Introduction:Conducting a dry-off period during the late lactation phase in dairy cows can reduce the incidence of clinical mastitis both during the dry period and after subsequent calving. The primary dry-off methods include the application of antibiotics alone or in combination with internal teat sealants. A thorough understanding of the mammary gland microbiota composition before and after dry-off is essential for developing scientifically sound dry-off protocols in practical dairy production. Methods:Five Holstein cows approaching dry-off were selected for this study. The day of calving is designated as Day 0. Milk samples were collected at three time points around the drying period for 16S rRNA gene sequencing to investigate the differences in mammary microbial composition during these stages. Including Group A (-95 to -67 days), Group B (1 to 2 days), and Group C (14 days). Results:The results showed that compared to Group A, the abundance of Firmicutes, Actinobacteria, and Bacteroides, as well as the genera Psychrobacter, Romboutsia, Clostridium sensu stricto 1, Turicibacter, Corynebacterium, Staphylococcus, Streptococcus, and Pseudonocardia (all Gram-positive bacteria) in the milk samples of Group B was significantly lower. In addition, the microbial diversity and richness in the milk samples of Groups A and C exhibited highly significant differences compared to those of Group B (p ≤ 0.001). However, no significant differences were found in the microbial communities of the milk samples between Groups A and C (p > 0.05). Furthermore, the abundance of beneficial bacterial genera such as Lactobacillus was also increased by antibiotic treatment. Conclusion:This exploratory study preliminarily indicates that a single dose of cefapirin benzathine administered via intramammary infusion before drying-off can effectively reduce the abundance of specific mammary pathogens, including Staphylococcus, Streptococcus, and Corynebacterium (all Gram-positive bacteria). Furthermore, the diversity and richness of the mammary microbiota generally recovered approximately 14 days after calving. These findings provide a temporal framework for the reconstruction of the mammary microbiota in dairy cows following the dry period.

Despite the widespread distribution of horses and their close interface with human environments, the equine antibiotic resistance gene (ARG) reservoir remains insufficiently characterized. This study aimed to comprehensively elucidate the equine resistome by analyzing 104 equine fecal metagenomes sampled across China, thereby establishing a critical baseline for antimicrobial stewardship in equine husbandry. Through a non-redundant gene catalog approach, we identified 3,264 unique ARG-related coding sequences (CDSs). To detect distant homologs, hidden Markov model (HMM) profiling was applied, revealing 2,420 putatively novel ARG-like sequences. Functional classification indicated that over 90

Lipid metabolism influences horse athletic performance, yet its genetic basis remains unclear. Here, we investigate the genetic basis of lipid metabolic traits by quantifying key metabolic parameters and sequencing the genomes of 298 horses. We identify a missense mutation (ECA19:9,198,989, G>C) in the BCHE gene that is strongly associated with lipid metabolism, especially butyrylcholinesterase (BChE) activity. The “C” allele is associated with increased BChE activity and reduced intracellular triglyceride levels, hepatic adiposity, and overall fat mass, highlighting a central role for BCHE in lipid hydrolysis and fat storage in horses. This variant evolved neutrally across most of the horse domestication history outside Asia, but underwent strong positive selection in Asia until the 13th century CE. This spatiotemporal divergence suggests that Asian and non-Asian horses experienced distinct selective pressures on lipid metabolism, possibly reflecting differences in management practices, environmental conditions, or energetic demands before the rise of the Great Mongolian Empire.

Fat metabolism plays a pivotal role in determining meat quality and economic value in domestic pigs, yet its underlying genetic mechanisms remain poorly understood. To clarify the molecular basis of adipose deposition, this study integrated whole-genome resequencing data from 226 individuals across 61 Eurasian pig breeds and applied population genetic and selective sweep analyses to identify and validate candidate genes involved in fat metabolism. Significant genetic divergence was observed between Asian and European domestic pigs, with higher linkage disequilibrium (LD) levels in domestic breeds likely driven by artificial selection during domestication. High-fat groups (backfat thickness: 39 mm; fat percentage: 35

