Animals doi: 10.3390/ani16213505

Authors: Jong-Yeol Park In-Kyu Lee Ji-Eun Son Ki-Woong Kim Kai-Qiong Zheng Cheng-Dong Yu Won-Bin Jeon Hyung-Kwan Jang Bai Wei Min Kang

H9N2 low-pathogenic avian influenza remains endemic in Korean poultry production, where commercial broilers rely largely on maternally derived antibodies (MDA) transferred from vaccinated breeder hens. This study quantified MDA transfer and decay and evaluated protection against homologous Y280-lineage H9N2 challenge. Paired sera were collected from breeder hens and day-old progeny on six commercial farms. MDA decay was monitored in 90 broilers from 1 to 22 days of age, and 32 birds with individual HI titers of 0, 2, 4, or 6 log2 were challenged intranasally at 7 days of age. Transfer efficiency averaged 18.2 ± 11.5%, and MDA had an estimated half-life of 3.2 days. Clinical signs occurred in 8/8, 8/8, 7/8, and 0/8 birds in the MDA 0, 2, 4, and 6 groups, respectively. Cecal tonsil virus detection decreased from 87.5% in the MDA 0 group to 37.5% in the MDA 6 group, although the pairwise difference was not statistically significant (p = 0.119). Oropharyngeal shedding occurred in all challenged birds at comparable titers. These findings suggest that higher MDA levels are associated with reduced clinical disease and lower cecal tonsil virus detection but do not prevent oropharyngeal shedding under the conditions tested.

Animals doi: 10.3390/ani16213504

Authors: Diego Amaya Jhones O. Sarturi Janghan Choi

This study investigated the temporal metabolic differences between sow colostrum and mature milk using an untargeted metabolomics approach. Nine multiparous Camborough sows were longitudinally sampled for colostrum and mature milk collection. Proximate analysis demonstrated that protein content was significantly greater in colostrum than in mature milk (p < 0.05). Untargeted metabolomic profiling successfully putatively annotated 3906 metabolites, with 560 significantly increased and 1083 decreased in colostrum compared to mature milk. Multivariate statistical analyses, including PCA and OPLS-DA, confirmed distinct metabolic separation between the two lactation stages. Furthermore, pathway enrichment and Differential Abundance score analyses revealed that diverse lipid metabolism pathways, fat digestion and absorption, thermogenesis, and glycerolipid synthesis were significantly elevated in mature milk. Physicochemical evaluation indicated that mature-milk-enriched metabolites exhibited significantly greater molecular weights and Log P values, reflecting enhanced structural complexity and lipophilicity. These comprehensive differences suggest that mature milk features higher-molecular-weight and lipophilic metabolites aligned with changing nutritional requirements during later lactation, whereas colostrum is enriched with simpler fractions characteristic of the early postpartum stage. Collectively, this study establishes a comprehensive comparative metabolic baseline of sow milk, providing a robust framework for understanding maternal metabolite dynamics with potential application for optimizing sow nutrition and milk quality.

Animals doi: 10.3390/ani16213503

Authors: Molthida Rungchaicharoenphai Patcharida Sriwicha Pheeraphong Phaengphairee Anusorn Cherdthong Sawitree Wongtangtintharn Benjamad Khonkhaeng Saroat Rawdkuen Chanon Suntara

This study evaluated the effects of protected and non-protected black soldier fly larvae oil (BSFLO), palm oil (PO), and rice bran oil (RBO) on ruminal fermentation characteristics and nutrient digestibility using an in vitro gas production technique. A completely randomized design with a 3 × 3 + 1 factorial arrangement was employed, consisting of three oil sources, three oil forms (non-protected, calcium soap, and potassium soap) supplemented at 4% of dry matter, and a control without oil. The results showed that oil supplementation without the basal diet reduced total volatile fatty acid (TVFA) production and ammonia nitrogen (NH3-N) concentration at 24 h, indicating an inhibitory effect of free fatty acids on rumen microorganisms. With the basal diet, TVFA concentrations at 24 h were higher in oil-supplemented treatments compared to the control. Specifically, protected BSFLO in both Ca-soap and K-soap forms showed the numerically highest TVFA concentrations (127.02 and 125.87 mmol, respectively), although these did not differ significantly from those of RBO Ca-soap and K-soap; BSFLO K-soap had a lower TVFA concentration than the control at 48 h. Oil supplementation significantly modified VFA profiles by decreasing the proportion of acetic acid, increasing propionic acid, and consequently lowering the acetate-to-propionate ratio. Ruminal pH was not affected by oil supplementation. While in vitro dry matter degradability was lower in the oil-supplemented treatments than in the control, IVDMD did not show differences between non-protected oil and protected oil treatments. Abomasal simulated digestion was not adversely affected by oil source or protection form. In conclusion, in the presence of a basal diet, BSFLO in Ca-soap and K-soap forms increased TVFA at 24 h, and BSFLO in all forms increased propionate proportions, without compromising rumen stability or abomasal simulated digestion under the conditions tested. These in vitro results indicate protected BSFLO’s potential as a lipid source in ruminant diets and warrant in vivo confirmation.

Animals doi: 10.3390/ani16213502

Authors: Amir Mohammad Shadjou Saghar Mohsenian Parham Vatankhah Farzam Hosseinpour Vardin Uttama Deb Fatemeh Taghvaei Dongyi Wang Marty Matlock

Artificial intelligence (AI) and machine learning (ML) are promoted as tools for improving the environmental sustainability of livestock production, but it remains unknown whether the models reported in this literature are used to support real-world decisions. We systematically reviewed studies that applied AI or ML to environmental sustainability outcomes in livestock production. Four databases were searched in January 2026, and 165 of 1994 screened records were included. The protocol was registered, but the main outcome was defined after data extraction, so the analysis is exploratory. A study counted as delivering decision support only if it named a specific decision and used its model to compare options or recommend an action. The authors of 96 studies (58.2%) stated that their work could support decisions, but only 26 studies (15.8%) met our definition. This gap appeared in 10 of 11 application areas. The gap held under both a broader and a stricter definition, and when a second coder repeated the coding. Only 30 studies (18.2%) tested their model outside the setting where it was developed, and 17 (10.3%) included an optimization step, which turns a prediction into a choice. Linking ML with mechanistic farm models is a promising approach that still needs testing. We also describe the main components a practical decision tool would need.

Animals doi: 10.3390/ani16213500

Authors: Gayatree Behera Sabine E. Hammer Astrid S. Holzer Mona Saleh

The assembly of the gut microbiome in fish is a dynamic and tightly regulated process influenced by host genetics, early-life environmental exposure, diet, and aquaculture practices. Increasing evidence indicates that deliberate manipulation of this assembly through probiotics, prebiotics, synbiotics, environmental microbiome management, and husbandry interventions can significantly alter host–microbe interactions and downstream health outcomes. This review synthesizes we have summarized the current understanding about the regulatory principles of gut microbiome assembly in fish and explored the impact of manipulating the microbiome assembly process. Emphasis will be given to the early-life microbial colonization process, which is an important time point for microbiota succession and plays an important role in the development of immunity and resistance against diseases. Aquaculture systems, like recirculating aquaculture system (RAS) and flow-through system, where there is variation in microbiome exposure and water quality, further influence the process of microbiome assembly and pathogen dynamics. Several manipulative techniques, such as live feed enrichment, the seeding of microbes, and the management of water microbiomes, have been successful in inducing the growth of beneficial microbes while suppressing opportunistic pathogens. However, the risk of microbiome dysbiosis, reduction in microbial resilience, and pathogen resistance due to microbiome manipulation cannot be overlooked. Understanding these trade-offs is essential for developing sustainable and effective strategies. By integrating insights from microbiology, immunology, and aquaculture science, this review carries out a systematic PRISMA analysis, highlights the consequences of microbiome manipulation on disease susceptibility, and provides a framework for designing microbiome-informed interventions to enhance fish health and productivity in modern aquaculture systems.

Animals doi: 10.3390/ani16213499

Authors: Mery Campalto Giammarco Assirelli Andrea Simonetto Elisa Mazzotta Arianna Brandimarti Marilena Carrino Francesca Crescenti Alda Natale Letizia Ceglie Laura Gagliazzo Alessia Franzoso Federica Obber Nicola Pozzato

Interfaces among humans, wildlife and domestic animals represent critical hotspots for the emergence and spread of infectious agents. The gray wolf (Canis lupus) and the golden jackal (Canis aureus) are expanding wild canid populations in Italy, whose increasing overlap with anthropized environments may facilitate pathogen transmission. This study investigated mammalian orthoreovirus (MRV), rotavirus A (RVA), canine coronavirus (CCoV), canine parvovirus (CPV2) and feline parvovirus (FPV) in wild canids from north-eastern Italy. The 101 carcasses (83 gray wolves and 18 golden jackals) obtained through passive wildlife surveillance were examined post-mortem. Tissue samples and swabs were screened using molecular assays, and positive samples were characterized and tested for virus isolation. MRV was detected in gray wolves (27.7%) and golden jackals (33.3%), representing the first report and successful isolation from both species. RVA prevalence was 60.2% in gray wolves and 61.1% in golden jackals. CCoV occurred exclusively in gray wolves (12.5%). CPV-2 was found in 10.8% of gray wolves and 35.3% of golden jackals, with FPV accounting for 66.7% of CPV-2-positive golden jackal samples. These findings provide valuable data on the circulation of MRV, RVA, CCoV, and CPV-2/FPV in free-ranging gray wolves and golden jackals and highlight the value of passive surveillance within a One Health framework overall.

Animals doi: 10.3390/ani16213501

Authors: Sihyeon Hwang Geonil Moon Young-Gwang Cho Chanil Park Inhyung Lee Tran Thi Kim Ngan Namsoo Kim

Patellar luxation is uncommon in horses, and traumatic lateral luxation in adult equids has rarely been reported. A 3-year-old, 75 kg female Shetland pony presented with severe right hindlimb lameness and minimal weight bearing approximately 2 weeks after a suspected traumatic event. The right patella was fixed lateral to the femoral trochlea and could not be manually reduced during conscious examination. Radiographs confirmed lateral patellar luxation without apparent osseous deformity. Following general anesthesia and surgical exposure, the patella was manually reduced. Treatment comprised lateral retinacular and fascial release, medial capsular imbrication and extra-articular polypropylene mesh augmentation. No osseous corrective procedure was performed. Radiographs obtained 2 days postoperatively confirmed maintenance of reduction. Partial weight bearing was observed approximately 10 days after surgery. No surgical site complications were observed during hospitalization, and the pony was discharged on postoperative day 12. At telephone follow-up 37 days after surgery, the owner reported that the pony was walking more frequently. An owner-provided video recorded on postoperative day 130 showed no overt lameness during walking; trotting was not assessed, and no further clinical examination or radiography was performed. Mesh-augmented soft-tissue reconstruction was technically feasible in this adult pony, although objective follow-up remained limited.

Animals doi: 10.3390/ani16213498

Authors: Barbara Wilka Leal Silva Carlos Gabriel Oliveira Freitas Davi Emanuel Ribeiro de Sousa Juliana Paula Martins Alves Camila de Oliveira Campos Ana Raquel Almeida Pinheiro Francisco Bastos Cavalcante Sobrinho Adriana de Queiroz Pinheiro Steffi Lima Araujo Yury Carantino Costa Andrade Francisco Antônio Félix Xavier Júnior Isaac Neto Goes da Silva

Zoonotic sporotrichosis caused by Sporothrix brasiliensis is an emerging public health threat in Brazil. Under a One Health perspective, this study described the temporal and geographic distribution of recorded feline sporotrichosis detections in Fortaleza (2022–2026), as well as their co-occurrence with human notifications. Additionally, the study explored associations with socio-environmental factors and evaluated predictive performance at both the neighborhood and individual-animal levels. Suspected cats underwent cytology, culture, and species-specific calmodulin PCR. Cases were mapped across 116 neighborhoods, and logistic regression and random forest models were evaluated exploratorily at the neighborhood (61 neighborhoods) and animal (239 cats) levels, using grouped nested cross-validation and Spearman correlations corrected for multiple testing. Of 246 cats, 120 were confirmed, and all 37 isolates analyzed were S. brasiliensis. Neighborhoods with cases rose from one (2022) to 34 (March 2026), with 68 human notifications in 25 neighborhoods. After grouped validation, none of the evaluated predictor–outcome contrasts met the significance criterion after BY adjustment, and the exploratory associations were subject to substantial uncertainty. Consequently, no model demonstrated meaningful discriminative performance (out-of-sample AUC-ROC 0.34–0.50 at the neighborhood level and 0.38–0.47 at the animal level). These results are consistent with the complex, multifactorial nature of the disease, in which host behavior, environmental persistence, and surveillance intensity likely interact. These findings confirm the urban establishment of S. brasiliensis and the need for more complete data and robust analytical approaches. Outbreak control requires integrated One Health strategies: expanded access to diagnosis, cat neutering, and strengthened surveillance.

Animals doi: 10.3390/ani16213497

Authors: Wasana Chaisri Duanghathai Saipinta Montira Intanon Noppason Pangprasit Anyaphat Srithanasuwan Witaya Suriyasathaporn

Microbial interactions control mammary gland inflammation and disease severity. This study examined bacterial dynamics, inflammatory responses, and oxidative stress following Staphylococcus hominis (SH) superinfection in cows with chronic subclinical mastitis. Mammary quarters were assigned to varying SH doses: COW1 received 106 and 105 CFU/mL (two quarters each); COW2 received 106 CFU/mL (two quarters) and PBS control (one quarter); COW3 received 104 and 105 CFU/mL (two quarters each); and COW4 received 104 CFU/mL (two quarters). On Day 0, quarters received 5 mL of their assigned preparations. Milk and clinical parameters were evaluated during a pre-challenge baseline and monitored for 8 days post-inoculation. Results showed post-infection mastitis severity depended heavily on prior infections. SH alone functioned as a mild pathogen, inducing short-term inflammation with elevated somatic cell counts and IL-6 levels primarily on Days 1 to 3, but did not independently cause severe tissue damage. The intense immune response, elevated malondialdehyde (MDA) levels, and severe tissue damage were primarily driven by pre-existing major mastitis pathogens. Although S. hominis is inherently a mild pathogen, high bacterial loads synergistically exacerbate oxidative stress and clinical mastitis symptoms in co-infected quarters.

Animals doi: 10.3390/ani16213496

Authors: Héctor A. Uribe Alejandro Velásquez Rodrigo Arias

Beef-on-dairy (BoD) crossbreeding, defined as the planned use of beef sires on dairy dams, has expanded as a strategy to increase the value of dairy-origin calves while maintaining herd replacement requirements. This review critically synthesizes research published since 2016 across six interconnected dimensions: genetics and genomics, management and productive efficiency, carcass performance and meat quality, farm economics, environmental sustainability, and animal welfare. The available evidence indicates that BoD can increase calf market value and improve selected growth and carcass traits, although the magnitude of these effects varies with sire genetic merit, dairy breed, reproductive performance, calf management, and finishing conditions. Genetic selection involves balancing calving ease and other functional traits against postnatal growth and carcass merit. Economic outcomes depend not only on calf prices but also on semen costs, reproductive efficiency, replacement requirements, and rearing and finishing expenses; therefore, improvements in individual production traits do not necessarily translate into greater whole-farm profitability. Environmental performance is similarly context-dependent and requires explicit consideration of system boundaries, functional units, emissions intensity, and potential soil carbon sequestration. Animal welfare must also be evaluated throughout the production chain rather than inferred from productive performance alone. A major limitation of the available evidence is its concentration in intensive and semi-intensive production systems, which restricts its transferability to permanent-pasture environments. Further research should prioritize longitudinal, farm-level evaluations from birth to slaughter under defined pasture-based conditions, integrating genetic and management decisions with productive, economic, environmental, and welfare outcomes.

Animals doi: 10.3390/ani16213495

Authors: Xin Li Zhengbang Chen Xuexi Wang Xinhua Chen Lianghua He Yinnan Mu Pan Qin

Siganus fuscescens, an economically important species in southern China, is noted for its rapid growth and high nutritional value. Nonetheless, the prevalence of darkening syndrome, a condition marked by skin discoloration and significant mortality rates, presents a challenge to aquaculture practices, with its etiology remaining largely unexplored. This study aimed to examine the impacts of varying dietary lipid levels and multivitamin supplementation on the incidence of darkening syndrome in S. fuscescens. Five experimental diets were developed: a low-fat diet containing 10% lipids; a moderate-fat diet with 13% lipids; a moderate-fat diet with 13% lipids supplemented with multivitamins; a high-fat diet containing 16% lipids; and a high-fat diet with 16% lipids supplemented with multivitamins. A 55-day feeding trial was conducted on S. fuscescens specimens. The results indicate that, in this experiment, increasing dietary fat content led to a higher cumulative incidence of darkening syndrome in S. fuscescens. Serological and histological findings suggested that in the 16% lipid diet group, S. fuscescens exhibited liver injury and oxidative stress. Notably, supplementation with multivitamins alleviated these adverse effects. Overall, we recommend using an appropriate dietary fat level and further supplementing with antioxidant multivitamins, which may represent an effective strategy to mitigate darkening syndrome in S. fuscescens.

Animals doi: 10.3390/ani16213494

Authors: Ruiqi Jiao Xin Xu Jun Ji Yanhua Wang Lunguang Yao

Canine circovirus (CCV) infection could cause immunosuppression in dogs, predisposing them to secondary complications. The virus also shows notable cross-species transmission potential. To investigate codon usage bias (CUB) and host adaptation, 102 CCV sequences from various hosts, including dogs, wolves, foxes, cats, and badgers, were analyzed. The mean GC% of CCV’s complete coding sequences was 50.31% (σ = 0.47), indicating no significant compositional bias. Furthermore, the replication protein (Rep) gene of CCV exhibited significant overrepresentation of T-ending codons and strongly avoided A-ending codons, whereas the capsid protein (Cap) gene displayed a clear codon bias toward A-ending codons and avoidance of G- or C-ending codons. Effective number of codons, parity rule 2, and neutrality analyses collectively indicated that natural selection acted as the primary evolutionary force governing the codon usage bias of CCV. Correspondence analysis (CoA) and correlation analysis demonstrated that other factors such as nucleotide composition and translational preferences could also affect CCV’s CUB. However, geography and genotype should not be ignored. The host adaptation analysis suggests that foxes are the most suitable hosts for CCV. This study provides a foundation for understanding CCV’s host adaptation mechanisms.

Animals doi: 10.3390/ani16213493

Authors: Maxime Vandersmissen Alexandre Charles Laurence Evrard Claudia Fraschetto Fabrice Audigié Valeria Busoni

Standing low-field magnetic resonance imaging (MRI) is widely used for subchondral bone (SCB) assessment of the equine proximal phalanx, but radiography remains the most common first-line imaging modality, and no direct comparison between the two is available for SCB sclerosis and resorption. Fifty-four fetlocks from 52 horses referred for standing MRI were retrospectively assessed at the medial glenoid, sagittal groove and lateral glenoid, using an ordinal scale for sclerosis and a four-type nominal classification for resorption. Seven readers of varied clinical backgrounds performed independent radiographic readings; two board-certified radiologists then established a consensus reading, blinded to MRI. Agreement was quantified with Gwet’s agreement coefficients (inter-reader), weighted and unweighted Cohen’s κ (reader-MRI) and symmetric metrics (consensus-MRI). Inter-reader agreement for sclerosis was location-dependent (Gwet’s agreement coefficient = 0.27–0.73, lowest medially) and substantial-to-almost-perfect for resorption (Gwet’s agreement coefficient ≥ 0.72). The radiographic consensus aligned closely with MRI at the sagittal groove but underestimated marked sclerosis medially and over-called it laterally; fracture-type resorption was reliably detected at the sagittal groove but misclassified medially. Radiography can be relied upon for sagittal groove lesions, but complementary MRI is warranted medially when resorption or clinical suspicion of fracture is present, and to confirm radiographic positive calls laterally.

