Impact of Cryopreservation Extenders on Epigenetic Changes in Bull Sperm: H19 and MEG3 Methylation

Cryopreservation of sperm is critical for livestock genetic improvement, yet its impact on epigenetic stability, especially the methylation of imprinted genes, remains unclear. This study aimed to compare the effects of soy lecithin (SLE) and egg yolk (EYE) extenders on sperm quality and the DNA methylation of the imprinted genes H19 and MEG3 following the cryopreservation process. We evaluated sperm motility parameters, membrane integrity and morphology using six Holstein-Friesian bull sperm samples. Total motility (TM) showed significant reductions after cryopreservation, decreasing from 91.6% ± 1.52% in fresh sperm to 79.9% ± 1.52% in the SLE group and 77.3% ± 1.52% in the EYE group post-thaw (p ≤ 0.05). Similarly, progressive motility (PM) decreased from 64.4% ± 1.8% in fresh sperm to 44.9% ± 1.8% in the (SLE) group and 39.6% ± 1.8% in the (EYE) group. Freezing and post-thawing processes resulted in significant reductions (p ≤ 0.05) in other motility parameters, including linearity (LIN), curvilinear velocity (VCL), straight-line velocity (VSL) and average path velocity (VAP). Following the freezing-thawing process, the SLE group exhibited a smaller reduction in PM and LIN compared to the EYE group. Notably, the SLE extender demonstrated a protective role in membrane integrity compared to the EYE extender (p ≤ 0.05). However, bisulphite sequencing revealed no significant differences in the methylation levels of H19 and MEG3 genes or in sperm morphology between the two extenders (p ≥ 0.05). This study highlights the importance of selecting appropriate extenders in cryopreservation protocols and their implications for future research on sperm quality and fertility.