higher relative abundance compared with Groups I–III,\nrespectively. Group VII also exhibited a 22.4% higher Bacteroidetes\nabundance than Group III.\n4 Discussion\n4.1 Effects on growth performance and\nprotein and lipid metabolism\nThe significant increase in ADG observed in the CCP group\n(Group IV: CCP 50 mg/kg BW) compared with the vaccinated\ncontrol (Group II) indicates that CCP alone was effective in\npromoting growth. The numerically highest ADG was observed\nin the blank control group (Group I), which is consistent with\nthe expected absence of vaccination-induced immune stress, a\nphysiological state known to divert nutrients away from growth.\nAlthough the high-dose combination group (Group VI) did not\n\nAlthough the high-dose combination group (Group VI) did not\nshow a statistically significant increase in ADG relative to Group\nII, its ADG did not differ significantly from that of Group IV,\nsuggesting that the addition of PE did not diminish the growth-\npromoting effect of CCP. This combined effect may be attributed\nto their complementary biological functions in the intestinal tract.\nPlant-derived active components, such as cinnamaldehyde and\ncarvacrol, have been widely reported to improve nutrient utilization\nand digestive efficiency in poultry (11, 12) and can upregulate\nintestinal nutrient transporter genes and enhance digestive enzyme\nactivities, thereby improving nutrient absorption (13). Meanwhile,\nas a prebiotic substrate, CCP can be fermented by gut microbiota\nto produce short-chain fatty acids (SCFAs), these metabolites\npresumably supply energy for intestinal epithelial cells and help\nmaintain intestinal barrier integrity (14).\nThe additives were administered via oral gavage in this study\nto ensure precise and uniform intake of the test substances. For\nlarge-scale production, these dose levels can be used as a reference\nfor in-feed inclusion via uniform mixing, with the gavage-derived\nresponses providing a basis for designing feeding regimens.\nAll treatment groups (IV–VII) exhibited numerically lower\nF/G ratios than the vaccinated control (Group II), though the\ndifference was not statistically significant, which is consistent\nwith the above mechanistic interpretation. The lack of significant\ndifferences may be associated with the genetic characteristics\nof Yandang chickens, the relatively short 42-day experimental\nperiod, or insufficient dosage optimization. This interpretation\nis consistent with previous reports that the efficacy of plant\nessential oils is regulated by multiple interacting factors (15).\nImproved growth performance was accompanied by favorable\nchanges in serum protein parameters. Compared with Group\nII, all treatment groups (IV–VII) had significantly higher serum\nTP and ALB levels and lower CREA content. Higher TP and\nALB levels are consistent with improved protein metabolism,\n\nCompared with Group\nII, all treatment groups (IV–VII) had significantly higher serum\nTP and ALB levels and lower CREA content.Higher TP and\nALB levels are consistent with improved protein metabolism,\nas reported for bioactive polysaccharides previously (16). The\nreduced serum CREA suggests more efficient protein turnover and\ndecreased nitrogen excretion, which may be closely related to the\nimprovement of intestinal microbial composition induced by the\nCCP-PE composite (17).\nFrontiers in Veterinary Science\n06\nfrontiersin.org\n\nLi et al.\n10.3389/fvets.2026.1878293\nTABLE 7 PERMANOVA results based on Bray—Curtis distance.\nSource\ndf\nSum of squares\nR2\nF\np-value\nGroup\n6\n0.528\n0.158\n1.718\n0.001\nResidual\n55\n2.815\n0.842\n—\n—\nTotal\n61\n3.342\n1.000\n—\n—\nFIGURE 3\nRelative abundance of cecal microbiota at the phylum level across\nexperimental groups of Yandang chickens. Firmicutes and\nBacteroidetes are the dominant phyla in the cecal ecosystem. Values\nare presented as the mean relative abundance (%) per treatment.\nGroups I–VII are defined in Table 2.\nSerum TC was significantly higher in the additive-treated\ngroups (Groups IV–VII) compared with Group II (p < 0.05),\nwhich may reflect enhanced lipid metabolism associated with\nCCP and PE supplementation. Serum UN and TG showed no\nsignificant differences among groups. The lower UN level in Group\nVI suggested higher amino acid utilization efficiency, while the\ndecreased TG tendency in Group V may reflect enhanced lipid\noxidation. Plant extracts are known to regulate lipid metabolism by\nmodulating key metabolic enzymes and signaling pathways (17, 18).\nCollectively, the increased serum TP and ALB, decreased\nCREA, and favorable trends in lipid metabolites were associated\nwith improved growth performance and favorable changes in\nserum protein and lipid parameters. These findings provide a\nrationale for further evaluation of this compound as a potential feed\nadditive candidate for antibiotic-free poultry production (19).\n4.2 Effects on serum antioxidant capacity\nThe present study revealed two notable findings regarding\nsystemic antioxidant status. First, Groups IV and VI exhibited\nenhanced systemic antioxidant defense compared with Groups II\nand III, as evidenced by elevated antioxidant enzyme activities and\nreduced MDA content. Notably, Group VI outperformed Group\nIV in T-AOC. Second, Groups I and II showed higher antioxidant\nactivities and lower MDA levels than Group III, suggesting that zinc\nbacitracin may have suppressed endogenous antioxidant function\n(20, 21). This contrast highlights a physiological advantage of\nphytogenic additives over antibiotics in protecting against cellular\noxidative injury.\nThe superior antioxidant profile observed in Group VI suggests\n\nThis contrast highlights a physiological advantage of\nphytogenic additives over antibiotics in protecting against cellular\noxidative injury.The superior antioxidant profile observed in Group VI suggests\na combined beneficial effect of CCP and PE. On one hand, CCP has\nbeen reported to activate the Nrf2-Keap1 signaling pathway, which\nmay contribute to the upregulation of downstream antioxidant\nenzymes such as SOD, CAT, and GSH-Px (22, 23), although this\npathway was not directly assessed in the present study. On the other\nhand, bioactive components in PE, including cinnamaldehyde\nand carvacrol, have been reported to possess direct free radical-\nscavenging capacities and may enhance cellular resistance to\noxidative stress (13, 17). The combination of these distinct yet\ncomplementary mechanisms may provide a more comprehensive\nantioxidant defense than either component alone.\nCollectively, the CCP-PE composite was associated with\nenhanced\nantioxidant\ncapacity,\nas\nevidenced\nby\nincreased\nantioxidant enzyme activities and reduced lipid peroxidation.\nFurthermore, the composite showed favorable redox-modulating\neffects compared with the antibiotic control, supporting its further\nevaluation as a candidate for antibiotic-free poultry production\n(19, 21).\n4.3 Effects on the gut microbiota\nThe gut microbiota plays a critical role in maintaining host\nhealth, regulating nutrient metabolism, and modulating immune\nfunction (24, 25). In the present study, 16S rRNA high-throughput\nsequencing was used to evaluate the effects of CCP and PE on cecal\nmicrobial community structure in Yandang chickens. Changes in\nmicrobial composition and diversity may help explain the observed\nimprovements in growth performance and antioxidant capacity\nfrom a gut microecological perspective.\n4.3.1 Analysis of gut microbial diversity\nFor alpha diversity, Group VII showed a numerically higher\nShannon index and a numerically lower Simpson index compared\nwith Groups I, II, and III, although these differences were not\nstatistically significant (p > 0.05). This trend suggests that PE at\nFrontiers in Veterinary Science\n07\nfrontiersin.org\n\nLi et al.\n10.3389/fvets.2026.1878293\n250 mg/kg BW may contribute to higher richness and evenness of\nthe cecal microbial community (26).\nPERMANOVA revealed significant differences in overall\nmicrobial community structure among groups. The relatively\nlow R2 value suggests that treatment effects accounted for a\nlimited proportion of the total variation. Samples from Group III\nwere more dispersed than those from other groups, indicating\nlower stability and higher inter-individual variation in microbial\ncommunity structure. These results are consistent with previous\nfindings that antibiotic treatment can disrupt gut microbiota\nhomeostasis and increase inter-individual variability (27, 28).\n4.3.2 Changes in gut microbiota composition\nAt the phylum level, Firmicutes and Bacteroidetes were\nthe dominant constituents of the cecal microbiota, collectively\naccounting for more than 70% of the total microbial community\n(29, 30). Notably, the high-dose combination group (Group VI)\nexhibited a markedly higher relative abundance of Bacteroidetes\ncompared with the control groups (Groups I–III), and Group VII\nalso showed an increase relative to Group III. For Firmicutes,\nGroup VII displayed a higher abundance relative to Groups I–III,\nand Group V showed an increase compared with Groups I and II.\nThe\nenrichment\nof\nBacteroidetes\nin\nGroup\nVI\nis\nof\nparticular interest. As a major phylum specialized in the\ndegradation\nof\ncomplex\npolysaccharides,\nBacteroidetes\npossesses extensive enzymatic machinery to ferment dietary\nand\nhost-derived\nglycans,\nproducing\nSCFAs\nas\nprimary\nmetabolic\nend-products\n(31).\nThese\nSCFAs—particularly\nacetate, propionate, and butyrate—are reported to serve as\nessential energy substrates for intestinal epithelial cells and\ncontribute to maintaining gut barrier integrity, regulating host\nmetabolism, and modulating systemic immune responses (32–\n35). The elevated Bacteroidetes abundance raises the plausible\npossibility that the CCP-PE blend improves intestinal carbohydrate\nfermentation capacity of the gut microbiota, providing a potential\nmicroecological explanation for the observed improvements\nin growth performance and systemic antioxidant capacity in\nGroup VI.\n\n4.3.3 Potential links between gut microbiota\nremodeling and improved host physiological\nstatus\nThe present study showed that the high-dose combination\nof\nCCP\nand\nPE\nwas\nassociated\nwith\nboth\nimproved\ngrowth\nperformance\nand\nsystemic\nantioxidant\ncapacity,\nalongside an increased relative abundance of Bacteroidetes\nin\nthe\ncecal\nmicrobiota.\nWhile\nthese\nassociations\ndo\nnot\nestablish\ncausality,\nthey\nraise\nthe\npossibility\nof\na\ngut\nmicrobiota-mediated\nsignaling\naxis\nthrough\nwhich\nthe\nCCP-PE\ncomposite\nmay\ncontribute\nto\nthe\nobserved\nphenotypic improvements.\nBacteroidetes is a major phylum specialized in the degradation\nof complex polysaccharides, fermenting dietary and host-derived\nglycans into SCFAs—predominantly acetate, propionate, and\nbutyrate (31, 34). These SCFAs serve as energy substrates for\nintestinal epithelial cells, help maintain gut barrier integrity,\nand can enter the portal circulation to reach distal organs\nincluding the liver and muscle (35). Through pathways such\nas the gut-liver axis, SCFAs can activate host cellular defense\nmechanisms, including the Nrf2-Keap1 antioxidant signaling\npathway (22), which in turn upregulates the expression of\nantioxidant enzymes such as SOD, CAT, and GSH-Px while\nsuppressing lipid peroxidation. This microbiota-metabolism-host\naxis, well-documented in the literature (21, 36), provides a\nplausible mechanistic framework linking the CCP-PE-induced\nenrichment of Bacteroidetes with the systemic benefits observed in\nGroup VI.\nSeveral\nlimitations\nof\nthe\npresent\nstudy\nshould\nbe\nconsidered\nwhen\ninterpreting\nthese\nfindings.\nFirst,\nSCFA\nconcentrations were not directly measured in cecal contents\nor\nserum;\nthe\nlink\nbetween\nBacteroidetes\nenrichment\nand\nenhanced\nSCFA\nproduction\nis\ntherefore\ninferred\nfrom\nestablished\nmicrobial\nphysiology.\nSecond,\nalthough\nPERMANOVA\nrevealed\nstatistically\nsignificant\ndifferences\nin\noverall\nmicrobial\ncommunity\nstructure,\nthe\nR2\nvalue\nwas low, suggesting that treatment effects accounted for a\nlimited proportion of the total variation. This may reflect\nthe limited sample size for sequencing or the 42-day trial\nduration\n(37).\nFuture\nstudies\nincorporating\ntargeted\nSCFA\nmetabolomics and direct assessment of intestinal barrier function\nand Nrf2 pathway activation are warranted to validate the\nproposed microbiota-mediatedmechanism.\n5 Conclusions\nThis study showed that dietary supplementation with CCP was\n\n5 Conclusions\nThis study showed that dietary supplementation with CCP was\nassociated with improved growth performance, while the CCP-\nPE composite, especially the high-dose combination, enhanced\nantioxidant capacity and modulated cecal microbiota in Yandang\nchickens under the conditions of this study.\nSupplementation with CCP at 50 mg/kg BW (Group IV)\nsignificantly increased ADG, while the high-dose combination\n(Group VI) did not differ significantly from Group IV in\nADG. Both Group IV and Group VI significantly elevated\nserum\nTP\nand\nALB\nlevels\nand\nreduced\nCREA\ncontent,\nfindings\nconsistent\nwith\nimproved\nprotein\nmetabolism.\nGroup\nVI\nexhibited\nthe\nmost\npronounced\nantioxidant\neffects, with significantly higher serum T-AOC, SOD, CAT,\nand GSH-Px activities and lower MDA content compared\nwith all other groups (p < 0.05), suggesting alleviation of\noxidative stress.\nFurthermore, the high-dose combination was associated with\nan increased relative abundance of Bacteroidetes in the cecum, a\nphylum known for its capacity to degrade complex polysaccharides.\nCollectively, the CCP-PE composite, especially the high-dose\ncombination, exhibited combined beneficial effects on antioxidant\nstatus and gut microbiota composition. These findings suggest that\nthe CCP-PE composite may be considered a promising natural feed\nadditive candidate for antibiotic-free poultry production. Further\nstudies incorporating direct SCFA measurement, intestinal barrier\nFrontiers in Veterinary Science\n08\nfrontiersin.org\n\nLi et al.\n10.3389/fvets.2026.1878293\nassessment, and in-feed supplementation trials are warranted to\nvalidate these observations.\nData availability statement\nThe 16S rRNA gene sequencing data have been deposited in\nthe NCBI Sequence Read Archive (SRA) under BioProject accession\nnumber PRJNA1464183. The other datasets generated and analyzed\nduring this study are available from the corresponding author upon\nreasonable request.\nEthics statement\nThe animal study was approved by Institutional Animal Care\nand Use Committee of the Zhejiang Academy of Agricultural\nSciences\n(Approval\nNo.\n2022ZAASLA59).\nAll\nefforts\nwere\nmade to minimize animal suffering, and humane treatment\nwas\nimplemented\nduring\nall\nprocedures,\nincluding\nsample\ncollection and euthanasia by cervical dislocation. The study\nwas conducted in accordance with the local legislation and\ninstitutional requirements.\nAuthor contributions\nRL: Data curation, Formal analysis, Funding acquisition,\nProject administration, Software, Validation, Visualization, Writing\n– original draft, Writing – review & editing. SL: Resources,\nSupervision, Writing – review & editing. LD: Validation, Writing –\nreview & editing. ZL: Methodology, Writing – review & editing. SX:\nResources, Writing – review & editing. TZ: Supervision, Writing –\nreview & editing. CL: Resources, Writing – review & editing. HM:\nResources, Writing – review & editing. YC: Resources, Supervision,\nWriting – review & editing.\nFunding\nThe author(s) declared that financial support was received\nfor this work and/or its publication. This work was supported\nby the Wenzhou Science and Technology Commissioner Project\n(Grant No. X2025071) and the 2025 Provincial General Project\nof\nHigher\nVocational\nEducation\nTeaching\nReform\n(Grant\nNo. JGCG2025228GZ).\nConflict of interest\nThe author(s) declared that this work was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declared that Generative AI was not used in the\ncreation of this manuscript.\nAny alternative text (alt text) provided alongside figures in\nthis article has been generated by Frontiers with the support of\n\nGenerative AI statement\nThe author(s) declared that Generative AI was not used in the\ncreation of this manuscript.Any alternative text (alt text) provided alongside figures in\nthis article has been generated by Frontiers with the support of\nartificial intelligence and reasonable efforts have been made to\nensure accuracy, including review by the authors wherever possible.\nIf you identify any issues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. 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+{"text": "【兽医文献】\n标题: article_509_A comparison of gonadotropin-releasing hormone and human cho\n\nDoes the extent of the surgical margin affect the \nlikelihood of local recurrence in Patnaik grade I or II \ncutaneous mast cell tumours?\n\nA Knowledge Summary by\n\nChristos Dorlis DVM AFHEA MRCVS 1*\n\n1 Medivet 24 Hour Enfield, 673 Hertford Road, Enfield, EN3 6NH \n* Corresponding Author (chrisdorlis@gmail.com)\n\nISSN: 2396-9776 \nPublished: 10 Mar 2022 \nin: Veterinary Evidence Vol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508 \nReviewed by: Jane Dobson (BVetMed DVetMed DipECVIM \nDipECVIM DipECVIM-CA FRCVS), Joanna Morris \n(BVSc BSc PhD FRCVS) and Adam Swallow (BVSc \nMRCVS) \nNext Review Date: 09 Nov 2023\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 2 of 18\n\nKNOWLEDGE SUMMARY\n\nClinical scenario  \nA 4 year old neutered male Pug presents with a 1 cm cutaneous mass in the left flank region. The owner \nreports that the mass has been there for 3 months and does not change in size. Cytologic examination \nsuggests a mast cell tumour (MCT). Blood work and abdominal ultrasound were unremarkable. What margin \nwould you ideally excise this mass?\n\nPICO question \nDoes the extent of the surgical margin affect the likelihood of local recurrence in Patnaik grade I or II \ncutaneous mast cell tumours?\n\nBlood work and abdominal ultrasound were unremarkable.What margin \nwould you ideally excise this mass?PICO question \nDoes the extent of the surgical margin affect the likelihood of local recurrence in Patnaik grade I or II \ncutaneous mast cell tumours?\n\nClinical bottom line \nCategory of research question \nTreatment \nThe number and type of study designs reviewed \nEight papers were critically reviewed. Five were retrospective case series, two prospective clinical trials, and \none prospective case series \nStrength of evidence \nLow \nOutcomes reported \nSequin et al. (2001) reported a local recurrence rate of 5%, but this study is 20 years old. In the studies of \nSimpson et al. (2004), Fulcher et al. (2006), Pratschke et al. (2013), Saunders et al. (2020), and Itoh et al. \n(2021), no local recurrence was observed in grade I and II mast cell tumours, while in the Milovancev et al. \n(2019) study, only 1/30 low-grade cutaneous mast cell tumors developed local recurrence. Therefore, there \nis some evidence that conservative surgical excision is sufficient to achieve local control with low \nrecurrence rates \nConclusion \nThere is increasing evidence in the literature for conservative surgical excision of grade I and II MCTs, but \nbecause the quality of evidence is low, no clear recommendations can be made. Further studies are needed \nto determine recommendations for surgical excision of cutaneous MCTs based on the biological \ncharacteristics of the tumour and the completeness of histologic margins\n\nFurther studies are needed \nto determine recommendations for surgical excision of cutaneous MCTs based on the biological \ncharacteristics of the tumour and the completeness of histologic margins\n\nHow to apply this evidence in practice \nThe application of evidence into practice should take into account multiple factors, not limited to: \nindividual clinical expertise, patient’s circumstances and owners’ values, country, location or clinic where \nyou work, the individual case in front of you, the availability of therapies and resources. \nKnowledge Summaries are a resource to help reinforce or inform decision making. They do not override the \nresponsibility or judgement of the practitioner to do what is best for the animal in their care.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 3 of 18\n\nThe evidence \nThe overall quality of evidence is low, consisting predominantly of retrospective observational studies (Sequin et al., \n2001; Simpson et al., 2004; Pratschke et al., 2013; Chu et al., 2020; Saunders et al., 2020; and Itoh et al., 2021) and \nno randomised prospective clinical trials. Two prospective observational clinical studies (Fulcher et al., 2006; and \nMilovancev et al., 2019) showed a low recurrence rate, but both consisted of a very small sample size (23 and 30 \ncutaneous MCTs, respectively).\n\nSummary of the evidence\n\nTwo prospective observational clinical studies (Fulcher et al., 2006; and \nMilovancev et al., 2019) showed a low recurrence rate, but both consisted of a very small sample size (23 and 30 \ncutaneous MCTs, respectively).Summary of the evidence\n\nSeguin et al. (2001) \nPopulation: Medical records of dogs with mast cell tumours (MCTs) were diagnosed \nat Colorado State University Veterinary Teaching Hospital (CSU-VTH) \nbetween January 1996 and August 1999. The study included only dogs \nwith grade II (Patnaik) cutaneous MCTs treated with surgery alone. 21 \nof the included tumours were scars from previously incompletely \nexcised MCTs. \nSample size: 55 dogs (60 MCTs). \nIntervention details: \n• \nSurgical margins: 2–3 cm lateral margins and one fascial plane \nas deep margin. \n• \nAdjuvant treatment: None. \n• \nTumour grading: Patnaik grading system. No Kiupel grading. \n• \nHistological margins: were categorised as complete, complete \nbut close (tumour cells within 1 mm of surgical margin), or \nincomplete (tumour cells at the surgical margin). \n• \nFollow-up method: Follow-up information was obtained via \nreassessment at the clinic or telephone calls to referring \nveterinarians or owners. \nStudy design: Retrospective case series. \nOutcome studied: \n• \nLocal recurrence. \n• \nCompleteness of excision. \n• \nTime of local recurrence. \n• \nSurvival time (time from surgery until death). \nMain findings: \n(relevant to PICO question): \n• \nTumour diameter: Median tumour diameter (range): 2 cm (1–4 \ncm). \n• \nTumour grading: \no \n39 grade II MCTs (Patnaik). \no \n21 surgical scars from previously incompletely excised \nMCTs. \n• \nTumour location: The tumours were located on the head and \nneck = 8 (13%), trunk = 29 (48%), limb =18 (30%), genital region \n= 3 (5%) and location were not available for 2 (3%) tumours. \n• \nHistological margins results: \no \ncomplete in 54 (90%) tumours, \no \ncomplete but close in 3 (5%) tumours, \no \nincomplete in 1 (2%) tumour, \no \ninformation not available in 2 (3%) tumours, \no \ntwo dogs had amputation for definite surgery, \no \nall 21 scar excision had complete histological margins. \n• \nLocal recurrence: three tumours (5%). \n• \nThe median time to local recurrence was 62 days (range, 51–\n252 days).\n\n• \nLocal recurrence: three tumours (5%).• \nThe median time to local recurrence was 62 days (range, 51–\n252 days).\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 4 of 18\n\n• \nMedian follow-up time 540 days (range, 77–1,804 days). \n46/55 (84%) dogs were free of MCT during the study period.\n\nLimitations: \n• \nSmall sample size – risk of type II error. \n• \nTwo dogs had amputation for definite treatment and local \nrecurrence was impossible to assess in these two cases. \n• \nNo clear definition of exact margins as a range was given (2–\n3 cm) and lacked assigned groups. \n• \nNon-objective follow-up in some cases (client follow-up) – \npotential risk of local recurrence underestimated. \n• \nDefinition of local recurrence in relation to the original scar \nwas not specified – the risk of some local recurrences being \nclassified as de novo. \n• \nRetrospective nature – some medical records are \nincomplete (e.g., tumour size) and therefore cannot provide \ninformation for tumours larger than 4 cm. \n• \nNo definite diagnosis of local recurrence with cytology or \nbiopsy. \n• \nPrognostic relationship between histopathological status of \nsurgical margins and recurrence could not be determined \nbecause the number of local recurrences was small (5%), \nand the classification system of histological margins differed \nfrom other studies and was therefore difficult to compare. \n• \nParticipants’ characteristics not homogeneous – surgery was \nperformed as revision in some cases (21 dogs).\n\nSimpson et al. (2004) \nPopulation: Client-owned dogs diagnosed with cutaneous mast cell tumours \n(MCTs) based on cytological examination of fine needle aspirates, at \nAnimal Medical Center, New York, USA – time interval not reported.\n\nSample size: 21 dogs (23 cutaneous grade I and II (Patnaik) MCTs).\n\nSample size: 21 dogs (23 cutaneous grade I and II (Patnaik) MCTs).\n\nIntervention details: \n• \nSurgical margins: MCT removed with 3 cm margins and one \nfascial plane as deep margin. \no Prior to removal, the skin was marked at 1, 2, and 3 \ncm from the tumour margin at 0°, 90°, 180°, and \n270°. \no Tumours were affixed to cardboard to recreate their \noriginal shape. \no Tissues were fixed in formalin and eight 1 cm long \nfull-thickness tissue samples were taken at 3 cm \nmargins. \no Four 1cm long full-thickness tissue sections were \nobtained at the 1cm and 2cm margins. \no All tissue margins were examined by one board-\ncertified pathologist. \n• \nAdjuvant treatment: One dog had radiation therapy after \nincomplete deep margin excision. \n• \nTumour grading: All tumours were graded by two \npathologists using the Patnaik system.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 5 of 18\n\n•\nHistological margins: were categorised as complete,\ncomplete but close (tumour cells within 1 mm of surgical\nmargin), or incomplete (tumour cells at the surgical margin).\n•\nFollow-up methods: Follow-up information was obtained via\nreassessment at the clinic or telephone calls to referring\nveterinarians.\n•\nLocal recurrence: Disease-free interval was defined as the\ninterval from the date of tumour excision to the date of local\nrecurrence.\n•\nSurvival time was defined as the interval from the date of\ntumour excision to the date of death.\nStudy design: Prospective case series.\nOutcome studied:\n•\nLocal recurrence.\n•\nSurvival time.\nMain findings:\n(relevant to PICO question):\n•\nTumour diameter: Mean tumour diameter was 1.9 cm\n(median, 1.7 cm; range, 0.35–5.0 cm).\n•\nTumour location: 15/23 (65%) tumours were located on the\ntrunk, 5/23 (22%) were located on the hind limbs, and 3/23\n(13%) were located on the neck.\n•\nTumour grading: There were three grade I and 20 grade II\ntumours.\n•\nHistological margins results:\no All grade I tumours were completely excised at all\nmargins.\no 15/20 (75%) of the grade II tumours were\ncompletely excised at the 1 cm margin.\no 20/20(100%) of grade II tumours were completely\nexcised at the 2 cm margin.\no 2/20 grade II tumours had incomplete deep margins.\n•\nLocal recurrence: No local recurrence was noted in any dog.\n•\nFollow-up time: The median follow-up time was 351 days\n(range, 78–603 days).\nLimitations:\n•\nThis study has a small sample size and is prone to type II\nerror.\n•\nThis study does not include MCTs on the face or inguinal and\nperineal regions, and no recommendation can be made for\nthese locations.\n•\nOne dog in which MCTs were detected within 1 mm of the\ndeep margin received full-course radiation therapy;\ntherefore direct comparison cannot be made for this\npatient.\n•\nAlthough there is no statistical correlation between tumour\ndiameter and completeness of excision, most tumours in\nthis study were small.\n•\nThe period during which participants were enrolled in the\nstudy was not mentioned.\n•\nInconsistent method of follow-up.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 6 of 18\n\nFulcher et al. (2006)\nPopulation: Client-owned dogs with one or more cutaneous mast cell tumours\n(MCTs) diagnosed with a cytological examination of fine needle\naspirates, at Animal Medical Center, New York, USA – time interval not\nreported.\nSample size: 16 dogs (23 cutaneous MCTs).\nIntervention details:\n•\nSurgical margins: MCT removed with 3 cm margins and one\nfascial plane deep margin.\no\nPrior to removal, the skin was marked at 1, 2, and 3 cm\nfrom the tumour margin at 0°, 90°, 180°, and 270°.\no\nTumours were affixed to cardboard to recreate their\noriginal shape.\no\nTissues were fixed in formalin and eight 1 cm long full-\nthickness tissue samples were taken at 3 cm margins.\no\nFour 1cm long full-thickness tissue sections were\nobtained at the 1cm and 2cm margins.\no\nAll tissue margins were examined by one board-\ncertified pathologist.\n•\nTumour grading: All tumours were graded by two pathologists\nusing the Patnaik system.\n•\nAdjuvant treatment: Some dogs had corticosteroids, but no\nmore information was given.\n•\nHistological margins were categorised as complete, complete\nbut close (tumour cells within 1 mm of surgical margin), or\nincomplete (tumour cells at the surgical margin).\n•\nFollow-up method: Follow-up information was obtained via\nreassessment at the clinic or telephone calls to referring\nveterinarians.\n•\nLocal recurrence was defined as the development of an MCT at\nor within 2 cm of the original surgical site.\n•\nDisease-free interval: time of surgery to local recurrence.\nStudy design: Prospective clinical trial.\nOutcome studied:\n•\nLocal recurrence.\n•\nDisease-free interval.\nMain findings:\n(relevant to PICO question):\n•\nTumour diameter: Mean tumour diameter was 1.3 cm\n(median, 1.1 cm; range, 0.4–3.1 cm).\n•\nTumour location: 10 (44%) located in the trunk, 7 (30%) on the\nhindlimbs, 3 (13%) on the forelimbs and 3 (13%) on the head\nand neck.\n•\nTumour grading:\no\n4/23 (17%) MCTs were grade I tumours,\no\n19/23 (83%) were grade II tumours.\n•\nHistological margins results:\no\nAll grade I tumours were completely excised at the 1-\nand 2cm margins.\no\n13/19 grade II MCTs, no MCTs were detected at the 1\ncm margin.\no\n17/19 tumours, no MCTs were detected at the 2cm\nmargin.\no\n\n•\nHistological margins results:\no\nAll grade I tumours were completely excised at the 1-\nand 2cm margins.o\n13/19 grade II MCTs, no MCTs were detected at the 1\ncm margin.o\n17/19 tumours, no MCTs were detected at the 2cm\nmargin.o\nOverall, 21/23 (91%) MCTs were completely excised.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 7 of 18\n\no There was no recurrence in two of the grade II \ntumours with incomplete margins, although one had \nrevision surgery 2 weeks after the first excision. \no All had complete deep margins. \n• \nLocal recurrence: None of the dogs had local recurrence. \n• \nFollow-up time: The median follow-up interval for all dogs \nwas 379 days (range, 51–538 days). \nLimitations: \n• \nThis study has small sample size and is prone to type II error. \n• \nThis study does not include MCTs on the face or inguinal and \nperineal regions, and no recommendation can be made for \nthese locations. \n• \nOne dog with incompletely excised MCT received additional \nsurgery in 2 weeks therefore direct comparison cannot be \nmade. \n• \nInconsistent method of follow-up (either clinical \nexamination or telephone update). \n• \nThe period when participants were enrolled in the study was \nnot mentioned.\n\n• \nInconsistent method of follow-up (either clinical \nexamination or telephone update).• \nThe period when participants were enrolled in the study was \nnot mentioned.\n\nPratschke et al. (2013) \nPopulation: Dogs with cutaneous and subcutaneous mast cell tumours (MCTs) \nwere diagnosed either with cytological or histopathological \nexamination at the University of Glasgow between 2008 and 2012. \nSample size: 40 dogs (47 MCT [41 cutaneous and six subcutaneous]). \nIntervention details: \n• \nSurgical margins: Lateral margins equivalent to the widest \nmeasured diameter of the tumour and a minimum depth of \none well-defined fascial plane. If tumour size exceeded 4 cm \nthen a fixed 4 cm lateral margin was taken. \n• \nTumour grading: Grading based on Patnaik and Kiupel \nsystem. \n• \nHistological margins: Margins were considered clear when \nthe distance between neoplastic cells and non-neoplastic \ntissue was > 1 mm; otherwise, they were considered \nincomplete. \n• \nFollow-up methods: retrieved from clinical records and \nthrough contact with the referring veterinarians and dog \nowners. \nStudy design: Retrospective case series. \nOutcome studied: Local recurrence. \nMain findings: \n(relevant to PICO question): \n• \nTumour diameter: Tumour diameter ranged from 5 mm to \n60 X 40 mm. \n• \nTumour location: 24/47 (51%) located in the limbs, 9/47 \n(19%) on the trunk, 9/47 (19%) on the head, and 5 (11%) the \ninguinal region or prepuce and genital area. \n• \nTumour grading: 21 tumours grade I, 18 tumours grade II, \ntwo grade III, and six subcutaneous. \n• \nAdjuvant treatments: Five low-grade MCTs (four cutaneous \nand one subcutaneous) had postoperative chemotherapy\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 8 of 18\n\n• \nAdjuvant treatments: Five low-grade MCTs (four cutaneous \nand one subcutaneous) had postoperative chemotherapy\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 8 of 18\n\n(all these five had nodal metastasis). One dog with \nincomplete margins had postoperative radiation therapy. \n• \nHistological margins results: \no 40/47 (85%) tumours with complete and 7/47 \ntumors (15%) with incomplete margins. \no Of the excised tumours with incomplete margins, \nfour had one incomplete lateral margin and three \nhad an incomplete deep margin. \no 5/7 tumours with incomplete margins were located \non the limbs. \no Of the dogs with incomplete excisional margins, one \nhad postoperative radiation therapy (disease free \ninterval [DFI]: 300 days) and one had chemotherapy \n(DFI: 510 days), one died from gastric dilatation-\nvolvulus the day after surgery, one developed local \nrecurrence and was euthanised 1.5 months later, \nand the other three had no adjunctive treatment \nand no identified recurrence at follow-up times of \n120, 300, and 330 days. \n• \nLocal recurrence: Only one local recurrence (dog with grade \nIII mast cell tumour completely excised). \n• \nFollow-up time: Follow-up intervals ranged from 0 to 1,140 \ndays after MCT excision (median, 420 days). \nLimitations: \n• \nRetrospective nature – incomplete medical records may \ninfluence the outcome. \n• \nLack of control group for comparison (a group with 3 cm \nmargins for example). \n• \nTwo dogs with incomplete margins have additional \ntreatments and the other died from another cause and this \nmay influence the low number of recurrences. \n• \nInconsistent follow-up methods (some cases had owner and \nreferring veterinary surgeon follow-up and other reviewing \nthe clinical records).\n\n• \nInconsistent follow-up methods (some cases had owner and \nreferring veterinary surgeon follow-up and other reviewing \nthe clinical records).\n\nMilovancev et al. (2019) \nPopulation: Client-owned dogs presented to the Veterinary Teaching Hospital at \nOregon State University for surgical treatment of mast cell tumours \n(MCTs) between August 1, 2014–July 31, 2016. The population \ninclude: 38 dogs (52 MCTs), 50 low-grade (30 cutaneous and 20 \nsubcutaneous), two MCTs high grade. MCTs diagnosed via \ncytological examination. The authors also looked at 19 soft tissue \nsarcoma cases, but they are not relevant to the PICO topic in this \nstudy, thus they will not be discussed further. \nSample size: 38 dogs (30 cutaneous low-grade MCTs). \nIntervention details: \n• \nSurgical margins: en bloc removal of a grossly normal \nsurgical margin of at least 1 mm beyond the palpable and \nvisible edge of the tumour (i.e., no grossly visible tumour at \nthe surgical margins). \n• \nTumour grading: based on Kiupel and Patnaik system.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 9 of 18\n\ne., no grossly visible tumour at \nthe surgical margins).• \nTumour grading: based on Kiupel and Patnaik system.Veterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 9 of 18\n\n• \nHistological margins: histological margins of 0 mm were \nconsidered as incomplete excision whereas histological \nmargins >0 mm (i.e., tumour cells do not reach the inked \nsurgical margin) were considered as complete excision, \n• \nAdjuvant treatment: 4 low-grade tumours had additional \nchemotherapy (three were clinical stage III and one was \nclinical stage IV). \n• \nFollow-up methods: 2 year postoperative active surveillance \nperiod with clinical outcome data. \n• \nLocal recurrence confirmed by cytological evaluation in a \nfollow-up period of 24 months. \nStudy design: Prospective clinical trial. \nOutcome studied: Local recurrence. \nMain findings: \n(relevant to PICO question): \n• \nTumour diameter: Median 15 mm, range 5–52 mm. \n• \nTumour grading: 30 low-grade MCTs (graded as II on Patnaik \nsystem and low-grade on Kiupel system). \n• \nNarrowest intraoperative margins: \no Two MCTs: 5 mm \no One MCT: 8 mm \no Four MCTs: 10 mm \no Two MCTs: 15 mm \no 18 MCTs: 20 mm \no One MCT: 25 mm \no Two MCTs: 30 mm \n• \nNarrowest histological margins: \no 0 mm: Four tumours \no <1 mm: 11 tumours \n• \nNumber of deep fascial planes: \no 0: Two tumours \no 1: 27 tumours \no 2: One tumour \n• \n1/30 low-grade MCT developed local recurrence. This was a \ncompletely excised (narrowest histological margin = 10.6 m) \ncutaneous MCT from the ventral abdomen, with local \nrecurrence noted at the 12 month postoperative recheck \n(confirmed via fine-needle-aspirate cytology). The dog was \neuthanised 1229 days postoperatively for progressive \nosteoarthritis. \nLimitations: \n• \nSmall number of cases – risk of type II error. \n• \nNo standardised margin of excision.\n\nThe dog was \neuthanised 1229 days postoperatively for progressive \nosteoarthritis.Limitations: \n• \nSmall number of cases – risk of type II error.• \nNo standardised margin of excision.\n\nChu et al. (2020) \nPopulation: Cutaneous mast cell tumours (MCTs) excised from dogs between \nJanuary 1, 2007, and December 31, 2017, at the Cornell University \nHospital for Animals were identified from a search of the pathology \ndepartment’s database. 623 cutaneous MCTs were identified (83 \ntumours grade I and II met the inclusion criteria). \nSample size: 68 dogs (83 tumours grade I and II [Patnaik grading system]).\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 10 of 18\n\nIntervention details: \n• \nSurgical margins: Tumours were assigned to groups. \no \nConservative-margin group (n = 46): if they had been \nexcised with lateral margins that were 2 cm for \ntumours ≥ 2 cm in diameter or equal to the tumour \ndiameter (proportional) for tumours < 2 cm in \ndiameter. \no \nWide-margin group (n = 37): if they had been excised \nwith lateral margins of 3 cm, regardless of tumour \ndiameter. \no \nA noninferiority margin of > 0.9 cm was used for the \nrisk ratio (probability of complete excision for the \nconservative- vs wide-margin group). \n• \nNeoadjuvant treatment: some dogs had preoperative \nprednisone administration. \n• \nTumour grading system: Patnaik and Kiupel grading system. \n• \nHistological margins: \no \nComplete excision: classified when results of \nmicroscopic examination revealed no MCTs with \ncharacteristics suggestive of neoplastic behaviour at \nthe lateral or deep edge of the section, whether \nindividually scattered or as part of the neoplasm \n(Pratschke et al., 2013). \no \nIncomplete excision: all the other instances was \nclassified as incomplete excision. \n• \nFollow-up method: assessment of histological margins for \ncompleteness of excision.\n\nStudy design: Retrospective observational study.\n\n, 2013).o \nIncomplete excision: all the other instances was \nclassified as incomplete excision.• \nFollow-up method: assessment of histological margins for \ncompleteness of excision.Study design: Retrospective observational study.\n\nOutcome studied: The margin of excision (complete vs incomplete excision as \nhistologically determined) was compared between conservative- and \nwide-margin groups.\n\nMain findings: \n(relevant to PICO question): \n• \nMedian (range) tumour diameter: \no \nWide-margin group: 1 cm (0.2–7.0 cm). \no \nConservative-margin groups :1.0 cm (0.1–4.0 cm). \n• \nTumour location: limbs (29), thoracic region (25), abdominal \nregion (11), head (five), and neck region (three). The study did \nnot specify the location of the remaining 10 tumours. \n• \nTumour grading: \no \nAll tumours were histologically classified as grade I or II \nby the Patnaik system. \no \nTumours in the wide-margin group were more likely to \nbe classified as grade II (vs grade I) than were tumours \nin the conservative-margin group. \n• \nAdjuvant treatments: One dog in the wide margin group and \ntwo in the conservative margin group had preoperative \nprednisone administration. \n• \nHistological tumour-free margins: \no \nConservative group: 43/46 (93%). \no \nWide margin group: 34/37 (92%). \n• \nThe risk ratio met the criterion for noninferiority.\n\nLimitations: \n• \nLack of randomisation: More tumours grade II in the wide \nmargins group which could induce bias to the study.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 11 of 18\n\n• \nThe outcome was assessed by histological complete / \nincomplete margins and there is no correlation with clinical \nrecurrence – further studies are needed to assess these \ngroups with clinical recurrence. \n• \nDue to the retrospective nature 473 dogs were excluded due \nto missing information which may introduce selection bias. \n• \nHistological margin assessment based on radial method only \nby different board-certified pathologists.\n\n• \nDue to the retrospective nature 473 dogs were excluded due \nto missing information which may introduce selection bias.• \nHistological margin assessment based on radial method only \nby different board-certified pathologists.\n\nSaunders et al. (2020) \nPopulation: Dogs presented to the Veterinary Oncology Specialists (Underwood, \nAustralia) between July and October 2016 and Animal Referral \nHospital Brisbane (Brisbane, Australia) between October 2016 and \nAugust 2018. \nSample size: 65 dogs (100 cutaneous mast cell tumours [MCTs]). \nIntervention details: \n• \nSurgical margins: The width of proportional margin excision \nwas based on the largest lateral diameter of the mass up to \na level of 2 cm. If the tumour diameter was greater than 2 \ncm, the lateral margins that were used to excise the mass \nwere limited to 2 cm. The tumours were excised in an en \nbloc manner with the proposed lateral surgical margins and \na minimum of at least one fascial plane as deep margin. \n• \nPreoperative neoadjuvant medications: prednisolone (0.5–\n1 mg/kg PO SID ± chlorpheniramine (0.5–1 mg/kg PO BID) \nfor 7–10 days prior to surgical intervention. \n• \nTumour grading: all tumours graded with Kiupel system. \n• \nTumour size: Authors created two subgroups based on \ntumour size: \no Of the low-grade MCTs, 42 were < 10 mm in \ndiameter, with the remaining 46 = 10 mm. \no Of the high-grade MCTs 5 were < 10 mm and seven \nwere = 10 mm groups. \n• \nTissues were fixed in formalin and were reviewed by a \nboard-certified pathologist. \n• \nHistological margins: Complete excision of the mass was \ndefined as both the lateral and deep margins being clear of \ninfiltration with MCTs. Incomplete excision was defined as \nmast cell infiltration in lateral or deep margins. \n• \nHistological margin size was grouped into three categories: \n≤2 mm, 2.1–5 mm, and > 5 mm. \n• \nFollow-up method: a follow-up period of at least 365 days \nby physical examination or owner-contact ± cytological \nevaluation when appropriate. \nStudy design: Retrospective cohort study. \nOutcome studied: Local recurrence. \nMain findings: \n(relevant to PICO question): \n• \nTumour diameter: \no Low-grade MCTs: 42 were < 10 mm in diameter, and \n46 being = 10 mm in diameter.\n\nStudy design: Retrospective cohort study.Outcome studied: Local recurrence.Main findings: \n(relevant to PICO question): \n• \nTumour diameter: \no Low-grade MCTs: 42 were < 10 mm in diameter, and \n46 being = 10 mm in diameter.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 12 of 18\n\no High-grade MCTs: five were <10mm and seven were\n=10mm\no Median overall tumour diameter =10.0 mm (range\nof 1–85 mm).\no Median high-grade tumour diameter = 15.0 mm\n(range of 3–85 mm).\no Median low-grade tumours diameter = 10.0 mm\n(range of 1–45 mm).\n•\nTumour location: (35%) limbs, (23%) flank and abdomen,\n(15%) thorax, (15%) the prepuce and / or perineal regions,\n(12%) the head and neck.\n•\nTumour grading:\no 88 low-grade MCT and 12 high-grade MCT.\no 11 dogs had high grade MCTs (one had two\ntumours).\no 54 dogs had low-grade MCTs (18 multiple).\n•\nHistological margins/completeness of excision:\no 95/100 (95%) MCTs were completely excised.\no Low-grade: 84/88 (96%).\no High grade:11/12 (92%).\no small tumours (< 10 mm): all had complete excision\n47/47 (100%).\no large tumours (≥ 10 mm): 48/53 (91%) had\ncomplete.\n•\nHistological tumour-free margins (HTFM) size:\no Median HTFM size of all complete excised tumours:\n5 mm (range 0.5–15 mm).\no Median HTFM size of high-grade tumours: 5 mm\n(range 2–14 mm).\no Median HTFM size of low-grade tumours: 5 mm\n(range 0.5–15 mm).\no No significant association was evident between\ntumour grade and HTFM size.\no No significant association was evident between the\ntumour size groups and the HTFM size groups.\n•\nRecurrence rate: 3/100 tumours (3%) with a median follow-\nup period of 593 days (range 180–1460 days) with none on\nthe low-grade group and three on the high-grade group\n3/12(25%).\n•\nOf the incompletely excised masses, four were low-grade,\nand one was high-grade and only the high grade recurred.\n•\n45 tumours had neoadjuvant prednisolone and\nchlorpheniramine administered but there was no significant\nassociation between the rates of complete excision between\ntumours that had neoadjuvant therapy compared to those\nthat did not.\nLimitations:\n•\nRetrospective nature and as such inability to standardise\nfollow-up assessment for recurrence.\n•\nSome dogs had neoadjuvant prednisolone which could\naffect tumour size, completeness of excision, and margin\nassessment.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 13 of 18\n\n• \n18 dogs were excluded from the margin assessment as no \nreport on histological margins in the clinical records. \n• \nTumour size may affect the completeness of excision as this \nrelationship was close to statistical significance – potentially \nwith increased sample size groups, the relationship would \nbe more obvious. \n• \nEight dogs lost to follow-up before the 365 days but all of \nthem were grade I. \n• \nLimitation is also the radial trimming of margins as less \nsensitive than the tangential method to detect residual \ntumours at the margins.\n\nItoh et al. (2021) \nPopulation: Dogs with cutaneous and subcutaneous mast cell tumours (MCTs) \npresented to Aoba Animal Hospital between November 2013 and \nSeptember 2018 and were treated with the proportional margin \nresection technique. 29 dogs had MCT surgery in this period. Six \ndogs were excluded because margins were wider or narrower than \nthe proportional diameter. The final population was 23 dogs (25 \nMCTs). From the 25 MCTs, the 24 were cutaneous and one \nsubcutaneous.\n\nSample size: 23 dogs (24 cutaneous MCTs). \nIntervention details: \n• \nSurgical margins: MCTs excised with margins equal to the \nproportional diameter of the tumour as described by \nPratschke et al. (2013) and at least one fascial plane as deep \nmargin. \n• \nTumour grading: All tumours graded based on Patnaik and \nKiupel system. \n• \nAdjuvant treatment: No dogs had adjuvant treatment. \n• \nHistological margins: Classified as complete, close (tumour \ncells within 1 mm of the margin), or incomplete (tumour \ncells in the margin). \n• \nFollow-up method: Review of the medical records.\n\nStudy design: Retrospective case series.\n\nOutcome studied: \n• \nLocal recurrence. \n• \nCompleteness of resection.\n\n• \nFollow-up method: Review of the medical records.Study design: Retrospective case series.Outcome studied: \n• \nLocal recurrence.• \nCompleteness of resection.\n\nMain findings: \n(relevant to PICO question): \n• \nTumour diameter: The tumour diameter range was 0.3–2.6 \ncm (Median: 0.9 cm). \n• \nTumour location: Trunk (12), extremities (seven), head \n(two), vagina / perineum (three), and tail (one). \n• \nTumour grading: \no All 24 cutaneous MCTs grades as low-grade (Kiupel). \no Five MCTs Patnaik grade I and 19 as Patnaik grade II. \n• \nHistological margins results: \no Complete in 20 cases. \no Close (deep margin) in two cases. \no Incomplete (deep margin) in two cases.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 14 of 18\n\n• \nLocal recurrence: No local recurrence in a follow-up period \nof 410–2,219 days (median: 990 days). \nLimitations: \n• \nLocal recurrence distance in relation to surgical scar was not \nspecified. \n• \nSmall sample size – prone to type II error. \n• \nOnly tumours with small size were included.\n\nAppraisal, application and reflection\n\nHistorically, it has been recommended that mast cell tumours (MCTs) independently of grade, must be excised \nwith 3 cm lateral margins and one fascial plane as deep margin. However, evidence to support this \nrecommendation is lacking, especially for grade I and II MCTs. Recent literature suggests that the removal of \nthese low-grade tumours with 2 cm lateral margins and one deep fascial plane, is sufficient to achieve local \ncontrol and does not increase the incidence of local recurrence (Simpson et al., 2004; Fulcher et al., 2006; Chu \net al., 2020; Milovancev et al., 2019; and Saunders et al., 2020).\n\n, 2004; Fulcher et al., 2006; Chu \net al., 2020; Milovancev et al., 2019; and Saunders et al., 2020).\n\nIn the study by Seguin et al. (2001), 2–3 cm margins were used to remove MCTs, however, the authors did not \ndivide the dogs into groups in terms of margins, so no direct comparison between conservative and wide \nexcision is possible. In this study, only 5% (three tumours) recurred locally; however, the authors based their \nfollow-up not only on clinical examinations but also on referring veterinarians or telephone updates from \nclients. There is a possibility that this local recurrence rate is underestimated, as some recurrences may be \nmissed by clients. The authors also included dogs with scar tissue of previously incompletely excised MCTs, \nwhich makes the study population not homogeneous. In addition, two dogs had an amputation as definite \ntreatment, and as such local recurrence could not be evaluated in these dogs. Other limitations of this study \ninclude its retrospective nature, which may mean that some information was not recorded in the medical \nrecords.\n\nTwo later studies, one by Simpson et al. (2004) and Fulcher et al. (2006), investigated the removal of grade I \nand II MCTs with 2 cm lateral margins. Both studies suffer from small sample sizes, which predispose them to \ntype II statistical error. When we combine both studies for a total number of 46 MCTs (seven grades I and 39 \ngrade II), 44/46 MCTs (95%) were completely excised at 2 cm lateral surgical margin. Only two dogs, in the \nFulcher et al. (2006) study, had incomplete histological margins and none of them suffered local recurrence; \nhowever, one dog received revision surgery 2 weeks later. In both studies, no local recurrence was noted at a \nmedian follow-up of 351 days and 379 days, respectively. Based on these studies, it was suggested that a \nsurgical dose of 2 cm lateral margins and one fascial plane deep should be sufficient to achieve local control \nwithout increased rates of local recurrence for grade I and II MCTs.\n\nBased on these studies, it was suggested that a \nsurgical dose of 2 cm lateral margins and one fascial plane deep should be sufficient to achieve local control \nwithout increased rates of local recurrence for grade I and II MCTs.\n\nPratschke et al. (2013) first described the approach of surgical excision based on the proportional margin of \nthe tumour. In this study, masses were resected with a lateral margin equal to their widest diameter, with a \nmaximum lateral margin of 4 cm and a well-defined deep fascial plane. Complete excision was achieved in \n40/47 (85%) of cases, and although 7/47 (15%) of excised tumours had dirty margins, only one recurred locally \nduring a median follow-up of 420 days. This was a completely excised, grade III MCT, highlighting the fact that \nachieving local control is not only influenced by the completeness of histological margins but also depends on \nthe biological behaviour of the tumour. This study had a small sample size and a relatively short median \nfollow-up time, which may have influenced the local recurrence rate. Furthermore, two of the dogs with \nincomplete margins had adjuvant treatments and another dog with incomplete margins died from gastric \ndilatation-volvulus 1 day post-surgery. One advantage of this study is that it included tumours in difficult \nlocations such as the face, distal extremities, inguinal and preputial regions.\n\nBased on the original work by Pratchke et al. (2013), two recent studies used a modified proportional margins \napproach for MCT excision. Chu et al. (2020) compared the completeness of excision (histologically free of \ntumour margins) after conservative surgical excision (excision with lateral margins equal to or smaller than 2 \ncm) and wide surgical excision (3 cm lateral margins) and showed a similar percentage of histologically free\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 15 of 18\n\norg/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 15 of 18\n\nmargins in both groups (93% and 92%, respectively). The authors concluded that this conservative approach \nwas not inferior to the wide approach in achieving histologically free margins. The main limitation of this study \nwas that the outcome measured was histologically free margins and not local recurrence. Milovancev et al. \n(2019) showed in a prospective clinical study with a follow-up of 2 years that narrow histological margins (1 \nmm) do not always correlate with local recurrence. In this study, 30 low-grade MCTs with a mean \nintraoperative margin of 20 mm were removed and only one recurred.\n\nSaunders et al. (2020) used a similar approach to Pratchke et al. (2013) and Chu et al. (2020), where they \nexcised MCTs based on the largest lateral diameter of the mass up to 2 cm and with 2 cm margins for masses \ngreater than 2 cm. This study contains the largest sample size with 100 MCTs but both low-grade (88) and \nhigh-grade (12) tumours were included. The local recurrence rate was 3% (3/100), similar to Pratchke et al’s. \n(2013) study 1/45 (2%) but occurred only in high-grade tumours. None of the low-grade mast cell tumours \nincluded in the study recurred locally.\n\nA correlation between conservative versus wide surgical excision and completeness of histological margins \ncould not be established in this review because the classification methods of histologic margins were \ninconsistent between studies and the sampling methods used by histopathologists were different. In addition, \nthe clinical significance of surgical margins and completeness of excision with the likelihood of metastatic \nspread could not be evaluated in this review.\n\nIn addition, \nthe clinical significance of surgical margins and completeness of excision with the likelihood of metastatic \nspread could not be evaluated in this review.\n\nIncreased tumour size is a significant risk factor for incomplete excision with a 1.4% increase in the risk of \nincomplete excision per cm2 (Monteiro et al., 2011). The studies in this review failed to show a significant \nassociation between tumour diameter and completeness of excision and clear recommendations regarding \nsurgical margins and large diameter tumours (>4 cm) cannot be made because most of the tumours in the \nincluded studies were small in diameter.\n\nMCTs in muzzle, prepuce, inguinal and perineal areas have been associated with aggressive behaviour (Hillman \net al., 2010; and Sfiligoi et al., 2005). In Simpson et al. (2004), the location of the MCT on the hind limb was \nsignificantly associated with incomplete excision and in Pratchke et al’s. (2013) study, 5/7 tumours with \nincomplete margins were located on the limbs. This review cannot evaluate surgical margins and recurrence \nrates in MCTs in difficult locations such as the muzzle, distal extremities, inguinal and perineal areas due to the \nsmall number of these tumours in the study populations.\n\nThis review cannot evaluate surgical margins and recurrence \nrates in MCTs in difficult locations such as the muzzle, distal extremities, inguinal and perineal areas due to the \nsmall number of these tumours in the study populations.\n\nThis review suggests that most grade I and II cutaneous MCTs can be safely excised without an increase in the \nrecurrence rate using either 2 cm lateral margins (Simpson et al., 2004; and Fulcher et al., 2006) or the \nproportional margin approach (Pratschke et al., 2013; Chu et al., 2020; and Saunders et al., 2020) instead of \nthe classic recommendation of wide surgical excision (3 cm lateral margins). Nevertheless, the overall evidence \nis low and further studies, preferably multi-institutional studies with larger study populations, are needed to \nprovide higher quality of evidence. These recommendations do not apply to high-grade MCTs, which are \nbiologically more aggressive, locally invasive, and more likely to metastasize. Similarly, these \nrecommendations must be used with caution for subcutaneous MCTs. There is no grading system for \nsubcutaneous MCTs and many times their actual margins are not easily distinguished due to their \nsubcutaneous origin and surrounding oedema. Studies by Pratchke et al. (2013) and Itoh et al. (2021) \ncontained subcutaneous MCTs, but due to their low numbers, no correlation between surgical margins and \nlocal recurrence could be made.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 16 of 18\n\nMethodology\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 16 of 18\n\nMethodology\n\nSearch strategy \nDatabases searched and dates \ncovered: \nCAB Abstracts accessed via OVID platform (1980–2021) \nPubMed database accessed via NCBI (1986–November 2021) \nSearch terms: CAB Abstracts: \n(Dog OR canine OR canines OR bitch OR canid) AND (mast cell \ntumour OR mast cell tumor) AND (surgical margin OR incomplete \nmargin OR dirty margin OR surgical resection OR surgical excision OR \nsurgery OR margin OR resection) AND (recurrence OR local control \nOR neoplasm recurrence)\n\nPubMed: \n(Dog OR canine OR canines) AND (mast cell tumour OR mast cell \ntumor) AND (surgical margin OR incomplete margin OR dirty margin \nOR surgical resection OR surgical excision OR surgery) AND \n(recurrence OR local control OR neoplasm recurrence) \nDates searches performed: 09 Nov 2021\n\nExclusion / Inclusion criteria \nExclusion: \n• \nSingle case reports, duplicate articles, review articles, book \nchapters, or sections. \n• \nArticles which did not directly evaluate the relationship \nbetween lateral surgical margins and local recurrence. \n• \nArticles that did not specify the surgical margins at the time \nof the surgery. \n• \nArticles not available in the English language. \nInclusion: Original peer-reviewed articles including case series, observational, \nor interventional studies which evaluate the relationship of lateral \nsurgical margins in MCT excision and local recurrence.\n\nSearch outcome \nDatabase \nNumber \nof results \nExcluded – \nNot relevant \nto PICO \nquestion \nExcluded – \nSurgical margins \nat the time of the \nsurgery not \nreported and \nstudies with only \nsubcutaneous \nMCTs \nExcluded – \nReviews, case \nreports and \nletters \nExcluded – \nNot in \nEnglish or \nnot available \nTotal \nrelevant \npapers \nCAB \nAbstracts \n31 \n9 \n5 \n10 \n1 \n6 \nPubMed \n99 \n58 \n14 \n19 \n0 \n8 \nTotal relevant papers when duplicates removed \n8\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 17 of 18\n\nCONFLICT OF INTEREST\n\nThe authors declare no conflict of interest.\n\nREFERENCES\n\n1. Chu, M., Hayes, G., Henry, J. & Oblak, M. (2020). Comparison of lateral surgical margins of up to two\ncentimeters with margins of three centimeters for achieving tumor-free histologic margins following\nexcision of grade I or II cutaneous mast cell tumors in dogs. Journal of the American Veterinary Medical\nAssociation. 256(5), 567–572. DOI: https://doi.org/10.2460/javma.256.5.567\n2. Fulcher, R., Ludwig, L., Bergman, P., Newman, S., Simpson, A. & Patnaik, A. (2006). Evaluation of a two-\ncentimeter lateral surgical margin for excision of grade I and grade II cutaneous mast cell tumors in\ndogs. Journal of the American Veterinary Medical Association. 228(2), 210–215.\nDOI: https://doi.org/10.2460/javma.228.2.210\n3. Hillman, L., Garrett, L., de Lorimier, L., Charney, S., Borst, L. & Fan, T. (2010). Biological behavior of oral\nand perioral mast cell tumors in dogs: 44 cases (1996–2006). Journal of the American Veterinary\nMedical Association. 237(8), 936–942. DOI: https://doi.org/10.2460/javma.237.8.936\n4. Itoh, T., Kojimoto, A., Uchida, K., Chambers, J. and Shii, H. (2021). Long-term postsurgical outcomes of\nmast cell tumors resected with a margin proportional to the tumor diameter in 23 dogs. Journal of\nVeterinary Medical Science. 83(2), 230–233. DOI: https://doi.org/10.1292/jvms.20-0281\n5. Milovancev, M., Townsend, K., Tuohy, J., Gorman, E., Bracha, S., Curran, K. and Russell, D. (2019).\nLong‐term outcomes of dogs undergoing surgical resection of mast cell tumors and soft tissue\nsarcomas: A prospective 2‐year‐long study. Veterinary Surgery. 49(1), 96–105.\nDOI: https://doi.org/10.1111/vsu.13225\n6. Monteiro, B., Boston, S. & Monteith, G. (2011). Factors influencing complete tumor excision of mast\ncell tumors and soft tissue sarcomas: a retrospective study in 100 dogs. Canadian Veterinary Journal.\n52(11), 1209–1214.\n7. Pratschke, K., Atherton, M., Sillito, J. & Lamm, C. (2013). Evaluation of a modified proportional margins\napproach for surgical resection of mast cell tumors in dogs: 40 cases (2008–2012). Journal of the\n\nCanadian Veterinary Journal.52(11), 1209–1214.7.Pratschke, K., Atherton, M., Sillito, J.& Lamm, C.(2013).Evaluation of a modified proportional margins\napproach for surgical resection of mast cell tumors in dogs: 40 cases (2008–2012).Journal of the\nAmerican Veterinary Medical Association. 243(10), 1436–1441.\nDOI: https://doi.org/10.2460/javma.243.10.1436\n8. Saunders, H., Thomson, M., O'Connell, K., Bridges, J. & Chau, L. (2020). Evaluation of a modified\nproportional margin approach for complete surgical excision of canine cutaneous mast cell tumours\nand its association with clinical outcome. Veterinary and Comparative Oncology. 19(4).\nDOI: https://doi.org/10.1111/vco.12630\n9. Seguin, B., Leibman, N., Bregazzi, V., Ogilvie, G., Powers, B., Dernell, W., Fettman, M. & Withrow, S.\n(2001). Clinical outcome of dogs with grade-II mast cell tumors treated with surgery alone: 55 cases\n(1996–1999). Journal of the American Veterinary Medical Association. 218(7), 1120–1123.\nDOI: https://doi.org/10.2460/javma.2001.218.1120\n10. Sfiligoi, G., Rassnick, K., Scarlett, J., Northrup, N. & Gieger, T. (2005). Outcome of dogs with mast cell\ntumors in the inguinal or perineal region versus other cutaneous locations: 124 cases (1990–\n2001). Journal of the American Veterinary Medical Association. 226(8), 1368–1374.\nDOI: https://doi.org/10.2460/javma.2005.226.1368\n11. Simpson, A., Ludwig, L., Newman, S., Bergman, P., Hottinger, H. and Patnaik, A. (2004). Evaluation of\nsurgical margins required for complete excision of cutaneous mast cell tumors in dogs. Journal of the\nAmerican Veterinary Medical Association. 224(2), 236–240.\nDOI: https://doi.org/10.2460/javma.2004.224.236\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 1 \nDOI: https://doi.org/10.18849/ve.v7i1.508  \nNext review date: 09 Nov 2023 \np a g e | 18 of 18\n\nIntellectual Property Rights \nAuthors of Knowledge Summaries submitted to RCVS Knowledge for publication will retain \ncopyright in their work, and will be required to grant RCVS Knowledge a non-exclusive license \nof the rights of copyright in the materials including but not limited to the right to publish, re-\npublish, transmit, sell, distribute and otherwise use the materials in all languages and all \nmedia throughout the world, and to license or permit others to do so.\n\nDisclaimer \nKnowledge Summaries are a peer-reviewed article type which aims to answer a clinical \nquestion based on the best available current evidence. It does not override the responsibility \nof the practitioner. 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+{"text": "【兽医文献】\n标题: RAA-CRISPR/Cas12a-driven two-tube, one-tube and one-tube-LFS for rapid detection of feline parvovirus\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nDietary supplementation of Citrus\nbioflavonoids improves lactation\nperformance in buffaloes during\nhot weather by regulating\nantioxidant capacity, immune\nfunction, and rumen microbes\nYinghui Li 1,2,3†, Hanxing Yao 2,3†, Chenglong Li 2,3, Mengwei Li 1,\nFengming Chen 4, Xingguo Huang 2,3* and Chengjian Yang 1*\n1 Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research\nInstitute, Nanning, Guangxi, China, 2 College of Animal Science and Technology, Hunan Agricultural\nUniversity, Changsha, Hunan, China, 3 Yuelushan Laboratory, Changsha, Hunan, China, 4 Hunan\nProvincial Key Laboratory of the TCM Agricultural Biogenomics, Changsha Medical University,\nChangsha, Hunan, China\nCitrus bioflavonoids (CB) are well recognized for their antioxidant, anti-inflammatory,\nand digestion-promoting properties. This study aimed to evaluate the effects of\ndietary CB supplementation on apparent nutrient digestibility, lactation performance,\nblood biochemical/immunological parameters, and the ruminal microbial community\nof dairy buffaloes during hot weather. A 35-day trial was conducted using 20\nMediterranean dairy buffaloes, which were randomly assigned to two groups: a\ncontrol group (CON) fed a basal diet, and a CB-supplemented group (CB) fed the\nbasal diet plus 20 g/d of CB. The results showed that compared with the CON\ngroup, CB supplementation significantly reduced (p < 0.05) the respiratory rate, and\nincreased (p < 0.05) the apparent digestibility of acid detergent fiber in buffaloes.\nRegarding lactation performance, CB supplementation significantly elevated (p < 0.05)\nmilk yield, 4% fat corrected milk, and the percentage of milk protein, lactose, and\nsolids-not-fat; it also increased the concentrations of polyunsaturated fatty acids\nand conjugated linoleic acids in milk while decreasing (p < 0.05) the concentration\nof saturated fatty acids. For blood parameters, the CB group exhibited significantly\nhigher (p < 0.05) blood catalase activity, immunoglobulin M concentration, and heat\n\n05) the concentration\nof saturated fatty acids.For blood parameters, the CB group exhibited significantly\nhigher (p < 0.05) blood catalase activity, immunoglobulin M concentration, and heat\nshock protein 70 concentration, as well as significantly lower (p < 0.05) concentrations\nof the pro-inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1β,\nand IL-6. In terms of ruminal function, CB supplementation significantly increased\n(p < 0.05) ruminal microbial crude protein concentration. There was a tendency\ntoward higher (0.05 < p < 0.10) microbial α-diversity indices (Chao 1, Ace, and\nShannon) in the CB group, accompanied by the enrichment of several bacterial\ngenera within the family Lachnospiraceae and other saccharolytic taxa, and the\nsuppression of the genus Segatella. In conclusion, dietary supplementation with\n20 g/d CB enhances the lactation performance of heat-stressed dairy buffaloes\nby improving the ruminal microenvironment and host health status.\nKEYWORDS\nCitrus bioflavonoids, lactation performance, rumen microbes, immune function, dairy\nbuffalo, heat stress\nOPEN ACCESS\nEDITED BY\nKai Wang,\nChinese Academy of Agricultural Sciences\n(CAAS), China\nREVIEWED BY\nXun Wang,\nQinghai University, China\nYanliang Bi,\nFeed Research Institute (CAAS), China\n*CORRESPONDENCE\nXingguo Huang\nhxg68989@hunau.edu.cn\nChengjian Yang\nycj0746@sina.com\n†These authors have contributed equally to\nthis work\nRECEIVED 17 September 2025\nACCEPTED 20 October 2025\nPUBLISHED 03 November 2025\nCITATION\nLi Y, Yao H, Li C, Li M, Chen F, Huang X and\nYang C (2025) Dietary supplementation of\nCitrus bioflavonoids improves lactation\nperformance in buffaloes during hot weather\nby regulating antioxidant capacity, immune\nfunction, and rumen microbes.\nFront. Vet. Sci. 12:1707093.\ndoi: 10.3389/fvets.2025.1707093\nCOPYRIGHT\n© 2025 Li, Yao, Li, Li, Chen, Huang and Yang.\nThis is an open-access article distributed\nunder the terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other forums is\npermitted, provided the original author(s) and\nthe copyright owner(s) are credited and that\n\nThis is an open-access article distributed\nunder the terms of the Creative Commons\nAttribution License (CC BY).The use,\ndistribution or reproduction in other forums is\npermitted, provided the original author(s) and\nthe copyright owner(s) are credited and that\nthe original publication in this journal is cited,\nin accordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Original Research\nPUBLISHED  03 November 2025\nDOI  10.3389/fvets.2025.1707093\n\nLi et al.\b\n10.3389/fvets.2025.1707093\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n1 Introduction\nGlobal climate change has intensified the frequency and\nseverity of heat waves, posing a significant threat to livestock\nproduction, particularly in tropical and subtropical regions where\nambient temperatures often exceed the thermoneutral zone of\nruminants. Heat stress disrupts the physiological homeostasis of\nanimals, triggering a cascade of adverse responses including\nelevated core body temperature, increased respiratory and heart\nrates, and suppressed feed intake (1, 2). Among ruminants,\nbuffaloes (Bubalus bubalis) are inherently more susceptible to heat\nstress due to their unique anatomical and physiological\ncharacteristics: they possess a lower density of sweat glands, a\nthicker epidermis, and a darker coat, all of which hinder efficient\nheat dissipation through evaporation and radiation (3, 4). Under\nprolonged heat stress, buffaloes experience heightened oxidative\nstress, as the overproduction of reactive oxygen species (ROS)\noverwhelms the endogenous antioxidant defense system,\ndamaging cellular lipids, proteins, and DNA (5). Concurrently,\nheat stress suppresses immune function by altering cytokine\nprofiles and reducing lymphocyte activity, thereby increasing\nsusceptibility to infectious diseases (6). These physiological\nimpairments, coupled with reduced nutrient utilization due to\naltered rumen fermentation, collectively contribute to poor\nlactation performance, making heat stress a critical constraint to\nsustainable buffalo dairy production in hot climates.\nCitrus aurantium L., commonly known as trifoliate orange in\ntraditional Chinese medicine, is a deciduous shrub whose dried\nimmature fruit (Fructus aurantii) has been widely used for\ncenturies to treat digestive and metabolic disorders (7). Modern\nphytochemical studies have identified Citrus bioflavonoids (CB)\nas the primary bioactive components of Fructus Aurantii,\nincluding hesperidin, naringin, neohesperidin, and quercetin,\nwhich are renowned for their antioxidant, anti-inflammatory, and\nimmunomodulatory properties (7). Studies focusing on citrus-\n\nStudies focusing on citrus-\nderived flavonoids have demonstrated their protective role in\nrumen and overall health of ruminants. Paniagua et al. (8, 9)\nreported that Citrus flavonoids supplementation can enhance feed\nefficiency, alter rumen macroscopic characteristics, and improve\nrumen wall health in Holstein bulls. Similarly, other studies have\nshown that Citrus flavonoid extracts reduce bacterial endotoxin\nand systemic inflammation in dairy cows, thereby improving\nimmune-metabolic status and modulating the hindgut\nmicrobiome (10, 11). These findings highlight the potential of CB\nas natural alternatives to synthetic anti-stress agents, aligning with\nthe growing demand for sustainable and antibiotic-free livestock\nproduction. However, research specifically investigating the\neffects and underlying mechanisms of CB on heat-stressed\nbuffaloes remains scarce.\nWe hypothesized that dietary supplementation with CB would\nimprove the fermentation profile, antioxidant status, and immune\nfunction of dairy buffaloes under hot weather conditions by\nregulating rumen microbes, resulting in a positive impact on their\nlactation performance. Therefore, the current study aimed to\nevaluate the effects of dietary CB supplementation on lactation\nperformance, blood antioxidant and immune indices, rumen\nfermentation profiles, and rumen microbial communities.\n2 Materials and methods\n2.1 Animal ethics statement\nAll animal experimental procedures were approved by the\nInstitutional Animal Care and Use Committee of the Guangxi Buffalo\nResearch Institute, Chinese Academy of Agricultural Sciences\n(Nanning, Guangxi, China), with the approval protocol No.\n20240801L.\n2.2 Citrus bioflavonoids\nThe Citrus extract used in this study was kindly provided by\nLianyuan Kanglu Biotechnology Co., Ltd. (Loudi, Hunan, China),\nwith a total flavonoid content of 22.28%. CB were extracted from\ndried bitter orange (Citrus aurantium L.) following the optimized\nprotocol described below. In brief, dried bitter orange was first\ncrushed and sieved through a 40-mesh sieve to obtain a uniform\n\n28%.CB were extracted from\ndried bitter orange (Citrus aurantium L.) following the optimized\nprotocol described below.In brief, dried bitter orange was first\ncrushed and sieved through a 40-mesh sieve to obtain a uniform\nCitrus powder firstly. 1 kg of the Citrus powder was mixed with 10 L\nof 70% (v/v) ethanol, and ultrasonic extraction was performed for\n35 ± 5 min. Then, the mixture after ultrasonic treatment was subjected\nto reflux extraction to maximize the recovery of flavonoids. The\nresulting extract was concentrated and lyophilized for 24 h to obtain\nthe crude CB extract. The total flavonoid content of the crude CB\nextract (22.28%) was determined by HPLC, including naringin\n(7.38%), hesperidin (5.62%), neohesperidin (3.05%), and nobiletin\n(1.26%).\n2.3 Animals, diets and experimental design\nThis study was conducted from August to September 2024 at the\nGuangxi Buffalo Research Institute, Nanning, South China\n(22°53′22.59″N, 108°21′51.19′′E). A total of 20 Mediterranean dairy\nbuffaloes with similar body weight (600 ± 25 kg), parity (2.69 ± 1.30),\nand lactation stage (120 ± 20 d) were enrolled in this study under a\ncompletely randomized design. Buffaloes were randomly assigned to\ntwo dietary treatments: basal diet (CON group) or basal diet\nsupplemented with 20 g/d of CB per buffalo (CB group). The dose of\nCB was determined based on a prior in  vitro rumen simulation\ntechnique experiment conducted by our team. In that in vitro study,\nsupplementing CB at 0.15% of dietary dry matter (DM) in total mixed\nration (TMR) yielded the optimal rumen fluid fermentation\nperformance for dairy buffaloes (unpublished data). For the current\nin vivo study, the CB dose was adjusted to 0.1% of the average dry\nmatter intake (DMI, approximately 14 kg/head·d) of dairy buffaloes,\nresulting in a final dose of 20 g/head·d.\nThe CB product was top-dressed daily and mixed with a small\namount of total mixed ration (TMR) during morning feeding (before\nmilking), and each buffalo was monitored for feed leftover. The\nexperiment lasted for 5 weeks, including a 1-week adaptation period.\n\nThe CB product was top-dressed daily and mixed with a small\namount of total mixed ration (TMR) during morning feeding (before\nmilking), and each buffalo was monitored for feed leftover.The\nexperiment lasted for 5 weeks, including a 1-week adaptation period.\nAll buffaloes were fed the basal TMR twice daily (05:00 and 15:00) for\nad libitum intake. The TMR was formulated to meet the nutritional\nrequirements of dairy buffaloes, consisting of Pennisetum purpureum\n(elephant grass), corn silage, brewer’s grains, and a concentrated feed\nmixture. Details of the chemical composition of the experimental diet\n\nLi et al.\b\n10.3389/fvets.2025.1707093\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nare given in Table 1. Individual DMI was calculated daily by weighing\nthe offered feed and orts for each buffalo. Fresh water was available ad\nlibitum via individual water troughs.\nAside from time for exercise and swimming, buffaloes were\nhoused individually in an open-sided shed. To exercise, the buffaloes\nwere set free in an adjacent open yard with a stocking density of\n15 m2/buffalo. Fans were installed in the buffalo barn to improve\nairflow. Buffaloes were allowed 30 min of swimming time before\nmilking. All buffaloes were machine-milked twice daily (05:00 and\n14:00), and individual milk yield was recorded manually immediately\nafter milking.\n2.4 Recording of meteorological data and\nphysiological parameters of buffalo\nAn online dust monitoring system (Shenzhen Greenforze\nEnvironmental Technology Co., Ltd., Shenzhen, China) was used to\nrecord daily meteorological data in real-time, mainly including air\ntemperature (AT, °C) and relative humidity (RH, %), at an interval of\n30 min. The device was installed at a height equal to the height of the\nanimal’s back and can measure a temperature range from −20 to 80 °C\nwith a precision of ±0.5 °C (error rate ±1%).\nRecording of physiological parameters was performed in the\nmorning and afternoon twice a week (on Monday and Thursdays).\nThe rectal temperature (RT, °C), body surface temperature (BST,\n°C), and respiratory rate (RR, breaths/min) of buffaloes were\nrecorded in the shed in the morning (8:00–9:00 a.m.) and\nafternoon (14:30–15:30 p.m.). The rectal temperature was\nrecorded using an animal rectal thermometer (GLA 700, GLA\nCorporation, San Luis Obispo, CA 93401, U.S.), and the stabilized\nmaximum temperature was taken as the rectal temperature value\nby keeping the thermometer in the rectum for 2 min. Body\nsurface temperature was detected using an animal infrared\nthermometer (HRQ-S60, Zhengzhou Haorunqi Electronic\nTechnology Co., Ltd., Zhengzhou, Henan, China), and the average\ntemperature value of three body sites (forehead, left chest, and\n\nBody\nsurface temperature was detected using an animal infrared\nthermometer (HRQ-S60, Zhengzhou Haorunqi Electronic\nTechnology Co., Ltd., Zhengzhou, Henan, China), and the average\ntemperature value of three body sites (forehead, left chest, and\nleft abdomen) was taken as the body surface temperature of the\nbuffalo. Respiratory rate (breathes/min) was recorded by manual\ncounting using a stopwatch, observation chest and abdomen\nmovements for 2 min, and the average value per minute was\ncalculated as the RR.\n2.5 Evaluation of thermal comfort state in\nbuffalo\nThe thermal comfort state of buffaloes was evaluated using the\npaired index models (E3 for environment conditions and P3 for\nphysiological status) proposed by Li et al. (12), which have been\nvalidated as practical and accurate for assessing the thermal\ncomfort of dairy buffaloes under hot and humid climate. The\ncalculation formulas for the two indices are as follows: E3\n(Environmental index model) = 1.016 × AT (°C) + 0.139 × RH\n(%); P3 (physiological index model) = 0.654 × BST (°C) + 0.381 ×\nRR (breaths/min). Thermal comfort levels of buffaloes are\ncategorized into four grades based on the E3 and P3 values, with\nthresholds and corresponding states defined as follows: comfort\n(E3 ≤ 37.15, P3 ≤ 25.30); danger (37.15 < E3 ≤ 44.06, 25.30 <\nP3 ≤ 28.64); stress (44.06 < E3 < 50.97, 28.64 < P3 < 31.98);\nemergency (E3 ≥ 50.97, P3 ≥ 31.98).\n2.6 Apparent total tract digestibility\n(ATTD) analysis\nFeed samples were collected weekly throughout the\nexperimental period and immediately stored at −20 °C. Samples\nwere composited by period, dried at 65 °C for 48 h, ground using\na multifunctional pulverizer (Old Bank Boou Hardware Factory,\nZhejiang, China), and sieved through a 40-mesh stainless steel\nsieve (manufactured by Tianxing Wusi Yarn Sieve Factory,\nShangyu City, Zhejiang, China). The ground sample was stored at\n−20 °C until subsequent nutrient analysis. The dry matter (DM),\ncrude protein (CP), ether extract (EE), acid detergent fiber (ADF),\nneutral detergent fiber (NDF), and acid-insoluble ash of feed were\n\nThe ground sample was stored at\n−20 °C until subsequent nutrient analysis.The dry matter (DM),\ncrude protein (CP), ether extract (EE), acid detergent fiber (ADF),\nneutral detergent fiber (NDF), and acid-insoluble ash of feed were\ndetermined according to the Association of Official Analytical\nChemists (AOAC, 1995).\nFecal samples were collected during the last 3 consecutive days\nof the experimental period, with two collections per day (07:00\nand 17:00). Feces were weighed and mixed daily, and 10 mL of\n10% H2SO4 was added to each daily mixed fecal sample (per 100 g\nof fresh feces) to fix nitrogen, then stored at −20 °C for further\nanalysis. Subsequently, thawed fecal samples were dried at 65 °C\nfor 48 h and ground to pass through a 40-mesh sieve.\nConcentrations of CP, EE, ADF, and NDF in feces were analyzed\nas described above.\nTABLE 1  Composition and nutritional level in the basal diet (DM basis).\nItems\nContent\nIngredients, %\nPennisetum purpureum Schumach.\n12.00\nCorn silage\n29.28\nBrewer’s grains\n40.51\nConcentrated feed mixture1\n17.21\nPremix2\n0.50\nNaCl\n0.50\nTotal\n100.00\nNutrient levels3, %\nGE, MJ/kg\n17.10\nCP\n18.94\nCrude ash\n7.18\nNDF\n45.16\nADF\n23.67\nCa\n1.83\nP\n0.37\n1The concentrated feed mixture was purchased from a livestock and poultry company in\nNanning, with the following nutritional composition (as-fed basis): DM 91.50%, CP 20.05%,\ncrude ash 7.93%, NDF 15.44%, ADF 6.52%, and GE 19.04 MJ/kg.\n2Per kg premix contained the following: VA 550,000 IU, VE 3,000 IU, VD 3150,000 IU, Fe (as\nferrous sulfate) 4.0 g, Cu (as copper sulfate) 1.3 g, Mn (as manganese sulfate) 3.0 g, Zn (as\nzinc sulfate) 6.0 g, Co (as Cobalt sulfate) 80.0 mg.\n3All the nutrient levels are measured values.\n\nLi et al.\b\n10.3389/fvets.2025.1707093\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n2.7 Milk yield and composition\nMorning (05:00) and afternoon (14:00) milk yield was\nrecorded daily for each buffalo, and milk samples for determination\nof milk composition were collected weekly. Fresh milk samples\nwere used to analyze milk composition, including milk protein, fat,\nlactose, milk urea nitrogen (MUN), total solids (TS), solids-not-fat\n(SNF), free fatty acids (FFA) for morning and afternoon separately\nusing\nMilkoScan™\nF120\n(FOSS,\nHillerød,\nDenmark).\nSubsequently, 4% fat-corrected milk (4% FCM) was calculated\naccording to NRC (2001) using the following equation: 4%\nFCM = 0.4 × milk yield (kg/d) + 0.15 × milk fat percentage\n(%) × milk yield (kg/d).\nMilk samples collected twice a day were mixed proportionally\nto morning and afternoon milk yield for each week and stored at\n−20 °C. The pooled samples were quickly sent to the Shanghai\nSensichip Biotech Co., Ltd. for analysis of medium- and long-chain\nfatty acid profile using chromatography-mass spectrometry (GC–\nMS), which was performed with a Thermo Scientific Trace 1,300\ngas chromatograph connected to a Thermo Scientific ISQ7000\nsingle quadrupole mass spectrometer selective detector\n(Trace1300- ISQ7000, Thermo Fisher Scientific, Waltham, MA,\nUSA). A DB-5MS chromatographic column was used\n(60 m × 0.25 mm inner diameter and 0.25 μm film thickness; J&W\nScientific, USA).\n2.8 Blood samples collection and analysis\nBefore morning feeding on the last day of the experimental\nperiod, blood samples (approximately 10 mL) were collected from\nthe coccygeal vein of buffaloes using disposable vacuum blood\ncollection tubes (non-anticoagulant tubes) (Shandong Aosaite\nMedical Devices Co., Ltd.). The blood samples were then\ncentrifuged at 4 °C (1,500 × g) for 15 min, and serum was\nharvested and stored at −80 °C until further analysis.\nSimultaneously, 10 mL blood samples were collected using heparin\nsodium-anticoagulated tubes and centrifuged at 4 °C (3,500 × g)\nfor 15 min. The resulting plasma was then stored at −80 °C until\nfurther analysis.\n\nSimultaneously, 10 mL blood samples were collected using heparin\nsodium-anticoagulated tubes and centrifuged at 4 °C (3,500 × g)\nfor 15 min.The resulting plasma was then stored at −80 °C until\nfurther analysis.\nSerum concentrations of total protein (TP), albumin (ALB), alanine\ntransaminase (ALT), aspartate aminotransferase (AST), alkaline\nphosphatase (ALP), creatine kinase (CK), lactate dehydrogenase (LDH),\nurea nitrogen (UN), glucose (GLU), total cholesterol (TC), triglyceride\n(TG), immunoglobulin G (IgG), IgM, IgA, insulin (INS),\ntriiodothyronine (T3), and tetraiodothyronine (T4) were determined\nusing an automatic blood biochemical analyzer (Cobas c311, Roche,\nSwitzerland), according to the manufacturer’s instructions (Roche\nBiochemical Kit). Plasma superoxide dismutase (SOD) and catalase\n(CAT) activities, as well as total antioxidant capacity (T-AOC) and\nmalondialdehyde (MDA) levels, were measured using commercial kits\n(Nanjing Jiancheng Bioengineering Institute) in strict compliance with\nthe operating procedures. The concentrations of serum amyloid A\n(SAA), heat shock protein 70 (HSP70), tumor necrosis factor-α\n(TNF-α), interferon-γ (IFN-γ), interleukin-1beta (IL-1β), interleukin-2\n(IL-2), and interleukin-6 (IL-6) were detected using ELISA kits provided\nby Jiangsu Jingmei Biotechnology according to the manufacturers’\nguidelines.\n2.9 Collection of ruminal contents and\nprocessing\nOn the last day of the experiment before morning feeding,\nruminal contents samples (500 mL) were collected in sterilized plastic\nbottles from buffaloes using a stainless-steel stomach tube. After\ncollection, samples were immediately transferred to the laboratory for\nfurther analysis. Subsequently, the ruminal contents were strained\nthrough two layers of cheesecloth and subsamples of ruminal contents\nfor determination of volatile fatty acids (VFA), ammonia nitrogen\n(NH3-N) and microbial crude protein (MCP) were stored at\n−20 °C. Subsamples for DNA extraction were stored at −80 °C until\nfurther processing.\n2.10 Determination of rumen fermentation\nparameters\nAfter the collection of ruminal contents, pH was measured\n\nSubsamples for DNA extraction were stored at −80 °C until\nfurther processing.2.10 Determination of rumen fermentation\nparameters\nAfter the collection of ruminal contents, pH was measured\nimmediately using a pH meter (HI 9024C; HANNA Instruments,\nWoonsocket, RI, USA). A subsample of rumen fluid (4 mL) was\nacidified with 4 mL of 0.2 mol/L HCl and stored at −20 °C for\ndetermination of NH3-N using the indophenols method. MCP\ncontent was analyzed with a spectrophotometer at 595 nm using\n1 mg/mL bovine serum albumin solution (Sigma-Aldrich Co., LLC,\nSt. Louis, MO, USA) as the standard. The concentrations of VFA,\nincluding acetate, propionate, isobutyrate, butyrate, isovalerate, and\nvalerate, were measured using a gas chromatography (GC) system\n(Agilent 7890A, Agilent Technologies, USA).\n2.11 DNA extraction from ruminal contents\nand sequencing of the 16S rRNA gene\nTotal genomic DNA from rumen fluid was extracted using an\nE.Z.N.A Soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA).\nDNA concentration and purity were determined using a\nNanoDrop\n2000\nspectrophotometer\n(Thermo\nScientific,\nWilmington, DE, USA). High throughput (Illumina MiSeq)\nsequencing of the 16S rRNA gene was carried out using barcoded\nprimers targeting the V3–V4 region. Amplicons were extracted\nfrom 2% agarose gels and purified using the QIAquick PCR\nPurification Kit (Qiagen, Hilden, Germany), and library\nconstruction was performed according to Illumina’s instructions.\nPaired-end reads were assigned to the samples based on their\nunique barcodes and truncated by removing the barcodes and primer\nsequences. The raw read pairs were overlapped and merged using\nFLASH (v1.2.11), then imported into QIIME 2 for demultiplexing.\nQuality control, denoising, removal of chimeric sequences, and\ngeneration of amplicon sequencing variants (ASVs) were performed\nusing the QIIME2 DADA2 plugin. The SILVA database (version 138)\nwas used for ASV classification and annotation analysis, and the\nmicrobial species composition at different taxonomic levels (phylum,\nclass, order, family, genus, and species) was obtained. Several\n\nThe SILVA database (version 138)\nwas used for ASV classification and annotation analysis, and the\nmicrobial species composition at different taxonomic levels (phylum,\nclass, order, family, genus, and species) was obtained.Several\nα-diversity indices, including Chao 1, Ace, Shannon, and Simpson\nindices, were calculated using QIIME 2. Microbial β-diversity was\ndetermined using distance matrices generated from Bray-Curtis\nanalysis, principal coordinate analysis (PCoA), and analysis of\n\nLi et al.\b\n10.3389/fvets.2025.1707093\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nsimilarities (ANOSIM). Linear discriminant analysis effect size\n(LEfSe) was used to identify significantly different taxa between the\ntwo treatment groups.\n2.12 Statistical analysis\nData were analyzed using independent samples t-test with SPSS\nversion 23 (IBM Corp., Armonk, NY, USA). Differences in microbial\ncomposition and α-diversity between the two groups were compared\nusing Welch’s t-test, and p values were corrected by false-discovery\nrate adjustment. All results are reported as means ± standard error of\nthe mean (SEM), with significant differences declared at p < 0.05 and\na trend of difference defined at 0.05 ≤ p < 0.10.\n3 Results\n3.1 Thermal comfort state in dairy buffalo\nduring the experiment\nDuring the experimental period, the E3 ranged from 40.71 to\n41.30, indicating that the thermal comfort state of the buffaloes\nwas in the “danger” grade; meanwhile, the P3 varied from 31.30\nto 38.00, with most values exceeding the “emergency” threshold\n(P3 ≥ 31.98), indicating that the dairy buffaloes were primarily in\nan emergency state (Figure 1). Notably, compared with E3 (which\nintegrates AT and RH), P3 (combining BST and RR) more\nintuitively reflected the actual heat stress status of the buffaloes,\nas it directly mirrors the host’s physiological responses to thermal\nstress. Collectively, the E3 and P3 data confirmed that the dairy\nbuffaloes in this experiment experienced relatively severe and\npersistent heat stress throughout the trial.\n3.2 Physiological indices\nAs shown in Table 2, the RR of buffaloes in the CB group was\nsignificantly lower (p < 0.05) than that in the CON group, with a\nreduction of 17.0%. Meanwhile, compared to the CON group, CB\nsupplementation tended to decrease the BST of the dairy buffaloes\n(p = 0.07). In contrast, dietary CB supplementation had no effect on\nthe RT of the dairy buffaloes (p > 0.05).\n3.3 Feed intake and nutrient apparent\ndigestibility\nResults revealed no significant effect (p > 0.05) of CB\nsupplementation on the DMI of the dairy buffaloes (Table 3). Similarly,\n\n05).3.3 Feed intake and nutrient apparent\ndigestibility\nResults revealed no significant effect (p > 0.05) of CB\nsupplementation on the DMI of the dairy buffaloes (Table 3).Similarly,\nno differences were observed between the CON and CB groups in the\napparent digestibility of nutrients including CP, EE, and NDF\n(p > 0.05). Nevertheless, the apparent digestibility of ADF in the CB\ngroup was 6.89% higher (p < 0.05) than that in the CON group\n(Table 3).\n3.4 Milk production and composition\nThe effects of CB supplementation on milk production and\ncomposition of dairy buffaloes during hot weather are summarized in\nTable  4. Compared to the CON group, CB supplementation\nsignificantly increased (p < 0.05) milk yield, 4% FCM, and several\nmilk composition parameters including milk protein, milk lactose,\nSNF, and FFA with. However, no differences in milk fat, MUN, or TS\ncontents were observed between the CON and CB groups (p > 0.05).\n3.5 Milk fatty acid composition\nIn the present study, we quantified 38 major fatty acids including\n15 saturated fatty acids (SFA), 10 monounsaturated fatty acids\n(MUFA), and 13 polyunsaturated fatty acids (PUFA) via GC–MS\nanalysis. Our study revealed that the major SFA in buffalo milk were\nC12:0, C21:0, and C10:0, while the major unsaturated fatty acid (UFA)\nwere C18:2 (10-TRANS, 12-CIS), C18:1n9t, and C20:5n3\n(eicosapentaenoic acid, EPA) (Table 5). Compared to the CON group,\nthe CB group exhibited a significant decrease in the contents of major\nSFA (C14:0, C15:0, C16:0, and C18:0) and a significant increase in the\ncontents of MUFA (C20:1 and C22:1n9) and PUFA (C18:2(10-\nTRANS, 12-CIS), C20:3n3, and C22:5n3) in milk (p  < 0.05).\nConsequently, dairy buffaloes receiving CB had lower total SFA\ncontents and SFA/UFA ratio in milk, as well as higher total UFA,\nPUFA, and CLA contents, than those in the CON group (p < 0.05).\n3.6 Blood biochemical indices, hormone\nlevels, oxidative status and immune indices\nTable  6 shows that, compared to the CON diet, the CB diet\nsignificantly increased (p < 0.05) the serum concentration of TP in\n\n05).3.6 Blood biochemical indices, hormone\nlevels, oxidative status and immune indices\nTable  6 shows that, compared to the CON diet, the CB diet\nsignificantly increased (p < 0.05) the serum concentration of TP in\ndairy buffaloes; serum ALB concentration also tended to increase\n(p = 0.072) with CB supplementation. However, no significant\nFIGURE 1\nEnvironmental index and physiological index models of dairy\nbuffaloes during the experiment. The environment variables and the\nbuffalo physiological index variables can be derived from this index\nmodel (practical model) as follows: E3 (Environmental index\nmodel) = 1.016 × AT (°C) + 0.139 × RH (%); P3 (physiological index\nmodel) = 0.654 × BST (°C) + 0.381 × RR (breaths/min). Buffalo status\nis comfort (E3 ≤ 37.15; P3 ≤ 25.30), danger (37.15 < E3 ≤ 44.06;\n25.30 < P3 ≤ 28.64), stress (44.06 < E3 < 50.97; 28.64 < P3 < 31.98),\nemergency (E3 ≥ 50.97; P3 ≥ 31.98). AT, air temperature; RH, relative\nhumidity; BST, body surface temperature; RR, respiratory rate.\n\nLi et al.\b\n10.3389/fvets.2025.1707093\nFrontiers in Veterinary Science\n06\nfrontiersin.org\ndifferences were observed in other detected blood biochemical\nparameters between buffaloes fed the CON and CB diets (p > 0.05).\nSimilarly, dietary CB failed to influence serum levels of hormones\nsuch as INS, T3, and T4 in dairy buffaloes (p > 0.05). In addition,\ncompared to the CON group, CB supplementation significantly\nincreased the plasma concentration of CAT (p < 0.01); however,\nplasma SOD activity and T-AOC and MDA levels were not affected\nby CB supplementation (p > 0.05). Regarding serum immune indices,\ndietary CB supplementation significantly increased serum\nconcentrations of IgM and HSP70, while significantly decreasing\nserum concentrations of TNF-α, IL-1β, and IL-6 (p < 0.05).\n3.7 Fermentation profile in the ruminal\ncontents\nRegarding the ruminal fermentation profile, the concentrations of\nTVFA, acetate, propionate, isobutyrate, butyrate, isovalerate, and\nvalerate, along with the A:P value were not significantly altered by CB\nsupplementation (p > 0.05; Table 7). Moreover, CB supplemention had\nno significant effect on ruminal fluid pH or NH3-N concentration\n(p  > 0.05), whereas it significantly increased (p  < 0.05) ruminal\nMCP concentration.\n3.8 Taxonomic configuration of ruminal\nbacteria\nThe effect of CB supplementation on ruminal microbes in dairy\nbuffaloes was evaluated using 16S rRNA high-throughput sequencing.\nAs shown in Figures 2A–D, the α-diversity indices of CB group,\nincluding species richness (Chao 1 and Ace indices), tended to\nincrease (p = 0.071 and p = 0.072, respectively) compared to the CON\ngroup, and bacterial diversity (Shannon index) exhibited a similar\ntrend. No differences were found in the Simpson index between the\nCON and CB groups (p > 0.10). Furthermore, β-diversity of rumen\nbacteria was determined via PCoA, which explained 22.42 and 12.31%\nof the variance, respectively, however, no distinct separation between\nthe CON and CB groups was observed (Figure 2E).\nAs illustrated in Figure  2F, Bacteroidota (62.35%), Bacillota\n(28.74%), Pseudomonadota (3.18%), Spirochaetota (1.61%), and\n\n42 and 12.31%\nof the variance, respectively, however, no distinct separation between\nthe CON and CB groups was observed (Figure 2E).As illustrated in Figure  2F, Bacteroidota (62.35%), Bacillota\n(28.74%), Pseudomonadota (3.18%), Spirochaetota (1.61%), and\nFibrobacterota (1.35%) were the dominant bacterial phyla in both\ngroups, with no significant differences in phyla relative abundance\nbetween the CON and CB groups (p > 0.05, Table 8). At the genus\nlevel, the ruminal microbiome was dominated by Xylanibacter\n(35.31%), norank_f__F082 (6.13%), Rikenellaceae_RC9_gut_group\n(6.09%), Christensenellaceae_R-7_group (3.65%), NK4A214_group\n(3.43%), norank_f__Muribaculaceae (3.28%), and Prevotellaceae_\nUCG-001 (3.08%) (Figure 2G). Compared to the CON group, CB\nsupplementation significantly decreased the relative abundance of\nSegatella in dairy buffaloes (p < 0.01; Table 9). Notably, the CB group\nexhibited an approximately 83.16% increase in the relative abundance\nof Fibrobacterota (phyla level; Table 8) as well as an 82.11% increase\nin the relative abundance of Fibrobacter (genus level; Table  9)\ncompared to the CON group, although these increases did not reach\nstatistical significance.\nAdditionally, LEfSe analysis (LDA > 2.5, p < 0.05) was used to\nidentify significantly differential bacterial taxa between the CON and\nCB groups, with 12 biomarkers identified. Increased abundance of\nmembers of the dominant Bacteroidota phylum was evident in\nresponse\nto\nCB\nsupplementation,\nincluding\nthe\nfamily\nTABLE 2  Effects of dietary with Citrus bioflavonoids (CB)\nsupplementation on physiological indices of dairy buffaloes during hot\nweather.\nItems\nTreatment groups1\np-value\nCON\nCB\nBST, °C\n35.93 ± 0.04\n35.77 ± 0.07\n0.070\nRT, °C\n38.62 ± 0.06\n38.74 ± 0.07\n0.221\nRR, breaths/\nmin\n32.10 ± 1.09a\n27.57 ± 1.30b\n0.015\nBST, body surface temperature; RR, respiratory rate; RT, rectal temperature.\na,bMean values within a row with different superscripts differed (p < 0.05).\n1CON, control diet with no CB supplementation; CB, control diet with 20 g/d CB\nsupplementation for per buffalo.\nTABLE 3  Effects of dietary with Citrus bioflavonoids (CB)\n\na,bMean values within a row with different superscripts differed (p < 0.05).1CON, control diet with no CB supplementation; CB, control diet with 20 g/d CB\nsupplementation for per buffalo.TABLE 3  Effects of dietary with Citrus bioflavonoids (CB)\nsupplementation on the dry matter intake and nutrient apparent\ndigestibility of dairy buffaloes during hot weather.\nItems\nTreatment groups1\np-value\nCON\nCB\nDMI, kg/d\n15.03 ± 0.30\n15.16 ± 0.80\n0.882\nCP, %\n58.56 ± 1.84\n59.53 ± 1.500\n0.685\nEE, %\n60.98 ± 2.48\n62.57 ± 2.52\n0.658\nADF, %\n33.66 ± 1.35b\n40.55 ± 1.97a\n0.010\nNDF, %\n58.11 ± 1.05\n57.18 ± 1.80\n0.663\nDMI, dry matter intake; CP, crude protein; EE, ether extract; ADF, acid detergent fiber; NDF,\nneutral detergent fiber.\na,bMean values within a row with different superscripts differed (p < 0.05).\n1CON, control diet with no CB supplementation; CB, control diet with 20 g/d CB\nsupplementation for per buffalo.\nTABLE 4  Effects of dietary with Citrus bioflavonoids (CB)\nsupplementation on milk production and composition in dairy buffaloes\nduring hot weather.\nItems\nTreatment groups1\np-value\nCON\nCB\nMilk yield, kg/d\n6.76 ± 0.03b\n7.32 ± 0.04a\n< 0.01\n4%FCM2, kg/d\n10.66 ± 0.05b\n11.74 ± 0.07a\n< 0.01\nMilk protein, %\n4.59 ± 0.02b\n4.77 ± 0.02a\n< 0.01\nMilk fat, %\n7.85 ± 0.46\n8.03 ± 0.37\n0.772\nMilk lactose, %\n5.01 ± 0.02b\n5.17 ± 0.02a\n0.017\nMUN, mg/dL\n21.80 ± 0.88\n20.55 ± 0.81\n0.312\nTS, %\n18.34 ± 0.50\n18.51 ± 0.40\n0.800\nSNF, %\n10.26 ± 0.03b\n10.44 ± 0.02a\n< 0.01\nFFA, mmol/L\n0.41 ± 0.01b\n0.49 ± 0.01a\n< 0.01\nMUN, milk urea nitrogen; TS, total solids; SNF, milk solids-not-fat; FFA, Free fatty acids;\nFCM, fat corrected milk.\na,bMean values within a row with different superscripts differed (p < 0.05).\n1CON, control diet with no CB supplementation; CB, control diet with 20 g/d CB\nsupplementation for per buffalo.\n24%FCM = 0.4 × milk yield (kg/d) + 0.15 × milk fat (%) × milk yield (kg/d).\n\nLi et al.\b\n10.3389/fvets.2025.1707093\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nTABLE 5  Effects of dietary with Citrus bioflavonoids (CB) supplementation on milk fatty acids composition in dairy buffaloes during hot weather.\nItems\nTreatment groups1\np-value\nCON\nCB\nC8:0, μg/mL\n13.65 ± 2.04\n17.36 ± 1.70\n0.143\nC10:0, μg/mL\n313.32 ± 28.25\n338.17 ± 27.25\n0.535\nC11:0, μg/mL\n8.62 ± 1.75\n9.37 ± 1.18\n0.728\nC12:0, μg/mL\n602.12 ± 44.10\n587.57 ± 39.83\n0.809\nC13:0, μg/mL\n56.08 ± 7.99\n60.81 ± 6.30\n0.648\nC14:1n5, μg/mL\n24.46 ± 3.67\n29.88 ± 3.38\n0.291\nC14:0, μg/mL\n304.46 ± 25.82a\n87.79 ± 9.39b\n< 0.01\nC15:1n5, μg/mL\n148.97 ± 5.56\n134.17 ± 9.34\n0.194\nC15:0, μg/mL\n217.75 ± 15.89a\n168.28 ± 15.22b\n0.037\nC16:1n7, μg/mL\n262.56 ± 27.15\n339.91 ± 25.07\n0.051\nC16:0, μg/mL\n352.73 ± 41.30a\n176.45 ± 10.59b\n< 0.01\nC17:1n7, μg/mL\n6.69 ± 0.97\n5.95 ± 0.78\n0.561\nC17:0, μg/mL\n185.33 ± 21.67\n217.89 ± 15.73\n0.240\nC18:3n6, μg/mL\n140.62 ± 21.09\n144.02 ± 22.00\n0.912\nC18:3n3, μg/mL\n155.86 ± 12.15\n169.13 ± 14.26\n0.487\nC18:2n6c, μg/mL\n17.06 ± 2.27\n21.84 ± 2.17\n0.146\nC18:2 (9-CIS, 11-TRANS), μg/mL\n209.18 ± 22.78\n210.73 ± 21.94\n0.961\nC18:2 (10-TRANS, 12-CIS), μg/mL\n411.54 ± 25.98b\n557.61 ± 20.71a\n< 0.01\nC18:1n9c, μg/mL\n280.23 ± 47.01\n262.61 ± 41.80\n0.783\nC18:1n9t, μg/mL\n379.41 ± 48.63\n449.88 ± 65.07\n0.397\nC18:0, μg/mL\n331.22 ± 24.91a\n236.15 ± 25.34b\n0.015\nC20:5n3 (EPA), μg/mL\n332.66 ± 30.20\n340.06 ± 15.55\n0.831\nC20:4n6 (ARA), μg/mL\n11.11 ± 2.19\n12.58 ± 1.47\n0.584\nC20:3n3, μg/mL\n8.97 ± 1.29b\n18.44 ± 1.02a\n< 0.01\nC20:3n6, μg/mL\n92.46 ± 9.34\n116.89 ± 11.79\n0.122\nC20:2, μg/mL\n198.61 ± 17.17\n229.30 ± 12.14\n0.162\nC20:1, μg/mL\n31.86 ± 5.44b\n66.81 ± 5.68a\n< 0.01\nC20:0, μg/mL\n84.64 ± 7.81\n86.01 ± 7.87\n0.903\nC21:0, μg/mL\n358.07 ± 24.92b\n432.45 ± 24.02a\n0.045\nC22:6n3 (DHA), μg/mL\n53.05 ± 5.13\n50.09 ± 6.46\n0.724\nC22:5n3 (DPA), μg/mL\n14.08 ± 1.25b\n25.27 ± 2.10a\n< 0.01\nC22:5n6, μg/mL\n32.13 ± 3.04\n39.31 ± 3.98\n0.169\nC22:1n9, μg/mL\n25.65 ± 2.98b\n42.40 ± 3.21a\n< 0.01\nC22:1 T, μg/mL\n16.94 ± 1.26\n20.55 ± 2.63\n0.232\nC22:0, μg/mL\n165.36 ± 14.03\n161.28 ± 14.72\n0.843\nC23:0, μg/mL\n97.28 ± 8.68\n100.20 ± 11.42\n0.841\nC24:1n9, μg/mL\n15.54 ± 1.44\n19.74 ± 2.41\n0.156\nC24:0, μg/mL\n108.88 ± 9.53\n115.27 ± 10.58\n0.659\nSFA, μg/mL\n3199.52 ± 141.26a\n2795.05 ± 116.15b\n0.040\nUFA, μg/mL\n2869.63 ± 99.43b\n3307.17 ± 106.74a\n< 0.01\nSFA/UFA\n1.12 ± 0.05a\n0.85 ± 0.03b\n< 0.01\nMUFA, μg/mL\n1192.31 ± 69.41\n1371.89 ± 85.60\n0.121\nPUFA, μg/mL\n1677.32 ± 54.80b\n1935.28 ± 45.57a\n< 0.01\nCLA2, μg/mL\n620.72 ± 43.43b\n768.34 ± 28.42a\n0.011\n\n52 ± 141.26a\n2795.05 ± 116.15b\n0.040\nUFA, μg/mL\n2869.63 ± 99.43b\n3307.17 ± 106.74a\n< 0.01\nSFA/UFA\n1.12 ± 0.05a\n0.85 ± 0.03b\n< 0.01\nMUFA, μg/mL\n1192.31 ± 69.41\n1371.89 ± 85.60\n0.121\nPUFA, μg/mL\n1677.32 ± 54.80b\n1935.28 ± 45.57a\n< 0.01\nCLA2, μg/mL\n620.72 ± 43.43b\n768.34 ± 28.42a\n0.011\nSFA, saturated fatty acids; UFA, unsaturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; CLA, conjugated linoleic acids.\na,bMean values within a row with different superscripts differed (p < 0.05).\n1CON, control diet with no CB supplementation; CB, control diet with 20 g/d CB supplementation for per buffalo.\n2CLA included the C18:2 (9-CIS, 11-TRANS) and C18:2 (10-TRANS, 12-CIS) fatty acids.\n\nLi et al.\b\n10.3389/fvets.2025.1707093\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nTABLE 6  Effects of dietary with Citrus bioflavonoids (CB)\nsupplementation on blood biochemical indices, hormone levels,\noxidative status, and immune indices of dairy buffaloes during hot\nweather.\nItems\nTreatment groups1\np-value\nCON\nCB\nBiochemical indices\nTP, g/L\n72.16 ± 1.79b\n77.76 ± 1.14a\n0.018\nALB, g/L\n37.03 ± 0.93\n39.04 ± 0.53\n0.072\nGLO, g/L\n35.12 ± 1.55\n37.73 ± 1.45\n0.236\nA:G\n1.09 ± 0.05\n1.04 ± 0.05\n0.504\nALT, U/L\n52.38 ± 2.51\n53.90 ± 3.02\n0.713\nAST, U/L\n137.00 ± 5.66\n147.50 ± 7.08\n0.270\nAST:ALT\n2.56 ± 0.14\n2.82 ± 0.22\n0.353\nALP, U/L\n173.44 ± 16.75\n190.38 ± 23.06\n0.555\nCK, U/L\n168.78 ± 13.21\n162.89 ± 10.26\n0.729\nLDH, U/L\n596.88 ± 19.31\n602.80 ± 41.27\n0.899\nUN, mmol/L\n6.21 ± 0.28\n6.34 ± 0.29\n0.750\nGLU, mmol/L\n2.32 ± 0.22\n2.57 ± 0.13\n0.839\nTC, mmol/L\n2.98 ± 0.10\n2.93 ± 0.15\n0.798\nTG, mmol/L\n0.23 ± 0.04\n0.23 ± 0.03\n0.933\nHormone levels\nINS, uIU/mL\n7.01 ± 0.70\n6.83 ± 074\n0.865\nT3, ng/mL\n1.14 ± 0.04\n1.18 ± 0.11\n0.771\nT4, ug/dL\n4.24 ± 0.28\n3.95 ± 0.27\n0.471\nOxidative status\nSOD, U/mL\n23.01 ± 2.57\n26.24 ± 0.62\n0.447\nT-AOC, nmol/\nmL\n799.40 ± 20.43\n807.73 ± 13.10\n0.736\nCAT, U/mL\n1.80 ± 0.21b\n2.91 ± 0.26a\n< 0.01\nMDA, nmol/\nmL\n1.75 ± 0.21\n1.70 ± 0.13\n0.810\nImmune indices\nIgG, mg/dL\n0.088 ± 0.003\n0.079 ± 0.005\n0.157\nIgM, mg/dL\n0.50 ± 0.05b\n0.62 ± 0.03a\n0.048\nIgA, mg/dL\n0.044 ± 0.002\n0.047 ± 0.003\n0.522\nSAA, μg/mL\n2.63 ± 0.15\n2.64 ± 0.11\n0.978\nHSP70, pg./mL\n35.92 ± 1.93b\n42.42 ± 1.36a\n0.012\nTNF-α, pg./mL\n17.69 ± 0.51a\n14.62 ± 0.69b\n< 0.01\nIFN-γ, pg./mL\n347.75 ± 20.65\n342.33 ± 14.71\n0.831\nIL-1β, pg./mL\n143.32 ± 4.28a\n126.60 ± 5.87b\n0.035\nIL-2, pg./mL\n56.42 ± 6.31\n55.07 ± 3.12\n0.851\nIL-6, pg./mL\n167.62 ± 8.98a\n140.15 ± 6.15b\n0.024\nTP, total protein; ALB, albumin; GLO, globulin; A:G, albumin-to- globulin ratio; ALT,\nalanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; CK,\ncreatine kinase; LDH, lactate dehydrogenase; UN, urea nitrogen; GLU, glucose; TC, total\ncholesterol; TG, triglyceride; INS, insulin; T3, triiodothyronine; T4, tetraiodothyronine;\nSOD, superoxide dismutase; T-AOC, total antioxidant capacity; CAT, catalase; MDA,\nmalondialdehyde; IgG, immunoglobulin G; IgM, immunoglobulin M; IgA, immunoglobulin\nA; SAA, serum amyloid-A; HSP70, heat shock protein 70; TNF-α, tumor necrosis factor-α;\nIFN-γ, interferon-γ; IL-1β, interleukin-1β; IL-2, interleukin-2; IL-6, interleukin-6.\n\na,bMean values within a row with different superscripts differed (p < 0.05).\n1CON, control diet with no CB supplementation; CB, control diet with 20 g/d CB\nsupplementation for per buffalo.\nBacteroidetes_BD2-2 as well as genera such as norank_f__\nBacteroidetes_BD2-2 and norank_f__Weeksellaceae. Besides, the\ndominant Bacillota phylum was also enriched in the CB-supplemented\ngroup, including genera such as norank_f__Lachnospiraceae, probable_\ngenus_10, [Eubacterium]_xylanophilum_group, and Oribacterium, all\nbelonging to the family Lachnospiraceae. Moreover, the order\nBradymonadales and its members (including f__norank_o__\nBradymonadales and g__norank_o__Bradymonadales) were enriched\nin the CB group. In contrast, the CON group exhibited a higher\ndifferential abundance of the genera Segatella and Schwartzia\n(Figure 2H).\n4 Discussion\nOur laboratory’s previous findings showed that the most accurate\nmodel, based on BST and RR, effectively indicates thermal comfort\nin dairy buffaloes under hot and humid climates, and this model was\ndeveloped accordingly (13). Using this established thermal comfort\nindex, we  confirmed that the dairy buffaloes in the present\nexperiment suffered from relatively severe and persistent heat stress.\nPhytogenics-derived flavonoids, such as those from mulberry leaves\nand propolis extract, have emerged as promising modulators for\nalleviating heat stress responses in ruminants, with accumulating\nevidence supporting their ability to mitigate physiological\nperturbations induced by heat stress (4, 14, 15). Heat stress triggers a\nseries of adaptive mechanisms in livestock, including increased RR,\na primary thermoregulatory response to dissipate excess body heat\n(15). As expected, dietary supplementation with CB reduced the RR\nand BST of dairy buffaloes under hot weather conditions in the\npresent study, and such reductions are indicative of heat stress\nTABLE 7  Effects of dietary with Citrus bioflavonoids (CB)\nsupplementation on fermentation profile in the rumen of dairy buffaloes\nduring hot weather.\nItems\nTreatment groups1\np-value\nCON\nCB\npH\n6.76 ± 0.10\n6.89 ± 0.08\n0.346\nNH3-N, mg/dL\n8.71 ± 0.44\n7.20 ± 1.03\n0.233\n\nItems\nTreatment groups1\np-value\nCON\nCB\npH\n6.76 ± 0.10\n6.89 ± 0.08\n0.346\nNH3-N, mg/dL\n8.71 ± 0.44\n7.20 ± 1.03\n0.233\nMCP, mg/dL\n12.25 ± 0.33b\n14.36 ± 0.60a\n0.015\nTVFA, mmol/L\n84.32 ± 1.46\n78.16 ± 7.36\n0.455\nAcetate, mmol/L\n56.86 ± 2.15\n50.96 ± 4.11\n0.214\nPropionate,\nmmol/L\n14.55 ± 0.51\n14.22 ± 1.59\n0.852\nIsobutyrate,\nmmol/L\n1.21 ± 0.07\n1.35 ± 0.17\n0.503\nButyrate, mmol/L\n8.95 ± 0.44\n8.78 ± 1.17\n0.894\nIsovalerate,\nmmol/L\n1.49 ± 0.09\n1.62 ± 0.24\n0.634\nValerate, mmol/L\n1.25 ± 0.03\n1.23 ± 0.18\n0.918\nA: P\n3.95 ± 0.26\n3.63 ± 0.14\n0.324\nNH3-N, ammonia-nitrogen; MCP, microbial crude protein; TVFA, total volatile fatty acid;\nA:P, acetate-to-propionate ratio.\na,bMean values within a row with different superscripts differed (p < 0.05).\n1CON, control diet with no CB supplementation; CB, control diet with 20 g/d CB\nsupplementation for per buffalo.\n\nLi et al.\b\n10.3389/fvets.2025.1707093\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nFIGURE 2\nEffects of dietary with Citrus bioflavonoids (CB) supplementation on diversity and composition of the rumen bacterial community of dairy buffaloes \nduring hot weather. (A) Chao1 index. (B) Ace index. (C) Shannon index. (D) Simpson index. (E) Beta diversity based on the principal coordinate analysis \n(PCoA) using the distance matrices generated from Bray-Curtis analysis. (F) Relative abundance of bacteria community at the phylum level. (G) Relative \nabundance of bacteria community at the genus level. (H) Linear discriminant analysis effect size approach identifying biomarker genera between two \ngroups. CON, control diet with no CB supplementation; CB, control diet with 20 g/d CB supplementation for per buffalo.\n\nLi et al.\b\n10.3389/fvets.2025.1707093\nFrontiers in Veterinary Science\n10\nfrontiersin.org\nmitigation (16). Regrettably, however, this study only tested a single\nCB dose without multi-dose groups, we cannot identify the optimal\nCB dose for heat-stressed dairy buffaloes or clarify whether CB exert\ndose-dependent effects on heat stress alleviation. In terms of nutrient\ndigestibility, our study demonstrated that CB supplementation\nimproved the apparent digestibility of ADF without affecting DMI of\ndairy buffaloes, which is in accordance with others studies, who\nreported that feeding Citrus extracts did not alter DMI of dairy cows\n(11, 17). The absence of DMI changes in our study suggests that the\nimproved ADF digestibility and subsequent benefits to buffaloes’\nlactation performance are not driven by increased feed consumption\nbut rather by enhanced nutrient utilization efficiency. Given that ADF\ndigestion relies on the synergistic action of enzymes and rumen\nmicroorganisms, the improved ADF digestibility in our study may\nreflect CB-induced modifications in cellulase activity or rumen\nmicrobial communities.\nHeat stress poses a major challenge to dairy animals, leading to\ndeteriorated lactation performance and altered metabolic processes.\nThis is typically accompanied by reductions in milk protein, milk fat,\nlactose, SNF, and TS contents, along with increased MUN levels,\ncollectively resulting in diminished milk quality (18–20).\nAccumulating evidence has demonstrated that feeding Citrus extract\nexerts positive effects on milk production and composition in dairy\ncows (11, 21). Consistent with these findings, our study observed\nhigher milk yield, 4%FCM, milk protein, milk lactose, and SNF in the\nCB-supplemented group. These results indicate that dietary CB\nsupplementation significantly improves the milk quality of dairy\nbuffaloes under high-temperature conditions. However, in contrast to\nour findings, Ying et al. (17) reported no significant effects of Citrus\nextract supplementation on milk yield or composition. Notably, the\nsupplementation level employed in their research (4 g/d) was\n\nHowever, in contrast to\nour findings, Ying et al.(17) reported no significant effects of Citrus\nextract supplementation on milk yield or composition.Notably, the\nsupplementation level employed in their research (4 g/d) was\nsubstantially lower than that used in ours, and this dosage discrepancy\nmay well explain the differing outcomes regarding lactation\nperformance. Currently, studies investigating the effects of flavonoid-\nrich plant extracts on the milk fatty acids profile of ruminants remain\nlimited. Dietary phytogenic compounds can optimize milk fatty acid\ncomposition by regulating microbial biohydrogenation in the rumen\n(22). In the present study, CB treatment resulted in an approximately\n15% increase in UFA and a 24% increase in CLA, coupled with\nreduced levels of major SFA, including myristic acid (C14:0) and\npalmitic acid (C16:0). These changes are desirable from a human\nhealth perspective, with potential benefits particularly in terms of\ncardiovascular protection and metabolic regulation. These findings\ncan be reasonably explained by the ability of flavonoids to selectively\nmodulate specific rumen microorganisms, thereby reducing the\nbiohydrogenation of ingested fatty acids and increasing the proportion\nof UFA (23). For instance, condensed tannins, a class of flavonoid-\nbased polymers, have been shown to partially inhibit the final step of\nC18:3 biohydrogenation in the Rumen Simulation Technique system\n(24). The observed decrease in stearic acid (C18:0) alongside increased\nCLA content in our study is consistent with earlier research, which\nreported similar results in dairy ewes supplemented with tannins (25).\nIt is generally hypothesized that tannins enhance the activity of\nstearoyl-CoA desaturase (SCD), an enzyme that mediates the\nconversion of stearic acid to oleic acid and vaccenic acid to CLA. This\nimplies that flavonoids can increase milk UFA levels not only by\nTABLE 8  Effects of dietary with Citrus bioflavonoids (CB)\nsupplementation on bacterial abundance in rumen of dairy buffaloes\nduring hot weather (phylum level, %).\nItems\nTreatment groups1\np-value\nCON\nCB\nBacteroidota\n60.37 ± 4.53\n64.32 ± 0.65\n\nItems\nTreatment groups1\np-value\nCON\nCB\nBacteroidota\n60.37 ± 4.53\n64.32 ± 0.65\n0.449\nBacillota\n30.85 ± 4.10\n26.63 ± 0.79\n0.382\nPseudomonadota\n3.64 ± 0.97\n2.72 ± 0.42\n0.417\nSpirochaetota\n1.34 ± 0.38\n1.87 ± 0.40\n0.370\nFibrobacterota\n0.95 ± 0.25\n1.74 ± 0.43\n0.166\nPatescibacteria\n0.96 ± 0.21\n0.94 ± 0.23\n0.943\nCyanobacteriota\n0.66 ± 0.15\n0.69 ± 0.27\n0.910\nVerrucomicrobiota\n0.56 ± 0.19\n0.37 ± 0.07\n0.396\nThermodesulfobacteriota\n0.15 ± 0.04\n0.20 ± 0.03\n0.356\nunclassified_k__\nnorank_d__Bacteria\n0.14 ± 0.02\n0.15 ± 0.02\n0.957\n1CON, control diet with no CB supplementation; CB, control diet with 20 g/d CB\nsupplementation for per buffalo.\nTABLE 9  Effects of dietary with Citrus bioflavonoids (CB)\nsupplementation on bacterial abundance in rumen of dairy buffaloes\nduring hot weather (genus level, %).\nItems\nTreatment groups1\np-value\nCON\nCB\nXylanibacter\n35.88 ± 5.39\n34.74 ± 3.01\n0.860\nnorank_f__F082\n5.78 ± 0.66\n6.47 ± 0.85\n0.543\nRikenellaceae_RC9_gut_\ngroup\n5.74 ± 0.78\n6.44 ± 0.63\n0.517\nChristensenellaceae_R-7_\ngroup\n3.93 ± 0.84\n3.37 ± 0.45\n0.579\nNK4A214_group\n4.25 ± 1.06\n2.60 ± 0.38\n0.193\nnorank_f__Muribaculaceae\n2.36 ± 0.36\n4.20 ± 2.24\n0.505\nPrevotellaceae_UCG-001\n2.52 ± 0.29\n3.64 ± 0.75\n0.211\nnorank_f__UCG-011\n2.32 ± 0.31\n1.56 ± 0.21\n0.090\nUCG-005\n2.10 ± 0.37\n1.42 ± 0.18\n0.153\nSuccinivibrionaceae_UCG-\n002\n1.46 ± 0.16\n1.38 ± 0.33\n0.830\nSucciniclasticum\n1.89 ± 0.15\n1.69 ± 0.33\n0.600\nAcetobacter\n1.51 ± 0.81\n0.69 ± 0.25\n0.373\nRuminococcus\n1.73 ± 0.44\n1.11 ± 0.21\n0.243\nnorank_o__Bacteroidales\n1.24 ± 0.12\n1.46 ± 0.14\n0.284\nFibrobacter\n0.95 ± 0.25\n1.73 ± 0.44\n0.167\nTreponema\n1.18 ± 0.37\n1.67 ± 0.39\n0.406\nPrevotellaceae_UCG-003\n1.00 ± 0.18\n1.32 ± 0.14\n0.222\nunclassified_f__\nPrevotellaceae\n0.93 ± 0.19\n1.21 ± 0.16\n0.303\nSaccharofermentans\n0.93 ± 0.06\n0.91 ± 0.10\n0.833\nSegatella\n0.72 ± 0.06a\n0.36 ± 0.03b\n< 0.01\na,bMean values within a row with different superscripts differed (p < 0.05).\n1CON, control diet with no CB supplementation; CB, control diet with 20 g/d CB\nsupplementation for per buffalo.\n\nLi et al.\b\n10.3389/fvets.2025.1707093\nFrontiers in Veterinary Science\n11\nfrontiersin.org\nregulating rumen biohydrogenation but also by enhancing SCD\nactivity (25, 26). However, due to the lack of data on fatty acid content\nin rumen microbial biomass in the current study, we were unable to\nestablish a direct link between microbial abundance and milk fatty\nacid profiles. This aspect warrants further investigation in future\nresearch. From the perspective of application prospects, CB show\ngreat potential as a green feed additive in future livestock farming, its\nability to alleviate heat stress and improve lactation performance\nmakes it particularly suitable for high-yielding lactating ruminants in\ntropical/subtropical regions, where heat stress often limits production.\nThe CB supply did not interfere with the most of biochemical\nparameters, except for TP and ALB, whose concentrations were higher\nin the CB group compared to the CON group. Consistent with our\nfindings, previous studies have also reported that flavonoid-rich\nextracts, such as those from mulberry leaves (13) or propolis (27),\nexert little to no impact on the blood concentrations of common\nbiochemical parameters in heat-stressed dairy cows or buffaloes.\nHowever, in contrast to the current study, which observed no\nsignificant effects on metabolic hormones, some prior research has\ndocumented flavonoids-induced changes in hormonal parameters.\nFor instance, mulberry leaf flavonoids supplementation in buffaloes\nwas shown to significantly alter serum insulin, T3, and T4 levels (13).\nThis discrepancy suggests that the effects of flavonoids on hormonal\nparameters may be influenced by factors such as flavonoid type/\ndosage or the supplementation duration. Under normal environmental\nconditions, the oxidant-antioxidant system maintains a delicate\nbalance to sustain bodily homeostasis. However, exposure to elevated\nambient temperature disrupts this equilibrium, triggering oxidative\nstress. Heat stress induces an excessive accumulation of ROS in\nanimals, leading to severe oxidative stress that subsequently accelerates\n\nHowever, exposure to elevated\nambient temperature disrupts this equilibrium, triggering oxidative\nstress.Heat stress induces an excessive accumulation of ROS in\nanimals, leading to severe oxidative stress that subsequently accelerates\nlipid peroxidation and impairs immune responses (28, 29). Therefore,\nmaintaining oxidant-antioxidant balance is critical to mitigating the\nadverse effects of oxidative stress. In dairy animals, particularly dairy\nbuffaloes, the impact of oxidative stress is more pronounced, as milk\nproduction, a metabolically active process, generates additional heat,\nexacerbating the stress burden. Under oxidative stress conditions, the\nbody’s defense system fails to timely scavenge excessive free radicals,\nprimarily due to reduced activities of antioxidant enzymes such as\nCAT, SOD, and GSH-Px during chronic heat stress (30, 31).\nConsequently, the activity of these antioxidant enzymes in ruminants\nis widely used as physiological indicator to assess the severity of\noxidative and heat stress (30). As a comprehensive marker of the\nantioxidant system, plasma T-AOC reflects the cumulative effect of all\nantioxidants in the body, with higher levels indicating stronger\nantioxidant capacity (32). Conversely, MDA, a byproduct of lipid\nperoxidation, serves as a key indicator of oxidative damage, where\nlower plasma MDA concentrations signify reduced oxidative stress\n(33). Previous studies have confirmed that numerous traditional\nChinese medicinal materials can enhance the antioxidant capacity of\ndairy ruminants under heat stress. For instance, dietary supplementing\nof dairy cows with Radix bupleuri or Lonicera japonica has been\nshown to increase plasma SOD activity and T-AOC levels, while\nsimultaneously reducing MDA concentrations (34, 35). Similarly, a\nstudy on multiparous Murrah buffaloes demonstrated that mulberry\nflavonoids inhibited MDA in a dose-dependent manner, exerting a\npositive effect on alleviating oxidative stress (12). Consistent with\nthese findings, the present study observed a remarkable increase (up\nto 61.67%) in plasma CAT activity following CB supplementation,\n\nConsistent with\nthese findings, the present study observed a remarkable increase (up\nto 61.67%) in plasma CAT activity following CB supplementation,\nindicating CB’s potential to alleviate heat-induced oxidative stress.\nThis effect is likely attributed to the ability of flavonoids to act as\nreducing agents and hydrogen donors, which neutralize ROS and\nscavenge hydrogen peroxide and superoxide ions (36). HSP70, known\nas a cellular “thermometer” for mediating heat stress, is pivotal for\nthermotolerance and plays critical roles in cellular heat resistance,\nimmune modulation, and heat stress responses (37). In the present\nstudy, under heat stress conditions, CB supplementation significantly\nincreased serum HSP70 content in buffaloes compared to the CON\ngroup. Earlier studies using a mouse model reported that quercetin\ncan effectively upregulate HSP70 expression by mediating the ERK/\nPPARγ signaling pathways (38). Biomarkers of inflammatory status\nprovide direct insights into homeostasis and health in dairy ruminants\n(39). Indeed, the elevation and release of proinflammatory cytokines\nare hallmarks of oxidative stress, associated with mitochondrial\ndysfunction and increased free radical production (40, 41). Notably,\nthe observed increase in serum IgM levels, alongside reduced\npro-inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6) suggests\nthat CB supplementation can enhance immune function while\nalleviating inflammatory response in buffaloes during hot weather.\nThis aligns with recent studies on Citrus extract (containing hesperidin\nand naringin) in dairy cows, which demonstrated reduced systemic\ninflammation, evidenced by lower serum endotoxin, IL-1β and TNF-α\nlevels, indicating the potential anti-inflammatory effects of Citrus\nextract and improved immune-metabolic status in dairy cows (10, 11).\nIt is widely accepted that VFA act as the primary carbon source\nfor ruminal microbes and provide 60% ~ 80% of the digestible energy\nrequired by ruminants (42). Furthermore, the concentration and\ncomposition of VFA are key indicators of rumen fermentation status\n\nIt is widely accepted that VFA act as the primary carbon source\nfor ruminal microbes and provide 60% ~ 80% of the digestible energy\nrequired by ruminants (42).Furthermore, the concentration and\ncomposition of VFA are key indicators of rumen fermentation status\nand microbial community structure. A growing body of evidence has\nshown that dietary supplementation with flavonoid-rich extracts can\nalter the concentration or composition of rumen VFA (43–45).\nHowever, our observations revealed that CB supplementation had no\nsignificant effect on either total or individual VFA concentrations in\nbuffaloes under hot climatic conditions. These findings add to the\naccumulating literature indicating that plant-derived flavonoids exert\nvariable effects on rumen fermentation, with such inconsistencies\nlikely stemming from differences in flavonoid sources, dosage, animal\nspecies, and experimental conditions. Encouragingly, our results\ndemonstrated that CB supplementation increased ruminal MCP\nconcentration in heat-stressed buffaloes, suggesting that the effects of\nCB are more specific to microbial protein synthesis rather than broad\nfermentation changes. To our knowledge, flavonoid-rich extracts have\nbeen associated with elevated MCP concentrations, potentially by\ncreating a favorable microenvironment for ruminal microbiota\ninvolved in protein metabolism or serving as an energy substrate for\nmicrobial growth, thereby enhancing nitrogen utilization efficiency\n(46, 47). This is supported by the present study, where CB treatment\nsignificantly increased both MCP and milk protein contents in the\nexperimental buffaloes.\nA stable and diverse rumen microbial community is fundamental\nto the health and productivity of ruminants, as it provides functional\nredundancy and enhances resilience against dietary or environmental\nperturbations (48). In our study, three key microbial α-diversity\nindices, Chao 1, ACE, and Shannon, all exhibited an upward trend.\nThis observation is particularly vital, as it implies that the rumen\nmicrobial community in the CB group was not only richer in species\n\nIn our study, three key microbial α-diversity\nindices, Chao 1, ACE, and Shannon, all exhibited an upward trend.This observation is particularly vital, as it implies that the rumen\nmicrobial community in the CB group was not only richer in species\nbut also more evenly distributed in species abundance. A more evenly\n\nLi et al.\b\n10.3389/fvets.2025.1707093\nFrontiers in Veterinary Science\n12\nfrontiersin.org\ndistributed microbial community is generally regarded as more stable,\nprimarily because it is less prone to collapse if one or two dominant\nspecies are adversely affected. Notably, this CB-induced enhancement\nof microbial diversity contrasts sharply with the effects of some potent\nantimicrobial feed additives (e.g., monensin). Monensin is known to\noccasionally reduce overall rumen microbial diversity by specifically\ntargeting gram-positive bacteria (49). The effect of CB aligns more\nclosely with certain other flavonoids that have been shown to\nmodulate the rumen microbiome without compromising its overall\ndiversity, thereby fostering a healthier and more robust fermentation\nsystem (50).\nBeyond improving diversity, the >80% increase in the relative\nabundance of the phylum Fibrobacterota, along with its primary\ngenus, Fibrobacter, in the CB group is of paramount importance.\nFibrobacter species are recognized as keystone cellulolytic bacteria in\nthe rumen, equipped with highly efficient enzyme systems that enable\nthe degradation of crystalline cellulose, one of the most recalcitrant\ncomponents of plant cell walls (51). The enrichment of Fibrobacter\nstrongly suggests that CB enhance the host’s capacity to digest\nlow-quality forage, which in turn facilitates greater energy release in\nthe form of VFA. This effect is particularly valuable for high-\nproducing lactating ruminants, where energy demand is a primary\nlimiting factor for milk production. Furthermore, LEfSe analysis\nfurther validated the pro-fibrolytic role of CB by identifying the\nphylum Bacillota, and specifically the family Lachnospiraceae, as\nsignificantly enriched in the CB group. Within Lachnospiraceae, the\n[Eubacterium]_xylanophilum_group, is specialized in degrading\nxylan, a major component of hemicellulose. Its enrichment\ncomplements the cellulolytic activity of Fibrobacter, ensuring the\ncomprehensive breakdown of complex plant fibers. Similarly, the\nfamily Bacteroidetes_BD2-2 and the genus norank_f__Weeksellaceae,\n\nIts enrichment\ncomplements the cellulolytic activity of Fibrobacter, ensuring the\ncomprehensive breakdown of complex plant fibers.Similarly, the\nfamily Bacteroidetes_BD2-2 and the genus norank_f__Weeksellaceae,\nboth members of the phylum Bacteroidota, were also enriched in the\nCB group. Bacteroidota is well-documented for its extensive\nrepertoire of carbohydrate-active enzymes, which enable the\ndegradation of a broad range of polysaccharides (52). Collectively, the\nenrichment of these taxa further reinforces the hypothesis that CB\nenhance the overall glycan-degrading capacity of the rumen\nmicrobiome, a mechanism that may partially explain the improved\nADF digestibility observed in CB-supplemented dairy buffaloes. In\naddition to promoting fibrolytic bacteria, our data show that CB\nselectively suppress specific microbial groups, particularly those\ninvolved in protein and amino acid metabolism. LEfSe analysis\nrevealed that the genera Segatella and Schwartzia were significantly\nmore abundant in the CON group. By suppressing Segatella, CB may\nhelp preserve dietary protein, thereby increasing the flow of MCP and\nbypass protein to the small intestine. This, in turn, supports more\nefficient nitrogen absorption and utilization for milk protein\nsynthesis. This mechanism aligns with findings from studies on other\nflavonoids, such as biochanin A (a key isoflavone in the pasture\nlegume red clover), which have been shown to inhibit hyper-\nammonia-producing bacteria and improve nitrogen utilization\nefficiency in ruminants (53). Overall, dairy CB the supplementation\norchestrates a beneficial and comprehensive shift in the rumen\nmicrobial ecosystem of dairy buffaloes. To further elucidate CB’s\nmechanisms, future research should integrate multi-omics\napproaches (e.g., metagenomics, metabolomics) to validate microbial\nfunctional pathways and their links to host metabolic changes.\n5 Conclusion\nIn this study, we demonstrated that CB exert a distinct beneficial\neffect on the lactation performance of dairy buffaloes during hot\nweather, primarily by enhancing their antioxidant capacity and\n\n5 Conclusion\nIn this study, we demonstrated that CB exert a distinct beneficial\neffect on the lactation performance of dairy buffaloes during hot\nweather, primarily by enhancing their antioxidant capacity and\nregulating immune function. Meanwhile, dietary CB supplementation\nwas associated with specific shifts in the ruminal microbial\ncommunities of dairy buffaloes, namely, the enrichment of fiber-\ndegrading taxa and the suppression of the genus Segatella. This\nmicrobial shift is likely linked to the observed improvements in ADF\ndigestibility and ruminal MCP concentration. In summary, CB exhibit\nconsiderable potential as a natural feed supplement for dairy buffaloes,\nas it can alleviate heat stress, thereby optimizing ruminal function and\nfurther improving lactation performance. Despite these prospects, the\nlarge-scale application of CB in livestock farming still faces practical\nchallenges that require addressing, particularly in terms of extraction\nefficiency, stability during feed processing, and production\ncost control.\nData availability statement\nThe datasets presented in this study can be  found in online\nrepositories. The names of the repository/repositories and accession\nnumber(s) can be found in the article/supplementary material.\nEthics statement\nThe animal study was approved by the Institutional Animal Care\nand Use Committee of the Guangxi Buffalo Research Institute. The\nstudy was conducted in accordance with the local legislation and\ninstitutional requirements.\nAuthor contributions\nYL: Data curation, Formal analysis, Writing – original draft. HY:\nData curation, Formal analysis, Investigation, Writing – review &\nediting. CL: Investigation, Validation, Writing – review & editing. ML:\nMethodology, Resources, Validation, Writing – review & editing. FC:\nMethodology, Resources, Validation, Writing – review & editing. XH:\nConceptualization, Project administration, Writing  – review &\nediting. CY: Conceptualization, Funding acquisition, Project\nadministration, Writing – review & editing.\nFunding\nThe author(s) declare that financial support was received for the\n\nXH:\nConceptualization, Project administration, Writing  – review &\nediting.CY: Conceptualization, Funding acquisition, Project\nadministration, Writing – review & editing.Funding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. This research was funded\nby the National Natural Science Foundation of China (NSFC) Interna-\ntional (Regional) Cooperation and Exchange Program, Grant No.\n32361143788; the Guangxi Natural Science Foundation General\nProject, Grant No. 2024GXNSFAA010387; and the Guangxi Dairy\nBuffalo Innovation Team of National Modern Agricultural Industry\nTechnology System, Grant No. nycytxgxcxtd-2021-21-03.\n\nLi et al.\b\n10.3389/fvets.2025.1707093\nFrontiers in Veterinary Science\n13\nfrontiersin.org\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. 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(2020) 15:e0229200. doi:\n10.1371/journal.pone.0229200\n\nLi et al.\b\n10.3389/fvets.2025.1707093\nFrontiers in Veterinary Science\n15\nfrontiersin.org\nGlossary\nROS - Reactive oxygen species\nTMR - Total mixed ration\nAT - Air temperature\nRH - Relative humidity\nRT - Rectal temperature\nBST - Body surface temperature\nRR - Respiratory rate\nATTD - Apparent total tract digestibility\nDM - Dry matter\nCP - Crude protein\nEE - Ether extract\nADF - Acid detergent fiber\nNDF - Neutral detergent fiber\nMUN - Milk urea nitrogen\nTS - Total solids\nSNF - Solids-not-fat\nFFA - Free fatty acids\nTP - Total protein\nALB - Albumin\nALT - Alanine transaminase\nAST - Aspartate aminotransferase\nALP - Alkaline phosphatase\nCK - Creatine kinase\nLDH - Lactate dehydrogenase\nUN - Urea nitrogen\nGLU - Glucose\nTC - Total cholesterol\nTG - Triglyceride\nIgG - Immunoglobulin G\nIgM - Immunoglobulin M\nIgA - Immunoglobulin A\nINS - Insulin\nT3 - Triiodothyronine\nT4 - Tetraiodothyronine\nSOD - Superoxide dismutase\nCAT - Catalase\nT-AOC - Total antioxidant capacity\nMDA - Malondialdehyde\nSAA - Serum amyloid A\nHSP70 - Heat shock protein 70\nTNF-α - Tumor necrosis factor-α\nIFN-γ - Interferon-γ\nIL-1β - Interleukin-1beta\nIL-2 - Interleukin-2\nIL-6 - Interleukin-6\nVFA - Volatile fatty acids\nNH3-N - Ammonia nitrogen\nMCP - Microbial crude protein\nASV - Amplicon sequencing variants\nSFA - Saturated fatty acids\nPUFA - Polyunsaturated fatty acids\nUFA - Unsaturated fatty acid\nMUFA - Monounsaturated fatty acids\nSCD - Stearoyl-CoA desaturase\n\n---"}
+{"text": "【兽医文献】\n标题: Case Report: Presumptive spinal embryonal tumor in a cat\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nUnderstanding the motivations,\ndeterrents, and incentives for\nrural Albertan veterinary practice\nHanna Mughal 1, Thomas A. O’Neill 1*, Lena Le Huray 1,\nMegan Bergman 2, John Remnant 3, Angelica M. Galezowski 3,\nKent G. Hecker 3 and Robert McCorkell 3\n1 Department of Psychology, University of Calgary, Calgary, AB, Canada, 2 Alberta Veterinary Medical\nAssociation, Edmonton, AB, Canada, 3 Faculty of Veterinary Medicine, University of Calgary, Calgary,\nAB, Canada\nIntroduction: The shortage of rural veterinarians is a growing concern globally.\nThis shortage increases the risk of significant negative impacts on livestock\nmanagement, agriculture, and public health in rural and remote communities.\nTo provide concrete solutions to sustain our rural veterinarian workforce,\nwe examine motivations, incentives, and deterrents to rural veterinary practice\n(RVP). We  do this through a qualitative study in Alberta, Canada, which is a\ngeographically unique and understudied context.\nMethods: We surveyed veterinary students and practicing veterinarians,\nobtaining 124 responses. Data were analyzed using thematic analysis.\nResults: Results revealed key motivating factors that influence attraction and\nretention included personal and family considerations that require living in rural\ncontexts, the nature of strong relationships that develop in rural communities,\nexperiencing a range in work factors that enhances professional development,\nfeeling fulfilled by rural veterinary work, and exposure during veterinary school\nleading to a strong interest in rural settings. Deterrents included limited\nresources and supports in rural contexts, personal and family needs that require\nliving in urban settings, and challenges inherent to rural communities and\nenvironmental characteristics. Finally, key incentives included better salary and\nbenefits, financial incentives, tuition/debt forgiveness, enhanced mentorship,\nfewer on-call duties, and tailored incentives.\nDiscussion: Strong alignment between student and practicing veterinarian\n\nFinally, key incentives included better salary and\nbenefits, financial incentives, tuition/debt forgiveness, enhanced mentorship,\nfewer on-call duties, and tailored incentives.Discussion: Strong alignment between student and practicing veterinarian\nmotivations, deterrents, and incentives was observed, extending previous\nfindings that only look at the perceptions of a single group. The results\ncorroborated previous findings, while revealing that the same motivations and\ndeterrents remained important for students and PVs in Alberta’s geographically\nunique context. Finally, they provided key insights to inform policy, practice,\nand education developments to enhance attraction and retention rates of rural\nveterinarians, contributing to a path forward for addressing the rural shortage of\nveterinary services.\nKEYWORDS\nrural practice, rural attraction, rural retention, veterinary shortage, policy, education,\npractice\nOPEN ACCESS\nEDITED BY\nLászló Ózsvári,\nUniversity of Veterinary Medicine Budapest,\nHungary\nREVIEWED BY\nPhilip A. Robinson,\nHarper and Keele Veterinary School,\nUnited Kingdom\nSarah Wood,\nUniversity of Bristol, United Kingdom\n*CORRESPONDENCE\nThomas A. O’Neill\ntoneill@ucalgary.ca\nRECEIVED 22 May 2025\nACCEPTED 19 August 2025\nPUBLISHED 18 September 2025\nCITATION\nMughal H, O’Neill TA, Le Huray L, Bergman M,\nRemnant J, Galezowski AM, Hecker KG and\nMcCorkell R (2025) Understanding the\nmotivations, deterrents, and incentives for\nrural Albertan veterinary practice.\nFront. Vet. Sci. 12:1633149.\ndoi: 10.3389/fvets.2025.1633149\nCOPYRIGHT\n© 2025 Mughal, O’Neill, Le Huray, Bergman,\nRemnant, Galezowski, Hecker and McCorkell.\nThis is an open-access article distributed\nunder the terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other forums is\npermitted, provided the original author(s) and\nthe copyright owner(s) are credited and that\nthe original publication in this journal is cited,\nin accordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Original Research\nPUBLISHED  18 September 2025\nDOI  10.3389/fvets.2025.1633149\n\nMughal et al.\b\n10.3389/fvets.2025.1633149\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n1 Introduction\nThere is a global shortage of veterinarians in rural contexts, as\nevidenced by high attrition rates of new graduates entering rural\npractice (1) and a decrease in rural interest over career progress (2).\nThis trend is echoed in Canadian rural contexts and is reaching a crisis\nlevel (3). This reflects a systemic issue that rural Canadian\ncommunities consistently face regarding access to health care,\nchildcare, talent attraction and retention, and affordable housing – all\nof which affect economic development in rural communities (4, 5).\nVeterinarians are a key component of this larger rural\nresource challenge.\nThe lack of rural veterinarians has significant implications for the\nhealth of animals and people within these communities and beyond.\nPracticing veterinarians (PVs) meet the needs of rural communities\nby providing services for both large and food animals, as well as the\nincreasing number of companion pets (6, 7). Their impact reaches\nbeyond rural communities; veterinarians play an integral role in food\nsafety and public health and support the agricultural sector and\ntherefore the economy (6, 7).\nMoreover, the shortage may also negatively impact veterinarians\nwho remain in rural practice; while community veterinary needs\nendure, remaining rural practitioners may need to increase their\nworkload to accommodate this shortfall. Heavy workloads are a\nknown source of stress and burnout in veterinarians (8) and can\nincrease the likelihood of career attrition (9). In Alberta specifically,\nveterinarians are known to have the highest prevalence of veterinarians\nworking long hours, suggesting heavy workloads (3) and an increased\nrisk for burnout. The systemic impacts of the rural PV shortage\ndemand attention as they put animal and public health at risk and\nfurther jeopardize the sustainability of rural veterinarian\npractice (RVP).\nDespite this system-wide concern, only a handful of studies on the\nfactors that influence attraction and retention rates for rural\n\nDespite this system-wide concern, only a handful of studies on the\nfactors that influence attraction and retention rates for rural\nveterinarians have been conducted in Canadian contexts (2, 10, 11).\nEvidence from international contexts highlights motivating factors\nthat influence the attraction of new graduates to RVP include: having\na rural background (2, 7, 12); growing up working or living on a farm\n(2, 7, 12); previous experience and interest in large animals (7, 12);\nexposure to RVP during veterinary education (7, 13); and having a\nmentor from RVP (10, 14). Motivating factors that encourage\nexperienced PVs to stay in RVP include strong ties with the\ncommunity (13); strong relationships with clients (15, 16); supportive\nemployers and a good work-life balance (15, 16); financial incentives\n(17); and opportunities for professional development (15).\nAdditionally, addressing deterrents and implementing incentives\nto rural practice may be another route to improving both attraction\nand retention rates. Potential deterrents to consider that have been\nhighlighted in international studies include financial constraints (18,\n19), high risk of injuries and burnout (9, 15), and work-life balance\n(15, 20). In turn, potential incentives from international studies\ninclude financial support (e.g., bonuses) (17), support for professional\ndevelopment (e.g., mentorship) (13, 15), and improved working\nconditions (e.g., support for work-life balance) (16, 17).\nDespite these international findings, the need to uncover the\ncurrent and most important motivating and deterring factors in\nCanadian rural veterinary contexts persists, and whether these factors\ndiffer meaningfully from findings in international settings that have\ndifferent environments. We  utilized the province of Alberta as a\nrepresentative example of Canadian rural contexts and the challenges\npresented by Canadian geography, climate, and remote communities.\nConsulting the individuals at the center of this issue in Alberta is\nnecessary to make recommendations to the Alberta Veterinary\n\nConsulting the individuals at the center of this issue in Alberta is\nnecessary to make recommendations to the Alberta Veterinary\nAssociation, governments, educators, and other partners regarding\neffective and impactful solutions. As such, our study aimed to achieve\nthe following objectives: (1) understand the motivations for choosing\na career in RVP; (2) identify deterrents preventing PVs and students\nfrom pursuing RVP; (3) and explore incentives to make RVP more\nattractive. The research question guiding this study was: How can\nwe  attract and retain veterinary professionals in rural settings? To\nachieve these objectives, we employed a mixed-method survey design,\nincorporating both demographic and open-ended questions to gather\ncomprehensive insights.\n2 Materials and methods\n2.1 Sample\nParticipants consisted of both veterinary students and practicing\nveterinarians (PVs) in Alberta. Student participants were recruited\nfrom the University of Calgary Faculty of Veterinary Medicine and\nprofessional networks by an email sent by the Faculty to students, and\nPVs were recruited by an email sent by the Alberta Veterinary Medical\nAssociation (ABMVA).\n2.2 Ethics statement\nThis research was approved by the University of Calgary Conjoint\nFaculties Research Ethics Board. Potential participants were provided\nprior informed consent, assured that their participation would\nbe  entirely voluntary, and that their data would be  anonymous\nand confidential.\n2.3 Measures\nTo assess the factors influencing attraction and retention in RVP,\nwe relied on previous studies (21, 22) to develop two equivalent,\nparallel surveys that included both demographic questions and open-\nended responses (see Supplementary Materials 1, 2). The demographic\nquestions provided quantitative insights into the backgrounds, career\npaths, and current work environments of participants, whereas the\nopen-ended questions allowed for a deeper exploration of their\npersonal motivations, challenges, and experiences in RVP. The survey\nwas piloted and tested internally with members of the research team.\n2.4 Analysis\n\nThe survey\nwas piloted and tested internally with members of the research team.2.4 Analysis\nWe utilized descriptive statistics to report on frequencies and\ndemographics. In addition, we employed thematic analysis (23) to\nidentify themes in the open-ended questions. The steps of thematic\nanalysis include: (1) familiarizing with the data; (2) generating codes\n(building blocks of analysis); (3) constructing themes (clusters of\n\nMughal et al.\b\n10.3389/fvets.2025.1633149\nFrontiers in Veterinary Science\n03\nfrontiersin.org\ncodes that create meaning); (4) reviewing potential themes; (5)\ndefining and naming themes, which was done through discussion by\nthree of the authors; and (6) producing the report. We relied on an\ninductive approach with semantic and conservative coding. A primary\ncoder first created the coding classifications, assigned codes, and\ndiscussed these with another researcher. Subsequently, a trained coder\nverified that codes were assigned accurately. Where discrepancies\nsurfaced, they were resolved through discussion. Finally, we  also\nemployed a review process where a member of the research team\nreviewed half of the codes per theme and provided feedback.\nConsensus was reached on any points of disagreement.\n3 Results\nAn invitation to complete the survey was sent to 159 University of\nCalgary Faculty of Veterinary Medicine students. There were 48\nstudent responses (response rate = 30.19%). Furthermore, an\ninvitation was sent to all members of the ABVMA by email listserv,\ncontaining approximately 2,390 practicing veterinarians (PVs). There\nwere 76 practicing veterinarians who completed the survey (response\nrate = 3.18%). This generated 124 completed surveys that were\nincluded in the analysis.\n3.1 Student demographics\nFull demographic results for the 48 students can be found in\nSupplementary Material 3. Among the students surveyed, the majority\nwere in their third year (46%), and almost all identified as white (79%)\nand as women (94%). Students’ practice intentions were divided into\nthree roughly even groups: 40% intended to pursue rural practice, 33%\nintended to pursue urban practice, and 27% were undecided. Most\nstudents indicated they would like to focus on companion animals\n(40%) or mixed animal (33%) practice. Note that rural communities\nwere defined as any community with a population equal to or below\n24,999 residents.\n3.2 PV demographics\nFull demographic results for the 76 PVs can be  found in\nSupplementary Material 3. Among the PVs surveyed, most had been\npracticing for over 10 years (75%), and most identified as white (96%)\n\n3.2 PV demographics\nFull demographic results for the 76 PVs can be  found in\nSupplementary Material 3.Among the PVs surveyed, most had been\npracticing for over 10 years (75%), and most identified as white (96%)\n(Supplementary Table S1) and as women (70%). Most PVs worked in\na rural setting (91%) and indicated they worked with mixed\nanimals (48%).\nBelow are the results of the thematic analysis by students and PVs\n(Figure 1).\n3.3 Veterinary students’ results\n3.3.1 Motivations for RVP\nThe motivations to work in a rural setting of the students who\nintended to pursue rural practice generated three themes that\nincluded: (1) community and lifestyle, (2) work-related factors, and\n(3) personal and family considerations. The first theme, community\nand lifestyle, identified relationship-based and lifestyle factors that\ndrew students to RVP, outlining that students desired to be part of a\nclose-knit community where they felt a strong sense of belonging.\nStudents felt a sense of duty to provide a needed service in these\ncommunities, seeing it as an opportunity to make a meaningful\nimpact. They also noted how they appreciated the quieter, slower-\npaced lifestyle in rural settings, and financial freedom due to a lower\ncost of living.\n“I think the role we have as a veterinarian in these settings is very\nrelationship-based. To me, this makes it a lot more rewarding. I also\nknow that I one day want to raise my family in a rural community.”\n(Theme 1).\nThe work-related factors theme highlighted professional aspects of\nRVP that were attractive to students. These included the preference to\nwork with diverse animal populations typically found in rural practice,\nand the variety of cases and challenges were seen as valuable for\nprofessional and clinical skills development. In addition, the trusting\nand loyal relationships with clients that tend to develop in rural\nsettings were attractive to students.\n“I wanted to work with a variety of animals rather than just cats and\ndogs.” (Theme 2).\nLastly, the personal and family considerations theme illustrated\nthat prior rural experiences and having a rural background created a\n\n“I wanted to work with a variety of animals rather than just cats and\ndogs.” (Theme 2).Lastly, the personal and family considerations theme illustrated\nthat prior rural experiences and having a rural background created a\nsense of familiarity with the lifestyle, prompting students to be more\ninclined to return to these contexts. A sense of belonging and family\nties within rural communities aligned with this, also observed to be an\nimportant motivator.\n“I grew up in a rural community and I want to eventually [settle]\ndown in that same community.” (Theme 3).\n3.3.2 Deterrents for RVP\nTwelve deterrents were identified for the remaining urban/\nundecided students who stated they had not considered a rural setting\n(Supplementary Material 3). The top five deterrents included: low rate\nof financial return for hours worked, on-call and after-hours demands,\ndanger of working with large animals, location of job opportunities,\nand lack of social activities. To contextualize these results, three\nthemes were generated, including: (1) environmental and community\nfactors, (2) personal factors, and (3) resource and support limitations.\nThe first theme, environmental and community factors, revealed that\nstudents’ perceptions of rural environments deterred them from rural\npractice. One important perception was the lack of diversity that could\nimpact their personal and professional experiences. Students\nexpressed concerns about encountering greater discrimination and\ndifferent viewpoints compared to urban centers. Students also\nemphasized that entering rural practice would result in their removal\nfrom their current community and would significantly limit their\naccess to social and recreation activities in urban centers.\n“Lack of diversity, fear of perceived bias or discrimination (especially\nas a neurodivergent female), different political ideologies, different\nviews on animal use (especially in rodeos) …I think I’m too different\nto ever fit in or be accepted in the community.” (Theme 1).\n\nMughal et al.\b\n10.3389/fvets.2025.1633149\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nPersonal considerations, including preferences for companion\nanimals and personal values of animal treatment and welfare,\npresented in the second theme also influenced decreased\ninterest in RVP.\n“I want to be  a small animal surgeon and there would not\nbe  enough of a case load for me in a rural community.”\n(Theme 2).\nLastly, resource and support limitations that are characteristic of\nrural settings acted as deterrents to RVP. Students expressed\nreservations about the lack of professional and mental health support\nin rural areas, and isolation. They were concerned about potential\nfinancial constraints due to lower income potential in rural areas\ncompared to urban centers. Finally, the lack of infrastructure, services,\nand social and recreation activities was another factor that deterred\nstudents from RVP.\n“Lack of amenities and activities such as nice swimming pools, 24 h\nrestaurants, nice restaurants, arts and culture, festivals, large and\ndiverse population.” (Theme 3).\n3.3.3 Incentives for students with RVP interest\nA third (33%) of the students indicated they intended to pursue a\ncareer in an urban setting, while just under a third (27%) indicated\nthey were undecided. Of the students who were undecided or intended\nto pursue an urban setting, 79% said they had considered rural\npractice, while 21% said they had not. Incentives are tangible offerings\nthat can influence RVP considerations and intentions. As such, the top\nfive incentives of the students who had not considered rural practice\nand who were undecided included: better salary and benefits, financial\nincentives, tuition/debt forgiveness, mentorship and additional\nsupport, and tailored incentives based on personal characteristics.\n3.4 PV results\n3.4.1 Initial motivations for choosing RVP\nFour themes were generated that represented the initial\nmotivations to begin their career in rural practice of the PVs who\ncurrently work in a rural setting. These themes included: (1)\npersonal and family considerations, (2) professional opportunities and\nFIGURE 1\n\nThese themes included: (1)\npersonal and family considerations, (2) professional opportunities and\nFIGURE 1\nTopics and themes from qualitative data.\n\nMughal et al.\b\n10.3389/fvets.2025.1633149\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nfulfillment, (3) community and relationships, and (4) education and\nexposure. The first theme, personal and family considerations,\nrevealed that personal considerations like preferences for a quiet\nlifestyle, quality of life inherent to rural contexts, and favoring the\nnatural environment motivated PVs to choose RVP initially.\nFurther, feelings of comfort with rural contexts due to growing up\nin rural areas or having positive past experiences were motivating\nfactors. They expressed the desire to be close to family, to raise\nchildren in a rural environment, and to accommodate\nspousal employment.\n“Grew up in rural Alberta, all I knew about were mixed animal\npractices. It is my roots where I’m the happiest.” (Theme 1).\nRural contexts offer unique professional opportunities and\nfulfillment, which were attractive to PVs. Such opportunities were\ndescribed as diverse animals to treat, a broad scope of practice, a\nhigher potential for ownership, higher job availability, more control\nover their work schedule, the ability to utilize advanced techniques,\nand cases that allowed PVs to develop a wide variety of clinical skills.\n“I wanted to practice large animal medicine and most of these\npractices are in more rural settings so following my career goals\nnaturally lead toward a more rural environment.” (Theme 2).\nThe third theme, community and relationships, highlighted that\nstrong connections within the rural community was a strong\nmotivator and was seen as essential for both professional success and\npersonal satisfaction. The sense of belonging within the community,\ndeveloped through the personal relationships with clients, co-workers,\nand community members, was another strong motivating factor for\nPVs. Further, the ability to make a meaningful impact by meeting the\ncommunity’s veterinary needs in areas where veterinary services were\nlimited or lacking was another important aspect of why PVs wanted\nto work in rural spaces initially.\n“I enjoy forming ties to the community and getting to know my\n\n“I enjoy forming ties to the community and getting to know my\nclients (longer appointment times, farm visits, etc.).” (Theme 3).\nLastly, the fourth theme, education and exposure, highlighted that\ngaining exposure through internships or rotations provided PVs with\na realistic understanding of rural practice, reinforcing their interest\nand decisions to pursue a career in these settings. The rural interests\nand passions of professors and classmates also influenced PVs to want\nto practice rurally.\n“I began working in rural practices during my summers in veterinary\nschool and developed an interest in the beef industry specifically.”\n(Theme 4).\n3.4.2 Motivations to stay in RVP\nIn terms of the motivators that influenced rural PVs to remain in\nthis setting, four themes were generated: (1) personal and family\nconsiderations, (2) community and relationships, (3) professional\nfulfillment and opportunities, and (4) desire to leave. Personal and\nfamily considerations in the first theme included a preference for the\nrural lifestyle and quality of life, love for the outdoors, and general\nenjoyment and satisfaction from living and working rurally. PVs\nvalued the ability to raise children in rural areas, to stay close to\nfamily, and to be near partners’ jobs in the area.\n“…rural settings offer a freedom, privacy, and independence that is\nnot easily fostered in urban settings.” (Theme 1).\nThe second theme, community and relationships, underscored the\nimportance of trusting and loyal relationships with clients, co-workers,\nand community members to maintain dedication to RVP. It was also\nseen as essential to professional success and personal satisfaction.\nAgain, those who grew up rurally continued to feel a strong sense of\nconnection and comfort in rural areas, further grounding them to\nthe environment.\n“I’ve always loved working with livestock and poultry producers, as\nthey work very hard to take care of their animals … They appreciate\nwhen we work with them to improve/resolve their disease outbreak\nsituations they may be having.” (Theme 2).\nProfessional fulfillment and opportunities highlighted in the third\n\n” (Theme 2).Professional fulfillment and opportunities highlighted in the third\ntheme made it clear that the diversity in workload and animals found\nin rural practice, along with the chance to develop a range of skills,\nwere important motivators. Increased opportunities for practice\nownership, continuous skill development, and a steady flow of work\nproviding job security were related motivators.\n“This type of practice is fantastic, rewarding (but also very\nchallenging), and provides something different every day.”\n(Theme 3).\nFinally, the desire to leave did not go unacknowledged; some\nstruggled to remain motivated, and challenges such as long work\nhours, on-call duties, and a desire to work more with small animals\nwere referenced as factors that made PVs consider leaving.\n“Used to be passion for equine medicine, now finding it very hard to\nstay motivated to stay in equine practice, pay and hours and not\nsustainable long term to build a family and have quality of life…”\n(Theme 4).\n3.4.3 Reasons for leaving\nFor the seven urban-based veterinarians who had left rural\npractice, there were several factors that influenced their decision to\nleave. One factor was the nature of referral practices for large animals,\nwhich are predominantly based in suburban areas. This geographic\nlimitation made it challenging for veterinarians to find the necessary\nsupport and resources in rural settings. Additionally, personal and\nfamily considerations played a role. For example, one respondent\nhighlighted the need to be closer to family as they planned to have\nchildren and required family support to manage both professional and\npersonal responsibilities.\nAnother factor was related to employment conditions and support\nwithin the practice. Some veterinarians were asked to switch to part-\ntime roles when new graduates were hired, likely due to overstaffing\nand the need to reduce hours. This was not financially viable for those\nwho were the primary income earners, which forced them to seek new\njob opportunities elsewhere. Additionally, the lack of support from\npractice owners to reduce their workload and collaborative support\n\nMughal et al.\b\n10.3389/fvets.2025.1633149\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nfrom fellow veterinarians contributed to reported feelings of burnout\nand dissatisfaction. On-call duties, long work hours, and insufficient\npay were commonly cited issues, likely creating challenges for\nmanaging family life and professional obligations. The absence of\nadequate mentorship and the strain of balancing on-call\nresponsibilities with family life led many to seek positions in urban\nareas where support and resources were more readily available.\nIncentives may have persuaded these PVs to stay. These top five\nincentives (from most to least important) were: fewer on-call duties,\nmentorship and additional support, financial incentives, better salary\nand benefits, and tailored incentives based on personal characteristics.\n3.4.4 Challenges for rural veterinarians\nChallenges in rural contexts can impact rural PVs’ motivations.\nThe top five challenges (from most to least) stated were: on-call and\nafter-hours demands, physically tiring nature of working with large\nanimals, dangers of working with large animals, low rate of return for\nhours\nworked,\nand\nissues\nwith\nstaff\nmanagement\n(Supplementary Material 3). For those who chose “other,” the written\nresponses included: lack of peer/colleague support, on-call pressures,\nreferral challenges, hiring difficulties, client expectations and\nboundaries, poor salary, high workload, lack of anonymity, childcare,\nisolation, and technological limitations such as poor cell service in\nrural locations.\n3.5 Preparing students for RVP\nPVs were asked to provide recommendations on how veterinary\nschools can better prepare students for rural practice. From these data,\nthree themes were generated: (1) recruitment and awareness, (2)\ncurriculum and training, and (3) support systems. The first theme,\nrecruitment and awareness, identified strategies to enhance admission\nand recruitment procedures to attract more individuals interested in\nRVP. Such strategies included targeting applicants with a rural\nbackground or passion for rural settings for admission and developing\n\nSuch strategies included targeting applicants with a rural\nbackground or passion for rural settings for admission and developing\nmore realistic expectations of what rural practice entails throughout\nschool, mainly in relation to the challenges and rewards such as\non-call duties and the physically demanding nature of the work.\nSchools should also emphasize the unique benefits of RVP to students,\nincluding job opportunities, diversity of cases, and strong\ncommunity relationships.\n“Focus on admitting students that are from a rural area. Interview\nstudents to see if they actually want to practice on large animals/in\na rural area or are just saying what they need to on the application\nto make it seem like they do… Do not rely on just academic scores\nor MCAT…” (Theme 1).\nThe second theme, curriculum and training, outlined a need for\nmore comprehensive education and practical training to prepare\nveterinarians for future practice. Increased exposure to rural settings\nduring school through internships, externships, and rotations, along\nwith improving the knowledge and skills in students that are necessary\nto cover the full spectrum of care required in rural practice, were\nidentified as necessary improvements to veterinary education.\nStrengthening the foundation of basic veterinary knowledge, in\naddition to offering advanced training opportunities, were additional\nsuggested strategies. They believed that these approaches would\nimprove interest and dedication to RVP.\n“I believe all students should have to do multiple rural placements\nprior to graduation. It allows them to see that there is more than just\ncity life.” (Theme 2).\nLastly, providing ongoing support to rural veterinarians was\nidentified as a necessary method to bolster student preparation after\ngraduation. Improving access to veterinary resources, increasing staff,\nand increasing wages were seen as strong steps forward to providing\nmore assistance. Establishing mentorship programs to increase\ncomfort and reduce isolation, plus fostering collaboration with rural\ncolleagues, were all believed to be necessary supports after graduation\n\nEstablishing mentorship programs to increase\ncomfort and reduce isolation, plus fostering collaboration with rural\ncolleagues, were all believed to be necessary supports after graduation\nto address retention rates.\n“When I graduated, mentorship was not a thing – you either swam\nat the front with everyone else, or you got out of the water…I think\nthe awareness of the need to mentor new grads is much better now.”\n(Theme 3).\n4 Discussion\nThis study aimed to identify PVs’ and students’ perceptions of\nmotivators and barriers to rural practice in Alberta, whether they\naligned, and whether the unique geography of rural Alberta is related\nto different challenges when compared to other geographic locations.\nMost research on this topic focuses on either students or PVs. A novel\ncontribution of this study was the investigation of both groups\nsimultaneously, allowing us to compare their perceptions and\nexperiences. Interestingly, there was consistent overlap between the\nmotivations of students considering RVP, the initial motivations of\nPVs, and the motivations that support retention in RVP. Motivators\nidentified by the participants reinforced previous findings (1, 11, 13,\n15, 17, 19, 21, 22, 24). Notably, the importance placed on community\nand relationships by students and PVs suggests the importance of this\nfactor (11). In addition, there was a moderate overlap in deterrents for\nboth groups. Many deterrents identified by our study (e.g., on-call\nburdens, lack of resources) are corroborated by previous research (15,\n16, 18, 19), emphasizing the continued need to address these concerns.\nFinally, both groups strongly agreed on incentives for rural practice,\nwhich suggests that the same incentives are likely effective in\nimproving attraction and retention for both students and PVs, despite\ngenerational and other group differences. Overall, this strong\nalignment between the two groups is promising for interventions, as\nboth groups are likely receptive to the same interventions.\nThese findings confirm that decisions of students and PVs in\nAlberta align with those of participants in published work in other\n\nThese findings confirm that decisions of students and PVs in\nAlberta align with those of participants in published work in other\ncontexts (13, 15–17), while also providing a rich descriptive summary\nof the ways these factors influence decision-making. Interestingly, the\nsame motivators and deterrents appeared in our study as in previous\nresearch, despite Alberta’s unique setting. Rural Alberta is geographically\nvery different than many of the settings where previous studies were\nconducted, which often included samples from countries in Western\nEurope. For instance, rural Alberta is often more remote, experiences\nmore extreme weather patterns, and rural clients are more likely to\nrequire practitioners with a mixed practice skill set. Yet, despite these\n\nMughal et al.\b\n10.3389/fvets.2025.1633149\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nmajor contextual differences, students and PVs in Alberta highlight that\nthey are motivated and deterred from rural practice for the same reasons\nas students and PVs in a Western European country. This finding reveals\ninsight into the consistency of individual perceptions and experiences\nacross varied geographies, resulting in important implications for the\napplication of interventions across different locations.\n4.1 Implications\nThere were several suggested strategies suggested by our results\nthat may address some of the root issues with RVP attraction and\nretention. While untested in this study, these strategies – grounded in\nconsultation with the people at the heart of the issue  – provide\nopportunities for future pilots. Therefore, any strategies that are\nimplemented must be  rigorously evaluated to ensure that the\nintervention results in the desired outcomes.\nFirst, the results suggest certain key educational opportunities that\nmay help prepare students for RVP, including providing targeted\nhands-on experiences, coursework that covers the full spectrum of\nrural care required, and increasing exposure to rural environments\nthrough externships and rotations. Participants also believed that\nhighlighting the unique benefits of rural practice may help attract\nstudents. While these strategies align with current efforts to improve\nthe attraction and retention of rural health care practitioners (25),\ncurrent evidence from the healthcare sector suggests that their\neffectiveness is mixed (26, 27). Thus, these strategies should be tested\nto identify their effectiveness.\nSecond, the findings highlighted the necessity for policy initiatives\nto address rural-specific challenges. Participants believed that policies\nthat provide financial incentives, such as loan forgiveness programs,\nscholarships, and competitive salaries, may make rural practice more\nviable and attractive. Although aligned with similar initiatives in health\ncare (4), evidence on the effectiveness of financial incentives in healthcare\n\nAlthough aligned with similar initiatives in health\ncare (4), evidence on the effectiveness of financial incentives in healthcare\nis inconsistent and warrants further investigation (26–29). Findings also\nsuggest that mentorship programs and professional development\nopportunities may support retention rates, but testing is also required as\nthere are limited studies to corroborate this approach (27).\nThird, regarding practices, the results suggested several strategies\nthat may improve retention rates, including fostering a supportive and\ninclusive work environment, reducing on-call demands, providing\nbetter access to resources and support systems, and promoting work-life\nbalance. Building strong community connections and improving\nprofessional fulfillment were also seen as promising strategies to test. As\nnoted, these strategies should be  carefully evaluated as healthcare\nresearch does not provide any insight into their effectiveness (27).\nFinally, encouraging collaboration and teamwork within the practice\nmay also help mitigate isolation and burnout. This approach is supported\nby research in healthcare and may be a rewarding solution to trial (29).\n4.2 Limitations and future research\ndirections\nThere is little evidence on the efficacy of potential strategies to\naddress rural attraction and retention rates, even in the health care\nliterature, so a crucial next step is to measure their actual impacts. Our\nstudy provides potential solutions to include in pilot applications that\nshould be  rigorously evaluated. Specifically, mentorship and\nrelationships (in community and practices) emerged as strategies that\nshould be prioritized in pilots.\nOur findings also provided valuable insights from PVs who left\nrural for urban practice, however, the response rate from this group\nwas low. Future research should further investigate their experiences\nto ensure that these insights are representative of all PVs who left\nfor urban practice. Further, though our study was open to PVs in\nall contexts, we received no responses from PVs strictly in urban\n\nFuture research should further investigate their experiences\nto ensure that these insights are representative of all PVs who left\nfor urban practice.Further, though our study was open to PVs in\nall contexts, we received no responses from PVs strictly in urban\nsettings. Consulting this population in future research to\nunderstand their motivations and deterrents is crucial to address\nrural attraction.\nFinally, gathering qualitative data through a survey may have\npresented limitations such as participants experiencing survey\nfatigue throughout the survey and/or from being frequently asked\nto complete surveys, in addition to surveys being a less engaging\nqualitative data collection method than interviews or focus groups.\nWhile we were able to gain detailed experiences and perceptions\nfrom a wide range of voices, we recognize that data from focus\ngroups or interviews would strengthen and expand these findings.\nFuture research should consider consulting students and PVs in\nfocus groups and interviews.\nData availability statement\nThe de-identified aggregate data supporting the conclusions of this\narticle will be made available by the authors, without undue reservation.\nEthics statement\nThe studies involving humans were approved by University of\nCalgary Research Ethics Board. The studies were conducted in\naccordance with the local legislation and institutional requirements.\nThe participants provided their written informed consent to\nparticipate in this study.\nAuthor contributions\nHM: Data curation, Formal analysis, Writing – original draft. TAO:\nConceptualization, Methodology, Project administration, Writing  –\nreview & editing. LLH: Conceptualization, Data curation, Formal analysis,\nMethodology, Writing – original draft. MB: Conceptualization, Resources,\nWriting – review & editing. JR: Conceptualization, Resources, Writing –\nreview & editing. AMG: Conceptualization, Resources, Writing – review\n& editing. KGH: Methodology, Writing  – review & editing. RM:\nResources, Writing – review & editing.\nFunding\nThe author(s) declare that no financial support was received for\n\nAMG: Conceptualization, Resources, Writing – review\n& editing.KGH: Methodology, Writing  – review & editing.RM:\nResources, Writing – review & editing.Funding\nThe author(s) declare that no financial support was received for\nthe research and/or publication of this article.\n\nMughal et al.\b\n10.3389/fvets.2025.1633149\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. Any product that may be evaluated in this article, or\nclaim that may be made by its manufacturer, is not guaranteed or\nendorsed by the publisher.\nSupplementary material\nThe Supplementary material for this article can be found online\nat: https://www.frontiersin.org/articles/10.3389/fvets.2025.1633149/\nfull#supplementary-material\nReferences\n1.\tHeath T. Longitudinal study of veterinarians from entry to the veterinary course to\nten years after graduation: career paths. Aust Vet J. (2002) 80:468–73. doi:\n10.1111/j.1751-0813.2002.tb12467.x\n2.\tJelinski MD, Campbell JR, Naylor JM, Lawson KL, Derkzen D. Factors associated\nwith the career path choices of veterinarians in western Canada. Can Vet J. 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+{"text": "【兽医文献】\n标题: Adipose-derived mesenchymal stem cell exosomes ameliorate copper metabolism dysregulation and reduce cuproptosis caused by liver IRI\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nTYPE  Original Research\nPUBLISHED  21 July 2026\nDOI  10.3389/fvets.2026.1893535\nOPEN ACCESS\nEDITED BY\nJianzhu Liu,\nShandong Agricultural University, China\nREVIEWED BY\nMei Mei,\nJiangsu Academy of Agricultural\nSciences (JAAS), China\nMonu Karki,\nGuru Angad Dev Veterinary and Animal\nSciences University, India\n*CORRESPONDENCE\nChangbo Ou\nouchangbo2004@163.com\nYang Li\nliyang@cahec.cn\nAnding Zhang\nandye8019@mail.hzau.edu.cn\n†These authors have contributed equally\nto this work and share first authorship\nRECEIVED 28 May 2026\nREVISED 30 June 2026\nACCEPTED 30 June 2026\nPUBLISHED 21 July 2026\nCITATION\nYang H, Yuan L, Chen H, Liu Y, Xu A,\nLing Z, Xuan X, Ma S, Feng S, Wang Y,\nSi H, Li G, Li Y, Zhang A and Ou C (2026)\nGenomic and pathogenic\ncharacterization of a highly pathogenic\nchicken infectious anemia virus strain in\nChina.\nFront. Vet. Sci. 13:1893535.\ndoi: 10.3389/fvets.2026.1893535\nCOPYRIGHT\n© 2026 Yang, Yuan, Chen, Liu, Xu, Ling,\nXuan, Ma, Feng, Wang, Si, Li, Li, Zhang\nand Ou. This is an open-access article\ndistributed under the terms of the\nCreative Commons Attribution License\n(CC BY). The use, distribution or\nreproduction in other forums is\npermitted, provided the original\nauthor(s) and the copyright owner(s) are\ncredited and that the original publication\nin this journal is cited, in accordance\nwith accepted academic practice. No\nuse, distribution or reproduction is\npermitted which does not comply with\nthese terms.\nGenomic and pathogenic\ncharacterization of a highly\npathogenic chicken infectious\nanemia virus strain in China\nHongchun Yang 1†, Liuyang Yuan 1†, Hao Chen 1†, Yue Liu 2†,\nAobo Xu 1, Ziqi Ling 1, Xiaoran Xuan 1, Shuangshuang Ma 1,\nShufeng Feng 1, Yaping Wang 1, Hongbin Si 1, Gonghe Li 1,\nYang Li 3*, Anding Zhang 4* and Changbo Ou 1,5,6*\n1College of Animal Science and Technology, Guangxi University, Nanning, China, 2Guangxi Yuanfeng\nGroup Pastoral Industry Co. Ltd, Lingshan, China, 3China Animal Health and Epidemiology Center,\nQingdao, Shandong, China, 4College of Veterinary Medicine, Huazhong Agricultural University, Wuhan,\nChina, 5Guangxi Key Laboratory of Animal Reproduction, Breeding and Disease Control, Nanning,\n\nChina, 6Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics,\nNanning, China\nChicken infectious anemia virus (CIAV) is a major immunosuppressive patho­\ngen of poultry, causing aplastic anaemia, lymphoid atrophy, and severe hae­\nmatopoietic dysfunction in young chicks, thereby posing a substantial threat to\nglobal poultry health and production. In this study, a novel highly pathogenic\nCIAV strain, designated CIAV-GDHY230813, was isolated from young Mahuang\nchickens in China and subjected to comprehensive molecular and pathogenic\ncharacterization. Whole-genome sequencing and phylogenetic analysis revealed\nthat CIAV-GDHY230813 belongs to I-a branch. Notably, the VP1 protein har­\nbored a glutamine residue at position 394, a molecular marker strongly asso­\nciated with high virulence, together with multiple amino acid substitutions,\ninsertions, and deletions across the coding regions. Pathogenicity experiments\nin specific-pathogen-free (SPF) chicks demonstrated that infection with CIAV-\nGDHY230813 resulted in pronounced growth retardation, severe damage to\nimmune organs, and increased mortality. From 3 to 21 days post-infection (dpi),\nbody weights of infected chicks were significantly lower than those of the con­\ntrol group (p < 0.01). Marked thymic and bursal atrophy, splenomegaly, and sig­\nnificantly reduced haematocrit levels were observed, indicating severe anaemia.\nFurthermore, CIAV infection led to a marked reduction in antibody titres against\nNewcastle disease virus vaccination by 4-fold to 8-fold, reflecting substantial\nsuppression of humoral immune responses. Quantitative analysis of viral distribu­\ntion showed significantly elevated viral loads in blood, liver, thymus, spleen, and\nbursa of Fabricius at both 14 and 21 dpi (p < 0.001), with peak levels detected at\n21 dpi. Collectively, these findings demonstrate the strong replicative capacity\nand high pathogenic potential of CIAV-GDHY230813, providing valuable insights\ninto the molecular epidemiology and pathogenic mechanisms of CIAV in China,\n\nCollectively, these findings demonstrate the strong replicative capacity\nand high pathogenic potential of CIAV-GDHY230813, providing valuable insights\ninto the molecular epidemiology and pathogenic mechanisms of CIAV in China,\nsupporting improved surveillance and control strategies, and laying a solid foun­\ndation for the development of effective vaccines against CIAV.\nKEYWORDS\nchicken infectious anemia virus, genomic characterization, immunosuppression,\npathogenicity, virulence\n\nYang et al.\b\n10.3389/fvets.2026.1893535\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n1 Introduction\nChicken infectious anemia (CIA), caused by CIAV, is a highly con­\ntagious immunosuppressive disease that poses a substantial threat to\nthe global poultry industry. The virus primarily induces severe immu­\nnosuppression in young chicks, characterized by aplastic anemia, sys­\ntemic lymphoid atrophy, and hematopoietic dysfunction (1, 2). In\nadult chickens, CIAV infection is generally subclinical; however, it\nmarkedly compromises production performance and induces persis­\ntent immunosuppression, thereby reducing vaccine efficacy and\nincreasing susceptibility to secondary infections by a wide range of\npathogenic microorganisms, thereby threatening the economic sus­\ntainability of poultry operations (3). Consequently, CIAV represents a\nmajor challenge to the sustainability and economic viability of modern\npoultry production systems. CIAV was first isolated in Japan in 1979,\nand efficient in vitro propagation was subsequently achieved using\nMDCC-MSB1 cells (4). Since then, the virus has disseminated world­\nwide and becomes endemic in nearly all major poultry-producing\nregions, emerging as one of the most important immunosuppressive\npathogens affecting poultry.\nCIAV can be transmitted both vertically and horizontally. Vertical\ntransmission from infected breeder hens to their offspring results in\nearly infection and severe disease in chicks, whereas horizontal trans­\nmission via feces, secretions, and other excreta facilitates rapid spread\nwithin flocks (5, 6). In China, CIAV was first reported in Heilongjiang\nProvince in 1992 and has since spread extensively, becoming prevalent\nin most poultry-producing provinces nationwide (7). Recent serologi­\ncal surveys indicate persistently high CIAV seroprevalence rates in\ncommercial chicken farms across Jiangsu, Zhejiang, Anhui, Guangxi,\nand other regions, exceeding 70% in some areas, which confirms the\nstable endemic status of CIAV in Chinese poultry populations (8).\nNotably, CIAV frequently co-infects chickens together with other\n\nNotably, CIAV frequently co-infects chickens together with other\nimmunosuppressive viruses, including Marek’s disease virus (MDV),\nreticuloendotheliosis virus (REV), subgroup J avian leukosis virus\n(ALV-J), and fowl adenovirus (FAdV). Such co-infections exacerbate\nclinical manifestations, increase morbidity and mortality, severely\nimpair host immune function, and substantially reduce the protective\nefficacy of routine vaccinations, thereby resulting in significant eco­\nnomic losses to the poultry industry (9–11).\nAccording to the International Committee on Taxonomy of\nViruses (ICTV), CIAV is classified within the genus Gyrovirus of\nthe family Anelloviridae (12, 13). It is a small, non-enveloped,\nsingle-stranded circular DNA virus with icosahedral virions\napproximately 23.5 nm in diameter (14). The CIAV genome is typi­\ncally 2,298 bp in length, although some strains extend to 2,319 bp\ndue to the presence of an additional 21-bp direct repeat (DR). The\nnumber of DRs is closely associated with viral adaptability to host\ncells (15, 16). The viral genome contains three partially overlap­\nping open reading frames (ORFs) encoding VP1 (1,350 bp,\n51.6 kDa), VP2 (651 bp, 24.0 kDa), and VP3 (366 bp, 13.6 kDa)\n(17). VP1, the sole structural protein, is essential for virion assem­\nbly and harbors major antigenic determinants, forming neutral­\nizing epitopes together with VP2. VP2 functions as a scaffold\nprotein required for proper VP1 folding and virion assembly and\nalso exhibits protein phosphatase activity critical for viral replica­\ntion. VP3, also known as apoptin, selectively induces apoptosis in\ntransformed and tumor cells and plays an important role in CIAV-\ninduced immunosuppression, hemorrhage, and anemia (18–20).\nThe VP2 and VP3 coding regions are fully overlapping, while the\nVP1 coding region partially overlaps with VP2. A 300-bp non-\ncoding region (NCR) upstream of VP2 contains promoters, tran­\nscriptional regulatory elements, polyadenylation signals, and\ntranscription factor binding sites, which play a pivotal role in regu­\nlating viral transcription (21).\n\nA 300-bp non-\ncoding region (NCR) upstream of VP2 contains promoters, tran­\nscriptional regulatory elements, polyadenylation signals, and\ntranscription factor binding sites, which play a pivotal role in regu­\nlating viral transcription (21).\nDespite significant advances in CIAV surveillance, molecular\ncharacterization, and epidemiological studies in China, effective con­\ntrol of CIAV remains challenging. Currently, no commercially avail­\nable CIAV vaccine has been approved for use in China, and prevention\nstrategies rely mainly on biosecurity measures and routine pathogen\nmonitoring. However, these approaches have proven insufficient to\neffectively curb viral transmission under intensive production condi­\ntions (22). Although single CIAV infection typically results in rela­\ntively low mortality, the severe immunosuppression induced by the\nvirus, together with frequent co-infections and reduced vaccine effi­\ncacy against Marek’s disease and infectious bursal disease, leads to\nsubstantial economic losses (23). Therefore, systematic isolation of\ncirculating CIAV strains and comprehensive investigations into their\ngenomic characteristics, evolutionary dynamics, and pathogenic\ndiversity are essential for enriching molecular epidemiological data,\nfacilitating viral tracing and evolutionary studies, and providing a sci­\nentific basis for the development of targeted control strategies, novel\nvaccines, and optimized diagnostic tools. In this study, we isolated and\ncharacterized a CIAV strain from commercial chickens in South\nChina and conducted detailed analyses of its genomic features, evolu­\ntionary relationships, and pathogenicity. Our findings aim to elucidate\nthe molecular epidemiology and pathogenic mechanisms of CIAV\ncirculating in this region, thereby providing theoretical and technical\nsupport for effective disease control and prevention strategies in the\nChinese poultry industry.\n2 Materials and methods\n2.1 Clinical samples\nIn August 2023, young Mahuang chickens at a farm in Heyuan\nCity of Guangdong Province, China, developed symptoms of anemia,\n\n2 Materials and methods\n2.1 Clinical samples\nIn August 2023, young Mahuang chickens at a farm in Heyuan\nCity of Guangdong Province, China, developed symptoms of anemia,\nwhich was suspected of being infected with CIAV.\n2.2 CIAV detection\nTissue samples were collected from diseased chickens, includ­\ning the bursa of Fabricius and spleen. The tissues were\nhomogenised and subjected to three freeze–thaw cycles at −80 °C,\nfollowed by centrifugation at 5,000 rpm for 10 min to obtain the\nsupernatants. Viral genomic DNA was extracted using the\nFastPure Viral DNA/RNA Mini Kit (Vazyme, Nanjing, China), in\naccordance with the manufacturer’s instructions. Specific primers\nwere designed based on the genomic sequence of the CIAV refer­\nence strain Cuxhaven-1 (GenBank accession number M55918.1)\nusing Primer-BLAST, as follows: forward primer: 5′-\nAATGAACGCTCTCCAAGAAG-3′;\nreverse\nprimer:\n5′-\nAGCGGATAGTCATAGTAGAT -3′. PCR amplification was\nperformed using 2 × Rapid Taq Master Mix (Vazyme, Nanjing,\nChina). The expected amplicon size was 582 bp. The PCR cycling\n\nYang et al.\b\n10.3389/fvets.2026.1893535\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nconditions were as follows: initial denaturation at 95 °C for 5 min;\n35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for\n15 s, and extension at 72 °C for 15 s; followed by a final extension\nat 72 °C for 5 min. Finally, 5 μL of each PCR product was analysed\nby electrophoresis on a 1% agarose gel and visualized under ultra­\nviolet illumination.\n2.3 Virus isolation\nThe supernatant derived from PCR-positive tissues was fil­\ntered through a 0.22 μm sterile membrane filter and subsequently\ntreated with penicillin–streptomycin (2,000 IU/mL; Solarbio,\nBeijing, China) at 4 °C for approximately 1 h. Thereafter, 200 μL\nof a ten-fold dilution was inoculated into the yolk sac of 7-day-old\nSPF chicken embryos. The embryos were incubated at 37 °C, and\nthose that died within 24 h post-inoculation were excluded from\nfurther analysis. At 16 days of embryonation, the liver, spleen, and\ndecapitated and de-limbed embryo bodies were aseptically col­\nlected, homogenised in sterile PBS, and subjected to three freeze–\nthaw cycles at −80 °C. Following centrifugation at 5,000 rpm for\n5 min, the supernatants were harvested and subjected to three\nconsecutive blind passages for virus isolation.\n2.4 Viral genome sequencing\nViral genomic DNA was extracted using the FastPure Viral\nDNA/RNA Mini Kit (Vazyme, Nanjing, China), in accordance\nwith the manufacturer’s instructions. The full-length CIAV\ngenome was amplified using three pairs of primers (Table 1),\ndesigned based on the reference strain Cuxhaven-1 (GenBank\naccession no. M55918.1) with Primer-BLAST. The purified PCR\nproducts were ligated into the pMD18-T vector (TaKaRa, Beijing,\nChina) and subjected to commercial sequencing (Sangon Biotech,\nShanghai, China). Sequence assembly and analysis were per­\nformed using SeqMan software (Lasergene v7.1, DNASTAR,\nMadison, WI, USA) to obtain the complete CIAV genome.\n2.5 Homology and phylogenetic analysis\nThe assembled CIAV gene sequences were processed using\nSeqMan (Lasergene 7.1). Based on whole-genome CIAV sequences\n\n1, DNASTAR,\nMadison, WI, USA) to obtain the complete CIAV genome.2.5 Homology and phylogenetic analysis\nThe assembled CIAV gene sequences were processed using\nSeqMan (Lasergene 7.1).Based on whole-genome CIAV sequences\navailable in GenBank, 49 reference strains from different periods\nand regions were selected (Supplementary Table S1). Nucleotide\nsequence similarity between the isolate and reference strains was\nanalyzed with MEGA 7.0. Phylogenetic trees were constructed\nfrom whole-genome sequences. Sequence alignment of whole\ngenomes and specific genes was performed using the Clustal W\nalgorithm in MEGA 7.0, and trees were generated via iqtree3.1.1\nusing maximum likelihood, the optimal nucleotide substitution\nmodel was automatically selected as TN + F + R2 with 1,000 boot­\nstrap replicates. Amino acid sequences of the isolate and reference\nstrains were compared to identify mutations in key functional\nregions.\n2.6 Pathogenicity of the CIAV isolate to SPF\nchicks\nFifty-nine one-day-old SPF chicks were randomly allocated into\nfour experimental groups and one control group. Group A served as\nthe negative control (n = 20) and received an intramuscular injection\nof 0.2 mL sterile 0.9% saline into the leg muscle at one day of age.\nGroup B constituted the CIAV-infected group (n = 15) and was inocu­\nlated intramuscularly in the leg with 0.2 mL of the CIAV-\nGDHY230813 isolate at one day of age. Group C was the NDV LaSota\nvaccine-immunised group (n = 12), which received 0.2 mL of the\nLaSota vaccine via the intranasal and intraocular routes at seven days\nof age. Group D was the CIAV and NDV LaSota co-infection group\n(n = 12); chicks were inoculated intramuscularly with 0.2 mL of\nCIAV-GDHY230813 at one day of age and subsequently immunised\nwith the LaSota vaccine (0.2 mL per bird) via the intranasal and intra­\nocular routes at seven days of age. All experimental birds were housed\nunder independent isolation conditions. Groups A and B were main­\ntained until 21 days of age, while Groups C and D were reared until\n28 days of age. Birds were provided with ad libitum access to feed and\n\nAll experimental birds were housed\nunder independent isolation conditions.Groups A and B were main­\ntained until 21 days of age, while Groups C and D were reared until\n28 days of age.Birds were provided with ad libitum access to feed and\nwater throughout the experimental period. Clinical signs were moni­\ntored daily, and mortality was recorded.\nAt 7, 14, and 21 days of age, five birds were randomly selected\nfrom each of groups A and B. From each bird, 2 mL of anticoagulated\nblood and 2 mL of non-anticoagulated blood were collected via the\nwing vein. White blood cell counts, red blood cell counts, and haema­\ntocrit values were determined using an automated haematology analy­\nser. Pharyngeal and cloacal swabs were collected for the detection of\nviral shedding. Tissue samples from the thymus, trachea, spleen, liver,\nkidney, proventriculus, femoral bone marrow, bursa of Fabricius, and\ncaecal tonsils were collected for histopathological examination and\nviral load quantification. In addition, immune organs (thymus, bursa\nof Fabricius, and spleen) were harvested from another five randomly\nselected chicks and weighed. The immune organ index was calculated\nas immune organ weight (mg) divided by body weight (g) to evaluate\nthe impact of CIAV infection on primary immune organs.\nTo evaluate the effect of CIAV infection on antibody responses\nfollowing routine vaccination, at 14, 21, and 28 days of age, three\nchicks were randomly selected from groups C and D, and non-antico­\nagulated blood was collected from the jugular vein to separate serum.\nSerum antibody levels were assessed through both a conventional\nhemagglutination inhibition (HI) assay.\nTo further assess growth performance and survival, an addi­\ntional 30 one-day-old SPF chicks were randomly assigned to two\nequal groups. The control group received an intramuscular injec­\ntion of 0.2 mL sterile saline into the leg at one day of age, whereas\nthe challenged group was inoculated intramuscularly with 0.2 mL\nTABLE 1  Primers for PCR detection of CIAV VP1, VP2, and VP3.\nName\nSequence (5′-3)\nLength\n(bp)\nCIAV-F-1\nATTCCGAGTGGTTACTATTCC\n861\nCIAV-R-1\nCGTCTTGCCATCTTACAGTCTTAT\n\n2 mL\nTABLE 1  Primers for PCR detection of CIAV VP1, VP2, and VP3.Name\nSequence (5′-3)\nLength\n(bp)\nCIAV-F-1\nATTCCGAGTGGTTACTATTCC\n861\nCIAV-R-1\nCGTCTTGCCATCTTACAGTCTTAT\nCIAV-F-2\nCGAGTACAGGGTAAGCGAGCTAAA\n990\nCIAV-R-2\nTGCTATTCATGCAGCGGACTT\nCIAV-F-3\nACGAGCAACAGTACCCTGCTAT\n806\nCIAV-R-3\nCTGTACGTGCTCCACTCGTT\n\nYang et al.\b\n10.3389/fvets.2026.1893535\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nof the CIAV-GDHY230813 isolate. All birds were reared in isolation\nuntil 21 days of age with unrestricted access to feed and water.\nClinical signs, body weight, and mortality were recorded daily.\nGrowth curves were generated based on body weight measure­\nments, deceased chicks were subjected to immediate necropsy, and\nsurvival rates were calculated for each group. All animal experi­\nments were performed in strict compliance with the institution’s\nguidelines on animal welfare.\n2.7 Histopathological examination\nThe collected thymus, spleen, bursa of Fabricius and liver were\nfixed in 10% formalin and then embedded in paraffin, sectioned,\nstained with hematoxylin and eosin (H&E), and observed using a light\nmicroscope.\n2.8 Viral load determination in tissues\nBased on the Diagnostic Techniques for Chicken infectious\nanemia virus (Industry Standard, NY/T 1187–2019), the viral load of\neach organ was detected by SYBR Green I Real-time PCR to analyze\nthe replication of the CIAV-GDHY230813 strain. The specific primers\n(forward primer: 5’-GCAGGGGCAAGTAATTTCAA-3′; reverse\nprimer: 5’-GCCACACAGCGATAGAGTGA-3′) were synthesized by\nSangon Biological Technology (Shanghai, China). Total DNA was\nextracted from anticoagulated blood and from the thymus, spleen,\nliver, and bursa of Fabricius tissues collected from Groups A and B at\n7, 14, and 21 days of age were extracted using the TIANamp Genomic\nDNA Kit (TIANGEN, Beijing, China). Amplification was performed\nusing ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing,\nChina) on a TGreat Real 96 Real-Time PCR System (TIANGEN)\nunder the following conditions: incubation at 95 °C for 30 s; 35 cycles\nof 95 °C for 10 s and 60 °C for 30 s; the instrument’s default melting\ncurve program was applied for data collection.\n2.9 Statistical analysis\nGraphPad Prism (version 8.3.0) and SPSS (version 27.0) software\nwere used for statistical analysis, and the following symbols were used\nto indicate significant differences between groups: *p < 0.05,\n**p < 0.01, ***p < 0.001, ns, not significant.\n3 Results\n\n3.0) and SPSS (version 27.0) software\nwere used for statistical analysis, and the following symbols were used\nto indicate significant differences between groups: *p < 0.05,\n**p < 0.01, ***p < 0.001, ns, not significant.3 Results\n3.1 Isolation and identification of CIAV\nThe CIAV-positive tissue homogenate supernatant was inocu­\nlated into the yolk sac of 7-day-old SPF chicken embryos and\nincubated for 9 days. After three blind passages, viral DNA was\nextracted from homogenized liver, spleen, and embryo tissues for\nPCR detection (Figure 1A). The amplified target fragments were\nsubsequently cloned into the pMD18-T vector and transformed\ninto Escherichia coli DH5α competent cells. Positive clones were\nscreened by PCR, and recombinant plasmids were purified and\nsequenced by Sangon Biotech (Shanghai, China). BLAST analysis\nconfirmed the isolate as CIAV, and the strain was designated\nCIAV-GDHY230813.\nThe viral genome was subjected to PCR identification, and a\nCIAV-specific band was detected at the position of 582 bp. The detec­\ntion results for common poultry viruses such as NDV, IBV, ALV,\nFADV, ARV, MDV, ILTV, AIV, and IBDV showed that, except for\nCIAV, no other common poultry pathogens were detected (Figure 1B).\nTo determine the complete genome sequence, three pairs of spe­\ncific primers were used to amplify overlapping fragments covering the\nentire viral genome. Agarose gel electrophoresis confirmed successful\namplification of three fragments with expected sizes of 861 bp, 990 bp,\nand 806 bp, respectively (Figure 1C). These PCR products were\nFIGURE 1\nPCR detection for CIAV. The genomic DNA extracted from the clinical samples were detected by PCR using specific primers of CIAV (A) PCR detection\nresults of clinical samples. M: DNA Marker DL2000, 1–4: Samples, 5: Positive Control, 6: Negative Control. (B) Detection Results of Exogenous Viruses\nin Poultry by PCR. 1: CIAV, 2: CIAV Positive Control, 3: CIAV Negative Control, 4–12: NDV, IBV, ALV, FADV, ARV, MDV, ILTV, AIV, IBDV. (C) Whole genome\n\n(B) Detection Results of Exogenous Viruses\nin Poultry by PCR.1: CIAV, 2: CIAV Positive Control, 3: CIAV Negative Control, 4–12: NDV, IBV, ALV, FADV, ARV, MDV, ILTV, AIV, IBDV.(C) Whole genome\namplification of CIAV-GDHY230813 isolate. M: DNA Marker DL2000, 1–3: Samples, 4–5: Negative Control.\n\nYang et al.\b\n10.3389/fvets.2026.1893535\nFrontiers in Veterinary Science\n05\nfrontiersin.org\ngel-purified, cloned individually into the pMD18-T vector, and\nsequenced. The resulting sequences were assembled using SeqMan\nsoftware, yielding a complete genome of 2,298 bp.\n3.2 CIAV-GDHY230813 belongs to the I-a\nbranch\nThe complete genome sequence of CIAV-GDHY230813 was\naligned with those of 49 reference CIAV strains retrieved from GenBank\nusing MegAlign software (Figure 2A). Nucleotide sequence identity\nranged from 96.0 to 99.7%. The highest homology (99.7%) was observed\nwith strain GX1804 (MK484615.1), whereas the lowest identity (96.0%)\nwas shared with strain CIAV-F10 (KU845735.1).\nUsing the maximum likelihood (ML) method with iqtree 3.1.1,\nthe optimal nucleotide substitution model was automatically selected\nas TN + F + R2. Based on the topology of the phylogenetic tree con­\nstructed from 50 reference sequences, the virus was divided into two\nmajor groups (I and II): Group I further subdivided into subgroups\nI-a, I-b, and I-c, while Group II was further divided into subgroups\nII-a and II-b. CIAV-GDHY230813 belongs to the I-a branch and\nexhibits close genetic relationships with the GD-103 and FJ21425\nstrains, whereas it diverges significantly from vaccine strains such as\nCux-1 and 26P4, indicating a distant phylogenetic relationship\n(Figure 2B). These findings suggest that CIAV-GDHY230813 likely\nrepresents a wild-type strain circulating in the wild.\n3.3 CIAV-GDHY230813 has highly\npathogenic sequence characteristics\nVP1 is a structural protein with relatively low conservation in\nthe CIAV genome, and it directly influences viral replication rate\nand cellular infectivity. Sequence alignment revealed that the\nhypervariable region (amino acids 139–157) of VP1 in CIAV-\nGDHY230813 was completely conserved compared with reference\nstrains (Supplementary Figure S1). Residues at positions 139 and\n144 were lysine (K) and glutamic acid (E), respectively, which are\nassociated with high infectivity in MDCC-MSB1 cells.\nImportantly, residue 394 was glutamine (Q), a molecular marker\n\nResidues at positions 139 and\n144 were lysine (K) and glutamic acid (E), respectively, which are\nassociated with high infectivity in MDCC-MSB1 cells.Importantly, residue 394 was glutamine (Q), a molecular marker\nstrongly correlated with high virulence, suggesting that CIAV-\nGDHY230813 is highly pathogenic. In addition, amino acids at\npositions 75, 89, 125, and 141 were valine (V), threonine (T), leu­\ncine (L), and glutamine (Q), respectively, consistent with the\nmolecular profile of highly virulent strains (Table 2). Two addi­\ntional amino acid substitutions, D192G and L376I, were also\nnoted. Since they lie outside the classical hypervariable region,\nthey were consequently omitted from the Table.\nVP2, a nonstructural protein essential for viral replication and\nassembly, displayed a high degree of sequence conservation. Only\na single amino acid substitution (G24E) was observed in CIAV-\nGDHY230813, while residues at positions 95 and 97 remained\ncysteine (C), indicating preservation of key functional domains\n(Supplementary Figure S2). VP3, also known as apoptin, contrib­\nutes to viral pathogenicity by inducing apoptosis in host cells. No\namino acid mutations were detected in VP3 of CIAV-\nGDHY230813 (Supplementary Figure S3), suggesting that its\nstructure and function are likely intact and may support efficient\nviral replication and pathogenicity.\nCollectively, these sequence features, particularly those at\nestablished virulence-determining sites, indicate that CIAV-\nGDHY230813 possesses a molecular profile characteristic of\nhighly pathogenic strains.\n3.4 The CIAV-GDHY230813 strain was\nhighly pathogenic to SPF chickens\nFollowing intramuscular inoculation of 1-day-old SPF chicks\nwith CIAV-GDHY230813, the challenged group gradually exhib­\nited clinical signs including lethargy, ruffled feathers, reduced\nFIGURE 2\nHomology comparison and phylogenetic analysis of the CIAV isolate. (A) Homology analysis was performed on the complete genome sequence of the\nCIAV-GDHY230813 isolate and 49 reference strains (including both domestic and international strains) retrieved from the GenBank database using the\n\n(A) Homology analysis was performed on the complete genome sequence of the\nCIAV-GDHY230813 isolate and 49 reference strains (including both domestic and international strains) retrieved from the GenBank database using the\nMegAlign program in the Lasergene 7.1 software package. (B) Phylogenetic tree based on the complete genome. Phylogenetic trees were constructed\nfrom whole-genome sequences. Sequence alignment of whole genomes and specific genes was performed using the Clustal W algorithm in MEGA 7.0,\nand trees were generated via iqtree3.1.1 using maximum likelihood, the optimal nucleotide substitution model was automatically selected as\nTN + F + R2 with 1,000 bootstrap replicates.\n\nYang et al.\b\n10.3389/fvets.2026.1893535\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nfeather luster, and growth retardation, whereas control chicks\nremained clinically normal throughout the observation period.\nBody weights in the challenged group were consistently lower than\nthose in the control group and were significantly reduced at 3, 6,\n9, 12, 15, 18, and 21 days of age (p < 0.01). Moreover, the magni­\ntude of weight difference increased progressively over time, indi­\ncating that CIAV-GDHY230813 infection severely impaired chick\ngrowth and development (Figure 3A). No mortality occurred in\nthe control group, yielding a survival rate of 100%. In contrast,\nmortality in the challenged group was primarily observed within\nthe first 5 dpi, approximately 6.7% mortality in SPF chicks\n(Figure 3B).\nHistopathological examination revealed marked pathological\nalterations in the thymus, spleen, bursa of Fabricius, and liver of\ninfected chicks. The thymus exhibited capsule shrinkage, reduced\ncortical and medullary lymphocyte density, indistinct corticomedul­\nlary boundaries, widened interlobular septa, and connective tissue\nhyperplasia. The spleen showed disruption of normal architecture,\nblurring of red and white pulp boundaries, expansion of red pulp,\nextensive lymphocyte necrosis and depletion, and reticular cell pro­\nliferation. The bursa of Fabricius displayed severe lymphoid\ndepletion, follicular atrophy, widened interfollicular spaces, and\nreticular cell hyperplasia. Hepatic lesions included hepatocellular\ndegeneration, interstitial hemorrhage, and infiltration of lymphocytes\nand monocytes. No remarkable pathological changes were observed\nin tissues from the control group (Figure 3C).\n3.5 CIAV-GDHY230813 induces severe\nAnemia in infected chickens\nRoutine hematological analyses were conducted to evaluate\nthe impact of CIAV-GDHY230813 on hematopoietic function.\nCompared with controls, infected chicks exhibited significantly\nreduced red blood cell (RBC) and white blood cell (WBC) counts\n(Figures 4A,B). Hematocrit (HCT) values in the control group\nremained above 30% throughout the experimental period. In con­\n\nCompared with controls, infected chicks exhibited significantly\nreduced red blood cell (RBC) and white blood cell (WBC) counts\n(Figures 4A,B).Hematocrit (HCT) values in the control group\nremained above 30% throughout the experimental period.In con­\ntrast, HCT levels in the challenged group were significantly\ndecreased at 7 and 21 days of age (p < 0.05). Although the differ­\nence at 14 days did not reach statistical significance, HCT values\nin infected chicks remained consistently lower than those of con­\ntrols. The most pronounced anemia was observed at 7 dpi,\nTABLE 2  The known virulence determining sites in VP1 of the isolates.\nIsolates/amino\nacidsites\n75\n89\n125\n139\n141\n144\n157\n287\n394\nMajority virulent\nstrains\nV\nT\nI\nQ\nQ\nQ\nV\nT\nQ\nLow virulent strains\nI\nA\nL\nK\nE\nE\nM\nS\nH\nCIAV-GDHY230813\nV\nT\nL\nK\nQ\nE\nV\nS\nQ\nFIGURE 3\nPathogenicity analysis of CIAV-GDHY230813 on SPF chickens. (A) Changes in body weight of SPF chickens. (B) Survival rate of SPF chickens.\n(C) Histopathological lesions change in the thymus, spleen, bursa of Fabricius and liver the control group and the CIAV-GDHY230813 group on d 7\npostinfection. Lesions are marked with a black arrow in the CIAV-GDHY230813 group (Thymus ×100, Spleen ×100, bursa of Fabricius × 200, Liver ×\n200).\n\nYang et al.\b\n10.3389/fvets.2026.1893535\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nfollowed by partial recovery at later time points; however, HCT \nlevels remained below 30% throughout the study (Figure 4C).\n3.6 CIAV-GDHY230813 causes atrophy of \nimmune organs and suppresses immune \nresponses in chickens\nTo assess the effects of CIAV-GDHY230813 on immune organ \ndevelopment, thymus, spleen, and bursa of Fabricius indices were \ndetermined in randomly selected chicks from each group at 7, 14, \nand 21 dpi. The thymus index in the challenged group was \nsignificantly reduced compared with that in the control group \n(p < 0.05), indicating marked suppression of thymic development \n(Figure 5A). The spleen index was generally higher in infected \nchicks, reflecting splenic enlargement, although a slight decrease \nwas observed at 14 dpi, which did not reach statistical significance \n(Figure 5B). Bursa index was significantly lower in the challenged \ngroup at 7 dpi (p < 0.05), suggesting early inhibition of bursal \ndevelopment, whereas no significant differences were detected at \n14 or 21 days (Figure 5C).\nTo evaluate the immunosuppressive effects of CIAV-\nGDHY230813 on vaccine-induced immunity, serum samples were \nFIGURE 4\nEffects of CIAV-GDHY230813 on White Blood Cells, Red Blood Cells and Hematocrit in SPF Chickens. (A) Red blood cell numbers of chickens in \ndifferent groups. (B) White blood cell numbers of chickens in different groups. (C) Hematocrit of chickens in different groups.\nFIGURE 5\nEffect of CIAV-GDHY230813 on the Immune Organ Index and Immune Response of SPF Chickens. (A) Thymus index. (B) Spleen index. (C) bursal index. \n(D) Effect of CIAV-GDHY230813 infection on antibody levels after NDV vaccine. The y-axis represents log2-transformed antibody titres, *indicates \nsignificant difference between CIAV-infected/vaccinated group and vaccinated-only group, p < 0.05.\n\nYang et al.\b\n10.3389/fvets.2026.1893535\nFrontiers in Veterinary Science\n08\nfrontiersin.org\ncollected at 14 and 21 days following immunization with\nNewcastle disease virus (NDV) vaccine, and antibody titers were\nmeasured using the hemagglutination inhibition (HI) assay. At\n14 days of age, NDV-specific antibody titers in the infected group\nwere significantly lower than those in the non-infected immu­\nnized group (p < 0.05), indicating a pronounced inhibitory effect\nof CIAV on humoral immune responses. Although antibody levels\nincreased at 21 days of age, titers in the challenged group remained\nsignificantly lower than those of the control group, demonstrating\nsustained immunosuppression induced by CIAV infection\n(Figure 5D).\n3.7 Virus shedding status and viral load in\ndifferent tissues of SPF chickens\nPharyngeal and cloacal swabs were collected from infected\nand control chicks at 7, 14, and 21 days of age and analyzed by\nPCR to assess viral shedding (Table 3). All samples from the con­\ntrol group remained negative throughout the experiment. In the\nchallenged group, viral detection rates were low at 7 days, with\nonly 25% positivity in both pharyngeal and cloacal swabs. By\n14 days, detection rates increased to 75% in pharyngeal swabs and\n100% in cloacal swabs. At 21 days, both sample types exhibited\n100% positivity, indicating active and persistent viral shedding.\nTo further characterize viral distribution, blood, thymus, liver,\nspleen, and bursa of Fabricius samples were collected at 7, 14, and\n21 days post-infection and subjected to qPCR (Figure 6). At\n7 days, viral loads were generally low; however, significantly\nhigher copy numbers were detected in the spleen and bursa of\ninfected chicks compared with controls (p < 0.05), whereas no\nsignificant differences were observed in blood, liver, or thymus\n(p > 0.05). As infection progressed, viral loads increased markedly\nin all examined tissues. At 14 and 21 days, viral copy numbers in\nblood and all sampled organs of the challenged group were sig­\nnificantly higher than those of controls (p < 0.001), reaching peak\n\n05).As infection progressed, viral loads increased markedly\nin all examined tissues.At 14 and 21 days, viral copy numbers in\nblood and all sampled organs of the challenged group were sig­\nnificantly higher than those of controls (p < 0.001), reaching peak\nlevels at 21 days. These findings indicate that CIAV-GDHY230813\nis capable of sustained and efficient replication in peripheral\nblood and major immune organs, demonstrating strong replica­\ntive capacity and systemic dissemination.\n4 Discussion\nRecent epidemiological investigations have demonstrated that\nCIAV infection, either alone or in combination with other pathogens,\nis widespread in commercial poultry flocks, posing a substantial threat\nto the sustainable development of the poultry industry in China. In\nthe present study, a CIAV strain, designated CIAV-GDHY230813, was\nsuccessfully isolated from clinically suspected samples collected in\nsouthern China. Following inoculation into the yolk sacs of SPF\nchicken embryos, the virus was efficiently propagated, and complete\ngenome sequencing revealed a genome length of 2,298 bp.\nHomology analysis indicated that CIAV-GDHY230813 shares\n96.0–99.7% nucleotide sequence similarity with domestic and inter­\nnational reference strains. It exhibited the highest similarity (99.7%)\nwith strain GX1804 (MK484615.1) and the lowest (96.0%) with strain\nCIAV-F10 (KU845735.1). Phylogenetic trees based on the complete\ngenome were largely consistent. Among 50 strains classified into two\nmajor groups (I and II), CIAV-GDHY230813 fell within Group I-a,\nshowing close genetic relatedness to circulating strains from southern\nChina such as GD-103 (Guangdong) and FJ21425 (Fujian), but distant\nfrom foreign vaccine strains including 26P4 and Cux-1. These findings\nsuggest that CIAV-GDHY230813 likely represents a wild-type strain\ncirculating in the wild (8). Although recombination analysis revealed\nno genetic recombination in CIAV-GDHY230813, previous studies\nhave reported isolates from contaminated attenuated vaccines that are\nclosely related to foreign strains (3). Thus, the potential for increased\n\nAlthough recombination analysis revealed\nno genetic recombination in CIAV-GDHY230813, previous studies\nhave reported isolates from contaminated attenuated vaccines that are\nclosely related to foreign strains (3).Thus, the potential for increased\ngenetic diversity in CIAV warrants attention, and vigilance against\nemerging recombinant viruses remains necessary.\nThe VP1 protein, the sole structural component of CIAV, is also\nits most variable region. VP1 serves as the major capsid protein and\nkey immunogen, located on the virion surface and subject to host\nimmune selection pressure, which drives high sequence variability\n(24). The region spanning amino acids 39–157 is considered hyper­\nvariable. Substitutions at positions 139 and 144 to glutamine (Q) have\nbeen shown to markedly reduce viral replication efficiency and infec­\ntivity in MDCC-MSB-1 cells (25). Moreover, position 394 is an\nimportant virulence determinant; mutation from glutamine (Q) to\nhistidine (H) significantly attenuates pathogenicity. In CIAV-\nGDHY230813, residues 139 and 144 were identified as lysine and\nglutamic acid, respectively, and residue 394 remained glutamine, a\nmolecular profile consistent with highly pathogenic strains recently\nisolated in Guangdong and Guangxi provinces, furthermore, residues\n75, 89, 125, 141 and 144 are identical to those of known highly viru­\nlent strains (3). Genetic analysis identified the Q394 mutation on\nVP1, a well-established molecular marker of highly pathogenic CIAV\nstrains. Combined with obvious clinical symptoms and severe atro­\nphy of immune organs in infected chickens, we preliminarily defined\nthis strain as highly pathogenic. Nevertheless, the mortality rate in\nthis study was only 6.7%, which was lower than the mortality caused\nby several classic hypervirulent CIAV strains reported previously.\nThis relatively low mortality does not deny the intrinsic high patho­\ngenicity of the isolate. Multiple experimental conditions collectively\nalter the final pathogenic phenotype of CIAV infection, including the\nintramuscular inoculation route, uniform genetic background of SPF\n\nMultiple experimental conditions collectively\nalter the final pathogenic phenotype of CIAV infection, including the\nintramuscular inoculation route, uniform genetic background of SPF\nchickens, and the use of 1-day-old chicks as infection hosts. These\ninfluencing factors may alleviate the lethal outcome of infection and\npartially explain the low mortality observed in our animal challenge\ntest. Notably, unlike certain prevalent Guangxi strains, the VP1\nhypervariable region of CIAV-GDHY230813 exhibited complete\nsequence conservation, and key residues associated with virulence\nremained unchanged. This high degree of conservation may reflect\nstrong functional constraints and could partly explain the pro­\nnounced pathogenicity observed in experimental infections.\nNevertheless, several amino acid substitutions and a single deletion\nTABLE 3  The virus shedding of experimental groups after CIAV-GDHY230813 strain infection.\nGroups\nThroat\nCloaca\n7\ndpi\n14\ndpi\n21\ndpi\n7\ndpi\n14\ndpi\n21\ndpi\nControl\n0/4\n0/4\n0/4\n0/4\n0/4\n0/4\nCIAV-\nGDHY230813\n1/4\n3/4\n4/4\n1/4\n4/4\n4/4\n\nYang et al.\b\n10.3389/fvets.2026.1893535\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nwere identified outside the hypervariable region, indicating that\nmutations beyond traditionally recognised variable domains may\nalso contribute to viral fitness, replication dynamics, and pathogenic\npotential, meriting further structural and functional investigation.\nThe VP2 and VP3 proteins are relatively conserved, assisting in the\ncorrect folding of VP1 during viral particle assembly and playing an aux­\niliary role in inducing neutralizing antibody production (2). VP2 func­\ntions as a dual-specificity protein phosphatase essential for proper VP1\nfolding and virion assembly, while VP3, also known as apoptin, induces\napoptosis in host immune cells, particularly thymocytes. Although several\namino acid substitutions, insertions, and a deletion were observed in VP2,\nno alterations were detected within its highly conserved catalytic motif,\nsuggesting that its phosphatase activity and functional integrity are likely\npreserved (26). In contrast, the VP3 sequence of CIAV-GDHY230813\nshowed complete conservation, implying that the virus retains its capacity\nto induce apoptosis in lymphoid and haematopoietic tissues, thereby con­\ntributing to immunosuppression and anaemia.\nThis study systematically evaluated the pathogenicity of the CIAV-\nGDHY230813 strain isolated from southern China. The results demon­\nstrated that the body weight of chicks in the challenged group was\nsignificantly lower than that of the control group from 3 dpi, indicating a\npronounced inhibitory effect on growth performance. Furthermore, the\nmortality rate in the challenged group reached approximately 7%, with\ndeaths occurring predominantly within the first 5 dpi, a pattern consistent\nwith the infection characteristics of highly virulent CIAV strains reported\npreviously. Infected birds also exhibited conspicuous point-like and\npatchy haemorrhages in the pectoral and leg muscles, as well as in subcu­\ntaneous tissues, reflecting the severe pathological damage induced by\nCIAV infection (27), which may contribute to the development of anae­\n\nmia through the destruction of erythrocytes and myeloid-derived special­\nized progenitor cells (28). No dermatitis or blue wing syndrome was\nobserved following challenge with CIAV-GDHY230813; however, severe\nthymic and bursal atrophy, splenomegaly, and a marked reduction in hae­\nmatocrit levels were evident, indicating profound anaemia, whether there\nis a persistent anemia status requires further long-term monitoring.\nCIAV can disseminate within chicken flocks through both\nhorizontal and vertical transmission, with infection of the gastro­\nintestinal tract generally regarded as the primary route of horizon­\ntal spread (29). Following challenge with CIAV-GDHY230813, the\ndetection rate in anal swabs reached 100% as early as 14 days of\nage, and both anal and pharyngeal swabs consistently exhibited a\n100% positivity rate at 21 days of age, indicating a high capacity for\nhorizontal transmission. Accordingly, cloacal swabs represent the\npreferred sample type for the clinical detection and epidemiologi­\ncal surveillance of CIAV. These findings further suggest that, in the\nabsence of specific antiviral agents and highly effective vaccines,\nCIAV is exceedingly difficult to eradicate once introduced into a\npoultry flock.\nAnalysis of viral tissue distribution and load further eluci­\ndated the tissue tropism of CIAV-GDHY230813. During the early\nstage of infection, viral loads in the spleen and bursa of Fabricius\nwere significantly higher than those observed in the control\ngroup. As the duration of infection increased, viral loads in the\nblood, liver, and multiple immune organs continued to rise, indi­\ncating that CIAV-GDHY230813 is capable of sustained and effi­\ncient replication in vivo. These findings are consistent with\nprevious reports demonstrating that CIAV primarily targets lym­\nphoid tissues, particularly thymic lymphocytes, and that the virus\ncan persist in the host for prolonged periods, rendering it diffi­\ncult to achieve complete neutralization by host antibodies (30–\n32). Following CIAV infection, the virus can be released from the\n\nFollowing CIAV infection, the virus can be released from the\nthymus into the systemic circulation, subsequently disseminating\nand replicating in multiple tissues and organs throughout the\nhost, which may account for its extensive tissue distribution.\nVP3 induces apoptosis in thymic CD4+/CD8+ immature T precur­\nsors, depleting peripheral helper T cells and abolishing costimulatory\nsignals and IL-4/IL-6 critical for B-cell activation. This disrupts germi­\nnal center reactions, blocks somatic mutation and IgM to IgY switch­\ning, causing persistent dual immunosuppression, poor vaccine efficacy,\nand frequent secondary infections in chicks (28, 31). The observed\nFIGURE 6\nVirus load in different tissues of SPF chickens after CIAV infection. On 7, 14, and 21 days of age of SPF checks, blood (A) spleen (B) thymus (C), bursa of\nFabricius (D) and liver (E) samples were collected from chicks, after extracting tissue DNA, qPCR was used to detect the CIAV viral load in each tissue.\n\nYang et al.\b\n10.3389/fvets.2026.1893535\nFrontiers in Veterinary Science\n10\nfrontiersin.org\natrophy of the thymus and bursa of Fabricius indicates damage to cen­\ntral immune organs, suggesting profound suppression of humoral\nimmunity. Serum antibody titres against NDV in the CIAV-\nGDHY230813-infected group were significantly lower than those in\nthe control group at both 14 and 21 days of age, indicating that this\nstrain effectively suppresses the humoral immunity elicited by NDV\nvaccination. These findings are consistent with previous reports dem­\nonstrating that both single CIAV infection and co-infection with ALV-J\nsignificantly inhibit antibody production following vaccination against\nH9N2 and FAdV-. By suppressing host immune function, CIAV com­\npromises the efficacy of conventional vaccines in poultry flocks,\nthereby rendering chickens more susceptible to secondary infections.\nConsequently, stringent screening for CIAV contamination is of critical\nimportance in both vaccine production and clinical application.\n5 Conclusion\nIn conclusion, this study successfully isolated a highly patho­\ngenic CIAV strain, designated CIAV-GDHY230813, from 56-day-\nold Mahuang chickens in Heyuan, Guangdong Province, China.\nPhylogenetic analysis classified this isolate within I-a, with a com­\nplete genome length of 2,298 bp and a glutamine (Q) residue at\nposition 394 of VP1, a molecular marker associated with high\nvirulence. Experimental infection demonstrated that CIAV-\nGDHY230813 induced severe anaemia, marked immune organ\natrophy, growth retardation, and approximately 6.7% mortality in\nSPF chicks. Furthermore, the virus exhibited sustained replication\nin the bloodstream and major immune organs, significantly\nimpaired the protective efficacy of Newcastle disease vaccination,\nand displayed efficient horizontal transmission. Collectively, these\nfindings expand the genomic dataset of CIAV circulating in China\nand provide valuable insights into its molecular epidemiology and\npathogenic mechanisms, thereby contributing to the development\nof more effective prevention and control strategies.\nData availability statement\n\nData availability statement\nThe data presented in the study are deposited in the National\nCenter for Biotechnology Information repository, accession number\nPX496712.1.\nEthics statement\nThe animal study was approved by Committee on Ethics of\nAnimal Experiments of Guangxi University. The study was con­\nducted in accordance with the local legislation and institutional\nrequirements.\nAuthor contributions\nHY: Visualization, Writing – original draft, Writing – review & edit­\ning. LY: Writing  – original draft, Writing  – review & editing. HC:\nVisualization, Writing – original draft, Writing – review & editing. YuL:\nWriting – review & editing. AX: Writing – review & editing. ZL: Writing –\nreview & editing. XX: Writing – review & editing. SM: Writing – review\n& editing. SF: Writing – review & editing. YW: Writing – review & edit­\ning. HS: Writing – review & editing. GL: Writing – review & editing. YaL:\nSupervision, Writing – review & editing. AZ: Supervision, Writing –\nreview & editing. CO: Conceptualization, Funding acquisition, Resources,\nSupervision, Writing – review & editing.\nFunding\nThe author(s) declared that financial support was received for this\nwork and/or its publication. The project is supported by the Guangxi\nAgriculture Research System Innovation Team (nycytxgx­\ncxtd-2024-19), Guangxi Science and Technology Program (GUIKE\nAD23049005) and National Natural Science Foundation of China\n(32260876).\nAcknowledgments\nWe acknowledge Qilu Animal Health Products Co. LTD and\nBeijing Centre Company for kindly providing the clinical sample,\nwhich was utilized to isolate the virus.\nConflict of interest\nYuL was employed by Guangxi Yuanfeng Group Pastoral Industry\nCo. Ltd.\nThe remaining author(s) declared that this work was conducted\nin the absence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declared that Generative AI was not used in the\ncreation of this manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\n\nGenerative AI statement\nThe author(s) declared that Generative AI was not used in the\ncreation of this manuscript.Any alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the review­\ners. Any product that may be evaluated in this article, or claim that\nmay be made by its manufacturer, is not guaranteed or endorsed\nby the publisher.\n\nYang et al.\b\n10.3389/fvets.2026.1893535\nFrontiers in Veterinary Science\n11\nfrontiersin.org\nSupplementary material\nThe Supplementary material for this article can be found online\nat: https://www.frontiersin.org/articles/10.3389/fvets.2026.1893535/\nfull#supplementary-material\nSUPPLEMENTARY FIGURE S1\nAmino acid sequence analysis of CIAV-GDHY0813 VP1.\nSUPPLEMENTARY FIGURE S2\nAmino acid sequence analysis of CIAV-GDHY0813 VP2.\nSUPPLEMENTARY FIGURE S3\nAmino acid sequence analysis of CIAV-GDHY0813 VP3.\nReferences\n1.\tRosenberger JK, Cloud SS. 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+{"text": "【兽医文献】\n标题: Jaw tone dynamics across varying depths of anesthesia in a swine model\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nTYPE  Case Report\nPUBLISHED  24 July 2026\nDOI  10.3389/fvets.2026.1858804\nOPEN ACCESS\nEDITED BY\nAlejandro Casas Alvarado,\nNational Autonomous University of\nMexico, Mexico\nREVIEWED BY\nPalagan Senopati Sewoyo,\nUdayana University, Indonesia\nYalcin Alper Ozturan,\nAdnan Menderes University, Türkiye\n*CORRESPONDENCE\nDae-Hyun Kim\nvet1982@cnu.ac.kr\n†These authors have contributed equally\nto this work\nRECEIVED 17 April 2026\nREVISED 27 June 2026\nACCEPTED 29 June 2026\nPUBLISHED 24 July 2026\nCITATION\nPark J, Lee W-J and Kim D-H (2026)\nCase Report: Fascia lata transposition for\nchronic recurrent perineal hernia\nfollowed by laparoscopic colopexy for\nsubsequent rectal prolapse in a dog.\nFront. Vet. Sci. 13:1858804.\ndoi: 10.3389/fvets.2026.1858804\nCOPYRIGHT\n© 2026 Park, Lee and Kim. This is an\nopen-access article distributed under\nthe terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other\nforums is permitted, provided the\noriginal author(s) and the copyright\nowner(s) are credited and that the\noriginal publication in this journal is\ncited, in accordance with accepted\nacademic practice. No use, distribution\nor reproduction is permitted which does\nnot comply with these terms.\nCase Report: Fascia lata\ntransposition for chronic\nrecurrent perineal hernia\nfollowed by laparoscopic\ncolopexy for subsequent rectal\nprolapse in a dog\nJiyoung Park 1,2†, Won-Jong Lee 2† and Dae-Hyun Kim 2*\n1Ulsan S Animal Medical Center, Ulsan, Republic of Korea, 2Department of Veterinary Surgery, College\nof Veterinary Medicine, Chungnam National University, Daejeon, Republic of Korea\nThis case report describes a surgical management of chronic, recurrent, compli­\ncated perineal hernia following bilateral herniorrhaphy in a 6-kg castrated male\nmixed-breed dog, estimated to be over 10 years old. The patient presented with\nrecurrent right-sided perineal hernia and constipation associated with rectal sac­\nculation. During the herniorrhaphy attempted, severe atrophy of pelvic mus­\nculature was identified; therefore, a fascia lata transposition was performed.\n\nDuring the herniorrhaphy attempted, severe atrophy of pelvic mus­\nculature was identified; therefore, a fascia lata transposition was performed.\nPostoperatively, the dog developed a rectal prolapse, which was successfully\naddressed through laparoscopic colopexy. The dog recovered uneventfully,\nmaintaining a good appetite and activity levels. There were no complications\nrelated to urination, defecation, gait, or wound healing following either proce­\ndure. At the 27-month follow-up, the dog remained in good condition without\nclinical signs. Fascia lata transposition is effective for complicated perineal hernias\nlacking of musculature for reconstruction. Laparoscopic colopexy is also a clini­\ncally practical option for rectal prolapse in small-breed dogs.\nKEYWORDS\ncolopexy, dog, fascia lata transposition, laparoscopy, perineal herniorrhaphy, rectal\nprolapse, recurrent perineal hernia\n1 Introduction\nPerineal hernia (PH) occurs when the pelvic diaphragm fails to support the rectal wall due\nto muscular weakness, leading to the dilatation or deviation of the rectum and the herniation\nof pelvic or abdominal organs (1, 2). While the exact pathogenesis of PH is not fully under­\nstood, it is believed to be multifactorial. Contributing factors include tenesmus associated with\nconstipation, chronic prostatic disease, or rectal disease; myopathy; gonadal hormonal imbal­\nances; the effects of relaxin; and neurogenic atrophy of the pelvic diaphragm muscles (1–3).\nPH frequently occurs in middle-aged, intact male dogs, with the levator ani muscle being the\nmost commonly affected (2, 4). The severity of PH varies; complicated cases are often associ­\nated with recurrence, significant rectal dilatation, concurrent prostatic disease requiring sur­\ngery, and retroflexed bladder (5).\nCanine PH is primarily a surgical condition requiring reconstruction of the pelvic dia­\nphragm. Various methods are employed, including appositional herniorrhaphy, autografts\n\nPark et al.\b\n10.3389/fvets.2026.1858804\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n(such as internal obturator, superficial gluteal, semitendinosus, fascia\nlata, and tunica vaginalis communis muscles), allografts (canine small\nintestinal submucosa), xenografts (porcine small intestinal submu­\ncosa, porcine dermal collagen), use of synthetic polypropylene mesh\nor collagen based sponge, and sacroischial sling technique (1, 2, 5–7).\nIn cases of severe atrophy of the levator ani or coccygeus muscles,\nappositional herniorrhaphy is contraindicated due to excessive tension\non the external anal sphincter. Internal obturator muscle transposition\n(IOMT) is the most widely used technique in such cases, sometimes\nincorporating the sacrotuberous ligament for lateral repair (3, 5).\nOrganopexy (colopexy, cystopexy, or vasopexy) combined with castra­\ntion can serve as an effective alternative or adjunctive treatment for\nPH, yielding satisfactory outcomes by reducing recurrence rates\n(2, 5, 8).\nRectal prolapse (RP) occurs immediately after herniorrhaphy in\n7–42% of cases and is associated with straining due to misplaced\nsutures in the rectal lumen, postoperative pain, underlying rectal dis­\nease, or injury to the external anal sphincter nerve (5, 9). Furthermore,\nRP may also develop particularly in dogs presenting with bilateral\nhernias complicated by concurrent rectal sacculation (10). Colopexy\nis generally performed for chronic RP, and PH to prevent caudal dis­\nplacement of the colon and rectum (11, 12). This procedure creates a\npermanent adhesion between the descending colon and the left\nabdominal wall, reducing the contents within the hernia and prevent­\ning recurrent RP (11). While a ventral midline or left paramedian\nceliotomy is standard, laparoscopic approaches have also been utilized\n(12). Successful outcomes depend on avoiding suture penetration into\nthe colonic lumen and preventing excessive colonic tension (5, 12).\nThese precautions help prevent complications such as local wound\ninfection and peritonitis, which might necessitate revision surgery (5,\n\nThese precautions help prevent complications such as local wound\ninfection and peritonitis, which might necessitate revision surgery (5,\n12). Additionally, any underlying causes of straining must be\naddressed (12).\nFascia lata has been extensively utilized in both human and vet­\nerinary medicine for various reconstructive purposes due to its dura­\nbility, minimal retraction, availability, and low vascularity\nrequirements (13). Autologous fascia lata grafts are frequently used for\ntendon, ligament, urethral, and joint capsule repairs, as well as abdom­\ninal wall reconstructions and cranioplasties (13–15). This report\ndetails a fascia lata transposition (FLT) for a complicated PH with\nrectal sacculation, followed by laparoscopic colopexy for subsequent\nRP, including the clinical course and long-term prognosis.\n2 Case description\nA 6-kg castrated male mixed-breed dog, estimated to be over\n10 years old, presented for surgical treatment of PH. The dog, rescued\n4 months prior, had a body condition score of 6 out of 9 and was\nbright and alert. Shelter staff reported constipation and a firm, non-\npainful bulge on the right perineum (Figure 1A). Surgical scars were\nevident on both sides of the perineum, with subcutaneous foreign\nmaterial palpable on the left and suture materials on the right.\nComprehensive screening tests, including physical examination, rectal\npalpation, thoracic and abdominal radiography, abdominal ultraso­\nnography, complete blood count, serum chemistry, and electrolytes,\nrevealed no specific findings except for intestinal fecal accumulation\nand PH with fecal impaction in the rectal sacculation without rectal\nwall defect or thinning (Figures 1B,C). The patient was initially man­\naged with manual expression and oral lactulose (0.5 mL/kg body\nweight [BW], twice daily [BID], Dulackhan Easy Syrup, JW\nPharmaceutical, Korea) to alleviate tenesmus.\nThree weeks later, perineal exploration and herniorrhaphy—ini­\ntially planned as an IOMT—were attempted under general anesthesia\nwith isoflurane inhalation (< 2%, Ifran, Hana Pharm, Korea) without\n\nThree weeks later, perineal exploration and herniorrhaphy—ini­\ntially planned as an IOMT—were attempted under general anesthesia\nwith isoflurane inhalation (< 2%, Ifran, Hana Pharm, Korea) without\nmechanical ventilation. Premedication and induction were achieved\nusing midazolam (0.2 mg/kg BW, intravenously [IV]; Bukwang\nMidazolam, Bukwang Pharm, Korea) and propofol (4 mg/kg BW, slow\nIV; Provive, Pharmbio Korea, Korea), respectively. Cefazolin (20 mg/\nkg BW, IV; Cefazoline, Chongkundang, Korea) and meloxicam\n(0.2 mg/kg BW, IV; Metacam, Boehringer Ingelheim Vetmedica\nKorea, Korea) were administered preoperatively, and a transdermal\nfentanyl patch (25 μg/h, Durogesic D-trans Patch, Janssen Korea,\nKorea) was attached 6 h before surgery. Intraoperative fluid therapy\nwas administered using Lactated Ringer’s solution (5 mL/kg/h), and\ncontinuous patient monitoring throughout the procedure included\nheart rate, respiratory rate, pulse oximetry, non-invasive blood pres­\nsure, tidal volume, end-tidal carbon dioxide, inspiratory pressure, and\nFIGURE 1\nA right perineal bulging (A) and fecal impaction in rectal sacculation (B: lateral view, C: dorsoventral view) of a recurrent perineal hernia in a dog. Black\narrow; subcutaneous foreign material, *; herniated rectum, dotted line; previous surgical scar on the right perineum.\n\nPark et al.\b\n10.3389/fvets.2026.1858804\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nbody temperature. The isoflurane concentration was adjusted accord­\ning to the anesthetic depth. The patient was positioned in sternal\nrecumbency with the hind limbs extending beyond the edge of the\nsurgical table. In anticipation of a potential autograft using FLT, the\nsurgical field was extended to include the right caudolateral thigh,\nwhich was also prepared aseptically.\nUpon exploration of the right perineal region following a skin\nincision, inflamed subcutaneous tissues, perianal fat, and the hernia\nsac were found to be interwoven, accompanied by a clear, mildly vis­\ncous, and slippery discharge (Figure 2A). During dissection and\ndebridement, a synthetic mesh and non-absorbable suture materials\nwere identified around the ischiatic border and penile root, which\nwere removed along with the inflammatory tissues (Figure 2B). A pro­\ntrusion of the rectal sacculation, which was the only herniated organ,\nwas also observed without any wall tearing (Figure 2C). The pelvic\nmusculature was challenging to distinguish, with no evident muscular\nstructures identified except for the external anal sphincter. The hernia­\ntion was bordered by the external sphincter, sacrotuberous ligament,\nischiatic table, and sacrum. The internal obturator muscle was barely\nvisible and thus not a viable option for reconstructing the pelvic\ndiaphragm.\nAn additional incision was made on the right lateral thigh for FLT\n(Figure 2D). After dissecting the subcutaneous tissue, a fascia lata flap\nwas created. The borders of the flap were defined by the sartorius\nmuscle (cranially), the cranial border of the biceps femoris muscle\n(caudally), and the patella (distally). Vertical incisions along the cra­\nnial and caudal margins were extended proximally to the level of the\ntensor fascia lata, with the flap remaining attached at its proximal\norigin. The flap, including the tensor fascia lata, was then rotated\ncaudo-medially and passed through a subcutaneous tunnel into the\nperineal region (Figure 2E). It was sutured to the external anal sphinc­\n\nThe flap, including the tensor fascia lata, was then rotated\ncaudo-medially and passed through a subcutaneous tunnel into the\nperineal region (Figure 2E).It was sutured to the external anal sphinc­\nter, ischiourethralis, periosteum of the ischial table/ischial border,\nsacrotuberous ligament, and rectal wall using 3–0 polydioxanone\n(PDS II, Ethicon, USA) (Figure 2F). The redundant distal portion of\nthe flap was trimmed and removed. Both surgical fields were lavaged\nand closed routinely after bupivacaine infiltration. An active drain\nusing a butterfly needle and a vacuum tube was placed at the perineal\nwound. Postoperative rectal palpation confirmed a well-reconstructed\npelvic diaphragm supporting the rectal wall.\nThe patient recovered uneventfully from anesthesia, with a good\nappetite and no signs of lameness. A seven-day course of oral medica­\ntion, including amoxicillin/clavulanic acid (12.5 mg/kg BW, BID;\nAmocla, Kuhnil, Korea), famotidine (0.5 mg/kg BW, BID; Famotidine,\nNelson Korea, Korea), and meloxicam (0.1 mg/kg BW, once daily),\nwas prescribed. A cold pack was recommended to be applied to the\nsurgical sites (right perineum and right lateral thigh) twice daily for\n3 days to alleviate potential acute pain, swelling, and inflammation.\nOn the night of surgery, however, a 3 cm full-thickness RP devel­\noped. A temporary purse-string suture was performed after manual\nreduction, followed by laparoscopic colopexy the next day (postopera­\ntive day 1 [POD 1]). Three 5-mm ports were used: the primary port\nfor the 5 mm, 0° telescope (1,488 HD, Stryker, Portage, Michigan,\nUSA) was placed cranial to the umbilicus, and two instrumental ports\nwere placed right paramedian, caudal to the primary port. Intra-\nabdominal pressure was maintained at < 10 mmHg throughout the\nprocedure. The caudal abdomen and pelvic cavity were explored, and\nthe distal colon was retracted cranially, revealing dilatation of the\ndistal colon (Figure 3A). The selected site for colopexy was the inter­\nsection of the left paramedian line of the rectus abdominis muscle and\n\nThe selected site for colopexy was the inter­\nsection of the left paramedian line of the rectus abdominis muscle and\nthe transverse line of the preputial end. A 2 cm longitudinal incision\nwas made in the peritoneum using a J-hook for laparoscopic cauter­\nization (Figure 3B). The colon was lifted to the abdominal wall, and 4\nintracorporeal, simple interrupted sutures were placed under lower\nintra-abdominal pressure (6 mmHg) using 3–0 polydioxanone (PDS\nFIGURE 2\nFascia lata transposition to repair recurrent perineal hernia in a dog. (A) Right perineal region showing inflamed tissues with clear, viscous, and slippery\ndischarge, (B) previously implanted synthetic mesh (m), (C) herniated rectum with sacculation (Rs), (D) fascia lata (FL) being dissected, (E) fascia lata flap\n(FLF) before being passed through the subcutaneous tunnel (white arrow), (F) completed fascia lata transposition. Black arrows; ischial table, bf; biceps\nfemoris.\n\nPark et al.\b\n10.3389/fvets.2026.1858804\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nII, Ethicon, USA), taking care not to penetrate the colonic lumen\n(Figures 3C–F). The abdominal wall was sutured first, followed by the\nseromuscular layer of the colon. Sutures were placed in a single row\nfrom tail to head, with more than 5 mm between each suture. One\nsuture (the third) failed due to tearing of the seromuscular layer of the\ncolon during knot tightening and was reinforced with an additional\npercutaneous anchoring suture. The extracorporeal knot was buried\nsubcutaneously through a deep stab incision in the skin. A mild bleed­\ning point from puncturing a vessel on the serosal surface was easily\nmanaged by compression with a hemostatic agent (Surgicel®,\nEthicon, USA).\nThe patient recovered well, showing normal activity, appetite, uri­\nnation, and defecation. The active drain at the perineal site and the\nfentanyl patch were removed on POD 3 and POD 4, respectively. The\nhighest C-reactive protein (CRP) value was 82 mg/L (reference range:\n0–20 mg/L) on POD 2, which subsided over time. The surgical sites\nappeared normal grossly (Figure 4). Abdominal radiography and\nultrasonography performed on POD 10 and 17 confirmed that the\ncolopexy site remained in its intended position, with no evidence of\ncomplications such as peritonitis, gastrointestinal dysfunction, or\nwound infection. Lactulose administration was discontinued on POD\n20. The patient exhibited no signs of straining, and the feces were of\nnormal length and shape. Rectal palpation revealed no\nabnormalities.\nThe patient remained hospitalized under institutional care after\nsurgery until being adopted on POD 26. During a follow-up interview\nwith the new owner, it was reported that the patient remained clini­\ncally well for 27 months after surgery, with no recurrence of PH or RP,\nand no associated clinical signs.\n3 Discussion\nComplications of perineal herniorrhaphy include wound dehis­\ncence/infection (5–45%), seroma formation, persistent tenesmus\n(2–8%), urinary incontinence (4–8%), fecal incontinence (3–15%),\n\n3 Discussion\nComplications of perineal herniorrhaphy include wound dehis­\ncence/infection (5–45%), seroma formation, persistent tenesmus\n(2–8%), urinary incontinence (4–8%), fecal incontinence (3–15%),\nneuropraxia, and recurrence (0–70%) (2, 5). Recurrence rates are\ninfluenced by various factors, including the surgeon’s experience, the\nchosen surgical method, a history of previous herniorrhaphy, local\ntissue strength, the type and tension of the suture material, predispos­\ning factors, the severity of clinical signs, overactivity, surgical site\ninfection, tenesmus, the development of fecal incontinence, underly­\ning prostatic disease, and castration status (2, 16).\nAlthough PH predominantly occurs in intact males, a definitive\ncorrelation between the disease and gonadal hormones, such as tes­\ntosterone or estradiol, remains unestablished despite suspected hor­\nmonal influences (6). According to Hatch et al., a strong correlation\nexists between neutering status and the reccurrence of PH; specifically,\ndogs that underwent castration prior to PH development faced a 4.4-\nfold increase in recurrence odds, while those neutered concurrently\nwith herniorrhaphy showed a protective effect (17). In contrast,\nFerrari et al. identified no significant relationship between castration\nstatus and recurrence (16). The patient in the present case was a res­\ncued, castrated male dog with no available medical history, presenting\nwith bilateral perineal scars. A synthetic mesh inserted during a previ­\nous surgery was removed from the right side, while a subcutaneous\nforeign body, presumed to be the migrated mesh, was identified in the\nleft perineal region. These findings suggest a chronic and recurrent\ncondition following surgical repair involving bilateral synthetic mesh\nimplantation, regardless of whether castration was performed prior to\nor concurrently with PH repair. The absence of a visible internal obtu­\nrator muscle makes it unclear whether IOMT had been performed at\nthe time of mesh implantation or whether the muscle had simply\n\nThe absence of a visible internal obtu­\nrator muscle makes it unclear whether IOMT had been performed at\nthe time of mesh implantation or whether the muscle had simply\nseverely atrophied. Reported recurrence rates of PH after IOMT alone\nand IOMT combined with mesh implantation are up to 36% and\napproximately 12.5%, respectively (5, 18).\nRegarding the repair of recurrent PH, an appositional suture tech­\nnique could be used occasionally; however, transposition of peri-\nlesional or distant tissue, or implantation of synthetic or biomaterials,\nis usually required due to the massive atrophy or defect of the pelvic\nmusculature. In this case of recurrent PH, conventional appositional\nFIGURE 3\nLaparoscopic colopexy for the treatment of rectal prolapse subsequent to perineal herniorrhaphy in a dog. (A) Exploration of the caudal abdomen and\ndilated distal colon (*), (B) peritoneal opening at the site to be pexied, (C) distal colon being retracted cranially and lifted up to the abdominal wall,\n(D) seromuscular bite of the colonic wall, (E) colopexy with two caudal sutures, (F) completed colopexy. The black arrows in panel (A,C) indicate the\nsame location in the distal colon.\n\nPark et al.\b\n10.3389/fvets.2026.1858804\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nherniorrhaphy and IOMT were not viable due to insufficient pelvic\ndiaphragm muscles for proper apposition or suture anchoring. Thus,\na salvage procedure was necessary, and FLT was selected over semi­\ntendinosus muscle transposition, as fascia closure provides a stronger\nrepair compared to muscular closure, while utilizing fresh, robust\ntissue from a distant site rather than the compromised tissue at the\nlesion (4, 14). The ischiatic periosteum and sacrotuberous ligament\nwere also used for anchoring the flap. The use of synthetic mesh was\navoided due to the local inflammatory condition of the previous surgi­\ncal site (though bacterial culture was omitted) and the surgeon’s pref­\nerence for utilizing native tissue.\nA fascia lata flap, rather than a graft, was created while preserving\nits muscular attachment to provide robust lateral fixation for the her­\nniorrhaphy. If the flap length had been insufficient to cover the hernia\ndefect extending to the medioventral aspect, its attachment could have\nbeen transected and used as a free graft. This technique has been\nreported to be easy to perform and handle, requiring minimal tissue\ndissection compared with semitendinosus muscle transposition, and\nresulting in low postoperative lameness scores (4, 14). Generally, fascia\nlata is large enough to cover hernia defects even in large-breed dogs,\nand in FLT, the base of the flap can provide lateral and ventral support\nsuccessfully without suture placement (4, 14). Moreover, it does not\nform a nidus for persistent infection, and its autogenous nature mini­\nmizes the risks of immunologic foreign body reaction and associated\nsurgical site infection, which are major causes of reconstruction failure\nin synthetic mesh implantation (4, 14). Autogenous tunica vaginalis\ngrafting might be considered, but it was not possible in this case as this\ntechnique can be performed only in intact male dogs (19).\nDespite this being the authors’ first experience with FLT, harvest­\ning and handling the flap were accomplished with ease. However, the\n\nDespite this being the authors’ first experience with FLT, harvest­\ning and handling the flap were accomplished with ease.However, the\nrelatively narrow and caudally exposed surgical field positioned the\nskin incision near the cranial border of the biceps femoris muscle,\nslightly hindering the cranial dissection of the flap. A more cranially\nlocated and longer skin incision would have facilitated the flap cre­\nation. Therefore, it is recommended that the sterile surgical field be\nsufficiently wide, covering at least the entire surface of the lateral\nthigh. Alternatively, employing a hanging-leg technique during aseptic\npreparation and draping to expose the entire right hindlimb could also\nenhance the freedom of flap harvesting.\nThe characteristics of FLT, as mentioned above, make this tech­\nnique suitable for recurrent PH (4), as observed in this case.\nMeanwhile, Sutthiprapa et al. suggested a sacroischial sling technique\nas another practical, low-complication alternative for recurrent PH\nthat is cost-effective, straightforward, and comparable to IOMT while\noffering a shorter surgical time (7).\nLarge rectal sacculation and diverticulum—defined as rectal\ndilatation with or without disruption of the rectal muscularis layer\nleading to herniation of the mucosa through the defect—are\ncommon in chronic PH. These persistent lesions may induce\nexcessive straining and tenesmus to expel impacted feces, ulti­\nmately resulting in PH repair failure and recurrence (2, 6–8, 20).\nIn a recent study (16), histological evaluation of the rectal wall in\npatients with PH revealed significantly higher inflammation and\nfibrosis scores. Furthermore, PH recurrence was significantly\nassociated with both a high rectal dilatation score—measured\nboth immediately postoperatively and 60 days after herniorrha­\nphy—and a high fibrosis score. Accordingly, some surgeons rec­\nommend plication or lateral resection with inverting sutures prior\nto herniorrhaphy to prevent recurrence (6, 8, 9, 20). Intriguingly,\nin contrast to the aforementioned inability to defecate, another\nstudy also reported that patients developing postoperative fecal\n\nIntriguingly,\nin contrast to the aforementioned inability to defecate, another\nstudy also reported that patients developing postoperative fecal\nincontinence exhibited 3.4 times higher odds of PH recur­\nrence (17).\nTwo previous studies using fascia lata for 21 complicated PHs in\n18 dogs reported no recurrence of PH or development of RP during a\nmean follow-up of 5.8 months (n = 12 dogs, fascia lata graft for 15\nPHs) and 24 months (n = 6 dogs, FLT for 6 PHs) (4, 14). Recurrent\nPH was identified in five among them, and rectal diseases included 14\nrectal sacculations, 1 rectal deviation, and 3 rectal herniations.\nAlthough 2 dogs showed transient, manually reducible anal mucosal\nprolapses postoperatively, none of the 18 dogs required colopexy, cys­\ntopexy, or vasopexy. Accordingly, in the present patient, only hernior­\nrhaphy using FLT was initially performed and colopexy was not\ncarried out.\nHowever, an acute RP developed on the night of FLT, even\nthough the patient did not show marked signs of straining. In a\nprevious study, colopexy, although ineffective in reducing rectal\nsacculation or diverticulum, restored the linear tubular structure\nof the colorectum and reduced rectal diameter, thereby decreasing\nfecal accumulation in the dilated rectum and consequently lower­\ning the pressure on the pelvic diaphragm (5). Therefore, preemp­\ntive colopexy is recommended for complicated PH presenting\nFIGURE 4\nPortal sites for laparoscopic colopexy (A) and gross appearance at 3 weeks after fascia lata transposition for recurrent perineal hernia in a dog (B: caudal\nview, C: lateral view, D: dorsal view). a; camera port, b/c; instrumental ports, d; one simple interrupted skin suture to bury percutaneous anchoring\nsuture, dotted line; scar of lateral skin incision to create fascia lata flap.\n\nPark et al.\b\n10.3389/fvets.2026.1858804\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nwith obvious rectal dilatation and recurrent RP (14). The develop­\nment of RP in this case could be attributed to various factors that\naltered intra-pelvic pressure after herniorrhaphy without colo­\npexy. These include the persistently dilated, deviated, and redun­\ndant rectum, despite the restoration of its intra-pelvic position, as\nwell as postoperative discomfort or pain. Following the recon­\nstruction of the pelvic diaphragm, the pressure previously dissi­\npated through the hernia defect could no longer be released,\nsubsequently leading to the RP. This necessitated surgical fixation\nto prevent further rectal expulsion driven by the increased intra-\npelvic pressure. In terms of managing postoperative discomfort,\nimplementing epidural anesthesia may serve as a valuable adjunc­\ntive strategy.\nWhile a temporary purse-string suture alone might have been suf­\nficient, a colopexy was chosen considering the complicated chronic\ncondition of recurrent PH with rectal sacculation as previously\ndescribed (20). Additionally, the patient was planned for adoption\npost-discharge, necessitating a more definitive treatment. When colo­\npexy was performed alone, without excision or plication of the rectal\nsacculation, laparoscopy provided magnified visualization, which\nfacilitated selective incorporation of only the seromuscular layer\nduring colonic suturing in a minimally invasive manner. Lowering the\nintra-abdominal pressure during suturing was helpful in preventing\noverexpansion of the abdominal wall and excessive tension on the\ncolon, particularly during placement of the first suture. Of the four\nsimple interrupted sutures, one failed due to excessive tension caused\nby inadvertent manipulation; however, it was successfully secured\nwith an additional percutaneous suture. To minimize such suture\nbreakage, placing sutures in a cranio-caudal (head-to-tail) direction,\nrather than a caudo-cranial (tail-to-head) direction, could be benefi­\ncial. Conversion to laparotomy was not needed, and postoperative\n\nTo minimize such suture\nbreakage, placing sutures in a cranio-caudal (head-to-tail) direction,\nrather than a caudo-cranial (tail-to-head) direction, could be benefi­\ncial.Conversion to laparotomy was not needed, and postoperative\ncomplications such as colonic perforation were not observed.\nLaparoscopic or laparoscopic-assisted colopexy in dogs and cats\nhas been reported in a limited number of publications (10, 12, 21, 22).\nNotably, significantly lower postoperative serum CRP levels have been\nobserved in laparoscopic colopexy compared to conventional open\ncolopexy in dogs, indicating less tissue inflammation (21).\nConventionally, fixation sutures are recommended to be placed in two\nparallel rows with 5 to 8 sutures in each row, in an interrupted, con­\ntinuous, or horizontal mattress suture pattern. Although laparoscopic\ncolopexy requires skilled intracorporeal suturing, it has been reported\nto achieve stable adhesion at the pexy site on POD 30 in an experi­\nmental study using a total of six sutures arranged in two rows, with\nthree sutures per row (21). However, in two previous reports using\nlaparoscopic approaches, fixation with only 3 to 5 simple interrupted\nsutures in a single row was found to be sufficient (12, 22).\nImplementing five bites of a simple continuous suture pattern using\nbarbed suture material was also effective in three cats, providing a\nstable adhesion (10).\nAs demonstrated in the present case, a combined surgical\napproach including herniorrhaphy and organopexy is highly effec­\ntive for the management of complicated and recurrent\nPH. Although the two procedures were performed separately in\nthis patient, when considering a combined surgical approach from\nthe outset, Brissot et al. recommended a two-stage surgery with\norganopexy preceding herniorrhaphy to empty the perineal space,\nresolve rectal deviation, and facilitate herniorrhaphy (4, 5). This\napproach could benefit the herniorrhaphy procedure by providing\nadvantages such as better anatomical visualization, easier suture\nplacement, a decreased risk of trauma to the anal sphincter and\n\nThis\napproach could benefit the herniorrhaphy procedure by providing\nadvantages such as better anatomical visualization, easier suture\nplacement, a decreased risk of trauma to the anal sphincter and\ncaudal rectal nerves, and a subsequent decrease in postoperative\nfecal incontinence and recurrence rates. In their study of compli­\ncated PH in 41 dogs, IOMT and organopexy, including 41 colo­\npexies, were performed, reporting a mean follow-up of\n26.6 months, a success rate of 92%, and a recurrence rate of 10%;\nall PH relapses occurred within 6 months.\nIn this case report, the combination of FLT and laparoscopic colo­\npexy for recurrent PH complicated by rectal sacculation and subse­\nquent RP demonstrated a favorable outcome, with no recurrence of\nPH or RP observed up to 27 months postoperatively. FLT is an effec­\ntive option for complicated PH characterized by a deficiency in the\npelvic musculature for reconstruction, although the potential develop­\nment of RP should be considered. Consequently, it is strongly recom­\nmended that colopexy be performed, either prior to or concurrently\nwith FLT.\nData availability statement\nThe original contributions presented in the study are included in\nthe article, further inquiries can be directed to the correspond­\ning author.\nEthics statement\nEthical approval was not required for the studies involving ani­\nmals in accordance with the local legislation and institutional require­\nments because this report describes a clinical case that occurred\nduring routine veterinary care. Written informed consent was\nobtained from the participants for the publication of this case report.\nAuthor contributions\nJP: Investigation, Methodology, Writing – original draft, Writing –\nreview & editing. W-JL: Data curation, Writing – review & editing.\nD-HK: Supervision, Writing – review & editing.\nFunding\nThe author(s) declared that financial support was not received for\nthis work and/or its publication.\nConflict of interest\nThe author(s) declared that this work was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\n\nPark et al.\b\n10.3389/fvets.2026.1858804\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nGenerative AI statement\nThe author(s) declared that Generative AI was not used in the\ncreation of this manuscript.\nAny alternative text (alt text) provided alongside figures in this article\nhas been generated by Frontiers with the support of artificial intelligence\nand reasonable efforts have been made to ensure accuracy, including review\nby the authors wherever possible. If you identify any issues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the review­\ners. Any product that may be evaluated in this article, or claim that\nmay be made by its manufacturer, is not guaranteed or endorsed\nby the publisher.\nReferences\n1.\tGill SS, Barstad RD. A review of the surgical management of perineal hernias in dogs.\nJ Am Anim Hosp Assoc. (2018) 54:179–87. doi: 10.5326/JAAHA-MS-6490\n2.\tAronson LR. \"Perineal hernia\". In: Tobias KM and Johnston SA, editors. Veterinary\nSurgery: small animal. St. Louis, MO: Saunders (2012). p. 1589–600.\n3.\tCinti F, Rossanese M, Pisani G. A novel technique to incorporate the sacrotuberous ligament\nin perineal herniorrhaphy in 47 dogs. Vet Surg. (2021) 50:1023–31. doi: 10.1111/vsu.13574\n4.\tGuerios SD, Monteiro JF, da Silva DM. Fascia lata flap to repair perineal hernia in dogs:\na preliminary study. Turk J Vet Anim Sci. (2017) 41:686–91. doi: 10.3906/vet-1703-32\n5.\tBrissot HN, Dupré GP, Bouvy BM. Use of laparotomy in a staged approach for resolu­\ntion of bilateral or complicated perineal hernia in 41 dogs. Vet Surg. (2004) 33:412–21.\ndoi: 10.1111/j.1532-950X.2004.04060.x\n6.\tOzturan YA, Gursel A, Akin I. Combined use of polypropylene mesh and collagen\nsponge in the surgical repair of perineal hernia with rectal diverticulum in a dog. Acta\nVet Hung. (2025) 73:167–71. doi: 10.1556/004.2025.01181\n7.\tSutthiprapa W, Budsayaplakorn P, Koatsang N, Theerapan W, Thengchaisri N.\n\nCombined use of polypropylene mesh and collagen\nsponge in the surgical repair of perineal hernia with rectal diverticulum in a dog.Acta\nVet Hung.(2025) 73:167–71.doi: 10.1556/004.2025.01181\n7.Sutthiprapa W, Budsayaplakorn P, Koatsang N, Theerapan W, Thengchaisri N.\nRetrospective evaluation of surgical outcomes using traditional, internal obturator muscle\nflap, and sacroischial sling technique for canine perineal hernia repair. J Adv Vet Anim\nRes. (2025) 12:1421–9. doi: 10.5455/javar.2025.l1002\n8.\tPekcan Z, Besalti O, Sirin YS, Caliskan M. Clinical and surgical evaluation of perineal hernia\nin dogs: 41 cases. Kafkas Univ Vet Fak Derg. (2010) 16:573–8. doi: 10.9775/KVFD.2009.1149\n9.\tMann FA, Constantinescu GM, Anderson MA. \"Surgical techniques for treatment of\nperineal hernia\". In: Bojrab MJ, Waldron DR and Toombs JP, editors. Current Techniques\nin small animal Surgery. Jackson, WY: Teton NewMedia (2014). p. 569–84.\n10.\tGuadalupi M, Piemontese C, Stabile M, Dizonno R, Staffieri F, Lacitignola L. Total\nlaparoscopic colopexy for the treatment of recurrent rectal prolapses in three cats. Vet Sci.\n(2024) 11:355. doi: 10.3390/vetsci11080355\n11.\tSmeak DD. \"Colopexy\". In: Monnet E and Smeak DD, editors. Gastrointestinal\nSurgical Techniques in small Animals. Hoboken, NJ: John Wiley & Sons (2020). p. 231–3.\n12.\tPark J, Moon C, Kim DH, Lee HB, Jeong SM. Laparoscopic colopexy for recurrent\nrectal prolapse in a Maltese dog. Can Vet J. (2022) 63:593–6.\n13.\tFerreira AP, Oliveira AL, Mostachio GQ, Ferreira JZ, Fernandez S, Souza TFB, et al.\nCranioplasty using autologous fasciae latae graft for nasal bone fracture repair in a dog.\nActa Sci Vet. (2017) 45:1–3.\n14.\tBongartz A, Carofiglio F, Balligand M, Heimann M, Hamaide A. Use of autogenous\nfascia lata graft for perineal herniorrhaphy in dogs. Vet Surg. 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Laparoscopic-assisted colopexy and sterilization in\nmale dogs: short-term results and physiologic consequences. Vet Surg. (2011)\n40:500–8. doi: 10.1111/j.1532-950X.2011.00835.x\n\n---"}