Animals doi: 10.3390/ani16213492

Authors: Ahlem Boubakri Hichem Kacem Slaheddine Selmi Xavier Roca-Geronès Jordi Miquel

Psammomys obesus is a diurnal herbivorous rodent widely distributed across the arid regions of North Africa and the Arabian Peninsula. Although it has ecological, agricultural, public health, and biomedical importance, its helminth fauna remains poorly known. This study provides a comprehensive inventory of helminth parasites infecting P. obesus in southern Tunisia and examines variations in parasitism according to host sex, age, body condition, and sampling region. A total of 152 specimens of P. obesus were captured between September 2023 and March 2026 in the Tamaghza and Djebel Dahar regions. Parasitological examination revealed four helminth species: three cestodes, Gerbillitaenia psammomi, Sudarikovina aegyptica, and Mesocestoides sp. larvae, and one nematode, Syphacia sp. Their respective prevalences were 63.8%, 12.5%, 0.7%, and 16.4%. The species are morphologically described, and genetic data and a phylogenetic tree are provided for Syphacia sp. The findings of Mesocestoides and Syphacia species represent new records for P. obesus. Furthermore, S. aegyptica constitutes a new record for Tunisia. Host age and sampling region significantly influenced the occurrence and abundance of G. psammomi, as adults were more likely to be infected and harbored a significantly higher parasite burden than juveniles. In contrast, neither host sex nor body condition was significantly associated with parasite occurrence or abundance. No significant association between parasite occurrence or abundance and the host’s sex, age, or body condition was found for S. aegyptica and Syphacia sp.

Animals doi: 10.3390/ani16213491

Authors: Birgit Deboutte Luc Peelman Bart J.G. Broeckx

Managers of closed or semi-closed animal breeding programs must balance production targets with the preservation of genetic diversity, yet they often lack training in population genetics and access to integrated, user-friendly tools. RAPPORT (Relatedness Analysis and Population Planning, Organisation, Reporting and Tracking) is a freely available, open-source R Shiny application that combines pedigree-based genetic monitoring with operational breeding management in a single no-code interface. The application computes inbreeding coefficients and pairwise kinship, estimates rate of inbreeding (ΔF) and effective population size (Ne), and visualises inbreeding trends with forward projections. Production planning tools include dam scheduling, litter projections, and phenotype-integrated breeder selection with mean kinship ranking. Mating recommendations are filtered by a per-mating inbreeding increase threshold and user-defined health criteria. Pedigree and phenotypic data are uploaded in Excel format, and any user-defined traits are automatically incorporated into downstream analyses. Using a test case with anonymised data from a Belgian assistance dog breeding program (852 pedigree entries, 65 litters), the numerical correctness of RAPPORT’s pedigree-based genetic metrics was compared and confirmed against established R packages, and RAPPORT’s capacity to identify a production shortfall and support breeding decisions integrating genetic and phenotypic criteria was illustrated. RAPPORT makes population-level genetic management accessible to non-specialist breeding program managers.

Animals doi: 10.3390/ani16213490

Authors: Xuhui Ye Lilu Huang Yuyan Li Zhong Li Yan Chen

To address background redundancy in detection boxes and identity switches caused by variable body orientations, mutual occlusion, and high appearance similarity among group-housed pigs, a multi-object tracking method based on oriented bounding box detection was proposed to improve the accuracy of pig detection and trajectory association in farming environments. First, YOLO11m-OBB was adopted as the baseline detector. Its original backbone was replaced with EfficientViT-M0, and a dual-teacher knowledge distillation framework based on the DINOv3 vision foundation model was constructed, resulting in a lightweight pig detection model named PIG-YOLO. Second, an AdaptiveSORT tracking algorithm was developed within the tracking-by-detection framework. Camera motion compensation, adaptive state prediction, and multi-scale feature extraction modules were incorporated into the multi-object tracking process, together with a three-stage association strategy and a dedicated track management mechanism. Finally, detection, tracking, and cross-dataset generalization experiments were conducted using both public and self-collected pig video datasets. PIG-YOLO achieved a precision of 94.7%, a recall of 88.3%, an mAP50 of 93.1%, and an mAP50–95 of 85.6%, representing improvements of 3.7, 9.9, 8.0, and 8.9 percentage points over the baseline model, respectively. Meanwhile, the number of parameters, floating-point operations, and model size were reduced by 37.7%, 51.5%, and 36.2%, respectively. On the public test dataset, the combination of PIG-YOLO and AdaptiveSORT achieved HOTA, MOTA, and IDF1 scores of 96.32%, 98.82%, and 99.41%, respectively. On the private dataset, the corresponding scores were 94.98%, 97.31%, and 98.86%. In the cross-dataset generalization experiment, the proposed method achieved HOTA, MOTA, and IDF1 scores of 71.231%, 76.043%, and 83.008%, respectively, outperforming the other comparative tracking algorithms in overall performance. The proposed method reduces the computational complexity of the detection model while improving the accuracy of pig detection and the stability of trajectory association. It therefore provides technical support for pig behavior analysis and intelligent monitoring in large-scale pig farms.

Animals doi: 10.3390/ani16213487

Authors: Zhuoyu Yang Peiwen Shi Kaiting Wang Min Ma Yuriko Enomoto Tomoko Suda Junyou Li

Although condensed tannins have shown antiparasitic, antioxidant, and microbiota-modulating effects, their long-term effects on growing–finishing pigs remain unclear. In this study, 24 pigs were randomly assigned at 43 days of age to a control group fed a basal diet or a treatment group supplemented with 0.75% commercial quebracho tannin (MGM-P) for 16 weeks. The effects on A. suum infection, growth performance, fecal microbial community structure, and pork quality were evaluated. The results showed that egg counts, adult worm counts, and the number of infected pigs were not significantly different between groups (p > 0.1). No differences were observed between groups in growth performance, meat texture, juiciness, or fatty acid composition (p > 0.1). Denaturing gradient gel electrophoresis (DGGE) analysis indicated altered fecal microbial community structure in the MGM-P group. The longissimus dorsi from the MGM-P group showed lower lightness and hue angle (p < 0.05), and free amino acids associated with bitter taste, including valine and leucine, also tended to decrease (0.05 < p < 0.1). These findings indicate that long-term supplementation with 0.75% MGM-P did not influence growth performance or overall meat quality in growing–finishing pigs; its efficacy against A. suum infection was limited under the natural infection conditions of this study.

Animals doi: 10.3390/ani16213489

Authors: Yuyang Wang Lifang Wen Wenjin He Jia Fu Panyang Zheng Yuxuan Chen Mingshan Song Baoying Guo Weifeng Wang

Larimichthys crocea is an important marine aquaculture fish in China, but its farming industry has long been threatened by viral diseases. MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression post-transcriptionally, typically by inducing mRNA degradation or translational repression. And they have emerged as essential players in orchestrating antiviral-related cellular responses in fish. In the present study, the large yellow croaker kidney cell line (PCK) was used as an in vitro model. Through transfection of miR-122 mimics and inhibitors, combined with poly(I:C) immune stimulation, transcriptome, dual-luciferase reporter assays and qPCR validation were employed to systematically investigate the immune regulatory mechanism of miR-122. Transcriptome analysis revealed that 1995 and 1577 genes were differentially expressed after miR-122 overexpression and inhibition, respectively, and they were significantly enriched in pathways such as ECM–receptor interaction, focal adhesion, PPAR signaling, and TGF-β signaling. In these DEGs, prkci and slc40a1 were confirmed as direct target genes of miR-122 in large yellow croaker via target gene prediction and dual-luciferase reporter assays. Furthermore, miR-122 overexpression significantly inhibited poly(I:C)-induced superoxide dismutase (SOD) activity, suggesting its involvement in the regulation of oxidative stress. These results support miR-122-responsive transcriptional and redox changes in poly(I:C)-stimulated PCK cells, mediated by targeting prkci and slc40a1 and by affecting SOD-mediated redox balance. This study provides new insights into the miRNA-mediated immune regulatory network in teleosts and offers potential molecular targets for disease-resistant breeding and the development of disease prevention and control strategies in large yellow croaker.

Animals doi: 10.3390/ani16213488

Authors: Sutthida Thiwato Tanom Tathong Anusorn Cherdthong Kampanat Phesatcha

This study evaluated the effects of Mitragyna speciosa leaf powder (MSP) and Saccharomyces cerevisiae (SC) supplementation on nutrient intake and digestibility, rumen fermentation, microbial populations, and microbial protein synthesis in Thai native beef cattle. Four female Thai native cattle (130.0 ± 7.4 kg body weight at the start of the experiment) were randomly assigned in a 4 × 4 Latin square design to four dietary treatments: (T1) total mixed ration (TMR) alone (control); (T2) TMR + MSP at 10 g/head/day; (T3) TMR + SC at 10 g/head/day; and (T4) TMR + MSP at 5 g/head/day + SC at 5 g/head/day. A TMR containing 14% crude protein was offered ad libitum throughout four 21-day periods. Total dry matter intake and nutrient intakes did not differ among treatments (p > 0.05), although the planned contrast between the control and the supplemented treatments indicated a small shortfall in the control for dry matter intake (p = 0.041). Apparent total-tract digestibility of dry matter, organic matter, crude protein, and acid detergent fiber was higher in supplemented animals than in the control (p < 0.05), and neutral detergent fiber digestibility tended to increase (p = 0.052). Ruminal pH increased, significantly so in the two treatments containing SC (p = 0.001), whereas ruminal ammonia nitrogen and blood urea nitrogen decreased with supplementation (p < 0.05). Total volatile fatty acid concentration was unchanged, but the propionate proportion increased and estimated methane production decreased (p < 0.05). Total bacterial counts increased and protozoal counts decreased with supplementation (p < 0.05), while fungal counts were unaffected. Urinary purine derivative excretion and absorption increased with supplementation, and microbial nitrogen supply rose from 24.66 to 34.21–35.93 g N/d (p = 0.002). The efficiency of microbial nitrogen supply rose from 15.44 to 19.30–20.07 g N/kg of organic matter digested in the rumen (p = 0.007). Supplementation with MSP and SC, alone or in combination at 5 g of each, therefore enhances nutrient digestibility and rumen fermentation efficiency in Thai native beef cattle without depressing intake.

Animals doi: 10.3390/ani16213486

Authors: Michael Patrignani Jean-Marie Graïc Tommaso Gerussi Valentina Vadori Livio Finos Enrico Grisan Antonella Peruffo

Neurohistological analysis has evolved from descriptive microscopy and classical staining toward quantitative, standardized, and increasingly automated approaches. This narrative review traces this evolution in domestic animals, such as dogs, cats, horses, cattle, pigs, sheep, and goats, with humans and common laboratory animals as methodological comparators, based on the literature retrieved from PubMed, Scopus, and Web of Science. Gross dissection, selective staining, immunohistochemistry, and neuronal tracing progressively expanded the structural and neurochemical information obtainable from the brain, while macroscopic and microscopic morphometry, including design-based stereology, enabled unbiased estimation of brain volumes and cell numbers. Digital microscopy made histological slides computable, and machine learning and deep learning now allow automated segmentation directly from raw images, while magnetic resonance imaging and spatial transcriptomics link histology to connectivity and gene expression. However, direct evidence in domestic animals remains restricted to a few species and brain regions: annotated collections are scarce, data are poorly integrated, and most tools are calibrated on human, mouse, or rat brains. Open resources such as the CytoDArk0 dataset and the CISCA segmentation method are early steps forward. Progress will require species-specific brain atlases, shared protocols, and validated, interpretable methods that treat anatomical differences between species as biological information rather than noise.

Animals doi: 10.3390/ani16213485

Authors: Na Li Fan Li Mingqi Zhang Fanfan Zhang Chengcheng Wu Lizhen Hu Jun Xu

This study evaluated testicular and rumen microbial responses to selenium-enriched malt in heat-exposed Simmental bulls. Eighteen bulls received a basal diet alone or supplemented to provide target supplemental Se levels of 0.3 or 0.5 mg/kg dietary DM. Metagenomics included six bulls per group, host assays four per group, and RNA sequencing three per group. Compared with controls, the high-dose group had higher serum antioxidant indices and reproductive hormone concentrations, lower inflammatory markers, higher Johnsen scores, and lower TUNEL positivity. Gene-level FDR control identified 23, 1, and 4 differentially expressed genes in the low-dose versus control, high-dose versus control, and high-dose versus low-dose contrasts, respectively. Provider-reported GSEA showed negative normalized enrichment scores for steroid biosynthesis in the control-versus-low-dose and control-versus-high-dose contrasts, indicating enrichment toward the supplemented side under the provider’s contrast convention. qPCR showed lower CYP11A1, CYP17A1, SCARB1, and LDLR expression at the low dose, whereas Bax, Bcl-2, and CYP17A1 protein abundance did not differ. Rumen microbial diversity and community composition differed among groups, and two post hoc selected cofactor-related pathways had lower abundance at the low dose. None of the 160 microbiome–testis or 720 microbiome–serum associations survived FDR correction. Supplementation was associated with favorable systemic and testicular histological responses and dose-specific molecular and microbial changes under heat exposure.

Animals doi: 10.3390/ani16213484

Authors: Wenli Hu Wenbin Lei Xilong Jia Xueyan Wang Ting Liu Xiaojuan Wang

To develop unconventional feed resources, this study investigated the effects of adding HHRM to the diet on carcass traits, meat quality, amino acids, and fatty acids in male Hu sheep lambs. Forty-eight healthy, 3-month-old lambs (24.33 ± 1.56 kg) were randomly divided into four groups (n = 12): CON (0%), LC (3%), MC (6%), and HC (9%). The trial included a 5-day adaptation period followed by an 80-day main trial period. Compared with the CON group, the HC group showed higher levels of GR, crude protein, EAAs, and several individual amino acids, while simultaneously reducing drip loss and improving cooking yield (p < 0.05). However, compared with the CON group, the HC group had a higher pH24 h value but lower b*24 h values, hardness, elasticity, cohesiveness, and chewiness; it also had the highest SFA content and the lowest PUFA, UFA, and n-6 contents. The LC and MC groups increased intramuscular fat content and reduced shear force (p < 0.05); among them, the MC group had the highest n-3 content (p < 0.05) and the lowest shear force. There were no significant differences in MUFA content. In summary, it is recommended that 6% HHRM be used as the appropriate addition level.

Animals doi: 10.3390/ani16213483

Authors: Mario Alberto González Rafael Barrera-Chacón Joaquín Jiménez-Fragoso Beatriz Macías-García Javier Fernández-Cotrina María Gil-Molino Nicolás Roberto Robles Francisco Eduardo Martín-Cano Francisco Javier Duque

The tolerability and efficacy of treatment with allogeneic adipose-derived mesenchymal stem cells (aAMSCs) were evaluated in dogs with kidney disease secondary to canine leishmaniosis (CanL). In this non-blinded, single-center, 3-month study, twenty dogs received either conventional treatment (CT; n = 10) or conventional treatment plus an intravenous dose of aAMSCs (CT + aAMSCs; n = 10). Efficacy was assessed at days 0, 30, and 90 using plasma and urinary biomarkers. In both groups (CT, n = 6; CT + aAMSCs, n = 7), glomerular filtration rate was estimated by scintigraphy, and urinary proteomics was performed. No serious adverse events were observed, although two dogs experienced transient gastrointestinal events. No significant between-group differences occurred in renal parameters at time points. Intragroup analyses showed significant reductions in uNGAL/c in both groups and UPCR only in CT + aAMSCs. Longitudinal mixed-effects analyses revealed no significant group × time interactions for any of the evaluated variables. Urinary proteomics identified four protein entries with higher abundance in the CT + aAMSCs group at T90, although UMAP showed no separation between groups. A single intravenous aAMSCs dose was well tolerated but provided no renal benefits over conventional treatment. Further studies should evaluate alternative dosing protocols.

Animals doi: 10.3390/ani16213482

Authors: Mingkang Zhu Tianyou Wu Li Liu Kui Li Zhen Chen Jie Wu Miao Lin Jiakun Wang Ruihua Zhang Xingkai Zhang Huizeng Sun Zihai Wei

This study aimed to evaluate the impact of moderately elevated dietary starch levels and the partial replacement of dietary protein source with canola meal (CM) on lactation performance, methane (CH4) emissions, ruminal fermentation parameters, and serum biochemical parameters in dairy cows. Initially, two in vitro gas production trials were conducted: Trial 1 involved diets formulated with starch levels of 24.0% (Low starch), 27.4% (Medium starch), and 30.8% (High starch) on a dry matter (DM) basis. In Trial 2, compared with a control diet without CM (CONin_vitro; 25.7% starch, the mean of 24.0% and 27.4%), a diet with moderately increased starch content and the inclusion of CM at 10.73% of DM (HSCMin_vitro; 29.1% starch, the mean of 27.4% and 30.8%) on a DM basis was formulated. In the subsequent in vivo experiment, thirty mid-lactation Holstein cows were randomly allocated to two groups: a control diet (CON, comprising 26.1% starch without CM, on a DM basis) or an experimental diet (HSCM, containing 28.9% starch with 7.68% CM, on a DM basis). In vitro results in Trial 1 showed that increasing dietary starch linearly decreased CO2 production (p < 0.01) and CH4 production (p = 0.03), but no significant differences between groups were detected. In Trial 2, a 3.4% increase in dietary starch combined with partial inclusion of CM showed a tendency to reduce CH4 (p = 0.06) and CO2 production (p = 0.08) compared to the CON diet. For the in vivo experiment, cows on the HSCM diet exhibited a tendency for greater dry matter intake (DMI; p = 0.06) and feed efficiency (milk yield/DMI; p = 0.09), and significantly improved milk yield (p < 0.01), energy-corrected milk (ECM; p < 0.01), and fat- and protein-corrected milk (FPCM; p < 0.01). The yields of milk fat (p < 0.01), protein (p < 0.01), lactose (p = 0.03), and total solids (p < 0.01) were also significantly improved, while the milk urea nitrogen (MUN) concentration was decreased (p < 0.01). The HSCM diet significantly decreased CH4 production by 14.1% (p = 0.02), as well as CH4 yield (p < 0.01) and CH4 emission intensity (p < 0.01) in terms of g/kg milk yield, ECM, and FPCM. Rumen fermentation parameters indicated lower molar proportions of isobutyrate and isovalerate in the HSCM group (p < 0.01), with no differences in other rumen parameters between groups. Additionally, the HSCM group exhibited a higher serum total protein (TP) concentration (p = 0.02) and a decreasing trend in blood urea nitrogen (BUN; p = 0.09). These findings demonstrate that a moderate increase in dietary starch content and partial replacement of dietary protein source with CM is an effective nutritional strategy for simultaneously improving lactation performance and mitigating CH4 emissions. However, as starch and CM were altered simultaneously in the in vivo experiment, the independent contribution of each factor cannot be determined and warrants further investigation.

Animals doi: 10.3390/ani16213481

Authors: Monika Budzyńska Joanna Kapustka

Human–animal interaction is considered one of the key determinants not only of animal welfare but also of the safety and ease of contact with humans. The aim of this paper is to review current knowledge on tools of assessment and improvement in human–animal interaction from an alpaca welfare perspective. Because alpaca-specific evidence is limited, the scientific evidence for some of the mechanisms and management strategies discussed is derived from other camelids or other animal species rather than directly from alpacas. Various factors affect the HAI quality, including early human contact, husbandry and veterinary procedures, handling, and training, as well as genetically influenced traits such as temperament variations. The methods used to evaluate the quality of HAI in alpacas mainly included three types of assessment tools: behaviour observation, measuring physiological parameters, and using questionnaires. Tools used to improve the quality of HAI in alpacas can include various techniques based on animal behaviour and learning knowledge, such as socialisation, desensitisation and operant conditioning. Considering human–animal interactions in alpaca husbandry (animals kept for fibre production, animal-assisted interventions or recreational activities) is crucial because it can ensure animal well-being, human safety and production/intervention effectiveness. Negative human–animal interactions can impair alpaca welfare through fear and stress as underlying mechanisms; however, practical applications (proper socialisation, handling, training) to achieve positive HAI and a positive perception of humans by alpacas could be better recognised and used, thereby benefiting their welfare.