Streptococcus agalactiae is an opportunistic Gram-positive pathogen that can infect humans, dairy cattle, and other animals, posing a potential zoonotic risk. In this study, whole-genome sequencing was performed on 40 GBS strains isolated from dairy cows. The results showed high homology among strains collected from the same farm. Based on these findings, two representative dairy cattle-derived strains and one human-derived strain were selected for comparative whole-genome analysis to identify differences in antibiotic resistance and virulence genes. Additionally, we characterized the antibiotic resistance phenotypes, hemolytic phenotypes, invasion capacity, and the levels of oxidative stress markers and inflammatory factors in bovine mammary epithelial cells (BMECs) during infection for all three strains. The three strains HB27, HB31, and RW exhibited close phylogenetic relationships. All showed strong resistance to sulfonamide and aminoglycoside antibiotics but remained susceptible to cephalosporins, quinolones, and macrolides. The mefA gene was present in RW but absent in HB27 and HB31. RW exhibited higher hemolytic activity and cylE mRNA expression levels than HB27 and HB31, and the pilA and pilB genes were also present only in RW. HB31 demonstrated significantly faster invasion rates than HB27 and RW before the 2‑h invasion time point. The effects of the three strains on antioxidant markers and the protein and mRNA expression of inflammatory factors in BMECs were consistent, indicating that the human-derived S. agalactiae possesses the capacity to infect BMECs. Although human- and dairy cattle-derived GBS differ in virulence-associated genes and hemolytic activity, the human strain invades BMECs in vitro and induces host responses similar to those of dairy cattle strains. These findings suggest that human-derived GBS may have the potential for cross-species infection. To date, most studies on human- and dairy cattle-derived GBS have focused primarily on epidemiology. Even when addressing whether GBS is a zoonotic pathogen, direct evidence of cross-species transmission mechanisms remains limited. This study provides in vitro evidence that human-derived GBS can directly infect BMECs. However, whether cross-species transmission between human- and dairy cattle-derived GBS requires repeated exposure or can occur following a single exposure requires further investigation.

Cattle are integral to global food security, yet the molecular architecture of their complex traits remains poorly understood. Here, we present the Cattle Genotype–Tissue Expression (CattleG-TEx) Phase 1 resource (https://cattlegtex.farmgtex.org/), a substantial expansion of the pilot study. By leveraging 12,422 RNA-seq profiles across 43 tissues and 82 breeds, we characterized 433,972 primary and 161,428 non-primary regulatory effects spanning seven molecular phenotypes. This high-resolution atlas resolves 75% of GWAS signals for 44 complex traits, significantly addressing the "missing regulation" in livestock. We propose a genetic regulatory model demonstrating how variants across multiple biological layers interact with specific biological contexts to shape pheno-typic variation. Furthermore, CattleGTEx elucidates mechanisms underlying adaptive evolution between Bos taurus and Bos indicus, as well as artificial selection in dairy and beef breeds. Finally, by mapping evolutionary constraints on these regulatory effects, we demonstrate the translational value of this resource for prioritizing causal variants in human complex diseases. Together, Phase 1 of CattleGTEx provides a transformative framework for functional genomics, precision breeding, and comparative genetics.

The selection of horses has been instrumental in shaping long-range mobility, advancing warfare strategies, and diversifying equine phenotypes. However, the structural variants (SVs) favored by this process remain largely unexplored. Utilizing high-fidelity Pacific Biosciences sequencing, we assembled three high-quality horse genomes, incorporating 237 Mb (9.48%) of novel sequences relative to the existing reference genome. These assemblies achieved an average Benchmarking Universal Single-Copy Orthologs completeness of 95.1%. By employing a graph-based approach, we genotyped 23,163 high-confidence, non-redundant SVs, which include 11,714 insertions and 11,449 deletions, across 390 horse genomes representing 61 breeds worldwide. Our findings provide evolutionary insights into the origins of diverse horse populations, through both SVs and single nucleotide polymorphisms. Notably, we identified a 273-bp deletion in the THSD7A gene and a 98-bp long terminal repeat insertion in the OPCML gene, both of which are significantly associated with racing ability and body index in horses (P = 3.87E-05 and 4.08E-05). These genes are linked to bone metabolism and racing ability, respectively. Luciferase assays demonstrated the regulatory potential of these sequences, showing they can significantly modulate the transcriptional activity of their associated genes. In summary, our findings highlight the potential of SVs as genetic markers and functional elements that shape equine phenotypes.

