Integrated microbiome and metabolome revealing new insight into the combination of lactic acid bacteria in preventing postpartum metritis of dairy cows.

Probiotics, particularly composite lactic acid bacteria (CLAB), are emerging as a potential alternative to antibiotics for managing bovine metritis. While prior studies have highlighted CLAB's anti-inflammatory effects, limited data exist on its impact on vaginal and uterine microbiomes and systemic metabolism. In this study, Holstein cows received deep vaginal infusions of CLAB (a blend of Lactobacillus rhamnosus, Pediococcus acidilactici and Lactobacillus reuteri; at a standardized 4.5 × 1010 CFU/dose and a proportion of 25/25/2, respectively) at 20 and 10 days prepartum. We analyzed changes in vaginal and uterine microbiota, plasma metabolomes, and assessed the incidence of metritis, conception rate, and lactation performance postpartum. CLAB administration significantly altered the vaginal microbiota by reducing opportunistic pathogens (Prevotella heparinolytica, Bacteroides and Fusobacteria) and promoting beneficial taxa (Akkermansia and Prevotellaceae). In the uterus, CLAB enriched Rikenellaceae, Christensenellaceae and Lachnospiraceae, while suppressing pathogenic genera such as Cutibacterium and Fournierella. Metabolomic analysis identified pyruvic acid, L-glutamine and L-valine as hub metabolites, with KEGG enrichment revealing involvement in amino acid metabolism and immunomodulatory pathways. Although CLAB infusion did not significantly reduce metritis incidence (5.00 % vs. 5.52 %) or improve conception rate (47.50 % vs. 45.00 %), it showed promising trends without affecting milk production. These findings suggest that prepartum vaginal CLAB infusion modulates reproductive tract microbiota and systemic metabolism, potentially contributing to uterine health maintenance in dairy cows. A key limitation of this study was the absence of significant reductions in metritis incidence and improvements in conception rates, likely attributable to the small sample size and limited study period. Further large-scale studies are warranted to validate its efficacy during high-risk seasons.