CN1748494A - Porgy embryo particulate glass freezing and storing method - Google Patents
Porgy embryo particulate glass freezing and storing method Download PDFInfo
- Publication number
- CN1748494A CN1748494A CN 200410050415 CN200410050415A CN1748494A CN 1748494 A CN1748494 A CN 1748494A CN 200410050415 CN200410050415 CN 200410050415 CN 200410050415 A CN200410050415 A CN 200410050415A CN 1748494 A CN1748494 A CN 1748494A
- Authority
- CN
- China
- Prior art keywords
- embryo
- porgy
- freezing
- storing method
- particulate glass
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The invention belongs to the marine biotechnology field, specifically freezing by particle the porgy embryo, liquid nitrogen is preserved and the wash-out that thaws, and obtains the process of bringing back to life the embryo.The present invention is to porgy heart beat period embryo, by adding antifreeze, adopts steps such as quick particle glass freezing, liquid nitrogen are preserved, thawed, wash-out, obtains the resurrection embryo.There is significance quality saving, genetic diversity and aspects such as breeding and sustainable breed that the porgy embryo cryopreservation is preserved for porgy.
Description
Technical field
The invention belongs to the marine biotechnology field, specifically freezing by particle the porgy embryo, liquid nitrogen is preserved and the wash-out that thaws, and obtains the process of bringing back to life the embryo.
Background technology
The freezing preservation research of fish embryo has 20 years history approximately.Chinese scholars has been carried out big quantity research to the aspects such as micromechanism of damage, frozen cooling speed, the rewarming that thaws, antifreeze kind and toxicity of the freezing preservation of fish embryo, and obtains remarkable progress.The cyprinid fish embryo has obtained more stable resurrection rate (Zhang Kejian etc. in the freezing preservation of temperature more than-100 ℃ at present.The research of three kinds of freshwater fish embryo low temperature preservations and cooling and rewarming speed.Aquatic product journal 1997,21 (4): 366-372), salmon trout class embryo also breaks through-25 ℃ boundary (Erdahl, D.A., Preservation of spermatozoa and ova from freshwater fish.Thesis of the Universityof Minnesota 1986).Carp (Zhang, X.S., et al.A study on the cryopreservation of commoncarp embryos.Cryo Letters, 1989,10:271-278), loach (Zhang Kejian etc., the same) obtained indivedual freezing resurrection embryos in liquid nitrogen region, although above experiment also is difficult to repetition, can be described as present reasonable result.Since fish embryo especially seawater fish embryo volume big, contain characteristics such as a large amount of yolk and moisture, preserve to embryo cryopreservation and cause very big difficulty.The particulate glass method is based on that the instrument heat exchange area is big more, the thermal conductivity factor at interface is high more, the method for vitrification that cooling rate designs with regard to fast more principle, with its embryo independent freezing, cooling rate is fast, convenient to operation, equipment requires characteristics such as simple and showed good prospects for application.The embryo cryopreservation of porgy is preserved significant to quality saving, genetic diversity protection and breeding and the aquaculture sustainable development thereof of the famous and precious fish of this seawater of porgy.
Summary of the invention
The object of the present invention is to provide porgy embryo particulate glass freezing and storing method.
For achieving the above object, the technical scheme taked of the present invention is:
A kind of porgy embryo particulate glass freezing and storing method, select the porgy heart beat period to the membrane pre-embryo, two steps balance precooling in the antifreeze of variable concentrations, directly the embryo is dripped on the copper foil that places on the liquid nitrogen surface with glass pipette, snap frozen forms the vitrifying particle, directly immerses in the liquid nitrogen freezingly then, directly frozen particle is put into when thawing in 37 ℃ the sucrose solution of 0.6M and is carried out, behind wash-out, change in the seawater and cultivate, obtain the resurrection embryo.
When selecting porgy heart beat period embryo, porgy embryo heartbeat number of times 60-140 time/minute.
When using antifreeze, antifreeze is a glycerine, is base fluid with balanced salt solution Cortland, and the glycerol concentration of two step balances is respectively the first step 20% volumetric concentration and second step, 40% volumetric concentration.
