Alterations in Vaginal Microbiota Associated with Pregnancy Outcomes in Dairy Cows Revealed by 16S rRNA Gene Sequencing

Early embryonic loss remains a major limitation to reproductive efficiency in dairy cows, with approximately 30–40% of embryonic mortality occurring within 21 days after artificial insemination (AI), ultimately reducing conception rates and economic profitability in dairy production. Increasing evidence suggests that reproductive tract microorganisms may contribute to pregnancy establishment; however, their functions during early gestation in dairy cows remains poorly understood. In this study, 16S rRNA gene amplicon sequencing was used to characterize vaginal microbiota profiles in multiparous Holstein dairy cows at 7.5 and 21 days after AI, with comparisons performed between pregnant and non-pregnant cows. Significant differences in microbial community structure were observed between pregnancy states. At 7.5 days post-AI, distinct differences in microbial composition were detected at the phylum level, with Bacillota, Bacteroidota, and Pseudomonadota identified as dominant phyla. The relative abundance of Bacillota was higher in pregnant cows, whereas Pseudomonadota was enriched in non-pregnant cows. At the genus level, Ureaplasma was significantly increased in pregnant cows, while Streptococcus and Actinobacillus were significantly enriched in non-pregnant cows. By 21 days after AI, beta diversity analysis revealed a highly significant separation in microbial community structure between pregnant and non-pregnant cows (p < 0.01). Volcano plot and LEfSe analyses further identified representative microbial taxa associated with pregnancy status. Notably, inflammatory-associated genera, including Streptococcus and Actinobacillus, were predominantly enriched in non-pregnant cows, whereas pregnancy-associated taxa, including Ureaplasma and Bifidobacterium, were enriched in pregnant cows. Collectively, early alterations in the vaginal microbiota after AI were closely associated with pregnancy outcomes. These findings provide insight into the microbial associations related to early pregnancy outcomes and may facilitate the identification of potential microbial biomarkers for predicting pregnancy success in dairy cows.