Effects of various combinations of cryoprotectants and cooling speed on the survival and further development of mouse oocytes after vitrification

OBJECTIVE The objectives of this study were to analyze efficacy of immature and mature mouse oocytes after vitrification and warming by applying various combinations of cryoprotectants (CPAs) and/or super-rapid cooling using slush nitrogen (SN(2)). METHODS Four-week old ICR female mice were superovulated for GV- and MII-stage oocytes. Experimental groups were divided into two groups. Ethylene glycol (EG) only group: pre-equilibrated with 1.5 M EG for 2.5 minutes and then equilibrated with 5.5 M EG and 1.0 M sucrose for 20 seconds. EG+dimethylsulfoxide (DMSO) group: pre-equilibrated with 1.3 M EG+1.1 M DMSO for 2.5 minutes and equilibrated with 2.7 M EG+2.1 M DMSO+0.5 M sucrose for 20 seconds. The oocytes were loaded onto grids and plunged into SN(2) or liquid nitrogen (LN(2)). Stored oocytes were warmed by a five-step method, and then their survival, maturation, cleavage, and developmental rates were observed. RESULTS The EG only and EG+DMSO groups showed no significant difference in survival of immature oocytes vitrified after warming. However, maturation and cleavage rates after conventional insemination were greater in the EG only group than in the EG+DMSO group. In mature oocytes, survival, cleavage, and blastocyst formation rates after warming showed no significant difference when EG only or EG+DMSO was applied. Furthermore, cleavage and blastocyst formation rates of MII oocytes vitrified using SN(2) were increased in both the EG only and EG+DMSO groups. CONCLUSION A combination of CPAs in oocyte cryopreservation could be formulated according to the oocyte stage. In addition, SN(2) may improve the efficiency of vitrification by reducing cryoinjury.