Vaginal and Uterine Bacterial Communities in Postpartum Lactating Cows

The vaginal and uterine microbiome of cattle could provide important indicators of fertility pregnancy success; however only recently has next-generation sequencing made it possible to deeply interrogate these microbial communities. The objective of this study was to determine the bacteriome of the vaginal and uterine environments in lactating Angus cows (n=30) undergoing estrus synchronization prior to timed artificial insemination (TAI). On D-2 prior to TAI, uterine and vaginal flushes were performed. Vaginal flushes consisted of 60mL of saline solution injected into the vaginal cavity, rectally massaged, and fluid collected using sterile 50mL conical tubes. Uterine flushes were conducted using a Foley catheter, flushed with 180mL of saline solution, and collected in sterile 50mL conical tubes. After initial pregnancy check on Day 30 post TAI, 10 open cows were chosen based on successful flush, presence of corpus luteum on D-9, and ovarian activity throughout. DNA was extracted and the V1-V3 hypervariable regions of the 16S rRNA gene were targeted and sequenced for bacterial community analysis. Sequences were cleaned and processed using QIIME-1.8.0 software package. Differences in bacterial community characteristics by normally distributed variables (Shannon index) were statistically analyzed by a one-way analysis of variance (ANOVA) for multiple independent groups. Differences for not normally distributed variables and all multiple-group comparisons (observed OTUs, Chao1, relative abundances of taxonomic profiles) were completed using the Kruskal-Wallis H test with Benjamini-Hochberg FDR multiple test correction. For all analyses, the significance level was set at 0.05. The vagina contained a significantly greater number of operational taxonomic units (OTU; p=0.002) than did the uterus, where 482 OTU were shared between the two. The vagina was significantly greater in phylogenetic diversity (Shannon Diversity Index; p=0.029), and both environments were distinct in phylogenetic distribution utilizing UniFrac distance matrices (p=0.005). The most abundant bacterial phyla in the uterus and vagina, respectively, were Firmicutes (31.3 ± 5.6%; 65.9 ± 4.1%), Protebacteria (22.9± 7.7 %; 7.4± 3.8 %), Actinobacteria (13.2± 6.7 %; 2.3± 0.8 %), and Bacteriodetes (8.5± 1.9 %; 16.8± 2.7 %). The vaginal and uterine bacteriome determined in this study provide the basis to evaluate potential roles microbiota may play in affecting reproductive outcomes.