N-Acetyl-L-Cysteine Promotes Porcine Oocyte In Vitro Maturation and Subsequent Embryonic Development by Regulating Oxidative Stress.

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.