Extracellular vesicles from the follicular fluid of competent oocytes improve blastocyst yields when supplemented during simulated physiological oocyte maturation in cattle

The objectives of this work were to evaluate: i) the miRNA profile of extracellular vesicles (EVs) isolated from bovine follicular fluid (FF) of follicles enclosing competent (FF-C) and non-competent (FF-NC) oocytes; ii) the effect of incubating bovine cumulus-oocyte complexes (COCs) during a simulated physiological oocyte maturation system (SPOM) with EVs derived from FF-C and FF-NC on developmental capacity, iii) the effect of miR-143, one of the most abundant miRNAs found in EVs isolated from FF-C, during SPOM on oocyte competence. FF was collected from individual follicles of abattoir-derived ovaries and retrospectively categorized based on the developmental outcome of corresponding oocytes: blastocyst (B), cleaved-arrested (C), and uncleaved (U). EVs were isolated from FF pools by ultracentrifugation, characterized by Nanosight, TEM, and Western Blot, and analyzed for miRNA profile. The EVs from competent follicles (B) were larger and more concentrated than those from non-competent (U) ones, and carried distinct miRNA cargo, including higher levels of miR-143. Bovine COCs were matured in SPOM with 0, 1, 10, or 100 × 10⁶ EV/mL corresponding to FF-C or FF-NC. Only EVs from FF-C improved blastocyst rates, with the optimal response at 10 × 10⁶ EV/mL (59.1 ± 4.1 vs 41.8 ± 6.4 %). Then, SPOM was supplemented with miR-143 mimic, inhibitor, or corresponding negative controls. While the mimic did not significantly enhance development, inhibition of miR-143 reduced blastocyst rates, suggesting a modulatory role. These findings indicate that EVs from FF-C can enhance oocyte developmental competence during SPOM, while miR-143 alone is not effective.