CN109258623A - A kind of diluting boar semen agent and application - Google Patents
A kind of diluting boar semen agent and application Download PDFInfo
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- CN109258623A CN109258623A CN201811089980.1A CN201811089980A CN109258623A CN 109258623 A CN109258623 A CN 109258623A CN 201811089980 A CN201811089980 A CN 201811089980A CN 109258623 A CN109258623 A CN 109258623A
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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
- A01N1/0231—Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
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Abstract
The invention belongs to the technology of artificial insemination fields of pig, more particularly, to a kind of diluting boar semen agent and its application.The diluent is using glucose as nutritional agents；Using citric acid monohydrate sodium, EDTAP dipotassium ethylene diamine tetraacetate and anhydrous sodium bicarbonate as buffer；Using trehalose as protective agent；Using jamaicin as antibacterial agent；Using aurantiin as antioxidant, make sperm from peroxide injury and protection plasmalemmae of sperms using its stronger anti-radical action.Using diluent of the invention, pig semen, which saves 3 days, reaches 85% through detecting sperm motility, saves 7 days and still ensures that its sperm motility reaches 75%.
Description
Technical field
The invention belongs to the technology of artificial insemination fields of pig, more particularly, to a kind of diluting boar semen agent and application.
Background technique
The artificial insemination of pig is a routine techniques of pig large-scale cultivation, can be improved the utilization rate of outstanding boar, is protected
The high-quality for demonstrate,proving market pig, increases the economic benefit of raiser.With the extensive use of technology of artificial insemination, the fresh-keeping skill of sperm
Art is also evolving.The external fresh-keeping key point of sperm is to slow down the metabolic intensity of sperm, to reach extension essence
The purpose of sub time-to-live in vitro.
Fresh-keeping mode includes that semen cryopreservation and semen at normal temperature save to common pig semen in vitro.Semen cryopreservation skill
Art is mainly to inhibit the metabolic intensity of sperm using low temperature, to achieve the effect that extending sperm in vitro saves.But
Semen cryopreservation has that frozen sperm survival rate is low, refrigeration operation is complicated, freezen protective is at high cost, is unfavorable for essence
Liquid it is external fresh-keeping.Being stored at room temperature for sperm mainly saves sperm under normal temperature conditions using semen diluent.Sperm is dilute
Releasing the ingredient in agent includes that the functional components molecular structure such as can stablize and maintain sperm amplifying nucleic acid, protein, enzyme and amino acid
Substance, also comprising be able to suppress the microorganisms such as bacterium growth antibacterial material.It is fresh-keeping using the semen at normal temperature of semen diluent
Method is low to preservation condition requirement, and easy to operate, living spermatozoa percentage is higher, is more suitable for the external fresh-keeping of sperm.Therefore, into
When the artificial insemination operations of row pig, porcine semen at normal temperature store method is more applicable for Large-scale pig farm, is also more worth energetically
It promotes.
Currently, diluting boar semen agent a part used in China pig farm is raiser oneself preparation, semen at normal temperature is protected
It is poor to deposit effect, preservation effective time is short, is typically only 2-3 days；Another part is external import diluent, but diluent cost
Height, and using effect is different.Therefore, develop suitable for China pig farm use it is of good preservation effect, the holding time is long, sexual valence
Have great importance than high long-acting diluting boar semen agent.
Enrofloxacin and Ciprofloxacin conduct is respectively adopted in patent (CN107372460A) and patent (CN103518703A)
Antibacterial agent, although playing certain antibacterial effect, Enrofloxacin and Ciprofloxacin are antibiotic drug, using antibiosis
Element is used as pig semen antibacterial agent, and there are poor biocompatibilities, and people and animals are generated with the defect of contamination hazard.
Summary of the invention
Aiming at the above defects or improvement requirements of the prior art, it the present invention provides a kind of diluting boar semen agent and its answers
With the diluent is using glucose as nutritional agents；With citric acid monohydrate sodium, EDTAP dipotassium ethylene diamine tetraacetate and anhydrous sodium bicarbonate work
For buffer；Using trehalose as protective agent；Using jamaicin as antibacterial agent；It is stronger using it using aurantiin as antioxidant
Anti-radical action make sperm from peroxide injury and protection plasmalemmae of sperms.Natural antibacterial agent barberry used in it
Alkali and natural aurantiin are entirely to be extracted from natural plants, do not generate pollution generally to people and animals and environment
Harm, good biocompatibility.Thus it is poor to solve porcine semen at normal temperature preservation effect, saves that effective time is short, antibiotic bio-compatible
Property difference and to people and animals generate contamination hazard the technical issues of.
