Cooled-storage of equine semen does not induce major changes in sperm DNA methylation

A decrease in fertility of equine semen during cooled-storage so far has mainly been attributed to changes in sperm membrane function. In the present study we hypothesized that cooled-storage also changes the sperm DNA methylation level. For this purpose, semen was collected from 10 fertile stallions and processed for cooled-storage at 5 °C. Two final concentrations, 50 × 10⁶ and 100 × 10⁶ cells/mL, were used. Semen was analyzed for total motility, progressive motility, membrane integrity, phosphatidylserine translocation (PST), mitochondrial membrane potential and chromatin condensation, immediately after processing and at 24 h-intervals until 72 h of storage. DNA cytosine methylation was assessed by ELISA after DNA extraction and denaturation. DNA methylation did neither change over time nor was affected by semen concentration. Total motility, progressive motility, membrane integrity, PST, mitochondrial membrane potential and chromatin condensation changed over storage time, but no differences between semen concentrations could be demonstrated. The results demonstrate that cooled-storage of equine semen does not induce changes in sperm DNA cytosine methylation. In cooled-semen of good quality, a concentration of 100 × 10⁶ sperm/mL does not affect semen longevity. It can be concluded that a better fertility of cooled-stored than cryopreserved stallion semen is at least in part a result of only minor influences of cooled-storage on DNA integrity and methylation.