CN104604845A - Hucho taimen seminal fluid ultralow-temperature cryopreservation fluid and hucho taimen seminal fluid freezing and preserving method - Google Patents
Hucho taimen seminal fluid ultralow-temperature cryopreservation fluid and hucho taimen seminal fluid freezing and preserving method Download PDFInfo
- Publication number
- CN104604845A CN104604845A CN201510063790.2A CN201510063790A CN104604845A CN 104604845 A CN104604845 A CN 104604845A CN 201510063790 A CN201510063790 A CN 201510063790A CN 104604845 A CN104604845 A CN 104604845A
- Authority
- CN
- China
- Prior art keywords
- seminal fluid
- sieve salmon
- wise
- cryopreservation
- wise sieve
- 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.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a hucho taimen seminal fluid ultralow-temperature cryopreservation fluid and a hucho taimen seminal fluid cryopreservation method, and relates to a fish seminal fluid ultralow-temperature cryopreservation fluid and a hucho taimen seminal fluid cryopreservation method, which are used for solving the problems that in an existing hucho taimen sperm long-term freezing technique, the motility rate is low, viability of alive sperms is low, and the activity ratio is reduced greatly. In the hucho taimen seminal fluid ultralow-temperature cryopreservation fluid, the molar ratio of K<+> to Na<+> is smaller than 1 to 16, and the concentration of K<+> in the hucho taimen seminal fluid ultralow-temperature cryopreservation fluid is 0.00480mol/L to 0.00682mol/L. The cryopreservation method comprises the following steps: firstly, collecting fresh hucho taimen seminal fluids; secondly, preparing a seminal cryopreservation mixed solution; thirdly, subpackaging the seminal fluid cryopreservation mixed solution; fourthly, performing cryopreservation; and fifthly, unfreezing by adopting a rapid water bath unfreezing method. The method is simple and feasible, and has the advantages of long cryopreservation time, high sperm quality after unfreezing.
Description
Technical field
The present invention relates to the freezing and storing method of a kind of fish sperm cryogenic freezing conserving liquid and wise sieve salmon seminal fluid.
Background technology
Wise man sieve salmon belongs to salmon shape order, salmon section, is under the jurisdiction of wise sieve salmon genus; Threatened level is easily endanger.Because wise sieve salmon is nutritious, fine and tender taste, meat flavour is delicious, deeply favors by consumers in general; Therefore, the demand of market to wise sieve salmon constantly increases in recent years.Although the commercialization cultivation of wise sieve salmon and breeding cause obtain greatly develop; but it is always out in the cold in the protection carrying out germ plasm resource in seed selection work; cause inbreeding kind matter in wise sieve salmon crossbreeding process to be degenerated to grow in intensity; genetic diversity reduces; quality declines, and growth performance is low, and the defence capability of most of breed variety to disease and environment-stress reduces; disease takes place frequently, and thus causes huge economic loss to breeding enterprise.
Since eighties of last century fifties, British scholar Blaxter first successfully to fish spermary carry out freezing since, external many scholars preserve the long-term frozen of fish sperm and have also carried out correlative study and exploration, not only significant to the life process of fish, and also huge profound influence will be produced to cultivation fish new varieties and commercialization cultivation thereof.
Although, long-term frozen technology had successful case at other fingerlings, but there is motility rate low (about 30%) after the sperm freezing of wise sieve salmon thaws again, the problem that the vigor of survival sperm is low, activity ratio (motion ratio is less than 20%) declines to a great extent, causes the sperm of wise sieve salmon but still cannot be preserved by long-term frozen technology at present.
Summary of the invention
The motility rate that the object of the invention is to solve existing wise sieve salmon sperm long-term frozen technology existence is low, the problem that the vigor of survival sperm is low, activity ratio declines to a great extent, and the freezing and storing method of a kind of wise sieve salmon seminal fluid Ultra-cryofreezing preservation liquid provided and wise sieve salmon seminal fluid.
