209 MATURATION KINETICS AFTER HOLDING EQUINE OOCYTES IN EMBRYO HOLDING MEDIUM

Abstract

It has been reported that immature, equine oocytes can be maintained in meiotic arrest at 24°C. To evaluate a commercial equine embryo holding medium for storage of equine oocyte at 24°C and to determine the effect of holding on maturation kinetics, cumulus‐oocyte complexes (COC) were recovered from slaughtered mares and placed in Syngro® Embryo Holding Solution at 22–25°C for 18–20 h (OH Group) or placed directly in DMEM-F12-based in vitro maturation (IVM) conditions (D-Mat Group) at 5% CO₂ in air at 38.5°C. Maturation rate (metaphase II percentage; MII%) was assessed (presence of polar body under stereomicroscope) after denudation at 22, 24, and 28 h. After assessment, the denuded oocytes that were considered immature were placed back in IVM, reassessed at 24 and 28 h, and MII% was compared with that of oocytes remaining uninterrupted in IVM for 24 and 28 h. One-way ANOVA was used to compare dependent variable in different groups using PROC ANOVA (SAS, version 9.2, SAS Institute, Cary, NC, USA). A random selection of mature oocytes from both groups were fertilised using intracytoplasmic sperm injection (ICSI). A total of 250 injected oocytes were cultured in DMEM-F12 with 10% FCS. Blastocyst rates in OH and D-mat groups were similar (7.1% v. 6.3%). At 22 h, significantly more oocytes reached the MII stage in the OH group than in the D-Mat group, but MII% was similar in both groups at 24 and 28 h (Table 1). Denuded, immature oocytes reached similar maturation rate as the undenuded oocytes in the same group. Our data suggest that oocytes can be held in Syngro® Embryo Holding Solution at 22–25°C for 18–20 h without compromising oocyte developmental competence. Overnight holding of oocytes accelerates maturation with similar maturation rate at 22, 24, and 28 h of IVM in the OH group. Denudation of immature oocytes after 22 h of IVM and returning the denuded oocytes to IVM does not affect the progression of maturation. In subsequent experiment, overnight held oocytes were fixed and stained (Hoechst 33342) and MII% was evaluated after 20, 22, and 28 h of IVM. Chromatin configuration confirmed that stored oocytes reach the MII stage at 22 h. Maturation rates were significantly lower at 20 h, suggesting that 22 h of IVM is required for stored oocytes.

Table 1.  Maturation rates (% in MII stage) at 22, 24, and 28 h of IVM for equine oocytes held in Syngro^® Embryo Holding Medium before IVM (OH) and oocytes placed directly in IVM (D-Mat)

Thanks to I. Lemahieu and P. Van Damme. Study was supported by the Special Research Fund at UGent.