CN103947584B - A kind of defreezing method of Acipenser Sinensis glass frozen essence - Google Patents
A kind of defreezing method of Acipenser Sinensis glass frozen essence Download PDFInfo
- Publication number
- CN103947584B CN103947584B CN201410134054.7A CN201410134054A CN103947584B CN 103947584 B CN103947584 B CN 103947584B CN 201410134054 A CN201410134054 A CN 201410134054A CN 103947584 B CN103947584 B CN 103947584B
- Authority
- CN
- China
- Prior art keywords
- essence
- capsule
- water
- defrosting
- acipenser sinensis
- 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.)
- Active
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
A kind of defreezing method of Acipenser Sinensis glass frozen essence, it is to take out being loaded with the capsule freezing essence in Dewar tank from liquid nitrogen, put in water-bath, keeping the temperature of water in water-bath is 28 DEG C, 40 42 seconds time, then take out the capsule equipped with defrosting seminal fluid, the defrosting seminal fluid in capsule is poured in Sheng essence bottle, Acipenser Sinensis female parent can be carried out in vitro fertilization.The present invention takes the mode of constant water-bath that Acipenser Sinensis is frozen the row defrosting that progresses greatly, and allows the sperm thawed be suitable for suitable use environment, it is possible to be effectively improved the survival activity freezing essence.After defrosting, the vigor of sperm is close to fresh essence, inseminates in fresh ovum with it, and its fresh feritilization of ovum rate is up to 80 90%, and fetal development is good, reduces the production cost of commercial application, improves the use value freezing essence preservation.
Description
Technical field
The present invention relates to the profound hypothermia thawing technology of a kind of fish hybridization breeding, the defreezing method of a kind of Acipenser Sinensis glass frozen essence.
Background technology
Save the application of biological heredity multiformity and cross-breeding from damage, select less than 170 DEG C Cryopreserved sperms to carry out the procreation of Fish in aquaculture, be the development trend of biotechnology innovation and application.But at present, deep-bed drying fish sperm is also the highly developed country method for laboratory, and practical ranges is the most very limited, its main cause, is owing to many kind fish sperms can occur extensive damage in process of freezing and thawing.The degree of infringement depends on freezing rate, frozen solution and thawing method.
Many scholars propose, if can realize vitrification during deep-bed drying freezes essence, would be even more beneficial to biological heredity multiformity and save the preservation of the research with cross-breeding and application, beneficially rare animal from damage.The method using glass freezing, if cryopreservation hereditary material can successfully be realized, will bring benefit to the mankind.
The existing slow freezing method of freezing of semen guard method easily forms ice crystal; the outer ice of born of the same parents and intracellular ice can be resulted in and damaging cells, in addition the crystallization recrystallization in frozen-thaw process, affect its survival rate; making this biological Cryopreservation Technology cost intensive, application efficiency is extremely low.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, and the defreezing method of a kind of Acipenser Sinensis glass frozen essence is provided.
The technical scheme is that the defreezing method of a kind of Acipenser Sinensis glass frozen essence, take out being loaded with the capsule freezing essence liquid nitrogen in Dewar tank, put in water-bath, keeping the temperature of water in water-bath is 28 DEG C-36 DEG C, second time 40-50, then take out the capsule equipped with defrosting seminal fluid, the defrosting seminal fluid in capsule is poured in Sheng essence bottle, Acipenser Sinensis female parent can be carried out in vitro fertilization.
The present invention further technical scheme is: the Acipenser Sinensis of defrosting is frozen essence and adopts freezing with the following method: after needing frozen Acipenser Sinensis sperm and the ratio uniform dilution of freeze proof protection liquid by volume part 100:90 110, add the freshly-slaughtered poultry egg yolk of 8-10 parts by volume, the arachidonic acid of 46-48 parts by volume, the lipid of 6-8 parts by volume, contain with freezing capsule after mixing, it is placed in drawer type freezing compartment freezing, freeze speed for-58 to-68 DEG C/min, after being chilled to-56 to-70 DEG C, it is discharged in filling the capsule freezing essence in the Dewar tank filling liquid nitrogen again, control to freeze speed for-148 to-152 DEG C/min, after being chilled to-190 to-196 DEG C frozen.
