Extracellular vesicles from small-follicle follicular fluid improve redox balance and embryo development in naturally heat-stressed bovine oocytes.

Heat stress is a major factor impairing cattle fertility by reducing oocyte competence and embryo development leading to significant economic losses. Follicular fluid-derived extracellular vesicles (FF-EVs) are key mediators of intercellular communication within the ovarian follicle and may improve oocyte competence. However, their potential to mitigate naturally occurring seasonal heat stress remains unclear. This study evaluated the impact of supplementing the in vitro maturation medium with FF-EVs from small (3-5 mm) or large (>9 mm) follicles from normothermic conditions on bovine oocytes collected during summer. EVs were isolated by size exclusion chromatography and characterized by nanoparticle tracking analysis, cryo-electron microscopy, and flow cytometry. Oocyte competence was evaluated through oxidative status and DNA fragmentation, while embryo development and quality were assessed by developmental rates, redox status, differential cell count, apoptosis, and lipid content. Supplementation with FF-EVs derived from small follicles (Small FF-EVs) reduced intracellular reactive oxygen species (ROS) levels and DNA fragmentation in oocytes, while significantly increasing blastocyst rates. In addition, Small FF-EVs enhanced embryonic redox balance and reduced lipid content. In contrast, EVs from large follicles increased oxidative stress and DNA damage in oocytes, although they reduced ROS levels and lipid droplet volume in blastocysts without affecting embryo development rates. No significant differences were observed in total cell number or apoptosis rates. Overall, FF-EVs exert differential effects on oocyte competence and embryo development according to their follicular origin. In particular, Small FF-EVs enhance oocyte capacity to withstand naturally occurring heat stress and represent a promising strategy for improving reproductive outcomes under challenging environmental conditions.