59 post-thaw survival of vitrified, in vitro-produced porcine embryos: comparisons of vitrification solutions, solution temperature, and blastocoele collapse methods.

Current post-thaw survival rates of vitrified in vitro-produced porcine blastocysts are much lower than those of other domestic species. The main reason for this is the suboptimal development of in vitro-produced porcine embryos, and the endemically high lipid content of porcine oocytes. Currently, there are several vitrification protocols that have been used to successfully vitrify blastocysts from other species, but many of these have not been used to vitrify porcine embryos. Furthermore, the practice of collapsing the blastocoele cavity before vitrification is used routinely in human clinics and in other domestic animals; however, there has been little data published regarding the collapse of porcine blastocysts before vitrification. In this study we compared several different vitrification protocols containing different constituents and altered constituent concentrations. We also compared 2 methods of blastocoele collapse before vitrification. The aim of this study was to determine the optimum conditions for cryopreserving porcine embryos. All experiments were performed on Day 7 in vitro-produced porcine blastocysts. In experiment 1 embryos were vitrified in either a standard solution (17% ethylene glycol + 17% dimethyl sulfoxide + 0.4 M sucrose) or 17% ethylene glycol + 17% propandiol + 0.4 M sucrose. In experiment 2 embryos were vitrified in either the standard solution or 17% ethylene glycol + 17% dimethyl sulfoxide + 0.4 M trehalose. In experiment 3 embryos were vitrified in the standard solution at 38.5°C or at room temperature. In experiment 4 embryos were collapsed before vitrification by micro-pipetting or via 2 sucrose solutions. Survival of embryos was determined by the presence of a blastocoele cavity 24 h after thaw. Data were subjected to ANOVA and an appropriate post-hoc test when differences were found. The post-thaw survival of embryos vitrified in the standard solution (58%) and the propandiol solution (61%) did not differ. Also, the survival rates of blastocysts vitrified using sucrose (72%) and trehalose (72%) were the same. The survival rate of embryos vitrified in warmed media was significantly lower than embryos vitrified in media at room temperature (54 and 71%, respectively; P < 0.05). In experiment 4 the post-thaw survival of noncollapsed and embryos collapsed via micro-pipetting was similar (55 and 44%, respectively); however, survival tended to decrease if embryos were collapsed using sucrose before vitrification (25%; P = 0.093). These findings indicate there was little impact on post-thaw survival rates due to the vitrification solutions used. However, the temperature of the solutions appears to influence post-thaw survival. Contrary to findings in other species, collapsing the blastocoele cavity before vitrification did not improve cryosurvivability; it had no effect or, in the case of sucrose, a negative effect on survival. This may be due to the inherent sensitivity of in vitro-produced porcine embryos to manipulations.