To achieve the above object, according to one aspect of the present invention, a kind of diluting boar semen agent is provided, the diluent packet
Containing following components: glucose, citric acid monohydrate sodium, EDTAP dipotassium ethylene diamine tetraacetate, anhydrous sodium bicarbonate, trehalose, aurantiin and
Jamaicin；Wherein,
The glucose is used as nutritional agents；
The citric acid monohydrate sodium, EDTAP dipotassium ethylene diamine tetraacetate and anhydrous sodium bicarbonate are used as buffer；
The trehalose is used as protective agent；
The aurantiin is used as antioxidant；
The jamaicin is used as antibacterial agent.
Preferably, the aurantiin is to extract to obtain from the fruit of Citrus perfume (or spice) garden, and purity is 95% or more.
Preferably, the fragrant garden fruit is trifoliate orange sparrow.
Preferably, the jamaicin is to extract to obtain from from Chinese Drug Rhizomes of Coptis, and purity is 95% or more.
Preferably, glucose 20.00-45.00g, citric acid monohydrate sodium 2.00- are contained in each liter diluent
10.00g, EDTAP dipotassium ethylene diamine tetraacetate 1.00-6.00g, anhydrous sodium bicarbonate 0.05-2.00g, trehalose 0.05-4.00g, shaddock
Skin glycosides 0.05-0.5g and jamaicin 0.10-1.00g.
Preferably, glucose 27.00-37.20g, citric acid monohydrate sodium 4.00- are contained in each liter diluent
6.00g, EDTAP dipotassium ethylene diamine tetraacetate 2.00-4.00g, anhydrous sodium bicarbonate 1.00-1.25g, trehalose 1.50-2.00g, shaddock ped
Glycosides 0.15-0.30g and jamaicin 0.25-0.50g.
Preferably, solvent is water in the diluent.
Other side according to the invention provides the application of diluting boar semen agent described in one kind, every 1 milliliter of pig essence
The 4-8 milliliters of diluents are added in liquid, for being stored at room temperature for pig semen.
In general, through the invention it is contemplated above technical scheme is compared with the prior art, can obtain down and show
Beneficial effect:
(1) in long-acting diluting boar semen agent of the invention, the physiological activity of sperm is maintained using glucose as nutritional agents
It needs；Tie up the pH value of pig semen using citric acid monohydrate sodium, EDTAP dipotassium ethylene diamine tetraacetate and anhydrous sodium bicarbonate as buffer
It holds between 6.5-7.0, reduces sperm metabolism and sports level, and provide a relatively suitable osmotic pressure for sperm；With
Trehalose forms protective film in pig sperm cell surface as protective agent, is effectively protected protein molecule invariance inactivation；With from
The natural component jamaicin isolated and purified in Chinese Drug Rhizomes of Coptis inhibits the growth and metabolism of microorganism as antibacterial agent；With from Shan Nan
The natural products aurantiin extracted in the trifoliate orange sparrow fruit of area plantation is antioxidant, is made using its stronger anti-radical action
Sperm is from peroxide injury and protection plasmalemmae of sperms.
(2) long-acting diluting boar semen agent of the invention joined natural broad spectrum antimicrobial agent jamaicin and naturally anti-oxidant
Agent aurantiin, the two are not admitted to chemicals, are extracted from natural plants completely, do not generate generally to people and animals and environment
Contamination hazard, good biocompatibility.To the of good preservation effect of pig semen, the holding time is long, cost performance is high, is suitable for China's scale
Change pig farm and carry out artificial insemination operations, individual raiser in China's is also suitble to carry out the artificial insemination operations of pig.
(3) each component cooperates in diluting boar semen agent provided by the invention, shares out the work and help one another, each component can not be replaced arbitrarily
It changes, it is indivisible, diluting boar semen agent organic whole is collectively formed, Boar spermatozoa cell is protected, is provided for Boar spermatozoa suitable
Suitable living environment, guarantees the normal physiological activity of Boar spermatozoa cell, and can effectively remove during Boar spermatozoa saves and generate
Extra free radical, improve the anti-oxidation stress ability and preservation effect of pig semen, ensure that Boar spermatozoa survival rate, Jin Erti
The high breeding rate and cultivation effect of sow.
