Rapid warming improves blastocyst production of vitrified bovine oocytes while significantly affecting mitochondrial activity

Improvement of bovine oocyte vitrification focuses mainly on the cooling step, while warming steps receive little attention. Recent studies report improved in vitro embryo production (IVEP) efficiency in vitrified rodent embryos warmed rapidly at high temperatures (50-60°C). This effect has not yet been tested on bovine oocytes; therefore, this study aimed to evaluate rapid warming's influence on mitochondrial functionality, nuclear DNA integrity, and IVEP efficiency. Three groups of in vitro matured oocytes were included: 1) control, 2) SW - vitrified oocytes warmed slowly, and 3) RW - vitrified oocytes warmed rapidly. Oocytes were assessed in two experiments for: (1) embryo production and (2) mitochondrial functionality and DNA integrity using MitoTracker® Red CMXRos and Hoechst33342 dyes. All vitrified-warmed oocytes were examined for cytoplasm expansion and rupture after warming. In Experiment 1, cleavage rates differed significantly among all groups, with RW showing higher rates than SW. In Experiment 2, the strongest MitoTracker® Red CMXRos fluorescence was observed in the control group, followed by SW and RW. No significant differences were found between SW and RW in DNA degeneration, cytoplasm expansion, or rupture. Although mitochondrial mass was lowest in RW, higher temperature did not increase DNA damage or affect cytoplasm expansion compared to SW. Moreover, RW achieved the best cleavage and blastocyst rates among vitrified-warmed groups. These findings indicate that rapid warming does not compromise oocyte integrity and may improve IVEP outcomes. The data suggests potential for incorporating rapid warming into vitrification protocols for mature bovine oocytes.