Effects of Cryoprotectant Combinations on Post-Thawed Sperm Quality, Biomolecular Changes, DNA Methylation, and Pregnancy Rates in Boer Goat Semen

Abstract

This study investigated the effects of various cryoprotectant combinations on post-thaw sperm quality, biomolecular changes, DNA methylation, and pregnancy rates using Boer goat semen. Synchrotron-based Fourier-transform infrared spectroscopy (SR-FTIR) was used to assess biomolecular changes. A Tris-based extender supplemented with 5% glycerol was used in combination with different concentrations of cryoprotectants, including 1% and 3% soybean lecithin and 10% and 18% egg yolk, with Andromed® serving as the control. SR-FTIR analysis revealed that the combination of 5% glycerol and 18% egg yolk (T4) resulted in significantly higher levels of lipids, ester lipids, and secondary protein structures (α-helix) compared with those under the other treatments (p < 0.05). Analysis of the principal component analysis (PCA) score plot and correlation loadings revealed a positive association between the cryoprotectant combination of T4 and increased levels of lipids and ester lipids, as well as enhanced sperm motility, progressive motility, and viability. Furthermore, this combination achieved a pregnancy and parturition rate of 66.67%, which was notably higher than the rate achieved with Andromed® (37.50%). Moreover, T4 did not show a significant difference in DNA methylation levels compared to Andromed® and fresh sperm (p > 0.05). Overall, the results indicated that specific cryoprotectant combinations play a key role in enhancing the biomolecular and functional integrity of freeze-thawed Boer goat semen.