Animals doi: 10.3390/ani16213480

Authors: Haolun Fan Zhikai Zheng Xi Zheng

Human pressures may be associated with predator space use directly or through prey, with variation by season and zoning context. We combined cameras, source-specific patrol observations, and environmental covariates across 18,243 km grids in Northeast China Tiger and Leopard National Park. Hierarchical models estimated predator spatial-use probabilities and prey use intensities using 2024 monthly inputs. Tigers used cameras and patrols, whereas leopard states used cameras only. Spatial-error piecewise SEMs used 500 independently paired state draws and 400 conditional multivariate coefficient simulations per pair. No direct path and two single-prey chains passed the extended descriptive screening rule during snow, compared with four direct paths and one chain in snow-free conditions. The four directional hypotheses received limited, pressure- and species-specific support; seasonal and B–H–M comparisons remained exploratory. Negative net indirect estimates sometimes arose from component signs contrary to the proposed resource-depletion pathway. Opposing farmland–leopard estimates produced an uncertain total rather than evidence of exact cancellation. Six park-wide alternatives examined estimation, covariate, and network assumptions; the original B–H–M models described four snow-free contexts. These conditional patterns support field assessment rather than causal mediation or intervention prescriptions.

Animals doi: 10.3390/ani16213478

Authors: Byron Xavier Muñoz Sara Benavides Alba Fireman Cristiano Bortoluzzi Ivânio José Martins Bueno Lucas Alves Rodrigues Raimundo Netto Mário Lopes Vitor Pais Giovana Longhini Melany Lovon Cristiane Araujo Lucio Araujo

This study evaluated the effects of increasing dietary inclusion levels of branched-chain volatile fatty acids, also called isoacids (ISO), on the growth performance, carcass traits, intestinal health, and breast muscle metabolomic profile of broiler chickens. A total of 700 one-day-old male Cobb 500 chicks were assigned to five corn–soybean meal-based diets with 10 replicate pens (14 birds/pen) in a completely randomized design: A Positive Control (PC) diet formulated to meet nutritional requirements was supplemented with 0.0, 0.10, 0.20, 0.40, or 0.60% of ISO (supplied as a blend of isobutyrate and 2-methylbutyrate). Data were analyzed using General Linear Models with orthogonal polynomial contrasts, PERMANOVA for microbiome beta diversity and ANOVA with FDR correction for metabolomics. Growth performance was monitored across feeding phases. At day 42, carcass traits, breast meat quality, intestinal permeability, cecal short-chain fatty acids (SCFAs), and cecal and ileal microbiota were assessed, alongside liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) metabolomics of the Pectoralis major muscle. Dietary ISO significantly improved average daily weight gain (p < 0.0001) and feed conversion ratio (p < 0.0001) during the first 21 days (early-life), with an optimal inclusion level of 0.40%. While overall carcass and breast yields were unaffected, ISO supplementation significantly and quadratically reduced abdominal fat deposition (p < 0.0001), with an optimal inclusion range between 0.10% and 0.40%. ISO did not compromise intestinal permeability or alter microbial alpha diversity, but it significantly restructured the cecal microbiota (beta diversity; Bray–Curtis p = 0.0213) and linearly increased cecal isobutyric acid accumulation in the cecal content (p = 0.0443). Breast muscle metabolomics revealed a dose-dependent, nonlinear response; the 0.40% inclusion level maximized the relative abundance of galactolipids, whereas the highest dose (0.60% ISO) upregulated sphingomyelins, lysophospholipids, and acylcarnitines. In conclusion, a specific dietary ISO supplementation dose of 0.4% serves as an effective nutritional strategy to boost early feed efficiency and reduce abdominal adiposity. Doses exceeding this optimal level, such as 0.60%, offer no further productive benefits and may trigger cellular stress.

Animals doi: 10.3390/ani16213479

Authors: Xin Du

Rearing Whitmania pigra and Hirudo nipponia relieves the hirudin feedstock shortage, and the rack sets its cost and the animals’ environment; earlier designs were judgement-based. Six husbandry requirements become load and geometric inputs; limits (SFy ≥ 2, SFb ≥ 2, and δ ≤ L/200) screen 36 tube and rank 17 bar schemes. The machine is three tiers of one pan, sized by the credible accident (pans full to their rims, 1417 L each, 47.1 kN), not by the 20–60 mm operating depth, where nothing approaches a limit. The three-ring frame is 74.9% of the baseline, so tubes and bars can save at most 25.1%; the tube number governs the mass (61.26%) and stress (74.32%), and strength governs 34 of 36 schemes. Three 30 × 30 × 2.5 mm tubes with eight 5 mm bars weigh 101.34 kg, 18.4% below baseline, at SFy = 2.09 and SFb = 3.35 on the 235 MPa minimum for Q235; the lightest section breaks at 315 mm and this one at 481 mm. The 1.1% lighter 25 × 25 mm section clears the criterion only where the capacity and scaling both favour it; the ±8.3% band on the connection factor exceeds the 4.6% margin, its lower end falling below 2.0; one measurement closes it.

Animals doi: 10.3390/ani16213477

Authors: Lina Nyberg Mette Pökelmann Malin Connysson Susanne Lundesjö Kvart Elke Hartmann

Within harness racing, the concept of driveability has received little scientific attention despite its apparent relevance to training, racing, and horse welfare. This study explored, through semi-structured interviews, how eight Swedish harness-racing trainers and drivers conceptualise driveability and relate it to performance, rein tension, equipment use, and horse welfare. The data were analysed with reflexive thematic analysis. Three themes were developed: (1) calm training is key, (2) one cannot always influence driveability, and (3) equipment is a tool. Participants described a driveable horse as calm, attentive, and responsive to the driver’s signals. The findings suggest that driveability is best understood as a relational and context-dependent concept, shaped by training and ongoing interaction between driver and horse, while also being influenced by individual horse characteristics, situational conditions, and equipment use. Participants described both potential alignment and tension between welfare and performance. These perceived relationships require further investigation through independent assessment of practices and welfare outcomes.

Animals doi: 10.3390/ani16213476

Authors: Zhen Chen Zhiqiang Cheng Yuan Gao Shiwei Yang Xiaoqing Hong Heng Wang Xuan Hu Kang Zhan Yongjiu Huo Miao Lin Yimin Zhuang Guoqi Zhao Yihui Zhang

Coated slow-release urea (SRU) may provide an alternative source of supplemental nitrogen in dairy cattle diets, but its ruminal release behavior can vary among formulations. This study compared three palm-oil-coated SRU products with different nominal release specifications using nonlactating, nonpregnant, rumen-cannulated Chinese Holstein cows. Three cows were used in an in situ experiment to characterize dry matter (DM) and urea disappearance from 0.5 to 12 h, and four cows were assigned to a 4 × 4 Latin square feeding trial. The control treatment consisted of a basal diet plus 500 g/d supplemental soybean meal (SBM), whereas the SRU treatments received the same basal diet plus 100 g/d SRU I, SRU II, or SRU III as approximately isonitrogenous supplemental nitrogen alternatives. SRU I consistently showed slower early DM and urea disappearance than SRU II and SRU III, whereas differences between SRU II and SRU III were less consistent across incubation times. Dietary treatment did not significantly affect DM, crude protein, or neutral detergent fiber intake, apparent total-tract digestibility, ruminal pH, total or individual volatile fatty acid concentrations, or hematological variables. Ruminal ammonia nitrogen differed among treatments (p = 0.04), with SRU III having a higher concentration than the control and SRU I and SRU II showing intermediate values. These results demonstrate formulation-dependent in situ disappearance patterns among coated SRU products and suggest that they can serve as alternative supplemental nitrogen sources under the conditions tested.

Animals doi: 10.3390/ani16213475

Authors: Jing Zhang Jiaqi Jiang Feixiang Li Xiancun Zhou Ye Zhang Yadong Yang Shijian Zheng Xianmeng Meng

To address low efficiency in manual counting and high costs of data collection and labeling in Landes goose farming, this study developed a simulation-driven video detection and counting method. A three-dimensional simulation environment generated goose scenes under varied densities, perspectives, and lighting conditions, with automatic bounding-box labeling. A YOLO26 detector was pre-trained on the 6000-image training subset of a 10,000-image simulated dataset, which was divided into training, validation, and test subsets at a ratio of 6:2:2 (6000, 2000, and 2000 images, respectively), and was then fine-tuned using 500 real images. A bottom-center trajectory anchor and line-crossing rules were combined with BoT-SORT tracking to construct a detection-tracking-counting pipeline. Evaluation on three real video sequences comprising 1780 reference crossing events yielded an event-level counting accuracy of 99.16% and an aggregate net-count accuracy of 99.38%, with a mean absolute error of 3.7 and a root mean square error of 5.3 across the sequences. In the dense sequence, the predicted count was 1021 against 1030 reference crossings, corresponding to a net-count accuracy of 99.13%. These findings support simulation-assisted training and non-contact line-crossing counting for the evaluated Landes goose passage. Further validation is needed before extending the method to other farm layouts or production environments.

Animals doi: 10.3390/ani16213474

Authors: Bianca da Costa Tavares da Silva Leonila Ester Reinert Raspantini Silvana Lima Górniak

In both aquaculture and scientific experimentation, fish handling often requires anesthesia to facilitate immobilization and minimize handling-associated disturbances. This study aimed to compare the anesthetic performance of different anesthetic agents in Astyanax lacustris, assessing induction, recovery, and safety to support protocol selection for tropical freshwater fish. A total of 114 yellowtail tetra Astyanax lacustris were used, including 54 fish in a preliminary experiment and 60 fish in the main experiment, with 10 fish per concentration in each anesthetic treatment during the main experiment. Fish were exposed to different concentrations of propofol (5.0 and 6.5 mg L−1), eugenol (40 and 50 mg L−1), and MS-222 (120 and 130 mg L−1). Anesthetic efficacy and complementary physiological responses were assessed through induction and recovery times, responsiveness during recovery, opercular beat rate (OBR), hematological parameters, and plasma concentrations of glucose, lactate, and cortisol. Data were analyzed using analysis of covariance (ANCOVA) followed by appropriate post hoc tests (p < 0.05). All anesthetics successfully induced stage IV anesthesia at the evaluated concentrations, although distinct induction and recovery profiles were observed. MS-222 promoted significantly faster progression to deep anesthesia and shorter recovery times, whereas propofol induced prolonged recovery and marked reduction in OBR, with residual ventilatory depression after recovery. Eugenol showed an intermediate anesthetic profile associated with pronounced excitation during induction and an altered recovery pattern. Hematological parameters and plasma lactate concentrations did not differ significantly among anesthetic types or concentrations. Plasma glucose concentrations increased at higher anesthetic concentrations, whereas plasma cortisol concentrations differed significantly between propofol and eugenol groups (p < 0.05). Overall, the findings demonstrate that propofol, eugenol, and MS-222 produce distinct anesthetic and ventilatory responses in A. lacustris, highlighting the importance of selecting anesthetic protocols according to the intended experimental or handling procedure.

Animals doi: 10.3390/ani16213473

Authors: Éva Kissné Váradi Jakub Vozaf Jaromír Vašíček Jozef Berčik Andrej Baláži Peter Chrenek Árpád Drobnyák Zsuzsa Szabó Éva Török Krisztina Liptói Barbara Végi

Sperm cryopreservation is the most effective method for the long-term preservation of genetic resources. However, rabbit sperm cryopreservation remains challenging. One of the key factors influencing freezing success is the choice of semen diluent; however, no specific diluent has been established for rabbit semen. In this study, we compared the effectiveness of modified Lake’s diluent and evaluated the cryosurvival of semen from the native Hungarian breed and a native Slovak rabbit breed. Both breeds were subjected to the same static freezing procedure. The following parameters were evaluated: motility, viability, plasma membrane integrity, mitochondrial activity, acrosomal status, and intracellular reactive oxygen species. In fresh samples, viability (93.5% vs. 77.4%), total motility (84.6% vs. 67.0%), and progressive motility (76.1% vs. 47.0%) were significantly higher for the Hungarian Giant than in the Chrabrany rabbit samples. Following cryopreservation, viability (56.4% vs. 43.4%), total (39.2% vs. 20.5%) and progressive (20.6% vs. 8.5%) motility, acrosomal status (72.2% vs. 44.9%) and ROS activity (33.8% vs. 24.6%) were higher in samples from the Chrabrany breed. The results suggest that the modified Lake’s diluent may also be suitable for other rabbit breeds and highlight the importance of developing breed-specific cryopreservation protocols.

Animals doi: 10.3390/ani16213472

Authors: Anne Steinemann

Muscovy Ducks (Cairina moschata), one of the world’s oldest domesticated waterfowl, inhabit nearly all continents. Considered non-native across most of the United States, Muscovy Ducks are often described as “unprotected” or “invasive,” terms that may be used to justify harmful actions. The question is whether these descriptions accurately reflect their legal protection and wildlife classification. This study addresses that question through the first nationwide analysis of anti-cruelty statutes and invasive-species classifications at the federal and state levels. Results show that every state prohibits intentional cruelty toward Muscovy Ducks, subject to exemptions. While considered “unprotected” for conservation purposes, they remain protected under anti-cruelty statutes. No federal agency formally classifies Muscovy Ducks as “invasive.” Although two of the 50 states list Muscovy Ducks as invasive, each listing lacks science-based substantiation of harm in the state. This study corrects claims that Muscovy Ducks are legally unprotected or invasive. Misuse and misinterpretation of terms can obscure the reality that intentional cruelty against Muscovy Ducks is unlawful nationwide.

Animals doi: 10.3390/ani16213471

Authors: Mette Uldahl Jan Dahl David J. Mellor

Assessment of oral mucosal colour is widely used in equine clinical and welfare evaluations, but inconsistent terminology limits comparison between clinical observations and research studies. This study evaluates, as an initial proof-of-concept, the Uldahl Standard for systematically describing mucosal colour and saturation. Gingival and lingual mucosae were assessed in clinically normal horses, healthy horses sedated for routine dental procedures, and horses with severe systemic circulatory compromise. Photographic colour samples validated against in vivo observations were classified using Munsell notation, ISCC-NBS terminology and the Uldahl Standard. Three illustrative cases of tongue discolouration were additionally assessed. Descriptive analyses characterised colour and saturation across physiological states, while clinically normal and sedated horses were compared using exact chi-square tests and logistic regression. Overall, 42 unique colour-saturation combinations were identified. Chromatic diversity increased with physiological compromise. Primary colour differed significantly between clinically normal and sedated horses for both gingival (p = 0.05) and lingual (p = 0.004) mucosae, whereas secondary colour and saturation did not. Grey was significantly associated with physiological state in both gingival (p = 0.05) and lingual (p = 0.03) mucosae. It showed high specificity but low sensitivity, including 100% specificity for lingual grey. Purple occurred only in sedated and systemically compromised horses, while blue occurred only as a secondary colour in systemically compromised horses. The findings demonstrate the feasibility of applying the Uldahl Standard to equine oral mucosal assessment and suggest that primary colour, particularly grey, may provide greater discriminatory information than secondary colour or saturation. Further studies are required to establish the diagnostic relevance of broader colour-saturation patterns.

Animals doi: 10.3390/ani16213470

Authors: Yo Han Choi Jae-beom Park Tae-kyeong Kim Hyeong-sik Won Hyun Ju Park Hong-jun Kim Jo Eun Kim Yong Dae Jeong Jin-soo Kim Hyun-sik Ham Hyun-chong Cho

Accurate pregnancy diagnosis is important for reproductive management in pig farming. This study evaluated a computer-aided diagnosis workflow using portable low-frequency ultrasound videos from 1895 sows at two sites. Xception frame predictions were aggregated into video-level decisions. Original, Preprocessing (wavelet denoising and contrast-limited adaptive histogram equalization), Mixup, and their combination (Combined) were compared across ten repeated sow-level splits. Combined achieved mean accuracies of 84.5% and 85.3% in Datasets A and B, respectively, with pregnant-sow videos restricted to days 18–25 post-insemination. Compared with Original, mean specificity increased by 1.3 and 2.4 percentage points and Youden’s index by 1.7 and 2.3 points, while mean sensitivity was numerically similar. Paired 95% confidence intervals for specificity and Youden’s index differences excluded zero only in Dataset B, without adjustment for multiple comparisons. Paired comparisons of the area under the curve (AUC) did not establish improved discrimination. Mixup yielded the highest mean specificity and AUC, whereas Combined yielded higher mean sensitivity than Mixup. This trade-off varied across partition subsets. Cross-site and leave-one-session-out evaluations showed performance variability across acquisition settings, with no consistently best condition. These comparisons identify configuration-dependent trade-offs requiring further evaluation for diagnostic support.

Animals doi: 10.3390/ani16213469

Authors: Emadeldien Mohamed Ibrahim Mohamed Shehab-El-Deen Ibrahim H. Algherair A. A. AbdElhakeam Yassin Kassim

Thirty Ossimi ewes and twelve Ossimi rams were used to evaluate the effects of supplementing a low-selenium (Se) basal diet with Se nanoparticles (Se-NP) on nutrient digestibility, hemato-biochemical indices, and reproductive-lactational performance. Ewes and rams were randomly allocated to two groups: a control group fed the basal diet (estimated Se content 0.005–0.014 mg/kg dry matter, DM) and a Se-NP group fed the same basal diet supplemented with 0.3 mg Se-NP/kg DM. Repeated measurements were analyzed with linear mixed models. In rams, dietary Se-NP increased (p < 0.001) ejaculate volume, sperm concentration, progressive motility, and serum testosterone levels compared with the control. In the Se-NP breeding group, the conception rate was numerically higher (93.3%) compared with 60.0% in the control group. Dietary Se-NP increased (p < 0.001) total antioxidant capacity, blood Se concentration, serum triiodothyronine (T3), the T3/thyroxine (T4) ratio, nutrient digestibility coefficients and nutritive values in both rams and ewes. Maternal Se-NP supplementation also increased (p < 0.001) milk yield, fat corrected milk, milk composition and daily yields of milk fat, protein, and lactose, as well as the birth weight, weaning weight and average daily gain of suckling lambs. Under the conditions of this study, Se-NP supplementation of a low-Se diet during the breeding period was associated with improved semen characteristics, lactational, and digestive performance; because ram and ewe supplementation were applied jointly within breeding groups and the sample size was limited, these findings should be confirmed in larger studies.

Animals doi: 10.3390/ani16213468

Authors: Mohammed Babatunde Sadiq Siti Zubaidah Ramanoon Z. A. Jelan

Digital dermatitis (DD) contributes to lameness, posing major animal welfare and economic concerns. Corrective trimming, topical antibiotics, and bandaging are widely practised in managing DD. However, the challenges associated with conventional bandaging include water retention and a moist environment, which affect the healing process. This study evaluated the effectiveness of a Cow Hoof Cover® (CHC) for the treatment of DD in dairy cows. A quasi-experimental study was conducted, involving cows affected with active stages of DD (M1 and M2), allocated to CHC (n = 29) and conventional bandaging (n = 27) groups. The standard treatment entailed a Dutch five-step hoof trimming, wound management, application of topical antibiotic containing oxytetracycline (Engemycin 25 mg/mL), and wrapping of the digit using either the CHC or conventional bandaging. All cows received an intramuscular injection of flunixin meglumine (2.2 mg/kg) for pain relief. Primary outcomes were healing of DD lesions (transition from active stages to M0) and restoration of normal gait (locomotion score of 1). All cows were observed weekly until Week 4 after treatment. While alterations in locomotion score were not significantly different between the groups (p = 0.23) during the study period, the CHC group had significantly higher (p < 0.01) proportions of cured cows at week 3 (n = 6, 20.6%) and week 4 (n = 17; 58.6%) compared to the control. None of the CHC-wrapped lesions progressed into the chronic hyperkeratotic or proliferative stage (M4), but such transitions were recorded in four cows in the control group. Overall, the CHC had significantly higher probability of cure (HR = 2.79; 95% CI 1.30–5.97) than the control group. Excluding the treatment group, none of the tested animal characteristics and treatment-related variables influenced the probability of cure. Overall, the CHC offers practical advantages over conventional bandaging; it is quick to apply, durable, and water-resistant, supporting its use for field application and large-scale herd management. However, histological investigation and robust clinical trials are recommended to ascertain whether the CHC promotes a complete cure of active DD lesions.

