CN102379279A - Refrigerating fluid for storing Tibetan spaniel sperm - Google Patents
Refrigerating fluid for storing Tibetan spaniel sperm Download PDFInfo
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- CN102379279A CN102379279A CN2011103657168A CN201110365716A CN102379279A CN 102379279 A CN102379279 A CN 102379279A CN 2011103657168 A CN2011103657168 A CN 2011103657168A CN 201110365716 A CN201110365716 A CN 201110365716A CN 102379279 A CN102379279 A CN 102379279A
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Abstract
The invention relates to refrigerating fluid for storing Tibetan spaniel sperm. The refrigerating fluid is used for refrigerating and artificially inseminating the Tibetan spaniel sperm. By the refrigerating fluid, vigor, a resuscitation rate, an acrosome integrity rate and the like of frozen sperm can be improved, and the conception rate and the litter farrowing average number of Tibetan spaniels reach or approach to a natural mating level, so the refrigerating fluid has popularization value.
Description
Technical field
The present invention relates to a kind of freezing liquid that the Tibetan mastiff sperm is preserved that is used for, be used for the freezing and artificial insemination of Tibetan mastiff seminal fluid.
Background technology
Tibetan mastiff is mainly raised and breeds in the Qinghai-Tibet pastoral area of China, and Tibetan mastiff receives the eco-environmental impact of high height above sea level, low temperature, hypoxemia amount for a long time, and it is tall and big to possess build, and characteristics such as the resolute and steadfast ferociousness of personality receive China even world Tibetan mastiff fan's praise deeply.Over nearly 20 years, Tibetan mastiff culture, economy have obtained development fast, and Tibetan mastiff is cultured and made the herdsman form the industry of getting rich, have brought number with ten million income to the herdsman.Because factors such as extensive management does not carry out special seed selection improvement, adds the loss of changes of environment and good variety population, and fertility of Tibetan mastiff itself and reproductive survival rate are low make Tibetan mastiff be in endangered condition.
Scholars have carried out further investigation to the Tibetan mastiff sperm freezing technology, with the improvement Tibetan mastiff, improve the reproduction rate of Tibetan mastiff through artificial insemination.Yet the Tibetan mastiff semen quality is a vital ring to Tibetan mastiff improvement and raising reproduction rate, discovers that a lot of inherences and external factor all can influence the quality of Tibetan mastiff seminal fluid, also has a lot of an open questions still need carry out comparatively deep research.It also is very difficult wanting to seek a purebred Tibetan mastiff.Secondly the Tibetan mastiff breeding is seasonal strong, and female large fierce dog for once oestruses every year, has restricted breeding and the cultivation of purebred Tibetan mastiff.And the sperm of Tibetan mastiff sperm and other canid is similar, in frozen-thaw process to the resistance of low temperature injury a little less than, sperm viability descends after the freeze thawing, pregnancy rate and the nest average number average of farrowing is lower than natural propagation.The key that influences the freezing success or not of dog sperm is the dilution prescription, the good and bad success or failure that directly influence Tibetan mastiff sperm freezing and artificial insemination of dilution quality.
Summary of the invention
The present invention provides a kind of freezing liquid that the Tibetan mastiff sperm is preserved that is used for, and has improved the percentage of head rice that freezes smart vigor, anabiosis rate, acrosome etc., thereby conception rate and nest farrowing mean are obviously improved, and reaches or approaching selfing level.
The technical scheme that the present invention adopts is:
Be used for the freezing liquid that the Tibetan mastiff sperm is preserved, the preparation method of this dilution is:
Each component and the weight of A liquid are: calcium chloride 0.22-0.58g, potassium chloride 0.11-0.45g, zinc chloride 0.25-0.75g, sodium chloride 0.9-1.2g, sodium dihydrogen phosphate 0.14-0.70g, sodium acetate 0.21-0.56g, astragalus polyose 0.015-0.045g, add distilled water and be settled to 500-1000mL and form;
Each component and the weight of B liquid are: get A liquid 35-55mL, little Tibetan mastiff serum 20-30mL, soybean oil 20-25mL, glycerine 5-10mL, Total Ginkgo Flavone-Glycoides 0.01-0.02g mix the freezing liquid that is used for the preservation of Tibetan mastiff sperm, and the pH value is 6.5-7.0.
Astragalus polyose, Total Ginkgo Flavone-Glycoides are from Tianjin spike company.
