62 COMPARISON OF SUGARS, COMBINATIONS OF PERMEABLE CRYOPROTECTANTS, AND EQUILIBRATION REGIMENS FOR THE SOLID SURFACE VITRIFICATION OF IMMATURE PORCINE OOCYTES
Authors: Somfai T. , Men N. T. , Kaneko H. , Noguchi J. , Haraguchi S. , Nagai T. , Kikuchi K. 
Published: December 2014

Cryotop and solid surface vitrification are frequently used methods for the cryopreservation of porcine oocytes. These methods differ not only in the vitrification carrier but also in the cryoprotectant (CPA) treatment including the type of sugar, permeable CPA (pCPA) combinations, and the equilibration regimen. This study compared the distinct points of CPA treatment of these 2 methods to determine the optimum CPA treatment for the solid surface vitrification of immature porcine oocytes. We vitrified and warmed follicular cumulus-oocyte complexes by our method (Somfai et al. 2014 PLoS One 9, e97731). In each experiment, the vitrification solution consisted of 50 mg mL–1 polyvinyl pyrrolidone, 0.3 M of the actual sugar, and 35% [v/v] in total of the actual pCPA combination (depending on the experiment). After warming, the cumulus-oocyte complexes were subjected to in vitro maturation, IVF, and embryo culture (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). Oocyte survival was assessed after IVF by morphological evaluation, and live oocytes were subjected to in vitro embryo culture. Cleavage and blastocyst rates were calculated from cultured oocytes on Day 2 (Day 0 = IVF) and Day 6, respectively. Each experiment was replicated at least 3 times. Results were analysed by ANOVA. In Experiment 1, we compared trehalose (n = 416) and sucrose (n = 440) as supplementations during vitrification and warming (0.3 M and 0.4 M of each, respectively). There was no significant difference between oocytes vitrified with trehalose or sucrose in terms of survival, cleavage, and blastocyst development (83.2% v. 80.3%, 39.7% v. 42.4%, and 3.6% v. 5.9%, respectively). Thus, vitrification and warming media were supplemented with sucrose thereafter. In Experiment 2, we compared 1 : 1 combinations of ethylene glycol with propylene glycol (EG+PG group, n = 452) and ethylene glycol with dimethyl sulfoxide (EG+DMSO group, n = 465) used as pCPA for equilibration (4% [v/v] pCPA in total for 15 min) and vitrification (35% [v/v] pCPA in total for 30 s). Oocyte survival rate was higher (P < 0.05) in the EG+PG group compared with the EG+DMSO group (73.8% v. 51.1%, respectively); however, cleavage and blastocyst development rates of surviving oocytes were not significantly different between the 2 groups (30.5% v. 44.5% and 4.1% v. 6.3%, respectively). In Experiment 3, we compared an equilibration treatment in 4% [v/v] of EG+PG for 13 to 15 min (regimen A, n = 368) with an equilibration in 15% [v/v] of EG+PG for 5 to 7 min (regimen B, n = 363) for oocyte vitrification. Survival, cleavage, and blastocyst development rates were higher (P < 0.01) for oocytes vitrified using regimen A compared with those vitrified using regimen B (82.5% v. 22.7%, 24.0% v. 7.7%, and 3.2% v. 0%, respectively). In conclusion, trehalose and sucrose are equally effective during vitrification and warming, the combination of EG+PG as pCPA is superior to EG+DMSO, and equilibration in 4% pCPA for 13 to 15 min is superior to that in 15% pCPA for 5 to 7 min for the vitrification of immature porcine oocytes.This work was partly supported by JSPS KAKENHI Grant Number 26870839.