CN101084926A - Diluent for preserving frozen red deer semen - Google Patents
Diluent for preserving frozen red deer semen Download PDFInfo
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- CN101084926A CN101084926A CNA2007100181613A CN200710018161A CN101084926A CN 101084926 A CN101084926 A CN 101084926A CN A2007100181613 A CNA2007100181613 A CN A2007100181613A CN 200710018161 A CN200710018161 A CN 200710018161A CN 101084926 A CN101084926 A CN 101084926A
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- diluent
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Abstract
The invention discloses a dilution liquid for preserving refrigerated sperm of deer, which comprises distilled water 80-150ml, laevulose 0.5-3g, Tris 2-7g, citric acid 1-5g, GSH 5-100mg, BSA 50-300mg, ATP 5-100mg, VC 100-1000mg, penicillin 100-1000 thousands, and streptomycin 50-500 thousands.
Description
Technical field
The present invention relates to the diluent of seminal fluid in a kind of deer technology of artificial insemination improvement, particularly a kind of diluent that is used for the deer testicular fluid freezing.
Background technology
Fine and soft deer is the important special economic animal of China, and its major product Cornu Cervi Pantotrichum has high medical value.The fine and soft deer aquaculture management method of long-term artificial domestication causes inbreeding, deer group downgrade, and technology of artificial insemination can improve outstanding kind of buck breeding efficiency greatly, break timeliness and region that buck is used, solved the not enough geographic female animal breeding problem of kind buck effectively, using artificial insemination and particularly frozen semen carry out breed of variety and the improvement of deer, can effectively contain this degradation trend.From Zhao's generation in 1961 attain etc. the people first with artificial vagina adopt the Cervus nippon Temminck seminal fluid, be defeated by Cervus elaphus linnaeus, conception rate 33%, people such as Bei Shiao (1968) by animal in deer family adopt seminal fluid, zoo, Guangdong in 1976 and Guangdong Province science and technology pilot plant is obtained deer electro-ejaculation success, and Heilungkiang special product institute in 1988 obtains conception rate more than 60% to the artificial insemination of Cervus elaphus linnaeus.Conventional cattle is frozen the smart diluent of producing can not obtain deer frozen semen up to standard, the at present domestic special-purpose diluent that is used for the preservation of deer frozen semen of developing that does not yet have autonomous fully success, so domestic now freeze-extender that uses French Etablissements Caillau and German Mi Nitu company more, its price height and refrigerating effect are general, have restricted the development of China's deer industry.
Summary of the invention
At problems of the prior art with not enough, the object of the present invention is to provide a kind of refrigerating effect good, produce the diluent that adult is low, method simply is used for the preservation of deer frozen semen.
The technical scheme that realizes the foregoing invention purpose is: a kind of diluent that is used for the preservation of deer frozen semen, and it mainly is to be made in proportion by following material:
Distilled water: 100ml
Fructose: 0.5-3g
Trihydroxy aminomethane (Tris): 2-7g
Citric acid: 1-5g
Glutathion (GSH): 5-100mg
Bovine serum albumin (BSA): 50-300mg
Adenosine triphosphate (ATP): 5-100mg
Vitamin C (V
C): 100-1000mg
Penicillin: 10-100 ten thousand
Streptomycin: 5-50 ten thousand
Yolk: 15-30ml
Glycerol: 3-30ml
Reach optimum efficiency in order to make diluent of the present invention be used for the freezing of semen preservation, can also add glycerol 3-30ml.
The preparation method of diluent that the present invention is used for preserving frozen red deer semen is very simple, described raw material is directly mixed get final product.
The diluent that the present invention is used for preserving frozen red deer semen has two purposes, and the one, be used for the seminal fluid cryopreservation, another purposes is to be used for freezing of semen to preserve.
The present invention is basal liquid with TRIS; meta-alkalescence and stronger buffer capacity is arranged; helping improving motility of sperm yolk and glycerol is mainly in sperm cooling and the refrigerating process as the universaling component that freezes smart diluent protection is provided; improve the stability of sperm and the vigor after the recovery; added and frozen the smart diluent of producing as deer behind ATP, BSA, VC, the CP composition and can obtain ideal and stable result; and low price, the effect stability of being impregnated.
