Abstract
The toxicity of cryoprotectant agents is one of the critical factors for the successful vitrification of mammalian embryos, which depends on the concentration, time and temperature of exposure to the cryoprotectant. Moreover, embryos from different species or stages of development have distinct levels of tolerance to cryoprotectant agents. This study aimed to evaluate the in vitro and in vivo survivals of mouse embryos after distinct times of exposure to two cryoprotectant concentrations prior to vitrification. In Experiment 1, compact morulas, blastocysts and expanded blastocysts were exposed to 10% glycerol for 10 min (group 1) or to 25% glycerol for 10, 5 and 2.5 min (groups 2, 3, and 4, respectively) prior to their exposure to the vitrification solution containing 45% glycerol for 1 min at 20°C before immersion in liquid nitrogen. Embryos were thawed in a water bath at 20°C for 20 sec, and the cryoprotectant was diluted in 1 M sucrose for 10 min. Then, embryos were morphologically evaluated following in vitro culture for 1 and 48 h. In vitro survivals of compact morulas and blastocysts were not affected by glycerol concentration and/or equilibration time prior to vitrification. However, expanded blastocysts demonstrated a lower survivability to vitrification. In Experiment 2, fresh and vitrified (according to procedures in group 1, Experiment 1) compact morulas and blastocysts were transferred to recipients following in vitro culture for 1 h at room temperature. Pregnancy rates, based on the proportion of viable fetuses, were similar between vitrified and fresh compact morulas (27% and 33%, respectively). However, vitrified blastocysts demonstrated a lower in vivo survival than controls (9% vs. 52%, respectively).
Highlights
Since the successful cryopreservation of the mouse embryo [30,32], many freezing methods have been devised in a number of species in an attempt to promote cell survival and reduce ice crystal formation, osmotic stress, and cell damage during the freezing process
In vitro survival at 1h post-thawing was similar among treatments, but the exposure for 10 min in 10% or 25% glycerol (Groups 1 or 2) prior to vitrification promoted higher rates of development to the expanded or hatched blastocyst stages after 48 hours of in vitro culture (IVC) than the exposure for 2.5 min at 25% glycerol (Group 4)
Our results indicated that the evaluation of vitrified compact morulas 1 h post-thawing was accompanied by a high rate of in vitro survival after IVC and a similar rate of in vivo development as observed for vitrification in 45% glycerol
Summary
Since the successful cryopreservation of the mouse embryo [30,32], many freezing methods have been devised in a number of species in an attempt to promote cell survival and reduce ice crystal formation, osmotic stress, and cell damage during the freezing process. Stated, freezing is the solidification of a liquid [13], which can occur either by crystallization, involving an ordered molecule arrangement, or by vitrification, by the extreme elevation in viscosity during cooling, with no ice crystal formation [8,12]. Cells are pre-dehydrated prior to cooling by their exposure to concentrated cryoprotectant solutions. Potential injuries due to the lack of ice crystal formation during freezing are minimized [12,20]. Cryoprotectant toxicity and osmotic injuries to embryos still may occur during vitrification. In this regard, the use of cryoprotectants of lower toxicity, and the determination of optimal cryoprotectant concentrations, and time, volume and temperature of exposure are strategies commonly used to minimize harmful effects to cells [20]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
