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

Abstract

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 > 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 < 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.

The effect of vitrification in open pulled straws on pregnancy rates after transfer of in vivoproduced bovine embryos

“…On the other hand pregnancy rates following transfer of programmable frozen compact morulae, early blastocysts, blastocysts and expanded blastocysts regardless of quality grades were 29%, 50%, 33% and 50%, respectively. Analogous to our observations, the studies of Vajta et al (1997bVajta et al ( , 1998aVajta et al ( , 1999 and Lazar et al (2000) demonstrated that advanced stages of in vitro produced bovine embryos can be vitrified successfully with hatching rates reaching 60% to 94%. Even in vitro produced embryos vitrified at the hatched blastocyst stage reached up to 81% re-expansion rate after 24 h of culture.…”

“…In addition vitrification is suitable for freezing of bovine embyos in the precompaction stage (Vajta, 2003) or advanced stages of embryos as in vivo or in vitro produced hatched blastocysts (Vajta et al, 1997b). Use of vitrification has been increased by the development of vitrification in open pulled straws (OPS).…”

Effect on Survival of Micromanipulating the Zona Pellucida of Bovine Embryos

“…The results of our in vitro and in vivo experiments are consistent with those published by Schiewe et al (1995) who used a mouse model and found higher hatching rates (p < 0.01), but no significant difference in implantation rates after AH treatment. Significant differences in the development between embryos with mechanically or laser-treated and non-treated zona were reported by Park et al (1999) and Vajta et al (1997) who cultured bovine blastocysts in vitro (IVM/IVF/IVC). We assume that the equal survival rate found in our experiments for zona-slit and nontreated fresh grade 1 embryos is attributable to the cell number and the quality of the embryonic cell complex with the natural expansion and hatching potentials.…”

Cryopreservation of Human Pluripotent Stem Cells: Are We Going in the Right Direction?

“…This is accomplished by direct immersion into liquid nitrogen of open-pulled straws containing small droplets (typically 1-20 µl) of vitrification solution within which the colony fragments (< 10) are held. Straws are then generally transferred to liquid nitrogen for long term storage (Vajta et al, 1997;Vajta et al, 1998).…”