40 THE EFFECT OF EXPOSURE TIME ON TOXICITY OF VITRIFICATION SOLUTION ON PORCINE CUMULUS–OOCYTE COMPLEXES BEFORE IN VITRO MATURATION

Abstract

Although offspring have been produced from porcine cumulus-oocyte complexes (COC) vitrified at the immature stage (Somfai et al. 2014 PLoS One 9, e97731), embryo development rates have remained low. Numerous vitrification protocols are reported with a wide variation in the applied exposure time to the vitrification solution. Because cryoprotectants in the vitrification solution can be detrimental to the oocytes and their subsequent development, it is important to verify the effect of their exposure time to the COC. In this study, we compared the development of a control group with 3 toxicity control (TC) groups in which COC were exposed to the vitrification solution for 30 s, 1 min, or 1.5 min (TC1, TC2, and TC3, respectively) at 38.5°C. Before exposure, the COC were rinsed and equilibrated in 7 µg mL⁻¹ cytochalasin B. The equilibration solution consisted of 2% (vol/vol) ethylene glycol + 2% (vol/vol) propylene glycol and the vitrification solution contained 17.5% (vol/vol) ethylene glycol + 17.5% (vol/vol) propylene glycol, 50 mg mL⁻¹ polyvinylpyrrolidone and 0.3 M sucrose. The COC were not exposed to liquid nitrogen. After washing in a warming solution of 0.4 M sucrose at 42°C, COC were washed in a sucrose gradient from 0.2 to 0.0 M. Subsequently, the COC were subjected to in vitro maturation in porcine oocyte medium. During the first 20 h of in vitro maturation, the porcine oocyte medium was supplemented with 10 IU mL⁻¹ eCG, 10 IU mL⁻¹ hCG, 1 mM dibutyryl cAMP, and 10 ng mL⁻¹ epidermal growth factor. Then, the medium was replaced with dibutyryl cAMP-free porcine oocyte medium for an additional 28 h. After in vitro maturation, oocytes were parthenogenetically activated (Day 0) and cultured for 7 days in porcine zygote medium. Survival, nuclear maturation, cleavage, and blastocysts rates (Days 6 and 7) were assessed. All parameters were statistically analysed by binary logistic regression. Only the survival rate of TC3 was significantly lower than that of the control group (89.2 v. 95.6%). Exposure to cryoprotectants significantly decreased maturation rates in TC1, TC2, and TC3 compared with the control (72.6%, 75.2%, 76.3% v. 86.1%). Cleavage rates were significantly lower in TC2 and TC3 than that in the control (82.8% and 81.7% v. 92.9%). Concerning blastocyst rates on Day 6 and Day 7 of in vitro culture, only TC1 could reach the same level as the control, expressed on the total number of activated oocytes (54.6% v. 67.7%, and 64.0% v. 72.9%, respectively) as well as expressed on the cleaved oocytes (61.4% v. 72.4% and 72.0% v. 78.0%, respectively). Consequently, despite the reduced maturation rate, TC1 provides the same quantity of blastocysts from matured oocytes as the control. In conclusion, exposure to the vitrification solution for longer than 30 s has toxic effects on COC and therefore is not recommended for vitrification.

R. Appeltant is an International Research Fellow of the JSPS Japan (P15402).