Comparison of the Efficacy of Conventional Slow Freezing and Rapid Cryopreservation Methods for Bovine Embryos

Abstract

Day 7 bovine morulae and early blastocysts were randomly assigned to one of four cryopreservation methods: (i) a modified conventional controlled slow freezing and stepwise dilution after thawing; and three methods which enable direct transfer of the embryo into the recipient upon thawing: (ii) conventional controlled slow freezing and a modification of a one-step procedure, (iii) vitrification with 6.5 M glycerol plus 6% BSA (w/v), and (iv) vitrification with 25% glycerol (v/v) and 25% propanediol (v/v). In a comparative in vitro study, the percentage of grade 1 and 2 embryos developing into expanded blastocysts in culture for cryopreservation methods 1-4 were, respectively, 53% (29/55), 33% (20/61), 44% (26/59), and 51% (17/33). Method 2 yielded a significantly lower survival rate than methods 1 ( P < 0.1) and 4 ( P < 0.05) and was excluded from a subsequent test of in vivo development. Pregnancy rates (Day 60) after transfer of embryos cryopreserved by methods 1, 3, and 4 were, respectively, 59% (20/34), 43% (17/40), and 24% (5/21). Method 4 yielded a significantly lower pregnancy rate than method 1 ( P < 0.05). Method 3, however, did not yield a statistically different pregnancy rate ( P > 0.1) when compared to method 1. Method 3 has considerable promise in providing a successful method for the cryopreservation of bovine embryos that (i) reduces the time required for equilibration and cooling, (ii) provides for simple and rapid one-step dilution of cryoprotectant after thawing, and (iii) enables more embryos to be thawed and transferred per unit time.