RNA Sequencing of Sperm from Healthy Cattle and Horses Reveals the Presence of a Large Bacterial Population.

RNA molecules within ejaculated sperm can be characterized through whole-transcriptome sequencing, enabling the identification of pivotal transcripts that may influence reproductive success. However, the profiling of sperm transcriptomes through next-generation sequencing has several limitations impairing the identification of functional transcripts. In this study, we explored the nature of the RNA sequences present in the sperm transcriptome of two livestock species, cattle and horses, using RNA sequencing (RNA-seq) technology. Through processing of transcriptomic data derived from bovine and equine sperm cell preparations, low mapping rates to the reference genomes were observed, mainly attributed to the presence of ribosomal RNA and bacteria in sperm samples, which led to a reduced sequencing depth of RNAs of interest. To explore the presence of bacteria, we aligned the unmapped reads to a complete database of bacterial genomes and identified bacteria-associated transcripts which were characterized. This analysis examines the limitations associated with sperm transcriptome profiling by reporting the nature of the RNA sequences among which bacterial RNA was found. These findings can aid researchers in understanding spermatozoal RNA-seq data and pave the way for the identification of molecular markers of sperm performance.