Influence of Slow-rate Freezing and Vitrification on Mouse Embryos

Abstract

Hredzák, R., A. Ostró, I. Maraãek, J. Kaãmárik, V. Îdilová, J. Veselá: 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% CO 2 . 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.

Closed-system solid surface vitrification versus slow programmable freezing of mouse 2-cell embryos

“…Slow programmable freezing in our study gave a comparable survival and blastocyst development rate to other studies (77.6% versus 50%-87.6% and 58.6% versus 32.8%-76.4%, respectively) [13,15,16]. For our vitrification system, the survival (95.7%) and blastocyst development rates (79.3%) were higher than those reported in the conventional vitrification methods (60.3%-85% and 22%-38%) [13,15,17,18].…”

“…For our vitrification system, the survival (95.7%) and blastocyst development rates (79.3%) were higher than those reported in the conventional vitrification methods (60.3%-85% and 22%-38%) [13,15,17,18]. Although the cooling rate achieved by our system (−964°C/min) was much lower than those of the container-less systems, such as the open-pulled straw (OPS; −2,000°C/min), the survival and blastocyst development rates were comparable to those reported in the literature (87.5-96.3% and 62.2-68.9%, respectively) [18].…”

“…Mouse 2-cell stage was specifically selected because it corresponds best to human 4-to 8-cell stage, with regard to morphology and size of the blastomeres [13]. Moreover, the 2-cell stage can be considered as a sub-optimal developmental stage [14], and it is the stage more sensitive to cryodamage than any other cleavage stage [15].…”

Ultra-Structural Alterations inIn VitroProduced Four-Cell Bovine Embryos Following Controlled Slow Freezing or Vitrification

“…The optimal developmental stage and protocol for cryopreservation is still not determined. Similar cryopreservation protocols applied to multiple species may result in different results (Rall, ; Hredzák et al., ; Pereira and Marques, ).…”

An Attempt of Cryopreservation of Mouse Embryos at the ACTREC Laboratory Animal Facility in India

“…These plates were kept in a CO 2 incubator (model 3111; Thermo Scientific, Waltham, MA, USA) at 37°C in 5% CO 2 for 24 h. Survival of the 8-cell-to morula-stage embryos was assessed by their ability to develop into fully expanded blastocysts with a blastocoel cavity. Twenty to twenty-five fully developed blastocysts were surgically transferred into the uterotubal junction of the left uterine horn in anesthetized 3.5-day-old pseudopregnant females [6].…”