CN107980766A - A kind of method of donkey testicular fluid freezen protective - Google Patents
A kind of method of donkey testicular fluid freezen protective Download PDFInfo
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- CN107980766A CN107980766A CN201711377558.1A CN201711377558A CN107980766A CN 107980766 A CN107980766 A CN 107980766A CN 201711377558 A CN201711377558 A CN 201711377558A CN 107980766 A CN107980766 A CN 107980766A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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Abstract
The present invention relates to a kind of method of donkey testicular fluid freezen protective.This method includes：The configuring of dilution, fresh smart processing, cooling and balance, freezing and defrosting.In the present invention, freezing liquid have selected forms the damage caused by sperm containing certain density graphene oxide and glycerine combination addition to suppress ice crystal, and carry out the cooling treatment of 20 40min in advance to sperm using -80 DEG C of ultra low temperature freezers before freezing of semen, simplify the difficulty that expensive program frigorimeter must be applied to carry out sperm cooling treatment during conventional freezing of semen, 60 DEG C of water-bath is selected after freezing to thawing of semen 6 seconds, effectively increase the sperm motility rate after donkey testicular fluid freezen protective, it is suitable for grass-roots unit using the popularization and application for freezing essence development donkey artificial insemination.
Description
Technical field
The invention belongs to biological technical field, is specifically a kind of method of donkey testicular fluid freezen protective.
Background technology
The cultivation quantity of China donkey there are about 10,000,000, and a species more than 30, is mainly dispersed in the ground such as Shan, Henan, Shandong, new, sweet
Area.The cultivation quantity of China donkey continues to decline in recent years, and main cause is cultivated with most of China industrialization and supported with pig, ox, sheep
Based on growing, the variety source of donkey is few, reproductive efficiency is low related.The purpose of Semen routine is to extend depositing for sperm to greatest extent
Live time and maintain its fertility.For the sperm of preservation by the transport between different regions, can expand sperm uses model
Enclose, increase the utilization ratio for by the quantity with animal, improving sire, enable the breeding potential and prepotency of excellent sire
It is not fully exerted.At present, the correlative study of equus Semen routine is also fewer, and the related data report of donkey is less.With
The rise and continuous development of donkey industry, carries out the research that donkey testicular fluid preserves, can not only solve China's donkey breeding stock and persistently subtract
The problem of increasingly being reduced with foster donkey scale less, also can further promote the development of foster donkey production.Sperm preserves room temperature preservation
(15-25 DEG C), Cord blood (4 DEG C) and freezen protective (- 196 DEG C or -79 DEG C), wherein only freezen protective can realize sperm
The purpose preserved for a long time, but sperm motility rate is relatively low after freezing preservation seminal fluid defrosting, and the conception rate of artificial insemination is relatively low.Thus,
There is an urgent need for carrying out the freezen protective research of donkey testicular fluid in production, the donkey testicular fluid freezing and storing method applied is established in a set of suitable production
And technical system.
The content of the invention
The purpose of the present invention is in view of the deficiencies of the prior art, and propose a kind of method of donkey testicular fluid freezen protective, solve
Scale supports donkey or grass-roots unit using the artificial insemination for freezing essence development donkey, to improve the utilization ratio of kind of jack ass, accelerates donkey
Group expansion is numerous and breed improvement.
The present invention solves its technical problem and takes following technical scheme to realize：
A kind of method of donkey testicular fluid freezen protective, it is as follows that the method comprising the steps of：
(1) dilution is configured
1. basal liquid configures：The basal liquid includes the following components formed by mass fraction：3.04 parts of Tris, lemon
1.70 parts of acid, 1.50 parts of glucose, 1.50 parts of fructose, 0.06 part of penicillin, 0.10 part of streptomysin, 100 parts of distilled water will be above-mentioned
Component constant volume is configured to basal liquid；
2. dilute the configuration of I liquid：85 parts of basal liquid is taken by volume, takes fresh 15 parts of yolk liquid, is fully mixed, is diluted
I liquid；
3. dilute the configuration of II liquid：Above-mentioned 80 parts of I liquid of dilution is taken by volume, is taken 20 parts of 0.5mg/mL graphene oxides, is filled
Divide and mix, obtain II liquid of dilution；
4. freezing liquid configures：Above-mentioned 46 parts of II liquid of dilution is taken by volume, takes 4 parts of glycerine, is fully mixed, is freezed
Liquid；
(2) fresh smart processing
1. using heat jenny ass as platform donkey, Pseudopyloric metaplasia gathers kind of a jack ass sperm, and the sperm of collection is sterile with 6-8 layers immediately
Filtered through gauze is simultaneously collected into sterile beaker and seals, while is more than with microexamination sperm concentration and motility rate, sperm motility rate
0.70 is standard compliant fresh essence；
2. standard compliant fresh essence and II liquid of dilution are placed in 35 DEG C of constant incubators and maintain 1-2h in the lump, then,
Draw the fresh essences of 5mL to be transferred in 15mL sterile test tubes, 2500rpm centrifugation 5min, abandon supernatant, it is dilute to be slowly added to 5mL along tube wall
II liquid is released, is uniformly mixed and obtains diluting fresh essence；
(3) cooling of sperm and balance
The fresh essence of the dilution of above-mentioned good seal is put into the beaker for filling 35 DEG C of water in the lump with freeze-extender, puts 4 DEG C of ice
Case cooling 6-10h, then, the freeze-extender for being cooled to 4 DEG C is drawn in the sperm after 5mL is slowly added to cooling, mixing is equal
4 DEG C of refrigerators are put back to after even, when balance 2 is small, the sperm after being balanced；
(4) freezing and defrosting of sperm
1. freezing of semen, the sperm after above-mentioned balance is fitted into the straw of 0.25mL under the conditions of 4 DEG C, puts -80 DEG C and surpass
20-40min in low temperature refrigerator, then puts into liquid nitrogen frozen and preserves；
2. semen thawing, takes the sperm after freezing to be put into 6 seconds in 60 DEG C of water-baths and thaws, after defrosting, put 37 DEG C of loadings
The motility rate of (400 ×) observation sperm under the microscope of platform.