During precooling, carry out in two steps by precooling in refrigerator for the porgy embryo, and temperature is 0～4 ℃, and precooling is respectively the first step 14～16min, second step, 25～35s.
In the freezing process, having used thickness is that 12 microns copper foil is as refrigerant.
In the freezing process, the embryo directly drips on the low temperature copper foil, forms the vitrifying particle, particle volume 0.005-0.01cm
3
In the process of freezing preservation, the embryo is freezing separately, be stored in the liquid nitrogen with the form of vitrifying particle.
When thawing, thaw and remove anti frozen liquid and carry out synchronously, the solution that thaws is the 0.6mol/L sucrose solution.
When water-bath was thawed, 37 ℃ of thaw points, the solution that thaws were the 0.6mol/L sucrose solution, melted to particle.
During wash-out, in 0.6mol/L sucrose, soaked 4～6 minutes.
The invention has the advantages that:
(1) the porgy heart beat period to the membrane pre-embryo with other in period stage embryo compare, the strongest to the tolerance of low temperature, antifreeze toxicity;
(2) to form ability strong in the glycerine vitrifying, and the freeze proof protective effect to embryo's particle in freezing is good.
(3) balance has reduced the toxicity of antifreeze to the embryo in two steps, can satisfy the requirement of vitrifying process high concentration simultaneously.
(4) good heat conductivity of copper foil can improve cooling velocity greatly;
(5) particle drips, and volume is little, and area of dissipation is big, does not have the obstruct of refrigerated container, helps snap frozen;
(6) wash-out with thaw synchronously, the secondary wash-out has been avoided the toxic action of antifreeze to the embryo of thawing;
The present invention is to porgy heart beat period embryo, by adding antifreeze, adopts steps such as quick particle glass freezing, liquid nitrogen are preserved, thawed, wash-out, obtains the resurrection embryo.There is significance quality saving, genetic diversity and aspects such as breeding and sustainable breed that the porgy embryo cryopreservation is preserved for porgy.
Embodiment
Protection scope of the present invention not only is confined in the following example.
(1) fish for batch natural birth porgy fertilized egg, place interior cultivation of hatching pail of 18 ± 1 ℃ of ocean temperatures, little inflation, to the heart beat period embryo, the heartbeat number of times is (60-140 time/minute) more than 60 times/minute;
(2) a certain amount of liquid nitrogen of incubator of packing into, the high about 10cm of liquid level lies in copper foil (thick 12 microns) on the liquid nitrogen surface and fixes.
(3) fish for the embryo with 80 mesh sieve tulles, gently dip in suction with filter paper and remove surperficial seawater, put into glycerine volumetric concentration 20% antifreeze, balanced salt solution Cortland (is surplus, pH=8.28), soaks 15min as base fluid, change over to then in glycerine volumetric concentration 40% antifreeze, balanced salt solution Cortland (is surplus, pH=8.28), carries out freezing behind the 30s fast as base fluid.Immersion process carries out in 0～4 ℃ of refrigerator.
(4) draw an embryo who soaked with the glass pipette of the about 1mm of diameter, drip on copper foil fast, can form the about 0.005-0.01cm of volume immediately
3The clear glass particle.
(5) it is freezing that repeated multiple times is carried out above particle, frozen particle imported the Copper Foil film trap that places liquid nitrogen;
When (6) thawing the particle in the Copper Foil film trap is poured into temperature and is in 37 ℃ the sucrose solution of 0.6M, the very fast thawing of particle, then the embryo is come out with screen filtration, change seawater over to after putting into the sucrose solution room temperature wash-out 5min of 0.6M again, observe embryo morphology and survival condition.
(7) in 48 particle embryos frozen, bringing back to life the embryo is 16, and the resurrection rate is that 33.3%, 11 incubation of membrane is grown to prelarva incubation rate: 68.8%.
In addition, through test, during precooling, temperature is 0～4 ℃ to the porgy embryo in refrigerator, and pre-cool time of two steps can be respectively the first step 14～16 minutes, second 25～35 seconds steps; During wash-out, in 0.6mol/L sucrose, soaked 4～6 minutes, also can reach the object of the invention, because its enforcement is comparatively simple, so repeat no more.
