99 Diversity of vaginal bacteria in lactating dairy cattle on pregnancy establishment
Authors: Soffa Dallas R , Dodd Ashton , Spencer Jennifer A , Poole Rebecca K 
Published: March 2024

Abstract
Establishment and maintenance of pregnancy has a substantial economic impact within the dairy industry each year. Recent research has indicated a potential role of the reproductive microbiome on impacting pregnancy status as a result of hormone concentrations and breeding strategies. However, the association between alpha (within sample metric) and beta (between samples metric) bacterial diversity with pregnancy establishment has not been extensively evaluated in dairy cattle. Thereby, the objective of this study was to analyze vaginal bacterial diversity in lactating dairy cows 1) between day of artificial insemination (AI; day 0) and maternal recognition of pregnancy (MRP; d 18), and 2) between resulting Open and Pregnant females. Lactating Holstein dairy cows (n = 53) were sampled of day of AI and day of MRP. Pregnancy determination occurred on day 32 via transrectal ultrasonography. Sterile swabs were inserted into the vagina, rotated eight times against the vaginal wall, and stored in sterile microcentrifuge tubes at -80°C. Swab samples were utilized for subsequent bacterial diversity analyses targeting the V4 hypervariable region of the 16S rRNA gene. Cows underwent transrectal ultrasonography on gestational day 32 for pregnancy determination. Sample quality was determined by FastQC before being processed with the qiime2 pipeline for taxonomic classification. Alpha diversity metrics were analyzed with Kruskal-Wallis and beta diversity was measured utilizing PERMANOVA in the qiime2 pipeline. At day 32 of gestation, the pregnancy rate was 47.2%. According to Faith’s Phylogenetic Diversity metric for alpha diversity, there was no significant difference in diversity richness (i.e., number of different bacterial species within a sample) between day of AI and MRP or between cows diagnosed as Open or Pregnant (P &gt;0.05). Pielou’s Evenness metric (i.e., distribution or balance of bacterial species within a sample) for alpha diversity indicated greater diversity within samples at day of AI compared with day of MRP (P = 0.02). Shannon’s diversity index metric (i.e., richness and evenness within a sample) for alpha diversity also indicated greater diversity at day of AI compared with day of MRP (P = 0.04). However, there was no difference between resulting Open and Pregnant females for Pielou’s or Shannon’s metrics (P &gt; 0.05). Distinctions between samples (beta diversity) were compared utilizing pairwise comparisons of the unweighted and weighted Unifrac distance metrics. Specifically, day of AI indicated lower diversity compared with day of MRP for both metrics (P &lt; 0.01). However, upon comparisons of Open and Pregnant status, there was no difference between samples for unweighted or weighted Unifrac distances (P &gt; 0.05). Based on these results, bacterial diversity within and between vaginal samples appear to shift between day of AI and day of MRP. Additional studies are needed to elucidate the mechanisms altering bacterial diversity during early gestation in lactating dairy cows.