Animals

13 pages, 356 KB

Open AccessFeature PaperArticle

In Vitro Assessment of Selected Postbiotic Substances Against Methicillin-Resistant and Methicillin-Susceptible Staphylococcus spp. and Mammaliicoccus spp. of Bovine Mastitis Origin

by Mariola Bochniarz, Joanna Kowalik, Aneta Nowakiewicz, Aleksandra Trościańczyk, Agata Hahaj-Siembida, Katarzyna Michalak, Dorota Pietras-Ożga, Łukasz Adaszek and Andrea Lauková

Animals 2026, 16(9), 1422; https://doi.org/10.3390/ani16091422 - 6 May 2026

Cited by 2 | Viewed by 712

Abstract

The growing problem of antibiotic resistance poses one of the greatest threats to modern medicine, making the search for new, alternative strategies to treat bacterial infections a matter of priority. The aim of the study was to evaluate the antimicrobial activity of selected [...] Read more.

The growing problem of antibiotic resistance poses one of the greatest threats to modern medicine, making the search for new, alternative strategies to treat bacterial infections a matter of priority. The aim of the study was to evaluate the antimicrobial activity of selected postbiotic substances (PS) and nisin against methicillin-susceptible (MS) and methicillin-resistant (MR) strains of coagulase-negative staphylococci (NAS) and mammaliicocci. The study was conducted on 24 strains of coagulase-negative staphylococci and mammaliicocci isolated from milk collected from Holstein-Friesian (HF) cows with subclinical mastitis. In vitro, PS/EMo (100–400 AU/mL) and PS/Eas (100–200 AU/mL) were the most effective, inhibiting the growth of all MS and MR strains of NAS and Mammaliicoccus spp. (100.0%). A high percentage of strains were also sensitive to postbiotic substances produced by Lactococcus lactis subsp. lactis PS/MK2/8 (400–1600 AU/mL), PS/MK1/3 (100–400 AU/mL), and nisin (100–25,600 AU/mL), both in MR strains (94.1%, 82.4%, 88.2%, respectively) and in MS strains (100.0%, 85.7% and 100%, respectively). PS/Esach (100–25,600 AU/mL) inhibited the growth of all strains, including the mecA gene-positive mammaliicocci strains (100.0%), but was slightly less effective against the MS strains (64.7%). The lowest activity was observed with postbiotic substances produced by Enterococcus faecium, PS/4231 (100–1600 AU/mL) and PS/9296 (100–6400 AU/mL), which inhibited the growth of 3 out of 17 MR strains (17.65%). Among the Mammaliicoccus strains with the mecA gene, postbiotic substances derived from strains PS/4231 and PS/9296 inhibited the growth of 42.9% and 28.6% of the strains, respectively. Four tested strains were sensitive to all postbiotic substances (MSC9, MSC11, MSC12 and SCH3). This study confirms that postbiotic substances and nisin inhibit the growth of staphylococci isolated from the milk of cows with subclinical mastitis, including methicillin-resistant strains. Full article

(This article belongs to the Special Issue Antimicrobial Resistance and Innovative Strategies in Veterinary Medicine)

11 pages, 257 KB

Open AccessArticle

Hunting and Stray Dogs as Reservoirs of Echinococcus Species

by Nikola Betić, Milica Kuručki, Katarina Breka, Aleksandra Penezić, Neda Bogdanović, Ilija Pantelić, Ana Vuletić, Tijana Kukurić, Jelena Karanović, Jovana Raičević, Aleksandar Bajčić, Ivana Klun and Aleksandra Uzelac

Animals 2026, 16(9), 1421; https://doi.org/10.3390/ani16091421 - 6 May 2026

Viewed by 634

Abstract

In Serbia, human echinococcosis cases are predominantly diagnosed as the cystic form, caused by Echinococcus granulosus sensu lato (s. l.) complex tapeworms. Alveolar echinococcosis, a more severe disease caused by Echinococcus multilocularis, emerged recently. Domestic dogs have been implicated in [...] Read more.

In Serbia, human echinococcosis cases are predominantly diagnosed as the cystic form, caused by Echinococcus granulosus sensu lato (s. l.) complex tapeworms. Alveolar echinococcosis, a more severe disease caused by Echinococcus multilocularis, emerged recently. Domestic dogs have been implicated in the transmission of E. granulosus sensu stricto G1/3 to humans. In an ongoing screening of definitive hosts for the presence of Echinococcus spp. to discover reservoirs of E. multilocularis, fecal samples of hunting (n = 29) and stray dogs (n = 66) were examined. Taeniid eggs were collected using flotation and mesh filtration. The presence of Cyclophyllidea, E. granulosus s. l. and E. multilocularis DNA was detected by specific PCR assays. In total, 60% of the fecal samples contained cyclophyllidean DNA, while Echinococcus spp. DNA was detected in 7.4% of the samples. E. canadensis G6–8,10 DNA was present in 4.2% and E. multilocularis in 3.2% of the samples. E. multilocularis DNA was detected only in stray dog feces, suggesting that strays are a reservoir and possibly an important link in the transmission chain. To the best of our knowledge, this study provides the first molecular identification of Echinococcus spp. in dogs in Serbia. Full article

(This article belongs to the Special Issue Parasite Epidemiology and Molecular Identification in Wild, Domestic, and Companion Animals: 2nd Edition)

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21 pages, 1169 KB

Open AccessArticle

Development and Validation of LC-MS/MS Methods for Quantification of Fluralaner and Moxidectin in Cat Plasma and Its Application in a Pharmacokinetic Study

by Xiaolin Liu, Tianqi Huang, Xinggang Tang, Qianni Ye, Minggui Yuan, Zehua Cui, Yuqiao Ma, Junheng He and Rong Xiang

Animals 2026, 16(9), 1420; https://doi.org/10.3390/ani16091420 - 6 May 2026

Cited by 1 | Viewed by 820

Abstract

This study was conducted to support the development of a novel generic veterinary formulation by performing a comparative pharmacokinetic analysis against an approved reference product (Bravecto^® Plus). Sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed and validated for the [...] Read more.

This study was conducted to support the development of a novel generic veterinary formulation by performing a comparative pharmacokinetic analysis against an approved reference product (Bravecto^® Plus). Sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed and validated for the quantification of fluralaner and moxidectin in cat plasma. A single topical dose (40 mg/kg fluralaner and 2 mg/kg moxidectin) of either the test or reference formulation was administered to 20 healthy cats in a parallel study. A validated LC-MS/MS method was used to determine plasma concentrations. Key pharmacokinetic parameters—including peak plasma concentration (C_max), time to C_max (t_max), and area under the curve (AUC_0–∞)—were calculated and statistically compared. The C_max of fluralaner was 1527.62 ± 905.94 ng/mL, t_max was 14.00 (interquartile range (IQR): 5.00–28.00) days and AUC_0–∞ was 1,415,464.8 ± 822,873.6 ng·h/mL. The C_max of moxidectin was 27.85 ± 38.78 ng/mL, t_max was 6.00 (IQR: 1.00–14.00) days, AUC_0–∞ was 16,807.2 ± 12,885.6 ng·h/mL. Compared with the reference veterinary drug (Bravecto^® Plus), the relative bioavailability (F) of both fluralaner (118.89% (90% CI: 96.5–145.3%)) and moxidectin (102.00% (90% CI: 85.2–118.8%)) exceeded 100%, and there was no statistical difference in the time to peak concentration (t_max). The test and reference formulations exhibit similar pharmacokinetic profiles in cats. These results provide critical pharmacokinetic data supporting the bioequivalence and therapeutic potential of the new generic formulation, thereby facilitating its further development and regulatory evaluation. Full article

(This article belongs to the Special Issue Advances in Veterinary Pharmacology: Improving Treatment Modalities for Animal Pathology)

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15 pages, 1793 KB

Open AccessArticle

Longitudinal Change in Rumen-Associated Bacterial Communities Following Aso Limonite Supplementation in Japanese Brown Cattle

by Kentaro Harakawa and Kazuhiko Imakawa

Animals 2026, 16(9), 1419; https://doi.org/10.3390/ani16091419 - 6 May 2026

Viewed by 427

Abstract

Aso limonite, a natural volcanic mineral, has been used as a cattle supplement in the Aso region of Japan; however, its effects have not been well-characterized. This study examined whether Aso limonite supplementation from 105 days prepartum to 90 days postpartum changed rumen-associated [...] Read more.

