Effect of Sampling Method on Detection of the Equine Uterine Microbiome during Estrus

SIMPLE SUMMARY: Bacterial endometritis is among the most common causes of subfertility in mares and has a major economic impact on the equine breeding industry. The sensitivity of detecting microbes using culture-based methods, irrespective of the sample collection method (whether double-guarded swab, low-volume lavage [LVL], or endometrial biopsy) is low, leading to a high rate of false negative samples. Here, using 16S rDNA sequencing, we found that the equine uterus does harbour a distinct microbiome during the estrus phase of the cycle. The microbial community was similar in composition as well as relative abundance at both phyla (Proteobacteria, Firmicutes and Bacteroidota) and genus (Klebsiella, Mycoplasma, and Aeromonas) level, regardless of the sampling technique. The present information about the equine endometrial microbiome could pave the way for innovative treatment methods for endometrial disease and assist sub-fertile mares. This, in turn, could lead to a reduction in the routine use of antibiotics in the equine breeding industry. ABSTRACT: 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.