N- Carbamoylglutamate enhances bull spermatogenesis via Paraprevotella-Mediated vitamin B6 biosynthesis in rumen microbiota

N-carbamoylglutamate (NCG), a functional analog of the arginine precursor, shows strong potential in enhancing spermatogenesis in bulls. In this study, dietary NCG supplementation significantly increased sperm density and motility in XiangXi yellow bulls, the local beef cattle. Metagenomic and serum metabolomic analyses revealed that NCG altered the composition of rumen microbiota, notably increasing the abundance of Paraprevotella and elevating serum vitamin B6 levels (p < .05), suggesting a possible microbiota-associated metabolic modulation underlying its reproductive benefits. To explore this mechanism, a busulfan-induced mouse model of impaired spermatogenesis was established. Mice received transplants of either rumen microbiota from NCG-treated bulls or the differential genus Paraprevotella. Both treatments alleviated reproductive damage and increased vitamin B6 levels in serum and testis. Mechanistic investigation indicated that Paraprevotella was associated with upregulated expression of 3-phosphoserine aminotransferase (SerC), a key enzyme involved in vitamin B6 biosynthesis. Subsequent vitamin B6 supplementation experiments showed increased testicular glutathione levels and reduced thiobarbituric acid-reactive substances level (TBARS, expressed as MDA equivalents). These experiments supported a contributory role of vitamin B6 in promoting spermatogenesis, including increased sperm count and enhanced expression of spermatogenic cell markers. In summary, this study demonstrated that NCG enhanced spermatogenesis in bulls by reshaping the rumen microbiota, particularly through enrichment of Paraprevotella, which was associated with increased systemic vitamin B6 levels and contributed to reproductive improvement. These findings provide further insights into the application of NCG in improving fertility in ruminants.