The present invention further technical scheme is: the preparation method of freeze proof protection liquid is: is joined in medical saline by the dimethyl phenol of 10% according to proportioning and is mixed evenly, adds mannitol, be mixed evenly；The weight proportion of freeze proof protection liquid is: medical saline: the dimethyl phenol of 10%: mannitol=100:8-10:6-8.
Due to the fact that the as above method that uses, compared with prior art, have the advantage that
Acipenser Sinensis is frozen the row that progresses greatly and thaws by the mode 1, taking constant water-bath, allows the sperm thawed be suitable for suitable use environment.Motility of sperm after defrosting, close to fresh essence, is inseminated in fresh ovum, and its fresh feritilization of ovum rate is up to 80-90%.And fetal development is good, reduce the production cost of commercial application, improve the use value freezing essence preservation.
2, the Acipenser Sinensis owing to thawing freezes essence is to use the method freezing of fast cooling; with freeze proof protection liquid as antifreezing agent; make effectively to change during ice phase change the area of ice microgranule; the ice-like form formed during greatly changing spermatozoa cryopreservation; both weakened and suppressed to freeze the generation of essence ice pellets, the most more effectively reduced ice microgranule solubility in aqueous medium, it is to avoid crystallization and recrystallization; avoid the injury freezing essence formation intracellular ice ice outer with born of the same parents, be effectively increased the survival activity freezing essence.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the detailed construction of the present invention is further described.
Embodiment 1:
A kind of defreezing method of Acipenser Sinensis glass frozen essence, it is to take out being loaded with the capsule freezing essence in Dewar tank from liquid nitrogen, put in water-bath, the temperature of water in water-bath is kept to be 34-36 DEG C, second time 40-42, then take out capsule, the defrosting seminal fluid in capsule is poured in Sheng essence bottle, Acipenser Sinensis female parent can be carried out in vitro fertilization.
Embodiment 2:
A kind of defreezing method of Acipenser Sinensis glass frozen essence, it is to take out being loaded with the capsule freezing essence in Dewar tank from liquid nitrogen, put in water-bath, the temperature of water in water-bath is kept to be 32-34 DEG C, second time 42-45, then take out capsule, the defrosting seminal fluid in capsule is poured in Sheng essence bottle, Acipenser Sinensis female parent can be carried out in vitro fertilization.
Embodiment 3:
The defreezing method of a kind of Acipenser Sinensis glass frozen essence, is to take out being loaded with the capsule freezing essence in Dewar tank from liquid nitrogen, puts in water-bath, keep the temperature of water in water-bath to be 30-32 DEG C, second time 45-48；Then take out capsule, the defrosting seminal fluid in capsule is poured in Sheng essence bottle, Acipenser Sinensis female parent can be carried out in vitro fertilization.
Embodiment 4:
The defreezing method of a kind of Acipenser Sinensis glass frozen essence, is to take out being loaded with the capsule freezing essence in Dewar tank from liquid nitrogen, puts in water-bath, keep the temperature of water in water-bath to be 28-30 DEG C, second time 48-50；Then take out capsule.Defrosting seminal fluid in capsule is poured in Sheng essence bottle, Acipenser Sinensis female parent can be carried out in vitro fertilization.
