Temporal optimization of meiotic arrest for enhancing oocyte maturity during in vitro maturation of porcine median antral follicles

During in vitro maturation (IVM), median antral follicles (MAFs) were mechanically aspirated from the porcine ovarian cortex, and this process causes an early disconnection of follicular somatic cells from oocytes within antral follicles before the formation of graafian follicles. Thus, nuclear maturation is accelerated ahead of the completion of cytoplasmic maturation. Dibutyryl-cAMP (dbcAMP), a well-known cAMP modulator, is used to inhibit the resumption of meiosis in immature oocytes. However, there is no information on the optimal timeframe for sustaining meiotic arrest to enhance oocyte maturity during IVM. To determine the optimal duration of meiotic arrest, immature cumulus-oocyte complexes (COCs) from MAFs were cultured with 1 mM dbcAMP for 0, 4, 8, 12, 16, or 22 h, followed by further IVM without dbcAMP for 44, 40, 36, 32, 28, or 22 h. Subsequently, nuclear maturation, cumulus cell expansion score, perivitelline space size, glutathione (GSH) and reactive oxygen species (ROS) levels, and preimplantation development of parthenogenetic and in vitro-fertilized embryos were assessed in oocytes from each group. The results showed that a 16-h treatment with 1 mM dbcAMP within the 44-h IVM process yielded the highest oocyte maturity. Accordingly, we established an advanced IVM protocol for producing oocytes with superior maturity from porcine MAFs by achieving nuclear maturation 36 h after initiating IVM, using a 16-h treatment with 1 mM dbcAMP within the 44-h IVM process.