+{"text": "【兽医文献】\n标题: 10.1136_vetreco-2017-000250_Pilot_study_evaluating_the_monitoring_of_canine_di\n\n\b\n1\nMshelbwala FM, et al. Vet Rec Open 2017;4:e000247. doi:10.1136/vetreco-2017-000247\nAbstract\nThis study was carried out to identify the Salmonella\nserotypes causing high mortality in chickens in Lagos,\nOgun and Oyo states, Nigeria. Chickens presented for\npostmortem examination during disease outbreaks\nthat were characterised by high mortality (40 per cent\nto 80 per cent) in poultry farms in the study area were\nexamined from January to December, 2013. Samples of\nthe lungs, heart, liver, spleen, kidneys, proventriculus,\nintestine and caecum were collected from suspected\ncases of salmonellosis, for bacterial culture and\nidentification. Salmonella isolates were confirmed using\nPCR and serotyped using the Kauffman-White scheme.\nTwenty-six day-old pullets were raised to two weeks and\ninoculated orally with 0.2 mL of 1×108 colony forming\nunits of Salmonella Zega identified in the present study\nto determine their pathogenicity, while another 26\nserved as control. The Salmonella serotypes were S\nZega (n=13; 35.14 per cent), Salmonella Kentucky (n=9;\n24.32 per cent), Salmonella Herston (n=6; 16.22 per\ncent), Salmonella Nima (n=4; 10.81 per cent), Salmonella\nTelelkebir (n=3; 8.11 per cent), Salmonella Colindale (n=1;\n2.70 per cent) and Salmonella Tshiongwe (n=1; 2.70\nper cent). Clinical signs in both natural and experimental\ninfections were acute (70 per cent) and chronic (30\nper cent), and included weakness, anorexia, yellowish\ndiarrhoea, pasted vents, somnolescence and mortality,\nwhile gross lesions showed marked pulmonary congestion\nand oedema, necrotic foci in the myocardium; the liver,\nspleen and kidneys were markedly enlarged and had\nsubcapsular multifocal necrosis. There were catarrhal\nproventriculitis and enteritis, and haemorrhagic typhlitis.\nWhile most of the serotypes identified in the present study\nhave been isolated from poultry sources from commercial\nfarms in Nigeria, to the best of the authors' knowledge,\nthey have not been previously reported to cause high\nmortality in chickens in the study area.\nIntroduction\nPoultry farming has contributed to the\neconomy of many countries and accounts\n\nIntroduction\nPoultry farming has contributed to the\neconomy of many countries and accounts\nfor 25 per cent of local meat production in\nNigeria.1 2 However, productivity of poultry is\nfaced with various disease challenges.3 4 Among\nthe poultry diseases, salmonellosis caused by\nSalmonella enterica is one of the most impor-\ntant bacterial diseases posing serious chal-\nlenge to poultry production worldwide.3–5\nSalmonella\nspecies\nare\nGram-negative,\naerobic or facultative anaerobic, motile by\nmeans of peritrichous flagella or non-mo-\ntile and non-spore forming rods.6 7 They\nare divided into serotypes based on the\nlipopolysaccharide (O), flagellar (H) and\nsometimes capsular (vi) antigens.8 There\nare more than 2500 known serotypes.6 7 The\nserotypes that are host-specific to poultry\nare non-motile which are Salmonella Galli-\nnarum and Salmonella Pullorum.9–11 While\nSalmonella Typhi is a host-restricted Salmonella\nserotype which causes enteric fever exclu-\nsively in human beings, Salmonella Enteritidis\nand Salmonella Typhimurium are commonly\nreported to cause disease in both poultry and\nhuman beings, and therefore, are of public\nhealth importance. However, there are many\nother motile Salmonella serotypes that can\ninfect poultry and the distribution of these\nserotypes vary according to geographical loca-\ntion.9 10 12–14 Poultry is the main reservoir for\nthis classic zoonotic foodborne pathogen and\nthis organism can survive for a long time in\nthe environment including poultry houses.15\nAlthough a tentative diagnosis of fowl\ntyphoid can be carried out by considering\nflock history, a clinical sign, mortality and\npathological lesions, a definitive diagnosis by\nisolation and identification of the organism is\nrequired for most cases.9\nSince vaccination is still the best preventive\nmeasure, characterising the different Salmo-\nnella serotypes in the study area will help in\ndeveloping an effective vaccine and provide\na better control programme. Therefore,\nthere is the need to isolate and characterise\nthe different Salmonella field strains to their\nserotypes.\nMotile Salmonella serotypes causing\nhigh mortality in poultry farms in three\nSouth-Western States of Nigeria\n\nTherefore,\nthere is the need to isolate and characterise\nthe different Salmonella field strains to their\nserotypes.Motile Salmonella serotypes causing\nhigh mortality in poultry farms in three\nSouth-Western States of Nigeria\nFakilahyel Musa Mshelbwala,1 Najume Doguwar-Giginya Ibrahim,2\nShehu NaAllah Saidu,3 Aminullah Ajiyobiojo Azeez,1 Paul Akinniyi Akinduti,4\nClara Nna Kwanashie,5 Amina Kinta Fakilahyel Kadiri,3 Maryam Muhammed,6\nIdowu Oluwabunmi Fagbamila,6 Pam Dachung Luka7\nFood/farmed animals\n1Department of Veterinary\nPathology, Federal University of\nAgriculture, Abeokuta, Nigeria\n2Department of Veterinary\nPathology, Ahmadu Bello\nUniversity, Zaria, Nigeria\n3Department of Veterinary\nMedicine, Ahmadu Bello\nUniversity, Zaria, Nigeria\n4Department of Veterinary\nMicrobiology and Parasitology,\nFederal University of Agriculture,\nAbeokuta, Nigeria\n5Department of Veterinary\nMicrobiology, Ahmadu Bello\nUniversity, Zaria, Nigeria\n6Department of Bacteriology,\nSalmonella Research Division,\nNational Veterinary Research\nInstutute, Jos, Plateau State,\nNigeria\n7Department of Biotechnology,\nNational Veterinary Research\nInstitute, Jos, Plateau State,\nNigeria\nCorrespondence to\nDr Fakilahyel Musa Mshelbwala; ​\nmshelbwalafakilahyel@​yahoo.​\ncom\nTo cite: Mshelbwala FM,\nIbrahim ND-G, Saidu SNaA, et al.\nMotile Salmonella serotypes\ncausing high mortality in\npoultry farms in three South-\nWestern States of Nigeria.\nVeterinary Record Open\n2017;4:e000247. doi:10.1136/\nvetreco-2017-000247\nReceived 2 August 2017\nRevised 2 November 2017\nAccepted 11 November 2017\n\nOpen Access\n2\nMshelbwala FM, et al. Vet Rec Open 2017;4:e000247. doi:10.1136/vetreco-2017-000247\nIn the present study, there were reports of outbreaks\nof diseases of unconfirmed aetiology that were charac-\nterised by high mortality in commercial poultry farms\nin the study area. Postmortem examinations on the\ncarcases  suggested avian salmonellosis. The Salmonella\nserotypes that were associated with the high mortality in\ncommercial and backyard poultry farms in Lagos, Ogun\nand Oyo states, within south-western Nigeria, were identi-\nfied, which were not known to cause mortality in chickens\nin the study area. The disease was then reproduced in an\nexperimental trial, following Koch’s postulate, thereby\nconfirming their pathogenicity.\nMaterials and methods\nStudy area\nThe study was conducted in Lagos, Ogun and Oyo states\nwithin south-western Nigeria, which lies between longi-\ntude 30oE and 7oE and latitude 4oN and 9oN in the west of\nthe lower Nigeria and south of the Niger Trough.16\nPostmortem examination\nChickens that died during outbreaks of avian diseases\nin commercial and backyard poultry farms in the study\narea were submitted for postmortem examination in the\nDepartment of Veterinary Pathology, College of Veteri-\nnary Medicine, Federal University of Agriculture, Abeo-\nkuta. The flock sizes of the farms ranged from 5000 to\n160,000 birds. The study was carried out over a period\nof one year (January to December, 2013) during which 8\nto 13 birds from each disease outbreak were examined.\nThe total number of outbreaks, chickens, their ages,\nbreed and type of chickens were recorded according to\nthe type of farm, month of examination and the State\nfrom which they were submitted. In each suspected case\nof salmonellosis outbreak investigated, samples of the\nlungs, heart, liver, spleen, kidneys, proventriculus, small\nintestine, caecum and bile were collected from one bird.\nTwo hundred and seventy samples were collected from 30\ndifferent suspected cases of salmonellosis in commercial\npoultry farms, and 54 from six cases were collected from\nbackyard farms, making a total of 324 samples from 36\n\nTwo hundred and seventy samples were collected from 30\ndifferent suspected cases of salmonellosis in commercial\npoultry farms, and 54 from six cases were collected from\nbackyard farms, making a total of 324 samples from 36\n(out of 55) salmonellosis outbreaks. The samples were\naseptically collected in separate sterile sample bottles,\nfor bacterial culture and identification. In 80 per cent\nof the cases, the process of bacteriology were started on\nthe same day postmortem samples were collected, while\nin 20 per cent, the samples were refrigerated at 4°C for\n24 hours.\nBacterial culture, isolation and Identification\nBacterial culture and identification were carried out in\nthe Department of Veterinary Microbiology and Para-\nsitology of the Federal University of Agriculture, Abeo-\nkuta. Swabs from aseptically sectioned tissue samples\nof the organs and bile collected at postmortem exami-\nnation were taken and separately applied into nutrient\nbroth and buffered peptone water for pre-enrichment,\nand incubated at 37°C for 24 hours. Two mL  of the\nsamples from the pre-enrichment media were inocu-\nlated into 50 mL of Rappaport-Vassiliades broth (Oxoid\nBasingstoke, UK) and onto Tetrathionate glucose broth\n(Oxiod, Basingstoke, UK) following the pre-enrich-\nment for selective enrichment and incubated at 37°C\nfor 24 hours. Using a sterile wire loop, a loopful of each\nincubated broth culture was inoculated onto xylose lysine\ndeoxycholate (XLD) agar (Oxiod, Basingstoke, UK) and\nincubated at 37°C for 24 hours. The plates were examined\nfor typical colonies of Salmonella. The colonies from these\nplates were further subcultured on XLD agar and incu-\nbated at 37°C for 24 hours. The plates from the subcul-\nture were also observed for typical colonies of Salmonella\nas described by Doughlas and others.17 Suspected Salmo-\nnella colonies were inoculated onto MacConkey agar for\npurification.\nThe prevalence of salmonellosis was calculated as\npercentage of isolation of Salmonella serotype from each\norgan.\nPCR procedure\nForty-eight representative samples of the suspected Salmo-\n\nThe prevalence of salmonellosis was calculated as\npercentage of isolation of Salmonella serotype from each\norgan.PCR procedure\nForty-eight representative samples of the suspected Salmo-\nnella isolates from the 36 outbreaks were put on nutrient\nslant, freeze-dried and sent to the National Veterinary\nResearch Institute, Vom, Plateau State for confirmation\nusing conventional PCR as follows:\nOligonucleotide primers\nA set of primer pair 139–141 specific for the invA gene\nfrom Salmonella species was used.18 The primer sequences\nwere as follows: invAf 5-31-​GTG​AAAT​TATC​GCCA​CGTT​\nCGGGCAA (sequence length of 26 bases) and invAr\n5–31–​TCA​TCGC​ACCG​TCAA​AGGAACC\n(sequence\nlength of 22 bases) with amplification product (base\npair) of 284. DNA extraction kit ZR Fungal/Bacterial\nDNA Miniprep, catalogue number D6005 was obtained\ncommercially from Zymo Research, USA. Extraction of\nDNA was done according to the manufacturer’s instruc-\ntions.\nDNA amplification\nPCR amplifications were performed in a final volume\nof 50 µL containing 10 µL of DNA template, 25 uL of 2×\nPCR master mix (x10 buffer, 3 µL of 1.5 Mm MgCl2, 3 µL\neach of 2.50 mM deoxynucleoside triphosphate, 0.2 µL\ncontaining 1.25 unit Taq polymerase), 2 mM forward and\nreverse primers and make up the volume to nuclease-free\nwater. Amplifications were carried out in a GeneAMP\nPCR Applied Biosystem 9700 thermocycler (Thermacy-\ncler Applied Biosystem (AB) 9700, USA) using condi-\ntions described by Galan and others.18 Initial denatura-\ntion was at 94°C for 5 minutes, followed by 35 cycles of\ndenaturation at 94°C for 1 second, annealing at 55°C\nfor 1 second and final extension at 72°C for 7 minutes.\nDNA Amplicons were sized by electrophoresis on 1:2 per\ncent agarose gel containing 5 µg/ml ethidium bromide\nwith 100 bp and 50 bp ladders (GibcoBRL) as molecular\nweight markers as described by various workers.19–23\n\nOpen Access\n3\nMshelbwala FM, et al. Vet Rec Open 2017;4:e000247. doi:10.1136/vetreco-2017-000247\nSalmonella species serotyping\nThirty-seven Salmonella isolates, one from each of 35\noutbreaks and 2 from 1, that were confirmed by PCR to\nbe Salmonella species were inoculated into nutrient agar\nslope, freeze-dried and sent to the World Organization for\nAnimal Health, Italian Reference Laboratory for Salmo-\nnella, Istuto Zooprofilatico Sperimentale Dellevenzie,\nPodava, Italy for serotyping using the Kauffman-White\nscheme.24\nExperimental study\nAn experimental study in line with Koch’s postulate of\nreproducing the disease experimentally and re-isolation\nof putative pathogen in order to demonstrate that the\nserotypes isolated in the present study were the cause of\nthe natural salmonellosis in chicken in the commercial\nand backyard poultry farms was carried out.\nSource of birds and bacteria (S Zega)\nA total of 52 day-old ISA brown chicks were used for the\nexperimental study. They were purchased from a certi-\nfied hatchery (Agritet, Yola, Adamawa State, Nigeria)\nand housed using deep litter management system, in the\nPoultry Pen of the Department of Veterinary Anatomy,\nFaculty of Veterinary Medicine, Ahmadu Bello University\n(ABU) Zaria. The poultry pen was previously cleaned,\ndisinfected with formalin and fumigated with potassium\npermanganate (KMNO4) before the arrival of the birds.\nThe birds were infected at two weeks of age after they\nwere examined to certify being Salmonella-free by taking\ncloacal swab samples from them before infection with S\nZega. The cloacal swabs were cultured in the Department\nof Public Health and Preventive Medicine, Faculty of\nVeterinary Medicine, ABU, Zaria which confirmed that\nthey were Salmonella-free, when no growth of the organ-\nisms was observed from the culture.\nThe S Zega used in this experimental study was among\nother Salmonella serotypes isolated from the natural cases\nof avian salmonellosis in the present study. It was used for\nthe experiment because it was the most predominantly\nisolated serotype.\nStandardisation of the inoculation dose of S Zega\n\nIt was used for\nthe experiment because it was the most predominantly\nisolated serotype.Standardisation of the inoculation dose of S Zega\nThe inoculation dose (colony forming units (cfu)) was\ndetermined using Sensititre nephelometer (TREK Diag-\nnostic Systems, UK) according to the method described\nby Goldman and Green.25 The pure culture of S Zega\nisolated was brought from an 18-hour plate culture.\nSterile wire loop was used to pick two to three colonies\nof the S Zega and emulsified in 50 mL of sterile normal\nsaline and incubated at 37°C for 10 min to allow for\nbacterial growth. Two mL of the suspension containing\nthe bacteria was put in a sterile tube and inserted into\nthe Sensititre nephelometer after calibration, and the\nconcentration of the bacteria was taken from the calibra-\ntion. The procedure was repeated after 10 minutes each\nof incubation at 37°C until a concentration of 1×108 was\nobtained.\nThe birds were grouped into infected (group A) and\nuninfected control (group B) as follows:\nGroup A consisting of 26 birds and each was inoculated\nwith 0. 2 ml of 1×108 cfu of the S Zega orally and were fed\ncommercial poultry feed and administered water (Spring\nWaters Nigeria Limited, Kerang Mangu local government\narea (LGA)) ad libitum.\nGroup B consisting of 26 birds was not innoculated but\nwas fed commercial poultry feed and administered water\n(Spring Waters Nigeria Limited, Kerang Mangu LGA)\nad libitum and was used as a control group.\nAll the birds in the four groups were monitored daily,\nfrom the day of treatment to day 12 post-infection (pi) to\ndetermine the incubation period and clinical signs of the\ndisease. Two birds from each group were sacrificed on\nday zero before infection and continued pi at days 1 pi,\n2 pi, 3 pi, 4 pi, 5 pi, 6pi, 7 pi, 8 pi, 9 pi, 10 pi, 11 pi and\n12 pi and postmortem examinations were carried out on\nthe carcases to determine the development of patholog-\nical lesions according to days pi. Those birds that died\nnaturally from the disease were collected and examined\nat postmortem as for the rest of the birds under the study.\nSwabs from aseptically sectioned visceral organs were\n\nThose birds that died\nnaturally from the disease were collected and examined\nat postmortem as for the rest of the birds under the study.Swabs from aseptically sectioned visceral organs were\ntaken from both infected chicks and uninfected chicks,\nfor bacterial culture and identification to determine the\nday from which S Zega can be isolated.\nBacterial culture and identification of putative pathogen(S\nZega)\nBacterial culture and identification of the putative path-\nogen (S Zega) was carried out using the tissue swabs\ncollected at postmortem, according to the method\ndescribed above.\nResults\nNatural cases of avian salmonellosis in commercial poultry\nfarms\nOne thousand and sixty-four birds from 104 outbreaks of\ndiseases were submitted from commercial poultry farms\nwhile 123 from 14 outbreaks were from backyard farms,\nmaking a total of 1187 birds from 118 outbreaks that were\nexamined. This comprised 748 birds from 72 outbreaks\nsubmitted from Ogun state; 273 from 27 outbreaks from\nLagos state and 166 from 17 outbreaks from Oyo state.\nOut of the total number of birds examined, 547 birds\nfrom 55 outbreaks were diagnosed with salmonellosis.\nOut of the 118 cases of disease outbreaks submitted\nfor postmortem examination during the study period,\n16 representing 13.56 per cent were recorded in birds\nwithin the one to three weeks age bracket. There were 28\noutbreaks representing 23.73 per cent among those birds\nwithin the ages of four to eight weeks. Birds within the\nages of nine weeks and above had the highest number\n(n=74; 62.71 per cent) of outbreaks. The ISA  brown\nbreed had the highest number of outbreaks (n=63; 53.39\nper cent), followed by Nera black (n=35; 29.66 per cent),\nwhile other exotic breeds had 13 representing 11.02 per\n\nOpen Access\n4\nMshelbwala FM, et al. Vet Rec Open 2017;4:e000247. doi:10.1136/vetreco-2017-000247\ncent outbreaks. The local breed had the least number of\noutbreaks (n=7; 5.93). The type of bird which had the\nhighest number of outbreaks were layers (n=66; 55.93\nper cent) followed by broilers (n=27; 11.02 per cent),\nthen pullets and cockerels, (n=13; 11.02 per cent) and\n(n=11; 9.32 per cent), respectively. Breeders had the least\noutbreak (n=1; 0.85 per cent).\nThe highest number of disease outbreaks in chicken in\nwhich carcasses were presented for postmortem examina-\ntion were recorded in January which had 14 representing\n11.86  per cent outbreaks, followed by April (n=13; 11.02\nper cent), May (n=12; 10.17 per cent), June (n=12;\n10.17 per cent), March (11; 9.32 per cent), July (n=10;\n8.47 per cent), October (n=9; 7.63 per cent), November\n(n=9; 7.63 per cent), December (n=8; 6.78 per cent),\nFebruary (n=7; 5.93 per cent), August (n=7; 5.93 per\ncent). The least number of outbreaks was recorded in\nNovember (n=6; 5.08 per cent).\nClinical signs\nThe disease presented with an acute form in most cases\n(n=39; 70 per cent) while in some cases, they showed\nchronic manifestation (n=16; 30 per cent). The clinical\nsigns were in acute salmonellosis weakness, anorexia,\nyellowish diarrhoea, pasted vents, somnolescence and\nmortality of 40 per cent to 80 per cent. The chronic form\nof the disease showed lethargy, yellowish diarrhoea with\npasted vents and severe emaciation.\nPostmortem findings\nThe disease presented with acute (n=39; 70 per cent)\nand chronic (n=16; 30 per cent) pathological changes in\nvisceral organs. In the acute form of the disease, the lungs\nwere severely congested and oedematous; there were\nnecrotic foci on the myocardium. The livers were mark-\nedly enlarged and had multifocal subcapsular necrosis in\nthe visceral and parietal surfaces (Fig 1). The spleen and\nthe kidneys were severely congested and enlarged. The\nproventriculus contained a large amount of mucus and\nthe mucous membrane was hyperaemic. The intestines\ncontained mucus admixed with diarrhoeic faeces and\nthe mucous membranes were necrotic and hyperaemic.\n\nThe\nproventriculus contained a large amount of mucus and\nthe mucous membrane was hyperaemic.The intestines\ncontained mucus admixed with diarrhoeic faeces and\nthe mucous membranes were necrotic and hyperaemic.\nThe mucous membranes of the caeca were necrotic and\nthe lumen contained dark red caecal cores. While in the\nchronic form of the disease, most of the changes observed\nin the acute form have resolved, but the carcases were\nemaciated with pasted vents. There were foci of necrosis\nin the visceral organs.\nPrevalence of Salmonella serotypes\nDuring the period of this study, 36 samples each of the\nlungs, heart, liver, spleen, kidneys, proventriculus, small\nintestine, caecum and bile were collected from 36 (out of\nthe 55) suspected cases of salmonellosis, making a total\nof 324 samples, for bacterial isolation and identification.\nOut of the 36 outbreaks, 21 were from Ogun state, 9 from\nLagos state and 6 from Oyo state (Table 1). Of the 324\nsamples of the visceral organs collected, the lungs showed\nthe highest percentage of isolation (n=36; 86.1 per cent)\nfollowed by the kidneys (n=36; 77.8 per cent) and the bile\n(n=36; 77.8 per cent). The proventriculus had the lowest\npercentage of isolation (n=36; 55.6 per cent) (Table 2).\nThe overall percentage of isolation was 86.1 per cent\n(n=324) and Salmonella species were isolated in all the\nmonths of examination.\nFIG 1:  Liver of a two-week-old pullet with natural\nSalmonella Zega infection showing enlargement with\nmultifocal subcapsular necrosis.\nTABLE 1:  Percentage of isolation of Salmonella species\nfrom chickens submitted from Lagos, Ogun and Oyo states,\nNigeria for postmortem examination in 2013\nState\nNumber of outbreaks\nNumber of isolation (%)\nLagos\n9\n7 (77.8)\nOgun\n21\n20 (95.2)\nOyo\n6\n4 (66.7)\nTotal\n36\n31 (86.1)\nTABLE 2:  Organ-specific prevalence of Salmonella species\nfrom chicken submitted from Lagos, Ogun and Oyo states,\nNigeria for postmortem examination in 2013\nOrgan\nNumber of\nsample\nNumber of\nisolation\nPercentage\nLung\n36\n31\n86.1\nHeart\n36\n23\n63.9\nLiver\n36\n25\n69.4\nSpleen\n36\n24\n66.7\nKidney\n36\n28\n77.8\nProventriculus\n36\n20\n55.6\nIntestine\n36\n24\n66.7\nCaecum\n36\n25\n69.4\nGall bladder (bile)\n36\n28\n77.8\nTotal\n324\n228\n70.4\n\nOpen Access\n5\nMshelbwala FM, et al. Vet Rec Open 2017;4:e000247. doi:10.1136/vetreco-2017-000247\nPCRs\nIn this study, 48 representatives of suspected Salmonella\nisolates from the 36 natural cases of avian salmonel-\nlosis were tested using PCR. Out of the 48 presumed\nSalmonella isolates, 41 were confirmed to be Salmonella\nspecies. The PCR result showed that Salmonella-posi-\ntive samples and the positive control, S Enteritidis\ncorrespond to 284 bp on the ladder of the molecular\nmarker, which is the base pair unique for all Salmonella\nspecies.\nSalmonella serotypes\nThirty-seven Salmonella isolates were serotyped in\nwhich seven serotypes were identified. They included S\nZega, Salmonella Kentucky, Salmonella Herston, Salmo-\nnella  Nima, Salmonella  Telelkebir, Salmonella  Colin-\ndale, and Salmonella  Tshiongwe. The predominant\nserotype was S Zega (n=13; 35.14 per cent) followed\nby S Kentucky (n=9; 24.32 per cent), then S Herston\n(n=6; 16.22 per cent), S Nima (n=4; 10.81 per cent), S\nTelelkebir (n=3; 8.11 per cent), S Colindale (n=1; 2.70\nper cent) and S Tshiongwe (n=1; 2.70 per cent). Out\nof the 37 serotypes, 13.51 per cent were isolated from\nLagos state, 78.38 per cent from Ogun state and 8.11\nper cent from Oyo state. All the S Hertson, S Nima,\nS Telelkebir as well as the S Tshiongwe and S Colindale\nserotypes were isolated from Ogun state (Table  3).\nSome of the S  Zega and S Kentucky were isolated from\nboth commercial and backyard poultry farms whereas\nthe remaining isolates were isolated from commercial\npoultry farms.\nExperimental studies\nClinical signs\nThe chicks in group D (uninfected control) showed no\nclinical signs. The infected chicks showed signs of weak-\nness and anorexia by day four pi while somnolescence\nwere observed from day five pi. Yellowish diarrhoea and\nmortality were recorded from day six pi (Fig 2).\nPostmortem findings\nGross lesions were pulmonary congestion and oedema,\nthe heart had necrotic foci on the myocardium. The liver\nwas markedly enlarged and had multifocal necrosis in the\nvisceral and parietal surfaces from day five pi till the end\nof the experiment (Fig 3). The spleen and kidneys were\n\nThe liver\nwas markedly enlarged and had multifocal necrosis in the\nvisceral and parietal surfaces from day five pi till the end\nof the experiment (Fig 3).The spleen and kidneys were\nTABLE 3:  Salmonella serotypes isolated from chicken\nsubmitted from Lagos, Ogun and Oyo states, Nigeria for\npostmortem examination, in 2013\nSalmonella serotypes\nState\nLagos\nOgun\nOyo\nTotal\nN (%)\nSalmonella\nZega\n2 (40.00)\n9 (31.03)\n2 (66.67)\n13 (35.14)\nSalmonella\nKentucky\n3 (60.00)\n5 (17.24)\n1 (33.33)\n9 (24.32)\nSalmonella\nHerston\n0 (0.00)\n6 (20.69)\n0 (0.00)\n6 (16.22)\nSalmonella\nNima\n0 (0.00)\n4 (13.79)\n0 (0.00)\n4 (10.81)\nSalmonella\nTelelkebir\n0 (0.00)\n3 (10.34)\n0 (0.00)\n3 (2.70)\nSalmonella\nTshiongwe\n0 (0.00)\n1 (3.45)\n0 (0.00)\n1 (2.70)\nSalmonella\nColindale\n0 (0.00)\n1 (3.45)\n0 (0. 00)\n1 (2.70)\nOverall\n5 (13.51)\n29 (78.38)\n3 (8.11)\n37 (100)\nN, number of serotypes\nFIG 2:  Three-week-old chicks infected orally with\nSalmonella Zega showing somnolescence and mortality, six\ndays pi.\nFIG 3:  Liver of a three-week-old chick infected orally with\nSalmonella Zega (left) showing enlargement with multifocal\nnecrosis 10 days postinfection; and normal liver from an\nuninfected chick (right).\n\nOpen Access\n6\nMshelbwala FM, et al. Vet Rec Open 2017;4:e000247. doi:10.1136/vetreco-2017-000247\nmarkedly congested, enlarged with subcapsular foci of\nnecrosis. The proventriculus contained a large amount\nof mucus and the mucous membranes were hyperaemic.\nThe intestines were distended with diarrhoeic faeces\nadmixed with mucus and the mucous membranes were\nhyperaemic, while the caecum contained reddish caecal\ncore and the mucous membranes were hyperaemic.\nBacterial culture and identification\nS Zega was re-isolated from the infected chicks and iden-\ntified as described earlier.\nDiscussion\nMotile Salmonella serotypes causing high mortality in\ncommercial poultry farms in Lagos, Ogun and Oyo\nstates, Nigeria, were identified in the present study, and\nthe pathogenicity of S Zega was confirmed experimen-\ntally. The Salmonella serotypes reported in this study were\nisolated from outbreaks of salmonellosis in all age groups,\nbreeds and types of chickens, with older birds and layers\nbeing the most affected, in both backyard and commer-\ncial poultry farms. Poppe and others26 reported a high\noccurrence of diseases in older birds among commercial\npoultry farms in Canada. The reason may be attributed to\nthe longer time the older birds have stayed on the farms,\npersistent contamination in poultry houses for long\nperiods of time, consecutive generation of chickens that\nare being kept on the farm and laying stress in layers.13 26\nThe low percentage of diseases among young chicks may\nsuggest that the hatcheries where day-olds were procured\nhad strict prevention and control measures. All the seven\nserotypes were associated with the outbreaks in commer-\ncial poultry farms while only S Zega and S Kentucky were\nisolated from outbreaks in both backyard and commer-\ncial poultry farms. This may probably be due to the fact\nthat they were the most predominant serotypes isolated\nin this study. S Kentucky has been reported to be the most\npredominantly isolated serotype in commercial layer\nfarms in Nigeria.27 It may also be due to the less number\nof samples collected from backyard farms compared with\n\nS Kentucky has been reported to be the most\npredominantly isolated serotype in commercial layer\nfarms in Nigeria.27 It may also be due to the less number\nof samples collected from backyard farms compared with\nthe ones from commercial poultry farms. Similarly, the\nisolation of S Zega and S Kentucky in all the two states\nmay be attributed to their predominance in the study area\nas seen in this study; whereas the isolation of all the seven\nserotypes in Ogun state may be due to the larger number\nof samples collected from the state. S Zega was the most\ncommonly isolated serotype in the present study and has\nbeen isolated from human sources in Zaire.28 However, S\nKentucky was the second most frequently isolated serotype\nand has been reported to infect chickens worldwide.29\nS Kentucky was also reported to be the most common\nserotype isolated from litters, dust, faeces, feed and water\nin Nigerian commercial chicken layer farms across the\ncountry.27 In other separate studies, S Herston has been\nreported to be associated with diarrhoeal cases in chil-\ndren from the Niger Republic; and S Nima has caused\nan outbreak of salmonellosis in human beings through\ncontaminated chocolates, in many countries.30 Whereas\nS Colindale was one of the most common Salmonella sero-\ntypes isolated from fresh lettuce in Burkina Faso.31 Barua\nand others32 also reported a high prevalence of motile\nSalmonella in commercial layer farms in Bangladesh.\nHowever, most of these serotypes of Salmonella have not\nbeen reported to cause high mortality in poultry. With\nthe exception of S Kentucky, information appears to be\nscanty on the prevalence of these serotypes on commer-\ncial poultry farms worldwide, and reports in the litera-\nture on their pathogenicity to avian species are very rare.\nThe result was contrary to the expectation that S Galli-\nnarum and S Pullorum may be associated with the natural\ncases, since they are host-specific and highly adapted to\nchickens.33 34 While these serotypes have been reported to\ncause disease in human beings, the mortality most of them\ncause in chickens is unusual. However, motile Salmonella\n\n33 34 While these serotypes have been reported to\ncause disease in human beings, the mortality most of them\ncause in chickens is unusual.However, motile Salmonella\nserotypes other than the ones isolated in this study have\nbeen reported by several workers to be the cause of serious\nmortality and pathological lesions in commercial poultry\nfarms.6 12  35 Foley and others15 and Baumler and others36\nreported that the niche created by the eradication of S\nGallinarum and S Pullorum in commercial poultry farms\nin the USA and Canada are likely to be filled by S Ente-\nritidis, Salmonella Heidelberg and S Kentucky. Ogunleye\nand others (2006) reported that Salmonella Paratyphi,\nmotile Salmonella isolated during a fulminating outbreak\nof salmonellosis on commercial poultry farms in Debiwise\nfarms, Ibadan, Oyo state, Nigeria were highly pathogenic\nin pullets that were experimentally infected. While there\nis paucity of information on the pathogenicity of some of\nthe Salmonella serotypes identified in the present study,\nthe availability of draft genomes of these serotypes have\nearlier been reported.37\nThe isolation of motile Salmonella serotypes from\nthe outbreak of salmonellosis on commercial poultry farms\nis of significant public health concern.15 38 Motile Salmo-\nnella are known to infect a wide range of hosts including\nhuman  beings15 39–42 and account for approximately\n93.8  per cent  of million human cases of gastroenteritis\nand 155,000 of deaths worldwide each year.38 Contact with\ninfected poultry and consumption of contaminated poultry\nproducts, especially undercooked meat and raw eggs are\nimportant sources of infection by this major foodborne\npathogen worldwide.15 39–42\nThe clinical signs and gross lesions observed in the\nexperimental study were similar to the ones described in\nthe natural cases in the present study and are consistent\nwith those earlier reported in avian salmonellosis.4 5 9 The\nfinding on the clinical signs and pathology of S Zega in\nexperimentally infected pullets following Koch’s postu-\nlate of reproducing the natural disease in experimental\n\n4 5 9 The\nfinding on the clinical signs and pathology of S Zega in\nexperimentally infected pullets following Koch’s postu-\nlate of reproducing the natural disease in experimental\ninfection, which was fully established proves its pathoge-\nnicity, and this suggests that the other Salmonella sero-\ntypes identified from the natural cases in the present\nstudy may equally be highly pathogenic in chickens, since\nthey present with similar clinical signs and lesions in the\nnatural cases.\n\nOpen Access\n7\nMshelbwala FM, et al. Vet Rec Open 2017;4:e000247. doi:10.1136/vetreco-2017-000247\nIn conclusion, motile Salmonella serotypes were the\ncause of high mortality in commercial poultry farms.\nAlthough the serotypes identified in the present study\nhave been previously isolated from poultry sources\nfrom commercial poultry farms in Nigeria, to the best\nof the  authors' knowledge, this is the first report on\nthem causing high mortality in chickens in the study\narea.\nAcknowledgements  The authors thank the Department of Salmonella Research,\nNational Veterinary Research Institute, Vom, Plateau State, Nigeria for sending\nthe Salmonella isolates to the World Organization for Animal Health/OIE, Italian\nReference Laboratory for Salmonella, Istituto Zooprofilatico Sperimentale\nDellevenzie, Podava, Italy, where they were serotyped.\nContributors  Conception and design: FMM, ND-GI, SNS. Acquisition of data: FMM,\nAAA. Data Analysis: AKFK. Interpretation of data: FMM, ND-GI, SNS, MM. Drafting\nthe work: FMM, PDL. Revising the work critically: ND-GI, SNS, CNK. Final approval\nof the version to be published: FMM. Postmortem diagnosis: FMM, ND-GI, SNS.\nCulture and isolation of the Salmonella: PAA, FMM, AAA. Confirmation by PCR: PDL.\nParticipation in serotyping work: IOF. Supervision: ND-GI, SNS, CNK, MM.\nCompeting interests  None declared.\nProvenance and peer review  Not commissioned; externally peer reviewed.\nData sharing statement  The prevalence study, the pathological and\nimmunohistochemical studies in both natural and experimental trials are not\nincluded in this article.\nOpen Access  This is an Open Access article distributed in accordance with the\nCreative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which\npermits others to distribute, remix, adapt, build upon this work non-commercially,\nand license their derivative works on different terms, provided the original work is\nproperly cited and the use is non-commercial. See: http://​creativecommons.​org/​\nlicenses/​by-​nc/​4.​0/\n© British Veterinary Association (unless otherwise stated in the text of the article)\n\nSee: http://​creativecommons.​org/​\nlicenses/​by-​nc/​4.​0/\n© British Veterinary Association (unless otherwise stated in the text of the article)\n2017. All rights reserved. No commercial use is permitted unless otherwise\nexpressly granted.\nReferences\n1\t Sonaiya EB, Swan ESJ. Small scale poultry production technical\nguide. Animal Production and Health. Rome Italy:  FAO of United\nNations, 2004:114–5.\n2\t Agbaje M, Davies R, Oyekunle MA, et al. Observation on the\noccurrence and transmission pattern of Salmonella Gallinarum in\ncommercial poultry farms in Ogun State, South Western Nigeria. Afr\nJ Microbiol Res 2010;4:796–800.\n3\t Saidu L, Abdu PA, Umoh JU, et al. Diseases of Nigerian indigenous\nchickens. Bull Anim Health Prod Afr 1994;42:19–23.\n4\t Abdu PA. Pullorum Disease and Fowl typhoid. Abdu PA, ed. 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+{"text": "【兽医文献】\n标题: Avoiding drug residues: a multivariate approach to estimating withdrawal intervals in edible tissues of goats following extra label administration of flunixin meglumine\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nCase Report: Anaesthetic\nmanagement in a canine patient\nwith severe atrioventricular septal\ndefect and pulmonary\nhypertension undergoing\nnon-cardiac surgery\nClaire Nicol *, Karin Kriström  and Vanessa Bettembourg\nEvidensia Södra Animal Hospital Kungens Kurva, Stockholm, Sweden\nIntroduction: A 9-year-old, 6.8-kg intact male Petit Brabançon with a\ncongenital partial atrioventricular septal defect (AVSD) presented for dental\nextractions. Pre-anaesthetic echocardiographic assessment revealed a large\nostium primum atrial septal defect with bidirectional interatrial shunting, severe\nright atrial and ventricular enlargement, abnormal atrioventricular valves with\nsevere tricuspid regurgitation, mild relative pulmonic stenosis, and moderate\npulmonary hypertension (PH). Anaesthetic goals were to minimize increases in\npulmonary vascular resistance (PVR), preserve right ventricular perfusion and\ncardiac output (CO), and avoid alterations in intracardiac shunt dynamics that\ncould promote right-to-left shunting. Premedication consisted of methadone\n[0.2 mg/kg intramuscularly (IM)], lidocaine (1 mg/kg intravenously (IV)) and\nmidazolam (0.3 mg/kg IV). Continuous infusions of lidocaine (30–50 μg/kg/\nmin) and remifentanil (10–30 μg/kg/h) were used as inhalant minimum alveolar\nconcentration (MAC)-sparing analgesic adjuncts. Anaesthesia was induced with\nketamine (1 mg/kg IV) and propofol (2 mg/kg IV) and maintained with sevoflurane\nin oxygen and air. Controlled ventilation targeted normocapnia and the fraction\nof inspired oxygen (FiO₂) was titrated to maintain pulse oximeter (SpO₂) values\nabove 95%, while avoiding unnecessary hyperoxia. A norepinephrine infusion\n(0.2–0.4 μg/kg/min) supported mean arterial pressure (MAP) > 65 mmHg.\nRegional dental nerve blocks supplemented analgesia. Fourteen teeth were\nextracted during a 64-min anaesthetic. Recovery was complicated by transient\nhypoxemia attributed to upper airway obstruction, resolving with oxygen\nsupplementation and nebulized adrenaline.\nOutcome: No arrhythmias or sustained right-to-left shunting occurred. The\n\nRecovery was complicated by transient\nhypoxemia attributed to upper airway obstruction, resolving with oxygen\nsupplementation and nebulized adrenaline.Outcome: No arrhythmias or sustained right-to-left shunting occurred.The\ndog was discharged the same day and returned to normal activity. Survival\nafter the procedure was 14 months before euthanasia for acute respiratory\ndecompensation.\nClinical relevance: This report highlights practical, physiology-guided strategies\nto conduct anaesthesia in non-cardiac procedures in dogs with severe AVSD\nand PH, in hospital settings without advanced equipment such as intra-operative\ntransesophageal echocardiography or direct CO measurements. It also serves as\na reminder not to let the anaesthetic risk in patients with severe cardiac disease\nbe the reason for refraining from quality of life improving dental procedures.\nKEYWORDS\ncardiac anaesthesia, case report, congenital defect, dental procedure, dog\nOPEN ACCESS\nEDITED BY\nCarolina Palacios Jimenez,\nRoyal Veterinary College, United Kingdom\nREVIEWED BY\nFederico Corletto,\nUniversity of Nottingham, United Kingdom\nAlfonso Rodriguez,\nBristol Vet Specialists, United Kingdom\n*CORRESPONDENCE\nClaire Nicol\nClaire.nicol@evidensia.se\nRECEIVED 30 October 2025\nREVISED 12 January 2026\nACCEPTED 13 January 2026\nPUBLISHED 29 January 2026\nCITATION\nNicol C, Kriström K and Bettembourg V (2026)\nCase Report: Anaesthetic management in a\ncanine patient with severe atrioventricular\nseptal defect and pulmonary hypertension\nundergoing non-cardiac surgery.\nFront. Vet. Sci. 13:1736215.\ndoi: 10.3389/fvets.2026.1736215\nCOPYRIGHT\n© 2026 Nicol, Kriström and Bettembourg.\nThis is an open-access article distributed\nunder the terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other forums is\npermitted, provided the original author(s) and\nthe copyright owner(s) are credited and that\nthe original publication in this journal is cited,\nin accordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Case Report\nPUBLISHED  29 January 2026\nDOI  10.3389/fvets.2026.1736215\n\nNicol et al.\b\n10.3389/fvets.2026.1736215\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nIntroduction\nAtrioventricular septal defect is a rare congenital cardiac anomaly\ncaused by incomplete fusion of the endocardial cushions during cardiac\ndevelopment. The defect ranges from partial to complete forms. In\npartial AVSD, incomplete fusion produces an ostium primum atrial\nseptal defect and an atrioventricular valve malformation of variable\nseverity, which may include a common atrioventricular (AV) junction\nwith two functional orifices. In complete AVSD, the deficiency also\ninvolves the inlet portion of the ventricular septum and results in a single\ncommon AV valve overlying both atrial and ventricular septal defects (1).\nLarge left-to-right shunts in either form, together with regurgitation\nfrom both AV valve components, increase atrial and ventricular volume,\ncause chamber dilation, and promote pulmonary overcirculation (2).\nShunt magnitude and direction are influenced by ventricular compliance,\nany systemic or pulmonary outflow tract obstruction, and the balance\nbetween pulmonary and systemic vascular resistance (SVR).\nDevelopmental factors such as myocardial stiffness, pulmonary vascular\nmaturation, and cardiopulmonary reserve may further modify shunt\nvolume, particularly in young animals. Over time, as pulmonary vascular\nresistance rises, shunting may become bidirectional or reverse to right-\nto-left once pulmonary pressures approach systemic values (3–6).\nShunt flow also varies during the cardiac cycle. Left-to-right flow\ntypically predominates during late ventricular systole and early diastole,\nand minor reversal of shunt flow may be seen in early systole and early\ndiastole (7, 8). These cyclical fluctuations contribute to right-sided\nvolume loading and may influence peri-anaesthetic haemodynamic\nstability.\nPulmonary hypertension increases right ventricle (RV) afterload,\npredisposing to RV hypertrophy (concentric and eccentric), dilatation,\nand hypoxemia, as well as arrhythmia and right-sided heart failure (9,\n10). Pulmonary hypertension is defined in human medicine as a mean\n\nPulmonary hypertension is defined in human medicine as a mean\npulmonary artery pressure (mPAP) > 20 mmHg at rest by right heart\ncatheterization, a threshold recently lowered from the former cutoff of\n≥ 25 mmHg (11, 12). In veterinary medicine, invasive measurements\nare rarely performed. Instead, the American College of Veterinary\nInternal Medicine consensus defines PH as an increase in pulmonary\narterial pressure sufficient to cause hemodynamic consequences and\nrecommends integrating clinical signs with Doppler echocardiographic\nestimates, primarily tricuspid regurgitation (TR) velocity, and\nsupportive echocardiographic indicators of right-sided pressure\noverload to assess PH probability and severity. A TR velocity ≥ 3.4 m/s,\nroughly corresponding to a systolic pulmonary arterial pressure of\n~50 mmHg assuming normal right-atrial pressure, is considered a key\nthreshold supporting a diagnosis of clinically relevant PH (13).\nTo the authors knowledge, there are no previous case reports\nspecifically focusing on the anaesthetic management in canine patients\nwith significant AVSD and PH undergoing non-cardiac surgery. We\ndescribe the peri-operative concerns and a successful anaesthetic\nbased on pathophysiological considerations in such a patient.\nCase description\nSignalment and history\nA 9-year-old, intact male Petit Brabançon (6.8 kg, body condition\nscore: 7/9) with congenital AVSD and a common atrium diagnosed at\n6 months of age, presented for dental extractions due to painful,\nadvanced periodontal disease. Comorbidities included brachycephalic\nobstructive airway syndrome, chronic large-bowel disease managed\nwith diet, and hyperadrenocorticism that remained incompletely\ncontrolled (polydipsia and polyuria) despite medical therapy. There\nwas no history of regurgitation related to brachycephaly. The owners\nreported increased respiratory effort and exertional cyanosis. The\ncurrent medications were pimobendan (0.3 mg/kg orally every 12 h)\nand trilostane (3 mg/kg orally every 24 h). Pimobendan was\nadministered the morning of anaesthesia.\nPre-anaesthetic evaluation\n\nThe owners\nreported increased respiratory effort and exertional cyanosis.The\ncurrent medications were pimobendan (0.3 mg/kg orally every 12 h)\nand trilostane (3 mg/kg orally every 24 h).Pimobendan was\nadministered the morning of anaesthesia.Pre-anaesthetic evaluation\nOn examination, the dog was quiet, alert and responsive. The\nmucous membranes were pink at rest with mild cyanosis when\nexcited. The respiratory rate was 30/min with mild effort and there\nwere increased respiratory sounds on auscultation. A grade 2/6\nsystolic murmur was auscultated on the right side and the heart rate\nwas 110/min with regular rhythm. Pulse quality was satisfactory. No\narrhythmias could be seen on lead-II electrocardiogram (ECG).\nOscillometric blood pressure was 140/80 mmHg (MAP 100 mmHg).\nPre-operative blood work included a cephalic venous blood gas\nsample, packed cell volume and total solids (Table 1). Oxygen was\nprovided via face mask during blood sampling. Full blood work\nincluding biochemistry and haematology had been done a month\nprior. These were within normal limits except for moderately elevated\nphosphate and liver enzymes.\nEchocardiographic examination\nThe echocardiographic examination was performed by a board-\ncertified specialist in cardiology (KK). The dog was conscious and\nTABLE 1  Venous blood sample results from a dog with severe\natrioventricular septal defect and pulmonary hypertension before\nundergoing anaesthesia.\npH\n7.33\n7.32–7.4\npCO2\n54.9\n39.8–46.2 mmHg\npO2\n60.3\n35–45 mmHg\nSO2\n89.5\n50–60%\nHCO3\n29.1\n18–26 mmol/L\nBase excess\n3.2\n–5 - –1 mmol/L\nNa+\n155\n140–159 mmol/L\nK+\n3.8\n3.5–5.5 mmol/L\niCa2+\n1.24\n1.12–1.42 mmol/L\nCl-\n109\n105–115 mmol/L\nGlucose\n6.0\n3.9–6.1 mmol/L\nLactate\n3.2\n0–1.5 mmol/L\nPacked cell volume\n(PCV)\n42\n43.3–59.3%\nTotal solids (TS)\n7.2\n6–7.5 g/dL\nHCO3, bicarbonate; pCO2, partial pressure of carbon dioxide; pO2, partial pressure of\noxygen; SO2, oxygen saturation.\n\nNicol et al.\b\n10.3389/fvets.2026.1736215\nFrontiers in Veterinary Science\n03\nfrontiersin.org\ngently\nrestrained\nin\nlateral\nrecumbency.\nTransthoracic\nechocardiography was performed using an ultrasound unit\nequipped with 5.0–9.2 MHz phased-array transducers (EPIQ CVX;\nPhilips Ultrasound, Bothell, WA, USA). The examination included\ntwo-dimensional (2D), M-mode, and color flow Doppler imaging\nand was conducted as previously described (14). A simultaneous\nECG tracing was recorded during the examination, showing normal\nsinus rhythm. A large interatrial communication consistent with an\nostium primum was visualized. A common AV valve with two\nseparate orifices was present (Figure 1). The interventricular septum\nwas abnormal, but a thin membrane, appearing to separate the\nventricles, was present. Severe dilation of the right atrium and right\nventricle was observed, with mild systolic and diastolic flattening of\nthe interventricular septum. Right ventricular systolic function was\nsubjectively reduced. The left ventricle was small, with a left\nventricular internal diameter in diastole (LVIDd) normalized to\nbody weight of 0.9 (15). The caudal vena cava was moderately\ndilated with reduced collapsibility. Bidirectional shunting was\nobserved across the interatrial communication on agitated-saline\ncontrast study [maximal velocity 0.8 meter per second (m/s)]. No\nflow was identified between the ventricles on color Doppler,\nexcluding a ventricular septal defect. Turbulent flow with increased\nvelocity was noted across the pulmonic valve (maximal velocity\n2.2 m/s). No structural abnormalities were identified in the\npulmonary artery, indicating that the pulmonic stenosis was\nrelative. Severe tricuspid and moderate mitral regurgitation were\npresent, with maximal velocities of 3.8 m/s (tricuspid) and 4.8 m/s\n(mitral). Based on the tricuspid regurgitation velocity in\ncombination with right-sided structural abnormalities, moderate\npulmonary hypertension was suspected. Thoracic radiographs\nrevealed severe cardiomegaly, generalized interstitial pulmonary\npattern, and hepatomegaly compressing the lungs.\nAnaesthetic management\n\nThoracic radiographs\nrevealed severe cardiomegaly, generalized interstitial pulmonary\npattern, and hepatomegaly compressing the lungs.Anaesthetic management\nFood was withheld for 6 h prior to anaesthesia. A 22 gauge\nintravenous cannula was placed in the left cephalic vein. He was\npremedicated with methadone (0.2 mg/kg IM), and midazolam\n(0.3 mg/kg IV). Approximately ten minutes before anaesthesia\ninduction, a bolus of lidocaine at 1 mg/kg IV was given, followed by a\ncontinuous lidocaine infusion (30 μg/kg/min). Lidocaine and\nremifentanil (10 μg/kg/h) infusions were used to provide adjunct\nanalgesia and reduce inhalant requirements and were subsequently\ntitrated intra-operatively to 50 μg/kg/min and 30 μg/kg/h, respectively.\nRemifentanil was chosen due to its rapid titratability. Anaesthesia was\ninduced with ketamine (1 mg/kg IV) and propofol (2 mg/kg IV)\nfollowing 3 min of pre-oxygenation via face-mask. Because of the\nbreed-related increased risk of regurgitation and aspiration, the head\nwas elevated during the induction process. The trachea was intubated\nwith a 4.5 mm ID PVC endotracheal tube with some difficulty due to\nan elongated soft palate.\nAnaesthesia was maintained with sevoflurane (end-tidal\nconcentration: 1.1–1.4%) delivered in an oxygen/air mixture via a circle\nbreathing system (GE Carestation 650), with FiO₂ titrated to maintain\nSpO₂ > 95%. During spontaneous breathing, the dog hypoventilated\n(end-tidal CO2 (EtCO₂) 57 mmHg), likely due to opioid administration,\nbrachycephaly, and reduced thoracic compliance associated with\nhepatomegaly and interstitial lung disease. Pressure-controlled\nventilation was instituted to avoid hypercapnia, using inspiratory\nFIGURE 1\nRight parasternal long axis four-chamber echocardiographic view on a dog with severe atrioventricular septal defect and pulmonary hypertension.\nShowing eccentric right ventricular hypertrophy, right atrial enlargement, and a large ostium primum defect (*). Arrows show the abnormal\natrioventricular valves. LV, left ventricle; RV, right ventricle.\n\nNicol et al.\b\n10.3389/fvets.2026.1736215\nFrontiers in Veterinary Science\n04\nfrontiersin.org\npressures of approximately 10 cmH₂O, positive end-expiratory pressure\n(PEEP) of 4 cmH₂O, a respiratory rate of 12–23 breaths/min and an\ninspiration:expiration ratio of 1:2.5 to maintain EtCO₂ within\n35–45 mmHg, with care taken to limit excessive intrathoracic\npressures. Just prior to initiating mechanical ventilation a transient\nepisode of hypoxaemia (SpO2 91%) was seen which resolved with\nincreased FiO₂ (0.6 to 0.9) and improved ventilation.\nNorepinephrine (0.2–0.4 μg/kg/min) was infused to maintain\nMAP above 65 mmHg. Bilateral maxillary nerve blocks were\nperformed using bupivacaine (0.3 mg/kg total; 0.2 mL per site) via a\nsubzygomatic approach to provide regional analgesia. Multiparameter\nmonitoring (GE B125M) included ECG, pulse oximetry, capnography,\ninspired/expired gas analysis, spirometry, oscillometric blood\npressure, as well as Doppler blood pressure and rectal temperature.\nPoint-of-care transthoracic Doppler ultrasound was available.\nHowever, the short episode of hypoxemia seen, resolved rapidly with\ninitiation of mechanical ventilation and increased FiO₂, and its\ntransient nature did not raise concern for right-to-left shunting\nwarranting ultrasonographic confirmation. Invasive arterial blood\npressure monitoring was unfortunately unavailable due to accidental\ndislodgement of the arterial cannula during positioning of the dog.\nIsotonic crystalloids (1–2 mL/kg/h) were administered, and total fluid\nvolume including infusions did not exceed 3 mL/kg/h.\nThe procedure lasted 64 min. Fourteen teeth were extracted. No\narrhythmia or signs of sustained right-to-left shunting occurred. Mild\nhypotension (MAP 55 mmHg) responded to norepinephrine titration.\nEarly intra-operative tachycardia (peak 150/min, baseline 100/min)\nassociated with inadequate anaesthetic depth resolved after increasing\nthe vaporizer setting and adjusting remifentanil and lidocaine\ninfusions. End-tidal sevoflurane remained between 1.1–1.4% with the\nsupport of MAC-sparing infusions and effective regional anaesthesia.\n\nEnd-tidal sevoflurane remained between 1.1–1.4% with the\nsupport of MAC-sparing infusions and effective regional anaesthesia.\nAnalgesic infusions were tapered and stopped prior to emergence.\nTemperature was maintained using active warming, and normothermia\nwas preserved. Core temperature at the end of anaesthesia was 37.8 °C.\nPost-operative analgesia consisted of robenacoxib (2 mg/kg\nsubcutaneously). Pain scoring every 2 h using the Glasgow Composite\nPain Scale revealed no need for rescue analgesia. Due to elongated soft\npalate and stenotic nares, the endotracheal tube remained in place for\n15 min during recovery until the dog no longer tolerated it. Mild post-\nextubation hypoxemia (SpO₂ 90%) resolved with mask oxygen\nsupplementation and nebulized adrenaline. Apart from the brief\nhypoxemia in recovery, no other events related to brachycephaly such\nas regurgitation or increased vagal tone were seen. The dog recovered\nuneventfully in the intensive care unit and was monitored\ncontinuously with multiparameter monitoring over 4 h without\nrespiratory compromise or cardiovascular instability.\nThe owner reported normal behavior that evening and improved\ncomfort within 48 h. The dog survived a further 14 months before\neuthanasia due to acute respiratory distress with cyanosis.\nDiscussion\nThis case illustrates a pragmatic, physiology-guided anaesthetic\nfor a non-cardiac procedure in a dog with a severe AVSD and PH. The\nlarge septal defect posed a risk of decreased CO, right sided heart\nfailure and hypoxemia. Other anaesthetic considerations potentially\naffecting ventilation included obesity, hyperadrenocortisicm with\nhepatomegaly, and suspected interstitial lung disease. The dog was\nalso brachycephalic which meant there was an increased risk of\nregurgitation, increased vagal tone, difficult intubation and\nhypoxemia. Our aims were to preserve RV perfusion and CO, avoiding\nincreases in PVR, and minimising inhalant requirements using\nmultimodal analgesia and regional anaesthesia to support\nSVR and CO.\nDuring anaesthesia, a failing RV is vulnerable to decreases in\n\nOur aims were to preserve RV perfusion and CO, avoiding\nincreases in PVR, and minimising inhalant requirements using\nmultimodal analgesia and regional anaesthesia to support\nSVR and CO.During anaesthesia, a failing RV is vulnerable to decreases in\ncoronary perfusion pressure. A recommendation from human\nmedicine is to maintain a MAP of 65–70 mmHg to preserve RV\ncoronary perfusion (16). However, coronary perfusion is more\ncomplex than maintaining a fixed MAP target. Coronary blood flow\nis determined by multiple interacting factors, including myocardial\noxygen demand, intrinsic coronary autoregulation, heart rate (which\nmodulates diastolic filling time), and autonomic nervous system tone\n(17). Increases in oxygen consumption, tachycardia, or sympathetic\nactivation can significantly reduce effective RV perfusion despite an\napparently adequate MAP. The systolic function of the failing RV is\nmarkedly sensitive to changes in afterload thus even minor increases\nin PVR can cause large decreases in stroke volume (16).\nMost inhalation anaesthetics cause dose-dependent vasodilation\nand reductions in SVR, which can unfavourably alter shunt\ndynamics and compromise CO (2). To combat vasodilation,\nvasopressors are often needed. Norepinephrine was chosen due to\nits vasopressor and ionotropic properties, and its minimal\nchronotropic effect. This allowed preservation of coronary perfusion\npressure without inducing tachycardia-associated reductions in\ndiastolic perfusion time (18).\nIn patients with intracardiac shunts and PH, the balance between\nSVR and PVR is central to determining shunt magnitude and\ndirection. If SVR rises disproportionately relative to PVR, blood may\npreferentially shunt toward the right heart, exacerbating RV volume\noverload and failure. Conversely, if SVR falls below PVR, right to left\nshunting may occur and impaired oxygen delivery may follow.\nAnaesthetic management therefore aimed at preventing adverse\neffects on shunt physiology by maintaining MAP around 65–70 mmHg\nand avoiding factors that would increase PVR (2, 8, 16).\n\nAnaesthetic management therefore aimed at preventing adverse\neffects on shunt physiology by maintaining MAP around 65–70 mmHg\nand avoiding factors that would increase PVR (2, 8, 16).\nFactors known to raise PVR such as hypothermia, hypercapnia,\nhypoxaemia, high mean airway pressure, acidosis and noxious\nstimulation were prevented (3, 8). Active warming was provided\nthroughout the procedure, and normothermia was maintained.\nControlled mechanical ventilation was initiated when hypercapnia\nwas seen. Pressure-controlled ventilation was preferred over volume-\ncontrolled ventilation because it allows more predictable limitation of\npeak and mean airway pressures. In contrast, volume-controlled\nventilation may generate injuriously high airway pressures in the\npresence of reduced pulmonary compliance, increasing intrathoracic\npressure, impairing preload, and elevating PVR (19). Although PEEP\ncan improve oxygenation, excessive levels may increase PVR and\nreduce venous return; therefore, the lowest PEEP compatible with\nadequate oxygenation was selected (16).\nThe FiO₂ was titrated to achieve a SpO₂ of ~95% to support\noxygen delivery while minimising potential absorption atelectasis and\ndecreased coronary perfusion as well as hyperoxia-induced V/Q\nmaldistribution\ndue\nto\ndepressing\nhypoxic\npulmonary\nvasoconstriction (20–22). Readiness to increase FiO₂ in case of\nhypoxaemia, while correcting reversible contributors (airway\nobstruction, secretions, positioning), was crucial.\n\nNicol et al.\b\n10.3389/fvets.2026.1736215\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nIn our effort to minimize inhalant dose and to dampen\nsympathetic surges from nociception, we combined systemic analgesic\ninfusions with locoregional techniques to improve haemodynamic\nstability and limit PVR triggers (23, 24). To further preserve\ncardiovascular stability, we aimed to reduce the required dose of each\ninduction agent by co-inducing anaesthesia with ketamine and\npropofol. Ketamine has been reported to increase PVR under some\nconditions. However, contemporary perioperative data suggest any\nPVR effect typically is small and may be offset by ketamine’s\nsympathetic support of SVR and RV contractile performance (25, 26).\nIn addition to afterload management, preload was considered. A\nhypertrophied and pressure-loaded RV is highly preload dependent,\nand reductions in venous return can rapidly reduce CO. Fluid therapy\nwas administered cautiously with the aim to maintain preload without\ncausing RV volume overload or worsening tricuspid regurgitation (16).\nWe avoided α₂-agonists and acepromazine for premedication\nbecause their haemodynamic effects (pulmonary vasoconstriction/\nbradycardia and systemic vasodilation, respectively) can be\ndisadvantageous in patients with PH and intracardiac shunt\nphysiology (18). However, in human medicine, dexmedetomidine\nhas been used successfully and without increased complications in\npatients with atrial septal defect (ASD) (27). Of the case reports the\nauthors found for cardiac corrective surgery in dogs with ASD,\nthere were no reports of dexmedetomidine being used. The\nanaesthetic protocols mostly comprised of premedication with an\nopioid, fentanyl and lidocaine infusions throughout the procedure,\ninduction with etomidate, propofol, alfaxalone and ketamine, and\nco-induction with midazolam. Both inhalant anaesthesia and total\nintravenous anaesthesia with propofol were described (28–32).\nAlthough total intravenous anaesthesia can limit systemic\nvasodilation, we elected to maintain anaesthesia with a low\nconcentration of inhalant to facilitate rapid depth adjustments and\n\nAlthough total intravenous anaesthesia can limit systemic\nvasodilation, we elected to maintain anaesthesia with a low\nconcentration of inhalant to facilitate rapid depth adjustments and\na controlled recovery in a brachycephalic patient, meanwhile\nMAC-sparing infusions and vasopressor support were used to blunt\ninhalant-related hypotension.\nIntra-operative monitoring emphasized trend recognition and rapid\nintervention. Invasive arterial pressure monitoring would have been\nideal for continuous beat-to-beat assessment, but due to dislodgement\nof the arterial cannula, we relied on combined non-invasive blood\npressure modalities, capnography, spirometry and gas analysis to guide\ntherapy. Where available, intra-operative echocardiography and arterial\nblood gases would further refine management by quantifying shunt\nbehavior, oxygenation and acid–base status.\nThe management of this case has its limitations. Invasive\nhaemodynamic measurements and direct quantification of shunt\nfraction were not obtained, and protocol generalization should be\nmade cautiously. Nevertheless, the approach anchored in maintaining\nMAP and SVR, preventing PVR surges, and reducing inhalant\nrequirements with multimodal analgesia and regional techniques, is\ntransferable to similar patients, also in settings without advanced\nimaging or inhaled pulmonary vasodilators.\nPractical takeaways: treat dogs with significant atrial\ncommunications and PH as RV-fragile. Maintain MAP and avoid\ntachyarrhythmias and ischaemia, prevent PVR surges by ensuring\noxygenation, normocapnia, normothermia, adequate analgesia, and\ncautious ventilator pressures. Usage of MAC-sparing strategies and\nregional blocks to limit vasodilation from inhalants can be\nbeneficial.\nData availability statement\nThe original contributions presented in the study are included in\nthe article/supplementary material, further inquiries can be directed\nto the corresponding author/s.\nEthics statement\nWritten informed consent was obtained from the participant/\npatient(s) for the publication of this case report.\nAuthor contributions\nCN: Writing – original draft, Writing – review & editing. KK:\n\nEthics statement\nWritten informed consent was obtained from the participant/\npatient(s) for the publication of this case report.Author contributions\nCN: Writing – original draft, Writing – review & editing.KK:\nWriting – review & editing, Writing – original draft. VB: Writing –\noriginal draft, Writing – review & editing.\nFunding\nThe author(s) declared that financial support was received for this\nwork and/or its publication. We thank the IVC Evidensia Research\nGroup for their generous contribution covering publishing fees.\nAcknowledgments\nWe extend our sincere gratitude to the owner for granting\npermission to share this case. We also thank the clinical teams in\ndentistry and cardiology for their close collaboration and invaluable\ncontributions to the management of this patient.\nConflict of interest\nThe author(s) declared that this work was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declared that Generative AI was not used in the\ncreation of this manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. Any product\nthat may be evaluated in this article, or claim that may be made by its\nmanufacturer, is not guaranteed or endorsed by the publisher.\n\nNicol et al.\b\n10.3389/fvets.2026.1736215\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nReferences\n1.\tBussadori C. Textbook of cardiovascular medicine in dogs and cats. Milan: Edra\nPublishing; 2023. p. 221–229.\n2.\tAkiyama M, Tanaka R, Maruo K, Yamane Y. Surgical correction of a partial\natrioventricular septal defect with a ventricular septal defect in a dog. J Am Anim Hosp\nAssoc. 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+{"text": "【兽医文献】\n标题: Prevalence of Trypanosoma cruzi infection in dogs in the United States: a systematic review and meta-analysis of observational studies (2000–2024)\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nEffects of neutral detergent fiber\nlevels on apparent nutrient\ndigestibility and intestinal\nmicrobiota composition and\nfunction in forest musk deer\nQindan Dai 1†, Yu He 2†, Jie Wu 1, Lei Zhou 1, Guimei Jiang 1* and\nFeng Chen 1\n1 Sichuan Institute for Drug Control (Sichuan Testing Center of Medical Devices, Sichuan Institute of\nMusk Deer Breeding), Chengdu, China, 2 Chengdu Century Investment Co., Ltd., Chengdu, China\nThis experiment was conducted to explore the effects of different neutral\ndetergent fiber (NDF) levels on nutrient apparent digestibility and intestinal\nmicrobiota composition and function in adult male forest musk deer (FMD)\n(Moschus berezovskii). A total of 18 adult male forest musk deer (FMD) (aged\n4–10 years) with an initial average body weight of 7.09 ± 0.82 kg were selected\nand randomly divided into three groups with different NDF levels: L: 21.60%, M:\n25.14%, and H: 28.47%. The FMD were used in a 50-day trial. The results showed\nthat the apparent digestibility of NDF and acid detergent fiber (ADF) first increased\nand then decreased as NDF levels rose, with the M group showing the highest\ndigestibility (p < 0.05). The H group exhibited significantly higher (p < 0.05) Chao1\nand ACE indices compared to the L group. In addition, at the phylum level (the\nrelative abundance > 0.5%), no significant differences were observed among the\nthree groups, except for Mycoplasmatota, which showed higher (p < 0.05) relative\nabundance in the M group compared to the L group. At the genus level (the relative\nabundance > 1%), the three groups did not change (p > 0.05) significantly. In the\nKEGG function analysis, differentially expressed genes were primarily enriched\nin pathways related to organismal systems and human diseases. In the CAZy\nfunctional analysis, significant differences (p < 0.05) were observed in glycoside\nhydrolases (GHs) and carbohydrate-binding modules (CBMs), with the M group\nshowing clear enrichment in fiber-degrading enzymes. Overall, the M group\ndemonstrated superior NDF apparent digestibility and enhanced fiber degradation\n\n05) were observed in glycoside\nhydrolases (GHs) and carbohydrate-binding modules (CBMs), with the M group\nshowing clear enrichment in fiber-degrading enzymes.Overall, the M group\ndemonstrated superior NDF apparent digestibility and enhanced fiber degradation\ncapacity. Therefore, a dietary NDF level of approximately 25% is recommended\nas optimal for adult male FMD.\nKEYWORDS\nforest musk deer, neutral detergent fiber, nutrient digestibility, metagenome, gut\nmicroflora\n1 Introduction\nThe forest musk deer (FMD) (Moschus berezovskii), the smallest species within the\nMoschus genus, is a ruminant under first-class protection in China (1). The musk secreted\nby male forest musk deer (FMD) holds great economic and social value, particularly in\nthe production of high-end perfumes and traditional Asian medicine (2, 3). However, due\nto long-term illegal hunting and habitat fragmentation over the past 70 years—especially\nduring the 1980s—the number of FMD has sharply declined (4). Although China initiated\nOPEN ACCESS\nEDITED BY\nRavikanthreddy Poonooru,\nUniversity of Missouri, United States\nREVIEWED BY\nYujia Tian,\nTianjin Agricultural University, China\nMoyosore Joseph Adegbeye,\nUniversity of Africa, Toru-Orua, Nigeria\n*CORRESPONDENCE\nGuimei Jiang\nlg.xnz.hy1@163.com\n†These authors have contributed equally to\nthis work\nRECEIVED 02 July 2025\nACCEPTED 13 August 2025\nPUBLISHED 11 September 2025\nCITATION\nDai Q, He Y, Wu J, Zhou L, Jiang G and\nChen F (2025) Effects of neutral detergent\nfiber levels on apparent nutrient digestibility\nand intestinal microbiota composition and\nfunction in forest musk deer.\nFront. Vet. Sci. 12:1658189.\ndoi: 10.3389/fvets.2025.1658189\nCOPYRIGHT\n© 2025 Dai, He, Wu, Zhou, Jiang and Chen.\nThis is an open-access article distributed\nunder the terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other forums is\npermitted, provided the original author(s) and\nthe copyright owner(s) are credited and that\nthe original publication in this journal is cited,\nin accordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\n\nNo use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Original Research\nPUBLISHED  11 September 2025\nDOI  10.3389/fvets.2025.1658189\n\nDai et al.\b\n10.3389/fvets.2025.1658189\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nan artificial musk deer farming program in 1958, more than\n60 years later (5), the captive breeding of FMD continues to face\nsubstantial challenges due to the species’ physiological constraints.\nFor example, their heightened susceptibility to stress potentially\ncontributes to elevated captive morbidity (35 ~ 62%) and mortality\nrates (18 ~ 30%), primarily due to gastrointestinal and respiratory\ndiseases (6). Additionally, there are critical knowledge gaps\nregarding the nutritional requirements and feeding standards of\nFMD. This complex issue has resulted in markedly slower progress\nin breeding technology compared to other ungulate species (cattle,\nsheep, and deer, among others). Current husbandry practices for\nFMD remain largely experience-based, lacking scientific validation\nregarding optimal dietary composition. The nutritional adequacy\nof diets for captive FMD remains uncertain, potentially posing the\nrisks of both deficiency and excess. Generally, wild FMD exhibit\nseasonal dietary diversity through selective browsing of varied\nplant species, but captive FMD are typically fed a restricted diet\nconsisting primarily of concentrates formulated from conventional\nruminant feedstuffs, supplemented with a limited variety of leaves\nand vegetables obtained through manual collection. However, this\nfeeding approach lacks established nutritional standards, making\nit difficult to determine whether it meets the nutritional\nrequirements or increases the body’s burden for FMD. Meanwhile,\nthe nutritional requirements of FMD cannot be fully based on\nthose of other ruminants because of special feeding ecology. This\nnutritional paradigm gap may contribute to the high incidence of\ndigestive disorders in captive FMD, and appropriate nutritional\nprovision is critical for the physiological maintenance and growth\nof captive FMD.\nOptimal nutritional levels are essential for establishing stable\ngut microbial communities, which, in turn, promote host health.\nGong et  al. (7), through a comprehensive analysis of gut\n\nOptimal nutritional levels are essential for establishing stable\ngut microbial communities, which, in turn, promote host health.Gong et  al.(7), through a comprehensive analysis of gut\nmicrobiome composition, determined that growing male FMD\nrequire a dietary protein level of 13.37%. Even so, reports on the\nnutritional requirements of FMD remain scarce. Neutral detergent\nfiber (NDF) serves as a critical nutritional indicator in ruminant\nfeeding systems; suitable NDF levels are essential for ruminal\nhealth maintenance, production performance, high feed intake,\nand digestibility (8, 9). Given the stringent conservation policies\ngoverning FMD husbandry in China—which strictly prohibit any\ninvasive procedures that may cause harm or stress—this study\nadopted non-invasive fecal sampling to evaluate the effects of\nvarying NDF levels, using nutrient digestibility and fecal\nmicrobiome profiling indices. Nutrient digestibility coefficients\nserve as fundamental indicators of feeding efficiency in FMD,\ndirectly reflecting the animal’s capacity to utilize dietary\ncomponents. The gut microbiome serves as a critical interface\nbetween diet and host physiology, with particular relevance for\nFMD conservation, and is frequently studied in forest musk deer\nresearch.\nFurthermore,\nfecal\nmicrobial\nprofiles\nprovide\nmultidimensional insights into health status, immune competence,\nnutritional adaptation, and evolutionary fitness (10, 11). Therefore,\nthis study systematically evaluated the effects of dietary NDF levels\non nutrient apparent digestibility and the structure and function\nof intestinal microbiota in FMD. The findings recommend the\nappropriate NDF levels for the diet of adult male FMD to provide\na valuable reference for scientifically formulating their\nnutritional plan.\n2 Materials and methods\n2.1 Ethics committee approval\nAll experimental procedures involving animals were reviewed and\napproved by the Institutional Animal Care and Use Committee at the\nSichuan Institute of Musk Deer Breeding, Chengdu, Sichuan, China\n(SCYS-E2024002). All studies were conducted in accordance with the\n\nAll studies were conducted in accordance with the\nguidelines outlined in the Ethical Treatment of Experimental Animals\nof China.\n2.2 Experimental design and animals\nThis study was conducted at the Musk Deer Farm of the Sichuan\nInstitute of Musk Deer Breeding, located in Marcon County, Aba\nTibetan and Qiang Autonomous Prefecture, Sichuan Province, China.\nA total of 18 adult male FMD (years 4–10) with similar body\nconditions (weighing 7.09 ± 0.82 kg) were selected. These FMD were\nrandomly divided into three groups, with six replicates per group and\none FMD in each replicate. The experiment lasted for 50 days. All\nanimals were housed individually, and their sheds were thoroughly\ncleaned before the experiment.\n2.3 Diet and feeding management\nIn this experiment, each FMD was fed the total mixed ration\n(TMR) at 9:00 a.m. and 16:00 p.m. every day. Considering the\nphysiological activity patterns of FMD (crepuscular habits), only\n20–30% of the total daily feed ration was provided in the morning,\nwith the remainder fed in the afternoon. The TMR consists of dried\nleaves (a mixture of several kinds of leaves), concentrate, and fresh\nvegetables, and the leaves and vegetables were pre-treated by\ncutting them into pieces approximately 1–3 cm in size before\nmixing. The NDF contents of the three TMR were 21.6% (L group),\n25.14% (M group), and 28.47% (H group), respectively. The dietary\ncomposition of the experimental groups is presented in Table 1. All\nFMD were provided with ration and water ad libitum, and the\ncleanliness of the enclosure was maintained.\n2.4 Data and sample collection\nOn day 16 of the experimental period, daily feed provision and\nresidues were recorded for each FMD, with refusal samples collected\ndaily. Then, the samples were dried at 105 °C for 4 h in a forced-air\noven, equilibrated in a desiccator, and weighed for DMI analysis.\nA total of 0.5 kg of the TMR from the different experimental\ngroups was collected, packed into ziplock bags, marked, and stored in\nthe refrigerator at −20 °C for later use. After mixing the dietary\nsamples collected from each group, samples of appropriate portions\n\nA total of 0.5 kg of the TMR from the different experimental\ngroups was collected, packed into ziplock bags, marked, and stored in\nthe refrigerator at −20 °C for later use.After mixing the dietary\nsamples collected from each group, samples of appropriate portions\nwere obtained and dried to a constant weight in an oven at 105 °C. The\ndried samples were then ground using a pulverizer and passed\nthrough a 40-mesh sieve for further analysis.\nDuring days 43–49, fresh feces from all FMD were collected\ninto ziplock bags by the total fecal collection method for the\ndigestibility test. Fecal samples were collected and pooled based on\n\nDai et al.\b\n10.3389/fvets.2025.1658189\nFrontiers in Veterinary Science\n03\nfrontiersin.org\na per-FMD individual basis. A representative 10% aliquot was \nobtained from the thoroughly mixed fecal matter and homogenized \nwith a 10% sulfuric acid solution at a ratio of 10 mL acid per 100 g \nof the fecal sample. These samples were then stored with the \nresidual fecal samples at −20 °C for subsequent determination of \napparent nutrient digestibility.\nOn the final experimental day, fresh fecal samples were \ncollected prior to morning feeding, which were immediately snap-\nfrozen in liquid nitrogen and stored at −80 °C for subsequent \nmetagenomic analysis.\n2.5 Nutrient digestibility and digestive \nenergy\nThe feed and fecal samples were dried at 105 °C to a constant \nweight to determine dry matter (DM) content for subsequent \nanalysis. Crude protein (CP) was determined using a Kjeldahl \napparatus (KDN-2008, Shanghai Xianjian, China). NDF and acid \ndetergent fiber (ADF) contents were analyzed using a fiber analyzer \n(ANKOM-A2000i, Ankom Technology, United  States). Ether \nextract (EE) was determined using a fat analyzer (SZC-C, Shanghai \nXianjian, China). Gross energy (GE) and fecal energy (FE) were \nmeasured using an oxygen bomb calorimeter (Parr6400, Parr \nInstrument Company, Champaign, IL, United States). Nutrient \napparent digestibility and digestive energy (DE) were calculated \nusing the following formulas:\n\n( )\n(\n)\n(\n)\nApparent digestibility %\nNutrient in feed\nNutrient in feces / Nutrient in feed\n100\n=\n−\n×\n\n(\n)\n(\n)\n(\n)\nDigestive energy MJ/ kg\nIntake gross energy GE\nFecal energy FE\n=\n−\n2.6 DNA extraction and high-throughput\nsequencing\nThe 18 samples collected were sent to Rhonin Bioscience Co., Ltd.\n(Chengdu, Sichuan, China) for metagenomic sequencing and\nfunctional analysis. Microbial genomic DNA was extracted from each\nsample using a PowerFecalTM Fecal DNA Kit (MOBIO, Carlsbad,\nCA, United States) following the manufacturer’s instructions. The\nsample gDNA was purified using Zymo Research BIOMICS DNA\nMicroprep Kit (Cat# D4301, United  States). The quality of the\nextracted DNA was assessed using fluorescence quantitative PCR and\nagarose gel electrophoresis. After quality control, the DNA was\nutilized to construct sequencing libraries with the TruSeq™ DNA\nPCR-Free Sample Prep Kit (Illumina, Inc., San Diego, CA,\nUnited  States) following the manufacturer’s protocols. Second-\ngeneration sequencing (Next-Generation Sequencing, NGS) was\nperformed using the PE150 sequencing method on the Illumina\nNovaSeq 6,000 (Illumina, Inc., San Diego, CA, United States). The\nobtained data underwent quality control and were used for\nbioinformatics analysis.\n2.7 Taxonomy profiling\nRaw data were preprocessed using Trimmomatic (version 0.36)\n(12) to obtain high-quality sequences. Kraken2 was used for\ntaxonomic annotation of each sequence (13), and species counts\nwere compiled to construct an operational taxonomic unit (OTU)\ntable. Using R software (version 4.4.2), the abundance data were\nstatistically analyzed at taxonomic levels, which culminated in\nspecies composition and abundance distribution results. α diversity\nindices, including Shannon, Simpson, Chao1, and ACE, were\ncalculated using the “Vegan” package (version 2.6.2) in R software\n(version 4.4.2) to evaluate species diversity and richness within each\nsample’s gut microbiota (14). β diversity was assessed using Bray–\nCurtis distance algorithms with the “Vegan” package in R software\n(version 4.4.2) to obtain distance matrices for principal coordinates\nanalysis (PCoA) (15). The adonis function was used to perform\nnon-parametric multivariate analysis of variance (PERMANOVA)\n\n4.2) to obtain distance matrices for principal coordinates\nanalysis (PCoA) (15).The adonis function was used to perform\nnon-parametric multivariate analysis of variance (PERMANOVA)\n(16) in order to examine the significance of similarities and\ndifferences in the structure of gut microbial community among the\nsamples and groups.\n2.8 Assembly and gene prediction\nThe clean reads obtained after quality control were used for\nmetagenomic assembly with SPAdes (17), and the resulting contigs\nTABLE 1  Composition and nutritional levels of the experimental diet (DM\nbasis).\nItem\nGroup\nL\nM\nH\nIngredients (%)\nDry leaves\n30\n40\n35\nAlfalfa meal\n1\n6\n20\nSucculent feeda\n19\n14\n15\nConcentrateb\n50\n40\n30\nTotal\n100\n100\n100\nNutrient levelsc\nCP (%)\n17.09\n16.59\n16.23\nNDF (%)\n21.60\n25.14\n28.47\nADF (%)\n13.43\n17.61\n20.42\nEE (%)\n6.24\n6.06\n5.82\nGE (MJ/kg)\n16.46\n16.53\n16.32\nAsh (%)\n9.05\n8.98\n9.34\nCalcium (%)\n1.06\n1.21\n1.33\nPhosphorus (%)\n0.49\n0.43\n0.40\nL = low NDF (21.6%); M = middle NDF (25.14%); H = high NDF (28.47%); CP = crude\nprotein; NDF = neutral detergent fiber; ADF = acid detergent fiber; EE = ether extract;\nGE = gross energy.\naThe succulent feed composition (on a fresh weight basis) consisted of 55% lettuce, 15%\ncarrot, 15% pumpkin, and 15% cabbage.\nbThe concentrate composition (on an air-dry basis) consisted of 57% corn, 19.5% soybean\nmeal, 10% bran, 7% extruded soybean, 2.5% Angel yeast, 1.5% sodium bicarbonate, and 2.5%\npremix.\ncNutrient levels were based on measured values.\n\nDai et al.\b\n10.3389/fvets.2025.1658189\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nwere used to evaluate the assembly effect using QUAST (18). Gene\nprediction was performed on the assembled contigs using Prodigal\n(version 2.6.3) (19), and the predicted nucleic acid sequence and\namino acid sequence of the coding gene were obtained.\nNon-redundant gene clusters were generated using MMseqs2\n(version 13.45111) (20), with the following parameters: identity\n> = 95% and coverage > = 90 as the clustering threshold and the\nlongest sequence selected as the representative sequence. All\nrepresentative sequences formed a non-redundant gene set for\nsubsequent quantitative analysis. The non-redundant gene sets\nobtained were annotated using the KEGG (21, 22) and CAZy (23)\nfunctional databases and were statistically analyzed in combination\nwith the quantitative results of the genes. Linear discriminant\nanalysis effect size (LEfSe) analysis of CAZy families was performed\nusing the LEfSe online platform.1\n2.9 Data analysis\nAll experiment results had at least three replicates and were\nexpressed as mean ± standard error (SEM). ANOVA (SPSS 20.0) was\nused for statistical analysis, and a p-value of < 0.05 was considered\nstatistically significant.\n3 Result\n3.1 Intake, digestibility, and digestive\nenergy\nAs shown in Table 2, the DM digestibility of the L group was\nhigher (p < 0.05) compared to the H group. Moreover, the FMD fed\nwith medium NDF levels had significantly higher (p < 0.05) NDF and\nADF apparent digestibility compared to the H group. Similarly, NDF\ndigestibility initially increased but then declined with increasing NDF\nlevels. Nevertheless, there were no significant differences in DMI,\nOM digestibility, and DE among the three experimental groups\n(p > 0.05).\n1  https://huttenhower.sph.harvard.edu/galaxy/\n3.2 Profiling of the intestinal metagenome\nMetagenome sequencing analysis was performed on the fecal\nsamples obtained from the 18 FMD, and a total of 630,074,687 raw\nreads were obtained. After filtering the host genome data,\n628,138,962 clean reads were obtained, averaging 34,896,609\nper sample.\n\nAfter filtering the host genome data,\n628,138,962 clean reads were obtained, averaging 34,896,609\nper sample.\nA total of 4 kingdoms, 82 phyla, 178 classes, 338 orders, 764\nfamilies, and 3,081 genera were obtained. At the kingdom level, the\nrelative abundance, from highest to lowest, was as follows: Bacteria\n(90.69 ~ 97.74%),\neukaryotes\n(1.24 ~ 3.79%),\narchaea\n(0.57 ~ 4.29%), and viruses (0.07 ~ 0.18%). In addition, no\nsignificant differences were observed among the three treatments\n(p > 0.05) (Figure 1).\n3.3 Comparison of intestinal microbial\ndiversity\nIn the α-diversity analysis results (Figure  2), the Chao1\n(Figure 2A) and ACE (Figure 2B) indices in the H group were higher\n(p < 0.05) than those in the L group. However, no significant difference\n(p > 0.05) for the Shannon (Figure 2C) and Simpson (Figure 2D)\nindices was found among the three groups. Furthermore,\nPERMANOVA of β diversity revealed no significant differences in\nfecal microbial communities among the three groups (p > 0.05),\nalthough a trend toward a difference was observed between the L and\nM groups (p = 0.067) (Figure 3; Table 3).\n3.4 Comparison of intestinal microbiome\nAt the phylum level, the dominant phyla across the three groups\nincluded Bacillota (L = 35.62%; M = 36.16%; H = 33.83%) and\nBacteroidota (L = 28.93%; M = 31.30%; H = 30.69%), followed by\nPseudomonadota\n(L = 16.65%;\nM = 15.42%;\nH = 16.44%),\nActinomycetota (L = 8.08%; M = 5.74%; H = 7.27%), Euryarchaeota\n(L = 2.17%; M = 2.04%; H = 1.43%), and Chordata (L = 1.20%;\nM = 1.77%; H = 1.63%) (Figure 4A; Supplementary Table S1). With\nan increase in the NDF level, the relative abundance of Bacillota and\nBacteroidota first increased and then decreased. There was no\nsignificant difference in microbial relative abundance among the three\nTABLE 2  Effects of the different NDF levels on feed intake, apparent digestibility, and digestible energy.\nItem\nGroup\nP-value\nL\nM\nH\nDMI (g/d)\n405.48 ± 87.58\n482.69 ± 121.35\n410.77 ± 116.95\n0.191\nDM (%)\n73.76 ± 1.31a\n71.19 ± 1.08ab\n70.19 ± 0.57b\n0.079\nOM (%)\n75.85 ± 1.46\n73.97 ± 1.10\n73.14 ± 0.47\n0.237\nCP (%)\n59.50 ± 1.83\n59.96 ± 1.37\n59.94 ± 0.97\n0.969\nNDF (%)\n44.63 ± 1.36ab\n50.18 ± 2.46a\n41.42 ± 1.56b\n0.018\nADF (%)\n45.01 ± 2.03ab\n50.11 ± 2.60a\n\nItem\nGroup\nP-value\nL\nM\nH\nDMI (g/d)\n405.48 ± 87.58\n482.69 ± 121.35\n410.77 ± 116.95\n0.191\nDM (%)\n73.76 ± 1.31a\n71.19 ± 1.08ab\n70.19 ± 0.57b\n0.079\nOM (%)\n75.85 ± 1.46\n73.97 ± 1.10\n73.14 ± 0.47\n0.237\nCP (%)\n59.50 ± 1.83\n59.96 ± 1.37\n59.94 ± 0.97\n0.969\nNDF (%)\n44.63 ± 1.36ab\n50.18 ± 2.46a\n41.42 ± 1.56b\n0.018\nADF (%)\n45.01 ± 2.03ab\n50.11 ± 2.60a\n40.44 ± 1.44b\n0.021\nDE (MJ/kg)\n11.76 ± 0.25\n11.45 ± 0.19\n11.37 ± 0.10\n0.354\nL = low NDF (21.6%); M = middle NDF (25.14%); H = high NDF (28.47%); DMI = dry matter intake; DM = dry matter; OM = organic matter; CP = crude protein; NDF = neutral detergent\nfiber; ADF = acid detergent fiber; DE = digestible energy. Within a row, values with different lowercase letters indicate significant differences among groups (p < 0.05).\n\nDai et al.\b\n10.3389/fvets.2025.1658189\nFrontiers in Veterinary Science\n05\nfrontiersin.org\ngroups (p > 0.05). In addition, the relative abundance of\nMycoplasmatota in the M group was significantly higher compared to\nthe L group (p < 0.05).\nAt the genus level, species with over 1% average relative\nabundance in gut microbial communities among the three groups\nare presented (Figure 4B; Supplementary Table S2). Among these\ngenera, Bacteroides (L = 9.34%; M = 7.97%; H = 8.78%) exhibited the\nhighest relative abundance, followed by Phocaeicola (L = 3.89%;\nM = 4.08%; H = 4.69%) and Paenibacillus (L = 3.90%; M = 4.33%;\nH = 4.59%). However, no significant differences in the relative\nabundance at the genus level were observed among the three\ntreatments (p > 0.05).\n3.5 Functional gene annotation analysis\n3.5.1 KEGG functional annotation\nThe KEGG database was used to annotate the pathways of\ndifferent levels and abundances. A total of 1,048,575 UniGenes were\nannotated, covering 426 pathways across six major categories. The\ncategories ranked by the relative abundance of enriched genes from\nhighest to lowest were as follows (Figure 5): metabolism (64.19%),\ngenetic\ninformation\nprocessing\n(13.81%),\nenvironmental\ninformation processing (8.02%), cellular processes (5.55%),\norganismal systems (3.19%), and human diseases (5.24%). The level\n2 classification results showed that carbohydrate metabolism\n(19.89%) had the highest relative abundance, followed by amino\nacid metabolism (12.48%), energy metabolism (7.17%), replication\nand repair (6.12%), and metabolism of cofactors and vitamins\n(5.31%).\nThe distribution plot of the relative abundance differences in the\nKEGG functional annotation is shown in Figure  6. The relative\nabundance of neurodegenerative disease in the L group was\nsignificantly higher (p < 0.05) than in the M group. In addition, the\nabundances of drug resistance: antineoplastic, infectious disease:\nbacterial, and excretory system in the L group were significantly\nhigher (p < 0.05) than those in the H group. The abundances of\nenvironmental adaptation and neurodegenerative disease were\n\n05) than those in the H group.The abundances of\nenvironmental adaptation and neurodegenerative disease were\nsignificantly lower (p < 0.05) in the M group compared to the H group,\nwhile the abundance of the excretory system was higher than that in\nthe H group (p < 0.05).\n3.5.2 CAZy functional annotation\nThe column chart illustrating the number of carbohydrate-active\nenzyme genes was generated by comparison with the CAZy database.\nAs shown in Figure 7, a total of 269,885 carbohydrate-active enzymes\nwere predicted. Among the six types of enzyme molecules, glycoside\nhydrolases (GHs) accounted for the highest proportion at 48.53%,\nwhile auxiliary oxidoreductases (AAs) accounted for the lowest\nproportion at 0.04%.\nTo further analyze the differences in carbohydrate-active enzymes\ncaused by varying NDF levels, the LEfSe analysis results shown in\nFigure 8 indicated that the LDA scores of seven biomarkers among the\nthree groups were greater than 2. Notably, according to the LDA\nscores, GH177 was significantly enriched in the H group (LDA ≥ 2;\np < 0.05), while GH78, GH120, and GBM5 were enriched in the M\nFIGURE 1\nInfluence of the different NDF levels on relative abundance at the intestinal kingdom level. L = low NDF (21.6%); M = middle NDF (25.14%); H = high\nNDF (28.47%). The “ns” indicates that the difference was not significant (p > 0.05).\n\nDai et al.\b\n10.3389/fvets.2025.1658189\nFrontiers in Veterinary Science\n06\nfrontiersin.org\ngroup (LDA ≥ 2; p < 0.05). In contrast, GH13, GH6, and GH2 showed\nsignificant enrichment in the L group (LDA ≥ 2; p < 0.05).\n4 Discussion\nTo ensure experimental precision, this study employed a TMR\nfeeding system instead of the conventional separate feeding of\nconcentrate and roughage. The palatability of the TMR influenced\nintake patterns, although no statistically significant differences in DMI\nwere observed across the NDF levels, and it is possible that when the\nNDF content exceeds a certain level, it may enhance the animal’s satiety,\nthereby reducing DMI. Notably, the M group exhibited the highest DMI\nvalues, suggesting a potential acceptability for balancing fiber content\nand feed intake. Nutrient digestibility reflects the efficiency of nutrient\nutilization by animals, and high nutrient digestibility is beneficial for\ngrowth performance. Although the L group exhibited higher DM\ndigestibility, its DMI was low. The elevated DM digestibility compared\nto the H group may be attributed to the lower NDF content, which\nallowed rumen microbes—normally adherent to fiber—to preferentially\nferment carbohydrates and proteins, thereby contributing to the higher\nDM digestibility in the L group. Similarly, a study in cattle reported that\nthe low-fiber group exhibited higher DM digestibility but lower NDF\ndigestibility compared to the high-fiber group (24). In addition, the\ndigestibility of NDF showed significant quadratic relationships with\nincreasing dietary NDF levels. The quadratic regression analysis of the\nNDF levels and NDF digestibility showed that the regression equation\nwas Y = − 0.761 × X2 + 37.188 × X − 400.781 (R2 = 0.549). In the\nformula, Y is NDF digestibility and X is the NDF level, and maximum\npredicted NDF digestibility occurred at an NDF level of 24.43%. With\nreference to previous studies in other ruminant species, the dietary NDF\nlevel for Tarim red deer was 37 ~ 47% (25). For growing lambs, an NDF\nlevel of 280 g/kg of ration was suggested (26). In peak-lactation dairy\n\n43%.With\nreference to previous studies in other ruminant species, the dietary NDF\nlevel for Tarim red deer was 37 ~ 47% (25).For growing lambs, an NDF\nlevel of 280 g/kg of ration was suggested (26).In peak-lactation dairy\ncows, a dietary NDF level of 28% was recommended (27). In contrast,\nas a specialized small ruminant with distinct browsing preferences\n(Moschus berezovskii preferentially consumes tender leaves while\navoiding graminoids), the FMD exhibits markedly lower fiber tolerance.\nThis dietary behavior helps explain why the experimentally determined\nNDF level, based on NDF digestibility, was lower compared to\nother herbivores.\nFIGURE 2\nInfluence of different NDF levels on α diversity. (A) Chao1, (B) ACE, (C) Shannon, and (D) Simpson. Different lowercase letters denote significant\ndifferences among groups (p < 0.05). L = low NDF (21.6%); M = middle NDF (25.14%); H = high NDF (28.47%).\n\nDai et al.\b\n10.3389/fvets.2025.1658189\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nThe gastrointestinal microbiome constitutes a fundamental\ncomponent of ruminant physiology, playing a key role in nutrient\ndigestion and utilization, host immunity, intestinal barrier\nmaintenance, and intestinal epithelium differentiation (10, 28).\nIn this study, the α-diversity analysis revealed significantly higher\nChao 1 and ACE indices in the H group compared to the L group,\nand the PERMANOVA revealed a tendency for separation\nbetween the L group and M group, indicating that gut microbial\nrichness progressively increased with elevated dietary NDF levels.\nZhang et al. (29) observed that rumen microbial diversity had a\npositive association with NDF levels. The H group’s diet,\ncharacterized by higher structural carbohydrate content,\npresumably enhanced rumen distension, and this dietary\ncomposition likely led to increased retention of undigested\nstructural carbohydrates in the rumen. The associated ruminal\nmicrobiota, adhering to these fibrous substrates, were\nsubsequently conveyed to the intestinal tract via the digesta\npassage, which may explain the demonstrated elevation in\nmicrobial α diversity. The microbial community was\npredominantly composed of Bacillota and Bacteroidetes, which\nwere the dominant phyla, consistent with previous findings in\nFMD (30, 31). Bacillota, as primary cellulolytic bacteria, degrade\nfiber into short-chain fatty acids (SCFAs) for host utilization,\nwhile Bacteroidetes specialize in carbohydrate and protein\ndegradation, promoting gastrointestinal immune system\ndevelopment (32, 33). Although the differences were not\nstatistically significant, the M group exhibited the highest relative\nabundance of these two predominant bacterial phyla. Evidently,\nMycoplasmatota (formerly Tenericutes) showed significantly\nhigher relative abundance in the M group than in the L group.\nSimilar to our findings, a significant quadratic correlation was\nobserved between physically effective NDF (peNDF1.18) levels and\nTenericutes abundance in the rumen of goats (34). Moreover,\n\nSimilar to our findings, a significant quadratic correlation was\nobserved between physically effective NDF (peNDF1.18) levels and\nTenericutes abundance in the rumen of goats (34).Moreover,\ndietary fiber supplementation was associated with increased\nTenericutes abundance in the porcine cecum (35). As\nFIGURE 3\nInfluence of the different NDF levels on β diversity. L = low NDF (21.6%); M = middle NDF (25.14%); H = high NDF (28.47%).\nTABLE 3  PERMANOVA of the gut microbiota under the different NDF\nlevels.\nGroup\nR2\nP-value\nL vs. M\n0.129\n0.067\nL vs. H\n0.080\n0.554\nM vs. H\n0.069\n0.814\nL vs. M vs. H\n0.122\n0.380\nL = low NDF (21.6%); M = middle NDF (25.14%); H = high NDF (28.47%).\n\nDai et al.\b\n10.3389/fvets.2025.1658189\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nFIGURE 4\nBar charts showing the relative abundance of the intestinal microbiota at the phylum classification level (A) and the genus classification level (B). \nL = low NDF (21.6%); M = middle NDF (25.14%); H = high NDF (28.47%).\nFIGURE 5\nColumn chart of annotation classification based on the KEGG database (Level 1 and Level 2).\n\nDai et al.\b\n10.3389/fvets.2025.1658189\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nFIGURE 6\nDifferential analysis diagram of the KEGG functional annotations (Level 2) under the different NDF levels. L = low NDF (21.6%); M = middle NDF \n(25.14%); H = high NDF (28.47%).\nFIGURE 7\nBar chart showing the number of carbohydrate-active enzyme genes annotated based on the CAZy database.\n\nDai et al.\b\n10.3389/fvets.2025.1658189\nFrontiers in Veterinary Science\n10\nfrontiersin.org\ncell-wall-deficient bacteria, Mycoplasmatota may exhibit unique\ninteractions with the host intestinal epithelium (36). Marine\nstudies further suggest its potential involvement in DNA\nmetabolism (37, 38). However, its ecological role in mammalian\ngut systems remains poorly characterized. Therefore, further\ninvestigation is required to elucidate whether Mycoplasmatota\nparticipates in fiber degradation. In mammalian systems,\nalthough transient dietary alterations provoke immediate\nmicrobial shifts, attainment of a stabilized gut microbiota\nconfiguration generally requires an extended adaptation period\nthat ranges from 30 days to multiple months (39, 40). The\nabsence of intergroup differences at the genus level may reflect\nthe limitation of the 50-day experimental period; however,\nsubsequent metagenomic functional analyses revealed distinct\ncharacteristics among the three groups.\nKEGG pathway analysis demonstrated that metabolism\ndominated the functional potential of the gut microbiota,\nwhereas organismal systems were minimally represented,\nconsistent with previous findings (41). Among the three\ntreatment groups, differences in the KEGG pathways were\nprimarily observed in organismal systems and human disease\ncategories. The significant enrichment of the human disease\npathways in the L group suggested a potential predisposition to\ndisease development or manifestation of inflammatory\nconditions. Interestingly, the H group exhibited environmental\nadaptation enrichment under high NDF levels and low DMI\nconditions. This phenomenon may be attributed to the animal’s\nadaptive self-regulation in response to stress induced by external\nfactors under conditions of reduced total energy intake. A\nprevious study indicated that intestinal barrier disruption and gut\nmicrobiota dysbiosis are associated with reduced activity of\nexcretory system pathways in mouse models (42). Consistently,\nthe enrichment of the excretory system implied that the moderate\ndietary NDF level could enhance digestive and metabolic\n\nConsistently,\nthe enrichment of the excretory system implied that the moderate\ndietary NDF level could enhance digestive and metabolic\nefficiency in FMD, potentially optimizing digestion and waste\nexcretion processes. LEfSe analysis based on the CAZy database\nidentified differential microbial biomarkers across the three\ntreatment groups, with the majority belonging to GHs. All known\nagar-degrading bacteria possess at least one conserved GH117\nenzyme, which is essential for their polysaccharide utilization\ncapabilities (43). GH78 has been demonstrated to hydrolyze ester\nbonds between lignin and polysaccharides in plant cell walls, and\nit exhibits robust α-L-rhamnosidase activity, which is implicated\nin the degradation of hemicellulose and pectin chains (44).\nGH120 functions as a β-xylosidase, catalyzing the hydrolysis of\nxylooligosaccharides into monomeric xylose units (45, 46).\nCarbohydrate-binding modules (CBMs) primarily facilitate\nsubstrate conversion, with CBM5 specifically acting as a\nchitinase-binding module that enhances chitinase activity on\nfermentative substrates (47). The GH13 family includes starch-\ndebranching enzymes that specifically and efficiently hydrolyze\nα-1,6-glycosidic bonds at starch branching points, thereby\nenhancing starch utilization efficiency (48). GH6 comprises\nglucanases and cellobiohydrolases, constituting a specialized\nenzyme family involved in cellulose degradation (49). GH2 plays\na pivotal role in oligosaccharide degradation (50). These results\nindicate that the L group was primarily involved in degrading\nnon-structural carbohydrates (e.g., starch and oligosaccharides),\nwhereas the M group predominantly targeted the fibrous\ncomponents of plant cell walls, which aligns with the high NDF\ndigestibility observed in this study.\n5 Conclusion\nBased on the apparent nutrient digestibility results, an NDF level\nof 24.43% is recommended for optimal fiber degradation in FMD. Gut\nmicrobiome structure and function analysis further demonstrated that\nthe M group (NDF level of 25.14%) exhibited superior digestive\n\n43% is recommended for optimal fiber degradation in FMD.Gut\nmicrobiome structure and function analysis further demonstrated that\nthe M group (NDF level of 25.14%) exhibited superior digestive\nsystem performance, with higher enzymatic activity for structural\ncarbohydrate degradation. Therefore, we recommend maintaining\ndietary NDF at approximately 25% for adult male FMD under the\nconditions of this experiment.\nData availability statement\nRaw sequencing reads have been deposited into the National\nCenter for Biotechnology Information Sequence Read Archive (SRA)\ndatabase (accession number: PRJNA1266873).\nFIGURE 8\nLEfSe analysis bar chart showing the differences in the carbohydrate-active enzymes of the intestinal microbiota. L = low NDF (21.6%); M = middle NDF\n(25.14%); H = high NDF (28.47%).\n\nDai et al.\b\n10.3389/fvets.2025.1658189\nFrontiers in Veterinary Science\n11\nfrontiersin.org\nEthics statement\nThe animal study was approved by Sichuan Institute for Drug\nControl (Sichuan Testing Center of Medical Devices, Sichuan Institute\nof Musk Deer Breeding). The study was conducted in accordance with\nthe local legislation and institutional requirements.\nAuthor contributions\nQD: Investigation, Writing – review & editing, Writing – original\ndraft, Conceptualization, Visualization. YH: Writing  – review &\nediting, Investigation, Visualization. JW: Validation, Investigation,\nWriting – review & editing. LZ: Investigation, Writing – review &\nediting. GJ: Data curation, Project administration, Conceptualization,\nSupervision, Writing – review & editing, Funding acquisition. FC:\nWriting – review & editing, Investigation.\nFunding\nThe author(s) declare that financial support was received for\nthe research and/or publication of this article. This research was\nfinancially supported by integration and promotion of healthy and\nhigh-efficiency feed formulation technology for the breeding of\nforest musk deer (2024ZHYS0013).\nConflict of interest\nYH was employed by Chengdu Century Investment Co., Ltd.\nThe remaining authors declare that the research was conducted in\nthe absence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declare that no Gen AI was used in the creation of\nthis manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. 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+{"text": "【兽医文献】\n标题: An overview of the benefits of animal-assisted interventions in medical and therapeutic contexts for human health: cognitive mechanisms, sensory perception and welfare considerations\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nCase Report: Severe wound\nformation following intratumoral\ntigilanol tiglate treatment\nresulting in limb amputation in a\n10-year-old male dog\nLiam Kitson , Jeffrey J. Runge * and Mathew Shay\nDepartment of Surgery, Guardian Veterinary Specialists, Brewster, New York, NY, United States\nTigilanol tiglate (TT; Stelfonta) is a novel intratumoral injection approved for the\ntreatment of non-metastatic canine mast cell tumors (MCTs). Although the major­\nity of reported adverse effects are localized and self-limiting, this case describes\nsevere systemic and wound complications in a 10-year-old male Vizsla treated\nwith a single 2 mL intratumoral injection for a subcutaneous mast cell tumor on\nthe left lateral hock. Within 24 h, the dog developed vomiting, diarrhea, hypovo­\nlemic shock, and pancreatitis, requiring hospitalization and intensive supportive\ncare. The cause of these systemic complications could not be definitively linked\nto Stelfonta, as multiple alternative diagnoses were also possible. Local tissue\nnecrosis and wound infection subsequently progressed despite repeated surgi­\ncal debridement, open wound management, and antibiotic therapy, culminat­\ning in distal limb necrosis and coxofemoral amputation 8 weeks post-injection.\nHistopathology confirmed proximal cicatrix formation, necrosuppurative inflam­\nmation, and fibrosis without residual neoplasia. This case highlights that while TT\ncan be effective for local tumor control, clinicians must recognize the potential\nfor rare but severe localized complications that may require surgical intervention.\nKEYWORDS\ncanine (dog), case report, Stelfonta, systemic adverse effects, tigilanol tiglate\n1 Introduction\nMast cell tumors (MCTs) are the third most common type of tumor and the most common\nmalignant skin tumor in dogs (1). They arise from bone marrow-derived mast cells, which\nnormally participate in immune defense and tissue repair through the release of inflammatory\nmediators such as histamine, heparin, and proteases. These masses can show highly variable\n\nThey arise from bone marrow-derived mast cells, which\nnormally participate in immune defense and tissue repair through the release of inflammatory\nmediators such as histamine, heparin, and proteases.These masses can show highly variable\nbehavior, ranging from benign lesions to aggressive, metastatic disease. Prognosis is influenced\nby factors such as histologic grade, tumor location, and clinical stage, making thorough staging\nand careful management and treatment essential (2). Treatment of canine mast cell tumors\ntypically involves surgical excision, with radiation therapy, chemotherapy, and tyrosine kinase\ninhibitors such as toceranib or masitinib used in cases with incomplete margins, high-grade\ndisease, or metastasis.\nTigilanol tiglate (TT; Stelfonta) is a novel small-molecule agent labeled for use in veterinary\nmedicine for the local treatment of non-metastatic cutaneous mast cell tumors located any­\nwhere on the body and non-metastatic subcutaneous MCTs at or distal to the elbow or hock.\nThe drug is administered via direct intratumoral injection, with the dosage based on the\nTYPE  Case Report\nPUBLISHED  16 April 2026\nDOI  10.3389/fvets.2026.1757258\nOPEN ACCESS\nEDITED BY\nNarda Gail Robinson,\nCuraCore Integrative Medicine and\nEducation Center, United States\nREVIEWED BY\nPaul Reddell,\nQBiotics Group Limited, Australia\nSakurako Neo,\nAzabu University, Japan\nTiago Ferreira,\nState University of Ceará, Brazil\n*CORRESPONDENCE\nJeffrey J. Runge\njeffreyjrunge@gmail.com\nRECEIVED 30 November 2025\nREVISED 26 February 2026\nACCEPTED 27 February 2026\nPUBLISHED 16 April 2026\nCITATION\nKitson L, Runge JJ and Shay M (2026)\nCase Report: Severe wound formation\nfollowing intratumoral tigilanol tiglate\ntreatment resulting in limb amputation\nin a 10-year-old male dog.\nFront. Vet. Sci. 13:1757258.\ndoi: 10.3389/fvets.2026.1757258\nCOPYRIGHT\n© 2026 Kitson, Runge and Shay. This is\nan open-access article distributed under\nthe terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other\nforums is permitted, provided the\noriginal author(s) and the copyright\nowner(s) are credited and that the\n\nThe use,\ndistribution or reproduction in other\nforums is permitted, provided the\noriginal author(s) and the copyright\nowner(s) are credited and that the\noriginal publication in this journal is\ncited, in accordance with accepted\nacademic practice. No use, distribution\nor reproduction is permitted which does\nnot comply with these terms.\n\nKitson et al.\b\n10.3389/fvets.2026.1757258\nFrontiers in Veterinary Science\n02\nfrontiersin.org\ncalculated tumor volume. It destroys tumors through oncolysis by\nactivating protein kinase C (PKC). This activation leads to rapid\nimmunogenic tumor cell death through vascular disruption and direct\ncytotoxicity, which promote further local and systemic antitumor\nimmune responses (3, 4). This “tumor agnostic” method of action has\nprompted research into its effectiveness in other types of cancer and\nin other species, including two horses with non-resectable masses (5)\nand human cancer cell lines grafted onto mice (6). These studies have\ndemonstrated the effectiveness of local chemoablation provided by\nTT, regardless of the tumor type.\nThe reported side effects of Stelfonta in canines are typically local­\nized. These may include swelling, pain, erythema, and wound forma­\ntion at the injection site due to tumor sloughing (10). These effects are\ntypically self-limiting and directly related to the drug’s mode of action.\nThe reported systemic adverse events are rare but usually related to\nmast cell degranulation, including lethargy, loss of appetite, vomiting,\nand even anaphylaxis. In humans, the intratumoral use of this medica­\ntion for various types of tumors has reportedly caused adverse events\nsuch as respiratory arrest and sepsis (7). Concomitant medications\n(steroids and antihistamines) are recommended to minimize the risk\nof mast cell degranulation and its associated complications.\nThis case report describes the acute, progressive, and severe local­\nized wound that ultimately led to hind limb amputation. The wound\ndeveloped following a single intratumoral dose of Stelfonta (2 mg)\nadministered to a 21.6 kg, 10-year-old intact male Vizsla. The injection\ntargeted a subcutaneous MCT on the left lateral hock. Despite\nrepeated attempts at management, the condition necessitated\nhindlimb amputation.\n2 Case description\nA 10-year-old intact male Vizsla was referred to the oncology ser­\nvice at a privately owned veterinary specialty hospital for a subcutane­\n\nDespite\nrepeated attempts at management, the condition necessitated\nhindlimb amputation.2 Case description\nA 10-year-old intact male Vizsla was referred to the oncology ser­\nvice at a privately owned veterinary specialty hospital for a subcutane­\nous haired MCT on the lateral aspect of the left tarsus. The mass was\noriginally diagnosed via fine-needle aspiration (FNA) on 26 February\n2025 by the primary veterinarian, who started the patient on oral\ndiphenhydramine (2.2 mg/kg, twice daily), omeprazole (1 mg/kg,\ntwice daily), and prednisone (1 mg/kg, twice daily for 10 days, then\nonce daily). At the oncology consultation, repeat FNA confirmed a\nlow cytologic grade MCT. Staging included three-view thoracic radio­\ngraphs, abdominal ultrasound, CBC, serum biochemistry, urinalysis,\nand a Nu. Q® cancer screen. No evidence of metastasis was identified.\nMild neutropenia (3.38 K/μL) and a subjectively coarse hepatic\nechotexture were noted but considered clinically insignificant. The\ntumor met the labeled inclusion criteria for intratumoral tigilanol\ntiglate treatment (non-metastatic, low-grade subcutaneous MCT\ndistal to the hock). The patient was then scheduled to return in 8 days\n(day 0) for Stelfonta treatment and was discharged on oral famotidine\n(0.5 mg/kg, twice daily), diphenhydramine (2 mg/kg, twice daily),\nprednisone (0.5 mg/kg, twice daily) and gabapentin (14 mg/kg, to be\ngiven on the morning of treatment).\nOn 20 May 2025 (Day 0), an IV catheter was placed, and the\npatient was sedated with 5 μg/kg of Dexdomitor and 0.2 mg/kg of\nTorbugesic (Zoetis). The tumor volume was estimated at 4cm3, and\nan intratumoral injection (2 mL) of Stelfonta was administered into\nthe MCT site at the proximolateral aspect of the left tarsus. Sedation\nwas then reversed with 0.2 mL of Antisedan (Zoetis) IM. The\npatient recovered uneventfully from sedation and was discharged\nwith instructions to continue famotidine for 8 more days, adjust\ndiphenhydramine to every 8 h for 4 days, then every 12 h for 4 days.\nPrednisone was prescribed on a tapering schedule: 10 mg every 12 h\n\nPrednisone was prescribed on a tapering schedule: 10 mg every 12 h\nfor 5 days, then once daily for 7 days, followed by a 1/2 tablet once\ndaily for 7 days, and finally a 1/2 tablet every other day for\nthree doses.\nApproximately 41 h post-injection (Day 2), the owner reported\nacute diarrhea and vomiting, without dietary indiscretion. The owner\nwas advised to bring the dog to the emergency service, and the patient\npresented 8 h later. On examination, the dog was quiet to dull, dehy­\ndrated, and hypotensive, with pale mucous membranes, tachycardia\n(230 bpm), prolonged capillary refill time (>2 s), weak femoral pulses,\nand a systolic blood pressure of 100 mmHg (Doppler). Point-of-care\ntesting revealed a lactate of 8.3 mmol/L. spec cPL was 1273.6 ng/\nmL. CBC demonstrated leukopenia (WBC 3.2 K/μL), and biochem­\nistry showed mild ALP elevation and mild hyperglycemia. Abdominal\nultrasound demonstrated mildly hyperechoic peripancreatic fat. A\ndiagnosis of hypovolemic shock with clinicopathologic and ultraso­\nnographic findings consistent with pancreatitis was made.\nThe patient received two 20 mL/kg IV boluses of Ringer’s lactate\nsolution, which resolved the tachycardia. He was subsequently hos­\npitalized under internal medicine for supportive care, including IV\nLRS (90 mL/kg/day, adjusted to maintain normotension), maropi­\ntant (1 mg/kg IV once daily), methadone (0.2 mg/kg IV every 6 h),\nfamotidine (0.5 mg/kg IV twice daily), dexamethasone SP (0.1 mg/\nkg IV once daily), ampicillin/sulbactam (30 mg/kg IV every 8 h),\nand diphenhydramine (2 mg/kg IM twice daily), with close moni­\ntoring. Vomiting resolved, and the fluid deficit was corrected within\n24 h. Appetite returned by day 5 post-injection (Day of discharge),\nand normal, formed stool was observed at home on day 7.\nThe patient’s one-week oncology recheck coincided with his fifth\nand final day of hospitalization, during which the oncology team\ndetermined that the treatment site was appropriately debriding and\nprogressing. The patient was discharged with instructions to continue\n\nThe patient was discharged with instructions to continue\npreviously prescribed medications. In addition, the patient was dis­\npensed Proviable (Nutramax) to be given daily, along with as-needed\noral medications for vomiting and diarrhea: Maropitant (2 mg/kg\nonce daily), metoclopramide (0.25 mg/kg), and Canalevia-CA1 (one\ntablet twice daily for 3 days) (Table 1).\n3 Diagnostics, therapeutic\ninterventions, follow-up, and\noutcomes\n3.1 Initial wound formation\nWithin 24 h of intratumoral injection, localized erythema and\nedema developed at the injection site on the proximolateral aspect of\nthe left hock. A total of 6 days after injection, the area developed\nsevere circumferential edema and erythema around the injection site.\nNo discharge or exposed deep layers were noted at this time (Figure 1).\nOn day 9, the patient returned to the emergency service due to\nowner-reported non-weight-bearing lameness of the affected limb,\n\nKitson et al.\b\n10.3389/fvets.2026.1757258\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nas well as chewing and licking at the site. Physical examination\nrevealed a wound stable in size (7×10 cm, with circumferential\ninvolvement of 270° around the tarsus) but with marked tissue\nnecrosis and sloughing (Figure 1). Circumferential eschars were\nnoted along wound edges. Odorous, white, purulent discharge was\nobserved oozing from the lateral side of the distal wound edges,\nwhere exposed muscle was also observed. The samples were sub­\nmitted for aerobic culture and sensitivity. These cultures grew\nE. coli, Enterobacter sp., and Klebsiella aerogenes. While the E. coli\nand Klebsiella isolates were broadly susceptible to different antibi­\notic classes, it should be noted that the Enterobacter isolate was\ncarbapenem-resistant (CRE) and reportedly susceptible only to\naminoglycosides. Antibiotic therapy was adjusted accordingly once\nthe culture results became available.\nThe patient was once again hospitalized under the care of the\ninternal medicine service. Empiric antibiotic therapy was started with\nIV ampicillin/sulbactam (30 mg/kg IV every 8 h) and cefazolin\n(22 mg/kg IV twice daily), along with IV crystalloids (LRS, 90 mL/kg/\nday, adjusted to maintain normotension). Previously prescribed sup­\nportive medications were continued in injectable form: Maropitant\n(1 mg/kg IV q24h), methadone (0.2 mg/kg IV q4–6h), famotidine\n(0.5 mg/kg IV q12h), and dexamethasone SP (0.1 mg/kg IV once).\nTopical manuka honey was applied twice daily.\n3.2 Wound deterioration and infection (day\n8–13 post-injection)\nBetween days 8 and 10, the wound area expanded to 8 × 10 cm,\nencompassing approximately 270° of the distal limb circumference\nwith deep dermal and subcutaneous necrosis. Edema of the distal\nextremity increased, and the patient became non-weight-bearing.\nPurulent exudate with a foul odor was also observed. Cytology of the\nwound exudate revealed mixed cocci and rods; aerobic culture grew\nE. coli, Enterobacter sp., and Klebsiella aerogenes, with the latter two\nresistant to β-lactams but susceptible to aminoglycosides. Broad-\n\nCytology of the\nwound exudate revealed mixed cocci and rods; aerobic culture grew\nE.coli, Enterobacter sp., and Klebsiella aerogenes, with the latter two\nresistant to β-lactams but susceptible to aminoglycosides.Broad-\nspectrum ampicillin/sulbactam (30 mg/kg IV q8h) was continued\npending sensitivity results. Once sensitivity results were available,\nampicillin/sulbactam was discontinued, and enrofloxacin (9 mg/kg\nPO twice daily) was instituted. Systemic signs included mild pyrexia\n(103.8 °F) and leukocytosis (WBC = 26 × 109/L). The decision was\nmade to proceed with surgical debridement.\n3.3 First surgical debridement\nThe first debridement (Figure 2) was performed on day 13 post-\ninjection. The large circumferential eschar (7 × 11 cm), surrounding\nthe wound from the mid-tibia to the metatarsals, was removed. All\nnecrotic tissue was excised using sharp debridement; the wound bed\nwas mechanically debrided with sterile gauze and a #10 blade until\ncapillary bleeding was observed. The wound was lavaged with 3 L of\nsterile saline using a SurgiLav system. Partial closure of the caudo­\nproximal quadrant was achieved with 2–0 nylon tacking sutures, fol­\nlowed by application of honey-impregnated gauze for autolytic\ndebridement. The limb was supported with a lightly padded Robert\nJones bandage. Samples were submitted for repeat aerobic culture and\nsensitivity, which grew Enterobacter sp. and Klebsiella pneumoniae.\nThe Enterobacter isolate was consistent with the previous culture\n(CRE, susceptible only to aminoglycosides), while the Klebsiella isolate\nwas susceptible to marbofloxacin, amikacin, and carbapenem. Therapy\nwas adjusted to initiate meropenem (8 mg/kg SQ twice daily) and\nclindamycin (15 mg/kg PO twice daily). Pain was managed with\nmethadone (0.2 mg/kg IV q6h) and gabapentin (10 mg/kg PO q8h).\nThe patient remained hospitalized for monitoring and analgesia, with\nbandages changed every 12 h, and partial granulation was expected\nonce the necrotic tissue demarcated.\n3.4 Ongoing open wound management and\nrepeat debridement\nOpen wound management and supportive care were continued\n\n3.4 Ongoing open wound management and\nrepeat debridement\nOpen wound management and supportive care were continued\nduring hospitalization, with three additional rounds of surgical\ndebridement performed.\nDay 15 (Second debridement): Dried, crusted discharge and\ndebris were present on the superficial surface of the wound. The\nwound edges were healthy and indicated progressive contraction. The\nwound extended 360° around the tarsus. After cleaning with a chlo­\nrohexidine scrub, mild proliferative granulation tissue was noted\nacross the entire wound, which still measured approximately 7×11 cm;\na central fibrinous plaque persisted. Overall, the leg edema was\ndecreasing. The wound surface was mechanically debrided for the\nsecond time using a combination of sterile gauze and a #20 blade until\ncapillary bleeding was achieved. Debridement continued until the\nTABLE 1  Timeline of disease progression and treatments.\nDate/Day post-\ninjection\nClinical event\n02/26/25\nInitial diagnosis of subcutaneous MCT by the\nprimary veterinarian; started prednisone,\ndiphenhydramine, and omeprazole.\n05/12/25\nOncology consultation; low-grade MCT confirmed;\nstaging unremarkable; Stelfonta treatment\nscheduled.\n05/20 (Day 0)\nIntratumoral injection of tigilanol tiglate (2 mL).\n05/21–05/25 (Days\n1–5)\nOnset of vomiting, diarrhea, and hypovolemic\nshock; hospitalization; pancreatitis diagnosed.\nDay 9\nSevere circumferential necrosis identified, with\nmild pyrexia and leukocytosis; wound cultures\npositive for E. coli, Enterobacter, and Klebsiella spp.;\nenrofloxacin instituted; beginning of\nhospitalization.\nDay 13\nFirst surgical debridement; culture and sensitivity\nresults guided discontinuation of enrofloxacin and\ninitiation of meropenem and clindamycin.\nDay 15\nSecond surgical debridement.\nDay 26\nThird surgical debridement.\nDay 36\nFourth surgical debridement; cicatrix removal;\nreleasing incision.\nDay 53\nDischarged after 44 days of hospitalization.\nDay 59\nDeclining QOL; coxofemoral amputation\nperformed for non-salvageable limb.\nPost-operative\nUneventful recovery; normal ambulation on three\nlegs within 1 week; return to normal appetite and\nenergy within 1 month.\n\nKitson et al.\b\n10.3389/fvets.2026.1757258\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nFIGURE 1\nProgression of the left hindlimb MCT site wound at day 1 post-injection (left), day 6 (center), and day 9 (right).\nFIGURE 2\nDay 13: Stages of the first surgical debridement. Before debridement (A), removal of eschar during debridement (B), after lavage (C), and a partially \nclosed caudoproximal aspect of the wound (D).\n\nKitson et al.\b\n10.3389/fvets.2026.1757258\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nwound bed appeared cleaner and healthier. The wound bed was then\nlavaged, and the edges were reassessed.\nDay 26 (Figure 3): Persistent exudate and delayed granulation\ntissue prompted the third surgical debridement, during which bio­\nfilms and debris were noted on the superficial surface of the wound.\nAt this time, the wound measured approximately 6×10 cm and still\nextended 360° around the tarsus. The wound was once again debrided\nusing a combination of sterile gauze and a #20 blade. Debridement\ncontinued until the wound bed appeared healthier with capillary\nbleeding exposed.\nA shave biopsy was taken from the superficial layer of the cranio­\nlateral aspect of the wound bed, just proximal to the hock (the previ­\nous MCT site), to ensure no recurrence of neoplasia. Histopathology\nof the biopsy revealed fibrin and fibroplasia with a mixed lymphoplas­\nmacytic and neutrophilic infiltrate. Repeat culture and sensitivity per­\nformed at this debridement grew Staphylococcus pseudintermedius,\nwhich was sensitive only to aminoglycosides, amphenicols, and mupi­\nrocin. Chloramphenicol was added (50 mg/kg per os every 8 h) and\ncontinued for the remainder of the hospitalization.\nDay 36: The fourth and final debridement (Figure 4) was per­\nformed in the same manner as previous sessions, except it included\nremoval of a significant circumferential cicatrix extending ~1 cm deep\nat the proximal aspect of the wound bed and the creation of a 2.5 cm\nlinear releasing incision running distal to proximal along the lateral\naspect of the proximal wound edge. After this, the wound area was\nreduced to 6×8 cm, with improved drainage and minimal edema.\n3.5 Late course and decision for amputation\nOn day 53 post-injection (after 44 days of hospitalization), the\npatient was discharged with instructions to continue antibiotics\n(Chloramphenicol 50 mg/kg per os every 8 h), trazodone (5 mg/kg\nper os every 8–12 h), OWM, and physical therapy at home. Despite\nfour aggressive wound debridements, broad-spectrum antibiotics, and\n\nDespite\nfour aggressive wound debridements, broad-spectrum antibiotics, and\nsupportive care, epithelization failed, and the patient continued to\nexperience persistent distal edema and non-weight-bearing lameness.\nA total of 6 days after discharge, after seeking second opinions from\nother surgical specialists, the owner determined that, despite previous\ntreatments, the condition had not improved. Therefore, the patient’s\nquality of life was greatly compromised, and due to the chronic infec­\ntion risk and poor perfusion, amputation was elected on day 59\npost-injection.\nOn presentation for amputation, the limb was diffusely edema­\ntous, with raised circumferential wound margins and induration\nextending circumferentially at the proximal edge. At coxofemoral dis­\narticulation, diffuse necrosuppurative inflammation and fibrosis were\nconfirmed grossly. The procedure was uneventful, and postoperative\nanalgesia included an epidural morphine/bupivacaine combination\nand oral carprofen (2 mg/kg PO q12h). Histopathology of the limb\nconfirmed necrosuppurative inflammation and fibrosis, with no resid­\nual mast cells or bacteria detected.\n3.6 Outcomes and follow-up\nThe patient recovered uneventfully from amputation, resumed\nambulation within 1 week, and demonstrated normal appetite and\nenergy by 1 month. No further systemic illness or wound complica­\ntions were reported. At the time of writing, the patient was doing well,\nwith gradual improvements in comfort and mobility at home and no\nlingering adverse effects.\n4 Discussion\nA 10-year-old neutered male dog treated with intratumoral\nStelfonta for a left hindlimb MCT ultimately developed progressive\nlocal necrosis with a circumferential cicatrix, systemic inflammatory\nshock, and pancreatitis, necessitating left hindlimb amputation. The\nadverse events described in this case highlight a more severe outcome\nthan is typically reported following intratumoral injection of Stelfonta.\nThis patient developed profound local necrosis, likely resulting from\na tourniquet effect secondary to a circumferential cicatrix. It should\n\nThis patient developed profound local necrosis, likely resulting from\na tourniquet effect secondary to a circumferential cicatrix.It should\nbe noted that initial superficial and subcutaneous necrosis and slough­\ning were the expected consequences of the Stelfonta injection.\nFIGURE 3\nDay 26: Caudomedial view of the wound after debridement (left). Caudal view (center). Lateral view (right).\n\nKitson et al.\b\n10.3389/fvets.2026.1757258\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nThe underlying etiology of the extensive necrosis and systemic\neffects (pancreatitis and shock) in this patient is likely multifactorial. The\nlocal effects are closely related to the mechanism of action of tigilanol\ntiglate. The drug induces rapid vascular disruption and oncolysis, pro­\ncesses that inherently cause local tissue destruction and trigger damage-\nassociated molecular patterns (DAMPs) (3). As demonstrated in human\nresearch (8) and in this case, it is plausible that these inflammatory\nmediators propagated beyond the local site, triggering a systemic inflam­\nmatory cascade. Potential mast cell degranulation cannot be ignored and\nis also a plausible primary cause of the systemic disease. These pathways\nmay have acted synergistically, further exacerbating vascular leakage and\ntissue swelling caused by Stelfonta and amplifying the subsequent sys­\ntemic inflammatory response, thereby creating a cycle of local compro­\nmise and distant organ dysfunction.\nThe decision to continue prolonged wound management versus\nproceeding with early amputation represents a difficult clinical and\nethical consideration. The manufacturer recommends minimal inter­\nvention in wound care. However, in this case, more than 7 weeks of\nintensive treatment were pursued prior to limb removal, during which\nthe patient experienced persistent pain, infection, immobility, and\nrecurrent hospitalization. Although amputation can be a definitive\nintervention that may prolong survival in some otherwise non-resect­\nable neoplasms when performed early (9), it was not a preferred option\nfor this owner early in the treatment process. Stelfonta was elected by\nthe owner after the patient met the labeled criteria for appropriate case\nselection, including initial tumor size, location, and cytologic grade.\nEarly recognition of signs such as progressive necrosis, circumfer­\nential scarring, escalating pain, or distal ischemia should prompt\nimmediate reassessment of the therapeutic approach. Amputation\n\nEarly recognition of signs such as progressive necrosis, circumfer­\nential scarring, escalating pain, or distal ischemia should prompt\nimmediate reassessment of the therapeutic approach.Amputation\nshould be considered when wounds fail to heal within 4–6 weeks post-\ninjection, infection persists despite appropriate antimicrobial therapy,\nor quality-of-life markers such as systemic compromise, refractory\npain, or significant functional loss develop. Based on the findings of\nthis report, monitoring for systemic inflammation and pancreatitis\nthrough clinical and laboratory assessments is likely a good idea,\nalongside the use of culture-directed antimicrobial therapy to ensure\ntargeted infection control.\nTo the best of the authors’ knowledge, this is the first report of\npancreatitis and systemic inflammatory shock temporally associated\nwith tigilanol tiglate in a canine patient. Clinicians utilizing this medi­\ncation should remain vigilant for the possibility of unusually severe\nlocal tissue reactions and systemic inflammatory complications and\nshould consider early surgical intervention when objective criteria for\nlimb compromise or systemic risk are met.\n5 Conclusion\nThis case is unique in demonstrating the development of severe sys­\ntemic illness, progressive distal extremity necrosis, and eventual limb\namputation following a single intratumoral injection of Stelfonta. TT can\ninduce severe local reactions and may also have the potential to cause\nsystemic complications. Regardless of the underlying cause, this report\nemphasizes the principle that clinicians should closely monitor patients\nfor systemic illness and wound complications, particularly in distal limb\ntumors where circumferential scarring can compromise perfusion.\nData availability statement\nThe original contributions presented in the study are included in\nthe article/Supplementary material, further inquiries can be directed\nto the corresponding author/s.\nEthics statement\nWritten informed consent was obtained from the participants for\nthe publication of this case report.\nAuthor contributions\n\nEthics statement\nWritten informed consent was obtained from the participants for\nthe publication of this case report.Author contributions\nLK: Data curation, Writing – original draft, Writing – review &\nediting, Investigation. JR: Resources, Conceptualization, Writing –\noriginal draft, Project administration, Supervision, Data curation,\nFIGURE 4\nLeft hindlimb before (left) and after (right) the fourth and final debridement, including cicatrix removal and releasing incision.\n\nKitson et al.\b\n10.3389/fvets.2026.1757258\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nWriting – review & editing. MS: Investigation, Resources, Writing –\nreview & editing, Writing – original draft, Visualization, Methodology.\nFunding\nThe author(s) declared that financial support was not received for\nthis work and/or its publication.\nConflict of interest\nThe author(s) declared that this work was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declared that Generative AI was not used in the\ncreation of this manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organiza­\ntions, or those of the publisher, the editors and the reviewers. Any\nproduct that may be evaluated in this article, or claim that may be\nmade by its manufacturer, is not guaranteed or endorsed by the\npublisher.\nSupplementary material\nThe Supplementary material for this article can be found online\nat: https://www.frontiersin.org/articles/10.3389/fvets.2026.1757258/\nfull#supplementary-material\nReferences\n1.\tDe Nardi AB, Dos Horta RS, Fonseca-Alves CE, De Paiva FN, Linhares LCM, Firmo\nBF, et al. Diagnosis, prognosis and treatment of canine cutaneous and subcutaneous mast\ncell Tumors. Cells. (2022) 11:618. doi: 10.3390/cells11040618\n2.\tVail DM, Thamm DH, Liptak JM. Withrow and Macewen’s Small Animal Clinical\nOncology. Amsterdam, Netherlands: Elsevier (2020).\n3.\tCullen JK, Yap PY, Ferguson B, Bruce ZC, Koyama M, Handoko H, et al. Tigilanol\ntiglate is an oncolytic small molecule that induces immunogenic cell death and enhances\nthe response of both target and non-injected tumors to immune checkpoint blockade.\n\n3.Cullen JK, Yap PY, Ferguson B, Bruce ZC, Koyama M, Handoko H, et al.Tigilanol\ntiglate is an oncolytic small molecule that induces immunogenic cell death and enhances\nthe response of both target and non-injected tumors to immune checkpoint blockade.\nJournal for ImmunoTherapy of. Cancer. (2024) 12:e006602. doi: 10.1136/jitc-2022-006602\n4.\tDe Ridder TR, Campbell JE, Burke-Schwarz C, Clegg D, Elliot EL, Geller S, et al.\nRandomized controlled clinical study evaluating the efficacy and safety of intratumoral\ntreatment of canine mast cell tumors with tigilanol tiglate (EBC-46). J Vet Intern Med.\n(2021) 35:415–29. doi: 10.1111/jvim.15806\n5.\tDe Ridder T, Ruppin M, Wheeless M, Williams S, Reddell P. Use of the Intratumoural\nanticancer drug Tigilanol Tiglate in two horses. Front. Vet. Sci. (2020) 7:639. doi: 10.3389/\nfvets.2020.00639\n6.\tBarnett CME, Broit N, Yap PY, Cullen JK, Parsons PG, Panizza BJ, et al. Optimising\nintratumoral treatment of head and neck squamous cell carcinoma models with the diter­\npene ester Tigilanol tiglate. Investig New Drugs. (2019) 37:1–8. doi: 10.1007/\ns10637-018-0604-y\n7.\tPanizza BJ, De Souza P, Cooper A, Roohullah A, Karapetis CS, Lickliter JD. Phase I\ndose-escalation study to determine the safety, tolerability, preliminary efficacy and phar­\nmacokinetics of an intratumoral injection of tigilanol tiglate (EBC-46). EBioMedicine.\n(2019) 50:433–41. doi: 10.1016/j.ebiom.2019.11.037\n8.\tBortolotti P, Faure E, Kipnis E. Inflammasomes in tissue damages and immune disor­\nders after trauma. Front Immunol. 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+{"text": "【兽医文献】\n标题: Barriers to dog bite case management and dog rabies vaccination in Soroti district, Uganda\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nTYPE  Review\nPUBLISHED  13 April 2026\nDOI  10.3389/fvets.2026.1814057\nOPEN ACCESS\nEDITED BY\nStefan-Gregore Ciornei,\nIasi, University of Life Sciences (IULS),\nRomania\nREVIEWED BY\nMuloongo C. Sitali,\nUniversity of Zambia, Zambia\nNacu Gherasim,\nIasi, University of Life Sciences (IULS),\nRomania\n*CORRESPONDENCE\nMustafa Hitit\nmuhitit@pvamu.edu\nErdogan Memili\nermemili@pvamu.edu\nRECEIVED 19 February 2026\nREVISED 26 March 2026\nACCEPTED 30 March 2026\nPUBLISHED 13 April 2026\nCITATION\nKaya A, Murray R, Bodu M, Hitit M and\nMemili E (2026) Sperm capacitation in\nbulls, rams, bucks, and boars: molecular\nmechanisms and species-specific\ndifferences.\nFront. Vet. Sci. 13:1814057.\ndoi: 10.3389/fvets.2026.1814057\nCOPYRIGHT\n© 2026 Kaya, Murray, Bodu, Hitit and\nMemili. This is an open-access article\ndistributed under the terms of the\nCreative Commons Attribution License\n(CC BY). The use, distribution or\nreproduction in other forums is\npermitted, provided the original\nauthor(s) and the copyright owner(s) are\ncredited and that the original publication\nin this journal is cited, in accordance\nwith accepted academic practice. No\nuse, distribution or reproduction is\npermitted which does not comply with\nthese terms.\nSperm capacitation in bulls, rams,\nbucks, and boars: molecular\nmechanisms and species-specific\ndifferences\nAbdulkadir Kaya 1,2, Raheem Murray 2, Mustafa Bodu 3,\nMustafa Hitit 2* and Erdogan Memili 2*\n1Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Kırıkkale\nUniversity, Kırıkkale, Türkiye, 2College of Agriculture, Food, and Natural Resources, Prairie View\nUniversity, Prairie View, TX, United States, 3Department of Reproduction and Artificial Insemination,\nFaculty of Veterinary Medicine, Selcuk University, Konya, Türkiye\nCapacitation is the process by which sperm undergoes physical, biochemical,\nand molecular changes to acquire the ability to fertilize an oocyte. The first stage\noccurs after ejaculation with the removal of cholesterol from the cell membrane,\nreorganization of the cell membrane, and permission of ion entry into the cell.\n\nThe first stage\noccurs after ejaculation with the removal of cholesterol from the cell membrane,\nreorganization of the cell membrane, and permission of ion entry into the cell.\nIntracellular alkalization leads to signaling, resulting in the basic capacitation\ncascade (sAC-cAMP-PKA-PTP). Consequently, the process of hyperactivation\nand preparation for the acrosome reaction is completed. Significant interspecies\ndifferences are observed in farm animals, and identifying these differences will\nmake important contributions to the development of reproductive technologies.\nBull sperm is mostly dependent on heparin-like glycosaminoglycans; however, in\nram, seminal plasma proteins are shown to play important roles during capaci­\ntation. In goat, the rapid and stress-sensitive capacitation mechanism and the\ntight zinc-dependent in vivo control of boar sperm are important. Furthermore,\nearly and premature capacitation caused by cryopreservation is called cryoca­\npacitation and directly affects fertilization success. Species-specific capacitation\nmechanisms have been investigated; however, understanding differences will\nbe beneficial using omics approaches in fertilization prediction and in develop­\ning cryopreservation strategies. Moreover, this review compares the molecular\nmechanisms of sperm capacitation in bulls, rams, bucks, and boars. It highlights\nspecies-specific differences and their impact on reproductive technologies.\nKEYWORDS\nbull, capacitation, comparative reproduction, goat, livestock, signal transduction,\nsperm\n1 Introduction\nSperm capacitation refers to the process in which sperm undergo physical, biochemical,\nand molecular transformations after ejaculation, then acquire the ability to fertilize. The cas­\ncade of capacitation consists of consecutive events; the removal of cholesterol from the plasma\nmembrane, increased membrane fluidity, influx of bicarbonate and calcium, activation of\nsoluble adenylyl cyclase (sAC), cAMP-dependent protein kinase A (PKA) signaling, and final\nprotein tyrosine phosphorylation (PTP) (1, 2) (Table 1). Consequently, sperm gains the capac­\n\nConsequently, sperm gains the capac­\nity to bind to the zona pellucida, participate in the acrosome reaction, penetrate the oocyte via\nhyperactivation, and finally achieve gamete fusion (3). Recently, some methodologies like, flow\n\nKaya et al.\b\n10.3389/fvets.2026.1814057\nFrontiers in Veterinary Science\n02\nfrontiersin.org\ncytometry (4), time-lapse epifluorescence imaging of zinc ion dynam­\nics (5), and morphometric analyses based on combined fluorescence-\nelectron microscopy (6) have strengthened our understanding of the\nmolecular and structural events underlying the capacitation cascade.\nDespite numerous studies on sperm capacitation, key differences\nbetween species remain incompletely understood, particularly among\nlivestock species with high economic value. Differences in the sperm\nmembrane, signaling pathways, and sensitivity to capacitating factors\nsuggest that capacitation is regulated in a species-specific manner. A\ncomparative understanding of these differences is essential for\nimproving assisted reproductive technologies and cryopreservation\nstrategies. Therefore, the objective of this review is to compare the\nregulation and timing of sperm capacitation in bull (Bos taurus), ram\n(Ovis aries), buck (Capra hircus), and boar (Sus domesticus). These\nspecies represent economically important farm animals and differ in\ntheir physiological and molecular characteristics during sperm capaci­\ntation (Figure 1).\n2 Mechanism of capacitation cascade\n2.1 Early stage of capacitation\nEarly stage of capacitation is characterized by the removal of cho­\nlesterol from the plasma membrane and the rearrangement of mem­\nbrane structure through lipid raft reorganization in the female\nreproductive system by the influence of albumin and high-density\nlipoproteins (7–9). As a result, cholesterol depletion induces the redis­\ntribution and aggregation of lipid rafts, which are dynamic micro-\nspaces rich in cholesterol and sphingolipids that serve as platforms for\nsignaling molecules (10). Sphingolipids and glycolipids in the cell\nmembrane play a critical role in lipid raft organization associated with\nhyperactivation and acrosome exocytosis (11). Thus, a more fluid cell\nmembrane allows calcium and bicarbonate ions to enter the cell,\nenabling sAC activation, increased intracellular cAMP, and PKA acti­\nvation (7, 9).\n2.2 Ion flux and intracellular signaling phase\n\nThus, a more fluid cell\nmembrane allows calcium and bicarbonate ions to enter the cell,\nenabling sAC activation, increased intracellular cAMP, and PKA acti­\nvation (7, 9).2.2 Ion flux and intracellular signaling phase\nThe generation of the capacitation signal is dependent on an\nincrease in intracellular bicarbonate (HCO₃−) and calcium (Ca2+)\n(12–14). Following ejaculation, sperm cells entering the female\nreproductive system are exposed to high bicarbonate concentrations\noriginating from both seminal plasma and the oviduct environment,\nwhich then initiates the signaling pathways necessary for capacitation\n(2, 15). Increased membrane fluidity facilitates bicarbonate entry into\nthe sperm cell, leading to an elevation of intracellular pH and initia­\ntion of capacitation. This bicarbonate influx is mediated by Na+/\nHCO₃− transporters and Cl−/HCO₃− exchangers (1, 13, 15). On the\nother hand, the Cystic Fibrosis Transmembrane Conductivity\nRegulator (CFTR) plays a role in regulating signaling, and carbonic\nanhydrase (CA) catalyzes the hydration of CO₂ to bicarbonate (2, 14,\n15). Alkalization is a prerequisite for the activation of CatSper chan­\nnels (16), a pH-sensitive, voltage-gated ion channel located in the\nflagellum plays a role in calcium influx into the cell (17, 18). The\nCatSper channel also senses signals from progesterone, prostaglan­\ndins, and zona pellucida proteins, and links extracellular stimuli from\nthe female reproductive system to intracellular sperm Ca2+ dynamics\n(16, 18, 19). Therefore, the uptake of bicarbonate and calcium into\nthe cell is an important factor in maintaining capacitation. Dilution\nand cryopreservation, which are known to affect the sperm cell mem­\nbrane, have been reported to disrupt the mechanism of capacitation\nand induce premature activation (20).\nTABLE 1  Key molecular events involved in sperm capacitation.\nMolecular event\nMain pathway/molecule(s)\nFunctional outcome\nReferences\nCholesterol efflux\nAlbumin, HDL, heparin-like\nglycosaminoglycans; BSP/PDC-109 proteins\nIncreased membrane fluidity; redistribution of\nlipid rafts; initiation of capacitation\n(14, 94)\nBicarbonate influx\n\nHCO₃− → soluble adenylyl cyclase (sAC)\nRapid increase in intracellular cAMP; activation\nof downstream signaling\n(14)\nPhysiological ROS generation\nH₂O₂, NO; mitochondrial and membrane-\nassociated oxidases\nRedox-dependent signaling supporting\ncapacitation without oxidative damage\n(57, 95)\ncAMP production and PKA activation\ncAMP–PKA–AKAP signaling complexes\nPhosphorylation cascades coordinating motility\nand capacitation\n(12, 96)\nProtein tyrosine phosphorylation\nPKA-dependent activation of tyrosine kinases\nAcquisition of fertilizing ability; preparation for\nhyperactivation and acrosome reaction\n(94, 96)\nCa2+ influx\nCatSper channels; membrane depolarization\nInduction of hyperactivated motility; regulation\nof flagellar beating\n(16)\nActin cytoskeleton remodeling\nF-actin polymerization; actin-binding proteins\nStabilization of sperm head; prevention of\npremature acrosome reaction\n(29, 38)\nDisplacement of seminal plasma–\nderived inhibitory proteins\nBSP/PDC-109, RSVP14/20, SPINK3,\nSERPINE2; albumin-mediated dissociation and\nsperm surface protein reorganization\nRelief of inhibitory constraints on cAMP/\nPKA-dependent signaling, allowing full\nestablishment of the capacitated state\npermissive for zona pellucida binding and\nacrosome reaction\n(8, 44, 53)\n\nKaya et al.\b\n10.3389/fvets.2026.1814057\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nIon influx and alkalization hyperpolarize the cell membrane, sta­\nbilizing membrane potential and structure via CFTR-Epithelial Na+\nChannel (ENaC) interaction, leading to sAC activation (2).\nMechanistically, calcium (Ca2+) influx works synergistically with\nHCO₃− to stabilize ATP binding on the sAC and increase cAMP syn­\nthesis (21–23). Furthermore, calcium is a key ion in the process from\nthe initiation of capacitation to zona pellucida penetration and gamete\nfusion (19, 23, 24). Consequently, the increase in both ions leads to the\nconversion of ATP to cAMP and inorganic pyrophosphate (PPi) (13,\n21, 22). It has been shown that the capacitation cascade can be stimu­\nlated by exogenous cAMP analogs, demonstrating the key role of\ncAMP (25, 26). Intracellular cAMP accumulation is a crucial step in\nsperm capacitation and leads to the activation of two main down­\nstream effectors in which (1) cAMP-activated exchange protein\n(EPAC) regulates processes such as Ca2+ signaling and acrosome reac­\ntion and (2) protein kinase A (PKA), whose importance we will\nemphasize in the next section, activates protein phosphorylation,\nwhich underlies hyperactivation and acrosome reaction (13, 22, 27).\n2.3 Final stage of capacitation\nProtein phosphorylation is a key regulatory mechanism during\nsperm capacitation, and this process is characterized by a progressive\nincrease in protein tyrosine phosphorylation. Most tyrosine-phos­\nphorylated proteins are localized in the fibrous sheath of the sperm\nflagellum, where they regulate hyperactivated motility and support\nenergy metabolism (28, 29). In addition to the flagellum, tyrosine\nphosphorylation is also observed in the sperm head, particularly in\nthe acrosomal and equatorial regions, where it is associated with zona\npellucida recognition and preparation for acrosomal exocytosis (30).\nIn this way, phosphorylation plays an integral role in the coordination\nof motility, metabolism, and fertilization (31, 32).\nPKA, via cAMP, is a key regulatory element at the functional\n\nIn this way, phosphorylation plays an integral role in the coordination\nof motility, metabolism, and fertilization (31, 32).PKA, via cAMP, is a key regulatory element at the functional\nremodeling in sperm through ionic signaling. PKA alters both the\nstructural properties and functional activities of target proteins by\nadding a phosphate group (–PO₄) to serine (Ser) and threonine (Thr)\nresidues, and serine/threonine phosphorylation is one of the early and\nprerequisite molecular events that occur during capacitation (12, 14,\n24). At the same time, motor proteins such as dynein and tubulin, as\nwell as various signaling proteins, are affected by this phosphorylation,\nundergoing reorganization in sperm motility and energy utiliza­\ntion (33).\nOn the other hand, PKA is to initiate a second wave of phosphory­\nlation in the capacitation process by activating tyrosine kinases such\nas Src family kinases and Pyk2. Subsequently, transition allows for the\nextension of serine/threonine-based signaling through tyrosine phos­\nphorylation. During this stage, protein phosphatase-1 (PP1) is inhib­\nited via CaMKII, thus maintaining the phosphorylation status of\ndownstream proteins. PP1 inhibition acts as a critical mechanism for\nensuring signal continuity during capacitation. The ionic signaling of\nPKA is spatially organized by α-kinase binding proteins (AKAPs),\nwhich enable localized and controlled phosphorylation by fixing PKA\nto specific subcellular regions (32, 34). While kinase plays a role in\nregulating sperm energy metabolism through phosphorylation, the\nAKAP4 protein binds PKA to the flagellum, allowing the signaling\npathway to exert flagellum-specific effects (35, 36).\nIn addition, the activation of ERK1/2 and p38 proteins is initiated\nvia the mitogen-activated protein kinase (MAPK) pathway. PKA acti­\nvation increases calcium influx into the cell by contributing to the\nphosphorylation of specific Ca2+ channels along with their associated\nFIGURE 1\nSchematic overview of the capacitation cascade in mammalian sperm highlighting membrane, ionic, and phosphorylation-dependent events (created\n\nKaya et al.\b\n10.3389/fvets.2026.1814057\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nregulatory proteins. Increased Ca2+ levels activate kinases such as\nCaMKII and PKC, which, in coordination with the chaperone protein\nHsp90, thereafter facilitate the activation of the MAPK signaling path­\nway. Meanwhile, activation of the MAPK cascade leads to increased\nsperm flagellum motility while simultaneously achieving the cellular\npreparatory process necessary for the acrosome reaction (37).\nFocal adhesion kinase (FAK) in the cytoskeleton, is activated in\nthis process, then stimulating ERK2. FAK-mediated signaling enables\nthe conversion of cellular mechanical stimuli into biochemical\nresponses. ERK2 phosphorylates GEF-H1, subsequently triggering the\nactivation of small GTP-binding proteins. Rho GTPases play a central\nrole in regulating cytoskeletal dynamics of sperm cell, and activation\nof RhoA and Rac1 activates LIM kinase and phosphorylates cofilin\n(24, 29, 38). Consequently, it suppresses the degradation of actin fila­\nments, promotes polymerization, and ensures the formation of a stable\nactin scaffold necessary for acrosomal exocytosis (38, 39).\nFinally, PTP supports cytoskeletal remodeling and actin polym­\nerization, then the resulting hyperactivation facilitates sperm-egg\ninteraction through protein regulation in the cell membrane and pre­\npares sperm for the acrosome reaction (12, 40, 41).\n3 Comparative molecular analysis of\nspecies-specific differences\n3.1 Bull (cattle)\nBull sperm exhibits a distinct capacitation response to glycosami­\nnoglycans (GAGs), particularly heparin and structurally analogous\nmolecules, which are widely used as physiological inducers of capaci­\ntation (42, 43). Nevertheless, capacitation in bull sperm is not exclu­\nsively dependent on GAGs, and additional factors such as albumin,\nbicarbonate, calcium, and seminal plasma proteins (e.g., BSP proteins)\nalso contribute to the regulation of this process (1, 44, 45).\nAfter ejaculation, heparin starts functioning at the sperm plasma\nmembrane, especially in bull sperm, where it is often used to help\n\ng., BSP proteins)\nalso contribute to the regulation of this process (1, 44, 45).After ejaculation, heparin starts functioning at the sperm plasma\nmembrane, especially in bull sperm, where it is often used to help\nsperm become more active. Heparin first attaches to a certain group\nof seminal plasma proteins that are all called “binder of sperm” (BSP)\nproteins. PDC-109 (BSP-A1/A2), BSP-A3 (PDC-86), BSP-30 kDa\n(BSP-30A/B), and BSP5 (BSP-A5) are all members of this protein\nfamily (44–46). Consequently, PDC-109 extensively covers the sperm\nsurface, which helps cholesterol and phospholipids leave the plasma\nmembrane (44, 45). Lipid efflux greatly increases the fluidity and dis­\norder of membranes. This process prepares bull spermatozoa for\ndownstream signaling related to capacitation, rather than being a\nmechanism that is shared by all mammals (42).\nPhosphorylation signaling events during capacitation are well-\ndefined, with heparin-induced capacitation acting as a reliable and\nspecies-specific experimental model. Chamberland et al. (42) were the\nfirst to find a sperm protein that was about 50 kDa and only under­\nwent intense phosphorylation during heparin-induced capacitation.\nThis was a signaling event that had not been seen in other species.\nLater studies showed that this protein was made up of partial proteo­\nlytic fragments of AKAP4, which is a key structural part of the flagel­\nlar fibrous sheath and an important scaffold for PKA-mediated\nsignaling in sperm (31, 32). Mostek-Majewska et al. (47) also found\nmore PKA-phosphorylated targets that are only found in bull sperm\ncapacitation. They also saw that tyrosine phosphorylation of\nPRKAR1A, the regulatory subunit of PKA, supports the idea that\nthere is a bull-specific autoregulatory loop that keeps PKA signaling\ngoing during capacitation (47).\nIn accordance with these capacitation-specific signatures, numer­\nous proteins have established connections to bull fertility that intersect\nwith the capacitation-associated proteome. Protamine-1 (PRM1) is\nessential for chromatin condensation and DNA integrity, with\n\nProtamine-1 (PRM1) is\nessential for chromatin condensation and DNA integrity, with\nincreased levels associated with diminished DNA fragmentation and\nenhanced fertility outcomes (48, 49). HSP-70 aids in cryo-tolerance,\nmitochondrial stability, and membrane integrity, functions that are\nespecially important for processing semen. Additionally, AKAP4, in\naddition to its signaling role during capacitation, has recently been\nconfirmed as a dependable marker of sperm motility and fertilization\npotential (50). We also found that bulls with high fertility have higher\nlevels of structural and oocyte-activating proteins like ODF2 and\nPAWP, which help with hyperactivation, the acrosome reaction, and\nthe fusion of sperm and oocytes (51).\n3.2 Ram (sheep)\nRam sperm, particularly when compared to the bull, whose capac­\nitation mechanism is significantly dependent on GAGs, rely on its\nproteomic structure, where immune-protective seminal plasma pro­\nteins are critical for capacitation (42, 43). Ram sperm are also more\ndependent on seminal plasma factors that stabilize membranes, act as\ndecapacitating agents, and modulate immune responses, thereby pre­\nventing premature capacitation along the female reproductive tract\n(vagina, cervix, and uterus) and ensuring that capacitation is com­\npleted at the appropriate site within the oviduct (52, 53).\nRSVP14 and RSVP20 are seminal plasma proteins identified in\nram semen that act as decapacitating factors. These proteins bind to\nthe sperm surface and help maintain a non-capacitated state by pre­\nventing the increase in protein tyrosine phosphorylation during\ncapacitation (53). RSVP20, on the other hand, maintains sperm mem­\nbrane integrity by binding to heparin-like molecules and plays a dual\nrole in both inhibiting and fine-tuning the process by increasing\nsperm-zona pellucida binding (52–54). Similarly, members of the BSP\nfamily show distinct functional effects on sperm physiology. BSP1 is\nknown to interact with sperm membranes and participate in lipid\nremodeling processes associated with capacitation, whereas BSP5 has\n\nSimilarly, members of the BSP\nfamily show distinct functional effects on sperm physiology.BSP1 is\nknown to interact with sperm membranes and participate in lipid\nremodeling processes associated with capacitation, whereas BSP5 has\nbeen reported to modulate sperm viability and capacitation dynamics\nunder certain conditions (46). Another protein involved in preventing\npremature capacitation is serine protease inhibitor Kazal-type 3\n(SPINK3), a seminal plasma protein that binds to the sperm surface\nand stabilizes the plasma membrane, thereby helping maintain sperm\nin a non-capacitated state and modulating calcium influx (55).\nSeminal plasma proteins play important roles in stabilizing the\nsperm membrane, regulating ion channels, and controlling oxidative\nand apoptotic processes (52, 56). These proteins also help maintain\nsperm viability during storage and transport by regulating signaling\npathways involved in capacitation (52, 56). Signaling pathways such\nas c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein\nkinase (p38 MAPK) regulate cellular stress and apoptosis, and their\nexcessive activation may negatively affect sperm capacitation and sur­\nvival (56).\nDespite having a well-developed antioxidant defense system, ram\nsperm are highly susceptible to oxidative damage (57). However, con­\ntrolled production of reactive oxygen species (ROS) is essential for\n\nKaya et al.\b\n10.3389/fvets.2026.1814057\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nsperm capacitation and contributes to membrane remodeling and\nprotein tyrosine phosphorylation (57, 58). However, excessive ROS\nproduction can lead to lipid peroxidation, protein oxidation, DNA\nfragmentation, and premature capacitation or the acrosome reaction\n(59, 60). The high sensitivity of ram sperm to ROS is largely related to\nthe polyunsaturated fatty acid (PUFA)-rich composition of the plasma\nmembrane and their limited antioxidant capacity, which mainly\ndepends on enzymes such as superoxide dismutase (SOD), glutathi­\none peroxidase (GPX), and peroxiredoxins (PRDXs) (61).\n3.3 Buck (goat)\nCapacitation in buck sperm occurs faster than in many other live­\nstock species. This feature influences fertilization efficiency and makes\nbuck sperm particularly sensitive to environmental and biochemical\nconditions during semen handling and storage. Under in vitro condi­\ntions, functional changes associated with capacitation can be detected\nrapidly (62, 63), and these changes may appear within approximately\none hour after induction (64). When compared with other species\nsuch as bull and ram, the duration of capacitation in bucks is generally\nshorter (62, 64). Although this rapid response can facilitate fertiliza­\ntion, it may also limit the effective use of frozen–thawed semen by\ncausing premature capacitation (65).\nShort-term storage of buck sperm, at 5 °C in glucose-based\nextenders, resulted in faster rates of capacitation and acrosome reac­\ntions after 48–72 h, accompanied by increased lipid peroxidation and\ndecreased membrane integrity. However, the addition of fructose to\nthe extender was beneficial in reducing above mentioned effect (66).\nIt was also shown that adding arginine to the extender enhanced\ncapacitation and acrosome reactions and upregulated motility and\ncapacitation-related genes (67). Similarly, pyridoxine supplementation\nhas been reported to enhance motility, promote in vitro capacitation,\nand elevate acrosome reactions during cryopreservation (68).\n\nSimilarly, pyridoxine supplementation\nhas been reported to enhance motility, promote in vitro capacitation,\nand elevate acrosome reactions during cryopreservation (68).\nA study examining the proteomic profiles of ram and buck sperm\nidentified more than 2,100 proteins, of which 238 were differentially\nexpressed between the two species (69). Among these, a higher abun­\ndance of capacitation-inhibitory proteins, such as SERPINE2, a serine\nprotease inhibitor known to delay the capacitation cascade, was detected\nin goat sperm (69). In contrast, ram sperm showed lower SERPINE2\nexpression despite a generally high abundance of capacitation-regulatory\nproteins, suggesting the presence of species-specific regulatory mecha­\nnisms (69). Furthermore, the relatively rapid capacitation observed in\nbuck sperm indicates that certain inhibitory regulatory pathways may\nbe downregulated or modulated by biochemical and signaling stimuli.\n3.4 Boar (pig)\nIn boar, sperm capacitation generally follows the conserved mam­\nmalian signaling cascade involving bicarbonate and calcium influx,\nactivation of the sAC–cAMP/PKA pathway, and subsequent hyperac­\ntivation and acrosomal priming (70). However, boar sperm differ from\nother farm animal species by their marked sensitivity to in vitro condi­\ntions, due to a plasma membrane enriched in PUFA and low choles­\nterol content, which accelerated capacitation (70, 71). Despite this\ntendency, premature capacitation is physiologically restrained in vivo\nthrough zinc signature–dependent mechanisms that regulate sperm\nattachment to the oviduct and control the timing of acrosomal remod­\neling (72). In addition, seminal plasma components such as fibronec­\ntin-1 (FN1) and post-transcriptional regulation by sperm-derived\nmicroRNAs contribute to membrane stabilization, redox balance, and\nresistance to cryo- and in vitro–induced capacitation, collectively\ndefining a boar-specific capacitation profile (73–75).\nThe PUFA-rich, highly fluid plasma membrane predisposes boar\nsperm to rapid in vitro capacitation, necessitating tight physiological\n\nThe PUFA-rich, highly fluid plasma membrane predisposes boar\nsperm to rapid in vitro capacitation, necessitating tight physiological\nregulation in vivo. Zinc signatures reflect distinct Zn2+ distribution pat­\nterns associated with functional capacitation states; early signatures (Sig-\n1/2) stabilize membranes and suppress premature capacitation, whereas\nlater signatures (Sig-3/4) permit membrane remodeling and acrosomal\npriming near ovulation. Proteomic profiles indicated a zinc-centered\nregulatory network, with dynamic modulation of zinc-interacting pro­\nteins involved in motility, metabolism, and signaling, accordingly dis­\ntinguishing boar capacitation from that of other species (72, 76, 77).\nRecent studies have identified FN1, Izumo sperm–egg fusion pro­\ntein 1 (IZUMO1), and acrosin-binding protein (ACRBP) as key pro­\nteins involved in the regulation of boar sperm capacitation (78–80).\nHigh-fertility boars exhibited elevated FN1 levels in seminal plasma\nand spermatozoa, associating FN1 to sperm durability and fertilizing\ncapacity under stress (73, 75, 81). IZUMO1 is localized in the sperm\nhead and has been associated with capacitation-related processes, con­\ntributing to the preparation of sperm for the acrosome reaction (78).\nIn addition, ACRBP is localized in the acrosomal region of the sperm\nhead and has been reported to contribute to acrosomal stability during\ncapacitation through the ubiquitin–proteasome system (79).\nAlso, small non-coding RNAs, particularly microRNAs\n(miRNAs), have emerged as regulatory molecules involved in sperm\ncapacitation and may contribute to species-dependent differences in\ncapacitation-associated signaling pathways (67, 82). Several miRNAs,\nmiR-21-5p, miR-15a, miR-34c, and miR-185 have been directly asso­\nciated with capacitation potential and membrane stability in boar\nsemen. Notably, miR-21-5p targets vinculin, a cytoskeletal linker\ninvolved in sperm–oocyte interaction, thereby suppressing premature\ncapacitation and stabilizing the plasma membrane under in vitro con­\nditions (82). Importantly, boar sperm with high cryotolerance exhib­\n\nImportantly, boar sperm with high cryotolerance exhib­\nited miRNA profiles that limit oxidative stress and spontaneous\ncapacitation through PI3K–Akt and mTOR signaling, potentially dis­\ntinguishing miRNA signatures as functional markers of cryocapacita­\ntion and capacitation control (80, 83, 84).\n4 The impact of cryopreservation on\ncapacitation\nCryopreservation induces a capacitation-like state in spermato­\nzoa, marked by enhanced membrane fluidity and lipid peroxidation\n(85), subsequently linked to excessive reactive oxygen species (ROS)\nproduction and aberrant intracellular signaling (65). Changes occur­\nring during cryopreservation may disrupt the normal regulation and\ntiming of sperm capacitation, leading to premature capacitation-like\nchanges known as cryocapacitation (65, 85). The degree of cryocapaci­\ntation varies among species. Bull and ram sperm are not very sensitive\n(42, 52), but buck sperm are somewhat sensitive (64). Boar sperm, on\nthe other hand, are very sensitive to cryocapacitation (71) because\ntheir plasma membranes are high in polyunsaturated fatty acids and\nlow in cholesterol (86) (Table 2).\nAt the molecular level, cryopreservation causes sudden changes\nin temperature and osmotic pressure, which cause sperm plasma\nmembrane phase transitions that mess up the organization of lipids\n\nKaya et al.\b\n10.3389/fvets.2026.1814057\nFrontiers in Veterinary Science\n06\nfrontiersin.org\n(87, 88). Changes in the sperm plasma membrane cause cholesterol\nefflux, lipid raft reorganization, and increased membrane fluidity.\nThese changes are similar to the early stages of physiological capacita­\ntion and start capacitation-related phosphorylation cascades (85).\nNevertheless, in contrast to physiological capacitation, cryocapacita­\ntion occurs in an uncontrolled and heterogeneous way. Excessive\nROS production after thawing, caused by challenges with mitochon­\ndria and the activation of NADPH oxidase, decreases antioxidant\ndefenses and accelerates lipid peroxidation, which makes the mem­\nbrane even less stable (89–91). Because of this, oxidative imbalance\nchanges signaling related to capacitation toward premature acroso­\nmal destabilization and stops the energy-dependent control of capaci­\ntation by lowering the mitochondrial membrane potential and ATP\navailability (92, 93).\nSperm plasma membrane instability and abnormal calcium sig­\nnaling also change where and how key proteins involved in capacita­\ntion, such as AKAP3, CABYR, and CatSper, work. These alterations\nlead to hyperactivation and acrosome exocytosis; however, they dis­\nrupt the temporal coordination of the capacitation cascade, resulting\nin fertilization failure (71, 85). To prevent cryocapacitation, it’s neces­\nsary to use methods that keep the membrane stable and the redox\nbalance. To prevent premature capacitation of sperm before freezing\nand to improve post-thaw function, optimization of cooling and\nwarming rates, careful selection of cryoprotectants, and the addition\nof antioxidants are recommended. Recent proteomic and lipidomic\ninvestigations underscore biomarkers such as fibronectin 1 (FN1),\nosteopontin (OPN), and glutathione peroxidases 4 and 5 (GPX4/5) as\nprospective targets for enhancing cryopreservation protocols designed\nto preserve fertilization competence (80, 86).\n5 Conclusions and future directions\nSperm capacitation is a highly regulated biochemical and bio­\nphysical process that facilitates fertilization via membrane remodel­\ning,\nbicarbonate\nand\n\n5 Conclusions and future directions\nSperm capacitation is a highly regulated biochemical and bio­\nphysical process that facilitates fertilization via membrane remodel­\ning,\nbicarbonate\nand\ncalcium\ninflux,\nactivation\nof\nthe\nsAC–cAMP–PKA pathway, and subsequent protein phosphorylation.\nThe core signaling cascade is preserved across farm animals; however,\nsignificant interspecies variations exist in regulatory mechanisms,\ntiming, and susceptibility to oxidative stress and cryopreservation. To\nimprove artificial insemination, cryopreservation, and in vitro fertil­\nization protocols, it is important to know what regulatory features are\nunique to each species. Future research should focus on using integra­\ntive methods to track the progress of capacitation from early mem­\nbrane remodeling to the ability to fertilize, as well as combining\nreal-time imaging with omic-based analyses. Nevertheless, this review\nhas several limitations. The available literature on sperm capacitation\nis not equally represented among livestock species. In addition, dif­\nferences in experimental designs, capacitation protocols, and analyti­\ncal methodologies across studies may limit direct comparisons\nbetween species. Therefore, further studies using standardized\napproaches are needed to clarify species-specific capacitation\nmechanisms.\nAuthor contributions\nAK: Conceptualization, Formal analysis, Investigation, Project\nadministration, Resources, Visualization, Writing – original draft,\nWriting – review & editing. RM: Visualization, Writing – original\ndraft. MB: Formal analysis, Writing – original draft, Writing –\nreview & editing. MH: Conceptualization, Formal analysis,\nFunding\nacquisition,\nProject\nadministration,\nResources,\nSupervision, Validation, Writing – original draft, Writing – review\n& editing. EM: Conceptualization, Formal analysis, Funding\nacquisition, Resources, Visualization, Writing – original draft,\nWriting – review & editing.\nFunding\nThe author(s) declared that financial support was received for\nthis work and/or its publication. Partial funds were provided\nthrough the TÜBİTAK 2219 – International Postdoctoral Research\n\nFunding\nThe author(s) declared that financial support was received for\nthis work and/or its publication.Partial funds were provided\nthrough the TÜBİTAK 2219 – International Postdoctoral Research\nFellowship Programme of the Scientific and Technological Research\nCouncil of Türkiye (Project No: 1059B192500151) to AK. This study\nwas funded by the USDA-National Institute of Food and Agriculture\n(Grant No. 2024-38821-42115) and Evans Allen (Accession\nNumber: 7006455 to MH and EM).\nTABLE 2  Comparative overview of sperm capacitation mechanisms across farm animal species.\nSpecies\nApproximate\ncapacitation time\nDominant trigger(s)\nKey molecular and\nbiochemical features\nReferences\nBull (Bos taurus)\n3–6 h\nBicarbonate (HCO₃−) sensitivity\nand heparin-like\nglycosaminoglycans\nTyrosine phosphorylation and\nCatSper-mediated Ca2+ influx\n(44, 45)\nRam (Ovis aries)\n4–6 h\nCholesterol efflux and protein\ntyrosine phosphorylation\nHigh susceptibility to oxidative\nstress; seminal plasma proteins\n(46, 53, 69)\nBuck (Capra hircus)\n1–4 h (early changes within\nabout 1 h)\nHCO₃−/Ca2+-dependent\nactivation of the sAC–cAMP–\nPKA pathway\nRapid and highly sensitive\ncapacitation\n(64, 69)\nBoar (Sus domesticus)\n1–2 h (rapid)\nStrong bicarbonate influx,\ncholesterol efflux, and Ca2+ entry\nHighly fluid and fragile\nmembrane\n(70, 72, 81)\n\nKaya et al.\b\n10.3389/fvets.2026.1814057\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nConflict of interest\nThe author(s) declared that this work was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declared that Generative AI was not used in the\ncreation of this manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. 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(2022) 193:103–13. doi: 10.1016/j.theriogenology.2022.09.014\n\nKaya et al.\b\n10.3389/fvets.2026.1814057\nFrontiers in Veterinary Science\n09\nfrontiersin.org\n83.\tDeng C-Y, Lv M, Luo B-H, Zhao S-Z, Mo Z-C, Xie Y-J. The role of the PI3K/AKT/\nmTOR signalling pathway in male reproduction. Curr Mol Med. (2021) 21:539–48. doi:\n10.2174/1566524020666201203164910\n84.\tTang X, Chen Y, Luo H, Bian Q, Weng B, Yang A, et al. miR-126 controls the apoptosis\nand proliferation of immature porcine sertoli cells by targeting the pik3r2 gene through\nthe PI3K/AKT signaling pathway. Animals. (2021) 11:2260. doi: 10.3390/ani11082260\n85.\tBenko F, Ďuračka M, Ba��as Š, Lukáč N, Tvrdá E. Biological relevance of free radicals\nin the process of physiological capacitation and cryocapacitation. Oxygen. (2022)\n2:164–76. doi: 10.3390/oxygen2020014\n86.\tYeste M. Sperm cryopreservation update: Cryodamage, markers, and factors affecting\nthe sperm freezability in pigs. Theriogenology. (2016) 85:47–64. doi: 10.1016/j.\ntheriogenology.2015.09.047\n87.\tSaha A, Asaduzzaman M, Bari FY. Cryopreservation techniques for ram sperm. Vet\nMed Int. (2022) 2022:7378379. doi: 10.1155/2022/7378379\n88.\tXu B, Wang R, Wang Z, Liu H, Wang Z, Zhang W, et al. Evaluation of lipidomic\nchange in goat sperm after cryopreservation. Front Vet Sci. (2022) 9:1004683. doi:\n10.3389/fvets.2022.1004683\n89.\tIrigoyen P, Mansilla S, Castro L, Cassina A, Sapiro R. Mitochondrial function and\nreactive oxygen species production during human sperm capacitation: unraveling key\nplayers. FASEB J. (2024) 38:e23486. doi: 10.1096/fj.202301957rr\n90.\tLuna C, Serrano E, Domingo J, Casao A, Pérez-Pé R, Cebrián-Pérez J, et al.\nExpression, cellular localization, and involvement of the pentose phosphate pathway\nenzymes in the regulation of ram sperm capacitation. Theriogenology. (2016) 86:704–14.\ndoi: 10.1016/j.theriogenology.2016.02.024\n91.\tShi H, Li Q-Y, Li H, Wang H-Y, Fan C-X, Dong Q-Y, et al. ROS-induced oxidative\nstress is a major contributor to sperm cryoinjury. Hum Reprod. (2024) 39:310–25. doi:\n10.1093/humrep/dead250\n92.\tKadirvel G, Kathiravan P, Kumar S. Protein tyrosine phosphorylation and zona bind­\n\nShi H, Li Q-Y, Li H, Wang H-Y, Fan C-X, Dong Q-Y, et al.ROS-induced oxidative\nstress is a major contributor to sperm cryoinjury.Hum Reprod.(2024) 39:310–25.doi:\n10.1093/humrep/dead250\n92.Kadirvel G, Kathiravan P, Kumar S.Protein tyrosine phosphorylation and zona bind­\ning ability of in vitro capacitated and cryopreserved buffalo spermatozoa. Theriogenology.\n(2011) 75:1630–9. doi: 10.1016/j.theriogenology.2011.01.003\n93.\tKadirvel G, Kumar S, Kumaresan A. Lipid peroxidation, mitochondrial membrane\npotential and DNA integrity of spermatozoa in relation to intracellular reactive oxygen\nspecies in liquid and frozen-thawed buffalo semen. Anim Reprod Sci. (2009) 114:125–34.\ndoi: 10.1016/j.anireprosci.2008.10.002\n94.\tCormier N, Bailey JL. A differential mechanism is involved during heparin-and\ncryopreservation-induced capacitation of bovine spermatozoa. Biol Reprod. (2003)\n69:177–85. doi: 10.1095/biolreprod.102.011056\n95.\tde Lamirande E, Gagnon C. Impact of reactive oxygen species on spermatozoa: a\nbalancing act between beneficial and detrimental effects. Hum Reprod. (1995) 10:15–21.\ndoi: 10.1093/humrep/10.suppl_1.15\n96.\tVisconti PE, Bailey JL, Moore GD, Pan D, Olds-Clarke P, Kopf GS. Capacitation\nof mouse spermatozoa: I. Correlation between the capacitation state and protein\ntyrosine phosphorylation. Development. (1995) 121:1129–37. doi: 10.1242/\ndev.121.4.1129\n\n---"}
+{"text": "【兽医文献】\n标题: Comparative design of artificial ochratoxin A antigens: conjugation strategies and immunogenicity evaluation for antibody production\n\nTYPE Original Research\nPUBLISHED 10 February 2026\nDOI 10.3389/fvets.2026.1733620\nOPEN ACCESS\nEDITED BY\nPaul Mandigers,\nUtrecht University, Netherlands\nREVIEWED BY\nKoen Santifort,\nIVC Evidensia Small Animal Referral Hospital\nArnhem, Neurology, Netherlands\nValentina Lorenzo,\nNeurología Veterinaria, Spain\n*CORRESPONDENCE\nFranziska Spohn\nFranziska.spohn@tiho-hannover.de\nRECEIVED 27 October 2025\nREVISED 06 January 2026\nACCEPTED 12 January 2026\nPUBLISHED 10 February 2026\nCITATION\nSpohn F, Lesta G, Wang-Leandro A, Kajin F,\nVolk HA and Nessler JN (2026) Cerebrospinal\nfluid markers and magnetic resonance\nimaging lesion volume predicting relapse in\ncanine meningoencephalitis of unknown\norigin. Front. Vet. Sci. 13:1733620.\ndoi: 10.3389/fvets.2026.1733620\nCOPYRIGHT\n© 2026 Spohn, Lesta, Wang-Leandro, Kajin,\nVolk and Nessler. This is an open-access\narticle distributed under the terms of the\nCreative Commons Attribution License (CC\nBY). The use, distribution or reproduction in\nother forums is permitted, provided the\noriginal author(s) and the copyright owner(s)\nare credited and that the original publication\nin this journal is cited, in accordance with\naccepted academic practice. No use,\ndistribution or reproduction is permitted\nwhich does not comply with these terms.\nCerebrospinal fluid markers and\nmagnetic resonance imaging\nlesion volume predicting relapse\nin canine meningoencephalitis of\nunknown origin\nFranziska Spohn1*, Gloria Lesta2, Adriano Wang-Leandro1,\nFilip Kajin3, Holger A. Volk1 and Jasmin N. Nessler1\n1Department of Small Animal Medicine and Surgery, University of Medicine Hannover, Foundation,\nHannover, Germany, 2DOS Software-Systeme GmbH, Wolfsburg, Germany, 3Clinic for Internal\nMedicine, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia\nIntroduction: Meningoencephalitis of unknown origin (MUO) is a potentially\nlethal neurological disease in dogs with a high relapse rate. Prognostic factors for\nrelapse based on neurological examination, magnetic resonance imaging (MRI),\nor cerebrospinal fluid (CSF) examination are inconsistently reported.\nMethods: This retrospective single center study included 35 dogs with MUO.\n\nPrognostic factors for\nrelapse based on neurological examination, magnetic resonance imaging (MRI),\nor cerebrospinal fluid (CSF) examination are inconsistently reported.Methods: This retrospective single center study included 35 dogs with MUO.\nBrain MRI, CSF findings, clinical signs at diagnosis and during follow-up MRI\n(routine or relapse-related) were analyzed. Lesion volumes were calculated using\nthe Cavalieri method. Relapse predictors were evaluated for routine follow- up\nMRI examinations using logistic regression and ROC/AUC.\nResult: Only higher CSF albumin (p = 0.0413) and lymphocyte proportion (p\n= 0.0288) at routine follow-up examination were predictive of future relapse.\nROC analyses identified thresholds of 9.64 mg/dl for CSF albumin (AUC:\n0.75; sensitivity 86.7%, specificity 61.1%) and 74.0% for CSF lymphocytes (AUC\n0.76; sensitivity 66.7%, specificity 64.3%). Total lesion volume and volume of\ncontrast-enhancing lesions decreased after treatment and increased again at\nrelapse. Increased lesion volume or normal MRI on routine follow-up MRI\ndid not reliably predict future relapse, although increased lesion volume in\nT1-weighted contrast enhancement and Fluid-attenuated inversion recovery\n(FLAIR) was observed at or after relapse. Lesion volume, lesion number, and\nlesion localization in different sequences was associated with neurodisability\nscale (NDS) and epileptic seizures.\nDiscussion:\nIn this study, CSF albumin and lymphocyte proportion were\nidentified as predictors of future relapse. Routine follow-up MRI was not\npredictive of future relapse, but useful for detection of an active inflammation.\nKEYWORDS\ncanine, CSF-albumin, meningoencephalitis of unknown origin (MUO), MRI, relapse\n1 Introduction\nMeningoencephalitis of unknown origin (MUO) is an umbrella term for a group of\nnon-infectious inflammatory diseases of the central nervous system (CNS) in dogs (1).\nThe clinical diagnosis is based on clinical signs of focal or multifocal lesions in the CNS,\nmagnetic resonance imaging (MRI) findings, cerebrospinal fluid (CSF) pleocytosis, and\n\nThe clinical diagnosis is based on clinical signs of focal or multifocal lesions in the CNS,\nmagnetic resonance imaging (MRI) findings, cerebrospinal fluid (CSF) pleocytosis, and\nthe absence of detectable infectious diseases (2, 3). Nevertheless, the gold standard for\ndiagnosis is histopathological diagnosis (4).\nFrontiers in Veterinary Science\n01\nfrontiersin.org\n\nSpohn et al.\n10.3389/fvets.2026.1733620\nImmunosuppression with glucocorticoids remains the of\ncornerstone initial therapy, often in combination with other\nimmunosuppressive drugs such as ciclosporin or cytarabine (5, 6).\nPlease replace the sentence with: Nevertheless, 26%−33% of the\ndogs die despite therapy in the first 7 days after diagnosis (3, 7, 8).\nOf the dogs that survive, up to 65% experience a recurrence of\nclinical signs (3).\nSeveral studies have been conducted to identify prognostic\nfactors associated with improved outcomes in dogs diagnosed with\nMUO (5, 8). The occurrence of seizures or decreased mentation\nare clinical signs associated with a poorer prognosis in the first\nweek after diagnosis (7, 9, 10). Furthermore, some studies have\nidentified CSF parameters related to shorter survival time, such\nas an increased neutrophil cell count (7) and elevated CSF lactate\nlevels (11). Additionally, MRI findings that may correlate with a\npoor short-term prognosis include loss of cerebral sulci, foramen\nmagnum herniation, and mass effect (3, 12). Long-term prognosis\nmight be worse with higher T2-weighted (T2w) lesion burden,\nand risk of a relapse seems increased with higher T1-weighted\n(T1w) post contrast lesion burden (8). A normal control MRI\nexamination after 3 months is reported to be associated with a\nbetter outcome (3).\nRepeated MRI examinations are helpful for monitoring dogs\nwith MUO. Lowrie et al. (3), recommended a follow-up MRI\nexamination 3 months after diagnosis, in combination with\nCSF analysis to evaluate the success of treatment. However,\nimage interpretation is currently still purely subjective. In order\nto objectify this, recent studies have investigated quantitative\napproaches, such as measuring the total lesion burden (8) and\nassessment of brain atrophy based on the level of interthalamic\nadhesion in relation to brain volume (13), which may have a\npotential impact on prognosis.\nThe Cavalieri method is a well-established, reliable stereological\nmeasurement procedure. With this method it is possible to estimate\nthe volume of three-dimensional structures on the basis of two-\n\nThe Cavalieri method is a well-established, reliable stereological\nmeasurement procedure.With this method it is possible to estimate\nthe volume of three-dimensional structures on the basis of two-\ndimensional structures (14). One study showed that the Cavalieri\nmethod can estimate brain compartment volumes from MRI, with\ngood agreement to postmortem reference measurements (15).\nThe aim of this study was to investigate MRI, CSF, and clinical\nfindings in dogs with MUO during immunosuppressive therapy\nand to identify potential predictors of relapse. Focus was placed\non longitudinal changes in lesion volume, the appearance of new\nlesions, CSF markers, and how these relate to neurological status\nand treatment decisions.\n2 Materials and methods\n2.1 Data collection\nThis is a retrospective, single-center cohort study of the\nSmall Animal Hospital of the University of Veterinary Medicine\nHannover including dogs presented for the diagnosis of MUO\nbetween 2013 and 2024. The medical records of the dogs were\nanalyzed, and dogs were included in the study if the following\ncriteria were met: more than one MRI examination, clinical signs\nsuggestive of focal or multifocal CNS lesions, single or multiple\nintra-axial hyperintense lesions on T2w on the diagnostic MRI\n(present in all included dogs), initial CSF examination showing an\nincreased cell count, exclusion of possible infectious agents (2), and\nif treatment with immunosuppressive therapy resulted in initial\nclinical improvement. Based on available records, no dogs with a\nnormal diagnostic MRI underwent a second MRI examination and\ntherefore did not meet the inclusion criteria.\nCollected data included age, weight, sex, neuter status,\nbreed, date of death, cause of death, number and date of\nexaminations, occurrence of generalized tonic-clonic and focal\nseizures, status epilepticus and cluster seizures, results of the CSF\nexamination (protein mg/dl, albumin in mg/dl, Qalb (ratio of\nCSF albumin and albumin of the blood) [commercially available\nphotometric assay test adapted for dogs, cobas c311 analyzer,\n\nHitachi, Roche, Mannheim, Germany], amount of white blood\ncells/3 μl (WBC), proportion of lymphocytes in %, segmented\nnucleated neutrophil granulocytes in %, and macrophages in\n%, treatment (immunosuppressive drugs, dosage, and change\nin medication including add-on therapy). Recorded neurological\nsigns were used to determine the neurodisability scale (NDS)\n(1). Although retrospective assessment of the NDS may be less\nreliable than prospective application (1, 16). The NDS was used\nas a standardized clinical assessment tool to enable consistent\ncomparisons across neurological examinations. This approach also\nallowed a uniform definition of clinical relapse. Relapse was defined\nas an increased of NDS at a follow-up examination compared with\nthe preceding examination.\n2.2 MRI evaluation\nMRI examinations were performed using a 3.0 Tesla MRI\nScanner (Achieva, Philips Medical Systems, Best, The Netherlands).\nTransverse T2w, FLAIR and T1w after intravenous injection of\ngadolinium-based contrast agent (Gadoteric acid; - Dotarem R⃝,\nGuerbet GmbH Sulzbach, Germany) were obtained.\nTo objectify MRI findings, volume, quantity, and localization of\nthe lesions was recorded. Lesion localization was recorded as within\nthe brainstem, the forebrain (including: the hippocampus, the\ncingulate gyrus, the piriform lobe, the frontal lobe), the cerebellum,\nand the meninges. Furthermore, caudal transtentorial herniation,\nloss of sulci, or the presence of new lesions compared to the last\nMRI were noted.\nAll\nMRI\nevaluations\n(including\nlesion\nidentification,\nmeasurements, and lesions volume quantification) were performed\nby the first author not blinded to the clinical information.\nAs a parameter to assess brain atrophy, the interthalamic\nadhesion\nheight\nwas\nmeasured\non\nsagittal\nT2w\nsequence.\nMeasurements were performed on the slice showing the maximum\ninterthalamic adhesion height (17). A commercially available\nDICOM viewer was used for this purpose (Easy Image; VetZ GmbH\nIsernhagen, Germany).\nFor brain volume analysis an artificial intelligence (AI) based\nbrain extraction was used to first create a 3D volume mask of the\n\nA commercially available\nDICOM viewer was used for this purpose (Easy Image; VetZ GmbH\nIsernhagen, Germany).For brain volume analysis an artificial intelligence (AI) based\nbrain extraction was used to first create a 3D volume mask of the\nbrain parenchyma by stripping the skull (18) from T2-weighted\nimages: the DICOM sequences are read by the AI and converted\ninto a 3D image with isometric voxels. Based on 3D model, the\nAI uses segmentation to create a mask in Nifti format, which\ndistinguishes brain areas from other image content (18). The\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n\nSpohn et al.\n10.3389/fvets.2026.1733620\ngenerated brain masks were saved and imported into 3D Slicer,\nwhere they were reviewed, manually corrected if necessary by\nthe first author, and used for final brain volume calculation with\nOpen-Source-Software 3D Slicer [version 5.8.0 for macOS from the\nNational Institutes of Health (NIH) was used; https://www.slicer.\norg].\nLesion volume was determined using the image editing\nprogram ImageJ [National Institute of Health (NIH), based in\nBethesda, Maryland USA] (19). Each lesion was manually outlined\nfor every MRI in the transverse T2w, FLAIR and T1w post contrast\nimages. A cross-counting grid (between 12 and 24 mm2) was\napplied, and grid points located within a brain lesion were counted\nfor each slices.\nWith the total of the crosses for each brain lesion the volume\nof each lesion was determined using the Cavalieri Method. The\nlesion volume was estimated with the following formula as used in\nWatson et al. (14):\nˆV = Apm\n′t\n\u0002 n\n\u0003\ni=1\nPi\n\u0004\nAp is the area that each point represents. m\n′ represents the\nspacing between MRI slices. t represents the thickness of cross\nsection (if every MRI sequences were counted would be one). Pi\nrepresents the number of grid points counted (14).\nTotal lesion volume was defined as the sum of the volume of all\nlesions per MRI sequence. The relative lesion volume was calculated\nas the ratio of total lesion volume to brain volume.\n2.3 Data analysis\nData were analyzed using the statistical software SAS Studio\nEnterprise, Version 3.8.2 (SAS Institute Inc., Cary, North\nCarolina, USA).\nFor statistical analysis, data were first grouped according to\nMRI examination timepoint (first to fourth examination) to find\ndifferences in clinical, CSF, and MRI variables over repeated\nexaminations. Dogs were additionally classified into relapse-related\ngroups (before relapse, no relapse, relapse and after relapse)\nto identify differences in routine follow-up examinations and\nexaminations at or after relapse.\nThis grouping approach was used to evaluate whether findings\nobtaining routine follow-up MRI examinations differed between\n\nThis grouping approach was used to evaluate whether findings\nobtaining routine follow-up MRI examinations differed between\ndogs that subsequently relapsed and dogs that did not relapse.\nTo evaluate whether findings from routine follow-up MRI\nexaminations were predictive of future relapse, MRI examinations\nperformed at diagnosis (first MRI) and those conducted at or\nafter relapse were excluded from this analysis. The remaining\nroutine follow-up examination were compared between dogs that\nsubsequently relapsed and dogs that did not relapse.\nFor all statistical analyses, a p-value ≤0.05 was considered\nstatistically significant. Continuous variables were summarized\nas mean ± standard deviation (SD) for normally distributed\ndata, or as median and interquartile range (IQR) for non-\nnormally distributed data. Categorical variables were described as\nfrequencies with 95% confidence intervals (CI).\nNormality was assessed using the Shapiro–Wilk test and by\nvisual inspection of histograms and boxplots. Correlations between\ncontinuous variables were examined using Pearson’s correlation\ncoefficient\nwhen\nboth\nvariables\nwere\nnormally\ndistributed;\notherwise, Spearman’s rank correlation coefficient was used.\nLinear regression was performed to explore associations\nbetween\ncontinuous\nvariables.\nTo\nidentify\npredictors\nof\nrelapse, binary logistic regression was conducted. Variables\nwere\nselected\nbased\non\nunivariable\nscreening\nand\nclinical\nrelevance. Specifically, variables that were statistically significant\nin univariate analyses were considered for inclusion; in addition,\nvariables that were considered clinically relevant were also\nincluded regardless of the univariate p-value, as they may be\nimportant indicators of disease activity and are commonly\nused in clinical decision-making. Results are reported as odds\nratios (OR) with corresponding 95% CIs. Receiver operating\ncharacteristic\n(ROC)\nanalyses\nwere\nadditionally\nperformed\nfor CSF albumin and CSF lymphocytes. The optimal cut-\noffwas determined using the Youden index, which selects\nthe threshold with the highest combined value of sensitivity\nand specificity.\n2.4 Ethic committee\n\nThe optimal cut-\noffwas determined using the Youden index, which selects\nthe threshold with the highest combined value of sensitivity\nand specificity.2.4 Ethic committee\nAs this was a retrospective study, no ethical approval was\nrequired in accordance with local legislation and institutional\nrequirements. Only previously collected data were used. Written\ninformed consent for the use of these data was obtained from\nthe owners prior to the examinations, in accordance with the\nUniversity’s guidelines.\n3 Results\nA\ntotal\nof\n35\ndogs\nwere\nincluded\nin\nthe\nstudy.\nOf\nthese,\n18\ndogs\n(51%)\nwere\nmale\n(14/18\nintact\nand\n4/18\nneutered). The remaining 17 dogs (49%) were female (10/17\nintact and 7/17 neutered). The following breeds were included:\nchihuahua (n = 5), French Bulldog (n = 5), mixed breed\n(n = 5), Labrador Retriever (n = 2), Jack Russel Terrier\n(n = 2), Yorkshire Terrier (n = 2) and one each Elo,\nAiredale Terrier, Australian Shepherd, Beagle, Biewer Terrier,\nBolognese,\nGolden\nRetriever,\nHavanese,\nPug,\nPygmy\nSpitz,\nSmall Münsterländer, Swiss Mountain Dog, West Highland\nWhite Terrier.\nThe median age at the time of diagnosis was 4.00 years\n(0.35 to 11.1 years). Of the 35 dogs nine dogs were euthanized\nafter a median of 790 days (185 to 2,376 days) after the first\nMRI. 19/35 dogs were still alive at the time of data collection\n(2023, 2024), while no further information was available for\n7/35 dogs.\n3.1 Findings in follow-up examinations\nA total of 86 examinations were analyzed, including MRI,\nCSF, clinical signs (expressed as NDS), and medication. Not all\nFrontiers in Veterinary Science\n03\nfrontiersin.org\n\nSpohn et al.\n10.3389/fvets.2026.1733620\nFIGURE 1\nFlow chart showing the number of the magnet resonance imaging\n(MRI) examinations performed (n = 86). The initial MRI examination\n(n = 35) and follow-up examinations (n = 51) were shown. The\nfollow-up MRIs are further subdivided according to the number of\nMRI examinations performed (second to fourth MRI). These were\nfurther subdivided into routine follow-up MRIs examinations and\nMRI examinations due to a relapse or after a relapse.\nFIGURE 2\nBar chart for number of patients in relation to the number of\nmagnetic resonance imaging (MRI) examination performed and\ndifferentiated by clinical relapse groups. Definition of relapse: in a\nfollow-up examination a higher neurodisabilty scale (NDS) in one or\nmore points, compared to a previous examination.\ndata and MRI sequences were available for all dogs at every\ntime point. The first MRI was performed at diagnosis in all 35\ndogs. The remaining 51 MRIs were follow-up examinations. By\ninclusion criteria, all dogs (35/35) underwent at least two MRI\nexaminations; 13/35 had three, and 3/35 had four examinations\n(Figure 1).\nThe second MRI was performed at or after relapse in 4/35\ncases, the third in 4/13 cases, and the fourth in 2/3 cases. The\nremaining 41/51 follow-up MRIs were routine controls without\nsigns of relapse at the time of imaging (Figure 2).\nFIGURE 3\nBox plot shows the distribution of the neurodisability scale (NDS) by\nthe number of magnetic resonance imaging (MRI) sequences. Boxes\nshow the interquartile range (25th to 75th percentiles) with a line at\nthe median; diamond inside boxes indicate the arithmetic mean;\nwhiskers show the most extreme values within or 1.5× interquartile\nrange, and observation beyond (outliners) are plotted as points.\n3.2 Comparison of findings according to\nMRI examination timepoint\nFor detailed results, see Supplementary Table 1. After the\nfirst MRI, each dog received a median dose of prednisolone\nof 1.04 mg/kg/day (0.84–3.28 mg/kg/day). At the second MRI\nexamination, the median prednisolone dose was 0.71 mg/kg/day\n(0.00–1.25 mg/kg/day). At later MRI examinations, when available,\n\nAfter the\nfirst MRI, each dog received a median dose of prednisolone\nof 1.04 mg/kg/day (0.84–3.28 mg/kg/day).At the second MRI\nexamination, the median prednisolone dose was 0.71 mg/kg/day\n(0.00–1.25 mg/kg/day).At later MRI examinations, when available,\nthe median prednisolone dose was 0.49 mg/kg/day (0.00–2.66\nmg/kg/day) at the time of the third MRI, and 0.78 mg/kg/day\n(0.00 −1.11 mg/kg/day) at the time of the fourth MRI. During\nthe disease course, 29 dogs received one or multiple add-on\nimmunomodulatory drugs (n = 19/29 cytarabine arabinose s.c., n\n= 18/29 ciclosporin p.o., n = 2/29 mofetil mycophenolate p.o.).\nThe neurodisability scale (NDS) differed significantly between\nMRI examinations timepoints (p < 0.0001). NDS at the first MRI\nwas significantly higher than that at the second (p < 0.0001) and\nthird MRI (p = 0.0393), but did not differ significantly the fourth\nMRI examination (p > 0.05; Figure 3).\nTotal and relative lesion volume in T2w, FLAIR and\ncontrast enhancement were significantly different between the MRI\nexaminations (all p < 0.02). Pairwise comparisons demonstrated\nsignificant differences between the first and the second MRI (p <\n0.02) and in FLAIR and contrast enhancement between the first\nand third MRI among dogs with three MRI examinations available\n(p < 0.05), but not in comparison with the fourth MRI (p > 0.05;\nFigure 4).\nWhile the number of lesions in T2w was not significantly\ndifferent between the MRI examinations (p > 0.05), the number\nof lesions in FLAIR and contrast enhancement was significantly\ndifferent between MRI examinations (p < 0.01), with the first MRI\nand the fourth MRI showing the highest lesion numbers.\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n\nSpohn et al.\n10.3389/fvets.2026.1733620\nFIGURE 4\nBox plot shows the distribution of lesion volume in mm3 by magnetic resonance imaging (MRI) sequences, Number of MRI and relapse groups.\nDefinition of relapse: in a follow-up examination a higher neurodisability scale (NDS) in one or more points, compared to a previous examination. T2\nweighted (T2w): lesion volume in first MRI to before relapse were significant different (p = 0.0023). FLAIR: lesion volume in first MRI to before relapse\n(p = 0.0003) and no relapse (p = < 0.0001) were significant different. T1 post contrast: lesion volume in first MRI to before relapse (p = 0.0005) and\nno relapse (p = 0.0017) were significant different. Boxes show the interquartile range (25th to 75th percentiles) with a line at the median; circle inside\nboxes indicate the arithmetic mean; whiskers show the most extreme values within or 1.5× interquartile range, and observation beyond (outliners)\nare plotted as points.\nThe MRI examinations differed significantly for the numbers\nof new lesions in FLAIR, T2w, and contrast enhancement\n(p\n<\n0.0001).\nIn\nT2w\nor\nFLAIR,\n10/35\nand\n6/32\ndogs\ndeveloped new lesions at the second MRI examination compared\nto the previous MRI. 6/13 dogs developed new lesions on\ntheir third MRI examination comapred to the previous. 2/3\nand 1/2 dogs developed new lesions on their fourth MRI\nexamination compared to the previous MRI. In the second\nMRI no new T1 contrast enhancing lesions were found (n\n= 0/32), while in the third 3/11 and in the fourth MRI\n1/2 dogs showed new contrast enhancing lesions compared to\nthe previous.\nCSF findings, namely WBC count, protein, albumin, and Qalb,\nwere significantly different between examinations (all p < 0.002)\nand improved after the initial examination. The proportions of\nlymphocytes and macrophages did not differ significantly between\nexaminations (p > 0.05). In contrast, the proportion of neutrophilic\ngranulocytes differed significantly between MRI time points (p =\n0.0274) and decreased after the first MRI and was again higher at\nthe time of the fourth MRI examination.\n3.3 Comparison of findings according to\nthe reason for MRI examination\n\n0274) and decreased after the first MRI and was again higher at\nthe time of the fourth MRI examination.3.3 Comparison of findings according to\nthe reason for MRI examination\nFor detailed results, see Supplementary Table 2. The initial MRI\nwas performed at diagnosis of MUO (n = 35/86) on day 0. Routine\nfollow-up examinations to monitor therapy in dogs without relapse\nwere conducted in 41/86 cases at a median of 120 days (65–\n443 days) after diagnosis. In 10/86 cases with relapse, MRI was\nperformed either at the time of relapse (n = 4) a median of 312.5\ndays (183–483 days) after the initial diagnosis, or after relapse\n(n = 6) at a median of 1,123.5 days (329–1,528 days) after the\ninitial diagnosis.\nNDS\ndiffered\nsignificantly\nbetween\nMRI\nexaminations\nperformed for clinical reasons (p < 0.0001), being lower at routine\nfollow-up than at diagnosis (p < 0.0001) and higher after relapse\ncompared to routine follow- up (p = 0.0019; Figure 5).\nTotal and relative lesion volumes in T2w, FLAIR, and\ncontrast-enhanced sequences differed significantly between MRI\nexaminations (all p < 0.01). Pairwise comparisons showed\nsignificant differences between routine follow-up MRIs and the\nFrontiers in Veterinary Science\n05\nfrontiersin.org\n\nSpohn et al.\n10.3389/fvets.2026.1733620\ninitial MRI at MUO diagnosis (all p < 0.005), as well as\nbetween MRIs performed at or after relapse and the initial MRI\n(all p < 0.05).\nFIGURE 5\nBox plot shows the distribution of the neurodisability scale (NDS) by\nthe relapse groups and the number of magnetic resonance imaging\n(MRI). Definition of relapse: in a follow-up examination a higher\nneurodisability scale (NDS) in one or more points, compared to a\nprevious examination. Boxes show the interquartile range (25th to\n75th percentiles) with a line at the median; circle inside boxes\nindicate the arithmetic mean; whiskers show the most extreme\nvalues within or 1.5× interquartile range, and observation beyond\n(outliners) are plotted as points.\nRepresentative\nexamples\nillustrating\nlesion\nappearance\nand the relative lesion volume measurements are shown in\nFigure 6.\nThe number of lesions in T2w images did not differ significantly\nbetween MRI examinations (p > 0.05). In contrast, lesion numbers\nin FLAIR and contrast-enhanced sequences differed significantly\nbetween examinations (p < 0.0001). The initial MRI had a\nsignificantly higher lesion numbers than routine follow-up MRIs\n(median: FLAIR 3 vs. 1 lesion; T1w post contrast 3 vs. 0 lesions, all\np < 0.001). Routine follow-up MRIs also differed significantly in\nthe number of lesions in FLAIR images at time of relapse (median\n1 vs. 4) and in post contrast images after relapse (median: 0 vs. 2.5;\nboth <0.05; Figure 7).\nIn T2w and FLAIR sequences, dogs developed new lesions at\nthe routine follow-up MRIs compared to the previous MRI in 11/41\nand 6/38 MRI examinations, respectively. At relapse, new lesions\nwere observed in both T2w and FLAIR sequences in 2/4 dogs. After\na relapse, new lesions were identified in 5/6 dogs in T2w and 5/5\ndogs in FLAIR (p < 0.0001).\nNo new T1 contrast-enhancing lesions were detected during\nroutine follow-up (0/37 dogs). Conversely, at or after relapse, 1/4\nand 3/4 dogs, respectively, showed new contrast-enhancing lesions\ncompared to the previous MRI (p < 0.0001; Figure 7).\nBrain volume or interthalamic adhesion height as signs of\n\nConversely, at or after relapse, 1/4\nand 3/4 dogs, respectively, showed new contrast-enhancing lesions\ncompared to the previous MRI (p < 0.0001; Figure 7).Brain volume or interthalamic adhesion height as signs of\nbrain atrophy, did not differ significantly when examinations were\ngrouped by MRI time point (p > 0.05). Both parameters were\npositively correlated with body weight (r = 0.30, p = 0.0081 and r\n= 0.90, p < 0.0001). However, when examinations were grouped by\nrelapse status, interthalamic adhesion height differed significantly\nFIGURE 6\nTransverse T2 weighted (T2w) brain magnet resonance images of two different dogs with meningoencephalitis of unknown origin (MUO). These\nimages illustrating lesion appearance and the lesion volume assessment workflow. Lesions were manually outline (yellow line) and a 12 mm2\npoint-counting grid was applied with (cyan crosses) for lesion estimation (Cavalieri method). (A–C) and (D–F) shows example examinations from two\ndogs with MUO.\nFrontiers in Veterinary Science\n06\nfrontiersin.org\n\nSpohn et al.\n10.3389/fvets.2026.1733620\nFIGURE 7\nBox plots and bar chart of certain values for cerebrospinal fluid (CSF) and magnetic resonance imaging (MRI) for the relapse groups. The MRI values\nare additionally divided into the MRI Sequences. Definition of relapse: in a follow-up examination a higher neurodisability scale (NDS) in one or more\npoints, compared to a previous examination. Boxes show the interquartile range (25th−75th percentiles) with a line at the median; circle inside boxes\nindicate the arithmetic mean; whiskers show the most extreme values within or 1.5x interquartile range, and observation beyond (outliners) are\nplotted as points. T2W, T2-weighted; FLAIR, fluid attenuated inversion recovery; T1 post contrast: T1-weigthed post contrast.\nbetween groups (p = 0.0255) and was smaller after relapse than at\ndiagnosis (p = 0.0397; Figure 8).\nCSF findings, including WBC count, protein, albumin,\nand Qalb, differed significantly between examinations (all p\n< 0.05) and were mostly different between the diagnostic\nexamination for MUO and routine follow-up examinations (all\np < 0.01; Figure 7). The percentage of lymphocytes did not\ndiffer significantly between groups (p > 0.05). In contrast,\nFrontiers in Veterinary Science\n07\nfrontiersin.org\n\nSpohn et al.\n10.3389/fvets.2026.1733620\nFIGURE 8\nSagittal magnetic resonance imaging (MRI) T2w brain image of a 5 years old neutered female Maltese dog with measuring of the interthalamic\nadhesion high. (A) Interthalamic adhesion (6.10 mm) height at time of diagnosis. (B, C) Interthalamic adhesion (5.43/5.57 mm) height in a routine\nfollow up MRI. (D) interthalamic adhesion (4.48 mm) height after a relapse.\nthe proportions of neutrophilic granulocytes and macrophages\ndiffered significantly between groups (all p < 0.05), although\npairwise comparisons between individual examinations revealed no\nsignificant differences (p > 0.05).\n3.4 Predictors of relapse\nFor\ndetailed\nresults,\nsee\nSupplementary Table 4.\nRelapse\noccurred at a median of 351 days (83–1,527 days) after initial MRI\nexamination in 17/35 dogs.\nAt diagnosis, neither examination results nor signalment\ndiffered significantly between dogs that relapsed and those without\nclinical relapse (all p > 0.05).\nA total of 41 routine follow-up MRI examinations were\nincluded in the analysis. Of these, 17/41 examinations (from 13\ndogs) were followed by relapse, while 24/41 examinations (from 18\ndogs) were not followed by any relapse.\nIn routine follow-up examinations, absolute and relative T2w\nlesion volumes were significantly different between MRIs followed\nby relapse and those not followed by relapse (both p < 0.05), with\nlower median lesion volumes observed in MRIs followed by relapse.\nA morphologically normal brain MRI was an uncommon\nfinding in both groups: only 2/17 MRIs were normal in dogs\nwith subsequent relapse, and 1/24 MRIs in dogs without relapse\nwere classified as normal, with no significant difference between\ngroups (p > 0.05). No other MRI finding differed significantly\nbetween routine follow-up scans with subsequent relapse and\nthose without.\nCSF albumin was significantly higher in routine follow-\nup examinations followed by relapse compared with those not\nfollowed by relapse (p = 0.0222), whereas QAlb did not differ\nsignificantly between groups (p > 0.05). CSF albumin levels were\npositively associated with QAlb (p < 0.0001). The proportion of\n\n0222), whereas QAlb did not differ\nsignificantly between groups (p > 0.05).CSF albumin levels were\npositively associated with QAlb (p < 0.0001).The proportion of\nCSF lymphocytes were also significantly different between these\ngroups (p = 0.0329; Figure 9).\nGroups did not differ significantly regarding treatment. Only\nthe decrease in medication after the MRI examination was\nsignificantly different (p = 0.0496), with a more frequent reduction\nin medication in dogs without relapse.\nA binary logistic regression model identified predictors of\nrelapse at routine follow-up examinations. Higher CSF albumin\nlevels (p = 0.0413) and a higher proportion of CSF lymphocytes\n(p = 0.0288) were associated with an increased risk of relapse. In\ncontrast, a reduction in medication after MRI was associated with a\ndecreased risk of relapse (p = 0.0476; Table 1).\nFIGURE 9\nBox plots of cerebrospinal fluid (CSF) albumin (mg/dl) and\nlymphocytes (%) measured in routine follow-up examination in dogs\nwith subsequent relapse and without a relapse. CSF lymphocytes (p\n= 0.0329) and CSF albumin (p = 0.0222) were significantly higher in\ndogs with a subsequent relapse (BR) compared to those without\nrelapse. Definition of relapse: in a follow-up examination a higher\nneurodisability scale (NDS) in one or more points, compared to a\nprevious examination. Boxes show the interquartile range (25th to\n75th percentiles) with a line at the median; diamont inside boxes\nindicate the arithmetic mean; whiskers show the most extreme\nvalues within or 1.5× interquartile range, and observation beyond\n(outliners) are plotted as points.\nROC analysis with Youden index optimization yielded a\nthreshold value of 9.64 mg/dl for CSF albumin (sensitivity of\n86.7% and a specificity of 61.1%) and 74.0% for CSF lymphocytes\n(sensitivity of 66.7% and a specificity of 64.3%; Figure 10).\nFrontiers in Veterinary Science\n08\nfrontiersin.org\n\nSpohn et al.\n10.3389/fvets.2026.1733620\nTABLE 1 Logistic regression of predictors of relapse.\nVariable\nOdds ratio\np-value\n95% Confidence interval\nCoefficient (β)\nCSF\nWBC (cells/3 μl)\n0.936\n0.2541\n0.835–1.049\n−0.0665\nTotal protein (mg/dl)\n1.075\n0.1466\n0.975–1.185\n0.0721\nAlbumin (mg/dl)\n1.151\n0.0413\n1.006–1.318\n0.1409\nQalb (mg/dl)\n1.570\n0.0958\n0.923–2.668\n0.4508\nNeutrophilic granulocytes (%)\n0.966\n0.1744\n0.918–1.016\n−0.0351\nLymphocytes (%)\n1.045\n0.0288\n1.005–1.087\n0.0438\nLarge monocytes (%)\n0.977\n0.2538\n0.938–1.017\n−0.0237\nMedication\nIncreased medication\n3.0667\n0.5598\n0.255–36.878\nDecreased medication\n0.2339\n0.0476\n0.0594–0.9207\nAdd on medication\n1.3382\n0.7364\n0.802–3.042\nDose prednisone\n7.495\n0.0762\n0.809–69.450\n2.0143\nLogistic regression of possible predictors in routine examination [at the time of magnetic resonance imaging (MRI) examination] associated with a risk of a relapse between group no relapse\nand before relapse.\nSignificant level for p < 0.05. Bold values indicate statistical significance (p < 0.05).\nFIGURE 10\nReceiver operating characteristic (ROS) curves for cerebrospinal fluid (CSF) albumin (mg/dl) and lymphocytes (%) measured at routine follow-up.\nOptimal threshold (Youden index): CSF albumin 9.64 mg/dl (sensitivity of 86.7% and a specificity of 61.1%); CSF lymphocytes 74.0% (sensitivity of\n66.7% and a specificity of 64.3%). BR, before relapse [Examinations before deterioration of the neurodisability scale (NDS)].\n3.5 Association of clinical signs with MRI\nfindings\nNDS was significantly associated with total and relative lesion\nvolume in T2w (total volume: p = 0.0027; relative volume: p =\n0.0027) and FLAIR (total volume: p = 0.0034; relative volume: p\n= 0.0005) and with the number of T1 contrast enhancing lesions (p\n= 0.0006; Table 2).\nHigher NDS was associated with the presence of lesions in the\nbrainstem in T2w (p = 0.0114) or FLAIR (p = 0.0213), visible\nlesions in the meninges (p = 0.0059), forebrain (p = 0.0381), and\ncerebellum (p = 0.0064) in FLAIR, and loss of sulci (p < 0.0001).\nHigher NDS was also associated with contrast enhancement of\nthe forebrain (p = 0.0020), the piriform lobe (p = 0.0114), the\nbrainstem (p < 0.0001), and the cerebellum (p = 0.0203; Table 2).\n\n0381), and\ncerebellum (p = 0.0064) in FLAIR, and loss of sulci (p < 0.0001).Higher NDS was also associated with contrast enhancement of\nthe forebrain (p = 0.0020), the piriform lobe (p = 0.0114), the\nbrainstem (p < 0.0001), and the cerebellum (p = 0.0203; Table 2).\nThe risk of epileptic seizures increased with a higher total lesion\nvolume (p = 0.0485) and a higher relative lesion volume (p =\n0.0170) in T2w. Lesions in the piriform lobe (T2w: p = 0.0001;\nFLAIR: p < 0.0001) and in the frontal lobe (T2w: p = 0.0102;\nFLAIR: p = 0.0218), and a higher relative volume of contrast\nenhancement (p = 0.0309) or contrast enhancement in the frontal\nlobe (p = 0.0151; Table 3).\n4 Discussion\nThis study investigated longitudinal changes in MRI findings,\nneurological status (NDS), and CSF parameters in dogs with MUO\nundergoing immunosuppressive therapy. Follow-up evaluations,\nincluding MRI, neurological examination, and CSF analysis, were\ncompared between routine follow-up visits and examinations\nperformed at or after clinical relapse to identify potential predictors\nof relapse.\nIn our cohort, newly appearing lesions on T2w and FLAIR\nimages, as well as new contrast enhancing lesions occurred\nFrontiers in Veterinary Science\n09\nfrontiersin.org\n\nSpohn et al.\n10.3389/fvets.2026.1733620\nTABLE 2 Factors associated with neurodisabilty scale (NDS).\nVariable\nCoefficient\n(β)\nStandard\nError (SE)\np-\nvalue\nTotal lesion volume, absolute\nT2w\n−0.00061\n0.000186\n0.0027\nFLAIR\n−0.00048\n0.000148\n0.0034\nT1w contrast\nenhancement\n−0.00040\n0.000245\n0.1131\nTotal lesion volume, relative (%)\nT2w\n0.5921\n0.1552\n0.0006\nFLAIR\n0.4897\n0.1227\n0.0005\nT1w contrast\nenhancement\n0.4279\n0.2150\n0.0558\nNumber of lesions\nT2w\n0.3926\n0.1143\n0.0018\nFLAIR\n0.6961\n0.1220\n0.5730\nT1w contrast\nenhancement\n0.6592\n0.1725\n0.0006\nLesion in cerebellum\nT2w\n−0.6416\n0.6809\n0.3473\nFLAIR\n−1.3588\n0.4924\n0.0064\nT1w contrast\nenhancement\n−1.3105\n0.5580\n0.0203\nLesion in brainstem\nT2w\n−1.1229\n0.4395\n0.0114\nFLAIR\n−0.9117\n0.3922\n0.0213\nT1w contrast\nenhancement\n−2.6295\n0.5461\n<0.0001\nLesion in forebrain\nT2w\n−0.2760\n0.4724\n0.5597\nFLAIR\n−0.8862\n0.4239\n0.0381\nT1w contrast\nenhancement\n−1.6403\n0.5202\n0.0020\nLesion in piriform lobe\nT2w\n−0.1865\n0.4497\n0.6788\nFLAIR\n0.5029\n0.4701\n0.2863\nT1w contrast\nenhancement\n−3.2682\n1.2747\n0.0114\nLesion meninges\nT2w\n−0.1835\n0.3612\n0.6121\nFLAIR\n−1.2890\n0.4620\n0.0059\nT1w contrast\nenhancement\n0.5724\n0.5499\n0.2998\nLoss of sulci (T2w)\n−1.7162\n0.3700\n<0.0001\nNegative coefficient for binary nominal variables (reference: lesion present) indicates that\ndogs without a lesion in this brain area have a decreased NDS.\nPositive coefficient for continuous variables indicates that dogs with higher values are\nassociated with an increased NDS.\nSignificant level for p < 0.05 is labeled in bold.\nTABLE 3 Factors associated with seizures.\nVariable\nOdds\nratio\np-\nvalue\n95%\nConfidence\ninterval\nCoefficient\n(β)\nTotal lesion volume, absolute\nT2w\n1.000\n0.0485\n1.000–1.000\n0.000167\nFLAIR\n1.000\n0.0997\n1.000–1.000\n0.000100\nT1w contrast\nenhancement\n1.000\n0.0618\n1.000–1.000\n0.000173\nTotal lesion volume, relative (%)\nT2w\n1.194\n0.0170\n1.032–1.381\n0.1773\nFLAIR\n1.108\n0.0525\n0.999–1.230\n0.1028\nT1w contrast\nenhancement\n1.184\n0.0309\n1.016–1.318\n0.0309\nInterthalamic\nadhesion size\n(mm)\n0.431\n0.0130\n0.222–0.837\n0.0130\nBrain volume\n(mm3)\n1.000\n0.2367\n1.000–1.000\n−0.00002\nLesion in piriform lobe\nT2w\n0.113\n0.0001\n0.037–0.342\n−2.1833\nFLAIR\n0.091\n<0.0001\n0.030–0.278\n−2.3998\nT1w contrast\nenhancement\n0.955\n0.9712\n0.081–1.327\n−0.0456\nLesion in frontal lobe\nT2w\n0.209\n0.0102\n0.064–0.690\n−1.5632\nFLAIR\n0.238\n0.0218\n0.070–0.811\n−1.4352\nT1w contrast\nenhancement\n0.185\n0.0151\n0.047–0.721\n−1.6896\nBinar logistic regression based on the estimation of the probability of a seizure occurring\n(seizure = “Yes”).\nNegative regression coefficient in nominal variables (reference category: lesion present)\nindicates that dogs without a lesion have a decreased risk of seizure.\n\nNegative regression coefficient in nominal variables (reference category: lesion present)\nindicates that dogs without a lesion have a decreased risk of seizure.\nPositive regression coefficient in continuous variables indicates that increased values are\nassociated with an increased risk of seizure.\nSignificant level for p < 0.05 is labeled in bold.\nsignificantly more often in examinations performed after a relapse\nthan during follow-up MRI examinations (p ≤0.0029), supporting\nthese MRI changes as markers of current disease activity and\npotentially reduced treatment response. Newly occurring lesions\nalso indicated disease progression. Beyond imaging, CSF albumin\nand lymphocytes emerged as the most clinically relevant predictors\nof a subsequent relapse.\n4.1 Findings at follow up examinations\nRoutine\nfollow-up\nMRI\nexaminations\n(predominantly\nMRI examination two and three, performed in the absence\nof clincal relapse) showed a continuous reduction in lesion\nvolume across T2w, FLAIR, and T1 post-contrast sequences\nduring immunosuppressive therapy. In contrast, lesion counts\ndid not decrease consistently over time, suggesting that lesion\nFrontiers in Veterinary Science\n10\nfrontiersin.org\n\nSpohn et al.\n10.3389/fvets.2026.1733620\nvolume is a more sensitive marker of treatment response and\ninflammatory activity than lesion number. This supports the use\nof volumetric assessment in follow-up imaging to better evaluate\ndisease regression.\nCSF analysis supported the imaging and clinical findings\nby demonstrating a reduction in inflammatory activity during\ntreatment. Routine follow-up examinations showed lower WBC\ncounts, protein, albumin, and QAlb compared with baseline,\nconsistent with therapeutic suppression of CNS inflammation.\nParallel improvement in neurological status, reflected by lower\nNDS, mirrored the reduction in lesion volume. This association\nbetween lesion load and clinical course accords with previous\nfindings, where higher lesion volume at the time of diagnosis was\nlinked to higher NDS (8).\n4.1.1 Imaging features associated with relapse\nIn dogs with a fourth MRI examination, lesion volumes\nincreased again across all sequences, reversing the gradual\nreduction observed during earlier follow-ups. This was most\nlikely because MRI was predominantly performed at or after\nclinical relapse in the population examined at a fourth time point,\nwhen renewed inflammatory activity was present. Active relapse\nwas reflected by increased lesion burden, the reappearance of\ncontrast enhancement, and higher NDS values, indicating a parallel\ndeterioration in clinical status. While lesion counts in T2w images\ndid not differ significantly across examinations, lesion numbers in\nFLAIR and contrast-enhanced sequences were significantly higher,\nwith the MRI examination at a fourth time point and the initial MRI\nshowing the greatest counts. These findings underscore that both\nlesion volume and specific sequence-based lesion counts, alongside\nclinical scoring, provide complementary information to confirm\nrelapse and guide treatment adjustments.\nThis is more clearly seen when MRI examinations are\ncategorized according to the reason for imaging (initial diagnosis,\nroutine follow-up, or at/after relapse). Under immunosuppressive\ntherapy, routine follow-up MRIs showed a reduction in both\n\nThis is more clearly seen when MRI examinations are\ncategorized according to the reason for imaging (initial diagnosis,\nroutine follow-up, or at/after relapse).Under immunosuppressive\ntherapy, routine follow-up MRIs showed a reduction in both\nabsolute and relative lesion volumes, accompanied by clinical\nimprovement and reduced relapse activity. In contrast, MRIs\nperformed at or after relapse revealed increased lesion volumes,\nparticularly in dogs examined after relapse, which paralleled\nworsening NDS scores. The number of lesions and the number of\nnew lesions were also higher at relapse and after relapse compared\nwith the preceding MRI, with the most pronounced increases seen\nin new contrast-enhancing lesions. These findings indicate that\nrelapse is associated with renewed active inflammation, likely due\nto reactivation of existing lesions and/or the formation of new\nlesions. This is consistent with findings by Soulé et al. (20). In their\nstudy, dogs experiencing relapse after 6 months had more lesions\noverall, both newly developed and pre-existing lesions that had\nenlarged, and showed more frequent contrast enhancement. The\nauthors proposed that this pattern reflects an ongoing or reactive\ninflammatory process and noted that individual lesions may\nrespond differently to immunosuppressive therapy (20). Similarly,\nGonçalves et al. (16) reported that, while the mere presence of\ncontrast-enhancing lesions was not associated with relapse, the risk\nof relapse increased with a higher number of such lesions.\n4.1.2 Brain atrophy during follow-up and after\nrelapse\nDuring treatment, no significant changes consistent with brain\natrophy were detected across examinations time points. However,\nwhen grouped by relapse status, brain volume decreased slightly,\nas indicated by a reduction in interthalamic adhesion height.\nThis may indicate that the brain is swollen at diagnosis or that\natrophy develops following relapse. Interpretation is limited, as\ninterthalamic adhesion height is positively correlated with both\nbrain volume and body weight. Previous studies have shown\n\nThis may indicate that the brain is swollen at diagnosis or that\natrophy develops following relapse.Interpretation is limited, as\ninterthalamic adhesion height is positively correlated with both\nbrain volume and body weight.Previous studies have shown\nthat interthalamic adhesion height, alone or in combination with\nother parameters, can serve as a marker for brain atrophy in\ndogs with cognitive dysfunction (17, 21). In addition, a recent\nstudy investigating brain atrophy in MUO used various volumetric\nmeasures, including interthalamic adhesion thickness-to-brain\nheight ratio, and found that atrophy occurs in dogs with MUO, with\nmore severe atrophy associated with poorer prognosis (13). These\nfindings suggest that interthalamic adhesion height may also serve\nas a useful marker for brain atrophy in dogs with MUO.\n4.2 Predictors of relapse\n4.2.1 MRI derived predictors of relapse\nNormal MRI during follow-up examination was rare in this\nstudy. Only three follow-up MRIs were normal, two of which\nwere followed by subsequent relapse. While numbers are small,\nthis observation suggests that a normal follow-up MRI may not\nreliably indicate relapse free disease. In contrast, another study\ndemonstrated that dogs with a normal MRI at time of diagnosis\nhad better long-term prognosis than dogs with abnormal MRIs\n(22). These results suggest that normal MRI at time of diagnosis\nmay indicate a good long-term prognosis, whereas a normal MRI\nduring follow-up should be interpreted cautiously and does not\nimply relapse freedom.\nAbsolute and relative lesion volumes on T2-weighted images\nwere significantly higher in dogs that did not experience a\nsubsequent relapse compared to those that did. One possible\nexplanation is that the visibly larger lesions led veterinarians\nto be more cautious in tapering immunosuppressive therapy,\nwhich may have helped prevent relapse. No other MRI findings\ndiffered between dogs that did not experience a subsequent relapse\ncompared to those that did at the timepoint of routine follow-\nup examinations.\nNew lesions on routine follow-up MRIs in T2w or FLAIR\n\nNo other MRI findings\ndiffered between dogs that did not experience a subsequent relapse\ncompared to those that did at the timepoint of routine follow-\nup examinations.New lesions on routine follow-up MRIs in T2w or FLAIR\nsequences did not necessarily predict future relapse. Dogs without\nrelapse even showed an increase in new lesions, particularly in\nFLAIR sequences (5/22 dogs compared to 1/16 dogs), although\nthis difference was not statistically significant. None of the\ndogs undergoing routine follow-up MRI developed new contrast-\nenhancing lesions. It is possible that the detection of new lesions\nduring follow-up imaging influenced clinical decision-making,\nleading veterinarians to prolong prednisone treatment or avoid\ntherapy reduction. Such an effect could have reduced the risk\nof relapse in these cases, although this cannot be confirmed\nstatistically in the present study.\nFrontiers in Veterinary Science\n11\nfrontiersin.org\n\nSpohn et al.\n10.3389/fvets.2026.1733620\n4.2.2 CSF derived predictors of relapse\nCSF analysis at routine follow-up examinations revealed\nsignificantly\nhigher\nalbumin\nconcentrations\nin\ndogs\nwith\nsubsequent relapse (median 11.72 mg/dl) compared to those\nwithout relapse (median 8.19 mg/dl). In contrast, QAlb—a marker\nof blood–brain barrier (BBB) integrity—did not differ significantly\nbetween groups, although it was correlated with CSF albumin\nlevels. This suggests that total albumin concentration, rather than\nBBB disruption alone, may play a role in the pathophysiology\nof MUO or MUO relapse. Experimental evidence indicates that\nalbumin can activate glial cells via the transforming growth factor\nbeta receptor (TGFβR) and the canonical TGFβR–smad signaling\npathway, increasing phosphorylation of smad2 and smad3 and\npromoting nuclear translocation of smad4 (23, 24). Albumin\nalso induces the pro-inflammatory cytokine IL-1β in astrocytes\nand microglia through TGFβR-dependent but smad-independent\nmechanisms (24). These effects, together with TGFβR-mediated\ncytotoxicity and other signaling changes, support a role for\nalbumin in perpetuating CNS inflammation (24). In our logistic\nregression model, each 1 mg/dl increase in CSF albumin was\nassociated with a 15% higher probability of relapse and might\ntherefore be a more valuable prognostic marker than MRI. The\nROC analysis identified a CSF albumin concentration above\n9.64 mg/dl as being associated with relapse, with a sensitivity of\n86.7% and a specificity of 61.1%. The relatively high sensitivity\nsuggests that this threshold may be useful for identifying dogs at\nincreased risk during routine follow-up, whereas the moderate\nspecificity indicates that false-positive results can occur. Further\nstudies are needed to replicate this cut-offvalue of 9.64 mg/dl in an\nindependent cohort. Until then, this cut-offcould be used as part\nof an integrated clinical assessment together with MRI findings,\nneurological status, and treatment history.\nCerebrospinal\nfluid\nlymphocyte\nproportions\nwere\nalso\nsignificantly\nhigher\nat\nfollow-up\nin\ndogs\nwith\nsubsequent\nrelapse compared to those without. This may indicate that\n\nCerebrospinal\nfluid\nlymphocyte\nproportions\nwere\nalso\nsignificantly\nhigher\nat\nfollow-up\nin\ndogs\nwith\nsubsequent\nrelapse compared to those without.This may indicate that\nthese dogs still had an active adaptive immune response at\nthe time of CSF sampling or were experiencing a recurrence\nof neuroinflammatory activity. Supporting this interpretation,\nour binary logistic regression analysis showed that each one\npercentage-point increase in CSF lymphocytes was associated\nwith an increased risk of relapse. The ROC analysis revealed\na threshold of 74.0% CSF lymphocytes as the value at which\nthe risk of relapse is increased, with a sensitivity of 66.7% and\na specificity of 64.3%. The moderate sensitivity and specificity\nsuggest that this threshold may support risk stratification during\nroutine follow-up, but that misclassification can occur when\nused in isolation. Further validation of this cut-offis required to\nconfirm the robustness. Meanwhile, it should be used as supportive\ninformation in combination with other clinical and imaging\nfindings. These findings are consistent with immunohistochemical\nstudies showing a predominance of CD3+ T-lymphocytes in\nactive neuroinflammatory lesions in MUO, particularly in cases\nof granulomatous meningoencephalitis (25). Conversely, dogs\nwithout subsequent relapse may have already entered a reparative\nphase, characterized by declining lymphocyte counts and a\npredominance of macrophages or neutrophils, which function in\ntissue remodeling and repair.\nElevated CSF WBC counts were not predictive of relapse in\nthis study. One possible explanation is that in dogs with high WBC\ncounts, the treating veterinarian may have refrained from tapering\nmedication or may have intensified treatment as a precautionary\nmeasure. This is supported by our logistic regression findings,\nwhich suggested a reduced relapse risk in dogs whose medication\nwas increased or maintained rather than reduced after follow-\nup MRI.\nAlthough therapy type did not differ significantly between\nrelapse and non-relapse groups, dogs without relapse more often\nhad medication reduced, likely due to favorable MRI findings.\n\nAlthough therapy type did not differ significantly between\nrelapse and non-relapse groups, dogs without relapse more often\nhad medication reduced, likely due to favorable MRI findings.\nTreatment decisions may have influenced outcomes: new or\nenlarged lesions often prompted maintaining therapy, whereas\nmarked MRI improvement encouraged tapering. This aligns with\nour finding that reduction of medication was associated with lower\nrelapse risk.\n4.3 Association of clinical signs with MRI\nfindings\n4.3.1 NDS and MRI findings\nThis study found that higher absolute or relative lesion volumes\nin T2w and FLAIR images were associated with worse NDS. Loss\nof sulci also correlated with higher NDS, likely reflecting acute,\ndiffuse, and severe inflammation with brain swelling, which can\nimpair multiple neurological functions. This accords with previous\nfindings linking loss of sulci to increased mortality (3) and higher\nNDS at diagnosis to greater relapse risk (16).\nLesions in the brainstem on any MRI sequence were also\nassociated with increased NDS, as many NDS-assessed functions,\nsuch as cranial nerve function, consciousness, balance, gait,\nand proprioception, depend on brainstem integrity. Contrast\nenhancement\nin\nthe\nbrainstem,\ncerebrum,\ncerebellum,\nor\nmeninges was significantly linked to higher NDS, supporting the\ninterpretation that these represent acute inflammatory rather than\nchronic lesions.\nAlthough brain herniation is known to be associated with\npoor prognosis (3), it was not assessed in our study because\nnone of the dogs showed brain herniation on the first MRI, likely\nreflecting selection bias, as such dogs may have died before follow-\nup imaging. This may have resulted in a study population with less\nsevere clinical disease.\n4.3.2 Seizure and MRI localization\nSeizures were observed primarily in dogs with lesions in\nthe piriform and frontal lobes, regions previously described\nas epileptogenic centers. MRI studies during seizures have\nidentified abnormalities in the cingulate gyrus, piriform lobe,\nand hippocampus (26). Another study found that lesions in the\ntemporal lobe (including the piriform lobe), frontal lobe, parietal\n\nMRI studies during seizures have\nidentified abnormalities in the cingulate gyrus, piriform lobe,\nand hippocampus (26).Another study found that lesions in the\ntemporal lobe (including the piriform lobe), frontal lobe, parietal\nlobe, and olfactory bulb significantly increased seizure risk in\ndogs with neoplasia (27). Furthermore, MUO lesions in the\nhippocampus and the occurrence of acute symptomatic seizures\npredicted the development of post encephalitic epilepsy (28). Taken\nFrontiers in Veterinary Science\n12\nfrontiersin.org\n\nSpohn et al.\n10.3389/fvets.2026.1733620\ntogether, these data support that, the localization of MUO lesions\non MRI reflects the occurrence of seizures and contributes to\nexplaining the observed clinical symptoms.\nThis study has several limitations that should be considered\nwhen interpreting the results. As a retrospective study, there was\nno standardized design, and the interval between initial diagnosis\nand follow-up examinations varied. Patients also received different\nimmunosuppressive treatments, which may have influenced disease\nprogression. Lesion volume measurements were performed with\na degree of standardization, for example, by counting all crosses\nwithin a lesion. However, MRI evaluations were conducted by a\nsingle observer, introducing the potential for subjective bias.\nThe study was based on three Tesla MRI scans from a single\ninstitution, which may limit generalizability.\nThe\nNDS\nis\nvalidated,\nbut\nretrospective\napplication\nis\nless\nreliable\n(1,\n16),\nespecially\nif\nclinical\nrecords\nlack\ndetailed neurological descriptions; however, well-documented\nexaminations could make NDS use more consistent in such\nsettings. Additional sources of bias may include survival bias, as\ndogs with severe disease (e.g., brain herniation) may not have\nsurvived to undergo follow-up imaging. In addition, the small\nnumbers of dogs that underwent a fourth MRI examination\nmight limit the statistical power of comparisons involving this\ntime point.\nDogs with normal MRI findings on initial diagnostic MRI\nwere not included to reduce diagnostic ambiguity, even though\nnormal MRI findings have been reported in a minority of dogs\nwith MUO (2). Consequently, our findings and proposed cut-off\nvalues may not apply to MUO dogs with a normal diagnostic\nMRI and should be evaluated in future studies. Morphologically\nnormal findings on some routine follow-up MRI examinations\nin this study likely reflect radiological improvement under\ntreatment and do not contradict the absence of normal MRIs\nat diagnosis.\nObserver bias in lesion identification, differences in MRI\nsequence quality, and variations in the timing of follow-up imaging\nrelative to relapse onset could also have influenced results.\n5 Conclusion\n\nObserver bias in lesion identification, differences in MRI\nsequence quality, and variations in the timing of follow-up imaging\nrelative to relapse onset could also have influenced results.5 Conclusion\nDuring follow-up examination CSF albumin and lymphocytes\nwere the strongest predictors of an impending relapse. However,\nImaging remains essential to identifying active lesions, especially\nthe appearance of new contrast- enhancing lesions should be\ninterpreted as a marker of active inflammation and ongoing relapse.\nThese results support cautious reduction of immunosuppressive\nmedication and only after objective stabilization has been\nconfirmed by both imaging and CSF findings.\nData availability statement\nThe original contributions presented in the study are included\nin the article/Supplementary material, further inquiries can be\ndirected to the corresponding authors.\nEthics statement\nEthical approval was not required for the studies involving\nanimals in accordance with the local legislation and institutional\nrequirements because this was a retrospective study. Only\npreviously collected data were used. Written informed consent\nfor the use of these data was obtained from the owners prior to\nthe examinations, in accordance with the University’s guidelines.\nWritten informed consent was obtained from the owners for the\nparticipation of their animals in this study.\nAuthor contributions\nFS:\nWriting\n–\nreview\n&\nediting,\nSoftware,\nWriting\n–\noriginal draft, Formal analysis, Methodology, Visualization, Data\ncuration, Investigation. GL: Software, Writing – review & editing.\nAW-L: Conceptualization, Writing – review & editing. FK:\nWriting – review & editing, Data curation. HV: Writing –\nreview & editing, Supervision, Conceptualization. JN: Project\nadministration, Formal analysis, Supervision, Writing – review &\nediting, Conceptualization, Writing – original draft.\nFunding\nThe author(s) declared that financial support was received for\nthis work and/or its publication. This manuscript is financially\nsupported by the University of Veterinary Medicine, Hannover,\nthrough Open Access Publishing funding.\nConflict of interest\n\nFunding\nThe author(s) declared that financial support was received for\nthis work and/or its publication.This manuscript is financially\nsupported by the University of Veterinary Medicine, Hannover,\nthrough Open Access Publishing funding.Conflict of interest\nGL was employed by DOS Software-Systeme GmbH.\nThe remaining author(s) declared that this work was conducted\nin the absence of any commercial or financial relationships that\ncould be construed as a potential conflict of interest.\nThe handling editor PM declared a past co-authorship with the\nauthors HV and JN.\nThe authors HV and JN declared that they were an editorial\nboard member of Frontiers, at the time of submission. This had no\nimpact on the peer review process and the final decision.\nGenerative AI statement\nThe author(s) declared that generative AI was used in the\ncreation of this manuscript. Artificial intelligence was used to\nimprove the correct spelling and grammar of the manuscript:\nDeepL Translator and DeepL Write, Colon, Germany. Access via\nhttps://www.deepl.com/en/write. ChatGPT (OpenAI, Version 4\nand 5), San Francisco, CA, United States. Access via https://chatgpt.\ncom.\nAny alternative text (alt text) provided alongside figures in\nthis article has been generated by Frontiers with the support of\nartificial intelligence and reasonable efforts have been made to\nFrontiers in Veterinary Science\n13\nfrontiersin.org\n\nSpohn et al.\n10.3389/fvets.2026.1733620\nensure accuracy, including review by the authors wherever possible.\nIf you identify any issues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. Any product that may be evaluated in this article, or\nclaim that may be made by its manufacturer, is not guaranteed or\nendorsed by the publisher.\nSupplementary material\nThe Supplementary Material for this article can be found\nonline at: https://www.frontiersin.org/articles/10.3389/fvets.2026.\n1733620/full#supplementary-material\nReferences\n1. Gonçalves R, Maddox TW, Phillipps S, Nagendran A, Cooper C, Orlandi R, et al.\nDevelopment of a reliable clinical assessment tool for meningoencephalitis in dogs: the\nneurodisability scale. J Vet Intern Med. (2023) 37:1111–8. doi: 10.1111/jvim.16717\n2. Granger N, Smith PM, Jeffery ND. Clinical findings and treatment of non-\ninfectious meningoencephalomyelitis in dogs: a systematic review of 457 published\ncases from 1962 to 2008. Vet J. (2010) 184:290–7. doi: 10.1016/j.tvjl.2009.03.031\n3. 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+{"text": "【兽医文献】\n标题: Mapping the flow of commercial broiler day-old chicks in Kenya\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nEvaluation of a caudal midline\nglossectomy on tongue volume\nand upper airway cross-sectional\nareas in brachycephalic dogs: a\ncadaveric study\nValeria T. Colberg , Raymond K. Kudej *, Nicole Moyer ,\nJoshua A. Peters  and William M. Karlin\nDepartment of Veterinary Clinical Sciences, Tufts University, Cummings School of Veterinary\nMedicine, North Grafton, MA, United States\nIntroduction: Relative macroglossia may contribute to brachycephalic\nobstructive airway syndrome, the pathologic disorder associated with\nrespiratory dysfunction commonly seen in brachycephalic dogs. Recent studies\non brachycephalic dogs have demonstrated a relative macroglossia along with\nreduced air volume in the upper airway compared to non-brachycephalic dogs.\nTongue reduction glossectomy may be a surgical option to address upper\nairway obstruction secondary to macroglossia. The objective of this study\nwas to evaluate the effects of a caudal midline glossectomy (CMG) on tongue\nvolume and upper airway cross-sectional areas.\nMethods: Cadaveric brachycephalic dogs (n = 6) were positioned with the\ntongue retracted and jaw nearly closed. Computed tomography was performed\nto evaluate tongue volume and cross-sectional areas of tongue, oropharynx,\npalatal soft tissue and nasopharynx at two levels, the caudal aspect of the hard\npalate and pterygoid hamulae. A standardized CMG was performed. Positioning\nand CT scan were repeated.\nResults: CMG resulted in a 20% decrease in tongue volume (from 87,546 ± 21,121\nto 70,259 ± 17,586 mm3; p < 0.01). CMG resulted in a 20 to 25% decrease in cross-\nsectional area of the tongue at both hard palate (from 1662 ± 311 to 1339 ± 254\nmm2; p < 0.01) and pterygoid hamulae (from 1425 ± 222 to 1041 ± 150 mm2;\np < 0.01), and 2 to 3-fold increase in cross-sectional area of the oropharynx\nat both hard palate (from 226 ± 68 to 595 ± 138 mm2; p < 0.01) and pterygoid\nhamulae (from 110 ± 64 to 351 ± 37 mm2; p < 0.01).\nDiscussion: This study provides preliminary guidelines toward the feasibility\nand potential benefit of CMG in select cases of macroglossia-associated upper\n\n01) and pterygoid\nhamulae (from 110 ± 64 to 351 ± 37 mm2; p < 0.01).Discussion: This study provides preliminary guidelines toward the feasibility\nand potential benefit of CMG in select cases of macroglossia-associated upper\nairway obstruction.\nKEYWORDS\nBOAS, brachycephalic, brachycephalic airway obstruction syndrome, macroglossia,\nglossectomy\nIntroduction\nArtificial selection of genetic mutations linked to skull shape in dogs has resulted in breeds\nwith varying degrees of facial retrusion, the shortened snout and widened hard palate common\nto brachycephalic dogs (1). These skeletal changes obstruct air flow in the nasal passages;\nhowever, the entire upper airway is further compromised by a relative excess of all the\nOPEN ACCESS\nEDITED BY\nBryden Stanley,\nMichigan State University, United States\nREVIEWED BY\nKathleen Ham,\nThe Ohio State University, United States\nKelley Thieman,\nTexas A&M University, United States\n*CORRESPONDENCE\nRaymond K. Kudej\nraymond.kudej@tufts.edu\nRECEIVED 08 April 2025\nACCEPTED 14 July 2025\nPUBLISHED 04 August 2025\nCITATION\nColberg VT, Kudej RK, Moyer N, Peters JA and\nKarlin WM (2025) Evaluation of a caudal\nmidline glossectomy on tongue volume and\nupper airway cross-sectional areas in\nbrachycephalic dogs: a cadaveric study.\nFront. Vet. Sci. 12:1607711.\ndoi: 10.3389/fvets.2025.1607711\nCOPYRIGHT\n© 2025 Colberg, Kudej, Moyer, Peters and\nKarlin. This is an open-access article\ndistributed under the terms of the Creative\nCommons Attribution License (CC BY). The\nuse, distribution or reproduction in other\nforums is permitted, provided the original\nauthor(s) and the copyright owner(s) are\ncredited and that the original publication in\nthis journal is cited, in accordance with\naccepted academic practice. No use,\ndistribution or reproduction is permitted\nwhich does not comply with these terms.\nTYPE  Original Research\nPUBLISHED  04 August 2025\nDOI  10.3389/fvets.2025.1607711\n\nColberg et al.\b\n10.3389/fvets.2025.1607711\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nassociated soft tissue structures which did not scale down\nproportionately with the skull, e.g., nostrils, turbinate mucosa, soft\npalate, tonsils and tongue (2).\nEpidemiological studies have found that extreme brachycephalic\nbreeds (Bulldog, French bulldog and Pug) die younger than other breeds,\nwith a higher proportion of deaths related to upper respiratory tract\ndysfunction (3). Brachycephalic obstructive airway syndrome (BOAS) is\na general term used to describe the full spectrum of structural and\nfunctional aberrations seen in some clinically affected brachycephalic\ndogs. Numerous primary anatomic abnormalities and deleterious\nsecondary sequalae have been associated with BOAS (4, 5). Primary\nanatomic abnormalities include stenotic nares, elongated soft palate,\naberrant nasopharyngeal turbinates, hypoplastic trachea, and\nmacroglossia (5–11). Secondary sequelae include progressive laryngeal\ncollapse, esophagitis, regurgitation, hiatal herniation, aspiration\npneumonia, bronchial collapse, heart base tumors, altered blood\nparameters, and sleep apnea (6, 12–17). Consequently, numerous surgical\nprocedures have been described to improve the airflow and welfare of\nthese animals including alarplasty, staphylectomy, laryngeal sacculectomy,\nlaser-assisted turbinectomy, and tonsillectomy (18, 19).\nInterestingly, recent studies have determined that macroglossia may\nbe a contributing factor to upper airway obstruction in dogs with BOAS\n(20, 21). Using computed tomography (CT), Jones et al. (20) demonstrated\na relative macroglossia with reduced air volume in the upper airway in\nbrachycephalic compared to mesaticephalic dogs, specifically at the level\nof the caudal hard palate and hamulae of the pterygoid bone. The\nconclusion of their study was that a relative macroglossia in brachycephalic\nbreeds may contribute to upper airway obstruction. Seidenburg and\nDupre used CT scans to compare total tongue volume and cross-sectional\nareas of the tongue, oropharynx, soft palate, and nasopharyngeal airways\n\nSeidenburg and\nDupre used CT scans to compare total tongue volume and cross-sectional\nareas of the tongue, oropharynx, soft palate, and nasopharyngeal airways\nin three common brachycephalic breeds, pugs and French and English\nbulldogs. Their study noted that normalized tongue volumes and cross-\nsectional areas were smaller in the pug, and that this finding should\nbe taken into account if surgical correction of a relative macroglossia is\nconsidered in this breed.\nVery little information is available in the veterinary literature on the\ntopic of tongue reduction glossectomy, e.g., feasibility and utility. Canine\nBOAS and human sleep apnea have many similarities, e.g., disordered\nbreathing and episodes of oxygen desaturation associated with abnormal\nupper airway anatomy (9). Interestingly, caudal midline glossectomy\n(CMG) tongue reduction surgery is used for humans with severe sleep\napnea persistent after failure of palatal surgery (22–24). Although CMG\nhas a therapeutic role in human medicine, its potential role in relieving\nupper airway obstruction secondary to macroglossia in brachycephalic\ndogs remains undetermined.\nTo assess the feasibility and potential benefit in select cases of\nmacroglossia-associated upper airway obstruction, the first objective\nof this study was to evaluate the effect of a standardized CMG\nprocedure on tongue volume, as well as cross-sectional areas of the\nupper airway in brachycephalic dog cadavers. Additionally,\nwe intended to assess the amount of caudal tongue muscle removed\nas well as proximity to the lingual artery.\nMaterials and methods\nSix donated, cadaveric brachycephalic dogs were used within 48 h\nof euthanasia. Dogs were held in a refrigerated room (4–8°C) prior\nto study and held at room temperature during setup time\n(approximately 2 h). None of the dogs had a clinical history of BOAS\nor prior airway surgery performed. All of the dogs were euthanized\nfor conditions unrelated to BOAS. Upper airway and oral\nexaminations were performed post-mortem to confirm the lack of\nprior airway surgery or obvious abnormalities of the airway or\ntongue, e.g., tumors.\n\nAll of the dogs were euthanized\nfor conditions unrelated to BOAS.Upper airway and oral\nexaminations were performed post-mortem to confirm the lack of\nprior airway surgery or obvious abnormalities of the airway or\ntongue, e.g., tumors.\nCadavers were positioned on the patient table using the same\nprotocol: sternal recumbency, maxilla suspended with tape behind the\nmaxillary canines and the hard palate parallel to the table. Initial\npositioning of the mandible and tongue for each dog was with the\ntongue retracted and the jaw nearly closed. Care was taken to maintain\nprecise initial positioning throughout the experimental protocol for\neach cadaver, i.e., (1) between the rostral tongue and mandibular\nincisors, and (2) distance between the upper  and lower incisors.\nRadiolucent foam was placed below the rostral mandible to maintain\nits position (Figure 1). Computed tomography was then performed of\nthe entire skull. Following a caudal, midline reduction glossectomy,\ncadavers were replaced into the gantry in the exact same position and\nCT scans were repeated similarly.\nComputed tomography examination\nA 16-slice CT scanner (Aquilion 16, Toshiba Medical Systems,\nOtawara, Japan) was used to perform all imaging. Volumetric data\nwas acquired in 0.5-1 mm slice thickness from a level rostral to\nthe nasal planum to a level caudal to the larynx by using a pitch\nof 0.69, 120 kVp, 100 mA, and a tube rotation time of 0.5 s. Sagittal\nand transverse images were reconstructed in either 0.5- (n = 1),\n1- (n = 1), 2- (n = 3) or 3-mm (n = 1) slice thickness using a soft\ntissue algorithm.\nFIGURE 1\nPositioning of dog for computed tomography. Note that the maxilla\nis suspended with tape behind the canines and the hard palate is\nparallel to the table. Radiolucent foam was used ventral to the rostral\nmandible to maintain consistent distance between upper and lower\nincisors.\n\nColberg et al.\b\n10.3389/fvets.2025.1607711\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nDigital Images and Communication in Medicine (DICOM) files\nfrom the computed tomography were uploaded and segmented using\nMimics Innovation Suite 26 (Materialise NV, Leuven Belgium) with\nthresholds set at −1,023 to −75 Hounsfield units (HU) and − 200 to 150\nHU focused on soft tissue segmentation (20). Additional segmentation\nof the transitional areas was performed as previously described (20).\nAssessments of the total tongue volume and cross-sectional areas of the\nupper airway were performed as previously described (20, 21). Briefly,\nthe tongue was segmented in all planes to ensure accurate volume\nrendering. Total tongue volume was measured according to the following\nguidelines for determining borders: ventrally-geniohyoid, mylohyoid,\nand hyoid muscles; dorsally-oral cavity and oropharynx; laterally-\nmandibular teeth, digastricus muscle, and hyoid apparatus; and caudally-\nbasihyoid bone. Positioning was verified in transverse, dorsal, and\nsagittal planes. If the hard palate was not parallel to the CT table,\nreformatted transverse images were created within the Mimics software\nso that the transverse planes were perpendicular to the plane of the hard\npalate to ensure accurate cross-sections. Tongue, oropharynx,\nnasopharynx, and palatal soft tissue areas were evaluated at two\ntransverse areas, the caudal hard palate (HP) and pterygoid hamulae\n(PH). HP was determined as the very last transverse CT slice in which\nthe caudal nasal spine was visible. PH was determined as the very last\ntransverse CT slice in which the hamulae of the pterygoid were visible.\nCT images were also used to measure the skull index (skull width/skull\nlength ratio) to categorize the cadaveric skull conformation in our\ncadaveric dogs (25). The width was determined as the greatest distance\nbetween the outer borders of the zygomatic arches on transverse view.\nThe length was determined as the most rostral aspect of the incisive bone\nby the caudal border of the occipital bone (dorsal border of the foramen\nmagnum) on sagittal view.\n\nThe length was determined as the most rostral aspect of the incisive bone\nby the caudal border of the occipital bone (dorsal border of the foramen\nmagnum) on sagittal view.\nCaudal midline reduction glossectomy\nThe patient was placed in sternal recumbency with the maxilla\nsuspended with gauze for visualization of the oral cavity. Caudal midline\nreduction glossectomy was performed (22, 23). A rectangular midline\nsegment of tongue was excised using a #10 blade, beginning at the base\nof the epiglottis and extending rostrally approximately 60% of the\ntongue length (Figure 2). The CMG tissue dimensions used in this study\nwere standardized (33% width and 50% depth of the caudal tongue and\n60% of the total tongue length) and based on literature descriptions,\ninitial CT measurements (sagittal and transverse images) and lingual\nartery anatomic position. The glottal defect was closed using a simple\ncontinuous suture pattern of 2–0 nylon suture. After completion of post-\nglossectomy CT imaging, the tongue was dissected from the cadaver\nand tissue measurements and weights were performed.\nStatistical analysis\nAge, bodyweight, body condition score (BCS; 1–9 scale) and skull\nconformation data were reported with median values. Continuous\nnumerical variables assessed included tongue volume and dimensions,\nand cross-sectional areas (nasopharynx, palatal soft tissue,\noropharynx, tongue). Continuous variables were reported as mean ±\nstandard error of the mean. Paired data were examined statistically for\nall comparisons. Comparisons between the pre-and post-glossectomy\ndata were made by Student’s t-test, with p < 0.05 taken as the level\nfor significance.\nResults\nBrachycephalic breeds included English bulldog (n = 2), American\nbulldog (n = 1), Pug (n = 2), and Boston terrier (n = 1). The median\nage was 9.5 years, bodyweight was 13.4 kg, and BCS was 6.0. The\nmedian L: W index and skull index values were 1.09 and 0.92,\nrespectively (Table 1).\nThe CMG resulted in a decrease (p < 0.001) in mean cross-\nsectional area of the tongue at both HP (20 ± 3%) and PH (25 ± 5%).\n\n5 years, bodyweight was 13.4 kg, and BCS was 6.0.The\nmedian L: W index and skull index values were 1.09 and 0.92,\nrespectively (Table 1).The CMG resulted in a decrease (p < 0.001) in mean cross-\nsectional area of the tongue at both HP (20 ± 3%) and PH (25 ± 5%).\nConversely, CMG resulted in a 2.5-fold increase (p < 0.005) in mean\nFIGURE 2\nCaudal midline reduction glossectomy was performed beginning at the base of the epiglottis and extending rostrally approximately 60% of the tongue\nlength.\n\nColberg et al.\b\n10.3389/fvets.2025.1607711\nFrontiers in Veterinary Science\n04\nfrontiersin.org\ncross-sectional area of the oropharynx at HP and a 3-fold increase\n(p < 0.005) at PH (Figure 3). There was no significant difference in\nmean areas of the nasopharynx, palatal soft tissue or total area\nbetween pre-glossectomy and post-glossectomy at either level\n(Table 2).\nThere was a 21 ± 3% decrease (p < 0.01) in mean tongue volume\nbetween pre-and post-glossectomy (Table 3). Mean tongue volume\npre-and\npost-glossectomy\nfor\nthe\nbulldogs\n(n = 3)\nwas\n132,176 ± 18,543 and 106,920 ± 16,564  mm3, respectively. Mean\ntongue volume pre-and post-glossectomy for the pugs and Boston\nterrier (n = 3) was and 42,916.1 ± 3,736 and 33,599 ± 5,392 mm3,\nrespectively. Mean tongue volume/bodyweight pre- and post-\nglossectomy for the bulldogs was 5,420 ± 243 and 4,367 ± 94 mm3/kg,\nrespectively. Mean tongue volume/bodyweight pre- and post-\nglossectomy for the pugs and Boston terrier was 5,450 ± 884 and\n4,277 ± 940 mm3/kg, respectively.\nThere was no significant difference in inter-arterial (lingual)\ndistance/tongue width pre- versus post-glossectomy at either HP\n(45 ± 2 versus 49 ± 4%) or PH (50 ± 2 versus 45 ± 4%). There was no\nsignificant difference in lingual artery depth/tongue depth pre- versus\npost-glossectomy at either HP (68 ± 2 versus 69 ± 1%) or PH (66 ± 3\nversus 67 ± 5%) (Table 4).\nPercent CMG tissue/total tongue data from excised tissue\nfollowing study completion for weight, width, depth and length were\n19 ± 2%, 35 ± 1%, 49 ± 1% and 53 ± 4%, respectively (Table 5).\nDiscussion\nThe current cadaveric study demonstrated a potential benefit for\nCMG to reduce tongue volume in dogs with macroglossia-related\nobstruction of the caudal oral cavity and pharynx. The procedure used\nin the study involved removing approximately 33% width and 50%\ndepth of the caudal tongue (tongue root extending rostrally into body)\nand 60% of the total tongue length. This standardized glossectomy\nprocedure averaged a 20% reduction in total tongue volume. From\nfixed, baseline positions of the tongue and mandible determined by\nexperimental design, the midline glossectomy averaged a 20–25%\n\nThis standardized glossectomy\nprocedure averaged a 20% reduction in total tongue volume.From\nfixed, baseline positions of the tongue and mandible determined by\nexperimental design, the midline glossectomy averaged a 20–25%\nreduction of the cross-sectional area of the caudal tongue within the\npharynx. Additionally, the glossectomy width and depth kept the\nlingual artery at a reasonably safe distance.\nThe dimensions used for CMG in this cadaveric study are not\nprovided as a universal recommendation for all clinical procedures to\nreduce the tongue, only as a solid frame of reference. Each case may\nhave individual variations in tongue anatomy which will need to\nbe  accounted for using a pre-operative contrast CT for surgical\nplanning. A CMG procedure is not described in the veterinary\nliterature. Interestingly, descriptions of CMG techniques in human\nliterature are often poorly detailed, i.e., do not explicate any parameters\nor guidelines for the extent of tissue excision. Limiting factors\nmentioned relate to the preservation of blood supply and muscular\nfunction of the tongue, i.e., lingual arteries and genioglossus muscle.\nThe lingual arteries are lateral, medial and adjacent to the genioglossus,\nhyoglossus and inferior longitudinal muscles, respectively. Human\nliterature describes the use of intra-op ultrasound or placing only\nsuperficial sutures to prevent damage to a lingual artery during CMG\n(22, 23). An additional surgical concern was the depth of the\nglossectomy be  limited to prevent functional deficits of the\ngenioglossus muscle and tongue movement (23, 24). The genioglossus\nis an extrinsic muscle which forms the bulk of the tongue (middle and\nventral). Given the lack of specific guidelines in the literature, the\nextent of the standardized technique used in this cadaveric study was\nbased on subjective assessment of diagrams and concern for\ngenioglossus muscle function, the position of the lingual arteries on\nour transverse CT sections of the caudal tongue, and our CT sagittal\nviews showing the prominent areas of dorsal incursion of the tongue\n\ninto the pharynx and caudal oral cavity. It seems reasonable that a\npre-operative contrast CT and similar guidelines would dictate the\nextent of CMG performed for a clinical case.\nMean L: W index and skull index for all the dogs in our study were\nwithin the range for brachycephalism, i.e., below 1.44 (26) and above\n0.81 (27), respectively, but less severe than in recent clinical studies\nexamining tongue volume and upper airway cross-sectional area\nmeasurements in brachycephalic dogs (20, 21). Skull index has been\nshown previously to have a significant association with BOAS status\nin pugs and bulldogs, but not all brachycephalic dogs, and other\nfactors are involved (10). However, our data are consistent with such\nan association in that none of our dogs had a history of clinical signs,\nprior BOAS surgery or any stage of laryngeal collapse on post-\nmortem examination.\nCross-sectional areas of the tongue decreased (p < 0.01) and\noropharynx increased (p < 0.01) following CMG at both levels of the\npharynx. These changes resulted in an approximately 3-fold increase\n(p < 0.01) in airway/tongue and airway/tissue ratios at both the HP\nand PH. As discussed in Seidenberg and Dupre, no consensus exists\nregarding appropriate normalization methods for cross-sectional areas\nTABLE 1  Signalment, bodyweight, body condition score, and skull conformation.\nBreed\nGender\nAge (years)\nWeight (kg)\nBCS (1–9)\nSkull\nLength\n(cm)\nSkull\nWidth\n(cm)\nL: W\nindex\nSkull\nindex\nEnglish bulldog\nMN\n8.0\n18.0\n5.0\n14.8\n12.7\n1.17\n0.86\nEnglish bulldog\nMN\n9.0\n26.0\n7.0\n13.3\n14.3\n0.93\n1.08\nAmerican\nbulldog\nF\n9.0\n29.8\n6.0\n16.4\n13.1\n1.25\n0.80\nPug\nF\n15.0\n7.0\n6.0\n9.1\n8.9\n1.02\n0.98\nPug\nM\n14.0\n8.3\n6.0\n9.5\n8.6\n1.10\n0.91\nBoston Terrier\nM\n10.0\n8.7\n5.0\n10.8\n10.0\n1.08\n0.93\nMedian\n9.5\n13.4\n6.0\n12.1\n11.4\n1.09\n0.92\n\nColberg et al.\b\n10.3389/fvets.2025.1607711\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nor tissue volumes of parts of the skull in small animals. Therefore,\nwe included normalization indices used in prior studies (20, 21).\nHowever, an important consideration for interpreting or comparing\nupper airway cross-sectional areas is the beam angle of the CT relative\nto the rostro-caudal axis of the hard palate, tongue and mandible.\nInterestingly, these angles were markedly different between each of the\nrecent brachycephalic airway studies, i.e., Jones et al. (20), Seidenberg\nand Dupre, and the current study. Briefly, Jones et al. (20) positioned\nthe hard palate parallel to the table and angled the mandible and\ntongue downward. Seidenberg and Dupre held the mandible and\ntongue parallel to the table and angled the hard palate upward. In our\nstudy the hard palate was held parallel to the table, but the mandible\nand tongue were also placed near parallel. Each of these different head\npositions will result in different relative sizes of the adjacent areas of\ninterest, i.e., the measured cross-sectional area will increase the farther\nthe structure axis deviates from parallel to the patient table. Depending\non what is being analyzed, standard or consensus positioning would\nbe an important consideration for future studies.\nDespite the positioning differences, it is interesting to compare the\ncross-sectional area proportion of air (oropharyngeal and\nFIGURE 3\nCT images of a representative cadaveric dog pre-glossectomy (left column) and post-glossectomy (right column). Midline sagittal images (A,B)\nillustrate the transverse levels at the caudal hard palate (HP) and pterygoid hamulae (PH) with red lines. Transverse images (C,D) are at the HP;\nTransverse images (E,F) are at the level PH. Highlighted areas represent the nasopharynx (pink), palatal tissues (aqua), oropharynx (royal blue), and\ntongue (green). In all images, note the decreased tongue tissue and markedly increased oropharyngeal air post-glossectomy (right) compared with\npre-glossectomy (left).\n\nIn all images, note the decreased tongue tissue and markedly increased oropharyngeal air post-glossectomy (right) compared with\npre-glossectomy (left).\nTABLE 2  Cross sectional areas at hard palate and pterygoid hamulae.\nArea\nHard palate\nPterygoid hamulae\nPre-glossectomy\nPost-\nglossectomy\np-value\nPre-glossectomy\nPost-\nglossectomy\np-value\nTongue (mm2)\n1,662 ± 311\n1,339 ± 254*\n0.005\n1,425 ± 222\n1,041 ± 150*\n0.009\nOropharynx (mm2)\n225 ± 68\n595 ± 138*\n0.004\n110 ± 64\n351 ± 37*\n0.001\nSoft Palate (mm2)\n145 ± 32\n140 ± 36\n0.751\n358 ± 73\n370 ± 73\n0.224\nNasopharynx\n(mm2)\n111 ± 25\n106 ± 25\n0.225\n90 ± 19\n98 ± 19\n0.141\nTotal area (mm2)\n2,143 ± 419\n2,179 ± 426\n0.178\n1984 ± 344\n1860 ± 263\n0.253\nAirway-total (mm2)\n337 ± 93\n701 ± 163*\n0.005\n201 ± 79\n449 ± 53*\n0.001\nTissue-total (mm2)\n1806 ± 332\n1,478 ± 273*\n0.004\n1783 ± 290\n1,411 ± 219*\n0.010\nAirway/Tongue (%)\n0.19 ± 0.04\n0.53 ± 0.07*\n0.001\n0.13 ± 0.04\n0.44 ± 0.03*\n0.001\nAirway/Tissue (%)\n0.17 ± 0.03\n0.46 ± 0.05*\n0.0003\n0.10 ± 0.03\n0.33 ± 0.002*\n0.002\nValues are mean ± SEM.\n*Significant difference from pre-glossectomy using paired Student’s t-test.\n\nColberg et al.\b\n10.3389/fvets.2025.1607711\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nnasopharyngeal)/soft tissue (tongue and palatal) at the hard palate and\npterygoid hamuli levels between studies. The relative positions of the\nhard palate and mandible used in the current study, compared to tongue\nand airway measurements in prior clinical studies (20, 21), more closely\nresembled those of a dog with tongue retracted and jaw closed. Indeed,\nour incisor/mandibula opening angles pre- and post-glossectomy were\n10.6 ± 1.1 and 10.7 ± 1.3 degrees, respectively, more acute than those\nreported by Siedenburg and Dupre (24.5 to 29.6 degrees). Thus, due to\nexperimental design, our mean baseline air/soft tissue ratios at these\nlevels (0.17 ± 0.03 and 0.10 ± 0.03) were much smaller than the averaged\nbrachycephalic medians reported by either Jones et al. (20) (0.60 and\n0.28) or Siedenburg and Dupre (0.54 and 0.26). However, after CMG our\nmean baseline air/soft tissue ratios at the HP and PH (0.46 ± 0.05 and\n0.33 ± 0.01) were nearer to previously published data, especially those for\nthe English bulldog (0.48 and 0.15) (21). Considering our baseline data,\nthese latter comparisons are consistent with a potential beneficial effect\nof CMG to increase upper airway air/soft tissue ratios.\nOur baseline tongue volumes were similar to prior studies, including\nbreed differences (20, 21). After CMG, our group values for average\ntongue volume and volume/body weight ratio were 70,259 ± 17,586 mm3\nand 4,322 ± 346 mm3/kg, respectively. It is worth noting that our mean\nvolume/body weight ratio after CMG was similar to the median values\npreviously reported for mesaticephalic breeds (4,454 mm3/kg) (20) and\npugs (4,362 mm3/kg) (21).\nSurprisingly, CMG resulted in no change in either lingual artery\ninter-arterial distance/tongue width or arterial depth from the dorsal\nsurface of the tongue/tongue depth ratio measured at both the hard\npalate and pterygoid hamuli. In other words, any CMG related change\nin an arterial measurement occurred with a similar change in the\ncorresponding tongue measurement. These corresponding changes are\n\nIn other words, any CMG related change\nin an arterial measurement occurred with a similar change in the\ncorresponding tongue measurement.These corresponding changes are\nmost easily recognized at the level of the pterygoid hamulus. For\nexample, the decrease in pre- versus post-CMG inter-arterial distance\n(24.9 ± 2.3 versus 20.7 ± 1.8 mm) coincided with a decrease in tongue\nwidth (50.2 ± 4.4 versus 44.6 ± 4.2 mm). Importantly, the extent of the\nCMG used in the current study did not damage the lingual arteries in\nany of our cadavers, as assessed by the CT cross-sectional images and\nsubsequent tongue dissection after each experiment. Although lingual\nartery position on CT cross-sectional images was determined from\ncadaveric tongues, their anatomic position is lateral and ventral to the\ngenioglossus and intrinsic musculature, respectively, each readily\nidentifiable on our transverse CT images. The methodology used to\ndetermine the position of the lingual arteries was reasonable, consistent\nand supported by tissue dissection. However, planning for and\nperforming this procedure on a clinical patient would necessitate a\nthorough knowledge of tongue anatomy and a pre-operative contrast CT\nstudy including an accurate measurement of lingual artery position.\nAfter completion of each study, the tongue was excised from the\ncadaver, tissue measurements were recorded, and the tongue was further\ndissected to check for lingual artery position and integrity.\nNotwithstanding dissection error and loss of anatomic position and\nmoisture, examination of the excised tissues provided a reasonable\nassessment of relative proportions related to the CMG, i.e., removal of\napproximately 20% tongue volume, 33% width, 50% depth and 60%\nlength (Table 5), and further dissection supported CT assessed lingual\nartery position and maintenance of integrity.\nThis cadaveric feasibility study has several limitations in addition\nto those already mentioned. It is not possible to directly extrapolate\npost-mortem measurements or results directly to performing the\nprocedure on a clinical patient, as cadaveric tissues do not have\n\nIt is not possible to directly extrapolate\npost-mortem measurements or results directly to performing the\nprocedure on a clinical patient, as cadaveric tissues do not have\nidentical properties or response compared to viable tissue. Additionally,\nwe closed our cadaver CMG with a single layer, simple continuous\nsuture pattern of 2–0 nylon instead of a multiple layer closure technique\nthat would be used in a clinical procedure (28). Post-operative swelling,\nperi-operative management and clinical outcomes may preclude any\napparent cadaveric utility or feasibility. However, this technique is used\nin humans and has been used successfully in a clinical case of\nmacroglossia in a dog secondary to stable muscular dystrophy (RK).\nThis study had a limited sample size (n = 6); however, results were\npaired, consistent and statistical significance was achieved. As with\nprevious similar studies (20, 21), our imaging software required specific\nair and soft tissue areas to be  identified on CT images of tissue.\nHowever, our studies were performed on cadavers within 24–48 h of\neuthanasia, and tongue structure delineation was consistently\ndemonstrable. Prior to CT analysis, it was important to become\nfamiliar with the anatomy and relative positioning of tongue structures,\ne.g., with an imaging-based canine anatomy atlas and reviewing\ncontrast CT images of clinical patients. We used consistent anatomical\nguidelines for each structure and area, similar to prior studies (20, 21).\nWe also used similar methodology to minimize overlap and ensure that\nair was not included in soft tissue measurements and vice versa (20).\nAdditionally, the methodology used in the current study was consistent\nfor all dogs and, therefore, should not have markedly altered the data\nor deterred from the purpose of this feasibility study. Importantly, our\nCT findings were corroborated with tongue tissue dissection after the\nexperimental protocol.\nThe findings of this study were consistent with CMG potentially\nbeing feasible and beneficial as an adjunctive surgical procedure in\n\nImportantly, our\nCT findings were corroborated with tongue tissue dissection after the\nexperimental protocol.The findings of this study were consistent with CMG potentially\nbeing feasible and beneficial as an adjunctive surgical procedure in\nselect BOAS patients, e.g., have Stage 1 laryngeal collapse, are\nminimally to non-responsive to standard surgical techniques, and have\nprofound relative macroglossia. This technique could also be employed\nfor other select clinical cases where macroglossia is causing moderate\nto severe dyspnea, or possibly dysphagia, e.g., a dog with stable\nmuscular dystrophy. Although this procedure is used in humans and\nappears to be feasible and potentially beneficial for select dogs, its\nclinical use would require a pre-operative contrast CT and thorough\nknowledge of tongue anatomy. Additionally, peri-operative\nmanagement must account for and accommodate temporary post-\noperative tissue swelling, i.e., compromised airway or ability to\nswallow. Given the apparent feasibility and potential benefits, the\ncurrent study should help to establish reasonable guidelines and\nexpectations when performing CMG. We believe that it was important\nTABLE 3  Tongue volumes pre- and post-glossectomy.\nParameter\nPre-\nglossectomy\nPost-\nglossectomy\np-\nvalue\nTotal tongue\nvolume (mm3)\n87,546 ± 21,121\n70,259 ± 17,586*\n<0.01\nTongue volume/\nbodyweight\n5,435 ± 335\n4,322 ± 346*\n<0.001\nTongue volume/\nLW index\n89,014 ± 20,647\n71,011 ± 17,059*\n<0.001\nTongue volume/\nskull length\n724 ± 115\n575 ± 99*\n<0.002\nValues are Mean ± SEM.\n*Significant difference from pre-glossectomy using paired Student’s t-test.\n\nColberg et al.\b\n10.3389/fvets.2025.1607711\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nto see the expected utility of this procedure in a cadaver prior to\nclinical use.\nData availability statement\nThe original contributions presented in the study are included in\nthe article/supplementary material, further inquiries can be directed\nto the corresponding author.\nEthics statement\nThe animal studies were approved by Institutional Animal Care\nand Use Committee, Tufts University, Cummings School of Veterinary\nMedicine. The studies were conducted in accordance with the local\nlegislation and institutional requirements. Written informed consent\nwas obtained from the owners for the participation of their animals in\nthis study.\nAuthor contributions\nVC: Writing – original draft, Writing – review & editing, Data\ncuration, Formal analysis, Investigation, Visualization, Methodology.\nRK: Conceptualization, Formal analysis, Funding acquisition,\nInvestigation, Methodology, Writing – original draft, Writing – review\n& editing, Project administration, Supervision. NM: Writing – original\ndraft. JP: Investigation, Writing  – review & editing. WK: Data\ncuration, Formal analysis, Investigation, Visualization, Writing  –\nreview & editing, Software, Supervision.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. This study was funded by\nthe Companion Animal Health Fund, Tufts University, Cummings\nSchool of Veterinary Medicine.\nAcknowledgments\nWe would like to thank the Companion Animal Health Fund and\nall the dog owners for their participation in this study.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\n\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. Any product that may be evaluated in this article, or\nclaim that may be made by its manufacturer, is not guaranteed or\nendorsed by the publisher.\nTABLE 4  Lingual artery and tongue measurements from CT cross-sectional images.\nMeasurement\nHard palate\nPterygoid hamulae\nPre-\nglossectomy\nPost-\nglossectomy\n%\nreduction\np-\nvalue\nPre-\nglossectomy\nPost-\nglossectomy\n%\nreduction\np-\nvalue\nInter-arterial\ndistance (mm)\n22.9 ± 2.6\n22.9 ± 2.4\n−1.9 ± 7.9\n0.982\n24.9 ± 2.3\n20.7 ± 1.8*\n16.2 ± 3.0\n0.004\nTongue width (mm)\n50.3 ± 4.1\n47.3 ± 3.9*\n5.9 ± 1.6\n0.018\n50.2 ± 4.4\n44.6 ± 4.2*\n11.5 ± 1.3\n<0.001\nArterial distance/\nTongue width\n45.2 ± 2.4\n48.8 ± 3.9\n−8.1 ± 7.4\n0.280\n49.6 ± 1.9\n46.7 ± 1.0\n5.4 ± 3.3\n0.130\nLingual artery depth\n(mm)\n21.1 ± 2.9\n18.4 ± 2.4*\n12.3 ± 3.9\n0.032\n18.9 ± 1.6\n15.1 ± 0.8\n18.1 ± 7.4\n0.079\nTongue depth (mm)\n31.0 ± 3.4\n26.8 ± 3.6*\n14.3 ± 3.8\n0.003\n28.7 ± 2.3\n23.1 ± 1.9*\n19.5 ± 0.7\n<0.001\nArterial depth/\nTongue depth\n67.5 ± 2.2\n68.9 ± 0.6\n−2.5 ± 3.1\n0.499\n66.0 ± 3.1\n66.5 ± 4.7\n−2.0 ± 9.5\n0.943\nValues are mean ± SEM.\n*Significant difference from pre-glossectomy using paired Student’s t-test.\nTABLE 5  Excised tongue measurements and weights.\nMeasurement\nWeight\n(grams)\nWidth\n(mm)\nDepth\n(mm)\nLength\n(mm)\nTissue\n13.2 ± 3.4\n17.0 ± 1.5\n13.3 ± 1.5\n67.0 ± 5.8\nTongue\n71.2 ± 17.9\n48.3 ± 3.8\n27.0 ± 3.0\n130.8 ± 14.3\nTissue/Tongue %\n19.1 ± 2.1\n35.1 ± 0.8\n49.4 ± 1.4\n52.7 ± 4.4\nValues are mean ± SEM.\n\nColberg et al.\b\n10.3389/fvets.2025.1607711\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nReferences\n1.\tMarchant TW, Johnson EJ, McTeir L, Johnson CI, Gow A, Liuti T, et al. 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+{"text": "【兽医文献】\n标题: Mechanism and application of yeast and its culture in regulating intestinal antioxidant defense in ruminants\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nFree thyroxine measured by\nchemiluminescence and\nequilibrium dialysis is frequently\nbelow the reference interval in\nknown euthyroid dogs with\nnonthyroidal illness syndrome\nTimothy A. Bolton 1*, Christina L. Marino 2, Maxime G. Derré 3,\nGeorge E. Moore 4, Julie Piccione 5 and Malcolm L. Delovio 5\n1 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West\nLafayette, IN, United States, 2 Small Animal Veterinary Specialists, Wyndmoor, PA, United States,\n3 College of Veterinary Medicine, Purdue University, West Lafayette, IN, United States, 4 Department of\nVeterinary Administration, College of Veterinary Medicine, Purdue University, West Lafayette, IN,\nUnited States, 5 Texas A&M Veterinary Medical Diagnostic Laboratory, College Station, TX,\nUnited States\nIntroduction: Measuring serum free thyroxine (fT4) concentration is\nrecommended to distinguish hypothyroidism from nonthyroidal illness\nsyndrome (NTIS) in dogs having a serum total thyroxine (TT4) concentration\nbelow the reference interval (RI). Serum fT4 concentration can be measured\nby equilibrium dialysis (fT4ED) or chemiluminescent immunoassay (fT4CLIA),\nneither of which have been extensively evaluated in known euthyroid dogs with\nNTIS. The objectives of this study were to: (1) determine agreement between\nfT4ED and fT4CLIA in serum samples from established euthyroid dogs with\nNTIS, (2) evaluate how frequently each fT4 assay is within the RI when the serum\nTT4 concentration is within and below the RI, and (3) evaluate concordant and\ndiscordant fT4ED and fT4CLIA results in dogs with a serum TT4 concentration\nbelow the RI.\nMethods: This retrospective study used 116 banked serum samples from 38 known\neuthyroid dogs with NTIS. On each sample, fT4ED and fT4CLIA were measured, and\nagreement assessed by a Bland–Altman plot. Using the hospital admission serum\nTT4 concentration, dogs were stratified into two groups: TT4 within the RI (n = 16)\nand TT4 below the RI (n = 22). The frequency that each fT4 assay was within the RI\n\nUsing the hospital admission serum\nTT4 concentration, dogs were stratified into two groups: TT4 within the RI (n = 16)\nand TT4 below the RI (n = 22).The frequency that each fT4 assay was within the RI\nwas calculated in both groups. In dogs having a serum TT4 concentration below\nthe RI, concordant and discordant fT4 results were evaluated.\nResults: Assay comparison showed agreement, with no significant systematic or\nproportional bias. When the serum TT4 concentration was within the RI, fT4ED\nand fT4CLIA were within the RI in 100% (95% CI: 79.4–100%) and 94% (95% CI:\n69.8–99.8%) of dogs, respectively; however, when the serum TT4 concentration\nwas below the RI, fT4ED and fT4CLIA were within the RI in 41% (95% CI: 20.7–\n63.6%) and 45% (95% CI: 24.4–67.8%) of dogs, respectively. Concordant fT4ED\nand fT4CLIA were present in 77% (95% CI: 54.6–92.2%) of dogs with a serum\nTT4 concentration below the RI.\nDiscussion: The fT4ED and fT4CLIA were regularly below the RI in dogs with\nNTIS and a serum TT4 concentration below the RI, potentially resulting in a\nOPEN ACCESS\nEDITED BY\nRemo Lobetti,\nVeterinary Specialists Ireland, Ireland\nREVIEWED BY\nAndrea Corsini,\nUniversity of Parma, Italy\nVictoria Travail,\nConsultant, Adelaide, South Australia,\nAustralia\n*CORRESPONDENCE\nTimothy A. Bolton\ntabolton@purdue.edu\nRECEIVED 01 July 2025\nACCEPTED 16 September 2025\nPUBLISHED 10 October 2025\nCITATION\nBolton TA, Marino CL, Derré MG, Moore GE,\nPiccione J and Delovio ML (2025) Free\nthyroxine measured by chemiluminescence\nand equilibrium dialysis is frequently below\nthe reference interval in known euthyroid\ndogs with nonthyroidal illness syndrome.\nFront. Vet. Sci. 12:1657215.\ndoi: 10.3389/fvets.2025.1657215\nCOPYRIGHT\n© 2025 Bolton, Marino, Derré, Moore,\nPiccione and Delovio. This is an open-access\narticle distributed under the terms of the\nCreative Commons Attribution License\n(CC BY). The use, distribution or reproduction\nin other forums is permitted, provided the\noriginal author(s) and the copyright owner(s)\nare credited and that the original publication\nin this journal is cited, in accordance with\naccepted academic practice. No use,\n\nThe use, distribution or reproduction\nin other forums is permitted, provided the\noriginal author(s) and the copyright owner(s)\nare credited and that the original publication\nin this journal is cited, in accordance with\naccepted academic practice.No use,\ndistribution or reproduction is permitted\nwhich does not comply with these terms.\nTYPE  Original Research\nPUBLISHED  10 October 2025\nDOI  10.3389/fvets.2025.1657215\n\nBolton et al.\b\n10.3389/fvets.2025.1657215\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nmisdiagnosis of hypothyroidism. This raises concerns about the ability of both\nfT4 assays to distinguish hypothyroidism from NTIS in this subset of dogs.\nKEYWORDS\neuthyroid sick syndrome, hypothyroidism, thyroid hormone assays, dog,\nendocrinology\n1 Introduction\nThyroid function in the dog is evaluated by measuring serum\nthyroid hormone concentrations, particularly total thyroxine (TT4),\nfree thyroxine (fT4), and thyroid-stimulating hormone (TSH). Up to\n35% of euthyroid dogs with nonthyroidal illness syndrome (NTIS)\ndemonstrate a decrease in serum TT4 concentration below the\nreference interval (RI), an alteration that frequently results in the\nincorrect diagnosis of hypothyroidism and unnecessary thyroxine\n(T4) supplementation in a euthyroid dog (1–3). Measuring serum fT4\nconcentration is recommended to distinguish NTIS from\nhypothyroidism in dogs with a low serum TT4 concentration since it\nmore accurately reflects the functional status of the thyroid gland by\nquantifying the amount of T4 available to the tissues (4).\nEquilibrium dialysis (ED) is considered the most accurate stand-\nalone test for the measurement of serum fT4 concentration since it\nseparates the free fraction of T4 from serum binding proteins, protein\nbinding inhibitors, and autoantibodies, such as T4 autoantibodies,\nthat falsely increase the serum TT4 concentration, and is less affected\nby NTIS (1–3, 5–8). Despite these advantages, ED is technically\ndemanding, time-consuming, and expensive, making it a cumbersome\nassay for use in veterinary laboratories processing large numbers of\nsamples (6). Conversely, measurement of the serum fT4 concentration\nwith a chemiluminescent immunoassay (CLIA) decreases the\ntechnical demand, time to results, and expense. Rather than dialysis\nto separate the free fraction of T4 from circulating proteins, CLIA\nemploys a radioligand analog that has minimal interaction with these\nproteins (9). A veterinary-specific fT4 CLIA (fT4CLIA) purports to\nhave a similar accuracy, but superior precision and turnaround time\n\nA veterinary-specific fT4 CLIA (fT4CLIA) purports to\nhave a similar accuracy, but superior precision and turnaround time\ncompared to fT4 measured by ED (fT4ED) (10). A recent study\ncomparing fT4ED and fT4CLIA in dogs with NTIS revealed\nsubstantial agreement (Cohen’s kappa coefficient 0.63 [95% CI: 0.47–\n0.79]) between the 2 assays (11). In this study, greater than 90% of\ndogs had a serum fT4 concentration within the RI with both assays\nwhen the serum TT4 concentration was also within the RI; however,\nthe fT4ED and fT4CLIA results were within the RI in only 43 and 12%\nof dogs, respectively, when the serum TT4 concentration was below\nthe RI (11). These findings suggest that in dogs with NTIS and a low\nserum TT4 concentration, fT4ED is less reliable than previously\nreported at differentiating NTIS from hypothyroidism and that the\nfT4CLIA appears inferior to the fT4ED at such a differentiation (2, 3,\n12). However, the inability to unambiguously exclude hypothyroid\ndogs when evaluating fT4 assays in dogs with NTIS is a limitation of\nthe previous studies (2, 3, 11). The most recent study comparing\nfT4ED and fT4CLIA might have unintentionally included hypothyroid\ndogs and knowingly included dogs administered medications that can\nfalsely lower the serum fT4 concentration, possibly contributing to the\nhigh percentage of serum fT4 concentrations below the RI (11). To\naddress this limitation, a comparable study investigating these fT4\nassays in established euthyroid dogs with NTIS was needed. Because\neuthyroid dogs with NTIS and serum TT4 and fT4 concentrations\nbelow the RI are frequently misdiagnosed and mistreated as\nhypothyroid in clinical practice, reassessing the capability of the\nfT4ED and fT4CLIA to distinguish NTIS from hypothyroidism in\nknown euthyroid dogs with a serum TT4 concentration below the RI\nis clinically relevant.\nThe objectives of this study were three-fold: (1) to determine the\nagreement between fT4ED and fT4CLIA in serum samples from\nknown euthyroid dogs with NTIS, (2) to evaluate how frequently each\nfT4 assay is within the RI when the serum TT4 concentration is within\n\nthe RI and below the RI, and (3) to assess concordant and discordant\nfT4ED and fT4CLIA results in dogs with a serum TT4 concentration\nbelow the RI. Our first hypothesis was that the fT4 assays would agree\nand not be statistically different. Our second hypothesis was that each\nfT4 assay would have a result within the RI with high frequency and\nlow frequency when the serum TT4 concentration was within and\nbelow the RI, respectively; however, since only euthyroid dogs were\nincluded in this study, the frequency of fT4 results within the RI when\nthe serum TT4 concentration was below the RI would be higher than\nin a previous study (11).\n2 Materials and methods\n2.1 Current study\nThis was a retrospective study utilizing banked serum samples\ncollected from client-owned dogs that participated in a prospective,\nobservational study performed at the Virginia-Maryland College of\nVeterinary Medicine (12). Banked samples from the original study\nwere included in the current study if serum of sufficient volume was\navailable. The current study was exempt from institutional animal care\nand use committee review since only banked samples were used.\nOne hundred sixteen banked samples from 38 euthyroid dogs\nwith NTIS had a sufficient volume of serum remaining to\nsimultaneously measure fT4ED and fT4CLIA in the current study.\n2.2 Original study\nThe original study received approval by the Virginia-Maryland\nCollege of Veterinary Medicine Institutional Animal Care and Use\nCommittee, and informed owner consent was obtained prior to\nenrollment. Dogs were eligible for inclusion in the prospective,\nobservational study to evaluate the alterations and time to resolution\nof thyroid function tests in dogs with NTIS if they suffered from acute\nillness (≤1-week duration) sufficiently severe to require hospitalization\n(12). Dogs fulfilling the inclusion criteria had a serum TT4\nconcentration measured at hospital admission. If the serum TT4\nconcentration was within the RI, no additional blood samples were\n\nBolton et al.\b\n10.3389/fvets.2025.1657215\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nobtained and the dog was excluded from the study. If the serum TT4\nconcentration was below the RI, further blood samples were obtained\nevery 24 h after hospital admission until hospital discharge (acute\nphase) and at 2 weeks and 4 weeks following hospital discharge\n(recovery phase).\nExclusion criteria (no blood sample collected) included\nsighthounds, a history of chronic disease (prior disease diagnosis\nrequiring ongoing treatment for management of clinical signs or\ncurrent illness >1-week duration), and administration of a\nmedication(s) known to affect thyroid function within 60 days of\nhospital admission. Excluded medications were acetylsalicylic acid,\namiodarone, glucocorticoids, phenobarbital, sulfonamides, thyroid\nhormone replacement, toceranib, tricyclic antidepressants, trilostane,\nand zonisamide.\nDuring the study, each blood sample collected was immediately\nplaced into a siliconized glass vacutainer tube containing no\nanticoagulant and centrifuged at 3,000 g for 10 min. Serum was\nharvested, aliquoted into two separate polypropylene red top tubes\n(1 mL serum into one tube and the remaining serum into a second\ntube), frozen at −80 °C within 1 h of collection, and stored at −80 °C\nuntil analysis. As each dog completed the study, batch submission of\nserum samples (tubes containing 1 mL serum) for the measurement\nof serum concentrations of TT4, fT4, TSH, and total triiodothyronine\nwas performed. All dogs analyzed in the study were deemed euthyroid\nbased on the resolution of all serum thyroid hormone abnormalities\nfound in both phases of illness, including low serum TT4, low serum\nfT4, and/or high serum TSH concentrations. Dogs excluded at the\ntime of hospital admission due to a serum TT4 concentration within\nthe RI were also deemed euthyroid.\nOne hundred banked samples from 22 euthyroid dogs with NTIS\nand a serum TT4 concentration below the RI at hospital admission\n(1–8 samples per dog obtained at 24 h to 2 week intervals) evaluated\nin the original study had a sufficient volume of serum remaining for\n\nuse in the current study and 16 banked samples from 16 euthyroid\ndogs with NTIS (1 sample per dog) excluded from the original study\nbecause of a serum TT4 concentration within the RI at hospital\nadmission had a sufficient volume of serum remaining for use in the\ncurrent study.\n2.3 Clinicopathologic analyses\n2.3.1 Serum fT4ED and fT4CLIA concentrations\nOne hundred sixteen banked serum samples were submitted to a\nsingle American Association of Veterinary Laboratory Diagnosticians\n(AAVLD) accredited laboratory (Texas A&M Veterinary Medical\nDiagnostic Laboratory (TVMDL), College Station, Texas) for\nconcurrent measurement of serum fT4 concentration using fT4ED\nand fT4CLIA. Serum samples included in the present study were\ncollected from January 2020 to December 2022 and were analyzed in\nJanuary 2024. Because the aliquots used in the current study were\nstored at −80 °C until analysis, all samples only underwent a single-\nfreeze–thaw cycle after they were batch shipped on dry ice to\nthe TVMDL.\nOn each serum sample, simultaneous measurement of serum fT4\nconcentration by fT4ED (Vet Direct fT4 by ED, IVD Technologies,\nSanta Ana, California) and fT4CLIA (Immulite 2000 XPi, Veterinary\nfT4, Siemens, Erlangen, Germany) was performed. The fT4ED assay\nwas performed in duplicate on each serum sample and an average\nwas reported.\nThe fT4ED assay is a commercially available kit validated in\naccordance with AAVLD standards through onboarding verification,\nongoing proficiency testing, and daily control charting. The assay has\nreported limits of detection and quantification of <1.29 pmol/L and\n2.57 pmol/L, respectively, average mean accuracy of 94.6%, analytical\nrange of 2.57 to 164.76 pmol/L, intra-assay variations of 14.3, 11.2, and\n6.5% at mean serum fT4 concentrations of 10.30 pmol/L,\n23.17 pmol/L, and 55.35 pmol/L, respectively, and inter-assay\nvariations of 16.2, 10.5, and 8.4% at mean serum fT4 concentrations\nof 10.30 pmol/L, 21.88 pmol/L, and 50.20 pmol/L, respectively (13).\nThe assay RI of 7.20 to 36.70 pmol/L was determined by TVMDL in\naccordance with the American Society for Clinical Pathology\n\n2, 10.5, and 8.4% at mean serum fT4 concentrations\nof 10.30 pmol/L, 21.88 pmol/L, and 50.20 pmol/L, respectively (13).The assay RI of 7.20 to 36.70 pmol/L was determined by TVMDL in\naccordance with the American Society for Clinical Pathology\n(ASVCP) Quality Assurance and Laboratory Standards guidelines (14).\nThe fT4CLIA assay is a commercially available kit whose accuracy\nhas been evaluated in both euthyroid and hypothyroid dogs (15). It\nhas been validated in accordance with AAVLD standards through\nonboarding verification, ongoing proficiency testing, and daily control\ncharting. The assay has reported limits of detection and quantification\nof 1.29 pmol/L and 3.86 pmol/L, respectively, analytical range of 3.86\nto 77.20 pmol/L, intra-assay variations of 10.8, 5.9, and 3.5% at mean\nserum fT4 concentrations of 7.59 pmol/L, 24.20 pmol/L, and\n75.56 pmol/L, respectively, and inter-assay variations of 11.9, 6.5, and\n4.3% at similar mean serum fT4 concentrations as listed for the intra-\nassay variation (16). The assay RI of 7.10 to 32.70 pmol/L was\ndetermined by TVMDL in accordance with the ASVCP Quality\nAssurance and Laboratory Standards guidelines (14).\n2.3.2 Serum TT4 concentration\nThe serum TT4 concentration measured on each hospital\nadmission sample in the original study was used in the present study to\nseparate the 38 dogs into two groups based on whether the serum TT4\nconcentration was within or below the RI. In the original study, the\nserum TT4 concentration was measured with a CLIA (Immulite 2000\nXPi, Siemens, Erlangen, Germany) at the Virginia-Maryland College of\nVeterinary Medicine clinical pathology laboratory with a RI of 12.8 to\n36.3 nmol/L and lower limit of quantification of 6.4 nmol/L (17).\n2.4 Statistical analysis\n2.4.1 Agreement between fT4ED and fT4CLIA\nThe fT4ED and fT4CLIA results from all 116 banked serum\nsamples were included in objective 1. Agreement between serum\nconcentrations of fT4ED and fT4CLIA was assessed using a Bland–\nAltman plot and the calculation of systematic and proportional bias.\nSystematic bias was measured as the arithmetic mean difference, with\n\nAgreement between serum\nconcentrations of fT4ED and fT4CLIA was assessed using a Bland–\nAltman plot and the calculation of systematic and proportional bias.Systematic bias was measured as the arithmetic mean difference, with\nits 95% limits of agreement (±1.96 standard deviation), between\nmethods of the concentrations of the same sample. Proportional bias\nwas measured as the slope, with its 95% confidence interval, of the\nregression equation of method differences plotted against the mean of\nthe two methods. All calculations were performed using commercial\nstatistical software (MedCalc Statistical Software, Ostend, Belgium,\n2013). p-values < 0.05 were significant.\nFor statistical purposes, when the serum fT4 concentration\nobtained by ED or CLIA was below the limits of quantification, a value\nof 2.56 pmol/L or 3.85 pmol/L was assigned, respectively.\n\nBolton et al.\b\n10.3389/fvets.2025.1657215\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n2.4.2 Frequency of fT4ED and fT4CLIA within the\nRI\nThe fT4ED and fT4CLIA results from 38 banked serum samples,\nrepresenting each dog’s sample at hospital admission, were included\nin objective 2. The serum concentrations of fT4ED, fT4CLIA, and TT4\non each serum sample were documented as below, within, or above\nthe RI. The frequency of fT4 results within the RI for each fT4 assay\nwas calculated when the serum TT4 concentration was within and\nbelow the RI.\n2.4.3 Concordance and discordance of fT4ED\nand fT4CLIA\nFor each of the 22 dogs having a serum TT4 concentration below\nthe RI at hospital admission, fT4ED and fT4CLIA results from only\nthe hospital admission sample were included in objective 3. When\nfT4ED and fT4CLIA results were similar (both within the RI or below\nthe RI), they were deemed concordant, and when fT4ED and fT4CLIA\nresults were different (one within the RI and one below the RI), they\nwere deemed discordant. Magnitude of disparity from the RI and\ndisease diagnoses in the dogs were evaluated.\n3 Results\n3.1 Agreement between fT4ED and fT4CLIA\nNo significant systematic bias (arithmetic mean difference,\n0.3 pmol/L; 95% limits of agreement, −11.5 to 12.2 pmol/L; p = 0.54)\nor proportional bias (slope, −0.03; 95% CI, −0.18 to 0.12; p = 0.70)\nwas identified when comparing fT4ED to fT4CLIA (Figure 1).\n3.2 Frequency of fT4ED and fT4CLIA within\nthe RI\nUpon hospital admission, 16 dogs had a serum TT4 concentration\nwithin the RI and 22 dogs had a serum TT4 concentration below the\nRI. In dogs with a serum TT4 concentration within the RI, fT4ED and\nfT4CLIA were within the RI in 100% (16/16; 95% CI: 79.4–100%) and\n94% (15/16; 95% CI: 69.8–99.8%) of dogs, respectively. One dog had\na fT4CLIA result above the RI while the corresponding fT4ED result\nwas within the RI. In dogs with a serum TT4 concentration below the\nRI, fT4ED and fT4CLIA were within the RI in 41% (9/22; 95% CI:\n20.7–63.6%) and 45% (10/22; 95% CI: 24.4–67.8%) of dogs,\nrespectively. Conversely, fT4ED and fT4CLIA were below the RI in\n\nIn dogs with a serum TT4 concentration below the\nRI, fT4ED and fT4CLIA were within the RI in 41% (9/22; 95% CI:\n20.7–63.6%) and 45% (10/22; 95% CI: 24.4–67.8%) of dogs,\nrespectively.Conversely, fT4ED and fT4CLIA were below the RI in\n59% (13/22; 95% CI: 36.4–79.3%) and 55% (12/22; 95% CI: 32.2–\n75.6%) of dogs, respectively, with a serum TT4 concentration\nbelow the RI.\n3.3 Concordance and discordance of\nfT4ED and fT4CLIA\nIn the 22 dogs with a serum TT4 concentration below the RI at\nhospital admission, concordant results were present in 77% (17/22;\n95% CI: 54.6–92.2%) of dogs. Both assays were below the RI in 10\ndogs (dogs 1–10; Table 1) and within the RI in 7 dogs (dogs 16–22;\nTable 1). Of the 10 dogs with serum fT4 concentrations below the RI\nby both assays, the fT4ED was undetectable in 2 dogs (dogs 1 and 2;\nTable 1), ≥1.70 pmol/L below the RI in 7 dogs (dogs 3–9; Table 1), and\n1.00 pmol/L below the RI in 1 dog (dog 10; Table 1). In the same 10\ndogs, the fT4CLIA was undetectable in 5 dogs (dogs 1–5; Table 1),\n>2.10 pmol/L below the RI in 3 dogs (dogs 6–8; Table 1), and within\n0.45 pmol/L of the low end of the RI in 2 dogs (dogs 9 and 10; Table 1).\nDiscordant results were present in 23% (5/22; 95% CI: 7.8–45.4%) of\ndogs. The fT4ED was below the RI and the fT4CLIA was within the\nRI in 3 dogs (dogs 11–13; Table 1), whereas the fT4ED was within the\nRI and the fT4CLIA was below the RI in 2 dogs (dogs 14 and 15;\nTable 1). Regardless of fT4 assay, all results below the RI among the\ndiscordant dogs were >1.90 pmol/L below the RI (dogs 11–15;\nTable 1). The disease diagnoses for the concordant and discordant\nfT4ED and fT4CLIA results are presented in Table 1.\n4 Discussion\nA serum fT4 concentration measured by ED has been considered\nthe most accurate stand-alone test for distinguishing NTIS from\nhypothyroidism (1–3); however, prior studies present conflicting data\non the frequency that fT4ED and fT4CLIA are within the RI in sick\ndogs with a serum TT4 concentration below the RI (2, 3, 11, 12). In\nour cohort of established euthyroid dogs with NTIS, the fT4ED and\n\nIn\nour cohort of established euthyroid dogs with NTIS, the fT4ED and\nfT4CLIA demonstrated agreement and had no significant bias;\nnevertheless, the small systematic bias of 0.3 pmol/L and wide 95%\nlimits of agreement could influence clinical decision making,\nespecially when assay results are near the low end of the RI. While\n>90% of dogs with a serum TT4 concentration within the RI also had\na serum fT4 concentration within the RI by either assay, <50% of dogs\nwith a serum TT4 concentration below the RI had a serum fT4\nconcentration within the RI by either assay. Most of the dogs with a\nfT4ED, fT4CLIA, or both below the RI had an inflammatory disease\nthat likely affected assay results. In dogs with NTIS caused by an acute,\nresolvable disease, a clinician should exercise caution when\ninterpreting concurrent serum TT4 and fT4ED or fT4CLIA\nFIGURE 1\nBland–Altman plot depicting the comparison of means to\ndifferences between 2 methods of serum free thyroxine\nmeasurement (fT4ED and fT4CLIA). Open circles represent the 116\nserum samples. The solid line represents the arithmetic mean\ndifference; dashed lines represent the 95% limits of agreement. The\ndotted line would indicate perfect agreement between methods.\nfT4CLIA, free thyroxine by chemiluminescent immunoassay; fT4ED,\nfree thyroxine by equilibrium dialysis.\n\nBolton et al.\b\n10.3389/fvets.2025.1657215\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nconcentrations below the RI because it could result in a misdiagnosis\nof hypothyroidism.\nWhen comparing fT4ED and fT4CLIA, neither significant\nsystematic nor proportional bias was present; however, this does not\nmean these assays are interchangeable. The small systematic bias of\n0.3 pmol/L means that, on average, fT4ED measures 0.3 pmol/L lower\nthan fT4CLIA. A visual inspection of the Bland–Altman plot\ndemonstrates larger differences between fT4ED and fT4CLIA at\nserum fT4 concentrations >10 pmol/L and smaller differences\nbetween fT4ED and fT4CLIA at serum fT4 concentrations\n<10 pmol/L. Furthermore, the 95% limits of agreement are wide in\norder to include the large differences between fT4ED and fT4CLIA at\nhigher serum fT4 concentrations. Luckily, large differences between\nfT4ED and fT4CLIA at higher serum fT4 concentrations does not\nimpact clinical decision making when attempting to exclude\nhypothyroidism; nonetheless, the small and statistically insignificant\nsystematic bias near the low end of the RI may be  of sufficient\nmagnitude to falsely lower the serum fT4 concentration below the\nRI. Consequently, a serum fT4 concentration just below the low end\nof the RI measured by either assay should be interpreted with caution\nand in light of the clinical picture.\nIn euthyroid dogs with NTIS and a serum TT4 concentration\nbelow the RI, fT4ED and fT4CLIA were also below the RI in 59 and\n55% of dogs, respectively. While these findings highlight the limited\nability of these assays to reliably exclude hypothyroidism in this\ncohort, it is notable that almost 50% of the dogs had a serum fT4\nconcentration within the RI by either assay. In such cases, a serum fT4\nconcentration might still provide useful clinical guidance on the true\nthyroid status of the dog, particularly when gold-standard testing (i.e.,\nTSH stimulation or thyroid scintigraphy) is unavailable. However, a\nrecent study reported that roughly 50% of dogs in first opinion\npractices receiving levothyroxine were likely being administered this\n\ne.,\nTSH stimulation or thyroid scintigraphy) is unavailable.However, a\nrecent study reported that roughly 50% of dogs in first opinion\npractices receiving levothyroxine were likely being administered this\nmedication unnecessarily, supporting the notion that hypothyroidism\nis overly and incorrectly diagnosed (18). Reasons for this include\ninherent limitations to the thyroid function tests and the influence of\nexternal factors on such tests, such as NTIS and medications (18).\nUnnecessary thyroid hormone supplementation subjects dogs to\nrepeat and lifelong blood sampling for monitoring purposes and can\ndelay the diagnosis of the true underlying disease. Given the frequency\nof serum TT4 and fT4 concentrations below the RI in euthyroid dogs\nwith NTIS, clinicians should only perform thyroid function testing in\nthose dogs with a high clinical suspicion for the disease.\nSerum fT4 assays were developed for more accurate measurement\nof T4, either via separating the free fraction of T4 from serum proteins\n(ED) or using a radioligand analog that binds directly to T4 without\ninterference from serum proteins (CLIA). Having intraassay variations\nTABLE 1  Concordant and discordant fT4ED and fT4CLIA results in dogs with a low serum total thyroxine concentration.\nRI: 7.20–36.70 pmol/L\nRI: 7.10–32.70 pmol/L\nDog\nDisease diagnosis\nfT4ED\nfT4CLIA\nConcordant/\ndiscordant\n1\nGastrointestinal obstruction\n<2.57 (L)\n<3.86 (L)\nConcordant\n2\nAcute gastroenteritis\n<2.57 (L)\n<3.86 (L)\nConcordant\n3\nBacterial bronchopneumonia\n3.61 (L)\n<3.86 (L)\nConcordant\n4\nParvoviral enteritis\n4.29 (L)\n<3.86 (L)\nConcordant\n5\nPyometra\n4.70 (L)\n<3.86 (L)\nConcordant\n6\nBacterial bronchopneumonia\n4.80 (L)\n4.62 (L)\nConcordant\n7\nPyometra\n5.26 (L)\n4.07 (L)\nConcordant\n8\nAcute gastroenteritis\n5.49 (L)\n4.98 (L)\nConcordant\n9\nAcute gastroenteritis\n5.50 (L)\n6.67 (L)\nConcordant\n10\nBacterial prostatitis/epididymitis\n6.20 (L)\n6.81 (L)\nConcordant\n11\nAcute gastroenteritis\n4.84 (L)\n7.99 (N)\nDiscordant\n12\nTraumatic pneumothorax\n5.11 (L)\n8.38 (N)\nDiscordant\n13\nGastrointestinal obstruction\n5.28 (L)\n11.20 (N)\nDiscordant\n14\nGastrointestinal obstruction\n9.31 (N)\n<3.86 (L)\nDiscordant\n15\nPyothorax\n11.26 (N)\n4.74 (L)\nDiscordant\n16\nGastrointestinal obstruction\n9.48 (N)\n7.50 (N)\nConcordant\n17\n\n11 (L)\n8.38 (N)\nDiscordant\n13\nGastrointestinal obstruction\n5.28 (L)\n11.20 (N)\nDiscordant\n14\nGastrointestinal obstruction\n9.31 (N)\n<3.86 (L)\nDiscordant\n15\nPyothorax\n11.26 (N)\n4.74 (L)\nDiscordant\n16\nGastrointestinal obstruction\n9.48 (N)\n7.50 (N)\nConcordant\n17\nShock of unknown etiology\n11.70 (N)\n8.56 (N)\nConcordant\n18\nAcute gastroenteritis\n13.32 (N)\n16.10 (N)\nConcordant\n19\nDrug-induced hepatopathy\n13.39 (N)\n8.76 (N)\nConcordant\n20\nLung lobe torsion\n19.30 (N)\n15.80 (N)\nConcordant\n21\nGallbladder mucocele\n24.30 (N)\n15.80 (N)\nConcordant\n22\nGastrointestinal obstruction\n25.01 (N)\n18.10 (N)\nConcordant\nfT4ED, free thyroxine by equilibrium dialysis; fT4CLIA, free thyroxine by chemiluminescent immunoassay.