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Hredzak, R., A. Ostro, I. Maraaek, J. Kaamarik, V. Idilova, J. Vesela: Influence of Slow-rate Freezing and Vitrification on Mouse Embryos. Acta Vet. Brno 2005, 74: 23-27. The aim of the study was to compare the effect of two different methods of embryo freezing (slow-rate freezing employing programmable freezing equipment and ultra-rapid freezing by vitrification) on developmental capacity of two-cell mouse embryos on the basis of their development to blastocyst stage and implantation rate of blastocysts. Two-cell embryos were obtained from superovulated female mice and divided to three groups. The first group of embryos was frozen by the slow controlled-rate method using a programmable freezing equipment with propanediol as a cryoprotectant. Embryos from the second group were vitrified employing ethylene glycol as a cryoprotectant. The third group of embryos was cultivated in vitro without cryopreservation in a cultivation medium in an atmosphere of 95% air and 5% CO2. After thawing, the embryos from the first two groups were cultivated in vitro under conditions identical to those used for fresh embryos. The blastocysts that developed in vitro from embryos of all three groups were transferred to uteri of pseudo-gravid female mice to determine their implantation capacity. The percentage of vitrified embryos that developed into blastocysts was significantly lower than that of the fresh and slow-rate frozen embryos. The morphological appearance of embryos from all three groups was the same. The implantation rate of blastocysts that developed from vitrified embryos was significantly lower compared to the fresh and slow-rate frozen embryos. The results obtained indicate that freezing of embryos affects negatively their further development the negative effect of vitrification being more detrimental. As a “universal” vitrification protocol has not yet been defined, additional studies are needed to achieve its optimisation. Cryopreservation, cryoprotectant, implantation, slow-rate freezing, vitrification

The aim of the study was to determine the relationship between the rate of cooling of eight-cell mouse embryos to the temperature of liquid nitrogen (-196°C) and their developmental capacity after thawing on the basis of their ability to leave the zona pellucida ('hatching') during in vitroculturing. Eight-cell embryos were obtained from superovulated female mice and divided into three experimental and one control group. Embryos from the experimental groups were cryopreserved by the vitrification method using ethylene glycol as cryoprotectant. The vitrification protocols used in the study differed in the rate of cooling of the cryoprotectant solution. Embryos from the first group were frozen in conventional 0.25-ml plastic straws, those from the second group in pipetting 'tips', and embryos from the third group, placed in vitrification solution, were introduced dropwise directly into liquid nitrogen. The control group of embryos was cultured in vitrowithout freezing in a culturing medium in an environment consisting of 95% air and 5% CO2. The developmental capacity of thawed embryos was assessed on the basis of their ability to leave the zona pellucida ('hatching') after three days of in vitroculturing. In the control group 95.1% of embryos 'hatched'. A significantly higher number of embryos that 'hatched' after thawing was observed in the group introduced dropwise directly into liquid nitrogen (60.0%) compared to the group frozen in pipetting 'tips' (37.9%). The group frozen in straws yielded significantly the lowest proportion of 'hatching' embryos (8.1%). These results showed that increasing cooling rates during vitrification of embryos improved their survival.