(4) component that diluting boar semen agent of the invention is selected is common substance, good biocompatibility, to biomaterial
It is pollution-free non-stimulated, antibiotic-free class chemical substance；Constituent species are few, and effect is good, and economic cost is low, and biological value is high, tool
Have the advantages that safety, source are wide, without side-effects, and there is good buffering, antibacterial, antioxidation, long-term effect can be with
International advanced diluting boar semen agent effect compares favourably, even more excellent, convenient for promoting the use of in production.
(5) diluting boar semen agent provided by the invention is easily mixed preparation, and condition is controllable, and production cost is low, and performance is stablized
Property is good, has a good application prospect.
Detailed description of the invention
Fig. 1 is the pig essence vigor of diluent described in embodiment 1 and the agent of import diluting boar semen under the different holding times.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.As long as in addition, technical characteristic involved in the various embodiments of the present invention described below
Not constituting a conflict with each other can be combined with each other.
The present invention provides a kind of diluting boar semen agent, which includes following components: glucose, citric acid monohydrate
Sodium, EDTAP dipotassium ethylene diamine tetraacetate, anhydrous sodium bicarbonate, trehalose, aurantiin and jamaicin；Wherein, each component in this diluent
Main function it is as follows:
The glucose is used as nutritional agents, to maintain the physiological activity needs of sperm；
The citric acid monohydrate sodium, EDTAP dipotassium ethylene diamine tetraacetate and anhydrous sodium bicarbonate make the pH of pig semen as corrosion inhibiter
Value maintains between 6.5-7.0, reduces sperm metabolism and sports level, and provide a relatively suitable infiltration for sperm
Pressure；
The trehalose is used as protective agent and forms protective film in pig sperm cell surface, and it is constant to be effectively protected protein molecule
Property inactivation；
The aurantiin be used as antioxidant, using its stronger anti-radical action make sperm from peroxide injury with
And protection plasmalemmae of sperms；Aurantiin is a kind of Flavanones compound, with good antioxidant activity, anti-inflammatory effect,
Antivirus action and anti-allergic effects, the aurantiin that the present invention uses preferably are extracted from the fruit of Citrus perfume (or spice) garden and are obtained,
Purity can be met the requirements in 95wt% or more.Preferably, it for example can extract, adopt from the trifoliate orange sparrow fruit that Southern Shausi is planted
Isolate aurantiin crude extract from trifoliate orange sparrow fruit with solvent extraction, recycle high performance liquid chromatography to aurantiin crude extract into
Row is further purified.
The jamaicin is used as antibacterial agent, inhibits the growth and metabolism of microorganism.Jamaicin is also known as berberine, is from Chinese medicine
It extracts and obtains in the coptis, purity can meet use demand of the present invention in 95wt% or more.Using solvent extraction from the coptis
Jamaicin crude extract is isolated, recycles high performance liquid chromatography that jamaicin crude extract is further purified.
Long-acting diluting boar semen agent provided by the invention, it is preferable that contain glucose in each liter diluent
20.00-45.00g, citric acid monohydrate sodium 2.00-10.00g, EDTAP dipotassium ethylene diamine tetraacetate 1.00-6.00g, anhydrous sodium bicarbonate
0.05-2.00g, trehalose 0.05-4.00g, aurantiin 0.05-0.5g and jamaicin 0.10-1.00g.
It is further preferred that containing glucose 27.00-37.20g, citric acid monohydrate sodium in each liter diluent
4.00-6.00g, EDTAP dipotassium ethylene diamine tetraacetate 2.00-4.00g, anhydrous sodium bicarbonate 1.00-1.25g, trehalose 1.50-
2.00g, aurantiin 0.15-0.30g and jamaicin 0.25-0.50g.
Solvent is water in diluting boar semen agent of the present invention, is preferably distilled water.When the diluent concrete application, according to essence
Protonatomic mass and density are diluted, and the 4-8 milliliters of diluents are added in every 1 milliliter of pig semen, and the room temperature for pig semen is protected
It deposits, suitable storage temperature range is 15-25 DEG C, preferably 15-20 DEG C.
The invention discloses a kind of long-acting diluting boar semen agent, the artificial insemination suitable for pig.Pig disclosed by the invention
Semen diluent is by following material composition: glucose, citric acid monohydrate sodium, anhydrous sodium bicarbonate, trehalose, ethylenediamine tetra-acetic acid
Dipotassium, aurantiin, jamaicin.It joined the natural products barberry extracted, purified from Chinese Drug Rhizomes of Coptis in diluent of the invention
Alkali is as antibacterial agent, and joined the natural products aurantiin for extracting, purifying from the trifoliate orange sparrow fruit that Southern Shausi is planted
As antioxidant.With diluent of the invention, pig semen, which saves 3 days, reaches 85% through detecting sperm motility, saves 7 days and remains to
Guarantee that its sperm motility reaches 75%.
