31 Correlation of the Reproductive Microbiome and Cytokine Profiles with Plasma Progesterone Concentrations in Postpartum Beef Cows Prior to Timed Artificial Insemination
Authors: Smith Molly S , Hickman-Brown Kyle J , McAnally Brooke E , Dalmaso Gabriela , Oliveira Ramiro V , Pohler Ky , Poole Rebecca K 
Published: May 2023

Abstract
Female sex steroid hormones have been shown to interact with cytokines within the immune system to help influence fertility. Previous research has indicated that shifts in commensal bacteria are associated with compromised reproductive performance during the postpartum period, yet evaluation of how the female sex hormone progesterone (P4) influences these microbial shifts is not fully understood. The objectives of this study were to determine 1) the relationship between P4 concentrations and cytokine concentrations, and 2) the relationship between P4 concentrations and the reproductive microbiome. Twenty Bos indicus-influenced beef cows (~60 days postpartum and free of health issues) were weighed, body condition scored and then subjected to the Bos indicus prostaglandin 5 day (d) + CIDR protocol beginning on d -8 and timed artificially inseminated on d 0. Blood samples were collected on d-3, -1, 0, and 28. Cytokines and reproductive microbiome were evaluated using vaginal and uterine flushes collected on d -3 and d-1. Experimental groups were assigned by d 28 pregnancy status, determined by transrectal ultrasonography, resulting in Open (n = 13) and Pregnant (n = 7) females. Bacterial community analyses were conducted targeting the V4 hypervariable region of the 16S rRNA gene. Plasma P4 concentrations on d -3 and d -1 were quantified per manufacturers’ instructions using a double-antibody RIA kit. Cytokine analyses of plasma and flushes were conducted using the RayBiotech Quantibody Bovine Cytokine Array Q1 kit per the manufacturer’s instructions. Statistical analyses were conducted using the GLM procedure and CORR procedure in SAS. Plasma P4 concentrations did not differ by pregnancy status (P &gt; 0.05) but did decrease from d -3 to d -1 (0.89 vs. 0.08 ± 0.23 ng/mL, respectively; P = 0.02). There were no significant correlations between plasma P4 and plasma cytokines (P &gt; 0.05). Interferon-alpha (IFNα) and P4 concentrations were positively correlated in Open and Pregnant uterine flushes (r = 0.41; P = 0.01). Concentrations of P4 interleukin (IL)-1F5 were negatively correlated in Open and Pregnant vaginal flushes (r = -0.32; P = 0.04). The relative abundance of genus Methanobrevibacter in uterine flushes was positively correlated with P4 concentrations in both experimental groups (r = 0.35; P = 0.02). Within vaginal flushes, there was a tendency for a negative correlation between the relative abundance of genus Bacteroides and P4 concentrations (r = -0.31; P = 0.05). Alternatively, there is a positive correlation between P4 concentration and relative abundance of genus Streptococcus (r = 0.47; P = 0.01) and genus Methanobrevibacter (r = 0.37; P = 0.02) in vaginal flushes. These results suggest there may be relationships between several cytokines and circulating P4 concentrations; therefore, potentially suggesting immunomodulatory activities of P4. Overall, this research alludes to P4 having a vital role in reproductive microbial shifts and associated immune responses. Further investigation on the local effects of P4 within the reproductive tract on immune responses and bacterial shifts could provide greater insight into mechanisms to improve fertility in beef cattle.