K in molar ratio in the present invention wise man sieve salmon seminal fluid Ultra-cryofreezing preservation liquid
+︰ Na
+<1 ︰ 16, K in wise sieve salmon seminal fluid Ultra-cryofreezing preservation liquid
+concentration is 0.00480mol/L ~ 0.00682mol/L.
Wise man sieve salmon seminal fluid Ultra-cryofreezing preservation liquid is by NaCl, the KCl of 0.325 ~ 0.475g, the CaCl of 0.12 ~ 0.15g of 7.95 ~ 8.07g
2, 0.349 ~ 0.351g NaHCO
3, 0.059 ~ 0.061g KH
2pO
4, the glucose of 0.30 ~ 0.38g and 1000ml distilled water composition.
Above-mentioned wise sieve salmon seminal fluid Ultra-cryofreezing preservation liquid freezen protective wise man sieve salmon seminal fluid is adopted to carry out according to the following steps:
One, wise sieve salmon fresh semen is gathered;
Two, in 7.92ml wise man sieve salmon seminal fluid Ultra-cryofreezing preservation liquid, 0.27ml glycerine is added, fully mix with eddy blending machine, then 4 DEG C of environment balances are placed in 15 minutes, put into fresh wise sieve salmon seminal fluid again to mix, abundant mixing be placed in 4 DEG C of environment balance 1 minute, add again afterwards 0.27ml glycerine fully mix be put in 4 DEG C of environment balance 1 minute, then add 0.54ml glycerine again, abundant mixing, namely obtains semen cryopreservation mixed liquor;
Three, semen cryopreservation mixed liquor micropipettor is sub-packed in straw, is then positioned in 4 DEG C of environment for subsequent use;
Four, liquid nitrogen is imported in bubble chamber with cover, semen cryopreservation mixed liquor in immigration straw is at distance liquid nitrogen ullage 8cm place's stop 5 ~ 15min, then at distance liquid nitrogen ullage 4cm place's stop 3 ~ 8min, then by medium-term and long-term for straw direct plunge into Liquid Nitrogen preservation;
Five, thaw adopt quick water-bath freezing process; Wherein, in step 2, the addition of fresh wise sieve salmon seminal fluid is 3ml.
Wise man sieve salmon is different from other seawater fishs and temperate water fishes, the sperm of wise man sieve salmon only could keep static be not activated (not doing straight line or vortex movement) in the solution of particular ion concentration, but also will keep K in Ultra-cryofreezing preservation liquid simultaneously
+with Na
+between special ratios relation can enough realize.
The present invention wise man sieve salmon seminal fluid Ultra-cryofreezing preservation liquid is by K
+concentration is designed to 0.00480mol/L ~ 0.00682mol/L, keeps that wise sieve salmon sperm is static not to be activated; And the energy of wise sieve salmon sperm is high after thawing, motion ratio>=50%, survival rate>=60%.
Adopt freezing and storing method of the present invention wise man sieve salmon sperm can preserve 3 ~ 15 years, the even longer time.High, motion ratio >=50% of the energy of wise sieve salmon sperm after wise man sieve salmon sperm cryopreservation thaws for 3 years, survival rate >=60%.
The inventive method is simple, has the freezen protective time long, the advantage that after thawing, sperm quality is high.
Different for breeding period by solving wise sieve salmon to wise sieve salmon sperm cryopreservation, the problem of breeding cannot be joined between each breeding populations; Especially the hybridization between the strain that genetic distance is far away, kind.Wise man sieve salmon sperm cryopreservation also makes trans-regional crossbreed obtain and realize, and reduces cost of transportation, it also avoid the risk that in transportation, live body death causes.
The present invention wise man sieve salmon sperm freezing protects technology to following commercial aquaculture kind, avoids natural breeding, inbreeding or microcommunity breeding to be significant.Also can effectively prevent the kind matter that wise sieve salmon gene is lost and hereditary transmuting causes from degenerating.