The method for extraction and purification of little Tibetan mastiff serum:
(1) adopts little Tibetan mastiff venous blood, isolate serum: under aseptic condition, adopt 6 months with interior little Tibetan mastiff venous blood, be put in 4 ℃ of refrigerator overnight; Natural separation goes out a part of serum, remainder under aseptic condition, 10000rpm; 4 ℃ centrifugal 25 minutes, it is subsequent use to isolate serum;
(2) ethanol removes albumen: according to ethanol and serum volume ratio is that 2: 1 amount adds 95% ethanol in serum, and stirring was left standstill 100 minutes, made protein denaturation precipitation, stayed supernatant to abandon deposition after centrifugal (6000 rev/mins, 10 minutes);
(3) remove ethanol: utilize the rotary evaporation in vacuo appearance, 37 ℃, 1 atmospheric pressure decompression removes ethanol;
(4) coarse filtration:, collect filtrating with the hollow fiber column coarse filtration of molecular cut off 100000D;
(5) enzymolysis: in filtrating, add compound protease, per 10 liters of serum add compound protease 9-10mg, and wherein cellulose protease and pepsic ratio are 400: 1;
(6) centrifugal: as under 4 ℃, 10000rpm condition centrifugal 30 minutes, to abandon deposition and stay supernatant;
(7) ultrafiltration:, collect filtrating with the hollow fiber column ultrafilter of molecular cut off 5000D;
(8) purifying: with the desalination of cross-link dextran G-50 gel chromatography (chromatographic solution is the Tris-Hcl buffer solution of pH 6.8), collect 280nm wavelength ultraviolet radiation absorption part, transferring pH is 7.6; Select DEAE-Sephadex-50 gel chromatography (ratio of glue bed height and diameter is 20: 1, and column diameter is selected 4.5cm for use) then for use, eluent is a Tris-Hcl buffer solution; The concentration that contains NaCl is 0.07mol/l, and 1.0 mol/l, pH are 7.9; Flow velocity is 3-5ml/min, uses the 280nm UV detection, discards 0.07mol/l NaCl wash-out part; Collect 1.0 mol/l NaCl wash-out parts, concentrating under reduced pressure is 1.07-1.09 to relative density;
(9) inactivation of virus: adopt miillpore filter filtration+short-wave ultraviolet light irradiation to carry out inactivation of virus; The miillpore filter that adopts is 0.025 micron; Use the short wave ultraviolet of wavelength as 254nm, intensity is 1000 μ w/cm
2, the direct irradiation time is 10 seconds; Obtain little Tibetan mastiff serum product.
The method for extraction and purification of little Tibetan mastiff serum: can prepare according to the method for the method for extraction and purification of calf serum, it is just passable only need raw material to be changed to little Tibetan mastiff venous blood by calf venous blood.
The present invention is used for the freezing liquid that the Tibetan mastiff sperm preserves has following purposes, and the one, be used for Tibetan mastiff seminal fluid low temperature and preserve, if remove cryoprotective agent such as glycerine, the normal temperature that then also can be used for the Tibetan mastiff seminal fluid is preserved.
Specific embodiment
Embodiment 1
The present invention is used for the freezing liquid that the Tibetan mastiff sperm is preserved:
Each component and the weight of A liquid are: calcium chloride 0.22g, potassium chloride 0.11g, zinc chloride 0.25g, sodium chloride 0.9g, sodium dihydrogen phosphate 0.14g, sodium acetate 0.21g, astragalus polyose 0.015g, add distilled water and be settled to 500mL and form;
Each component and the weight of B liquid are: get A liquid 35mL, little Tibetan mastiff serum 20mL, soybean oil 20mL, glycerine 5mL, Total Ginkgo Flavone-Glycoides 0.01g mix freezing liquid of the present invention, and the pH value is 6.5.
Embodiment 2
The present invention is used for the freezing liquid that the Tibetan mastiff sperm is preserved:
Each component and the weight of A liquid are: calcium chloride 0.58g, potassium chloride 0.45g, zinc chloride 0.75g, sodium chloride 1.2g, sodium dihydrogen phosphate 0.70g, sodium acetate 0.56g, astragalus polyose 0.045g, add distilled water and be settled to 1000mL and form;
Each component and the weight of B liquid are: get A liquid 55mL, little Tibetan mastiff serum 30mL, soybean oil 25mL, glycerine 10mL, Total Ginkgo Flavone-Glycoides 0.02g mix freezing liquid of the present invention, and the pH value is 7.0.
Embodiment 3
In January, 2009, the freezing liquid of use embodiment 2 Tibetan mastiff sperms is after the semen collection of large fierce dog base is supported in the mountain area, Lanzhou; Carry out the equality of temperature dilution in 1:1 ratio and Tibetan mastiff seminal fluid; Put into 0 ℃ incubation chamber, at once make aircraft to return Shanghai, the female Tibetan mastiff that oestruses in Shanghai is carried out the artificial insemination result of the test:
| Semen collection number (only) | Become pregnant and count (only) | Conception rate (%) | Nest farrowing mean (only) |
| 20 | 10 | 85 | 6 |
Obviously, the conception rate of dilution of the present invention and nest farrowing mean reaches the natural mating level.