Animals doi: 10.3390/ani16213467

Authors: Monif AlRashidi Ahmed Saleh Alali

Biparental cooperation during incubation is particularly important in harsh environments. We quantified sex-specific incubation contributions and body orientation in Black-winged Stilts (Himantopus himantopus) from one-minute images at 12 nests monitored for 24 h in Saudi Arabia. Incubation partitioning, environmental associations, and sex differences in body orientation relative to the sun and wind were evaluated using nest-level resampling and generalized linear mixed models, with Holm correction for environmental tests. Male incubation share averaged 50.72% (95% CI 47.21–54.35%), without clear evidence of deviation from 50% (p = 0.697). Male contributions varied among two-hour periods (cyclic-shift p = 0.042; parametric-bootstrap likelihood-ratio p = 0.004), whereas the daylight–night difference was inconclusive (p = 0.234). No environmental predictor of incubation partitioning was supported after correction. No clear sex difference was detected in solar-relative orientation (p = 0.154). Although the primary four-direction (p = 0.015) and exploratory eight-direction (p = 0.038) models yielded nominal sex-related differences in wind-relative orientation, these findings were not consistently corroborated by compass-preference-adjusted and episode-onset sensitivity analyses and therefore do not establish a sex-specific wind response. Sex-by-wind-speed interactions were inconclusive (p = 0.071 and 0.172). Overall, near-equal daily incubation contributions can mask temporal variation in parental duties across two-hour periods.

Animals doi: 10.3390/ani16213466

Authors: Wilmer Cuervo María Galeano Cassie Flannagan Elliot Hayhurst Vidalejandra Araujo

(1) Background: Beef cattle production is essential for Montana’s economy. These operations rely on fibrous diets promoting enteric methane (CH4) emissions, resulting in dietary energy inefficiency and environmental impacts. Concurrently, noxious weeds invade approximately 10% of state rangeland. Yet, some species produce plant secondary metabolites (PSMs) that could be repurposed for CH4 mitigation strategies. (2) Methods: This study evaluated seven invasive weeds from South-Central Montana using in vitro incubations with ruminal fluid from cannulated Angus Cows (590 ± 18 kg). Experiment 1 screened whole plants added at 20% of incubated dry matter (IDM) against a control (hay), then, experiment 2 evaluated ethanolic extracts from leaves and inflorescences from top candidates identified in experiment 1. (3) Results: In the screening phase, common burdock (BURD) and Curly dock (DOCK) modified fermentation dynamics. BURD leaf extract achieved a 25.2% reduction in CH4 yield (mL/g fermented organic matter) without compromising in vitro organic matter digestibility (IVOMD) or ammonia (NH3-N). Conversely, DOCK extracts demonstrated the strongest CH4 volume reduction but at the cost of a dramatic reduction in IVOMD and NH3-N depletion. Bioactivity of PSM remained consistent across anatomical structures. (4) Conclusions: Specific weeds (BURD, DOCK) represent a biochemical repository with potential as a feed additive for roughage-based diets. Leaves from BURD exhibited optimal potential to mitigate CH4 emission without impairing diet digestibility.

Animals doi: 10.3390/ani16213465

Authors: Fangshan Wang Yawen Wang Jing Ren Xue Zhao Zelin Liu Tairan Sun Lihua Cheng Jinhai Huang Qinye Song

Porcine epidemic diarrhea virus (PEDV), the etiological agent of porcine epidemic diarrhea (PED), remains a major threat to swine production worldwide. The genetic variation and the co-circulation of multiple genotypes of this virus may compromise vaccine-induced protection, creating an urgent need for practical serological tools for herd-level monitoring. A blocking enzyme-linked immunosorbent assay (blocking ELISA) was developed using a recombinant truncated PEDV S1 protein as the coating antigen and a horseradish peroxidase (HRP)-labeled anti-PEDV S1 monoclonal antibody as the competing detector. No cross-reactivity was detected with the tested antisera against porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), classical swine fever virus (CSFV), or porcine circovirus type 2 (PCV2). The intra-assay and inter-assay coefficients of variation were 3.02% and 3.76%, respectively. Receiver operating characteristic analysis yielded an optimal cutoff of 31.76%, with a sensitivity of 98% and a specificity of 99%. Among 33 clinical serum samples, blocking-ELISA classifications were fully concordant with infected-cell indirect immunofluorescence assay (IFA) results (33/33, 100%), which were interpreted as a measure of general PEDV seroreactivity. Comparison with virus neutralization testing of 98 sera showed category-specific concordance rates of 81.25%, 90.91%, and 100% for neutralizing antibody titers of 1:64, 1:128, and ≥1:256, respectively. All sera in the ≤1:16 category were negative by blocking ELISA. A continuous correlation analysis between the blocking percentage and neutralizing antibody titer was performed (ρ = 0.484, 95% CI 0.315–0.623, p = 5.1 × 10−7; n = 97). Compared to traditional neutralization tests, this method features simpler operation, faster detection, and higher efficiency, rendering it suitable for large-scale monitoring of vaccine efficacy in swine herds and offering robust technical support for precise evaluation of PEDV immunity.

Animals doi: 10.3390/ani16213464

Authors: Lianguang Jiang Meiqi Wang Junzhao Xu Huaxin Niu Jingfeng Yang

In this study, three bovine-derived lactic acid bacteria (LAB) strains were isolated from feces of healthy Simmental calves: Lacticaseibacillus paracasei (Lp), Enterococcus hirae (Eh), and Enterococcus faecalis (Ef). Bacterial suspensions were inoculated into pasteurized milk at a 2% inoculation ratio and anaerobically fermented at 37 °C for 24 h to obtain three kinds of LAB-fermented milks: Lacticaseibacillus paracasei fermented milk (LpM), Enterococcus hirae fermented milk (EhM), and Enterococcus faecalis fermented milk (EfM). Their probiotic potential was comprehensively evaluated through a series of in vitro assays, and in vitro colonic anaerobic fermentation was conducted using fecal inoculum from four Simmental calves (three biological replicates per group) to investigate the regulatory effects of these strains on the colonic microbiota and short-chain fatty acids (SCFAs). The three strains exhibited similar growth trends during raw milk fermentation. Compared with Eh and Ef, Lp showed significantly greater utilization of galactooligosaccharides (GOS) and lactooligosaccharides (LOS) (p < 0.01), whereas no significant difference in proteolytic activity was observed among the three strains (p = 0.192). LpM exhibited significantly higher water-holding capacity and cell-surface hydrophobicity than EhM and EfM (p < 0.01), along with a higher auto-aggregation rate (p < 0.01). Conversely, Eh and Ef displayed stronger co-aggregation with Staphylococcus aureus, Escherichia coli, and Salmonella, as well as greater antibacterial activity, with Ef being the most effective (p < 0.01). All three strains were highly sensitive to kanamycin, streptomycin, tetracycline, and doxycycline, whereas Eh and Ef showed intermediate sensitivity or resistance to several antibiotics. After fermentation, LpM and EhM retained IgG more effectively, while EfM consumed the least lactose. EhM produced the most acetic acid, LpM was dominant in propionic and butyric acid production, and both LpM and EhM generated more lactic acid than EfM (p < 0.01). The fermented milks displayed distinct 2,2-Diphenyl-1-picrylhydrazyl(DPPH) and hydroxyl-radical-scavenging capacities; LpM possessed the strongest antioxidant activity, followed by EhM, while EfM showed the weakest antioxidant performance (p < 0.01). During in vitro fermentation, both LpM and EhM produced more SCFAs than EfM. Through 16S rRNA gene sequencing it was revealed that LpM increased the abundance of Butyricicoccus pullicaecorum, Lactococcus, Streptococcaceae, and Lachnospiraceae. Although Eh and Ef could enrich genera such as Bacillus to inhibit diarrhea-associated pathogens, they also coincided with an increase in opportunistic pathogens including Escherichia-Shigella, Klebsiella, and Enterococcus. Therefore, Lp represents a promising candidate strain of bovine-derived lactic acid bacteria for fermented milk aimed at preventing calf diarrhea. Notably, Eh and Ef also enriched opportunistic pathogens, and further safety assessment is required before practical application.

Animals doi: 10.3390/ani16213463

Authors: Elena Infante González-Mohino Alfonso Balmori-de la Puente Manuel Collado-Cuadrado Alberto Gil-Abad Elena Carretón Rodrigo Morchón Iván Rodríguez-Escolar

Zoonotic leishmaniasis, caused by Leishmania infantum, is an emerging public health concern in Southern Europe, where climate change is altering vector phenology. Traditional static risk maps fail to capture critical seasonal transmission dynamics. This study analyzed the monthly risk maps of L. infantum infection across Spain and Portugal, including the Balearic, Canary, Madeira, and Azores archipelagos. We developed Ecological Niche Models (MaxEnt) for the primary vector, Phlebotomus perniciosus, and integrated them with a temperature-dependent parasite development model to generate high-resolution monthly risk maps. The models exhibited high predictive power. In the Iberian Peninsula, human footprint was the primary driver, revealing a marked seasonal expansion of risk towards northern latitudes and higher altitudes during summer (June–September), with potential transmission windows extending into autumn. Conversely, in Macaronesia, temperature was the dominant factor. The Canary Islands and Madeira showed sustained high risk in coastal areas year-round, while the Azores, historically considered non-endemic, displayed climatic suitability hotspots in central islands. These findings confirm the tropicalization of transmission patterns and identify cryptic risk periods. This dynamic approach offers a robust tool for veterinary and public health authorities to transition from static, calendar-based prophylaxis to adaptive, evidence-based control strategies, optimizing resources in a changing climate.

Animals doi: 10.3390/ani16213462

Authors: Jona Danaj Dimitra V. Liagka Konstantinos Tousis Artemis Konstantinou Konstantinos Marinakos Dimitrios Kantas Elisavet Giamouri Vasiliki Spyrou Natalia G. C. Vasileiou Maria Spilioti

The global pet food market is influenced by the growing number of cats and dogs, the humanization of pets and the changing expectations of dog and cat owners. This study examines the role of marketing strategies in the dog and cat food market, focusing on the attitudes of Greek survey respondents, recognition of specific food categories, and factors associated with purchasing behavior. Data were collected through an online survey of 526 pet owners and guardians (n = 249 dog owners and n = 277 cat owners). The behavior toward pet foods containing selected premium ingredients was significantly linked to pet type (p = 0.021), the importance attributed to food ingredients (p < 0.001), veterinarian recommendations (p = 0.009), and the availability of scientifically substantiated information (p < 0.001). Among all factors, the importance of ingredients emerged as the factor displaying the strongest statistical association with consumer behavior (p < 0.001). Based on these findings, the study proposes a targeted marketing mix addressing product transparency, pricing, distribution, and promotion. The results contribute to the literature on pet humanization and provide practical guidance for pet food businesses, while highlighting the need for clearer regulatory definitions and scientific substantiation of marketing claims.

Animals doi: 10.3390/ani16213461

Authors: Rebel Skirving Cynthia D. K. Bottema Do Thi Hue Richard Laven Kiro R. Petrovski

Passive transfer of immunity through colostrum is known to improve short-term health in neonatal dairy calves, but its long-term effects remain poorly understood. Lactational programming proposes that bioactive components in milk and colostrum induce epigenetic changes that influence future performance, although this concept has received limited attention in dairy cattle. The aim of this study was to investigate potential associations between neonatal colostrum intake, as estimated by serum total protein (STP), with subsequent growth, reproductive performance, and milk production in 2661 dairy heifers from five pasture-based dairy herds in South Australia. Blood samples collected within the first 1–7 days of life were used to measure STP as an indicator of passive immunity transfer, and consequently, colostrum intake. Heifers with adequate passive transfer (STP > 50 g/L) produced 0.5, 2.4, and 0.7 kg more milk per day during their first, second, and third lactations, respectively, than heifers with inadequate passive transfer (STP ≤ 50 g/L). In contrast, no meaningful associations were identified between neonatal STP and yearling weight, age at first or subsequent calvings or calving intervals. These findings suggest that adequate colostrum intake enhances lifetime milk production and provide evidence supporting lactocrine programming in dairy cattle.

Animals doi: 10.3390/ani16213460

Authors: Jiamei Liu Weili Liang Wanhong Li Xiangpeng Yue Fadi Li

Cryptorchidism is a common male reproductive disorder generally characterized by spermatogenic failure. Nevertheless, the molecular mechanisms underlying infertility induced by cryptorchidism in sexually mature rams remain insufficiently characterized. N6-methyladenosine (m6A), the most prevalent internal RNA modification, plays a dynamic role in testicular development and spermatogenesis. In the present study, testicular tissues collected from normal and cryptorchid Tianhua mutton sheep were used to construct a transcriptome-wide m6A methylation atlas of the testis. Histological observation showed significantly reduced numbers of Sertoli and Leydig cells and diminished seminiferous tubule diameters in cryptorchid testes. RNA sequencing (RNA-seq) results demonstrated the dysregulated tissue-level mRNA expression of m6A regulators in cryptorchid testes. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) revealed 2080 differentially methylated m6A peaks, with 1449 hypermethylated and 631 hypomethylated m6A peaks in cryptorchid testes. The majority of these peaks were concentrated in the CDS region, followed by the stop codons and 3′ UTR. The integrative analysis of MeRIP-seq and RNA-seq data revealed that differentially expressed and m6A methylated genes were enriched in the apoptosis, AMPK, FoxO, TNF, and MAPK signaling pathways. Seven candidate genes, including RRM2, GTSE1, ATM, GORAB, INSR, HSPA1L, and MAPK9 were screened. In conclusion, this study identified differences in the mRNA abundance of m6A regulators and regional m6A enrichment between normal and cryptorchid sheep testes. These tissue-level differences may partly reflect changes in cellular composition. The findings provide a basis for further investigation of the potential involvement of m6A regulation in cryptorchidism-associated spermatogenic impairment.

Animals doi: 10.3390/ani16213458

Authors: Pan Yang Jiaman Pang Bangli Yang Yongqiang Liu Min Song Dun Deng Yusheng Lu Ting Rong

During weaning, piglets are exposed to multiple nutritional, environmental, pathogenic, and psychological stressors and are therefore susceptible to structural and functional intestinal injury. The resulting damage can lead to diarrhea, impaired growth, and increased susceptibility to disease, seriously compromising piglet health and the efficiency of pig production. Establishing stable and reliable experimental models that match specific research objectives is essential for elucidating the mechanisms of intestinal injury in piglets and for evaluating nutritional interventions and prevention and control strategies. Current research on porcine intestinal injury uses a multilevel framework centered on porcine intestinal epithelial cells, porcine intestinal organoids, and in vivo piglet models. These models offer distinct advantages in experimental controllability, tissue-level physiological relevance, and whole-organism biological fidelity, respectively, while each has a defined scope of application. Focusing on intestinal injury in piglets, this review comprehensively summarizes the construction methods, injury-induction approaches, and evaluation systems used across these model levels and analyzes the key factors affecting model stability and the comparability of research findings. This review is intended to inform mechanistic studies of intestinal injury in piglets, model standardization, and the evaluation of nutritional intervention strategies. On this basis, the review concludes that porcine intestinal epithelial cell lines are most appropriate for high-throughput mechanistic screening, organoids for questions that require epithelial diversity and donor- or region-specific context, and in vivo piglet models for confirming physiological and production relevance. Because model reproducibility depends strongly on challenge dose, animal or donor age, culture conditions, and assessment endpoints, a coordinated sequence from cell lines to organoids and finally to in vivo validation should be combined with standardized reporting of model parameters.

Animals doi: 10.3390/ani16213459

Authors: Zifan Li Yitong Yin Yashi Wu Yanchao Duan Zhigang Chen Yuandong Niu Nannan Gong Yu Kang Yuyu Niu Chenyang Si

Animals derived from embryo splitting have been successfully produced in livestock farming, and their milk and meat products have entered the human food market. However, although monkeys have been obtained through the same technique, their adult health status remains poorly studied. Here, we performed a comprehensive and systematic evaluation of five adult cynomolgus and two rhesus monkeys derived from embryo splitting, including hematological and biochemical analyses, glucose metabolism assessment, cardiovascular evaluation, bone mineral density examination, brain MRI, and reproductive performance assessment. No significant differences were found in measured parameters between sp-monkeys and controls in cynomolgus monkeys (5 vs. 4), with similar trends in rhesus monkeys. Because only two rhesus sp-monkeys and three rhesus controls were available, the rhesus data were not statistically compared. These results provide no evidence of obvious adverse effects on adult health and offer preliminary support for the safety of this technique in biomedical research.

Animals doi: 10.3390/ani16213457

Authors: Vinciane Roger Dominique Grandjean Milagros Benito

Decisions regarding neutering in athletic sled dogs involve a complex balance between health, behaviour, performance, breeding objectives, and management considerations. However, evidence regarding neutering practices and their relationship with health outcomes in actively competing sled dogs remains limited. To identify factors associated with neutering status and musher-reported motivations for neutering or maintaining dogs intact, and to evaluate the occurrence and distribution of clinical findings according to reproductive status in sled dogs participating in the 2023 Lekkarod® event. A total of 599 sled dogs registered for the 2023 Lekkarod® event, including participants in the main endurance race and the associated Kraft tournament, were included. Information regarding age, sex, breed, neutering status, and owner-reported motivations was collected using a structured questionnaire. Multivariable logistic regression was used to identify factors associated with neutering status. Clinical findings recorded during veterinary examinations conducted throughout the main Lekkarod race were classified and analysed according to reproductive status. Overall, 28.0% (168/599) of dogs were neutered. Neutering was significantly associated with female sex, older age, and breed category, whereas team position was not associated with reproductive status. The most frequently reported reasons for neutering were reproductive management, behavioural management, and disease prevention, whereas breeding purposes, preservation of genetic lines, performance-related considerations, and ethical concerns were the principal motivations for maintaining dogs intact. A total of 82 clinical findings were recorded, corresponding to 13.7% of the study population. Reproductive status was not associated with the overall occurrence of clinical abnormalities. However, the distribution of clinical finding categories differed significantly according to reproductive status (χ2 = 7.564, p = 0.023). Intact dogs presented proportionally more metabolic/systemic abnormalities, whereas neutered dogs presented proportionally more musculoskeletal findings. The observed distribution of musculoskeletal findings was not explained by age alone. In this population of actively competing European sled dogs, neutering was relatively uncommon and reflected a combination of reproductive management, health, breeding, and performance-related considerations. Although reproductive status was not associated with the overall occurrence of clinical abnormalities during competition, it was associated with differing patterns of clinical findings. Intact dogs presented proportionally more metabolic abnormalities, whereas neutered dogs presented proportionally more musculoskeletal findings. These findings support an individualised approach to reproductive management in canine athletes and warrant further prospective investigation of the relationships among reproductive status, metabolic adaptation, musculoskeletal health, and athletic performance.

Animals doi: 10.3390/ani16213456

Authors: Motswapo Esther Phoko Jones Wilfred Ng’ambi Tlou Julius Tjelele

This study assessed the effects of replacing lucerne meal with Seriphium (S.) plumosum meal on growth performance, methane emissions, rumen fermentation characteristics, and rumen bacterial community composition of Nguni steers. Twenty-eight 22-month-old Nguni steers (average body weight 300 ± 10 kg) were randomly assigned to diets containing 0%, 10%, 20%, or 30% of S. plumosum meal in a completely randomized design. The diets were formulated to be isocaloric to meet the nutrient requirements of growing Nguni steers. Feed intake, nutrient digestibility, and live weight gain were measured over a 90-day feeding period. Methane emissions were recorded using a laser methane detector, while rumen fermentation characteristics were evaluated in vitro. Rumen microbial communities were characterized using 16S rRNA gene sequencing. Increasing S. plumosum inclusion had no effect (p > 0.05) on dry matter intake, nutrient digestibility, live weight gain, or methane emissions, indicating that replacement of lucerne meal did not compromise production performance. However, dietary treatments influenced (p < 0.05) gas production throughout the incubation period (3–48 h). The 10% diet (T2) produced the highest gas volumes, with values declining at 20% (T3) and 30% (T4). Cumulative gas production after 48 h followed a similar pattern. Significant linear, quadratic, and cubic responses (p < 0.01) indicated a non-linear response of gas production to increasing dietary inclusion of S. plumosum. In vitro organic matter digestibility, ammonia–nitrogen concentration, volatile fatty acid profiles, and the acetate-to-propionate ratio, demonstrated changes (p < 0.05) in rumen fermentation dynamics. Heatmap analysis of the rumen microbiota revealed clear shifts in bacterial community composition with increasing S. plumosum inclusion in the diet. Therefore, S. plumosum meal has potential as a locally available alternative forage resource for partial replacement of lucerne meal in Nguni steer diets.

