L-citrulline protects testicular Sertoli cell function by mitigating DNA damage via the gut-testis axis of sheep fed a high-concentrate diet.

Mounting evidence underscores that high-concentrate diets (HCD) significantly impair male reproductive health, leading to infertility, and are closely associated with dysregulation of the gut microbiome and metabolome. However, effective therapeutic strategies targeting these alterations remain elusive. Notably, compelling evidence implicates the gut-testis axis as a critical mediator in the etiology of poor semen quality. Gut-derived metabolites, as key players in the gut-testis axis, warrant in-depth investigation as potential therapeutic targets for addressing male infertility caused by environmental factors, particularly dietary stressors. In this study, through an integrated multi-omics approach employing 10× Genomics single-cell mRNA sequencing, 16S ribosomal DNA sequencing and metabolomic profiling, we demonstrate that HCD induces DNA damage in Sertoli cells and disrupts the integrity of the blood-testis barrier (BTB), resulting in a significant decline in spermatozoa quality. Moreover, HCD impairs gut microbiota homeostasis and arginine biosynthesis, particularly leading to a remarkable decrease in L-citrulline levels. Additionally, fecal microbiota transplantation (FMT) experiments confirm that gut microbiota dysbiosis contributes to Sertoli cell DNA damage and BTB dysfunction. Interestingly, the effect of HCD-induced aberrant Sertoli cells function can be rescued by supplementation with L-citrulline. Collectively, these findings highlight the therapeutic potential of L-citrulline in protecting male reproductive health under dietary stress conditions, particularly through its action on the gut-testis axis.