The present invention has profound influence to the preserving seed of the improved seeds of wise sieve salmon, genetic diversity conservation, genetic breeding, artificial propagation.Make the frozen sperm storehouse building wise sieve salmon, form genetic background archives and become possibility.Can the threat that wise sieve salmon species are caused such as corresponding burst disease, natural calamity or water pollution better.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises any combination between each embodiment.
Embodiment one: K in molar ratio in present embodiment wise man sieve salmon seminal fluid Ultra-cryofreezing preservation liquid
+︰ Na
+<1 ︰ 16, K in wise sieve salmon seminal fluid Ultra-cryofreezing preservation liquid
+concentration is 0.00480mol/L ~ 0.00682mol/L.
Embodiment two: the difference of present embodiment and embodiment one is: wise sieve salmon seminal fluid Ultra-cryofreezing preservation liquid is by NaCl, the KCl of 0.325 ~ 0.475g, the CaCl of 0.12 ~ 0.15g of 7.95 ~ 8.07g
2, 0.349 ~ 0.351g NaHCO
3, 0.059 ~ 0.061g KH
2pO
4, the glucose of 0.30 ~ 0.38g and 1000ml distilled water composition.Other is identical with embodiment one.
HCO in present embodiment
3 -and H
2pO
4 -there is weakly ionized effect, weak base effect can be formed, improve the fertility of sperm; And the sperm viability of wise sieve salmon can be increased.
Glucose not only can provide external source energy to sperm; diaphragm can also be formed at sperm skin; in the energy loss that freezing state suppresses alkaline environment to cause wise sieve salmon sperm; and glucose can be water-soluble rapidly after thawing; remove and the sugar-coat of wise sieve salmon sperm is protected, sperm is better played a role at alkaline environment.Therefore, even the energy in weakly alkaline freezen protective liquid in wise sieve salmon sperm head still can be retained at prolonged cold environment, be not consumed.
Glucose not only solves the damaging action of other ion pairs wise man sieve salmon sperm, also solves the toxic action reducing antifreeze.
Embodiment three: the difference of present embodiment and embodiment one is: wise sieve salmon seminal fluid Ultra-cryofreezing preservation liquid is by the CaCl of KCl, 0.14g of NaCl, 0.4g of 8.01g
2, 0.35g NaHCO
3, 0.06g KH
2pO
4, the glucose of 0.34g and 1000ml distilled water composition.Other is identical with embodiment one.
Embodiment four: present embodiment wise man sieve salmon seminal fluid carries out freezen protective according to the following steps:
One, wise sieve salmon fresh semen is gathered;
Two, in 7.92ml wise man sieve salmon seminal fluid Ultra-cryofreezing preservation liquid, 0.27ml glycerine is added, fully mix with eddy blending machine, then 4 DEG C of environment balances are placed in 15 minutes, put into fresh wise sieve salmon seminal fluid again to mix, abundant mixing be placed in 4 DEG C of environment balance 1 minute, add again afterwards 0.27ml glycerine fully mix be put in 4 DEG C of environment balance 1 minute, then add 0.54ml glycerine again, abundant mixing, namely obtains semen cryopreservation mixed liquor;
Three, semen cryopreservation mixed liquor micropipettor is sub-packed in straw, is then positioned in 4 DEG C of environment for subsequent use;
Four, liquid nitrogen is imported in bubble chamber with cover, semen cryopreservation mixed liquor in immigration straw is at distance liquid nitrogen ullage 8cm place's stop 5 ~ 15min, then at distance liquid nitrogen ullage 4cm place's stop 3 ~ 8min, then by medium-term and long-term for straw direct plunge into Liquid Nitrogen preservation;
Five, thaw adopt quick water-bath freezing process; Wherein, in step 2, the addition of fresh wise sieve salmon seminal fluid is 3ml.
In present embodiment, wise sieve salmon seminal fluid Ultra-cryofreezing preservation liquid is by the CaCl of KCl, 0.14g of NaCl, 0.4g of 8.01g
2, 0.35g NaHCO
3, 0.06g KH
2pO
4, the glucose of 0.34g and 1000ml distilled water composition.