\n(L), below the reference interval; (N), within the reference interval; RI, reference interval.\n\nBolton et al.\b\n10.3389/fvets.2025.1657215\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nof 14.3 and 10.8% for the fT4ED and fT4CLIA, respectively, when\nserum fT4 concentrations are near the low end of the RI, assay results\n<1.03 pmol/L below the low end of the RI for fT4ED and <0.77 pmol/L\nbelow the low end of the RI for fT4CLIA would fall within the\npublished intra-assay variation for each assay. If repeated, the serum\nfT4 concentration may fall within the RI. Since only 1 dog (dog 10;\nTable 1) with a fT4ED result and 2 dogs (dogs 9 and 10; Table 1) with\na fT4CLIA result fell within the intra-assay variation for each assay, it\nappears unlikely that serum fT4 concentrations below the RI by either\nassay are a result of inherent assay imprecision and are more likely due\nto factors in each dog related to the severity and specific illnesses\npresent. Interestingly, most dogs having a serum fT4 concentration\nbelow the RI by one or both assays were diagnosed with an\ninflammatory disease (Table 1). In an inflammatory disease, acute\nphase proteins (APPs) increase by more than 25% in the serum and\ncan consequently lower T4 measurement (19, 20). Furthermore, it has\nbeen demonstrated that fT4ED and fT4CLIA have decreasing\nconcentrations as the severity of the illness increases, possibly due to\nthe presence of serum proteins that interfere with the fT4 assays, such\nas APPs (2, 11). Because all dogs in the present study with a serum\nTT4 concentration below the RI were hospitalized, indicating, at\nminimum, a moderate severity of illness based on a disease severity\nstratification published in a previous study (2), it is plausible that APPs\ninterfered with the ability of both assays to accurately measure T4 in\ndogs with a fT4ED, fT4CLIA, or both below the RI (dogs 1–15;\nTable 1).\nIn this study, 45% of dogs with NTIS and a serum TT4\nconcentration less than the RI had a fT4CLIA result within the RI\ncompared to just 12% in a previous study (11). While the fT4CLIA\nperformed better in the present study, both results are unacceptably\npoor in clinical practice when used to rule out hypothyroidism in this\n\nWhile the fT4CLIA\nperformed better in the present study, both results are unacceptably\npoor in clinical practice when used to rule out hypothyroidism in this\ncohort of dogs. The findings of this study corroborate those of\nprevious studies whereby the fT4CLIA (same methodology) is of little\nadditional diagnostic value both in dogs with NTIS and a serum TT4\nconcentration below the RI (11), and in dogs being investigated for\npotential hypothyroidism (21). While still poor, the superior\nperformance of the fT4CLIA in the present study could be, in part,\ndue to the inclusion of only known euthyroid dogs and exclusion of\ndogs administered medications that are known to lower the serum fT4\nconcentration (11, 22). The severity and specific illnesses present\nbetween the 2 studies are likely different and can explain the\ndiscordant finding; however, a direct comparison between studies is\ndifficult due to the lack of data on these variables in the earlier study\n(11). Of note, while the fT4CLIA used in these studies was the same,\nthe laboratory performing the analysis in the present study internally\nvalidated the fT4CLIA assay and generated a laboratory-specific RI,\nnone of which was reported in the prior study (11).\nEarlier studies evaluating the fT4ED assay in dogs with NTIS\ndiscovered that greater than 80% of dogs had a serum fT4\nconcentration within the RI (1–3). This high percentage created a\nfavorable opinion of the fT4ED assay, perpetuating the assertion that\nit is the most accurate stand-alone diagnostic test for differentiating\nNTIS and hypothyroidism; however, it is important to understand that\nthis high percentage represents an aggregate of dogs with serum TT4\nconcentrations within and below the RI without separating out the\nlatter group of dogs (1–3). Since measurement of the serum fT4\nconcentration is usually reserved for the differentiation of NTIS and\nhypothyroidism in dogs with a serum TT4 concentration below the\nRI, evaluation of the fT4ED in this cohort is more clinically relevant.\nIn 4 previous studies evaluating dogs with NTIS using fT4ED, the\n\nIn 4 previous studies evaluating dogs with NTIS using fT4ED, the\nserum fT4 concentration was within the RI when the serum TT4\nconcentration was below the RI in 43% (11), 55% (2), 86% (12), and\n97% (3) of dogs. Notably, in the previous study (12) using the same\nserum samples, 86% of dogs with a low serum TT4 concentration had\na serum fT4 concentration by ED within the RI, compared to only\n41% in the current study. All prior studies (2, 3, 11, 12) used the same\nED assay (Antech Diagnostics, Irvine, CA), whereas the current study\nemployed a different ED assay (IVD Technologies, Santa Ana, CA).\nThis decision introduced methodology variation; however, it was\nmade to ensure consistency in sample handling and reduce\npre-analytical variation. Given that the serum fT4 concentration is\nstable in human serum samples stored frozen for a long period of\ntime, sample degradation is unlikely to explain the discrepancy (23).\nInstead, it is possible that the ED assay used in the current study\nunderperformed compared to the previously used assay. Nonetheless,\nthe clinical consequence of all these findings is that the fT4ED assay\ncan correctly diagnose NTIS and exclude hypothyroidism in 41–97%\nof dogs with illness. Taking all of this into consideration, the reliability\nof fT4ED at differentiating NTIS from hypothyroidism in dogs with a\nserum TT4 concentration below the RI, the clinically relevant group,\nis questionable.\nOverall, ED assay results are extremely variable, with up to 90%\nvariability in humans when the serum fT4 concentration is at the\nlower end of the RI (24). Because of this, human laboratories have key\nstandardized components when performing the ED assay, particularly\nusing a buffer that mimics the ionic environment of the serum,\nensuring a constant temperature of the dialysate, using an undiluted\nserum sample, and obtaining a standard serum pH prior to performing\nthe ED assay (25). Despite these standardizations, variation in the\nserum fT4 concentration of as much as 50% is expected when the\nserum sample is analyzed at a different reference laboratory (26). As\n\nDespite these standardizations, variation in the\nserum fT4 concentration of as much as 50% is expected when the\nserum sample is analyzed at a different reference laboratory (26).As\nof right now in veterinary medicine, standardization does not occur;\nhowever, using an AAVLD accredited laboratory with robust quality\nguidelines, validation requirements, standard operating procedures,\nand duplicate assay runs limits result variation. Nonetheless, analyzing\nserum fT4 concentrations at different veterinary reference laboratories\ncan still result in variation of up to 25% in the ED assay result (27).\nThe inferior performance of the fT4ED in the present study as\ncompared to prior studies (2, 3, 11, 12) could be because it was a\ndifferent ED assay run at a different laboratory or the result of assay\nperformance in the face of illness. While the dogs with a serum TT4\nconcentration below the RI in the present study were not stratified\nbased on severity of disease, all dogs required hospitalization for\ninclusion in the study from which these samples originated, meaning\nthey had, at minimum, illness of moderate severity (2). Because\nincreasing severity of illness results in an incrementally lower serum\nfT4 concentration, the inferior performance of the fT4ED in this study\nand others (2, 11) could be due to the severity or even type of illnesses\npresent. Thus, the ability of the fT4ED assay to rule out hypothyroidism\nin an ill dog having a serum TT4 concentration below the RI (likely a\ndog with moderate to severe illness) is low and clinically unacceptable.\nTo limit some of the variation seen with the fT4ED assay, however, the\nsubmission of all serum samples for a single patient to the same\nreference laboratory is recommended.\nOur study had some limitations. First, serum samples were stored\nat −80 °C for up to 4 years prior to analysis making sample\n\nBolton et al.\b\n10.3389/fvets.2025.1657215\nFrontiers in Veterinary Science\n07\nfrontiersin.org\ndegradation a possibility. Although no data is available on the effect of\nlong-term storage at −80 °C on serum thyroid hormone\nconcentrations in dogs with NTIS, the serum fT4 concentration is\nstable and can therefore be reliably analyzed in human serum samples\nstored for up to 23 years at −25 °C (23). Additionally, all serum\nsamples were kept frozen until analysis so only endured one freeze–\nthaw cycle to limit potential degradation.\nSecond, a different ED assay was used in this study compared to\n4 previous studies (2, 3, 11, 12). This decision was made to minimize\npre-analytical variability by selecting an AAVLD-accredited laboratory\nable to perform both the fT4ED and fT4CLIA assays concurrently on\nthe same serum aliquot with only a single freeze–thaw cycle. While\nthis approach improved consistency in sample handling, it required\nthe use of a different ED assay kit, as the assay used in the prior studies\nis not readily available to most laboratories. The decrease in assay\nperformance in this study compared to the previous study (12) using\nthese same serum samples may be secondary to the ED assay used;\nhowever, this reflects the reality of clinical practice where clinicians\ntypically cannot choose the ED assay used when submitting serum\nsamples. Because fT4ED results in the original study (12) were\nobtained at a different time and under different conditions, we did not\ninclude them in this study, as doing so would have introduced\nadditional confounding variables.\nThird, our study investigated dogs with acute resolvable illness\naffecting limited body systems and for which stratification based on\ndisease severity was not performed due to the criteria under which\nthese samples were obtained (12), unlike previous studies where dogs\nwith both acute and chronic illness affecting more than 10 body\nsystems and stratified based on disease severity were evaluated (1–3,\n11). While this might limit the applicability of our findings to all dogs\nwith NTIS, the study design guaranteed that only known euthyroid\ndogs were included.\n\nWhile this might limit the applicability of our findings to all dogs\nwith NTIS, the study design guaranteed that only known euthyroid\ndogs were included.\nFourth, despite a large number of serum samples available for the\nmethod comparison portion of our study (objective 1), each sample did\nnot represent a unique dog and the range of serum fT4 concentrations\nassociated with these samples might be  considered narrow.\nUnfortunately, because our study was retrospective, these limitations\nwere unavoidable; however, unlike correlation or agreement coefficients,\nBland–Altman analysis is preferred for assessing agreement between two\nmethods (28), and serum samples from the same dog represented\ndifferent time points within that particular dog’s illness with each sample\ncollected at least 24 h apart. Alternatively, assessing agreement between\nED and CLIA with one sample per dog would have substantially\ndecreased the number of samples and range of serum fT4 concentrations\nanalyzed, potentially resulting in type II error. Therefore, a decision was\nmade to pursue the current analysis.\nFinally, the number of dogs used to evaluate the frequency that\neach fT4 assay was within the RI and subsequently diagnose the dog\nwith NTIS and not hypothyroidism was small. Additional studies with\na larger group of known euthyroid dogs with NTIS with more diseases\nis warranted.\nIn conclusion, while fT4ED and fT4CLIA measured with these\nspecific assays in this cohort of dogs were not statistically different,\ncareful interpretation of a serum fT4 concentration just below the\nlower limit of the RI is recommended. In dogs with a serum TT4\nconcentration below the RI, the serum fT4 concentration was within\nthe RI in less than 50% of dogs, demonstrating limited reliability for\nruling out hypothyroidism in dogs with NTIS. However, both assays\nwere capable of appropriately ruling out hypothyroidism in almost\nhalf of the dogs, suggesting that when using these particular assays,\nneither needs to be preferentially selected over the other. When both\nserum TT4 and fT4 concentrations are below the RI, clinical context\n\nWhen both\nserum TT4 and fT4 concentrations are below the RI, clinical context\nbecomes crucial, especially if gold-standard testing is not feasible or\navailable. In these cases, rechecking thyroid function tests after\nresolution of the illness may provide diagnostic clarity.\nData availability statement\nThe original contributions presented in the study are included in\nthe article/supplementary material, further inquiries can be directed\nto the corresponding author.\nEthics statement\nThe animal studies were approved by Virginia-Maryland College\nof Veterinary Medicine Institutional Animal Care and Use Committee.\nThe studies were conducted in accordance with the local legislation\nand institutional requirements. Written informed consent was\nobtained from the owners for the participation of their animals in\nthis study.\nAuthor contributions\nTB: Formal analysis, Writing – review & editing, Data curation,\nConceptualization, Writing – original draft. CM: Conceptualization,\nWriting – original draft, Writing – review & editing, Data curation,\nFormal analysis. MGD: Writing – review & editing, Writing – original\ndraft. GM: Formal analysis, Methodology, Writing – review & editing.\nJP: Writing – review & editing. MLD: Writing – review & editing.\nFunding\nThe author(s) declare that no financial support was received for\nthe research and/or publication of this article.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\n\nBolton et al.\b\n10.3389/fvets.2025.1657215\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the reviewers.\nAny product that may be evaluated in this article, or claim that may\nbe made by its manufacturer, is not guaranteed or endorsed by\nthe publisher.\nReferences\n1.\tPeterson ME, Melián C, Nichols R. Measurement of serum total thyroxine,\ntriiodothyronine, free thyroxine, and thyrotropin concentrations for diagnosis of\nhypothyroidism in dogs. J Am  Vet Med Assoc. (1997) 211:1396–402. doi:\n10.2460/javma.1997.211.11.1396\n2.\tKantrowitz LB, Peterson ME, Melián C, Nichols R. 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+{"text": "【兽医文献】\n标题: Multi-target synergistic mechanisms of flavonoid compounds from traditional Chinese medicine in non-alcoholic fatty liver disease: insights for human and veterinary medicine\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nTYPE  Original Research\nPUBLISHED  09 June 2026\nDOI  10.3389/fvets.2026.1804735\nOPEN ACCESS\nEDITED BY\nP. K. Malik,\nNational Institute of Animal Nutrition\nand Physiology (ICAR), India\nREVIEWED BY\nIulian Alexandru Grosu,\nNational Research Development\nInstitute for Animal Biology and\nNutrition, Romania\nWu Qiang,\nYibin Vocational and Technical College,\nChina\n*CORRESPONDENCE\nHaizhen Wang\n20201018@llu.edu.cn\nJinlong Huo\njinlonghuo973@163.com\n†These authors have contributed equally\nto this work and share first authorship\nRECEIVED 05 February 2026\nREVISED 17 May 2026\nACCEPTED 26 May 2026\nPUBLISHED 09 June 2026\nCITATION\nZhang X, Huo H, Hu L, Yang F, Hu X,\nDeng Y, Feng C, Wang H and\nHuo J (2026) Dietary Lonicera japonica\nsupplementation modulates cecal gut\nmicrobial composition and metabolomic\nprofiles in weaned piglets.\nFront. Vet. Sci. 13:1804735.\ndoi: 10.3389/fvets.2026.1804735\nCOPYRIGHT\n© 2026 Zhang, Huo, Hu, Yang, Hu,\nDeng, Feng, Wang and Huo. This is an\nopen-access article distributed under\nthe terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other\nforums is permitted, provided the\noriginal author(s) and the copyright\nowner(s) are credited and that the\noriginal publication in this journal is\ncited, in accordance with accepted\nacademic practice. No use, distribution\nor reproduction is permitted which does\nnot comply with these terms.\nDietary Lonicera japonica\nsupplementation modulates cecal\ngut microbial composition and\nmetabolomic profiles in weaned\npiglets\nXia Zhang 1†, Hailong Huo 2†, Lijuan Hu 1,3†, Fuhua Yang 4†,\nXiaojing Hu 1, Yunze Deng 1, Caiping Feng 1, Haizhen Wang 1* and\nJinlong Huo 5*\n1Department of Biological and Food Engineering, Lyuliang University, Lvliang, Shanxi, China, 2Yunnan\nOpen University, Kunming, Yunnan, China, 3College of Animal Science and Technology, Shanxi\nAgricultural University, Taiyuan, Shanxi, China, 4Yunnan Academy of Animal Husbandry and Veterinary\nSciences, Kunming, Yunnan, China, 5College of Animal Science and Technology, Yunnan Agricultural\nUniversity, Kunming, Yunnan, China\nWeaning is a critical developmental stage in piglets and is often associated with\n\nintestinal dysbiosis, metabolic disturbances, and impaired gut barrier function.\nPhytogenic feed additives have emerged as promising natural alternatives to\nantibiotics for improving gut health. Lonicera japonica, a traditional medicinal\nand edible plant rich in bioactive compounds, exhibits well-documented antimi­\ncrobial, antioxidant, and immunomodulatory properties; however, its effects on\nthe gut microbiota-metabolite axis in weaned piglets remain poorly understood.\nIn this study, weaned piglets were fed either a basal diet (control group) or a\nLonicera japonica-supplemented diet (experimental group). Cecal contents were\ncollected for metagenomic sequencing to characterize gut microbial composi­\ntion and for untargeted LC–MS-based metabolomic profiling. Functional path­\nway enrichment and microbe-metabolite correlation network analyses were\nconducted to elucidate potential mechanisms. Lonicera japonica supplementa­\ntion significantly improved evenness in terms of microbial species richness and\nreshaped microbial community structure, characterized by the enrichment of\nbeneficial taxa, including Firmicutes and Eubacterium coprostanoligenes, and a\nconcomitant reduction in opportunistic pathogens such as Proteobacteria and\nEscherichia coli. KEGG pathway analysis revealed the upregulation of micro­\nbial pathways related to translation, replication, and energy metabolism, along­\nside the downregulation of stress-response-associated pathways. Metabolomic\nprofiling demonstrated distinct metabolic signatures between groups, with\nelevated levels of unsaturated fatty acids, amino acid derivatives, and organic\nacids, and reduced bile acid intermediates in the Lonicera japonica-treated pig­\nlets. Correlation network analysis further revealed strong positive correlations\nbetween SCFA-producing bacteria and beneficial metabolites, underscoring a\nreinforced microbiota-metabolite axis. Collectively, these findings indicate that\nLonicera japonica supplementation promotes a healthier and more stable gut\necosystem in weaned piglets through coordinated modulation of microbial com­\n\nCollectively, these findings indicate that\nLonicera japonica supplementation promotes a healthier and more stable gut\necosystem in weaned piglets through coordinated modulation of microbial com­\nposition, functional potential, and metabolic outputs. This study provides novel\ninsights into microbiota-metabolite interactions underlying phytogenic interven­\ntions and supports the use of Lonicera japonica as a natural feed additive to\nenhance intestinal health and resilience during weaning.\n\nZhang et al.\b\n10.3389/fvets.2026.1804735\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nKEYWORDS\ncecal contents, Lonicera japonica supplementation, metabolomics, metagenomics,\nweaned piglets\n1 Introduction\nThe weaning period represents one of the most critical and\nvulnerable stages in the life of piglets. Following weaning, piglets\nare exposed to multiple challenges, including the abrupt transition\nfrom sow’s milk to solid feed, environmental and social stressors,\nand an immature immune system. These stressors frequently\nresult in reduced feed intake, growth retardation, diarrhea, and\nincreased mortality rates (1). From a physiological perspective,\nweaning stress damages the intestinal barrier, alters gut microbi­\nota, and disrupts lipid, carbohydrate, and amino acid metabolism,\nleading to energy metabolic imbalance (2). Lipid metabolism dys­\nregulation is strongly linked to energy imbalance, intestinal dys­\nfunction, and weakened immunity, and is a key factor in reduced\ngrowth after weaning (3). The gut microbiota plays a central role\nin the regulating of lipid metabolism by influencing fatty acid\nabsorption and synthesis, modulating bile acid metabolism (4).\nAlterations in the relative abundance of specific bacterial taxa,\nsuch as Firmicutes and Bacteroidetes, can directly affect energy\nharvest efficiency and fat deposition (5, 6). In addition, short-\nchain fatty acids (SCFAs) produced by the gut microbiota-such as\nacetate, propionate, and butyrate-play an important role in regu­\nlating lipid metabolism in the liver and adipose tissue (7).\nConsequently, targeting the gut microbiota to alleviate lipid meta­\nbolic disturbances in weaned piglets has emerged as a major focus\nin animal nutrition research.\nAmong natural plant-derived feed additives, Lonicera japonica\nhas attracted increasing scientific attention due to its multifunc­\ntional biological properties. Lonicera japonica is rich in bioactive\ncompounds, including chlorogenic acid, luteolin, quercetin, fla­\nvonoids, saponins, and phenolic acids (8). These constituents\nexhibit potent antioxidant, anti-inflammatory, antibacterial, and\n\nLonicera japonica is rich in bioactive\ncompounds, including chlorogenic acid, luteolin, quercetin, fla­\nvonoids, saponins, and phenolic acids (8).These constituents\nexhibit potent antioxidant, anti-inflammatory, antibacterial, and\nimmunomodulatory activities. They act mainly by inhibiting of\nkey signaling pathways such as the nuclear factor kappa B (NF-kB)\nand mitogen-activated protein kinase (MAPK) pathways, while\nalso enhancing endogenous antioxidant defense systems (9).\nPrevious studies have shown that dietary supplementation with\nLonicera japonica extracts significantly improves intestinal mor­\nphology, increases the relative abundance of lactic acid bacteria,\nand suppresses pathogenic microorganisms in pigs (10).\nConsistently, Lonicera japonica supplementation has been\nreported to promote beneficial microbial populations while inhib­\niting pathogens such as Escherichia coli, contributing to improved\nintestinal microbial homeostasis and feed conversion efficiency\n(11). In addition to its microbiota-modulating effects, Lonicera\njaponica has been demonstrated to upregulate the expression of\nintestinal epithelial tight junction proteins, including zonula\noccludens-1 (ZO-1), occludin, and claudin-1. This enhancement\nof barrier function subsequently alleviates intestinal inflammation\nand oxidative stress in the host (12, 13). Its major active compo­\nnents, chlorogenic acid and luteolin, further contribute to intesti­\nnal immune regulation by promoting secretory immunoglobulin\nA (slgA) production and modulating the Treg/Th17 cell balance,\nthus maintaining intestinal immune homeostasis (14). Moreover,\nLonicera japonica has been implicated in regulating hepatic and\nplasma lipid profiles through its influence on fatty acid metabo­\nlism and cholesterol biosynthesis pathways (15). Studies in aquatic\nspecies have shown that dietary Lonicera japonica supplementa­\ntion enhances immune function, improves lipid metabolic bal­\nance, and stabilizes intestinal microbial communities (16).\nCollectively, these findings suggest that Lonicera japonica may\n\nCollectively, these findings suggest that Lonicera japonica may\nexert beneficial effects on lipid metabolism via modulation of the\ngut microbiota-host metabolic axis.\nHowever, despite accumulating evidence supporting the func­\ntional application of Lonicera japonica in livestock and poultry\nproduction, its precise role and underlying mechanisms in regu­\nlating lipid metabolism in weaned piglets remain incompletely\nunderstood. In particular, it remains unclear whether Lonicera\njaponica can restore metabolic homeostasis by reshaping the intes­\ntinal microecological environment and modulating lipid meta­\nbolic pathways. Given the complexity of the interactions among\ngut microorganisms, metabolites, and host physiological pro­\ncesses, single-omics approaches are often insufficient to compre­\nhensively elucidate these mechanisms. Therefore, an integrative\nmulti-omics strategy is warranted (17). The combined application\nof metagenomics and untargeted metabolomics provides a power­\nful framework for deciphering the mechanistic links between\nmicrobial functions and metabolic processes. Metagenomic analy­\nsis enables comprehensive profiling of gut microbial taxonomic\ncomposition and functional gene potential. Untargeted metabo­\nlomics allows systematic characterization of host- and microbiota-\nderived metabolic alterations. Integrating these complementary\napproaches enables the identification of changes in the gut micro­\nbiota and their links to host metabolic remodeling (18). Recent\nstudies\nemploying\nintegrated\nmetagenomic-metabolomic\napproaches have demonstrated that weaning stress induces pro­\nfound alterations in gut microbial composition and associated\nmetabolic pathways in piglets. (19). Notably, interventions with\nprobiotics or plant-derived bioactive compounds have been\nshown to partially or fully reverse these adverse effects, under­\nscoring the pivotal role of the gut microbiota in regulating fat\ndeposition and energy metabolic homeostasis (20).\nHere, we systematically investigated the effects of Lonicera\njaponica supplementation on intestinal microbial structure, func­\n\nHere, we systematically investigated the effects of Lonicera\njaponica supplementation on intestinal microbial structure, func­\ntional gene composition, and lipid metabolism in weaned piglets\nusing an integrated metagenomic and untargeted metabolomic\napproach. The specific objectives were to: (1) elucidate the regula­\ntory effects of Lonicera japonica supplementation on the composi­\ntion and functional metabolic potential of the intestinal microbiota;\n(2) identify key metabolites and metabolic pathways associated with\nlipid metabolism; and (3) construct a microbiota-metabolite inter­\naction network to elucidate the potential mechanisms by which\nLonicera japonica modulates lipid metabolism. This study provides\na theoretical basis for utilizing Lonicera japonica as a sustainable\nfeed additive for weaned piglets. It also yields novel multi-omics\ninsights into how natural plant compounds in host metabolic\nhomeostasis.\n\nZhang et al.\b\n10.3389/fvets.2026.1804735\nFrontiers in Veterinary Science\n03\nfrontiersin.org\n2 Materials and methods\n2.1 Experimental animals and feeding\nmanagement\nAll piglets were routinely vaccinated and dewormed throughout\nthe experimental period. The experimental and control groups were\nraised in different enclosures under standard commercial husbandry\nconditions. Piglets had ad libitum access to feed, and water were fed\nthree times daily at 07:00, 12:00, and 17:00. All management, housing,\nand vaccination procedures were conducted in accordance with stan­\ndard practices for commercial pig production. A total of 20 healthy\n21-day-old weaned Duroc × Landrace × Yorkshire crossbred piglets\nwith similar initial body weights (no significant difference; p > 0.05)\nwere selected for the study. Piglets were randomly assigned to two\nexperimental groups (n = 10 per group). During the 35-day experi­\nmental period, the control group (CON) was fed a basal diet, whereas\nthe experimental group (FLJ) received the same basal diet supple­\nmented with Lonicera japonica flower. The basal diet was supple­\nmented with 1,000 mg/kg dried Lonicera japonica powder, which was\nthoroughly homogenized with the feed prior to administration. The\npowder contained 7.87% moisture, while the concentrations of the\nprincipal bioactive were quantified as 1.535% chlorogenic acid and\n0.0541% luteolin. The ingredient composition and nutrient levels of\nthe basal diet (air-dried basis) are presented in Supplementary Table S1.\n2.2 Sample collection\nOn day 35 of the experimental period, six piglets were randomly\nselected from each group (provided that there was no significant dif­\nference in weight at the start of the experiment) as target animals for\nmulti-omics analysis. The piglets were anesthetized and then\nhumanely euthanized by exsanguination via the jugular vein. All of\nthe research was authorized and approved by the ethical committee of\nLyuliang University (SHANXI Lvliang, China). Every effort was made\nto minimize suffering. Immediately following euthanasia (within\n2–3 min), the abdominal cavity was opened and the cecum was rap­\n\nEvery effort was made\nto minimize suffering.Immediately following euthanasia (within\n2–3 min), the abdominal cavity was opened and the cecum was rap­\nidly isolated. Subsequently, the entire dissection and content-collec­\ntion procedure was performed on an ice plate under strictly controlled\nlow-temperature conditions, thereby minimizing potential metabolic\nalterations and microbial perturbations. The collected cecal contents\nwere promptly aliquoted into sterile cryovials, snap-frozen in liquid\nnitrogen, and stored at −80 °C until further analysis.\n2.3 Growth performance, diarrhea rate and\nserum biochemical index detection\nBody weight was recorded for each piglet at both the beginning\nand the end of the experiment, while feed intake per pen was continu­\nously monitored throughout the trial. Subsequently, Average daily gain\n(ADG), average daily feed intake (ADFI), and feed-to-gain ratio (F/G)\nwere calculated. Fecal consistency was visually assessed daily using a\nstandardized five-point scoring system, as previously described (21),\nthereby enabling calculation of the diarrhea rate. In addition, piglet\nhealth status and mortality were monitored and recorded daily\nthroughout the experiment period. Following overnight fasting, 5 mL\nof blood was collected from the anterior vena cava into anticoagulant\ntubes and analyzed using an automated hematology analyzer (Mindray\nBC-30 Vet, China). Additionally, 10 mL of blood was collected for\nserum preparation, centrifuged at 3,500 rpm for 10 min, and stored at\n−20 °C until subsequent analysis. Serum biochemical parameters were\ndetermined using an automated biochemical analyzer (DOTOP-8018).\n2.4 Metagenomic sequencing and data\nanalysis\nTotal microbial DNA was extracted from cecal content samples\n(n = 6 per group) following the manufacturer’s protocols. DNA was\nextracted using the CretMag™ Power Soil DNA Kit (27100-4-EP,\nCretBiotech, China) in accordance with the manufacturer’s instruc­\ntions. For mechanical lysis, samples were homogenized using a\nTGrinder H24 tissue homogenizer (OSE-TH-01, Tiangen Biotech),\n\nDNA was\nextracted using the CretMag™ Power Soil DNA Kit (27100-4-EP,\nCretBiotech, China) in accordance with the manufacturer’s instruc­\ntions.For mechanical lysis, samples were homogenized using a\nTGrinder H24 tissue homogenizer (OSE-TH-01, Tiangen Biotech),\nwith bead-beating parameters set at 6.0 m/s for 30 s, followed by a 30 s\npause, for a total of two cycles. Subsequently, the quality and concen­\ntration of the extracted DNA were assessed, and qualified DNA sam­\nples were stored at −80 °C until further processing. DNA libraries\nwere constructed, fragmented, and subjected to quality control before\nsequencing. Qualified libraries were sequenced on the NovaSeq 6,000\nplatform (Illumina, San Diego, CA, USA) using a 150-bp paired-end\nstrategy. Raw sequencing reads were processed using Trimmomatic\n(version 0.40) (22) to remove adapter sequences and low-quality\nreads, and host-derived sequences were filtered using BMTagger (ver­\nsion 3.101), yielding high-quality clean reads for downstream analy­\nses. The resulting high-quality, host-depleted reads were de novo\nassembled using MEGAHIT with the parameters—min-contig-len\n500—k-min 21—k-max 141—k-step 12. Contigs with lengths ≥\n500 bp were retained for downstream analyses. Assembly quality was\nsubsequently evaluated using QUAST with default parameters.\nProtein-coding genes were predicted from assembled contigs using\nProdigal in metagenomic mode (−p meta), thereby generating both\nnucleotide and amino acid sequences. Thereafter, predicted gene\nsequences from all samples were pooled and clustered using CD-HIT\nwith the parameters -c 0.95 -aS 0.9 -g 1 -d 0 to construct a non-redun­\ndant gene catalog. Finally, clean reads from each sample were mapped\nback to this catalog using Bowtie 2 in end-to-end mode with the\nparameter—very-sensitive, and gene abundance profiles were quanti­\nfied using SAMtools and featureCounts with the parameters -p -t CDS\n-g ID, thereby enabling downstream functional and statistical analyses.\nTaxonomic annotation was performed to determine the relative\nabundances of microbial taxa at the phylum, genus, and species\n\nTaxonomic annotation was performed to determine the relative\nabundances of microbial taxa at the phylum, genus, and species\nlevels. Functional annotation of the representative sequences was\nperformed using DIAMOND v0.9.19 (23) with an E-value threshold\nof 1e-5. Specifically, sequence annotation was conducted against the\nNCBI NR database using the blastp algorithm, while taxonomic clas­\nsification was carried out against the eggNOG database, and path­\nway-level annotation was performed using the Kyoto Encyclopedia\nof Genes and Genomes (KEGG) database (version 94.2). Stacked bar\nplots were generated using the ggplot2 package (version 3.4.4) within\nthe tidyverse framework in R (version 4.3.1). Differences in microbial\ncommunity composition between groups were assessed using non-\nparametric statistical methods. Alpha diversity indices were com­\npared using the Kruskal-Wallis test, while beta diversity was evaluated\nbased on Bray–Curtis dissimilarity, with group differences tested by\npermutational multivariate analysis of variance (PERMANOVA).\nPrincipal coordinate analysis (PCoA) was conducted to visualize\nmicrobial community similarities among samples. Functional profil­\ning was conducted based on KEGG (24) pathway annotation.\nPathway enrichment analysis was performed using the clusterProfiler\n\nZhang et al.\b\n10.3389/fvets.2026.1804735\nFrontiers in Veterinary Science\n04\nfrontiersin.org\npackage (version 4.10.0) in R (version 4.3.1). Differentially expressed\ngenes were identified based on an absolute log2 fold change > 1 and\nan adjusted p < 0.05. Significantly enriched pathways were deter­\nmined using a hypergeometric test with Benjamini-Hochberg correc­\ntion, and pathways with adjusted p < 0.05 were considered statistically\nsignificant. Enrichment results were visualized using clusterProfiler\nand ggplot2. Linear discriminant analysis effect size (LEfSe) (25) was\napplied to identify differentially abundant taxa between groups. The\nKruskal-Wallis test was used to detect taxa with significant differ­\nences followed by linear discriminant analysis (LDA) to estimate\neffect size. Taxa with an LDA score (log10) ≥ 2.0 and p < 0.05 were\nconsidered significantly enriched. Results were visualized as LDA\nscore histograms.\n2.5 Untargeted metabolomics sequencing\nand data analysis\nSamples were separated using an Agilent 1,290 UHPLC system\nequipped with a HILIC column, with the column temperature main­\ntained at 25 °C, a flow rate of 0.5 mL/min, and an injection volume of\n2 μL. The mobile phase comprised solvent A (water containing 25 mM\nammonium acetate and 25 mM ammonia) and solvent B (acetoni­\ntrile). Subsequently, following UHPLC separation, the analytes were\nsubjected to mass spectrometric detection using an AB Triple TOF\n6600 system (AB SCIEX), operating in both positive and negative elec­\ntrospray ionization (ESI) modes. Thereby enabling comprehensive\nmetabolic coverage. The ESI source parameters were configured as\nfollows: Gas1 = 60 psi, Gas2 = 60 psi, CUR = 30 psi, ion source tem­\nperature = 600 °C, and spray voltage = ±5,500 V. Raw LC–MS/MS\ndata were processed using XCMS (v3.12.0) (26, 27) for peak detection,\nretention time alignment, and signal normalization. Metabolite anno­\ntation was performed by matching accurate mass, retention time, and\nMS/MS fragmentation patterns against the Human Metabolome\nDatabase (HMDB) (28), KEGG, and METLIN databases. QC-based\n\nMetabolite anno­\ntation was performed by matching accurate mass, retention time, and\nMS/MS fragmentation patterns against the Human Metabolome\nDatabase (HMDB) (28), KEGG, and METLIN databases.QC-based\nsignal correction and missing value imputation were applied to ensure\nhigh-quality datasets for subsequent analyses.\nMultivariate statistical analyses, including principal component\nanalysis (PCA) and orthogonal partial least squares discriminant\nanalysis (OPLS-DA), were conducted using MetaboAnalyst (29) to\nevaluate metabolic differences between groups. Differential metabo­\nlites were identified based on variable importance in projection (VIP)\nscores (VIP > 1.0), univariate statistical analysis (p < 0.05), and fold\nchange thresholds. KEGG pathway enrichment analysis was subse­\nquently performed to identify significantly altered metabolic pathways\nassociated with treatment. Heatmaps, volcano plots, and pathway\nenrichment visualizations were generated using the R package ggplot2\n(version 3.4.4) and pheatmap (version 1.0.12).\n2.6 Statistical analysis\nData normality was assessed a priori prior to statistical analyses to\nensure appropriate test selection. Intergroup comparisons were con­\nducted using Student’s t-test for normally distributed data, whereas\ndifferential metabolites were identified using the Wilcoxon rank-sum\ntest, with false discovery rate (FDR) correction applied to account for\nmultiple testing. Microbe–metabolite associations were evaluated\nusing Spearman’s rank correlation coefficient. All statistical analyses\nwere performed using R (v4.3.2) and GraphPad Prism (v10.2.1), with\nstatistical significance defined as a two-tailed p-value or FDR < 0.05.\n3 Results\n3.1 Growth performance, diarrhea rate, and\nserum biochemical results\nAt baseline, no significant difference in initial body weight was\ndetected between the two groups (p > 0.05). By contrast, on days 14\nand 35, piglets in the FLJ group exhibited significantly higher body\nweight than those in the CON group (p < 0.05), accompanied by a\nconcomitant increase in average daily gain (ADG) (p < 0.05)\n\n05).By contrast, on days 14\nand 35, piglets in the FLJ group exhibited significantly higher body\nweight than those in the CON group (p < 0.05), accompanied by a\nconcomitant increase in average daily gain (ADG) (p < 0.05)\n(Supplementary Table S2). Regarding serum biochemical parameters,\nthe FLJ group demonstrated significantly elevated total protein (TP)\nand globulin (GLO) levels relative to the CON group (p < 0.05),\nwhereas alanine aminotransferase (ALT) and total bilirubin (TBIL)\nwere significantly reduced (p < 0.05). No significant differences were\ndetected in the remaining biochemical indices (p > 0.05)\n(Supplementary Table S3). Collectively, these findings suggested that\nFLJ supplementation effectively improved growth performance in pig­\nlets, while being associated with improved immune-related biochemi­\ncal profiles and ameliorated hepatic function, without exerting a\ndiscernible effect on feed intake.\n3.2 Overview of metagenomic sequencing\ndata\nAfter quality control and filtering, high-quality metagenomic\nsequencing data were obtained for all samples. Detailed sequencing\nstatistics are summarized in Supplementary Table S4. In total,\n531,773,478 raw reads were generated, corresponding to 76.00 Gb of\nraw data. Following quality filtering, 70.33 Gb of clean data were\nretained, with each sample yielding more than 5.33 Gb of valid\nsequences. The average GC content of the clean reads was 48.26%, and\nthe Q20 and Q30 values reached 97.55 and 93.68%, respectively, indi­\ncating high sequencing accuracy and data reliability.\nTo further evaluate assembly quality for downstream analyses, the\ndistribution of contig lengths was examined for each sample\n(Supplementary Figure S1). The majority of assembled contigs ranged\nfrom 500 to 1,000 bp, accounting for an average of 67.04% in the FLJ\ngroup and 65.25% in the CON group. Contigs with lengths between\n1,000 and 1,500 bp represented 15.14 and 15.41% of the assemblies in\nthe FLJ and CON groups, respectively. Overall, these results indicate\nthat the metagenomic assemblies were of sufficient quality for subse­\n\n25% in the CON group.Contigs with lengths between\n1,000 and 1,500 bp represented 15.14 and 15.41% of the assemblies in\nthe FLJ and CON groups, respectively.Overall, these results indicate\nthat the metagenomic assemblies were of sufficient quality for subse­\nquent microbial diversity and functional analyses.\n3.3 Microbial diversity analysis of cecal\ncontents\nAlpha diversity analysis was performed to evaluate microbial\nrichness, diversity, and evenness within individual samples. The\nShannon diversity index showed no significant difference in overall\nspecies richness between the FLJ and CON groups (p = 0.1205),\nalthough the FLJ group exhibited a numerically higher diversity trend\nwith reduced intra-group variability. In contrast, the Simpson index\nrevealed a significant increase in community evenness in the FLJ\ngroup compared with the CON group (p = 0.0004). Consistently, the\nInverse Simpson index was significantly higher in the FLJ group\n(p = 0.0009), indicating an increased effective species number and\nreduced dominance of individual taxa. Collectively, these alpha diver­\nsity indices showed that there was no significant difference in species\n\nZhang et al.\b\n10.3389/fvets.2026.1804735\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nrichness (Shannon index) among the FLJ groups; however, microbial\nevenness was significantly improved. (Figure 1A). Detailed alpha\ndiversity metrics are provided in Supplementary Table S5.\nBeta diversity analysis was conducted to assess differences in\nmicrobial community composition between groups. Principal coordi­\nnate analysis (PCoA) based on Bray–Curtis dissimilarity revealed a\nclear separation between the FLJ and CON groups, with the first two\naxes (PC1 and PC2) explaining 22.67 and 19.26% of the total varia­\ntion, respectively. Principal component analysis (PCA) showed a simi­\nlar clustering pattern, with PC1 and PC2 accounting for 33.10 and\n21.55% of the variance. Non-metric multidimensional scaling\n(NMDS) analysis further supported the group-wise separation\n(Figure 1B).\nBased on the Bray-Curtis distance calculated from relative abun­\ndance at the species level, PERMANOVA showed a significant dif­\nference in gut microbiota structure between the FLJ and CON\ngroups (F = 2.34, R2 = 0.190, p = 0.028). The intragroup dispersion\ntest was not significant (F = 0.16, p = 0.698), indicating that the\ncommunity differences were mainly driven by differences in inter­\ngroup community composition (centroid) rather than intragroup\ndispersion (Supplementary Table S6). Together, these analyses indi­\ncate that the overall structure of the cecal microbiota differed\nbetween groups.\n3.4 Bacterial community structure in cecal\ncontents\nTaxonomic profiling of the metagenomic data identified a\ntotal of 14,385 bacterial species, classified into 147 phyla, 233\nclasses, 387 orders, 757 families, and 2,754 genera. To characterize\ndifferences in microbial composition between groups, relative\nabundances were analyzed at the phylum, genus, and species\nlevels. At the phylum level, the cecal microbiota of both groups\nwas dominated by Firmicutes, Bacteroidota, Proteobacteria,\nActinobacteria, and Spirochaetes (Figure 2A). Compared with the\nCON group, the FLJ group exhibited higher relative abundances\n\nAt the phylum level, the cecal microbiota of both groups\nwas dominated by Firmicutes, Bacteroidota, Proteobacteria,\nActinobacteria, and Spirochaetes (Figure 2A).Compared with the\nCON group, the FLJ group exhibited higher relative abundances\nof Firmicutes (60.00% vs. 54.95%) and Actinobacteria (4.75% vs.\n4.01%), accompanied by lower abundances of Bacteroidota\n(24.83% vs. 25.47%) and Proteobacteria (7.90% vs. 12.93%). At the\ngenus level, the predominant taxa included Prevotella, Clostridium,\nBacteroides, Eubacterium, and Escherichia (Figure 2B). The FLJ\ngroup showed higher relative abundances of Clostridium and\nEubacterium, whereas Prevotella, Bacteroides, and Escherichia\nwere reduced compared with the CON group. At the species level,\ndominant taxa included Firmicutes bacterium CAG110, Escherichia\ncoli, Phascolarctobacterium succinatutens, Prevotella sp. P2-180,\nand Desulfovibrio piger (Figure 2C). The relative abundances of\nthese dominant species were generally lower in the FLJ group than\nin the CON group, reflecting differences in microbial composition\nat the species level.\n3.5 Analysis of differentially abundant\nmicrobes between FLJ and CON groups\nAt the phylum level, the FLJ group showed higher relative abun­\ndances of Firmicutes and Actinobacteria, while lower abundances of\nBacteroidota and Proteobacteria compared with the CON group, and\ndifferences were also observed in several low-abundance phyla,\nFIGURE 1\nDiversity of cecal content community composition: (A) Alpha diversity (Shannon index, Simpson index, inverse Simpson index). (B) Beta diversity\nanalyses based on principal coordinates analysis (PCoA), principal component analysis (PCA), and non-metric multidimensional scaling (NMDS).\n\nZhang et al.\b\n10.3389/fvets.2026.1804735\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nincluding Thermodesulfobacteria and p_Candidatus_Kaiserbacteria\n(Figure 3A). At the genus level, the dominant genera in the FLJ group\nwere Eubacterium, Alloprevotella, and Lachnospiraceae, whereas those\nin the control group (CON) were Prevotella, Escherichia, and\nBacteroides. Higher relative abundances of Eubacterium and other\nbeneficial genera while reducing potentially pathogenic genera such\nas Escherichia and several other genera in the FLJ group, whereas\nPrevotella and Escherichia were enriched in the CON group.\nSignificant differences were also detected for Collinsella,\nMogibacterium, Eubacterium, Dorea, and Blautia (Figure 3B). At the\nspecies level, Eubacterium_coprostanoligenes and Firmicutes_bacte­\nrium CAG110 were enriched in the FLJ group, whereas Escherichia_\ncoli and Prevotella_sp_P2-180 were enriched in the CON group, with\nEubacterium_coprostanoligenes and uncultured_Eubacterium_sp\nexhibiting the highest LDA scores (Figure 3C). Overall, Lonicera\njaponica treatment markedly modulated the cecal microbiota, pro­\nmoting beneficial bacteria while suppressing potential pathogens, and\nexerted multi-level regulatory effects on non-dominant microbial taxa.\n3.6 Functional potential of the cecal\nmicrobiota revealed by KEGG enrichment\nanalysis\nTo explore differences in the functional potential of the cecal\nmicrobiota between groups, metagenomic reads were annotated\nagainst the KEGG database, and functional enrichment analyses\nwere performed. At KEGG level 2, microbial functions were primar­\nily associated with metabolism-related categories, including carbo­\nhydrate metabolism, amino acid metabolism, energy metabolism,\nand nucleotide metabolism (Figure 4A). LEfSe analysis identified\ndistinct functional signatures between the FLJ and CON groups at\nthis level (Figure 4B). Pathways related to translation, replication\nand repair, nucleotide metabolism, glycan biosynthesis and metabo­\nlism, and energy metabolism were enriched in the FLJ group. In\ncontrast, functions associated with membrane transport, signal\n\nPathways related to translation, replication\nand repair, nucleotide metabolism, glycan biosynthesis and metabo­\nlism, and energy metabolism were enriched in the FLJ group.In\ncontrast, functions associated with membrane transport, signal\ntransduction, carbohydrate metabolism, cell motility, and sensory\nsystems were enriched in the CON group. At KEGG level 3, more\nrefined pathway analysis further supported these functional shifts\n(Figure 4C). The FLJ group was enriched in pathways involved in\nribosome biogenesis, DNA replication, homologous recombination,\npyrimidine metabolism, and mismatch repair (Figure 4D).\nConversely, the CON group showed enrichment in two-component\nsystems, ABC transporters, flagellar assembly, bacterial chemotaxis,\nand biofilm formation, including pathways associated with\nEscherichia coli. Overall, KEGG functional enrichment analysis\nrevealed distinct differences in microbial functional profiles between\nthe FLJ and CON groups. These differences were characterized by\nenrichment of biosynthesis- and replication-related pathways in the\nFLJ group and enrichment of transport- and motility-related path­\nways in the CON group.\n3.7 Composition of metabolites\nAfter database matching and compound annotation, a total of\n97 metabolites were identified across all samples. These metabo­\nlites were classified according to their chemical taxonomy, and\ntheir\noverall\ndistribution\nis\nsummarized\nin\nSupplementary Figure S2. Among the identified metabolites, lipids\nand lipid-like molecules constituted the largest category, account­\ning for approximately one-third of the total metabolite pool, fol­\nlowed by organic acids and derivatives, which together represented\nanother substantial proportion. Other metabolite classes, includ­\ning organic heterocyclic compounds, benzenes, organic oxygen\ncompounds, and phenylpropanoids and polyketides, were present\nat moderate levels, while nucleotides, alkaloids, lignans, and sul­\nfur-containing compounds accounted for relatively minor frac­\ntions. At a more detailed classification level, fatty acyls, steroids\nand steroid derivatives, prenol lipids, and glycerophospholipids\n\nAt a more detailed classification level, fatty acyls, steroids\nand steroid derivatives, prenol lipids, and glycerophospholipids\nwere the predominant subclasses within the lipid category, whereas\ncarboxylic acids and derivatives represented the major component\nof organic acids. Overall, the metabolite composition was charac­\nterized by a predominance of lipid-and organic acid-related com­\npounds, which were the main metabolite classes detected in the\ncecal contents.\nFIGURE 2\nMicrobial community composition at phylum, genus, and species levels. (A–C) Stacked bar plots showing the relative abundance of microbial\ncommunities at the phylum (A), genus (B), and species (C) levels in the FLJ group (n = 6) and the CON group (n = 6).\n\nZhang et al.\b\n10.3389/fvets.2026.1804735\nFrontiers in Veterinary Science\n07\nfrontiersin.org\n3.8 Principal component analysis of\nmetabolites\nPrincipal component analysis (PCA) was applied to assess\nglobal metabolic differences between the FLJ and CON groups. In\npositive ion mode, the first two principal components (PC1 and\nPC2) explained 30.1 and 18.0% of the total variance, respectively,\nwhereas in negative ion mode they accounted for 32.8 and 18.7% of\nthe variance (Figures 5A,B). PCA score plots showed a tendency\ntoward separation between the two groups in both ionization\nmodes, reflecting differences in overall metabolic profiles. To fur­\nther evaluate group discrimination, orthogonal partial least squares\ndiscriminant analysis (OPLS-DA) was performed. The OPLS-DA\nmodels for both positive and negative ion modes were validated\nusing 200 permutation tests. As demonstrated by the results, the Q2\nvalues of all permuted models were consistently lower than those of\nthe corresponding original models, thereby indicating that the\nobserved discriminatory performance was unlikely to arise from\nrandom permutations. More importantly, the regression lines of the\nQ2 values intersected the Y-axis at negative intercepts (Q2 = −0.236\nfor negative ion mode and −0.1283 for positive ion mode), further\nsubstantiating model validity and effectively excluding overfitting.\nThe OPLS-DA score plots demonstrated a clear separation between\nthe FLJ and CON groups in both positive and negative ion modes\n(Figures 5C,D), indicating distinct metabolite profiles between\ngroups. Together, the PCA and OPLS-DA analyses revealed clear\ngroup-level differences in the global metabolite profiles of cecal\ncontents, providing a basis for subsequent identification of differ­\nential metabolites and pathway analyses.\n3.9 Identification of differentially abundant\nmetabolites\nUntargeted metabolomic analysis detected a total of 9,151\nmetabolic features in positive ion mode, among which 1,203\nfeatures were significantly increased, and 663 were decreased in\nthe FLJ group compared with the CON group. In negative ion\n\nIn negative ion\nmode, 8,367 metabolic features were detected, including 672\nupregulated and 706 downregulated features (Figures 6A,B).\nFollowing metabolite annotation, 16.8% of the detected features\nin positive ion mode were successfully identified as known metab­\nolites. Among these annotated metabolites, 459 compounds\n(5.0%) were associated with lipid metabolism, and 420 com­\npounds (4.5%) were classified as organic acids and derivatives. In\nnegative ion mode, 9.0% of the detected features were annotated,\nincluding 303 lipid-related metabolites (3.6%) and 152 organic\nacids and derivatives (1.8%) (Figures 6C,D). Collectively, these\nresults revealed extensive differences in the cecal metabolite pro­\nfiles between the FLJ and CON groups, with a large proportion of\ndifferential metabolites related to lipid metabolism and organic\nacid pathways.\nFurther characterization of differential metabolites revealed\npronounced metabolic alterations induced by Lonicera japonica\nsupplementation in both positive and negative ion modes. In posi­\ntive ion mode, metabolites with higher abundances in the FLJ\ngroup were predominantly enriched in amino acid derivatives, pep­\ntides, unsaturated fatty acids, prostaglandins, and triterpenoids,\nwhereas metabolites with lower abundance were mainly associated\nwith sugars, amino acid derivatives, polyphenols, alkaloids, and\nprostaglandin-related compounds (Supplementary Table S7). In\nnegative ion mode, metabolites enriched in the FLJ group were\nprimarily involved in fatty acid metabolism, amino acid metabo­\nlism, steroid hormone biosynthesis, porphyrin metabolism, and\neicosanoid signaling. In contrast, metabolites enriched in the CON\ngroup were largely associated with bile acid metabolism, steroid\nhormone conjugation, carbohydrate metabolism, and xenobiotic-\nrelated pathways (Supplementary Table S8). Comparative analysis\nof the top five significantly enriched and depleted metabolites in\nboth ion modes further confirmed consistent group-level differ­\nences in metabolite abundance between the FLJ and CON groups\nFIGURE 3\n\nComparative analysis\nof the top five significantly enriched and depleted metabolites in\nboth ion modes further confirmed consistent group-level differ­\nences in metabolite abundance between the FLJ and CON groups\nFIGURE 3\nAnalysis of microbial community differences among groups (A–C). Differences in bacterial species between the FLJ and CON groups at the phylum\nlevel, the genus level, species level (Wilcoxon rank-sum test). ** indicates p < 0.01, * indicates p < 0.05.\n\nZhang et al.\b\n10.3389/fvets.2026.1804735\nFrontiers in Veterinary Science\n08\nfrontiersin.org\n(Figures 7A,B). Violin plot analysis revealed clear distributional \nseparation of representative differential metabolites between \ngroups, demonstrating the robustness of the metabolomic data \n(Figures 7C,D). Overall, differential metabolite analysis showed \ncoordinated \nchanges \nin \nmultiple \nmetabolic \ncategories \nbetween groups.\nFIGURE 4\nFunctional pathway differences of the cecal microbiota between the FLJ and CON groups based on KEGG analysis. (A) Relative abundance of KEGG \nfunctional categories at LEVEL2. (B) Differentially enriched KEGG LEVEL2 pathways between the FLJ and CON groups identified by LEfSe analysis (LDA \nscore >2.0, p < 0.05). (C) Relative abundance of KEGG functional pathways at LEVEL3. (D) Differentially enriched KEGG LEVEL3 pathways between the \nFLJ and CON groups identified by LEfSe analysis (LDA score > 2.0, p < 0.05).\n\nZhang et al.\b\n10.3389/fvets.2026.1804735\nFrontiers in Veterinary Science\n09\nfrontiersin.org\n3.10 Cluster analysis of differential\nmetabolites\nBased on positive ion mode data, a hierarchical clustering heat­\nmap was constructed using the top 10 differential metabolites ranked\nby VIP value. These metabolites were classified into six major chemical\ncategories (Figure 8A). Notably, several lipid-related and heterocyclic\ncompounds, including arachidonic acid-derived metabolites, steroid\nhormone precursors, and L-thyronine, exhibited distinct abundance\npatterns between the FLJ and CON groups. In addition, three bioactive\nshort peptides (e.g., Cys-Pro-Arg and Lys-Trp-Lys) were identified,\nshowing clear group-specific clustering patterns, indicating pro­\nnounced differences in peptide-related metabolism. Similarly, hierar­\nchical clustering analysis based on negative ion mode data revealed\nclear separation between the FLJ and CON groups using the top 10\ndifferential metabolites (Figure 8B). These metabolites mainly\nbelonged to steroid hormones, prostaglandins, phospholipids, amino\nacids and their derivatives, quinoline compounds, and microcystins,\nwith a predominance of acidic and highly polar metabolites. Among\nthem, multiple prostaglandin derivatives (e.g., 15-cyclohexylpentanor­\nprostaglandin F2α and 17-trifluoromethylphenyltrinorprostaglandin\nF2α), epoxy fatty acids (cis-9,10-epoxystearic acid), and\nphosphatidylserine-related lipids (2-oleoyl-1-palmitoyl-sn-glycero-\n3-phosphoserine) displayed marked differences in relative abundance\nbetween groups. Overall, hierarchical clustering analysis demonstrated\nthat differential metabolites in both ionization modes exhibited con­\nsistent and distinct grouping patterns between the FLJ and CON\ngroups, with lipid- and inflammation-associated metabolites emerging\nas prominent clustered features.\n3.11 KEGG pathway enrichment analysis of\ndifferential metabolites\nTo comprehensively characterize the metabolic pathways\naffected by Lonicera japonica supplementation, KEGG pathway\nenrichment analysis was conducted based on the identified differ­\n\n3.11 KEGG pathway enrichment analysis of\ndifferential metabolites\nTo comprehensively characterize the metabolic pathways\naffected by Lonicera japonica supplementation, KEGG pathway\nenrichment analysis was conducted based on the identified differ­\nential metabolites. Pathway enrichment was evaluated using the\nRich Factor, defined as the proportion of significantly altered\nmetabolites relative to the total number of metabolites annotated\nwithin each pathway. As shown in the bubble plot (Figure 9A), dif­\nferential metabolites were predominantly enriched in pathways\nassociated with lipid metabolism (biosynthesis of unsaturated fatty\nacids, steroid hormone biosynthesis, bile secretion), as well as path­\nways related to nucleotide and energy metabolism (nucleotide\nFIGURE 5\nPCA and OPLS-DA of cecal metabolites in weaned piglets. (A,B) PCA score plots in positive and negative ion modes, respectively. (C,D) OPLS-DA score\nplots in positive and negative ion modes, respectively, showing group-level separation between the FLJ and CON groups.\n\nZhang et al.\b\n10.3389/fvets.2026.1804735\nFrontiers in Veterinary Science\n10\nfrontiersin.org\nmetabolism and pyrimidine metabolism) and membrane transport\nprocesses (ABC transporters).\nKEGG annotation identified the top nine significantly enriched\npathways, involving 39 differential metabolites. These pathways\ncould be broadly categorized into three functional groups: lipid\nmetabolism-related pathways, nucleotide metabolism-related path­\nways, and transport or signaling-associated pathways. Among\nthem, the biosynthesis of unsaturated fatty acids was enriched with\nsix metabolites, five of which were upregulated, including cis-\n4,7,10,13,16,19-docosahexaenoic acid, cis-7,10,13,16,19-docosa­\npentaenoic acid, arachidic acid, behenic acid, and erucic acid, while\nlinoleic acid was downregulated. Steroid hormone biosynthesis\npathway was enriched with eight metabolites, showing increased\nlevels of hydrocortisone, aldosterone, progesterone, 21-hydroxy­\npregnenolone, and 11β-hydroxyprogesterone, along with decreased\nlevels of androsterone glucuronide and estrone glucuronide. In\naddition, the bile secretion pathway was characterized primarily by\ndownregulated levels of bile acid-related metabolites, including\nchenodeoxycholate, deoxycholic acid, taurolithocholic acid sulfate,\nand taurochenodeoxycholate. Pathways related to nucleotide\nmetabolism and transport were also significantly affected.\nPyrimidine metabolism and nucleotide metabolism showed enrich­\nment of multiple upregulated nucleosides and nucleobases, includ­\ning uridine, thymine, uracil, and adenosine, whereas cytidine and\nxanthine were downregulated. The ABC transporter pathways\ninvolved both upregulated metabolites (uridine, glutamine, adenos­\nine, and His-Lys) and downregulated carbohydrate-related sub­\nstrates, such as D-mannose, D-fructose, maltotriose, and\nN-acetylglucosamine, indicating alterations in substrate transport\nand utilization.\nTo further visualize pathway-level changes, a Differential\nAbundance Score (DAScore) analysis was performed. At the pri­\nmary KEGG classification level, pathways related to metabolism-\n\nTo further visualize pathway-level changes, a Differential\nAbundance Score (DAScore) analysis was performed.At the pri­\nmary KEGG classification level, pathways related to metabolism-\nparticularly lipid metabolism, nucleotide metabolism, and\nenergy metabolism-showed an overall upregulation trend\nin the FLJ group compared with the CON group, whereas path­\nways associated with neuroactive signaling exhibited a relative\ndownregulation (Figure 9B). At the secondary classification level,\nbiosynthesis of unsaturated fatty acids and steroid hormone bio­\nsynthesis were consistently grouped under lipid metabolism, fur­\nther supporting the metabolite-level enrichment results\n(Figure 9C).\nIn addition, metabolic set enrichment analysis (MSEA) revealed\nthat the most significantly enriched pathways in the FLJ group were\nprimarily associated with lipid metabolic regulation. These included\ncofactor-providing pathways such as pantothenate and coenzyme A\nbiosynthesis, signaling pathways involved in lipid metabolism regu­\nlation, including the cAMP signaling pathway and aldosterone-reg­\nulated sodium reabsorption, as well as pathways associated with\nlipid-related metabolic disorders, such as endocrine resistance and\nFIGURE 6\nVolcano plots of differential metabolic features and annotated metabolites in the cecal contents of weaned piglets. (A,B) Volcano plots of differential\nmetabolic features in positive and negative ion modes, respectively, identified based on variable importance in projection (VIP) scores. (C,D) Volcano\nplots of annotated differential metabolites in positive and negative ion modes, categorized according to chemical superclasses. Differential features and\nmetabolites were defined by VIP > 1.0, fold change (FC) > 1.5, and p < 0.05.\n\nZhang et al.\b\n10.3389/fvets.2026.1804735\nFrontiers in Veterinary Science\n11\nfrontiersin.org\ndiabetic cardiomyopathy (Figure 9D). Collectively, these results \nindicate that Lonicera japonica supplementation is associated with \nmulti-level modulation of lipid-related metabolic pathways, \naccompanied by coordinated changes in nucleotide metabolism and \nmembrane transport processes in the cecal metabolic profile of \nweaned piglets.\nFIGURE 7\nComparison of the top five significantly upregulated and downregulated metabolites between the FLJ and CON groups. (A,B) Relative abundance of \nthe top five upregulated and downregulated metabolites identified in positive and negative ion modes, respectively. (C,D) Statistical comparison of the \nrelative abundance of the top five upregulated and downregulated metabolites between the FLJ and CON groups in positive and negative ion modes. \nData are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.\n\n(C,D) Statistical comparison of the \nrelative abundance of the top five upregulated and downregulated metabolites between the FLJ and CON groups in positive and negative ion modes.Data are presented as mean ± SEM.*p < 0.05, **p < 0.01, ***p < 0.001.\n\nZhang et al.\b\n10.3389/fvets.2026.1804735\nFrontiers in Veterinary Science\n12\nfrontiersin.org\n3.12 Correlation network between gut \nmicrobiota and metabolites\nTo further explore the associations between gut microbial taxa \nand metabolic alterations following Lonicera japonica supplementa­\ntion, Spearman’s correlation analysis was performed between differ­\nentially abundant microbial species and metabolites. The resulting \ncorrelation matrix was visualized as a clustered correlation heatmap \n(Figure 10). Overall, distinct clustering patterns were observed \nbetween bacterial taxa and metabolites, with correlation coefficients \nranging from approximately −1 to 1, indicating both positive and \nnegative associations within the gut microbiota–metabolite network. \nPrevotella sp. CAG279, Prevotella sp. CAG891, Clostridium sp. \nCAG678 and Eubacterium coprostanoligenes exhibited strong positive \nFIGURE 8\nCluster heat map of differential metabolites between the FLJ and CON groups. (A) Hierarchical clustering heat map of the top 10 differential \nmetabolites in the positive ion mode. (B) Hierarchical clustering heat map of the top 10 differential metabolites in the negative ion mode. Differential \nmetabolites were selected based on VIP > 1.0 and p < 0.05. Color intensity represents normalized relative abundance (Z-score) of metabolites.\n\nZhang et al.\b\n10.3389/fvets.2026.1804735\nFrontiers in Veterinary Science\n13\nfrontiersin.org\ncorrelations with multiple differential metabolites, which were\nmainly involved in amino acid metabolism and SCFA-related path­\nways. Additionally, certain members of the phylum Firmicutes\nshowed negative associations with these metabolites, further high­\nlighting group-specific differences in microbe-metabolite interaction\npatterns. Collectively, these results demonstrate that Lonicera japon­\nica supplementation was associated with a distinct restructuring of\nthe gut microbiota-metabolite correlation network, potentially con­\ntributing to the observed metabolic remodeling in weaned piglets.\n4 Discussion\nThe present study provides comprehensive, integrated multi-\nomics evidence that dietary supplementation with Lonicera japonica\nprofoundly reshaped the gut microbial composition and repro­\ngrammed the cecal metabolic landscape of weaned piglets. Weaning\nis recognized as a critical and stressful developmental transition that\ndisrupts gut homeostasis, often leading to microbial dysbiosis and\ninflammation (30, 31). In this study, Lonicera japonica supplementa­\ntion was found to significantly increased microbial evenness,\nenriched beneficial bacterial taxa, enhanced microbial functional\npotential, and potentially induce coordinated alterations in lipid- and\namino acid-related metabolic pathways. Furthermore, correlation\nnetwork analysis revealed a relatively more structured and tightly\ncoordinated microbe-metabolite interaction pattern in the treatment\ngroup, characterized by potentially strengthened associations\nbetween beneficial microbes and key metabolic pathways.\nCollectively, these findings support the hypothesis that Lonicera\njaponica may acts as a phytogenic functional feed additive capable of\nstabilizing the gut ecosystem during early-life nutritional transitions\nthrough modulation of the gut microbiota-metabolite axis (32, 33).\n4.1 Effects of Lonicera japonica on gut\nmicrobial community structure\nGut microbial diversity is a key determinant of intestinal resilience\n\n4.1 Effects of Lonicera japonica on gut\nmicrobial community structure\nGut microbial diversity is a key determinant of intestinal resilience\nand host health (34). In this study, Lonicera japonica supplementation\nwas observed to significantly improved alpha diversity, particularly\nFIGURE 9\nIntegrated pathway enrichment and functional analysis of differential metabolites. (A) KEGG pathway enrichment bubble plot based on significantly\ndifferential metabolites, where bubble size represents the number of metabolites and color indicates enrichment significance. (B,C) DAScore plots at\nthe primary (H1) and secondary (H2) KEGG classification levels, illustrating overall upregulation or downregulation trends of metabolic pathways\nbetween the FLJ and CON groups. (D) MSEA showing significantly enriched metabolic pathways associated with Lonicera japonica supplementation.\n\nZhang et al.\b\n10.3389/fvets.2026.1804735\nFrontiers in Veterinary Science\n14\nfrontiersin.org\nmicrobial evenness, indicating a more balanced microbial community\nstructure. Higher microbial diversity has been consistently associated\nwith relatively greater ecological stability and resistance to pathogenic\ninvasion, particularly during the weaning period (35). Taxonomic\nprofiling revealed a pronounced restructuring of the bacterial com­\nmunity. Specifically, the relative abundances of Firmicutes and\nActinobacteria were elevated, whereas Proteobacteria were markedly\nreduced in the Lonicera japonica group. Within the Firmicutes\nphylum, the enrichment of SCFA-producing genera (such as\nFaecalibacterium and Eubacterium) is widely regarded as a hallmark\nof a more balanced and functionally robust gut ecosystem (36).\nThe observed increase in Firmicutes abundance was primarily\ndriven by the enrichment of SCFA-producing genera within this\nphylum, such as Faecalibacterium and Eubacterium. These genera are\nbelieved to play important roles in maintaining mucosal integrity, pro­\nviding energy for colonocytes, and modulating host immune\nresponses (37, 38). In contrast, increased abundance of Proteobacteria-\nparticularly Escherichia coli-is commonly considered to be a potential\nhallmark of gut dysbiosis and intestinal inflammation in weaned pig­\nlets (39). The observed reduction in these taxa suggests that Lonicera\njaponica may help suppress opportunistic pathogens, thereby poten­\ntially alleviating dysbiosis associated with weaning stress, potentially\nthrough direct antimicrobial activity or by enhancing colonization\nresistance via beneficial microbes expansion.\nNotably, the enrichment of Prevotella_sp_CAG279 and Prevotella_\nsp_CAG891 suggests a potentially enhanced capacity for dietary fiber\nand complex carbohydrate fermentation. Prevotella species are key\ndegraders of dietary fibers and phytochemicals and are frequently\nassociated with SCFA production, improved mucosal health, and\nmetabolic flexibility (40). Together, these microbial shifts indicate that\nLonicera japonica supplementation may promote a gut microbial\n\nTogether, these microbial shifts indicate that\nLonicera japonica supplementation may promote a gut microbial\necosystem characterized by enhanced functional redundancy and\nresilience, thereby supporting intestinal homeostasis during wean­\ning stress.\n4.2 Functional shifts in microbial\nmetabolism\nBeyond taxonomic restructuring, Lonicera japonica supplementa­\ntion also appeared to induce substantial shifts in the functional meta­\nbolic potential of the gut microbiome. Functional annotation based\non KEGG pathways revealed significant enrichment of pathways\nrelated to ribosome biogenesis, DNA replication, translation, energy\nmetabolism, and amino acid biosynthesis in the treatment group.\nThese functions are typically associated with enhanced microbial\ngrowth capacity, efficient substrate utilization, and increased biosyn­\nthetic activity, which collectively reflect a more stable ecological state\n(41). In contrast, pathways related to bacterial chemotaxis, two-com­\nponent regulatory systems, and environmental sensing and stress\nresponses were relatively more abundant in the control group, suggest­\ning a microbial community potentially adapted to fluctuating or\nstressful conditions (42).\nSimilar functional transitions have been reported in previous\nstudies demonstrating that dietary phytogenic additives promote a\nmetabolically active yet less inflammatory gut microbial environment\n(43). The bioactive constituents of Lonicera japonica, including chlo­\nrogenic acid, flavonoids, and iridoids, may directly modulate micro­\nbial metabolic activity or indirectly reshape microbial niches. These\ncompounds are known to possess prebiotic, antioxidant, and antimi­\ncrobial properties, which may selectively stimulate beneficial taxa\nwhile constraining opportunistic bacteria. Collectively, these func­\ntional enhancements likely contribute to the reprogramming of\nFIGURE 10\nCorrelation network between gut microbiota and metabolites. Spearman correlation heatmap between the top 20 differential bacterial species and\nmetabolites.\n\nZhang et al.\b\n10.3389/fvets.2026.1804735\nFrontiers in Veterinary Science\n15\nfrontiersin.org\nmicrobial metabolic strategies and stabilization of the gut ecosystem\nduring the weaning transition (44).\n4.3 Remodeling of cecal metabolomic\nprofiles\nUntargeted metabolomics revealed distinct metabolic separation\nbetween the treatment and control groups, indicating substantial\npotential remodeling of metabolites related to lipid metabolism,\norganic acids, and nitrogen-containing compounds. One of the most\nnotable findings was the significant reduction of bile acid-related\nmetabolites, including taurochenodeoxycholate (TCDCA) and deoxy­\ncholic acid (DCA), in the Lonicera japonica group. As a highly hydro­\nphobic secondary bile acid, excessive accumulation of DCA has been\nreported to exert cytotoxic and pro-inflammatory effects, thereby\ncontributing to intestinal mucosal barrier disruption (45). In parallel,\nTCDCA, serving as a ligand for receptors such as FXR and TGR5, has\nbeen implicated in immune dysregulation when aberrantly elevated\n(46). Notably, the production of both metabolites is contingent upon\nmicrobial enzymatic activity. Accordingly, the concomitant reduction\nin DCA and TCDCA suggests that Lonicera japonica may not directly\nperturb hepatic primary bile acid synthesis, but it may act by reshap­\ning the gut microbiota community, thereby suppressing the excessive\ngeneration of cytotoxic secondary bile acids at their microbial origin.\nThis regulatory effect may reduce intestinal inflammation and improve\noverall lipid metabolism homeostasis while maintaining basic lipid\nabsorption processes (47).\nConversely, Lonicera japonica supplementation was associated\nwith an increased the abundance of unsaturated fatty acids and steroid\nhormone-related metabolites, which are known to potentially support\nepithelial repair, immune modulation, and energy homeostasis (48).\nElevated levels of amino acid-derived metabolites and microbial fer­\nmentation-associated organic acids further suggest a potentially\nenhanced microbial fermentation capacity, consistent with the\n\nElevated levels of amino acid-derived metabolites and microbial fer­\nmentation-associated organic acids further suggest a potentially\nenhanced microbial fermentation capacity, consistent with the\nobserved enrichment of Prevotella and Clostridium. Such metabolic\nremodeling may likely contributes to improved nutrient utilization\nand optimized energy metabolism, thereby potentially mitigating\npost-weaning stress and intestinal metabolic homeostasis during\nearly-life development (49).\n4.4 Microbiota-metabolite interaction\nnetwork\nCorrelation network analysis provided deeper insight into the\nfunctional coupling between microbial taxa and metabolic out­\nputs. Beneficial genera such as Clostridium, and Eubacterium\nexhibited strong positive correlations with SCFA- and amino acid-\nrelated metabolites, whereas Escherichia and Corynebacterium\nshowed negative associations with lipid- and bile acid-related\nmetabolites (50). These patterns align with the concept of the gut\nmicrobiota-metabolite axis, wherein dietary interventions reshape\nmicrobial communities and, consequently, their metabolic\nproducts.\nSCFAs play pivotal roles in maintaining epithelial barrier integrity,\nregulating mucosal immunity, and modulating host energy metabo­\nlism (51). By promoting SCFA-producing taxa, Lonicera japonica\nsupplementation likely enhances these protective mechanisms.\nSimultaneously, the attenuation of associations involving opportunis­\ntic pathogens and potentially harmful bile acid metabolites suggests\nthat Lonicera japonica may interrupt deleterious microbe–metabolite\nfeedback loops that exacerbate dysbiosis during weaning (52). This\nnetwork-level reorganization reflects a more resilient, coordinated,\nand metabolically balanced intestinal ecosystem.\n4.5 Implications for pig health and\nproduction\nThe observed microbial and metabolic alterations carry important\npractical implications for pig health and production performance.\nPost-weaning diarrhea, impaired growth, and increased morbidity\nremain major challenges in modern pig production systems (53).\n\nPost-weaning diarrhea, impaired growth, and increased morbidity\nremain major challenges in modern pig production systems (53).\nTraditional interventions often rely on antibiotics or pharmacological\ndoses of zinc oxide, both of which face increasing regulatory restric­\ntions and environmental concerns. In this context, phytogenic feed\nadditives such as Lonicera japonica represent a promising, sustainable,\nand antibiotic-free alternative.\nThese microbial and metabolic shifts, including the regulation of\nSCFA and bile acid profiles, may play a role in maintaining intestinal\nhomeostasis. Consequently, Lonicera japonica supplementation holds\npromise as a strategy to favorably influence gut health, thereby sup­\nporting better feed efficiency and growth performance (54). These\nfindings are consistent with growing evidence that plant-derived bio­\nactive compounds can modulate the gut microbiota-metabolite axis\nand improve production traits, offering an environmentally friendly\nand sustainable nutritional strategy for animal health management in\nintensive pig production systems (55).\n5 Conclusion\nThis study suggests that dietary supplementation with Lonicera\njaponica can modulate the cecal gut ecosystem of weaned piglets.\nIntegrated metagenomic and metabolomic analyses revealed a rela­\ntively increased microbial diversity, enrichment of beneficial taxa,\nand enhanced microbial functions related to energy, lipid, and\namino acid metabolism. These microbial changes were accompa­\nnied by a relatively marked remodeling of the cecal metabolomic\nprofile, particularly involving unsaturated fatty acids, steroid hor­\nmone–related metabolites, and bile acid-associated pathways.\nCorrelation network analysis further showed that Lonicera japonica\nmay have strengthened beneficial microbiota-metabolite interac­\ntions while potentially weakening associations linked to opportu­\nnistic pathogens, suggesting improved intestinal ecological stability.\nCollectively, these findings highlight the gut microbiota-metabolite\naxis as a potentially key mechanism underlying the beneficial effects\n\nCollectively, these findings highlight the gut microbiota-metabolite\naxis as a potentially key mechanism underlying the beneficial effects\nof Lonicera japonica and support its potential use as a natural phy­\ntogenic feed additive to promote gut health during the post-wean­\ning period.\nData availability statement\nThe datasets presented in this study can be found in online\nrepositories. The names of the repository/repositories and accession\nnumber(s) can be found at: https://www.ncbi.nlm.nih.gov/,\nPRJNA1417956.\n\nZhang et al.\b\n10.3389/fvets.2026.1804735\nFrontiers in Veterinary Science\n16\nfrontiersin.org\nEthics statement\nThe animal study was approved by Department of Biological and\nFood Engineering, Lyuliang University, Lvliang, Shanxi, China. The\nstudy was conducted in accordance with the local legislation and insti­\ntutional requirements.\nAuthor contributions\nXZ: Data curation, Funding acquisition, Investigation,\nValidation, Visualization, Writing  – original draft. HH:\nConceptualization, Data curation, Methodology, Validation,\nWriting  – original draft. LH: Data curation, Investigation,\nValidation, Writing  – original draft. FY: Data curation,\nInvestigation, Validation, Writing – original draft. XH: Data cura­\ntion, Investigation, Validation, Writing  – original draft. YD:\nInvestigation, Validation, Writing – original draft. CF: Data cura­\ntion, Writing – original draft. HW: Conceptualization, Resources,\nWriting  – review & editing, Writing  – original draft. JH:\nConceptualization, Resources, Writing – review & editing.\nFunding\nThe author(s) declared that financial support was received for\nthis work and/or its publication. This work was supported by funds\nfrom the 2023 Lyuliang Development Zone’s Plan to Introduce\nHigh-Level Scientific and Technological Talents (no. 2023RC26 to\nXZ), the Fundamental Research Program of Shanxi Province (no.\n202403021222371 to XZ), the Key Laboratory of Specialty\nLivestock and Poultry Healthy Breeding and Deep Processing of\nProducts in Lyuliang City (no. 2025SYS05 to CF), the Project of\nAcademician and Expert’s Workstation in Yunnan Province (no.\n202605AF350075 to JH), the Agricultural Joint Project of the\nYunnan Province Science and Technology Department (no.\n202401BD070001-004 to JH), the Basic Research Key Project of\nYunnan Province, China (202501AS070041 to JH), and the\nResearch Start-up Fund for High-level Recruited Talent of Yunnan\nOpen University (to HH).\nConflict of interest\nThe author(s) declared that this work was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\n\nConflict of interest\nThe author(s) declared that this work was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.Generative AI statement\nThe author(s) declared that Generative AI was not used in the\ncreation of this manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. 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+{"text": "【兽医文献】\n标题: Differential organ responses to fumonisins in rabbits: kidney, liver, and spleen membrane fatty acid composition, oxidation markers, and histopathology\n\nTYPE Original Research\nPUBLISHED 29 August 2025\nDOI 10.3389/fvets.2025.1599721\nOPEN ACCESS\nEDITED BY\nKennedy Kapala Mwacalimba,\nZoetis, United States\nREVIEWED BY\nLászló Ózsvári,\nUniversity of Veterinary Medicine\nBudapest, Hungary\nPerle Zhitnitskiy,\nUniversity of Minnesota, United States\n*CORRESPONDENCE\nCharlotte Teixeira Costa\nc.teixeira-costa@rezoolution.fr\nRECEIVED 21 May 2025\nACCEPTED 04 August 2025\nPUBLISHED 29 August 2025\nCITATION\nTeixeira Costa C, Lebret A, Comer C and\nRose N (2025) Evidence-based veterinary\nmedicine perception by swine veterinarians: a\nEuropean survey across diverse practitioner\nprofiles. Front. Vet. Sci. 12:1599721.\ndoi: 10.3389/fvets.2025.1599721\nCOPYRIGHT\n© 2025 Teixeira Costa, Lebret, Comer and\nRose. This is an open-access article\ndistributed under the terms of the Creative\nCommons Attribution License (CC BY). The\nuse, distribution or reproduction in other\nforums is permitted, provided the original\nauthor(s) and the copyright owner(s) are\ncredited and that the original publication in\nthis journal is cited, in accordance with\naccepted academic practice. No use,\ndistribution or reproduction is permitted\nwhich does not comply with these terms.\nEvidence-based veterinary\nmedicine perception by swine\nveterinarians: a European survey\nacross diverse practitioner\nprofiles\nCharlotte Teixeira Costa1*, Arnaud Lebret1, Cl´ementine Comer2\nand Nicolas Rose3\n1Rezoolution, Pig Consulting services, Noyal-Pontivy, France, 2IRISSO-INRAE, Université\nParis-Dauphine, Paris, France, 3ANSES, Epidemiology, Health and Welfare Research Unit, Ploufragan,\nFrance\nEvidence-Based Veterinary Medicine (EBVM) enhances the quality of care by\nguiding clinical decisions with robust scientific data, leading to more effective\ndisease management. Evaluating the levels of knowledge and use is crucial\nfor identifying gaps and training needs, ultimately promoting the adoption of\nevidence-based practices and optimizing herd health and productivity. This study\naimed to give an overview of EBVM perception, with the goal of raising awareness\nof this concept and highlighting reservations they face in applying it to decision-\n\nThis study\naimed to give an overview of EBVM perception, with the goal of raising awareness\nof this concept and highlighting reservations they face in applying it to decision-\nmaking process in swine veterinary practice. A cross-sectional online survey was\nconducted among European swine veterinarians. Respondent characteristics\nand knowledge of EBVM were analyzed using descriptive statistics. Associations\nbetween demographic variables (country, specialization) and EBVM knowledge\nwere assessed using Chi-square tests with Yates’ continuity correction where\nappropriate. A significant level of 0.05 was applied. Results showed that 68%\nof veterinarians were familiar with EBVM, with knowledge levels varying by\ncountry (p < 0.05) and educational background (p<0.01). While 82% recognized\nits practical value, barriers such as limited time, difficulty accessing relevant\ndata, and a lack of decision-support tools hindered implementation. Younger\nveterinarians and those with less experience expressed more reluctance toward\nEBVM. Despite these challenges, 90% of respondents believed EBVM improved\ndecision-making, professional confidence, and communication with farmers.\nHowever, opinions differed regarding how well EBVM is currently integrated and\ndeveloped in everyday veterinary practice. These findings highlight the need for\nenhanced education and structured methodologies to bridge the gap between\ntheoretical knowledge and practical application in herd health management.\nKEYWORDS\nevidence-based,\nevidence-based\nveterinary\nmedicine,\nswine\nhealth,\nEurope,\nveterinarian\n1 Introduction\nOver the past decade, there has been a growing societal expectation that diagnostic\nand therapeutic decisions made by practitioners are based on evaluations of high-quality\nscientific literature (1). First described by Sackett et al. (2) as the “conscientious, explicit,\nand judicious use of current best evidence to make the best possible decision about a\nFrontiers in Veterinary Science\n01\nfrontiersin.org\n\nTeixeira Costa et al.\n10.3389/fvets.2025.1599721\npatient,” Evidence-Based Veterinary Medicine (EBVM) offers\na framework for veterinary practitioners to integrate critical\nappraisal skills and clinical evidence into their decision-making\nprocesses (30). Kochevar and Fajt (30) further elaborated the\nEBVM process into five key steps: (1) converting the need for\ninformation into an answerable question; (2) locating the best\nevidence to answer the question; (3) critically appraising the\nevidence for validity, impact, and applicability; (4) integrating\nthe appraisal with clinical expertise and the patient’s unique\nbiology, values, and circumstances; and (5) evaluating the\neffectiveness and efficiency of these steps and seeking ways\nto improve both the process and clinical outcomes. EBVM is\ndirectly inspired by Evidence-Based Medicine (EBM) in human\nhealthcare, though its adoption in veterinary practice is still\nevolving (3).\nWhile the adoption of EBVM in clinical practice is increasing\namong veterinary practitioners, it remains an evolving process\nrequiring the development of specific skills and tools (4–6).\nIncorporating EBVM principles into veterinary education is\nessential to equip practitioners with the necessary competencies\nfor evidence-based decision-making. For example, Huntley et al.\n(7) conducted an international survey, including veterinarians\nprimarily working in small animal practice across the UK, USA,\nCanada, and Australia. They reported that EBVM awareness often\noriginated during veterinary school or university, underlining\nthe importance of education in promoting EBVM principles.\nFor instance, young veterinarians and those who have recently\ncompleted further training tend to be more familiar with EBVM\nprinciples. (7). However, regardless of age, some veterinarians may\nfeel less confident engaging with scientific research methods due\nto limited training in epidemiology, statistics, or evidence appraisal\nduring their veterinary education (8).\nWhile previous research has addressed EBVM awareness\nand practices among veterinarians in general or in companion\n\nWhile previous research has addressed EBVM awareness\nand practices among veterinarians in general or in companion\nanimal medicine, very few studies have explored its application\nin the context of herd health management. This represents a\nsignificant gap, given the increasing emphasis on evidence-based\npractices in animal production systems. Compared to individual\nanimal medicine, herd health management introduces unique\nchallenges for EBVM implementation. Swine veterinarians often\noperate within complex, multifactorial production environments\nthat involve collective decision-making, time constraints, economic\npressures, and the need to balance population-level outcomes\nwith individual animal welfare. These factors complicate the\nstraightforward application of research findings and require a\nbroader and more integrated approach to evidence use.\nFurthermore,\nfew\nstudies\nhave\nexplored\nveterinarians’\nknowledge of EBVM practices, revealing varying levels of\nunderstanding. While most practitioners are aware of the concept,\nmany struggle with its practical application in daily practice (9, 10).\nEBVM was originally introduced in individual animal veterinary\nmedicine, where evidence-based practices follow a structured,\nunidirectional flow of information from research to assessment\nand ultimately to clinical application (11). This model, rooted in\nrandomized controlled trials (RCTs), prioritizes methodological\nrigor and clearly defined clinical endpoints. However, as Karriker\n(11) also noticed, this approach becomes more complex in\nthe context of production animal medicine, especially swine\nhealth. Blinded RCTs—considered the gold standard of clinical\nresearch—are particularly difficult to implement in pig barns due to\nethical, logistical, and economic constraints. As a result, the type of\nevidence generated in swine medicine is often perceived as weaker,\neven if it remains relevant and valuable for field application.\nThis reality influences not only the availability of high-quality\nevidence but also veterinarians’ confidence in its applicability to\nherd-level decision-making. Unlike individual animal medicine,\n\nThis reality influences not only the availability of high-quality\nevidence but also veterinarians’ confidence in its applicability to\nherd-level decision-making.Unlike individual animal medicine,\nwhich focuses on single-patient diagnoses and treatments, herd\nhealth management relies on a population-level approach that\nincorporates multifactorial influences and collective decision-\nmaking. This shift in decision-making necessitates adapting EBVM\nprinciples to population-level dynamics, economic constraints,\nand the involvement of multiple stakeholders (12). The application\nof these principles varies across production systems, such as pig\nfarming. Furthermore, there is limited consensus on core outcome\nmeasures in swine medicine, which hinders the comparability\nof studies and the development of a robust evidence base. As\nhighlighted by Sargeant et al. (13), establishing standardized\ncore outcome sets would improve consistency across trials and\nstrengthen the evidence supporting EBVM in swine practice.\nMoreover, the demanding nature of managing health across\nnumerous pig farms leaves veterinarians with limited time to\nengage with scientific literature, attend continuing education\ncourses, or participate in conferences. When assessing how\nveterinarians worldwide are accessing information, a recent study\nshowed that both practitioners and non-practitioners consult a\nwide array of journals and electronic resources (14). Examples of\nsuch electronic resources include online databases, professional\nforums, and veterinary-specific websites. This suggests that\nveterinarians are actively consulting a variety of journals and\ndigital resources to stay informed. If this information is made\naccessible and practical, veterinarians could effectively translate\nand communicate evidence-based health management strategies\ndirectly to producers. However, beyond access to information, the\npractical implementation of EBVM principles requires a structured\napproach. This includes the ability to critically appraise scientific\nevidence, integrate it with clinical expertise and farm-specific\n\nHowever, beyond access to information, the\npractical implementation of EBVM principles requires a structured\napproach.This includes the ability to critically appraise scientific\nevidence, integrate it with clinical expertise and farm-specific\nconstraints, and apply it through standardized decision-making\nframeworks. Encouraging veterinarians to adopt a methodological\napproach, such as formulating answerable clinical questions,\nsystematically searching for evidence, and objectively evaluating\nstudy outcomes, could help close the gap between theoretical\nknowledge and practical application in herd health management.\nIn this context, the objectives of this study were to: (1) assess\nthe level of knowledge of European swine veterinarians regarding\nEBVM and how it impacts their practice, (2) raise their awareness\nof this concept, and (3) evaluate the obstacles European swine\nveterinarians face in applying EBVM to swine health management\nstrategies in practice.\n2 Material and methods\n2.1 Survey design\nThe survey was designed to evaluate veterinarians’ knowledge\nof EBVM and their level of engagement in its application (see\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n\nTeixeira Costa et al.\n10.3389/fvets.2025.1599721\nSupplementary File 1). It consisted of 67 questions, integrated\ninto a broader questionnaire on porcine reproductive and\nrespiratory syndrome virus (PRRSV) and related practices, totaling\n197 questions. The questionnaire consisted predominantly of\nmultiple-choice questions (∼85%), complemented by short-\nanswer questions (∼15%) that allowed respondents to provide\nmore detailed feedback or explanations where appropriate. The\nquestionnaire was piloted with three swine veterinarians from\ndifferent European countries to ensure clarity and comprehension.\nBased on their feedback, minor changes were made to the wording\nand structure of some questions, particularly multiple-choice\nitems. The estimated time to complete the entire questionnaire\nwas ∼30 min. Most questions were closed-ended to enhance the\ncomparability of responses. A brief introduction provided the\ncontext for the study. Emphasis was placed on the anonymity\nof responses and the importance of this survey in improving\nand facilitating swine veterinary practice. The first question\naimed to determine the level of knowledge of veterinarians\nregarding EBVM. Subsequent questions explored key aspects of\nEBVM, including barriers to its adoption, perceived usefulness,\ncommonly used tools, and existing knowledge gaps. Additionally,\nan open-ended question allowed respondents to share their\nconcerns or fears regarding EBVM implementation. Veterinarians\nwere asked to rank both the perceived relevance of EBVM\nand their perception of how developed or established EBVM\npractices are within their daily clinical work. Furthermore, nine\nadditional questions were included to identify the tools and\ndata sources veterinarians rely on in their professional activities.\nLastly, a section of the questionnaire was dedicated to collecting\ngeneral demographic and professional information about the\nrespondents, such as gender, age, degree, type of workplace (size,\nproportion of veterinarians and technicians), country, and years\nof experience.\n2.2 Data collection and analysis\nThe target population included vet practitioners in Europe\n\n2.2 Data collection and analysis\nThe target population included vet practitioners in Europe\nwho dedicated at least 50% of their practice to pig health. To\nensure respondent eligibility, an open-ended question at the\nbeginning of the survey asked: “What percentage of your working\ntime is dedicated to swine health?” Only responses indicating\n50% or more were included in the analysis. All respondents\nin the final dataset met this requirement. Additionally, the\nsurvey was distributed exclusively through professional networks\ninvolved in swine health, helping to ensure the relevance\nof the sample. Open-ended responses collected in the survey\nwere analyzed using thematic content analysis. Two researchers\nindependently coded the qualitative data to identify recurring\nthemes related to barriers, concerns, and perceptions of EBVM\namong swine veterinarians. Discrepancies in coding were resolved\nthrough discussion to reach consensus. This approach ensured\na rigorous and repeatable analysis of the qualitative feedback\nprovided by respondents. The questionnaire was developed as\nan online survey using Sphinx iQ3 (Version 8.2.2). It was\ndistributed via email. All contact emails were collected through\nthe French Association for Pig Health (ANSP), the French\nInterprofessional Pork Council (INAPORC), the European College\nof Porcine Health Management (ECPHM), colleagues, and, in some\ncases, through contacts within pharmaceutical companies. Data\ncollection took place between October 2nd, 2023 and March 1st,\n2024, after extending the initial deadline by 1 month. Respondents’\ncharacteristics and EBVM knowledge were analyzed quantitatively.\nThe responses’ dataset was transferred to a Microsoft Excel V.14.0.6\n(2010, Microsoft) spreadsheet for data management. Descriptive\nstatistics, including frequencies and percentages, were calculated\nto summarize respondents’ characteristics and their knowledge of\nEBVM. Associations between categorical variables such as country,\nage, gender, and EBVM knowledge were assessed using Chi-\nsquare tests with Yates’ continuity correction where appropriate.\n\nAssociations between categorical variables such as country,\nage, gender, and EBVM knowledge were assessed using Chi-\nsquare tests with Yates’ continuity correction where appropriate.\nA significance level of 0.05 was applied. For theses analysis,\nno missing data was encountered in the questions analyzed,\nas most questions were closed-ended with forced responses\nor based on predefined lists, ensuring complete datasets for\nstatistical analysis.\n3 Results\n3.1 Study sample\nThe survey was sent to 657 veterinarians in 24 countries across\nEurope. A total of 108 veterinarians from 22 countries filled in the\nquestionnaire, which corresponds to a response rate of ∼16.6%.\nThe characteristics of respondents are summarized in Table 1. Most\nrespondents were male (60%), salaried employees (54%), with over\n10 years of professional experience (80%). Approximately 30% of\nrespondents were from France, with others representing various\nEuropean countries. Over half of the respondents reported having\neither a national or European specialization (53%). Although the\ntarget was veterinary practitioners who spent at least 50% of their\ntime monitoring pig farms, our sample was composed almost\ncompletely of swine veterinarians dedicating 100% of their time to\npig practice.\n3.2 Basic knowledge about EBVM\nSixty-eight percent of veterinarians answered “yes,” meaning\nthey had already heard of the EBVM concept before being\ncontacted (Figure 1). Despite the limited number of veterinarians\nin some countries, we observed that 100% of veterinarians\nwho completed the questionnaire in Poland and Romania\nwere unfamiliar with EBVM. Similarly, a large proportion of\nveterinarians in Greece (75%, n = 3), Denmark (70%, n = 7), and\nCroatia (67%, n = 2) had never encountered the concept. In other\ncountries, at least 50% of respondents were already aware of EBVM.\nOur results showed a significant association between veterinarians\nwho knew the concept and the country of the respondent (p <\n0.05) or the fact of having a European or national specialist diploma\n(p < 0.01). While, most veterinarians became aware of EBVM\n\nOur results showed a significant association between veterinarians\nwho knew the concept and the country of the respondent (p <\n0.05) or the fact of having a European or national specialist diploma\n(p < 0.01).While, most veterinarians became aware of EBVM\nthrough veterinary schools (42%, n = 31), others learned about the\nconcept through training programs and conferences (16%, n = 12),\nFrontiers in Veterinary Science\n03\nfrontiersin.org\n\nTeixeira Costa et al.\n10.3389/fvets.2025.1599721\nTABLE 1 Main characteristics of survey respondents (n = 108 European\npig veterinarians).\nVariable\nCategory\nSample (n = 108)\nNumber\n(Percentage)\nGender\nMale\n63 (58.3%)\nFemale\n45 (41.7%)\nAge\n<30 y/o\n8 (7.4%)\n30–39 y/o\n24 (22.2%)\n40–49 y/o\n37 (34.3%)\n50–59 y/o\n27 (25.0%)\n≥60 y/o\n12 (11.1%)\nStatus\nSalaried practitioner\n58 (53.7%)\nIndependent practitioner\n50 (46.3%)\nNumber of\nemployees in the\ncompany\nLess than 10\n34 (31.5%)\nBetween 10 and 50\n42 (38.9%)\nMore than 50\n32 (29.6%)\nProportion of\nveterinarians\namong the total\nnumber of\nemployees (in %)\n≤20%\n16 (14.8%)\n21–79%\n36 (33.3%)\n≥80%\n56 (51.9%)\nExperience of\nveterinarians in pig\nhealth\nLess than 10 years\n23 (21.3%)\nMore than 10 years\n85 (78.7%)\nOther diploma\nEuropean specialist\n11 (10.2%)\nNational specialization\n47 (43.5%)\nOther1\n5 (4.6%)\nNone\n45 (41.7%)\nCountry\nAustria\n5 (1.9%)\nBelgium\n4 (4.0%)\nCroatia\n3 (3.1%)\nDenmark\n10 (5.2%)\nEngland + Scotland\n1 (1.7%)\nFinland\n1 (1.0%)\nFrance\n34 (29.3%)\nGermany\n2 (2.3%)\nGreece + Cyprus\n4 (3.7%)\nHungary\n5 (6.4%)\nItaly\n4 (4.1%)\nLuxembourg\n1 (0.7%)\nMalta\n1 (1.1%)\nThe Netherlands\n9 (7.6%)\nNorway\n1 (1.2%)\nPoland\n2 (2.6%)\nPortugal\n2 (1.9%)\nRepublic of Ireland\n4 (6.2%)\n(Continued)\nTABLE 1 (Continued)\nVariable\nCategory\nSample (n = 108)\nNumber\n(Percentage)\nRomania\n1 (1.6%)\nSpain\n6 (6.2%)\nSweden\n5 (6.4%)\nSwitzerland\n3 (3.7%)\n1The category “other” includes other diploma with the same level as osteopathy, agricultural\ndiploma, etc.\nprofessional associations (7%, n = 10), scientific literature (19%, n\n= 14), or colleagues (8%, n = 6).\n3.3 European veterinarians’ opinions on the\nutility of EBVM\nMore than 82% of veterinarians recognized the practical\nvalue of the EBVM approach, even if they were unfamiliar\nwith the concept itself. However, 19 out of 108 veterinarians\nconsidered EBVM to be either not useful or only conditionally\nuseful, depending on factors such as the type of pathology, the\nspecific challenges encountered in the field, or the availability of\nreliable scientific data. Many respondents highlighted the variable\napplicability of EBVM, noting that while it can be a valuable\ncomplement in uncommon cases, its relevance is more limited\nin routine practice. One practitioner remarked, “Sometimes we\napply things without scientific proof that they work, we simply don’t\n\nOne practitioner remarked, “Sometimes we\napply things without scientific proof that they work, we simply don’t\nhave enough data.” Others (n = 19) emphasized the importance\nof pragmatic considerations, with one stating, “If it benefits\nthe farmer, that’s what matters.” Additionally, a certain mistrust\ntoward the European scientific community was expressed, with\nsome veterinarians questioning the reliability of the research\navailable. Some practitioners also admitted using EBVM selectively,\nas a way to validate their existing practices rather than as a\nframework for change. Despite some reservations expressed by\na minority of respondents, the majority acknowledged that the\nusefulness of EBVM depends on the clinical context, with its\nrelevance varying from moderate in routine cases to more critical\nin complex or uncommon situations. This contextual nuance\nemerged particularly from the open-ended responses explaining\nwhy some veterinarians viewed EBVM as conditionally useful or\nless applicable in daily practice.\n3.4 Definitions and features regarding\nEBVM according to swine veterinarians\nFor the majority of respondents, the EBVM approach was\nprimarily seen as a way to rely on evidence to make better decisions,\nwith nearly 90% agreeing on this point, regardless of their level\nof knowledge (Figure 2). Prior familiarity with EBVM significantly\ninfluenced respondents’ perceptions, with those already acquainted\nwith the concept more likely to recognize its practical value\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n\nTeixeira Costa et al.\n10.3389/fvets.2025.1599721\nFIGURE 1\nEBVM knowledge level by country of practice (n = 108).\nand benefits in clinical practice compared to those unfamiliar\nwith it (p < 0.01). This suggests that experience or education\non EBVM enhances appreciation of its usefulness. Additionally,\nmore than 50% of respondents viewed EBVM as a tool to\nincrease job satisfaction, while over 40% believed it helped them\nfeel more confident when interacting with farmers. This latter\nperception was strongly influenced by employment status (p <\n0.01): 86% of independent practitioners felt that EBVM made\nthem more comfortable in their role, compared to only 14% of\nsalaried veterinarians.\n3.5 Obstacles and barriers regarding EBVM\nuse\n3.5.1 Swine veterinarians’ requirements\nA total of 24 out of 108 veterinarians (22%) reported\nencountering obstacles when trying to implement EBVM in\ntheir practice. Among them, over 46% believed that EBVM\nrequires more time than they can afford (Figure 3). Additionally,\naround 33% assumed that accessing relevant data is difficult,\nwith many reporting challenges in retrieving scientific articles.\nMoreover, nearly 40% of respondents felt that there is a lack\nof decision-support tools to effectively apply EBVM. Finally,\napproximately 25% found EBVM challenging to implement due\nto a lack of support or resistance from colleagues, making its use\nuncomfortable in their daily practice.\n3.5.2 Impact of age and veterinary experience on\nconcerns about EBVM\nResistance\nto\nEBVM\nwas\nstrongly\nassociated\nwith\na\nveterinarian’s limited experience in swine health, namely among\nthose with <10 years of practice (p < 0.001). Indeed, 48% (n = 11)\nof veterinarians with <10 years of experience expressed reluctance\ntoward using EBVM, compared to only 15% of those with more\nthan 10 years of experience. In the same way, age also played a\nsignificant role: younger veterinarians (<30 years old) were more\nhesitant to use EBVM in practice, with 60% expressing concerns,\ncompared to <30% in older age groups (p < 0.001).\n3.6 Impact of EBVM on practitioners’\nconfidence\nOver 90% of all respondents felt that their relationship\n\n001).3.6 Impact of EBVM on practitioners’\nconfidence\nOver 90% of all respondents felt that their relationship\nwith farmers was easier to establish when they could present\nevidence-based support for their recommendations. Among these\nrespondents, 73% (n = 72) reported monitoring regularly the farms\nunder their care. Additionally, over 30% felt that their arguments\nbecame more persuasive as a result (Figure 4). This trend appeared\nto be influenced by gender, with a higher proportion of men\nreporting greater confidence in their communication compared\nto women (p < 0.10). Similarly, veterinarians working in larger\ncompanies tended to express higher self-confidence compared to\nthose working in smaller companies (p < 0.10).\n3.7 Is EBVM sufficiently developed in swine\npractice?\nResponses regarding the development of the EBVM concept\nin practice were mitigated (Figure 5). More than 47% (n = 51) of\nrespondents believed that the approach is sufficiently developed,\nwhile 53% disagreed. Among the 108 veterinarians surveyed, 18\nFrontiers in Veterinary Science\n05\nfrontiersin.org\n\nTeixeira Costa et al.\n10.3389/fvets.2025.1599721\nFIGURE 2\nOpinions of swine veterinarians on the definition of EBVM depending on whether they had previously heard of the concept (A) or not (B) (n = 108).\n(A) shows responses from veterinarians familiar with EBVM, while (B) presents responses from those unfamiliar with the concept.\nFIGURE 3\nBarriers to EBVM implementation in daily practice (n = 108).\n(17%) felt that EBVM is not developed at all in practice, whereas\n11 (10%) completely agreed that it is sufficiently established.\nAdditionally, women tended to be more likely to agree that EBVM\nis adequately developed in practice (p < 0.10).\n4 Discussion\nThis study provides new baseline data on European swine\nveterinarians’ knowledge and perceptions of EBVM. To our\nknowledge, it is the first European-wide study focusing on\nveterinarians who dedicate at least 50% of their practice to pig\nhealth. While EBVM has been widely discussed in veterinary\nliterature (15–17, 29), few studies have specifically explored\nits application in rural or swine-focused veterinary contexts.\nCompared to these studies, our findings reveal a lower level of\nEBVM awareness among swine practitioners, despite a generally\npositive attitude toward its principles. This discrepancy could\nbe due to limited integration of EBVM into swine-focused\ncurricula or continuing education programs. In contrast to studies\nwhere EBVM was well established among clinicians (18, 19), our\nresults suggest that swine veterinarians face unique structural\nand informational barriers, reinforcing the need for targeted\neducational and decision-support interventions in this field.\nFrontiers in Veterinary Science\n06\nfrontiersin.org\n\nTeixeira Costa et al.\n10.3389/fvets.2025.1599721\nFIGURE 4\nVeterinarians’ opinions regarding the influence of EBVM on farmer’s relationship (n = 108).\nThis survey represents the first phase of a broader project\non the EBVM concept and its application to veterinary decision-\nmaking for PRRSV control. This context may have influenced\nparticipants’ responses by framing EBVM primarily within the\nscope of infectious disease management. As such, respondents\nmay have viewed EBVM more through the lens of disease control\nstrategies, potentially underrepresenting its perceived usefulness in\nother aspects of swine veterinary practice. It was designed as a\nEuropean-wide study targeting veterinarians with at least 50% of\ntheir practice dedicated to pig health. Email contact was chosen as\nthe primary communication method to maximize reach. Although\nresponse rates in other studies using similar outreach methods\nwere generally higher (10, 14), our survey remains satisfactory\ngiven its European scope and the specificity of its target audience—\na relatively small population of swine practitioners. However,\nthe results must be interpreted with caution. Moreover, although\nthe study was conducted across 24 countries, the number of\nrespondents per country varied considerably, with some countries\nhaving only one or two participants. As a result, our findings\nshould not be interpreted as representative at the national level. The\nsampling strategy was based on voluntary participation through\nprofessional networks, which may have led to selection bias\nparticularly favoring veterinarians with a pre-existing interest in\nEBVM or PRRSV. This self-selection may influence the perceived\nlevel of EBVM awareness or motivation for its use.\nWhile our sample was diverse in terms of gender, status,\nage, and experience, nearly 30% of respondents were based in\nFrance, suggesting a potential overrepresentation of this group.\nFurthermore, due to the absence of comprehensive demographic\ndata on the European swine veterinarian population, we cannot\nconfidently assess the overall representativeness of our sample.\n\nFurthermore, due to the absence of comprehensive demographic\ndata on the European swine veterinarian population, we cannot\nconfidently assess the overall representativeness of our sample.\nNevertheless, we found no published data to assess it. Additionally,\nthere is a potential selection bias, as respondents may have been\nthose with a greater interest in this issue, such as practitioners\nactively dealing with PRRSV and seeking decision-making tools\nFIGURE 5\nSwine practitioners’ opinion on the development of EBVM in their\npractice (n = 108)\nfor disease control. Despite these biases, the survey provides\nvaluable insights into pig veterinarians’ knowledge of EBVM.\nOur results reveal substantial knowledge gaps regarding EBVM\namong swine veterinarians across Europe. The relatively low\nfamiliarity with the concept may reflect disparities in veterinary\ncurricula or continuing education opportunities. Interestingly,\nthe countries with the highest awareness may reflect stronger\nacademic emphasis on EBVM or more active involvement in\nprofessional training networks. These findings support the idea\nthat EBVM dissemination remains uneven across Europe and\nhighlight the need for harmonized educational efforts at the\nEuropean level. Notably, education was the primary source of\nknowledge on EBVM, reinforcing previous findings that formal\neducation plays a crucial role in informing veterinary practitioners\nFrontiers in Veterinary Science\n07\nfrontiersin.org\n\nTeixeira Costa et al.\n10.3389/fvets.2025.1599721\n(8, 20, 21). For instance, Block (2024) recently reported that\nEBVM remains insufficiently emphasized in veterinary curricula.\nAlthough the study did not focus specifically on European\ninstitutions, the findings likely reflect a broader trend that\nmay also apply to veterinary education in Europe, thereby\ncontributing to the ongoing challenges in integrating EBVM\ninto daily practice. Similarly, Keay et al. (22) conducted an\ninternational survey of swine veterinarians identifying barriers\nand knowledge gaps related to epidemiology, statistical methods,\nand research interpretation, which likely impact evidence-based\nveterinary practice across various countries, including Europe.\nMoreover, our survey revealed that respondents with additional\ndiplomas (e.g., in agriculture or osteopathy) tended to be better\ninformed about EBVM. This outcome might be expected, as\nthese individuals may have broader educational backgrounds and\ngreater exposure to scientific literature and professional networks\n(13, 23). Our results highlighted the crucial role of conferences,\nseminars, and professional training events in fostering EBVM\nawareness. This is particularly evident among specialists, as the\nEuropean College of Porcine Health management (ECPHM)\nguidelines mandate that graduates demonstrate their professional\ncompetence by practicing evidence-based veterinary medicine (The\nEuropean College of Porcine Health management, Constitution\nand Bylaws, article 4, section 10, ECPHM 2024). Thus, our survey\nresults highlight the importance of continuously educating swine\npractitioners on evidence-based decision-making to enhance their\nclinical practices.\nOur findings indicate an association between reservations\ntoward EBVM and demographic factors such as age and years\nof professional experience, which are closely related, making\nit difficult to separate their individual effects. While it is\noften assumed that more experienced veterinarians may be\nless receptive to new approaches, such as EBVM, our data\nalso suggest that younger professionals encounter challenges\n\nWhile it is\noften assumed that more experienced veterinarians may be\nless receptive to new approaches, such as EBVM, our data\nalso suggest that younger professionals encounter challenges\nin applying EBVM. Specifically, younger and less experienced\nveterinarians more frequently reported lack of time as a significant\nbarrier to implementing EBVM in their daily work. These\nchallenges may be influenced by workplace structure: early-\ncareer veterinarians often handle high-volume, routine cases\nand have limited decision-making authority. These structural\nand organizational factors could reduce their opportunities to\nengage with evidence-based approaches, despite having recently\nacquired theoretical knowledge. This complexity points to the need\nfor further investigation into how demographic, organizational,\nand educational factors interact in shaping EBVM adoption in\nveterinary practice.\nOur study also highlighted the need for swine veterinarians to\nbetter understand and properly implement the EBVM approach in\ntheir daily practice. Our findings suggest that developing decision-\nmaking tools and facilitating access to relevant data could enhance\ntheir ability to integrate this concept into their work. Improving\nveterinary practices, particularly in terms of facilitating diagnosis\nand clinical decision-making, will depend on both the specific\ndisease and the availability of relevant literature on the topic.\nTherefore, gathering additional input from veterinarians regarding\ntheir specific needs and gaps is essential to support better decision-\nmaking when faced with a disease (24).\nMoreover, veterinarians are recognized as key actors in\ntransmitting health knowledge to farmers (25–27). Given their\ngenerally positive perception of EBVM, they could serve as an\neffective knowledge transfer channel between researchers and\nfarmers. However, our results indicate that practitioners often lack\ntime to stay up to date with recent literature, highlighting the\nneed for researchers to explore more efficient ways of formalizing\nand disseminating information. Future improvements should focus\n\nHowever, our results indicate that practitioners often lack\ntime to stay up to date with recent literature, highlighting the\nneed for researchers to explore more efficient ways of formalizing\nand disseminating information.Future improvements should focus\non strengthening communication and knowledge transfer between\nresearch and veterinary practice. Since EBVM remains relatively\nunknown, ongoing research projects aim to better understand the\nobstacles to its implementation and its practical benefits. Regular\nupdates on these findings should be shared with practitioners\nto ensure they remain informed about the method and its\nadvantages. To this end, an initial factsheet summarizing European\nswine veterinarians’ perspectives on EBVM will be developed\nand distributed.\nAdditionally, our survey was part of a broader project aimed\nat evaluating the role of the “Evidence-Based Medicine” concept in\nveterinary decision-making within organized production systems,\nspecifically in the context of controlling PRRS in pig farming.\nThus, our present results should be analyzed within the context\nof veterinarians’ attitudes toward the EBVM concept and\nits application to PRRSV control. Indeed, the questionnaire\ninvestigated more deeply three other main areas: (i) Regulatory\nframework (the country-specific regulations governing PRRSV\ncontrol and the resources available for its management). (ii) Disease\nmanagement knowledge, including understanding the interactions\nwithin PRRSV control systems. (iii) Decision-making tools [among\nother thing, technology-assisted decision-making tools as described\nby Zhou et al. (28)] including digital platforms, data analytics,\nand monitoring technologies that support PRRSV prevention and\ncontrol efforts. In this context, veterinarians’ responses may likely\nbe influenced not only by their level of knowledge of EBVM but\nalso by their individual PRRSV approach. Further studies will be\nnecessary to determine whether veterinarians are both willing and\nable to implement PRRSV control and surveillance plans using\ntools derived from the EBVM approach.\n5 Conclusion\n\nFurther studies will be\nnecessary to determine whether veterinarians are both willing and\nable to implement PRRSV control and surveillance plans using\ntools derived from the EBVM approach.5 Conclusion\nOur baseline study revealed significant knowledge gaps\nregarding EBVM among swine practitioners that need to be\naddressed. Targeted educational initiatives should be implemented\nto raise veterinarians’ awareness of EBVM and its practical\napplications. Before launching a PRRSV control plan based\non EBVM-driven decision-making tools, further research is\nrequired to assess the specific needs for this objective. Such data\nwould provide valuable insights into veterinarians’ perspectives,\nenabling a more customized approach and adapted specifically\nto different practitioner profiles (for example, early-career vs.\nexperienced veterinarians). Supporting early-career veterinarians\nwith mentoring, practical resources, and simplified access to\nevidence summaries could improve their ability to implement\nEBVM in daily practice. Finally, collaboration between veterinary\nfaculty,\nprofessional\nassociations,\nand\nindustry\nstakeholders\nFrontiers in Veterinary Science\n08\nfrontiersin.org\n\nTeixeira Costa et al.\n10.3389/fvets.2025.1599721\nis essential to ensure that training initiatives are aligned\nwith field realities and help create optimal conditions for\nevidence-based disease control on pig farms. This, in turn,\nwould help guide efforts to overcome specific barriers and\ncreate\noptimal\nconditions\nfor\neffective\ndisease\ncontrol\non\npig farms.\nData availability\nThe raw data supporting the conclusions of this article will be\nmade available by the authors, without undue reservation.\nEthics statement\nAs this study involved a voluntary, anonymous survey\nof\nveterinary\nprofessionals\nand\ndid\nnot\ncollect\nsensitive\npersonal\ndata,\nit\ndoes\nnot\nfall\nunder\nthe\ncategory\nof\nhuman\nsubject\nresearch\nrequiring\nInstitutional\nReview\nBoard\n(IRB)\napproval.\nHowever,\nparticipants\nwere\nfully\ninformed about the study’s objectives and gave their consent\nbefore participating.\nAuthor contributions\nCT:\nMethodology,\nSoftware,\nWriting\n–\nreview\n&\nediting, Supervision, Writing – original draft, Investigation,\nConceptualization, Resources, Formal analysis, Visualization,\nFunding\nacquisition,\nProject\nadministration,\nData\ncuration,\nValidation.\nAL:\nProject\nadministration,\nValidation,\nConceptualization, Writing – review & editing, Supervision,\nMethodology. CC: Writing – review & editing, Methodology.\nNR:\nMethodology,\nValidation,\nConceptualization,\nProject\nadministration, Supervision, Writing – review & editing.\nFunding\nThe author(s) declare that no financial support was received for\nthe research and/or publication of this article.\nAcknowledgments\nThis work is part of a CIFRE PhD program supported by\nRezoolution and the ANSES agency. In this context, we would\nlike to express our gratitude to all those involved in this large-\nscale project. The authors also wish to thank the 108 participating\nveterinarians for their valuable contribution to this study.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declare that no Gen AI was used in the creation\nof this manuscript.\nAny alternative text (alt text) provided alongside figures in\nthis article has been generated by Frontiers with the support of\n\nGenerative AI statement\nThe author(s) declare that no Gen AI was used in the creation\nof this manuscript.Any alternative text (alt text) provided alongside figures in\nthis article has been generated by Frontiers with the support of\nartificial intelligence and reasonable efforts have been made to\nensure accuracy, including review by the authors wherever possible.\nIf you identify any issues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. 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+{"text": "【兽医文献】\n标题: Community perspectives on dogs, health risks, and veterinary care impacts in rural Alaska\n\nTYPE Editorial\nPUBLISHED 20 May 2025\nDOI 10.3389/fvets.2025.1602545\nOPEN ACCESS\nEDITED AND REVIEWED BY\nHussein M. El-Husseiny,\nTokyo University of Agriculture and\nTechnology, Japan\n*CORRESPONDENCE\nSokol Duro\ndurosokol@ubt.edu.al\nRECEIVED 29 March 2025\nACCEPTED 02 May 2025\nPUBLISHED 20 May 2025\nCITATION\nDuro S, G¨undemir O and Szara T (2025)\nEditorial: The integration of clinical veterinary\nanatomy and diagnostic imaging.\nFront. Vet. Sci. 12:1602545.\ndoi: 10.3389/fvets.2025.1602545\nCOPYRIGHT\n© 2025 Duro, G¨undemir and Szara. This is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY). The use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\naccordance with accepted academic practice.\nNo use, distribution or reproduction is\npermitted which does not comply with these\nterms.\nEditorial: The integration of\nclinical veterinary anatomy and\ndiagnostic imaging\nSokol Duro\n1*, Ozan G¨undemir2 and Tomasz Szara3\n1Department of Morphofunctional Modules, Faculty of Veterinary Medicine, Agricultural University,\nTirana, Albania, 2Department of Anatomy, Faculty of Veterinary Medicine, Istanbul\nUniversity—Cerrahpa¸sa, Istanbul, Türkiye, 3Department of Morphological Sciences, Institute of\nVeterinary Medicine, Warsaw University of Life Sciences, Warsaw, Poland\nKEYWORDS\nanatomical structures, diagnostic imaging, innovative MRI techniques, spectral CT, shear\nwave elastography, vascular anatomy\nEditorial on the Research Topic\nThe integration of clinical veterinary anatomy and diagnostic imaging\nThe integration of clinical veterinary anatomy and diagnostic imaging has significantly\nadvanced veterinary medicine, enhancing diagnostic precision, surgical planning, and\ntherapeutic strategies. Veterinary anatomy provides the fundamental framework for\nunderstanding the structure and function of animal bodies. At the same time, imaging\nmodalities offer non-invasive tools to visualize these structures in both normal and\n\nVeterinary anatomy provides the fundamental framework for\nunderstanding the structure and function of animal bodies.At the same time, imaging\nmodalities offer non-invasive tools to visualize these structures in both normal and\npathological states. The synergy between these fields has improved the accuracy of\ndisease diagnosis and paved the way for more refined interventional procedures. With\ncontinuous advancements in imaging technologies, including higher-resolution imaging,\nthree-dimensional reconstructions, and quantitative analysis, veterinary medicine is\nexperiencing an unprecedented transformation. The Research Topic, “The integration of\nclinical veterinary anatomy and diagnostic imaging,” presents a collection of 14 studies\nthat exemplify this interdisciplinary approach, contributing to a deeper understanding of\nanatomical variations, pathological conditions, and novel diagnostic methodologies.\nThe evolution of imaging technologies has been instrumental in veterinary diagnostics.\nTechniques such as computed tomography (CT), magnetic resonance imaging (MRI), and\nultrasonography have become indispensable tools, providing detailed anatomical insights\nthat facilitate the identification of pathologies challenging to detect through traditional\nmethods. These modalities have expanded the diagnostic capabilities of veterinarians,\nallowing for early disease detection, improved treatment monitoring, and more precise\ntherapeutic interventions. Several studies within this Research Topic highlight the practical\napplications of integrating anatomy and imaging:\nMagnetic resonance imaging (MRI) is an exceptionally valuable tool in veterinary\nmedicine, providing detailed visualization of musculoskeletal, nervous, and lymphatic\nstructures. Several studies in this collection explore innovative MRI techniques and their\npotential applications in diagnosing and monitoring various conditions. They offer new\napproaches for early detection, disease characterization, and improved treatment planning.\nBunzendahl et al. investigated the magnetic properties of canine menisci using ex\n\nThey offer new\napproaches for early detection, disease characterization, and improved treatment planning.Bunzendahl et al.investigated the magnetic properties of canine menisci using ex\nvivo MRI techniques to explore age-related joint degeneration. Their study assessed\nmagnetization transfer, T1, and T2∗relaxation times as potential cartilage and meniscal\nhealth biomarkers. By analyzing meniscal tissue’s structural and biochemical composition,\nFrontiers in Veterinary Science\n01\nfrontiersin.org\n\nDuro et al.\n10.3389/fvets.2025.1602545\nthe researchers identified patterns associated with degenerative\nchanges, which could serve as early indicators of osteoarthritis.\nTheir findings provide a foundation for developing non-invasive\ndiagnostic techniques that allow veterinarians to track joint\ndeterioration over time. The study’s results emphasize the potential\nof MRI-based biomarkers in guiding therapeutic interventions to\npreserve joint function and mitigate osteoarthritic progression in\naging dogs.\nLee et al. applied two-point and six-point Dixon MRI\nfor fat fraction analysis in healthy dogs’ lumbar vertebral\nbodies and paraspinal muscles, comparing the results with\nmagnetic resonance spectroscopy. The study demonstrated that\nDixon MRI provides a reliable and non-invasive method for\nquantifying fat infiltration in muscle tissue. It is a valuable\ntool for assessing muscle health and detecting early signs of\natrophy. Their research suggests that this imaging technique\ncould be beneficial for monitoring metabolic disorders and\nneuromuscular conditions that affect muscle composition. By\neliminating the need for invasive biopsies, Dixon MRI offers\na practical alternative for routine veterinary assessments and\nlong-term health monitoring. The study further validates Dixon\nMRI as a faster, clinically feasible alternative to traditional\nmagnetic resonance spectroscopy, increasing its applicability in\nveterinary practice.\nDuPont\nand\nBoudreau\nmeasured\ncanine\nmedial\nretropharyngeal lymph nodes using T2 spin-echo sequences\nat 3T MRI to establish baseline values for normal lymph node\nsize and texture. The study provides critical reference data for\ndistinguishing between physiological variations and pathological\nenlargement due to lymphoma, infection, or metastatic disease. By\ndefining normal morphometric parameters, this research improves\nthe diagnostic utility of MRI in clinical settings, allowing for more\naccurate assessments of lymphadenopathy. Their findings highlight\nthe importance of advanced imaging techniques in early disease\ndetection, ultimately aiding in timely therapeutic decision-making.\n\nTheir findings highlight\nthe importance of advanced imaging techniques in early disease\ndetection, ultimately aiding in timely therapeutic decision-making.\nThe study also explores the potential correlation between lymph\nnode size and systemic disease, further expanding the role of MRI\nin clinical veterinary oncology.\nKim et al. detailed the MRI characteristics of an atypical\ncervical glioma with a predominant extramedullary distribution\nin a dog. The case study underscored the importance of MRI\nin differentiating spinal tumors based on their location, extent,\nand tissue characteristics, which directly influence treatment\nplanning. Their imaging findings provided crucial insights into\nthe compressive effects of the tumor on surrounding neural\nstructures, guiding the surgical approach and postoperative\nmanagement. The study further demonstrated the potential\nfor significant neurological recovery following the intervention,\neven in severe spinal cord compression cases, emphasizing\nthe importance of early and accurate imaging for optimizing\ntreatment outcomes. By combining advanced MRI findings with\nhistopathological confirmation, this case highlights the evolving\nrole of MRI in diagnosing and managing complex spinal tumors\nin veterinary patients.\nIn addition to MRI, CT remains a highly effective tool in\nveterinary diagnostic imaging. By utilizing advanced computed\ntomography (CT) modalities—including spectral CT, cone beam\nCT (CBCT), multidetector CT (MDCT), and CT angiography—\nresearchers are enhancing diagnostic accuracy, refining treatment\nstrategies, and improving non-invasive assessment techniques.\nThese innovations provide a more precise understanding of\nanatomical and pathological variations, ultimately optimizing\nclinical decision-making in veterinary medicine.\nJi et al. examined the echocardiographic and CT features\nof congenital bronchoesophageal artery hypertrophy and fistula\nin a dog. Their study highlighted the complementary roles of\nechocardiography and CT angiography in diagnosing complex\nvascular anomalies, which are often challenging to detect using a\n\nTheir study highlighted the complementary roles of\nechocardiography and CT angiography in diagnosing complex\nvascular anomalies, which are often challenging to detect using a\nsingle imaging modality. A 4-year-old beagle underwent routine\nmedical screening, revealing a right-sided continuous murmur.\nFurther imaging identified multiple systemic-to-pulmonary shunts,\nwhich were surgically ligated. Postoperative imaging confirmed\nreduced ventricular overload and decreased shunt flow. This\nstudy provides valuable insight into the imaging features and\nsurgical\nmanagement\nof\nmulti-origin\nsystemic-to-pulmonary\nshunts, emphasizing the role of multimodal imaging in diagnosis\nand intervention.\nUmh et al. conducted a detailed morphologic study of PDA in\n25 dogs using computed tomography imaging, providing an in-\ndepth characterization of anatomical variations in this congenital\ndefect. By evaluating differences in PDA morphology, the study\nhelps refine surgical and catheter-based closure techniques,\nensuring better procedural outcomes. Their findings revealed three\ndistinct PDA morphologies, with significant correlations between\nanatomical dimensions and body weight. The distinctive cross-\nsectional configuration observed via CT imaging offers valuable\npre-procedural planning insights, potentially aiding the design\nof new occlusion devices. This research enhances interventional\ncardiology approaches, allowing for more tailored and effective\ntreatment strategies.\nHagenbach et al. compared cone beam computed tomography\n(CBCT) with multidetector computed tomography (MDCT) for\nimaging the equine carpal region. Their study demonstrated\nthat CBCT provides detailed visualization of bone and soft\ntissue structures with reduced radiation exposure compared to\nMDCT. In a study of 28 forelimbs from 15 horses, CBCT was\neffective for assessing osseous structures and some intraarticular\nligaments, particularly after contrast enhancement. However,\nMDCT provided superior imaging of soft tissue structures and\ncartilage. These findings position CBCT as a reliable diagnostic\ntool for equine orthopedic evaluations, particularly useful in\n\nHowever,\nMDCT provided superior imaging of soft tissue structures and\ncartilage.These findings position CBCT as a reliable diagnostic\ntool for equine orthopedic evaluations, particularly useful in\ndetecting early-stage musculoskeletal injuries such as stress\nfractures and osteoarthritis.\nMiki´c et al. evaluated virtual non-contrast spectral CT\nimages compared to accurate unenhanced images in rabbits,\ndemonstrating that spectral CT offers a viable alternative to\nconventional imaging with potentially lower radiation doses. By\nanalyzing 219 regions of interest in 20 rabbits, the study confirmed\nthat attenuation values between virtual and accurate non-contrast\nimages were highly comparable, particularly in the spleen, liver,\nmusculature, and renal cortices. Additionally, the elimination of\nmotion artifacts and reduction in radiation exposure make this\nmethod particularly beneficial for fragile or repeatedly imaged\npatients. While the technique has shown promise for normal\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n\nDuro et al.\n10.3389/fvets.2025.1602545\ntissues, further research is needed to validate its applicability in\ndiseased organs, paving the way for broader veterinary applications.\nShear Wave Elastography (SWE) has emerged as a valuable\nnon-invasive diagnostic tool for assessing reproductive organ\nhealth in veterinary medicine. Zappone et al. investigated testicular\nstiffness in healthy and fertile male dogs using qualitative (2D-\nSWE) and quantitative (pSWE, 2D-SWE) techniques. Their study\nestablished baseline values for normal testicular stiffness in\nmedium-to-large breed dogs, finding relatively uniform stiffness\nwith minor variations across anatomical regions but no significant\ndifferences between testes, breeds, or age groups. Additionally,\nbody weight showed a correlation with stiffness in 2D-SWE\nmeasurements. These findings highlight the potential of SWE for\ndetecting early testicular abnormalities, improving reproductive\nmanagement, and aiding in diagnosing subclinical infertility in\nbreeding dogs. Further research with larger and more diverse\ncanine populations will help refine its clinical applications.\nSimilarly, Uçmak et al. explored the use of SWE in monitoring\npostpartum reproductive organ recovery in Kivircik ewes. Their\nstudy quantified shear wave speed (SWS) and stiffness in the uterine\ncervix, caruncles, and vulvar labia, alongside Power Doppler\nultrasonography to assess blood flow changes in caruncles. They\nobserved significant time-dependent differences in cervical stiffness\nand caruncular regression, providing key insights into uterine\ninvolution. The findings demonstrate the potential of SWE in\ntracking postpartum recovery and fertility status in livestock,\noffering a new non-invasive approach to reproductive health\nassessment in ewes.\nTogether, these studies underscore the growing role of\nelastography in veterinary reproductive medicine. By enabling\nprecise, real-time evaluation of tissue stiffness in different\nreproductive structures, SWE is promising to improve breeding\nmanagement, early disease detection, and overall reproductive\nefficiency in companion animals and livestock.\n\nBy enabling\nprecise, real-time evaluation of tissue stiffness in different\nreproductive structures, SWE is promising to improve breeding\nmanagement, early disease detection, and overall reproductive\nefficiency in companion animals and livestock.\nKleiner et al. and Wolf et al. investigated advanced techniques\nfor sacroiliac luxation repair in feline cadaveric models. Both\nstudies utilized CT-based neuronavigation to enhance surgical\naccuracy and safety. Kleiner et al. compared fluoroscopy-controlled\nfreehand drilling to computer-assisted navigation, demonstrating\nimproved\nscrew\nplacement\nprecision\nwith\nneuronavigation.\nSimilarly, Wolf et al. assessed the feasibility of minimally invasive\ncomputer-assisted drilling (MICA) compared to the traditional\nfluoroscopy-controlled approach. Their study highlighted a steep\nlearning curve but showed that computer-assisted techniques\nreduced surgical inaccuracies over time, improving accuracy\ndeviations from 4.2 mm to 0.9 mm after five procedures. Moreover,\nWolf et al. introduced a new patient reference array placement\nmethod, which improved safety by preventing violations of vital\nstructures. These findings emphasize the role of CT-based image\nguidance in orthopedic procedures, enhancing surgical precision\nwhile minimizing neurovascular risks.\nGodlewska et al. presented a case report on a nodular lesion\nin the ventral neck of a rat, utilizing radiography, ultrasound,\nand echocardiography alongside histopathology to establish\na differential diagnosis. The study highlighted the challenges\nof assessing subcutaneous tumors in small mammals due to\ntheir rapid growth and varied etiologies. Their comprehensive\nmultimodal imaging approach facilitated a more accurate lesion\ncharacterization,\nenabling\ntargeted\nbiopsy\nand\nsubsequent\ntherapeutic planning. The case emphasizes the importance of\ncombining different imaging modalities in diagnosing rare or\nambiguous masses in exotic animal practice.\nReinitz et al. utilized CT-based angiography with a barium\ncontrast agent to generate a 3D reconstruction of the vascular\nanatomy in an African elephant’s hindfoot. This study provides\n\nReinitz et al.utilized CT-based angiography with a barium\ncontrast agent to generate a 3D reconstruction of the vascular\nanatomy in an African elephant’s hindfoot.This study provides\ncrucial insights into the unique blood supply to the digital cushion,\nsupporting in the diagnosis, treatment, and prevention of foot\ndiseases, which are a leading cause of morbidity in captive and\nwild elephants.\nIn conclusion, this Research Topic of studies underscores\nthe pivotal role of advanced imaging techniques in modern\nveterinary medicine. By integrating detailed anatomical knowledge\nwith cutting-edge imaging modalities, veterinary professionals can\nachieve more accurate diagnoses, refine surgical techniques, and\nimprove patient outcomes across various species and conditions.\nThe studies within this Research Topic illustrate how imaging\nis essential for diagnosing diseases and advancing anatomical\nknowledge, surgical precision, and therapeutic approaches in\nveterinary science.\nAuthor contributions\nSD: Writing – original draft, Writing – review & editing,\nConceptualization. OG: Writing – original draft, Writing – review\n& editing. TS: Writing – review & editing, Writing – original draft,\nConceptualization, Supervision.\nAcknowledgments\nThe Topic Editors appreciate the contributions of all authors to\nthis Research Topic.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. Any product that may be evaluated in this article, or\nclaim that may be made by its manufacturer, is not guaranteed or\nendorsed by the publisher.\nFrontiers in Veterinary Science\n03\nfrontiersin.org\n\n---"}
+{"text": "【兽医文献】\n标题: Arctic-like canine distemper virus in partially immunized puppies\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nTYPE  Original Research\nPUBLISHED  22 May 2026\nDOI  10.3389/fvets.2026.1824857\nOPEN ACCESS\nEDITED BY\nTugay Ayasan,\nOsmaniye Korkut Ata University, Türkiye\nREVIEWED BY\nIlias Giannenas,\nAristotle University of Thessaloniki,\nGreece\nAhmed Samy,\nNational Research Centre, Egypt\n*CORRESPONDENCE\nMoustafa Amin Osman\nmoustafaamin17@gmail.com\nMohammed Al-Rasheed\nmalrasheed@kfu.edu.sa\nRECEIVED 09 March 2026\nREVISED 24 April 2026\nACCEPTED 27 April 2026\nPUBLISHED 22 May 2026\nCITATION\n​Al-Ardhi SA, ​Al-Waeli SK, Osman MA, ​\nAl-Shahari EA, Siddig NH, Ahmed AE,\nHazzazi Y, Sumayli M, ​Al-Rasheed M and\nAlanazi IMM (2026) Modeling of growth\nperformance, physiological response,\nand intestinal microbiota shift in growing\nJapanese quail fed olive leaf powder.\nFront. Vet. Sci. 13:1824857.\ndoi: 10.3389/fvets.2026.1824857\nCOPYRIGHT\n© 2026 Al-Ardhi, Al-Waeli, Osman,\nAl-Shahari, Siddig, Ahmed, Hazzazi,\nSumayli, Al-Rasheed and Alanazi. This is\nan open-access article distributed under\nthe terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other\nforums is permitted, provided the\noriginal author(s) and the copyright\nowner(s) are credited and that the\noriginal publication in this journal is\ncited, in accordance with accepted\nacademic practice. No use, distribution\nor reproduction is permitted which does\nnot comply with these terms.\nModeling of growth performance,\nphysiological response, and\nintestinal microbiota shift in\ngrowing Japanese quail fed olive\nleaf powder\nSaad A. Al-Ardhi 1, Saad K. Al-Waeli 1, Moustafa Amin Osman 2*,\nEman A. Al-Shahari 3, Nawal H. Siddig 4, Ahmed Ezzat Ahmed 5,\nYehia Hazzazi 6, Mari Sumayli 6, Mohammed Al-Rasheed 7* and\nIbrahim Mufadhi M. Alanazi 8\n1Department of Animal Production, College of Agriculture, Al-Muthanna University, Al-Muthanna, Iraq,\n2Regional Centre for Food and Feed, Agricultural Research Centre, Ministry of Agriculture and Land\nReclamation, Giza, Egypt, 3Biology Health Specialties, Basic Sciences and Their Applications Unit, Applied\nCollege at Muhayil Asir, King Khalid University, Abha, Saudi Arabia, 4Department of Mathematical Science,\n\nCollege of Science, Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia, 5Biology\nDepartment, College of Science, King Khalid University, Abha, Saudi Arabia, 6Department of Biology,\nCollege of Science, Jazan University, Jazan, Saudi Arabia, 7Department of Clinical Sciences, College of\nVeterinary Medicine, King Faisal University, Al-Ahsa, Saudi Arabia, 8Department of Pharmacology and\nToxicology, Faculty of Medicine, Umm Al-Qura University, Makkah, Saudi Arabia\nIntroduction: Due to the anti-inflammatory, antioxidant, and antibacterial proper­\nties of olive leaf powder (OLP), it may serve as a beneficial feed supplement for\nbirds. This study aimed to evaluate the effect of adding OLP to feed on growth\nefficiency, carcass traits, blood parameters, antioxidant activity, and cecal microbial\nload in growing Japanese quail.\nMethods: Three hundred and seventy-five one-day-old quail chicks were ran­\ndomly assigned to five experimental groups, each containing five replicates of 15\nbirds: a control group fed a basal feed and four experimental groups given 3, 4,\n5, and 6% OLP per kg of quail diets for the 6 weeks.\nResults: The results showed a significant (P < 0.05) improvement in growth per­\nformance, with increased live body weight (LBW) and an insignificantly improved\nfeed conversion ratio in the 6% OLP group. Carcass yield and total edible meat also\nsubstantially improved (P < 0.05). Blood parameters showed improved (P < 0.05)\nprotein and lipid levels and increased liver enzyme activity; moreover, the use of\nthe OLP led to a decrease in TC, TG, LDL, and VLDL levels, as well as an increase in\nHDL. Additionally, the activity of antioxidant enzymes increased (P < 0.001), along\nwith higher levels of GSH and SOD activity. All meat quality attributes, including\ncooking loss %, water-holding capacity, PH values, and meat color, improved\n(P < 0.05) with the use of OLP. Furthermore, intestinal microbiota analysis showed\na significant improvement in Lactobacillus count and a decrease in pathogenic\nbacteria (total bacterial count, coliforms, E. coli, and Salmonella). However, the\n\n05) with the use of OLP.Furthermore, intestinal microbiota analysis showed\na significant improvement in Lactobacillus count and a decrease in pathogenic\nbacteria (total bacterial count, coliforms, E.coli, and Salmonella).However, the\nBacillus count was not significantly affected by OLP supplementation.\nDiscussion: The findings of this study demonstrate that OLP is a promising natural\nfeed additive for growing Japanese quail, enhancing growth performance and the\ngut microbiome, thereby establishing it as a safe and natural growth promoter.\nKEYWORDS\nblood indicators, cecal microbiota, growth performance, olive leaf powder, quail\n\nAl-Ardhi et al.\b\n10.3389/fvets.2026.1824857\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n1 Introduction\nThe steady population growth has led to an increasing demand for\nanimal protein, necessitating plans to increase production in efficient\nand economic ways (1, 2). Poultry meat, particularly quail meat, is a\nmajor source of protein due to its low cost, rapid production, and\nnutritional quality (3). However, the poultry industry faces challenges\nin producing antibiotic-free and environmentally safe meat, leading\nto a search for natural feed additives that enhance productivity and\nreduce disease and mortality (4, 5). As a result, replacing antibiotics\nused as growth promoters with effective natural alternatives, such as\nplant products and herbal extracts, is a global trend (6, 7).\nIn this context, natural additives and their extracts are rich in\nactive ingredients that act as antibacterial and growth promoters, pro­\nviding nutritional and health benefits for poultry (8, 9). For example,\nOlive trees are characterized by their high content of natural antioxi­\ndants such as oleuropein, dimethyloleuropein, ligstroside, and oleo­\nside, as well as phenolic compounds such as tyrosol, caffeic acid,\ntocopherols, catechol, rutin, and hydroxytyrosol, in addition to flavo­\nnoids such as kaempferol, apigenin, and luteolin; all these compounds\npossess significant medicinal potential (10).\nBeyond growth promotion, OLP is a natural alternative to antibi­\notics for bacterial infections in poultry, especially Salmonella. Its bio­\nactive compounds, such as oleuropein and hydroxytyrosol, work by\nbreaking down bacterial cell membranes, stopping biofilm formation,\nand slowing bacterial growth (11). Thanks to its potent antimicrobial,\nanticoccidial, antioxidant, and anti-inflammatory properties (12),\nolive leaf extract shows promise in improving poultry health and\nreducing the risk of Salmonella enterica contamination of eggs\nand meat.\nPrevious research has demonstrated the potential benefits of olive\nleaf products in poultry diets. Adding olive leaf extract (OLE) to quail\n\nPrevious research has demonstrated the potential benefits of olive\nleaf products in poultry diets.Adding olive leaf extract (OLE) to quail\ndiets resulted in increased body weight, improved feed conversion\nratio, decreased MDA levels, and enhanced glutathione reductase and\nSOD activity in both the liver and heart (13). Furthermore, Bahshwan\net al. (14) observed an improvement in intestinal microbial load, char­\nacterized by an increase in lactic acid bacteria and a decrease in\nSalmonella and E. coli, when olive leaf extract was added to the diets\nof Japanese quail. Similarly, a significant decrease in HDL levels and\nE. coli count in the cecum was also observed in broiler chickens when\nolive leaf extract was added to their diets at varying ratios (15).\nFurthermore, the addition of 150 mg/kg of OLE enhanced the lipid\nlevels, growth performance characteristics, antioxidant activity, and\nimmunity of laying hens (16).\nPrevious research has shown that olive OLP can be good for poul­\ntry, but there are still some important gaps in our knowledge.\nAlthough OLP holds potential as growth promoters and alternative\nfeed additives in poultry nutrition, further investigation is necessary\nto fully clarify their efficacy, mechanisms of action, and antimicrobial\nproperties. Most of the research that has been done so far has been on\nbroiler chickens or laying hens, and there is not much data on growing\nJapanese quail. Moreover, most studies have utilized olive leaf extract\ninstead of the more cost-effective powdered form, and dose–response\nrelationships across a broad spectrum of inclusion levels (from 3 to\n6%) have not been systematically assessed in this species. Furthermore,\nthe simultaneous evaluation of growth performance, blood biochem­\nistry, antioxidant status, meat quality, and cecal microbiota within a\nsingle experimental model is still limited. This study aimed to inves­\ntigate the effects of four different doses of OLP on growth,\nhematological parameters, antioxidant levels, immune response, and\ngut microbiota composition in Japanese quail, thereby offering a thor­\n\nThis study aimed to inves­\ntigate the effects of four different doses of OLP on growth,\nhematological parameters, antioxidant levels, immune response, and\ngut microbiota composition in Japanese quail, thereby offering a thor­\nough assessment of OLP as a potential natural feed additive for this\nspecies.\n2 Materials and methods\n2.1 Ethical approval\nThis study was performed on a private poultry farm in the Sharqia\nGovernorate and at the Animal Production Research Institute labora­\ntory in Dokki, Giza, Egypt, with ethical approval from the Institute’s\nResearch Ethics Committee, reference number IACUC-2023. The\ncommittee approved all experimental procedures, including the\nbreeding and management of birds.\n2.2 Preparation of olive leaf powder (OLP)\nand extract (OLE)\nThe study utilized olive leaves acquired from a commercial source.\nFor chemical analysis, employ reagents such as gallic acid, quercetin,\nmethanol, aluminum chloride, foline, ethanol, and DPPH (2,2-diphe­\nnyl-1-picrylhydrazyl) sourced from Merck, Darmstadt, Germany.\nOlive leaves were first washed with distilled water and air-dried,\nthen oven-dried at 40–45 °C until weight stabilized. The dried leaves\nwere ground into a fine powder (OLP) using a laboratory mill and\nstored in airtight containers at room temperature until incorporation\ninto the quail diets at levels of 3, 4, 5, and 6%.\nFor the sole purpose of determining total phenolic (TP) and total\nflavonoid (TF) content and antioxidant activity, a methanolic extract\nwas prepared. Exactly 10 g of OLP were mixed with 200 mL of 70%\nmethanol (v/v) in a 500 mL Erlenmeyer flask, yielding a solvent-to-\nsample ratio of 20:1 (mL/g). The mixture was stirred continuously\nusing a magnetic stirrer at 500 rpm for 3 h at room temperature\n(25 ± 2 °C) in the dark to prevent photodegradation of sensitive phe­\nnolic compounds. After extraction, the mixture was filtered through\nWhatman No. 1 filter paper under vacuum. The filtrate was collected,\nand the methanol was removed under reduced pressure using a rotary\nevaporator (Büchi Rotavapor R-300, Flawil, Switzerland) at 45 °C and\n\nAfter extraction, the mixture was filtered through\nWhatman No.1 filter paper under vacuum.The filtrate was collected,\nand the methanol was removed under reduced pressure using a rotary\nevaporator (Büchi Rotavapor R-300, Flawil, Switzerland) at 45 °C and\n100 mbar pressure. The dried extract (OLE) was weighed to calculate\nextraction yield, then transferred into amber glass vials, flushed with\nnitrogen gas to prevent oxidation, and stored at −20 °C until analysis\n(17, 18).\nAccording to Altıok et al. (19), the TP components were analyzed\nby HPLC, while total TF content was estimated using a method by\nOrdonez et al. (20). Furthermore, antioxidant activity was measured\nthrough the DPPH assay (20). Table 1 presents the TP content (mg GAE\nTABLE 1  Total phenols, flavonoids, and antioxidant activity of the methanolic extract acquired\nfrom olive leaves.\nItems\nContent\nTP (mg GAE g−1 extract)\n147.28\nTF (mg QE g−1 extract)\n41.70\nDPPH activity (SC50; mg mL−1)\n60.89\nTP, total phenol; TF, total flavonoid; DPPH, 2,2-diphenyl-1-picrylhydrazyl.\n\nAl-Ardhi et al.\b\n10.3389/fvets.2026.1824857\nFrontiers in Veterinary Science\n03\nfrontiersin.org\ng−1 extract), TF content (mg QE g−1 extract), and DPPH activity (SC50;\nmg mL−1) of the methanolic extract (OLE) derived from olive leaves.\n2.3 Birds, management, and design\nThree hundred and seventy-five one-day-old, unsexed Japanese\nquail chicks with a similar average initial live weight were randomly\ndistributed into five groups of 75 chicks each, with five replicates of 15\nbirds each; Unsexed chicks were used to reflect standard commercial\nproduction practices, as sexing at hatch is not routinely performed due\nto the lack of easily distinguishable sexual characteristics until\n2–3 weeks of age. The basal diet was formulated according to the rec­\nommendations of the National Research Council (21). Table 2 shows\nthe components and chemical composition of the basal diet. The quail\nchicks in the control group were fed the basal diet without additives,\nwhile the diets of the other four groups (OLP1-OLP4) were supple­\nmented with OLP at ratios of 3, 4, 5, and 6%, respectively, for six\nweeks. During the experiment, the birds were reared in electrically\nheated battery cages with a constant supply of water and feed, and a\ncontinuous lighting program was implemented.\n2.4 Data collection\nAll birds were individually weighed at 0, 2, 4, and 6 weeks of age\nto record LBW. Body weight gain (BWG) was calculated as the differ­\nence between the final and initial body weight over each period. Daily\nfeed intake (DFI) was calculated by subtracting the remaining feed\nfrom the amount dispensed. The feed conversion ratio (FCR) was cal­\nculated as DFI (g) / BWG (g). Both BWG, FI, and FCR were calculated\non a cumulative basis over defined experimental periods (1–15, 16–28,\n29–42 days, and overall, 1–42 days).\nAt the end of the experiment, five birds from each group were\nisolated and kept without feed overnight, with free access to water. The\nbirds were then weighed, slaughtered according to traditional Islamic\nmethods by cutting the jugular vein, and bled for approximately\n2–3 min. All carcass characteristics were calculated as a percentage of\n\nThe\nbirds were then weighed, slaughtered according to traditional Islamic\nmethods by cutting the jugular vein, and bled for approximately\n2–3 min.All carcass characteristics were calculated as a percentage of\nLBW (live body weight). Blood samples were also collected from each\nbird during the bleeding process.\n2.5 Blood indicators\nTwo blood samples were collected from each slaughtered bird. The\nfirst sample was collected in a heparin-free tube, and the serum was\ncentrifuged at 3000 rpm for 15 min. The serum was kept at −20 °C\nuntil its biochemical properties were measured. The activity of the liver\nenzymes alanine aminotransferase (ALT) and aspartate aminotransfer­\nase (AST) in the serum was measured using calorimetry. Total protein\n(TP), albumin (ALB), globulin (GLO), triglycerides (TG), total choles­\nterol (TC), high-density lipoprotein (HDL), and low-density lipopro­\ntein (LDL) levels were estimated according to the manufacturer’s\ninstructions (22, 23). Oxidation status parameters, including reduced\nglutathione (GSH), malondialdehyde (MDA), and superoxide dis­\nmutase (SOD), were determined using standard biochemical methods.\nThe second blood specimen was collected in a tube containing\nheparin and stored at 4 °C until blood parameters were checked.\nUsing a Neubauer hemocytometer improved Natt & Herrick’s solution\nas a diluent, allowing for the counting of white blood cells (24).\n2.6 Meat quality\nThree specimens of chicken breast fillet were prepared and cut\ninto small pieces (3 cm). The color indices of the meat samples were\nassessed as follows: brightness (L), redness (a), and yellowness (b)\n(25). The pH of the breast meat samples was measured using a\nHANNA pH meter (Won Sookit, USA). Approximately 10 g of the\nmuscle samples were cooked in an aluminum pan placed in an electric\noven (preheated to 200 °C) for 15 min or until the oven temperature\nreached approximately 80 °C. The percentage of weight loss during\ncooking was estimated by assessing the weight differences between the\ncooked and raw meat samples, expressed as a percentage of the\n\nThe percentage of weight loss during\ncooking was estimated by assessing the weight differences between the\ncooked and raw meat samples, expressed as a percentage of the\nuncooked beef samples after a 30-min cooling period at 15 °C. Water\nretention capacity (WHC) was assessed according to the methodology\ndescribed by Hoff-Lonergan and Lonergan (26).\n2.7 Cecal microbial load\nThe total bacterial count (TBC), along with the counts of coli­\nforms, Lactobacillus, Salmonella, and E. coli, was determined using ten\ngrams of ileum samples obtained from the birds in each replicate on\nslaughtering day; subsequently, the cecal bacterial load was deter­\nmined as described by (27, 28).\n2.8 Data statistical analysis\nThe collected data were analyzed by using General Linear Models\n(GLM), one-way analysis (SPSS software, 2019). Significant variations\nTABLE 2  Basal diet composition and calculated analysis.\nIngredients\n%\nMaize corn\n53.50\nSoybean meal (44%)\n30.50\nCorn gluten meal (60%)\n9.50\nWheat bran\n1.50\nVegetable oil\n0.50\nDL-methionine\n0.20\nL-Lysine HCl\n0.30\nNaCl\n0.50\nLimestone\n1.0\nMineral premix and vitaminsa\n0.50\nDi calcium phosphate (DCP)\n2.0\nCalculated analysis\nMetabolizable energy (kcal/kg)\n2900.0\nCrude protein %\n24.00\nCrude fiber %\n3.60\nCa %\n1.25\nAvailable phosphorus %\n0.40\nLysine\n1.36\nMethionine\n0.63\nMethionine + Cystine\n0.88\naPremix provided per kg of diet: vitamin A, 12.000 IU; vitamin D3, 2.400 IU; vitamin E,\n30 mg; vitamin K3, 4 mg; vitamin B1, 3 mg; vitamin B2, 7 mg; vitamin B6, 5 mg; vitamin\nB12, 15 μg; niacin, 25 mg, Fe, 80 mg; folic acid, 1 mg; pantothenic acid, 10 mg; biotin, 45 mg;\ncholine, 125,000 mg; Cu, 5 mg; Mn, 80 mg; Zn, 60 mg; Se, 150 μg.\n\nAl-Ardhi et al.\b\n10.3389/fvets.2026.1824857\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nacross treatment means were assessed utilizing Duncan’s multiple \nrange test (29); moreover, statistical significance was declared at \np < 0.05. Orthogonal polynomial contrasts were utilized to assess the \nlinear and quadratic impacts of OLP. Graphs were produced utilizing \nGraphPad Prism version 8.0. In addition, the normality of the data \ndistribution was assessed using the Shapiro–Wilk test, and the homo­\ngeneity of variances was evaluated using Levene’s test. All data met \nthese assumptions; hence, no transformations were required. The fol­\nlowing statistical model was employed:\n\nµ\n=\n+\n+\nij\ni\nij\nY\nT\ne\nWhere: Yij = the ith observation; μ = the overall means; Ti = effect\nof OLP treatments; eij = the random experimental error.\n3 Results\n3.1 Growth efficiency\nThe outcomes in Table 3 illustrated that OLP addition in quail\ndiets substantially (p < 0.05) improved LBW, BWG, and FI compared\nto the control. The inclusion of OLP at the 6% diet level yielded the\nbest results of LBW (224.65 g), BWG (5.10 g), and FI (15.78)\ncompared to other levels of OLP and those of the control. For FCR, no\nsignificant differences were detected among treatment groups for the\noverall experimental period (p > 0.05), although numerically lower\n(improved) FCR values were observed in the 4, 5, and 6% OLP groups\ncompared to the control.\n3.2 Carcass characteristics\nTable 4 showed that varying levels of OLP incorporation signifi­\ncantly (p < 0.05) influenced most carcass features. As shown, quails of\nthe OLP 6 group yielded the highest carcass%, heart%, and liver% in\ncomparison to other groups. On the other hand, the control group’s\nquails displayed the highest spleen percentage.\n3.3 Blood indicators\nThe data presented in Table 5 illustrate a significant difference\n(p < 0.05) between treatments that were detected in most protein frac­\ntions, with the highest levels of TP and GLO recorded in the control\ngroup compared to the other OLP groups. Additionally, the results\nshow lower levels of the enzymes ALT and AST in OLP groups 5 and\n6, respectively, compared to the other treatments. The introduction of\nOLP into the quail diets led to improvements in their lipid profile,\nparticularly in the OLP group 6, which exhibited the lowest levels of\nTG, TC, VLDL, and LDL, along with the highest levels of HDL com­\npared to the control group.\nTABLE 3  Illustrate the impact of diet supplementation with OLP on quails’ growth performance.\nItems\nTreatments\nSEM\np-value\nControl\nOLP 3%\nOLP 4%\nOLP 5%\nOLP 6%\nT\nL\nQ\nLBW (g)\nInitial\n10.48\n10.41\n10.48\n10.43\n10.50\n0.03\n0.956\n0.848\n0.677\nLBW 2 W\n37.86b\n37.53b\n51.35a\n45.27a\n47.28a\n1.61\n0.001\n0.001\n0.057\nLBW 4 W\n107.24b\n108.98ab\n109.85ab\n131.51a\n124.90ab\n3.71\n0.110\n0.025\n0.878\nLBW 6 W\n189.86c\n197.82bc\n207.84b\n201.13bc\n224.65a\n3.59\n0.004\n0.001\n0.427\nBWG (g)\nBWG 1–15 d\n1.95b\n1.93b\n2.92a\n2.49a\n2.62a\n0.11\n0.002\n0.153\n0.437\nBWG 16–28 d\n4.95ab\n5.10ab\n4.17b\n6.16a\n5.54ab\n0.24\n\n86b\n37.53b\n51.35a\n45.27a\n47.28a\n1.61\n0.001\n0.001\n0.057\nLBW 4 W\n107.24b\n108.98ab\n109.85ab\n131.51a\n124.90ab\n3.71\n0.110\n0.025\n0.878\nLBW 6 W\n189.86c\n197.82bc\n207.84b\n201.13bc\n224.65a\n3.59\n0.004\n0.001\n0.427\nBWG (g)\nBWG 1–15 d\n1.95b\n1.93b\n2.92a\n2.49a\n2.62a\n0.11\n0.002\n0.153\n0.437\nBWG 16–28 d\n4.95ab\n5.10ab\n4.17b\n6.16a\n5.54ab\n0.24\n0.102\n0.153\n0.437\nBWG 29–42 d\n5.90ab\n6.34ab\n7.00b\n4.97b\n7.12a\n0.29\n0.101\n0.548\n0.725\nBWG 1–42 d\n4.27c\n4.46bc\n4.70b\n4.54bc\n5.10a\n0.08\n0.004\n0.001\n0.431\nFI (g)\nFI 1–15 d\n7.99bc\n6.77d\n10.80a\n8.67b\n7.20cd\n0.39\n0.000\n0.766\n0.000\nFI 16–28 d\n16.09\n18.33\n13.03\n16.38\n19.44\n1.29\n0.644\n0.643\n0.390\nFI 29–42 d\n19.63ab\n22.07a\n18.80ab\n16.03b\n20.70ab\n0.77\n0.122\n0.421\n0.390\nFI 1–42 d\n14.56\n15.72\n14.21\n13.69\n15.78\n0.56\n0.768\n0.929\n0.593\nFCR (g/g)\nFCR 1–15 d\n4.10a\n3.54a\n3.71a\n3.51a\n2.74b\n0.56\n0.022\n0.003\n0.376\nFCR 16–28 d\n3.19\n3.56\n3.01\n2.74\n3.52\n0.20\n0.730\n0.913\n0.560\nFCR 29–42 d\n3.44\n3.50\n2.85\n3.27\n2.91\n0.18\n0.782\n0.390\n0.893\nFCR 1–42 d\n3.42\n3.52\n3.02\n3.04\n3.09\n0.13\n0.726\n0.295\n0.738\na,b,c,d,eMeans within the same row with different superscripts are significantly different (p ≤ 0.05).\nOLP, olive leaf powder; LBW, live body weight; BWG, body weight gain; FI, feed intake; FCR, feed conversion ratio; SEM, standard error of the mean; T, treatment; L, linear; Q, quadratic.\n\nAl-Ardhi et al.\b\n10.3389/fvets.2026.1824857\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nTable 6 shows the effects of OLP on the antioxidant activity of\nJapanese quail. All parameters were significantly affected (p < 0.05) by\nthe OLP addition. It is obvious that the OLP groups, especially\nOLP6%, yielded the highest levels of GSH, GPx, SOD, and CAT com­\npared to the untreated groups.\n3.4 Meat quality\nTable 7 shows the effect of adding different levels of OLP to quail\ndiets on meat quality characteristics. All meat quality characteristics\nimproved (p < 0.05) with the use of OLP. Compared to the control\ngroup and other treatments, the diet containing 6% extract achieved\nthe best pH value. Furthermore, this diet exhibited the lowest values\nfor brightness (L*) and yellowness (b*) and the highest values for red­\nness (a*). Additionally, the diet containing 6% OLP demonstrated the\nhighest water retention capacity and the lowest water loss during\ncooking.\n3.5 Microbiological examination of the\ncecum\nThe OLP treatment substantially influenced (p < 0.05) all caecal\nmicrobiota, except the Bacillus count, as illustrated in Figure 1. The\nOLP groups had the lowest levels of TBC, coliforms, E. coli, and\nSalmonella in comparison to the control. While OLP levels exerted\nmarked effects (p < 0.05) on Lactobacillus counts, OLP treatments\nelevated these counts, particularly at concentrations of 4 and 6%,\nwhich exhibited the highest numbers.\n4 Discussion\nThe olive leaf is a key component in phytotherapy, with Bucciantini\net al. (30) indicating phenolic compounds from olive oil and leaves as\nTABLE 4  Impact of diet supplementation with OLP on quails’ carcass traits.\nItems\nTreatments\nSEM\np-value\nControl\nOLP 3%\nOLP 4%\nOLP 5%\nOLP 6%\nT\nL\nQ\nPre SW (g)\n193.33d\n212.33bc\n206.66c\n213.33b\n221.66a\n2.61\n0.000\n0.000\n0.240\nCarcass %\n68.64d\n68.91cd\n72.06ab\n72.79a\n70.46bc\n0.48\n0.001\n0.001\n0.003\nLiver %\n3.44a\n3.09a\n2.61b\n3.30a\n3.35a\n0.09\n0.013\n0.959\n0.004\nHeart %\n0.75c\n0.81b\n0.89a\n0.81b\n0.88a\n0.01\n0.000\n0.000\n0.034\nGizzard %\n2.04\n2.11\n1.99\n2.08\n1.97\n0.03\n0.808\n0.569\n0.681\nT. edible %\n5.48a\n5.27a\n4.72b\n5.41a\n5.44a\n0.09\n0.038\n0.892\n0.016\nSpleen %\n0.23a\n0.11bc\n0.10c\n0.22ab\n0.10c\n0.02\n0.038\n0.183\n0.367\nBursa %\n0.12\n0.09\n0.09\n0.12\n0.09\n0.01\n0.774\n0.722\n0.699\n\n44a\n3.09a\n2.61b\n3.30a\n3.35a\n0.09\n0.013\n0.959\n0.004\nHeart %\n0.75c\n0.81b\n0.89a\n0.81b\n0.88a\n0.01\n0.000\n0.000\n0.034\nGizzard %\n2.04\n2.11\n1.99\n2.08\n1.97\n0.03\n0.808\n0.569\n0.681\nT.edible %\n5.48a\n5.27a\n4.72b\n5.41a\n5.44a\n0.09\n0.038\n0.892\n0.016\nSpleen %\n0.23a\n0.11bc\n0.10c\n0.22ab\n0.10c\n0.02\n0.038\n0.183\n0.367\nBursa %\n0.12\n0.09\n0.09\n0.12\n0.09\n0.01\n0.774\n0.722\n0.699\na,b,c,d,eMeans within the same row with different superscripts are significantly different (p ≤ 0.05).\nOLP, olive leaf powder; Pre-SW, preslaughter; T. edible, total edible part; SEM, standard error of the mean; T, treatment; L, linear; Q, quadratic.\nTABLE 5  Effect of diet supplementation with OLP on quails’ blood biochemicals.\nItems\nTreatments\nSEM\np-value\nControl\nOLP 3%\nOLP 4%\nOLP 5%\nOLP 6%\nT\nL\nQ\nTC\n261.00a\n182.36c\n233.41b\n204.59c\n192.24c\n8.24\n0.000\n0.001\n0.101\nTG\n345.46a\n254.00b\n205.74bc\n253.36b\n161.54c\n19.30\n0.008\n0.002\n0.370\nHDL (mg/dL)\n26.95c\n57.55a\n37.94b\n40.64b\n53.05a\n3.11\n0.000\n0.003\n0.208\nLDL (mg/dL)\n181.62b\n74.01c\n220.98a\n152.28ab\n101.22bc\n18.02\n0.000\n0.040\n0.033\nVLDL (mg/dL)\n69.09a\n50.80ab\n41.15b\n69.34a\n34.31b\n4.45\n0.006\n0.0.024\n0.852\nCRT (g/dL)\n0.75a\n0.46c\n0.72ab\n0.55bc\n0.46c\n0.03\n0.008\n0.019\n0.861\nUrea (g/dL)\n3.81bc\n4.62bc\n11.13a\n7.01b\n1.87c\n0.94\n0.001\n0.677\n0.000\nTP (g/dL)\n7.16a\n7.08a\n4.54bc\n5.76ab\n3.93c\n0.39\n0.002\n0.000\n0.886\nAlb (g/dL)\n2.46b\n3.29a\n2.37b\n2.62b\n2.10b\n0.12\n0.004\n0.021\n0.029\nGlo (g/dL)\n4.80a\n3.91ab\n2.28bc\n3.24abc\n1.94c\n0.33\n0.014\n0.003\n0.369\nA/G ratio\n0.65b\n0.95ab\n1.15ab\n0.97ab\n1.36a\n0.09\n0.157\n0.031\n0.800\nAST (U/L)\n184.16a\n144.82b\n182.65a\n145.29b\n117.43c\n7.33\n0.000\n0.000\n0.088\nALT(U/L)\n23.44a\n8.44c\n16.60b\n15.75b\n12.85bc\n1.44\n0.001\n0.018\n0.026\na,b,c,d,eMeans within the same row with different superscripts are significantly different (p ≤ 0.05).\nOLP, olive leaf powder; TC, total cholesterol; TG, triglyceride; ALT, alanine transaminase; AST, aspartate aminotransferase; CRT, creatinine; TP, total protein; Alb, albumin; Glo, globulin; HDL,\nhigh-density lipoprotein; LDL, low-density lipoprotein; SEM, standard error of the mean; T, treatment; L, linear; Q, quadratic.\n\nAl-Ardhi et al.\b\n10.3389/fvets.2026.1824857\nFrontiers in Veterinary Science\n06\nfrontiersin.org\npotentially beneficial to health, especially with prolonged consump­\ntion. The results found that supplementing quail diets with 6% OLP\nsignificantly enhanced LBW, BWG, and FI. However, the magnitude\nof improvement in BWG was relatively small, suggesting limited bio­\nlogical relevance despite statistical significance. This modest effect\nmay be because the basal diet already met or exceeded NRC (21) rec­\nommendations, leaving limited room for further growth enhance­\nment. While FCR improvements were not statistically significant\nduring the overall experimental period (p > 0.05), numerically lower\nFCR values were observed in the 4, 5, and 6% OLP groups compared\nto the control. The lack of statistical significance may be attributed to\nthe relatively short 6-week experimental duration, the possible satiety\neffect of dietary fiber in OLP, or the adequacy of the basal diet. These\noutcomes are consistent with the results of El-Kelawy and Refaie (31),\nwho found marked enhancements in quails’ growth efficiency, LBW,\nFI, FCR, and carcass traits with the inclusion of olive pomace in their\ndiets at concentrations of 5 or 10%. Likewise, Adel et al. (32), who\nassessed the inclusion of OLE at variance levels (0.015, 0.03, and\n0.06%) for growing quails’ diet, noted a notable improvement in previ­\nously estimated traits.\nThis boost can be ascribed to the multiple active compounds\ninvolved in OLE, including oleuropein and its derivatives, hydroxy­\ntyrosol, vanillic acid, caffeic acid, rutin, and vanillin, which may\nconfer specified health benefits (33). Parallel results were recorded\nin broilers of the Sasso breed by Nassar et al. (34), who found that\nthe best values of FCR were obtained from birds fed an OLP-\nsupplemented diet at a level of 2%. OLE’s antimicrobial features\nmake it a safe and sustainable growth promoter for poultry (35, 36).\nNevertheless, the lack of significant improvement in FCR suggests\nthat the bioactive compounds in OLP may exert their effects\nprimarily through physiological and health-related pathways, rather\n\nNevertheless, the lack of significant improvement in FCR suggests\nthat the bioactive compounds in OLP may exert their effects\nprimarily through physiological and health-related pathways, rather\nthan through a direct enhancement of dietary efficiency. This aligns\nwith recent evidence suggesting that phytogenic additives function\npredominantly as modulators of oxidative status and gut health\nrather than direct growth promoters (37). Consequently, such mech­\nanisms may yield moderate performance responses while pro­\nfoundly influencing health-related parameters. This suggests that the\nobserved improvements in LBW and BWG are likely mediated by\nenhanced intestinal health and antioxidant status rather than a sub­\nstantial increase in nutrient absorption. Further research utilizing\ndirect digestibility coefficients is warranted to validate these impacts.\nMoreover, OLE has been shown to positively alter the gut microbi­\nota in poultry, further supporting its role as a functional feed\nadditive.\nThe findings showed that varying concentrations of OLP incorpo­\nration significantly affected carcass features. Quails in the OLP 6 group\nexhibited the highest percentages of carcass, heart, and liver, while the\ncontrol had the highest spleen. The findings align with Erener et al.\n(15), who noted that administering 0.6 g of olive leaf extract/kg of feed\nresulted in carcass weight and dressing in broiler chickens. The per­\ncentage of abdominal fat was decreased in untreated birds relative to\nall animals administered olive leaf extract. These findings imply that\nOLP may influence carcass improvement through metabolic modula­\ntion rather than substantial growth enhancement, consistent with\nRichards (38), who observed that FI is a pivotal determinant affecting\nanimal growth rate and body composition during their lifespan.\nAlmuhayawi et al. (37) noted that broiler chicks administered OLP\nexhibited decreased abdominal fat formation and a marked rise in net\ncarcass weight and breast muscle mass. Moreover, Vasilopoulou et al.\n(39) indicated that OLP, especially at elevated doses, can enhance\n\n(37) noted that broiler chicks administered OLP\nexhibited decreased abdominal fat formation and a marked rise in net\ncarcass weight and breast muscle mass.Moreover, Vasilopoulou et al.(39) indicated that OLP, especially at elevated doses, can enhance\nTABLE 6  Impact of diet supplementation with OLP on quails’ antioxidant enzymes.\nItems\nTreatments\nSEM\np-value\nControl\nOLP 3%\nOLP 4%\nOLP 5%\nOLP 6%\nT\nL\nQ\nGSH (ng/mL)\n0.14c\n0.22bc\n0.35a\n0.26b\n0.42a\n0.02\n0.000\n0.000\n0.611\nGPx (ng/mL)\n0.13c\n0.27b\n0.27b\n0.24b\n0.35a\n0.02\n0.000\n0.000\n0.247\nSOD (U/mL)\n0.18c\n0.22c\n0.34b\n0.24c\n0.43a\n0.01\n0.000\n0.000\n0.030\nCAT (mg/dL)\n0.10d\n0.18c\n0.30a\n0.24b\n0.35a\n0.02\n0.000\n0.000\n0.513\na,b,c,d,eMeans within the same row with different superscripts are significantly different (p ≤ 0.05).\nOLP, olive leaf powder; GSH, Glutathione reduced; GPX, Glutathione peroxidase; SOD, superoxide dismutase; CAT, catalase; SEM, standard error of the mean; T, treatment; L, linear; Q,\nquadratic.\nTABLE 7  The effect of diet supplementation with OLP on quails’ meat quality traits.\nItems\nTreatments\nSEM\np-value\nControl\nOLP 3%\nOLP 4%\nOLP 5%\nOLP 6%\nT\nL\nQ\nCooking loss %\n19.95a\n19.24b\n19.00c\n17.62d\n17.52d\n0.25\n0.000\n0.000\n0.713\nWHC\n24.36e\n24.92d\n25.74b\n25.48c\n26.74a\n0.21\n0.000\n0.000\n0.000\nPH\n7.35a\n7.00b\n6.94c\n6.76d\n6.72e\n0.05\n0.000\n0.000\n0.000\nProteolytic\n24.16a\n20.76ab\n19.06ab\n20.08ab\n15.66b\n1.04\n0.117\n0.018\n0.928\nLightness (L*)\n23.88e\n32.61c\n38.91b\n40.79a\n30.80d\n1.61\n0.000\n0.000\n0.000\nRedness (a*)\n6.64ab\n7.24a\n7.34a\n6.19b\n5.04c\n0.24\n0.000\n0.000\n0.000\nYellowness (b*)\n6.63ab\n6.64a\n6.62b\n6.17c\n5.09d\n0.16\n0.000\n0.000\n0.000\na,b,c,d,eMeans within the same row with different superscripts are significantly different (p ≤ 0.05).\nOLP, olive leaf powder; WHC, water-holding capacity; SEM, standard error of the mean; T, treatment; L, linear; Q, quadratic.\n\nAl-Ardhi et al.\b\n10.3389/fvets.2026.1824857\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nintestinal growth, as demonstrated by the augmented ratio of intesti­\nnal weight to carcass weight.\nThe outcomes are probably attributable to bioactive polyphenolic \ncompounds and flavonoids prevalent in olive leaves, which demon­\nstrate dose-dependent antioxidant properties, fostering cardiovascular \nand hepatic protection, thus augmenting organ growth while \nimproving overall carcass productivity in Japanese quail (40). \nMoreover, Noreen et al. (41) indicated that such effects are increas­\ningly linked to reduced systemic inflammation and oxidative burden, \nwhich may lower immune-related energy expenditure and redirect \nnutrients toward productive tissues. The enlarged spleen size in the \ncontrol group aligns with recent research demonstrating that \nFIGURE 1\nEffect of diet supplementation with OLP on quails’ cecal microbiota (Log10CFU/ml). a,bMeans within the same row with different superscripts are \nsignificantly different (p ≤ 0. 05). OLP, olive leaf powder; TBC, total bacterial count; CFU, colony-forming units.\n\nAl-Ardhi et al.\b\n10.3389/fvets.2026.1824857\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nunfortified diets lead to larger lymphoid organs due to chronic oxida­\ntive stress or subclinical inflammation, contrasting with the immuno­\nmodulatory benefits of optimal oleuropein levels, which normalize\nspleen size without compromising immunity (37). This trend shows\nthat OLP is a beneficial long-term plant supplement for quail nutri­\ntion. It improves carcass characteristics at moderate to high inclusion\nrates (2–6%) and reduces organ hypertrophy that is linked to base\ndiets (42).\nThe findings indicate a notable difference in protein fractions\nacross treatments, with the control group having higher TP and GLO\nlevels. This reduction in TP and GLO in OLP-supplemented groups\nmay reflect a shift in protein metabolism or reduced inflammatory\nstimulation, rather than a negative physiological impact, as globulins\nare often elevated under immune or stress conditions (37, 41).\nMoreover, the ALT and AST enzyme levels were lower in OLP groups\n5 and 6;this decline strongly suggests a hepatoprotective effect of OLP,\nindicating improved liver integrity and reduced cellular leakage of\ntransaminases, which are key biomarkers of hepatic stress (43, 44).\nIncorporating OLP into quail diets improved the lipid profile, particu­\nlarly in group 6, which displayed the lowest TG, TC, VLDL, and LDL,\nalongside the highest HDL levels compared to the control. Such hypo­\nlipidemic effects are biologically relevant, as they indicate improved\nlipid metabolism and reduced risk of fat deposition, which aligns with\nenhanced carcass traits observed in this study (37, 45). Additionally,\nall indicators of antioxidants were substantially impacted by OLP\naddition, with OLP groups exhibiting the lowest GSH, GPx, SOD, and\nCAT levels, particularly the OLP 3%, which had the lowest values\namong all groups. However, this statement requires clarification, as\nthe data (Table 6) actually demonstrate a significant increase in anti­\noxidant enzyme activities with increasing OLP levels, particularly at 4\n\nHowever, this statement requires clarification, as\nthe data (Table 6) actually demonstrate a significant increase in anti­\noxidant enzyme activities with increasing OLP levels, particularly at 4\nand 6%, indicating enhanced oxidative defense capacity rather than\nsuppression (10, 46). Our results are consistent with the results of\nBahshwan (14), which indicate that the dietary addition of OLE mark­\nedly improves lipid profiles and liver function in quails. A diet con­\ntaining 0.03% OLE resulted in significant reductions in TC, VLDL,\nTG, and LDL, while increasing HDL. Additionally, OLE levels of 0.01,\n0.02, and 0.03% contributed to notable improvements in ALT and\nAST. Furthermore, Nassar et al. (34) showed a significant increase in\nTP and GLO, whereas the A/G ratio declined in Sasso broilers fed a\ndiet with 2% OLP compared to the control. This discrepancy with the\npresent study may be attributed to differences in inclusion level, spe­\ncies-specific responses, or the bioavailability of phenolic compounds\nin powdered versus extracted forms (33, 37). Moreover, Nafea and\nHussein (47) noted a substantial increase in blood TP and GLO levels\nin broilers with 2% olive leaves, alongside a decrease in TG and\nTC. Sarica and Toptas (48) reported a reduction in blood TC and LDL\nafter a dietary addition of 0.2 g OLP/kg of quail feed. Almuhayawi et\nal. (37) found that the injection of OLP enhanced the lipid profiles and\nALT or AST in broilers compared to the control.\nAgah et al. (43) identified a reduction in TC, TG, ALT, and AST\nlevels in broilers fed diets with 0.2 or 0.4 g OLP/kg diet. Parsaei et al. (49)\nobserved a significant decrease in blood TC, HDL, LDL, and VLDL\nlevels in broilers supplemented with an OL-diet at levels of 0.5 and 0.75%,\nrelative to the untreated group. However, unlike some previous studies\n(50), which reported that OLE insignificantly influenced serum TC,\nLDL, and HDL, our study found significant lipid-lowering effects. This\ninconsistency highlights the importance of dose-dependent responses\nand suggests that higher inclusion levels (e.g., 6% OLP) may be required\n\nThis\ninconsistency highlights the importance of dose-dependent responses\nand suggests that higher inclusion levels (e.g., 6% OLP) may be required\nto elicit measurable metabolic changes in quail (37, 50). Also, this\ndiscrepancy may be explained by differences in dosage (6% OLP vs.\nlower levels), bird species (quail vs. broilers), or the phenolic profile of\nthe olive leaf powder used. Variability in oleuropein and hydroxytyrosol\ncontent due to harvest time, drying method, and storage conditions may\nalso contribute. The hepatoprotective and hypolipidemic effects observed\nin the present study may be mechanistically linked to the ability of olive\npolyphenols to modulate lipid metabolism and reduce oxidative stress\nvia redox-sensitive signaling pathways such as Nrf2 Soleimani et al. (44).\nActivation of Nrf2 signaling enhances the transcription of antioxidant\nand detoxifying enzymes, thereby improving hepatic resilience and sys­\ntemic metabolic homeostasis (44, 51). Compounds found in OLP may\npossess the capacity to suppress the release of inflammatory mediators\nand cytokines, consequently reducing inflammation in the kidneys and\nliver (52). Moreover, the antioxidants present in OLP can safeguard these\norgans from damage linked to oxidative stress; the application of OLP\ncan maintain the normal functionality of renal and hepatic cells by\ndiminishing oxidative stress and inflammation. This unified antioxidant\nand anti-inflammatory mechanism offers a clear rationale for the con­\ncurrent enhancement of liver enzymes and lipid profile (53, 54).\nChemicals in olive leaf (OL) may enhance liver metabolism of harmful\nsubstances, reducing kidney workload (53). Oleuropein, a significant\nantioxidant polyphenol in OL, reduces lipid peroxidation and cholesterol\nlevels by neutralizing free radicals (54). OL may lower dietary cholesterol\nabsorption in birds by binding cholesterol and promoting bile acid\nmetabolism, with polyphenols increasing bile acid production, thereby\naiding in cholesterol elimination (45). Collectively, these mechanisms\n\nOL may lower dietary cholesterol\nabsorption in birds by binding cholesterol and promoting bile acid\nmetabolism, with polyphenols increasing bile acid production, thereby\naiding in cholesterol elimination (45).Collectively, these mechanisms\nindicate that OLP acts not merely as a dietary additive but as a metabolic\nmodulator influencing hepatic function, lipid turnover, and systemic\noxidative balance (10, 37, 44).\nAmong the key antioxidant enzymes are reduced GSH and SOD,\nwith MDA serving as a biomarker for lipid damage due to oxidative\nstress (51). The study indicates that OLP treatments significantly enhance\nthe quail’s antioxidant status, aligning with findings by Jemai et al. (46)\nthat hens given OLE show increased SOD levels and reduced MDA con­\ncentrations. This enhancement is strongly supported by recent studies\ndemonstrating that olive leaf polyphenols upregulate endogenous anti­\noxidant enzymes through transcriptional activation of antioxidant\ndefense systems (10). OLP may strengthen the body’s defense against\noxidative stress, supporting the production of antioxidant enzymes and\nstabilizing cell membranes, thus lowering MDA levels (55). Also, a recent\nstudy by Al-Shammari et al. (56) demonstrated that compounds like\noleuropein and hydroxytyrosol neutralize reactive oxygen species, miti­\ngating oxidative damage. Additionally, research shows increased T-SOD\nand GPx levels in broilers consuming diets with enzymatically fermented\nOP, attributed to its phenolic components (57). These improvements\nemphasize a stronger impact of OLP on metabolic health and oxidative\nstatus than on growth performance.\nOLP usage significantly enhanced all meat quality characteristics.\nThe diet containing 6% extract yielded the best pH, the lowest L* and\nb*, and the highest a*. It also exhibited superior water retention capac­\nity and minimized water loss during cooking. Moreover, the findings\nalign with Xie et al. (50) and Vasilopoulou et al. (39), indicating that\nthe incorporation of 0.3% OLE into the diet diminished breast meat\nshear force, elevated GSH levels, enhanced T-SOD and GSH-Px activi­\n\nMoreover, the findings\nalign with Xie et al.(50) and Vasilopoulou et al.(39), indicating that\nthe incorporation of 0.3% OLE into the diet diminished breast meat\nshear force, elevated GSH levels, enhanced T-SOD and GSH-Px activi­\nties, and decreased MDA levels. This indicates that lipid peroxidation\nand protein denaturation were diminished, resulting in enhanced soft­\nness, color fidelity, and moisture retention. Likewise, 1% oleuropein-\nenriched OLE improved the color of thigh meat, increased tenderness,\nand maintained intestinal health without hindering growth, supporting\n\nAl-Ardhi et al.\b\n10.3389/fvets.2026.1824857\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nthe dose-dependent effectiveness of polyphenols in regulating post­\nmortem glycolysis for ideal pH reduction and myoglobin preservation\nagainst metmyoglobin development, thereby enhancing sustainable\nmeat quality through natural feed additives (58, 59). The polyphenols\nfound in OLP can reduce the drop in muscle pH after slaughter by\nregulating microbial growth and metabolic processes, often leading to\na more stable final meat pH and slowing down spoilage (60). In addi­\ntion, these improvements in meat quality when OLP or extract is added\nto poultry feed are due to biochemical mechanisms, as the polypheno­\nlic antioxidants found in olive leave help reduce the oxidation of fats\nand proteins, stabilize meat quality after slaughter, and improve water\nretention, leading to improved muscle condition and reduced losses\nduring cooking.\nThe OLP groups showed the lowest concentrations of TBC, coli­\nforms, E. coli, and Salmonella compared to the control. OLP treat­\nments significantly increased Lactobacillus counts, especially at 4 and\n6%, which were the highest. This shift in gut microbiota indicates that\nOLP may function as a natural antimicrobial and prebiotic agent,\ncontributing to improved intestinal health rather than direct growth\nenhancement. However, OLP supplementation did not significantly\naffect the Bacillus count, indicating that not all beneficial bacterial\ngenera respond equally to olive leaf polyphenols. These outcomes\nalign with the results Adel et al. (32) demonstrated that 0.03 and\n0.06% OLP enhance the proliferation of Lactobacilli and diminish the\nprevalence of pathogenic bacterial species in the ceca of growing\nquail. OLP contains many bioactive chemicals with strong antibacte­\nrial properties that can compromise bacterial cell membranes, result­\ning in cell death and the release of internal components or impair\ntheir enzymatic systems (61, 62). Additionally, several bioactive\nchemicals included in OL may impede bacterial communication, or\nquorum sensing, hence decreasing the capacity of pathogens to\n\nAdditionally, several bioactive\nchemicals included in OL may impede bacterial communication, or\nquorum sensing, hence decreasing the capacity of pathogens to\ndevelop biofilms and propagate illnesses (63). Also, Plamada and\nVodnar (64) indicated that the fiber and polyphenol content in olive\noil functions as prebiotics, promoting the multiplication of beneficial\nbacteria such as Lactobacillus and Bifidobacterium. Probiotic-\nenhanced fermentation improves the production of SCFA, such as\nbutyrate, which fortifies intestinal cells and suppresses pathogen pro­\nliferation (65).\n5 Conclusion\nIn the context of the current 6-week experiment, the integration of\nOLP into the quails’ diet yielded significant advantages for the Japanese\nquail growth, performance, and production. The results revealed an\nincrease in growth performance, improvements in carcass attributes,\nenhancements in hematological parameters, a further boost in antioxi­\ndant activity, and a superior composition of intestinal microbiota.\nHowever, the improvements in growth performance were modest and\nshould be interpreted with caution in terms of biological relevance.\nAmong the investigated inclusion levels, 6% OLP proved most favor­\nable in improving growth performance, lipid profile, antioxidant\nenzyme activity, and meat quality traits, but both 4 and 6% OLP com­\nparably increased the cecum Lactobacillus population. Accordingly,\ninclusion of 6% OLP is advised as the ideal dietary intake based on the\nparameters of the current study. The results of this study indicate that\nOLP demonstrates potential as a sustainable and safe feed supplement\nfor Japanese quail production. Overall, OLP appears to act primarily as\na functional feed additive that supports physiological health, antioxi­\ndant status, and microbial balance rather than a strong growth pro­\nmoter. Further investigation is necessary to discover the biological\nmechanisms of the bioactive compounds in OLP, estimate their long-\nterm impacts on quail products, and ascertain their economic feasibil­\nity in commercial quail farming enterprises.\nData availability statement\n\nData availability statement\nThe raw data supporting the conclusions of this article will be\nmade available by the authors, without undue reservation.\nEthics statement\nThis study was performed on a private poultry farm in the\nSharqia Governorate and at the Agricultural Research Center labo­\nratory in Dokki, Giza, Egypt, with ethical approval from the\nCenter’s Research Ethics Committee, reference number (ARC-\nIACUC), which assessed and approved all experimental protocols\n(ARC-CLAR-5129). The committee approved all experimental pro­\ncedures, including the breeding and management of birds. The\nstudy was conducted in accordance with the local legislation and\ninstitutional requirements.\nAuthor contributions\nSA-A: Data curation, Formal analysis, Writing – original draft,\nConceptualization. SA-W: Writing – original draft, Formal analysis,\nData curation, Conceptualization. MO: Writing  – original draft,\nMethodology, Resources, Conceptualization, Data curation, Writing –\nreview & editing, Investigation. EA-S: Writing  – original draft,\nResources, Investigation, Software, Formal analysis. NS: Resources,\nValidation, Investigation, Formal analysis, Writing – original draft. AA:\nVisualization, Validation, Resources, Investigation, Writing – original\ndraft, Writing – review & editing. YH: Data curation, Visualization,\nSoftware, Investigation, Writing – review & editing. MS: Software,\nVisualization, Investigation, Writing – review & editing, Validation.\nMA-R: Funding acquisition, Writing – review & editing, Supervision,\nWriting – original draft, Resources, Validation, Project administration,\nVisualization. IA: Validation, Investigation, Formal analysis, Writing\n– original draft.\nFunding\nThe author(s) declared that financial support was received for this\nwork and/or its publication. Princess Nourah bint Abdulrahman\nUniversity Researchers Supporting Project number (PNURSP2026R924),\nPrincess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.\nKing Khalid University under the large research grant group number\n(R.G.P.2/129/46). The deanship of Scientific Research, Vice Presidency\n\nKing Khalid University under the large research grant group number\n(R.G.P.2/129/46).The deanship of Scientific Research, Vice Presidency\nfor Graduate Studies and Scientific Research, King Faisal University,\nSaudi Arabia (Grant No. 261166).\n\nAl-Ardhi et al.\b\n10.3389/fvets.2026.1824857\nFrontiers in Veterinary Science\n10\nfrontiersin.org\nAcknowledgments\nThis work was supported by Princess Nourah bint Abdulrahman\nUniversity (Riyadh, Saudi Arabia); the Deanship of Research and\nGraduate Studies at King Khalid University; and the Deanship of\nScientific Research, Vice Presidency for Graduate Studies and\nScientific Research at King Faisal University (Saudi Arabia). The\nauthors acknowledge and appreciate the financial and institutional\ncontributions of these bodies, which enabled the successful comple­\ntion of this study.\nConflict of interest\nThe author(s) declared that this work was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nThe reviewer AS declared a past co-authorship with the author\nMA-R to the handling editor.\nGenerative AI statement\nThe author(s) declared that Generative AI was not used in the\ncreation of this manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. Any product\nthat may be evaluated in this article, or claim that may be made by its\nmanufacturer, is not guaranteed or endorsed by the publisher.\nReferences\n1.\tKishawy AT, Al-Khalaifah HS, Nada HS, Roushdy EM, Zaglool AW, Ismail TA, et al.\nBlack pepper or radish seed oils in a new combination of essential oils modulated broiler\nchickens’ performance and expression of digestive enzymes, lipogenesis, immunity, and\nautophagy-related genes. Vet Sci. (2022) 9:43. doi: 10.3390/vetsci9020043\n2.\tAl-Khalaifah HS, Al-Nasser A, Surrayai T. 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+{"text": "【兽医文献】\n标题: Key regulators of lactation performance in Xinjiang Brown cattle and Chinese Holstein cattle unraveled by multi-omics integration\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nTYPE  Original Research\nPUBLISHED  11 June 2026\nDOI  10.3389/fvets.2026.1808217\nOPEN ACCESS\nEDITED BY\nAndreas Erich Zautner,\nUniversity Hospital Magdeburg,\nGermany\nREVIEWED BY\nMaribel Jiménez-Edeza,\nAutonomous University of Sinaloa,\nMexico\nJames Jacob Sasanya,\nJoint FAO/IAEA Programme of Nuclear\nTechniques in Food and Agriculture,\nAustria\n*CORRESPONDENCE\nMartina Ludewig\nmartina.ludewig@vetmeduni.ac.at\nRECEIVED 10 February 2026\nREVISED 10 May 2026\nACCEPTED 18 May 2026\nPUBLISHED 11 June 2026\nCITATION\nLudewig M, Firth CL, Roschitz M,\nHinterleithner SH and\nKäsbohrer A (2026) Evaluation of\nß-lactamase-induced inactivation of\npenicillin G and cefoperazone in spiked\nultra-high temperature and raw\nunpasteurized milk.\nFront. Vet. Sci. 13:1808217.\ndoi: 10.3389/fvets.2026.1808217\nCOPYRIGHT\n© 2026 Ludewig, Firth, Roschitz,\nHinterleithner and Käsbohrer. This is an\nopen-access article distributed under\nthe terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other\nforums is permitted, provided the\noriginal author(s) and the copyright\nowner(s) are credited and that the\noriginal publication in this journal is\ncited, in accordance with accepted\nacademic practice. No use, distribution\nor reproduction is permitted which does\nnot comply with these terms.\nEvaluation of\nß-lactamase-induced inactivation\nof penicillin G and cefoperazone\nin spiked ultra-high temperature\nand raw unpasteurized milk\nMartina Ludewig 1*, Clair L. Firth 2, Michael Roschitz 1,\nSophie H. Hinterleithner 1 and Annemarie Käsbohrer 2,3\n1Clinical Department for Farm Animals and Food System Transformation, Centre for Food Science,\nUniversity of Veterinary Medicine Vienna, Vienna, Austria, 2Clinical Department for Farm Animals and\nFood System Transformation, Centre for Veterinary Public Health & One Health, University of\nVeterinary Medicine Vienna, Vienna, Austria, 3Department for Biological Safety, Federal Institute for\nRisk Assessment, Berlin, Germany\nIntroduction: In Austria, veterinary medicinal products (VMPs) containing peni­\ncillin G or cefoperazone are frequently used to treat dairy cows, which leads to\n\nthe production of non-saleable waste milk. This study aimed to determine the\nloss of detectable antimicrobial activity of penicillin G and cefoperazone using a\ncommercial ß-lactamase preparation, Antipen®.\nMethods: Ultra-high-temperature (UHT) processed and raw unpasteurized milk\nwere spiked with penicillin G and cefoperazone (both pure pharmaceutical sub­\nstances and VMPs), and the effects of temperature (37 °C, 25 °C, and 10 °C), stor­\nage time, and pH on Antipen® treatment on antibiotic activity were investigated.\nThe aerobic mesophilic count (AMC) and Enterobacteriaceae before and after\nenzyme treatment in raw milk were also examined to assess the microbiological\nquality of milk that is potentially fed to calves.\nResults: The VMPs containing penicillin G (Vanapen®) or cefoperazone\n(Peracef®) in 50 mL of UHT or raw milk showed a higher stability after Antipen®\ntreatment compared to pure antibiotic substances. Concentrations of 300 μg/mL\nof Vanapen® tested negative by Delvotest® T under all experimental conditions\n(12- and 6-h treatment, at natural pH and pH 5.5, at 37–10 °C). Treatments of milk\nwith natural pH spiked with 10 μg/mL of Peracef® tested negative for the anti­\nbiotic after 12 and 6 h at 37 °C, while after acidification of the milk to pH 5.5, 10\nμg/mL Peracef® was undetectable at all temperatures and time periods. In milk\nwith natural pH treated with Antipen® for 12 h at 10–37 °C, the values of AMC\nalways exceeded 5.0-log cfu/mL, indicating that the milk is not suitable for use as\ncalf feed without prior acidification or subsequent pasteurization. Reducing the\nexposure time to 6 h or acidifying the milk to a pH of 5.5 during 12-h treatment\nsignificantly reduced bacterial growth.\nDiscussion: This study demonstrated that Antipen® can effectively degrade\npenicillin, although its efficacy is lower and slower on cephalosporins. Since the\nstudy was conducted in a laboratory setting, extrapolation of the effective treat­\nment durations to on-farm application requires validation in practical, real-world\nconditions.\nKEYWORDS\n\nSince the\nstudy was conducted in a laboratory setting, extrapolation of the effective treat­\nment durations to on-farm application requires validation in practical, real-world\nconditions.KEYWORDS\nantibiotics, enzymes, microbiological counts, pH, ß-lactamase, waste milk\n\nLudewig et al.\b\n10.3389/fvets.2026.1808217\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n1 Introduction\nOn dairy farms, antibiotics are frequently used to treat systemic\nand udder-associated diseases. Mastitis treatment is the most common\nindication for antibiotic use in dairy cows, with ß-lactam antibiot­\nics–penicillin (systemic and intramammary) and third-generation\ncephalosporins (intramammary)–frequently used in Austria (1, 2).\nFollowing the administration of medicinal products (such as antibiot­\nics) during lactation, waste milk that cannot be sold is produced.\nDuring the treatment of lactating cows with medicinal products and\nwithin the licensed withdrawal period, the milk produced is classified\nas waste milk and is unfit for human consumption due to potential\ndrug residues (3, 4). However, research has shown a controversial\nassociation between this practice and negative health effects, including\nissues related to antibiotic resistance (5, 6). As summarized in a scien­\ntific opinion published by the European Food Safety Authority\n(EFSA), the selection pressure for antibiotic resistance in gut micro­\nbiota and the possible risk of resistance genes or residues being\nreleased into the environment are of significant concern from a One\nHealth perspective (7). Several studies reported that feeding waste\nmilk had similar or positive effects on daily weight gain, health, or\nperformance of dairy calves compared with bulk milk or milk replacer\n(8–10). On the other hand, the practice of feeding waste milk and the\nnumber of antibiotic applications appear to correlate with the preva­\nlence\nof\nextended-spectrum\nß-lactamase-(ESBL)-producing\nEscherichia coli on farms (11) and led to a higher proportion of anti­\nbiotic resistant E. coli in calf feces compared to those fed bulk milk or\nmilk replacer (3, 8, 12). Unpasteurized waste milk may contain signifi­\ncantly higher counts of aerobic mesophiles than pasteurized or bulk\nmilk and may pose a risk for transmission of potential pathogens (8,\n13, 14). Furthermore, it has been associated with an increased likeli­\n\nUnpasteurized waste milk may contain signifi­\ncantly higher counts of aerobic mesophiles than pasteurized or bulk\nmilk and may pose a risk for transmission of potential pathogens (8,\n13, 14).Furthermore, it has been associated with an increased likeli­\nhood of diarrhea and a reduction in fecal microbiota diversity in pre-\nweaned calves, with a shift toward potential pathogens (15, 16). A few\nstudies showed that even low concentrations of antibiotic residues in\nwaste milk promote the development of resistance to those drugs in\nthe calf’s intestine, which might negatively affect the health of animals\n(9, 17, 18). For this reason, it is important to investigate the degrada­\ntion of antibiotic residues in waste milk. Various methods such as\ninactivation by heat, the use of ß-lactamase enzymes, microbial fer­\nmentation, and acidification or alkalinization of waste milk have been\ntested. Several studies indicated that penicillin and cephalosporins are\nrelatively heat-stable in milk (19–22). Inactivation of antimicrobial\nresidues was observed only at high temperatures and long exposure\ntimes, for example, penicillin: 4.0 μg/kg at 95 °C for 120 min and cefo­\nperazone: 5.0 μg/kg at 120 °C for 20 min (19, 22). ß-lactamase\nenzymes are effective in decreasing penicillin residues in milk within\n2–8 h, whereas the reduction of cephalosporin residues takes 24 h\n(22–24). Furthermore, adjusting the natural milk pH to lower (4.0–\n5.5) or higher (9.0–10.0) values can significantly increase the effective­\nness of thermal activity in reducing residues (21, 22, 25, 26).\nIn the present study, the effect of temperature, storage times,\nand pH of treatments with the commercially available enzyme\npreparation\nAntipen®\nwas\ninvestigated\nat\ndifferent\nconcentrations of penicillin G and cefoperazone (both pure phar­\nmaceutical substances and veterinary medicinal products, VMP)\nin (i) ultra-high temperature processed and (ii) raw unpasteur­\nized milk using a microbial growth inhibition assay to assess the\nloss of antibiotic activity after enzyme treatment. The milk was\n\nThe milk was\nspiked with high and low concentrations of antibiotics to deter­\nmine the limits of ß-lactamase activity in laboratory-scale experi­\nments. Additionally, for each experimental combination in raw\nmilk, the counts of aerobic mesophiles and Enterobacteriaceae\nbefore and after enzyme treatment were examined to assess the\nmicrobiological quality of milk potentially fed to calves. The\noverall aim of the present study was to identify, through labora­\ntory-scale experiments, the most effective treatment combina­\ntions for reducing or eliminating penicillin G and cefoperazone\nresidues in milk. Cefoperazone, a third-generation cephalospo­\nrin, was included in the study, as it was, at the time, still approved\nand a frequently used intramammary therapeutic agent in Austria\n(27). With the introduction of new Austrian drug legislation, its\nuse no longer licensed in the country, although it remains\napproved for this indication in other European Union Member\nstates (28, 29).\n2 Materials and methods\n2.1 Antibiotics, ß-lactamase, and milk\nsamples\nIn preliminary test series carried out in 1 mL of ultra-high-\ntemperature (UHT) processed milk, the loss of detectable antimi­\ncrobial activity of the pure pharmaceutical substances penicillin G\n(PENG; procaine benzylpenicillin, pharmaceutical reference stan­\ndard, Merck KGaA, Darmstadt, Germany) and cefoperazone (CEF;\ncefoperazone dihydrate, Vetranal, analytical standard, Merck\nKGaA) was analyzed. UHT milk was initially spiked with 2,500 μg/\nmL PENG and then diluted serially to a concentration of 0.0025 μg/\nmL. Initial CEF concentration was 200 or 100 μg/mL milk, diluted\nto 0.02 or 0.01 μg/mL.\nFurther laboratory-scale experiments were performed with the\nveterinary medicinal products (VMPs) Vanapen® (VAN; active sub­\nstance: benzylpenicillin procaine monohydrate, 300 mg/mL, intended\nfor intramuscular use; Vana GmbH, Vienna, Austria) and Peracef®\n(PER; active substance: cefoperazone, 10 mg/mL, intended for intra­\nmammary use; Zoetis Deutschland GmbH, Berlin, Germany) in\n50 mL of UHT or raw milk. The initial spiking concentration for VAN\n\nThe initial spiking concentration for VAN\nwas 3,000 μg/mL, serially diluted to 0.003 μg/mL; for PER, it was\n100 μg/mL, diluted to 0.01 μg/mL.\nFor the degradation of these antibiotic substances in milk,\nAntipen® (Vetcare Oy, Helsinki, Finland) was used, which is a com­\nmercially available β-lactamase developed for the degradation of peni­\ncillin G, ampicillin, and amoxicillin in waste milk. Antipen® is\nmarketed in Finland, where veterinary antibiotic treatment guidelines\nprimarily recommend penicillin for mastitis in dairy cattle (30). In\naddition to ß-lactamase, Antipen® also contains a potassium phos­\nphate buffer, methyl parahydroxybenzoate, glycerin, and a food colo­\nrant additive to dye the treated milk blue. Experiments were conducted\naccording to the manufacturer’s instructions with a volume of\n25 μL/L. As Antipen® has not been evaluated for the degradation of\nAbbreviations: AMC, aerobic mesophilic count; cfu, colony-forming units; EB,\nEnterobacteriaceae; CEF, cefoperazone; PENG, penicillin G; PER, peracef®; UHT,\nultra-high-temperature processed; VAN, vanapen®; VMP, veterinary medicinal\nproduct.\n\nLudewig et al.\b\n10.3389/fvets.2026.1808217\nFrontiers in Veterinary Science\n03\nfrontiersin.org\ncephalosporins, CEF concentrations in the range of 200–2.0 μg/mL\nwere tested with both 25 and 250 μL Antipen®/L of milk.\nThe pH of the milk was adjusted with apple cider vinegar (5%\nacetic acid; standardized according to Codex Alimentarius\nAustriacus), as commonly applied in calf milk feeding, and measured\nwith a Hanna pH meter (Professional pH & Temperature Meter\nHI98165, Hanna Instruments Inc., Woonsocket, United States) (31).\nUHT milk (containing 3.5% fat and 3.3% protein) was purchased\nfrom retail markets, and raw unpasteurized milk (containing natural\nfat content 3.8–3.9%; and 3.4% protein, milked on the day of sam­\npling) was collected directly from a milk filling station at a local farm.\nBefore each experiment, the pH levels were carefully monitored,\nremaining between 6.5 and 6.7 in both UHT and raw milk, consistent\nwith the natural pH (NpH) of fresh milk. Raw milk was stored at 5.0\n°C for 12 to 24 h before being used for analysis.\n2.2 Detection of antibiotic residues\nThe commercial broad-spectrum antibiotic test kit Delvotest® T,\nampoules format, (DSM Firmenrich. Delft, Netherlands) with\nGeobacillus stearothermophilus var. calidolactis as an indicator organ­\nism, was used to assess the antimicrobial activity of antibiotic residues\nin UHT and raw milk following treatment with the β-lactamase prepa­\nration Antipen®. Delvotest® T is used as a screening method for the\nqualitative detection of antibiotic residues in milk in accordance with\nImplementing Regulation (EU) 2021/808 and enables detection of the\nmaximum residue limits defined in Commission Regulation (EU) No\n37/2010 (4 μg/kg for PENG and 50 μg/kg for CEF) (32, 33). The\nmethod has also been evaluated by the Association française de nor­\nmalization (AFNOR), with detection limits of 0.003 μg for PENG and\n0.02 μg for CEF per 1 mL of milk. In Austria, the use of Delvotest® T\nfor residue control in milk is strongly recommended in accordance\nwith the national legislation (34). Delvotest® T allows the simultane­\n\n003 μg for PENG and\n0.02 μg for CEF per 1 mL of milk.In Austria, the use of Delvotest® T\nfor residue control in milk is strongly recommended in accordance\nwith the national legislation (34).Delvotest® T allows the simultane­\nous analysis of a large number of samples and was therefore selected\nas the method of choice in this study.\nBefore the experiments, the test system was carefully assessed.\nUHT milk was spiked with antibiotic substances (such as pure sub­\nstances in 1 mL milk or VMPs in 50 mL milk) at initial concentrations\nand then diluted to the respective detection limits. The system was\nfirst verified in milk with NpH to ensure compliance with the required\ndetection limits and was subsequently assessed in milk with experi­\nmentally modified pH values (6.0 and 5.5), followed by neutralization\nwith 1.0 M sodium hydroxide solution (Merck KGaA), to evaluate\nperformance under altered matrix conditions.\nThe UHT or raw milk batches were tested for the absence of anti­\nbiotic residues using Delvotest® T prior to each assay. An unspiked\nUHT or raw milk sample was used as a blank, in accordance with the\nmanufacturer’s instructions, to determine the correct incubation time\nfor the test. Furthermore, a control sample containing the final\nenzyme concentration and adjusted to the corresponding pH level was\nincluded in each trial. For subsequent experiments in acidified milk,\nthe pH of the control and sample solutions was readjusted to 6.6 using\n1.0 M sodium hydroxide before use in the detection assay. After com­\npletion of the predefined enzymatic incubation time, all samples were\nplaced in crushed ice until inoculation of the ampoules with milk\nsamples to halt further reactions.\nIn samples where the antibiotic concentration had degraded\nbelow the detection limit of the assay, growth of the test organism was\nindicated by a color change from purple to yellow. This color change\nwas compared to the manufacturer’s reference scale and used for final\nevaluation.\n2.3 Microbiological quantification of raw\nunpasteurized milk\nMilk samples were mixed thoroughly, and the initial homogenate\n\nThis color change\nwas compared to the manufacturer’s reference scale and used for final\nevaluation.2.3 Microbiological quantification of raw\nunpasteurized milk\nMilk samples were mixed thoroughly, and the initial homogenate\nwas serial decimal diluted (to 10−8) in Ringer’s solution (B. Braun,\nMelsungen, Germany) according to ISO 6887-1:2017 (35). Analyses\nof aerobic mesophilic counts (AMC) were performed according to\nISO 4833-2:2013/Amd 1:2022 (36) on Trypto–Casein–Soy agar\nmedium with 0.6% yeast extract (TSAYE, Biokar, Groupe Solabia,\nPantin Cedex, France). Enterobacteriaceae (EB) were enumerated on\nBrilliance E. coli/Coliform Selective Agar (BECA, Thermo Fisher\nScientific Inc., Oxoid, Waltham, MA, United States). Typical colony\nmorphologies (1–3 isolates) for EB (colorless), coliforms (pink), and\nE. coli (purple) were selected and sub-cultivated on TSAYE for further\nconfirmation by catalase (3%; Merck KgaA) and cytochrome-oxidase\ntesting (BioMérieux, Marcy-l’Etoile, Frankreich). For the final E. coli\nconfirmation, the ability to split indole from tryptophan was tested\nwith the BactiDrop Spot Indol test on BECA (Thermo Fisher Scientific\nInc., Oxoid). Counts for aerobic mesophiles, EB, and E. coli were cal­\nculated according to ISO 7218:2024 (37). The mean increase of AMC\nwas calculated by the difference between the initial AMC of the\nuntreated milk and the mean of the final AMC, comprising the sam­\nples with 300 μg/mL VAN, 10 μg/mL PER, and the control milk after\nall treatment procedures. The results were assigned to three categories,\nincluding “low” (<0.5 log Cfu/mL), “moderate” (0.5–1.5 log Cfu/mL),\nand “high” (>1.5 log Cfu/mL) increase, consistent with the assessment\nof microbial growth for shelf-life determination as outlined in the\nEFSA guidelines (38).\n2.4 Statistics\nDescriptive statistics (such as mean, standard deviation, and mini­\nmum and maximum values) of microbial counts were performed\nusing Prism version 11.0.0 software for Windows (GraphPad Software,\nBoston, Massachusetts, United States, https://www.graphpad.com/).\nLogarithmically transformed microbial counts resulted in normally\n\n0.0 software for Windows (GraphPad Software,\nBoston, Massachusetts, United States, https://www.graphpad.com/).Logarithmically transformed microbial counts resulted in normally\ndistributed data, which were assessed using the Kolmogorov–Smirnov\ntest. A two-way analysis of variance (ANOVA) was conducted to\nassess the effects of storage temperature and the experimental condi­\ntions (such as time and pH) on microbial counts. In case of significant\ninteractions, post hoc comparisons of simple effects were performed\nusing the Tukey’s test. A p-value of <0.05 was considered statistically\nsignificant.\n2.5 Experimental design\nThe experimental program of the study is illustrated in Table 1.\nThe model matrix for waste milk was UHT or raw unpasteurized milk\nspiked either with a pure antibiotic substance or an antibiotic\nVMP. The initial spiking concentrations of the antibiotics were\nselected to approximate the respective therapeutic dose levels when\nreferenced to 1 L of milk and were then serially diluted to the esti­\nmated detection limits. The preliminary tests evaluated the effective­\nness of ß-lactamase Antipen® to inactivate the two pure antibiotic\nsubstances, PENG or CEF, in 1 mL UHT milk under different treat­\nment conditions (such as storage temperature and duration, and pH\n\nLudewig et al.\b\n10.3389/fvets.2026.1808217\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nof the milk). In further laboratory-scale experiments, the effectiveness\nof Antipen® for the inactivation of commercial preparations of veteri­\nnary antibiotic preparations containing PENG (VAN) or CEF (PER)\nwas tested in 50 mL UHT and raw milk each. Parallel to experiments\non antibiotic activity in unpasteurized raw milk, the microbiological\ncounts for AMC and EB were determined in the control milk samples\nand milk spiked with VAN (300 μg/mL) or PER (10 μg/mL).\n3 Results\n3.1 Preliminary tests on the activity of pure\nantibiotics in 1 mL of UHT milk using\nAntipen®\nIn all experiments, PENG at concentrations of 2,500–0.025 μg/\nmL, treated with 0.025 μL Antipen® in 1 mL of UHT milk, tested\nnegative in the Delvotest® T after 6 and 12 h at NpH, pH 6.0, and pH\n5.5, and at 10, 25, and 37 °C. At shorter storage periods (4 and 2 h),\n2,500 μg/mL PENG could not be consistently reduced below the\ndetection limit (Table 2A). Concentrations of 200 μg/mL CEF were\nreduced by Antipen® (0.25 μL/mL) after 12 h (NpH and pH 5.5) and\nafter 6 h (NpH), at 37 °C (Table 2B). CEF concentrations of 100 μg/\nmL were not detectable in UHT milk stored at 25 and 37 °C, at pH 5.5\nfor 10 h. In UHT milk spiked with lower CEF concentrations (20–\n0.1 μg/mL), Antipen® (0.25 μL/mL) reduced the antimicrobial sub­\nstance to below the detection limit of Delvotest® T in all experiments\n(Table 2B).\n3.2 Activity of antibiotic drugs in 50 mL of\nUHT and raw unpasteurized milk using\nAntipen®\nIn laboratory-scale experiments, the effects of Antipen® (0.025 μL/\nmL for VAN and 0.25 μL/mL for PER) on the stability of VAN and\nPER in 50 mL of milk were evaluated under different conditions.\nSamples were stored for 6 or 12 h at NpH or pH 5.5 and at 10, 25, or\n37 °C. The results are presented in Tables 3A,B. High PER\nconcentrations (100 μg/mL) remained detectable using Delvotest® T\nunder all tested conditions. In contrast, high VAN concentrations\n(3,000 μg/mL) were no longer detectable after Antipen® treatment\nwithin 12 h at 37 °C (NpH) and at 25 °C (pH 5.5). Lower VAN con­\n\nIn contrast, high VAN concentrations\n(3,000 μg/mL) were no longer detectable after Antipen® treatment\nwithin 12 h at 37 °C (NpH) and at 25 °C (pH 5.5).Lower VAN con­\ncentrations (300–0.03 μg/mL) showed no detectable activity under\nany experimental conditions (Table 3A). Treatments of milk (NpH)\nspiked with a lower concentration of PER (10 μg/mL) tested negative\nusing Delvotest® T after 12 and 6 h at 37 °C. After acidification of the\nmilk to pH 5.5, PER at 10 μg/mL was consistently undetectable\n(Table 3B).\n3.3 Microbiological quantification in raw\nunpasteurized milk during storage\nexperiments with Antipen®\nIn the untreated raw milk, the initial AMC ranged from 2.6 to 4.9\nlog cfu/mL, and counts of EB were between 1.0 and 3.0 log cfu/\nmL. The mean AMC obtained in raw milk without and with antibiot­\nics (VAN 300 μg/mL or PER 10 μg/mL) after 12 h of treatment with\nAntipen® at NpH was in the range of 5.3 cfu/mL at 10 °C and 7.4 log\ncfu/mL at 37 °C (p < 0.05), while in milk at pH 5.5 the mean AMC was\nestimated to be between 3.9 log cfu/mL at 10 °C and 4.8 log cfu/mL at\n37 °C (p < 0.05; Supplementary Table 1). After 6 h of storage, the raw\nmilk samples treated with Antipen® showed a mean AMC of 4.3 (10\n°C) to 4.7 log cfu/mL (37 °C) at NpH and 4.1 (10 °C) to 3.6 log cfu/\nmL (37 °C) at pH 5.5 (p > 0.05; Supplementary Table 1). In general,\nthe mean counts of EB were lower than those of AMC in all experi­\nments. The highest microbial load (6.1 log cfu/mL) was found in raw\nmilk (NpH) stored for 12 h at 37 °C, and the lowest loads (0.7–2.3 log\ncfu/mL) were observed with the experimental combination 6 h and\npH 5.5 at 10, 25, and 37 °C (p < 0.05; Figure 1B, Supplementary Table 1).\nHigher counts (such as 5.3 and 3.3 log cfu/mL) of potential E. coli were\nonly determined with the combination 37 °C, 12 and 6 h, NpH\n(Supplementary Table 1). The analysis showed that the time/pH com­\nbinations influenced the mean count of AMC and Enterobacteriaceae\nin a temperature-dependent manner and that the magnitude of the\ntemperature effect varied among the experimental conditions\n(p < 0.05; Figures 1A,B).\n\nThe analysis showed that the time/pH com­\nbinations influenced the mean count of AMC and Enterobacteriaceae\nin a temperature-dependent manner and that the magnitude of the\ntemperature effect varied among the experimental conditions\n(p < 0.05; Figures 1A,B).\nTABLE 1  Experimental program–degradation of antibiotics by ß-lactamase enzymes in UHT and raw unpasteurized milk.\nPreliminary tests: degradation of pure pharmaceutical\nsubstances by Antipen® in 1 mL UHT milk\nLaboratory-scale experiments: degradation of\nveterinary medical products by Antipen® in 50 mL\nUHTb or unpasteurized raw milk\nStorage temperatures: 37, 25, and 10 °C\nPenicillin Ga\nCefoperazonea\nVanapen®a\nPeracef®a\n12 h, NpH (1)\n12 h, NpH (2)\n12 h, NpH (4)\n12 h, NpH (4)\n12 h, pH 6.0 (1)\n12 h, pH 5.5 (3)\n12 h, pH 5.5 (4)\n12 h, pH 5.5 (4)\n12 h, pH 5.5 (1)\n10 h, pH 5.5 (2)\n6 h, NpH (3)\n6 h, NpH (3)\n6 h, NpH (3)\n6 h, NpH (1)\n6 h, pH 5.5 (3)\n6 h, pH 5.5 (3)\n4 h, NpH (2)\n4 h, NpH (1)\n2 h, NpH (3)\nUHT: ultra-high temperature processed, NpH: natural pH of milk, aStorage time, pH (number of replicates).\nbA single series of analytical tests was carried out using UHT milk.\n\nLudewig et al.\b\n10.3389/fvets.2026.1808217\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nThe microbiological increase in raw milk treated with\nAntipen® stored for 12 and 6 h at different temperatures and pH\nvalues is illustrated in Figure 2, with the results of detectable anti­\nbiotic residues assayed by Delvotest® T. In general, the AMC\nincrease was significantly influenced by the interaction between\ntemperature and the time/pH combination (p < 0.05; Figure 2).\nThe AMC found in milk after enzyme treatment for 12 h (NpH) at\n25 and 37 °C was high (>5.0 log cfu/mL) and increased by more\nthan 1.5 log cfu/mL. The AMC increase for the other test combina­\ntions was “moderate” (≥0.5–1.5 log cfu/mL) to “low” (<0.5 log cfu/\nmL), with a high final AMC (>5.0 log cfu/mL) in all or single\nsamples at combination: 10 °C, 12 h (NpH); 25 °C, 6 h (NpH); and\n37 °C, 12 h (pH 5.5) and 6 h (NpH) (Figure 2, Supplementary\nTable 1). High EB counts (>5.0 log cfu/mL) with an increase of\nmore than 1.5 log cfu/mL were only observed after treatment at 37\n°C, 12 h (NpH). The categories “low” to “moderate” increases in\nAMC correspond to VAN levels (300 μg/mL) below the detection\nlimit after 12 h (pH 5.5) and 6 h (NpH, pH 5.5) and PER (10 μg/\nmL) after 12 and 6 h (pH 5.5) at 10, 25, and 37 °C. However, PER\nwas still detectable in samples after treatment at 10 and 25 °C for\n6 h, at NpH.\n4 Discussion\nThe study aimed to identify a combination of treatment condi­\ntions suitable for practical use in farms to decrease residue concentra­\ntions in waste milk, focusing on two important antibiotic substances,\nincluding penicillin G and cefoperazone (PENG and CEF), frequently\nused in Austrian mastitis therapy. Antibiotics of the ß-lactam family\nare characterized by the ß-lactam ring in their chemical structure and\ninclude aminopenicillins and cephalosporins. These antibiotics can be\ninactivated by the hydrolysis of ß-lactamase enzymes, which break the\nring open (39).\nThe results of preliminary tests in 1 mL of UHT milk showed that\nthe commercially available β-lactamase Antipen®, at the concentra­\n\nThese antibiotics can be\ninactivated by the hydrolysis of ß-lactamase enzymes, which break the\nring open (39).The results of preliminary tests in 1 mL of UHT milk showed that\nthe commercially available β-lactamase Antipen®, at the concentra­\ntion recommended by the manufacturer (25 μL/L), reduced high\nTABLE 2  Degradation of (A) penicillin G (PENG) and (B) cefoperazone (CEF) in 1 mL spiked UHT milk by Antipen®.\n(A) Storage temperature\n°C\n10\n25\n37\n10\n25\n37\n10\n25\n37\nAntipen®\nμL/mL\nPENG\nμg/mL\n12 h, NpHa\n12 h, pH 6.0a\n12 h, pH 5.5a\n0.025\n2,500\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n250\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n25\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n2.5\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n1/1\n0.25\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n0.025\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n0/1\n6 h, NpHa\n4 h, NpHa\n2 h, NpHa\n0.025\n2,500\n0/3\n0/3\n0/3\n1/2\n1/2\n0/2\n2/3\n2/3\n2/3\n250\n0/3\n0/3\n0/3\n0/2\n0/2\n0/2\n0/3\n0/3\n0/3\n25\n0/3\n0/3\n0/3\n0/2\n0/2\n0/2\n0/3\n0/3\n0/3\n2.5\n0/3\n0/3\n0/3\n0/2\n0/2\n0/2\n0/3\n0/3\n0/3\n(B) Storage temperature\n°C\n10\n25\n37\n10\n25\n37\n10\n25\n37\nAntipen®\nμL/mL\nCEF μg/\nmL\n12 h, NpHa\n12 h, pH 5.5a\n6 h, NpHa\n0.25\n200\n2/2\n1/2\n0/2\n2/3\n2/3\n0/3\n1/1\n1/1\n0/1\n20\n0/2\n0/2\n0/2\n0/3\n0/3\n0/3\n0/1\n0/1\n0/1\n2\n0/2\n0/2\n0/2\n0/3\n0/3\n0/3\n0/1\n0/1\n0/1\n0.2\n0/2\n0/2\n0/2\n0/3\n0/3\n0/3\n0/1\n0/1\n0/1\n10 h, pH 5.5a\n4 h, NpHa\n0.25\n100\n2/2\n0/2\n0/2\n1/1\n1/1\n0/1\n10\n0/2\n0/2\n0/2\n0/1\n0/1\n0/1\n1\n0/2\n0/2\n0/2\n0/1\n0/1\n0/1\n0.1\n0/2\n0/2\n0/2\n0/1\n0/1\n0/1\naNumber of samples positive for antibiotics/number of assayed by Delvotest® T. Degraded samples are highlighted in bold. Limit of detection: PENG 0.0025/CEF 0.02 μg/mL.\nMilk stored at: 10 °C, 25 °C, and 37 °C (A) for 12 h, at natural pH (NpH) and pH 6.0 and 5.5, and for 6 h, 4 h, and 2 h, at NpH and (B) for 12 h, 6 h, and 4 h, at natural pH (NpH), for 12 h and\n10 h, at pH 5.5.\n\nLudewig et al.\b\n10.3389/fvets.2026.1808217\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nlevels of pure PENG (2,500 μg/mL) within 6–12 h and over a broad\ntemperature range from 10 to 37 °C below the detection limit of\nDelvotest® T.\nFurthermore, the acidification of milk to pH of 6.0 and 5.5 for\n12 h had no additional effect on detectable PENG residues by\nAntipen®. The highest concentration (200 μg/mL) of pure CEF could\nonly be inactivated at 37 °C in 1 mL UHT milk with NpH within 6\nand 12 h and in acidified milk after 12 h. However, as Antipen® is not\nevaluated to inactivate cephalosporins, it is not surprising that a\n10-fold dose of the enzyme was required to degrade CEF. Previous\nstudies (22, 23) reported complete ß-lactamase degradation of PENG\n(1 μg/mL and 25 μg/g) within 2 and 8 h at 20–37 °C. The fourth-gen­\neration cephalosporin, cefquinome (2 μg/mL) was eliminated after\n24 h at 10 °C (24). However, it is important to note that these studies\ninvestigated the inactivation of lower antibiotic concentrations com­\npared to this study.\nThis study observed that experiments with the VMPs (VAN,\nactive antibiotic substance PENG and PER, active antibiotic substance\nCEF) in 50 mL of raw unpasteurized or UHT milk showed a higher\nstability of these substances after treatment with Antipen® compared\nto pure antibiotics. One possible explanation is the excipients used in\nthese formulations: povidone in VAN, which exhibits binding and\nstabilizing properties, and peanut oil in PER, which reduces solubility\n(40, 41).\nFurthermore, CEF was more stable in all experiments, and\nboth the pure substance and the corresponding drug were less\nreduced by ß-lactamase than PENG. This was expected, as cepha­\nlosporins are more resistant to opening their ß-lactam ring than\nPENG (39). Acidification of milk to pH 5.5 improved the reduction\nof both drugs compared to milk with NpH. High levels of the VMP\nVAN (PENG 3000 μg/mL) in acidified milk were reduced below\nthe detection limit at 25 °C after 12 h with ß-lactamase, and PER\n(CEF) at 10 μg/mL was reliably reduced after 12 and 6 h treatment\n\nHigh levels of the VMP\nVAN (PENG 3000 μg/mL) in acidified milk were reduced below\nthe detection limit at 25 °C after 12 h with ß-lactamase, and PER\n(CEF) at 10 μg/mL was reliably reduced after 12 and 6 h treatment\nwith Antipen® at pH 5.5 and 10–37 °C. In the study presented by\nRenner (42), ß-lactamase hydrolysis of PENG was extended in\nmilk acidified to pH 5.0 and 5.5, and the enzyme was no longer\nactive in milk with a pH below 4.5. However, Li et al. (23) only\nobserved complete degradation of PENG by ß-lactamase in milk\nwith NpH. For cephalosporins, comparable studies have not been\npublished to date. Hydrolysis of cephalosporins in pure milk\nincreased when the NpH was changed to an alkaline pH of 9.0–\n10.0 (21, 24, 25); however, the palatability of alkalinized milk to\ncalves remains questionable (21, 24).\nA few studies have previously determined that residue levels in\nwaste milk from the same antibiotic can vary significantly. In waste\nmilk from various farms, the maximum residue concentration\nranged from 0.026–2.5 mg/L for PENG and 1.7–4.6 mg/L for cefqui­\nnome, a fourth-generation cephalosporin, and the maximum level\nreported for cefoperazone was 0.15 mg/kg (9, 18, 43). In the study\nconducted by Pereira et al. (44), waste milk contained most fre­\nquently PENG but in very low concentrations, with a mean level of\n0.008 mg/L, and the third-generation cephalosporin (ceftiofur;\nmean 0.15 mg/L). It has also been noted that milk on the first day\nafter systemic administration contains a very low concentration of\nPENG (0.092 mg/L), and within 7 days it decreased below the\nassigned maximum residue level of 4 μg/kg (45). PENG was elimi­\nnated from milk below this MRL within 72–96 h (depending on\nmilking frequency) after three intramammary treatments (46).\nCefoperazone (limit of detection 0.002 μg/mL, MRL 50 μg/kg in\nmilk) was detectable between 60 and 84 h following a single intra­\nmammary administration (47). Both the injectable VMP VAN and\nthe intramammary formulation PER used in this study had a legal\n\nCefoperazone (limit of detection 0.002 μg/mL, MRL 50 μg/kg in\nmilk) was detectable between 60 and 84 h following a single intra­\nmammary administration (47).Both the injectable VMP VAN and\nthe intramammary formulation PER used in this study had a legal\nTABLE 3  Degradation of (A) Vanapen® (VAN) and (B) Peracef® (PER) in 50 mL spiked UHT or raw unpasteurized milk by Antipen®.\n(A) Storage\ntemperature °C\n10\n25\n37\n10\n25\n37\n10\n25\n37\n10\n25\n37\nAntipen®\nμL/mL\nVAN μg/\nmL\n12 h, NpHa\n12 h, pH 5.5a\n6 h, pH 5.5a\n6 h, pH 5.5a\n0.025\n3,000\n4/4\n3/4\n0/4\n3/4\n0/4\n1/4\n3/3\n3/3\n3/3\n3/3\n3/3\n3/3\n300\n0/4\n0/4\n0/4\n0/4\n0/4\n0/4\n0/3\n0/3\n0/3\n0/3\n0/3\n0/3\n30\n0/4\n0/4\n0/4\n0/4\n0/4\n0/4\n0/3\n0/3\n0/3\n0/3\n0/3\n0/3\n3\n0/4\n0/4\n0/4\n0/4\n0/4\n0/4\n0/3\n0/3\n0/3\n0/3\n0/3\n0/3\n0.3\n0/4\n0/4\n0/4\n0/4\n0/4\n0/4\n0/3\n0/3\n0/3\n0/3\n0/3\n0/3\n0.03\n0/4\n0/4\n0/4\n0/4\n0/4\n0/4\n0/3\n0/3\n0/3\n0/3\n0/3\n0/3\n(B) Storage\ntemperature °C\n10\n25\n37\n10\n25\n37\n10\n25\n37\n10\n25\n37\nAntipen®\nμL/mL\nPER μg/\nmL\n12 h, NpHa\n12 h, pH 5.5a\n6 h, pH 5.5a\n6 h, pH 5.5a\n0.25\n100\n4/4\n3/4\n0/4\n3/4\n0/4\n1/4\n3/3\n3/3\n3/3\n3/3\n3/3\n3/3\n10\n3/4\n2/4\n0/4\n0/4\n0/4\n0/4\n3/3\n1/3\n0/3\n0/3\n0/3\n0/3\n1\n0/4\n0/4\n0/4\n0/4\n0/4\n0/4\n0/3\n0/3\n0/3\n0/3\n0/3\n0/3\n0.1\n0/4\n0/4\n0/4\n0/4\n0/4\n0/4\n0/3\n0/3\n0/3\n0/3\n0/3\n0/3\naNumber of samples positive for antibiotics/number assayed by Delvotest® T. Degraded samples are highlighted in bold. Limit of detection: VAN 0.003/PER 0.01 μg/mL.\nMilk stored at: 10 °C, 25 °C, and 37 °C for 12 h and 6 h, at natural pH (NpH) and pH 5.5.\n\nLudewig et al.\b\n10.3389/fvets.2026.1808217\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nmilk withdrawal period in the EU of 5 days after the last treatment \n(48, 49).\nIt is also important to note that the VMP for PENG, including \nVAN, was a solution for intramuscular injection in dairy cattle rather \nthan an intramammary formulation, which would, therefore not nec­\nessarily be present in this form in the udder and/or milk. Comparable \npenicillin-containing products for intramammary injections do exist; \nhowever, these were not commercially available at the time of the \nstudy. The VMP containing CEF, namely PER, was an intramammary \nformulation, although it should still be considered that cows are not \ngenerally milked immediately after intramammary tube application, \nmeaning that some pharmacokinetic changes would be expected \nin vivo.\nThe experimental findings in the laboratory suggest that a concen­\ntration of 300 μg/mL VAN (active antibiotic substance PENG) or \n10 μg/mL PER (active antibiotic substance CEF) can be successfully \nreduced below the detection limit of Delvotest® T by the ß-lactamase \nAntipen® within a reasonable time period. Experimental pharmaco­\nkinetic studies provide indicative estimates of residual concentrations \nexpected in waste milk; however, milking intervals and the applied \nFIGURE 1\nMean microbiological counts ± standard deviation of (A) aerobic mesophilic count (AMC) and (B) Enterobacteriaceae in raw unpasteurized milk treated \nwith Antipen® after 12 and 6 h of storage at 10 °C, 25 °C, and 37 °C, at natural pH (NpH) and pH 5.5. The mean was calculated for each experimental \ncombination based on counts in the control sample and milk spiked with Vanapen® (VAN 300 μg/mL) or Peracef® (PER 10 μg/mL). Different subscript \nletters above the bars indicate statistically significant differences among experimental conditions within each temperature group (p < 0.05).\n\nLudewig et al.\b\n10.3389/fvets.2026.1808217\nFrontiers in Veterinary Science\n08\nfrontiersin.org\ndose and treatment intervals under experimental conditions do not\nalways reflect those used in clinical practice. A study investigating the\nexcretion of PENG reported a mean residual milk concentration of\n103.5 μg/mL 12 h after a single intramammary administration of the\ntherapeutic dose (50). For CEF, the calculated concentration excreted\ninto milk 12 h after administration of a single high intramammary\ndose of 1,200 mg ranged between 47.7 and 525 μg/mL in cows with\ninfected udders (47). However, in practice, these CEF concentrations\nare unlikely to be achieved, as it is not standard practice to treat all\nfour quarters of the udder for mastitis, and 10 mL of PER contains\nonly 100 mg of CEF for one application.\nBased on reported residue levels and evaluating concentrations\nobtained in pharmacokinetic studies of both antibiotics, higher con­\ncentrations are not expected to occur in waste milk on farms, particu­\nlarly since waste milk represents a mixture of milk collected at\ndifferent withdrawal times.\nMicrobial quality is an extremely important aspect of waste milk\nfed to calves. The requirements for AMC in raw waste milk fed to\ncalves should be the same as for raw unpasteurized milk used for\nhuman consumption according to Regulation (EC) No 853/2004 (51):\nThe limit for this is 5.0 log cfu/mL, as also recommended by Godden\net al. (52). High AMC poses a health risk for calves, as mastitis-causing\nbacteria or other potentially pathogenic bacteria (such as\nStaphylococcus spp., Streptococcus spp., Listeria monocytogenes,\nCampylobacter spp., Shiga toxin-producing E. coli, and Salmonella\nspp.) may occur in raw milk and could increase during unrefrigerated\nstorage (14, 53–55).\nIn this study, milk with NpH treated with Antipen® for 12 h, at\n10–37 °C showed AMC values exceeding the limit of 5.0 log cfu/\nmL. This long-period treatment in NpH milk can therefore not be\nrecommended without additional pasteurization. In addition, very\nhigh EB and E. coli counts (>7.0 log cfu/mL) are to be expected at very\n\n0 log cfu/\nmL.This long-period treatment in NpH milk can therefore not be\nrecommended without additional pasteurization.In addition, very\nhigh EB and E.coli counts (>7.0 log cfu/mL) are to be expected at very\nhigh ambient temperatures (37 °C). Reducing the exposure time to\n6 h, or alternatively acidifying the milk to pH 5.5 with a 12-h treat­\nment, resulted in a significant reduction in bacterial growth (p < 0.05),\nwith only a small number of individual samples reaching AMC above\nthe specified limit. The initial bacterial load in waste milk is difficult\nto estimate, as it varies considerably dependent on mastitis pathogen\nand the cow’s immune response to infection. Values of 3.3 to 6.9 log\ncfu/mL have been reported in the literature, and higher counts may\nbe associated with mastitis (8, 10, 14, 56).\nThe present study found that acidification of the milk with apple\ncider vinegar to pH 5.5, combined with a ß-lactamase treatment for\n12 h at 10 and 25 °C, and for 6 h at 10–37 °C, resulted in an increase\nof AMC of less than 0.5 log cfu/mL (categorized as “low”). Acidified\nmilk has long been known to prevent bacterial growth in normal and\nwaste milk (57) and has also been reported to have positive effects on\ncalf health status (16, 58).\nIncreases in microbial counts in waste milk are influenced by mul­\ntiple factors, including intrinsic and extrinsic conditions (such as tem­\nperature, pH, and storage time), the initial bacterial load, variability\nin microbial growth optima, and variability in antibiotic residue con­\ncentrations. Concurrently, the values of 300 μg/mL VAN and 10 μg/\nmL PER could be reduced below the detection limit of the Delvotest®\nT by Antipen® treatment. The blue colorant of Antipen® prevents\nmisuse and possible addition of treated waste milk into the food chain.\nOne limitation of this study is that Delvotest® T is a qualitative\nscreening method and therefore does not provide quantitative infor­\nmation on antibiotic residue concentrations in milk. However, the test\nis validated by AFNOR, and the detection limits for PENG and CEF\n\nHowever, the test\nis validated by AFNOR, and the detection limits for PENG and CEF\nare below the MRLs assigned by the EU (32). A further limitation of\nthis study is that milk acidification was performed using apple cider\nvinegar. Other commercially available acidifying agents may have\nmore complex compositions, including additives such as sugars and\ndextrin, which could influence both the degradation of antibiotic resi­\ndues and the increase of microbial count. These laboratory-scale\nexperiments provide a basis for potential application on farms.\nValidation for larger milking volumes and practical farm conditions\nis still required.\n5 Conclusion\nExpected residue levels in waste milk depend on the antibiotic\ntype, route of administration (such as systemic or intramammary),\nnumber of treatments, milking frequency, udder health, and post-\ntreatment period. Overall, these results show that veterinary drugs\ncontaining penicillin or cefoperazone can be successfully reduced\nbelow the detectable antimicrobial activity in milk by ß-lactamases.\nThe efficacy of the enzyme treatment is influenced by several factors,\nincluding exposure time, milk pH, and treatment temperature. These\nexperiments with the VMPs, penicillin (VAN), and cefoperazone\n(PER), suggest that the exposure time to degrade residues of these two\nantibiotics should be at least 6 h. Acidifying the milk to pH 5.5\nimproved the inactivation process and prevented a large increase in\nmicrobial load in raw milk during enzyme treatment. If ß-lactamase\ntreatment is performed without acidification, the milk should always\nbe pasteurized after enzyme exposure; otherwise, bacterial growth\nmay pose a risk to calf health.\nAuthor’s note\nThe commercial enzyme, Antipen® was provided to the authors for\nresearch purposes at a reduced wholesale price by the Finnish company,\nFIGURE 2\nHeat maps illustrating the mean increase of the aerobic mesophilic\ncount (AMC) in raw unpasteurized milk by categoryA after 12 and 6 h\nof storage at 10 °C, 25 °C, and 37 °C, at natural pH (NpH), pH 5.5, and\ntreated with Antipen®. The increase was calculated by the difference\n\n5, and\ntreated with Antipen®.The increase was calculated by the difference\nbetween the initial AMC in untreated milk and the mean of the final\nAMC comprising samples with Vanapen® (VAN 300 μg/mL), Peracef®\n(PER 10 μg/mL), and the control milk. Bp/n—milk positive/negative for\nantibiotics assayed by Delvotest® T. Different subscript letters in a cell\nindicate statistically significant differences in AMC increase among\nexperimental conditions for each temperature (p < 0.05).\n\nLudewig et al.\b\n10.3389/fvets.2026.1808217\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nVetCare Oy. However, employees of this company were not involved at any\nstage in the planning or execution of this experimental study, nor in the\nwriting of this manuscript.\nData availability statement\nThe raw data supporting the conclusions of this article will be\nmade available by the authors, without undue reservation.\nAuthor contributions\nML: Data curation, Methodology, Supervision, Validation,\nVisualization, Writing – original draft. CF: Writing – review & editing,\nConceptualization. MR: Data curation, Formal analysis, Investigation,\nValidation, Writing – original draft. SH: Data curation, Formal analysis,\nInvestigation, Validation, Writing – original draft. AK: Funding acquisition,\nProject administration, Supervision, Writing  – review & editing,\nConceptualization.\nFunding\nThe author(s) declared that financial support was received for this\nwork and/or its publication. This study was conducted within the\nCOMET-Project D4Dairy (Digitalisation, Data integration, Detection\nand Decision support in Dairying) that is supported by BMK\n(Austrian Federal Ministry of Climate Action, Environment, Energy,\nMobility, Innovation and Technology), BMDW (Austrian Federal\nMinistry of Digital and Economic Affairs) and the provinces of\nNiederösterreich and Vienna in the framework of COMET-\nCompetence Centers for Excellent Technologies. The COMET pro­\ngram is handled by the FFG (grant number 872039).\nConflict of interest\nThe author(s) declared that this work was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declared that Generative AI was not used in the\ncreation of this manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\n\nIf you identify any\nissues, please contact us.Publisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. Any product\nthat may be evaluated in this article, or claim that may be made by its\nmanufacturer, is not guaranteed or endorsed by the publisher.\nSupplementary material\nThe Supplementary material for this article can be found online\nat: https://www.frontiersin.org/articles/10.3389/fvets.2026.1808217/\nfull#supplementary-material\nReferences\n1.\tWerner T, Käsbohrer A, Wasner B, Köberl-Jelovcan S, Vetter SG, Egger-Danner C, et al.\nAntimicrobial resistance and its relationship with antimicrobial use on Austrian dairy\nfarms. Front Vet Sci. 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+{"text": "【兽医文献】\n标题: Pseudolaric acid B induces G2/M phase arrest in canine mammary tumor cells by targeting CDK1\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nAttitudes of pet owners in coastal\nOaxaca, Mexico, towards pet\nownership and access to care\nRachael Schulte 1*, Guillermo Arcega Castillo 1 and\nMelinda J. Wilkins 2*\n1 Center for Animal Health and Food Safety, College of Veterinary Medicine, University of Minnesota,\nSaint Paul, MN, United States, 2 Veterinary Population Medicine, College of Veterinary Medicine,\nUniversity of Minnesota, Saint Paul, MN, United States\nFree-roaming dogs pose risks to human, animal, and environmental health, through\nzoonotic disease transmission, contribution to parasite life cycles, and predation\non vulnerable species. Sterilization campaigns are a common method to reduce\nfree-roaming dog populations. A questionnaire was developed to assess the\nattitudes and practices of dog owners in coastal Oaxaca, Mexico, regarding pet\nownership and access to veterinary care. The primary reasons for owning dogs were\ncompanionship and protection, and the most common barriers to care reported\nwere cost and access. The professionalism, knowledge, and communication\nof veterinarians was rated highly, suggesting veterinarians are a trusted source\nof information and should play an important role in education efforts around\nzoonotic diseases and animal care, including population management. Short-term\nsterilization campaigns are effective at reducing free-roaming dog populations and\nhave demonstrated benefits to local wildlife species through reducing predation.\nThese campaigns, however, do not create sustainable change on their own;\nbuilding local veterinary surgical capacity is an important need in rural Oaxaca.\nKEYWORDS\nfree-roaming dogs, sterilization campaigns, zoonotic diseases, veterinary access to\ncare, veterinary capacity building, public health education, sea turtle conservation,\ndog sterilization\n1 Introduction\nA survey conducted in 2021 by the National Statistics Institute of Mexico (INEGI)\nestimated that the population of pet dogs in Mexico was 42,625,010, and in Oaxaca the\npopulation of pet dogs was estimated to be 1,394,967 (1). Estimates of the stray dog population\n\nEstimates of the stray dog population\nwere not available, but it is estimated that only 30% of the dog population in Mexico are owned\n(1). Free-roaming owned and unowned dogs are common in Mexico, but lack of access to\naffordable veterinary care presents risks to the health of dogs and humans (2–6). Risks include\ntransmission of parasites and zoonotic diseases, dog aggression towards humans, and\necosystem effects such as predation on sensitive wildlife species like sea turtles (2–5, 7–12).\nLow rates of sterilization due to cost and cultural beliefs contribute to free-roaming dog\npopulations, with owners of bitches who have litters frequently giving the puppies away rather\nthan increasing the number of dogs in their own household (3–5, 7, 13). The Mexican\ngovernment administers an annual rabies vaccination campaign, which has resulted in high\nrates of rabies vaccination among dogs and a marked decrease in rabies cases in the country;\nhowever, even vaccinated free-roaming dogs can cause severe trauma and infections through\nbites (3–7, 9, 13–15).\nOPEN ACCESS\nEDITED BY\nGustavo Viozzi,\nNational University of Comahue, Argentina\nREVIEWED BY\nEstevam Lux Hoppe,\nSão Paulo State University, Brazil\nSebastian Ballari,\nCONICET Patagonia Norte, Argentina\n*CORRESPONDENCE\nRachael Schulte\nschu4994@umn.edu\nMelinda J. Wilkins\nwilki510@umn.edu\nRECEIVED 09 June 2025\nACCEPTED 30 July 2025\nPUBLISHED 17 September 2025\nCITATION\nSchulte R, Arcega Castillo G and\nWilkins MJ (2025) Attitudes of pet owners in\ncoastal Oaxaca, Mexico, towards pet\nownership and access to care.\nFront. Vet. Sci. 12:1644080.\ndoi: 10.3389/fvets.2025.1644080\nCOPYRIGHT\n© 2025 Schulte, Arcega Castillo and Wilkins.\nThis is an open-access article distributed\nunder the terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other forums is\npermitted, provided the original author(s) and\nthe copyright owner(s) are credited and that\nthe original publication in this journal is cited,\nin accordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Brief Research Report\n\nNo use, distribution or reproduction\nis permitted which does not comply with\nthese terms.TYPE  Brief Research Report\nPUBLISHED  17 September 2025\nDOI  10.3389/fvets.2025.1644080\n\nSchulte et al.\b\n10.3389/fvets.2025.1644080\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nIn 2001, the Palmarito Sea Turtle Rescue in Mazunte, Mexico,\nestablished the Mazunte Project, an annual sterilization campaign that\nprovides ovariohysterectomy and gonadectomy services to coastal\nOaxacan communities at no charge for dogs and cats (3, 11).\nAnecdotal evidence from local residents and returning veterinary\nvolunteers of the project report fewer dogs seen on the beaches than\nin previous years, suggesting that the project’s goals of protecting sea\nturtles through reduction in stray dog populations are being realized\n(email communications with Pierre DePorre, DVM, Palmarito Sea\nTurtle Rescue University Liaison, 2/2/2025 and Rich Rodgers, DVM,\nannual volunteer surgeon, 5/1/2025, conversation with J Adán Ruiz,\nRVT, Founder and Executive Director of Biological Reserve Cerro\nHermoso, 5/7/2025, and WhatsApp with Adriana Cortés-Gómez,\nDVM, PhD, Director of Latin America Programs, SEE Turtles,\n5/2/2025). The long-term goals of the campaign include creating more\nsustainable access to affordable veterinary care in coastal Oaxaca, by\nproviding training to Mexican veterinary students and new graduates,\nestablishing a permanent mobile clinic, and establishing a teaching\nhospital in the area (11), (conversation with J Adán Ruiz, RVT,\nFounder and Executive Director of Biological Reserve Cerro Hermoso,\n5/7/2025).\nShifts in reasons for dog ownership have been previously reported,\nfrom predominantly guardian or working dogs to companions (3, 6,\n7, 13); as attitudes shift, the demand for veterinary care is also likely\nto shift, and patterns of dog ownership are likely to change. The\nobjective of this survey was to establish a baseline for owner\nperceptions of access to and necessity of veterinary care in coastal\nOaxaca, as well as to investigate owner attitudes towards\ndog ownership.\n2 Methods\n2.1 Questionnaire development and\nadministration\nA questionnaire was developed using Qualtrics software (Provo,\nUT) (see Supplementary Data Sheet 1). The questionnaire included 44\n\n2 Methods\n2.1 Questionnaire development and\nadministration\nA questionnaire was developed using Qualtrics software (Provo,\nUT) (see Supplementary Data Sheet 1).The questionnaire included 44\nquestions in 8 sections: demographic information (8 questions); care\nof unowned animals (1 question); questions pertaining to the animal\nbrought to the campaign (3 questions); attitudes and practices around\nanimal ownership (8 questions), vaccination (4 questions), sterilization\n(5 questions), and parasite prevention (6 questions); and access to care\n(9 questions). The questionnaire employed a mix of structured and\nsemi-structured questions; the majority of questions were multiple\nchoice or multiple select with text entry boxes for respondents to\nprovide additional detail (see Supplementary Data Sheet 1). The\nUniversity of Minnesota Institutional Review Board (IRB) determined\nthe survey qualified for IRB exemption.\nThe purpose of this questionnaire was to understand attitudes and\npractices around pet ownership and provide a baseline for local\nveterinarians and public health officials to use when planning public\nhealth interventions. Questionnaires were developed in English, then\ntranslated into Spanish by a member of the research team and verified\nby a veterinarian local to coastal Oaxaca. The questionnaire was\nadministered by interviewers on paper  and tablets in Spanish at\nlocations where free sterilization clinics were being offered in coastal\nOaxaca in the first 2 weeks of January, 2025 (see Figure 1). Spanish-\nspeaking volunteers were recruited to act as interviewers. Interviewers\nwere trained on-site on the objectives and methodology of the survey\nand on survey-taking practices. The average duration of interviews\nwas 30 min. Response rate was not recorded.\nNo questions were required to be  answered to complete the\nsurvey; missing answers to questions were excluded from analysis and\nreported percentages are valid percents (missing values were excluded\nfrom the calculation). One question regarding income was removed\nfrom the survey after the first day of survey administration.\n\nOne question regarding income was removed\nfrom the survey after the first day of survey administration.\n2.2 Participants\nA convenience sampling strategy was utilized, with the survey\nbeing administered to persons taking advantage of free sterilization\nclinics. Ninety-five surveys were administered. The exclusion criterion\nwas failing to answer any questions related to animal ownership or\nveterinary care (only answering demographic questions); one survey\nwas excluded, resulting in a final sample size of 94 respondents.\n2.3 Statistical analysis\nSurvey analysis was performed initially on Qualtrics (Provo, UT,\nUSA RRID: SCR_016728) and Excel (Redmond, WA, USA RRID:\nSCR_016137), and finalized in SPSS (Armonk, NY, USA RRID:\nSCR_016479). Descriptive statistics were used to analyze the results,\nand the relationships between selected variables were analyzed using\nchi-square tests due to the small sample size.\n3 Results\nOut of 94 respondents, four were under 18; they took the survey\non behalf of related adults, whose ages were not recorded. Of the other\n90 responses, 15.6% were 18–24, 15.6% were 25–34, 31.1% were\n35–44, 15.6% were 45–54, 8.9% were 55–64, and 13.3% were over\n65 years old. As expected, the majority of respondents lived in a rural\nenvironment (81.3%, 74/91), with the rest living in an urban\nenvironment (18.7%, 17/91). A similar percentage owned their own\nhome (84%, 79/94), while 9.6% (9/94) rented, and 6.4% (6/94) selected\n“other”; five of those respondents provided text answers, which all\nreported living with an older related generation, and were 18–32 years\nold. Less than half of respondents reported someone in their\nhousehold was under 5 years or over 65 years (40.2%, 37/92). When\nasked about education level, 25% reported not finishing primary\nschool (23/92), 33.7% reported entering secondary school (31/92),\n29.3% completed preparatory or technical education (27/92), 10.9%\ncompleted a university degree (10/92), and 1.1% completed post-\ngraduate education (1/92).\nThe majority of respondents brought an owned animal in to the\n\n3% completed preparatory or technical education (27/92), 10.9%\ncompleted a university degree (10/92), and 1.1% completed post-\ngraduate education (1/92).The majority of respondents brought an owned animal in to the\nsterilization campaign, either their own or a relative/neighbor’s animal\n(91.3%, 84/92), while 4.3% of respondents (4/92) brought in a stray\nanimal, and the remaining 4.3% did not bring an animal to the\ncampaign. If the sterilization campaign was not available, 63% (58/92)\nstated they would have had their animal sterilized, 19.6% (18/92)\nstated they might have their animal sterilized, and 17% (16/92) stated\nthey would not have their animal sterilized if the campaign was not\noffered. When asked about bite history, 97.9% of respondents (92/94)\nstated none of their dogs or cats had bitten a person hard enough to\n\nSchulte et al.\b\n10.3389/fvets.2025.1644080\nFrontiers in Veterinary Science\n03\nfrontiersin.org\ndraw blood out of aggression or fear in the past 2 years, and only 2.1%\n(2/94) affirmed that their dog or cat had a recent bite history. Mixed\nbreed dogs were owned by the majority of respondents (82.4%, 75/91),\n9.6% owned only purebred dogs (9/91), and 7.7% owned both mixed\nand purebreds (7/91). Dog ownership was reported by 96.8% of\nrespondents (91/94), with a mean of 3.4 and a range of 1 to 9 dogs\nowned (n = 73). Cat ownership was reported by 61.7% of respondents\n(58/94), with a mean of 2.5 and a range of 1 to 7 cats owned (n = 48).\nForty-four respondents reported owning chickens (46.8%, n = 36).\nLivestock ownership (cattle, goats, sheep, or pigs) was reported by\n13.8% of respondents (13/94, n = 9). Four respondents (4.3%)\nreported ownership of other animals; two owned parrots, and two\nowned turkeys.\nWhen asked if they provide care to dogs they do not own, 52.1%\n(49/94) stated that they do not, while 44.7% (42/94) stated that they feed\ndogs they do not own. Less than 13% let dogs they do not own into their\nhouse or outbuildings, or provide medical care to those dogs. The majority\nof respondents reported that their dogs and cats were given to them as a gift\n(54.3%, 51/94); 24.5% (23/94) reported finding their pets, while 23.4%\n(22/94) reported their pets being born in their house. Less than 20%\nreported buying or adopting their pets. The majority of respondents (85.1%,\n80/94) stated that they always or sometimes allowed their dogs in the house,\nwhile 14.9% (14/94) stated that they never let their dogs in the house or only\nsome of their dogs were permitted in the house. Most respondents reported\ntheir dogs are in a fenced yard when outside (70.2%, 66/94), while 42.6%\n(41/94) reported their dogs roam freely for at least part of the day; less than\nFIGURE 1\nMap of coastal Oaxaca showing the state capital, major tourist destinations and major urban centers. The inset shows survey locations in rural Oaxaca.\n\nSchulte et al.\b\n10.3389/fvets.2025.1644080\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n13% of respondents reported leash walking or tying their dogs when\noutside. Companionship was rated as the most important reason for\nowning dogs by 81.8% (72/88) of respondents, followed by protection\n(54.8%, 45/82) and vermin control (23.3%, 17/73) (see Figure 2). The most\ncommon dog-animal interactions were neighbor’s animals (82.3%, 65/79),\nfollowed by stray dogs (26.6%, 20/79) and wildlife (19%, 15/79).\nVaccination rates were reported to be high, with 74.4% (67/90) of\nrespondents stating all their dogs were vaccinated, and only 12.2% (11/90)\nstating none of their dogs were vaccinated. The most common barriers to\nvaccination were reported to be cost (55.6%, 10/18) and access to services\n(55.6%, 10/18) (see Figure 3). Rabies was the most common vaccine given\n(90.4%, 75/83), followed by canine distemper (62.7%, 52/83); Bordetella\nand Leptospirosis were both reported at 9.6% (8/83). The most common\nprovider of vaccination was a government initiative (65.9%, 54/82),\nfollowed by private veterinarians (57.3%, 47/82); less than 10% of\nrespondents vaccinated their dogs at home.\nSterilization was less common than vaccination, with 42.4% (39/92)\nof respondents stating none of their dogs were sterilized, and only 20.7%\n(19/92) claiming all their dogs were sterilized. Cost (51.9%, 27/52) and\naccess (34.6%, 18/52) were reported to be the most common barriers to\nsterilization; notably, 25% of respondents did not believe sterilization was\nnecessary (13/52) (see Figure 3). Most respondents did not believe it is\nnecessary to wait to sterilize a bitch until she has had a litter (72.1%,\n62/86). The most common provider of sterilization was sterilization\ncampaigns (81.7%, 49/66), followed by private veterinarians (28.3%,\n17/66). Sterilization campaigns were considered beneficial to the\ncommunity by 100% of respondents (91/91).\nFlea and tick ectoparasiticides were most commonly given every\n3–4 months (44.9%, 40/89) or when external parasites were seen on pets\nor in the house (24.7%, 22/89). Anthelmintics were given most commonly\n\nFlea and tick ectoparasiticides were most commonly given every\n3–4 months (44.9%, 40/89) or when external parasites were seen on pets\nor in the house (24.7%, 22/89).Anthelmintics were given most commonly\nevery 3–4 months (47.7%, 41/86) or seasonally (18.6%, 16/86); the most\ncommon barriers to ectoparasiticides and anthelmintics was cost (68.8%,\n11/16, and 71.4%, 15/21, respectively) (see Figure 3). The majority of\nrespondents reported picking up their dog’s feces in their yard (88.8%,\n79/89), while only 22.5% (20/89) picked up their dog’s feces in the street.\nHandwashing between handling pets and eating was common, reported\nby 96.7% of respondents (89/92).\nThe majority of respondents visited private veterinarians for\nmedical advice, care, or products (53.3%, 49/92), while 26.1% (24/92)\nvisited both a veterinarian and a granero1. Those who bought products\nfrom the granero bought ectoparasiticides (82.5%, 52/63) or\nanthelmintics (79.4%, 50/63) more commonly than other products or\nservices. The most common services purchased from veterinarians\nwere vaccines (63.6%, 49/77), anthelmintics (57.1%, 44/77), and care\nfor mild illnesses (41.6%, 32/77).\nRespondents were asked to rate local veterinarians’ professionalism,\nknowledge, and communication on a scale of 1–10, with 1 being poor\nand 10 being excellent. The mean scores were 8.87 for professionalism\n(n = 55); 8.76 for knowledge (n = 54); and 9 for communication\n(n = 54). The range for all three was 5–10, and the mode for all three was\n10. Respondents were also asked how much they were willing to pay for\nvaccines, anti-parasitic medications, sterilization, and emergency care\nfor their pets (see Figure 4). The most common veterinary care provided\nat home by respondents was deworming (77.8%, 70/90), followed by\nbasic wound care (44.4%, 40/90) and vaccinations (30%, 27/90).\nHaving at least 6 years of education was significantly correlated\nwith having at least some of their dogs vaccinated (Chi-square 5.256,\np < 0.05, n = 88). Living in a rural environment was significantly\ncorrelated with owning dogs for protection (Chi-square 16.304,\n\nHaving at least 6 years of education was significantly correlated\nwith having at least some of their dogs vaccinated (Chi-square 5.256,\np < 0.05, n = 88).Living in a rural environment was significantly\ncorrelated with owning dogs for protection (Chi-square 16.304,\np < 0.001, n = 80), having at least some of their dogs being vaccinated\n(Chi-square 6.145, p < 0.05, n = 87) and sterilized (Chi-square 8.771,\np < 0.05, n = 89) (see Supplementary Table 1). No significance was\nfound when comparing education or living environment (urban vs.\nrural) to willingness to seek sterilization outside a campaign, allowing\ndogs in the house, breed owned, reasons for owning dogs, willingness\nto seek other veterinary care (vaccines, ectoparasiticides,\nanthelmintics), seeking care from a veterinarian vs. granero, or\nwillingness to pay more or less for veterinary services. No correlation\n1  A local agricultural supply store that sells animal products such as balanced\nfeed, veterinary medicines, vaccines, and pest control products, sometimes\nwithout direct veterinary oversight.\nFIGURE 2\nReasons for owning dogs, ranked from most important to least important, by percentage of respondents.\n\nSchulte et al.\b\n10.3389/fvets.2025.1644080\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nwas found when comparing the presence of children or seniors in the \nhome with bite history, allowing dogs in the house, reasons for owning \ndogs, or veterinary care metrics.\n4 Discussion\nAlthough living in close proximity to dogs and cats provides many \nhealth benefits (16), it also increases the risk of zoonotic diseases. \nImmunocompromised persons are at higher risk for complications \nsecondary to zoonotic diseases (17, 18), including children under \n5 years and adults over 65 years. Steps to reduce the risk of zoonotic \ntransmission include using anti-parasitic medications appropriately, \nappropriate disposal of feces, and appropriate hand hygiene. Although \nhigh rates of fecal pick up were reported in people’s own yards, the \nrates dropped off for picking up dog’s feces on the street; this is likely \nin part due to dogs being allowed to roam freely, and owners not \nseeing where their dogs defecate. This contributes to the lifecycle of \nparasites in the environment, especially on beaches, where people \noften walk barefoot and may become infected (2, 8, 19, 20). The \nFIGURE 3\nReasons why specific veterinary services were not obtained for dogs, by percent of respondents. No respondent indicated a belief that vaccines were \nunnecessary.\nFIGURE 4\nPercent of respondents willing to pay defined amounts for various veterinary services, in Mexican pesos. The amounts listed for vaccines are amount \nper pet per year, anti-parasitic medications are amount per pet per month, and sterilization is per procedure.\n\nSchulte et al.\b\n10.3389/fvets.2025.1644080\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nmajority of respondents used both ectoparasiticides and anthelmintics\nevery 3–4 months; the similar schedule may be in part due to using\ncombination products. This schedule is unlikely to fully control\nestablished flea infestations, but does align with recommendations for\nanthelmintic administration (21, 22); the schedule may also be in part\ndue to barriers to using anti-parasitic medications, of which cost and\naccess to care were most common. In Puerto Escondido, Oaxaca,\nMexico, the most prevalent intestinal parasites were Toxocara canis,\nwhich was found in 48% of samples (23); in the nearby state Tabasco,\nMexico, the prevalence of ticks on dogs was 22% and fleas was 8.7%\n(24), exemplifying the need for parasite control in this region.\nVeterinarians and local public health officials should encourage\nappropriate use of anti-parasitic medications and other steps to\nprevent zoonotic diseases, including appropriate disposal of feces. The\nassessment showed high rates of handwashing, the importance of\nwhich should continue to be  emphasized in conversations about\nparasites and zoonotic risk. Most respondents reported that they\nwould or might have their pets sterilized even if the campaign was not\noffered; this suggests the community recognizes the value of\nsterilization of pets. However, the actual rate of sterilization is lower\nthan the rate of vaccine uptake, and a belief that sterilization was\nunnecessary for their pets was expressed by some community\nmembers. Those who believed sterilization was unnecessary\ncommented that they planned to breed their dogs, or their bitches\nwere spayed so it was unnecessary to sterilize their male dogs;\neducation on reducing unwanted behavior exhibited by intact males,\nsuch as urine marking and unplanned mating may be beneficial (2, 5,\n25). Such education should take into account a cultural reluctance to\nsterilize male dogs, and the belief that female dogs should have at least\none litter before being sterilized (3, 7, 26).\nInteractions with wildlife were reported by numerous\n\nSuch education should take into account a cultural reluctance to\nsterilize male dogs, and the belief that female dogs should have at least\none litter before being sterilized (3, 7, 26).Interactions with wildlife were reported by numerous\nrespondents, but were likely underreported, as free-roaming dogs\nwould have numerous opportunities to interact with wildlife while\nunsupervised. Local veterinary and wildlife conservation\nprofessionals, and returning volunteers, stated that fewer dogs have\nbeen seen on beaches; they stated that more sea turtle hatchlings are\nsuccessfully reaching the ocean, due to the campaign, education of\ncommunity members on keeping dogs confined, and efforts to\nprotect nests before hatching (email communications with Pierre\nDePorre, DVM, Palmarito Sea Turtle Rescue University Liaison,\n2/2/2025, and Rich Rodgers, DVM, annual volunteer surgeon,\n5/1/2025, conversation with J Adán Ruiz, RVT, Founder and\nExecutive Director of Biological Reserve Cerro Hermoso, 5/7/2025,\nand WhatsApp with Adriana Cortés-Gómez, DVM PhD, Director of\nLatin America Programs, SEE Turtles, 5/2/2025). One location that\nthe sterilization campaign has only been offered at more recently has\nnot seen as noticeable an effect on dog populations on beaches, but\na noticeable decrease in the number of litters and an increase in the\nnumber of community members taking advantage of the campaign\nhas been noted (email communication with Luis Ángel Rojas Cruz,\n5/9/2025), suggesting the campaign’s impacts on sea turtle\nconservation are not always immediate. Alternatively, in 2001, the\nfirst year of the campaign, an estimated 70–80 dogs were noted on\nLa Escobilla beach at hatchling emergence; within a year, less than\nhalf that number were noted, and now fewer than 5 dogs predate on\nsea turtles on that beach (email communication with Rich Rodgers,\nDVM, annual volunteer surgeon, 5/1/2025). This may be due in part\nto the sterilization campaign decreasing the dog population, shifts\nin attitudes around pet ownership and free-roaming dogs, or\nother causes.\nThe most common provider of sterilization to this community was\n\nThis may be due in part\nto the sterilization campaign decreasing the dog population, shifts\nin attitudes around pet ownership and free-roaming dogs, or\nother causes.The most common provider of sterilization to this community was\nfree campaigns, and 100% of respondents believe these campaigns\nprovide a benefit. However, external aid provides fewer and shorter-term\nbenefits than community driven efforts (27). The sterilization campaign\nonly operates in communities that do not have access to veterinary\nsurgical services, and stopped operating in one community in 2023 to\navoid competing with local veterinarians. In 2025, however, that\ncommunity reached out asking for the campaign to resume visits. This\nsuggests efforts to act as a bridge only until private veterinarians can begin\noffering services for long-term population management are not fully\neffective. One ongoing challenge is the limited surgical training in many\nMexican veterinary schools. Students often graduate with little hands-on\nsurgical experience, so sterilization campaigns are one of the few\nopportunities for them to practice, build confidence, and develop surgical\ncompetence. Such capacity building efforts are an integral part of the\nMazunte project, with local veterinary students and new graduates\nparticipating in the campaigns to learn surgical and anesthetic skills (11,\n28); additionally, future plans to build a teaching hospital in Oaxaca (11)\nwill provide more sustainable benefits to the community.\nHigh rates of rabies vaccination were reported, likely due to\ngovernment rabies vaccination campaigns, which report vaccinating\n80% of dogs and cats (14); combined with robust surveillance (15), the\nrisk of rabies zoonosis is low. No cases of canine-transmitted human\nrabies have occurred since 2006, and Mexico was the first country\nrecognized by the WHO as rabies free (29, 30). Wildlife, especially\nbats, continue to act as reservoirs, and have been implicated in recent\nhuman and canine cases (29–32). Additionally, the low bite history\nreported decreases the risk further, though unwitnessed bites from\n\nWildlife, especially\nbats, continue to act as reservoirs, and have been implicated in recent\nhuman and canine cases (29–32).Additionally, the low bite history\nreported decreases the risk further, though unwitnessed bites from\nfree-roaming dogs is a risk (4), and all dog bites pose a risk for severe\ntrauma and infection (9). The percentage of respondents that allow\ntheir dogs to roam freely was high, which is supported by the large\nnumber of dogs roaming freely through the community witnessed by\nthe authors; previous studies have shown that dogs in coastal Oaxaca\nfrequently roam free, and are often fed by other community members\nin addition to their owners (3), which is supported by nearly half of\nrespondents feeding dogs they do not own. A high percentage of\nrespondents also reported keeping their dogs in a fenced yard, but\nauthors witnessed dogs easily escaping fenced yards while in Oaxaca,\nsuggesting that at least some fenced dogs may contribute to the free-\nroaming dog population.\nPrevious work has demonstrated the most common reasons for\nowning dogs in Oaxaca, Mexico, are protection and companionship\n(3), which is supported in this work (see Figure  2), where\ncompanionship and protection were most frequently rated as the most\nimportant reason to own dogs. This likely exemplifies a shift noted in\nareas with high tourism towards viewing dogs as companions, rather\nthan solely as protectors, as has been previously reported (3, 13).\nIncreased zooeyia, or health benefits associated with owning animals,\nis expected to accompany these shifts (16).\nThe most common barriers to care were cost and access to\nveterinary services (see Figure 3); this is unsurprising, as the campaign\ntargets rural communities without veterinary care, and previous\nresearch in the state of Hidalgo, Mexico, also showed low rates of\nveterinary care and inability to afford sterilization procedures (33) as\ndoes broader work on veterinary care accessibility (34, 35). Recent\nlegislation at the federal level mandates publicly funded veterinary\n\nSchulte et al.\b\n10.3389/fvets.2025.1644080\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nclinics to reduce cost and increase access to veterinary care, but\nveterinarians in Mexico stated a lack of enforcement and funding\nprovisions reduced its efficacy; many veterinarians were unaware of\nthe legislation a year after it took effect (26, 36, 37), demonstrating\nfurther work on accessibility is needed. The authors found no publicly\nfunded clinics or hospitals in Oaxaca.\nA perceived lack of necessity of certain types of care was also\nnoted, which should be  addressed by local public health officials\nplanning public health education campaigns, and local veterinarians\neducating clients in the course of providing care. Additional reasons\nfor not pursuing vaccination and sterilization for pets was a belief that\ntheir dogs were too young; dog ages were not collected in this survey,\nbut previous studies found community members in the city of Mexicali,\nBaja California believed dogs under 1 year to be  too young for\nvaccination (13), highlighting an area for education by public health\nofficials and veterinarians. Overall, the professionalism, knowledge,\nand communication skills of local veterinarians was rated highly,\nwhich corresponds with high levels of trust in veterinarians in other\ncountries (38, 39), suggesting they are well positioned to lead education\nefforts related to animal health and zoonotic disease prevention.\nThe willingness of respondents to pay for veterinary services\ntended toward a mid-high range of cost for lower-cost items (vaccines,\nanti-parasitic medications), and toward lower ranges for high-cost\nitems (sterilization, emergency care) (see Figure 4). These results are\nparticularly interesting, as a survey in Hidalgo in 2022 showed nearly\nhalf of community members were unwilling to pay more than $100\nMXN for sterilization (33), while our data suggests close to 40% of\nrespondents are willing to pay $500–$1,000 MXN for sterilization.\nProximity to Mazunte, a small tourist center, may influence perceptions\nof welfare (3)(see Figure 1), which could contribute to an increased\n\nProximity to Mazunte, a small tourist center, may influence perceptions\nof welfare (3)(see Figure 1), which could contribute to an increased\nwillingness to pay higher prices for sterilization in this study location.\nThe annual sterilization campaign may have also demonstrated the\nbenefits of sterilization, which could contribute to increased willingness\nto pay for services. This information could be used as a baseline by\nlocal veterinarians and veterinary distributors for cost setting.\nCorrelation was only significant between a few variables, likely\ndue to our small sample size; statistical power was therefore limited.\nRespondents reporting completing at least 6 years of education were\nmore likely to have some or all of their dogs vaccinated; this may\nbe due to greater awareness of public health practices, or different\nsocioeconomic conditions improving access to veterinary care. Living\nin a rural environment was correlated with having some or all of their\ndogs vaccinated and sterilized, and with owning dogs for protection;\nthis is likely due to using dogs in rural settings as guardians for\nlivestock or property. This aligns with research done in 2012, which\ndemonstrated protection was the predominant reason for dog\nownership in both rural and urban Oaxaca (3), and may indicate a\ndivergence between rural and urban attitudes toward dog ownership.\nSeveral limitations were present in this assessment. Due to the\nsmall sample size, the significance of correlation between variables\nmay have been under or overestimated. Convenience sampling was\nused, with participants recruited from community members\nbringing pets to a sterilization campaign for dogs and cats. This\nresulted in selecting for people who are more likely to own pets,\nseek veterinary care for pets, and believe that sterilization of pets\nserves a purpose; this likely resulted in selection bias. Social\ndesirability bias may have also played a role in respondents’\nanswers to questions on hygiene and animal care practices. This\ndata should not be used to estimate animal ownership in the area,\n\nSocial\ndesirability bias may have also played a role in respondents’\nanswers to questions on hygiene and animal care practices.This\ndata should not be used to estimate animal ownership in the area,\nand is not generalizable to the greater public.\nThis assessment has several notable strengths. Administering\nquestionnaires at sterilization campaign sites provided the\nopportunity to engage with a wide range of pet owners and\ncommunity members, providing insight into animal health\npractices and attitudes toward pet ownership in the region. The\nquestionnaire also captured a wide range of variables related to\npet ownership, access to care, preventive practices, and attitudes\ntoward sterilization, enabling a comprehensive descriptive\nanalysis. Finally, this assessment evaluated context-specific\nfactors relevant to rural coastal communities, such as the\ninteraction between free-roaming dogs and local wildlife,\nincluding sea turtles, which are often overlooked in broader\nnational studies. This assessment can be used to inform local\npublic health practice and educational strategies.\n5 Conclusion\nThe high levels of trust reported in veterinarians position them\nas essential leaders in both delivering care and educating\ncommunities about zoonotic disease risks and the health benefits\nof sterilization. Although survey respondents believed the free\nsterilization campaigns provided benefits to the community, such\nbenefits can only be short-term until local veterinary capacity is\nbuilt-up. Efforts to manage the population with campaigns should\ncontinue, but with a greater emphasis on providing opportunities\nfor local veterinarians and veterinary students to develop the\nsurgical skills necessary to provide surgical sterilization services to\nthe community, to improve public health and animal welfare, and\nto protect vulnerable wildlife species.\nData availability statement\nThe original contributions presented in the study are publicly\navailable. This data can be found in the Data Repository for the\nUniversity of Minnesota (DRUM), https://doi.org/10.13020/\n6HYG-AS52 and in the Supplementary materials.\n\nData availability statement\nThe original contributions presented in the study are publicly\navailable.This data can be found in the Data Repository for the\nUniversity of Minnesota (DRUM), https://doi.org/10.13020/\n6HYG-AS52 and in the Supplementary materials.\nAuthor contributions\nRS: Project administration, Formal analysis, Methodology,\nWriting – review & editing, Investigation, Writing – original draft,\nConceptualization, Resources, Visualization, Data curation. GA:\nConceptualization, Investigation, Writing  – review & editing,\nVisualization. MW: Supervision, Writing  – review & editing,\nMethodology, Conceptualization.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article.\n\nSchulte et al.\b\n10.3389/fvets.2025.1644080\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nAcknowledgments\nThe authors gratefully acknowledge the veterinarians, veterinary\ntechnicians, and other volunteers whose work ensures the success of\nthe Mazunte Project. Special acknowledgement is extended to the\nPalmarito Sea Turtle Rescue board and volunteers and Drs. Pierre\nDePorre, Marcelino López Reyes, Leonel Ramos, and Adriana\nA. Cortés-Gómez. We  are deeply grateful to our questionnaire\nadministrators for their work on data collection. We acknowledge the\ntravel funding provided by the University of Minnesota College of\nVeterinary Medicine which made this work possible. We would also\nlike to acknowledge Zoetis™ for generously donating medications\nutilized in the sterilization campaigns.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. 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Mexico news daily.\n(2024) Available online at:https://mexiconewsdaily.com/news/public-free-veterinary-\nclinics-to-be-established-across-mexico/\n37.\tCámara de Diputados [Internet]. [cited 2025 May 8]. DOF publica decreto con reforma\npara promover establecimiento de clínicas veterinarias públicas. (2003). Available online at:\nhttp://comunicacionsocial.diputados.gob.mx/index.php/notilegis/dof-publica-decreto-con-\nreforma-para-promover-establecimiento-de-clinicas-veterinarias-publicas\n38.\tRoyal College of Veterinary Surgeons. Vets amongst the most trusted\nprofessionals, according to survey. (2019). Available online at: https://www.rcvs.\norg.uk/news-and-views/news/vets-amongst-the-most-trusted-professionals-\naccording-to-rcvs/\n39.\tProfessionals [Internet]. Vets amongst the most trusted professionals, according\nto survey. Available online at:https://www.rcvs.org.uk/news-and-views/news/vets-\namongst-the-most-trusted-professionals-according-to-rcvs/\n\n---"}
+{"text": "【兽医文献】\n标题: A comprehensive study on freshwater pufferfish Pao baileyi: profiles of toxin possession, accumulation, and uptake\n\nTYPE Correction\nPUBLISHED 29 June 2026\nDOI 10.3389/fvets.2026.1897588\nOPEN ACCESS\nAPPROVED BY\nFrontiers Editorial Office,\nFrontiers Media SA, Switzerland\n*CORRESPONDENCE\nWidjiati Widjiati\nwidjiati@fkh.unair.ac.id\nRECEIVED 02 June 2026\nREVISED 02 June 2026\nACCEPTED 03 June 2026\nPUBLISHED 29 June 2026\nCITATION\nWidjiati W, Luqman EM, Darsini N,\nAulanni’am A, Hendrawan VF,\nKurniawati DY and Jaafar WNFBW (2026)\nCorrection: IGF-1 supplementation\nimproves in vitro maturation quality of\nCapra hircus oocytes via PDE3A\nactivation, gap junction remodeling,\napoptosis suppression, and kisspeptin\nsignaling. Front. Vet. Sci. 13:1897588.\ndoi: 10.3389/fvets.2026.1897588\nCOPYRIGHT\n© 2026 Widjiati, Luqman, Darsini,\nAulanni’am, Hendrawan, Kurniawati and\nJaafar. This is an open-access article\ndistributed under the terms of the\nCreative Commons Attribution License\n(CC BY). The use, distribution or\nreproduction in other forums is\npermitted, provided the original author(s)\nand the copyright owner(s) are credited\nand that the original publication in this\njournal is cited, in accordance with\naccepted academic practice. No use,\ndistribution or reproduction is permitted\nwhich does not comply with these terms.\nCorrection: IGF-1\nsupplementation improves\nin vitro maturation quality of\nCapra hircus oocytes via PDE3A\nactivation, gap junction\nremodeling, apoptosis\nsuppression, and kisspeptin\nsignaling\nWidjiati Widjiati1*, Epy Muhammad Luqman1, Ninik Darsini2,\nAulanni’am Aulanni’am3, Viski Fitri Hendrawan4,5,\nDevia Yoanita Kurniawati5 and Wan Nor Fitri Bin Wan Jaafar6\n1Department of Veterinary Anatomy, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya,\nIndonesia, 2Department of Medical Biology, Faculty of Medicine, Universitas Airlangga, Surabaya,\nIndonesia, 3Department of Biochemistry, Faculty of Veterinary Medicine, Universitas Brawijaya, Malang,\nIndonesia, 4Department of Veterinary Reproduction, Faculty of Veterinary Medicine, Universitas\nBrawijaya, Malang, Indonesia, 5Doctoral Program of Veterinary Sciences, Faculty of Veterinary\nMedicine, Universitas Airlangga, Surabaya, Indonesia, 6Department of Farm and Exotic Animal Medicine\n\nand Surgery, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia\nKEYWORDS\napoptosis, gap junction, IGF-1, in vitro maturation, kisspeptin, oocyte, PDE3A, Zero\nHunger\nA Correction on\nIGF-1 supplementation improves in vitro maturation quality of Capra\nhircus oocytes via PDE3A activation, gap junction remodeling, apoptosis\nsuppression, and kisspeptin signaling\nby Widjiati, W., Luqman, E. M., Darsini, N., Aulanni’am, A., Hendrawan, V. F., Kurniawati, D. Y.,\nand Jaafar, W. N. F. B. W. (2026). Front. Vet. Sci. 13:1821251. doi: 10.3389/fvets.2026.1821251\nFunding information from the Indonesian Endowment Fund for Education (LPDP), on\nbehalf of the Indonesian Ministry of Higher Education, Science and Technology, managed\nunder the EQUITY Program, was erroneously omitted from the Funding statement. The\ncorrect Funding statement reads:\n“The author(s) declared that financial support was received for this work and/or its\npublication. This study was supported by Universitas Airlangga through the Airlangga\nResearch Fund, Airlangga Flagship Research Scheme/Penelitian Unggulan Airlangga 2024\n(Contract No. 358/UN3.LPPM/PT.01.03/2024). This paper is a part of activities funded\nby the Indonesian Endowment Fund for Education (LPDP) on behalf of the Indonesian\nMinistry of Higher Education, Science and Technology and managed under the EQUITY\nProgram (Contract No. 4300/B3/DT.03.08/2025 and 297/UN3/HK.07.00/2025).”\nFrontiers in Veterinary Science\n01\nfrontiersin.org\n\nWidjiati et al.\n10.3389/fvets.2026.1897588\nIn the Acknowledgments, the required Acknowledgments\nstatements\nfor\nthe\nAirlangga\nResearch\nFund\nand\nthe\nLPDP-funded\nEQUITY\nProgram\nwere\nomitted.\nThe\ncorrected\nAcknowledgments\nsection\nshould read:\n“We acknowledge the technical assistance provided by the\nstaffinvolved in sample collection and laboratory support.\nThe authors also acknowledge Universitas Airlangga through\nthe Airlangga Research Fund, Airlangga Flagship Research\nScheme/Penelitian\nUnggulan\nAirlangga\n2024\n(Contract\nNo.\n358/UN3.LPPM/PT.01.03/2024), and the Indonesian Endowment\nFund for Education (LPDP) on behalf of the Indonesian Ministry\nof Higher Education, Science and Technology, managed under\nthe EQUITY Program (Contract No. 4300/B3/DT.03.08/2025\nand\n297/UN3/HK.07.00/2025),\nfor\nsupporting\nthis\nresearch activity.”\nThe original version of this article has been updated.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. Any product that may be evaluated in this article, or\nclaim that may be made by its manufacturer, is not guaranteed or\nendorsed by the publisher.\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n\n---"}
