Vitrication of in vitro produced bovine embryos at different ages using one- and three-step addition of cryoprotective additives

Abstract

The effect of embryo age on development and ratio of live : dead cells after vitrication and warming was examined. One-step and three-step addition of cryoprotectants in vitrification solution (40% ethylene glycol, 0·3 M trehalose and 12% polyvinylpyrrolidone) were compared usingin vitro produced (IVP) bovine blastocysts and expanded blastocysts. Rates of development and hatching were 74 ·2% and 41· 9% for Day 7, 57·8% and 23· 8% for Day 8, 33· 7% and 6·1% for Day 9 embryos with one-step addition. In three-step addition, those rates were 86·2% and 77·3% for Day 7, 72·3% and 39·0% for Day 8, 47·3% and 10·5% for Day 9 embryos. Day 7 embryos showed highest (P < 0·01) development and hatching rates with one exception. Hatching rate of Day 7 embryos with three-step addition was higher (P < 0·01) than with one-step addition. The ratio of live : dead cells differed between one-step (94%) and three-step (97%) additions for Day 7 embryos (P < 0· 05). The results indicate the higher resistance of younger IVP bovine embryos against vitrication and the potential for three-step addition of cryoprotectants to yield a higher survival rate after warming than with one-step addition.