CN104585164A - Improved diluent for long-term preservation of semen of ruminant - Google Patents
Improved diluent for long-term preservation of semen of ruminant Download PDFInfo
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- CN104585164A CN104585164A CN201510065462.6A CN201510065462A CN104585164A CN 104585164 A CN104585164 A CN 104585164A CN 201510065462 A CN201510065462 A CN 201510065462A CN 104585164 A CN104585164 A CN 104585164A
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Abstract
The invention discloses improved diluent for long-term preservation of semen of ruminant. The improved diluent is characterized in that per 100ml of double distilled water contains 3 to 9g of tris (hydroxymethyl) aminomethane, 5 to 25ml of yolk, 0.5 to 3g of trehalose, 1 to 4g of fructose, 10 to 90mg of glutathione, 60 to 280mg of bovine serum albumin, 20 to 150mg of triphosadenine, 100 to 800mg of vitamin C, 0.8 to 2.8g of citric acid, 100000 to 800000 units of penicillin, 50000 to 300000 units of streptomycin, and 2 to 10mg of superoxide dismutase. The diluents has a good effect on preserving semen of ruminants at low temperature, such as sheep, cattle and deer; in addition, the production method is simple, the production cost is low, and the popularization and application are convenient.
Description
Technical field
The present invention relates to the dilution of the long-acting preservation of seminal fluid in a kind of ruminant technology of artificial insemination, particularly a kind of dilution for sheep, ox, the long-acting preservation of deer testicular fluid.
Background technology
The ruminants such as camel, deer, alpaca, antelope, ox, sheep are the important economic animals of China, these ruminants have high meat, breast with and medical value.Extensive and casual household raises the technology of artificial insemination that uses to accelerate ruminant breed improvement more now, improves and breeds speed and set up large-scale cultivation management system.
Semen cryopreservation is one of test-tube key technology, application frozen semen carries out the restriction that artificial insemination can not be subject to time, region, kind and individuality, and a large amount of raising quantity reducing male animal, reduce production cost, improve the availability of good species male animal, improve the economic benefit of aquaculture.Simultaneously this technology kind is introduced, crossbreeding and improvement, and improve reproductive performance, Disease epizootic and conservation genetics Protection of Diversity aspect and also have great importance, therefore how as far as possible for a long time preserving spermatozoon activity is one of problem of very paying close attention to of this area.
The dilution of present domestic use exists when preserving seminal fluid that timeliness is short, weak effect problem, the seminal fluid once gathered causes waste because preservation effect is undesirable after dilution, cannot maximum using good species male animal fertility, this constrains the application of technology of artificial insemination and the development of China's livestock culturing industry undoubtedly.
Chinese patent application file (application number: disclose milch goat freezing seminal fluid dilution and preparation method thereof and dilution process 201310491965.0), wherein, the basic dilution of freeze-extender is added water be made into by Tris, citric acid, glucose, trehalose, Vc, GSH, BSA, ATP, composite antibiotic, yolk.This invention achieves good effect to milch goat seminal fluid Cord blood and freezen protective, but there is abundant unsaturated fatty acid owing to plasmalemmae of sperms maintaining the membrane fluidity very easily oxidized and asthenospermia that causes and the higher problem of cost under aerobic conditions in the semen diluent of this disclosure of the invention.
Summary of the invention
For the deficiency of depositing in prior art, applicant has invented a kind of dilution for the long-acting preservation of ruminant seminal fluid by research, this dilution can effectively ensure sperm quality, and there is the advantages such as production cost is lower, the holding time is long, preparation method is simple, be applicable to the long-acting preservation of the ruminant class seminal fluid such as camel, deer, alpaca, antelope, ox, sheep.
The technical solution adopted in the present invention is: a kind of dilution for the long-acting preservation of ruminant seminal fluid, containing 3-9g trihydroxy aminomethane, 5-25ml yolk, 0.5-3g trehalose, 1-4g fructose, 10-90mg glutathione, 60-280mg bovine serum albumin(BSA), 20-150mg adenosine triphosphate, 100-800mg vitamin C, 0.8-2.8g citric acid, 10-80 ten thousand units of Penicillin, 5-30 ten thousand unit streptomycin, 2-10mg superoxide dismutase in every 100ml distilled water.
Applicant have extensively studied the impact of yolk Ingredient Amount on seminal fluid activity, and preferably adopt the yolk of 15-25ml, in this amount ranges, sperm motility rate is not less than 0.4; Be more preferably the yolk of 20ml, sperm motility rate is not less than 0.45.
On this basis, the invention discloses described dilution preserving the purposes in ruminant seminal fluid, in use motility rate being not less than the seminal fluid of 0.6, with this diluted 2-10 times, cooling makes dilution reach 0 DEG C, preserves at 0 DEG C.