+{"text": "【兽医文献】\n标题: Experimental comparison of African swine fever virus exposure through contaminated feed and infected pig organ homogenate\n\nTYPE Original Research\nPUBLISHED 28 May 2026\nDOI 10.3389/fvets.2026.1830161\nOPEN ACCESS\nEDITED BY\nAlexandre Rodrigues Silva,\nFederal University Rural Semi-Arid, Brazil\nREVIEWED BY\nRomário Parente Dos Santos,\nFederal University Rural Semi-Arid, Brazil\nHerlon Silva,\nState University of Ceará, Brazil\n*CORRESPONDENCE\nViola Zappone\nviola.zappone@unime.it\nRECEIVED 13 March 2026\nREVISED 05 May 2026\nACCEPTED 05 May 2026\nPUBLISHED 28 May 2026\nCITATION\nZappone V, Alonge S, Angelastri R,\nTroisi A, Marcoccia R, Quartuccio M and\nAiudi GG (2026) The impact of\nsupplementation with calcium and\nnaloxone to TRIS and Lepus extenders\non the quality of refrigerated rabbit\nsemen. Front. Vet. Sci. 13:1830161.\ndoi: 10.3389/fvets.2026.1830161\nCOPYRIGHT\n© 2026 Zappone, Alonge, Angelastri,\nTroisi, Marcoccia, Quartuccio and Aiudi.\nThis is an open-access article distributed\nunder the terms of the Creative\nCommons Attribution License (CC BY).\nThe use, distribution or reproduction in\nother forums is permitted, provided the\noriginal author(s) and the copyright\nowner(s) are credited and that the\noriginal publication in this journal is\ncited, in accordance with accepted\nacademic practice. No use, distribution\nor reproduction is permitted which does\nnot comply with these terms.\nThe impact of supplementation\nwith calcium and naloxone to\nTRIS and Lepus extenders on the\nquality of refrigerated rabbit\nsemen\nViola Zappone1*, Salvatore Alonge2, Roberta Angelastri3,\nAlessandro Troisi4, Romina Marcoccia4, Marco Quartuccio1 and\nGiulio Guido Aiudi3\n1Department of Veterinary Medicine, University of Messina, Messina, Italy, 2Società Veterinaria “Il\nMelograno” Srl, Sesto Calende, Italy, 3Department of Veterinary Medicine, University of Bari “Aldo\nMoro”, Bari, Italy, 4Department of Bioscience, University of Camerino, Camerino, Italy\nMaintaining sperm quality during storage is essential for optimizing reproductive\nefficiency in rabbit breeding. This study investigated the effects of modulating\ncalcium metabolism and the endogenous opioid system on rabbit sperm\nfunction. Two commercial extenders (TRIS and Lepus) were supplemented\nwith calcium chloride (CaCl2) and naloxone (Nx), either individually or in\n\nThis study investigated the effects of modulating\ncalcium metabolism and the endogenous opioid system on rabbit sperm\nfunction.Two commercial extenders (TRIS and Lepus) were supplemented\nwith calcium chloride (CaCl2) and naloxone (Nx), either individually or in\ncombination. Semen was collected from 20 New Zealand White (NZW) males,\npooled, diluted, and assigned to eight experimental treatments. Sperm motility,\nkinematic parameters, vitality, and morphology were assessed after storage, and\ndata were analyzed by two-way ANOVA. The results showed that treatment\nwas the main factor affecting sperm quality parameters, whereas the effect of\ndiluent was significant only for selected variables. The results revealed significant\ndifferences among experimental groups, with naloxone-treated samples (alone\nor combined with CaCl2) showing the highest sperm quality. In particular,\ntotal motility (MOT) increased from approximately 56–71% in control and\nCaCl2 groups to about 77–85% in Nx-treated groups, while progressive motility\n(PMOT) improved from approximately 44–57% to 73–82%. Similarly, kinetic\nparameters (e.g., VAP and VCL) were markedly enhanced in Nx and Nx + CaCl2\ngroups (reaching ∼60–69 μm/s and ∼150–159 μm/s, respectively), whereas\nCaCl2 alone consistently reduced all motility-related parameters. Sperm vitality\nalso improved in Nx-containing treatments (up to ∼0.95–0.96), compared\nto lower values in CaCl2-only groups (∼0.77–0.92), while the percentage of\nabnormalities was reduced, particularly in TRIS-based and Nx-supplemented\nsamples (down to ∼2–4%). The combined Nx + CaCl2 treatments consistently\nshowed the best overall performance across most parameters. The synergistic\neffect observed in the Nx + CaCl2 groups suggests that naloxone may\nregulate Ca2+ influx and intracellular homeostasis, preventing detrimental\neffects associated with calcium overload. In conclusion, pharmacological\nmodulation of the opioid system, particularly through low concentrations of\nnaloxone, significantly improves rabbit semen quality and, when combined with\n\nIn conclusion, pharmacological\nmodulation of the opioid system, particularly through low concentrations of\nnaloxone, significantly improves rabbit semen quality and, when combined with\nCa2+, further enhances sperm functional parameters. These findings provide\nFrontiers in Veterinary Science\n01\nfrontiersin.org\n\nZappone et al.\n10.3389/fvets.2026.1830161\nnew perspectives for optimizing artificial insemination (AI) protocols in rabbit\nproduction systems.\nKEYWORDS\ncalcium ions, cooled semen, extender, ion homeostasis, naloxone, rabbit reproduction\n1 Introduction\nArtificial\ninsemination\n(AI)\nis\nthe\nmost\nwidely\nused\nreproductive biotechnology in farm animals and is essential\nfor improving genetic quality and production performance (1). In\nrabbits, AI has quickly become standard practice in commercial\nproduction systems, particularly for high-performance breeds such\nas New Zealand White (NZW) and Californian rabbits. These\nbreeds are selected for their fertility, semen quality, and ability\nto adapt to intensive farming conditions (2, 3). Currently, most\ncommercial rabbit farms use fresh or chilled semen as this method\nensures high sperm viability while remaining more cost-effective\nthan cryopreservation (4–6).\nSpermatozoa are highly specialized cells that have evolved\nto transfer the paternal genome to the oocyte. This process\nrequires a substantial amount of energy, primarily in the form\nof adenosine triphosphate (ATP), which supports key functions\nsuch as motility, capacitation, and acrosome reaction. These\nfunctions are all essential for fertilization (7). Among the factors\nregulating these processes, calcium ions (Ca2+) play a central\nrole in sperm physiology (8, 9). In human spermatozoa, low\nintracellular Ca2+ levels have been associated with reduced\npremature capacitation, thereby prolonging cell viability (10).\nConversely,\nhigh\nconcentrations\nmay\ninduce\nspontaneous\nacrosomal exocytosis and other detrimental effects (11). However,\nits role in mammals remains only partially understood. In\nmammalian spermatozoa, Ca2+ acts as a key regulator of multiple\nfunctional\nprocesses\nthrough\ntightly\ncontrolled\nspatial\nand\ntemporal signaling. Variations in intracellular Ca2+ concentration\nmodulate flagellar activity and are essential for the transition from\nprogressive motility (PMOT) to hyperactivation, mainly through\nCatSper channel-mediated influx and its interaction with cAMP-\ndependent signaling pathways (12–14). During capacitation, Ca2+\n\nDuring capacitation, Ca2+\nexerts a concentration-dependent regulatory role, coordinating\nintracellular signaling pathways, protein phosphorylation and\nmetabolic activity required for fertilization competence (15, 16).\nIn addition, the acrosome reaction depends on a Ca2+-driven\ncascade involving both intracellular store release and extracellular\ninflux (14).\nRecent evidence in mammals indicates that Ca2+ homeostasis\nis essential for maintaining sperm function during liquid storage,\nas ion channels and Ca2+-ATPases contribute to the regulation\nof intracellular Ca2+ balance and metabolic stability. Similarly,\ncontrolled calcium regulation has been shown to preserve sperm\nmetabolic activity and functionality during liquid storage in pig\nsemen, highlighting the importance of calcium balance rather than\nabsolute concentration (17).\nSeveral studies have investigated the practical effects of\ncalcium supplementation in semen extenders, demonstrating that\ncontrolled extracellular Ca2+ addition can improve sperm motility\nand viability, whereas unregulated Ca2+ influx may be detrimental.\nIn vitro experiments have shown that calcium salts such as CaCl2\ncan increase progressive motility (approximately 6–16%) and\nviability (4–11%), while calcium ionophores or channel antagonists\nsignificantly impair sperm function, highlighting the importance of\nregulated Ca2+ entry (18, 19). In addition, extender composition\nhas been shown to influence intracellular Ca2+ dynamics during\ncooling and thawing, with consequences on acrosome integrity and\nsperm survival (20). In practical applications, supplementation of\nextenders with CaCl2, in combination with metabolic modulators,\nhas improved fertility outcomes in swine by increasing post-thaw\nsperm functionality and reproductive performance (21). More\nrecent studies also confirm that Ca2+ homeostasis is essential\nfor maintaining sperm metabolic activity and functional integrity\nduring liquid storage in mammals, further supporting the relevance\nof calcium regulation in semen preservation (17).\nIn addition to Ca2+, increasing evidence indicates that the\n\nIn addition to Ca2+, increasing evidence indicates that the\nendogenous opioid system may modulate sperm physiology\nthrough mechanisms involving energy metabolism and motility\nregulation. However, the effects of opioids on sperm function are\ncontroversial, with conflicting results reported in different species,\nincluding humans and horses (22–24).\nThe presence of μ-, δ- and κ-opioid receptors has been\ndocumented in the spermatozoa of various species, including\nhumans, horses, pigs, sheep and fish (24–29). These receptors\nare mainly distributed across the acrosomal membrane, the\nequatorial region, the neck and the tail. These areas are\nfunctionally relevant for motility and the acrosome reaction (25,\n27, 29, 30). Activation of the μ-receptor by agonists such as\nmorphine has been shown to reduce sperm motility, an effect\nthat can be reversed by the antagonist naloxone (Nx) (31).\nImportantly, naloxone activity appears to be dose-dependent, as\nlow concentrations may enhance motility through μ-receptor\nantagonism, while higher concentrations may inhibit motility via\ninterference with δ-receptors (24). Despite these findings, the\napplication of naloxone in semen extender formulations remains\npoorly characterized. Although opioid receptors have been shown\nto influence sperm motility in different species, including boar and\nequine spermatozoa (24, 29), and naloxone has been evaluated\nunder post-thaw conditions, current evidence does not consistently\nsupport its beneficial effect on sperm preservation, suggesting that\nits action is species- and protocol-dependent.\nOpioid receptors can interact with calcium channels and\ninfluence the entry of calcium into cells, which in turn affects\nATP production and sperm motility (32, 33). This suggests\na possible interaction between the opioid system and calcium\nmetabolism in regulating sperm function. Based on this evidence,\nthe present study aimed to evaluate the effects of calcium chloride\n(CaCl2), naloxone (Nx), and their combination on rabbit sperm\nquality during liquid storage, using two different commercial\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n\nZappone et al.\n10.3389/fvets.2026.1830161\nextenders (TRIS and Lepus), in order to assess how modulation\nof calcium homeostasis and opioid signaling influences sperm\nmotility, kinematics, vitality, and morphology.\n2 Materials and methods\n2.1 Ethical approval\nAll treatments, as well as the housing and care of the animals,\nwere carried out in accordance with EU Directive 2010/63/EU\non the protection of animals used for scientific purposes. The\nEthics Committee of the Department of Veterinary Medicine at the\nUniversity of Bari “Aldo Moro”, Italy (protocol no. 5082 - III/13)\napproved the protocol and procedures.\n2.2 Animals and breeding conditions\nThis study was conducted on New Zealand White male rabbits\nthat were bred in a semi-intensive meat production facility in\nPoggio Imperiale, Foggia, Italy. Twenty clinically healthy breeding\nsubjects, aged between 8 and 16 months with an average weight of\napproximately 4 kg, were selected. The animals underwent regular\nveterinary checks and were kept under uniform environmental\nand feeding conditions throughout the experiment to minimize\nvariability in semen parameters between individuals.\n2.3 Collection of seminal material and\npreliminary assessments\nThe semen was collected using a standardized procedure\ninvolving an Amantea artificial vagina and constant temperature\nconditions. Immediately after collection, the total volume of\nthe ejaculate was measured using a graduated container. Sperm\nconcentration and motility parameters were assessed using\na\ncomputer-assisted\nsperm\nanalysis\nsystem\n(AndroScope\nR⃝,\nMinitube, Berlin, Germany) that was specifically calibrated for\nrabbits. The system was used with the standard calibration settings\nprovided by the manufacturer, and no additional adjustments\nor custom configurations were applied. This provided the sperm\nconcentration, expressed in millions of spermatozoa/ml, as well as\nthe percentage of total and progressive motility.\nThe pH of each sample was measured using a digital pH meter\n(HI9810402HAL02, Hanna Instruments, Italy).\nSperm vitality and morphology were analyzed through\ndifferential staining with eosin and nigrosin to determine the\n\nThe pH of each sample was measured using a digital pH meter\n(HI9810402HAL02, Hanna Instruments, Italy).Sperm vitality and morphology were analyzed through\ndifferential staining with eosin and nigrosin to determine the\npercentage of live, dead, and morphologically abnormal cells by\nexamining at least 200 spermatozoa per slide.\n2.4 Preparation of the semen pool\nAll ejaculates were collected from each animal at a single time\npoint and subsequently pooled to create a homogeneous sample\nrepresentative of the 20 donors, following standard procedures\ncommonly adopted in rabbit artificial insemination. A second\nquantitative and qualitative assessment was performed on this pool,\nthe results of which were comparable to the average values of\nthe individual samples. This confirmed the homogeneity of the\nmaterial used for subsequent experimental tests. The entire process\nof collection, preliminary evaluation, and pooling was performed\nwithin a short time frame and under controlled conditions to\nminimize potential alterations in sperm quality.\n2.5 Dilution and experimental treatments\nThe semen pool had a total volume of 13.5 ml. This was\ndivided into two equal aliquots of approximately 6.7 ml each. One\nof these was intended for use with one commercial diluent and\nthe other with a different one. The first aliquot was diluted with\nLepus diluent (MEDI Chimica, Reggio Emilia, Italy), characterized\nby a pH between 6.9 and 7.2. This diluent contains bicarbonate\n(0.005% in 50 kg of raw material) and is free of calcium in any\nform. The second was treated with a TRIS buffer solution prepared\naccording to the formulation described by Dal Bosco and Castellini\n(57). This consisted of Tris-amino-methane (3.028 g), citric acid\nmonohydrate (1.675 g), fructose (1.250 g), and sterile distilled water\nto make up 100 ml. The extender was subsequently supplemented\nwith 20% fresh egg yolk.\nEach extender sample was further divided into four treatment\nsamples, each containing approximately 1.6 ml of semen: a control\ncontaining only the diluent; a formulation containing Naloxone\n\nThe extender was subsequently supplemented\nwith 20% fresh egg yolk.Each extender sample was further divided into four treatment\nsamples, each containing approximately 1.6 ml of semen: a control\ncontaining only the diluent; a formulation containing Naloxone\n(Nx) at a concentration of 10−8 M selected based on previous\nstudies demonstrating beneficial effects of low-dose naloxone\non sperm motility through opioid receptor antagonism (24); a\nformulation containing calcium chloride (CaCl2) at a dose of\n16.615 mg per 100 ml; and a formulation containing a combination\nof Nx and CaCl2 at the same concentrations. The amount of\nCaCl2 used provided a total of approximately 6 mg of Ca2+\nions, corresponding to the physiological concentration present in\n100 ml of rabbit ejaculate. All samples were diluted at a ratio of\n1:20, resulting in a final sperm concentration of approximately\n18 × 106 spermatozoa/mL in each experimental group. This\nresulted in eight distinct experimental groups (Figure 1). For each\nexperimental group, three analytical replicates were evaluated. For\neach replicate, a 2 μL aliquot of seminal material was placed on a\nLeja chamber (SC 10-01-04-B, Leja, GN Nieuw-Vennep, NL), pre-\nwarmed on a laboratory heating plate, and analyzed with the aid\nof a Nikon Eclipse Ni phase contrast optical microscope, equipped\nwith a heated stage, 10 ×/0.25 Ph1 phase contrast objective, and\ncomputerized automatic semen analysis system (CASA) were used.\nAll semen parameters were therefore evaluated in triplicate for each\ntreatment combination.\n2.6 Storage and analysis of seminal material\nAfter collection, preliminary evaluation, and pooling, semen\nsamples\nwere\nplaced\nin\na\nportable\ntemperature-controlled\nrefrigerator to ensure controlled cooling conditions during\ntransport (cooled at an approximate rate of −0.5\n◦C/min,\nFrontiers in Veterinary Science\n03\nfrontiersin.org\n\nZappone et al.\n10.3389/fvets.2026.1830161\nFIGURE 1\nExperimental design.\nreaching +4 ◦C within 2 h). Upon arrival at the Andrology Clinic\nlaboratories at the Veterinary Hospital of the University of Bari\n“Aldo Moro”, samples were gently rewarmed in a water bath to\n37 ◦C prior to analysis.\nA computerized sperm analysis system [Computer Assisted\nSperm Analysis (CASA)] using IVOS-Sperm CASA version\n12.3 equipment (Hamilton Thorne, USA) was used to perform\nthe kinetic analyses. Using the rabbit-specific setup (software\nsettings: frame rate 60 Hz; minimum contrast 50; minimum\ndata points 7; path velocity threshold 100 μm/s−1, average\npath velocity (VAP) cut-off9.0 μm/s−1, medium VAP cut-\noff20 μm/s−1; straightness threshold 70%), the following\nparameters\nwere\nevaluated:\nsperm\nconcentration\n(CONC,\n106 ml−1), total motility (MOT, %; >5 μm/s), progressive\nmotility (PMOT, %; > 20 μm/s), VAP (average path velocity,\nμm/s), VCL (curvilinear velocity, μm/s), VSL (straight-line\nvelocity,\nμm/s),\nDAP\n(average\npath\ndistance,\nμm),\nDCL\n(curvilinear distance, μm), DSL (straight-line distance, μm),\nSTR\n(straightness,\nVSL/VAP),\nLIN\n(linearity,\nVSL/VCL),\nALH (amplitude of lateral head displacement), and BCF (beat\ncross frequency).\n2.7 Statistical analysis\nThe data distribution was preliminarily verified using the\nShapiro–Wilk normality test at a significance level of α =\n0.05. Data were considered normally distributed since the\nvalue of p was greater than α for all the variables that\nwere analyzed.\nDifferences among experimental conditions were analyzed\nusing a two-way ANOVA in a 2 × 4 factorial design, with\ndiluent (Lepus vs. TRIS) and treatment (control, Nx, CaCl2, and\nNx + CaCl2) as fixed factors, including their interaction. When\nsignificant main effects or interactions were detected (p < 0.05),\npairwise comparisons were performed using Tukey’s honestly\nsignificant difference (HSD) post hoc test. The results are expressed\nas mean ± standard deviation (SD), and effect size was estimated\nusing the η2 coefficient.\n3 Results\nThe semen parameters of the 20 male rabbits included in the\nstudy were all within the normal range for the species, as shown in\n(Table 1).\n\n3 Results\nThe semen parameters of the 20 male rabbits included in the\nstudy were all within the normal range for the species, as shown in\n(Table 1).\nThe semen pool obtained from the 20 breeding rabbits showed\nfavorable semen quality parameters, as indicated by high sperm\nmotility and vitality levels (Table 2).\n3.1 Effect of diluent and treatments on\nsperm quality\nTwo-way ANOVA showed that treatment was the main factor\naffecting all analyzed sperm quality parameters. In contrast, the\nmain effect of the diluent was significant only for certain variables,\nwhile the diluent × treatment interaction was significant only for\ncertain traits. This suggests that the effect of supplementation was\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n\nZappone et al.\n10.3389/fvets.2026.1830161\nTABLE 1 The semen parameters of the 20 male rabbits used in the study are reported below.\nRabbit\nVolume\n(ml)\npH\nSperm ×\n106/ml\nTotal\nmotility\n(%)\nProgressive\nmotility (%)\nNumber of\nalive (%)\nNumber of\ndead (%)\nAbnormalities\n(%)\n1\n0.6\n7\n324\n76\n63\n81\n14\n5\n2\n0.58\n7.5\n426\n64\n51\n72\n21\n7\n3\n1.2\n7.4\n253\n71\n63\n77\n17\n6\n4\n0.9\n7.3\n485\n65\n52\n71\n18\n11\n5\n0.49\n7.6\n368\n60\n54\n84\n14\n2\n6\n0.55\n7.4\n346\n62\n55\n81\n16\n3\n7\n0.78\n7.5\n397\n67\n56\n70\n20\n10\n8\n0.94\n7.5\n443\n69\n57\n87\n11\n2\n9\n1\n7.6\n286\n72\n68\n81\n18\n1\n10\n0.53\n7.6\n361\n75\n65\n70\n20\n10\n11\n0.74\n7.7\n352\n61\n55\n71\n19\n10\n12\n0.69\n7.4\n423\n67\n58\n80\n16\n4\n13\n0.48\n7.6\n486\n68\n57\n83\n16\n1\n14\n0.56\n7.5\n376\n82\n73\n79\n17\n4\n15\n0.62\n7.6\n314\n81\n71\n83\n14\n3\n16\n0.52\n7.4\n349\n64\n54\n71\n21\n8\n17\n0.55\n7.5\n374\n73\n62\n88\n8\n4\n18\n0.61\n7.6\n339\n71\n63\n80\n14\n6\n19\n0.53\n7.5\n358\n62\n54\n70\n22\n8\n20\n0.63\n7.7\n367\n79\n63\n81\n15\n4\nMedia\n0.67894737\n7.495\n371.35\n69.45\n59.7\n78\n16.55\n5.45\nSt. dev.\n0.19977473\n0.15719582\n59.4875263\n6.668662\n6.317061275\n6.01751829\n3.53143776\n3.18673236\nThe following parameters are reported: ejaculate volume (ml), pH, sperm concentration, total and progressive motility (%); Percentage of live and dead spermatozoa (%); and percentage of\nmorphological abnormalities. The mean values and standard deviations (SD) are shown at the bottom of the table.\nTABLE 2 The semen parameters of a pool obtained from 20 male breeding rabbits are reported.\nTotal volume\n13.5 ml\npH\n7.5\nSperm concentration\n368 × 106/ml\nTotal motility\n68%\nProgressive motility\n59%\nPercentage of live/dead spermatozoa\n78/16.55%\nAbnormalities\n5.4%\nThe\nfollowing\nare\nreported:\ntotal\nvolume;\npH;\nsperm\nconcentration;\ntotal\nand\nprogressive motility; percentage of live and dead spermatozoa; and percentage of\nmorphological abnormalities.\nconsistent across extenders, but depended on the specific parameter\n(Table 3).\nFor total motility (MOT%), the main effect of treatment was\nhighly significant (p < 0.05), whereas neither diluent (p > 0.05) nor\nthe interaction between diluent and treatment (p > 0.05) reached\nsignificance. Similarly, for progressive motility (PMOT%), only\ntreatment showed a significant effect (p < 0.05), while diluent (p\n> 0.05) and the interaction term (p > 0.05) were not significant. In\nboth extenders, samples supplemented with naloxone, either alone\nor in combination with CaCl2, showed the highest MOT% and\nPMOT% values, whereas CaCl2 alone significantly reduced both\nparameters compared with the respective controls.\nA similar pattern was observed for the kinetic parameters.\nTreatment significantly affected DAP (p < 0.05), DCL (p < 0.05),\nDSL (p < 0.05), VAP (p < 0.05), VCL (p < 0.05), and VSL (p\n< 0.05). The main effect of diluent was significant for DCL (p <\n0.05) and VAP (p < 0.05), but not for DAP (p > 0.05), DSL (p\n\nTreatment significantly affected DAP (p < 0.05), DCL (p < 0.05),\nDSL (p < 0.05), VAP (p < 0.05), VCL (p < 0.05), and VSL (p\n< 0.05).The main effect of diluent was significant for DCL (p <\n0.05) and VAP (p < 0.05), but not for DAP (p > 0.05), DSL (p\n> 0.05), VCL (p > 0.05), or VSL (p > 0.05). A significant diluent\n× treatment interaction was detected only for DAP (p < 0.05),\nwhereas the interaction was not significant for DCL (p > 0.05),\nDSL (p > 0.05), VAP (p > 0.05), VCL (p > 0.05), or VSL (p\n> 0.05). Overall, naloxone-containing treatments were associated\nwith higher kinetic values, whereas CaCl2 alone resulted in the\nlowest values in both extenders. In particular, the Nx + CaCl2\ngroups showed the highest values for several kinematic traits.\nRegarding trajectory indices, treatment significantly affected\nboth STR (p < 0.05) and LIN (p < 0.05). The main effect of diluent\nwas significant for STR (p > 0.05), but not for LIN (p > 0.05).\nMoreover, a significant diluent × treatment interaction was found\nfor both STR (p < 0.05) and LIN (p < 0.05), indicating that the\nFrontiers in Veterinary Science\n05\nfrontiersin.org\n\nZappone et al.\n10.3389/fvets.2026.1830161\nTABLE 3 Results for the different parameters expressed as the mean value in the 2 × 4 factorial arrangement [2 extenders (LEPUS and TRIS) × 4 treatments (diluent alone, diluent + Nx, Diluent + CaCl2; diluent + Nx + CaCl2)].\nParameter\nTreatment\ngroup\nDiluent\nDiluent + Nx\nDiluent +\nCaCl2\nDiluent + Nx +\nCaCl2\nTwo-way anova results\nFactor\np-value\nF-test\nη2\nValue\nInterpretation\nMOT%\nL\n70.93 (5.99)a\n77.07 (1.27)b\n56.37 (1.02)c\n84.58 (1.19)b\nGroup\np > 0.05\n2.2609\n0.12\nMedium\nTreatments\np < 0.05\n242.6258\n0.97\nLarge\nT\n71.2 (1.05)a\n80.83 (1.72)b\n57.30 (0.95)c\n85.48 (0.81)b\nInteraction\np > 0.05\n0.6435\n0.11\nMedim\nPMOT%\nL\n57.37 (0.76)a\n73.37 (1.46)b\n44.67 (1.96)c\n81.77 (1.36)d\nGroup\np > 0.05\n0.2539\n0.16\nMedium\nTreatments\np < 0.05\n364.88\n0.99\nLarge\nT\n57.07 (4.77)a\n75.53 (0.86)b\n44.27 (1.96)c\n82.08 (1.35)d\nInteraction\np > 0.05\n0.4523\n0.07\nMedium\nDAP\nL\n24.53 (1.59)a\n31.20 (0.78)b\n17.23 (3.25)c\n29.00 (2.23)a\nGroup\np > 0.05\n3.099\n0.16\nLarge\nTreatments\np < 0.05\n70,653\n0.93\nLarge\nT\n32.00 (1.00)b\n29.20 (1.25)b\n15.87 (1.16)c\n30.0 (2.51)b\nInteraction\np < 0.05\n7.4654\n0.58\nLarge\nDCL\nL\n40.53 (0.75)a\n53.97 (1.47)b\n31.2 (1.40)c\n70.23 (0.91)d\nGroup\np < 0.05\n9.8713\n0.38\nLarge\nTreatments\np < 0.05\n331.64\n0.98\nLarge\nT\n42.53 (1.40)a\n58.07 (1.50)b\n37.07 (2.70)ac\n69.16 (4.36)d\nInteraction\np > 0.05\n2.9676\n0.58\nLarge\nDSL\nL\n16.47 (0.83)a\n20.27 (1.19)ac\n11.47 (2.14)b\n21.80 (2.88)c\nGroup\np > 0.05\n0.5264\n0.03\nSmall\nTreatments\np < 0.05\n43.90\n0.89\nLarge\nT\n15.27 (0.90)a\n19.73 (0.47)ac\n12.33 (0.91)b\n20.80 (1.73)c\nInteraction\np > 0.05\n0.5246\n0.09\nMedium\nVAP\nL\n51.47 (0.74)a\n54.00 (1.49)a\n29.60 (2.56)b\n68.73 (0.92)d\nGroup\np < 0.05\n6.0082\n0.27\nLarge\nTreatments\np < 0.05\n124.6478\n0.96\nLarge\nT\n54.03 (0.70)a\n59.67 (0.91)ad\n34.43 (5.12)c\n69.17 (7.30)d\nInteraction\np > 0.05\n0.7334\n0.12\nMedium\nVCL\nL\n98.23 (0.85)a\n116.13 (3.73)a\n61.10 (14.48)b\n152.53 (8.90)c\nGroup\np > 0.05\n0.0585\n0.003\nNegligible\nTreatments\np < 0.05\n164.2211\n0.97\nLarge\nT\n105.90 (3.84)a\n111.43 (11.75)a\n54.73 (3.43)b\n158.90 (2.16)c\nInteraction\np > 0.05\n1.3515\n0.2\nLarge\nVSL\nL\n37.27 (0.86)ab\n42.80 (1.49)ac\n19.07 (0.153)b\n53.00 (8.86)c\nGroup\np > 0.05\n3.7384\n0.19\nLarge\nTreatments\np < 0.05\n67.1239\n0.93\nLarge\nT\n40.00 (2.19)a\n14.47 (2.25)a\n26.17 (4.86)b\n57.10 (3.18)c\nInteraction\np > 0.05\n1.1547\n0.18\nLarge\nSTR\nL\n0.68 (0.02)a\n0.67 (0.02)a\n0.54 (0.06)b\n0.72 (0.05)a\nGroup\np < 0.05\n6.135\n0.28\nLarge\n(Continued)\nFrontiers in Veterinary Science\n06\nfrontiersin.org\n\nZappone et al.\n10.3389/fvets.2026.1830161\nTABLE 3 (Continued)\nParameter\nTreatment\ngroup\nDiluent\nDiluent + Nx\nDiluent +\nCaCl2\nDiluent + Nx +\nCaCl2\nTwo-way anova results\nFactor\np-value\nF-test\nη2\nValue\nInterpretation\nTreatments\np < 0.05\n15.6922\n0.75\nLarge\nT\n0.68 (0.03)a\n0.69 (0.01)a\n0.65 (0.03)a\n0.72 (0.04)a\nInteraction\np < 0.05\n3.7603\n0.42\nLarge\nLIN\nL\n0.37 (0.01)a\n0.33 (0.01)a\n0.28 (0.01)b\n0.33 (0.07)a\nGroup\np > 0.05\n0.0059\n0.00037\nNegligible\nTreatments\np < 0.05\n8.9321\n0.63\nLarge\nT\n0.28 (0.01)b\n0.35 (0.03)a\n0.28 (0.01)b\n0.38 (0.01)a\nInteraction\np < 0.05\n7.8623\n0.60\nLarge\nALH\nL\n3.83 (0.35)a\n4.03 (0.21)ab\n3.13 (0.61)ac\n4.97 (1.01)b\nGroup\np > 0.05\n2.1576\n0.16\nLarge\nTreatments\np < 0.05\n16.0174\n0.75\nLarge\nT\n4.53 (0.25)b\n4.59 (0.60)ab\n2.90 (0.45)ac\n5.20 (0.10)b\nInteraction\np > 0.05\n0.9479\n0.12\nMedium\nBSF\nL\n22.47 (0.49)a\n27.53 (0.70)a\n19.73 (2.76)a\n29.97 (2.36)b\nGroup\np < 0.05\n21.2636\n0.57\nLarge\nTreatments\np < 0.05\n41.9133\n0.88\nLarge\nT\n31.43 (0.40)b\n30.50 (1.75)a\n19.93 (1.54)a\n31.47 (2.58)b\nInteraction\np < 0.05\n6.868\n0.56\nLarge\nVitality\nL\n0.87 (0.01)a\n0.90 (0.01)a\n0.77 (0.02)c\n0.90 (0.01)a\nGroup\np < 0.05\n160.7424\n0.91\nLarge\nTreatments\np < 0.05\n32.6616\n0.86\nLarge\nT\n0.94 (0.01)b\n0.95 (0.01)b\n0.92 (0.04)b\n0.96 (0.01)b\nInteraction\np < 0.05\n11.5707\n0.68\nLarge\nAbnormalities\nL\n8.33 (0.58)a\n8.00 (1.00)a\n7.67 (1.53)a\n6.00 (0.58)ac\nGroup\np < 0.05\n129.94\n0.89\nLarge\nTreatments\np < 0.05\n14.6471\n0.73\nLarge\nT\n1.67 (0.58)b\n4.33 (0.58)c\n6.33 (0.8)ac\n2 (0.22)b\nInteraction\np < 0.05\n10.098\n0.65\nLarge\nStatistical analysis summary for the two-way Anova reports p-value, F-test and η2. Different superscripts denote the statistical differences obtained by the post-hoc test through pairwise comparisons within each parameters. The following parameters were evaluated:\ntotal motile spermatozoa (%, MOT > 5 μm/s) and progressive motile spermatozoa (%, PMOT > 20 μm/s), velocity of average path (VAP, μm/s), velocity of curved line (VCL, μm/s), velocity of straight line (VSL, μm/s), distance of average path (DAP, μm), distance\nof curved line (DCL, μm), distance of straight line (DSL, μm), straightness (STR, VSL:VAP ratio), linearity (LIN, VSL:VCL ratio), ALH (lateral head displacement) and beat cross frequency (BCF). Different lowercase superscript letters within the same row indicate\n\nDifferent lowercase superscript letters within the same row indicate\nstatistically significant differences among treatment groups (p < 0.05), whereas values sharing the same letter are not significantly different.\nFrontiers in Veterinary Science\n07\nfrontiersin.org\n\nZappone et al.\n10.3389/fvets.2026.1830161\neffect of supplementation on sperm trajectory partly depended on\nthe extender used. In general, the lowest STR and LIN values were\nobserved in CaCl2-treated samples, whereas naloxone, particularly\nwhen combined with CaCl2, maintained values comparable to or\nhigher than those of the corresponding controls.\nFor ALH, treatment had a significant effect (p < 0.05), whereas\nneither diluent (p > 0.05) nor the interaction term (p > 0.05) was\nsignificant. By contrast, BSF was significantly affected by diluent (p\n< 0.05), treatment (p < 0.05), and their interaction (p < 0.05). In\ngeneral, naloxone-containing treatments, especially Nx + CaCl2,\nshowed higher ALH and BSF values, whereas CaCl2 alone was\nassociated with lower values.\nSperm vitality was significantly influenced by diluent (p < 0.05),\ntreatment (p < 0.05), and their interaction (p < 0.05). In general,\nTRIS-based samples showed higher vitality values than LEPUS-\nbased samples, and naloxone-containing treatments yielded the\nbest overall results. The lowest vitality was observed in the LEPUS\n+ CaCl2 group, whereas the highest value was recorded in the TRIS\n+ Nx + CaCl2 group.\nSimilarly, sperm abnormalities were significantly affected by\ndiluent, treatment, and their interaction, as shown in Table 3.\nOverall, TRIS-based groups showed lower abnormality rates\nthan LEPUS-based groups, and naloxone supplementation was\nassociated with reduced abnormalities, whereas higher values\nwere generally observed in LEPUS-based samples and in CaCl2-\ntreated groups.\nEffect size analysis confirmed that treatment was the main\nsource of explained variance for all analyzed parameters, with\nconsistently large η2-values. By contrast, the contribution of diluent\nand of the interaction term varied according to the trait considered,\nranging from negligible or small effects for some variables to large\neffects for DAP, STR, LIN, BSF, vitality, and abnormalities.\n4 Discussion\nThis study evaluated the effects of calcium chloride (CaCl2),\nnaloxone (Nx), and their combination on rabbit sperm quality\nduring short-term storage in two different extenders, TRIS and\n\n4 Discussion\nThis study evaluated the effects of calcium chloride (CaCl2),\nnaloxone (Nx), and their combination on rabbit sperm quality\nduring short-term storage in two different extenders, TRIS and\nLepus. The two-way ANOVA confirmed that treatment was the\nmain source of variation for all analyzed sperm quality parameters,\nwhereas the effect of diluent was significant only for selected\nvariables and the interaction between diluent and treatment\nemerged only for specific traits.\nThe success of artificial insemination largely depends on the\nuse of appropriate sperm extenders, which are designed to preserve\nthe vitality and fertilizing capacity of sperm during storage. Such\nextenders typically contain organic buffers such as Tris, TES or\nsodium citrate to stabilize the pH within the physiological range of\n6.8–7.2; sugars such as glucose or fructose to provide energy; solutes\nto control osmotic pressure; and antibiotics to restrict bacterial\ngrowth. Numerous studies have demonstrated that adding specific\nadditives can enhance structural stability and sperm function in\nvarious species. In rabbits in particular, the use of molecules\nsuch as heparin and buserelin has had a positive effect on semen\nquality (34–40).\nIn this context, ionic homeostasis, particularly that of Ca2+,\nplays a fundamental role in maintaining sperm function during\nsemen storage. Several ion channels and pumps, including voltage-\ndependent anion channels (VDACs) and sarco/endoplasmic\nreticulum\nCa2+-ATPase\n(SERCA),\nhave\nbeen\nidentified\nas\nkey regulators of the balance of calcium ions inside cells,\nthereby contributing to the metabolic and functional stability of\nspermatozoa (41, 42). Studies in horses have demonstrated that\nadding Ca2+ chelating agents to freezing media can significantly\nenhance sperm cryotolerance (43). In pigs, Ca2+ has been shown\nto regulate intracellular ATP levels and protein phosphorylation,\nthereby directly influencing sperm motility (44).\nThe results of this study confirm that, while calcium (Ca2+)\nis essential for sperm physiology, an uncontrolled increase in\nits extracellular availability can be detrimental. Adding CaCl2\n\nThe results of this study confirm that, while calcium (Ca2+)\nis essential for sperm physiology, an uncontrolled increase in\nits extracellular availability can be detrimental.Adding CaCl2\nalone led to a significant decrease in motility, kinematic and\nvitality parameters, likely due to excessive intracellular Ca2+ levels.\nThis condition is known to induce increased ATP consumption,\npremature capacitation and, in some cases, spontaneous acrosome\nreaction triggering, resulting in reduced sperm longevity and\nfunction (8–11). These findings are also consistent with recent\nobservations in other species, such as poultry, where modulation of\ncalcium metabolism has been shown to be crucial for maintaining\nfertility during semen storage (12). Another key regulator of Ca2+\nflow is the CatSper channel, which is the primary calcium channel\nfound in spermatozoa. First identified in mice and humans in\nthe early 2000s (45, 46), the CatSper channel consists of four\nmain subunits (CatSper1–4) and several accessory subunits and\nhas since been found in numerous vertebrate species. It plays a\ncrucial role in the processes of capacitation and hyperactivation,\nwhich are essential for spermatozoa to acquire fertilization\ncompetence (47–49).\nIn this complex context of ionic regulation, the endogenous\nopioid system acts as an additional modulator of sperm function.\nAsthenozoospermia, a condition characterized by impaired sperm\nmotility, is frequently encountered in individuals with opioid\ndependence, suggesting an inhibitory effect of opioids on sperm\nmotility (50). Furthermore, males with reduced sperm motility have\nbeen observed to exhibit decreased activity of aminopeptidase N,\nan enzyme involved in the metabolism of enkephalins. Enkephalin\nexpression in male germ cells has also been widely documented\n(51, 52). These findings collectively support the hypothesis of direct\ninvolvement of the opioid system in reproductive processes.\nOne of the main findings of the present study was the beneficial\neffect of naloxone on sperm quality in both extenders. Samples\nsupplemented with naloxone, either alone or in combination with\n\nOne of the main findings of the present study was the beneficial\neffect of naloxone on sperm quality in both extenders.Samples\nsupplemented with naloxone, either alone or in combination with\nCaCl2, showed the highest values for total motility, progressive\nmotility and most kinematic parameters. The two-way ANOVA\ndemonstrated that treatment had a significant effect on MOT%\nand PMOT%, whereas neither the main effect of diluent nor\nthe interaction term was significant for these two variables. This\nindicates that the positive action of naloxone on the main motility\ntraits was consistent across both extenders.\nThe increase in MOT% and PMOT%, together with the\nimprovement observed in several kinetic parameters, suggests that\nantagonism of opioid receptors, particularly μ-receptors, may\nremove a physiological inhibitory mechanism affecting sperm\nFrontiers in Veterinary Science\n08\nfrontiersin.org\n\nZappone et al.\n10.3389/fvets.2026.1830161\nmotility. These results are in agreement with findings reported\nin humans, horses and pigs, where opioid receptor activation\nhas been associated with reduced sperm motility, while naloxone\nadministration has been shown to restore or improve movement\n(24, 29, 31, 52).\nThe localization of μ-, δ- and κ-opioid receptors in the\nacrosomal membrane, the neck and the flagellum provide further\nsupport for the hypothesis that the opioid system directly\ncontributes to the controls sperm motility (25, 27). Therefore,\nthe observed effect of naloxone may be interpreted as the\nresult of competitive antagonism of endogenous opioids, which\nallows for more efficient restoration of energy processes and\nflagellar movement.\nAnother relevant findings of this study was the observed\nsynergistic effect in groups treated with a combination of naloxone\nand CaCl2. The motility, kinematics and vitality parameters in these\nsamples were higher than in the control group and exceeded those\nin the naloxone-only group. These results suggest that naloxone\nmay modulate the entry and intracellular distribution of Ca2+,\nthereby preventing the cytosolic peaks that are responsible for\nthe negative effects observed in samples treated with CaCl2 alone.\nPrevious studies have shown that opioid receptors can interact\nwith L-type calcium channels and intracellular signaling pathways\ninvolved in energy metabolism, such as protein kinase C (PKC)\n(23, 24), supporting this hypothesis. In this context, naloxone could\nhelp to maintain balanced Ca2+ turnover, thereby preserving ATP\nproduction and supporting flagellar activity, even when there is\nincreased availability of extracellular Ca2+.\nPrevious studies have demonstrated that diluent composition\nsignificantly impacts sperm motility, membrane integrity, and DNA\nintegrity (6). A comparison of the two extenders showed that the\nmain effect of diluent was more limited than that of treatment,\nbut remained significant for selected parameters, including DCL,\nVAP, STR, BSF, vitality and abnormalities. In general, TRIS-\nbased samples showed higher vitality and lower abnormality rates\n\nIn general, TRIS-\nbased samples showed higher vitality and lower abnormality rates\nthan Lepus-based samples, suggesting that the composition of the\nextender contributed to sperm protection during storage. However,\nbecause the diluent effect was not significant for several of the\nmajor motility and kinematic variables, the present data indicate\nthat extender composition played a secondary role compared with\nthe supplementation. Therefore, it would be more appropriate to\nstate that TRIS performed better than Lepus only for selected traits,\nrather than concluding that it was globally superior for all sperm\nquality parameters. The effect size analysis further supports this\ninterpretation. Across all analyzed variables, η²-values associated\nwith treatment were consistently large, indicating that treatment\nexplained the highest proportion of variance. By contrast, the\ncontribution of diluent and of the interaction term varied according\nto the trait considered, ranging from negligible or small effects\nfor some variables to large effects for DAP, STR, LIN, BSF, vitality\nand abnormalities.\nPrevious\nstudies\nhave\nshown\nthat\nnaloxone\nactivity\nis\ndose-dependent,\nwith\ndifferent\neffects\nreported\nat\nvarying\nconcentrations. However, in the present study, naloxone was used\nat a single concentration (10−8 M), and all analyses were performed\non refrigerated semen samples (4 ◦C). The results of this study\nsuggest that pharmacological modulation of the opioid system,\nthrough the use of naloxone at the tested concentration, may be\na promising strategy for improving the quality of refrigerated\nsemen intended for artificial insemination in rabbits. The observed\nsynergistic effect with Ca2+ also indicates that optimizing the\nionic and metabolic balance of spermatozoa through a combined\napproach could improve reproductive efficiency in intensive\nbreeding systems.\nThe present findings should also be interpreted considering\nthe storage conditions. All semen samples were refrigerated at\n+4 ◦C, and chilled storage is known to alter sperm metabolic\nactivity, membrane fluidity and ion transport mechanisms. Cooling\n\nThe present findings should also be interpreted considering\nthe storage conditions.All semen samples were refrigerated at\n+4 ◦C, and chilled storage is known to alter sperm metabolic\nactivity, membrane fluidity and ion transport mechanisms.Cooling\nis known to reduce sperm metabolic rate, leading to decreased\nATP production and a general slowdown of cellular processes,\nas demonstrated in several species including rabbits (53). This\nreduction in metabolic activity is associated with changes in\nmotility and energy availability, which are closely linked to\nintracellular ATP levels (54). In addition, chilled storage can\ninduce alterations in sperm membrane composition and fluidity,\nincreasing susceptibility to cold-shock damage and oxidative stress\n(55, 56). These temperature-dependent changes may affect ion\ntransport mechanisms, including calcium fluxes, and consequently\ninfluence sperm motility regulation and functional stability. Under\nthese conditions, the effects of calcium supplementation and opioid\nreceptor modulation may be partially dependent on the reduced\nmetabolic and functional state of the spermatozoa. In particular,\nthe lower metabolic demand at reduced temperatures may mitigate\nthe detrimental effects of Ca2+ overload, while enhancing the\nregulatory role of naloxone on calcium homeostasis and energy\nbalance. Therefore, the observed treatment effects should be\ninterpreted in the context of refrigerated storage, which may have\ncontributed to stabilizing sperm function and modulating the\ninteraction between calcium dynamics and the opioid system.\nThis study demonstrates that naloxone significantly improves\nrabbit sperm quality by enhancing motility, kinematics and vitality,\nwhile reducing abnormalities. While the addition of Ca2+ can be\ndetrimental if not properly modulated, combining it with naloxone\nhelps to further enhance sperm function. These results support\nthe hypothesis of a close functional interaction between the opioid\nsystem and calcium metabolism in spermatozoa, offering new\ninsights into the optimisation of artificial insemination protocols\nin rabbits.\n5 Conclusion\n\nThese results support\nthe hypothesis of a close functional interaction between the opioid\nsystem and calcium metabolism in spermatozoa, offering new\ninsights into the optimisation of artificial insemination protocols\nin rabbits.5 Conclusion\nThis study demonstrates that modulation of the endogenous\nopioid system through naloxone significantly improves rabbit\nsemen quality during short-term storage, enhancing motility,\nkinematics, and vitality, while reducing sperm abnormalities. In\ncontrast, calcium supplementation alone negatively affected sperm\nperformance, whereas its combination with naloxone resulted in\na synergistic effect, leading to the highest overall sperm quality.\nFrom an applied perspective, the addition of naloxone at the\ntested concentration (10−8 M) may represent a practical strategy\nto improve the quality of refrigerated semen used in artificial\ninsemination protocols in rabbits.\nFrontiers in Veterinary Science\n09\nfrontiersin.org\n\nZappone et al.\n10.3389/fvets.2026.1830161\nHowever, this study has some limitations, including the in\nvitro experimental design and the absence of direct fertility\noutcomes. Therefore, further studies are needed to evaluate in vivo\nreproductive performance and to better elucidate the molecular\nmechanisms underlying the interaction between opioid receptors\nand calcium metabolism. Nevertheless, these results must be\nvalidated in vivo fertility tests, and the molecular mechanisms\ninvolved must be investigated, particularly the interaction between\nopioid receptors, calcium channels and mitochondrial energy\nmetabolism. Future studies will also need to evaluate the\nmedium- to long-term effects and those under conditions of\nprolonged storage.\nData availability statement\nThe original contributions presented in the study are included\nin the article/supplementary material, further inquiries can be\ndirected to the corresponding author.\nEthics statement\nThe animal studies were approved by Department of Veterinary\nMedicine at the University of Bari Aldo Moro, Italy (protocol no.\n5082 - III/13). The studies were conducted in accordance with the\nlocal legislation and institutional requirements. Written informed\nconsent was obtained from the owners for the participation of their\nanimals in this study.\nAuthor contributions\nVZ: Investigation, Methodology, Writing – original draft,\nWriting – review & editing. SA: Formal analysis, Methodology,\nWriting – review & editing. RA: Investigation, Methodology,\nWriting\n–\noriginal\ndraft.\nAT:\nMethodology,\nSupervision,\nValidation, Writing – review & editing. RM: Investigation,\nMethodology, Writing – original draft. MQ: Methodology,\nSupervision, Writing – review & editing. GA: Conceptualization,\nMethodology, Supervision, Writing – review & editing.\nFunding\nThe author(s) declare that financial support was received for\npublication of this article. The publication costs were funded by the\nUniversity of Messina through the APC initiative.\nAcknowledgments\nThe authors acknowledge support from the University of\nMessina through the APC initiative.\nConflict of interest\nSA is employed by Società Veterinaria “Il Melograno” Srl.\n\nAcknowledgments\nThe authors acknowledge support from the University of\nMessina through the APC initiative.Conflict of interest\nSA is employed by Società Veterinaria “Il Melograno” Srl.\nThe remaining author(s) declared that this work was conducted\nin the absence of any commercial or financial relationships that\ncould be construed as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declared that generative AI was not used in the\ncreation of this manuscript.\nAny alternative text (alt text) provided alongside figures in\nthis article has been generated by Frontiers with the support of\nartificial intelligence and reasonable efforts have been made to\nensure accuracy, including review by the authors wherever possible.\nIf you identify any issues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. 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+{"text": "【兽医文献】\n标题: Usefulness of cerebrospinal fluid analysis in dogs and cats with suspected intracranial disease and normal magnetic resonance imaging\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nExploring potential cytokine\nprofiles as diagnostic biomarkers\nfor brucellosis in Mediterranean\nBuffaloes\nGiulia Franzoni 1*, Federica Signorelli 2, Anna Donniacuo 3,\nLorena Schiavo 3, Michele Napoletano 3, Giovanna De Matteis 2,\nFrancesco Grandoni 2, Susanna Zinellu 1, Vincenzo Bove 3,\nSilvia Dei Giudici 1, Esterina De Carlo 3, Giorgio Galiero 3,\nFrancesco Napolitano 2 and Alessandra Martucciello 3\n1 Department of Animal Health, Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Italy,\n2 Council for Agricultural Research and Economics (CREA)- Research Centre for Animal Production\nand Aquaculture, Monterotondo (RM), Italy, 3 National Reference Centre for Hygiene and Technologies\nof Mediterranean Buffalo Farming and Productions, Istituto Zooprofilattico Sperimentale del\nMezzogiorno, Salerno, Italy\nBrucellosis is a zoonotic disease, with an important economic impact on the\nlivestock industry and public health worldwide. Both Brucella abortus and Brucella\nmelitensis can infect Mediterranean Buffalo (Bubalus bubalis), leading to infertility and\nabortion. In ruminants, the standard diagnostic approach involves two serological\ntests, the Rose Bengal Test and the Complement Fixation Test, applied in parallel,\nthough their specificity requires improvement. Cytokines play a crucial role in\ncoordinating immune responses through complex networks and can serve as\nbiomarkers for various diseases. This study explored the potential use of cytokines\nas immunological biomarkers for Brucella infection in Mediterranean Buffalo.\nFor this purpose, we  included 18 healthy and 20 Brucella-infected buffaloes\nin our analysis. Heparinized blood samples were stimulated with the Brucella\nantigen, with PBS as nil control and PWM as lymphocyte viability control. After\n16–24 h, plasma levels of IL-1α, IL-1β, IL-4, IL-6, IL-10, IL-17, IL-36Ra, MIP-1α,\nMIP-1β, MCP-1, CXCL8, IP-10, IFN-γ, TNF, and VEGF-A were measured using\nmultiplex ELISA. Our results showed that infected animals released significantly\n\nAfter\n16–24 h, plasma levels of IL-1α, IL-1β, IL-4, IL-6, IL-10, IL-17, IL-36Ra, MIP-1α,\nMIP-1β, MCP-1, CXCL8, IP-10, IFN-γ, TNF, and VEGF-A were measured using\nmultiplex ELISA.Our results showed that infected animals released significantly\nhigher levels of IFN-γ, IP-10, MCP-1 in response to Brucella antigen compared\nto healthy controls. Conversely, healthy animals released instead higher levels\nof IL-1α, IL-1β, IL-6 and IL-10 following antigen stimulation compared to infected\nanimals. Finally, sequential canonical discriminant analyses were performed to\ngenerate predictive cytokine profiles for each group. The findings indicated that\na combination of five cytokines (IFN-γ, IP-10, IL-1α, IL-1β, IL-6) can effectively\ndistinguished infected from healthy buffaloes. Overall, this study suggests that\nincorporating these key immune cytokines could improve the diagnostic accuracy\nof brucellosis in Mediterranean Buffalo.\nKEYWORDS\nBrucella, biomarkers, IFN-γ, cytokines, Mediterranean Buffaloes\nOPEN ACCESS\nEDITED BY\nCamila Hamond,\nUniversity of Connecticut, United States\nREVIEWED BY\nMirinda Van Kleef,\nAgricultural Research Council of South Africa\n(ARC-SA), South Africa\nAman Ullah Khan,\nUniversity of Veterinary and Animal Sciences,\nPakistan\n*CORRESPONDENCE\nGiulia Franzoni\ngiulia.franzoni@izs-sardegna.it\nRECEIVED 26 February 2025\nACCEPTED 11 April 2025\nPUBLISHED 08 May 2025\nCITATION\nFranzoni G, Signorelli F, Donniacuo A,\nSchiavo L, Napoletano M, De Matteis G,\nGrandoni F, Zinellu S, Bove V, Dei Giudici S,\nDe Carlo E, Galiero G, Napolitano F and\nMartucciello A (2025) Exploring potential\ncytokine profiles as diagnostic biomarkers for\nbrucellosis in Mediterranean Buffaloes.\nFront. Vet. Sci. 12:1583858.\ndoi: 10.3389/fvets.2025.1583858\nCOPYRIGHT\n© 2025 Franzoni, Signorelli, Donniacuo,\nSchiavo, Napoletano, De Matteis, Grandoni,\nZinellu, Bove, Dei Giudici, De Carlo, Galiero,\nNapolitano and Martucciello. This is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY). The use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\n\nThis is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY).The use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\naccordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Original Research\nPUBLISHED  08 May 2025\nDOI  10.3389/fvets.2025.1583858\n\nFranzoni et al.\b\n10.3389/fvets.2025.1583858\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n1 Introduction\nBrucellosis is a zoonotic disease caused by bacteria belonging to\nthe genus Brucella (1). This disease has significant economic impact\nfor the livestock industry and pose a serious public health concern.\nBrucella are Gram-negative coccobacilli and intracellular\npathogens capable of surviving and replicating within macrophages (1).\nBoth Brucella abortus (B. abortus) and Brucella melitensis (B. melitensis)\ncan infect buffalo species (Bubalus bubalis) (2). In Italy, B. abortus is the\nmost prevalent Brucella species affecting cattle and buffaloes, with\nseveral outbreaks reported in recent years, particularly in the province\nof Caserta (3). In Mediterranean Buffaloes, the infection leads to\ninfertility and abortion, resulting in considerable economic losses (1).\nIn humans, B. abortus infection causes a severe, chronic and debilitating\ndisease (4), primarily transmitted through contaminated milk and raw\ndairy products (5) or direct contact with infected animals (6).\nItaly is currently running a compulsory eradication and surveillance\nprogram for brucellosis in cattle and Mediterranean Buffaloes. In\nregions not yet declared free of the disease, surveillance is conducted\nthrough serological testing of all animals over 1 year old (7). To enhance\nspecificity, at least two diagnostic tests should be used in parallel (8).\nA widely accepted diagnostic approach combines the Rose Bengal\nTest (RBT) with the Complement Fixation Test (CFT) (8). Nevertheless,\nthese tests present some limitations, particularly in cattle and buffaloes,\ndue to their low specificity. False-positive serological reactions (FPSR)\nmight result from exposure to cross reacting microorganisms (9). Both\nRBT and CFT can detect antibodies produced to the S-LPS and false\npositive results might occur in animals exposed to Gram-negative\nbacteria with LPS O-chains similar to those of brucellae. These bacteria\ninclude Escherichia coli O:157, Salmonella group N (O:30), and Yersinia\n\nThese bacteria\ninclude Escherichia coli O:157, Salmonella group N (O:30), and Yersinia\nenterocolitica O:9. Notably, Y. enterocolitica O:9 is a major cause of\nFPSR in diagnosis of brucellosis in bovine and buffaloes (9, 10).\nThe lack of Brucella specific antigen highlights the need to develop\nnew diagnostic methods, in order to improve the efficacy of\neradication strategies and to avoid un-necessary animal sacrifices. In\naddition, in Italy Mediterranean buffaloes are regarded as a national\nlivestock heritage (7, 11). Accurate diagnosis is essential for an\neffective surveillance in brucellosis-free areas and for achieving the\nfinal stages of eradication (11). Advancing our understanding of host\nimmune responses to Brucella is crucial for developing improved\ndiagnostic strategies and ultimately eliminating the disease from\naffected infected regions.\nIn our previous studies, we demonstrated the effectiveness of the\ninterferon-gamma (IFN-γ) assay test for diagnosing Mycobacterium\nbovis (M. bovis) infection in Mediterranean Buffaloes (12, 13). More\nrecently, we  identified additional cytokines that could enhance\nM. bovis diagnostic accuracy in this species (14). Building on this\nknowledge, we investigated the potential of key immune cytokines as\ndiagnostic\nbiomarkers\nfor\nBrucella\ninfection\nin\nMediterranean Buffaloes.\n2 Materials and methods\n2.1 Ethical statements\nMediterranean Buffaloes used in this study were analyzed\nwithin\nthe\ncontext\nof\nNational\nand\nRegional\nbuffalo\nbrucellosis-surveillance program, provided by the EU Delegates\nRegulations 2020/689 and Campania Region Regional Regulation\n(DGRC 104/2022) (15, 16).\nNo animal was harmed or killed for the specific purpose of this\nstudy and the experimental procedure was carried out in compliance\nwith the European Directive 210/63/UE and the Italian regulation D\nLgs n26/2014.\n2.2 Animals and study design\nThirty-eight Mediterranean Buffaloes were enrolled in the study\nand were divided in two groups: healthy (N = 18) and infected\n(N = 20).\nInfected animals were selected from herds with confirmed\nbrucellosis outbreaks. Infection status was determined according to\n\nInfected animals were selected from herds with confirmed\nbrucellosis outbreaks.Infection status was determined according to\ncurrent legislation using serological tests RBT and CFT, following the\nguidelines outlined in the WOAH Manual of Diagnostic Tests and\nVaccines for Terrestrial Animals (8). RBT is used as a screening test\n(carried out every 6 months in all herds of Campania region), while\nCFT is used to confirm RBT positive cases. If an animal tests positive\non CFT, the herd is classified as a brucellosis outbreak, then it is\nsubsequently monitored every 21 days using both RBT and CFT (8).\nSeropositive animals were culled in compliance with national and\nregional regulations. Brucella presence in selected organs was assessed\nthrough PCR and culture isolation (see Section 2.3). Animals included\nin the infected group were seropositive, with Brucella DNA detected\nin target organs (Supplementary Table S1). Brucella was isolated from\nat least one animal per outbreak.\nHealthy animals were selected from Officially Brucellosis-Free\n(OTF) herds in the Campania region (Southern Italy). These animals\ntested negative during the annual serological screening tests\nperformed in the last 6 years (Supplementary Table S1).\n2.3 PCR and culture isolation\nLymph nodes (retropharyngeal, supra-mammary, iliac, and\nmandibular lymph nodes) and other organs (spleen, uterus, mammary\ngland) were collected and sent to the IZSME (Portici, Italy) for\nBrucella detection though PCR and culture isolation.\nPCR analysis was performed to detect Brucella DNA in the\ncollected tissues. Genomic DNA was extracted using the QIAamp\nDNA MINI KIT (QIAGEN) following the manufacturer’s instructions,\nas previously described (17). Then, the identification of the Brucella\nspp. was performed though a real-time PCR assay using TaqMan\nprobes and targeting IS711 (18). The presence of viable Brucella\nbacteria was assessed through culture isolation, according to the\nWOAH Manual of Diagnostic Tests and Vaccines for Terrestrial\nAnimals (8). Bacterial isolates were further identified using the VITEK\n2 system (3).\n\nThe presence of viable Brucella\nbacteria was assessed through culture isolation, according to the\nWOAH Manual of Diagnostic Tests and Vaccines for Terrestrial\nAnimals (8).Bacterial isolates were further identified using the VITEK\n2 system (3).\n2.4 Whole blood stimulation and evaluation\nof cytokines release\nBlood samples were collected from the jugular vein and heparin\nwas used as anticoagulant and processed within 8 h of collection.\n\nFranzoni et al.\b\n10.3389/fvets.2025.1583858\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nWhole blood from each animal was dispensed in aliquots of\n1 mL, using a 24 well-plate and stimulated with Phosphate-buffered\nsaline (PBS, nil Control Antigen) or Brucella antigen (Brucellergene®\nOCB) (100 μL per well, corresponding to 1,500–2,500 UI) or\nPokeweed Mitogen (PWM, final concentration 1 μg/mL, used as a\nlymphocyte viability control). After incubation for 16–24 h at 37°C\nin a humidified atmosphere, plasma samples were collected. Then,\nlevels of 15 key immune cytokines (IFN-γ, IL-1α, IL-1β, IL-4, IL-6,\nIL-10, IL17, IL-36Ra, MIP-1α, MIP-1β, MCP-1, IP-10, CXCL8, TNF,\nVEGF-A) were measured using Bovine Cytokine/Chemokine\nMagnetic Bead Panel Multiplex assay (Merck Millipore, Darmstadt,\nGermany) and a Bioplex MAGPIX Multiplex Reader (Bio-Rad,\nHercules, CA, United  States), as previously described (14). All\nsamples were tested in duplicate (two technical replicates).\nTABLE 1  Cytokine production in whole blood from healthy and Brucella-infected Mediterranean Buffaloes.\nPBS\nBRC\nPWM\nPBS-BRC\nPBS-PWM\nLSM ± SE\nLSM ± SE\nLSM ± SE\np-value\np-value\nHealthy\nCytokines\n\u0007IFN-γ\n6 ± 102\n11 ± 102\n1,158 ± 102\n0.9993\n0.0001\n\u0007IL-17\n9 ± 149\n35 ± 149\n1,106 ± 149\n0.9887\n0.0001\n\u0007IP-10\n1,611 ± 179\n1,692 ± 179\n3,721 ± 179\n0.9887\n0.0001\n\u0007IL-6\n1,085 ± 210\n1,430 ± 210\n1,436 ± 210\n0.4109\n0.3984\n\u0007TNF\n1,772 ± 604\n3,943 ± 604\n5,142 ± 604\n0.0261\n0.0005\n\u0007IL-1ɑ\n34 ± 13\n70 ± 13\n91 ± 13\n0.1097\n0.0069\n\u0007IL-1β\n321 ± 295\n2,218 ± 295\n891 ± 295\n0.0001\n0.2992\n\u0007IL-4\n90 ± 26\n89 ± 26\n269 ± 26\n0.9995\n0.0001\n\u0007CXCL8\n3,628 ± 355\n3,451 ± 366\n4,122 ± 346\n0.9159\n0.5091\n\u0007IL-10\n380 ± 148\n469 ± 148\n1,819 ± 148\n0.8771\n0.0001\n\u0007MIP-1ɑ\n2,328 ± 481\n3,914 ± 481\n5,506 ± 481\n0.0434\n0.0001\n\u0007MIP-1β\n322 ± 60\n582 ± 60\n871 ± 60\n0.0064\n0.0001\n\u0007IL-36Ra\n480 ± 55\n452 ± 55\n467 ± 55\n0.9074\n0.9781\n\u0007VEGF\n188 ± 19\n233 ± 19\n140 ± 19\n0.1827\n0.1475\n\u0007MCP-1\n5,075 ± 175\n4,602 ± 175\n5,212 ± 175\n0.1092\n0.8036\nInfected\nCytokines\n\u0007IFN-γ\n16 ± 120\n861 ± 120\n1,133 ± 120\n0.0001\n0.0001\n\u0007IL-17\n2 ± 51\n23 ± 51\n364 ± 51\n0.9429\n0.0001\n\u0007IP-10\n2,111 ± 246\n3,093 ± 246\n3,109 ± 246\n0.0126\n0.0111\n\u0007IL-6\n1,316 ± 187\n1,005 ± 187\n1,159 ± 187\n0.3957\n0.7768\n\u0007TNF\n2,163 ± 718\n2,790 ± 718\n5,860 ± 718\n0.7625\n0.0011\n\u0007IL-1ɑ\n44 ± 25\n52 ± 25\n156 ± 25\n0.9608\n0.0055\n\u0007IL-1β\n291 ± 175\n755 ± 175\n1,702 ± 175\n0.1193\n0.0001\n\u0007IL-4\n82 ± 18\n85 ± 18\n115 ± 18\n0.9867\n0.3391\n\u0007CXCL8\n3,351 ± 312\n3,412 ± 321\n3,193 ± 331\n0.9868\n0.9165\n\u0007IL-10\n378 ± 99\n334 ± 99\n1,311 ± 99\n0.9337\n0.0001\n\u0007MIP-1ɑ\n2,553 ± 411\n3,494 ± 411\n5,000 ± 411\n0.1929\n0.0002\n\u0007MIP-1β\n466 ± 76\n727 ± 76\n946 ± 76\n0.0348\n0.0001\n\u0007IL-36Ra\n297 ± 25\n296 ± 25\n\n9867\n0.3391\n\u0007CXCL8\n3,351 ± 312\n3,412 ± 321\n3,193 ± 331\n0.9868\n0.9165\n\u0007IL-10\n378 ± 99\n334 ± 99\n1,311 ± 99\n0.9337\n0.0001\n\u0007MIP-1ɑ\n2,553 ± 411\n3,494 ± 411\n5,000 ± 411\n0.1929\n0.0002\n\u0007MIP-1β\n466 ± 76\n727 ± 76\n946 ± 76\n0.0348\n0.0001\n\u0007IL-36Ra\n297 ± 25\n296 ± 25\n292 ± 25\n0.9997\n0.9867\n\u0007VEGF\n194 ± 23\n224 ± 23\n177 ± 23\n0.5691\n0.8385\n\u0007MCP-1\n4,964 ± 173\n4,914 ± 173\n5,268 ± 173\n0.9704\n0.3634\nWhole blood samples were stimulated with PBS (nil control) or Brucellergene® OCB antigen (BRC), or PWM (positive control). The concentration of 15 cytokines were measured using\nmultiplex ELISA. The table presents the Least Squares Mean (LSM), Standard Estimated Error (SEE), and statistical differences (p-value) between conditions. p-value < 0.05 were considered\nstatistically significant and are marked in bold.\n\nFranzoni et al.\b\n10.3389/fvets.2025.1583858\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nFIGURE 1\nAntigen-specific release of T-cell cytokines in Brucella-Infected buffaloes. Blood samples from Brucella-infected (n = 20) and healthy (n = 18) \nMediterranean Buffaloes were collected using heparin as anticoagulant. Whole blood was stimulated with PBS (nil control) or Brucellergene® OCB \nantigen (BRC). After 16–24 h of incubation, plasma was collected, and levels of T-cell cytokines (IFN-γ, IL-17, IL-4, IL-10) were determined using \nmultiplex ELISA. Brucella-specific cytokines values were calculated by subtracting baseline cytokines levels (PBS) from those in antigen condition. \nDifferences between groups are shown, with statistical significance set at p < 0.05.\nAntigen-specific cytokine in responses were calculated by \nsubtracting baseline cytokines concentrations (PBS, nil control) from \nthose of the antigen condition (Brucellergene® OCB).\n2.5 Statistical analysis\nLevels of the 15 tested cytokines were analyzed using the general \nlinear model (GLM) to estimate the mean of each trait per stimulus \n(PBS, BRC, and PWM) within groups (healthy and infected):\n\n2.5 Statistical analysis\nLevels of the 15 tested cytokines were analyzed using the general \nlinear model (GLM) to estimate the mean of each trait per stimulus \n(PBS, BRC, and PWM) within groups (healthy and infected):\n\n= µ +\n+\njk\nj\njk\nY\nG\ne\nwhere Yjk is the trait measured for each animal, μ is the overall \nmean, Gj is the fixed effect of the stimuli (j = 3 levels: PBS, BRC, and \nPWM), and ejk is the random residual effect of each observation.\nThe statistical significance of all traits and least-square means were \nassessed by Dunnet’s multiple test in the GLM procedure.\nAdditionally, the difference (∆_cytokine) between the level of each \nspecific cytokine measured in the Brucella antigen condition (BRC) and \nits baseline concentration (PBS) was analyzed and displayed by GraphPad \nPrism 10.01 (GraphPad Software Inc., La Jolla, CA, United States).\nA multivariate approach was conducted using canonical \ndiscriminant analysis (CDA) on 15, 7 and finally 5 ∆_cytokines by the \nCANDISC Procedure.\nThe significance level for both statistical analyses was set at a \np-value < 0.05.\nThe CDAs were conducted by categorizing animals prior to \nhealthy and infected. The CANDISC method was utilized to estimate \nlinear functions of all quantitative variables that best discriminated \nagainst the groups while minimizing the variation within each group.\nAll statistical analyses were performed with SAS software \nversion 9.4.\n3 Results\nWhole blood samples from healthy (N = 18) and infected (N = 20) \nMediterranean Buffaloes were stimulated with a specific Brucella \nantigen (Brucellergene® OCB), alongside controls, and then the \nrelease of key immune cytokines was determined.\nIn both infected and healthy animal groups, PWM triggered the \nrelease of IFN-γ, IL-17, IL-10, and TNF indicating that T cells viability \nand functionality were not altered by treatments that could have \naffected the analyses (e.g., corticosteroid administration) (Table 1). \nPWM triggered also the release of chemokines IP-10, MIP-1α, and \nMIP-1β (Table 1), suggesting that the viability of other immune cells \n(e.g., monocytes) was not altered as well.\nIn the infected group, but not in the healthy group, stimulation \nwith Brucella antigen (BRC) led to a significantly higher release of\n\nFranzoni et al.\b\n10.3389/fvets.2025.1583858\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nIFN-γ and IP-10 compared to the nil control (Table 1). Both groups\nexhibited an increase in MIP-1β levels in response to Brucella antigen.\nIn the healthy group, additional pro-inflammatory cytokines (TNF,\nIL-1β, MIP-1α) were also released upon antigen stimulation, suggesting\nan innate immune response to Brucella components in naïve animals.\nThen, differences between the two groups (healthy and infected)\nin terms of antigen-specific cytokine releases were assessed. Baseline\ncytokines levels (PBS, nil control) were subtracted from those in the\nantigen condition (Brucellergene® OCB) and then differences between\ngroups were analyzed.\nIn Figure 1, the results of four key T cell cytokines are presented:\nIFN-γ (Th1 response marker), IL-4 (Th2 response marker), IL-17\n(mainly released by Th17), IL-10 (Treg-associated immunosuppressive\ncytokine) (19). Infected animals released higher levels of antigen-\nspecific IFN-γ compared to healthy animals (p < 0.0001), whereas no\nsignificant differences were observed for IL-4 and IL-17. Conversely,\nhealthy animals produced higher antigen-specific IL-10 levels\ncompared to -infected subjects (p = 0.0184) (Figure 1).\nIn Figure 2, the results of four key pro-inflammatory cytokines\nare shown: IL-1α, IL-1β, IL-6, TNF (19). Infected animals released\nlower levels of antigen-specific IL-1α, IL-1β, IL-6 compared to\nhealthy subjects, whereas TNF levels did not differ significantly\nbetween groups (Figure 2).\nThe results of five key chemokines are presented in Figure 3.\nNo differences between groups were observed for MIP-1α and\nMIP-1β, whereas infected animals released higher levels of\nantigen-specific IP-10 (p < 0.0001) and MCP-1 (p = 0.0277)\ncompared to healthy subjects. Regarding CXCL8, 5 out of 38\nbuffaloes presented cytokine levels higher than the detection limit\nof the kit, so they were excluded from the analysis. In the other\nanimals (17 infected vs. 16 healthy), no differences were observed\nbetween groups (Figure 3).\nThe antigen-specific release of IL-36Ra (receptor antagonist) and\n\nIn the other\nanimals (17 infected vs.16 healthy), no differences were observed\nbetween groups (Figure 3).The antigen-specific release of IL-36Ra (receptor antagonist) and\nVEGF-A (growth factor) was also investigated, but no significant\ndifferences between groups were observed (Figure 4).\nCanonical discriminant analysis was then used to generate\npredictive cytokine profiles distinguishing healthy and infected\nanimals as potential diagnostic biomarkers. First, a CDA was\nperformed with the 15 cytokines analyzed in the study. As presented\nin Figure 5, these 15 cytokines can clearly differentiate between the\ntwo groups under evaluation. Table 2A reports the factor loading\n(FL) for each cytokine in the canonical variable (Can 1), showing a\npositive and high correlation with ∆_IFN-γ, ∆_IP-10 (FL ≥ 0.70),\ncharacterizing the infected group, and a negative correlation with\n∆_IL-6, ∆_IL-10, ∆_IL1-β, and ∆_IL1-α (FL ≥ 0.50), characterizing\nthe healthy one.\nFIGURE 2\nAntigen-specific release of pro-inflammatory cytokines in Brucella-infected buffaloes. Blood samples from Brucella-infected (n = 20) and healthy\n(n = 18) Mediterranean Buffaloes were collected using heparin as anticoagulant. Whole blood was stimulated with PBS (nil control) or Brucellergene®\nOCB antigen (BRC). After 16–24 h of incubation, plasma was collected, and levels of pro-inflammatory cytokines (IL-1α, IL-1β, IL-6, TNF) were\nmeasured using multiplex ELISA. Brucella-specific cytokines values were determined by subtracting baseline cytokines levels (PBS) from those in the\nantigen condition. Differences between the groups are shown, with statistical significance set at p < 0.05.\n\nFranzoni et al.\b\n10.3389/fvets.2025.1583858\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nFIGURE 3\nAntigen-specific release of chemokines in Brucella-Infected buffaloes. Blood samples from Brucella-infected (n = 20) and healthy (n = 18)\nMediterranean Buffaloes were collected using heparin as anticoagulant. Whole blood was stimulated with PBS (nil control) or antigen Brucellergene®\nOCB antigen (BRC). After 16–24 h of incubation, plasma was collected, and levels of key chemokines (IP-10, MIP-1α, MIP-1β, MCP-1, CXCL8) were\nmeasured using multiplex ELISA. Brucella-specific cytokines values were calculated by subtracting baseline cytokines levels (PBS) from those in antigen\ncondition. Differences between the groups are shown, with statistical significance set at p < 0.05.\nThen, to identify the most discriminating combination of\ncytokines, two other CDAs were conducted. The first one was\nperformed with the 7 ∆_cytokine levels that significantly differentiate\nthe two groups (IFN-γ, IP-10, MCP-1, IL-6, IL1-β, IL-1α, IL-10). As\npresented in Figure 6, this model clearly discriminated against the\ntwo groups, except of one healthy animal (Healthy_2). Table 2B\nreports the cytokine FL in this new canonical function, revealing a\nhigh correlation with ∆_IFN-γ, ∆_IP-10 (0.80–0.79, respectively)\nand a medium one with IL-6, ∆_IL1-β, and ∆_IL1-α (FL ≥ 0.50).\nFinally, the third CDA (Table 2C) was conducted using only the\n5 cytokines with an FL ≥ 0.50 in the previous Can 1: IFN-γ, IP-10,\nIL-6, IL1-β, and IL1-α. As presented in Figure 7, the use of these 5\ncytokines can clearly differentiate the two groups under evaluation\n(healthy-infected).\n4 Discussion\nBrucella is a globally distributed zoonotic pathogen that affects\nvarious\ndomestic\nand\nwild\nanimals\nspecies,\nincluding\nMediterranean Buffalo (3, 20). Early and accurate detection of\nBrucella infection in this specie is crucial for effective\ndisease control.\nA widely accepted diagnostic approach for Brucella in cattle and\nbuffaloes combines two serological assays (the RBT with the CFT),\nalthough these test present some disadvantages, such as low\n\nA widely accepted diagnostic approach for Brucella in cattle and\nbuffaloes combines two serological assays (the RBT with the CFT),\nalthough these test present some disadvantages, such as low\nspecificity. False-positive serological reactions (FPSR) might result\nfrom exposure to cross reacting microorganisms, especially Gram-\nnegative bacteria with LPS O-chains similar to those of Brucella (8,\n9). There is a need to develop new diagnostic methods, in order to\n\nFranzoni et al.\b\n10.3389/fvets.2025.1583858\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nimprove the efficacy of eradication strategies and to avoid \nunnecessary animal sacrifices. Brucella is an intracellular pathogen, \nwhich primarily targets macrophages and dendritic cells, employing \nsophisticated mechanism to both innate and adaptive immune \nresponse, enabling its survival and persistence within host cells \n(20). Resistance to intracellular bacterial pathogens relies on \nFIGURE 4\nAntigen-specific release of IL-36Ra and VEGF-A in Brucella-Infected buffaloes. Blood samples from Brucella-infected (n = 20) and healthy (n = 18) \nMediterranean Buffaloes were collected using heparin as anticoagulant. Whole blood was stimulated with PBS (nil control) or Brucellergene® OCB \nantigen (BRC). After 16–24 h of incubation, plasma was collected, and levels of IL-36Ra and VEGF-A were determined through multiplex ELISA. \nBrucella-specific cytokines values were quantified by subtracting baseline cytokines levels (PBS) from those in the antigen condition. Differences \nbetween the groups are shown, with statistical significance set at p < 0.05.\nFIGURE 5\nPlot from canonical discriminant analysis. Canonical discriminant analysis on 15 Δ_cytokines by the CANDISC Procedure. Animals belonging to the \ntwo groups are displayed based on the canonical function Can1.\n\nFranzoni et al.\b\n10.3389/fvets.2025.1583858\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nTABLE 2  Correlations between canonical function (Can 1) and original\nvariables.\nA\n∆_cytokine\nCan 1\n∆_IFN-γ\n0.74\n∆_IP-10\n0.73\n∆_IL-6\n−0.59\n∆_IL-10\n−0.59\n∆_IL-1β\n−0.56\n∆_IL-1α\n−0.50\n∆_MCP1\n0.44\n∆_TNF\n−0.36\n∆_MIP-1α\n−0.28\n∆_CXCL8\n0.23\n∆_IL-36Ra\n0.19\n∆_VEGF\n−0.18\n∆_IL4\n0.11\n∆_IL-17\n−0.03\n∆_MIP-1β\n−0.02\nB\n∆_cytokine\nCan 1\n∆_IFN-γ\n0.8\n∆_IP-10\n0.79\n∆_IL-6\n−0.61\n∆_IL-1β\n−0.56\n∆_IL-1α\n−0.54\n∆_IL-10\n−0.45\n∆_MCP1\n0.42\nC\n∆_cytokine\nCan 1\n∆_IFN-γ\n0.82\n∆_IP-10\n0.80\n∆_IL-6\n−0.63\n∆_IL-1β\n−0.58\n∆_IL-1α\n−0.55\nThe difference (∆_cytokine) between the level of each specific cytokine measured in the\nantigen condition (Brucellergene® OCB) and its baseline cytokine concentration (PBS), and\ntheir weight in the Can 1, are shown: A—the 15 tested cytokines. B—the seven cytokines that\nsignificantly differentiate the healthy and infected groups. C—the five cytokines with a\nFL ≥ 0.50 in the previous Can 1 (B). The heavier correlation coefficients are marked in bold.\ncell-mediated immunity, and it was reported that an adequate Th1\nimmune response is critical for the clearance of Brucella infection.\nIn humans, IFN-γ is the key cytokine involved in the immune\nresponse against Brucella (20, 21). Cytokines are crucial mediators\nof immune responses and their quantification provides insights into\nphysiological and pathological processes, aiding diagnosis and\ntreatment, thus they have been widely studied as biomarkers for\nmany diseases. Cytokine testing has the potential to support\ndiagnosis also due to its lack of invasiveness and relative low\ncost (22).\nBiomarker development is a multistep process that starts with its\ndiscovery in a pathophysiological context and progresses through\nvarious validation phases. This study provides preliminary insights\ninto the role of key immune cytokines as biomarkers for Brucella\ninfection in Mediterranean Buffaloes, with the aim to implement the\ndiagnosis of Brucella in this specie. Understating cytokine responses\nto antigen-stimulation could offer also a more comprehensive picture\nof host immunity against this pathogen.\nIFN-γ is an antiviral cytokine released mainly by NK and activated\nT cells; it is regarded as a hallmark of a Th1 response, which is\n\nIFN-γ is an antiviral cytokine released mainly by NK and activated\nT cells; it is regarded as a hallmark of a Th1 response, which is\nassociated with resistance to intracellular pathogens (23, 24). Our\nfinding indicates that Brucella-infected buffaloes release higher levels\nof antigen-specific IFN-γ compared to uninfected controls. Similar\nobservations have been reported in cattle infected with B. abortus,\nwhere infected animals exhibited elevated IFN-γ levels upon antigen\nstimulation (25, 26). Likewise, in B. melitensis infected sheep,\nincreased IFN-γ secretion was noted following antigen exposure (27,\n28). Overall, our data suggest that antigen-specific IFN-γ release could\nserve as a potential diagnostic marker for Brucella infection in\nMediterranean Buffaloes.\nWe recently observed that Mediterranean Buffaloes infected with\nB. abortus presented lower levels of circulating T and B cells compared\nto healthy controls (29) and this might result in a different T-cell\ncytokine pattern in response to antigen stimulation. IL-17 and IL-4\nreleases were therefore investigated. IL-17 and IL-14 are regarded as\nhallmark of the Th17 and Th2 response, respectively (19, 30). IL-17\npromotes inflammation and a granulocyte-dependent response to\npathogens; it triggers the release of pro-inflammatory cytokines and\nenhances the recruitment of neutrophils (30). IL-4 promotes instead\ntissue regeneration and the differentiation of Th2 cells (19). Despite\nthe important role of these cytokines in the immune response to\nseveral pathogens, little is known about their function during Brucella\ninfection. In humans, the IL-17 serum levels were significantly higher\nin subjects with brucellosis compared to healthy controls and the\nlevels of this cytokine decreased in patients after specific treatment\n(31). However, in our study, no differences in antigen-specific IL-17\nor IL-4 secretion were observed between infected and healthy\nbuffaloes, suggesting that these cytokines may not be  reliable\ndiagnostic markers for brucellosis in this specie.\nIL-10 is a cytokine with strong anti-inflammatory action, with the\n\nIL-10 is a cytokine with strong anti-inflammatory action, with the\nability to reduce or terminate inflammation (32). It promotes the\nexpansion and persistence of T reg cells, which prevent autoimmunity\nand limit chronic inflammatory diseases (33). Our data revealed that\ninfected buffaloes release lower levels of antigen-specific IL-10\ncompared to healthy subjects. Similarly, we  observed that\nMediterranean Buffaloes with brucellosis release lower levels of three\npro-inflammatory cytokines than healthy controls: IL-1α, IL-1β, IL-6.\nThese cytokines are typically released early during infection to initiate\ninflammation, with IL-10 acting to balance their effects (19, 32).\nComparable findings have been reported in human patients with\nbrucellosis, where monocytes exhibited diminished IL-1β, IL-6, and\nIL-10 secretion in response to LPS stimulation (34). Brucella employs\nimmune evasion strategies, including inflammasome downregulation\nand inhibition of macrophage polarization toward a pro-inflammatory\nphenotype (M1) (34, 35). Our results suggest that Brucella infection\nimpairs monocyte ability to release pro-inflammatory cytokines in\n\nFranzoni et al.\b\n10.3389/fvets.2025.1583858\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nresponse to pathogen associated molecular patterns, such as those\ncontained in the antigen Brucellergene® OCB (protein extract of\nB. melitensis strain BB15).\nTNF is another pro-inflammatory cytokine, which triggers the\nrelease of pro-inflammatory chemokines, effectively recruiting\nleukocytes to the inflammatory site (36). Studies in humans\nreported that monocytes and dendritic cells from patients with\nbrucellosis released lower levels of TNF in response to external\nstimuli compared to healthy controls (34, 37). However, in our\nstudy, no differences in antigen-specific TNF secretion were\nobserved between infected and healthy buffaloes. This discrepancy\ncould be attributed to the involvement of other immune cells, such\nas CD4+ and CD8+ T cells, in TNF production (38), equalizing the\ndifference between groups in terms of total TNF-release in response\nto antigen-stimulation. Our previous research suggested that TNF\nmight be a useful biomarker for identifying buffaloes infected with\nM. bovis (14), but the current data indicate that TNF is not a\nsuitable biomarker for Brucella infection.\nWe also examined the antigen-specific secretion of key\nchemokines: IP-10, MCP-1, MIP-1α, MIP-1β, CXCL8. Chemokines\nplay a crucial role in immune cell recruitment to infected tissues\n(39). IP-10 is secreted by monocytes, macrophages, endothelial cells\nand fibroblasts and its release is sharply enhanced by IFN-γ (40).\nThis chemokine triggers the recruitment of monocytes, macrophages,\nNK cells, and activated T cells to the inflammatory site (40). It has\nbeen identified as a promising biomarker for M. bovis infection in\ncattle and buffaloes (14, 41–44). In this study, we observed that\nBrucella-infected animals release high levels of antigen-specific\nIP-10 compared to uninfected controls. These data are in agreement\nwith what observed in a mouse models of brucellosis: immune cells\nof mice immunized with a B. melitensis attenuated strains (WR201)\nrelease high levels of antigen-specific IP-10 compared to healthy\n\nThese data are in agreement\nwith what observed in a mouse models of brucellosis: immune cells\nof mice immunized with a B.melitensis attenuated strains (WR201)\nrelease high levels of antigen-specific IP-10 compared to healthy\ncontrols (45). Overall, our preliminary data suggest that this\nchemokine could be  a promising biomarker of brucellosis in\nbuffaloes. MCP-1 is a strong chemoattractant for monocytes and it\nis released mainly by monocytes and macrophages (46). In a mouse\nmodel of brucellosis, immune cells of Brucella-infected mice release\nhigh levels of antigen-specific MCP-1 compared to healthy controls\n(45). In agreement, in our study we observed that Brucella-infected\nanimals release high levels of antigen-specific MCP-1 compared to\nuninfected controls, suggesting that this chemokine could be  a\npromising biomarker of brucellosis in buffaloes. MIP-1α and MIP-1β\nare pro-inflammatory chemokines mainly produced by monocytes/\nmacrophages (47). They promote recruitment of diverse cell types\n(chemotaxis of monocytes, dendritic cells, T cells, NK cells, and\ngranulocytes) to the inflammatory sites (47). Previous studies\nsuggest that these chemokines were involved in the immune\nresponse to other intracellular bacteria infecting Mediterranean\nBuffaloes, such as M. bovis (14). Our data revealed that infected and\nhealthy buffaloes release similar levels of antigen-specific MIP-1α\nand MIP-1β, suggesting that these two chemokines could not\nbe useful biomarkers of brucellosis in buffaloes. CXCL8, also known\nFIGURE 6\nPlot from canonical discriminant analysis. Canonical discriminant analysis on 7 Δ_cytokines by the CANDISC Procedure. Animals belonging to the two\ngroups are displayed based on the canonical function Can1.\n\nFranzoni et al.\b\n10.3389/fvets.2025.1583858\nFrontiers in Veterinary Science\n10\nfrontiersin.org\nFIGURE 7\nPlot from canonical discriminant analysis. Canonical discriminant analysis on 5 Δ_cytokines by the CANDISC Procedure. Animals belonging to the two\ngroups are displayed based on the canonical function Can1.\nas IL-8, is a strong neutrophil chemoattractant, which triggers the\nrecruitment of neutrophils and other granulocytes to the site of\ninfection, promotes neutrophil degranulation and enhancements in\ntheir phagocytic functions (39, 48). This chemokine is released by\nneutrophils and macrophages in response to Brucella infection (49,\n50), but infection results also in a lower ability of macrophages to\npresent antigen to T cells and to release pro-inflammatory cytokines\nin response to external stimuli (34, 51). Our data revealed that there\nwere no differences between groups, suggesting that the evaluation\nof CXCL8 will not improve the diagnosis of brucellosis in\nMediterranean Buffaloes.\nSubsequently, we  investigated the antigen-specific release of\nIL-36Ra and VEGF. IL-36Ra is the receptor antagonist of the\npro-inflammatory interleukin IL-36 and it is secreted to prevent the\ndevelopment of an exacerbated inflammatory response to stressors\n(52). VEGF is a growth factor, and it possesses pro-angiogenic\nactivity, promoting endothelial cell survival, cell migration and\nincreasing vascular permeability (53). We observed no differences\nbetween infected and healthy animals, suggesting that they are\nunlikely to serve as diagnostic biomarkers for Brucella infection\nin buffaloes.\nFinally, we aimed to identify the cytokine set which better\ndiscriminate Brucella-infected and healthy Mediterranean\nBuffaloes. Three canonical analyses were performed sequentially\nto reduce the number of cytokines while ensuring effective\ndiscrimination between the two groups of animals. From the\ninitial 15, we narrowed it down to five: our analysis showed that\nthe quantitative determination of IFN-γ, in parallel with the\nchemokine IP-10 and three pro-inflammatory cytokines (IL-6,\n\nFrom the\ninitial 15, we narrowed it down to five: our analysis showed that\nthe quantitative determination of IFN-γ, in parallel with the\nchemokine IP-10 and three pro-inflammatory cytokines (IL-6,\nIL-1α and IL-1β) could be useful in the diagnosis of brucellosis in\nMediterranean Buffaloes. The infected group is characterized\nby high values of IFN-γ and IP-10, unlike the other three\npro-inflammatory markers, which have higher values in\nhealthy subjects.\nOverall, our data suggest that IFN-γ, IP-10, IL-1a, IL-1ß, IL-6\ncould enhance brucellosis diagnosis in Mediterranean Buffaloes.\nThese preliminary observation should be validated on a larger set\nof samples, in order to properly establish the sensitivity and\nspecificity of these ELISAs. In particular, samples from buffaloes\ninfected with other Gram-negative bacteria should be included in\nthe analysis, to evaluate the specificity of these potential\nbiomarkers and to implement the diagnosis of Brucella in this\nspecie, in order to avoid un-necessary animal sacrifices. In\nconclusion, these preliminary findings provide a foundation for\ndeveloping cytokine-based diagnostic tools for Brucella infection\nin Mediterranean Buffaloes.\n\nFranzoni et al.\b\n10.3389/fvets.2025.1583858\nFrontiers in Veterinary Science\n11\nfrontiersin.org\nData availability statement\nThe raw data supporting the conclusions of this article will\nbe made available by the authors, without undue reservation.\nEthics statement\nThe animal study was approved by the Institutional Ethics\nCommittee of the Istituto Zooprofilattico Sperimentale del\nMezzogiorno. Mediterranean Buffaloes used in this study were\nanalyzed within the context of official eradication program, therefore\nwere not considered experimental animals. The study was conducted\nin accordance with the local legislation and institutional requirements.\nAuthor contributions\nGF: Conceptualization, Data curation, Formal analysis,\nInvestigation, Writing – original draft. FS: Conceptualization, Data\ncuration, Formal analysis, Investigation, Writing – review & editing.\nAD: Investigation, Methodology, Writing  – review & editing. LS:\nInvestigation, Methodology, Writing  – review & editing. MN:\nInvestigation, Methodology, Writing – review & editing. GM: Data\ncuration, Investigation, Writing – review & editing. FG: Data curation,\nInvestigation, Writing  – review & editing. SZ: Investigation,\nMethodology, Writing  – review & editing. VB: Investigation,\nMethodology, Writing  – review & editing. SD: Formal analysis,\nInvestigation, Methodology, Writing  – review & editing. EC:\nInvestigation, Resources, Writing – review & editing. GG: Investigation,\nResources, Writing – review & editing. FN: Conceptualization, Data\ncuration, Formal analysis, Investigation, Writing – review & editing.\nAM: Conceptualization, Funding acquisition, Investigation, Project\nadministration, Resources, Writing – review & editing.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. This research was funded\nby the Italian Ministry for Health (grant number Research Project RC\nIZSME 11/21 RC).\nAcknowledgments\nThe authors would like to acknowledge the Veterinary Services for\ntheir help in sample collection.\nConflict of interest\nThe authors declare that the research was conducted in the\n\nAcknowledgments\nThe authors would like to acknowledge the Veterinary Services for\ntheir help in sample collection.Conflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nThe author(s) declared that they were an editorial board member\nof Frontiers, at the time of submission. This had no impact on the peer\nreview process and the final decision.\nGenerative AI statement\nThe author(s) declare that no Gen AI was used in the creation of\nthis manuscript.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. Any product\nthat may be evaluated in this article, or claim that may be made by its\nmanufacturer, is not guaranteed or endorsed by the publisher.\nSupplementary material\nThe Supplementary material for this article can be found online\nat: https://www.frontiersin.org/articles/10.3389/fvets.2025.1583858/\nfull#supplementary-material\nReferences\n1.\tFranc KA, Krecek RC, Häsler BN, Arenas-Gamboa AM. Brucellosis remains a\nneglected disease in the developing world: a call for interdisciplinary action. BMC Public\nHealth. 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+{"text": "【兽医文献】\n标题: article_51_In small animal surgery are alcoholic hand rubs superior to \n\nWhen treating medial patellar luxation in dogs is a \nblock trochleoplasty superior over a wedge \ntrochleoplasty?\n\nA Knowledge Summary by\n\nMaria Norell Candetoft DVM 1*\n\n1 Djursjukhuset AniCura Albano, Rinkebyvägen 21A, 182 36 Danderyd, Sweden \n* Corresponding Author (maria.norell.candetoft@anicura.se)\n\nISSN: 2396-9776 \nPublished: 15 Jul 2022 \nin: Veterinary Evidence Vol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.517  \nReviewed by: Andy Morris (BSc[Hons] BVSc CertAVP[GSAS] \nMRCVS) and Maureen O'Mara (PhD MGIS) \nNext Review Date: 22 Nov 2023\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.517  \nNext review date: 22 Nov 2023 \np a g e | 2 of 13\n\nKNOWLEDGE SUMMARY\n\nClinical scenario  \nYou have been working for a long time in a large veterinary hospital and over the years performed many block \nrecession trochleoplasties as part of the surgical treatment of medial patellar luxation, and you have been very \nsatisfied with the postoperative results. Now you have recently been employed by another veterinary hospital \nwhere all veterinary surgeons perform wedge recession trochleoplasty and they want you to switch your \nPICO question \nAs part of the surgical correction for medial patellar luxation in dogs, which procedure results in a better \noutcome for the patient: block or wedge recession trochleoplasty?\n\nClinical bottom line \nCategory of research question \nTreatment \nThe number and type of study designs reviewed \nThree studies satisfied the inclusion criteria for answering the PICO; one cadaver study, one retrospective \nobservational study and one clinical case series \nStrength of evidence \nWeak \nOutcomes reported \nPostoperative complications including reluxation rates. \nEx vivo: Trochlear groove depth, patella articular contact, percentage of recessed trochlear surface area, \nresistance to medial patella luxation \nConclusion \nThere is only weak evidence to support block recession trochleoplasty over wedge recession trochleoplasty \nas part of the surgical correction for medial patella luxation in dogs. Both procedures are associated with a \ngood clinical outcome. There are some proposed benefits to trochlear block recession made from an ex \nvivo study comparing the two procedures. These include an increased patellar volume under the trochlear \nridges when the stifle is extended. The articular contact and recessed trochlear surface area were also \nincreased in the trochlear block recession group when compared to trochlear wedge recession. However, \nthe clinical relevance of these perceived benefits remains unproven. In practice, and until prospective \nrandomised controlled trials are carried out, veterinary surgeon preference and previous experience \nremain relevant factors in choosing which procedure to perform\n\nHow to apply this evidence in practice \nThe application of evidence into practice should take into account multiple factors, not limited to: \nindividual clinical expertise, patient’s circumstances and owners’ values, country, location or clinic where \nyou work, the individual case in front of you, the availability of therapies and resources. \nKnowledge Summaries are a resource to help reinforce or inform decision making. They do not override the \nresponsibility or judgement of the practitioner to do what is best for the animal in their care.\n\nKnowledge Summaries are a resource to help reinforce or inform decision making.They do not override the \nresponsibility or judgement of the practitioner to do what is best for the animal in their care.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.517  \nNext review date: 22 Nov 2023 \np a g e | 3 of 13\n\nmethod since that they think that wedge recession is superior. You decide to read about the topic to learn \nmore regarding the evidence between the two methods. To do a block or a wedge, that is the question.\n\nThe evidence \nThe literature search uncovered only three papers that addressed the PICO question and fitted the inclusion \nand exclusion criteria applied. The three studies described the outcome of wedge recession trochleoplasty and \nblock recession trochleoplasty; one study in cadavers (Johnson et al., 2001), one retrospective observational \nstudy (Rossanese et al., 2019) and one case series study (Ballatori et al., 2005). The number of cases included \nin the cadaver study by Johnson et al. (2001) was low, 12 cases with 24 stifles, and only four cases with eight \nstifles were included in the case series study. The latter study by Ballatori et al. (2005), was included despite \nthe fact that it had both lateral and medial patellar luxations, although only the results applying to the medial \npatellar luxation (two dogs) were included. The study that included most animals was Rossanese et al. (2019) \nwith 87 cases and a total of 100 stifles\n\nThe study that included most animals was Rossanese et al.(2019) \nwith 87 cases and a total of 100 stifles\n\nIn the study by Johnson et al. (2001) the difference in patellar depth, patellar articular contact, percentage of \nrecessed trochlear surface area, and resistance to medial patellar luxation in stifles treated with trochlear \nblock recession or trochlear wedge recession were described. In the specimens, the block technique was \nsuperior to the wedge technique as it had a deeper proximal patellar depth, a greater patellar articular contact \nin the extended stifle (P< 0.01), a larger percentage of recessed trochlear surface and a greater resistance to \nmedial patellar luxation. Since the study was performed on cadavers with specimens mounted in a position \ndevice, it is difficult to interpret the results in live animals. Also, all dogs were large breed dogs, and it is \nunclear whether or not the results can be applied also to small breed dogs.\n\nIn the study by Rossanese et al. (2019) femoral trochleoplasty was performed in 90 stifles and included a \ntrochlear wedge recession in 68 stifles (76%) and a trochlear block recession in 22 stifles (24%). Results from \nthis study showed no significant difference in complication rates between the trochlear wedge technique and \nthe trochlear block technique. The type of trochleoplasty performed (block vs wedge) was not associated with \nthe occurrence of postoperative complications.\n\nValid conclusions may not be drawn from the study by Ballatori et al. (2005) as only four dogs and eight stifles \nwere included, and two dogs had medial patellar luxation and two dogs had lateral patellar luxation.\n\nSummary of the evidence\n\nValid conclusions may not be drawn from the study by Ballatori et al.(2005) as only four dogs and eight stifles \nwere included, and two dogs had medial patellar luxation and two dogs had lateral patellar luxation.Summary of the evidence\n\nJohnson et al. (2001) \nPopulation: Normal, large-breed canine cadavers. \nThe dogs were euthanised for reasons unrelated to the study and \nwere determined to be free of stifle disease via orthopaedic \nexamination and radiographs. \nMean weight was 31.9 kg (25.0–38.6 kg). \nSample size: 24 stifles from 12 dogs. \nIntervention details: \n• \nSpecimens consisting of femur, tibia and intact stifle joint \nwere removed bilaterally from each cadaver. Soft tissues \nwere dissected free, leaving only the stifle joint capsule and \nligaments intact. The quadriceps tendon of insertion on the \npatella was transected 1.5 cm proximal to the patella. \n• \nBilateral pelvic limb specimens were mounted on two \nidentical position devices so that the legs could be examined \nand tested in standardised methods. \n• \nTo simulate a shallow trochlea, the trochlear ridges of each \nspecimen were reduced using a high-speed drill and burr.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.517  \nNext review date: 22 Nov 2023 \np a g e | 4 of 13\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.517  \nNext review date: 22 Nov 2023 \np a g e | 4 of 13\n\nA plastic guiding template was used to make the trochlea \nridges at the same (low) height on each specimen. \n• \nThe joint capsule was opened laterally (to mimic a stretched \nlateral joint capsule) and a mechanic arm applied 40° of \ninternal tibial rotation in both flexion and extension. The \nmedially internal rotation made all of the patellas to luxate \nmedially within 40° of internal tibial rotation in each trial. \n• \nTrochlear block recession (TBR) and trochlear wedge \nrecession (TWR) was performed on opposite stifles of paired \nspecimens, selection of right or left leg in each pair was \nmanaged by flipping a coin. \n• \nDepth and position of TWR were standardised between \nspecimens using a custom-made template that directed the \nsaw blade in the same way in all the trochleas. \n• \nThe position of the TBR was also standardised between \nspecimens based on anatomic landmarks. \n• \nEach specimen was evaluated before and after \ntrochleoplasty (TBR or TWR) with computed tomography \n(CT) and biomechanical testing with the stifle held in \nextension (148° to evaluate the patella within the proximal \ntrochlea) and in flexion (113° to evaluate the patella within \nthe central trochlea). The depth of the trochlea in the \ncentral and proximal portion was measured as well as the \npatellar coverage percent. \n• \nThe patellar articular contact with the recessed trochlea was \nalso determined postoperatively. \n• \nThe percentage of recessed trochlear surface area was \ndetermined for each specimen.\n\nStudy design: In vitro CT and biomechanical evaluation using a cadaver model.\n\nOutcome studied: To compare TBR to TWR with regards to patellar depth (percentage \nof patellar volume under the trochlear ridges), patellar articular \ncontact, percentage of recessed trochlear surface area, and \nresistance to patellar luxation.\n\nOutcome studied: To compare TBR to TWR with regards to patellar depth (percentage \nof patellar volume under the trochlear ridges), patellar articular \ncontact, percentage of recessed trochlear surface area, and \nresistance to patellar luxation.\n\nMain findings: \n(relevant to PICO question): \n• \nNo significance in the depth of trochlear recession existed \nbetween groups. \n• \nPostoperative patellar depth in the extended stifle position \nwas significantly greater in the TBR group compared with \nthe TWR group. \n• \nIn the extended stifle position, postoperative patellar \narticular contact was significantly greater in the TBR group \ncompared with the TWR group. \n• \nPostoperative recessed trochlear surface area was \nsignificantly greater in the TBR group (76.5%) compared with \nthe TWR group (49.9%). \n• \nPostoperatively patellar luxation did not occur in any \nspecimen in the flexed stifle but in the extended stifle (with \nthe leg held in 40° internal rotation) the patella luxated in \n8% (1/12) after TBR and in 42% (5/12) after TWR. The \ndifference was not statistically significant (P = 0.06).\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.517  \nNext review date: 22 Nov 2023 \np a g e | 5 of 13\n\nThe \ndifference was not statistically significant (P = 0.06).Veterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.517  \nNext review date: 22 Nov 2023 \np a g e | 5 of 13\n\nLimitations: \n• \nAlthough more versatile, CT is a less sensitive imaging \nmodality for visualising articular cartilage compared with \nmagnetic resonance imaging (MRI). However, to investigate \nthe groove depth of the trochleoplasty it is easier to make \nmeasurements from CT reconstruction / multiplanar image \nmanipulation compared to MRI. \n• \nThis was a cadaver study so we do not know how to \ncorrectly interpret the data in live animals. The authors did \nnot attempt to approximate physiologic forces placed on the \npatella by the quadriceps muscle group. \n• \nThe model also does not account for tibial or femoral \ntorsional deformities commonly seen in dogs with patellar \nluxation. \n• \nThe test was only performed in specimens from large breed \ndogs so we do not know if the results would be the same for \nsmall breed dogs.\n\n• \nThe test was only performed in specimens from large breed \ndogs so we do not know if the results would be the same for \nsmall breed dogs.\n\nRossanese et al. (2019) \nPopulation: Dogs <20 kg surgically treated for medial patellar luxation between \n2011–2016. \nThe preoperative luxation grade was classified as Grade I in one \nstifle Grade II in 51 stifles, Grade III in 42 stifles and Grade IV in six \nstifles. \nMedian weight 8.2 kg (total range 1.2–20.00 kg). \nSample size: 87 dogs met the inclusion criteria and a total of 100 surgical \nprocedures for medial patellar luxation were performed. \nIntervention details: \n• \nSurgery was performed by an experienced surgeon in 70 \ncases and by a resident under supervision in 30 cases. \n• \nNo dogs in the study required corrective osteotomy \ntechniques in the distal femur. \n• \nAll surgical procedures included lateral tibial tuberosity \ntransposition (TTT). \n• \nMedial soft tissue release was performed in 41 stifles (41%). \n• \nLateral imbrication was performed in 81 stifles (81%). \n• \nFemoral trochleoplasty was performed in 90 stifles (90%) \nand included a trochlear wedge recession in 68 stifles (76%) \nand a trochlear block recession in 22 stifles (24%). \n• \nMedial surgical time was 75 minutes (total range 25–195 \nminutes). \nStudy design: Retrospective observational study. \nOutcome studied: Complications in dogs weighing <20 kg surgically treated for medial \npatellar luxation and to determine risk factors associated with these \ncomplications. \nMain findings: \n(relevant to PICO question): \n• \n37 stifle joints developed postoperative complications. 12 \nwere considered minor (patellar reluxation Grade I, tibial \ntuberosity fracture, skin irritation), and 25 complications \nwere considered major (patellar luxation Grade II, surgical \nsite infection, wound dehiscence, removal of K-wires\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.517  \nNext review date: 22 Nov 2023 \np a g e | 6 of 13\n\norg/10.18849/ve.v7i3.517  \nNext review date: 22 Nov 2023 \np a g e | 6 of 13\n\nbecause of pin-fracture, pin-migration or seroma). Results \nrelevant to the PICO question included four cases of Grade I \npatellar luxation that did not require reoperation, and two \ncases of Grade II patella luxation that required revision \nsurgery. \n• \nResults from the study showed no significant difference in \ncomplication rates between the trochlear wedge technique \nand the trochlear block technique. \nLimitations: \n• \nRetrospective observational studies are considered to \nprovide low-level scientific evidence. \n• \nThe data reported relies on the accuracy of the medical \nrecord entries. The article identified long-term complications \nby reviewing the referring veterinarian clinical records and it \nmight be possible that some long-term complications were \nnot noticed by the referring veterinarian. We do not know if \nthe wedge trochleoplasty has a greater tendency to reluxate \nafter several years, when the dog is older and has less \nmuscle mass compared with the block trochleoplasty or vice \nversa, or if none of the techniques reluxates later on in life. \n• \nThe study did not address the progression of osteoarthritis. \n• \nThere were no standardised protocols to compare the \ndifferent techniques. All of them performed transposition of \nthe tibial tuberosity, but in some cases imbrication of the \njoint capsule and / or medial release of the joint capsule was \nalso performed. \n• \nSince the article only reviewed dogs less than 20 kg of body \nweight, we do not know if either of the two different \ntrochleoplasty techniques are more or less suitable for \nheavier dogs. \n• \nAs surgical procedures cannot be clearly differentiated from \nthe surgeons performing them, we do not know whether or \nnot we are investigating the surgeons or the procedures.\n\n• \nAs surgical procedures cannot be clearly differentiated from \nthe surgeons performing them, we do not know whether or \nnot we are investigating the surgeons or the procedures.\n\nBallatori et al. (2005) \nPopulation: Dogs with bilateral patellar luxation Grade I–IV brought to the \ndepartment with a complaint about secondary lameness, between \nthe years 2002–2004. \nSample size: Eight stifles in four dogs. Two dogs had medial patellar luxation and \ntwo dogs had lateral patellar luxation. \nIntervention details: \n• \nRadiographic examination was performed to exclude severe \nmalformation of the femur and tibia. \n• \nTwo types of surgeries were performed in the same dog: \ntrochlear block recession (TBR) in the right stifle and \ntrochlear wedge recession (TWR) in the left stifle. Both \nprocedures were performed in the same operation. \n• \nBoth of the dogs with medial patellar luxation also had tibial \ntubercle transposition (transposition of tibial tuberosity). \n• \nThe dogs had a postoperative period of physiotherapy with \ngradual return to normal activity in about 2 months.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.517  \nNext review date: 22 Nov 2023 \np a g e | 7 of 13\n\n• \nFollow-up period: 10–15 days postoperatively, 2–3 months \npostoperative and one case 12 months postoperatively.\n\nStudy design: Clinical case series.\n\norg/10.18849/ve.v7i3.517  \nNext review date: 22 Nov 2023 \np a g e | 7 of 13\n\n• \nFollow-up period: 10–15 days postoperatively, 2–3 months \npostoperative and one case 12 months postoperatively.Study design: Clinical case series.\n\nOutcome studied: Immediate postoperative computed tomography (CT): \n• \nDirect after the surgical procedure the stifles were examined \nwith CT in ventrodorsal position with semi-flexed pelvic \nlimbs. \n• \nThe CT images was used to estimate trochlear depth and \npatellar depth. \n• \nA line between the trochlear ridges and the centre of the \ntrochlear surface provided the measurement for the \ntrochlear depth. \n• \nThe patellar depth was measured as the percentage of the \nentire patellar volume positioned under the trochlear ridges. \nClinical evaluation 10–15 days postoperative: \n• \n10–15 days postoperative the Robert Jones bandage was \nremoved and clinical articular evaluation was assessed. \nClinical evaluation 2–3 months postoperative: \n• \n2–3 months after surgery the dogs were evaluated clinically \nregarding distribution of weight between the two hindlegs, \ntolerance during activity, muscular growth, patellar stability \nand pain. \nOne dog was examined with CT 12 months after surgery.\n\nOne dog was examined with CT 12 months after surgery.\n\nMain findings: \n(relevant to PICO question): \nCT findings: \n• \nThe CT showed correct autograft positioning and an \nadequate patellar lodging into the trochlear groove in all \ncases, independently of surgical technique used. \n• \nIn the central trochlea both TBR and TWR allowed the \nachievement of a good trochlear depth, but the patellar \ndepth was greater in the knee with rectangular recession. \n• \nThe proximal trochlear sulcus was wider and deeper with \nTBR than with TWR. \n• \nThe distal part of the subchondral wedge reached deeper in \nthe caudal portion of the femoral trochlea but the proximal \npart of the autograft wedge is entirely excavated in the \ncranial portion of the trochlea. \nFindings in the clinical evaluation: \n• \nIn the postoperative period no complications or relapses in \nany of the dogs were seen. \n• \n15 days after surgery the patients showed a fairly good \nability to distribute weight on both operated limbs but there \nwas more lameness and pain in articulations with TBR. \n• \nThe postoperative clinical picture 2–3 months after surgery \nwas similar in both joints, with the difference that the \npatella was stable in the flexed position but in extended \nposition a more lateromedial patellar instability was \nrecognised in stifles treated with trochlear wedge recession.\n\nLimitations: \n• \nVery small sample size. \n• \nCase series which is lower in evidence hierarchy.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.517  \nNext review date: 22 Nov 2023 \np a g e | 8 of 13\n\nLimitations: \n• \nVery small sample size.• \nCase series which is lower in evidence hierarchy.Veterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.517  \nNext review date: 22 Nov 2023 \np a g e | 8 of 13\n\n• \nThe group was not homogenous: the dogs with medial \npatellar luxation were of different age (18 months and 7 \nyears), different breeds (Pinscher and Springer Spaniel) the \ngrade of patellar luxation was not the same (ranging from \nGrade I–IV) and it was not the same grade between the legs \nin the same patient. \n• \nNo observer standardised clinical examination was \ndescribed, introducing observer bias. \n• \nIt does not say if the patellas luxated postoperatively, only \nthat the patella was more unstable in a lateromedial \ndirection in the trochlear wedge technique. \n• \nNo description of inclusion or exclusion criteria (except for \nradiographic examination for malformation of femur / tibia). \n• \nNo long-term follow-up was available to examine if any \nsurgical method acquired less degenerative joint disease.\n\nAppraisal, application and reflection\n\nOutcome as a measure of success might be difficult to objectively assess as it is influenced by many different \nfactors. When surgically correcting a medial patellar luxation one seldom only corrects the trochlea but most \noften also amends the soft tissues (imbrication of the joint capsule, release incision of the retinaculum or \ndesmotomi) in combination with longitudinal realignment of the tibial tuberosity relative to the trochlear \ngroove, by performing a tibial tuberosity transposition (Arthurs & Langley-Hobbs, 2006). Thus, it is difficult to \ndetermine if a trochlear block recession is superior to a wedge recession technique as the two different \nsurgical methods are not performed in isolation.\n\nThus, it is difficult to \ndetermine if a trochlear block recession is superior to a wedge recession technique as the two different \nsurgical methods are not performed in isolation.\n\nOnly three studies were identified addressing the PICO question; one biomechanical cadaver study, one \nretrospective observational study with 100 stifles and one case series including only two dogs. Thus, the \nevidence base for answering the query if block recession trochleoplasty is superior to wedge recession \ntrochleoplasty when treating medial patellar luxation is indeed very limited.\n\nOf the three studies, the biomechanical cadaver study (Johnson et al., 2001) has a more impressive \nimplementation with ex vivo testing of a stifle model that mimics patellar luxation, objective interobserver \nassessment and distinct variable measurements obtained from the computed tomography. The main \nlimitation with the cadaver study, when using it to answer the PICO question, is that results might not be \napplicable to live animals. Also, long-term effects cannot be evaluated.\n\nThe main \nlimitation with the cadaver study, when using it to answer the PICO question, is that results might not be \napplicable to live animals.Also, long-term effects cannot be evaluated.\n\nThe retrospective observational study by Rossanese et al. (2019) described complications following surgical \ncorrection of medial patellar luxation in dogs < 20 kg. In the study including 87 dogs and 100 stifles with \ndifferent degree of patellar luxation, complications were recognised in 37 stifles; 12 minor and 25 major. The \noutcome relevant to the PICO question showed no significant difference in complication rates between \ntrochlear wedge resection and trochlear block resection. One of the main limitations with this study, apart \nfrom it being a retrospective study, is that it lacks long-term follow-up. We do not know if any of the \ntrochleoplasty techniques had worse or more favourable outcome after several years. Also, to use this article \nto answer the question whether or not one surgical method is superior to another is difficult because, there \nare no kinematics, no description of repeated clinical examinations, no owner questionnaires, or second-look \narthroscopy.  However, a second look arthroscopy may not be of added value to the animal and thus not \nconsidered to be ethical.\n\nIn addition, time records to perform each procedure are not published. It is therefore not possible to comment \nif one procedure is less time consuming. This would be an interesting factor to compare.\n\nGenerally, retrospective case series are regarded low on the hierarchy of the evidence scale. In the case series \nby Ballatori et al. (2005), the number of dogs studied are by far too low to allow a meaningful comparison\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.517  \nNext review date: 22 Nov 2023 \np a g e | 9 of 13\n\nbetween the treatment methods as only two dogs had medial patellar luxation and also with different luxation \ndegree in the individual hindleg.\n\norg/10.18849/ve.v7i3.517  \nNext review date: 22 Nov 2023 \np a g e | 9 of 13\n\nbetween the treatment methods as only two dogs had medial patellar luxation and also with different luxation \ndegree in the individual hindleg.\n\nThe difference between the two surgical methods judged by the use of computed tomography is a result of the \ngeometry of the block and wedge. The articular width of the block and wedge are similar in the centre of the \ntrochlea, but the block maintains the articular surface width along the entire length of the trochlea compared \nto the wedge, which tapers to a point proximally and distally. After trochlear wedge recession, as the stifle is \nextended and the patella moves proximally, the patella may articulate with the non-recessed proximal femoral \ntrochlea instead of the recessed articular cartilage of the wedge, resulting in decreased patellar depth. The \nreduction in proximal patellar height could be essential in the clinical treatment of patellar luxation since the \npatella most often luxate in the proximal trochlea when the stifle is extended (Talcott et al., 2000, and Johnson \net al., 2001).\n\nIn dogs with medial patellar luxation, block trochlear recession results in a proximal deeper trochlear groove \nand a larger contact area between the proximal trochlea and patella, compared with the wedge trochlear \nrecession. In the extended stifle, the patella lies deeper in the proximal part of the joint (Johnson et al., 2001; \nand Talcott et al., 2000). Since no long-term studies comparing the amount of degenerative changes in the \nstifle joint and re-luxation rates between the two methods exist, and since the scientific information that \nactually compares the methods are very limited, no conclusion can be made from the existing evidence \nregarding whether or not a trochlear block recession is superior to trochlear wedge recession technique, when \ntreating medial patellar luxation in dogs.\n\nSince there are few reports comparing the two surgical methods, studies describing outcomes and\ncomplication rates with each technique might provide useful information when choosing between the two\nsurgical methods: Slocum et al. (1982) performed trochlear wedge surgery and showed excellent results in\n13/17 stifles and good results in the remaining cases. The follow-up period in that study was 12–29 months.\nTalcott et al. (2000) described 100 dogs after trochlear block surgery with positive short-term results: the\njoints were considered free of crepitus, limb function was improved and patellar stability achieved in the 6\nweek follow-up period. However, no long-term radiographic studies, histological analyses or second-look\narthroscopy was performed in that study.  A retrospective case series by Gallegos et al. (2016) described\nbilateral wedge trochleoplasty in 50 small breed dogs (100 stifle joints) with medial patellar luxation. In the\nstudy, 5/50 dogs (10%) had reluxation (Grade I) none of the dogs showed clinical lameness postoperatively.\nThe median follow-up time was 8 weeks. Arthurs & Langley-Hobbs (2006) reported retrospectively the clinical\noutcome in 109 dogs undergoing surgery because of lateral or medial patellar luxation. In the study, 74/107\n(69%) had trochlear wedge recession and 8/107 (7%) had trochlear block recession. However, no comparison\nwas made between the methods. In another retrospective study by Gibbons et al. (2006) trochlear wedge or\ntrochlear block was performed to treat patellar luxation in 70 large breed dogs (>15 kg). As no comparisons\nwere made between the two methods, no conclusions regarding the superiority of either method can be\ndrawn. Cashmore et al. (2014) reported a retrospective study regarding complications and risk factors\nassociated with surgical correction of medial patellar luxation in 124 dogs. Major complications (implant\nassociated, patellar luxation and persisting lameness, patellar tendon rupture etc.) occurred in 24/124 (19%) of\n\nMajor complications (implant\nassociated, patellar luxation and persisting lameness, patellar tendon rupture etc.) occurred in 24/124 (19%) of\ndogs. Although no comparisons were made between methods, a case of trochlear wedge displacement was\nreported. In a pilot study (Blackford-Winders et al., 2021) of 10 dogs, where the trochlear block recession\ntechnique was performed, the block autograft fractured in three cases. In a case report by Ellis & House (2021)\nthe trochlear block migrated distally 7 days postoperatively. In another case report (Chase & Farrell, 2010) a\nfracture of the lateral trochlear ridge following trochlear block recession was described.\n\nOne plausible reason for recurrent postoperative patellar luxation may, at least in part, be caused by \ninadequate appreciation of the underlying skeletal deformity and subsequent selection and application of \ncorrective surgery. Accurately measuring anatomic conformational abnormalities, for example identifying an \nextensive varus deformity or torsion in the distal femur, to better understand the deformities and \nsubsequently better tailor corrective surgery by performing different kind of osteotomies than \ntrochleoplasties, of the distal femur or proximal tibia, may result in lower frequency of reluxation.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.517  \nNext review date: 22 Nov 2023 \np a g e | 10 of 13\n\nIn conclusion, further studies are needed to evaluate both short-term and long-term clinical outcome in small \nbreed dogs with medial patellar luxation undergoing either trochlear wedge recession or trochlear block \nrecession. On one hand, the block technique might be considered to be more physiologically or anatomically \nappropriate creating a deeper trochlea proximally in the joint and a more profound femoropatellar contact. On \nthe other hand, the wedge technique is less invasive and thus conceivably associated with lower risk of \ncomplications. Whether or not the degree of subsequent osteoarthrosis might be influenced by the choice of \nsurgical technique is of major importance for the individual dog. The preference and previous experiences of \nthe veterinary surgeon are relevant issues when choosing which operation to perform until randomised and \ncontrolled trials in live animals and in comparable groups are performed.\n\nMethodology\n\nThe preference and previous experiences of \nthe veterinary surgeon are relevant issues when choosing which operation to perform until randomised and \ncontrolled trials in live animals and in comparable groups are performed.Methodology\n\nSearch strategy \nDatabases searched and dates \ncovered: \nCAB Abstracts on the OVID interface 1973–2021 week 46 \nPubMed accessed via the NCBI website 1920–Nov 2021 \nSearch terms: CAB Abstracts: \n1. (dog or dogs or canine or canines or canis or bitch or bitches \nor puppy or puppies).mp. or exp dogs/ or exp bitches/ or \nexp puppies/ or exp canidae/ or exp canis/ \n2. (lux* or MPL or dislocat*).mp. or exp dislocation/ \n3. ((trochle* or sulcoplas*) and (wedge or block or \nrecession)).mp. \n4. 1 and 2 and 3\n\nPubMed: \n1. dog or canine or bitch or puppy \n2. luxation or MPL or dislocation \n3. (trochleoplasty or sulcoplasty or trochlear) and (wedge or \nblock or recession) \n4. 1 and 2 and 3 \nDates searches performed: 22 Nov 2021\n\nExclusion / Inclusion criteria \nExclusion: Non-English language. \nInclusion: Articles concerning canine stifles with medial patellar luxation that \nhave undergone either trochlear block recession or trochlear wedge \nrecession.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.517  \nNext review date: 22 Nov 2023 \np a g e | 11 of 13\n\nSearch outcome \nDatabase \nNumber \nof results \nExcluded – \nWrong \nspecies \nExcluded – \nNot in \nEnglish \nExcluded – \nNot relevant \nto the PICO \nquestion \nTotal relevant \npapers \nCAB Abstracts \n48 \n2 \n4 \n40 \n2 \nPubMed \n20 \n1 \n0 \n18 \n1 \nTotal relevant papers when duplicates removed \n3\n\nCONFLICT OF INTEREST\n\nThe author declares no conflicts of interest.\n\nREFERENCES\n\n1. Arthurs, G.I. & Langley-Hobbs, S.J. (2006). Complications associated with corrective surgery for patellar\nluxation in 109 dogs. Veterinary Surgery. 35(6), 559–566. DOI: https://doi.org/10.1111/j.1532-\n950x.2006.00189.x\n2. Ballatori, C., Modenato, M. & Sbrana, S. (2005). Comparison of trochlear block recession and trochlear\nwedge recession in four dogs with bilateral patellar luxation. Annali della Facolta di Medicina\nVeterinaria di Pisa. 58, 295–303. [online]. Available\nfrom: http://eprints.adm.unipi.it/209/1/295.pdf [Accessed 26 May 2021].\n3. Blackford-Winders, C. L., Daubert, M., Rendahl, A. K. & Conzemius, M. G. (2021). Comparison of Semi-\nCylindrical Recession Trochleoplasty and Trochlear Block Recession for the Treatment of Canine Medial\nPatellar Luxation: A Pilot Study. Veterinary and Comparative Orthopaedics and Traumatology. 34(3),\n183–190. DOI: https://doi.org/10.1055/s-0040-1721107\n4. Cashmore, R. G., Havlicek, M., Perkins, N. R., James, D. R., Fearnside, S. M., Marchevsky, A. M. & Black,\nA. P. (2014). Major complications and risk factors associated with surgical correction of congenital\nmedial patellar luxation in 124 dogs. Veterinary and Comparative Orthopaedics and Traumatology.\n27(4), 263–270. DOI: https://doi.org/10.3415/vcot-13-08-0100\n5. Chase, D. & Farrell, M. (2010). Fracture of the lateral trochlear ridge after surgical stabilisation of\nmedial patellar luxation. Veterinary and Comparative Orthopaedics and Traumatology. 23(3), 203–208.\nDOI: https://doi.org/10.3415/vcot-09-11-0117\n6. Ellis, L.F. & House, A.K. (2021). Trochlear block recession sulcoplasty migration in a dog. Journal of\nSmall Animal Practice. 62(9),823. DOI: https://doi.org/10.1111/jsap.13340\n7. Gallegos, J., Unis, M., Roush, J. K. & Agulian, L. (2016). Postoperative Complications and Short-Term\nOutcome Following Single-Session Bilateral Corrective Surgery for Medial Patellar Luxation in Dogs\nWeighing <15 kg: 50 Cases (2009–2014). Veterinary Surgery. 45(7), 887–\n892.DOI: https://doi.org/10.1111/vsu.12525\n\n(2016).Postoperative Complications and Short-Term\nOutcome Following Single-Session Bilateral Corrective Surgery for Medial Patellar Luxation in Dogs\nWeighing <15 kg: 50 Cases (2009–2014).Veterinary Surgery.45(7), 887–\n892.DOI: https://doi.org/10.1111/vsu.12525\n8. Gibbons, S.E., Macias, C., Tonzing, M.A., Pinchbeck, G.L. & McKee, W.M. (2006). Patellar luxation in 70\nlarge breed dogs. The Journal of Small Animal Practice. 47(1), 3–9.\nDOI: https://doi.org/10.1111/j.1748-5827.2006.00004.x\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.517  \nNext review date: 22 Nov 2023 \np a g e | 12 of 13\n\n9. Johnson, A.L., Probst, C.W., Decamp, C.E., Rosenstein, D.S., Hauptman, J.G., Weaver, B.T. & Kern, T.L. \n(2001). Comparison of trochlear block recession and trochlear wedge recession for canine patellar \nluxation using a cadaver model. Veterinary Surgery. 30(2), 140–150. \nDOI: https://doi.org/10.1053/jvet.2001.21391 \n10. Talcott, K.W., Goring, R.L. & de Haan, J.J. (2000). Rectangular Recession Trochleoplasty for Treatment \nof Patellar Luxation in Dogs and Cats. Veterinary and Comparative Orthopaedics and Traumatology. \n13(1), 39-43. DOI: https://doi.org/10.1055/s-0038-1632628 \n11. Slocum, B., Slocum, D.B., Devine, T. & Boone, E. (1982). Wedge recession for treatment of recurrent \nluxation of the patella: a preliminary report. Clinic Orthopaedics and Related Research. (164), 48–53. \n12. Rossanese, M., German, A.J., Comerford, E., Pettitt, R., Tomlinson, A. & de Vicente, F. (2019). \nComplications Following Surgical Correction of Medial Patellar Luxation in Small-to-Medium-Size \nDogs. Veterinary and Comparative Orthopaedics and Traumatology. 32(4), 332–\n340. DOI: https://doi.org/10.1055/s-0039-1683872\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 7, Issue 3 \nDOI: https://doi.org/10.18849/ve.v7i3.517  \nNext review date: 22 Nov 2023 \np a g e | 13 of 13\n\nIntellectual Property Rights \nAuthors of Knowledge Summaries submitted to RCVS Knowledge for publication will retain \ncopyright in their work, and will be required to grant RCVS Knowledge a non-exclusive license \nof the rights of copyright in the materials including but not limited to the right to publish, re-\npublish, transmit, sell, distribute and otherwise use the materials in all languages and all \nmedia throughout the world, and to license or permit others to do so.\n\nDisclaimer \nKnowledge Summaries are a peer-reviewed article type which aims to answer a clinical \nquestion based on the best available current evidence. It does not override the responsibility \nof the practitioner. 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+{"text": "【兽医文献】\n标题: Combined cross-sectional and tangential margin evaluation of different tumor types in dogs and cats\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nDog attacks on livestock: insights\nfrom Swedish news articles and\nexperiences of farmers and\ninspectors\nSirkku Sarenbo * and Marie Doane\nDepartment of Biology and Environment, Faculty of Health and Life Sciences, University of Linnaeus,\nKalmar, Sweden\nIntroduction and purpose: Wild carnivore predation on Swedish livestock has\nbeen meticulously recorded, but dog attacks on livestock tend to be overlooked.\nThis study aimed to map the nature of dog attacks on livestock.\nMaterial and methods: Data was collected from Swedish news media articles,\nRovbase records in the region Västra Götaland, and electronic online surveys\nof farmers and official inspectors. In addition to descriptive statistics, monthly\nindices of dog attacks on livestock were calculated using data from Rovbase and\nnews media articles.\nResults: Half of the inspectors had inspected livestock attacked by dogs. Dogs\naccounted for 3.8% of all predator attacks inspected in Västra Götaland 2004–\n2024. Most dog attacks occurred during summer and fall. Sheep were most often\nsubjected to dog attack, followed by horses. Sheep were most often attacked by\ndogs on pastures, hens in their enclosed barnyard, and horses when ridden or\ndriven. The attacking dogs were most often unknown, loose, or unsupervised.\nDiscussion: Dog attacks impact both animal welfare and societal interests. The\nfarmers expressed emotional distress after the dog attacks. Misidentification can\nworsen wolf-related conflicts and misdirect public funds, while livestock owners\nmay face economic losses despite the dog owner’s sole responsibility. Further\nresearch is needed to assess dog attacks on livestock nationwide. However,\ntargeted measures such as predator-deterrent fencing, prolonged mandatory\nleashing of dogs, and enhanced monitoring of grazing livestock during hunting\nseasons could already be implemented.\nKEYWORDS\ndog attack, livestock protection, wild predators, public awareness, supervision of dogs\nIntroduction\nOur relationship with dogs (Canis lupus domesticus) is complex, and how dogs are\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nDog attacks on livestock: insights\nfrom Swedish news articles and\nexperiences of farmers and\ninspectors\nSirkku Sarenbo * and Marie Doane\nDepartment of Biology and Environment, Faculty of Health and Life Sciences, University of Linnaeus,\nKalmar, Sweden\nIntroduction and purpose: Wild carnivore predation on Swedish livestock has\nbeen meticulously recorded, but dog attacks on livestock tend to be overlooked.\nThis study aimed to map the nature of dog attacks on livestock.\nMaterial and methods: Data was collected from Swedish news media articles,\nRovbase records in the region Västra Götaland, and electronic online surveys\nof farmers and official inspectors. In addition to descriptive statistics, monthly\nindices of dog attacks on livestock were calculated using data from Rovbase and\nnews media articles.\nResults: Half of the inspectors had inspected livestock attacked by dogs. Dogs\naccounted for 3.8% of all predator attacks inspected in Västra Götaland 2004–\n2024. Most dog attacks occurred during summer and fall. Sheep were most often\nsubjected to dog attack, followed by horses. Sheep were most often attacked by\ndogs on pastures, hens in their enclosed barnyard, and horses when ridden or\ndriven. The attacking dogs were most often unknown, loose, or unsupervised.\nDiscussion: Dog attacks impact both animal welfare and societal interests. The\nfarmers expressed emotional distress after the dog attacks. Misidentification can\nworsen wolf-related conflicts and misdirect public funds, while livestock owners\nmay face economic losses despite the dog owner’s sole responsibility. Further\nresearch is needed to assess dog attacks on livestock nationwide. However,\ntargeted measures such as predator-deterrent fencing, prolonged mandatory\nleashing of dogs, and enhanced monitoring of grazing livestock during hunting\nseasons could already be implemented.\nKEYWORDS\ndog attack, livestock protection, wild predators, public awareness, supervision of dogs\nIntroduction\nOur relationship with dogs (Canis lupus domesticus) is complex, and how dogs are\n\nKEYWORDS\ndog attack, livestock protection, wild predators, public awareness, supervision of dogs\nIntroduction\nOur relationship with dogs (Canis lupus domesticus) is complex, and how dogs are\nperceived may vary depending on cultural, geographical, historical, and religious contexts (1).\nThere were 1.156 million registered dogs and 0.835 million registered dog owners in Sweden\nas of 31st December 2024 (2). These dogs are primarily used for companionship or hunting,\nbut they also play a role in agriculture, the tourism industry, healthcare, military, police, and\nSwedish Customs Service. To date, there are no known packs of feral dogs in Sweden (3).\nDog attacks on livestock have been documented in several studies; however, attacks carried\nout by dogs are still sometimes attributed to wolves and other wild predators (4, 5). The attacks\nhave been documented across the European continent, from northern to southern regions (6).\nHowever, accurate estimations of the total number of dog attacks remain challenging due to\nthe absence of a centralized database for reporting such events. Moreover, the topic has received\nOPEN ACCESS\nEDITED BY\nVerónica Roxana Flores,\nNational University of Comahue, Argentina\nREVIEWED BY\nAna Paola Yusti,\nAutonomous University of Chile, Chile\nEstela Maris,\nConicet Bahía Blanca, Argentina\n*CORRESPONDENCE\nSirkku Sarenbo\nsirkku.sarenbo@lnu.se\nRECEIVED 16 May 2025\nACCEPTED 15 September 2025\nPUBLISHED 31 October 2025\nCITATION\nSarenbo S and Doane M (2025) Dog attacks\non livestock: insights from Swedish news\narticles and experiences of farmers and\ninspectors.\nFront. Vet. Sci. 12:1629966.\ndoi: 10.3389/fvets.2025.1629966\nCOPYRIGHT\n© 2025 Sarenbo and Doane. This is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY). The use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\naccordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Original Research\n\nKEYWORDS\ndog attack, livestock protection, wild predators, public awareness, supervision of dogs\nIntroduction\nOur relationship with dogs (Canis lupus domesticus) is complex, and how dogs are\nperceived may vary depending on cultural, geographical, historical, and religious contexts (1).\nThere were 1.156 million registered dogs and 0.835 million registered dog owners in Sweden\nas of 31st December 2024 (2). These dogs are primarily used for companionship or hunting,\nbut they also play a role in agriculture, the tourism industry, healthcare, military, police, and\nSwedish Customs Service. To date, there are no known packs of feral dogs in Sweden (3).\nDog attacks on livestock have been documented in several studies; however, attacks carried\nout by dogs are still sometimes attributed to wolves and other wild predators (4, 5). The attacks\nhave been documented across the European continent, from northern to southern regions (6).\nHowever, accurate estimations of the total number of dog attacks remain challenging due to\nthe absence of a centralized database for reporting such events. Moreover, the topic has received\nOPEN ACCESS\nEDITED BY\nVerónica Roxana Flores,\nNational University of Comahue, Argentina\nREVIEWED BY\nAna Paola Yusti,\nAutonomous University of Chile, Chile\nEstela Maris,\nConicet Bahía Blanca, Argentina\n*CORRESPONDENCE\nSirkku Sarenbo\nsirkku.sarenbo@lnu.se\nRECEIVED 16 May 2025\nACCEPTED 15 September 2025\nPUBLISHED 31 October 2025\nCITATION\nSarenbo S and Doane M (2025) Dog attacks\non livestock: insights from Swedish news\narticles and experiences of farmers and\ninspectors.\nFront. Vet. Sci. 12:1629966.\ndoi: 10.3389/fvets.2025.1629966\nCOPYRIGHT\n© 2025 Sarenbo and Doane. This is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY). The use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\naccordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Original Research\n\nNo use, distribution or reproduction\nis permitted which does not comply with\nthese terms.TYPE  Original Research\nPUBLISHED  31 October 2025\nDOI  10.3389/fvets.2025.1629966\n\nNo use, distribution or reproduction\nis permitted which does not comply with\nthese terms.TYPE  Original Research\nPUBLISHED  31 October 2025\nDOI  10.3389/fvets.2025.1629966\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nlimited attention within the scientific literature. However, dogs pose a\nthreat to livestock and should be considered in mitigation plans when\nassessing the impact of predation by different predator species,\nincluding wild ones. Dogs can act both alone and in groups, and they\ncan be escaped pet dogs or feral dogs that form packs (5, 7). Dogs\nbehave differently than wild predators do. They generally run up the\nside of the prey animal and bite many times wherever they can, thighs,\nlegs, back, neck, usually low down, while wild predators are\nsignificantly more skilled and cause fewer bites on specific attack areas,\nusually higher than dog bites (8–10). Bite wounds may appear minor\non the surface, but they can cause significant internal damage and\ninfections in the underlying tissues that are not immediately visible. As\na result, bite injuries almost always require prompt antibiotic\ntreatment—or, in some cases, euthanasia—to ensure the animal’s\nwelfare (8). Sweden is so far free from rabies, but it has been shown that\nimported dogs from risk areas lack protection against rabies, which\nmay be due to the vaccination not being carried out correctly (11).\nThe relationship between rural communities and native\ncarnivores, such as wolves (Canis lupus), is complex and often\ncontentious. While wolves play a key ecological role, their presence\nposes significant challenges to rural livelihoods, especially in livestock\nfarming. Notably, their ecological importance tends to be more widely\nrecognized by urban societies, which can lead to differing perspectives\nand priorities (12). Recently, debates have intensified across Europe\nand the EU regarding the appropriate presence and population size of\nthe grey wolf (Canis lupus) (13–16). Political interests can intensify\ntensions by exaggerating the perceived impact of large carnivores (14),\nincreasing the potential for conflict with livestock producers.\nA Swedish legislative inquiry into competitive livestock\nproduction practices that ensure good animal welfare is currently\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nlimited attention within the scientific literature. However, dogs pose a\nthreat to livestock and should be considered in mitigation plans when\nassessing the impact of predation by different predator species,\nincluding wild ones. Dogs can act both alone and in groups, and they\ncan be escaped pet dogs or feral dogs that form packs (5, 7). Dogs\nbehave differently than wild predators do. They generally run up the\nside of the prey animal and bite many times wherever they can, thighs,\nlegs, back, neck, usually low down, while wild predators are\nsignificantly more skilled and cause fewer bites on specific attack areas,\nusually higher than dog bites (8–10). Bite wounds may appear minor\non the surface, but they can cause significant internal damage and\ninfections in the underlying tissues that are not immediately visible. As\na result, bite injuries almost always require prompt antibiotic\ntreatment—or, in some cases, euthanasia—to ensure the animal’s\nwelfare (8). Sweden is so far free from rabies, but it has been shown that\nimported dogs from risk areas lack protection against rabies, which\nmay be due to the vaccination not being carried out correctly (11).\nThe relationship between rural communities and native\ncarnivores, such as wolves (Canis lupus), is complex and often\ncontentious. While wolves play a key ecological role, their presence\nposes significant challenges to rural livelihoods, especially in livestock\nfarming. Notably, their ecological importance tends to be more widely\nrecognized by urban societies, which can lead to differing perspectives\nand priorities (12). Recently, debates have intensified across Europe\nand the EU regarding the appropriate presence and population size of\nthe grey wolf (Canis lupus) (13–16). Political interests can intensify\ntensions by exaggerating the perceived impact of large carnivores (14),\nincreasing the potential for conflict with livestock producers.\nA Swedish legislative inquiry into competitive livestock\nproduction practices that ensure good animal welfare is currently\n\nA Swedish legislative inquiry into competitive livestock\nproduction practices that ensure good animal welfare is currently\nopen to consultation. During this consultation, the presence of wolves\nwas cited as a factor deterring animal producers from expanding their\noperations (17). However, the involvement of dogs in the predation of\nsheep and other livestock has not been documented in Sweden as it\nhas in Italy (18–21), Spain (22), Poland (7) and Estonia (5), probably\nobscuring a more complex predation problem that need to\nbe addressed before promoting the expansion of livestock production.\nHere, we need to note that the term “predation” refers to the\ncondition in which an animal, a predator, kills and wholly or partially\nconsumes another animal (23). Feral dogs have been shown to hunt\nand consume their prey, which can consist of wild and domesticated\nanimals (24), while domestic dogs kept by humans as pets or for other\npurposes rarely consume their prey after hunting and killing them\n(8–10). Therefore, we used the term “attack” instead of “predation.”\nThere is strict liability for Swedish dog owners or keepers, which\nmeans that damage caused by a dog shall be compensated by its owner or\nkeeper, even if he or she did not cause damage [Section 19 Swedish Act\n(2007:1150) on the supervision of dogs and cats]. The owner or keeper is\nentitled to recover any damage paid by the owner from the person who\ncaused the damage. Section 16 states that during the period from March\n1st to August 20th, dogs must be kept under such supervision that they are\nprevented from running loose in areas with wildlife. During the rest of the\nyear, dogs must be kept under the supervision that they are prevented from\nchasing or pursuing wildlife when they are not used for hunting.\nExceptions from Section 16 are provided in Sections 16–19 of the Hunting\nordinance (1987:905): Hunting dogs that are suitable for their respective\nhunting purposes may be used by the hunting rights holder or by another\nperson with the hunting rights holder’s consent during permitted hunting\n\nA Swedish legislative inquiry into competitive livestock\nproduction practices that ensure good animal welfare is currently\nopen to consultation. During this consultation, the presence of wolves\nwas cited as a factor deterring animal producers from expanding their\noperations (17). However, the involvement of dogs in the predation of\nsheep and other livestock has not been documented in Sweden as it\nhas in Italy (18–21), Spain (22), Poland (7) and Estonia (5), probably\nobscuring a more complex predation problem that need to\nbe addressed before promoting the expansion of livestock production.\nHere, we need to note that the term “predation” refers to the\ncondition in which an animal, a predator, kills and wholly or partially\nconsumes another animal (23). Feral dogs have been shown to hunt\nand consume their prey, which can consist of wild and domesticated\nanimals (24), while domestic dogs kept by humans as pets or for other\npurposes rarely consume their prey after hunting and killing them\n(8–10). Therefore, we used the term “attack” instead of “predation.”\nThere is strict liability for Swedish dog owners or keepers, which\nmeans that damage caused by a dog shall be compensated by its owner or\nkeeper, even if he or she did not cause damage [Section 19 Swedish Act\n(2007:1150) on the supervision of dogs and cats]. The owner or keeper is\nentitled to recover any damage paid by the owner from the person who\ncaused the damage. Section 16 states that during the period from March\n1st to August 20th, dogs must be kept under such supervision that they are\nprevented from running loose in areas with wildlife. During the rest of the\nyear, dogs must be kept under the supervision that they are prevented from\nchasing or pursuing wildlife when they are not used for hunting.\nExceptions from Section 16 are provided in Sections 16–19 of the Hunting\nordinance (1987:905): Hunting dogs that are suitable for their respective\nhunting purposes may be used by the hunting rights holder or by another\nperson with the hunting rights holder’s consent during permitted hunting\n\nand hunting training. Additionally, special provisions regarding the\nsupervision of dogs are found in the Reindeer Husbandry act (1971:437).\nThere are also regulations aimed at protecting livestock. If a dog is found\nloose in an area where livestock are present, and if the dog cannot\nbe captured and it is necessary to prevent harm to the animals, the dog\nmay be killed by a person who owns or cares for the animals. The person\nwho killed the dog is required to report this to the police as soon as\npossible [Section 14 Act (2007:1150) on the supervision of dogs and cats].\nThe Swedish Wildlife Damage Center (VSC) provides annual\nstatistics on the damage caused by large grazing birds and large\npredators such as gray wolf (Canis lupus), lynx (Lynx lynx), brown\nbear (Ursus arctos), golden eagle (Aquila chrysaetos), and wolverine\n(Gulo gulo) (25). Dogs injured or killed by large carnivores are\nincluded in the VSC’s statistics and scientific studies (13) but cases\nwhere dogs are suspected to lie behind injuries or kill livestock are not\nsystematically tracked. We find it important that wild predators are\nnot held responsible for injuries caused by domestic dogs, and vice\nversa. It is equally important not to exaggerate the impact of wild\npredators on livestock losses. The role and number of dogs involved\nin attacks on livestock in Sweden are currently unknown and should\nbe investigated to ensure accurate and adequate mitigation efforts for\nattacks caused by domesticated dogs. In this study, information\ngathered from available sources was used to characterize the problem\nof dogs attacking, injuring, and killing livestock.\nThe aim of this study was to map the extent and nature of dog\nattacks on livestock in Sweden using four different information\nsources. Using multiple sources to study a new area increases the\ncredibility of the results and reduces the risk of error compared to\nrelying on a single source. It also provides a broader perspective and\nthus a more nuanced understanding of the issue and helps to confirm\nany emerging trends or patterns. For example, farmers offer first-hand\n\nand hunting training. Additionally, special provisions regarding the\nsupervision of dogs are found in the Reindeer Husbandry act (1971:437).\nThere are also regulations aimed at protecting livestock. If a dog is found\nloose in an area where livestock are present, and if the dog cannot\nbe captured and it is necessary to prevent harm to the animals, the dog\nmay be killed by a person who owns or cares for the animals. The person\nwho killed the dog is required to report this to the police as soon as\npossible [Section 14 Act (2007:1150) on the supervision of dogs and cats].\nThe Swedish Wildlife Damage Center (VSC) provides annual\nstatistics on the damage caused by large grazing birds and large\npredators such as gray wolf (Canis lupus), lynx (Lynx lynx), brown\nbear (Ursus arctos), golden eagle (Aquila chrysaetos), and wolverine\n(Gulo gulo) (25). Dogs injured or killed by large carnivores are\nincluded in the VSC’s statistics and scientific studies (13) but cases\nwhere dogs are suspected to lie behind injuries or kill livestock are not\nsystematically tracked. We find it important that wild predators are\nnot held responsible for injuries caused by domestic dogs, and vice\nversa. It is equally important not to exaggerate the impact of wild\npredators on livestock losses. The role and number of dogs involved\nin attacks on livestock in Sweden are currently unknown and should\nbe investigated to ensure accurate and adequate mitigation efforts for\nattacks caused by domesticated dogs. In this study, information\ngathered from available sources was used to characterize the problem\nof dogs attacking, injuring, and killing livestock.\nThe aim of this study was to map the extent and nature of dog\nattacks on livestock in Sweden using four different information\nsources. Using multiple sources to study a new area increases the\ncredibility of the results and reduces the risk of error compared to\nrelying on a single source. It also provides a broader perspective and\nthus a more nuanced understanding of the issue and helps to confirm\nany emerging trends or patterns. For example, farmers offer first-hand\n\nIt also provides a broader perspective and\nthus a more nuanced understanding of the issue and helps to confirm\nany emerging trends or patterns.For example, farmers offer first-hand\naccounts of attacks on their livestock, describing in their own words\nhow both they and their animals were affected. The County\nAdministrative Board, as an official authority, provides an institutional\nperspective on these incidents. Meanwhile, media coverage serves as\na complementary source, highlighting the social relevance of the issue\nand illustrating how it is reported over time, potentially revealing\nbroader trends. The research questions are as follows:\n•\t In what situations do dogs attack livestock?\n•\t What characteristics do dog attacks have, and which livestock\nspecies are the targets of the attacks?\n•\t Do the attacks occur by chance, or is there a discernible pattern?\nAre there similarities with studies from other countries?\n•\t What consequences did the attack have on animals subjected to\ndog attacks?\n•\t What are the possible societal consequences of dog attacks\non livestock?\nThese results could serve as a foundation for larger-scale studies,\nsuggest mitigation strategies, and support development of official\nstatistics on dog attacks on livestock and horses.\nMaterials and methods\nInformation about dog attacks on livestock species alpaca\n(Vicugna pacos), cattle (Bos taurus), goat (Capra hircus), horse (Equus\n\nIt also provides a broader perspective and\nthus a more nuanced understanding of the issue and helps to confirm\nany emerging trends or patterns.For example, farmers offer first-hand\naccounts of attacks on their livestock, describing in their own words\nhow both they and their animals were affected. The County\nAdministrative Board, as an official authority, provides an institutional\nperspective on these incidents. Meanwhile, media coverage serves as\na complementary source, highlighting the social relevance of the issue\nand illustrating how it is reported over time, potentially revealing\nbroader trends. The research questions are as follows:\n•\t In what situations do dogs attack livestock?\n•\t What characteristics do dog attacks have, and which livestock\nspecies are the targets of the attacks?\n•\t Do the attacks occur by chance, or is there a discernible pattern?\nAre there similarities with studies from other countries?\n•\t What consequences did the attack have on animals subjected to\ndog attacks?\n•\t What are the possible societal consequences of dog attacks\non livestock?\nThese results could serve as a foundation for larger-scale studies,\nsuggest mitigation strategies, and support development of official\nstatistics on dog attacks on livestock and horses.\nMaterials and methods\nInformation about dog attacks on livestock species alpaca\n(Vicugna pacos), cattle (Bos taurus), goat (Capra hircus), horse (Equus\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n03\nfrontiersin.org\ncaballus), pig (Sus scrofa), poultry (Gallus gallus, Meleagris gallopavo,\nAnser anser f. domesticus, Anas platyrhynchos), sheep (Ovis aries) was\ncollected from four sources: Swedish news media articles, an online\nelectronic survey to animal keepers, an online electronic survey to\ncounty administrative board inspectors who inspect predator-infested\ndomestic animals, and inspection records from the database\n“Rovbase.” The study was conducted between October 2023 and\nJanuary 2025. All data were processed using Microsoft Excel\n(Microsoft 365), which was also employed to produce descriptive\nstatistics and visual representations.\nSwedish news media articles\nSwedish news media articles were searched using three sources:\nGoogle, Media Archive, and Swedish Newspapers. The search was\nconducted using the term “dog attack” combined with different types\nof livestock. Google search generated varying numbers of articles and\nother media. To limit the scope, only the first 100 results were included\nfor each combination of search words. The Media Archive is Nordic’s\nlargest media database. It includes all major daily newspapers and\nmost regional newspapers as well as radio and television (26).\nSwedish Newspapers comprise a collection at the Swedish\nNational Library, including almost all newspapers published in\nSweden since 1645 (27). It allows browsing and free text search of\nSwedish newspapers (N = 1,470) from the 17th century onwards. The\nfollowing information was collected from the articles: the attacking\ndog (breed or type, number of dogs, owner known or unknown,\nowner present or absent during the attack), the victim (species,\nnumber of animals, consequences of the attack), date and time of the\nyear, and circumstances during the attack.\nSurveys for farmers and inspectors\nOur study do not include any information that can identify a\nperson or an animal. Separate surveys for farmers and inspectors were\ncreated using Artologik® Survey & Report software. The written\ninformed consent process is as follows. Both surveys contained an\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n03\nfrontiersin.org\ncaballus), pig (Sus scrofa), poultry (Gallus gallus, Meleagris gallopavo,\nAnser anser f. domesticus, Anas platyrhynchos), sheep (Ovis aries) was\ncollected from four sources: Swedish news media articles, an online\nelectronic survey to animal keepers, an online electronic survey to\ncounty administrative board inspectors who inspect predator-infested\ndomestic animals, and inspection records from the database\n“Rovbase.” The study was conducted between October 2023 and\nJanuary 2025. All data were processed using Microsoft Excel\n(Microsoft 365), which was also employed to produce descriptive\nstatistics and visual representations.\nSwedish news media articles\nSwedish news media articles were searched using three sources:\nGoogle, Media Archive, and Swedish Newspapers. The search was\nconducted using the term “dog attack” combined with different types\nof livestock. Google search generated varying numbers of articles and\nother media. To limit the scope, only the first 100 results were included\nfor each combination of search words. The Media Archive is Nordic’s\nlargest media database. It includes all major daily newspapers and\nmost regional newspapers as well as radio and television (26).\nSwedish Newspapers comprise a collection at the Swedish\nNational Library, including almost all newspapers published in\nSweden since 1645 (27). It allows browsing and free text search of\nSwedish newspapers (N = 1,470) from the 17th century onwards. The\nfollowing information was collected from the articles: the attacking\ndog (breed or type, number of dogs, owner known or unknown,\nowner present or absent during the attack), the victim (species,\nnumber of animals, consequences of the attack), date and time of the\nyear, and circumstances during the attack.\nSurveys for farmers and inspectors\nOur study do not include any information that can identify a\nperson or an animal. Separate surveys for farmers and inspectors were\ncreated using Artologik® Survey & Report software. The written\ninformed consent process is as follows. Both surveys contained an\n\nSeparate surveys for farmers and inspectors were\ncreated using Artologik® Survey & Report software.The written\ninformed consent process is as follows.Both surveys contained an\nintroduction letter in which the research project, project leader, and\naim of the project were presented. They were also informed that\nanswering the questions in the survey was voluntary and that no\npersonal data were collected. It was possible to answer only once from\nan IP address. Certain questions were mandatory to answer in order\nto proceed with or complete the survey (see Appendices 1 and 2). It\nwas possible to cancel participation in the survey at any time; however,\nbut a submitted reply could not be withdrawn.\nThe introduction letter of the survey for farmers explained that\n“dog attack” referred to an event where one or more animals were\ndisturbed/molested, injured or killed by one or more dogs (this did\nnot include, for example, intentional work or herding of animals with\ndogs). Additionally, if several dog attacks have affected the farmers’\nanimals, the response should be based on the attack that the farmer\njudged to be the most serious.\nBoth open and closed questions were included (Appendix 1). The\nsurvey was sent to the Swedish Sheep Breeding Association, who\npublished it in a weekly letter. It was also sent to Swedish alpaca\nowners through social media. The comment fields in several questions\noffered an opportunity to provide further comments if the event was\nnot covered by the options. The survey was open from 8th July to 15th\nSeptember 15, 2024.\nThe survey for inspectors (Appendix 2) was directed at the inspection\npersonnel on county administrative boards. A link to the survey was sent\nby email to the 21 county administrative boards of Sweden and to email\naddresses listed as inspection personnel at the Swedish Wildlife Damage\nCentre. The survey was open between 13th and 31st of January 2025.\nRovbase records from Västra Götaland\n2001–2024\nWe chose to investigate the occurrence of dog attacks in Västra\nGötaland because this county accounted for approximately one-third\n\nSeparate surveys for farmers and inspectors were\ncreated using Artologik® Survey & Report software.The written\ninformed consent process is as follows.Both surveys contained an\nintroduction letter in which the research project, project leader, and\naim of the project were presented. They were also informed that\nanswering the questions in the survey was voluntary and that no\npersonal data were collected. It was possible to answer only once from\nan IP address. Certain questions were mandatory to answer in order\nto proceed with or complete the survey (see Appendices 1 and 2). It\nwas possible to cancel participation in the survey at any time; however,\nbut a submitted reply could not be withdrawn.\nThe introduction letter of the survey for farmers explained that\n“dog attack” referred to an event where one or more animals were\ndisturbed/molested, injured or killed by one or more dogs (this did\nnot include, for example, intentional work or herding of animals with\ndogs). Additionally, if several dog attacks have affected the farmers’\nanimals, the response should be based on the attack that the farmer\njudged to be the most serious.\nBoth open and closed questions were included (Appendix 1). The\nsurvey was sent to the Swedish Sheep Breeding Association, who\npublished it in a weekly letter. It was also sent to Swedish alpaca\nowners through social media. The comment fields in several questions\noffered an opportunity to provide further comments if the event was\nnot covered by the options. The survey was open from 8th July to 15th\nSeptember 15, 2024.\nThe survey for inspectors (Appendix 2) was directed at the inspection\npersonnel on county administrative boards. A link to the survey was sent\nby email to the 21 county administrative boards of Sweden and to email\naddresses listed as inspection personnel at the Swedish Wildlife Damage\nCentre. The survey was open between 13th and 31st of January 2025.\nRovbase records from Västra Götaland\n2001–2024\nWe chose to investigate the occurrence of dog attacks in Västra\nGötaland because this county accounted for approximately one-third\n\nThe survey was open between 13th and 31st of January 2025.Rovbase records from Västra Götaland\n2001–2024\nWe chose to investigate the occurrence of dog attacks in Västra\nGötaland because this county accounted for approximately one-third\nof all predator attacks that received government compensation in 2023\n(25). Rovbase is a database that records the inventories of brown bears\n(Ursus arctos), wolverines (Gulo gulo), eurasian lynxes (Lynx lynx), gray\nwolves (Canis lupus), and golden eagles (Aquila chrysaetos) in Sweden\nand Norway. It also documents the damage these animals cause to\ndomestic animals, as well as details of hunting and other causes of\nmortality (28). When a suspected predator attack on livestock is\nreported to the county administrative board, an inspector inspects the\ncarcass, injuries and surrounding clues. The report is the basis for\ncompensation that the Swedish state pays to the livestock owner for the\ndamage caused by a wild predator. The inspectors recorded the type of\npredator, physical damage to the livestock or dog, and other relevant\nfacts. However, there were no statistical records or official monitoring\nof dog attacks. Some inspectors do note in the report and enter it into\nRovbase if they believe that the attack was performed by a dog instead\nof a wild predator (Mia Bisther, personal communication). To illustrate\nhow dog attacks on livestock may contribute to societal conflict\nsurrounding wolves, cases of dog attacks were included and compared\nalongside confirmed instances of wolf predation.\nMonthly indices of dog attacks\nUsing data from Rovbase and Swedish Media News, monthly\nindices of dog attacks on livestock were calculated: the average\nnumber of attacks per month was divided by the overall monthly\naverage across all months:\n\nThe survey was open between 13th and 31st of January 2025.Rovbase records from Västra Götaland\n2001–2024\nWe chose to investigate the occurrence of dog attacks in Västra\nGötaland because this county accounted for approximately one-third\nof all predator attacks that received government compensation in 2023\n(25). Rovbase is a database that records the inventories of brown bears\n(Ursus arctos), wolverines (Gulo gulo), eurasian lynxes (Lynx lynx), gray\nwolves (Canis lupus), and golden eagles (Aquila chrysaetos) in Sweden\nand Norway. It also documents the damage these animals cause to\ndomestic animals, as well as details of hunting and other causes of\nmortality (28). When a suspected predator attack on livestock is\nreported to the county administrative board, an inspector inspects the\ncarcass, injuries and surrounding clues. The report is the basis for\ncompensation that the Swedish state pays to the livestock owner for the\ndamage caused by a wild predator. The inspectors recorded the type of\npredator, physical damage to the livestock or dog, and other relevant\nfacts. However, there were no statistical records or official monitoring\nof dog attacks. Some inspectors do note in the report and enter it into\nRovbase if they believe that the attack was performed by a dog instead\nof a wild predator (Mia Bisther, personal communication). To illustrate\nhow dog attacks on livestock may contribute to societal conflict\nsurrounding wolves, cases of dog attacks were included and compared\nalongside confirmed instances of wolf predation.\nMonthly indices of dog attacks\nUsing data from Rovbase and Swedish Media News, monthly\nindices of dog attacks on livestock were calculated: the average\nnumber of attacks per month was divided by the overall monthly\naverage across all months:\n\n=\nm\nm\nTotal\nI\nÃ\n/ Ã\nIm = Index value for month m.\nÃm = Average number of attacks in month m.\nÃTotal = Overall average number of attacks per month across \nall months.\nResults\nSwedish news media\nGoogle generated 752 media articles, of which 49 were included in \nthe results. The Media Archive search generated 172 articles, of which \n23 were included in the results. A search in Swedish Newspapers for\n\n=\nm\nm\nTotal\nI\nÃ\n/ Ã\nIm = Index value for month m.\nÃm = Average number of attacks in month m.\nÃTotal = Overall average number of attacks per month across \nall months.\nResults\nSwedish news media\nGoogle generated 752 media articles, of which 49 were included in \nthe results. The Media Archive search generated 172 articles, of which \n23 were included in the results. A search in Swedish Newspapers for\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n‘dog attack’ from 2000 to 2024 yielded 2,886 pages (articles) in 100\nnewspapers, and a search with combined keywords and exclusion of\nnon-relevant articles and duplicates, 20 articles remained (Table 1).\nNinety-two news media articles about dog attacks on livestock\nwere found in three Swedish news media sources, involving eight\ndomestic animal species, totaling 507 animals (Figs. 1-2). Three articles\nreported that two different domestic animal species (cattle and pig;\ngeese and duck; geese and hens) were subjected to a dog attack on the\nsame occasion. The data from the news media articles shows a trend of\nincreasing dog attacks over time from 2008, while survey answers from\nsheep farmers are mixed, with a noticeable spike in 2023 (Figure 1).\nThe livestock species most often described in the news media\narticles were sheep (n = 38), followed by horses (n = 21), and hens\n(n = 20), and the livestock species most frequently attacked by dogs\nwere sheep, followed by hens and cattle (Figure 2).\nThe location of the dog attacks varied. Sheep were most often\nattacked in the pasture and hens in their enclosed barnyard or when\nthey were loose. Horses (21 attacks, 23 animals) were most frequently\nattacked outdoors when ridden or driven (Figure 3). In four cases, the\ndogs also turned on the rider or driver during the attack, causing bite\ninjuries to humans. In one case where the rider was saved by a\npasserby, the bite injuries on the rider were life-threatening.\nThe type and identity of 35% (n = 30) of the attacking dogs was\nknown. Thirteen percent (n = 12) were of fighting dog type (i.e., “Bull\ntype terrier,” according to the FCI nomenclature), e.g., American\nStaffordshire terrier “Amstaff,” Pitbull, American Bully, American\nBulldog, Staffordshire Bull terrier, or the article identified the dog as\na Bull type, e.g., Pitbull. Ten percent (n = 9) were of hunting type (e.g.,\nHound, Swedish Elkhound, Norwegian Elkhound, Drever, English\nPointer, or the article identified the dog as hunting dog). Seven of the\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n‘dog attack’ from 2000 to 2024 yielded 2,886 pages (articles) in 100\nnewspapers, and a search with combined keywords and exclusion of\nnon-relevant articles and duplicates, 20 articles remained (Table 1).\nNinety-two news media articles about dog attacks on livestock\nwere found in three Swedish news media sources, involving eight\ndomestic animal species, totaling 507 animals (Figs. 1-2). Three articles\nreported that two different domestic animal species (cattle and pig;\ngeese and duck; geese and hens) were subjected to a dog attack on the\nsame occasion. The data from the news media articles shows a trend of\nincreasing dog attacks over time from 2008, while survey answers from\nsheep farmers are mixed, with a noticeable spike in 2023 (Figure 1).\nThe livestock species most often described in the news media\narticles were sheep (n = 38), followed by horses (n = 21), and hens\n(n = 20), and the livestock species most frequently attacked by dogs\nwere sheep, followed by hens and cattle (Figure 2).\nThe location of the dog attacks varied. Sheep were most often\nattacked in the pasture and hens in their enclosed barnyard or when\nthey were loose. Horses (21 attacks, 23 animals) were most frequently\nattacked outdoors when ridden or driven (Figure 3). In four cases, the\ndogs also turned on the rider or driver during the attack, causing bite\ninjuries to humans. In one case where the rider was saved by a\npasserby, the bite injuries on the rider were life-threatening.\nThe type and identity of 35% (n = 30) of the attacking dogs was\nknown. Thirteen percent (n = 12) were of fighting dog type (i.e., “Bull\ntype terrier,” according to the FCI nomenclature), e.g., American\nStaffordshire terrier “Amstaff,” Pitbull, American Bully, American\nBulldog, Staffordshire Bull terrier, or the article identified the dog as\na Bull type, e.g., Pitbull. Ten percent (n = 9) were of hunting type (e.g.,\nHound, Swedish Elkhound, Norwegian Elkhound, Drever, English\nPointer, or the article identified the dog as hunting dog). Seven of the\n\ng., Pitbull.Ten percent (n = 9) were of hunting type (e.g.,\nHound, Swedish Elkhound, Norwegian Elkhound, Drever, English\nPointer, or the article identified the dog as hunting dog).Seven of the\n12 Bull type terrier owners were present at the attack, while all the\nother dog owners were absent, regardless of the type of dog. Almost\nall dogs (n = 32) that were known to the livestock owner, besides the\nBull type, were alone and loose when they attacked the animals. Also,\na suspected wolf/dog hybrid, an Irish rescue dog, an Alaskan\nmalamute and a herding-type dog were mentioned.\nSixty-five percent of the attacking dogs (n = 60) were unidentified\nor unknown. In 30 of these, news media articles provided no\nFIGURE 1\nDog attacks on livestock in Swedish news media articles 2006–2024 (n = 92) and dog attacks on livestock as reported by farmers (n = 25) in the survey\nfrom 2014 or earlier to 2024.\nTABLE 1  Article search from three news media sources.\nGoogle\nMedia archive\nSwedish newspapers\nSearch words in entire article\ndog attack AND pig OR swine OR cow OR horse OR sheep OR ewe OR lamb OR alpaca OR hen OR chicken OR goose OR duck\nNo of hits\n752\n172\n240\nExclusion criteria\ndog attack on other than livestock, wrong time span, not Swedish dog attack, non-relevant\nNo of relevant articles\n130\n44\n34\nExclusion criteria\nduplicate\nNo of relevant articles\n49\n23\n20\nTotal no of articles\n92\n\ng., Pitbull.Ten percent (n = 9) were of hunting type (e.g.,\nHound, Swedish Elkhound, Norwegian Elkhound, Drever, English\nPointer, or the article identified the dog as hunting dog).Seven of the\n12 Bull type terrier owners were present at the attack, while all the\nother dog owners were absent, regardless of the type of dog. Almost\nall dogs (n = 32) that were known to the livestock owner, besides the\nBull type, were alone and loose when they attacked the animals. Also,\na suspected wolf/dog hybrid, an Irish rescue dog, an Alaskan\nmalamute and a herding-type dog were mentioned.\nSixty-five percent of the attacking dogs (n = 60) were unidentified\nor unknown. In 30 of these, news media articles provided no\nFIGURE 1\nDog attacks on livestock in Swedish news media articles 2006–2024 (n = 92) and dog attacks on livestock as reported by farmers (n = 25) in the survey\nfrom 2014 or earlier to 2024.\nTABLE 1  Article search from three news media sources.\nGoogle\nMedia archive\nSwedish newspapers\nSearch words in entire article\ndog attack AND pig OR swine OR cow OR horse OR sheep OR ewe OR lamb OR alpaca OR hen OR chicken OR goose OR duck\nNo of hits\n752\n172\n240\nExclusion criteria\ndog attack on other than livestock, wrong time span, not Swedish dog attack, non-relevant\nNo of relevant articles\n130\n44\n34\nExclusion criteria\nduplicate\nNo of relevant articles\n49\n23\n20\nTotal no of articles\n92\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n05\nfrontiersin.org\ninformation about the dog’s owner or whether the dog was alone or \nloose during the attack. In 16 cases, the articles did not specify the breed \nor type of the attacking dog but mentioned that the dog was loose and \nalone. In eight cases, the owner was known, but the dog responsible for \nthe attack was not identified (Figure 4). News media articles indicated \nthat livestock and horses frequently suffer not only from bite injuries, \nbut also from being chased by dogs during dog attacks. For example, \nanimals were sometimes driven through barriers, resulting in injuries \nalso from their attempts to escape, such as running through fencing (not \nquantified in the study). In seven cases, dogs either broke free from the \nleash or escaped from their owners (e.g., from cars with open windows) \nand attacked the livestock with the owner present at the attack (Figure 4).\nFIGURE 2\nNumber of dog attacks (n = 95) and number of injured or killed animals per livestock species (in total 507 animals) according to Swedish news media \narticles (n = 92) 2006–2024. One attack can involve several livestock species and several injured or killed animals.\nFIGURE 3\nCircumstances during dog attacks on sheep, cattle, hen and horses according to Swedish news media articles (2006–2024).\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n05\nfrontiersin.org\ninformation about the dog’s owner or whether the dog was alone or \nloose during the attack. In 16 cases, the articles did not specify the breed \nor type of the attacking dog but mentioned that the dog was loose and \nalone. In eight cases, the owner was known, but the dog responsible for \nthe attack was not identified (Figure 4). News media articles indicated \nthat livestock and horses frequently suffer not only from bite injuries, \nbut also from being chased by dogs during dog attacks. For example, \nanimals were sometimes driven through barriers, resulting in injuries \nalso from their attempts to escape, such as running through fencing (not \nquantified in the study). In seven cases, dogs either broke free from the \nleash or escaped from their owners (e.g., from cars with open windows) \nand attacked the livestock with the owner present at the attack (Figure 4).\nFIGURE 2\nNumber of dog attacks (n = 95) and number of injured or killed animals per livestock species (in total 507 animals) according to Swedish news media \narticles (n = 92) 2006–2024. One attack can involve several livestock species and several injured or killed animals.\nFIGURE 3\nCircumstances during dog attacks on sheep, cattle, hen and horses according to Swedish news media articles (2006–2024).\n\nOne attack can involve several livestock species and several injured or killed animals.FIGURE 3\nCircumstances during dog attacks on sheep, cattle, hen and horses according to Swedish news media articles (2006–2024).\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nRovbase records from Västra Götaland \n2001–2024\nIn total, 1,497 inspection records of predator attacks were \ndocumented by county administrative board inspectors between 2001 \nand 2024 in the Region Västra Götaland. Dogs accounted for 3.8% \n(n = 57) of all attacks that were inspected (Figure 5). A single attack \ncould involve multiple domestic animal species, but the specific \nspecies detected concurrently were not specified in the report. \nInspectors reported an average of 2.59 ± 1.62 dog attacks per year, \nwith a median of two dog attacks (IQR 1).\nThe ratio between dog and wolf attacks on livestock reported in \nRovbase varied substantially between years in Västra Götaland (range \n0–50%, Figure  6). When wolf and dog attacks on livestock were \nFIGURE 4\nAttacks by unknown dogs (n = 60), including whether the owner was known and if the dog was alone and loose during the attack. Swedish news \nmedia articles 2006–2024.\nFIGURE 5\nLivestock attacked by dogs. Identification was performed by the county administrative board inspectors of Västra Götaland and recorded in Rovbase \n2004–2024 (n = 56). One attack on rabbits in 2015 is not included in the figure.\n\nOne attack can involve several livestock species and several injured or killed animals.FIGURE 3\nCircumstances during dog attacks on sheep, cattle, hen and horses according to Swedish news media articles (2006–2024).\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nRovbase records from Västra Götaland \n2001–2024\nIn total, 1,497 inspection records of predator attacks were \ndocumented by county administrative board inspectors between 2001 \nand 2024 in the Region Västra Götaland. Dogs accounted for 3.8% \n(n = 57) of all attacks that were inspected (Figure 5). A single attack \ncould involve multiple domestic animal species, but the specific \nspecies detected concurrently were not specified in the report. \nInspectors reported an average of 2.59 ± 1.62 dog attacks per year, \nwith a median of two dog attacks (IQR 1).\nThe ratio between dog and wolf attacks on livestock reported in \nRovbase varied substantially between years in Västra Götaland (range \n0–50%, Figure  6). When wolf and dog attacks on livestock were \nFIGURE 4\nAttacks by unknown dogs (n = 60), including whether the owner was known and if the dog was alone and loose during the attack. Swedish news \nmedia articles 2006–2024.\nFIGURE 5\nLivestock attacked by dogs. Identification was performed by the county administrative board inspectors of Västra Götaland and recorded in Rovbase \n2004–2024 (n = 56). One attack on rabbits in 2015 is not included in the figure.\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n07\nfrontiersin.org\ncombined, dog attacks accounted for an annual mean of 19.5%\n(SD = 13.5%), with a median of 16.5% (IQR 15.3), according to\nrecords from Rovbase in Västra Götaland.\nTime of the year for dog attacks\nBoth the Swedish news media articles and the records of the\ncounty administrative board inspectors in Rovbase show that most\ndog attacks on livestock occur during the summer and fall months\n(Figure 7). Data from Rovbase indicate seasonal peaks in activity, with\nmonthly indices (range 0.215–2.15) in June (1.92), August (1.33),\nOctober (2.15), and November (1.33), while the dog attacks in Swedish\nnews media articles have a strong peak in September (2.60) and a\nweaker peaks in May (1.29), July (1.17) and October (1.17).\nSurvey for inspectors\nThe survey of inspectors had a response rate of 47% (n = 56). The\ninspectors’ experience with inspections varied from having conducted\n1–10 inspections (n = 15, 25%) to more than 200 inspections (n = 9,\n16%), and 45% of the inspectors (n = 25) conducted between 11 and\n50 inspections. The inspectors had experience in inspecting a wide\nrange of animal species, including alpacas, bees (colonies), cats, cattle,\nhens, deer, dogs, ducks, horses and ponies, fallow deer, foxes, goats,\nmouflon sheep, rabbits, red deer, reindeer, sheep, and turkeys.\nSixty-four percent of the inspectors (n = 36) indicated that the\nlivestock species most attacked by dogs was sheep. However, 20\npercent of the inspectors (n = 11) omitted answering the question,\nmost of them referring to a lack of experience. While 55% of the\ninspectors attributed 0–10% of their inspections to dog attacks, 9%\nreported that 10–20% were due to dog attacks and 36% claimed to not\nhave inspected any dog attacks on livestock at all.\nSurvey for farmers\nTwenty-five farmers who had experienced dog attacks on their\nlivestock responded to the survey. Of these, 76% (n = 19) were\nsheep farmers, 20% (n = 5) were alpaca farmers, and 4% (n = 1)\nwere goat farmers. The herds subjected to dog attacks comprised\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n07\nfrontiersin.org\ncombined, dog attacks accounted for an annual mean of 19.5%\n(SD = 13.5%), with a median of 16.5% (IQR 15.3), according to\nrecords from Rovbase in Västra Götaland.\nTime of the year for dog attacks\nBoth the Swedish news media articles and the records of the\ncounty administrative board inspectors in Rovbase show that most\ndog attacks on livestock occur during the summer and fall months\n(Figure 7). Data from Rovbase indicate seasonal peaks in activity, with\nmonthly indices (range 0.215–2.15) in June (1.92), August (1.33),\nOctober (2.15), and November (1.33), while the dog attacks in Swedish\nnews media articles have a strong peak in September (2.60) and a\nweaker peaks in May (1.29), July (1.17) and October (1.17).\nSurvey for inspectors\nThe survey of inspectors had a response rate of 47% (n = 56). The\ninspectors’ experience with inspections varied from having conducted\n1–10 inspections (n = 15, 25%) to more than 200 inspections (n = 9,\n16%), and 45% of the inspectors (n = 25) conducted between 11 and\n50 inspections. The inspectors had experience in inspecting a wide\nrange of animal species, including alpacas, bees (colonies), cats, cattle,\nhens, deer, dogs, ducks, horses and ponies, fallow deer, foxes, goats,\nmouflon sheep, rabbits, red deer, reindeer, sheep, and turkeys.\nSixty-four percent of the inspectors (n = 36) indicated that the\nlivestock species most attacked by dogs was sheep. However, 20\npercent of the inspectors (n = 11) omitted answering the question,\nmost of them referring to a lack of experience. While 55% of the\ninspectors attributed 0–10% of their inspections to dog attacks, 9%\nreported that 10–20% were due to dog attacks and 36% claimed to not\nhave inspected any dog attacks on livestock at all.\nSurvey for farmers\nTwenty-five farmers who had experienced dog attacks on their\nlivestock responded to the survey. Of these, 76% (n = 19) were\nsheep farmers, 20% (n = 5) were alpaca farmers, and 4% (n = 1)\nwere goat farmers. The herds subjected to dog attacks comprised\n\nOf these, 76% (n = 19) were\nsheep farmers, 20% (n = 5) were alpaca farmers, and 4% (n = 1)\nwere goat farmers.The herds subjected to dog attacks comprised\nbetween 2 and 175 individuals, with a mean population size of\n19 ± 36.33 (SD). The total number of injured animals was 53, and\nthe mean affected livestock by these dog attacks were 2.1 ± 1.59\n(SD) animals with a median of 2 animals. Half of the farmers\n(n = 13, 52%) reported that animals were killed during the dog\nattack. In one case, 6–10 animals were killed; in the remaining\nfatal cases, between one and five animals were killed with a mean\n1.03 ± 0.88 (SD).\nEighty-four percent of the attacking dogs (n = 21) was\nowned by a neighbor. Twelve percent of farmers (n = 3) reported\nthat the dog owner was unknown. Most of the dogs reported\nby farmers in the survey as involved in attacks were identified\nFIGURE 6\nNumber of wolf (n = 265) and dog attacks (n = 56) recorded to the Rovbase by County Administrative Board Inspectors (Västra Götaland, 2001–2024),\nalong with the percentage of these attacks attributed to dogs, expressed as a share of the total (wolf and dog) attacks (dashed line).\n\nOf these, 76% (n = 19) were\nsheep farmers, 20% (n = 5) were alpaca farmers, and 4% (n = 1)\nwere goat farmers.The herds subjected to dog attacks comprised\nbetween 2 and 175 individuals, with a mean population size of\n19 ± 36.33 (SD). The total number of injured animals was 53, and\nthe mean affected livestock by these dog attacks were 2.1 ± 1.59\n(SD) animals with a median of 2 animals. Half of the farmers\n(n = 13, 52%) reported that animals were killed during the dog\nattack. In one case, 6–10 animals were killed; in the remaining\nfatal cases, between one and five animals were killed with a mean\n1.03 ± 0.88 (SD).\nEighty-four percent of the attacking dogs (n = 21) was\nowned by a neighbor. Twelve percent of farmers (n = 3) reported\nthat the dog owner was unknown. Most of the dogs reported\nby farmers in the survey as involved in attacks were identified\nFIGURE 6\nNumber of wolf (n = 265) and dog attacks (n = 56) recorded to the Rovbase by County Administrative Board Inspectors (Västra Götaland, 2001–2024),\nalong with the percentage of these attacks attributed to dogs, expressed as a share of the total (wolf and dog) attacks (dashed line).\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nas hunting dogs (52%; n = 13). Eighty-four percent of the farmers\n(n = 21) did not report dog attacks to any authority. Three\nfarmers (12%) reported a dog attack on the county administrative\nboard, and one farmer (4%) reported it to the police. Eighteen\nfarmers (72%) in the survey expressed emotions such as anger,\nsadness, and concern for their animals and how heartbreaking it\nwas to find a dead animal or a mutilated animal still fighting for\nits life.\nDiscussion\nIn what situations do dogs attack livestock\nand horses?\nA prerequisite for dog attacks is that dogs are given the opportunity\nto gain access to animals. For example, a large part of broiler and pork\nproduction takes place in enclosed and highly controlled spaces where\ndogs do not have access. Therefore, it is often a small-scale environment\nwhere animals that are affected by dog attacks are kept outdoors, in\naddition to animals used for recreation. According to news media\narticles, sheep are most often attacked by dogs in pastures, hen\noutdoors in enclosed barnyards, or loose in yards in backyard systems.\nPastures interspersed with trees or vegetative cover and flocks of sheep\nwithout supervision by either shepherds or guard dogs have been\nshown to be more vulnerable to predation (18), as could also be case in\nthe incidents presented in this study.\nOne fifth of the responding farmers were alpaca keepers. Dog\nattacks on alpacas warrants more attention since the alpaca industry\nin Sweden is currently expanding; the number of alpaca facilities\nincreased from 169 in December 2021 to 313 in July 2025, and the\nalpaca population grew from 2,113 to 3,313 during the same period\n(The Animal Registry Unit at the Swedish Board of Agriculture,\npersonal communication, 2025). Young alpacas (crias) are considered\nparticularly vulnerable to dog attacks, although adult alpacas may\nalso suffer significant harm. In addition to bite-related injuries,\nalpacas may develop stress- or capture-induced myopathy as a result\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nas hunting dogs (52%; n = 13). Eighty-four percent of the farmers\n(n = 21) did not report dog attacks to any authority. Three\nfarmers (12%) reported a dog attack on the county administrative\nboard, and one farmer (4%) reported it to the police. Eighteen\nfarmers (72%) in the survey expressed emotions such as anger,\nsadness, and concern for their animals and how heartbreaking it\nwas to find a dead animal or a mutilated animal still fighting for\nits life.\nDiscussion\nIn what situations do dogs attack livestock\nand horses?\nA prerequisite for dog attacks is that dogs are given the opportunity\nto gain access to animals. For example, a large part of broiler and pork\nproduction takes place in enclosed and highly controlled spaces where\ndogs do not have access. Therefore, it is often a small-scale environment\nwhere animals that are affected by dog attacks are kept outdoors, in\naddition to animals used for recreation. According to news media\narticles, sheep are most often attacked by dogs in pastures, hen\noutdoors in enclosed barnyards, or loose in yards in backyard systems.\nPastures interspersed with trees or vegetative cover and flocks of sheep\nwithout supervision by either shepherds or guard dogs have been\nshown to be more vulnerable to predation (18), as could also be case in\nthe incidents presented in this study.\nOne fifth of the responding farmers were alpaca keepers. Dog\nattacks on alpacas warrants more attention since the alpaca industry\nin Sweden is currently expanding; the number of alpaca facilities\nincreased from 169 in December 2021 to 313 in July 2025, and the\nalpaca population grew from 2,113 to 3,313 during the same period\n(The Animal Registry Unit at the Swedish Board of Agriculture,\npersonal communication, 2025). Young alpacas (crias) are considered\nparticularly vulnerable to dog attacks, although adult alpacas may\nalso suffer significant harm. In addition to bite-related injuries,\nalpacas may develop stress- or capture-induced myopathy as a result\n\nYoung alpacas (crias) are considered\nparticularly vulnerable to dog attacks, although adult alpacas may\nalso suffer significant harm.In addition to bite-related injuries,\nalpacas may develop stress- or capture-induced myopathy as a result\nof such attacks, a condition that in severe cases can be fatal (29).\nAttacks on horses are different as horses are more often attacked by\ndogs when ridden or driven than when grazing on pastures. In Sweden,\n76% of all horses and 71% of locations housing horses were in or near\nurban areas in 2016 (30). Therefore, it is reasonable to assume that\nencounters between riders and dogs are common in woodlands and\nrecreational areas near urban environments. Additional danger was\nFIGURE 7\nTime of the year dog attacks on livestock as reported in Swedish news media (n = 92) and in Rovbase, reported by county administrative board\ninspectors (n = 56).\n\nYoung alpacas (crias) are considered\nparticularly vulnerable to dog attacks, although adult alpacas may\nalso suffer significant harm.In addition to bite-related injuries,\nalpacas may develop stress- or capture-induced myopathy as a result\nof such attacks, a condition that in severe cases can be fatal (29).\nAttacks on horses are different as horses are more often attacked by\ndogs when ridden or driven than when grazing on pastures. In Sweden,\n76% of all horses and 71% of locations housing horses were in or near\nurban areas in 2016 (30). Therefore, it is reasonable to assume that\nencounters between riders and dogs are common in woodlands and\nrecreational areas near urban environments. Additional danger was\nFIGURE 7\nTime of the year dog attacks on livestock as reported in Swedish news media (n = 92) and in Rovbase, reported by county administrative board\ninspectors (n = 56).\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nthat the riders were also kicked and trampled by the horses. Dogs\nescaping from cars or breaking free from the leash must be considered\nhighly motivated and possibly different in behavior, and it could\nbe argued that they actively seek opportunities to attack. In contrast,\nmany livestock attacks may result from loose dogs that encounter\nlivestock by chance, turning the situation into an opportunity for attack.\nWhat characteristics do dogs that attack\nhave, and which animals are the targets of\nthe attacks?\nAmong farmers, most of the dogs reported were hunting dogs, while\nin the media, hunting dogs were less reported. Hunting dogs have been\nmentioned as aggressors in earlier publications (8, 10). The true number\nof hunting dogs involved in attacks is likely underreported. In news\nmedia articles, most attacking dogs were unidentified, and no\ninformation was available for half of those cases. Given how many dogs\nwere unidentifiable and lacked descriptive details, it’s probable that\nhunting dogs were involved more often than the data suggests. The\naffected livestock were found to be injured or dead without the animal\ncaregiver seeing the attacking dog. However, signs of dog attacks were\nalso evident. Dogs were identified in the remaining cases. Whether the\ndog owner was identified or known might have been kept out of the\nmedia, perhaps to maintain good neighbor relationships.\nAccording to all four sources of this study, sheep were the most\ncommon animals killed or injured by dogs. The proportion of livestock\nanimals killed by dogs, as reported by the different sources, ranged\nfrom 41 to 64%. This agrees with reports of dog attacks on livestock in\nother countries (31). Dogs are the primary cause of sheep and goat\nlosses on small-scale livestock farms in Chile (32). Damage by predators\nto adult horses is quite uncommon compared to that in sheep (10, 33,\n34). If rapid flight is not possible, horses can defend themselves against\ncanid predators with foreleg strikes (35). In several media articles, the\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nthat the riders were also kicked and trampled by the horses. Dogs\nescaping from cars or breaking free from the leash must be considered\nhighly motivated and possibly different in behavior, and it could\nbe argued that they actively seek opportunities to attack. In contrast,\nmany livestock attacks may result from loose dogs that encounter\nlivestock by chance, turning the situation into an opportunity for attack.\nWhat characteristics do dogs that attack\nhave, and which animals are the targets of\nthe attacks?\nAmong farmers, most of the dogs reported were hunting dogs, while\nin the media, hunting dogs were less reported. Hunting dogs have been\nmentioned as aggressors in earlier publications (8, 10). The true number\nof hunting dogs involved in attacks is likely underreported. In news\nmedia articles, most attacking dogs were unidentified, and no\ninformation was available for half of those cases. Given how many dogs\nwere unidentifiable and lacked descriptive details, it’s probable that\nhunting dogs were involved more often than the data suggests. The\naffected livestock were found to be injured or dead without the animal\ncaregiver seeing the attacking dog. However, signs of dog attacks were\nalso evident. Dogs were identified in the remaining cases. Whether the\ndog owner was identified or known might have been kept out of the\nmedia, perhaps to maintain good neighbor relationships.\nAccording to all four sources of this study, sheep were the most\ncommon animals killed or injured by dogs. The proportion of livestock\nanimals killed by dogs, as reported by the different sources, ranged\nfrom 41 to 64%. This agrees with reports of dog attacks on livestock in\nother countries (31). Dogs are the primary cause of sheep and goat\nlosses on small-scale livestock farms in Chile (32). Damage by predators\nto adult horses is quite uncommon compared to that in sheep (10, 33,\n34). If rapid flight is not possible, horses can defend themselves against\ncanid predators with foreleg strikes (35). In several media articles, the\n\nDamage by predators\nto adult horses is quite uncommon compared to that in sheep (10, 33,\n34).If rapid flight is not possible, horses can defend themselves against\ncanid predators with foreleg strikes (35).In several media articles, the\ndog attacked not only the horse but also the horseback rider. This\nmakes such incidents distinct, as both animals and humans were\nvictims. Cattle and cows are known to attack dogs even if they do not\nhave calves on their sides (36). Therefore, predation and injury in adult\ncattle are quite unusual. In the sheep farming sector, however, dog\nattacks may result in decreased productivity, underutilized pasture\nresources, and increased financial burdens for individual producers due\nto necessary investments in protective measures.\nDo the attacks occur by chance, or is there a\ndiscernible pattern – and are there similarities\nin similar studies from other countries?\nMost dog attacks occur during the summer and fall months\nwhen livestock are grazing outdoors or in paddocks. They are\nusually performed by individual dogs, but dogs can cooperate in\npairs or groups. Two dogs cooperated when killing four cattle\nduring the same attack in Italy (19). A New Mexican sheep flock,\nconsisting of 40 pregnant ewes, was also attacked by two dogs.\nDuring a dog attack, four ewes were killed, five later died due to\ncomplications from injuries, and over 75% of the remaining flock\nsustained injuries (37). In about 50% of the cases reported in\nSwedish news media articles, there was no information about\nattacking dogs. This suggests that livestock owners often find\ncarcasses and injured animals after an attack. Many of these\nattacks might be opportunistic, as attacking dogs are often loose\nand unsupervised. In Sweden, it is against the law to have dogs\nloose during the summer months [Section 16 Swedish Act\n(2007:1150) on the supervision of dogs and cats]. The fact that\nmany of the attacks occurred during these months is troublesome\nand illustrates a disregard for the law. It is also worth noting that\nnone of the farmers seemed to have exercised their right to kill\n\nDamage by predators\nto adult horses is quite uncommon compared to that in sheep (10, 33,\n34).If rapid flight is not possible, horses can defend themselves against\ncanid predators with foreleg strikes (35).In several media articles, the\ndog attacked not only the horse but also the horseback rider. This\nmakes such incidents distinct, as both animals and humans were\nvictims. Cattle and cows are known to attack dogs even if they do not\nhave calves on their sides (36). Therefore, predation and injury in adult\ncattle are quite unusual. In the sheep farming sector, however, dog\nattacks may result in decreased productivity, underutilized pasture\nresources, and increased financial burdens for individual producers due\nto necessary investments in protective measures.\nDo the attacks occur by chance, or is there a\ndiscernible pattern – and are there similarities\nin similar studies from other countries?\nMost dog attacks occur during the summer and fall months\nwhen livestock are grazing outdoors or in paddocks. They are\nusually performed by individual dogs, but dogs can cooperate in\npairs or groups. Two dogs cooperated when killing four cattle\nduring the same attack in Italy (19). A New Mexican sheep flock,\nconsisting of 40 pregnant ewes, was also attacked by two dogs.\nDuring a dog attack, four ewes were killed, five later died due to\ncomplications from injuries, and over 75% of the remaining flock\nsustained injuries (37). In about 50% of the cases reported in\nSwedish news media articles, there was no information about\nattacking dogs. This suggests that livestock owners often find\ncarcasses and injured animals after an attack. Many of these\nattacks might be opportunistic, as attacking dogs are often loose\nand unsupervised. In Sweden, it is against the law to have dogs\nloose during the summer months [Section 16 Swedish Act\n(2007:1150) on the supervision of dogs and cats]. The fact that\nmany of the attacks occurred during these months is troublesome\nand illustrates a disregard for the law. It is also worth noting that\nnone of the farmers seemed to have exercised their right to kill\n\nThe fact that\nmany of the attacks occurred during these months is troublesome\nand illustrates a disregard for the law.It is also worth noting that\nnone of the farmers seemed to have exercised their right to kill\ndogs that were harassing or injuring their animals. In Australia, it\nhas been reported that domestic dogs were more often seen or\nheard of attacking livestock because they attack during the\ndaytime (9). However, the witnesses did not destroy the attacking\ndog because of the absence of equipment, or because the dog was\ntheir own or belonged to a neighbor – most often living within a\nfew hundred meters from the livestock being subjected to\nattack (9).\nWhat consequences did the attack have for\nthe animals that were subjected to dog\nattacks?\nAccording to Gregory (38), suffering associated with predation\ncan be categorized into two distinct forms. First, there is acute\ndistress resulting from pursuit and physical assault by predators.\nSecond, chronic stress is induced by a persistent threat of\npredation. Furthermore, flocks that are subjected to regular\npredation exhibit heightened vigilance and become easily agitated,\nwhich can complicate their management during standard\nprocedures (38). In our study, the animals were frightened,\nchased, injured, and killed by dogs. The bite wounds and\nsubsequent infection caused suffering before they healed or until\nthe animal died or was euthanized.\nConsequences for the livestock owners\nThe death or injury of livestock caused by dogs can negatively\naffect livestock production capacity, resulting in economic losses for\nthe owner. If the inspection shows that damage to a domestic animal\nor dog is found to have been caused by a bear, lynx, wolf, golden eagle,\nor wolverine, the animal owner may receive compensation from the\ncounty administrative board. A trained inspector must be called if a\nlarge predator is suspected of having injured or killed a domestic\nanimal or dog, even if the veterinarian has already examined\nthe animal.\nDog attacks can be mistaken for wolf attacks (5, 7, 10, 39) and\nbites from large dogs can be easily confused with wolf bites (8),\n\nThe fact that\nmany of the attacks occurred during these months is troublesome\nand illustrates a disregard for the law.It is also worth noting that\nnone of the farmers seemed to have exercised their right to kill\ndogs that were harassing or injuring their animals. In Australia, it\nhas been reported that domestic dogs were more often seen or\nheard of attacking livestock because they attack during the\ndaytime (9). However, the witnesses did not destroy the attacking\ndog because of the absence of equipment, or because the dog was\ntheir own or belonged to a neighbor – most often living within a\nfew hundred meters from the livestock being subjected to\nattack (9).\nWhat consequences did the attack have for\nthe animals that were subjected to dog\nattacks?\nAccording to Gregory (38), suffering associated with predation\ncan be categorized into two distinct forms. First, there is acute\ndistress resulting from pursuit and physical assault by predators.\nSecond, chronic stress is induced by a persistent threat of\npredation. Furthermore, flocks that are subjected to regular\npredation exhibit heightened vigilance and become easily agitated,\nwhich can complicate their management during standard\nprocedures (38). In our study, the animals were frightened,\nchased, injured, and killed by dogs. The bite wounds and\nsubsequent infection caused suffering before they healed or until\nthe animal died or was euthanized.\nConsequences for the livestock owners\nThe death or injury of livestock caused by dogs can negatively\naffect livestock production capacity, resulting in economic losses for\nthe owner. If the inspection shows that damage to a domestic animal\nor dog is found to have been caused by a bear, lynx, wolf, golden eagle,\nor wolverine, the animal owner may receive compensation from the\ncounty administrative board. A trained inspector must be called if a\nlarge predator is suspected of having injured or killed a domestic\nanimal or dog, even if the veterinarian has already examined\nthe animal.\nDog attacks can be mistaken for wolf attacks (5, 7, 10, 39) and\nbites from large dogs can be easily confused with wolf bites (8),\n\nDog attacks can be mistaken for wolf attacks (5, 7, 10, 39) and\nbites from large dogs can be easily confused with wolf bites (8),\nleading to inaccurate reporting, unjustly killing of wolves, worsen\nrural–urban tensions, increased public fear and incorrect\ncompensation claims. It is also important to identify dogs that have\nattacked, injured, or even killed livestock in order to prevent further\nattacks by the same animal. Displaying hunting (or foraging)\nbehavior is considered self-rewarding for a dog (40), and the risk of\nrepeated attacks is high.\n\nDog attacks can be mistaken for wolf attacks (5, 7, 10, 39) and\nbites from large dogs can be easily confused with wolf bites (8),\nleading to inaccurate reporting, unjustly killing of wolves, worsen\nrural–urban tensions, increased public fear and incorrect\ncompensation claims. It is also important to identify dogs that have\nattacked, injured, or even killed livestock in order to prevent further\nattacks by the same animal. Displaying hunting (or foraging)\nbehavior is considered self-rewarding for a dog (40), and the risk of\nrepeated attacks is high.\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n10\nfrontiersin.org\nThere are many reasons why dog attacks on livestock are\nunderestimated. In Sweden, when a dog attacks livestock, the dog\nowner is solely legally responsible for the damage the dog causes\nto livestock. If the attacking dog is known, the dog owner and the\nlivestock owner often settle amicably in private, and no record\nexists of the event. This might seem to be a great outcome, with\nno government involvement or ease of action. Maintaining a good\nrelationship with one’s neighbors is very important, as bad\nrelationships can have a great impact on day-to-day life (9, 41).\nWill agreement and compensation be fair, or will it better reflect\nthe relationship between them? The most troublesome aspect is\nthat if the livestock owner and dog owner cannot settle on\ncompensation, then there is no available arbitration or other help\nfor the parties. In this study, the alpaca and the sheep owners\nidentified the neighbor as the owner of the attacking dog, and only\nthree of the 25 attacks were due to unknown dogs. However, in the\nstudy’s media search, 50% of the dog attacks were caused by\nunknown dogs and owners. No rights or compensation are\ngranted to the livestock owner for the loss of livestock. Elsewhere,\nlivestock owners experienced helplessness when they were unable\nto locate the dogs responsible, and on the other hand also fear of\nretribution from the dog owner once a dog is destroyed (9). The\nlivestock owners in our study felt emotional distress.\nThere is also a risk that dogs’ share of livestock predation is\nunderestimated because of error bias in field observations\nconducted by local officials (5). This is not unexpected,\nconsidering that compensation for livestock losses is only granted\nwhen predation is due to wildlife carnivores and not dogs. False\npredation can be  declared by livestock owners to obtain\ncompensation (4). In a study by Mattiello et al. (21), only 35% of\nthe predation incidents on sheep were verified as contributing to\nwolf predation, illustrating the difficulty in identifying the\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n10\nfrontiersin.org\nThere are many reasons why dog attacks on livestock are\nunderestimated. In Sweden, when a dog attacks livestock, the dog\nowner is solely legally responsible for the damage the dog causes\nto livestock. If the attacking dog is known, the dog owner and the\nlivestock owner often settle amicably in private, and no record\nexists of the event. This might seem to be a great outcome, with\nno government involvement or ease of action. Maintaining a good\nrelationship with one’s neighbors is very important, as bad\nrelationships can have a great impact on day-to-day life (9, 41).\nWill agreement and compensation be fair, or will it better reflect\nthe relationship between them? The most troublesome aspect is\nthat if the livestock owner and dog owner cannot settle on\ncompensation, then there is no available arbitration or other help\nfor the parties. In this study, the alpaca and the sheep owners\nidentified the neighbor as the owner of the attacking dog, and only\nthree of the 25 attacks were due to unknown dogs. However, in the\nstudy’s media search, 50% of the dog attacks were caused by\nunknown dogs and owners. No rights or compensation are\ngranted to the livestock owner for the loss of livestock. Elsewhere,\nlivestock owners experienced helplessness when they were unable\nto locate the dogs responsible, and on the other hand also fear of\nretribution from the dog owner once a dog is destroyed (9). The\nlivestock owners in our study felt emotional distress.\nThere is also a risk that dogs’ share of livestock predation is\nunderestimated because of error bias in field observations\nconducted by local officials (5). This is not unexpected,\nconsidering that compensation for livestock losses is only granted\nwhen predation is due to wildlife carnivores and not dogs. False\npredation can be  declared by livestock owners to obtain\ncompensation (4). In a study by Mattiello et al. (21), only 35% of\nthe predation incidents on sheep were verified as contributing to\nwolf predation, illustrating the difficulty in identifying the\n\nFalse\npredation can be  declared by livestock owners to obtain\ncompensation (4).In a study by Mattiello et al.(21), only 35% of\nthe predation incidents on sheep were verified as contributing to\nwolf predation, illustrating the difficulty in identifying the\npredating species and the risk of false attribution by the livestock\nowner. Perhaps part of the solution is to compensate for all canine\npredation, as some local administrations in Italy do to compensate\nfor wolf predation, regardless of whether wolves, dogs, or\nwolf-dog hybrids were responsible for the damage (4). In Poland,\nlivestock owners are compensated for livestock killed by wolves\nonly and not livestock killed by dogs (7). Similarly, interviews\nwith Finnish sheep farmers revealed that, surprisingly, dog\nowners were often not held accountable for the damage dogs\ncaused, and the costs fell entirely on sheep owners (42), increasing\nthe risk of conflict.\nIn Sweden, it is against the law to have dogs loose in nature\nfrom March 1 to August 20, but despite this, many of the dog\nattacks occurred during this time. The attacks mainly occurred\nfrom May to December, likely because of the increased outdoor\nactivity of livestock, dogs, and their owners during this period.\nRovbase records from inspectors do not necessarily end up in news\nmedia articles, and many articles during fall and late fall/early\nwinter may reflect seasons for moose and deer hunting where\nhunting with dogs is permissible and could perhaps explain part\nof the spike in the number of dog attacks identified in the news\nmedia articles. It is not uncommon for hunting dogs to enter\nsheepfolds during big game hunting when the driven animal (e.g.,\nan elk) runs through the sheep fence and tears it down. In this case,\nthe dog can leave the elk as hunting prey and instead attack the\nsheep (8). Small lambs are usually bitten on their backs and shaken\nuntil death (8).\nNearly 50 percent of the inspectors reported that they had not\ninspected a livestock victim of a dog attack. Perhaps not surprisingly,\nmany inspectors also stated a lack of experience. Considering the\n\nFalse\npredation can be  declared by livestock owners to obtain\ncompensation (4).In a study by Mattiello et al.(21), only 35% of\nthe predation incidents on sheep were verified as contributing to\nwolf predation, illustrating the difficulty in identifying the\npredating species and the risk of false attribution by the livestock\nowner. Perhaps part of the solution is to compensate for all canine\npredation, as some local administrations in Italy do to compensate\nfor wolf predation, regardless of whether wolves, dogs, or\nwolf-dog hybrids were responsible for the damage (4). In Poland,\nlivestock owners are compensated for livestock killed by wolves\nonly and not livestock killed by dogs (7). Similarly, interviews\nwith Finnish sheep farmers revealed that, surprisingly, dog\nowners were often not held accountable for the damage dogs\ncaused, and the costs fell entirely on sheep owners (42), increasing\nthe risk of conflict.\nIn Sweden, it is against the law to have dogs loose in nature\nfrom March 1 to August 20, but despite this, many of the dog\nattacks occurred during this time. The attacks mainly occurred\nfrom May to December, likely because of the increased outdoor\nactivity of livestock, dogs, and their owners during this period.\nRovbase records from inspectors do not necessarily end up in news\nmedia articles, and many articles during fall and late fall/early\nwinter may reflect seasons for moose and deer hunting where\nhunting with dogs is permissible and could perhaps explain part\nof the spike in the number of dog attacks identified in the news\nmedia articles. It is not uncommon for hunting dogs to enter\nsheepfolds during big game hunting when the driven animal (e.g.,\nan elk) runs through the sheep fence and tears it down. In this case,\nthe dog can leave the elk as hunting prey and instead attack the\nsheep (8). Small lambs are usually bitten on their backs and shaken\nuntil death (8).\nNearly 50 percent of the inspectors reported that they had not\ninspected a livestock victim of a dog attack. Perhaps not surprisingly,\nmany inspectors also stated a lack of experience. Considering the\n\nNearly 50 percent of the inspectors reported that they had not\ninspected a livestock victim of a dog attack.Perhaps not surprisingly,\nmany inspectors also stated a lack of experience.Considering the\nRovbase data from Västra Götaland, where 3.8% of inspections were\nattributed to dog attacks, an inspector can perform many inspections\nwithout coming across a case in which a dog was the aggressor.\nHowever, our results indicate that dogs contribute negatively to the\ncarnivore conflict in Sweden, as seen in statistics from\nVästra Götaland.\nMethod limitations\nDue to the lack of official statistics, we gathered data on dog\nattacks through Swedish news media articles and surveys of\nlivestock owners and official inspectors. While news articles have\nbeen used elsewhere as data source when there is limited data\n(43), only a fraction of incidents are likely reported. More targeted\nsearch terms might also have yielded additional results.\nSearch engines like Google present challenges due to their\nproprietary and evolving algorithms, which prioritize established\nsources and may exclude smaller outlets (44). This limits replicability\nand can skew news searches. To address this, we also consulted local\nnewspapers. The overlap between different news sources was\nsubstantial. Another limitation of the study was the very low response\nrate of the farmers. Perhaps the survey did not reach out to the farmers\nas expected, or for unknown reasons, only a few chose to answer. The\nresponse rate to a survey of Scottish sheep farmers was 21%\n(n = 9,148), which was considered high by researchers (45). In\ncontrast to our study, Murray et al. (45) study used a mixed-methods\napproach that included surveys and interviews. We  used a\nsurvey alone.\nThe survey of the inspectors had a higher response rate, suggesting\nthat the topic was perceived as important and relevant by the\ntarget group.\nThe Rovbase records have certain limitations. The accuracy of\ncarcass inspections conducted by board inspectors may be uncertain,\nand some dog attacks could be misattributed to other predators, or\n\nNearly 50 percent of the inspectors reported that they had not\ninspected a livestock victim of a dog attack.Perhaps not surprisingly,\nmany inspectors also stated a lack of experience.Considering the\nRovbase data from Västra Götaland, where 3.8% of inspections were\nattributed to dog attacks, an inspector can perform many inspections\nwithout coming across a case in which a dog was the aggressor.\nHowever, our results indicate that dogs contribute negatively to the\ncarnivore conflict in Sweden, as seen in statistics from\nVästra Götaland.\nMethod limitations\nDue to the lack of official statistics, we gathered data on dog\nattacks through Swedish news media articles and surveys of\nlivestock owners and official inspectors. While news articles have\nbeen used elsewhere as data source when there is limited data\n(43), only a fraction of incidents are likely reported. More targeted\nsearch terms might also have yielded additional results.\nSearch engines like Google present challenges due to their\nproprietary and evolving algorithms, which prioritize established\nsources and may exclude smaller outlets (44). This limits replicability\nand can skew news searches. To address this, we also consulted local\nnewspapers. The overlap between different news sources was\nsubstantial. Another limitation of the study was the very low response\nrate of the farmers. Perhaps the survey did not reach out to the farmers\nas expected, or for unknown reasons, only a few chose to answer. The\nresponse rate to a survey of Scottish sheep farmers was 21%\n(n = 9,148), which was considered high by researchers (45). In\ncontrast to our study, Murray et al. (45) study used a mixed-methods\napproach that included surveys and interviews. We  used a\nsurvey alone.\nThe survey of the inspectors had a higher response rate, suggesting\nthat the topic was perceived as important and relevant by the\ntarget group.\nThe Rovbase records have certain limitations. The accuracy of\ncarcass inspections conducted by board inspectors may be uncertain,\nand some dog attacks could be misattributed to other predators, or\n\nThe Rovbase records have certain limitations.The accuracy of\ncarcass inspections conducted by board inspectors may be uncertain,\nand some dog attacks could be misattributed to other predators, or\nvice versa. However, assessing animal carcasses by inspectors can\nbe challenging, especially if a long time has passed between the attack\nand inspection (10). Inspectors could benefit from additional training\nin recognizing signs of dog attacks and understanding the\ncharacteristic ways in which dogs handle prey. There are also\ndifferences in bite patterns depending on the size of the dog, and the\nanatomy of its skull (46).\nConclusion\nThis study provides valuable insights on prevalence and\nnature of dog attacks on livestock in Sweden, from multiple\nperspectives: news media articles, farmers and regional\nauthorities. Dog attacks appear opportunistic, often involving\nunsupervised, loose dogs targeting accessible livestock, primarily\nsheep, on pasture during summer and fall. Most attacks resulted\n\nThe Rovbase records have certain limitations.The accuracy of\ncarcass inspections conducted by board inspectors may be uncertain,\nand some dog attacks could be misattributed to other predators, or\nvice versa. However, assessing animal carcasses by inspectors can\nbe challenging, especially if a long time has passed between the attack\nand inspection (10). Inspectors could benefit from additional training\nin recognizing signs of dog attacks and understanding the\ncharacteristic ways in which dogs handle prey. There are also\ndifferences in bite patterns depending on the size of the dog, and the\nanatomy of its skull (46).\nConclusion\nThis study provides valuable insights on prevalence and\nnature of dog attacks on livestock in Sweden, from multiple\nperspectives: news media articles, farmers and regional\nauthorities. Dog attacks appear opportunistic, often involving\nunsupervised, loose dogs targeting accessible livestock, primarily\nsheep, on pasture during summer and fall. Most attacks resulted\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n11\nfrontiersin.org\nin the animals being killed, often displaying a characteristic\npattern of injuries. Dog attacks on equestrian units pose a\nsignificant threat to the life and health of both animals\nand humans.\nDog attacks on livestock can have serious societal consequences,\nsuch as being mistaken for wolf predation, and aggravating the\ninflamed human-wolf conflict. Dog owners have strict liability for\nthe damage caused by the dog, but unidentified dogs and dog\nowners often leave livestock owners without financial compensation\nand with emotional distress. Preventive measures against dog\nattacks on livestock should be tailored to seasonal patterns and the\nspecific target populations to ensure maximal effectiveness. Also,\nfurther research is warranted.\nSuggestions for the future\nThe consequences of dog attacks on livestock for farmers are\nboth financial and emotional. Although official statistics and\ncomprehensive data on the issue are lacking, the results presented\nhere are sufficiently compelling to justify the proposal of targeted\nmitigation strategies. One could, for example, conduct information\ncampaigns targeting specific groups such as alpaca farm visitors,\nhorse owners and riders, about the risks associated with dogs and\nhow to avoid dog attacks. Measures such as predator-deterrent\nfencing, currently used to prevent attacks by wild predators, also\nappear to be  effective against dog attacks (47). However,\nalternative funding mechanisms are required, for instance, a dog\ntax allocated to a dedicated fund from which livestock owners\nmay apply for financial support to install predator-deterrent\nfencing specifically aimed at preventing dog incursions on their\nproperties. Nonetheless, systematic data collection and further\nresearch are needed to deepen our understanding of the problem’s\nfull scope and to refine future interventions. Since dog owners are\nsolely responsible for their animals, it may also be advisable to\nextend the period when dogs must be kept on a leash to cover the\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n11\nfrontiersin.org\nin the animals being killed, often displaying a characteristic\npattern of injuries. Dog attacks on equestrian units pose a\nsignificant threat to the life and health of both animals\nand humans.\nDog attacks on livestock can have serious societal consequences,\nsuch as being mistaken for wolf predation, and aggravating the\ninflamed human-wolf conflict. Dog owners have strict liability for\nthe damage caused by the dog, but unidentified dogs and dog\nowners often leave livestock owners without financial compensation\nand with emotional distress. Preventive measures against dog\nattacks on livestock should be tailored to seasonal patterns and the\nspecific target populations to ensure maximal effectiveness. Also,\nfurther research is warranted.\nSuggestions for the future\nThe consequences of dog attacks on livestock for farmers are\nboth financial and emotional. Although official statistics and\ncomprehensive data on the issue are lacking, the results presented\nhere are sufficiently compelling to justify the proposal of targeted\nmitigation strategies. One could, for example, conduct information\ncampaigns targeting specific groups such as alpaca farm visitors,\nhorse owners and riders, about the risks associated with dogs and\nhow to avoid dog attacks. Measures such as predator-deterrent\nfencing, currently used to prevent attacks by wild predators, also\nappear to be  effective against dog attacks (47). However,\nalternative funding mechanisms are required, for instance, a dog\ntax allocated to a dedicated fund from which livestock owners\nmay apply for financial support to install predator-deterrent\nfencing specifically aimed at preventing dog incursions on their\nproperties. Nonetheless, systematic data collection and further\nresearch are needed to deepen our understanding of the problem’s\nfull scope and to refine future interventions. Since dog owners are\nsolely responsible for their animals, it may also be advisable to\nextend the period when dogs must be kept on a leash to cover the\n\nSince dog owners are\nsolely responsible for their animals, it may also be advisable to\nextend the period when dogs must be kept on a leash to cover the\nentire grazing season, when livestock are outdoors. This could\nbe a viable legal measure, provided it is properly enforced. Finally,\nlivestock owners should enhance the supervision of their animals\nduring peak hunting seasons and engage in collaborative\ndiscussions with local hunting associations to develop appropriate\nmitigation strategies.\nData availability statement\nThe raw data supporting the conclusions of this article will\nbe made available by the authors, without undue reservation.\nEthics statement\nEthical approval was not required for the study involving\nanimals in accordance with the local legislation and institutional\nrequirements because it does not involve any identifiable\ninformation, such as names, addresses, or ID numbers. This study\ndoes not include medical records or archived samples. All\nparticipants remain anonymous, and no identifiable information\nwas collected. Information in news media articles are freely\navailable and the personal information that could be referred to a\ncertain person was not used (names, addresses). Informed consent\nwas obtained from all survey respondents through their\nanonymous participation.\nAuthor contributions\nSS: Funding acquisition, Project administration, Investigation,\nWriting – review & editing, Writing – original draft, Methodology,\nConceptualization. MD: Investigation, Formal analysis, Writing –\noriginal draft, Writing – review & editing.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. This work was supported\nby the Marie-Claire Cronstedt Foundation under grant number\n2023/14 and Linnaeus University as regards the article processing\ncharge, APC.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that Gen AI was used in the creation of this\n\nSince dog owners are\nsolely responsible for their animals, it may also be advisable to\nextend the period when dogs must be kept on a leash to cover the\nentire grazing season, when livestock are outdoors. This could\nbe a viable legal measure, provided it is properly enforced. Finally,\nlivestock owners should enhance the supervision of their animals\nduring peak hunting seasons and engage in collaborative\ndiscussions with local hunting associations to develop appropriate\nmitigation strategies.\nData availability statement\nThe raw data supporting the conclusions of this article will\nbe made available by the authors, without undue reservation.\nEthics statement\nEthical approval was not required for the study involving\nanimals in accordance with the local legislation and institutional\nrequirements because it does not involve any identifiable\ninformation, such as names, addresses, or ID numbers. This study\ndoes not include medical records or archived samples. All\nparticipants remain anonymous, and no identifiable information\nwas collected. Information in news media articles are freely\navailable and the personal information that could be referred to a\ncertain person was not used (names, addresses). Informed consent\nwas obtained from all survey respondents through their\nanonymous participation.\nAuthor contributions\nSS: Funding acquisition, Project administration, Investigation,\nWriting – review & editing, Writing – original draft, Methodology,\nConceptualization. MD: Investigation, Formal analysis, Writing –\noriginal draft, Writing – review & editing.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. This work was supported\nby the Marie-Claire Cronstedt Foundation under grant number\n2023/14 and Linnaeus University as regards the article processing\ncharge, APC.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that Gen AI was used in the creation of this\n\nGenerative AI statement\nThe authors declare that Gen AI was used in the creation of this\nmanuscript. Microsoft 365 Copilot Chat was used for language and\ngrammar checks.\nAny alternative text (alt text) provided alongside figures\nin this article has been generated by Frontiers with the\nsupport of artificial intelligence and reasonable efforts\nhave been made to ensure accuracy, including review by the\nauthors wherever possible. If you  identify any issues, please\ncontact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. Any product that may be evaluated in this article, or\nclaim that may be made by its manufacturer, is not guaranteed or\nendorsed by the publisher.\n\nGenerative AI statement\nThe authors declare that Gen AI was used in the creation of this\nmanuscript. Microsoft 365 Copilot Chat was used for language and\ngrammar checks.\nAny alternative text (alt text) provided alongside figures\nin this article has been generated by Frontiers with the\nsupport of artificial intelligence and reasonable efforts\nhave been made to ensure accuracy, including review by the\nauthors wherever possible. If you  identify any issues, please\ncontact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. Any product that may be evaluated in this article, or\nclaim that may be made by its manufacturer, is not guaranteed or\nendorsed by the publisher.\n\nSarenbo and Doane\b\n10.3389/fvets.2025.1629966\nFrontiers in Veterinary Science\n12\nfrontiersin.org\nReferences\n1.\tBryce CM. Dogs as pets and pests: global patterns of canine abundance, activity,\nand health. Integr Comp Biol. (2021) 61:154–65. doi: 10.1093/icb/icab046\n2.\tSwedish Board of Agriculture. Statistik ur hundregistret [statistics from the dog\nregistry]. Jönköping: Swedish Board of Agriculture (2025).\n3.\tSarenbo S, Striwing H. Djur och rätt. Förvaltningslagen, djurskyddslagen och\ntillsynslagen m.m. [animals and the law. 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+{"text": "【兽医文献】\n标题: Thalidomide as a rescue protocol for treatment of multiple myeloma in dogs: preliminary data from a multicentre retrospective study\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nDiet supplemented with\nfermented onion improves\ngrowth performance, health\ncondition, meat quality, and\nmodifies rumen metabolite\nprofiles in Liangshan black sheep\nShengwang Jiang 1,2, Chaoyun Yang 1, Chen Ji 3, Chao Li 1,\nGang Lv 4, Hongwei Gao 4, Wei Zhang 5, Yi Zhang 1,2* and\nZengwen Huang 1,4*\n1 College of Animal Science, Xichang University, Xichang, Sichuan, China, 2 Key Laboratory of Local\nCharacteristic Goat at Xichang University of Sichuan Province, Xichang, Sichuan, China, 3 College of\nAgricultural Science, Xichang University, Xichang, Sichuan, China, 4 Xinjiang Taikun Group Co., Ltd.,\nChangji, Xinjiang, China, 5 College of Animal Science and Technology, Xinjiang Agricultural Vocational\nand Technical University, Changji, Xinjiang, China\nThis study aims to investigate the effect of fermented onion on Liangshan black\nsheep’s growth performance, health, meat quality, and rumen metabolite profiles.\nA total of 80 four-month-old female Liangshan black sheep were randomly divided\ninto four groups of five replicate pens (four sheep per pen). Sheep were fed a basal\ndiet supplemented with 0 (control), 10, 20% or 30% fermented onion. Compared\nto that of the control group, dietary supplementation with 20% fermented onion\nimproved final body weight, ADG and ADFI; enhanced GPT and GOT activities\nand increased IgA, IgG, IgM, C3, and C4 levels; increased the levels of IL-4, IL-10,\nTGF-β and decreased the levels of IL-1β, IL-6, TNF-α, IFN-γ; decreased MDA level\nand increased SOD, GST, CAT and GSH-Px activities; decrease the pH, L* value, b*\nvalue and shear force; increase the a* value and the content s of protein and fat;\nincrease the expression levels of FN1, TGFβ1, Myf5, FAS, PPARγ and FABP4; decrease\nthe expression levels of CPT1A and LPL. Metabolomic analysis revealed that 20%\nfermented onion supplementation significantly modified the metabolite profiles\nin the rumen liquid, with 44 downregulated metabolites and four upregulated\nmetabolites mainly enriched in purine metabolism, microbial metabolism, cutin, and\n\nsuberine biosynthesis pathways. Data from our study suggest that supplementation\nof fermented onion to the basal diet at 20% (w/w) can be used safely to increase\nmeat yield and to improve meat quality in the sheep industry.\nKEYWORDS\nfermented onion, Liangshan black sheep, growth performance, rumen metabolite\nprofiles, meat quality\n1 Introduction\nXichang city has a mild climate, fertile land, and a convenient irrigation system. Abundant\nlight, warm weather, and land resources create favorable conditions for onion cultivation.\nXichang onion has the characteristics of fresh color with tight structure and rich nutrients,\ncrisp and sweet taste with fragrant smell and tenderness, large fruit that is easy to store, and\nOPEN ACCESS\nEDITED BY\nTugay Ayasan,\nOsmaniye Korkut Ata University, Türkiye\nREVIEWED BY\nAli Ihsan Atalay,\nIğdır Üniversitesi, Türkiye\nÇağri Özkan,\nKahramanmaras Sütçü Imam University,\nTürkiye\n*CORRESPONDENCE\nYi Zhang\n1296033350@qq.com\nZengwen Huang\nxndaxue@126.com\nRECEIVED 29 August 2025\nACCEPTED 01 October 2025\nPUBLISHED 29 October 2025\nCITATION\nJiang S, Yang C, Ji C, Li C, Lv G, Gao H,\nZhang W, Zhang Y and Huang Z (2025) Diet\nsupplemented with fermented onion\nimproves growth performance, health\ncondition, meat quality, and modifies rumen\nmetabolite profiles in Liangshan black sheep.\nFront. Vet. Sci. 12:1695023.\ndoi: 10.3389/fvets.2025.1695023\nCOPYRIGHT\n© 2025 Jiang, Yang, Ji, Li, Lv, Gao, Zhang,\nZhang and Huang. This is an open-access\narticle distributed under the terms of the\nCreative Commons Attribution License\n(CC BY). The use, distribution or reproduction\nin other forums is permitted, provided the\noriginal author(s) and the copyright owner(s)\nare credited and that the original publication\nin this journal is cited, in accordance with\naccepted academic practice. No use,\ndistribution or reproduction is permitted\nwhich does not comply with these terms.\nTYPE  Original Research\nPUBLISHED  29 October 2025\nDOI  10.3389/fvets.2025.1695023\n\nJiang et al.\b\n10.3389/fvets.2025.1695023\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nresistant to transportation. It contains 17 amino acids, vitamin C, trace\nelement selenium, etc., plus garlic, prostaglandin A, and quercetin,\nwhich are unique to onion. Additionally, Xichang onion contains a\nvariety of substances that are bactericidal and anti-inflammatory, anti-\naging, lowering blood pressure, preventing cancer, improving\nimmunity, and preventing osteoporosis. Due to its unique properties,\nthe Xichang onion is greatly favored by consumers. In 2010, Xichang\nonion was certified as a national geographical indication product by\nAQSIQ (General Administration of Quality Supervision, Inspection\nand Quarantine of the People’s Republic of China).\nThe onion planting area in Xichang is about 5,000 hectares, and\nthe total output exceeds 250,000 tons. Xichang is regarded as one\nof the main onion planting areas in China. The production of\nonions and their by-products (such as onion stems and leaves) has\nsteadily increased yearly, resulting in high pressure on onion sales\nand sharp price fluctuations. In addition, a large amount of onion\nwaste is generated every year (1). Thus, it is an urgent and critical\nissue to explore the full utilization of onions and their\nby-products (2).\nOn July 1, 2020, the document No. 194 from the Ministry of\nAgriculture and Rural Affairs of the People’s Republic of China was\nimplemented, marking China official entry into the era of “feed\nprohibition” (no antibiotic is allowed in animal feeds). This makes\nanimal husbandry enterprises face a major challenge of frequent\ndisease. So, to promote the green and sustainable development of the\nlivestock and poultry industry, it is urgent to find high-quality,\nefficient, green, and safe new feed additives with antibacterial\nactivities. Studies have shown that onions contain a large number of\nnatural antibiotics, which have strong bactericidal activities that can\nkill or inhibit Staphylococcus aureus, Streptococcus, diphtheria,\ndysentery, tuberculosis, and Escherichia coli (3). In addition, onions\n\nIn addition, onions\nand their extracts, when used as feed additives, play an important role\nin improving the production performance, immunity, nutritional\ndigestibility, and meat quality of livestock and poultry (4–8). Onions\nare rich in carbohydrates, vitamin C, and trace elements such as\npotassium, calcium, and phosphorus. Studies have shown that the\nbioactive substances in onion extract, such as sulfur-containing\ncompounds, flavonoids, polysaccharides, and saponins, can improve\nthe body weight and feed conversion efficiency of broilers. Therefore,\nonions have potential antibacterial and growth-promoting value (9,\n10). Since fresh onions contain high levels of sulfur compounds (such\nas dipropyl disulfide, dipropyl trisulfide, etc.) in vacuoles, these sulfur\ncompounds are released and contacted with onion enzymes, resulting\nin the generation of volatile substances with a pungent smell once the\nchopped onions are eaten and chewed by animals. These volatile sulfur\ncompounds can irritate the eyes and respiratory nerves, causing\nreactions such as tears and coughing, thereby making unfermented\nonions much less palatable as a feed material for animals. By\nundergoing the natural fermentation after adding sugar and salt to\nchopped onions, most sulfides in fresh onions will be converted into\nsweet substances, reducing the spiciness and improving the\nonion’s palatability.\nLiangshan black sheep is one of the top 10 excellent germplasm\nresources of livestock and poultry in China. Due to its strong and tall\nphysique, symmetrical structure, high meat quality, genetic stability\nand strong adaptability, easy feeding, and the combined use of meat\nand hair characteristics, it has been certified as a national geographic\nindication product in 2020 (11, 12).\nHowever, the poor growth performance seriously restricted the\ndevelopment of the Liangshan black sheep industry. Because the\nLiangshan black sheep are mainly raised by grazing and supplementary\nfeeding, thus, the growth rate is slow and the breeding cycle is long.\nThe rumen, as the central site of microbial fermentation, is the critical\n\nBecause the\nLiangshan black sheep are mainly raised by grazing and supplementary\nfeeding, thus, the growth rate is slow and the breeding cycle is long.The rumen, as the central site of microbial fermentation, is the critical\ninterface where diet influences host metabolism. While the positive\noutcomes are clear, how fermented onion precisely alters the metabolic\nnetwork within the rumen to fuel superior growth and health is not\nunderstood. Therefore, this study was designed to make full use of\nXichang onion and Liangshan black sheep to investigate the effect of\nadding fermented onion to the basic diet on Liangshan black sheep’s\ngrowth performance, health, meat quality, and rumen metabolite\nprofiles. By generating new data and understanding the molecular\nmechanism of fermented onion, we aim to make full use of onion\nresources in Xichang and promote the development of the Liangshan\nblack sheep industry.\n2 Materials and methods\n2.1 Fermented onion preparation\nThe onion was peeled, with heads and roots being removed, and\nthe remaining portion was chopped into pieces of approximately\n1.5 cm × 1.5 cm in size. Subsequently, 5% sugar and 1% salt were\nadded to the chopped onion pieces, and the mixture was agitated at\n20–25 °C until the onion juice was released. The agitation took about\n2 h. Once the agitation was completed, the mixture was then\ntransferred to a food-grade fermentation container and placed in a\n15–20 °C environment. The container was incubated at room\ntemperature for a period of 25 days, during which time the\nfermentation process took place in an anaerobic environment. A\nsuccessfully fermented onion had the following characteristics: soft in\ntexture, exuding a sour smell and a robust aroma, and no pungent\nodor or evidence of mildew.\n2.2 Ethics statement\nThe experimental protocol and procedures were approved by the\nExperimental Animal Welfare and Ethics Committee of Xichang\nUniversity (permit number for conducting animal experiments: No.\nxcc2025001). All animal treatments and experiments were performed\naccording to the recommendations described in the guidelines for\n\nxcc2025001).All animal treatments and experiments were performed\naccording to the recommendations described in the guidelines for\nethical review of animal welfare in the national standards of the\nPeople’s Republic of China (151).\n2.3 Experimental design and animal\nfeeding management\nA single-factor test design was used for our experiment in this\nstudy. A total of 80 four-month-old female Liangshan black sheep\n(initial body weight: 23.40 ± 0.36 kg) in good health were selected\nand randomly divided into four groups, with a total of 20 animals\nper group and five replicates per group. Each replicate had four\nsheep in a pen. The control group (Con) was fed with the basic diet\n(Xinjiang Taikun Group Co., Ltd.), while the experimental groups\n\nJiang et al.\b\n10.3389/fvets.2025.1695023\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nwere fed with the basic diet supplemented with 10% (Trt1), 20%\n(Trt2), and 30% (Trt3) fermented onion, respectively. All diets were\nisonitrogenic and isoenergetic, and the ingredient composition and\nnutrient levels (measured values) of diets are listed in Table 1. The\npercent of fermented onion was calculated by weight (dry matter).\nFor example, 2 kg of fermented onion added to 8 kg of the basal\ndiet resulted in a diet with 20% fermented onion. The basal diet\nwas formulated to meet sheep nutrient requirements during the\nexperimental period. The pre-experiment lasted for 7 days, and the\nexperiment lasted for 70 days. All sheep were fed twice a day (08:30\nand 18:30) according to the daily management methods of the\nsheep farm and had ad libitum access to food and water. Sheep\npens were kept clean and dry, and disinfected regularly.\n2.4 Growth performance\nEach sheep was weighed before the experiment began (initial\nweight) and after the experiment was completed (final weight). All\nsheep were fasted for 12 h before weighing. Simultaneously, the feed\nintake and leftover feed of each pen were recorded daily. The average\ndaily gain (ADG) in body weight, average daily feed intake (ADFI),\nand feed conversion ratio (FCR) were calculated using the daily data\ncollected during the study period.\n2.5 Blood sample collection and analysis\nAt the end of feeding experiment, one sheep in each replicate\nwas randomly selected and euthanized, followed by bloodletting\nand slaughter. Blood samples (5 mL) were collected in 10 mL\nanticoagulant-free vacutainer tubes and immediately centrifuged at\n3,000 × g for 10 min at 4 °C to obtain serum. Serum samples were\nstored at −20 °C until further analysis (serum biochemical and\nimmunological parameters, serum inflammatory factors, and\nantioxidant capacity).\n2.5.1 Serum biochemical parameters\nThe contents of total protein (TP), albumin (ALB), globulin\n(GLB), glutamic pyruvic transaminase (GPT), and glutamic\noxaloacetic transaminase (GOT) in serum were determined using an\n\n2.5.1 Serum biochemical parameters\nThe contents of total protein (TP), albumin (ALB), globulin\n(GLB), glutamic pyruvic transaminase (GPT), and glutamic\noxaloacetic transaminase (GOT) in serum were determined using an\nautomatic biochemical analyzer (BK-280, Biobase, China).\n2.5.2 Serum immunological parameters\nThe levels of serum immunoglobulin A (IgA), immunoglobulin\nG (IgG), immunoglobulin M (IgM), complement 3 (C3), and\ncomplement 4 (C4) were determined using enzyme-linked\nimmunosorbent assay (ELISA) kits according to the manufacturer’s\ninstructions (Jiangsu Enzyme Exemption Industry Co., Ltd., China).\nAll samples were measured in quadruplicate.\nTABLE 1  Ingredient composition (%) and analytical composition in each diet.\nItems\nGroups\nCon\nTrt1\nTrt2\nTrt3\nIngredient composition (%)\nCorn\n33.00\n29.70\n26.40\n23.10\nCotton meal (42% CP)\n10.00\n9.00\n8.00\n7.00\nSpray corn husks\n10.00\n9.00\n8.00\n7.00\nWheat bran\n10.00\n9.00\n8.00\n7.00\nExtruded soybean\n2.00\n1.80\n1.60\n1.40\nCorn DDGS\n5.00\n4.50\n4.00\n3.50\nRice bran\n10.00\n9.00\n8.00\n7.00\nCorn stalks\n10.00\n9.00\n8.00\n7.00\nVitamin-mineral premix\n10.00\n9.00\n8.00\n7.00\nFermented onion\n0.00\n10.00\n20.00\n30.00\nTotal\n100\n100\n100\n100\nAnalytical composition in diet\nNet energy (NE, kcal/kg)\n2850.00\n2851.00\n2852.00\n2853.00\nDry matter (DM, %)\n88.69\n88.82\n88.95\n89.08\nCrude protein (CP, %)\n14.42\n14.23\n14.04\n13.84\nNeutral detergent fiber (NDF, %)\n26.00\n25.80\n25.60\n25.40\nAcid detergent fiber (ADF, %)\n16.20\n16.33\n16.46\n16.59\nCrude fiber (CF, %)\n11.40\n11.51\n11.62\n11.73\nCrude ash (CA, %)\n11.20\n11.08\n10.96\n10.84\nCalcium (Ca, %)\n0.65\n0.65\n0.64\n0.64\nTotal phosphorus (TP, %)\n0.55\n0.54\n0.53\n0.52\nSodium chloride (NaCl, %)\n0.33\n0.34\n0.34\n0.35\n\nJiang et al.\b\n10.3389/fvets.2025.1695023\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n2.5.3 Serum inflammatory factors\nThe levels of interleukin-1beta (IL-1β), interleukin-4 (IL-4),\ninterleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-\nalpha (TNF-α), interferon-gamma (IFN-γ), and transforming growth\nfactor-beta (TGF-β) in serum were determined using enzyme-linked\nimmunosorbent assay (ELISA) kits according to the manufacturer’s\ninstructions (Nanjing Jiancheng Bioengineering Institute, Nanjing,\nChina). All samples were measured in quadruplicate.\n2.5.4 Serum antioxidant capacity\nThe\ntotal\nantioxidant\ncapacity\n(T-AOC),\ncontent\nof\nmalondialdehyde (MDA), and the activities of superoxide dismutase\n(SOD), catalase (CAT), glutathione S-transferase (GST) and\nglutathione peroxidase (GSH-Px) in serum were measured by enzyme\nlabeling\ninstrument\n(SMR60047,\nUSCNK,\nChina)\nor\nspectrophotometer (721G, INESA, China) using commercially\navailable colorimetric diagnostic kits according to the manufacturer’s\nprotocol (Nanjing Jiancheng Bioengineering Institute, China).\n2.6 Muscle sample collection and analysis\nImmediately following exsanguination, the longissimus dorsi\n(LD) muscle samples (about 20 g) over the 12th–13th rib on the left\nside of the carcasses were collected within 15 min. Muscles were\nquickly cut into small pieces, stored in a frozen tube (RNase/DNase\nfree) immediately, snap frozen in liquid nitrogen, and stored at −80\n°C until RT-qPCR analysis of genes associated with muscle\ndevelopment and fat deposition. The LD muscle samples between 3th\nand 4th, 6th and 7th, 12th and 13th ribs on the right side of carcasses\nwere collected to evaluate meat quality (pH value, meat color,\nmarbling score, shear force, cooking loss) and chemical composition\nanalysis (moisture, ash, protein, fat).\n2.6.1 Assessment of meat quality\nThe pH of LD muscles was measured at 45 min and 24 h after\nslaughter by a portable pH meter (Testo 205, Testo Instrument Co., Ltd.,\nGermany). The L* (lightness), a* (redness), and b* (yellowness) of the\nLD muscles were recorded at 45 min after slaughter by the NR10QC\n\n, Ltd.,\nGermany).The L* (lightness), a* (redness), and b* (yellowness) of the\nLD muscles were recorded at 45 min after slaughter by the NR10QC\ncolorimeter (Guangdong 3nh Technology Co., Ltd., China). The pH and\nmeat color at three different sites of the LD muscles were measured, and\nthe average values were calculated. Marbling was scored according to\nthe 5-level scoring system compared to the American NPPC\ncolorimetric plate (1991 edition): trace fat (1), a little bit of fat (2),\nmedium fat (3), too much fat (4), super fat (5). 0.5 points are allowed\nbetween two levels, and at least three people participated in the marbling\nscoring for each LD sample. The meat tenderness meter (MAQC-12,\nNanjing) was used to measure the tenderness (shear force, N) of sheep\nLD muscle samples. The determination methods follow the agricultural\nindustry standard of the People’s Republic of China (NY/T 1180-2006\ndetermination of meat tenderness shear force method). In brief, samples\nwere cooked in an 80 °C water bath until the core temperature reached\n70 °C. Then, samples were removed from the water bath and cooled to\na central temperature of 4 °C in a refrigerator. Subsequently, the meat\nsamples were taken with a circular sampler (Φ1.27 cm) in a direction\nparallel to the muscle fibers, and the shear force was determined. The\nmuscle samples used for cooking loss were chilled at 4 °C for 24 h. Then,\ncooked in a water bath at 85 °C for 20 min. The cooking loss (%) was\ncalculated as a percentage of the weight change of the samples compared\nto the initial weight of the samples.\n2.6.2 Chemical composition analysis\nThe percentage content (w/w) of moisture, ash, protein and fat in\nLD muscle samples were analyzed according to GB5009.3-2016\nNational Food Safety Standard Determination of Moisture in Foods,\nGB5009.4-2016 National Food Safety Standard Determination of Ash\nin Foods, GB5009.5-2016 National Food Safety Standard\nDetermination of Protein in Foods, and GB5009.6-2016 National\nFood Safety Standard Determination of fat in Foods, respectively.\n2.7 RT-qPCR analysis of genes associated\nwith muscle development and fat\n\n5-2016 National Food Safety Standard\nDetermination of Protein in Foods, and GB5009.6-2016 National\nFood Safety Standard Determination of fat in Foods, respectively.2.7 RT-qPCR analysis of genes associated\nwith muscle development and fat\ndeposition\nThe expression levels of genes associated with muscle\ndevelopment, such as fibronectin 1 (FN1), transforming growth factor\nbeta 1 (TGFβ1), myogenic factor 5 (Myf5), myogenic differentiation 1\n(MyoD1), myogenin (MyoG), myostatin (MSTN), and fat deposition,\nsuch as fatty acid synthase (FAS), peroxisome proliferator activated\nreceptor gamma (PPARγ), fatty acid binding protein 4 (FABP4),\nacetyl-CoA carboxylase alpha (ACC), diacylglycerol O-acyltransferase\n1 (DGAT1), sterol regulatory element binding transcription factor 1\n(SREBF1), carnitine palmitoyltransferase 1A (CPT1A), lipoprotein\nlipase (LPL), hormone-sensitive lipase (HSL), were detected by real-\ntime quantitative reverse transcription PCR (RT-qPCR).\n2.7.1 RNA extraction and cDNA synthesis\nThe total RNA was extracted from LD muscle samples using a\ncommercially available animal tissue total RNA extraction kit\n(#DP451, Tiangen Biochemical Technology Co., Ltd., Beijing)\naccording to the manufacturer’s protocol. The RNA quantity and\npurity were determined using an ultraviolet visible spectrophotometer\n(#UV1800, RUNQEE, Shanghai). Stock cDNA was synthesized from\nhigh-quality RNA (260/280 ratio >1.98) using a commercially\navailable cDNA first chain synthesis kit (#KR103, Tiangen Biochemical\nTechnology Co., Ltd., Beijing) according to the manufacturer’s protocol.\n2.7.2 Primer design and RT-qPCR\nPrimers were based on mRNA sequences1 and designed using the\nPrimer-Blast program.2 Gene-specific primer sequences are listed in\nTable 2. RT-qPCR reaction system (total volume 10 μL) consists of\n5 μL 2 × SG Green qPCR Mix, 0.2 μL forward primer (10 μM), 0.2 μL\nreverse primer (10 μM), 1 μL cDNA, and 3.6 μL RNase-free water.\ncDNA was amplified in triplicate using a QuantStudio™ 7 Flex Real-\nTime qPCR system (ABI, United  States). The RT-qPCR reaction\n\n2 μL forward primer (10 μM), 0.2 μL\nreverse primer (10 μM), 1 μL cDNA, and 3.6 μL RNase-free water.cDNA was amplified in triplicate using a QuantStudio™ 7 Flex Real-\nTime qPCR system (ABI, United  States).The RT-qPCR reaction\nprocedure includes the following steps: Initial denaturation at 95 °C\nfor 2 min; 40 cycles with denaturing at 95 °C for 5 s, annealing and\nextension at 60 °C for 30 s. Melting curve analysis was performed by\nRT-qPCR after 40 cycles of amplification. Data were analyzed using\n2−ΔΔCT method.\n1  http://www.ncbi.nlm.nih.gov/nucleotide\n2  http://www.ncbi.nlm.nih.gov/tools/primer-blast/\n\nJiang et al.\b\n10.3389/fvets.2025.1695023\nFrontiers in Veterinary Science\n05\nfrontiersin.org\n2.8 Rumen fluid sample collection and\nmetabolomic analysis\nAt the end of feeding experiment, one sheep in each replicate\nwas randomly selected and euthanized, followed by bloodletting\nand slaughter. Rumen fluid samples (20 mL) were collected in\n50 mL anticoagulant-free vacutainer tubes, quickly cooled in\nliquid nitrogen, and immediately stored at −80 °C until\nmetabolomic analysis.\nFor\nmetabolomic\nprofiling,\nweighed\nsamples\nwere\nhomogenized in 400 μL of an internal standard-containing\nmethanol–water solution within 2 mL centrifuge tubes. The\nextraction protocol involved ultrasonic treatment, incubation, and\ncentrifugation, after which the clarified supernatants were\nharvested for analysis. A pooled quality control (QC) sample,\ncreated by combining equal volumes of all samples, was analyzed\nalongside the experimental samples to monitor instrument\nperformance. These extracts were analyzed using a Thermo\nUHPLC-Q Exactive HF-X Mass Spectrometer equipped with an\nESI source. The instrument operated in both positive and negative\nion modes with a source temperature of 425 °C, sheath/aux gas\nflows of 50/13 arb, and a spray voltage of 3,500 V. Data were\nacquired in DDA mode from m/z 70–1,050, using a stepped\ncollision energy (20–40–60 V) for fragmentation. The resolution\nwas set to 60,000 for MS1 scans and 7,500 for MS2 scans. The\nanalysis of metabolomic LC/MS data commenced with\npreprocessing in Progenesis QI (v4.3, Waters Corp.). The software\ngenerated a three-dimensional CSV matrix of sample information,\nmetabolite names, and spectral intensities. A data cleaning\npipeline was applied to filter out peaks from internal standards,\nfalse positives, and redundant signals. To correct for instrumental\nand handling variations, we normalized the dataset by the total\nsum of intensities, followed by a log10 transformation to stabilize\nvariance. For metabolite identification, features were cross-\nreferenced with the HMDB, Metlin, and Majorbio databases.\n2.9 Statistical analysis\nData analysis was performed by using SPSS 25.0 (SPSS Inc.,\n\nFor metabolite identification, features were cross-\nreferenced with the HMDB, Metlin, and Majorbio databases.2.9 Statistical analysis\nData analysis was performed by using SPSS 25.0 (SPSS Inc.,\nChicago, IL, United States). Statistical analysis of growth performance\nwas performed using one-way ANOVA followed by Fisher’s protected\nleast significant difference test, with individual sheep (n = 5) as the\nexperimental unit. For all other data, a Student’s t-test was performed.\nAll data are expressed as the mean ± standard error of the mean\n(SEM). Levels for significant differences were set at p < 0.05.\nThe curated data matrix from metabolomic LC/MS was subjected\nto further analysis on the Majorbio cloud platform. An initial\nexploratory assessment was performed via principal component\nanalysis (PCA) with the R package “ropls.” To explore the biological\ncontext, significant metabolic alterations were investigated through\npathway enrichment analysis against the KEGG (Kyoto Encyclopedia\nof Genes and Genomes) database.\n3 Results\n3.1 Diet supplemented with 20% fermented\nonion improves the growth performance\nof Liangshan black sheep\nAs shown in Table 3, compared with the Con group, starting at the\nsame initial weight, adding 20% fermented onion to the basal diet\n(Trt2 group) significantly increased the final body weight, ADG, and\nFCR of Liangshan black sheep (p < 0.05), while the ADFI in the Trt2\ngroup significantly decreased (p < 0.05). On the other hand, adding\n10% fermented onion to basal diet (Trt1 group) only significantly\nincreased the ADG of Liangshan black sheep (p < 0.05), and adding\n30% fermented onion to basal diet (Trt3 group) significantly reduced\nthe final weight, ADG, ADFI and FCR (p < 0.05) of Liangshan black\nsheep (p < 0.05). Thus, the basal diet supplemented with 20%\nfermented onion improves the growth performance of Liangshan\nTABLE 2  List of primer sequences for RT-qPCR.\nGene\nGene ID\nForward primer\nReverse primer\nProduct length\nFN1\n100,216,462\nCACGTGCTATGACGATGGGA\nCTGGTTGTAGGAGTGGGCAG\n178\nTGFβ1\n443,417\nCCTGTACAACCAGCACAACC\nAGGAGCGCACGATCATGTTG\n137\nMyf5\n443,159\nGAGTTCGGGGACGAGTTTGA\nCCTCTGGTTAGGGTTGGTCG\n273\nMyoD1\n443,405\n\nGene\nGene ID\nForward primer\nReverse primer\nProduct length\nFN1\n100,216,462\nCACGTGCTATGACGATGGGA\nCTGGTTGTAGGAGTGGGCAG\n178\nTGFβ1\n443,417\nCCTGTACAACCAGCACAACC\nAGGAGCGCACGATCATGTTG\n137\nMyf5\n443,159\nGAGTTCGGGGACGAGTTTGA\nCCTCTGGTTAGGGTTGGTCG\n273\nMyoD1\n443,405\nTGCACGTCTAGCAACCCAAA\nGCTGTAGTCCATCATGCCGT\n240\nMyoG\n443,158\nGAGCGTGATCTCCGCTACC\nTTGTGGGCATCTGTAGGGTC\n164\nMSTN\n281,187\nCAACTTTTGCCCAAGGCTCC\nACTCCGTGGGCATGGTAATG\n139\nFAS\n100,170,327\nCTTAACAGCACGTCCCCCAT\nTCCTCGGGCTTGTCTTGTTC\n149\nPPARγ\n443,513\nGATCTCCAGCGACATCGACC\nCTCGCCTTTGCTTTGGTCAG\n114\nFABP4\n100,137,067\nGTCCTTCAAATTGGGCCAGG\nGGTGGTTGATTTCCCATCCCA\n124\nACC\n443,186\nAAAAAGTACAGGCGGAGCGA\nAGATGCTATTCCGCAGGCTC\n90\nDGAT1\n100,126,245\nGGTCCGGGACACAGACAAG\nAGTTGCTGAAGCCACTGTCA\n113\nSREBF1\n100,329,218\nGCTCGTCTTCCTCTGTCTCTCC\nGTGGTTGATGCTGGTGGTGTC\n94\nCPT1A\n443,434\nTCACATCCAGGCGGCAAGAG\nAGCAGAGCGGAATCGTAGACC\n119\nLPL\n443,408\nAGACTCCAACGTCATCGTGG\nCTCGAAGTTAGGTCCAGCCG\n259\nHSL\n100,169,699\nAACAGCAGCGACACAACAGAC\nCTCAGACACTTCAGATTCATCCTCAG\n120\n\nJiang et al.\b\n10.3389/fvets.2025.1695023\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nblack sheep, while the effect of 10% fermented onion is minimal, and\neven the effect of the 30% fermented onion is negative. Based on the\ngrowth performance data, the Con group and Trt2 group were\nsubsequently selected for the following study. The Trt1 and Trt3\ngroups were not analyzed for other parameters.\nThe superscripts “a, b, c” indicate that, within each row, values\nwith different superscript letters differ significantly (p < 0.05), whereas\nvalues with no letters or the same superscript letters are not\nsignificantly different (p > 0.05).\n3.2 Diet supplemented with 20% fermented\nonion improves the health of Liangshan\nblack sheep\nAs shown in Table 4, all the serum biochemical parameters of the\nsheep in the two groups were within the normal range. Although there\nwas no significant difference between the experimental group and the\ncontrol group in TP, ALB, and GLB (p > 0.05), the values are slightly\nhigher in the experimental group. Meanwhile, the GPT and GOT in\nthe experimental group sheep were significantly higher than those of\nthe control group (p < 0.05), indicating that the sheep in the\nexperimental group had stronger immunity and metabolism.\nAs shown in Table 5, the levels of serum IgA, IgG, IgM, C3, and C4\nwere significantly increased in sheep fed with a diet supplemented with\n20% fermented onion than in those fed with the control diet (p < 0.05).\nAs shown in Table 6, the levels of serum IL-1β, IL-6, TNF-α, and\nIFN-γ were significantly decreased, and the levels of serum IL-4,\nIL-10, and TGF-β were significantly increased in sheep fed with the\nfermented onion-supplemented diet (p < 0.05).\nAs shown in Table 7, compared with the control diet, the diet\nsupplemented with 20% fermented onion decreased MDA\nconcentration (p < 0.05) but increased SOD, GST, CAT, and GSH-Px\nactivity (p < 0.05), and the T-AOC capacity in the serum (p < 0.05).\n3.3 Diet supplemented with 20% fermented\nonion improves the meat quality of\nLiangshan black sheep\nAs shown in Table  8, compared with the Con group, diet\n\n05), and the T-AOC capacity in the serum (p < 0.05).3.3 Diet supplemented with 20% fermented\nonion improves the meat quality of\nLiangshan black sheep\nAs shown in Table  8, compared with the Con group, diet\nsupplemented with 20% fermented onion decreased meat pH45min\nand pH24h of Liangshan black sheep (p < 0.05). In addition, the\nlightness and yellowness in the Trt2 group significantly decreased\n(p < 0.05), while the redness was significantly increased (p < 0.05).\nThe LD muscle in the Trt2 group had a higher marbling score and\nlower shear force (p < 0.05). Meanwhile, compared with the Con\ngroup, the diet supplemented with 20% fermented onion also\nsignificantly increased the contents of protein and fat in the\nLD muscle.\nTABLE 3  Effect of diet supplemented with fermented onion on growth\nperformance of Liangshan black sheep (n = 5).\nItems\nGroup (fermented onion level, %)\nCon (0)\nTrt1\n(10%)\nTrt2\n(20%)\nTrt3 (30%)\nInitial weight\n(kg)\n23.54 ± 0.57\n23.36 ± 0.38\n23.40 ± 0.61\n23.28 ± 0.33\nFinal weight (kg)\n30.94 ± 0.51b\n31.52 ± 0.56ab\n32.10 ± 0.60a\n28.44 ± 0.62c\nADG (g/d/sheep)\n211.43 ± 4.85c\n233.14 ± 5.93b\n248.57 ± 5.72a\n147.43 ± 14.65d\nADFI (kg/d/\nsheep)\n1.81 ± 0.05a\n1.86 ± 0.17a\n1.62 ± 0.09b\n1.66 ± 0.08b\nFCR (g/g)\n8.55 ± 0.22b\n8.00 ± 0.86b\n6.52 ± 0.39c\n11.37 ± 1.57a\nTABLE 4  Effect of diet supplemented with 20% fermented onion on\nserum biochemical parameters of Liangshan black sheep (n = 5).\nParameters\nCon\nTrt2\nReference\nrange\nTP (g/L)\n59.80 ± 6.52\n67.21 ± 5.76\n60.00–85.00\nALB (g/L)\n23.40 ± 2.91\n26.04 ± 0.67\n20.00–55.00\nGLB (g/L)\n36.40 ± 4.78\n41.17 ± 5.89\n20.00–45.00\nGPT (U/L)\n23.25 ± 0.69\n25.82 ± 2.34*\n0.50–47.00\nGOT (U/L)\n103.01 ± 2.57*\n106.93 ± 2.59*\n12.00–122.00\nThe superscript “*” means p < 0.05 compared with the Con group.\nTABLE 5  Effect of diet supplemented with 20% fermented onion on\nserum immunological parameters of Liangshan black sheep (n = 5).\nParameters\nCon\nTrt2\nIgA (μg/mL)\n27.17 ± 1.13\n35.02 ± 0.36*\nIgG (μg/mL)\n1416.06 ± 31.43\n1806.82 ± 34.85*\nIgM (μg/mL)\n13.74 ± 0.44\n16.40 ± 0.14*\nC3 (μg/mL)\n595.34 ± 16.96\n721.02 ± 10.20*\nC4 (μg/mL)\n241.74 ± 4.12\n282.06 ± 9.96*\nThe superscript “*” means p < 0.05 compared with the Con group.\nTABLE 6  Effect of diet supplemented with 20% fermented onion on\n\n06 ± 31.43\n1806.82 ± 34.85*\nIgM (μg/mL)\n13.74 ± 0.44\n16.40 ± 0.14*\nC3 (μg/mL)\n595.34 ± 16.96\n721.02 ± 10.20*\nC4 (μg/mL)\n241.74 ± 4.12\n282.06 ± 9.96*\nThe superscript “*” means p < 0.05 compared with the Con group.TABLE 6  Effect of diet supplemented with 20% fermented onion on\nserum inflammatory factors of Liangshan black sheep (n = 5).\nItems\nCon\nTrt2\nIL-1β (ng/L)\n150.02 ± 5.07\n128.07 ± 3.90*\nIL-4 (ng/L)\n107.88 ± 3.22\n151.72 ± 2.91*\nIL-6 (ng/L)\n229.28 ± 8.60\n182.26 ± 7.39*\nIL-10 (ng/L)\n199.64 ± 6.48\n262.83 ± 11.43*\nTNF-α (ng/L)\n1101.20 ± 29.30\n933.39 ± 24.02*\nIFN-γ (ng/L)\n66.59 ± 1.33\n51.16 ± 0.91*\nTGF-β (ng/L)\n377.68 ± 13.54\n443.75 ± 13.78*\nThe superscript “*” means p < 0.05 compared with the Con group.\nTABLE 7  Effect of diet supplemented with 20% fermented onion on\nserum antioxidant capacity of Liangshan black sheep (n = 5).\nParameters\nCon\nTrt2\nMDA (nmol/mL)\n7.17 ± 1.29\n4.83 ± 0.85*\nSOD (U/mL)\n62.73 ± 3.76\n71.35 ± 3.94*\nGST (U/mL)\n58.03 ± 7.31\n74.98 ± 11.72*\nCAT (U/mL)\n1.78 ± 0.09\n2.15 ± 0.18*\nGSH-Px (U/mL)\n120.00 ± 22.06\n240.86 ± 25.25*\nT-AOC (U/mL)\n2.59 ± 0.20\n3.21 ± 0.20*\nThe superscript “*” means p < 0.05 compared with the Con group.\n\nJiang et al.\b\n10.3389/fvets.2025.1695023\nFrontiers in Veterinary Science\n07\nfrontiersin.org\n3.4 Effect of diet supplemented with 20%\nfermented onion on the expression of\ngenes related to muscle development and\nfat deposition\nAs shown in Figure  1, compared with the Con group, the\nexpression levels of FN1, TGFβ1, and Myf5 were significantly\nincreased (p < 0.05), the levels of MyoD1 and MyoG showed an\nupward trend (p > 0.05), and the level of MSTN showed a downward\ntrend in the Trt2 group.\nAs shown in Figure  2, compared with the Con group, the\nexpression levels of FAS, PPARγ, and FABP4 were significantly\nincreased (p < 0.05), and the levels of ACC, DGAT1, and SREBF1\nshowed an upward trend (p > 0.05). In contrast, the levels of CPT1A\nand LPL were significantly decreased (p < 0.05), and the level of HSL\nshowed a downward trend in the Trt2 group.\n3.5 Effect of diet supplemented with 20%\nfermented onion on the rumen metabolite\nprofiles of Liangshan black sheep\nAfter the preprocessing steps, peaks with CV >30% in the QCs or\npresent in <80% of the samples were excluded. As a result, 1,143\nmetabolites were identified in the samples.\nThe PCA score plots (Figure  3A) illustrate the metabolite\nprofiles of rumen fluid from the Con and Trt2 groups. The\nvariance contribution, as indicated by the R-squared (R2) value,\nrepresents the extent to which group factors account for the\ndifferences among samples. The metabolite profiles of the Trt2\ngroup significantly differ from those of the control group (Con),\nwith PC1 (20.55%) and PC2 (17.48%) as the first and second\nprincipal component contributions for these differences,\nrespectively.\nThe overall distribution of metabolites with significant changes\nbetween the Con and Trt2 groups was depicted using a volcano plot\n(Figure 3B). In this plot, points were color-coded to denote their\nstatus: red for significant upregulation, blue for significant\ndownregulation, and gray for non-significance. The contribution of\neach metabolite, as measured by its VIP value, is represented by the\nsize of the corresponding point. It can be viewed that 44 metabolites\n\nThe contribution of\neach metabolite, as measured by its VIP value, is represented by the\nsize of the corresponding point.It can be viewed that 44 metabolites\nwere significantly downregulated and four were upregulated by the\n20% fermented onion supplementation.\nFurthermore, a cluster analysis of the differential metabolites was\nvisualized using a heatmap (Figure 3C). In this visualization, the\nvertical axis represents the clustered metabolites, the horizontal axis\ndisplays the individual samples, and the color scale indicates the\nrelative metabolite abundance. Consistent with the results from\nFigure 3B, the metabolite information from each group clusters well,\nin which a large portion of differential metabolites in the Trt2 group\nshows significant downregulation.\nTo elucidate the biological functions of the differential metabolites,\nwe performed pathway analysis using the Kyoto Encyclopedia of\nGenes and Genomes (KEGG) database. The compound names of the\ndifferential metabolites, identified in both positive and negative ion\nmodes, were mapped to the KEGG database to retrieve their associated\npathways. Subsequently, pathway enrichment analysis was conducted\nbased on a hypergeometric test to determine whether these\nmetabolites were significantly enriched in specific pathways. A p-value\nwas calculated for each pathway to assess the significance of the\nenrichment. As shown in Figure 3D, the results are visualized as a\nbubble plot, where purine metabolism, microbial metabolism, cutin,\nsuberine, and wax biosynthesis pathways were identified for these\ndifferential metabolites between the Con and Trt2 groups.\n4 Discussion\nIt was reported that dietary supplementation with onion extracts\nimproves the growth performance and meat quality of broiler\nchickens (13). However, to the best of our knowledge, no study has\nbeen conducted on the effects of a diet supplemented with fermented\nonion on sheep’s growth performance and health. One previous study\ndemonstrated that, after fermentation, the content of esters, alcohols,\nand aldehydes in onions was increased, producing a unique and more\n\nOne previous study\ndemonstrated that, after fermentation, the content of esters, alcohols,\nand aldehydes in onions was increased, producing a unique and more\npopular flavor (14). In addition, fermentation increased the quercetin\ncontent in onion and subsequently enhanced the antioxidative effect\nand anti-inflammatory activities (15–17). The appearance, texture,\njuiciness, flavor, water-holding capacity (WHC), and nutritional value\nof meat are key parameters in meat production. Color, pH, cooking\nloss, drip loss, and shear strength are the main indicators of meat\nquality (18, 19). In this study, the diet supplemented with 20%\nfermented onion improved the final weight, ADG, and FCR;\ndecreased the ADFI, pH45min, pH24h, lightness, yellowness, and shear\nforce; increased the redness, the marbling score, and the content of\nprotein and fat. In summary, the diet supplemented with 20%\nfermented onion improves growth performance and meat quality of\nLiangshan black sheep. This is consistent with previous studies in\nsheep (20). Fermented onion contains a large amount of quercetin,\nwhich acts as a potential prebiotic with several health-promoting\nproperties (16, 21), including anti-oxidation (15, 22), anti-\ninflammatory and immune-regulating effects (17, 23), which may\nbe responsible for promoting sheep growth and improving meat\nquality. However, the beneficial effects of fermented onion on growth\nTABLE 8  Effect of diet supplemented with 20% fermented onion on meat\nquality of Liangshan black sheep (n = 5).\nParameters\nCon (0)\nTrt2 (20%)\npH45min\n6.31 ± 0.01\n6.27 ± 0.02*\npH24h\n5.92 ± 0.03\n5.87 ± 0.03*\nLightness (L*)45min\n30.94 ± 0.23\n30.48 ± 0.25*\nRedness (a*)45min\n16.67 ± 0.37\n17.86 ± 0.98*\nYellowness (b*) 45min\n2.51 ± 0.26\n2.09 ± 0.29*\nMarbling score\n2.2 ± 0.27\n2.7 ± 0.27*\nShear force (N)\n66.80 ± 0.97\n65.33 ± 0.59*\nCooking loss (%)\n36.30 ± 1.58\n35.88 ± 0.32\nChemical compositions (%)\nMoisture\n73.14 ± 0.24\n72.91 ± 0.15\nAsh\n1.27 ± 0.08\n1.26 ± 0.06\nProtein\n21.88 ± 0.37\n23.14 ± 0.71*\nFat\n2.33 ± 0.34\n3.32 ± 0.72*\nThe superscript “*” means p < 0.05 compared with the Con group.\n\nJiang et al.\b\n10.3389/fvets.2025.1695023\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nperformance\nand\nthe\nunderlying\nmechanisms\nwarrant\nfurther investigation.\nThe serum biochemical parameters reflect the health condition of\nanimals and the metabolic status of nutrients. The TP content reflects\nthe intake of protein and the level of absorption and utilization of\nprotein. The increase in TP content is beneficial to promote the growth\nand development of animals and improve the FCR. ALB is a nutrient\ncarrier that maintains plasma osmotic pressure, repairs tissue, and\nprovides energy (24). GLB includes immunoglobulins such as IgA, IgG,\nand IgM, and complements such as C3, C4, with high content and\ndefensive function, which can enhance the body’s resistance and\nprevent infection (25). GPT and GOT are important indicators for\nevaluating liver health. The increase of these two transaminases within\na healthy and normal range indicates that the liver has a higher\nmetabolic capacity (26). In this study, it was found that the contents of\nTP, ALB, and GLB have an increasing trend, and GPT and GOT\nactivities were significantly increased in the Trt2 group. It is likely that\nthe bioactive substances in fermented onion, such as polysaccharides,\nmercaptan, polyphenols, and flavonoids, could promote the synthesis\nand metabolism of protein, stimulate the proliferation and\ndifferentiation of lymphocytes, and then enhance the immune and\nmetabolism functions (27, 28).\nSerum immunoglobulins (IgA, IgM, and IgG) and complement\nC3 Complement C4 are important components of the humoral\nimmune system and play critical roles in immune responses and gut\nepithelial\nprotection\nagainst\npathogens\n(29).\nExcessive\nimmunoglobulins stimulate complement components to enhance\nspecific immune mechanisms in birds and protect them against\ninfection (30). In the present study, the levels of IgA, IgM, IgG, C3, and\nC4 were increased in the serum of Liangshan black sheep fed with diet\nsupplemented with fermented onion.\nInflammatory cytokines play essential roles in immune\nresponses (31). Studies demonstrated that onion polysaccharides\n\nInflammatory cytokines play essential roles in immune\nresponses (31).Studies demonstrated that onion polysaccharides\nhave antioxidant, immunomodulatory, antimicrobial, and anti-\ninflammatory (32–34). Flavonoids extracted from onion inhibited\nthe release of pro-inflammatory factors (e.g., IL-1β, IL-6, TNF-α,\nIFN-γ) and reduced inflammation (17, 35). IL-4 is an anti-\ninflammatory factor that inhibits the synthesis and secretion of\npro-inflammatory factors such as IL-1, IL-6, and TNF-α. So, IL-4\nplays an impartment role in inhibiting the inflammatory response\n(36, 37). IL-10 is a pleiotropic cytokine with anti-inflammatory\nproperties and inhibits the expression of inflammatory cytokines\nsuch as TNF-α, IL-6, and IL-1 by activating macrophages. IL-10 can\nalso inhibit IFN-�� produced by NK cells (38). TGF-β is mainly\nFIGURE 1\nEffect of diet supplemented with 20% fermented onion on the expression of genes related to muscle development. The superscript “*” means p < 0.05\ncompared with the Con group.\n\nJiang et al.\b\n10.3389/fvets.2025.1695023\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nproduced by activated T cells; it inhibits macrophage activation and \nexhibits anti-inflammatory activity (39). In this study, sheep fed with \ndiet supplemented with fermented onion had low serum \nconcentrations of pro-inflammatory factors such as IL-1β, IL-6, \nTNF-α, IFN-γ, and high serum concentrations of anti-inflammatory \nfactors such as IL-4, IL-10, and TGF-β. Similar findings are reported \nFIGURE 2\nEffect of diets supplemented with 20% fermented onion on the expression of genes related to fat deposition. The superscript “*” means p < 0.05 \ncompared with the Con group.\n\nJiang et al.\b\n10.3389/fvets.2025.1695023\nFrontiers in Veterinary Science\n10\nfrontiersin.org\nby Elattar et al. (17), who demonstrate that onion extracts have\npromising anti-inflammatory potentials and can effectively suppress\nthe upregulation of pro-inflammatory markers in LPS-stimulated\nWBCs. Our results suggest that the inclusion of fermented onion in\ndiets enhances humoral immune responses in Liangshan\nblack sheep.\nExcessive reactive oxygen species (ROS) may disrupt\nmetabolism in animals, damage cell structures, and accelerate\noxidation, thereby causing various diseases (19, 40). SOD, GST,\nCAT, GSH-Px, and T-AOC are the primary parameters used to\nevaluate antioxidant levels in organisms. SOD participates in\noxygen metabolism, scavenging, and the reduction of superoxide to\nwater and molecular oxygen (19, 41). GST is involved in antioxidant\nprocesses that protect cells from free radical damage (42). CAT is a\nkey antioxidant enzyme in living organisms that catalyzes the\ndecomposition of toxic hydrogen peroxide (H₂O₂) into harmless\nwater and oxygen, protecting cells from oxidative damage (42).\nGSH-Px reduces lipid hydroperoxides to alcohols and free hydrogen\nperoxide to water (43). T-AOC refers to the total antioxidant level\ncomposed of various antioxidant substances and antioxidant\nenzymes. MDA levels indicate the degree of organic lipid\nperoxidation and are associated with cell damage (44). Our current\nstudy revealed that dietary supplementation with 20% fermented\nonion increased SOD, GST, CAT, GSH-Px activities and total\nantioxidant capacity, meanwhile, reduced the concentration of\nMDA in the serum of Liangshan black sheep. Many studies suggest\nthat onions are rich in quercetin, a strong antioxidant flavonoid that\ncan decrease lipid peroxidation, MDA, but enhance antioxidants\nsuch as SOD, CAT, GSH, GPx, and GST activities (33, 45, 46). The\nfindings of our study suggest that fermented onion may be used as\na natural antioxidant in sheep and that their antioxidant activity\nmay correlate with growth performance.\nFIGURE 3\n\nThe\nfindings of our study suggest that fermented onion may be used as\na natural antioxidant in sheep and that their antioxidant activity\nmay correlate with growth performance.FIGURE 3\nEffect of diets supplemented with 20% fermented onion on the rumen metabolite profiles in Liangshan black sheep. (A) PCA, (B) volcano analysis,\n(C) heatmap, and (D) KEGG pathway enrichment diagrams based on the metabolomic LC/MS data from the rumen liquid of the Liangshan black sheep\nwith (Trt2 group) or without (Con group) 20% fermented onion supplementation.\n\nJiang et al.\b\n10.3389/fvets.2025.1695023\nFrontiers in Veterinary Science\n11\nfrontiersin.org\nFN1 is a macromolecular structural glycoprotein that mediates\nvarious cell-extracellular matrix interactions and can promote cell\nadhesion, migration, growth, and differentiation (47). TGF-β can\npromote\ncell\nproliferation,\ncell\ndifferentiation,\nembryonic\ndevelopment, and tissue fibrosis (48). Myf5, MyoD, and MyoG all\nbelong to myogenic regulatory factors. They participate in muscle\ndevelopment and promote the proliferation and differentiation of\nmyoblasts (49). Conversely, MSTN is a protein that negatively\nregulates the growth of skeletal muscle, and inactivation of MSTN\nimproves the mass of skeletal muscle (50, 51). In our study, dietary\nsupplementation with 20% fermented onion increased the expression\nof FN1, TGFβ1, Myf5, MyoD, and MyoG but decreased the expression\nof MSTN. In summary, dietary supplemented with 20% fermented\nonion can promote muscle development. This might be one of the\nreasons why the growth performance and protein content of the sheep\nin the Trt2 group were higher.\nFAS catalyzes the synthesis of fatty acids from acetyl-CoA and\nmalonyl-CoA, thereby playing an important role in body fat\ndeposition (52). PPARγ promotes the differentiation of adipocyte\nprecursor cells into mature adipocytes and participates in lipid storage\n(53). FABP4 participates in the transportation, storage, and\nmetabolism of fatty acids as well as facilitates the absorption and\nutilization of water-insoluble dietary long-chain fatty acids (54, 55).\nACC is a rate-limiting enzyme that plays a key role in fatty acid\nsynthesis, which catalysis the first committed step of fatty acid\nsynthesis (56). DGAT1 transfers the acyl group from fatty acyl-\nCoenzyme A to diacylglycerol to synthesize triacylglycerol. It is the\nkey rate-limiting enzyme that controls the synthesis rate of\ntriacylglycerol (57). Triacylglycerol is an important component of\nintramuscular fat and the most significant form of energy storage in\neukaryotic cells (58). Intramuscular fat, which was also subjectively\n\nTriacylglycerol is an important component of\nintramuscular fat and the most significant form of energy storage in\neukaryotic cells (58).Intramuscular fat, which was also subjectively\nevaluated as a marbling score, represents an important meat quality\ntrait (59). Relatively higher intramuscular fat improves tenderness,\nmaking the meat more tender and easier to chew. Intramuscular fat\ncan also improve the water-holding capacity, reduce the cooking loss,\nmaintain the juiciness, and enhance the meat flavor. There are two\ntranscripts of SREBF1 known as SREBP-1a and SREBP-1c. SREBP-1a,\nwhich regulates the biosynthesis of cholesterol and fatty acid, exhibits\nthe highest transcriptional activity in the splenic organ and intestinal\ntract. SREBP-1c is highly expressed in the liver, adipose tissue and\nskeletal muscle, mainly regulating fatty acid synthesis and participating\nin the differentiation of adipocytes (60). CPT1 is the rate-limiting\nenzyme of fatty acid β-oxidation. CPT1 contains three isoforms:\nCPT1A, CPT1B, and CPT1C, and they are highly expressed in the\nliver, muscle, and brain, respectively. CPT1A transports long-chain\nfatty acids to mitochondria for β-oxidation, thereby providing energy\nfor cells (61, 62). LPL and HSL are two important enzymes involved\nin lipolysis. LPL decomposes triglyceride into glycerol and free fatty\nacids (63). Hormone-sensitive lipase (HSL) is responsible for releasing\nfree fatty acids from adipose tissue into circulation (64). In this study,\ndietary supplementation with 20% fermented onion increases the\nexpression of genes related to fat deposition (FAS, PPARγ, FABP4,\nACC, DGAT1, and SREBF1) while reducing the expression of genes\nrelated to fat breakdown (CPT1A, LPL, and HSL). This explains the\nreason why the marbling score and fat content of the mutton in the\nTrt2 group were higher.\nThe metabolomic analysis of rumen fluid provides a critical\nbiochemical lens through which to interpret the significant\nimprovements in growth performance observed in sheep\nsupplemented with 20% fermented onion. The profound shift in\n\nThe metabolomic analysis of rumen fluid provides a critical\nbiochemical lens through which to interpret the significant\nimprovements in growth performance observed in sheep\nsupplemented with 20% fermented onion.The profound shift in\nthe rumen metabolite profiles, characterized by the downregulation\nof 44 metabolites and upregulation of four, strongly suggests that\n20% fermented onion acts as a potent modulator of the rumen\nmicrobial ecosystem. The enrichment of these differential\nmetabolites in specific pathways, notably purine metabolism,\nmicrobial metabolism in diverse environments, and cutin/suberine\nbiosynthesis, offers a compelling mechanistic basis for the\nenhanced average daily gain (ADG) and final body weight. The\ndownregulation of numerous purine metabolites is particularly\nintriguing. In the context of the rumen, purines (e.g., adenine,\nguanine) and their degradation products (e.g., hypoxanthine,\nxanthine, uric acid) are central to microbial nucleic acid synthesis\n(65). A decrease in their free concentration in the rumen fluid may\nnot indicate reduced production, but rather a more efficient\ncapture and utilization by the microbial biomass for proliferation.\nThis heightened efficiency in microbial synthesis would lead to an\nincreased yield of microbial crude protein (MCP), which is the\nprimary source of high-quality protein for the host ruminant (66).\nThis proposed increase in MCP flow to the small intestine directly\nsupports the observed improvements in growth performance and\naligns with the upregulation of myogenic-related genes such\nas Myf5.