CN101353643B - Tibetan mastiff sperm freezing diluent - Google Patents
Tibetan mastiff sperm freezing diluent Download PDFInfo
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- CN101353643B CN101353643B CN2008101609863A CN200810160986A CN101353643B CN 101353643 B CN101353643 B CN 101353643B CN 2008101609863 A CN2008101609863 A CN 2008101609863A CN 200810160986 A CN200810160986 A CN 200810160986A CN 101353643 B CN101353643 B CN 101353643B
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Abstract
The invention relates to a cryogenic diluent of the semen of Tibetan mastiffs, which belongs to the technical field of animal reproduction and comprises 1 fluid and 2 fluid; wherein, the 1 fluid is prepared by the following steps: the raw materials comprise 0.20 to 0.50g of calcium chloride, 0.18 to 0.36g of potassium chloride, 0.22 to 0.45g of magnesium chloride, 3.5 to 5.85g of sodium chloride,0.30 to 0.55g of monosodium phosphate, 1.38 to 3.20g of sodium bicarbonate and 0.025 to 0.036g of sodium pyruvate, and double distilled water is added to obtain constant volume of 1L; the 2 fluid is prepared by the following steps: 35 to 55ml of the 1 fluid, 20 to 30ml of calf serum, 20 to 25ml of yolk, 5 to 10ml of glycerin and 1000IU/ml of penicillin are mixed to prepare to the diluent with thepenetrating pressure of 300 to 330mosmo/l and the pH value of 6.5 to 7.0; the diluent leads the activity of the cryogenic diluent of the semen of Tibetan mastiffs to reach 0.6 to 0.7, and the conception rate and the average number of litter size to respectively reach 77.3 percent to 84 percent and four to five so as to reach or near the selfing level, thus being a cryogenic diluent of the semen of Tibetan mastiff with practical application and promotion value.
Description
Technical field
The present invention relates to a kind of Tibetan mastiff sperm freezing diluent, be used for the freezing and artificial insemination of Tibetan mastiff seminal fluid, belong to the animal reproduction technical field.
Background technology
The semen freezing of dog has very high using value, uses the semen freezing of dog to have many superiority with technology of artificial insemination than natural propagation.As: can highly bring into play the utilization ratio of outstanding kind of dog; Reduce the propagation of the propagation, particularly reproductive system disease of various diseases; Not limited by space-time, introduce a fine variety convenient and swift, and cost poor benefit height; Germ plasm resource can effectively be preserved, and particularly kind protection and the development to China Tibetan mastiff has important practical significance.
At present, China studies less aspect Tibetan mastiff sperm freezing and artificial insemination, and does not see than more comprehensive document announcement in proliferation.Tibetan mastiff belongs to species of Qinghai-Tibet Platean, and the purebred Tibetan mastiff of legend is distributed in Gansu, Qinghai, first gulf, the Yellow River, boundary, Sichuan Province--the meander of China.The meander Tibetan mastiff is considered to true ancestor Tibetan mastiff product group, enjoys " the refreshing dog in east " good reputation.Tibetan mastiff is subjected to 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 are subjected to 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.But, because the herdsman protects consciousness weak to outstanding purebred Tibetan mastiff blood lineage, arbitrarily dealing, original Tibetan mastiff and local shepherd dog blood lineage mix endogamy etc., cause outstanding purebred Tibetan mastiff fewer and feweri, the crisis that this rare kind of dog is endangered.Hundred of the purebred Tibetan mastiff less thaies of China, even nowadays in the country of origin of Qinghai-Tibet Platean, it also is very difficult wanting to seek a purebred Tibetan mastiff according to statistics.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 Canis animals is similar, in frozen-thaw process to the resistance of low temperature injury a little less than, motility of sperm descends after the freeze thawing, conceptual quotient and the nest average number average of farrowing is lower than natural propagation.Conception rate during Tibetan mastiff natural propagation and nest farrowing mean number are lower than other kind of dog, are respectively 80%～85%, 5～7 approximately.Therefore, the how long-term purebred Tibetan mastiff seminal fluid of cryopreservation high-quality and to make full use of fine provenance be the extremely urgent problem that solves of needing in this area.
One of key factor that influences the freezing success or not of dog sperm is the diluent prescription, the good and bad success or failure that directly influence Tibetan mastiff sperm freezing and artificial insemination of diluent quality.Carry out both at home and abroad at present that most widely used diluent has two classes in the dog frozen semen research: a class is yolk-lactose diluent, and its main component is yolk, lactose, glycerine and antibiotics; Another kind of is yolk-tris diluent, and its main component is tris (trishydroxymethyl), citric acid, fructose, glycerine and yolk, and the antibiotic of use mostly is penicillin, Streptomycin sulphate and gentamicin.In two kinds of diluents of experiment confirm such as Pan Shou literary composition and Yu Xueli, yolk-tris diluent is better than yolk-lactose diluent.(Pan Shouwen etc., herding and animal doctor, 1986; (3): 105-106; Yu Xueli etc., Luoyang agricultural higher junior college journal 1999; (2); 20～23; University Of Science and Technology Of He'nan's journal, 2004; (2); 32-36).But just research level is compared with natural propagation at present, and also there are certain gap in conception rate and nest farrowing mean number.
Summary of the invention
In view of the deficiencies in the prior art, the invention provides a kind of Tibetan mastiff sperm freezing diluent, this diluent has further reduced the infringement that the Tibetan mastiff sperm was caused by cold stimulation in refrigerating process, improved the percentage of head rice that freezes smart vigor, anabiosis rate, acrosome etc., thereby make conception rate and nest the farrowing mean number obviously improve, reach or near the selfing level.
