96 Correlating vaginal bacteria abundance and interferon-stimulated genes in lactating dairy cattle

Abstract

Pregnancy relies on a variety of physiological factors and processes for successful establishment. The reproductive microbiome appears to influence pregnancy status, and previous research suggests that the expression of interferon-stimulated genes (ISGs) may play a role on pregnancy determination in cattle. However, the relationship between ISGs and reproductive microbiome has yet to be comprehensively evaluated in dairy cattle. Thus, the objective of this study was to analyze correlations between vaginal bacteria abundance and circulating ISGs in lactating dairy cattle in open and pregnant females. Lactating Holstein cows (n=27) were sampled on day of artificial insemination (AI) and day 18 post-AI. Sterile swabs were inserted into the vagina and rotated eight times along the vaginal wall prior to storage in sterile microcentrifuge tubes at -80°C. Swab samples were utilized for subsequent bacterial abundance analyses targeting the V4 hypervariable region of the 16S rRNA gene. Blood was collected via coccygeal venipuncture, placed on ice, and centrifuged at 2500 x g and 4°C for 20 minutes. Resulting buffy coats were collected, and samples stored at -80°C for real-time quantitative polymerase chain reaction (RT-qPCR) analysis. Cows underwent transrectal ultrasonography on day 32 post-AI for pregnancy determination. Selected target genes were interferon-stimulated gene 15 kDa (ISG15) and myxovirus resistance gene 2 (MX2). Mean relative mRNA expression of ISG15 was negatively correlated with phylum Tenericutes on day of AI (r = -0.43; P = 0.03). Concurrently, mean relative mRNA expression of MX2 was negatively correlated with phylum Bacteroidetes (r = -0.35; P = 0.08) and positively correlated with phyla Actinobacteria and Tenericutes (r = 0.41 and r = 0.41, respectively; P = 0.04). On day 18, MX2 mean relative mRNA expression was positively correlated with phylum Firmicutes (r = 0.39; P = 0.05). Regarding bacterial genera abundance, mean relative mRNA expression of ISG15 was negatively correlated with Ureaplasma on day of AI (r = -0.41; P = 0.04) and positively correlated with Sneathia on day 18 (r = 0.40; P < 0.05). MX2 mean relative mRNA expression was negatively correlated with Ruminococcus (r = -0.40; P = 0.05) and positively correlated with Ureaplasma (r = 0.42; P = 0.03) on day of AI. On day 18, mean relative mRNA expression of MX2 was positively correlated with Streptococcus (r = 0.42; P = 0.04). Interestingly, genera Ureaplasma and Sneathia have been previously associated with reproductive diseases in cattle, yet the current work suggests their bacterial load may not be indicative of inflammation. Further, the positive correlation of MX2 expression with phylum Actinobacteria may indicate potential pathogenicity as it has been previously associated with infertility in both women and cattle. Additional research is needed to further determine the relationship between reproductive microbiota and ISGs during pregnancy establishment.