CYTOLOGICAL OBSERVATIONS ON INDUCTION OF MITOGYNOGENESIS BY HEAT-SHOCKING OF THE EGGS IN GRASS CARP, CTENOPHARYNGODON IDELLUS

Mitogynogenesis referring to inducing gynogenesis by inhibiting the first cleavage could generate fully homozygous offspring and could product cloned fishes within short time. Generally, the optimum timing to induce mitogynogenesis have to be the metaphase of first cleavage, because at that time the chromosome sets have duplicated while the sister chromosomes have not separated yet. Since different species of fish needs different time for completing their copying of chromosome sets, obviously, a given species of fish should be suppressed the first cleavage in a specific time. Similarly, the suppressing intensity should be different for diferent species of fish. Therefor, in order to induce mitogynogenesis successfully for a given species of fish, the developmental procedure of the first cleavage must be understood fully and the proper parameters of suppression should be examined first. In this study, in order to induce mitogynogenesis in grass carp, Ctenopharyngodon idellus, effectively, the developmental procedure of first cleavage was investigated systematically and the optimum parameters for inducing mitogynogenesis by heat shocking of eggs were determined. The grass carp eggs were activated by UV-irradiated common carp sperm. The reason for choosing the common carp sperm is that the heterozygote of common carp sperm and grass carp eggs is absolutely lethal. The activated eggs were fixed with Smith's solution for every 3 minutes after activation 18 to 48min. Then the fixed samples were sectioned according to rule procedure and observed under microscope. Results show that when incubated at 24℃, the activated eggs entered the prophase after activation for 24min, the metaphase for 27—30min, the anaphase for 33min. Obviously, the optimum period for suppression of chromosome set separating of the grass carp is from 27 to 30min after the eggs were activated. At this time, the haploid chromosome set of the activated grass carp eggs had copied completely and had not separated yet. After the optimum period of heat shocking was determined, the activated eggs were treated with gradient shocking temperatures and shocked for variable times. Then these heat-shocked samples were fixed, sectioned and observed as described above. The observation revealed that no spindles of the activated eggs were destroyed by the heat shocking below and at 39℃ for 2min. If the activated eggs were heat shocked at 40℃ for 2min, the spindle was destroyed partly. Only when the temperature was at or over 41℃, then the spindles were destroyed completely. In experiment of the survival rate of embryos, the survival rate in the groups with the treatment of 41℃ was the highest. In the groups of 42℃ the mortality rate was very high. These results means that the optimum heat shocking temperature for suppressing the first cleavage of the grass carp eggs is 41℃ and the effective intensity of heat shocking is 41℃ for 2min at the optimum time. After the heat shocking was released, the reformation ratio of spindles of the heat shocking treated eggs is about 78.6% of the observed samples. This may be one of the reasons why the survival rate of the mitogynogenetic diploid is low in experiments.