Metagenomic characterization of the equine endometrial microbiome during anestrus

Recent studies have confirmed that healthy uteri, including the equine uterus, harbour a microbiome. Studies on mares have been performed in estrus and therefore under the influence of high estrogen concentrations. Multiple human studies investigating the vaginal, as well as the gut microbiome have shown that the microbiome is dynamic throughout the estrous cycle and is influenced by differences in estrogen levels. This is in contrast to a study performed on Arabian mares which indicated that the equine vaginal microbiome is stable throughout the estrous cycle.In this study the resident uterine microbiome of healthy anestrous mares was characterized by metagenomics from double-guarded endometrial swabs and compared to the microbiome in the same mares during estrus.Double guarded endometrial swabs from 15 healthy estrous mares were taken and stored at -80°C until extraction, in the following non-breeding season 8 mares from this group were sampled in similar fashion during anestrus. Blank samples were obtained during both sample period. Genomic DNA was extracted using Qiagen DNeasy PowerSoil extraction kits and 16S rRNA gene was amplified using primers 515f-806r. An amplicon sequence variant table was made and chimeras were removed and the genomic16S rRNA from the blank samples was substracted. Sequences were processed and analysed using 16S rRNA SILVA v138.1 database. Contaminating reads were removed from the samples by subtracting the reads from the blank samples using Microdecon and downstream analysis performed using the Phyloseq package. Alpha and beta diversity were calculated using microbiome, amplicon and vegan packages.Results suggest that the equine uterine microbiome in estrus has a low diversity, low richness, and a low abundance at phylum level. The equine uterine microbiome in anestrus has a higher diversity, higher richness, and higher abundance compared to the estrus uterine microbiome. However, both the estrus as well as the anestrus endometrial microbiome is dominated by Proteobacteria, Firmicutes, and Bacteroidota. PCoA analysis displays a strong dissimilarity between estrus and anestrus samples.The difference in the microbiome between estrus and anestrus can possibly be explained due to the variance in immune responsiveness of the endometrium. The estrous mare's endometrium is under the influence of estrogens, allowing for a high immune response and possibly less diverse microbiome.