Nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide (NAD), has been shown to improve oocyte quality in mammals. A healthy ovarian microenvironment is essential for oocyte quality and reproductive success in teleost fish. To investigate the effects of NMN on ovarian microenvironment parameters associated with oocyte quality in the spotted scat (Scatophagus argus), the levels of serum glucose, insulin, total cholesterol, triglycerides, 17β-estradiol (E), and ovarian NADwere analyzed following an intraperitoneal (IP) injection of NMN. Ovarian transcriptomic profiling was also conducted using RNA sequencing. The expression of genes related to steroidogenesis, apoptosis, and oxidative stress after NMN supplementation was analyzed by qRT-PCR. In vitro, ATP content, glutathione (GSH) levels, reactive oxygen species (ROS) accumulation, and mitochondrial membrane potential (ΔΨm) were quantified in follicles after NMN incubation. The results showed that NMN injection significantly increased ovarian NAD levels and serum Econcentrations. NMN improved glucose and lipid metabolism, as evidenced by elevated insulin levels and reduced serum glucose, total cholesterol, and triglycerides. KEGG enrichment analysis revealed significant enrichment of pathways related to metabolism, the stress response, and apoptosis. qRT-PCR demonstrated the upregulation of steroidogenesis-related genes (cyp17a1, hsd3b1, 20β-hsd, cyp19a1a, mmp2, and foxl2), antioxidant genes (gpx1a, gpx1b, and slc7a11), and the anti-apoptotic gene bcl2, along with downregulation of pro-apoptotic genes (bax and caspase3). NMN incubation reduced ROS accumulation and increased GSH levels, accompanied by enhanced mitochondrial function, as reflected by increased ΔΨm and ATP production. Collectively, these results suggest that NMN improves the ovarian microenvironment associated with oocyte quality in spotted scat by enhancing NADavailability, which subsequently promotes estrogen signaling, reprogrammed ovarian metabolism, mitigates oxidative stress, and inhibits apoptosis. + + + + 2 2
