CN107624752A - The preparation method of one boar freezing seminal fluid dilution - Google Patents
The preparation method of one boar freezing seminal fluid dilution Download PDFInfo
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- CN107624752A CN107624752A CN201710839170.2A CN201710839170A CN107624752A CN 107624752 A CN107624752 A CN 107624752A CN 201710839170 A CN201710839170 A CN 201710839170A CN 107624752 A CN107624752 A CN 107624752A
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Abstract
The invention discloses the preparation method of a boar freezing seminal fluid dilution, dilution includes the configuration step of following composition by weight and dilute solution：S1：Feeding, S2：Adjust pH value, S3：Add fresh yolk, S4：Centrifugation, S5：Stand, S6：Filtering, mysoinositol class compound of the present invention can effectively reduce the forming amount of ice crystal during freezing of semen, reduce mechanical injuries of the ice crystal to sperm, the technical indicators such as final motility rate, vigor, acrosomal integnity and the membrane integrity for improving pig animal frozen semen, wherein penicillin and streptomysin primarily serve sterilization, suppress growth of microorganism effect；Monose is used as main energy substance；Citric acid and trishydroxymethylaminomethane cooperate to be used mainly as cushioning liquid, sperm motility is reduced, so as to extend sperm life and keep fertility.
Description
Technical field
The present invention relates to the preparation method of pig frozen semen technical field, specially a boar freezing seminal fluid dilution.
Background technology
With the lifting of people's living standard, scientific culture can be very helpful to the cultivation of livestock industry, livestock semen
Freezen protective research has been carried out more than 50 years, but the sperm viability after thawing is only after 30%-50% or so, artificial insemination
Feelings rate and farrowing rate are not returned is substantially less than fresh and deepfreeze seminal fluid (seminal fluid of this friendship and normal temperature preservation).Pig is cultivated at me
Play the part of in livestock industry cultivation in essential role, but during boar semen, ice crystal pair that intraor extracellular is formed
Mechanical damage caused by animal sperm directly results in the decline of sperm motility after freezing, how to reduce the generation of ice crystal, reduces ice
The brilliant damage to spermatozoa is always the study hotspot of livestock reproduction technology and cryobiology research field.By changing essence
The constituent of liquid dilution can reduce damage of the freezing processing to sperm, and freezing of semen is to preserve genetic resources, establishes essence
The important measures of sub- gene pool.And the quality of sperm quality and the pregnancy rate, produce surviving of son rate etc. of domestic animal are closely related after freezing.At present
Untill, sperm motility of the Boar spermatozoa after freezing, acrosomal integrity, plasm membrane integrity etc. be not highly desirable, causes in commercialization
In Swine Production, using frozen semen carry out artificial insemination sow conception rate and farrowing rate it is relatively low and can not extensive use, sternly
The utilization rate of high-quality boar is reduced again, and feeding cost can not reduce, and the pollution of ecological environment is not more eased.
The content of the invention
It is an object of the invention to provide the preparation method of a boar freezing seminal fluid dilution, to solve above-mentioned background technology
The problem of middle proposition.
To achieve the above object, the present invention provides following technical scheme：
The preparation method of one boar freezing seminal fluid dilution, dilution include matching somebody with somebody for following composition by weight and dilute solution
Put step：Distilled water 100ml, 0.45~0.90g of trishydroxymethylaminomethane, 0.71~0.86g of citric acid, monose 5.0~
The u of 8.10g, the u of penicillin 00,000~1,000,000, streptomysin 0.20g~0.5g, 0.29~6.97g of flesh alcohol compound, glycerine 6.7~
9.5g, 25~33ml of fresh yolk；
S1：Feeding, 100ml distilled waters are taken to place in beaker, the trihydroxy methyl amino first more than addition matched into beaker
Alkane, citric acid, monose, penicillin, streptomysin, flesh alcohol compound and glycerine, then uniform stirring 20~30 minutes, are obtained molten
Liquid；
S2：PH value is adjusted, the pH value of citric acid regulating solution is added in above-mentioned solution, its pH value is 6.8~7.2；
S3：Fresh yolk is added, fresh yolk is added in the solution in S2, carries out uniform stirring 5~10 minutes；
S4：Centrifugation, the solution after being well mixed are placed at 3~5 DEG C and small with 15000 revs/min of speed centrifugation 1~1.5
When；
S5：Stand, the solution left standstill 20~30 minutes in S4, wherein temperature is 3~5 DEG C；
S6：Filtering, the supernatant obtained in S5 can be prepared by dilute solution in 0.45 μm of filter filtering.