Skeletal muscle development not only determines the growth performance and meat quality of livestock, but is also closely associated with human metabolic health. ZBED6 has been identified as a transcriptional repressor of IGF2, yet its role and molecular mechanism in skeletal muscle development remain unclear. In this study, we employed a ZBED6 knockout (KO) Bama pig model and found that loss of ZBED6 led to increased muscle mass and muscle fiber hypertrophy, suggesting that ZBED6 plays an inhibitory role in muscle growth. To further elucidate the underlying mechanisms, we performed integrated transcriptomic and functional analyses to investigate the ZBED6-CDKN1A axis. RNA sequencing identified differentially expressed genes across seven tissues from 8-month-old KO and wild-type pigs, and WGCNA revealed a skeletal muscle-specific module enriched in myogenic pathways. By integrating this module with transcriptome data from longissimus dorsi at 5 and 8 months of age and ChIP-seq data, CDKN1A was identified as a central hub gene. Dual-luciferase reporter assays demonstrated that ZBED6 binds to a conserved GCTCG motif in the CDKN1A promoters of pigs and mice to repress its transcription. Consistently, in C2C12 cells, ZBED6 knockdown upregulated the expression of myogenic markers (MyoD, MyoG, and MyHC) and promoted myotube formation, whereas silencing CDKN1A attenuated this effect. Conversely, ZBED6 overexpression reduced CDKN1A expression and suppressed differentiation, confirming its bidirectional regulatory role. Collectively, our data identify CDKN1A as a transcriptional target of ZBED6 and show that ZBED6 constrains skeletal muscle development by repressing CDKN1A.

The growth and maturation of skeletal muscle have a significant influence on the meat quality and quantity. N6-methyladenosine (m6A) is a dynamic and reversible modification that predominantly affects RNA stability, splicing, translation, nuclear export, and so on. M6A has been reported to be involved in the regulation of myogenesis. However, the mechanisms determining m6A modification in ovine skeletal muscle growth and development are still not well understood. We revealed the landscape of m6A modification in the skeletal muscle of the 70 day-old embryos (F70) and 40 day-old lambs (D40) using m6A-MeRIP-seq. In addition, we found that the m6A modification level of the membrane-associated guanylate kinase, WW, and PDZ domain containing 3 (MAGI3) gene significantly changed during skeletal muscle development. Further experimental verification showed that MAGI3 facilitated the proliferation and differentiation of ovine skeletal muscle satellite cells and C2C12 in vitro. Notably, YTHDC1 acts as an m6A reader to promote the degradation of the MAGI3 mRNA, thereby regulating myogenesis. These findings provide a comprehensive transcriptome profile of m6A methylation in ovine muscle at two key developmental stages. Additionally, MAGI3 modulated myogenesis in an m6A-YTHDC1-dependent manner.

Accurate breed identification serves is a crucial cornerstone for the conservation and utilization of livestock and poultry genetic resources. The identification of breeds based on a variety of information sources and analytical methods has been extensively applied in the domain of animal genetics and breeding. Recently, the integration of large-scale genomic data with machine learning has become increasingly prevalent for breed identification tasks. However, such projects typically require extensive sequencing data and expertise in bioinformatics. To address this, we introduce rPIMS, a comprehensive tool designed to simplify breed identification and genetic analysis. With intuitive modules for data input, dimensionality reduction, phylogenetic tree construction, population structure analysis, and machine learning-based classification, rPIMS has the capacity to streamlines the analytical process for researchers. It promotes collaboration, facilitates efficient data sharing, and enhances the ability to identify and report genetic diversity and evolutionary relationships among livestock breeds. We performed a validation analysis to confirm that rPIMS achieved 100% classification accuracy in distinguishing 10 breeds using only 860 SNPs. In summary, rPIMS significantly simplifies complex model-building processes, making breed classification and genetic structure visualization accessible and intuitive to users. rPIMS is a Shiny R application designed for breed identification in livestock using genomic data and machine learning, accessible through an intuitive graphical user interface. It is freely available under the GNU Public License on GitHub: https://github.com/Werewolfzy/rPIMS.

