Kai Hu

Semen diluents are crucial for the effective utilization of male germplasm resources and for enhancing the efficiency of artificial insemination (AI). However, reports on the in vitro low-temperature preservation of semen are still limited. This study comprised three separate experiments. Experiment 1 (semen storage): An ex vivo experiment using one ejaculate from each of five bulls, split into two aliquots per ejaculate, stored at 0 °C for 15 days, with repeated assessments of sperm quality (n = 5 biological replicates). Experiment 2 (IVF): An in vitro fertilization study using slaughterhouse-derived oocytes fertilized with day-15 stored semen from Experiment 1, conducted in five independent sessions (n = 5 sessions). Experiment 3 (AI): A non-randomized interventional field study involving 400 synchronized cows at China Hualing Ranch, allocated upon detection of estrus to receive either treatment or control semen (n = 400 cows). The primary outcome for Experiment 1 was total motility at day 15; for Experiment 2, blastocyst formation rate per oocyte; for Experiment 3, pregnancy rate at day 50 post-AI among all allocated cows. These findings suggest that the treatment formulation may offer advantages for liquid storage of bull semen at 0 °C.

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: 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.

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.

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.