Aso limonite, a natural volcanic mineral, has been used as a cattle supplement in the Aso region of Japan; however, its effects have not been well-characterized. This study examined whether Aso limonite supplementation from 105 days prepartum to 90 days postpartum changed rumen-associated bacterial communities and calf growth in the Kumamoto strain of Japanese Brown cattle. Buccal swab-derived bacterial profiles were regarded as proxies of examining rumen-associated bacterial communities. Diversity analysis detected prepartum differences between the control and limonite groups. Primary longitudinal screening further identified rumen-associated, oral-associated and archaea subsets from buccal swab-derived microbiota. Within the rumen-associated subset, Blastomonas and Denitromonas emerged as representative taxa showing interaction-associated signals at day −45 in the limonite group, whereas Adlercreutzia showed different pattern at day −15 in the control group. Additionally, heatmap and network analyses showed that these taxa were found in distinct broader rumen-associated bacterial communities rather than a single common taxon. In parallel, calves born to limonite-fed dams were heavier than those from control dams between days 28 to 84 postpartum. These findings indicate that maternal limonite supplementation was associated with selective changes in rumen-associated bacterial communities together with greater early calf body weight. Full article

(This article belongs to the Section Animal Nutrition)

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28 pages, 3929 KB

Open AccessArticle

Hermetia illucens and Tenebrio molitor as Supplements in Maize Silage for Potential Use in Waterfowl Diets: Effects on Nutritional Composition, Selected Microbial Indicators, and Selected Mycotoxins

by Matúš Džima, Miroslava Kačániová, Daniel Bíro, Milan Šimko, Branislav Gálik, Michal Rolinec, Ondrej Hanušovský and Miroslav Juráček

Animals 2026, 16(9), 1418; https://doi.org/10.3390/ani16091418 - 6 May 2026

Cited by 1 | Viewed by 518

Abstract

Alternative protein sources are increasingly considered in poultry nutrition, and insect meals may represent a promising strategy for modifying the compositional profile of maize silage in the context of waterfowl feeding. This study evaluated maize silage supplemented with Hermetia illucens (BAHI) or Tenebrio [...] Read more.

Alternative protein sources are increasingly considered in poultry nutrition, and insect meals may represent a promising strategy for modifying the compositional profile of maize silage in the context of waterfowl feeding. This study evaluated maize silage supplemented with Hermetia illucens (BAHI) or Tenebrio molitor (BATM) meal as alternatives to urea (BAUR) as a nitrogen source, including variants with lauric acid (BALA) and with the lactic acid bacteria (LAB) inoculant alone (BA), focusing on nutritional composition, microbiological quality, and selected mycotoxins (MT). Maize forage treated with LAB was ensiled for 8 weeks in five variants, with three independent silage replicates per treatment, and the silages were analyzed for nutrients, energy value, amino acids, fatty acids, microbial and MT content. Insect meals increased crude protein compared with BA (64.94–67.32 vs. 49.73 g/kg dry matter, DM) and nearly doubled ether extract in BAHI (40.08 vs. 21.74 g/kg DM), while apparent metabolizable energy for poultry ranged from 8.55 to 9.87 MJ/kg DM (BAHI). Urea elevated crude protein content to 66.37 g/kg DM without improving the essential amino acid index. Microbial indicators did not differ among treatments. In the MT profile, lower deoxynivalenol concentrations were observed in BAUR, BAHI, and BATM, and lower fumonisin B1 concentrations were observed in BATM and BALA, while aflatoxins and ochratoxin A were not detected. Overall, the results indicate that low-level inclusion of insect meals in combination with LAB improved the compositional characteristics of maize silage, while no significant differences were detected in the monitored microbial counts and only selective changes were observed in the mycotoxin profile. Full article

(This article belongs to the Special Issue Optimizing Alternative Protein Sources for Sustainable Poultry Diet)

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28 pages, 4529 KB

Open AccessArticle

Monitoring and Early Warning of the Cage-Rearing Broiler Farming Environment Based on an Inspection Robot

by Sai Luo, Xiangchao Kong, Xintong Xie, Wanchao Zhang, Pengshen Zheng, He Zhu, Deqi Hao, Jingkun Sun and Changxi Chen

Animals 2026, 16(9), 1417; https://doi.org/10.3390/ani16091417 - 6 May 2026

Cited by 1 | Viewed by 570

Abstract

In large-scale caged broiler houses, the combined effects of ventilation regulation, external meteorological disturbances, and changes in flock growth stages can easily cause the indoor thermal environment to deviate from the target settings, thereby increasing the difficulty of environmental control and production risks. [...] Read more.

In large-scale caged broiler houses, the combined effects of ventilation regulation, external meteorological disturbances, and changes in flock growth stages can easily cause the indoor thermal environment to deviate from the target settings, thereby increasing the difficulty of environmental control and production risks. Meanwhile, the broiler house environment exhibits pronounced spatial heterogeneity, while manual inspection is limited by low efficiency, high labor intensity, and delayed detection of anomalies. Therefore, this study proposes an inspection-robot-based method for predicting and providing early warnings for temperature deviation (TD) and temperature–humidity index (THI). In this method, TD is used to characterize the degree of deviation in environmental control, whereas THI is used to characterize the overall thermal environmental risk under the coupled effects of temperature and humidity. For the two prediction tasks, input variables were separately selected based on correlation analysis, and the same Wavelet–ECA–GRU model architecture was adopted for training to achieve short-term prediction over the next 30 min. The results show that the proposed model outperformed baseline models, including LSTM, TCN, and GRU. On the test set, the RMSE and R² for TD prediction were 0.2886 and 0.9238, respectively, while those for THI prediction were 0.4309 and 0.9287, respectively. Based on the TD classification results and THI risk thresholds, warning strategies for heat deviation, cold deviation, and humid-heat risk were established. The proposed method can identify potential thermal environmental anomalies in caged broiler houses in advance from the perspectives of environmental control deviation and comprehensive thermal environmental risk, thereby assisting farm managers in assessing whether timely adjustments to ventilation, heating, or cooling are needed. Full article

(This article belongs to the Section Poultry)

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13 pages, 1153 KB

Open AccessArticle

Relationship Between Rectal Temperature and Infrared Temperatures of a Goat’s External Body Surfaces

by Md. Niamot Ali, Abdulla Al Mamun Bhuyan, Mst. Ishrat Zerin Moni, Mst. Farzana Aktar, Md. Arafat Hossain, Dipa Ray, Mehedy Hasan Pranto, Md. Mahabubur Rashid and Jashim Uddin

Animals 2026, 16(9), 1416; https://doi.org/10.3390/ani16091416 - 6 May 2026

Cited by 1 | Viewed by 1594

Abstract

Measuring rectal temperature is a common method for assessing the body temperature in animals in response to changes in physiology, health, and emotions. However, non-invasive infrared thermography is a more advanced technique that reduces the stress associated with restraining animals and minimises risks [...] Read more.

Measuring rectal temperature is a common method for assessing the body temperature in animals in response to changes in physiology, health, and emotions. However, non-invasive infrared thermography is a more advanced technique that reduces the stress associated with restraining animals and minimises risks related to cross-contamination and disease transmission. The present study investigated the association between rectal temperature (RT) and infrared temperature (IRT) of the goat’s external body surfaces. A total of one hundred thirteen (113) goats were considered for this study, and those that showed signs of health deviations were excluded from the evaluation. Thus eighty-four (84) goats were used for the study. We recorded the RT of each goat, as well as the maximum (M) and average (A) IRT of the forehead, right and left ears, right and left eyes, right and left coronary bands of the forelimbs, right and left shoulders, and the mucous membrane of the mouth, simultaneously. The descriptive statistics revealed that the maximum IRT values were higher than RT, except for the right coronary band, left coronary band, right shoulder, and left shoulder. Pearson correlation showed RT has a positive association with all IRT measures, including maximum and average values. The highest association was observed between RT and right eye maximum IRT (REM) (r = 0.63). In addition, the regression analysis showed RT and REM have a moderate correlation (S = 0.36 and R² = 39.6%). In conclusion, the maximum IRT of the right eye could be used as a non-invasive measure of body temperature in goats. More research is needed to investigate the potential of eye IRT towards automated recording of body temperature in goats. Full article

(This article belongs to the Section Small Ruminants)

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19 pages, 2688 KB

Open AccessArticle

Effects of Marine Protein Hydrolysates as Squid-Liver Powder Replacements on Growth Performance, Digestive Capacity and Health Status of Pacific White Shrimp (Litopenaeus vannamei)

by Dachawat Poonnual, Siriporn Tola and Bundit Yuangsoi

Animals 2026, 16(9), 1415; https://doi.org/10.3390/ani16091415 - 6 May 2026

Viewed by 1105

Abstract

This study investigated the replacement of squid-liver powder (SLP) with different marine protein hydrolysates in diets for Pacific white shrimp (Litopenaeus vannamei) over an eight-week feeding period. A control diet contained 5% SLP, whereas four test diets replaced 1% of the [...] Read more.