Genetic mutation and drift, coupled with natural and human-mediated selection and migration, have produced a wide variety of genotypes and phenotypes in farmed animals. We here introduce the Farm Animal Genotype-Tissue Expression (FarmGTEx) Project, which aims to elucidate the genetic determinants of gene expression across 16 terrestrial and aquatic domestic species under diverse biological and environmental contexts. For each species, we aim to collect multiomics data, particularly genomics and transcriptomics, from 50 tissues of 1,000 healthy adults and 200 additional animals representing a specific context. This Perspective provides an overview of the priorities of FarmGTEx and advocates for coordinated strategies of data analysis and resource-sharing initiatives. FarmGTEx aims to serve as a platform for investigating context-specific regulatory effects, which will deepen our understanding of molecular mechanisms underlying complex phenotypes. The knowledge and insights provided by FarmGTEx will contribute to improving sustainable agriculture-based food systems, comparative biology and eventual human biomedicine.

Wool curvature is one of the most valuable characteristics of Zhongwei goat fur. As the goats grow, the curvature progressively diminishes, which has a substantial impact on the quality and market value of wool. Circular RNAs (circRNAs) are a class of noncoding RNA and play vital roles in animal growth and development. However, the expression and function of circRNAs in the wool curvature of Zhongwei goats are unclear. In our study, we conducted circRNA expression profiling of Zhongwei goat skin exhibiting divergent curvature wool phenotypes at two developmental stages using the RNA-seq. In total, 12,682 circRNAs and 158 differentially expressed circRNAs (DE circRNAs) were identified. KEGG analysis illustrated that host genes of DE circRNAs were significantly enriched in the signaling pathways of Ras, JAK/STAT5, and cAMP, which might affect wool curvature. We further validated five circRNAs using qRT-PCR, which were consistent with the sequencing results. Functional verification assay demonstrated that circRNA8782 regulated fibroblast proliferation. In addition, we constructed a regulatory competing endogenous RNA (ceRNA) network and predicted circRNA3173-miR-16b-5p-IGF1 axes involved in the regulation of wool curvature. Our result will provide the foundation for uncovering the regulatory mechanisms of underlying wool curvature patterns in goats.

Characterizing the impact of genomic variants on genome function and ultimately complex traits in livestock is essential for the development of sustainable precision agriculture and comparative genomics. Here, as part of the Farm animal Genotype-Tissue Expression (FarmGTEx) project, we present the pilot phase of the SheepGTEx resource through analyzing 6,761 RNA-sequencing samples of 51 primary tissues in a multi-breed population of sheep. We identify millions of regulatory variants associated with seven types of molecular phenotypes, and fine-map 322,467 primary and 113,968 non-primary effects, revealing a high degree of regulatory allelic heterogeneity. We systematically characterize the pleiotropic effects of these variants on molecular phenotypes, assess their context-specific regulatory patterns across tissues, breeds, sexes, and developmental stages, as well as explore their evolutionary constraints across mammals. Finally, we demonstrate the substantial potential of the SheepGTEx resource (https://sheepgtex.farmgtex.org), by providing examples of regulatory mechanisms underpinning 34 complex traits, population divergence between European and Asian breeds, and adaptive evolution in sheep over the past ten millennia.

Livestock germplasm resources are a strategic foundation for sustainable agricultural development, food security, and biodiversity conservation. Their security status directly influences the development of the livestock industry and the safety of the national seed sector. To systematically assess the current status and challenges of livestock germplasm conservation in China, as well as to enhance China's conservation capacities, an in-depth comparative analysis from a global perspective is urgently needed. Based on domestic and international databases and policy documents, and drawing on the results of the third national census of livestock germplasm resources, this study reviews the multiple values of China's livestock germplasm resources across economic, breeding, cultural, ecological, medical, and social dimensions. It compares conservation systems in China and other countries in terms of breed diversity, endangered status, and the current state of in situ and ex situin vivo conservation, unveiling global trends in livestock germplasm resources conservation. The results indicate that although China possesses abundant livestock germplasm resources and a conservation system of an initial scale, significant gaps remain in diversity monitoring, conservation coverage, and technical support compared with advanced international standards. Based on these findings, the study proposes recommendations for future conservation efforts in China. These include developing a population dynamics monitoring and evaluation system, improving the in situ and ex situin vivo conservation framework, and strengthening the development of critical technologies such as cryopreservation, thus to enhance the conservation coverage and resource utilization efficiency. This study provides theoretical support and policy guidance for understanding global trends in livestock resource conservation and advancing the high-quality construction and operation of China's conservation system.