Wherein, cooling and preserving type can be selected according to actual needs, normally water-bath cooling in refrigerator or other low-temperature receiver, and ensure 0 DEG C of preservation with mixture of ice and water in water bath containers.
The experiment display that applicant carries out, dilution of the present invention has good, stable motility rate after the long-time sperm preserved can be made to thaw.
Dilution of the present invention, liquid based on Tris, makes dilution meta-alkalescence and buffer capacity is comparatively strong, is conducive to improving sperm viability; The yolk of suitable consumption is protected for sperm provides and improves the stability of sperm; Adenosine triphosphate, vitamin C, bovine serum albumin(BSA), trehalose, penicillin, streptomycin provide energy and protection for sperm; Superoxide dismutase (SOD) alleviates the injury of longer-term storage Process of Peroxide to sperm viability; Adjust bovine serum albumin(BSA) and glutathione ratio and Antibiotics simultaneously, the basis keeping dilution effect reduces production cost.
Embodiment
Beneficial effect of the present invention is further illustrated below in conjunction with concrete preparation embodiment and product effect test example.
Embodiment 1
Distilled water 100mL and trehalose 3g, yolk 20mL, superoxide dismutase 10mg, fructose 4g, trihydroxy aminomethane 6g, citric acid 1.5g, glutathione 50mg, bovine serum albumin(BSA) 180mg, adenosine triphosphate 50mg, vitamin C 800mg, penicillin 800,000 unit, streptomycin 100,000 unit are mixed.
Embodiment 2
Distilled water 100mL and trehalose 2g, yolk 25mL, superoxide dismutase 5mg, fructose 1g, trihydroxy aminomethane 8g, citric acid 2.5g, glutathione 70mg, bovine serum albumin(BSA) 100mg, adenosine triphosphate 100mg, vitamin C 300mg, penicillin 500,000 unit, streptomycin 200,000 unit are mixed.
Embodiment 3
Distilled water 100mL and trehalose 3g, yolk 15mL, superoxide dismutase 7mg, fructose 2g, trihydroxy aminomethane 4g, citric acid 2.5g, glutathione 30mg, bovine serum albumin(BSA) 220mg, adenosine triphosphate 100mg, vitamin C 500mg, penicillin 300,000 unit, streptomycin 200,000 unit are mixed.
In order to study effect of the present invention, for the dilution of embodiment 1.Observing in laboratory the seminal fluid be collected by experiment and measure motility rate, density, is the percentage that canonical statistics is superior, good, the seminal fluid number of the sperm viability of standard accounts for 0.7 and 0.6.Select the seminal fluid of motility rate >=0.7, determine extension rate, use dilution of the present invention to dilute seminal fluid, preserve in 0 DEG C of environment, thaw in the water-bath of 35 DEG C 20s, microscopy, and the seminal fluid of motility rate more than 0.5 is used for semen deposition.
Contrast with common glucose-lemon acid-yolk diluent, result is as shown in table 1 simultaneously.
Table 1 different diluent is on the impact of sperm viability
The above results shows, and is that the seminal fluid of the same race of initial vigor is through diluted of the present invention and obviously different through conventional diluted preservation effect with vigor 0.8.Use conventional dilution to sheep, the motility rate of its seminal fluid after 24 hours declines obviously, can not reach semen deposition standard after 48 hours; And use dilution of the present invention still can obtain desirable and stable result in 168 hours, may be used for semen deposition.
Simultaneously applicant also to have extensively studied in dilution yolk consumption to the impact of motility rate, shown in table 2 specific as follows.
The screening of table 2 dilution yolk of the present invention concentration
The yolk all adding variable concentrations in semen diluent exempts from low temperature infringement to protect sperm.Yolk is rich in lecithin, unsaturated fatty acid, and yolk is a kind of osmolarity buffers, sperm can be made to tolerate hypotonic or height ooze, namely can increase extracellular osmotic pressure and promote that in sperm born of the same parents, moisture outflow prevents from forming ice crystal in cell, thus play protection perforatorium film, maintain the effect of sperm viability, but have a strong impact on sperm motility along with the increase yolk protein of yolk concentration and fragment also increase simultaneously, the constituent concentration being simultaneously conducive to sperm storage along with the increase of yolk concentration rises, but some can be breathed or reduce the composition of sperm motility rate and also increase by Inhibit sperm in yolk thereupon, to determine in dilution that yolk concentration needs a large amount of tests to carry out screening and determines thus.The sperm motility rate that result of the test shows in 5 kinds of yolk concentration 15% and 20% group reaches 0.45, comparatively other 3 groups of vigor are better, but 15% group of motility rate is 0.45 lower than 20% group, and its variation is larger, therefore select 20%, its result is comparatively stablized and can be obtained optimal motility rate.
Content of the present invention is not limited to cited by embodiment, and the conversion of those of ordinary skill in the art by reading specification of the present invention to any equivalence that technical solution of the present invention is taked, is claim of the present invention and contains.