In present embodiment, glycerine is antifreeze.
Present embodiment step one gathers wise sieve salmon fresh semen: pulled out from pond by wise sieve salmon milter of healthy and strong for body health, and working concentration is that the Phenoxyethanol aqueous solution of 5ml/L is anaesthetized; Treat that wise sieve salmon enters narcosis, its body surface and gonopore are cleaned with dry sterile gauze repeatedly; From the beginning powerful pressing upper abdomen (about above lateral line scales) repeatedly to tail direction afterwards, ripe seminal fluid flows out on a small quantity, continue the seminal fluid flowed out by 2ml suction pipe or 5ml syringe continuous acquisition, be filled and constantly put into large mouth container afterwards, and 4 DEG C of low temperature are kept in.Be the most frequently used method by manual compression belly collecting semen, noting will the pollution to seminal fluid such as anti-sealing, blood, epidermal mucus, urine and ight soil when gathering.
After gathering the fresh essence of wise sieve salmon, semen quality is identified: get appropriate seminal fluid, utilize the water of isothermal or hypotonic sodium salt solution to activate sperm, the vigor condition of microscopy sperm, after ripe activation of spermatozoa, rectilinear motion fast should be made immediately, or eddying motion; The seminal fluid of activity ratio more than 80% can carry out freezen protective.
Present embodiment forms mixture by wise sieve salmon seminal fluid Ultra-cryofreezing preservation liquid and the wise sieve salmon seminal fluid be saved; concurrent looks mutual effect; in the specific temperature-fall period of present embodiment seminal fluid itself with refining become branch alleviate run into freezen protective liquid and meet with hypotonic water suction may; along with the continuous fusion of refining composition and freezen protective liquid; make the effect of the steady seepage of the ions exert in freezen protective liquid pressure, protect sperm and reduce antifreeze toxicity.
Embodiment five: the difference of present embodiment and embodiment four is: liquid nitrogen liquid level distance case mouth 12cm ~ 15cm in step 4.Other step and parameter identical with embodiment four.
Embodiment six: the difference of present embodiment and embodiment four or five is: the quick water-bath freezing process of step 5: the straw of freezen protective is carried out water-bath and thaws, water-bath water temperature is 25 DEG C ~ 30 DEG C, thawing time is 15 ~ 18s, shakes straw in course of defrosting.Other step and parameter identical with embodiment four or five.
Embodiment 1
Wise man sieve salmon seminal fluid carries out freezen protective according to the following steps:
One, gather wise sieve salmon fresh semen, sperm motility is 85.4% after testing;
Two, in 7.92ml wise man sieve salmon seminal fluid Ultra-cryofreezing preservation liquid, 0.27ml glycerine is added, fully mix with eddy blending machine, then 4 DEG C of environment balances are placed in 15 minutes, put into fresh wise sieve salmon seminal fluid again to mix, abundant mixing be placed in 4 DEG C of environment balance 1 minute, add again afterwards 0.27ml glycerine fully mix be put in 4 DEG C of environment balance 1 minute, then add 0.54ml glycerine again, abundant mixing, namely obtains semen cryopreservation mixed liquor;
Three, semen cryopreservation mixed liquor micropipettor is sub-packed in straw, is then positioned in 4 DEG C of environment for subsequent use;
Four, imported by liquid nitrogen in bubble chamber with cover, the semen cryopreservation mixed liquor moved in straw stops 5min at distance liquid nitrogen ullage 8cm place, is then stopping 3min apart from liquid nitrogen ullage 4cm place, then by medium-term and long-term for straw direct plunge into Liquid Nitrogen preservation;
Five, the straw of freezen protective is carried out water-bath to thaw, water-bath water temperature is 25 DEG C ~ 30 DEG C, and thawing time is 15 ~ 18s, shakes straw in course of defrosting;
Wherein, in step 2, the addition of fresh wise sieve salmon seminal fluid is 3ml;
Wise man sieve salmon seminal fluid Ultra-cryofreezing preservation liquid is by the CaCl of KCl, 0.14g of NaCl, 0.4g of 8.01g
2, 0.35g NaHCO
3, 0.06g KH
2pO
4, the glucose of 0.34g and 1000ml distilled water composition;
Liquid nitrogen liquid level distance case mouth 13cm in step 4;
The straw of freezen protective is carried out water-bath thaw, water-bath water temperature is 25 DEG C ~ 30 DEG C, and thawing time is 15 ~ 18s, shakes straw in course of defrosting.