Animals doi: 10.3390/ani16213455

Authors: Larissa Y. Rodrigues Ricardo A. S. Faria Gustavo H. Alberto Carolina Zucatelle Pablo Dominguez-Castaño Josineudson A. II V. Silva

In the Brazilian Quarter Horse (QH), cutting points (CP) and monetary earnings (ME) are key success indicators, yet their genetic parameters remain unestablished. This study estimated heritability and breeding values for these traits to guide breeding decisions. Performance data from 1661 QHs competing in official Brazilian cutting events between 1981 and 2020 were analyzed for three traits: total lifetime cutting points (CPt), cutting points at four years of age (CP4), and lifetime monetary earnings (ME). Variance components and genetic parameters were estimated using single-trait Bayesian animal models. Heritability estimates were 0.110 ± 0.038 for CPt, 0.110 ± 0.048 for CP4, and 0.288 ± 0.091 for ME. Although overall genetic trends were positive, in the last decade CPt registered the highest annual genetic gain (0.116). The findings suggest that both CP and ME could be considered as potential selection criteria, and evaluating performance at four years of age (CP4) may favor early genetic progress. While ME shows greater potential for lifelong phenotypes due to its moderate heritability, both traits exhibited comparable selection accuracies; therefore, definitive recommendations require further studies on genetic correlations and expected responses.

Animals doi: 10.3390/ani16213454

Authors: V. Margarita Sanguinetti Nathan E. N. Erickson Barbara Wilhelm Colleen Pollock Su Hyun Lim Lianne McLeod Cheryl L. Waldner

Background: Bovine respiratory disease (BRD) drives antimicrobial use in cow–calf herds and feedlots. This rapid scoping review evaluated vaccination of North American beef dams or pre-weaning calves against respiratory pathogens and subsequent calf health. Methods: Five databases and gray literature sources were searched for English-language North American trials and observational studies published since 2000. Data were charted in Excel and independently reviewed. Results: Twenty-eight studies reported clinical outcomes. One of seven field trials of pre-weaning calf vaccination reported pre-weaning clinical benefit; two of six reported post-weaning benefit. Four of ten calf vaccination challenge trials reported clinical differences. Two of six observational studies of dam vaccination reported beneficial pre-weaning associations. Post weaning, one dam vaccination challenge trial reduced pathogen shedding without improving clinical outcomes, while one cohort reported favorable feedlot associations. Two of the three field trials with beneficial clinical findings evaluated vaccines targeting pathogens previously associated with disease in the study herd. Observational evidence from multicomponent programs could not isolate vaccination effects. Conclusions: Evidence did not identify a consistently effective vaccination protocol. Benefits may depend on herd disease history, pathogen targets, disease pressure, and management context. Trials should evaluate clearly defined protocols in relevant settings and report absolute and relative effects with confidence intervals.

Animals doi: 10.3390/ani16213453

Authors: Bakhit Baimenov Alma Dossova Stepan Khlyupko Vitaly Raketsky Aigul Tastemirova Vadim Ulyanov Tatyana Ulyanova

The Kustanay horse is a riding breed developed in Kazakhstan whose genetic diversity and differentiation remain insufficiently studied. We assessed genetic diversity and population structure using 16 autosomal STR loci from a 17-locus panel comprising 12 ISAG core markers. Of 765 genotyped horses, 703 remained after excluding duplicate records and close relatives; the X-linked LEX3 locus was excluded from population analyses. Five groups were studied: registered Kustanay (KST, n = 32), Kustanay from Qazaq Tulpary (KT, n = 36), Thoroughbred (TB, n = 31), Kustanay crossbred (CROSS, n = 9), and undocumented origin (UB, n = 595). Expected heterozygosity was high across groups (HE = 0.661–0.725). Overall FST was 0.0088, and AMOVA attributed 99.13% of molecular variance to variation within individuals. The Puechmaille estimators favored K = 2, whereas Evanno ΔK peaked at K = 4; neither solution revealed clear separation among groups. Nei’s genetic distances and PCoA were consistent with weak differentiation and similarity between registered Kustanay and undocumented horses. Unequal group sizes limit interpretation, and these findings do not establish breed identity for individual undocumented horses. The results inform pedigree recording and conservation planning.

Animals doi: 10.3390/ani16213452

Authors: Goutham Kumar Isai Kevin A. Drygas Brad Weeks

Background: Achieving complete surgical margins can be challenging for tumors involving the distal extremities due to limited surrounding soft tissue and the proximity of functionally important structures. Incompletely excised tumors may require adjuvant radiation therapy to treat suspected residual microscopic disease. Yttrium-90 phosphate hydrogel (IsoPet®) is an emerging injectable radiation modality that delivers localized beta radiation to a tumor bed or surgical margin. Case Description: An eight-year-old neutered male Bullmastiff was evaluated by the referring veterinarian for firm subcutaneous masses developed along the right pelvic limb. Fine-needle aspiration was initially suggestive of an inflammatory process; however, subsequent punch biopsies identified Grade II soft tissue sarcoma. The patient was referred for surgical oncology evaluation. Four right pelvic limb masses and the right popliteal lymph node were surgically excised. Histopathological examination of the excisional specimens identified poorly differentiated carcinoma involving the excised masses, with metastatic involvement of the right popliteal lymph node and incomplete surgical margins. Adjuvant Y-90 hydrogel was elected following complete surgical wound healing. On 20 November 2025, 8.46 mCi (313 MBq) of Y-90 phosphate hydrogel in a total volume of 4.7 mL was administered through 66 peri-incisional and incisional injections distributed across the previous right pelvic limb surgical beds using a grid-based technique. The procedure was completed without complications. Post-treatment follow-up demonstrated satisfactory wound healing, with only mild transient appetite reduction and minimal localized tissue irritation. After surgery plus adjuvant Y-90, no clinically detectable local recurrence was identified at the treatment sites during the 120-day documented follow-up period. Seventy-four days following Y-90 hydrogel treatment, computed tomography performed for persistent otic disease identified an aggressive osteolytic lesion involving the right C5 vertebra with extradural spinal cord compression. The relationship between this lesion and the previously treated pelvic limb neoplasia could not be definitively established. Despite the subsequent development of this cervical lesion, no clinically detectable recurrence was documented at the original treatment limb sites. This finding is an observation after combined surgery and Y-90, not evidence of Y-90-specific efficacy. The patient was subsequently managed supportively and was euthanized by the referring veterinarian approximately four months after identification of the cervical lesion. Conclusion: This single case demonstrates that peri-incisional Y-90 phosphate hydrogel could be administered to multiple distal pelvic-limb surgical beds after limb-sparing excision. The procedure was well tolerated. No clinically detectable local recurrence was identified at the treated sites during the 120-day documented follow-up period. Because gross disease had already been removed and there was no comparator, these observations cannot establish the independent efficacy of IsoPet®. Prospective studies are needed.

Animals doi: 10.3390/ani16213451

Authors: Mihaela Niculae Smaranda Crăciun Raluca Marica Maria Carmen Turcu Lucia Victoria Bel Andrei Răzvan Codea Raul Alexandru Pop Simon L. Priestnall

Chronic renal disease is recognized in geriatric companion mammals, but detailed clinicopathological descriptions in chinchillas remain limited. An 11-year-old neutered female pet chinchilla (Chinchilla lanigera) with a six-month history of progressive weight loss and hyporexia developed acute clinical deterioration following approximately 8 h of indoor heat exposure (estimated maximum 29 °C). Anorexia, lethargy, absence of fecal output, and progressive dyspnoea preceded death. Necropsy revealed severe bilateral chronic renal lesions, gastrointestinal gaseous distension, and severe pulmonary congestion. Histopathology documented chronic tubulointerstitial nephropathy with interstitial fibrosis supported by Masson–Goldner trichrome staining and mineral deposits demonstrated by von Kossa staining. Periodic acid–Schiff staining highlighted glomerular and tubular basement membranes. Marked pulmonary vascular congestion was confirmed histologically, whereas pulmonary oedema and established pneumonia were not demonstrated in the examined sections. Together with the progressive terminal dyspnoea, these findings indicated clinically important respiratory abnormalities, although their underlying mechanism and contribution to death remained uncertain. Pure cultures of Klebsiella pneumoniae were recovered from six anatomical sites, with all isolates exhibiting an identical antimicrobial susceptibility profile and a reported multidrug-resistant phenotype. No bacterial-associated inflammatory lesions were identified in the histologically examined tissues, providing no morphological evidence of established disseminated septic disease. Given the uncertain time of death and a possible post-mortem interval of up to approximately 6 h, ante-mortem infection could not be reliably distinguished from perimortem dissemination or post-mortem translocation. Environmental heat exposure may have contributed to acute deterioration in an animal with severe chronic disease, although heat stroke could not be established retrospectively. This case supports a multifactorial interpretation and highlights the potential One Health relevance of antimicrobial resistance in companion animals, without demonstrating transmission.

Animals doi: 10.3390/ani16213450

Authors: Quan-Gang Wang Qian Jing Guoxin Wu Hang Shu Zhong-Ying Liu Ronghua Ji Yao Guo Zhong-Hong Wu

This study compared a programmable logic controller (PLC) combining indoor temperature, outdoor temperature, and outdoor relative humidity with conventional temperature-triggered pad cooling and manual fan adjustment in two commercial rabbit houses in Leshan, China, a region characterized by a hot and humid climate. The 33-day summer trial included 108 selected Hyla rabbits aged 39 d per house. The PLC house had a mean air temperature 0.36 °C higher and relative humidity 4.34 percentage points lower than the control house. Final body weight and average daily gain were higher, while feed conversion did not differ significantly. Intra-abdominal temperature near the cooling pads was 0.29 °C lower. Estimated cooling pad recirculation was 81.0% lower and combined fan and pump electricity use was 21.2% higher. A partial-budget analysis at a standardized scale of 3000 retained rabbit production-equivalents estimated a benefit 2955.95 Chinese yuan (CNY) higher for the treatment house than for the control house under the specified prices and scaling assumptions. Each strategy was applied in one house, so these preliminary differences cannot be separated from house effects. These observations support further evaluation in independently replicated houses, with animal responses, resource use, and deployment costs assessed together.

Animals doi: 10.3390/ani16213448

Authors: Tarik Safak Elena Stancheva Toncho Penev Hafize Tuğba Yüksel-Dolgun Ali Risvanli Oznur Yılmaz-Koç Özgül Gülaydın Ruslan Salykov Ömer Deniz Özgür Kaynar Oğuzhan Dolgun

This pilot study characterised bacterial DNA profiles in 40 milk and 39 faecal samples from eight Kyrgyz mare–foal pairs collected at birth, 24 h, and days 7, 14, and 21 postpartum using V3–V4 16S rRNA gene sequencing. Permutation Friedman tests detected temporal differences in foal faecal Shannon diversity and observed feature counts (Holm-adjusted p = 0.020 for both), but not in milk. Faecal diversity was descriptively lower at 24 h and day 7 than at birth and increased at days 14–21. Animal-blocked weighted UniFrac PERMANOVA detected a temporal effect in faecal DNA profiles (Holm-adjusted p = 0.0028), but not in milk (p = 0.0956). Pair-blocked milk–faecal comparisons were significant at each time point (Holm-adjusted p = 0.0391), although differences in dispersion limit interpretation. Without sequenced negative controls, background contamination cannot be distinguished from genuine milk-associated bacterial DNA; therefore, milk profiles cannot be confidently attributed to resident milk microbiota. Observed taxonomic similarities do not demonstrate microbial transmission or milk-derived foal gut colonisation. Significance in faeces but not milk does not establish a difference between their temporal responses, which was not directly tested. These findings are exploratory and require controlled validation.

Animals doi: 10.3390/ani16213449

Authors: Jialin Gao Junhao Wang Ping Jiang Shaohui Tan

Accurate recognition of pig behavior is important for animal health monitoring and precision livestock farming. However, fine-grained behavior recognition in group-housed pigs is challenging because of individual occlusion, large posture variation, and subtle visual differences among similar behaviors. Here, an improved YOLOv8n-based model, EPP-YOLOv8n, was developed for fine-grained pig behavior recognition. An efficient multi-scale attention module was inserted after the spatial pyramid pooling-fast module (SPPF) to enhance behavior-related feature extraction. A behavior-factorized asymmetric head (BFA-Head) was designed with scale-adaptive mixed convolution to reduce detection-head complexity, while posture- and interaction-assisted supervision was incorporated to guide fine-grained feature learning. A partial-channel mixed convolution module (P5-selective PLC-Mix) was deployed within the Backbone P5 C2f module to reduce redundant computation, followed by task-aware structured pruning with recovery training for further model compression. On a dataset comprising 2713 images and 30,695 annotated instances after training-set augmentation, EPP-YOLOv8n achieved 98.13% precision, 94.50% recall, and 89.07% mean average precision at intersection over union (IoU) thresholds from 0.50 to 0.95 (mAP@0.5:0.95), representing increases of 2.05, 1.52, and 1.64 percentage points, respectively, over YOLOv8n. Parameter count and billion floating-point operations (GFLOPs) were reduced by 14.59% and 9.70%, respectively. These results indicate that the proposed model can improve fine-grained behavior recognition while reducing model complexity, supporting automated behavior monitoring in group-housed pigs.

Animals doi: 10.3390/ani16213447

Authors: Yuting Hu Guoqing Duan Huaxing Zhou Amei Liu Huan Wang

Parasinilabeo maculatus is a rare cyprinid endemic to China whose phylogenetic position and taxonomy are poorly understood. We present its first complete mitogenome to clarify its phylogenetic placement within Semilabeonini. The circular mitogenome is 16,606 bp and shows the typical vertebrate gene arrangement: 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes, and a control region, with the L-strand replication origin (OL) in the WANCY tRNA cluster. It has a pronounced AT bias (58.7%), with AT-skew (0.094) and GC-skew (−0.250), consistent with other cyprinids. Phylogenetic trees reconstructed using ML and BI methods based on concatenated PCGs show similar topologies: (1) placing P. maculatus within Semilabeonini, where it is only provisionally grouped with P. assimilis and P. longicorpus in the full PCG dataset. This placement receives low gene and site concordance (gCF = 15.38%, sCF = 40.57%) and conflicts with the first + second codon position tree, in which P. maculatus is sister to Hongshuia microstomata rather than to its congeners; (2) supporting the monophyly of Semilabeonini and of several genera (e.g., Ptychidio), whereas the monophyly of Parasinilabeo is tentatively resolved at best and is therefore treated here as tentative (see point 1); (3) preliminarily revealing non-monophyly of some genera (e.g., Sinocrossocheilus) based on the current sampling, suggesting that some relationships may not fully match morphological classifications. However, these inferences rest on mtPCGs and limited taxon sampling and require testing with broader taxonomic and genomic sampling. These mitogenomic data provide a genomic resource for taxonomic and conservation studies of Semilabeonini.

Animals doi: 10.3390/ani16213446

Authors: Katarzyna Żarczyńska

Milk stones (lactoliths) are poorly understood structures occurring within the ductal and cisternal systems of the bovine mammary gland. Although they have been described in the veterinary literature for more than 150 years, data on their formation, composition, prevalence, and clinical significance remain limited. This review synthesizes evidence identified through searches of PubMed, Scopus, Web of Science, and Google Scholar, supplemented by screening reference lists and historical veterinary sources. Historical concepts of their pathogenesis have included precipitation of milk minerals, aggregation or coagulation of proteins, and the formation of an organic core around which organic and mineral components may subsequently accumulate. Studies of corpora amylacea, secretory concretions, and mineralization of alveolar contents confirm that protein–mineral structures can form within the mammary gland; however, their relationship to clinically recognized lactoliths has not been established. Likewise, no direct association has been demonstrated between lactolith formation and prolonged milk accumulation, incomplete emptying of the gland, or mastitis. The clinical significance of lactoliths primarily results from their potential to cause partial, intermittent, or complete impairment of milk outflow. Ultrasonography enables their localization and morphological assessment and assists in differentiating them from other causes of obstruction; however, validated ultrasonographic criteria for the definitive identification of these structures are lacking. This review summarizes current knowledge of the terminology, possible mechanisms of formation, clinical significance, diagnosis, and management of lactoliths in cattle and identifies the most important directions for future research.

Animals doi: 10.3390/ani16213445

Authors: Liisa Siimon Tanel Kaart Merle Valdmann Jevgeni Kurykin Andres Waldmann

Inflammation is a major risk factor for cytological endometritis (CYTO) in dairy cows. This study evaluated plasma concentrations of positive acute phase proteins (APPs) including haptoglobin, serum amyloid A and ceruloplasmin, as well as the negative acute phase protein albumin and the metabolic markers cholesterol and insulin-like growth factor-1 during the transition period. Blood was collected from multiparous Holstein cows at −14 days prepartum and 7 and 21 days postpartum. Endometrial cytology samples were collected at 40 ± 2 days postpartum, and CYTO was diagnosed using a polymorphonuclear neutrophil threshold of >8%. Cows were classified according to CYTO status and the presence of clinical disease or calving-related disorder (DIS) as CYTO/DIS− (n = 14), CYTO/DIS+ (n = 22) or HEALTHY (n = 50). APP and metabolic marker profiles associated with CYTO differed according to concurrent disease/disorder status. Plasma haptoglobin concentrations at 7 days postpartum were higher and albumin concentrations at 21 days postpartum were lower in both CYTO groups than in healthy cows (p < 0.05), suggesting that these markers may have potential value for identifying cows at risk of CYTO, irrespective of concurrent clinical disease or calving-related disorder. Further studies are needed to validate their predictive performance.

Animals doi: 10.3390/ani16213444

Authors: Minghui Liu Terigele Wang Hang Zhang Ni Dan Zheming Ren Wenqi Liang Pengfei Sun

This study evaluated the effects of dietary conjugated linoleic acid (CLA) on serum metabolites, antioxidant capacity, and immune function of transition ewes, as well as on growth performance, serum biochemical indices, and hormone levels of their suckling lambs. Forty healthy Ujumqin ewes (body weight 56.62 ± 5.30 kg) were randomly assigned to four groups (n = 10/group) at 28 d before expected parturition: control (basal diet), CLA-1 (basal diet + 10 g/d CLA), CLA-2 (basal diet + 20 g/d CLA), and CLA-3 (basal diet + 30 g/d CLA). Blood samples were collected from ewes at 21 d prepartum, at delivery, and at 21 d postpartum, and from lambs at 7 and 42 d postpartum. Serum metabolites, antioxidant enzymes, and inflammatory cytokines were measured. In ewes, CLA supplementation did not significantly affect serum AST, β-HB, GLU, TG, NEFA, SOD, GPX, MDA, or T-AOC (p > 0.05), although β-HB, GLU, SOD, GPX, and MDA changed with time. However, CLA markedly decreased serum TNF-α, IL-8, IL-6, and IL-1β (p < 0.05). In lambs, maternal CLA did not affect birth or final body weight. Maternal CLA decreased serum GH and the GH/IGF-1 ratio and increased IGF-1 (p < 0.05), especially at 30 g/d. Serum SOD, GPX, and MDA did not differ among groups, whereas T-AOC showed a significant group × time interaction (p = 0.004). Maternal CLA also decreased serum TNF-α, IL-8, IL-6, and IL-1β in lambs (p < 0.05). In conclusion, perinatal CLA supplementation did not significantly improve most energy metabolic or antioxidant enzyme indices under these conditions, but it alleviated excessive inflammatory responses in ewes and suckling lambs and activated the GH-IGF-1 axis in lambs. The 30 g/d CLA dose showed the strongest immunomodulatory and endocrine effects. These findings suggest that CLA may serve mainly as an immunomodulatory strategy in perinatal sheep, and dose optimization warrants further study.