Preferably, the temperature of the distilled water is 32~38 DEG C.
Preferably, the monose both any mixtures between glucose, fructose and lactose.
Preferably, the flesh alcohol compound is 1,3- cyclohexanediols either Isosorbide-5-Nitrae-cyclohexanediol or 1,3,5- hexamethylenes three
Alcohol or the mixture of three.
Preferably, the fresh yolk passes through 50~60 DEG C, 25~35min inactivation treatment.
The beneficial effects of the invention are as follows：Mysoinositol class compound of the present invention can effectively reduce ice crystal during freezing of semen
Forming amount, reduces mechanical injuries of the ice crystal to sperm, and motility rate, vigor, the acrosome of final raising pig animal frozen semen are complete
The technical indicator such as property and membrane integrity, the ice crystal inhibitor using flesh alcohol compound as sperm freezing dilution liquid, has
Immune response will not be produced, the advantages that purity is guaranteed, cheap.Wherein penicillin and streptomysin primarily serve sterilization, suppression
Microorganism growth processed；Monose is as main energy substance；Citric acid and trishydroxymethylaminomethane cooperate main
Used as cushioning liquid, sperm motility is reduced, so as to extend sperm life and keep fertility.
Dilution of the present invention is prepared simply, and easily operation, and it is molten efficient can to produce desirable pig semen
Liquid, and then the survival rate of Boar spermatozoa is improved, to be convenient for people to large-scale cultivating pig, and then the cost of pig cultivation is reduced,
Using fresh yolk inactivation treatment, reduce fresh yolk Carried bacteria and infect Boar spermatozoa, improve the survival rate of Boar spermatozoa in dilution.
Embodiment
The technical scheme in the embodiment of the present invention will be clearly and completely described below, it is clear that described implementation
Example only part of the embodiment of the present invention, rather than whole embodiments.It is common based on the embodiment in the present invention, this area
The every other embodiment that technical staff is obtained under the premise of creative work is not made, belong to the model that the present invention protects
Enclose.
The present invention provides a kind of technical scheme：
Embodiment one
The preparation method of one boar freezing seminal fluid dilution, dilution include matching somebody with somebody for following composition by weight and dilute solution
Put step：Distilled water 100ml, trishydroxymethylaminomethane 0.45g, citric acid 0.71g, monose 5.0g, the u of penicillin 00,000, strepto-
Plain 0.20g, flesh alcohol compound 0.29g, glycerine 6.7g, fresh yolk 25ml；
S1：Feeding, 100ml distilled waters are taken to place in beaker, the trihydroxy methyl amino first more than addition matched into beaker
Alkane, citric acid, monose, penicillin, streptomysin, flesh alcohol compound and glycerine, then uniform stirring 20 minutes, obtain solution；
Wherein the temperature of distilled water is 32 DEG C, monose both any mixtures, flesh between glucose, fructose and lactose
Alcohol compound is the mixture of 1,3- cyclohexanediols either Isosorbide-5-Nitrae-cyclohexanediol or 1,3,5- phloroglucites or three；
Flesh alcohol compound in the present invention can effectively reduce the forming amount of ice crystal during freezing of semen, reduce ice crystal pair
The skill such as the mechanical injuries of sperm, final motility rate, vigor, acrosomal integnity and the membrane integrity for improving pig animal frozen semen
Art index, the ice crystal inhibitor using flesh alcohol compound as sperm freezing dilution liquid, immune response, pure will not be produced by having
Spend the advantages that guaranteed, cheap；
S2：PH value is adjusted, the pH value of citric acid regulating solution is added in above-mentioned solution, its pH value is 6.8；
S3：Fresh yolk is added, fresh yolk is added in the solution in S2, carries out uniform stirring 5 minutes, fresh yolk passes through 50
DEG C, 25min inactivation treatment, using fresh yolk inactivation treatment, reduce fresh yolk Carried bacteria to infect Boar spermatozoa, and then influence
The effect of the culture Boar spermatozoa of dilution；
S4：Centrifugation, the solution after being well mixed are placed at 3 DEG C and centrifuged 1 hour with 15000 revs/min of speed；
S5：Stand, the solution left standstill 20 minutes in S4, wherein temperature is 3 DEG C；
S6：Filtering, the supernatant obtained in S5 can be prepared by dilute solution in 0.45 μm of filter filtering.
