Comparison of the cryoprotectant properties of glycerol and ethylene glycol for early (day 6) equine embryos.

Abstract: Early (day 6) equine embryos (n=23) were assigned to four treatment groups to assess the cryoprotectant properties of glycerol and ethylene glycol and the effect of adding sucrose during removal of the cryoprotectant: (i) group GG (n=5) embryos were frozen and thawed using 1.5 mol glycerol l(-1) as the cryoprotectant, which was added at 22 degrees C in four steps (0.375, 0.75, 1.125 and 1.5 mol glycerol l(-1)), and removed after thawing in five steps (1.5, 1.125, 0.75, 0.375 and 0.0 mol glycerol l(-1)); (ii) group GS (n=6) embryos were frozen and thawed using 1.5 mol glycerol l(-1) as for group GG, except that 0.25 mol sucrose l(-1) was added during removal of the glycerol; (iii) group EE (n = 6) embryos were frozen and thawed using 1.5 mol ethylene glycol l(-1) as the cryoprotectant, which was added in three steps (0.5, 1.0 and 1.5 mol ethylene glycol l(-1)) and removed after thawing in four steps (1.5, 1.0, 0.5, 0.0 mol ethylene glycol l(-1)); and (iv) group ES (n = 6) embryos were frozen and thawed using 1.5 mol ethylene glycol l(-1) as for group EE, except that 0.25 mol sucrose l(-1) was added during removal of the ethylene glycol. After thawing, the embryos were incubated at 37 degrees C in Ham's F10 medium supplemented with 10% (v/v) fetal calf serum and antibiotics, in 5% CO2 in air for 6 h. The embryos were fixed in glutaraldehyde, serially sectioned and observed using light microscopy. None of the frozen-thawed embryos treated with ethylene glycol (groups EE and ES) had any viable cells. There were no lysed cells in the frozen-thawed embryos treated with glycerol (groups GG and GS) and the proportion of cells with pyknotic nuclei was low (group GG = 1.1 +/- 0.8% and group GS = 2.5 +/- 1.5%). There were no differences between embryos treated with cyroprotectant diluted with or without sucrose. The embryos were morulae or early blastocysts and either did not have a capsule or had a very thin capsule. The results of the present study confirm that ethylene glycol is a poor cryoprotectant for early equine embryos and that the use of sucrose during dilution of the cryoprotectant after thawing does not improve the morphology of the embryos. The results of this study also indicate that glycerol is an effective cryoprotectant for freezing equine embryos with intact zonae pellucidae and with either very thin or no capsules.