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Bacterial endometritis is among the most common causes of subfertility in mares. It has a major economic impact on the equine breeding industry. The sensitivity of detecting uterine microbes using culture-based methods, irrespective of the sample collection method, double-guarded endometrial swab, endometrial biopsy, or uterine low-volume lavage (LVL), is low. Therefore, equine bacterial endometritis often goes undiagnosed. Sixteen individual mares were enrolled, and an endometrial sample was obtained using each method from all mares. After trimming, quality control and decontamination, 3824 amplicon sequence variants were detected in the dataset. We found using 16S rRNA sequencing that the equine uterus harbors a distinct resident microbiome during estrus. All three sampling methods used yielded similar results in composition as well as relative abundance at phyla (Proteobacteria, Firmicutes, and Bacteroidota) and genus (Klebsiella, Mycoplasma, and Aeromonas) levels. A significant difference was found in alpha diversity (Chao1) between LVL and endometrial biopsy, suggesting that LVL is superior at detecting the low-abundant (rare) taxa. These new data could pave the way for innovative treatment methods for endometrial disease and subfertility in mares. This, in turn, could lead to more judicious antimicrobial use in the equine breeding industry.

• Characterization of the equine endometrial microbiome in anestrus using 16S rRNA. • Mare endometrial microbiome in anestrus has high diversity, richness and abundance. • Significant differences between endometrial microbiome of anestrus and estrus mares. The equine uterus is highly interrogated during estrus prior to breeding and establishing pregnancy. Many studies in mares have been performed during estrus under the influence of high estrogen concentrations, including the equine estrual microbiome. To date, it is unknown how the uterine microbiome of the mare is influenced by cyclicity; while, the equine vaginal microbiome is stable throughout the estrous cycle. We hypothesized that differences would exist between the equine endometrial microbiome of mares in estrus and anestrus. The aim of this study was two-fold: to characterize the resident endometrial microbiome of healthy mares during anestrus and to compare this with estrus. Double-guarded endometrial swabs were taken from healthy mares during estrus (n = 16) and in the following non-breeding season during anestrus (n = 8). Microbial population was identified using 16S rRNA sequencing. Our results suggest that the equine uterine microbiome in estrus has a low diversity and low richness, while during anestrus, a higher diversity and higher richness were seen compared to estrus. Despite this difference, both the estrus and anestrus endometrial microbiome were dominated by Proteobacteria, Firmicutes, and Bacteroidota. The composition of the microbial community between anestrus and estrus was significantly different. This may be explained by the difference in the composition of the endometrial immune milieu based on the stage of the cycle. Further research investigating the function of the equine endometrial microbiome and dynamics changes within the uterine environment is required. [ABSTRACT FROM AUTHOR]