Wherein penicillin and streptomysin primarily serve sterilization in the present invention, suppress growth of microorganism effect；Monose uses work
For main energy substance；Citric acid and trishydroxymethylaminomethane cooperate to be used mainly as cushioning liquid, reduces essence
Subactivity, so as to extend sperm life and keep fertility.
Dilution of the present invention is prepared simply, and easily operation, and it is molten efficient can to produce desirable pig semen
Liquid, and then the survival rate of Boar spermatozoa is improved, to be convenient for people to large-scale cultivating pig, and then the cost of pig cultivation is reduced,
Using fresh yolk inactivation treatment, reduce fresh yolk Carried bacteria and infect Boar spermatozoa, improve the survival rate of Boar spermatozoa in dilution.
Embodiment two
The preparation method of one boar freezing seminal fluid dilution, dilution include matching somebody with somebody for following composition by weight and dilute solution
Put step：Distilled water 100ml, trishydroxymethylaminomethane 0.75g, citric acid 0.79g, monose 6.40g, the u of penicillin 500,000, chain
Mycin 0.3g, flesh alcohol compound 4.84g, glycerine 8.56g, fresh yolk 30ml；
S1：Feeding, 100ml distilled waters are taken to place in beaker, the trihydroxy methyl amino first more than addition matched into beaker
Alkane, citric acid, monose, penicillin, streptomysin, flesh alcohol compound and glycerine, then uniform stirring 25 minutes, obtain solution；
Wherein the temperature of distilled water is 35 DEG C, monose both any mixtures, flesh between glucose, fructose and lactose
Alcohol compound is the mixture of 1,3- cyclohexanediols either Isosorbide-5-Nitrae-cyclohexanediol or 1,3,5- phloroglucites or three；
Flesh alcohol compound in the present invention can effectively reduce the forming amount of ice crystal during freezing of semen, reduce ice crystal pair
The skill such as the mechanical injuries of sperm, final motility rate, vigor, acrosomal integnity and the membrane integrity for improving pig animal frozen semen
Art index, the ice crystal inhibitor using flesh alcohol compound as sperm freezing dilution liquid, immune response, pure will not be produced by having
Spend the advantages that guaranteed, cheap；
S2：PH value is adjusted, the pH value of citric acid regulating solution is added in above-mentioned solution, its pH value is 7.0；
S3：Fresh yolk is added, fresh yolk is added in the solution in S2, carries out uniform stirring 8 minutes, fresh yolk passes through 55
DEG C, 30min inactivation treatment, using fresh yolk inactivation treatment, reduce fresh yolk Carried bacteria to infect Boar spermatozoa, and then influence
The effect of the culture Boar spermatozoa of dilution；
S4：Centrifugation, the solution after being well mixed are placed at 4 DEG C and centrifuged 1.3 hours with 15000 revs/min of speed；
S5：Stand, the solution left standstill 25 minutes in S4, wherein temperature is 4 DEG C；
S6：Filtering, the supernatant obtained in S5 can be prepared by dilute solution in 0.45 μm of filter filtering.
Wherein penicillin and streptomysin primarily serve sterilization in the present invention, suppress growth of microorganism effect；Monose uses work
For main energy substance；Citric acid and trishydroxymethylaminomethane cooperate to be used mainly as cushioning liquid, reduces essence
Subactivity, so as to extend sperm life and keep fertility.