Animals doi: 10.3390/ani16213440

Authors: Yanan Liu Lili Xu Qun Liu Sudong Xia Xuying Jia Yan Sun Wenli Zhou Dan Zhang

The Chinese mitten crab (Eriocheir sinensis) is an important freshwater aquaculture species in China, but high summer temperatures may disrupt its behavioral patterns and energy metabolism. In this study, male E. sinensis were maintained at 25 °C or 35 °C, and behavioral tracking, histological observation, biochemical assays, RT-qPCR, and untargeted metabolomics were combined to investigate the effects of thermal stress on swimming and aggressive behaviors and their associated metabolic responses. For swimming behavior, compared with 25 °C, crabs at 35 °C showed decreases in total moving distance, average speed, and the proportions of slow, medium, and fast-speed movement, together with a significant increase in inactive time (p < 0.05). For agonistic behavior, compared with 25 °C, both the overall proportion of agonistic intensity and the proportion of strong fighting decreased significantly at 35 °C (p < 0.05). According to agonistic behavior data, the crabs were classified as winners or losers, generating four groups: T25W, T25L, T35W, and T35L. Non-contact behaviors decreased significantly in both winners and losers, whereas contact behaviors also decreased significantly in winners (p < 0.05). Histopathological analysis revealed that, at 35 °C, E. sinensis exhibited disorganized muscle fibers and structural damage to the gills, including swelling of the distal lamellar tips and morphological abnormalities in epithelial and pillar cells. With respect to energy metabolism, muscle glycogen in winners was significantly higher than that in losers at both temperatures, whereas haemolymph lactate concentrations were significantly higher in winners only at 25 °C. Haemolymph glucose concentrations were significantly higher in winners at 25 °C but in losers at 35 °C (p < 0.05). Compared with 25 °C, exposure to 35 °C significantly decreased muscle succinate dehydrogenase (SDH) activity in both winners and losers and increased gill citrate synthase (CS) activity in winners. In losers, muscle pyruvate kinase (PK) activity decreased nonsignificantly; lactate dehydrogenase (LDH) activity increased (p < 0.05). Under 35 °C conditions, significant upregulation of AMPK, Sestrin, and SIRT1 expression and significant downregulation of mTOR expression (except in losers’ muscle) were exhibited in both muscle and gill tissues (p < 0.05). Untargeted metabolomics identified 297, 301, 484, and 393 differential metabolites in comparisons in T25W vs. T25L, T35W vs. T35L, T35W vs. T25W, and T35L vs. T25L, respectively. These differential metabolites were mainly associated with amino acid, nucleotide, and lipid metabolism, aminoacyl-tRNA biosynthesis, ABC transporters, and neuroactive ligand–receptor interaction. Collectively, acute exposure to 35 °C suppressed swimming and agonistic behavior, with this behavioral inhibition being associated with tissue damage and regulation of energy metabolism. This study provides candidate indicators and a scientific basis for the early assessment and monitoring of thermal stress in E. sinensis aquaculture.

Animals doi: 10.3390/ani16213443

Authors: Slawomir Gonkowski Paweł Świsłowski Nariste Kadyralieva Liliana Rytel Małgorzata Rajfur

In Kyrgyzstan, despite heavy environmental pollution, no assessment of dairy cow exposure to trace elements using hair sample analysis has been conducted so far. Therefore, this study aimed to assess the concentrations of selected trace elements, including the essential elements Cu, Fe, Mn, and Zn, as well as Cd, Pb, Hg, and Ni, in the hair of dairy cows in the vicinity of Bishkek—the largest city in Kyrgyzstan. The mean concentrations (±SD) and medians (given in parentheses) for individual elements were as follows (in mg/kg dry weight): Cd: 0.62 ± 0.19 (0.60), Cu: 5.63 ± 1.16 (5.69), Fe: 274.9 ± 169.8 (248.0), Mn: 7.81 ± 6.06 (6.12), Ni: mean and median below the limit of detection, Pb: 3.98 ± 1.47 (3.68), Hg: 0.0051 ± 0.0022 (0.0049), and Zn: 77.94 ± 12.91 (79.65). Statistically significant spatial differences were found for Cd, Fe, Hg, Mn, Pb, and Zn, whereas Cu concentrations did not differ significantly among districts. A statistically significant positive correlation with age was found only for Fe (Rs = 0.39, p = 0.006). The results demonstrate the occurrence and spatial variability of the analyzed elements in the hair of cows from the vicinity of Bishkek. However, further comprehensive studies are needed to determine the sources of contamination and the influence of the element concentrations noted in this study on animal health status, as well as to assess potential risks to human consumers of animal products.

Animals doi: 10.3390/ani16213441

Authors: Ramya Kota Shreya Neelagiri Goutham Kumar Isai Davia Brown Jayanta Banik Andrea Villa Rubio Phaneendra Batchu David Shapiro-Ilan Cristina Pisani Aftab Siddique Ajit K. Mahapatra Sudhanshu S. Panda Thomas H. Terrill

Bioelectrical impedance analysis (BIA) has been widely evaluated in livestock for body composition and physiological assessment, but its incremental value for anemia-risk monitoring in small ruminants remains unclear. This study evaluated BIA as part of an environmentally aware multimodal machine-learning framework for anemia-related monitoring in goats. Data from 81 Spanish male goats were restructured into animal-by-date observations and integrated with objective FAMACHA© classification, blood packed cell volume, gastrointestinal nematode fecal egg count, weather variables, temperature–humidity index, and raster-derived environmental covariates. Objective FAMACHA© was derived from packed cell volume and used as the primary classification endpoint. Binary, three-class, and four-class classification tasks were evaluated using animal-grouped cross-validation to reduce leakage from repeated observations. Binary low-risk versus high-risk classification was the most reliable task, whereas multiclass prediction showed limited resolution among adjacent FAMACHA© categories. A formal ablation analysis showed that BIA-only features achieved a ROC-AUC of 0.672, compared with 0.838 for fecal egg count plus weather/THI and 0.824 for the complete multimodal model. The full model did not significantly outperform the fecal egg count plus weather/THI model (ΔAUC = −0.013; 95% CI: −0.124 to 0.093; p = 0.844). A supplementary matched feature-block analysis likewise did not demonstrate a statistically reliable incremental improvement from adding BIA. Threshold analysis showed that a screening-oriented threshold detected 83.3% of high-risk observations with 71.3% specificity. These findings indicate that parasitological and environmental predictors provided the strongest discriminatory information, whereas the independent incremental predictive benefit of BIA remained unestablished. BIA should therefore be regarded as a potential supplementary physiological sensing modality rather than a validated primary predictor or replacement for established anemia-assessment methods.

Animals doi: 10.3390/ani16213442

Authors: Fujiang Hu Jiahao Xiao Yuancheng Guo Yang Mi Rong Huang Linsen Zan Wucai Yang Chunping Zhao

Low-protein diets supplemented with rumen-protected methionine and lysine (RPML) can optimize amino acid supply while maintaining milk performance and reducing environmental impacts. But responses of cows to RPML are inconsistent and modulating factors remain poorly understood. This meta-analysis tested whether RPML supplementation improves lactational performance and alters plasma amino acid profiles of dairy cows. Forty-two eligible studies published up to 1 June 2026 were retrieved from PubMed, Web of Science and ScienceDirect. Meta-analysis, subgroup analysis, correlation analysis and dose–response regression were adopted. Results showed that RPML supplementation significantly increased milk yield by 0.500 kg/d, energy-corrected milk by 1.378 kg/d, protein yield by 0.038 kg/d, protein concentration by 0.052%, fat yield by 0.030 kg/d, fat concentration by 0.043%, 3.5% fat-corrected milk by 0.778 kg/d, milk total nitrogen by 0.010%, and casein nitrogen by 0.007%. Plasma arginine, asparagine, isoleucine, leucine, lysine and methionine increased, while glycine declined. Subgroup analysis showed that cow breed, parity, lactation stage, basal diet and dietary crude protein were key moderators. Digestible methionine dosage correlated with milk fat concentration and plasma leucine, and digestible lysine dosage correlated with milk protein yield. Significant regression models confirmed the combined effects of methionine and lysine on multiple milk traits. These results indicate that supplementation with RPML represents an effective nutritional strategy for enhancing milk yield and milk quality in dairy cows. Such findings may assist producers and nutritionists in refining amino acid feeding programs to optimize dairy cow productive performance.

Animals doi: 10.3390/ani16213439

Authors: Gabija Džiaugienė Katja Schulz Marius Masiulis Carola Sauter-Louis Alvydas Malakauskas

Since 2014, Lithuania has been affected by African swine fever (ASF), with varying levels of disease occurrence. This study investigated factors associated with ASF persistence in wild boar populations across Lithuanian municipalities. Based on ASF occurrence patterns, municipalities were classified into three groups: endemic, non-endemic, and ‘no ASF detected’. Endemic municipalities had ASF cases reported for at least three consecutive years. Data from 2016 to 2022 were analysed, including numbers of wild boar hunted or found dead, environmental variables, and spatial characteristics. Analyses included non-parametric tests, correlation analyses, negative binomial regression, and longitudinal logistic regression using generalised estimating equations to assess associations with ASF occurrence. Differences were observed between municipality groups, with endemic areas showing higher numbers of ASF cases and found dead wild boar. The number of hunted wild boar was positively correlated with the number of ASF-positive hunted wild boar, likely reflecting hunting-related sampling effort. Forest cover was negatively associated with ASF case counts, while wetland coverage was positively associated with endemic status, case counts and ASF presence over time. The findings highlight the importance of surveillance strategies, especially those based on carcass detection and testing. Environmental factors, including wetland-associated conditions, may influence ASF occurrence. Results support the need for targeted, landscape-informed approaches to ASF surveillance and control in endemic regions.

Animals doi: 10.3390/ani16213438

Authors: Jinfeng Ma Qingtian Wei Peipei Niu Mengyu Li Chifeng Chen Xuemei Xiao Wangjun Wu Pinghua Li Ruihua Huang

Pigs weaned per sow per year (PSY) is the core indicator used to evaluate sow-herd reproductive efficiency and a key factor affecting production profitability in commercial pig farms. The traditional PSY calculation uses the average inventory of mated (or parous) sows as the denominator and ignores the resource consumption of non-productive inventory, such as unserved gilts and weaned sows awaiting culling, leading to overestimated reproductive efficiency, concealed costs, and misleading management decisions. The overestimation range of 8–15% has been indicative estimates on non-productive days of commercial sow herds, and the improved method achieved a robustness of ≤3% based on the validation of basic data from pig farming enterprises involved in this paper. In this study, an improved PSY accounting method was developed based on whole-herd resource accounting. Gilts ≥220 days of age and weaned sows not yet culled were incorporated into the basic sow inventory, a dynamic inventory-time-weighting approach was established, and the method was adaptively optimized for continuous and batch production, with mode-specific formulas, validation cases, and an implementation system. It should be noted that traditional PSY and improved whole-herd PSY measure different quantities: the former reflects reproductive efficiency per mated sow, while the latter reflects resource-weighted reproductive efficiency per unit of resource-weighted sow inventory. Case studies on two commercial pig farms showed that the improved method addressed the structural limitations of the traditional formula, produced PSY values of 20–30 piglets per sow per year, and remained robust to parameter variation, with PSY estimates fluctuating by no more than 3% across the feasible range of w (0.60–0.80) in these two cases, and supports refined on-farm cost accounting and standardized cross-farm comparison, although independent multi-farm validation is still required. This study provides a practical tool for reproductive efficiency assessment and refined management in pig farms.

Animals doi: 10.3390/ani16213436

Authors: Woo-Do Lee Hyun-Woo Cho Min Young Lee Sang-Yeob Lee Han Tae Bang Ju Lan Chun Seyeon Chang Won Yong Jung Kyoung-Min So

This nonrandomized sequential study evaluated nitrogen (N) balance, apparent nutrient digestibility, serum biochemistry, and indoxyl sulfate (IS)-related outcomes in ten healthy adult Beagles. Dogs received the crude protein (CP) 30% formulation for four weeks followed by the CP 18% formulation for four weeks; urine and feces were quantitatively collected during four consecutive days in each period. Analyzed dietary CP concentrations were 31.36% and 19.20% of dry matter. Daily urinary N excretion was lower in the second period (5.60 ± 0.35 vs. 3.34 ± 0.15 g/day; false discovery rate (FDR)-adjusted q < 0.001), as was calculated N balance (2.52 ± 0.39 vs. 1.04 ± 0.14 g N/day; paired p = 0.0022). The N retention percentages were 28.78 ± 4.45% and 20.66 ± 2.81%, respectively (p = 0.077; q = 0.132), and were not demonstrated to be equivalent. Apparent total tract digestibility of CP and most analyzed amino acids was lower in the second period. Exploratory daily urinary IS excretion was 3.08 ± 0.35 and 2.59 ± 0.36 mg/day, respectively (p = 0.394; q = 0.394). The analyzed indispensable amino acid concentrations of both diets met the dietary nutrient profile benchmarks evaluated in this study. However, the formulations differed in ingredients and predicted daily energy intake, and period was completely confounded with feeding order. These observations cannot establish an isolated effect of dietary protein, actual long-term body protein retention, or nutritional equivalence.

Animals doi: 10.3390/ani16213434

Authors: Qingyu Zhao Wanling He Fumiao Sun Fengyan Wang Jing Zhang Zhaoyang Wu Jiangtao Liu Zhibin Zhang Xiaoli Li

Soybean meal (SBM) is a major protein source in commercial layer diets; however, fluctuations in SBM prices and supply have increased the need for alternative protein sources in poultry production. Plant processing by-products are considered promising feed resources because of their abundant availability and nutritional value. However, the effects of partially replacing SBM with different plant-derived by-products on digestive function, gut microbiota, nutrient utilization, productive performance, and egg quality in aged laying hens remain insufficiently characterized. Therefore, this study aimed to evaluate the effects of partially replacing SBM with different plant-derived by-products on gastrointestinal digestive enzyme activities, cecal microbial community composition, apparent nutrient utilization, productive performance, and egg quality in aged laying hens. A total of 270 Hy-Line Brown laying hens at 80 weeks of age were randomly assigned to five dietary treatments, with six replicates per treatment and nine hens per replicate. Digestive enzyme activities and cecal microbiota were assessed using three birds per treatment. The control group was fed a corn-SBM basal diet, whereas the experimental groups were fed diets in which SBM was partially replaced with 10% palm kernel meak (PKM, T1), 12% rice bran meal (RBM, T2), 15% sunflower meal (SFM, T3), or 8% Zanthoxylum bungeanum seeds (ZBS, T4), respectively. The results showed that digestive enzyme activities varied among dietary treatments, with responses depending on the type of enzyme and gastrointestinal segment examined. Cecal microbiota analysis revealed no significant differences in alpha diversity among dietary treatments (p > 0.05), whereas overall microbial community composition differed significantly (PERMANOVA, p = 0.015). Differences in the relative abundances of individual bacterial taxa were also observed, but none remained significant after multiple-testing correction. Furthermore, the T3 group exhibited significantly higher apparent utilization of all six measured nutrients than the control group, whereas the other treatments showed mixed, nutrient-specific responses. Dietary treatments affected laying rate and eggshell thickness (p < 0.05), whereas average egg weight, average daily feed intake, feed-to-egg ratio, and other measured egg quality parameters were not significantly affected (p > 0.05). Overall, the four plant-derived by-products produced variable responses across the measured outcomes, suggesting ingredient-specific potential as partial substitutes for SBM under the dietary formulations and inclusion levels evaluated. However, their equivalent nutritional suitability cannot be established from the present findings, and further studies with larger sample sizes and multiple inclusion levels are warranted.

Animals doi: 10.3390/ani16213437

Authors: Iván Miralles-Plaza Marina Martín-Ambrosio-Francés Isabel del Portillo-Miguel Hayley-Rebecca McDonald Altin Cala Inmaculada Cerrada-Serra

Focal junctional tachycardia (FJT) is a regular, narrow QRS complex tachycardia with subtle cycle length irregularity, frequently associated with isorhythmic atrioventricular dissociation (IAVD). Its presentation may be paroxysmal or non-paroxysmal. This rhythm is well recognized in humans and dogs. However, to the best of the authors’ knowledge, FJT has not been previously published in cats. This case report describes a 9-year-old neutered male domestic shorthair cat presenting with signs of congestive heart failure, hyperthermia, and a non-paroxysmal FJT. Electrocardiography demonstrated a narrow QRS tachycardia with type I synchronization IAVD, consistent with suspected FJT. Transthoracic echocardiography revealed severe biatrial enlargement, concentric ventricular hypertrophy and echocardiographic measures suggestive of high probability of pulmonary hypertension. There was apparently preserved global systolic function and diastolic dysfunction. These findings were consistent with a non-specific cardiomyopathy phenotype stage C. Pleural fluid cytology initially suggested a lymphoproliferative disorder; however, postmortem histopathological examination confirmed diffuse lymphocytic myocarditis without evidence of neoplasia. Despite intensive supportive and antithrombotic therapy, the cat experienced sudden death. To the authors’ knowledge, this is the first description of suspected FJT with type I IAVD in a cat. This case highlights myocarditis as a critical differential diagnosis in cats presenting with a lymphocytic effusion, structural echocardiographic changes, and complex supraventricular arrhythmias. It also discusses the diagnostic challenges and prognostic implications associated with inflammatory myocardial disease in this species.

Animals doi: 10.3390/ani16213435

Authors: Narudee Kashemsant Panida Panitsillapakit Monchanok Vijarnsorn Chalermpol Lekcharoensuk Sarawut Yangtara Julaporn Pooliam B. Duncan X. Lascelles

Canine hip osteoarthritis (OA) requires multidimensional assessment. This randomized, double-blind, placebo-controlled trial evaluated 4-week treatment with meloxicam, PCSO-524, EAB-277, meloxicam plus PCSO-524, or placebo in 203 client-owned dogs with naturally occurring hip OA. The principal outcome was index-hindlimb peak vertical force (PVF) measured by force-plate gait analysis. Secondary outcomes included PVF responder status, orthopedic assessment score (OAS), Liverpool Osteoarthritis in Dogs (LOAD), Canine Brief Pain Inventory (CBPI), and safety assessments. The primary baseline-adjusted mean PVF analysis did not demonstrate treatment superiority among groups. A secondary responder analysis using an analysis-stage >5%BW PVF threshold showed that meloxicam had a nominally higher Week 4 responder probability than placebo (relative risk = 4.000; 95% confidence interval, 1.223 to 13.079; p = 0.022), interpreted as supportive rather than confirmatory. Within-group score reductions were observed in LOAD and CBPI pain interference for the combination group, LOAD for PCSO-524, and CBPI pain interference for EAB-277. OAS did not change significantly within any group. Between-group differences in LOAD and CBPI changes were not significant. No consistent group-level laboratory safety concern was detected. These findings support multidimensional assessment while emphasizing that secondary results do not establish treatment superiority.

Animals doi: 10.3390/ani16213433

Authors: Linghao Kong Yi Zhu Bowen Liu Hua Wei Yingdong Li

Geographic phenotypic variation is a widespread feature of natural populations, often driven by local environmental heterogeneity and trophic interactions. While the gut microbiome is recognized as a key mediator between environmental resources and host physiology, its role in the adaptive differentiation of amphibians remains insufficiently explored. In this study, we employed an integrative approach—combining morphological measurements, DNA metabarcoding for dietary analysis, and 16S rRNA sequencing for gut microbial profiling—to investigate three geographically structured populations (Dandong [DD], Fushun [FS], and Benxi [BX]) of the Dybowski’s frog (Rana dybowskii). The results showed that the morphological characteristics (body weight, head-body length, hindlimb length, etc.) of DD were significantly higher than those of FS and BX. Dietary analysis via gut content DNA metabarcoding identified distinct food structures; notably, the DD population consumed a higher proportion of Dipteran prey (e.g., Smittia). Correspondingly, the gut microbiota showed significant population-level clustering. We identified an enrichment of specific functional bacteria in the DD population, such as Arthrobacter, which is known for its chitin-degrading capabilities, aligning with the consumption of chitin-rich insect prey. Mantel tests further confirmed a strong correlation between dietary composition, microbial community structure, and host morphological traits. These findings suggest that diet-driven gut microbiome variation is consistent with a contributing role in the phenotypic differentiation of R. dybowskii across different habitats, although causal relationships remain to be tested directly. By integrating trophic ecology with microbial landscapes, this study provides new insights into the adaptive evolution and resource management of amphibians in heterogeneous environments.

