Cellular alteration after dilution of cryoprotective solutions used for the vitrification of in vitro-produced bovine embryos

Embryo quality of in vitro-produced bovine blastocysts was assessed at seve ral steps of a vitrification procedure in which glycerol and ethylene glyco l were used as cryoprotectants (3-step equilibration with cryoprotectants f ollowed by vitrification, dilution of the cryoprotectants in 0.85 M galacto se then in embryo transfer freezing medium [ETF], and finally co-culture fo r periods). To visualize cell membrane alterations, double staining was per formed using a cell permeant fluorochrome (bisbenzimide - BIS) and a nonper meant one (propidium iodide - PI). In Experiment 1, the effect of the vitri fication procedure on the hatching rate and total cell number was assessed 72 h after treatment. Hatching rate and the number of stained nuclei were d ecreased in comparison with untreated embryos when blastocysts were exposed to the whole procedure with or without vitrification (respectively 42 and 53% vs 76% for hatching and 128 +/- 17 and 141 +/- 17 vs 226 +/- 13 for sta ined nuclei). In Experiment 2, the effect of cryoprotectants and their dilu tion was evaluated on membrane permeability and total cell numbers at Vario us steps of the vitrification procedure. Blastocysts exposed only to cryopr otectant solutions and stained immediately after dilution in galactose show ed no modification. After dilution in ETF, the total number of stained nucl ei decreased, and the number of blastomeres showing membrane permeabilizati on (PI-stained) increased (respectively, 74 +/- 5 vs 110 +/- 5 and 32 +/- 2 % vs 0.1 +/- 1.8%). In Experiment 3, we demonstrated that the total number of stained nuclei after ethanol fixation (membrane permeabilization) was hi gher when embryos treated up to dilution in ETF were stained with PI than w hen the same embryos were stained with BIS. This suggests that, for unknown reasons, some nuclei of the treated embryos were not stained with BIS. Mem brane permeabilization and inability of BIS to stain some nuclei were the m ost obvious alterations probably induced by osmotic shock at dilution. This hypothesis is supported by the fact that the introduction of a further dil ution step in 0.42M galactose (Experiment 4) before dilution in ETF decreas ed the proportion of cells permeant to PI and increased the hatching rate a fter 72 h of co-culture. In conclusion, double staining with BIS and PI all owed for discrimination between different types of cellular injuries after the various steps of our vitrification protocol. It represents a useful too l for adjusting equilibration and dilution conditions during a cryopreserva tion procedure. (C) 1999 by Elsevier Science Inc.