The appropriate seminal fluid getting the present embodiment freezen protective after thawing activates, then microscopy observe and thaw after wise sieve salmon sperm.The present embodiment freezen protective wise sieve salmon sperm of 2 months thaws, activates rear survival rate is after testing 79.4%, and motion ratio reaches 75.8%; The present embodiment freezen protective wise sieve salmon sperm of 3 years thaws, activate rear survival rate is 79.3%, and motion ratio reaches 75.6%.
Get residue to activate wise sieve salmon sperm (freezen protective 3 years) and carry out artificial insemination experiment (every milliliter of seminal fluid can to 10,000 ovum fertilizations, 10,000 ovum about 1.25 liters), fertilization rate reached 86.8%.Illustrate that the present invention has high viability and high energy through wise sieve salmon essence tool of freezen protective.
Embodiment 2
Wise man sieve salmon seminal fluid carries out freezen protective according to the following steps:
One, gather wise sieve salmon fresh semen, sperm motility is 85.4% after testing;
Two, in 7.92ml wise man sieve salmon seminal fluid Ultra-cryofreezing preservation liquid, 0.27ml glycerine is added, fully mix with eddy blending machine, then 4 DEG C of environment balances are placed in 15 minutes, put into fresh wise sieve salmon seminal fluid again to mix, abundant mixing be placed in 4 DEG C of environment balance 1 minute, add again afterwards 0.27ml glycerine fully mix be put in 4 DEG C of environment balance 1 minute, then add 0.54ml glycerine again, abundant mixing, namely obtains semen cryopreservation mixed liquor;
Three, semen cryopreservation mixed liquor micropipettor is sub-packed in straw, is then positioned in 4 DEG C of environment for subsequent use;
Four, imported by liquid nitrogen in bubble chamber with cover, the semen cryopreservation mixed liquor moved in straw stops 10min at distance liquid nitrogen ullage 8cm place, is then stopping 5min apart from liquid nitrogen ullage 4cm place, then by medium-term and long-term for straw direct plunge into Liquid Nitrogen preservation;
Five, the straw of freezen protective is carried out water-bath to thaw, water-bath water temperature is 25 DEG C ~ 30 DEG C, and thawing time is 15 ~ 18s, shakes straw in course of defrosting;
Wherein, in step 2, the addition of fresh wise sieve salmon seminal fluid is 3ml;
Wise man sieve salmon seminal fluid Ultra-cryofreezing preservation liquid is by the CaCl of KCl, 0.14g of NaCl, 0.4g of 8.01g
2, 0.35g NaHCO
3, 0.06g KH
2pO
4, the glucose of 0.34g and 1000ml distilled water composition;
Liquid nitrogen liquid level distance case mouth 13cm in step 4;
The straw of freezen protective is carried out water-bath thaw, water-bath water temperature is 25 DEG C ~ 30 DEG C, and thawing time is 15 ~ 18s, shakes straw in course of defrosting.
The appropriate seminal fluid getting the present embodiment freezen protective after thawing activates, then microscopy observe and thaw after wise sieve salmon sperm.The present embodiment freezen protective wise sieve salmon sperm of 2 months thaws, activates rear survival rate is after testing 80.0%, and motion ratio reaches 74.5%; The present embodiment freezen protective wise sieve salmon sperm of 3 years thaws, activate rear survival rate is 80.1%, and motion ratio reaches 74.4%.
