Stage of pregnancy impacts the bacterial communities of reproductive and placental tissues in gilts

Bacterial communities within the female reproductive tract have previously been linked to fertility status in mammals, however, minimal research has focused on reproductive microbiota in swine. Therefore, the objectives of this study were to compare and analyze the composition and α- and β-diversity of bacterial populations of different reproductive tissues (i.e., vagina, cervix, endometrium, and chorion) and placental fluids (i.e., allantoic and amniotic) in cyclic or pregnant gilts throughout gestation. Crossbred gilts free of physical, health or reproductive-related issues were euthanized, hysterectomized, and sampled during diestrus (n = 5; cyclic) or on day 11 (n = 11; peri-implantation), 15 (n = 10; implantation), 60 (n = 6; mid-gestation) or 90 (n = 6; late-gestation) of pregnancy. A sterile swab was rotated 8 times to collect samples from the mucosal surface of individual tissues and immediately placed in microcentrifuge tubes for storage (-80°C) until sequencing. For allantoic and amniotic fluids, 5 mL samples were collected and placed immediately in a sterile 10 mL conical tube and stored (-80°C) for subsequent analyses. Bacterial DNA was extracted and genome sequencing targeting the V4 hypervariable region of the 16S rRNA gene was conducted. The relative abundance of Fusobacterium was more abundant (P < 0.05) in the vagina compared to all tissues and fluids across all days except in day 90 gilts, while the relative abundance of Lactobacillus was more abundant in allantoic fluid than the endometrium at day 60 (P < 0.05). Alpha-diversity (Shannon's and Simpson's index) was lower in amniotic fluid compared to other tissues (P < 0.05), but there were no differences in α-diversity by day of pregnancy or between cyclic and pregnant gilts (P > 0.05). Beta-diversity revealed distinct clustering by tissue type, with posterior tissues such as the vagina clustering separately from placental fluids, and by day, with cyclic, day 11, and 15 samples clustering separately from day 60 and 90 samples (P < 0.01). These findings suggest that bacterial communities within the reproductive tract are dynamic and vary by both anatomical location and stage of pregnancy in gilts.