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

## TL;DR

This study explores the bacterial communities in the uteruses of healthy horses and how they change during the reproductive cycle.

## Contribution

The study provides new insights into the stability and variation of the endometrial microbiota in healthy mares across estrus and diestrus.

## Key findings

- Bacterial diversity was higher during estrus compared to diestrus.
- The overall structure of the microbiota remained largely stable across the estrous cycle.
- Inter-individual differences between mares had a greater impact on microbiota variation than cycle phase.

## Abstract

Traditionally, the presence of bacteria in the mare’s uterus was interpreted as evidence of endometritis, as the uterine environment was long considered sterile. 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. How this microbial community changes during the reproductive cycle, however, is still not well understood. The aim of this study was to describe the uterine microbiota of healthy mares and evaluate whether it differs between estrus and diestrus. Samples were collected from the same mares during both phases and analyzed through a 16S rRNA amplicon sequencing technique. The most abundant genera were Staphylococcus, Acinetobacter, Sphingomonas, Corynebacterium, Streptococcus, Clostridium and Pseudomonas. Bacterial diversity was higher during estrus, while the overall structure of the microbiota remained largely stable across the cycle with phase-dependent and mare-specific fluctuations. These results provide new insight into the uterine microbiome of healthy mares.

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 Staphylococcus, Acinetobacter, Sphingomonas, Corynebacterium, Streptococcus, Clostridium, and Pseudomonas. 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.