\nFurthermore, the significant alterations within the “microbial\nmetabolism in diverse environments” pathway underscore a\nfundamental reshaping of the rumen’s metabolic network. Fermented\nonion, rich in prebiotics like fructans, probiotics from the fermentation\nprocess, and bioactive organosulfur compounds, likely selects for a\nmore efficient microbial community (67, 68). This could involve a shift\nfrom less efficient proteolytic fermentation, which can produce\nwasteful compounds like ammonia, to more robust saccharolytic\n\nThis could involve a shift\nfrom less efficient proteolytic fermentation, which can produce\nwasteful compounds like ammonia, to more robust saccharolytic\nfermentation, maximizing the production of volatile fatty acids\n(VFAs) (69). Although VFAs were not directly measured, they are the\nprimary energy source for ruminants (70), and an enhanced\nproduction would directly fuel the energy-demanding processes of\ngrowth and maintenance, explaining the increased average daily feed\nintake (ADFI) as the animals’ metabolic capacity expanded. The\nenrichment in the “cutin and suberine biosynthesis” pathway, which\ninvolves the breakdown of complex plant polymers, points towards an\nenhanced fibrolytic capability of the rumen microbiota. The\ndownregulation of certain intermediates in this pathway could signify\na more complete and efficient degradation of the basal diet’s fibrous\ncomponents, unlocking previously inaccessible energy and nutrients.\nCollectively, these metabolomic shifts paint a coherent picture of a\nrumen environment reprogrammed by fermented onion for superior\nnutrient conversion efficiency, providing both the protein building\nblocks (via enhanced MCP) and the energy (via enhanced VFA\nproduction and fiber digestion) necessary to drive the remarkable\nimprovements in the growth performance of Liangshan black\nsheep (71).\n5 Conclusion\nThis study revealed that diet supplemented with 20% fermented\nonion improved growth performance, health, and meat quality in\nLiangshan\nblack\nsheep\nthrough\nenhancing\nimmunity,\n\nJiang et al.\b\n10.3389/fvets.2025.1695023\nFrontiers in Veterinary Science\n12\nfrontiersin.org\nanti-inflammatory ability, antioxidant capacity, and modifying the\nrumen metabolite profiles.\nData availability statement\nThe raw data supporting the conclusions of this article will\nbe made available by the authors, without undue reservation.\nEthics statement\nThe animal study was approved by the Experimental Animal\nWelfare and Ethics Committee of Xichang University. The study was\nconducted in accordance with the local legislation and\ninstitutional requirements.\nAuthor contributions\nSJ: Writing – original draft. CY: Resources, Writing – review &\nediting. CJ: Data curation, Writing – review & editing. CL: Writing –\nreview & editing, Software. GL: Writing  – review & editing,\nInvestigation. HG: Writing  – review & editing, Resources. WZ:\nWriting  – review & editing, Methodology. YZ: Formal analysis,\nWriting – review & editing. ZH: Funding acquisition, Writing – review\n& editing.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. The authors are grateful for\nthe financial support provided by the Doctoral Research Project of\nXichang University (YBZ202254) and the Project of State Key\nLaboratory of Animal Nutrition and Feeding (2025SKLANF-CAU2).\nConflict of interest\nGL, HG, and ZH were employed by Xinjiang Taikun Group\nCo., Ltd.\nThe remaining authors declare that the research was conducted in\nthe absence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. 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+{"text": "【兽医文献】\n标题: Case Report: Inhaled salbutamol in the successful treatment of life-threatening acute hyperkalaemia in an anaesthetised horse\n\nTYPE Review\nPUBLISHED 04 September 2025\nDOI 10.3389/fvets.2025.1663577\nOPEN ACCESS\nEDITED BY\nGuanghui Zhao,\nNorthwest A&F University, China\nREVIEWED BY\nQingxia Wu,\nTibet Agricultural and Animal Husbandry\nUniversity, China\nAli Raza,\nUniversity of Engineering and\nTechnology, Pakistan\n*CORRESPONDENCE\nWenlong Wang\nwwl.imau@163.com\nRECEIVED 10 July 2025\nACCEPTED 20 August 2025\nPUBLISHED 04 September 2025\nCITATION\nWang T, Chen X, Yan X, Su Y, Gao W, Liu C and\nWang W (2025) Progress in serology and\nmolecular biology of equine parasite\ndiagnosis: sustainable control strategies.\nFront. Vet. Sci. 12:1663577.\ndoi: 10.3389/fvets.2025.1663577\nCOPYRIGHT\n© 2025 Wang, Chen, Yan, Su, Gao, Liu and\nWang. This is an open-access article\ndistributed under the terms of the Creative\nCommons Attribution License (CC BY). The\nuse, distribution or reproduction in other\nforums is permitted, provided the original\nauthor(s) and the copyright owner(s) are\ncredited and that the original publication in\nthis journal is cited, in accordance with\naccepted academic practice. No use,\ndistribution or reproduction is permitted\nwhich does not comply with these terms.\nProgress in serology and\nmolecular biology of equine\nparasite diagnosis: sustainable\ncontrol strategies\nTengyu Wang1, Xindi Chen1, Xu Yan2, Ya Su3, Wa Gao4,\nChunxia Liu5 and Wenlong Wang1*\n1Key Laboratory of Animal Disease Clinical Diagnosis and Treatment Technology, College of Veterinary\nMedicine, Inner Mongolia Agricultural University, Hohhot, China, 2National Key Laboratory of\nAgricultural Microbiology, Huazhong Agricultural University, Wuhan, China, 3Department of Husbandry\nand Veterinary, Ulanqab Vocational College, Ulanqab, China, 4Inner Mongolia Key Laboratory of\nTick-Borne Zoonotic Infectious Disease, Department of Medicine, Hetao College, Bavan Nur, China,\n5Key Laboratory of Animal Disease Clinical Diagnosis and Treatment Technology, College of Life\nScience, Inner Mongolia Agricultural University, Hohhot, China\nInternal parasitic infections are a persistent challenge for horse owners, in the\nabsence of effective vaccines and the growing challenge of drug resistance,\n\nleading many researchers to view current control strategies as unsustainable.\nDespite slow progress over the past two decades, effective parasitic diagnosis\nremains crucial for controlling infections and preventing the growing issue of\ndrug resistance. This review examines the research progress in serological and\nmolecular biological diagnostic methods for major equine parasites. Currently,\nmost diagnostic techniques are based on genes such as ITS1, ITS2, COI, and\nIGS, which have been applied to equine strongylids, including Strongylus spp.,\nCylicocyclus spp., and Cylicostephanus spp. These methods are particularly\nsuitable for large-scale epidemiological studies and rapid species identification.\nAlthough many diagnostic methods have been developed, most remain confined\nto laboratory research and have seldom been used for real-time field diagnostics.\nFuture research should prioritize precise diagnostic methods and clinically\napplicable alternatives. Additionally, whole genome sequencing has been widely\nused in eukaryotes for population genetics and the development of diagnostic\nmarkers. However, comprehensive genomic data on parasitic species infecting\nequines is still limited. With the decrease in sequencing costs in the post-\ngenomic era, a growing number of genome assemblies are expected to be\nreleased soon. These genome maps will offer comprehensive genomic data\nto identify specific genetic markers and variations associated with parasitic\ninfections, enabling more accurate and reliable diagnostic techniques. High-\nthroughput sequencing technologies will significantly accelerate progress in\nequine parasitology research and the development of diagnostic tools like\nenzyme-linked immunosorbent assay (ELISA) and TaqMan quantitative PCR\n(qPCR). At the same time, this paper also provides some insights into\nthe research direction of sustainable control programs and equine parasite\ndiagnostic methods.\nKEYWORDS\nhorses, parasites, serology, molecular biology, diagnosis, sustainable control\nFrontiers in Veterinary Science\n01\nfrontiersin.org\n\nWang et al.\n10.3389/fvets.2025.1663577\n1 Introduction\nIt is estimated that approximately 116 million equines\n(donkeys, horses, and mules) are distributed globally. According\nto FAO data, the number of equines has remained relatively\nstable over the past 50 years (1961–2019). World Mapper\nestimates that the global horse population will reach about\n60 million by 2024. Despite millions of years of biological\nevolution,\nEquus\nremains\nhighly\nsusceptible\nto\nmultiple\ninternal parasites due to its unique living habits and feeding\nconditions.\nMore\nthan\n60\ncommon\nparasites,\nincluding\nprotozoa, trematodes, cestodes, and nematodes, are known\nto cause parasitic infections (1). In most cases, equines exhibit\nsymptoms similar to those of other livestock when infected\nwith internal parasites, such as abdominal swelling, nutritional\ndeficiencies, and weight loss. These symptoms can be assessed\nthrough body condition scoring, and are often accompanied by\ndigestive disorders, poor appetite, and stunted growth, especially\nin\nimmunocompromised\nfoals\n(2).\nDuring\ngastrointestinal\nparasitic infections, equines may experience disruptions in\ntheir intestinal microbiota and impaired nutrient absorption,\nin addition to clinical signs. This results in increased feed\nconversion ratios and a higher demand for proteins and amino\nacids. Incorrect feeding of concentrates and changes in diet\nformulation may also result in the occurrence of “Friday disease”\n(3, 4).\nFor a long time, parasitic detection methods have traditionally\nrelied on microscopic examination and necropsy of collected\nparasite tissues (5, 6), fecal samples (7, 8), and blood smears\n(9–12), thus enabling the diagnosis of internal and external\nparasitic infections in livestock. Some equine parasites, such\nas Parascaris equorum (P. equorum), Anoplocephala perfoliata\n(A. perfoliate), and Oxyuris equi, expel their eggs with feces,\nwhile others, such as the horse stomach bot fly, are expelled\nonly when the larvae mature and the infection load becomes\nsubstantial. Furthermore, parasitic examinations of horses require\ntrained personnel to collect feces and perform microscopic\n\nFurthermore, parasitic examinations of horses require\ntrained personnel to collect feces and perform microscopic\nexamination, necessitating the individual restraint and examination\nof each horse, which makes it impractical for analyzing large\npopulations. Due to the lack of simple and effective internal\nparasite detection methods, deworming in equines is typically\nperformed 2–4 times per year, depending on their use. Since the\ndiscovery of anthelmintics, animal husbandry has largely relied\non the frequent use of chemical drugs, such as Benzimidazoles\n(BZDs), macrolides (MLs), and tetrahydropyrimidines, for year-\nround parasitic control (13, 14). Most competitive sport horses\nundergo deworming four or more times a year to enhance\nperformance, which has contributed to the growing issue of\nresistance to deworming drugs. Anthelmintic Resistance (AR)\nrefers to the inability of previously effective drugs to kill parasite\npopulations when exposed to therapeutic doses (15). Over time,\nsensitive parasites transmit drug resistance to their offspring\nthrough genetic mechanisms, leading to the loss of sensitivity in\npreviously susceptible populations (16). As the livestock industry\nhas rapidly developed, the prevalence of drug resistance has\nbecome particularly severe, especially in cattle (17, 18), sheep, and\ngoats (19, 20), which are economic animals. In recent years, an\nincreasing number of clinical and epidemiological reports have\ndescribed the occurrence of AR in various parasites of equines\n(21–23). Global equine parasitic epidemiological surveys report\nresistance to these four classes of anthelmintics/dewormers in over\n70 species of parasites, including nematodes, Gasterophilus spp.,\nand P. equorum (24–26), further highlighting the need to reassess\nparasite control strategies.\nWhile there are differing opinions on the mechanisms behind\nthe development of drug resistance, most researchers agree\non the importance of early monitoring of parasitic infections.\nEarly detection and control of parasitic infections and AR\nare critical. Many proposed deworming programs recommend\n\nEarly detection and control of parasitic infections and AR\nare critical.Many proposed deworming programs recommend\nregular population-wide testing using fecal samples; however,\nthis poses a challenge for equine-specific parasites that are not\ndetectable through fecal egg analysis. The rapid evolution of AR\nnecessitates the urgent development of efficient and standardized\ndiagnostic methods for internal parasites (27). Early and accurate\nidentification of parasitic infections in individuals, along with\nan understanding of their development mechanisms, will help\nimplement sustained and effective deworming programs and may\nassist in slowing the development of resistance. Additionally,\nthe development of in vitro diagnostic methods is mostly based\non the development of host-parasite immune-related protective\nantigens, which will also contribute to the development of new\nanthelmintic drugs and vaccines. In the foreseeable future, parasites\nin the body will still need to be controlled using chemical\ndrugs for a long time. Although many researchers are exploring\nsome alternative strategies to combat parasitic infections, such as\nbreeding resistant varieties, increasing protein supplementation,\nand implementing “low-parasite” pasture strategies, most methods\nto counter resistance focus on boosting the host’s immunity to\nenhance parasite resistance. Research has shown that adding certain\nbioactive substances to the diet can play a significant role in\nreducing parasitic infections and preventing AR development (28).\nHowever, while this approach can mitigate some of the impact\nparasites have on livestock traits, it may also lead to increased\nfeed consumption. Currently, many in vitro diagnostic methods\nare used in parasitic research on ruminants such as cattle and\nsheep, and these methods can also detect host AR. However,\nrelated studies have few examples of parasites in horses, such\nas P. equorum, Strongylus vulgaris (S. vulgaris) and Gastrophilus\nspp., which can also allow us to obtain a lot of reference from\nthe study of ruminants in the diagnostic test of horse parasites\n\nequorum, Strongylus vulgaris (S.vulgaris) and Gastrophilus\nspp., which can also allow us to obtain a lot of reference from\nthe study of ruminants in the diagnostic test of horse parasites\nand the development mechanism of AR. However, it is worth\nthinking that the delay of related research also further illustrates\nthat the development of related detection methods, with many\nareas in need of improvement. The purpose of this review is\nto systematically describe the diagnostic methods of common\nparasites in horses, focusing on in vitro detection techniques based\non serology and molecular biology, and to explain their application\nand scientific strategies in diagnosing horse parasites, aiming to\nsupport the prevention and control of AR in horses. Additionally,\nthis review presents opinions on the development of the horse\nindustry and the sustainable control of parasites in equine animals\nin the future.\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n\nWang et al.\n10.3389/fvets.2025.1663577\n2 Serological detection\n2.1 Overview of serological detection and\nits role in the diagnosis\nSerology mainly achieves the purpose of detection and\ndiagnosis by detecting antibodies in serum, plasma, saliva,\nsemen, or cerebrospinal fluid of the host (29, 30). In some\nclinical settings, serological testing remains the gold standard for\ndiagnosing parasitic infections when biological samples cannot\nbe directly obtained. Although serological analysis is similar to\nmicroscopic examination in terms of operational convenience\nand test turnaround time, for parasites such as Babesia and\nTheileria, which are difficult to accurately identify on blood\nsmears, or some horses infected with Trypanosoma cruzi, these\nindividuals may be mild or asymptomatic, serological analysis\ncan show higher sensitivity and specificity, which is crucial\nfor the diagnostic process (31). The parasitic stage in the host\ntriggers both nonspecific and specific immune responses, which\nare influenced by the location of the parasite within the host,\nthe host’s health condition, and the characteristics of the parasite\nantigens. Therefore, a deep understanding of the host’s immune\nresponse is critical in the development of serological diagnostic\nmethods (32). Equine parasites are typically obligate parasites that\nremain in the host for extended periods. Their life cycles primarily\noccur within the host, with eggs or adult parasites expelled in\nthe feces. Among the current diagnostic methods, serological\ndiagnostics serve as an effective alternative to parasitological\nexaminations and autopsy diagnoses. During the larvae are in\nthe migration stage in the host or fail to find signs of parasitic\ninfection on the body surface, serological detection is a simple\nand effective method for rapid diagnosis of parasitic infection. As\nlarge animals, horses typically do not experience significant pain\nor noticeable damage to the host’s body from internal parasitic\ninfections, making it difficult for horse owners to determine\nwhether their horses are infected with parasites through daily\n\nAs\nlarge animals, horses typically do not experience significant pain\nor noticeable damage to the host’s body from internal parasitic\ninfections, making it difficult for horse owners to determine\nwhether their horses are infected with parasites through daily\nobservation. However, during the early stages of parasitic infection\n(especially during the larval development stage), the establishment\nof early diagnostic methods could provide strong support for timely\ndrug treatment and intervention, preventing or mitigating the\ndevelopment of resistance.\n2.2 Host immune mechanisms in parasitic\ninfections\nAt present, studies on ruminants infected with liver flukes\nhave shown that during the parasitic infection of the host,\nboth humoral and cell-mediated immune responses occur, and\nthere is a complex interaction between the parasite and the\nhost’s immune system after infection (33). Common equine\nparasitic nematodes such as P. equorum, Strongyloides westeri, and\nGastrophilus spp. regulate the immune system by releasing soluble\nmediators. These mediators interact with the host’s immune cells\nand molecules, leading to the expression of secretory proteins\nand carbohydrates. In nematode studies, it has been clarified that\ncysteine and serine protease inhibitors (cystatins and serpins) can\nblock T-cell responses, regulate macrophage function, and alter\nthe activity of eosinophils and neutrophils (34). The regulation\nof the host immune system is both rapid and sustained, allowing\nparasites to persist for extended periods within the host. During\nthe process of maintaining immune system homeostasis, both\nparasites and host-derived damage-associated molecular patterns\nare crucial for protective immune responses and tissue repair\nmechanisms. In studies involving fly larvae parasites, the life\ncycle and parasitic stages of Hypoderma bovis (H. bovis) share\nmany similarities with certain obligate equine parasites, such as\nthe horse stomach bot fly. The larvae of H. bovis can penetrate\nthe host’s skin after hatching, triggering an immune response.\nCurrently, immunological techniques detecting excretory-secretory\n\nThe larvae of H.bovis can penetrate\nthe host’s skin after hatching, triggering an immune response.Currently, immunological techniques detecting excretory-secretory\nantigens (ESA) specific to parasites can identify anti-parasitic\nantibodies within 3–5 weeks post-infection (35, 36). Although\nnatural ESA are immunogenic, their accuracy and sensitivity\nare limited, especially in low-intensity infections, where they\nare particularly ineffective, especially in large animals like cattle\nand horses, where such methods provide only coarse diagnostic\nresults. In contrast, recombinant antigens exhibit greater sensitivity\nand specificity than natural crude antigens. Their standardized\nproduction not only improves yield but also has significant cost-\ncontrol implications (37). However, despite its diagnostic value,\nserological testing faces limitations in large-scale epidemiological\nsurveys. For instance, after horses undergo systematic deworming\ntreatment, antibody titers remain elevated for an extended period,\nwhich can significantly affect serological test results and lead to a\nconsiderable number of “false positives” (38).\n2.3 Limitations and challenges of current\nserological methods\nCurrently developed serological diagnostic methods include the\ncomplement fixation test (CFT), agglutination test (AT), indirect\nimmunofluorescence assay (IFA), Western blot, and enzyme-\nlinked immunosorbent assay (ELISA). These methods significantly\nenhance diagnostic sensitivity. A highly sensitive and specific\nELISA method for detecting Fasciola hepatica has been developed,\nbased on recombinant F. hepatica-specific antigens (39). These\nantigens include enzymes essential for the parasite’s survival within\nthe host, such as glutathione S-transferases (40–42). Research\non has shown that after initial infection with H. bovis larvae,\nfirst-stage larvae (L1) produce trypsin and chymotrypsin. These\nproteases regulate the immune response by inhibiting both specific\nand nonspecific host reactions (43, 44). IgG antibodies in cattle\ntypically appear around 45 days after H. bovis infection, peaking\n\nThese\nproteases regulate the immune response by inhibiting both specific\nand nonspecific host reactions (43, 44).IgG antibodies in cattle\ntypically appear around 45 days after H.bovis infection, peaking\nat approximately 18 weeks. This is critical for implementing early\nserological diagnostic strategies. Although the larvae emerge from\nthe host’s back skin during the third larval stage (L3), antibodies\nremain at a high level for about 4 months. Diagnosing during\nthis period is essential for formulating an effective, scientifically\ninformed deworming strategy (45). However, the prevalence of\nparasitic diseases is often associated with poor pasture management\nor prolonged exposure to parasitic-contaminated environments.\nMost horses on pastures or farms are wild and free-roaming,\nwhich presents significant challenges for tasks such as restraining\nFrontiers in Veterinary Science\n03\nfrontiersin.org\n\nWang et al.\n10.3389/fvets.2025.1663577\nhorses and collecting blood samples. The collection process may\nresult in varying degrees of injury to the horses. Additionally,\nthese diagnostic methods also have inherent limitations, such as\ncross-reactivity, which can lead to false-positive results.\n3 Molecular biology detection\n3.1 Limitations of traditional diagnostic\nmethods\nSince the advent of the optical microscope, microscopic\nexamination has been considered the most commonly used\nand economical gold standard for clinical parasitic diagnosis.\nParasitological research originated from taxonomic studies, where\nresearchers identified species by examining the morphological\ncharacteristics of parasites under the microscope. Although simple\nand cost-effective, this method has several limitations. For instance,\nit is labor-intensive, time-consuming, requires highly trained\npersonnel, and makes it difficult to distinguish subtle differences\nbetween closely related species (46), During the microscopic\nexamination of roundworms and nematodes, FEC values do not\nalways correlate with worm burden (47), leading parasitologists to\nincreasingly explore molecular biology techniques, such as gene\namplification. As previously mentioned, immunodiagnostic tests\nhave significant limitations. Currently, there are no commercially\navailable or FDA-approved antibody tests for diagnosing parasitic\ndiseases, such as cryptosporidiosis, schistosomiasis, and African\ntrypanosomiasis. Commonly used antigen preparations include\ncrude proteins, recombinant purified proteins, adult parasites,\neggs, and non-standardized test antigens, leading to significant\nvariability in experimental outcomes. In the development of\ndiagnostic methods for Theileria haneyi (T. haneyi), since T. equi\nand T. haneyi are closely related, the current equi merozoite antigen\n1 -based competitive ELISA used in the U.S. detects T. equi but\nnot T. haneyi. Previous studies on serological diagnostic methods\nhave found that immunoblot results show antigen cross-reactivity\nbetween species within the same genus, especially in regions where\n\nS.detects T.equi but\nnot T.haneyi.Previous studies on serological diagnostic methods\nhave found that immunoblot results show antigen cross-reactivity\nbetween species within the same genus, especially in regions where\nmore than one parasite is prevalent, leading to no results or false-\npositive results (48). Furthermore, when the number of parasites in\nthe host is low, serological tests tend to have lower sensitivity, which\nposes significant challenges for detection. Studies have shown that\ncirculating antigens may persist in the host for some time after\nsystematic deworming treatment, leading to false-positive results\nwhen compared with parasitic test outcomes. Therefore, the most\ncritical time to apply this diagnostic method is during the acute\nphase of parasitic infection, ensuring the accuracy and reliability\nof the test results (49–51).\n3.2 Emerging molecular techniques for\nparasitic diagnosis\nWith\nthe\nongoing\ndevelopment\nof\nmolecular\nbiology\ntechniques, polymerase chain reaction (PCR) technology has\nbeen increasingly applied to parasite detection. In addition to\ntraditional PCR, multiplex PCR and nested PCR techniques\nhave also emerged. Multiplex PCR can detect multiple sequences\nin a single reaction, enabling the simultaneous detection of\nvarious parasites (52). Several molecular biology-based diagnostic\nmethods, including amplified fragment length polymorphism,\nrestriction fragment length polymorphism (RFLP), and real-time\nquantitative PCR (qPCR), have been utilized in parasitic detection.\nLoop-mediated Isothermal Amplification (LAMP) and Luminex\nMulti-Analyte Profiling (xMAP) technologies are emerging as\nnovel methods for parasitic disease diagnosis. LAMP is a rapid,\nisothermal DNA amplification method that operates at a constant\ntemperature (60–65 ◦C) using Bst DNA polymerase. It forms\nstem-loop structures containing repeated target DNA sequences,\nenabling the amplification of DNA to high copy numbers within\n1 h. Nucleic acid-based molecular techniques provide superior\nsensitivity and specificity compared to traditional diagnostic\ntests. In microscopic parasitology, species identification based\n\nNucleic acid-based molecular techniques provide superior\nsensitivity and specificity compared to traditional diagnostic\ntests.In microscopic parasitology, species identification based\nsolely on egg observation is challenging, especially for certain\nparasite intermediate hosts. Equine fasciolosis is a significant\nre-emerging parasitic disease of horses, donkeys, and mules,\ncaused by trematodes of the Fasciola genus, infecting animals\nthrough the ingestion of encysted metacercariae found on plants.\nTo date, no diagnostic method is considered the gold standard for\ndetecting equine fasciolosis (53). Molecular biology techniques can\ndetect parasitic infections sensitively from small host samples, such\nas blood and saliva, even in asymptomatic hosts, making them\nhighly valuable for early diagnosis and the detection of subclinical\ninfections (54). qPCR uses fluorescently labeled probes or double-\nstranded DNA-specific fluorescent dyes to analyze the fluorescence\nsignal of amplification products in real time, eliminating the need\nfor complex electrophoresis steps (55). Due to its rapid speed, high\nspecificity, excellent sensitivity, broad dynamic range, and low risk\nof cross-contamination, qPCR is widely used in the development of\ndiagnostic methods for various diseases. It is particularly favored by\nresearchers (56, 57). Additionally, RT-qPCR can be used to study\nparasite vector capability, host infectivity, diagnostics, and drug\nefficacy (22). TaqMan probe-based qPCR utilizes a non-extendable\nDNA probe that hybridizes to a specific region within the amplicon,\nensuring high specificity and protecting quantitative precision by\npreventing the amplification of nonspecific molecules (58). This\nmethod enables accurate assessment of infection intensity by the\nsame parasite in different hosts, with exceptional specificity and\nquantitative capability. This characteristic makes it an ideal tool\nfor assessing host infection loads, providing a scientific basis for\ndeveloping individualized deworming treatment plans. Molecular\nbiology-based diagnostic techniques allow precise quantification\n\nThis characteristic makes it an ideal tool\nfor assessing host infection loads, providing a scientific basis for\ndeveloping individualized deworming treatment plans.Molecular\nbiology-based diagnostic techniques allow precise quantification\nof infection levels, providing important references for developing\nmore effective and accurate deworming treatment plans, thereby\nplaying a significant role in improving treatment efficacy and\nreducing drug misuse.\n4 Application of serological techniques\nin parasite diagnosis of equids\n4.1 Importance of serological techniques in\nfield diagnosis\nFor most equine parasites, the humoral immune response\ntypically precedes clinical symptoms during the initial infection.\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n\nWang et al.\n10.3389/fvets.2025.1663577\nHorses often display notable behavioral changes only when they\nexperience abdominal pain or more severe limb and hoof disorders.\nParasitic infections are challenging to diagnose through routine\nvisual inspection. During microscopic examination, parasites such\nas F. hepatica, Clonorchis sinensis, and Strongyloides can be detected\nin fecal samples. However, due to intermittent shedding or\nsampling limitations, eggs per gram (EPG) are typically very low\nwhen using the modified McMaster method (56). A retrospective\nstudy found that horses with EPG values below 500 had\nsignificantly fewer parasites in their intestines than those suggested\nby the fecal egg count (FEC). In summary, fecal egg tests have issues\nwith missed diagnoses and instability. Serological methods may\nexhibit cross-reactivity between phylogenetically closely related\nspecies, and combining immunochroma-tographic tests with rapid\nimmunoenzymatic assays (RIA) can greatly enhance the specificity\nand sensitivity of these tests, improving diagnostic accuracy (59,\n60). Most serological methods for detecting equine parasites rely\non ELISA (61).\n4.2 Serological diagnosis of Cyathostomins\nCyathostomins (small strongyles) are the most prevalent and\nsignificant parasites affecting horses (62). Over the past two\ndecades, the Jacqueline B. Matthews team at the Moredun\nResearch Institute in the UK has made significant progress\nin developing in vitro diagnostic methods for Cyathostomins.\nEarly studies identified two diagnostic antigens (20 and 25\nkDa) and developed an ELISA method based on serum-specific\nIgG(T) responses (63–65). Although this method exhibits low\ncross-reactivity with S. edentatus and S. vulgaris, it remains\nvaluable as a diagnostic antigen. Subsequent studies involved\nconstructing a cDNA library from mixed Cyathostomins species\nand immunoscreening, resulting in the identification of the\ngut-related larval antigen Cy-GALA protein, which acts as an\nimmune marker for the developmental larval stage. This antigen\nis exclusively expressed during the larval stage and is the first\n\nThis antigen\nis exclusively expressed during the larval stage and is the first\ndiagnostic method capable of detecting the latent phase of\nCyathostomins (66). Further research focused on recombinantly\nexpressing two proteins from 14 Cyathostomins species, leading\nto the selection of an antigen cocktail (Cocktail 3; CT3), which,\nbased on specific recombinant antigen IgG(T), provides a standard\ncurve to generate a “serum score” for each horse. The results\ndemonstrated excellent detection performance (receiver operating\ncharacteristic area under the curve values >0.9), making it\nsuitable for diagnosing horses infected with over 1,000–10,000\nCyathostomins in the mucosa and intestines, with sensitivity\nand specificity greater than 90 and 70%, respectively, making it\ncommercially viable (67).\n4.3 Serological diagnosis of A. perfoliate\nEarly studies on A. perfoliate involved serological research\non scolex antigens (68), whole worm extracts, and excretory-\nsecretory (ES) antigens (69). However, crude protein antigens,\nalthough easy to prepare, complicate the protein composition,\nwith at least 14 different proteins identified, predominantly low\nmolecular weight proteins in the 31–45 kDa range. Among the\ncrude antigen mixture, two highly immunogenic proteins (12\nand 13 kDa) were identified. Researchers measured the negative\nthreshold values for anti-12/13 kDa IgG and IgG(T) ELISA systems,\nwith critical OD values of 0.123 and 0.103, and sensitivity values\nof 56 and 62%, respectively. This suggests that anti-12/13 kDa\nIgG(T) may more accurately predict infection intensity in A.\nperfoliate (70). However, this method also yielded negative results\nin horses with low infection intensity, suggesting that diagnostic\nfactors in parasitic diseases are not solely based on the parasite\npresence (especially in large animals) but also depend on the\nhost’s infection intensity (71). Among common equine parasitic\ninfections, Anoplocephala spp. exhibit mixed infections, with A.\nperfoliata having significantly higher infection levels than other\nspecies. Immunological studies have yet to resolve antigen cross-\n\nAmong common equine parasitic\ninfections, Anoplocephala spp.exhibit mixed infections, with A.perfoliata having significantly higher infection levels than other\nspecies.Immunological studies have yet to resolve antigen cross-\nreactivity between A. perfoliata and A. magna (72). No cross-\nreactivity has been observed between 12/13 kDa ES antigens and\nother parasite antigens such as A. mamillana and Gastrophilus spp.\n(70). Currently, commercial diagnostic kits for equine tapeworm,\nincluding the Tapeworm Blood Test and Saliva Test launched in\n2014 by Austin Davis Biologics Ltd., are based on these 12/13 kDa\nES antigens. The saliva test has a sensitivity of 83% and specificity\nof 85%, with no misdiagnoses when detecting infections with more\nthan 20 adult tapeworms, yielding results comparable to serological\ntests (73).\n4.4 Serological diagnosis of Gastrophilus\nJin et al. developed an indirect ELISA detection method\nusing crude L3 antigens from Gastrophilus pecorum and salivary\ngland antigens. Only salivary gland antigens could be used for\nfecal sample detection, exhibiting high specificity (100%) and\nsensitivity (83.33%), while crude protein may non-specifically\nbind with other substances in the feces, hindering direct antigen\ndetection (74).\n4.5 Serological diagnosis of P. equorum\nP. equorum, a nematode that threatens foal health, begins\nshedding eggs approximately 10–15 weeks after the initial\ninfection (75). Serological diagnostic tools are crucial for assessing\nthe\nlikelihood\nof\nlatent\nP.\nequorum\ninfections.\nCurrently,\nmany parasite ES products from Ascaris species are utilized\nin antigen-based diagnostic development (76), including those\nfrom Ascaris. Lumbricoides (77) and Ascaris suum (78). In\nprevious studies on P. equorum ES products, the fifth stage\n(L5) of P. equorum has a molecular weight range of 12–\n189 kDa, while L2/L3 products range from 12 to 94 kDa.\nBlood samples from two naturally infected foals showed antigen\nrecognition at approximately 19, 22, 26, and 34 kDa for ES\nproducts in Silver-stained SDS-PAGE analysis (79). However,\nIgG(T) antibodies were not detected in foal serum prior to\ncolostrum ingestion.\n\nHowever,\nIgG(T) antibodies were not detected in foal serum prior to\ncolostrum ingestion.\nFrontiers in Veterinary Science\n05\nfrontiersin.org\n\nWang et al.\n10.3389/fvets.2025.1663577\n4.6 Challenges and considerations in\nserological testing\nAccording to the performance assessment by Gasser et al.\n(80), ES antigens, when used as the capture layer in ES-ELISA,\nshow significantly higher sensitivity than the other two antigens,\nalthough this increased sensitivity compromises antigen specificity.\nAcquiring a detailed treatment history is especially important for\nserological testing. Common deworming drugs, such as MLs, do\nnot provide prolonged deworming effects, and IgG levels typically\ndecrease over a period of 6 months following treatment (81, 82). It\nis generally recommended to conduct serological testing at least 4\nmonths after treatment. A key issue in serology is the potential for\nantibody cross-reactivity. Although current serological diagnostic\nmethods have achieved acceptable specificity, identifying and\naccurately expressing target antigens, as well as ensuring their high\nquality, are critical for their diagnostic application (83). We hope\nthat equine parasite serology will advance to the same level of\nadvancement as virology and bacteriology. Table 1 summarizes the\nadvantages of various serological detection methods in parasite\ndiagnosis and the main challenges faced by the current technology.\nAdditionally, the current research progress and factors influencing\nfuture developments are summarized.\n5 Application of molecular biology\ntechniques in parasite diagnosis of\nequids\n5.1 Traditional PCR-based molecular\ndiagnostic methods\nIn recent years, significant progress has been made in\nparasitic research of great public health and veterinary importance,\nparticularly in phylogenetic systematics and diagnostics, due\nto ongoing advancements in life sciences and technology. The\napplication of molecular biology has introduced new approaches\nand methods for the early diagnosis and precise control of parasites.\nStudies aimed at differentiating A. magna and A. perfoliata have\nshown that both parasites, whether in crude or recombinant\nprotein form, contain low molecular weight immunoreactive\ncomponents in their antigens. These components are recognized\n\nStudies aimed at differentiating A.magna and A.perfoliata have\nshown that both parasites, whether in crude or recombinant\nprotein form, contain low molecular weight immunoreactive\ncomponents in their antigens.These components are recognized\nby Anoplocephala-positive sera at the genus level, but not at the\nspecies level (84). Currently, no reliable serological assays are\navailable to differentiate A. magna from A. perfoliata, primarily\ndue to the absence of species-specific antigens. This diagnostic\nlimitation highlights the critical importance of molecular-based\napproaches in differentiating these closely related species. Several\nPCR-based diagnostic methods have been developed for equine\nparasite detection, primarily utilizing ITS1 and ITS2 sequence\nregions. These two sequences, employed as phylogenetic molecular\nmarkers in molecular classification and phylogenetic diagnosis,\nhave significantly corroborated taxonomic frameworks derived\nfrom traditional morphological and life history analyses (85). To\nconfirm infections in equines caused by S. vulgaris, S. equinus, A.\nperfoliata, and Parascaris species, fecal egg counts are commonly\nconducted, but the identification of the parasite species causing\nthe infection cannot be determined by egg morphology alone.\nAccurate diagnosis often requires culturing the collected feces for\na certain period to allow development to the third larval stage (L3).\nDNA fingerprinting techniques, such as restriction fragment length\npolymorphism (RFLP), single-strand conformation polymorphism\n(SSCP), and random amplified polymorphic DNA (RAPD), have\nbeen widely used in the development of molecular diagnostic\nmethods for equine parasites, due to their speed, cost-effectiveness,\nand the fact that they do not require complete genome\ninformation. The fundamental principle of RFLP involves the\ndigestion of DNA fragments from target genes using specific\nrestriction endonucleases. Genotypes are identified based on\nthe electrophoretic patterns of the resulting digestion products,\neliminating the need for further sequencing or sequence alignment\n\nGenotypes are identified based on\nthe electrophoretic patterns of the resulting digestion products,\neliminating the need for further sequencing or sequence alignment\nanalysis (86). In 1995, Campbell et al. first used PCR-RFLP\ntechnology to amplify the ITS2 region of Strongylus spp., and the\nresults indicated that the ITS2 sequence, as a diagnostic target gene,\nexhibits low intraspecific variation and high interspecific variation.\nThis study laid the foundation for the ITS2 region as the basis\nfor molecular identification. Additionally, RFLP technology in this\nstudy also provided a means of separating amplified products using\nagarose gel based on fragment size, allowing for rapid identification\nof Strongylus spp. without sequencing or sequence analysis (87). In\nsubsequent studies, Gasser and Monti optimized PCR-RFLP and\nPCR-SSCP methods to distinguish 16 species of equine strongyles\n(88, 89). PCR-RAPD technology has been widely used in the\nclassification of numerous parasites (90, 91). In equine parasitic\ndetection, this technology has been applied to define species of\ntwo Cylicocyclus species, such as C. insigne and C. elongatus,\nwhich are morphologically very similar but can be accurately\ndistinguished, further demonstrating that this method is suitable\nfor closely related species. Over the past 30 years, researchers\nhave primarily developed diagnostic methods for equine parasites\nbased on ribosomal RNA genes, particularly the ITS and IGS\n(Intergenic Spacer) regions. In studies of equine strongylids, by\ncomparing the performance of two molecular barcodes (COI gene\nand ITS-2 rDNA gene) in different biological type samples, it was\nfound that although the results of the two barcodes were fairly\nconsistent, the PCR amplification efficiency of the COI gene was\nlower than that of ITS2, particularly in certain strongyle species\n(e.g., Cylicostephanus calicatus and Cylicostephanus longibursatus),\nwhere the COI barcode amplification efficiency was less than\n70%. In group identification, the COI gene, due to its higher\ngenetic diversity and larger sequence variation, can lead to PCR\n\ng., Cylicostephanus calicatus and Cylicostephanus longibursatus),\nwhere the COI barcode amplification efficiency was less than\n70%.In group identification, the COI gene, due to its higher\ngenetic diversity and larger sequence variation, can lead to PCR\namplification bias, making it difficult to identify specific life\ncycle stages of some species and affecting the final sequencing\nresults (92).\n5.2 Emerging molecular diagnostic\napproaches\nHodgkinson et al. (93, 94) used Southern blot to validate\nthe design of digoxigenin (DIG)-labeled probes targeting the IGS\nconserved region of four highly prevalent species (C. ashworthi,\nC. nassatus, C. longibursatus, C. goldi), achieving molecular\nidentification of single eggs, L3 larvae, and L4 larvae. The research\nFrontiers in Veterinary Science\n06\nfrontiersin.org\n\nWang et al.\n10.3389/fvets.2025.1663577\nTABLE 1 Comparison of different serological methods.\nSerologic\nmethod\nCore principle\nApplication fields and\ntechnical advantages\nCurrent\nchallenges\nFuture development\ndirection and potential\nApplication\nexample\nELISA\nBased on\nantigen-antibody\nreaction,\nparasite-associated\nantibodies were detected\nby enzyme labeling\nreaction\nThe operation is simple, the\ndetection sample requirements are\nlow, the low concentration\ndetection is suitable for large-scale\nsample detection\nIt is not sensitive to acute\ninfection. It is difficult to\ndetect when the antibody\nconcentration is low. The\npreparation cost of\nmonoclonal antibody is\nhigh, and it needs to be\ndetected immediately\nImprove sensitivity and develop\nefficient detection reagents for low\nantibody titers\n(39, 48, 63, 67, 73,\n187–189)\nRIA\nRadioimmunoassay was\nused to detect antigen by\nradiolabeled antibody\nHigh specificity and sensitivity,\nsuitable for quantitative analysis\nThe use of radioactive\nmaterials has safety risks\nand complicated\noperation\nExplore non-radioactive\nalternative technologies to\nsimplify operations and expand\napplications\n(190–192)\nIFAT\nImmunofluorescence\nantibody method was\nused to detect parasite\nantibodies\nHigh sensitivity, suitable for\naccurate diagnosis\nNeed professional\nequipment, operation\nmust be set at the same\ntime the control, easy to\nother antigen cross\nreaction, the result may\nbe affected by the\noperator\nOptimize reagents and develop a\nsimpler detection platform\n(193–197)\nCFT\nSerological test,\ndiagnosis by detection of\nantibodies or antigens\nHigh sensitivity, suitable for\nlarge-scale detection\nIt is not sensitive to acute\ninfection, and the\ndetection process and\nresult analysis take a long\ntime. The non-specific\nbinding of complements\ncan easily lead to false\npositive results\nCombined with other\nimmunological techniques, a\ncomprehensive diagnostic method\nwas developed\n(198–201)\nteam later developed a PCR-ELISA method, which was completely\nconsistent with the Southern blot results and could identify\nlow-abundance species (e.g., C. catinatum, which accounted for\nonly 1.7%). This method was used to assess the therapeutic\nefficacy in horses treated with BZDs. Compared to the RFLP\nprobe method, it was more suitable for analyzing polymorphic\n\ng., C.catinatum, which accounted for\nonly 1.7%).This method was used to assess the therapeutic\nefficacy in horses treated with BZDs.Compared to the RFLP\nprobe method, it was more suitable for analyzing polymorphic\nfragments (95). The widely used Reverse Line Blot (RLB) detection\nmethod for diagnosing strongyloid species currently involves\nnon-radioactive hybridization of PCR amplicons with different\noligonucleotide probes in a single reaction, allowing for the\ndetection and identification of more than 16 strongyloid species\nsimultaneously (96–98). This method has also been widely applied\nto study the relationship between drug resistance and changes in\nCyathostomin population structure, thereby exploring the potential\nmechanisms behind the shortened egg reappearance period after\ntreatment (99, 100). Martins amplified the ITS2 DNA fragment\nof S. vulgaris by PCR and compared it with the results of fecal\nculture L3, demonstrating a lower consistency, further confirming\nthat PCR diagnostic methods have become the most effective\ntool for diagnosing S. vulgaris (101). Nielsen developed a dual-\nlabeled TaqMan qPCR assay speci���cally targeting S. vulgaris,\nwhich exhibited no cross-reactivity with other equine parasitic\nnematodes. This assay demonstrated a detection limit of 0.5 egg\nequivalents, providing a precise and efficient tool for diagnosing S.\nvulgaris infections. Similarly, Zhou et al. developed a novel real-\ntime quantitative PCR assay based on the TaqMan-MGB probe,\nspecifically for detecting T. haneyi, a pathogen causing equine\npiroplasmosis. The assay demonstrated excellent specificity and\nsensitivity, with a detection limit of 1 × 10² copies/µl, and was\nvalidated by comparison with nested PCR. This new diagnostic\nmethod promises to enhance early detection and control of T.\nhaneyi infections, complementing existing PCR techniques for\nTheileria equi (T. equi), Babesia caballi (B. caballi). The LAMP-\nbased diagnostic method for T. equi and B. caballi has been\ndeveloped, providing rapid on-site results within 1 h (102). Fecal\nsamples, the most accessible for on-site diagnosis, contain more\n\nequi), Babesia caballi (B.caballi).The LAMP-\nbased diagnostic method for T.equi and B.caballi has been\ndeveloped, providing rapid on-site results within 1 h (102).Fecal\nsamples, the most accessible for on-site diagnosis, contain more\npotential inhibitors than blood samples, which can reduce the\nsensitivity and accuracy of the LAMP assay. Furthermore, fecal\nsamples often require more rigorous pretreatment and ultrapure\nDNA extraction (103, 104). The advancement of microfluidic chip\ntechnology has enabled the successful application of isothermal\namplification methods for detecting various veterinary parasites\n(105–107). Chen et al. (108) developed a microfluidic LAMP system\nthat can identify five parasites. This method integrates sample\npretreatment, biological separation, biochemical reactions, and\nsignal analysis into microchips just a few square centimeters in\nsize, achieving both miniaturization and automation in parasite\ndetection (109, 110).\n5.3 Next-generation sequencing for\ncomprehensive parasitic diagnosis\nWith the ongoing development of high-throughput sequencing\ntechnology, next-generation sequencing (NGS) has become widely\napplied in the diagnosis and research of equine parasites. Mitchell\nused NGS technology to conduct qualitative and quantitative\nFrontiers in Veterinary Science\n07\nfrontiersin.org\n\nWang et al.\n10.3389/fvets.2025.1663577\nanalysis of the species and egg abundance in equine fecal samples,\nreplacing traditional larval culturing and necropsy methods. This\nalso marks the first recorded instance of the equine “nemabiome”\n(111). Ghazanfar et al. combined NGS technology with the\nmodified McMaster technique to study the prevalence and diversity\nof gastrointestinal nematodes in Australian racehorses. Compared\nto traditional microscopic examination or PCR methods, NGS\nproved more efficient and precise, identifying 23 strongylid species,\nincluding 18 cyathostomins and five large strongyle species. Using\nhigh-throughput data from NGS, the species abundance of various\nparasites in fecal samples was analyzed. The three most abundant\nspecies of small strongyles were Cylicostephanus longibursatus\n(28%), Cylicocyclus nassatus (23%), and Coronocyclus coronatus\n(23%), which together accounted for 95.8% of the entire parasite\ncommunity. The study also revealed the diversity of nematode\ncommunities across different climatic zones and age groups. In\nfoals and weanlings, the most common strongylid species was\nCylicostephanus longibursatus, with relative abundances of 35.3%\nin foals and 34.1% in weanlings. Nematode community diversity\nin the Non-Seasonal Rainfall Zone and Winter Rainfall Zone was\nsignificantly higher than in the Mediterranean Rainfall Zone (112).\nRecently, Hamad et al. employed ITS-2 rDNA metabolic barcoding\nsequencing technology with the Illumina MiSeq platform to\nstudy the strongylid nematode populations on a large equine\nfarm in Thailand. By analyzing fecal samples from 57 horses\non the farm, they successfully identified 14 strongylid species,\nincluding Cylicocyclus nassatus and Cylicostephanus longibursatus.\nThe study revealed the complexity of the strongylid nematode\ncommunity and the relative abundance of species, while also\nvalidated the quantitative potential of high-throughput technology\nin equine parasite detection. It provides an important molecular\ndata foundation for future drug resistance monitoring and parasite\nmanagement (113, 114). Table 2 summarizes the advantages of\n\nIt provides an important molecular\ndata foundation for future drug resistance monitoring and parasite\nmanagement (113, 114).Table 2 summarizes the advantages of\nwidely used molecular biological detection methods in parasite\ndiagnosis, along with the main challenges faced by current\ntechnologies. It also outlines the current research progress and\nfactors influencing future developments.\n6 Pasture management and\nsustainable control strategies for\nequine parasites\nThis review provides a clear summary of current serological\nand molecular biological methods for detecting equine parasites.\nTraditional fecal examination techniques remain a key component\nin the development of detection methods. Serological and\nmolecular biology-based diagnostic tools are crucial in the early\ndetection of parasitic infections in horses. However, these methods\nface several limitations regarding their development and practical\nfield application. Table 3 presents a comparative analysis of the two\ndiagnostic methods, highlighting key factors such as sensitivity,\nfield applicability, and commercial viability. These factors are vital\nfor the selection of appropriate clinical diagnostic tools.\nGastrointestinal parasites, particularly cyathostomins, are\namong the most common equine parasitic infections and are\nprevalent in global epidemiological surveys, with over 50 species\nidentified (115). In pastures where horses are free-ranging, the\ninfection rate can reach up to 100%, with most horses carrying 3–4\ndifferent internal parasites. Due to the misuse of deworming drugs\nand unscientific medication practices, numerous failures in control\nmeasures have occurred, and the failure of recommended treatment\nprotocols has indicated widespread resistance to deworming\nmedications. This poses new challenges in parasite control\nand resistance management. Effective parasite control requires\nplanning and management at each developmental stage. The use of\nbroad-spectrum dewormers, such as ivermectin, should be assessed\naccording to pasture management conditions, reducing deworming\nfrequency and drug dosages while avoiding frequent large doses.\n\nThe use of\nbroad-spectrum dewormers, such as ivermectin, should be assessed\naccording to pasture management conditions, reducing deworming\nfrequency and drug dosages while avoiding frequent large doses.\nScientific recommendations on whether horses need deworming\nshould be provided through in vitro diagnostic methods for equine\nparasites. Growing resistance has created new challenges for farm\nand ranch owners, urgently necessitating the re-evaluation of\nmanagement strategies and the formulation of scientifically sound\ncontrol plans. Large-scale fecal collection and analysis should be\nperformed, with control plans based on diagnostic results. Horses\ncan be grouped according to parasite species and number, and\nhigh- and low-risk areas should be established. High-risk areas\ninclude high-density breeding zones and young horses (2–5 years\nold) raised on low-quality forage, with high FEC and initial\nindications of AR. Low-risk areas consist of low-density breeding\nor individually housed horses, fed high-quality, clean pasture, and\nexhibiting low FEC as confirmed by parasite testing. Horses in\nlow-risk areas only need to maintain good feeding environments\nand fodder hygiene. With veterinary evaluation, the frequency of\nfecal egg count monitoring can be reduced in these areas. Grazing\nmanagement is especially critical for herds where parasite control is\nfailing. If rotational grazing is used, the pasture should be regularly\ncleaned to prevent overgrazing, which can effectively control the\nrisk of parasitic infection.\nFor the past half-century, the horse industry has depended\non the use of chemical drugs for deworming. In recent years, an\nincreasing amount of research shows that uncontrolled single or\nfrequent rotations of drugs accelerate the emergence and spread\nof resistant populations, rendering a limited number of drugs\nineffective (116). With the rapid development of the global equine\nand animal husbandry industries, sustainable pasture management\nstrategies have become a key focus. In ruminant research, many\nnatural plants or feed additives similar to probiotics have been\n\nWith the rapid development of the global equine\nand animal husbandry industries, sustainable pasture management\nstrategies have become a key focus.In ruminant research, many\nnatural plants or feed additives similar to probiotics have been\nfound to combat parasites, but research in equines is relatively\nlimited. Beg et al. administered water-soluble herbal extracts of\nNigella sativa (Kalwanji) seeds, Fumaria parviflora (Burgi Shatara)\nleaves, and Fumaria macrophylla (Jantaan) leaves via nasogastric\ntube on Day 0 and 18, with doses of 0.05, 0.10, and 0.15\ng/kg, respectively, and evaluated efficacy based on the eggs per\ngram of feces (EPG). The results showed that all herbal extracts\nsignificantly reduced EPG after the first treatment on Day 18,\nwith the best results at Day 28, demonstrating that N. sativa\nhas strong anthelmintic effects against P. equorum (117). Payne\net al. conducted crude extractions from 37 native Australian\nplants and tested their in vitro effects on the development of\nCyathostomins. Seven plants, including Acacia baileyana, Acacia\nmelanoxylon, Acacia podalyriifolia, Alectryon oleifolius, Duboisia\nFrontiers in Veterinary Science\n08\nfrontiersin.org\n\nWang et al.\n10.3389/fvets.2025.1663577\nTABLE 2 Comparison of different molecular biology methods.\nMolecular\nbiology\nmethods\nCore principle\nApplication fields and\ntechnical advantages\nCurrent\nchallenges\nFuture development\ndirection and potential\nApplication\nexample\nPCR\nUsing DNA\namplification\ntechnology to detect the\ngenetic material of\nparasites\nHigh sensitivity and specificity, can\ndetect low concentrations of\nparasitic infections\nSample processing\nrequirements are strict,\nonly qualitative, high\nequipment\nrequirements, may be\naffected by pollution\nDevelop portable PCR equipment\nto simplify operations and reduce\non-site requirements\n(52, 202–204)\nReal-time PCR\nReal-time quantification\nof DNA in the\namplification process\nAccurate detection, real-time\nquantification, suitable for viral\nload assessment\nHigh equipment\nrequirements, complex\noperation, sensitivity is\nlimited by the quality of\nthe sample\nHigh-throughput detection,\ncombined with portable devices,\nimproves real-time detection\ncapabilities\n(163, 205–207)\nLAMP\nAmplification of\nspecific DNA fragments\nby cyclic reaction\nRapid reaction, low equipment\nrequirements, suitable for on-site\nrapid detection, real-time\nevaluation results by naked eye\nSensitive to sample\ncontamination, there is\na cross-reaction, may\naffect the accuracy\nCombined with microfluidic chip,\nthe development of multiple\ntarget detection platform\n(208–211)\nMicroarray\nDetection of multiple\ntarget genes by\nmicroarray\nIt can detect the expression of\nmultiple parasite genes at the same\ntime, which is suitable for\ncomprehensive analysis and drug\nresistance detection of complex\nsamples\nThe data processing is\ncomplex, and only\nsamples with existing\nsequencing information\ncan be detected, with\nhigh cost and low\nsensitivity\nImprove chip sensitivity and\nthroughput, reduce costs, and\ndevelop convenient application\ntools\n(212–214)\nRFLP\nSpecific DNA fragments\nare analyzed by\nrestriction enzyme\ndigestion\nIt can be used to study the typing\nand genetic diversity of parasite\nspecies\nThe operation is\ncumbersome, the\namount of DNA\ntemplate is high, the\ntime is long, and the\ncost is high\nCombined with high-throughput\ntechnology, accelerate data\ncollection and analysis, and\nimprove application flexibility\n(87, 88, 215–219)\nRAPD\nBy using random\nprimers to amplify\nspecific DNA\nfragments, genetic\npolymorphisms can be\n\ndetected\nThe technology is simple, low cost,\nand does not require complete\ngenomic information, which is\nsuitable for rapid screening of\nmultiple samples\nThe sensitivity is\nunstable, mainly\naffected by primers, and\ncannot provide accurate\ngenomic location\ninformation\nCombined with high-throughput\ntechnology, the accuracy of\nprimer design is improved, which\nis suitable for gene diversity\nresearch\n(90, 91, 220)\nRLB\nBy using specific probes\nto hybridize with the\ntarget DNA, the DNA of\nmultiple parasites was\ndetected\nIt can detect multiple parasites at\nthe same time with high sensitivity\nand is suitable for clinical samples\nHigh-quality probes\nand samples are needed,\nand the control of\nhybridization\nconditions is high\nCombined with microfluidic\ntechnology, the operation is\nsimplified, the sensitivity is\nimproved, and the detection range\nis expanded\n(96–98, 100, 221,\n222)\nNext-\nGeneration\nSequencing\n(NGS)\nThe parasite genome\nsequence was detected\nby large-scale parallel\nsequencing technology\nHigh precision, can carry out\nwhole genome analysis of parasites,\nhelp to analyze the development\nprocess of drug resistance, and\nformulate scientific prevention and\ncontrol programs\nData analysis is\ncomplex, requires\nstrong computing\npower, high technical\nrequirements, high cost\nReduce costs, improve data\nprocessing efficiency, applied to a\nwide range of parasite diagnosis\n(111, 112, 223)\nhopwoodii, Eucalyptus gomphocephala, and Santalum spicatum,\ncompletely inhibited the development of Cyathostomins larvae,\nwith an IC50 value of just 30.9 µg/ml to achieve 50% developmental\ninhibition. Other tested plant extracts displayed varying degrees\nof anthelmintic activity in vitro (118). Alectryon oleifolius was one\nof the most effective plants in inhibiting larvae. The main active\ncomponent in the plant is Procyanidin A2, which, after purification,\ncompletely inhibited larval development at a low concentration\nof 50 µg/ml, with an IC50 value of 12.6 µg/ml. Compared\nto commonly used anthelmintics such as ivermectin (IC50 =\n0.22 ng/ml) and levamisole (IC50 = 115 ng/ml), Procyanidin A2\nexhibited excellent anthelmintic properties (119). Some studies\nsuggest that tannins and total phenols are considered the key\n\n6 µg/ml.Compared\nto commonly used anthelmintics such as ivermectin (IC50 =\n0.22 ng/ml) and levamisole (IC50 = 115 ng/ml), Procyanidin A2\nexhibited excellent anthelmintic properties (119).Some studies\nsuggest that tannins and total phenols are considered the key\nactive components in plants for combating parasites. Tannins can\nbind to proline, an amino acid in the nematode’s cuticle, causing\nlarval death (120–122). Moringa oleifera (M. oleifera) leaves contain\nabundant vitamin E and quercetin, which protect the plant from\nmicrobial damage during growth and possess strong antioxidant\nactivity (123–125). Mbogning-Tayo et al. (126) used M. oleifera leaf\nextract for egg hatching inhibition tests on Haemonchus contortus\neggs, showing that at concentrations of 3.75 and 5 mg/ml of M.\noleifera extract, the egg development was inhibited by 60.3% ± 8.2%\nand 92.8% ± 6.2%, respectively. Although research on M. oleifera\ncomponents is incomplete, the maximum anti-parasitic effect likely\nresults from the coordinated action of multiple components (127).\nBuono et al. extracted allicin, the active component of garlic,\nFrontiers in Veterinary Science\n09\nfrontiersin.org\n\nWang et al.\n10.3389/fvets.2025.1663577\nTABLE 3 comparison-of-serological-methods-and-molecular-biology-methods.\nFeature\nSerological methods\nMolecular biology methods\nSensitivity\nModerate sensitivity; antibody testing is less effective in the early stages\nbut more reliable in later stages. It may fail to detect latent or low-level\ninfections\nHigh sensitivity; capable of detecting early, latent, and low-level\ninfections\nSpecificity\nRelatively high; potential cross-reactivity among closely related species\nExtremely high (under strict contamination control), with\nminimal cross-reactivity\nDetection stages\nPrimarily used for chronic or asymptomatic infections, with detection\ndependent on the onset of immune response\nApplicable for detecting infections at any stage, particularly\neffective for acute or latent infections\nCost\nLow cost; suitable for use by non-specialized personnel\nHigh cost; requires specialized equipment, reagents, and trained\npersonnel\nField applicability\nSuitable for large-scale testing and field use, particularly in\nresource-limited settings. Requires minimal laboratory infrastructure,\nand the developed colloidal gold method is applicable for on-site rapid\ndetection\nLimited field applicability; sample processing requires a\nlaboratory setting. The method is highly dependent on\nenvironmental conditions, with strict temperature requirements\nfor reagent storage and transport, and specialized equipment\nneeded for detection\nEase of operation\nSimple and rapid; no technical training required, easy to implement in\nclinical practice\nMore complex; requires specialized training and dedicated\nequipment\nTime for results\nShort sample preparation and reaction time, suitable for rapid\nscreening\nLonger sample preparation and turnaround time, especially with\ncomplex protocols such as qPCR and NGS\nCommercial viability\nELISA is a well-established and widely used method, while the\ndevelopment of colloidal gold assays has a low entry barrier, making\nmass production feasible. Currently, commercial test kits are available\nfor various parasitic infections\nMolecular detection methods have a high development\n\nCurrently, commercial test kits are available\nfor various parasitic infections\nMolecular detection methods have a high development\nthreshold, requiring comprehensive omics data support. The\nproduction capacity of biological reagents is limited, with a high\ndependency on instruments and reagents. Commercial test kits\nfor equine parasites are limited in number\nCross-reactivity\nRelatively high, particularly among phylogenetically similar species,\nwhere pathogens of the same family or genus may share similar\nepitopes\nExhibits a low degree of species recognition, typically based on\nthe design of specific target sequences\nSample requirements\nRequires serum or other bodily fluids, which can be used directly or\nsubjected to simple centrifugation\nRequires blood, fecal, or tissue samples, typically requiring\ncomplex steps such as nucleic acid extraction\nImpact of treatment\nCan be affected by previous treatments (e.g., anti-parasitic drugs)\nNot affected by past treatments; reflects current infection status\nSource\n(36, 194, 224–234)\n(92, 161, 226, 229, 232, 235–240)\nand conducted egg hatching inhibition assay (EHA) and larval\nmigration inhibition tests on Cyathostominae eggs and L3 larvae,\nshowing inhibitory activity. Allicin also exhibited growth inhibition\nin in vitro studies of B. caballi and Theleria equi. However, in\nnaturally infected horses, it did not exhibit significant anti-parasitic\neffects (128–130). This suggests that garlic’s mechanism of action\nmay not directly kill the parasites, but rather enhance resistance\nby modulating the immune system. Other herbs, such as Acacia\nnilotica, Rumex abyssinicus, and Zingiber officinale, also exhibit\nstrong anti-parasitic effects (131).\nWith growing attention to the sustainable development\nof grassland ecosystems and livestock, biological control-based\nmanagement strategies are being increasingly implemented.\nDuddingtonia flagrans (D. flagrans) and Pochonia chlamydosporia\nhave been shown to be effective and feasible alternatives to chemical\ndewormers. Horses consuming these fungi can survive in the\ngastrointestinal tract without disrupting the existing gut microbiota\n\nDuddingtonia flagrans (D.flagrans) and Pochonia chlamydosporia\nhave been shown to be effective and feasible alternatives to chemical\ndewormers.Horses consuming these fungi can survive in the\ngastrointestinal tract without disrupting the existing gut microbiota\n(132). After excretion with feces, predatory fungi form capture\nnetworks that significantly reduce larval contamination of pastures\nand the environment, thus potentially affecting the survival of\nnematode larvae (133–135). Commercial products based on D.\nflagrans have already been launched on the market, primarily\napplied through feed supplementation. Following a period of\nresearch and use, these products have significantly reduced the\nnumber of parasites in pastures (132, 136–138). Research indicates\nthat equine gastrointestinal worm infections significantly disrupt\nthe host’s intestinal microbial (139, 140). Adding probiotic dietary\nsupplements to the diet is undoubtedly an effective way to reduce\nparasitic infections in horses (141).\nIn many pastures where horses are free-ranging, many horse\nowners engage in unscientific, irregular, and non-systematic\ndeworming practices. The need for parasite testing and guided\ndeworming should be emphasized through public education and\ngovernment regulatory oversight. A comprehensive, year-round\ncontrol strategy should be developed based on regional differences\nand parasite distribution. Regular cleaning of feces from horses\nand other livestock to maintain pasture hygiene has long been\nconsidered a fundamental method for controlling equine parasitic\ninfections. However, this task is time-consuming and labor-\nintensive, making it difficult for many pasture owners to adopt.\nDespite the current available parasite control strategies, these\nactions should still be undertaken.\n7 Conclusion and future directions\nOver the past 30 years, numerous diagnostic kits for identifying\nand diagnosing equine parasites have been developed, leading\nFrontiers in Veterinary Science\n10\nfrontiersin.org\n\nWang et al.\n10.3389/fvets.2025.1663577\nto significant advancements. However, most of these methods\ndetect the presence of infection in horses, often requiring\nexpensive instruments and reagents. Additionally, diagnosing\nparasitic diseases during their latent phase is challenging, which\nlikely explains why few findings have been widely implemented.\nAlthough current parasitic diagnostic technologies, such as ELISA\nand qPCR, offer highly sensitive and specific detection methods,\nthey still face several limitations in field applications, particularly\nwhen compared to traditional fecal egg detection methods.\nMain obstacles include high equipment costs, the need for\nspecialized training, and difficulties in transporting equipment\nand reagents to remote regions. In contrast, although FEC testing\nhas relatively lower sensitivity, it offers significant advantages in\ncost, examination time, and operational convenience, making it\nthe most widely used screening tool in resource-limited areas.\nBridging the gap between laboratory-based technologies and their\nfield applications requires the establishment of mobile diagnostic\nplatforms that integrate serology and molecular biology (142). The\nwidespread adoption of smartphones has led to the development\nof efficient smartphone-based parasitic diagnostic platforms,\nincluding those for egg identification, remote diagnosis, and\ncolorimetric assays (143–145). Additionally, expanding veterinary\ntraining programs is crucial to ensure on-site professionals become\nproficient in these diagnostic techniques, thereby enhancing their\neffectiveness in field applications. Automated mobile diagnostic\nplatforms can significantly reduce labor costs. Compared to\nmanual methods for sample extraction and preparation, automated\nplatforms use fewer reagents and exhibit smaller variability\nbetween groups (108). Point-of-care tests (POCTs) have emerged\nas a practical alternative and are rapidly growing in veterinary\ndiagnostics (146). Fecal egg count reduction tests are currently\nconsidered the “gold standard” for anti-resistance detection in\nhorses (147–149). In vitro tests, including egg hatching tests and\n\nFecal egg count reduction tests are currently\nconsidered the “gold standard” for anti-resistance detection in\nhorses (147–149).In vitro tests, including egg hatching tests and\nlarval development tests, are also commonly used as supplementary\nmethods for anti-resistance testing (27, 150, 151). However, it is\ncurrently difficult to define the AR profiles of various parasites in\nhorses, as each parasite may display resistance to different classes\nof anthelmintics, and the mechanisms of resistance associated with\neach drug may vary (152, 153). Attributing this resistance to specific\ngene SNP mutations is not always straightforward. Furthermore,\nmany resistance mechanisms remain poorly understood (154).\nResearch on resistance suggests that the development of parasitic\nresistance is likely influenced by multiple genes and various\nmechanisms of action (155–157). Current diagnostic methods\nface significant challenges in specifically identifying resistant\npopulations, resulting in prevention and control strategies lacking\nspecificity. The lack of related research is associated with horses\nbeing a special non-economic livestock species. The horse parasite\ngenomic information available from WormBase ParaSite and NCBI\nparasite genome databases is still limited compared to ruminant\nparasite genomic information, further reflecting the significant gap\nbetween research teams and funding investment.\nGenomic and transcriptomic data related to equine parasites\nare currently scarce. Whole-genome sequencing of different\nparasitic species, including both sensitive and drug-resistant\nstrains, would provide deeper insights into the quantity and\nmechanisms of resistance genes (158–161). Constructing genetic\nmaps of these species’ genomes to identify unique genetic markers\nfor species identification will aid in the development of early\ndiagnostic methods. Additionally, research on veterinary helminths\nhas confirmed that genetic markers of AR to BZDs, Levamisole\n(LEV), and Monepantel are identified using known SNP markers.\nExpanding the genomic maps of parasitic strains will enhance\n\nAdditionally, research on veterinary helminths\nhas confirmed that genetic markers of AR to BZDs, Levamisole\n(LEV), and Monepantel are identified using known SNP markers.Expanding the genomic maps of parasitic strains will enhance\nthe diagnosis and detection of resistance (154, 162). Serological\nand molecular biological techniques currently allow for the study\nof parasite species, population genetic diversity, and mutations\nbetween strains in different regions. In the future, multiplex qPCR\ndiagnostic methods can be developed for common or target parasite\nspecies on pastures (163–165). This method can detect infection\nstatus in horses and estimate infection quantities and ratios, thereby\nassessing the AR situation of specific parasites (166). LAMP has\ngood potential for on-site testing and has been widely applied in\nresearch on bacteria, viruses, and parasites (167–170). However,\nparasitic detection samples are often feces or eggs, which are\nsmall and contain numerous contaminants, posing challenges for\ndeveloping effective detection methods (171).\nWith the rapid advancement of artificial intelligence (AI),\nan increasing number of researchers are integrating parasitic\ndiagnostic techniques with artificial intelligence (AI) technology\nto enhance detection efficiency and accuracy. Li et al. developed\na low-cost, fully automated diagnostic system that integrates a\nportable robotic-assisted microscope with convolutional neural\nnetworks for the counting of parasite eggs in fecal samples.\nThe system was tested on eggs of Eimeria, Strongyles, and\nTrichuris spp., with an error margin of fewer than one egg per\nMcMaster counting chamber (172). Numerous machine learning\n(ML) and artificial neural network-based (ANN) models have\nbeen employed for the automated identification of parasite eggs\n(173–175) Januário et al. proposed an innovative approach that\nintegrates genomic selection, machine learning, and image analysis\nto optimize resistance to gastrointestinal nematodes (GIN) in sheep\n(176). This method classifies sheep into resistant, resilient, and\n\nproposed an innovative approach that\nintegrates genomic selection, machine learning, and image analysis\nto optimize resistance to gastrointestinal nematodes (GIN) in sheep\n(176).This method classifies sheep into resistant, resilient, and\nsusceptible categories using machine learning techniques such as\nmultinomial logistic regression, random forest (RF), and artificial\nneural networks (ANN), while evaluating susceptibility levels\nacross different farms based on fecal egg count (FEC), packed cell\nvolume, and Famacha© scores (177, 178). Additionally, the RF\nmodel was applied to segment ocular conjunctiva images of sheep,\nautomatically classifying anemia levels according to Famacha©\nscores, thus improving the accuracy of parasite infection detection.\nFor genetic analysis, Bayesian methods, including BayesA, BayesB,\nand Bayesian Lasso (BLASSO), were employed to estimate the\nbreeding values (EBVs) of traits related to resistance (179–\n181). Datasets that are continuously enriched will substantially\nenhance diagnostic efficiency and accuracy, and AI will offer\nmore efficient solutions for screening parasites and drug resistance\n(182–186). The development of these technologies facilitates large-\nscale parasite control strategies for equines, benefiting more key\nstakeholders. Additionally, specific immune antigens and drug\ntargets can be identified for the development of vaccines and other\nbiological products for major parasites, providing more effective\nmeans of safeguarding horse health.\nIn conclusion, while there are some ambiguities in the Swedish,\nDanish, Dutch, and ESCCAP guidelines concerning equine\nFrontiers in Veterinary Science\n11\nfrontiersin.org\n\nWang et al.\n10.3389/fvets.2025.1663577\nparasitism and farm management, and differences in deworming\ntiming and diagnostic testing methods due to certain policy\nfactors, there is a shared consensus on advocating monitoring-\nbased control programs. Countries and organizations should\nproactively offer continuing education webinars on parasite\ncontrol, providing horse owners and stable managers with up-\nto-date, evidence-based information to reduce confusion from\noutdated knowledge. There should also be a concerted effort\nto implement standardized parasite control strategies across\nthe\nindustry,\npromoting\ndiagnostic-driven\ndeworming\nover\ntraditional blanket deworming. Given the current prevalence\nof drug resistance, not all categories of deworming agents\nare available in all countries, and strengthening regulation on\ndeworming drug use is essential to preserving their efficacy.\nPolicies should encourage the development of new deworming\ndrugs and explore alternative control measures, especially through\nlong-term studies on combination drug strategies. Furthermore,\nfostering collaboration among veterinary schools, pharmaceutical\ncompanies, and regulatory agencies will ensure that, in the coming\nyears, parasite control guidelines become more unified and\nwidely adopted, effectively curbing resistance development and\nsafeguarding horse health.\nAuthor contributions\nTW:\nConceptualization,\nWriting\n–\noriginal\ndraft,\nWriting\n–\nreview\n&\nediting.\nXC:\nSupervision,\nWriting\n–\nreview\n&\nediting.\nXY:\nWriting\n–\nreview\n&\nediting.\nYS:\nWriting\n–\nreview\n&\nediting.\nWG:\nWriting\n–\nreview\n&\nediting.\nCL:\nSupervision,\nWriting\n–\nreview\n&\nediting.\nWW:\nConceptualization,\nSupervision,\nWriting\n–\nreview\n& editing.\nFunding\nThe author(s) declare that financial support was received for\nthe research and/or publication of this article. This research was\nfunded by the Basic scientific research operating expenses project\nof universities directly under the Inner Mongolia Autonomous\nRegion (BR231406), the National Natural Science Foundation\nof China (32360884), the Scientific Research Special Project for\nFirst-Class Disciplines of the Department of Education of Inner\nMongolia Autonomous Region (YLXKZX-NND-012).\nAcknowledgments\nWe\nwould\nlike\nto\nthank\nthe\nparasite\nresearch\nteam\n\nAcknowledgments\nWe\nwould\nlike\nto\nthank\nthe\nparasite\nresearch\nteam\nfrom the College of Veterinary Medicine, Inner Mongolia\nAgricultural University.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declare that no Gen AI was used in the creation\nof this manuscript.\nAny alternative text (alt text) provided alongside figures in\nthis article has been generated by Frontiers with the support of\nartificial intelligence and reasonable efforts have been made to\nensure accuracy, including review by the authors wherever possible.\nIf you identify any issues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. 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+{"text": "【兽医文献】\n标题: Isolation and genetic characterization of a novel recombinant HP-PRRSV strain in Jiangxi Province, China\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nCase Report: Selective hepatic\nresection guided by indocyanine\ngreen short-wave infrared\nfluorescence imaging in a dog\nwith multifocal liver lesions\nSoyoung Jang 1, Yujin Kim 1, Sunyoung Kim 2 and Sungin Lee 1*\n1 Department of Veterinary Surgery, College of Veterinary Medicine, Chungbuk National University,\nCheongju, Republic of Korea, 2 Department of Veterinary Clinical Sciences, College of Veterinary\nMedicine, Purdue University, West Lafayette, IN, United States\nAn 11-year-old female spayed Maltese was presented with multiple hepatic masses\nidentified on abdominal ultrasonography and triphasic computed tomography.\nLesions were distributed across the left lateral, left medial, quadrate, and caudate\nliver lobes. A large multicystic mass originating from the left lateral lobe was\nconsidered at high risk of rupture, leading to the palliative surgery. Indocyanine\ngreen (ICG) was intravenously administered at a dose of 0.5 mg/kg 24 h prior to\nsurgery. During laparotomy, the liver was assessed using a short-wave infrared\n(SWIR) fluorescence imaging system. Only the mass arising from the left lateral\nlobe exhibited ICG fluorescence, while no fluorescence was detected in the other\nlesions. Based on these findings, a left lateral lobectomy including the ICG-positive\nmass was performed, and punch biopsies were taken from the non-fluorescent\nlesions in other lobes. Histopathologic examination confirmed the mass from the\nleft lateral lobe as combined hepatocellular carcinoma-cholangiocarcinoma with\ntumor-free margins. The biopsied lesions were diagnosed as vacuolar hepatopathy.\nFollow-up evaluation over 6 months revealed no evidence of metastasis. This case\ndemonstrates the potential clinical utility of ICG-SWIR fluorescence imaging in not\nonly detecting and achieving complete resection of tumors, but also supporting\nreal-time intraoperative decision-making, which enables selective resection of\nmalignant tissue while sparing benign lesions.\nKEYWORDS\nindocyanine green, fluorescence imaging, image-guided surgery, hepatic tumors,\n\nKEYWORDS\nindocyanine green, fluorescence imaging, image-guided surgery, hepatic tumors,\nmultiple tumors, canine hepatocellular carcinoma-cholangiocarcinoma\n1 Introduction\nHepatic lesions in dogs can be classified into massive, multifocal, and diffuse types based\non their distribution pattern. Multifocal hepatic lesions are characterized by the presence of\nmultiple lesions across several hepatic lobes and can arise from various etiologies, including\ninflammatory changes, benign proliferations, and malignancies (1, 2). Surgical resection,\nincluding partial or complete liver lobectomy, is a treatment option for massive liver tumors\nand generally offers a favorable prognosis if complete excision is achieved (3–5). However,\nmultifocal or diffuse liver tumors involving multiple lobes typically have a poor surgical\noutcome due to the difficulty in achieving complete resection and the high metastatic\npotential (3, 4).\nOPEN ACCESS\nEDITED BY\nAshraf M. Abu-Seida,\nCairo University, Egypt\nREVIEWED BY\nNaoki Sakurai,\nNihon university, Japan\nTristan Wagner,\nUniversity Hospital of Cologne, Germany\n*CORRESPONDENCE\nSungin Lee\nsunginlee@cbnu.ac.kr\nRECEIVED 02 August 2025\nACCEPTED 22 September 2025\nPUBLISHED 10 October 2025\nCITATION\nJang S, Kim Y, Kim S and Lee S (2025) Case\nReport: Selective hepatic resection guided by\nindocyanine green short-wave infrared\nfluorescence imaging in a dog with multifocal\nliver lesions.\nFront. Vet. Sci. 12:1678363.\ndoi: 10.3389/fvets.2025.1678363\nCOPYRIGHT\n© 2025 Jang, Kim, Kim and Lee. This is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY). The use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\naccordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Case Report\nPUBLISHED  10 October 2025\nDOI  10.3389/fvets.2025.1678363\n\nJang et al.\b\n10.3389/fvets.2025.1678363\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nIn human medicine, several staging systems and surgical\nalgorithms for multifocal hepatic tumors have been developed\n(6–10). According to the Barcelona Clinic Liver Cancer (BCLC)\nguideline, surgical resection is recommended for patients with a\nsingle tumor or up to three small tumors, while liver\ntransplantation, locoregional therapies, or systemic treatments are\nfavored for those with multifocal lesions involving different lobes\n(7). However, recent studies have demonstrated that some patients\nwith multifocal hepatic tumors in clinical practice may benefit\nfrom hepatectomy, prompting discussion around expanding\nsurgical indications beyond those recommended by the BCLC\nguideline (6, 8–10). In veterinary medicine, there is no standardized\nstaging system or treatment guidelines for multifocal hepatic\ntumors, and treatment decisions are often individualized based on\neach patient’s condition. This underscores the need for systematic\nstudies to establish objective treatment criteria and evidence-based\nsurgical\nstrategies\nfor\nmultifocal\nhepatic\ntumors\nin\nveterinary patients.\nIndocyanine green (ICG) is the first fluorescent dye approved by\nthe U.S. Food and Drug Administration, emitting light at wavelengths\napproximately 830 nm when excited at 750–800 nm (11–13).\nFollowing intravenous (IV) injection, ICG rapidly binds to plasma\nproteins without altering their structure, and is therefore considered\nnon-toxic (11, 14). Indocyanine green is selectively absorbed by\nhepatocytes and excreted exclusively into the bile without metabolism\n(11, 15). In Ishizawa et al. (16) reported the intraoperative use of ICG\nfluorescence imaging for detecting liver tumors and highlighted\ndifferences in biliary excretion between normal hepatocytes and\nmalignant tissues. Malignant tissues are unable to excrete bile\nnormally, leading to intracellular accumulation of ICG and resulting\nin fluorescent signal emission (5, 15, 16).\nIn veterinary surgery, ICG fluorescence imaging has been used\nintraoperatively to aid in tumor detection and to ensure complete\n\nIn veterinary surgery, ICG fluorescence imaging has been used\nintraoperatively to aid in tumor detection and to ensure complete\nresection (5, 15). However, there have been no reports where surgical\ndecisions regarding tumor resection were made intraoperatively based\non the presence or absence of ICG fluorescence. This case report\ndescribes the application of the ICG-SWIR imaging system to assist\nintraoperative surgical decision-making in a dog with multifocal\nhepatic lesions, ultimately enabling the selective resection of a\nmalignant tumor.\n2 Case description\n2.1 Case description and preoperative\nfindings\nAn 11-year-old female spayed Maltese weighing 3.84 kg was\nreferred for evaluation of an intra-abdominal mass identified during\na health examination at a local veterinary hospital. Physical\nexamination revealed no abnormalities. A complete blood count\nshowed thrombocytosis (673,000/μL; reference range [Ref]: 148,000–\n484,000) and reticulocytosis (140,000/μL; Ref: 10,000–110,000).\nSerum chemistry showed elevated aspartate aminotransferase\n(76 IU/L; Ref: 23–66), alanine aminotransferase (221 IU/L; Ref:\n21–102), gamma-glutamyl transferase (12 IU/L; Ref: 1–10), and\nalkaline phosphatase (1,242 IU/L; Ref: 29–97). All other blood test\nresults were within normal limits.\nAbdominal radiography revealed a relatively well-defined soft\ntissue opacity mass with a round-to-amorphous shape in the\nmid-abdomen. Abdominal ultrasound identified a multicystic,\namorphous-shaped mass measuring approximately 7 cm in diameter,\nmost likely arising from the left lateral hepatic lobe. A heterogenous,\nround-shaped mass approximately 2.5 cm in diameter with\nsignificant blood flow was also detected, likely in the left medial lobe.\nTriphasic computed tomography (CT) imaging (Hi Speed QX/I, GE\nMedical Co., Milwaukee, WI, USA), which had been performed at a\nlocal hospital prior to referral, identified multiple hepatic masses. A\nheterogeneous, amorphous, pedunculated mass (#1) originating\nfrom the left lateral lobe measured 7.29 × 4.37 × 4.06 cm\n(W × H × L) and exhibited contrast enhancement in the arterial,\n\nA\nheterogeneous, amorphous, pedunculated mass (#1) originating\nfrom the left lateral lobe measured 7.29 × 4.37 × 4.06 cm\n(W × H × L) and exhibited contrast enhancement in the arterial,\nportal, and delayed phases, with rapid washout observed in the\ndelayed phase. The mass contained at least six well-defined\nnon-enhancing cystic lesions. A heterogeneous mass (#2) in the left\nmedial lobe measuring 3.12 × 2.02 × 2.97 cm (W × H × L) was\ncontrast-enhancing in the arterial and portal phases but\nnon-enhancing in the delayed phase. An ill-defined, non-enhancing\nnodule measuring approximately 2 mm was also identified in the left\nlateral lobe. A mass (#3) in the quadrate lobe measuring\n0.84 × 0.71 × 0.73 cm (W × H × L) was isoattenuating and\ndemonstrated contrast enhancement in the arterial and portal\nphases but not in the delayed phase. A mass (#4) in the caudate\nprocess of the caudate lobe measuring 0.92 × 0.36 × 0.66 cm\n(W × H × L) was isoattenuating and non-enhancing in all phases\n(Figure 1).\nThe largest mass (#1), which was pedunculated from the left\nlateral lobe and had a cystic internal structure, posed a high risk of\nrupture due to its size and structure. Palliative surgery was planned to\nremove it, without performing fine-needle aspiration (FNA) or biopsy.\nThe lesion in the left medial lobe was relatively larger than the others,\nprompting consideration of complete resection of the left hepatic\ndivision. Because surgery was already planned and biopsy is\nconsidered more sensitive than FNA among the diagnostic techniques\nfor hepatic lesions in dogs (17), FNA was not performed for the\nother lesions.\n2.2 Preoperative management and\nanesthesia\nIndocyanine green (25 mg; Cellbiongreen inj., Cellbion, Seoul,\nKorea) was dissolved in 5 mL of sterile water (5 mg/mL), then diluted\n1:10 with sterile saline to prepare a working solution (0.5 mg/mL).\nThe ICG solution was intravenously injected as a bolus at a dose of\n0.5 mg/kg, 24 h preoperatively. Vital signs were carefully monitored,\nalong with any cutaneous signs suggestive of an allergic reaction. No\nadverse events were noted over 24 h, and surgery was\n\nThe ICG solution was intravenously injected as a bolus at a dose of\n0.5 mg/kg, 24 h preoperatively.Vital signs were carefully monitored,\nalong with any cutaneous signs suggestive of an allergic reaction.No\nadverse events were noted over 24 h, and surgery was\nsubsequently performed.\nPremedication was administered with midazolam (0.2 mg/kg IV;\nBukwang midazolam inj., Bukwang Pharm, Seoul, Korea), followed\nby induction with propofol (4 mg/kg IV; Freepol-MCT Inj., Daewon\nPharm, Seoul, Korea) and maintenance of anesthesia using 1.3%\nisoflurane inhalation. Plasmalyte A solution (20–40 mL/h) and\nremifentanil (0–3 μg/kg/h; Tivare Inj., BC World Pharm, Gyeonggi-do,\nKorea) were administered by constant rate infusion (CRI) throughout\nthe surgery.\n\nJang et al.\b\n10.3389/fvets.2025.1678363\nFrontiers in Veterinary Science\n03\nfrontiersin.org\n2.3 Surgical procedure\nThe dog was placed in dorsal recumbency and stabilized using a\nvacuum positioning mat. A midline skin incision was made using a\n#10 scalpel blade, starting at the xiphoid process and extending\ncaudally toward the pubis.\nUpon laparotomy, the mass originating from the left lateral lobe\nwas immediately identified on gross inspection. After ensuring\nadequate exposure, a short-wave infrared (SWIR) fluorescence-guided\nimaging system (ZNI; Metaple Bio Co., Ltd., Seoul, Korea) was\nperformed. The mass exhibited increased ICG fluorescence with a\npartial fluorescence pattern. Adhesions around the mass and the left\nlateral lobe were carefully dissected using sterile cotton swabs and\nmonopolar electrocautery (Covidien, Mansfield, MA, USA). The\nentire left lateral lobe, including the mass, was ligated and resected\nusing a vascular/medium, 45 mm, Tri-Staple EndoGIA stapler\n(Covidien/Medtronic, Dublin, Ireland). In the left medial lobe,\nquadrate lobe, and caudate process of the caudate lobe, lesions were\nidentified grossly, but no ICG fluorescence was detected on the SWIR\nimaging system. A 6-mm punch biopsy was performed in each of\nthese areas, followed by application of a gelatin sponge (Spongostan;\nEthicon, a Johnson & Johnson company, NJ, USA) and oxidized\nregenerated cellulose (Surgicel; Ethicon, a Johnson & Johnson\ncompany, NJ, USA) for hemostasis (Figure 2). All other liver lobes\nwere visually inspected, palpated, and evaluated using ICG\nfluorescence to check for additional lesions, and no abnormalities\nwere found. After confirming the absence of intra-abdominal\nbleeding, abdominal closure was performed.\nPostoperative management included a CRI of Plasmalyte A\nsolution combined with vitamin B and taurine (0.5–1.5 mL/kg/h CRI)\nfor fluid and metabolic support, along with remifentanil (5–10 μg/\nkg/h CRI) for analgesia during the first 24 h. A fentanyl patch (12 μg/h;\nDurogesic D-trans Patch, Janssen Korea, Seoul, Korea) was applied on\npostoperative day 2 to maintain analgesia as remifentanil was\n\nA fentanyl patch (12 μg/h;\nDurogesic D-trans Patch, Janssen Korea, Seoul, Korea) was applied on\npostoperative day 2 to maintain analgesia as remifentanil was\ngradually tapered and discontinued by postoperative day 3. Additional\nmedications included famotidine (1 mg/kg IV, every 12 h; Gaster inj.,\nDong-A Pharm, Seoul, Korea), cefazolin (22 mg/kg IV, every 12 h;\nCephazolin sodium, Chong Kun Dang Pharmaceutical Corporation.,\nSeoul, Korea), vitamin K (1  mg/kg subcutaneously, every 24 h;\nVitami-K1 Inj., Dai Han Pharm, Seoul, Korea), and N-acetylcysteine\n(70 mg/kg IV, every 12 h; Muteran Inj., Han Wha Pharma,\nGangwon-do, Korea), all administered for 2 days postoperatively. In\nconsideration of the risk of hypoglycemic shock associated with\nimpaired hepatic function and hepatic lobectomy, a continuous\nglucose monitoring system (Libre; Abbott, Chicago, IL, USA) was\napplied. Blood glucose levels were monitored every 2 h postoperatively\nand ranged from 80 to 200 mg/dL, with no clinically significant\nabnormalities observed. The patient was discharged 3 days\npostoperatively. Ursodeoxycholic acid (UDCA; 10 mg/kg orally, every\n12 h; Ursa Tab., Dae Woong, Seoul, Korea) and S-adenosyl methionine\n(SAMe)/silybin (100 mg per dog PO every 24 h; Zentonil Advanced,\nVetoquinol, Lavaltrie, QC, Canada) were prescribed for 4 weeks.\n2.4 Histopathological findings and\nfollow-up\nThe resected left lateral lobe with the mass, along with the biopsy\nsamples, was preserved in 10% neutral buffered formalin and\nsubmitted for histopathologic evaluation (IDEXX Laboratories, Inc.,\nFIGURE 1\nPost-contrast arterial phase computed tomography images of multiple liver masses. (A) A large, amorphous, pedunculated, contrast-enhancing mass\n(#1, red arrow) with internal cavitations originating from the left lateral lobe, measuring 7.29 × 4.37 × 4.06 cm (W × H × L). (B) A heterogeneous,\ncontrast-enhancing mass (#2, green arrow) in the left medial lobe, measuring 3.12 × 2.02 × 2.97 cm (W × H × L). (C) An isoattenuating, contrast-\n\n29 × 4.37 × 4.06 cm (W × H × L).(B) A heterogeneous,\ncontrast-enhancing mass (#2, green arrow) in the left medial lobe, measuring 3.12 × 2.02 × 2.97 cm (W × H × L).(C) An isoattenuating, contrast-\nenhancing mass (#3, yellow arrow) in the quadrate lobe, measuring 0.84 × 0.71 × 0.73 cm (W × H × L). (D) An isoattenuating, non-enhancing mass (#4,\nblue arrow) in the caudate process of the caudate lobe, measuring 0.92 × 0.36 × 0.66 cm (W × H × L). W, width; H, height; L, Length.\n\nJang et al.\b\n10.3389/fvets.2025.1678363\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nUSA). Pathological analysis of the mass revealed a combined\nhepatocellular carcinoma-cholangiocarcinoma (cHCC-CCA), with a\nmitotic count of 0 per 10 high-power fields. The surgical margin was\ntumor-free, exceeding 15 mm, and no vascular invasion was observed.\nHistopathologic evaluation of the three biopsy specimens revealed\nvacuolar hepatopathy characterized by combined glycogen and lipid\naccumulation, which is considered a benign lesion (Figure 3).\nOn postoperative day 20, clinical evaluation showed no significant\nabnormalities. The surgical site had healed completely, allowing for\nsuture removal. At 6 weeks postoperatively, liver enzymes had further\ndecreased compared with the immediate postoperative values,\nindicating continued improvement in the biochemical profile.\nAspartate aminotransferase decreased from 161 to 38 IU/L (Ref:\n23–66), alanine aminotransferase from 312 to 133 IU/L (Ref: 21–102),\ngamma-glutamyl transferase from 22 to 15 IU/L (Ref: 1–10), and\nalkaline phosphatase from 2,345 to 974 IU/L (Ref: 29–97). Follow-up\nthoracic and abdominal radiographs revealed no signs of metastasis,\nand generalized hypertrophy of the right liver lobes was noted.\nAbdominal ultrasonography revealed multiple hyperechoic nodules\n(approximately 3 mm in diameter) throughout the right liver lobes,\nand a single hypoechoic, oval-shaped nodule (approximately 2 cm) in\nthe left medial lobe. These lesions had not been detected preoperatively.\nAt 6 months postoperatively, in addition to the previously identified\nlesions, a 1.6 × 1.7 cm nodule was observed in the right lateral lobe,\nalong with multiple hyperechoic nodules diffusely distributed across\nall hepatic lobes on ultrasound. FNA of the lesion in the right lateral\nlobe was performed and revealed vacuolar hepatopathy. As the FNA\nresults indicated a non-neoplastic lesion, the hepatic nodules were\nconsidered more likely benign than metastatic. Nonetheless, because\nmetastasis could not be  definitively ruled out, continued regular\nfollow-up was deemed necessary.\n3 Discussion\n\nAs the FNA\nresults indicated a non-neoplastic lesion, the hepatic nodules were\nconsidered more likely benign than metastatic.Nonetheless, because\nmetastasis could not be  definitively ruled out, continued regular\nfollow-up was deemed necessary.3 Discussion\nReal-time fluorescence imaging offers intraoperative guidance\nand greater sensitivity for tumor detection than visual inspection or\npalpation (13, 18). Notably, ICG-SWIR fluorescence imaging aids not\nonly in tumor identification but also in surgical planning and\nachieving tumor-free margins (19, 20). During laparoscopic surgery,\nthe limited surgical field and inability to palpate organs make the\nSWIR imaging system an essential modality for complete tumor\nresection (16). In human medicine, SWIR imaging with ICG has been\nwidely used for intraoperative tumor detection and margin\ndetermination in patients with various tumors (11, 19–24), and has\nsimilarly been applied in veterinary patients (5, 12, 14, 15, 18, 25–27).\nIn this case, ICG-SWIR fluorescence-guided resection enabled\ncomplete removal of the malignant lesion with tumor-free margins.\nPreoperative imaging and intraoperative visualization revealed\nmultiple hepatic lesions in this patient, but ICG fluorescence was\ndetected only in the mass originating from the left lateral lobe (28).\nHistopathology confirmed that the fluorescence-positive lesion was\nmalignant, whereas the non-fluorescent lesions were benign, further\nsupporting the utility of intraoperative ICG imaging in surgical\ndecision-making. Recent studies have suggested the potential of ICG\nfluorescence to differentiate benign from malignant lesions (15, 20,\n28). In human medicine, ICG fluorescence showed a sensitivity of 88%\nand a positive predictive value (PPV) of 77% for distinguishing\nmalignancies from benign or inflammatory lesions (20). In veterinary\nmedicine, a small-scale study reported a sensitivity of 71% and a PPV\nof 80% for differentiating HCC from nodular hyperplasia in dogs (15).\nIn addition, findings for nodular hyperplasia lesions have been\ninconsistent, with fluorescence observed in 11 of 12 lesions in one\n\nIn addition, findings for nodular hyperplasia lesions have been\ninconsistent, with fluorescence observed in 11 of 12 lesions in one\nstudy (5), but in only 1 of 6 lesions in another (15), indicating the need\nfor additional investigation. In this case, the non-fluorescent lesions\nwere diagnosed as vacuolar hepatopathy. To our knowledge, ICG\nfluorescence in vacuolar hepatopathy has not previously been\nevaluated, and thus the reason for the absence of fluorescence remains\nunclear. Given that vacuolar hepatopathy is reported not to involve\nalterations of the intracellular bile canaliculi (29), we suggest that bile\nexcretion may be preserved, which in turn could explain the absence\nof ICG accumulation, unlike in neoplastic hepatocytes. However,\nfurther studies are needed to determine the exact underlying\nmechanism. Despite these considerations, this case demonstrates that\nICG-SWIR fluorescence imaging provides real-time surgical guidance\nfor determining the extent of resection in patients with multiple\nhepatic lesions. This approach allows for the selective removal of\nmalignant tissue while preserving normal liver parenchyma.\nMinimizing hepatic resection helps preserve liver function and\nFIGURE 2\nIntraoperative view and visualization of the lesions using the short-\nwave infrared (SWIR) fluorescence imaging system. (A,B) The mass\nfrom the left lateral lobe (white dotted line) was grossly identified and\nexhibited increased indocyanine green (ICG) fluorescence on the\nSWIR imaging system, displaying a partial fluorescence pattern. (C–F)\nThe lesions in the left medial lobe (yellow dotted line), quadrate lobe\n(green dotted line), and caudate process of the caudate lobe (red\ndotted line) were grossly identified but showed no ICG fluorescence\non the SWIR imaging system.\n\nJang et al.\b\n10.3389/fvets.2025.1678363\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nreduces the risk of complications due to insufficient residual liver\nvolume (30).\ncHCC-CCA is a rare primary hepatic neoplasm in dogs,\naccounting for approximately 2% of all primary hepatobiliary\ntumors (31). Histopathologically, the tumor exhibits features of\nboth HCC and CCA (31, 32). In dogs, the median survival time\n(MST) after surgical resection ranges from 695 to over 1,460 days\nfor HCC, and is less than 6 months for CCA (31, 33). Interestingly,\ndogs diagnosed with cHCC-CCA have an MST of approximately\n700 days, comparable to that of HCC and notably longer than that\nof CCA (31). These findings suggest that surgical resection,\nwhether partial or complete liver lobectomy, can be  a viable\ntreatment option for cHCC-CCA in dogs (3, 34). When multiple\nlesions are identified intraoperatively in dogs with cHCC-CCA,\nan aggressive surgical approach may be  warranted to prevent\npostoperative metastasis (31). However, extensive liver lobectomy\nwas not feasible in this case because of masses in multiple lobes.\nAs demonstrated in this report, the SWIR imaging system with\nICG may offer real-time intraoperative guidance, supporting\nselective resection of suspicious lesions. Furthermore, a recent\nveterinary study on ICG fluorescence patterns in canine hepatic\ntumors reported that cHCC-CCA exhibited a characteristic partial\nfluorescence pattern (5). A similar pattern was observed in the\npresent case. Although fluorescence patterns alone may not\nbe  sufficient for definitive diagnosis, they aided surgeon to\ndifferentiate\nneoplasms\nin\nthis\nreport\nand\nwarrant\nfurther investigation.\nIn conclusion, the SWIR imaging system with ICG was effective\nin facilitating the selective resection of malignant hepatic tumors in a\ndog with multifocal liver lesions in this case. When multifocal liver\nlesions are present and immediate surgical intervention is required\ndue to limitations in preoperative diagnosis or the patient’s condition,\nthis imaging modality may serve as a valuable tool for real-time\n\nWhen multifocal liver\nlesions are present and immediate surgical intervention is required\ndue to limitations in preoperative diagnosis or the patient’s condition,\nthis imaging modality may serve as a valuable tool for real-time\nintraoperative decision-making. Further studies are required to\nvalidate the diagnostic accuracy of ICG fluorescence imaging in\ndifferentiating malignant from benign lesions and in identifying\ntumor subtypes in veterinary hepatic oncology.\nData availability statement\nThe original contributions presented in the study are included in\nthe article/supplementary material, further inquiries can be directed\nto the corresponding author.\nEthics statement\nEthical approval was not required for the studies involving\nanimals in accordance with the local legislation and institutional\nrequirements because this is the case report of a clinical patient, not\nan experimental research. Written informed consent was obtained\nfrom the owners for the participation of their animals in this study.\nWritten informed consent was obtained from the participants for the\npublication of this case report.\nAuthor contributions\nSJ: Conceptualization, Data curation, Investigation, Methodology,\nProject administration, Visualization, Writing  – original draft,\nWriting – review & editing. YK: Conceptualization, Data curation,\nFIGURE 3\nHistopathological findings of liver lesions. (A) Photomicrograph of the mass from the left lateral lobe showing features consistent with a\nhepatocellular-cholangiocellular carcinoma. The image highlights the junctional area with cholangiocellular differentiation (left) and hepatocellular\ndifferentiation (right). The neoplastic cells exhibit mild to moderate anisocytosis and anisokaryosis, with prominent nucleoli (hematoxylin and eosin\n[H&E] stain, Magnification ×200, Scale bar 50 μm). (B–D) Photomicrographs of the lesions from the left medial (B), quadrate (C), and caudate (D) lobes\nexhibit features consistent with vacuolar hepatopathy (H&E stain, Magnification ×200, Scale bar 50 μm).\n\nJang et al.\b\n10.3389/fvets.2025.1678363\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nInvestigation, Methodology, Project administration, Writing – review\n& editing. SK: Conceptualization, Supervision, Validation, Writing –\nreview & editing. SL: Conceptualization, Funding acquisition,\nSupervision, Validation, Writing – original draft, Writing – review &\nediting.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. This work was supported\nby Chungbuk National University Glocal30 project (2025). This work\nwas supported by the Korea Institute of Planning and Evaluation for\nTechnology in Food, Agriculture, and Forestry (IPET) through the\nCompanion Animal Life Cycle Industry Technology Development\nProgram, which is funded by the Ministry of Agriculture, Food, and\nRural Affairs (MAFRA) (322095-04).\nAcknowledgments\nThe authors would like to thank the owners of the dog included\nin this study. The authors would like to thank Sooyeon Cho for\nassistance in formatting the figures.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. 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+{"text": "【兽医文献】\n标题: article_118_Retrospective Study on External Canine Limb Prosthesis used \n\nDoes Adding Transdermal Nitroglycerine to Other \nTherapies Used for Management of Left-sided \nCongestive Heart Failure in Dogs Speed the Resolution \nof Clinical Signs?\n\nA Knowledge Summary by\n\nJenefer R Stillion DVM, DACVECC 1 \nSøren R Boysen DVM, DACVECC 2*\n\n1 Western Veterinary Specialist and Emergency Centre, Calgary, Canada \n2 Department of Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, \nUniversity of Calgary, Calgary, Canada \n* Corresponding Author (srboysen@ucalgary.ca)\n\nThere is an erratum to this paper published in Veterinary Evidence Vol 3, Issue 1 \n(2018): http://dx.doi.org/10.18849/ve.v3i1.168\n\nISSN: 2396-9776 \nPublished: \n  Updated: \n12 Dec 2017 \n01 Feb 2018 \nIn: Vol 2, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v2i4.115 \nReviewed by: Myra Forster-van Hijfte (CertVR CertSAM \nDipECVIM-cA FRCVS) and William Chandler \n(BVetMed, MRCVS) \nNext Review Date: 02 Jan 2019\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 2, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v2i4.115  \nnext review date: 02 Jan 2019 \np a g e | 2\ntotal pages: 8\n\nKNOWLEDGE SUMMARY\n\nQuestion \nDoes adding transdermal nitroglycerine to other therapies used for management of left-sided congestive heart \nfailure in dogs speed the resolution of clinical signs?\n\nClinical Scenario  \nTransdermal nitroglycerin is frequently recommended as an adjunct therapy in many canine treatment \nprotocols for left-sided congestive heart failure due to its preload and afterload reducing effects. Does adding \ntransdermal nitroglycerin to other therapies used for management of left-sided congestive heart failure in \ndogs speed the resolution of clinical signs?\n\nDoes adding \ntransdermal nitroglycerin to other therapies used for management of left-sided congestive heart failure in \ndogs speed the resolution of clinical signs?\n\nThe Evidence  \nThere is no evidence in the literature to suggest transdermal nitroglycerin speeds resolution of clinical signs \nin dogs with pulmonary oedema secondary to left-sided congestive heart failure. There is equivocal to weak \nevidence that transdermal nitroglycerin reduces systolic and/or mean arterial blood pressure in dogs. There is \nno evidence that transdermal nitroglycerin has any effect on resolution of any clinical signs of left-sided \ncongestive heart failure. Although there are no reports of adverse harm associated with transdermal \nnitroglycerin, there are insufficient clinical studies to be able to state nitroglycerin does not have any adverse \naffects in dogs.\n\nSummary of the evidence\n\nNakayama (2007) \nPopulation: mixed breed dogs with Seller’s grades 2+ or 3+ mitral regurgitation \nSample size: 9 dogs \nIntervention details: Mitral regurgitation was produced by surgical disruption of the \nmitral valves.  Five months after production of mitral regurgitation, \nleft atrial dimension and ventricular function were measured using \nechocardiography/Doppler and left ventricular micromanometry.  \nNine mixed breed dogs weighing 21-32 kg were administered \nbutorphanol (0.05 mg/kg IM) prior to the start of each study.  \nEnalaprilat, nitroglycerin, ouabain, milrinone or placebo (IV saline, \nThere was a one-week washout period between arms of the study to \nensure at least 10 half-lives between exposures. Recordings were \nobtained, along with an ECG, during baseline and 30 minutes after \nClinical bottom line  \nThere is very weak veterinary clinical and experimental evidence based upon a limited number of studies \nto indicate that adding transdermal nitroglycerine to other therapies used for management of left-sided \ncongestive heart failure in dogs speeds the resolution of clinical signs.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 2, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v2i4.115  \nnext review date: 02 Jan 2019 \np a g e | 3\ntotal pages: 8\n\ndogs received IV (in random sequence) 0.3 mg/kg enalaprilat, \n20mg/kg ouabain, or  100mg/kg  of  milrinonel;  or  2  inches  of  2% \nnitroglycerin paste applied to the skin of the inner thigh over an area \nof 16 cm2. All dogs also were given an IV (saline) and  a  transdermal  \nplacebo  (Vaseline). Dogs were studied 1 hour and 2 hours after \napplication.  \nStudy design: Randomised controlled clinical trial \nOutcome studied: The effect of 4 different cardioactive drugs on left ventricular \nfunction in dogs with mitral regurgitation of 5 months duration. \nParameters studied included intracardiac dimension, peak aortic \nflow, left atrial diastolic and systolic function.  \nMain findings: \n(relevant to PICO question): \n \nWhen compared to placebo (Vaseline), transdermal \nnitroglycerin failed to produce an effect on any of the \nparameters studied.  \nLimitations: Small sample size. Surgically induced mitral regurgitation may not \naccurately reflect clinical mitral endocardiosis. No animals had \nclinical signs of congestive heart failure.\n\nKanda  (1995)\n\nLimitations: Small sample size.Surgically induced mitral regurgitation may not \naccurately reflect clinical mitral endocardiosis.No animals had \nclinical signs of congestive heart failure.Kanda  (1995)\n\nPopulation: Healthy Beagle dogs \nSample size: 5 dogs in each group \nIntervention details: There were 3 groups; Nitroglycerin tape at 2.5 mg/kg (NT-l, 5 mg \nnitroglycerin per 5 x 10 cm2) with placebo capsule, nifedipine given \norally (3mg/kg) with placebo tape, and a control group that received \na placebo (blank) capsule orally and pacebo tape. Placebo and \nnitroglycerine tape were applied to a clipped area of skin on the \nchest of healthy beagle dogs. \nStudy design: Prospective study (non-randomised, non-blinded) \nOutcome studied: The effect of nitroglycerin tape compared to placebo tape on blood \npressure (systolic and diastolic), heart rate and coronary blood flow \nin healthy beagle dogs. Baseline values recorded for 1 hour before \ndrug administration and then at 30 min, 1 hour and every hour until \n9 hours.  Parameters were measured again 1 hours following \nremoval of tape.  \nMain findings: \n(relevant to PICO question): \n \nSystolic blood pressure was decreased by 10-15%,  \ncompared to baseline, with application of nitroglycerin tape \nin awake healthy instrumented beagle dogs. The decrease \nwas noted 1 hour after application, persisted for the \nduration of the study and returned to baseline values within \n1 hour of removing the tape.  \n \nCalculated (not directly measured) mean arterial pressure \ndecreased 4-5 %.  \n \nDiastolic blood pressure and heart rate were not affected.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 2, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v2i4.115  \nnext review date: 02 Jan 2019 \np a g e | 4\ntotal pages: 8\n\n \nChanges were noted 30 minutes after the application of \nnitroglycerin tape and remained constant for 8h.  \n \nNo changes from baseline parameters were noted in the \ncontrol group.\n\n18849/ve.v2i4.115  \nnext review date: 02 Jan 2019 \np a g e | 4\ntotal pages: 8\n\n \nChanges were noted 30 minutes after the application of \nnitroglycerin tape and remained constant for 8h. \nNo changes from baseline parameters were noted in the \ncontrol group.\n\nLimitations: Small sample size may have failed to detect true differences that \nexisted between groups or within groups. Decrease in blood \npressure provides indirect evidence of the efficacy of nitroglycerin \nwhich does not equate to clinical improvement. Dogs were healthy \nand did not have any clinical signs of congestive heart failure.\n\nMcKie (2014)\n\nDecrease in blood \npressure provides indirect evidence of the efficacy of nitroglycerin \nwhich does not equate to clinical improvement.Dogs were healthy \nand did not have any clinical signs of congestive heart failure.McKie (2014)\n\nPopulation: Male mongrel dogs with induced mild left ventricular dysfunction. \nSample size: Three study groups with 7 dogs in each group (intravenous M-ANP, \nnitroglycerin, and vehicle 0.9% normal saline) \nIntervention details: Mild left ventricular dysfunction was induced via right ventricular \npacing at 180 beats per minute for 10 days in male mixed mongrel \ndogs. RV pacing was terminated the day of the study and dogs were \nanesthetized with pentobarbital sodium (15mg/kg IV), intubated and \nventilated.  Hypertension was induced in instrumented dogs via IV \nadministration of Angiotensin II (40 mg/kg/1min).  Dogs were then \nadministered either M-ANP (30 pmol/kg/min), nitroglycerin (10 \nug/kg IV) or 0.9% normal saline at a rate of 1ml/min. \nStudy design: Prospective experimental study under anaesthesia (non-\nrandomised, non-blinded) \nOutcome studied: Blood pressure, PCWP, systemic vascular resistance, left ventricular \nfilling pressures, heart rate, cardiac output. \nMain findings: \n(relevant to PICO question): \n \nNitroglycerin significantly decreased mean arterial blood \npressure, PCWP, systemic vascular resistance and left \nventricular filling pressures.  \n \nNo effect on heart rate or cardiac output. \nLimitations: Nitroglycerin was administered IV, not topically. Sample size was \nsmall. Study was not blinded or randomised. Experimentally induced \nheart failure was studied, not naturally occurring heart failure. \nEvidence provided that IV nitroglycerin reduced preload and \nafterload but this does not equate to improvement in clinical signs.\n\nParameswaran (1999) \nPopulation: Healthy anaesthetised dogs \nSample size: 15 dogs \nIntervention details: Sonomicrometer crystals were applied to the spleen in each dog and \na pressure-measuring catheter was inserted into a splenic vein. A\n\nParameswaran (1999) \nPopulation: Healthy anaesthetised dogs \nSample size: 15 dogs \nIntervention details: Sonomicrometer crystals were applied to the spleen in each dog and \na pressure-measuring catheter was inserted into a splenic vein.A\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 2, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v2i4.115  \nnext review date: 02 Jan 2019 \np a g e | 5\ntotal pages: 8\n\n2.5cm strip of transdermal 2% nitroglycerin ointment was applied to \nthe inner surface of the pinna in 10 dogs and 5 additional dogs \n(control group) were given only petrolatum.  \nStudy design: Non-randomised clinical trial \nOutcome studied: Splenic dilatation in healthy dogs anaesthetised with alpha \nchloralose after transdermal application of nitroglycerin. Splenic \ndimension and venous pressure were measured for 10 minutes \nbefore application and time from application of transdermal \nnitroglycerin to the initial change in dimension and to the maximal \nchange in dimension (measured to the nearest second). \nMain findings: \n(relevant to PICO question): \n \nSplenic dimension increased significantly from baseline in all \n10 dogs receiving transdermal nitroglycerin. \n \nSplenic enlargement was noted within 482  652 seconds \nafter application of transdermal nitroglycerin with maximal \ndilatation at 861  632 seconds. \n \nSplenic venous pressure did not change significantly in dogs \nreceiving transdermal nitroglycerin or in control dogs.\n\nLimitations: Animals were healthy and anaesthetised with alpha chloralose, \nwhose physiologic effects are not well characterized in the literature \nfor any species. Results of the present study may not translate to un-\nanaesthetised dogs with left-sided congestive heart failure.\n\nAppraisal, application and reflection\n\nResults of the present study may not translate to un-\nanaesthetised dogs with left-sided congestive heart failure.Appraisal, application and reflection\n\nGiven studies are lacking with regards to the efficacy of transdermal nitroglycerin when included as an adjunct \ntreatment to other therapies in dogs with left-sided congestive heart failure, it’s use cannot be recommended \nin the management of these cases at this time. Future clinical studies are needed to evaluate the safety, \nefficacy and ideal dosage of transdermal nitroglycerin in the treatment of dogs with left-sided congestive heart \nfailure.\n\nMethodology Section\n\nSearch Strategy \nDatabases searched and dates \ncovered: \nPubmed Platform 1973- Week 1 2017], CAB Abstracts <1973 to 2016 \nWeek 51>\n\nSearch terms: \nPubmed \n1 \nSearch ((((glyceryl trinitrat*) OR glyceryltrinitrat*) OR \ntrinitrat glycerin*) OR nitro glycerin*) OR nitroglycerin*\n\n16518 \n2 \nSearch ((((((((heart) OR cardiac) OR CHF) OR cardio*) OR left \nventricular) OR left ventricle*) OR LV) OR heart diseases) OR \ncardiovascular diseases 2866548 \n3 \nSearch (((((dog) OR dogs) OR canine) OR bitch*) OR canis) \nOR bitches \n351249 \n4 \nSearch ((transdermal) OR cutaneous) OR ointment\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 2, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v2i4.115  \nnext review date: 02 Jan 2019 \np a g e | 6\ntotal pages: 8\n\n185703\n\nCAB Abstracts:  \n1. (\"glyceryl trinitrat*\" or glyceryltrinitrat* or \"trinitro \nglycerin*\" or trinitroglycerin* or \"nitro glycerin*\" or \nnitroglycerin*).mp. [mp=abstract, title, original title, broad \nterms, heading words, identifiers, cabicodes] (374) \n2. (heart or cardiac or CHF or cardio* or \"left ventricular\" or \n\"left ventricle*\" or \"left ventrical*\" or \"LV\").mp. or heart \ndiseases/ or cardiovascular diseases/ [mp=abstract, title, \noriginal title, broad terms, heading words, identifiers, \ncabicodes] (151135) \n3. (dog or dogs or canine or bitch*).mp. or dogs/ or canis/ or \nbitches/ (192085) \n4. 1 and 2 and 3 (16)\n\nDates searches performed: 22/12/2016 (CAB) 02/01/2017 (Pubmed)\n\n(dog or dogs or canine or bitch*).mp.or dogs/ or canis/ or \nbitches/ (192085) \n4.1 and 2 and 3 (16)\n\nDates searches performed: 22/12/2016 (CAB) 02/01/2017 (Pubmed)\n\nExclusion / Inclusion Criteria \nExclusion: Study design did not involve dogs and/or was not relevant to the \nresearch questions asked \nInclusion: Any relevant primary veterinary research or systematic review which \ncompared transdermal nitroglycerine to other therapies to answer \nthe following question; “Does adding transdermal nitroglycerine to \nother therapies used for management of left-sided congestive heart \nfailure in dogs speed the resolution of clinical signs?”\n\nSearch Outcome \nDatabase \nNumber \nof \nresults \nExcluded – \nDid not \naddress \nthe PICO \nExcluded – \nProceedings \nand review \narticles with no \nevidence \nExcluded – \nGiven IV, did \nnot compare \ngroups, and \ndid not \naddress \nclinical \nquestion \nExcluded – \nSingle case \nreport with \nno evidence \nto the \nquestion \nDuplicate \nresults \nTotal \nrelevant \npapers \nPubMed \n10 \n6 \n0 \n0 \n0 \n0 \n4 \nCAB \nAbstracts  \n16 \n0 \n6 \n6 \n2 \n2 \n0 \nTotal relevant papers when duplicates removed\n\n4\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 2, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v2i4.115  \nnext review date: 02 Jan 2019 \np a g e | 7\ntotal pages: 8\n\nCONFLICT OF INTEREST\n\nThe author declares no conflict of interest.\n\nREFERENCES\n\ndoi.org/10.18849/ve.v2i4.115  \nnext review date: 02 Jan 2019 \np a g e | 7\ntotal pages: 8\n\nCONFLICT OF INTEREST\n\nThe author declares no conflict of interest.REFERENCES\n\n1. Kanda A, Yoshida M, Kanou M, et al. (1995). Cardiovascular effects of NT-1, a new patch form of \nnitroglycerin, alone and in combination with nifedipine in conscious dogs. Journal of Pharmacy \nPharmacology. 47(12A): pp. 1021-1024. DOI: http://dx.doi.org/10.1111/j.2042-7158.1995.tb03289.x \n2. McKie PM, Cataliotti A, Ichiki T, et al. (2014). M-atrial natriuretic peptide and nitroglycerin in a canine \nmodel of experimental acute hypertensive heart failure: differential actions of 2 cGMP activating \ntherapeutics. Journal of the American Heart Association, 3(1):e000206. \nDOI: http://dx.doi.org/10.1161/JAHA.113.000206 \n3. Nakayama, T. Nishijima, Y.  Miyamoto, et al. (2007). Effects of 4 classes of cardiovascular drugs on \nventricular function in dogs with mitral regurgitation.  Journal of Veterinary Internal Medicine, 21(3), \npp. 445-450. PMID: 17552449 DOI: http://dx.doi.org/10.1111/j.1939-1676.2007.tb02988.x \n4. Parameswaran, N. Hamlin, R. L.  Nakayama, T.  Rao, S. S. (1999). Increased splenic capacity in response \nto transdermal application of nitroglycerin in the dog.  Journal of Veterinary Internal Medicine, 13(1), \npp. 44-46. PMID: 10052063 DOI: 1111/j.1939-1676.1999.tb02164.x \n5. Sellers R, Levy M, Amplatz K, Lillehei CW. Left retrograde cardioangiography in  acquired  cardiac  \ndisease:  Technic,  indications  and  interpretations  in  700  Am J  Cardiol  1964;14: 437–447 \nDOI: http://dx.doi.org/10.1016/0002-9149(64)90027-X\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 2, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v2i4.115  \nnext review date: 02 Jan 2019 \np a g e | 8\ntotal pages: 8\n\ndoi.org/10.1016/0002-9149(64)90027-X\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 2, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v2i4.115  \nnext review date: 02 Jan 2019 \np a g e | 8\ntotal pages: 8\n\nIntellectual Property Rights \nAuthors of Knowledge Summaries submitted to RCVS Knowledge for publication will retain \ncopyright in their work, and will be required to grant to RCVS Knowledge a non-exclusive \nlicense of the rights of copyright in the materials including but not limited to the right to \npublish, re-publish, transmit, sell, distribute and otherwise use the materials in all languages \nand all media throughout the world, and to license or permit others to do so.\n\nDisclaimer \nKnowledge Summaries are a peer-reviewed article type which aims to answer a clinical \nquestion based on the best available current evidence. 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Our \nambition is to become a global intermediary for evidence based veterinary knowledge by providing access to information \nthat is of immediate value to practicing veterinary professionals and directly contributes to evidence based clinical \ndecision-making. \nwww.veterinaryevidence.org\n\nRCVS Knowledge is a registered Charity No. 230886.  \nRegistered as a Company limited by guarantee in England and Wales No. 598443.\n\nRegistered Office: \nBelgravia House  \n62-64 Horseferry Road \n London SW1P 2AF\n\nwww.veterinaryevidence.org\n\nRCVS Knowledge is a registered Charity No.230886.Registered as a Company limited by guarantee in England and Wales No.598443.Registered Office: \nBelgravia House  \n62-64 Horseferry Road \n London SW1P 2AF\n\nThis work is licensed under a Creative Commons Attribution 4.0 International License.\n\n---"}
+{"text": "【兽医文献】\n标题: Modulation of growth, gut microbiota, and health markers in rabbits fed hydroponic barley with or without enzymes\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nSerum metabolomics reveal the\nmechanisms by which fermented\nbrewer’s spent grains promote\nintestinal development in\nwhite-feathered broilers\nYuanfeng Li *†, Zhiheng Meng † and Yiyuan Wang\nCollege of Agriculture and Biology, Liaocheng University, Liaocheng, China\nThis study aimed to investigate the effects of wet-fermented brewer’s grains\n(WFBGs) on gut development and serum metabolism in white-feathered broilers.\nA total of 192 one-day-old male broilers (initial body weight: 36.46 ± 0.93 g) were\nrandomly assigned to two treatment groups: the control group (0% WFBGs) and\nthe experimental group (20% WFBG inclusion), with 6 replicates of 16 birds per\nreplicate. The results of intestinal morphological parameters, quantified using\nImageJ software after hematoxylin–eosin (HE) staining, showed that compared\nwith the control group, broilers fed a diet supplemented with 20% WFBGs had\nsignificantly improved duodenal development. Specifically, the duodenal villus height\n(VH) increased by 10.2% (p < 0.05), and the villus height-to-crypt depth ratio (VH/\nCD) increased by 27.2% (p < 0.05)—both indicators reflecting enhanced duodenal\ndevelopment. Through untargeted metabolomics analysis for screening differentially\nexpressed metabolites (DEMs) from serum samples, 211 DEMs were identified,\nincluding 98 upregulated DEMs and 113 downregulated DEMs in the WFBG group.\nKEGG pathway enrichment analysis revealed that these DEMs were significantly\nassociated with key metabolic processes, including linoleic metabolic pathways,\nlinoleic acid metabolism, phenylalanine metabolism, and other relevant pathways.\nSpecifically, key DEMs involved in amino acid metabolism included significantly\nincreased phenylalanine levels and decreased 4-HPA and 3-HPA levels (p < 0.05).\nIn conclusion, the inclusion of 20% WFBGs in the diet of white-feathered broilers\nsignificantly promoted intestinal development. These favorable outcomes are tied to\nmodified serum metabolic profiles and shifts in lipid and amino acid metabolism in\n\n05).In conclusion, the inclusion of 20% WFBGs in the diet of white-feathered broilers\nsignificantly promoted intestinal development.These favorable outcomes are tied to\nmodified serum metabolic profiles and shifts in lipid and amino acid metabolism in\nbroilers, underscoring WFBG’s significance for boosting broiler intestinal development\nwhile also adding to the theoretical framework for poultry by-product-based feeds.\nKEYWORDS\nfermented brewer’s spent grains, intestinal development, broilers, metabolomics,\nmetabolic mechanism\n1 Introduction\nAgainst the backdrop of escalating geopolitical conflicts, the global food crisis is becoming\nincreasingly severe, and it is extremely urgent to find and develop alternative animal feed\nmaterials. China has a huge production of brewer’s grains, which are often directly discharged\nas waste, resulting in serious resource waste (1). It is essential to effectively apply brewer’s\ngrains to livestock and poultry production. Brewer’s grains are rich in amino acids, trace\nelements, and proteins, making them a high-quality feed material for animal production (2).\nPrevious studies have shown that dried brewer’s grains (DBGs) have been used in poultry\nOPEN ACCESS\nEDITED BY\nFelix Kwame Amevor,\nSichuan Agricultural University, China\nREVIEWED BY\nQianyun Zhang,\nGuangxi University, China\nSamuel Kumi Okyere,\nWayne State University, United States\nOdinaka C. Iwuozo,\nNorth Carolina Agricultural and Technical\nState University, United States\nYingjie Wang,\nSichuan Agricultural University, China\nGeorgeta Stefan,\nUniversity of Agronomic Sciences and\nVeterinary Medicine Bucharest, Romania\n*CORRESPONDENCE\nYuanfeng Li\nliyuanfeng@lcu.edu.cn\n†These authors have contributed equally to\nthis work\nRECEIVED 20 April 2025\nACCEPTED 31 August 2025\nPUBLISHED 10 October 2025\nCITATION\nLi Y, Meng Z and Wang Y (2025) Serum\nmetabolomics reveal the mechanisms by\nwhich fermented brewer’s spent grains\npromote intestinal development in\nwhite-feathered broilers.\nFront. Vet. Sci. 12:1614917.\ndoi: 10.3389/fvets.2025.1614917\nCOPYRIGHT\n© 2025 Li, Meng and Wang. This is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\n\nFront.Vet.Sci.12:1614917.doi: 10.3389/fvets.2025.1614917\nCOPYRIGHT\n© 2025 Li, Meng and Wang.This is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY). The use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\naccordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Original Research\nPUBLISHED  10 October 2025\nDOI  10.3389/fvets.2025.1614917\n\nLi et al.\b\n10.3389/fvets.2025.1614917\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n(3–5) and livestock (6), helping to modulate the gut microbiota\nbalance (7) and increase the apparent digestibility of nutrients (4),\nthus improving growth and reducing breeding costs (8). Wet brewer’s\ngrains (WBGs) have also been used in livestock and poultry feed (9).\nDue to their unique digestive and metabolic properties in the rumen,\nWBGs are more commonly used in ruminants (10) than in poultry\n(7). Fermentation technology can effectively preserve and improve the\nnutritional content of brewer’s grains, enhancing their value as animal\nfeed (1). After fermentation, the apparent digestibility of nutrients\nfrom DBGs was significantly increased in laying hens (9) or ducks (3).\nPrevious studies have demonstrated that probiotics and fermentation\nmetabolites derived from fermented wheat bran enhance intestinal\nbarrier function (11), regulate gut microbiota homeostasis (12), and\nalleviate intestinal inflammation (13). Other studies on broilers show\ncommon fibrous additives are typically included at 2–12% of the basal\ndiet (5), and ≥15% is generally considered “high-dose” for their\naltering feed intake, nutrient digestibility, or intestinal physiology (14,\n15). However, there is currently a lack of research on the application\nof high-dose wet-fermented brewer’s grains (WFBGs) to\nbroiler chickens.\nMetabolomics is commonly used as a technique for biomarker\ndiscovery, which can analyze metabolites in biological fluids, cells, and\ntissues (16). Abnormal changes in small molecule metabolites within\nthe body are usually the ultimate response of the organism to the effect\nof disease or stress (17). Metabolomics of stress-induced biological\nsamples may identify diagnostic biomarkers and reveal stress\nmechanisms. Clarifying these mechanisms through the investigation\nof intestinal development and serum metabolism may provide a bridge\nfor studies related to the regulation of WFBGs (18). Serum metabolites\ncan characterize the body’s metabolism and inflammatory response\n(18). Serum metabolomics can better demonstrate the correlation\n\nSerum metabolites\ncan characterize the body’s metabolism and inflammatory response\n(18).Serum metabolomics can better demonstrate the correlation\nbetween intestinal development and organs and pathways (19). Serum\nmetabolomics proves useful for capturing digestive efficiency,\nintestinal structural features, and dynamic diet-linked metabolic\nchanges in broilers and other livestock, thus serving as a more holistic\nand application-friendly sample type to assess how WFBGs in diets\nimpact intestinal development (20). In recent years, metabolomics\ntechnology has been widely used to evaluate fermented feed in pigs\n(21), ducks (22), and broilers (23). To the best of our knowledge, there\nare no reports investigating how metabolomics can identify serum\nmetabolites regulating intestinal development in WFBG-fed broilers.\nIn broiler production, the intestinal development of broiler chickens\ndirectly determines their growth rate in the later stages. Previous studies\nhave found that fermented feed or fiber raw materials can promote\nintestinal development through regulating gut microbiota (24, 25), but\nwhether high-dose addition causes damage to intestinal development\nhas not been reported. Therefore, it is hypothesized that the addition of\nWFBGs may promote the intestinal development of white-feathered\nbroilers, and thus, this study intends to explore the impacts of WFBGs\non their intestinal development and serum metabolism.\n2 Materials and methods\n2.1 Animals and treatments\nA total of 192 one-day-old (36.5 ± 0.93 g) white-feathered broilers\n(hybrid of Cobb breed cock and Hy-line brown laying hen) were\nrandomly divided into two groups (6 replicates of 16 birds each). The\ncontrol and experimental groups were fed a basic diet containing 0%\nWFBGs or 20% WFBGs (basic feed to WFBG ratio of 80:20),\nrespectively. This trial lasted for 21 days. All birds were housed in wire\ncages (70 cm × 80 cm × 40 cm). The birds were purchased from\nAoxiang Poultry Industry Co., Ltd., Liaocheng, Shandong Province,\nChina. During this study, all birds had free access to feed and water. The\n\nThis trial lasted for 21 days.All birds were housed in wire\ncages (70 cm × 80 cm × 40 cm).The birds were purchased from\nAoxiang Poultry Industry Co., Ltd., Liaocheng, Shandong Province,\nChina.During this study, all birds had free access to feed and water.The\nbasic feed for birds was purchased from a commercial feed enterprise\n(Haiding Feed Co., Ltd., Liaocheng, China). The WFBGs used in this\nexperiment were prepared in the laboratory of Liaocheng University\n(Liaocheng, Shandong, China) (WBG: corn: wheat bran: fermenting\nagent = 45:23:30:2, incubated at 37°C for 72 h, once a week). This study\nwas approved by the Animal Protocol Review Committee of Liaocheng\nUniversity, China (No: LCU20240016). The fermenting agent\n(composed of Lactobacillus acidophilus, Bifidobacterium spp., and\nBacillus subtilis) was purchased from Qingdao Genyuan Biotechnology\nGroup (Qingdao, Shandong Province, China). The contents of crude\nprotein and crude fiber of fermented WFBGs increased from 9.22 to\n10.23% and decreased from 8.40 to 7.20%, respectively.\n2.2 Sample collection\nOn day 21, one bird (fasted for 12 h, with body weight close to the\naverage of each replicate) was randomly selected from each replicate\nof the control and 20% WFBG groups for sample collection. Briefly,\n2–3 mL of blood samples were collected from broilers’ wing veins\nusing a sterile syringe, then left to stand at room temperature for 2 h\nfor natural coagulation. Subsequently, serum samples were separated\nby centrifugation at 5,000 g for 10 min (4°C) and then stored at −80°C\nfor metabolomics analysis. The birds were euthanized with\nexsanguination under sodium pentobarbital anesthesia (60 mg·kg−1)\nafter blood collection. Under strict aseptic procedures, the middle\nsegments of the duodenum, jejunum, and ileum (each approximately\n2 cm in length) were carefully removed and fixed in 4% formalin for\nsubsequent intestinal morphological observations.\n2.3 Intestinal morphology\nAfter being dehydrated and embedded in paraffin, the fixed\nintestinal tissues (5 μm thickness) were continuously sliced and stained\n\n2.3 Intestinal morphology\nAfter being dehydrated and embedded in paraffin, the fixed\nintestinal tissues (5 μm thickness) were continuously sliced and stained\nwith hematoxylin–eosin (26). The intestinal mucosal structure was\nobserved using a microscope and analyzed using Media Cybernetics\nimaging software (Image-Pro Plus 6.0, USA). The villus height (VH)\nand crypt depth (CD) of 16 intact villi per slice were measured, and 5\nslices were selected to calculate the average value of each tissue.\n2.4 Serum metabolite extraction\n2.4.1 Serum preparation for metabolomics\nBefore analysis, serum samples were thawed on ice and mixed\nthoroughly by vortexing for 10 s. A volume of 100 μL of serum was\nmixed with 400 μL of extraction solution (MeOH: ACN, 1:1 (v/v),\ncontaining deuterated internal standards). The mixture was vortexed\nfor 30 s, sonicated for 10 min in a 4°C water bath, and incubated at\n−40°C for 1 h to precipitate proteins. Samples were then centrifuged\nat 12000 rpm for 15 min at 4°C. The supernatant was transferred to a\nfresh glass vial and injected into the ultra-high liquid\n\nLi et al.\b\n10.3389/fvets.2025.1614917\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nchromatography–tandem mass spectrometry (UHPLC–MS/MS)\nsystem for analysis. An equal aliquot of the supernatant of all samples\nwas fixed to prepare the quality control (QC) sample.\n2.4.2 Metabolomics data capture\nFor positive and negative metabolites, LC–MS/MS analyses were\nperformed using a UHPLC system (Orbitrap Exploris, Thermo Fisher\nScientific, USA), coupled with UPLC BEH Amide (Waters ACQUITY,\n2.1 mm × 50 mm, 1.7 μm) and MS (Orbitrap Exploris 120, Thermo\nFisher Scientific, USA). The mobile phase consisted of solvent A\n(water containing 25 mmol/L of ammonium acetate and 25 mmol/L\nof ammonia hydroxide, pH = 9.75) and solvent B (ACN, 0.1% formic\nacid). Other parameters were as follows: autosampler temperature,\n4°C; autosampler mode, partial loop injection; sample loop volume,\n10 μL; injection volume, 2 μL. The MS (Orbitrap Exploris 120,\nThermo Fisher Scientific, USA) was used to evaluate the full scan MS\nspectrum in information-dependent acquisition mode, controlled by\nthe Xcalibur (V4.4) software (Thermo Fisher Scientific, USA).\nFurther details regarding metabolomics analysis are provided in\nSupplementary file S1.\n2.5 Metabolomics data analysis\nThe raw data were converted to the mzXML format using\nProteoWizard software (ProteoWizard Software Foundation,\nUSA) and processed with an in-house program (developed using\nR and based on XCMS). The R package and BiotreeDB (V3.0,\nChina) were applied to metabolite annotation (27). Candidate\nmetabolites (VIP > 1 and adjusted p < 0.05) were regarded as\npotential biomarkers.\n2.6 Statistical analysis\nData on intestinal morphology were analyzed using Student’s\nt-test in SPSS software (V23.0; SPSS Inc., Chicago, Illinois, USA) (28),\nwith the pen considered as a fixed effect. All data are expressed as\nmeans. p-values < 0.05 were considered statistically significant.\n3 Results\n3.1 Intestinal morphology\nCompared to the control group, the duodenum VH and VH/CD\nratio in the 20% WFBG group were significantly increased (p < 0.05,\n\nAll data are expressed as\nmeans.p-values < 0.05 were considered statistically significant.3 Results\n3.1 Intestinal morphology\nCompared to the control group, the duodenum VH and VH/CD\nratio in the 20% WFBG group were significantly increased (p < 0.05,\nTable 1). No differences were observed in CD in the duodenum, VH,\nCD, or VH/CD ratio in the jejunum or ileum (p > 0.05, Table 1).\n3.2 Serum untargeted metabolomics\nprofile of WFBGs\n3.2.1 Quality control of metabolite data from\nserum samples\nIn this study, pooled QC samples were used to validate the system\nperformance. These tightly clustered QC samples demonstrated that\nthe metabolomic method had good repeatability and stability\n(Figure 1A). Serum samples of broiler chickens revealed significant\nchanges in serum metabolic profiles of broiler chickens fed 20%\nWFBG diets (Figure 1A). OPLS-DA score plots (Figure 1B) revealed\ndistinct clustering patterns between the control and WFBG groups,\ndemonstrating the model’s robustness with parameters: R2X = 0.332,\nR2Y = 0.991, and Q2 = 0.477, indicating no overfitting.\n3.2.2 Differential metabolite analysis\nThe screening criteria used to identify differently expressed\nmetabolites (DEMs) in the serum of broiler chickens from the 20%\nWFBG and control groups were VIP score > 1, fold change (FC) > 1.2,\nor FC < 0.833, and p-value < 0.05. There were 211 DEMs in the serum\n(Figure 1C), including 98 upregulated DEMs and 113 downregulated\nDEMs in the 20% WFBG group compared to the control group.\nAmong these DEMs, dehydroalanine and N6-mmethyllysine, related\nto amino acid metabolism, were upregulated, while benzamide was\ndownregulated. In lipid metabolism and energy regulation,\npropionylcarnitine [Car(3:0)], 2-hydroxy-1-[hydroxy(7-methoxy-2-\noxo-2H-1-benzopyran-6-yl)methyl]-2-methylpropyl\nester,\n3-methylbutanoic acid, and 2-[(1-methyl-1H-pyrazolo[3,4-d]\npyrimidin-4-yl)amino]ethanol\nwere\nupregulated,\nwhereas\npalmitoylcarnitine [Car(16:0)], linoleoyl carnitine [Car(18:2)],\noleoylcarnitine [Car(18:1)], and 5α-pregnan-3α,17-diol-20-one\n3-sulfate were downregulated. For antioxidant and inflammatory\n\nFor antioxidant and inflammatory\nregulation, taxifolin and kaempferol 3-α-L-arabinoside were\nupregulated, while formoterol and apigetrin were downregulated. In\ndrug metabolism and xenobiotic handling, bezafibrate and 2-chloro-\n5-nitro-N-phenyl compound were upregulated, and 5-fluoro-1,3-\ndihydro-2H-benzimidazole-2-thione was downregulated. Tricin\nmethyl ether (6β,22E)-6-hydroxystigmasta-4,22-dien-3-one, (3E)-4-\n(2-carboxyphenyl)-2-oxobut-3-enoate, and benzamide, related to\nhormone signaling and nitrogen metabolism, were downregulated,\nTABLE 1  Effects of dietary WFBGs on the intestinal development in\nbroiler chickens.1\nItems2\nCON\ngroup3\n20%\nWFBG\ngroup3\nSEM4\nP-value\nDuodenum\nVH, mm\n1.27b\n1.40a\n0.057\n0.045\nCD, mm\n0.24\n0.20\n0.017\n0.052\nVH/CD\n5.48b\n6.97a\n0.524\n0.018\nJejunum\nVH, mm\n0.65\n0.76\n0.079\n0.208\nCD, mm\n0.15\n0.15\n0.021\n0.884\nVH/CD\n4.58\n5.17\n0.575\n0.331\nIleum\nVH, mm\n0.37\n0.36\n0.032\n0.908\nCD, mm\n0.11\n0.11\n0.011\n0.782\nVH/CD\n3.47\n3.41\n0.505\n0.908\n1Means represent 6 replicates with 16 birds per cage (n = 6/group).2VH, villus height; CD,\ncrypt depth. 3CON group: basal diet; 20% WFBG group: basal diet + 20% WFBG. 4Pooled\nstandard error of mean. Values within a row with different superscripts differ significantly at\naP < 0.05.\n\nLi et al.\b\n10.3389/fvets.2025.1614917\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nwhile N-acetyl-D-galactosaminitol, associated with carbohydrate\nmetabolism and glycoconjugates, was upregulated. Hierarchical\nclustering analysis according to the relative abundance of serum\nmetabolites was conducted to generate a heatmap (Figure 1D). The\ntop 10 DEMs in the 20% WFBG and control groups are shown in\nTable 2. More detailed results are shown in Supplementary Table 1.\n3.2.3 KEGG pathway analysis\nA total of 1987 metabolites in the positive and negative ion\nmodes were submitted to the KEGG pathway analysis, of which 211\nwere DEMs (Supplementary Table 1). Key functional impacts of\nenriched DEMs on metabolic pathways were determined by the\nMetaboAnalyst (V 6.0) (Figure 1E). The enriched DEMs between\nthe 20% WFBG and control groups had a significant effect on 51\nmetabolic pathways, such as metabolic pathways, biosynthesis of\namino acids, ABC transporters, 2-oxocarboxylic acid metabolism,\nbiosynthesis of cofactors, carbon metabolism, linoleic acid (LA)\nmetabolism, and phenylalanine metabolism (p < 0.05). There were\n32 metabolites involved in metabolic pathways, 8 metabolites\ninvolved in the biosynthesis of amino acids, 7 metabolites involved\nin ABC transporters, 5 metabolites involved in 2-oxocarboxylic\nacid metabolism, 5 metabolites in biosynthesis of cofactors, 4\nmetabolites in carbon metabolism, 1 metabolite involved in LA\nmetabolism, and 3 metabolites involved in phenylalanine\nmetabolism. Notably, linoleic acid (LA) metabolism and\nphenylalanine metabolism exhibited pathway impact values of\n1.000 and 0.436 (Figure 1F), respectively. These two pathways are\nlikely to exert pivotal effects on mediating how WFBGs regulate\nintestinal development in broilers. Specifically, LA metabolism\nserves as a core biosynthetic pathway for polyunsaturated fatty\nacids such as arachidonic acid. These lipid metabolites are capable\nof enhancing the structural integrity of intestinal epithelial tight\njunctions and mitigating intestinal inflammatory reactions by\ninhibiting the activation of the nuclear factor-κB (NF-κB) signaling\n\nThese lipid metabolites are capable\nof enhancing the structural integrity of intestinal epithelial tight\njunctions and mitigating intestinal inflammatory reactions by\ninhibiting the activation of the nuclear factor-κB (NF-κB) signaling\npathway, ultimately reinforcing the protective capacity of the\nintestinal barrier. In parallel, phenylalanine metabolism is\nintricately involved in the biosynthesis of two distinct classes of\nbioactive molecules: neurotransmitters (e.g., dopamine, which\nregulates gastrointestinal motility and mucosal nerve signaling)\nand antioxidants (e.g., melanin, a potent scavenger of reactive\noxygen species (ROS) in mucosal tissues). For broilers\nsupplemented with WFBGs, this dual biosynthetic role of\nphenylalanine metabolism could potentially alleviate oxidative\nstress in the intestinal mucosa and fine-tune the balance of\nintestinal mucosal immunity—a mechanism that ultimately\nsupports the maintenance of intestinal homeostasis. Therefore,\nwe  further investigated the changes in LA and phenylalanine\nmetabolites in the serum of broiler chickens fed 20% WFBG diets.\nThe detailed results of metabolic pathway analysis are shown in\nSupplementary Table 2.\n3.2.4 LA and phenylalanine targeted metabolomic\nstudies\nThe quantitative analysis results of phenylalanine metabolites and\nLA metabolites are presented in Figures 2A,B, respectively. Specifically,\nin the 20% WFBG group, the level of phenylalanine was significantly\nincreased (p < 0.05, Figure 2A), the levels of LA (p < 0.05, Figure 2B),\n4-hydroxyphenylacetic\nacid\n(p < 0.05,\nFigure\n2C),\nand\nFIGURE 1\nSerum metabolome analysis in broilers with WFBGs and normal birds. (A) PCA-DA plots of serum metabolites in the positive and negative ion modes.\n(B) OPLS-DA analysis of alterations in the metabolite profile. (C) Volcano plot of DEMs between the control and 20% WFBG groups (p-value of t-test <\n0.05 and fold change (FC) > 1.2, or FC < 0.833). Gray, no change; red, upregulation; green, downregulation. (D) Cluster heat map of DEMs: from green\n\n(C) Volcano plot of DEMs between the control and 20% WFBG groups (p-value of t-test <\n0.05 and fold change (FC) > 1.2, or FC < 0.833).Gray, no change; red, upregulation; green, downregulation.(D) Cluster heat map of DEMs: from green\n(less) to red (more). (E) The enrichment map of metabolic pathways of DEMs from the control and 20% WFBG group comparisons. X-axis, rich factor;\ny-axis, enriched pathway; size of the bubble, the number of DEMs enriched in the pathway; color, enrichment significance. CON, control group; SM1,\n20% WFBG group. (F) The analysis diagram of metabolic pathways. Each bubble is a metabolic pathway; the horizontal axis and size of the bubble\nrepresent the size of the influencing factor of the pathway in topological analysis. The vertical axis and the color of the bubble represent the negative\nnatural logarithm of the p-value of enrichment analysis.\n\nLi et al.\b\n10.3389/fvets.2025.1614917\nFrontiers in Veterinary Science\n05\nfrontiersin.org\n3-hydroxyphenylacetic\nacid\n(p < 0.05,\nFigure\n2D)\nwere\nsignificantly decreased.\n4 Discussion\n4.1 Intestinal development\nThe quality of intestinal development greatly affects animal\ngrowth. The small intestine is a crucial digestive and absorptive organ\nin the body, and an appropriate amount of fiber can alter the villous\nmorphology of the small intestine. The changes in villus morphology\ncan be reflected in the variations in VH and CD (27). Higher VH and\nCD values indicate a larger absorption area and higher cell\nproliferation activity, respectively. The VH/CD ratio is commonly\nused for the comprehensive evaluation of small intestine digestive\ncapacity. An increase in intestinal length will increase food retention\ntime in the intestine, thereby increasing the absorption of nutrients.\nThe increase in CD can enhance cell proliferation activity, while the\ndecrease in CD may reflect a slower rate of cell generation. Previous\nstudies demonstrated that a 2–10% dried BG diet did not promote\nintestinal development in broilers (29), and a 2–12% DBG diet had no\nTABLE 2  Identification results of top-10 DEMs in the serum between the 20% WFBG and control groups.\nNo.\nMetabolites\nm/z\nRT(s)\nVIP\nFC\nMode\nP-value\n1\nKaempferol\n3-.alpha.-L-arabinoside\n419.1\n169.8\n1.33\n1.79\nup\n0.0106\n2\nButanoic acid, 3-methyl-,\n2-hydroxy-1-[hydroxy(7-\nmethoxy-2-oxo-2H-1-\nbenzopyran-6-yl)methyl]-2-\nmethylpropyl ester\n379.18\n128.6\n2.1\n1.78\nup\n0.0048\n3\nBezafibrate\n362.11\n112\n1.99\n1.71\nup\n0.0067\n4\nTaxifolin\n303.05\n116.8\n1.2\n2.4\nup\n0.0054\n5\nBenzamide, 2-chloro-5-\nnitro-N-phenyl-\n275.02\n96.6\n1.9\n1.97\nup\n0.0327\n6\nN-Acetyl-D-galactosaminitol\n224.11\n146.1\n2.11\n1.74\nup\n0.0025\n7\nPropionylcarnitine\n[Car(3:0)]\n218.14\n171.1\n2.06\n1.91\nup\n0.0135\n8\n2-[(1-Methyl-1H-\npyrazolo[3,4-d]pyrimidin-4-\nyl)amino]ethanol\n194.1\n32.2\n2.01\n1.75\nup\n0.0118\n9\nN6-Methyllysine\n161.13\n288.7\n2.52\n1.81\nup\n0\n10\nDehydroalanine\n88.04\n231.8\n1.82\n2.54\nup\n0.0252\n11\nApigetrin\n431.1\n131.7\n2.56\n0.09\ndown\n0.0029\n12\n(6beta,22E)-6-\nHydroxystigmasta-4,22-\ndien-3-one\n427.36\n90\n2.6\n0.35\ndown\n0.0009\n13\nCar(18:1)\n426.36\n90\n2.56\n0.36\ndown\n0.0016\n14\nLinoleoylcarnitine\n[Car(18:2)]\n424.34\n93.4\n2.41\n0.2\ndown\n0.0043\n15\n5.alpha.-Pregnan-3.\nalpha.,17-diol-20-one\n3-sulfate\n413.2\n159.5\n2.06\n0.44\ndown\n0.0035\n16\nPalmitoylcarnitine\n[Car(16:0)]\n400.34\n95.4\n2.57\n0.33\ndown\n0\n17\nFormoterol\n345.18\n275.5\n1.96\n0.43\ndown\n0.0289\n18\nTricin methyl ether\n343.09\n25.3\n1.98\n0.39\ndown\n0.0178\n19\n(3E)-4-(2-Carboxyphenyl)-\n\n0043\n15\n5.alpha.-Pregnan-3.alpha.,17-diol-20-one\n3-sulfate\n413.2\n159.5\n2.06\n0.44\ndown\n0.0035\n16\nPalmitoylcarnitine\n[Car(16:0)]\n400.34\n95.4\n2.57\n0.33\ndown\n0\n17\nFormoterol\n345.18\n275.5\n1.96\n0.43\ndown\n0.0289\n18\nTricin methyl ether\n343.09\n25.3\n1.98\n0.39\ndown\n0.0178\n19\n(3E)-4-(2-Carboxyphenyl)-\n2-oxobut-3-enoate\n238.07\n99.4\n1.91\n0.33\ndown\n0.0435\n20\n5-Fluoro-1,3-dihydro-2H-\nbenzimidazole-2-thione\n167.01\n14\n1.56\n0.11\ndown\n0.0165\nWFBGs, wet fermented brewer’s grains; m/z, mass-to-charge ratio; RT, retention time; FC, fold change, 20% WFBG group vs. control group; VIP, variable importance in the projection; DEMs,\ndifferentially expressed metabolites.\n\nLi et al.\b\n10.3389/fvets.2025.1614917\nFrontiers in Veterinary Science\n06\nfrontiersin.org\neffect on the VH, CD, or VH/CD in slow-growing broiler chickens (5).\nOur previous study demonstrated that the jejunum VH and VH/CD\nratio in pullets fed with a 20% WFBG diet was significantly increased\n(unpublished data). In this study, compared with the control group,\nthe duodenal VH and VH/CD ratio in the 20% WFBG group were\nsignificantly increased (p < 0.05, Table 1), but no significant differences\nwere observed in the jejunum and ileum (p > 0.05, Table 1). The\nduodenum (the primary site for the initial digestion of carbohydrates,\nproteins, and lipids) exhibits heightened sensitivity to dietary stimuli.\nThis heightened responsiveness directly underlies the notable\nmorphological alterations observed in the duodenum, distinguishing\nit from other intestinal segments. In contrast, the jejunum (the main\nsite for absorbing amino acids, fatty acids, and vitamins) receives\nchyme that has already undergone partial digestion in the duodenum.\nThis prior digestion step reduces the intensity of WFBG-associated\nstimuli reaching the jejunum, thereby weakening potential\nmorphological effects. In parallel, the ileum functions as a post-\nabsorption segment, characterized by a well-developed mucosal\nbarrier and specialized immune cell populations. These features\nendow the ileum with a more robust functional buffering capacity to\ncounteract external dietary perturbations. Given these functional\ndifferences across intestinal segments, neither the jejunum nor the\nileum showed detectable significant morphological changes—a result\nthat aligns with observations from prior studies focusing on dietary\nfiber-induced intestinal adaptations in broiler chickens (30). WFBG\nmetabolites modulate duodenal pH to facilitate villus development.\nThey may weakly adjust jejunal digestive enzyme activity and ileal\nmicrobiota. These observations are hypothesized to stem from the\nappropriate fiber content in WFBGs, as the fiber promotes the\ndevelopment of duodenal mucosal structures while avoiding imposing\nan excessive metabolic burden on the broiler.\n\nThese observations are hypothesized to stem from the\nappropriate fiber content in WFBGs, as the fiber promotes the\ndevelopment of duodenal mucosal structures while avoiding imposing\nan excessive metabolic burden on the broiler.\n4.2 Serum untargeted metabolomics\nprofile of WFBGs\nWith the application of the WFBG diet in broilers, an untargeted\nUHPLC–MS/MS metabolomics study of serum was performed to\ninvestigate the growth-promoting mechanism of WFBGs. To\ninvestigate the growth-promoting mechanism of WFBGs in broilers,\nwe performed an untargeted metabolomic profiling using UHPLC–\nMS/MS to analyze the serum metabolome of broilers fed the WFBG\ndiet. A total of 211 DEMs were significantly observed in the WFBG\ngroup. These DEMs not only quantify WFBG’s regulatory effect on\nbroiler metabolism but also provide a key “metabolic signature” for\ndecoding its growth-promoting mechanisms. Biologically, this\nmetabolomic variation indicates that the WFBG drives systematic\nremodeling of the broiler serum metabolome, likely through its\nbioactive components (e.g., fiber and polyphenols) interacting with\nbroilers’ digestion, absorption, and hepatic metabolism processes.\nFIGURE 2\nConcentrations of serum metabolites involved in LA metabolism and phenylalanine metabolism by targeted analysis. LA, linoleic acid. *p < 0.05,\ncompared with the control group. CON, control group. 20% WFBG, basic feed to WFBG ratio of 80:20.\n\nLi et al.\b\n10.3389/fvets.2025.1614917\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nDownregulated DEMs may reduce metabolic burden and\ninflammation, while upregulated DEMs may enhance protein\nsynthesis—a process critical for broiler growth (31). Notably, these\nDEMs establish a link between WFBG intake and broiler\ngrowth phenotypes.\nSpecifically, dehydroalanine—upregulated in the WFBG group—\nsupports intestinal barrier repair and protein digestion, while\nN6-methyllysine (also upregulated) enhances functional protein\nactivity; both address the gut’s growth-related demands.\nDownregulated benzamide, by contrast, lowers the accumulation of\ntoxic ammonia, thereby protecting the intestinal mucosa. For energy\nmetabolism and gut health: upregulated propionylcarnitine boosts\nintestinal ATP supply, 3-methylbutanoic acid inhibits pathogens, and\nflavonoid ester reduces lipid oxidation. On the other hand,\ndownregulated long-chain acylcarnitines [Car(16:0), Car(18:2),\nCar(18:1)—common subtypes in broilers] and steroid metabolites\nease oxidative stress and excessive inflammation. In terms of oxidative\nstress regulation, upregulated taxifolin and kaempferol 3-α-L-\narabinoside activate the Nrf2/HO-1 pathway to clear ROS, which\nhelps improve the duodenal VH/CD ratio. Downregulated formoterol\nand apigetrin, meanwhile, prevent unwanted functional disruptions.\nOther key changes: upregulated bezafibrate and 2-chloro-5-nitro-N-\nphenyl compound strengthen xenobiotic clearance; downregulated\n5-fluoro-1,3-dihydro-2H-benzimidazole-2-thione reduces potential\ntoxicity. Additionally, downregulated tricin methyl ether and (6β,22E)-\n6-hydroxystigmasta-4,22-dien-3-one maintain hormonal and\nnitrogen balance, while upregulated N-acetyl-D-galactosaminitol\nsupports glycoconjugate synthesis to sustain intestinal function.\nPathway analysis showed that these metabolites were mainly involved\nin metabolic pathways, biosynthesis of amino acids, ABC transporters,\n2-oxocarboxylic acid metabolism, biosynthesis of cofactors, carbon\nmetabolism, LA metabolism, and phenylalanine metabolism. Changes\n\nChanges\nin these serum metabolites and the altered metabolic pathways may\nprovide new evidence to understand the action pathway of WFBGs.\nLipids, the main components of cell membranes, directly maintain\nthe physiological functions of cells and participate in the transport of\ntriglycerides (32). Growing evidence has shown that animal growth is\nassociated with the alteration of lipid metabolism (33). LA, n-6\npolyunsaturated fatty acids, plays an important role in inhibiting fatty\nacid synthesis by regulating the expression of enzymes or receptors\nrelated to fatty acid synthesis, thereby reducing fat production (34, 35).\nLA also promotes mitochondrial biogenesis and alleviates acute lung\ninjury (36) and has shown pro-tumorigenic (37) or anti-tumorigenic\neffects in multiple types of cancer (38). LA metabolism is an important\nmetabolic pathway involving many important physiological functions,\nincluding the oxidation of fatty acids to LA, activating glucose and\nlipid metabolism processes to maintain blood glucose stability and\npromote cellular metabolic activity. The most important function of\nLA metabolism is the control of blood sugar. LA metabolism can\npromote the oxidation of saturated fatty acids, thereby reducing the\nproduction of cholesterol and triglycerides, and ultimately lowering\nblood lipid levels. Furthermore, LA also strengthened the intestinal\nepithelial barrier by regulating the NF-κB/MLCK pathway in mice\n(39). It was reported that conjugated LA regulated gut microbiota-host\nmetabolic and immunomodulatory interactions (40). In this study, it\nwas found that LA metabolism was disturbed in broiler chickens fed\nwith 20% FBG, with a decrease in LA metabolites. Other metabolites\nclosely related to LA metabolism, such as arachidonic acid,\ngamma-linolenic acid, 13(S)-HODE, and 9-HPODE, were also\ndetected, but no significant changes were observed. These findings\nindicated that high doses of WFBGs may have a negative impact on\nlipid metabolism changes in broiler chickens. More animal\nexperiments are needed for further verification.\nIn addition to LA metabolites, the changed metabolites\n\nThese findings\nindicated that high doses of WFBGs may have a negative impact on\nlipid metabolism changes in broiler chickens.More animal\nexperiments are needed for further verification.In addition to LA metabolites, the changed metabolites\n(phenylalanine,\n4-hydroxyphenylacetic\nacid,\n4-HPA,\nand\n3-hydroxyphenylacetic acid, 3-HPA) in this study are also involved\nin the phenylalanine metabolism pathway. Phenylalanine is an\nimportant essential amino acid. Phenylalanine metabolism deficiency\ncan lead to various diseases. Phenylalanine in the plasma is mainly\nmetabolized and converted into tyrosine in the body, which is closely\nrelated to the formation of certain hormones and neurotransmitters.\nIf phenylalanine metabolism is disrupted, the conversion of\nphenylalanine to tyrosine is blocked. Increased phenylalanine\nconcentration and decreased tyrosine concentration in the blood, or\nan increase in the phenylalanine/tyrosine ratio, can cause various\ndiseases, especially damage to the nervous system. Qin et al. (38)\nreported that excessive phenylalanine reduced glucose utilization in\nthe liver, exacerbated pancreatic fat deposition, and induced\npancreatic injury and glucose metabolism disorders in growing and\nfattening pigs (38). It has been reported that remodeling gut\nmicrobiota and phenylalanine metabolism can effectively inhibit\npulmonary inflammatory damage induced by Mycoplasma\ngallisepticum in chickens (41). 4-HPA, a major microbiota-derived\nmetabolite of polyphenols, participates in antioxidant activity. 4-HPA\nmight regulate immune function and improve intestinal health in\nmeat pigeons (42). 3-HPA, formed by gut microbiota, might increase\nthe release of nitric oxide from the endothelial layer, causing\nvasodilation and ultimately lowering blood pressure in the body (43).\nIn this study, increased phenylalanine and decreased 4-HPA and\n3-HPA levels were observed in the serum from the 20% WFBG\ngroup, which might indicate that phenylalanine metabolism plays a\nsignificant role in the growth and development of broiler chickens.\nElevated phenylalanine levels may supply substrates for intestinal\n\nElevated phenylalanine levels may supply substrates for intestinal\ndevelopment by promoting amino acid transport and protein\nsynthesis in intestinal epithelial cells (44). Reduced 4-HPA and\n3-HPA—microbiota-derived metabolites—may alleviate inhibition\nof the mTOR pathway, activating villus cell proliferation signals (45).\nCollectively, these may synergistically promote intestinal\nmorphological development by enhancing amino acid utilization and\nregulating the cell cycle, though experimental validation of metabolic\npathway bidirectional regulation is warranted.\nWith the application of a UHPLC–MS/MS-based metabolomics\napproach, 211 serum DEMs involved in metabolic pathways,\nbiosynthesis of amino acids, ABC transporters, 2-oxocarboxylic acid\nmetabolism, biosynthesis of cofactors, carbon metabolism, LA\nmetabolism, and phenylalanine metabolism were identified, which\nindicated that WFBGs might affect amino acid metabolism and lipid\nmetabolism by regulating changes in GM. These DEMs may\nbe potential biomarkers for WFBGs promoting the growth of broiler\nchickens. However, the mechanistic exploration of how WFBGs link\nto metabolic and intestinal changes remains incomplete, preventing\nthe establishment of a causal chain between WFBG intake, gut\nmicrobial shifts, and downstream metabolic/intestinal responses.\nAdditionally, the long-term effects and potential safety risks of\nWFBGs were not evaluated, precluding assessment of their long-term\nimpacts on broiler health or product quality.\n\nLi et al.\b\n10.3389/fvets.2025.1614917\nFrontiers in Veterinary Science\n08\nfrontiersin.org\n5 Conclusion\nThis study shows that adding 20% WFBGs to broiler diets\neffectively promotes intestinal development. Metabolomic analysis\nreveals that WFBGs modulate LA and phenylalanine metabolism,\nboosting nutrient absorption and energy balance—laying a metabolic\nfoundation to clarify its gut health benefits. Sourced from brewery\nwaste, WFBGs are an economical feed ingredient fitting circular\neconomy principles. The 20% inclusion offers a scientific reference for\nfeed formulation, cutting reliance on conventional feeds and\nsupporting brewery waste’s industrial use in animal nutrition.\nData availability statement\nThe metabolic data generated in this study have been deposited in\nMetabolights under accession number MTBLS13078. The data are\npublicly available at https://www.ebi.ac.uk/metabolights/MTBLS13078.\nEthics statement\nThe animal studies were approved by the Institutional Animal\nCare and Use Committee of Liaocheng University. The studies were\nconducted in accordance with the local legislation and institutional\nrequirements. Written informed consent was not obtained from the\nowners for the participation of their animals in this study because the\nbirds were purchased from Aoxiang Poultry Industry Co., Ltd.,\nLiaocheng, Shandong Province, China.\nAuthor contributions\nYL: Conceptualization, Software, Funding acquisition, Writing –\nreview & editing, Resources, Investigation, Writing – original draft,\nProject administration, Supervision, Data curation, Formal analysis,\nMethodology. ZM: Software, Data curation, Writing  – review &\nediting, Conceptualization, Supervision, Investigation. YW: Data\ncuration, Methodology, Conceptualization, Software, Writing – review\n& editing, Investigation.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. This study was supported\nby the Doctoral Research Start-Up Fund of Liaocheng University\n(2024318052415).\nConflict of interest\nThe authors declared that the research was conducted in the\n\nThis study was supported\nby the Doctoral Research Start-Up Fund of Liaocheng University\n(2024318052415).Conflict of interest\nThe authors declared that the research was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. Any product\nthat may be evaluated in this article, or claim that may be made by its\nmanufacturer, is not guaranteed or endorsed by the publisher.\nSupplementary material\nThe Supplementary material for this article can be found online\nat: https://www.frontiersin.org/articles/10.3389/fvets.2025.1614917/\nfull#supplementary-material\nReferences\n1.\tZhao G, Wu H, Li Y, Li L, He J, Yang X, et al. Fermentation characteristics and\nmicrobial community composition of wet brewer's grains and corn Stover mixed silage\nprepared with cellulase and lactic acid bacteria supplementation. Animal Bioscience.\n(2024) 37:84–94. doi: 10.5713/ab.23.0177\n2.\tLi L, Xie X, Zhao G, He J, Zhang Y. 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+{"text": "【兽医文献】\n标题: A retrospective review of small intestinal intussusception in 126 cattle in Switzerland\n\nReceived: 12 October 2022\nAccepted: 21 November 2022\nDOI: 10.1002/vro2.53\nOR IG I NA L R E SE A RC H\nA retrospective study of choke (oesophageal obstruction) in \none-hump Dromedary camels (Camelus dromedarius) in Saudi\nArabia\nMohamed K. Zabady,\nTurke Shawaf\n1Department of Clinical Sciences, College of\nVeterinary Medicine, King Faisal University,\nAl-Ahsa, Saudi Arabia\n2Department of Surgery, Faculty of Veterinary\nMedicine, Cairo University, Giza, Egypt\nCorrespondence\nMohamed K. Zabady, Department of Clinical\nSciences, College of Veterinary Medicine, King\nFaisal University, PO Box 400, Al-Ahsa 31982,\nSaudi Arabia.\nEmail: mzabady@kfu.edu.sa\nFunding information\nDeanship of Scientific Reseach at King Faisal\nUniversity, Grant/Award Number: GRANT2141\nAbstract\nBackground: Choke (oesophageal obstruction) is an important oesophageal disorder in\nlarge domestic animals. Published studies on choke in the dromedary camel (Camelus\ndromedarius) are few in number and deal with small number of cases.\nMethods: Sixty-four camels with choke were presented to the Veterinary Teaching Hos-\npital, King Faisal University. History, breed, age, sex, duration of obstruction and clinical\nsigns were recorded. The diagnosis was established using examination with a stom-\nach tube, oesophageal radiography and endoscopy. Choke was managed either by using\nalligator forceps guided endoscope or by cervical oesophagotomy.\nResults: Choke was recorded in camels less than 1 year old (84.38%) more than camels\nmore than 1 year of age (15.62%) and complete obstruction more than partial. Most\ncases had obstruction involving the cervical oesophagus (96.87%). In the majority of\nobstructive masses, there were pieces of fabric (48.44%) and plastic bags (35.94%).\nMost obstructive masses were slightly radio-opaque (62.5%). Surgical and non-surgical\nmanagements were effective (91.3% and 94.44%, respectively) in resolving the choke.\nConclusions: Choke was most likely in animals less than 1 year of age with complete\nobstruction of the cervical oesophagus. Surgical and non-surgical methods were effec-\n\n3% and 94.44%, respectively) in resolving the choke.Conclusions: Choke was most likely in animals less than 1 year of age with complete\nobstruction of the cervical oesophagus.Surgical and non-surgical methods were effec-\ntive in resolving the choke in the dromedary camels. It was not practical to use forceps\nextraction in all adult camels due to the limited length of the alligator forceps.\nINTRODUCTION\nOesophageal obstruction has been described in cattle,1 horse,2\ndromedary camel calf3 and water buffalo.4 It has also been\ndescribed occasionally in sheep and goats.5 In cattle, food\nitems such as potato, beet root, radish, corn cobs and water-\nmelon rind, without sufficient mastication, have been asso-\nciated with oesophageal obstruction.6 In horses, choke may\nbe a sequel to intraluminal impaction with food material\nor ingesta.7,8 Choke has been described in South American\ncamelids.9,10 In alpaca and llama crias, vascular ring anoma-\nlies may present with clinical signs similar to those seen\nwith choke9; similarly with megaoesophagus in llamas.10 In\ndromedary camel calves, endoscopy has been reported as an\neffective method for dealing with oesophageal obstruction.11\nThere are limited numbers of published reports and case\ndescription of dromedary camels with choke12–15; including\ntwo small case series in calves from the authors’ institution.3,11\nThe aim of this study was to describe choke in dromedary\ncamels, including some observations on diagnostic methods,\nThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original\nwork is properly cited.\n© 2022 The Authors. Veterinary Record Open published by John Wiley & Sons Ltd on behalf of British Veterinary Association.\nthe nature of the obstructive masses; with some comments on\nthe methods of management.\nMATERIALS AND METHODS\nAnimals\nSixty-four dromedary camels (Camelus dromedarius) with a\ndiagnosis of choke were presented to the Veterinary Teach-\ning Hospital of the College of Veterinary Medicine-King Faisal\n\nMATERIALS AND METHODS\nAnimals\nSixty-four dromedary camels (Camelus dromedarius) with a\ndiagnosis of choke were presented to the Veterinary Teach-\ning Hospital of the College of Veterinary Medicine-King Faisal\nUniversity from 2009 to 2018. The history in all cases included\nregurgitation of food and/or milk. The breed (Majaheem,\nMagateer and Omani), age, sex and duration of clinical signs\nwere recorded. The age ranged from 10 days to 8 years; most\ncases were under 1 year old; consequently, age was designated\nas one of two groups (less than 1 year and more than 1 year\nold).\nThe type of oesophageal obstruction was designated\nas either complete or incomplete (partial) based on the\nVet Rec Open. 2022;9:e53.\nwileyonlinelibrary.com/journal/vro2\nof \nhttps://doi.org/10.1002/vro2.53\n\nof \nVeterinary Record Open\nhistory, oesophageal radiography and endoscopy. With com-\nplete obstruction, the history usually included sudden onset\nof dysphagia, regurgitation of food, milk and fluids, sali-\nvation and restlessness. With partial obstruction, there was\nregurgitation only of coarse and hard food. The dura-\ntion of clinical signs varied between 2–4 days (median 3\ndays) with complete obstruction and 1–5 months (median\n3 months) with partial obstruction. The clinical exam-\nination included passing a flexible stomach tube in all\ncases.\nRadiographic examination\nPlain oesophageal radiography was performed for all cases.\nAn oesophagogram was carried out in 30 camels using 200–\n300 ml and 500–600 ml barium sulphate solution (72%, w/v,\nwith water) in young and adult camels, respectively. The solu-\ntion was administered orally with a dose syringe or by stomach\ntube.\nEndoscopic examination\nEndoscopy was conducted in all camels under 1 year of age\n(N = 54) using a flexible endoscope (VetVu, a unit of Swiss\nPrecision Products), 8 mm diameter, 110 cm long, supported\nwith an inflation and irrigation system and light source.\nThe camel was secured in sternal recumbence and sedated\nwith xylazine 0.2 mg/kg intravenously (I/V) (Seton 2%; Lab-\noratorios Calier, S.A.C./Barcelonés, Barcelona-Espana). The\noral cavity of camel was kept open with a Gunther’s mouth\nGag (Eickeymeyer, Germany). The endoscope was inserted\nin the oral cavity, pharynx and oesophagus. Oesophageal\nfluid was aspirated with a surgical suction unit (New Askir,\nItaly). The oesophageal mucosa was examined and the lumen\nwas inspected for the nature of obstruction. The obstruc-\ntive area was washed through the channel of the endoscope\nusing 50 ml NaCl 0.9% solution to visualise the foreign\nbody.\nNon-surgical management (n = )\nAfter completion of the endoscopic examination, grasping\nalligator forceps (Eickeymeyer) with 80 cm long arms were\ninserted into the oral cavity and oesophagus to grasp the\nend of obstructive mass to attempt to remove it (Figure\nS1). The procedure was repeated up to five to six times\n\nThe procedure was repeated up to five to six times\ndepending on the type, size and location of obstructive mass.\nAfter removal of the obstructive mass, the oesophagus was\nflushed with 300 ml of saline to ensure patency of the\noesophagus.\nAfter the removal of the obstruction, camels received peni-\ncillin and streptomycin (Pen & Strep, Norbrook Laboratories,\nUK) intramuscularly (I/M) as 8 mg/kg procaine penicillin\nwith 10 mg/kg dihydrostreptomycin sulphate, equivalent to\n1 ml per 25 kg bodyweight, for 5 days; together with flunixin\nmeglumine (Finadyne solution, MSD Animal Health, North\nIreland) I/V, single daily dose of 1.1 mg/kg for 3 days. Camel\ncalves were given access to suckle milk after 12 h.\nSurgical management (n = )\nCervical oesophagotomy was carried out in 36 camels under\n1 year of age that had tightly impacted obstructive masses\nthat could not resolved with endoscopy and in 10 camels over\n1 year of age. The anaesthetic regimen comprised sedation\nwith xylazine 0.2 mg/kg I/V, followed by general anaesthe-\nsia with ketamine hydrochloride 10% (Ketamidor 10%, Richter\nPharma AG, Austria) 2 mg/kg I/V. Each camel was placed on\nits right side in lateral recumbence and a stomach tube was\nintroduced close to the obstruction. The distal third of the\nneck was prepared for surgery. A single incision (10 cm long)\nwas made at the ventrolateral aspect at skin and fascia; then,\nthe sternocephalicus and sternothyrohyoideus muscles were\nbluntly separated while preserving the jugular vein beneath\nthe sternocephalicus muscle. The oesophagus was located\ndorsolateral to the trachea and under the jugular vein; it was\nheld with two stay stitches. A longitudinal incision (5 cm\nlong) was made in the dorsolateral aspect of the oesoph-\nagus through the outer fibrous and muscular layers, then\nthrough the inner submucosal and mucosal layers, to expose\nthe lumen. Rochester-Carmalt forceps were used to extract\nthe obstructing foreign object (Figure S2a,b). The stomach\ntube was pushed towards the chest to check the patency of\nthe oesophagus and the surgical field was flushed using sterile\n\nRochester-Carmalt forceps were used to extract\nthe obstructing foreign object (Figure S2a,b).The stomach\ntube was pushed towards the chest to check the patency of\nthe oesophagus and the surgical field was flushed using sterile\nnormal saline solution. The oesophageal mucosa and sub-\nmucosa were sutured using simple interrupted pattern (in\nout–out in) using 1–0 polygalactine 910 (Surgicryl, Smi AG,\nHünningen 37 Belgium). The outer muscular and fibrous lay-\ners were opposed with a simple continuous pattern. Benzyl\npenicillin powder was dusted into the depth of the wound.\nThe fascia over the muscles was opposed using 1–0 polygalac-\ntine 910 in a simple continuous suture pattern. The skin was\nclosed using two silk (Lukens Medical, USA) in an interrupted\npattern.\nPostoperative care\nFood was withheld for 3 days and maintenance fluid ther-\napy (Lactate Ringer’s solution) was infused I/V at a rate of\n70 ml/kg/24 h. After 3 days, a soft diet such as milk was\nadvised. Camels received oxytetracycline (Tetracyn, Labora-\ntorios Argos, Santa Fe, Argentina) I/M, 20 mg/kg, every 72 h\nand flunixin meglumine I/V, once daily, 1.1 mg/kg, for 7 days.\nThe skin sutures were removed 14 days postoperation. The\ncases were followed up for 6 months via telephone.\nRESULTS\nChoke was more common in the Majaheem breed (67.19%; N\n= 43) than the Magateer and Omani breeds and in camels less\nthan 1 year of age (84.38%; N = 54) than camels more than 1\nyear (15.62%; N = 10) (see Table 1). The most common type of\nobstruction was complete (84.38%; N = 54).\nThe flexible stomach tube was valuable in determining the\nsite of obstruction in all cases. Attempts to push the obstruc-\ntive mass with the stomach tube always failed because it only\nmoved the mass several centimetres and then the mass was\nstuck in the oesophageal lumen.\n\nVeterinary Record Open\nof \nTA BL E \nThe number and percentage of breed, sex, age, type of\nobstruction, site of obstruction and nature of obstructive mass in 64\ndromedary camels with oesophageal obstruction\nItems\nVariable\nNo.\n%\nBreed\nMajaheem\n43\n67.19\nMagateer\n11\n17.19\nOmani\n10\n15.62\nSex\nMale\n26\n40.62\nFemale\n38\n59.38\nAge\n<1 year\n54\n84.38\n>1 year\n10\n15.62\nType of obstruction\nComplete\n54\n84.38\nPartial\n10\n15.62\nSite of obstruction\nCervical\n62\n96.87\nThoracic\n2\n3.13\nNature of obstructive mass\nPieces of fabric\n31\n48.44\nPlastic bags and ropes\n23\n35.94\nPhytobezoar\n6\n9.38\nTrichophytobezoar\n2\n3.12\nRubber material\n2\n3.12\nRadio-opacity of obstructive mass\nRadio-opaque\n8\n12.5\nSlightly radio-opaque\n40\n62.5\nRadiolucent\n16\n25\nManagement\nSurgical (n = 46)\nResolved cases\n42\n91.3\nComplicated cases\n4\n8.7\nNon-surgical (n = 18)\nResolved cases\n17\n94.44\nComplicated cases\n1\n5.56\nNote: Including radiographic findings and case outcome.\nOesophageal radiography revealed the site and the radio-\nopacity of the obstructive mass in all cases examined. Choke\nin the cervical oesophagus was more common that the tho-\nracic oesophagus (96.87%; N = 62; 3.13%; N = 2, respectively).\nIn terms of radio-opacity, the obstructive masses were radio-\nopaque, slightly opaque or radiolucent. Some obstructive\nmasses were radio-opaque (12.5%; N = 8) such as phyto-\nbezoars (Figure S3a), tightly impacted clothing fabric and\na piece of rubber. The majority of obstructive masses were\nslightly radio-opaque (62.5%; N = 40) including pieces of\nfabric (Figure S3b), trichophytobezoar (compacted hair and\nplant/food material) and some phytobezoars (compacted\nplant/food material). Some masses were radiolucent (25%; N\n= 16) (Figure S4a).\nIn all 30 cases, contrast oesophagram revealed that the\nbarium solution was retained at the site of obstruction in com-\nplete oesophageal obstruction, while in partial obstruction,\nincomplete filling of oesophageal lumen was observed (Figure\nS4b). Megaoesophagus was detected in one calf at the third\nintercostal space due to obstruction of the thoracic part of the\noesophagus. Endoscopy of camel calves assisted in recognis-\ning the nature of obstructive mass and assessing the integrity\nof oesophageal mucosa.\nNon-surgical management using the alligator forceps\n\nEndoscopy of camel calves assisted in recognis-\ning the nature of obstructive mass and assessing the integrity\nof oesophageal mucosa.Non-surgical management using the alligator forceps\nguided endoscope was successful in 17 camels under 1 year.\nThese camels had obstructive masses that were not firmly\nimpacted in the lumen of the oesophagus and were within\nthe range of the forceps. Inflammation of the oesophageal\nmucosa was observed in seven cases. One camel calf had an\noesophageal tear after removal of an obstructive mass and the\nowner advised to slaughter the animal. The tear was associated\nwith an attempt to retrieve an impacted trichophytobezoar.\nSurgical management (cervical oesophagotomy) was effec-\ntive in resolving choke of adult camels and calves with\ntightly impacted foreign bodies located beyond the length of\nthe forceps (N = 42; 91.30%). One adult camel had choke\nin the thoracic part of the oesophagus; this was resolved\nby oesophagotomy at the distal oesophagus and introduced\nlong forceps and endoscope from the oesophagotomy inci-\nsion. A further three camels developed complications with\noesophageal fistula in the second week postoperatively at\nthe oesophagotomy site. Food material discharged from the\noesophagotomy incision with phlegmon to the surrounding\ntissues.\nThe obstructive masses are listed in Table 1 (see Figure S5).\nDISCUSSION\nThe primary goal of treatment for choke is to relieve the\nobstruction with minimal trauma to the oesophagus and to\nlimit other complications. In keeping with previous reports,\nthe majority of the oesophageal obstructions in our study\noccurred in camel calves.3,11 This may be due to camel calves\ningesting inappropriate materials without adequate mastica-\ntion. Some greedy animals may bolt a mouthful of feed and\ntry to swallow it without chewing.16 The signs of oesophageal\nobstruction recorded were comparable to those reported in\ncamel calves,3,11 adult camels,12,14,15,17 llama,16 horses,7,18–20\ncattle1,6 and small ruminants.21,22\nBoth complete and partial oesophageal obstructions were\nrecorded in this study, similar to ruminants23 and llamas.16\n\n21,22\nBoth complete and partial oesophageal obstructions were\nrecorded in this study, similar to ruminants23 and llamas.16\nOne study only reported complete oesophageal obstruction\nin camel calves.3 Complete obstruction may be due to spasm\ninduced around the obstructing mass.24\nIn this study, the cervical part of the oesophagus was the\nmost common site of obstruction, especially its distal third,\ncranial to the thoracic inlet. In a previous report on camel\ncalves,3 the most common site was caudal to the thoracic inlet.\nFactors that may contribute to the location of the obstruction\nmay include the length of the cervical oesophagus, which is\nnearly twice that of the thoracic part. Moreover, oesophageal\ndiameter in the cervical portion is narrow (2.5 cm) compared\nwith thoracic portion (3.9 cm).25 Furthermore, camels have a\nlong vertical neck except at its distal third, where it becomes\nhorizontal to enter the chest; such a curvature may increase\nthe possibility of obstruction.3\nOesophageal radiography revealed that some obstructive\nmasses were radio-opaque or slightly opaque and could be\ndetected easily with plain radiography. Barium oesophagram\nwas useful for viewing radiolucent obstructive masses and\nevaluating the oesophageal condition. This is similar to horses\nwhere survey radiography of feed impaction or lodgment of\nmetallic foreign bodies can be a valuable diagnostic method.20\nPlain and contrast oesophageal radiography in horses19 and\ncattle6 can help to diagnose various oesophageal disorders.\nEndoscopic removal of oesophageal foreign bodies in the\ncamel calves was successful; similar to previous reports in\nHolstein bovine calves26 and camel calves.11 In adult camels,\n\nof \nVeterinary Record Open\nendoscopic removal of oesophageal foreign bodies could not\nbe performed because the length of alligator forceps was 80 cm\nand the oesophageal length is approximately 150 cm in adult\ncamels.17\nCervical oesophagotomy via a ventrolateral approach pro-\nvides access to the middle and distal cervical oesophagus\nand proved to be a convenient way for extraction of tightly\nimpacted oesophageal foreign bodies. The cervical part of the\ncamel oesophagus was most accessible to surgery given that\nit makes up over 60% of the total length of the oesophagus.\nIn horses, the cervical oesophagus constitutes over 50% of\nthe oesophageal length and a ventrolateral approach is more\ndesirable than a ventral approach due to the heavily devel-\noped ventral cervical musculature.20 Oesophagotomy has\nbeen described in camel calves,3 horses20 and goats.22 Three\ncamels developed an oesophageal fistula because of rapid\naccess to the food on the second day postoperatively, leading\nto food accumulation, infection at oesophagotomy incision\nand subsequent fistula formation. In horses, oesophageal\nfistula may result from healing of the oesophagotomy\nincision.27\nThe obstructive masses were similar to those reported in\nprevious studies of camels.3,12,13 It has been reported that pota-\ntoes and root crops occasionally cause choke in camels.3,28\nOne case was previously reported due to an injured and\nparalysed soft palate.29 Unlike in dromedary camels, the\noesophagus was frequently obstructed in cattle and horses by\nvarious feed items including apples, carrots, corn cobs and\nfibrous feed.6,23 Phytobezoars may cause choke in horses30\nand goats.22 This study has helped to extend the find-\nings of previous studies on choke in dromedary camels.\nIt also complements other studies of oesophageal disorders\nin dromedary camels which have, separately, reported that\nlesions of sarcocystosis are commonly found in oesophageal\ntissues.31\nAU T HOR C ON T R I BU T ION S\nMohamed K. Zabady collected the clinical cases, recorded the\nhistory, performed the examination, radiography, oesophago-\ntomy, wrote and revised the manuscript. Turke Shawaf com-\n\n31\nAU T HOR C ON T R I BU T ION S\nMohamed K.Zabady collected the clinical cases, recorded the\nhistory, performed the examination, radiography, oesophago-\ntomy, wrote and revised the manuscript.Turke Shawaf com-\npleted the examination with endoscope, wrote and revised the\nmanuscript.\nAC K NOW L E D G E M E N T S\nThe authors acknowledge the Deanship of Scientific Research\nat King Faisal University for their financial support under\nthe annual research (GRANT2141). The authors appreciate\nRamadan O. Ramadan, Ayman El-Nahas, Magdi Waheed\nand Abdul Wehab Elholabi for their kind cooperation in\nconducting this study.\nC ON F L IC T S OF I N T E R E ST\nThe authors declare that they have no conflicts of interest.\nDATA AVA I L A B I L I T Y STAT E M E N T\nThe data support the findings of this paper are available from\nthe corresponding author upon reasonable request. The data\nwill be anonymised and made available on reasonable request.\nET H IC S STAT E M E N T\nThe authors confirm that the ethical polices of the journal have\nbeen followed.\nORC I D\nMohamedK.Zabady\nhttps://orcid.org/0000-0002-7851-\n6280\nR E F E R E NC E S\n1. Scott PR, Penny CD, Macrae AI. Diseases of the digestive tract and\nabdomen. In: Cattle medicine. London, UK: Manson Publishing Ltd.;\n2011. p. 66–7.\n2. Chiavaccini L, Hassel DM. Clinical features and prognostic variables in\n109 horses with esophageal obstruction (1992–2009). J Vet Intern Med.\n2010;24(5):1147–52.\n3. Ahmed A. Esophageal obstruction in young camel calves (Camelus\ndromedarius). Res J Vet Sci. 2011;41:20–6.\n4. Marzok M, Moustafa A, El-Khodery S, Müller K. Esophageal obstruc-\ntion in water buffalo (Bubalus bubalis)—a retrospective study of 44 cases\n(2006–2013). Turk J Vet Anim Sci. 2015;39:233–40.\n5. Baird AN, Shipley CF. Oral-esophageal diseases. In: Pugh DG, Baird AN,\nEdmondson MA, Passler T, editors. Sheep, goat and cervid medicine. 3rd\ned. Elsevier; 2021. p. 59–61.\n6. Blowey RW, Weaver AD. Alimentary disorders. In: Color atlas of dis-\neases and disorders of cattle. 3rd ed. Edinburgh, UK: Mosby Elsevier;\n2011. p. 66–7.\n7. Feige K, Schwarzwald C, Furst A, Kaser-Hotz B. Esophageal obstruction\n\nSheep, goat and cervid medicine.3rd\ned.Elsevier; 2021.p.59–61.6.Blowey RW, Weaver AD.Alimentary disorders.In: Color atlas of dis-\neases and disorders of cattle.3rd ed.Edinburgh, UK: Mosby Elsevier;\n2011.p.66–7.7.Feige K, Schwarzwald C, Furst A, Kaser-Hotz B.Esophageal obstruction\nin horses: a retrospective study of 34 cases. Can Vet J. 2000;41(3):207–10.\n8. Sanchez LC. Disorders of the gastrointestinal system. In: Reed SM, Bayly\nWM, Sellon DC, editors. Equine internal medicine. 4th ed. St. Louis, MO,\nUSA: Elsevier; 2018. p. 796–800.\n9. McKenzie EC, Seguin B, Cebra CK, Margiocco ML, Anderson DE, Löhr\nCV. Esophageal dysfunction in four alpaca crias and a llama cria with\nvascular ring anomalies. J Am Vet Med Assoc. 2010;237(3):311–6.\n10. Watrous BJ, Pearson EG, Smith BB, Snyder SP, Blythe LL, Riebold TW,\net al. Megaesophagus in 15 llamas: a retrospective study (1985–1993). J Vet\nIntern Med. 1995;9(2):92–9.\n11. Shawaf T, Ramadan O, Elnahas A, Al Salman MF. Oesophagoscopy and\nendoscopic aided removal of esophageal foreign bodies in camel calves\n(Camelus dromedarius). J Camel Pract Res. 2017;24(1):35–9.\n12. Ramadan RO, Abdin-Bey MR. Obstruction of the esophagus in camels.\nIndian Vet J. 1990;674:363–4.\n13. Bolbol A. Esophageal ectasia in a female camel. In: Proceedings of the\nFirst International Camel Conference, Dubai. 1992. p. 419–21.\n14. Kuldeep S, Prem S, Ashok K. Esophageal obstruction in a camel: an\nunusual case. J Camel Pract Res. 2000;7:213.\n15. Singh P, Candoliya RK, Behi SM, Tayal R, Chawla SK, Bugalia NS. Sur-\ngical management of esophageal obstruction in camels—reports of 5\nclinical cases. J Camel Pract Res. 2011;18(2):347–9.\n16. Niehaus AJ, Mora DP. Digestive system and abdomen. In: Medicine and\nsurgery of camelids. 4th ed. Hoboken, NJ, USA: Wiley-Blackwell; 2022.\np. 336–90.\n17. Al-Ani FK, Qureshi A. The digestive system. In: Al-Ani FK, editor. Camel\nmanagement and diseases. Amman, Jordan: Al-Sharq Printing Press;\n2004. p. 197–218.\n18. Craig DR, Shivy DR, Pankowski RL, Erb HN. Esophageal disorders in\n61 horses. Results of nonsurgical and surgical management. Vet Surg.\n\nThe digestive system.In: Al-Ani FK, editor.Camel\nmanagement and diseases.Amman, Jordan: Al-Sharq Printing Press;\n2004.p.197–218.18.Craig DR, Shivy DR, Pankowski RL, Erb HN.Esophageal disorders in\n61 horses.Results of nonsurgical and surgical management.Vet Surg.\n1989;18(6):432–8.\n19. Sams AE, Weldon AD, Rakestraw P. Surgical treatment of intramural\nesophageal inclusion cysts in three horses. Vet Surg. 1993;22(2):135–9.\n20. Fubini SL. Esophagus. In: Auer JA, Stick JA, Kümmerle JM, Prange T,\neditors. Equine surgery. 5th ed. St. Louis, MO, USA: Elsevier; 2019.\np. 474–85.\n21. Anderson R, Bath GF, Thompson PN, Stadler MM. Oesophageal\nobstruction in Dorper ewes caused by impaction of a pelleted ration. J S\nAfr Vet Assoc. 2010;81(2):118–20.\n22. Sankar P, Kumar R, Lakshmi N. Surgical management of cervical\nesophageal obstruction in a goat (Capra hircus). Indian J Anim Res.\n2010;44:153–4.\n23. Rings DM. Esophageal obstructions/choke. In: Rings DM, Anderson DE,\neditors. Current Veterinary Therapy: Food Animal Practice. 5th ed. St.\nLouis, MO, USA: Saunders; 2009. p. 8–9.\n24. Jones SL, Blikslager AT. Disorders of the esophagus. In: Smith BP, Van\nMetre DC, Pusterla N, editors. Large animal internal medicine. 6th ed.\nSt. Louis, MO, USA: Elsevier; 2020. p. 3113–7.\n25. Sukon P, Timm K, Valentine B. Esophageal anatomy of llama (Lama\nglama). Int J Morph. 2009;27(3):811–7.\n\nVeterinary Record Open\nof \n26. Gomez DE, Cribb NC, Arroyo LG, Desrochers A, Fecteau G, Nichols\nS. Endoscopic removal of esophageal and ruminal foreign bodies in 5\nHolstein calves. Can Vet J. 2014;55(10):965–9.\n27. Stick JA. Surgery of equine esophagus. Vet Clin North Am Large Anim\nPract. 1982;4(1):33–59.\n28. Ramadan RO, Kock RA, Higgins AJ. Observations on the diagnosis and\ntreatment of surgical conditions in the camel. Br Vet J. 1986;142(1):75–89.\n29. Semieka M. Radiography of unusual foreign body in ruminants. Vet\nWorld. 2010;3(10):473–5.\n30. Orsini JA, Dikes N, Ruggles A, Charlton C, Perry R. Use of gastro-\ntomy to relieve esophageal obstruction in a horse. J Am Vet Med Assoc.\n1991;198(2):295–6.\n31. Metwally DM, Al-Otaibi TT, Al-Turaiki IM, El-Khadragy MF, Alajmi\nRA. Identification of Sarcocystis spp. in one-humped camels (Camelus\ndromedarius) from Riyadh and Dammam, Saudi Arabia, via histological\nand phylogenetic approaches. Animals. 2020;10(7):1108.\nSU PP ORT I NG I N F OR M AT ION\nAdditional supporting information can be found online in the\nSupporting Information section at the end of this article.\nHow to cite this article: Zabady MK, Shawaf T. A\nretrospective study of choke (oesophageal\nobstruction) in 64 one-hump Dromedary camels\n(Camelus dromedarius) in Saudi Arabia. Vet Rec\nOpen. 2022;9:e53. https://doi.org/10.1002/vro2.53\n\n---"}
+{"text": "【兽医文献】\n标题: article_207_The Use of Ultrasonography for Detection of Meniscal Damage \n\nDoes the Use of Intratesticular Blocks in Dogs \nUndergoing Orchiectomies Serve as an Effective \nAdjunctive Analgesic?\n\nA Knowledge Summary by\n\nErik Fausak MSLIS, MA, LVT, RLAT 1 \nElizabeth Rodriguez LVT, CVT 1 \nAnna Simle Student 1* \nNetwarat Merman Student MBA 1 \nDakota Cook Student 1\n\n1 Bel-Rea Institute of Animal Technology, 1681 S. Dayton St. Denver, CO 80247, USA  \n* Corresponding Author (asimle@gmail.com)\n\nISSN: 2396-9776 \nPublished: 10 Dec 2018 \nin: Vol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206 \nReviewed by: Dick White (BVetMed PhD DSAS DVR DipACVS \nDipECVS FRCVS) and Bruce Smith (BVSc MS \nFANZCVS DACVS) \nNext Review Date: Dec 10th 2020\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 2 of 15\n\nKNOWLEDGE SUMMARY\n\nClinical Scenario  \nWhile animals don’t appear to show a great deal of pain on our pain scale in post orchiectomies, does the use \nof intratesticular lidocaine work as a good adjunctive analgesic compared to not using it, and are there many \nsevere complications?\n\nSummary of the evidence\n\nSummary of the evidence\n\nStevens et al. (2013) \nPopulation: Shelter dogs at least 4 months of age (determined by dentition or \nrecord), in general good health without requiring additional \nprocedures and no signs of testicular or scrotal disease.  Mean age \nwas about 2 years old and average weight about 18 kg. \nSample size: 38 dogs were initially entered into the study but follow up data was \nlost on 5 dogs (they wanted 40 for a power of 0.8).  Dogs were \nseparated into two groups : \nPlacebo group 1 (n=16) and lidocaine/bupivacaine treatment group \n2 (n=17) \nIntervention details: All animals were induced and maintained with the same protocol: \nPremed: \nMorphine 0.5 mg/kg IM \nAcepromazine 0.025 mg/kg IM  \nInduction (20-45 minutes later): \nTiletamine and zolazepam 0.22 ml/kg \nIntubated \nMaintenance: \nPICO question \nIn dogs undergoing orchiectomy, does the use of intratesticular blocks reduce the pain in patients compared \nto not using intratesticular blocks? \nClinical bottom line \nLow level of evidence suggests that when compared to a pre-medication with pure-µ agonist opioids, \nintratesticular blocks do not appear to provide significant benefit.  However, based on our clinical scenario \nwhere pre-medication is with a partial-µ opioid like buprenorphine, there may be benefit in utilising \nintratesticular lidocaine or bupivicaine.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 3 of 15\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 3 of 15\n\nIsoflurane \nA certified veterinary technician recorded parameters and \ndocumented at 5 minute intervals.  Parameters included:  heart rate, \nrespiratory rate, mucous membrane colour, pulse oximetry, \nisoflurane concentration, and oxygen flow rate. \nA third party randomly assigned animals. \nGroup 1 (n=16) received an equivalent volume to body weight of \nsaline intratesticularly \nGroup 2 (n=17) received 1 mg/kg of bupivacaine and 1 mg/kg of \nlidocaine (combined) intratesticularly. \nEvery animal received 1/3 to 1/2 of the volume drawn up in each \ntesticle.  All parties involved in scoring were blinded to which \nreceived placebo or treatment by mixing volumes and delivering to \nall animals. \nIntratesticular technique:  22g 1 inch needles were used where the \nneedle was placed at the caudal pole of the testis and the needle \nwas directed toward the spermatic cord.  All syringes were aspirated \nand the needle was slowly pulled out while 1/3 to 1/2 of the syringe \nvolume was placed in each testicle. \nStandard castration was performed (technique not specified if \nprescrotal or scrotal) \nPost-operatively, every dog received 4.4 mg/kg carprofen SQ and \ndogs were evaluated for pain at 15 min, 1 hr, 2 hr, and 24 hr post-\noperatively.\n\nStandard castration was performed (technique not specified if \nprescrotal or scrotal) \nPost-operatively, every dog received 4.4 mg/kg carprofen SQ and \ndogs were evaluated for pain at 15 min, 1 hr, 2 hr, and 24 hr post-\noperatively.\n\nStudy design: Blinded randomised control trial \nOutcome studied: Perioperatively parameters were monitored and cremastor twitch \nresponse was recorded during the surgical procedure. \nDogs were evaluated for pain at 15 min, 1 hr, 2 hr, and 24 hr post-\noperatively.  Pain score evaluation was done utilising a modified VAS \npain scale used by Sammarco et al.  1996: \nScale was given on 6 criteria: comfort, movement, appearance, \nvocalisation, heart rate, and respiratory rate from a range of 0 – 12 \nwhere 12 is most painful. \nAny animal with a score equal or over six received rescue analgesia \nof either morphine (0.5 mg/kg IM) or tramadol (1-2 mg/kg PO). \nSites were also evaluated for bruising and swelling on a visual \nanalogue scale of 0-3, 3 being the most bruised or swollen.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 4 of 15\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 4 of 15\n\nMain findings: \n(relevant to PICO question): \n \n1/16 dog in the placebo group and 8/17 in the \nlidocaine/bupivacaine  did not demonstrate cremastor \ntwitch. \n \nNo statistical significance in any pain scorings between \ngroups (4/17 had successful (low) pain scores in \nlidocaine/bupivicaine group compared to 1-2/16 in control \ngroup. all groups had the exact same pain scores 120 \nminutes after surgery. \n \nSurgical bruising and edema did not have significance, group \n2 had one swelling score of 2 (highest level of the two \ngroups), but all other scores were similar.  Both groups did \nnot have bruising. \nLimitations: \n \nThe pain scale was not validated. \n \nRecovery times were not compared. \n \nBlood pressure was not recorded. \n \nIsoflurane setting was not recorded and compared \n \nNot sure what cremastor twitch is supposed to represent, \nthe efficacy of the intratesticular absorption of lidocaine and \nbupivacaine?  Or is this a reflection of pain response \nperioperatively? \n \nIf 40  dogs were needed for a good power (0.8), the sample \nsize falls well below that. \n \nEarly scores of analgesia, despite no statistical significance, \nwas clearly clinically relevant as they were two to four times \nbetter than the placebo group.\n\nRodriguez et al. (2016) \nPopulation: Client-owned dogs averaging 3 years with weights between 6 and 30 \nkg but averaging 18 kg. Exclusion criteria included cryptorchid, any \ntesticular abnormalities, brachycephalics, aggressive behaviour, or \nextreme anxiety, and any dogs that were not American Society of \nAnesthesiologists( ASA) anesthetic risk status of 1 or 2. \nSample size: 31 dogs\n\nExclusion criteria included cryptorchid, any \ntesticular abnormalities, brachycephalics, aggressive behaviour, or \nextreme anxiety, and any dogs that were not American Society of \nAnesthesiologists( ASA) anesthetic risk status of 1 or 2.Sample size: 31 dogs\n\nIntervention details: Group 1 (n=11)  intratesticular lidocaine at 2mg/kg (1 mg/kg for each \ntesticle) \nGroup 2 (n=10)  same dose of lidocaine, but intrafunicular instead of \nintratesticular. \nGroup 3 (n=10)  control group received saline equivalent to lidocaine \namount intratesticular.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 5 of 15\n\nAll animals were premedicated with: \nAcepromazine 0.2 mg/kg IM \nMeloxicam 0.3 mg/kg IV \nTramadol 3 mg/kg IV\n\nInduction and maintenance: \nKetamine induction 5 mg/kg and maintenance with ketamine \nboluses of 2.5 mg/kg.\n\n5 minutes after induction all animals had the scrotum aseptically \nprepared.  All animals received lidocaine (0.33mg/kg) SQ in the \nscrotal incision site. All groups had injections performed with 22g 1 \n1/2 “ needles. They were then subdivided into three groups: \nGroup 1 (n=11)  intratesticular lidocaine at 0.66 mg/kg of lidocaine in \neach testicle. Placed in the body of the testicle based on Mcmillan \n(2012). \nGroup 2 (n=10) same dose of lidocaine, but intrafunicular instead of \nintratesticular. Placed as close as possible to the external inguinal \ncanal from Suriano et al. (2014). \nGroup 3 (n=10) control group received saline equivalent to lidocaine \namount intratesticular. Placed in the body of the testicle based on \nMcMillan (2012). \nAll groups had sodium bicarbonate mixed with all injections at a ratio \n1:10 volume. \nDuring procedure rescue analgesics of fentanyl 5µg/kg IV was \ndelivered if ketamine was considered insufficient and the animal \nshowed significant changes in respiration, cardiovascular (heart rate \nor blood pressure), movement, and if vocalisation was present.\n\nStudy design: Randomised controlled, blinded trial. \nOutcome studied: Perioperatively monitored by veterinary staff with blood pressure \n(oscillometric), pulse oximetry, HR, and RR. \nTimes of monitoring T0 (5 minutes after induction), T1 (incision into \nscrotal body (left)), T2 (retraction of spermatic cord), T3 (pinching \nand cutting spermatic cord), T4 (incision into scrotum (right)), T5 \n(retraction of second testicle), T6 (Pinching and cutting of spermatic \ncord) recorded on excel spreadsheet.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 6 of 15\n\nMain findings: \n(relevant to PICO question): \nRespiratory, significant changes were noticed at T1 and T5.  \nIntrafunicular had the least change in respiration compared to the \nother techniques except the control group had the least change at \nT5. \nIn heart rate, the intratesticular group had the least changes except \nfor T2 where intrafunicular had the least change and intratesticular \nlidocaine had the greatest change. \nMean arterial pressure, the control group showed the least change \nin blood pressure with the exception of T2 where the intrafunicular \ngroup had the least change. \nKetamine and fentanyl were least supplemented in the lidocaine \nintratesticular (ketamine) and intrafunicular (fentanyl) groups and \nno significant difference between lidocaine intratesticular and \nintrafenicular groups were found, but there was a difference from \ncontrol groups. \nQualitative differences in terms of haematoma, bleeding, dysphoria, \nmovement on the table, and cremastor contraction were not \nsignificant.\n\nLimitations: \n \nThe graphs were difficult to read because they used percent \nchanges instead of absolute numbers.  \n \nDid no post-operative evaluation. \n \nNot described how the study was randomised\n\nLimitations: \n \nThe graphs were difficult to read because they used percent \nchanges instead of absolute numbers. \nDid no post-operative evaluation. \nNot described how the study was randomised\n\nMcMillan et al. (2012) \nPopulation: Client-owned 6 mos-8yo dogs receiving elective castration and \njudged healthy on physical exam by the lead investigator.  Exclusion \n– aggressive behavior, adverse reactions to NSAIDS, and previous \npainful condition.  \nSample size: 30 dogs \nIntervention details: All animals were premedicated with 0.03 mg/kg acepromazine, 0.02 \nmg/kg buprenorphine, and injected IM in cervical epaxial muscles.  \nAll animals had a 22 g catheter and 2-4 mg/kg propofol induction.  \nAll animals were intubated and maintained with isoflurane and put \non IV fluids at 10 ml/hr.  After induction, 4 mg/kg carprofen was \ngiven intravenously.  \nAfter intubation animals were prepared pre-scrotal castration, dogs \nwere assigned to two groups:\n\nLidocaine – 1 mg/kg lidocaine given into the body of each testes\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 7 of 15\n\nusing a one inch, 22 g needle. Lidocaine injection was stopped if \ntesticle was hard and firm.  \nControl – dogs received no additional treatment. \nPatients were warmed on a hot dog patient warming system.  Before \nskin closure lidocaine at 1mg/kg was administered as an incisional \nsplash block if they already received lidocaine.\n\nIsoflurane over 2.1% required rescue analgesia 1 µg/kg fentanyl IV, \nanimals were supposed maintained on 1.3% isoflurane.  \n0.02 mg/kg buprenorphine was given IV before patients were \ndischarged.\n\nIsoflurane over 2.1% required rescue analgesia 1 µg/kg fentanyl IV, \nanimals were supposed maintained on 1.3% isoflurane.0.02 mg/kg buprenorphine was given IV before patients were \ndischarged.\n\nStudy design: Randomised Control Trial \nOutcome studied: ECG, respired gasses, pulse oximetry, esophageal temperature, and \nDoppler blood pressure.  HR, RR, SBP, ETCO2, Iso concentration, and \nvaporizer setting was recorded.  \nT0 baseline \nT1 first skin incision \nT2  clamping first testicular pedacles \nT3 Clamping second testicular pedacles. \nT4 when 1st skin suture was placed \nUnblended postoperative pain scores were assessed.\n\nMain findings: \n(relevant to PICO question): \n \nNo statistically significant changes in isoflurane early on, but \nthe lidocaine group had lower isoflurane settings than the \nfirst group when castration was in process.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 8 of 15\n\n \nA spot of blood on all intratesticular injections and some \nhaematomas were noticed after testicular injection as \nnegative side effects of intratesticular injection.\n\n \nPost-operative pain scores were lowest in intratesticuar \nlidocaine groups 7/15 of the control required rescue \nanalgesia as opposed to 1/15 of the lidocaine group.\n\nLimitations: \n \nUnblended observation of pain score.\n\n \nInadequate blinding procedure with control group not \nreceiving any injection.\n\n \nStudent veterinarians doing the procedure may be \nunnecessarily longer compared to clinical practice.\n\nLimitations: \n \nUnblended observation of pain score. \nInadequate blinding procedure with control group not \nreceiving any injection. \nStudent veterinarians doing the procedure may be \nunnecessarily longer compared to clinical practice.\n\nPerez et al. (2013) \nPopulation: Healthy Male intact dogs from shelters aged 4mos-4 years old, \ngreater than 4.5 kg, Healthy based on physical exam, PCV and TP \nresults.  Aggressive and fearful animals were excluded.  Dogs with \nconditions to preclude epidural administration of drugs were also \nexcluded.  Surgeries greater than three hours or with a great deal of \ncomplications were excluded. \nSample size: 51 dogs \nIntervention details: All animals were premedicated with carprofen 4.4 mg/kg \nsubcutaneously, acepromazine 0.02 mg/kg, and hydromorphone 0.1 \nmg/kg were combined and given IM.  \nAll dogs were administered propofol induction 3.4 – 5.5 mg/kg and \nintubated.  All dogs were maintained with isoflurane, starting at 3% \nand adjusted accordingly.  \n3 treatment groups were arranged (randomisation of timestamps of \nanesthesia forms): \ncontrol group (n=17)– received nothing besides their premed and \ninduction with maintenance agents.  Saline was provided \nintratesticularly and epidurally. \nEpidural group (n=17) – morphine epidural was placed 0.1 mg/kg.  \nSaline was provided intratesticularly. \nIntratesticular group (n=17) – bupivacaine was injected 1 mg/kg \nintratesticularly. 22 g 2.2 cm needle was injected into the \nparenchyma of each testes after aspiration of each injection.  ½ the\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 9 of 15\n\n22 g 2.2 cm needle was injected into the \nparenchyma of each testes after aspiration of each injection.½ the\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 9 of 15\n\ndose was in each testicle.  Saline was provided epidurally. \nRescue analgesics (fentanyl 2 µg/kg) were provided intraoperatively \nwhen a noxious stimulus resulted in heart rate, respiratory rate, and \nmean blood pressure greater than or equal to 20% including \nanaesthetist experience.  \nPost-operative pain was evaluated by modified Glasgow pain scale \n(short form).  Pain was evaluated when the animal could raise its \nhead, or responded when incisional site is manipulated.  After initial \nexam, pain scores were evaluated at 1 hour and 4 hour interval. Pain \nscale higher than 5 received rescue analgesics of hydromorphone 0.1 \nmg/kg IV.  Dexmedetomidine would be delivered at 2 µg/kg if the \npain score did not go down and the animal would be removed from \nthe study.\n\nPain \nscale higher than 5 received rescue analgesics of hydromorphone 0.1 \nmg/kg IV.Dexmedetomidine would be delivered at 2 µg/kg if the \npain score did not go down and the animal would be removed from \nthe study.\n\nStudy design: Blinded Randomised Control Trial \nOutcome studied: SEM weight, age, anaesthesia duration and surgery duration were \nmeasured.  End Tidal Isoflurane was measured, time for epidural and \nintratesticular administration, rescue analgesics administered peri \nand post operatively.  Anesthetic values were utilised but not \npresented in study. Pain scores were recorded, as well. Serum \ncortisol was recorded in all patients prior to surgery, 15 minutes \nafter both testes were removed, and at 1 h and 4 h marks after \nextubation. \nMain findings: \n(relevant to PICO question): \nPain score at 0 and 1 hours were higher in the control group.  \nNo significant difference in anaesthetic values (HR, RR, Mean or \nsystolic pressures), no significant difference in HR or RR in post-\noperative group. Control group had higher blood pressures at the 25 \nminute intraoperative measurement.  \nSignificantly lower rescue analgesia occurred intraoperatively and \npostoperatively in both experimental groups.  Intratesticular group \nneeded a much larger quantity of post-operative rescue analgesia \nbut less intraoperatively compared to epidural anaesthesia. \n14/17 control group animals needed post-operative analgesia, 3/17 \nepidural group needed post-operative analgesia, and 7/17 in the \nintratesticular group needed post-operative analgesia.  \nSerum cortisol was significantly lower for the intratesticular group \nthan control.\n\nLimitations: \n \nCortisol is a stress hormone but may not be an accurate \nsurrogate outcome of pain.\n\n \nRandomisation was not well explained.\n\n \nAnaesthesia values were not displayed in the results.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 10 of 15\n\n \nRandomisation was not well explained. \nAnaesthesia values were not displayed in the results.Veterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 10 of 15\n\nHuuskonen et al. (2013) \nPopulation: Male client-owned dogs weighing on average 17.5 kg and average \nage 14.3 months.  Inclusion was normal testicular anatomy and \nrelatively healthy American Society of Anesthesiologists (ASA) status \ngrade 1.  Dogs were excluded if they needed additional procedures. \nSample size: 42  \nIntervention details: Animals were randomly assigned via random.org to: \nGroup L(n=19) – received intratesticular injection of 2% lidocaine at \n2 mg/kg receiving 1/3 of volume in each testis and 1/3 SQ in pre-\nscrotal surgical site. \nGroup S (n=23) – control group received intratesticular injection of \nsaline identical in volume to Group L \nAll injections were blinded to anaesthetist and surgeon, a syringe \nwas handed to the surgeon without their knowledge if it is saline or \nlidocaine.  All surgeons got a 23 x 1 1/4 inch needle to administer \ninjection.  \nAll animals were premedicated 30-45 minutes prior to induction with \n0.025 mg/kg acepromazine, 0.3 mg/kg morphine, mixed in the same \nsyringe and given IM lumbar and cervical epaxial muscles. \nAll animal were induced with 2-4 mg/kg propofol IV, to effect to \nallow endotracheal intubation.  All animals also received a \nmeloxicam (NSAID) dose of 0 .2 mg/kg IV after induction of \nanaesthesia. \nAll animals were maintained with isoflurane.  Procedures were \nstarted within 5 minutes of lidocaine injection.  Final year \nveterinary students performed procedure under supervision. \nStudy design: Double Blinded Randomised Control Prospective Study \nOutcome studied: Parameters monitored during the procedure every 5 minutes prior \nto surgery, every 3 minutes and during surgical events: \nintratesticular injection, first skin incision, exteriorisation of first \ntestes, tearing epididymis of first testes, and clamping spermatic of \nfirst testes cord, exteriorisation of second testes, tearing epididymis \nof second testes, and clamping spermatic of second testes: \nEnd-tidal isoflurane concentration\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 11 of 15\n\nCapnography \nHeart rate and rhythm \nArterial Oxygen saturation \nEsophageal temperature \nElectrocardiography \n12 dogs had systolic measured with Doppler and 30 dogs had \noscillometric with systolic, diastolic and mean pressures. \nPost-operative measurements were every half hour: \nShort form of Glasgow composite pain scale (6 or greater got rescue \nanalgesia of morphine and removed from further analysis)  - carried \nout by anaesthetist. \nMain findings: \n(relevant to PICO question): \n \nNo overall difference in heart rate \n \nMAP changed less in group L than S \n \nOverall, group L had lower heart rate and MAP scores \n \nRespiratory rate was significantly higher in group S at an \nearlier point than group L but both had similar respiratory \naverages \n \nOverall values were not dissimilar with the exception of \nfewer changes earlier in the procedure with the lidocaine \ngroup \n \n8/19 group L at later surgical times and 7/23 group S at \nearlier surgical times required additional propofol. \nIsoflurane was maintained at the same level in all cases with \nthe exception of one dog \n \nNo significant differences in intraoperative temperature. \n \n7/19 dogs in group L and 12/23 dogs in group S required \nrescue analgesia.\n\nIsoflurane was maintained at the same level in all cases with \nthe exception of one dog \n \nNo significant differences in intraoperative temperature. \n7/19 dogs in group L and 12/23 dogs in group S required \nrescue analgesia.\n\nLimitations: \n \nThe charts could have been better explained, particularly \ntable 1. \n \nBlood pressure measurement, Doppler and oscillometrics \nwere used that would cause an additional variable in values. \n \nANOVA (parametric) values showed no significance but \nwhen Bonferroni adjustments (non-parametric) were made, \nvalue was found.  It seems questionable application to make \nnumbers have value. \n \nStudent surgeons were doing the procedure which may not \nbe realistic to the clinical environment (in terms of \nprocedure duration) and lidocaine may have worn off before \nprocedure was complete. \n \nLidocaine duration was biggest hindrance in author’s view, \nwhere longer acting bupivacaine may have been preferred \nbut has risk of toxicity if given accidentally intravascularly.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 12 of 15\n\nAppraisal, application and reflection\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 12 of 15\n\nAppraisal, application and reflection\n\nExamination of the whole body of studies made the effect of lidocaine extremely inconsistent.  However, when \ntaking into account the type of premedication used in the studies, a clearer picture presented itself.  The most \nsignificant contribution to post-operative analgesia in routine castration of the dog and cat seems to be the \nanalgesic used in premedication.  In studies that used pure µ opioid agonists, there was less significance \nbetween control and intratesticular block groups in post-operative pain scores.  In all studies where a pure µ \nopioid agonist was not used, there was clinical significance in the use of intratesticular blocks (to favourable \neffect).  As our clinical scenario utilises buprenorphine, intratesticular lidocaine or bupivacaine may be \nindicated. \nAnother variable that should be further examined is the use of lidocaine versus bupivacaine as an \nintratesticular block.  The one study that utilised pure µ opioid premeds and had positive results was the study \nthat used bupivacaine instead of lidocaine as an intratesticular block.  While there is concern about \nintravascular bupivacaine toxicity, bupivacaine toxicity has not been noted in any of the two studies that \nutilised bupivacaine(Perez et al., 2013; Stevens et al., 2013).  \nA further limitation of these results is in our PICO, we were focusing on pain and the use of intratesticular \nblocks.  If this PICO were modified to isoflurane, propofol, and rescue analgesic sparing effect, our conclusion \nmay be different.  Only one study, measured isoflurane requirements based on different modalities of \nadjunctive analgesia(McMillan, Seymour and Brearley, 2012). \nOverall, blocks led to no significantly aversive events in any of the studies, it is relatively inexpensive, and fairly \neasy to administer.  In light of the low cost and minimal risk with inconsistent results, an argument could be \nmade that it is still worthwhile to administer in most clinical scenarios.\n\nIn light of the low cost and minimal risk with inconsistent results, an argument could be \nmade that it is still worthwhile to administer in most clinical scenarios.\n\nMethodology Section\n\nSearch Strategy \nDatabases searched and dates \ncovered: \nPubMed (1963 – 07/2017) \nVetMed Resource (CAB) (1973 – week 1 2017) \nCAB Abstracts on OVID Platform (1973- Week 1 2017) \nSearch terms: Orchiectomy AND intratesticular AND veterinary (pubmed 22 results \nand 9 relevant) , (dog OR cat) AND intratesticular (Pubmed 46 \nresults/5 relevant), lidocaine AND castration AND (dogs OR cats) \n(Pubmed 21/3 relevant), orchiectomy AND intratesticular (Vetmed \nResource and Cab Abstracts) 11 results/3 relevant\n\nDates searches performed: 17th July 2017\n\nExclusion / Inclusion Criteria \nExclusion: Anesthetic injections versus local blocks \nInclusion: English, French, Spanish, and Thai articles on intratesticular injection \nof local anesthetic to testicles.\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 13 of 15\n\nSearch Outcome \nDatabase \nNumber \nof \nresults \nExcluded – not \ninvolving lidocaine \nintratesticular \ninjection on dogs  \nExcluded – \nduplicate \nExcluded – not local \nanesthetic – lidocaine \nor bupivicaine \nTotal relevant \npapers \nPubmed \n89 \n77\n\n7 \n5 \nVetMed \nResource \n11 \n3 \n3\n\n0 \nCAB \nAbstracts \n11\n\n11\n\n0 \nTotal relevant papers when duplicates removed \n5\n\nCONFLICT OF INTEREST\n\nErik Fausak is a member of the editorial board of Veterinary Evidence. \nThis paper underwent a rigorous peer-review process as per our normal reviewing guidelines of inviting a \nminimum of two external reviewers. The identity of the Associate Editor handling the paper has not been \ndisclosed to the author. The final decision to accept this paper rested with the Editor-in-chief. \nAll other authors declare no conflict of interest.\n\nREFERENCES\n\nThe identity of the Associate Editor handling the paper has not been \ndisclosed to the author.The final decision to accept this paper rested with the Editor-in-chief.All other authors declare no conflict of interest.REFERENCES\n\n1. Huuskonen, V. et al. (2013) ‘Intratesticular lidocaine reduces the response to surgical castration in \ndogs’, Veterinary Anaesthesia and Analgesia, 40(1), pp. 74–82. doi: http://dx.doi.org/10.1111/j.1467-\n2995.2012.00775.x \n2. McMillan, M. W., Seymour, C. J. and Brearley, J. C. (2012) ‘Effect of intratesticular lidocaine on \nisoflurane requirements in dogs undergoing routine castration’, Journal of Small Animal Practice, \n53(7), pp. 393–397. doi: http://dx.doi.org/10.1111/j.1748-5827.2012.01233.x \n3. Perez, T. E. et al. (2013) ‘Effects of intratesticular injection of bupivacaine and epidural administration \nof morphine in dogs undergoing castration’, Journal of the American Veterinary Medical Association, \n242(5), pp. 631–642. doi: http://dx.doi.org/10.2460/javma.242.5.631 \n4. Rodriguez, A. R. Á., Gaviria, E. F. B. and Bonilla, D. F. E. (2016) ‘Evaluación del efecto analgésico de la\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 14 of 15\n\ndoi.org/10.2460/javma.242.5.631 \n4.Rodriguez, A.R.Á., Gaviria, E.F.B.and Bonilla, D.F.E.(2016) ‘Evaluación del efecto analgésico de la\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 14 of 15\n\nlidocaína vía intratesticular o intrafunicular en perros sometidos a orquiectomia electiva bajo un \nprotocolo de anestesia disociativa’, REDVET. Revista Electrónica de Veterinaria. Veterinaria \nOrganización, 17(9), pp. 1–16. \n5. SAMMARCO, J.L., CONZEMIUS, M.G., PERKOWSKI, S.Z., WEINSTEIN, M.J., GREGOR, T.P. and SMITH, \nG.K., 1996. Postoperative analgesia for stifle surgery: a comparison of intra‐articular bupivacaine, \nmorphine, or saline. Veterinary Surgery, 25(1), pp.59-69. doi: http://dx.doi.org/10.1111/j.1532-\n950X.1996.tb01377.x \n6. Stevens, B. J. et al. (2013) ‘Comparison of the effect of intratesticular lidocaine/bupivacaine vs. saline \nplacebo on pain scores and incision site reactions in dogs undergoing routine castration’, The \nVeterinary Journal, 196(3), pp. 499–503. doi: http://dx.doi.org/10.1016/j.tvjl.2012.11.019\n\nVeterinary Evidence \nISSN:2396-9776 \nVol 3, Issue 4 \nDOI: http://dx.doi.org/10.18849/ve.v3i4.206  \nnext review date: Dec 10th 2020 \np a g e | 15 of 15\n\nIntellectual Property Rights \nAuthors of Knowledge Summaries submitted to RCVS Knowledge for publication will retain \ncopyright in their work, and will be required to grant RCVS Knowledge a non-exclusive license \nof the rights of copyright in the materials including but not limited to the right to publish, re-\npublish, transmit, sell, distribute and otherwise use the materials in all languages and all \nmedia throughout the world, and to license or permit others to do so.\n\nDisclaimer \nKnowledge Summaries are a peer-reviewed article type which aims to answer a clinical \nquestion based on the best available current evidence. 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+{"text": "【兽医文献】\n标题: 10.1136_vetreco-2016-000179_Efficacy_of_recombinant_bovine_epidermal_growth_fa\n\nEvaluation of quality of life of carers of\nItalian spinoni with idiopathic epilepsy\nLuisa De Risio, Julia Freeman, Anita Shea\nTo cite: De Risio L,\nFreeman J, Shea A.\nEvaluation of quality of life of\ncarers of Italian spinoni with\nidiopathic epilepsy. Vet Rec\nOpen 2016;3:e000174.\ndoi:10.1136/vetreco-2016-\n000174\n▸Prepublication history for\nthis paper is available online.\nTo view these files please\nvisit the journal online\n(http://dx.doi.org/10.1136/\nvetreco-2016-000174).\nReceived 2 March 2016\nRevised 9 May 2016\nAccepted 28 May 2016\nNeurology/Neurosurgery\nService, Centre for Small\nAnimal Studies, Animal\nHealth Trust, Newmarket,\nSuffolk, UK\nCorrespondence to:\nDr Luisa De Risio;\nluisa.derisio@aht.org.uk\nABSTRACT\nThe carers of all UK Kennel Club registered Italian\nspinoni (IS) born between January 1, 2000 and\nDecember 31, 2011 were invited to participate in the\nstudy. The carers of 47 of 63 IS diagnosed with\nidiopathic epilepsy (IE) returned the questionnaire,\nwhich included numerous questions on various\naspects of IE including the effect of IE on the dog’s\ncarer’s quality of life. Median epileptic seizure number\nin the three months before study end or death was five\nepileptic seizures, 72 per cent of dogs had cluster\nseizures, 94 per cent of dogs were administered one or\nmore antiepileptic medications and 36 per cent of dogs\nwere euthanased due to poorly controlled IE. Seventy-\none per cent and 65 per cent of the participants were\nmoderately to extremely worried about the frequency\nand severity of their dog’s epileptic seizures,\nrespectively. Caring for an IS with IE caused conflict\nwith the carer’s work, education or daily activity often\nor very often in 50 per cent of the participants. Overall\nthe limitations on the carer’s life due to caring for an\nIS with IE were considered as very to extremely\nbothersome in 29 per cent of the participants, a little\nto moderately bothersome in 40 per cent of the\nparticipants and not at all bothersome in 31 per cent of\nthe participants.\nINTRODUCTION\nCanine idiopathic epilepsy (IE) has been\nrecently defined as two or more unprovoked\nepileptic seizures at least 24 hours apart, with\n\nINTRODUCTION\nCanine idiopathic epilepsy (IE) has been\nrecently defined as two or more unprovoked\nepileptic seizures at least 24 hours apart, with\nno identifiable underlying aetiology other\nthan a suspected genetic origin (Berendt\nand others 2015). The diagnosis of IE is cur-\nrently one of exclusion, and is made based\non\nthe\nage\nat\nepileptic\nseizure\nonset\n(between six months and six years), unre-\nmarkable interictal physical and neurological\nexaminations and exclusion of metabolic,\ntoxic and structural cerebral disorders by\nmeans of diagnostic investigations (De Risio\nand others 2015a). IE is the most common\nchronic neurological condition in domestic\ndogs, with an estimated prevalence of 0.6 per\ncent in the general canine population in the\nUK (Kearsley-Fleet and others 2013). Breed-\nspecific and country-specific prevalence of IE\nranges from 3.1 per cent to 18.3 per cent\n(Hülsmeyer and others 2015).\nCanine epilepsy can substantially reduce\nthe quality of life of the dog and can affect\nthe daily life of its carer (Packer and Volk\n2015). Caring for an epileptic dog is a life-\nlong emotional, time and financial commit-\nment. It involves regular administration of\nantiepileptic drugs (AEDs) (sometimes as\noften as every six to eight hours), maintain-\ning an accurate seizure diary, coping with\nand monitoring for adverse effects of the\nAEDs, emergency management of potentially\nlife-threatening\nepileptic\nseizure\nactivity\n(such as cluster seizures and status epilep-\nticus), regular veterinary check-ups as well as\nemergency\nvisits\nand\nassociated\nongoing\nfinancial costs. Epileptic seizures may be\nstressful to witness and manage, and may\nappear\nuncontrollable\n(Packer\nand\nVolk\n2015). Epileptic seizure occurrence is sudden\nand unpredictable, and long-term prognosis\nis\ndifficult\nto\npredict\nin\nthe\nindividual.\nTherefore the constant feeling of uncertainty\ncan cause emotional distress in the epileptic\ndog’s carer. In addition, the carer’s commit-\nment may not be matched by treatment\nsuccess (Potschka and others 2015) and this\nmay result in further frustration and stress.\nStudies in human medicine have shown that\ncaring for an epileptic child or adult has a\nsevere impact on the carer’s quality of life and\n\nStudies in human medicine have shown that\ncaring for an epileptic child or adult has a\nsevere impact on the carer’s quality of life and\npsychological health (Lv and others 2009,\nFerro and others 2012, Norup and Elklit\n2013, Reilly and others 2015).\nThere is limited veterinary literature on\nquality of life in epileptic dogs and their carers\n(Lord and Podell 1999, Chang and others\n2006, Wessmann and others 2012, Wessmann\nand others 2014) and no breed-specific study\nhas been performed. A previous study has\nidentified a set of key questions to evaluate\nquality of life in carers of dogs with IE\n(Wessmann and others 2014). There are breed-\nspecific\ndifferences\nin\nIE\nonset,\nclinical\ncharacteristics, epileptic seizure severity, disease\ncourse, response to antiepileptic treatment\nand survival (Hülsmeyer and others 2015).\nDe Risio L, Freeman J, Shea A. Vet Rec Open 2016;3:e000174. doi:10.1136/vetreco-2016-000174\n1\nCompanion or pet animals\n\nThese breed-specific IE features could affect the dog’s\ncarer’s perception of canine epilepsy and quality of life.\nThe authors have previously investigated the preva-\nlence, clinical characteristics and survival in Italian\nspinoni (IS) with IE (De Risio and others 2015b). The\naim of this study was to evaluate the quality of life of\ncarers of IS with IE.\nMATERIAL AND METHODS\nThe carers of all UK Kennel Club registered IS born\nbetween January 1, 2000 and December 31, 2011 were\nsent a letter inviting them to participate in the study,\nwhich involved two phases. The aim of phase I was to\nestimate the prevalence of IE in the IS in the UK (De\nRisio and others 2015b). Sixty-three (5.3 per cent) of\nthe 1192 IS whose carers responded to the phase I ques-\ntionnaire were diagnosed with IE based on revision of\nthe questionnaire and the primary veterinarian’s and,\nwhen available, the veterinary neurologist’s medical\nrecords. IE was defined as recurrent epileptic seizures\n(≥two epileptic seizures occurring >24 hours apart) with\nan onset between six months and six years of age in\ndogs with normal interictal physical and neurological\nexaminations, and results of haematology and biochem-\nistry\nprofiles\nwithin\nthe\nnormal\nreference\nranges.\nMedian follow-up time from first epileptic seizure to\nstudy end or death was 35 months (range, 5–124\nmonths) (De Risio and others 2015b). The carers of the\n63 IS diagnosed with IE were invited to complete a\nphase II questionnaire containing 110 questions on\nvarious aspects of epilepsy (De Risio and others 2015b)\nincluding the effect of IE on the dog’s carer’s quality of\nlife, and their perception of epileptic seizure severity,\nAED\nadverse\neffects\nand\nuse\nof\nrectal\ndiazepam\n(Table 1). The part of the questionnaire investigating\nthe epileptic dog’s carer’s quality of life and perspectives\non IE was developed based on a previously published\nveterinary study (Wessmann and others 2014). Epileptic\nseizure frequency was evaluated based on seizure diaries\nprovided by the epileptic IS’s carers. Cluster seizures\nwere defined as more than one epileptic seizure within\n24 hours with full recovery of consciousness between epi-\n\nEpileptic\nseizure frequency was evaluated based on seizure diaries\nprovided by the epileptic IS’s carers.Cluster seizures\nwere defined as more than one epileptic seizure within\n24 hours with full recovery of consciousness between epi-\nleptic seizures. Status epilepticus was defined as continu-\nous epileptic seizure activity lasting at least 5 minutes or\nat least two discrete epileptic seizures between which\nthere is incomplete recovery of consciousness. IS carers\nwere requested to provide information on AEDs admi-\nnistered, AED-related adverse effects, date and cause of\ndeath of their IS (when applicable).\nRESULTS\nData on the IS included in the study\nThe phase II questionnaire was returned for 47 IS.\nMean±sd age at onset of IE was 39±19 months (median\n35 months; range 11–72 months). Thirty-one (66 per\ncent) IS were male (of which 16 were neutered) and 16\n(34 per cent) were female (of which 11 were spayed).\nCluster seizures and status epilepticus occurred in 34\n(72 per cent) and 10 (21 per cent) of 47 IS included in\nthis study. Mean±sd number of epileptic seizures in the\nthree months before study end or death was 8±10\n(median 5 epileptic seizures, range 0–38 epileptic sei-\nzures). Only nine IS had been epileptic seizure-free for\nthree or more months before questionnaire completion\nor death. Information on epileptic seizure occurrence in\nthe three months before questionnaire completion or\ndeath was unknown in seven IS.\nThree (6 per cent) IS were not administered AEDs, 32\n(68 per cent) IS were treated with one or two AEDs and\n12 (26 per cent) IS were administered three or more\nAEDs. The numbers of IS administered phenobarbitone,\npotassium bromide, levetiracetam, imepitoin, gabapentin,\ntopiramate and zonisamide were 40, 26, 11, 2, 2, 2 and 1,\nrespectively. Of the 15 IS that received monotherapy, 14\nwere treated with phenobarbitone and 1 with imepitoin.\nAED-related adverse effects were reported in 36 (82\nper cent) of 44 IS on AED and persisted to some degree\nafter the first three weeks or three months (for potas-\nsium bromide) from AED initiation or dose increase in\n\nAED-related adverse effects were reported in 36 (82\nper cent) of 44 IS on AED and persisted to some degree\nafter the first three weeks or three months (for potas-\nsium bromide) from AED initiation or dose increase in\n30 (83 per cent) of 36 IS with adverse effects. The\nAED-related adverse effects most commonly reported\nwere polyphagia, polydipsia, weight gain, sedation and\nataxia.\nTwenty-two (47 per cent) IS were dead at the time of\ndata collection end. Of these 22 IS, 17 (77 per cent)\nhad been euthanased due to IE and 5 (23 per cent)\ndied for causes unrelated to IE. Mean survival of the 22\nIS that died was 31±27 months (median 27 months).\nMean survival of the 17 IS that were euthanased due to\nIE was 23±17 months (median 19 months). At the time\nof the carer filling out the phase II questionnaire, 3 IS\nwere alive and 19 IS were dead.\nEffect of IE on the IS carer’s quality of life\nThe answers of the 47 IS carers to the questions related\nto the effect of caring for an epileptic IS on their life\nare summarised in Table 1. Response rate to each ques-\ntion was incomplete ranging from 70 per cent to 96 per\ncent. When results are analysed as a per cent of the total\nnumber of participants for a specific question, caring for\nan IS with IE caused conflict with the carer’s work, edu-\ncation or daily activity often or very often in 50 per cent\n(20/40) of the participants and limited the carer’s social\nlife and independence often or very often in 36 per cent\n(14/39) and 41 per cent\n(16/39) of participants,\nrespectively. Overall the limitations on the carer’s life\ndue to caring for an IS with IE were considered as very\nto extremely bothersome in 29 per cent (13/45) of the\nparticipants, a little to moderately bothersome in 40 per\ncent (18/45) of the participants and not at all bother-\nsome in 31 per cent (14/45) of the participants. Only\n16 per cent (6/38) and 13 per cent (5/40) of the parti-\ncipants were not worried about epileptic seizure fre-\nquency and severity, respectively. Fifty-three per cent\n(20/38) and 48 per cent (19/40) of the participants\n2\n\nOnly\n16 per cent (6/38) and 13 per cent (5/40) of the parti-\ncipants were not worried about epileptic seizure fre-\nquency and severity, respectively.Fifty-three per cent\n(20/38) and 48 per cent (19/40) of the participants\n2\nDe Risio L, Freeman J, Shea A. Vet Rec Open 2016;3:e000174. doi:10.1136/vetreco-2016-000174\nOpen Access\n\nTABLE 1:\nSummary of questions and answers on the effect of caring for an epileptic IS on the carer’s life and their perception on epileptic seizure severity, AED adverse\neffects and use of rectal diazepam (data on 47 IS)\nQuestionnaire themes and questions\nEpileptic IS carer’s answers\nRestrictions on the carer’s life\nNever\nA few times\nSometimes\nOften\nVery often\nNot\nanswered\nIn the last three months, how often did you feel that caring for\nyour epileptic IS caused conflict with your work, education or\ndaily activities?\n12 (26%)\n4 (8%)\n4 (8%)\n6 (13%)\n14 (30%)\n7 (15%)\nIn the last three months, how often did you feel that caring for\nyour epileptic IS limited your social life?\n15 (32%)\n5 (11%)\n5 (11%)\n5 (11%)\n9 (19%)\n8 (17%)\nIn the last three months, how often did you feel that caring for\nyour epileptic IS limited your independence?\n19 (41%)\n1 (2%)\n3 (6%)\n6 (13%)\n10 (21%)\n8 (17%)\nFrustrations due to caring for an epileptic IS\nNot at all\nbothersome\nA little\nbothersome\nModerately\nbothersome\nVery\nbothersome\nExtremely\nbothersome\nNot\nanswered\nHow bothersome have the limitations on your work, education or\ndaily activities been due to caring for your epileptic IS?\n18 (38%)\n12 (26%)\n4 (9%)\n4 (9%)\n7 (15%)\n2 (4%)\nHow bothersome have the limitations on your social life been due\nto caring for your epileptic IS?\n17 (36%)\n11 (23%)\n5 (11%)\n5 (11%)\n7 (15%)\n2 (4%)\nOverall, how bothersome have the limitations on your life been\ndue to caring for your epileptic IS?\n14 (30%)\n12 (25%)\n6 (13%)\n7 (15%)\n6 (13%)\n2 (4%)\nCarer’s anxiety due to the seizures\nNot worried\nA little worried\nModerately\nworried\nVery worried\nExtremely\nworried\nNot\nanswered\nIn the last three months, how much have you been worried about\nthe frequency of the seizures in your epileptic IS?\n6 (13%)\n5 (11%)\n7 (15%)\n5 (11%)\n15 (32%)\n9 (19%)\nIn the last three months, how much have you been worried about\nthe severity of the seizures in your epileptic IS?\n5 (11%)\n9 (19%)\n7 (15%)\n1 (2%)\n18 (38%)\n7 (15%)\nCarer’s perception of seizure severity\nVery mild\nMild\nModerately\nsevere\nSevere\nVery severe\nNot\nanswered\nIn the last three months, the severity of your epileptic IS\nseizures has been\n0\n6 (13%)\n12 (25%)\n8 (17%)\n7 (15%)\n14 (30%)\nCarer’s perception of adverse effects of AEDs\nNot at all\nbothersome\nA little\nbothersome\nModerately\nbothersome\nVery\nbothersome\nExtremely\nbothersome\nNot\nanswered\n\nOverall how bothersome are the side effects of the AEDs on your\nepileptic IS?\n8 (18.2%)\n6 (13.6%)\n8 (18.2%)\n7 (15.9%)\n8 (18.2%)\n7 (15.9%)*\nCarer’s perception of rectal diazepam use for emergency\nmanagement of seizures at home\nNever\nA few times\nSometimes\nOften\nVery often\nNot\nanswered\nIf your vet advised to give rectal diazepam, have you ever been\nuncertain when to give it?\n14 (52%)\n2 (7%)\n7 (26%)\n1 (4%)\n1 (4%)\n2 (7%)†\nIf your vet advised to give rectal diazepam, have you ever been\nworried how much or how many times you are supposed to give\nit?\n16 (59%)\n1 (4%)\n6 (22%)\n1 (4%)\n1 (4%)\n2 (7%)†\n*AEDs were administered to 44 of the 47 IS\n†Rectal diazepam was used in 27 of the 47 IS\nAEDs, antiepileptic drugs; IS, Italian spinone\nDe Risio L, Freeman J, Shea A. Vet Rec Open 2016;3:e000174. doi:10.1136/vetreco-2016-000174\n3\nOpen Access\n\nwere very or extremely worried about epileptic seizure\nfrequency and severity, respectively. Epileptic seizure\nseverity was considered mild or moderate in 55 per cent\n(18/33) of the participants and severe to very severe in\n45 per cent (15/33) of the participants. Overall the\nadverse effects of the AEDs were considered very to\nextremely bothersome in 40 per cent (15/37) of the\nparticipants, a little to moderately bothersome in 38 per\ncent (14/37) of the participants and not at all bother-\nsome in 22 per cent (8/37) of the participants. Fifty-six\nper cent (14/25) and 64 per cent (16/25) of the partici-\npants knew when to administer rectal diazepam for\nemergency seizure management and were not worried\nabout diazepam dosage, respectively.\nDISCUSSION\nThis is the first study investigating the effect of caring\nfor an idiopathic epileptic dog focused on a specific\ncanine breed. There are breed-specific differences in\nthe prevalence, clinical characteristics and severity of\ncanine IE (Hülsmeyer and others 2015). The prevalence\nof IE in IS (5.3 per cent) is higher than in the general\ncanine population (0.6 per cent) in the UK, and IE in\nIS has a severe phenotype and low remission rate (De\nRisio and others 2015b). In the 47 IS included in this\nstudy, median epileptic seizure number in the three\nmonths before study end or death was five epileptic sei-\nzures, 72 per cent of dogs had cluster seizures, 26 per\ncent of dogs were administered three or more AEDs and\n36 per cent of dogs were euthanased due to poorly con-\ntrolled IE. Therefore it is not surprising that 71 per cent\nand 65 per cent, of the participants were moderately to\nextremely worried about the frequency and severity of\nthe epileptic seizures, respectively. Epileptic\nseizure\nseverity was perceived as moderately severe to very severe\nby the carers of 82 per cent IS. These results are in\nagreement with those of a previous study including 128\ndogs with IE from 42 breeds, in which 69 per cent and\n74 per cent of participants were moderately to extremely\nworried about the frequency and severity of the epileptic\nseizures, respectively (Wessmann and others 2012). In\n\nIn\nthis multibreed study, epileptic seizure frequency was\nhigher than a single epileptic seizure or cluster seizure\nevery three months in 63 per cent of the dogs, and epi-\nleptic seizure severity was considered moderate to severe\nby 67 per cent of the participants (Wessmann and\nothers 2012).\nCaring for an IS with IE caused conflict with the\ncarer’s work, education or daily activity and limited their\nsocial life and independence often or very often in 50\nper cent, 36 per cent and 41 per cent of participants,\nrespectively. A previous study including dogs of various\nbreeds selected from a referral population reported\nsimilar findings as 54 per cent (13/24) carers reported\nthat caring for an epileptic dog had caused them con-\nflicts with their work and day-to-day activities and 60 per\ncent (15/25) felt that caring for an epileptic dog had an\neffect on the organisation of their free time (Chang and\nothers\n2006).\nInterestingly,\nin\nthis\nprevious\nstudy,\napproximately 50 per cent of the carers indicated that\ntheir dog’s epilepsy had affected their ability to stay away\novernight as they were too worried to leave their epilep-\ntic dog, they were unable to find someone able to care\nfor an epileptic dog, or their dog usually had epileptic\nseizures when the carer was away or after return (Chang\nand others 2006). Conversely, in the only other veterin-\nary study which assessed quality of life in the epileptic\ndog’s carer, the carers felt that the work involved in\ncaring for an epileptic dog was not a burden or a sub-\nstantial detriment to their own quality of life (Lord and\nPodell 1999). This study included only 19 dogs of\nvarious breeds (of which 10 had IE and 9 had structural\nepilepsy). Of these 19 dogs, 12 (63 per cent) dogs had\nup to one epileptic seizure in 90 days, 3 (16 per cent)\ndogs had two epileptic seizures in 90 days and 4 (21 per\ncent) dogs had more than two epileptic seizures in\n90 days; phenobarbitone was the only administered AED\nand\nAED-related\nadverse\neffects\nwere\ninfrequent.\nOccurrence of cluster seizures and status epilepticus was\nnot mentioned (Lord and Podell 1999). In the present\n\nOccurrence of cluster seizures and status epilepticus was\nnot mentioned (Lord and Podell 1999).In the present\nstudy, median epileptic seizure frequency in all 47 IS was\nfive epileptic seizures in three months, cluster seizures\nand status epilepticus occurred in 72 per cent and 21\nper cent of IS, respectively, 61 per cent IS were adminis-\ntered two or more AEDs and AED-related adverse effects\npersisted in 83 per cent IS. Therefore the discrepancy in\nthe carers’ perception of their quality of life between\nthe present study and the study by Lord and Podell\n(1999) is likely to be due to differences in the dogs’ epi-\nleptic seizure frequency and severity, number of AEDs\nadministered and occurrence of AED-related adverse\neffects.\nThe adverse effects of the AEDs were considered mod-\nerately to extremely bothersome by 58 per cent of IS\ncarers who answered this specific question. Similarly, 49\nper cent (54/110) of participants in the study by\nWessmann were concerned about the adverse effects of\nthe AEDs (Wessmann and others 2012) and 58 per cent\n(7/12) of participants in the study by Chang considered\nAED-related adverse effects one of the reasons for a\ndecreased quality of life in their dogs. Prevalence and\ntype of adverse effects (e.g. polyphagia, polydipsia, sed-\nation and ataxia) were similar between the present study\nand\nthe\nstudy\nby\nChang\nand\nothers\n(2006).\nPhenobarbitone and/or potassium bromide were admi-\nnistered to all dogs included in the study by Chang and\nwere the most commonly used AEDs in the present\nstudy.\nAED-related adverse effects, epileptic seizure control\nand occurrence of status epilepticus have been reported\namongst the factors correlated with quality of life in\ncarers of children with epilepsy (Lv and others 2009).\nParents of children with epilepsy have a lower quality of\nlife and a higher incidence of anxiety and depression\nthan parents of healthy children (Lv and others 2009).\n4\nDe Risio L, Freeman J, Shea A. Vet Rec Open 2016;3:e000174. doi:10.1136/vetreco-2016-000174\nOpen Access\n\nThe levels of anxiety and depression are higher and the\nquality of life is lower in the parents of children with\npoorly controlled epilepsy compared with those of the\nparents of children with well-controlled epilepsy (Lv and\nothers 2009). In a prospective study of children with\nnew-onset epilepsy, 28 per cent of mothers without clin-\nically relevant levels of depressive symptoms at baseline\nwere at risk of clinical depression by 24 months after epi-\nlepsy onset (Ferro and others 2012). Parents of children\nwith epilepsy have to learn to cope with stress that is\nongoing, as epilepsy is a chronic illness and epileptic\nseizure\noccurrence\nis\nunpredictable\n(Duffy\n2011).\nParental adjustment to caring for a child with epilepsy\nwas negatively associated with the severity of the child’s\nepilepsy and positively correlated with satisfaction of\nsocial support (Pal and others 2002). Epileptic seizure\nfrequency and severity, AED-related adverse\neffects,\nanxiety and depression were amongst the variables\nplaying a significant role in the development of post-\ntraumatic stress disorder in partners living with an epi-\nleptic patient (Norup and Elklit 2013). High levels of\nsocial support decreased the level of traumatic stress\n(Norup and Elklit 2013).\nThirty-six per cent (17/47) of IS in this study were\neuthanased due to IE. This high mortality rate may\nreflect the severity of IE in IS as well as the impact of\nthe disease on the carer and the challenges in coping\nwith the demands of caring for an epileptic dog.\nOne limitation of this study is that response rate was\nnot 100 per cent for each of the questionnaire’s ques-\ntions, therefore the impact of each domain could be\nassessed in a subset of participants only and this may\nhave partly biased the results. The questions with the\nhigher response rate (96 per cent) were those to assess\nthe frustrations due to caring for an epileptic dog,\nwhereas the question about the carer’s perception of\nepileptic seizure severity achieved the lowest response\nrate (70 per cent). Another limitation of this study is\nthat the carers of 19 IS filled in the phase II question-\n\nAnother limitation of this study is\nthat the carers of 19 IS filled in the phase II question-\nnaire after their IS had died and this could have biased\ntheir responses.\nThis study did not evaluate various factors that could\nhave influenced the impact of IE on the dog’s carer\nsuch as the dog’s insurance status, the number of carers\ninvolved in the management of the epileptic dog, the\ncarer’s sex, age, physical and psychological health,\nemployment, financial, social, and education status, as\nwell\nas\npresence\nand\ntype\nof\nemotional\nsupport.\nInvestigating some of these aspects may have been per-\nceived as inappropriate or intrusive in the context of a\nveterinary study. The phase II questionnaire contained\n110 questions on various aspects of IE in IS and add-\nitional questions on the carer’s personal life may have\nnegatively affected the response rate. Participants were\nrecruited through the UK Kennel Club and did not\nmeet the investigators. This allowed a broad sample to\nbe obtained across the UK however there was not an\nestablished\npersonal\nrelationship\nbetween\nthe\ninvestigators and the dog’s carer which may have facili-\ntated obtaining additional information. The part of the\nquestionnaire investigating the epileptic dog’s carer’s\nquality of life was based on a previous study which identi-\nfied a set of key questions to evaluate quality of life in\ncarers of dogs with IE (Wessmann and others 2014).\nIn conclusion, the results of this study suggest that IE\nhas an impact in the majority of IS carers and the epi-\nleptic seizure frequency and severity are the greatest con-\ncerns.\nOptimising\ntreatment\nof\nIE\nand\nproviding\nadequate education on IE as well as emotional and logis-\ntic support to the carer is likely to improve quality of life\nof the epileptic dog’s carer.\nAcknowledgements The authors thank the Italian Spinone Club of Great\nBritain, the UK Kennel Club, and the owners, carers and veterinarians of the\nItalian spinoni who contributed to this study.\nContributors LDR designed and conducted the study and wrote the\nmanuscript. JF and AS assisted with data management and analysis and\nrevised the manuscript.\nCompeting interests None declared.\n\nContributors LDR designed and conducted the study and wrote the\nmanuscript.JF and AS assisted with data management and analysis and\nrevised the manuscript.Competing interests None declared.\nPatient consent Obtained.\nProvenance and peer review Not commissioned; externally peer reviewed.\nData sharing statement No additional data are available.\nOpen Access This is an Open Access article distributed in accordance with\nthe Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license,\nwhich permits others to distribute, remix, adapt, build upon this work non-\ncommercially, and license their derivative works on different terms, provided\nthe original work is properly cited and the use is non-commercial. See: http://\ncreativecommons.org/licenses/by-nc/4.0/\nREFERENCES\nBerendt M., Farquhar R. G., Mandigers P. G., Pakozdy A., Bhatti S. 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+{"text": "【兽医文献】\n标题: Multimodal digital sensing of early-life laying hens: a pilot study integrating thermal, acoustic, optical-flow and environmental data\n\nTYPE Perspective\nPUBLISHED 26 June 2026\nDOI 10.3389/fvets.2026.1796034\nOPEN ACCESS\nEDITED BY\nFrancisco Javier Salguero,\nUK Health Security Agency (UKHSA),\nUnited Kingdom\nREVIEWED BY\nNicole Dvorak,\nUniversity of Oxford, United Kingdom\n*CORRESPONDENCE\nWanxia Zhang\nzwx18789068092@163.com\nLong Chen\n19303029737@163.com\nRECEIVED 26 January 2026\nREVISED 24 May 2026\nACCEPTED 28 May 2026\nPUBLISHED 26 June 2026\nCITATION\nGuo W, Chen L, Chen L and Zhang W\n(2026) Animal models of\ndiabetes-related shoulder disorders.\nFront. Vet. Sci. 13:1796034.\ndoi: 10.3389/fvets.2026.1796034\nCOPYRIGHT\n© 2026 Guo, Chen, Chen and Zhang.\nThis is an open-access article distributed\nunder the terms of the Creative\nCommons Attribution License (CC BY).\nThe use, distribution or reproduction in\nother forums is permitted, provided the\noriginal author(s) and the copyright\nowner(s) are credited and that the\noriginal publication in this journal is\ncited, in accordance with accepted\nacademic practice. No use, distribution\nor reproduction is permitted which does\nnot comply with these terms.\nAnimal models of\ndiabetes-related shoulder\ndisorders\nWeifeng Guo1, Long Chen2*, Licheng Chen2 and Wanxia Zhang3*\n1Guangdong Provincial Second Hospital of Traditional Chinese Medicine, Guangzhou, China, 2Foshan\nHospital of Traditional Chinese Medicine, The Eighth Clinical Medical College of Guangzhou University\nof Chinese Medicine, Foshan, China, 3The First Dongguan Affiliated Hospital of Guangdong Medical\nUniversity, Dongguan, China\nDiabetic shoulder pathology constitutes a significant complication of diabetes\nmellitus, characterized by complex pathological mechanisms and a lack of\nsystematic research models. Existing animal models can simulate pathological\nprocesses such as frozen shoulder, rotator cuffrepair impairment, or natural\ndegeneration at different levels, yet they exhibit certain limitations. These include\ninconsistency with the metabolic background of human type 2 diabetes, a lack\nof dynamic observation of multi-tissue collaborative pathologies, and difficulty\nin replicating the natural clinical disease course. This article analyzes three\n\nThese include\ninconsistency with the metabolic background of human type 2 diabetes, a lack\nof dynamic observation of multi-tissue collaborative pathologies, and difficulty\nin replicating the natural clinical disease course.This article analyzes three\nmainstream animal models: the diabetes-combined frozen shoulder model, the\ndiabetes-combined rotator cuffinjury and repair model, and the type 2 diabetes-\ninduced natural degeneration model of the rotator cufftendon. By comparing the\nmodeling methodologies, pathological features, advantages, and disadvantages\nof these different models, potential improvements are proposed. An ideal diabetic\nshoulder model should simultaneously replicate the metabolic characteristics of\ntype 2 diabetes, simulate the progressive and collaborative pathology of multiple\nshoulder structures (tendon, joint capsule, subchondral bone), and incorporate\nbehavioral assessments for pain and functional impairment. Promoting a\nparadigm shift in animal models from “single-disease simulation” to “integrated\nsimulation” will provide an important platform for elucidating the pathogenesis\nof diabetic shoulder and developing novel therapeutic strategies.\nKEYWORDS\nanimal models, diabetes, frozen shoulder, rotator cufftendinopathy, shoulder disorders\nIntroduction\nDiabetes mellitus poses a significant global public health challenge, with its\ncomplications extending beyond traditional cardiovascular and renal diseases to include\nthe musculoskeletal system (1). Clinical studies indicate a substantially increased incidence\nof shoulder disorders among diabetic patients. Diabetes is recognized as a high-risk factor\nfor rotator cuffinjuries, with the prevalence of rotator cuffpathology ranging from\n10% to 30% and that of frozen shoulder (adhesive capsulitis) ranging from 20% to 30%\n(2, 3). These conditions severely impact patients’ quality of life and functional recovery,\nimposing a substantial healthcare burden (4). Shoulder pathology represents a common yet\nfrequently overlooked musculoskeletal complication of diabetes, manifesting as a complex\n\nThese conditions severely impact patients’ quality of life and functional recovery,\nimposing a substantial healthcare burden (4).Shoulder pathology represents a common yet\nfrequently overlooked musculoskeletal complication of diabetes, manifesting as a complex\nclinical picture that primarily includes rotator cufftendinopathy, adhesive capsulitis\n(frozen shoulder), and impaired tendon-to-bone healing (5–7). These disorders not only\nFrontiers in Veterinary Science\n01\nfrontiersin.org\n\nGuo et al.\n10.3389/fvets.2026.1796034\ncause shoulder pain, restricted mobility, and diminished quality of\nlife but also present a significant clinical challenge due to their often\ninsidious progression and suboptimal treatment response within\nthe hyperglycemic milieu (8). Although epidemiological research\nhas firmly established an association between diabetes and an\nelevated risk of shoulder disorders, the precise pathophysiological\nmechanisms underlying this link remain incompletely understood\n(9). It is widely accepted that persistent hyperglycemia disrupts\nthe structural and functional homeostasis of shoulder soft\ntissues—including tendons, the joint capsule, and the tendon-\nbone interface—through multiple interconnected pathways. These\ninclude the accumulation of advanced glycation end products\n(AGEs), chronic low-grade inflammation, microangiopathy, and\naberrant tissue remodeling (10–12). Research has demonstrated\nthat a hyperglycemic microenvironment impairs tendon-to-bone\nhealing following rotator cuffrepair, potentially mediated through\nmechanisms such as the let-7b-5p/CFTR pathway (13). Currently,\nexisting animal models lack a systematic understanding of the\ndiabetic shoulder as an integrated functional unit undergoing\nprogressive, multi-structural pathology. There is a particular\npaucity of experimental platforms capable of replicating the natural\nhistory of type 2 diabetes mellitus (T2DM) and dynamically\nobserving its synergistic effects on various shoulder structures,\nincluding tendons, the joint capsule, and subchondral bone.\nThis gap limits a holistic perspective on the pathogenesis of\ndiabetic shoulder and hinders the development of broad-spectrum\ntherapeutic strategies targeting common pathological mechanisms.\nA literature search was conducted in PubMed, Web of Science,\nand CNKI databases using keywords including “diabetes mellitus,”\n“shoulder,” “rotator cuff,” “frozen shoulder,” “tendinopathy,” and\n“animal model.” This search confirmed that only a limited number\nof in vivo studies have concurrently modeled diabetes and shoulder\npathology, highlighting a significant gap in the literature.\n\n” This search confirmed that only a limited number\nof in vivo studies have concurrently modeled diabetes and shoulder\npathology, highlighting a significant gap in the literature.\nConsequently,\ndeveloping\nanimal\nmodels\nthat\nintegrate\nmetabolic dysregulation with multi-structural pathological changes\nin the shoulder is crucial for advancing the investigation into\ndiabetic shoulder disorders. This passage aims to analyze and\nsystematically evaluate established animal models, focusing on\ntheir utility in elucidating the integrated pathology of diabetic\nshoulder and their potential to inform the development of\nmulti-target intervention strategies.\nDiabetes-combined frozen shoulder\nmodel (based on joint immobilization)\nModel establishment\nThe diabetes-combined frozen shoulder model is established\nusing a two-step protocol of “metabolic disturbance + mechanical\nrestriction,” simulating the clinical characteristic of diabetic patients\nbeing more prone to joint stiffness and fibrosis. The study by\nKim et al. (14) provides a standard paradigm for this model.\nAdult male Sprague Dawley rats are selected, and diabetes is\ninduced via intraperitoneal injection of streptozotocin (STZ) at\na dose of 65 mg/kg. Successful induction of the diabetic model\nis defined as a sustained blood glucose concentration exceeding\n300 mg/dL. Diabetes is successfully induced in rats 3 days\nafter STZ injection. Subsequently, under anesthesia (achieved by\nintraperitoneal injection of Zoletil 30 mg/kg and Rompun 10\nmg/kg), surgical immobilization of the right shoulder joint is\nperformed to induce stiffness: nylon sutures are passed through the\nscapula and looped around the humeral shaft to achieve complete\nimmobilization of the glenohumeral joint. The immobilization\nperiod lasts for 3 weeks, during which the left shoulder joint serves\nas an untreated internal control. After the immobilization period,\nthe sutures are removed. An intra-articular corticosteroid injection\nintervention group is added to the diabetic model cohort to further\ninvestigate therapeutic effects.\nPathological features and mechanisms\n\nAfter the immobilization period,\nthe sutures are removed.An intra-articular corticosteroid injection\nintervention group is added to the diabetic model cohort to further\ninvestigate therapeutic effects.Pathological features and mechanisms\nThe core pathological feature of this model is the synergistic\nworsening effect of diabetes and mechanical immobilization. In\nthe context of diabetes, joint immobilization not only leads to\na significant loss of shoulder joint range of motion but, more\ncritically, triggers progressive and irreversible fibrotic thickening\nof the joint capsule. The joint capsule area in the diabetic model\ngroup is increased compared to the non-diabetic group, resulting\nin reduced joint volume and mechanical restriction. Histologically,\nMasson’s trichrome staining reveals thickening of the joint\ncapsule accompanied by disordered collagen fiber arrangement,\nsignificantly increased density, and abnormal extracellular matrix\ndeposition, consistent with the pathological fibrosis driven by\nAGEs accumulation (14). Molecular mechanism studies indicate\nthat inflammation accompanied by fibrosis is a major pathological\nfeature of frozen shoulder. The binding of AGEs to their receptor\n(RAGE) activates the nuclear factor kappa B (NF-κB) pathway,\nexacerbating the local inflammatory response (15). Functional\nassessments reveal that the external rotation range of motion of\nthe shoulder joint in the diabetic group rats is reduced compared\nto the non-diabetic immobilized group, and the recovery rate is\nsignificantly slower. These findings are consistent with clinical\nobservations that diabetic patients have a higher risk of post-\ntraumatic joint stiffness and a longer recovery period (4). This\nmodel validates that corticosteroid intervention can effectively\nreverse this process. It not only simulates the pathological features\nof diabetic shoulder arthropathy but also reproduces key aspects\nof clinical treatment response, providing a solid foundation for\nclinical translation (16).\nAdvantages and limitations of the model\nThe core advantage lies in its successful simulation of key\n\nAdvantages and limitations of the model\nThe core advantage lies in its successful simulation of key\nclinical pathological features: the synergistic effect of hyperglycemia\nand mechanical restriction exacerbates fibrotic thickening of the\nshoulder joint capsule and loss of shoulder joint range of motion.\nThis provides a platform for studying the pathogenesis of diabetic\nshoulder and evaluating the efficacy of interventions such as\ncorticosteroids. However, this model has significant limitations: it\nis an “acute induction-acute immobilization” accelerated model,\nwhich shows a temporal discrepancy from the chronic and\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n\nGuo et al.\n10.3389/fvets.2026.1796034\ninsidious progression of the human disease; it focuses primarily on\nstiffness and generally lacks systematic assessment of pain-related\nbehaviors; and the STZ-induced type 1 diabetes model differs in its\nmetabolic basis from the clinical majority of patients with type 2\ndiabetes, limiting its applicability.\nDiabetes-combined rotator cuffinjury\nand repair model (based on surgical\nintervention)\nModel establishment\nThis model focuses on investigating the impact of diabetes on\nsoft tissue healing capacity. The rotator cufftransection and repair\nmodel established by Bedi et al. (12) simulates the repair challenges\nfaced by clinical diabetic patients using a two-step protocol of\n“metabolic disorder combined with surgical injury.”\nFirst,\ntype\n1\ndiabetes\nis\ninduced\nin\nmale\nLewis\nrats\nin the experimental group via intraperitoneal injection of\nstreptozotocin (STZ, 65 mg/kg), and the hyperglycemic state\nis\nconfirmed\nby\nglucose\ntolerance\ntesting.\nA\nnon-diabetic\ncontrol group was established by injecting citrate buffer only.\nSubsequently, both diabetic and non-diabetic control animals\nundergo unilateral supraspinatus tendon transection followed\nby immediate transosseous suture repair. Postoperative glycated\nhemoglobin (HbA1c) measurements are used to consistently\nconfirm the metabolic abnormality. Specimens are collected at\n1 and 2 weeks postoperatively for systematic evaluation of\nthe biomechanical strength, histomorphology, and molecular\npathological changes at the tendon-bone interface. This model\nsuccessfully reproduces the core pathological features of delayed\ntendon-bone healing and impaired mechanical performance under\ndiabetic conditions.\nPathological features and mechanisms\nThe core pathological feature of this model is a severe reduction\nin the mechanical strength of the tendon-bone interface and\nimpaired fibrocartilage formation. Histological analysis reveals\ndisordered collagen arrangement, incomplete fibrocartilage band\nformation, and reduced vascularization at the healing interface\nin diabetic animals (8). The underlying mechanism involves a\nvicious cycle driven by hyperglycemia: accumulation of AGEs\n\nThe underlying mechanism involves a\nvicious cycle driven by hyperglycemia: accumulation of AGEs\ninterferes with collagen assembly and activates the RAGE/NF-\nκB pathway, leading to chronic inflammation. This inflammatory\nmilieu ultimately impedes the normal formation of a functional\ntendon-bone interface.\nAdvantages and limitations\nThe primary advantage of this model lies in its accurate\nsimulation of the core clinical problem of impaired tendon-bone\nhealing in the context of diabetes. It provides stable and quantifiable\nbiomechanical and histological evaluation metrics, making it\nsuitable for mechanistic studies and therapeutic screening. Its\nlimitations are as follows: as an acute surgical model, its observation\nperiod is short and cannot reflect the chronic progression of the\nhuman disease; the STZ-induced type 1 diabetes model differs\nmetabolically from the more common clinical type 2 diabetes; and\nthe model focuses primarily on structural repair, with insufficient\nassessment of pain and functional behavioral outcomes.\nMetabolic-driven natural degeneration\nmodel of diabetic rotator cufftendon\nModel establishment\nThe natural progression model of type 2 diabetes-related\nrotator\ncuff\ntendinopathy,\ndeveloped\nby\nXu\net\nal.\n(17),\nsystematically simulates the progressive degeneration of the\nrotator cufftendon under the long-term influence of clinical\ntype 2 diabetic metabolic disorders. The model employs adult\nmale Sprague-Dawley rats and utilizes a protocol of “high-fat\ndiet-induced insulin resistance combined with low-dose chemical\ninjury” for modeling. Animals are first fed a high-fat diet for 4\nweeks to induce insulin resistance and a pre-metabolic syndrome\nstate. Subsequently, a low-dose intraperitoneal injection of\nstreptozotocin (40 mg/kg) is administered to impair pancreatic\nβ-cell function, establishing the core pathophysiology of type\n2 diabetes onset. Successful induction of diabetes is defined as\nthree consecutive measurements of fasting blood glucose ≥16.7\nmmol/L. After successful modeling, animals are maintained under\nhyperglycemic conditions without any surgical intervention to the\n\nSuccessful induction of diabetes is defined as\nthree consecutive measurements of fasting blood glucose ≥16.7\nmmol/L.After successful modeling, animals are maintained under\nhyperglycemic conditions without any surgical intervention to the\nshoulder joint. Serum and shoulder joint specimens are collected\nin batches at weeks 2, 4, 8, and 12. A comprehensive assessment\nis\nperformed\nusing\nenzyme-linked\nimmunosorbent\nassay,\nmultimodal histological staining, biomechanical tensile testing,\nand real-time quantitative PCR to evaluate the systemic impact\nof varying durations of diabetes on the structure, composition,\nmechanical properties, and molecular microenvironment of the\nsupraspinatus tendon. This model is the first to systematically\nconstruct, in a temporal dimension, the progressive pathology of\ndiabetes-induced rotator cufftendon degeneration, from early\ncollagen disorganization to late-stage structural disruption and\nmechanical failure.\nCore pathological mechanisms\nThe core pathological feature revealed by this model is the\nprogressive degeneration of the rotator cufftendon induced by\ndiabetes, characterized by time-dependent exacerbation of collagen\nstructure disruption, abnormal tissue remodeling, and loss of\nbiomechanical integrity. From sparse collagen arrangement and\nmicro-tears appearing 2 weeks post-modeling, it progresses to\nevident collagen rupture, scar hyperplasia, and neovascularization\nby week 12. Concurrently, the ultimate load of the tendon\ndecreases significantly starting from week 8, showing a significant\nnegative correlation with serum levels of AGEs. The intrinsic\nmechanism involves chronic abnormal remodeling driven by\nFrontiers in Veterinary Science\n03\nfrontiersin.org\n\nGuo et al.\n10.3389/fvets.2026.1796034\nthe hyperglycemic microenvironment: persistent hyperglycemia\nleads to AGEs deposition, which directly compromises tendon\nmechanical properties by disrupting collagen cross-linking and\nactivates inflammatory pathways. Simultaneously, it triggers a\ncascade of dysregulated repair responses, including upregulated\nexpression of pro-fibrotic proteins (e.g., Biglycan), increased\nactivity of matrix-degrading enzymes (e.g., MMP-3), release of pro-\nangiogenic factors, and aberrant expression of adipogenic genes\n(e.g., FABP4). These processes collectively establish a chronic, low-\ngrade pathological repair microenvironment. This disrupts the\nhomeostasis of the tendon extracellular matrix and prevents the\nmaintenance of normal mechanical structure, thereby simulating\nthe progressive degenerative process of human diabetic rotator\ncufftendinopathy.\nAdvantages and limitations\nThe prominent advantage of this model lies in its excellent\nclinical relevance: it accurately simulates the diabetic metabolic\nenvironment and provides a relatively complete representation\nof the progressive natural history of diabetic rotator cufftendon\ndegeneration, from early microscopic degradation to late-stage\nfunctional loss, offering an ideal window to elucidate the long-term\npatterns of disease onset and development. However, its significant\nlimitations are primarily reflected in the lengthy research cycle,\nhigh cost, and operational complexity, which impose considerable\ndemands on experimental resources. Furthermore, as a non-injury\nobservational model, it fails to incorporate key clinical events\nsuch as rotator cufftears, precluding the study of the interaction\nbetween injury and metabolic abnormalities. Moreover, in this\nmodel, the T2DM rats exhibited significantly lower body weight\nthan the control group, which may have reduced mechanical\nloading on the tendon and partially underestimated the true effect\nof hyperglycemia. Additionally, the inherent differences in shoulder\njoint mechanical loading between animals and humans remain a\npersistent translational bottleneck.\nDiscussion\nThe\nestablishment\nand\ndevelopment\nof\nanimal\nmodels\n\nAdditionally, the inherent differences in shoulder\njoint mechanical loading between animals and humans remain a\npersistent translational bottleneck.Discussion\nThe\nestablishment\nand\ndevelopment\nof\nanimal\nmodels\nfor diabetic shoulder pathology have evolved from simulating\nsingle pathological phenotypes to exploring complex metabolic-\nstructural interactions. The three core models reviewed in this\narticle—the “immobilization model” focusing on joint stiffness,\nthe “surgical model” focusing on repair impairment, and the\n“metabolic model” simulating natural degeneration—reveal the\nspecific damage inflicted by the hyperglycemic environment on\nshoulder joint tissues from different dimensions. Collectively,\nthese models validate the pathological characteristics of diabetic\nshoulder, demonstrating that persistent hyperglycemia disrupts\nshoulder\ntissue\nhomeostasis\nthrough\nmultiple\nmechanisms,\nincluding the AGEs-RAGE axis, chronic inflammation, and\nabnormal repair.\nHowever, the current model system exhibits certain limitations.\nFirstly, the simulated pathological features are relatively singular.\nThe “shoulder immobilization model” struggles to reflect rotator\ncuffdegeneration, while the “metabolic model” cannot simulate\nacute injury to shoulder tissues. This discrepancy does not align\nwith the clinical reality where patients often present with multi-\nstructural pathologies simultaneously. Secondly, most models\nutilize STZ-induced hyperglycemia, which differs fundamentally\nfrom type 2 diabetic patients in aspects such as insulin\nresistance and lipid metabolism disorders. Finally, existing research\npredominantly emphasizes structural degeneration, including\ntissue morphology and biomechanics, while neglecting functional\ndegeneration such as pain and functional impairment. This\noversight weakens the clinical translational value of these models.\nConsequently, future animal models for diabetic shoulder\nshould aim for integration as their objective. New models should\nemploy methods such as high-fat diets and genetic modification to\nsimulate the complete metabolic profile of type 2 diabetes. Building\n\nConsequently, future animal models for diabetic shoulder\nshould aim for integration as their objective.New models should\nemploy methods such as high-fat diets and genetic modification to\nsimulate the complete metabolic profile of type 2 diabetes.Building\non this foundation, interventions like surgery and joint fixation\nshould be combined to synchronously induce multiple pathological\nchanges, including joint capsule fibrosis, tendon degeneration,\nand impaired tendon-bone healing. Regarding model evaluation,\nwhile continuing to prioritize structural degeneration, such as\ntissue morphology and biomechanics, equal emphasis must be\nplaced on functional degeneration, including pain and functional\nimpairment, to construct a comprehensive assessment system.\nConclusion\nCurrent animal models for diabetic shoulder can simulate\nthe pathological process to a certain extent but are limited by\nthe singularity of the simulated pathological process and the\ndisconnection from the simulated metabolic background. Future\nefforts require the development of “integrated models” capable of\nsynchronously simulating the metabolic characteristics of type 2\ndiabetes, progressive multi-structural pathologies, and pain-related\nfunctional impairment.\nData availability statement\nThe original contributions presented in the study are included\nin the article/supplementary material, further inquiries can be\ndirected to the corresponding authors.\nAuthor contributions\nWG: Conceptualization, Data curation, Formal analysis,\nMethodology, Software, Validation, Writing – original draft,\nWriting – review & editing (most significant contributor). LoC:\nConceptualization, Data curation, Formal analysis, Methodology,\nSoftware, Validation, Writing – original draft, Writing – review\n& editing. LiC: Conceptualization, Data curation, Formal analysis,\nMethodology, Software, Validation, Visualization, Writing –\noriginal draft, Writing – review & editing. WZ: Conceptualization,\nFormal analysis, Methodology, Validation, Visualization, Writing –\noriginal draft, Writing – review & editing\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n\nGuo et al.\n10.3389/fvets.2026.1796034\nFunding\nThe author(s) declared that financial support was not received\nfor this work and/or its publication.\nConflict of interest\nThe author(s) declared that this work was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declared that Generative AI was used in the\ncreation of this manuscript. The author(s) confirm that language\npolishing tools (specifically Youdao Dictionary / NetEase Youdao)\nwere used to assist with sentence refinement and English expression\nin this manuscript.\nAny alternative text (alt text) provided alongside figures in\nthis article has been generated by Frontiers with the support of\nartificial intelligence and reasonable efforts have been made to\nensure accuracy, including review by the authors wherever possible.\nIf you identify any issues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. Any product that may be evaluated in this article, or\nclaim that may be made by its manufacturer, is not guaranteed or\nendorsed by the publisher.\nReferences\n1. Yameny AA. Diabetes mellitus overview 2024. J Biosci Appl Res. (2024) 10:641–5.\ndoi: 10.21608/jbaar.2024.382794\n2. Hsu CL, Sheu WHH. Diabetes and shoulder disorders. J Diabetes Investig. (2016)\n7:649. doi: 10.1111/jdi.12491\n3. Zreik NH, Malik RA, Charalambous CP. Adhesive capsulitis of the shoulder and\ndiabetes: a meta-analysis of prevalence. Muscles Ligaments Tendons J. (2016) 6:26–34.\ndoi: 10.11138/mltj/2016.6.1.026\n4. Struyf F, Mertens MG, Navarro-Ledesma S. Causes of shoulder dysfunction in\ndiabetic patients: a review of literature. Int J Environ Res Public Health. (2022) 19:6228.\ndoi: 10.3390/ijerph19106228\n5. Giha\nHA,\nSater\nMS,\nAlamin\nOA.\nDiabetes\nmellitus\ntendino-myopathy:\nepidemiology, clinical features, diagnosis and management of an overlooked diabetic\ncomplication. Acta Diabetol. (2022) 59:871–83. doi: 10.1007/s00592-022-01860-9\n\n(2022) 19:6228.doi: 10.3390/ijerph19106228\n5.Giha\nHA,\nSater\nMS,\nAlamin\nOA.Diabetes\nmellitus\ntendino-myopathy:\nepidemiology, clinical features, diagnosis and management of an overlooked diabetic\ncomplication.Acta Diabetol.(2022) 59:871–83.doi: 10.1007/s00592-022-01860-9\n6. Giri A, O’Hanlon D, Jain NB. Risk factors for rotator cuffdisease: a systematic review\nand meta-analysis of diabetes, hypertension, and hyperlipidemia. Ann Phys Rehabil\nMed. (2023) 66:101631. doi: 10.1016/j.rehab.2022.101631\n7. Liu C, Luo JW, Zhang KK, et al. Tendon-derived stem cell differentiation in\nthe degenerative tendon microenvironment. Stem Cells Int. (2018) 2018:2613821.\ndoi: 10.1155/2018/2613821\n8. Kim DH, Min SG, Kim HM, Kang HR, Choi JH, Lee HJ, et al. Comparison of\nthe characteristics of rotator cufftissue in a diabetic rat model. Orthopedics. (2022)\n45:e154–61. doi: 10.3928/01477447-20220128-08\n9. 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+{"text": "【兽医文献】\n标题: Effect of transport stress on apoptosis and autophagy in goat lung cells\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nOutbreak of recombinant lumpy\nskin disease virus in yaks: high\nmortality and systemic\npathogenesis in Qinghai-Tibet\nPlateau yak herds\nJianwu Hu 1,2†, Yan Li 1†, Xiaohu Zhang 1, Yaozhong Lu 1,\nZhibo Zeng 1, Mengen Xu 1, Siyang Mou 1, Shah Nawaz 1,\nDongjing Wang 3, Tianwu An 4, Xiaowei Li 5, Quan Mo 1* and\nJiakui Li 1,6*\n1 College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China, 2 Department of\nAgriculture and Rural Affairs of Tibet, Lhasa, China, 3 Institute of Animal Husbandry and Veterinary,\nTibet Autonomous Region Academy of Agricultural Sciences, Lhasa, China, 4 Sichuan Academy of\nGrassland Science, Chengdu, China, 5 Longri Livestock Breeding Farm, Aba Tibetan and Qiang\nAutonomous Prefecture, China, 6 College of Animals Husbandry and Veterinary Medicine, Tibet\nAgricultural and Animal Husbandry University, Linzhi, China\nThe emergence of recombinant Lumpy Skin Disease Virus (LSDV) strains in Asia\nhas led to outbreaks marked by severe skin nodules, high transmissibility, and\ntransboundary spread, resulting in significant economic losses to cattle industries in\nChina and neighboring countries. The Qinghai-Tibet Plateau, historically a natural\nbarrier against viral incursions, has recently experienced increasing LSDV cases in\nyaks (Bos grunniens). Current study elucidates the threat posed by recombinant\nLSDV strains to yaks through clinical, pathological, and molecular analyses. Field\nobservations revealed infected yaks exhibited fever, dyspnea, cutaneous pox\nlesions, lymphadenopathy, and mucosal lesions. Viral DNA detection showed\n100% positivity in skin samples (6/6), 53.33% (8/15) in nasal swabs, and 33.33%\n(5/15) in anal swabs, with an overall mortality rate of 46.67% (7/15). Necropsy\nidentified respiratory and digestive system lesions, including tracheal congestion,\npulmonary hemorrhagic plaques, and ruminal serosal hemorrhagic masses.\nHistopathology demonstrated dermal vasculitis, lymphocytic infiltration, and viral\ninclusion bodies. Immunohistochemistry localized viral antigens to hair follicle\n\nHistopathology demonstrated dermal vasculitis, lymphocytic infiltration, and viral\ninclusion bodies.Immunohistochemistry localized viral antigens to hair follicle\nepithelia and macrophages. Phylogenetic analysis positioned the yak-derived\nLSDV strain (LSDV/China/GS/Yak) within the Cluster 1.2 recombinant subclade\nwith high homology to recombinant strains circulating in East/Southeast Asia but\ndiffering from non-recombinant Indian Cluster 1.2 strains. The results emphasize\nincreased pathogenicity of recombinant LSDV in plateau yaks and convey the\ncritical need for region-specific control strategies.\nKEYWORDS\nLSDV, recombinant strain, Qinghai-Tibet Plateau, yak, prevention and control\nOPEN ACCESS\nEDITED BY\nVesna Milicevic,\nUniversity of Belgrade, Serbia\nREVIEWED BY\nDejan Bugarski,\nScientific Veterinary Institute Novi Sad, Serbia\nMilovan Milovanović,\nFriedrich-Loeffler-Institute, Germany\nAndrea Radalj,\nUniversity of Belgrade, Serbia\n*CORRESPONDENCE\nQuan Mo\nmoquan@mail.hzau.edu.cn\nJiakui Li\nlijk210@mail.hzau.edu.cn\n†These authors have contributed equally to\nthis work and share first authorship\nRECEIVED 28 February 2025\nACCEPTED 16 April 2025\nPUBLISHED 21 May 2025\nCITATION\nHu J, Li Y, Zhang X, Lu Y, Zeng Z, Xu M, Mou S,\nNawaz S, Wang D, An T, Li X, Mo Q and\nLi J (2025) Outbreak of recombinant lumpy\nskin disease virus in yaks: high mortality and\nsystemic pathogenesis in Qinghai-Tibet\nPlateau yak herds.\nFront. Vet. Sci. 12:1584975.\ndoi: 10.3389/fvets.2025.1584975\nCOPYRIGHT\n© 2025 Hu, Li, Zhang, Lu, Zeng, Xu, Mou,\nNawaz, Wang, An, Li, Mo and Li. This is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY). The use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\naccordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Original Research\nPUBLISHED  21 May 2025\nDOI  10.3389/fvets.2025.1584975\n\nHu et al.\b\n10.3389/fvets.2025.1584975\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n1 Introduction\nLumpy skin disease (LSD) is a transboundary infectious disease\ncaused by the lumpy skin disease virus (LSDV), primarily transmitted\nby insect vectors. It affects cattle including Water buffalo (Bubalus\nbubalis), giraffes (Giraffa camelopardalis), impalas (Aepyceros\nmelampus), Arabian oryx (Oryx leucoryx), and Indian gazelles\n(Gazella bennettii), among other species (1–6). In cattle, LSD is\ncharacterized by cutaneous nodules, fever, and systemic infection,\nleading to reduced milk production, compromised reproductive\nefficacy in bulls, and economic losses in beef cattle (7, 8).