This study investigated the replacement of squid-liver powder (SLP) with different marine protein hydrolysates in diets for Pacific white shrimp (Litopenaeus vannamei) over an eight-week feeding period. A control diet contained 5% SLP, whereas four test diets replaced 1% of the diet (dry-matter basis) with tuna hydrolysate liquid (TH), shrimp hydrolysate powder (SH), fish hydrolysate powder (FH), or salmon silage liquid (SS), with soybean meal adjusted to maintain dietary protein balance. Shrimp fed the TH diet showed higher final body weight (15.76 ± 0.24 g shrimp⁻¹) and specific growth rate (3.18 ± 0.02% day⁻¹) than those fed the other experimental diets (p < 0.05). TH also increased feed intake (20.96 ± 0.49 g shrimp⁻¹) and was associated with higher trypsin activity (3236 ± 226 unit h⁻¹ mg protein⁻¹) and improved protein digestibility. Histological observations indicated greater hepatopancreatic cell density and increased intestinal villus height in the TH group. In addition, survival of shrimp fed TH (90.0 ± 2.8%) was higher than that observed in the FH and SS groups (p < 0.05), and hemolymph from TH-fed shrimp showed stronger inhibitory activity against Vibrio parahaemolyticus. Overall, among the evaluated hydrolysates, 1% tuna hydrolysate (dry-matter basis) produced the most consistent improvements across growth performance, digestive capacity, and selected health-related indicators, supporting its feasibility as a partial substitute for squid liver powder in shrimp diets. Full article

(This article belongs to the Section Animal Nutrition)

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26 pages, 514 KB

Open AccessReview

The Equine Reproductive Microbiota: Composition, Dynamics, Dysbiosis, and Implications for Fertility in Mares and Stallions

by Urtė Pelenė, Artūras Šiukščius, Rasa Nainienė, Inga Merkelytė and Rūta Šveistienė

Animals 2026, 16(9), 1414; https://doi.org/10.3390/ani16091414 - 5 May 2026

Viewed by 1462

Abstract

The equine reproductive microbiota has emerged as an important factor influencing reproductive health and fertility in both mares and stallions. Traditionally, the equine uterus was considered sterile, and microbial presence was interpreted primarily in the context of infection. However, sequencing-based studies have demonstrated [...] Read more.

The equine reproductive microbiota has emerged as an important factor influencing reproductive health and fertility in both mares and stallions. Traditionally, the equine uterus was considered sterile, and microbial presence was interpreted primarily in the context of infection. However, sequencing-based studies have demonstrated that the reproductive tract, including the uterus and semen, contains detectable microbial communities or microbial DNA signatures, challenging this traditional paradigm. In mares, the vaginal microbiota is consistently dominated by Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, whereas the uterine environment represents a low-biomass niche in which interpretation is complicated by contamination risk and the inability of sequencing-based methods to distinguish viable from nonviable microorganisms. Culture-based studies consistently identify opportunistic pathogens such as Streptococcus equi subsp. zooepidemicus, Escherichia coli, and Pseudomonas aeruginosa in association with endometritis and persistent breeding-induced endometritis, while sequencing-based studies suggest broader community-level dysbiosis rather than simple pathogen presence. In stallions, semen is not sterile and commonly contains taxa such as Porphyromonas, Corynebacterium, Peptoniphilus, and other opportunistic bacteria that may influence sperm quality and microbial transmission to mares during breeding. However, most reported associations remain correlative, and direct longitudinal evidence for persistent stallion-to-mare microbial transmission is limited. This review synthesizes current evidence on microbial composition, hormonal influences, dysbiosis, and reproductive implications of the equine reproductive microbiota, integrating culture-based and sequencing-based findings while emphasizing methodological limitations associated with low-biomass samples. Improved understanding of these microbial ecosystems may support more evidence-based reproductive diagnostics and microbiome-informed fertility management in horses. Full article

(This article belongs to the Special Issue The Role of Reproductive Microbiota in Animal Fertility and Reproductive Health)

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20 pages, 8307 KB

Open AccessArticle

Effects of RNA Interference-Mediated Silencing of the Insulin-Like Androgenic Gland Hormone Gene on Growth and Gonad Development in the Swimming Crab (Portunus trituberculatus)

by Weiren Zhang, Ronghua Li, Chuan He, Changkao Mu, Chunlin Wang, Ce Shi and Weiwei Song

Animals 2026, 16(9), 1413; https://doi.org/10.3390/ani16091413 - 5 May 2026

Cited by 1 | Viewed by 800

Abstract

The insulin-like androgenic gland (IAG) gene is considered a crucial factor in the process of sexual differentiation in decapod crustaceans. In this study, we characterized the structural features and ontogenetic expression profile of the IAG gene in the swimming crab P. trituberculatus ( [...] Read more.

The insulin-like androgenic gland (IAG) gene is considered a crucial factor in the process of sexual differentiation in decapod crustaceans. In this study, we characterized the structural features and ontogenetic expression profile of the IAG gene in the swimming crab P. trituberculatus (Pt-IAG). To further elucidate its biological roles, we performed long-term RNA interference (RNAi) to evaluate the physiological impacts of sustained Pt-IAG suppression. Results showed that the gene structure of Pt-IAG is conserved with the characteristic B-C-A polypeptide arrangement that is representative of decapod IAGs. Transcripts of Pt-IAG were detectable as early as the zygote stage, which may imply its potential role during early-stage development. Although no completesex reversal was observed, continuous knockdown of Pt-IAG resulted in a decrease in body weight and a delay in gonad development in both sexes, underscoring its pleiotropic role in coordinating growth and maturation. Furthermore, the silencing of Pt-IAG led to a significant downregulation of insulin-like growth factor-binding protein (Pt-IGFBP), and a decrease in insulin-receptor (Pt-IR) expression was observed in males. Notably, the Pt-IR gene was not detected in female ovaries. These findings suggest that IAG may modulate male development through an insulin-like signaling axis; however, the absence of ovarian Pt-IR expression implies the potential existence of alternative regulatory pathways in females. Collectively, this study identifies a dual regulatory role of IAG in both growth and reproduction, providing a specific theoretical basis for developing sex-control and growth-optimization strategies in crab aquaculture. Full article

(This article belongs to the Section Aquatic Animals)

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15 pages, 769 KB

Open AccessArticle

Evaluation and Selection of Stable Reference Genes for qRT-PCR Analysis in Different Tissues of Mugilogobius chulae Under Pollutant Exposure

by Zhongdian Dong, Jiahao Gao, Xiaobin Li, Zhishan Chen, Jianjun Li, Jian Liao, Yanping Zhang, Zhongduo Wang, Yusong Guo and Ning Zhang

Animals 2026, 16(9), 1412; https://doi.org/10.3390/ani16091412 - 5 May 2026

Viewed by 666

Abstract

Reliable normalization is essential for accurate quantitative real-time PCR (qRT-PCR) analysis, yet suitable reference genes have not been systematically evaluated in Mugilogobius chulae, an emerging marine experimental fish used in physiological and environmental research. In this study, 17 candidate reference genes were [...] Read more.

Reliable normalization is essential for accurate quantitative real-time PCR (qRT-PCR) analysis, yet suitable reference genes have not been systematically evaluated in Mugilogobius chulae, an emerging marine experimental fish used in physiological and environmental research. In this study, 17 candidate reference genes were evaluated in five tissues (brain, gill, gonad, intestine, and liver) of sexually mature male and female M. chulae under solvent control (DMSO), bisphenol A (BPA), cadmium (Cd), and sulfamethazine (SMX) treatment conditions. Gene-expression stability was assessed using the comparative ΔCt method, NormFinder, geNorm, BestKeeper, and the integrated RefFinder ranking. The results showed that reference-gene stability was strongly tissue-specific and analysis-dependent. The integrated RefFinder analysis identified eif3h, rpl7, rps4x, stau1, and ef1y as the most stable genes in the pooled dataset, whereas ube2, hprt1l, and aldob were the least stable. geNorm analysis indicated that two reference genes were sufficient for brain, gill, intestine, and liver, whereas four were required for the gonad and six for cross-tissue comparisons. These findings provide the first systematic basis for reference-gene selection in M. chulae and establish an important methodological foundation for future qRT-PCR studies in this species, particularly in research on endocrine disruption, reproductive physiology, and marine ecotoxicology. Full article

(This article belongs to the Special Issue Sustainable Aquaculture: A Functional Genomic Perspective)

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19 pages, 6457 KB

Open AccessArticle

Protective Effect of Synbiotic Supplementation Against Salmonella Typhimurium Infection in Young Broiler Chickens

by Walter Rivera Pérez, Elías Barquero Calvo, Aida Chaves Hernández and Catalina Salas Duran

Animals 2026, 16(9), 1411; https://doi.org/10.3390/ani16091411 - 5 May 2026

Viewed by 918

Abstract

Salmonella infection in poultry remains a major concern due to its economic impact and public health risks. Synbiotics have emerged as a natural strategy to improve gut health and resistance to enteric pathogens. This study evaluated the immune response and early protective effects [...] Read more.

