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.

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.