Get residue to activate wise sieve salmon sperm (freezen protective 3 years) and carry out artificial insemination experiment (every milliliter seminal fluid can to 10,000 ovum fertilizations), fertilization rate reached 85.6%.Illustrate that the present invention has high viability and high energy through wise sieve salmon sperm of freezen protective.
Embodiment 3
Wise man sieve salmon seminal fluid carries out freezen protective according to the following steps:
One, gather wise sieve salmon fresh semen, sperm motility is 85.4% after testing;
Two, in 7.92ml wise man sieve salmon seminal fluid Ultra-cryofreezing preservation liquid, 0.27ml glycerine is added, fully mix with eddy blending machine, then 4 DEG C of environment balances are placed in 15 minutes, put into fresh wise sieve salmon seminal fluid again to mix, abundant mixing be placed in 4 DEG C of environment balance 1 minute, add again afterwards 0.27ml glycerine fully mix be put in 4 DEG C of environment balance 1 minute, then add 0.54ml glycerine again, abundant mixing, namely obtains semen cryopreservation mixed liquor;
Three, semen cryopreservation mixed liquor micropipettor is sub-packed in straw, is then positioned in 4 DEG C of environment for subsequent use;
Four, imported by liquid nitrogen in bubble chamber with cover, the semen cryopreservation mixed liquor moved in straw stops 15min at distance liquid nitrogen ullage 8cm place, is then stopping 8min apart from liquid nitrogen ullage 4cm place, then by medium-term and long-term for straw direct plunge into Liquid Nitrogen preservation;
Five, the straw of freezen protective is carried out water-bath to thaw, water-bath water temperature is 25 DEG C ~ 30 DEG C, and thawing time is 15 ~ 18s, shakes straw in course of defrosting;
Wherein, in step 2, the addition of fresh wise sieve salmon seminal fluid is 3ml;
Wise man sieve salmon seminal fluid Ultra-cryofreezing preservation liquid is by the CaCl of KCl, 0.14g of NaCl, 0.4g of 8.01g
2, 0.35g NaHCO
3, 0.06g KH
2pO
4, the glucose of 0.34g and 1000ml distilled water composition;
Liquid nitrogen liquid level distance case mouth 13cm in step 4;
The straw of freezen protective is carried out water-bath thaw, water-bath water temperature is 25 DEG C ~ 30 DEG C, and thawing time is 15 ~ 18s, shakes straw in course of defrosting.
The appropriate seminal fluid getting the present embodiment freezen protective after thawing activates, then microscopy observe and thaw after wise sieve salmon sperm.The present embodiment freezen protective wise sieve salmon sperm of 2 months thaws, activates rear survival rate is after testing 78.5%, and motion ratio reaches 73.6%; The present embodiment freezen protective wise sieve salmon sperm of 3 years thaws, activate rear survival rate is 78.2%, and motion ratio reaches 73.5%.
Get residue to activate wise sieve salmon sperm (freezen protective 3 years) and carry out artificial insemination experiment (every milliliter seminal fluid can to 10,000 ovum fertilizations), fertilization rate reached 84.3%.Illustrate that the present invention has high viability and high energy through wise sieve salmon sperm of freezen protective.
Adopt the inventive method freezen protective 2 months and freezen protective wise sieve salmon sperm quality of 3 years substantially not to change, illustrate that the method for freezen protective of the present invention wise man sieve salmon sperm is applicable to preserving wise sieve salmon sperm for a long time.
Claims (6)
1. wise sieve salmon seminal fluid Ultra-cryofreezing preservation liquid, to is characterized in that in wise sieve salmon seminal fluid Ultra-cryofreezing preservation liquid K in molar ratio
+︰ Na
+<1 ︰ 16, K in wise sieve salmon seminal fluid Ultra-cryofreezing preservation liquid
+concentration is 0.00480mol/L ~ 0.00682mol/L.