Salmonella infection in poultry remains a major concern due to its economic impact and public health risks. Synbiotics have emerged as a natural strategy to improve gut health and resistance to enteric pathogens. This study evaluated the immune response and early protective effects of a synbiotic in broiler chickens using a reduced in vivo model of Salmonella Typhimurium infection. A total of 44 broiler chickens were randomly assigned to four dietary treatments: control (CT), control plus synbiotic (CT + Syn), challenged (ST), and challenged plus synbiotic (ST + Syn). Birds were challenged at 7 days of age and euthanized at day 12 for sample collection. Synbiotic supplementation reduced bacterial colonization and adhesion in the ileum, as assessed by intestinal content isolation and immunofluorescence analysis. Additionally, the synbiotics mitigated lesion severity in the intestine, liver, and heart, increased anti-inflammatory IL-10 expression, and reduced pro-inflammatory cytokines IL-1β and TNF-α. Ileal histomorphometry showed increased epithelial height in the ST + Syn birds, while intestinal permeability was not affected. Improved body weight was also observed compared to the challenged birds without supplementation. In conclusion, synbiotic supplementation provided early protection against S. Typhimurium infection by modulating immune responses, preserving intestinal morphology, and supporting weight gain in broiler chickens. Full article

(This article belongs to the Special Issue Probiotics and Synbiotics in Poultry: Impacts on Gut Microbiota)

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14 pages, 902 KB

Open AccessFeature PaperArticle

Birth Weight and Early Weight Changes in Newborn Dogs: Analyzing Key Influencing Factors in Dobermann Puppies

by Jasmine Fusi, Roberta Bucci, Claudia Scabrosetti, Massimo Faustini and Maria Cristina Veronesi

Animals 2026, 16(9), 1410; https://doi.org/10.3390/ani16091410 - 5 May 2026

Viewed by 1773

Abstract

Birthweight (BW) and early weight changes are important prognostic factors in dog neonatology, but are influenced by the breed. The present study aimed to evaluate BW and early growth in a single breed, Dobermann, and single breeding facility for a better evaluation of [...] Read more.

Birthweight (BW) and early weight changes are important prognostic factors in dog neonatology, but are influenced by the breed. The present study aimed to evaluate BW and early growth in a single breed, Dobermann, and single breeding facility for a better evaluation of data and analysis of influencing factors. In 364 puppies born by elective cesarean section, the ROC curve and cut-off weight values associated with the risk for death within 15 days after birth were defined for BW and Day 1 weight (D1W). Both BW and D1W were influenced by newborn’s sex (p < 0.05), maternal age (p < 0.05) and maternal bodyweight (p < 0.05), litter size (p < 0.05), and “litter effect” (p < 0.05). The weight charts for the first 15 days after birth were constructed for female and male puppies, and weight percentage variation analysis of the first 5 days after birth showed that the nadir of weight loss occurred around 1 day after birth, followed by BW recovery within 3 days, and doubling BW around 12–14 days. Therefore, BW (p < 0.001), D1W (p < 0.001) and early weight changes are important factors for prognosis and growth monitoring, allowing the prompt detection of those puppies needing special care. However, the influences of sex-related differences and maternal factors must be considered. Full article

(This article belongs to the Section Veterinary Clinical Studies)

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17 pages, 1665 KB

Open AccessCommunication

Preoperative Cardiac Risk Stratification in Dogs with Mammary Tumors Using Two-Dimensional Speckle Tracking Echocardiography: A Pilot Study

by Didem Algan, Tuğba Varlik, Hüseyin Tan, Pelin Erden, Lina Hamabe, Ryou Tanaka and Zeki Yilmaz

Animals 2026, 16(9), 1409; https://doi.org/10.3390/ani16091409 - 4 May 2026

Viewed by 776

Abstract

Although breast cancer has been associated with subclinical left ventricular dysfunction in humans, the cardiac effects of CMTs remain poorly defined. This pilot, exploratory (communication) study compared clinical and echocardiographic parameters between dogs with CMTs and healthy controls and assessed the feasibility of [...] Read more.

Although breast cancer has been associated with subclinical left ventricular dysfunction in humans, the cardiac effects of CMTs remain poorly defined. This pilot, exploratory (communication) study compared clinical and echocardiographic parameters between dogs with CMTs and healthy controls and assessed the feasibility of combining myocardial deformation imaging with exploratory data-driven analysis for preoperative cardiac assessment. All dogs underwent a standardized clinical and echocardiographic assessment, including two-dimensional speckle-tracking echocardiography (2D-STE). Given the limited sample size, analyses were designed to generate hypotheses rather than to provide definitive predictive conclusions. Exploratory machine learning modeling (XGBoost), receiver operating characteristic (ROC) analysis, calibration, and decision curve analysis were performed as proof-of-concept approaches without external validation. Despite normal conventional systolic indices, dogs with CMTs exhibited reduced global longitudinal strain (GLS) and mitral annular plane systolic excursion (MAPSE) (p < 0.01), suggesting subclinical systolic dysfunction. Deformation-derived parameters appeared more sensitive for detecting subtle myocardial alterations within this cohort. The exploratory machine learning model demonstrated moderate discrimination (AUC-ROC = 0.75); however, these findings are preliminary and should not be interpreted as evidence of clinical predictive performance. Overall, these results suggest that conventional systolic indices may underestimate early myocardial changes in dogs with CMTs. This communication highlights the feasibility of integrating deformation imaging with exploratory analytical approaches and provides a basis for future large-scale, validated studies in veterinary cardio-oncology. Full article

(This article belongs to the Section Veterinary Clinical Studies)

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24 pages, 9986 KB

Open AccessArticle

Tea Saponin Exerts Dose-Dependent Dual Effects on Growth and Hepatic Health in Hybrid Grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) Fed a High-Lipid, Low-Protein Diet via Redox-Immune Regulation

by Shengrong Guo, Jun Yu, Lili Shi, Beiping Tan, Shuang Zhang and Xiaobo Yan

Animals 2026, 16(9), 1408; https://doi.org/10.3390/ani16091408 - 4 May 2026

Viewed by 773

Abstract

High-lipid, low-protein diets are economically advantageous in aquaculture costs but often induce hepatic damage, immunosuppression and metabolic disorders in carnivorous fish like hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂). Tea saponin (TS) is a bioactive triterpenoid from camellia seed meal [...] Read more.

High-lipid, low-protein diets are economically advantageous in aquaculture costs but often induce hepatic damage, immunosuppression and metabolic disorders in carnivorous fish like hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂). Tea saponin (TS) is a bioactive triterpenoid from camellia seed meal with excellent lipid-lowering and anti-inflammatory effects. The purpose of the current study was to investigate the dose-dependent impacts of TS supplementation at 0% (control, T0), 0.05% (T5), 0.10% (T10), 0.15% (T15), and 0.20% (T20) in diets formulated to contain 42% crude protein and 16% crude lipid on juvenile hybrid grouper (17.51 ± 0.03 g), with three replicate tanks per treatment, over a 4-week feeding trial. The results showed that low TS supplementation (0.05%) improved growth, with the effect limited to final average weight (FAW), whereas higher doses (≥0.15%) reduced weight gain. The T5 group exhibited the highest hepatic total antioxidant capacity and superoxide dismutase activity, consistent with the corresponding gene expression, and the lowest malondialdehyde and reactive oxygen species levels. Lysozyme activity and immunoglobulin M content were significantly higher in T5–T15 groups (p < 0.05). Furthermore, appropriate TS significantly upregulated anti-inflammatory cytokines (p < 0.05) and downregulated pro-inflammatory factors. Histologically, 0.10% TS reduced inflammatory infiltration, while high doses caused hepatocyte rupture and lipid vacuolation. Transcriptomic analysis further elucidated that the beneficial effects of low-dose TS were linked to the activation of PPAR signaling, fatty acid catabolism, and cellular quality control pathways, while high-dose TS triggered stress-related and biosynthetic programs. In conclusion, moderate TS supplementation (0.05–0.10%) ameliorated diet-induced oxidative stress and immunosuppression, improved growth performance and anti-inflammatory factor expression, whereas over-addition inhibited growth and exacerbated hepatic damage. Based on a quadratic model, the optimal dietary TS level for maximizing growth under this low-protein, high-lipid regimen is estimated at 0.055%. Full article

(This article belongs to the Special Issue Genomics, Evolution, and Biodiversity of Fishes)

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18 pages, 55736 KB

Open AccessArticle

Developmental Comparative Analysis of Enteroendocrine Hormone Immunoreactive Cells in the Abomasum and Small Intestine of Holstein–Friesian Bulls

by Ligia Janicka, Aleksandra Dajnowska, Katarzyna Woźniak, Cezary Osiak-Wicha, Katarzyna Kras, Iwona Łuszczewska-Sierakowska and Marcin B. Arciszewski

Animals 2026, 16(9), 1407; https://doi.org/10.3390/ani16091407 - 4 May 2026

Viewed by 705

Abstract

Within the complex regulatory network of the gastrointestinal tract, enteroendocrine cells play a fundamental role in coordinating digestive processes and maintaining metabolic balance. These cells produce a wide range of hormones that influence secretion, motility, and nutrient utilization, constituting an essential component of [...] Read more.

