Semester of Graduation

Spring 2018


Master of Science (MS)


Animal Science

Document Type



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 using fresh mature bovine oocytes and vitrified mature bovine oocytes. Oocytes were post-slaughter from mixed breed cows. After collection, oocytes were randomly assigned to three cryoprotectant treatment groups: dimethyl sulfoxide (DMSO), glycerol or phosphate buffered saline (PBS-control). Drops (50ml) 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 minutes before being transferred to VS2 for 45 seconds. All oocytes were washed in a common dilution solution (80% PBS, 20% calf serum, 0.025 M sucrose) for 5 minutes. Next, oocytes were moved to drops (50ml) of protease solution (0.1% protease) under mineral oil. Control oocytes were held in PBS for 5 minutes and 45 seconds before entering the dilution solution for 5 minutes, to represent the same time period as the vitrification procedure. The oocytes were observed until the zona pellucidae were completely digested and times were recorded for each oocyte. This protocol was repeated with the vitrified oocytes, which were vitrified in liquid nitrogen after exposure to the vitrification solutions. The first experiment using fresh oocytes included five replicates with a total of 117 oocytes used, 41 each in DMSO and glycerol and 35 in PBS. The data were analyzed using LSM and contrasts. The differences between DMSO and glycerol were significant (p = 0.0238) but, the differences between DMSO and PBS were not significant (p = 0.3325). However, both the differences between glycerol versus PBS and the average of DMSO and glycerol compared to PBS was significantly different (P-value = 0.0058 and P-value = 0.0262, respectively). The second experiment using vitrified ‘oocytes included three replicates with a total of 114 oocytes used, 37 in DMSO, 41 in glycerol and 36 in PBS. The data were analyzed using LSM and contrasts. In all of the comparisons, no statistical significance was observed (DMSO vs. Glycerol- P=0.5301; DMSO vs. PBS- P=0.1699; Glycerol vs. PBS- P= 0.3818; and DMSO and Glycerol vs. PBS- P=0.2004). These results show that with fresh oocytes, glycerol hardens the zona pellucida more than DMSO or PBS but, there is not enough evidence to determine if DMSO hardens the zona pellucida more than PBS. However, with vitrified oocytes, cryoinjury proved to be too great to accurately measure zona hardening. These data 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.



Committee Chair

Bondioli, Kenneth R.