(165) Unraveling the Influence of Estradiol on Reproductive Tract Microbiota and Fertility in Beef Cattle

Dallas R. Soffa¹; Michael F. Smith²; Julie A. Walker³; Gregory A. Johnson⁴; Thomas W. Geary⁵; George A. Perry⁶; Rebecca K. Poole¹

1. Department of Animal Science, Texas A&M University, College Station, USA


2. Department of Animal Sciences, University of Missouri, Columbia, USA


3. Department of Animal Science, South Dakota State University, Brookings, USA


4. Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, USA


5. Fort Keogh Livestock and Range Research Laboratory, USDA-ARS, Miles City, USA


6. Texas A&M AgriLife Research and Extension Center, Texas A&M University, Overton, USA

Reproductive failure within the first 30 days of gestation financially devastates the beef cattle industry. Previous research indicates that elevated preovulatory estradiol concentrations along with estrus expression increases pregnancy rates. Endogenous ovarian steroid hormones have also been associated with shifts in bacterial communities. However, minimal research has been dedicated towards linking alterations in the reproductive microbiome and establishment of pregnancy in cattle. Therefore, the objective of this work was to determine the impact of endogenous and exogenous estradiol at the time of ovulation on the uterine microbiome during early gestation in beef cattle. Crossbred beef cows (n=62) were synchronized utilizing the 7-day CO-Synch + CIDR protocol. Two days post-CIDR removal (day [d] 0), cows were allocated to one of three treatment groups: 1) Estrus Expression (ESTRUS), 2) No Estrus + induced ovulation with gonadotropin releasing hormone (GnRH; GNRH), 3) No Estrus + induced ovulation with GnRH and 0.1 mg estradiol 17β (GNRH+E2). All cows were artificially inseminated on d0 with semen from a single sire. Blood was collected via coccygeal venipuncture on d0 for estradiol, and one day prior to harvest for progesterone and pregnancy associated glycoproteins (PAGs). Circulating concentrations of estradiol and progesterone were quantified using radioimmunoassay, and a modified version of the IDEXX Bovine Pregnancy ELISA detected PAGs for diagnosis of pregnancy (NONPREG = nonpregnant; PREG = pregnant). 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 8 times along the endometrial mucosa, immediately placed in individual, sterile microcentrifuge tubes, and stored at -80°C. For microbiome analysis, the V4 hypervariable region of the 16S rRNA bacterial gene was targeted. Statistical analysis for relative abundance was conducted using nonparametric ANOVA in SAS with the independent variable of day, pregnancy status, or treatment. Progesterone, estradiol, and PAGs concentrations were analyzed using PROC GLM and correlations with bacterial communities using PROC CORR in SAS. There were no differences in circulating estradiol concentrations on d0 between treatments or pregnancy status (P>0.05). Progesterone (P=0.02) and PAGs (P=0.001) concentrations were greater in PREG cows compared to NONPREG cows. 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), all within Firmicutes, on d20 compared to later days of gestation within the IPS horn. For the CON horn, relative abundance for Firmicutes was greater on d32 compared to d26 (P=0.05). Phylum Euryarchaeota had lower relative abundance in CON for ESTRUS cows compared to the other two treatments (P=0.004). Phylum Bacteroidetes had greater relative abundance in PREG females compared to NONPREG cows in both IPS and CON horns (P=0.01). The uterine microbiome appears to shift during early gestation and may be driven by PAGs production by the developing conceptus. A decrease in Firmicutes within the gravid horn may play an imperative role in maintenance of pregnancy during early gestation. Additional research is required to understand the underlying mechanisms behind alterations in the uterine microbiome during pregnancy establishment in cattle. This research was funded by the 2024 SSR Emerging Investigator Grant titled “Unraveling the influence of estradiol on reproductive tract microbiota and fertility in beef cattle” and NIFA 2019-67015-29411.