Improving the efficiency of oocyte cryopreservation in veterinary medicine

Oocyte vitrification represents one of the most widely applied cryopreservation techniques, but several biological and technical challenges remain unresolved, particularly in veterinary medicine. This thesis investigated how vitrification influence oocyte quality and survival, focusing primarily on the equine and bovine species. The first study evaluated the effect of naloxone, an opioid antagonist with antioxidant properties, and the impact of an uncontrolled room-temperature holding period prior to vitrification on immature equine oocytes. Vitrification increased oxidative stress, apoptosis, and the proportion of mitochondria with high mitochondrial membrane potential. Overnight holding reduced oxidative stress but also lowered GSH levels after vitrification, potentially impairing developmental competence and altering the oocyte’s response to naloxone. Naloxone improved meiotic competence only in oocytes vitrified immediately after collection. Moreover, it showed antioxidant activity in oocytes vitrified after holding but an opposite effect in those vitrified immediately. The second study extended this investigation to held mature equine oocytes, using bovine oocytes as a comparative model. Overall, naloxone provided no meaningful protection during equine oocyte vitrification, although a slight reduction in ROS indicates limited antioxidant activity. Comparative experiments with bovine oocytes confirmed their value as a preliminary model but highlighted species-specific differences, particularly regarding naloxone’s interaction with the holding phase. The final study assessed a shortened, DMSO-free vitrification commercial protocol using bovine oocytes as a model for human applications. The modified protocol ensured high post-warming survival and stable redox balance across both closed and semi-open vitrification systems, with an observed increase in mitochondrial activity potentially linked to post-warming recovery processes. Collectively, these findings highlight the complexity of oocyte physiology and the influence of species-specific features, maturation stage, and protocol design on vitrification outcomes. The results contribute to refining current cryopreservation strategies and provide a foundation for developing safer, more efficient protocols applicable to animal assisted reproduction.