CN103098792B - Ultralow temperature cryopreservation method of Eriocheir sinensis embryo - Google Patents
Ultralow temperature cryopreservation method of Eriocheir sinensis embryo Download PDFInfo
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- CN103098792B CN103098792B CN201110355081.3A CN201110355081A CN103098792B CN 103098792 B CN103098792 B CN 103098792B CN 201110355081 A CN201110355081 A CN 201110355081A CN 103098792 B CN103098792 B CN 103098792B
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Abstract
An ultralow temperature cryopreservation method of Eriocheir sinensis embryo. The method is characterized by comprising the steps of: equilibrizing Eriocheir sinensis embryos at early nauplius stage in a vitrified solution containing 30% of 1, 2-propylene glycol and 20% of dimethyl formamide for 40min; equilibrizing in an ice bottle at -20 DEG C for 1 min; equilibrizing at a liquefied nitrogen port for 1 min and adding the embryos into the liquefied nitrogen; freezing for 40 min; rapidly lifting the embryos to the liquefied nitrogen port to equilibrize for 1 min; then quickly placing the embryos into a water bath at 38 DEG C for thawing; eluting by 0.25mol / L sucrose for 10 min; culturing the embryos at early nauplius stage in seawater with salinity of 15; equilibrizing Eriocheir sinensis embryos at zoea stage in a vitrified solution for 40 min; equilibrizing in an ice bottle at -20 DEG C for 1 min; equilibrizing at a liquefied nitrogen port for 1 min and adding the embryos into the liquefied nitrogen; freezing for 35 min; rapidly lifting the embryos to the liquefied nitrogen port to equilibrize for 1 min; then quickly placing the embryos into a water bath at 38 DEG C for thawing; eluting by 0.25mol / L sucrose for 10 min; and culturing the embryos at zoea stage in seawater with salinity of 15.
Description
Technical field
The present invention relates to Eriocheir sinensia embryo low temperature Techniques of preserving.
Background technology
Eriocheir sinensia, as the economic kind of important crustacean, occupies important economic status in China's culture fishery.In recent years; because overfishing, unreasonable fishing gear utilization etc. cause the decline of studies on germplasm in Chinese mitten crab, Erocheir sinensis resource; bio-diversity and genetic diversity reduce, and carry out Eriocheir sinensia embryo's superfreeze preservation research for the enhancement releasing of the protection exploitation of elite germplasm, resource and carry out commercialization to cultivate significant.
Preserve and have successfully report for mammal and vertebrate embryo's superfreeze at present, but still have many key technology bottlenecks for invertebrate embryo's freezing preservation, yet there are no crustacean embryo superfreeze and preserve successfully report.
Summary of the invention
The problem that the present invention need to solve is to create a kind of superfreeze preservation Eriocheir sinensia embryo method.
Technical scheme of the present invention is chosen the embryo of front nauplius stage of Eriocheir sinensia or protozoea larva phase, front nauplius stage embryo is 1 of concentration 30%, 2-propane diols+20% dimethyl formamide is balance 40min in vetrifying solution, then balance 1min in-20 DEG C of ice chests, again after liquid nitrogen mouth balance 1min in direct plunge into Liquid Nitrogen, front nauplius stage embryo is after 40min is freezing, Quick is to liquid nitrogen mouth balance 1min, putting into fast 38 DEG C of water-baths thaws again, through 0.25mol/L sucrose wash-out 10min, front nauplius stage embryo is placed in the seawater of salinity 15 and cultivates, or adopt protozoea larva phase embryo in 1 of concentration 30%, 2-propane diols+20% dimethyl formamide is balance 40min in vetrifying solution, then balance 1min in-20 DEG C of ice chests, again after liquid nitrogen mouth balance 1min in direct plunge into Liquid Nitrogen, the protozoea larva phase, embryo was after 35min is freezing, and Quick, to liquid nitrogen mouth balance 1min, then is put into fast 38 DEG C of water-baths and thawed, through 0.25mol/L sucrose wash-out 10min, then protozoea larva phase embryo is placed in the seawater of salinity 15 and cultivates.
Outstanding feature of the present invention is to have created a kind of method that crustacean embryo superfreeze is preserved, and has successfully preserved first Eriocheir sinensia embryo, and has obtained the membrane young, reaches leading in the world.
Embodiment
The present invention is by Eriocheir sinensia mating season, gather Eriocheir sinensia ovigerous crab, from ovigerous crab, carefully peel off fertilized egg, get respectively embryo's (cell division phase of 5 different times, blastula stage, gastrul stage, front nauplius stage and protozoea larva phase) as research object, the embryo in these 5 periods is put into respectively to three kinds of variable concentrations (10%, 15%, 20%) 4 kinds of antifreeze (methyl-sulfoxides, methyl alcohol, 1, 2-propane diols, dimethyl formamide) in after balance 30min, observe survival rate of embryo, be front nauplius stage and protozoea larva phase embryonic development period of having established the suitable preservation of Eriocheir sinensia, the antifreeze that has filtered out suitable preservation is 1, 2-propane diols and dimethyl formamide, on this Research foundation, 4 kinds of single-factor antifreezes are combined according to finite concentration, form 6 kinds of vetrifying solutions, different times embryo is put into 6 kinds of vetrifying solutions respectively according to two step balancing methods, three step balancing methods and five step balancing method balance 40min, wherein (1) two step method, vetrifying solution is diluted to 1/2 concentration with the seawater of salinity 15, the embryo in each period is difference balance 20min in 1/2 concentration and vetrifying solution, (2) three-step approach, is diluted to vetrifying solution respectively 1/4 and 1/2 concentration with the seawater of salinity 15, and the embryo in each period is the about 13min of each balance in 1/4,1/2 concentration and vetrifying solution, (3) five-step approach, is diluted to vetrifying solution respectively 1/4,1/3,1/2 and 2/3 concentration with the seawater of salinity 15, the embryo in each period balance 8min successively in each diluted concentration and vetrifying solution.
