A Study on the Viability of Bovine Blastocysts Derived from In Vitro Fertilization after Freezing and Thawing by Two Cryo-preservation Methods Adapted to Practical Use in the Field

Two cryopreservation methods adapted to field use were investigated with bovine blastocysts produced by in vitro technique. In Method I, embryos were equilibrated with 1.36M glycerol and 0.25M trehalose for 10-15 min in modified phosphate buffered saline (mPBS) supplemented with 20% cow serum. Embryos were loaded into 0.25-ml plastic straws (2 embryos/straw) in a small volume of freezing medium separated by 2 air bubbles from the medium (0.25M trehalose with 20% cow serum in mPBS) filling the rest of the straw. The straws were placed directly into a cooling chamber kept at -7C and then seeded followed by holding for 10 min at this temperature. The straws were then cooled to -25C at 0.45C/min before being plunged into liquid nitrogen.In Method II, embryos were equilibrated in a three-step procedure with 0.5M 1, 2-propanediol (PD) for 5 min, 1.0M PD for 5 min and 1.6M PD for 10-15 min in mPBS supplemented with 20% cow serum and loaded into straws separated by 2 air bubbles from the medium (0.2M sucrose with 20% cow serum in mPBS) filling the rest of the straw. The straws were placed directly into a cooling chamber kept at 0C and then cooled to -6C at 1C/min. After leaving for 10 min at this temperature, including seeding, the straws were then cooled to -30C at 0.3C/min. The straws were then left for 10 min at this temperature before being plunged into liquid nitrogen.The straws containing embryos were thawed at 30C and the embryos were directly transferred into cow uterus. Pregnancy rates of transferred embryos cryopreserved by Methods I and II were 12.5% (3/24) and 37.8% (17/45), respectively. In Methods I, one out of 3 heifers has delivered a calf. The remaining 2 heifers have been maintaining pregnancy. In Method II, 4 (including one set of twins) out of 16 heifers lost their pregnancy between 2 and 4 months of gestation. One heifer (carrying twins) died due to disease and the another one died due to bad weather within 1 month before the expected days of delivery. Thirteen calves (including 2 sets of twins) were born from the remaining 11 heifers.