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The purpose of this study was to establish an efficient combination of assisted hatching and cryopreservation procedures for producing bovine embryos in vitro. A total of 1312 day 7 blastocysts were subjected randomly to 14 different combinations of three factors: osmotic stress, assisted hatching and vitrification. Re-expansion, initiation and completion of the hatching process, as well as attachment to the culture dish, were analysed by SAS Genmod procedure. Incubation with sucrose was found to decrease survival rates; among the assisted hatching procedures used, zona fenestration resulted in higher survival rates compared with partial zona dissection and controls; and vitrification decreased survival and further development. The combined effect of sucrose incubation and vitrification decreased further development markedly, as did partial zona dissection followed by vitrification. Partial zona dissection performed in medium containing sucrose severely lowered embryo 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 controls (80%, 76% and 63%, respectively; P &gt; 0.05). Cell count analysis revealed a decrease in the total number of cells as a result of the assisted hatching and vitrification compared with controls (135 versus 202, respectively; P &lt; 0.0001). Although embryo transfer results (36% pregnancy rate and 30% calving rate) require further improvement, this combination of methods may prove useful in the commercial production of bovine embryos in vitro.

Survival and development of bovine blastocysts produced in vitro after assisted hatching, vitrification and in-straw direct rehydration