186. Award Talk: An interplay of internal and external factors on reproductive microbiota and immune signaling in cattle

Abstract Reproductive failure continues to exacerbate economic losses within both the dairy and beef cattle industries. Alterations of the vaginal and uterine environments in response to reproductive hormones and/or conceptus development may coincide with shifts in reproductive microbiota populations to influence fertility. To address this, our objectives aimed to 1) characterize shifts in the vaginal microbiota from time of artificial insemination to maternal recognition of pregnancy and analyze its relationship with interferon-stimulated gene (ISG) 15 in lactating dairy cows; 2) determine the impact of endogenous and exogenous estradiol at the time of ovulation on uterine microbiota during early gestation in beef cattle; and 3) detect and localize ISG15 at the bovine uteroplacental interface during early gestation in beef cattle. For objective 1, lactating Holstein cows (n = 27) were sampled on day (day [d] 0) of artificial insemination (AI) and day 18 post-AI. Sterile swabs were inserted into the vagina and utilized for subsequent bacterial abundance analyses targeting the V4 hypervariable region of the 16S rRNA gene. Blood was collected and resulting buffy coats utilized for real-time quantitative polymerase chain reaction (RT-qPCR) analysis targeting ISG15. Cows underwent transrectal ultrasonography on day 32 post-AI for pregnancy determination. All statistical analysis for relative abundance was conducted using nonparametric ANOVA, diversity using Wilcoxon or Kruskal-Wallis, hormone concentrations using PROC GLM, and correlations with bacterial communities using PROC CORR in SAS. Interestingly, there were no major differences due to the main effect of pregnancy status. However, between days, Bacteroidetes (13.80 ± 1.29 vs. 8.96 ± 1.24; P = 0.009) had a greater relative abundance on d0 compared to d18 in Open cows. Upon evaluation of diversity matrices within days, there was a difference in α-diversity between d0 and d18 for Simpson’s diversity index (P = 0.02) and Shannon’s diversity index (P = 0.009), and a difference between days for the weighted UniFrac β-diversity matrix (P = 0.005). When evaluated with ISG15, there were no significant correlations between the mean relative mRNA expression of ISG15 on d18 with phyla bacterial abundance. This uncertainty of progesterone’s role in shifting the vaginal microbiota during early gestation warranted further consideration as to whether these bacterial shifts are influenced by hormone concentrations. Thus, for objectives 2 and 3, crossbred beef cows (n = 62) were synchronized utilizing the 7-day CO-Synch + CIDR protocol. Two days post-CIDR removal (d0), cows were allocated to one of three treatment groups: 1) Estrus Expression, 2) No Estrus + induced ovulation with gonadotropin releasing hormone, 3) No Estrus + induced ovulation with GnRH and 0.1 mg estradiol 17β. All cows were artificially inseminated on d0 with semen from a single sire, and blood was collected for estradiol, progesterone, and pregnancy associated glycoproteins (PAGs). Cows were harvested and ovariohysterectomized on d20, d26, and d32. An incision was made in each uterine horn (ipsilateral [IPS] and contralateral [CON]), and sterile swabs were rotated along the endometrial mucosa for microbiota analysis. Cross-sections of the ipsilateral uterine horn of suspected pregnant tracts were collected for immunofluorescence analyses to colocalize ISG15 with epithelial cadherin (E-Cad). Interestingly, an increase in PAGs appears to be correlated with a decrease in phylum Firmicutes (r=-0.340; P = 0.03). This coincides with greater relative abundance of genera Blautia (P = 0.03), Caloramator (P = 0.05), Oscillospira (P = 0.03), and Ruminococcus (P = 0.04) on d20 compared to later days of gestation within the IPS horn. Similarly to our first study, there were differences for α- and β-diversity matrices compared between days (P ≤ 0.05), yet there were no differences between treatments, pregnancy status, and uterine horns (P > 0.05). Thus, the uterine microbiota appears to shift during early gestation in a time-dependent manner and may be driven by PAGs production by the developing conceptus. This potential role of the conceptus was further noted on d20, as ISG15 was localized to endothelial cells within the stratum compactum stroma of both caruncular and intercaruncular regions. Similar expression of ISG15 was detected on d26, with distinct localization in stromal areas beneath the uterine luminal epithelium (LE) more proximal to the developing conceptus. With conflicting results across the literature, additional research is needed to further elucidate the complex relationship between vaginal microbiota and interferon-stimulated genes during early pregnancy establishment. Understanding the requirements necessary in the establishment of healthy reproductive tract microbiota that are associated with successful pregnancy establishment is also essential for the future improvement of reproductive efficiency in cattle.