A study on cryoprotectant solution suitable for vitrification of rat two-cell stage embryos

• Published in: Cryobiology 2014-02, Vol.68 (1), p.147-151
• Main Authors: Eto, Tomoo, Takahashi, Riichi, Kamisako, Tsutomu, Hioki, Kyoji, Sotomaru, Yusuke
• Format: Article
• Language: English
• Subjects: Animals; Cell Membrane Permeability; Cell Survival - drug effects; Cryopreservation; Cryoprotectant solution; Cryoprotective Agents - pharmacology; Dimethyl Sulfoxide - pharmacology; Embryo, Mammalian - cytology; Embryo, Mammalian - drug effects; Embryo, Mammalian - physiology; Embryonic Development - drug effects; Ethylene Glycol - pharmacology; Female; Glycerol - pharmacology; Male; Osmolar Concentration; P10; PEPeS; Povidone - pharmacology; Propylene Glycol - pharmacology; Rat embryos; Rats; Silicon Dioxide - pharmacology; Strain preservation; Sucrose - pharmacology; Vitrification
• ISSN: 0011-2240; 1090-2392
• DOI: 10.1016/j.cryobiol.2014.01.011

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, 0.3mol 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 600s 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.