41 Effects of Dimethyl Sulfoxide- or Glycerol-Based Vitrification Protocols on Zona Pellucida Hardening in Mature Bovine Oocytes

Abstract

Zona pellucida hardening is a natural process that occurs after oocyte fertilization to prevent polyspermic fertilization and to protect embryonic development. Pre-fertilization hardening of the zona pellucida, however, decreases fertilization rates. Cryoprotectants have also been shown to negatively affect fertilization rates, one possible mechanism of which being through zona hardening. This experiment was conducted to determine the effect of different cryoprotectants on hardening of the zona pellucida of mature bovine oocytes. Oocytes were collected by ovum pick-up (OPU) by transvaginal ultrasound guided aspiration (TUGA) from mixed-breed cows. After collection, oocytes were randomly assigned to 3 cryoprotectant treatment groups: dimethyl sulfoxide (DMSO), glycerol, or PBS (control). Drops (50 µL) of each vitrification solution were placed under mineral oil. Vitrification solution 1 (VS1) contained 10% ethylene glycol (EG), either 10% DMSO or glycerol, and 0.5 M sucrose. Vitrification solution 2 (VS2) contained 20% EG, 20% DMSO or glycerol, and 0.5 M sucrose. All oocytes were held in VS1 for 5 min before being transferred to VS2 for 45 s. All oocytes were washed in a common dilution solution (80% PBS, 20% calf serum, 0.025 M sucrose) for 5 min. Next, oocytes were moved to 50-µL drops of protease solution (0.1% protease) under mineral oil. Control oocytes were held in PBS for ~10 min before entering the protease solution to represent the same period as the vitrification procedure. The oocytes were observed until the zonae pellucidae were completely digested and times were recorded for each oocyte. This experiment included 4 replicates with a total of 88 oocytes used, 32 each in DMSO and glycerol and 24 in PBS. The data were analysed using ANOVA. The DMSO group had the lower mean zona digestion time out of the 2 cryoprotectants at 15.75 min and glycerol had the highest mean digestion time at 19.3 min. The control group (PBS) had the lowest mean of the 3 treatments at 12.7 min. The differences between DMSO and glycerol, and between DMSO and PBS were not significant (P = 0.0654 and 0.1073, respectively). However, both glycerol versus PBS and the average of DMSO and glycerol versus PBS were significantly different (P-value = 0.0053 and 0.0119, respectively). These results suggest that glycerol hardens the zona pellucida more than DMSO or PBS; however, there is not enough evidence to determine whether DMSO hardens the zona pellucida compared with PBS. This would suggest that, in relation to zona hardening and ensuring proper fertilization, glycerol-based cryoprotectants may be a better option than DMSO-based ones. Further, these results may be important in embryo vitrification as zona hardening may prevent blastocyst hatching, suggesting that glycerol-based cryoprotectants should be investigated as the optimal cryoprotectant here also.