Objective: This study aimed to assess the impact of the dilution ratio of Tris diluent, storage at 0°C, and long-distance transportation on the spermatozoa of Simmental cattle. It also validated the feasibility of the regional distribution of fresh semen.Methods: In experiment 1, semen was diluted at four dilution ratios (1:6, 1:9, 1:12, and 1:15) to determine the optimal dilution ratio of Tris diluent. In experiment 2, we assessed sperm viability, progressive motility (objectively assessed by computer-assisted sperm analyzer), and acrosome intactness in Tris dilutions kept at constant 0°C for 1, 3, 6, 9, and 12 days. We compared them to Tianshan livestock dilutions (Commercial diluent). In experiment 3, semen was diluted using Tris diluent, and sperm quality was measured before and after long-distance transport. Artificial insemination of 177 Simmental heifers compared to 156 using Tianshan Livestock dilution.Results: The outcomes demonstrated that 1:9 was the ideal Tris diluent dilution ratio. The sperm viability, Progressive Motility, and acrosome integrity of both Tris and Tianshan dilutions preserved at 0°C gradually decreased over time. sperm viability was above 50% for both dilutions on d 9, with a flat rate of decline. The decrease in acrosome integrity rate was faster for Tianshan livestock dilutions than for Tris dilutions when stored at 0°C for 1 to 6 days. There was no significant difference (p>0.05) in sperm viability between semen preserved in Tris diluent after long-distance transportation and semen preserved in resting condition. The conception rates for Tris dilution and Tianshan livestock dilution were 49.15% and 46.15% respectively, with no significant difference (p>0.05).Conclusion: This shows that Tris diluent is a good long-term protectant. It has been observed that fresh semen can be successfully preserved for long-distance transport when stored under 0°C conditions. Additionally, it is feasible to distribute semen regionally.

This study investigates the genetic structure of Belgian Red (BR), Belgian Red and White (BWR), and Qinchuan (QinC) cattle, with a focus on identifying genes associated with reproductive functions, growth, and development. A total of 270 Belgian cattle (91 BR and 179 BWR) and 286 Qinchuan cattle were genotyped using the Illumina Bovine SNP 50K microarray. Data analysis was conducted using PLINK and Beagle 5.1 to estimate linkage disequilibrium (LD) and effective population size (Ne). Candidate SNP loci were identified by selecting the top 5% based on the weighted fixation index (Fst) and nucleotide diversity (θπ ratio), followed by gene annotation. The analysis revealed 160 candidate genes under selection between Qinchuan and Belgian Red cattle, and 98 candidate genes between Qinchuan and Belgian Red and White cattle. Key genes associated with reproductive functions, including NFKBIA, PTHLH, UGT2B10, TRPC4, and ALOX5AP, were identified. Additionally, genes involved in growth and muscle development were highlighted, particularly those influencing protein synthesis, fatty acid metabolism, and collagen synthesis. These findings provide valuable molecular markers for enhancing reproductive efficiency, growth, and meat production through genetic selection and selective breeding strategies.

Zygotic genome activation (ZGA) marks the critical transition from reliance on maternal transcripts to the initiation of embryonic transcription early in development. Despite extensive characterization in model species, the regulatory framework of ZGA in sheep remains poorly defined. Here, we applied single-cell RNA sequencing (Smart-seq2) to in vivo- and in vitro-derived sheep embryos at the 8-, 16-, and 32-cell stages. Differential expression analysis revealed 114, 1628, and 1465 genes altered in the 8- vs. 16-, 16- vs. 32-, and 8- vs. 32-cell transitions, respectively, with the core pluripotency factors SOX2, NANOG, POU5F1, and KLF4 upregulated during major ZGA. To uncover coordinated regulatory modules, we constructed a weighted gene co-expression network using WGCNA, identifying the MEred module as most tightly correlated with developmental progression (r = 0.48, p = 8.6 × 10⁻¹⁴). The integration of MERed genes into the STRING v11 protein–protein interaction network furnished a high-confidence scaffold for community detection. Louvain partitioning delineated two discrete communities: Community 0 was enriched in ER–Golgi vesicle-mediated transport, transmembrane transport, and cytoskeletal dynamics, suggesting roles in membrane protein processing, secretion, and early signaling; Community 1 was enriched in G2/M cell-cycle transition and RNA splicing/processing, indicating a coordinated network for accurate post-ZGA cell division and transcript maturation. Together, these integrated analyses reveal a modular regulatory architecture underlying sheep ZGA and provide a framework for dissecting early embryonic development in this species.

Objective: The X/Y sperm separation technique plays a crucial role in gender control. The objective of this experiment is to investigate the effect of polyvinylpyrrolidone (PVP) concentration (A: 0%, B: 1%, C: 3%, D: 5%, E: 7%, vol/vol) on Y sperm sorting efficiency, based on the specific binding of Resiquimod (R848) to toll-like receptor (TLR)7/8 receptors on the tail of X sperm.Methods: The different concentrations of PVP were added to the R848 sperm sorting solution to facilitate the separation of Y sperm. Subsequently, the isolated sperm were subjected to quantification and motility assessment using computer-assisted semen analysis system. The X/Y sperm ratio is then analyzed by flow cytometry. The sorted sperm were evaluated for acrosomal and plasma membrane integrity. The spermatozoa were then subjected to immunofluorescent staining through immunofluorescence (IF) techniques, which preceded the quantification of the negative sperm rate. The proportion of male embryos was determined through embryonic sex identification after in vitro fertilization.Results: Flow cytometry analysis results showed that when the PVP concentrations were 3%, 5% and 7%, the proportion of Y sperm was not statistically significant, (p≥0.05). However, these percentages were significantly elevated compared to those obtained with 0% and 1% PVP concentrations (p<0.05). The IF staining results demonstrated that the proportion of TLR7/8-negative sperm remained statistically unchanged across PVP concentrations of 3%, 5%, and 7% (p≥0.05). However, these percentages were significantly elevated compared to those obtained with 0% and 1% PVP concentrations (p<0.05). The generation of male blastocysts was significantly higher at a PVP concentration of 3% compared to 0% and 1% (p<0.05), but showed no significant difference from 5% and 7% (p≥0.05).Conclusion: Selecting a 3% PVP concentration not only ensures sufficient sperm yield but also promotes effective selection of Y-sperm. These findings provide empirical evidence supporting the high-efficiency separation of X/Y sperm in livestock.

The maturation of oocytes is a critical step in mammalian reproduction, involving dynamic regulation of gene expression. Therefore, investigating how gene expression varies during different stages of oocyte maturation is highly important. This study employed single-cell RNA sequencing (scRNA-seq) to analyze bovine oocytes at the germinal vesicle (GV) and metaphase II (MII) stages. The results identified 1787 differentially expressed genes (DEGs) between the two stages, with 1556 genes upregulated and 231 downregulated in the GV stage. Further investigation using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that the upregulated genes are mainly involved in mitochondrial functions and energy metabolism, whereas the downregulated genes are primarily associated with signaling pathways. Validation through RT-qPCR confirmed that COA4, TKT and GPX4 were significantly higher in GV-stage oocytes, while ISG15, MAP1LC3C and ZEB2 were notably downregulated. This study highlights significant gene expression differences between GV and MII bovine oocytes, underscoring the vital roles of genes related to energy metabolism and signaling during oocyte maturation. The expression patterns of these genes provide important molecular markers for further elucidating the mechanisms underlying oocyte maturation.