Technological advancements and decrease of costs of whole-genome sequencing approaches has made available a huge and ever increasing amount of resequencing data for many species. It is thus now possible to assemble large sized datasets encompassing the molecular variation of several species and/or populations or breeds. Nonetheless, these datasets can be extremely variable in terms of geographical provenance and sample sizes, with taxonomic groups varying from hundreds to just a few or even one single entry. In such circumstances, the application of standard filtering approaches may lead to the introduction of biases and to the under/over representation of some groups or gene pools. Commonly adopted variant filtering approaches relying on Minor Allele Frequency (MAF) and Linkage Disequilibrium (LD) may not be suitable to treat datasets representing broadscale diversity of multiple species, due to remarkable differences in LD structure and in the frequency of variants at the local vs. global scale. Thus, by exploiting the VarGoats 1000 goat genome project data as an optimal case study, we devised a novel approach based on within-population subsampling, Minor Allele Count (MAC) and marker spacing (bp-space), specifically designed to avoid biases introduced by standard filtering procedures and to adequately represent continental and species-specific variation. Starting from a quality-filtered dataset of >28M SNPs from 1372 animals, we obtained a dataset of <14M markers and 750 individuals, complying with the initial requirements and more handy for further computational steps. The dataset was validated by PCA, Neighbor Joining and Admixture analyses. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND:Effective treatments for skeletal muscle atrophy, a debilitating condition linked to ageing and glucocorticoid therapy, remain lacking. Zinc finger BED-type containing 6 (ZBED6), a transcriptional repressor, enhances muscle growth and protects against sepsis-induced atrophy, but its role in ageing- and dexamethasone (Dex)-induced muscle atrophy remains unknown. This study investigated the protective role of ZBED6 knockout (KO) against muscle atrophy through the Dkk3-Fbxo32 pathway. METHODS:The muscle mass, ratio and myofibrillar morphology of 5-day-old (wild-type (WT): KO, n = 5:3), 5-month-old (n = 8:9) and 8-month-old (n = 3:3) ZBED6-KO pigs and 18-month-old mice (n = 3:3) were analysed. A model of Dex-induced muscle atrophy was established using 3-month-old mice (n = 6:6) via intraperitoneal injections (15 mg/kg/day for 10 days). C2C12 myotubes were treated with 100 μM Dex for 24 h. Muscle morphology was analysed through H&E and immunofluorescence staining. Gene expression was assessed through RNA-seq, qRT-PCR and western blotting. The downstream targets were identified through ChIP-seq using anti-ZBED6 antibodies and RNA-seq analysis of the gastrocnemius muscle from ZBED6-KO and WT pigs. Dkk3 was overexpressed by injecting AAV9-myo2A-Dkk3 (2 × 1011) into the tibialis anterior muscle of 3-month-old ZBED6-KO mice (n = 4), which were harvested 1 month postinjection. ZBED6-KO C2C12 cells were generated via CRISPR/Cas9 and treated with Dex to assess the effects on myotube diameter and gene expression. RESULTS:The muscle mass and muscle-to-carcass ratio in ZBED6-KO pigs increased by 27% and 12%, respectively (p < 0.05), while the Dkk3-Fbxo32 pathway was suppressed by 50% (p < 0.01). ChIP-seq/RNA-seq identified Dkk3 as the most significant ZBED6 target (log2FC = -3.38, p < 0.01). The myofibrillar cross-sectional areas (CSAs) increased twofold in aged ZBED6-KO mice, while fibrosis and the Dkk3-Fbxo32 pathway were suppressed by 76% and 50%, respectively (all p < 0.01). Dkk3 overexpression reduced the tibialis anterior muscle weight and CSA in ZBED6-KO mice by 31% and 61%, respectively (p < 0.01). Dex reduced the CSA in WT mice (45%, p < 0.01), but ZBED6-KO mice resisted atrophy (CSA similar to untreated WT). ZBED6-KO increased myotube diameter by twofold (p < 0.01) and inhibited the activation of the Dkk3-Fbxo32 pathway (p < 0.01). Conversely, Zbed6 overexpression reduced the CSA and myotube diameter by 32% and 64%, respectively (p < 0.01) and rescued by Dkk3 silencing (50% recovery, p < 0.01). CONCLUSIONS:ZBED6 depletion mitigates ageing- and Dex-induced muscle atrophy via the Dkk3-Fbxo32 axis, highlighting its therapeutic potential.