Dilution of the present invention is prepared simply, and easily operation, and it is molten efficient can to produce desirable pig semen
Liquid, and then the survival rate of Boar spermatozoa is improved, to be convenient for people to large-scale cultivating pig, and then the cost of pig cultivation is reduced,
Using fresh yolk inactivation treatment, reduce fresh yolk Carried bacteria and infect Boar spermatozoa, improve the survival rate of Boar spermatozoa in dilution.
Embodiment three
The preparation method of one boar freezing seminal fluid dilution, dilution include matching somebody with somebody for following composition by weight and dilute solution
Put step：Distilled water 100ml, trishydroxymethylaminomethane 0.90g, citric acid 0.86g, monose 8.10g, the u of penicillin 1,000,000,
Streptomysin 0.5g, flesh alcohol compound 6.97g, glycerine 9.5g, fresh yolk 33ml；
S1：Feeding, 100ml distilled waters are taken to place in beaker, the trihydroxy methyl amino first more than addition matched into beaker
Alkane, citric acid, monose, penicillin, streptomysin, flesh alcohol compound and glycerine, then uniform stirring 30 minutes, obtain solution；
Wherein the temperature of distilled water is 38 DEG C, monose both any mixtures, flesh between glucose, fructose and lactose
Alcohol compound is the mixture of 1,3- cyclohexanediols either Isosorbide-5-Nitrae-cyclohexanediol or 1,3,5- phloroglucites or three；
Flesh alcohol compound in the present invention can effectively reduce the forming amount of ice crystal during freezing of semen, reduce ice crystal pair
The skill such as the mechanical injuries of sperm, final motility rate, vigor, acrosomal integnity and the membrane integrity for improving pig animal frozen semen
Art index, the ice crystal inhibitor using flesh alcohol compound as sperm freezing dilution liquid, immune response, pure will not be produced by having
Spend the advantages that guaranteed, cheap；
S2：PH value is adjusted, the pH value of citric acid regulating solution is added in above-mentioned solution, its pH value is 7.2；
S3：Fresh yolk is added, fresh yolk is added in the solution in S2, carries out uniform stirring 10 minutes, fresh yolk passes through
60 DEG C, 35min inactivation treatment, using fresh yolk inactivation treatment, fresh yolk Carried bacteria is reduced to infect Boar spermatozoa, Jin Erying
Ring the effect of the culture Boar spermatozoa of dilution；
S4：Centrifugation, the solution after being well mixed are placed at 5 DEG C and centrifuged 1.5 hours with 15000 revs/min of speed；
S5：Stand, the solution left standstill 30 minutes in S4, wherein temperature is 5 DEG C；
S6：Filtering, the supernatant obtained in S5 can be prepared by dilute solution in 0.45 μm of filter filtering.
Wherein penicillin and streptomysin primarily serve sterilization in the present invention, suppress growth of microorganism effect；Monose uses work
For main energy substance；Citric acid and trishydroxymethylaminomethane cooperate to be used mainly as cushioning liquid, reduces essence
Subactivity, so as to extend sperm life and keep fertility.
Dilution of the present invention is prepared simply, and easily operation, and it is molten efficient can to produce desirable pig semen
Liquid, and then the survival rate of Boar spermatozoa is improved, to be convenient for people to large-scale cultivating pig, and then the cost of pig cultivation is reduced,
Using fresh yolk inactivation treatment, reduce fresh yolk Carried bacteria and infect Boar spermatozoa, improve the survival rate of Boar spermatozoa in dilution.
Sperm survival rate during the survival rate of Boar spermatozoa is relatively other two groups in embodiment two in three groups of embodiments of the above is inclined
Height, and within the data area between embodiment one and embodiment three, Boar spermatozoa survival rate data model in the present invention relatively
Boar spermatozoa survival rate under the conditions of outside enclosing is higher.Therefore the present invention have developed the preparation side of a boar freezing seminal fluid dilution
Method, it can efficiently ensure the survival rate of Boar spermatozoa, to facilitate the cultivation to pig in domestic animal.
