Optimized protocols of meiotic arrest maintenance improved in vitro maturation of ovine oocytes

Context The developmental potential (DP) of in vitro-matured (IVM) oocytes is considerably lower than that of in vivo-matured oocytes, primarily owing to the premature resumption of meiosis without adequate cytoplasmic maturation during IVM. Evidence indicates that meiotic arrest maintenance (MAM) during IVM significantly enhances oocyte DP. However, studies on ovine oocytes remain scarce. Aims To optimize MAM protocols for improving maturation of ovine oocytes. Methods Ovine oocytes collected from abattoir-derived ovaries were subjected to MAM culture for 24 h, followed by assessment of MAM efficiency and oocyte DP. Key results When used individually, roscovitine (RSC), db-cAMP, and U0126 showed suboptimal MAM efficiency. However, their combined application at substantially reduced concentrations yielded markedly improved outcomes. A combination of 12.5 μM RSC, 1000 μM db-cAMP, and 10 μM U0126 achieved high MAM efficiency, and when MAM lasted for 18 h, it resulted in significantly higher oocyte DP than in control oocytes matured without MAM. Furthermore, oxidative stress was detected during MAM, and the addition of antioxidants during the 24 h MAM period enhanced oocyte DP beyond that of non-MAM controls. Conclusions High MAM efficiency and improved oocyte DP were achieved using a combination of RSC, db-cAMP, and U0126 at reduced concentrations, indicating that meiotic arrest in ovine oocytes is regulated through multiple signaling pathways. Antioxidant supplementation is essential for successful extended MAM. Implications Findings are critical for refining MAM and IVM protocols and for advancing understanding of mechanisms governing oocyte meiotic arrest and maturation.