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Background  The greatest impact on profitability of a commercial beef operation is reproduction. However, in beef heifers, little is known about the vaginal and fecal microbiota with respect to their relationship with fertility. To this end, we followed heifers through gestation to examine the dynamics of vaginal and fecal microbial composition throughout pregnancy.    Results  Heifers were exposed to an estrus synchronization protocol, observed over a 12-day period, artificially inseminated 12 h to 18 h after observed estrus, and subsequently exposed to bulls for a 50-day breeding season. Vaginal samples were taken at pre-breeding ( n  = 72), during the first ( n  = 72), and second trimester ( n  = 72) for all individuals, and third trimester for individuals with confirmed pregnancies ( n  = 56). Fecal samples were taken at pre-breeding ( n  = 32) and during the first trimester ( n  = 32), including bred and open individuals. Next generation sequencing of the V4 region of the16S rRNA gene via the Illumina MiSeq platform was applied to all samples. Shannon indices and the number of observed bacterial features were the same in fecal samples. However, significant differences in vaginal microbiome diversity between gestation stages were observed. No differences in beta-diversity were detected in vaginal or fecal samples regarding pregnancy status, but such differences were seen with fecal microbiome over time. Random Forest was developed to identify predictors of pregnancy status in vaginal (e.g.,  Histophilus , Clostridiaceae,  Campylobacter ) and fecal (e.g., Bacteroidales,  Dorea ) samples.    Conclusions  Our study shows that bovine vaginal and fecal microbiome could be used as biomarkers of bovine reproduction. Further experiments are needed to validate these biomarkers and to examine their roles in a female’s ability to establish pregnancy.

The vaginal and fecal microbiomes are related to pregnancy status in beef heifers