Fast Cryopreservation of the Mammalian Embryos in Different Developmental Stages by 0.25 mL Straws Vitrification with One Equilibration Step

Abstract

The aim of our study was to test the cryoprotective proprieties of 7 vitrification media, designed in our laboratory, using the 0.25 mL straws vitrification method, with one equilibration step. As biological material we used mouse females, age 2 months superovulated with 5UI PMSG (Pregnant Mare Serum Gonadotropine) and 5 UI hCG (human Corionic Gonadotropine). For freezing we used embryos in three developmental stages: 2 cells, morula and blastocyst. After recovery, the embryos were placed in equilibration media, after 5 minutes, the embryos were introduced in straws, in vitrification media and plugged directly into liquid nitrogen. After vitrification the straws were thawed in water bath at 37°C, the embryos were rehydrated for 5 minutes and then in vitro cultured. The percent of embryos that rehydrated, resumed development and hatched were registered. The best results were obtained with embryos in morula stage that had a hatching rate of 20.83% when MV1 was used for vitrification. None of the embryos in 2 cells and blastocyst stage hatched after thawing and in vitro culture, regardless of the vitrification media used. From the vitrification media tested, the worst results were obtained with MV4 and MV6, none of the embryos reached hatching stage, regardless of the development stage. The vitrification method in 0.25 mL straws, with one equilibration step can be used for cryopreservation of the morula stage embryos, but is ineffective for vitrification of the 2 cells and blastocyst stage embryo. Media VM4 and VM6 are not suited for vitrification in 0.25 mL straws, with one equilibration step, of mouse embryos.

Keywords

• mouse embryos
• vitrification media
• hatching rate
• cryoprotectors