Duration and temperature of culture medium equilibration affect frequency of blastocyst development

Hamster 2-cell embryos were cultured in 50 μl drops of chemically defined medium (HECM-9) under oil in 60 mm Petri dishes. In the first experiment, the dishes were equilibrated with 5% O2/10% CO2/85% N2 for 2 h either within sealed plastic bags or exposed directly to the same gas mixture in a tissue culture incubator. After culture of embryos for 48 h, there was no difference in development to the blastocyst stage. In the second experiment, the dishes were first equilibrated with 5% O2/10% CO2/85% N2 within sealed plastic bags, (A) at 4°C overnight (16-18 h), or (B) at 37.5°C overnight or (C) at 37.5°C for 2 h. Dishes in treatment A were placed in the incubator at 37.5°C for 2 h next day just before use. Two-cell embryos from a superovulated, mated female were equally distributed among the three treatments, then the dishes were sealed in fresh bags containing the same gas mixture and incubated at 37.5°C for 48 h. This experiment was replicated 13 times with a total of 20 females and 268-275 embryos/treatment. There was no significant difference among the treatments for development to the (combined) morula/blastocyst stages. However, the percentage of blastocysts that developed in culture dishes that had been equilibrated overnight at 37.5°C (treatment B) was significantly lower [50 ± 14% (SEM)] than in treatments A and C, which were not different from one another (67 ± 11 and 60 ± 17% respectively). These results indicate that when culture medium is incubated at 37.5°C overnight, chemical deterioration occurs that is detrimental to embryo development, and that this can be avoided by equilibrating dishes at 4°C overnight, followed by a brief period at 37.5°C to warm the medium before inserting embryos. This finding may have clinical relevance for human embryo culture. The study also demonstrates the utility and advantages of the sealed bag system for embryo culture.