\nLSDV is a member of the genus Capripoxvirus within the\nsubfamily chordopoxvirinae (family poxviridae), along with the\nsheeppox virus (SPPV) and goatpox virus (GTPV) (9, 10). It is an\nenveloped, double-stranded DNA virus with a 151 kb genome, sharing\nover 95% nucleotide homology with SPPV and GTPV, and exhibiting\ncross-immunogenicity among capripoxviruses (11). Current\ncommercial vaccines against LSDV include homologous vaccines\n(attenuated Neethling field strains) and heterologous vaccines (live-\nattenuated GTPV and SPPV) (1). Its vaccination is very effective and\nprovides potent protection from specific pathogens, but noteworthy\nis that mild “Neethling-like” signs can appear after immunization.\nWhile generally benign and harmless, there is valid reason to have\nreservations concerning the safety profile of live-attenuated\nimmunization overall. It is therefore essential in this context to closely\nmonitor the situation, followed by thorough investigation to improve\nimmunization strategies in the future (1, 12).\nHistorically, LSD was endemic to sub-Saharan Africa until the\n1990s (12, 13). The first African outbreaks outside the continent were\nreported in the Middle East in 1989, followed by sporadic cases (14–\n16). By 2014, LSD had spread to Balkan countries (17–19). In 2017, a\nvaccine-escape recombinant LSDV strain—derived from genetic\nrecombination between Neethling-like vaccine strains and field\n\nBy 2014, LSD had spread to Balkan countries (17–19).In 2017, a\nvaccine-escape recombinant LSDV strain—derived from genetic\nrecombination between Neethling-like vaccine strains and field\nisolates—was identified in Russia near the Kazakhstan border (20).\nOver the past decade, LSDV has expanded into Europe and Asia, now\ncirculating in East, South, and Southeast Asia (21, 22). Notably, two\ndistinct LSDV lineages co-circulate in Asia: (1) recombinant vaccine-\nlike strains (e.g., in China, Mongolia, Thailand, and Vietnam) and (2)\nclassical field strains (e.g., in Bangladesh, India, Nepal, and Myanmar)\n(18, 23–29). This dual viral pressure highlights the ongoing threat to\nAsian cattle populations.\nYaks (Bos grunniens), a cold-adapted ruminant species with a\nglobal population exceeding 20 million, are predominantly (>95%)\ndistributed across the high-altitude (3,000–5,000 m), hypoxic regions\nof China’s Qinghai-Tibet Plateau (30, 31). Dubbed the “ship of the\nplateau,” yaks serve as a critical livestock resource, providing meat,\nhides, and dairy products for local communities (32). LSDV outbreaks\nhave inflicted severe economic losses on global cattle industries (8).\nGiven the harsh environmental conditions and limited veterinary\ninfrastructure in yak habitats, the introduction of LSDV presents a\ncatastrophic risk to yak farming on the plateau. The correspondence\nof heightened susceptibility among yak populations and the high\ntransmissibility of recombinant LSDV strains requires in-depth\nunderstanding of viral pathogenesis in this novel host species. This\nstudy investigates the clinicopathological features and molecular\nepidemiology of LSDV infections in Qinghai-Tibet Plateau yaks,\nunderscoring the escalating threat to high-altitude ecosystems and the\nurgent need for cross-border surveillance. Longitudinal research\ninvolving next-generation molecular tools, including whole-genome\nsequencing and phylogenetic analysis, is essential to track viral\nevolution and the development of new recombinant strains to\nimplement preventive disease management and control strategies in\nthis vulnerable ecosystem.\n\n2 Materials and methods\n2.1 Sample collection and viral DNA\nextraction\nIn 2023, samples were collected from 15 yaks (Bos grunniens)\nexhibiting cutaneous nodules or pox-like lesions at a farm in Gannan\nTibetan Autonomous Prefecture, China, suspected of being infected\nwith LSDV. Samples included blood, nasal and anal swabs (15 from\neach animal), and 6 skin tissues. Nasal and anal swabs were\nimmediately placed in 800 μL phosphate-buffered saline (PBS), while\nblood specimens were permitted to stay at room temperature for 2 h\nto obtain serum and were subsequently centrifuged at 6,000 rpm for\n5 min to discard coagulated red cells. Specimens were shipped to the\nlaboratory under cold chain conditions to −20°C and were preserved\nat −80°C until further handling.\nApproximately 1 g of skin tissue was homogenized in 1 mL PBS\nusing an Automatic Sample Fast Grinder (Jingxin, Shanghai, China).\nThe homogenate, along with other samples, was centrifuged at\n9,000 rpm for 3 min at 4°C. Viral DNA was extracted from 400 μL of\neach sample using the VAMNE Magnetic Pathogen DNA/RNA Kit\n(Prepackaged) (Nanjing Vazyme, Nanjing, China) following the\nmanufacturer’s protocol.\n2.2 qPCR detection of LSDV DNA\nLSDV DNA was detected using quantitative polymerase chain\nreaction (qPCR) with primers targeting the GPCR gene (19, 33),\nmodified as follows: Forward primer: 5′-agtcgaatataaagtaatcagtc-3′,\nReverse primer: 5′-ccgcata-taatacaacttattatag-3′. Each 20 μL\nreaction contained: 10 μL Taq Pro Universal SYBR qPCR Master\nMix (Nanjing Vazyme, Nanjing, China), 0.4 μL each of forward and\nreverse primers (10 μM), 2 μL template DNA, 7.2 μL distilled\nH₂O. Thermal cycling conditions: 95°C for 30 s (pre-denaturation),\n40 cycles of 95°C for 5 s and 60°C for 30 S. Melt curve analysis was\nperformed using the default settings. All reactions were performed\non a LightCycler® 96 Instrument (Roche Diagnostics, Basel,\nSwitzerland) Melt curve analysis was performed using the\ndefault settings.\n2.3 Histopathology and\nimmunohistochemistry analysis\nFresh nodule tissues were fixed in 10% neutral buffered\n\n2.3 Histopathology and\nimmunohistochemistry analysis\nFresh nodule tissues were fixed in 10% neutral buffered\nformalin, washed with PBS, dehydrated through graded ethanol\n(70, 95, 100%), cleared in xylene, embedded in paraffin, and\nsectioned at 3 μm. Sections were stained with hematoxylin and\neosin\n(H&E)\nfor\nhistopathological\nanalysis.\nFor\nimmunohistochemistry (IHC), adjacent sections were incubated\n\nHu et al.\b\n10.3389/fvets.2025.1584975\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nwith mouse anti-LSDV126 protein monoclonal antibody\n(generously provided by Prof. Yuefeng Sun, Lanzhou Veterinary\nResearch Institute) following established protocols (34). Slides were\nscanned using an automated slide scanner (3DHistech, Budapest,\nHungary) and visualized with SlideViewer v2.7.1\n2.4 Whole-genome sequencing\nThe whole genome sequencing was performed on the Illumina\nNovaSeq 6,000 PE150 platform. Raw reads were quality-controlled\nusing Trimmomatic v0.39 (35), and clean reads were mapped to the\nLSDV\nreference\ngenome\n(China/GD01/2020;\nGenBank:\nMW355944.1) using Bowtie2 v2.5.2 (36). Contigs were assembled\nusing MEGAHIT v1.2.9 (37) and further polished with SeqMan\nmodule in DNASTAR Lasergene v112 to generate complete genomes.\nWe have submitted the genomic sequences to the GenBank database,\nGenBank No. MW355944.1.\n2.5 Phylogenetic analysis\nFull-genome, GPCR, and RPO30 sequences of LSDV, sheeppox\nvirus (SPPV), and goatpox virus (GTPV) were retrieved from the\nNCBI database.3 Sequence alignment was performed using MAFFT\nv7.505 (38) with the FFT-NS-1 (fast) strategy and default alignment\nparameters within the PhyloSuite v1.2.3 software package (39). The\nbest substitution model was selected via ModelFinder v2.2.0 (BIC\ncriterion) (40). Maximum likelihood trees were constructed using\nIQ-TREE v2.2.0 (1,000 bootstrap replicates) (41) and visualized using\nChiplot4 (42).\n3 Results\n3.1 Field observations\nObserved clinical signs of LSDV infection of yaks were tachypnea,\nhaemorrhagic diarrhea, fever, and skin lesions of characteristic\npox-like appearance. The lesions were most prominent on hairless or\nlightly covered skin surfaces, i.e., nasal planum, ventral cervical\nregion, perineum, and the inguinal region (Figures 1A–C). Initial\nlesions manifested as erythematous skin patches (Figure  1A),\nprogressing to raised nodules (Figure 1B), and eventually forming\nscabs that shed, leaving scars (Figure 1C). Enlarged, firm superficial\ncervical lymph nodes (lymphadenopathy) were observed in some yaks\n\nEnlarged, firm superficial\ncervical lymph nodes (lymphadenopathy) were observed in some yaks\n(Figure  1D). Soft periocular swelling (Figure  1E), hoof lesions,\ngingival mucosal erythema, and conjunctival hyperemia were noted\nin individual cases (Figures 1F–H).\n1  https://www.3dhistech.com/research/software-downloads/\n2  https://www.dnastar.com/software/lasergene/\n3  http://www.ncbi.nlm.nih.gov\n4  https://www.chiplot.online/tvbot.html; accessed on December 1, 2024.\n3.2 Viral detection and necropsy findings\nSamples were collected from 15 yaks displaying typical LSDV\nsymptoms. qPCR detected LSDV DNA in 53.33% (8/15) of nasal swabs,\n33.33% (5/15) of anal swabs, and 100% (6/6) of skin tissues. Seven yaks\nwith severe clinical signs died, resulting in a mortality rate of 46.67%\n(7/15) while the morbidity rate was recorded as 100% (15/15).\nPostmortem examination revealed lesions predominantly in the\nrespiratory and digestive systems. The trachea exhibited congestion,\nhemorrhage, and excessive mucus (Figure 2A). The lungs displayed\npatchy hemorrhagic foci protruding from the serosal surface\n(Figure  2B). The rumen serosa showed multifocal hemorrhagic\nnodules resembling pox lesions (Figure 2C). Hemorrhage was also\nobserved in the jejunal serosa, mirroring ruminal findings\n(Figure 2D).\n3.3 Histopathology and\nimmunohistochemistry\nH&E staining identified epidermal-dermal edema with disrupted\nrete ridge architecture (Figure 3A). Severe vasculitis in the dermis\nfeatured perivascular lymphocyte infiltration, vascular occlusion, and\neosinophilic viral inclusion bodies in affected keratinocytes\n(Figure 3B). Mid-dermal layers showed extensive lymphocytic and\nerythrocyte infiltration (Figure 3C). Deep dermal regions exhibited\nmarked arteriolar vasculitis, endothelial necrosis, and luminal\nobstruction (Figure 3D).\nImmunohistochemistry using anti-LSDV126 antibody highlighted\nviral antigen (brown staining) in hair follicle epithelial cells\n(Figure 4A), deep dermal macrophages (Figure 4B), and perivascular\nlymphocytes (Figure 4C).\n3.4 Phylogenetic analysis\nMaximum likelihood phylogenetic trees constructed from full-\n\n3.4 Phylogenetic analysis\nMaximum likelihood phylogenetic trees constructed from full-\ngenome sequences of LSDV, SPPV, and GTPV (NCBI database)\nrevealed three major LSDV clades: Cluster 1.1, Cluster 1.2, and Cluster\n1.2 recombinant (Figure  5). Cluster 1.1 included strains from\nSouth Africa (1954–2016) and Balkan countries. Cluster 1.2 comprised\nstrains from Africa, the Balkans, the Middle East, Eastern Europe, and\nSouth/Central Asia (1958–2023). Cluster 1.2 recombinant subclade\ncontained strains from East/Southeast Asia and Asian Russia (2017–\n2023). The yak-derived LSDV strain in this study (LSDV/China/GS/\nYak, GB No. MW355944.1) clustered within the Cluster 1.2\nrecombinant subclade, consistent with Chinese, Thai, and Russian\nrecombinant strains (19).\nIn contrast, Indian yak LSDV strains formed a distinct Cluster 1.2\nnon-recombinant branch (43). GPCR and RPO30 gene-based\nphylogenies corroborated these findings, with LSDV/China/GS/Yak\ngrouping within the recombinant subclade (Figure 6).\n4 Discussion\nRecombinant LSDV infections in yaks have been reported in\nChina’s Sichuan Province and India’s Himachal Pradesh near the\n\nHu et al.\b\n10.3389/fvets.2025.1584975\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nQinghai-Tibet Plateau (19, 43). Our study demonstrates active LSDV\ntransmission among yaks in this region, supported by severe clinical\nmanifestations (fever, hemorrhagic skin nodules, lymphadenopathy)\nand a 100% viral DNA detection rate in skin lesions. The high viral\nload in skin samples suggests potential environmental persistence via\nscab shedding critical concern given yaks’ year-round outdoor grazing\npractices (44). Concurrent respiratory and gastrointestinal lesions\n(tracheal hemorrhage, pulmonary necrosis, ruminal hemorrhagic\nplaques) indicate systemic viral spread, likely exacerbated by high-\naltitude hypoxia, contributing to the observed 46.67% mortality. Of\nparticular concern is the adaptation of the virus to cold climates (up\nto 3,600 m elevation in this study), raising alarms about its potential\ninvasion into ecologically fragile core yak habitats, which host over\n90% of the world’s 16 million yaks (45, 46). Traditional migratory\ngrazing and limited veterinary infrastructure may further accelerate\nsilent transmission.\nPhylogenetic analysis revealed a striking geographic dichotomy in\nLSDV evolution. Classical non-recombinant Cluster 1.2 strains\ndominate in Bangladesh (26), India (27), Nepal (28), and Myanmar\n(29), whereas the yak-derived strain in this study (LSDV/China/GS/\nYak) clusters within the emerging recombinant Cluster 1.2 sub-lineage\n(Neethling vaccine-like recombinants) circulating in China, Russia, and\nThailand since 2017. The dominance of this recombinant lineage across\nEast Asia suggests independent evolutionary trajectories or differential\nvaccine pressures. Notably, GPCR/RPO30 gene homology with strains\nfrom Russia (47) and Mongolia (24) implies undocumented cross-\nborder transmission via livestock movement along the Silk Road\nEconomic Belt. The rapid replacement of ancestral strains by\nrecombinant strains within approximately 6 years highlights the need\nto investigate potential adaptive advantages, such as immune evasion or\nchanges in vector competence.\nHistopathological\nfindings\nunderscore\nthe\n\nThe rapid replacement of ancestral strains by\nrecombinant strains within approximately 6 years highlights the need\nto investigate potential adaptive advantages, such as immune evasion or\nchanges in vector competence.Histopathological\nfindings\nunderscore\nthe\nheightened\npathogenicity of recombinant Cluster 1.2 LSDV in yaks. Severe\nvasculitis with endothelial necrosis and trans-mural lymphocyte\ninfiltration mirrors poxvirus-endothelial interactions in human\nsmallpox, raising questions about potential zoonotic barriers (48, 49).\nViral tropism for hair follicle epithelium may enhance environmental\npersistence via keratinocyte shedding—a key adaptation for survival\nin high-UV plateau environments.\nWhile LSD morbidity typically ranges from 3 to 85% (rarely\nreaching 100%) with mortality below 10% (4, 8, 21), Classical Cluster\n1.2 strains in India caused mild, non-fatal infections in yaks (43). In\ncontrast, our study and prior work (19) report 46.67–53.33% mortality\nin yaks infected with recombinant Cluster 1.2 LSDV, highlighting\nheightened susceptibility to emerging recombinants. Given yaks’\ngrazing patterns and limited veterinary resources, unchecked spread\nof recombinant strains across the Qinghai-Tibet Plateau could\nprecipitate catastrophic losses.\nFIGURE 1\nClinical manifestations of LSDV infection in yaks. (A–C) Cutaneous pox nodules; (D) Enlarged superficial cervical lymph nodes; (E) Periorbital skin\nedema; (F–H) Pox lesions on hooves, gingival swelling, and conjunctival hyperemia.\n\nHu et al.\b\n10.3389/fvets.2025.1584975\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nFIGURE 2\nPostmortem findings in yaks, fatally infected with LSDV. (A) Hemorrhage in tracheal longitudinal sections; (B) Near-circular hemorrhagic plaques on \nthe pulmonary pleura; (C) Pinpoint or patchy hemorrhage on the ruminal serosa; (D) Hemorrhage in the jejunal serosa. Blue arrows indicate \nhemorrhagic foci.\nFIGURE 3\nHistopathology of skin pox lesions in naturally LSDV-infected yaks (H&E staining). (A) Epidermal-dermal edema (arrow); (B) Dermal vasculitis (dashed \ncircle) with rare cytoplasmic inclusion bodies (arrow); (C) Lymphocytic and erythrocytic infiltration in the dermis (arrow); (D) Arteriolar vasculitis in the \ndermis (dashed circle).\n\n(A) Epidermal-dermal edema (arrow); (B) Dermal vasculitis (dashed \ncircle) with rare cytoplasmic inclusion bodies (arrow); (C) Lymphocytic and erythrocytic infiltration in the dermis (arrow); (D) Arteriolar vasculitis in the \ndermis (dashed circle).\n\nHu et al.\b\n10.3389/fvets.2025.1584975\nFrontiers in Veterinary Science\n06\nfrontiersin.org\n5 Conclusion\nLSDV represents an emerging threat to yaks in the Qinghai-\nTibet Plateau, demonstrating exceptional virulence with a 46.67% \ncase fatality rate. Histopathological evidence reveals dual pathogenic \nmechanisms: microcirculatory disruption and direct cytopathic \neffects. Phylogenetically, Chinese yak LSDV strains cluster within the \nrecombinant 1.2 sublineage, showing 99.2–99.4% homology with \nEast Asian and Russian variants. Notably, this contrasts with \nnon-recombinant Indian yak strains (Cluster 1.1), revealing \nFIGURE 4\nImmunohistochemical (IHC) staining of skin lesions from naturally LSDV-infected yaks. (A) IHC-positive hair follicle epithelial cells; (B) Deep dermal \nmacrophages; (C) Perivascular lymphocytes. Blue arrows highlight IHC-positive cells.\nFIGURE 5\nMaximum likelihood phylogenetic tree of LSDV based on full-genome sequences. The tree was constructed using PhyloSuite v1.2.3 (38) with IQ-TREE \nv2.2.0 (41), rooted with Sheeppox virus and Goatpox virus as outgroups. The best-fit substitution model (TVM + F + I + I + R2) was selected by BIC. \nBootstrap values are color-coded at branch nodes. GenBank accession numbers, host species (color-coded), and LSDV sublineages (innermost ring) \nare annotated. The middle ring denotes country of origin, and the outermost ring indicates collection year. Red stars and black circles mark yak-\nderived LSDV genomes from this study and prior work, respectively.\n\nHu et al.\b\n10.3389/fvets.2025.1584975\nFrontiers in Veterinary Science\n07\nfrontiersin.org\ndivergent evolutionary paths between South Asian and East\nAsian lineages.\nData availability statement\nThe datasets presented in this study can be found in online repositories.\nThe names of the repository/repositories and accession number(s) can\nbe found at: https://www.ncbi.nlm.nih.gov/genbank/, MW355944.\nEthics statement\nEthical approval not required for the studies involving animals\nbecause our study involved routine diagnostic sampling from\nfarmed yaks with owner consent, which falls under standard\nveterinary practice not in experimental animal research. According\nto Directive 2010/63/EU, US PHS Policy, and China’s Animal\nEpidemic Prevention Law, such sampling does not need formal\nethical approval as part of disease surveillance and clinical care.\nMoreover, field personnel were licensed veterinary practitioners\ncertified by the People’s Republic of China, and sampling procedures\nstrictly followed regulations. The studies were conducted in\naccordance with the local legislation and institutional requirements.\nWritten informed consent was obtained from the owners for the\nparticipation of their animals in this study.\nAuthor contributions\nJH: Writing  – original draft, Conceptualization, Data\ncuration, Formal analysis, Methodology. YLi: Conceptualization,\nData curation, Formal analysis, Methodology, Writing – original\ndraft. XZ: Methodology, Writing  – original draft. YLu:\nMethodology, Writing  – original draft. ZZ: Methodology,\nWriting – original draft. MX: Methodology, Writing – original\ndraft. SM: Methodology, Writing – original draft, Data curation.\nSN: Data curation, Conceptualization, Writing – review & editing.\nDW: Writing – original draft. TA: Writing – original draft. XL:\nWriting – original draft, Methodology, Resources. QM: Writing –\noriginal draft, Formal analysis, Project administration, Validation,\nWriting  – review & editing. JL: Writing  – review & editing,\nWriting  – original draft, Funding acquisition, Resources,\nSupervision.\nFIGURE 6\n\nQM: Writing –\noriginal draft, Formal analysis, Project administration, Validation,\nWriting  – review & editing.JL: Writing  – review & editing,\nWriting  – original draft, Funding acquisition, Resources,\nSupervision.FIGURE 6\nMaximum likelihood phylogenetic trees based on LSDV GPCR (A) and RPO30 (B) gene sequences. Trees were generated using PhyloSuite v1.2.3 (38)\nwith IQ-TREE v2.2.0 (41), rooted with Sheeppox virus and Goatpox virus. Substitution models (K3Pu + F + I for GPCR; HKY + F + I for RPO30) were\nselected by BIC. Bootstrap values <50 are omitted. Branch labels include strain metadata, with colors denoting host species or LSDV subtypes. Red\nstars and black circles mark yak-derived LSDV sequences from this study and previous work.\n\nHu et al.\b\n10.3389/fvets.2025.1584975\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. This study was supported by the\nNational\nKey\nR&D\nProgram\nof\nChina\n(2024YFD1800200,\n2024YFD1800201); Tibet Autonomous Region Science Fund and the\nChinese Agricultural Research Systems (Beef/yaks) (CARS-37), and The\nTibet Autonomous Region Science and Technology Department 2023 Key\nR&D Program (XZ202301ZY0016N).\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. 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+{"text": "【兽医文献】\n标题: article_29_Is there enough evidence to recommend surgical treatment as \n\nDevelopments in surgical fluid therapy rates in cats \nand dogs\n\nA Knowledge Summary by\n\nKristina Naden BVN1*\n\n1 Unitec Institute of Technology, 139 Carrington Road, Mount Albert, Auckland 1025, New Zealand \n* Corresponding Author (knaden@unitec.ac.nz)\n\nISSN: 2396-9776 \nPublished: 09 Jul 2020 \nin: Vol 5, Issue 3 \nDOI: 10.18849/VE.V5I3.299 \nReviewed by: Matt Gurney (BVSc CertVA PgCertVBM \nDipECVAA MRCVS) and Jodie Hughes (BVSc \nCertAVP(VA) Dip.ECVAA MRCVS) \nNext Review Date: 22 Jul 2021\n\nVeterinary Evidence \nISSN: 2396-9776 \nVol 5, Issue 3 \nDOI: 10.18849/VE.V5I3.299  \nnext review date: 22 Jul 2021 \np a g e | 2 of 15\n\nKNOWLEDGE SUMMARY\n\nPICO question \nIs there sufficient evidence to show surgical fluid therapy delivered at the recommended 3 mL/kg/hour for \ncats and 5 mL/kg/hour for dogs leads to a better outcome compared with widely accepted rates of 10 \nmL/kg/hour for both cats and dogs?\n\nClinical bottom line \nCategory of research question \nTreatment \nThe number and type of study designs reviewed \nFive studies were appraised. Two of these were opinion pieces, with one non-comparative prospective study, \none randomised controlled trial, and one case control study \nStrength of evidence \nWeak \nOutcomes reported \nCurrently there is limited evidence to show that the surgical fluid therapy recommendations made by the \n2013 Journal of the American Animal Hospital Association guidelines (Davis et al., 2013) for cats and dogs \nlead to a better outcome than accepted fluid therapy rates used. Fluid overload in humans can cause long-\nterm adverse effects, however the same effects have yet to be shown specifically in veterinary patients \nConclusion \nNo evidence was found that provides strong, conclusive evidence that the 2013 recommendations by the \nAmerican Animal Hospital Association and American Association of Feline Practitioners leads to a better \noutcome for both cats and dogs. The resulting research outlined below identifies a need to conduct clinical \nstudies on the effects of fluid therapy on cats and dogs, and identify clear monitoring protocols to minimise \nand ideally avoid, fluid overload. When adequate, valid clinical studies have been carried out, this will provide \nsufficient information for the development of evidence-based recommended rates of fluid therapy for \nveterinary medicine, in a range of contexts\n\nHow to apply this evidence in practice \nThe application of evidence into practice should take into account multiple factors, not limited to: individual \nclinical expertise, patient’s circumstances and owners’ values, country, location or clinic where you work, the \nindividual case in front of you, the availability of therapies and resources. \nKnowledge Summaries are a resource to help reinforce or inform decision-making. They do not override the \nresponsibility or judgement of the practitioner to do what is best for the animal in their care.\n\nKnowledge Summaries are a resource to help reinforce or inform decision-making.They do not override the \nresponsibility or judgement of the practitioner to do what is best for the animal in their care.\n\nVeterinary Evidence \nISSN: 2396-9776 \nVol 5, Issue 3 \nDOI: 10.18849/VE.V5I3.299  \nnext review date: 22 Jul 2021 \np a g e | 3 of 15\n\nThe evidence\n\nThe author of this Knowledge Summary was unable to find any specific evidence supporting the fluid rates \nrecommended by Davis et al. (2013). However, there was also no evidence found to support the widely \naccepted higher recommended rate of fluid (typically 10–20 mL/kg/hour) used for cats and dogs (Davis et al., \n2013; and Hopper et al., 2018).\n\nThere has, however, been some research around the effects of fluid therapy on the cardiovascular system. A \nsmall study carried out on dogs assessed the delivery of fluid therapy at 1 mL/kg/minute for 1 hour to healthy, \nnormovolaemic dogs when various depths of anaesthesia were induced. Dogs were maintained under light \nanaesthesia with an end tidal isoflurane (ETI) of 1.3%, (1.0 minimum alveolar concentration (MAC)) for at least \n15 minutes, then anaesthesia was increased to 3% ETI (2.3 MAC) for at least 15 minutes to induce a \nhypotensive state from anaesthesia, when fluid therapy was begun. This deep state of anaesthesia was \nmaintained for 45 minutes, until the last 15 minutes of fluid therapy when MAC was reduced to 1.2 (ETI of \n1.6%). This intervention impacted cardiovascular function, primarily stroke volume variation (SVV) (Valverde et \nal., 2012). SVV measures the change in volume of blood expelled from the left ventricle to the aorta, with each \nheartbeat, and was found to decrease by 67%, 60 minutes after the commencement of fluid therapy, with a \nconcurrent reduction in isoflurane concentration. Valverde et al. (2012) reports that SVV is well-documented \nto be a sensitive measure of responsiveness to fluid therapy, namely by demonstrating improvements to \nvenous return.\n\nValverde et al.(2012) reports that SVV is well-documented \nto be a sensitive measure of responsiveness to fluid therapy, namely by demonstrating improvements to \nvenous return.\n\nValverde et al. (2012) also noted that high rates of isotonic fluid therapy did not affect arterial blood pressure \nin the short-term (arterial blood pressure was measured up to 60 minutes following administration of fluids). \nThis result suggests that solely monitoring arterial blood pressure is not the most accurate measure of fluid \nbalance – a conclusion also noted in more recent studies that report hypovolaemia-induced hypotension \noccurring only after 30–40% of estimated blood volume has been lost (Drozdzynska et al., 2018; and Hopper et \nal., 2018).\n\nValverde et al. (2012) also noted that cardiac output and stroke volume did not increase at high rates of fluid \ntherapy (60 mL/kg/hour), when hypotension was due to deep anaesthesia. Packed cell volume (PCV) did \nhowever decrease significantly, due to plasma dilution. A PCV decrease of 16% at 30 minutes and 24% at 60 \nminutes was noted, and attributed to blood volume expansion of 32%. This change in PCV due to plasma \ndilution by fluid therapy is an important one to note, when interpreting results on an intra-operative \nhaematocrit of an animal receiving fluid therapy. A smaller PCV decrease would be expected with a lower rate \nof fluid therapy.\n\nUrine output was not influenced until up to 60 minutes after fluid therapy was begun, however this is more \nlikely due to the effects of a lighter anaesthetic plane also achieved at this point of the study (Valverde et al., \n2012).\n\nUrine output was not influenced until up to 60 minutes after fluid therapy was begun, however this is more \nlikely due to the effects of a lighter anaesthetic plane also achieved at this point of the study (Valverde et al., \n2012).\n\nBrodbelt et al. (2007) offered some evidence that fluid therapy in cats had increased mortality rates fourfold, \ncompared to cats not receiving fluid therapy. While this was a large study, with results provided by over 100 \nveterinary clinics, specific details around the data received from clinics was not fully described in the report. A \nlack of information in the study about the selection criteria for clinics, the fluid rates given to cats in the study, \nand any correlation between postoperative mortality and pre-anaesthetic health assessments, may have \nconfounded the data. It should also be remembered that cats are more susceptible to overhydration than \ndogs, due to the lower total blood volume of cats (approximately 170 mL for a 3 kg cat). Many clinics did not \nmeasure central venous pressure, or use infusion pumps – with the latter being a factor in accuracy of fluid \nrate delivery (Brodbelt, 2010).\n\nA number of human studies have identified increased mortality rates, and an increased incidence of acute \nkidney injury (AKI) due to fluid overload (McDermid et al., 2014; Ostermann et al., 2015; and Wang et al., \n2015). However, there is limited information currently available about the relationship between fluid overload\n\nVeterinary Evidence \nISSN: 2396-9776 \nVol 5, Issue 3 \nDOI: 10.18849/VE.V5I3.299  \nnext review date: 22 Jul 2021 \np a g e | 4 of 15\n\nand AKI in a veterinary context. Much information for veterinary patients has been extrapolated from human \ndata.\n\n18849/VE.V5I3.299  \nnext review date: 22 Jul 2021 \np a g e | 4 of 15\n\nand AKI in a veterinary context.Much information for veterinary patients has been extrapolated from human \ndata.\n\nResearch around the efficacy of fluid therapy in humans focuses on fluid therapy volumes, and often refers to \ngoal directed therapy (GDT) or zero-balance fluid therapy (Voldby & Brandstrup, 2016). Zero-balance fluid \ntherapy in humans focuses on not adding more than 1 kg of body weight to a patient following surgery with \nconcurrent fluid therapy (Voldby & Brandstrup, 2016). GDT uses parameters such as stroke volume and pulse \npressure variation analysis to determine appropriate fluid rates, in order to maximise cardiac output and \noxygen delivery (Drozdzynska et al., 2018; Licker et al., 2016; and Voldby & Brandstrup, 2016). In veterinary \nmedicine, current practice typically does not include measuring any of these parameters following clinical \nexamination, and of these parameters, weight is typically the only measurement taken (K. N. pers. obs.). The \npurpose of these two approaches to fluid therapy is to minimise tissue inflammation, wound dehiscence, and \npoor collagen regeneration, perioperative morbidity and mortality – all of which can be caused by excessive \npositive fluid balance (Drozdzynska et al., 2018; Licker et al., 2016; and Voldby & Brandstrup, 2016).\n\nThe complications detailed above were considered in depth by Kehlet (1997), who proposed a multimodal \napproach in human medicine to control general post-operative dysfunction – with avoidance of fluid overload \nspecifically noted to minimise cardiopulmonary complications. This concept, coined enhanced recovery after \nsurgery (ERAS), was revisited almost 20 years later, when collaboration was further emphasised between \nanaesthesiologists, surgeons, surgical nurses and post-surgical rehabilitation services such as physiotherapy, in \norder to minimise complications (Kehlet, 2015).\n\nThe approaches noted above to fluid therapy in humans should also be considered in two contexts – low-risk \npatients undergoing low-risk procedures, and high-risk patients undergoing higher risk procedures. In lower \nrisk human patients, high-volume crystalloid fluids (20–30 mL/kg, or 2 litres over 30 minutes for an average \nadult) proved beneficial to recovery, while high-risk patients, undergoing major procedures benefitted from a \nlower fluid rate, that was undefined by the authors but assumed an intraoperative urine output of 0.5–1.0 \nmL/kg/hour (Doherty & Buggy, 2012). These approaches may be applicable to veterinary medicine.\n\nRecommended fluid rates in human surgery range from not exceeding 6–8 mL/kg/hour for thoracic surgery \n(Licker et al., 2016) to 15 mL/kg/hour for the first hour of general surgery and then decreasing as required \n(Voldby & Brandstrup, 2016). Current trend in veterinary surgical practice is to provide a generic rate of fluid \ntherapy for a range of surgical procedures (Davis et al., 2013). This may vary in clinics where blood pressure, \n(in addition to other more standard parameters such as heart rate, and respiratory rate) is monitored intra-\noperatively, with fluid therapy levels adjusted in response to variance in this parameter (pers. obs.). This \nparameter may have limited uses as a measure of overhydration of animals, given fluid overload in humans is \nnot measurable via blood pressure (Voldby & Brandstrup, 2016). Kehlet’s (2015) principle of a multimodal \napproach to minimise postoperative complications in human medicine has been applied to veterinary \nmedicine, with recommendation that the ERAS programme be used in animals (Gurney, 2018). While Gurney \n(2018) does not recommend specifics around fluid therapy, it is noted that overload of fluids be avoided, and \nthe GDT approach be used in veterinary medicine, in order to avoid postoperative complications.\n\nWhile Gurney \n(2018) does not recommend specifics around fluid therapy, it is noted that overload of fluids be avoided, and \nthe GDT approach be used in veterinary medicine, in order to avoid postoperative complications.\n\nThis limited amount of evidence suggests further research is required to identify the impact of current \nrecommendations for fluid therapy in cats and dogs.\n\nVeterinary Evidence \nISSN: 2396-9776 \nVol 5, Issue 3 \nDOI: 10.18849/VE.V5I3.299  \nnext review date: 22 Jul 2021 \np a g e | 5 of 15\n\nSummary of the evidence\n\nDavis et al. (2013) \nPopulation: Cats and dogs \nSample size: n/a – recommendation \nIntervention details: n/a – recommendation \nStudy design: Opinion article \nOutcome studied: n/a – recommendation \nMain findings: \n(relevant to PICO question): \n \nFluid therapy should be individualised for each patient \n \nFluid selection may need to be adjusted from one type of \nfluid to another during hospitalisation \n \nWhile blood pressure is often used to assess tissue \nperfusion, intraoperative hypotension may not always be \nrelated to dehydration. Anaesthetic depth should be \nconsidered prior to administering or increasing fluid rates \n \nThere is limited evidence-based research for fluid therapy \nrates in a veterinary context \nLimitations: \n \nThe recommendations in the report are based on a limited \nnumber of research articles, providing minimal evidence to \nsupport these new recommendations \n \nThe recommendations used are based on human evidence \nof over-infusion of fluids, including damage to the kidneys \nand the endothelial glycocalyx. This may or may not hold \ntrue for cats and dogs \n \nThis recommendation is not based on clinical studies for cats \nand dogs\n\nThis may or may not hold \ntrue for cats and dogs \n \nThis recommendation is not based on clinical studies for cats \nand dogs\n\nValverdeet al. (2012)  \nPopulation: Normovolaemic adult dogs between the age of 1 and 4-years-old \nSample size: Six mixed-breed dogs (three male, three female), with a mean \nweight of 22.1 kg (±2.8) \nIntervention details: \n \nAnaesthesia was induced and maintained using gaseous \nagents of isoflurane and oxygen, until a steady \nhaemodynamic state was achieved at 1.3% end tidal \nisoflurane \n \nBaseline cardiovascular parameters (including heart rate, \narterial blood pressure, central venous pressure, stroke \nvolume variation and cardiac output) were taken at this \npoint, and again 10, 30 and 60 minutes following the \ncommencement of fluid therapy \n \nDiagnostic samples of urine and blood were collected to \nmeasure blood gases, electrolytes, PCV and plasma volume \nat 30 and 60 minutes after fluid therapy began\n\nVeterinary Evidence \nISSN: 2396-9776 \nVol 5, Issue 3 \nDOI: 10.18849/VE.V5I3.299  \nnext review date: 22 Jul 2021 \np a g e | 6 of 15\n\n18849/VE.V5I3.299  \nnext review date: 22 Jul 2021 \np a g e | 6 of 15\n\n \nA hypotensive state under deep anaesthesia (3% end tidal \nisoflurane) was induced and maintained for at least 15 \nminutes, with cardiovascular parameters taken again at this \npoint \n \nIsotonic fluids (Plasma-Lyte A solution) were administered at \na rate of 1 mL/kg/minute for 1 hour \n \nEnd tidal isoflurane was gradually reduced 45 minutes after \nfluid therapy began, to 1.6% \n \nDogs were recovered from anaesthesia and administered \nintravenous meloxicam 0.1 mg/kg  \nStudy design: Non-comparative prospective study \nOutcome studied: The effect of high-volume isotonic fluid therapy on normovolaemic \ndogs with isoflurane-induced hypotension \nMain findings: \n(relevant to PICO question): \n \nIntravenous fluid therapy at high volumes (15–80 \nmL/kg/hour) does not increase arterial blood pressure in \nanaesthetised normovolaemic patients, during \nnormotensive and hypotensive states \n \nIsotonic fluid therapy at 1 mL/kg/minute does not increase \ncardiac output or stroke volume, in dogs where hypotension \nis a result of a deep anaesethetic plane  \n \nPlasma volume increased by 41% at 30 minutes after fluid \ntherapy began, and by 49% at 60 minutes \nLimitations: \n \nNo control group was used to assess the cumulative \ncardiodepressive effects of 3% end tidal isoflurane  \n \nThe sample size used in this study was small, with only six \ndogs \n \nThe sample was a group of healthy, normovolaemic dogs, \nand application of any results is limited to this type of \npatient\n\nSilverstein et al. (2014) \nPopulation: Healthy client-owned female dogs undergoing elective \novariohysterectomy \nSample size: 48 dogs \nIntervention details: \n \nDogs were premedicated, anaesthesia was induced with \npropofol and diazepam, and maintained using isoflurane and \noxygen \n \nBaseline cardiovascular parameters were measured at \nintubation, including arterial blood pressure (using a Doppler \nflow detector), heart rate, mucous membrane colour, \ncapillary refill time, with tissue oxygen saturation data was \ncollected using pulse oximetry \n \nThe same cardiovascular parameters were measured 30 and \n60 minutes following induction of anaesthesia \n \nA video microscope was used to assess microcirculation in \nthe buccal mucosa dorsal to the maxillary canine of all dogs\n\nVeterinary Evidence \nISSN: 2396-9776 \nVol 5, Issue 3 \nDOI: 10.18849/VE.V5I3.299  \nnext review date: 22 Jul 2021 \np a g e | 7 of 15\n\n18849/VE.V5I3.299  \nnext review date: 22 Jul 2021 \np a g e | 7 of 15\n\nupon induction, then again 30 and 60 minutes following \ninduction \n \nDogs were randomly assigned one of three fluid therapy \ntreatments using Lactated Ringer’s Solution – none, 10 \nmL/kg/hour, or 20 mL/kg/hour, with 16 dogs in each \ntreatment group \n \nAll dogs were recovered successfully from anaesthesia  \nStudy design: Randomised controlled trial \nOutcome studied: The effects of intravenous fluid administration on microcirculatory \nblood flow to the oral mucosa in healthy anaesthetised dogs \nMain findings: \n(relevant to PICO question): \n \nBlood vessels < 20m in diameter show no change in vessel \ndensity for any of the three treatment groups  \n \nVessel diameter was found to be significantly different in \nvessels < 20m, between the group receiving 20 mL/kg/hour \nand the group receiving 0 mL/kg/hour of fluid \n \nDensity of vessels  20m (most likely venules and/or \narterioles) was significantly more in dogs receiving 20 \nmL/kg/hour compared with dogs receiving 0 mL/kg/hour of \nfluid \n \nOverall, no significant association was found between \nintravenous fluid administration and tissue perfusion \nLimitations: \n \nThe study reported primarily on one parameter only (tissue \nperfusion in the oral mucosa) and does not consider tissue \nperfusion of other organs \n \nThe fluid balance of the sample was not reported, giving no \nsuggestion if dogs were normovolaemic \n \nSmall sample size of 48 dogs (one dog excluded from the \nstudy due to excessive pigmentation of the oral mucosa \ninterfering with the video microscope’s ability to collect \ndata)\n\nBrodbelt et al. (2007)  \nPopulation: Cats admitted to 117 veterinary clinics in the United Kingdom for \nprocedures where sedation or anaesthesia was required \nSample size: 79,178 cats \nIntervention details: No specific interventions. Cats were already being \nsedated/anaesthetised and monitored to that veterinary clinic’s \nusual standards \nStudy design: \n \nCase control study \n \nDetails of patient, procedure, and perioperative \nmanagement for all cats in 117 participating clinics were \nrecorded, however not included in the report. The study was \ncarried out with a control group nested in the main cohort of \ncats \n \nThe ‘study’ cohort consisted of all cases, with details of each \ncase recorded for 48 hours post-anaesthesia or sedation.\n\nVeterinary Evidence \nISSN: 2396-9776 \nVol 5, Issue 3 \nDOI: 10.18849/VE.V5I3.299  \nnext review date: 22 Jul 2021 \np a g e | 8 of 15\n\nVeterinary Evidence \nISSN: 2396-9776 \nVol 5, Issue 3 \nDOI: 10.18849/VE.V5I3.299  \nnext review date: 22 Jul 2021 \np a g e | 8 of 15\n\nOutcomes for the main study cohort were limited to alive, \ndead or euthanised. Treatment types for the cohort were \nnot included \n \nA nested ‘control’ cohort consisted of randomly selected \ncats that were alive more than 48 hours post-anaesthesia or \nsedation. It was not noted if these cats received fluid \ntherapy or not. The ratio of study to control cases was 1:4. \nDemographics and procedure data of the control cohort was \ncompared to that of the study cohort \nOutcome studied: The frequency of risk (including death) for sedated or anaesthetised \ncats in British veterinary clinics \nMain findings: \n(relevant to PICO question): \n \nCats receiving fluid therapy experienced a mortality rate \nfour times that of cats not receiving fluid therapy \n \nCurrent usage of fluid therapy may have adverse effects on \nanaesthetised and/or sedated cats, and there is room for \nimprovement in the monitoring and general management of \nfluid therapy in cats \n \nThe potential for fluid overload in cats due to the lower fluid \nvolume in cats – careful administration and monitoring is \nrecommended \nLimitations: \n \nThis study focused on feline deaths in clinical practice, due \nto anaesthetic complications, with limited data provided on \nfluid therapy \n \nThe study did not identify causality, and authors suggested a \nlack of monitoring could be related to the increased \nmortality rate for cats receiving fluid therapy \n \nThe rate of fluid therapy (where given) was not stated, \nwhich could have assisted with understanding of the high \nmortality rate in cats receiving fluid therapy \n \nThe study did not identify how veterinary clinics were \nselected, or what criteria were required for participation in \nthe study \n \nMinimum monitoring and treatment standards were not \nidentified \n \nThe study did not specify or categorise cats according to risk \nfactor (for example, anaesthetic risk assessment [ASA] risk \nlevels), which may have had an impact on the outcome \nstudied\n\nThomovsky et al. (2016)  \nPopulation: n/a – review \nSample size: n/a – review \nIntervention details: n/a – review \nStudy design: Opinion article \nOutcome studied: Defining “fluid overload” in small animals, and means of avoiding \nthis in small animals\n\nVeterinary Evidence \nISSN: 2396-9776 \nVol 5, Issue 3 \nDOI: 10.18849/VE.V5I3.299  \nnext review date: 22 Jul 2021 \np a g e | 9 of 15\n\nMain findings: \n(relevant to PICO question): \n \nHeart rate and blood pressure do not change in dogs with \nfluid overload, demonstrating the limitations of these two \nparameters as measures of fluid overload \n \nFluid overload causes fluid to leave the vascular space and \nmove into the interstitial space, damaging the endothelial \nglycocalyx in the process \n \nOedema in abdominal organs (due to fluid overload) may \nadd pressure to abdominal blood vessels and the vena cava, \nimpeding blood flow to other organs and restricting venous \nreturn to the heart \n \nFluid overload can be avoided using the ROSE principle \n(Resuscitation, Optimisation, Stabilisation, Evacuation) for \nphases of fluid delivery \n \nFluid therapy should be considered with the same principles \nas delivery of drugs or other medical interventions \nLimitations: \n \nLimited methods of identifying fluid overload are provided  \n \nWhile a link between fluid overload, and increased mortality \nand acute kidney injury is noted, the article does not explore \nthe physiological pathway of mortality due to fluid overload \n \nThe article provides one example case of fluid overload (a \n10-years-old female desexed domestic shorthaired cat) and \none theoretical case (a 9-years-old entire male dog with \nparvovirus) showing a comparison of a traditional approach \nto fluid therapy vs the ROSE approach\n\nAppraisal, application and reflection\n\nThe concept of the need to replace lost body fluids, either via trauma, illness, or surgery, has existed for \nhundreds of years (Driessen & Brainard, 2006), and intravenous fluid therapy has been administered to \nhumans since 1832 when salt water solutions were first used by Robert Lewins to treat cholera (Stanzani & \nChan, 2010). In these early days of using fluid as a therapy to treat disease, Lewins was already considering the \ndecision over the volume of fluid that would be required. His objective was to replace the volume of serum \nlost, in order to return the patient to normovolaemia (Myburgh & Mythen, 2013).\n\nThe first use of fluid therapy in animals is not well documented, however studies on the use of crystalloid \nfluids on veterinary patients were carried out in the 1960s (Silverstein et al., 2014). Fluid therapy plays a vital \nrole in stabilising patients in shock, and improving cardiac output (Marshall et al., 2016).\n\n, 2014).Fluid therapy plays a vital \nrole in stabilising patients in shock, and improving cardiac output (Marshall et al., 2016).\n\nThere is currently a paucity of research that has been carried out in veterinary medicine in terms of the rate \nthat this fluid therapy should be delivered. Traditionally, a rate of 10 mL/kg/hour was delivered to \nanaesthetised veterinary patients (Davis et al., 2013; and Hopper et al., 2018) – how this rate was calculated is \nunclear, although may be based around human recommendations of 6–8 mL/kg/hour for patients undergoing \nthoracic or abdominal surgery (Borland & Bennett, 2018; and Licker et al., 2016), and 15 mL/kg/hour for \npatients undergoing other procedures (Voldby & Brandstrup, 2016). The most recent literature (Davis et al., \n2013), recommends lower rates of fluid therapy, with a rule of thumb guide given as 3 mL/kg/hour for cats, \nand 5 mL/kg/hour for dogs. Davis et al. (2013) also recommended the following: provide maintenance rate \nplus necessary replacement at less than 10 mL/kg/hour; adjust fluid rates based on patient assessment and \nmonitoring; patients with cardiovascular disease and renal disease should be administered a lower rate of \nfluid; and rates should be decreased if anaesthesia is more than one hour, with suggested reduction to be 25% \nper hour until fluid is being delivered at maintenance rate (assuming a stable patient). These\n\nVeterinary Evidence \nISSN: 2396-9776 \nVol 5, Issue 3 \nDOI: 10.18849/VE.V5I3.299  \nnext review date: 22 Jul 2021 \np a g e | 10 of 15\n\nThese\n\nVeterinary Evidence \nISSN: 2396-9776 \nVol 5, Issue 3 \nDOI: 10.18849/VE.V5I3.299  \nnext review date: 22 Jul 2021 \np a g e | 10 of 15\n\nrecommendations appear to be based on human research identifying excessive fluid therapy causing a range \nof postoperative complications mentioned earlier in this Knowledge Summary. While many clinics are adopting \nthis lower rate of fluid therapy, a recent survey of 113 New Zealand veterinary clinics found that more than \nhalf the respondents currently administer fluids at a rate of 10 mL/kg/hour during surgery (Sano et al., 2018). \nMembers of the Veterinary Information Network (VIN) stated that determining fluid rates for animals \nremained the most challenging aspect of fluid therapy (Hopper et al., 2018).\n\nTo determine optimal assessment methods of fluid therapy, it should be remembered that two of the key\ngoals of this therapy are to improve cardiac output and improve oxygen delivery (Licker et al., 2016; and\nMarshall et al., 2016). Several articles suggest monitoring of central venous pressure (CVP) (Lunn, 2011;\nMarshall et al., 2016; and Siemionow et al., 2012) as a good starting point to ascertain the risk of fluid\noverload, with Marshall et al. (2016) also suggesting some less invasive methods, however there is emerging\nstrong evidence that blood pressure is an inaccurate measure of fluid overload, and CVP is no longer\nconsidered an adequate method of measuring fluid responsiveness in an animal (Borland & Bennett, 2018;\nDrozdzynska et al., 2018; Fantoni et al., 2017; and O’Dwyer, 2011). Pulse pressure variation (PPV) is reported\nto be the most sensitive measure of fluid responsiveness, with changes in PPV occurring earlier than arterial\nblood pressure, heart rate or CVP (Celeita-Rodríguez et al., 2019; Drozdzynska et al., 2018; Fantoni & Shih,\n2017). PPV is expressed as a percentage, and measures the difference in maximum and minimum pulse\npressure in one respiratory cycle, which is then divided by the mean of these two values (Drozdzynska et al.,\n2018). PPV is measured non-invasively in humans, with a finger cuff that transmits data to a multi-parameter\nmonitor, and some recent studies have compared the use and accuracy of PPV measurement in anaesthetised\ndogs. In a study comparing the use of CVP and MAP with PPV, the latter was found to predict fluid\nresponsiveness to volume expansion, when CVP and MAP did not (Fantoni et al., 2017). This study used “non-\ninvasive methods” (a multi-parameter monitor) to calculate PPV on mechanically ventilated, hypotensive (MAP\n<60 mmHg), anaesthetised dogs administered a fluid challenge of 15 mL/kg over 15 minutes. Gonçalves et al.\n(2020) also compared the accuracy of invasive arterial blood pressure monitoring with the use of PPV and\n\nGonçalves et al.(2020) also compared the accuracy of invasive arterial blood pressure monitoring with the use of PPV and\nfound the latter method predicted fluid responsiveness in hypotensive anaesthetised, mechanically ventilated\ndogs (MAP <65 mmHg), administered a fluid challenge of 5 mL/kg over 15 minutes, reliably as invasive\nmethods. While this study stated PPV was assessed in a non-invasive manner, the placement of an arterial\ncatheter was still required for calculation of PPV via the multi-parameter monitor.\n\nAn additional method of assessing fluid responsiveness and guiding GDT is pleth variability index (PVI), a \nmethod that uses a pulse oximeter (equipment that is commonplace in veterinary clinics) to measure changes \nin the perfusion index, over one respiratory cycle (den Boogert et al., 2018). This method would be considered \ncompletely non-invasive, with no arterial catheterisation required. Human studies have found PVI and PPV \nboth optimised fluid preload outcome in patients undergoing low- to moderate-risk abdominal surgery \n(Coeckelenbergh et al., 2019). PVI has been shown to be less consistent in detecting fluid responsiveness in \npatients undergoing renal transplant (Coeckelenbergh et al., 2019), and is not suitable in critically ill or \nhaemodynamically unstable adults (den Boogert et al., 2018).\n\n, 2019).PVI has been shown to be less consistent in detecting fluid responsiveness in \npatients undergoing renal transplant (Coeckelenbergh et al., 2019), and is not suitable in critically ill or \nhaemodynamically unstable adults (den Boogert et al., 2018).\n\nThe choice to administer fluids in non-anaesthetised patients is usually based on signs of dehydration in cats \nand dogs: skin tenting, dry mucous membranes, oliguria, systolic hypotension. Many anaesthetised patients \nare administered fluids during surgery based on anaesthesia-induced hypotension, or blood loss. However, the \nquestion remains, what is an appropriate fluid rate, based on the physiological changes caused by anaesthesia \nand surgery? What is clear is that monitoring of fluid therapy is vital – the use of fluid therapy should be \nlikened to using substances such as antibiotics, opioid analgesics, or non-steroidal anti-inflammatory drugs on \ncats and dogs, and should be administered in such a way. Anaesthetised patients are monitored constantly to \nassess basic cardiovascular, neurological and respiratory functions. Monitoring currently often only focuses on \nthe anaesthetic depth of the patient, and this may be a very blinkered approach to our care of anaesthetised \npatients. This approach to monitoring omits the renal system entirely, and monitoring of the pulmonary \nsystem is often limited to respiratory rate and depth and end tidal carbon dioxide, with little consideration \noften given to actual blood loss volumes. Weighing saturated swabs, sponges and any blood removed via\n\nVeterinary Evidence \nISSN: 2396-9776 \nVol 5, Issue 3 \nDOI: 10.18849/VE.V5I3.299  \nnext review date: 22 Jul 2021 \np a g e | 11 of 15\n\nWeighing saturated swabs, sponges and any blood removed via\n\nVeterinary Evidence \nISSN: 2396-9776 \nVol 5, Issue 3 \nDOI: 10.18849/VE.V5I3.299  \nnext review date: 22 Jul 2021 \np a g e | 11 of 15\n\nsuction should also be considered as an additional assessment tool of blood loss. It should be remembered \nthat many of the fluid products commonly used during anaesthesia, do not replace the blood components lost \nas part of a surgical procedure. Blood products such as plasma, whole blood or packed red blood cells may be \nmore appropriate to use in some situations.\n\nDue to the limited amount of evidence in veterinary medicine, some research included in this Knowledge \nSummary is from human medicine. While this is valid in a human medical context, it should be remembered \nthat there are still physiological differences between animals and humans, which may impact the \ntransferability of results (Valverde et al., 2012; and Yozova et al., 2017).\n\nWhile this is valid in a human medical context, it should be remembered \nthat there are still physiological differences between animals and humans, which may impact the \ntransferability of results (Valverde et al., 2012; and Yozova et al., 2017).\n\nIt was challenging to find a range of comprehensive clinical studies that looked at the effect of fluid rates and \nhence fluid overload in a veterinary context, suggesting there is still a great need to carry out clinical research \nin this area of veterinary medicine. In the interim, robust pre-anaesthetic assessment of hydration status, type \nof surgical procedure (and therefore likely risk of blood and/or fluid loss), and pre-existing medical conditions \nof the patient should all be considered and should be used to determine the need for fluid therapy. This, \ncombined with intra-operative monitoring of patients on fluid therapy (non-invasive and invasive where \nequipment and expertise exists to carry this out) are currently the only available techniques in minimising \npotential physiological trauma to patients caused by excessive fluid therapy. In terms of specific rates for fluid \ntherapy in veterinary patients, it is difficult at this stage to definitively state a generic rate for a species, \nwithout considering comorbidities, procedures and existing hydration status. In the healthy cat or dog, 3 \nmL/kg and 5 mL/kg, respectively, may suffice as a starting point for fluid therapy. Veterinarians and veterinary \nnurses should consider a collaborative approach to fluid rates, based on an animal’s pre-, post and intra-\noperative hydration status, and underlying health conditions, and adjust rates accordingly. Investigation into \nthe use of non-invasive methods of assessing fluid responsiveness, such as PVI, would be recommended for \nthose practitioners providing fluid therapy on a regular basis.\n\nMethodology Section\n\nSearch Strategy \nDatabases searched and dates \ncovered: \nCAB Abstracts 1973–2019, week 28 \nPubMed Central 1950–2019 \nSearch terms: CAB Abstracts (using OVID):\n\nMethodology Section\n\nSearch Strategy \nDatabases searched and dates \ncovered: \nCAB Abstracts 1973–2019, week 28 \nPubMed Central 1950–2019 \nSearch terms: CAB Abstracts (using OVID):\n\n1. (dog or dogs or canine or canines or cat or cats or feline or \nfelines or 'small animal' or 'companion animal').mp. or exp \ndogs/ or exp cats/ fluid therapy.mp. or exp Fluid Therapy/ \n2. (surg* or intraoper* or perioper*).mp. \n3. (rate or overload).mp. \n4. 1 and 2 and (3 or 4)\n\nPubMed (via NCBI website):\n\n1. dog or dogs or canine or canines \n2. fluid therapy \n3. surgery or surgical or interoperative or perioperative \n4. rate or overload \n5. 1 and 2 and (3 or 4) \nDates searches performed: 22 Jul 2019\n\nVeterinary Evidence \nISSN: 2396-9776 \nVol 5, Issue 3 \nDOI: 10.18849/VE.V5I3.299  \nnext review date: 22 Jul 2021 \np a g e | 12 of 15\n\nExclusion / Inclusion Criteria \nExclusion: Non-English language journals, articles pre-2009, journals related to \nhuman medicine \nInclusion: Veterinary journals, peer-reviewed journals\n\nSearch Outcome \nDatabase \nNumber \nof \nresults \nExcluded – \nnon-peer \nreviewed \njournals \nExcluded – \nnon-English \nlanguage \nExcluded – \npre-2009 \nExcluded – \nnot from \nsmall animal \nveterinary \nspecific \nExcluded \n– not \nrelevant \nto PICO \nquestion \nTotal \nrelevant \npapers \nCAB Abs \n557 \n145 \n58 \n194 \n0 \n156 \n4 \nMedline \n508 \n5 \n15 \n266 \n2 \n217 \n3 \nTotal relevant papers when duplicates removed \n5\n\nCONFLICT OF INTEREST\n\nThe author declare no conflicts of interest.\n\nREFERENCES\n\nREFERENCES\n\n1. Borland, K. & Bennett, R. C. (2018). Perianaesthetic fluid therapy: an update. Companion Animal, 23(7), \n406–411. DOI: https://doi.org/http://dx.doi.org/10.12968/coan.2018.23.7.406 \n2. Brodbelt, D. (2010). Feline anesthetic deaths in veterinary practice. Topics in Companion Animal \nMedicine. DOI: https://doi.org/10.1053/j.tcam.2010.09.007 \n3. Brodbelt, D. C., Pfeiffer, D. U., Young, L. E. & Wood, J. L. N. (2007). Risk factors for anaesthetic-related \ndeath in cats: results from the confidential enquiry into perioperative small animal fatalities \n(CEPSAF). British Journal of Anaesthesia, 99, 617–623. DOI: https://doi.org/10.1093/bja/aem229 \n4. Celeita-Rodríguez, N., Teixeira-Neto, F. J., Garofalo, N. A., Dalmagro, T. L., Girotto, C. H., Oliveira, G. C. \nV. & Santos, I. F. (2019). Comparison of the diagnostic accuracy of dynamic and static preload indexes \nto predict fluid responsiveness in mechanically ventilated, isoflurane anesthetized dogs. 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The Veterinary Clinics of North America.\nSmall Animal Practice, 47(2), 423–434. DOI: https://doi.org/10.1016/j.cvsm.2016.11.004\n12. Fantoni, D. T., Ida, K. K., Gimenes, A. M., Mantovani, M. M., Castro, J. R., Patricio, G. C. F., Ambrósio, A.\nM. & Otsuki, D. A. (2017). Pulse pressure variation as a guide for volume expansion in dogs undergoing\northopedic surgery. Veterinary Anaesthesia and Analgesia, 44(4), 710–718.\nDOI: https://doi.org/http://dx.doi.org/10.1016/j.vaa.2016.11.011\n13. Gonçalves, L. A., Otsuki, D. A., Pereira, M. A., Nagashima, J. K., Ambrósio, A. M. & Fantoni, D. T. (2020).\n\nVeterinary Anaesthesia and Analgesia, 44(4), 710–718.DOI: https://doi.org/http://dx.doi.org/10.1016/j.vaa.2016.11.011\n13.Gonçalves, L.A., Otsuki, D.A., Pereira, M.A., Nagashima, J.K., Ambrósio, A.M.& Fantoni, D.T.(2020).\nComparison of pulse pressure variation versus echocardiography-derived stroke volume variation for\nprediction of fluid responsiveness in mechanically ventilated anesthetized dogs. Veterinary\nAnaesthesia and Analgesia, 47(1), 28–37. DOI: https://doi.org/10.1016/j.vaa.2019.08.047\n14. Gurney, M. (2018). A procedure-specific approach to recovery after surgery.\n15. Hopper, K., Garcia Rojas, A. & Barter, L. (2018). An Online Survey of Small Animal Veterinarians\nRegarding Current Fluid Therapy Practices in Dogs and Cats. Journal of the American Veterinary\nMedical Association, 252(5), 553–559. DOI: https://doi.org/10.2460/javma.252.5.553\n16. Kehlet, H. (1997). Multimodal approach to control postoperative pathophysiology and\nrehabilitation. British Journal of Anaesthesia, 78(5), 606–617.\nDOI: https://doi.org/10.1093/bja/78.5.606\n17. Kehlet, H. (2015). Enhanced Recovery After Surgery (ERAS): good for now, but what about the\nfuture? Canadian Journal of Anesthesia, 62(2), 99–104. DOI: https://doi.org/10.1007/s12630-014-\n0261-3\n18. Licker, M., Triponez, F., Ellenberger, C. & Karenovics, W. (2016). Fluid therapy in thoracic surgery: A\nzero-balance target is always best! Turk Anesteziyoloji ve Reanimasyon Dernegi Dergisi, 44(5), 227–\n229. DOI: https://doi.org/10.5152/TJAR.2016.006\n19. Lunn, K. F. (2011). The kidney in critically ill small animals. Veterinary Clinics of North America - Small\nAnimal Practice, 41(4), 727–744. DOI: https://doi.org/10.1016/j.cvsm.2011.03.020\n20. Marshall, K., Thomovsky, E., Johnson, P. & Brooks, A. (2016). A review of available techniques for\ncardiac output monitoring. Topics in Companion Animal Medicine, 31(3), 100–108.\nDOI: https://doi.org/10.1053/j.tcam.2016.08.006\n21. McDermid, R. C., Raghunathan, K., Romanovsky, A., Shaw, A. D. & Bagshaw, S. M. (2014).\nControversies in fluid therapy: Type, dose and toxicity. World Journal of Critical Care Medicine, 3(1),\n\nTopics in Companion Animal Medicine, 31(3), 100–108.DOI: https://doi.org/10.1053/j.tcam.2016.08.006\n21.McDermid, R.C., Raghunathan, K., Romanovsky, A., Shaw, A.D.& Bagshaw, S.M.(2014).Controversies in fluid therapy: Type, dose and toxicity.World Journal of Critical Care Medicine, 3(1),\n24. DOI: https://doi.org/10.5492/wjccm.v3.i1.24\n22. Myburgh, J. A. & Mythen, M. G. (2013). Resuscitation Fluids. New England Journal of\nMedicine, 369(13), 1243–1251. DOI: https://doi.org/10.1056/NEJMra1208627\n23. O’Dwyer, L. (2011). How to perform central venous pressure measurement. The Veterinary\nNurse, 2(10), 600–603.\n\nVeterinary Evidence \nISSN: 2396-9776 \nVol 5, Issue 3 \nDOI: 10.18849/VE.V5I3.299  \nnext review date: 22 Jul 2021 \np a g e | 14 of 15\n\n24. Ostermann, M., Straaten, H. M. O. & Forni, L. G. (2015). Fluid overload and acute kidney injury: cause\nor consequence? Critical Care (London, England), 19, 443. DOI: https://doi.org/10.1186/s13054-015-\n1163-7\n25. Sano, H., Barker, K., Odom, T., Lewis, K., Giordano, P., Walsh, V. & Chambers, J. P. (2018). A Survey of\nDog and Cat Anaesthesia in a Sample of Veterinary Practices in New Zealand. New Zealand Veterinary\nJournal. DOI: https://doi.org/10.1080/00480169.2017.1413959\n26. Siemionow, K., Cywinski, J., Kusza, K. & Lieberman, I. (2012). Intraoperative Fluid Therapy and\nPulmonary Complications. Orthopedics, 35(2), 184–191. DOI: https://doi.org/10.3928/01477447-\n20120123-06\n27. Silverstein, D. C., Cozzi, E. M., Hopkins, A. S. & Keefe, T. J. (2014). Microcirculatory effects of\nintravenous fluid administration in anesthetized dogs undergoing elective\novariohysterectomy. American Journal of Veterinary Research, 75(9), 809–817.\nDOI: https://doi.org/10.2460/ajvr.75.9.809\n28. Stanzani, G. & Chan, D. L. (2010). Controversies in Fluid Therapy. European Journal of Companion\nAnimal Practice, 24(2), 14–23.\n29. Thomovsky, E., Brooks, A. & Johnson, P. (2016). Fluid Overload in Small Animal Patients. Topics in\nCompanion Animal Medicine, 31(3), 94–99. DOI: https://doi.org/10.1053/j.tcam.2016.08.007\n30. Valverde, A., Gianotti, G., Rioja-Garcia, E. & Hathway, A. (2012). Effects of high-volume, rapid-fluid\ntherapy on cardiovascular function and hematological values during isoflurane-induced hypotension in\nhealthy dogs. Canadian Journal of Veterinary Research (Revue Canadienne de Recherche\nVeterinaire), 76(2), 99–108.\n31. Voldby, A. W. & Brandstrup, B. (2016). Fluid therapy in the perioperative setting-A clinical\nreview. Journal of Intensive Care, 4(1). DOI: https://doi.org/10.1186/s40560-016-0154-3\n32. Wang, N., Jiang, L., Zhu, B., Wen, Y. & Xi, X.-M. (2015). Fluid balance and mortality in critically ill\npatients with acute kidney injury: a multicenter prospective epidemiological study The Beijing Acute\nKidney Injury Trial (BAKIT) Workgroup. Critical Care (London, England), 19(371).\n\n, Zhu, B., Wen, Y.& Xi, X.-M.(2015).Fluid balance and mortality in critically ill\npatients with acute kidney injury: a multicenter prospective epidemiological study The Beijing Acute\nKidney Injury Trial (BAKIT) Workgroup.Critical Care (London, England), 19(371).\nDOI: https://doi.org/10.1186/s13054-015-1085-4\n33. Yozova, I. D., Howard, J., Sigrist, N. E. & Adamik, K.-N. (2017). Current Trends in Volume Replacement\nTherapy and the Use of Synthetic Colloids in Small Animals-An Internet-Based Survey (2016). Frontiers\nin Veterinary Science, 4, 140. 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+{"text": "【兽医文献】\n标题: Modulation of antiviral genes by African swine fever isolates of diverse virulence\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nCase Report: Diagnosis and\ntreatment of equine ascending\nplacentitis: compilation of 17 case\nreports\nElisabeth Hemberg 1 and Jane M. Morrell 2*\n1 Haddebo Seminstation, Hjortkvarn, Sweden, 2 Department of Clinical Sciences, Swedish University of\nAgricultural Sciences, Uppsala, Sweden\nPlacentitis (inflammation of the placenta) most commonly occurs during the last\ntrimester of pregnancy, frequently due to bacterial entry via the vulva. The outcome of\nthe pregnancy, i.e., prevention of abortion or the birth of a compromised foal, depends\non when treatment is initiated, the appropriate medication, and surgical correction of\nthe vulva to ensure an effective seal. In this study, during the period 2012–2024, 17\nmares were referred to the clinic, presenting with signs of placentitis, most commonly\npremature udder activity and/or discharge from the vulva. All mares maintained the\npregnancy after treatment, ultimately producing live foals that survived. The earliest\ncases involved mares with the most pronounced clinical signs; these mares received\ntreatment with poor perfusion into the placenta and delivered septicaemic foals. The\nremaining foals born exhibited only minor clinical signs or appeared healthy. For later\ncases, treatment with drugs providing good uterine perfusion continued until foaling,\nincluding trimethoprim sulphate twice a day and acetylsalicylic acid, a COX-1 anti-\ninflammatory drug, administered twice a day until parturition, if necessary. In addition,\nfor 16 mares, a vulvoplasty (Caslick’s operation) was performed, or was extended if\nthe mare had already undergone surgery.\nKEYWORDS\ncase report, equine placentitis, premature udder activity, vulval discharge, vulval\nconformation\n1 Introduction\nPlacentitis is inflammation of the placenta, most commonly due to a bacterial infection\n(1–3). The normal cervix and the lower reproductive tract, which includes the vagina, the\nvestibule, and the vulva (4), provide a physical barrier to the entry of foreign microorganisms\nto the foetoplacental unit (5). In ascending placentitis, the infection is caused by pathogens\n\nIn ascending placentitis, the infection is caused by pathogens\ngaining entry through the cervix (3). There are different types, depending on morphology and\npathogenesis: ascending via the vulva, haematogenous, and nocardioform (US). Regardless of\nthe cause, the infection results in an inflammatory reaction in the placenta and eventually in\nthe foetus. The most common cause is an ascending infection, with bacteria entering via the\nvulva (5), continuing through the cervical canal into the placenta and foetus.\nInsufficient sealing of the vulva can cause this problem since, in late pregnancy, the foetal\nweight can become heavy enough to render the vulval seal inadequate in some mares, allowing\nthe ingress of bacteria. Figure 1 shows some examples of an inadequate vulval seal while a\nnormal vulva is shown in Figure 2. The incidence of placentitis is estimated to be between 3\nand 7% (2). Most studies on placentitis in mares are induced rather than spontaneous and have\nbeen shown to be associated with uterine production of prostaglandins (PG) PGE2 and\nPGF2α. These can promote myometrial contraction and are a risk factor for premature\nOPEN ACCESS\nEDITED BY\nMallikarjun Bidarimath,\nNational Institute of Environmental Health\nSciences (NIH), United States\nREVIEWED BY\nHéctor Flores-Herrera,\nInstituto Nacional de Perinatología (INPER),\nMexico\nKalie Beckers,\nTulane University, United States\n*CORRESPONDENCE\nJane M. Morrell\njane.morrell@slu.se\nRECEIVED 11 March 2025\nACCEPTED 28 May 2025\nPUBLISHED 18 June 2025\nCITATION\nHemberg E and Morrell JM (2025) Case\nReport: Diagnosis and treatment of equine\nascending placentitis: compilation of 17 case\nreports.\nFront. Vet. Sci. 12:1591452.\ndoi: 10.3389/fvets.2025.1591452\nCOPYRIGHT\n© 2025 Hemberg and Morrell. This is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY). The use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\naccordance with accepted academic\npractice. No use, distribution or reproduction\n\nNo use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Case Report\nPUBLISHED  18 June 2025\nDOI  10.3389/fvets.2025.1591452\n\nHemberg and Morrell\b\n10.3389/fvets.2025.1591452\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nparturition. However, as there is a difference in the reaction of\ninflammatory blood parameters in induced placentitis compared to\nnaturally occurring placentitis (6), there is a need for reliable\ndiagnostic blood parameters for this condition.\nThe clinical signs of placentitis were described previously (5).\nPremature udder development and/or vulval discharge are regarded\nas indications for further investigation. In previous publications,\nudder development is not mentioned as a consistent sign of placentitis\n(2). If the influx of pathogens overcomes the natural barriers, an\ninflammatory reaction will ensue, (ascending placentitis), with or\nwithout a vulval discharge due to separation of placental tissue from\nthe endometrium. Thus, depending on the amount of placental\nseparation, vulval discharge may be an inconsistent finding (3).\nCommon diagnostic tools include an ultrasound examination of\nthe reproductive tract and examination of the cervix with the aid of a\nspeculum; these provide the clinician with further information on the\nextent of the inflammation. However, some authors advise against\nusing the speculum in placentitis cases (2) because of the risk of\nintroducing pathogens, while other authors suggest that it may\nbe acceptable if specifically indicated, such as in the presence of a\ndischarge (5). Nevertheless, examination with a speculum provides\nimportant information about the colour and the softening of the\ncervical portio. The healthy pregnant cervix should be closed to inhibit\nthe entry of foreign material and pathogens (7). The impact of the\npathogens may increase if no remedial action is taken, likely resulting\nin the loss of the pregnancy. The outcome of the pregnancy will\nbe favourable if treatment is provided as soon as clinical signs are\nobserved. However, many mare owners are unaware of the implications\nof the clinical signs, resulting in a delay before the mare is presented at\nthe clinic, and clinicians may not be certain about which treatment to\n\nHowever, many mare owners are unaware of the implications\nof the clinical signs, resulting in a delay before the mare is presented at\nthe clinic, and clinicians may not be certain about which treatment to\nuse. In general, treatment with antibiotics and anti-inflammatory\nagents known to cross the placenta is required; vulvoplasty (a “Caslick’s”\noperation) will reduce further ingress of bacteria.\nPoor vulval conformation can arise from a variety of causes such as\ncervical fibrosis, tears or adhesions, or from anatomical defects (8). A\nnon-sealed vulva can result in abortion of the foetus, most commonly at\n9–10 months of gestation, due to the increased weight of the foetus\nexacerbating the poor vulval seal (9). Therefore, it is surprising that so\nlittle attention is paid to vulval conformation for those mares that develop\nplacentitis, since correction of an inadequate vulval seal could prevent\nrecurrence of the infection later in pregnancy. Although some articles on\nthis subject are available, most describe induced placentitis with different\ndiagnostic methods, e.g., (10), and medical treatments (2, 3), focusing on\nwhich treatment is the most beneficial. There are different approaches\nregarding the duration of treatment and which antimicrobials and anti-\ninflammatory medications to use, e.g., trimethoprim sulfamethoxazole\n(TMS), alone or in combination with potassium penicillin G and\ngentamicin, or ceftiofur. Apart from TMS, these antibiotics have poor or\nno penetration into the foetal compartments; TMS has an allantoic fluid\nFIGURE 1\nSome examples of vulval conformation from mares presenting with placentitis in late pregnancy. (A) The mare described in Case 16, at day 310 of gestation.\nThe vulval plicae are angular and uneven, are not horizontal and do not have a uniform distance to each other from the top to the pelvic brim. (B) The mare\nin Case 17 at day 302 of gestation. The vulval plicae are more pronounced, showing the angular and uneven pattern clearly. (C) An 8-year-old trotting mare\n\n(B) The mare\nin Case 17 at day 302 of gestation.The vulval plicae are more pronounced, showing the angular and uneven pattern clearly.(C) An 8-year-old trotting mare\n(from 2025) at day 264, CTUP 11 mm, detachment of placenta, soft cervix, red discharge from vulva and an active udder. This vulval pattern has only minor\nangulation and unevenness, making the decision of the vulva sealing questionable, but the other clinical signs were indicative of a diagnosis of placentitis.\nThe clinical signs disappeared on treatment. This mare has not foaled yet and is therefore not included in the case report.\nFIGURE 2\nNormal vulva in late pregnant mares; note the regularly spaced, even\nplicae around the vulva. There was no udder activity and no\ndischarge in these mares but (A) (Case 5, day 296) had an increased\nCombined Thickness Uterus Placenta (CTUP), which was not caused\nby an ascending infection. (B) An 8-year-old North Swedish Draught\nHorse at day 255 of gestation, with no udder activity, no discharge\nand no increased CTUP. She has not foaled yet and is therefore not\nincluded in the case report.\n\nHemberg and Morrell\b\n10.3389/fvets.2025.1591452\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nconcentration equal to the concentration in serum (3). Of the anti-\ninflammatory medications, non-steroidal anti-inflammatory drugs such\nas flunixin meglumine or phenylbutazone are not known to penetrate into\nplacental fluid. Other NSAIDs that cross the placenta are acetylsalicylic\nacid and firocoxib (3). Synthetic progesterones, such as altrenogest\n(Regumate; Merck, Sharpe and Dohme Animal Health), alter the immune\nsystem systemically and locally within the endometrium (3). These\narticles do not mention the importance of the vulval seal; a defective\nvulval seal could be the main trigger for inducing placentitis.\nThe aim of the present report is to provide practitioners with\ndetailed clinical examples of the diagnosis and management of late-\nterm equine placentitis in the field, presenting with early development\nof the udder and/or discharge from the vulva. The treatments chosen\nin each case represent the best practice at the time, and changed\nsomewhat over time, reflecting increasing knowledge of drug\npharmacodynamics in the uterus.\n2 Diagnosis and treatment\nThe cases described here were mares with ascending placentitis,\nreferred to Haddebo Seminstation, Sweden, over a 12-year period (2012–\n2024). The 17 mares comprised 15 Swedish Halfbred and 2 Icelandic\nhorses. Seven of the mares were old maiden mares (mares that were\n>8 years old and had never had a foal). Four had previously aborted their\nfirst foetus and were now pregnant for the second time. The remaining six\nanimals were mares that had previously foaled. Their ages varied from 8\nto 20 years. A summary of each case is provided in Table 1.\nAll the mares presented with either premature mammary activity\nand/or vulval discharge. The vulval discharge was observed with\nplacenta detachment and appeared as dark blood-stained discharge,\neither as crystals on the vulval lips or dripping out through the vulva,\ndepending on the amount.\nOn arrival, a thorough examination of the vulva was carried out\nto assess its capacity to seal properly. The apposition of the vulval lips\n\nOn arrival, a thorough examination of the vulva was carried out\nto assess its capacity to seal properly.The apposition of the vulval lips\nwas regarded as if it were a zip. If there was any discontinuity in the\nlips of the vulva, i.e., in the edges of the vulval lips at the pelvic brim\nor folds at the edges, a deficient vulval closure was suspected, which\nwas likely to allow the ingress of bacteria (Figure 1).\nA gynaecological examination was performed to investigate the\nclosure of the cervix and to provide a measure of the combined thickness\nuterus placenta (CTUP) using ultrasound (11, 12). In mares with a soft\ncervix and increased CTUP for their gestational time, the cervix was\nexamined using a speculum (single-use speculum; Minitüb; Tiefenbach,\nGermany) after first washing the vulva thoroughly with betadine soap and\nusing a sterile vaginal cream. The appearance of the portio was noted; in\nsome cases, it was reddish and slightly open instead of pale and sealed.\nThe ultrasound examination also revealed the structure of the chorio-\nallantois and any detachment, the presence of a live foetus, and the\nappearance of the amniotic fluid. After 7 months of gestation, the CTUP\nis expected to be no more than 1 mm less than the month of pregnancy;\ni.e. if the mare is 9 months pregnant, the CTUP should be 8 mm (5).\n3 Individual case histories\nThe cases are presented in chronological order. For the earliest\ncases, no recommended evidence-based treatment protocols were\navailable. Cases 1–4 were treated with trimethoprim/sulfamethoxazole\n400/80 (TMS) intravenously at 15–30 mg/kg twice daily (Intervet/\nSchering-Plough Animal Health). Cases 2–4 were also given NSAID\n(Cronyxin; Bimeda; UK) 100 mg/mL, Flunixin (Rakshit, India),\n5–10 mL intramuscularly once daily.\n3.1 Case 1\nAn 8-year-old Icelandic mare had foaled the previous year but had\nbeen difficult to get in-foal again because of endometritis. The mare was\npresented at 311 days of gestation. There was a small amount of bloody\ndischarge from the vulva, and a full udder except for the teats. Placental\n\nThe mare was\npresented at 311 days of gestation.There was a small amount of bloody\ndischarge from the vulva, and a full udder except for the teats.Placental\ndetachment was evident on ultrasound examination; upon viewing with\na speculum, the cervix appeared red and was not properly sealed. The\nmare was given TMS intravenously for 1 day, and thereafter the owners\ncontinued oral treatment twice daily until foaling. Pneumovagina was\npresent; therefore, a vulvoplasty was performed, despite being so close to\nparturition. The mare started to produce milk on day 315 of gestation and\nfoaled the next day. The placenta was retained. The body of the placenta\nhad patches of atrophic villi, and the detached area was clearly visible close\nto the cervical star. The foal was bright and alert, with a Foalcheck score\nof >>8 g/L (SNAP Foal IgG, Idexx Laboratories, Holland). However, it\nneeded an enema for meconium constipation.\n3.2 Case 2\nThis 12-year-old mare was presented at 270 days of gestation. She\nhad respiratory problems due to sensitivity to being indoors. She was\ngiven additional Clenbuterol (Ventipulmin; Boehringer Ingelheim\nAnimal Health) to facilitate her breathing and to avoid contractile\nactivity in the uterus. The blood work showed the following results:\nSAA (<5 mg/L), WBC (8.2), and fibrinogen (3.2 g/L), all of which\nwere within normal values. The CTUP was 17 mm, and her udder was\nactive. She was given TMS twice daily intravenously and NSAID\nintramuscularly (Flunixin). Administration of Flunixin was stopped\nat 315 days as the mare was becoming more sensitive to injections.\nThere was no total regression of udder activity, and there was a small\namount of dark bloody discharge from her vulva. Two days before\nfoaling, the udder was leaking milk. The placenta had two detached\nareas, one close to the cervical star and the other at the base of the\npregnant horn. The umbilicus measured 95 cm. At birth, the\ncolostrum had a low IgG level (40 g/L); the foal was lethargic, the\nmucous membranes were slightly injected, but the blood work was\n\nThe umbilicus measured 95 cm.At birth, the\ncolostrum had a low IgG level (40 g/L); the foal was lethargic, the\nmucous membranes were slightly injected, but the blood work was\nwithin normal values (WBC 8,000, fibrinogen 2.7 g/L, SAA < 5 mg/L,\nFoalcheck 6 g/L). An indwelling catheter was sutured into the jugular\nvein. The foal was given 1 L hyperimmune plasma and antibiotics\n(benzyl penicillin four times daily and gentamicin (Ceva Animal\nHealth) once daily for 4 days). At this time, the umbilicus was dripping\nurine and was swollen, and the foal’s temperature was increasing.\nTherefore, the antibiotic was changed to ceftiofur for 10 days, with\ndaily washing of the umbilicus with chlorhexidine. Bacteriological\nculture from the umbilicus indicated the presence of Staphylococcus\nhemolyticum, which was sensitive only to enrofloxacin. At the time of\nthe bacterial result, the umbilicus was almost dry. All antibiotics were\nstopped as the blood results were within normal values, although local\ntreatment with chlorhexidine was continued for a further 2 days.\n\nHemberg and Morrell\b\n10.3389/fvets.2025.1591452\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nTABLE 1  Summary of mares presenting with ascending placentitis in chronological order from 2012 to 2024: clinical signs, treatment and outcome.\nCase\nAge of \nmare \n(years)\nGestation \nlength\nAnamnesis\nUdder \nactivity\nCTUP \nmm\nDischarge\nTreatment\nDay \nfoaling\nFoal \nstatus\nPlacenta\n1\n8\n311\nF\n++\nCTUP \nnot \nknown, \ndetached \na-c\n++\nTMS\nCaslick\n316\nMeconium \nretention\nRet sec, \npatches of \nnecrotic villi \nthroughout \nthe entire \nbody of the \nplacenta, \ncervical star \ndemarcation \nzone of the \nlocation of \ndetachment\n2\n12\n270\nF\n+++\n17\n+\nTMS iv NSAID\nVentipulmin\nCaslick \nextended\n333\nSepsis+\nPlasma\nLow IgG\nSeveral \ndetached \nareas with \nblood clots \nlocated at \nplacenta \nbody\n3\n15\n1. 265\n2. 295\nF, aborted first \nfoal previous \nyear\n1. +++\n2. 0\n1. 7\n2. 22\n3. Large \ndetached \na–c\n1. +++\n2. +\nTMS, NSAID\nVentipulmin, \nCaslick \nextended\n305\nSepsis+++\n30% body \nnecrotic villi \nmucous pus \nsmear, \ndemarcation \nzone, \nfunisitis\n4\n13\n1. 288\n2. 296\nOM\n++\n1. 7\n2. 10 \ndetached \na–c\nNAD\nTMS, NSAID\nCaslick \nextended\n323\nSepsis++\nCervical star \nweak \ndemarcation \nzone, surface \nmucous pus\n5\n13\n296\nF\n+++ \nunweaned \nfoal\n11\nNAD\nNo treatment\n333\nDysmature,\nPlasma\nlow IgG\nLymphatic \noedema \nplacenta, \numbilical \ncord 145 cm\n6\n9/10\n1. 120,\n2. 274\nOM\n2. +\n1. 9\n2. 14\nNAD\n1. TMS Caslick\n2. TMSX1\n329\nNormal\nNAD\n7\n13\n256\nOM\n++\n14\ndetached \na–c\n++\nTMS ASA\nCaslick\n342\nSepsis +\nRet sec, \ncervical star \noedema \nnecrotic villi\n8\n16/17\n1. 65,\n2. 175,\n3. 301\nF, d 28 poor \nblood \ncirculation and a \nthin umbilicus, \nd135 amnion \nfluid patches\n++ d 301\n1. 8,\n2. 13,\n3. 9.5\nNAD\nTMS ASA d \n65–142,\nCaslick \nextended\nd175–325\n325\nSepsis, \nplasma\nUterene wall \nvery \noedematous\n(Continued)\n\nHemberg and Morrell\b\n10.3389/fvets.2025.1591452\nFrontiers in Veterinary Science\n05\nfrontiersin.org\n3.3 Case 3\nDuring pregnancy in the previous year, this 15-year-old mare\nproduced a bloody discharge from the vulva, culminating in the loss of\nthe foal. She was presented at 265 days of the current gestation with\nbloody fluid issuing from the vagina. Examination with a speculum\nrevealed an inflamed cervix exuding reddish fluid. The udder was active,\nbut it was cold and floppy. Treatment with TMS x2 intravenously was\ncontinued for 31 days, with an additional treatment of benzyl penicillin\n3 g and gentamicin 100 mg/mL (10 mL mixed in 100 mL sterile water),\ninfused into the vagina. Flunixin (Cronyxin 10 mL) was administered\nintramuscularly for 10 days, 5 mL for 15 days, and 3.5 mL for 6 days; in\ntotal, the treatment was given for 31 days. After 2 days, the udder and\nvulval discharge regressed; on the 4th day of treatment, there was no\nvulval discharge and the udder was less tense. However, the clinical signs\nresumed after 2 days. The vaginal infusion was then stopped. In addition,\nclenbuterol was also administered orally twice a day for 31 days. The\nblood work showed a white blood cell count (WBC) of 7.2, differential\nneutrophils/lymphocytes 5.0/1.6, SAA < 5 mg/L, and fibrinogen\n2.2 g/L. On ultrasound examination, there was a large area (20 cm) of\nplacental detachment, but the foetus was lively. On examination with a\nspeculum on the 8th day after treatment started, the cervix was pink and\nclosed, with no bloody discharge. On Day 14 after treatment started, the\nudder had regressed completely and there was no discharge from the\nvulva. However, on ultrasound examination on Day 26, the CTUP was\n22 mm, and the foetus was not as active. Pus was noticed from the vulva\non Day 25, with the bacterial growth identified as Enterococcus faecalis\nand yeast. By Day 28, the udder was developing normally, and no pus was\nseen from the vagina. On Day 30, procaine penicillin (Ethacillin vet) was\nadministered intramuscularly for 12 days. On Day 34, there was pus\n\nBy Day 28, the udder was developing normally, and no pus was\nseen from the vagina.On Day 30, procaine penicillin (Ethacillin vet) was\nadministered intramuscularly for 12 days.On Day 34, there was pus\ncoming from the vagina again; by Day 40, pentoxifylline (Trental, no\nlonger available) was added to the treatment, given orally at 10 tablets\ntwice a day. The mare foaled the same day at 305 days of gestation. The\nentire placenta was hyperaemic and 30% of the placental body was\nTABLE 1  (Continued)\nCase\nAge of\nmare\n(years)\nGestation\nlength\nAnamnesis\nUdder\nactivity\nCTUP\nmm\nDischarge\nTreatment\nDay\nfoaling\nFoal\nstatus\nPlacenta\n9\n12\n294\nOM\n+++\n14.5\nNAD\nTMS ASA\nCaslick\n321\nNormal\nplasma low\nIgG\nNAD\n10\n11\n315\nF\n+\n11.05\n++\nTMS ASA\nCaslick\n332\nNormal\nplasma low\nIgG\nNAD\n11\n20\n180\nF, first foal.\nSeptic foal that\ndied two years\npreviously\nNAD\n4.7\n++\nTMS\nCaslick\n328\nNormal\nNAD\n12\n13\n282\nF, First foal “red\nbag” with dead\nfoal 2 yrs. before\n++\n10\nNAD\nTMS ASA\nCaslick\n326\nNormal,\nplasma\nNAD\n13\n17\n262\nOM\n++\n15\nNAD\nTMS ASA\nCaslick\n320\nMeconium\nretention\nNAD\n14\n15\n0.300\nF\n++\n10–14\ndetached\na–c\nNAD\nTMS ASA\nCaslick\n322\nNormal\nNAD\n15\n16\n251\nOM\n+\n11\n++\nTMS ASA\nCaslick\nextended\n324\nNormal\nNAD\n16\n14\n310\nF Aborted first\nfoal 2 years\npreviously\n+++\n17\n+\nTMS ASA\nCaslick\n333\nNormal\nplasma\nNAD\n17\n17\n302\nF\n++\n11.5\nNAD\nTMS ASA\nCaslick\n345\nNormal\nplasma\nNAD\nGestation length: days of pregnancy at examination.\nOM = Old maiden mare, F = mare at foot.\nUdder activity; + = teats swollen, ++ = teats and udder swollen, +++ = full udder, milk; NAD = no abnormality detected.\nCTUP = combined thickness uterus and placenta (mm); detachment of allanto-chorion (a–c) is noted.\nDischarge: + sparse reddish discharge, ++ moderate reddish discharge, +++ profuse discharge. 0 = no discharge.\nTreatment: TMS = trimethoprim sulphate, NSAID = non-steroidal anti-inflammatory drug; Flunixin meglumin, ASA = acetylsalicylic acid, Caslick = vulvoplasty.\nFoaling days: gestational day when the mare foaled.\nFoal status; ++ = moderate-severely affected, foal cannot keep a normal feeding schedule, moderate-high inflammatory blood results.\n+++ very sick foal, needs help to stand up, cannot keep normal feeding times, high inflammatory results.\nPlacenta: Ret sec = retained placenta, the mare did not expel the placenta within 3 h postpartum, 0 = normal placenta.\n\nHemberg and Morrell\b\n10.3389/fvets.2025.1591452\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nFIGURE 3\nPlacenta from Case 3. (a) Shows the cervical star and its surrounding area, the effect of the necrotising inflammation of the chorioallantois and the\ndemarcation zone to somewhat more healthy-looking tissue. (b) Shows the thick mucus exudate that covered large areas of the chorion.\ncovered in mucus; there was an abundance of thick, necrotic villi and a\nsharp demarcation zone to the more normal placenta (Figure 3a); the\numbilical cord was thick and hyperaemic. The villi of the placenta were\ncovered with a thick mucus smear (Figure 3b).\nThe foal was very weak, with violet–blue mucous membranes,\nopisthotonic seizures and omphalitis. An intravenous catheter was\nsutured into the jugular vein; the foal was given 5 mg/kg Ceftiofur\n(Excenel; Zoetis Sweden) twice within 10 min after birth and 1 L of\nhyperimmune plasma within 40 min after birth, as the colostrum IgG was\nonly 30 g/L. The foal was fed milk from a bottle every 20 min to encourage\nthe suckling reflex. The foal drank readily and gained strength quickly,\nbeing able to stand by itself after 12 h, although it could not maintain its\nfeeding schedule. Therefore, the foal was encouraged to get up every\n20 min. The Foalcheck was >8 g/L after plasma infusion, WBC 9.7,\ndifferential 7.9/1.0, fibrinogen 4.9 g/L, SAA < 5 mg/L. An ultrasound\nexamination of the umbilicus showed thickening from the urethra to the\nbladder, resulting in the foal urinating small amounts frequently. On the\nsecond day, omeprazole (Gastrogard) was added to the medication list;\nthe colour of the mucous membranes was improving to a more pinkish\nhue, but the foal still needed help to maintain its feeding schedule. On the\nthird day, the foal was taken out for a short walk to improve the strength\nof its joints. Blood work on the third day showed that the WBC was 10.4,\ndifferential 8.5/1.3, fibrinogen 4.3 g/L, SAA < 5 mg/L. A foul-smelling\ndiarrhoea started on the fourth day, with the foal being alternately\n\nBlood work on the third day showed that the WBC was 10.4,\ndifferential 8.5/1.3, fibrinogen 4.3 g/L, SAA < 5 mg/L.A foul-smelling\ndiarrhoea started on the fourth day, with the foal being alternately\nlethargic and very bright. The diarrhoea was treated with Diarsanyl (oral\npaste; CEVA, France), along with intravenous Ringer’s acetate and glucose\naccording to the packed cell volume (PCV) and blood glucose level.\nOn the 7th day, gentamicin (100 mg/mL) at 6.6 mg/kg was added to\nthe treatment. The blood work showed WBC 10.5, differential 7.6/1.8,\nfibrinogen 5.4 g/L, SAA < 5 mg/L, and creatinine 85 μmol/L. The culture\nfrom the umbilicus showed growth of Staph vitulinus, Staph hemolyticum,\nand Enterococcus faecalis. On the 8th day, the umbilicus started to drip\nurine again, but the diarrhoea had stopped. On day 10, the umbilicus was\nstill swollen and dripping urine. The intravenous treatment was changed\non day 11 to Clarithromycin (Klaritromycin tablet; Orion Pharma,\nSweden) 500 mg twice daily and Rifampicin (Rimactan; Sandoz, Denmark)\n600 mg once daily per orally for 8 days, according to the bacterial\nsensitivity. The swelling of the umbilicus decreased significantly within\n2 days. Blood work showed WBC 11.6, differential 8.0/2.7, SAA < 5 mg/L,\nand fibrinogen 3.2 g/L. The amount of omeprazole was kept the same in\norder to decrease the dose per kilogram as the foal’s weight increased. By\nday 19, the foal was judged to be healthy; at this time, the WBC was 9.1,\ndifferential 6.0/2.4, fibrinogen 3.1 g/L, and SAA < 5 mg/L.\n3.4 Case 4\nA 13-year-old mare had an active udder at 288 days of gestation\nand was treated with NSAID Flunixin (Cronyxin) once a day and TMS\ntwice a day. The udder regressed 5 days after treatment started. The\nFlunixin was given for a total of 16 days, at a high dose (10 mL) only\non the first day and thereafter at a lower dose (5 mL) for 13 days,\nfollowed by 3.5 mL for the last 2 days. The TMS 400/80 intravenously\nwas continued for 23 days. At foaling, the mare showed poor abdominal\ncontractions. The foal was born in a twisted position, was cold and\n\n5 mL for the last 2 days.The TMS 400/80 intravenously\nwas continued for 23 days.At foaling, the mare showed poor abdominal\ncontractions.The foal was born in a twisted position, was cold and\nalmost lifeless, with injected mucous membranes. It was treated with\nCeftiofur (Excenel injectable; Zoetis Sweden) at a dose of 10 mg/kg\ntwice a day, with the first injection being given <10 min after birth, and\nwith plasma administered within 40 min after birth. The Foalcheck\nshowed >>8 g/L, WBC 17.4 with a differential of neutrophils at 16.1,\nSAA 282 mg/L, and fibrinogen 2.9 g/L. The suckling reflex was poor\ninitially but became stronger after 2 h. The foal’s posture was initially\nhypotonic, and it needed assistance to rise. After 12 h, it could get up\nwith help to maintain 20-min feeding intervals, and it was observed\n\nHemberg and Morrell\b\n10.3389/fvets.2025.1591452\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nnursing well. It was given 400 mg DMSO in 1 L of 5% glucose on the\nfirst and second days of life, omeprazole orally (Gastrogard oral paste;\nBoehringer Ingelheim, Germany) for 10 days, and diarsanyl 2–3 times\na day for 7 days. On the third day, it was taken out to pasture; it could\nrise unaided and maintained its feeding times every 20 min with the\nhelp of the mare. The foal was much brighter and more alert. On the\nfifth day, it was coughing and had a fast respiratory rate. Ultrasound\nexamination of the chest revealed three broken ribs, although no\npneumonia was detected, with respiratory crackles only from the\ntrachea. The SAA was 399 mg/L. A tracheal wash was performed. The\nantibiotic was changed to benzyl penicillin (Sandoz GmbH) four times\na day and gentamycin once a day intravenously for 2 days until the\nresults of the bacteriological culture were ready, showing a pure culture\nof β-haemolytic Streptococcus. Treatment continued with procaine\npenicillin (Merck, Sharpe and Dohme Animal Health) at a dose of\n300 mg/mL, 3.5 mL once a day for 6 days intramuscularly. The\nincreased respiratory rate was judged to be caused by the broken ribs.\nThe foal was given box rest with the mare for 3 weeks. All treatments\nwere finished on day 11, as the blood work was normal at that time.\n3.5 Case 5\nThis 13-year-old mare arrived at 296 days of gestation for foaling. She\nhad milk in the udder, and the owner was unsure when the previous year’s\nfoal had been weaned. On ultrasound, there was oedema in the placenta,\nCTUP 11 mm; the cervix was hard and closed, and the vulva was sealing\nvery well. Since the timing of weaning was unknown, it was decided to\nwait to see if the udder would regress naturally, which occurred within\n3 days. The mare foaled at 333 days. The placenta was excessively\noedematous, with large patches of fluid outside the vessels, both on the\nplacenta itself (Figure 4a) and on the umbilical cord (Figure 4b). The\numbilical cord was 145 cm, which could have caused the oedema of the\n\nThe placenta was excessively\noedematous, with large patches of fluid outside the vessels, both on the\nplacenta itself (Figure 4a) and on the umbilical cord (Figure 4b).The\numbilical cord was 145 cm, which could have caused the oedema of the\nplacenta (Sandra Wilsher, personal communication to EH). The foal was\ndysmature, with yellowish mucous membranes, and the joints on both\nfront and hind legs were weak. The foal needed help to get up. It nursed\nvery hungrily. The mare did not have any colostrum, producing only thin\nmilky fluid (40 mg/mL); therefore, the foal received an infusion of\nhyperimmune plasma, S-LD 3.5 mmol/L. The foal was given TMS\nintramuscularly for 3 days. By Day 3, the S-LD was 1.2 mmol/L, and the\nfoal was alert and judged not to require further treatment.\n3.6 Case 6\nA 9/10-year-old maiden mare presented at day 120 of gestation.\nUpon arrival at the clinic, the mare had a swollen vulva with faecal\ntraces inside the vagina. There was no udder development, but some\nventral oedema close to the udder; CTUP was 9 mm, with separation\nof the placenta ventral to the cervical star. A vulvoplasty was\nperformed, and oral antibiotic treatment with TMS twice daily\n(Elanco, Denmark) was initiated. The mare returned for a check-up\nafter 12 days. The vulva and the ventral oedema had disappeared; the\nCTUP was 5.4 mm, and the vulvoplasty was elongated at this time.\nThree months later, at 274 days of gestation, the mare kicked the\nowner badly and was subsequently given TMS only once daily until\nfoaling. Udder development prior to foaling proceeded normally. The\nmare foaled at day 329, with a healthy foal.\n3.7 Case 7\nA 13-year-old maiden mare was presented at day 256 with a full\nudder (3+), including the teats, which had developed over the last month,\nand a red discharge from the vulva. On ultrasound, the CTUP was\n14.3 mm, there was ventral detachment of the placenta and allantoic folds,\nand the cervix was not properly closed and was soft. A vulvoplasty was\nperformed, and treatment was initiated with oral TMS twice daily. Two\nweeks later, there was no discharge from the vulva, and the CTUP\n\nA vulvoplasty was\nperformed, and treatment was initiated with oral TMS twice daily.Two\nweeks later, there was no discharge from the vulva, and the CTUP\nmeasured 18 mm close to the cervix, although it was only 8 mm ventrally;\nthe cervix was closed, and the allantoic fluid contained flares.\nAcetylsalicylic Acid (ASA; Bamyl, tablets, DrPfleger Arzneimittel GmbH,\nGermany) was added to the treatment 10 g twice daily orally, together\nwith TMS until foaling. The udder was not tense but had increased in size.\nAt 305 days, the mare returned to the clinic to stay until foaling; the CTUP\nwas 7 mm, the cervix was softer, and there was a sparse bloody discharge.\nOne week later, at 312 days of gestation, the CTUP was 9.8 mm, the\nallantoic folds could not be seen, the cervix was closed, and the udder had\nalmost completely regressed. Two weeks later, at day 326 of gestation,\nnormal pre-parturition udder development began. She foaled at 342 days.\nThe placenta was retained, the cervical star was thick, and the body and\npregnant horn showed abundant necrotic villi (Figure 5). The foal was\nable to stand within 1 h and nursed in <2 h, but it was unable to maintain\na normal feeding schedule after 6 h and was lethargic. The Foalcheck\nresult was >>8 g/L, S-LD 7.2 g/L, SAA < 5 mg/L, WBC 15,300, and\nfibrinogen 1.8 g/L, indicating septicaemia. The mucous membranes were\npale greyish. An indwelling catheter was sutured in place, and antibiotics\n(benzyl penicillin four times and gentamicin once) and hyperimmune\nplasma were administered. The foal appeared brighter after 2 h. By the\nsecond day, the foal was extremely hungry, and on the third day, it could\nmaintain its feeding times (three times per hour). The blood work showed\nSAA < 5 g/L, WBC 9.4, differential 6.3/2.6, and fibrinogen 2.8 g/L. The\nantibiotic treatment was concluded after day 5.\n3.8 Case 8\nA 16/17-year-old mare with a history of being barren for 5 years was\ndiagnosed with pituitary pars intermedia dysfunction (PPID; previously\nknown as Cushing’s syndrome) at another equine clinic and was treated\n\n3.8 Case 8\nA 16/17-year-old mare with a history of being barren for 5 years was\ndiagnosed with pituitary pars intermedia dysfunction (PPID; previously\nknown as Cushing’s syndrome) at another equine clinic and was treated\nwith the dopamine agonist Pergolide (Boehringer Ingelheim Animal\nHealth, tablet 1 mg). The mare had endometritis at that time and required\ntreatment to become pregnant. She was then presented at the author’s\nclinic at day 28 of gestation. The umbilical cord was considered to be thin,\nand treatment with TMS once daily was initiated until day 40; at day 65,\nthe placenta and the amnion wall were oedematous with folds. The TMS\ntreatment was increased to twice daily and prolonged. By day 91, the\ncervix was tight, and the foetus size was within the normal parameters of\na 90-day pregnancy. Treatment was supplemented with TMS and ASA\nand continued for another 3 weeks; it was stopped at day 111. The mare\nreturned for a check-up at day 142, at which stage the CTUP was 4 mm,\nand the cervix was closed. When the mare returned 6 weeks later (day\n182), the CTUP was 13 mm, there was oedema around the cervix, the\ncervix was soft, and there were moderate flares in the amniotic fluid.\nTreatment with TMS twice daily and ASA twice daily was initiated again.\nThree months later (day 272), she was presented for a check-up; the\nCTUP was 8.9 mm, there were no flares in the amniotic fluid, and the\ncervix was closed. Treatment was continued until she foaled. Udder\n\nHemberg and Morrell\b\n10.3389/fvets.2025.1591452\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nFIGURE 4\nThe placenta from Case 5. (a) Shows the extra vascular fluid around the placenta vessel on the allantoic surface. (b) Shows the umbilical cord, which\nwas 145 cm and also had large pockets of extra vascular fluid.\ndevelopment started at 303 days, at which time the CTUP was 9.5 mm.\nThe mare foaled at 329 days. The endometrium was heavily oedematous\nafter foaling. The foal’s mucous membranes were brick-coloured, and it\nwas lethargic; sepsis was suspected. An indwelling catheter was sutured\ninto the jugular vein; antibiotic treatment with crystalline penicillin four\ntimes daily and gentamicin once daily was started, and 1 L hyperimmune\nplasma was given. The Foalcheck result was 8 g/L, S_LD 3.3 mmol/L,\nwhich is above the normal value (1.2–2.6 mmol/L). Since the foal was\nborn during a weekend, other bloodwork was postponed until the\nbeginning of the following week. Two days later, the S-LD was 1.2 mmol/L,\nWBC was 6.3, diff 4.9/1.2, fibrinogen 3.1 g/L, and SAA < 5 mg/L. The foal\nwas able to keep his feeding times and was alert. The antibiotic treatment\nwas stopped after 5 days. The foal had a loose stool and was therefore\ngiven omeprazole paste for 5 days.\n3.9 Case 9\nA 12-year-old mare was presented at 294 days of gestation with a\nfull udder and teats, with crystallised fluid at the end of the teats. On\nultrasound, the CTUP was 14 mm, and the cervix was not closed. A\nvulvoplasty was performed, and TMS x2/day and AS Ax2/day were\nadministered until foaling. Three days later, the udder was dripping\nmilk. The mare foaled at 321 days, producing a healthy foal. The\nFoalcheck was <<8 g/L according to the attending veterinarian, and\nthe foal was therefore given 1 L of plasma in the jugular vein.\n3.10 Case 10\nThis 11-year-old mare was presented at 315 days with swollen teats\nand a red mucous discharge from the vulva. On ultrasound, the CTUP\nwas 11.4 mm, the cervix was not closed, and the right vulval lip above the\npelvic rim had a tear that had not been repaired from a previous foaling.\n\nOn ultrasound, the CTUP\nwas 11.4 mm, the cervix was not closed, and the right vulval lip above the\npelvic rim had a tear that had not been repaired from a previous foaling.\nA vulvoplasty was performed, and TMS twice daily and ASA twice daily\nwere administered until foaling. She foaled 17 days later, at 332 days. The\nfoal was healthy, and the Foalcheck result was >8 g/L.\n3.11 Case 11\nAn Icelandic mare, 20 years old at presentation, had a history of\nproducing a septic foal 2 weeks early at an equine hospital 2 years\npreviously, which subsequently died. The mare was presented at 180 days\nof gestation, with a yellow discharge from the vulva of 1 week’s duration;\nthe CTUP was 4.7 mm, and the cervix was open towards the portio. On\nFIGURE 5\nPlacenta from Case 7, showing the chorionic side with an abundance\nof necrotic villi which covered a large portion of the placenta.\n\nHemberg and Morrell\b\n10.3389/fvets.2025.1591452\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nexamination with a speculum, the vagina was inflamed, and the cervix\nwas open 1(+); at the cervix, there was marmorated sticky secretion. There\nwere old tears on both edges of the vulva. The secretion was sampled and\nsent to a certified laboratory, where a small amount of growth of\nEnterococcus faecalis was found. A vulvoplasty was performed, and\ntreatment with TMS x2/day was initiated. The mare returned for a\ncheck-up 3 weeks later, at day 188 of gestation; the vulvoplasty needed\nelongation. The CTUP was 5.7 mm; on examination with a speculum, the\ncervix was closed, and there was no secretion. The owner preferred to\nadminister TMS x1, but 4 days later, the mare had a yellow vulval\ndischarge again, and TMS was increased to x2/day. The mare foaled at day\n328 of gestation. The Foalcheck result was >>8 g/L, WBC 8.2, differential\n5.5/2.4, SAA < 5 g/L, fibrinogen 2 g/L. The foal was healthy.\n3.12 Case 12\nA 13-year-old mare presented at 282 days of gestation with a swollen\nudder (except for the teats) of 1 day’s duration. Her first foal, 2 years\npreviously, had been a “red bag” delivery, i.e., premature detachment of\nthe placenta. The foal died shortly after birth; the mare was treated for\nendometritis due to Escherichia coli infection, and vulvoplasty was\nperformed. She was barren that year. Now, she was showing similar\nclinical signs as in the previous pregnancy. On ultrasound examination,\nthe CTUP was 10 mm and the cervix was soft. Treatment with TMS twice\ndaily and ASA twice daily was started. The vulvoplasty was extended.\nWhen re-examined at 303 days of gestation, the udder was much smaller\nbut still active, and the CTUP was 12 mm. Ten days later, the udder was\nsecreting transparent fluid, the CTUP was 10 mm, and the cervix was\nnow closed. The mare foaled 13 days later at 326 days of gestation. The\nplacenta had necrotic villi. The colostrum IgG, measured using a Brix\nrefractometer, correlated to an IgG concentration of 28 g/L, which is\n\nThe mare foaled 13 days later at 326 days of gestation.The\nplacenta had necrotic villi.The colostrum IgG, measured using a Brix\nrefractometer, correlated to an IgG concentration of 28 g/L, which is\nregarded as an adequate level (13). The foal was bright and alert. After\nplasma infusion, the Foalcheck was >>8 g/L, WBC 8.4 diff 5.6/2.4,\nfibrinogen 2 g/L, SAA 7 mg/L. The foal remained healthy.\n3.13 Case 13\nA 17-year-old maiden mare was presented with an active udder;\nthe CTUP was 15.7 mm, there was oedema at the entrance of the\nallantois, and the cervix was soft. On examination with a speculum,\nthe vagina was red, and the cervix was open (2+). A vulvoplasty was\nperformed, and the mare was treated with TMS twice daily and ASA\ntwice daily until foaling. Three weeks later, the udder had regressed,\nand there was no vulval discharge. The mare produced a dysmature\nfoal at 320 days. The foal had poor muscle development, weak fetlocks,\nan umbilical hernia, and meconium constipation. The Foalcheck result\nwas >>8 g/L, SAA 38 mg/L, WBC 8.3 diff 65.5/1.4, fibrinogen\n2.8 g/L. The constipation was treated with an enema of acetylcysteine,\nbaking soda, and water. The foal remained healthy.\n3.14 Case 14\nThis mare, 15 years old, had produced a stillborn foal the previous\nyear, which was revived after intensive resuscitation. The mare arrived\nat day 300 of the current gestation, with a cold and active udder. There\nwas a CTUP of 14 mm with oedema and ventral separation of the\nplacenta. On examination with a speculum, the cervix was closed but\nhad a red colour. Treatment of TMS twice daily and ASA twice daily\nwas administered until foaling. A week after starting the treatment,\nthe udder had regressed completely. The mare foaled a healthy foal at\n322 days. The Foalcheck result was 8 g/L, and all blood parameters\nwere normal: WBC 12,200, SAA < 5 g/L, and fibrinogen 2.2 g/L.\n3.15 Case 15\nA 16-year-old maiden mare that had previously received a\nvulvoplasty was presented at day 251 with a bloody vulval discharge and\nan active udder; the CTUP was 11 mm. On ultrasound examination,\n\n2 g/L.3.15 Case 15\nA 16-year-old maiden mare that had previously received a\nvulvoplasty was presented at day 251 with a bloody vulval discharge and\nan active udder; the CTUP was 11 mm.On ultrasound examination,\nthe cervix appeared tight but was not closed when examined with a\nspeculum. The vulvoplasty was extended. The mare was treated with\nTMS x2/day and ASA x2/day until she foaled at 324 days. The foal was\nhealthy at birth; the Foalcheck result was >>8 g/L, WBC 9.1, differential\n7.8/0.9, fibrinogen 2.4 g/L, SAA > 5 mg/L.\n3.16 Case 16\nA 14-year-old mare, which aborted a dead foal 2 years previously, was\npresented at day 310 of gestation, with a well-filled udder and small crystal\ndrops around the vulval edges. The owner reported that the mare had\nexhibited similar signs when she aborted in the previous pregnancy. The\nCTUP was 17 mm, with fluid accumulation ventral to the cervix. On\nexamination with a speculum, the cervix was reddish and open. Since\npneumovagina was diagnosed (Figure 4A), a vulvoplasty was performed\nand treatment with TMS twice daily and ASA twice daily was initiated.\nThe udder started to regress a week after treatment began. The mare\nfoaled at home at 333 days of gestation. The Foalcheck result was >8 g/L,\nbut hyperimmune plasma was administered as well as antibiotics,\nCeftiofur, for 6 days by the attending veterinarian due to the mare’s\nprevious history.\n3.17 Case 17\nA 17-year-old maiden mare was presented at 302 days of gestation\nwith an active udder of 1 day’s duration and a small amount of reddish\nfluid coming from the vulva (Figure 4B). The CTUP was 11.5 mm. The\nvulva was sutured, and treatment with TMS twice daily and ASA twice\ndaily was initiated. She foaled at day 345. The foals’ blood work was\nnormal; the Foalcheck result was >>8 g/L. WBC 7.5, differential\n4.8/2.5, SAA < 5 g/L, fibrinogen 2.1 g/L. Hyperimmune plasma\ninfusion was given at the owner’s request.\n4 Summary of case histories\nIn these 17 cases, clinical signs were observed at an average of day 279\nof pregnancy. Sixteen of the mares received trimethoprim sulphate twice\n\n1 g/L.Hyperimmune plasma\ninfusion was given at the owner’s request.4 Summary of case histories\nIn these 17 cases, clinical signs were observed at an average of day 279\nof pregnancy.Sixteen of the mares received trimethoprim sulphate twice\ndaily either intravenously or orally. One difficult mare was only given TMS\norally once daily. Three mares were also given NSAIDs intramuscularly\nonce daily, and 10 mares were given acetylsalicylic acid twice daily until\nthey foaled. The two Icelandic Horses were not given anti-inflammatory\ntreatment at the owners’ request. For the five mares that had already been\noperated on, their vulvoplasty was extended. A vulvoplasty was carried out\n\nHemberg and Morrell\b\n10.3389/fvets.2025.1591452\nFrontiers in Veterinary Science\n10\nfrontiersin.org\nin 11 mares that had not previously been operated on. None of these\npregnant mares were negatively affected by the use of a speculum for the\nvaginal examination, in contrast to the suggestion of (2).\nOne mare was not treated with vulvoplasty as her vulva had a perfect\nclosure; her CTUP was increased for her gestational age, and her active\nudder was a result of a late-weaned foal the day before her arrival. This\nwas confirmed as the correct diagnosis when she subsequently foaled at\n333 days, although her placenta showed generalised oedema with free\nfluid around the placental and umbilical cord vessels. Furthermore, the\numbilical cord measured 145 cm (Figures 3a,b). The foal was dysmature,\ni.e., it did not exhibit the usual behavioural patterns for a newborn foal,\nsuch as getting up within an hour, nursing within 2 h, and showing a brisk\npresentation at 4 h (14), but did not have an infection.\nAll the referred mares, except for two, foaled at more than 320 days,\nwhich is regarded as being within the normal range of foaling (15). The\nplacenta appeared normal upon expulsion in 10 cases. One of these (as\nalready mentioned) had an excessive amount of oedema outside the\nplacental vessels and the umbilical vessels. Seven mares were previously\nfoaling mares, four mares (cases 3, 11, 12, 16) had never given birth to\na live foal, and six mares were old maiden mares. Of the 17 mares, 16\nwere observed to have some degree of premature udder development\non arrival. Case 11 was the only mare without premature udder\ndevelopment. Five mares had fully developed udders and were\nproducing milk. Of these, one foal had been weaned just prior to\nsending the mare to the clinic. Eight mares had some fluid discharge\nfrom the vulva, two of which also showed placental detachment. The\ndischarge was reddish in all mares.\nThe presenting signs observed in these mares and foals are shown\nin Table 1. The length of pregnancy ranged from 175 to 320 days. The\nCTUP measured 5–17 mm (mean 13 mm), with mares pregnant at\n\nThe\ndischarge was reddish in all mares.The presenting signs observed in these mares and foals are shown\nin Table 1.The length of pregnancy ranged from 175 to 320 days.The\nCTUP measured 5–17 mm (mean 13 mm), with mares pregnant at\n175 days (<6 months) having a CTUP of 5–7 mm, at 255–295 days\n(8.5–10 months) 11–22 mm, and at 300–320 days (10–<11 months)\n7–17 mm (11). Of the 17 mares, 5 had undergone vulvoplasty (Caslick),\nwhich had been performed in the autumn of the previous year at their\npregnancy check. In five mares, the placentas exhibited necrotic villi and\ndetached areas, with a demarcation zone close to the cervical star and/\nor oedema in the placenta.\nFive foals were septic at birth, two had meconium constipation, and\nfour mares produced colostrum with low IgG. The most septic foals\nwere those from mares that had exhibited clinical signs of placentitis the\nlongest before being presented and did not receive any anti-\ninflammatory drug that could penetrate the placenta. All five foals\nreceived intravenous antibiotics via an indwelling catheter sutured in\nplace 20���min after parturition. They also received plasma within 40 min\nof birth. All five foals had a weak suckling reflex but drank readily. They\nwere bottle-fed every 20 min and encouraged to drink as much as they\nwanted until they could drink unaided and nurse regularly. Ten foals\nwere born healthy, of which four received plasma at the owner’s request.\nThe blood results could not be obtained for two foals directly after\nfoaling because it was the weekend, or the mares foaled at another\nlocation (cases 6 and 16) where such investigations were not performed.\nOne foal (Case 3) showed a high level of fibrinogen within the first day,\nindicating that the inflammation started in utero, as it takes 48 h for\nfibrinogen to react (16). When the mares foaled over the weekend\nwithout access to a laboratory, a field device, Lactate Pro 2 (Arkray\nFactory Inc., Japan), was used to measure the L-lactate (cases 5, 7, 8).\nIncreased blood or plasma L-lactate can be  useful in predicting\nmorbidity and mortality in septic foals (17).\n\n, Japan), was used to measure the L-lactate (cases 5, 7, 8).Increased blood or plasma L-lactate can be  useful in predicting\nmorbidity and mortality in septic foals (17).\nAll 17 foals survived and went home healthy; they later became\nriding horses. For most mares arriving with an active udder, with or\nwithout milk, a regression of udder activity was noted a week after the\nstart of treatment.\nCases 2, 3, and 4 were the foals most severely affected by\nsepticaemia. These were the mares presenting with the worst clinical\nsigns. Their placentas had large areas of detachment, necrotic villi, a\nmucous pus smear on the surface, and a demarcation zone between\nthe affected and healthy tissue at or close to the cervical star.\n5 Discussion\nThe following reflections arose from the management of these\ncases and are presented here with the aim of assisting other\npractitioners. The purpose of the treatment was to obtain an early\nresolution of clinical signs to maintain the pregnancy past 320 days\nand to deliver a viable foal. Moreover, the aim of the vulvoplasty was\nto prevent the ingress of bacteria via an incompletely sealing vulva and\nto prevent further inflammatory reactions. The anti-inflammatory\ndrugs that can penetrate the placenta are COX 1 (ASA) and COX 2\n(Firocoxib). All the mares responded well to TMS (twice daily),\ndespite the previous administration of other antibiotics at the time of\ncovering or artificial insemination. None of these mares were treated\nwith Altrogenest, although there is a recommendation to administer\nthis product to help maintain the pregnancy (2, 3).\nAt the time of the first cases (2012–17), little was known among\nhorse owners about placentitis, and these mares may have shown signs\nfor a prolonged period before being presented at the clinic. Bernard\net al. (18) also reported that mares with placentitis are often presented\nlate in the course of the condition. Furthermore, during the earlier\ncases, there was a paucity of knowledge about which drugs could\npenetrate the placenta. Therefore, these mares were treated with a\n\n(18) also reported that mares with placentitis are often presented\nlate in the course of the condition.Furthermore, during the earlier\ncases, there was a paucity of knowledge about which drugs could\npenetrate the placenta.Therefore, these mares were treated with a\nnon-steroidal anti-inflammatory drug (NSAID) to complement\ntrimethoprim sulphate (TMS), which most likely did not provide an\nappropriate anti-inflammatory effect. These mares foaled at 305, 316,\n324, and 333 days. Their foals showed signs of prematurity with high\ninflammatory blood parameters and needed intensive care to survive.\nAcetylsalicylic acid (ASA) was shown to be an effective COX 1\nanti-inflammatory with penetration into the placenta (19). It was also\nshown to increase blood flow into the placenta, which could\nbe favourable in a pregnancy in an old maiden mare. Treatment with\nCOX 1 has been suggested for women for its vasoactive properties\n(19); administration to mares could reduce the risk of intrauterine\ngrowth restriction (IUGR), commonly occurring in foals from old\nmaiden mares (20). However, ulceration in the gastrointestinal (GI)\ntract could be a complication of long-term medication, and uterine\ncontractions could be  negatively affected. None of the 10 mares\ndescribed here showed any signs of ulcers in the GI tract during\ntreatment and had no problems during foaling.\nSome mares, which had received a vulvoplasty during the breeding\nseason, still presented with signs of placentitis and needed an elongation\nof the original vulvoplasty. The mares with a shorter duration of signs gave\nbirth to healthy foals. One mare that had milk in her udder at 10 months\nwas shown to have weaned her foal the day before arriving, and she\nexhibited an increased CTUP, but her vulval conformation was perfect.\nThis mare’s placenta was very oedematous. Therefore, it is important to\ninform the owner, in the case of an older multiparous mare or a high-risk\n\nHemberg and Morrell\b\n10.3389/fvets.2025.1591452\nFrontiers in Veterinary Science\n11\nfrontiersin.org\nmare, to observe the udder and vulva closely to detect the first signs of\nplacentitis. If there is any early reaction of the udder or discharge from the\nvulva, an ultrasound examination should be implemented immediately for\nCTUP measurement, and the vulval conformation should be inspected.\nAll the mares gave birth to live foals, although some were\ncompromised, including needing assistance to stand and nurse\nregularly. These foals required intensive care. However, they survived,\ndemonstrating the importance of early antibiotic treatment and the\ninitiation of early (and regular) bottle feeding. In all but seven cases,\nthe placenta appeared normal on expulsion, confirming the resolution\nof the placentitis following effective treatment. Although difficult to\nprove, and therefore a limitation of this study, it is likely that these\npregnancies would have been lost without treatment.\nSince ascending placentitis is the most frequent cause, vulval\nconformation should be  included in the examination of mares\nshowing signs of placentitis. One reason for vulval conformation not\nbeing included could be because of previous recommendations to use\nthe Caslick index (CI) when deciding if a mare required a vulvoplasty.\nThis index is calculated from the effective length of the vulva\nmultiplied by its angle (21). However, one study showed that even in\nmares with a high CI score, there were no signs of placentitis, and the\nCI was found to change with the body condition score (22). Therefore,\na better understanding is required of which vulval conformation does\nnot seal well enough to prevent pathogens from entering later in\npregnancy. Photographs of vulvas with poor closure and vulvas with\nnormal closure are shown in Figures 1, 2, respectively. Photograph 1:3\nwas taken at 264 days of gestation. Figure 2b is a normal vulva at\n255 days of pregnancy for comparison.\nThe vulva is regarded as the first seal between the foetus and the\noutside world. The seal can be likened to an elastic “zip,” similar to a\n\nPhotograph 1:3\nwas taken at 264 days of gestation.Figure 2b is a normal vulva at\n255 days of pregnancy for comparison.The vulva is regarded as the first seal between the foetus and the\noutside world.The seal can be likened to an elastic “zip,” similar to a\nrubber band. The elasticity of the vulva should expand enough to allow\nthe birth of the foal to take place and, shortly after, contract sufficiently\nto provide a seal against bacteria from the outside. If there is any\ndiscontinuity in the “zip,” it will not seal properly. Continuous suction\ndue to a weakness in the elasticity of the vulva’s edge will result in a\ndefective seal and allow an inflow of bacteria, which is the most\ncommon cause of ascending placentitis (5). This can be observed as the\nplicae of the edges of the vulval lips become deeper and irregularly\nordered, similar to the lines around the lips of a smoker. Therefore, the\nCI according to Pascoe (21) may not provide sufficient indication of the\npotential effectiveness of vulval closure later in pregnancy (22).\nFurthermore, an increased CTUP as the only presenting sign is not\nindicative of placentitis per se. Several clinical signs taken together, such\nas premature udder development, vaginal discharge, and increased\nCTUP, are needed to make an accurate diagnosis of placentitis.\nHowever, if there is an ascending infection via the vulva during\npregnancy, it is likely that the sealing of the vulva is insufficient, and an\nexamination of vulval conformation should always be conducted to\ndetermine if a vulvoplasty is required.\nExamples are presented as Figures 1a–c. The mare in Figure 1c is an\n8-year-old trotting mare, at day 265 of gestation, with a CTUP of\n7–11 mm, placental detachment, a soft cervix, a slight reddish discharge\nfrom the vulva, and an active udder. She was given 52 g TMS twice daily,\n5 g ASA twice daily, and a vulvoplasty was performed. Figure 2b is from\nan 8-year-old North Swedish Draught Horse, at day 255 of gestation,\nwith no clinical signs indicating placentitis. The vulva of the two mares\n\nShe was given 52 g TMS twice daily,\n5 g ASA twice daily, and a vulvoplasty was performed.Figure 2b is from\nan 8-year-old North Swedish Draught Horse, at day 255 of gestation,\nwith no clinical signs indicating placentitis.The vulva of the two mares\nis very similar in appearance, highlighting the importance of conducting\na thorough clinical examination before proceeding with treatment or\nperforming a vulvoplasty.\n6 Conclusion\nThe first signs of an ascending infection and placentitis are premature\nudder development and/or vulval discharge. In cases with ascending\nplacentitis, the ingress of bacteria occurs through an inadequately sealed\nvulva. The cases presented here illustrate the importance of early\ndiagnosis of placentitis to achieve resolution of the infection, as well as\ncorrection of vulval conformation to establish an effective vulval seal. The\nlatter is crucial to inhibit the continued ingress of bacteria and to prevent\nfurther infection. Therefore, examination of the vulva must be included\nin the initial gynaecological evaluation of a mare presenting with\npremature udder development and/or vaginal discharge in late\npregnancy. Effective treatment of the infection can be achieved with\ntrimethoprim sulphate twice a day and acetylsalicylic acid, a COX-1 anti-\ninflammatory drug, until foaling. Sickly foals respond well to intensive\ncare. When performing vulvoplasty, clinicians should be encouraged to\nensure that the resulting tissue bridge will be sufficient to provide an\neffective vulval seal for the entire pregnancy. The COX2 inhibitor\n(Firocoxib), which has been recommended recently, was not used by the\nauthor since the results with COX1 (ASA) were satisfactory, and most of\nthe mares were older and would benefit from treatment with COX1.\nData availability statement\nThe original contributions presented in the study are included in\nthe article/supplementary material, further inquiries can be directed\nto the corresponding author.\nEthics statement\nInformed consent was obtained from the owners for the publication\n\nEthics statement\nInformed consent was obtained from the owners for the publication\nof any identifiable data in this study. Written informed consent was\nobtained from the participants for the publication of this case report.\nAuthor contributions\nEH: Conceptualization, Data curation, Resources, Visualization,\nWriting – original draft. JM: Resources, Writing – review & editing.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. JM is funded by the Swedish\nUniversity of Agricultural Sciences.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\n\nHemberg and Morrell\b\n10.3389/fvets.2025.1591452\nFrontiers in Veterinary Science\n12\nfrontiersin.org\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. Any product that may be evaluated in this article, or\nclaim that may be made by its manufacturer, is not guaranteed or\nendorsed by the publisher.\nReferences\n1.\tGiles RC, Donahue JM, Hong CB, Tuttle PA, Petrites-Murphy MB, Poonacha KB,\net al. Causes of abortion, stillbirth, and perinatal death in horses: 3,527 cases\n(1986-1991). J Am Vet Med Assoc. (1993) 203:1170–5.\n2.\tGovaere J, Roles K, Ververs C, van de Velde M, de Lange V, Gerrits I, et al.\nAscending placentitis in the mare. Vlaams Diergeneeskd Tijdschr. (2018) 87:115–26. doi:\n10.21825/vdt.v87i3.16074\n3.\tCrabtree JR. Update on diagnosing and treating ascending placentitis in horses. In\nPract. (2023) 45:420–8. doi: 10.1002/inpr.353\n4.\tHemberg E, Lundeheim N, Einarsson S. Retrospective study on vulvar\nconformation in relation to endometrial cytology and fertility in thoroughbred mares.\nJ Vet Med A. (2005) 52:474–7. doi: 10.1111/j.1439-0442.2005.00760.x\n5.\tCummins C, Carrington S, Fitzpatrick E, Duggan V. Ascending placentitis in the\nmare: a review. Ir Vet J. (2008) 61:307–13. doi: 10.1186/2046-0481-61-5-307\n6.\tFeijo LS, Wolfsdorf KE, Canisso IF, Parry S, Felippe JB. Application of blood\nparameters for the early diagnosis of natural ascending placentitis in pregnant mares.\nTheriogenology. (2024) 228:37–53. doi: 10.1016/j.theriogenology.2024.07.025\n7.\tKatila T. The equine cervix. Pferdeheilkunde. (2012) 28:35–8. doi:\n10.21836/PEM20120108\n8.\tLeBlanc MM. Ascending placentitis in the mare: an update. Reprod Domest Anim.\n(2010) 45:28–34. doi: 10.1111/j.1439-0531.2010.01633.x\n9.\tPlatt H. Growth and maturity in the equine fetus. J R Soc Med. (1978) 71:658–61.\ndoi: 10.1177/014107687807100906\n10.\tCanisso IF, Ball BA, Esteller-Vico A, Williams NM, Squires EL, Troedsson MH.\n\nReprod Domest Anim.(2010) 45:28–34.doi: 10.1111/j.1439-0531.2010.01633.x\n9.Platt H.Growth and maturity in the equine fetus.J R Soc Med.(1978) 71:658–61.doi: 10.1177/014107687807100906\n10.Canisso IF, Ball BA, Esteller-Vico A, Williams NM, Squires EL, Troedsson MH.\nChanges in maternal androgens and oestrogens in mares with experimentally-induced\nascending placentitis. Equine Vet J. (2017) 49:244–9. doi: 10.1111/evj.12556\n11.\tLöf HK, Gregory JW, Neves AP, Jobim MIM, Gregory RM, Mattos RC. Combined\nthickness of the uterus and placenta (CTUP) as indicator of placentitis in Thoroughbred\nmares. Pferdeheilkd Equine Med. (2014) 30:37–41. doi: 10.21836/PEM20140105\n12.\tMcCue PM. Combined thickness of the uterus and placenta In: JJ Dascanio and\nPM McCue, editors. Equine reproductive procedures. Wiley Blackwell. (2014).\n13.\tCash RSG. Colostral quality determined by refractometry. Equine Vet Educ. (1999)\n11:36–8. doi: 10.1111/j.2042-3292.1999.tb00916.x\n14.\tRossdale PD. Clinical studies on the newborn thoroughbred foal: I. perinatal\nbehaviour. Br Vet J. (1967) 123:470–81. doi: 10.1016/S0007-1935(17)39702-6\n15.\tClothier J, Hinch G, Brown W, Small S. Equine gestational length and location: is\nthere more that the research could be telling us? Aust Vet J. (2017) 95:454–46. doi:\n10.1111/avj.12653\n16.\tLong A, Nolen-Walston. Equine inflammatory markers in the twenty-first\ncentury. Vet Clin North Am  Equine Pract. (2020) 36:147–60. doi:\n10.1016/j.cveq.2019.12.005\n17.\tBorchers A, Wilkins PA, Marsh PM, Axon JE, Read J, Castagnetti C, et al.\nAssociation of admission L-lactate concentration in hospitalised equine neonates with\npresenting complaint, periparturient events, clinical diagnosis and outcome: a\nprospective multicentre study. Equine Vet J Suppl. (2012) 44:57–63. doi:\n10.1111/j.2042-3306.2011.00509.x\n18.\tBernard M, Donnelly CG, Miller A, Diel de Amorim M. Diagnosis and\nmanagement of placentitis with severe funisitis in a multiparous Warmblood mare.\nJEVS. (2024) 137:105075. doi: 10.1016/j.jevs.2024.105075\n\nEquine Vet J Suppl.(2012) 44:57–63.doi:\n10.1111/j.2042-3306.2011.00509.x\n18.Bernard M, Donnelly CG, Miller A, Diel de Amorim M.Diagnosis and\nmanagement of placentitis with severe funisitis in a multiparous Warmblood mare.JEVS.(2024) 137:105075.doi: 10.1016/j.jevs.2024.105075\n19.\tSielhorst J, Roggel-Buecker U, Neudeck K-C, Kahler A, Rohn K, Luettgenau J, et al.\nEffect of acetylsalicylic acid on uterine blood flow, gestation length, foal birth weight\nand placental weight in pregnant thoroughbred mares – a clinical pilot study. J Equine\nVet Sci. (2022) 118:104107. doi: 10.1016/j.jevs.2022.104107\n20.\tElliot C, Morton J, Chopin J. Factors affecting birthweight in thoroughbred horses.\nTheriogenology. (2008) 71:683–9. doi: 10.1016/j.theriogenology.2008.09.041\n21.\tPascoe RR. Observations on the length and angle of declination of the vulva and\nits relation to fertility in the mare. J Reprod Fertil Suppl. (1979) 27:299–305.\n22.\tMorais de Souza A, Zimmermann Winter GH, Weber Gregory J, Mascarenhas\nJobim MI, Gregory RM, Costa Mattos R. The combined thickness of uterus and placenta\n(CTUP) and vulvar conformation of the mare. Pferdeheilkunde Equine Medicine. (2014)\n30:652–6. doi: 10.21836/PEM20140601\n\n---"}
+{"text": "【兽医文献】\n标题: article_8_The Evidence Behind the Diagnostic Investigation of Canine I\n\n__\nThis Open Access work is\ndistributed under a Creative\nCommons Attributions 4.0\nInternational License. You\nare free to share (copy and\nredistribute the material in\nany medium or format) or\nadapt (remix, transform,\nand build upon the material\nfor any purpose, even\ncommercially), provided you\nfully cite this original work.\n__\nVeterinary Evidence is an\nonline, open access, peer-\nreviewed journal owned\nand published by RCVS\nKnowledge.\nVeterinary Evidence (2026) Vol 11 Iss 3 | Page 1 of 4\nDOI: https://doi.org/10.18849/ve.v11i3.754\nEDITORIAL\nKeywords: EVIDENCE-BASED VETERINARY MEDICINE; CONTEXTUALISED CARE\nEvidence-based veterinary medicine and\ncontextualised care\nPeter Cockcroft, MA, MSc, MBA, Vet MB, DVM&DCHP, Dip ECBHM1*\nEditor-in-Chief\n1 School of Veterinary Medicine, University of Surrey, Guildford, GU2 7AL\n* Corresponding author email: eic@veterinaryevidence.org\nVol 11, Issue 3 (2026)\nPublished: 06 July 2026\nAbstract\nThe welfare and the quality of life of the patient are central to the practice of veterinary medicine.\nWith this in mind there is a growing number of treatment options available and factors related to the\npatient, the owner/carer, the type of consultation, the profession, the practice, the wider society, and\nsustainability both environmental and financial that should be considered when making a decision\nabout the management of a patient. The similar terms contextualised care and spectrum of care\nhave been used to describe these considerations.\nDecision making in the practice of contextualised care/spectrum of care, whichever options are\nunder consideration, should be informed by the best current evidence from scientific research.\nThe primary role of the Veterinary Evidence journal is to provide the scientific evidence from\npublished research in a form that can be interpreted by veterinary professionals and which\ncan used to inform their decision making.\nContextualised care and the spectrum of care\nThe welfare and the quality of life of the patient are central to the practice of veterinary\nmedicine. Advances in technology, an increase in the range of diagnostic tests available, and\n\nContextualised care and the spectrum of care\nThe welfare and the quality of life of the patient are central to the practice of veterinary\nmedicine.Advances in technology, an increase in the range of diagnostic tests available, and\nthe expansion of referral practice in the profession have focused attention on the range of\ndifferent patient management options available and additional factors that need to be taken\ninto consideration (Englar, 2023a, 2023b; Skipper et al., 2024; Fredesco, 2025). The terms\ncontextualised care (Skipper et al., 2024) and spectrum of care (Brown et al., 2021; Fingland,\n2021) have been used to describe these considerations.\nContextualised veterinary care takes into account the aims, knowledge, experiences, and\ncircumstances of individual animal caregivers and veterinary professionals, acknowledging\nthe wider contexts of each clinical encounter, to deliver the most appropriate welfare focused\ncare for every animal (Skipper et al., 2024). Factors related to the patient, owner/carer, type\nof consultation, profession, wider society, and sustainability should be considered (RCVS\nKnowledge, 2025).\nThe spectrum of care similarly refers to the wide range of care options veterinarians can provide and\ntailoring the care options based on contextual factors, such as client goals, abilities, and resources,\nas well as patient, veterinarian, and practice factors, while considering available evidence (Englar,\n2023a, 2023b). These factors have been described in detail (Englar, 2023a, 2023b).\nThe Veterinary Evidence journal has recently published the findings of a survey investigating barriers\nto the delivery of contextualised care in UK small animal veterinary practice (Everitt et al., 2026). In\nparallel, RCVS Knowledge has developed a roadmap which describes the support that is needed to\nimplement contextualised care (RCVS Knowledge, 2025).\nRole of the journal in supporting contextual care/spectrum of care\nThe primary purpose of the Veterinary Evidence journal is to publish Knowledge Summaries that crit­\n\nRole of the journal in supporting contextual care/spectrum of care\nThe primary purpose of the Veterinary Evidence journal is to publish Knowledge Summaries that crit­\nically appraise scientific research related to important clinical questions which have an impact on\n\npatient care. Critical appraisal enables the strength of evidence provided by a study to be defined \nand the validity of the reported outcomes to be established. This information supports the practice \nof evidence-based veterinary medicine (EBVM) by veterinarians and veterinary nurses, and facil­\nitates the translation of research into clinical practice to enhance the quality of care provided to \nanimals.\nThere are 5 steps described in the practice of EBVM (adapted from Sackett, 1997):\n1.\t\nDefining a clinically relevant question\n2.\t\nSearching for the best available evidence\n3.\t\nCritically appraising the evidence\n4.\t\nApplying the evidence\n5.\t\nEvaluating the performance\nA Knowledge Summary performs steps 1, 2, and 3. The responsibility for step 4, applying \nthe evidence in clinical decision-making, and step 5, evaluating the performance, rests with \nthe veterinarian. The relationship between scientific evidence, evidence-based veterinary \nmedicine and contextualised care/spectrum of care is shown in Fig. 1.\nFigure 1: Venn diagram illustrating the relationship between scientific evidence, evidence-based \nveterinary medicine and contextualised care/spectrum of care.\nDecision making in the practice of contextualised care/spectrum of care, whichever options are \nunder consideration, should be informed by the best current evidence from scientific research.\nThe role of the Veterinary Evidence journal is to provide the scientific evidence from published \nresearch in a form that can be interpreted by veterinary professionals and inform their decision \nmaking. Contextualised care and spectrum of care prompt us to consider what we know to be \ntrue and why, so that we actively re-examine evidence-based options, and the way we deliver \nthose options to clients (Englar, 2023a).\nORCiD\nPeter Cockcroft: https://orcid.org/0000-0003-0090-07060\nVeterinary Evidence (2026) Vol 11 Iss 3 | Page 2 of 4\n\nCockcroft | Page 3 of 4\nContribute to the evidence\nThere are two main ways you can contribute to the evidence base while also enhancing your\nCPD:\n•\nTell us your information need\n•\nWrite a Knowledge Summary\nEither way, you will be helping to add to the evidence base, and strengthen the decisions that\nveterinary professionals around the world make to give animals the best possible care. Learn\nmore here: https://veterinaryevidence.org/index.php/ve/author-hub\nReferences\n1.\nBrown, C.R., Garrett, L.D., Gilles, W.K., Houlihan, K.E., McCobb, E., Pailler, S., Putnam, H.,\nScarlett, J.L., Treglia, L., Watson, B. & Wietsma, H.T. (2021). Spectrum of care: more than\ntreatment options. Journal of the American Veterinary Medical Association. 259(7), 712–717.\nDOI: https://doi.org/10.2460/javma.259.7.712\n2.\nEnglar, R.E. (2023a). Recasting the gold standard – part I of II: delineating healthcare options\nacross a continuum of care. Journal of Feline Medicine and Surgery. 25(12), 1098612X231209855.\nDOI: https://doi.org/10.1177/1098612X231209855\n3.\nEnglar, R.E. (2023b). Recasting the gold standard – part II of II: communicating\nhealthcare options along a continuum of care. Journal of Feline Medicine and Surgery.\n25(12), 1098612X231215639. DOI: https://doi.org/10.1177/1098612X231215639\n4.\nEveritt, S., Taylor, A., Doorly, A., Cropley, K., Osterberg, A. & Mantell, K. (2026). Barriers\nto the delivery of contextualised care in UK small animal veterinary practice. Veterinary\nEvidence. 11(2), 1–19. DOI: https://doi.org/10.18849/ve.v11i2.752\n5.\nFedesco, H.N., Brodsky, J.E., Warman, S.M. & Khosa, D.K. (2025). The American Association of\nVeterinary Medical Colleges Spectrum of Care Initiative: supporting spectrum of care preparation\nin veterinary education programs. Journal of the American Veterinary Medical Association. 263(S3),\nS85–S91. DOI: https://doi.org/10.2460/javma.25.05.0358\n6.\nFingland, R.B., Stone, L.R., Read, E.K. & Moore, R.M. (2021). Preparing veterinary students\nfor excellence in general practice: building confidence and competence by focusing on\n\nJournal of the American Veterinary Medical Association.263(S3),\nS85–S91.DOI: https://doi.org/10.2460/javma.25.05.0358\n6.Fingland, R.B., Stone, L.R., Read, E.K.& Moore, R.M.(2021).Preparing veterinary students\nfor excellence in general practice: building confidence and competence by focusing on\nspectrum of care. Journal of the American Veterinary Medical Association. 259(5), 463–470. DOI:\nhttps://doi.org/10.2460/javma.259.5.463\n7.\nRCVS Knowledge. (2025). Contextualised care: a road map, [online]. Available at: https://s47944.pcdn.\nco/wp-content/uploads/RCVS-Knowledge-Contextualised-care-roadmap-November-2025.pdf\n[Accessed 01 June 2026].\n8.\nSackett, D.L. (1997). Evidence-based medicine. Seminars in Perinatology. 21(1), 3–5. DOI:\nhttps://doi.org/10.1016/S0146-0005(97)80013-4\n9.\nSkipper, A., O’Neill, D., Serlin, R., Davidson, J., Elwood, C. & Gray, C. (2024). Contextualised\ncare: faddish or foundational? Veterinary Record, 195(3), 117. DOI: https://doi.org/10.1002/\nvetr.4567\nLicence\nCopyright (c) 2026 Peter Cockcroft\n\nVeterinary Evidence (2026) Vol 11 Iss 3 | Page 4 of 4\nIntellectual property rights\nAuthors of articles submitted to RCVS Knowledge for publication will retain copyright in their \nwork, and will be required to grant to RCVS Knowledge a non-exclusive licence of the rights of \ncopyright in the materials including but not limited to the right to publish, re-publish, transmit, \nsell, distribute and otherwise use the materials in all languages and all media throughout the \nworld, and to licence or permit others to do so.\nDisclaimer\nAny opinions expressed in articles and other publication types published in Veterinary Evidence \nare the author’s own and do not necessarily reflect the view of the RCVS Knowledge. Veterinary \nEvidence is a resource to help inform, and the content herein should not override the responsibility \nof the practitioner. Practitioners should also consider factors such as individual clinical expertise \nand judgement along with patient’s circumstances and owners’ values. Authors are responsible \nfor the accuracy of the content. While the Editor and Publisher believe that all content herein are \nin accord with current recommendations and practice at the time of publication, they accept \nno legal responsibility for any errors or omissions, and make no warranty, express or implied, with \nrespect to material contained within. For further information please refer to our Terms of Use.\n\n---"}
+{"text": "【兽医文献】\n标题: Implementation of LABSTER virtual lab in immunology for innovative teaching and improved learning in veterinary degree\n\nTYPE Original Research\nPUBLISHED 07 August 2025\nDOI 10.3389/fvets.2025.1603458\nOPEN ACCESS\nEDITED BY\nDaniela Bertotto,\nUniversity of Padua, Italy\nREVIEWED BY\nJames Edward Brereton,\nSparsholt College, United Kingdom\nGuilherme Rizzoto,\nGhent University, Belgium\n*CORRESPONDENCE\nLucrezia Forte\nlucrezia.forte@uniba.it\nAlice Carbonari\nalice.carbonari@uniba.it\nRECEIVED 31 March 2025\nACCEPTED 15 July 2025\nPUBLISHED 07 August 2025\nCITATION\nForte L, Burgio M, Rizzo A, Maggiolino A,\nCarbonari A, De Palo P, Lacalandra GM and\nCicirelli V (2025) Heat stress on sperm quality\nin dogs: effect of natural antioxidant\nsupplementation. Front. Vet. Sci. 12:1603458.\ndoi: 10.3389/fvets.2025.1603458\nCOPYRIGHT\n© 2025 Forte, Burgio, Rizzo, Maggiolino,\nCarbonari, De Palo, Lacalandra and Cicirelli.\nThis is an open-access article distributed\nunder the terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other forums is\npermitted, provided the original author(s) and\nthe copyright owner(s) are credited and that\nthe original publication in this journal is cited,\nin accordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nHeat stress on sperm quality in\ndogs: effect of natural\nantioxidant supplementation\nLucrezia Forte*, Matteo Burgio, Annalisa Rizzo,\nAristide Maggiolino, Alice Carbonari*, Pasquale De Palo,\nGiovanni Michele Lacalandra and Vincenzo Cicirelli\nDepartment of Veterinary Medicine, University of Bari Aldo Moro, Bari, Italy\nIntroduction: Heat stress negatively affects canine reproductive health by\ninducing oxidative stress and impairing sperm quality. This study assessed the\nefficacy of a polyphenolic extract from Loblolly pine (Pinus taeda) lignin (PTHL),\nin mitigating heat stress-induced oxidative damage and preserving sperm quality\nin dogs.\nMethods: Forty clinically healthy male dogs were divided into two groups: a\ncontrol (CON) group receiving a standard diet and a PTHL-supplemented group\nfor 90 days. During summer, dogs were exposed to natural heat stress, and key\nparameters, including serum biochemical markers, oxidative stress indicators,\n\nDuring summer, dogs were exposed to natural heat stress, and key\nparameters, including serum biochemical markers, oxidative stress indicators,\nantioxidant enzyme activity, and sperm quality were evaluated. Mixed-effects\nmodels analyzed group, time, and interaction effects.\nResults and discussion:\nPTHL supplementation significantly reduced plasma\nTBARS and seminal d-ROMs levels (P < 0.01), indicating lower lipid peroxidation\nand confirming its protective effects. Antioxidant capacity improved in the PTHL\ngroup, with increased activities of antioxidant enzyme activity (SOD, CAT, and\nGSPx) and elevated plasma Ferric Reducing Antioxidant Power (FRAP) levels (P\n< 0.01). Notably, the PTHL group demonstrated higher progressive motility (P <\n0.01) and a greater percentage of rapid-movement sperm (P < 0.01) at 90 days,\nindicating improved sperm function under heat stress.\nConclusion: These findings suggest that PTHL enhances antioxidant defenses,\nmitigating heat stress-induced reproductive impairment. This natural strategy\nmay improve fertility in dogs facing environmental challenges. Future studies\nshould explore different dosages and extended supplementation to optimize\nits benefits.\nKEYWORDS\nthermal stress, dogs, antioxidants, semen quality, biochemical profile\n1 Introduction\nHeat stress is a critical environmental factor affecting animal physiology and\nreproduction, particularly in mammals with limited thermoregulatory capacities, such as\ndogs (1). Elevated ambient temperatures disrupt homeostasis by inducing oxidative stress,\naltering metabolic pathways, and impairing organ function (2, 3). In male dogs, heat\nstress has been associated with a decline in sperm quality, compromised spermatogenesis,\nand reduced reproductive efficiency, leading to transient or even prolonged infertility (4).\nFrontiers in Veterinary Science\n01\nfrontiersin.org\n\nForte et al.\n10.3389/fvets.2025.1603458\nUnderstanding the mechanisms underlying these adverse effects\nand developing mitigation strategies is essential for preserving\ncanine fertility, especially in breeding programs and working dogs\nexposed to challenging climatic conditions. In this context, the\nThermo-Hygrometric Index (THI) is a valuable tool for assessing\nenvironmental heat load (5); in dogs, THI values above 72–\n74 are generally recognized as critical thresholds, beyond which\nphysiological signs of heat stress begin to manifest (4). One of the\nprimary pathways through which heat stress affects reproductive\nfunction is oxidative stress, characterized by excessive production\nof reactive oxygen species (ROS) overwhelming endogenous\nantioxidant defenses (6). ROS, including superoxide anions,\nhydrogen peroxide, and hydroxyl radicals, are natural byproducts\nof aerobic metabolism and play dual roles in biological systems\n(7). While they are involved in essential processes such as sperm\ncapacitation and immune responses, their overproduction leads\nto lipid peroxidation, protein oxidation, and DNA fragmentation,\nultimately impairing sperm function (2). Due to their high\npolyunsaturated fatty acid (PUFA) content and low intrinsic\nantioxidant capacity, spermatozoa are particularly vulnerable\nto oxidative damage, which can manifest as reduced motility,\nstructural abnormalities, and apoptosis (8). Heat stress not only\nincreases oxidative stress but also affects systemic physiology,\nleading to metabolic alterations that can further compromise\nreproductive\nfunction.\nStudies\nhave\nshown\nthat\nprolonged\nexposure to high temperatures reduces testosterone production,\nimpairs Sertoli and Leydig cell function, and disrupts the\nspermatogenic cycle (9–14). Additionally, elevated temperatures\npromote peripheral vasodilation, leading to increased glucose\nuptake and subsequent hypoglycemia, which may exacerbate\ncellular stress responses (15). The combined impact of oxidative\nand metabolic stressors underscores the need for effective\ninterventions aimed at preserving sperm integrity and function\nunder heat stress conditions.\n\nThe combined impact of oxidative\nand metabolic stressors underscores the need for effective\ninterventions aimed at preserving sperm integrity and function\nunder heat stress conditions.\nDietary antioxidant supplementation has emerged as a\npromising strategy to counteract oxidative damage and support\nmale reproductive health (16, 17). Antioxidants, particularly\npolyphenols, exhibit strong free radical-scavenging properties,\nmodulating cellular redox balance and enhancing enzymatic\nantioxidant\ndefenses\n(18).\nAmong\nthese,\nlignin-derived\npolyphenolic compounds from Loblolly pine (Pinus taeda)\nhave gained interest due to their ability to improve antioxidant\nstatus and mitigate oxidative damage in various physiological\ncontexts (17, 19–21).\nThe selection of Pinus taeda hydroxylated lignin (PTHL) for\nthis study is based on previous findings from our group, in\nwhich dietary supplementation with this compound improved\nsemen quality in healthy dogs under normal environmental\nconditions. Specifically, PTHL enhanced total antioxidant capacity\nand improved sperm parameters such as motility, concentration,\nand semen volume, likely through the upregulation of endogenous\nantioxidant enzymes such as superoxide dismutase, catalase, and\nglutathione peroxidase (17). Considering that heat stress has been\nshown to exert the opposite effect on these same parameters,\nreducing semen quality through oxidative and metabolic stress\n(17), we identified PTHL as a biologically plausible candidate for\na targeted intervention. This rationale supports the hypothesis\nthat PTHL may counterbalance the deleterious effects of elevated\ntemperatures on male fertility. However, its protective effects under\nheat stress conditions remain unexplored.\nThis study aims to assess the efficacy of PTHL dietary\nsupplementation in mitigating oxidative stress and preserving\nsperm quality in dogs subjected to heat stress. We hypothesize that\nPTHL supplementation enhances the antioxidant defense system,\nreduces oxidative damage, and improves semen characteristics\ncompared to non-supplemented controls. By addressing this\n\nWe hypothesize that\nPTHL supplementation enhances the antioxidant defense system,\nreduces oxidative damage, and improves semen characteristics\ncompared to non-supplemented controls.By addressing this\nknowledge gap, our research provides novel insights into the role of\ndietary antioxidants in safeguarding reproductive function under\nadverse environmental conditions, with potential implications for\ncanine breeding management and welfare.\n2 Materials and methods\nThis prospective, randomized clinical study was conducted\nin compliance with the ethical guidelines of the Animal Welfare\nCommittee. Approval was granted by the Institutional Review\nBoard (approval number: 656/18 – III/13). Informed consent was\nobtained from all dog owners.\n2.1 Animals\nThe study was conducted from May to September 2024 in\nsouthern Italy. A total of 40 male mixed-breed dogs, aged 2–\n4 years and weighing between 15 and 25 kg, participated in the\ntrial. All dogs were owned, always kept outdoors, and housed\nindividually. They were fed a standardized diet [10 g/kg of body\nweight (BW) daily, composition detailed in Table 1] twice a day,\nstarting 30 days before the trial commenced. To ensure their health\nstatus, all dogs underwent a clinical examination 30 days before\nthe start of the study. Each animal was subjected to both general\nand specialized reproductive tract assessments. Additionally, an\nultrasound examination of the reproductive system was performed,\nand all dogs were confirmed to respond positively to sperm\ncollection via digital manipulation. Exclusion criteria included\nobesity (weight exceeded its ideal weight by more than 30%) (22),\nwhite or long hair, and any medication use within the previous\n30 days.\n2.2 Experimental protocol\nThe dogs were randomly assigned to one of two equal-sized\ngroups (n = 20) using www.randomizer.org. One group served as\nthe experimental group (PTHL), while the other functioned as the\ncontrol group (CON). The PTHL group received a daily oral dose\nof 1 g per dog (about 50 mg/kg/day) of a supplement containing\nPinus taeda hydrolyzed lignin (Oxyphenol R⃝, I-Green, Padua, Italy)\n\nThe PTHL group received a daily oral dose\nof 1 g per dog (about 50 mg/kg/day) of a supplement containing\nPinus taeda hydrolyzed lignin (Oxyphenol R⃝, I-Green, Padua, Italy)\n(Table 2) for the entire 120-day trial (4). The supplement was\nadministered in powder form according to the manufacturer’s\nguidelines, which were based on empirical trials (I-Green, Padua,\nItaly). The CON group did not receive any supplementation but\nwas fed the same standardized diet as the experimental group.\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n\nForte et al.\n10.3389/fvets.2025.1603458\nTABLE 1 Composition of diet commercial feed.\nItem\nOn 100 g of product\nMoisture\n10 g\nRaw protein\n28 g\nFat\n15 g\nRaw fibers\n3 g\nAshes\n9 g\nVitamin A\n1,500 IU\nVitamin D\n100 IU\nVitamin E acetate (alpha-tocopherol 91%)\n12.5 mg\nVitamin B2\n2.6 mg\nVitamin B6 (pyridoxine hydrocloride)\n0.5 mg\nVitamin B1 (thiamine mononitrate)\n0.6 mg\nCholine chloride\n75 mg\nIodine (anhydrous calcium iodate)\n0.075 mg\nD-panthotenic acid\n1 mg\nVitamin H (Biotin D)\n0.05 mg\nCalcium\n0.5 g\nVitamin K3 (menadione)\n0.125 mg\nVitamin PP (nicotine acid)\n2.5 mg\nVitamin B12\n0.0035 mg\nFolic acid\n0.1 mg\nCobalt (basic cobalt carbonate)\n0.015 mg\nIron (ferrous carbonate)\n2 mg\nManganese (manganous oxide)\n4 mg\nCopper (Copper sulfate, pentahydrate)\n1 mg\nSelenium (sodium selenite)\n0.01 mg\nZinc (zinc oxide)\n3 mg\nAll owners were provided with a temperature and humidity\nmonitoring system (Tinytag from Data Loggers, Gemini Data\nLoggers Ltd, West Sussex, United Kingdom), which was placed\nwithin the outdoor area. The data loggers were programmed to\nrecord temperature and humidity hourly. During each semen and\nblood sampling session, the data loggers were transported to the\nObstetric, Gynecological, and Andrological Clinic of the Veterinary\nMedicine Department at the “Aldo Moro” University of Bari\n(Italy), where the recorded data were downloaded to calculate the\nTemperature-Humidity Index (THI).\nThis dataset was employed to compute the hourly THI utilizing\nthe formula outlined by (23):\nTHI = (1.8 × AT + 32) −(0.55 −0.0055 × RH)\n× [(1.8 × AT + 32) −58]\nwhere AT is the environmental temperature expressed in\ndegrees Celsius, so that the term (1.8 × AT + 32) represents the\nTABLE 2 Phenolic composition and antioxidant activity of Pinus taeda\nHydrolyzed LigninA.\nComponent\ng/100g\nDetermined composition (g/100 g)\nVanillin\n26.4\nEriodictyol\n3.4\nQuercetin\n2.7\nIsorhamnetin\n1.6\nRosmarinic acid\n1.4\nQuercetin ramnoside\n13.9\nMethylgallate retunoside\n42.3\nEpigallocatechin-3-methylgallate\n1.5\nFerulic acid derivates\n6.7\nAntioxidant activity (µmol TEB g−1 DWC)\nTrolox equivalent antioxidant capacity\n23.9\nOxygen radical absorbance capacity\n122.4\nAAccording to Gerardi et al. (63) and Blando et al. (61).\nBTrolox equivalents.\nCDry weight.\nconversion of temperature data in degrees Fahrenheit, and RH is\n\n9\nOxygen radical absorbance capacity\n122.4\nAAccording to Gerardi et al.(63) and Blando et al.(61).BTrolox equivalents.CDry weight.conversion of temperature data in degrees Fahrenheit, and RH is\nthe relative humidity as a fraction of unit.\nThe trial lasted 120 days, after 30 days of adaptation to new diet,\nthere was the starting time point (T0) and semen and blood samples\ncollection were performed every 30 days (T30, T60, and T90) until\nthe end of the trial. The experimental design is reported in Figure 1.\n2.3 Blood samples collection and analysis\nBlood samples were aseptically collected via cephalic vein\npuncture using disposable 22G needles and a 4 ml vacuum tube\nsystem for serum (without anticoagulant) and plasma (with 15\nUSP U/ml of heparin) (Becton, Dickinson Canada Inc, Vacutainer\n1, Oakville, Canada). Plasma tubes were immediately centrifuged\nat 1,500 × g for 10 min, while serum tubes were left to clot at a\nrefrigerated temperature for 10 min before undergoing the same\ncentrifugation process. All plasma and serum aliquots were then\nstored at −80◦C until analysis.\n2.3.1 Clinical biochemical analysis\nClinical biochemistry parameters were assessed from serum\nsamples using an automated biochemistry analyzer (CS-300B;\nDirui, Changchun, China) as described by De Palo et al. (24).\nThe parameters analyzed included alanine aminotransferase (ALT),\naspartate aminotransferase (AST), creatine phosphokinase (CPK),\nlactate dehydrogenase (LDH), alkaline phosphatase (ALP), glucose\n(Glu), blood urea nitrogen (BUN), creatinine (Crea), total serum\nprotein (TP), albumin (Alb), cholesterol (Chol), triglycerides\n(Trig), non-esterified fatty acids (NEFA), calcium (Ca), phosphate\n(P), magnesium (Mg), and chloride (Cl) (Gesan Production Kit,\nCampobello di Mazara, Trapani, Italy). Additionally, globulin\nFrontiers in Veterinary Science\n03\nfrontiersin.org\n\nForte et al.\n10.3389/fvets.2025.1603458\nFIGURE 1\nExperimental design. Timing of activities, feeding and sampling in the CON and PTHL groups during their heat stress period.\n(Glob) levels and the albumin/globulin ratio (Alb/Glob) were\nderived from total protein and albumin values. Before each\nanalytical session, the multi-parameter analyzer was calibrated\nusing\nstandard\nassay\nkits\n(Seracal,\nGesan\nProduction\nKit,\nCampobello di Mazara, Trapani, Italy). Internal accuracy was\nverified using two multi-parameter control sera, a normal and\na pathological one (Seracontrol N and Seracontrol P, Gesan\nProduction Kit, Campobello di Mazara, Trapani, Italy), and was\nconsidered acceptable when deviations did not exceed 3.00% from\nthe manufacturer’s declared values. Each sample was analyzed in\ntriplicate, with the arithmetic mean of the three readings used for\nstatistical analysis.\n2.3.2 Oxidative stress assessment\nPlasma\nsamples\nwere\nused\nto\nevaluate\noxidative\nstress\nparameters\nand\nantioxidant\nenzyme\nactivities.\nThiobarbituric acid reactive substances (TBARS) were quantified\nspectrophotometrically\nfollowing\nthe\nmethod\ndescribed\nby\nMaggiolino et al. (20), where 100 µl of plasma were mixed\nwith a 3.7 µl/ml thiobarbituric acid solution. Reactive carbonyl\nderivative (RCD) levels in plasma were assessed according to\nFaure and Lafond (25) using the DNPH carbonyl reagent. Plasma\nsamples (200 µl) were combined with 1 ml of water and 2 ml of\n200 µl/ml trichloroacetic acid before being centrifuged at 1,000\n× g for 10 min. The resulting pellet was resuspended in 1 ml\nof 10 mmol/L DNPH and incubated at 37.8◦C for 60 min. A\ncontrol sample was prepared by replacing DNPH with 1 ml of 1\nmol/L hydrochloric acid. Following incubation, 1 ml of 200 µl/ml\ntrichloroacetic acid was added, and samples were centrifuged\nagain at 1,000 × g for 10 min. The pellet was then washed with a\n1:1 ethanol-ethyl acetate solution, centrifuged, and resuspended\nin 1 ml of 6 mol/L guanidine (diluted in 20 mmol/L dihydrogen\nphosphate, pH 2.3), followed by a final incubation at 37.8◦C for\n40 min. Absorbance was measured at 380 nm. Hydroperoxide\n(Hy) levels were determined spectrophotometrically using an\n\n3), followed by a final incubation at 37.8◦C for\n40 min.Absorbance was measured at 380 nm.Hydroperoxide\n(Hy) levels were determined spectrophotometrically using an\niodometric method described by Maggiolino et al. (26). Plasma\naliquots (90 µl) were distributed into eight microcentrifuge vials\n(1.5 ml), with 10 µl of 10 mM TPP in methanol added to four\nvials to reduce ROOHs, creating a set of blank samples. The\nremaining four vials received 10 µl of methanol, serving as test\nsamples. After vortexing and incubating at room temperature for\n30 min, 900 µl of FOX2 reagent were added. The mixture was\nincubated for another 30 min before centrifugation at 2,400 × g\nfor 10 min using a swing-out rotor (Hettich Rotenta/RP centrifuge,\nHettich-Zentrifugen, Tuttlingen, Germany). The absorbance of\nthe supernatant was recorded at 560 nm using an Ultraspec 2000\nspectrophotometer (Pharmacia Biotech, Uppsala, and Sweden),\nand the ROOH concentration was determined based on the mean\nabsorbance difference between blank and test samples. Protein\ncarbonyls (PC) levels were determined spectrophotometrically as\nreported by Salzano et al. (27). The superoxide dismutase (SOD)\n(EC 1.15.1.1) activity was examined according to Misra (28), and\nthe enzymatic activity was based on the 50% inhibition rate of\nepinephrine autooxidation at 480 nm (29). The SOD activity was\nassessed as the 50% inhibition rate of epinephrine auto-oxidation\nat 480 nm. The epinephrine autoxidation stimulation by traces\nof heavy metals present as contaminants in the reagents was\nprevented by adding 10–4 M EDTA in the buffer to chelate those\nions. The SOD activity was expressed as U/ml. The catalase (CAT)\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n\nForte et al.\n10.3389/fvets.2025.1603458\n(EC 1.11.1.6) activity was assayed by the method of Clairborne (30)\nas described by Tateo et al. (31). The amount of enzyme required\nto degrade 1 µmol of H2O2 in 60 s was defined as 1 unit of enzyme\nactivity by following the decrease in absorbance of H2O2 at 240 nm\n(e = 40 M−1 cm−1). Its activity was expressed as U/mg of protein.\nThe glutathione peroxidase (GPx) (EC1.11.1.9.) activity was\nmeasured according to Gunzler (32) as described by Dinardo et al.\n(33). The analysis was based on the measure of the rate of reduced\nglutathione oxidation by tertbutyl hydroperoxide, catalyzed by\nGPx. The constant concentration of reduced glutathione was\nensured by the addition of exogenous glutathione reductase and\nNADPH, which converted the oxidized glutathione to reduced\nglutathione. The rate of oxidized glutathione formation was then\nmeasured by the change in the absorbance of NADPH at 340 nm.\nIts activity was expressed as nmol of NADPH oxidized/min per ml.\n2.4 Semen collection and analysis\nSemen samples were collected using an artificial vagina\nthrough manual stimulation while the dogs were exposed to\nswabs containing estrous bitch pheromones. The ejaculate was\nfractionated into three separate Falcon tubes, corresponding to\nthe urethral, spermatic, and prostatic fractions (34, 35). All\nsemen analyses were performed by an operator who was blind to\nthe treatment.\n2.4.1 Ejaculate analysis\nEjaculate analysis was conducted following the methodology\ndescribed by Alonge et al. (36). The second seminal part was\nanalyzed by the Computer Assisted Sperm Analyzer (37), (CASA,\nIVOS-Sperm CASA system, Version 12.3, Hamilton Thorne, MA,\nUSA). The CASA software (IVOS 12.3 version) was calibrated\nfor canine semen-specific parameters, as outlined in Table 3.\nAccording to the manufacturer’s instructions, each semen sample\nwas diluted fivefold in a Tris-Fructose extender, and a 3 µl drop\nwas placed on a Leja slide with four 20 µm chambers (Leja\nProducts B.V., Nieuw Vennep, The Netherlands). The slide was\npositioned in the CASA microscope chamber, allowing the sample\n\nV., Nieuw Vennep, The Netherlands).The slide was\npositioned in the CASA microscope chamber, allowing the sample\nto stabilize before analysis. The software scanned five random,\nnon-consecutive microscopic fields to assess semen quality. The\nfollowing parameters were measured: ejaculate volume, sperm\nconcentration, total motility, progressive motility, velocity average\npathway (VAP), straight-line velocity (VSL), curvilinear velocity\n(VCL), amplitude of lateral head displacement (ALH), beat-cross\nfrequency (BCF), straightness (STR), linearity (LIN), and total\nsperm count (TSC). VAP, expressed in µm/s, was calculated\nas the average velocity of a smoothed sperm path. Based on\nvelocity thresholds, the sperm population was categorized into\nfour subgroups: rapid spermatozoa (VAP > MVV), medium\nspermatozoa (LVV < VAP < MVV), slow spermatozoa (VAP <\nLVV), and static spermatozoa, which consisted of non-motile cells\nduring the analysis (38).\nTABLE 3 IVOS 12,3 software settings for dog semen parameters.\nParameters\nCut-offvalue\nFrames per second (Fps)\n30\nFrequency\n60 Hz\nTemperature of analysis\n37◦C\nMinimum contrast\n75\nMinimum cell size\n4 pixels\nProgressive cell cut-off\n100 µm/s; 75% STR\nLow VAP cut-off\n9 µm/s\nLow VSL cut-off\n20 µm/s\n2.4.2 Oxidative stress\nProstatic fluid was collected for the determination of reactive\noxygen metabolites (d-ROMs) and biological antioxidant potential\n(BAP). The d-ROMs were measured using a free radical\nelective evaluator (FREE Carpe Diem; Diacron International srl,\nGrosseto, Italy), which includes a spectrophotometric reader and\nmeasurement kits (d-ROMs test, Wismerll Co. Ltd., Tokyo, Japan)\noptimized for the FREE Carpe Diem R⃝system, following the\nmanufacturer’s protocol. For d-ROMs assessment, a 20 µl aliquot\nof seminal plasma was mixed with 1 ml of buffered solution (R2 kit\nreagent, pH 4.8) in a cuvette. Subsequently, 20 µl of chromogenic\nsubstrate (R1 kit reagent) was added. The mixture was thoroughly\nblended and incubated in the analyzer’s thermostatic block at\n37◦C for 5 min, after which absorbance at 505 nm was recorded.\nResults were expressed in arbitrary units (U/CARR), where one unit\n\nThe mixture was thoroughly\nblended and incubated in the analyzer’s thermostatic block at\n37◦C for 5 min, after which absorbance at 505 nm was recorded.Results were expressed in arbitrary units (U/CARR), where one unit\ncorresponds to 0.8 mg/L of hydrogen peroxide. BAP determination\nwas carried out using the same spectrophotometric reader and\ncorresponding measurement kits (BAP test, Wismerll Co. Ltd.,\nTokyo, Japan), adhering to the manufacturer’s instructions. In\nthis assay, 50 µl of chromogenic substrate (R2 kit reagent) was\ncombined with 1 ml of reactive solution (R1 kit reagent) in a\ncuvette, and absorbance at 505 nm was initially recorded. A 10 µl\nsample of seminal plasma was then introduced into the cuvette,\nfollowed by thorough mixing. The mixture was incubated at 37◦C\nfor 5 min in the thermostatic block of the analyzer, after which a\nsecond absorbance reading at 505 nm was obtained. Results were\nexpressed as mmol/L of reduced ferric ions (39).\n2.4.3 Structural membrane integrity\nAs\npreviously\ndescribed\nin\nsimilar\nclinical\nstudies\n(36), structural membrane integrity was assessed using the\nEosin/Nigrosin staining method. The proportion of spermatozoa\nwith an intact cytoplasmic membrane was determined by\nstaining with Eosin/Nigrosin and analyzing at least 100 sperm\ncells per sample under 400× magnification. Spermatozoa with\ncompromised membrane integrity, including dead cells and those\nexhibiting structural alterations, appeared with a red or intensely\npink head, whereas live sperm cells remained pale white. Since\ndead spermatozoa have a damaged membrane with perforations\nand loss of integrity, they become permeable to the dye, resulting\nin coloration during the vitality test. Conversely, live spermatozoa\nFrontiers in Veterinary Science\n05\nfrontiersin.org\n\nForte et al.\n10.3389/fvets.2025.1603458\nretain an intact membrane, preventing dye penetration and thus\nremaining unstaine (40).\n2.4.4 Functional membrane integrity\nThe functional integrity of the spermatozoa’s cytoplasmic\nmembrane was assessed using the hypo-osmotic swelling (HOS)\ntest. In this assay, sperm cells were exposed to a hypo-\nosmotic solution, allowing fluid to enter the cells. The test was\nperformed by mixing 25 µl of semen with 250 µl of a hypo-\nosmotic solution (containing 25 mM sodium citrate dihydrate and\n75 mM D-fructose) at 37◦C, followed by incubation for 30 min.\nAfter incubation, a 10 µl aliquot was placed on a microscope\nslide, covered with a coverslip, and examined under an optical\nmicroscope at 400× magnification. At least 100 sperm cells were\nevaluated across five different fields, focusing on the presence\nof coiled tails. Spermatozoa with intact cytoplasmic membranes\nexhibited tail swelling due to water influx, whereas those with\ndamaged membranes showed no such morphological changes (41).\nSperm morphology was further analyzed, and the number of cells\nwith naturally coiled tails was subtracted from those that exhibited\ncoiling after incubation.\n2.5 Data analysis\nEach animal represented an experimental unit. All data sets\nwere tested for normal distribution using the Shapiro-Wilk test and\nfor homogeneity of variance using the Bartlett test. All parameters\nwere analyzed using analysis of variance (ANOVA) according to\nthe General Linear Model (GLM) procedure, following the model\nreported below:\nyijk = µ + αi + Gj + Tk + (G × T)jk + εijkl,\n(1)\nwhere yijk represents all dependent variables; µ is the overall\nmean; αi is the constant of the individual dog random effect (i\n= 1,..., 40); G represents the effect of the jth group (j = 1, 2), T\nwas the effect of the kth time (k = 1, ..., 4), G × T represents the\nbinary interaction between the jth group and the kth time (1, ...,\n8). Significance was set at P < 0.05, and the results were expressed\nas predicted means and mean standard errors. All the analysis was\nperformed using SAS software (42).\n3 Results\n3.1 Clinical biochemical results\n\n..,\n8).Significance was set at P < 0.05, and the results were expressed\nas predicted means and mean standard errors.All the analysis was\nperformed using SAS software (42).3 Results\n3.1 Clinical biochemical results\nAverage maximum THI values registered for each experimental\ngroup were reported in Figure 2. All biochemical parameters of all\ndogs were in the reference value (62). The fixed effects of group,\ntime and their interaction on serum biochemical-clinical profile are\nshown in Table 4. Total protein, Albumin, Glcemia, ALT, AST, ALP\n(P < 0.01), and Urea (P < 0.05) were affected by the interaction\nof group and time. Total protein levels decreased contantly until\n60 days in CON group (P < 0.01) and then remained constant,\nwhile in PTHL group showed at 90 days lower values then 0 days\n(P < 0.01). Moreover, PTHL group reported higher serum calues\nthan CON group at 60 and 90 days of trial (P < 0.01). Albumin\nserum levels decreased in CON group after 60 days (P < 0.01)\nremaining constant until 90, while decreased at 90 days in PTHL\ngroup (P < 0.05), showing no differences between groups within\neach experimental time (P > 0.05). The Urea level decreased at 60\nFIGURE 2\nAverage maximum THI registered for the two experimental groups during the trial.\nFrontiers in Veterinary Science\n06\nfrontiersin.org\n\nForte et al.\n10.3389/fvets.2025.1603458\nTABLE 4 Total protein, albumin, urea, uric acid, creatinine, bilirubin, triglyceride, glycemia, alanine amino transferase (ALT), aspartate amino transferase\n(AST), alkaline phosphatase (ALP) and cholesterol serum concentration in heat-stressed dogs over 90 days, with (PTHL) or without (CON) dietary\nsupplementation of Pinus taeda hydrolyzed lignin.\nParameter\nGroup\nTime (days)\nSEM1\nF and P-value\nReference\nvalue4\n0\n30\n60\n90\nG2\nT3\nG × T\nTotal protein\n(g/dL)\nCON\n6.78A\n6.02B\n5.42C,X\n5.22C,X\n0.27\nF = 20.1 <0.001\nF = 32.6 <0.001\nF = 9.8 <0.001\n5.4–7.1\nPTHL\n6.65A\n6.17\n6.12Y\n6.02B,Y\nAlbumin\n(g/dL)\nCON\n3.08A\n2.71\n2.26B\n2.18B\n0.12\nF = 0.18 0.671\nF = 19.7 <0.001\nF = 8.4 <0.001\n2.6–3.3\nPTHL\n3.11a\n3.06\n2.96\n2.68b\nUrea\n(mmol/L)\nCON\n58.73A\n55.42A\n43.17B,x\n40.57B,x\n8.71\nF = 15.4 <0.001\nF = 14.6 <0.001\nF = 3.4 0.021\n21–60\nPTHL\n57.73A\n56.49\n50.47y\n46.91B,y\nUric acid\n(mg/dL)\nCON\n0.65\n0.66\n0.84\n0.89\n0.12\nF = 1.5 0.236\nF = 1.3 0.302\nF = 0.9 0.434\n0.0–2.0\nPTHL\n0.69\n0.65\n0.71\n0.73\nCreatinine\n(mg/dL)\nCON\n1.36\n1.32\n1.28\n1.24\n0.11\nF = 0.9 0.347\nF = 1.5 0.228\nF = 0.6 0.621\n0.5–1.5\nPTHL\n1.39\n1.25\n1.31\n1.29\nBilirubine\n(mg/dL)\nCON\n0.29\n0.23\n0.30\n0.28\n0.06\nF = 0.4 0.516\nF = 0.2 0.675\nF = 0.1 0.801\n0.1–0.5\nPTHL\n0.27\n0.29\n0.26\n0.34\nTriglyceride\n(mg/dL)\nCON\n34.35\n33.67\n31.38\n31.34\n9.105\nF = 1.1 0.337\nF = 1.8 0.179\nF = 0.6 0.542\n30.1–38.1\nPTHL\n35.93\n34.35\n33.52\n33.32\nGlycemia\n(mg/dL)\nCON\n99.05A\n81.29B,X\n71.69BC,X\n66.79C,X\n5.56\nF = 19.9 <0.001\nF = 33.7 <0.001\nF = 12.5 <0.001\n65–118\nPTHL\n101.83A\n98.14Y\n89.44Y\n86.12B,Y\nALT5\n(IU/L)\nCON\n32.25A\n49.93B,X\n65.73C,X\n67.18C,X\n6.12\nF = 25.2 <0.001\nF = 29.4 <0.001\nF = 10.3 <0.001\n21–102\nPTHL\n34.68A\n36.23Y\n44.99B,Y\n44.67B,Y\nAST6\n(IU/L)\nCON\n23.36A\n41.15B,X\n46.53B,X\n47.68B,X\n3.34\nF = 22.1 <0.001\nF = 28.6 <0.001\nF = 11.2 <0.001\n0.0–66.0\nPTHL\n22.25A\n26.77Y\n31.91B,Y\n33.67B,Y\nALP7\n(IU/L)\nCON\n23.62A\n29.87\n34.22B\n37.34B\n3.61\nF = 1.8 0.178\nF = 15.7 <0.001\nF = 3.9 0.008\n20–156\nPTHL\n24.06A\n26.45\n28.71\n32.53B\nCholesterol\n(mg/dL)\nCON\n178.93\n179.37\n203.14\n197.87\n22.33\nF = 0.2 0.689\nF = 0.9 0.442\nF = 0.7 0.603\n135–270\nPTHL\n170.4\n180.76\n186.12\n194.66\n1Standard error of the means.\n2Group.\n3Time.\n4Kaneko et al. (62).\n5Alanine aminotransferase.\n6Aspartate aminotransferase.\n7Alkaline phosphatase.\nDifferent letters on the same parameter of the same group show statistical differences: A, B, C = P < 0.01. Different letters at the same time for the same parameter show statistical differences:\nX, Y = P < 0.01; x, y = P < 0.05.\ndays in CON group and at 90 days in PTHL group (P < 0.01),\n\n01.Different letters at the same time for the same parameter show statistical differences:\nX, Y = P < 0.01; x, y = P < 0.05.days in CON group and at 90 days in PTHL group (P < 0.01),\nreporting higher values in PTHl grop at 60 and 90 days (P <\n0.05). Clycemia decreased in CON group at 30 days (P < 0.01)\nand then again at 90 days (P < 0.01), while decreased directly at\n90 days in PTHL grooup (P < 0.01), showing in this last group\nhigher values than CON one from 30 to 90 days of trial (P <\n0.01). ALT serum levels inreased at 30 and 60 days in CON group\n(P < 0.01), while increased at 60 days in PTHL one (P < 0.01),\nreporting in this last one costantly lower values than CON group\nfrom 30 to 90 days (P < 0.01). Differently, AST increased at 30\ndays in CON group and at 60 days in PTHL group (P < 0.01),\nremaining constant until 90 days in both groups but showing\nhigher values in CON one from 30 to 90 days (P < 0.01). ALP\nlevels increased at 60 days in CON group and at 90 days in PTHL\none (P < 0.01), with no differences between groups within each\nexperimental time (P > 0.05). The fixed effects of group, time\nand their interaction on chloride, sodium, potassium, magnesium,\nphosphorous and calcium serum concentration are presented in\nTable 5. Chloride was affected by the interaction of group and\ntreatment. After 90 days it showed lower values than 0 and 30 days\nin CON group (P < 0.01) and than PTHL group at the sema time\n(P < 0.01).\nFrontiers in Veterinary Science\n07\nfrontiersin.org\n\nForte et al.\n10.3389/fvets.2025.1603458\nTABLE 5 Chloride, sodium, potassium, magnesium, phosphorus and calcium serum concentration in heat-stressed dogs over 90 days, with (PTHL) or\nwithout (CON) dietary supplementation of Pinus taeda hydrolyzed lignin.\nParameter\nGroup\nTime (days)\nSEM1\nP-value\nReference\nvalue4\n0\n30\n60\n90\nG2\nT3\nG × T\nChloride\n(mEq/L)\nCON\n113.82A\n112.63A\n98.71\n92.15B,X\n10.91\nF = 20.5 <0.001\nF = 28.7 <0.001\nF = 9.2 <0.001\n105.0–115.0\nPTHL\n113.47\n110.85\n111.63\n113.23Y\nSodium\n(mmol/L)\nCON\n144.72\n149.79\n145.99\n142.52\n13.88\nF = 0.16 0.689\nF = 0.13 0.912\nF = 0.57 0.591\n141.0–152.0\nPTHL\n147.47\n146.17\n144.95\n145.83\nPotassium\n(mg/dL)\nCON\n4.48\n4.46\n4.51\n4.51\n0. 35\nF = 0.06 0.819\nF = 0.43 0.711\nF = 0.69 0.507\n4.4–5.3\nPTHL\n4.52\n4.49\n4.53\n4.55\nMagnesium\n(mg/dL)\nCON\n2.16\n2.08\n2.13\n2.11\n0.22\nF = 0.04 0.840\nF = 0.35 0.765\nF = 0.66 0.554\n1.8–2.4\nPTHL\n2.26\n2.22\n2.14\n2.24\nPhosphorus\n(mg/dL)\nCON\n4.32\n4.66\n4.41\n4.78\n0.48\nF = 0.09 0.771\nF = 0.44 0.697\nF = 0.23 0.817\n2.6–6.2\nPTHL\n4.03\n4.27\n4.33\n4.29\nCalcium\n(mg/dL)\nCON\n10.55\n10.65\n11.07\n10.82\n1.29\nF = 0.96 0.336\nF = 0.41 0.677\nF = 0.52 0.752\n9–12\nPTHL\n10.43\n9.95\n10.31\n10.27\n1Standard error of the means.\n2Group.\n3Time.\n4Kaneko et al. (62).\nDifferent letters on the same parameter of the same group show statistical differences: A, B = P < 0.01. Different letters at the same time for the same parameter show statistical differences: X,\nY = P < 0.01.\n3.2 Oxidative stress results\nThe fixed effects of group, time and their interaction on the\noxidative profile are shown in Table 6. FRAP, SOD, CAT and GSPx\nwere affected by the interaction of group and treatment (P < 0.01),\nTBARs were affected by time (P < 0.05). TBARs plasma levels\nincreased in at 30 days in CON group (P < 0.01) and then remained\nconstant until 90 days, while in PTHL group TBARs plasma levels at\n90 days were higher than 0 days (P < 0.05). At 30, 60 (P < 0.05) and\n90 (P < 0.01) days, CON group showed higher TBARs levels than\nPTHL one. FRAP plasma levels increased in PTHL group at 30 days\nand then at 90 days (P < 0.01), reporting higher values than CON\ngroup from 30 to 90 days of trial (P < 0.01). SOD plasma levels at\n30 days decreased in CON group and increased in PTHL group (P\n< 0.01), showing lower values in CON group from 30 to 90 days\nof trial (P < 0.01). The CAT levels increased at 60 and then at 90\n\n01).SOD plasma levels at\n30 days decreased in CON group and increased in PTHL group (P\n< 0.01), showing lower values in CON group from 30 to 90 days\nof trial (P < 0.01).The CAT levels increased at 60 and then at 90\ndays in CON group (P < 0.01), while in PTHL group shoved higher\nvalues at 90 days compared to 0 days (P < 0.01). Moreover, the PL\ngrop showed higher CAT values at 60 and 90 days (P < 0.01). The\nGSPx plasma levels decreased in CON group at 60 days, reporting\nlower values tha PTHL group at 60 and 90 days (P < 0.01).\n3.3 Semen results\nThe fixed effects of group, time and their interaction on sperm\nparameters are shown in Table 7. Volume, concentration, TSN,\nSTR, LIN, HOS (P < 0.01), VCL, and Sperm viability (P < 0.05)\nwhere affected by the interaction of group and time. Semen volume\ndecreased at day 90 compared to day 0 in both CON (P < 0.01) and\nPTHL (P < 0.05) groups, with higher values observed in the latter\n(P < 0.05). Sperm concentration decreased at 30 (P < 0.05), 60 and\n90 days in CON group (P < 0.01) and at 60 days in PTHL group (P\n< 0.01), showing in this last one higher values at 60 and 90 days (P\n< 0.01). The TSN decreased at 30 (P < 0.05), 60 and 90 (P < 0.01)\ndays in CON group, while only at 60 days in PTHL one (P < 0.01),\nshowing in this last group higher values at 60 and 90 days compared\nto CON group (P < 0.01). VCL values decreased in CON group at\n30 days (P < 0.05) and from 30 to 90 days remained lower than\nPTHL group (P < 0.05). STR values of CON group decreased at 60\ndays (P < 0.01), showing lower values tha PTHL group at 60 (P <\n0.05) and 90 (P < 0.01) days. LIN and HOS values of CON group at\n90 days were lower than 0 days (P < 0.01) and HOS showed lower\nvalues at 90 days than PTHL group (P < 0.01). Also sperm viability\nshowed lower values at 90 days compared to 0 days in CON group\n(P < 0.05). The fixed effects of group, time and their interaction\non spermatozoa motility parameters are reported in Table 8. Total\nmotility, progressive motility, rapid, slow and static values where\naffected by the interaction of group and time (P < 0.01). Both total\n\n05).The fixed effects of group, time and their interaction\non spermatozoa motility parameters are reported in Table 8.Total\nmotility, progressive motility, rapid, slow and static values where\naffected by the interaction of group and time (P < 0.01).Both total\nand progressive sperm motility in CON group decreased at 60 days\ncompared to day 0 (P < 0.01), with a further decline at 90 days\n(P < 0.01). Additionally, at 90 days, CON group revealed lower\ntotal motility (P < 0.01), and at both 60 (P < 0.05) and 90 (P <\n0.01) days, lower progressive motility compared to PTHL group.\nThe percentage of rapid sperm movements decreased at 30 and\n60 days in CON dogs (P < 0.01) and at day 90 in PTHL dogs (P\n< 0.01) compared to their respective day 0 values. Additionally,\nthe percentage of rapid movements was higher in PTHL group at\n60 and 90 days (P < 0.01). The percentage of slow movements\nincreased in both groups from 30 to 90 days (P < 0.01) compared\nto 0 days (P < 0.01), but it was higher in CON group than in\nPTHL one from 30 to 90 days (P < 0.01). Moreover, the percentage\nFrontiers in Veterinary Science\n08\nfrontiersin.org\n\nForte et al.\n10.3389/fvets.2025.1603458\nTABLE 6 Thiobarbituric acid reactive substances (TBARs), hydroperoxides, protein carbonyls, Ferric Reducing Antioxidant Power (FRAP), superoxide\ndismutase (SOD), catalase (CAT) and glutathione peroxidase (GSPx) serum concentration in heat-stressed dogs over 90 days, with (PTHL) or without\n(CON) dietary supplementation of Pinus taeda hydrolyzed lignin.\nParameter\nGroup\nTime (days)\nSEM1\nP-value\n0\n30\n60\n90\nG2\nT3\nG × T\nTBARs 4\n(mmol/mL)\nCON\n1.29A\n2.36B,x\n2.85B,x\n3.09B,X\n0.18\nF = 1.5 0.223\nF = 3.1 0.042\nF = 0.5 0.622\nPTHL\n1.33a\n1.68y\n1.66y\n1.82b,Y\nHydroperoxides\n(mmol/mL)\nCON\n5.15\n5.23\n5.17\n5.34\n0.51\nF = 0.2 0.671\nF = 1.0 0.369\nF = 0.1 0.706\nPTHL\n4.98\n5.09\n4.84\n4.91\nProtein carbonyls\n(µmol/mg Protein)\nCON\n93.88\n89.23\n92.94\n88.01\n10.18\nF = 0.6 0.452\nF = 0.6 0.662\nF = 0.5 0.467\nPTHL\n94.03\n90.56\n89.78\n93.21\nFRAP5\n(µmol TE/mL)\nCON\n47.64\n45.92X\n46.27X\n42.35X\n0.41\nF = 50.2 <.0001\nF = 55.3 <.0001\nF = 48.7 <.0001\nPTHL\n46.01A\n58.25B,Y\n58.93B,Y\n61.53C,Y\nSOD6\n(U/mL)\nCON\n44.39A\n36.81B,X\n32.20B,X\n34.01B,X\n1.140\nF = 45.8 <.0001\nF = 60.1 <.0001\nF = 40.3 <.0001\nPTHL\n43.98A\n49.72B,Y\n51.31B,Y\n50.57B,Y\nCAT 7\n(U/mL)\nCON\n2.24A\n2.18AB\n1.71B,X\n1.55C,X\n0.07\nF = 30.5 <.0001\nF = 25.7 <.0001\nF = 28.4 <.0001\nPTHL\n2.26A\n2.38\n2.41Y\n2.70B,Y\nGSPx 8\n(nmol NADPH ox/mL)\nCON\n2.41A\n2.33\n1.80B,X\n1.71B,X\n0.09\nF = 33.2 <.0001\nF = 42.5 <.0001\nF = 35.6 <.0001\nPTHL\n2.32\n2.43\n2.49Y\n3.52Y\n1Standard error of the means.\n2Group.\n3Time.\n4TBARS - Thiobarbituric Acid Reactive Substances.\n5FRAP - Ferric Reducing Antioxidant Power.\n6SOD - Superoxide Dismutase.\n7CAT - Catalase.\n8GSPx - Glutathione Peroxidase.\nDifferent letters on the same row show statistical differences during time in the same group: A, B, C, D = P < 0.01; Different letters on the same column show statistical differences between\ngroups at the same aging time: X, Y = P < 0.01.\nof static movements increased only in CON dogs starting from\n30 days compared to 0 days (P < 0.01) and was higher than in\nPTHL dogs from 30 to 90 days (P < 0.01). Figure 3 illustrates\nthe concentrations of d-ROMs and BAP in the seminal plasma. d-\nROMs levels increased only in the seminal plasma of CON group\nat 60 and 90 days compared to 0 days (P < 0.01), and at both\ntime points, their concentrations were higher than those in PTHL\ngroup (P < 0.01). In contrast, BAP levels in the seminal plasma of\n\nd-\nROMs levels increased only in the seminal plasma of CON group\nat 60 and 90 days compared to 0 days (P < 0.01), and at both\ntime points, their concentrations were higher than those in PTHL\ngroup (P < 0.01).In contrast, BAP levels in the seminal plasma of\nCON group decreased at 60 and 90 days compared to 0 days (P <\n0.01), and at 90 days, they were lower than those in PTHL group (P\n< 0.01).\n4 Discussion\nThis\nstudy\nevaluated\nthe\nefficacy\nof\nPTHL\ndietary\nsupplementation in mitigating oxidative stress and preserving\nsperm quality in dogs subjected to heat stress conditions. While\nprior study demonstrated the benefits of PTHL in enhancing\nantioxidant status and sperm quality in clinically healthy dogs\n(17), this research extended the inquiry to heat stress scenarios,\nyielding new insights into its protective effects under adverse\nenvironmental conditions. This study provides novel evidence that\ndietary PTHL supplementation can attenuate the physiological\nand reproductive impacts of heat stress, supporting its potential\napplication in canine breeding programs. Environmental stressors,\nincluding heat, significantly affect hematological parameters in\nvertebrates, often signaling stress conditions (43). Blood, as a\ndynamic biomarker, revealed notable alterations in this study.\nSpecifically, reductions in serum total protein, albumin, urea,\nand glucose levels, coupled with increases in ALT, AST, and\nALP levels, highlighted the physiological strain imposed by heat\nstress. These changes, observed in both CON and PTHL groups,\nappeared later and were less pronounced in PTHL-supplemented\ndogs, underscoring the potential mitigating role of PTHL. The\nreduction in total protein and albumin levels in heat-stressed\ndogs is consistent with previous findings (4, 44, 45). These\nchanges likely reflect cellular damage, coupled with increased\nmetabolic activity and oxygen consumption, which together can\ntrigger widespread cellular necrosis through protein denaturation\n(46). Concurrently, elevated AST and ALT levels point to liver\ndysfunction, which aligns with prior research suggesting impaired\n\nConcurrently, elevated AST and ALT levels point to liver\ndysfunction, which aligns with prior research suggesting impaired\nhepatic blood flow and oxygen delivery during thermal stress (45).\nThe observed hypoglycemia likely arises from the dual impact of\nincreased glucose utilization to meet thermoregulatory demands\nand enhanced insulin absorption due to peripheral vasodilation\n(47, 48). Alterations in serum urea and chloride concentrations\nunder heat stress could be attributed to compensatory mechanisms\nFrontiers in Veterinary Science\n09\nfrontiersin.org\n\nForte et al.\n10.3389/fvets.2025.1603458\nTABLE 7 Volume, concentration, total sperm number (TSN), velocity average pathway (VAP), straight line velocity (VLS), curvilinear velocity (VCL),\namplitude of lateral head displacement (ALH), beat-cross frequency (BCF), straightness (STR), linearity (LIN), hypo-osmotic swelling test (HOS) and\nsperm viability of semen in heat-stressed dogs over 90 days, with (PTHL) or without (CON) dietary supplementation of Pinus taeda hydrolyzed lignin.\nParameter\nGroup\nTime (days)\nSEM1\nP-value\n0\n30\n60\n90\nG2\nT3\nG × T\nVolume\n(mL)\nCON\n3.35A\n3.12\n2.78\n2.37B,x\n0.26\nF = 4.2 0.045\nF = 4.6 0.0085\nF = 6.1 0.0003\nPTHL\n3.33a\n3.16\n3.02\n2.96b,y\nConcentration\n(M/mL)\nCON\n254.41Aa\n232.50Ab\n206.32B,x\n180.16B,x\n18.35\nF = 6.2 0.0201\nF = 30.5 <.0001\nF = 10.5 0.0013\nPTHL\n248.33a\n236.66\n223.22by\n226.21b,y\nTSN\n(M)\nCON\n869.34Aa\n736.11Ab\n584.22B,X\n448.09C,X\n52.85\nF = 25.3 <.0001\nF = 40.2 <.0001\nF = 20.7 <.0001\nPTHL\n854.49A\n751.99\n668.07B,Y\n658.44B,Y\nVAP\n(µm/s)\nCON\n96.31\n88.44\n84.22\n81.16\n10.35\nF = 0.2 0.6652\nF = 0.8 0.4701\nF = 0.5 0.6229\nPTHL\n95.33\n92.15\n93.62\n88.61\nVLS\n(µm/s)\nCON\n95.18\n90.66\n90.14\n88.22\n11.25\nF = 0.3 0.6225\nF = 0.7 0.3328\nF = 0.4 0.5383\nPTHL\n96.41\n93.45\n94.19\n92.77\nVCL\n(µm/s)\nCON\n173.33a\n156.71b,x\n154.40b,x\n155.19bx\n13.88\nF = 5.3 0.0257\nF = 3.1 0.0446\nF = 3.7 0.0184\nPTHL\n178.16\n173.36y\n170.17y\n169.09y\nALH\n(µ)\nCON\n5.22\n5.54\n5.72\n5.59\n0.84\nF = 0.5 0.7745\nF = 0.6 0.5769\nF = 0.3 0.6106\nPTHL\n5.71\n5.88\n5.49\n6.01\nBCF\n(Hz)\nCON\n24.36\n25.12\n23.49\n24.81\n3.62\nF = 0.6 0.5913\nF = 0.4 0.5116\nF = 0.7 0.4289\nPTHL\n26.31\n26.86\n25.37\n26.21\nSTR\n(VSL/VAP)\nCON\n84.81A\n79.21\n76.19Bx\n74.88BX\n7.33\nF = 30.1 <.0001\nF = 35.7 <.0001\nF = 28.4 <.0001\nPTHL\n85.12\n82.53\n83.16y\n81.41Y\nLIN\n(VSL/VCL)\nCON\n59.31A\n56.82\n53.21\n49.83BX\n4.18\nF = 9.5 0.0030\nF = 12.8 0.0002\nF = 15.2 <.0001\nPTHL\n60.66\n59.33\n56.61\n57.47Y\nHOS\n(% cureled)\nCON\n94.33A\n90.44\n88.61\n85.95B\n6.31\nF = 1.5 0.2214\nF = 7.1 0.0007\nF = 6.8 0.0004\nPTHL\n93.66\n91.93\n90.21\n89.89\nSperm viability\n(Eo-Nig) (%)\nCON\n92.49a\n90.12\n88.26\n86.66b\n4.49\nF = 0.2 0.6443\nF = 1.1 0.3221\nF = 5.9 0.0159\nPTHL\n93.01\n92.12\n91.44\n90.40\n1Standard error of the means.\n2Group × Time.\n3G is group and T is time.\nDifferent letters on the same line show statistical differences during time in the same group: A, B = P < 0.01; a, b = P < 0.05; Different letters on the same row show statistical differences\nbetween groups at the same aging time: X, Y = P < 0.01; x, y = P < 0.05.\nfor maintaining hydration. Excessive water intake driven by heat\nexposure often results in overhydration, leading to alterations\n\n05; Different letters on the same row show statistical differences\nbetween groups at the same aging time: X, Y = P < 0.01; x, y = P < 0.05.for maintaining hydration.Excessive water intake driven by heat\nexposure often results in overhydration, leading to alterations\nin these parameters (49). Heat stress is a well-documented\ntrigger for oxidative stress, disrupting the balance between the\nproduction of ROS and the antioxidant defense mechanisms (1).\nOxidative stress induced by heat stress is well-documented in\nseveral species (50–52). ROS, such as superoxide anions, hydrogen\nperoxide, and hydroxyl radicals, are natural by-products of aerobic\nmetabolism with dual roles. On the one hand, they contribute\nto essential processes such as immune defense and intracellular\nsignaling. On the other hand, excessive ROS accumulation causes\noxidative damage by reacting with cellular macromolecules,\nincluding lipids, proteins, and DNA. This damage occurs when\nthe rate of ROS production surpasses the neutralizing capacity\nof endogenous antioxidants, which include enzymatic (e.g., SOD,\nCAT) and non-enzymatic (e.g., glutathione, vitamins C and E)\ncomponents (1). Although research on the effects of heat stress in\ndogs is limited, Burgio, Forte, Prete, Maggiolino, De Palo, Aiudi,\nRizzo, Carbonari, Lacalandra and Cicirelli (4) provided evidence\nsupporting the impact of heat stress on oxidative stress markers\nin this species. Our findings confirm these effects, demonstrating\nincreased TBARS and ROS levels while antioxidant enzyme activity\nwas reduced. Specifically, exposure to heat stress led to a marked\nimbalance in redox homeostasis: plasma TBARS and ROS levels\nwere significantly elevated, while the activities of key antioxidant\ndefenses—SOD, CAT and GSPx—and overall antioxidant capacity\n(as measured by FRAP) were significantly suppressed compared\nto dogs maintained under thermoneutral conditions. Conversely,\ndietary antioxidant supplementation has been shown to enhance\nthe bioavailability of polyphenolic and other bioactive compounds\nFrontiers in Veterinary Science\n10\nfrontiersin.org\n\nForte et al.\n10.3389/fvets.2025.1603458\nTABLE 8 Total motility, progressive motility, and the distribution of rapid, medium, slow, and static movements of spermatozoa in heat-stressed dogs\nover 90 days, with (PTHL) or without (CON) dietary supplementation of Pinus taeda hydrolyzed lignin.\nParameter\nGroup\nTime (days)\nSEM1\nP-value\n0\n30\n60\n90\nGroup\nTime\nG × T2\nTotal motility\n(%)\nCON\n91.82A\n87.87AB\n82.41B\n76.19CX\n7.98\nF = 50.4 <.0001\nF = 45.7 <.0001\nF = 38.2 <.0001\nPTHL\n89.66\n88.33\n86.66\n87.66Y\nProgressive motility\n(%)\nCON\n71.37A\n69.44A\n63.04Bx\n57.56CX\n6.12\nF = 5.0 0.0315\nF = 40.9 <.0001\nF = 35.6 <.0001\nPTHL\n72.22\n70.33\n68.52y\n67.91Y\nRapid\n(%)\nCON\n91.26A\n85.55B\n72.21C,X\n67.18C,X\n5.58\nF = 60.2 <.0001\nF = 55.1 <.0001\nF = 45.9 <.0001\nPTHL\n90.33A\n88.64\n85.66Y\n81.51B,Y\nMedium\n(%)\nCON\n9.47\n9.33\n10.19\n9.89\n1.17\nF = 0.3 0.4401\nF = 0.4 0.5992\nF = 0.6 0.6914\nPTHL\n9.88\n10.28\n9.29\n9.46\nSlow\n(%)\nCON\n4.42A\n15.12B,X\n16.28B,X\n17.62B,X\n0.43\nF = 50.3 <.0001\nF = 48.7 <.0001\nF = 42.2 <.0001\nPTHL\n5.02A\n9.19B,Y\n10.31B,Y\n10.49B,Y\nStatic\n(%)\nCON\n2.41A\n4.83B,X\n5.61B,X\n5.44B,X\n0.44\nF = 10.6 0.0015\nF = 12.3 0.0002\nF = 20.4 <.0001\nPTHL\n2.04\n2.64Y\n2.82Y\n2.70Y\n1Standard error of the means.\n2Group × Time.\nDifferent letters in the same row show statistical differences during time in the same group: A, B = P < 0.01; a, b = P < 0.05; Different letters on the same column show statistical differences\nbetween groups at the same aging time: X, Y = P < 0.01; x, y = P < 0.05.\nin target tissues, resulting in a robust, measurable increase in\ntotal antioxidant capacity across multiple organ systems (53).\nThis effect is particularly relevant for polyphenolic compounds\nfound in plants, which possess potent antioxidant properties\n(54). Our study extends previous research by showing that\nPTHL supplementation not only enhances antioxidant status in\nhealthy dogs but also plays a protective role under heat stress\nconditions. However, considering the strong connection between\nheat stress and oxidative stress, such dietary interventions could\noffer a promising approach to alleviate the negative effects of heat\nstress by enhancing the antioxidant defense system and reducing\noxidative damage (54, 55). Our findings reveal a significant increase\nin plasma TBARS levels of CON dogs as early as 30 days into the\ntrial. In contrast, PTHL dogs showed a delayed rise in TBARS\n\nOur findings reveal a significant increase\nin plasma TBARS levels of CON dogs as early as 30 days into the\ntrial.In contrast, PTHL dogs showed a delayed rise in TBARS\nlevels, which became apparent only at 90 days and remained\nnotably lower than those observed in the CON group. These results\nsuggest that PTHL supplementation not only mitigated the extent\nof lipid oxidation but also effectively delayed its onset. Moreover,\nthe concentrations of antioxidant enzymes, including SOD, CAT,\nand GSPx, were consistently higher in PTHL group compared\nto CON dogs. Additionally, the plasma of PTHL dogs showed\nsignificantly higher FRAP values than that of CON dogs. Together,\nthese findings highlight the potential of PTHL supplementation to\nfortify the plasma’s antioxidant defense system, providing effective\nprotection against the oxidative challenges imposed by heat stress.\nHeat stress negatively affects not only the oxidative status\nbut also sperm quality of male dogs, reducing reproductive\nperformance. In dogs, elevated temperatures are associated\nwith reduced libido, impaired spermatogenesis, lower sperm\nconcentration, poor sperm quality, decreased testicular weight,\nand a temporary period of partial or total infertility (54,\n55). Sperm parameters, including motility and vigor, may be\nnegatively impacted, while morphological defects such as acrosome\ndegradation, proximal cytoplasmic droplets, bent tails at the\nhead, small heads, and isolated heads become more prevalent,\nas observed in bulls (56). In this study, heat stress worsened\nthe semen characteristics. In both groups, before the warmer\nmonths (T0), the parameters examined (volume, motility, vigor,\nsperm concentration, total sperm count and morphology) were\nwithin the normal range. Afterwards, with an increase in THI\n(in July and August), the semen characteristics have worsened.\nVolume, concentration and TSN progressively decreased during\nthe study, as did the motility parameters. The volume decreased\nin both groups, reaching statistically significant differences at T90.\nThis reduction was more evident in the CON group than in\n\nVolume, concentration and TSN progressively decreased during\nthe study, as did the motility parameters.The volume decreased\nin both groups, reaching statistically significant differences at T90.This reduction was more evident in the CON group than in\nthe PTHL group. Concentration and TSN, instead, were already\nreduced at T30 in the CON group, while in the PTHL group\nthis occurred at T60. At T90, both parameters were worse in the\nCON group than in the PTHL group. Probably, these negative\neffects are due to ROS produced by the animals’ exposure to\nhigh temperatures, which negatively affect spermatogenesis and\ncan cause cellular damage and apoptosis (57). This hypothesis\nis also supported by the results of sperm viability, where a\nreduction in live spermatozoa was observed in the control animals\ncompared to the treated group at T90. Spermatozoa viability\nworsened significantly only in the CON group, while the PTHL\ngroup did not show any significant decline. However, in the\nPTHL group, the seminal parameters worsened less during the\nsummer months than in the CON group. This is likely due to\nthe antioxidant effect of a polyphenolic mixture of substances\nderived from the hydroxylation of Pinus taeda lignin. Specifically,\nPTHL supplementation can significantly increase semen volume\nand sperm motility, as demonstrated in healthy dogs under normal\nenvironmental conditions and temperatures (17). Furthermore, in\nFrontiers in Veterinary Science\n11\nfrontiersin.org\n\nForte et al.\n10.3389/fvets.2025.1603458\nFIGURE 3\nReactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP) concentrations in canine seminal plasma (third fraction). Different\nletters of the same colors show statistical differences among times in the same group: A, B = P < 0.01; ** = P < 0.01 showed statistical differences\nbetween groups in the same experimental time.\nthis study, total motility decreased in both groups but at different\ntimes: at T30 in the CON group and at T90 in the PTHL group. In\nthe PTHL group, progressive motility and the percentage of rapidly\nmoving sperm did not worsen significantly, and the proportion\nof slow and static sperm did not increase as observed in the\ncontrol group in August. This was happening probably on the\nantioxidant power of polyphenols contained in PTHL, which can\nimprove sperm quality at the beginning of the sperm differentiation\nand development. Itis well known that oxidative stress is one\nof the major issues associated with sperm function (58) and\nsperm motility is one of the factors limiting male fertility. In fact,\nspermatozoa are sensitive to oxidative stress because they are well\nendowed with polyunsaturated fatty acids, which are vulnerable\nto free radical attack at the alpha methylene carbons adjacent to\nthe carbon–carbon double bonds. In addition, the spermatozoa’s\ncapacity for antioxidative defense is relatively low/vulnerable\nin comparison to other cells/tissues that are susceptible (59).\nFurthermore, spermatozoa are attacked by the oxidative effect of\nleukocytes, such as neutrophils, which are present in the male\ngenitals for infection and other causes. The literature shows that\nantioxidants may play a primary role in protecting male germ cells\nagainst oxidative action (60). Our results encourage considering\nthe alimentary approach of balanced food supplementation for the\nsolution of dog subfertility. Balanced feed supplementation and\nintegration could mitigate the negative impact of heat stress in\ncanine breeding.\nFrontiers in Veterinary Science\n12\nfrontiersin.org\n\nForte et al.\n10.3389/fvets.2025.1603458\n5 Conclusions\nThis study provides compelling evidence supporting the\nefficacy of PTHL dietary supplementation in mitigating the\ndetrimental effects of heat stress on oxidative status and sperm\nquality in dogs. PTHL supplementation effectively delayed and\nreduced lipid peroxidation while enhancing enzymatic antioxidant\nactivity\n(SOD,\nCAT,\nand\nGSPx)\nand\nFRAP.\nFurthermore,\nit preserved seminal parameters such as progressive motility\nand the percentage of rapid-movement sperm, which were\nsignificantly compromised in CON group. Incorporating dietary\nsupplementation into breeding programs, PTHL presents a\npromising natural strategy to alleviate the negative impacts of\nheat stress, safeguard reproductive health, and improve fertility\noutcomes in dogs. In light of these findings, PTHL dietary\nsupplementation proves to be a natural, sustainable and effective\nstrategy to strengthen the antioxidant defenses of dogs, enhance\ntheir overall well being, and safeguard sperm quality, even under\nthe challenging conditions of heat stress. While our findings\nhighlight the benefits of PTHL supplementation, certain limitations\nmust be acknowledged. Variations in individual responses to heat\nstress and supplementation could not be fully accounted for.\nAdditionally, the study duration may not reflect the cumulative\neffects of prolonged heat stress exposure. Future research should\ninvestigate different dosages, longer supplementation periods, and\npotential interactions with other dietary antioxidants to optimize\nreproductive performance in heat-stressed dogs.\nData availability statement\nThe raw data supporting the conclusions of this article will be\nmade available by the authors, without undue reservation.\nEthics statement\nThe animal studies were approved by Institutional Review\nBoard (approval number: 656/18 – III/13). The studies were\nconducted\nin\naccordance\nwith\nthe\nlocal\nlegislation\nand\ninstitutional\nrequirements.\nWritten\ninformed\nconsent\nwas\nobtained from the owners for the participation of their animals in\nthis study.\nAuthor contributions\nLF: Data curation, Formal analysis, Methodology, Writing –\n\nWritten\ninformed\nconsent\nwas\nobtained from the owners for the participation of their animals in\nthis study.Author contributions\nLF: Data curation, Formal analysis, Methodology, Writing –\noriginal draft. MB: Data curation, Formal analysis, Methodology,\nWriting\n–\noriginal\ndraft.\nAR:\nConceptualization,\nFunding\nacquisition,\nValidation,\nWriting\n–\nreview\n&\nediting.\nAM:\nConceptualization, Funding acquisition, Investigation, Project\nadministration, Validation, Writing – review & editing. AC: Formal\nanalysis, Methodology, Software, Writing – original draft. PD:\nSupervision, Validation, Visualization, Writing – review & editing.\nGL: Supervision, Visualization, Writing – review & editing. VC:\nConceptualization, Investigation, Project administration, Writing\n– review & editing.\nFunding\nThe author(s) declare that no financial support was received for\nthe research and/or publication of this article.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declare that no Gen AI was used in the creation\nof this manuscript.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. 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+{"text": "【兽医文献】\n标题: Transcriptome and HS-SPME-GC-MS analysis of key genes and flavor components associated with beef marbling\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nEffects of Pueraria extracts on\ngrowth performance, immune\nfunction, and immune-related\ngene expression of Wuzhishan\npiglets\nHongzhi Wu 1†, Weiqi Peng 1†, Xilong Yu 2, Xiaoyu Zhang 3,\nFengjie Ji 1, Qian Shen 4 and Renlong Lv 1*\n1 Tropical Crops Genetic Resources Research Institute, Chinese Academy of Tropical Agricultural\nSciences, Haikou, China, 2 College of Animal Science and Technology, Northeast Agricultural\nUniversity, Harbin, China, 3 College of Animal Science and Technology, Henan University of Science\nand Technology, Luoyang, China, 4 Hainan Xuhuai Technology Co. Ltd., Haikou, China\nPueraria extracts contain phytoestrogens, particularly isoflavones such as daidzein\nand genistein, which have been used in traditional Chinese medicine for centuries\ndue to their various health benefits. Wuzhishan pig is a small pig unique to China,\nnative to Wuzhishan, Hainan Province. Pueraria extracts have been widely\nutilized in the production of various animals. However, there are relatively few\nreports regarding their application in piglets, particularly in local pig breeds. The\nexperiment aimed to investigate the effects of dietary Pueraria extracts on the\ngrowth performance, immune function, and immune-related gene expression of\nWuzhishan piglets. Forty-eight piglets were randomly divided into four groups,\neach with six replicates and two piglets in each replicate. The control group (CON)\nwas fed the basal diet, and the other groups were given 400, 600, and 800 mg/kg\nPueraria extracts (PE1, PE2, and PE3, respectively). Compared with the CON, (1)\nthe dietary Pueraria extracts increased the average daily gain and decreased the\nfeed conversion ratio in PE2 and PE3 (p < 0.05); (2) the dietary Pueraria extracts\nincreased the serum immunoglobulin M and complement 3 and 4 contents in\nPE2 (p < 0.05); (3) the serum ALB and ALP contents in groups treated with the\nPueraria extracts were higher (p < 0.05); (4) the ALT and GOT contents were lower\n(p < 0.05) in groups treated with Pueraria extracts; (5) the dietary Pueraria extracts\n\n05); (3) the serum ALB and ALP contents in groups treated with the\nPueraria extracts were higher (p < 0.05); (4) the ALT and GOT contents were lower\n(p < 0.05) in groups treated with Pueraria extracts; (5) the dietary Pueraria extracts\nincreased the Bacillus acidi lactici and Saccharomycetes contents, and decreased\nthe Escherichia coli and Salmonella contents in the jejunum in PE1, PE2, and PE3\n(p < 0.05); (4) the dietary Pueraria extracts ameliorated the relative expression of\nInterleukin-10, Tumor necrosis factor-α, Mucoprotein 2, Transforming growth\nfactor-β1, Zonula occludens-1 in jejunum mucosa (p < 0.05). In this study, adding\n600 mg/kg dietary Pueraria extracts to the Wuzhishan piglets’ diets improved the\ngrowth performance, immune function, and immune-related gene expression.\nThis study laid a theoretical foundation for popularizing and applying Pueraria\nextracts in local pig production.\nKEYWORDS\nPueraria extracts, Wuzhishan piglets, growth performance, immune function,\nimmune-related gene expression\nOPEN ACCESS\nEDITED BY\nMoyosore Joseph Adegbeye,\nUniversity of Africa, Nigeria\nREVIEWED BY\nXihong Zhou,\nChinese Academy of Sciences (CAS), China\nHongbin Pan,\nYunnan Agricultural University, China\nZipeng Jiang,\nZhejiang University, China\n*CORRESPONDENCE\nRenlong Lv\nlvrenlong@aliyun.com\n†These authors have contributed equally to\nthis work\nRECEIVED 04 September 2024\nACCEPTED 15 May 2025\nPUBLISHED 30 May 2025\nCITATION\nWu H, Peng W, Yu X, Zhang X, Ji F,\nShen Q and Lv R (2025) Effects of Pueraria\nextracts on growth performance, immune\nfunction, and immune-related gene\nexpression of Wuzhishan piglets.\nFront. Vet. Sci. 12:1491130.\ndoi: 10.3389/fvets.2025.1491130\nCOPYRIGHT\n© 2025 Wu, Peng, Yu, Zhang, Ji, Shen and Lv.\nThis is an open-access article distributed\nunder the terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other forums is\npermitted, provided the original author(s) and\nthe copyright owner(s) are credited and that\nthe original publication in this journal is cited,\nin accordance with accepted academic\npractice. No use, distribution or reproduction\n\nNo use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Original Research\nPUBLISHED  30 May 2025\nDOI  10.3389/fvets.2025.1491130\n\nWu et al.\b\n10.3389/fvets.2025.1491130\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n1 Introduction\nPlant extracts, recognized for their natural and safe properties,\nhave increasingly become a valuable feed additive in livestock and\npoultry breeding, driven by their wide-ranging benefits (1–3). These\nbenefits extend across multiple dimensions of animal production, such\nas enhancing growth performance, boosting immune function,\nimproving carcass traits, and elevating meat quality (2, 3). In piglet\nnutrition, plant extracts have garnered particular attention in recent\nyears. Research has shown that specific plant extracts can optimize\npiglets’ growth performance and overall health by modulating gut\nmicrobiota and enhancing immune responses (4, 5). Essential oils\nderived from plants like oregano and thyme have demonstrated\nantimicrobial properties, which can effectively reduce the incidence of\ngastrointestinal infections in piglets, potentially diminishing the\nreliance on antibiotics (6). Additionally, Galanopoulos et al. explored\nthe antioxidant and anti-inflammatory effects of plant extracts, such as\nflaxseed oil, which can improve systemic and gut immunity in piglets\n(7). These findings underscore the necessity of further investigating the\napplications of plant extracts in piglet nutrition to foster sustainable\nand healthy livestock production practices. Pueraria extracts are\nderived from the root of the Pueraria lobata plant, native to parts of\nAsia, including China, Japan, and Korea (8–10). Pueraria extracts\ncontain phytoestrogens, particularly isoflavones such as daidzein and\ngenistein, which have been used in traditional Chinese medicine for\ncenturies due to their various health benefits (9, 11, 12). Wang et al.\n(13) found that Puerarin improves the immune response, antioxidant\ncapacity, and intestinal morphology of pigeons. Liu et al. (14) reported\nthat the Pueraria crude extracts significantly changed the fecal\nmicrobiota of finishing pigs. Guo et al. (15) found that the Pueraria\nextracts enhanced the antioxidant status and intestinal integrity of\n\nLiu et al.(14) reported\nthat the Pueraria crude extracts significantly changed the fecal\nmicrobiota of finishing pigs.Guo et al.(15) found that the Pueraria\nextracts enhanced the antioxidant status and intestinal integrity of\nbroilers. Zhang et al. (16) studied the Pueraria lobata extracts, which\nremediated intestinal dysbiosis and promoted bile acid biosynthesis.\nPueraria extracts have been widely utilized in the production of various\nanimals. However, there are relatively few reports regarding their\napplication in piglets, particularly in local pig breeds.\nThe Wuzhishan pig is a diminutive swine species endemic to\nChina, originating specifically from Wuzhishan in Hainan Province\n(17). Characterized by its small stature and robust and compact\nphysique, this pig has a relatively small and slightly elongated head,\nsmall and thin ears, and an irregular black and white fur distribution.\nMost individuals exhibit a black back and a white belly, earning them\nthe colloquial name mouse pig (18, 19). The Wuzhishan pig is highly\ninbred, rendering it an invaluable genetic resource for elucidating the\ngenetic underpinnings of various traits. Owing to its anatomical,\nphysiological, and pathological similarities to humans, it is an ideal\nbiomedical research model. Moreover, the Wuzhishan pig is a crucial\nbreed for the development of high-quality pork products. Its unique\ncharacteristics and potential applications underscore the practical\nsignificance of studying this pig breed.\nThe primary aim of this study is to systematically investigate the\nmultifaceted impacts of Pueraria extracts on Wuzhishan piglets.\nSpecifically, we hypothesize that Pueraria extracts can significantly\nenhance growth performance metrics, such as weight gain and feed\nefficiency. Additionally, we posit that these extracts can bolster immune\nfunction by upregulating key immune-related genes and improving\nbiochemical blood indicators. To achieve these goals, the study will (a)\nmeticulously assess growth parameters and immune responses in piglets\nadministered varying doses of Pueraria extracts; and (b) evaluate the\n\nTo achieve these goals, the study will (a)\nmeticulously assess growth parameters and immune responses in piglets\nadministered varying doses of Pueraria extracts; and (b) evaluate the\npractical applicability of Pueraria extracts in piglet production settings\nthrough pilot trials. By focusing on these targeted objectives, the study\naims to provide robust scientific evidence supporting the integration of\nPueraria extracts into pig production practices, thereby laying a solid\ntheoretical foundation for their widespread adoption.\n2 Materials and methods\n2.1 Experiment material\nForty-eight Wuzhishan piglets (sows or castrated boars) with an\naverage body weight of 10.00 ± 1.00 kg, the same genetic background,\nand similar birth dates were used.\nPueraria extracts (PE) were purchased from Shaanxi Baichuan\nBiotechnology Co., LTD., Xian, China. The active ingredient contents\nwere 4.37% puerarin, 1.62% daidzin, 1.05% puerarin apigenin, and\n0.27% genistin, detected in Liquid chromatography–tandem\nmass spectrometry.\nSerum biochemical kits were purchased from Shanghai Sangon\nBiotechnology Co., Ltd., Shanghai, China.\n2.2 Experiment design and sample\ncollection\nThe amount of Pueraria extracts added was a single influencing\nfactor in this study. Forty-eight Wuzhishan piglets were randomly\nallocated into four groups, with six replicates and two piglets per\nreplicate. The basal experimental diets for piglets were corn-soybean\nmeal-type diets formulated according to the NCR (2012) standards,\nand the composition of the basal experimental diets and nutritional\nlevels are shown in Table 1. The control group (Con) was fed basal\nexperimental diets, and the experimental groups were fed 400, 600,\nand 800 mg/kg Pueraria extracts in powder (PE1, PE2, and PE3,\nrespectively) according to recommended usage. The experiment\nperiod was 90 days. The piglet house was maintained at a temperature\nof 28–30°C and 60–70% humidity. Lighting was provided for 16 h\ndaily. Fecal waste was cleared twice daily, and the house was disinfected\nweekly with a 0.10% povidone-iodine solution to ensure hygiene.\n\nThe piglet house was maintained at a temperature\nof 28–30°C and 60–70% humidity.Lighting was provided for 16 h\ndaily.Fecal waste was cleared twice daily, and the house was disinfected\nweekly with a 0.10% povidone-iodine solution to ensure hygiene.\nAt the end of the experiment, all piglets were given 12 h of\nforbidden feeding and were allowed to drink freely. We chose one\npiglet that was close to the average weight for slaughter from each\nreplicate. We put one piglet that will be euthanized into a carbon\ndioxide euthanasia chamber (LC-800-S1, 80 × 70 × 60 cm3, Shanghai,\nChina). Once it is calm, the air in the chamber will be replaced with a\nmixture of 90% carbon dioxide and 10% air at a rate of 15% of the\nchamber’s volume per minute until the piglet is in a deep state of\nunconsciousness. The entire process is expected to take about 45 min.\nBefore slaughter, five mL of blood was collected from each pig’s ear\nvein and centrifuged at 3000 r/min for 15 min. The super serum was\ncollected and divided into 1.50 mL Ep tubes and stored at −20°C for\nlater use. Two grams of jejunum contents were taken from each piglet\nin 2.00 mL frozen storage tubes and stored in a − 80°C refrigerator for\nfuture use. Another section of the jejunum was taken and cut\nlengthwise to expose the intestinal cavity. Sterile saline was used to\nclean the intestinal cavity and gently remove the surface intestinal\n\nWu et al.\b\n10.3389/fvets.2025.1491130\nFrontiers in Veterinary Science\n03\nfrontiersin.org\ncontents. A sterile slide was used to scrape the intestinal mucosa\ngently, and this process should avoid penetrating the basement\nmembrane. The intestinal mucosa was stored in 2.00 mL frozen\nstorage tubes and placed in a − 80°C refrigerator for later use.\n2.3 Growth performance\nThe feed intake of piglets was carefully recorded. The amount of\nfeed offered and the amount wasted by the piglets in each replicate were\nprecisely measured and recorded daily. The average daily feed intake\n(ADFI) was calculated by subtracting the weight of leftover feed from\nthe initial feed provided. Additionally, each pig was weighed at the end\nof the experiment to determine the average daily gain (ADG). ADFI,\ng/d = Total feed intake, g/ Experiment period, d. ADG, g/d = (Final\nweight, kg - Initial weight, kg) * 1,000/Experiment period, d. Feed\nconversion ratio (FCR) = ADFI, g/d/ADG, g/d.\n2.4 Immune function\nThe immunoglobulin A (IgA), immunoglobulin G (IgG),\nimmunoglobulin M (IgM), complement 3 (C3), complement 4 (C4),\ntotal protein (TP), albumin (ALB), alkaline phosphatase (ALP),\nalanine transaminase (ALT), glutamic oxalacetic transaminase (GOT),\nand urea nitrogen (UN) in the serum were determined using\ncorresponding commercial assay kits, Shanghai Sangon Biotechnology\nCo., Ltd., Shanghai, China. The determining process is strictly\naccording to the kit instructions.\n2.5 Jejunal microorganism\nIn a sterile super-clean workbench, two grams of jejunum contents\nwere taken and diluted to 10−7 with sterile normal saline. Then the\ntotal bacteria, Escherichia coli, Bacillus acidi lactici, Salmonella, and\nSaccharomycetes were identified and cultured with LB culture,\nMacconkey AGAR culture, MRS culture, SS AGAR culture with\nbrilliant green, and Potato glucose AGAR with antibiotics, respectively.\nThe number of various microorganisms was counted using the plate\ncounting method.\n2.6 RNA extraction and quantitative\nanalysis of mRNA with real-time PCR\nJejunum mucosa samples of soybean size were taken, and RNA was\n\nThe number of various microorganisms was counted using the plate\ncounting method.2.6 RNA extraction and quantitative\nanalysis of mRNA with real-time PCR\nJejunum mucosa samples of soybean size were taken, and RNA was\nextracted according to the instructions of the animal tissue RNA\nTABLE 1  Composition (kg/100 kg) of the basal experimental diets1 for Wuzhishan piglets.\nIngredients\nContents\nNutrient levels, on an air-dry basis\nContents\nCorn, %\n32.80\nDigestible energy3, DE, MJ/kg\n13.61\nSoybean meal, %\n15.40\nCrude protein4, CP, %\n18.00\nWheat, %\n8.00\nCalcium4, Ca, %\n0.95\nLow protein whey powder, %\n6.00\nTotal Phosphorus4, P, %\n0.75\nFish meal, %\n3.00\nAvailable phosphorus4, AP, %\n0.53\nCorn gluten meal, %\n10.00\nLysine4, Lys, %\n1.25\nWheat bran, %\n3.00\nMethionine4, Met, %\n0.35\nSoybean oil, %\n5.00\nThreonine4, Thr, %\n0.78\nMountain flour, %\n0.80\nTryptophan4, Try, %\n0.29\nCalcium hydrogen phosphate, %\n2.00\nSodium chloride, %\n0.30\nL-Lysine hydrochloride, %\n0.60\nThreonine, Thr, %\n0.10\nTryptophan, Try, %\n0.10\nSaccharose, %\n2.50\nGlucose, %\n2.50\nPremix2, %\n2.00\nFeed mold inhibitor, %\n1.00\nSweetening agent, %\n0.30\nZeolite powder, %\n4.60\nTotal, %\n100.00\n1Based on the NRC (2012) nutrient requirements for piglets.\n2The premix provided the following per kg of diet: VA 2,000 IU, VD 200 IU, VE 45.00 IU, VK 0.50 mg, VB1 1.00 mg, pantothenic acid 12.00 mg, nicotinic acid 10.25 mg, VB6 3.85 mg, VB12\n15.00 ug, folic acid 1.35 mg, biotin 0.21 mg, VC 200 mg, Mn (Manganese sulfate) 20.00 mg, Fe (Ferrous sulfate) 80.00 mg, Cu (Copper sulfate) 5.00 mg, I (Potassium iodide) 0.14 mg, Se\n(Sodium selenite) 0.15 mg.\n3Calculated value (NRC, 2012).\n4Analysed content.\n\nWu et al.\b\n10.3389/fvets.2025.1491130\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nextraction kit. The quality of RNA was assessed by a 2% agarose gel\nelectrophoresis. An ultra-microspectrophotometer (NanoPhotometer,\nImplen German) was used to evaluate the total RNA concentration and\npurity (A260/A280 ratio). The primer sequences are shown in Table 2,\nand the primers corresponding to the sow gene sequence were\nsynthesized using Sangon (Shanghai, China). The cDNA was\nsynthesized by PrimeScript® RT regent Kit With gDNA Eraser\n(TaKaRa, Dalian, China) reverse transcription kit. DNA was removed\nfrom the sample using a reaction system as 2 μL 5 × gDNA Eraser\nBuffer, 1 μL gDNA Eraser, 1 μg RNA, then replenished the volume with\nRNase Free ddH2O to 10 μL (20). The system of reverse transcription\nwas 10 μL reaction liquid, 1 μL RT Primer Mix, 4 μL RNase Free\nddH2O, 4 μL 5 × PrimeScript® Buffer 2, PrimeScript® RT Enzyme\nMixI. The reaction procedure was 37°C constant temperature for\n15 min, 85°C constant temperature for 5 s; 4°C store briefly. The\nobtained cDNA was stored at −20°C (17). The housekeeping gene of\nGlyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used and the\nrelative mRNA levels of Interleukin-10 (IL-10), Tumor necrosis factor-α\n(TNF-α), Mucoprotein 2 (MUC2), Transforming growth factor-β1 (TGF-\nβ1), Zonula occludens-1 (ZO1), Proliferating Cell Nuclear Antigen\n(PCNA) were calculated using the 2-ΔΔCt method.\n2.7 Statistical analysis\nStatistical analyses were conducted using SPSS 29.0 statistics\nsoftware (NY, USA). The sample size for this study was determined based\non the anticipated effect size, statistical power, and significance level.\nDrawing on prior research and pilot data, we set the expected effect size\nat 0.50, the significance level at 0.05, and the statistical power at 0.80.\nUtilizing G*Power software for the calculation, we determined that a\nminimum of six piglets per group was required. Accordingly, the study\nwas structured with six replicates per group, each comprising two piglets,\nfor a total of 48 piglets. This design ensures the robustness of our findings\n\nAccordingly, the study\nwas structured with six replicates per group, each comprising two piglets,\nfor a total of 48 piglets.This design ensures the robustness of our findings\nand achieves the desired statistical power. The Kolmogorov–Smirnov test\nwas used to check if all data in this study followed a normal distribution.\nData were expressed as mean ± SEM. Statistical comparisons between\ntreatments were conducted using one-way ANOVA or Welch ANOVA\nfollowing Kolmogorov–Smirnov and variance homogeneity tests\n(Barteet’s or Levene’s test). To mitigate the risk of Type I errors due to\nmultiple comparisons, we employed the Bonferroni correction method\nto adjust the p-values. The test results of all analyses were considered\nsignificant at p < 0.05.\n3 Results\n3.1 Effects of Pueraria extracts on the\ngrowth performance of Wuzhishan piglets\nThere were no significant differences (p > 0.05) in initial weight\nand ADFI among groups. The final weight and ADG were higher\n(p < 0.05), and the FCR was lower (p < 0.05) in PE2 and PE3 compared\nwith the Con. (Table 3).\n3.2 Effects of Pueraria extracts on serum\nimmune indices of Wuzhishan piglets\nThere was no significant difference (p > 0.05) in lgA contents among\ngroups. The lgG contents in PE3 were higher (p < 0.05) compared with\nthe Con. The lgM and C3 contents in PE2 and PE3 were higher (p < 0.05)\nthan in the Con. The C4 contents were higher (p < 0.05) among groups\ntreated with Pueraria extracts compared with the Con. (Table 4).\n3.3 Effects of Pueraria extracts on serum\nbiochemical indices of Wuzhishan piglets\nThe serum TP and UN contents among groups were not significantly\ndifferent (p > 0.05). The serum ALB and ALP contents in groups treated\nwith the Pueraria extracts were higher (p < 0.05) compared with the\nTABLE 2  Primer sequence list.\nGene\nGene name\nForward and reverse primers\nProduct size\nAccession No.\nIL-10\nInterleukin-10\nF:5’-GTGGCAGCCAGCATTAAGTC-3′\n100\nNM_214041.1\nR:5’-AACTCTTCACTGGGCCGAAG-3’\nTNF-α\nTumor necrosis factor-α\nF: 5’-CCAGACCAAGGTCAACCTCC-3’\n89\nNM_214022.10\nR: 5’-TCCCAGGTAGATGGGTTCGT-3’\nMUC2\nMucoprotein 2\nF: 5’-CTGTGCGACTACAACTTCGC-3’\n142\nXM_021082584.1\nR: 5’-AGATGGTGTCGTCCTTGACC-3’\n\n1\nR:5’-AACTCTTCACTGGGCCGAAG-3’\nTNF-α\nTumor necrosis factor-α\nF: 5’-CCAGACCAAGGTCAACCTCC-3’\n89\nNM_214022.10\nR: 5’-TCCCAGGTAGATGGGTTCGT-3’\nMUC2\nMucoprotein 2\nF: 5’-CTGTGCGACTACAACTTCGC-3’\n142\nXM_021082584.1\nR: 5’-AGATGGTGTCGTCCTTGACC-3’\nTGF-β1\nTransforming growth\nfactor-β1\nF: 5’-GGACCTTATCCTGAATGCCTT-3’\n134\nXM_021093503.1\nR: 5’-TAGGTTACCACTGAGCCACAAT-3’\nZO1\nZonula occludens-1\nF: 5’-CTGAGGGAATTGGGCAGGAA-3’\n178\nXM_021098896.1\nR: 5’-TCACCAAAGGACTCAGCACG-3’\nPCNA\nProliferating Cell Nuclear\nAntigen\nF: 5’-GTGATTCCACCACCATGTTC-3’\n123\nNM_001291925.1\nR: 5’-TGAGACGAGTCCATGCTCTG-3’\nGAPDH\nGlyceraldehyde-3-\nphosphate dehydrogenase\nF: 5’-GTCGGAGTGAACGGATTTGG-3’\n76\nNM_001206359.1\nR: 5’-CAATGTCCACTTTGCCAGAGTTAA-3’\n\nWu et al.\b\n10.3389/fvets.2025.1491130\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nCon. The ALT and GOT contents were lower (p < 0.05) in groups treated\nwith Pueraria extracts than in the Con. (Table 5).\n3.4 Effects of Pueraria extracts on the\njejunum flora of Wuzhishan piglets\nThere was no significant difference (p > 0.05) in total bacteria\ncontents among groups. The Escherichia coli and Salmonella contents\namong groups treated with Pueraria extracts were lower (p < 0.05)\ncompared with the Con. The Saccharomycetes and Bacillus acidi\nlactici contents among groups treated with Pueraria extracts were\nhigher (p < 0.05) than in the Con. (Table 6).\n3.5 Effects of Pueraria extracts on\nexpression of immune-related genes in\nWuzhishan piglets\nThe relative expressions of IL-10, ZO1, and MUC2 in jejunum\nmucosa in groups treated with Pueraria extracts were higher (p < 0.05)\nthan those in the Con, and the relative expressions of IL-10 and ZO1\nTABLE 3  Effects of Pueraria extracts on the growth performance of Wuzhishan piglets.\nItems\nCon\nPE1\nPE2\nPE3\nP-value\nInitial weight, kg\n10.00 ± 1.01\n10.00 ± 0.98\n10.00 ± 0.96\n10.00 ± 1.02\n0.1206\nFinal weight, kg\n24.62 ± 0.35b\n25.12 ± 0.46b\n26.56 ± 0.16a\n26.51 ± 0.22a\n0.0352\nADFI, g/d\n583 ± 10.26\n580 ± 10.26\n570 ± 6.92\n572 ± 8.12\n0.2315\nADG, g/d\n162 ± 5.76b\n168 ± 3.05b\n184 ± 5.06a\n183 ± 6.23a\n0.0238\nFCR\n3.60 ± 0.12a\n3.45 ± 0.10a\n3.10 ± 0.10b\n3.12 ± 0.11b\n0.0389\na,bValues with different small letter superscripts in the same column mean a significant difference (p < 0.05). ADFI: average daily feed intake; ADG: the average daily gain; FCR: feed conversion\nratio.\nTABLE 4  Effects of Pueraria extracts on serum immune indice of Wuzhishan piglets.\nItems\nCon\nPE1\nPE2\nPE3\nP-value\nIgA, g·L−1\n0.03 ± 0.01\n0.04 ± 0.01\n0.04 ± 0.02\n0.03 ± 0.02\n0.0968\nIgG, g·L−1\n3.12 ± 0.21c\n3.56 ± 0.29b\n3.97 ± 0.12ab\n4.13 ± 0.17a\n0.0452\nIgM, g·L−1\n0.88 ± 0.07b\n0.90 ± 0.03b\n1.12 ± 0.01a\n1.11 ± 0.02a\n0.0311\nC3, mg·L−1\n30.52 ± 1.56b\n31.99 ± 2.01b\n36.25 ± 1.26a\n36.86 ± 1.37a\n0.0205\nC4, mg·L−1\n26.59 ± 1.23b\n31.26 ± 1.56a\n32.23 ± 1.75a\n32.46 ± 2.01a\n0.0289\na,b,cValues with different small letter superscripts in the same column mean a significant difference (p < 0.05). IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M; C3,\ncomplement 3; C4, complement 4.\n\n59 ± 1.23b\n31.26 ± 1.56a\n32.23 ± 1.75a\n32.46 ± 2.01a\n0.0289\na,b,cValues with different small letter superscripts in the same column mean a significant difference (p < 0.05).IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M; C3,\ncomplement 3; C4, complement 4.\nTABLE 5  Effects of Pueraria extracts on serum biochemical indices of Wuzhishan piglets.\nItems\nCon\nPE1\nPE2\nPE3\nP-value\nTP, g·L−1\n80.17 ± 16.03\n83.23 ± 8.95\n81.19 ± 9.12\n83.05 ± 10.12\n0.0859\nALB, g·L−1\n36.03 ± 2.12b\n39.29 ± 1.06a\n39.72 ± 1.07a\n39.00 ± 2.61a\n0.0416\nALP, U·L−1\n230 ± 5.12c\n270 ± 10.56b\n262 ± 10.39b\n290 ± 6.31a\n0.0357\nALT, U·L−1\n99.02 ± 5.87a\n80.12 ± 6.95b\n76.25 ± 6.12bc\n67.19 ± 4.12c\n0.0267\nGOT, U·L−1\n190 ± 10.12a\n156 ± 10.92b\n120 ± 10.11c\n117 ± 9.32c\n0.0379\nUN, mmol·L−1\n5.87 ± 0.42\n5.32 ± 1.25\n5.27 ± 2.17\n5.00 ± 2.29\n0.9150\na,b,cValues with different small letter superscripts in the same column mean a significant difference (P < 0.05). TP, total protein; ALB, albumin; ALP, alkaline phosphatase; ALT, alanine\ntransaminase; GOT, glutamic oxalacetic transaminase; UN, urea nitrogen.\nTABLE 6  Effects of Pueraria extracts on jejunum flora of Wuzhishan piglets.\nItems\nCon\nPE1\nPE2\nPE3\nP-value\nTotal bacteria, lgCFU·g−1\n11.62 ± 0.18\n11.65 ± 0.12\n11.62 ± 0.16\n11.63 ± 0.25\n0.0951\nEscherichia coli, lgCFU·g−1\n6.59 ± 0.25a\n5.56 ± 0.26b\n5.00 ± 1.13c\n5.02 ± 1.09c\n0.0326\nBacillus acidi lactici, lgCFU·g−1\n6.24 ± 0.16c\n7.15 ± 0.19b\n7.20 ± 0.20b\n7.56 ± 0.18a\n0.0215\nSalmonella, lgCFU·g−1\n5.12 ± 0.08a\n4.67 ± 0.10b\n4.32 ± 0.09c\n4.33 ± 0.01c\n0.0456\nSaccharomycetes, lgCFU·g−1\n5.00 ± 0.16c\n5.36 ± 0.10b\n5.69 ± 0.12a\n5.72 ± 0.14a\n0.0411\na,b,cValues with different small letter superscripts in the same column mean a significant difference (P < 0.05).\n\nWu et al.\b\n10.3389/fvets.2025.1491130\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nin jejunum mucosa were higher (p < 0.05) in PE2 and PE3 compared\nwith PE1. The relative expressions of TNF-α and TGF-β1 in jejunum\nmucosa were lower (p < 0.05) in PE1, PE2, and PE3 compared with\nthe Con. There was no significant difference (p > 0.05) in the relative\nexpressions of PCNA in jejunum mucosa among groups (Figure 1).\n4 Discussion\nGrowth performance is a key indicator of animal growth, which\nis significant for evaluating animal growth environment and feed\nnutrition (21–23). The isoflavones in Pueraria extract, such as\npuerarin, daidzein, and genistein, have essential medicinal value; they\nhave anti-inflammatory, antioxidant, anti-tumor, and improve\nimmunity (24, 25). Guo et al. (15) found that the Pueraria extract\nsupplementation decreased the average daily gain during the whole\nperiod and improved the feed conversion ratio of broiler chickens.\nGong et al. (26) added the Pueraria lobata leaf powder to the feed of\nbroiler chickens and found it did not affect the growth and slaughter\nperformance. Wang et al. (13) found that the puerarin treatment did\nnot significantly alter the growth performance of pigeons.\nTiyasatkulkovit et  al. (27) studied the effect of Pueraria Mirifica\nextract and puerarin on the healthy growth of primary baboon\nosteoblasts. In this study, the Pueraria extract improved the final body\nweight, the average daily gain, and the feed conversion ratio of\nWuzhishan piglets, possibly because Pueraria extract affects mammals\nmore than birds. At the same time, the isoflavones in Pueraria extracts\nmay improve the growth performance of pigs through multiple\nmechanisms, such as regulating metabolic pathways and hormone\nlevels, improving gut health, and exerting antioxidant and anti-\ninflammatory effects.\nThe animal immune system is a set of complex biological\nmechanisms that enable the animal to recognize and defend against\nforeign pathogens, such as bacteria, viruses, parasites, etc., while also\nremoving damaged or aging cells in the body and maintaining the\n\nThe animal immune system is a set of complex biological\nmechanisms that enable the animal to recognize and defend against\nforeign pathogens, such as bacteria, viruses, parasites, etc., while also\nremoving damaged or aging cells in the body and maintaining the\nstability of the internal environment (28–30). Plant extracts have\nreceived attention for their naturalness, versatility, and potential\nanimal health and immunity benefits. They contain various bioactive\nsubstances, such as alkaloids, polyphenols, flavonoids, polysaccharides,\nand volatile oils, which are closely related to immune function (2, 3).\nImmunoglobulins are a class of globulins with antibody activity or\nmolecular chemical structure similar to antibodies, and they play an\nessential role in the animal immune system. B lymphocytes secrete\nimmunoglobulin after being stimulated by an antigen, mainly in\nserum, body fluid, exocrine fluid, and some cell membranes, and have\nthe efficacy and effect of anti-infection, improving immunity, and\npreventing and treating diseases (31–33). The complement C3 and\nC4 in serum are critical immune components that play a key role in\nanimal immune response and inflammation. Complement C3 is the\nmost abundant complement component in serum, while C4 is\ninvolved in the classical activation pathway of complement, and their\ncontent can reflect inflammation and immune system problems (34–\n36). Gong et al. (26) found that Pueraria lobata leaf powder improved\nthe thymus organ index, antioxidant performance, and immune\nfunctions of broiler chickens. Wang et al. (13) found that the puerarin\ntreatment afforded a significant linear enhancement in the thymus\nindex and significantly increased serum immunoglobulin A and\nimmunoglobulin M levels in pigeons in a linear manner of pigeons.\nFIGURE 1\nEffects of Pueraria extract on the expression of immune-related genes in jejunum mucosa of Wuzhishan piglets. (A) The data of relative expression\nlevels of IL-10 (Interleukin-10) in Con, PE1, PE2, and PE3 were 1.00 ± 0.01c, 1.32 ± 0.03b, 1.47 ± 0.05a, 1.48 ± 0.07a, individually, p = 0.0237; (B) The data\n\n(A) The data of relative expression\nlevels of IL-10 (Interleukin-10) in Con, PE1, PE2, and PE3 were 1.00 ± 0.01c, 1.32 ± 0.03b, 1.47 ± 0.05a, 1.48 ± 0.07a, individually, p = 0.0237; (B) The data\nof relative expression levels of TNF-α (Tumor necrosis factor-α) in Con, PE1, PE2, and PE3 were 1.00 ± 0.01a, 0.83 ± 0.02b, 0.81 ± 0.01b, 0.81 ± 0.02b,\nindividually, p = 0.0429; (C) The data of relative expression levels of MUC2 (Mucoprotein 2) in Con, PE1, PE2, and PE3 were 1.00 ± 0.01b, 1.57 ± 0.03a,\n1.60 ± 0.04a, 1.61 ± 0.05a, individually, p = 0.0436; (D) The data of relative expression levels of TGF-β1 (Transforming growth factor-β1) in Con, PE1, PE2,\nand PE3 were 1.00 ± 0.01a, 0.97 ± 0.04b, 0.72 ± 0.03c, 0.74 ± 0.05c, individually, p = 0.0268; (E) The data of relative expression levels of ZO1 (Zonula\noccludens-1) in Con, PE1, PE2, and PE3 were 1.00 ± 0.01c, 1.57 ± 0.05b, 1.92 ± 0.04a, 1.90 ± 0.07a, individually, p = 0.0357; (F) The data of relative\nexpression levels of PCNA (Proliferating Cell Nuclear Antigen) in Con, PE1, PE2, and PE3 were 1.00 ± 0.01, 0.99 ± 0.01, 0.98 ± 0.01, 1.01 ± 0.01,\nindividually, p = 0.0689. a,b,cValues with different small letter superscripts in the same column mean a significant difference (p < 0.05).\n\nWu et al.\b\n10.3389/fvets.2025.1491130\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nJung et al. (37) studied the effects of Lactobacillus casei HY2782 and\nPueraria lobata root extract complex, and they found that it\nsignificantly reduced Th2/Th17-derived cytokines, immunoglobulin\nE, and leukotriene C4 in bronchoalveolar lavage fluid and serum in\nmice. In this study, the Pueraria extract improved the serum\nimmunoglobulins (G and M) and complements (C3 and C4) of\nWuzhishan piglets, which suggests that Pueraria extract can improve\nthe immune performance of piglets by increasing the immunoglobulins\nand complements in the body.\nAlbumin, alkaline phosphatase, alanine aminotransferase, and\nglutamic oxalate aminotransferase are all critical indicators of animal\nimmune function (38–40). Albumin is the most abundant protein in\nplasma, which can combine with many small insoluble molecules of\norganic matter and inorganic ions in the body to form soluble\ncomplexes and become the transport form of these substances in\nblood circulation (38). Albumin levels can reflect the nutritional status\nand immune function in animals, and low levels may indicate\nmalnutrition or impaired immune function (39). Alkaline phosphatase\nis a nonspecific phosphohydrolase that can catalyze the hydrolysis of\nmonophosphate and transfer phosphate groups (40). Alanine\naminotransferase and glutamic oxalate aminotransferase are\ncommonly used to assess liver health because they are highly active in\nliver cells (40). When liver cells are damaged, these enzymes are\nreleased into the blood, resulting in elevated serum aminotransferase\nactivity (39, 40). In this study, the Pueraria extracts increased the\nalbumin and alkaline phosphatase contents, and they decreased\nalanine aminotransferase and glutamic oxalate aminotransferase\ncontents, which suggests that Pueraria extract has the function of\nprotecting the animal liver, thereby directly or indirectly improving\nanimal immune function.\nEscherichia coli, Lactobacillus, Salmonella, and Saccharomycetes\naffect the balance of the gut microbiota through interactions with\n\nEscherichia coli, Lactobacillus, Salmonella, and Saccharomycetes\naffect the balance of the gut microbiota through interactions with\nhost cells, which impact the immune system (41–44). Probiotics such\nas Lactobacillus and Saccharomycetes usually help to enhance the\nimmune response (45). In contrast, pathogens such as Salmonella and\npathogenic Escherichia coli may activate the immune response to\nclear the infection (46, 47). Kwon et al. (48) found that Pueraria\nlobata extract increased the Lactobacillus rhamnosus in human fecal\nspecimens. Xu et al. (49) found that the polyphenols extracted from\nPueraria lobata root improved the gut microbiota in mice, and Chen\net al. (50) reported that the polysaccharides from Pueraria lobata\nsignificantly increased the abundance of beneficial bacteria, including\nOscillospira and Anaerotruncus. In this study, Pueraria extracts\nsignificantly increased the Saccharomycetes content while decreasing\nthe content of Escherichia coli and Salmonella in the jejunum of\nWuzhishan piglets. This finding directly demonstrates that Pueraria\nextracts have the ability to inhibit the growth of harmful bacteria\nwhile simultaneously promoting the growth of beneficial bacteria.\nThese changes may indirectly enhance the immune function of pigs\nby improving gut barrier function and reducing the risk of pathogen\ninfection, thereby improving the immunity of piglets, which is\nconsistent with the improved immune indicators described above.\nInterleukin-10 (IL-10) is a pleiotropic cytokine with\nimmunosuppressive or immunostimulating effects (51). Mucin 2\n(MUC2) is a significant component of the intestinal mucus layer,\nwhich has the functions of lubricating the intestinal tract, providing\nantimicrobial proteins and adhesion sites for commensal flora, and\nresisting the invasion of pathogenic bacteria and harmful substances\n(52). Zonula occludens 1 (ZO1) is a tight junction protein that plays\na vital role in forming and maintaining tight junctions between cells\n(53). Tumor necrosis factor-α (TNF-α) is an important cytokine that\nregulates inflammation, apoptosis, and immune cell activity (54).\n\nTumor necrosis factor-α (TNF-α) is an important cytokine that\nregulates inflammation, apoptosis, and immune cell activity (54).\nTransforming growth factor-β1 (TGF-β1) is a multifunctional\ncytokine synthesized by almost all cells and has the functions of\nimmune regulation and promoting fibrosis (55). IL-10, MUC2, ZO1,\nTNF-α, and TGF-β1 play essential roles in the animal immune system\nand intestinal health, and they are involved in regulating the immune\nresponse and maintaining the integrity of the intestinal barrier\nthrough different mechanisms. In this study, the relative expressions\nof IL-10, MUC2, and ZO1 were increased, and the relative expressions\nof TNF-α and TGF-β1 were decreased in the groups treated with\nPueraria extracts, which may be because the changes in the microbial\ncommunity described above, by improving gut barrier function and\nreducing the risk of pathogen infection, indirectly affected the\nfunction of the immune system. It can be  speculated that the\nalterations in immune-related gene expression are likely associated\nwith the changes in the gut microbial community, which will\nbe further explored in future studies.\nThe Wuzhishan pig, a local pig breed, is distinguished by its small\nsize and relatively slow growth rate, resulting in a longer growth cycle\ncompared to other pig breeds. Typically, it requires a longer period to\nreach market weight. Although the study period allowed for the\nobservation of significant improvements in growth performance and\nimmune function of Wuzhishan pigs due to Pueraria extracts, it may\nnot fully reflect the potential long-term impacts of prolonged use of\nPueraria extracts on piglets. In this study, Pueraria extracts\nsignificantly enhanced the average daily gain and feed conversion ratio\nof Wuzhishan pigs. In the long term, these improvements may help\npiglets maintain better growth efficiency throughout their entire\ngrowth cycle, thereby reducing feeding costs. Pueraria extracts also\nincreased the immunoglobulins and complement levels in piglets\nwithin a short period, suggesting that they may have a positive impact\n\nPueraria extracts also\nincreased the immunoglobulins and complement levels in piglets\nwithin a short period, suggesting that they may have a positive impact\non the long-term immune function of piglets. It can be inferred that\nlong-term use of Pueraria extracts may enhance the immune system\nof piglets, reduce disease incidence, and consequently improve the\noverall health status of the pig herd. Moreover, Pueraria extracts\nimproved the number of beneficial bacteria in the piglets’ intestines\nwhile reducing the number of harmful bacteria in the short term.\nThese changes may further optimize the gut microbiota structure and\nenhance gut barrier function over the long term, thereby reducing the\nincidence of intestinal diseases. However, while Pueraria extracts have\nshown positive effects on growth and immunity in the short term,\nlong-term use may alter the piglets’ requirements and absorption of\nother nutrients. This could lead to the development of tolerance to\nPueraria extracts, thereby reducing their effectiveness in later stages.\nTherefore, further research is needed to fully understand the long-\nterm effects and safety of Pueraria extracts in piglet diets.\n5 Conclusion\nIn this study, adding dietary Pueraria extracts at 600 mg/kg to the\ndiets of Wuzhishan piglets significantly improved growth performance\nby increasing final weight and average daily gain, while decreasing the\nfeed conversion ratio. These findings indicate that Pueraria extracts\ncan enhance feed efficiency and promote piglet growth, potentially\n\nWu et al.\b\n10.3389/fvets.2025.1491130\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nleading to economic benefits for pig farming. Additionally, Pueraria\nextracts enhanced immune function by boosting the levels of\nimmunoglobulin G and M and complement 3 and 4, and by promoting\nbeneficial bacteria such as Lactobacillus and Saccharomycetes in the\njejunum. These results suggest that Pueraria extracts can be used as a\nnatural and safe feed additive to improve the health status of piglets,\nthereby reducing the risk of diseases and the need for antibiotics.\nFuture research endeavors will be dedicated to unraveling the intricate\nmechanisms through which Pueraria extracts exert their beneficial\neffects. This will involve investigating their role in regulating metabolic\npathways and modulating the gut microbiota.\nData availability statement\nThe raw data supporting the conclusions of this article will\nbe made available by the authors, without undue reservation.\nEthics statement\nThe animal study was approved by All procedures involving care\nand management were approved by the Institutional Animal Care and\nUse Committee of the Chinese Academy of Tropical Agricultural\nSciences (Approval No.: CATAS-20231101-23). The study was\nconducted in accordance with the local legislation and\ninstitutional requirements.\nAuthor contributions\nHW: Conceptualization, Data curation, Formal analysis, Funding\nacquisition, Investigation, Methodology, Project administration,\nResources, Software, Supervision, Validation, Visualization, Writing –\noriginal draft, Writing – review & editing. WP: Conceptualization, Data\ncuration, Formal analysis, Funding acquisition, Investigation,\nMethodology, Project administration, Resources, Software, Supervision,\nValidation, Visualization, Writing – original draft, Writing – review &\nediting. XY: Conceptualization, Data curation, Formal analysis,\nFunding\nacquisition,\nInvestigation,\nMethodology,\nProject\nadministration, Resources, Software, Supervision, Validation,\nVisualization, Writing – original draft. XZ: Conceptualization, Data\ncuration, Formal Analysis, Funding acquisition, Investigation,\n\nXZ: Conceptualization, Data\ncuration, Formal Analysis, Funding acquisition, Investigation,\nMethodology, Project administration, Resources, Software, Supervision,\nValidation, Visualization, Writing – original draft. FJ: Conceptualization,\nData curation, Formal analysis, Funding acquisition, Investigation,\nMethodology, Project administration, Resources, Software, Supervision,\nValidation,\nVisualization,\nWriting\n–\noriginal\ndraft.\nQS:\nConceptualization, Data curation, Formal analysis, Funding\nacquisition, Investigation, Methodology, Project administration,\nResources, Software, Supervision, Validation, Visualization, Writing –\noriginal draft. RL: Conceptualization, Data curation, Formal analysis,\nFunding\nacquisition,\nInvestigation,\nMethodology,\nProject\nadministration, Resources, Software, Supervision, Validation,\nVisualization, Writing – original draft, Writing – review & editing.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. This study was supported\nby the National Key Research and Development Program of China\n(No. 2024YFE0214700), the Central Public-interest Scientific\nInstitution Basal Research Fund (No. 1630032024020), the Hainan\nModern Agricultural Production Research System (HNARS-02-G03),\nthe Joint Research on Modern Agricultural Technology Between\nChina and Congo (B) (No. 1630032025001), and the China\nScholarship Council (CSC) National Public Graduate Program for\nBuilding Highly Qualified Universities.\nAcknowledgments\nWe extend our sincere gratitude to Ting Cao and Hanfeng Li from\nthe Tropical Crops Genetic Resources Research Institute, Chinese\nAcademy of Tropical Agricultural Sciences, Euphrème Ipemba and\nGhislain Boungou Bakala from the National Centre for Crop Disease\nControl under the Ministry of Agriculture, Animal Husbandry and\nFisheries of the Republic of Congo, and Lessebe Gambou Dieu Leveut\nfrom\nthe\nChina-aid-Congo\n(B)\nAgricultural\nTechnology\nDemonstration Center of the Republic of Congo, for their valuable\nsupport in writing guidance and the provision of materials for\nthis study.\nConflict of interest\nThe authors declare that the research was conducted in the\n\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. 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+{"text": "【兽医文献】\n标题: Neurological examination of Pacific harbor seal (Phoca vitulina richardii) pups: development and assessment of a protocol\n\nTYPE Original Research\nPUBLISHED 02 January 2026\nDOI 10.3389/fvets.2025.1656345\nOPEN ACCESS\nEDITED BY\nBin Zhou,\nNanjing Agricultural University, China\nREVIEWED BY\nBabatunde Olanrewaju Motayo,\nDepartment of Medical Laboratory Science\nChrisland University, Ajebo, Nigeria\nHongliang Zhang,\nChinese Academy of Agricultural\nSciences, China\n*CORRESPONDENCE\nAlvaro Ruiz-Garrido\naruiz@udec.cl\n†These authors have contributed equally to\nthis work and share first authorship\nRECEIVED 30 June 2025\nREVISED 06 November 2025\nACCEPTED 13 November 2025\nPUBLISHED 02 January 2026\nCITATION\nLarenas-Mu˜noz F, De Barbieri P, Mellado C,\nReyes F, Toledo J, Neira V, Lamazares E and\nRuiz-Garrido A (2026) Assessment of a\nporcine circovirus type 2 vaccine prototype\nthrough anatomopathological analysis and its\ncorrelation with blood viral load.\nFront. Vet. Sci. 12:1656345.\ndoi: 10.3389/fvets.2025.1656345\nCOPYRIGHT\n© 2026 Larenas-Mu˜noz, De Barbieri, Mellado,\nReyes, Toledo, Neira, Lamazares and\nRuiz-Garrido. This is an open-access article\ndistributed under the terms of the Creative\nCommons Attribution License (CC BY). The\nuse, distribution or reproduction in other\nforums is permitted, provided the original\nauthor(s) and the copyright owner(s) are\ncredited and that the original publication in\nthis journal is cited, in accordance with\naccepted academic practice. No use,\ndistribution or reproduction is permitted\nwhich does not comply with these terms.\nAssessment of a porcine\ncircovirus type 2 vaccine\nprototype through\nanatomopathological analysis\nand its correlation with blood\nviral load\nFernanda Larenas-Mu˜noz1†, Paloma De Barbieri1†,\nC´esar Mellado1, F´atima Reyes2, Jorge Toledo3, Víctor Neira4,\nEmilio Lamazares3 and Alvaro Ruiz-Garrido1*\n1Departamento de Patología y Medicina Preventiva, Facultad de Ciencias Veterinarias, Universidad de\nConcepción, Chillán, Chile, 2Departamento de Farmacología, Facultad de Ciencias Biológicas,\nUniversidad de Concepción, Concepción, Chile, 3Laboratorio de Biotecnología y Biofármacos,\nDepartamento de Fisiopatología, Facultad de Ciencias Biológicas, Universidad de Concepción,\nConcepción, Chile, 4Departamento de Medicina Preventiva Animal, Facultad de Ciencias Veterinarias y\n\nPecuarias, Universidad de Chile, Santiago, Chile\nPorcine circovirus type 2 (PCV2) is an endemic pathogen of global relevance,\nresponsible for porcine circovirus-associated diseases (PCVAD). Consequently,\nvaccination against PCV2 is a standard practice in intensive swine production\nsystems. This study aimed to evaluate the effect of a prototype vaccine on\nPCV2 viral load and associated lesions. Thirty-nine pigs from a high-health-\nstatus farm were randomly assigned to three experimental groups (n = 13\nper group), corresponding to a placebo group, a commercial vaccine group\nand a prototype vaccine group. Viral load was assessed by qPCR from serum\nsamples, while lesions were evaluated through necropsy and histopathological\nanalysis of lymph nodes and lungs tissue. No statistically significant differences\nin viral loads were observed among the three groups, and most animals did not\nexhibit detectable viremia. However, the placebo group showed more numerous\nand severe lesions in lymph nodes and lungs compared to the vaccinated\ngroups, with the commercial vaccine group showing milder lesions than the\nprototype vaccine group. These findings suggest that the absence of viremia in\nmost animals may reflect the timing of infection or effective containment by\nhost immunity. Both the commercial and prototype vaccines were associated\nwith reduced lesion severity, although the prototype vaccine demonstrated\nan intermediate performance between the placebo and commercial vaccine\ngroups. Further development and optimization of the prototype formulation are\nwarranted to enhance its protective efficacy.\nKEYWORDS\nPCV2, porcine circovirus, pigs, pathogenesis, viremia, PCVAD\nFrontiers in Veterinary Science\n01\nfrontiersin.org\n\nLarenas-Mu˜noz et al.\n10.3389/fvets.2025.1656345\nIntroduction\nPorcine circovirus type 2 (PCV2) is a virus of the circovirus\ngenus, and it is considered one of the most important pathogens\nin the swine industry due to the high number of syndromes and\ndiseases associated with it. The PCV2 virus, which affects growing\nand fattening piglets, is recognized as the main etiological agent\ncausing Postweaning Multisystemic Wasting Syndrome (PMWS)\n(1–4). Several retrospective epidemiological studies have shown\nthat PCV2 is a pathogenic agent present in a large part of the world\n(3). Twenty-five years ago, PCV2 was considered one of the most\nimportant pathogens in swine diseases, since it was identified as the\nmain causative agent of wasting syndrome, reproductive diseases,\ndermopathies and nephropathies, and respiratory diseases (5, 6).\nThe pigs are considered as natural hosts for PCV2, which\nremain chronically infected upon infection (6). PCV2 associated\ndisease are multifactorial, requiring additional intrinsic or extrinsic\nfactors to initiate clinical disease (7). The PCV2 genome\ncontain eleven open reading frames (ORFs). ORF1 the most\nconserved region, encodes replication-associated proteins, while\nORF2 encodes the capsid protein (Cap), the major viral antigen\nthat elicits the strongest host immune response. Because ORF2\nexhibits genetic variability, it serves as a key determinant for\ndifferentiating PCV2 genotypes. Several other ORFs are involved\nin cytokine and interleukin expression, cell apoptosis, and viral\nstructure formation (7). Based on ORF2 variability, nine genotypes\nhave been identified, among which PCV2a, PCV2b and PCV2d\nare the most prevalent and widely distributed worldwide (8),\naccounting for ∼16, 46 and 34% reported genotypes, respectively.\nHowever, in South America and Chile, the most prevalent genotype\nis PCV2b (9). Several studies have shown that nearly all farms\nworldwide are seropositive for PCV2, indicating that the virus\nis present in all intensive swine production farms (10), where\nhorizontal transmission through the oro-nasal route represents the\n\nSeveral studies have shown that nearly all farms\nworldwide are seropositive for PCV2, indicating that the virus\nis present in all intensive swine production farms (10), where\nhorizontal transmission through the oro-nasal route represents the\nmain route of viral entry, although vertical transmission can also\npossible, albeit less frequently and with variable occurrence (7).\nOnce the virus enters the host, it infects it primarily targets antigen-\npresenting cells notably macrophages and dendritic cells, which\nphagocytize the virus following infection (11). Viral entry into\ncells occurs through endocytosis, after which virions accumulate\nin phagolysosomes and form intracytoplasmic inclusion bodies.\nDuring this process, viral particles may associate with mitochondria\nand migrate toward the nucleus. Within the nucleus, virions\ncluster in intranuclear inclusion bodies, where they replicate and\nare encapsidated into new virions (7, 11). Upon release into the\ncytoplasm, newly formed virions aggregate into intracytoplasmic\ninclusion bodies and exit the cell either via exocytosis or through\nlytic release, processes that are associated with marked cellular\ndisruption and death (7, 11).\nPCV2 impairs the host’s immune response compromising the\nfunctionality of dendritic cells and macrophages, reducing their\nability to present antigens and secrete key cytokines such as IFN-\nα and TNF-α (12–14). Additionally, PCV2 infection upregulates\nIL-10 and SOCS3 expression, which suppress proinflammatory\nresponses and support viral replication (12, 14, 15). The virus\nalso promotes the expansion of regulatory T cells (Tregs) via the\nTGF-β/Smad3 signaling pathway, inducing immune tolerance and\nimpairing antiviral defenses (16). Moreover, Foxp3 has been shown\nto inhibit viral replication by reducing the ATPase activity of the\nviral Rep protein (17).\nFollowing\ninfection,\nmacrophages\nand\ndendritic\ncells\ndisseminate the virus through the lymphoid tissue, infiltrating\nand displacing resident lymphocytes and serving as the main\nvehicles system spread (12, 18). Infected mononuclear cells\n\nFollowing\ninfection,\nmacrophages\nand\ndendritic\ncells\ndisseminate the virus through the lymphoid tissue, infiltrating\nand displacing resident lymphocytes and serving as the main\nvehicles system spread (12, 18).Infected mononuclear cells\ntransport the virus via blood to peripheral organs such as lungs,\nkidneys and intestines where it replicates in epithelial cells,\nproducing lesions like pneumonia, nephritis and enteritis (8, 19).\nClinical disease grouped under PCVAD, requires cofactors such\nas genetic susceptibility or co-infections, notably with porcine\nparvovirus (PPV), which enhance immune activation and viral\nload (13, 15, 18).\nSubclinical infections detected mainly by serology are the most\nfrequent presentation, affecting 70%−96% of infected pigs, with no\nevident gross lesions and mild lymphoid depletion microscopically;\nviral load is typically below 1 × 107 viral particles/ml (20, 21).\nAmong clinical forms, PMWS or PCV2-SD is the most common,\nwith morbidity of 4%−30% and mortality of 4%−20% (20). It\nis characterized by weight loss, pale skin, respiratory distress,\nand lymphadenopathy, with histologic findings of granulomatous\ninflammation, cytoplasmic inclusions, and lymphoid depletion (18,\n20, 22). Lung lesions include interstitial pneumonia and necrotizing\nbronchiolitis, while spleen and liver may show vasculitis, infarcts, or\nfibrosis; in rare cases, brain lesions with necrotizing vasculitis occur\n(1, 20, 23, 24).\nPCV2 lung disease (PCV2-LD) and enteric disease (PCV2-\nED) manifest as respiratory distress and diarrhea, respectively\nwith bronchointerstitial pneumonia or granulomatous enteritis\naffecting Peyer’s patches (1, 20). Quantitative PCR is the preferred\ndiagnostic tool for early detection (25). Vaccination remains the\nmain preventive strategy, most based on the PCV2a genotype and\ntargeting the Cap protein (pCap) (26, 27). Despite high efficacy,\noccasional clinical cases occur in vaccinated pigs (9). According\nto the World Organization for Animal Health (WOAH) (28), five\ncommercially vaccines are available worldwide: one inactivated\n\nDespite high efficacy,\noccasional clinical cases occur in vaccinated pigs (9).According\nto the World Organization for Animal Health (WOAH) (28), five\ncommercially vaccines are available worldwide: one inactivated\nvaccine, three Cap -derived, and one chimeric formulation.\nVaccines against porcine circovirus infection aim to prevent\nthe appearance of severe macroscopic and microscopic lesions.\nA prototype vaccine was developed based on chimeric antigens\nderived from the PCV2b genotype, with the aim of providing\neffective protection against PCV2 infection (29). In this context,\nthe present study aimed to evaluate the effect of this novel vaccine\nprototype on PCV2-associated histopathological lesions in selected\ntissues and to assess its correlation with the viral load detected in\nthe vaccinated animals.\nMaterials and methods\nAnimal selection and experimental design\nPiglets used in this study were obtained at weaning age (21 days\nof age), coming from a high-health-status intensive production\nfarm free of major, globally recognized swine diseases. The animals\nshowed no clinical or subclinical signs of illness at time of\nacquisition. Piglets were raised under controlled experimental\nconditions as a part of a study evaluating a chimeric PCV2\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n\nLarenas-Mu˜noz et al.\n10.3389/fvets.2025.1656345\nexperimental vaccine developed within the framework of the\nFONDEF IT21I0001 project. The trial was conducted at the\nexperimental unit of the Center of Biotechnology and Biomedicine\nSpa in Quinchamalí, Chile.\nThe facilities (Supplementary Figure 1) comprised three rooms\nwith a personnel area divides into a dirty zone equipped with a\nfootbath, a shower transition zone, and a clean area containing\nbiosafety equipment. Adjacent to the main room were dedicated\nstorage areas for feed and materials. The animal handling area\nconsisted of three pens (each measuring 12 m2), equipped with\nRotecna\nR⃝plastic slap flooring, Cristal Spring\nR⃝feeders, and\nad libitum access to drinking water via nipple drinkers. Each pen\ncontained a wooden stall (200 × 90 × 80 cm) with a rubber\nfloor, infrared heating (175 W InterHead 3G infrared bulb), and a\nthermo-hygrometer (HRC-2) to monitor environmental and stall\ntemperature. The wooden stall was used only during the first weeks\nof the experiment and was subsequently then removed.\nUpon arrival, pigs were allowed a 4-day acclimatization period\nbefore trial initiation (day 0). The selection criteria included 21-\nday-old PIC terminal hybrids piglets of both sexes, clinically\nhealthy, PCV2 by ELISA, and qPCR-negative. All piglets were\nunvaccinated against PCV2 prior to the experiment.\nBody weight was recorded at three specific time points: at\nthe start of the study (day 0), after 7 weeks, and at 17 weeks\n(previous euthanasia). The weights were used to monitor growth\nperformance and to ensure the general health status of the animals\nthroughout the study period. Clinical signs, including liveliness,\nrespiratory distress, and anorexia, were observed daily to detect any\ndeviation from normal behavior or signs of disease.\nAnimals were fed ad libitum with phase-specific diets\nformulated for intensive swine production and were randomly\ndivided into three groups (n = 13 each): placebo group (PL),\nreceiving an intramuscular placebo solution; commercial vaccine\ngroup (VAC-C), administered an inactivated aqueous-based PCV2\n\nvaccine containing the VQ2610 strain (1 × 109 genome copies);\nand prototype vaccine group (VAC-PR), receiving the oil-based\nrecombinant chimeric vaccine developed by Lamazares et al. (29),\nformulated with the Qm1 gene and Montanide ISA 50 V2 adjuvant\n(SEPPIC, Paris, France). All groups received a first intramuscular\ndose on day 4 post-acclimatization (∼25 days of age) and a booster\ndose 3 weeks later (Figure 1).\nThree weeks after booster [day 0 post-inoculation (dpi)], all\nanimals were challenged with PCV2b by intranasal (2 ml) and\nintramuscular (1 ml) routes at a concentration of 4.3 × 107 viral\nparticles/ml, as described by Segalés (14). This was combined with\n1 ml of porcine parvovirus (PPV) diluted 1:50 (cycle threshold =\n28) provided by Dr. Victor Neira (University of Chile), as described\nby Reséndiz et al. (21). Animals remained in the facility until\nthe end of the trial (11.3 weeks post-inoculation), approximating\nslaughter age.\nSerological analysis\nSpecific\nantibodies\nagainst\nPCV2\nwere\ndetected\nusing\nINgezim\nR⃝\nCirco\nIgG\n11.PCV.K1\nELISSA\ntest\n(Ingenasa\nLaboratories,\nMadrid,\nSpain)\nfollowing\nthe\nmanufacturer’s\ninstructions. Animal were tested upon arrival and at the end of\nvaccination period to evaluate seroconversion.\nSample collection and histopathological\nevaluation\nPigs were necropsied according the protocol described by\nSegalés and Domingo (30). Euthanasia was performed with an\nintravenous overdose of sodium thiopental (5%, 100 mg/kg),\nadministered through the jugular vein, in accordance with\nAmerican Veterinary Medical Association (AVMA) guidelines\n(31). Macroscopic lesions were described, and tissue samples\nfrom lungs and mediastinal lymph nodes were collected for\nhistopathological analysis. Blood samples were obtained prior to\nnecropsy to assess PCV2 viral load.\nTissue samples\nFor histopathological analysis, tissue samples were fixed in\n10% neutral buffered formalin (NBF) and routinely processed\nembedding into paraffin wax and subsequent histopathology\naccording to the standard protocol established by Luna (32). After\nthis, sections were stained with hematoxylin and eosin (H&E)\nfollowing the standard protocols of the diagnostic laboratory. Slides\n\nAfter\nthis, sections were stained with hematoxylin and eosin (H&E)\nfollowing the standard protocols of the diagnostic laboratory.Slides\nwere examined using an Axioskop 40 microscope equipped with an\nintegrate Canon 1200 camera.\nViral load quantification by real-time PCR\nTo determine the viral load in blood, serum samples\nwere used, and DNA extraction was performed using the\ncommercial QIAamp\nR⃝\nDNA Mini Kit (Qiagen, Germany),\nfollowing\nthe\nmanufacturer’s\ninstructions.\nSemi-quantitative\nreal-time PCR was performed using SensiMixTM SYBR\nR⃝Hi-\nROX Kit and oligonucleotides described by Opriessnig et al.\n(33)\n(forward:\n5′-TGGCCCGCAGTATTCTGATT-3′;\nreverse:\n5′-CAGCTGGGACAGCAGTTGAG-3′). Reactions were run on\nABI Prism Primer Express software (V1.5, PE Applied Biosystems,\nFoster City, CA, USA). Each 50 μl reaction consisted of 25 μl of\nSensiMixTM SYBR\nR⃝, 0.5 μl of each primer, 5 μl of template DNA,\nand 19 μl of nuclease-free water. A standard curve was constructed\nusing five 10-fold serial dilutions of PCV2 genomic DNA, starting\nat 1 × 108 copies, and viral load was expressed as genome copies\nper ml of serum.\nSamples with Ct value corresponding to viral loads ≤1.0 ×\n105 copies/ml were considered negative for PCV2 infection, while\nvalues ≥1.0 × 106 copies/ml were considered positive (33).\nMacroscopic and microscopic lesions\nclassification\nMacroscopic\nlesions\nassociated\nwith\nPCV2\nfocused\non\nenlargement and hyperemia of mediastinal lymph nodes, which\nFrontiers in Veterinary Science\n03\nfrontiersin.org\n\nLarenas-Mu˜noz et al.\n10.3389/fvets.2025.1656345\nFIGURE 1\nTimeline of the experimental design used in the trial.\nwere scored as follows: (0 = absent, 1 = mild, 2 = moderate, 3 =\nsevere). Lungs were assessed for collapse and edema.\nMicroscopic lesions were evaluated according to the criteria\ndescribed by Segalés (20) and Segalés et al. (34). In lymphoid tissues,\nlymphoid depletion and granulomatous infiltrate were scored based\non lesion severity: 0 = none, 1 = mild, 2 = moderate, 3 =\nsevere. Presence of multinucleated giant cells (MGCs) was recorded\nas 0 = absent, 1 = present. In lung tissues, septal thickening\nand peribronchiolar/peribronchial (BALT) infiltrates were similarly\nscored from 0 to 3 (0 = none, 1 = mild, 2 = moderate, 3 = severe)\naccording to the extent of the lesion.\nStatistics analysis\nSample size was calculated using log10-transformed viral load\ndata to account for the log-normal distribution typical of qPCR\nresults. The estimation was performed at 95% confidence level,\nresulting in 13 animals per experimental group (35).\nDifferences in mean viral load between groups were analyzed\nusing a paired samples t-test for repeated measures, applying a\n95% confidence level. Associations between categorical variables, as\ndefined in the lesion scoring tables, were evaluated using the chi-\nsquare test or Fisher’s exact test when expected frequencies were\nbelow five. A 95% confidence level was applied for all analyses (36).\nThe association between viral load and lesions severity was assessed\nusing Fisher’s exact test (36).\nTo assess statistical significance, GraphPad Prism version\n10.3.0 (217) for windows (GraphPad Software, LLC) was used for\ngraphical representations and statistical analyses. Normality was\nevaluated using the D’Agostino and Pearson omnibus normality\ntest. Since data did not follow a normal distribution, statistical\ndifferences were assessed by the Kruskal-Wallis nonparametric test.\nA p-value below 0.05 was considered statistically significant and\nwas indicated as follows: with p ≤0.05 (∗), p ≤0.01 (∗∗), and\np ≤0.001 (∗∗∗).\nResults\nBody weight gain\n\nA p-value below 0.05 was considered statistically significant and\nwas indicated as follows: with p ≤0.05 (∗), p ≤0.01 (∗∗), and\np ≤0.001 (∗∗∗).Results\nBody weight gain\nAt the time of challenge, all animals appeared clinically healthy.\nBody weight gain was evaluated to determine the potential impact\nof vaccination on growth performance during the experimental\nFIGURE 2\nAverage weight gain in pigs from the three experimental groups\nthroughout the trial. PL (green), VAC-C (blue), and VAC-P (orange)\ngroups. No statistical differences (p > 0.05).\nperiod. The three experimental groups started the trial with similar\nmean initial body weights (p > 0.05). At the end of the trial, both\nvaccinated groups showed higher average weight gains compared\nwith the placebo group (Figure 2). Notably, pigs in the VAC-PR\ngroup exhibited a marked improvement in weight gain, comparable\nto that of the VAC-C group. In contrast, the Placebo (PL) group\nshowed the lowest weight gain across the trial, although these\ndifferences were not statistical significance (p > 0.05).\nViral load\nTo determine the development of viremia, blood samples were\ntaken from the day of challenge until the end of the experiment\nand analyzed by qPCR viral loads ≤1.0 × 105 copies/ml were\nconsidered negative, whereas values ≥1.0 × 106 copies/ml were\nclassified positive. All animals tested negative by qPCR at day 0. At\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n\nLarenas-Mu˜noz et al.\n10.3389/fvets.2025.1656345\nthe end of trial, most animals across the three experimental groups\nshowed no detectable viral genome copies in serum (Table 1).\nHowever, two pigs from PL group (1.0 × 107 copies/ml) and\none from VAC-PR group (1.0 × 107 copies/ml) presented viral\ngenome copies compatible with infection, while no viral genomes\nwere detected in pigs VAC-C group. No statistically significant\ndifferences were observed among the three groups in regarding\nblood viral loads (p > 0.1; Figure 3).\nLesions in the lungs and lymph nodes\nMacroscopic\nexamination\nrevealed\nmoderate\nlymphadenopathy of the mediastinal lymph nodes in the PL\ngroup. This same lesion was also identified in both vaccinated\ngroups, though with lower intensity. In contrast, no apparent\npulmonary lesions were observed either vaccinated groups.\nHowever, animals from the PL group exhibited pulmonary\ncongestion and edema in 50% of the cases.\nTABLE 1 Results of blood viral load of porcine circovirus type 2 per ml of\nserum observed by real-time polymerase chain reaction for each\nexperimental group and the end of trial.\nGroup\nBlood viral load/ml\nNo Ct\n≤105\n106\n≥107\nPL\n7\n4\n0\n2\nVAC-C\n9\n4\n0\n0\nVAC-PR\n8\n4\n0\n1\nCt, threshold cycle; PL, placebo group; VAC-C, commercial vaccine group; VAC-PR,\nprototype vaccine group.\nFIGURE 3\nPCV2 serum viral load was quantified in PL (green), VAC-C (blue),\nand VAC-P (orange) groups. No statistical differences (p > 0.05).\nMicroscopically lung section from all three experimental\ngroups (Figure 4) showed mild thickening of the alveolar septa,\nmainly due to mononuclear inflammatory infiltrates composed\npredominantly of macrophages, lymphocytes, and plasma cells\n(Figures 5A–C). Macrophages were the most abundant cell type,\nfrequently located within the alveolar septa and peribronchiolar\nregions. Lymphocytes were distributed diffusely throughout\nthe interstitial and perivascular areas, while peribronchial and\nperibronchiolar\nregions\nexhibited\nhyperplasia\nof\nbronchus-\nassociated lymphoid tissue (BALT), characterized by lymphoid\nfollicle formation with prominent germinal centers (Figures 5D–\nF). The PL group showed the most severe lesions, characterized by\n\nThe PL group showed the most severe lesions, characterized by\ndiffuse alveolar septal thickening, congestion, and accumulation\nof mononuclear cells within the peribronchial and perivascular\nregions. In contrast, lungs from VAC-C and VAC-PR groups\nshowed lower histological changes, consisting of focal to multifocal\nFIGURE 4\nHistopathological scoring lesion in lungs. Mean severity scores for\neach experimental group, were graded as 0 (absent), 1 (mild), 2\n(moderate), and 3 (severe). Representative features include\nthickening of alveolar septa (green) and peribronchial inflammatory\ninfiltrate (blue).\nFrontiers in Veterinary Science\n05\nfrontiersin.org\n\nLarenas-Mu˜noz et al.\n10.3389/fvets.2025.1656345\nFIGURE 5\n(A–C) Microscopic lesion severity in the lungs. Representative interstitial septal thickening in animals from the (A) placebo group, (B) commercial\nvaccine, and (C) prototype vaccine group. Hematoxylin and eosin. Scale bars: 200 μm. Representative peribronchiolar inflammatory infiltrates\n(arrows) from animals in the (D) placebo group, (E) commercial vaccine and (F) prototype vaccine group with hemorrhages (arrowhead).\nHematoxylin and eosin. Scale bars: 50 μm.\ninterstitial thickening with scant peribronchiolar mononuclear\ninfiltrates and minimal congestion.\nOverall, the lesions were less extensive and less intense in\nboth vaccinated groups compared with the PL group, indicating\na protective effect of vaccination on the pulmonary parenchyma.\nNo evidence of suppurative bronchopneumonia, vasculitis, or\nsecondary bacterial infection was detected in any group.\nIn all three experimental groups, lymphoid depletion in the\nlymph nodes was generally mild (Figure 6). However, the PL\ngroup exhibited a higher number of animals with moderate to\nmarked lymphoid depletion, particularly affecting the cortical\nand paracortical regions (Figures 7A–C). In these animals, loss\nfollicular architecture and reduction of germinal centers were\nfrequently observed.\nFrontiers in Veterinary Science\n06\nfrontiersin.org\n\nLarenas-Mu˜noz et al.\n10.3389/fvets.2025.1656345\nFIGURE 6\nHistopathological scoring of lesion in mediastinal lymph nodes.\nMean severity scores for each experimental group were graded as 0\n(absent), 1 (mild), 2 (moderate), and 3 (severe). Representative\nfeatures include histiocytic inflammatory infiltrate (green) and\nlymphoid depletion (blue).\nA clear difference was also noted in the peritrabecular and\nsubcapsular histiocytic infiltrates between groups. The PL group\n(Figure 7D) showed dense accumulations of histiocytes, often\naccompanied by MGCs within the parafollicular zone, consistent\nwith the granulomatous pattern commonly associated with PCV2\ninfection. In contrast, both vaccinated groups (VAC-C and VAC-\nPR) exhibited milder histiocytic infiltration, limited mainly to the\nsubcapsular sinus (Figures 7E, F).\nInterestingly, in the VAC-C group, the histiocytic infiltrate\nfrequently contained numerous eosinophils interspersed among\nmononuclear cells (Figure 7E). The VAC-PR group showed\ncomparable findings but with fewer eosinophils and less disruption\nof lymphoid structure (Figure 7F).\nOverall, the lymphoid depletion and histiocytic reaction\nwere more pronounced in the PL group, while vaccinated\nanimals maintained better follicular organization, indicating a\nprotective effect of vaccination on lymphoid integrity and\nimmune homeostasis.\nDiscussion\nEffective control of PCV2 requires a multifaceted approach,\nwith vaccines playing a critical role. Despite the widespread use\nof commercial vaccines, sporadic cases of infection and variable\nimmune responses continue to be reported, emphasizing the need\nto better understand vaccine efficacy and host–virus interactions\nunder experimental conditions.\nIn this study, we evaluated the efficacy of a prototype\nintramuscular PCV2 vaccine in pigs experimentally challenged\nwith a virulent PCV2 strain. The analysis focused on virological and\nhistopathological parameters to determine the level of protection\ninduced by the prototype formulation in comparison with a\ncommercial vaccine and a placebo control. Previous studies have\nreported that between 70 and 96% of infected animals do not\n\nPrevious studies have\nreported that between 70 and 96% of infected animals do not\ndevelop clinical disease or show detectable viral loads in the blood,\nnor do they present macroscopic lesions characteristic with PMWS\n(20). Therefore, it was expected that 70%−90% of the animals in the\ntrial would not display detectable viral loads or lesions compatible\nwith the disease, which was confirmed by the study results. Indeed,\nat least 50% of the pigs across all experimental groups did not\nexhibit viral loads >105 copies/ml or show severe lesions associated\nwith PMWS.\nIn seven animals of the PL group, no viral replication was\ndetected. It is hypothesized that viremia did not persist in these\npigs due to two main factors. First, the expected morbidity of this\ndisease which not exceed 40% even under experimental conditions\n(13, 14, 19, 20) and second, the adequate dietary supplementation\nprovided throughout the trial, which may have contributed to an\neffective immune response against infected cells, ultimately leading\nto a reduction in blood viral load by the end of the study. Zhai\net al. (37) reported that oxidative stress enhances viral replication\nin leukocytes and promotes PCV2 viremia, whereas antioxidant\ncompounds reduce oxidative stress and suppress viral replication.\nTherefore, a nutrient rich diet high in antioxidants may play a\ncritical role in controlling viral persistence and limiting PCV2-\ninduced damage in vaccinated pigs.\nRegarding the VAC-C group, it was expected that they would\nnot replicate the viral genome or that they would be presented a\nviral load equal to or <105 copies/ml, as vaccination reduces the\namount of virus circulating in the organism. Accordingly, a notable\nreduction in the intensity of macroscopic and histological lesions\nwas observed, primarily in the lymph nodes and lungs, indicating\nthat the VAC-C vaccine was highly effective in reducing PCV2-\nassociated lesions. Interestingly, a predominance of eosinophilic\ngranulomatous inflammation was observed in the lymph nodes,\nmacroscopically evidenced by enlarged mediastinal lymph nodes.\n\nInterestingly, a predominance of eosinophilic\ngranulomatous inflammation was observed in the lymph nodes,\nmacroscopically evidenced by enlarged mediastinal lymph nodes.\nThis finding suggests a differential local immune activation pattern\nrather than a pathological hypersensitivity reaction. Although the\nexact mechanism remains to be elucidated, it could reflect a Th2-\nskewed immune response, possibly influenced by the adjuvant\ncomposition of the commercial vaccine (38–41).\nThis interpretation should be regarded as preliminary, as no\ndirect immunophenotypic or cytokine analyses were performed\nFrontiers in Veterinary Science\n07\nfrontiersin.org\n\nLarenas-Mu˜noz et al.\n10.3389/fvets.2025.1656345\nFIGURE 7\nRepresentative histopathological features in mediastinal lymph nodes. (A–C) lymphoid depletion (arrows) in pigs from the (A) placebo, (B)\ncommercial vaccine and (C) prototype vaccine groups. The lymphoid depletion was more pronounced in the placebo group with few lymphoid\nfollicles (arrows), whereas lymphoid architecture was better preserved in the prototype vaccine group (arrows). Hematoxylin and eosin. Scale bars:\n200 μm. (D–F) Histiocytic infiltration (arrows) in with scattered eosinophilic infiltration (arrowheads) in (D) placebo, (E) commercial vaccine, and (F)\nprototype vaccine groups. Hematoxylin and eosin. Scale bars: 10 μm.\nto confirm eosinophil recruitment pathways. Therefore, while a\nmild hypersensitivity-type reaction cannot be entirely ruled out, the\nabsence of clinical manifestations consistent with hypersensitivity\nin vaccinated pigs supports a non-adverse immunomodulatory\norigin (42, 43).\nFurther\nstudies\nare\nwarranted\nto\ncharacterize\nthe\nimmunological polarity of this eosinophilic response, including\nthe quantification of Th2 cytokines such as IL-4, IL-5, IL-13\nand histochemical markers of eosinophil activation, to clarify\nwhether this phenomenon represents an adjuvant-induced bias\nFrontiers in Veterinary Science\n08\nfrontiersin.org\n\nLarenas-Mu˜noz et al.\n10.3389/fvets.2025.1656345\nor a beneficial immunoregulatory feature of the commercial\nformulation (41, 44).\nAccording to Zachary (45), the chronic phase of a type I\nhypersensitivity reaction is responsible for the recruitment of\neosinophils, a mechanism observed in this experimental group.\nMoreover, this response is usually focal, and its severity depends\non the individual’s susceptibility to histamine, which, in this case,\ndid not trigger an intense reaction. On the other hand, Batista-\nDuharte et al. (46) indicate that certain types of adjuvants can\ncause local adverse side effects, including delayed recruitment of\nimmune cells that release proinflammatory cytokines, resulting\nin a delayed hypersensitivity reaction. It is hypothesized that the\nadjuvants present in the commercial vaccine may have induced the\neosinophilic infiltrate observed in the lymph nodes, consistent with\na local and mild hypersensitivity reaction.\nThe\nresults\nshow\nthat\nVAC-PR\npig\ngroup\npresented\nintermediate outcomes between the PL and VAC-C groups.\nTheir viral loads, as well as their macroscopic and histological\nlesions, were comparable to those observed in the VAC-C group.\nHowever, one pig in the VAC-PR group showed a viral genome\ncopy number consistent with clinical PCV2 infection, although\nits lesions resembled those of the subclinical form of the disease.\nThese results suggest that the VAC-PR provides partial protection\nagainst viremia; however, since it is still under development, its\nperformance is not yet fully comparable to that of a VAC-C, as\nsome animals showed blood viral loads indicative of the disease.\nAccording to Batista-Duharte et al. (46), adjuvants contribute\nsignificantly to the potency, quality, and duration of the immune\nresponse. In this context, it is presumed that VAC-C achieved\na stronger response against blood viral loads and tissue lesions\nbecause its aqueous-based adjuvants were specifically designed\nto optimize its efficacy, whereas VAC-PR used commercial\noil-based adjuvants.\nRegarding to this, although Porcine parvovirus (PPV) is\nrecognized as a potential cofactor that can enhance the replication\n\nRegarding to this, although Porcine parvovirus (PPV) is\nrecognized as a potential cofactor that can enhance the replication\nand pathogenicity of PCV2 during the acute phase of infection\n(47), the present study demonstrated that both the experimental\nchimeric vaccine (VAC-PR) and the commercial vaccine (VAC-\nC) provided effective protection against the deleterious effects\ntypically associated with such co-infections. Both vaccinated\ngroups exhibited reduced viremia and milder microscopic lesions\ncompared with the unvaccinated group, indicating that vaccination\ncould mitigate the synergistic impact usually observed between\nPPV and PCV2.\nImportantly, no adverse systemic reactions or clinical signs\nsuggestive of vaccine-induced hypersensitivity or exacerbated\npathology were recorded, supporting the safety and tolerability\nof\nboth\nformulations\nunder\nco-infection\nconditions.\nThis\ncontrasts with classical experimental co-infection models, where\nunprotected pigs developed pronounced lymphoid depletion,\npulmonary inflammation, and elevated viral loads (47, 48). The\nabsence of these effects in vaccinated animals strongly suggests that\nimmunization effectively modulated the host immune response,\npreventing\nthe\namplification\nof\nPCV2\nreplication\ntypically\ntriggered\nby\nPPV-induced\nimmunomodulation.\nTherefore,\nthe results of this study indicate that both vaccines were\neffective in reducing PCV2 replication under experimental\nchallenge conditions.