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.

Sperm proteins play vital roles in improving sperm freezing resilience in domestic animals. However, it remains poorly defined which proteins regulate the freezing resilience of spermatozoa in rams (Ovis aries). Here, we compared the proteome of ram sperm with a high cryopreservation recovery ratio (HCR) with that of ram sperm with a low cryopreservation recovery ratio (LCR) using a tandem mass tag-based quantitative proteomics approach. Bioinformatic analysis was performed to evaluate differentially expressed proteins (DEPs). A total of 2464 proteins were identified, and 184 DEPs were screened. Seventy-two proteins were higher in the LCR group. One hundred and twelve proteins were more abundant in the HCR group, and they were mainly involved in the regulation of oxidative phosphorylation and thermogenesis pathways. Proteins in high abundance in the HCR group included the S100A family, such as S100A8, S100A9, S100A14, and S100A16, effectively controlling for CA²⁺ and maintaining flagella structure; HYOU1 and PRDX1, which participate in antioxidant protection and anti-apoptosis to prevent cell death; and HSP90B1, which maintains cell activity and immune response. Our results could help illuminate the molecular mechanisms underlying cryopreservation of ram semen and expand the potential direction of cryopreservation of high-quality semen.