Abstract
Pig hatched blastocysts, especially those which had been developed in vitro from expanding blastocysts, had a higher tolerance to cooling compared with pre-hatching stage embryos. The blastocysts which underwent hatching in vivo (vivo-HB) and in vitro (vitro-HB) were subjected to cooling compared with control pre-hatching stage embryos: expanding blastocysts, zona-removed blastocysts, early blastocysts and morulae. The embryos suspended in Dulbecco's PBS supplemented with 16% fetal calf serum were cooled in 0.25 ml plastic straws to 6°C for 1 hr or 16 hr. Survival of the embryos were assessed by testing the ability to develop in culture for 24 hr. All of the 26 (100%) vitro-HB and 51 of 65 (78.5%) vivo-HB, which had been exposed to 6°C for 1 hr, were survived. When the length of exposure to 6°C was prolonged from 1 hr to 16 hr, a remarkable difference (p<0.05) of the survival rate was found between the vitro-HB (20/21, 95.2%) and vivo-HB (22/41, 53.7%). This suggests that the vitro-HB were more tolerant of the cooling than the vivo-HB. The survival rate of the expanding blastocysts was significantly lower than that of the vitro-HB (p<0.001) and vivo-HB (p<0.05). None of the other control embryos showed any signs of survival, except for one small vesicle derived from one of the 15 zona-removed blastocysts.
Transfer of 18 vitro-HB, which had been cooled to 11°C, resulted in a normal fetus development, but none of the 22 morulae and 18 early blastocysts could survive this short exposure.
