The Microbiome of Reproductive Tissues, and Amniotic and Allantoic Fluid of Pregnant Gilts During Mid- and Late Gestation

Kyle J Hickman-Brown, Molly S Smith, Brooke E McAnally, Joe Cain, Heewon Seo, Fuller W Bazer, Greg Johnson, Jeffrey G Wiegert, Rebecca K Poole

The differences between the microbial communities in reproductive tissues, and amniotic and allantoic fluids of cattle have previously been investigated. Our objective was to provide insight into microbial differences across reproductive tissues in gilts during mid- and late-gestation. Specifically, we characterized the microbiomes of the vagina, cervix, endometrium, chorion, and allantoic and amniotic fluids. Duroc x Landrace x Yorkshire gilts (n = 12) free of physical, health or reproductive-related issues were euthanized and hysterectomized at either mid-gestation (d 60, n = 6) or late-gestation (d 90, n = 6). 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. To sample the endometrium and cervix, a 1 cm incision was made to expose the endometrium and interdigitating prominences, whereas for vaginal sampling, sterile swabs were inserted 6 inches past the vulva and then rotated. For collection of samples of chorion, an incision was made in the endometrium, exposing the chorioallantois to allow sampling of both the chorion-endometrial and chorion-allantois sides. 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 by FERA Diagnostics and Biologicals Corp. Data were analyzed statistically using the GLM procedure in SAS. Across all samples, abundances of the phylum Firmicutes was affected by stage of pregnancy with greater relative abundance at d 90 compared with d 60 (52.23 ± 2.85% vs 42.60 ± 3.14%, respectively; P = 0.03). Within this phylum and across all samples, the relative abundance of the genus Lactobacillus differed due to stage of pregnancy with a greater relative abundance at d 90 compared with d 60 (19.33 ± 2.37% vs 12.14 ± 2.61%, respectively; P = 0.05). Lactobacillus was most abundant in allantoic fluid when compared with all other samples (P < 0.01). The relative abundance of the genus Streptococcus, within the phylum Firmicutes, was less in amniotic and allantoic fluid compared with the cervix and vagina (P < 0.01). The phylum Bacteroidetes tended to differ by sample type (P = 0.06) with amniotic fluid having the lowest relative abundance. Within this phylum, the relative abundance of the genus Prevotella was also least in amniotic fluid (P < 0.01). These results suggest that microbial communities of reproductive tissues and fluids differ between mid- and late-gestation in pregnant gilts. Additional research on reproductive microbiomes in swine, and other livestock species, is required to develop methods to modulate bacteria and improve reproductive performance and successful outcomes of pregnancy.