Maintaining normal functions of ovarian granulosa cells (GCs) is essential for oocyte development and maturation. The dysfunction of GCs impairs nutrition supply and estrogen secretion by follicles, thus negatively affecting the breeding capacity of farm animals. Impaired GCs is generally associated with declines in Nicotinamide adenine dinucleotide (NAD⁺) levels, which triggers un-controlled oxidative stress, and the oxidative stress, thus, attack the subcellular structures and cause cell damage. β-nicotinamide mononucleotide (NMN), a NAD⁺ precursor, has demonstrated well-known antioxidant properties in several studies. In this study, using two types of ovarian GCs (mouse GCs (mGCs) and human granulosa cell line (KGN)) as cell models, we aimed to investigate the potential effects of NMN on gene expression patterns and antioxidant capacity of both mGCs and KGN that were exposed to hydrogen peroxide (H₂O₂). As shown in results of the study, mGCs that were exposed to H₂O₂ significantly altered the gene expression patterns, with 428 differentially expressed genes (DEGs) when compared with those of the control group. Furthermore, adding NMN to H₂O₂-cultured mGCs displayed 621 DEGs. The functional enrichment analysis revealed that DEGs were mainly enriched in key pathways like cell cycle, senescence, and cell death. Using RT-qPCR, CCK8, and β-galactosidase staining, we found that H₂O₂ exposure on mGCs obviously reduced cell activity/mRNA expressions of antioxidant genes, inhibited cell proliferation, and induced cellular senescence. Notably, NMN supplementation partially prevented these H₂O₂-induced abnormalities. Moreover, these similar beneficial effects of NMN on antioxidant capacity were confirmed in the KGN cell models that were exposed to H₂O₂. Taken together, the present results demonstrate that NMN supplementation protects against H₂O₂-induced impairments in gene expression pattern, cell cycle arrest, and cell death in ovarian GCs through boosting NAD⁺ levels and provide potential strategies to ameliorate uncontrolled oxidative stress in ovarian GCs.

With estrus confined to three winter months, early pregnancy detection is essential for reproductive management in farmed sika deer. However, the development of reliable non-invasive early pregnancy detection techniques has been hindered by limited understanding of their reproductive physiology. To identify pregnancy-specific biomarkers in sika deer, we performed RNA-sequencing (RNA-Seq) on maternal peripheral blood collected on days 0, 7, 15, and 20 after artificial insemination. Using time-series clustering analysis and weighted gene co-expression network analysis (WGCNA), we identified key genes and pathways at each stage. Notably, maternal-fetal recognition-related interferon-stimulated genes (ISGs; IFNAR1/2, STAT1/2, MX1/2, and RSAD2), anti-apoptotic and immune-regulatory genes (BCL2, XIAP, and IL10), and cysteine metabolism genes (CTH, CBS, GCLC, and GCLM) were upregulated by day 7, suggesting their role in supporting corpus luteum development through immune regulation and redox homeostasis. By days 15–20, upregulated genes were enriched in pathways related to mitochondrial function, cell adhesion, and cell cycle regulation, indicating their involvement in embryo adhesion and syndesmochorial placentation. In conclusion, this study demonstrates that ISGs, immune-regulatory genes and cysteine metabolism genes are detectable as early as day 7 post-insemination, highlighting their promise as early pregnancy biomarkers and providing a molecular basis for non-invasive diagnostic development in sika deer.