The effect of equilibration time on survival and development rates of mouse pronuclear-stage embryos vitrified in solid surface (SSV) and convential straws: In vitro and In vivo evaluations

Abstract

The objective of this study was to improve the efficiency of cryopreservation of pronuclear-stage (PN) mouse embryos. A novel vitrification technique (solid surface vitrification, SSV) was compared with a convential one in straws both for cryosurvival and obtaining progeny from cryopreserved PN mouse embryos. In the SSV method, 15-20 PN embryos were exposed to vitrification solutions for approximately 20 sec after equilibration, and then they were dropped in 2 microl drops onto a pre-cooled (-150 to -180 degrees C) metal surface. In the straws method, groups of 5-10 PN embryos were loaded in a single straw after equilibration. In experiment I, it was compared the effect of the vitrification solutions alone, without vitrification. No reduction was detected in survival, cleavage and blastocysts rates and the lowest development rate was obtained from hatched blastocyst for 20 min equilibration (24.5%). In experiment II, SSV method resulted in significantly higher survival and cleavage rates than that of in-straw vitrified 15-20 min group (87% vs. 60%, 83% vs. 67%, respectively; P < 0.05). There were no statistical differences among any of the blastocyts groups. However, there was a statistical difference in hatched blastocysts between 15 to 5, 10, and 20 min (P < 0.05). In experiment III, it was found no major effect among equilibration time periods in toxicity groups according to the mean cell number of blastocysts developed from PN embryos. But, there was a significant differences between 15 min SSV and 10 min in straw vitrified according to the mean cell number of blastocysts developed from PN embryos following vitrification (P < 0.05). The good results were obtained from 15 min equilibration group for SSV and 10 min equilibration group for straw vitrification. In the last experiment, embryo transfer after vitrification and toxicity was investigated. There were significant differences between SSV and straw just on the rate of pups born (30% and 20.5% respectively; P < 0.05). In conclusion, vitrification of PN mouse embryos by SSV can result in high rates of in vitro development to expanded and hatched blastocyst stage and in vivo development to live pups.