Characterization of the Endometrial Microbiota of Healthy Mares Across the Estrous Cycle

In the past, bacteria detected in the mare's uterus were generally interpreted as a sign of endometritis, since the uterus was considered a sterile environment. This assumption has been challenged by the introduction of culture-independent molecular techniques, particularly 16S rRNA amplicon sequencing, which have demonstrated that healthy mares harbor an endometrial microbiota. The aim of this study was to characterize the endometrial microbiota of healthy mares and to determine whether microbial composition differs between estrus and diestrus. Endometrial samples were collected from eleven healthy Standardbred mares during estrus and diestrus and analyzed by sequencing the V1-V2 region of the bacterial 16S rRNA gene. A total of 24 bacterial phyla and 599 genera were identified. At the phylum level, Firmicutes, Proteobacteria, Bacteroidota, and Actinobacteriota accounted for most of the relative abundance, while the most abundant genera were <i>Staphylococcus</i>, <i>Acinetobacter</i>, <i>Sphingomonas</i>, <i>Corynebacterium</i>, <i>Streptococcus</i>, <i>Clostridium</i>, and <i>Pseudomonas</i>. Alpha diversity was significantly higher during estrus, likely due to hormonally mediated changes in cervical opening and local immunity. Beta diversity analyses showed substantial overlap between estrus and diestrus samples. The phase of the cycle had a weak effect on microbiota structure, while inter-individual differences between mares explained a larger proportion of the observed variation. These findings suggest that the uterine microbiota of healthy mares is largely stable across the estrous cycle, with phase-dependent and mare-specific fluctuations in microbial composition.