\nThe lack of statistically significance differences in viral loads\nbetween the vaccinated groups and the PL group is likely due\nto the challenges of maintaining sustained viral replication under\nexperimental conditions over an extended period. This limitation\nhindered the successful replication of infection until the end of the\ntrial in most of the animals in the PL group, as previously reported\nby Segalés (20). Nevertheless, a notable reduction in viral load was\nobserved in the vaccinated groups compared to the unvaccinated\ngroup, indicating a trend toward viral control associated with\nvaccination. Although no statistically significant differences in\nweight gain were detected among the groups, vaccinated animals,\n\nAlthough no statistically significant differences in\nweight gain were detected among the groups, vaccinated animals,\nin special VAC-PR group, showed a higher average weight gain\ncompared to the unvaccinated group. This tendency suggests that\nboth vaccines may help reduce the productive impact of PCV2\ninfection, with the prototype vaccine demonstrating promising\nperformance. These findings support the potential of the VAC-PR\ncandidate as an effective alternative for improving both health and\nproductivity outcomes in swine. Additionally, while several animals\nwith high viral loads were identified in the PL group, only one pig\nin the VAC-PR group exhibited a high viral load. These findings\nconfirm that viral replication persisted until the end of the trial in\nthose specific cases.\nConclusion\nThe findings of this study demonstrate that the VAC-PR\nprovided partial protection against PCV2-associated viremia and\nhistopathological damage. Although VAC-PR showed promise in\nreducing the histopathological impact of PCV2, its protective\nefficacy was slightly lower than that of the commercial vaccine,\nlikely due to differences in adjuvant formulation. This suggests\nthat further optimization of the adjuvant system or antigen\ndesign may enhance the immunogenicity and consistency of the\nprototype vaccine.\nData availability statement\nThe original contributions presented in the study are included\nin the article/Supplementary material, further inquiries can be\ndirected to the corresponding author.\nEthics statement\nThe animal study was approved by Comité de Ética,\nBioética y Bioseguridad de la Vicerrectoría de Investigación\ny\nDesarrollo\nde\nlaUniversidad\nde\nConcepción.\nThe\nstudy\nwas conducted in accordance with the local legislation and\ninstitutional requirements.\nAuthor contributions\nFL-M: Writing – review & editing, Formal analysis, Writing\n– original draft, Visualization, Investigation. PD: Investigation,\nWriting – review & editing, Writing – original draft, Formal\nanalysis, Visualization. CM: Writing – review & editing, Formal\nFrontiers in Veterinary Science\n09\nfrontiersin.org\n\nLarenas-Mu˜noz et al.\n10.3389/fvets.2025.1656345\nanalysis. FR: Writing – review & editing, Investigation. JT:\nMethodology, Writing – review & editing, Conceptualization.\nVN: Writing – review & editing, Resources. EL: Supervision,\nConceptualization, Writing – review & editing. ÁR-G: Resources,\nFunding\nacquisition,\nConceptualization,\nSupervision,\nProject\nadministration, Methodology, Writing – review & editing.\nFunding\nThe author(s) declare that financial support was received\nfor\nthe\nresearch\nand/or\npublication\nof\nthis\narticle.\nThe\nmethodology and procedures used in this study have the\napproval of the Ethics, Bioethics and Biosafety Committee\nof the Vice Rector’s Office for Research and Development of\nthis university, number CEBB 1155-2022 dated April 2022,\nbecause this study is part of the FONDEF project IT21I0001,\nentitled\n“Development\nof\na\nrecombinant\nchimeric\nvaccine\nagainst PCV2 and validation of its effectiveness under controlled\nconditions.” This study was financed by FONDEF project, code\nFONDEF IT21I0001.\nAcknowledgments\nWe express our appreciation to Cecilia Contreras Muñoz\nand\nDaniela\nRojas\nMansilla\nfor\ntheir\ntechnical\nassistance.\nThe\ntechnical\nsupport\noffered\nby\nthe\nPorcine\nLaboratory\nand Histopathology Laboratory of University of Concepcion,\nChillán (Chile).\nConflict of interest\nThe\nauthors\ndeclare\nthat\nthe\nresearch\nwas\nconducted\nin the absence of any commercial or financial relationships\nthat\ncould\nbe\nconstrued\nas\na\npotential\nconflict\nof interest.\nGenerative AI statement\nThe author(s) declare that no Gen AI was used in the creation\nof this manuscript.\nAny alternative text (alt text) provided alongside figures in\nthis article has been generated by Frontiers with the support of\nartificial intelligence and reasonable efforts have been made to\nensure accuracy, including review by the authors wherever possible.\nIf you identify any issues, please contact us.\nPublisher’s note\nAll\nclaims\nexpressed\nin\nthis\narticle\nare\nsolely\nthose\nof the authors and do not necessarily represent those of\ntheir\naffiliated\norganizations,\nor\nthose\nof\nthe\npublisher,\nthe editors and the reviewers. Any product that may be\nevaluated in this article, or claim that may be made by\nits\nmanufacturer,\nis\nnot\nguaranteed\nor\nendorsed\nby\nthe\npublisher.\nSupplementary material\nThe Supplementary Material for this article can be found\n\nAny product that may be\nevaluated in this article, or claim that may be made by\nits\nmanufacturer,\nis\nnot\nguaranteed\nor\nendorsed\nby\nthe\npublisher.Supplementary material\nThe Supplementary Material for this article can be found\nonline at: https://www.frontiersin.org/articles/10.3389/fvets.2025.\n1656345/full#supplementary-material\nSUPPLEMENTARY FIGURE 1\nLayout of the facilities. Entry to the premises through a footbath (red arrow),\nfollowed by a transition area equipped with a shower and biosafety gear,\nwhich provides access to the main room (blue arrow). (1) To the right, a\nnursing room containing disposable materials. (2) At the end of the corridor,\na feed storage room. (3) To the left, three pig pens, each measuring 12 m2.\nReferences\n1. Segalés J, Allan GM, Domingo M. Porcine circovirus diseases. Anim Health Res\nRev. (2005) 6:119–42. doi: 10.1079/AHR2005106\n2. Alpízar A. Hallazgos histopatológicos e implicación del PCV2, PoRV y PRRSV en\nlechones con desmedro en granjas de México (master’s thesis). México: Universidad\nAutónoma del Estado de México (2015). Spanish.\n3. Neira V, Ramos N, Tapia R, Arbiza J, Neira-Carrillo A, Quezada M, et al.\nGenetic analysis of porcine circovirus type 2 from pigs affected with PMWS in Chile\nreveals intergenotypic recombination. Virol J. (2017) 14:191. doi: 10.1186/s12985-017-0\n850-1\n4. Noriega J, Reyes P, Bucarey S. Circovirus porcino: un virus pequeño que\ngenera un gran problema. Avances en Ciencias Veterinarias. (2007) 22:62–71.\nSpanish. doi: 10.5354/0716-260X.2007.918\n5. Figueras-Gourgues S, Fraile L, Segalés J, Hernández-Caravaca I, López-Úbeda R,\nGarcía-Vázquez FA, et al. 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+{"text": "【兽医文献】\n标题: Onset and progression of postmortem histological changes in the kidneys of RccHanTM:WIST rats\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nCase Report: Idiopathic\nintramural hematoma of the\njejunum in a French Bulldog\nYeon-Jin Kim 1, Dong-Min Choi 1, Jong-Il Kang 2,\nDong-Yeong Kim 2, Chan-Sik Nam 1, Kwang-Sup Lee 1 and\nHee-Myung Park 1*\n1 Department of Veterinary Internal Medicine, College of Veterinary Medicine, Konkuk University,\nSeoul, Republic of Korea, 2 Choonghyun Animal Hospital, Seoul, Republic of Korea\nThis case report describes clinical and diagnostic features of an idiopathic jejunal\nintramural hematoma in a dog presenting with non-specific gastrointestinal symptoms.\nA 2-year-old neutered male French Bulldog was presented with vomiting, abdominal\npain, and chronic soft stools persisting for 2 weeks, with no history of trauma\nor dietary indiscretion. Physical examination revealed abdominal distension and\npalpable discomfort. The complete blood count (CBC) showed leukocytosis with\nneutrophilia, accompanied by signs of dehydration. Serum chemistry revealed no\nsignificant abnormalities, although mild electrolyte imbalances were observed.\nThe canine pancreatic lipase immunoreactivity (cPLI) test was within the reference\nrange and the C-reactive protein (CRP) level was slightly elevated. Ultrasonography\nrevealed a heterogeneous mass in the jejunal region, which was suspected to be a\nhematoma. An exploratory laparotomy was performed, and the affected jejunal\nsegment was surgically resected. Histopathological examination confirmed the\nmass as an intramural hematoma. Additionally, coagulation profiles revealed no\nremarkable findings. The cause of the hematoma was determined to be idiopathic\nafter excluding other possible causes. This case highlights the importance of\nconsidering intramural hematomas in the differential diagnosis for dogs presenting\nwith non-specific gastrointestinal symptoms, even in the absence of trauma and\ncoagulation abnormalities.\nKEYWORDS\nintramural hematoma, idiopathic, diarrhea, vomiting, case report, French bulldog,\njejunum\nIntroduction\nChronic diarrhea is a common clinical symptom in dogs, resulting from either primary or\n\nKEYWORDS\nintramural hematoma, idiopathic, diarrhea, vomiting, case report, French bulldog,\njejunum\nIntroduction\nChronic diarrhea is a common clinical symptom in dogs, resulting from either primary or\nsecondary causes. Primary enteropathies include infectious, neoplastic, mechanical, toxic, and\nnon-infectious inflammatory diseases. In contrast, secondary enteropathies are associated with\nsystemic diseases affecting the pancreas, liver, kidneys, endocrine system, cardiovascular\nsystem, and central nervous system (1). According to a previous study published in 2017 (2),\n90% of dogs presenting with chronic diarrhea were diagnosed with primary enteropathy, of\nwhich 79% were classified as chronic inflammatory enteropathy (CIE). Among the different\nforms of CIE, food-responsive enteropathy was the most prevalent, followed by antibiotic-\nresponsive enteropathy and idiopathic inflammatory bowel disease. Neoplastic causes including\nintestinal lymphoma and intestinal adenocarcinoma were identified in only 4% of the cases.\nIntestinal hematomas are well-documented in human medicine, with traumatic abdominal\ninjury being the primary cause. Other reported etiologies include pancreatic diseases,\nanticoagulant therapy, intestinal biopsy, or injections (3). In contrast, occurrences of intramural\nOPEN ACCESS\nEDITED BY\nRemo Lobetti,\nVeterinary Specialists Ireland, Ireland\nREVIEWED BY\nIzabela Janus-Ziółkowska,\nWroclaw University of Environmental and Life\nSciences, Poland\nMiltiadis Markou,\nAristotle University of Thessaloniki, Greece\n*CORRESPONDENCE\nHee-Myung Park\nparkhee@konkuk.ac.kr\nRECEIVED 17 February 2025\nACCEPTED 24 April 2025\nPUBLISHED 20 May 2025\nCITATION\nKim Y-J, Choi D-M, Kang J-I, Kim D-Y, Nam\nC-S, Lee K-S and Park H-M (2025) Case\nReport: Idiopathic intramural hematoma of\nthe jejunum in a French Bulldog.\nFront. Vet. Sci. 12:1577989.\ndoi: 10.3389/fvets.2025.1577989\nCOPYRIGHT\n© 2025 Kim, Choi, Kang, Kim, Nam, Lee and\nPark. This is an open-access article\ndistributed under the terms of the Creative\nCommons Attribution License (CC BY). The\nuse, distribution or reproduction in other\n\nFront.Vet.Sci.12:1577989.doi: 10.3389/fvets.2025.1577989\nCOPYRIGHT\n© 2025 Kim, Choi, Kang, Kim, Nam, Lee and\nPark.This is an open-access article\ndistributed under the terms of the Creative\nCommons Attribution License (CC BY).The\nuse, distribution or reproduction in other\nforums is permitted, provided the original\nauthor(s) and the copyright owner(s) are\ncredited and that the original publication in\nthis journal is cited, in accordance with\naccepted academic practice. No use,\ndistribution or reproduction is permitted\nwhich does not comply with these terms.\nTYPE  Case Report\nPUBLISHED  20 May 2025\nDOI  10.3389/fvets.2025.1577989\n\nKim et al.\b\n10.3389/fvets.2025.1577989\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nhematomas in companion animals have been extremely rarely\nreported (4–7). To date, only five cases in dogs and one case in a cat\nhave been reported across four studies. Among the reported canine\ncases, two were associated with chronic pancreatitis, one was related\nto a foreign body, and two were classified as undetermined. The single\nfeline case involved a colonic hematoma of unknown origin (4–7).\nTherefore, given the rarity of such cases, this case report aims to\ndocument the diagnostic process of an idiopathic jejunal intramural\nhematoma in a 2-year-old castrated male French Bulldog, emphasizing\nthe importance of including intramural hematoma in the differential\ndiagnosis for dogs presenting with non-specific gastrointestinal\nsymptoms, even in the absence of trauma and coagulation abnormalities.\nCase description\nA 2-year-old neutered male French Bulldog was presented with\nclinical signs of vomiting, abdominal pain, and chronic soft stools\npersisting for 2 weeks. The owner reported no dietary indiscretions,\nrecent trauma, or administration of any medications. Physical\nexamination revealed that the dog was alert but lethargic, with mildly dry\nmucous membranes and a capillary refill time of 2 s. Rectal temperature\nwas 38.5°C. The heart rate was 120 beats per minute, and respiratory rate\nwas 24 breaths per minute, both within normal limits. Mild abdominal\ndistension and discomfort on palpation were also noted in the\nmid-abdomen. No external signs of trauma, such as bruising, lacerations,\nor abrasions, were observed. To facilitate the differential diagnosis of\nthese gastrointestinal symptoms, hematological and biochemical profiles\nwere performed to rule out systemic diseases.\nThe complete blood count showed mild leukocytosis (12.7 × 109/L;\nreference range: 6–12), with neutrophilia (11.6 × 109/L; reference range:\n3–10), elevated hemoglobin levels (20.2 g/dL; reference range: 12–18), and\nan increased hematocrit (60.7%; reference range: 37–55). The red blood\n\n7 × 109/L;\nreference range: 6–12), with neutrophilia (11.6 × 109/L; reference range:\n3–10), elevated hemoglobin levels (20.2 g/dL; reference range: 12–18), and\nan increased hematocrit (60.7%; reference range: 37–55).The red blood\ncell count was at the upper limit of the reference range (8.49 × 1012/L;\nreference range: 5.5–8.5), while the platelet count remained within normal\nlimits (363 × 109/L; reference range: 200–500). Serum chemistry revealed\nno significant abnormalities except for a slightly elevated C-reactive\nprotein (CRP) level at 7 mg/L (reference range: 0–7). Mild electrolyte\nimbalances were noted, with hyponatremia (141 mmol/L; reference\nrange: 145–151), hypokalemia (3.63 mmol/L; reference range: 3.9–5.1),\nand hypochloremia (102 mmol/L; reference range: 110–119). These\nfindings suggested no significant systemic abnormalities but indicated\nmild dehydration and electrolyte imbalances, likely due to gastrointestinal\nlosses. Additionally, the canine pancreatic lipase immunoreactivity (cPLI)\ntest was performed due to the chronicity of gastrointestinal symptoms\npersisting for 2 weeks. The results were within the normal range (<50 ng/\nmL; reference range: 0–200) (Table 1).\nRadiographic examination indicated the presence of food retention\nwithin the stomach, with no significant abnormalities noted in the\nintestinal tract or other abdominal organs (Figures 1A,B). Ultrasound\nexamination was performed using a Vivid E90 ultrasound system (GE\nHealthcare, Chicago, IL, USA) equipped with a 9–12 MHz linear\ntransducer. Ultrasonography revealed a 3.5 cm mass in the distal\nduodenum, which was not well demarcated from the intestinal wall. The\nmass exhibited heterogeneous echogenicity and fluid accumulation\nobserved within the lesion, consistent with features of an intramural\nlesion, as shown in the ultrasound image. No ultrasonographic signs of\nperforation or free abdominal fluid were observed (Figures 1C,D).\nSubsequently, computed tomography (CT) was recommended to assess\ntumor staging and potential metastasis; however, the owner declined\n\nNo ultrasonographic signs of\nperforation or free abdominal fluid were observed (Figures 1C,D).Subsequently, computed tomography (CT) was recommended to assess\ntumor staging and potential metastasis; however, the owner declined\nfurther anesthesia or sedation solely for diagnostic purposes, and thus\nCT imaging was not performed.\nDue to the size and obstructive potential of the jejunal mass\nidentified on ultrasound, the dog owner consented to an exploratory\nlaparotomy for surgical resection of the intramural jejunal mass. The\npatient was premedicated with cephradine (20 mg/kg IV; Hankook\nKorus Pharm, Chuncheon, Korea), tramadol (1.25 mg/kg IV; Jeil\nPharm, Daegu, Korea), maropitant (1 mg/kg IV; Cerenia®, Zoetis,\nParsippany, NJ, USA), and glycopyrrolate (0.01 mg/kg IV; Myungmoon\nPharm, Seoul, Korea). Anesthesia induction was performed using\npropofol (1 mg/kg IV; Daewon Pharm, Seoul, Korea), and anesthesia\nwas maintained with isoflurane (Forane®, Hana Pharm, Seoul, Korea)\nin 100% oxygen. The concentration of isoflurane was individualized to\n1–3%, maintaining minimum alveolar concentration (MAC) of 1.3–1.5.\nDuring the surgical procedure, particularly during the more painful\nstages such as intestinal resection and anastomosis, more than 2% of\nisoflurane was administered.\nSurgical intervention was undertaken with the dog positioned in\ndorsal recumbency. A standard midline celiotomy was carried out,\nextending from approximately 4–5 cm caudal to the xiphoid process\nto 4–5 cm caudal to the umbilicus. Upon exploratory laparotomy, no\nTABLE 1  Results of a complete blood count (CBC), serum chemistry, and\nelectrolytes (WBC = 12.7 × 109/L [6–12], NEU = 11.6 × 109/L [3–10]).\nParameters\nResults\nReference range\nComplete blood count (CBC)\nRBC (M/uL)\n8.49\n5.5–8.5\nHCT (%)\n60.7\n37.0–55.0\nHGB (g/dL)\n20.2\n12–18\nPLT (K/μL)\n363\n200–500\nSerum Chemistry\nGLU (mg/dL)\n119\n75–128\nCREA (mg/dL)\n0.66\n0.4–1.4\nBUN (mg/dL)\n13.5\n9.2–29.2\nTP (g/dL)\n6.5\n5–7.2\nALB (g/dL)\n3.7\n2.6–4.0\nGLOB (g/dL)\n2.8\n1.6–3.7\nALT (U/L)\n70\n17–78\nAST (U/L)\n35\n17–44\nALP (U/L)\n146\n47–254\nCRP (mg/L)\n7\n0–7\ncPLI (ng/ml)\n<50\n0–200\nElectrolytes\nNa+ (mmol/L)\n141\n145–151\nK+ (mmol/L)\n3.63\n3.9–5.1\nCl− (mmol/L)\n102\n110–119\nRBC, red blood cells; HCT, hematocrit; HGB, hemoglobin; PTL, platelet; GLU, glucose;\n\n63\n3.9–5.1\nCl− (mmol/L)\n102\n110–119\nRBC, red blood cells; HCT, hematocrit; HGB, hemoglobin; PTL, platelet; GLU, glucose;\nCREA, creatinine; BUN, blood urea nitrogen; TP, total protein; ALB, albumin; GLOB,\nglobulin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline\nphosphatase; CRP, c-reactive protein; cPLI, canine pancreatic lipase immunoreactivity; Na+,\nsodium Ion; K+, potassium Ion; Cl−, chloride Ion. Bold values indicate abnormal findings.\n\nKim et al.\b\n10.3389/fvets.2025.1577989\nFrontiers in Veterinary Science\n03\nfrontiersin.org\ngross lesions suggestive of trauma were identified in the abdominal\norgans or along the abdominal wall. Additionally, there were no\nobservable abnormalities such as bowel wall edema, mesenteric\nthickening, or venous and lymphatic congestion within the\nmesentery. However, an intramural hematoma was identified in the\njejunal segment. The affected jejunal loop was exteriorized and\nisolated from adjacent. The mesenteric vessels supplying the segment\ndesignated for resection were ligated using 2–0 monofilament\nabsorbable suture (PDS II, Ethicon, Somerville, NJ, USA). A surgical\nmargin of at least 4 cm was secured on both the proximal and distal\nsides of the lesion. Atraumatic intestinal clamps were placed\napproximately 6 cm proximal and distal to the lesion to temporarily\nocclude the intestinal lumen (Figure 2A). Stay sutures were placed on\nthe antimesenteric borders of the proximal and distal intestinal loops\nnear the intended transection sites using 3–0 polypropylene suture\n(Prolene, Ethicon, Somerville, NJ, USA) to facilitate alignment. The\nmass and adjacent tissues were subsequently resected. An end-to-end,\nappositional anastomosis was performed using 3–0 monofilament\nabsorbable suture (PDS II, Ethicon, Somerville, NJ, USA). The\nanastomosis was initiated by placing three simple interrupted sutures\nin a triangular pattern to align the lumen, followed by a simple\ncontinuous pattern to complete the closure. A leaking test was\nconducted following anastomosis, and no leakage was observed.\nOmentum draping was performed over the anastomotic site following\nlavage with sterile saline to promote healing. Routine three-layer\nclosure of the abdominal wall was performed. The linea alba was\nclosed with simple interrupted sutures using 2–0 monofilament\nabsorbable suture (PDS II, Ethicon, Somerville, NJ, USA). The\nsubcutaneous tissues were approximated using 3–0 monofilament\nabsorbable suture (Monocryl, Ethicon, Somerville, NJ, USA) in a\nsimple interrupted pattern. Finally, the skin was closed using 3–0\n\nThe\nsubcutaneous tissues were approximated using 3–0 monofilament\nabsorbable suture (Monocryl, Ethicon, Somerville, NJ, USA) in a\nsimple interrupted pattern.Finally, the skin was closed using 3–0\nnon-absorbable monofilament suture (Prolene, Ethicon, Somerville,\nNJ, USA) in a simple interrupted pattern. After the anastomosis, the\ndog recovered well from anesthesia without any postoperative\ncomplications. The excised jejunal mass, measuring 3.5 cm in length,\nwas prepared for histopathological evaluation (Figure 2B). Blood\nclots were observed in the dissected mass (Figure 2C).\nThe excised jejunal mass was submitted to Kopath Co., Ltd.\n(Seoul, Republic of Korea) for histopathological evaluation.\nHistological examination revealed extensive blood retention within a\nsubstantial portion of the muscular layer (Figure  3A), while the\nmucosal layer exhibited lymphatic dilation and blood congestion\n(Figure 3B). In the mesentery, granulomatous inflammation, increased\nconnective tissue, and the formation of newly developed blood vessels\nwere observed (Figure 3C). However, no other remarkable findings,\nsuch as infectious pathogens or neoplastic changes, were identified.\nFIGURE 1\nRadiographic and ultrasonographic images of the abdomen. (A) Right lateral abdominal radiograph showing food retention in the stomach (asterisk)\nand multiple gas-filled loops of the small intestine. However, no radiographic abnormalities suggestive of an intestinal lesion are identified.\n(B) Ventrodorsal abdominal radiograph also demonstrates food retention within the stomach (asterisk) and some intestinal gas patterns, but no specific\nabnormalities indicative of intestinal pathology. (C, D) Ultrasonographic images of the abdomen reveal a heterogeneous mass (asterisk) in the jejunal\narea, distal to the duodenum, measuring approximately 3.5 cm in length. The mass exhibited mixed echogenicity with internal fluid retention and\nshowed an indistinct demarcation from the bowel wall layers. Loss of the normal wall structure was observed within the mass. However, no evidence\n\n5 cm in length.The mass exhibited mixed echogenicity with internal fluid retention and\nshowed an indistinct demarcation from the bowel wall layers.Loss of the normal wall structure was observed within the mass.However, no evidence\nof small bowel perforation or ascites was observed.\n\nKim et al.\b\n10.3389/fvets.2025.1577989\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nPotential causes of hematoma in the intestine, such as anticoagulant\nuse, traumatic injury, foreign body, intussusception, and coagulopathy\nwere considered. However, in this case, there was no history of\nanticoagulant use or traumatic injury, and given the dog’s young age,\nneoplastic processes were also unlikely. Additionally, no lesions\nsuggestive of foreign body or intussusception were identified during\nexploratory laparotomy or histopathological examination, allowing\nthese causes to be ruled out. Consequently, coagulation testing was\nwarranted to evaluate platelet function and potential coagulation factor\ndeficiencies, including von Willebrand disease (vWD). Since the platelet\ncount was normal on pre-surgical testing, thrombocytopenia was ruled\nout as a cause. Postoperative evaluations of prothrombin (PT) and\nactivated partial thromboplastin time (APTT) were within normal\nranges, and the vWD factor test results were also normal. Therefore, this\ncase was diagnosed as a hematoma caused by idiopathic etiology. For\n6 months follow-up period, there was no recurrence of symptoms such\nas vomiting, abdominal pain, or soft stools.\nDiscussion\nThe dog presented clinical signs of vomiting, chronic soft stools,\nand abdominal pain, which are commonly associated with a broad\nrange of gastrointestinal disorders, from benign dietary indiscretion\nto severe life-threatening conditions. The lack of a history of dietary\nindiscretion or trauma complicated the diagnosis, necessitating a\nmore in-depth evaluation. The persistence of vomiting for 2 weeks\nraised particular concern, as prolonged vomiting can lead to\ndehydration and electrolyte disturbances, complicating the clinical\nassessment (8).\nFIGURE 2\nGross features of the jejunal mass. (A) Intraoperative photograph showing the jejunal mass identified during surgery. The mass’s appearance was\nconsistent with hematoma. The proximal and distal sections of the jejunum were secured with atraumatic intestinal clamp forceps before resection.\n\n(A) Intraoperative photograph showing the jejunal mass identified during surgery.The mass’s appearance was\nconsistent with hematoma.The proximal and distal sections of the jejunum were secured with atraumatic intestinal clamp forceps before resection.\n(B) The excised jejunal mass, approximately 3.5 cm in size, is prepared for histopathological examination. (C) The bisected jejunal mass, examined for\ninternal contents, reveals clotted blood within the lesion.\n\nKim et al.\b\n10.3389/fvets.2025.1577989\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nThe development of leukocytosis in this case could be  due to\nchronic hematoma, possibly indicating an ongoing inflammatory\nresponse, which is typically associated with unresolved or prolonged\nhematomas. This chronic inflammation may result in a sustained\nelevation in white blood cell count as the body attempts to manage the\ninjury and repair the tissue over time (9). Platelet levels may either\nremain normal or show mild thrombocytosis as a response to injury and\nhealing. Blood loss, however, could prevent a significant increase in\nplatelet counts. Dehydration, due to the chronic nature of the condition,\nmay result in an elevation of red blood cell, hematocrit and hemoglobin\nlevels, reflecting fluid loss. While hematoma may cause mild anemia,\nthe chronic progression of the condition could lead to increased\nhematocrit and hemoglobin levels, a pattern consistent with this\ncase (10).\nThus, based on the hematological evaluation and signs of\ndehydration, it is likely that the lesion had been present for a prolonged\nperiod, as reflected in the history of chronic soft stools and vomiting.\nThese findings may vary depending on the hematoma’s severity and any\nassociated complications, such as secondary infection or fluid shifts (8).\nIn veterinary practice, when abdominal ultrasonography reveals\nmixed echogenicity, it can be associated with various conditions. In\ncases of hematomas, early stages may exhibit a combination of\nhyperechoic and hypoechoic areas (11). In abscesses, the hypoechoic\narea generally represents fluid accumulation, while the hyperechoic area\nmay indicate tissue response or inflammation (12). Neoplasms,\nparticularly malignant tumors, may also present with mixed\nechogenicity due to necrosis or hemorrhage within the tumor (13).\nCysts with hemorrhage or infection can show mixed echogenicity due\nto the presence of blood or infectious material (14, 15). Similarly,\nfibrosis or scar tissue resulting from previous injuries may demonstrate\na mixed echogenic pattern (16). To confirm these diagnoses, further\n\nSimilarly,\nfibrosis or scar tissue resulting from previous injuries may demonstrate\na mixed echogenic pattern (16).To confirm these diagnoses, further\nimaging, biopsy, or laboratory testing may be required.\nIn this case, the owner strongly opposed performing fine needle\naspiration cytology due to concerns about the patient’s aggressive\nbehavior and the potential for stress-induced complications such as\nrespiratory distress or hyperthermia. The owner also expressed\nsignificant anxiety regarding the risks of sedation or anesthesia in\nbrachycephalic breeds like French Bulldogs. As a result, they declined\nany additional diagnostic procedures requiring sedation or anesthesia\nthat were not directly related to treatment. However, since\nhistopathological examination of the mass was already planned\nfollowing surgical removal, the necessity for additional procedures such\nas fine needle aspiration cytology was considered relatively low.\nBased on histopatholgical findings, granulomatous inflammation\nwas observed in the mesentery adjacent to the site of the jejunal\nintramural hematoma. While granulomatous inflammation is typically\nassociated with infectious, autoimmune, or foreign-body reactions, it\nmay also develop as a secondary response to tissue injury (17). In\nparticular, an intramural hematoma can act as an inert substance that is\nnot readily cleared by the host, thereby inducing a foreign body-type\ngranulomatous reaction. This pattern is consistent with non-infectious\ngranulomatous inflammation and is often accompanied by\nneovascularization and early fibrosis, supporting the interpretation of a\nchronic reactive process rather than a primary granulomatous disease\n(18). Therefore, the mesenteric granulomatous inflammation identified\nin this case was most likely a secondary response to localized tissue\ninjury caused by the hematoma. This interpretation is further supported\nby the absence of identifiable infectious organisms on histochemical\nstaining and the lack of clinical or histological evidence of infectious or\nautoimmune disease.\nHistopathological examination confirmed the diagnosis of an\n\nHistopathological examination confirmed the diagnosis of an\nintramural hematoma, validating both the diagnostic and therapeutic\ndecisions, particularly in light of the challenging clinical presentation.\nGiven the non-specific nature of the symptoms, radiographic and\nultrasonographic imaging were utilized for further evaluation of the\ngastrointestinal tract. While radiographs revealed food retention in the\nstomach without significant findings in this case, ultrasonography\nidentified a not well demarcated and heterogeneous mass in the jejunal\nregion, consistent with intramural hematoma (3). This case underscores\nthe limitations of radiographs in detecting certain gastrointestinal issues\nand highlights the importance of ultrasonography, particularly when\ninitial diagnostic tests are necessary.\nAccording to a prior study, the most common cause of intramural\nhematoma in the small intestine of children is abdominal trauma (19).\nThe increased risk of jejunal hematomas in children is attributed to\nanatomical differences, including underdeveloped abdominal\nmusculature and a shallower anteroposterior depth of the abdominal\nFIGURE 3\nHistopathological examination of the mass. (A) A substantial portion\nof the muscular layer was extensively composed of retained blood\n(white asterisk), and granulomatous inflammation (black arrow) was\nobserved in the mesentery; H&E stain; scale bar, 500 μm. (B) In the\nmucosal layer, lymphatic vessel dilation (black arrow) and congestion\n(blue arrow) were noted, with retained blood also evident in the\nmuscular layer (white asterisk); H&E stain; scale bar, 100 μm. (C) In\nthe mesentery, granulomatous inflammation (black asterisk) was\nobserved, along with increased connective tissue and the formation\nof some newly developed blood vessels (red arrow). No other\nnotable findings, such as infectious pathogens or neoplastic\nchanges, were observed; H&E stain; scale bar, 100 μm.\n\nKim et al.\b\n10.3389/fvets.2025.1577989\nFrontiers in Veterinary Science\n06\nfrontiersin.org\ncavity compared to adults (20). Given the patient’s aggressive nature,\ntrauma could not be completely ruled out. However, the owner was\nhighly attentive and careful in managing the patient, and reported no\nincidents that could have resulted in significant trauma. Moreover,\nphysical examination revealed no external signs of trauma, such as\nbruising, lacerations, or abrasions. In addition, no trauma-associated\nlesions were identified in other abdominal organs or the abdominal wall\nduring exploratory laparotomy. These findings collectively rule out\ntrauma as the underlying cause in this case.\nThe possibility of a transient intussusception that had spontaneously\nresolved was also considered. Such cases generally present with acute\nabdominal pain or sudden anorexia due to intermittent obstruction. In\ncontrast, the patient in this case exhibited a more insidious and continuous\nprogression of symptoms, which did not align with the typical episodic\nclinical pattern associated with self-limiting intussusception (21).\nMoreover, careful intraoperative inspection of the entire abdominal cavity,\nincluding all segments of the gastrointestinal tract, failed to reveal any\nmacroscopic evidence typically observed in prior intussusception, such\nas segmental bowel wall swelling, mesenteric thickening, or venous and\nlymphatic engorgement within the affected region (22).\nThe potential for foreign body-induced intestinal injury was included\nin the differential assessment. Typically, such trauma is confined to the\nmucosa and manifests as localized inflammation, mucosal erosion, or\nulcer formation (23). However, in this case, histological evaluation did not\nidentify any signs of epithelial disruption or ulceration indicative of\nmechanical injury. The hematoma was restricted to the muscularis layer,\nwhile the mucosal surface showed only vascular congestion and lymphatic\ndilation without associated tissue damage. These findings are not\ncharacteristic of a foreign body-related etiology. The owner was generally\n\nThe hematoma was restricted to the muscularis layer,\nwhile the mucosal surface showed only vascular congestion and lymphatic\ndilation without associated tissue damage.These findings are not\ncharacteristic of a foreign body-related etiology.The owner was generally\nattentive to the dog’s behavior and condition and reported no recent\nepisodes of dietary indiscretion or foreign body ingestion.\nOther causes of jejunal hematomas include hepatopathy,\nanticoagulant use, and coagulopathies like hemophilia (24). In this case,\nbased on the owner’s statements and laboratory tests, hepatopathy,\ntrauma, and medication use were ruled out. Moreover, due to normal\ncoagulation testing, coagulopathies could be  ruled out as a\ncontributing factor.\nWhile this case had a successful immediate outcome, monitoring\nover a 1–2 years period would be beneficial to evaluate resolution or\nrecurrence, as suggested by some studies on intramural hematomas (6,\n25). However, no recurrence of the hematoma was observed during the\n6-month follow-up period after resection in this case.\nThis case highlights the importance of considering rare conditions like\nnon-traumatic intramural hematomas in the differential diagnosis of\nprolonged gastrointestinal symptoms, even in animals without a history of\ntrauma or dietary indiscretion. Additionally, it emphasizes the diagnostic\nvalue of advanced imaging techniques, particularly ultrasonography, in\nidentifying intestinal wall abnormalities that may be  missed on\nconventional radiography. While CT imaging was not used in this case, it\ncould be considered in future cases for further characterization of the\nhematoma and potential identification of underlying causes.\nIn summary, this case describes a rare instance of a non-traumatic\nintramural hematoma in the jejunum of a 2-year-old neutered male\nFrench Bulldog. The dog was presented with non-specific clinical signs,\nincluding vomiting, soft stools, and abdominal pain. The owner\nreported that the dog had consumed only its regular diet and had no\nhistory of dietary indiscretion or trauma, nor had any medications that\n\nThe dog was presented with non-specific clinical signs,\nincluding vomiting, soft stools, and abdominal pain.The owner\nreported that the dog had consumed only its regular diet and had no\nhistory of dietary indiscretion or trauma, nor had any medications that\ncould affect coagulation been administered. These findings underscore\nthe importance of thoroughly investigating gastrointestinal symptoms,\neven when there are no obvious external risk factors.\nConclusion\nThis case underscores the importance of including intramural\nhematomas in the differential diagnosis for dogs presenting with\nnon-specific gastrointestinal symptoms, even in the absence of trauma\nand coagulation abnormalities.\nData availability statement\nThe original contributions presented in the study are included in\nthe article, further inquiries can be directed to the corresponding\nauthor.\nEthics statement\nEthical approval was not required for the studies involving animals in\naccordance with the local legislation and institutional requirements\nbecause we  obtained consent from the owner to use the patient’s\ninformation, and since it was a case report, ethical approval was not\nrequired. Written informed consent was obtained from the owners for the\nparticipation of their animals in this study. Written informed consent was\nobtained from the participant for the publication of this case report.\nAuthor contributions\nY-JK: Writing  – original draft, Writing  – review & editing,\nConceptualization. D-MC: Data curation, Writing – original draft,\nWriting – review & editing. J-iK: Data curation, Writing – original\ndraft, Writing – review & editing. D-YK: Data curation, Writing –\noriginal draft, Writing – review & editing. C-SN: Writing – review &\nediting. K-SL: Writing  – review & editing. H-MP: Supervision,\nWriting – original draft, Writing – review & editing.\nFunding\nThe author(s) declare that no financial support was received for\nthe research and/or publication of this article.\nConflict of interest\nThe authors declare that the research was conducted without any\ncommercial or financial relationships that could be construed as a\n\nConflict of interest\nThe authors declare that the research was conducted without any\ncommercial or financial relationships that could be construed as a\npotential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\n\nKim et al.\b\n10.3389/fvets.2025.1577989\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. Any product that may be evaluated in this article, or claim\nthat may be made by its manufacturer, is not guaranteed or endorsed\nby the publisher.\nReferences\n1.\tGerman AJ. Diseases of the small intestine In: JW Simpson and MJ Day, editors.\nBSAVA manual of canine and feline gastroenterology. 2nd ed. Gloucester: British Small\nAnimal Veterinary Association (2005). 176–202.\n2.\tVolkmann M, Steiner JM, Fosgate GT, Zentek J, Hartmann S, Kohn B. Chronic\ndiarrhea in dogs–retrospective study in 136 cases. J Vet Intern Med. (2017) 31:1043–55.\ndoi: 10.1111/jvim.14739\n3.\tGuzman C, Bousvaros A, Buonomo C, Nurko S. Intraduodenal hematoma\ncomplicating intestinal biopsy: case reports and review of the literature. Am J\nGastroenterol. (1998) 93:2547–50. doi: 10.1016/S0002-9270(98)00597-8\n4.\tPadalkar M, Savage M, Cohen EB. Small intestinal intramural hematoma secondary\nto a migrating wire foreign body in a dog. Vet Radiol Ultrasound. 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+{"text": "【兽医文献】\n标题: In silico performance of a targeted enriched metagenomics approach to infer Mycoplasma bovis strains in milk\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nAn evaluation of a canine dry\nwipe decontamination kit\nRobert G. Buntz 1, Meghan K. Haselden 2, Kiran P. Badola 2,\nJulia L. Sharp 3, Kelly A. Mann 2,4 and Brian France 5*\n1Department of Small Animal Primary Care, Midwestern University, Glendale, AZ, United States, 2Mantel\nTechnologies, Inc., Fort Collins, CO, United States, 3Sharp Analytics LLC, Fort Collins, CO, United\nStates, 4Department of Clinical Sciences, Colorado State University, Fort Collins, CO, United States,\n5TDA Research, Inc., Golden, CO, United States\nIntroduction: Decontamination of working dogs at the point of exposure is criti­\ncal for military, law enforcement, and search and rescue operations in austere\nor hazardous environments where water may be scarce and conventional wet\ndecontamination poses logistical and safety challenges. Prototype dry (waterless)\nwipe decontamination kits were evaluated for efficacy in removing a surrogate\ncontaminant (GloGerm™) when used by canine handler teams performing total\nbody field decontaminations. The prototype kit procedures employed a sequence\nof three microfiber towel types: (1) dry, to remove gross contaminants; (2) wet,\nsaturated with a surfactant to clean away residual contaminants; and, (3) dry,\nto remove final contamination and residual surfactant. Several variables were\ninvestigated to refine the final design, test the effectiveness of decontamination at\ndifferent anatomical locations on the canine, and evaluate the handlers’ success\nin removing the surrogate contaminant after reading the instructional pamphlet\nalone versus having the instructions and receiving in-person training on kit use.\nMethods: Three form factors (single towel, multi-towel small, and multi-towel\nlarge) and simulated contamination at three anatomical regions (back, inguinal,\nand forelimb) were tested by 64 canine handler teams randomized to “trained”\n(15-min verbal instruction plus pamphlet) or “untrained” (pamphlet only) groups.\nResults: Across all form factors and training groups, mean removal efficien­\n\nResults: Across all form factors and training groups, mean removal efficien­\ncies were highest on the back (91.38% ± 0.18), followed by the forelimb\n(82.04% ± 0.26) and inguinal region (70.15% ± 0.31). No statistically significant\ndifferences were observed among the form factors or training variables. Average\ndecontamination time was 9 min and 24 s (range = 3 min 4 s to 26 min 0 s).\nDiscussion: Dry wipe decontamination can provide rapid and effective removal of\ncontaminants without water, improving canine and handler safety and enabling\nearly intervention at the point of exposure prior to additional wet decontamina­\ntion or medical treatment when necessary. Personnel with minimal training can\neffectively conduct dry wipe decontamination.\nKEYWORDS\ncanine, decontamination, dry decon, military working dog, multi-purpose canine\n1 Introduction\nEmergency preparedness for the effective decontamination of a working dog (WD) at the\npoint of exposure is a necessity for military, law enforcement, and search and rescue (SAR)\ncanine teams. Their missions often occur in austere, limited resource environments, or in the\nimmediate aftermath of man-made or natural disasters where hazardous materials exposures\nmay be encountered unexpectedly (1). There is also the possibility of deliberate attacks using\nTYPE  Original Research\nPUBLISHED  12 February 2026\nDOI  10.3389/fvets.2026.1770205\nOPEN ACCESS\nEDITED BY\nErin Beth Perry,\nSouthern Illinois University Carbondale,\nUnited States\nREVIEWED BY\nKelly Bender,\nSouthern Illinois University Carbondale,\nUnited States\nAlison G. Simon,\nOklahoma State University Center for\nHealth Sciences, United States\n*CORRESPONDENCE\nBrian France\nbfrance@tda.com\nRECEIVED 17 December 2025\nREVISED 18 January 2026\nACCEPTED 26 January 2026\nPUBLISHED 12 February 2026\nCITATION\nBuntz RG, Haselden MK, Badola KP,\nSharp JL, Mann KA and France B (2026)\nAn evaluation of a canine dry wipe\ndecontamination kit.\nFront. Vet. Sci. 13:1770205.\ndoi: 10.3389/fvets.2026.1770205\nCOPYRIGHT\n© 2026 Buntz, Haselden, Badola, Sharp,\nMann and France. This is an\nopen-access article distributed under\nthe terms of the Creative Commons\n\nFront.Vet.Sci.13:1770205.doi: 10.3389/fvets.2026.1770205\nCOPYRIGHT\n© 2026 Buntz, Haselden, Badola, Sharp,\nMann and France.This is an\nopen-access article distributed under\nthe terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other\nforums is permitted, provided the\noriginal author(s) and the copyright\nowner(s) are credited and that the\noriginal publication in this journal is\ncited, in accordance with accepted\nacademic practice. No use, distribution\nor reproduction is permitted which does\nnot comply with these terms.\n\nBuntz et al.\b\n10.3389/fvets.2026.1770205\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nchemical, biological, or nuclear agents to disrupt protected human\npopulations like the commuting public or military personnel. Limited\nprotective equipment exists for WDs and available equipment may\ninterfere with the dog’s ability to complete their job, increasing the risk\nof exposure (2).\nCurrent canine decontamination procedures involve copious\namounts of soap and water (i.e., “wet decontamination”) to remove\ncontaminants (3–5). Access to safe, potable water is one of the most\nconstrained resources in austere environments and creates a substan­\ntial logistical burden. Typically, the canine must be brought to a static\nwet decontamination lane location, delaying time from exposure to\ndecontamination, allowing for transdermal absorption, and increasing\nhuman team members’ exposure risks via transference from the canine.\nWhen a dog is exposed to hazardous or toxic agents, their fur or\nhair coat acts as the first line of defense. Materials and chemicals that\ncontact the fur must penetrate through the coat before contacting and\nbeing absorbed through the underlying skin (6, 7). Use of high-pres­\nsure soap and water as the first step in decontamination can provide\nan avenue for fur penetration, transdermal absorption, and potential\nharm to a previously unaffected dog (8–10).\nThe use of soap and water has been shown to enhance the percu­\ntaneous absorption of lipophilic chemicals (6, 11) and repeated wash­\ning with soaps has been shown to damage the barrier function of the\nskin against external infectious or chemical agents (12). Additionally,\nrepeated soap and water bathing may result in a pruritic inflammatory\nresponse, increasing the likelihood of licking and scratching behaviors\nand elevating the risk of absorption through skin penetration and oral\ncontamination (13). SAR dogs will often work several weeks at the\nsame disaster site, requiring repeated decontamination procedures\nwhich increases these risks during subsequent exposures (14).\nHuman procedures for entering a wet decontamination lane\n\nSAR dogs will often work several weeks at the\nsame disaster site, requiring repeated decontamination procedures\nwhich increases these risks during subsequent exposures (14).Human procedures for entering a wet decontamination lane\ninvolve stripping off their outer layer of clothing, which removes up to\n90% of the contaminant (15). As quadrupeds, canines may be exposed\nto significantly more contaminants than their human counterpart\nbecause they are at ground level and cannot remove their contami­\nnated fur upon entering the decontamination lane (16). All of these\nfactors, combined with normal dog behavior like tail wagging, shak­\ning, and rolling on ground, cause significant cross contamination in\nthe environment which increases risks to other animals and people in\nthe area, and increases logistical management of contaminated water\ndisposal. Furthermore, high pressure washing increases the risk of\naerosolization of contaminants, which is exacerbated by the potential\nfor a high contaminant load within the canine fur or hair coat (17, 18).\nA dry (waterless) wipe decontamination kit and simple point of\nexposure procedures offer many benefits to current canine decontami­\nnation recommendations. Waterless decontamination with the proto­\ntype decontamination wipes used in this study and the “pinch and\npull” technique was developed and studied under laboratory condi­\ntions for the removal of chemical warfare agents from canine cadaver\nskin/fur samples (7). The objective of this study was to evaluate the use\nand efficacy of prototype canine field decontamination kits (TDA\nResearch, Inc., Golden, CO; Figure 1) in removing a safe, surrogate\ncontaminant (GloGerm™ MIST Spray, Glo Germ Company, Moab,\nUT) from a working dog by their handler. This practical evaluation, in\naddition to live agent laboratory testing on canine fur samples (7), is\nimportant in assessing the kit form factors and effectiveness prior to\npotential adoption by military, federal and civilian working dog han­\ndlers, Chemical, Biological, Radiological, and Nuclear personnel, disas­\n\nter response groups, first responders, and veterinary medical\nprofessionals.\nLive agent studies using chemical warfare and other life-threaten­\ning contaminants are important in assessing the effectiveness of\ndecontamination equipment and procedures; however, they have sev­\neral limitations. As these contaminants cause severe life-threatening\neffects, they cannot safely be used on active WDs by their handlers.\nLive agent testing is limited to controlled environments where bio­\nsafety personnel can safely conduct tests on cadaver fur and tissue\nsamples. Combining the results from laboratory live agent studies and\non-dog simulant contaminant removal studies allows for a more com­\nplete assessment of the effectiveness and limitations of canine decon­\ntamination procedures (19). Practical evaluation with canine handler\nteams encompasses important aspects such as canine restraint and\ndecontamination on a non-static target, canine behaviors toward a\nnovel stimulus of this decontamination type, difference in fur length\nand composition across individual dogs, non-ideal angle and position­\ning for the handler to reach differing anatomic locations during\ndecontamination, and feasibility of decontaminating the entirety of a\ndog (as opposed to smaller laboratory fur samples). The outcome of\ninterest in this study was the evaluation of laboratory-developed\ndecontamination techniques in real world environments, the percent­\nage of simulant removed helps us understand the challenges and vari­\nables faced during decontamination events. The following variables\nwere assessed in this study:\nAnatomical Location/Hair Type Effectiveness: Three anatomical\nlocations were chosen based on their differences in hair types and in\nhandler access during decontamination: (1) the back (dorsum) with\nits longer primary or guard hairs and ease of handler access, (2) the\ninguinal (inner thigh) region with its shorter, thinner secondary hairs\nand more difficult to reach location, and (3) the forelimb below the\nelbow with its shorter guard hairs.\nForm Factor Effectiveness: Three form factors were evaluated in\n\nForm Factor Effectiveness: Three form factors were evaluated in\nthis study, looking primarily at the wipe size and number of wipes\nneeded to decontaminate a canine. The form factors tested included:\n(1) “single towel,” a single ‘large’ microfiber towel (40 × 40-cm) stored\nin vacuum-sealed packages were used for each step (e.g., Wipe A Dry\nWipe, Wipe B Wet Wipe, and Wipe C Dry Wipe; Figure 1), (2) “multi-\ntowel small,” multiple (5 each) small towels (20 × 20-cm) in each\nvacuum-sealed package, and (3) “multi-towel large,” multiple (3 each)\nlarge towels (40 × 40-cm) in each vacuum-sealed package. This study\ncompared the decontamination effectiveness among these form fac­\ntors, allowing for evidence-based selection of the form factor for opti­\nmization of the final kit through downselection.\nTrained Versus Untrained Effectiveness: A one-page instructional\npamphlet was included in each prototype canine field decontamina­\ntion kit. In order to assess the decontamination effectiveness of the\ninstructions, a trained group (provided with both the instructional\npamphlet and verbal instruction by the research team) was compared\nto an untrained group (provided an instruction pamphlet only).\nAbbreviations: CBRN, Chemical, Biological, Radiological, and Nuclear; DOD,\nDepartment of Defense; HD, sulfur mustard, a chemical warfare agent that causes\nblistering of the skin and mucous membranes; IACUC, Institutional Animal Care\nand Use Committee; IRB, Institutional Review Board; MOPP, Mission Oriented\nProtective Posture; MPC, multi-purpose canine; MWD, military working dog; PPE,\npersonal protective equipment; SAR, search and rescue; SME, subject matter\nexpert; U.S., United States; UV, ultraviolet; VX, venomous agent X, a chemical\nwarfare agent classified as a nerve agent; WD, working dog.\n\nBuntz et al.\b\n10.3389/fvets.2026.1770205\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nHandlers included in the study encompassed a wide variety of work­\ning dog groups including military, special operations, law enforcement, \nand civilian SAR groups, with the purpose of capturing how persons of \ndifferent backgrounds and experience levels would be able to effectively \nemploy the prototype canine field decontamination kit. Additionally, \nhandler feedback was collected and used to assess the kit’s function in \nproposed real-world scenarios and to optimize future kit design.\n2 Materials and methods\n2.1 Participants\nInstitutional Animal Care and Use Committee (IACUC) and \nInstitutional Review Board (IRB) approval was obtained from \nColorado State University prior to the initiation of this study. \nParticipants were WDs and their handlers from military, law enforce­\nment, or SAR backgrounds. Permissible breeds were German, Belgian \n(e.g., Groenendael, Tervuren, Malinois, Laekenois), and Dutch \nShepherds, or mixes of the aforementioned breeds. The canines were \n1–10 years of age, either male or female, neutered or intact. The \ncanines could be excluded from the IACUC protocol based on aggres­\nsive temperament, active dermatologic conditions, or subjective exclu­\nsion by their attending veterinarian, unit commander, or supervisor \nfor any reason without explanation or fear of retribution.\n2.2 Application of contaminant\nThe simulant contaminant used was a commercial product, \nGloGerm™. GloGerm™ is available in powder, spray and gel formu­\nlations which are well known in the safety training community and \nFIGURE 1\nPrototype canine field decontamination kit. Three vacuum-sealed microfiber towel packages labeled Wipe A Dry Wipe, Wipe B Wet Wipe, and Wipe C \nDry Wipe, protective non-sterile gloves, examples of the microfiber wipes, and the complete prototype canine field decontamination kit in overwrap \npackaging with the instruction sheet facing outwards. Not pictured: two slip leads, scissors, and M8 paper.\n\nBuntz et al.\b\n10.3389/fvets.2026.1770205\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nhave been employed by other working dog research teams (3, 14, 16,\n19, 20). In this study, colorless, GloGerm™ MIST Spray droplets were\nused to simulate a potentially lethal toxin exposure in a safe non-\nthreatening environment for the canine handler team. The simulant\nwas applied in the presence of the handler, however the GloGerm™\nwas not visible to the unaided eye once applied, making the exact\nlocation of the contamination less obvious. GloGerm™ MIST Spray\n(Figure 2) was applied topically to each canine at three anatomical\nlocations: dorsal midline behind the shoulder blades (“back”); the\ninner aspect of either hindlimb above the knee where the abdomen\nmeets the inner thigh (“inguinal”); and the cranial aspect of either\nforelimb between the carpus and elbow (“forelimb”). A plastic barrier\nwith a cutout (circle, 3.2-cm diameter) for the application of the\nGloGerm™ (Figure 2) was utilized to prevent overspray and standard­\nize the area of GloGerm™ application. The plastic template was held\nagainst the dog and GloGerm™ was sprayed once over the cutout to\nproduce consistent application size at all three locations on the indi­\nvidual dog as well as for each canine participant in the study. Before\nthe application of GloGerm™, a pre-contamination photo was\nobtained under ultraviolet (UV) illumination in a darkened room.\nAfter the application of GloGerm™, photographic images using UV\nillumination were obtained in a darkened room before and after the\nhandler performed the decontamination procedure using the pro­\nvided prototype kit. A camera standoff (Figure 2) provided for a con­\nsistent 13-cm distance between the camera lens and the dog when\neach photograph was taken as well as a consistent 1-cm reference\nmeasurement within each photograph in order to quantify the area of\nsimulant fluorescence during analysis. After initial photography and\nsimulant application, the handler was instructed to conduct a full\ncanine decontamination while being observed and timed by a research\n\nAfter initial photography and\nsimulant application, the handler was instructed to conduct a full\ncanine decontamination while being observed and timed by a research\nteam member. After the handler completed the decontamination pro­\ncedure, the locations where simulant had been applied were re-pho­\ntographed and analyzed to determine effectiveness of the kit materials\nand the procedure to remove the simulant.\n2.3 Prototype canine field decontamination\nkit and wipe procedures\nThe waterless, non-reactive decontamination procedure recom­\nmended in the prototype canine field decontamination kits uses a\nsequence of three microfiber towel types: (1) dry to initially remove\ngross contaminants from the fur (e.g., package labeled Wipe A Dry\nWipe); (2) wet which is saturated with a surfactant to clean away\nresidual contaminants on the fur (e.g., package labeled Wipe B Wet\nWipe); and, (3) dry to remove final contamination and residual sur­\nfactant (e.g., package labeled Wipe C Dry Wipe; Figure 1). The proce­\ndures recommended in the kit instructions were designed to transfer\ncontaminants from the canine’s fur to the microfiber towels while\nkeeping contaminants off and away from the canine’s skin.\nThis study was conducted in small groups at individual canine\nhandler team locations based on their schedule and availability. Each\nparticipating canine handler team was randomly assigned to one of\nthree possible form factors: single towel, multi-towel small, and multi-\ntowel large. Each handler was randomized into a trained or untrained\ncategory and one of the three possible form factor subcategories. The\ntrained group participants received approximately 15 min of interac­\ntive instruction from a research team member in addition to the kit\nuse instruction sheet and specific instruction on how to implement\nFIGURE 2\nApplication of contaminant. GloGerm™ MIST Spray, plastic stencil for uniform distribution of simulant, and camera with standoff for uniform image\nacquisition (left). Photo acquisition after “contamination” with GloGerm™ on the forelimb (right).\n\nBuntz et al.\b\n10.3389/fvets.2026.1770205\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nthe “pinch and pull” technique (Figure 3). The untrained group par­\nticipants received just the kit use instruction sheet to read at their own\npace with no additional interactive training from the research team.\nAll participants received identical instruction sheets.\n2.4 Image analysis\nAnalysis of photos was performed with FIJI image software (based\non ImageJ, originally developed by the National Institutes of Health).\nThe “particle count” tool was used with a mask subtraction technique\nto objectively quantify the quantity of fluorescent particles removed\nby the handlers using the prototype canine field decontamination kits.\nThree photos from the same dog and same anatomical location (one\npre-contamination photo, one post-contamination photo, and one\npost-decontamination photo) were captured and uploaded to FIJI. In\norder to measure particle count, these three images were cropped to\ninclude only the simulant contaminated region of interest (e.g.,\nexcluded the 1-cm fiducial for photographic scaling). These images\nwere then compiled into a ‘stack’ on FIJI and converted from color to\n8-bit black and white images. From there, the threshold of all images\nwas adjusted at once. The ‘pre’ image was selected to view as the\nthreshold is adjusted, as there should be no contamination visible on\nthe ‘pre-contamination’ image. The threshold was adjusted so that\nthere were no visible particles (e.g., dust and dander can fluoresce\nunder UV light) in the ‘pre-contamination’ images, and that same\nthreshold was applied to all the images in the stack. This allowed the\nimages to be analyzed by comparing the quantified particle count for\nthe ‘post-contamination’ and ‘post-decontamination’ images. The fluo­\nrescent particle counts were calculated by selecting ‘analyze particles’\non FIJI. This computes a fluorescence particle count for each image,\nand these values were recorded for further analysis to calculate the\npercent particle count decontamination. Similarly, the FIJI software\n\nThis computes a fluorescence particle count for each image,\nand these values were recorded for further analysis to calculate the\npercent particle count decontamination.Similarly, the FIJI software\n“size” tool was used with a mask subtraction technique to objectively\nmeasure the area (cm2) of fluorescent simulant reduction in the same\nimages. The results from the area reduction method did not differ\nsubstantially from the particle count method and were excluded from\nthis report in support of continued use of the quantitative particle\ncount method in this and future studies.\n2.5 Mitt form factor\nEarly in this investigation, a mitt form factor was evaluated.\nDespite positive feedback from end users, the mitt form factor was\nfound to be underperforming compared to the other form factors\n(single towel, multi-towel small, multi-towel large) and presented\nlogistical burdens in both manufacturing and utilization. Due to the\nstatic position on the hand, the surface area in direct contact with the\ndog’s coat was limited. Only the area between the fingertips and the\npalm on one side of the mitt made direct contact with the fur during\nthe “pinch and pull” method and the back of the mitt never contacted\nthe fur. The additional thickness of the mitt increased the size and\nweight of the kit which was an important consideration raised in feed­\nback from all end users. Due to poor early performance, difficulties in\nmanufacturing, and limited adaptability for use with different per­\nsonal protective equipment (PPE) and individual hand sizes, along\nwith the increase in kit size/weight, the mitt form factor was excluded\nfrom further canine handler team evaluation and from data analysis.\n2.6 Statistical analysis\nDescriptive statistics were computed for the percentage of decon­\ntamination for both “size” (area; cm2) calculated with the FIJI software\narea tool and “particle count” (FIJI software image subtraction tool)\nreduction considered across all variables (trained versus untrained,\ntowel form factors, anatomical location). Histograms and dot plots\n\nHistograms and dot plots\nwere constructed to visualize the distribution of percentage decon­\ntamination “particle count” and “size.” For each anatomical location\nand form factor combination, the distribution of the number of dogs\nin the quartile categories was compared between the trained and\nuntrained groups using Fisher’s Exact Test. Similarly, for each location\nFIGURE 3\nPinch and pull technique used with all wipes to remove contaminant without pushing it deeper to contact the skin.\n\nBuntz et al.\b\n10.3389/fvets.2026.1770205\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nand training group, the distribution of the number of dogs in the quar­\ntile categories was compared among the kit combinations using\nFisher’s Exact Test. When differences in either percentage “particle\ncount” or “size” decontamination between training groups were not\nfound, training groups were combined to compare the kit combina­\ntions at each location using Fisher’s Exact Test. A significance level of\n0.05 was used for all tests of significance. Data was also evaluated\nusing the above methods as “pass” versus “fail,” with <75% of contami­\nnant removed denoting a failure, in order to consider areas that were\noverlooked by the handler or inadequately decontaminated by the\nwipe procedures and not substantially contacted with the decontami­\nnation wipes.\n3 Results\nA total of 64 canine handler teams evaluated the prototype canine\nfield decontamination kits. Ten participants each in the untrained\nsingle towel and untrained multi-towel large categories and eleven\nparticipants each in the trained single towel, untrained and trained\nmulti-towel small, and trained multi-towel large categories. The means\nand standard deviations of percentage of decontamination based on\nparticle count for all kit combinations and both training groups are in\nTable 1. For all form factor combinations and trained and untrained\ngroups, the mean percentage decontamination particle count for the\nback was higher than the forelimb and the inguinal locations.\nThe number of observations that had 75–100% particle count\nreduction was largest for all kit form factors, training categories, and\nall anatomical locations (Table 2; Figures 4, 5). For all locations and\nboth training groups, there was not sufficient evidence that the distri­\nbution of dogs across the percentage particle count decontaminated\nquartiles differed among the three kit combinations (p-value range\n0.157 and 1.000; Table 3). There was not sufficient evidence that the\ndistribution of dogs exceeding the 75% pass criteria differed among\n\n157 and 1.000; Table 3).There was not sufficient evidence that the\ndistribution of dogs exceeding the 75% pass criteria differed among\nthe three kit combinations (p-value range 0.387 to 1.000; Table 3).\nAlso, there was not sufficient evidence that the distribution of dogs\nacross the percentage particle count decontaminated quartiles differed\nbetween the trained and untrained groups (p-value range 0.113 to\n1.000; Table 4) or that the distribution of dogs exceeding the 75% pass\ncriteria differed between the trained and untrained groups (p-value\nrange 0.387 to 1.000; Table 4).\n4 Discussion\n4.1 Anatomical location effectiveness\nThe highest decontamination efficacy based on mean particle\ncount reduction was noted on the back (91.38% ± 0.18). The guard\nhairs at this anatomical location are longer, which allows for more\nefficient performance of the recommended “pinch and pull” decon­\ntamination technique, are the most easily accessible for the handler,\nand have more intrinsic protective qualities when compared to other\nhair types. The forelimb and inguinal areas had less contaminant\nremoved across the form factors and both training groups. The fore­\nlimb had a mean fluorescent particle count reduction of 82.04% ± 0.26.\nDue to the short guard hairs at this anatomical location, decontamina­\ntion using the “pinch and pull” method is difficult and less practical in\na moving canine. Some handlers disregarded the recommended\n“pinch and pull” technique in the instructions and utilized a “circum­\nferential rub” technique. Observationally, this may have improved\ntheir efficiency of GloGerm™ removal and time spent decontaminat­\ning the limbs. This technique may be added to future kit instructions\nand dry wipe decontamination procedures. When spraying\nGloGerm™ on the shorter or thinner haired areas on the limbs of\nsome dogs, the GloGerm™ droplets penetrated the fur and directly\ncontacted the skin. This is noted because the goal is to avoid skin con­\ntact with the contaminant since this is the point at which the canine\ncould become poisoned. Also, contaminated skin is anticipated to be\n\nThis is noted because the goal is to avoid skin con­\ntact with the contaminant since this is the point at which the canine\ncould become poisoned.Also, contaminated skin is anticipated to be\nthe most challenging location to decontaminate because the overlying\nhair may block direct physical contact with the wipe.\nThe inguinal area had a mean reduction of 70.15% ± 0.31 for\nfluorescent particle count across all form factors and training groups.\nThis represents failed decontamination of this anatomical region\nbased on the 75% cutoff used in the study. The hairs in this anatomi­\ncal location are considered undercoat, lacking the protective quality\nof guard hairs present in other areas. The inguinal area is also the\nmost difficult to reach and most easily overlooked by the handler.\nAdditionally, the hairs vary in length and density and in some indi­\nvidual dogs there was GloGerm™ penetration to the skin. Some\nmembers of the canine community hypothesize the inguinal area is\nless likely to be contaminated due to its anatomically protected loca­\ntion. However, discussion with subject matter experts (SME) during\nmilitary decontamination training events provided observational\nevidence to the contrary. For example, a dog walking in tall grass or\nlying down on a surface will easily have droplets transfer from the\ncontaminated vegetation or surface to their ventrum. Peer-reviewed\nliterature has also shown the ventral surfaces of the dog are more\nlikely to be contaminated and more likely to be missed during wet\ndecontamination (16). While the Perry et al. (16) study focuses on\nenvironmental contamination from standing liquid hazards which\nmay differ from the dispersion of a chemical warfare agent, the find­\nings reinforce the need for proper decontamination of ventral ana­\ntomical areas. In other decontamination training events, lifting of the\nTABLE 1  The percentage of decontamination based on particle count for all kit combinations\nand both training groups.\nPercentage of decontamination based on particle\ncount\nCategory\nBack\nForelimb\nInguinal\nSingle towel\ntrained\n98.61%\n(±2.81)\n72.10%\n(±39.4)\n81.54%\n(±23.64)\nSingle towel\nuntrained\n\nPercentage of decontamination based on particle\ncount\nCategory\nBack\nForelimb\nInguinal\nSingle towel\ntrained\n98.61%\n(±2.81)\n72.10%\n(±39.4)\n81.54%\n(±23.64)\nSingle towel\nuntrained\n87.28%\n(±23.09)\n85.21%\n(±24.58)\n61.83%\n(±36.98)\nMulti-towel small\ntrained\n99.69%\n(±0.75)\n84.87%\n(±19.19)\n62.90%\n(±35.17)\nMulti-towel small\nuntrained\n88.51%\n(±22.67)\n72.36%\n(±31.06)\n82.38%\n(±30.60)\nMulti-towel large\ntrained\n78.52%\n(±32.67)\n70.09%\n(±34.24)\n74.32%\n(±32.02)\nMulti-towel large\nuntrained\n95.48%\n(±13.55)\n83.81%\n(±19.80)\n64.88%\n(±35.32)\n\nBuntz et al.\b\n10.3389/fvets.2026.1770205\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nTABLE 2  The number of observations of percentage of particle count reduction for all kit form factors, training categories, and all anatomical locations.\nForm factor\nTraining \ncategory\nLocation\nPercentage particle count reduction\n0–25%\n25–50%\n50–75%\n75–100%\nForm factor A\nTrained (N = 10)\nBack\n10\nForelimb\n2\n1\n7\nInguinal\n1\n3\n6\nUntrained (N = 11)\nBack\n1\n1\n9\nForelimb\n1\n1\n9\nInguinal\n3\n2\n1\n5\nForm factor C\nTrained (N = 11)\nBack\n11\nForelimb\n1\n3\n7\nInguinal\n3\n3\n5\nUntrained (N = 11)\nBack\n1\n1\n9\nForelimb\n1\n2\n2\n6\nInguinal\n1\n2\n8\nForm factor D\nTrained (N = 11)\nBack\n2\n1\n8\nForelimb\n2\n4\n5\nInguinal\n2\n1\n1\n7\nUntrained (N = 10)\nBack\n1\n9\nForelimb\n1\n2\n7\nInguinal\n2\n2\n1\n5\nFIGURE 4\nDot plot of individual decontamination results (percent reduction in particle count) across anatomical locations (back, forelimb, inguinal) and form \nfactors (single towel, multi-towel small, multi-towel large). Means represented by horizontal lines.\n\nBuntz et al.\b\n10.3389/fvets.2026.1770205\nFrontiers in Veterinary Science\n08\nfrontiersin.org\ncanine onto a table or tailgate improved the handler’s ability to access\nand decontaminate the ventrum and inguinal areas of the canine. For\nthese reasons, kit use instructions and training recommendations\nmust highlight frequently encountered human errors in order to opti­\nmize decontamination efficacy. Proper restraint techniques to ensure\nthe physical safety of the handler and other personnel assisting with\ncanine decontamination as well as proper wear of PPE must be\nemphasized to avoid contaminant transference to additional person­\nnel and equipment. Finally, sedation may be required in cases of\nmedically symptomatic or overtly aggressive canines to allow a han­\ndler to implement the “pinch and pull” and rub or wipe techniques\nin difficult to reach and often overlooked anatomical areas.\n4.2 Form factor effectiveness\nThere was no statistically significant difference between the single\ntowel, multi-towel small, and multi-towel large form factors in percent­\nage of fluorescent particle reduction, percentage of area reduction, or\nquartile reduction categories (e.g., 0–25, 26–50, 51–75, 76–100%).\nCombining objective data collection with handler and SME feedback\ninformed several kit improvement recommendations. For example, the\nmulti-towel large form factor was much larger in size and weight, and\nthere was not a significant improvement in handler decontamination\nefficacy, therefore this kit form factor was excluded from production\nconsideration. The larger towels were often noted to be unwieldy as\nhandlers tried to use the clean portions of the towel without cross con­\ntamination of the dog or themselves from unclean portions of the\ntowel. Handlers preferred to use and discard multiple small towels. The\nsingle towel and multi-towel small kits both had advantages: the single\ntowel was the most compact and lightweight of all the evaluated form\nfactors, giving it a significant advantage in handler feedback; however,\none towel was not adequate for whole body decontamination (e.g.,\n\ng.,\nquickly covered with dirt, loose fur) and shared similar cross contami­\nnation issues described for large towels. Mass animal decontamination\nin disaster response preparedness is a growing area of concern. The\nsingle large towel form factor may be the most beneficial in mass casu­\nalty scenarios where kits could be provided to pet owners prior to going\nthrough wet decontamination. This would allow for earlier contami­\nnant removal, decreased wicking of contaminants to the dog’s skin, and\ndecreased cross contamination. For individual canine handler use at\nthe point of exposure, the multi-towel small form factor has the advan­\ntage of using a separate towel for specific regions of the body to avoid\ntracking contamination to clean and sensitive areas. The multi-towel\nFIGURE 5\nHistogram depicting the decontamination results (percent reduction in particle count) across anatomical locations (back, forelimb, inguinal) and form\nfactors (single towel, multi-towel small, multi-towel large) separated into quartiles.\nTABLE 3  Fisher’s exact test p-values comparing the distribution of percentage particle count\nquartiles among the form factors for each location and training group found no significant\ndifferences.\nComparison among form factor particle count\nLocation\nTraining\ngroup\np-\nvalue\nAbove 75%\ndecontamination\nthreshold p-value\nBack\nTrained\n0.157\n0.091\nUntrained\n1\n1\nForelimb\nTrained\n0.193\n0.552\nUntrained\n0.757\n0.408\nInguinal\nTrained\n0.489\n0.742\nUntrained\n0.87\n0.449\n\nBuntz et al.\b\n10.3389/fvets.2026.1770205\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nsmall form factor was more favorably viewed by medical personnel for\nthis reason. Training handlers in a decontamination pattern based on\nanatomical region may improve the percentage removal of contami­\nnants. For example, use the first towel on the head and neck region and\ndiscard the towel, use the second towel on the back and discard, etc.\n4.3 Trained versus untrained effectiveness\nThere was no statistically significant difference found between the\n“trained” and “untrained” variables. The lack of significant difference\nbetween these two groups provides evidence that the dry wipe decon­\ntamination procedures are intuitive and that the written instructions\nwere clear, easy to understand, and easy to implement by working dog\nhandlers.\n4.4 Average time for kit use\nThe average time for decontamination using the prototype canine\nfield decontamination kit was 9 min 24 s (range = 3 min 4 s to 26 min\n0 s) across all handler backgrounds, trained and untrained groups, and\nkit form factors. However, handlers were not wearing full Mission\nOriented Protective Posture (MOPP) gear or PPE. This average time\ncan be used as a baseline for planning purposes in a hasty decontami­\nnation or limited exposure scenario. A significant time difference is\nexpected for decontamination in lethal environments with personnel\noperating in Joint Service Lightweight Integrated Suit Technology\novergarments or Advanced Chemical Protective Garments.\n4.5 Inter-study comparison\nWaterless decontamination with the prototype canine field decon­\ntamination kits used in this study and the “pinch and pull” technique\nwas developed and studied under laboratory conditions for the\nremoval of sulfur mustard (HD; blister agent) and venomous agent X\n(VX; nerve agent) from canine cadaver skin/fur samples (7). The\nresults of handler GloGerm™ decontamination efficacy on the canine\nback were similar to the live agent laboratory decontamination results.\nThe lower mean particle count reduction on the back by the handler\n\nThe\nresults of handler GloGerm™ decontamination efficacy on the canine\nback were similar to the live agent laboratory decontamination results.The lower mean particle count reduction on the back by the handler\n(91.38%) versus mean reduction of VX (97.9%) and HD (99.6%) from\ncanine cadaver fur samples may be attributed to the research team’s\nvisual observation of 11% of handlers omitting an area when\ndecontaminating the entire dog versus conditions in a controlled labo­\nratory setting where the location of contaminant was known. This\ninter-study comparison lends strength to the necessity and ability of a\ncanine handler to initiate decontamination at the point of exposure.\nAdditionally, the levels of effective decontamination demonstrated by\nthe handlers in this study provides evidence that untrained individuals\ncan effectively utilize the dry wipe decontamination method in a real­\nistic scenario.\n4.6 Limitations\nThe training guidelines were created early in the study design\nbased on published canine decontamination literature and laboratory\nstudies. As the study progressed, discussions with professional work­\ning dog personnel and observations of handler kit use and effective­\nness led to productive insights. This information was not implemented\nduring the study to avoid affecting data collection. However, new rec­\nommendations were discussed with handlers and SMEs after data col­\nlection at each testing site with favorable feedback. Additionally,\nhandlers were not required to wear full MOPP gear or PPE during\ndecontamination procedures. In real-world scenarios when wearing\nthis gear, it is expected that performing decontamination procedures\nand canine handling will increase in difficulty, increasing the time\nrequired to fully decontaminate a WD. Individual handler experiences\nwith other decontamination methods was unknown in this study.\nEach handler received instructions as described but then were allowed\nto conduct the decontamination procedure to their satisfaction with­\nout further guidance from the research team. Future experimental\n\nEach handler received instructions as described but then were allowed\nto conduct the decontamination procedure to their satisfaction with­\nout further guidance from the research team.Future experimental\ndesigns could evaluate handler-related variables like prior training and\ndeployment experience as well as the conditions necessary to improve\nefficiency and efficacy in trained decontamination procedures. This\nstudy included only German, Belgian, and Dutch Shepherd breeds.\nFuture research should include breeds with a wider variety of fur tex­\ntures and thicknesses.\n4.7 Advantages of field decontamination\nUsing the canine field decontamination kit allows for debulking\nprior to and in conjunction with soap and water decontamination\nprotocols. Dry wipe decontamination leverages the intrinsic protec­\ntive mechanisms of the canine’s fur coat, and prevents initial wetting\nTABLE 4  Fisher’s exact test p-values comparing the distribution of percentage particle count quartiles between trained and untrained groups for all locations and each form factor found no\nsignificant differences.\nComparison between trained and untrained groups particle count\nLocation\nForm factor\np-value\nAbove 75% decontamination threshold p-value\nBack\nSingle towel\n1\n0.476\nMulti-towel small\n0.476\n0.476\nMulti-towel large\n0.724\n0.587\nForelimb\nSingle towel\n0.672\n0.635\nMulti-towel small\n1\n1\nMulti-towel large\n0.329\n0.387\nInguinal\nSingle towel\n0.316\n0.67\nMulti-towel small\n0.113\n0.387\nMulti-towel large\n0.906\n0.67\n\nBuntz et al.\b\n10.3389/fvets.2026.1770205\nFrontiers in Veterinary Science\n10\nfrontiersin.org\nof the coat which has been shown to cause transport of the contami­\nnant to the skin and transdermal absorption. Field decontamination\ncompliments current water-based decontamination protocols by\nreducing aerosolization risks, reducing excessive contaminated water,\nand reducing environmental and cross contamination through its\nmore controlled collection of contaminants within the microfiber\ntowels. The contaminated wipes can then be disposed of as a solid\nwaste whereas soap and water wash decontamination methods create\na significant logistical challenge to dispose of up to 40 L of contami­\nnated water per canine (8). The dry wipe decontamination technique\nlimits contact with the skin and the prototype kit contains a proprie­\ntary surfactant (not a soap) proven to be non-irritating for both\ncanine and human skin. These factors limit the impact of decontami­\nnation on the natural skin barrier, making dry wipe decontamination\nmore suitable when repeated decontamination procedures are neces­\nsary (e.g., daily SAR work in a contaminated environment) (13, 21).\nThe prototype canine field decontamination kit has been designed\nand tested for single handler use, reducing stress on the canine and\npreventing additional personnel exposures. It allows for canine decon­\ntamination to be performed at the point of contamination and, when\npossible, away from large crowds and loud equipment. By reducing\nstress of the canine, a more thorough decontamination can be per­\nformed. This reduces the human health hazards associated with an\nincomplete decontamination of the canine, re-exposure of personnel\nto contaminants, PPE suit tear risks, and/or physical injury to the\ncanine or personnel.\nIn a mass contamination scenario, using the prototype canine\nfield decontamination kit, handlers can simultaneously decontami­\nnate their canines as opposed to a one at a time system of a wet soap\nand water lane. This allows early decontamination, early medical\nintervention, decreased cross contamination, and overall improve­\n\nThis allows early decontamination, early medical\nintervention, decreased cross contamination, and overall improve­\nment in canine and human outcomes and survivability. In civilian\ncanine contamination scenarios, field decontamination can be per­\nformed by untrained personnel like pet owners or disaster relief\nvolunteers.\n4.8 Future research and recommendations\nThe canine field decontamination kit is non-reactive, surfactant-\nbased, and may be effective for a variety of chemical, biological, and\nnuclear contaminants, but because of their inherent dangers, indi­\nvidual contaminant types must be specifically tested in controlled\nlaboratory studies. Individual training at the unit-level as well as com­\nplex multiagency events should include hands-on decontamination\nscenarios using GloGerm™ or other traceable simulants to allow\nhandlers to practice a variety of decontamination techniques while\nthey experience and discover their own lessons learned (e.g., circum­\nferential rub technique for the forelimb, elevation of the canine to\nreach the ventrum, a step-wise approach to the multiwipe kits, com­\nmonly missed anatomical areas) while improving their confidence,\nefficiency and efficacy.\n5 Conclusion\nThe use of a canine field decontamination kit can provide rapid\nand effective removal of contaminants without water and fills a\nneeds gap in current canine decontamination protocols. Personnel\nwith minimal training can effectively conduct dry wipe decontami­\nnation. A compact, lightweight, waterless kit allows the canine han­\ndler to initiate decontamination at the point of exposure, decreasing\ncanine absorption and transference risks, and allowing for decon­\ntamination prior to transportation to a wet decontamination site or\nmedical care. Implementation of field decontamination procedures\ncan significantly improve the prognosis for contaminated canine\nsurvival and lessen the risk of dangerous toxin transference to their\nhuman teammates.\nAuthor’s note\nThis manuscript uses “military working dog” as the encompassing\nterm for MWDs, Multi-Purpose Canines, and other categories of\n\nAuthor’s note\nThis manuscript uses “military working dog” as the encompassing\nterm for MWDs, Multi-Purpose Canines, and other categories of\nworking dogs employed by DOD components and interagency\npartners.\nData availability statement\nThe raw data supporting the conclusions of this article will be\nmade available by the authors, without undue reservation.\nEthics statement\nThe animal studies were approved by Colorado State University\nInstitutional Animal Care and Use Committee and the Defense Health\nAgency Animal Care and Use Review Office. The studies were con­\nducted in accordance with the local legislation and institutional\nrequirements. Written informed consent was obtained from the\nowners for the participation of their animals in this study.\nAuthor contributions\nRB: Visualization, Investigation, Writing  – review & editing,\nValidation, Writing – original draft, Methodology. MH: Validation,\nMethodology, Writing – review & editing, Investigation, Visualization.\nKB: Investigation, Writing – review & editing, Writing – original draft.\nJS: Data curation, Formal analysis, Methodology, Visualization,\nWriting – review & editing. KM: Project administration, Supervision,\nInvestigation, Writing – review & editing, Resources, Writing – origi­\nnal draft. BF: Conceptualization, Funding acquisition, Writing – origi­\nnal draft, Resources, Writing – review & editing.\nFunding\nThe author(s) declared that financial support was received for this\nwork and/or its publication. This material is based upon work sup­\nported by the U.S. Army Small Business Innovation Research Program\nand the Army Research Office under Contract No. W911NF-\n21-C-0024. The funder was not involved in the study design, collec­\ntion, analysis, interpretation of data, the writing of this article, or the\ndecision to submit it for publication.\n\nBuntz et al.\b\n10.3389/fvets.2026.1770205\nFrontiers in Veterinary Science\n11\nfrontiersin.org\nAcknowledgments\nThe authors thank all the kennel masters, canine trainers, canine\nowners, and handlers for their time, expertise and feedback as well as\nall the canine participants without whom this research would never\nbe possible.\nConflict of interest\nBF holds USPTO number 12,220,497 for “Materials and Methods\nfor Decontaminating Animal Integument” and is a Chief Scientist at\nTDA Research, Inc. RB, MH, KB, and KM are research veterinarians\nand technicians at Mantel Technologies, Inc., a distributor of the\nCanine Field Decontamination Kit. JS was employed by Sharp\nAnalytics LLC.\nGenerative AI statement\nThe author(s) declared that Generative AI was not used in the\ncreation of this manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. Any product\nthat may be evaluated in this article, or claim that may be made by its\nmanufacturer, is not guaranteed or endorsed by the publisher.\nAuthor disclaimer\nThe opinions, findings and conclusions or recommendations\nexpressed in this manuscript are those of the authors and do not nec­\nessarily reflect the views of the U.S. Army Small Business Innovation\nResearch Program or the Army Research Office.\nReferences\n1.\tCengiz S. Possible hazards and risks that search and rescue (SAR) dogs may face in\nCBRN incidents. Gümüşhane Üniversitesi Sağlık Bilimleri Dergisi. (2022) 11:300–7. doi:\n10.37989/gumussagbil.1003880\n2.\tClements J. 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+{"text": "【兽医文献】\n标题: Protective effects of chlorogenic acid against LPS-induced intestinal oxidative injury in mice via activation of the PI3K/Akt-Nrf2/HO-1 signaling axis\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nTYPE  Original Research\nPUBLISHED  20 July 2026\nDOI  10.3389/fvets.2026.1870669\nOPEN ACCESS\nEDITED BY\nLauren Virginia Schnabel,\nNorth Carolina State University,\nUnited States\nREVIEWED BY\nKimberly Young,\nNorth Carolina State University,\nUnited States\nMichala De Linde Henriksen,\nNorth Carolina State University,\nUnited States\n*CORRESPONDENCE\nAmanda B. Davis\nDavis.8097@osu.edu\nRECEIVED 01 May 2026\nREVISED 17 June 2026\nACCEPTED 30 June 2026\nPUBLISHED 20 July 2026\nCITATION\nDavis AB, Stewart ML and\nHickox AA (2026) Safety of\nsubconjunctival allogeneic platelet-rich\nplasma on normal canine eyes.\nFront. Vet. Sci. 13:1870669.\ndoi: 10.3389/fvets.2026.1870669\nCOPYRIGHT\n© 2026 Davis, Stewart and Hickox. This\nis an open-access article distributed\nunder the terms of the Creative\nCommons Attribution License (CC BY).\nThe use, distribution or reproduction in\nother forums is permitted, provided the\noriginal author(s) and the copyright\nowner(s) are credited and that the\noriginal publication in this journal is\ncited, in accordance with accepted\nacademic practice. No use, distribution\nor reproduction is permitted which does\nnot comply with these terms.\nSafety of subconjunctival\nallogeneic platelet-rich plasma on\nnormal canine eyes\nAmanda B. Davis *, Mia L. Stewart  and Abby A. Hickox\nDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University,\nColumbus, OH, United States\nPurpose: To determine the safety and tolerance of an allogeneic, pooled freeze-\ndried platelet-rich plasma (PRP; VetStem PrecisePRP™ Canine) product admin­\nistered via subconjunctival injection two weeks apart in clinically normal canine\neyes.\nMethods: Allogeneic PRP was reconstituted with sterile water. With topical anes­\nthesia and gentle manual restraint of each dog, 0.2 mL (500 × 106 platelets/mL)\nof the reconstituted preparation was injected under the dorsal bulbar conjunc­\ntiva of the right eye. The procedure was repeated with 0.2 mL of sterile saline\n(control) under the dorsal bulbar conjunctiva of the left eye. A complete blood\ncount, chemistry profile, and comprehensive physical and ophthalmic examina­\n\nThe procedure was repeated with 0.2 mL of sterile saline\n(control) under the dorsal bulbar conjunctiva of the left eye.A complete blood\ncount, chemistry profile, and comprehensive physical and ophthalmic examina­\ntions were conducted on all dogs at multiple time points to monitor for potential\nadverse reactions.\nResults: No significant adverse effects associated with physical examination,\nhematologic or ocular parameters were noted at any time point during the\nstudy. All allogeneic PRP treated eyes developed a mild to moderate conjunctival\nhyperemia and chemosis that self-resolved within 48–72 h. Additionally, one eye\ndeveloped a mild periocular hyperemia that self-resolved within 72 h. No adverse\nreaction was noted in any saline treated eye.\nConclusion: Allogeneic PRP administered via subconjunctival injection in normal\ncanine eyes appears to be safe and well-tolerated with only transient conjunctival\nhyperemia and chemosis noted. This study allows for further investigation into\nthe clinical efficacy of subconjunctival allogeneic PRP for canine ocular surface\ninflammatory disorders.\nKEYWORDS\nallogeneic platelet-rich plasma, canine, platelet-rich plasma injection, subconjunctival\ninjection, veterinary ophthalmology\nIntroduction\nOcular surface diseases, such as keratoconjunctivitis sicca, are frequently encountered in\nveterinary patients and may leave the ocular surface particularly susceptible to chronic inflam­\nmation, immune activation/dysregulation, and tissue damage (1, 2). Most therapeutic interven­\ntions for these inflammatory disorders are palliative in nature; therefore, strategies aimed at\nmodulating and repairing the local immune environment may offer significant benefit for these\ncommon and occasionally unmanageable conditions.\nIn recent years, platelet-rich plasma (PRP) has emerged as a promising therapeutic option\nin both human and veterinary medicine for the management of a broad range of conditions.\nPRP is the plasma fraction of whole blood with a platelet concentration above physiologic\n\nDavis et al.\b\n10.3389/fvets.2026.1870669\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nbaseline (3). It contains serum, platelets, leukocytes, and an array of\ngrowth factors, including vascular endothelial growth factor (VEGF),\nplatelet-derived growth factor (PDGF), transforming growth factor-\nbeta (TGF-β), epidermal growth factor (EGF), insulin-like growth\nfactor (IGF), and fibroblast growth factor (FGF) (4, 5). These bioactive\nmolecules promote collagen synthesis, angiogenesis, cytokine altera­\ntion, epithelial differentiation, endothelial cell proliferation, and other\nreparative processes (4–6).\nDue to its regenerative and anti-inflammatory properties, PRP has\nbeen heavily utilized in the treatment of musculoskeletal disorders;\nhowever, PRP treatment of the ocular surface has been and continues\nto be heavily investigated. PRP can be administered in many ophthal­\nmic forms such as topical drops, subconjunctival injections, and clot\napplication which has a particular focus in the treatment of corneal\nand ocular surface disorders (4, 7). Preliminary evidence supports the\nuse of PRP in the treatment of dry eye disease, corneal chemical and\nthermal injuries, epithelial defects, keratitis, and corneal ulceration in\nboth veterinary and human patients (4, 5, 8, 9). A recent systematic\nreview of PRP within the veterinary ophthalmic literature notes that\nPRP is associated with a reduction of inflammation in corneal healing\nand animal ocular surface disease (OSD) scores; however, there are\nlimitations in study design and variability in PRP preparation (10).\nPRP can be derived autologously (from the patient) or allogenei­\ncally (from a donor of the same species). Autologous PRP has been\nwidely studied and shown to be safe, inexpensive, and effective in\nregenerative applications (4, 8, 11). However, significant variability\nexists in autologous PRP preparation methods, including differences\nin anticoagulants, centrifugation practices, and collection volumes.\nThese variables affect platelet and leukocyte concentrations and con­\nsequently alter the therapeutic potential of the final PRP product (4,\n\nThese variables affect platelet and leukocyte concentrations and con­\nsequently alter the therapeutic potential of the final PRP product (4,\n5, 12). Even commercially available PRP preparation systems lack\nstandardization across manufacturers (13). Additionally, intrinsic\npatient factors, such as hydration status, medication use, age, etc. and\nvariation in protocol may further influence final platelet yield (4, 14).\nThis lack of consistency is one of the primary limitations in under­\nstanding the efficacy of autologous PRP in both human and veterinary\nstudies (5). Processing time also creates practical challenges as prepa­\nration of autologous PRP commonly requires up to one hour. This may\nbe impractical in a high-volume clinical setting (11).\nAllogeneic PRP, while less extensively researched, has demon­\nstrated comparable safety and efficacy profiles in the management of\nmusculoskeletal conditions (4, 5, 15–19). Allogeneic formulations\noffer advantages over autologous, including a consistent product with\nknown platelet concentrations, improved quality control, reduction in\npatient stress and visit times, and the potential for higher and more\nstandardized platelet and growth factor concentrations. Notably, one\nhuman study reported significantly higher platelet counts and at least\ntwo-fold greater growth factor levels in allogeneic PRP compared to\nautologous PRP (20).\nAn FDA-reviewed, leucoreduced, canine allogeneic PRP product\n(VetStem PrecisePRP™ Canine) is already available to licensed vet­\nerinarians and has undergone thorough safety and quality testing for\nintra-articular use (21). Donor dogs are screened according to criteria\nfrom the Food and Drug Administration CVM Guidance #254. The\nproduct is shipped in freeze-dried vials, allowing convenient storage\nand reconstitution. An allogeneic PRP product offers the advantage of\na standard composition and consistent quality of platelets to every\npatient, which allows wider access to this treatment option and\nimproves its practicality in clinical use.\nNumerous allogeneic PRP formulations have been evaluated as\n\nNumerous allogeneic PRP formulations have been evaluated as\ntopical ophthalmic preparations in both human and veterinary models\nto assess their tolerability and efficacy in the management of various\nocular surface disorders (22–28). Allogeneic PRP eye drops do not\nappear to induce adverse reactions due to their low immunogenicity.\nHowever, PRP treatment often requires frequent administration (4–6\ntimes daily) over multiple weeks to achieve the desired effect (29, 30).\nAt this frequency, a topical ophthalmic PRP preparation may be\nimpractical for many veterinary clients, resulting in low compliance\nand inconsistent long-term results.\nA subconjunctival route of PRP administration may alleviate\ntreatment variability and reduce the degree of home care required.\nAdditionally, a subconjunctival approach may enable more sustained\ndelivery to the intended site and enhance regenerative potential\nthrough a depot-like effect that increases the local bioavailability of\nimportant growth factors (31, 32). Autologous subconjunctival PRP\nhas already shown efficacy for a variety of ophthalmic disorders (7, 8,\n33, 34). For the reasons discussed above, an allogeneic product may\nprovide superior benefit; however, the safety of allogeneic PRP\ninjected into the subconjunctival space has yet to be evaluated. The\npurpose of this study is to verify the safety and tolerance of an alloge­\nneic PRP product (VetStem PrecisePRP™ Canine) injected into the\nsubconjunctival space of normal canine patients. Due to its low\nimmunogenicity and proven tolerance via other routes, we hypothe­\nsize that allogeneic PRP is safe with no significant adverse effects\ndemonstrable when administered in this manner. Ultimately, if well-\ntolerated, this would translate into evaluating the efficacy of subcon­\njunctival allogeneic PRP for the treatment of a variety of canine ocular\nsurface conditions.\nMaterials and methods\nAll care and use of research animals in this study was approved\nand monitored by the Ohio State University Institutional Animal Care\nand Use Committee (IACUC; protocol #2025A00000066).\n\nMaterials and methods\nAll care and use of research animals in this study was approved\nand monitored by the Ohio State University Institutional Animal Care\nand Use Committee (IACUC; protocol #2025A00000066).\nAll examination parameters were obtained and documented, and\nall injections were performed by a board-certified ophthalmologist\n(AD) or an ophthalmology resident (AH) under direct supervision of\na board-certified ophthalmologist (AD). For this study, four healthy,\n1-year-old intact female mixed large breed dogs were obtained and\ngroup-housed with two dogs to an enclosure in a climate-controlled\nenvironment. All dogs underwent comprehensive baseline physical\nexaminations, including assessment of temperature, heart rate, respi­\nratory rate and mucous membrane color with capillary refill time.\nAdditionally, complete blood counts and serum chemistry profiles\nwere obtained for all dogs. A complete ophthalmic examination was\nperformed to include menace response, dazzle and pupillary light\nreflexes, Schirmer tear testing (Merck Animal Health, Madison, NJ,\nUSA), fluorescein staining (JorVet, Loveland, CO, USA), tonometry\n(Tonometer, Dan Scott and Associates, Westerville, OH, USA), slit-\nlamp biomicroscopy (SL-17 Kowa Company Torrance, CA, USA) and\nindirect ophthalmoscopy. Dogs were excluded if they had evidence of\nophthalmic or systemic disease.\nAllogeneic, pooled freeze-dried platelet-rich plasma (PRP;\nVetStem PrecisePRP™ Canine) was reconstituted with 8 mL of sterile\nwater as per label instructions. Approximately 0.1–0.2 mL of 0.5%\nproparacaine hydrochloride ophthalmic solution (Alcon Laboratories,\n\nDavis et al.\b\n10.3389/fvets.2026.1870669\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nFort Worth, TX, USA) was administered topically to both eyes of each\ndog. Subsequently, 0.2 mL (500 × 106 platelets/mL) of the reconsti­\ntuted PRP preparation was drawn through a 22-gauge needle into a\n1 mL syringe following gentle agitation. A 27-gauge needle was\napplied to the 1 mL syringe and, with gentle manual restraint of the\ndog, the platelets were injected just under the dorsal bulbar conjunc­\ntiva in the subconjunctival space of the right eye. The procedure was\nrepeated for the left eye; however, 0.2 mL of sterile saline was admin­\nistered into the dorsal bulbar subconjunctival space in place of PRP to\nserve as a control.\nFull physical and ophthalmic examinations were performed in\nthe same manner as baseline examinations at one hour and twenty-\nfour hours post-injection. Standardized clinical photographs were\nobtained of all eyes. If any notable ocular adverse effects were still\nnoted, slit-lamp biomicroscopic examination and photographs were\nrepeated daily until clinical signs were resolved. Any adverse effects\nwere graded according to the semi-quantitative preclinical ocular\ntoxicology scoring criteria (SPOTS) generally accepted for stan­\ndardizing slit lamp biomicroscopic findings in laboratory species\n(35). Daily observation was performed to assess general health\n(including temperature, heart rate and respiration), appetite, and\nattitude.\nFourteen days after the first injection, a comprehensive physi­\ncal examination and ophthalmic examination, identical to the\nbaseline examination, was repeated. A second injection was com­\npleted in the same manner as described above (0.2 mL PRP sub­\nconjunctival injection in the right eye and 0.2 mL sterile saline\nsubconjunctival injection in the left eye) for each dog with gentle\nrestraint and topical anesthesia. Full physical and ophthalmic\nexaminations were performed at one hour and at minimum, one\nday post-injection. Standardized clinical photographs were\nobtained of all eyes. If any notable ocular adverse effects remained\n\nFull physical and ophthalmic\nexaminations were performed at one hour and at minimum, one\nday post-injection.Standardized clinical photographs were\nobtained of all eyes.If any notable ocular adverse effects remained\npresent, slit-lamp biomicroscopic examination and photographs\nwere repeated daily until clinical signs were fully resolved.\nComprehensive bloodwork (complete blood count and chemistry\nprofile) was repeated one day after the second injection. Daily\nobservation was performed to assess general health (including\ntemperature, heart rate and respiration), appetite, and attitude\nuntil 7 days after the second injection, at which point the study was\nconcluded.\nResults\nNo clinically significant, serious adverse effects were observed\nat any time point. (0/4; exact 95% CI 0–57%) Physical examina­\ntion findings and hematologic parameters remained within\nnormal limits throughout the study period. All dogs developed a\ndiffuse, nonpainful chemosis and conjunctival hyperemia in the\nPRP-treated eye within one hour of both the first and second\ninjections (Figure 1). For all dogs (4), both conjunctival hyper­\nemia and chemosis/conjunctival swelling were given a score of 2\naccording to the SPOTS criteria (35). The degree of chemosis and\nconjunctival hyperemia did not differ between the first and\nsecond injections. In three out of four dogs, the chemosis and\nhyperemia fully resolved within 48 h without any intervention\n(Figures 2, 3). In one dog, periocular hyperemia, most prominent\nnasally, was noted in addition to chemosis and conjunctival\nhyperemia (Figure 4). This fully resolved by 72 h post-injection\nwithout any intervention. No signs of ocular discomfort as defined\nby blepharospasm, discharge, pawing or rubbing at the eye or\nbehavioral changes were noted at any time point. All other oph­\nthalmic findings (including Schirmer tear testing, fluorescein\nstain testing, tonometry, slit-lamp biomicroscopy and indirect\nophthalmoscopy) were normal at all time points. No adverse\neffects were noted at any time point in the saline-treated eye.\nFIGURE 1\n\nNo adverse\neffects were noted at any time point in the saline-treated eye.FIGURE 1\n(A,B) Representative images of mild to moderate diffuse chemosis\nand hyperemia noted in all dogs one hour after the first PRP\nsubconjunctival injection (A) and similarly, one hour after the second\nPRP subconjunctival injection performed 2 weeks later (B).\nFIGURE 2\nRepresentative images (A,B) of mild chemosis and hyperemia that\npersisted 24 hours post-PRP injection. Note that PRP injection was\ngiven in the subconjunctival space of the right eye only. The left eye\nreceived an equal volume of sterile saline.\nFIGURE 3\nRepresentative image of the right eye 48 h after subconjunctival PRP\ninjection. Note complete resolution of the chemosis and hyperemia.\nThree out of four dogs developed complete resolution at this time\npoint with one dog showing complete resolution of clinical signs by\n72 h post-PRP injection.\n\nDavis et al.\b\n10.3389/fvets.2026.1870669\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nDiscussion\nIn this preliminary safety study, repeated subconjunctival admin­\nistration of allogeneic, pooled freeze-dried platelet-rich plasma (PRP;\nVetStem PrecisePRP™ Canine) appears to be well-tolerated by normal\ndogs. Diffuse conjunctival hyperemia and chemosis were noted in all\neyes receiving PRP injections; however, this biologic reaction to PRP\nappeared nonpainful with no signs of discomfort noted at any time\npoint after injection. Additionally, conjunctival hyperemia and che­\nmosis resolved within 48 to 72 h post-injection in all dogs without\nnecessitation of further intervention.\nAllogeneic PRP theoretically carries increased risk of undesired\ninflammatory or hypersensitivity reactions compared to autologous\nPRP. Previous studies have evaluated allogeneic PRP to classify its\nimmunogenic potential. An in vitro study by Papait et al. (36) pro­\nposed that allogeneic PRP elicits a minimal immunogenic response by\ninducing monocyte differentiation and promoting an anti-inflamma­\ntory microenvironment. The immunogenicity of allogeneic PRP was\nfurther studied in vivo by Zhang et al. (16). Rabbits receiving local,\nintramuscular injections of allogeneic PRP did not exhibit a severe or\nchronic immune response, as demonstrated by assessment of periph­\neral blood lymphocyte populations and histological evaluation of\nmuscle tissue at the injection site. Further studies have verified the\nclinical tolerability and efficacy of allogeneic PRP for its use in a vari­\nety of veterinary and human conditions such as chronic wounds and\ninflammatory orthopedic diseases and found no adverse reactions\n(37–41). The low immunogenicity of local, targeted allogeneic PRP\ntherapy is thought to be due to a variety of factors including minimal\ninteraction with host antibodies, degradation of PRP over several\nweeks leaving little opportunity for development of a chronic immune\nresponse, and possible modification of platelet antigen structure\nduring platelet activation (37, 41).\nThe transient changes observed likely represent an expected, mild\n\nThe transient changes observed likely represent an expected, mild\ninflammatory response to the biologic activity of PRP, rather than an\nadverse pathologic reaction (42). The findings in the present study are\nconsistent with those of previous reports investigating adverse reac­\ntions associated with allogeneic PRP therapy. Garbin et al. (18) per­\nformed a safety analysis of freeze-dried allogeneic PRP in normal\nequine joints and noted a mild, but significant self-limiting inflamma­\ntory response in synovial fluid defined by an increase in nucleated cell\ncounts, prostaglandin E2, and total protein when compared to autolo­\ngous PRP. This did not cause lameness or other overt adverse clinical\nsigns. Additionally, allogeneic PRP has been utilized for intra-articular\nuse in canine patients with no apparent adverse effects noted in both\nlaboratory and clinical patients (21). More recently, Kooy et al. (43)\ncompared local and systemic effects of a single intra-articular injection\nof equine leucoreduced allogeneic pooled freeze-dried PRP to a pla­\ncebo control (saline) in normal, healthy equine joints. No differences\nbetween groups for joint swelling, joint circumference, heat scores or\npassive flexion were noted post-injection. Topical allogeneic PRP eye\ndrops have also been successfully used in both human and veterinary\nspecies for the treatment of various ocular surface conditions, most\nimportantly, dry eye disease, with no significant adverse effects noted\n(10, 23, 25–27).\nInterestingly, autologous PRP has been shown to cause similar,\nmild adverse reactions to those described for allogeneic PRP. A sys­\ntematic review of the current literature by Cardona-Ramirez et al. (44)\nevaluated 18 studies utilizing PRP for intra-articular use in canines.\nWhile there was variability in study design and PRP composition, all\nstudies noted improvement in function and pain post-PRP intra-\narticular injection. Six of these studies reported adverse effects to\ninclude self-limiting local pain, inflammation and lameness that\nresolved 48–72 h post-injection.\n\nSix of these studies reported adverse effects to\ninclude self-limiting local pain, inflammation and lameness that\nresolved 48–72 h post-injection.\nSubconjunctival autologous PRP injections performed in canine\npatients for various ocular surface conditions have demonstrated no\nserious adverse effects (7, 8, 33, 34). Similar findings have been\nreported for subconjunctival autologous PRP injections in human\npatients with dry eye disease; however, a small number of cases exhib­\nited subconjunctival hyperemia or hemorrhage, similar with the\nobservations in this study (31, 45). Additionally, mild ocular irritation\nhas been reported with both injection and topical application of autol­\nogous PRP for human use (31, 46).\nSubconjunctival injection may be a preferred route of ocular\nadministration in veterinary species due to a variety of factors. Topical\nadministration is most effective when performed frequently (4–6\ntimes daily) over at least several weeks (29, 30) which is impractical\nfor many pet owners. Subconjunctival administration can provide\nmore concentrated PRP delivery, which has been shown to have faster\nand more significant improvement in dry eye disease in humans when\ncompared to a topical PRP formulation (31). An allogeneic PRP prep­\naration may also provide more consistent dosing with a higher con­\ncentration of platelets and associated growth factors compared to\nautologous PRP. Therefore, subconjunctival administration of alloge­\nneic PRP may, in theory, provide a better formulation and delivery\nmethod, thereby enhancing its overall efficacy for ophthalmic use.\nAdditionally, subconjunctival injections are generally well tolerated by\nveterinary patients. In our study population, only minimal physical\nrestraint and topical anesthesia were required to perform an injection.\nThis observation is consistent with the authors’ clinical experience;\nhowever, select patients may require light sedation based on\ntemperament.\nMore recently, subconjunctival injection of autologous platelet-\nrich fibrin (PRF) for the treatment of severe dry eye disease has been\n\nMore recently, subconjunctival injection of autologous platelet-\nrich fibrin (PRF) for the treatment of severe dry eye disease has been\nstudied in humans (47). PRF may provide a more sustained benefit\nwhen compared to PRP due to the gradual release of bioactive factors\nthrough a fibrin matrix. To the authors’ knowledge, an allogeneic PRF\nproduct is not commercially available. Additionally, PRF must be\ninjected within the first 15–20 min following a reduced centrifugation\nprocess, while it remains in its liquid state prior to polymerization.\nFIGURE 4\nPhotograph of one dog that developed periocular hyperemia one\nhour post-PRP injection in the subconjunctival space of the right eye.\nAdditionally, consistent with the other dogs, is evidence of chemosis\nand conjunctival hyperemia of the right eye. All clinical signs,\nincluding periocular hyperemia, resolved without treatment within\n72 h post-injection.\n\nDavis et al.\b\n10.3389/fvets.2026.1870669\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nDue to these limitations, PRP was chosen over PRF for the purposes\nof this study and future clinical applications.\nWhile results of this study show promise in the safety of canine\nsubconjunctival allogeneic PRP, there are several limitations worth\nnoting. The sample size in this study was small, and inclusion of a larger\nnumber of normal dogs may have demonstrated unanticipated, adverse\noutcomes not identified in the current study. Additionally, this was an\nopen-label study, and both examiners were aware of which eye received\nPRP vs. saline. This may have inadvertently introduced a potential for\nbias into the measured outcome parameters. However, all efforts were\nmade to use objective criteria and standardized clinical photographs\nwhen evaluating eyes. Importantly, this study evaluated reactions to\nsubconjunctival PRP injections in normal eyes. It is possible that the\nminimal adverse effects seen in this study may be more pronounced in\ncanine patients who already have evidence of ocular surface inflamma­\ntory disease causing hyperemia and irritation. Therefore, translating\nthis data to clinical patients with underlying ocular inflammatory dis­\nease may be challenging. Further classification of the transient, inflam­\nmatory reaction noted in this study may be useful in understanding the\ntype of reaction caused by PRP. Objective data such as conjunctival\ncytokine analysis could provide more insight into the type of inflamma­\ntory reaction observed post-injection and is a future consideration.\nIn conclusion, allogeneic PRP is safe, technically straightforward,\nand well-tolerated when delivered as subconjunctival injections admin­\nistered 2 weeks apart in normal canine eyes. No significant adverse\neffects were noted other than a non-painful, transient, mild to moder­\nate conjunctival hyperemia and chemosis. Findings from this safety\nstudy support future clinical investigation into the efficacy of allogeneic\nPRP for the treatment of a variety of chronic canine ocular surface\n\nFindings from this safety\nstudy support future clinical investigation into the efficacy of allogeneic\nPRP for the treatment of a variety of chronic canine ocular surface\ninflammatory diseases, further highlighting its potential as a safe and\nuseful regenerative therapeutic option in veterinary ophthalmology.\nData availability statement\nThe raw data supporting the conclusions of this article will be\nmade available by the authors, without undue reservation.\nEthics statement\nThe animal study was approved by Ohio State University\nInstitutional Animal Care and Use Committee. The study was con­\nducted in accordance with the local legislation and institutional\nrequirements.\nAuthor contributions\nAD:\nConceptualization,\nData\ncuration,\nInvestigation,\nMethodology, Project administration, Writing  – original draft,\nWriting – review & editing. MS: Writing – original draft, Writing –\nreview & editing. AH: Data curation, Investigation, Project adminis­\ntration, Writing – review & editing.\nFunding\nThe author(s) declared that financial support was received for this\nwork and/or its publication. AD received start-up funding from The\nOhio State University College of Veterinary Medicine.\nConflict of interest\nThe author(s) declared that this work was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declared that Generative AI was not used in the\ncreation of this manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. 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+{"text": "【兽医文献】\n标题: Creating partnerships between veterinarians and underserved producers: getting started with establishing veterinary client–patient relationships\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nEvaluation of clopidogrel\nresponsiveness using the Platelet\nFunction Analyzer-200 (PFA-200)\nin dogs\nJoon-Ho Shin 1 and Hyun-Jung Han 1,2*\n1 Department of Veterinary Emergency and Critical Care Medicine, College of Veterinary Medicine,\nKonkuk University, Seoul, Republic of Korea, 2 KU Center for Animal Blood Medical Science, Konkuk\nUniversity, Seoul, Republic of Korea\nWe aimed to evaluate the prevalence of clopidogrel resistance in hypercoagulable\ndogs using the Platelet Function Analyzer-200 (PFA-200) P2Y cartridge; further,\nwe aimed to assess the utility of hematocrit (HCT), platelet count (PLT), prothrombin\ntime (PT), activated partial thromboplastin time (aPTT), thromboelastography\n(TEG) parameters, and D-dimer level as indicators of clopidogrel efficacy. Forty\nhealthy dogs underwent single measurements of P2Y closure time (CT), HCT, PLT,\nPT, aPTT, TEG parameters, and D-dimer levels, while thirty hypercoagulable dogs\nunderwent two measurements of these parameters before and after clopidogrel\ntreatment. The reference interval for P2Y CT in healthy dogs was 40.0–141.5 s, with\na mean of 63.9 ± 26.82 s. Hypercoagulable dogs showed a mean baseline P2Y CT\nof 77.4 ± 37.6 s. Moreover, 23 (76.67%) and 7 (23.33%) showed responsiveness and\nresistance to the initial clopidogrel dose, respectively. The mean P2Y CT of the\nclopidogrel-resistant group after clopidogrel administration was 182.71 ± 78.43 s.\nIncreasing the maintenance dose successfully overcame clopidogrel resistance in\nthese seven dogs. Among the assessed parameters, only D-dimer levels showed\na significant decrease in the clopidogrel-responder group (p < 0.05), suggesting\nits potential utility in evaluating responsiveness. In conclusion, the PFA-200 P2Y\ncartridge effectively detects clopidogrel resistance in dogs and can guide therapeutic\nadjustments such as dose escalation.\nKEYWORDS\nPlatelet Function Analyzer, PFA, P2Y cartridge, clopidogrel resistance, dogs\n1 Introduction\nClopidogrel is a common antiplatelet agent in small animal practice and a second-\n\nKEYWORDS\nPlatelet Function Analyzer, PFA, P2Y cartridge, clopidogrel resistance, dogs\n1 Introduction\nClopidogrel is a common antiplatelet agent in small animal practice and a second-\ngeneration thienopyridine ADP (adenosine diphosphate) receptor (P2Y12) antagonist that\nrequires hepatic activation via cytochrome P450-dependent oxidation (1). In humans,\nclopidogrel response variability has been widely investigated (2, 3). Additionally, clopidogrel\nresponse variability has been demonstrated in dogs and cats (4–7). Accordingly, intended\ntherapeutic outcomes may not be consistently achieved, and thus it is important to monitor\ntreatment efficacy. In humans, platelet function tests identify failure to achieve the expected\nantiplatelet effect of clopidogrel (clopidogrel resistance) (3). In humans, clopidogrel resistance\nis common, with prevalence rates of 4–30% (3). Although there is limited veterinary research\non clopidogrel resistance, studies have identified clopidogrel resistance in dogs using\nthromboelastography (TEG) with platelet mapping and multiplate, as well as in cats using light\ntransmission aggregometry (LTA), Platelet Function Analyzer (PFA)-100, and multiplate (4–7).\nOPEN ACCESS\nEDITED BY\nValeria Pasciu,\nUniversity of Sassari, Italy\nREVIEWED BY\nCorrin John Boyd,\nMurdoch University, Australia\nDale Adam Lauver,\nMichigan State University, United States\n*CORRESPONDENCE\nHyun-Jung Han\nab1234@konkuk.ac.kr\nRECEIVED 01 April 2025\nACCEPTED 16 June 2025\nPUBLISHED 11 July 2025\nCITATION\nShin J-H and Han H-J (2025) Evaluation of\nclopidogrel responsiveness using the Platelet\nFunction Analyzer-200 (PFA-200) in dogs.\nFront. Vet. Sci. 12:1595147.\ndoi: 10.3389/fvets.2025.1595147\nCOPYRIGHT\n© 2025 Shin and Han. This is an open-access\narticle distributed under the terms of the\nCreative Commons Attribution License\n(CC BY). The use, distribution or reproduction\nin other forums is permitted, provided the\noriginal author(s) and the copyright owner(s)\nare credited and that the original publication\nin this journal is cited, in accordance with\naccepted academic practice. No use,\ndistribution or reproduction is permitted\n\nNo use,\ndistribution or reproduction is permitted\nwhich does not comply with these terms.\nTYPE  Original Research\nPUBLISHED  11 July 2025\nDOI  10.3389/fvets.2025.1595147\n\nShin and Han\b\n10.3389/fvets.2025.1595147\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nThe PFA-100/200 system (Siemens Healthcare, Germany) is a\ncartridge-based point-of-care assay that evaluates platelet function in\nhigh-shear-stress conditions by measuring the closure time (CT) of a\nmembrane aperture (Figure 1). The PFA P2Y cartridge can assess the\nantiplatelet effects of clopidogrel. Although the current gold standard\nfor platelet function testing is LTA, the PFA is simpler and faster, and\nthus more suitable for in-hospital testing (8). Additionally, compared\nto TEG with platelet mapping, the PFA is more cost-effective and\noffers quicker assessment.\nWe aimed to establish a reference interval (RI) for P2Y CT in\nhealthy dogs and observe changes in P2Y CT following clopidogrel\nadministration in order to assess clopidogrel responsiveness in dogs.\nAdditionally, we evaluated the utility of conventional coagulation-\nrelated markers, including hematocrit (HCT), platelet count (PLT),\nprothrombin time (PT), activated partial thromboplastin time\n(aPTT), TEG parameters, and D-dimer level, as indicators of\nclopidogrel efficacy.\n2 Materials and methods\n2.1 Animals\nThis prospective study included hypercoagulable dogs diagnosed\nvia TEG at Konkuk Veterinary Medical Teaching Hospital and\nhealthy donor dogs from the KU I’M DOgNOR Blood Donation\nCenter. Ethical approval was granted by the Institutional Animal\nCare and Use Committee at Konkuk University, under approval\nnumber [KU23191, KU24249]. Dogs exhibiting hypercoagulability,\ndefined by TEG parameters with a maximum amplitude (MA) > 69\n(9, 10) and a coagulation index (CI) > 4 (11), were included.\nExclusion criteria included the use of drugs that could potentially\ninterfere with P2Y CT, including antiplatelet agents such as\nclopidogrel, aspirin, cilostazol, and tirofiban; anticoagulants such as\nheparin, rivaroxaban, and warfarin; and thrombolytic agents such as\nstreptokinase, as well as a history of bleeding disorders or a platelet\ncount of <100 × 109/L. The healthy group comprised dogs without\nunderlying diseases and normal findings in complete blood count,\n\nThe healthy group comprised dogs without\nunderlying diseases and normal findings in complete blood count,\nelectrolytes (sodium, chloride, potassium), serum chemistry\n(albumin, alkaline phosphatase, alanine aminotransferase, blood\nurea nitrogen, creatinine, glucose, total protein), and coagulation\ntests (PT, aPTT).\n2.2 Study design\nHealthy dogs underwent TEG, PFA P2Y, and D-dimer tests.\nHypercoagulable dogs underwent tests for P2Y CT, complete blood\ncount, PT, aPTT, and D-dimer levels at diagnosis. Subsequently, they\nreceived 10 mg/kg clopidogrel (loading dose), followed by 2 mg/kg\nonce daily for ≥4 days. Blood samples were obtained 5–7 days after\ninitiation and 12–14 h following the last dose for re-assessments of the\naforementioned parameters.\n2.3 Blood collection\nTo minimize mechanical stress on platelets during sample\ncollection, blood was drawn from the jugular vein using needles no\nsmaller than 21 gauge. After collection, the needle was removed from\nthe\nsyringe\nbefore\ngently\ntransferring\nthe\nblood\ninto\nethylenediaminetetraacetic acid and citrate tubes. We collected 0.3 mL\nof blood into tubes containing ethylenediaminetetraacetic acid for\ncomplete blood count analysis, and 2.7 mL of blood into 3.2% sodium\ncitrate tubes using an exact 1:9 ratio of anticoagulant to blood for\nTEG, PFA P2Y, PT, aPTT, and D-dimer testing.\n2.4 TEG test procedure\nWhole blood collected in 3.2% sodium citrate was gently mixed\nand then rested at room temperature (20–25°C) for 15 min in\naccordance with the manufacturer’s instructions. One milliliter of\ncitrated whole blood was transferred into a proprietary kaolin tube\n(Haemonetics®,\nBraintree,\nMA,\nUSA)\ncontaining\nkaolin,\nphospholipids, and stabilizers. From this, 340 μL was placed into a\nTEG cup and recalcified with 20 μL of 200 mM calcium chloride.\nFIGURE 1\nThe Platelet Function Analyzer −200 device used to assess the\nantiplatelet effects of clopidogrel. Citrate-treated blood is dispensed\ninto the P2Y cartridge.\nAbbreviations: ADP, adenosine diphosphate; ASVCP, American Society for\nVeterinary Clinical Pathology; aPTT, activated partial thromboplastin time; CI,\n\nCitrate-treated blood is dispensed\ninto the P2Y cartridge.Abbreviations: ADP, adenosine diphosphate; ASVCP, American Society for\nVeterinary Clinical Pathology; aPTT, activated partial thromboplastin time; CI,\ncoagulation index; CT, closure time; HCT, hematocrit; K, coagulation time; LTA,\nlight transmission aggregometry; MA, maximum amplitude; PFA, Platelet Function\nAnalyzer; PLT, platelet count; PT, prothrombin time; R, reaction time; RI, reference\ninterval; TEG, thromboelastography; α, alpha angle.\n\nShin and Han\b\n10.3389/fvets.2025.1595147\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nStandard TEG parameters (reaction time [R], alpha angle [α], \ncoagulation time [K], and MA) were recorded. The CI was calculated \nas follows (12):\n\n( )\n( )\n(\n)\n( )\n=\n+\n+\nα\nCI\n0.1227 R\n0.0092 K\n0.1655 MA –0.0241\n–5.0220\n2.5 PFA P2Y test procedure\nAs with the TEG assay, whole blood collected in 3.2% sodium\ncitrate was gently mixed and then rested without agitation at room\ntemperature (20–25°C) for 15 min, in accordance with the\nmanufacturer’s instructions. Using the PFA-200, 800–900 μL of\ncitrated whole blood was pipetted into a prewarmed P2Y test\ncartridge. P2Y CT was measured with a maximum threshold of 300 s;\nwith values exceeding 300 s being reported as non-closure and\nrecorded as 300 s. Dogs exhibiting non-closure in P2Y CT after\nclopidogrel administration were classified as the clopidogrel-\nresponder group, whereas those with a P2Y CT of less than 300 s were\nclassified as the clopidogrel-resistant group. Samples were tested\nwithin 1 h after venipuncture.\n2.6 Other blood test procedures\nHCT and PLT were measured using an automated hematology\nanalyzer (IDEXX ProCyte Dx, IDEXX Laboratories, Inc., Westbrook,\nME, USA). PT and aPTT were measured with a coagulation analyzer\n(IDEXX Coag Dx, IDEXX Laboratories, Inc., Westbrook, ME, USA).\nD-dimer levels were determined by a fluorescent immunoassay (Vet\nChroma™, ANIVET Diagnostic Inc., Chuncheon, Kangwon, South\nKorea), with a detection range of 50–10,000 ng/mL. Values <50 ng/\nmL and >10,000 ng/mL were recorded as 50 ng/mL and 10,000 ng/\nmL, respectively.\n2.7 Statistical analysis\nAll statistical analyses were performed using SPSS software\n(version 29.0, IBM Corp., Armonk, NY, USA). Statistical significance\nwas defined as p < 0.05. Normality was assessed by the Shapiro–Wilk\ntest. For normally distributed data, independent samples t-tests were\nused for between-group comparisons and paired t-tests for pre- and\npost-treatment comparisons. For non-normally distributed data, the\nMann–Whitney U and Wilcoxon signed-rank tests were used.\nReference Value Advisor v2.1 was used to calculate the RI for P2Y CT\n(13). Although robust methods are recommended by the American\nSociety for Veterinary Clinical Pathology (ASVCP) guidelines (14),\ntheir application in this data set yielded unreliable estimates, likely\n\n1 was used to calculate the RI for P2Y CT\n(13).Although robust methods are recommended by the American\nSociety for Veterinary Clinical Pathology (ASVCP) guidelines (14),\ntheir application in this data set yielded unreliable estimates, likely\ndue to a limited sample size. Therefore, a nonparametric percentile\nmethod was used as a more reliable alternative. Outliers were\nevaluated using Tukey’s method, which defines outliers as values\nfalling below the first quartile minus 1.5 times the interquartile range\n(IQR) or above the third quartile plus 1.5 times the IQR. All identified\noutliers were assessed for potential analytical or clinical exclusion in\naccordance with ASVCP guidelines.\n3 Results\n3.1 Animals\nA total of 70 dogs were included in this study, consisting of 40\nhealthy dogs and 30 dogs diagnosed with hypercoagulability. The\ncharacteristics of the dogs in both the healthy control and\nhypercoagulable groups, including sex, breed, weight, and age are\npresented in an electronic spreadsheet database (Supplementary Table 1).\nThe hypercoagulable dogs had a variety of underlying conditions, such\nas immune-mediated diseases, inflammation, and hemodynamic\ncompromise. The medical information, including the diagnoses, was\norganized in the Supplementary Table 1.\n3.2 PFA P2Y test results\nThe mean P2Y CT in the healthy group was 63.9 ± 26.82 s\n(minimum, 40 s; maximum, 142 s), which was shorter than in the\nhypercoagulable group (77.37 ± 37.61 s; minimum, 41 s; maximum,\n244 s; p < 0.05; Figure 2). The RI for P2Y CT in the healthy group was\ndetermined to be  40.0–141.5 s using a nonparametric percentile\nmethod. The 90% confidence intervals for the lower and upper RI\nlimits, calculated by bootstrap resampling, were 40.0–41.0 s and\n119.6–142.0 s, respectively. Outlier analysis using Tukey’s method\nidentified one value exceeding the upper fence, but this value was\nretained in the final RI as no clinical or analytical justification for\nexclusion was identified.\nIn the hypercoagulable group, 23 (76.67%) and 7 (23.33%) dogs\nwere classified as the clopidogrel-responder and clopidogrel-resistant\n\nIn the hypercoagulable group, 23 (76.67%) and 7 (23.33%) dogs\nwere classified as the clopidogrel-responder and clopidogrel-resistant\ngroups, respectively (Table 1). In the clopidogrel-responder group, the\nmean P2Y CT significantly increased from 77.48 ± 41.67 s (41.00–\n244.00) to >300 s (p < 0.05); similarly, it increased from 77.00 ± 21.63 s\n(55.00–118.00) s to 182.71 ± 78.43 s (70.00–273.00) in the clopidogrel-\nresistant group (p < 0.05).\nIn the clopidogrel-resistant group, the maintenance dose was\ninitially increased from 2 mg/kg to 4 mg/kg, which was administered\nfor 5 days, followed by P2Y CT remeasurement. If the P2Y CT\nremained <300 s, the dose was further increased to 6 mg/kg, followed\nby to 8 mg/kg, until the P2Y CT exceeded 300 s. In this group, five,\none, and one dog achieved a P2Y CT > 300 s at 4 mg/kg, 6 mg/kg, and\n8 mg/kg once daily, respectively. In the hypercoagulable group, 76.67,\n16.67, 3.33, 3.33% of the dogs showed clopidogrel responsiveness at\n2 mg/kg, 4 mg/kg, 6 mg/kg, and 8 mg/kg once daily, respectively\n(Figure 3).\n3.3 Hematological (HCT, PLT) and\ncoagulation (PT, aPTT, D-dimer, R, K, α, MA,\nCI) test results\nIn the healthy group, the mean HCT, PLT count, PT, and aPTT\nwere 48.63 ± 4.55% (40.20–60.80), 207.95 ± 42.61 K/μL (150.00–\n300.00), 14.58 ± 1.71 s (12.00–17.00), and 82.68 ± 7.12 s (72.00–\n101.00),\nrespectively.\nThe\nmean\nD-dimer\nlevels\nwas\n474.26 ± 1110.54 ng/mL (50.00–6634.98). For TEG parameters, the\nmean R, K, α, MA, and CI were 2.38 ± 0.99 min (0.70–4.90),\n\nShin and Han\b\n10.3389/fvets.2025.1595147\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nFIGURE 2\nBox-and-Whisker plots of P2Y closure time (CT) in the healthy and hypercoagulable groups. The boxes indicate the interquartile ranges (25th–75th \npercentile) and lines within the boxes indicate the median values. Whiskers indicate either 1.5 times the interquartile range or the range limit for the \ndata, whichever is less. Circles represent mild outliers, which are values exceeding 1.5 times the interquartile range below the 25th percentile or above \nthe 75th percentile. Asterisks represent extreme outliers, which are values exceeding three times the interquartile range beyond these percentiles. The \nhypercoagulable group shows prolonged P2Y CT compared to the healthy group (p < 0.05, Mann–Whitney U test).\nTABLE 1  Comparison of P2Y closure times before and after treatment with clopidogrel in clopidogrel-responder and clopidogrel-resistance groups.\nVariables\nClopidogrel responder (n = 23)\nClopidogrel-resistance (n = 7)\nBefore\nAfter\np-value\nBefore\nAfter\np-value\nP2Y CT (s)\n77.48 ± 41.67 (41.00–244.00)\n300.00 ± 0.00 (300.00–300)\n* < 0.001\n77.00 ± 21.63 (55.00–118.00)\n182.71 ± 78.43 (70.00–273.00)\n*0.018\nMean ± SD (range), CT; closure time. *p-value<0.05 indicates significance.\nFIGURE 3\nPie chart of proportion of dogs by responsive dosage. Among the 30 dogs in the hypercoagulable group, 76.67, 16.67, 3.33, 3.33% of the dogs showed \nclopidogrel responsiveness at 2 mg/kg, 4 mg/kg, 6 mg/kg, and 8 mg/kg once daily, respectively.\n\nShin and Han\b\n10.3389/fvets.2025.1595147\nFrontiers in Veterinary Science\n05\nfrontiersin.org\n4.64 ± 2.78 min\n(0.80–13.50),\n53.54 ± 14.29\n(22.80–81.70),\n51.88 ± 6.72 mm (35.80–62.50), and 2.60 ± 0.86 (0.94–4.00),\nrespectively.\nTable 2 presents the hematological and coagulation test results. In\nthe clopidogrel-responder group (n = 23), mean HCT did not\nsignificantly differ, changing from 35.06 ± 8.28% (22.60–57.70) to\n36.01 ± 6.04% (25.90–45.80) (p = 0.456) (Table 2). The mean PLT\nsignificantly increased from 356.30 ± 142.73 K/μL (100.00–591.00) to\n450.17 ± 184.52 K/μL (227.00–792.00) (p < 0.05). PT remained stable,\nwith values of 13.48 ± 1.12 s (11.00–16.00) before and 13.09 ± 1.50 s\n(11.00–16.00) after treatment (p = 0.441). Mean aPTT significantly\ndecreased from 96.17 ± 10.28 s (78.00–119.00) to 88.09 ± 6.98 s\n(76.00–100.00) (p < 0.05). D-dimer levels showed a significant\nreduction from 2476.95 ± 3185.40 ng/mL (50.00–10000.00) to\n775.29 ± 880.31 ng/mL (50.00–3786.00) (p < 0.05). For TEG\nparameters, the mean R did not significantly differ, changing from\n3.14 ± 1.19 min (1.60–6.40) to 3.27 ± 1.29 min (1.50–5.80) (p = 0.681),\nK showed no meaningful difference, changing from 1.14 ± 0.48 min\n(0.80–2.70) to 1.43 ± 0.94 min (0.80–3.70) (p = 0.38), α did not\nsignificantly differ, changing from 73.30 ± 8.02 (43.60–82.70) to\n70.7 ± 9.85° (48.20–81.40) (p = 0.336), MA did not change\nsignificantly, changing from 77.59 ± 3.87 mm (70.10–84.20) to\n74.52 ± 7.24 mm (55.30–83.70) (p = 0.176), and CI did not differ\nsignificantly, changing from 6.37 ± 0.78 (4.2–7.33) to 6.02 ± 1.02\n(3.38–7.35) (p = 0.069).\nIn the clopidogrel-resistant group (n = 7), the mean HCT showed\nno meaningful difference, changing from 33.89 ± 8.79% (17.60–41.80)\nto 33.43 ± 11.37% (21.40–50.80) (p = 1.00) (Table 2). The mean PLT\ndid not significantly differ, changing from 443.57 ± 151.22 K/μL\n(308.00–720.00) to 429.86 ± 148.43 K/μL (259.00–683.00) (p = 0.612).\nPT did not change significantly, changing from 13.57 ± 2.07 s (11.00–\n16.00) to 13.86 ± 1.86 s (13.00–18.00) (p = 1.00). Mean aPTT did not\n\n00) (Table 2).The mean PLT\ndid not significantly differ, changing from 443.57 ± 151.22 K/μL\n(308.00–720.00) to 429.86 ± 148.43 K/μL (259.00–683.00) (p = 0.612).PT did not change significantly, changing from 13.57 ± 2.07 s (11.00–\n16.00) to 13.86 ± 1.86 s (13.00–18.00) (p = 1.00).Mean aPTT did not\ndiffer significantly, changing from 95.00 ± 12.60 s (77.00–112.00) to\n91.86 ± 16.04 s (72.00–113.00) (p = 0.499). D-dimer levels did not\nsignificantly differ, changing from 681.56 ± 523.00 ng/mL (127.70–\n1780.21) to 782.23 ± 810.40 ng/mL (62.60–1925.10) (p = 0.735). For\nTEG parameters, mean R showed no meaningful difference, changing\nfrom 3.69 ± 0.96 min (2.80–5.20) to 22.89 ± 1.21 min (1.40–4.70)\n(p = 0.116), K did not significantly differ, changing from\n1.21 ± 0.47 min (0.80–2.10) to 1.34 ± 0.71 min (0.80–2.70) after\n(p = 1.00), α did not change significantly, changing from 72.13 ± 6.9\n(59.70–78.80) to 72.70 ± 8.00 (58.00–79.90) (p = 0.735), MA did not\ndiffer significantly, changing from 78.49 ± 6.07 mm (70.40–85.10) to\n75.36 ± 8.75 mm (62.40–84.60) (p = 0.352), and CI did not\nsignificantly differ, changing from 6.19 ± 1.19 (4.24–7.58) to\n6.30 ± 1.13 (4.24–7.64) (p = 0.109).\n4 Discussion\nAntiplatelets have been traditionally used to prevent arterial\nthrombosis, with emerging evidence suggesting platelets also\ncontribute to venous thrombosis (15–18). In humans, antiplatelet\nagents have demonstrated efficacy in preventing venous thrombosis\n(19–21), suggesting that the application of clopidogrel in veterinary\nmedicine can be expanded. Additionally, advancements in coagulation\ntests to facilitate hypercoagulability diagnosis (22, 23), as well as\nadvances in interventional cardiology and open-heart surgery in\nTABLE 2  Hematological and coagulation test results before and after clopidogrel administration in the clopidogrel-responder and clopidogrel-resistance groups.\nVariables\nReference\ninterval\nClopidogrel responder\nClopidogrel-resistance\nBefore\nAfter\np-value\nBefore\nAfter\np-value\nHematocrit (%)\n37.3–61.7\n35.06 ± 8.28 (22.60–57.70)\n36.01 ± 6.04 (25.90–45.80)\n0.456\n33.89 ± 8.79 (17.60–41.80)\n33.43 ± 11.37 (21.40–50.80)\n1.00\n\nVariables\nReference\ninterval\nClopidogrel responder\nClopidogrel-resistance\nBefore\nAfter\np-value\nBefore\nAfter\np-value\nHematocrit (%)\n37.3–61.7\n35.06 ± 8.28 (22.60–57.70)\n36.01 ± 6.04 (25.90–45.80)\n0.456\n33.89 ± 8.79 (17.60–41.80)\n33.43 ± 11.37 (21.40–50.80)\n1.00\nPlatelet (K/μL)\n148–484\n356.30 ± 142.73 (100.00–591.00)\n450.17 ± 184.52 (227.00–792.00)\n*0.01\n443.57 ± 151.22 (308.00–720.00)\n429.86 ± 148.43 (259.00–683.00)\n0.612\nPT (s)\n11–17\n13.48 ± 1.12 (11.00–16.00)\n13.09 ± 1.50 (11.00–16.00)\n0.441\n13.57 ± 2.07 (11.00–16.00)\n13.86 ± 1.86 (13.00–18.00)\n1.00\naPTT (s)\n72–102\n96.17 ± 10.28 (78.00–119.00)\n88.09 ± 6.98 (76.00–100.00)\n*0.011\n95.00 ± 12.60 (77.00–112.00)\n91.86 ± 16.04 (72.00–113.00)\n0.499\nD-dimer (ng/mL)\n< 250\n2476.95 ± 3185.40 (50.00–10000.00)\n775.29 ± 880.31 (50.00–3786.00)\n*0.01\n681.56 ± 523.00 (127.70–1780.21)\n782.23 ± 810.40 (62.60–1925.10)\n0.735\nR (minute)\n1.8–8.6\n3.14 ± 1.19 (1.60–6.40)\n3.27 ± 1.29 (1.50–5.80)\n0.681\n3.69 ± 0.96 (2.80–5.20)\n2.89 ± 1.21 (1.40–4.70)\n0.116\nK (minute)\n1.3–5.7\n1.14 ± 0.48 (0.80–2.70)\n1.43 ± 0.94 (0.80–3.70)\n0.38\n1.21 ± 0.47 (0.80–2.10)\n1.34 ± 0.71 (0.80–2.70)\n1.00\nα (°)\n36.9–74.6\n73.30 ± 8.02 (43.60–82.70)\n70.70 ± 9.85 (48.20–81.40)\n0.336\n72.13 ± 6.90 (59.70–78.80)\n72.70 ± 8.00 (58.00–79.90)\n0.735\nMA (mm)\n42.9–67.9\n77.59 ± 3.87 (70.10–84.20)\n74.52 ± 7.24 (55.30–83.70)\n0.176\n78.49 ± 6.07 (70.40–85.10)\n75.36 ± 8.75 (62.40–84.60)\n0.352\nCI\n-4–4\n6.37 ± 0.78 (4.20–7.33)\n6.02 ± 1.02 (3.38–7.35)\n0.069\n6.19 ± 1.19 (4.24–7.58)\n6.30 ± 1.13 (4.24–7.64)\n0.109\nMean ± SD (range). HCT; hematocrit, PLT; platelet count, PT; prothrombin time, aPTT; activated partial thromboplastin time, R; reaction time, K; coagulation time, α; alpha angle, MA; maximum amplitude, CI; coagulation index. *p-value<0.05 indicates significance.\n\nShin and Han\b\n10.3389/fvets.2025.1595147\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nveterinary medicine, have led to an increase in clopidogrel\nprescriptions (24, 25).\nClopidogrel resistance is defined as insufficient platelet\ninhibition in vitro following drug administration (3). In humans, the\nCYP2C19 gene, which is essential for clopidogrel metabolism,\nexhibits various polymorphisms that lead to significant variability\nin enzyme activity, which is the primary cause of clopidogrel\nresistance (26–28). Other known mechanisms of clopidogrel\nresistance in humans include differences in intestinal absorption,\ngenetic polymorphisms of ABC1 as well as GpIIb-IIIa, GPIa-IIa and\nP2Y12 receptors, and clinical factors such as diabetes and\noverweightness (29). In cats, mutations in the P2RY1 gene, one of\nthe platelet ADP receptor genes, excessively activates the P2Y1\nreceptor in response to ADP, contributing to clopidogrel resistance\n(30). The mechanisms underlying clopidogrel resistance in dogs\nremains unclear. Nonetheless, clopidogrel resistance in dogs is\nconsidered to involve genetic polymorphisms in either platelet ADP\nreceptor genes (P2RY1, P2RY12) or the cytochrome P450\nenzyme gene.\nIn humans, clopidogrel resistance is clinically associated with an\nincreased risk of thrombosis (2, 31, 32). Although therapeutic\nstrategies for clopidogrel resistance remain inconclusive in humans,\nincreasing the loading dose, using a higher maintenance dose, or\nswitching to alternative thienopyridines such as ticlopidine or\nprasugrel, may enhance platelet inhibition in clopidogrel-resistant\npatients (29, 33). In veterinary medicine, there remains scarce research\non treatment strategies related to clopidogrel resistance. Therefore, it\nis important to identify patients with inadequate responses to\nclopidogrel and implement alternative therapeutic strategies in order\nto improve prognosis and survival in veterinary patients.\nThere remain no established standards for evaluating the efficacy\nof antiplatelet therapy in dogs, which is further complicated by\ninconsistencies across platelet function tests and the lack of an\n\nThere remain no established standards for evaluating the efficacy\nof antiplatelet therapy in dogs, which is further complicated by\ninconsistencies across platelet function tests and the lack of an\nestablished RI for treatment monitoring. Platelet function tests\nmeasure the inhibition of ADP-induced platelet aggregation to\nevaluate the efficacy and resistance of clopidogrel; however, the\nmethods differ across devices (8). LTA and turbidimetric-based\noptical detection systems both assess platelet aggregation by detecting\nchanges in light transmission, using platelet-rich plasma and whole\nblood, respectively. Another method, electrical aggregometry, assesses\nplatelet aggregation by measuring changes in electrical impedance as\nplatelets adhere to electrodes in whole blood. TEG with platelet\nmapping measures platelet aggregation and thrombus formation in\nreal-time, indirectly confirming clopidogrel inhibition by observing a\ndecrease in clot strength. Flow cytometry evaluates the expression and\nactivation status of platelet surface receptors to assess the effectiveness\nof clopidogrel-mediated P2Y12 receptor inhibition. Furthermore, the\nPFA P2Y test is a useful tool for assessing the antiplatelet effects of\nclopidogrel. This cartridge-based point-of-care assay involves\naspirating whole blood through a small aperture (150-μm diameter)\nin a membrane coated with 20 μg of ADP, 5 ng of prostaglandin E1,\nand 459 μg of calcium chloride. Under high shear conditions, these\nplatelet activators induce platelet plug formation, eventually occluding\nthe aperture, with the time until full occlusion being measured.\nLTA is considered the gold standard for evaluating the antiplatelet\neffect of clopidogrel. However, ADP-induced LTA may be slightly\ninappropriate for evaluating clopidogrel’s effect since ADP can induce\nplatelet aggregation through both the P2Y12 and P2Y1 receptors,\nmeaning that using ADP alone may lack the specificity required to\naccurately assess clopidogrel’s effect (3, 34, 35). LTA requires larger\nsample volumes and a high level of technical expertise, which impedes\n\nLTA requires larger\nsample volumes and a high level of technical expertise, which impedes\nits practicability in routine clinical settings, especially in veterinary\nmedicine. Additionally, electrical aggregometry and flow cytometry\nalso require a high level of technical expertise, while TEG with platelet\nmapping is relatively expensive and time-consuming.\nContrastingly, the PFA is simpler and only requires small blood\nvolumes; therefore, it is widely used in clinical practice. The PFA\nhas three cartridge types, including the collagen/epinephrine\ncartridge (C/Epi), which uses collagen and epinephrine as platelet\nactivators; the collagen/adenosine diphosphate cartridge (C/ADP),\nwhich uses collagen and adenosine diphosphate; and the P2Y\ncartridge, which is coated with prostaglandin E1. In humans, both\nC/Epi and C/ADP cartridges are used to evaluate platelet function,\nshowing particular sensitivity to von Willebrand disease, and the C/\nEpi cartridge is especially sensitive to aspirin therapy (36). Both\ncartridges are useful for evaluating primary hemostasis disorders in\ndogs (37); however, the role of epinephrine remains unclear in\ncanine platelet aggregation, which differs from humans (38, 39).\nAccordingly, the use of the C/Epi cartridge in assessing canine\nplatelet function remains controversial. Contrastingly, ADP is\nhighly sensitive to platelet aggregation in dogs, making the C/ADP\ncartridge useful for evaluating platelet function (40–42). Specifically,\nP2Y cartridge is considered more suitable for evaluating clopidogrel\nefficacy since it inhibits P2Y1-mediated aggregation using\nprostaglandin E1  in the cartridge, facilitating more specific\nevaluation of P2Y12-mediated aggregation, which is directly\ninfluenced by clopidogrel. In cats, the P2Y cartridge is more\nsensitive and effective than the C/ADP cartridge in evaluating\nclopidogrel efficacy, expanding the potential applications of the PFA\nin veterinary medicine (43). We performed the PFA P2Y test using\na relatively small volume of whole blood (approximately 800 μL),\nmaking the testing procedure simple and quick (5–10 min). These\n\nWe performed the PFA P2Y test using\na relatively small volume of whole blood (approximately 800 μL),\nmaking the testing procedure simple and quick (5–10 min).These\nfactors contribute to the practicality and ease of applying this\nmethod in veterinary clinical settings. Consequently, via this\nclinical utility, we established the RI for P2Y CT using P2Y cartridge\nand observed that some P2Y CT parameters were significantly\nlonger in the hypercoagulable group than in the healthy group.\nFurther, 76.67% of the clopidogrel-administered dogs showed\nclopidogrel responsiveness, with a P2Y CT > 300 s.\nNotably, the PFA P2Y test has several limitations, including a\ncapped measurement limit of 300 s for CT, beyond which values are\nnot recorded, and the influence by various hematologic factors. If a\npatient’s P2Y CT prior to clopidogrel administration already exceeds\n300 s, it may be  impossible to use the PFA P2Y test to monitor\ntreatment responsiveness. Additionally, human studies on C/Epi and\nC/ADP cartridges have showed that significant reductions in PLT or\nHCT, lower von Willebrand factor levels, and the use of higher-\nconcentration sodium citrate tubes all led to prolonged CT (44–46).\nFurthermore, these factors may similarly affect the P2Y cartridge,\nmaking it unsuitable for dogs with low PLT, HCT, or von Willebrand\nfactor levels. In our study, all blood samples for the PFA P2Y test were\nuniformly treated with 3.2% sodium citrate, which eliminated the\neffects of variations in sodium citrate concentration. Therefore, the\nrelatively low HCT in the hypercoagulable group may have contributed\nto the prolonged P2Y CT. Furthermore, one hypercoagulable dog,\nwith a P2Y CT of 244 s outside the normal reference range, presented\n\nShin and Han\b\n10.3389/fvets.2025.1595147\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nwith thrombocytopenia (100 × 109/L). This suggests that the low\nplatelet count may have contributed to the prolonged CT.\nIn dogs, a P2Y CT < 300 s after clopidogrel administration is\nconsidered indicative of clopidogrel resistance (47). The rate of\nclopidogrel resistance in our study was 23.33%, which is consistent\nwith a previously reported value of 25% (4) and comparable to that in\nhumans (4–30%) (3), although its clinical implications may differ.\nUnlike in humans, where clopidogrel is administered at a fixed dose\nof 300 mg on the first day, followed by 75 mg/day, dogs are typically\ndosed based on body weight. Additionally, the broader dosing range\nfor clopidogrel in dogs (1–4 mg/kg) (48–52) and the variability in the\nuse of a loading dose impedes determination of an optimal\npersonalized dose without inducing resistance or causing adverse\neffects. These challenges in dogs emphasize the need for monitoring\nclopidogrel efficacy.\nOur findings showed that the effective clopidogrel dosage varies\nacross dogs, highlighting the importance of confirming clopidogrel\nefficacy. Furthermore, increasing the maintenance dose helped\novercome resistance. In this study, we implemented a personalized\nantiplatelet therapy inspired by therapeutic strategies from human\nmedicine for managing clopidogrel resistance (33). To date, the\nhighest maintenance dose of clopidogrel reported in the veterinary\nliterature is 4 mg/kg (48). Therefore, we established 8 mg/kg, which\nis twice that amount, as the maximum maintenance dose in our\nprotocol. To minimize the risk of adverse effects associated with high-\ndose escalation, an intermediate step of 6 mg/kg was incorporated.\nFor dogs receiving doses exceeding the standard maintenance\nregimen, consent was obtained from the owners after providing a\ncomprehensive explanation of the rationale, potential benefits, and\npossible adverse effects of the proposed treatment. Safety monitoring\nconsisted of complete blood count assessments and thorough clinical\n\nSafety monitoring\nconsisted of complete blood count assessments and thorough clinical\nevaluations for signs of bleeding, including petechiae, ecchymosis,\nepistaxis, melena, and hematochezia. These evaluations were\nconducted on days 2 and 5 following clopidogrel administration. No\nadverse events were observed in any dog throughout the study\nperiod. All dogs exhibiting resistance responded when the dose was\nincreased to a maximum of 8 mg/kg once daily. However, for dogs\nthat continue to exhibit resistance despite this dose adjustment, other\nthienopyridine therapies, similar to those used in humans, could\nbe  an alternative. Nonetheless, further studies are warranted to\nvalidate these approaches in veterinary medicine.\nAmong the conventional coagulation markers, only PLT, aPTT, and\nD-dimer levels showed significant pre-post differences in the clopidogrel-\nresponder group. Following clopidogrel administration, the increase in\nPLT and shortening of aPTT is suggestive of enhanced coagulation.\nHowever, no significant changes were observed in TEG parameters, and\nP2Y CT was prolonged after clopidogrel administration. Therefore, the\nincrease in PLT and shortening of aPTT were not considered significant\nrisk factors for hypercoagulability or increased thrombus formation\nfollowing clopidogrel administration. Although D-dimer levels cannot\nbe considered a specific indicator of thrombus formation, a decrease in\nD-dimer level reflects reduced thrombin generation and fibrin turnover,\nwhich is associated with a decreased risk of thrombus-related\ncardiovascular events in humans (53). Consequently, D-dimer levels\nonly significantly decreased in the clopidogrel-responder group,\nsuggesting that clopidogrel may have contributed to reducing blood clot\nformation. Among the conventional markers, D-dimer could be useful\nin evaluating clopidogrel responsiveness.\nThis study has several limitations. First, the relatively small sample\nsize, particularly in the clopidogrel-resistant group, may have impeded\nthe statistical robustness and the generalizability of the findings.\n\nThis study has several limitations.First, the relatively small sample\nsize, particularly in the clopidogrel-resistant group, may have impeded\nthe statistical robustness and the generalizability of the findings.\nAnemia, characterized by low HCT, may influence the accuracy of CT\nmeasurements. A previous study in human medicine has reported that\nHCT levels below 25% can result in CT prolongation (54). In the\npresent study, 18 of the 30 dogs in the hypercoagulable group were\nanemic (HCT < 36%), yet 17 of these had P2Y CT values within the\nestablished reference interval (40.0–141.5 s). Given that the primary\nobjective of this study was to evaluate within-individual pre- and post-\ntreatment changes, these cases were retained in the analysis.\nFurthermore, a prior canine study has shown that the extent of CT\nprolongation associated with low HCT is relatively modest (55).\nNonetheless, in dogs with moderate to severe anemia (HCT < 30%), the\npotential impact of HCT on P2Y CT values should be recognized as a\nlimitation of this study, warranting cautious interpretation and further\ninvestigation. The healthy group mainly comprised large-breed dogs,\nwhile the hypercoagulable group was composed primarily of small-\nbreed dogs, introducing potential breed-related variability. Additionally,\nthe underlying causes of hypercoagulability and their treatment\nstrategies differed across the dogs, which may have influenced the\nresults. However, in the hypercoagulable group, no dogs were receiving\nany antiplatelet or anticoagulant therapy except for clopidogrel.\nAnother consideration is that the mean D-dimer concentration in\nthe healthy group was unexpectedly high. The median D-dimer\nconcentration in this group was 152.29 ng/mL, indicating that most\nhealthy dogs had physiologically normal levels. However, three dogs\nexhibited markedly elevated values of 2199.84, 2287.67, and\n6634.98 ng/mL, which significantly influenced the group mean.\nVigorous physical activity prior to blood sampling, such as playing\nfetch, may have contributed to this finding, as reported in human\n\n84, 2287.67, and\n6634.98 ng/mL, which significantly influenced the group mean.Vigorous physical activity prior to blood sampling, such as playing\nfetch, may have contributed to this finding, as reported in human\nstudies demonstrating exercise-induced increases in D-dimer levels\n(56). While this variability may influence interpretation, the PFA P2Y\ncartridge is less likely to be affected by such factors, as it specifically\nevaluates the P2Y12 receptor pathway and minimizes the impact of\nnon-specific physiological changes. Importantly, our analysis focused\non within-individual changes, and a significant reduction in D-dimer\nlevels was still observed in responders. Nevertheless, as D-dimer is a\nnon-specific marker, reliance on absolute values should be avoided, and\nthis should be acknowledged as a potential limitation in interpreting\nthe study findings. Finally, although we identified clopidogrel resistance\nin some dogs, we did not evaluate long-term clinical outcomes such as\nthromboembolic events or bleeding complications. Given the role of\ngenetic factors, such as CYP2C19 and P2Y12 receptor polymorphisms,\nin clopidogrel resistance in humans, future studies investigating genetic\nvariability in dogs are warranted to improve our understanding of the\nunderlying mechanisms of clopidogrel resistance and to optimize\nantiplatelet therapy in this species.\n5 Conclusion\nOur findings confirmed individual variability in response to\nclopidogrel, emphasizing the importance of monitoring clopidogrel\nefficacy in dogs. The PFA P2Y test effectively assessed clopidogrel\nefficacy, and thus is a highly useful tool for veterinarians to objectively\nmonitor clopidogrel responsiveness in dogs and inform\ntherapeutic strategies.\n\nShin and Han\b\n10.3389/fvets.2025.1595147\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nData availability statement\nThe original contributions presented in the study are included in\nthe article/Supplementary material, further inquiries can be directed\nto the corresponding author.\nEthics statement\nThe animal studies were approved by Institutional Animal Care\nand Use Committee at Konkuk University. The studies were conducted\nin accordance with the local legislation and institutional requirements.\nWritten informed consent was obtained from the owners for the\nparticipation of their animals in this study.\nAuthor contributions\nJ-HS: Writing – original draft, Writing – review & editing. H-JH:\nWriting – original draft, Writing – review & editing.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. This study was conducted\nas a contracted research project without a specific grant number, with\nfinancial support from Siemens Healthineers Ltd. The sponsor had no\ninvolvement in the study design, data collection, analysis,\ninterpretation, or in the preparation and writing of this manuscript.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. Any product that may be evaluated in this article, or claim\nthat may be made by its manufacturer, is not guaranteed or endorsed\nby the publisher.\nSupplementary material\nThe Supplementary material for this article can be found online\nat: https://www.frontiersin.org/articles/10.3389/fvets.2025.1595147/\nfull#supplementary-material\nReferences\n1.\tAl-Abcha A, Radwan Y, Blais D, Mazzaferri EL, Boudoulas KD, Essa EM, et al.\n\nfrontiersin.org/articles/10.3389/fvets.2025.1595147/\nfull#supplementary-material\nReferences\n1.Al-Abcha A, Radwan Y, Blais D, Mazzaferri EL, Boudoulas KD, Essa EM, et al.\nGenotype-guided use of P2Y12 inhibitors: a review of current state of the art. Front\nCardiovasc Med. (2022) 9:850028. doi: 10.3389/fcvm.2022.850028\n2.\tKim Y, Weissler EH, Pack N, Latz CA. A systematic review of clopidogrel resistance\nin vascular surgery: current perspectives and future directions. Ann Vasc Surg. 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+{"text": "【兽医文献】\n标题: Chemical reprogramming regulates Tip60 expression to improve cleavage rates in somatic cell nuclear transfer reconstituted embryos of cashmere goats\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nTranscriptomic insights into the\neffects of tyrosine on\nsub-Columbian plumage in H line\nchickens\nXinlei Wang 1,2†, Liheng Zhang 1†, Liyu Yang 1, Pengkun Yang 1,2,\nYingying Qiao 1, Zhanbing Han 1, Jiaying Fan 1,2, Qiang Li 1,\nDingding Zhang 1, Zhuanjian Li 3,4, Xiangtao Kang 3,4, Juan Du 5\nand Ruiting Li 1*\n1College of Animal Science and Technology, Henan University of Animal Husbandry and Economy,\nZhengzhou, China, 2Henan Key Laboratory of Healthy Breeding and Efficient Reproduction of\nLivestock and Poultry, Zhengzhou, China, 3College of Animal Science and Technology, Henan\nAgricultural University, Zhengzhou, China, 4Key Research Project of the Shennong Laboratory,\nZhengzhou, China, 5Zhengzhou Health College, Zhengzhou, China\nTyrosine is known to influence melanin generation; however, its involvement in\nmelanin production in chicken feathers is unknown. We evaluated the feather color\nof H-line chickens fed diets containing different concentrations of tyrosine (0, 0.4,\n0.6, 0.8, and 1.0%). The results indicated that a diet containing 1.0% tyrosine fed\nfor 40 days significantly increased melanin deposition in the feathers (p < 0.05).\nFollowing this observation, we collected feather follicle tissue from chickens fed\neither 0% or 1.0% tyrosine at the 40-day time point for transcriptome sequencing.\nRNA-seq analysis identified a total of 314 DEGs, comprising 116 upregulated and\n198 downregulated genes. KEGG analysis of feather follicle tissue revealed that\n7 DEGs (EDNRB2, WNT3, POMC, INS, FLT3, CACNA2D3, and CACNA1I) mapped\nto melanin-related pathways, including the melanogenesis, MAPK signaling and\nWnt signaling pathways. We also identified specific protein interactions within the\nmelanin pathway, including EDNRB2–MLPH and WNT3–FGF16 interactions. Notably,\nthe expression level of the EDNRB2 gene reached its peak at 10 weeks within the\n0–12 week growth period in H-line chickens. In primary chicken melanocytes,\nEDNRB2 expression was quantified following tyrosine supplementation and was\n\nNotably,\nthe expression level of the EDNRB2 gene reached its peak at 10 weeks within the\n0–12 week growth period in H-line chickens.In primary chicken melanocytes,\nEDNRB2 expression was quantified following tyrosine supplementation and was\nfound to be markedly elevated at a concentration of 10−6 mol/L, significantly\nhigher than the control and other treatment groups (p < 0.05). Overall, our findings\nsuggest the significant involvement of the tyrosine-induced EDNRB2 regulatory\nnetwork in melanin levels in sub-Columbian plumage. Taken together, these\nfindings increase our understanding of the molecular mechanisms that regulate\ntyrosine-mediated melanin deposition in chicken plumage.\nKEYWORDS\nchicken, sub-Columbian plumage, EDNRB2, tyrosine, RNA-seq\n1 Introduction\nThe chicken, with diverse plumage color combinations and patterns, ranks among the\nmost colorful terrestrial vertebrates, thereby capturing the interest of researchers (1). Feather\ncolor not only attracts the opposite sex but also deters predators from chickens. Feather color\nis an important external characteristic in chickens (2). It not only is a significant identifying\nfeature for different breeds but also influences consumers’ choices when selecting high-quality\nOPEN ACCESS\nEDITED BY\nMiguel H. A. Santana,\nUniversity of São Paulo, Brazil\nREVIEWED BY\nZhanwei Teng,\nHenan Institute of Science and Technology,\nChina\nDandan Wang,\nHenan Institute of Science and Technology,\nChina\nXiangyong Qu,\nHunan Agricultural University, China\n*CORRESPONDENCE\nRuiting Li\nliruiting210@163.com\n†These authors have contributed equally to\nthis work and share first authorship\nRECEIVED 08 October 2025\nREVISED 13 November 2025\nACCEPTED 14 November 2025\nPUBLISHED 28 November 2025\nCITATION\nWang X, Zhang L, Yang L, Yang P, Qiao Y,\nHan Z, Fan J, Li Q, Zhang D, Li Z, Kang X,\nDu J and Li R (2025) Transcriptomic insights\ninto the effects of tyrosine on sub-Columbian\nplumage in H line chickens.\nFront. Vet. Sci. 12:1720520.\ndoi: 10.3389/fvets.2025.1720520\nCOPYRIGHT\n© 2025 Wang, Zhang, Yang, Yang, Qiao, Han,\nFan, Li, Zhang, Li, Kang, Du and Li. This is an\nopen-access article distributed under the\n\nFront.Vet.Sci.12:1720520.doi: 10.3389/fvets.2025.1720520\nCOPYRIGHT\n© 2025 Wang, Zhang, Yang, Yang, Qiao, Han,\nFan, Li, Zhang, Li, Kang, Du and Li.This is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY). The use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\naccordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Original Research\nPUBLISHED  28 November 2025\nDOI  10.3389/fvets.2025.1720520\n\nWang et al.\b\n10.3389/fvets.2025.1720520\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nlive poultry in markets, so it holds economic significance (3). With the\nrapid development of integrated agriculture, improving feed nutrition\nto maintain and improve feather color is essential. Altering the\nplumage color phenotype by adding nutrients to the feed, with the aim\nof identifying key genes that influence this phenotype, has become an\nimportant approach in poultry breeding.\nChicken feather color is determined by the quantity and\ndistribution of melanin, which are dependent on the interaction\nbetween genotype and nutrition (4). Modern technological advances\nand scientific research have resulted in the identification of numerous\nmutations in genes affecting chicken feather color, including dominant\nwhite (I), recessive white (c), barring (B), spots (mo), extended black\n(E), dark brown (DB), and Columbia, among others (5–9). Nutritional\nfactors can also affect melanin deposition in animals (10). For\nexample, a combination of vitamin C and vitamin E supplementation\ncan be used to treat age spots or melasma in humans (11). Additional\nstudies have demonstrated a notable decrease in UV-induced skin\nredness and enhancements in skin hydration and elasticity after the\noral administration of lutein (12). Similarly, goldfish fed spirulina and\nastaxanthin-enriched brine shrimp presented improvements in\npigmentation and skin redness indices (13). However, there have been\nno reports on the impact of exogenous nutrient supplementation on\nchicken feather color.\nMelanin synthesis is performed by melanocytes in melanosomes\n(14), which then transfer melanin granules to adjacent keratinocytes,\nresulting in melanin accumulation and, in chickens, the production of\npigmented feathers (15). In mature melanocytes, tyrosine is acted\nupon by tyrosinase and tyrosinase-related proteins to produce DOPA,\nwhich subsequently undergoes further oxidation to dopaquinone.\nFinally, after further reactions, dopaquinone is converted to melanin,\nwhich affects fur color, body color, and skin color phenotypes (16, 17).\n\nFinally, after further reactions, dopaquinone is converted to melanin,\nwhich affects fur color, body color, and skin color phenotypes (16, 17).\nAt present, some reports indicate that the addition of moderate\namounts of tyrosine can promote the formation of melanin in animals.\nFor example, Li reported that adding tyrosine to the diet can enhance\nmelanin deposition in the breast muscles of Xichuan black-boned\nchickens (18). A tyrosine-deficient diet can cause black cat fur to\nbecome reddish-brown (19). By adjusting the concentrations of\ncopper and specific amino acids, such as phenylalanine and tyrosine,\nin the diet, the deposition of red pigments in the white fur of dogs can\nbe significantly increased (20). Additionally, increasing the intake of\ntyrosine in the diet can increase the deposition of black pigments in\ndog fur (21). However, the effects of a tyrosine diet on melanin\ndeposition in chicken feathers and the underlying mechanism are\nstill unclear.\nThree “Yufen I” hybrid lines were established from Chinese\ncommercial laying hens by Henan Agricultural University. The H line\nis characterized by mostly white feathers, accompanied by black\nbarring on the primary and secondary feathers, as well as the hackles\nand tail. While this coloration resembles the Columbian plumage, it\ninvolves black barring rather than verticle stripes and is thus termed\n“sub-Columbian” (22). Theoretical studies on the Columbian plumage\npattern began as early as 1955 (23). Previous studies in our laboratory\nhave shown that sub-Columbian plumage is influenced by SLC45A2\nand CDKN2A, which are inherited as dominant companions (22).\nMoreover, transcriptome sequencing of the sub-Columbian feathers,\nincluding black and white follicles from the dorsal neck region and\nwhite follicles from the abdominal neck, revealed that MED23 and\nGNAQ are essential for melanin accumulation (24). Additionally, by\nanalyzing transcriptome data from feather follicles from differently\ncolored chickens (yellow, sub-Columbian, and silver) in their necks\nand wings, it was determined that elevated SLC45A2 and GPNMB\n\nAdditionally, by\nanalyzing transcriptome data from feather follicles from differently\ncolored chickens (yellow, sub-Columbian, and silver) in their necks\nand wings, it was determined that elevated SLC45A2 and GPNMB\nlevels could enhance melanin deposition in feathers from H-line\nchickens (25). Although the genetic foundation of feather color in H\nline chickens is relatively well understood, the regulatory molecular\nmechanisms underlying melanin deposition in feathers through the\naddition of tyrosine in feed are not widely known. EDNRB2, a gene\ncrucial for melanoblast differentiation and migration along the\ndorsolateral pathway, plays a significant role in controlling melanin\ndeposition in the muscles of black-boned chickens (18, 26, 27).\nHowever, whether the expression of EDNRB2 is regulated by tyrosine,\nthereby influencing chicken plumage coloration, remains an\nunanswered question. This study focuses on H line hens as the\nresearch subjects, establishing a no-tyrosine group and groups with\nvarying concentrations of tyrosine in the feed. Samples were collected\nat different times to determine the time and concentration of tyrosine\naddition that significantly affect feather color changes. The\ntranscriptomes of neck follicles from untreated controls and tyrosine-\ntreated chickens were sequenced, and the differentially expressed\ngenes (DEGs) affecting feather color changes in follicles under\ntyrosine supplementation were screened. Using these data, we\nanalyzed the temporal expression characteristics of the candidate gene\nEDNRB2 in different stages of H line hens, as well as the expression\npatterns before and after the addition of tyrosine to melanocytes\n(Figure 1). This study aimed to determine the optimal tyrosine\nsupplementation in feed to increase melanin deposition in the feather\ncolor of H line hens and to elucidate the underlying molecular\nmechanisms involved.\n2 Materials and methods\n2.1 Sample collection\nThe H line chickens were provided by the Center of Poultry\nGermplasm Resources of Henan Agricultural University. A total of\n\n2 Materials and methods\n2.1 Sample collection\nThe H line chickens were provided by the Center of Poultry\nGermplasm Resources of Henan Agricultural University.A total of\n200 20-week-old H-line laying hens were randomly assigned to five\ndietary treatments: one control group and four treatment groups\n(Groups I-IV). Each group consisted of 4 biological replicates, with 10\nhens per replicate. The control group was fed a standard basal diet\n(Supplementary Table S1) and the four treatment groups received the\nsame basal diet supplemented with tyrosine at concentrations of 0.4,\n0.6, 0.8, and 1.0%, respectively. The tyrosine (purity 99%) used for\nsupplementation was obtained from Swire Coca-Cola Drinks Co., Ltd.\n(Zhengzhou, Henan Province, China). The tyrosine was thoroughly\nincorporated into the basal diet using a commercial feed mixer to\nensure uniform distribution within each formulated feed type. All\nhens were housed under identical environmental conditions. They\nwere provided with unrestricted access to water and were fed twice\ndaily according to the standard feeding regimen for the laying period.\nOn the 1st, 12th, 22nd and 40th days after tyrosine addition, the\nblack spots of the nuchal follicle tissue of each group of hens (5 groups\nof 40) were scored using a YN-15 precision colorimeter (ThreeNH\nTechnology, China), which operates on the CIE L*a*b* color space.\nPrior to measurement, the instrument was calibrated according to the\nmanufacturer’s protocol using a standard white calibration tile. In the\nCIE L*a*b* model, the L* value represents lightness on a scale from 0\n\nWang et al.\b\n10.3389/fvets.2025.1720520\nFrontiers in Veterinary Science\n03\nfrontiersin.org\n(pure black) to 100 (pure white), while a* and b* values represent the\ngreen-red and blue-yellow axes, respectively. As the plumage in this\nstudy exhibited a black-to-white spectrum, only the L* value was\nadopted for analysis. For each chicken, three feathers from the dorsal\nneck region were carefully selected. On each feather, a consistent\nmeasurement location near the tip was identified. Three replicate\nmeasurements were taken at this same spot per feather, ensuring the\nmeasuring aperture was in full and perpendicular contact with the\nfeather surface to avoid ambient light interference. This procedure\nyielded nine raw L* readings per chicken, from which a mean value\nwas calculated and recorded as the definitive neck feather lightness\n(L*) for the individual.\nBased on the analysis results of the L* values, the treatment group\nwith the most significant differences in feather color due to the\naddition of tyrosine was selected. From this treatment group and the\ncontrol group, 3 hens were randomly selected from each group (a total\nof 6) to collect samples of the follicular tissue from their necks. Feather\nsurfaces were wiped with cotton soaked in 75% alcohol, and tweezers\nwere used to clamp the feather root; the follicular tissue was then\nseparated using small forceps and placed in liquid nitrogen.\n2.2 RNA sequencing\nSix follicular tissues from the control (C) and treatment (T)\ngroups were sent to Huada Gene (Shenzhen) for transcriptome\nsequencing (3 biological replicates in each group). Extraction of total\nRNA was performed using TRIzol (Invitrogen, United States). A\nfragment analyzer (Agilent 5200) was used to measure quality\nparameters such as RNA sample concentration and sample integrity.\nSix RNA-seq libraries (C1, C2, C3, T1, T2 and T3) were constructed\nafter library purification (1.5% agarose gel electrophoresis). Library\nsequencing was performed on an Illumina HiSeq 4000 platform.\nFollowing the removal of adapter sequences and low-quality data, the\n\n5% agarose gel electrophoresis).Library\nsequencing was performed on an Illumina HiSeq 4000 platform.Following the removal of adapter sequences and low-quality data, the\nraw and high-quality sequence lengths were calculated, along with the\nQ20 and Q30 values. Cutadapt was used to remove sequences that did\nnot meet the quality criteria to obtain high-quality sequence data,\nwhich were then evaluated thoroughly before further analyses\nwere conducted.\n2.3 Bioinformatics analyses\nClean reads were aligned with the reference genome (Gallus_\ngallus-5.0) using HISAT (28). Transcript expression levels were\ndetermined using RSEM1 (29), with quantification as fragments per\nkilobase million (FPKM) values. EdgeR software (version 3.14.0) was\nutilized to assess expression variations among the groups to identify\nDEGs, using the criteria of p-value<0.05 and |log2FC (fold\nchange)| > 1.5.\nGO and KEGG analyses of the DEGs were performed using\nclusterProfiler in R (v3.6.1), using p  < 0.05 as the significance\nthreshold for enrichment.\nProtein–protein interaction networks (PPI) were constructed to\nfurther investigate functional interactions between these DEGs. DEGs\nwere mapped using STRING,2 and significant interactions with a\n1  http://deweylab.biostat.wisc.edu/rsem/rsem-calculate-expression.html\n2  http://www.string-db.org\nFIGURE 1\nFlow chart for screening and validating the effects of adding tyrosine to the diet on plumage color-associated genes in H line chickens.\n\nWang et al.\b\n10.3389/fvets.2025.1720520\nFrontiers in Veterinary Science\n04\nfrontiersin.org\ncombined score of >0.4 were selected for further analysis. Cytoscape\n(version 3.4.0) was utilized for network visualization.\n2.4 Spatiotemporal expression pattern\nanalysis\nSix healthy one-day-old H line hens were selected from the Henan\nAgricultural University resource field and raised in individual cages.\nThe neck feather follicle tissues were collected from the animals at 1d,\n2, 4, 6, 8, 10, and 12 weeks of age, separated, and frozen in liquid\nnitrogen for preservation.\n2.5 Tyrosine treatment of chicken\nmelanocytes\nChicken primary melanocytes were isolated and cultured as\ndescribed previously (25). Melanocytes were isolated from the\nperitoneum of Xichuan black-bone chickens at 20 embryonic days.\nThe tissue was washed with PBS containing antibiotics, minced, and\ndigested with dispase II and trypsin–EDTA at 37 °C for 1 h. The\ndigestion was halted by adding supplemented Medium 254. The cell\nsuspension was then filtered through a series of meshes, centrifuged,\nand the resulting pellet was resuspended to obtain the melanocytes,\nwhich were cultured at 37 °C with 5% CO₂. Cells (1 × 104/ml) were\ngrown in normal melanocyte medium, whereas experimental\nmelanocytes were grown in media supplemented with various\nconcentrations of tyrosine (10−9, 10−8, 10−7, and 10−6) mol/L for 3 days.\nThe medium was replaced daily with fresh medium containing the\ncorresponding tyrosine concentrations. After the treatment period,\ncells were collected and stored at −80 °C.\n2.6 qRT–PCR\nRNA was extracted from cells and tissues using TRIzol as above\nand was reverse-transcribed to cDNA using the Qiagen Reverse\nTranscription Kit. Primer Premier 6.0 was utilized for primer design;\nprimers were synthesized by Shanghai Sangon Bioengineering\nCorporation Ltd. (Shanghai, China) and the sequences are given in\nSupplementary Table S2. The internal reference was GAPDH. A series\nof qRT–PCRs were conducted using 10 ul of SYBR master mix\n(Vazyme, Nanjing, China), 2 ul of cDNA, 7 ul of ultrapure water, and\n0.5 ul of each primer (10 mM). The qRT–PCR program comprised\n\nThe internal reference was GAPDH.A series\nof qRT–PCRs were conducted using 10 ul of SYBR master mix\n(Vazyme, Nanjing, China), 2 ul of cDNA, 7 ul of ultrapure water, and\n0.5 ul of each primer (10 mM).The qRT–PCR program comprised\npredenaturation for 30 s at 95 °C, 40 3-s cycles at 95 °C and 30 s at\n60 °C. The results were calculated as relative expression levels using\nthe 2−ΔΔCt method (30).\n2.7 Statistical analysis\nGraphPad Prism 9 was utilized to calculate, analyze, and plot all\nthe data. Data are expressed as the mean ± standard error of the mean\n(S. E. M.). One-way ANOVA and Student’s t-test were used for\nstatistical analysis. Data are shown as means ± standard errors with\np < 0.05 considered statistically significant.\n3 Results\n3.1 Feather color analysis after tyrosine\nfeeding to H line chickens\nAn experiment in which tyrosine was added to the feed of H line\nhens revealed that a higher tyrosine concentration in the feed was\nassociated with a lower feather L* value, that is, darker black spots. In\naddition, a longer duration of tyrosine feeding was correlated with a\nlower feather color L* value (Table 1). Feather melanin spot L* values\nwere significantly lower in chickens fed a 1.0% tyrosine diet compared\nto those on a tyrosine-free diet after 40 days (p < 0.05) (Figure 2).\nFurthermore, within the 1.0% tyrosine group, the L* value decreased\nprogressively over time, with the value at 40 days being significantly\nlower than at the start of the experiment (p < 0.05). This study found\nthat feeding H line chickens with a diet containing 1.0% tyrosine for\n40 days significantly increased the deposition of melanin in their\nfeathers (p < 0.05).\n3.2 Descriptive statistics of the RNA-seq\ndata\nFollicle tissue gene expression in the C1, C2, C3, T1, T2, and T3\ngroups were analyzed by RNA-seq, and 43.04 ~ 44.07 MB of filtered\nclean reads were obtained. The per-sample mapping rate against the\nreference genome was between 82.40 and 86.95%. The average Q20\nTABLE 1  Effects of dietary tyrosine concentration and supplementation duration on feather melanin spot L* values.\nContent\nTime\n40d\n40d\n40d\n40d\n0\n69.380 ± 7.397\n68.110 ± 6.714\n\n40 and 86.95%.The average Q20\nTABLE 1  Effects of dietary tyrosine concentration and supplementation duration on feather melanin spot L* values.Content\nTime\n40d\n40d\n40d\n40d\n0\n69.380 ± 7.397\n68.110 ± 6.714\n64.683 ± 7.526\n65.447 ± 6.207A\n0.4%\n64.258 ± 9.188\n63.899 ± 9.448\n60.198 ± 10.579\n58.801 ± 8.835AB\n0.6%\n70.033 ± 9.334a\n64.581 ± 8.718ab\n61.232 ± 10.377b\n58.764 ± 10.688bAB\n0.8%\n66.801 ± 10.682a\n63.375 ± 11.080ab\n61.150 ± 12.767ab\n57.130 ± 12.945bB\n1.0%\n67.438 ± 9.035a\n61.072 ± 7.268ab\n56.889 ± 9.197b\n54.572 ± 5.715cB\nValues in the table represent the mean L* (lightness) of melanin spots measured by a colorimeter (n = 200, 40 per group). L* represents the intensity of brightness; a higher value indicates a\nless blackness, and vice versa. In the same row, values with different small letter superscripts mean significant difference (p < 0.05); While with the same small letter superscripts mean no\nsignificant difference (p > 0.05). In the same column, values with different uppercase letters superscripts mean significant difference (p < 0.05); While with the same uppercase letter\nsuperscripts mean no significant difference (p > 0.05).\n\nWang et al.\b\n10.3389/fvets.2025.1720520\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nwas\n96.47%,\nand\nthe\naverage\nQ30\nwas\n87.72%\n(Supplementary Table S3). Thus, the quality of the data was sufficient\nfor further analysis.\n3.3 DEG analysis\nTo clarify the pathways underlying the effects of tyrosine on\nfeather color, DEGs between the control and tyrosine-fed follicles were\nanalyzed. Overall, 13,956 genes were detected, from which 314 DEGs\nwere identified between the C and T groups (p < 0.05). Of these, 116\nwere upregulated and 198 were downregulated in the T group\n(Figure 3). Notably, these DEGs included two marker genes of melanin\ndeposition, EDNRB2 and MLPH.\n3.4 Verification by qRT-PCR\nThe accuracy and reliability of the RNA-seq data were verified by\nrandomly selecting six genes (WNT11, WNT3, GNAQ, PVALB,\nEDNRB2, and PRKCB) for qRT-PCR analysis. Assessment of\nexpression and comparison with the RNA-seq data demonstrated the\ncredibility of the RNA-seq data (Figure 4).\n3.5 GO analysis\nThe functions of the DEGs were analyzed further by GO\nenrichment. The GO classification system encompasses three primary\ncategories, namely, biological process (BP), cellular component (CC),\nand molecular function (MF). Enrichment in the BP category\nprimarily involved “cellular process,” “cell,” and “cell part,” while in MF,\nthe most enriched subcategories were “binding,” “protein binding,”\nand “organic cyclic compound binding,” and in CC, “cell,” “cell part,”\nand “membrane” (Figure 5A). Notably, the DEGs observed among H\nline hens fed tyrosine were enriched mainly in BP (cellular process,\nbiological regulation and regulation of biological process). Moreover,\nthe DEGs showed marked enrichment in multicellular biological\nprocesses, transmembrane signaling receptor activity, and signaling\nreceptor activity (p < 0.05) (Figures 5B,C).\n3.6 KEGG analysis\nAs shown in Figure 6A, 72 DEGs were enriched in 5 KEGG Class\nA pathways and 16 KEGG Class B pathways. The KEGG enrichment\ncircle plot revealed that the DEGs were the most enriched in\nneuroactive ligand–receptor interactions, the MAPK pathway and\ncytokine–cytokine receptor interactions (Figure 6B). Specifically,\n\nThe KEGG enrichment\ncircle plot revealed that the DEGs were the most enriched in\nneuroactive ligand–receptor interactions, the MAPK pathway and\ncytokine–cytokine receptor interactions (Figure 6B).Specifically,\ntyrosine supplementation resulted in significant changes in 4 pathways\n(p  < 0.05), namely, “neuroactive ligand–receptor interactions,”\n“cytokine–cytokine receptor interactions,” “the intestinal immune\nnetwork for IgA production,” and “valine, leucine and isoleucine\nbiosynthesis” (Figure 6C). As indicated in Supplementary Table S4,\nthe DEGs were associated with melanin-related pathways such as\nmelanogenesis, the MAPK, Wnt, cAMP, PI3K-Akt, Notch, TGF-β,\ncalcium, and mTOR pathways, as well as adrenergic signaling in\ncardiomyocytes. Notably, the network plot revealed relationships\namong the melanin-associated pathways involved in melanogenesis,\nMAPK and Wnt signaling; therefore, we focused on the 7 DEGs in\nthese pathways (EDNRB2, WNT3, POMC, INS, FLT3, CACNA2D3,\nand CACNA1I; Figure 6D), which were likely to be involved in\ntyrosine-mediated feather color development (Figure 7).\n3.7 DEG PPI network\nThe network consisted of 160 proteins and 172 interactions\n(Figure 8), among which the CD79B, IL21R, CXCR5, POU2AF1,\nCD72AG, CD72, PAX5, and VPREB3 proteins participated in more\nthan 10 interactions. Notably, these networks are enriched in proteins\ninvolved in several melanin-related pathways. For example, POMC\ninteracts with NPFFR1, INS, SSTR1, AGT, SSTR3, POU1F1, and\nTAC1, of which POMC participates in melanogenesis and INS\nparticipates in the MAPK and mTOR pathways. In addition, EDNRB2\ninteracted with MLPH, and WNT3 interacted with FGF16; among\nFIGURE 2\nPhenotypic effect of tyrosine on feather color. Representative feathers from the dorsal neck of hens fed a control (0% tyrosine) (A) or a 1.0% tyrosine-\nsupplemented (B) diet for 40 days.\n\nWang et al.\b\n10.3389/fvets.2025.1720520\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nthese, EDNRB2 and WNT3 showed enrichment in melanogenesis,\nwhereas FGF16 showed enrichment in the MAPK pathway. Moreover,\nACTC1, which is enriched in adrenergic signaling in cardiomyocytes,\ninteracts with MYOG, CENPA, MYH1A, and MYBPC2; GRIN1,\nwhich is enriched in the calcium signaling pathway, interacts with\nGRIN2B, ARC, GRM3, and CAMKV. In addition, CACNA2D3 and\nFLT3, which are enriched in the MAPK signaling pathway, are\nconnected to TENM4 and FGF16, respectively.\n3.8 Temporal expression analysis of\nEDNRB2\nOn the basis of the enrichment results of the KEGG pathways and\nPPIs related to the DEGs, we observed that EDNRB2 is not only\nannotated in the melanogenesis pathway but also interacts with the\nmelanosome transport protein MLPH. To investigate the temporal\nexpression characteristics of EDNRB2 at 1 d, 2 w, 4 w, 6 w, 8 w, 10 w,\nand 12 w in H line hens, we performed qRT–PCR with cDNA samples\nfrom neck follicle tissues at each time point (Figure 9). Notably, the\nexpression levels of EDNRB2 were the highest at 10 W, and the\nexpression levels were significantly different from those at other time\npoints (p < 0.01).\n3.9 Analysis of EDNRB2 expression in\nchicken melanocytes after tyrosine\nsupplementation\nIn this study, chicken primary melanocytes were selected, and the\nexpression of EDNRB2 was verified by adding different concentrations\nof tyrosine to the cells. It was found that EDNRB2 levels in chicken\nmelanocytes were highest when the tyrosine concentration was\n10−6 mol/L, and the differences were markedly greater than those of\nthe control (0 mol/L) and 10−9, 10−8, and 10−7 mol/L treatment groups\n(p < 0.05) (Figure 10).\n4 Discussion\nThe color of chicken feathers is influenced not only by genetic\nfactors but also by nutritional factors. Although many of the genes\nrelated to feather colors have been characterized, the molecular\nmechanisms controlling melanin deposition in feathers in relation to\ndietary nutrient intake have not yet been fully explored. Tyrosine is\nknown to be associated with melanin formation. Studies have shown\n\nTyrosine is\nknown to be associated with melanin formation.Studies have shown\nthat feeding tyrosine can increase the melanin content in animals, but\nthere is no relevant research indicating that tyrosine can increase\nmelanin in chicken feathers. In this study, we allocated chickens with\nFIGURE 3\nFollicle tissue DEGs between the C and T groups. (A) Up- and down-regulated gene numbers. (B) Volcano plot of genes identified by RNA-seq,\nincluding both upregulated and downregulated genes.\nFIGURE 4\nqRT–PCR verification of the RNA-seq data (n = 3).\n\nWang et al.\b\n10.3389/fvets.2025.1720520\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nsub-Columbian plumage to different tyrosine supplementation groups\nto determine the optimal dosage and feeding time for inducing black\nspots in feathers. The results indicated that dietary tyrosine affects the\nmelanin content in feathers in a concentration-dependent manner.\nSpecifically, when the tyrosine content in the feed is 1.0% and the\nfeeding duration is 40 days, it can significantly increase the melanin\ncontent in the follicles (p < 0.05). Similarly, in previous work, dietary\nsupplementation with 0.8% tyrosine was found to promote the\ndeposition of melanin in the pectoral muscles of the Xichuan black-\nbone chicken (18). This study provides guidance for the nutritional\nregulation of feather color in the H line chickens and the breeding of\nexcellent germplasm. These findings provide a theoretical foundation\nfor the development and utilization of tyrosine in improving melanin\ndeposition in chicken feathers.\nIndeed, deciphering the effects of tyrosine-mediated gene\nupregulation or downregulation on feather follicle melanin deposition\nrepresents an interesting topic. The elucidation of the mechanisms\nresponsible for melanin deposition in chicken feathers through\ntyrosine is extremely important for the development and utilization of\nsub-Columbian plumage. Transcriptome sequencing is a high-\nthroughput method used to examine RNA molecules in tissues, with\na focus on gene expression patterns and changes. Because black spots\nare present on the necks of chickens with sub-Columbian plumage, we\nselected a blank control group and an optimal feeding group of\nchicken neck follicle tissues for transcriptome sequencing to analyze\nthe molecular mechanism through which tyrosine promotes melanin\ndeposition in feathers. The marker gene MLPH, which is associated\nwith melanin deposition, was detected in our transcriptome\nsequencing data. MLPH is a structural protein that facilitates the\ntransport of mature melanosomes in melanocytes, which can cause\nmelanosomes to aggregate in the dendrites of melanocytes and\n\nMLPH is a structural protein that facilitates the\ntransport of mature melanosomes in melanocytes, which can cause\nmelanosomes to aggregate in the dendrites of melanocytes and\nthereby regulate animal skin and coat color (31, 32). The expression\nof MLPH varies across tissues, as mature melanosomes are transported\nto dendritic terminals within melanocytes for short-distance\nmovement, which is correlated with the expression levels and activity\nof the Rab27a, MLPH, and MyoVa ternary complex (33). Mutations\nin human MLPH can lead to Griscelli syndrome, which is\ncharacterized by abnormal skin pigmentation and light silver–white\nFIGURE 5\nGO enrichment analysis of DEGs. (A) DEG GO classifications. (B) Bubble diagram showing top 20 enriched GO terms. (C) GO enrichment circle.\n\nWang et al.\b\n10.3389/fvets.2025.1720520\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nFIGURE 6\nKEGG analysis of DEGs. (A) DEG enrichment in KEGG Class A and B pathways. (B) KEGG enrichment circle. (C) Bubble diagram showing top 20 KEGG \npathways. (D) Network plot of the interactions between different pathways.\nFIGURE 7\nThe mechanism of action of tyrosine on feather coloration. Red color indicates DEGs, while black represents non-DEGs.\n\nWang et al.\b\n10.3389/fvets.2025.1720520\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nhair starting in infancy (34). The lavender diluted feather phenotype\nin chickens, akin to that of Japanese quail, is due to mutations in\nMLPH (35, 36). Combined with previous results, we hypothesized that\nthe MLPH expression level is associated with tyrosine-mediated\nmelanin deposition in the chicken.\nEnrichment analyses were performed to elucidate DEG\nfunctions. KEGG pathway analysis revealed that the DEGs\nEDNRB2, WNT3, POMC, INS, FLT3, CACNA2D3, and CACNA1I\nwere involved in melanogenesis and MAPK and Wnt signaling, all\nof which are known to be interconnected with each other.\nMelanogenesis is a complex biosynthetic process in melanocytes\nthat leads to the production of melanin pigments through a series\nof enzymatic and chemically catalyzed reactions (37). EDNRB2 and\nPOMC both showed enrichment in the melanogenesis pathway,\nand their involvement in melanin production is well-documented.\nPOMC is mainly secreted in the hypothalamus and acts as a\nprecursor polypeptide hormone and is associated with the\nmorphological differentiation and the production of melanin in\nB16-F10 melanoma cells (38, 39). Moreover, POMC is converted\ninto hormones derived from it, including ACTH and α-MSH (39).\nα-MSH increases peroxide levels in melanocytes to stimulate the\nsynthesis of melanin (40). MITF is a melanocyte-specific\ntranscription factor that can directly bind and activate the\ntyrosinase promoter, upregulating tyrosinase levels and promoting\nmelanin production (41). The Wnt axis is known to regulate MITF\nexpression (42). This involves Wnt binding to the G-protein\ncoupled receptor Frizzled, inactivating GSK3β and ultimately\nresulting in β-catenin accumulation (43–46). This β-catenin\nundergoes nuclear translocation where it binds to Lef-TCF, thereby\nincreasing MITF expression and stimulating melanin synthesis (47,\nFIGURE 8\nPPI network of DEGs. Nodes in the PPI network are colored on the basis of degree, with darker nodes indicating a greater degree.\n\nWang et al.\b\n10.3389/fvets.2025.1720520\nFrontiers in Veterinary Science\n10\nfrontiersin.org\n48). Wnt3 is part of a group of approximately 20 ligands involved\nin the Wnt axis, influencing cell proliferation and differentiation\n(49). Therefore, we speculate that WNT3 may promote the\nproliferation of melanocytes through the WNT signaling pathway.\nIn addition, an interaction between Wnt3 and FGF16 was found\nwhen comparing the C and T groups. FGF family proteins are\ntypically involved in cell proliferation, differentiation, and survival,\nand it is thus suggested that an interaction between Wnt3 and\nFGF16 may promote proliferation (50). The MEK and ERK kinases\nassociated with the MAPK axis are known to activate melanocyte\nreceptors (51). Ligands bind to the extracellular domain of the\nreceptor, activating a cascade (Ras–Raf–MEK–ERK) that leads to\nthe upregulation of MITF (51). P38 phosphorylation induces MITF\nexpression, in turin upregulating proteins associated with melanin\nproduction and thus melanin synthesis (52). The INS gene encodes\ninsulin, which activates the insulin signaling pathway during the\nprocess of cell differentiation, thereby promoting cell maturation\n(53). FLT3 is a type III receptor tyrosine kinase, and its ligand can\ninduce dimerization and activate its intrinsic tyrosine kinase\nactivity, leading to autophosphorylation and the initiation of\nseveral signaling cascades, including the MAPK signaling pathway\n(54, 55). CACNA2D3 and CACNA1I are both gene families\nassociated with calcium channels that play key roles in the\nregulation of intracellular calcium ion concentration, influencing\ncell proliferation and differentiation by affecting the MAPK\nsignaling pathway (56). Thus, we speculate that these DEGs are\ninvolved in melanin production, although the precise genetic\nprocess requires further research.\nAmong the DEGs identified here, EDNRB2 is worthy of\nattention because it not only participates in melanin-related\npathways but also interacts with the MLPH protein. EDNRB2, an\nEDNRB paralog, encodes a seven-transmembrane-domain G\nprotein-coupled receptor (26, 27, 57, 58). EDNRB2 dysfunction\n\nEDNRB2, an\nEDNRB paralog, encodes a seven-transmembrane-domain G\nprotein-coupled receptor (26, 27, 57, 58).EDNRB2 dysfunction\naffects pigment synthesis, leading to white fur and pink skin (59–\n61). EDNRB2 is also associated with the migration and\ndifferentiation of melanocytes and has been linked to plumage color\nin quail, chickens, and domestic ducks (7, 27, 61, 62). Chicken\nfeathers from the embryonic stage to adulthood undergo 3 ~ 4\ngenerations of replacement, and we call this phenomenon\nproduction molting; after many molts, the feather color becomes\nstable. At 55 days of age, the third-generation feathers (young\nfeathers) of chickens begin to appear, and the feather color on the\nbody surface of chickens is mature or nearly mature and tends to be\nstable (63). Our own observations in the H-line chickens are\nconsistent with this timeline: starting from yellow chick down, the\nchickens develop their characteristic sub-Columbian plumage\npattern through successive molting, with the definitive coloration\nbecoming stable by approximately 10 weeks of age. This\ndevelopmental timeline explains the peak in EDNRB2 expression at\n10 weeks within the 0-12w expression profile, suggesting a crucial\nrole for EDNRB2 in the formation of the sub-Columbian plumage\npattern and the associated melanin deposition process.\nThe black pigment in the feathers of chickens is produced in\nmelanocytes through a series of biochemical reactions involving\ntyrosine, and many studies have indicated that adding tyrosine to\nmelanocytes can promote melanin deposition (64). For example,\nthe addition of tyrosine to culture medium promoted melanin\nsynthesis in mouse B16 melanoma cells (65). Adding\n400 μM L-tyrosine to Ham’s F-10 medium led to the induction of\nmelanin pigment production in human melanoma cells (66).\nTreating in vitro-induced primary chicken melanocytes with\nvarying amounts of tyrosine promoted melanin proliferation and\nincreased the tyrosinase content in the cells (18). Here, marked\nincreases in the levels of EDNRB2 were observed after the addition\nof 10−6 mol/L tyrosine to chicken primary melanocytes. However, a\n\nHere, marked\nincreases in the levels of EDNRB2 were observed after the addition\nof 10−6 mol/L tyrosine to chicken primary melanocytes.However, a\nprevious study reported that the highest expression level of\nEDNRB2 in chicken primary melanocytes was observed when\n10−9  mol/L tyrosine was added, indicating that the optimal\nconcentration for increasing EDNRB2 may be related to the cell\nstate and culture conditions (18). In summary, we confirmed that\nexogenously added tyrosine can promote the expression of EDNRB2\nat the cell level, thereby facilitating melanin deposition.\nFIGURE 9\nExpression profile of EDNRB2 in the follicle tissue of H line hens at\ndifferent stages. Data are expressed as mean ± SD (n = 6). Different\nuppercase letters within groups indicate highly significant differences\n(p < 0.01).\nFIGURE 10\nExpression profile of EDNRB2 in primary melanocytes of chickens\ntreated with different concentrations of tyrosine. Data are expressed\nas mean ± SD (n = 3). Different lowercase letters within groups\nindicate significant differences (p < 0.05).\n\nWang et al.\b\n10.3389/fvets.2025.1720520\nFrontiers in Veterinary Science\n11\nfrontiersin.org\n5 Conclusion\nThe results of our study reveal that a 40-day diet with 1.0%\ntyrosine can significantly intensify the darkness of black spots on\nthe feathers of sub-Columbian (p < 0.05). To further explore the\nmechanism of action, we conducted transcriptomic sequencing\nanalysis on the feather follicle tissues of the control group and the\ntyrosine treatment group (which received a diet supplemented\nwith 1.0% tyrosine for 40 days) using RNA-seq technology.\nTyrosine induction of melanin deposition in follicular tissues of\nchickens with sub-Columbian plumage might be significantly\naffected by the EDNRB2-mediated regulatory network. These\nresults advance the knowledge of the molecular regulation of\nmelanin deposition in sub-Columbian plumage, establishing a\nbasis for feeding and breeding practices to modulate melanin\nlevels in H-line chickens.\nData availability statement\nThe datasets supporting the conclusions of this article are included\nwithin the article and its additional files. Transcriptome sequencing\ndata were deposited in the NCBI SRA database (SRA accession:\nPRJNA578584).\nEthics statement\nAll animal experimental procedures were approved by the\nExperimental Animal Ethics Review Committee (HNUAHEER\n2425105) at Henan University of Animal Husbandry and Economy.\nThe studies were conducted in accordance with the local legislation\nand institutional requirements. Written informed consent was\nobtained from the owners for the participation of their animals in\nthis study.\nAuthor contributions\nXW: Data curation, Project administration, Validation, Formal\nanalysis, Methodology, Conceptualization, Writing  – original\ndraft, Investigation, Writing – review & editing. LZ: Supervision,\nWriting – review & editing, Project administration, Data curation,\nConceptualization. LY: Data curation, Writing – review & editing,\nMethodology, Validation, Formal analysis, Conceptualization. PY:\nConceptualization, Investigation, Writing  – review & editing,\nData curation, Validation, Methodology. YQ: Data curation,\n\nLY: Data curation, Writing – review & editing,\nMethodology, Validation, Formal analysis, Conceptualization.PY:\nConceptualization, Investigation, Writing  – review & editing,\nData curation, Validation, Methodology.YQ: Data curation,\nConceptualization, Formal analysis, Writing – review & editing,\nValidation,\nMethodology.\nZH:\nInvestigation,\nProject\nadministration, Writing  – review & editing, Data curation,\nMethodology. JF: Data curation, Conceptualization, Writing –\nreview & editing, Methodology, Investigation. QL: Supervision,\nWriting  – review & editing, Formal analysis, Methodology,\nInvestigation, Conceptualization. DZ: Writing – review & editing,\nMethodology, Investigation, Conceptualization, Validation,\nFormal analysis. ZL: Methodology, Data curation, Project\nadministration,\nConceptualization,\nFunding\nacquisition,\nResources, Writing  – review & editing, Supervision. XK:\nInvestigation, Conceptualization, Resources, Funding acquisition,\nWriting – review & editing, Project administration, Methodology,\nSupervision. JD: Formal analysis, Validation, Methodology,\nWriting – review & editing, Conceptualization. RL: Data curation,\nMethodology, Conceptualization, Validation, Investigation,\nWriting  – review & editing, Writing  – original draft, Project\nadministration, Resources.\nFunding\nThe author(s) declare that financial support was received for\nthe research and/or publication of this article. This work was\nsupported by the China Agriculture Research System of MOF and\nMARA (CARS-40), the Key Research Project of the Shennong\nLaboratory (SN01-2022-05), Modern Agricultural Industry\nTechnology System of Henan Province (HARS-22-18-G3), and the\nScience and Technology Project of Henan province of China\n(242102110069).\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nThe reviewer DW declared a past co-authorship with the authors\nZL, XK to the handling editor.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\n\nThe reviewer DW declared a past co-authorship with the authors\nZL, XK to the handling editor.Generative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the\nauthors and do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. Any product that may be evaluated in this article, or\nclaim that may be made by its manufacturer, is not guaranteed or\nendorsed by the publisher.\nSupplementary material\nThe Supplementary material for this article can be found online\nat: https://www.frontiersin.org/articles/10.3389/fvets.2025.1720520/\nfull#supplementary-material\n\nWang et al.\b\n10.3389/fvets.2025.1720520\nFrontiers in Veterinary Science\n12\nfrontiersin.org\nReferences\n1.\tMata-Estrada, A, González-Cerón, F, Pro-Martínez, A, Torres-Hernández, G,\nBautista-Ortega, J, Becerril-Pérez, CM, et al. Comparison of four nonlinear growth\nmodels in creole chickens of Mexico. Poult Sci. 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+{"text": "【兽医文献】\n标题: Schisandrin B alleviates high-glucose diet-induced oxidative stress, inflammation, and tissue injury in zebrafish and is associated with alterations in intestinal microbiota\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nTemperature-responsive hydrogel\nof oclacitinib maleate\nadministered via the rectum in\nrabbit\nPanPan Guo 1†, Yang Yang 1†, Zhongsheng Zhang 1, Yuxin Guo 1,\nMuhammad Mohsin 2, Haiyan Wu 1, Dengfeng Wang 1,\nGuangwen Yin 1* and Lei Wang 1*\n1College of Animal Sciences, Fujian Agriculture and Forestry University, Fuzhou, China, 2Academy of\nIntegrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, China\nBackground: The extensive and intense itching caused by skin diseases can sig­\nnificantly diminish the quality of life for companion animals. Oral administration\nof oclacitinib maleate is recognized as an important treatment for alleviating\nitching. Oral medications are affected by the animal’s temperament and swal­\nlowing ability.\nMethods: In this study, we developed a temperature-responsive hydrogel con­\ntaining oclacitinib maleate for rectal administration, and we evaluated its thera­\npeutic potential for treating itching through pharmacokinetic analysis using\nrabbits as test subjects.\nResults: Pharmacokinetic results indicated that the transrectal administration of\nOM T-R Hydrogel effectively increased the blood concentration of oclacitinib\nmaleate (Tmax = 87 min, Cmax = 37 ng/mL, t1/2λz = 762 min). Furthermore, the OM\nT-R Hydrogel demonstrated no rectal mucosal irritation, confirming its safety for\nclinical use.\nConclusion: In conclusion, this paper successfully investigated a promising novel\ndrug delivery system to alleviate pruritus in dogs and cats.\nKEYWORDS\nhydrogel, oclacitinib maleate, pharmacokinetics, rectal administration,\ntemperature-responsive\n1 Introduction\nSkin diseases are among the most prevalent clinical conditions observed in pets. The etiol­\nogy of these diseases is complex and includes factors such as dermatophytes, parasites, and\nautoimmune responses (1–3). Pruritus is one of the most common symptoms associated with\ndermatological conditions in pets, and widespread, intense itching significantly impacts their\noverall quality of life (4, 5). Therefore, pruritus management is crucial for ensuring animal\n\nPruritus is one of the most common symptoms associated with\ndermatological conditions in pets, and widespread, intense itching significantly impacts their\noverall quality of life (4, 5).Therefore, pruritus management is crucial for ensuring animal\nwelfare. In the pet clinic, the use of anti-itch medications is the most common method of\ncontrolling itching (6). Oclacitinib maleate (OM, Apoquel®), a Janus kinase (JAK) inhibitor,\nhas demonstrated efficacy in alleviating itching associated with various allergies, including flea,\nfood, and contact allergies, by down-regulating the expression of several inflammatory cyto­\nkines linked to JAK 1 (7–9). In veterinary practice, oral administration of OM is commonly\nemployed due to its simplicity and stability as a drug delivery method (10, 11). Frequent oral\ndosing of OM in pets with severe itching creates notable obstacles related to convenience and\nTYPE  Original Research\nPUBLISHED  11 February 2026\nDOI  10.3389/fvets.2026.1767033\nOPEN ACCESS\nEDITED BY\nArturo Anadón,\nComplutense University of Madrid, Spain\nREVIEWED BY\nLeah Swanson,\nPet Poison Helpline, United States\nRunan Zuo,\nAnhui Agricultural University, China\n*CORRESPONDENCE\nGuangwen Yin\nyinguangwen000@sina.com\nLei Wang\ndkwangl@fafu.edu.cn\n†These authors have contributed equally\nto this work\nRECEIVED 13 December 2025\nREVISED 21 January 2026\nACCEPTED 26 January 2026\nPUBLISHED 11 February 2026\nCITATION\nGuo P, Yang Y, Zhang Z, Guo Y,\nMohsin M, Wu H, Wang D, Yin G and\nWang L (2026) Temperature-responsive\nhydrogel of oclacitinib maleate\nadministered via the rectum in rabbit.\nFront. Vet. Sci. 13:1767033.\ndoi: 10.3389/fvets.2026.1767033\nCOPYRIGHT\n© 2026 Guo, Yang, Zhang, Guo, Mohsin,\nWu, Wang, Yin and Wang. This is an\nopen-access article distributed under\nthe terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other\nforums is permitted, provided the\noriginal author(s) and the copyright\nowner(s) are credited and that the\noriginal publication in this journal is\ncited, in accordance with accepted\nacademic practice. No use, distribution\nor reproduction is permitted which does\nnot comply with these terms.\n\nGuo et al.\b\n10.3389/fvets.2026.1767033\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nease of administration (12). However, it is essential to consider that\nthe temperament and swallowing abilities of dogs and cats are signifi­\ncant factors influencing the success of oral administration (13).\nSpecifically, irritable or sensitive pets may resist oral medication and\nspit out the drug, while those with oral or esophageal diseases, or neu­\nrological disorders that impair the swallowing reflex, are also unable\nto receive adequate medication via the oral route (14, 15). On the\nother hand, in cases involving irritable animals, the risk of bites to the\nveterinarian increases substantially (16). As a result, rectal administra­\ntion has emerged as a viable alternative for drug delivery that warrants\nattention from veterinarians (17–19).\nIn contrast to humans, animals lack conscious control of the\nexternal anal sphincter during rectal drug delivery, leading to a higher\nchance of medication leakage. Temperature-Responsive Hydrogel\n(T-R Hydrogel) is a specialized type of hydrogel that can alter its phys­\nical state in response to temperature stimuli (20). Due to its unique\nphysicochemical properties, the T-R hydrogel finds extensive applica­\ntions in the field of biomaterials (21–23). In this study, we developed\nan Oclacitinib Maleate T-R Hydrogel (OM T-R Hydrogel), which\nexists as a liquid at room temperature and transitions to a semi-solid\nstate at a phase change temperature of 38 °C. Our objective was to\nleverage the bioadhesive properties of semi-solid hydrogels to mini­\nmize drug leakage during rectal administration. To further investigate\nthe feasibility of OM T-R Hydrogel for clinical use in veterinary medi­\ncine, we conducted an exploratory study utilizing rabbits as experi­\nmental animal. Following the injection of OM T-R Hydrogel into the\nrectum via a catheter, venous blood samples were collected at various\ntime intervals. Moreover, the concentration of OM in rabbits was\ndetermined by liquid chromatography-mass spectrometry.\n2 Materials and methods\n2.1 Animals and reagents\n\nMoreover, the concentration of OM in rabbits was\ndetermined by liquid chromatography-mass spectrometry.2 Materials and methods\n2.1 Animals and reagents\nRabbits and mice were obtained from the Meat Rabbit Farm of\nFujian Agriculture and Forestry University and HFK Bio-Technology\nCo., Ltd. (Beijing, China), respectively, and the animal experiment was\napproved by the Ethics Committee of Fujian Agriculture and Forestry\nUniversity (FAFU-2023-0065). Oclacitinib Maleate, Poloxamer 407\n(P407), Poloxamer 188 (P188), Hydroxypropyl methyl cellulose\n(HPMC), Rhodamine B were purchased from Beijing Psaitong\nBiotechnology Co., Ltd. (Beijing, China). Hydroxypropyl-β-\nCyclodextrin (HP-β-CD) was purchased from Sangon Biotechnology\nCo., Ltd. (Shanghai, China).\n2.2 Preparation and characterization of OM\nT-R hydrogel\nA total of 30 mg of OM was accurately weighed and dissolved in\n30 mL of ultrapure water to prepare an aqueous solution of OM at a\nconcentration of 1 mg/mL. Subsequently, 0.132 g (0.44%, w/v) of\nHPMC was weighed as a mucosal adhesive, and 0.75 g (2.5%, w/v) of\nHP-β-CD was weighed as an absorption enhancer; both were thor­\noughly dissolved in the aqueous OM solution (24, 25). Following this,\n5.4 g (18%, w/v) of P407 and 1.2 g (4%, w/v) of P188 were added. The\nmixture was refrigerated at 4 °C for 24 h to ensure complete dissolu­\ntion, resulting in the formation of the OM hydrogel.\nThe prepared T-R Hydrogel and OM T-R Hydrogel were lyophi­\nlized separately for 48 h. Subsequently, they were subjected to further\nobservation using scanning electron microscopy and Fourier trans­\nform infrared spectroscopy. Additionally, we conducted temperature\nresponsiveness validation of the OM T-R Hydrogel, which included\nboth in vitro and in vivo assessments. The in vitro temperature respon­\nsiveness validation was executed by establishing a temperature gradi­\nent of 37 °C to 42 °C using a water bath, with three batches undergoing\nmultiple repetitions. The in vivo temperature responsiveness valida­\ntion involved the injection of rhodamine B, dissolved in the OM T-R\n\nThe in vivo temperature responsiveness valida­\ntion involved the injection of rhodamine B, dissolved in the OM T-R\nHydrogel, into the rectum of rabbits, followed by observation for any\nleakage within a 10 min period.\n2.3 Animal experiment\nPrior to the commencement of the animal experiments, we col­\nlected 2 mL of venous blood for backup purposes. Subsequently, eight\nrabbits were weighed and randomly assigned to two groups: the oral\ndrug group (O group, n = 3) and the rectal drug group (R group,\nn = 5). The OM was administered at a dosage of 1 mg/kg. Prior to drug\nadministration, the rabbits were fasted for 18 h, and it was confirmed\nvia X-ray that no fecal residue was present in the colonic and rectal\nsegments. Following drug administration, a specific volume of air was\ninjected into the drug delivery tube using a syringe to expel the drug\nas thoroughly as possible.\nAt 25 min, 50 min, 1.5 h, 3 h, 6 h, 8 h, 10 h, and 12 h post-drug\nadministration, we collected venous blood into EDTA-K2 anticoagula­\ntion tubes. After centrifugation at 3500 g for 10 min at 4 °C, 50 μL of\nplasma was separated into polyethylene tubes. Protein precipitation\nwas achieved by adding 100 μL of ice-cold acetonitrile at −20 °C. The\nsamples were vortexed for 5 min and subsequently centrifuged at\n20000 g for 10 min at 4 °C. Following centrifugation, the supernatant\nwas transferred to a conical polypropylene tube designed for liquid\nchromatography-mass spectrometry (LC–MS), which was then\ncapped and placed in an autosampler for injection.\n2.4 Establishment of relevant parameters of\nliquid chromatography-mass spectrometry\nand standard curves\nThis method was established based on the study conducted by\nFerrer et al. (26) The chromatographic column used was a ThermoFisher\nAccucore C18 (2.6 μm, 4.1 × 150 mm) with a column temperature of\n40 °C and an autosampler set at 8 °C. The mobile phase flow rate was\nmaintained at 0.8 mL/min and consisted of a gradient mixture of ace­\ntonitrile and water, both containing 0.1% formic acid, in the following\nratios: the initial condition was 5% acetonitrile and 95% water. After the\n\nThe mobile phase flow rate was\nmaintained at 0.8 mL/min and consisted of a gradient mixture of ace­\ntonitrile and water, both containing 0.1% formic acid, in the following\nratios: the initial condition was 5% acetonitrile and 95% water.After the\ninjection of 4 μL, the ratio changed linearly to 60% acetonitrile within\n4 min, then increased to 90% acetonitrile within 0.1 min, which was\nheld for 6 min. Finally, it was returned to the initial condition at 6.1 min\nand held for 1.9 min. Under these conditions, the retention time of OM\nwas 3.62 min. The assay was conducted using positive electrospray ion­\nization (ESI+), and the quantitative analysis was monitored for transi­\ntions from 338.2 to 149.2 atomic mass units (amu).\nA precisely weighed 12 mg of OM was dissolved in 10 mL of dis­\ntilled water to prepare a stock solution. Subsequently, 1 mL of this\nstock solution was diluted to a final volume of 100 mL with distilled\nwater to obtain the working solution. A multiple dilution method was\nemployed to prepare various concentrations of the drug solution. Add\n\nGuo et al.\b\n10.3389/fvets.2026.1767033\nFrontiers in Veterinary Science\n03\nfrontiersin.org\n50 μL of blank serum to each of the different concentrations of drug\nsolutions. Mix thoroughly to prepare 10 concentration gradients of\nstandard solutions: 2000 ng/mL, 1,000 ng/mL, 500 ng/mL, 250 ng/\nmL, 125 ng/mL, 62.5 ng/mL, 31.25 ng/mL, 15.625 ng/mL, 7.8125 ng/\nmL, and 3.905 ng/mL. Subsequently, the standard solutions were pre­\npared according to the procedure outlined in section 2.3 for injection.\nConsidering the specific conditions of this test, a concentration range\nof 250 ng/mL to 3.905 ng/mL was ultimately selected to establish a\nstandard curve, which included a total of seven concentration points.\n2.5 Acute toxicity test of OM T-R hydrogel\nFor seven consecutive days, 0.5 mL of OM T-R Hydrogel was\nwithdrawn and administered rectally in mice, during which spillage\nof the hydrogel from the anus was observed. Tissue sections from the\nanal end of the rectum were prepared to histologically assess the irri­\ntation caused by OM T-R Hydrogel at the site of administration.\nAdditionally, serum samples were collected to measure ALT, AST,\nCREA, BUN, TBIL, and ALP to evaluate the hepatic and renal effects\nof OM T-R Hydrogel.\n2.6 Statistical analysis\nInter-group differences were assessed using one-way analysis of\nvariance (ANOVA) by Dunnett’s test. The experimental data pre­\nsented in this study are expressed as the mean ± standard deviation\n(SD) derived from a minimum of three independent experiments.\nStatistical significance was defined as *p < 0.05, **p < 0.01. The data\nwere analyzed and processed using Phoenix, Excel, and Origin 2024\nsoftware.\n3 Results\n3.1 Characterization of OM T-R hydrogel\nAt room temperature, the OM T-R Hydrogel exhibited a uniform\ntexture and good flowability (Figure 1A). The transformation of the\nT-R Hydrogel into a semi-solid state was successfully achieved by\nmaintaining it at 38 °C for 30 s. However, the OM T-R Hydrogel\nrequired 39 s at 38 °C to effectively accomplish the phase transition\n(Supplementary data). The results from the in vivo temperature respon­\n\nHowever, the OM T-R Hydrogel\nrequired 39 s at 38 °C to effectively accomplish the phase transition\n(Supplementary data).The results from the in vivo temperature respon­\nsiveness test indicated that the rabbits’ anal region was dry, and the\nsurrounding fur maintained a normal color without any signs of leak­\nage, based on the condition that the rabbits had not defecated. Scanning\nelectron microscopy (SEM) images revealed that T-R Hydrogel pos­\nsesses a three-dimensional mesh architecture characterized by numer­\nous pores and channels of varying sizes (Figure 1B), which is\nadvantageous for drug loading and diffusion. Fourier Transform\nInfrared Spectroscopy (FTIR) results indicated that both OM T-R\nHydrogel and T-R Hydrogel exhibited characteristic absorption bands\ncorresponding to -OH stretching vibrations (3200–3,600 cm−1), C-O\nstretching vibrations (1000–1,250 cm−1), C-H stretching vibrations\n(2800–3,000 cm−1), and bound water peaks. Furthermore, the wave­\nlengths of the other absorption peaks for both hydrogels were found to\nbe consistent (Figure 1C). This observation suggests that T-R Hydrogel\ndoes not chemically react with OM to form new chemical bonds,\nimplying that T-R Hydrogel functions solely as a delivery system.\n3.2 Pharmacokinetics\nThis exploratory study was conducted in 8 healthy rabbits. Prior\nto the study, clinical physical examination revealed no significant\nabnormalities. All rabbits showed no adverse effects at the end of the\nanimal experiment.\nTable 1 shows the changes in the plasma concentrations (ng/mL)\nof OM over time in 8 rabbits under oral administration (n = 3) and\nrectal administration (n = 5), respectively. The changes in the average\nconcentrations over time for the two groups are shown in Figure 2.\nThe main pharmacokinetic data for oral and rectal administration are\npresented in Table 2. The results indicate that for the oral administra­\ntion group (Group O), the Tmax was 42 min, the Cmax was 94 ng/mL,\nthe t1/2λz was 522 min, and the Cl-F was 163 mL/min; for the rectal\nadministration group (Group R), the Tmax was 87 min, the Cmax was\n\nThe results indicate that for the oral administra­\ntion group (Group O), the Tmax was 42 min, the Cmax was 94 ng/mL,\nthe t1/2λz was 522 min, and the Cl-F was 163 mL/min; for the rectal\nadministration group (Group R), the Tmax was 87 min, the Cmax was\n37 ng/mL, the t1/2λz was 762 min, and the Cl-F was 316 mL/min.\n3.3 Hepatorenal toxicity and rectal mucosal\nirritation associated with OM T-R hydrogel\nAfter the experiment, there were no significant abnormalities\nobserved in the mental status of the mice across all groups. We further\nvalidated the hepato- and nephrotoxicity associated with the overdose of\nOM T-R Hydrogel in mice by analyzing serum biomarkers, with results\npresented in Figure 3. When the mice were administered an overdose of\nOM T - R Hydrogel, the serum levels of ALT, AST, and CREA in the OM\nT - R Hydrogel group were all significantly increased (p < 0.05).\nAdditionally, the levels of ALP, BUN, and TBIL also showed an upward\ntrend. These findings strongly indicate that an overdose of OM T-R\nHydrogel can lead to liver and kidney damage. In the group that received\nFIGURE 1\nCharacterization of OM T-R hydrogel. (A) Temperature\nresponsiveness of OM T-R hydrogel; (B) SEM of OM T-R hydrogel;\n(C) FTIR analysis.\n\nGuo et al.\b\n10.3389/fvets.2026.1767033\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nan overdose of T-R Hydrogel, these indicators were elevated to varying\ndegrees. The macroscopic examination of the isolated rectum revealed\nno signs of bleeding, necrosis, or other lesions. Histologically (Figure 4),\nthe rectum exhibited elongated and straight intestinal glands, a signifi­\ncant presence of goblet cells, and a clearly defined structure across all\nlayers, with no apparent lesions. Furthermore, there were no significant\ndifferences in histomorphology observed among the three groups.\n4 Discussion\nThe main mode of OM absorption in the rectum is through the\nrectal veins. Drugs absorbed via the superior rectal veins undergo\nhepatic first-pass metabolism, while those absorbed via the middle or\ninferior rectal veins enter the systemic circulation directly through the\ninferior vena cava, which can effectively avoid the first-pass effect (27).\nThe results of this study demonstrate that T-R Hydrogel serves as an\neffective drug carrier for the transrectal delivery of OM. In the phar­\nmacokinetic analysis, it was observed that rectal administration of OM\nT-R Hydrogel does not induce a rapid elevation in systemic OM levels;\ninstead, it results in a gradual increase in blood concentrations. At\n50 min, the mean blood concentration in group R exhibited a relatively\nsmooth decline after reaching its peak. Additionally, t₁/₂λz was longer in\ngroup R compared to group O, indicating that OM T-R Hydrogel has\nthe capacity for sustained drug release. These results indicate that rectal\nadministration of OM T-R Hydrogel extends the dosing interval and\ndecreases dosing frequency. Meanwhile, an analysis of Tmax and Cmax\nvalues for individual rabbits in Group R revealed substantial variability\nin drug absorption. Previous research has demonstrated that variable\ndrug absorption remains one of the primary limitations of rectal drug\ndelivery (27). In Group R rabbits, substantial variability was observed\nin Cmax and Tmax, likely attributed to inter-individual metabolic dispari­\n\nIn Group R rabbits, substantial variability was observed\nin Cmax and Tmax, likely attributed to inter-individual metabolic dispari­\nties and feces. Rabbits were fasted for 16 h, and X-ray examination was\nperformed to assess fecal content in the intestinal tract before the\nanimal experiment. The cecum of rabbits is extremely well-developed,\nand they defecate frequently with no fixed timing (28). Therefore,\nduring the experiment, we can only ensure that there is no feces in the\nTABLE 1  Plasma O-M concentrations (ng/mL) over time in eight rabbits following treatment.\nTime\\\nAnimal ID\n25 min\n50 min\n1.5 h\n3 h\n6 h\n8 h\n10 h\n12 h\nRabbit A1\n27.00\n79.21\n42.32\n15.87\n7.48\n8.73\n7.16\n1.53\nRabbit A2\n90.56\n105.04\n60.27\n11.25\n5.91\n4.98\n2.57\n1.50\nRabbit A3\n96.98\n90.88\n49.32\n13.75\n12.40\n6.09\n3.45\n1.54\nMean\n71.51\n91.71\n50.64\n13.63\n8.59\n6.60\n4.39\n1.52\nSD\n31.58\n10.56\n7.39\n1.89\n2.76\n1.57\n1.99\n0.01\nRabbit B1\n33.09\n17.84\n8.46\n5.91\n4.70\n3.25\n2.77\n1.97\nRabbit B2\n18.05\n45.09\n7.22\n19.02\n8.57\n5.77\n2.18\n1.48\nRabbit B3\n8.56\n15.06\n23.36\n10.69\n6.21\n4.53\n2.33\n1.34\nRabbit B4\n12.36\n41.84\n51.70\n21.03\n13.95\n6.44\n2.17\n1.45\nRabbit B5\n9.39\n10.97\n11.08\n31.26\n7.94\n5.23\n2.38\n1.40\nMean\n16.29\n26.16\n20.36\n17.58\n8.27\n5.04\n2.37\n1.53\nSD\n9.04\n14.33\n16.68\n8.77\n3.14\n1.10\n0.22\n0.23\nRabbit A1-3 using oral administration; Rabbit B1-5 using rectal administration.\nFIGURE 2\nPlasma O-M concentration-time curve.\nTABLE 2  Pharmacokinetic parameters of oral (n = 3) and rectal (n = 5) administration.\nPharmacokinetic\nparameter\nOral\nadministration\nMean ±\nStandard\nRectum\nadministration\nMean ±\nStandard\nDose (mg/kg)\n1\n1\nTmax (min)\n41.67 ± 11.79\n87.00 ± 52.69\nCmax (ng/mL)\n93.74 ± 10.79\n36.90 ± 10.15\nt1/2λz (min)\n521.89 ± 100.13\n761.71 ± 356.57\nAUC0-last (min*ng/mL)\n13957.03 ± 566.44\n8267.64 ± 2106.43\nAUC0-inf (min*ng/mL)\n15103.53 ± 727.39\n10061.81 ± 1765.75\nMRTlast (min)\n284.95 ± 51.95\n378.73 ± 53.32\nMRT0-inf (min)\n431.36 ± 16.44\n752.35 ± 518.92\nCl-F (mL/min)\n163.50 ± 7.47\n315.70 ± 154.60\nTmax indicates the time to reach maximum plasma concentration; Cmax indicates the\nmaximum plasma concentration; t1/2λz indicates the terminal elimination half-life; Cl-F\nindicates the clearance divided by absolute bioavailability.\n\nGuo et al.\b\n10.3389/fvets.2026.1767033\nFrontiers in Veterinary Science\n05\nfrontiersin.org\ncolon and rectum segments. Unfortunately, this still cannot completely\neliminate the interference of feces on the drug absorption. However,\nthe defecation time of dogs and cats is relatively fixed, and there is less\ninterference from feces in rectal management.\nMost notably, drug accumulation can raise safety concerns associated\nwith long-term drug use (29). Relevant studies indicate that prolonged use\nof OM does not result in significant liver or kidney damage (30). A study\nby Lopes et al. demonstrated that when cats received oral administration\nof OM at a dosage of 2 mg/kg for 28 consecutive days, only a small number\nof cats exhibited vomiting and diarrhea, and these symptoms cannot be\ndirectly attributed to OM (30). However, our findings from the acute tox­\nicity test of OM T-R Hydrogel indicate that an overdose leads to hepatore­\nnal injury, a result that is consistent with the effects observed from an\noverdose of T-R Hydrogel. These results indicated that overdosage of T-R\nHydrogel leads to its accumulation in the body, which ultimately manifests\nas hepatorenal toxicity. HPMC, HP-β-CD, P407, and P188 are all materials\nwith excellent biocompatibility, and no significant toxic effects are antici­\npated when used at low dosages (31–34). Therefore, it is sufficiently safe\nto use OM T-R Hydrogel according to the recommended dose.\nThe primary limitation of this study is the absence of an efficacy\nassessment to establish the effective blood concentration of rectally\ndelivered drugs. Previous studies have shown that the Cmax of oral OM\n(1 mg/kg) in cats is 1631.6 ng/mL (26). However, in this study, the Cmax\nin rabbits after oral administration of 1 mg/kg OM was only 93.7 ng/\nmL. This indicates that under the same administration route and dose\nof OM, there is a significant difference in Cmax among different species,\nwhich is related to species-specific variations in drug bioavailability\n(35, 36). Currently, the effective blood concentration of OM remains\n\nCurrently, the effective blood concentration of OM remains\nunknown. Oral administration of 0.4 mg/kg OM in dogs has been\nconfirmed effective by the FDA, with a Cmax of 259 ng/mL. In this\nstudy, the Cmax of OM T-R Hydrogel following rectal administration\nin rabbits was 36.9 ng/mL. Assuming that the efficacy of OM across\ndifferent animal species depends on achieving or maintaining the\ndrug’s blood concentration, increasing the administered dose would\nbe necessary to achieve therapeutic effects in rabbits. To further vali­\ndate the effective blood concentration of OM T-R Hydrogel in rabbits,\nit is necessary to establish an itching model to assess therapeutic effi­\ncacy and determine the effective blood concentration.\nFIGURE 3\nSerum biochemical indices in different experimental groups. A comparison is conducted between the experimental group and the blank group.\n*p < 0.05; **p < 0.01.\nFIGURE 4\nHistological section of the rectum (anal end) tissue. (A) Blank group; (B) treatment with 0.9% NaCl; (C) treatment with OM T-R hydrogel.\n\nGuo et al.\b\n10.3389/fvets.2026.1767033\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nNotably, this exploratory study only conducted relevant trials in\nrabbits, ultimately confirming that OM can effectively enter the sys­\ntemic circulation following rectal administration of OM T-R Hydrogel.\nThe clinical efficacy of OM T-R Hydrogel in dogs and cats needs to be\ndefinitively established through pharmacokinetic studies and efficacy\nevaluations in these species.\n5 Conclusion\nIn this study, T-R Hydrogel was used as a drug delivery system for\nthe transrectal administration of OM, resulting in a gradual increase in\nthe plasma concentration of OM in rabbits. In conclusion, this explor­\natory study suggests that OM T-R Hydrogel may offer a novel therapeutic\napproach for pruritus management in dogs and cats. Future studies are\nalso needed to evaluate its safety, optimal dosing, and therapeutic effec­\ntiveness across different breeds and severity levels of pruritus.\nData availability statement\nThe original contributions presented in the study are included in\nthe article/Supplementary material, further inquiries can be directed\nto the corresponding authors.\nEthics statement\nThe animal study was approved by the Ethics Committee of Fujian\nAgriculture and Forestry University (FAFU-2023-0065). The study\nwas conducted in accordance with the local legislation and institu­\ntional requirements.\nAuthor contributions\nPG: Methodology, Writing – original draft, Conceptualization,\nData curation, Visualization. YY: Methodology, Writing – original\ndraft, Investigation. ZZ: Software, Validation, Writing – original draft.\nYG: Formal analysis, Writing – original draft. MM: Writing – review\n& editing. HW: Validation, Writing – original draft. DW: Supervision,\nWriting  – original draft. GY: Funding acquisition, Supervision,\nWriting – review & editing. LW: Funding acquisition, Project admin­\nistration, Supervision, Writing – review & editing.\nFunding\nThe author(s) declared that financial support was received for this\nwork and/or its publication. This research was funded by the National\n\nLW: Funding acquisition, Project admin­\nistration, Supervision, Writing – review & editing.Funding\nThe author(s) declared that financial support was received for this\nwork and/or its publication.This research was funded by the National\nNatural Science Foundation of China (grant no. 32102734 to LW) and\nSpecial Fund Project for Science and Technology Innovation, Fujian\nAgriculture and Forestry University (grant no. KFB25012A to GY).\nAcknowledgments\nThe authors thank Analysis and Testing Center of Fujian\nAgriculture and Forestry University for their help in the experiments.\nConflict of interest\nThe author(s) declared that this work was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declared that Generative AI was not used in the\ncreation of this manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. Any product\nthat may be evaluated in this article, or claim that may be made by its\nmanufacturer, is not guaranteed or endorsed by the publisher.\nSupplementary material\nThe Supplementary material for this article can be found online\nat: https://www.frontiersin.org/articles/10.3389/fvets.2026.1767033/\nfull#supplementary-material\nSUPPLEMENTARY TABLE S1\nThe gelation behavior of T-R Hydrogel at 37°C and 38°C.\nReferences\n1.\tFrymus T, Gruffydd-Jones T, Pennisi MG, Addie D, Belak S, Boucraut-Baralon C, et al.\nDermatophytosis in cats: ABCD guidelines on prevention and management. J Feline Med\nSurg. 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+{"text": "【兽医文献】\n标题: Equine-assisted learning and leadership transformation: an exploratory qualitative study of workplace behavior\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nTranscriptome sequencing reveals\nthe key genes associated with\nhair follicle development in\nQianhua Mutton Merino\nZhiyun Qin 1, Xinming Sun 1,2, Limin Sun 2, Meng Yu 3 and\nHuaizhi Jiang 1*\n1Key Laboratory of Evaluation and Utilization of Livestock and Poultry Resources (Sheep), Ministry of\nAgriculture and Rural Affairs, Jilin Agricultural University, Changchun, China, 2Northeast Agricultural\nResearch Center of China, Jilin Academy of Agricultural Sciences, Changchun, China, 3Liaoning\nAnyou Biotechnology Co., ltd., Shenyang,  Liaoning, China\nBackground: The aim is to compare the morphological changes and gene\nexpression profiles of hair follicles in newborn and one-year-old Qianhua\nMutton Merino sheep, explore the changes in gene expression and related\nregulatory pathways during the process from incomplete development to full\ndevelopment of skin hair follicles, and identify the key genes influencing hair\nfollicle development and maturation. This study holds significant theoretical\nimportance for revealing the molecular regulatory mechanism of hair follicle\ndevelopment in Qianhua Mutton Merino sheep.\nMethods: A total of five newborn (1-day-old) and five yearling (12-month-old)\nQianhua Mutton Merino sheep were selected for this study. Skin tissue samples\nwere collected for histological analysis using HE staining. Transcriptome\nsequencing was performed on the Illumina NovaSeq 6000 platform, and\ndifferentially expressed gene (DEG) analysis was conducted using the R package\ndegeR. GO and KEGG pathway enrichment analyses were performed on the\nDEGs to identify genes associated with hair follicle development. Ten DEGs\nwere randomly selected for the validation of the transcriptome sequencing\nresults using realtime quantitative PCR. The KRT27 gene was further validated\nthrough immuno-histochemistry and quantitative PCR.\nResults: The results from HE staining indicated that with an increase in the\nage of the Qianhua Mutton Merino sheep, the density of both primary and\nsecondary hair follicles significantly decreased (p < 0.05), while the S/P ratio\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nTranscriptome sequencing reveals\nthe key genes associated with\nhair follicle development in\nQianhua Mutton Merino\nZhiyun Qin 1, Xinming Sun 1,2, Limin Sun 2, Meng Yu 3 and\nHuaizhi Jiang 1*\n1Key Laboratory of Evaluation and Utilization of Livestock and Poultry Resources (Sheep), Ministry of\nAgriculture and Rural Affairs, Jilin Agricultural University, Changchun, China, 2Northeast Agricultural\nResearch Center of China, Jilin Academy of Agricultural Sciences, Changchun, China, 3Liaoning\nAnyou Biotechnology Co., ltd., Shenyang,  Liaoning, China\nBackground: The aim is to compare the morphological changes and gene\nexpression profiles of hair follicles in newborn and one-year-old Qianhua\nMutton Merino sheep, explore the changes in gene expression and related\nregulatory pathways during the process from incomplete development to full\ndevelopment of skin hair follicles, and identify the key genes influencing hair\nfollicle development and maturation. This study holds significant theoretical\nimportance for revealing the molecular regulatory mechanism of hair follicle\ndevelopment in Qianhua Mutton Merino sheep.\nMethods: A total of five newborn (1-day-old) and five yearling (12-month-old)\nQianhua Mutton Merino sheep were selected for this study. Skin tissue samples\nwere collected for histological analysis using HE staining. Transcriptome\nsequencing was performed on the Illumina NovaSeq 6000 platform, and\ndifferentially expressed gene (DEG) analysis was conducted using the R package\ndegeR. GO and KEGG pathway enrichment analyses were performed on the\nDEGs to identify genes associated with hair follicle development. Ten DEGs\nwere randomly selected for the validation of the transcriptome sequencing\nresults using realtime quantitative PCR. The KRT27 gene was further validated\nthrough immuno-histochemistry and quantitative PCR.\nResults: The results from HE staining indicated that with an increase in the\nage of the Qianhua Mutton Merino sheep, the density of both primary and\nsecondary hair follicles significantly decreased (p < 0.05), while the S/P ratio\n\nResults: The results from HE staining indicated that with an increase in the\nage of the Qianhua Mutton Merino sheep, the density of both primary and\nsecondary hair follicles significantly decreased (p < 0.05), while the S/P ratio\nexhibited a remarkable increase (p < 0.05). The transcriptome sequencing\nresults identified a total of 1,637 differentially expressed genes, of which 1,134\nwere upregulated and 503 were downregulated. GO and KEGG enrichment\nanalyses of DEGs related to hair follicle development revealed that these genes\nwere enriched in signaling pathways associated with hair follicle development,\nsuch as the PI3K-Akt, MAPK, and Estrogen signaling pathways. The results of\nreal-time quantitative PCR were consistent with the transcriptome sequencing\nresults. The KRT27 gene was expressed in the inner root sheath region of\nhair follicles, with a significant upregulation observed at 12 months of age\n(p < 0.05).\nConclusion: In conclusion, this study identified the KRT27, IGF-2, FGF21,\nVEGF-D, KRT25, and KRT26 genes as important candidate genes in the\ndevelopment of hair follicles in Qianhua Mutton Merino sheep through\ntranscriptome sequencing analysis, providing a theoretical basis for further\nOPEN ACCESS\nEDITED BY\nGuangbin Zhou,\nSichuan Agricultural University, China\nREVIEWED BY\nWenlin Bai,\nShenyang Agricultural University, China\nChunqiang Wang,\nLiaoning Medical University, China\n*CORRESPONDENCE\nHuaizhi Jiang\njianghz6806@126.com\nRECEIVED 05 September 2025\nREVISED 31 October 2025\nACCEPTED 11 November 2025\nPUBLISHED 04 December 2025\nCITATION\nQin Z, Sun X, Sun L, Yu M and Jiang H (2025)\nTranscriptome sequencing reveals the key\ngenes associated with hair follicle\ndevelopment in Qianhua Mutton Merino.\nFront. Vet. Sci. 12:1699868.\ndoi: 10.3389/fvets.2025.1699868\nCOPYRIGHT\n© 2025 Qin, Sun, Sun, Yu and Jiang. This is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY). The use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\n\nResults: The results from HE staining indicated that with an increase in the\nage of the Qianhua Mutton Merino sheep, the density of both primary and\nsecondary hair follicles significantly decreased (p < 0.05), while the S/P ratio\nexhibited a remarkable increase (p < 0.05). The transcriptome sequencing\nresults identified a total of 1,637 differentially expressed genes, of which 1,134\nwere upregulated and 503 were downregulated. GO and KEGG enrichment\nanalyses of DEGs related to hair follicle development revealed that these genes\nwere enriched in signaling pathways associated with hair follicle development,\nsuch as the PI3K-Akt, MAPK, and Estrogen signaling pathways. The results of\nreal-time quantitative PCR were consistent with the transcriptome sequencing\nresults. The KRT27 gene was expressed in the inner root sheath region of\nhair follicles, with a significant upregulation observed at 12 months of age\n(p < 0.05).\nConclusion: In conclusion, this study identified the KRT27, IGF-2, FGF21,\nVEGF-D, KRT25, and KRT26 genes as important candidate genes in the\ndevelopment of hair follicles in Qianhua Mutton Merino sheep through\ntranscriptome sequencing analysis, providing a theoretical basis for further\nOPEN ACCESS\nEDITED BY\nGuangbin Zhou,\nSichuan Agricultural University, China\nREVIEWED BY\nWenlin Bai,\nShenyang Agricultural University, China\nChunqiang Wang,\nLiaoning Medical University, China\n*CORRESPONDENCE\nHuaizhi Jiang\njianghz6806@126.com\nRECEIVED 05 September 2025\nREVISED 31 October 2025\nACCEPTED 11 November 2025\nPUBLISHED 04 December 2025\nCITATION\nQin Z, Sun X, Sun L, Yu M and Jiang H (2025)\nTranscriptome sequencing reveals the key\ngenes associated with hair follicle\ndevelopment in Qianhua Mutton Merino.\nFront. Vet. Sci. 12:1699868.\ndoi: 10.3389/fvets.2025.1699868\nCOPYRIGHT\n© 2025 Qin, Sun, Sun, Yu and Jiang. This is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY). The use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\n\nThe use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\naccordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Original Research\nPUBLISHED  04 December 2025\nDOI  10.3389/fvets.2025.1699868\n\nThe use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\naccordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Original Research\nPUBLISHED  04 December 2025\nDOI  10.3389/fvets.2025.1699868\n\nQin et al.\b\n10.3389/fvets.2025.1699868\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nrevealing the molecular mechanisms underlying the formation of wool traits in\nQianhua Mutton Merino sheep.\nKEYWORDS\nQianhua mutton Merino sheep, hair follicle development, DEGs, KRT, S/P\n1 Introduction\nThe hair follicles of sheep are the “source” of their fleece and\nthe only organs that undergo periodic changes. Based on their\nmorphological structure and developmental timeline, hair follicles\ncan be categorized into primary follicles (PFs), which produce\nmyelinated hairs, and secondary follicles (SFs), which produce\nunmyelinated hairs. Several secondary hair follicles form clustered\nfollicle groups of around one to seven primary hair follicles.\nTherefore, the characteristics and tissue structure of sheep hair\nfollicles determine the quality and yield of wool (1). As hair\nfollicles rely on periodic cyclical growth to achieve fleece renewal,\ntheir cyclical changes dictate the growth and shedding of the fleece.\nThe periodic growth and development of hair follicles are\ninfluenced by various factors and regulated by a series of signaling\nmolecules. With the widespread application of second-generation\nhigh-throughput sequencing technology, it has been discovered\nthat multiple signaling pathways, including Wnt (2), TGF-β (3, 4),\nPI3K-Art (5, 6), Notch (7), MAPK (8, 9), and BMP (10), play\ncrucial regulatory roles in the morphological development of hair\nfollicles. The Qianhua Mutton Merino sheep is a dual-purpose\nbreed developed in China in 2018, characterized by its large body,\nrapid daily weight gain, high meat production performance, and\nexcellent meat quality. Additionally, it primarily produces\nhomogeneous fine wool with a count of 66 (11). Although extensive\nresearch exists on sheep hair follicle development, the signaling\npathways governing this process exhibit species and breed\nspecificity. This prior knowledge provides a crucial foundation for\nexploring the pathways and key genes in Qianhua Mutton Merino\nsheep. Previous studies have shown that the number of primary\nhair follicles in Qianhua Mutton Merino does not change\n\nQin et al.\b\n10.3389/fvets.2025.1699868\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nrevealing the molecular mechanisms underlying the formation of wool traits in\nQianhua Mutton Merino sheep.\nKEYWORDS\nQianhua mutton Merino sheep, hair follicle development, DEGs, KRT, S/P\n1 Introduction\nThe hair follicles of sheep are the “source” of their fleece and\nthe only organs that undergo periodic changes. Based on their\nmorphological structure and developmental timeline, hair follicles\ncan be categorized into primary follicles (PFs), which produce\nmyelinated hairs, and secondary follicles (SFs), which produce\nunmyelinated hairs. Several secondary hair follicles form clustered\nfollicle groups of around one to seven primary hair follicles.\nTherefore, the characteristics and tissue structure of sheep hair\nfollicles determine the quality and yield of wool (1). As hair\nfollicles rely on periodic cyclical growth to achieve fleece renewal,\ntheir cyclical changes dictate the growth and shedding of the fleece.\nThe periodic growth and development of hair follicles are\ninfluenced by various factors and regulated by a series of signaling\nmolecules. With the widespread application of second-generation\nhigh-throughput sequencing technology, it has been discovered\nthat multiple signaling pathways, including Wnt (2), TGF-β (3, 4),\nPI3K-Art (5, 6), Notch (7), MAPK (8, 9), and BMP (10), play\ncrucial regulatory roles in the morphological development of hair\nfollicles. The Qianhua Mutton Merino sheep is a dual-purpose\nbreed developed in China in 2018, characterized by its large body,\nrapid daily weight gain, high meat production performance, and\nexcellent meat quality. Additionally, it primarily produces\nhomogeneous fine wool with a count of 66 (11). Although extensive\nresearch exists on sheep hair follicle development, the signaling\npathways governing this process exhibit species and breed\nspecificity. This prior knowledge provides a crucial foundation for\nexploring the pathways and key genes in Qianhua Mutton Merino\nsheep. Previous studies have shown that the number of primary\nhair follicles in Qianhua Mutton Merino does not change\n\nThis prior knowledge provides a crucial foundation for\nexploring the pathways and key genes in Qianhua Mutton Merino\nsheep.Previous studies have shown that the number of primary\nhair follicles in Qianhua Mutton Merino does not change\nsignificantly after rebirth. Newborn (1 day old) Qianhua Mutton\nMerino are in the young stage, and their hair follicles are in the\nprocess of growth and development, not yet fully mature. However,\nyearling (12 months old) Qianhua Mutton Merino use have fully\ndeveloped hair follicles. Studying the skin hair follicles of newborn\nand one-year-old Qianhua Mutton Merino is helpful for exploring\nthe complete development of hair follicles from immaturity to\nmaturity. As Qianhua Mutton Merino sheep are a dual-purpose\nbreed for both meat and wool, the two critical time points for\nfollicle development are at birth and 1 year of age. The specific\ngenes that play a central role during these two developmental\nstages, as well as the expression dynamics of these genes, require\nfurther in-depth study. In order to investigate the key genes\ninvolved in the development of hair follicles in Qianhua Mutton\nMerino sheep at birth and 1 year of age, histological and\ntranscriptomic analyses were conducted on the skin tissues of\nnewborn and one-year-old Qianhua Mutton Merino sheep.\nDifferential genes at different develop-mental stages were screened,\nand their related functions were analyzed, providing a theoretical\nbasis for further elucidating the molecular mechanisms underlying\nthe formation of wool traits.\n2 Materials and methods\n2.1 Laboratory animals\nSamples were collected from the core breeding group of Qian’an\nZhihua Sheep Breeding Co., Ltd. (National Core Breeding Farm for\nSheep). Skin samples were taken from the left scapular posterior edge\nof 5 newborn (M1) and 1-year-old (M12) ewes, and the sampling area\nmeasured 2cm × 2cm. The samples were divided into two parts: one\npart was preserved in 4% paraformaldehyde fixation solution, while\nthe other part was transported back to the laboratory in liquid\nnitrogen and stored at −80 °C for future use.\n2.2 Reagents and instruments\n\nThis prior knowledge provides a crucial foundation for\nexploring the pathways and key genes in Qianhua Mutton Merino\nsheep.Previous studies have shown that the number of primary\nhair follicles in Qianhua Mutton Merino does not change\nsignificantly after rebirth. Newborn (1 day old) Qianhua Mutton\nMerino are in the young stage, and their hair follicles are in the\nprocess of growth and development, not yet fully mature. However,\nyearling (12 months old) Qianhua Mutton Merino use have fully\ndeveloped hair follicles. Studying the skin hair follicles of newborn\nand one-year-old Qianhua Mutton Merino is helpful for exploring\nthe complete development of hair follicles from immaturity to\nmaturity. As Qianhua Mutton Merino sheep are a dual-purpose\nbreed for both meat and wool, the two critical time points for\nfollicle development are at birth and 1 year of age. The specific\ngenes that play a central role during these two developmental\nstages, as well as the expression dynamics of these genes, require\nfurther in-depth study. In order to investigate the key genes\ninvolved in the development of hair follicles in Qianhua Mutton\nMerino sheep at birth and 1 year of age, histological and\ntranscriptomic analyses were conducted on the skin tissues of\nnewborn and one-year-old Qianhua Mutton Merino sheep.\nDifferential genes at different develop-mental stages were screened,\nand their related functions were analyzed, providing a theoretical\nbasis for further elucidating the molecular mechanisms underlying\nthe formation of wool traits.\n2 Materials and methods\n2.1 Laboratory animals\nSamples were collected from the core breeding group of Qian’an\nZhihua Sheep Breeding Co., Ltd. (National Core Breeding Farm for\nSheep). Skin samples were taken from the left scapular posterior edge\nof 5 newborn (M1) and 1-year-old (M12) ewes, and the sampling area\nmeasured 2cm × 2cm. The samples were divided into two parts: one\npart was preserved in 4% paraformaldehyde fixation solution, while\nthe other part was transported back to the laboratory in liquid\nnitrogen and stored at −80 °C for future use.\n2.2 Reagents and instruments\n\nThe samples were divided into two parts: one\npart was preserved in 4% paraformaldehyde fixation solution, while\nthe other part was transported back to the laboratory in liquid\nnitrogen and stored at −80 °C for future use.2.2 Reagents and instruments\nThe following were obtained: anhydrous ethanol (Tianjin\nChemical Reagent Co., Ltd.), paraformaldehyde (Tianjin No.3\nChemical Reagent Factory), Trizol (Invitrogen), neutral gum (Tianjin\nNo.3 Chemical Reagent Factory), Tris-EDTA (Saiwen Innovation\n(Beijing) Biotechnology Co., Ltd.), a biological tissue embedding\nmachine (Jinhua Yidi Medical Equipment Co., Ltd., YD-6D), a Rotary\nMicrotome (Leica Instruments AG, Germany, Leica RM2235), a\nbiological tissue baking machine (Jinhua Yidi Medical Equipment Co.,\nLtd., YD-AB), and a biological microscope (Leica Instruments AG,\nGermany, Leica DM 1000).\n2.3 Paraffin-embedded skin tissue sections\nwere used for hematoxylin and eosin (HE)\nstaining\nSkin samples were fixed with formaldehyde and embedded in\nparaffin, and continuous skin sections were prepared with a\nthickness of 4 μm. Hematoxylin and eosin staining was performed,\nfollowed by resin embedding to observe the structural organization\nof skin follicle tissues (12). Skin tissue exhibits a phenomenon in\nwhich it contracts in tissue fluid; therefore, it is necessary to correct\nthe skin contraction rate. The skin contraction rate = the stained\nskin area / in vivo sample skin area. The skin contraction rates at\nbirth and at 12 months of age were 0.61 and 0.78, respectively. All\nthe skin data presented were adjusted for skin contraction rates. In\nthe continuous sections obtained, transverse sections near the\nprimary hair follicle sebaceous glands were selected to observe the\nnumber of primary and secondary hair follicles in each field of\nvision and calculate the follicular density and S/P. During\n\nThe samples were divided into two parts: one\npart was preserved in 4% paraformaldehyde fixation solution, while\nthe other part was transported back to the laboratory in liquid\nnitrogen and stored at −80 °C for future use.2.2 Reagents and instruments\nThe following were obtained: anhydrous ethanol (Tianjin\nChemical Reagent Co., Ltd.), paraformaldehyde (Tianjin No.3\nChemical Reagent Factory), Trizol (Invitrogen), neutral gum (Tianjin\nNo.3 Chemical Reagent Factory), Tris-EDTA (Saiwen Innovation\n(Beijing) Biotechnology Co., Ltd.), a biological tissue embedding\nmachine (Jinhua Yidi Medical Equipment Co., Ltd., YD-6D), a Rotary\nMicrotome (Leica Instruments AG, Germany, Leica RM2235), a\nbiological tissue baking machine (Jinhua Yidi Medical Equipment Co.,\nLtd., YD-AB), and a biological microscope (Leica Instruments AG,\nGermany, Leica DM 1000).\n2.3 Paraffin-embedded skin tissue sections\nwere used for hematoxylin and eosin (HE)\nstaining\nSkin samples were fixed with formaldehyde and embedded in\nparaffin, and continuous skin sections were prepared with a\nthickness of 4 μm. Hematoxylin and eosin staining was performed,\nfollowed by resin embedding to observe the structural organization\nof skin follicle tissues (12). Skin tissue exhibits a phenomenon in\nwhich it contracts in tissue fluid; therefore, it is necessary to correct\nthe skin contraction rate. The skin contraction rate = the stained\nskin area / in vivo sample skin area. The skin contraction rates at\nbirth and at 12 months of age were 0.61 and 0.78, respectively. All\nthe skin data presented were adjusted for skin contraction rates. In\nthe continuous sections obtained, transverse sections near the\nprimary hair follicle sebaceous glands were selected to observe the\nnumber of primary and secondary hair follicles in each field of\nvision and calculate the follicular density and S/P. During\n\nQin et al.\b\n10.3389/fvets.2025.1699868\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nmeasurement, the microscope was set to 100 times magnification,\nand the field-of-vision area was 2.9225 mm2.\n2.4 RNA extraction, library construction,\nand sequencing\nTotal RNA was extracted, isolated, and purified from skin tissue\nsamples using TRIzol. Quality control was performed using NanoDrop\nND-1000, Bioanalyzer 2100, and agarose gel electrophoresis, ensuring\nthat the concentration was > 50 ng/μL, the RIN value was > 7.0, the\nOD260/280 was > 1.8, and total RNA was > 1 μg before proceeding to\ndownstream experiments. PolyA tail mRNA was captured through\ntwo rounds of purification using oligo(dT) magnetic beads, followed\nby fragmentation using a high-temperature magnesium ion\nfragmentation kit. cDNA was synthesized via reverse transcription,\nand subsequent steps included double-strand synthesis, end\nprocessing, adapter ligation, magnetic bead selection, and purification.\nThe UDG enzyme was used to digest the double strands, and PCR\namplification was performed to construct a library of 300 bp ± 50 bp.\nFinally, PE150 paired-end sequencing was conducted using the\nIllumina Novaseq™ 6000.\n2.5 Bioinformatics analysis\nThe differentially expressed mRNAs were selected using\nthresholds of a fold change > 2 or < 0.5 and a parametric F-test\ncomparing nested linear models (p value < 0.05) via the R package\nedgeR.1 We performed STRING protein–protein interaction analysis\non differentially expressed mRNA to predict the protein interaction\nrelationships of DEGs using the STRING database.2 The genes with\nthe parameter of false discovery rate (FDR) below 0.05 and absolute\nfold change > 2were considered differentially expressed genes. Finally\nusing DAVID software3 to GO and KEGG gene enrichment analysis.\nThis analysis aimed to explore the interaction relationships of genes\nrelated to hair follicle development.\n2.6 Real-time fluorescence quantitative\nPCR verification\nTotal RNA was extracted and reverse-transcribed into cDNA\nwith a total reaction volume of 20 μL: nuclease-free water up to 20\n\nQin et al.\b\n10.3389/fvets.2025.1699868\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nmeasurement, the microscope was set to 100 times magnification,\nand the field-of-vision area was 2.9225 mm2.\n2.4 RNA extraction, library construction,\nand sequencing\nTotal RNA was extracted, isolated, and purified from skin tissue\nsamples using TRIzol. Quality control was performed using NanoDrop\nND-1000, Bioanalyzer 2100, and agarose gel electrophoresis, ensuring\nthat the concentration was > 50 ng/μL, the RIN value was > 7.0, the\nOD260/280 was > 1.8, and total RNA was > 1 μg before proceeding to\ndownstream experiments. PolyA tail mRNA was captured through\ntwo rounds of purification using oligo(dT) magnetic beads, followed\nby fragmentation using a high-temperature magnesium ion\nfragmentation kit. cDNA was synthesized via reverse transcription,\nand subsequent steps included double-strand synthesis, end\nprocessing, adapter ligation, magnetic bead selection, and purification.\nThe UDG enzyme was used to digest the double strands, and PCR\namplification was performed to construct a library of 300 bp ± 50 bp.\nFinally, PE150 paired-end sequencing was conducted using the\nIllumina Novaseq™ 6000.\n2.5 Bioinformatics analysis\nThe differentially expressed mRNAs were selected using\nthresholds of a fold change > 2 or < 0.5 and a parametric F-test\ncomparing nested linear models (p value < 0.05) via the R package\nedgeR.1 We performed STRING protein–protein interaction analysis\non differentially expressed mRNA to predict the protein interaction\nrelationships of DEGs using the STRING database.2 The genes with\nthe parameter of false discovery rate (FDR) below 0.05 and absolute\nfold change > 2were considered differentially expressed genes. Finally\nusing DAVID software3 to GO and KEGG gene enrichment analysis.\nThis analysis aimed to explore the interaction relationships of genes\nrelated to hair follicle development.\n2.6 Real-time fluorescence quantitative\nPCR verification\nTotal RNA was extracted and reverse-transcribed into cDNA\nwith a total reaction volume of 20 μL: nuclease-free water up to 20\n\n2.6 Real-time fluorescence quantitative\nPCR verification\nTotal RNA was extracted and reverse-transcribed into cDNA\nwith a total reaction volume of 20 μL: nuclease-free water up to 20\nμL, 4 μL of 5 × RT Buffer, 1 μL of RT Enzyme Mix, 1 μL of Primer\nMix, and 2 μg of RNA. The reaction was denatured at 65 °C for 5\nmin, followed by a reverse transcription reaction at 37 °C for 15 min\nand, finally, treatment at 98 °C for 5 min. Based on the mRNA\nsequences of the sheep, KRT27, CPT1A, MT3, MRC1, MYLPF,\nKRT25, KRT71, FCGR2B, IL18, HSD11B1, and GSTA1, obtained\nfrom the NCBI database, primers for the cloning and amplification\nof the CDS region were designed using Primer 5.0 software. These\nprimers were synthesized by GeneChem Co., Ltd. (Shanghai, China),\n1  https://bioconductor.org/packages/release/bioc/html/edgeR.html\n2  https://cn.string-db.org/\n3  https://david.ncifcrf.gov/\nand the primer sequences are shown in Table 1. Using cDNA as a\ntemplate, the reaction system was set as follows 20μL: 6.4μL of\ndistilled water; 10μL of SYBR Green Realtime PCR Master Mix;\n0.8μL of primers; and 2μL of cDNA. The PCR reaction program\nconsisted of an initial denaturation at 94 °C for 3 min, followed by\n40 cycles of denaturation at 94 °C for 15 s and annealing at 60 °C for\n30 s. Finally, an extension step was performed at 72 °C for 20 s, with\na gradual increase from 65 °C to 95 °C over 5 s to obtain the melting\ncurve. All samples were measured in triplicate. The resulting data\nwere normalized using β-actin and calculated using the\n2-ΔΔCt method.\n2.7 Immunohistochemistry\nFor the immunohistochemical staining of 5μm thick tissue\nsections, the sections were deparaffinized using xylene and washed\nwith PBS four times. The sections were stained according to the\nimmunostaining reagents, with the KRT27 antibody (dilution 1:500)\nincubated at 4 °C for 12 h, followed by three washes with PBS. The\nsamples were counterstained with hematoxylin (Merck, Darmstadt,\nGermany) for 30 s and immediately washed with water. Finally, the\nsamples were mounted with neutral resin and covered with a\n\n2.6 Real-time fluorescence quantitative\nPCR verification\nTotal RNA was extracted and reverse-transcribed into cDNA\nwith a total reaction volume of 20 μL: nuclease-free water up to 20\nμL, 4 μL of 5 × RT Buffer, 1 μL of RT Enzyme Mix, 1 μL of Primer\nMix, and 2 μg of RNA. The reaction was denatured at 65 °C for 5\nmin, followed by a reverse transcription reaction at 37 °C for 15 min\nand, finally, treatment at 98 °C for 5 min. Based on the mRNA\nsequences of the sheep, KRT27, CPT1A, MT3, MRC1, MYLPF,\nKRT25, KRT71, FCGR2B, IL18, HSD11B1, and GSTA1, obtained\nfrom the NCBI database, primers for the cloning and amplification\nof the CDS region were designed using Primer 5.0 software. These\nprimers were synthesized by GeneChem Co., Ltd. (Shanghai, China),\n1  https://bioconductor.org/packages/release/bioc/html/edgeR.html\n2  https://cn.string-db.org/\n3  https://david.ncifcrf.gov/\nand the primer sequences are shown in Table 1. Using cDNA as a\ntemplate, the reaction system was set as follows 20μL: 6.4μL of\ndistilled water; 10μL of SYBR Green Realtime PCR Master Mix;\n0.8μL of primers; and 2μL of cDNA. The PCR reaction program\nconsisted of an initial denaturation at 94 °C for 3 min, followed by\n40 cycles of denaturation at 94 °C for 15 s and annealing at 60 °C for\n30 s. Finally, an extension step was performed at 72 °C for 20 s, with\na gradual increase from 65 °C to 95 °C over 5 s to obtain the melting\ncurve. All samples were measured in triplicate. The resulting data\nwere normalized using β-actin and calculated using the\n2-ΔΔCt method.\n2.7 Immunohistochemistry\nFor the immunohistochemical staining of 5μm thick tissue\nsections, the sections were deparaffinized using xylene and washed\nwith PBS four times. The sections were stained according to the\nimmunostaining reagents, with the KRT27 antibody (dilution 1:500)\nincubated at 4 °C for 12 h, followed by three washes with PBS. The\nsamples were counterstained with hematoxylin (Merck, Darmstadt,\nGermany) for 30 s and immediately washed with water. Finally, the\nsamples were mounted with neutral resin and covered with a\n\nThe\nsamples were counterstained with hematoxylin (Merck, Darmstadt,\nGermany) for 30 s and immediately washed with water.Finally, the\nsamples were mounted with neutral resin and covered with a\ncoverslip, and the staining was observed under an optical\nmicroscope.\nTABLE 1  The primer information used in this investigation.\nGene\nPrimer Sequence 5′ to 3′\nExpected\nproduct\nsize\nKRT27\nF: GGGACTGGGCGGAGGAAGTG\n108 bp\nR: GCCAGGCGGTCATTGAGGTTC\nCPT1A\nF: CGGTTGCTGATGACGGCTATGG\n114 bp\nR: TCCCGAAGCGATGCGAGTCC\nMT3\nF: TGCACCTGCTCCGACTCCTG\n150 bp\nR: GCAGCCGCACTTCTTCTCCTC\nMRC1\nF: AGGGATGCGGGAAAGTGGATGG\n87 bp\nR: ACTCGGCAAGGAAGGGTCAGG\nMYLPF\nF: AGAGAAGGGCAGCAGCAGAGG\n148 bp\nR: GCAAAGGTGTCCCGCAGGTC\nKRT25\nF: CAACGCTGACCTGGAGCAGAAG\n150 bp\nR: TTGGCATTGCTGGTGGTGGAAG\nKRT71\nF: AGCTGCTGGAGAGTGAGGAGTG\n81 bp\nR: CGCTGGTGCTGCTGATGATGG\nKCGR2B\nF: AGCCTGCGTGGATCAATGTGC\n86 bp\nR: CGTGGTGAGGTTGCCTGCATAG\nIL18\nF: TGGCGAAGACCTGGAATCAGATC\n96 bp\nR: CCCTGGCTAATGAAGAGAACTTGG\nHSD11B1\nF: GCCAGCAAGGGAATCGGAAGAG\n116 bp\nR: TCCAGGCAGCGGGATACCAC\nGSTA1\nF: GACGCCAAGCTGACACCAATCC\n100bp\nR: TGCCCACCAGGTAGTCTTGTCC\n\nThe\nsamples were counterstained with hematoxylin (Merck, Darmstadt,\nGermany) for 30 s and immediately washed with water.Finally, the\nsamples were mounted with neutral resin and covered with a\ncoverslip, and the staining was observed under an optical\nmicroscope.\nTABLE 1  The primer information used in this investigation.\nGene\nPrimer Sequence 5′ to 3′\nExpected\nproduct\nsize\nKRT27\nF: GGGACTGGGCGGAGGAAGTG\n108 bp\nR: GCCAGGCGGTCATTGAGGTTC\nCPT1A\nF: CGGTTGCTGATGACGGCTATGG\n114 bp\nR: TCCCGAAGCGATGCGAGTCC\nMT3\nF: TGCACCTGCTCCGACTCCTG\n150 bp\nR: GCAGCCGCACTTCTTCTCCTC\nMRC1\nF: AGGGATGCGGGAAAGTGGATGG\n87 bp\nR: ACTCGGCAAGGAAGGGTCAGG\nMYLPF\nF: AGAGAAGGGCAGCAGCAGAGG\n148 bp\nR: GCAAAGGTGTCCCGCAGGTC\nKRT25\nF: CAACGCTGACCTGGAGCAGAAG\n150 bp\nR: TTGGCATTGCTGGTGGTGGAAG\nKRT71\nF: AGCTGCTGGAGAGTGAGGAGTG\n81 bp\nR: CGCTGGTGCTGCTGATGATGG\nKCGR2B\nF: AGCCTGCGTGGATCAATGTGC\n86 bp\nR: CGTGGTGAGGTTGCCTGCATAG\nIL18\nF: TGGCGAAGACCTGGAATCAGATC\n96 bp\nR: CCCTGGCTAATGAAGAGAACTTGG\nHSD11B1\nF: GCCAGCAAGGGAATCGGAAGAG\n116 bp\nR: TCCAGGCAGCGGGATACCAC\nGSTA1\nF: GACGCCAAGCTGACACCAATCC\n100bp\nR: TGCCCACCAGGTAGTCTTGTCC\n\nQin et al.\b\n10.3389/fvets.2025.1699868\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n2.8 Data analysis and statistics\nAn analysis of variance (One-Way ANOVA) for differences\namong different groups was conducted using SPSS 22.0 software.\nGraphs were generated using GraphPad Prism 9.5 software. The\nresults are represented as the mean ± standard error, with p < 0.05\nindicating a significant difference.\n3 Results\n3.1 The morphological structure of hair\nfollicle tissues in Qianhua Mutton Merino\nsheep at different ages\nThrough HE staining, we made morphological observations of the\nskin follicle structure in newborn (M1) and one-year-old (M12)\nQianhua Mutton Merino sheep (Figures 1A,B). Structures such as\nprimary follicles (PFs), secondary follicles (SFs), and sebaceous glands\n(SGs) were observed in both periods. The diameter of primary hair\nfollicles is larger than that of secondary hair follicles, and they are\nsurrounded by sebaceous and sweat gland structures. In the skin tissue\nof newborn Qianhua Mutton Merino sheep, the number of primary hair\nfollicles is greater than that of secondary hair follicles, and at this time,\nsecondary hair follicles are not densely distributed around primary hair\nfollicles. The hair follicles of newborn Qianhua Mutton Merino sheep\nfor meat use are still in the developmental stage, not fully mature, and\nhave not yet entered the cyclical development of hair follicles.\nIn the skin tissue of the one-year-old Qianhua Mutton Merino\nsheep, the number of secondary hair follicles is greater than that of\nprimary hair follicles, and they are distributed more densely, forming\na hair follicle cluster. In this study, the skin samples of one-year-old\nQianhua Mutton Merino sheep were collected in March. At this time,\nthe hair follicles had undergone a complete hair follicle cycle and were\nin the resting phase. The Skin structure parameters of Qianhua\nMutton Merino sheepin newborn and one-year-old sheep is presented\nin Table 2. The number of primary and secondary follicles in Qianhua\nMutton Merino sheep shows a gradual decrease with increasing age.\n\nQin et al.\b\n10.3389/fvets.2025.1699868\nFrontiers in Veterinary Science\n04\nfrontiersin.org\n2.8 Data analysis and statistics\nAn analysis of variance (One-Way ANOVA) for differences\namong different groups was conducted using SPSS 22.0 software.\nGraphs were generated using GraphPad Prism 9.5 software. The\nresults are represented as the mean ± standard error, with p < 0.05\nindicating a significant difference.\n3 Results\n3.1 The morphological structure of hair\nfollicle tissues in Qianhua Mutton Merino\nsheep at different ages\nThrough HE staining, we made morphological observations of the\nskin follicle structure in newborn (M1) and one-year-old (M12)\nQianhua Mutton Merino sheep (Figures 1A,B). Structures such as\nprimary follicles (PFs), secondary follicles (SFs), and sebaceous glands\n(SGs) were observed in both periods. The diameter of primary hair\nfollicles is larger than that of secondary hair follicles, and they are\nsurrounded by sebaceous and sweat gland structures. In the skin tissue\nof newborn Qianhua Mutton Merino sheep, the number of primary hair\nfollicles is greater than that of secondary hair follicles, and at this time,\nsecondary hair follicles are not densely distributed around primary hair\nfollicles. The hair follicles of newborn Qianhua Mutton Merino sheep\nfor meat use are still in the developmental stage, not fully mature, and\nhave not yet entered the cyclical development of hair follicles.\nIn the skin tissue of the one-year-old Qianhua Mutton Merino\nsheep, the number of secondary hair follicles is greater than that of\nprimary hair follicles, and they are distributed more densely, forming\na hair follicle cluster. In this study, the skin samples of one-year-old\nQianhua Mutton Merino sheep were collected in March. At this time,\nthe hair follicles had undergone a complete hair follicle cycle and were\nin the resting phase. The Skin structure parameters of Qianhua\nMutton Merino sheepin newborn and one-year-old sheep is presented\nin Table 2. The number of primary and secondary follicles in Qianhua\nMutton Merino sheep shows a gradual decrease with increasing age.\n\nThe Skin structure parameters of Qianhua\nMutton Merino sheepin newborn and one-year-old sheep is presented\nin Table 2.The number of primary and secondary follicles in Qianhua\nMutton Merino sheep shows a gradual decrease with increasing age.\nThere were no significant differences (p > 0.05) in the diameter, depth\nof the primary hair follicles of Qianhua Mutton Merino sheep at birth\nand at one - year - old. However, significant differences (p < 0.05) were\nobserved in the diameter and depth of the secondary hair follicles.\nThis indicates that the development of primary hair follicles had been\ncompleted at birth, while the secondary hair follicles were still in the\ndevelopmental stage. In addition, the difference in the diameter of\nwool fibers grown from secondary hair follicles (p < 0.05) in the fleece\nwas also consistent with these findings. At birth, the density of PF and\nSF is significantly higher than that at 1 year of age (p < 0.05). However,\nat 1 year of age, the S/P ratio is significantly higher than that during\nthe newborn period (p < 0.05).\n3.2 Analysis of transcriptome sequencing\ndata obtained from Qianhua Mutton\nMerino sheep skin tissues at different ages\nTen skin tissue samples from Qianhua Mutton Merino sheep at\ndifferent ages were subjected to library construction and sequencing.\nAfter preprocessing and filtering the raw sequencing data, we obtained\nhigh-quality effective reads. The total raw read counts ranged from\n36,147,820 to 48,395,978 bp, with a base count between 5.42G and\n7.26G. After filtering, the effective read counts were between 34,809,696\nand 45,987,934 bp, with a base count ranging from 5.22G to 6.90G. The\nproportion of effective reads exceeded 95% (95.02–97.12%), retaining a\nlarge number of high-quality reads. The Q20 (base accuracy ≥ 99%) and\nQ30 (base accuracy ≥ 99.9%) proportions were in the range of 99.72–\n99.97% and 97.40–98.12%, respectively, indicating extremely high data\naccuracy. The GC content ranged from 47 to 50.5%, distributed evenly\nand in accordance with species characteristics. These metrics\n\nThe Skin structure parameters of Qianhua\nMutton Merino sheepin newborn and one-year-old sheep is presented\nin Table 2.The number of primary and secondary follicles in Qianhua\nMutton Merino sheep shows a gradual decrease with increasing age.\nThere were no significant differences (p > 0.05) in the diameter, depth\nof the primary hair follicles of Qianhua Mutton Merino sheep at birth\nand at one - year - old. However, significant differences (p < 0.05) were\nobserved in the diameter and depth of the secondary hair follicles.\nThis indicates that the development of primary hair follicles had been\ncompleted at birth, while the secondary hair follicles were still in the\ndevelopmental stage. In addition, the difference in the diameter of\nwool fibers grown from secondary hair follicles (p < 0.05) in the fleece\nwas also consistent with these findings. At birth, the density of PF and\nSF is significantly higher than that at 1 year of age (p < 0.05). However,\nat 1 year of age, the S/P ratio is significantly higher than that during\nthe newborn period (p < 0.05).\n3.2 Analysis of transcriptome sequencing\ndata obtained from Qianhua Mutton\nMerino sheep skin tissues at different ages\nTen skin tissue samples from Qianhua Mutton Merino sheep at\ndifferent ages were subjected to library construction and sequencing.\nAfter preprocessing and filtering the raw sequencing data, we obtained\nhigh-quality effective reads. The total raw read counts ranged from\n36,147,820 to 48,395,978 bp, with a base count between 5.42G and\n7.26G. After filtering, the effective read counts were between 34,809,696\nand 45,987,934 bp, with a base count ranging from 5.22G to 6.90G. The\nproportion of effective reads exceeded 95% (95.02–97.12%), retaining a\nlarge number of high-quality reads. The Q20 (base accuracy ≥ 99%) and\nQ30 (base accuracy ≥ 99.9%) proportions were in the range of 99.72–\n99.97% and 97.40–98.12%, respectively, indicating extremely high data\naccuracy. The GC content ranged from 47 to 50.5%, distributed evenly\nand in accordance with species characteristics. These metrics\n\n9%) proportions were in the range of 99.72–\n99.97% and 97.40–98.12%, respectively, indicating extremely high data\naccuracy.The GC content ranged from 47 to 50.5%, distributed evenly\nand in accordance with species characteristics.These metrics\ndemonstrate that the quality of the sequencing data is excellent,\nproviding a reliable foundation for subsequent analyses.\n3.3 Sequencing data filtering and reference\ngenome alignment\nThe distribution of reads compared to the reference genome was\nprimarily concentrated in the exon, intron, and intergenic regions. In all\nFIGURE 1\n(A) 100 × the structure of newborn skin hair follicles; (B) 100 × the structure of 12-year-old skin hair follicles. PF, primary follicle; SF, secondary follicle;\nSG, sebaceous gland.\n\n9%) proportions were in the range of 99.72–\n99.97% and 97.40–98.12%, respectively, indicating extremely high data\naccuracy.The GC content ranged from 47 to 50.5%, distributed evenly\nand in accordance with species characteristics.These metrics\ndemonstrate that the quality of the sequencing data is excellent,\nproviding a reliable foundation for subsequent analyses.\n3.3 Sequencing data filtering and reference\ngenome alignment\nThe distribution of reads compared to the reference genome was\nprimarily concentrated in the exon, intron, and intergenic regions. In all\nFIGURE 1\n(A) 100 × the structure of newborn skin hair follicles; (B) 100 × the structure of 12-year-old skin hair follicles. PF, primary follicle; SF, secondary follicle;\nSG, sebaceous gland.\n\nQin et al.\b\n10.3389/fvets.2025.1699868\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nthe samples, the vast majority of reads were distributed in the exon\nregions, accounting for between 75.96 and 78.28% (Figures 2A,B),\nindicating that the sequencing data mainly originate from coding\nregions, a finding consistent with the expectations of mRNA sequencing.\nThe next largest proportion was found in the intron regions, ranging\nfrom 9.68 to 14.08%, which may reflect unspliced precursor mRNA or\nother transcriptional fragments derived from introns. The proportion\nof reads in the intergenic regions was relatively low, ranging from 5.21\nto 14.35%, a finding that may be due to non-coding RNA or background\nnoise. Overall, the proportion of reads in the exon regions across all\nsamples was relatively consistent, validating the reliability of the\nsequencing data and the accuracy of the reference genome alignment,\nthereby providing foundational data support for subsequent expression\nquantification and functional analysis.\n3.4 Differentially expressed gene analysis\nTo screen for candidate genes related to hair follicle development\nat different stages in Qianhua Mutton Merino sheep, we compared two\ncritical time points: newborn (M1) and 1 year old (M12). A total of\n15,852 genes were identified in the M1 group, while 15,696 genes were\nidentified in the M12 group, with a total of 15,091 common genes\n(Figure 3B). Using thresholds of a fold change > 2 or < 0.5 and a p value\n< 0.05, we detected 1,637 DEGs, including CCL21, TPSB2, GSTA1,\nMRC1, ND1, MYLP, IL18, ND4L, GHRHR, ITGAM, LEPR, MMP2,\nANKRD1, PLIN5, and DIRAS3. Of these, 1,134 were upregulated and\n503 were downregulated, with the number of upregulated DEGs\nsignificantly exceeding the number of downregulated DEGs\n(Figures 3A,C). Ten genes were randomly selected from the DEGs for\nRT-qPCR validation (Figure 3D). The expression levels of CPT1A,\nKRT25, KRT71, MRC1, and MYLPF were significantly upregulated in\nthe M12 group (p < 0.01), while GSTA1, FCGR2B, HSD11B1, IL18, and\nMT3 showed significantly downregulated expression levels in the M12\n\nQin et al.\b\n10.3389/fvets.2025.1699868\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nthe samples, the vast majority of reads were distributed in the exon\nregions, accounting for between 75.96 and 78.28% (Figures 2A,B),\nindicating that the sequencing data mainly originate from coding\nregions, a finding consistent with the expectations of mRNA sequencing.\nThe next largest proportion was found in the intron regions, ranging\nfrom 9.68 to 14.08%, which may reflect unspliced precursor mRNA or\nother transcriptional fragments derived from introns. The proportion\nof reads in the intergenic regions was relatively low, ranging from 5.21\nto 14.35%, a finding that may be due to non-coding RNA or background\nnoise. Overall, the proportion of reads in the exon regions across all\nsamples was relatively consistent, validating the reliability of the\nsequencing data and the accuracy of the reference genome alignment,\nthereby providing foundational data support for subsequent expression\nquantification and functional analysis.\n3.4 Differentially expressed gene analysis\nTo screen for candidate genes related to hair follicle development\nat different stages in Qianhua Mutton Merino sheep, we compared two\ncritical time points: newborn (M1) and 1 year old (M12). A total of\n15,852 genes were identified in the M1 group, while 15,696 genes were\nidentified in the M12 group, with a total of 15,091 common genes\n(Figure 3B). Using thresholds of a fold change > 2 or < 0.5 and a p value\n< 0.05, we detected 1,637 DEGs, including CCL21, TPSB2, GSTA1,\nMRC1, ND1, MYLP, IL18, ND4L, GHRHR, ITGAM, LEPR, MMP2,\nANKRD1, PLIN5, and DIRAS3. Of these, 1,134 were upregulated and\n503 were downregulated, with the number of upregulated DEGs\nsignificantly exceeding the number of downregulated DEGs\n(Figures 3A,C). Ten genes were randomly selected from the DEGs for\nRT-qPCR validation (Figure 3D). The expression levels of CPT1A,\nKRT25, KRT71, MRC1, and MYLPF were significantly upregulated in\nthe M12 group (p < 0.01), while GSTA1, FCGR2B, HSD11B1, IL18, and\nMT3 showed significantly downregulated expression levels in the M12\n\nThe expression levels of CPT1A,\nKRT25, KRT71, MRC1, and MYLPF were significantly upregulated in\nthe M12 group (p < 0.01), while GSTA1, FCGR2B, HSD11B1, IL18, and\nMT3 showed significantly downregulated expression levels in the M12\ngroup (p < 0.01). The results of the differential gene RT-qPCR\nvalidation were largely consistent with the expression trends observed\nin the transcriptome sequencing analysis, thereby confirming the\nreliability of the data obtained from transcriptome sequencing.\n3.5 KEGG pathway enrichment and GO\nfunctional annotation analysis for DEGs\nTo further analyze the functions of differentially expressed genes\n(DEGs) in the skin tissues of Qianhua Mutton Merino sheep at\ndifferent ages, enrichment analysis of DEGs was conducted using the\nDAVID software product (Table 3). The results of the GO enrichment\nanalysis showed (Figure 4A) that in Biological Processes (BPs), the\nmost significantly enriched terms were the positive regulation of the\nERK1 and ERK2 cascade (GO:0070374) and PI3K-Akt (GO:0051897).\nIn Cellular Components (CCs), the most significantly enriched terms\nwere membrane (GO:0016020), plasma membrane (GO:0005886), and\ncytoplasm (GO:0005829). In Molecular Function (MF), the most\nabundant terms were protein binding (GO:0005515) and metal ion\nbinding (GO:0046872). Enrichment analysis revealed that several\ndifferentially expressed genes (DEGs) were enriched in entries related\nto hair follicle development (Figures 4C,F), such as structural molecule\nactivity, hair follicle morphogenesis (GO:0031069), keratinocyte\ndifferentiation (GO:0030216), keratinization (GO:0031424), and skin\ndevelopment (GO:0043588). These functions are crucial for the\nformation, maturation, and establishment of the stratum corneum of\nhair follicles. The enrichment of the hair cycle (GO:0042633) suggests\nthat some DEGs may be involved in the dynamic regulation of the hair\nanagen (growth) phase, catagen (regression) phase, and telogen\n(resting) phase. In the context of structural molecule activity, the\ndifferentially expressed genes (DEGs) include KRT18, KRT23, KRT25,\n\nThe expression levels of CPT1A,\nKRT25, KRT71, MRC1, and MYLPF were significantly upregulated in\nthe M12 group (p < 0.01), while GSTA1, FCGR2B, HSD11B1, IL18, and\nMT3 showed significantly downregulated expression levels in the M12\ngroup (p < 0.01). The results of the differential gene RT-qPCR\nvalidation were largely consistent with the expression trends observed\nin the transcriptome sequencing analysis, thereby confirming the\nreliability of the data obtained from transcriptome sequencing.\n3.5 KEGG pathway enrichment and GO\nfunctional annotation analysis for DEGs\nTo further analyze the functions of differentially expressed genes\n(DEGs) in the skin tissues of Qianhua Mutton Merino sheep at\ndifferent ages, enrichment analysis of DEGs was conducted using the\nDAVID software product (Table 3). The results of the GO enrichment\nanalysis showed (Figure 4A) that in Biological Processes (BPs), the\nmost significantly enriched terms were the positive regulation of the\nERK1 and ERK2 cascade (GO:0070374) and PI3K-Akt (GO:0051897).\nIn Cellular Components (CCs), the most significantly enriched terms\nwere membrane (GO:0016020), plasma membrane (GO:0005886), and\ncytoplasm (GO:0005829). In Molecular Function (MF), the most\nabundant terms were protein binding (GO:0005515) and metal ion\nbinding (GO:0046872). Enrichment analysis revealed that several\ndifferentially expressed genes (DEGs) were enriched in entries related\nto hair follicle development (Figures 4C,F), such as structural molecule\nactivity, hair follicle morphogenesis (GO:0031069), keratinocyte\ndifferentiation (GO:0030216), keratinization (GO:0031424), and skin\ndevelopment (GO:0043588). These functions are crucial for the\nformation, maturation, and establishment of the stratum corneum of\nhair follicles. The enrichment of the hair cycle (GO:0042633) suggests\nthat some DEGs may be involved in the dynamic regulation of the hair\nanagen (growth) phase, catagen (regression) phase, and telogen\n(resting) phase. In the context of structural molecule activity, the\ndifferentially expressed genes (DEGs) include KRT18, KRT23, KRT25,\n\nIn the context of structural molecule activity, the\ndifferentially expressed genes (DEGs) include KRT18, KRT23, KRT25,\nKRT39, CRYAB, KRT27, KRT33B, KRT24-1, CLDN4, CLDN23, and\nCLDN8. In the hair follicle morphogenesis pathway, the DEGs are\nKRT71, KRT25, KRT27, and GLI2. Within the PI3K-Akt pathway, the\nDEGs include CCL21, IL18, LRP2, IGF-2, MFHAS1, STOX1, HB-EGF,\nNRG1, and CHI3L1. The results of KEGG enrichment analysis\n(Figures 4B,D,E) showed that differentially expressed genes (DEGs)\nwere significantly enriched in multiple key biological pathways. Among\nthese pathways, the significantly enriched pathways include ECM–\nreceptor interaction, the PI3K-Akt signaling pathway, focal adhesion,\nthe estrogen signaling pathway, and the relaxin signaling pathway,\nindicating the role of hormonal regulation in hair follicle development.