Horsepower revolutionized human history through enhanced mobility, transport, and warfare. However, the suite of biological traits that reshaped horses during domestication remains unclear. We scanned an extensive horse genome time series for selection signatures at 266 markers associated with key traits. We detected a signature of positive selection at ZFPM1 —known to be a modulator of behavior in mice—occurring ~5000 years ago (ya), suggesting that taming was one of the earliest steps toward domestication of horses. Intensive selection at GSDMC began ~4750 ya with the domestication bottleneck, leading regulatory variants to high frequency by ~4150 ya. GSDMC genotypes are linked to body conformation in horses and to spinal anatomy, motor coordination, and muscular strength in mice. Our results suggest that selection on standing variation at GSDMC was crucial for the emergence of horses that could facilitate fast mobility in human societies ~4200 ya.

Global geographical, climatic, and ecological diversity has given rise to a wealth of domestic animals, which are essential for food security and agricultural sustainability. Since the 1960s, these critical genetic resources have declined significantly due to overdevelopment, ecological degradation, and climate change, posing a serious threat to global food security. In the face of these challenges, we emphasize the critical importance of promoting indigenous livestock and poultry germplasm resources in biodiversity conservation to enhance the adaptability and resilience of agricultural systems. To promote the sustainable management and conservation of genetic resources, a multistakeholder international cooperation framework is needed. Globally, many national and international institutions have initiated a variety of conservation measures, legislation, and technical strategies. In particular, genebanks play an indispensable role in the conservation of important livestock and poultry genetic resources. These banks not only aid in maintaining biodiversity but also provide valuable genetic material for future breeding programmes and scientific research. Through systematic collection, conservation and evaluation, genebanks ensure the long-term availability and sustainable use of genetic resources and provide an important foundation for addressing global environmental change and agricultural challenges.

BACKGROUND:Sheep (Ovis aries) are economically important agricultural animals, which provide meat, wool, and fur for humans. The growth and development of embryonic skeletal muscle are crucial for ovine muscle quality and yield. Increasing evidence has shown the three-dimensional (3D) genome structure is involved in gene transcriptional regulation and a variety of biological processes. Although the 3D genome structure in various species and cell types has been characterized, the dynamic remodeling of chromatin architecture during ovine skeletal muscle development remain poorly understood. RESULTS:Using Hi-C, RNA-seq, and ChIP-seq methods, we systematically explored the dynamics of the 3D genome structure, transcriptome, and epigenome in ovine skeletal muscle tissue from 90-day-old (D90) and 120-day-old fetuses (D120). Compared to the D90, the D120 exhibited a decrease in slow muscle fibers and an increase in fast muscle fibers. Additionally, we observed significant reorganization in chromatin compartments, topologically associating domains (TADs), and loop structures during muscle development. Notably, 18.07 % of A/B compartments switched, with A compartments transitioning to B compartments (9.09 %) and B compartments shifting to A compartments (8.98 %). The number of TADs was slightly lower in D120 (6937) compared to D90 (7036). Additionally, the number of loops increased from 7201 in D90 to 10,008 in D120. We also identified distal regulatory elements of SOX6 and PIK3R1, which play crucial roles in regulating sheep muscle development through loop structures. These findings provide valuable insights into the 3D genome architecture and its role in muscle development. CONCLUSIONS:Our study provides the dynamic 3D chromatin structure of ovine middle and late fetuses skeletal muscle and revealed 3D genome structure is involved in gene expression of fetus skeletal muscle during development. In addition, we identified several potential cis-regulatory elements that regulate gene expression through long range chromatin interactions. We present the first 3D genome data for ovine fetuses skeletal muscle, providing valuable information for epigenome studies. This study offers novel insights into the regulatory mechanism underlying growth and development of ovine muscles.