Milk-derived extracellular vesicles enhance oocyte competence and embryo development during bovine in vitro...

Extracellular vesicles (EVs) have been explored as bioactive components across various fields, including tissue repair, cell culture systems, and therapeutic delivery. In reproductive biology, EVs are investigated as regulators of oocyte maturation and embryo development; however, the effects of milk-derived EVs on bovine oocyte maturation remain poorly understood. This study evaluated milk-derived EV supplementation during bovine in vitro maturation (IVM) on oocyte competence and subsequent embryonic development. Cumulus-oocyte complexes were matured in IVM medium supplemented with milk-derived EVs at 1 × 10⁷, 1 × 10⁸, or 1 × 10⁹ particles/mL, and effects were assessed by oocyte maturation, embryo development, gene expression, intracellular oxidative status, apoptosis- and autophagy-related responses, and developmental kinetics. EV-associated fluorescence signals were observed within cumulus-oocyte complexes after co-incubation with labeled milk-derived EVs. Milk-derived EV supplementation increased first polar body extrusion and cleavage rates, while blastocyst formation and hatching rates were most consistently improved in the 1 × 10⁸ particles/mL group. The 1 × 10⁸ group also showed broad upregulation of oocyte competence-related genes, reduced H₂O₂ and NO levels, increased GSH levels, and enhanced expression of antioxidant defense-related genes. In addition, caspase-3 activity and the expression of apoptosis-related genes, including CTSB and BAD, were decreased, whereas ATG5 and ATG7 expression was increased. Time-lapse analysis showed that embryos derived from the 1 × 10⁸ group reached the 8-cell stage and start of blastulation earlier than control embryos. In conclusion, milk-derived EV supplementation during bovine IVM enhanced bovine oocyte maturation and subsequent embryonic development and was accompanied by favorable changes in intracellular redox status and stress-related cellular responses.