Claims (10)
1, a kind of porgy embryo particulate glass freezing and storing method, it is characterized in that: select the porgy heart beat period to the membrane pre-embryo, two steps balance precooling in the antifreeze of variable concentrations, directly the embryo is dripped on the copper foil that places on the liquid nitrogen surface with glass pipette, snap frozen forms the vitrifying particle, directly immerses in the liquid nitrogen freezingly then, directly frozen particle is put into when thawing in 37 ℃ the sucrose solution of 0.6M and is carried out, behind wash-out, change in the seawater and cultivate, obtain the resurrection embryo.
2, according to the described porgy embryo of claim 1 particulate glass freezing and storing method, it is characterized in that: when selecting porgy heart beat period embryo, porgy embryo heartbeat number of times 60-140 time/minute.
3, according to the described porgy embryo of claim 1 particulate glass freezing and storing method, it is characterized in that: when using antifreeze, antifreeze is a glycerine, with balanced salt solution Cortland is base fluid, and the glycerol concentration of two step balances is respectively the first step 20% volumetric concentration and second step, 40% volumetric concentration.
4, according to the described porgy embryo of claim 1 particulate glass freezing and storing method, it is characterized in that: during precooling, carry out in two steps by precooling in refrigerator for the porgy embryo, and temperature is 0～4 ℃, and precooling is respectively the first step 14～16min, second step, 25～35s.
5, according to the described porgy embryo of claim 1 particulate glass freezing and storing method, in the freezing process, it is characterized in that: having used thickness is that 12 microns copper foil is as refrigerant.
6, according to the described porgy embryo of claim 1 particulate glass freezing and storing method, it is characterized in that: in the freezing process, the embryo directly drips on the low temperature copper foil, forms the vitrifying particle, particle volume 0.005-0.01cm
3
7, according to the described porgy embryo of claim 1 particulate glass freezing and storing method, it is characterized in that: in the process of freezing preservation that the embryo is freezing separately, be stored in the liquid nitrogen with the form of vitrifying particle.
8, according to the described porgy embryo of claim 1 particulate glass freezing and storing method, it is characterized in that: when thawing, thaw and remove anti frozen liquid and carry out synchronously, the solution that thaws is the 0.6mol/L sucrose solution.
9, according to the described porgy embryo of claim 1 particulate glass freezing and storing method, it is characterized in that: when water-bath was thawed, 37 ℃ of thaw points, the solution that thaws were the 0.6mol/L sucrose solution, melted to particle.
10, according to the described porgy embryo of claim 1 particulate glass freezing and storing method, it is characterized in that: during wash-out, in 0.6mol/L sucrose, soaked 4～6 minutes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100504156A CN100348096C (en) | 2004-09-15 | 2004-09-15 | Vitrifiation freezing preservation method for genine porgy embryo particles |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100504156A CN100348096C (en) | 2004-09-15 | 2004-09-15 | Vitrifiation freezing preservation method for genine porgy embryo particles |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1748494A true CN1748494A (en) | 2006-03-22 |
| CN100348096C CN100348096C (en) | 2007-11-14 |
Family
ID=36604297
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100504156A Expired - Fee Related CN100348096C (en) | 2004-09-15 | 2004-09-15 | Vitrifiation freezing preservation method for genine porgy embryo particles |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100348096C (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100459861C (en) * | 2007-06-18 | 2009-02-11 | 北京锦绣大地农业股份有限公司 | Livestock embryo vitrifying freeze process on metal surface |
| CN104012521A (en) * | 2014-06-10 | 2014-09-03 | 西安交通大学 | Non-contact type liquid drop method refrigerating device and refrigerating method |
| CN104982501A (en) * | 2015-06-25 | 2015-10-21 | 浙江万里学院 | Method for liquid nitrogen glassy state freezing preservation of Penaeus vannamei |