The specific embodiment
The concrete preparation embodiment that provides below in conjunction with invention, the making embodiment and the product effect test example of straw frozen semen further specify beneficial effect of the present invention.
The diluent preparation of embodiment 1 deer frozen semen of the present invention preservation
With distilled water 100ml and fructose 0.5g, Tris5g, citric acid 3g, GSH 100mg, BSA 200mg, ATP 20mg, VC500mg, penicillin 400,000, streptomycin 400,000, yolk 25ml mix homogeneously promptly.
Embodiment 2 deer 12% glycerol diluent preparations of the present invention
With distilled water 100ml and fructose 2g, Tris7g, citric acid 2g, GSH30mg, BSA150mg, ATP50mg, V
c200mg penicillin 600,000, streptomycin 600,000, yolk 25ml mix homogeneously are got mixed diluting liquid 88ml and are added 12ml glycerol and prepare 12% glycerol freeze-extender.
The cryopreservation of embodiment 3 seminal fluid
Being sent to laboratory with gathering good seminal fluid, observing and also measure motility rate, density, is the percent that the seminal fluid umber of the motility of sperm of superior under the canonical statistics varying environment temperature, good standard accounts for 0.7 and 0.6.Select the seminal fluid of motility rate 〉=0.8, determine extension rate, semen dilution is cooled to 0 ℃ to predetermined dilution multiple refrigerator water-bath 1.5h with 20% yolk diluent, 0 ℃ of environment is preserved 7-10d, the 20s that thaws in 35 ℃ the water-bath, microscopy, the seminal fluid of motility rate more than 0.5 is used for semen deposition.
The making of embodiment 4 straw frozen semens
Being sent to laboratory with gathering good seminal fluid, observing and also measure motility rate, density, is the percent that the seminal fluid umber of the motility of sperm of superior under the canonical statistics varying environment temperature, good standard accounts for 0.7 and 0.6.Select the seminal fluid of motility rate 〉=0.8, determine extension rate, with 20% yolk diluent with semen dilution to a times of predetermined dilution multiple, will slowly add along test tube wall for preceding 1 time with dilution seminal fluid equivalent, isothermal 12% glycerol diluent balance, mixing makes the glycerol final concentration reach 6%.Refrigerator water-bath 1.5h cools to 0 ℃.Water-bath cooling balance 3h in 0 ℃ of refrigerator.Seminal fluid that balance is good sucks the 0.25ml tubule in refrigerator, and the polyvinyl alcohol powder seals, and places on freezing of liquid nitrogen surface 2cm, builds container cover, and freezing 5min drops in the liquid nitrogen.Pack into gauze bag liquid nitrogen preservation of the sampling and the 20s that in 35 ℃ water-bath, thaws, microscopy, the motility rate seminal fluid labelling 0.35 or more.
Test example 1 different freeze-extenders are to freezing the influence of smart vigor
The different freeze-extenders of table 1 are to freezing the influence of smart vigor
| Freeze-extender | Sample number (individual) | Motility rate (%) |
| Cattle sucrose-yolk-glycerol diluent (5% glycerol) TRIS basal liquid (5% glycerol) diluent of the present invention (5% glycerol) | 13 11 22 | 8.34±5.62 a 24.72±13.37 b 45.13±21.22 c |
Table 1 result shows, the routine of Tienshan wapiti being used cattle is frozen the smart diluent of producing, can not obtain frozen semen up to standard, using TRIS basal liquid (5% glycerol) and freeze the smart diluent of producing as Cervus elaphus linnaeus and can obtain result preferably, is that the diluent of the present invention (5% glycerol) of basal liquid improvement freezes the smart diluent of producing as Cervus elaphus linnaeus and can obtain ideal and stable result with TRIS.