Claims (6)
1. a defreezing method for Acipenser Sinensis glass frozen essence, is characterized in that to be loaded with the capsule freezing essence from Du
Taking out in liquid nitrogen in crock, put in water-bath, keeping the temperature of water in water-bath is 28 DEG C-36 DEG C,
Second time 40-50, then take out the capsule equipped with defrosting seminal fluid, pour the defrosting seminal fluid in capsule into Sheng
In essence bottle, Acipenser Sinensis female parent can be carried out in vitro fertilization；The Acipenser Sinensis sperm of thawing be adopt the coldest
Freeze: the ratio uniform of frozen Acipenser Sinensis sperm and freeze proof protection liquid by volume part 100:90 110 will be needed
After dilution, add the freshly-slaughtered poultry egg yolk of 8-10 parts by volume, the arachidonic acid of 46-48 parts by volume, 6-8
The lipid of parts by volume, is mixed into seminal fluid mixed liquor, contains with freezing capsule, is placed in drawer type freezing compartment
Freezing, freezing speed is-58 to-68 DEG C/min, after being chilled to-56 to-70 DEG C, then will be loaded with seminal fluid mixing
The capsule of liquid is discharged in the Dewar tank filling liquid nitrogen, and controlling to freeze speed is-148 to-152 DEG C/min, freezing
To frozen after-190 to-196 DEG C.
The defreezing method of Acipenser Sinensis glass frozen the most according to claim 1 essence, is characterized in that freeze proof guarantor
The preparation method protecting liquid is: is joined by the dimethyl phenol of 10% in medical saline according to proportioning and mixes
Uniformly, add mannitol, be mixed evenly；The weight proportion of freeze proof protection liquid is: medical physiology
Saline: the dimethyl phenol of 10%: mannitol=100:8-10:6-8.
The defreezing method of Acipenser Sinensis glass frozen the most according to claim 1 essence, is characterized in that Dewar
It is loaded with in tank and freezes the capsule of essence and takes out from liquid nitrogen, put in water-bath, the temperature of water in holding water-bath
Degree, for 34-36 DEG C, second time 40-42, then takes out the capsule equipped with defrosting seminal fluid, by the solution in capsule
Freeze seminal fluid to pour in Sheng essence bottle, Acipenser Sinensis female parent can be carried out in vitro fertilization.
The defreezing method of Acipenser Sinensis glass frozen the most according to claim 1 essence, is characterized in that: will Du
It is loaded with in crock and freezes the capsule of essence and takes out from liquid nitrogen, put in water-bath, water in holding water-bath
Temperature is 32-34 DEG C, second time 42-45, then takes out the capsule equipped with defrosting seminal fluid, by capsule
Defrosting seminal fluid is poured in Sheng essence bottle, Acipenser Sinensis female parent can be carried out in vitro fertilization.
The defreezing method of Acipenser Sinensis glass frozen the most according to claim 1 essence, is characterized in that: will Du
It is loaded with in crock and freezes the capsule of essence and takes out from liquid nitrogen, put in water-bath, water in holding water-bath
Temperature is 30-32 DEG C, second time 45-48, then takes out the capsule equipped with defrosting seminal fluid, by capsule
Defrosting seminal fluid is poured in Sheng essence bottle, Acipenser Sinensis female parent can be carried out in vitro fertilization.
