64 INCREASED REACTIVE OXYGEN SPECIES ACTIVITY IN PORCINE OOCYTES AND ZYGOTES VITRIFIED BY SOLID SURFACE VITRIFICATION

Abstract

This study investigated the effect of reactive oxygen species (ROS) activity on the viability and fertilization rate of porcine IVM oocytes and on the developmental competence of IVF zygotes after solid surface vitrification (SSV). The SSV was performed essentially as we described earlier (Gupta MK et al. 2007 Theriogenology 67, 238–248). Briefly, matured oocytes having first polar body or zygotes having second polar body were equilibrated in TCM-199 medium supplemented with 20% (v/v) fetal bovine serum (FBS), 2% (v/v) dimethyl sulfoxide (DMSO), and 2% (v/v) ethylene glycol (EG) for 10 to 15 min. Equilibrated oocytes/zygotes were then rinsed 3 times (<30 s) in vitrification solution consisting of 17.5% (v/v) EG, 17.5% DMSO, 5% (w/v) polyvinylpyrrolidone, 13.7% (v/v) sucrose, and 20% (v/v) FBS in HEPES-buffered TCM-199 medium and then placed in groups of 10 to 15 oocytes/zygotes per 1- to 2-μL droplet onto aluminum foil kept directly over the liquid nitrogen. Vitrified oocytes/zygotes were then directly plunged in liquid nitrogen. Warming was performed in 2 steps in 0.4 and 0.2 m sucrose solution, each for 5 min. Nonvitrified, untreated oocytes/zygotes were used as controls whereas oocytes/zygotes treated with cryoprotectants only were used as toxicity controls (TC). The viability was evaluated by morphological appearance and fluorescein diacetate staining, and ROS level was assessed by 2′,7′-dichlorofluorescene fluorescence staining. Results showed that the percentages of live oocytes (75.0 ± 1.3%) and zygotes (85.5 ± 1.4%) in the SSV group were significantly lower than those of control (100.0 ± 0.0%, 100.0 ± 0.0%) and TC groups (97.3 ± 2.4%, 96.2 ± 0.6%), respectively. Similarly, the ROS levels were significantly greater in SSV groups than in control groups (P < 0.05). Addition of antioxidant (50 μm β-mercaptoethanol) to vitrification and culture media reduced the level of ROS in both oocyte and zygote groups, but did not improve the percentage of live oocytes (73.4 ± 2.2%) and zygotes (88.1 ± 3.7%) after warming. No beneficial effect of antioxidant was observed on the fertilization rate of vitrified oocytes. However, it did improve the cleavage rates of vitrified oocytes (30.0 ± 0.3% v. 21.3 ± 0.8%) and zygotes (64.2 ± 0.6% v. 54.3 ± 0.7%). These data suggest, therefore, that an increase in ROS activity may be associated with poor developmental competence of vitrified oocytes and embryos.