Within the complex regulatory network of the gastrointestinal tract, enteroendocrine cells play a fundamental role in coordinating digestive processes and maintaining metabolic balance. These cells produce a wide range of hormones that influence secretion, motility, and nutrient utilization, constituting an essential component of gastrointestinal physiology in ruminants. Despite the growing interest in the role of endocrine regulation in digestive system function, comprehensive studies evaluating enteroendocrine cell populations across different segments of the gastrointestinal tract in cattle remain limited. In particular, the organization of these cell populations in key digestive segments such as the abomasum and small intestine requires further investigation to better understand their functional significance. The aim of the present study was to provide a comparative characterization of selected enteroendocrine cell populations in the abomasum and small intestine of young and adult Holstein–Friesian bulls. Tissue samples collected from the abomasum, duodenum, jejunum, and ileum were subjected to histological and immunohistochemical analyses. Chromogranin A (CgA) was used as a general marker of enteroendocrine cells, and the number of cells immunoreactive to CgA as well as to gastrin, secretin, somatostatin (SOM), glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide-1 (GLP-1) was quantified in the examined segments of the gastrointestinal tract. All types of immunoreactive cells were counted using a standardized quantitative method based on the assessment of positively stained cells in randomly selected microscopic fields of histological sections. The results revealed clear differences in the number of enteroendocrine cells between the analyzed segments of the gastrointestinal tract as well as between the studied age groups. These findings indicate regional and age-related variation in enteroendocrine cell populations within the gastrointestinal tract and highlight their association with the functional organization of individual segments of the digestive system. Full article

(This article belongs to the Special Issue Feed Additives for Farm Animals: Gut Microbiome, Metabolism, Neuroendocrine Regulation, and Product Quality)

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28 pages, 12533 KB

Open AccessReview

Research Progress on Mammalian Oocyte Vitrification: From Damage Mechanisms to Optimization Strategies

by Kelin Song, Li Wang, Feng Yang, Hongqian Zhu, Qiuyu Meng, Xuelei Han, Ruimin Qiao, Jun Bai, Shuangbao Gun, Tong Yu and Xinjian Li

Animals 2026, 16(9), 1406; https://doi.org/10.3390/ani16091406 - 3 May 2026

Cited by 2 | Viewed by 1252

Abstract

With the continuous advancement in reproductive biology, oocyte vitrification has become a critical technology for preserving female germplasm and protecting it from environmental disruptions. This technique also eliminates temporal and spatial constraints in animal embryo engineering research. However, during the vitrification of animal [...] Read more.

With the continuous advancement in reproductive biology, oocyte vitrification has become a critical technology for preserving female germplasm and protecting it from environmental disruptions. This technique also eliminates temporal and spatial constraints in animal embryo engineering research. However, during the vitrification of animal oocytes, exposure to low temperatures and high concentrations of cryoprotectants can cause various forms of damage, including cytoskeletal disruption, spindle abnormalities, mitochondrial dysfunction, apoptosis, oxidative stress and epigenetic modifications. These issues are now understood to severely restrict the subsequent developmental competence of oocytes, resulting in lower cleavage and blastocyst formation rates than those of fresh oocytes. Currently, the mechanisms of cryodamage in vitrified oocytes remain poorly understood, and standardized strategies to enhance vitrification efficiency have yet to be firmly established. This review provides a formal overview of the physiological factors underlying oocyte sensitivity to vitrification, alongside the mechanisms of cryodamage and the variables influencing post-thaw survival and reproductive success. It evaluates strategies for mitigating vitrification-induced stress, compares interspecies differences, and addresses current research limitations. By identifying future directions, this review offers new insights for optimizing mammalian oocyte cryopreservation techniques. Full article

(This article belongs to the Special Issue Advances in Cryopreservation of Livestock Oocytes and Embryos)

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14 pages, 442 KB

Open AccessArticle

Pre-Intervention Assessment of Toxocara Infection in Dogs in Vietnam: A Community-Based Cross-Sectional Study

by Minh-Trang Thi Hoang, Dinh Ng-Nguyen, Ketsarin Kamyingkird, Van-Phuong Ngo and Tawin Inpankaew

Animals 2026, 16(9), 1405; https://doi.org/10.3390/ani16091405 - 3 May 2026

Viewed by 791

Abstract

Dogs are key reservoirs of zoonotic infections, including Toxocara canis, a widely distributed parasite of major public health concern. In Vietnam, the parasite is highly prevalent in dog populations and humans. Epidemiological studies assessing infection and associated factors are essential to better [...] Read more.

Dogs are key reservoirs of zoonotic infections, including Toxocara canis, a widely distributed parasite of major public health concern. In Vietnam, the parasite is highly prevalent in dog populations and humans. Epidemiological studies assessing infection and associated factors are essential to better understand transmission and to inform effective control strategies. We conducted a cross-sectional baseline survey to assess Toxocara infection in dogs in rural Vietnam. Fecal samples from 371 dogs were examined using centrifugal flotation (Sheather’s solution, specific gravity 1.2) and conventional polymerase chain reaction (PCR), alongside structured questionnaires on dog demographics and management. Using combined copromicroscopic and molecular methods, the overall prevalence of Toxocara infection was 44.7% (95% CI: 39.6–50.0). By microscopy alone, 29.9% (95% CI: 25.4–34.9) of samples were positive, while PCR detected Toxocara DNA in 41.2% (95% CI: 36.2–46.5) of dogs. Molecular analysis identified T. canis in 35.9% (95% CI: 31.0–41.0) and T. cati in 10.5% (95% CI: 7.7–14.2) of tested dogs. Dog age and deworming status were independently associated with PCR-detected T. canis infection. The elevated likelihood of infection among dogs that have never been dewormed highlights the importance of canine deworming. Questionnaire findings indicating suboptimal dog care and management highlight the need for community public health education to promote responsible ownership and reduce transmission risk. This baseline assessment provides essential evidence to inform targeted interventions and improve understanding of Toxocara transmission in endemic settings. Full article

(This article belongs to the Special Issue Zoonotic Parasites at the Human-Animal Interface: Advances in Epidemiology, Transmission, Diagnosis, and Treatment)

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15 pages, 837 KB

Open AccessFeature PaperArticle

Native Fish Inclusion Promotes Nutrient Retention and Productivity in a Biofloc-Based Aquaponic System

by Adolfo Jatobá, Bruno Corrêa da Silva, Felipe Boéchat Vieira, Marco Shizuo Owatari, Leonardo Alexander Krause, Amanda Dartora, Maísa de Lima Lasala, Keren Fagundes Morais and Jaqueline I. A. de Andrade

Animals 2026, 16(9), 1404; https://doi.org/10.3390/ani16091404 - 3 May 2026

Cited by 1 | Viewed by 635

Abstract

The integration of multiple species has been proposed as a strategy to improve resource use efficiency in intensive aquaculture systems. This study evaluated the inclusion of a native fish species, yellowtail lambari (Astyanax bimaculatus), in a biofloc-based aquaponic system co-cultivating Nile [...] Read more.

