Small non coding RNA from frozen bull sperm cells: A Biomarkers panel of male fertility

New sperm function in embryo development have emerged recently, relying on their small noncoding RNAs (sncRNAs) content. Indeed, involvement of sperm-borne miRNAs in mouse epigenetic inheritance has been evidenced and paternal sncRNAs have been shown to be dispensable for fertilization but crucial for the development of zygotes and 2 cells-embryos. Our study was conducted to unravel the sncRNA content from bull frozen sperm cells and identify miRNA associated with fertility. Total RNA was extracted from 40 ejaculates originating from Holstein and Montbeliard bulls with contrasting fertility, using a novel enhanced protocol. The two quality controls were done on RNA to measure RNAs quantities (Qubit technology) and check whether a reference miRNA (miR125) could be detected by RTqPCR. NGS sequencing libraries were prepared using small RNA (<200 nucleotides) and sequenced at modest depth (40 million 50 bp single reads, Illumina HiSeq; Exiqon). Two thirds of the reads could be annotated as miRNA (16%), rRNA (13%), tRNA (7.5%), long non coding RNA (7.2%), as mitochondrial RNA (6.5%) and mRNA (17%). The remaining unknown sequences are consistent in terms of size with piRNA, which are known to be expressed spermatocytes, spermatids and sperm cells. By the use of miRDeep2 software, 3196 miRNA expressed in all samples have been identified, including 583 known and 2613 putative miRNAs. Statistical analysis carried out using the DESeq2 package, emphasized 47 differentially expressed miRNA between fertility groups. This study highlights the potential of sperm cells’ sncRNA as biomarkers for bull fertility.