Effects of long-term cryopreservation on sperm quality and oxidative stress in Manchega rams (Ovis aries)

In brief: This study evaluated the impact of long-term cryopreservation on oxidative stress and sperm quality in Manchega rams, suggesting that sperm quality decline may be due primarily to cryopreservation duration rather than genetic effects. Abstract: Cryopreservation plays an essential role in artificial insemination protocols and in the establishment of germplasm banks. However, cryopreservation is a key factor contributing to the increase in oxidative stress, potentially leading to deterioration of sperm quality over time. This study examined whether oxidative stress levels differed in Manchega sheep sperm samples stored for up to 25 years. It was hypothesised that these differences could be due to storage time or genetic selection focused on productive traits, given the antagonism between productive and reproductive traits. Frozen sperm samples from 145 rams, all cryopreserved at the same age and processed under identical conditions to minimise variation, were analysed. Sperm viability, intracellular reactive oxygen species production, lipid peroxidation, and chromatin integrity were assessed. The impact of cryopreservation storage duration on sperm quality was evaluated, and genetic trends were estimated using pedigree and genomic data to explore potential associations with reproductive genes. Our results revealed minimal changes in sperm quality parameters across generations. A negative trend was observed in oxidative stress indicators, while sperm viability exhibited a slight positive trend, suggesting that oxidative stress may have a greater impact on older frozen samples. Genetic trend analysis revealed no significant differences, and no associations with reproductive genes were found, which indicates that the deterioration in sperm quality was primarily due to storage time rather than genetic selection. However, sperm quality is influenced by multiple genes not examined in this study, and a genetic effect cannot be completely discarded.