Characterization of Small Non-Coding RNAs in the Seminal Plasma of Beef Bulls with Predicted High and Low Fertility

MicroRNAs (miRNAs) are a family of small non-coding RNAs (sRNA) that play a key role in regulating gene expression by binding to their complementary mRNA and altering translation. It has been reported that this down-regulation of specific areas of the genome plays a role in male fertility. Piwi-interacting RNAs (piRNAs) are another class of germline-specific non-coding RNAs that form silencing complexes to maintain germline genome integrity. In bovine seminal plasma, piRNAs have not previously been characterized. Thus, the objective of this study was to investigate a potential relationship between expression of these sRNAs and fertility in beef bulls and to characterize piRNA expression in bull seminal plasma. Semen samples were collected from 35 beef bulls and analyzed for Arachis hypogaea/peanut agglutinin (PNA) and Lens culinaris agglutinin (LCA) scores. PNA reveals acrosomal damage or malformation and LCA indicates an altered sperm surface. LCA and PNA are both correlated with ubiquitin, a negative protein biomarker found on sperm cells, which indicates poor overall fertility. Divergent cohorts consisting of 3 bulls with high PNA/LCA scores (H) and 3 bulls with low PNA/LCA scores (L) were chosen. The sRNAs were extracted from the seminal plasma of these bulls. Sequence libraries were prepared using the Illumina TruSeq Small RNA preparation kit and sequenced on an Illumina MiSeq. Using the NCBI database of known human, porcine, mouse, and bovine miRNAs and known human piRNAs, expressed sequences were identified and mapped to the Bovine genome (Bta_4.6.1). A weighted FDR-corrected t-type test statistic was used to identify differentially expressed sRNAs between the H and L groups. Of the 617 sRNAs that were discovered, 9 miRNAs that were differentially expressed (FDR