Nicotinic and Folic Acid Supplementation Improves Cryopreserved Ram Sperm Quality, Potentially Through Modulation of OGG1 and ROMO1 Expression and DNA Methylation.

This study aimed to evaluate the effects of nicotinic acid (NA) and folic acid (FA) as antioxidants on post-thaw spermatological characteristics and OGG1/ROMO1 gene expression and methylation. Collected ram semen was pooled and divided into four equal aliquots, diluted with different dose groups: Control, 10 mM NA with no FA (NA10), 10 mM NA + 50 nM FA (NA10FA50) and 20 mM NA + 50 nM FA (NA20FA50). Although not statistically significant (p > 0.05), the NA10FA50 (53.34%) group showed the highest numerical mean motility, whereas the control group (43.44%) showed the lowest numerical mean motility. There were no statistical differences in terms of plasma membrane and DNA integrity, malondialdehyde and total antioxidant capacity rates. The lowest acrosomal damage was obtained in the NA10FA50 group (36.50%), which was only statistically different from the control (48.00%) (p < 0.05). The lowest mitochondrial membrane potential was observed in the control group (13.80%) (p < 0.05). NA10FA50 and NA20FA50 altered OGG1 gene expression, which was accompanied by corresponding changes in DNA methylation, suggesting a potential epigenetic regulatory relationship. ROMO1 expression was significantly higher in the NA10 compared to the NA10FA50 (p < 0.01), accompanied by differences in DNA methylation patterns (p < 0.05). This study suggests that the addition of 10 mM NA or a combination with 50 nM FA showed beneficial effects on the post-thaw quality of ram semen, particularly with respect to acrosomal integrity and mitochondrial membrane potential and selected molecular markers.