Effect of three vitrification methods on development of two-cell mouse embryos

The purpose of this study was to investigate the effect of three vitrification procedures [conventional straw (CS), open pulled straw (OPS) and closed pulled straw (CPS)] on the development of two-cell mouse embryos. Two-cell mouse embryos were randomly divided into vitrified and non-vitrified control groups. Embryos in the vitrified group were cryopreserved within a combination of 5.5 M ethylene glycol and 1M sucrose as cryoprotectants, loaded within three different straws (CS, OPS and CPS) and warmed in stepwise sucrose solutions. The survived embryos from each procedure were cultured in human tubal fluid (HTF). The non-vitrified control embryos were also cultured in the same manner. The rates of the development in all the groups were daily determined and statistically compared. On day 4 of the cultivation period, several expanded blastocysts from each group were randomly selected and stained either with propidium iodide (PI) and bisbenzimide or with terminal transferase- mediate DNA end labeling (TUNEL) Technique. The mean number of the inner cell mass (ICM), trophoectoderm (TE), necrotic and apoptotic cells were counted and statistically compared. The survival rate of embryos in CPS was significantly higher than that in OPS and CS. The rate of hatched blastocysts did not differ in the three vitrification procedures, but in comparison with that of the control, CS and OPS showed a significant reduction. The mean number of ICM and TE decreased in CS and OPS, whereas in CPS it was almost identical to that of the control. The incidence of apoptosis and necrosis appeared to be almost similar in all the groups. In conclusion, CPS seems to be an effective, easy and rapid method for the cryopreservation of two-cell mouse embryos.