231 The association of the seminal microbiome to flow cytometry assays and breeding soundness exam metrics in yearling bulls.

Abstract The microbiome in bull semen remains largely underexplored, particularly in relation to fertility metrics. This study aimed to characterize the seminal microbiome by integrating a comprehensive panel of flow cytometry assays along with the traditional breeding soundness exam (BSE) measurements. Two ejaculates at the BSE were collected via electroejaculation from yearling virgin beef bulls (n = 32). Microbiome samples from each ejaculate were used for 16s rRNA analysis. Flow cytometry (FC) assays for oxidative stress, DNA integrity, mitochondria membrane potential, viability, acrosome integrity, and capacitation status were performed on the ejaculate with the greatest progressive motility. Spermatozoa were incubated, after which motility, viability, capacitation status, and acrosome integrity were evaluated again. Alpha diversity increased with fertility across multiple categories. Most notably, bulls with greater mitochondrial membrane potential exhibited greater (P < 0.01) Shannon diversity. Similarly, bulls classified as fertile based on spermatozoa progressive and total motility were positively associated with microbial richness, Faith’s phylogenetic diversity, and Shannon diversity (P < 0.05). Evenness was positively associated with acrosome integrity at both FC evaluations (P < 0.02). Sire of bulls had an association to all alpha and beta diversity metrics (P < 0.01), suggesting a potential role of seminal microbiome transmission from sire to offspring. Microbiome between ejaculates differed (P < 0.03) when compared on a per bull basis, but did not differ (P >0.13) for the whole population. This suggests the first ejaculate removes microorganisms from the reproductive tract but does not change the overall composition of the microbiome in the population. Bray-Curtis distances were positively correlated (P < 0.04) with the abnormal morphology classifications of distal midpiece reflex, bowed midpiece, and bent principle piece, but there were no correlations (P > 0.13) with normal morphology. Additionally, Bray-Curtis had a positive relationship with acrosome integrity markers of dead disrupted, total disrupted, and total intact at both FC evaluations (P < 0.04). Limited number of taxa were identified as potential biomarkers, but bulls with greater scrotal circumference had enriched species, such as Canibacter oris, present (P < 0.02). We conclude that relationships exist between the seminal microbiome and fertility measures of the BSE and spermatozoa functional measures assessed via FC, but additional data is needed to understand any causal effects or potential biomarkers of microbial populations on bull field fertility.