The screening of test example 2 diluent glycerol concentrations of the present invention
The screening of table 2 diluent glycerol concentration of the present invention
| Diluent glycerol final concentration of the present invention (%) | Sample number (individual) | Motility rate (%) |
| 3 4 5 6 7 8 | 9 13 22 16 16 14 | 13.65±11.52 a 28.33±12.67 b 45.13±19.22 c 48.13±15.19 c 40.51±13.80 d 31.46.±14.12 b |
Glycerol is most widely used permeability freezeproof protectant, and the concentration of glycerol and the dilution interpolation in the temperature-fall period directly determines refrigerating effect opportunity.It can infiltrate cell and alleviate cell permeability swelling in the freezing and rewarming process, slow down cell shrinkage speed, alleviate the shrinkage degree, reduce the formation of ice crystal, still, the higher permeability of glycerol easily causes the damage of spermatid again, glycerol concentration is high more, the easy more quilt of cell injures, and the requirement to glycerol concentration when various animal spermatids are freezing is different, and the glycerol optium concentration of Cervus elaphus linnaeus freezing of semen needs a large amount of tests to screen definite.Result of the test shows, 5% and 6% group the essence of freezing reaches 0.45 in 6 kinds of glycerol concentrations, than other each concentration groups higher motility rate is arranged, all of 6% group are frozen smart motility rate all more than 0.3, its refrigerating effect is best, 5% group to freeze smart motility rate also up to standard substantially, but variation is bigger, other each concentration groups can not obtain motility rate up to standard freeze essence.The diluent of 6% glycerol final concentration can obtain optimal motility rate.
The screening of test example 3 diluents of the present invention (6% glycerol) diluent joining day
Table 3 contains the screening of 6% glycerol diluent joining day
| Contain the glycerol diluent joining day | Sample number (individual) | Motility rate (%) |
| Once add before the cooling | 16 | 48.13±15.19 a |
| Once add before adding balance before the cooling and before the balance at twice | 15 15 | 47.89±16.08 a 45.37±17.76 b |
Glycerol diffuses into comparatively difficulty of cell at 0 ℃, and the adding of glycerin liquid is preferably in to add before cooling is finished and makes glycerol in the cell internal and external equilibrium.Table 3 result shows, before the cooling once group add with cooling before and before the balance adding group at twice to freeze smart motility rate all up to standard, two groups refrigerating effect ideal and difference are not remarkable, the refrigerating effect of once adding group is not as preceding two groups before the balance.This results suggest, the Cervus elaphus linnaeus sperm has tolerance more by force to the glycerol of higher concentration, and it is longer that the glycerol infiltration reaches the time that balance needs, and glycerol once adds before cooling and reaches 6% final concentration and not only simplified operating procedure but also can obtain best refrigerating effect.
Claims (2)
1. diluent that is used for preserving frozen red deer semen is characterized in that it mainly is to be made in proportion by following material:
Distilled water: 100ml
Fructose: 0.5-3g
Trihydroxy aminomethane: 2-7g
Citric acid: 1-5g
Glutathion: 5-100mg
Bovine serum albumin: 50-300mg
Adenosine triphosphate: 5-100mg
Vitamin C: 100-1000mg
Penicillin: 10-100 ten thousand
Streptomycin 5-50 ten thousand
Yolk 15-30ml
2. a kind of diluent that is used for preserving frozen red deer semen according to claim 1 is characterized in that adding glycerol 3-30ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200710018161A CN100581553C (en) | 2007-07-02 | 2007-07-02 | Diluent for preserving frozen red deer semen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200710018161A CN100581553C (en) | 2007-07-02 | 2007-07-02 | Diluent for preserving frozen red deer semen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101084926A true CN101084926A (en) | 2007-12-12 |
