Holding equine oocytes in a commercial embryo-holding medium: New perspective on holding temperature and maturation time

In the present study, we examined the effect of holding equine oocytes in Syngro embryo holding medium (EHM) overnight at either 4 °C, 17 °C, or 22 °C to 25 °C, on the time to maturation and developmental competence. We also examined the effect of placing denuded oocyte without extruded polar body back in maturation condition on subsequent maturation rate. In experiment 1, cumulus-oocyte complexes (COCs) were recovered postmortem and placed in EHM at 22 °C to 25 °C for 18 to 20 hours (OH) or placed directly in maturation (DM). The maturation rate was assessed after 22, 24, or 28 hours of culture. After denuding cumulus cells at 22 or 24 hours, oocytes without obvious polar body were placed back into culture and reassessed at subsequent time points. At 22 hours, a higher proportion of oocytes placed in OH achieved nuclear maturation than those placed in DM (63% and 37%, respectively, P = 0.008). At 24 and 28 hours, no significant differences in the % MII stage oocytes were observed between OH and DM. The nuclear maturation rate for OH oocytes was similar at 22, 24, and 28 hours, indicating that the maximum maturation rate was reached at an earlier time than that in DM. Oocytes fertilized by intracytoplasmic sperm injection resulted in a 7.1% and 6.3% blastocyst rate for OH and DM, respectively. Denuding oocytes after 22 hours or more of culture did not have an adverse effect on the final nuclear maturation rate. After 28 hours of culture, the same nuclear maturation rate (MII) was reached for nondenuded oocytes and oocytes denuded after 22 hours of 24 hours of culture. In experiment 2, COCs were held overnight at room temperature in EHM, then placed in maturation for 20, 22, and 28 hours. Nuclear maturation rate was significantly lower at 20 hours than 22 and 28 hours of culture and was similar at 22 and 28 hours, suggesting that at least 22 hours of culture is required to reach maximal maturation rate for stored oocytes (43%, 62%, and 65% at 20, 22, and 28 hours, respectively. P