The following are embodiments:
Embodiment 1
Take glucose 35g, citric acid monohydrate sodium 6g, anhydrous sodium bicarbonate 1.2g, trehalose 1.5g, ethylenediamine tetra-acetic acid two
Potassium 2g, aurantiin 0.3g, jamaicin 0.40g are added in 950mL distilled water and stir evenly, and adjust PH to 6.8, then constant volume to 1L,
Obtain diluting boar semen agent.
Be diluted when use by 5 milliliters of above-mentioned diluents are added in every 1 milliliter of pig semen, when dilution sperm temperature with it is dilute
Release only 1 DEG C of temperature difference.By the semen storage diluted in 17 DEG C of insulating boxs, sperm is mixed once every 12 hours, is prevented
Only Sperm pellets and lead to death.
Fig. 1 is the pig essence vigor of diluent described in embodiment 1 and the agent of import diluting boar semen under the different holding times.
Import diluting boar semen agent 1 is that III type boar semen dilution powder of Germany meter Ni Tu is prepared to obtain, and import diluting boar semen agent 2 is Hangzhou
Richer pig room temperature semen dilution preservative agent-II (former Japan's boar semen dilution powder), which is prepared, to be obtained.Embodiment 1 as seen from Figure 1
The diluent is good compared to import diluent pig semen sperm motility protecting effect.Using diluent of the invention, pig
Reach 85% through detecting sperm motility within Semen routine 3 days, saves 7 days and still ensure that its sperm motility reaches 75%.
Comparative example 1
Take glucose 35g, citric acid monohydrate sodium 6g, anhydrous sodium bicarbonate 1.2g, trehalose 1.5g, ethylenediamine tetra-acetic acid two
Potassium 2g, aurantiin 0.3g are added in 950mL distilled water and stir evenly, and adjust PH to 6.8, then it is dilute to obtain pig semen to 1L for constant volume
Release agent.
Be diluted when use by 5 milliliters of above-mentioned diluents are added in every 1 milliliter of pig semen, when dilution sperm temperature with it is dilute
Release only 1 DEG C of temperature difference.By the semen storage diluted in 17 DEG C of insulating boxs, sperm is mixed once every 12 hours, is prevented
Only Sperm pellets and lead to death.
Comparative example 2
Take glucose 35g, citric acid monohydrate sodium 6g, anhydrous sodium bicarbonate 1.2g, trehalose 1.5g, ethylenediamine tetra-acetic acid two
Potassium 2g, jamaicin 0.40g are added in 950mL distilled water and stir evenly, and adjust PH to 6.8, then it is dilute to obtain pig semen to 1L for constant volume
Release agent.
Be diluted when use by 5 milliliters of above-mentioned diluents are added in every 1 milliliter of pig semen, when dilution sperm temperature with it is dilute
Release only 1 DEG C of temperature difference.By the semen storage diluted in 17 DEG C of insulating boxs, sperm is mixed once every 12 hours, is prevented
Only Sperm pellets and lead to death.
Compared to diluent described in embodiment 1, diluent described in comparative example 1 is not added small diluent described in comparative example 1
Bark of a cork tree alkali, remaining component content are identical.
For diluent described in comparative example 2 compared to diluent described in embodiment 1, shaddock is not added in diluent described in comparative example 2
Skin glycosides, remaining component content are identical.
Table 1 be respectively using after comparative example 1, comparative example 2 and diluent described in embodiment 1 to pig semen sperm concentration,
The comparison result of sperm motility, rate of teratosperm and forward momentum.
Do not add the comparative example 1 of jamaicin in the 6th day pig semen bacterial infection as can be seen from Table 1, sperm motility declines,
There is dead essence；Not plus the comparative example 2 of aurantiin declined in the 6th day sperm motility, dead essence occurred within the 7th day；And jamaicin is added
And the embodiment 1 of aurantiin still has 75% or more sperm motility in the 7th day pig semen, sperm storage works well.
The different embodiment diluting boar semen agent of table 1 are to pig semen sperm concentration, sperm motility, rate of teratosperm and forward direction
The influence of power
Embodiment 2
Take glucose 20g, citric acid monohydrate sodium 2g, anhydrous sodium bicarbonate 0.05g, trehalose 0.05g, ethylenediamine tetra-acetic acid
Dipotassium 1g, aurantiin 0.05g, jamaicin 1g are added in 950mL distilled water and stir evenly, and adjust PH to 7.0, then fixed with distilled water
Hold 1L, obtains diluting boar semen agent.
