Multi-omics analysis links heat stress-associated vaginal microbial alterations with serotonin metabolism and reproductive endocrine changes in Simmental cattle

In this study, Simmental cattle at different physiological stages were evaluated for heat stress based on the THI and physiological parameters, and serum biochemical profiling, 16S rRNA gene sequencing, metagenomic sequencing, and LC–MS/MS-based untargeted metabolomics were integrated to characterize heat stress-associated changes in production performance, immune and oxidative stress indices, reproductive hormones, the vaginal microbiota, and vaginal and serum metabolites, with Spearman correlation analysis used to integrate microbial, metabolic, and serum variables. A mouse model was further used to evaluate the biological effects of a key differentially abundant bacterial taxon. The results showed that heat stress was associated with reduced rumination time, activity, and milk yield, together with alterations in immune and oxidative stress indices and decreased serum levels of LH, FSH, PRL, P4, and GnRH. Vaginal microbiota analysis revealed differences in several α-diversity indices and the abundance of specific bacterial taxa between heat-stressed and non-heat-stressed cattle, whereas separation of the overall microbial community structure was limited. Vaginal and serum metabolomic analyses revealed distinct metabolic profiles between heat-stressed and non-heat-stressed conditions, with elevated serum 5-HT levels observed in heat-stressed cattle. Multi-omics correlation analysis further showed that the abundance of Pseudomonas syringae ( P. syringae ) was negatively correlated with GnRH, P4, PRL, and LH levels and positively correlated with serum 5-HT levels, revealing an association among vaginal microbial alterations, 5-HT metabolism, and changes in reproductive hormones. Following intraperitoneal inoculation of mice with P. syringae , serum 5-HT and MT levels increased, whereas FSH and GnRH levels decreased, with no significant change in LH levels. Collectively, these findings indicate that heat stress in Simmental cattle is accompanied by coordinated alterations in production performance, reproductive endocrine profiles, the vaginal microbiota, and host metabolism. This study provides a novel perspective for understanding the reproductive endocrine changes associated with heat stress in Simmental cattle.