Heat stress (HS) has become a common stressor, owing to the increasing frequency of extreme high-temperature weather triggered by global warming, which has seriously affected the reproductive capacity of important livestock such as sheep. However, little is known about whether HS reduces sperm motility by inducing circadian rhythm disorders in rumen microorganisms and metabolites in sheep. In this study, the year-round reproduction of two-year-old Hu rams was selected, and the samples were collected in May and July 2022 at average environmental temperatures between 18.71 °C and 33.58 °C, respectively. The experiment revealed that the mean temperature-humidity index was 86.34 in July, indicating that Hu rams suffered from HS. Our research revealed that HS significantly decreased sperm motility in Hu rams. Microbiome analysis further revealed that HS reshaped the composition and circadian rhythm of rumen microorganisms, leading to the circadian disruption of microorganisms that drive cortisol and testosterone synthesis. Serum indicators further confirmed that HS significantly increased the concentrations of cortisol during the daytime and decreased the testosterone concentration at the highest body temperature. Untargeted metabolomics analysis revealed that the circadian rhythm of rumen fluid metabolites in the HS group was enriched by the cortisol and steroid synthesis pathways. Moreover, HS downregulated metabolites, such as kaempferol and L-tryptophan in rumen fluid and seminal plasma, which are associated with promotion of spermatogenesis and sperm motility; furthermore, these metabolites were found to be strongly positively correlated with Veillonellaceae_UCG_001. Overall, this study revealed the relationship between the HS-induced circadian rhythm disruption of rumen microorganisms and metabolites and sperm motility decline. Our findings provide a new perspective for further interventions in enhancing sheep sperm motility with regard to the circadian time scale.

Sleep deprivation (SD) disrupts circadian rhythms; however, its effects on SD and the mechanisms involved require further investigation. Previous studies on SD were mainly conducted on rodents, such as mice, with few studies on its effects on the liver of large diurnal animals, such as sheep. In this study, we used a Tibetan sheep model for the first time to investigate the effects of SD on the liver by exposing Tibetan sheep (Ovis aries) to 7 days of SD (6 h/day) and performed transcriptome sequencing analysis on liver samples taken at 4 h intervals over 24 h. The results revealed that SD significantly altered the circadian expression of genes and their expression patterns in the liver of Tibetan sheep. Enrichment analysis of the circadian rhythm-altered genes revealed changes in the pathways related to lipid metabolism in the liver. Further evidence from serum markers and gene expression analyses using qualitative real-time polymerase chain reaction and Oil Red O and apoptosis staining indicated that SD leads to abnormal lipid metabolism in the liver, potentially causing liver damage. Therefore, our results suggest that SD disrupts the circadian rhythms of metabolism-related genes in the Tibetan sheep liver, thereby affecting metabolic homeostasis.