Rapid cryopreservation of sheep embryos by direct transfer into liquid nitrogen vapour at -180 degrees C

Abstract

The in vitro survival of control and rapidly cryopreserved sheep embryos was examined as a function of the duration of exposure to a vitrification medium (25% glycerol + 25% propylene glycol). Embryos in late morula to late blastocyst stages were permeated by a mixture of 10% glycerol + 20% propylene glycol for 10 min at 18-23 degrees C and then exposed to the vitrification medium for 0.5, 1, 2 or 4 min at 18-23 or 4-12 degrees C. The cryoprotectants were removed without cryopreservation (control embryos) or after rapid cryopreservation by direct transfer into liquid nitrogen vapour at -180 degrees C. The duration of exposure to the vitrification medium at 18-23 degrees C affected the in vitro survival rate of control embryos (P = 0.06) but had no effect on the survival of rapidly cryopreserved embryos. However, at 4-12 degrees C the duration of exposure affected the survival of cryopreserved embryos (0.5 min: 64%, 18/28; 1-4 min: 43%, 34/80; P = 0.074). Overall, the in vitro survival rate of control and cryopreserved embryos increased with advancing development from late morulae (36%) to late blastocysts (70%). The in vivo survival of embryos that had been exposed to the vitrification medium for 0.5 min at 4-12 degrees C and then vitrified was tested. The rate of development to term was 11% (4/35) for late morulae or early blastocysts and 32% (6/19; P greater than 0.1) for blastocysts to hatching blastocysts. These results showed that sheep embryos can be successfully cryopreserved by a simple, rapid procedure.