A close association exists among testicular function, gut microbiota regulation, and organismal metabolism. In this study, serum and seminal plasma metabolomes, and the rumen microbiome of sheep with significant differences in sperm viability, were explored. Serum and seminal plasma metabolomes differed significantly between high-motility (HM) and low-motility (LM) groups of sheep, and 39 differential metabolites closely related to sperm motility in sheep were found in seminal plasma metabolomes, while 35 were found in serum samples. A 16S rRNA sequence analysis showed that the relative abundance of HM and LM rumen microorganisms, such as Ruminococcus and Quinella, was significantly higher in the HM group, whereas genera such as Rikenellaceae_RC9_gut_group and Lactobacillus were enriched in the mid-LM group. Serum hormone assays revealed that serum follicle-stimulating hormone (FSH) and MT levels were significantly lower in the LM group than in the HM group, whereas serum glucocorticoid (GC) levels were higher in the LM group than in the HM group, and they all affected sperm motility in sheep. Ruminococcus and other rumen microorganisms were positively correlated with sperm motility, whereas Lactobacillus was negatively correlated with FSH and GCs levels. Our findings suggest that rumen microbial activity can influence the host metabolism and hormone levels associated with fertility in sheep.
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