2. one according to claim 1 wise sieve salmon seminal fluid Ultra-cryofreezing preservation liquid, is characterized in that NaCl, the KCl of 0.325 ~ 0.475g, the CaCl of 0.12 ~ 0.15g of wise sieve salmon seminal fluid Ultra-cryofreezing preservation liquid by 7.95 ~ 8.07g
2, 0.349 ~ 0.351g NaHCO
3, 0.059 ~ 0.061g KH
2pO
4, the glucose of 0.30 ~ 0.38g and 1000ml distilled water composition.
3. one according to claim 1 wise sieve salmon seminal fluid Ultra-cryofreezing preservation liquid, is characterized in that the CaCl of wise sieve salmon seminal fluid Ultra-cryofreezing preservation liquid by KCl, 0.14g of NaCl, 0.4g of 8.01g
2, 0.35g NaHCO
3, 0.06g KH
2pO
4, the glucose of 0.34g and 1000ml distilled water composition.
4. the freezing and storing method of wise sieve salmon seminal fluid, is characterized in that wise sieve salmon seminal fluid carries out freezen protective according to the following steps:
One, wise sieve salmon fresh semen is gathered;
Two, in 7.92ml wise man sieve salmon seminal fluid Ultra-cryofreezing preservation liquid, 0.27ml glycerine is added, fully mix with eddy blending machine, then 4 DEG C of environment balances are placed in 15 minutes, put into fresh wise sieve salmon seminal fluid again to mix, abundant mixing be placed in 4 DEG C of environment balance 1 minute, add again afterwards 0.27ml glycerine fully mix be put in 4 DEG C of environment balance 1 minute, then add 0.54ml glycerine again, abundant mixing, namely obtains semen cryopreservation mixed liquor;
Three, semen cryopreservation mixed liquor micropipettor is sub-packed in straw, is then positioned in 4 DEG C of environment for subsequent use;
Four, liquid nitrogen is imported in bubble chamber with cover, semen cryopreservation mixed liquor in immigration straw is at distance liquid nitrogen ullage 8cm place's stop 5 ~ 15min, then at distance liquid nitrogen ullage 4cm place's stop 3 ~ 8min, then by medium-term and long-term for straw direct plunge into Liquid Nitrogen preservation;
Five, thaw adopt quick water-bath freezing process;
Wherein, in step 2, the addition of fresh wise sieve salmon seminal fluid is 3ml.
5. the freezing and storing method of wise sieve salmon seminal fluid according to claim 4, is characterized in that liquid nitrogen liquid level distance case mouth 12cm ~ 15cm in step 4.
6. the freezing and storing method of wise sieve salmon seminal fluid according to claim 4, it is characterized in that the quick water-bath freezing process of step 5: the straw of freezen protective is carried out water-bath and thaws, water-bath water temperature is 25 DEG C ~ 30 DEG C, and thawing time is 15 ~ 18s, shakes straw in course of defrosting.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510063790.2A CN104604845A (en) | 2015-02-06 | 2015-02-06 | Hucho taimen seminal fluid ultralow-temperature cryopreservation fluid and hucho taimen seminal fluid freezing and preserving method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510063790.2A CN104604845A (en) | 2015-02-06 | 2015-02-06 | Hucho taimen seminal fluid ultralow-temperature cryopreservation fluid and hucho taimen seminal fluid freezing and preserving method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104604845A true CN104604845A (en) | 2015-05-13 |
Family
ID=53139721
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510063790.2A Pending CN104604845A (en) | 2015-02-06 | 2015-02-06 | Hucho taimen seminal fluid ultralow-temperature cryopreservation fluid and hucho taimen seminal fluid freezing and preserving method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104604845A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105941391B (en) * | 2016-05-25 | 2018-11-09 | 广东海洋大学 | A kind of hybridization snakehead spermatozoa preservative fluid and its application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102986652A (en) * | 2012-12-25 | 2013-03-27 | 中国水产科学研究院黑龙江水产研究所 | Anti-freezing liquid for preserving pink salmon sperm at ultralow temperature in freezing manner and method for preserving pink salmon sperm at ultralow temperature by utilizing same |
