With the continuous advancement in reproductive biology, oocyte vitrification has become a critical technology for preserving female germplasm and protecting it from environmental disruptions. This technique also eliminates temporal and spatial constraints in animal embryo engineering research. However, during the vitrification of animal oocytes, exposure to low temperatures and high concentrations of cryoprotectants can cause various forms of damage, including cytoskeletal disruption, spindle abnormalities, mitochondrial dysfunction, apoptosis, oxidative stress and epigenetic modifications. These issues are now understood to severely restrict the subsequent developmental competence of oocytes, resulting in lower cleavage and blastocyst formation rates than those of fresh oocytes. Currently, the mechanisms of cryodamage in vitrified oocytes remain poorly understood, and standardized strategies to enhance vitrification efficiency have yet to be firmly established. This review provides a formal overview of the physiological factors underlying oocyte sensitivity to vitrification, alongside the mechanisms of cryodamage and the variables influencing post-thaw survival and reproductive success. It evaluates strategies for mitigating vitrification-induced stress, compares interspecies differences, and addresses current research limitations. By identifying future directions, this review offers new insights for optimizing mammalian oocyte cryopreservation techniques.

The reproductive efficiency of breeding boars substantially influences swine industry productivity. Sperm viability during ambient-temperature storage is critically affected by environmental factors, including microbial activity. This study aimed to elucidate the dynamics and interactions between the seminal microbiome and metabolome during boar semen storage at 17 °C. Using integrated 16S rRNA sequencing and untargeted metabolomics, we analyzed semen samples from six healthy boars (31–33 months old) collected at day 0 (control), 2, 4, and 6 of storage. Our results demonstrate that storage leads to a marked decline in microbial diversity, progressive enrichment of the opportunistic genus Proteus, depletion of key antioxidant and cofactor metabolites such as vitamin B6, and extensive metabolic reprogramming—including alterations in short-chain fatty acid, purine, and lipid oxidation pathways. Multi-omics correlation analysis further revealed strong associations between microbial succession and metabolic shifts, highlighting their combined role in driving sperm functional decline. These findings provide a mechanistic basis for improving semen preservation strategies through microbiome and metabolite-targeted interventions.

Early embryonic loss remains a major limitation to reproductive efficiency in dairy cows, with approximately 30–40% of embryonic mortality occurring within 21 days after artificial insemination (AI), ultimately reducing conception rates and economic profitability in dairy production. Increasing evidence suggests that reproductive tract microorganisms may contribute to pregnancy establishment; however, their functions during early gestation in dairy cows remains poorly understood. In this study, 16S rRNA gene amplicon sequencing was used to characterize vaginal microbiota profiles in multiparous Holstein dairy cows at 7.5 and 21 days after AI, with comparisons performed between pregnant and non-pregnant cows. Significant differences in microbial community structure were observed between pregnancy states. At 7.5 days post-AI, distinct differences in microbial composition were detected at the phylum level, with Bacillota, Bacteroidota, and Pseudomonadota identified as dominant phyla. The relative abundance of Bacillota was higher in pregnant cows, whereas Pseudomonadota was enriched in non-pregnant cows. At the genus level, Ureaplasma was significantly increased in pregnant cows, while Streptococcus and Actinobacillus were significantly enriched in non-pregnant cows. By 21 days after AI, beta diversity analysis revealed a highly significant separation in microbial community structure between pregnant and non-pregnant cows (p < 0.01). Volcano plot and LEfSe analyses further identified representative microbial taxa associated with pregnancy status. Notably, inflammatory-associated genera, including Streptococcus and Actinobacillus, were predominantly enriched in non-pregnant cows, whereas pregnancy-associated taxa, including Ureaplasma and Bifidobacterium, were enriched in pregnant cows. Collectively, early alterations in the vaginal microbiota after AI were closely associated with pregnancy outcomes. These findings provide insight into the microbial associations related to early pregnancy outcomes and may facilitate the identification of potential microbial biomarkers for predicting pregnancy success in dairy cows.

In vitro maturation (IVM) of oocytes is a critical initial step in mammalian in vitro production (IVP), and its quality directly influences the developmental competence of subsequent embryos. Oxidative stress is a major constraint on oocyte quality, which can be mitigated by exogenous antioxidants. In this study, porcine oocytes were matured in IVM medium supplemented with the antioxidant N-acetyl-L-cysteine (NAC; 0, 0.5, 1.5, and 4 mM) to evaluate its effects on maturation rate, antioxidant capacity, mitochondrial function, vitrification–warming survival rate, post-warming ROS levels, and early embryonic developmental rate. To elucidate the molecular mechanisms underlying the effects of NAC on porcine oocyte maturation, we conducted single-cell transcriptome sequencing. The results showed that, versus the control, 1.5 mM NAC substantially enhanced the IVM rate (p < 0.05), reduced ROS levels (p < 0.05), and increased mitochondrial activity, as assessed by MitoTracker, mitochondrial membrane potential (MMP), and ATP content (p < 0.05). These results suggest that 1.5 mM NAC relieves oxidative stress in oocytes and improves mitochondrial function. However, NAC addition showed no significant differences in vitrification–warming survival rate and post-warming ROS levels relative to the control group (p > 0.05). In addition, 1.5 mM NAC markedly improved the cleavage rate and blastocyst rate of porcine oocytes after in vitro fertilization (IVF, p < 0.05), and also enhanced the cleavage rate after parthenogenetic activation (PA, p < 0.05). Single-cell transcriptome sequencing revealed that, versus the control, differentially expressed genes (DEGs) identified after 1.5 mM NAC supplementation were mainly enriched in pathways related to oxidative phosphorylation (OXPHOS), spliceosome, and ubiquinone/terpenoid-quinone biosynthesis. Among these, the OXPHOS pathway showed the most significant enrichment, with upregulated expression of pathway-related genes such as COX6C, CYCS, and SDHD. The accuracy of the transcriptomic results was further validated by qPCR. In conclusion, supplementing with 1.5 mM NAC relieved oxidative stress, improved mitochondrial function, and thereby promoted oocyte maturation and improved oocyte quality, ultimately facilitating subsequent IVF early embryonic development.