NAD⁺ Promotes Superovulation of Huaxi Cattle Through Regulation of Cumulus Cell Proliferation and Oocyte Maturation

Superovulation and embryo transfer are key technologies to improve the reproductive ability of female animals and enhance the efficiency of livestock production. However, poor-quality oocytes or abnormal fluctuations of hormone levels caused by superovulation affect the embryonic development environment, which may lead to a significant decline in the number and quality of transferable embryos, thus reducing the efficiency of superovulation. In this study, nicotinamide adenine dinucleotide (NAD⁺) was injected into Huaxi cows during the superovulation period to observe the proliferation and apoptosis of transplanted embryos. We examined the proliferation, apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential of cumulus cells and oocytes directly treated with NAD⁺ and investigated the potential mechanism of NAD⁺ to improve the superovulation efficiency by serum metabolomics and single-cell RNA sequencing. The results show that the addition of NAD⁺ significantly increased the quantity and quality of transferable embryos after superovulation. Differential metabolites during estrus synchronization and embryo flushing are enriched in glycerophospholipid metabolic pathways, suggesting that NAD⁺ can regulate lipid metabolic pathways. We found that NAD⁺ optimized the secretion levels of the steroid hormone estradiol (E2) and progesterone (P4) during superovulation by regulating the activity of cumulus cells. In oocytes, we found that NAD⁺ can inhibit apoptosis, scavenge ROS, and enhance mitochondrial function, thereby promoting oocyte maturation and enhancing embryo developmental potential. In conclusion, NAD⁺ significantly improved the superovulation ability of Huaxi cattle and provides an effective way for animal husbandry to improve the yield of high-quality embryos.