The present study was performed to develop a suitable cryoprotectant solution for cryopreservation of rat two-cell stage embryos. First, we examined the cell permeability of several cryoprotectants; propylene glycol had the fastest permeability compared to dimethyl sulfoxide, ethylene glycol, and glycerol. Embryos were then exposed to a solution containing propylene glycol to evaluate its effects on fetal development. As the development was similar to that of fresh embryos, P10 (10% v/v propylene glycol in PB1) was used as a pretreatment solution. Next, the effects of the vitrification solution components (sucrose, propylene glycol, ethylene glycol, and Percoll) were examined by observing the vitrification status; 10% v/v propylene glycol, 30% v/v ethylene glycol, 0.3 mol sucrose, and 20% v/v Percoll in PB1 (PEPeS) was the minimum essential concentration for effective vitrification without the formation of ice crystals or freeze fractures. A new vitrification method using P10 and PEPeS was tested using rat embryos. The survival rate of vitrified embryos after exposure to P10 for 120, 300, or 600 s ranged from 95.9% to 98.3%. The fetal developmental rate ranged from 57.7% to 65.2%, which was not significantly different from that of fresh embryos. The experimental results indicated that vitrification using a combination of P10 and PEPeS was suitable for cryopreservation of rat early stage embryos.
Keywords: Cryopreservation; Cryoprotectant solution; P10; PEPeS; Rat embryos; Strain preservation; Vitrification.
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