Extreme temperature during summer may lead to heat stress in cattle and compromise their productivity. It also poses detrimental impacts on the developmental capacity of bovine budding oocytes, which halt their fertility. To mitigate the adverse effects of heat stress, it is necessary to investigate the mechanisms through which it affects the developmental capacity of oocytes. The primary goal of this study was to investigate the impact of heat stress on the epigenetic modifications in bovine oocytes and embryos, as well as on oocyte developmental capacity, reactive oxygen species, mitochondrial membrane potential, apoptosis, transzonal projections, and gene expression levels. Our results showed that heat stress significantly reduced the expression levels of the epigenetic modifications from histone H1, histone H2A, histone H2B, histone H4, DNA methylation, and DNA hydroxymethylation at all stages of the oocyte and embryo. Similarly, heat stress significantly reduced cleavage rate, blastocyst rate, oocyte mitochondrial-membrane potential level, adenosine-triphosphate (ATP) level, mitochondrial DNA copy number, and transzonal projection level. It was also found that heat stress affected mitochondrial distribution in oocytes and significantly increased reactive oxygen species, apoptosis levels and mitochondrial autophagy levels. Our findings suggest that heat stress significantly impacts the expression levels of genes related to oocyte developmental ability, the cytoskeleton, mitochondrial function, and epigenetic modification, lowering their competence during the summer season.

Oocyte vitrification is crucial for livestock reproduction, germplasm conservation, and human-assisted reproduction, but the overabundance of lipids is highly detrimental to oocyte development. It is necessary to reduce the lipid droplet content of oocytes before cryopreservation. This study analyzed the impact of β-nicotinamide mononucleotide (NMN), berberine (BER), or cordycepin (COR) on various aspects of bovine oocytes, including lipid droplet content and the expression levels of genes related to lipid synthesis in bovine oocytes, development ability, reactive oxygen species (ROS), apoptosis, and the expression levels of genes associated with endoplasmic reticulum (ER) stress, and mitochondrial function in vitrified bovine oocytes. The results of our study indicated that 1 μM NMN, 2.5 μM BER, and 1 μM COR were effective in reducing the lipid droplet content and suppressing the expression levels of genes involved in lipid synthesis in bovine oocytes. Our findings showed that the vitrified bovine oocytes treated with 1 μM of NMN had a significantly higher survival rate and better development ability compared to the other vitrified groups. Additionally, 1 μM NMN, 2.5 μM BER, and 1 μM COR decreased the levels of ROS and apoptosis, decreased the mRNA expression levels of genes involved in ER stress and mitochondrial fission but increased the mRNA expression levels of genes associated with mitochondrial fusion in the vitrified bovine oocytes. Our study results suggested that 1 μM NMN, 2.5 μM BER, and 1 μM COR effectively decreased the lipid droplet content and enhanced the development ability of vitrified bovine oocytes by lowering ROS levels, reducing ER stress, regulating mitochondrial function, and inhibiting apoptosis. Furthermore, the results showed that 1 μM NMN was more effective than 2.5 μM BER and 1 μM COR.

The improvement of in vitro embryo development is a gateway to enhance the output of assisted reproductive technologies. The Wnt and Hippo signaling pathways are crucial for the early development of bovine embryos. This study investigated the development of bovine embryos under the influence of a Hippo signaling agonist (LPA) and a Wnt signaling inhibitor (DKK1). In this current study, embryos produced in vitro were cultured in media supplemented with LPA and DKK1. We comprehensively analyzed the impact of LPA and DKK1 on various developmental parameters of the bovine embryo, such as blastocyst formation, differential cell counts, YAP fluorescence intensity and apoptosis rate. Furthermore, single-cell RNA sequencing (scRNA-seq) was employed to elucidate the in vitro embryonic development. Our results revealed that LPA and DKK1 improved the blastocyst developmental potential, total cells, trophectoderm (TE) cells and YAP fluorescence intensity and decreased the apoptosis rate of bovine embryos. A total of 1203 genes exhibited differential expression between the control and LPA/DKK1-treated (LD) groups, with 577 genes upregulated and 626 genes downregulated. KEGG pathway analysis revealed significant enrichment of differentially expressed genes (DEGs) associated with TGF-beta signaling, Wnt signaling, apoptosis, Hippo signaling and other critical developmental pathways. Our study shows the role of LPA and DKK1 in embryonic differentiation and embryo establishment of pregnancy. These findings should be helpful for further unraveling the precise contributions of the Hippo and Wnt pathways in bovine trophoblast formation, thus advancing our comprehension of early bovine embryo development.