Embodiment 4
In January, 2009, the freezing liquid of use embodiment 2 Tibetan mastiff sperms is after the semen collection of large fierce dog base is supported in the mountain area, Lanzhou; Carry out equality of temperature dilution in 1:1 ratio and Tibetan mastiff seminal fluid ,-5 ℃ preserved 11 months after, in December, 2009; Put into 0 ℃ incubation chamber; Deliver to Beijing, thaw, the female Tibetan mastiff that is oestrused in the Pekinese carries out the artificial insemination result of the test:
| Semen collection number (only) | Become pregnant and count (only) | Conception rate (%) | Nest farrowing mean (only) |
| 20 | 10 | 70 | 4 |
Obviously, after preserving 11 months, the conception rate of dilution of the present invention and nest farrowing mean are near the natural mating level.
Claims (3)
1. be used for the freezing liquid that the Tibetan mastiff sperm is preserved, it is characterized in that: the preparation method of this dilution is:
Each component and the weight of A liquid are: calcium chloride 0.22-0.58g, potassium chloride 0.11-0.45g, zinc chloride 0.25-0.75g, sodium chloride 0.9-1.2g, sodium dihydrogen phosphate 0.14-0.70g, sodium acetate 0.21-0.56g, astragalus polyose 0.015-0.045g, add distilled water and be settled to 500-1000mL and form;
Each component and the weight of B liquid are: get A liquid 35-55mL, little Tibetan mastiff serum 20-30mL, soybean oil 20-25mL, glycerine 5-10mL, Total Ginkgo Flavone-Glycoides 0.01-0.02g mix freezing liquid of the present invention, and the pH value is 6.5-7.0.
2. be used for the freezing liquid that the Tibetan mastiff sperm is preserved, it is characterized in that: the preparation method of this dilution is:
Each component and the weight of A liquid are: calcium chloride 0.22g, potassium chloride 0.11g, zinc chloride 0.25g, sodium chloride 0.9g, sodium dihydrogen phosphate 0.14g, sodium acetate 0.21g, astragalus polyose 0.015g, add distilled water and be settled to 500mL and form;
Each component and the weight of B liquid are: get A liquid 35mL, little Tibetan mastiff serum 20mL, soybean oil 20mL, glycerine 5mL, Total Ginkgo Flavone-Glycoides 0.01g mix freezing liquid of the present invention, and the pH value is 6.5.
3. be used for the freezing liquid that the Tibetan mastiff sperm is preserved, it is characterized in that: the preparation method of this dilution is:
Each component and the weight of A liquid are: calcium chloride 0.58g, potassium chloride 0.45g, zinc chloride 0.75g, sodium chloride 1.2g, sodium dihydrogen phosphate 0.70g, sodium acetate 0.56g, astragalus polyose 0.045g, add distilled water and be settled to 1000mL and form;
Each component and the weight of B liquid are: get A liquid 55mL, little Tibetan mastiff serum 30mL, soybean oil 25mL, glycerine 10mL, Total Ginkgo Flavone-Glycoides 0.02g mix freezing liquid of the present invention, and the pH value is 7.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103657168A CN102379279A (en) | 2011-11-17 | 2011-11-17 | Refrigerating fluid for storing Tibetan spaniel sperm |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103657168A CN102379279A (en) | 2011-11-17 | 2011-11-17 | Refrigerating fluid for storing Tibetan spaniel sperm |
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| CN102379279A true CN102379279A (en) | 2012-03-21 |
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| CN2011103657168A Pending CN102379279A (en) | 2011-11-17 | 2011-11-17 | Refrigerating fluid for storing Tibetan spaniel sperm |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107494520A (en) * | 2017-09-25 | 2017-12-22 | 江苏农牧科技职业学院 | A kind of dog freezing of semen diluent and freezing method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1305705A (en) * | 2001-01-08 | 2001-08-01 | 卢杲 | Method for permanently preserving all species genes and biological samples in the world |
| CN1351484A (en) * | 1999-04-28 | 2002-05-29 | 孙华燮 | Compositions for freezing dog sperm, method of freezing the dog sperm utilizing the compositions and artificial insemination method employing the frozen dog sperm |
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2011
- 2011-11-17 CN CN2011103657168A patent/CN102379279A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1351484A (en) * | 1999-04-28 | 2002-05-29 | 孙华燮 | Compositions for freezing dog sperm, method of freezing the dog sperm utilizing the compositions and artificial insemination method employing the frozen dog sperm |
| CN1305705A (en) * | 2001-01-08 | 2001-08-01 | 卢杲 | Method for permanently preserving all species genes and biological samples in the world |
Non-Patent Citations (1)
| Title |
|---|
| 刘景汉 等: "抗氧化剂对提高血液保存质量的作用", 《中国实验血液学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107494520A (en) * | 2017-09-25 | 2017-12-22 | 江苏农牧科技职业学院 | A kind of dog freezing of semen diluent and freezing method |
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Application publication date: 20120321 |