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

## TL;DR

This study compares soy lecithin and egg yolk extenders in preserving bull sperm quality and epigenetic stability after freezing.

## Contribution

The study evaluates the impact of cryopreservation extenders on imprinted gene methylation and sperm quality in bull sperm.

## Key findings

- Soy lecithin extender preserved sperm motility and membrane integrity better than egg yolk extender.
- No significant differences in H19 and MEG3 gene methylation were observed between the two extenders.
- Cryopreservation reduced sperm motility parameters regardless of the extender used.

## Abstract

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.

This study compared the cryoprotective effects of soy lecithin (SLE) and egg yolk (EYE) extenders on post‐thaw bull sperm quality and the methylation status of imprinted genes H19 and MEG3.

The visual abstract outlines the experimental workflow: Bull sperm collection, dilution with either SLE or EYE extenders, followed by cryopreservation (symbolized by ice crystals and liquid nitrogen) and thawing (represented by a warming water bath).

Key results are presented visually and categorically:

Sperm quality analysis: Microscope‐based evaluation revealed that SLE was superior to EYE in preserving total motility, progressive motility and membrane integrity, as indicated by upward and downward directional arrows.

Epigenetic analysis: Bisulphite sequencing showed no significant differences in the DNA methylation patterns of the H19 and MEG3 genes between the two extenders, depicted by identical DNA helices.

The conclusion demonstrates that the soy lecithin‐based extender offers enhanced protection for sperm quality parameters without inducing significant epigenetic alterations in key imprinted genes.