Animals doi: 10.3390/ani16213432

Authors: Baili Ren Xiaobo Guo Minfeng Ding Xin Li Jun Chen Tiande Zou Xingping Chen Jinming You

Camellia oleifera seed meal (CSM) is a protein-rich by-product from camellia oil production, but its utilization in animal diets is restricted by antinutritional compounds, especially tea saponins. This study evaluated the effects of solid-state fermented CSM, produced using Bacillus subtilis, Aspergillus niger, and Trichoderma longibrachiatum, on growth performance, antioxidant capacity, immune status, and intestinal health in weaned piglets, and identified the optimal dietary inclusion level. A total of 84 piglets (15.46 ± 0.10 kg) were randomly allocated to four treatments: a basal diet (control) or the basal diet supplemented with 2.5%, 5.0%, or 7.5% fermented CSM for 48 d. Compared with the control, 2.5% fermented CSM maintained normal growth performance while improving neutral detergent fiber digestibility, antioxidant activity, immune-related indices, jejunal morphology, and expression of tight junction proteins and MUC-2. The relative abundance of butyrate-producing Collinsella was altered, and colonic butyric acid showed a significant quadratic response to graded FCSM supplementation; meanwhile, isocaproic acid concentration was reduced. In contrast, supplementation at 5.0% or above negatively affected growth, nutrient utilization, and intestinal health. Overall, piglets exhibited an inverted U-shaped response to fermented CSM supplementation, with 2.5% identified as the most suitable inclusion level. This study provides a theoretical basis for the application of fermented CSM as a potential partial replacer of conventional feed ingredients in nursery pig diets.

Animals doi: 10.3390/ani16213431

Authors: Ayesha Tanveer Noor Khan Mahroze Fatima Hafiz Abdullah Shakir Wazir Ali Ayesha Khizar Samuel A. M. Martin Simon J. Davies

Probiotics and exogenous enzymes play essential roles in the growth and health of aquatic animals. This study explored the interactive effects of probiotics (Prob) and exogenous protease (Prot) on the growth indices, digestive and antioxidant enzyme activities, body composition, hematoimmune parameters and serum biochemical indices of striped catfish (n = 600, ≈10.17 ± 0.03 g). In this context, ten diets were formulated with a crude protein level of 35.23% by incorporating Prob (0.0, 1.0, 2.0, 3.0, and 4.0 g/kg) at each Prot level (0.5 and 1.0 g/kg). After a 14-day acclimatization period, the fish were stocked in ten groups, each in triplicate (20 fish/aquaria (40 × 40 × 70 cm, i.e., width × depth × length)) in a 2 × 5 factorial design and fed for 90 days at 3% of their body weight. The results of the current study revealed significant improvements in growth performance with Prob supplementation at both Prot levels (0.5 and 1.0 g/kg). At 0.5 g/kg Prot, FBW and AWG increased with increasing Prob supplementation, reaching their highest mean values at 4.0 g/kg Prob. However, WG% was highest (498.49%), and FCR was lowest (1.33) at 2.0 g/kg Prob, with no substantial further improvement at higher Prob levels. In contrast, at 1.0 g/kg Prot, the optimal Prob level shifted to 1.0 g/kg, resulting in a WG% of 486.99% and an FCR of 1.37. Similarly, the main effect and interactive effect were significant for digestive and antioxidant enzymes in response to dietary Prot and Prob. However, the levels of malondialdehyde (MDA), white blood cells (WBCs), and liver enzymes significantly decreased, reversing the growth trend. Furthermore, hematoimmunological parameters, including red blood cell (RBC) count, hemoglobin (Hb) concentration, hematocrit (HCT), lysozyme activity, and immunoglobulin M (IgM), showed the highest mean values at 4.0 g/kg Prob with 0.5 g/kg Prot, although the differences among the 2.0, 3.0, and 4.0 g/kg Prob treatments were not statistically significant. At 1.0 g/kg Prot, these parameters generally showed the highest mean values at 1.0–2.0 g/kg Prob. Moreover, the proximate analysis (crude protein, crude fat, moisture and ash) was not significantly (p > 0.05) affected by either supplement. The polynomial broken-line regression analysis based on WG% indicated that the optimum Prob level with 0.5 g/kg protease was 1.46 g/kg, whereas at 1.0 g/kg protease supplementation, the optimal probiotic level was estimated at 1.39 g/kg. In conclusion, dietary supplementation with Prob and Prot enhances the growth and health status of P. hypophthalmus, suggesting a sustainable and economical approach for intensive catfish farming.

Animals doi: 10.3390/ani16213430

Authors: Jin Wang Huiming Zhang Yu Fan Qingmin Li Jingsong Sun Jifeng Ma Qing Ma Yuxiang Zhou

Wolfberry (Lycium barbarum) sprouts are harvested on a large scale in Ningxia, and the residual tender stems and leaves constitute an abundant but largely unused by-product. This experiment was conducted to investigate the effects of dietary supplementation with different proportions of wolfberry (Lycium barbarum) sprout stems and leaves (WSSLs) on rumen fermentation and the rumen bacterial community of fattening Tan sheep, and to reveal the relationships between rumen bacteria and fermentation and growth traits. Forty-eight healthy 3-month-old weaned Tan rams with similar body weight ((22.75 ± 0.52) kg) were randomly divided into four groups of 12 sheep; the control group (G0) was fed a basal diet, while the G4, G8 and G12 groups were fed basal diets in which 4%, 8% and 12% of the corn straw was replaced by WSSLs. The adaptation period lasted 10 days and the experimental period 60 days. At the end of the experiment, six sheep per group (24 in total) were slaughtered and rumen contents were collected for fermentation measurements, and the V3–V4 region of the bacterial 16S rRNA gene was sequenced on the Illumina NovaSeq platform. Ruminal pH, ammonia nitrogen (NH3-N) and microbial crude protein (MCP) did not differ among groups (p > 0.05), whereas total volatile fatty acid (VFA) concentration differed (30.15–56.27 mmol/L; p = 0.025), together with acetate (p = 0.032) and butyrate (p = 0.010); these VFA differences were driven mainly by the high and variable values in the control group and showed no dose–response. Average daily gain was higher in G8 than in G0 and G4 (p = 0.010), and live weight before slaughter and carcass weight increased with the dietary WSSL level (p = 0.011 and 0.013). Seventeen phyla and 210 genera were identified, with Bacteroidota and Bacillota dominant; the relative abundance of Bacteroidota decreased while that of Bacillota tended to increase with the WSSL level, and Xylanibacter was the most abundant genus (15.85–18.18%). Alpha diversity indices did not differ among groups (p > 0.05), and principal coordinate analysis (PCoA) based on Bray–Curtis distance showed limited separation (Adonis R2 = 0.157, p = 0.002). Nine genera showed nominal between-group variation (uncorrected p < 0.05); however, after Benjamini–Hochberg FDR correction across all 210 genera tested, none remained significant (all q = 1.00). Howardella was negatively correlated with acetate, propionate, butyrate and total VFA (p < 0.05), U29-B03 was positively correlated with live weight before slaughter and carcass weight (p < 0.05), and bacteria of Christensenellaceae were positively correlated with propionate, butyrate and total VFA (p < 0.05); none of these associations survived FDR correction. Dietary supplementation with 4–12% WSSLs did not depress growth performance and did not significantly alter the abundance of any individual taxon after correction for multiple testing; it moderately alters rumen bacterial community composition, and WSSLs can be used safely at inclusion levels of 4–12% as an unconventional feed resource for fattening Tan sheep.

Animals doi: 10.3390/ani16213429

Authors: Hongliang Guan Tian Hua Jialei Xue Hao Bai Yuliang Gao Guobin Chang Bin Li

Automated monitoring of broiler feeding can improve management, but occlusion and scale variation hinder reliable detection in commercial multi-tier cages. This study developed a lightweight vision model for feeding behavior detection (RE-FB) and a feeding activity level (FAL) indicator for group-level assessment. The 2300 annotated images of yellow-feathered broilers were divided 8:1:1 for training, validation, and testing. RE-FB combines reparameterized multi-branch feature extraction, attention, and bidirectional multi-scale fusion to detect feeding birds and segment cages and feed troughs. On the test set, RE-FB-m achieved 89.7% precision, 87.3% recall, 92.1% box mAP@0.5, and 99.6% mask mAP@0.5, with 8.1 million parameters and a reported model inference speed of 158 FPS on the test platform. RE-FB-m achieved the highest box mAP@0.5 for feeding behavior detection among the evaluated Faster R-CNN, RT-DETR, and YOLO models. RE-FB was then applied to videos of four fixed groups monitored from 11 to 17 days of age. FAL was defined as the time-averaged proportion of the total birds in each monitored group detected as feeding, expressed as a percentage. Age-specific mean FAL values across the four groups ranged from 7.95% to 8.74%, with no significant age effect detected (p = 0.175). After controlling for group effects, the association between FAL and same-day weight gain was not statistically significant (partial r = −0.106, p = 0.614). RE-FB accurately and efficiently detects feeding behavior in caged broilers and generates group-level FAL records by aggregating detections over time. This approach provides a technical basis for non-contact monitoring of feeding activity in broiler production and can provide quantitative behavioral information for feeding management.

Animals doi: 10.3390/ani16213428

Authors: Hongshu Chi Ying Pan Xiaodong Liu Nengfeng Lin Qiuhuan Jiang Hui Gong Ziping Zhang

Cryptocaryon irritans, an obligate ectoparasite, causes cryptocaryoniasis and heavy economic losses to marine aquaculture. Cathepsin B (CatB) is highly expressed in C. irritans theronts and hypothesized to participate in parasite host invasion, yet its biochemical properties and pathogenic roles remain poorly defined. In this study, we performed bioinformatic analysis of the CiCatB gene. Following codon optimization, CiCatB was heterologously expressed in Pichia pastoris, and the biologically active recombinant CiCatB protein was purified. Subcellular localization analysis revealed that CiCatB predominantly localized in the cytoplasm of theronts, with partial signals detected at the cell membrane and in the extracellular space. Enzymatic assays showed that yCiCatB hydrolyzed gelatin and type I collagen from large yellow croaker, with optimum activity at pH 5.0 and stable performance from 28 to 37 °C. Mg2+ increased enzyme activity, while Cu2+ almost completely abolished catalytic activity. Both E-64 and anti-CiCatB serum inhibited enzyme activity. Functional blocking using inhibitor or antiserum markedly reduced parasite recovery following challenge. Our results suggest that CiCatB may contribute to host invasion by degrading host structural proteins, advancing our understanding of C. irritans pathogenicity. CiCatB represents a potential target for exploring control strategies against cryptocaryoniasis.

Animals doi: 10.3390/ani16213427

Authors: Can Zhang Wenqing Lu Juntao Li

High-fiber unbalanced amino acid (AA) profiles and toxic free gossypol restrict cottonseed by-products for swine diets, while solid-state microbial fermentation can upgrade their nutrition. We prepared 103 fermented batches with diverse microbes under aerobic or anaerobic conditions, selected 10 representative groups via multivariate statistics for scaled-up fermentation and chemical analysis, and conducted two Youden square pig trials to determine available energy and standardized ileal digestible (SID) AAs before establishing relevant prediction equations. The metabolism experiment used 24.10 kg pigs to detect total tract digestibility and energy values, and an ileal cannula trial on 60.88 kg barrows adopted a nitrogen-free diet to correct endogenous amino acid losses. Fermentation largely lowered free gossypol, and short-term aerobic Aspergillus niger fermentation yielded the highest digestibility of most nutrients and essential amino acids. Digestible and metabolizable energy was positively related to crude protein (CP) and ash but was negatively linked to fiber fractions. SID lysine was negatively correlated with CP and ash yet was positively associated with fiber, while SID methionine and tryptophan displayed identical positive correlations with CP and negative correlations with fiber and free gossypol. Stepwise regression models (R2 = 0.57–0.93) allow rapid nutritional assessment via routine chemical analysis to guide precise pig feed production.

Animals doi: 10.3390/ani16213426

Authors: Lili Hou Shuaipeng Xu Ming Xu

Understanding dietary relationships between wild ungulates and sympatric livestock is important for conserving threatened species in alpine grassland ecosystems. Przewalski’s gazelle (Procapra przewalskii) is an endangered ungulate endemic to the Qinghai Lake Basin, but its dietary relationship with sympatric domestic sheep across multiple subpopulations remains poorly understood, particularly at finer taxonomic scales. Using fecal microhistology of April 2023 samples from eight study sites during the late winter grazing season, we compared dietary composition, diversity, evenness, niche breadth, and overlap at the family, genus, and species levels. More plant taxa were identified in the fecal samples of Przewalski’s gazelle than in those of domestic sheep, and estimates of dietary diversity, evenness, and niche breadth were generally higher for gazelles. Both herbivores relied largely on graminoids, indicating a shared dietary foundation. However, dietary overlap was higher at the family level (Pianka’s index = 0.830–0.976) than at the genus (0.718–0.946) and species (0.629–0.946) levels, with substantial variation among subpopulations. These results suggest a shared broad dietary foundation but apparent differentiation in resource use at finer taxonomic scales during late winter. These findings can inform Przewalski’s gazelle conservation and grassland management in the Qinghai Lake Basin.

Animals doi: 10.3390/ani16213425

Authors: Xiwei Cui Yanyan Li Yuanyuan Guo Xiaotian Qiu Chousheng Liu Shengyue Gao Kexin Sun Peng Gai Xuankai Huang Peng Zuo Zhipeng Wang

Artificial insemination is used widely in the swine industry, resulting in increasing focus on the traits of boar semen and methods for their genetic improvement. This study analyzed 21,160 phenotypic records of fresh semen derived from 492 Large White boars and 231 Landrace boars. The semen traits included the semen volume (VOL), sperm concentration (CON), sperm motility (MOT), and abnormal sperm rate (ABN). Single-trait and multi-trait repeatability models were used to assess the overall genetic parameters of semen traits, while random regression models were applied to examine trends in heritability changes over the collection period. In addition, multi-trait repeatability models were also used to estimate genetic and phenotypic correlations among the traits. The single-trait repeatability model indicated heritability estimates of VOL, MOT, CON, and ABN of 0.23, 0.09, 0.22, and 0.07, respectively, for Large White boars, with corresponding values for Landrace boars of 0.42, 0.06, 0.16, and 0.17. The respective values calculated by the multi-trait repeatability model were 0.23, 0.08, 0.22 and 0.09 for Large White boars and 0.43, 0.06, 0.20 and 0.17 for Landrace boars. Sperm motility, sperm concentration, and abnormal sperm rate showed low-to-moderate heritability. Semen volume showed moderate heritability in Large White boars and high heritability in Landrace boars. The random regression results indicated heritability estimates for VOL, MOT, CON, and ABN of 0.24–0.53, 0.05–0.08, 0.13–0.23, and 0.03–0.31, respectively, for Large White boars and 0.08–0.31, 0.02–0.15, 0.26–0.53, and 0.12–0.17 for Landrace boars. Significant negative genetic correlations were found between VOL and CON in both breeds (−0.54 for Large White and −0.73 for Landrace), and between VOL and MOT in Landrace. These findings will facilitate more precise selection to improve boar semen quality and reproductive efficiency in the swine industry.

Animals doi: 10.3390/ani16213424

Authors: Yufei Song Linran Chen Wenqing Lu Yixin Luo Naiyi Xu Haiwei Wang Guangxin E

To elucidate the genetic basis underlying chicken adaptation to diverse environmental selection pressures, this study performed a genome-wide analysis of copy number variations (CNVs) using publicly available whole-genome resequencing data from global chicken populations. Population differentiation (FST) and nucleotide diversity (π ratio) were integrated to identify signatures of selection associated with thermal adaptation, domestication, and high-altitude adaptation, followed by the identification of candidate genes and enriched biological pathways. In total, 6862, 9348, and 9384 high-confidence copy number variations (CNVs) were identified in the heat adaptation group, domestication group, and high-altitude adaptation group, respectively. Candidate genes associated with heat adaptation include AQP9, SLC4A8, CAT, SMPD2, and CLOCK, which are mainly enriched in biological processes related to water and ion homeostasis, antioxidant defense, and circadian rhythm regulation. Candidate genes related to domestication, such as IL18R1, HTR2C, GRIN2A, PRKAG2, and FABP1, are mainly involved in neuroactive ligand-receptor interaction, energy metabolism, immune regulation, and melanogenesis. In contrast, candidate genes identified in high-altitude populations include EDNRB, ITPR1, OGDH, PPARA, and ADCY6, which are significantly enriched in vascular smooth muscle contraction, the tricarboxylic acid (TCA) cycle, the PPAR signaling pathway, and pathways related to hypoxic metabolic adaptation. The above findings indicate that distinct environmental selection pressures and artificial selection pressures have shaped population-specific CNV patterns in the chicken genome and promoted the formation of phenotypic adaptation through regulating water–salt balance, neural function, energy metabolism, vascular homeostasis, and hypoxic response pathways.

Animals doi: 10.3390/ani16213423

Authors: Jianing Rao Rongxuan Pan Yuanming Wang Jingjie Feng Xintong Li Kefeng Li

During discharge from high dams, the high-velocity water flows mix with air, causing downstream total dissolved gas (TDG) supersaturation, threatening fish survival. However, a systematic understanding on fish response patterns under the coupled influence of multiple environmental and biological factors remains limited. This study synthesized historical experimental data to explore fish tolerance and avoidance in response to TDG supersaturation in the upper Yangtze River. Economic fish species demonstrated overall stronger tolerance than the rare fish species under all TDG levels. Significant interspecific differences in economic fish tolerance were observed with increasing TDG levels. Aristichthys nobilis demonstrated stronger tolerance than Hypophthalmichthys molitrix. For the rare fish, Procypris rabaudi showed the weakest tolerance. Exposure time, TDG level, species and water temperature were identified as the primary factors influencing fish mortality. Based on these, mortality prediction formulas for rare and economic fish were established, achieving an R2 over 0.59. The avoidance ability of fish increased significantly with increasing TDG levels and reached 50% at 130% TDG. Significant interspecific differences in fish avoidance were observed at a 140% TDG level or above, and Schizothorax prenanti showed the weakest avoidance ability. TDG level was the dominant factor influencing fish avoidance ability, followed by body length. Accordingly, a prediction formula for fish avoidance rates was proposed with an R2 of 0.88. The findings offer critical insights for ecological risk management and the development of fish conservation strategies downstream of high dams.

Animals doi: 10.3390/ani16213419

Authors: Ligia G. S. Amorim Vincent Prémel Lucas Aosf Karina Nunes Kasperoviczus Selma Maria de Almeida-Santos

Reproductive behaviors in secretive Neotropical pitvipers are rarely documented under natural conditions, limiting empirical assessments of sexual selection, locomotor mechanics, and mating systems. Here, we present focal videography and photographic documentation of free-ranging Amazonian Lanceheads (Bothrops atrox) from coastal French Guiana during the rainy season. A prolonged copulation (~3 h) revealed a remarkable bimodal locomotor sequence: the pair moved from terrestrial litter across a 20 m-wide forest stream and back to land without cloacal separation. Video analysis demonstrates female-driven locomotion, wherein the substantially larger female pulls the coupled male through the water column against his counter-directional swimming movements. This behavior illustrates female locomotor dominance under sexual size dimorphism and the widely mechanical resilience of the spine-bearing crotaline hemipenis during forced aquatic displacement. Sequential photographic documentation captures ritualized male–male combat with synchronized vertical displays, anterior-trunk swaying, neck hooking, and topping attempts, confirming that stereotypic motor patterns described in western Amazonia are conserved in eastern Guiana Shield populations. These postural dynamics represent transient physical contests over spatial priority rather than permanent dominance hierarchies. Together, this evidence indicates that empirical validation for female behavioral control during copulation and expands the geographic framework supporting the prolonged mate-searching polygyny hypothesis in B. atrox.

Animals doi: 10.3390/ani16213422

Authors: George-Emanuel Harcotă Andreea-Mădălina Ciucă

This study investigates cetacean occurrence and stranding records along the Romanian Black Sea coast between 2021 and 2025, focusing on the three resident subspecies: Tursiops truncatus ponticus, Delphinus delphis ponticus, and Phocoena phocoena relicta. A total of 1392 live individuals were documented across 188 sighting events, with T. t. ponticus and D. d. ponticus together representing 88.3% of all recorded individuals. The monthly distribution of recorded sighting events differed significantly among subspecies (p < 0.001), as did the distance of recorded sightings from the coastline (p < 0.001). Median distances were 4.40 km for T. t. ponticus, 61.78 km for D. d. ponticus, and 30.44 km for P. p. relicta. During the same period, 76 stranded cetaceans were recorded along the Romanian Black Sea coast, of which 58 were identified to subspecies level. P. p. relicta accounted for 70.7% of identified stranded individuals, compared with 11.7% of live individuals recorded during research cruises. Stranding records were most frequent in August and May, although their monthly distribution did not differ significantly among subspecies. Although both datasets covered the same geographical region and study period, differences in observation methods and sampling effort limit direct comparisons and prevent inferences about mortality rates or population trends. Nevertheless, the observed differences in the spatial and monthly distribution of live sightings, together with the high representation of P. p. relicta among stranded individuals, provide complementary information to support future cetacean monitoring, stranding investigations, and conservation efforts in the Romanian Black Sea.

