Vitrification of Mouse and Sheep Embryos

Vitrification is a method of storing cells in a supercooled state, and is particularly useful for the cryostorage of embryos. With a supply of liquid nitrogen and simple apparatus, vitrification in the field situation is feasible and is potentially simpler and cheaper than the standard freezing processes. However, survival of embryos after vitrification has not yet proved better than after freezing. These experiments tested the relative efficacy of two vitrification methods upon both mouse and sheep embryos. A `standard' method, originally developed by Rall & Wood (1994) and called the VS3a method, was used with minor modifications (Rao et al., 1997) and was compared with a technically simpler method (M; Mermillod et al., 1997). Mouse and sheep embryos at the morula and blastocyst stages were obtained from superovulated females and vitrified using each method (mouse n = 180 and 162 for VS3a and M respectively; sheep n = 15 and 35 for VS3a and M). Mouse embryos were vitrified in 6 batches over a period of 6 months and stored at –196°C for at least 1 week before rewarming. Sheep embryos were vitrified in a single batch. Re-warmed embryos were cultured in a bicarbonate buffered Krebs-Ringer medium (M16: Whittingham, 1972) (mouse) and modified synthetic oviduct fluid (Tervit et al 1972) (sheep) for 3 days. Survival was defined as development through to either late expanded blastocyst or to a hatched blastocyst for both sheep and mouse embryo cultures. A logit binary logistic regression was performed on the data with rewarmed embryos classified as either having developed or not developed. For mouse embryos there was no overall difference between methods, but further analysis of the data set showed a significant method and batch first order interaction (p < 0.05), indicating that M is a better method for an inexperienced operator and that VS3a may be better overall with increased operator experience. The mouse data were therefore split into two groups, one with perceived significant operator inexperience influence, and the other with predominantly treatment effect. Sheep embryos were vitrified by an experienced operator. The smaller sample sizes with the sheep embryos resulted in inconclusive data and results of analysis; it is probable that ovine embryos do not survive the vitrification process as well as murine embryos, though the possibility exists that the culture system for testing this was inadequate. This research has shown that a simple method can be used for effective vitrification and that that simplest vitrification methods result in the highest embryonic viabilities for the inexperienced user. Vitrification has the potential to be used in the area of reproductive biology and technology as a simple alternative to conventional freezing methods.