Moreover, basal liquid is taken by volume in 2., the 3. and 4. step of the step (1), fresh yolk liquid is taken, takes dilution I
Liquid, take 0.5mg/mL graphene oxides, take dilution II liquid and take glycerine be using 20mL asepsis injectors draw.
The advantages and positive effects of the present invention are：
Freezing liquid in the present invention have selected to be added to suppress ice containing certain density graphene oxide and glycerine combination
Damage caused by the paired sperm of crystalline form, and 20- is carried out in advance to sperm using -80 DEG C of ultra low temperature freezers before freezing of semen
The cooling treatment of 40min, simplifying must apply expensive program frigorimeter to carry out sperm cooling during conventional freezing of semen
The difficulty of processing, selects 60 DEG C of water-bath to thawing of semen 6 seconds, after effectively increasing donkey testicular fluid freezen protective after freezing
Sperm motility rate.
Embodiment
Present invention implementation is further described below, following embodiments are descriptive, are not limited, it is impossible to
Protection scope of the present invention is limited with this.
A kind of method of donkey testicular fluid freezen protective, it is as follows that the method comprising the steps of：
(1) dilution is configured
1. basal liquid configures：Tris 3.04g, citric acid 1.70g, glucose 1.50g, fruit are accurately weighed with electronic balance
Sugared 1.50g, penicillin 0.06g, streptomysin 0.10g, adds distilled water 100mL constant volumes to be configured to basal liquid；
2. dilute the configuration of I liquid：Draw basal liquid common 85mL to 100mL sterilizing volumetric flasks by several times with 20mL asepsis injectors
In, spend the fresh yolk liquid of syringe needle 20mL asepsis injectors absorption and be slowly added in volumetric flask, be settled to 100mL, fully mix；
3. dilute the configuration of II liquid：I liquid of dilution being settled in 100mL volumetric flasks is drawn with 20mL asepsis injectors
20mL, abandons this 20mL and dilutes I liquid, and then drawing 0.5mg/mL graphene oxides with 20mL asepsis injectors is settled to 100mL,
Fully mix；
4. freezing liquid configures：Draw 46mL by several times with 20mL asepsis injectors and dilute II liquid into 50mL volumetric flasks, then
The 20mL asepsis injectors absorption glycerine for spending syringe needle is settled to 50mL, fully mixes；
(2) fresh smart processing
1. using heat jenny ass as platform donkey, Pseudopyloric metaplasia gathers kind of a jack ass sperm, and the sperm of collection is sterile with eight layers immediately
Filtered through gauze is simultaneously collected into sterile beaker and seals, and fast transfer to use for laboratory 10uL micropipettors draws sperm extremely
On glass slide, covered, to 37 DEG C of objective table test under microscope sperm motility rates, sperm motility rate is more than 0.70 to meet mark
Accurate adaptable fresh essence；
2. standard compliant fresh essence and II liquid of dilution are placed in 35 DEG C of constant incubators and maintain 1-2h in the lump, then,
Draw the fresh essences of 5mL to be transferred in 15mL sterile test tubes, 2500rpm centrifugation 5min, abandon supernatant, it is dilute to be slowly added to 5mL along tube wall
II liquid is released, is uniformly mixed and obtains diluting fresh essence；
(3) cooling of sperm and balance
The fresh essence of above-mentioned dilution is put into the 500mL beakers for filling 400mL warm water (35 DEG C) in the lump with freeze-extender, is put
4 DEG C of Temperature drop in refrigerator 6-10h, then, the 5mL freeze-extenders for being cooled to 4 DEG C are slowly added in the sperm after cooling, mixing is equal
4 DEG C of refrigerators are put back to after even, when balance 2 is small；
(4) freezing and defrosting of sperm
1. freezing of semen, the sperm after balance is fitted into the straw of 0.25mL under the conditions of 4 DEG C, puts -80 DEG C of ultralow temperature ices
20-40min in case, then puts into liquid nitrogen frozen and preserves；
2. semen thawing, takes the sperm after freezing to be put into 6 seconds in 60 DEG C of water-baths and thaws, after defrosting, put 37 DEG C of loadings
The motility rate of (400 ×) observation sperm, finds sperm motility rate up between 0.40-0.50, with conventional freezing ratio under the microscope of platform
It is obviously improved compared with sperm motility rate (between 0.25-0.40).
