P-26: The Effect of Zygote and 2-cell Development Stages on Vitrification Process of Mouse Embryo

Background: While it is possible to routinely cryopreserve embryos from several mammalian species, the cryopreservation of embryos has largely been limited by their high sensitivity to chilling injury. Many factors such as the stage of embryonic development, cryoprotectant toxicity, the composition of the vitrification solution and cooling and warming rates can influence survival of embryos after vitrification. The aim of the present work was to compare the survival rates of mouse embryos cryopreserved at zygote and 2-cell developmental stages of embryos. Materials and Methods: In the present study, weaned female mice (strain NMRI) aged 6-10 weeks were taken for hormonal treatments and male mice aged 8-12 were obtained. The females were given 7.5 IU PMSG i.p. and 48 hours later were given 7.5 IU hCG i.p. and 12 hours later, females and males were humanely killed by cervical dislocation. After IVF in culture medium (HTF solution consisted of 4 mg/mL BSA), embryos at various development stages (zygotes, 2-cell embryos) were vitrified using the modified droplet vitrification method. Embryos were vitrified in two vitrification solutions, VS-1 and VS-2 consisted of DMSO and EG. The embryos and vitrification solution were aspirated into a pipette. The pipette was held above the liquid nitrogen level and the drop was vitrified and sank into liquid nitrogen immediately. One month later, thawing and removal of cryoprotectants were performed by placing the vitrified drop directly into dilution medium and then they were incubated into above mentioned culture medium in CO2 atmosphere for 4 hours. Results: This study confirmed that almost all of the vitrified embryos were recovered, and most recovered embryos were morphologically normal in all developmental stages (zygotes, 2-cell embryos) However, a quite low proportion of recovered embryos developed to blastocysts in culture, but 2-cell embryos better than zygotes in this development. Conclusion: The present results highly support the idea that development stages of zygotes and 2-cell embryos aren’t proper for vitrification and recovering; perhaps the other development stages constitute better than them for this purpose.