Although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of changes, modification can be carried out to these embodiments, replace without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (5)
1. the preparation method of a boar freezing seminal fluid dilution, it is characterised in that dilution includes following composition by weight and dilute
Release the configuration step of solution：Distilled water 100ml, 0.45~0.90g of trishydroxymethylaminomethane, 0.71~0.86g of citric acid, list
It is the u of 5.0~8.10g of sugar, the u of penicillin 00,000~1,000,000, streptomysin 0.20g~0.5g, 0.29~6.97g of flesh alcohol compound, sweet
6.7~9.5g of oil, 25~33ml of fresh yolk；
S1：Feeding, 100ml distilled waters are taken to place in beaker, trishydroxymethylaminomethane, the lemon more than addition matched into beaker
Lemon acid, monose, penicillin, streptomysin, flesh alcohol compound and glycerine, then uniform stirring 20~30 minutes, obtain solution；
S2：PH value is adjusted, the pH value of citric acid regulating solution is added in above-mentioned solution, its pH value is 6.8~7.2；
S3：Fresh yolk is added, fresh yolk is added in the solution in S2, carries out uniform stirring 5~10 minutes；
S4：Centrifugation, the solution after being well mixed are placed at 3~5 DEG C and centrifuged 1~1.5 hour with 15000 revs/min of speed；
S5：Stand, the solution left standstill 20~30 minutes in S4, wherein temperature is 3~5 DEG C；
S6：Filtering, the supernatant obtained in S5 can be prepared by dilute solution in 0.45 μm of filter filtering.
2. the preparation method of boar freezing seminal fluid dilution according to claim 1, it is characterised in that：The distilled water
Temperature be 32~38 DEG C.
3. the preparation method of boar freezing seminal fluid dilution according to claim 1, it is characterised in that：The monose is
Both any mixtures between glucose, fructose and lactose.
4. the preparation method of boar freezing seminal fluid dilution according to claim 1, it is characterised in that：The inositol class
Compound is the mixture of 1,3- cyclohexanediols either Isosorbide-5-Nitrae-cyclohexanediol or 1,3,5- phloroglucites or three.
5. the preparation method of boar freezing seminal fluid dilution according to claim 1, it is characterised in that：The fresh yolk
By 50~60 DEG C, 25~35min inactivation treatment.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108782545A (en) * | 2018-08-30 | 2018-11-13 | 贵州省种畜禽种质测定中心 | One boar freezes essence pre-dilution liquid |
| CN109287621A (en) * | 2018-11-16 | 2019-02-01 | 翼城县富华养殖有限公司 | A kind of extender of freezing semen improving milk cow conception rate |
| CN110115264A (en) * | 2018-02-05 | 2019-08-13 | 天津农学院 | A kind of diluent and preparation method thereof and the application in pig semen cryo-conservation |
| CN110432261A (en) * | 2019-09-02 | 2019-11-12 | 李娜 | A kind of micro sperm freezing and defreezing method and its automatic fixture device |
| CN114210235A (en) * | 2021-12-15 | 2022-03-22 | 吉林省农业科学院 | Device and method for preparing pig frozen semen diluent |
| CN114651815A (en) * | 2022-04-12 | 2022-06-24 | 江苏农牧科技职业学院 | Method for preserving frozen boar semen |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110115264A (en) * | 2018-02-05 | 2019-08-13 | 天津农学院 | A kind of diluent and preparation method thereof and the application in pig semen cryo-conservation |
| CN108782545A (en) * | 2018-08-30 | 2018-11-13 | 贵州省种畜禽种质测定中心 | One boar freezes essence pre-dilution liquid |
| CN109287621A (en) * | 2018-11-16 | 2019-02-01 | 翼城县富华养殖有限公司 | A kind of extender of freezing semen improving milk cow conception rate |
| CN110432261A (en) * | 2019-09-02 | 2019-11-12 | 李娜 | A kind of micro sperm freezing and defreezing method and its automatic fixture device |
| CN114210235A (en) * | 2021-12-15 | 2022-03-22 | 吉林省农业科学院 | Device and method for preparing pig frozen semen diluent |
| CN114651815A (en) * | 2022-04-12 | 2022-06-24 | 江苏农牧科技职业学院 | Method for preserving frozen boar semen |
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