Definition of fusion medium and electric parameters for efficient zygote electrofusion in the Pacific oyster (Crassostrea gigas).

Cell electrofusion has been widely used in the induction of tetraploidy in mammals, but little attention has been paid in molluscs. This work pursued the establishment of fusion medium (ionic vs. non-ionic) and electric parameters in the electrofusion of Pacific oyster zygotes (prior to the completion of the first mitotic division), minimizing all deleterious effects possible to D-larval stage. The tested combinations of electric field intensity (Vcm(-1)) and number of square DC pulses applied (for 50 micros each) were (Voltage x N degrees pulses): 400 x 1, 400 x 2, 400 x 3 and 600 x 1, 600 x 2, 600 x 3. When pulses were applied for first time, it was determined that an ionic fusion medium (microfiltered seawater) offered better conditions than the non-ionic fusion media previously used (0.6 M sucrose or 0.6 M mannitol) in terms of embryo survival and lysis rates. In this fusion medium, two different combinations of electric parameters (3 square DC pulses of 400 Vcm(-1) for 50 micros each at 26 degrees C and 1 square DC pulse of 600 Vcm(-1) for 50 micros at 26 degrees C) offered the best technical results of fusion (57 and 79% respectively) and survival until D-larva (44 and 41% respectively). In conclusion, these electric parameters could be established, using seawater as electrofusion medium, for further approaches to evaluate individual ploidy and survival beyond spat.