PS2-8. Reproductive Microbiota Shifts Across Days Following Embryo Transfer in Beef Heifers.

Abstract Utilization of heat stress abatement methods is essential for limiting negative impacts on reproductive efficiency in cattle. Associations between reproductive microbiota and heat stress may influence pregnancy establishment. However, minimal work has assessed these associations or determined their influence at and following time of embryo transfer. Therefore, we aimed to evaluate vaginal and uterine microbiota during and succeeding embryo transfer in beef heifers exposed to environmental stress conditions. Beef heifers (n = 18) were randomly assigned to treatment groups: 1) thermoneutral (TN; n = 10) or 2) heat-stressed (HS; n = 8). All heifers housed within the TN pens received 24 h cooling fans with full shaded coverage, while the HS heifers were housed without cooling fans and half shaded coverage. Heifers were synchronized utilizing the 7-day CO-Synch protocol, before undergoing embryo transfer on day (d) 8 post-estrus. All heifers were ovariohysterectomized on d26. Climatic data was obtained for calculation of Temperature-humidity index (THI) values. Vaginal and uterine body swab samples were collected for microbiota analysis on d8 and d26, where sterile swabs were rotated along the vaginal or endometrial mucosa, respectively. Microbiota analysis targeted the V3-V4 hypervariable regions of the 16S rRNA bacterial gene. Taxon analysis was processed in the Qiime2 pipeline with resulting files exported for diversity analysis using the R package ‘phyloseq’. Alpha diversity was determined with Wilcoxon tests, and beta diversity was determined using PERMANOVA. Alpha diversity analysis included observed ASVs and Chao1 index (within sample richness), and Shannon and Simpson indices (within sample richness and evenness). Beta diversity matrices included unweighted UniFrac (between sample bacterial presence or absence) and weighted UniFrac (between sample bacterial presence or absence and abundance). Heifers were exposed to thermoneutral conditions for 19 h per day on average. No conceptuses were identified upon necropsy. Minimal microbiota differences were observed by treatment. Phylum Firmicutes was greater in relative abundance on d26 within the vagina, while Bacteroidetes was greater in relative abundance on d8 (P ≤ 0.05). Oppositely, Bacteroidetes had greater relative abundance on d26 compared to d8 within the uterine body (P ≤ 0.05). For alpha-diversity within the vagina, d26 was lower for Observed ASVs, Chao1, and Simpson’s diversity index (P ≤ 0.01). Additionally, there were differences within the vagina for both unweighted and weighted UniFrac β-diversity matrices between d8 and d26 (P ≤ 0.05). Within the uterine body, there was a difference between TN and HS heifers, regardless of day, for unweighted UniFrac β-diversity matrix (P ≤ 0.05). Thus, a temporal relationship in changes of reproductive microbiota may factor into reproductive efficiency, where certain shifts in bacterial ratios may not be conducive to pregnancy establishment in cattle. These results enhance our understanding of bovine reproductive microbiota and its ability to dynamically shift in response to time, providing insights into potential mechanisms in understanding reproductive efficiency in cattle.