Cryopreservation of Porcine Blastocysts by Vitrification

Abstract

The objective of this study was to assess the possibility of cryopreservation of porcine expanded and hatched blastocysts by vitrification. The following four types of vitrification solutions were applied in this study: (i) EFT, a mixture of 7.2 M ethylene glycol, 0.003 M ficoll, and 0.3 M trehalose; (ii) DAP213, 2 M dimethyl sulfoxide, 1 M acetamide, and 3 M propylene glycol; (iii) DAP213-T, DAP213 supplemented with 0.3 M trehalose; and (iv) EPT, 4 M ethylene glycol, 3.1 M propylene glycol and 0.3 M trehalose. The embryos collected on Days 5 to 7 (Day 0 = onset of estrus) were allocated to eight experimental groups according to the types of vitrification solution and the developmental stage. In the toxicity test, the embryos were equilibrated in the respective vitrification solutions either in a single step or in four steps and then transferred to 1 M sucrose in a single step at 22°C without cooling. As a whole, the viabilities of embryos equilibrated in the solutions were lower than those of control embryos. The stepwise equilibration was superior to a single-step equilibration in the in vitro survival of, especially, expanded blastocysts after dilution. No significant difference was observed between the vitrification solutions for the four-step method. In the vitrification test, the embryos were equilibrated in the solutions by the four-step method, loaded into a 0.25-ml plastic straw, and plunged into liquid nitrogen. Although viable embryos were obtained after warming from all of the combinations except the hatched blastocyst-EPT group, viabilities were further reduced by cooling (range of reduction rates: 60 to 100%). The possible cause of low survival after warming is also discussed concerning the cryophysical properties of vitrification solutions.