Claims (4)
1., for a dilution for the long-acting preservation of ruminant seminal fluid, it is characterized in that in every 100ml distilled water containing 3-9g trihydroxy aminomethane, 5-25ml yolk, 0.5-3g trehalose, 1-4g fructose, 10-90mg glutathione, 60-280mg bovine serum albumin(BSA), 20-150mg adenosine triphosphate, 100-800mg vitamin C, 0.8-2.8g citric acid, 10-80 ten thousand units of Penicillin, 5-30 ten thousand unit streptomycin, 2-10mg superoxide dismutase.
2. the dilution for the long-acting preservation of ruminant seminal fluid according to claim 1, is characterized in that: described yolk consumption is 15-25ml.
3. the dilution for the long-acting preservation of ruminant seminal fluid according to claim 1, is characterized in that: described yolk consumption is 20ml.
4. the dilution for the long-acting preservation of ruminant seminal fluid of claim 1 is preserving the purposes in ruminant seminal fluid, motility rate is not less than the seminal fluid of 0.6, with diluted 2-10 times in test tube, is cooled to dilution seminal fluid and reaches 0.℃，0。DEG C long-term to preserve.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105532648A (en) * | 2016-02-16 | 2016-05-04 | 云南省畜牧兽医科学院 | Small ruminant semen freezing dilution solution |
| CN106857500A (en) * | 2017-02-06 | 2017-06-20 | 张志刚 | The store method and its special fresh essence of a kind of fresh essence of animal preserve dilution |
| CN110301430A (en) * | 2018-03-27 | 2019-10-08 | 高庆华 | A kind of no animal originality additive sheep sperm efficient cryogenic preservation Dilution for powder liquid |
| CN110463687A (en) * | 2019-07-10 | 2019-11-19 | 安徽农业大学 | A kind of the preservation liquid and store method of sheep sperm |
| CN111053080A (en) * | 2019-11-01 | 2020-04-24 | 中国农业大学 | Semen diluent for improving semen low-temperature preservation quality and application method thereof |
| CN111053079A (en) * | 2016-06-29 | 2020-04-24 | 许红喜 | Preparation method of animal sperm diluent |
| CN111657270A (en) * | 2020-07-21 | 2020-09-15 | 张良斌 | Normal-temperature preservative for livestock semen and preparation method and use method thereof |
| CN111771872A (en) * | 2020-07-29 | 2020-10-16 | 天津市畜牧兽医研究所 | Bovine semen freezing diluent and preparation method and application thereof |
| CN114304138A (en) * | 2022-01-17 | 2022-04-12 | 阿拉善盟畜牧兽医技术推广中心(阿拉善盟动物疫病预防控制中心) | Method for freezing semen of bactrian camel |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105532648A (en) * | 2016-02-16 | 2016-05-04 | 云南省畜牧兽医科学院 | Small ruminant semen freezing dilution solution |
| CN105532648B (en) * | 2016-02-16 | 2018-01-12 | 云南省畜牧兽医科学院 | A kind of small ruminant sperm freezing dilution liquid |
| CN111053079A (en) * | 2016-06-29 | 2020-04-24 | 许红喜 | Preparation method of animal sperm diluent |
| CN111053079B (en) * | 2016-06-29 | 2022-02-01 | 许红喜 | Preparation method of animal sperm diluent |
| CN106857500A (en) * | 2017-02-06 | 2017-06-20 | 张志刚 | The store method and its special fresh essence of a kind of fresh essence of animal preserve dilution |
| CN110301430A (en) * | 2018-03-27 | 2019-10-08 | 高庆华 | A kind of no animal originality additive sheep sperm efficient cryogenic preservation Dilution for powder liquid |
| CN110463687A (en) * | 2019-07-10 | 2019-11-19 | 安徽农业大学 | A kind of the preservation liquid and store method of sheep sperm |
| CN110463687B (en) * | 2019-07-10 | 2021-07-27 | 安徽农业大学 | Preservation solution and preservation method for sheep semen solution |
| CN111053080A (en) * | 2019-11-01 | 2020-04-24 | 中国农业大学 | Semen diluent for improving semen low-temperature preservation quality and application method thereof |
| CN111657270A (en) * | 2020-07-21 | 2020-09-15 | 张良斌 | Normal-temperature preservative for livestock semen and preparation method and use method thereof |
| CN111771872A (en) * | 2020-07-29 | 2020-10-16 | 天津市畜牧兽医研究所 | Bovine semen freezing diluent and preparation method and application thereof |
| CN114304138A (en) * | 2022-01-17 | 2022-04-12 | 阿拉善盟畜牧兽医技术推广中心(阿拉善盟动物疫病预防控制中心) | Method for freezing semen of bactrian camel |
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