In vitro embryo production has become a popular breeding technique in horses, but suboptimal conditions for in vitro maturation and oocyte cryopreservation limit its full potential. When in vivo matured oocytes are used, high embryo development rates can be achieved with both fresh and vitrified oocytes, indicating the presence of crucial signaling factors in the natural environment, the growing follicle. Extracellular vesicles (EVs), present in the follicular fluid, carry regulatory molecules, such as microRNAs, playing a key role in oocyte maturation. Therefore, we hypothesize that supplementation of the in vitro maturation medium with EVs from follicular fluid of healthy mares can improve developmental competence of fresh and vitrified equine oocytes. In this project, we will assess three groups of follicular EVs, representing follicular dynamics during oocyte maturation. Molecular mechanisms underlying improved embryo development will be unravelled by profiling the microRNAs in beneficial EVs, as well as the direct and indirect effects on the transcriptome of the cumulus cells and the resulting embryos. Finally, the functional effect of promising EV-derived candidate miRNAs will be validated using mimics and inhibitors during in vitro maturation. The ultimate goal is to identify key molecules for successful in vitro maturation and oocyte vitrification, providing fundamental knowledge, with the potential for clinical application and translation to other species.