Animals doi: 10.3390/ani16213421

Authors: Sherief M. Abdel-Raheem El Said Yehia Mohammed Rania Elsaid Mahmoud Mahmoud Fathy El Gamal Hend S. Nada Waleed Rizk El-Ghareeb Mohamed Marzok Ahmed M. A. Meligy Mohamad Abdulmohsen Hesham Ismail Doaa Ibrahim Asmaa T. Y. Kishawy

There was an error in the original publication [...]

Animals doi: 10.3390/ani16213420

Authors: Ketian Chen Le Liu Qiang Zhang Zhiqi Dai Lu Luo Chunmei Li

Four laboratory-scale biofilters were designed and evaluated for treating trimethylamine (TMA). Each biofilter was measured as being 68 cm in height and 15 cm in diameter. The filter media consisted of various mixtures of dairy manure compost, dairy barn bedding, crushed corn cobs, wood chips, and sawdust. This study aimed to compare the removal efficiencies of TMA achieved by biofilters packed with different media compositions, as well as to analyze the succession patterns in the structure of microbial communities on the media surfaces during operation. Contaminated air streams were artificially generated by bubbling air through a TMA solution, resulting in inlet concentrations ranging from 1.88 to 4.23 mg/m3. Under empty bed residence times (EBRTs) ranging from 42.7 to 28.0 s, all four biofilters achieved removal efficiencies exceeding 60%. However, the performance varied among the biofilters. The biofilter packed with a mixture of matured compost, wood chips, and crushed corn cobs at a volume ratio of 10:9:1 exhibited the highest removal efficiency (>83%). Ammonia was detected at the outlet of the biofilters as a byproduct of microbial TMA degradation, accompanied by the accumulation of ammonium nitrogen in the filter media. Microbial community analysis of the high-performance biofilter revealed a progressive increase in microbial complexity and diversity over 95 days of operation. Glycomyces was the dominant genus throughout the operation and showed a significant positive association with TMA removal efficiency, but its specific role in TMA degradation remains to be experimentally confirmed.

Animals doi: 10.3390/ani16213418

Authors: Wirot Likittrakulwong Manatsanun Nopparatmaitree Payungsuk Intawicha Sukree Yoosuk Juan J. Loor Tossaporn Incharoen

This study evaluated extruded cassava-based corn analogs as a dietary replacement for yellow corn in laying hens. A total of 180 Hy-Line Brown hens (60 weeks old) were allocated to four dietary treatments for 10 weeks. Treatments included a standard corn diet (T1), 100% non-extruded analog (T2), 50% extruded analog (T3), and 100% extruded analog replacement (T4). Thermomechanical extrusion significantly increased starch gelatinization to 88.50% and decreased water activity to 0.42. Hens fed the T4 diet achieved the highest apparent ileal starch digestibility (92.1%) and digestible energy (3063 kcal/kg). The T4 group also exhibited increased jejunal villus height compared to the T2 group. Metabolically, this coincided with elevated circulating serum triglycerides and total cholesterol, without disrupting glucose homeostasis. Consequently, the T4 group achieved the highest average egg weight, while maintaining egg mass, yolk weight, and feed conversion ratio at levels statistically comparable to the T1 control. In contrast, the T2 diet resulted in lower starch digestibility, reduced jejunal villus height, and increased cecal volatile fatty. Complete replacement with extruded cassava-based analogs maintains nutrient assimilation and productive performance relative to standard corn. This strategy provides a viable alternative to diversify global feed ingredient sourcing.

Animals doi: 10.3390/ani16213416

Authors: Tian Wang Yuanchen Mei Qiang Yang

The intergenic regions of porcine convergent gene pairs frequently maintain an open chromatin configuration with a permissive transcriptional microenvironment, rendering them promising candidates for genomic safe harbors (GSHs). However, the molecular features and promoter activities of the convergent gene pair ABCF1 and PPP1R10 remain uncharacterized, impeding the utilization of their potential as GSHs. To address this, we systematically predicted cis-regulatory elements (including CpG islands) of the two genes in silico, evaluated the transcriptional activities of serially truncated promoter fragments by dual-luciferase reporter assays in porcine fetal fibroblasts (PFFs) and PK-15 cells, compared their promoter regions across seven species, and characterized the protein architectures and flanking genomic landscapes. The ABCF1 promoter contained canonical TATA, CCAAT, and GC boxes alongside three CpG islands, and its shortest fragment, P217 (−217 to −1 bp), exhibited the highest activity in both cell lines. The PPP1R10 promoter possessed a triple TATA-box arrangement without detectable CpG islands, and its core fragment P211 (−211 to −1 bp) exhibited the highest activity among all truncated fragments in both cell lines and showed numerically higher activity than the simian virus 40 (SV40) promoter in PK-15 cells. No canonical oncogenes were identified within 100 kb upstream or downstream of the intergenic region between the two genes. Both ABCF1 and PPP1R10 are intracellular soluble proteins lacking transmembrane domains. ABCF1 is predominantly composed of α-helices, localizes in the cytoplasm and at the nuclear membrane, and modulates cytoplasmic translation. By contrast, PPP1R10 is enriched in random coils, specifically associates with nuclear chromatin, and participates in the PP1 phosphatase regulatory network. Taken together, our study delineates the core active promoter regions of both genes and highlights the potential of this chromosomal interval as a candidate genomic safe-harbor locus, a hypothesis awaiting further experimental validation.

Animals doi: 10.3390/ani16213417

Authors: Nicole Huamán Nicolas Tarmeño Ruperto Severino David Lopez Luis A. Ñacari Jefferson Yunis-Aguinaga Celso L. Cruces Germán Augusto Murrieta-Morey Jhon D. Chero

Clinostomum chaacci Sereno-Uribe, López-Jiménez, González-García, Ortega-Olivares & García-Varela, 2025 is a recently described clinostomid trematode whose metacercariae have been molecularly identified in freshwater fishes from Mexico and South America. In October 2025, 29 siluriform fishes, comprising 18 Pimelodella cristata (Müller & Troschel, 1849) (Heptapteridae) and 11 Rineloricaria morrowi Fowler, 1940 (Loricariidae), were examined from the Nanay River, Loreto, Peru. Eight metacercariae were found encysted in the cephalic integument. The specimens were characterized morphologically and identified through Bayesian phylogenetic analyses of the mitochondrial Cox1 gene and the nuclear 28S rDNA. The new Cox1 sequences from both hosts were identical and nested within the C. chaacci clade, differing by 0.42–5.27% from conspecific isolates, whereas the 28S rDNA sequence differed by only 0–0.08% from reference sequences of C. chaacci. Morphometric data partially overlapped with those of C. chaacci metacercariae previously reported from Peru. The records from P. cristata and R. morrowi represent two new second intermediate hosts and add Loricariidae to the known host spectrum of C. chaacci, providing further evidence that a taxonomically diverse assemblage of fishes participates in its transmission in the western Amazon Basin.

Animals doi: 10.3390/ani16213415

Authors: Haoliang Chai Dexin Zhao Taibiao Wang Guanyu Hou Liguang Shi Maoyuan Wang Euphrème Ipemba Lessebe Gambou Dieu Leveut Hanfeng Li Hongzhi Wu Ting Cao

This study investigated the effects of graded dietary curcumin supplementation on production performance, antioxidant capacity, immune status, intestinal barrier function, and cecal microbiota in late-laying hens. A total of 480 61-week-old Jingfen No. 1 laying hens were randomly allocated to four groups with six replicates of 20 birds each: a basal diet control (CG) and diets supplemented with 100, 200, or 400 mg/kg curcumin (CN1, CN2 and CN3) for 35 days. Curcumin supplementation significantly increased average egg weight (p < 0.05). Serum catalase concentration and immunoglobulin levels (IgM, IgA, and IgY) were elevated, whereas malondialdehyde, interleukin-6, interleukin-10, and tumor necrosis factor-α levels were reduced in a dose-dependent manner (p < 0.05). Similar improvements were observed in the jejunum, including increased glutathione peroxidase and catalase concentrations and reduced inflammatory cytokines. Birds in the CN3 group exhibited greater villus height and villus-to-crypt ratio, lower crypt depth, and upregulated mRNA expression of tight junction proteins (ZO-1, Occludin, and Claudin-1) (p < 0.05). Curcumin also modulated the cecal microbiota by enriching beneficial butyrate-producing bacteria (Lactobacillales, Faecalibacterium, Odoribacter, Roseburia, and Phascolarctobacterium) and reducing pathogenic Staphylococcus and Bilophila. Overall, dietary curcumin improved egg quality, antioxidant and immune functions, intestinal health, and microbial composition in late-laying hens.

Animals doi: 10.3390/ani16213414

Authors: Cong Wei Shiyang Weng Yanchao Liu Jianchuan Li

Gut microbiota influence nutrient acquisition, immune function, and pathogen resistance, yet are rarely considered in ex situ planning. We compared gut bacterial (16S ribosomal RNA; 16S rRNA) and fungal (internal transcribed spacer 1; ITS1) communities of the Tibetan eared pheasant (Crossoptilon harmani), a protected Galliform endemic to the Qinghai–Tibet Plateau, across a management gradient: fully captive (n = 3), wild (n = 7), and free-ranging monastery birds receiving supplementary barley (n = 6); samples were not linked to identified donors, so the kingdoms are compared at group level only. Monastery birds had the numerically highest bacterial Shannon diversity (5.79), above wild (5.54) and captive birds (2.93), although the among-group test was not significant (p = 0.13). Permutational multivariate analysis of variance (PERMANOVA) showed significant bacterial differentiation (Bray–Curtis R2 = 0.309, p = 0.001), driven by the captive group, which showed Lactobacillus dominance (51.4%). Fungal communities also differed significantly (R2 = 0.194, p = 0.008), with no compositional turnover (Jaccard p = 0.447). Birds in natural habitat with supplementary feeding were no more dissimilar from wild birds than wild birds were among themselves, whereas fully captive birds were clearly separated; this pattern warrants testing in replicated, multi-site studies.

Animals doi: 10.3390/ani16213412

Authors: Zeyu Li Shuang Zhao Zhi Cao Jiuzhou Song Hongyan Sun Qisheng Zuo Wei Han Guohong Chen Bichun Li Kai Jin

Highly pathogenic avian influenza virus undergoes continuous evolution with strengthened cross-species transmission capacity, posing a severe threat to global public health security. Host acidic nuclear phosphoprotein 32A (ANP32A) acts as a critical host cofactor for activating influenza viral RNA-dependent RNA polymerase, and plays a central role in viral replication and host adaptation. This review systematically elaborates the molecular features of ANP32A, as well as the mechanism by which its leucine-rich repeat (LRR) domain specifically binds to the polymerase basic protein 2 (PB2) subunit and its low-complexity acidic region (LCAR) interacts with polymerase acidic protein (PA) to facilitate the assembly and functional activation of viral polymerase complexes. It also summarizes the influences of species-specific sequence variations and splicing isoforms of ANP32A on the host restriction barrier of cross-species transmission. Further analyses reveal that ANP32A indirectly modulates the transcriptional levels of genes related to interferons, NF-κB and NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasomes via chromatin epigenetic modification and transcriptional resource competition, remodeling the cellular antiviral microenvironment as a nuclear transcriptional regulator. ANP32A and acidic nuclear phosphoprotein 32B (ANP32B) exert complementary functions to support viral polymerase activity, while acidic nuclear phosphoprotein 32E (ANP32E) only provides weak auxiliary effects on a small subset of mutant influenza strains with its primary physiological function centered on chromatin regulation; the three proteins jointly participate in the regulatory network of viral replication. Drawing on recent advances in cryo-electron microscopy structural biology and CRISPR/Cas9 gene-editing technologies, which are discussed narratively in this review, this review highlights the pivotal hub role of ANP32A in viral host adaptation and cross-species spillover.

Animals doi: 10.3390/ani16213413

Authors: Adrián Halvoník Nina Moravčíková Ivan Pavlík Ján Tomka Luboš Vostrý Hana Vostrá-Vydrová Radovan Kasarda

The Slovak Askanian Merino is a small, endangered sheep population maintained on two farms in Slovakia. This study assessed its genomic diversity, population structure and the value of genomics-informed management. After quality control, 491 individuals and 45,695 SNPs were retained for within-population analyses, while 927 individuals and 18,143 SNPs were used for comparisons with other Merino and Merino-derived breeds. Observed and expected heterozygosity were 0.411 and 0.412, respectively, while genomic inbreeding based on homozygous-by-descent segments and runs of homozygosity was 0.025 and 0.019. Segments longer than 16 Mb contributed most to inbreeding, indicating recent inbreeding. Effective population size estimates ranged from 46 to 112. Population structure analyses separated Slovak Askanian Merino from other Merino-derived breeds and revealed farm-related subdivision. Forward simulations showed that optimized mating allocation slowed inbreeding accumulation over ten generations. By generation 10, optimized scenarios reached genomic inbreeding values of 0.042–0.044 based on homozygous-by-descent segments longer than 4 Mb, compared with 0.069–0.072 under random mating. Restricting each used ram to 15–50 ewes provided the best compromise between reducing inbreeding and practical feasibility. These findings show that genomic monitoring combined with optimized mating can support the long-term conservation and sustainable management of small endangered livestock populations.

Animals doi: 10.3390/ani16213411

Authors: Leshan Guo Xuguang Yang Junyan Bai Xiangdong Ding Xin Qi Xinjian Yang Xuewen Gong Yang Liu Renjie Liang

Growth traits are key economic indicators in pig breeding, and identifying molecular markers associated with body weight, backfat thickness, loin eye depth, body length, chest girth, and chest width is of great importance for improving the efficiency of marker-assisted selection (MAS). In this study, 400 French Large White sows and 40 French Landrace sows were used as experimental populations. Single nucleotide polymorphisms (SNPs) within the ARHGEF37 gene were genotyped using PCR amplification and sequencing. Genotype frequencies, allele frequencies, and genetic diversity parameters were systematically analyzed, and associations between different genotypes, haplotypes, and growth traits were evaluated using one-way analysis of variance (ANOVA). Several variants showed nominal associations with growth traits in the French Large White population, although none remained significant after FDR correction; ARHGEF37 is therefore proposed as a candidate gene requiring validation in larger populations. Additionally, haplotype analysis revealed that certain haplotype combinations exhibited synergistic improvements in economically important traits including body weight, backfat thickness, and chest circumference, providing potential genetic targets for marker-assisted selection.

Animals doi: 10.3390/ani16213409

Authors: Cristina Sánchez Dany Elzahaby Olivier Louis Levionnois

An 8-year-old, 600 kg, female Simmental cow and a 6-year-old, 662 kg, pregnant Hérens cow were referred for surgical repair of mandibular symphyseal fractures following trauma in two unrelated events. Pre-anaesthetic blood tests revealed mild hypokalaemia in the Simmental cow and mild hypocalcaemia in the Hérens cow. Anaesthesia was induced with xylazine, ketamine, and lidocaine, and maintained with isoflurane combined with a ketamine constant rate infusion. Throughout most of the procedures, physiological parameters remained stable, although mild hypercapnia and progressive bradycardia were noted. During surgical closure, both cows developed sudden severe bradycardia, followed by ventricular escape beats and short periods of asystole. Arterial blood gas analysis revealed acute severe hyperkalaemia, with potassium concentrations of 6.8 mmol/L in the Simmental cow and 8.66 mmol/L in the Hérens cow. Immediate treatment with a calcium–magnesium–glucose solution was initiated, resulting in rapid improvement in heart rate, restoration of sinus rhythm, and reduction in potassium concentrations. Both cows recovered uneventfully, with normal electrolyte values over the following 72 h. This report describes acute intra-anaesthetic hyperkalaemia in two cattle, including a pregnant animal, occurring without an identifiable predisposing factor. Continuous monitoring and rapid recognition of changes in cardiovascular status and electrolyte concentrations allowed prompt treatment and restoration of normal cardiac rhythm.

Animals doi: 10.3390/ani16213410

Authors: Zengwen Huang Jing Wang Jun Li Dayong Si Zengpeng Lü Gang Shu Zhiqiang Hu Chaoyun Yang

This study aimed to characterize the mitochondrial (MT) genome of Tibetan fragrant chickens along an altitudinal gradient and to relate it to meat quality. Eighteen 150-day-old male chickens were selected from three groups (LX, 1500 m; MX, 2800 m; HX, 3500 m; n = 6 each; six birds were randomly chosen from 30 reared per group) and subjected to whole-genome resequencing (reference: Gallus gallus GCA_000002315.5). Ninety MT variants (85 polymorphic; transition/transversion 83:7) were identified, comprising 58 coding (42 synonymous, 16 non-synonymous), 20 D-loop/non-coding, 10 rRNA and 2 tRNA sites; no MT insertions/deletions or structural variants were found. The 18 birds clustered into three maternal lineages (A, B, C) that were only partially structured by altitude (A more frequent at low altitude; B at mid and high altitude). A neighbour-joining tree with 12 Gallus mitogenomes placed the population close to Gallus gallus gallus and Chinese local breeds. Fifteen heteroplasmic sites were detected, with 16S rRNA nt3946 (G/C/A) heteroplasmic in all birds. Relative mtDNA copy number did not differ among groups (p = 0.52). Meat quality varied with altitude (highest protein and lowest fat at 2800 m; highest inosine monophosphate at 3500 m) but not with maternal lineage, suggesting that the observed phenotypic differences are associated mainly with altitude of rearing rather than with maternal mitochondrial lineage, and that the MT genome contributes standing genetic variation rather than a direct functional basis for these differences.

Animals doi: 10.3390/ani16213408

Authors: Alyssa C. Freeman Jonathan K. Warner Justine L. Smith John M. Tomeček

Understanding interactions between native and invasive species is crucial for effective wildlife management, as predation by invasive species can significantly impact native populations. This study investigated the factors influencing wild pig (Sus scrofa) predation on 28 American alligator (Alligator mississippiensis) nests in Texas coastal marshes between 2022 and 2023. We examined variables including nest location, wetland type, soil type, landcover, distance to roads, distance to management headquarters (i.e., human infrastructure), distance to levees, distance to trees, and salinity levels to determine their relationship with nest predation. We analyzed data using generalized linear regression models and evaluated the models’ performance using AICc values and the Hosmer–Lemeshow goodness-of-fit test. Our results revealed that proximity to roads was negatively associated with nest predation risk (β = −0.01, p = 0.05), while increased distance from management headquarters was positively correlated with predation (β = 0.0004, p = 0.09). This study’s findings indicate that distance to roads influence predation dynamics, with potential implications for wildlife managers seeking to mitigate wild pig depredation of alligator nests and potentially other ground-nesting reptiles and birds. These insights can guide habitat management strategies and inform conservation efforts by identifying high-risk areas and enhancing protective measures for alligator nests.

Animals doi: 10.3390/ani16213407

Authors: Yongyuan Li Guangzhi Chen Mengru Xie Yihao Fang Feng Qin Wenyu Song

In the original publication [...]

Animals doi: 10.3390/ani16213406

Authors: LeeAnn M. Perry Justin Shmalberg Ryan W. Honaker

This exploratory cross-sectional study analyzed owner-reported survey data from 5653 cats belonging to current and prospective customers of a UK fresh cat food company to identify factors associated with body condition in companion cats and to compare owner classifications made using a structured body condition score (BCS) tool with those made by unguided judgment. Owners classified fewer cats as ideal using the structured BCS tool than by unguided judgment, with lower agreement for underweight than for overweight cats. Multivariable logistic regression models were developed for overweight, underweight, and ideal body condition. A higher proportion of fresh food was associated with greater odds of ideal body condition (OR 1.22, 95% CI: 1.04–1.42), while dry food was associated with overweight (OR 1.83, 95% CI: 1.33–2.51) and wet food with underweight (OR 1.51, 95% CI: 1.06–2.16), though this association was less robust to differing model specifications. Further associations included certain additional calorie sources, feeding practices, treat type, activity, enrichment, household composition, and established demographic factors. These hypothesis-generating findings highlight a gap between structured and unguided owner assessment and identify associations that merit prospective evaluation in a more generally representative population.