Oocyte maturation is a critical step in the completion of female gametogenesis in the ovary; thus, for subsequent fertilization and embryogenesis. Vitrification of embryo also has been shown to be closely associated with oocyte maturation. To improve the quality and developmental potential of bovine oocytes derived from in vitro maturation (IVM), Pre-IVM with C-type natriuretic peptide (CNP), melatonin (MT) and in combination, IGF1, FGF2, LIF (FLI) were supplemented in the IVM medium. In this current study, we cultured bovine oocytes in Pre-IVM with CNP for 6 h before transferring them to the IVM medium supplemented with MT and FLI. The developmental potential of bovine oocytes was then investigated by measuring the reactive oxygen species (ROS), the intracellular glutathione (GSH) and ATP levels, the transzonal projections (TZP), the mitochondrial membrane potential (ΔΨm), cacline-AM, and the expression of related genes (cumulus cells (CCs), oocytes, blastocysts). The results revealed that oocytes treated with a combination of CNP, MT, and FLI had dramatically improved the percentage of oocytes developed to blastocyst, ATP content, GSH levels, TZP intensity, the ΔΨm, cacline-AM fluorescence intensity, and considerably reduced ROS levels of oocytes. Furthermore, the survival rate and the hatched rate after vitrification of the CNP+MT+FLI group were significantly higher than those other groups. Thus, we speculated that CNP+MT+FLI increases the IVM of bovine oocytes. In conclusion, our findings deepen our understanding and provide new perspectives on targeting the combination of CNP, MT and FLI to enhance the quality and developmental potential of bovine oocytes.

Porcine embryo cryopreservation remains challenging due to high lipid content, oxidative stress, and ice recrystallization that compromise post-thaw survival and developmental competence. We evaluated an integrated vitrification approach combining antioxidants (berberine, melatonin), iron oxide (Fe₃O₄) nanoparticles, and antifreeze protein I (AFP I) with post-thaw interventions (glutathione and zona pellucida digestion) to synergistically improve cryosurvival and developmental competence of porcine parthenogenetic embryos. In vitro-matured parthenogenetic embryos were vitrified on Cryotop using a protocol that included berberine and melatonin in embryo culture, Fe₃O₄ nanoparticles and AFP I in cryoprotectant solutions, and post-warming treatment with glutathione and a brief zona pellucida digestion. Survival, hatching, adenosine triphosphate (ATP) content, reactive oxygen species (ROS) levels, cytoskeletal integrity, and the expression of BAX, BCL2, OCT4, and SOX2 genes were measured. Both the dual antioxidant (berberine + melatonin) and nanoparticle + AFP interventions produced greater improvements than individual additives. Fully integrating all components yielded the highest post-thaw viability, with ~94% survival and ~90% hatching, values statistically equivalent to those of fresh embryos. Treated embryos also showed significantly higher ATP levels, lower ROS accumulation (approaching levels in fresh embryos), and preserved microtubule structure (~91% normal). Vitrification alone upregulated BAX and downregulated BCL2, OCT4, and SOX2, whereas the integrated protocol restored their expression levels to near control levels. This multi-component antioxidant, nanoparticle, antifreeze strategy synergistically enhances the cryotolerance and developmental competence of vitrified porcine embryos by mitigating oxidative stress and cryoinjury. Post-thaw viability and molecular markers were restored to near-fresh conditions, demonstrating a promising approach to improve embryo cryopreservation outcomes in swine and potentially other species.

The Bazhou yak, a major native meat yak breed in Xinjiang, China, is renowned for its fast growth rate, strong adaptability, and particularly high intramuscular fat (IMF) content. However, limited knowledge regarding its phylogenetic history and genomic composition has hindered its long-term conservation and utilization. This study evaluated the genetic diversity, population phylogenetics, and genome-wide selective sweep analysis (GWSA) of 100 newly obtained Bazhou yaks through genome resequencing, as well as 340 public yak genomes from nine other populations on the Qinghai–Tibet Plateau. The results revealed moderate diversity, lower genomic inbreeding levels, and rapid linkage disequilibrium (LD) decay in Bazhou yaks. Principal component analysis (PCA) and phylogenetic analysis showed a clear separation of Bazhou yaks from other yak populations, indicating the Bazhou yak as an independent genetic population. Furthermore, less genetic differentiation was found between the Bazhou yak and the Huanhu yak, while ADMIXTURE analysis revealed a common ancestral lineage between Bazhou yaks and Huanhu yaks, indicating an important genetic contribution of the Qinghai yak population to Bazhou yaks. The GWSA identified a total of 833 selected genes in Bazhou yaks using the top 5% interaction windows of both parameters (F_ST, Pi ratio, and XP-EHH). A significant number of these genes are related to fat synthesis and deposition, such as MTOR, APOA1, and GPAT4. In summary, this study sheds light on the phylogenetic status and distinctive genomic features of Bazhou yaks, which facilitates our understanding of the genetic basis of the IMF phenotype in Bazhou yaks.