Vaginal and uterine microbiomes in beef cattle at artificial insemination and associations with pregnancy outcomes

The female reproductive tract harbors complex microbial communities that may influence reproductive success. In previous work using 16S rRNA gene sequencing, we identified bacterial taxa in the vagina and uterus of beef cattle associated with pregnancy outcomes, but taxonomic resolution and functional inference was limited. Here we used shotgun metagenomic sequencing to characterize the taxonomic composition, functional potential, and antimicrobial resistome of vaginal and uterine microbiomes at the time of artificial insemination (AI) in cows that subsequently became pregnant or remained open. Vaginal (pregnant n = 54; open n = 7) and uterine (pregnant, n = 41; open, n = 9) samples were collected prior to AI. Microbial community structure did not differ between pregnancy outcome groups in either anatomical site (PERMANOVA; P > 0.05). However, cows that remained open showed significantly greater species-level richness and diversity in the vaginal microbiome (P < 0.05). No diversity differences were observed in the uterine microbiome. In contrast, significant differences were detected between anatomical sites, with distinct dominant taxa and functional profiles. Vaginal microbiomes were enriched in pathways related to genetic information processing, whereas uterine microbiomes exhibited greater representation of metabolic pathways. A total of 105 ARGs spanning 11 antimicrobial classes were identified, with tetracycline resistance genes [tet(Q), tet(W), and tet(M)] predominating, and blaTEM-116 more abundant in the uterine microbiome. Overall, while vaginal and uterine microbiomes were compositionally and functionally distinct, no robust pregnancy-associated taxonomic or functional signatures were detected, likely reflecting limited statistical power and challenges inherent to low-biomass metagenomic datasets. IMPORTANCEUnderstanding the role of the reproductive tract microbiome in fertility could improve reproductive efficiency in cattle. We used shotgun metagenomic sequencing to characterize the taxonomic composition, functional potential, and antimicrobial resistome of vaginal and uterine microbiomes at the time of artificial insemination in cows that subsequently became pregnant or remained open. Using paired samples from the same animals, we directly compared microbial communities between the upper and lower reproductive tract to identify shared and site-specific features. Although no distinct microbial signatures associated with pregnancy outcomes were detected, this may reflect limited statistical power and low microbial biomass inherent to these samples. Despite these challenges, our study provides high-resolution insights into the composition, functional potential, and resistome of bovine reproductive microbiomes and highlights important technical considerations for studying low-biomass microbial ecosystems.