27 Bacterial Community Diversity in Fresh Extended Boar Semen
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AbstractStatus of bacterial diversity within the male seminal microbiome has previously been linked to fertility status in mammals. However, minimal research has focused on the reproductive microbiome in boar semen. The objective of this study was to analyze the diversity of bacterial communities within fresh extended boar semen samples 1) between studs, 2) over 5 consecutive days, and 3) between pooled and single-sire doses. Eight single-sire (n = 4 per stud) and eight pooled (n = 4 per stud) fresh extended semen doses (80 mL) were obtained from two boar studs (A vs. B). Pooled doses were the composite of boars in single-sire doses. Doses were subsampled (16 mL) for 5 days post-collection. Negative controls remained unopened until the last day. Each day, 5 mL of each subsample were flash frozen and stored at -80C for bacterial analysis targeting the V4 hypervariable region of the 16S rRNA gene for Illumina sequencing. Quality of all sequences was determined by FastQC before being processed for taxonomic analysis using the qiime2 pipeline. Alpha diversity (difference within sample metric) was analyzed using Kruskal-Wallis test, whereas PERMANOVA was performed for beta diversity analysis (differences between sample metric). According to alpha diversity metrics, Chao1 (P > 0.01) and Shannons index (P > 0.01) indicated contrasts in diversity among all days and the negative controls. This was further confirmed with a pairwise comparison between individual groups, in which day 1 exhibited lower alpha diversity when compared with days 2, 3, 4, and 5 using Chao1 (P > 0.01) and Shannons index (P > 0.01). Interestingly, reduced diversity was observed for day 1 compared with the negative controls with Chao1 (57.4 6.6 vs. 132.4 22.1, P > 0.01, Q > 0.01; respectively) and Shannons index (4.8 0.2 vs. 6.1 0.3, P > 0.01, Q > 0.01; respectively). No differences between boar stud or sire-type were detected for alpha diversity metrics. For beta diversity analyses, both PCoA plots and PERMANOVA for weighted unifrac metric demonstrated no significant clustering between sampling days. However, distinctions between all days and negative controls were identified using PERMANOVA for unweighted unifrac metric. Specifically, differences were discovered between day 1 and days 3, 4, and 5 (P > 0.01, Q > 0.01). PERMANOVA for both weighted (P > 0.01) and unweighted (P > 0.01) unifrac metrics revealed distinct clustering by boar stud. However, there were no differences in beta diversity between pooled and single-sire samples for both weighted and unweighted unifrac metrics. Based on these results, bacterial diversity within a semen sample may be altered over time and could occur between boar stud operations. Additional studies analyzing the reproductive microbiome of semen could help improve the characterization of fertility status in boars.
