Heat Shock-Induced Apoptosis in Preimplantation Bovine Embryos: MATERIALS AND METHODS(2)

MATERIALS AND METHODS(2)

In Vitro Production of Embryos

Embryos were produced using procedures described earlier except that the culture medium for embryos was different. Briefly, cu-mulus-oocyte complexes (COCs) were obtained by slicing 2- to 10-mm follicles on the surface of the ovaries obtained from slaughtered cows (a mixture of beef and dairy cattle). COCs that had at least one layer of compact cumulus cells were washed two times and used for subsequent steps. Groups of 10 COCs were placed in 50-|xl drops of oocyte maturation medium overlaid with mineral oil. For most experiments, COCs were matured for 22 h at 38.5°C in an atmosphere of 5% (v/v) CO2 in humidified air. A longer time for oocyte maturation (24-26 h) was used for the experiment with caspase activity for technical reasons to ensure that the fluorescence microscope required was available when needed. COCs were removed from maturation drops and washed one time in Hepes-TALP. For in vitro fertilization, groups of 30 COCs were transferred to 4-well plates containing 600 |xl IVF-TALP and 25 |xl PHE (0.5 mM penicillamine, 0.25 mM hypotaurine, and 25 |xM epinephrine in 0.9% [w/v] NaCl) per well and fertilized with ~1 X 106 Percoll-purified spermatozoa.

After 8-10 h at 38.5°C and 5% (v/v) CO2 in humidified air, presumptive zygotes were removed from fertilization wells, denuded of cumulus cells by vortexing in 30 |xl of Hepes-TALP for 5 min in a microcentrifuge tube, washed two to three times in Hepes-TALP and placed in groups of 25-30 in 50-|xl drops of modified KSOM overlaid with mineral oil at 38.5°C and 5% CO2 (v/v) in humidified air. Embryos were harvested from culture at different times according to the specific experimental design. Two-cell embryos were harvested at 28-30 h after insemination; 4-cell embryos at 37-39 h; 8- to 16-cell embryos on Day 3 or 4 after insemination, and embryos >16 cells on Day 5 after insemination.

Using this system of embryo production, the cleavage rate ranged from 77% to 100% across all the experiments. In several replicates, one plate of embryos was not subjected to treatment or any staining procedure, and embryos were allowed to develop to the blastocyst stage to verify adequacy of culture conditions. In these plates, the percentage of putative zygotes developing to blastocyst by Day 8 after insemination ranged from 33% to 59%.

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