The integration of multiple species has been proposed as a strategy to improve resource use efficiency in intensive aquaculture systems. This study evaluated the inclusion of a native fish species, yellowtail lambari (Astyanax bimaculatus), in a biofloc-based aquaponic system co-cultivating Nile tilapia (Oreochromis niloticus) and lettuce (Lactuca sativa var. capitata). The experiment was conducted over 35 days using eight experimental units with two treatments (with and without lambari) and four replicates. Water quality, zootechnical performance, lettuce growth, hematological parameters of tilapia, and nitrogen and phosphorus retention were assessed. The presence of lambari was associated with lower total ammonia nitrogen, toxic ammonia, and total suspended solids, particularly during the final stage of the experimental period (p < 0.05), as well as reduced pH and alkalinity, likely reflecting increased microbial activity. Lettuce cultivated in the lambari treatment showed higher final weight, leaf height, and total biomass (p < 0.05), resulting in increased system productivity. No significant differences were observed in growth performance or hematological parameters of Nile tilapia (p > 0.05). In addition, nitrogen and phosphorus retention at the system level were higher in the lambari treatment (p < 0.05), although no differences were detected when fish and plants were evaluated separately. These results indicate that the inclusion of a native fish species can influence nutrient retention and productivity in biofloc-based aquaponic systems without compromising the performance of the primary cultured species. Full article

(This article belongs to the Special Issue Innovations in Aquaculture: New Technologies, Culture Systems and Integration of Emerging Species)

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15 pages, 2006 KB

Open AccessArticle

Sustainable Upcycling of Swine Wastewater Sludge: Using Thermal and Citrate Pretreatment to Enhance Volatile Fatty Acid Production

by Wei-Chen Chen and Jung-Jeng Su

Animals 2026, 16(9), 1403; https://doi.org/10.3390/ani16091403 - 3 May 2026

Viewed by 583

Abstract

The sustainable management of intensive swine farming is currently bottlenecked by the difficult valorization of metal-rich wastewater sludge. The structural rigidity of this sludge, stabilized by divalent cation bridging, severely limits its anaerobic digestion and overall resource recovery. To optimize the manure management [...] Read more.

The sustainable management of intensive swine farming is currently bottlenecked by the difficult valorization of metal-rich wastewater sludge. The structural rigidity of this sludge, stabilized by divalent cation bridging, severely limits its anaerobic digestion and overall resource recovery. To optimize the manure management chain, this study comprehensively evaluated various physical and chemical pretreatments to identify the most effective disintegration strategy for enhanced volatile fatty acid (VFA) production. Among the tested conditions, the coupling of thermal hydrolysis with citrate chelation (T/SC) was the most effective, achieving the highest disintegration degree (12.37%) and biopolymer solubilization. Mechanism analysis revealed that, unlike traditional alkaline treatments, which are limited by the severe reprecipitation of magnesium and phosphate, citrate effectively sequestered bridging cations (Ca²⁺ and Mg²⁺) via ligand exchange. This synergistic disintegration accelerated the fermentation kinetics, enhancing the total VFA yield 2-fold (1293 mg/L) compared to the control group while maintaining a high-value, butyrate-dominant product profile. These findings demonstrate that targeting ionic bridges via ligand-promoted dissolution provides a highly practical and sustainable strategy to maximize resource recovery and nutrient cycling from metal-laden livestock wastes. Full article

(This article belongs to the Special Issue Strategies for Integrating Waste Recycling and Resource Recovery in Animal Farming)

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16 pages, 9331 KB

Open AccessArticle

Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

by Liangkai Liu, Maohua Xia, Chengyao Hou, Danyu Chen, Chengyao Li, Xinggui Chen, Qinyuan Chu, Yue Sun, Shujun Liu, Yuqing Li, Hanlin Wang, Yan Zhu, Mengfang Yang, Hongning Wang, Caiwu Li and Xin Yang

Animals 2026, 16(9), 1402; https://doi.org/10.3390/ani16091402 - 3 May 2026

Viewed by 872

Abstract

Although canine parvovirus (CPV) vaccination has been widely implemented, CPV continues to circulate in dog populations and poses a potential cross-species transmission risk to wildlife, including giant pandas. Recent increases in CPV-2c detection in China highlight the need for molecular surveillance and standardized [...] Read more.

Although canine parvovirus (CPV) vaccination has been widely implemented, CPV continues to circulate in dog populations and poses a potential cross-species transmission risk to wildlife, including giant pandas. Recent increases in CPV-2c detection in China highlight the need for molecular surveillance and standardized immunoreagents for diagnosis and epitope mapping. This study aimed to isolate a representative CPV-2c strain from China and develop VP2-targeted nanobody-based recognition molecules to support antigen monitoring and detection optimization. Canine and giant panda samples were collected in Sichuan Province, and CPV was isolated in F81 cells, followed by VP2 gene sequencing and phylogenetic analysis. A secretion expression system in Bacillus subtilis was established to produce VP2-targeting nanobodies, and a canine Fc-fused format of Nb10 (Nb10-Fc) was constructed. Immunoreactivity was evaluated via immunoassays, and structural modeling and molecular docking were performed to predict binding interfaces. The results showed that CPV-2c was the dominant genotype in Sichuan, with CPV L4 being a representative strain that exhibited 100% identity in VP2 with a giant panda-derived CPV-2c strain. Nb10 and Nb10-Fc demonstrated strong reactivity in Western blotting and immunofluorescence assays. The Fc-fusion improved detection sensitivity, offering potential in vivo application benefits. This study provides a standardized VP2-specific nanobody and molecular system for CPV-2c surveillance, antigenic studies, and diagnostic optimization. Full article

(This article belongs to the Section Companion Animals)

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30 pages, 6172 KB

Open AccessArticle

Negative Phonotaxis Behavior of Juvenile Grass Carp (Ctenopharyngodon idella) to Different Acoustic Stimuli in Natural Aquatic Environments

by Jiaxin Li, Shenwei Zhang, Xuan Wang, Ji Yang, Guoyong Liu and Lixiong Yu

Animals 2026, 16(9), 1401; https://doi.org/10.3390/ani16091401 - 3 May 2026

Viewed by 749

Abstract

Hydraulic engineering structures can threaten freshwater fish by entraining them into hazardous areas. Acoustic barriers have been proposed as a non-physical method to guide fish away from these zones. In this study, we investigated the behavioral responses of juvenile grass carp to different [...] Read more.

Hydraulic engineering structures can threaten freshwater fish by entraining them into hazardous areas. Acoustic barriers have been proposed as a non-physical method to guide fish away from these zones. In this study, we investigated the behavioral responses of juvenile grass carp to different acoustic stimuli under semi-natural conditions using outdoor net cages. Four sound types were tested: a 1000 Hz pure tone and three broadband sounds, including Alligator sinensis hissing, pile-driving noise, and outboard motor noise. Behavioral responses were quantified using response frequency, total midline crossings, first-response time, maximum swimming speed, and average swimming speed. The results showed that Alligator sinensis hissing elicited the highest number of midline crossings, representing the strongest behavioral response among all tested sounds. In addition, both Alligator sinensis hissing and outboard motor noise induced significantly stronger avoidance responses than the pure tone or pile-driving noise, as indicated by higher response frequency and faster swimming speeds. Furthermore, manipulation of pulse repetition intervals in the most effective deterrent sounds generated a novel broadband sound, which altered fish distribution patterns and elicited avoidance behavior. These findings indicate that both sound type and temporal structure influence negative phonotaxis behavior in grass carp and provide experimental evidence for the optimization of acoustic barriers in fish management. Full article

(This article belongs to the Section Aquatic Animals)

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13 pages, 8649 KB

Open AccessFeature PaperArticle

Impact of Dietary Inclusion with Cocrystal Essential Oil on Growth Performance, Nutrient Digestibility, Intestinal Morphology, and Antioxidant Status in Weaned Piglets

by Yifei Sun, Jun Chen, Qiuting Yin, Pengbo Liang, Jinming You and Tiande Zou

Animals 2026, 16(9), 1400; https://doi.org/10.3390/ani16091400 - 3 May 2026

Viewed by 730

Abstract

This study assessed the impact of cocrystal essential oil (CEO) inclusion on growth performance, nutrient digestibility, intestinal morphology, and antioxidant status in weaned piglets. Ninety-six weaned piglets were assigned to four groups (n = 8, three piglets per pen). The piglets in the [...] Read more.