| CN105163579A (en) * | 2013-04-09 | 2015-12-16 | 楼伟 | Biological sample vitrification carrier and usage thereof |
| CN115052479A (en) * | 2019-12-10 | 2022-09-13 | 耶达研究及发展有限公司 | Cryopreserved insects and methods of producing same |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1133369C (en) * | 2000-05-08 | 2004-01-07 | 中国农业大学 | One-step process for embryo vitrifying freeze storage |
-
2004
- 2004-09-15 CN CNB2004100504156A patent/CN100348096C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100459861C (en) * | 2007-06-18 | 2009-02-11 | 北京锦绣大地农业股份有限公司 | Livestock embryo vitrifying freeze process on metal surface |
| CN105163579A (en) * | 2013-04-09 | 2015-12-16 | 楼伟 | Biological sample vitrification carrier and usage thereof |
| CN104012521A (en) * | 2014-06-10 | 2014-09-03 | 西安交通大学 | Non-contact type liquid drop method refrigerating device and refrigerating method |
| CN104982501A (en) * | 2015-06-25 | 2015-10-21 | 浙江万里学院 | Method for liquid nitrogen glassy state freezing preservation of Penaeus vannamei |
| CN104982501B (en) * | 2015-06-25 | 2018-09-28 | 浙江万里学院 | A kind of method that Penaeus Vannmei liquid nitrogen glassy state freezes preservation |
| CN115052479A (en) * | 2019-12-10 | 2022-09-13 | 耶达研究及发展有限公司 | Cryopreserved insects and methods of producing same |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100348096C (en) | 2007-11-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1295956C (en) | Practicalization method for frozen preserving sperm of fish | |
| CN104054697B (en) | A kind of acipenser dabryanus sperm cryopreservation liquid and preparation method and application | |
| CN110326610B (en) | Ultralow temperature cryopreservation method for sea cucumber sperms | |
| Tsai et al. | Sugars as supplemental cryoprotectants for marine organisms | |
| CN112741078B (en) | Hexagrammos otakii sperm productive cryopreservation method | |
| CN115708506B (en) | Low-temperature preservation method of Pinctada martensii Bei Jingzi | |
| CN114258911B (en) | Cryopreservation solution and cryopreservation method for sparus latus sperms | |
| US20060252026A1 (en) | Cryopreservation method for bivalve oocytes | |
| Yang et al. | Review of molluscan larval cryopreservation and application to germplasm cryobanking and commercial seed production | |
| CN114903032A (en) | Preparation method of frozen semen of Dongfrui raw milk sheep | |
| CN100348096C (en) | Vitrifiation freezing preservation method for genine porgy embryo particles | |
| CN111587876B (en) | Rapid vitrification cryopreservation and recovery method for sea urchin intermedium embryo | |
| US9005886B2 (en) | Method for cryopreservation of human spermatozoa free from seminal plasma using a fast and simple aseptic vitrification-devitrification process; portable kit for carrying out the method; and use of the same for treatment of disorders related to reproductive failures | |
| US20060105317A1 (en) | Method and solutions for cryopreserving oocytes, especially fresh human oocytes | |
| JP6983452B2 (en) | Ultra-low temperature cryopreservation method for sea cucumber sperm | |
| CN112075415B (en) | Cryoprotectant and ultra-low temperature cryopreservation method for stichopus japonicus sperms | |
| CN104336004A (en) | Method for collection and ultralow temperature refrigeration preservation of high-quality pacific oyster sperms | |
| CN116076485A (en) | Ultra-low temperature freezing protection liquid for grape dental oyster sperm and ultra-low temperature freezing preservation method | |
| CN1263378C (en) | Red sea bream embryo freezing preservation method | |
| Narida et al. | First successful production of adult corals derived from cryopreserved larvae | |
| CN1749392A (en) | Porgy straw vitrification freezing and storing method | |
| Okubo et al. | Sexual reproduction in transplanted coral fragments of Acropora nasuta | |
| CN117717066B (en) | Embryo preservation solution, cryopreservation method thereof and application of embryo preservation solution in cryopreservation of Babylonia embryos | |
| CN116034992B (en) | Low-temperature stichopus japonicus sperm preservation solution and application and stichopus japonicus sperm preservation method | |
| CN1944636A (en) | Freeze preservating method for domestic pig skin tissue |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20071114 Termination date: 20130915 |