| CN100581553C CN100581553C (en) | 2010-01-20 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200710018161A Expired - Fee Related CN100581553C (en) | 2007-07-02 | 2007-07-02 | Diluent for preserving frozen red deer semen |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102318596A (en) * | 2011-07-12 | 2012-01-18 | 青岛康大食品有限公司 | Diluent for preserving rabbit sperms at normal temperature and preparation method thereof |
| CN102613168A (en) * | 2012-03-05 | 2012-08-01 | 深圳市安多福动物药业有限公司 | Disinfectant for animal semen and preparation method thereof |
| CN103120155A (en) * | 2013-02-05 | 2013-05-29 | 新疆维吾尔自治区畜牧科学院畜牧科学研究所 | Cryopreservation solution of horse sperms and freezing method thereof |
| CN103518706A (en) * | 2013-10-21 | 2014-01-22 | 西北农林科技大学 | Diluent of frozen semen of milk goat, and preparation method and diluting method thereof as well as preparation method of frozen semen of milk goat in thin pipe |
| CN103548812A (en) * | 2013-10-30 | 2014-02-05 | 西北农林科技大学 | Normal-temperature and low-temperature preservation diluents of milk goat seminal fluid |
| CN104585164A (en) * | 2015-02-06 | 2015-05-06 | 塔里木大学 | Improved diluent for long-term preservation of semen of ruminant |
| 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 |
| CN110463688A (en) * | 2019-07-10 | 2019-11-19 | 安徽农业大学 | A kind of dilution and store method of sheep sperm |
| CN111657270A (en) * | 2020-07-21 | 2020-09-15 | 张良斌 | Normal-temperature preservative for livestock semen and preparation method and use method thereof |
-
2007
- 2007-07-02 CN CN200710018161A patent/CN100581553C/en not_active Expired - Fee Related
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102318596A (en) * | 2011-07-12 | 2012-01-18 | 青岛康大食品有限公司 | Diluent for preserving rabbit sperms at normal temperature and preparation method thereof |
| CN102613168A (en) * | 2012-03-05 | 2012-08-01 | 深圳市安多福动物药业有限公司 | Disinfectant for animal semen and preparation method thereof |
| CN103120155B (en) * | 2013-02-05 | 2014-07-16 | 新疆维吾尔自治区畜牧科学院畜牧科学研究所 | Cryopreservation solution of horse sperms and freezing method thereof |
| CN103120155A (en) * | 2013-02-05 | 2013-05-29 | 新疆维吾尔自治区畜牧科学院畜牧科学研究所 | Cryopreservation solution of horse sperms and freezing method thereof |
| CN103518706B (en) * | 2013-10-21 | 2015-04-01 | 西北农林科技大学 | Diluent of frozen semen of milk goat, and preparation method and diluting method thereof as well as preparation method of frozen semen of milk goat in thin pipe |
| CN103518706A (en) * | 2013-10-21 | 2014-01-22 | 西北农林科技大学 | Diluent of frozen semen of milk goat, and preparation method and diluting method thereof as well as preparation method of frozen semen of milk goat in thin pipe |
| CN103548812A (en) * | 2013-10-30 | 2014-02-05 | 西北农林科技大学 | Normal-temperature and low-temperature preservation diluents of milk goat seminal fluid |
| CN103548812B (en) * | 2013-10-30 | 2015-05-13 | 西北农林科技大学 | Normal-temperature and low-temperature preservation diluents of milk goat seminal fluid |
| CN104585164A (en) * | 2015-02-06 | 2015-05-06 | 塔里木大学 | Improved diluent for long-term preservation of semen of ruminant |
| 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 |
| CN110463688A (en) * | 2019-07-10 | 2019-11-19 | 安徽农业大学 | A kind of dilution and store method of sheep sperm |
| CN110463688B (en) * | 2019-07-10 | 2021-07-27 | 安徽农业大学 | Diluent of sheep semen and preservation method |
| CN111657270A (en) * | 2020-07-21 | 2020-09-15 | 张良斌 | Normal-temperature preservative for livestock semen and preparation method and use method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100581553C (en) | 2010-01-20 |
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Free format text: CORRECT: ADDRESS; FROM: 712100 ROOM 161, BUILDING 10, WAN AN GARDEN RESIDENTIAL QUARTER, SHENNONG ROAD, YANGLING DEMONSTRATION ZONE, XIANYANG CITY, SHAANXI PROVINCE TO: 843300 COLLEGE OF ANIMAL SCIENCE, TARIM UNIVERSITY, ALAER CITY, XINJIANG |
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Effective date of registration: 20110303 Address after: 843300 Xinjiang College of Animal Sciences, Tarim University Alar City Patentee after: Tarim University Address before: 712100, room 10, building 161, Wan An garden area, Shennong Road, Yangling demonstration area, Shaanxi, Xianyang Patentee before: Gao Qinghua |
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Granted publication date: 20100120 Termination date: 20120702 |