SURVIVAL AND DEVELOPMENT OF BOVINE BLASTOCYSTS PRODUCED IN-VITRO AFTER ASSISTED HATCHING, VITRIFICATION AND IN-STRAW DIRECT REHYDRATION

The purpose of this study was to establish an efficient combination of assisted hatching and cryopreservation procedures for producing bovin e embryos in vitro. A total of 1312 day 7 blastocysts were subjected r andomly to 14 different combinations of three factors: osmotic stress, assisted hatching and vitrification. Re-expansion, initiation and com pletion of the hatching process, as well as attachment to the culture dish, were analysed by SAS Genmod procedure. Incubation with sucrose w as found to decrease survival rates; among the assisted hatching proce dures used, zona fenestration resulted in higher survival rates compar ed with partial zona dissection and controls; and vitrification decrea sed survival and further development. The combined effect of sucrose i ncubation and vitrification decreased further development markedly, as did partial zona dissection followed by vitrification. Partial zona d issection performed in medium containing sucrose severely lowered embr yo survival. Zona fenestration without sucrose incubation followed by vitrification did not compromise further embryo development: 86%, 84% and 79% of the blastocysts initiated, completed hatching and attached to the bottom, respectively. These data were not different from the co ntrols (80%, 76% and 63%, respectively; P> 0.05). Cell count analysis revealed a decrease in the total number of cells as a result of the as sisted hatching and vitrification compared with controls (135 versus 2 02, respectively; P< 0.0001). Although embryo transfer results (36% pr egnancy rate and 30% calving rate) require further improvement, this c ombination of methods may prove useful in the commercial production of bovine embryos in vitro.