158 Characterization of the extent and composition of the bull reproductive microbiome

Two studies were conducted to assess whether organs of the bull reproductive system contain microbial communities and to characterize their composition. In Study 1, samples of rumen fluid, semen, and testicular tissue were collected from mature Holstein bulls (n = 14, mean age = 6.4 years) at a commercial semen collection facility. In Study 2, reproductive tracts were obtained from mature crossbred beef bulls (n = 14) at a commercial abattoir. Tissue samples were collected from the testis, cauda epididymis, and seminal vesicles and swab samples were obtained from the penile and pelvic urethra. For both experiments, genomic DNA was extracted, the V4 hypervariable region of the 16S rRNA gene was amplified by PCR, and 16S rRNA gene sequencing was subsequently performed using the Illumina MiSeq platform. Alignment of amplicon sequence variants (ASVs) was performed using SEPP via q2-fragment insertion, and the rooted phylogenic tree was used to classify ASVs to SILVA 99% full-length sequences using a 95% confidence interval. The core microbiome was generated in QIIME2 using feature-based filtering to identify ASVs that represented at least 1% of the microbial population and were present in at least 50% of samples. In Study 1, microbial diversity was affected (Shannon’s Diversity: P < 0.001) by sample type, with rumen fluid being richer than both semen and testicular tissue. Evenness of microbial communities was also affected by sample type (Pielou’s Evenness: P < 002), with each sample type statistically differing from one another (Q < 0.001). Measures of β diversity were also affected (Jaccard Distance and Weighted Unifrac: P < 0.001) by sample type and differed statistically between each of the three sample types (Q <0.001). Core microbiome analysis identified 440 ASVs with 434 of these unique to one of the three sample types. Semen and testicular tissue only had four ASVs in common, while rumen fluid only shared one ASV with semen and testicular tissue, respectively. In Study 2, microbial diversity (Shannon’s Diversity: P < 0.001) and evenness (Pielou’s Evenness: P < 0.001) were affected by sample type. The microbial diversity and evenness of the penile urethra was similar to the pelvic urethra, but was more diverse and even than all other sample types (P < 0.05). Measures of β diversity (Jaccard Distance and Weighted Unifrac) were also affected by sample type (P < 0.05), and each of the sample types differed from one another (Q < 0.01), except that there was no difference in Jaccard Distance between the penile and pelvic urethra. Analysis of the core microbiome identified 341 ASVs, with 137 being unique to one of the five sample types assessed. The penile and pelvic urethra shared 72 ASVs, while the penile urethra, pelvic urethra, and epididymis had 10 ASVs in common. Collectively, results of the present studies indicate that organs of the bull reproductive system and urogenital tract, including the testes and epididymis, contain microbial populations and that several of these microbial niches contain features unique to each. Further work is necessary to determine whether differences in the composition of such microbial populations can affect sperm viability and bull fertility.

Funding for these studies was provided by the NAAB Doak Graduate Fellowship.