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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. 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. [ABSTRACT FROM AUTHOR]

• 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]

Intracytoplasmic sperm injection (ICSI) is the only method for in vitro embryo production (IVP) in horses. Besides oocyte quality and competence, the outcome of IVP is also highly dependent on sperm quality. Therefore, it is essential to employ superior methods of selecting high quality sperm to maximize IVP outcomes. Clinical sperm selection techniques for stallions are generally limited to swim-up (SU), density gradient centrifugation (DGC), density gradient-swim up combination (DG-SU), and variations of these methods, despite the rapidly expanding use of novel techniques in humans and other species. One novel method of sperm selection is a microfluidic (MF) chip, which utilizes natural rheotactic behaviors of sperm to select for a highly motile and viable population of cells that would be optimal for IVP. However, no comprehensive comparison between MF and the conventional methods of DGC and DG-SU has been performed for stallions. Thus, we aimed to compare conventional equine sperm selection techniques to the novel method of MF sorting. Ejaculates (n = 22) were partitioned into fresh (∼23°C, 0 h; n = 12) and cooled (∼4°C, 24 h; n = 10) groups, and processed by wash-only, SU, DGC, DG-SU, and MF (ZyMōt™ Multi, 850µL, Sperm Separation Device™, DxNow, Gaithersburg, MD). Sperm motility, progressive motility, viability, and normal morphology,parameters used as basic estimates of sperm quality, were evaluated for both the unprocessed fraction and post-selected fractions. Among the compared methods of sperm selection, MF was user-friendly and effective in selecting high quality sperm as determined by individual parameters and by a summative sperm quality index (SQI) that combined individual factors. MF selected sperm with greater percentages of motility, progressive motility, viability, and normal morphology than unprocessed, wash-only, and SU-selected groups (P < 0.0001). Additionally, sperm selected by MF had numerically greater motility, progressive motility, and SQI, in comparison to DGC and DG-SU groups (P > 0.05). Cell viability and morphology parameters were comparable among MF, DGC, and DG-SU. Thus, MF-selected sperm not only exhibited numerically higher quality than DG-SU and DGC-selected sperm, but also the method did not use potentially harmful centrifugation steps. Therefore, MF may pose additional advantages over conventional methods. In conclusion, to select for superior sperm within a total ejaculate, implementation of MF technologies could be of interest to equine ICSI laboratories. This study was funded by the Theriogenology Foundation and the Center for Equine Health at UC Davis. [ABSTRACT FROM AUTHOR]