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.

Dihydroorotate dehydrogenase (DHODH) is a rate-limiting enzyme of de novo biosynthesis of pyrimidine. Although the involvement of DHODH in resisting ferroptosis has been successively reported in recent years, which greatly advanced the understanding of the mechanism of programmed cell death (PCD), the genetic sequence of the yak DHODH gene and its roles in ferroptosis are still unknown. For this purpose, we firstly cloned the coding region sequence of DHODH (1188 bp) from yak liver and conducted a characterization analysis of its predictive protein that consists of 395 amino acids. We found that the coding region of the yak DHODH gene presented high conservation among species. Second, the expression profile of the DHODH gene in various yak tissues was investigated using RT-qPCR. The results demonstrated that DHODH was widely expressed in different yak tissues, with particularly high levels in the spleen, heart, and liver. Third, to investigate the involvement of DHODH in regulating ferroptosis in cells, yak skin fibroblasts (YSFs) were isolated from fetuses. And then, bisphenol S (BPS) was used to induce the in vitro ferroptosis model of YSFs. We observed that BPS decreased the cell viability (CCK8) and membrane potential (JC-1) of YSFs in a dose-dependent manner and induced oxidative stress by elevating reactive oxygen species (ROS). Simultaneously, it was evident that BPS effectively augmented the indicators associated with ferroptosis (MDA and BODIPY staining) and reduced GSH levels. Importantly, the co-administration of Ferrostatin-1 (Fer), a potent inhibitor of ferroptosis, significantly alleviated the aforementioned markers, thereby confirming the successful induction of ferroptosis in YSFs by BPS. Finally, overexpression plasmids and siRNAs of the yak DHODH gene were designed and transfected respectively into BPS-cultured YSFs to modulate DHODH expression. The findings revealed that DHODH overexpression alleviated the occurrence of BPS-induced ferroptosis, while interference of DHODH intensified the ferroptosis process in YSFs. In summary, we successfully cloned the coding region of the yak DHODH gene, demonstrating its remarkable conservation across species. Moreover, using BPS-induced ferroptosis in YSFs as the model, the study confirmed the role of the DHODH gene in resisting ferroptosis in yaks. These results offer valuable theoretical foundations for future investigations into the functionality of the yak DHODH gene and the underlying mechanisms of ferroptosis in this species.

Enramycin is used as a dietary antimicrobial in young animals, but its effects on rabbit colonic ecology remain insufficiently characterized. This study evaluated the effects of enramycin in 60 weaned rabbits randomly assigned to a control or enramycin-treated group (60 mg/kg) for 14 days (n = 30/group). Clinical outcomes were evaluated in all rabbits, whereas paired colonic multi-omics profiling was performed in six randomly selected survivors per group with no recorded diarrhea. Enramycin reduced diarrhea incidence without affecting body-weight development, organ indices, or mortality. Metagenomic analysis revealed unchanged alpha diversity but significant alterations in microbial community structure, including decreased Verrucomicrobiota, Akkermansia, and Alistipes and increased Cyanobacteria and Avigastranaerophilus. Untargeted metabolomics identified 332 features meeting the predefined screening criteria, including 143 upregulated and 189 downregulated metabolites. Prominent pathway annotations included phenylpropanoid biosynthesis and arachidonic acid metabolism. Resistome analysis demonstrated that treatment drove shifts in streptothricin-, aminoglycoside- and glycopeptide-related resistance signatures. The integrated analysis identified associations between four selected genera and the top 30 differential metabolites. Overall, dietary enramycin was associated with a short-term reduction in diarrhea incidence and concurrent changes in selected colonic microbial, metabolic, and resistance gene features. These findings extend enramycin evaluation beyond conventional clinical outcomes and highlight the importance of considering broader ecological effects when assessing dietary antimicrobial exposure in weaned rabbits.