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.

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.

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.

The Lop sheep (LOP), a unique local breed from Xinjiang, exhibits remarkable resilience to the harsh conditions of a desert arid climate and frequent sandstorms, alongside notable fecundity characteristics. This study aims to investigate the adaptability of LOP within this challenging environment by collecting whole blood samples from 110 LOP individuals in the Lop Nur region of Xinjiang for genome resequencing. The resulting data will be compared with whole genome resequencing information from 22 local sheep breeds worldwide to analyze the origin and evolution of LOP. Additionally, comparisons will be made with HUS sheep from warm and humid regions to identify genomic differences through selection signal analysis, thereby assessing the impact of a desert arid climate on the extreme living conditions of LOP. Finally, qPCR was used to preliminarily analyse the impact of the desert arid climate on the genome of the Bactrian sheep. Genetic diversity results indicate that LOP exhibits a relatively stable genetic structure alongside high genetic diversity. The results of population structure analysis and gene flow indicate that we can tentatively posit that LOP is a breed that originated from the Middle East, subsequently mixing with MGS upon its arrival in Xinjiang. Chinese local sheep breeds trace their origins to AMS, with the gene flow evolving from west to east, progressing through mountainous hills (BSBS), basins (LOP, HTS, CLHS, DLS), plains (MGS, TANS), and coastal areas (HUS). LOP is associated with ALTS, BSBS, HTS, CLHS, and DLS, as well as with MGS, HUS, TANS, WDS, and SSSP, in a context of gene exchange, with the degree of exchange diminishing in that order. Selection signal analysis revealed that the candidate genes identified are closely related to adaptation to desert arid climates and disease resistance (PDGFD, NDUFS3, ATP1B2, ITGB8, and CD79A), using HUS as the reference group. qPCR results demonstrated that LOP was significantly upregulated in cardiac, splenic, and lung tissues compared to HUS, suggesting that LOP plays a crucial role in cardiac function, immune response, and respiratory capacity.

Objective: Semen quality plays a crucial role in embryo formation in cattle. This study aims to compare the effects of 0°C refrigerated semen and liquid nitrogen-frozen semen on the quality of in vitro fertilized embryos in Simmental cattle.Methods: Semen from six bulls was devidedt equally into two groups. One was diluted with cold storage solution and stored at 0°C for 72 h; the other with cryopreservation solution and preserved in liquid nitrogen for 72 h. Post-storage, sperm quality (motility, progressive motility via computer-assisted sperm analysis, and acrosome/plasma membrane integrity) was assessed, along with malondialdehyde, reactive oxygen species, adenosine triphosphate, and mitochondrial membrane potential levels. For in vitro fertilisation (IVF), oocytes from Simmental cows (>50 per group) were inseminated with the two semen groups. At 45 h post-IVF, embryo cleavage rate was recorded; on day 8, blastocyst rate was determined. Embryos then underwent BrdU staining (proliferation) and TUNEL staining (apoptosis), with proliferation and apoptosis levels quantified via marker genes.Results: The results showed that compared with liquid nitrogen-frozen semen, 0°C refrigerated semen exhibited significantly higher sperm motility, forward motility rate, acrosome integrity, and plasma membrane integrity (p<0.05). in vitro fertilized embryos also exhibited significant increases in quality and quantity (cleavage rate and blastocyst rate) (p<0.05), and embryos derived from 0°C refrigerated semen contained significantly more proliferating cells (p<0.05) and fewer apoptotic cells (p<0.05) than those from frozen semen.Conclusion: These findings suggest that 0°C refrigerated semen is superior to liquid nitrogen frozen semen in terms of sperm quality parameters and in vitro embryo development.