Enhancing developmental potential of vitrified in vitro matured bovine oocytes using extracellular vesicles from large follicles

This study investigates the differential effects of extracellular vesicles (EVs) derived from bovine follicular fluid (FF) on bovine oocytes cryotolerance, focusing on EVs from small (3-5 mm) and large (> 9 mm) follicles, with the aim of optimizing vitrification protocols. FF-EVs were isolated and characterized for size and concentration. Oocytes were in vitro matured with FF-EVs prior to vitrification. Post-warming assessment included spindle morphology, DNA integrity, embryo development, total cell count, and apoptotic index of resulting blastocysts. EVs uptake by cumulus-oocyte complexes was confirmed via confocal microscopy. While percentages of oocytes at the metaphase II stage were similar across groups, vitrification significantly reduced normal spindle configurations, except in oocytes supplemented with large FF-EVs (VIT Large), which resembled fresh oocytes. DNA fragmentation was significantly lower in the VIT Large group, whereas vitrification alone increased fragmentation. Blastocyst, expansion and hatching rates were higher in VIT Large, which also preserved inner cell mass quality similar to fresh counterparts. These findings suggest that FF-EVs from large follicles mitigate vitrification-induced damage, improving oocyte cryopreservation outcomes and embryo quality. To the best of our knowledge, this is the first study to demonstrate that FF-EVs can improve the cryotolerance of bovine oocytes, establishing a novel approach for enhancing oocyte vitrification outcomes.