Differentially methylated CpG sites in bull spermatozoa revealed by human DNA methylation arrays and bisulfite analysis.

The methylation status of sperm DNA differs between individual bulls. However, the relationship between methylation status and bull sperm parameters is not well elucidated. The present study investigated genome-wide methylation profiles at 450,000 CpG sites in bull spermatozoa by using a human DNA methylation microarray. Semen samples from three adult Japanese Black bulls with different in vitro fertilization (IVF) results and from a young Holstein bull through sexual maturation (at ages 10, 10.5, 15, and 25 months) were used for the analysis. The heatmap displaying the results of microarray analysis shows inter- and intra-individual differences in methylation profiles. After setting a cut-off of 0.2 for differences between ages (10, 10.5 vs. 15, 25 months) or between IVF results (developed to the blastocyst-stage, > 20% vs. < 10%), different methylation levels were detected at approximately 100 CpGs. We confirmed the different DNA methylation levels of CpG sites by using combined bisulfite restriction analysis (COBRA); five of the CpG sites reflected methylation levels similar to those detected by the microarray. One of the CpG sites was thought to reflect an age-related increase in methylation levels, which was confirmed by COBRA and bisulfite sequencing. However, the relationship between methylation status and IVF results could not be shown here. In conclusion, methylation profiles of individual and age-related alterations in bull spermatozoa can be revealed using a human microarray, and methylation changes in some CpG sites can be easily visualized using COBRA. Combined analysis of DNA methylation levels and sperm parameters could be considered an effective approach for assessing bull fertility in the future.