To establish a simple method for incorporating materials into fertilized medaka eggs by applying electric pulses including nanosecond pulsed electric fields (nsPEFs), we have tested pulse parameters as pulse width, pulse intensity, number of pulses, combination of several pulses, interval of pulses, and polarities of pulses. Improvement in incorporation efficiency was achieved with a new pulse system, named asymmetric burst system, which could generate a short rectangular pulse (from ten to several hundreds nanosecond) with high intensity (several kilovolt) and asymmetric bipolar burst pulses (in a range of microsecond to millisecond) with low intensity (from several volt to several tens volt). To gain the total efficiency for incorporation, it is necessary to investigate other factors in addition to the pulse parameters. In this paper, the effects of solutions that surrounds eggs are investigated. Among the combinations of pulsing and immersed surrounding solutions, we found that the electrical and experimental deleterious effects were strongly reduced under the condition that eggs were pulsed in a 6 mS/cm NaCl solution and kept in the same solution or put into and kept in an isotonic solution for 2 hours. This might be due to an appropriate electrical conductivity and an osmotic pressure difference between the inside of an egg and the surrounding solution. Moreover the adverse effects of cycloheximide on embryogenesis appeared clearly using the same condition.
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