| The number of germplasm resources has been decreased, due to factors such as changed ecological environment, artificial overfishing and so on. So research the sperm and embryo cryopreservation technology which is of great significance for germplasm resources is necessary. C urrently the target of fish embryo cryopreservation is the survival rate, but fish embryo hatchability is low in liquid nitrogen. So there is no widely used in fish production and the related molecular mechanism study is very necessary. In this paper, using the techniques of fish sperm and embryo cryopreservation studied marbled flounder(Pseudopleuronectes yokohamae) and seawater medaka(Oryzias melastigma). Selecting housekeeping genes of Japanese flounder(Paralichthys olivaceus) embryos at 0℃ low temperature condition and studying three related genes expression in the condition of low temperature.In present study, the series of experiments for sperm diluents, cryoprotectant, the suitable salinity of seawater and frozen semen artificial insemination were selected using the mature male marbled flounder(Pseudopleuronectes yokohamae). The results indicated that the antifreeze effect of sperm cryopreservation was good, using MFs-3 with respectively 20% PG and EG, which the sperm motility was respectively(95.26+0.39)% and(95.15+0.41)%. It indicates the diluent MFs-3 suits for marbled flounder sperm cryopreservation, and the best cryoprotectants are PG and EG. In the experiment of selecting of the suitable salinity of seawater, the frozen sperm were activated with 10~50‰ seawater,showed that the sperm motility was the highest at 30‰ and there was no significant difference compared with fresh sperm but there was significant difference between others and fresh sperm(P<0.05). And sperm motility has been gradually increased from(86.13+1.44)% to(95.07+0.69)% when the salinity is 16.7‰~30‰; Sperm vitality has been gradually decreased from(95.07+0.69) % to(84.09+2.61)% with 30‰-45‰ sea water. Lower than 16.7‰ and higher than 45‰ of sea water, sperm is almost no movement. The thawed sperm with PG and EG cryopreservation and marbled flounder eggs made fertilization experiments, fertilization rate and hatching rate respectively was(80.08+0.68)%,(77.44+1.76)% and(80.17+0.45)%,(77.92+1.33)%. It was also used computer aided sperm analysis(CASA) to analyze and test for motion parameters from frozen sperm sample in MSs+20%PG and MSs+20%EG including curvilinear velocity(VCL), straight line velocity(VSL), beat cross frequency(BCF), linearity(LIN) and str aightness(STR) and so on, showed that there was no significant difference between them. In present study, the sperm from marble flounder could be effective cryopreserved with MSs-3+20%PG and MSs-3+20%EG. This study has been successfully preserved marbled flounder sperm with 20% MFs-3+PG and MFs-3+20% EG at-196℃. And the frozen sperm motility, fast moving time and life time is of no significance with the fresh sperm. The technology has preserved the marbled flounder sperm cryopreservation, which has set a solid foundation for artificial cross breeding and genetic resources center. The research result firstly provide important the theory and technology basis.The experiment used the seawater medaka(Oryzias melastigma) in order to study the sea fish embryo cryopreservation. Medaka is one of the model organisms,and it has a most advantage which is spawning quantity will not be affected as long as the control of constant temperature and provide reasonable nutrition and light cycle. Observing medaka embryonic de velopment, and selecting key embryonic periods which are respectively the archenteron stage, nerve embryonic stage, 22- 28 sarcomere stage, tail bud stage, heart stage and body rotation stage. Screening different kinds and concentrations of antifreeze in vitrification solution and the purpose is to filter out the optimal vitrification formula. As a result, the body rotation stage has got an ideal hatchability used with the 50% PM vitrification solution, and hatchability of other embryonic stages is low. Three permeability antifreeze which are PG, MeOH and Gly configured solution in the proportion of PM(PG: MeOH=3:2): Gly=1:1, 2:1, 3:1 and 4:1, finding that PMG2 vitrification solution has the highest hatchability in different ratio vitrifications. Using different kinds and concentrations of carbohydrates deal with body rotation embryos, finding that glucose is the lowest toxicity in all carbohydrates. And when the glucose concentration arrived at 4% embryo hatching rate was the highest. Using the selected vit rification solution PMG2G4 cryopreserved a total of 309 body rotation embryos and 122 kept an intact embryo shapes.The article studied gene expression of Japanese embryos under low temperature condition from the molecular level and laid a molecular founda tion for the fish embryo cryopreservation. Sarcomere stage, tail bud stage and heart embryo were respectively studied in 0℃ and 16.5℃ temperature, analyzing 18 S r RNA, ACTB, GAPDH and EF1α four housekeeping genes stability by the GeNorm and NorFinder software. Results showed that the stability of four housekeeping genes is 18 S rRNA=EF1α>ACTB>GAPDH when using the GeNorm software. The stability of four housekeeping genes is 18 S r RNA>ACTB>EF1α>GAPDH while using the NorFinder software. 18 S r RNA is be supposed to the most stable housekeeping gene in 0℃ Japanese flounder embryos through comprehensive analysis of two software GeNorm and NorFinder.In order to study the influence of Japanese flounder embryos gene expression on low temperature, selecting the tail bud embryos do relative research which are strong adaptation to low temperature. Selecting embryos which are growth in 16.5℃ as control group. The article selected the three genes CIRP, S0X9 and HSP70 which are expressive in embryonic development as research genes. Then use q RT-PCR method to analyze gene expression in embryos. The study found that CIRP mRNA expressive levels was decreased within 30 min and there was an increased expressive levels in 60~120min. And CIRP mRNA expressive levels increased to the maximum at 120 min while CIRP mRNA expressive levels was decreased at 180 min and had no significant difference with control group. S0X9 mRNA expressive levels decreased within 30 min. Unlike the CIRP, S0X9 mRN A expressive levels was reduced in 120~180min and had a significant difference with the control group. And HSP70 mRNA expressive levels showed a rise in the whole process of low temperature treatment. And there was a significant difference with the control group. The study found that low temperature can really change the CIRP, S0X9 and HSP70 gene expression. |
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