The impact of the estrous cycle on the vaginal microbiota of dairy cows.

The present study investigated the vaginal microbiota of dairy cows throughout the estrous cycle and early gestation, under natural physiological conditions and without hormonal manipulation. Vaginal swabs were collected at estrus (D0), metestrus (D3), diestrus (D15), and Day 19 post-insemination (representing proestrus for non-pregnant cows or early gestation for pregnant cows). Pregnancy diagnosis was performed on day 31 post-insemination. Microbial characterization was performed using 16S rRNA gene sequencing, followed by analyses of alpha- and beta-diversity, community composition, and differential abundance. Overall, the vaginal microbiota exhibited high inter-individual variability and only subtle shifts across reproductive phases. Beta diversity analysis revealed modest but significant compositional rearrangements between estrus vs. proestrus (strictly within the non-pregnant cohort) and estrus vs. diestrus. Differential abundance testing identified two taxa enriched during pregnancy (Muribaculum and Hoministercoradaptatus), indicating potential microbial biomarkers associated with the gestational environment. These findings demonstrate that, in naturally cycling cows, physiological hormonal oscillations induce only mild microbial changes, whereas individual factors appear to play a dominant role in shaping the vaginal community. The work contributes to defining baseline microbial patterns in non-manipulated animals and highlights the importance of distinguishing natural variation from hormonally induced effects in reproductive microbiome research.