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Genomic selection in modern farming demands sufficient semen production in young bulls. Factors affecting semen quality and production capacity in young bulls are not well understood; DNA methylation, a complicated phenomenon in sperm cells, is one such factors. In this study, fresh and frozen-thawed semen samples from the same Norwegian Red (NR) bulls at both 14 and 17 months of age were examined for sperm chromatin integrity parameters, ATP content, viability, and motility. Furthermore, reduced representation bisulfite libraries constructed according to two protocols, the Ovation ®  RRBS Methyl-Seq System (Ovation method) and a previously optimized gel-free method and were sequenced to study the sperm DNA methylome in frozen-thawed semen samples. Sperm quality analyses indicated that sperm concentration, total motility and progressivity in fresh semen from 17 months old NR bulls were significantly higher compared to individuals at 14 months of age. The percentage of DNA fragmented sperm cells significantly decreased in both fresh and frozen-thawed semen samples in bulls with increasing age. Libraries from the Ovation method exhibited a greater percentage of read loss and shorter read size following trimming. Downstream analyses for reads obtained from the gel-free method revealed similar global sperm DNA methylation but differentially methylated regions (DMRs) between 14- and 17 months old NR bulls. The majority of identified DMRs were hypomethylated in 14 months old bulls. Most of the identified DMRs (69%) exhibited a less than 10% methylation difference while only 1.5% of DMRs exceeded a 25% methylation difference. Pathway analysis showed that genes annotated with DMRs having low methylation differences (less than 10%) and DMRs having between 10 and 25% methylation differences, could be associated with important hormonal signaling and sperm function relevant pathways, respectively. The current research shows that RRBS in parallel with routine sperm quality analyses could be informative in reproductive capacity of young NR bulls. Although global sperm DNA methylation levels in 14 and 17 months old NR bulls were similar, regions with low and varying levels of DNA methylation differences can be identified and linked with important sperm function and hormonal pathways.

Sperm DNA Hypomethylation Proximal to Reproduction Pathway Genes in Maturing Elite Norwegian Red Bulls