\nThe enrichment of the MAPK signaling pathway and calcium signaling\npathway plays a role in cellular energy metabolism, stress response, and\nother physio-logical processes. In the estrogen signaling pathway, the\ndifferentially expressed genes (DEGs) include KRT18, MMP2, ADCY2,\nKRT23, ESR1, CREB3L3, KRT25, KRT39, KRT26, and KRT27. In the\nPI3K-Akt pathway, the DEGs are VEGF-D, LAMA2, PDGFRA,\nCREB3L4, ITGA8, COL4A3, ITGA9, TNXB, FLT-4, CREB3L3, and\nFGF21. In the MAPK signaling pathway, the DEGs consist of VEGF-D,\nPDGFRA, CSF1R, FLT-4, DUSP10, MAPK23, CD14, FGF21, MAPK12,\nand MAPK13.\n3.6 STRING protein–protein interaction\nanalysis\nThrough STRING protein–protein interaction network analysis\n(Figure 5), it was found that the KRT27 gene serves as a core node\namong differentially expressed genes related to hair follicle\ndevelopment, closely interacting with several keratin genes as-sociated\nwith hair follicle development, such as KRT26, KRT71, KRT39, and\nKRT25. Moreover, the KRT27 gene is significantly enriched in the\nestrogen signaling pathway, as well as in the entries for structural\nmolecule activity and hair follicle morphogenesis, indicating that it\nmay play an important role in hair follicle development.\nTABLE 2  Skin structure parameters of Qianhua Mutton Merino sheep.\n\nIn the context of structural molecule activity, the\ndifferentially expressed genes (DEGs) include KRT18, KRT23, KRT25,\nKRT39, CRYAB, KRT27, KRT33B, KRT24-1, CLDN4, CLDN23, and\nCLDN8. In the hair follicle morphogenesis pathway, the DEGs are\nKRT71, KRT25, KRT27, and GLI2. Within the PI3K-Akt pathway, the\nDEGs include CCL21, IL18, LRP2, IGF-2, MFHAS1, STOX1, HB-EGF,\nNRG1, and CHI3L1. The results of KEGG enrichment analysis\n(Figures 4B,D,E) showed that differentially expressed genes (DEGs)\nwere significantly enriched in multiple key biological pathways. Among\nthese pathways, the significantly enriched pathways include ECM–\nreceptor interaction, the PI3K-Akt signaling pathway, focal adhesion,\nthe estrogen signaling pathway, and the relaxin signaling pathway,\nindicating the role of hormonal regulation in hair follicle development.\nThe enrichment of the MAPK signaling pathway and calcium signaling\npathway plays a role in cellular energy metabolism, stress response, and\nother physio-logical processes. In the estrogen signaling pathway, the\ndifferentially expressed genes (DEGs) include KRT18, MMP2, ADCY2,\nKRT23, ESR1, CREB3L3, KRT25, KRT39, KRT26, and KRT27. In the\nPI3K-Akt pathway, the DEGs are VEGF-D, LAMA2, PDGFRA,\nCREB3L4, ITGA8, COL4A3, ITGA9, TNXB, FLT-4, CREB3L3, and\nFGF21. In the MAPK signaling pathway, the DEGs consist of VEGF-D,\nPDGFRA, CSF1R, FLT-4, DUSP10, MAPK23, CD14, FGF21, MAPK12,\nand MAPK13.\n3.6 STRING protein–protein interaction\nanalysis\nThrough STRING protein–protein interaction network analysis\n(Figure 5), it was found that the KRT27 gene serves as a core node\namong differentially expressed genes related to hair follicle\ndevelopment, closely interacting with several keratin genes as-sociated\nwith hair follicle development, such as KRT26, KRT71, KRT39, and\nKRT25. Moreover, the KRT27 gene is significantly enriched in the\nestrogen signaling pathway, as well as in the entries for structural\nmolecule activity and hair follicle morphogenesis, indicating that it\nmay play an important role in hair follicle development.\nTABLE 2  Skin structure parameters of Qianhua Mutton Merino sheep.\n\nTABLE 2  Skin structure parameters of Qianhua Mutton Merino sheep.\nProject\nM1\nM12\nPF /mm2\n8.15 ± 1.04a\n4.38 ± 0.21b\nPF diameter/μm\n90.62 ± 7.13\n91.68 ± 7.24\nPF depth /μm\n865.09 ± 42.53\n889.64 ± 61.63\nSF /mm2\n61.81 ± 3.45a\n55.31 ± 5.21b\nSF diameter/μm\n80.61 ± 7.07b\n85.65 ± 6.45a\nSF depth /μm\n702.05 ± 31.41b\n846.45 ± 51.62a\nWFD/μm\n20.65 ± 1.42b\n21.61 ± 1.09a\nS/P\n7.58 ± 0.64b\n12.64 ± 1.02a\nDifferent letters in the same row indicate significant differences (p﹤0.05), and the same\nletters indicate non-significant differences (p﹥0.05).\n\nTABLE 2  Skin structure parameters of Qianhua Mutton Merino sheep.\nProject\nM1\nM12\nPF /mm2\n8.15 ± 1.04a\n4.38 ± 0.21b\nPF diameter/μm\n90.62 ± 7.13\n91.68 ± 7.24\nPF depth /μm\n865.09 ± 42.53\n889.64 ± 61.63\nSF /mm2\n61.81 ± 3.45a\n55.31 ± 5.21b\nSF diameter/μm\n80.61 ± 7.07b\n85.65 ± 6.45a\nSF depth /μm\n702.05 ± 31.41b\n846.45 ± 51.62a\nWFD/μm\n20.65 ± 1.42b\n21.61 ± 1.09a\nS/P\n7.58 ± 0.64b\n12.64 ± 1.02a\nDifferent letters in the same row indicate significant differences (p﹤0.05), and the same\nletters indicate non-significant differences (p﹥0.05).\n\nQin et al.\b\n10.3389/fvets.2025.1699868\nFrontiers in Veterinary Science\n06\nfrontiersin.org\n3.7 Detection of the expression level of the \nKRT27 gene in hair follicle tissues at \ndifferent ages\nIn this experiment, we employed immunohistochemistry (IHC) \nmethods to ex-amine the skin tissues of newborn and one-year-old \nQianhua Mutton Merino, aiming to investigate the tissue localization \nof KRT27 in the hair follicles of Qianhua Mutton Merino at different \nages (Figures 6A,B,D,E). The hair follicle structures in the M1 group \nwere relatively closely arranged, with clear structures and a more \nregular morphology, indicating that they were in the stage of \nincomplete maturation. In contrast, the hair follicles in the M12 group \nFIGURE 2\n(A) Comparison of the regional distribution of group M1 and that of the reference genome; (B) comparison of the regional distribution of group M12 \nand that of the reference genome.\nFIGURE 3\n(A) Volcano plot of differential mRNA; (B) Venn diagram of genes; (C) downregulation and upregulation of differential genes; (D) RT-qPCR validation.\n** indicates significance at p < 0.01.\n\nQin et al.\b\n10.3389/fvets.2025.1699868\nFrontiers in Veterinary Science\n06\nfrontiersin.org\n3.7 Detection of the expression level of the \nKRT27 gene in hair follicle tissues at \ndifferent ages\nIn this experiment, we employed immunohistochemistry (IHC) \nmethods to ex-amine the skin tissues of newborn and one-year-old \nQianhua Mutton Merino, aiming to investigate the tissue localization \nof KRT27 in the hair follicles of Qianhua Mutton Merino at different \nages (Figures 6A,B,D,E). The hair follicle structures in the M1 group \nwere relatively closely arranged, with clear structures and a more \nregular morphology, indicating that they were in the stage of \nincomplete maturation. In contrast, the hair follicles in the M12 group \nFIGURE 2\n(A) Comparison of the regional distribution of group M1 and that of the reference genome; (B) comparison of the regional distribution of group M12 \nand that of the reference genome.\nFIGURE 3\n(A) Volcano plot of differential mRNA; (B) Venn diagram of genes; (C) downregulation and upregulation of differential genes; (D) RT-qPCR validation.\n** indicates significance at p < 0.01.\n\nQin et al.\b\n10.3389/fvets.2025.1699868\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nexhibited a more complex morphology, with some curvature and\nbranching and a relatively loose arrangement of cells, indicating that\nthe hair follicles had fully matured. After DAB staining of the sections\n(Figures 6B,E), it was observed that the KRT27 positive expression\nareas were brownish-yellow, a trait specifically expressed in the inner\nroot sheath of the hair follicles. This finding indicates that KRT27 is\nlocalized in the inner root sheath region of the skin hair follicles of\nboth newborn and one-year-old Qianhua Mutton Merino.\nThe results of the RT-qPCR validation (Figure 6F) indicate that\nKRT27 is expressed in both tissue groups, with a lower expression level\nin the M1 group compared to the M12 group, and the M12 group’s\nexpression level was significantly upregulated (p < 0.01).\nThe analysis of the average optical density and cumulative optical\ndensity values from immunohistochemistry revealed that the expression\nlevel of the KRT27 gene in M12 is higher than that in M1 (Figure 6C).\n4 Discussion\n4.1 The tissue structure of the skin hair\nfollicles of Qianhua Mutton Merino sheep\nSkin hair follicles are where wool is produced in wool-bearing\nsheep. As one of the few periodically growing micro-organs in a\nTABLE 3  Kegg enrichment analysis of DEGs.\nPathway name\nDEGs\nECM-receptor interaction\nsignaling pathway\nLAMA2, ITGA8, COL4A3, ITGA9, TNXB, LAMC3, PG4, α4(IV) chain, α6(IV) chain, LAMA5, LAMA11, LAMC1, α3(IV) chain, HSPG2,\nTSP1, α1(IV) chain, α2(IV) chain, LAMB1, THBS3, FN1, ITGB3, CD36, ITGA4, LAMB2, ITGA10, RELN, TSP2\nPI3K-Akt signaling pathway\nVEGF-D, LAMA2, PDGFRA, CREB3L4, ITGA8, COL4A3, ITGA9, TNXB, LAMC3, CSF1R, α4(IV) chain, GHR, FLT – 4, CREB3L3,\nPIK3R6, α6(IV) chain, LAMA5, LAMA11, LAMC1, FGF21, IL-6R, α3(IV) chain, Cyclin E1, NR4A1, MYB, TSP1, α1(IV) chain, α2(IV)\nchain, LAMB1, THBS3, FN1, ITGB3, ITGA4, LAMB2, PDGFRβ, ITGA10, RELN, LPAR1, PP2A Bγ, GYS-1, TSP2, IL7, erbB – 4, CREB3L2\nFocal adhesion signaling\npathway\n\nQin et al.\b\n10.3389/fvets.2025.1699868\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nexhibited a more complex morphology, with some curvature and\nbranching and a relatively loose arrangement of cells, indicating that\nthe hair follicles had fully matured. After DAB staining of the sections\n(Figures 6B,E), it was observed that the KRT27 positive expression\nareas were brownish-yellow, a trait specifically expressed in the inner\nroot sheath of the hair follicles. This finding indicates that KRT27 is\nlocalized in the inner root sheath region of the skin hair follicles of\nboth newborn and one-year-old Qianhua Mutton Merino.\nThe results of the RT-qPCR validation (Figure 6F) indicate that\nKRT27 is expressed in both tissue groups, with a lower expression level\nin the M1 group compared to the M12 group, and the M12 group’s\nexpression level was significantly upregulated (p < 0.01).\nThe analysis of the average optical density and cumulative optical\ndensity values from immunohistochemistry revealed that the expression\nlevel of the KRT27 gene in M12 is higher than that in M1 (Figure 6C).\n4 Discussion\n4.1 The tissue structure of the skin hair\nfollicles of Qianhua Mutton Merino sheep\nSkin hair follicles are where wool is produced in wool-bearing\nsheep. As one of the few periodically growing micro-organs in a\nTABLE 3  Kegg enrichment analysis of DEGs.\nPathway name\nDEGs\nECM-receptor interaction\nsignaling pathway\nLAMA2, ITGA8, COL4A3, ITGA9, TNXB, LAMC3, PG4, α4(IV) chain, α6(IV) chain, LAMA5, LAMA11, LAMC1, α3(IV) chain, HSPG2,\nTSP1, α1(IV) chain, α2(IV) chain, LAMB1, THBS3, FN1, ITGB3, CD36, ITGA4, LAMB2, ITGA10, RELN, TSP2\nPI3K-Akt signaling pathway\nVEGF-D, LAMA2, PDGFRA, CREB3L4, ITGA8, COL4A3, ITGA9, TNXB, LAMC3, CSF1R, α4(IV) chain, GHR, FLT – 4, CREB3L3,\nPIK3R6, α6(IV) chain, LAMA5, LAMA11, LAMC1, FGF21, IL-6R, α3(IV) chain, Cyclin E1, NR4A1, MYB, TSP1, α1(IV) chain, α2(IV)\nchain, LAMB1, THBS3, FN1, ITGB3, ITGA4, LAMB2, PDGFRβ, ITGA10, RELN, LPAR1, PP2A Bγ, GYS-1, TSP2, IL7, erbB – 4, CREB3L2\nFocal adhesion signaling\npathway\n\nVEGF-D, LAMA2, PDGFRA, MYLPF, ITGA8, COL4A3, ITGA9, TNXB, FYN, LAMC3, α2(IV) chain, FLT – 4, PIPKβ, MLCK2, α6(IV)\nchain, LAMA5, LAMA11, LAMC1, Cav-3, Shc2, α3(IV) chain, TSP1, α1(IV) chain, LAMB2, filamin C, THBS3, FN1, LAMB3, ITGA4,\nPDGFRβ, ITGA10, RELN, TSP2\nCell adhesion molecules\nsignaling pathway\nITGA8, ITGA9, ITGAM, ALCAM, PCDH15, JAM3, Siglec – 1, VTCN1, CD34, ICAM1, VCAM – 1, HLA, SLA – DQA, NCAM2, CLDN4,\nCLDN 23, CLDN 8, CLDN 3, CLDN7, selectin L, ITGA4, CLDN 6, JAM2, CLDN 17\nMAPK signaling pathway\nVEGF-D, PDGFRA, CSF1R, FLT – 4, DUSP10, MAP2K3, CD14, FGF21, MNK1, RasGRP3, CACNA2D3, NR4A1, TGFβR2, GADD45γ,\nFLNC, PDGFRβ, MAPK12, VGCCs, MAPK13, RasGRP4, PLA2G4E, erbB – 4, STMN1, MEF2C\nEstrogen signaling pathway\nCREB3L4, KRT18, PLCβ1, MMP2, ADCY2, KRT23, ESR1, CREB3L3, KRT25, KRT39, KRT26, KRT27, SHC2, NESP55, KRT33B, HB – EGF,\nKRT40, KRT48 kDa,8C-1, KRT16, CaM, ADCY4, K33, V15, CREB3L2\nFIGURE 4\n(A) GO enrichment bar plot; (B) KEGG enrichment bar plot; (C) DEGs in the KEGG pathway related to hair follicle development; (D) KEGG enrichment\nscatter plot; (E) GO en-richment scatter plot; (F) DEGs in the GO pathway related to hair follicle development.\n\nVEGF-D, LAMA2, PDGFRA, MYLPF, ITGA8, COL4A3, ITGA9, TNXB, FYN, LAMC3, α2(IV) chain, FLT – 4, PIPKβ, MLCK2, α6(IV)\nchain, LAMA5, LAMA11, LAMC1, Cav-3, Shc2, α3(IV) chain, TSP1, α1(IV) chain, LAMB2, filamin C, THBS3, FN1, LAMB3, ITGA4,\nPDGFRβ, ITGA10, RELN, TSP2\nCell adhesion molecules\nsignaling pathway\nITGA8, ITGA9, ITGAM, ALCAM, PCDH15, JAM3, Siglec – 1, VTCN1, CD34, ICAM1, VCAM – 1, HLA, SLA – DQA, NCAM2, CLDN4,\nCLDN 23, CLDN 8, CLDN 3, CLDN7, selectin L, ITGA4, CLDN 6, JAM2, CLDN 17\nMAPK signaling pathway\nVEGF-D, PDGFRA, CSF1R, FLT – 4, DUSP10, MAP2K3, CD14, FGF21, MNK1, RasGRP3, CACNA2D3, NR4A1, TGFβR2, GADD45γ,\nFLNC, PDGFRβ, MAPK12, VGCCs, MAPK13, RasGRP4, PLA2G4E, erbB – 4, STMN1, MEF2C\nEstrogen signaling pathway\nCREB3L4, KRT18, PLCβ1, MMP2, ADCY2, KRT23, ESR1, CREB3L3, KRT25, KRT39, KRT26, KRT27, SHC2, NESP55, KRT33B, HB – EGF,\nKRT40, KRT48 kDa,8C-1, KRT16, CaM, ADCY4, K33, V15, CREB3L2\nFIGURE 4\n(A) GO enrichment bar plot; (B) KEGG enrichment bar plot; (C) DEGs in the KEGG pathway related to hair follicle development; (D) KEGG enrichment\nscatter plot; (E) GO en-richment scatter plot; (F) DEGs in the GO pathway related to hair follicle development.\n\nQin et al.\b\n10.3389/fvets.2025.1699868\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nFIGURE 5\nSTRING protein–protein interaction analysis.\nsheep’s body, a hair follicle relies on its cyclical growth to facilitate the\ngrowth and shedding of wool (13). Therefore, the characteristics and\ntissue structure of the hair follicle determine the quality and yield of\nwool in wool-producing animals. The skin hair follicles of fine wool\nsheep can be classified into primary hair follicles and secondary hair\nfollicles based on their developmental timing and tissue structure (14).\nPrimary hair follicles develop between 50 and 60 days of gestation and\ncomplete their development by the time of birth. These follicles are\ncharacterized by larger hair bulbs and longer and thicker follicles, and\nthey possess two well-developed sebaceous glands. The hair shafts\nproduced typically contain a medulla. In contrast, secondary hair\nfollicles appear between 80 and 90 days of gestation. They are\ncharacterized by smaller hair bulbs and shorter and finer follicles, and\nthey either lack or possess only one underdeveloped sebaceous gland.\nThe hair shafts from these follicles do not contain a medulla (15–17).\nIn the follicular characteristics of fine-wool sheep, the S/p value (the\nratio of secondary follicles to primary follicles) is a trait with moderate\nheritability (0.3–0.4), being minimally influenced by non-genetic\nfactors. It is one of the important indicators reflecting the genetic\ncharacteristics of sheep breeds and is closely related to the yield and\nquality of wool (18, 19). In their study of semi-fine hair in Yunnan,\nHong Qionghua et al. found that the number of primary hair follicles\nat birth and at 1 year old was 10.50/mm 2 and 3.55/mm 2, respectively;\nthe number of secondary hair follicles was 36.94/mm 2 and 26.26/mm\n2, respectively; and the S/P ratios were 3.52 and 7.39, respectively (20).\nThe number of primary hair follicles of cashmere goats at birth and in\nadulthood was 9.97/mm2 and 4.01/mm2, respectively; the number of\nsecondary hair follicles was 60.75/mm2 at birth and 46.83/mm2 in\n\nQin et al.\b\n10.3389/fvets.2025.1699868\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nFIGURE 5\nSTRING protein–protein interaction analysis.\nsheep’s body, a hair follicle relies on its cyclical growth to facilitate the\ngrowth and shedding of wool (13). Therefore, the characteristics and\ntissue structure of the hair follicle determine the quality and yield of\nwool in wool-producing animals. The skin hair follicles of fine wool\nsheep can be classified into primary hair follicles and secondary hair\nfollicles based on their developmental timing and tissue structure (14).\nPrimary hair follicles develop between 50 and 60 days of gestation and\ncomplete their development by the time of birth. These follicles are\ncharacterized by larger hair bulbs and longer and thicker follicles, and\nthey possess two well-developed sebaceous glands. The hair shafts\nproduced typically contain a medulla. In contrast, secondary hair\nfollicles appear between 80 and 90 days of gestation. They are\ncharacterized by smaller hair bulbs and shorter and finer follicles, and\nthey either lack or possess only one underdeveloped sebaceous gland.\nThe hair shafts from these follicles do not contain a medulla (15–17).\nIn the follicular characteristics of fine-wool sheep, the S/p value (the\nratio of secondary follicles to primary follicles) is a trait with moderate\nheritability (0.3–0.4), being minimally influenced by non-genetic\nfactors. It is one of the important indicators reflecting the genetic\ncharacteristics of sheep breeds and is closely related to the yield and\nquality of wool (18, 19). In their study of semi-fine hair in Yunnan,\nHong Qionghua et al. found that the number of primary hair follicles\nat birth and at 1 year old was 10.50/mm 2 and 3.55/mm 2, respectively;\nthe number of secondary hair follicles was 36.94/mm 2 and 26.26/mm\n2, respectively; and the S/P ratios were 3.52 and 7.39, respectively (20).\nThe number of primary hair follicles of cashmere goats at birth and in\nadulthood was 9.97/mm2 and 4.01/mm2, respectively; the number of\nsecondary hair follicles was 60.75/mm2 at birth and 46.83/mm2 in\n\n52 and 7.39, respectively (20).The number of primary hair follicles of cashmere goats at birth and in\nadulthood was 9.97/mm2 and 4.01/mm2, respectively; the number of\nsecondary hair follicles was 60.75/mm2 at birth and 46.83/mm2 in\nadulthood; and the S/P ratio at birth was 6.10, while the S/P ratio in\nadulthood was 11.82 (21). This study found that, at birth, Qianhua\nMutton Merino sheep had an average of 8.15 ± 1.04 primary hair\nfollicles/mm2 and 61.81 ± 3.45 secondary hair follicles/mm2, with an\nS/P ratio of 7.58 ± 0.64. By 1 year of age, these values changed to\n4.38 ± 5.21 primary hair follicles/mm2, 55.31 ± 5.21 secondary hair\nfollicles/mm2, and an S/P ratio of 12.64 ± 1.02. Compared to fine-wool\nsheep and wool-producing fine-wool sheep, the number of primary\nhair follicles in Qianhua Mutton Merino sheep at birth is lower than\nthat of the corresponding breeds both domestically and internationally,\nwhile the number of secondary hair follicles and the S/P ratio are\nhigher than those of the corresponding breeds. At 1 year of age, the\nnumber of primary hair follicles is higher than that of the\ncorresponding breeds both domestically and internationally, and the\n\n52 and 7.39, respectively (20).The number of primary hair follicles of cashmere goats at birth and in\nadulthood was 9.97/mm2 and 4.01/mm2, respectively; the number of\nsecondary hair follicles was 60.75/mm2 at birth and 46.83/mm2 in\nadulthood; and the S/P ratio at birth was 6.10, while the S/P ratio in\nadulthood was 11.82 (21). This study found that, at birth, Qianhua\nMutton Merino sheep had an average of 8.15 ± 1.04 primary hair\nfollicles/mm2 and 61.81 ± 3.45 secondary hair follicles/mm2, with an\nS/P ratio of 7.58 ± 0.64. By 1 year of age, these values changed to\n4.38 ± 5.21 primary hair follicles/mm2, 55.31 ± 5.21 secondary hair\nfollicles/mm2, and an S/P ratio of 12.64 ± 1.02. Compared to fine-wool\nsheep and wool-producing fine-wool sheep, the number of primary\nhair follicles in Qianhua Mutton Merino sheep at birth is lower than\nthat of the corresponding breeds both domestically and internationally,\nwhile the number of secondary hair follicles and the S/P ratio are\nhigher than those of the corresponding breeds. At 1 year of age, the\nnumber of primary hair follicles is higher than that of the\ncorresponding breeds both domestically and internationally, and the\n\nQin et al.\b\n10.3389/fvets.2025.1699868\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nnumber of secondary hair follicles and the S/P ratio are also higher\nthan those of the corresponding breeds. It is speculated that this may\nbe due to the Qianhua Mutton Merino sheep being a dual-purpose\nbreed for meat and wool, with a tendency toward meat production\nand relatively rapid body development, resulting in an increased body\nsurface area. Consequently, the density of hair follicles is lower than\nthat of breeds primarily used for wool or those that are dual-purpose\nfor both wool and meat.\n4.2 The genes and signaling pathways\nrelated to the development of skin hair\nfollicles in Qianhua Mutton Merino sheep\nHair follicle formation is jointly regulated by a series of related\nfactors and signaling pathways. There has been considerable research\nrelated to hair follicle development, and it has been found that various\nmolecular signaling pathways, including Wnt, BMP, TGF-β, PI3K-Akt,\nand MAPK, are involved in this process (22). In addition, with the\ncontinuous development of high-throughput sequencing technology,\nnumerous studies have indicated that various non-coding RNAs, such as\nlncRNA, miRNA, and circRNA, are also involved in the process of hair\nfollicle development (23–25). Different signaling pathways, non-coding\nRNAs, and other important regulatory factors form a complex molecular\nregulatory network that jointly regulates the development of hair follicles.\nAmong various signaling pathways, the PI3K-Akt signaling pathway\nplays a crucial role in the development of hair follicles by promoting\ncommunication between epidermal and dermal cells (26). The PI3K-Akt\nsignaling pathway can activate the Wnt signaling pathway by increasing\nthe phosphorylation levels of β-catenin and inhibiting the\nphosphorylation of glycogen synthase kinase-3b, thereby promoting the\ngrowth of hair follicles (27). Xiaoyang Lv et al. found that many genes,\nsuch as FGF12 and ATP1B4, are enriched in the PI3K-Akt signaling\npathway related to hair follicle growth and development (28). In this\n\nQin et al.\b\n10.3389/fvets.2025.1699868\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nnumber of secondary hair follicles and the S/P ratio are also higher\nthan those of the corresponding breeds. It is speculated that this may\nbe due to the Qianhua Mutton Merino sheep being a dual-purpose\nbreed for meat and wool, with a tendency toward meat production\nand relatively rapid body development, resulting in an increased body\nsurface area. Consequently, the density of hair follicles is lower than\nthat of breeds primarily used for wool or those that are dual-purpose\nfor both wool and meat.\n4.2 The genes and signaling pathways\nrelated to the development of skin hair\nfollicles in Qianhua Mutton Merino sheep\nHair follicle formation is jointly regulated by a series of related\nfactors and signaling pathways. There has been considerable research\nrelated to hair follicle development, and it has been found that various\nmolecular signaling pathways, including Wnt, BMP, TGF-β, PI3K-Akt,\nand MAPK, are involved in this process (22). In addition, with the\ncontinuous development of high-throughput sequencing technology,\nnumerous studies have indicated that various non-coding RNAs, such as\nlncRNA, miRNA, and circRNA, are also involved in the process of hair\nfollicle development (23–25). Different signaling pathways, non-coding\nRNAs, and other important regulatory factors form a complex molecular\nregulatory network that jointly regulates the development of hair follicles.\nAmong various signaling pathways, the PI3K-Akt signaling pathway\nplays a crucial role in the development of hair follicles by promoting\ncommunication between epidermal and dermal cells (26). The PI3K-Akt\nsignaling pathway can activate the Wnt signaling pathway by increasing\nthe phosphorylation levels of β-catenin and inhibiting the\nphosphorylation of glycogen synthase kinase-3b, thereby promoting the\ngrowth of hair follicles (27). Xiaoyang Lv et al. found that many genes,\nsuch as FGF12 and ATP1B4, are enriched in the PI3K-Akt signaling\npathway related to hair follicle growth and development (28). In this\n\nXiaoyang Lv et al.found that many genes,\nsuch as FGF12 and ATP1B4, are enriched in the PI3K-Akt signaling\npathway related to hair follicle growth and development (28).In this\nstudy, it was also found that 14 DEGs were enriched in the PI3K-Akt\nsignaling pathway. The trend of changes in these genes may be closely\nrelated to the development and growth of hair follicles. MAPK is another\nimportant signaling pathway in regulating hair follicle development. It\ncontributes to the formation of the hair follicle substrate and the cyclical\ngrowth of hair follicles by participating in the mechanisms of regulating\napoptosis, differentiation, proliferation, and stress (29). In this study, it\nwas found that 24 DEGs were enriched in MAPK, indicating that the\nMAPK signaling pathway is involved in the development of skin hair\nfollicles in Qianhua Mutton Merino sheep.\nThis study found that the secondary hair follicles of Qianhua\nMutton Merino sheep are still in the process of development at birth.\nThere are extremely significant differences between birth and age in\nterms of the number of secondary hair follicles, the diameter and depth\nof the hair follicles, and the diameter of the wool fibers growing from\nthe secondary hair follicles. It can be seen that secondary hair follicles\nare still in dynamic development after birth. In this study, DEGs related\nto hair follicle development were identified as IGF-2, FGF21, VEGF-D,\nand KRT16, KRT25, KRT26, KRT27, KRT33, and KRT39 of the KRT\nfamily. The above-mentioned genes are enriched in hair follicle-\ndevelopment-related signaling pathways such as PI3K-Akt, MAPK and\nEstrogen. Fibroblast growth factor 21 (FGF21) is a key regulatory factor\nin hair follicle development and cycling (30). Liu et al. found that\nFGF21 induces AKT expression, subsequently leading to the\nAKT-mediated phosphorylation of target molecules, thereby\ninfluencing the intricate signaling network that regulates hair growth\n(31). Basharat Bhat et al. found that insulin-like growth factor 2 (IGF2)\npromotes hair follicle development during the growth phase of goats\nFIGURE 6\n\nXiaoyang Lv et al.found that many genes,\nsuch as FGF12 and ATP1B4, are enriched in the PI3K-Akt signaling\npathway related to hair follicle growth and development (28).In this\nstudy, it was also found that 14 DEGs were enriched in the PI3K-Akt\nsignaling pathway. The trend of changes in these genes may be closely\nrelated to the development and growth of hair follicles. MAPK is another\nimportant signaling pathway in regulating hair follicle development. It\ncontributes to the formation of the hair follicle substrate and the cyclical\ngrowth of hair follicles by participating in the mechanisms of regulating\napoptosis, differentiation, proliferation, and stress (29). In this study, it\nwas found that 24 DEGs were enriched in MAPK, indicating that the\nMAPK signaling pathway is involved in the development of skin hair\nfollicles in Qianhua Mutton Merino sheep.\nThis study found that the secondary hair follicles of Qianhua\nMutton Merino sheep are still in the process of development at birth.\nThere are extremely significant differences between birth and age in\nterms of the number of secondary hair follicles, the diameter and depth\nof the hair follicles, and the diameter of the wool fibers growing from\nthe secondary hair follicles. It can be seen that secondary hair follicles\nare still in dynamic development after birth. In this study, DEGs related\nto hair follicle development were identified as IGF-2, FGF21, VEGF-D,\nand KRT16, KRT25, KRT26, KRT27, KRT33, and KRT39 of the KRT\nfamily. The above-mentioned genes are enriched in hair follicle-\ndevelopment-related signaling pathways such as PI3K-Akt, MAPK and\nEstrogen. Fibroblast growth factor 21 (FGF21) is a key regulatory factor\nin hair follicle development and cycling (30). Liu et al. found that\nFGF21 induces AKT expression, subsequently leading to the\nAKT-mediated phosphorylation of target molecules, thereby\ninfluencing the intricate signaling network that regulates hair growth\n(31). Basharat Bhat et al. found that insulin-like growth factor 2 (IGF2)\npromotes hair follicle development during the growth phase of goats\nFIGURE 6\n\nBasharat Bhat et al.found that insulin-like growth factor 2 (IGF2)\npromotes hair follicle development during the growth phase of goats\nFIGURE 6\nThe tissue expression levels of KRT27 in the skin hair follicles of sheep of different ages. (A) The control group of longitudinal sections of newborn skin\ntissue; (B) the experimental group of longitudinal sections of newborn skin tissue; (C) IOD and AOD of the KRT27 gene; (D) the one-year-old group of\nlongitudinal sections of newborn skin tissue; (E) the experimental group of longitudinal sections of newborn skin tissue; (F) RT-qPCR.\n\nBasharat Bhat et al.found that insulin-like growth factor 2 (IGF2)\npromotes hair follicle development during the growth phase of goats\nFIGURE 6\nThe tissue expression levels of KRT27 in the skin hair follicles of sheep of different ages. (A) The control group of longitudinal sections of newborn skin\ntissue; (B) the experimental group of longitudinal sections of newborn skin tissue; (C) IOD and AOD of the KRT27 gene; (D) the one-year-old group of\nlongitudinal sections of newborn skin tissue; (E) the experimental group of longitudinal sections of newborn skin tissue; (F) RT-qPCR.\n\nQin et al.\b\n10.3389/fvets.2025.1699868\nFrontiers in Veterinary Science\n10\nfrontiersin.org\nthrough MAPK signaling (32). Vascular endothelial growth factor\n(VEGF) plays a key role in promoting angiogenesis and supporting hair\nfollicle growth (33). Keratin, a member of the intermediate filament\nsuperfamily, is the primary structural component of hair and hair\nfollicles. The specific types and quantities of keratin not only determine\nhair characteristics but are also vital for maintaining its structural\nstability (34). Wen et al. identified 57 differentially abundant proteins\n(DAPs) in the skin of goat necks through multi-omics studies. These\nproteins from the keratin family are highly expressed during both the\ngrowth and regression phases (35). Therefore, the keratin family plays\na role in determining the morphology of hair follicles and fibers.\nResearch by Yu et al. indicates that the asymmetric expression of the\nKRT27 gene is associated with hair whorl deviation and follicle\ncurvature, playing a role in the de-termination of hair follicle\nmorphology (36). Haoran Sun et al. discovered that lncRNA targets the\nestrogen signaling pathway and the PI3K-Akt signaling pathway\nthrough the KRT27 gene in Dorper sheep (10). As a type I keratin gene,\nthe expression level of KRT27 in different sheep breeds is significantly\nrelated to the distribution density of hair follicles in specific areas (37).\nTherefore, it was inferred that the KRT27 gene could serve as a\ncandidate for exerting regulatory functions.\n5 Conclusion\nThe number of primary and secondary hair follicles in\nQianhua Mutton Merino sheep at birth was significantly higher\nthan that at the age of one, while the opposite was true for the S/P\nvalue. Qianhua Mutton Merino DEGs in the skin tissues of\nnewborn and one-year-old sheep, including IGF-2, FGF21, and\nVEGF-D, as well as KRT16, KRT25, KRT26, KRT27, KRT33, and\nKRT39 of the KRT family, are significantly enriched in signaling\npathways related to hair follicle development, such as the PI3K-\nAkt, MAPK, and Estrogen signaling pathways, indicating that the\nabove mentioned signaling pathways are involved in hair\n\nQin et al.\b\n10.3389/fvets.2025.1699868\nFrontiers in Veterinary Science\n10\nfrontiersin.org\nthrough MAPK signaling (32). Vascular endothelial growth factor\n(VEGF) plays a key role in promoting angiogenesis and supporting hair\nfollicle growth (33). Keratin, a member of the intermediate filament\nsuperfamily, is the primary structural component of hair and hair\nfollicles. The specific types and quantities of keratin not only determine\nhair characteristics but are also vital for maintaining its structural\nstability (34). Wen et al. identified 57 differentially abundant proteins\n(DAPs) in the skin of goat necks through multi-omics studies. These\nproteins from the keratin family are highly expressed during both the\ngrowth and regression phases (35). Therefore, the keratin family plays\na role in determining the morphology of hair follicles and fibers.\nResearch by Yu et al. indicates that the asymmetric expression of the\nKRT27 gene is associated with hair whorl deviation and follicle\ncurvature, playing a role in the de-termination of hair follicle\nmorphology (36). Haoran Sun et al. discovered that lncRNA targets the\nestrogen signaling pathway and the PI3K-Akt signaling pathway\nthrough the KRT27 gene in Dorper sheep (10). As a type I keratin gene,\nthe expression level of KRT27 in different sheep breeds is significantly\nrelated to the distribution density of hair follicles in specific areas (37).\nTherefore, it was inferred that the KRT27 gene could serve as a\ncandidate for exerting regulatory functions.\n5 Conclusion\nThe number of primary and secondary hair follicles in\nQianhua Mutton Merino sheep at birth was significantly higher\nthan that at the age of one, while the opposite was true for the S/P\nvalue. Qianhua Mutton Merino DEGs in the skin tissues of\nnewborn and one-year-old sheep, including IGF-2, FGF21, and\nVEGF-D, as well as KRT16, KRT25, KRT26, KRT27, KRT33, and\nKRT39 of the KRT family, are significantly enriched in signaling\npathways related to hair follicle development, such as the PI3K-\nAkt, MAPK, and Estrogen signaling pathways, indicating that the\nabove mentioned signaling pathways are involved in hair\n\nfollicle development.\nData availability statement\nThe original contributions presented in the study are publicly\navailable. This data can be found here: The data has been uploaded to the\nSRA of NCBI,  Link: http://www.ncbi.nlm.nih.gov/bioproject/1366756.\nEthics statement\nThe animal studies were approved by Laboratory Animal\nWelfare and Ethics Committee of Jilin Agricultural University. The\nstudies were conducted in accordance with the local legislation\nand institutional requirements. Written informed consent was\nobtained from the owners for the participation of their animals in\nthis study.\nAuthor contributions\nZQ: Validation, Writing  – original draft. XS: Visualization,\nWriting – review & editing. LS: Writing – review & editing, Formal\nanalysis. MY: Supervision, Software, Writing – review & editing. HJ:\nWriting – review & editing, Project administration, Methodology.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. This research was funded\nby National Key research and Development Project, grant\nnumber 2021YFF1000702.\nConflict of interest\nMY was employed by Liaoning Anyou Biotechnology Co., Ltd.\nThe remaining authors declare that the research was conducted in\nthe absence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. Any product that may be evaluated in this article, or claim\nthat may be made by its manufacturer, is not guaranteed or endorsed\nby the publisher.\nReferences\n\nfollicle development.\nData availability statement\nThe original contributions presented in the study are publicly\navailable. This data can be found here: The data has been uploaded to the\nSRA of NCBI,  Link: http://www.ncbi.nlm.nih.gov/bioproject/1366756.\nEthics statement\nThe animal studies were approved by Laboratory Animal\nWelfare and Ethics Committee of Jilin Agricultural University. The\nstudies were conducted in accordance with the local legislation\nand institutional requirements. Written informed consent was\nobtained from the owners for the participation of their animals in\nthis study.\nAuthor contributions\nZQ: Validation, Writing  – original draft. XS: Visualization,\nWriting – review & editing. LS: Writing – review & editing, Formal\nanalysis. MY: Supervision, Software, Writing – review & editing. HJ:\nWriting – review & editing, Project administration, Methodology.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. This research was funded\nby National Key research and Development Project, grant\nnumber 2021YFF1000702.\nConflict of interest\nMY was employed by Liaoning Anyou Biotechnology Co., Ltd.\nThe remaining authors declare that the research was conducted in\nthe absence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated\norganizations, or those of the publisher, the editors and the\nreviewers. Any product that may be evaluated in this article, or claim\nthat may be made by its manufacturer, is not guaranteed or endorsed\nby the publisher.\nReferences\n\nAny product that may be evaluated in this article, or claim\nthat may be made by its manufacturer, is not guaranteed or endorsed\nby the publisher.References\n1.\tLi, X, Hao, F, Hu, X, Wang, H, Dai, B, Wang, X, et al. Generation of Tβ4 knock-in\ncashmere goat using CRISPR/Cas9. Int J Biol Sci. (2019) 15:1743–54. doi: 10.7150/ijbs.34820\n2.\tLiu, G, Cheng, G, Zhang, Y, Gao, S, Sun, H, Bai, L, et al. Pyridoxine regulates hair\nfollicle development via the PI3K/Akt, Wnt and notch signalling pathways in rex rabbits.\nAnim Nutr. 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+{"text": "【兽医文献】\n标题: Artificial intelligence, epistemic authority, and emerging risks in veterinary clinical decision-making\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nTYPE  Original Research\nPUBLISHED  14 July 2026\nDOI  10.3389/fvets.2026.1861004\nOPEN ACCESS\nEDITED BY\nVeasna Duong,\nInstitut Pasteur du Cambodge,\nCambodia\nREVIEWED BY\nAdeniyi Charles Adeola,\nChinese Academy of Sciences (CAS),\nChina\nFrancisca Bravo-Risi,\nUniversity of Texas Health Science\nCenter at Houston, United States\n*CORRESPONDENCE\nAndrea L. Grindeland Panter\nandrea@mclaughlinresearch.org\nRECEIVED 20 April 2026\nREVISED 26 June 2026\nACCEPTED 29 June 2026\nPUBLISHED 14 July 2026\nCITATION\nSeerley AL, Rothfuss MT, Gray BM,\nSebogo MA, Manakelew BA, Pounder JI,\nBowler BE, Leavens MJ and Grindeland\nPanter AL (2026) Identification and initial\ncharacterization of novel prion protein\ngene (PRNP) variants in wild Montana\nmule deer.\nFront. Vet. Sci. 13:1861004.\ndoi: 10.3389/fvets.2026.1861004\nCOPYRIGHT\n© 2026 Seerley, Rothfuss, Gray, Sebogo,\nManakelew, Pounder, Bowler, Leavens\nand Grindeland Panter. This is an\nopen-access article distributed under\nthe terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other\nforums is permitted, provided the\noriginal author(s) and the copyright\nowner(s) are credited and that the\noriginal publication in this journal is\ncited, in accordance with accepted\nacademic practice. No use, distribution\nor reproduction is permitted which does\nnot comply with these terms.\nIdentification and initial\ncharacterization of novel prion\nprotein gene (PRNP) variants in\nwild Montana mule deer\nAlyssa L. Seerley 1, Michael T. Rothfuss 2, Bridget M. Gray 1,\nMylene A. Sebogo 1, Bethelhem A. Manakelew 1, June I. Pounder 1,\nBruce E. Bowler 2, Moses J. Leavens 1 and\nAndrea L. Grindeland Panter 1*\n1Weissman Hood Institute at Touro University, McLaughlin Research Institute for Biomedical Sciences,\nGreat Falls, MT, United States, 2Department of Chemistry and Biochemistry, University of Montana,\nMissoula, MT, United States\nChronic Wasting Disease (CWD) is a transmissible spongiform encephalopathy\n(TSE) of cervids (elk, deer, moose, and reindeer) that is increasing in prevalence\nand expanding to new geographical areas. TSEs, commonly referred to as prion\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nTYPE  Original Research\nPUBLISHED  14 July 2026\nDOI  10.3389/fvets.2026.1861004\nOPEN ACCESS\nEDITED BY\nVeasna Duong,\nInstitut Pasteur du Cambodge,\nCambodia\nREVIEWED BY\nAdeniyi Charles Adeola,\nChinese Academy of Sciences (CAS),\nChina\nFrancisca Bravo-Risi,\nUniversity of Texas Health Science\nCenter at Houston, United States\n*CORRESPONDENCE\nAndrea L. Grindeland Panter\nandrea@mclaughlinresearch.org\nRECEIVED 20 April 2026\nREVISED 26 June 2026\nACCEPTED 29 June 2026\nPUBLISHED 14 July 2026\nCITATION\nSeerley AL, Rothfuss MT, Gray BM,\nSebogo MA, Manakelew BA, Pounder JI,\nBowler BE, Leavens MJ and Grindeland\nPanter AL (2026) Identification and initial\ncharacterization of novel prion protein\ngene (PRNP) variants in wild Montana\nmule deer.\nFront. Vet. Sci. 13:1861004.\ndoi: 10.3389/fvets.2026.1861004\nCOPYRIGHT\n© 2026 Seerley, Rothfuss, Gray, Sebogo,\nManakelew, Pounder, Bowler, Leavens\nand Grindeland Panter. This is an\nopen-access article distributed under\nthe terms of the Creative Commons\nAttribution License (CC BY). The use,\ndistribution or reproduction in other\nforums is permitted, provided the\noriginal author(s) and the copyright\nowner(s) are credited and that the\noriginal publication in this journal is\ncited, in accordance with accepted\nacademic practice. No use, distribution\nor reproduction is permitted which does\nnot comply with these terms.\nIdentification and initial\ncharacterization of novel prion\nprotein gene (PRNP) variants in\nwild Montana mule deer\nAlyssa L. Seerley 1, Michael T. Rothfuss 2, Bridget M. Gray 1,\nMylene A. Sebogo 1, Bethelhem A. Manakelew 1, June I. Pounder 1,\nBruce E. Bowler 2, Moses J. Leavens 1 and\nAndrea L. Grindeland Panter 1*\n1Weissman Hood Institute at Touro University, McLaughlin Research Institute for Biomedical Sciences,\nGreat Falls, MT, United States, 2Department of Chemistry and Biochemistry, University of Montana,\nMissoula, MT, United States\nChronic Wasting Disease (CWD) is a transmissible spongiform encephalopathy\n(TSE) of cervids (elk, deer, moose, and reindeer) that is increasing in prevalence\nand expanding to new geographical areas. TSEs, commonly referred to as prion\n\nTSEs, commonly referred to as prion\ndiseases, are fatal neurodegenerative diseases that occur in a variety of mam­\nmals, including humans, and typically exhibit species-specific characteristics. This\nstudy reports the sequencing of the prion protein gene (PRNP) in retropharyngeal\nlymph node samples from 358 Montana mule deer (Odocoileus hemionus) and\nthe identification of 36 PRNP genetic variants, many of which have not been\nreported previously. Further investigations tracked spatiotemporal characteristics\nof variants to hunting districts, year of harvest, and CWD status. PRNP polymor­\nphisms V12F, D20G, R40Q, and S225F were examined with EmCAST computa­\ntional predictions to determine the relationship between sequence and structural\nvariations providing further insights into mechanisms affecting CWD outcomes.\nEmCAST predictions suggest the novel variant V12F phenotype is attributable to\nfunctional changes such as altered protein–protein interactions that might be\nlinked to the CWD positive status of the samples. Notably, the analysis of S225F\nby EmCAST predicted that S225F is a neutral variation for folded PrP and incom­\npatible with fibril PrP, suggesting a potential structural mechanism for reduced\nfibril PrP formation, which is consistent with the previously proposed protective\nrole of this variant. The CWD-positive samples harboring PRNP variants were\nexamined with the prion RT-QuIC assay, including the novel variant V12F, which\nresulted in prion seeding activity.\nKEYWORDS\nchronic wasting disease, computational modeling, CWD, prions, RT-QuIC\n1 Introduction\nTransmissible spongiform encephalopathies (TSEs), known as prion diseases, are a class\nof rare, fatal neurodegenerative diseases that slowly progress over time and have no cure (1).\nThese diseases can occur in a variety of mammals, including humans, and are typically known\nfor exhibiting species-specific characteristics (1, 2). TSEs can be acquired by three mechanisms:\nInherited genetic mutations, spontaneous occurrence with no known etiology, or acquisition\n\nTSEs, commonly referred to as prion\ndiseases, are fatal neurodegenerative diseases that occur in a variety of mam­\nmals, including humans, and typically exhibit species-specific characteristics. This\nstudy reports the sequencing of the prion protein gene (PRNP) in retropharyngeal\nlymph node samples from 358 Montana mule deer (Odocoileus hemionus) and\nthe identification of 36 PRNP genetic variants, many of which have not been\nreported previously. Further investigations tracked spatiotemporal characteristics\nof variants to hunting districts, year of harvest, and CWD status. PRNP polymor­\nphisms V12F, D20G, R40Q, and S225F were examined with EmCAST computa­\ntional predictions to determine the relationship between sequence and structural\nvariations providing further insights into mechanisms affecting CWD outcomes.\nEmCAST predictions suggest the novel variant V12F phenotype is attributable to\nfunctional changes such as altered protein–protein interactions that might be\nlinked to the CWD positive status of the samples. Notably, the analysis of S225F\nby EmCAST predicted that S225F is a neutral variation for folded PrP and incom­\npatible with fibril PrP, suggesting a potential structural mechanism for reduced\nfibril PrP formation, which is consistent with the previously proposed protective\nrole of this variant. The CWD-positive samples harboring PRNP variants were\nexamined with the prion RT-QuIC assay, including the novel variant V12F, which\nresulted in prion seeding activity.\nKEYWORDS\nchronic wasting disease, computational modeling, CWD, prions, RT-QuIC\n1 Introduction\nTransmissible spongiform encephalopathies (TSEs), known as prion diseases, are a class\nof rare, fatal neurodegenerative diseases that slowly progress over time and have no cure (1).\nThese diseases can occur in a variety of mammals, including humans, and are typically known\nfor exhibiting species-specific characteristics (1, 2). TSEs can be acquired by three mechanisms:\nInherited genetic mutations, spontaneous occurrence with no known etiology, or acquisition\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nof infectious forms of the prion protein (3–6). Examples of TSEs\ninclude Creutzfeldt-Jakob Disease (CJD), Fatal Familial Insomnia\n(FFI), Gerstmann-Straussler-Scheinker syndrome (GSS), bovine\nspongiform encephalopathy (BSE), scrapie, feline spongiform enceph­\nalopathy, exotic ungulate spongiform encephalopathy, and chronic\nwasting disease (CWD) (1).\nPrion diseases occur when the normal cell-surface glycoprotein,\nwith an alpha helical conformation (PrPC), is converted into a disease-\nassociated conformation containing a high beta-sheet content (PrPSc).\nThis conversion then acts as a template, with PrPC misfolding into\nPrPSc aggregates, ultimately leading to neurodegeneration (7–9). PrPC\nexists in all mammals where it is expressed throughout the body in\norgans, tissues, and the central and peripheral nervous systems (9).\nStructurally, PrPC has a flexible N-terminal domain, and a C-terminus\ncontaining three alpha-helices and two anti-parallel beta-strands\nflanking the helix (10, 11). While the functional role of the normal\nform of the prion protein is not well understood, PrPSc has been exten­\nsively studied because of its role in TSEs. An important factor in this\nprocess for disease conversion, is the homology between the host’s\nprimary PrP sequence in comparison to the incoming PrPSc.\nMismatches between PrPC and PrPSc sequences may explain prion\ndisease species barriers, however, occasionally prions have been\nknown to “jump” to a different species, including zoonotic transmis­\nsion (8). The most notable example of this was the rise of variant CJD\n(vCJD) in the United Kingdom resulting from oral ingestion of bovine\nspongiform encephalopathy contaminated meat (7).\nChronic wasting disease is a prion disease that was first recog­\nnized in 1967 in a captive deer in Colorado (2). This disease affects\ncervids, which includes, elk, deer, reindeer, and moose, and is the most\ninfective of all prion diseases (2). CWD is spread among cervids via\nperipheral shedding of PrPSc from infected animals through bodily\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nof infectious forms of the prion protein (3–6). Examples of TSEs\ninclude Creutzfeldt-Jakob Disease (CJD), Fatal Familial Insomnia\n(FFI), Gerstmann-Straussler-Scheinker syndrome (GSS), bovine\nspongiform encephalopathy (BSE), scrapie, feline spongiform enceph­\nalopathy, exotic ungulate spongiform encephalopathy, and chronic\nwasting disease (CWD) (1).\nPrion diseases occur when the normal cell-surface glycoprotein,\nwith an alpha helical conformation (PrPC), is converted into a disease-\nassociated conformation containing a high beta-sheet content (PrPSc).\nThis conversion then acts as a template, with PrPC misfolding into\nPrPSc aggregates, ultimately leading to neurodegeneration (7–9). PrPC\nexists in all mammals where it is expressed throughout the body in\norgans, tissues, and the central and peripheral nervous systems (9).\nStructurally, PrPC has a flexible N-terminal domain, and a C-terminus\ncontaining three alpha-helices and two anti-parallel beta-strands\nflanking the helix (10, 11). While the functional role of the normal\nform of the prion protein is not well understood, PrPSc has been exten­\nsively studied because of its role in TSEs. An important factor in this\nprocess for disease conversion, is the homology between the host’s\nprimary PrP sequence in comparison to the incoming PrPSc.\nMismatches between PrPC and PrPSc sequences may explain prion\ndisease species barriers, however, occasionally prions have been\nknown to “jump” to a different species, including zoonotic transmis­\nsion (8). The most notable example of this was the rise of variant CJD\n(vCJD) in the United Kingdom resulting from oral ingestion of bovine\nspongiform encephalopathy contaminated meat (7).\nChronic wasting disease is a prion disease that was first recog­\nnized in 1967 in a captive deer in Colorado (2). This disease affects\ncervids, which includes, elk, deer, reindeer, and moose, and is the most\ninfective of all prion diseases (2). CWD is spread among cervids via\nperipheral shedding of PrPSc from infected animals through bodily\n\nThis disease affects\ncervids, which includes, elk, deer, reindeer, and moose, and is the most\ninfective of all prion diseases (2).CWD is spread among cervids via\nperipheral shedding of PrPSc from infected animals through bodily\nfluids and excrement such as saliva, urine, blood, feces, and additional\ntissues, including antler velvet before clinical signs of disease appear\n(1, 2, 12–16). Additionally, infectious misfolded prions can persist in\nthe environment long-term independent of a host, bind to vegetation,\nand potentially infect animals that graze on contaminated foliage (17,\n18). The type of vegetation and soil may influence binding properties\nof the prions, further complicating detection and decontamination in\nthe environment (17, 19–22). These properties make CWD difficult to\nmanage in wild cervids and may increase difficulty as CWD epidemics\nprogress (23). Consequently, CWD has been spreading throughout\nwild populations of cervids and according to the United States\nGeological Survey (USGS), it is now found in 36 states and 5 Canadian\nprovinces (24).\nPolymorphisms in the prion protein gene (PRNP) have been\nshown to affect host CWD susceptibility, progression of disease, and\nincubation periods through their encoding of variations in amino\nacid sequence, informing protein conformation, and thus impacting\nthe conversion of PrPC to the disease-causing form, PrPSc (2, 25–30).\nStructural elements of PrP differ significantly between genetic vari­\nants, with the PRNP gene providing templates for prion growth and\nconformational basis for the propagation of different strains and\nphenotypes of disease (2, 16, 31). Notably, investigations into PRNP\ngenetics have historically been useful for designing disease control\nprograms. Researchers discovered that a homozygous genotype for\nA136R154R171 in sheep and goats conferred resistance to scrapie, an\nobservation that subsequently led to successful eradication of the\ndisease in domestic sheep through breeding programs (2, 32–34).\nWith CWD posing substantial management challenges, researchers\n\nThis disease affects\ncervids, which includes, elk, deer, reindeer, and moose, and is the most\ninfective of all prion diseases (2).CWD is spread among cervids via\nperipheral shedding of PrPSc from infected animals through bodily\nfluids and excrement such as saliva, urine, blood, feces, and additional\ntissues, including antler velvet before clinical signs of disease appear\n(1, 2, 12–16). Additionally, infectious misfolded prions can persist in\nthe environment long-term independent of a host, bind to vegetation,\nand potentially infect animals that graze on contaminated foliage (17,\n18). The type of vegetation and soil may influence binding properties\nof the prions, further complicating detection and decontamination in\nthe environment (17, 19–22). These properties make CWD difficult to\nmanage in wild cervids and may increase difficulty as CWD epidemics\nprogress (23). Consequently, CWD has been spreading throughout\nwild populations of cervids and according to the United States\nGeological Survey (USGS), it is now found in 36 states and 5 Canadian\nprovinces (24).\nPolymorphisms in the prion protein gene (PRNP) have been\nshown to affect host CWD susceptibility, progression of disease, and\nincubation periods through their encoding of variations in amino\nacid sequence, informing protein conformation, and thus impacting\nthe conversion of PrPC to the disease-causing form, PrPSc (2, 25–30).\nStructural elements of PrP differ significantly between genetic vari­\nants, with the PRNP gene providing templates for prion growth and\nconformational basis for the propagation of different strains and\nphenotypes of disease (2, 16, 31). Notably, investigations into PRNP\ngenetics have historically been useful for designing disease control\nprograms. Researchers discovered that a homozygous genotype for\nA136R154R171 in sheep and goats conferred resistance to scrapie, an\nobservation that subsequently led to successful eradication of the\ndisease in domestic sheep through breeding programs (2, 32–34).\nWith CWD posing substantial management challenges, researchers\n\nWith CWD posing substantial management challenges, researchers\nhave begun to investigate the potential for genetic resistance within\nsusceptible species. Previous studies in mule deer (Odocoileus\nhemionus) have established genetic variations in PRNP at amino\nacid positions 20, 131, 225, 226, and 247 (2, 8, 32, 35–37), some of\nwhich influence susceptibility and progression of disease (8, 32).\nFor example, mule deer have been documented to have a polymor­\nphism at codon position 225, expressing either serine (S) or phenyl­\nalanine (F), and this is implicated in variable disease prevalence,\naltered time course of disease progression, and even variations in\ndisease neuropathology (28, 38–40). A survey of Wyoming and\nColorado mule deer show that mule deer with the 225SS PRNP\nvariant were 30 times more likely to be CWD positive compared to\nthe animals carrying the 225SF polymorphism (32). Recently, addi­\ntional polymorphic PRNP variants have been identified in mule\ndeer at codon 20, 96, 98, 116, and 159 (30); however, the down­\nstream PrP structural effects and specific phenotypes are not well\nunderstood. As CWD continues to increase in prevalence, the need\nto investigate emerging genotypes which influence disease progres­\nsion and species barriers is critical.\nIn this study we: 1) Identified and cataloged novel and previously\nknown PRNP polymorphisms present in Montana’s wild-mule deer\npopulation in years 2017, 2018, and 2022; 2) performed a prevalence\nanalysis on the variants using spatiotemporal and CWD data; 3) mod­\neled protein stability and misfolding probability of a subset of the\nnovel variants through EmCAST computational analyses; and 4) con­\nfirmed PrP seeding activity of CWD positive protein variants relative\nto wild-type protein via the use of the Real-Time Quaking-Induced\nConversion assay (RT-QuIC).\n2 Materials and methods\n2.1 Cervid samples\nSamples of retropharyngeal lymph nodes of wild mule deer\n(Odocoileus hemionus) were provided by Montana Fish, Wildlife &\nParks (MT FWP). The 358 samples that were analyzed for this study\nwere collected at MT FWP CWD sampling stations during the 2017,\n\nWith CWD posing substantial management challenges, researchers\nhave begun to investigate the potential for genetic resistance within\nsusceptible species. Previous studies in mule deer (Odocoileus\nhemionus) have established genetic variations in PRNP at amino\nacid positions 20, 131, 225, 226, and 247 (2, 8, 32, 35–37), some of\nwhich influence susceptibility and progression of disease (8, 32).\nFor example, mule deer have been documented to have a polymor­\nphism at codon position 225, expressing either serine (S) or phenyl­\nalanine (F), and this is implicated in variable disease prevalence,\naltered time course of disease progression, and even variations in\ndisease neuropathology (28, 38–40). A survey of Wyoming and\nColorado mule deer show that mule deer with the 225SS PRNP\nvariant were 30 times more likely to be CWD positive compared to\nthe animals carrying the 225SF polymorphism (32). Recently, addi­\ntional polymorphic PRNP variants have been identified in mule\ndeer at codon 20, 96, 98, 116, and 159 (30); however, the down­\nstream PrP structural effects and specific phenotypes are not well\nunderstood. As CWD continues to increase in prevalence, the need\nto investigate emerging genotypes which influence disease progres­\nsion and species barriers is critical.\nIn this study we: 1) Identified and cataloged novel and previously\nknown PRNP polymorphisms present in Montana’s wild-mule deer\npopulation in years 2017, 2018, and 2022; 2) performed a prevalence\nanalysis on the variants using spatiotemporal and CWD data; 3) mod­\neled protein stability and misfolding probability of a subset of the\nnovel variants through EmCAST computational analyses; and 4) con­\nfirmed PrP seeding activity of CWD positive protein variants relative\nto wild-type protein via the use of the Real-Time Quaking-Induced\nConversion assay (RT-QuIC).\n2 Materials and methods\n2.1 Cervid samples\nSamples of retropharyngeal lymph nodes of wild mule deer\n(Odocoileus hemionus) were provided by Montana Fish, Wildlife &\nParks (MT FWP). The 358 samples that were analyzed for this study\nwere collected at MT FWP CWD sampling stations during the 2017,\n\nThe 358 samples that were analyzed for this study\nwere collected at MT FWP CWD sampling stations during the 2017,\n2018, and 2022 hunting seasons. Samples were screened for CWD via\nenzyme-linked immunosorbent assay (ELISA) and in most cases, sus­\npect positives were confirmed positive via immunohistochemistry. All\nsamples used in this study were collected from hunting districts 401,\n600, 670, 640, 502, and 555 in Montana with known CWD positive\ncases (Supplementary Figure S1). Due to the early timeframe of dis­\nease in the state of Montana, and the purposes of this study, the sam­\npling strategy for PRNP sequencing is one of availability rather than a\ntruly representative one. This means that the study included both\nsexes of deer (2 male: 1 female) equally distributed through each hunt­\ning district at all years if samples were available. All CWD positive\nsamples obtained in those years were included in the dataset.\n2.2 Transgenic mouse model samples\nAll animal procedures were conducted in compliance with the\nguidelines outlined in the US National Research Council’s Guide for\n\nThe 358 samples that were analyzed for this study\nwere collected at MT FWP CWD sampling stations during the 2017,\n2018, and 2022 hunting seasons. Samples were screened for CWD via\nenzyme-linked immunosorbent assay (ELISA) and in most cases, sus­\npect positives were confirmed positive via immunohistochemistry. All\nsamples used in this study were collected from hunting districts 401,\n600, 670, 640, 502, and 555 in Montana with known CWD positive\ncases (Supplementary Figure S1). Due to the early timeframe of dis­\nease in the state of Montana, and the purposes of this study, the sam­\npling strategy for PRNP sequencing is one of availability rather than a\ntruly representative one. This means that the study included both\nsexes of deer (2 male: 1 female) equally distributed through each hunt­\ning district at all years if samples were available. All CWD positive\nsamples obtained in those years were included in the dataset.\n2.2 Transgenic mouse model samples\nAll animal procedures were conducted in compliance with the\nguidelines outlined in the US National Research Council’s Guide for\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nthe Care and Use of Laboratory Animals (41) and the US Public Health\nService’s Policy on Humane Care and Use of Laboratory Animals. The\nstudy protocol was reviewed and approved by the Institutional Animal\nCare and Use Committee (IACUC) at the McLaughlin Research\nInstitute. Mice were housed in the Animal Resource Center at the\nWeissman Hood Institute at Touro University, a facility exclusively for\nmice and accredited by the Association for Assessment and\nAccreditation of Laboratory Animal Care. This research was approved\nby the Weissman Hood Institute at Touro University; McLaughlin\nResearch Institutional Animal Care and Use Committee under proto­\ncol number 2023-AG-100 and biosafety protocol number\n2022-AG-IBC5.\nThe Tg1536+/+ mouse line, commonly denoted as “cervidized\nmouse line” was used for controls in the RT-QuIC assay (1, 42).\nThese mice are transgenic homozygotes expressing wild type\n(WT) cervid prion protein (PrP) and maintained on the FVB/NJ\nbackground with endogenous mouse PrP removed. Previous lit­\nerature reported that the Tg136+/− line contains PrP at a five-fold\nlevel of expression and homozygotes resulted in a doubling of the\nlevel of expression of CerPrPC (42). At 8 weeks old, a group of\nthese mice were inoculated via intracranial injection with 30uL of\n1% CWD infected brain homogenate or normal brain homogenate\n(NBH). CWD and NBH inoculum were mule deer in origin and\nconfirmed wild-type, lacking PRNP polymorphisms. Mice were\nplaced under isoflurane anesthesia for inoculation, immediately\ngiven meloxicam after procedure, placed on warmers, and moni­\ntored until deemed safe and alert. The Tg1536+/+ mice were har­\nvested at CWD endpoint, 9.4 months of age, with confirmation of\ndisease via presence of PK-resistant PrP (Supplementary\nFigure S2A). The mice in this line inoculated with CWD were\nused as a prion-positive control to identify the seeding activity\nparameters in an overexpressing model. The animals inoculated\nwith NBH were utilized as an additional CWD-negative control.\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nthe Care and Use of Laboratory Animals (41) and the US Public Health\nService’s Policy on Humane Care and Use of Laboratory Animals. The\nstudy protocol was reviewed and approved by the Institutional Animal\nCare and Use Committee (IACUC) at the McLaughlin Research\nInstitute. Mice were housed in the Animal Resource Center at the\nWeissman Hood Institute at Touro University, a facility exclusively for\nmice and accredited by the Association for Assessment and\nAccreditation of Laboratory Animal Care. This research was approved\nby the Weissman Hood Institute at Touro University; McLaughlin\nResearch Institutional Animal Care and Use Committee under proto­\ncol number 2023-AG-100 and biosafety protocol number\n2022-AG-IBC5.\nThe Tg1536+/+ mouse line, commonly denoted as “cervidized\nmouse line” was used for controls in the RT-QuIC assay (1, 42).\nThese mice are transgenic homozygotes expressing wild type\n(WT) cervid prion protein (PrP) and maintained on the FVB/NJ\nbackground with endogenous mouse PrP removed. Previous lit­\nerature reported that the Tg136+/− line contains PrP at a five-fold\nlevel of expression and homozygotes resulted in a doubling of the\nlevel of expression of CerPrPC (42). At 8 weeks old, a group of\nthese mice were inoculated via intracranial injection with 30uL of\n1% CWD infected brain homogenate or normal brain homogenate\n(NBH). CWD and NBH inoculum were mule deer in origin and\nconfirmed wild-type, lacking PRNP polymorphisms. Mice were\nplaced under isoflurane anesthesia for inoculation, immediately\ngiven meloxicam after procedure, placed on warmers, and moni­\ntored until deemed safe and alert. The Tg1536+/+ mice were har­\nvested at CWD endpoint, 9.4 months of age, with confirmation of\ndisease via presence of PK-resistant PrP (Supplementary\nFigure S2A). The mice in this line inoculated with CWD were\nused as a prion-positive control to identify the seeding activity\nparameters in an overexpressing model. The animals inoculated\nwith NBH were utilized as an additional CWD-negative control.\n\nThe mice in this line inoculated with CWD were\nused as a prion-positive control to identify the seeding activity\nparameters in an overexpressing model.The animals inoculated\nwith NBH were utilized as an additional CWD-negative control.\nIn addition to the CWD and NBH inoculated mice expressing\ncervid PrP previously mentioned, a control expressing no PrP as\na knockout (KO) of the PRNP gene was included in the RT-QuIC\nassay (Supplementary Figure S2). These samples were denoted as\n“PrP KO mouse brain” and clearly lack any expression of PrP\nwhen compared to FVB/NJ (Supplementary Figure S2B).\n2.3 Sample preparation for prion RT-QuIC\non mule deer lymph nodes and transgenic\nmouse brain homogenates\nFor the transgenic mouse samples, hippocampal and cortex tis­\nsues were microdissected from the left hemisphere (43). Samples were\nweighed and diluted to 10% w/v using RIPA buffer (25 mM TRIS,\n150 mM NaCl, 0.1% (w/w) SDS, 0.5% (w/w) Sodium deoxycholate,\n1% (w/w) Triton X-100) made in house with protease inhibitor cock­\ntail (Sigma Aldrich, Darmstadt, Germany). Tissues were then homog­\nenized using a Bead Mill 4 on #5 speed setting for 60 s and aliquoted\nand stored at – 80 °C. The deer lymph node tissues were prepped\nsimilarly with the exception of being weighed and diluted to 20% w/v\nusing RIPA and protease inhibitor cocktail. The 20% w/v stock\nhomogenates were diluted using 1x PBS to 10% w/v, and then 10-fold\ndilutions were prepared with 1x PBS/1x N-2 (Gibco, ThermoFisher\nScientific, Waltham, MA).\n2.4 Sanger sequencing and analysis\nFrozen lymph node samples were processed for sequencing by\nstandard methods and guidelines as previously reported (44). 180\nmicroliters (μL) of cell lysis buffer (Tris-Cl pH 8.0, EDTA, NaCl, 0.2%\n(w/v) sodium dodecyl sulfate) and 20 μL of 10 mg/mL Proteinase K\n(PK) were added to each well in a 96 well plate containing a small\nfrozen lymph node sample approximately 0.5 cm by 0.5 cm. Samples\nwere then briefly vortexed and incubated at 50 °C for 6 h. PK diges­\ntion was terminated by heating samples at 95 °C for 15 min. PCR\n\nThe mice in this line inoculated with CWD were\nused as a prion-positive control to identify the seeding activity\nparameters in an overexpressing model.The animals inoculated\nwith NBH were utilized as an additional CWD-negative control.\nIn addition to the CWD and NBH inoculated mice expressing\ncervid PrP previously mentioned, a control expressing no PrP as\na knockout (KO) of the PRNP gene was included in the RT-QuIC\nassay (Supplementary Figure S2). These samples were denoted as\n“PrP KO mouse brain” and clearly lack any expression of PrP\nwhen compared to FVB/NJ (Supplementary Figure S2B).\n2.3 Sample preparation for prion RT-QuIC\non mule deer lymph nodes and transgenic\nmouse brain homogenates\nFor the transgenic mouse samples, hippocampal and cortex tis­\nsues were microdissected from the left hemisphere (43). Samples were\nweighed and diluted to 10% w/v using RIPA buffer (25 mM TRIS,\n150 mM NaCl, 0.1% (w/w) SDS, 0.5% (w/w) Sodium deoxycholate,\n1% (w/w) Triton X-100) made in house with protease inhibitor cock­\ntail (Sigma Aldrich, Darmstadt, Germany). Tissues were then homog­\nenized using a Bead Mill 4 on #5 speed setting for 60 s and aliquoted\nand stored at – 80 °C. The deer lymph node tissues were prepped\nsimilarly with the exception of being weighed and diluted to 20% w/v\nusing RIPA and protease inhibitor cocktail. The 20% w/v stock\nhomogenates were diluted using 1x PBS to 10% w/v, and then 10-fold\ndilutions were prepared with 1x PBS/1x N-2 (Gibco, ThermoFisher\nScientific, Waltham, MA).\n2.4 Sanger sequencing and analysis\nFrozen lymph node samples were processed for sequencing by\nstandard methods and guidelines as previously reported (44). 180\nmicroliters (μL) of cell lysis buffer (Tris-Cl pH 8.0, EDTA, NaCl, 0.2%\n(w/v) sodium dodecyl sulfate) and 20 μL of 10 mg/mL Proteinase K\n(PK) were added to each well in a 96 well plate containing a small\nfrozen lymph node sample approximately 0.5 cm by 0.5 cm. Samples\nwere then briefly vortexed and incubated at 50 °C for 6 h. PK diges­\ntion was terminated by heating samples at 95 °C for 15 min. PCR\n\n5 cm by 0.5 cm.Samples\nwere then briefly vortexed and incubated at 50 °C for 6 h.PK diges­\ntion was terminated by heating samples at 95 °C for 15 min.PCR\namplification of the targeted PRNP region resulted in a 907 bp ampli­\ncon.\nThe\nforward\nprimer\nwas\n5-“GCAGTCATTCATT\nATGCTGCAG-3” and the reverse primer was 5-“GCAGGTAGAT\nACTCCCTC-3”. Each PCR well contained 13.5 μL of 2x GoTaq cock­\ntail (Promega, Madison, WI), 0.405 μL of each primer (20 μM), and\n13.5 μL of diluted DNA (5 μL of lysate in 180 μL of water). The PCR\nprogram had 1 cycle of initial denaturation at 95 °C for 3 min. Then\n35 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s,\nand elongation at 72 °C for 30 s. A final elongation step was done at\n75 °C for 5 min. To check for amplicons, samples were run on a 1.5%\nagarose gel (0.5X Tris Borate EDTA buffer and Ethidium bromide) at\n225 V for 35 min. Results were visualized and photographed on an\nultraviolet transillumination box. Following the PCR amplification,\n20 μL of the samples were transferred to a 96 well plate for Sanger\nsequencing by the contract laboratory service provider, Psomagen.\nSequence data received from Psomagen was analyzed using Geneious\nPrime (version 2024.0.2) by aligning and comparing with mule deer\nsequence GenBank accession number AY228473.1. The sample iden­\ntification numbers containing single nucleotide polymorphisms\n(SNPs) which differed from wildtype (WT) PRNP were tracked.\n2.5 Spatiotemporal distribution of PRNP\nvariants with CWD disease status\nThe samples expressing the PRNP variants were integrated with\ndata provided by MT FWP such as CWD status, hunting district and\nyear of harvest to provide a spatiotemporal assessment. CWD binomi­\nnal probabilities were calculated for a 95% confidence interval using\na calculator that relies on the Clopper-Pearson (exact) method due to\nsmall sample sizes (np < 5).\n2.6 Computational structural analysis of\nmule deer PrP variants\nEmCAST (empirical Cα stabilization) software models protein\nsequence-structure relationships in the absence of tertiary interac­\n\n5 cm by 0.5 cm.Samples\nwere then briefly vortexed and incubated at 50 °C for 6 h.PK diges­\ntion was terminated by heating samples at 95 °C for 15 min.PCR\namplification of the targeted PRNP region resulted in a 907 bp ampli­\ncon.\nThe\nforward\nprimer\nwas\n5-“GCAGTCATTCATT\nATGCTGCAG-3” and the reverse primer was 5-“GCAGGTAGAT\nACTCCCTC-3”. Each PCR well contained 13.5 μL of 2x GoTaq cock­\ntail (Promega, Madison, WI), 0.405 μL of each primer (20 μM), and\n13.5 μL of diluted DNA (5 μL of lysate in 180 μL of water). The PCR\nprogram had 1 cycle of initial denaturation at 95 °C for 3 min. Then\n35 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s,\nand elongation at 72 °C for 30 s. A final elongation step was done at\n75 °C for 5 min. To check for amplicons, samples were run on a 1.5%\nagarose gel (0.5X Tris Borate EDTA buffer and Ethidium bromide) at\n225 V for 35 min. Results were visualized and photographed on an\nultraviolet transillumination box. Following the PCR amplification,\n20 μL of the samples were transferred to a 96 well plate for Sanger\nsequencing by the contract laboratory service provider, Psomagen.\nSequence data received from Psomagen was analyzed using Geneious\nPrime (version 2024.0.2) by aligning and comparing with mule deer\nsequence GenBank accession number AY228473.1. The sample iden­\ntification numbers containing single nucleotide polymorphisms\n(SNPs) which differed from wildtype (WT) PRNP were tracked.\n2.5 Spatiotemporal distribution of PRNP\nvariants with CWD disease status\nThe samples expressing the PRNP variants were integrated with\ndata provided by MT FWP such as CWD status, hunting district and\nyear of harvest to provide a spatiotemporal assessment. CWD binomi­\nnal probabilities were calculated for a 95% confidence interval using\na calculator that relies on the Clopper-Pearson (exact) method due to\nsmall sample sizes (np < 5).\n2.6 Computational structural analysis of\nmule deer PrP variants\nEmCAST (empirical Cα stabilization) software models protein\nsequence-structure relationships in the absence of tertiary interac­\n\n2.6 Computational structural analysis of\nmule deer PrP variants\nEmCAST (empirical Cα stabilization) software models protein\nsequence-structure relationships in the absence of tertiary interac­\ntions using overlapping tetrad fragments (45). The method is highly\nsensitive to sequence/structure and can be used to predict short\nstructures, stability changes caused by mutations, and structural dis­\ntortions caused by mutations (45). EmCAST uses a sequence-to-\nstructure fragment database to model context-dependent effects in\nprimary structure and to evaluate the different conformations a\nsequence may encode. Energy calculations are performed for a target\nprotein structure by comparing the population of fragments with\nmatching geometry to the population of fragments with non-match­\ning geometry.\n\n2.6 Computational structural analysis of\nmule deer PrP variants\nEmCAST (empirical Cα stabilization) software models protein\nsequence-structure relationships in the absence of tertiary interac­\ntions using overlapping tetrad fragments (45). The method is highly\nsensitive to sequence/structure and can be used to predict short\nstructures, stability changes caused by mutations, and structural dis­\ntortions caused by mutations (45). EmCAST uses a sequence-to-\nstructure fragment database to model context-dependent effects in\nprimary structure and to evaluate the different conformations a\nsequence may encode. Energy calculations are performed for a target\nprotein structure by comparing the population of fragments with\nmatching geometry to the population of fragments with non-match­\ning geometry.\n\nln\nmatching\nmatching\ntotal P\np\nG\nRT\nP\n−\n\n\n\n\n∆\n=\n⋅\n\n\n\n\nThe EmCAST sequence to structure algorithm identifies popula­\ntion peaks for each tetrad (typically 2–4 geometries) and assembles a\nCα backbone using every possible combination. Geometric combina­\ntions that produce backbone collisions are discarded. The number of\ngenerated backbones increases exponentially with sequence length;\nresidues 1–18 of mule deer PrP (MDPrP) produced 98,304 backbones.\nThe EmCAST energy function is then applied to all backbone confor­\nmations to sort them from lowest to highest energy, as described pre­\nviously (45).\nTo model a larger section of the N-terminal region of MDPrP\n(Residues 1–44), predictions were performed for three smaller, over­\nlapping fragments (MVKSHIGSWILVLFVAMW, VAMWSDV\nGLCKKRPKPG, and PKPGGGWNTGGSRYPGQ). Tetrads with few\npopulation peaks (VAMW and PKPG) were chosen as the break\npoints. The 50 lowest energy conformations for the three fragments\nwere combined and rescored by EmCAST to generate a predicted\nlowest energy conformation for residues 1–44. Mutation induced\nstability changes were calculated using this structure on the EmCAST\nwebsite1 with the temperature set to 25 °C. The sequence to structure\nprocess was repeated for the variants predicted to destabilize MDPrP.\n2.7 Preparation of bank vole prion plasmid\nfor RT-QuIC\ncDNA encoding Bank vole prion sequence was manufactured by\nGenscript (Piscataway NJ, United States) and placed in the pET 30a\n(+) vector (Supplementary Table S1). The cDNA prion sequence was\nconfirmed by Genscript.\n2.8 Bank vole recombinant prion expression\nand purification for RT-QuIC\nBank vole prion protein is the universal substrate, demonstrating\nsusceptibility to most types of prions, and thus is an ideal substrate for\nprion seed amplification RT-QuIC assays (46). Prion RT-QuIC assay\nwas performed as previously described (47) with modifications as\ndescribed below. The cDNA encoding Bank vole recombinant prion\n(rPrP) was expressed from the pET 30a (+) vector by transformation\ninto BL21 E. coli competent cells (New England Biolabs, Ipswich\nMA,). We used our previous protocol to express rPrP from E. coli (48).\n\nln\nmatching\nmatching\ntotal P\np\nG\nRT\nP\n−\n\n\n\n\n∆\n=\n⋅\n\n\n\n\nThe EmCAST sequence to structure algorithm identifies popula­\ntion peaks for each tetrad (typically 2–4 geometries) and assembles a\nCα backbone using every possible combination. Geometric combina­\ntions that produce backbone collisions are discarded. The number of\ngenerated backbones increases exponentially with sequence length;\nresidues 1–18 of mule deer PrP (MDPrP) produced 98,304 backbones.\nThe EmCAST energy function is then applied to all backbone confor­\nmations to sort them from lowest to highest energy, as described pre­\nviously (45).\nTo model a larger section of the N-terminal region of MDPrP\n(Residues 1–44), predictions were performed for three smaller, over­\nlapping fragments (MVKSHIGSWILVLFVAMW, VAMWSDV\nGLCKKRPKPG, and PKPGGGWNTGGSRYPGQ). Tetrads with few\npopulation peaks (VAMW and PKPG) were chosen as the break\npoints. The 50 lowest energy conformations for the three fragments\nwere combined and rescored by EmCAST to generate a predicted\nlowest energy conformation for residues 1–44. Mutation induced\nstability changes were calculated using this structure on the EmCAST\nwebsite1 with the temperature set to 25 °C. The sequence to structure\nprocess was repeated for the variants predicted to destabilize MDPrP.\n2.7 Preparation of bank vole prion plasmid\nfor RT-QuIC\ncDNA encoding Bank vole prion sequence was manufactured by\nGenscript (Piscataway NJ, United States) and placed in the pET 30a\n(+) vector (Supplementary Table S1). The cDNA prion sequence was\nconfirmed by Genscript.\n2.8 Bank vole recombinant prion expression\nand purification for RT-QuIC\nBank vole prion protein is the universal substrate, demonstrating\nsusceptibility to most types of prions, and thus is an ideal substrate for\nprion seed amplification RT-QuIC assays (46). Prion RT-QuIC assay\nwas performed as previously described (47) with modifications as\ndescribed below. The cDNA encoding Bank vole recombinant prion\n(rPrP) was expressed from the pET 30a (+) vector by transformation\ninto BL21 E. coli competent cells (New England Biolabs, Ipswich\nMA,). We used our previous protocol to express rPrP from E. coli (48).\n\nThe cDNA encoding Bank vole recombinant prion\n(rPrP) was expressed from the pET 30a (+) vector by transformation\ninto BL21 E.coli competent cells (New England Biolabs, Ipswich\nMA,).We used our previous protocol to express rPrP from E.coli (48).\nSterile 2xYT media (1 L) was inoculated with 10 mL of suspended\ntransformed E. coli cells and 1 mL of 50 mg/mL kanamycin and put in\na 2.8 L Fernbach flask. Cells were grown in an orbital shaker at\n250 rpm and 37 °C. One mM final IPTG (GoldBio, St. Louis, MO) was\nadded when OD600 was in the range of 0.6–0.8. Cells continued to\ngrow for ~15 h at 30 °C and 150 rpm. Media was spun for 15 min,\n7,500 rpm at 4 °C, and cells were frozen at −20 °C. Cells were lysed\nwith 50 mL of 0.5 M NaCl, 0.05 M Tris, pH 8.0 to 5 g cells, placed on\nice with 0.5 mg/mL lysozyme, and put on stir plate for 30 min, with\nsonication on ice for 5 min (30 s on/30 s off, 35% AMPL) using a\nprobe sonicator (Sonics VibraCell, Newtown, CT). Lysate was spun\n1  www.emcast.org\nfor 30 min at 12,500 rpm and 4 °C, and supernatant was separated\nfrom the pellet. The pellet was vigorously disrupted and mixed with\n~50 mL of wash buffer 1 (1% Triton X-114 in 50 mM HEPES pH 8.0),\nwash buffer 2 (20 mM HEPES 2 M NaCl, pH 8.0), and wash buffer 3\n(20 mM HEPES pH 8.0), with a 30 min, 12,500 rpm (Sorvall RC5B\nPlus centrifuge, Sorvall SS-34 rotor), 4 °C spin between each wash\nbuffer step. After the spin step with wash buffer 3, the supernatant was\ndiscarded, and the pellet was denatured overnight at room tempera­\nture in 8 M GuHCl, 20 mM Tris, pH 8.0. The next day, the pellet was\nvisually solubilized and spun at 4 °C, 30 min, 12,000 rpm. The super­\nnatant was then added to 20 mL of Ni-NTA resin (ThermoScientific,\nWaltham, MA) in a 3 cm (wide) x 30 cm (length) Bio-Rad gravity flow\ncolumn. After the flow through was collected, the column was washed\nwith 10 column volumes of refolding buffer (100 mM sodium phos­\nphate pH 8.0) and then washed with 10 column volumes of buffer B\n(100 mM sodium phosphate, 50 mM imidazole, pH 5.8). For the elu­\n\nThe cDNA encoding Bank vole recombinant prion\n(rPrP) was expressed from the pET 30a (+) vector by transformation\ninto BL21 E.coli competent cells (New England Biolabs, Ipswich\nMA,).We used our previous protocol to express rPrP from E.coli (48).\nSterile 2xYT media (1 L) was inoculated with 10 mL of suspended\ntransformed E. coli cells and 1 mL of 50 mg/mL kanamycin and put in\na 2.8 L Fernbach flask. Cells were grown in an orbital shaker at\n250 rpm and 37 °C. One mM final IPTG (GoldBio, St. Louis, MO) was\nadded when OD600 was in the range of 0.6–0.8. Cells continued to\ngrow for ~15 h at 30 °C and 150 rpm. Media was spun for 15 min,\n7,500 rpm at 4 °C, and cells were frozen at −20 °C. Cells were lysed\nwith 50 mL of 0.5 M NaCl, 0.05 M Tris, pH 8.0 to 5 g cells, placed on\nice with 0.5 mg/mL lysozyme, and put on stir plate for 30 min, with\nsonication on ice for 5 min (30 s on/30 s off, 35% AMPL) using a\nprobe sonicator (Sonics VibraCell, Newtown, CT). Lysate was spun\n1  www.emcast.org\nfor 30 min at 12,500 rpm and 4 °C, and supernatant was separated\nfrom the pellet. The pellet was vigorously disrupted and mixed with\n~50 mL of wash buffer 1 (1% Triton X-114 in 50 mM HEPES pH 8.0),\nwash buffer 2 (20 mM HEPES 2 M NaCl, pH 8.0), and wash buffer 3\n(20 mM HEPES pH 8.0), with a 30 min, 12,500 rpm (Sorvall RC5B\nPlus centrifuge, Sorvall SS-34 rotor), 4 °C spin between each wash\nbuffer step. After the spin step with wash buffer 3, the supernatant was\ndiscarded, and the pellet was denatured overnight at room tempera­\nture in 8 M GuHCl, 20 mM Tris, pH 8.0. The next day, the pellet was\nvisually solubilized and spun at 4 °C, 30 min, 12,000 rpm. The super­\nnatant was then added to 20 mL of Ni-NTA resin (ThermoScientific,\nWaltham, MA) in a 3 cm (wide) x 30 cm (length) Bio-Rad gravity flow\ncolumn. After the flow through was collected, the column was washed\nwith 10 column volumes of refolding buffer (100 mM sodium phos­\nphate pH 8.0) and then washed with 10 column volumes of buffer B\n(100 mM sodium phosphate, 50 mM imidazole, pH 5.8). For the elu­\n\nAfter the flow through was collected, the column was washed\nwith 10 column volumes of refolding buffer (100 mM sodium phos­\nphate pH 8.0) and then washed with 10 column volumes of buffer B\n(100 mM sodium phosphate, 50 mM imidazole, pH 5.8).For the elu­\ntion step, 10 column volumes of buffer C (100 mM sodium phosphate,\n300 mM imidazole, pH 5.8) was added and eluant fractions were col­\nlected in ~14 mL aliquots. Fractions were checked for A280 and frac­\ntions that contained the highest A280 (ε = 62,005 M−1 cm−1, Thermo\nScientific NanoDrop One) were collected and put in 10,000 MWCO\ndialysis tubing (ThermoScientific, Waltham, MA) overnight at room\ntemperature for ~18 h in 10 mM sodium phosphate pH 5.8. The next\nday, dialysate was spun for 30 min, 20 °C, and 12,000 rpm. Supernatant\nwas concentrated in a 10,000 MWCO Amicon centrifugal concentra­\ntor (Millipore Sigma, Burlington, MA) and protein purity and identity\nwas assessed with electrospray ionization LC–MS yielding the\nexpected monomeric mass of 22,914.26 Da (Supplementary Figure S3)\nand no heat SDS-PAGE stained with Coomassie (Supplementary\nFigure S4). It should be noted that the expected monomeric mass of\nrPrP from Bank vole is 22,914.26 Da, while the gel produced a mono­\nmeric band at ~22 kD, the mass spec data was collected later on and\nproduced a major peak at 20,846.9768 Da, which may reflect cleavage\nfrom the N-terminus near the Tyr-Pro-Gly sequence via low pH\nduring purification producing a major peak at 20,846.9 Da.\n2.9 RT-QuIC\nThe prion RT-QuIC buffer solution was prepared with a 50 mL\nvolumetric flask using 5 mL of 10x phosphate buffer (Boston\nBioProducts, Milford, MA), 45 mL of high-performance chromatog­\nraphy grade water (Sigma Aldrich, Burlington, MA), 10 μM of EDTA\n(FisherBiotech Wembley, Australia), 300 mM NaCl (Sigma Aldrich,\nBurlington, MA), 1 mg of SDS (Thermo Scientific, Waltham, MA),\nand 50 mg of potassium phosphate dibasic (FisherBiotech, Wembley,\nAustralia) to obtain a pH equal to 6.7. For the prion RT-QuIC reaction\nmix, a stock 5 mM ThT (Thermo Scientific, Waltham, MA) solution\n\nAfter the flow through was collected, the column was washed\nwith 10 column volumes of refolding buffer (100 mM sodium phos­\nphate pH 8.0) and then washed with 10 column volumes of buffer B\n(100 mM sodium phosphate, 50 mM imidazole, pH 5.8).For the elu­\ntion step, 10 column volumes of buffer C (100 mM sodium phosphate,\n300 mM imidazole, pH 5.8) was added and eluant fractions were col­\nlected in ~14 mL aliquots. Fractions were checked for A280 and frac­\ntions that contained the highest A280 (ε = 62,005 M−1 cm−1, Thermo\nScientific NanoDrop One) were collected and put in 10,000 MWCO\ndialysis tubing (ThermoScientific, Waltham, MA) overnight at room\ntemperature for ~18 h in 10 mM sodium phosphate pH 5.8. The next\nday, dialysate was spun for 30 min, 20 °C, and 12,000 rpm. Supernatant\nwas concentrated in a 10,000 MWCO Amicon centrifugal concentra­\ntor (Millipore Sigma, Burlington, MA) and protein purity and identity\nwas assessed with electrospray ionization LC–MS yielding the\nexpected monomeric mass of 22,914.26 Da (Supplementary Figure S3)\nand no heat SDS-PAGE stained with Coomassie (Supplementary\nFigure S4). It should be noted that the expected monomeric mass of\nrPrP from Bank vole is 22,914.26 Da, while the gel produced a mono­\nmeric band at ~22 kD, the mass spec data was collected later on and\nproduced a major peak at 20,846.9768 Da, which may reflect cleavage\nfrom the N-terminus near the Tyr-Pro-Gly sequence via low pH\nduring purification producing a major peak at 20,846.9 Da.\n2.9 RT-QuIC\nThe prion RT-QuIC buffer solution was prepared with a 50 mL\nvolumetric flask using 5 mL of 10x phosphate buffer (Boston\nBioProducts, Milford, MA), 45 mL of high-performance chromatog­\nraphy grade water (Sigma Aldrich, Burlington, MA), 10 μM of EDTA\n(FisherBiotech Wembley, Australia), 300 mM NaCl (Sigma Aldrich,\nBurlington, MA), 1 mg of SDS (Thermo Scientific, Waltham, MA),\nand 50 mg of potassium phosphate dibasic (FisherBiotech, Wembley,\nAustralia) to obtain a pH equal to 6.7. For the prion RT-QuIC reaction\nmix, a stock 5 mM ThT (Thermo Scientific, Waltham, MA) solution\n\n7.For the prion RT-QuIC reaction\nmix, a stock 5 mM ThT (Thermo Scientific, Waltham, MA) solution\nin ddH2O was added to obtain a 10 μM final ThT in prion RT-QuIC\nbuffer with 0.1 mg/mL (~4 μM) final concentration of 100 kD filtered\nrPrP substrate. Ninety-eight microliters of the prion RT-QuIC reac­\ntion mix was added to each well in clear bottom 96-well microplates\n(Thermo-Scientific Nunc 96-well optical bottomed black polystyrene\nplates w/Lid, Waltham, MA) and seeded with 2 μL of 10−3 tissue\nhomogenate in each well. Because recombinant substrate can sponta­\nneously convert to amyloid during RT-QuIC reaction conditions, we\nincluded rPrP substrate in reaction mix alone as an additional control\nin the 96 well plate (Supplementary Figure S5). Plates were sealed with\ntape (Thermo-Scientific, clear polyolefin, non-sterile, Waltham, MA)\n\n7.For the prion RT-QuIC reaction\nmix, a stock 5 mM ThT (Thermo Scientific, Waltham, MA) solution\nin ddH2O was added to obtain a 10 μM final ThT in prion RT-QuIC\nbuffer with 0.1 mg/mL (~4 μM) final concentration of 100 kD filtered\nrPrP substrate. Ninety-eight microliters of the prion RT-QuIC reac­\ntion mix was added to each well in clear bottom 96-well microplates\n(Thermo-Scientific Nunc 96-well optical bottomed black polystyrene\nplates w/Lid, Waltham, MA) and seeded with 2 μL of 10−3 tissue\nhomogenate in each well. Because recombinant substrate can sponta­\nneously convert to amyloid during RT-QuIC reaction conditions, we\nincluded rPrP substrate in reaction mix alone as an additional control\nin the 96 well plate (Supplementary Figure S5). Plates were sealed with\ntape (Thermo-Scientific, clear polyolefin, non-sterile, Waltham, MA)\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nand placed in a FLUOstar Omega reader (BMG Labtech, Germany) at \n42 °C, 700 rpm, with 60 s of shaking and 60 s of resting on a double \norbital setting. ThT fluorescence intensity measurements were col­\nlected every 40 min with 448 nm excitation and 482 nm emission \nusing a gain setting of 1,600. Each sample was replicated in quadru­\nplicate as is standard in this testing method (46). During the assay, the \nRT-QuIC technician was blinded to the CWD status and PRNP geno­\ntype of all wild mule deer samples.\n2.10 Prion RT-QuIC data analysis\nAfter the prion RT-QuIC experiment ended, the BMG Omega \nMars software. RUC file was exported to Microsoft Excel to process \ndata via SigmaPlot 14.5 or 15.0 (Systat software) for model fitting of \nprion RT-QuIC kinetic data. We used equation 1, below, as described \npreviously to model the data (48). In Equation 1,\n\nτ\n−\n\n\n−\n\n\n\n= \n\n\n\n+\n\n\n\n\n\n\n0\nA\n1\nx x\ny\ne\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n05\nfrontiersin.org\nand placed in a FLUOstar Omega reader (BMG Labtech, Germany) at \n42 °C, 700 rpm, with 60 s of shaking and 60 s of resting on a double \norbital setting. ThT fluorescence intensity measurements were col­\nlected every 40 min with 448 nm excitation and 482 nm emission \nusing a gain setting of 1,600. Each sample was replicated in quadru­\nplicate as is standard in this testing method (46). During the assay, the \nRT-QuIC technician was blinded to the CWD status and PRNP geno­\ntype of all wild mule deer samples.\n2.10 Prion RT-QuIC data analysis\nAfter the prion RT-QuIC experiment ended, the BMG Omega \nMars software. RUC file was exported to Microsoft Excel to process \ndata via SigmaPlot 14.5 or 15.0 (Systat software) for model fitting of \nprion RT-QuIC kinetic data. We used equation 1, below, as described \npreviously to model the data (48). In Equation 1,\n\nτ\n−\n\n\n−\n\n\n\n= \n\n\n\n+\n\n\n\n\n\n\n0\nA\n1\nx x\ny\ne\n\n(1)\nA is ThT fluorescence amplitude, x0 is the time to reach midpoint\nThT fluorescence, and τ is a fibril time constant where lag phase equals\nx0—2τ (48). Raw kinetic traces were fit to this model using no con­\nstraints to estimate x0, τ, and A. The mean ± standard deviation of\nprion RT-QuIC parameters (ThT fluorescence amplitude midpoint,\nThT FAm and lag phase) was then calculated. The cutoff for RT-QuIC\nsignal was established at 15 h as no negative controls had positive ThT\nsignal prior to this timepoint. Any sample showing signal after this\ntimepoint was determined negative.\nFor each sample that was ThT positive by RT-QuIC (4/4 wells\nThT+), we fit each replicate curve to Equation 1 to extract ThT fluo­\nrescence amplitude, time to midpoint ThT fluorescence, and a fibril\ntime constant, as previously described (48). A representative figure\ndemonstrating this is shown in Supplementary Figure S6. After fitting\nto Equation 1, we used the prion RT-QuIC parameters (ThT fluores­\ncence amplitude, time to midpoint ThT fluorescence, and the fibril\ntime constant) to calculate lag phase (x0-2τ) and half ThT fluorescence\namplitude (in Equation 1, when x = x0, y = A/2). With the computed\nthe mean and standard deviation for lag phase and half ThT fluores­\ncence amplitude and then graphed midpoint ThT fluorescence ampli­\ntude versus lag phase for each ThT positive PrPSc biospecimen at 10−3\ndilution.\n3 Results\n3.1 PRNP sequencing and identification of\nsingle nucleotide variants\nThe PRNP sequences in 358 mule deer samples obtained by MT\nFWP in the 2017, 2018, and 2022 hunting seasons, were acquired\nand analyzed resulting in the discovery of 36 polymorphisms. Of\nthese, 25 were non-synonymous PRNP genetic variants, many of\nwhich, to our knowledge, have not been previously reported in\nmule deer. These non-synonymous PRNP variants resulting in an\nalternative amino acid at the specified locations were identified in\n15.1% of the samples (Table 1). Eleven additional single nucleotide\nvariants identified resulted in synonymous substitutions which\nresulted\nin\nno\namino\nacid\ntranslational\nchanges\n(Supplementary Table S2).\n\n(1)\nA is ThT fluorescence amplitude, x0 is the time to reach midpoint\nThT fluorescence, and τ is a fibril time constant where lag phase equals\nx0—2τ (48). Raw kinetic traces were fit to this model using no con­\nstraints to estimate x0, τ, and A. The mean ± standard deviation of\nprion RT-QuIC parameters (ThT fluorescence amplitude midpoint,\nThT FAm and lag phase) was then calculated. The cutoff for RT-QuIC\nsignal was established at 15 h as no negative controls had positive ThT\nsignal prior to this timepoint. Any sample showing signal after this\ntimepoint was determined negative.\nFor each sample that was ThT positive by RT-QuIC (4/4 wells\nThT+), we fit each replicate curve to Equation 1 to extract ThT fluo­\nrescence amplitude, time to midpoint ThT fluorescence, and a fibril\ntime constant, as previously described (48). A representative figure\ndemonstrating this is shown in Supplementary Figure S6. After fitting\nto Equation 1, we used the prion RT-QuIC parameters (ThT fluores­\ncence amplitude, time to midpoint ThT fluorescence, and the fibril\ntime constant) to calculate lag phase (x0-2τ) and half ThT fluorescence\namplitude (in Equation 1, when x = x0, y = A/2). With the computed\nthe mean and standard deviation for lag phase and half ThT fluores­\ncence amplitude and then graphed midpoint ThT fluorescence ampli­\ntude versus lag phase for each ThT positive PrPSc biospecimen at 10−3\ndilution.\n3 Results\n3.1 PRNP sequencing and identification of\nsingle nucleotide variants\nThe PRNP sequences in 358 mule deer samples obtained by MT\nFWP in the 2017, 2018, and 2022 hunting seasons, were acquired\nand analyzed resulting in the discovery of 36 polymorphisms. Of\nthese, 25 were non-synonymous PRNP genetic variants, many of\nwhich, to our knowledge, have not been previously reported in\nmule deer. These non-synonymous PRNP variants resulting in an\nalternative amino acid at the specified locations were identified in\n15.1% of the samples (Table 1). Eleven additional single nucleotide\nvariants identified resulted in synonymous substitutions which\nresulted\nin\nno\namino\nacid\ntranslational\nchanges\n(Supplementary Table S2).\n\n1% of the samples (Table 1).Eleven additional single nucleotide\nvariants identified resulted in synonymous substitutions which\nresulted\nin\nno\namino\nacid\ntranslational\nchanges\n(Supplementary Table S2).\nNotably, we frequently discovered multiple polymorphisms within\na single deer PRNP sequence in our dataset, which is a common\noccurrence in white-tailed deer (2, 25), yet less frequently reported in\nmule deer. In fact, all 25 non-synonymous variants were found in a\ncollection of nine deer (Supplementary Table S3). This representative\ngroup\nis\nlisted\nwith\nGenBank\naccession\nnumbers\n(Supplementary Table S3).\n3.2 Spatiotemporal distribution of PRNP\nnon-synonymous variants\n3.2.1 PRNP polymorphisms identified and\nspatiotemporal data\nThe non-synonymous PRNP polymorphisms identified in this\nstudy were integrated with harvest year and specific Montana hunt­\ning region of origin to track how the variant distribution changed\nover time. Synonymous polymorphisms were not included in the fol­\nlowing datasets. In this spatiotemporal analysis, hunting seasons\n2017/2018 and 2018/2019 were combined to constitute an “early”\ntimepoint, while hunting season 2022/2023 represents a “late” time­\npoint. The prevalence of PRNP polymorphisms was determined by\nnormalizing the number of samples with variants to the number of\nsamples sequenced in that given hunting district and year. In the\nearly timepoint, both regions 401 and 640 had a similar variant prev­\nalences of 6.5% (4/61) and 6.6% (2/30) respectively. Regions 600 and\n670 had variant prevalences of 14% (4/28,) while region 502 had a\nprevalence of 20% (7/35), respectively. The region with the highest\nprevalence in the early timepoint was 555 with 33% (10/30)\n(Figure 1A). In the late timepoint, half the regions had similar rates\nas the early timepoint with region 600, 670, and 502 having 13.6%\n(3/22), 14.2% (4/28), and 18.5% (5/27) respectively. Region 640 had\nan increased prevalence of 11% (2/18). The biggest increase was in\nregion 401 that had a variant prevalence of 43.3% (13/30) and the\nbiggest decrease was region 555 with the prevalence of 22.2% (4/18)\n(Figure 1B).\n\n1% of the samples (Table 1).Eleven additional single nucleotide\nvariants identified resulted in synonymous substitutions which\nresulted\nin\nno\namino\nacid\ntranslational\nchanges\n(Supplementary Table S2).\nNotably, we frequently discovered multiple polymorphisms within\na single deer PRNP sequence in our dataset, which is a common\noccurrence in white-tailed deer (2, 25), yet less frequently reported in\nmule deer. In fact, all 25 non-synonymous variants were found in a\ncollection of nine deer (Supplementary Table S3). This representative\ngroup\nis\nlisted\nwith\nGenBank\naccession\nnumbers\n(Supplementary Table S3).\n3.2 Spatiotemporal distribution of PRNP\nnon-synonymous variants\n3.2.1 PRNP polymorphisms identified and\nspatiotemporal data\nThe non-synonymous PRNP polymorphisms identified in this\nstudy were integrated with harvest year and specific Montana hunt­\ning region of origin to track how the variant distribution changed\nover time. Synonymous polymorphisms were not included in the fol­\nlowing datasets. In this spatiotemporal analysis, hunting seasons\n2017/2018 and 2018/2019 were combined to constitute an “early”\ntimepoint, while hunting season 2022/2023 represents a “late” time­\npoint. The prevalence of PRNP polymorphisms was determined by\nnormalizing the number of samples with variants to the number of\nsamples sequenced in that given hunting district and year. In the\nearly timepoint, both regions 401 and 640 had a similar variant prev­\nalences of 6.5% (4/61) and 6.6% (2/30) respectively. Regions 600 and\n670 had variant prevalences of 14% (4/28,) while region 502 had a\nprevalence of 20% (7/35), respectively. The region with the highest\nprevalence in the early timepoint was 555 with 33% (10/30)\n(Figure 1A). In the late timepoint, half the regions had similar rates\nas the early timepoint with region 600, 670, and 502 having 13.6%\n(3/22), 14.2% (4/28), and 18.5% (5/27) respectively. Region 640 had\nan increased prevalence of 11% (2/18). The biggest increase was in\nregion 401 that had a variant prevalence of 43.3% (13/30) and the\nbiggest decrease was region 555 with the prevalence of 22.2% (4/18)\n(Figure 1B).\n\n2% (4/28), and 18.5% (5/27) respectively.Region 640 had\nan increased prevalence of 11% (2/18).The biggest increase was in\nregion 401 that had a variant prevalence of 43.3% (13/30) and the\nbiggest decrease was region 555 with the prevalence of 22.2% (4/18)\n(Figure 1B).\n3.2.2 PRNP polymorphisms identified and CWD\nstatus\nA number of our identified PRNP variants were found to be\nCWD positive, however due to the small sample size, correlations\nbetween PRNP polymorphism and CWD cannot be determined at\nthis time (Table 1). Further investigations were performed, however,\non a subset of samples containing both novel and previously reported\nvariants. Our specific criteria for inclusion were: samples containing\na variant found in a higher prevalence in the population (three or\nmore) such as codons 20 and 40; samples containing the previously\nreported variant 225 which has been identified to alter disease phe­\nnotypes in the literature (1, 32, 40), and polymorphisms which\nappeared in the central signaling peptide area of the prion protein at\ncodons 2–17.\nWe examined the overall CWD status of the samples in this con­\ndensed dataset which included samples with PRNP variants at codons\n\n2% (4/28), and 18.5% (5/27) respectively.Region 640 had\nan increased prevalence of 11% (2/18).The biggest increase was in\nregion 401 that had a variant prevalence of 43.3% (13/30) and the\nbiggest decrease was region 555 with the prevalence of 22.2% (4/18)\n(Figure 1B).\n3.2.2 PRNP polymorphisms identified and CWD\nstatus\nA number of our identified PRNP variants were found to be\nCWD positive, however due to the small sample size, correlations\nbetween PRNP polymorphism and CWD cannot be determined at\nthis time (Table 1). Further investigations were performed, however,\non a subset of samples containing both novel and previously reported\nvariants. Our specific criteria for inclusion were: samples containing\na variant found in a higher prevalence in the population (three or\nmore) such as codons 20 and 40; samples containing the previously\nreported variant 225 which has been identified to alter disease phe­\nnotypes in the literature (1, 32, 40), and polymorphisms which\nappeared in the central signaling peptide area of the prion protein at\ncodons 2–17.\nWe examined the overall CWD status of the samples in this con­\ndensed dataset which included samples with PRNP variants at codons\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nTABLE 1  Non-synonymous PRNP polymorphisms (novel and previously reported) identified in Montana mule deer within the study.\nPolymorphic\ncodon\nReference\namino acid\nVariant\namino acid\nReference\nsequence\nVariant\nsequence\nVariant\nheterozygote\nfrequency (%)\nVariant\nhomozygote\nfrequency (%)\nCWD\npositive (%)\nN (out\nof 358)\nBinomial\nprobability\nconfidence\ninterval (95%)\n2\nValine (V)\nGlycine (G)\nGTG\nGGG\n100\n0\n0\n1\n12\nValine (V)\nPhenylalanine (F)\nGTT\nTTT\n100\n0\n100\n2\n0.00068–0.02003\n*12\nValine (V)\nIsoleucine (I)\nGTT\nATT\n100\n0\n100\n1\n0.00007–0.01546\n16\nAlanine (A)\nProline (P)\nGCC\nCCC\n100\n0\n100\n1\n0.00007–0.01546\n17\nMethionine (M)\nLeucine (L)\nATG\nTTG\n100\n0\n100\n1\n0.00007–0.01546\n*20\nAspartic Acid (D)\nGlycine (G)\nGAC\nGGC\n87\n13\n14\n39\n0.00617–0.03612\n40\nArginine (R)\nGlutamine (Q)\nCGA\nCAA\n100\n0\n0\n3\n52\nTyrosine (Y)\nSerine (S)\nTAT\nTCT\n100\n0\n100\n1\n0.00007–0.01546\n*96\nGlycine (G)\nSerine (S)\nGGT\nAGT\n0\n100\n0\n1\n*98\nThreonine (T)\nIsoleucine (I)\nACC\nATC\n0\n100\n0\n1\n195\nThreonine (T)\nSerine (S)\nACC\nTCC\n100\n0\n0\n1\n204\nThreonine (T)\nAsparagine (N)\nACT\nAAT\n100\n0\n0\n1\n209\nMethionine (M)\nArginine (R)\nATG\nAGG\n100\n0\n50\n2\n0.00007–0.01546\n210\nGlutamic acid (E)\nAspartic acid (D)\nGAG\nGAT\n100\n0\n0\n1\n213\nValine (V)\nGlycine (G)\nGTG\nGGG\n100\n0\n100\n1\n0.00007–0.01546\n217\nCysteine (C)\nArginine (R)\nTGC\nCGC\n100\n0\n0\n1\n217\nCysteine (C)\nSerine (S)\nTGC\nTCC\n100\n0\n100\n1\n0.00007–0.01546\n221\nTyrosine (Y)\nSerine (S)\nTAC\nTCC\n100\n0\n100\n1\n0.00007–0.01546\n*225\nSerine (S)\nPhenylalanine (F)\nTCC\nTTC\n100\n0\n0\n2\n225\nSerine (S)\nTyrosine (Y)\nTCC\nTAC\n100\n0\n0\n1\n226\nGlutamine (Q)\nArginine (R)\nCAG\nCGG\n100\n0\n100\n1\n0.00007–0.01546\n227\nAlanine (A)\nGlycine (G)\nGCT\nGGT\n100\n0\n0\n1\n229\nTyrosine (Y)\nPhenylalanine (F)\nTAC\nTTC\n100\n0\n100\n1\n0.00007–0.01546\n237\nLeucine (L)\nPhenylalanine (F)\nCTC\nTTC\n100\n0\n100\n1\n0.00007–0.01546\n238\nPhenylalanine (F)\nLeucine (L)\nTTC\nTTA\n100\n0\n0\n1\nPreviously reported variants were denoted with an asterisk. Variant heterozygote, homozygote frequency and CWD status is reported as a percentage within the polymorphic codon dataset. Sample number reflects the number of samples identified carrying the polymorphic\ncodon out of a total of 358 samples. Binomial probability confidence interval was reported for the CWD positive samples using the Clopper-Pearson (exact) method.\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nTABLE 1  Non-synonymous PRNP polymorphisms (novel and previously reported) identified in Montana mule deer within the study.\nPolymorphic\ncodon\nReference\namino acid\nVariant\namino acid\nReference\nsequence\nVariant\nsequence\nVariant\nheterozygote\nfrequency (%)\nVariant\nhomozygote\nfrequency (%)\nCWD\npositive (%)\nN (out\nof 358)\nBinomial\nprobability\nconfidence\ninterval (95%)\n2\nValine (V)\nGlycine (G)\nGTG\nGGG\n100\n0\n0\n1\n12\nValine (V)\nPhenylalanine (F)\nGTT\nTTT\n100\n0\n100\n2\n0.00068–0.02003\n*12\nValine (V)\nIsoleucine (I)\nGTT\nATT\n100\n0\n100\n1\n0.00007–0.01546\n16\nAlanine (A)\nProline (P)\nGCC\nCCC\n100\n0\n100\n1\n0.00007–0.01546\n17\nMethionine (M)\nLeucine (L)\nATG\nTTG\n100\n0\n100\n1\n0.00007–0.01546\n*20\nAspartic Acid (D)\nGlycine (G)\nGAC\nGGC\n87\n13\n14\n39\n0.00617–0.03612\n40\nArginine (R)\nGlutamine (Q)\nCGA\nCAA\n100\n0\n0\n3\n52\nTyrosine (Y)\nSerine (S)\nTAT\nTCT\n100\n0\n100\n1\n0.00007–0.01546\n*96\nGlycine (G)\nSerine (S)\nGGT\nAGT\n0\n100\n0\n1\n*98\nThreonine (T)\nIsoleucine (I)\nACC\nATC\n0\n100\n0\n1\n195\nThreonine (T)\nSerine (S)\nACC\nTCC\n100\n0\n0\n1\n204\nThreonine (T)\nAsparagine (N)\nACT\nAAT\n100\n0\n0\n1\n209\nMethionine (M)\nArginine (R)\nATG\nAGG\n100\n0\n50\n2\n0.00007–0.01546\n210\nGlutamic acid (E)\nAspartic acid (D)\nGAG\nGAT\n100\n0\n0\n1\n213\nValine (V)\nGlycine (G)\nGTG\nGGG\n100\n0\n100\n1\n0.00007–0.01546\n217\nCysteine (C)\nArginine (R)\nTGC\nCGC\n100\n0\n0\n1\n217\nCysteine (C)\nSerine (S)\nTGC\nTCC\n100\n0\n100\n1\n0.00007–0.01546\n221\nTyrosine (Y)\nSerine (S)\nTAC\nTCC\n100\n0\n100\n1\n0.00007–0.01546\n*225\nSerine (S)\nPhenylalanine (F)\nTCC\nTTC\n100\n0\n0\n2\n225\nSerine (S)\nTyrosine (Y)\nTCC\nTAC\n100\n0\n0\n1\n226\nGlutamine (Q)\nArginine (R)\nCAG\nCGG\n100\n0\n100\n1\n0.00007–0.01546\n227\nAlanine (A)\nGlycine (G)\nGCT\nGGT\n100\n0\n0\n1\n229\nTyrosine (Y)\nPhenylalanine (F)\nTAC\nTTC\n100\n0\n100\n1\n0.00007–0.01546\n237\nLeucine (L)\nPhenylalanine (F)\nCTC\nTTC\n100\n0\n100\n1\n0.00007–0.01546\n238\nPhenylalanine (F)\nLeucine (L)\nTTC\nTTA\n100\n0\n0\n1\nPreviously reported variants were denoted with an asterisk. Variant heterozygote, homozygote frequency and CWD status is reported as a percentage within the polymorphic codon dataset. Sample number reflects the number of samples identified carrying the polymorphic\ncodon out of a total of 358 samples. Binomial probability confidence interval was reported for the CWD positive samples using the Clopper-Pearson (exact) method.\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n07\nfrontiersin.org\n12, 20, 40, 225, and wild type (Figure 2 and Supplementary Table S4).\nThe R40Q and the codon 225 variants were not observed in any\nCWD positive samples; in contrast, all the samples harboring a PRNP\nvariant at codon 12 were CWD positive (Figure 2 and\nSupplementary Table S4). The CWD status for the D20G PRNP vari­\nants and the wild-type genotype groups were varied, as shown in\nFigure 2 and Supplementary Table S4.\n3.2.3 PRNP polymorphisms, CWD status, and\nspatiotemporal data\nA brief analysis was conducted in our condensed dataset to inte­\ngrate the PRNP polymorphisms discovered, hunting district, time­\npoint, and CWD status in relation to the total number of samples\n(358) to better understand the longitudinal trajectories of these vari­\nants in wild Montana mule deer.\nIt was observed that 14 samples were CWD positive in the\nearly timepoint. 10 of these were wild type while the remaining\nfour samples contained one V12F variant and three D20G vari­\nants. (Supplementary Table S4). 135 samples were CWD negative\nwith 113 of them being wild type. Variant S225F appeared two\ntimes,\nR40Q\none\ntime,\nand\nD20G\n19\ntimes\n(Supplementary Table S4). The D20G variant appeared in all hunt­\ning districts in the early time point group and consisted of approx­\nimately 3.5–12.5% of the total samples within each hunting district\n(Figure 2).\nThe late time point group contained 27 total CWD positive samples,\n23 of which were wild type. Variant V12F was present once, V12I once,\nand D20G was present twice (Supplementary Table S4). One sample\nwith D20G variant was suspected to be CWD positive but not con­\nfirmed with further diagnostics. A total of 68 CWD negative samples\nwere within the late group. Forty-nine of those were wild-type, variant\nR40Q was identified once, while the D20G polymorphism was identi­\nfied 18 times (Supplementary Table S4). The D20G variant, was again\nidentified in all hunting districts in the late timepoint ranging from\n2–10% of total samples within each district (Figure 2). Hunting districts\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n07\nfrontiersin.org\n12, 20, 40, 225, and wild type (Figure 2 and Supplementary Table S4).\nThe R40Q and the codon 225 variants were not observed in any\nCWD positive samples; in contrast, all the samples harboring a PRNP\nvariant at codon 12 were CWD positive (Figure 2 and\nSupplementary Table S4). The CWD status for the D20G PRNP vari­\nants and the wild-type genotype groups were varied, as shown in\nFigure 2 and Supplementary Table S4.\n3.2.3 PRNP polymorphisms, CWD status, and\nspatiotemporal data\nA brief analysis was conducted in our condensed dataset to inte­\ngrate the PRNP polymorphisms discovered, hunting district, time­\npoint, and CWD status in relation to the total number of samples\n(358) to better understand the longitudinal trajectories of these vari­\nants in wild Montana mule deer.\nIt was observed that 14 samples were CWD positive in the\nearly timepoint. 10 of these were wild type while the remaining\nfour samples contained one V12F variant and three D20G vari­\nants. (Supplementary Table S4). 135 samples were CWD negative\nwith 113 of them being wild type. Variant S225F appeared two\ntimes,\nR40Q\none\ntime,\nand\nD20G\n19\ntimes\n(Supplementary Table S4). The D20G variant appeared in all hunt­\ning districts in the early time point group and consisted of approx­\nimately 3.5–12.5% of the total samples within each hunting district\n(Figure 2).\nThe late time point group contained 27 total CWD positive samples,\n23 of which were wild type. Variant V12F was present once, V12I once,\nand D20G was present twice (Supplementary Table S4). One sample\nwith D20G variant was suspected to be CWD positive but not con­\nfirmed with further diagnostics. A total of 68 CWD negative samples\nwere within the late group. Forty-nine of those were wild-type, variant\nR40Q was identified once, while the D20G polymorphism was identi­\nfied 18 times (Supplementary Table S4). The D20G variant, was again\nidentified in all hunting districts in the late timepoint ranging from\n2–10% of total samples within each district (Figure 2). Hunting districts\n\nThe D20G variant, was again\nidentified in all hunting districts in the late timepoint ranging from\n2–10% of total samples within each district (Figure 2).Hunting districts\n401, 502, and 555 in both timepoints had the greatest number of these\nvariants with 17 out of 25 variants in the early timepoint and 17 out of\n24 in the later timepoint (Figure 2 and Supplementary Table S4).\nIn addition to examining timing and locality of variants, it\nwas notable to observe that the PRNP variants at codon 12 were\nidentified in three different samples that were obtained from\nthree different sample years and three distinct remote areas in\nMontana, all of which were CWD positive (Figure 2). From all\n3 years we sequenced 91 samples in hunting region number 401,\nand the only CWD positive sample contained our V12F variant.\nSamples expressing the novel variant, R40Q, were also discov­\nered in different years and hunting regions (Figure 2 and\nSupplementary Table S4).\n3.3 Predictions of protein stability changes\nbased on PRNP genetic variant\nWe next used computational prediction modeling to probe\nthe stability and structural influence of the single nucleotide\nalterations which occurred in multiple samples. The variants\nV12F, D20G, and R40Q occur in the unstructured region of mule\ndeer PrP (MDPrP). EmCAST, a recent tool designed to analyze\nprotein sequence-structure relationships in the absence of tertiary\ninteractions, was used to predict the structural effects of these\nvariants (45). Sequence-to-structure modeling of the N-terminal\nregion (Residues 1–44) was performed using the wild-type\nsequence to generate a reference structure (Figure 3). A hydro­\nphobic alpha-helix is predicted to span residues 1–27; the remain­\ning residues form a disordered loop. The modeled wild-type\nstructure was used by EmCAST to predict stability changes for\nthe MDPrP variants: V12F (+0.114 kcal/mol), D20G (−0.725 kcal/\nmol), and R40Q (−0.207 kcal/mol) with positive values indicative\nof stabilizing impact. Only D20G was predicted to have an impact\non stability of the protein specifically. Sequence-to-structure\n\nThe D20G variant, was again\nidentified in all hunting districts in the late timepoint ranging from\n2–10% of total samples within each district (Figure 2).Hunting districts\n401, 502, and 555 in both timepoints had the greatest number of these\nvariants with 17 out of 25 variants in the early timepoint and 17 out of\n24 in the later timepoint (Figure 2 and Supplementary Table S4).\nIn addition to examining timing and locality of variants, it\nwas notable to observe that the PRNP variants at codon 12 were\nidentified in three different samples that were obtained from\nthree different sample years and three distinct remote areas in\nMontana, all of which were CWD positive (Figure 2). From all\n3 years we sequenced 91 samples in hunting region number 401,\nand the only CWD positive sample contained our V12F variant.\nSamples expressing the novel variant, R40Q, were also discov­\nered in different years and hunting regions (Figure 2 and\nSupplementary Table S4).\n3.3 Predictions of protein stability changes\nbased on PRNP genetic variant\nWe next used computational prediction modeling to probe\nthe stability and structural influence of the single nucleotide\nalterations which occurred in multiple samples. The variants\nV12F, D20G, and R40Q occur in the unstructured region of mule\ndeer PrP (MDPrP). EmCAST, a recent tool designed to analyze\nprotein sequence-structure relationships in the absence of tertiary\ninteractions, was used to predict the structural effects of these\nvariants (45). Sequence-to-structure modeling of the N-terminal\nregion (Residues 1–44) was performed using the wild-type\nsequence to generate a reference structure (Figure 3). A hydro­\nphobic alpha-helix is predicted to span residues 1–27; the remain­\ning residues form a disordered loop. The modeled wild-type\nstructure was used by EmCAST to predict stability changes for\nthe MDPrP variants: V12F (+0.114 kcal/mol), D20G (−0.725 kcal/\nmol), and R40Q (−0.207 kcal/mol) with positive values indicative\nof stabilizing impact. Only D20G was predicted to have an impact\non stability of the protein specifically. Sequence-to-structure\n\n114 kcal/mol), D20G (−0.725 kcal/\nmol), and R40Q (−0.207 kcal/mol) with positive values indicative\nof stabilizing impact.Only D20G was predicted to have an impact\non stability of the protein specifically.Sequence-to-structure\nmodeling for the D20G sequence predicts D20G to introduce a\nkink into the N-terminal helix (Figure 3). For the S225F variant,\nEmCAST was run using folded (PDB: 6FNV) and fibril (PDB:\n9DMY) structures of MDPrP; EmCAST predicted the S225F vari­\nant to be neutral (+0.128 kcal/mol) in the folded structure and\nFIGURE 1\nPercentage of mule deer with non-synonymous PRNP polymorphisms in relation to hunting district location and time. Prevalence (%) of deer displayed\nin a heat map carrying a non-synonymous variant in each hunting district from the (A) early timepoint hunting seasons (2017/2018 and 2018/2019) and\n(B) late timepoint hunting seasons (2022/2023).\n\n114 kcal/mol), D20G (−0.725 kcal/\nmol), and R40Q (−0.207 kcal/mol) with positive values indicative\nof stabilizing impact.Only D20G was predicted to have an impact\non stability of the protein specifically.Sequence-to-structure\nmodeling for the D20G sequence predicts D20G to introduce a\nkink into the N-terminal helix (Figure 3). For the S225F variant,\nEmCAST was run using folded (PDB: 6FNV) and fibril (PDB:\n9DMY) structures of MDPrP; EmCAST predicted the S225F vari­\nant to be neutral (+0.128 kcal/mol) in the folded structure and\nFIGURE 1\nPercentage of mule deer with non-synonymous PRNP polymorphisms in relation to hunting district location and time. Prevalence (%) of deer displayed\nin a heat map carrying a non-synonymous variant in each hunting district from the (A) early timepoint hunting seasons (2017/2018 and 2018/2019) and\n(B) late timepoint hunting seasons (2022/2023).\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nincompatible with fibril structure (-Inf kcal/mol). It is worth \nnoting these predictions are based on the currently known fibril \nstructure; therefore, they should be interpreted with appropriate \ncaution. However, inclusion of tools such as EmCAST in wildlife \nstudies may be useful where the downstream phenotypic conse­\nquences of genetic variation are often difficult or impossible to \ntrack directly.\n3.4 Prion seeding activity in Montana mule \ndeer lymph nodes expressing novel \nCWD-linked PrP polymorphisms\nAn exploratory investigation was performed to detect and assess \nthe impacts of a subset of our novel PRNP polymorphisms, both with \nand without CWD, on prion seeding activity. We included a set of \nFIGURE 2\nPrevalence of the PRNP polymorphisms found to be CWD positive, suspect, and negative within each hunting district and timeframe. PRNP non-\nsynonymous variants identified in the hunting districts. Results are shown as percentages of total samples within each hunting district. Red bar graph \nindicates CWD positive samples, green indicates CWD negative samples, and orange indicates suspect CWD status. This dataset contains a subset of \nsamples of interest; synonymous variants and less common variants were not included.\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nincompatible with fibril structure (-Inf kcal/mol). It is worth \nnoting these predictions are based on the currently known fibril \nstructure; therefore, they should be interpreted with appropriate \ncaution. However, inclusion of tools such as EmCAST in wildlife \nstudies may be useful where the downstream phenotypic conse­\nquences of genetic variation are often difficult or impossible to \ntrack directly.\n3.4 Prion seeding activity in Montana mule \ndeer lymph nodes expressing novel \nCWD-linked PrP polymorphisms\nAn exploratory investigation was performed to detect and assess \nthe impacts of a subset of our novel PRNP polymorphisms, both with \nand without CWD, on prion seeding activity. We included a set of \nFIGURE 2\nPrevalence of the PRNP polymorphisms found to be CWD positive, suspect, and negative within each hunting district and timeframe. PRNP non-\nsynonymous variants identified in the hunting districts. Results are shown as percentages of total samples within each hunting district. Red bar graph \nindicates CWD positive samples, green indicates CWD negative samples, and orange indicates suspect CWD status. This dataset contains a subset of \nsamples of interest; synonymous variants and less common variants were not included.\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nnovel variant CWD negative samples to both confirm the ELISA\nresults reported for an accurate assessment of the spatiotemporal\ndata, and to investigate the unknown effects the novel variant may\nhave on protein seeding activity. To determine whether PrPSc was\npresent in a condensed mule deer lymph node dataset expressing the\nPrP polymorphisms V12F, V12I, D20G, R40Q, and S225F we used\nthe prion RT-QuIC assay to probe lymph node samples along with\nappropriate controls (Table 2) (47). Samples were chosen based on\nthe presence of our variant of interest; however, we were limited by\nthe samples obtained from the wild specimens. Occasionally, the\nsamples contained more than one polymorphism (Table 2). We did\nnot perform RT-QuIC analysis on samples with synonymous single\nnucleotide polymorphisms, as the amino acid in PrP would be the\nsame as wild-type. We included five controls: 1) brain homogenates\nfrom both CWD infected and 2) uninfected brain homogenates of\ntransgenic mice with wild-type cervid PRNP (Tg1536+/+) (42), 3)\nCWD-positive and 4) CWD-negative mule deer brain homogenates,\nand 5) PRNP knock-out mice. At 10−3 dilution, we observed substan­\ntial ThT fluorescence in all CWD positive biospecimens (brain or\nlymph nodes) relative to tissue matched non-CWD controls (Table 2;\nFigure 4 and Supplementary Figure S5).\nUnsurprisingly, we observed that the CWD positive transgenic\nmouse brain expressing deer PRNP gave the highest midpoint ThT\nfluorescence amplitude signal (63,300 ± 27,453) and the shortest lag\nphase (7.6 ± 1), similar to its kinetic data (Control CWD 10, Table 2;\nFigure 4 and Supplementary Figure S6). With respect to CWD posi­\ntive mule deer lymph nodes, the WT, V12F, V12I, and D20G, all\ndisplayed significant prion seeding activity via ThT fluorescence\namplitude (Student’s t-test, p < 0.05) relative to lymph node controls\nthat were ThT negative (Figures 4, 5). The CWD positive mule deer\nlymph nodes had lag phase values that ranged from 10–14 h, with\nmidpoint ThT fluorescence amplitude values ranging from 40,000–\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n09\nfrontiersin.org\nnovel variant CWD negative samples to both confirm the ELISA\nresults reported for an accurate assessment of the spatiotemporal\ndata, and to investigate the unknown effects the novel variant may\nhave on protein seeding activity. To determine whether PrPSc was\npresent in a condensed mule deer lymph node dataset expressing the\nPrP polymorphisms V12F, V12I, D20G, R40Q, and S225F we used\nthe prion RT-QuIC assay to probe lymph node samples along with\nappropriate controls (Table 2) (47). Samples were chosen based on\nthe presence of our variant of interest; however, we were limited by\nthe samples obtained from the wild specimens. Occasionally, the\nsamples contained more than one polymorphism (Table 2). We did\nnot perform RT-QuIC analysis on samples with synonymous single\nnucleotide polymorphisms, as the amino acid in PrP would be the\nsame as wild-type. We included five controls: 1) brain homogenates\nfrom both CWD infected and 2) uninfected brain homogenates of\ntransgenic mice with wild-type cervid PRNP (Tg1536+/+) (42), 3)\nCWD-positive and 4) CWD-negative mule deer brain homogenates,\nand 5) PRNP knock-out mice. At 10−3 dilution, we observed substan­\ntial ThT fluorescence in all CWD positive biospecimens (brain or\nlymph nodes) relative to tissue matched non-CWD controls (Table 2;\nFigure 4 and Supplementary Figure S5).\nUnsurprisingly, we observed that the CWD positive transgenic\nmouse brain expressing deer PRNP gave the highest midpoint ThT\nfluorescence amplitude signal (63,300 ± 27,453) and the shortest lag\nphase (7.6 ± 1), similar to its kinetic data (Control CWD 10, Table 2;\nFigure 4 and Supplementary Figure S6). With respect to CWD posi­\ntive mule deer lymph nodes, the WT, V12F, V12I, and D20G, all\ndisplayed significant prion seeding activity via ThT fluorescence\namplitude (Student’s t-test, p < 0.05) relative to lymph node controls\nthat were ThT negative (Figures 4, 5). The CWD positive mule deer\nlymph nodes had lag phase values that ranged from 10–14 h, with\nmidpoint ThT fluorescence amplitude values ranging from 40,000–\n\n05) relative to lymph node controls\nthat were ThT negative (Figures 4, 5).The CWD positive mule deer\nlymph nodes had lag phase values that ranged from 10–14 h, with\nmidpoint ThT fluorescence amplitude values ranging from 40,000–\n55,000 relative fluorescence units (RFU) (Table 2 and\nSupplementary Figures S6, S9). Importantly, the CWD positive\nmule deer lymph nodes expressing the p. V12F and p. V12I, p. A16P,\np. M17L, p. Y52S, demonstrated that these novel polymorphisms\nhad prion seeding activity (CWD samples 4, 8, 3 Table 2 and\nFigure 5).\nThe majority of samples that were determined to be CWD nega­\ntive via an ELISA, did not show prion seeding activity by RT-QuIC,\nincluding a PRNP knock-out control (Control 9, Table 2,\nSupplementary Figure S5); however, there were exceptions. Two\nELISA-negative mule deer lymph nodes had lag phase and midpoint\nThT fluorescence amplitude values that clustered with lymph node\nsamples that were CWD positive via ELISA (Table 2; Figure 5;\nSupplementary Figures S8, S10). One of these two lymph node speci­\nmens expressed wild-type PrP (control 11) and the other expressed\nthe PrP variant R40Q (control 10), which to our knowledge, is a novel\nmutation in mule deer that has been previously reported in sheep (49).\nThe WT PrP (control 11) had ThT positive signal with a lag phase of\n13.1 ± 2 and midpoint ThT fluorescence amplitude of 39,708 ± 6,752\nRFU, while the PrP R40Q (control 10) had a lag phase of 12.4 ± 2, and\na half ThT fluorescence amplitude of 40,705 ± 6,901 RFU\n(Supplementary Figure S8).\n4 Discussion\nWe report the sequencing of the PRNP gene of wild Montana\nmule deer lymph nodes. We discovered that over half of the mule deer\nsamples possessed at least one of 36 PRNP polymorphisms (25 non-\nsynonymous and 11 synonymous) with many samples containing\nmultiple polymorphisms. In this study, we focused on further analysis\nof a subset of the non-synonymous variants. Nonetheless, we acknowl­\nedge that synonymous variants may not only relay information on\ngene evolution but also may alter protein expression, mRNA splicing\n\n05) relative to lymph node controls\nthat were ThT negative (Figures 4, 5).The CWD positive mule deer\nlymph nodes had lag phase values that ranged from 10–14 h, with\nmidpoint ThT fluorescence amplitude values ranging from 40,000–\n55,000 relative fluorescence units (RFU) (Table 2 and\nSupplementary Figures S6, S9). Importantly, the CWD positive\nmule deer lymph nodes expressing the p. V12F and p. V12I, p. A16P,\np. M17L, p. Y52S, demonstrated that these novel polymorphisms\nhad prion seeding activity (CWD samples 4, 8, 3 Table 2 and\nFigure 5).\nThe majority of samples that were determined to be CWD nega­\ntive via an ELISA, did not show prion seeding activity by RT-QuIC,\nincluding a PRNP knock-out control (Control 9, Table 2,\nSupplementary Figure S5); however, there were exceptions. Two\nELISA-negative mule deer lymph nodes had lag phase and midpoint\nThT fluorescence amplitude values that clustered with lymph node\nsamples that were CWD positive via ELISA (Table 2; Figure 5;\nSupplementary Figures S8, S10). One of these two lymph node speci­\nmens expressed wild-type PrP (control 11) and the other expressed\nthe PrP variant R40Q (control 10), which to our knowledge, is a novel\nmutation in mule deer that has been previously reported in sheep (49).\nThe WT PrP (control 11) had ThT positive signal with a lag phase of\n13.1 ± 2 and midpoint ThT fluorescence amplitude of 39,708 ± 6,752\nRFU, while the PrP R40Q (control 10) had a lag phase of 12.4 ± 2, and\na half ThT fluorescence amplitude of 40,705 ± 6,901 RFU\n(Supplementary Figure S8).\n4 Discussion\nWe report the sequencing of the PRNP gene of wild Montana\nmule deer lymph nodes. We discovered that over half of the mule deer\nsamples possessed at least one of 36 PRNP polymorphisms (25 non-\nsynonymous and 11 synonymous) with many samples containing\nmultiple polymorphisms. In this study, we focused on further analysis\nof a subset of the non-synonymous variants. Nonetheless, we acknowl­\nedge that synonymous variants may not only relay information on\ngene evolution but also may alter protein expression, mRNA splicing\n\nIn this study, we focused on further analysis\nof a subset of the non-synonymous variants.Nonetheless, we acknowl­\nedge that synonymous variants may not only relay information on\ngene evolution but also may alter protein expression, mRNA splicing\nand other key properties (50). Thus, there may be value to further\nanalysis of both the non-synonymous variants not included in our\ncondensed subset and the synonymous variants in future studies.\nIt is also imperative to note that the experimental sampling design\nwas one of availability and due to low sample size of some variants,\nthis study was inherently constrained to what was collected during\nMT FWP CWD surveillance. While this study provides new data that\nis of importance to the understanding of CWD, the results are solely\nhypothesis-generating observations and further studies with higher\npowered sample sizes would be required to make statistically mean­\ningful associations between variants and the implications they may\nhave on disease.\nPrior to the study, we predicted that a positive correlation\nwould occur between the prevalence of CWD and the prevalence\nof PRNP genetic variants. In other words, if CWD prevalence is\nhigher in a region, then selection for PRNP variants should be\nhigher if they decrease CWD susceptibility or increase survival\ntime. In this dataset, this hypothesis was not observed, as regions\n600, 670 and 640 had the highest prevalences of CWD, yet did not\nhave significantly higher prevalences of PRNP variants (Figure 2).\nHowever, at the time these samples were procured, detections of\ndisease were relatively new in MT which led to limited sampling\nand the outcome of this study does not have any indication or\nimplication for how selection of PRNP variants will evolve over\nmore time. Future studies such as this may determine protective\ngenetic variants from CWD driven genetic selection in Montana.\nInterestingly, rather than identifying beneficial variants, our data\nrevealed a number of novel PRNP variants that were CWD posi­\ntive, modeled for protein stability, and were confirmed to have PrP\n\nIn this study, we focused on further analysis\nof a subset of the non-synonymous variants.Nonetheless, we acknowl­\nedge that synonymous variants may not only relay information on\ngene evolution but also may alter protein expression, mRNA splicing\nand other key properties (50). Thus, there may be value to further\nanalysis of both the non-synonymous variants not included in our\ncondensed subset and the synonymous variants in future studies.\nIt is also imperative to note that the experimental sampling design\nwas one of availability and due to low sample size of some variants,\nthis study was inherently constrained to what was collected during\nMT FWP CWD surveillance. While this study provides new data that\nis of importance to the understanding of CWD, the results are solely\nhypothesis-generating observations and further studies with higher\npowered sample sizes would be required to make statistically mean­\ningful associations between variants and the implications they may\nhave on disease.\nPrior to the study, we predicted that a positive correlation\nwould occur between the prevalence of CWD and the prevalence\nof PRNP genetic variants. In other words, if CWD prevalence is\nhigher in a region, then selection for PRNP variants should be\nhigher if they decrease CWD susceptibility or increase survival\ntime. In this dataset, this hypothesis was not observed, as regions\n600, 670 and 640 had the highest prevalences of CWD, yet did not\nhave significantly higher prevalences of PRNP variants (Figure 2).\nHowever, at the time these samples were procured, detections of\ndisease were relatively new in MT which led to limited sampling\nand the outcome of this study does not have any indication or\nimplication for how selection of PRNP variants will evolve over\nmore time. Future studies such as this may determine protective\ngenetic variants from CWD driven genetic selection in Montana.\nInterestingly, rather than identifying beneficial variants, our data\nrevealed a number of novel PRNP variants that were CWD posi­\ntive, modeled for protein stability, and were confirmed to have PrP\n\nInterestingly, rather than identifying beneficial variants, our data\nrevealed a number of novel PRNP variants that were CWD posi­\ntive, modeled for protein stability, and were confirmed to have PrP\nseeding activity. It is well established in the prion field that certain\nstrains show differential molecular compatibility with amplifica­\ntion substrates, as demonstrated for CJD subtypes defined by\nPRNP codon 129 polymorphisms (51, 52). Likewise, in CWD\nstudies, RT-QuIC is commonly used as one of many methods to\ndetermine prion strains via differences in prion seeding activity\nFIGURE 3\nPredicted structures for the N-Terminal region of PrP (Residues 1–44)\nusing WT, R40Q, and D20G sequences in EmCAST. Positions with\nselect variants are rendered as spheres: V12 (magenta), D20 (green),\nand R40 (blue). Residues 39 and 40–44 are colored orange in the\nR40Q structure. Hydrophobic residues in the N-terminal helix are\ncolored magenta.\n\nInterestingly, rather than identifying beneficial variants, our data\nrevealed a number of novel PRNP variants that were CWD posi­\ntive, modeled for protein stability, and were confirmed to have PrP\nseeding activity. It is well established in the prion field that certain\nstrains show differential molecular compatibility with amplifica­\ntion substrates, as demonstrated for CJD subtypes defined by\nPRNP codon 129 polymorphisms (51, 52). Likewise, in CWD\nstudies, RT-QuIC is commonly used as one of many methods to\ndetermine prion strains via differences in prion seeding activity\nFIGURE 3\nPredicted structures for the N-Terminal region of PrP (Residues 1–44)\nusing WT, R40Q, and D20G sequences in EmCAST. Positions with\nselect variants are rendered as spheres: V12 (magenta), D20 (green),\nand R40 (blue). Residues 39 and 40–44 are colored orange in the\nR40Q structure. Hydrophobic residues in the N-terminal helix are\ncolored magenta.\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n10\nfrontiersin.org\n(53–55). Without further information on the wild cervid samples,\nanalysis on this data set was limited to binary confirmation of\nCWD status. However, studies such as ours underline the poten­\ntial use of the RT-QuIC assay in CWD diagnostic and surveillance\nprograms. With complete epidemiological and demographic\ninformation, RT-QuIC could provide insight into disease staging\nincluding early stages of disease that otherwise go undetected\ngiving crucial knowledge to how cervid populations are managed\nand surveyed.\nHere, we highlight a few polymorphisms investigated in this study\nand discuss the respective results and observations found through\nspatiotemporal analysis, computational protein modeling, and\nRT-QuIC.\n4.1 PRNP polymorphisms within the\nsignaling peptide area of the protein\n4.1.1 V12F\nThe novel genetic variant V12F is intriguing due to the loca­\ntion of the substitution in the center of the signaling peptide, and\nthe discovery that both deer in which it was identified were CWD\npositive (Figure 6). Two different PRNP V12F samples were dis­\ncovered, and they were obtained from different sample years and\ndistinct remote areas in Montana. An explanation for this could\nbe either that it has arisen multiple times spontaneously, a product\nof the movement of the wild mule deer, or that it is present in\nTABLE 2  RT-QuIC prion seeding activity on mule deer biospecimens harboring PRNP polymorphisms.\nPrP variant\nRT-QuIC\nsample\nnumber/\nAnimal ID\nTissue type\nCWD status\nLag phase\n(h)\nThT FAm (RFU)\nPrion RT-QuIC\noutcome\n[ThT+/Total\nwells (4)]\np. D20G\nControl 1/4000 (C1)\nLymph node\nNegative\n—\n—\n0/4\np. D20G\nControl 2/295 (C2)\nLymph node\nNegative\n—\n—\n1/4\np. D20G\nControl 3/3489 (C3)\nLymph node\nNegative\n—\n—\n0/4\np. R40Q, p. D20G\nControl 4/47482 (C4)\nLymph node\nNegative\n—\n—\n0/4\np. R40Q, p. V2G\nControl 5/48237 (C5)\nLymph node\nNegative\n—\n—\n0/4\np. R40Q\nControl 10/1358\n(C10)\nLymph node\nELISA Negative/\nRT-QuIC positive\n—\n—\n3/4\np. S225F\nControl 6/2536 (C6)\nLymph node\nNegative\n—\n—\n0/4\np. S225F, p. T195S,\np. E210D, p.\nC217R, p. F238L\nControl 7/608 (C7)\nLymph node\nNegative\n—\n—\n0/4\np. WT\nControl 8/1006 (C8)\nLymph node\nNegative\n—\n—\n0/4\np. WT\nControl 11/497 (C11)\nLymph node\nELISA Negative/\nRT-QuIC Positive\n13.1 ± 2\n39,708 ± 6,752\n3/4\nPrP Knockout\nControl 9/691 (C9)\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n10\nfrontiersin.org\n(53–55). Without further information on the wild cervid samples,\nanalysis on this data set was limited to binary confirmation of\nCWD status. However, studies such as ours underline the poten­\ntial use of the RT-QuIC assay in CWD diagnostic and surveillance\nprograms. With complete epidemiological and demographic\ninformation, RT-QuIC could provide insight into disease staging\nincluding early stages of disease that otherwise go undetected\ngiving crucial knowledge to how cervid populations are managed\nand surveyed.\nHere, we highlight a few polymorphisms investigated in this study\nand discuss the respective results and observations found through\nspatiotemporal analysis, computational protein modeling, and\nRT-QuIC.\n4.1 PRNP polymorphisms within the\nsignaling peptide area of the protein\n4.1.1 V12F\nThe novel genetic variant V12F is intriguing due to the loca­\ntion of the substitution in the center of the signaling peptide, and\nthe discovery that both deer in which it was identified were CWD\npositive (Figure 6). Two different PRNP V12F samples were dis­\ncovered, and they were obtained from different sample years and\ndistinct remote areas in Montana. An explanation for this could\nbe either that it has arisen multiple times spontaneously, a product\nof the movement of the wild mule deer, or that it is present in\nTABLE 2  RT-QuIC prion seeding activity on mule deer biospecimens harboring PRNP polymorphisms.\nPrP variant\nRT-QuIC\nsample\nnumber/\nAnimal ID\nTissue type\nCWD status\nLag phase\n(h)\nThT FAm (RFU)\nPrion RT-QuIC\noutcome\n[ThT+/Total\nwells (4)]\np. D20G\nControl 1/4000 (C1)\nLymph node\nNegative\n—\n—\n0/4\np. D20G\nControl 2/295 (C2)\nLymph node\nNegative\n—\n—\n1/4\np. D20G\nControl 3/3489 (C3)\nLymph node\nNegative\n—\n—\n0/4\np. R40Q, p. D20G\nControl 4/47482 (C4)\nLymph node\nNegative\n—\n—\n0/4\np. R40Q, p. V2G\nControl 5/48237 (C5)\nLymph node\nNegative\n—\n—\n0/4\np. R40Q\nControl 10/1358\n(C10)\nLymph node\nELISA Negative/\nRT-QuIC positive\n—\n—\n3/4\np. S225F\nControl 6/2536 (C6)\nLymph node\nNegative\n—\n—\n0/4\np. S225F, p. T195S,\np. E210D, p.\nC217R, p. F238L\nControl 7/608 (C7)\nLymph node\nNegative\n—\n—\n0/4\np. WT\nControl 8/1006 (C8)\nLymph node\nNegative\n—\n—\n0/4\np. WT\nControl 11/497 (C11)\nLymph node\nELISA Negative/\nRT-QuIC Positive\n13.1 ± 2\n39,708 ± 6,752\n3/4\nPrP Knockout\nControl 9/691 (C9)\n\nT195S,\np.E210D, p.C217R, p.F238L\nControl 7/608 (C7)\nLymph node\nNegative\n—\n—\n0/4\np.WT\nControl 8/1006 (C8)\nLymph node\nNegative\n—\n—\n0/4\np.WT\nControl 11/497 (C11)\nLymph node\nELISA Negative/\nRT-QuIC Positive\n13.1 ± 2\n39,708 ± 6,752\n3/4\nPrP Knockout\nControl 9/691 (C9)\nPrP KO mouse\nbrain\nNegative\n—\n—\n0/4\np. WT\nControl 12 deer brain\n(C12)\nDeer brain\nNegative\n—\n—\n0/4\np. WT\nControl 13 Tg mouse\nbrain (C13)\nTg cervidized\nmouse brain*\nNegative\n—\n—\n0/4\np. V12I, p. A16P, p.\nM17L, p. Y52S\nCWD 3 (P3)\nLymph node\nPositive\n11.9 ± 0.7\n54,300 ± 8,991\n4/4\np. V12F\nCWD 4 (P4)\nLymph node\nPositive\n14.2 ± 2\n45,200 ± 5,664\n4/4\np. V12F\nCWD 8 (P8)\nLymph node\nPositive\n12.4 ± 1\n54,100 ± 10,889\n4/4\np. D20G\nCWD 2 (P2)\nLymph node\nPositive\n13.7 ± 0.9\n55,047 ± 8,288\n4/4\np. D20G\nCWD 6 (P6)\nLymph node\nPositive\n10.5 ± 0.4\n48,075 ± 10,056\n4/4\np. D20G\nCWD 7 (P7)\nLymph node\nPositive\n11.8 ± 1\n46,000 ± 5,885\n4/4\np. WT\nCWD 1 (P1)\nLymph node\nPositive\n10.2 ± 0.3\n43,046 ± 4,005\n4/4\np. WT\nCWD 5 (P5)\nLymph node\nPositive\n10.0 ± 0.6\n46,368 ± 6,890\n4/4\np. WT\nControl CWD 9 (P9)\nDeer brain\nPositive\n10.4 ± 0.5\n41,147 ± 3,108\n4/4\np. WT\nControl CWD 10\n(P10)\nTg cervidized\nmouse brain*\nPositive\n7.6 ± 1\n63,300 ± 27,453\n4/4\n*Transgenic mouse brain expressing wild-type cervid PRNP (Tg1536+/+). Sample Control CWD 10 is a Tg1536+/+ mouse that was inoculated with wild-type CWD positive mule deer brain\nhomogenate and harvested at ~230dpi. ThT FAm, ThT fluorescence amplitude at the midpoint. CWD status is reported based on previous ELISA results.\n\nT195S,\np.E210D, p.C217R, p.F238L\nControl 7/608 (C7)\nLymph node\nNegative\n—\n—\n0/4\np.WT\nControl 8/1006 (C8)\nLymph node\nNegative\n—\n—\n0/4\np.WT\nControl 11/497 (C11)\nLymph node\nELISA Negative/\nRT-QuIC Positive\n13.1 ± 2\n39,708 ± 6,752\n3/4\nPrP Knockout\nControl 9/691 (C9)\nPrP KO mouse\nbrain\nNegative\n—\n—\n0/4\np. WT\nControl 12 deer brain\n(C12)\nDeer brain\nNegative\n—\n—\n0/4\np. WT\nControl 13 Tg mouse\nbrain (C13)\nTg cervidized\nmouse brain*\nNegative\n—\n—\n0/4\np. V12I, p. A16P, p.\nM17L, p. Y52S\nCWD 3 (P3)\nLymph node\nPositive\n11.9 ± 0.7\n54,300 ± 8,991\n4/4\np. V12F\nCWD 4 (P4)\nLymph node\nPositive\n14.2 ± 2\n45,200 ± 5,664\n4/4\np. V12F\nCWD 8 (P8)\nLymph node\nPositive\n12.4 ± 1\n54,100 ± 10,889\n4/4\np. D20G\nCWD 2 (P2)\nLymph node\nPositive\n13.7 ± 0.9\n55,047 ± 8,288\n4/4\np. D20G\nCWD 6 (P6)\nLymph node\nPositive\n10.5 ± 0.4\n48,075 ± 10,056\n4/4\np. D20G\nCWD 7 (P7)\nLymph node\nPositive\n11.8 ± 1\n46,000 ± 5,885\n4/4\np. WT\nCWD 1 (P1)\nLymph node\nPositive\n10.2 ± 0.3\n43,046 ± 4,005\n4/4\np. WT\nCWD 5 (P5)\nLymph node\nPositive\n10.0 ± 0.6\n46,368 ± 6,890\n4/4\np. WT\nControl CWD 9 (P9)\nDeer brain\nPositive\n10.4 ± 0.5\n41,147 ± 3,108\n4/4\np. WT\nControl CWD 10\n(P10)\nTg cervidized\nmouse brain*\nPositive\n7.6 ± 1\n63,300 ± 27,453\n4/4\n*Transgenic mouse brain expressing wild-type cervid PRNP (Tg1536+/+). Sample Control CWD 10 is a Tg1536+/+ mouse that was inoculated with wild-type CWD positive mule deer brain\nhomogenate and harvested at ~230dpi. ThT FAm, ThT fluorescence amplitude at the midpoint. CWD status is reported based on previous ELISA results.\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n11\nfrontiersin.org\ngeographical areas that link the distinct regions but was not\ndetected. Because this variant is rare in our dataset, and because\nwe only sampled in several geographically distinct areas, we\ncannot reliably comment on the distribution or frequency of this\nvariant across the rest of Montana or its association with\nCWD. Though, intriguingly, our newly identified V12F variant was\nthe only CWD positive sample out of 91 samples in hunting dis­\ntrict 401.\nWe hypothesized that these single non-synonymous nucleotide\nsubstitutions may alter the PrP folding characteristics, and therefore,\ndisease outcomes. Investigations into PrP indicates that amino acids\n12, 15, and 16 reside in the N-terminal region of the immature protein\nprior to cleavage during posttranslational modifications which is a\nregion with limited data regarding CWD and its impact on protein\nmisfolding (Figure 6) (56). Immature PrP has an N-terminal signal\npeptide at amino acids 1–23 that codes for entry into the endoplasmic\nreticulum of the cell (11, 57, 58). The N-terminal region of PrP is\nnatively unstructured and its function is in endocytic trafficking of\nPrP; this region also includes a sequence that is essential for toxicity\nof PrP (59). Alterations to the protein structure in the N-terminus may\naffect intracellular trafficking, may induce specific biochemical\nchanges resulting in PrP misfolding, or may lower the threshold for\nPrP corruption from another CWD positive animal. Deer with the\nV12F PRNP variant were heterozygous, expressing phenylalanine in\none allele and the wild-type valine in the other allele. Valine is known\nto contain a small hydrophobic side chain as opposed to phenylalanine\nwith a large hydrophobic aromatic side chain which likely disrupt\ninteractions with a binding partner important for proper trafficking\n(60). Thus, this variant could disrupt structure causing steric clashes,\ncause enhanced interactions with other hydrophobic residues that\npromote conversion, and/or alter protein stability, all leading to mis­\nfolding (61).\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n11\nfrontiersin.org\ngeographical areas that link the distinct regions but was not\ndetected. Because this variant is rare in our dataset, and because\nwe only sampled in several geographically distinct areas, we\ncannot reliably comment on the distribution or frequency of this\nvariant across the rest of Montana or its association with\nCWD. Though, intriguingly, our newly identified V12F variant was\nthe only CWD positive sample out of 91 samples in hunting dis­\ntrict 401.\nWe hypothesized that these single non-synonymous nucleotide\nsubstitutions may alter the PrP folding characteristics, and therefore,\ndisease outcomes. Investigations into PrP indicates that amino acids\n12, 15, and 16 reside in the N-terminal region of the immature protein\nprior to cleavage during posttranslational modifications which is a\nregion with limited data regarding CWD and its impact on protein\nmisfolding (Figure 6) (56). Immature PrP has an N-terminal signal\npeptide at amino acids 1–23 that codes for entry into the endoplasmic\nreticulum of the cell (11, 57, 58). The N-terminal region of PrP is\nnatively unstructured and its function is in endocytic trafficking of\nPrP; this region also includes a sequence that is essential for toxicity\nof PrP (59). Alterations to the protein structure in the N-terminus may\naffect intracellular trafficking, may induce specific biochemical\nchanges resulting in PrP misfolding, or may lower the threshold for\nPrP corruption from another CWD positive animal. Deer with the\nV12F PRNP variant were heterozygous, expressing phenylalanine in\none allele and the wild-type valine in the other allele. Valine is known\nto contain a small hydrophobic side chain as opposed to phenylalanine\nwith a large hydrophobic aromatic side chain which likely disrupt\ninteractions with a binding partner important for proper trafficking\n(60). Thus, this variant could disrupt structure causing steric clashes,\ncause enhanced interactions with other hydrophobic residues that\npromote conversion, and/or alter protein stability, all leading to mis­\nfolding (61).\n\nThus, this variant could disrupt structure causing steric clashes,\ncause enhanced interactions with other hydrophobic residues that\npromote conversion, and/or alter protein stability, all leading to mis­\nfolding (61).\nWhen we turned to modeling structural changes, the analysis\npredicted that the amino acid change from valine to phenylalanine\nat amino acid 12 was slightly stabilizing; however, this individual\namino acid change is not the only factor to consider in protein fold­\ning/misfolding predisposition. EmCAST modeled p. V12F to be a\nstructurally neutral mutation in a hydrophobic alpha-helix suggest­\ning that the V12F phenotype might be attributable to functional\nchanges such as altered protein–protein interactions that might be\nlinked to the CWD positive status of all V12F-containing samples.\nAdditionally, the increased hydrophobic nature of phenylalanine\nenhances the likelihood of conversion to a misfolded protein, as it\nis an important driver for endoplasmic signal peptide recognition\nand any changes to this may have an impact on PrP processing\n(62, 63).\nWhen we directly tested whether there was disease-associated\nprion seeding activity in the lymph nodes from Montana mule deer\nexpressing the PrP variant V12F, we found prion seeding activity. The\ndetection of prion seeding activity in mule deer lymph nodes express­\ning this V12F polymorphism via cell-free seed amplification (i.e.,\nRT-QuIC) analysis has not been previously reported. We also recog­\nnize that the sample size of this variant is low (N = 2) and that further\ninvestigations are needed to determine CWD susceptibility in rela­\ntionship to this variant.\nFIGURE 4\nPrion RT-QuIC kinetic data of representative samples by variant group including controls. CWD pos deer lymph nodes expressing novel PrP variants p.\nV12I, P. A16P, p. M17L, p. Y52S (aqua stars), p. V12F (blue triangles) along with the previously reported variant p. D20G (pink triangles) displaying ThT\nfluorescent signal. CWD neg novel variant p. R40Q, p. V2G (purple circles), p. D20G CWD neg (green crosses), WT lymph node samples (orange\n\nThus, this variant could disrupt structure causing steric clashes,\ncause enhanced interactions with other hydrophobic residues that\npromote conversion, and/or alter protein stability, all leading to mis­\nfolding (61).\nWhen we turned to modeling structural changes, the analysis\npredicted that the amino acid change from valine to phenylalanine\nat amino acid 12 was slightly stabilizing; however, this individual\namino acid change is not the only factor to consider in protein fold­\ning/misfolding predisposition. EmCAST modeled p. V12F to be a\nstructurally neutral mutation in a hydrophobic alpha-helix suggest­\ning that the V12F phenotype might be attributable to functional\nchanges such as altered protein–protein interactions that might be\nlinked to the CWD positive status of all V12F-containing samples.\nAdditionally, the increased hydrophobic nature of phenylalanine\nenhances the likelihood of conversion to a misfolded protein, as it\nis an important driver for endoplasmic signal peptide recognition\nand any changes to this may have an impact on PrP processing\n(62, 63).\nWhen we directly tested whether there was disease-associated\nprion seeding activity in the lymph nodes from Montana mule deer\nexpressing the PrP variant V12F, we found prion seeding activity. The\ndetection of prion seeding activity in mule deer lymph nodes express­\ning this V12F polymorphism via cell-free seed amplification (i.e.,\nRT-QuIC) analysis has not been previously reported. We also recog­\nnize that the sample size of this variant is low (N = 2) and that further\ninvestigations are needed to determine CWD susceptibility in rela­\ntionship to this variant.\nFIGURE 4\nPrion RT-QuIC kinetic data of representative samples by variant group including controls. CWD pos deer lymph nodes expressing novel PrP variants p.\nV12I, P. A16P, p. M17L, p. Y52S (aqua stars), p. V12F (blue triangles) along with the previously reported variant p. D20G (pink triangles) displaying ThT\nfluorescent signal. CWD neg novel variant p. R40Q, p. V2G (purple circles), p. D20G CWD neg (green crosses), WT lymph node samples (orange\n\nY52S (aqua stars), p.V12F (blue triangles) along with the previously reported variant p.D20G (pink triangles) displaying ThT\nfluorescent signal.CWD neg novel variant p.R40Q, p.V2G (purple circles), p.D20G CWD neg (green crosses), WT lymph node samples (orange\ncrosses) did not display ThT fluorescent signal. CWD positive transgenic cervidized mouse brain (black stars), was used as a positive control. Tg\ncervidized mouse brain CWD neg (gray crosses) showed low ThT signal at ~15 h; samples after this timepoint were considered negative such as p.\nS225F (yellow boxes) which displayed low signal after that timepoint.\nFIGURE 5\nThT fluorescence amplitude (ThT FAm, RFU at 14.6 h minus initial\nreading, 0 h) for samples in study along with controls. Sample\nidentification and abbreviations shown here are denoted in Table 2.\n\nY52S (aqua stars), p.V12F (blue triangles) along with the previously reported variant p.D20G (pink triangles) displaying ThT\nfluorescent signal.CWD neg novel variant p.R40Q, p.V2G (purple circles), p.D20G CWD neg (green crosses), WT lymph node samples (orange\ncrosses) did not display ThT fluorescent signal. CWD positive transgenic cervidized mouse brain (black stars), was used as a positive control. Tg\ncervidized mouse brain CWD neg (gray crosses) showed low ThT signal at ~15 h; samples after this timepoint were considered negative such as p.\nS225F (yellow boxes) which displayed low signal after that timepoint.\nFIGURE 5\nThT fluorescence amplitude (ThT FAm, RFU at 14.6 h minus initial\nreading, 0 h) for samples in study along with controls. Sample\nidentification and abbreviations shown here are denoted in Table 2.\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n12\nfrontiersin.org\n4.1.2 V12I, A16P, M17L, Y52S\nNotably, another PRNP genetic variant was discovered in a single\nsample at codon 12 which included the four polymorphisms V12I,\nA16P, M17L, Y52S and was CWD positive by ELISA and\nRT-QuIC. Three of these prion protein variants in this sample, V12I,\nA16P, M17L, occur within the center of the signaling peptide of PrP\n(Figure 6). All three samples with polymorphisms in this area of the\nprotein were CWD positive, providing further evidence that these\nvariants might disrupt proper trafficking resulting in failure or pertur­\nbations of normal PrP signal peptide cleavage. Prion seeding activity\nwas confirmed in the RT-QuIC assay, although the seeding activity\nand CWD status cannot be attributed to any single variant, only to the\nhaplotype.\nDue to the small sample size, it is unknown at this time if the\nPRNP variants found in the signaling peptide acquired bona-fide\nCWD or if a spontaneously misfolding event occurred. Future inves­\ntigations are critical in determining the relationship of polymorphisms\nin this area of the prion protein and CWD.\n4.1.3 D20G\nThis variant is also located within the signaling peptide of PrP\nand was the most commonly found non-synonymous polymor­\nphism, appearing in 12% of the samples (Figure 6). The D20G poly­\nmorphism was present in all hunting districts from each season\n(Figure 2). Previous mule deer reports of the D20G variant are con­\nflicting as to whether CWD susceptibility is altered due to the single\nnucleotide polymorphism at this location (2, 32, 37, 64). In our\nstudy, the disease prevalence within the D20G group at 16.2% was\nsimilar to wild type at 20.3% suggesting this is a neutral substitution\npertaining to CWD. In other words, substitutions in the signal pep­\ntide outside of the central area may be trafficked properly and the\nsignal peptide is cleaved off, therefore, is processed similarly as\nthe WT PrP.\nDeer with at least one copy of the allele of the common genotype\nexpressing aspartic acid were less likely to test positive in studies con­\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n12\nfrontiersin.org\n4.1.2 V12I, A16P, M17L, Y52S\nNotably, another PRNP genetic variant was discovered in a single\nsample at codon 12 which included the four polymorphisms V12I,\nA16P, M17L, Y52S and was CWD positive by ELISA and\nRT-QuIC. Three of these prion protein variants in this sample, V12I,\nA16P, M17L, occur within the center of the signaling peptide of PrP\n(Figure 6). All three samples with polymorphisms in this area of the\nprotein were CWD positive, providing further evidence that these\nvariants might disrupt proper trafficking resulting in failure or pertur­\nbations of normal PrP signal peptide cleavage. Prion seeding activity\nwas confirmed in the RT-QuIC assay, although the seeding activity\nand CWD status cannot be attributed to any single variant, only to the\nhaplotype.\nDue to the small sample size, it is unknown at this time if the\nPRNP variants found in the signaling peptide acquired bona-fide\nCWD or if a spontaneously misfolding event occurred. Future inves­\ntigations are critical in determining the relationship of polymorphisms\nin this area of the prion protein and CWD.\n4.1.3 D20G\nThis variant is also located within the signaling peptide of PrP\nand was the most commonly found non-synonymous polymor­\nphism, appearing in 12% of the samples (Figure 6). The D20G poly­\nmorphism was present in all hunting districts from each season\n(Figure 2). Previous mule deer reports of the D20G variant are con­\nflicting as to whether CWD susceptibility is altered due to the single\nnucleotide polymorphism at this location (2, 32, 37, 64). In our\nstudy, the disease prevalence within the D20G group at 16.2% was\nsimilar to wild type at 20.3% suggesting this is a neutral substitution\npertaining to CWD. In other words, substitutions in the signal pep­\ntide outside of the central area may be trafficked properly and the\nsignal peptide is cleaved off, therefore, is processed similarly as\nthe WT PrP.\nDeer with at least one copy of the allele of the common genotype\nexpressing aspartic acid were less likely to test positive in studies con­\n\nDeer with at least one copy of the allele of the common genotype\nexpressing aspartic acid were less likely to test positive in studies con­\nducted in Canada and Nebraska, as opposed to deer sampled in\nWyoming and Colorado where they report the substitution at codon\n20 appeared to have no definitive correlation with CWD status (2, 37).\nThe copy number of the D20G polymorphisms was not discussed in\nthese studies. Notably, our investigation identified five homozygote\nanimals out of a total of 39 carrying this variant. We observed that\ncopy number did not appear to influence CWD disease status as both\npositive and negative samples were discovered within the homozygous\nsamples.\nTo better understand the D20G variant effect on CWD status (2,\n32, 37, 64), we modelled the polymorphism using EmCAST. The mod­\neling predicted that this variant introduces a kink at the C-terminus\nof the helix while leaving the hydrophobic region of the helix mostly\nunperturbed (Figure 3). This variant is not known to affect properties\nsuch as protein–protein interactions in this region, which may explain\ninconsistencies between previous studies.\nOur RT-QuIC data confirmed prion seeding activity was present\nin all of our CWD infected samples expressing the PrP D20G variant\nand was not observed in the CWD negative samples harboring the\np. D20G.\n4.2 R40Q\nAnother novel PRNP variant discovered in this study is at amino\nacid 40 that substitutes an arginine for glutamine and was identified\nin three samples. The PRNP R40Q variant has been reported in Awassi\nsheep, but to date, it has not been reported in mule deer populations\n(49). Due to the small sample size containing this variant, strong con­\nclusions cannot be made. However, it is interesting to note that only\none sample with this variant was identified in 2017 whereas two were\nidentified in 2022. The different samples with this variant were\nobtained from different hunting regions, two in the south (from years\n2017 and 2022) and one in the north (from 2022). While not defini­\ntive, this implies that the variant was not genetically inherited between\n\nDeer with at least one copy of the allele of the common genotype\nexpressing aspartic acid were less likely to test positive in studies con­\nducted in Canada and Nebraska, as opposed to deer sampled in\nWyoming and Colorado where they report the substitution at codon\n20 appeared to have no definitive correlation with CWD status (2, 37).\nThe copy number of the D20G polymorphisms was not discussed in\nthese studies. Notably, our investigation identified five homozygote\nanimals out of a total of 39 carrying this variant. We observed that\ncopy number did not appear to influence CWD disease status as both\npositive and negative samples were discovered within the homozygous\nsamples.\nTo better understand the D20G variant effect on CWD status (2,\n32, 37, 64), we modelled the polymorphism using EmCAST. The mod­\neling predicted that this variant introduces a kink at the C-terminus\nof the helix while leaving the hydrophobic region of the helix mostly\nunperturbed (Figure 3). This variant is not known to affect properties\nsuch as protein–protein interactions in this region, which may explain\ninconsistencies between previous studies.\nOur RT-QuIC data confirmed prion seeding activity was present\nin all of our CWD infected samples expressing the PrP D20G variant\nand was not observed in the CWD negative samples harboring the\np. D20G.\n4.2 R40Q\nAnother novel PRNP variant discovered in this study is at amino\nacid 40 that substitutes an arginine for glutamine and was identified\nin three samples. The PRNP R40Q variant has been reported in Awassi\nsheep, but to date, it has not been reported in mule deer populations\n(49). Due to the small sample size containing this variant, strong con­\nclusions cannot be made. However, it is interesting to note that only\none sample with this variant was identified in 2017 whereas two were\nidentified in 2022. The different samples with this variant were\nobtained from different hunting regions, two in the south (from years\n2017 and 2022) and one in the north (from 2022). While not defini­\ntive, this implies that the variant was not genetically inherited between\n\nThe different samples with this variant were\nobtained from different hunting regions, two in the south (from years\n2017 and 2022) and one in the north (from 2022).While not defini­\ntive, this implies that the variant was not genetically inherited between\nthe animals. Following the prevalence of this variant will provide\ninsight into its relationship with CWD disease susceptibility.\nEmCAST computational predictions placed R40 in a disordered\nloop with only minor perturbations predicted for the R40Q variant.\nThis polymorphism occurs in a disordered region of the protein,\nmaking it more difficult to predict structural impact, and producing\nconflicting results regarding whether the variant renders the protein\nmore or less stable. The R40Q mutation removes a charged residue. It\nis noteworthy that charge patterning can affect the properties of dis­\nordered protein regions in ways that may cause the disordered region\nto be more susceptible to aggregation or conversion (65, 66). With all\nsamples (n = 3) being initially identified as CWD negative by ELISA,\nfurther investigations on impacts of the change in PrP structure in\nrelation to CWD are warranted.\nThe R40Q variant samples were particularly noteworthy pertain­\ning to the prion seeding activity data. One of the samples containing\nthe R40Q variant tested negative for CWD via ELISA however it\nshowed ThT signals with lag phases and amplitudes similar to known\nCWD-positive samples (Table 2; Figure 5; Supplementary Figure S10).\nThese results could be a testament to the highly sensitive nature of the\nRT-QuIC assay and warrant future studies investigating the use of this\nassay in early staging of disease as well as determining if the R40Q\nFIGURE 6\nLinear schematic representation of the cellular prion protein (PrP) highlighting key regions, structures, and where all 25 non-synonymous variants are\nfound. Created in BioRender. https://BioRender.com/tltdstc.\n\nThe different samples with this variant were\nobtained from different hunting regions, two in the south (from years\n2017 and 2022) and one in the north (from 2022).While not defini­\ntive, this implies that the variant was not genetically inherited between\nthe animals. Following the prevalence of this variant will provide\ninsight into its relationship with CWD disease susceptibility.\nEmCAST computational predictions placed R40 in a disordered\nloop with only minor perturbations predicted for the R40Q variant.\nThis polymorphism occurs in a disordered region of the protein,\nmaking it more difficult to predict structural impact, and producing\nconflicting results regarding whether the variant renders the protein\nmore or less stable. The R40Q mutation removes a charged residue. It\nis noteworthy that charge patterning can affect the properties of dis­\nordered protein regions in ways that may cause the disordered region\nto be more susceptible to aggregation or conversion (65, 66). With all\nsamples (n = 3) being initially identified as CWD negative by ELISA,\nfurther investigations on impacts of the change in PrP structure in\nrelation to CWD are warranted.\nThe R40Q variant samples were particularly noteworthy pertain­\ning to the prion seeding activity data. One of the samples containing\nthe R40Q variant tested negative for CWD via ELISA however it\nshowed ThT signals with lag phases and amplitudes similar to known\nCWD-positive samples (Table 2; Figure 5; Supplementary Figure S10).\nThese results could be a testament to the highly sensitive nature of the\nRT-QuIC assay and warrant future studies investigating the use of this\nassay in early staging of disease as well as determining if the R40Q\nFIGURE 6\nLinear schematic representation of the cellular prion protein (PrP) highlighting key regions, structures, and where all 25 non-synonymous variants are\nfound. Created in BioRender. https://BioRender.com/tltdstc.\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n13\nfrontiersin.org\nvariant is associated with subclinical shedding or altered diagnostic\nsensitivity (47, 67–69). The result could also represent a false positive\nor spontaneous seeding activity (70) though spontaneous activity is\nunlikely, as additional negative controls in the RT-QuIC runs\nremained negative. However, without orthogonal confirmation (e.g.,\nIHC on additional tissue or a second RT-QuIC with a different sub­\nstrate), the disease status of this sample remains ambiguous.\n4.3 S225F\nThe S225F variant is well documented and has been reported to\nalter disease phenotypes in mule deer (2, 32, 40). Specifically, in previ­\nous studies, animals expressing the S225F polymorphism have been\nshown to have reduced probability of acquiring CWD and an altered\nphenotype of disease, suggesting increased stability of the PrP folding.\nThis variant was present in our population; however, the prevalence\nwas extremely low. In our Montana deer sample set, only two deer\nwere identified with this p. S225F polymorphism, which is in line with\nprevious studies that report this variant as rare (30), especially when\nthe disease is new to an area. Both deer, one female and one male were\nfound in the 502 districts of Montana during the 2017–2018 season.\nGiven that this variant has been shown to provide CWD resistant\nproperties, further investigations into its characteristics could provide\nhelpful insight into how this variant might influence population level\nCWD dynamics, further informing CWD management. Notably, pre­\nvious investigations into the mechanisms of PrP misfolding due to\nsequence and structural variations in the S225F variant are limited\n(71). However, analysis of S225F by EmCAST predicted that S225F is\na neutral mutation for folded PrP and incompatible with fibril PrP,\nsuggesting a potential structural mechanism for reduced fibril forma­\ntion (2, 32, 40). These preliminary observations would be highly ben­\nefited by further research with a larger data set.\n4.4 WT\nOverall, the seeding activity of the PRNP wild-type samples\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n13\nfrontiersin.org\nvariant is associated with subclinical shedding or altered diagnostic\nsensitivity (47, 67–69). The result could also represent a false positive\nor spontaneous seeding activity (70) though spontaneous activity is\nunlikely, as additional negative controls in the RT-QuIC runs\nremained negative. However, without orthogonal confirmation (e.g.,\nIHC on additional tissue or a second RT-QuIC with a different sub­\nstrate), the disease status of this sample remains ambiguous.\n4.3 S225F\nThe S225F variant is well documented and has been reported to\nalter disease phenotypes in mule deer (2, 32, 40). Specifically, in previ­\nous studies, animals expressing the S225F polymorphism have been\nshown to have reduced probability of acquiring CWD and an altered\nphenotype of disease, suggesting increased stability of the PrP folding.\nThis variant was present in our population; however, the prevalence\nwas extremely low. In our Montana deer sample set, only two deer\nwere identified with this p. S225F polymorphism, which is in line with\nprevious studies that report this variant as rare (30), especially when\nthe disease is new to an area. Both deer, one female and one male were\nfound in the 502 districts of Montana during the 2017–2018 season.\nGiven that this variant has been shown to provide CWD resistant\nproperties, further investigations into its characteristics could provide\nhelpful insight into how this variant might influence population level\nCWD dynamics, further informing CWD management. Notably, pre­\nvious investigations into the mechanisms of PrP misfolding due to\nsequence and structural variations in the S225F variant are limited\n(71). However, analysis of S225F by EmCAST predicted that S225F is\na neutral mutation for folded PrP and incompatible with fibril PrP,\nsuggesting a potential structural mechanism for reduced fibril forma­\ntion (2, 32, 40). These preliminary observations would be highly ben­\nefited by further research with a larger data set.\n4.4 WT\nOverall, the seeding activity of the PRNP wild-type samples\n\nThese preliminary observations would be highly ben­\nefited by further research with a larger data set.4.4 WT\nOverall, the seeding activity of the PRNP wild-type samples\nreflected the CWD status reported by ELISA, however, one of our\nsamples was reported ELISA-negative but had ThT signal via the\nRT-QuIC assay. Most replicates (3/4) of this sample resulted in posi­\ntive ThT signal with lag phase and amplitudes congruent to known\nCWD positive samples. While further testing would be needed to rule\nout spontaneous seeding, assay noise, or false positives, RT-QuIC is\nreported to be a more sensitive test than ELISA in cervid retropharyn­\ngeal lymph nodes (69), and it may have detected a low level of CWD\nthat the ELISA could not. Sample control 8, which was also a WT\nCWD negative sample, did not contain seeding activity.\n5 Conclusion\n•\t The value of studying PRNP diversity in a newly endemic popula­\ntion is three-fold. First, the opportunity to establish a baseline\ndatabase of PRNP variant emergence prior to strong CWD-driven\nselection at newly endemic timepoints is a unique opportunity.\nThis baseline window will narrow as CWD becomes more widely\nestablished and disease-driven selection increasingly shapes\nPRNP allele frequencies. Second, the chance to observe variant\nemergence over time, as CWD epidemiologic factors are still not\ncompletely understood among wild cervid populations. Third, the\nopportunity to identify emerging PRNP polymorphisms which\ncan be studied further in the laboratory to better determine if\nthese substitutions might change disease phenotypes or harbor\nnew prion strains.\n•\t While research is underway for innovative approaches to reduce\nthe spread of CWD through practical applications of improving\ndiagnostics and treatments, disease management tools are cur­\nrently limited (30, 72). Development of genetic databases such as\nours provide wildlife managers with information which may be\nutilized in CWD management decisions, potentially making a\nbigger impact in newly endemic regions with lower prevalences\nthan in regions with higher disease prevalence numbers.\n\nThese preliminary observations would be highly ben­\nefited by further research with a larger data set.4.4 WT\nOverall, the seeding activity of the PRNP wild-type samples\nreflected the CWD status reported by ELISA, however, one of our\nsamples was reported ELISA-negative but had ThT signal via the\nRT-QuIC assay. Most replicates (3/4) of this sample resulted in posi­\ntive ThT signal with lag phase and amplitudes congruent to known\nCWD positive samples. While further testing would be needed to rule\nout spontaneous seeding, assay noise, or false positives, RT-QuIC is\nreported to be a more sensitive test than ELISA in cervid retropharyn­\ngeal lymph nodes (69), and it may have detected a low level of CWD\nthat the ELISA could not. Sample control 8, which was also a WT\nCWD negative sample, did not contain seeding activity.\n5 Conclusion\n•\t The value of studying PRNP diversity in a newly endemic popula­\ntion is three-fold. First, the opportunity to establish a baseline\ndatabase of PRNP variant emergence prior to strong CWD-driven\nselection at newly endemic timepoints is a unique opportunity.\nThis baseline window will narrow as CWD becomes more widely\nestablished and disease-driven selection increasingly shapes\nPRNP allele frequencies. Second, the chance to observe variant\nemergence over time, as CWD epidemiologic factors are still not\ncompletely understood among wild cervid populations. Third, the\nopportunity to identify emerging PRNP polymorphisms which\ncan be studied further in the laboratory to better determine if\nthese substitutions might change disease phenotypes or harbor\nnew prion strains.\n•\t While research is underway for innovative approaches to reduce\nthe spread of CWD through practical applications of improving\ndiagnostics and treatments, disease management tools are cur­\nrently limited (30, 72). Development of genetic databases such as\nours provide wildlife managers with information which may be\nutilized in CWD management decisions, potentially making a\nbigger impact in newly endemic regions with lower prevalences\nthan in regions with higher disease prevalence numbers.\n\nIdentifying novel and previously described PRNP variants may\nsupport\nmore\ntargeted\nCWD\nmanagement\ndecisions.\nIncorporating PRNP sequencing as an additional tool could help\nprioritize areas for CWD surveillance sampling. Because PRNP\npolymorphisms have been shown to alter disease susceptibility\nand modify phenotypes in cervid populations (72), knowledge of\nlocation and prevalence of these variants alongside other epide­\nmiologic factors may also improve predictions of CWD trajecto­\nries and be incorporated into wildlife management strategies.\nOur study contributes valuable information on PRNP variants\nidentified in wild mule deer, the timing and geographical location of\nthe variants, and the effects on prion protein folding and seeding\nactivity. In addition to the identification of 36 polymorphisms, this\nstudy highlights the need for further investigations into RT-QuIC\ndetection of early stages of disease. Both of these findings have the\npotential to greatly influence and expand the current understand of\nCWD nationwide as well as provides relevant and valuable context for\nmanagement decisions in Montana.\nData availability statement\nThe genetic sequence data can be found in the GenBank reposi­\ntory by the listed accession numbers found in Supplementary Table S3.\nAll other data in the study are available from the corresponding\nauthor, AG, upon reasonable request.\nEthics statement\nThe animal study was approved by Weissman Hood Institute at\nTouro University; McLaughlin Research Institutional Animal Care\nand Use Committee under protocol number 2023-AG-100 and bio­\nsafety protocol number 2022-AG-IBC5. The study was conducted in\naccordance with the local legislation and institutional requirements.\nAuthor contributions\nAS: Conceptualization, Data curation, Formal analysis,\nInvestigation, Methodology, Project administration, Supervision,\nValidation, Visualization, Writing – original draft, Writing – review &\n\nIdentifying novel and previously described PRNP variants may\nsupport\nmore\ntargeted\nCWD\nmanagement\ndecisions.\nIncorporating PRNP sequencing as an additional tool could help\nprioritize areas for CWD surveillance sampling. Because PRNP\npolymorphisms have been shown to alter disease susceptibility\nand modify phenotypes in cervid populations (72), knowledge of\nlocation and prevalence of these variants alongside other epide­\nmiologic factors may also improve predictions of CWD trajecto­\nries and be incorporated into wildlife management strategies.\nOur study contributes valuable information on PRNP variants\nidentified in wild mule deer, the timing and geographical location of\nthe variants, and the effects on prion protein folding and seeding\nactivity. In addition to the identification of 36 polymorphisms, this\nstudy highlights the need for further investigations into RT-QuIC\ndetection of early stages of disease. Both of these findings have the\npotential to greatly influence and expand the current understand of\nCWD nationwide as well as provides relevant and valuable context for\nmanagement decisions in Montana.\nData availability statement\nThe genetic sequence data can be found in the GenBank reposi­\ntory by the listed accession numbers found in Supplementary Table S3.\nAll other data in the study are available from the corresponding\nauthor, AG, upon reasonable request.\nEthics statement\nThe animal study was approved by Weissman Hood Institute at\nTouro University; McLaughlin Research Institutional Animal Care\nand Use Committee under protocol number 2023-AG-100 and bio­\nsafety protocol number 2022-AG-IBC5. The study was conducted in\naccordance with the local legislation and institutional requirements.\nAuthor contributions\nAS: Conceptualization, Data curation, Formal analysis,\nInvestigation, Methodology, Project administration, Supervision,\nValidation, Visualization, Writing – original draft, Writing – review &\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n14\nfrontiersin.org\nediting. MR: Formal analysis, Investigation, Writing – original draft,\nWriting – review & editing. BG: Conceptualization, Investigation,\nWriting – original draft, Writing – review & editing. MS: Formal\nanalysis, Investigation, Writing – review & editing. BM: Formal analy­\nsis, Writing – review & editing. JP: Conceptualization, Data curation,\nInvestigation, Methodology, Project administration, Resources,\nValidation, Writing – review & editing. BB: Formal analysis, Funding\nacquisition, Investigation, Resources, Software, Writing – original\ndraft, Writing – review & editing. ML: Conceptualization, Formal\nanalysis, Investigation, Resources, Validation, Visualization, Writing –\noriginal draft, Writing – review & editing. AG: Conceptualization,\nData curation, Formal analysis, Funding acquisition, Investigation,\nMethodology, Project administration, Resources, Supervision,\nValidation, Visualization, Writing – original draft, Writing – review &\nediting.\nFunding\nThe author(s) declared that financial support was received for this\nwork and/or its publication. Research reported in this publication was\nsupported by the National Institute of General Medical Sciences of the\nNational Institutes of Health under Award Number P20GM152335,\nthe Weissman Hood Institute; McLaughlin Research Institute institu­\ntional start-up funding, and the Jorgensen Foundation (AG).\nEmCAST analysis was supported by the National Institute of General\nMedical Sciences of the National Institutes of Health under Award\nNumber R01GM148610 (BB)\nAcknowledgments\nThe authors would like to acknowledge Montana Fish, Wildlife and\nParks for providing the tissues and associated demographic and disease\ndata. We thank Emily Almberg, Bevin McCormick, and Austin\nWieseler for discussions regarding the manuscript content and Sam\nTreece and Austin Wieseler for assistance with sample organization and\nacquisition. We would like to thank Renee Reijo Pera and the COBRE\nadministrative core for thoughtful discussions regarding this manu­\n\nSeerley et al.\b\n10.3389/fvets.2026.1861004\nFrontiers in Veterinary Science\n14\nfrontiersin.org\nediting. MR: Formal analysis, Investigation, Writing – original draft,\nWriting – review & editing. BG: Conceptualization, Investigation,\nWriting – original draft, Writing – review & editing. MS: Formal\nanalysis, Investigation, Writing – review & editing. BM: Formal analy­\nsis, Writing – review & editing. JP: Conceptualization, Data curation,\nInvestigation, Methodology, Project administration, Resources,\nValidation, Writing – review & editing. BB: Formal analysis, Funding\nacquisition, Investigation, Resources, Software, Writing – original\ndraft, Writing – review & editing. ML: Conceptualization, Formal\nanalysis, Investigation, Resources, Validation, Visualization, Writing –\noriginal draft, Writing – review & editing. AG: Conceptualization,\nData curation, Formal analysis, Funding acquisition, Investigation,\nMethodology, Project administration, Resources, Supervision,\nValidation, Visualization, Writing – original draft, Writing – review &\nediting.\nFunding\nThe author(s) declared that financial support was received for this\nwork and/or its publication. Research reported in this publication was\nsupported by the National Institute of General Medical Sciences of the\nNational Institutes of Health under Award Number P20GM152335,\nthe Weissman Hood Institute; McLaughlin Research Institute institu­\ntional start-up funding, and the Jorgensen Foundation (AG).\nEmCAST analysis was supported by the National Institute of General\nMedical Sciences of the National Institutes of Health under Award\nNumber R01GM148610 (BB)\nAcknowledgments\nThe authors would like to acknowledge Montana Fish, Wildlife and\nParks for providing the tissues and associated demographic and disease\ndata. We thank Emily Almberg, Bevin McCormick, and Austin\nWieseler for discussions regarding the manuscript content and Sam\nTreece and Austin Wieseler for assistance with sample organization and\nacquisition. We would like to thank Renee Reijo Pera and the COBRE\nadministrative core for thoughtful discussions regarding this manu­\n\nWe would like to thank Renee Reijo Pera and the COBRE\nadministrative core for thoughtful discussions regarding this manu­\nscript. We would thank Teresa Gunn and the McLaughlin Research\nInstitute Gene Editing and Mouse Models Assessment (GEMMA) Core\nFacility within the Center for Integrated Biomedical and Rural Health\nResearch, RRID: SCR_027045, 1P20GM152335 for sequencing analysis\ntraining and conversations about our investigations on the relationship\nbetween PRNP genetics and PrP protein misfolding. Additionally,\nthanks to Macie Frans for RT-QuIC figure creation. We also thank\nBrent Race for scientific mentoring and discussions regarding CWD\nand PRNP polymorphisms. This manuscript is dedicated to Tim\nWylder, our good friend who believed in this study. Our time together\nspent discussing this project and other topics will be profoundly missed.\nConflict of interest\nThe author(s) declared that this work was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declared that Generative AI was not used in the\ncreation of this manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. Any product\nthat may be evaluated in this article, or claim that may be made by its\nmanufacturer, is not guaranteed or endorsed by the publisher.\nSupplementary material\nThe Supplementary material for this article can be found online\nat: https://www.frontiersin.org/articles/10.3389/fvets.2026.1861004/\nfull#supplementary-material\nReferences\n1.\tCook M, Hensley-McBain T, Grindeland A. Mouse models of chronic wasting disease:\n\nWe would like to thank Renee Reijo Pera and the COBRE\nadministrative core for thoughtful discussions regarding this manu­\nscript. We would thank Teresa Gunn and the McLaughlin Research\nInstitute Gene Editing and Mouse Models Assessment (GEMMA) Core\nFacility within the Center for Integrated Biomedical and Rural Health\nResearch, RRID: SCR_027045, 1P20GM152335 for sequencing analysis\ntraining and conversations about our investigations on the relationship\nbetween PRNP genetics and PrP protein misfolding. Additionally,\nthanks to Macie Frans for RT-QuIC figure creation. We also thank\nBrent Race for scientific mentoring and discussions regarding CWD\nand PRNP polymorphisms. This manuscript is dedicated to Tim\nWylder, our good friend who believed in this study. Our time together\nspent discussing this project and other topics will be profoundly missed.\nConflict of interest\nThe author(s) declared that this work was conducted in the\nabsence of any commercial or financial relationships that could be\nconstrued as a potential conflict of interest.\nGenerative AI statement\nThe author(s) declared that Generative AI was not used in the\ncreation of this manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. Any product\nthat may be evaluated in this article, or claim that may be made by its\nmanufacturer, is not guaranteed or endorsed by the publisher.\nSupplementary material\nThe Supplementary material for this article can be found online\nat: https://www.frontiersin.org/articles/10.3389/fvets.2026.1861004/\nfull#supplementary-material\nReferences\n1.\tCook M, Hensley-McBain T, Grindeland A. Mouse models of chronic wasting disease:\n\nfrontiersin.org/articles/10.3389/fvets.2026.1861004/\nfull#supplementary-material\nReferences\n1.Cook M, Hensley-McBain T, Grindeland A.Mouse models of chronic wasting disease:\na review. Front Virol. (2023) 3:1055487. doi: 10.3389/fviro.2023.1055487\n2.\tMoazami-Goudarzi K, Andréoletti O, Vilotte JL, Béringue V. Review on PRNP genetics\nand susceptibility to chronic wasting disease of Cervidae. Vet Res. (2021) 52:128. doi:\n10.1186/s13567-021-00993-z\n3.\tBonda DJ, Manjila S, Mehndiratta P, Khan F, Miller BR, Onwuzulike K, et al. 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Prion protein: the molecule of many forms and faces. Int J\nMol Sci. (2022) 23:1232. doi: 10.3390/ijms23031232\n10.\tAbskharon R, Wang F, Wohlkonig A, Ruan J, Soror S, Giachin G, et al. Structural\nevidence for the critical role of the prion protein hydrophobic region in forming an\ninfectious prion. PLoS Pathog. (2019) 15:e1008139. doi: 10.1371/journal.ppat.1008139\n\nfrontiersin.org/articles/10.3389/fvets.2026.1861004/\nfull#supplementary-material\nReferences\n1.Cook M, Hensley-McBain T, Grindeland A.Mouse models of chronic wasting disease:\na review. Front Virol. (2023) 3:1055487. doi: 10.3389/fviro.2023.1055487\n2.\tMoazami-Goudarzi K, Andréoletti O, Vilotte JL, Béringue V. Review on PRNP genetics\nand susceptibility to chronic wasting disease of Cervidae. Vet Res. (2021) 52:128. doi:\n10.1186/s13567-021-00993-z\n3.\tBonda DJ, Manjila S, Mehndiratta P, Khan F, Miller BR, Onwuzulike K, et al. Human\nprion diseases: surgical lessons learned from iatrogenic prion transmission. Neurosurg\nFocus. (2016) 41:E10. doi: 10.3171/2016.5.FOCUS15126\n4.\tNafe R, Arendt CT, Hattingen E. Human prion diseases and the prion protein—what\nis the current state of knowledge? Transl Neurosci. (2023) 14:20220315. doi: 10.1515/\ntnsci-2022-0315\n5.\tPiñar-Morales R, Barrero-Hernández F, Aliaga-Martínez L. Human prion diseases: an\noverview. Med Clin (Barc). (2023) 160:554–60. doi: 10.1016/j.medcli.2023.03.001\n6.\tPrusiner SB. Human prion diseases and neurodegeneration. Curr Top Microbiol\nImmunol. (1996) 207:1–17. doi: 10.1007/978-3-642-60983-1_1\n7.\tPrusiner SB. Prions. Proc Natl Acad Sci USA. (1998) 95:13363–83. doi: 10.1073/\npnas.95.23.13363\n8.\tArifin MI, Hannaoui S, Chang SC, Thapa S, Schatzl HM, Gilch S. Cervid prion protein\npolymorphisms: role in chronic wasting disease pathogenesis. Int J Mol Sci. (2021)\n22:2271. doi: 10.3390/ijms22052271\n9.\tKovač V, Čurin Šerbec V. Prion protein: the molecule of many forms and faces. Int J\nMol Sci. 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+{"text": "【兽医文献】\n标题: Comparative evaluation of point-of-care blood gas analysis in rabbit disease models\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nMNQ derivative D19 alleviates\nLPS-induced inflammation and\noxidative stress in sheep follicular\ngranulosa cells through the\nGPX4-mediated ferroptosis\nChunlu Chen 1, Jia Lu 2, Yuhan Qu 1, Jianhua Dong 1, Yihao Hong 1,\nYongping Ren 1, Shouqing Jiang 1, Rafael Baptista 3, Dong Wang 4,\nLuis Aj Mur 5 and Lihua Lyu 1*\n1 College of Animal Science, Shanxi Agricultural University, Jinzhong, China, 2 College of Software,\nShanxi Agricultural University, Jinzhong, China, 3 Institute of Biological, Environmental and Rural\nSciences, Penglais Campus, Aberystwyth University, Aberystwyth, Wales, United Kingdom, 4 Institute of\nAnimal Science, Chinese Academy of Agricultural Sciences, Beijing, China, 5 Department of Biology\nand Health, Aberystwyth University, Aberystwyth, United Kingdom\nIntroduction: 2-methoxy-1,4-naphthoquinone (MNQ), a compound derived\nfrom Impatiens balsamina L., is recognized for its anti-inflammatory and\nantioxidant properties. However, the effects of D19, a derivative of MNQ, remain\nunexplored. This study aimed to elucidate the protective effect of D19 against\nlipopolysaccharide (LPS)-induced follicular granulosa cells (GCs) dysfunction in\nsheep and its underlying molecular mechanisms.\nMethods: An in  vitro model of GCs injury was established using LPS to\ninduce inflammation and oxidative stress. The effects of D19 were evaluated\nby examining inflammatory response, oxidative stress, ferroptosis and\nsteroidogenesis following treatment. Gene interference was applied to knock\ndown GPX4 expression to validate its role in the protective mechanism of D19.\nResults: D19 attenuated LPS-induced ferroptosis in GCs by restoring the\nexpression of the key ferroptosis regulator GPX4. Subsequently, interfering\nwith GPX4 activated NF-κB and upregulated the expression of inflammatory\nfactors (TNF-α, IL-1β, IL-6) while disrupting NRF2 and inhibiting the expression\nof antioxidant-related factors (CAT, GSH-PX, SOD2). D19 effectively protected\nGCs from GPX4 deficiency-induced inflammation and oxidative damage.\n\nD19 effectively protected\nGCs from GPX4 deficiency-induced inflammation and oxidative damage.\nFurthermore, D19 mitigated ferroptosis caused by GPX4 deficiency and\nmaintained iron metabolic homeostasis by restoring the morphology of GCs,\nincreasing mitochondrial membrane potential, decreasing the accumulation\nof Fe2+ and lipid peroxides, and promoting the expression of GPX4 and FTH1.\nD19 also improved steroid hormone secretion abnormalities caused by GPX4\ndeficiency.\nDiscussion: These results demonstrate that D19 protects sheep follicular GCs\nfrom LPS-induced damage by modulating the GPX4-mediated ferroptosis\nsignaling pathway, providing new potential drugs and therapeutic targets for\naddressing GCs dysfunction and follicular developmental abnormalities.\nKEYWORDS\nlipopolysaccharide, granulosa cells, inflammation, oxidative stress, ferroptosis\nOPEN ACCESS\nEDITED BY\nAmal M. Aboelmaaty,\nNational Research Centre, Egypt\nREVIEWED BY\nSherif Dessouki,\nCairo University, Egypt\nVenância Antônia Nunes Azevedo,\nFederal University of Ceará, Brazil\n*CORRESPONDENCE\nLihua Lyu\nlihualvsxau@126.com\nRECEIVED 01 May 2025\nACCEPTED 18 August 2025\nPUBLISHED 01 October 2025\nCITATION\nChen C, Lu J, Qu Y, Dong J, Hong Y, Ren Y,\nJiang S, Baptista R, Wang D, Mur LA and\nLyu L (2025) MNQ derivative D19 alleviates\nLPS-induced inflammation and oxidative\nstress in sheep follicular granulosa cells\nthrough the GPX4-mediated ferroptosis.\nFront. Vet. Sci. 12:1621738.\ndoi: 10.3389/fvets.2025.1621738\nCOPYRIGHT\n© 2025 Chen, Lu, Qu, Dong, Hong, Ren,\nJiang, Baptista, Wang, Mur and Lyu. This is an\nopen-access article distributed under the\nterms of the Creative Commons Attribution\nLicense (CC BY). The use, distribution or\nreproduction in other forums is permitted,\nprovided the original author(s) and the\ncopyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\naccordance with accepted academic\npractice. No use, distribution or reproduction\nis permitted which does not comply with\nthese terms.\nTYPE  Original Research\nPUBLISHED  01 October 2025\nDOI  10.3389/fvets.2025.1621738\n\nChen et al.\b\n10.3389/fvets.2025.1621738\nFrontiers in Veterinary Science\n02\nfrontiersin.org\n1 Introduction\nThe global farming industry has historically prioritized enhancing\nproductivity. However, a significant contemporary challenge\nconfronting this sector is the prevalence of bacterial contamination,\nparticularly affecting the reproductive system. Around 90% of animals\ndevelop uterine bacterial infections after parturition, leading to\nfertility decline, ovarian dysfunction, slower follicle development, and\nimpaired steroidogenesis (1). Lipopolysaccharide (LPS), a significant\ncomponent of Gram-negative bacteria, is a source of pathogenicity\ndue to its release in large quantities during bacterial death or lysis and\nhas been shown to cause inflammation, oxidative stress, and\ndisruption of testosterone secretion in testicular macrophages of sheep\n(2). Pigs, mice, and humans exposed to bacterial contamination have\nshown higher concentrations of LPS in serum and follicular fluid (3).\nThe causative agent of polycystic ovarian syndrome has also been\nfound to be closely related to abnormal LPS levels (4).\nAs an essential component of the follicle, follicular granulosa cells\n(GCs) are responsible for providing nutrients to the oocyte and regulating\nsteroid hormone synthesis, and their abnormal function is a major factor\nleading to follicular atresia. Extant data substantiate that LPS instigates\naberrant expression of inflammatory factors, mitochondrial dysfunction,\noxidative damage, and steroid-related hormone secretion disorders in\nfollicular GCs, profoundly impacting the normal development of follicles\n(5). Consequently, based on the above research background, it is\nimperative to investigate the precise mechanism of LPS to develop\neffective therapeutic strategies for treating follicular atresia.\nFerroptosis is a unique mode of cell death, the initiation mechanism\nof which involves intracellular iron overload, ROS production, lipid\nperoxidation processes, mitochondrial membrane densification\naccompanied by volume reduction, rupture of the outer membrane, and\n\nreduction or disappearance of mitochondrial cristae, accompanied by a\nsignificant decrease in the content of reduced glutathione (GSH) and loss\nof the activity of glutathione peroxidase 4 (GPX4), which is significantly\ndifferent from the traditional modes of cell death such as autophagy,\napoptosis, and necroptosis. The GPX family is widely present in mammals,\nbut its member, GPX4, is an indispensable antioxidant enzyme due to its\nunique amino acid sequence and spatial structure (6) and has been\ndemonstrated to be an important target for the treatment of ferroptosis\n(7). GPX4 deficiency compromises the antioxidant system in GCs, leading\nto intracellular oxidative stress, ferroptosis induction, and disruption of\nsteroid hormone synthesis and secretion, ultimately resulting in abnormal\novulation (8, 9). The proteomic analysis revealed that LPS induced\nferroptosis through the NRF2/GPX4 axis, accompanied by oxidative\nstress and inflammatory responses (10). Ferroptosis-mediated oxidative\nstress-inflammatory response, as evidenced by the down-regulation of\nNRF2/GPX4 and the up-regulation of NF-KB, has been identified in\nyoung rats presenting with premature ovarian failure (11). LPS inhibited\nthe expression of GPX4, which activated NF-kB signaling and promoted\nthe release of inflammation-associated factors IL-1β, IL-6, and TNF-α, as\nwell as a large accumulation of intracellular ROS levels, whereas the\nintracellular GSH content was noticeably downregulated (12). In light of\nthese findings, we  hypothesize that LPS-induced inflammation and\noxidative stress may occur through the GPX4-mediated ferroptosis\npathway, severely limiting the reproductive efficiency of the animals.\nTherefore, there is an urgent need to find novel therapeutic interventions\nto address this issue.\nSince the long-term use of conventional antibiotics can lead to\nproblems such as drug resistance and drug residues within livestock\nproducts, more and more scholars are focusing on highly effective and\nlow-toxicity natural herbal treatments. Impatiens balsamina L. contains a\n\nImpatiens balsamina L.contains a\nkey active component, 2-methoxy-1,4-naphthoquinone (MNQ), which\nhas been scientifically proven to possess a variety of bioactive properties,\nincluding antipruritic, anti-inflammatory, antimicrobial, anticancer, and\nantiallergenic effects (13). Available studies have shown that MNQ\npossesses potent antibacterial properties, effective against both Gram-\npositive (Staphylococcus aureus) and Gram-negative (Escherichia coli)\nbacteria, as well as Helicobacter pylori and several fungal species, such as\nPenicillium and Fusarium (14, 15). Treatment with MNQ promoted cell\ncycle progression from S phase to G2/M phase in olfactory ensheathing\ncells (OECs), stimulated mitotic division, enhanced proliferative capacity,\nand activated the NRF2-mediated antioxidant defense system (16).\nPrevious studies have shown that MNQ mediates the TNF signaling\npathway to alleviate inflammation and functional impairment in bovine\nfollicular GCs and regulates steroid hormone synthesis (17).\nBased on the chemical structure of MNQ, we synthesized its\nderivative, D19, a compound whose functions have never been\nreported (Figure 1). Our team’s proteomic sequencing data suggests\nthat the GPX4-mediated ferroptosis signaling pathway plays a key role\nin MNQ alleviation of LPS-induced functional impairment of GCs.\nTherefore, the present study aimed to explore whether GPX4-regulated\nferroptosis is the underlying mechanism by which D19 exerts its\nprotective effects against LPS-induced inflammation, oxidative stress,\nand steroid hormone synthesis disorders.\nFIGURE 1\nMolecular structures of MNQ and its derivative D19.\n\nChen et al.\b\n10.3389/fvets.2025.1621738\nFrontiers in Veterinary Science\n03\nfrontiersin.org\n2 Materials and methods\n2.1 Ovary collection and GCs cultivation\nIn this experiment, all sheep were grown to sexual maturity on the\nfarm and transported to the local slaughterhouse for slaughter\n(Jinzhong, Shanxi, China). Meanwhile, we collected the ovaries on-site\nand preserved them in DPBS, sterilized at 4 °C with 100 IU/mL of\npenicillin and 100 mg/mL of streptomycin (Solarbio, Beijing, China),\nand brought them back to the laboratory. The ovaries were sterilized\nwith 75% alcohol, and follicles with a diameter of 3–5 mm were\nselected to remove the GCs for in vitro culture. Specific culturing\nmethods were carried out as previously described (18). MNQ was\nisolated from the stems and leaves of Impatiens balsamina L., and its\nderivative D19 was synthesized based on the molecular structure of\nMNQ following previously reported methods (19). The structure of\nD19 was characterized by 1H nuclear magnetic resonance (1H NMR,\n400 MHz, DMSO-d6), with characteristic peaks observed at δ 8.53 (s,\n1H), 7.97–7.89 (m, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.73–7.68 (m, 1H),\n7.57 (t, J = 7.5 Hz, 1H), 6.71 (dd, J₁ = 14.7 Hz, J₂ = 3.3 Hz, 1H), 6.58–\n6.44 (m, 2H), 4.28 (s, 1H), 4.20–4.00 (m, 3H), and 3.95 (s, 1H).\n2.2 Test groups for GCs\nGCs were evenly suspended in a complete medium [89% DMEM-F12\n(BOSTER, Wuhan, China), 10% fetal bovine serum (Cellmax, Beijing,\nChina), and 1% penicillin–streptomycin (BOSTER, Wuhan, China)],\ninoculated in cell culture plates of different sizes and cultured in a 37 °C\nand 5% CO2 humidified incubator. The inoculum densities of GCs in\n96-well and 6-well plates were 1 × 104 cells/well and 1 × 106 cells/well,\nrespectively (20). GCs were treated with D19 (prepared as a 10 mM stock\nsolution in DMSO; final DMSO concentration <0.1%) at concentrations\nof 0, 0.01, 0.1, 1, 10, 100, and 200 μM. Control groups (CK) received\nequivalent DMSO concentrations without D19. All groups were incubated\nfor 24 h and 48 h, respectively (17, 21). GCs were incubated with different\n\n1%) at concentrations\nof 0, 0.01, 0.1, 1, 10, 100, and 200 μM.Control groups (CK) received\nequivalent DMSO concentrations without D19.All groups were incubated\nfor 24 h and 48 h, respectively (17, 21).GCs were incubated with different\nconcentrations of LPS (0, 10, 25, 50, 100, 200, 400 μg/mL) (Solarbio,\nBeijing, China) for 12 h and 24 h (22). Control siRNA (si-NC: sense,\n5′-UUCUCCGAACGUGUCACGUTT-3′; antisense, 5′-ACGUGACAC\nGUUCGGAGAATT-3′) and GPX4-targeting siRNA (si-GPX4: sense,\n5′-AAGAGUUCGCUGCUGGCUA-3′; antisense, 5′-UAGCCAGCA\nGCGAACUCUU-3′) were synthesized by Shanghai Sangon Biotech. GCs\nwere transfected with 20 μM of either si-NC or si-GPX4 using an RNA\ntransfection reagent (Sangon, Shanghai, China). Transfection was\nperformed for 12, 24, 36, or 48 h. The experimental groups included: (1)\nCK, (2) LPS (cells treated with LPS for 12 h), (3) LPS + D19 (cells treated\nwith LPS for 12 h, followed by D19 treatment for an additional 24 h), (4)\nsi-NC (cells transfected with control siRNA for 36 h), (5) si-GPX4 (cells\ntransfected with GPX4-targeting siRNA for 36 h), and (6) si-GPX4 + D19\n(0.1 μM) (cells transfected with GPX4-targeting siRNA for 12 h, followed\nby D19 treatment for an additional 24 h).\n2.3 Immunofluorescence\nThe immunofluorescence experiments were performed as\npreviously described (23). Anti-FSHR (Bioworld, Minnesota,\nUnited States) and anti-GPX4 (Sangon Biotech, Shanghai, China)\nwere used as primary antibodies, and goat anti-rabbit IgG (BOSTER,\nWuhan, China) was used as the secondary antibody. The negative\ncontrol group was incubated with PBS instead of the primary antibody.\nSpecifically, GCs were inoculated into six-well plates, and the medium\nwas removed and replaced with 4% paraformaldehyde fixation when\nthe cells had fused to 60–70%, and GCs were permeabilized with 1%\nTritonX-100 (Solarbio, Beijing, China) after washing with PBS,\nfollowed by sealing with 1% BSA (Solarbio, Beijing, China). GCs were\nincubated with anti-FSHR and anti-GPX4 antibodies overnight at\n4 °C. Hoechst 33342 was used to stain the nuclei of the cells, and the\n\nGCs were\nincubated with anti-FSHR and anti-GPX4 antibodies overnight at\n4 °C.Hoechst 33342 was used to stain the nuclei of the cells, and the\nantigen–antibody conjugate reaction was performed with anti-rabbit\nIgG, and then placed under a fluorescence microscope to observe the\nspecific protein expression.\n2.4 MTT analysis\nThe toxic effects of different concentrations of D19 and LPS, as\nwell as si-GPX4 on the cells at different time intervals, were performed\nin strict accordance with the instructions of the MTT kit (Solarbio,\nBeijing, China). GCs viability of each experimental group was\ncalculated using the OD detected at 490 nm.\n2.5 SA-β-galactosidase staining analysis\nThe senescence of GCs in each treatment group was detected\naccording to the instructions of the SA-β-galactosidase (SA-β-Gal) staining\nkit (Beyotime, Shanghai, China). Specifically, GCs were removed from the\nculture and added to β-galactosidase staining fixative, fixed at room\ntemperature for 15 min, washed with PBS, and then the prepared working\nsolution (1% β-galactosidase staining solution A, 1% β-galactosidase\nstaining solution B, 93% β-galactosidase staining solution C, and 5% X-Gal\nsolution) was incubated overnight at 37 °C. The senescent cells were\nvisualized under a light microscope to produce the Dark blue color results.\n2.6 Glucose and LDH assays\nGlucose content and LDH activity in treated GCs were assayed in\nstrict accordance with the Glucose kit (Njjcbio, Nanjing, China) and\nLactate dehydrogenase assay kit (Njjcbio, Nanjing, China).\n2.7 Transmission electron microscopy\nThe experimental procedure was performed as previously\ndescribed (24). Briefly, the treated GCs were pre-fixed with 3%\nglutaraldehyde for about 24 h and 1% osmium tetroxide for another\n2 h; dehydrated and osmotically embedded according to the\nconcentration gradient, and then stained after ultrathin sectioning\nusing an ultrathin sectioning machine, and finally the images were\ncaptured and analyzed in the electron microscope.\n2.8 Mitochondrial membrane potential\nassay\nCultured GCs (treated) complete medium was replaced with 2 μM\n\n2.8 Mitochondrial membrane potential\nassay\nCultured GCs (treated) complete medium was replaced with 2 μM\nJC-1 (MCE, Shanghai, China) and continued to be cultured in the\n\nChen et al.\b\n10.3389/fvets.2025.1621738\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nmedium for 15–20 min, washed with PBS, and then the staining\nresults were observed by fluorescence microscopy (green fluorescence:\nEx/Em = 510/527 nm; red fluorescence: Ex/Em = 585/590 nm).\n2.9 Measurement of androstenedione,\nestradiol, and progesterone, TNF-α, and\nIL-1β using ELISA\nThe levels of steroid hormones (A4, E2, and P4) and inflammatory\nfactors (TNF-β, IL-1β) in the treated GCs were measured according to\nthe instructions of the ELISA kit (MEIMIAN, Jiangsu, China). Specific\nexperimental steps were performed as previously described (25). Briefly,\nthe diluted GCs supernatant was labeled with horseradish peroxidase\n(HRP), incubated for 1 h at a constant temperature of 37 °C, washed\nfive times with washing solution, and then reacted with substrates A\nand B simultaneously for 15 min. The reaction was terminated by\nadding a termination solution. Then, the OD value at 450 nm was\nmeasured in each well, and the final concentration was calculated\naccording to the standard curve. It is worth noting that the procedure\nwas the same for all five assays, all concentrations were normalized to\n2.5 × 106 cells, and all experiments were repeated at least three times.\n2.10 Antioxidant assays\nThe antioxidant capacity of GCs in different treatment groups\nwas detected by using Nanjing Jianjian Bioengineering Institute\nkits (including SOD, GSH, GSH-PX, MDA, total antioxidant\ncapacity, CAT, and ROS) and Lipid Peroxidation Assay Kit\n(BODIPY 581/591 C11) (Beyotime, Shanghai, China) and operated\naccording to the specifications. All experiments were repeated at\nleast three times.\n2.11 Ferrous ion (Fe2+) detection\nA ferrous ion content assay kit (Solarbio, Beijing, China) was used\nto measure Fe2+ levels in GCs after adding different treatments. The OD\nvalue at 593 nm was substituted into the standard curve to calculate the\nfinal results, and each set of experiments was repeated at least three times.\n2.12 qRT-PCR analysis\nTotal RNA was extracted from GCs using the Mei5bio RNA\nextraction kit (Mei5bio, Beijing, China), and cDNA synthesis was\n\n2.12 qRT-PCR analysis\nTotal RNA was extracted from GCs using the Mei5bio RNA\nextraction kit (Mei5bio, Beijing, China), and cDNA synthesis was\nperformed according to the protocol provided with the PrimeScript\nRT Reagent Kit (Takara, Tokyo, Japan). Quantitative real-time PCR\n(qRT-PCR) was conducted on a Bio-Rad CFX instrument using the\nTransGen PerfectStart® Green qPCR SuperMix kit (TransGen, Beijing,\nChina), following the manufacturer’s instructions. Primer sequences\nwere designed and synthesized by Shanghai Sangon Biotech (see\nTable  1 for details). The qRT-PCR protocol included an initial\ndenaturation step at 95 °C for 60 s, followed by 40 cycles of\namplification (95 °C for 30 s, 95 °C for 5 s, and annealing at the\nprimer-specific Tm for 30 s) (25). β-actin was used as an internal\ncontrol, and each experiment was performed in triplicate to ensure\nreproducibility. Relative gene expression levels were calculated using\nthe 2−ΔΔCt method.\n2.13 Western blotting analysis\nTotal GC proteins were extracted with ready-to-use lysate (98%\nRIPA, 1% PMSF, 1% broad-spectrum phosphorylated protease\ninhibitor), centrifuged, and added to the protein sample buffer at a\nratio of 4:1. After SDS-PAGE electrophoresis (80 V for 40 min, 120 V\nfor 90 min), the proteins were transferred to NC membranes (120 V,\nice bath for 90 min). The membrane was blocked with skimmed milk\npowder for 1 h, the primary antibody was incubated overnight at 4 °C,\nand the secondary antibody (LI-COR, Lincoln, NE, United States) was\nincubated at room temperature and protected from light for 1 h. The\nabove reagents were purchased from BioTech. Protein bands were\nimaged using the Odyssey Infrared Imaging System and ImageJ to\nanalyze relative changes. β-actin was used as an internal reference\nprotein. Antibody information is shown in Table 2.\nTABLE 1  Primer sequences for real-time PCR.\nGene\nSequence (5′→3′)\nProduct\nsize (bp)\nIL-6\nF: CAATCTGGGTTCAATCAGGCGA\nR: TGCTCTGCAACTCCATGACAG\n130\nIL-1β\nF: CGTCTTCCTGGGACGTTTTAG\nR: CTGCGTATGGCTTCTTTAGGG\n85\nTNF-α\nF: GTAGCCCACGTTGTAGCCAA\nR: TGAGGTAAAGCCCGTCAGTG\n136\nNF-KB\nF: CTCCTGGAGCCTCAAACCTG\nR: TCTACAGGGAAAACTGAATCTTTCT\n121\nSOD2\n\nF: GTGGAGAACCCAAAGGGGAAT\nR: GCAGCAATCTGTAAGCGTCC\n160\nGPX4\nF: TCGCTGCTGGCTATAACGTC\nR: CCATTTGATGGCGTTTCCCA\n134\nCAT\nF: GCCTTCTGCCCTGGAACATA\nR: TAGAAATCCCGCACCTGAGTG\n96\nNRF2\nF: AAGTCAGGGAGAAGCGAGTTC\nR: TGTCAATCAAATCCATGTCCTGC\n199\nFTH1\nF: GCCATCAACCGCCAGATCAA\nR: GAAACTCGGCTCCCATGGACA\n70\nSTAR\nF: GCATCCTCAAAGACCAGGAG\nR: CTTGACACTGGGGTTCCACT\n194\n3β-HSD\nF: GGAGACATTCTGGATGAGCAG\nR: TCTATGGTGCTGGTGTGGA\n200\nCYP11A1\nF: GTTTCGCTTTGCCTTTGAGTC\nR: ACAGTTCTGGAGGGAGGTTGA\n158\nCYP19A1\nF: GCACTCTGGAAAGCTGTTCG\nR: CACGTCCACATAGCCCAAGT\n147\nHSD17B4\nF: ACGTGTCGAGATTCAAGGCA\nR: CCAGTTCCTTGGACCTTGGTT\n127\nβ-actin\nF: GCAAAGACCTCTACGCCAAC\nR: GGGCAGTGATCTCTTTCTGC\n90\n\nChen et al.\b\n10.3389/fvets.2025.1621738\nFrontiers in Veterinary Science\n05\nfrontiersin.org\n2.14 Statistical analysis\nData were analyzed statistically using SPSS 22.0, subject to\nnormality and chi-square: one-way ANOVA with Tukey’s-b or\nDunnett’s post-hoc multiple comparisons between groups; t-tests for\nindependent samples were used between groups. GraphPad Prism 8.0\nwas used for graphical presentation. Data are presented as mean ±\nSEM. p < 0.05 was the threshold for statistical significance, and all\nexperiments were repeated at least three times.\n3 Result\n3.1 Ameliorative effect of D19 treatment on\nLPS-induced reduction in viability of GCs\nImmunofluorescence staining showed that the FSHR protein\ndisplayed particular expression in sheep follicle GCs, demonstrating\nthat the GCs used in this study possess high purity and activity and\nare appropriate for subsequent experiments (Figure 2A).\nFigure 2B demonstrates a concentration-dependent effect of LPS\non GC viability, with the optimal survival rate observed at 25 μg/mL\n(p < 0.05). Then, the survival rate of the cells decreased with the\nincrease in the concentration. When the concentration of LPS reached\n400 μg/mL, the GCs were stressed, and some cells died. The results of\nGCs treated with different concentrations of D19 showed that low\nconcentrations of D19 (0–0.1 μM) accelerated the activity of GCs\n(Figure 2C). The survival rate of GCs was significantly increased\n(p < 0.05) after treatment with 0.1 μM D19 for 24 h and 48 h. On the\ncontrary, high concentrations (1–200 μM) of D19 caused dose-\ndependent cytotoxicity. Approximately 80% of GCs at 200 μM showed\nmorphological abnormalities and were detached (p < 0.05). Since\n400 μg/mL LPS would damage the activity of GCs, the present study\nwas carried out to investigate the optimal therapeutic concentration\nand safety range of D19 at different concentrations. Our findings\nindicate that 0.1 μM D19 is the optimal therapeutic concentration,\neffectively mitigating LPS-induced damage in GCs (p < 0.05).\nHowever, D19 exhibited a narrow therapeutic window, with\n\nOur findings\nindicate that 0.1 μM D19 is the optimal therapeutic concentration,\neffectively mitigating LPS-induced damage in GCs (p < 0.05).However, D19 exhibited a narrow therapeutic window, with\nconcentrations exceeding 100 μM failing to provide any protective\nbenefit and instead demonstrating significant toxicity (Figure 2D).\n3.2 D19 alleviates LPS-mediated\ninflammatory response\nTo explore the mitigating effect of D19 on LPS-induced\ninflammation in GCs, the mRNA expression levels of IL-6, IL-1β, and\nTNF-α in GCs treated with different concentrations of LPS were first\ndetected. The results showed that LPS dose-dependently increased the\nmRNA expression of the above inflammatory factors compared with CK\n(p < 0.05), with the maximum observed at a concentration of 400 μg/mL\n(Figure 3A). In subsequent experiments, 400 μg/mL LPS was used to\ninduce inflammation in GCs. Further studies revealed that D19 was able\nto inhibit the LPS-induced inflammatory response of GCs. Compared\nwith the LPS group, 0.1 μM D19 significantly reduced the mRNA levels\nof IL-6, IL-1β, and TNF-α (p < 0.05), and the therapeutic effect at this\nconcentration was better than that of other concentrations (Figures 2D,\n3B). In addition, 100 μM of D19 was the maximum safe concentration\nfor treating inflammation in combination with cell activity assay\n(Figures 2D, 3B). D19 was found to significantly inhibit the LPS-induced\nelevation of IL-1β and TNF-α levels in GCs by ELISA kit assay (p < 0.05)\n(Figure 3D), and the results of protein levels were consistent with the\nabove findings (Figures 3B,C). Notably, SA-β-Gal staining revealed that\nthe dark blue products in GCs were significantly reduced in the\nLPS + D19-0.1 μM and LPS + D19-100 μM groups compared to the LPS\ngroup, indicating that D19 alleviated LPS-induced cellular senescence\n(Figure 3E). Moreover, the effect of D19 was more pronounced at\n0.1 μM than at 100 μM (Figure 3E). The above studies confirmed that\nD19 reversed LPS-induced inflammatory injury in sheep follicular GCs.\n3.3 D19 protects GCs from LPS-induced\noxidative damage\n\nMoreover, the effect of D19 was more pronounced at\n0.1 μM than at 100 μM (Figure 3E).The above studies confirmed that\nD19 reversed LPS-induced inflammatory injury in sheep follicular GCs.3.3 D19 protects GCs from LPS-induced\noxidative damage\nAs shown in Figure 4A, 400 μg/mL LPS significantly inhibited the\nexpression of antioxidant-related genes GPX4, CAT, and SOD2 in\nsheep follicular GCs (p < 0.05). D19-0.1 μM effectively upregulated\nthe expression levels of the genes mentioned above, with its effective\nconcentration extending up to 100 μM (p < 0.05) (Figure 4B). Western\nblotting analysis (Figure  4C) confirmed that 0.1 μM D19 could\neffectively alleviate the LPS-induced reduction in the protein\nexpression of GPX4, CAT, GSH-PX, and SOD2 (p < 0.05). 100 μM\nD19 also had antioxidant effects, but the effect was weaker than that\nof 0.1 μM (p < 0.05). In addition, 400 μg/mL LPS effectively inhibited\nthe total antioxidant capacity, SOD activity, GSH-PX activity, CAT\nactivity, and GSH level of GCs, and significantly increased MDA\ncontent, glucose content, LDH level, and ROS production. D19 could\nsignificantly reverse the above LPS-induced weakening of antioxidant\nindexes in GCs (Figures 4D,E). These data demonstrate that D19\nprotects sheep follicular GCs from LPS-induced oxidative damage.\nTABLE 2  Antibodies information.\nAntibodies\nCat no.\nCompany\nSource\nDilution\nratio\nIL-1β\nD220820\nSangon\nRabbit\n1:500\nTNF-α\nCPA9458\nCohesion\nbiosciences\nRabbit\n1:500\nNF-KB\n380172\nZenbio\nRabbit\n1:500\npNF-KB\n310013\nZenbio\nRabbit\n1:500\nGPX4\nD290599\nSangon\nRabbit\n1:1000\nNRF2\n380773\nZenbio\nRabbit\n1:500\nSOD2\nCY5977\nAbways\nRabbit\n1:500\nGSH-Px\nCY8714\nAbways\nRabbit\n1:500\nCAT\nD122036\nSangon\nRabbit\n1:500\nFTH1\nCY5648\nAbways\nRabbit\n1:1000\nHSD17B4\nD122505\nSangon\nRabbit\n1:500\n3β-HSD\nCY8791\nAbways\nRabbit\n1:1000\nCYP11A1\nbs-10099R\nBioss\nRabbit\n1:500\nCYP19A1\nbs-0114R\nBioss\nRabbit\n1:500\nSTAR\nA16432\nABclonal\nRabbit\n1:500\nβ-actin\nAC038\nABclonal\nRabbit\n1:10000\n\nChen et al.\b\n10.3389/fvets.2025.1621738\nFrontiers in Veterinary Science\n06\nfrontiersin.org\n3.4 D19 inhibits LPS-induced ferroptosis in\nGCs\nGiven that GPX4, a core molecule in ferroptosis regulation, has\nbeen significantly inhibited by LPS in this study, and combining\nthe above findings, we speculate that the protective effect of D19\nagainst LPS-induced functional impairment of GCs may be realized\nthrough the GPX4-mediated ferroptosis signaling pathway. As\ndepicted in Figure 5A, LPS treatment significantly decreased the\nmitochondrial membrane potential, as evidenced by enhanced\ngreen fluorescence, whereas the mitochondrial membrane\npotential in the LPS + D19-0.1 μM and LPS + D19-100 μM groups\nwas close to the normal level. TEM results (Figure 5B) revealed\nthat GCs in the LPS-treated group displayed significant\nultrastructural abnormalities compared with the CK group. These\nabnormalities included mitochondrial condensation, reduced\nvolume, reduction and coarsening of cristae, widening of the inter-\ncristae lumen, and increased membrane and electron densities.\nFurthermore, autophagic lysosomes and a small number of\nvacuoles were observed. Compared with the LPS group,\nLPS + D19-0.1 μM\nsignificantly\nimproved\nmitochondrial\nmorphology, with most of the mitochondria restored to an\nelliptical shape, uniform matrix electron density, and well-defined\nand straight cristae. The morphology of LPS + D19-100 μM cells\nwas slightly abnormal, and the mitochondria showed a slight\ncondensation, accompanied by a mild expansion of the rough\nendoplasmic reticulum. Autophagic lysosomes were still visible in\nthe cytoplasm. In addition, the large accumulation of Fe2+ in\nLPS-treated cells was significantly reversed by D19 treatment\n(Figure 5C), which also successfully alleviated the inhibitory effect\nof LPS on FTH1 mRNA and protein expression levels (p < 0.05)\n(Figures 5D,E). Taken together, these results suggest that D19 can\neffectively alleviate the process of LPS-induced ferroptosis in sheep\nfollicular GCs and mitigate cellular damage.\n3.5 D19 restores LPS-suppressed steroid\nhormone production in GCs\n\n05)\n(Figures 5D,E).Taken together, these results suggest that D19 can\neffectively alleviate the process of LPS-induced ferroptosis in sheep\nfollicular GCs and mitigate cellular damage.3.5 D19 restores LPS-suppressed steroid\nhormone production in GCs\nTo assess the effect of D19 on LPS-induced steroid hormone\nsynthesis in GCs, we examined the levels of A4, E2, and P4 in GCs.\nThe ELISA results showed that LPS significantly inhibited the\nsynthesis of these three hormones in GCs compared to CK (p < 0.05),\nwhereas D19 treatment effectively reversed the inhibitory effect of\nLPS and significantly elevated the A4, E2, and P4 expression levels\n(p < 0.05) (Figure 6A). The expression of steroid synthesis-related\ngenes (HSD17B4, CYP19A1, 3β-HSD, CYP11A1, and STAR) and\ntheir proteins were also greatly inhibited by LPS, and the expression\nlevels of these genes and proteins were significantly increased by D19\ntreatment (p < 0.05) (Figures  6B,C). Based on these findings,\nFIGURE 2\nD19 treatment effectively mitigates the effects of LPS damage on the viability of GCs. (A) Cellular immunofluorescence showing FSHR-specific\nexpression in GCs. (B) MTT assays were performed to evaluate cell viability following 12 and 24 h treatment with LPS (0, 10, 25, 50, 100, 200, and\n400 μg/mL). (C) Cell viability was assessed by MTT assay after 24 and 48 h of D19 treatment (0, 0.01, 0.1, 1, 10, 100, 200 μM). (D) Mitigating the impact\nof different concentrations of D19 on LPS-induced decrease in cell viability of GCs. Data from at least three independent experiments are presented as\nmean ± SEM. Significant differences (p < 0.05) are denoted by different letters.\n\nChen et al.\b\n10.3389/fvets.2025.1621738\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nIL-6\nTNF-\nIL-1\nIL-6\nIL-1\nTNF-\nFIGURE 3\nD19 alleviates LPS-mediated GCs inflammatory response. (A) Relative expression of IL-6, IL-1β, and TNF-α in GCs under different LPS concentrations. \n(B) Effects of varying concentrations of D19 on IL-6, IL-1β, and TNF-α levels induced by LPS (400 μg/mL). (C) Western blotting analysis of IL-1β and \n(Continued)\n\nChen et al.\b\n10.3389/fvets.2025.1621738\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nwe conclude that D19 effectively mitigates LPS-induced impairment\nof steroid hormone synthesis in sheep follicular GCs.\n3.6 D19 suppresses inflammation caused\nby GPX4 deficiency in GCs\nGPX4 protein is specifically expressed in sheep follicular GCs\n(Figure 7A). si-GPX4 effectively inhibited GPX4 mRNA and protein\nexpression (p < 0.05) (Figures 7B,C) and significantly reduced cell\nviability at multiple time points (p < 0.05) (Figure 7D). However,\n0.1 μM D19 effectively alleviated the inhibitory effect of si-GPX4 on\nGC activity (p < 0.05) (Figure 7D). To further study the effect of\nGPX4 deletion on the inflammatory response of GCs and to clarify\nthe role of D19, we obtained the following findings: ELISA results\n(Figure  7E) revealed that D19 effectively reversed the si-GPX4-\nmediated upregulation of IL-1β and TNF-α levels in GCs (p < 0.05).\nMechanistically, we found that si-GPX4 activated the NF-κB signaling\npathway, indicated by a significant increase in NF-κB mRNA\nexpression and p65 phosphorylation, subsequently promoting the\nexpression of TNF-α, IL-6, and IL-1β (p < 0.05) (Figures  7F,G).\nNotably, total p65 protein levels remained unchanged across all\ngroups (p > 0.05) (Figure 7G). The protein levels of TNF-α and IL-1β\nwere consistent with those of mRNA (p < 0.05) (Figures  7F,G).\nInterestingly, after D19 treatment, the expression levels of NF-κB,\nTNF-α, IL-6, and IL-1β were significantly lower than those in the\nsi-GPX4 group (p < 0.05) and returned to near-normal cell levels.\nMeanwhile, Western blotting results showed that the trends of protein\nlevels of p65 NF-κB, TNF-α, and IL-1β were consistent with the\nmRNA results (Figure 7G). Therefore, we conclude that D19 can\nTNF-α protein expression in GCs. (D) ELISA quantification of IL-1β and TNF-α levels in different GCs treatment groups. (E) Senescence levels of GCs\nwere assessed using the SA-β-Galactosidase Staining kit. Data originating from at least three independent experiments are presented as mean ± SEM.\n\n(D) ELISA quantification of IL-1β and TNF-α levels in different GCs treatment groups.(E) Senescence levels of GCs\nwere assessed using the SA-β-Galactosidase Staining kit.Data originating from at least three independent experiments are presented as mean ± SEM.\nSignificant differences (p < 0.05) are denoted by different letters.\nFIGURE 3 (Continued)\nSOD2\nGPX4\nCAT\nSOD2\nCAT\nGPX4\nFIGURE 4 (Continued)\n\nChen et al.\b\n10.3389/fvets.2025.1621738\nFrontiers in Veterinary Science\n09\nfrontiersin.org\neffectively alleviate si-GPX4-induced decrease in cell viability and\ninflammatory injury.\n3.7 Effect of D19 on oxidative damage in\nGCs induced by GPX4 deficiency\nTo investigate whether D19 protects against LPS-induced\noxidative damage via GPX4, we suppressed GPX4 expression in\nsheep follicular GCs and assessed oxidative stress and D19’s effects.\nCompared with GCs transfected with si-NC, antioxidant indices\n(total antioxidant capacity, SOD activity, CAT activity, GSH-PX\nactivity, and GSH level) were significantly decreased (p < 0.05),\nwhile oxidative damage and metabolism-related indices (ROS level,\nMDA content, glucose level, and LDH activity) were significantly\nincreased (p < 0.05) in GCs with GPX4 knockdown (Figures 8A,B).\nSi-GPX4 significantly reduced mRNA and protein expression levels\nof antioxidant-related genes (NRF2, CAT, SOD2, and GPX4)\ncompared to si-NC (p < 0.05), as shown in Figures 8C,D. After the\nD19 treatment, the above antioxidant indexes were significantly\nrestored (Figure  8B), oxidative damage and metabolism-related\nindexes were reduced (Figures 8A,B), and the expression levels of\nantioxidant-related genes and proteins were effectively up-regulated\n(p < 0.05) (Figures 8C,D). These data demonstrate that D19 has\ntherapeutic potential for treating si-GPX4-induced oxidative\ndamage in GCs.\n3.8 D19 mediates ferroptosis in GCs by\nregulating GPX4\nEvidence indicates that intracellular inflammatory responses and\noxidative stress are strongly linked to ferroptosis caused by GPX4\ndownregulation. Thus, we investigated whether D19 alleviates GPX4\ndeficiency-induced inflammation and oxidative damage in GCs by\nmodulating ferroptosis. Suppressing GPX4 dramatically elevated Fe2+\nconcentration in GCs compared to the si-NC group (p < 0.05), as\ndemonstrated in Figure 9A. However, D19 treatment in si-GPX4-\ntransfected GCs normalized Fe2+ levels, indicating D19’s ability to\nrescue GCs from GPX4 deficiency-induced iron overload (see\nFIGURE 4\n\n05), as\ndemonstrated in Figure 9A.However, D19 treatment in si-GPX4-\ntransfected GCs normalized Fe2+ levels, indicating D19’s ability to\nrescue GCs from GPX4 deficiency-induced iron overload (see\nFIGURE 4\nD19 protecting GCs from LPS-induced oxidative damage. (A) qRT-PCR analysis of relative GPX4, CAT, and SOD2 mRNA expression in GCs exposed to\ndifferent LPS concentrations. (B,C) The effects of varying concentrations of D19 on the mRNA and protein levels of GPX4, CAT, and SOD2 induced by\nLPS (400 μg/mL) were analyzed using qRT-PCR and Western blotting. (D,E) Total antioxidant capacity, SOD activity, MDA levels, GSH-Px activity, CAT\nactivity, GSH levels, glucose concentration, LDH levels, and ROS levels in GCs under different treatments, as measured using antioxidant assay kits.\nData from at least three independent experiments are presented as mean ± SEM. Significant differences (p < 0.05) are denoted by different letters.\n\nChen et al.\b\n10.3389/fvets.2025.1621738\nFrontiers in Veterinary Science\n10\nfrontiersin.org\nFTH1\nFIGURE 5\nD19 inhibiting LPS-induced ferroptosis in GCs. (A) The effect of D19 on LPS-induced mitochondrial membrane potential changes was assessed using \nJC-1 staining. (B) TEM was used to examine the ultrastructural morphology of GCs in different treatment groups. Nucleus (N), nucleolus (No), \n(Continued)\n\nChen et al.\b\n10.3389/fvets.2025.1621738\nFrontiers in Veterinary Science\n11\nfrontiersin.org\nmitochondria (Mi), rough endoplasmic reticulum (RER); microvilli (↑, yellow arrows), autolysosomes (↑, green arrows). Scale bars: TEM × 8,000, 2 μm; \nTEM × 20,000, 500 nm. (C–E) The effects of D19 (0.1 μM, 100 μM) on LPS-induced changes in ferrous ion levels and FTH1 gene and protein expression \nwere evaluated. Data from at least three independent experiments are presented as mean ± SEM. Significant differences (p < 0.05) are denoted by \ndifferent letters.\nFIGURE 5 (Continued)\n3 -HSD\nHSD17B4\nCYP11A1\nCYP19A1\nSTAR\nFIGURE 6\nD19 restoring LPS-suppressed steroid hormone production in GCs. (A) ELISA measurement of A4, E2, and P4 levels in GCs. (B,C) qRT-PCR and Western \nblotting were employed to detect the relative expression levels of mRNA and protein, respectively, for genes involved in steroid hormone synthesis \n(HSD17B4, CYP19A1, 3β-HSD, CYP11A1, and STAR). Data from at least three independent experiments are presented as mean ± SEM. Significant \ndifferences (p < 0.05) are denoted by different letters.\n\nChen et al.\b\n10.3389/fvets.2025.1621738\nFrontiers in Veterinary Science\n12\nfrontiersin.org\nFigure  9A). As shown in Figure  9B, si-GPX4-transfected GCs\nexhibited a significantly increased green fluorescence signal compared\nto the si-NC group, indicating elevated lipid peroxide levels. D19\ntreatment attenuated this increase, resulting in a red fluorescence\nsignal distinct from the si-GPX4 group, suggesting that D19 effectively\nreduced lipid peroxide levels. TEM analysis showed that si-GPX4 GCs\nexhibited significant mitochondrial dysfunction, indicated by\ndecreased membrane potential, cristae loss, increased membrane\ndensity, and elevated autophagosome numbers. D19 treatment\nmitigated these abnormalities, restoring mitochondrial morphology\nand membrane potential (Figures 9C,D). In addition, the gene and\nprotein expression of FTH1 in the GCs of the si-GPX4 group was\nsignificantly lower than that of the si-NC group (p < 0.05), whereas\nthe expression level of FTH1 in the si-GPX4 + D19 group was\nsignificantly higher than that of the si-GPX4 group (p < 0.05)\n(Figures 9E,F). Therefore, we conclude that D19 can effectively inhibit\nthe ferroptosis process induced by GPX4 deficiency and maintain the\niron metabolism homeostasis in GCs.\nGPX4\nNF-kB\nTNF-\nIL-6\nIL-1\nFIGURE 7\nD19 suppressing inflammation caused by GPX4 deficiency in GCs. (A) Immunofluorescence was utilized to detect the expression of GPX4 protein in\nGCs. (B,C) qRT-PCR and Western blotting were used to analyze the interference efficiency of si-GPX4. (D) D19–0.1 μM reversed the si-GPX4-induced\ndecline in the cell viability of GCs. (E) ELISA measurement of IL-1β and TNF-α levels in treated GCs. (F) Relative mRNA expression of NF-κB, TNF-α, IL-6,\nand IL-1β in GCs, as determined by qRT-PCR. (G) Western blotting was conducted to investigate the effect of D19 on LPS-induced changes in the\nprotein levels of p65 NF-κB, phosphorylated p65 NF-κB (p-p65 NF-κB), TNF-α, and IL-1β in GCs. Data are expressed as mean ± SEM (n ≥ 3). Different\nletters indicate significant differences at p < 0.05, while ** and *** denote p < 0.01 and p < 0.001, respectively.\n\nChen et al.\b\n10.3389/fvets.2025.1621738\nFrontiers in Veterinary Science\n13\nfrontiersin.org\nNRF2\nGPX4\nCAT\nSOD2\nFIGURE 8\nEffect of D19 on oxidative damage in GCs induced by GPX4 deficiency. (A,B) D19 alleviated the effects of LPS on antioxidant-related indicators. \n(C) Relative mRNA expression of NRF2, GPX4, CAT, and SOD2 in GCs from different treatment groups, as determined by qRT-PCR. (D) Relative protein \nexpression of NRF2, GPX4, CAT, GSH-Px, and SOD2 in GCs from different treatment groups, as determined by Western blotting. Data are presented as \nmean ± SEM (n ≥ 3). Different letters denote significant differences (p < 0.05).\n\nChen et al.\b\n10.3389/fvets.2025.1621738\nFrontiers in Veterinary Science\n14\nfrontiersin.org\n3.9 D19 prevents GPX4 deficiency-induced\ndisruption of steroidogenesis in GCs\nThe critical role of GCs in follicular development is realized\nthrough the synthesis of steroid hormones. However, when GCs were\nexposed to an environment in which GPX4 was inhibited, the\nsynthesis of E2 and P4 was significantly suppressed compared with the\nsi-NC group (p < 0.05) (Figure 10A), while the mRNA and protein\nexpression levels of steroid hormone synthesis-associated factors\n(HSD17B4, CYP19A1, 3β-HSD, CYP11A1, and STAR) were also\nsignificantly downregulated (p < 0.05) (Figures 10B,C). Noticeably, the\nlevels of E2 and P4, as well as the expression of steroid hormone\nsynthesis-related genes and proteins, were significantly up-regulated\nin the si-GPX4 + D19 group compared with the si-GPX4 group\n(p < 0.05) (Figures  10A–C). These results suggest that D19 can\neffectively alleviate the inhibition of steroid hormone synthesis\nfunction in GCs by GPX4 deletion and thus repair the functional\ndamage of GCs.\n4 Discussion\nDysfunction of follicular GCs in sheep is one of the key factors\naffecting normal follicular development and ovulation. LPS has been\nshown to induce inflammatory responses, oxidative stress, and steroid\nhormone synthesis disorders in GCs, leading to impaired GCs\nfunction. Increasing evidence demonstrates that the exposure of\nanimals to LPS induces a range of reproductive disorders. Higher\nlevels of LPS were detected in the blood, follicular fluid, and milk of\ncows with endometritis (26), accompanied by diminished ovarian\nfunction (27). Several studies have indicated that LPS not only inhibits\nGCs’ proliferation (28) but also triggers an inflammatory response\nin vitro in human follicular GCs, characterized by the upregulation of\ninflammatory cytokines, including TNF-α, IL-1β, and IL-6 (29). LPS\nalso significantly increased SA-β-gal activity, a cellular senescence\nmarker, in macrophages (30). MNQ, belonging to the\nnaphthoquinones class of compounds, is considered the most\n\nLPS\nalso significantly increased SA-β-gal activity, a cellular senescence\nmarker, in macrophages (30).MNQ, belonging to the\nnaphthoquinones class of compounds, is considered the most\nprincipal and representative active component of Impatiens balsamina\nL. Studies have confirmed that MNQ exhibits dose-dependent\ncytotoxicity, effectively killing various cancer cells at high\nconcentrations while displaying anti-inflammatory and antioxidant\nactivities at low concentrations (31). MNQ also promotes the\nproliferation of OECs at concentrations ranging from 0 to 1 μM,\nwhereas a concentration of 10 μM is highly cytotoxic, significantly\nreducing cell viability (16). Our results demonstrate that low\nconcentrations of D19 (0–100 μM) significantly ameliorated the\nLPS-induced decrease in GCs viability, whereas high concentrations\nof D19 exhibited toxicity similar to that of MNQ (32). Furthermore,\nMNQ has been reported to participate in host defense and exert\nimmunomodulatory effects (33), as well as exhibit anti-\nneuroinflammatory activity by reducing nitric oxide (NO) production\nin LPS-stimulated BV-2 cells (34). Naphthoquinones extracted from\nSinningia canescens effectively reduced the elevation of inflammatory\nfactors TNF-α, IL-6, and IL-1β in mice blood induced by LPS (35).\nConsistent with these findings, our results demonstrate that the MNQ\nderivative D19 also exerts anti-inflammatory effects by downregulating\nthe LPS-induced expression of the inflammation-related genes TNF-α,\nIL-6, and IL-1β in sheep follicular GCs.\nLPS is recognized as a potent inducer of both inflammation\nand oxidative stress in cellular models in vitro. LPS led to a high\naccumulation of ROS in lung epithelial cells, thereby triggering\noxidative stress (36). LPS up-regulated MDA levels and down-\nregulated antioxidant capacity in human follicular GCs, as well as\ninhibited the expression of SOD, CAT, and GPx genes (5).\nInterestingly, we  observed a similar phenomenon in sheep\nfollicular GCs: LPS significantly suppressed total antioxidant\ncapacity, SOD activity, GSH-PX activity, and CAT activity, and\n\nInterestingly, we  observed a similar phenomenon in sheep\nfollicular GCs: LPS significantly suppressed total antioxidant\ncapacity, SOD activity, GSH-PX activity, and CAT activity, and\nreduced GSH levels in GCs, while increasing MDA and ROS levels.\nIn addition, the transcriptional and translational levels of CAT,\nGSH-PX, and SOD2 were also greatly inhibited by LPS, whereas the\nlevels of LDH and glucose were significantly increased, a result that\nis consistent with the findings in BEAS-2B cells (37). The above\nresults suggest that LPS can induce oxidative stress in sheep\nfollicular GCs. Studies indicate that naphthoquinones (plumbagin,\njuglone, menadione, etc.) possess strong antioxidant capabilities by\nscavenging ROS and inducing the expression of antioxidant genes\nlike SOD (38). Previous research has revealed potent antioxidant\nactivity in certain extracts of Impatiens balsamina L. (39). However,\nstudies on MNQ and its derivatives mainly focus on anticancer,\nantibacterial, and anti-inflammatory effects, while their antioxidant\neffects in animals are more limited (40). Interestingly, our\nexperimental results revealed that D19 treatment significantly\nmitigated the LPS-induced impairment of antioxidant capacity in\nGCs, contributing to a more robust antioxidant defense and\nenriching the understanding of MNQ and its derivative D19’s\nantioxidant effects.\nSteroid hormones are key regulators in maintaining follicular\ndevelopment and normal ovulation of oocytes. Within the steroid\nhormone synthesis pathway, P4 is converted to A4, which is further\nconverted to E2, and these hormones collectively regulate the overall\nprocess (41). CYP19A1 has been widely recognized as a key enzyme\nin estrogen synthesis (42). Previous studies have demonstrated that\nLPS-induced suppression of CYP19A1 expression in bovine (43),\nmouse, and porcine follicular GCs leads to reduced E2 secretion and\ndisruption of oocyte meiotic progression (44). CYP11A1, 3β-HSD,\nand STAR are involved in the early stages of steroid hormone\nsynthesis, where CYP11A1 catalyzes the conversion of cholesterol to\n\nCYP11A1, 3β-HSD,\nand STAR are involved in the early stages of steroid hormone\nsynthesis, where CYP11A1 catalyzes the conversion of cholesterol to\npregnenolone, and STAR is responsible for the transport of\ncholesterol to androgens. STAR is responsible for transporting\ncholesterol into mitochondria for CYP11A1 utilization (22).\nExposure of human follicular GCs to LPS also leads to a significant\nreduction in E2 and P4 levels, accompanied by a significant down-\nregulation of CYP19A1, CYP11A1, and STAR gene expression (45).\nHSD17B4 is reported to function in testosterone synthesis by\ncatalyzing the conversion of androstenedione into testosterone (46).\nTherefore, the reduction of these key enzymes inevitably leads to an\noverall decrease in steroid hormone synthesis. The results of the\npresent study are consistent with previous studies, which found that\nexposure of sheep follicular GCs to LPS significantly reduced the\ngene and protein expression levels of HSD17B4, CYP19A1,\nCYP11A1, 3β-HSD, and STAR, resulting in significant inhibition of\nE2, P4, and A4 synthesis. Our team’s previous research results showed\nthat MNQ derivative D21 could alleviate the steroid hormone\nsynthesis disorder caused by LPS (21). Encouragingly, the present\nstudy demonstrated that D19 exhibits a similar effect to D21 in\nmitigating LPS-induced steroid hormone synthesis disorder, further\n\nChen et al.\b\n10.3389/fvets.2025.1621738\nFrontiers in Veterinary Science\n15\nfrontiersin.org\nFTH1\nFIGURE 9\nD19 mediates ferroptosis in GCs by regulating GPX4. (A,B) D19 protects GCs from LPS-induced abnormalities in Fe2+ and lipid peroxide levels. (C,D) \nMitochondrial membrane potential and cellular ultrastructure of GCs in different treatment groups were assessed using JC-1 staining and TEM, \nrespectively. Nucleus (N), mitochondria (Mi), autolysosomes (↑ green arrows). Scale bars: TEM × 8,000, 2 μm; TEM × 20,000, 500 nm. (E,F) Relative \nmRNA and protein expression levels of FTH1 in GCs from different treatment groups. All experiments were performed in triplicate, and data are \npresented as mean ± SEM. Different letters indicate statistically significant differences (p < 0.05).\n\nChen et al.\b\n10.3389/fvets.2025.1621738\nFrontiers in Veterinary Science\n16\nfrontiersin.org\nexpanding the potential application of MNQ derivatives in\nenhancing follicular GCs function.\nGPX4, a key negative regulator of the ferroptosis pathway,\neffectively inhibits ferroptosis through GSH-dependent scavenging of\nlipid peroxides and is a potential target for the treatment of ferroptosis-\nrelated diseases (11). Previous studies have shown that LPS inhibits\nGPX4 expression in BEAS-2B cells, leading to intracellular Fe2+\naccumulation and disruption of iron metabolic homeostasis (47). In\naddition, lower-than-normal levels of GPX4 and FTH1 in both human\nand mouse ovarian tissues from patients with ovarian cysts suggest\nferroptosis (48). In the present study, we similarly observed that LPS\nsignificantly inhibited the expression of GPX4 and FTH1 in sheep\nfollicular GCs, leading to Fe2+ accumulation, which was significantly\nreversed by D19 treatment. Following D19 treatment, the mRNA and\nprotein expression levels of GPX4 and FTH1 in GCs were markedly\nincreased compared to the LPS group, alleviating LPS-induced iron\noverload and further suppressing the ferroptosis process in GCs.\nResearchers have found that LPS induced mitochondrial\nmorphological abnormalities and a decline in membrane potential in\nHT-22 cells (10). Interestingly, we also observed that D19 significantly\nprotected sheep follicular GCs from LPS-induced mitochondrial\ndamage and loss of membrane potential. Taken together with our\nteam’s previous sequencing data, we speculate that the core mechanism\nby which D19 alleviates LPS-induced inflammation, oxidative stress,\nand steroid hormone synthesis disorders in sheep follicular GCs may\nbe closely related to the GPX4-mediated ferroptosis signaling pathway.\nIt has been reported that GPX4 is recognized as a key inhibitor of\nferroptosis in various tissues and plays a crucial role in cell survival.\nEarly studies demonstrated that GPX4 deficiency induces ferroptosis\nin murine embryonic fibroblasts (49). Furthermore, GPX4 interacts\nsynergistically with the NF-κB signaling pathway to co-regulate\n\nEarly studies demonstrated that GPX4 deficiency induces ferroptosis\nin murine embryonic fibroblasts (49).Furthermore, GPX4 interacts\nsynergistically with the NF-κB signaling pathway to co-regulate\napoptosis and ferroptosis in KGN cells (8). MNQ can modulate cell\nproliferation, differentiation, and apoptosis in Raji cells, potentially\nmediating the suppression of inflammatory responses, angiogenesis,\nHSD17B4 CYP19A1 3 -HSD CYP11A1\nSTAR\nFIGURE 10\nD19 preventing GPX4 deficiency-induced disruption of steroidogenesis in GCs. (A) Levels of E2 and P4 in different treatment groups were measured\nusing ELISA. (B) The relative mRNA and protein expression levels of steroid hormone synthesis-related genes (HSD17B4, CYP19A1, 3β-HSD, CYP11A1,\nand STAR) were detected by qRT-PCR and Western blotting, respectively. All experiments were performed in triplicate, and data are presented as mean\n± SEM. Different letters indicate statistically significant differences (p < 0.05).\n\nChen et al.\b\n10.3389/fvets.2025.1621738\nFrontiers in Veterinary Science\n17\nfrontiersin.org\nand tumor metastasis via the NF-κB signaling pathway (50). In the\npresent study, we  observed that GPX4 knockdown significantly\nreduced viability in sheep follicular GCs, an effect that D19 markedly\nattenuated. Overexpression of GPX4 inhibits the NF-κB signaling\npathway, thereby downregulating the expression of pro-inflammatory\ncytokines such as TNF-α and IL-6, ultimately protecting the rat heart\nfrom inflammatory damage (51). 5,8-Dimethoxy-1,4-naphthoquinone\n(DMNQ) derivatives significantly attenuated the LPS-induced\nexpression of NO, ROS, and inflammatory cytokines in BV-2\nmicroglial cells by modulating the MAPK/NF-κB signaling pathway\n(52). In agreement with these findings, our experiments demonstrated\nthat D19 significantly mitigated the activation of the NF-κB pathway\nand the subsequent upregulation of pro-inflammatory cytokines\n(TNF-α, IL-1β, and IL-6) induced by GPX4 interference in sheep\nfollicular GCs. These results indicate that D19 treatment reversed this\nGPX4 deficiency-mediated inflammatory response.\nOxidative stress resulting from GPX4 deficiency significantly\ncompromises reproductive efficiency in animals. GPX4 is highly\nexpressed in the testes and sperm, where it regulates spermatogenesis,\nmaintains chromatin integrity, and counteracts oxidative stress to\nensure male fertility (53). Its deficiency leads to abnormal sperm\ndevelopment, germ cell apoptosis, and reduced fertilization capacity\n(54), thereby significantly diminishing reproductive efficiency.\nNumerous studies have established GPX4 as a transcriptional target of\nNRF2, with the two interacting to regulate cellular oxidative stress,\nferroptosis, and iron metabolism (55). In HepG2 cells, LPS may\ninduce oxidative stress by suppressing the activity of the NRF2/GPX4\naxis, while in the D-GalN/LPS-induced acute liver injury model,\nMaR1 alleviates oxidative stress and ferroptosis-related liver damage\nby activating the NRF2/HO-1/GPX4 pathway, inhibiting ROS and\nMDA production, and increasing reduced GSH levels (56).\n\nAdditionally, MNQ has been shown to inhibit glucose and LDH\nproduction in triple-negative breast cancer cells, concurrently\nsuppressing cellular glycolytic activity and the expression of related\nmolecules (32). In agreement with these findings, the present study\ndemonstrated that D19 effectively ameliorated oxidative damage in\nsheep follicular GCs resulting from GPX4 deficiency. Specifically,\ncompared to the si-GPX4 group, D19 significantly upregulates\nantioxidant-related indicators (total antioxidant capacity, SOD\nactivity, GSH-PX, CAT, and reduced GSH) in GCs, while\ndownregulating ROS, MDA, glucose, and LDH levels. Consistently,\nGPX4 exerts significant antioxidant activity within mitochondria. The\nupregulation of NRF2, CAT, and SOD2 in mouse follicular GCs\nprotects against premature ovarian failure (POF) (57). Our study\nrevealed that D19 significantly enhanced the expression of NRF2,\nGPX4, CAT, GSH-PX, and SOD2 at both transcriptional and\ntranslational levels in the si-GPX4 group. These findings highlight the\ncrucial role of D19 in improving GCs oxidative damage through the\nNRF2/GPX4 pathway.\nMechanistically, iron overload impairs NRF2 binding to\nantioxidant response elements (AREs), resulting in downregulated\nGPX4 expression, elevated mitochondrial ROS production, and\ndiminished mitochondrial membrane potential, ultimately promoting\nlipid peroxidation and ferroptosis (58). Consistent with these prior\nobservations, we found that the downregulation of GPX4 in sheep\nfollicular GCs resulted in significantly elevated levels of lipid peroxides\nand Fe2+ concurrent with a marked reduction in mitochondrial\nmembrane potential and the presence of abnormal mitochondrial\nmorphology, all characteristic features of ferroptosis (59, 60).\nImportantly, the upregulation of GPX4 has been shown to inhibit\nferroptosis and delay the senescence of spermatogenic cells in aged\nmice (61). FTH1 primarily functions to store intracellular iron and\nreduce free iron levels. As evidence, FTH1 downregulation induces\nferroptosis in bladder cancer cells (62). In the present study, si-GPX4\n\nFTH1 primarily functions to store intracellular iron and\nreduce free iron levels.As evidence, FTH1 downregulation induces\nferroptosis in bladder cancer cells (62).In the present study, si-GPX4\nsignificantly decreased FTH1 mRNA and protein expression levels in\nsheep follicular GCs, suggesting that GPX4 depletion is sufficient to\ninitiate ferroptosis in these cells. Research has shown that\nmenaquinone-4, a form of vitamin K, exerts a protective role in a\nGPX4-deficiency-induced murine hepatocyte ferroptosis model by\ndiminishing lipid peroxidation, cell death, and inflammation (63).\nRemarkably, we found that D19 effectively reversed ferroptosis in GCs\ncaused by aberrant GPX4 expression, highlighting a potential new\napproach for promoting GCs function.\nGiven our previous findings that D19 alleviates si-GPX4-induced\nferroptosis and the associated inflammatory and oxidative damage in\nsheep follicular GCs, we hypothesized that D19 might also regulate\nsi-GPX4-induced steroid hormone synthesis disorders. Evidence has\nestablished a close link between GPX4-mediated ferroptosis and\nsteroidogenesis (64). GPX4, acting as a key antioxidant in testicular\nLeydig cells, safeguards steroidogenesis by modulating STAR-\nmediated cholesterol delivery to CYP11A1 and the subsequent\nCYP11A1-catalyzed conversion of cholesterol into pregnenolone,\nensuring normal testosterone secretion (65). Similarly, in our\nexperiments, we observed that si-GPX4 significantly suppressed the\nexpression of CYP11A1 and STAR, along with other steroidogenesis-\nrelated genes (HSD17B4, CYP19A1, and 3β-HSD) in sheep follicular\ngranulosa cells, resulting in decreased E2 and P4 production. However,\nD19 treatment effectively reversed this si-GPX4-mediated\nsteroidogenic dysfunction (17), potentially contributing to the\nmaintenance of normal GCs function.\n5 Conclusion\nIn conclusion, we  synthesized the MNQ derivative D19 and\nsystematically investigated its biological activities. Our findings indicate\nthat D19 exhibits lower cytotoxicity than MNQ and effectively\nattenuates LPS-induced inflammation, oxidative stress, and\n\nOur findings indicate\nthat D19 exhibits lower cytotoxicity than MNQ and effectively\nattenuates LPS-induced inflammation, oxidative stress, and\nsteroidogenesis impairment, potentially via a mechanism involving the\nGPX4-ferroptosis axis. These results provide a theoretical foundation\nand a potential drug target for developing novel anti-inflammatory and\nantioxidant natural plant extracts to treat follicular developmental\ndisorders and reproductive system diseases.\nData availability statement\nThe original contributions presented in the study are included in\nthe article/supplementary material, further inquiries can be directed\nto the corresponding author.\nEthics statement\nAll animal experiments were conducted in compliance with the\nguidelines of the China Council on Animal Care and were approved\n\nChen et al.\b\n10.3389/fvets.2025.1621738\nFrontiers in Veterinary Science\n18\nfrontiersin.org\nby the Institutional Animal Care and Use Committee of Shanxi\nAgricultural University [Permit No. 2020(058)]. The study was\nconducted in accordance with the local legislation and\ninstitutional requirements.\nAuthor contributions\nCC: Conceptualization, Data curation, Formal analysis, Software,\nVisualization, Writing – original draft, Writing – review & editing. JL:\nData curation, Formal analysis, Writing – original draft. YQ: Data\ncuration, Resources, Visualization, Writing  – original draft. JD:\nResources, Visualization, Writing – original draft. YH: Data curation,\nResources, Visualization, Writing – original draft. YR: Data curation,\nFormal analysis, Resources, Writing – original draft. SJ: Investigation,\nMethodology, Writing  – original draft. RB: Conceptualization,\nWriting – original draft. DW: Conceptualization, Supervision,\nValidation, Writing  – review & editing. LM: Conceptualization,\nSupervision, Validation, Writing  – review & editing. LL:\nConceptualization, Funding acquisition, Project administration,\nSupervision, Validation, Writing – review & editing.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. This study was supported\nby the National Key R&D Program of China (No. 2024YFD1301004),\nthe National Key R&D Program of China (No. 2023YFD1300601), the\nJinzhong National Agricultural High District 2023 Science and\nTechnology Innovation Sub-Project, the earmarked fund for the\nModern Agro-industry Technology Research System in Shanxi\nProvince, and Shanxi Key Laboratory of Animal Genetics Resource\nUtilization and Breeding.\nAcknowledgments\nThe authors collectively express their gratitude to Lei Shi and\nsenior experimentalist Liying Qiao for their technical guidance and\nassistance with this study.\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\n\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.Generative AI statement\nThe authors declare that Gen AI was used in the creation of this\nmanuscript. Deepseek, ChatGPT, and Grammarly were used to check\nthe grammar of this article.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. If you identify any\nissues, please contact us.\nPublisher’s note\nAll claims expressed in this article are solely those of the authors\nand do not necessarily represent those of their affiliated organizations,\nor those of the publisher, the editors and the reviewers. Any product\nthat may be evaluated in this article, or claim that may be made by its\nmanufacturer, is not guaranteed or endorsed by the publisher.\nReferences\n1.\tPrice JC, Bromfield JJ, Sheldon IM. 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(2024) 82:213–22. doi: 10.1007/s12013-023-01194-5\n\nChen et al.\b\n10.3389/fvets.2025.1621738\nFrontiers in Veterinary Science\n21\nfrontiersin.org\nGlossary\nMNQ - 2-methoxy-1,4-naphthoquinone\nGCs - Granulosa cells\nLPS - Lipopolysaccharide\nqRT-PCR - Quantitative reverse transcription polymerase \nchain reaction\nGPX4 - Glutathione peroxidase 4\nE2 - Estradiol\nP4 - Progesterone\nA4 - Androstenedione\nNO - Nitric oxide\nOECs - Olfactory ensheathing cells\nKGN cells - Human granulosa-like tumor cell line\nROS - Reactive oxygen species\nPOF - Premature ovarian failure\nDMSO - Dimethyl sulfoxide\nTNF-α - Tumor necrosis factor alpha\nNF-kB - Nuclear factor kappa-light-chain-enhancer of activated B cells\nIL-1β - Interleukin-1 beta\nIL-6 - Interleukin-6\nCAT - Catalase\nGSH-PX - Glutathione peroxidase\nSOD2 - Superoxide dismutase 2\nNRF2 - Nuclear factor erythroid 2–related factor 2\nFTH1 - Ferritin heavy chain 1\nLDH - Lactate dehydrogenase\nTEM - Transmission electron microscopy\nMDA - Malondialdehyde\nHSD17B4 - Hydroxysteroid 17-beta dehydrogenase 4\nCYP19A1 - Cytochrome p450 family 19 subfamily A member 1\n3β-HSD - 3 beta-hydroxysteroid dehydrogenase\nCYP11A1 - Cytochrome p450 family 11 subfamily A member 1\nSTAR - Steroidogenic acute regulatory protein\nSA-β-Gal - SA-β-galactosidase\n\n---"}
+{"text": "【兽医文献】\n标题: The impact of age on the Na:K ratio: observations from a general canine population\n\nFrontiers in Veterinary Science\n01\nfrontiersin.org\nMolecular characteristics of a\npotentially novel BVDV (Pestivirus\nbovis) genotype 1b isolate from\ncommercial fetal bovine serum\nJuanjuan Pan 1†, Jianfeng Jiang 2†, Shijiang Mi 3†, Xintong Chen 1,\nRuli Duan 1, Shandian Gao 4, Ling Kuang 1, Panpan Tong 1 and\nJinxin Xie 1,5*\n1 Laboratory of Animal Etiology and Epidemiology, College of Veterinary Medicine, Xinjiang\nAgricultural University, Urumqi, China, 2 State Key Laboratory for Zoonotic Diseases, Key Laboratory\nfor Zoonosis Research of the Ministry of Education, Institute of Zoonosis, College of Veterinary\nMedicine, Jilin Universitygrid, Changchun, China, 3 Changchun Research Veterinary Institute, Chinese\nAcademy of Agricultural Sciences, Changchun, China, 4 State Key Laboratory for Animal Disease\nControl and Prevention, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary\nResearch Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China, 5 Xinjiang Key\nLaboratory of New Drug Research and Development for Herbivores, Urumqi, China\nBovine viral diarrhea virus (BVDV) is commonly detected in biological products such\nas tissues and serum. This study identified BVDV contamination in a commercially\navailable fetal bovine serum (FBS) sample. To determine whether the detected virus\nwas infectious or merely genetic material, the FBS was inoculated into Madin-\nDarby bovine kidney (MDBK) cells. Following six serial passages, both indirect\nimmunofluorescence assay results and electron microscopic visualization of viral\nparticles confirmed the presence of infectious BVDV. The isolated strain, designated\nBI-2023, had a complete genome length of 12,273 nucleotides. Comparative\nsequence analysis showed that the 5′ untranslated region (UTR) and full genome of\nBI-2023 shared 87.4–97.1% and 92.4–95.2% nucleotide identity, respectively, with\nreference strains of BVDV1 (Pestivirus bovis) genotype 1b. Phylogenetic analyses\nbased on the 5′UTR and whole genome placed BI-2023 within the genotype 1b\ncluster of Pestivirus bovis. Several amino acid substitutions were identified in\n\nPhylogenetic analyses\nbased on the 5′UTR and whole genome placed BI-2023 within the genotype 1b\ncluster of Pestivirus bovis.Several amino acid substitutions were identified in\nthe E2 and Erns proteins of the BI-2023 regions involved in immune evasion and\nviral secretion. This suggests this strain may represent a distinct variant within\nthe genotype 1b group. These results highlight the critical need for routine viral\nscreening in commercial FBS preparations.\nKEYWORDS\nPestivirus bovis, genotype 1b, fetal bovine serum, contamination, novel strain\nIntroduction\nFetal bovine serum (FBS) is an essential supplement in cell culture media, widely used in\nmanufacturing vaccines and other biologics. However, the adventitious agents, particularly\nviruses, can significantly compromise the safety and quality of these biological products (1–3).\nBovine serum is known to potentially carry a variety of contaminating viruses, including\nbluetongue virus (BTV), adenovirus (BAV), poliovirus (BPV), respiratory syncytial virus\n(BRSV), viral diarrhea virus (BVDV), rabies virus (RV), parainfluenza virus type III (PIV III),\nand infectious bovine rhinotracheitis virus (IBRV), among others. BVDV is one of the most\ncommonly detected contaminants in bovine serum samples (1–13).\nOPEN ACCESS\nEDITED BY\nVeasna Duong,\nInstitut Pasteur du Cambodge, Cambodia\nREVIEWED BY\nMassimo Giangaspero,\nIndependent Researcher, Teramo, Italy\nJames McConville,\nAgri-Food and Biosciences Institute,\nUnited Kingdom\nFlorence Hugues,\nUniversity of Concepcion, Chile\n*CORRESPONDENCE\nJinxin Xie\nxiejinxin198683@163.com\n†These authors have contributed equally to\nthis work\nRECEIVED 15 May 2025\nACCEPTED 08 August 2025\nPUBLISHED 24 September 2025\nCITATION\nPan J, Jiang J, Mi S, Chen X, Duan R, Gao S,\nKuang L, Tong P and Xie J (2025) Molecular\ncharacteristics of a potentially novel BVDV\n(Pestivirus bovis) genotype 1b isolate from\ncommercial fetal bovine serum.\nFront. Vet. Sci. 12:1629211.\ndoi: 10.3389/fvets.2025.1629211\nCOPYRIGHT\n© 2025 Pan, Jiang, Mi, Chen, Duan, Gao,\nKuang, Tong and Xie. This is an open-access\narticle distributed under the terms of the\nCreative Commons Attribution License\n\nFront.Vet.Sci.12:1629211.doi: 10.3389/fvets.2025.1629211\nCOPYRIGHT\n© 2025 Pan, Jiang, Mi, Chen, Duan, Gao,\nKuang, Tong and Xie.This is an open-access\narticle distributed under the terms of the\nCreative Commons Attribution License\n(CC BY). The use, distribution or reproduction\nin other forums is permitted, provided the\noriginal author(s) and the copyright owner(s)\nare credited and that the original publication\nin this journal is cited, in accordance with\naccepted academic practice. No use,\ndistribution or reproduction is permitted\nwhich does not comply with these terms.\nTYPE  Original Research\nPUBLISHED  24 September 2025\nDOI  10.3389/fvets.2025.1629211\n\nPan et al.\b\n10.3389/fvets.2025.1629211\nFrontiers in Veterinary Science\n02\nfrontiersin.org\nThe BVDV is a member of the Flaviviridae family and belongs to\nthe genus Pestivirus. It is the primary causative agent of bovine viral\ndiarrhea and mucosal disease in cattle, conditions characterized by\nclinical signs such as fever, diarrhea, decreased milk yield, and\nreproductive dysfunctions (14). BVDV is a single-stranded, positive-\nsense RNA virus with a genome approximately 12.3 kb in length (15).\nGlobally, two major genotypes of BVDV have been identified: BVDV-1\nand BVDV-2. According to the International Committee on Taxonomy\nof Viruses (ICTV), these are now classified as Pestivirus bovis (BVDV-1)\nand Pestivirus tauri (BVDV-2), respectively (15). Within Pestivirus\nbovis, at least 25 distinct genotypes (designated 1a to 1.25) have been\nrecognized (16–22). Furthermore, BVDV exists in two biotypes:\ncytopathogenic (CP) and non-cytopathogenic (NCP), differentiated by\ntheir ability to induce cytopathic effects (CPE) in cultured cells.\nIn this study, a non-cytopathogenic strain of BVDV was\nsuccessfully isolated from commercially available fetal bovine serum.\nMultiple amino acid substitutions were identified in the E2 and Erns\nprotein regions that play key roles in immune evasion and viral\nsecretion. These mutations suggest the isolate may represent a novel\nBVDV1 (Pestivirus bovis) genotype 1b group variant. This discovery\nunderscores the genetic diversity of circulating BVDV strains and\nhighlights the critical need for routine surveillance to ensure the safety\nof bovine-derived biological materials.\nMaterials and methods\nBVDV detection\nFour commercial fetal bovine serum (FBS) samples originating\nfrom Israel were obtained from Biological Industries (LOT: 2153440)\nand ExCell Bio (LOT: 12B052). Viral RNA was extracted from 200 μL\nof each FBS sample using the Geneaid extraction kit, following the\nmanufacturer’s protocol. Complementary DNA (cDNA) was\nsynthesized using PrimeScript II Reverse Transcriptase (Takara,\nChina) under the following thermal conditions: 65°C for 5 min, 42°C\nfor 60 min, and 95°C for 5 min. The 5′-UTR of BVDV was then\n\nComplementary DNA (cDNA) was\nsynthesized using PrimeScript II Reverse Transcriptase (Takara,\nChina) under the following thermal conditions: 65°C for 5 min, 42°C\nfor 60 min, and 95°C for 5 min.The 5′-UTR of BVDV was then\namplified by PCR using the primer pair 1F1 and 1R462 (Table 1).\nAmplification was performed with 2 × TransStart® FastPfu Fly PCR\nSuperMix (TransGen Biotech, Beijing, China) under the following\ncycling parameters: initial denaturation at 95°C for 2 min, followed by\n35 cycles of denaturation at 95°C for 20 s, annealing at 52°C for 20 s,\nand extension at 72°C for 30 s, with a final extension step at 72°C for\n5 min. The PCR products were analyzed via electrophoresis on a 1%\nagarose gel.\nCell culture and virus isolation\nMadin-Darby bovine kidney (MDBK) cells, confirmed to be free\nof BVDV by the PCR described above, were kindly provided by the\nlaboratory of Tu Changchun. MDBK cells were exposed to BVDV-\npositive fetal bovine serum for 2 h at 37°C in a humidified incubator\nfor viral inoculation with 5% CO2. Following incubation, the inoculum\nwas removed, and the cells were cultured in Dulbecco’s Modified Eagle\nMedium (DMEM) supplemented with 2% BVDV-negative FBS. The\ncultures were maintained under these conditions for 96 h, after which\nthe cells underwent three freeze–thaw cycles to release intracellular\nvirus particles. Six additional cell passages were performed to enhance\nviral propagation. For electron microscopy, virus particles were\npurified via sucrose density gradient centrifugation and negatively\nstained using 2% phosphotungstic acid.\nIndirect immunofluorescence assay (IFA)\nMDBK cells were seeded into 12 wells of a 96-well culture plate and\nallowed to grow until they reached 70–80% confluency. Four wells were\ninoculated with the previously prepared virus solution, while the\nremaining eight were negative controls. The cells were then incubated for\n48 h. Following incubation, the cells were fixed with 4% formaldehyde\nfor 30 min, then blocked with 1% bovine serum albumin (BSA) at room\ntemperature for 30 min. A mouse monoclonal antibody targeting the\n\nThe cells were then incubated for\n48 h.Following incubation, the cells were fixed with 4% formaldehyde\nfor 30 min, then blocked with 1% bovine serum albumin (BSA) at room\ntemperature for 30 min.A mouse monoclonal antibody targeting the\nBVDV E2 protein (IgG2a isotype; kindly provided by Prof. Changchun\nTu) was applied at a 1:1000 dilution, and the plates were incubated for 1 h\nat 37°C. After washing with phosphate-buffered saline containing Tween\n20 (PBST), the cells were incubated with Alexa Fluor 488-conjugated\ndonkey anti-mouse IgG (Invitrogen) at a 1:500 dilution for 2 h at\n37°C. Following a final wash with PBST, fluorescence was visualized\nusing an Olympus CKX53 fluorescence microscope at 5 × magnification.\nComplete genomic amplification and\nsequencing\nThe PCR assays were established to amplify the BVDV genomic\nsequence using primer pairs detailed in Table 1. RNA extraction and\nreverse transcription-PCR (RT-PCR) were carried out following the\nsame protocol described previously for BVDV detection. Positive PCR\nTABLE 1  Primers used in the present study.\nPrimer\nname\nSequence (5′-3′)\nProduct\nsize (bp)\n1F1\nGTATACGAGGTTAGGCAAGTTCTCG\n462\n1R462\nCTTGGTCGTATACTGGTTCCTCCAC\n2F469\nGTAATCCTTTGTTTGGAGAAAGAGG\n1,269\n2R1737\nGGTAGTGAGCTGTGTCTTGAATCAC\n3F1708\nCTAGTGTGATTCAAGACACAGCTC\n1,475\n3R3182\nAGAAGTCTCATCCACTTGTCTGTAG\n4F3131\nCAAATGCAGACTGAAGAATGAGAG\n1,645\n4R4775\nGTAGACTTCTTCCTCCTCATACCAG\n5F4722\nCATAAAGTTAGGAACCAGACTGTAG\n1,469\n5R6189\nCCACTCTTATGCTCTCAGAAAATC\n6F6164\nCAGATTTTCTGAGAGCATAAGAGTG\n1,469\n6R7659\nGTGTAGCGAATGCAGTTTCATG\n7F7641\nGAAACTGCATTCGCTACACTAGTG\n1,540\n7R9180\nCTGGTTTTATAGTCCCGATATCTTC\n8F9159\nGATATCGGGACTATAAAACCAGTAC\n1,581\n8R10739\nCCAATCATCACTGACATCTCTCTTC\n9F10715\nGAAGAGAGATGTCAGTGATGATTGG\n1,559\n9R12273\nTGGGGCTGTTAAGGGTTTTCCCTAG\n\nPan et al.\b\n10.3389/fvets.2025.1629211\nFrontiers in Veterinary Science\n03\nfrontiersin.org\nproducts were cloned into the pESI-T vector (Yeasen Biotech) and\ntransformed into E. coli DH5α chemically competent cells (Weidi\nBiotech). Three independent clones from each amplicon were selected\nfor Sanger sequencing (Sangon Biotech).\nPhylogenetic analyses\nA total of 23 BVDV reference strains representing diverse genotypes\nfrom the United States, Japan, Germany, and China were retrieved from\nGenBank. Detailed information, including GenBank accession numbers,\nis presented in Figures 3, 4. Sequence similarity analyses were performed\nusing MegAlign software (Lasergene v7.1). Phylogenetic trees were\nconstructed using the maximum-likelihood method based on the\nTamura–Nei model. To assess the robustness of the inferred tree\ntopologies, bootstrap analysis with 1,000 replicates was conducted (23).\nResults\nVirus isolation and identification\nRT-PCR analysis revealed a specific 462 bp amplicon corresponding\nto BVDV in all FBS samples obtained from Biological Industries, but not\nin those from ExCell Bio. The two BVDV-positive commercial FBS was\nthen used to inoculate MDBK cells, which were monitored daily. After\nsix successive passages, no apparent cytopathic effects were observed in\nthe infected cells. However, BVDV RNA was detected by RT-PCR in one\nof the two culture supernatant, indicating the presence of an NCP\nBVDV strain. This isolate was designated BVDV BI-2023. The presence\nof BVDV BI-2023 in the commercial FBS was further confirmed by\nindirect immunofluorescence assay (Figures 1A,B), and its identity was\nvalidated via transmission electron microscopy (TEM) (Figure 2).\nSequence analyses of 5’-UTR\nTo determine the genotype of BVDV BI-2023, phylogenetic\nanalysis was performed using the maximum likelihood method\nbased on its 5’-UTR sequence, alongside 20 BVDV reference strains\n(Figure 3). The results showed that BVDV BI-2023 clustered within\nthe BVDV1 (Pestivirus bovis) genotype 1b group. Sequence\nalignment revealed that the new isolate shared 87.4–97.1%\nnucleotide identity with known BVDV1 (Pestivirus bovis) genotype\n\nThe results showed that BVDV BI-2023 clustered within\nthe BVDV1 (Pestivirus bovis) genotype 1b group.Sequence\nalignment revealed that the new isolate shared 87.4–97.1%\nnucleotide identity with known BVDV1 (Pestivirus bovis) genotype\n1b strains, including USMARC/PI-91-21/CD46GE, USMARC/\nPI-91-21/MDBK, AU526, CP7, CC13B, and JL-1. However, its\nsimilarity with other BVDV1 (Pestivirus bovis) genotype ranged\nfrom 68.1 to 88.6% (Table  2). These findings support the\nclassification of BVDV BI-2023 as a member of the BVDV1\n(Pestivirus bovis) genotype 1b lineage.\nWhole-genome sequence comparisons\nand phylogenetic analyses\nTo investigate the molecular characteristics of the isolate, the full-\nlength genome of BVDV BI-2023 was amplified and sequenced,\nyielding a sequence of 12,273 nucleotides, which has been submitted\nto GenBank under accession number OR753412. Comparative\nanalysis revealed that BI-2023 shared 92.4–95.2% nucleotide identity\nwith BVDV1 (Pestivirus bovis) genotype 1b reference strains (Table 3).\nIn particular, it showed 95.2% identity with the BVDV1 (Pestivirus\nbovis) genotype 1b reference strain CP7, confirming its classification\nas a BVDV1 (Pestivirus bovis) genotype 1b group member (Table 3).\nFurther multiple sequence alignments revealed that BVDV BI-2023\nisolated in the present study shared 90.7–97.0% nucleotide (Table 4)\nand 90.1–100% amino acid (Table 5) identities for the Npro, Core, Erns,\nE1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B genes, with\nBVDV1 (Pestivirus bovis) genotype 1b reference strain CC13B, CP7,\nJL-1, USMARC/PI-91-21/CD46GE, USMARC/PI-91-21/MDBK, and\nTJ2018 etc.\nFurthermore, phylogenetic analysis based on the complete\ngenome sequence demonstrated that BVDV BI-2023 is closely\naligned with established BVDV1 (Pestivirus bovis) genotype 1b\nreference strains, including CC13B, CP7, JL-1, USMARC/\nPI-91-21/CD46GE, and TJ2018. As depicted in Figure 4, BI-2023\nclusters within the genotype 1b lineage, highlighting its strong\ngenetic relatedness to these strains and further supporting its\nclassification as a member of BVDV1 (Pestivirus bovis) genotype\n1b (Figure 4).\nFIGURE 1\n\nAs depicted in Figure 4, BI-2023\nclusters within the genotype 1b lineage, highlighting its strong\ngenetic relatedness to these strains and further supporting its\nclassification as a member of BVDV1 (Pestivirus bovis) genotype\n1b (Figure 4).FIGURE 1\nIFA for BVDV. (A) IFA detection of BVDV-infected MDBK cells using an anti-BVDV monoclonal antibody. (B) MDBK cells negative for BVDV were used as\nthe control.\n\nPan et al.\b\n10.3389/fvets.2025.1629211\nFrontiers in Veterinary Science\n04\nfrontiersin.org\nBVDV BI-2023 encodes E2, and Erns has\nseveral unique mutations in functional\ndomains\nA comparative analysis of the E2 and Erns amino acid sequences\namong BVDV1 (Pestivirus bovis) genotype 1b strains revealed\ndistinct differences in BVDV BI-2023. Residues F6, I16, I/T55,\nV77, and D/N83 within domain DA, as well as L/T/R252 and\nO263 in domain DC of the E2 protein, were conserved across all\nanalyzed BVDV1 (Pestivirus bovis) genotype 1b reference strains\nexcept in BVDV BI-2023 (Figure 5A). Furthermore, two unique\nsubstitutions were identified in the Erns protein: Q150K within one\nof the seven known linear epitopes, and S190R in the C-terminal\ndomain. These findings suggest that BVDV BI-2023 may represent\na novel variant within the BVDV1 (Pestivirus bovis) genotype 1b\ngroup (Figure 5B).\nDiscussion\nBovine serum has been an essential component in cell culture\nsystems for over five decades, yet it continues to be frequently\ncontaminated with adventitious viruses. There are numerous viral\nagents in bovine serum and its derivatives, with BVDV being the\nmost commonly detected contaminant (1–13). In this study, an\nNCP strain of BVDV was successfully detected and isolated from\ncommercial FBS. However, this analysis did not extend to other\npossible adventitious agents or pathogens, which may also pose\nsignificant risks to the safety of biological products. This\nhighlights the critical importance of thorough screening for viral\nFIGURE 3\nPhylogenetic analysis of the new isolates with 23 BVDV reference strains based on the 5′-UTR nucleotide sequences. The 5′-UTR nucleotide\nsequences of the new isolates were aligned with 23 reference BVDV strains, and phylogenetic analysis was carried out by the maximum-likelihood (ML)\nmethod using MEGA7 software (1,000 bootstrap replicates). Fixed circles represent the BVDV BI-2023 strain isolated in this study. The 5′-UTR\nnucleotide sequences of the reference BVDV strains were obtained from the GenBank data.\nFIGURE 2\nThe virions of BVDV BI-2023 display distinct morphologies.\n\nPan et al.\b\n10.3389/fvets.2025.1629211\nFrontiers in Veterinary Science\n05\nfrontiersin.org\ncontaminants in commercial serum and related biological\nmaterials before using.\nWhile PCR offers specific and sensitive detection for known\nviruses, it may fail to identify uncharacterized variants or closely\nrelated viral species. Commercial serum products may harbor\nundetected contaminants without comprehensive testing, adversely\naffecting experimental reliability. NCP-type BVDV, in particular, can\npersistently infect cultured cells without producing overt cytopathic\neffects, making it difficult to detect and potentially compromising\nexperimental results, diagnostic assays, and clinical trial outcomes.\nAdvanced techniques such as next-generation sequencing provide a\nmore robust approach for identifying these hidden viral contaminants.\nBased on 5′-UTR sequence comparisons, BVDV strains are classified\ninto two species: Pestivirus bovis (BVDV-1) and Pestivirus tauri (BVDV-\n2). Pestivirus bovis has been subdivided into 25 genotypes (1a to 1.25)\n(16–22). Previous unpublished sequence data have reported the presence\nof BVDV1 (Pestivirus bovis) genotype 1b in various regions of Israel (24).\nIn the current study, a putative BVDV1 (Pestivirus bovis) genotype 1b\nstrain was identified in commercial FBS sourced from Israel, suggesting\nthat this virus may be  circulating within Israeli cattle populations.\nFurther investigations are warranted to screen additional batches or\nsources of FBS to assess the extent of viral contamination.\nThe FBS is an essential raw material for cell culture applications\n(1–3). However, if contaminated with viruses, it can transmit these\npathogens into cell culture-derived vaccines and other biological\nproducts (1–3). Previous studies have demonstrated that commercial\nbovine serum products from various regions in China frequently\ncontain at least one type of viral contaminant (4–9). In a 1991\ninvestigation, the National Animal Disease Center identified 93 viral\nisolates from 190 batches of commercial FBS (5). Multiple reports have\nalso documented the presence of BVDV1 in imported commercial\n\nIn a 1991\ninvestigation, the National Animal Disease Center identified 93 viral\nisolates from 190 batches of commercial FBS (5).Multiple reports have\nalso documented the presence of BVDV1 in imported commercial\nFBS, highlighting the widespread issue of BVDV contamination in\nboth domestic and imported serum sources (4, 6–9).\nTABLE 2  Homology analysis of the BVDV BI-2023 5’UTR gene in this\nstudy.\nIsolate\nNucleotides (%)\nBVDV1b CC13B, CP7, JL-1, and TJ2108 etc.\n87.4–97.1\nBVDV1q SD0803\n83.8\nBVDV1m SD-15\n82.6\nBVDV1o IS2601ncp\n83.8\nBVDV1n Shitara02/06\n88.6\nBVDV1k SuwaNcp\n83.3\nBVDV1d 10JJ-SKR\n85.5\nBVDV1c Bega-like\n68.1\nBVDV1j KS86-1ncp\n85.3\nBVDV1a NADL\n86.7\nBVDV1e Carlito\n83.1\nBVDV2a SH-28\n72.0\nBVDV2b SH2210-23\n71.5\nBVDV2c SD1301\n71.0\nBVDV3 LV168/36/16RN\n67.6\nTABLE 3  Homology analysis of the full genome of BVDV BI-2023 in this\nstudy.\nIsolate\nNucleotides (%)\nBVDV1b CC13B, CP7, JL-1, and TJ2108 etc.\n92.4–95.2\nBVDV1q SD0803\n79.0\nBVDV1m SD-15\n79.1\nBVDV1o IS2601ncp\n78.8\nBVDV1n Shitara02/06\n78.7\nBVDV1k SuwaNcp\n79.5\nBVDV1d 10JJ-SKR\n80.3\nBVDV1c Bega-like\n80.3\nBVDV1j KS86-1ncp\n80.4\nBVDV1a NADL\n79.7\nBVDV1e Carlito\n79.8\nBVDV2a SH-28\n70.1\nBVDV2b SH2210-23\n70.1\nBVDV2c SD1301\n69.8\nBVDV3 LV168/36/16RN\n67.6\nTABLE 4  Homology analysis of the BVDV BI-2023 and BVDV1 (Pestivirus\nbovis) genotype 1b reference strains 11 genes in this study.\nGene\nNucleotides (%)\nNpro\n91.5–97.0\nCore\n90.7–93.6\nErns\n92.4–95.9\nE1\n91.3–94.4\nE2\n90.5–93.8\np7\n91.0–93.3\nNS2-NS3\n94.1–96.6\nNS4A\n93.4–97.0\nNS4B\n91.7–95.2\nNS5A\n90.7–94.1\nNS5B\n93.6–95.4\nTABLE 5  Homology analysis of the BVDV BI-2023 and BVDV1 (Pestivirus\nbovis) genotype 1b reference strains 11 proteins in this study.\nProtein\nAmino acid (%)\nNpro\n94.6–97.0\nCore\n92.3–95.2\nErns\n96.5–98.7\nE1\n93.3–96.4\nE2\n90.1–92.2\np7\n91.4–94.3\nNS2-NS3\n96.4–98.0\nNS4A\n100\nNS4B\n95.6–96.8\nNS5A\n91.3–95.4\nNS5B\n96.4–97.4\n\nPan et al.\b\n10.3389/fvets.2025.1629211\nFrontiers in Veterinary Science\n06\nfrontiersin.org\nFurthermore, a novel putative pestivirus species provisionally named\n“HoBi-like” and currently classified by the International Committee on\nTaxonomy of Viruses as Pestivirus brazilense was initially detected in\nEurope in FBS imported from Brazil (25, 26). Contaminated serum can\ninterfere with the diagnosis of viral infection, and using such contaminated\nserum to produce vaccines can lead to seroconversion or illness in\nvaccinated animals (4–6). These findings underscore the frequent\noccurrence of BVDV contamination in cattle-derived materials.\nCurrently, China restricts the import of FBS to countries certified\nfree of bovine spongiform encephalopathy, such as Australia,\nNew Zealand, and Uruguay. However, the circulation of American\nFBS with unverified origins in the domestic market raises concerns\nabout the effectiveness of animal disease prevention and control\nefforts in China (6). In this study, the BVDV BI-2023 strain identified\nin the FBS was an NCP type, posing a potential risk of\nwidespread infection and the birth of persistently infected animals. As\na result, laboratories must screen FBS for exogenous viruses and\ndetermine their genotypes before use.\nGenome sequencing of the BVDV BI-2023 strain revealed a total of\n12,273 nucleotides encoding a polyprotein comprising four structural and\nseven non-structural proteins. Based on 5’-UTR sequence comparison,\nthis isolate was classified as a BVDV1b genotype. A previous study by Zhu\net al. proposed that the dominant BVDV1 (Pestivirus bovis) genotype 1b\nstrains circulating in China (e.g., CC13B and JL-1) likely originated from\nEurope (27). In the present study, alignment analyses using the full\ngenome, 5’-UTR, structural, and non-structural protein sequences\nshowed that BVDV BI-2023 shared high sequence identity with several\nforeign reference strains, including USMARC/PI-91-21/CD46GE,\nUSMARC/PI-91-21/MDBK, and AU526, all originally isolated in the\nUnited States. Phylogenetic analysis of the full genome and the 5’-UTR\n\nPhylogenetic analysis of the full genome and the 5’-UTR\nfurther confirmed a closer genetic relationship between BVDV BI-2023\nand these United States-derived strains than with other BVDV1 (Pestivirus\nbovis) genotype 1b isolates. These findings support the hypothesis that the\nBVDV1 (Pestivirus bovis) genotype 1b strains currently reported in China\nmay have been introduced abroad, highlighting the need to monitor this\ngenotype in imported FBS and domestic cattle populations.\nGlycoprotein E2 is the primary envelope protein displayed on the\nsurface of BVDV virions and plays a key role in eliciting neutralizing\nantibody responses during infection (28). Structurally, the E2 protein\nis organized into four domains: DA (residues 4–87), DB (residues\n88–164), DC (residues 165–271), and DD (residues 272–333) (28).\nThere are three immunodominant regions in the BVDV E2 protein,\nlocated at residues 16–26, 71–74, and 142–150 (29–31), all of which\nare within the DC domain and contribute to neutralization. In this\nstudy, a comparison of BVDV BI-2023 with other BVDV1 (Pestivirus\nbovis) genotype 1b reference strains indicated that the BVDV BI-2023\nE2 protein had one unique mutations located at residue I16V of\nimmunodominant regions (Figure 5A). The unique mutation may\nreduce antibody-binding affinity, potentially enabling BVDV BI-2023\nto evade host immune recognition.\nErns is another envelope glycoprotein involved in virus\nneutralization and is distinguished by its intrinsic ribonuclease\n(RNase) activity (28). Seven linear epitopes of the Erns protein from\nBVDV1 (Pestivirus\nbovis) genotype 1b reference strains\n31GIWPEKIC38, 65NYTCCKLQ72, 127QARNRPTT134, 145SFAGTVIE152,\n161VEDILY166, 114CRYDKNTDVNV124, and 116YDKNTDVNV124 have\nFIGURE 4\nPhylogenetic analysis of the new isolates with pestiviruses based on the full-length nucleotide sequences. The genomic sequences of the new isolates\nwith pestiviruses were based on the full-length nucleotide sequences. The genomic sequences of the new isolates were generated and aligned with\n\nThe genomic sequences of the new isolates\nwith pestiviruses were based on the full-length nucleotide sequences.The genomic sequences of the new isolates were generated and aligned with\nrepresentative pestiviruses, including Pestivirus bovis, Pestivirus tauri, and Pestivirus brazilense, in this study. Phylogenetic analysis was carried out using the\nmaximum-likelihood (ML) method using MEGA7 software (1,000 bootstrap replicates). Fixed circles represent the BVDV BI-2023 strain isolated in this study.\n\nPan et al.\b\n10.3389/fvets.2025.1629211\nFrontiers in Veterinary Science\n07\nfrontiersin.org\nbeen identified as key antibody-interacting regions (28). In the current \nstudy, BVDV BI-2023 showed a unique mutation within the fourth \nlinear epitope, altering 145SFAGTVIE152 to 145SFAGTVQ151KE152 \n(Figure  5B), implying potential differences in antibody-binding \nproperties between BVDV BI-2023 and reference genotype 1b strains. \nFurthermore, the C-terminal region of Erns is critical for its secretion \nand intracellular retention, with residues 183, 190, and 208 playing \nimportant roles (28). Sequence analysis revealed a unique R190S \nsubstitution in BVDV BI-2023 (Figure  5B), suggesting that this \nmutation might affect the protein’s secretion dynamics. These findings \nindicate that BVDV BI-2023 shows molecular differences in Erns \ncompared to established genotype 1b reference strains.\nConclusion\nIn conclusion, this study identified a potentially novel strain of \nBVDV1 (Pestivirus bovis) genotype 1b from commercially imported \nFIGURE 5\nAnalysis of the E2 and Erns protein sequence. Analysis of the BVDV1b BI-2023 E2 protein sequence (A). Analysis of the BVDV1b BI-2023 Erns protein \nsequence (B). Sequence identity with the BVDV1b BI-2023 isolate (CLC Sequence Viewer 8) is indicated by dots.\n\nPan et al.\b\n10.3389/fvets.2025.1629211\nFrontiers in Veterinary Science\n08\nfrontiersin.org\nFBS. Given the risk of viral contamination in cell cultures and\nbiological products and the potential for broader dissemination, the\nimplicated serum batch should be discarded. These findings highlight\nthe urgent need for regulatory authorities to implement routine viral\nscreening protocols for all imported serum batches to safeguard\nlaboratory research and animal health.\nData availability statement\nThe datasets presented in this study can be  found in online\nrepositories. The names of the repository/repositories and accession\nnumber(s) can be found in the article/supplementary material.\nAuthor contributions\nJP: Investigation, Software, Writing – original draft. JJ: Investigation,\nSoftware, Writing – original draft. SM: Investigation, Writing – original\ndraft. XC: Investigation, Resources, Writing  – original draft. RD:\nInvestigation, Resources, Writing  – original draft. SG: Investigation,\nWriting – original draft, Funding acquisition. LK: Writing – original draft,\nInvestigation, Resources. JX: Conceptualization, Funding acquisition,\nWriting – original draft, Writing – review & editing. PT:Investigation,\nSoftware, Resources, Writing – original draft.\nFunding\nThe author(s) declare that financial support was received for the\nresearch and/or publication of this article. The Major Special Science\nand Technology Project of Xinjiang Uyghur Autonomous Region\n(grant no. 2023A02007-2) and the Key Research and Development\nProgram of Gansu Province (grant no. 22YF7NA030).\nConflict of interest\nThe authors declare that the research was conducted in the\nabsence of any commercial or financial relationships that could\nbe construed as a potential conflict of interest.\nGenerative AI statement\nThe authors declare that no Gen AI was used in the creation of\nthis manuscript.\nAny alternative text (alt text) provided alongside figures in this\narticle has been generated by Frontiers with the support of artificial\nintelligence and reasonable efforts have been made to ensure accuracy,\nincluding review by the authors wherever possible. 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