Embryo observes survival rate situation after distinct methods balance, and establishing 30%1,2-propane diols+20% dimethyl formamide is best vetrifying solution formula, and five step balancing methods are optimum balance method.Front nauplius stage embryo is adopted in this vetrifying solution to five-step approach balance 40min, then balance 1min in-20 DEG C of ice chests, again after liquid nitrogen mouth balance 1min in direct plunge into Liquid Nitrogen, after 40min is freezing, embryo's Quick, to liquid nitrogen mouth balance 1min, then is put into fast to 38 DEG C of water-baths and thawed, through 0.25mol/L sucrose wash-out 10min, embryo is placed in the seawater of salinity 15 and cultivates, survival rate of embryo is 9.3 ± 2.5%; Protozoea larva phase embryo is adopted to five-step approach balance 40min in above-mentioned vetrifying solution, then balance 1min in-20 DEG C of ice chests, again after liquid nitrogen mouth balance 1min in direct plunge into Liquid Nitrogen, after 35min is freezing, by embryo's Quick to liquid nitrogen mouth balance 1min, putting into fast 38 DEG C of water-baths thaws again, through 0.25mol/L sucrose wash-out 10min, embryo is placed in the seawater of salinity 15 and cultivates, survival rate of embryo is 11.3 ± 3.6%, be cultured to the 7th day, the complete incubation of membrane of embryo.
Claims (1)
1. Eriocheir sinensia embryo cryopreservation method, it is characterized in that choosing the embryo of front nauplius stage of Eriocheir sinensia or protozoea larva phase, front nauplius stage embryo is 1 of concentration 30%, 2-propane diols+20% dimethyl formamide is in vetrifying solution, to adopt five-step approach balance 40min, vetrifying solution is diluted to respectively to 1/4 with the seawater of salinity 15, 1/3, 1/2 and 2/3 concentration, embryo balance 8min successively in each diluted concentration and vetrifying solution, then balance 1min in-20 DEG C of ice chests, again after liquid nitrogen mouth balance 1min in direct plunge into Liquid Nitrogen, front nauplius stage embryo is after 40min is freezing, Quick is to liquid nitrogen mouth balance 1min, putting into fast 38 DEG C of water-baths thaws again, through 0.25mol/L sucrose wash-out 10min, front nauplius stage embryo is placed in the seawater of salinity 15 and cultivates, or adopt protozoea larva phase embryo in 1 of concentration 30%, 2-propane diols+20% dimethyl formamide is in vetrifying solution, to adopt above-mentioned five-step approach balance 40min, then balance 1min in-20 DEG C of ice chests, again after liquid nitrogen mouth balance 1min in direct plunge into Liquid Nitrogen, the protozoea larva phase, embryo was after 35min is freezing, Quick is to liquid nitrogen mouth balance 1min, putting into fast 38 DEG C of water-baths thaws again, through 0.25mol/L sucrose wash-out 10min, then protozoea larva phase embryo is placed in the seawater of salinity 15 and cultivates.
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103704204B (en) * | 2014-01-06 | 2016-04-06 | 中国科学院海洋研究所 | A kind of Pacific oyster embryo cryopreservation method |
| CN103947584B (en) * | 2014-04-04 | 2016-09-28 | 湖南苗王生物科技有限公司 | A kind of defreezing method of Acipenser Sinensis glass frozen essence |
| DK3445167T3 (en) * | 2016-04-18 | 2021-04-12 | Planktonic As | Cryopreservation of young stages of creeping feet |
| CN111587876B (en) * | 2020-05-25 | 2023-03-21 | 大连海洋大学 | Rapid vitrification cryopreservation and recovery method for sea urchin intermedium embryo |
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| US4155331A (en) * | 1977-04-01 | 1979-05-22 | Baust John G | Method for cryopreservation of multicellular organisms |
| DE4205386C1 (en) * | 1992-02-19 | 1992-12-10 | Rainer Dipl.-Biol. 1000 Berlin De Bock | Cryo:preservation and revitalisation of fish eggs, crab larvae etc. - involves treating with e.g. DMSO, freezing and revitalising by warming |
| CN1600098A (en) * | 2003-09-28 | 2005-03-30 | 中国水产科学研究院东海水产研究所 | Method for freezing embryo of fish |
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Patent Citations (3)
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|---|---|---|---|---|
| US4155331A (en) * | 1977-04-01 | 1979-05-22 | Baust John G | Method for cryopreservation of multicellular organisms |
| DE4205386C1 (en) * | 1992-02-19 | 1992-12-10 | Rainer Dipl.-Biol. 1000 Berlin De Bock | Cryo:preservation and revitalisation of fish eggs, crab larvae etc. - involves treating with e.g. DMSO, freezing and revitalising by warming |
| CN1600098A (en) * | 2003-09-28 | 2005-03-30 | 中国水产科学研究院东海水产研究所 | Method for freezing embryo of fish |
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| C. JEYALECTUMIE，等.Cryopreservation of Spermatophores and Seminal Plasma of the Edible Crab Scylla serrata.《Biological Bulletin》.1989,第177卷(第2期),247-253. * |
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