-
2015
- 2015-02-06 CN CN201510063790.2A patent/CN104604845A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102986652A (en) * | 2012-12-25 | 2013-03-27 | 中国水产科学研究院黑龙江水产研究所 | Anti-freezing liquid for preserving pink salmon sperm at ultralow temperature in freezing manner and method for preserving pink salmon sperm at ultralow temperature by utilizing same |
Non-Patent Citations (2)
| Title |
|---|
| 徐革锋等: "鲑鳟鱼类精子超低温保存研究进展", 《水产学杂质》 * |
| 田永胜等: "额尔齐斯河江鳕精子冷冻保存研究", 《农业生物技术学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105941391B (en) * | 2016-05-25 | 2018-11-09 | 广东海洋大学 | A kind of hybridization snakehead spermatozoa preservative fluid and its application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Muchlisin et al. | Exploration of natural cryoprotectants for cryopreservation of African catfish, Clarias gariepinus, Burchell 1822 (Pisces: Clariidae) spermatozoa. | |
| Muchlisin | Current status of extenders and cryoprotectants on fish spermatozoa cryopreservation | |
| Maria et al. | Extenders and cryoprotectants for cooling and freezing of piracanjuba (Brycon orbignyanus) semen, an endangered Brazilian teleost fish | |
| CN104521943A (en) | Ultralow-temperature refrigeration preservation and recovery method for oxyeleotris marmorata semen | |
| CN103026994B (en) | Factory hybrid breeding method of epinephelus fuscoguttatus and epinephelus lanceolatus | |
| CN105123580A (en) | Industrialized hybrid fry breeding method for saladfish and lanceolatus | |
| CN102273439A (en) | Cryopreservation and application method of Convict grouper sperms | |
| CN112741078B (en) | Hexagrammos otakii sperm productive cryopreservation method | |
| CN108244099A (en) | A kind of greenling sperm high-efficiency ultralow temperature freezing and storing method | |
| CN103348966A (en) | Method for efficient ultralow temperature cryopreservation of turbot sperms | |
| Pan et al. | Development of cryopreservation for maintaining yellow catfish Pelteobagrus fulvidraco sperm | |
| CN104145944A (en) | Ultralow-temperature cryopreservation and activation method of sperm of scapharca broughtonii sckrenck | |
| CN103891711B (en) | The processing method of flower perch seminal fluid | |
| CN103931531B (en) | A kind of utilize freezing point before low critical temperature preserve the method for Hong Kong oyster eyebot larvae for a long time | |
| CN114258911B (en) | Cryopreservation solution and cryopreservation method for sparus latus sperms | |
| CN105360109A (en) | Ultralow-temperature freezing and storing method for sperms of yellow catfishes | |
| CN110786321A (en) | Freeze preservation method for pseudosciaena crocea sperms | |
| Alvarez et al. | High fry production rates using post-thaw silver carp (Hypophthalmichthys molitrix) spermatozoa under farming conditions | |
| CN106818708B (en) | Ultralow temperature cryoprotectant for epinephelus coioides semen and preservation method thereof | |
| CN104604845A (en) | Hucho taimen seminal fluid ultralow-temperature cryopreservation fluid and hucho taimen seminal fluid freezing and preserving method | |
| Du et al. | Cryopreservation of Plagiognathops microlepis sperm | |
| CN102217590A (en) | Pomfret sperm cryopreservation method | |
| CN104304234B (en) | Leopard line gill sour jujube perch sperm freezing protection liquid and store method thereof | |
| CN103404508B (en) | Ultra-low temperature cryopreservation and resuscitation method of hemibarbus maculates sperms | |
| CN103348932B (en) | Method for carrying out interspecies cross artificial insemination on frozen semen of paralichthys dentatus and paralichthys olivaceus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150513 |
|
| RJ01 | Rejection of invention patent application after publication |