This study assessed the impact of cocrystal essential oil (CEO) inclusion on growth performance, nutrient digestibility, intestinal morphology, and antioxidant status in weaned piglets. Ninety-six weaned piglets were assigned to four groups (n = 8, three piglets per pen). The piglets in the four groups were fed basal diets added with 0, 120, 180, or 240 mg/kg of CEO, respectively, over a 28-day trial period. Results showed that during weeks 1–2, piglets in the 240 mg/kg CEO group exhibited a lower diarrhea rate and diarrhea index compared to the control group (p < 0.05). In weeks 3–4, the 180 and 240 mg/kg CEO groups demonstrated a reduced diarrhea rate and diarrhea index compared to the control group (p < 0.05). Relative to the control group, the apparent total tract digestibility (ATTD) of dry matter was elevated in piglets fed diets added with 120, 180, or 240 mg/kg CEO at both day 14 and day 28 (p < 0.05). Additionally, the ATTD of crude protein was elevated in the 120 mg/kg CEO group at day 14 and in the 180 mg/kg CEO group at day 28 (p < 0.05). Regarding intestinal morphology, supplementation with 180 or 240 mg/kg CEO increased jejunal villus height (VH) and the villus height to crypt depth (VH/CD) ratio compared with the control group (p < 0.05). Furthermore, 240 mg/kg CEO supplementation augmented the ileal VH/CD ratio relative to the control group (p < 0.05). For antioxidant status, 180 mg/kg CEO supplementation elevated serum glutathione peroxidase (GSH-Px) activity in piglets relative to the control group (p < 0.05). Importantly, no differences were found between the 180 mg/kg and 240 mg/kg CEO groups across all measured parameters (p > 0.05). In conclusion, dietary inclusion with 180 mg/kg CEO is recommended for weaned piglets, given its comprehensive benefits in alleviating diarrhea, improving nutrient digestibility, enhancing intestinal morphology, and bolstering antioxidant status. Full article

(This article belongs to the Section Pigs)

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19 pages, 2134 KB

Open AccessArticle

Multiscale Structural Evolution and Digestion Kinetics of Starch in Commercial Swine Compound Feed as Affected by Steam Conditioning Temperature and Retention Time

by Junhua Wu, Fanglei Zou, Wei Wang, Liangju Wang and Hongying Wang

Animals 2026, 16(9), 1399; https://doi.org/10.3390/ani16091399 - 3 May 2026

Viewed by 922

Abstract

Steam conditioning is a key hydrothermal process for ensuring the quality of pelleted commercial swine compound feed. However, the molecular evolution of starch during processing remains poorly understood, particularly under the industrial constraints of limited moisture and complex substrates. This study quantified the [...] Read more.

Steam conditioning is a key hydrothermal process for ensuring the quality of pelleted commercial swine compound feed. However, the molecular evolution of starch during processing remains poorly understood, particularly under the industrial constraints of limited moisture and complex substrates. This study quantified the effects of conditioning temperature (60–90 °C) and retention time (2–4 min) on starch structural reorganization across multiple length scales and in vitro digestion kinetics in pig diets. Structural evolution exhibited a nonlinear, temperature-driven pattern, with the temperature region around 80 °C representing a critical transition in structural reorganization. Although high-temperature conditioning (90 °C) increased the initial digestion rate, a nonlinear structure–digestion response was observed: final digestibility decreased, and resistant starch content rebounded. Microstructural analyses revealed that severe heat treatment was associated with the possible formation of amylose–lipid complexes (V-type crystallinity) and a more continuous protein-associated matrix, which may have imposed additional physical constraints on enzymatic hydrolysis. Consequently, maximizing starch gelatinization does not necessarily guarantee optimal in vitro digestibility. These findings integrate multiscale structural analysis with digestion kinetics, providing a mechanistic basis for balancing energy consumption, pellet quality, and nutritional value in the feed industry. Full article

(This article belongs to the Section Animal System and Management)

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27 pages, 4942 KB

Open AccessArticle

Ancestral BG1 Alleles and Structural Conservation Ensure Immune-Related Genetic Resilience in Southeast Asian Chicken Lineages

by Anh Huynh Luu, Trifan Budi, Worapong Singchat, Chien Tran Phuoc Nguyen, Thitipong Panthum, Nivit Tanglertpaibul, Kanithaporn Vangnai, Aingorn Chaiyes, Chotika Yokthongwattana, Chomdao Sinthuvanich, Orathai Sawatdichaikul, Kyudong Han, Narongrit Muangmai, Darren K. Griffin, Prateep Duengkae, Ngu Trong Nguyen and Kornsorn Srikulnath

Animals 2026, 16(9), 1398; https://doi.org/10.3390/ani16091398 - 3 May 2026

Viewed by 1151

Abstract

Chicken (Gallus gallus domesticus) domestication, likely associated with dry-rice farming in central Thailand, has led to substantial loss of ancestral immune-related genetic diversity in commercial chicken lineages. This study addresses allelic loss by providing the first comprehensive analysis of the highly [...] Read more.

Chicken (Gallus gallus domesticus) domestication, likely associated with dry-rice farming in central Thailand, has led to substantial loss of ancestral immune-related genetic diversity in commercial chicken lineages. This study addresses allelic loss by providing the first comprehensive analysis of the highly polymorphic BG1 gene, an MHC-linked marker across the wild–domestic interface in Thailand and Vietnam, using high-depth Illumina amplicon sequencing. Genomic DNA from 47 Thai and Vietnamese chicken populations was extracted using a salting-out protocol following ethical sampling. Allelic variation was examined by targeting the BG1 intron 15–exon 16 region using triplicate PCR and Salus Pro NGS sequencing. Evolutionary dynamics and selection pressures were analyzed using AmpliSAS, MrBayes, and Datamonkey, while AlphaFold 3 was used to predict and validate 3D protein structures. We identified 98 novel alleles and 172 polymorphic sites within the BG1 intron 15–exon 16 region encoding an Ig-like domain. Extensive allele sharing between indigenous chickens and red junglefowl indicated strong balancing selection and trans-species polymorphism. Selection analyses showed that purifying selection conserved structural integrity at codons 9, 13, and 18, while variation at other sites enhanced immune recognition. AlphaFold 3 modeling confirmed conservation of the β-sandwich fold across variants, maintaining stability of the Immunoreceptor Tyrosine-based Inhibition Motif (ITIM). Thus, despite the regional gene flow, geographic isolation has shaped distinct signatures, as evidenced by the presence of 38 unique Thai and 9 unique Vietnamese alleles in addition to breed-specific private markers in the Betong (BG1*TH88), Decoy (BG1*TH91), and Tre (BG1*VN54) populations. A notable adaptive outlier under positive selection (ω = 1.357) was detected in the Dong Tao population, suggesting a recent selective sweep. These findings support the mission of the Siam Chicken Bioresource Project (SCBP) to utilize indigenous breeds as genetic reservoirs and provide a molecular basis for restoring resilience traits in domestic poultry to enhance global food security. Full article

(This article belongs to the Section Animal Genetics and Genomics)

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18 pages, 4618 KB

Open AccessArticle

Establishment of a Quadruplex RT-qPCR Method for the Detection of All Lineages of PPRV

by Jiao Xu, Jiani Li, Qinghua Wang, Jiamin Zhou, Shuang Liu, Yingli Wang, Jiarong Yu, Jingyue Bao and Lin Yang

Animals 2026, 16(9), 1397; https://doi.org/10.3390/ani16091397 - 3 May 2026

Cited by 1 | Viewed by 905

Abstract

Peste des petits ruminants (PPR) is an infectious disease with high morbidity and mortality rates, and four distinct lineages have been discovered in different regions globally. In this study, a quadruplex RT-qPCR method capable of differentiating all four lineages of PPRV was established. [...] Read more.

Peste des petits ruminants (PPR) is an infectious disease with high morbidity and mortality rates, and four distinct lineages have been discovered in different regions globally. In this study, a quadruplex RT-qPCR method capable of differentiating all four lineages of PPRV was established. By screening specific conserved regions across all viral genomes, we designed primers, as well as four TaqMan probes capable of distinguishing all lineages. The established method underwent validation of its relevant characteristics. The sensitivity of the detection method was determined by testing plasmid serial dilutions ranging from 10⁸ to 10⁰ copies/μL; results showed that the method could detect as few as 10 copies per microliter of PPRV. No cross-reactivity was observed among the four probes or with other common pathogens of goats and sheep. The coefficient of variation (CV) values for inter-assay and intra-assay repeatability of each probe were both below 2% (intra-assay: 0.11% to 0.98%; inter-assay: 0.18% to 1.95%), demonstrating excellent repeatability. Testing of 62 clinical samples also confirmed that the method could effectively detect and differentiate clinical samples of different PPR lineages. This method, for the first time, enabled the differentiation of all PPRV lineages in a single reaction, improving the detection efficiency of the PPR virus and providing robust technical support for the global PPR eradication program. Full article

(This article belongs to the Collection Diseases of Small Ruminants)

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18 pages, 6275 KB

Open AccessReview

Respiratory Anatomy and Physiology of Reptiles

by Javier G. Nevarez

Animals 2026, 16(9), 1396; https://doi.org/10.3390/ani16091396 - 2 May 2026

Cited by 1 | Viewed by 3549

Abstract

Reptiles have varied lung anatomy with three main lung types: unicameral, transitional, and multicameral. Crocodilians have a well-developed bronchial tree, while all other species have either a limited number of bronchi or no bronchi at all. The primary gas exchange structures are the [...] Read more.

