| Different from mammals, the early embryos of fish can not be preserved for the long period at the very low temperature(-196 ).Therefore, three methods were usually applied to cryogenic preservation of the fine and rare species of fish :1) perserving fish spermatozoon in cryogenic condition. Researchers have had systematically studied on this technique for many years, and this technique has been utilized in application and made a lot of effects;2) Combining with the techniques of cell engineering (nuclear transplantation and electric fusion etc.), and through the process of culturing histiocyte of fish, cryopreservation and re-culture after thawing, carrying out somatic cell breeding of fish. The past studies showed that the nucleolus of somatic cells of fish have totipotency . with the development of the technology of cell culturing and cell engineering, so it is possible to preserve fish species for a very long time.3) using embryonic stern cells( ES).Fish embryonic cell has the totipotency and is better for breeding than somatic cell, but it can not so strong to resist the very low temperature. Except for some developmental potential studies on cryopreservation and growth by a few researchers, there are no systematical reports on cryopreservation culture of fish embryocells. For the cryogenic preservation of fish ,in this paper we made the primary culture of the kidney of allotetroploid crucian carp and primary studies were carried out on cryopreservation culture offish embryo cells derived from misgurnus auguillicaudatus or grass carp, the results of the experiments are as follows:The primary cell culture of the kidney tissue derived from allotetroploid crucian carp was carried out using tissue adherent culture, The primary observations of the growth conditions and morphology of the primary culture and subculture cells which originally come from the kidney tissue were also made. The results showed that :when cultured in the medium of M199, supplemented with 20% bovine serum containing a moderate amount of antibiotics, the incubtion PH 7.2-7.4,the culture temperature 28 .the primary culture cells formed THE dense single wall within three weeks ,the generation time of the subculture cells was 5-6 days, most cultured cells were fibroblastic-like cells with few epithelial-like cells. The karyotye analysis was made on the 5th passage, the number of the chromosomes ranged from 187 to 200,and no heteroploid cell was found.Using misgurnus auguillicaudatus as material, we made a primary study of embryonic cell preparation, cryopreservation, thawing and primary cell culture., the primary results showed: using M199 as diluents containing 20% bovine serum, It is better to freeze the cells slowly freezing at fist then increase freezing speed (for example, from 0 to -6 freezing speed is about -0.05 a minute, from -6 to -40 ,freezing speed is about -0.5 a minute) ,studies on effect of various concentration of DMSO demonstrate that about 12.5% DMSO gave the highest post-thaw percentage of viable cells. The concentration of bovine serum had no different effect on the percentage of the viable embryo cells of misgurnus auguillicaudatus .The embryo cells derived6from the later stage of blastula offish is more resistant to the cryogen than the cells of early stage of blastula. The cells preserved in liquid nitrogen at -196 were thawed and cultivated, a few cells were found adhere to the surface of culture vessel when the percentage of viable cell was more than 30%.the cells in only two culture vessels were found to proliferated and gave rise to many small morphologically undifferentiated cells. Using M199 containing 20% calf bovine serum and 11% DMSO as the diluent and by the methods using in cryopreservation of embryo cells of misgurnus auguillicaudatus, Two groups of cells derived from blastula of grass carp were preserved in liquid nitrogen at -196 .After 6 days cryopreservation, one group of cells were thrawed and the percentage of viable cell was about 72%;the other, cryopreserved for 13 days,was 52...
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