The defreezing method of Acipenser Sinensis glass frozen the most according to claim 1 essence, is characterized in that: will Du
It is loaded with in crock and freezes the capsule of essence and takes out from liquid nitrogen, put in water-bath, water in holding water-bath
Temperature is 28-30 DEG C, second time 48-50, then takes out the capsule equipped with defrosting seminal fluid, by capsule
Defrosting seminal fluid pour into Sheng essence bottle in, Acipenser Sinensis female parent can be carried out in vitro fertilization.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410134054.7A CN103947584B (en) | 2014-04-04 | 2014-04-04 | A kind of defreezing method of Acipenser Sinensis glass frozen essence |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410134054.7A CN103947584B (en) | 2014-04-04 | 2014-04-04 | A kind of defreezing method of Acipenser Sinensis glass frozen essence |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103947584A CN103947584A (en) | 2014-07-30 |
| CN103947584B true CN103947584B (en) | 2016-09-28 |
Family
ID=51325059
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410134054.7A Active CN103947584B (en) | 2014-04-04 | 2014-04-04 | A kind of defreezing method of Acipenser Sinensis glass frozen essence |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103947584B (en) |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030077566A1 (en) * | 2001-09-10 | 2003-04-24 | Palasz Andrzej T. | Method for cryopreserving mammalian cells and tissues |
| 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 |
| CN101003791A (en) * | 2007-01-11 | 2007-07-25 | 山东省农业科学院畜牧兽医研究所 | Refrigeration method for preserving embryo and oocyte by vitrification of glasscapillary |
| 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 |
| CN101836615B (en) * | 2010-05-27 | 2013-04-24 | 中国长江三峡集团公司中华鲟研究所 | Acipenser sinensis ultralow-temperature frozen semen artificial insemination method |
| CN103098792B (en) * | 2011-11-10 | 2014-09-10 | 中国水产科学研究院东海水产研究所 | Ultralow temperature cryopreservation method of Eriocheir sinensis embryo |
| CN103184189A (en) * | 2011-12-29 | 2013-07-03 | 北京富通华生物科技有限公司 | Cultivation method of cross-bred wagy |
| CN102640745B (en) * | 2012-05-04 | 2014-05-21 | 南京林业大学 | Ultralow-temperature cryopreservation and recovery method for embryonic materials |
-
2014
- 2014-04-04 CN CN201410134054.7A patent/CN103947584B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103947584A (en) | 2014-07-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104663649B (en) | Human oocytes cryoprotective agent | |
| CN104054697B (en) | A kind of acipenser dabryanus sperm cryopreservation liquid and preparation method and application | |
| CN104145944B (en) | A kind of stalwart blood clam sperm super-low temperature freezing is preserved and Activiation method | |
| CN103348966B (en) | Method for efficient ultralow temperature cryopreservation of turbot sperms | |
| CN105052894B (en) | A kind of GV phases egg mother cell freezen protective liquid and freezing and storing method | |
| CN105532639B (en) | A kind of sub- Large Copacity of epinephelus lanceolatus milt, the method for high-efficiency ultralow temperature freezen protective | |
| CN104521943A (en) | Ultralow-temperature refrigeration preservation and recovery method for oxyeleotris marmorata semen | |
| Parks | Processing and handling bull semen for artificial insemination—Don't add insult to injury | |
| CN104322484A (en) | Freezing and unfreezing method for boar semen | |
| CN103141472B (en) | Ultra-low-temperature freezing preservation and activation method for Hong Kong oyster sperm | |
| CN109468269A (en) | Cell vitrifying thawing solution and defreezing method | |
| CN105360109B (en) | A kind of Pelteobagrus fulvidraco sperm super-low temperature freezing store method | |
| CN106070184A (en) | The freezing of a kind of pig semen and defreezing method | |
| CN103262837B (en) | Livestock seminal fluid cryoprotectant and application thereof | |
| CN103947584B (en) | A kind of defreezing method of Acipenser Sinensis glass frozen essence | |
| CN106818708A (en) | Epinephelus fuscoguttatus sperm super-low temperature freezing protective agent and its store method | |
| CN106857497A (en) | A kind of seven methods preserved with grouper sperm super-low temperature | |
| CN116235847A (en) | Low-temperature cryopreservation tube, and grouper low-temperature cryopreservation method and application | |
| CN102217590A (en) | Pomfret sperm cryopreservation method | |
| CN103931607A (en) | Vitrification freezing method for sturgeon sperms | |
| CN106993605A (en) | A kind of down producing goat freezing seminal fluid dilution formula | |
| CN103931606A (en) | Preparation method for vitrification freezing-thawing protection liquid of sturgeon sperms | |
| US7011937B2 (en) | Method and solutions for cryopreserving oocytes, especially fresh human oocytes | |
| US20080113432A1 (en) | Methods and compositions for reanimating cryopreserved oocytes | |
| Otoi et al. | Developmental competence of bovine oocytes frozen at different cooling rates |
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 |