It is diluted when use by 4 milliliters of above-mentioned diluting boar semen agent are added in every 1 milliliter of pig semen, sperm temperature when dilution
Only 1 DEG C of temperature difference of degree and dilution.By the semen storage diluted in 20 DEG C of insulating boxs, sperm is mixed one every 12 hours
It is secondary, it prevents Sperm pellets and leads to death.
Embodiment 3
Take glucose 27g, citric acid monohydrate sodium 6g, anhydrous sodium bicarbonate 1g, trehalose 2g, EDTAP dipotassium ethylene diamine tetraacetate
2g, aurantiin 0.2g, jamaicin 0.25g are added in 950mL distilled water and stir evenly, and adjust PH to 7.0, then with distilled water constant volume
To 1L.
It is diluted when use by 5 milliliters of preservations liquid are added in every 1 milliliter of pig semen, sperm temperature and dilution are warm when dilution
Only 1 DEG C of degree difference.By the semen storage diluted in 17 DEG C of insulating boxs, sperm is mixed once every 12 hours, prevents essence
Son precipitates and leads to death.
Embodiment 4
Take glucose 45g, citric acid monohydrate sodium 10g, anhydrous sodium bicarbonate 2g, trehalose 4g, EDTAP dipotassium ethylene diamine tetraacetate
6g, aurantiin 0.5g, jamaicin 0.1g are added in 950mL distilled water and stir evenly, and adjust PH to 7.0, then with distilled water constant volume
To 1L, diluting boar semen agent is obtained.
It is diluted when use by 8 milliliters of above-mentioned diluting boar semen agent are added in every 1 milliliter of pig semen, sperm temperature when dilution
Only 1 DEG C of temperature difference of degree and dilution.By the semen storage diluted in 24 DEG C of insulating boxs, sperm is mixed one every 12 hours
It is secondary, it prevents Sperm pellets and leads to death.
As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, not to
The limitation present invention, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should all include
Within protection scope of the present invention.
Claims (8)
1. a kind of diluting boar semen agent, which is characterized in that the diluent includes following components: glucose, citric acid monohydrate sodium, second
Ethylenediamine tetraacetic acid (EDTA) dipotassium, anhydrous sodium bicarbonate, trehalose, aurantiin and jamaicin；Wherein,
The glucose is used as nutritional agents；
The citric acid monohydrate sodium, EDTAP dipotassium ethylene diamine tetraacetate and anhydrous sodium bicarbonate are used as buffer；
The trehalose is used as protective agent；
The aurantiin is used as antioxidant；
The jamaicin is used as antibacterial agent.
2. diluting boar semen agent as described in claim 1, which is characterized in that the aurantiin is from the fruit of Citrus perfume (or spice) garden
Extraction obtains, and purity is 95% or more.
3. diluting boar semen agent as claimed in claim 2, which is characterized in that the perfume garden fruit is trifoliate orange sparrow.
4. diluting boar semen agent as described in claim 1, which is characterized in that the jamaicin is to extract from from Chinese Drug Rhizomes of Coptis
It obtains, purity is 95% or more.
5. diluting boar semen agent as described in claim 1, which is characterized in that contain glucose in each liter diluent
20.00-45.00g, citric acid monohydrate sodium 2.00-10.00g, EDTAP dipotassium ethylene diamine tetraacetate 1.00-6.00g, anhydrous sodium bicarbonate
0.05-2.00g, trehalose 0.05-4.00g, aurantiin 0.05-0.5g and jamaicin 0.10-1.00g.
6. diluting boar semen agent as described in claim 1, which is characterized in that contain glucose in each liter diluent
27.00-37.20g, citric acid monohydrate sodium 4.00-6.00g, EDTAP dipotassium ethylene diamine tetraacetate 2.00-4.00g, anhydrous sodium bicarbonate
1.00-1.25g, trehalose 1.50-2.00g, aurantiin 0.15-0.30g and jamaicin 0.25-0.50g.
7. diluting boar semen agent as described in claim 1, which is characterized in that solvent is water in the diluent.
8. a kind of application of diluting boar semen agent as described in any one of claim 1 to 7, which is characterized in that every 1 milliliter of pig essence
The 4-8 milliliters of diluents are added in liquid, for being stored at room temperature for pig semen.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117296830A (en) * | 2023-09-25 | 2023-12-29 | 仲恺农业工程学院 | Boar semen diluent and preparation method and application thereof |
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