Semen quality plays a crucial role in bovine in vivo embryo production. This study aimed to compare the effects of 0 °C-refrigerated semen and liquid nitrogen-frozen semen on embryo quality in Simmental cattle. Semen collected from five bulls was equally divided into two groups: one diluted with a 0 °C refrigeration solution and stored at 0 °C, and the other diluted with a cryopreservation solution and stored in liquid nitrogen for 24 h. We evaluated sperm motility, progressive motility (assessed via a computer-assisted sperm analyzer), acrosome integrity, and plasma membrane integrity in both groups. Fifty superovulated Simmental cows were artificially inseminated with semen from both groups. Embryos were non-surgically flushed on day seven, followed by BrdU proliferation staining and TUNEL apoptosis staining. Proliferation and apoptosis levels were quantified using marker genes. Results showed that 0 °C-refrigerated semen exhibited significantly higher sperm motility, progressive motility, acrosome integrity, and plasma membrane integrity compared to liquid nitrogen-frozen semen (p < 0.05). While total embryo numbers showed no significant difference between groups (p ≥ 0.05), embryos from 0 °C-refrigerated semen contained significantly more proliferative cells (p < 0.05) and fewer apoptotic cells (p < 0.05) than those from frozen semen. These findings demonstrate that 0 °C-refrigerated semen outperforms liquid nitrogen-frozen semen in both sperm quality parameters and resultant embryo quality.

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.

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.

(1) Background: The proliferation and differentiation of antler mesenchymal stem cells (MSCs) are vital for antler growth. Our group found that BVES is downregulated during peak antler growth but is highly expressed in mesenchymal tissues, suggesting its role in regulating antler homeostasis. (2) Methods: We manipulated BVES expression in antler MSCs using lentiviral plasmids and siRNA, and then assessed its effects on MSC proliferation via CCK-8 and EdU assays, while also analyzing changes in Wnt pathway gene expression. (3) Results: Overexpression of BVES significantly decreased the CCK-8 OD value of antler MSCs (p < 0.05), and EdU assays revealed a significantly lower cell positivity rate compared to the control group (p < 0.01). In contrast, suppression of BVES resulted in a significant increase in the OD value (p < 0.05) and a significantly higher cell positivity rate than the control group (p < 0.05). Additionally, overexpression of BVES significantly downregulated the expression of key Wnt signaling pathway genes (LRP6, DVL3, β-catenin, c-Myc), while suppression of BVES led to their upregulation. (4) Conclusions: In summary, the BVES gene inhibits the proliferation of antler MSCs by suppressing the Wnt signaling pathway. It may serve as a key gene in maintaining antler MSC homeostasis and regulating antler growth, providing a new perspective for understanding the mechanisms of antler growth.

Background: Chinese Red Steppe cattle (CRS) combine indigenous environmental resilience with moderate dairy performance, whereas Holstein cattle (HOL), despite their high milk yield, suffer reduced genetic diversity and compromised adaptation. A comparative analysis of their population genetic architecture and selection signatures can reveal valuable targets for CRS dairy improvement. Methods: We genotyped 61 CRS and 392 HOL individuals using the Illumina GGP Bovine 100K SNP array and performed stringent quality control. Population structure was assessed via principal component analysis, neighbor-joining trees, and sparse nonnegative matrix factorization. Historical effective population size (Ne) and divergence time were inferred with SMC++. Genome-wide selection scans combined Fixation Index (FST) and Cross-Population Composite Likelihood Ratio test (XP-CLR); overlapping high-confidence regions were annotated and subjected to GO and KEGG enrichment analyses. Results: CRS and HOL were clearly separated along PC1 (explaining 57.48% of variance), with CRS exhibiting high internal homogeneity and weak substructure, versus greater diversity and complex substructure in HOL. SMC++ indicated a split approximately 3500 years ago (700 generations) and a pronounced recent decline in Ne for both breeds. Joint selection mapping identified 767 candidate genes; notably, the ACSM1/2B/3/4 cluster on chromosome 25—key to butanoate metabolism—showed the strongest signal. Enrichment analyses highlighted roles for proteasome function, endoplasmic reticulum stress response, ion homeostasis, and RNA processing in regulating milk fat synthesis and protein secretion. Conclusion: This study delineates the genetic divergence and demographic history of CRS and HOL, and pinpoints core genes and pathways—particularly those governing butanoate metabolism and protein quality control—underlying dairy traits. These findings furnish molecular markers and theoretical guidance for precision breeding and sustainable utilization of Chinese Red Steppe cattle.

This study investigates the recombinant Tarim red deer hepatocyte growth factor (HGF) in a mouse model to develop an HGF/c-Met-based regenerative therapy for alcoholic liver disease. We constructed a recombinant HGF fusion protein and expressed and purified it in Escherichia coli. The recombinant protein was administered via intravenous injection to treat mice with alcoholic liver disease induced by chronic alcohol feeding followed by acute alcohol gavage (NIAAA model). The therapeutic effects were evaluated based on liver tissue histology and biochemical indicators. The recombinant Tarim red deer HGF protein successfully reduced serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in mice, increased serum albumin (ALB) levels, decreased hepatic steatosis and triglyceride (TG) levels, lowered hepatic malondialdehyde (MDA) levels, and increased the levels of the antioxidants glutathione (GSH) and superoxide dismutase (SOD) in the liver. Additionally, it enhanced the proliferation capacity of liver cells, thereby promoting liver regeneration. In conclusion, our study demonstrates that recombinant Tarim red deer HGF effectively reduces liver damage in a mouse model of alcoholic liver disease.