Claims (2)
- A kind of 1. method of donkey testicular fluid freezen protective, it is characterised in that it is as follows that the method comprising the steps of：(1) dilution is configured1. basal liquid configures：The basal liquid includes the following components formed by mass fraction：3.04 parts of Tris, citric acid 1.70 parts, 1.50 parts of glucose, 1.50 parts of fructose, 0.06 part of penicillin, 0.10 part of streptomysin, 100 parts of distilled water, by above-mentioned group Part constant volume is configured to basal liquid；2. dilute the configuration of I liquid：85 parts of basal liquid is taken by volume, takes fresh 15 parts of yolk liquid, is fully mixed, and obtains I liquid of dilution；3. dilute the configuration of II liquid：Above-mentioned 80 parts of I liquid of dilution is taken by volume, takes 20 parts of 0.5mg/mL graphene oxides, it is fully mixed It is even, obtain II liquid of dilution；4. freezing liquid configures：Above-mentioned 46 parts of II liquid of dilution is taken by volume, takes 4 parts of glycerine, is fully mixed, is obtained freezing liquid；(2) fresh smart processing1. using heat jenny ass as platform donkey, Pseudopyloric metaplasia collection kind jack ass sperm, the sperm of collection uses 6-8 layers of sterile gauze immediately Filter and be collected into sterile beaker and seal, while be more than 0.70 with microexamination sperm concentration and motility rate, sperm motility rate For standard compliant fresh essence；2. standard compliant fresh essence and II liquid of dilution to be placed in 35 DEG C of constant incubators and maintain 1-2h in the lump, then, draw The fresh essences of 5mL are transferred in 15mL sterile test tubes, 2500rpm centrifugation 5min, abandon supernatant, and 5mL dilutions II are slowly added to along tube wall Liquid, is uniformly mixed and obtains diluting fresh essence；(3) cooling of sperm and balanceThe fresh essence of the dilution of above-mentioned good seal is put into the beaker for filling 35 DEG C of water in the lump with freeze-extender, puts 4 DEG C of refrigerator drops Warm 6-10h, then the freeze-extender for being cooled to 4 DEG C is drawn in the sperm after 5mL is slowly added to cooling, put after mixing 4 DEG C of refrigerators are returned, when balance 2 is small, the sperm after being balanced；(4) freezing and defrosting of sperm1. freezing of semen, the sperm after above-mentioned balance is fitted into the straw of 0.25mL under the conditions of 4 DEG C, puts -80 DEG C of ultralow temperature 20-40min in refrigerator, then puts into liquid nitrogen frozen and preserves；2. semen thawing, takes the sperm after freezing to be put into 6 seconds in 60 DEG C of water-baths and thaw, after defrosting, 37 DEG C of objective tables are put The motility rate of (400 ×) observation sperm under microscope.
- 2. the method for donkey testicular fluid freezen protective according to claim 1, it is characterised in that：The step (1) 2., 3. and 4. basal liquid is taken in step by volume, fresh yolk liquid is taken, takes I liquid of dilution, take 0.5mg/mL graphene oxides, take II liquid of dilution And taking glycerine is drawn using 20mL asepsis injectors.
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| CN201711377558.1A CN107980766A (en) | 2017-12-19 | 2017-12-19 | A kind of method of donkey testicular fluid freezen protective |
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| CN201711377558.1A CN107980766A (en) | 2017-12-19 | 2017-12-19 | A kind of method of donkey testicular fluid freezen protective |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110959607A (en) * | 2019-12-24 | 2020-04-07 | 扬州大学 | Formula and dilution method of diluent for storing Hu sheep semen at normal temperature |
| CN111226907A (en) * | 2020-01-21 | 2020-06-05 | 扬州大学 | Preparation method of diluent for normal-temperature preservation of sheep semen |
| CN111374124A (en) * | 2020-05-15 | 2020-07-07 | 辽宁省现代农业生产基地建设工程中心 | Formula and preparation method of donkey frozen semen diluent |
| CN111919839A (en) * | 2020-09-11 | 2020-11-13 | 扬州大学 | Preparation method and use method of Hu sheep semen low-temperature preservation diluent formula |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110959607A (en) * | 2019-12-24 | 2020-04-07 | 扬州大学 | Formula and dilution method of diluent for storing Hu sheep semen at normal temperature |
| CN111226907A (en) * | 2020-01-21 | 2020-06-05 | 扬州大学 | Preparation method of diluent for normal-temperature preservation of sheep semen |
| CN111374124A (en) * | 2020-05-15 | 2020-07-07 | 辽宁省现代农业生产基地建设工程中心 | Formula and preparation method of donkey frozen semen diluent |
| CN111919839A (en) * | 2020-09-11 | 2020-11-13 | 扬州大学 | Preparation method and use method of Hu sheep semen low-temperature preservation diluent formula |
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