Reptiles have varied lung anatomy with three main lung types: unicameral, transitional, and multicameral. Crocodilians have a well-developed bronchial tree, while all other species have either a limited number of bronchi or no bronchi at all. The primary gas exchange structures are the faveoli and ediculae, which can have either homogeneous or heterogeneous distribution within the lung parenchyma. The physiology of reptile respiration is also complex, varied, and at times contradictory across species. A basic understanding of anatomy and physiology is essential for proper diagnostic and therapeutic approaches to reptile patients. Therefore, a paradigm shift is needed to start thinking of the anatomy and physiology of individual species rather than believing that a single concept applies to all reptiles equally. As such, future research should aim to duplicate concepts across the more common species encountered in clinical practice. Full article

(This article belongs to the Special Issue Advances in Exotic Pet Medicine)

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15 pages, 2253 KB

Open AccessArticle

Breeding Biology of the Twite Linaria flavirostris in the North-Eastern Qinghai–Tibet Plateau, with Special Reference to Life-History Variation Across Latitudes and Altitudes

by Shuai Yan, Bowen Zhang and Shaobin Li

Animals 2026, 16(9), 1395; https://doi.org/10.3390/ani16091395 - 2 May 2026

Viewed by 1034

Abstract

In 2024 and 2025, researchers investigated the breeding ecology of the Twite Linaria flavirostris in riparian shrubland habitats at an elevation of 3400 m in the northeastern Qinghai–Tibet Plateau. This species lays eggs from late June to mid-July, capitalizing on the region’s brief [...] Read more.

In 2024 and 2025, researchers investigated the breeding ecology of the Twite Linaria flavirostris in riparian shrubland habitats at an elevation of 3400 m in the northeastern Qinghai–Tibet Plateau. This species lays eggs from late June to mid-July, capitalizing on the region’s brief warm season. Nests are typically open-cup structures built in Hippophae spp. shrubs. The population predominantly exhibits monogamous mating, with a mean clutch size of 4.7 ± 0.49 (3~5). Incubation is performed solely by the female and lasts 11.52 ± 1.65 days. Both parents provision the nestlings, and the nestling period lasts 12.43 ± 2.39 days. Morphological measurements of nestling body mass and external organs all fit well to the Logistic growth curve equation. By fledging, tarsus length and bill length reach over 90% of adult values, conferring substantial terrestrial mobility. However, flight-related feathers, primaries and rectrices, remain markedly underdeveloped compared to adults, resulting in extremely poor flight capability; further post-fledging development is thus required. Based on reproductive outcomes from this single breeding season, a total of 121 eggs were laid, of which 81 successfully hatched, and ultimately 79 fledglings survived to leave the nest. The overall hatching success was 66.94%, fledging success (among hatchlings) was 97.53%, and overall offspring survival (from eggs to fledglings) was 65.29%. The apparent nesting success rate was 76.0%, based on a total of 50 nests monitored over two years. Daily nest survival rates were estimated using Mayfield’s method and program MARK, resulting in nest success probabilities of 0.587 and 0.219, respectively. Comparing populations across different geographic regions, the results indicate that Twites breeding in environments with higher levels of environmental stress produce smaller clutch sizes and larger eggs, and exhibit a prolonged nestling period. This life-history strategy likely represents an evolutionary adaptation to spatially variable environmental conditions. Full article

(This article belongs to the Section Animal Reproduction)

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22 pages, 2817 KB

Open AccessArticle

Classification of Goat Vocalization via Lightweight Machine Learning and High-Dimensional Acoustic Features

by Daniel Alexander Méndez and Salvador Calvet Sanz

Animals 2026, 16(9), 1394; https://doi.org/10.3390/ani16091394 - 2 May 2026

Cited by 1 | Viewed by 1497

Abstract

Continuous monitoring of livestock vocalizations offers a non-invasive tool for welfare assessment, but deploying current deep learning models in resource-constrained farm environments remains challenging due to high computational demands. This study proposes a feature-based machine learning pipeline optimized for edge computing to classify [...] Read more.

Continuous monitoring of livestock vocalizations offers a non-invasive tool for welfare assessment, but deploying current deep learning models in resource-constrained farm environments remains challenging due to high computational demands. This study proposes a feature-based machine learning pipeline optimized for edge computing to classify caprine vocalizations. Using the VOCAPRA dataset, which comprises 4147 labeled caprine vocalizations categorized into eight distinct welfare states and contexts, a hybrid feature extraction framework was applied to derive 156 spectral, temporal, and bioacoustic descriptors. Dimensionality reduction and a comprehensive comparative screening of 18 algorithms identified the CatBoost Classifier and a Multilayer Perceptron (MLP) as the optimal models. The CatBoost ensemble achieved a robust accuracy of 85.2%, while the optimized MLP reached 87.2% overall accuracy. An edge deployment benchmark revealed that the MLP was the best candidate with for real-time application, featuring a memory footprint of just 0.639 MB and near-instantaneous inference speeds of under 0.005 milliseconds per sample. Furthermore, feature importance and SHAP analyses revealed that mel-frequency cepstral coefficients heavily drove model decisions, particularly for identifying extreme physical distress and maternal reunion. The proposed methodology achieves competitive classification performance while dramatically reducing pre-processing and computational loads compared to image-based deep learning approaches, demonstrating the viability of lightweight-model, energy-efficient, real-time bioacoustic monitoring for precision livestock farming. Full article

(This article belongs to the Special Issue The Application of Artificial Intelligence (AI) in Animal Behavior, Emotion and Health)

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30 pages, 1498 KB

Open AccessArticle

Appropriate Dietary Levels of Soybean Lecithin and Krill Oil Phospholipids Promote Growth, Antioxidant Capacity, and Lipid Metabolism While Reducing Lipid Deposition in Atlantic Salmon (Salmo salar) Fry

by Yuting Zhang, Qingli Gong, Jinghua Chen and Ming Liu

Animals 2026, 16(9), 1393; https://doi.org/10.3390/ani16091393 - 2 May 2026

Cited by 1 | Viewed by 1308

Abstract

This study evaluated the effects of dietary phospholipid (PL) source and supplementation level on growth performance, antioxidant capacity, and lipid metabolism in Atlantic salmon (Salmo salar) fry. A 56-day feeding trial was conducted using a basal diet containing 1.76% PL and [...] Read more.

This study evaluated the effects of dietary phospholipid (PL) source and supplementation level on growth performance, antioxidant capacity, and lipid metabolism in Atlantic salmon (Salmo salar) fry. A 56-day feeding trial was conducted using a basal diet containing 1.76% PL and six experimental diets with an additional 1.5%, 3.0%, or 4.5% PL provided by soybean lecithin (SL) or krill oil phospholipids (KOP). Dietary supplementation with 3.0–4.5% SL and 1.5–4.5% KOP significantly improved growth performance, whereas feed conversion ratio was significantly reduced in the 3.0–4.5% SL and 3.0% KOP groups (p < 0.05). At equivalent inclusion levels, no significant differences were observed between SL and KOP in growth performance parameters (p > 0.05). PL supplementation also reduced whole-body lipid deposition and enhanced visceral lipase activity in all groups except the 1.5% SL group, while antioxidant capacity was improved in all PL-supplemented groups (p < 0.05). SL had no significant effect on whole-body fatty acid composition (p > 0.05), whereas moderate to high levels of KOP significantly altered the fatty acid profile, characterized by reduced monounsaturated fatty acids and n-6 polyunsaturated fatty acids, along with increased eicosapentaenoic acid (EPA) levels (p < 0.05). Transcriptomic analysis indicated that PL supplementation affected hepatic lipid metabolism, with both PL sources downregulating apoa2-like, while KOP induced stronger hepatic transcriptional responses related to lipid utilization and innate immune signaling than SL (p_adj < 0.05). However, gut microbiota analysis revealed no significant differences in the relative abundances of the dominant phyla or in α- and β-diversity among the control, 3.0% KOP, and 4.5% SL groups (p > 0.05). Overall, dietary PL supplementation promoted growth, improved antioxidant capacity, enhanced lipid metabolism, and reduced lipid deposition in Atlantic salmon fry, with KOP exerting stronger effects than SL on fatty acid composition and hepatic gene expression. Full article

(This article belongs to the Special Issue Optimizing Farm-Raised Fish Production: Nutritional Requirements, Precision Feed Formulations, Sustainable Feeding Strategies, and Health and Welfare)

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Animals, Volume 16, Issue 9 (May-1 2026) – 142 articles

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