79 A COMBINATION OF ETHYLENE GLYCOL AND PROPYLENE GLYCOL IS SUPERIOR TO INDIVIDUAL CRYOPROTECTANTS FOR THE VITRIFICATION OF IMMATURE PORCINE OOCYTES

Abstract

We compared the feasibility of ethylene glycol (EG) and propylene glycol (PG) for the vitrification of immature porcine cumulus–oocyte complexes (COC). Porcine COC collected from 3- to 6-mm follicles of slaughterhouse-derived ovaries were subjected to solid-surface vitrification (Somfai et al. 2010 Theriogenology 73, 147–156) either in 35% (v/v) EG or 35% (v/v) PG or in the mixture of 17.5% (v/v) EG and 17.5% (v/v) PG. After warming, the COC were subjected to in vitro maturation, IVF, and embryo culture according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041). Oocyte survival and maturation rates were assessed after in vitro maturation by evaluating membrane integrity and the extrusion of the first polar body. All live oocytes were subjected to IVF and in vitro culture. Cleavage and blastocyst rates were calculated from the total number of oocytes subjected to IVF on Day 2 (Day 0 = IVF) and Day 7, respectively. Total-cell (blastomeres) numbers in blastocysts were recorded on Day 7 after staining with Hoechst 33342. In Experiment 1, competence parameters of oocytes vitrified either in EG-based (EG group; n = 310) or a PG-based (PG group; n = 265) vitrification media were compared with those in the nonvitrified control (n = 160). The experiment was replicated 4 times. In Experiment 2, the competence parameters of oocytes vitrified with the combination of 17.5% EG and 17.5% PG (EG+PG group; n = 397) were compared with those in nonvitrified control (n = 245) and toxicity control (TC, exposed to cryoprotectants without cooling; n = 245) groups. Five replications were performed. Results were analyzed by ANOVA. Differences with P < 0.05 were considered significant. In Experiment 1, the mean survival rate of vitrified oocytes was significantly higher (P < 0.05) in 35% PG compared with that in 35% EG (73.3 and 25.9%, respectively). Maturation rates of surviving oocytes did not differ among vitrified (PG and EG) and nonvitrified control groups (71.1, 62.4, and 64.0%, respectively). After IVF of surviving oocytes, blastocyst formation rate in the group vitrified in EG was higher (P < 0.05) compared with that vitrified in PG but was lower (P < 0.05) compared with control (10.8, 2.0, and 25.0%, respectively). Mean cell numbers in blastocysts did not differ among EG, PG, and control groups (50.5, 47.7, and 48.7, respectively). In Experiment 2, survival of immature oocytes in the EG+PG group was 42.6%. After IVF, 10.7% of oocytes developed to the blastocyst stage in the EG+PG group, which was lower (P < 0.05) than those of the control (18.1%) and TC (23.3%) groups. Blastocyst rates in the control and TC groups were not statistically different. Mean cell numbers in blastocysts did not differ significantly among the EG+PG, control, and TC groups (61.6, 59.3, and 53.3, respectively). In conclusion, 35% PG provided a higher oocyte survival rate after vitrification compared with 35% EG. However, presumably due to toxic effects, 35% PG greatly reduced the development competence of oocytes. The combination of 17.5% EG and 17.5% PG yielded higher survival rates than did 35% EG, without any toxic effect on oocytes.