Conrado A. Valdez

Effects of equilibration time, precooling and developmental stage on the survival of mouse embryos cryopreserved by vitrification

Factorial experiments were carried out to examine the effects of equilibration time, precooling and developmental stage on the postthaw in vitro survival of vitrified mouse embryos. Eight-cell embryos, compacted morulae, or blastocysts were cryopreserved using vitrification Solution 1 (VS1; 10% glycerol + 20% propylene glycol), and vitrification Solution 2 (VS2; 25% glycerol + 25% propylene glycol) in phosphate buffered saline + 10% calf serum. Each embryo stage group was first equilibrated in VS1 for 5, 10 or 20 min and then exposed to either a precooled ( approximately 4 degrees C) or nonprecooled ( approximately 20 degrees C) VS2 in a 0.25-ml straw before they were plunged directly into liquid nitrogen. Results of this study showed an interaction between precooling, equilibration time and developmental stage which affect significantly post-thaw embryo survival (P< 0.05). High survival rates were obtained after 10 min equilibration in VS1 irrespective of the embryo developmental stage. Precooling of the VS2 significantly improved the survival mainly of blastocysts. However, eight-cell and morula-stage embryos also showed high survival rates when they were exposed to precooled VS2 after 5 min equilibration in VS1. It was further observed that morulae usually exhibit high survival rates, and vitrification conditions are more critical for early and advanced stage embryo development.

Quick freezing of mouse embryos using ethylene glycol with lactose or sucrose

Abstract Mouse compacted morulae were quickly frozen in liquid nitrogen (LN2) vapor at approximately −170°C after 5 min equilibration in 2 M or 3 M ethylene glycol or glycerol in the presence of either 0.25 M lactose or sucrose to examine their post-thaw viability. After thawing in a water bath at 37°C and a one-step dilution of the cryoprotectant, the embryos were cultured for 48 h. Significantly higher in vitro survival rates were obtained with 3 M ethylene glycol than with 2 M ethylene glycol or 2 or 3 M glycerol irrespective of the sugar used (P