PSIII-21 Vaginal microbiome population and cytokines concentration associated with fertility in beef cows subjected to a 14-day implant synchronization protocol.

Abstract Synchronization protocols with progesterone (P4) implants for 7 days contribute to shifts in vaginal microbial populations. However, these alterations have yet to be characterized when utilizing extended implant time protocols. The objective of this study was to determine the diversity and composition of vaginal bacteria and characterize circulating cytokines in pregnant (P) vs. open (OP) beef cows submitted to a 14-day implant (7&7) synchronization protocol. Beef cows (n=133) were enrolled across two farms. Cows were swabbed with a double-guarded culture swab (Jorgensen Laboratories, CO) by spinning on the cranial vagina on days (d) 0, and 14 of the protocol. Additionally, blood samples were collected on d0, d14, and at artificial insemination (AI). On d14, cows received an estrus detection patch (Estrotect™, Rockway INC, WI) and detection was performed every 6h during the daytime, starting 24h after implant pull. Cows underwent artificial AI 12-18h after patch activation. Cows that expressed heat at 54h and up after implant removal were bred at the fixed time (66 ± 2h) and received a concomitant GnRH injection. Pregnancy diagnosis was performed by ultrasonography at d32 post-AI. Cows were grouped post-hoc based on pregnancy status, and then 12 P and 12 OP cows were randomly selected for microbiome and cytokine analysis. Cytokines were analyzed using RayBiotech Quantibody Bovine Cytokine Array Q1 and MyBioSource high-sensitivity ELISA kits for IL6 and TGFβ per the manufacturer’s instructions. Microbiome data were generated from Illumina MiSeq (2x300) sequencing of PCR amplicons targeting the V1-V3 region of the 16 rRNA gene. Sequences were analyzed using custom Perl scripts and software (Mothur v.1.40, RDP classifier, and NCBI Blast). Analysis of species-level operational taxonomic units (OTUs) was performed on the samples. Statistical analysis of microbial abundances used Kruskal-Wallis’s and Pairwise Wilcox’s test. Cytokines data was analyzed with Proc Mixed in Sas 9.4. The topmost abundant OTU, RP1-00038, closely related to Citrobacter gillenii (99.43%), decreased in abundance by d14 (OP:d0=68.6%, OP:d14=9.9%, P:d0=71.6%, P:d14=5.5%; P≤0.00003). OTU RP4-08239, closely related to Helcococcus ovis (99.60%), had abundance increased from d0 to d14 according to status (OP:d0=0.6%, OP:d14=18.4%, P:d0=0.5%, P:d14=24.1%; P< 0.0001). The OTUs RP1-00038 and RP4-08239 were similar between statuses on the same day (P >0.05). Cytokines IFNαA (d0 418.29±109.44, d14 564.34±110.24, AI 561.08±124.19 pg/ml), IL13 (d0 2589.24±404.25, d14 3869.99±601.42, AI 3692.72±402.75 pg/ml), and IL21 (d0 1540.35±227.05, d14 2180.29±318.93, AI 2123.44±208.401 pg/ml) increased from d0 to d14 (P< 0.05) but not from d14 to AI (P >0.05). In conclusion, these results indicate a significant shift in the microbiome population associated with the 14 days of intravaginal implant. In addition, cytokines concentrations were altered in the blood, indicating a potential immune response reaction linked to the presence of the implant for 14 days.