| The development of effective cryopreservation protocols for fish embryos has important potential for facilitating the conservation of endangered populations of fish, management of stocks in fisheries, creation of gene banks for fish species and bioassay in ecotoxicological studies. Despite many efforts, cryopreservation of teleost eggs and embryos have not been successful. Therefore, this paper studied that embryonic development of Medaka (Oryzias latipes), the effects of temperature on embryonic development of Medaka, and toxicity and cryopreservation effects of different cryoprotectants on the Medaka Embryos, which could provide groundwork data for study on cryopreservation of Medaka embryos and its damage mechanism.1. The course of embryonic development was observed consecutively under Olympus microscope. The total development time of every development stage was noted and calculated and the characteristic images were taken using Olympus camera and analyzed. The results showed that the characteristics and time of 39 embryonic development stages from the fertilization of Medaka to the hatching after 9 days at 26℃.2. This paper depicted the effect of temperature on the embryonic development of Medaka (Oryzias latipes). The experimental result showed that the embryo could be hatched out at the temperatrue range of 19.69℃-36.03℃. With rising of the water temperature the time of the hatching was shorten. The most proper water temperature was at 26.03℃-33.90℃. The rate of hatching at 27.58℃was achieved 83%, and the survival rate of larvae was 90.9%. When the water temperature below 17.64℃, the embryo developed very slowly, only 1 larvae was hatched out, the hatching rate was 1%. However, the embryos were all dead before hatching at temperature over 38.67℃. The academic accumulative temperature was 112.62d·℃and the developmental threshold temperature was 17.55℃.3. This paper analyzed the toxicity of cryoprotectants (EFS40 and EFS60) on the survival and incubation rate of medaka embryos at five developmental stages. The results showed that endurance of embryos at blastula stage on EFS40 and EFS60 was the worst; and after exposed in EFS40 and EFS60 for 10 minutes, the survival of the embryos were 27.04% and 24.07% respectively,and incubation rate of the embryos were 13.70% and 14.07% respectively; Compared with the control group, the differences of survival and incubation rate were very prominent (P<0.01). The endurances of embryos at 6 somite stage, 16 somite stage and 24 somite stage on EFS40 and EFS60 were strengthened in order. The endurance of embryos at eyed stage was the best in tested embryos. The survival and incubation rate of embryos at eyed stage were 93.33% and 77.27% respectively after exposed in EFS40 for 30 minutes, 75.93% and 70.60% after exposed in EFS60 for 20 minutes, whereas the survival and incubation rate of embryos at other stages reduced remarkably (P<0.01). The results demonstrated that with the increase of exposure time to both EFS40 and EFS60, the survival and incubation rate of embryos decreased, and the EFS60 showed higher toxicity to the embryos than the EFS40.4. Medaka embryos at eyed stage were cryopreserved in EFS40 or EFS60 in liquid nitrogen for 1h with electron microscope grids (EMG) or straws. The result shows that when use EFS40 as the vitrifying solution, the embryos became opaque during cooling and thawing, while embryos still transparent in EFS60; the integrity rate of embryos when used EFS40 is lower than EFS60 (P<0.01); after cryopreserved embryos with electron microscope grids, the integrity rate was higher than when with the straws (P<0.01); but none of the embryos in our experiments get survival after vitrification and thawing,...
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