For achieving the above object, the technical solution used in the present invention is: a kind of Tibetan mastiff sperm freezing diluent, this diluent is based on 1 liquid, get 1 liquid, 35～55mL, join calf serum 20～30mL, yolk 20～25mL, glycerine 5～10mL, penicillin 1000IU/mL mix, and the osmotic pressure of this diluent is 300～330mosmo/L, and the pH value is 6.5～7.0; Wherein each component of 1 liquid and weight are: calcium chloride 0.20～0.50g, Repone K 0.18～0.36g, magnesium chloride 0.22～0.45g, sodium-chlor 3.5～5.85g, SODIUM PHOSPHATE, MONOBASIC 0.30～0.55g, sodium bicarbonate 1.38～3.20g, Sodium.alpha.-ketopropionate 0.025～0.036g, add distilled water and be settled to 1L and form.
The present invention preferably fills a prescription: a kind of Tibetan mastiff sperm freezing diluent, this diluent is got 1 liquid 50mL based on 1 liquid, joins calf serum 25mL, yolk 20mL, glycerine 5mL, penicillin 1000IU/mL mix, and the osmotic pressure of this diluent is 325mosmo/L, and the pH value is 6.8; Wherein each component of 1 liquid and weight are: calcium chloride 0.30g, Repone K 0.25 g, magnesium chloride 0.32 g, sodium-chlor 5.85 g, SODIUM PHOSPHATE, MONOBASIC 0.45g, sodium bicarbonate 2.38g, Sodium.alpha.-ketopropionate 0.025g, add distilled water and be settled to 1L and form.For the survival of Tibetan mastiff sperm provides the optimum substrate condition.
Tibetan mastiff sperm freezing diluent of the present invention has following purposes, and the one, be used for Tibetan mastiff seminal fluid cryopreservation, another purposes is to be used for Tibetan mastiff sperm freezing to 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.
The present invention is according to the physiological properties such as absorption pattern of Tibetan mastiff spermoblast nutritional need, osmotic pressure, pH value and energy matter, with the nutritive substance sugar in the diluent prescription of traditional dog, as: glucose, sucrose, fructose, lactose, raffinose etc. substitute with calf serum, source as the spermoblast energy metabolism, and other ionogen have been done bigger adjustment, to keep the osmotic pressure balance of sperm internal and external environment.In addition, this diluent is made buffer reagent with SODIUM PHOSPHATE, MONOBASIC and sodium bicarbonate, also adds sterilant such as penicillin simultaneously, protective materials such as glycerine, the cryoprotection effect of raising diluent.The osmotic pressure that makes this diluent is 300～330mosmo/L, the pH value is 6.5～7.0, for the survival of Tibetan mastiff sperm provides the optimum substrate condition, further reduced the infringement that the Tibetan mastiff sperm was caused by cold stimulation in refrigerating process, thereby make the smart vigor that freezes, anabiosis rate, the percentage of head rice of acrosome, indexs such as the conception rate of frozen sperm and nest farrowing mean number reach 0.65 ± 0.05 respectively, 82.3 ± 5.55,51.2 ± 1.23,84% and 5, compare 0.30 ± 0.03 of traditional yolk-lactose diluent and yolk-Tris diluent, 3 8.40 ± 4.56,29.4 ± 1.29,22% and 2 only reach 0.47 ± 0.04,59.58 ± 4.63,31.9 ± 1.60,57.1% and 3 (Yu Xueli etc., Luoyang agricultural higher junior college journal, 1999; (2): 20-23) all increase significantly, and in experiment in recent years, every index tends towards stability more, as following table.
2007-2008 two annual Tibetan mastiff frozen semen and artificial insemination test-results
| Year | Freeze smart vigor | Semen deposition number (only) | Become pregnant and count (only) | Conception rate (%) | Nest farrowing mean number (only) |
| 07 year | 0.6±0.03 | 22 | 17 | 77.3 | 4 |
| 08 year | 0.65±0.05 | 25 | 21 | 84 | 5 |
Obviously, diluent of the present invention makes the vigor of Tibetan mastiff frozen semen can reach 0.6～0.7, conception rate and nest farrowing mean number can reach 77.3%～84% and 4～5 respectively, reach or near the selfing level, so the present invention is a kind of Tibetan mastiff sperm freezing diluent with practical application and promotional value.
Embodiment
Chemical reagent used among the present invention is all selected analytical pure.
Embodiment 1.A kind of Tibetan mastiff sperm freezing diluent, this diluent are based on 1 liquid, under aseptic condition, get 1 liquid 50mL, join calf serum 25mL, yolk 20mL, glycerine 5mL, penicillin 1000IU/mL mix, and the osmotic pressure of this diluent is 325mosmo/L, the pH value is 6.8; Wherein each component of 1 liquid and weight are: calcium chloride 0.30g, Repone K 0.25g, magnesium chloride 0.32g, sodium-chlor 5.85g, SODIUM PHOSPHATE, MONOBASIC 0.45g, sodium bicarbonate 2.8g, Sodium.alpha.-ketopropionate 0.025g, add distilled water and be settled to 1L and form, and heating for dissolving, with this area ordinary method filter-sterilized, reserved filtrate is 1 liquid.Described diluent is splendid to the Tibetan mastiff sperm freezing preservation effect.During use, carry out the equality of temperature dilution in 1: 2 ratio and Tibetan mastiff seminal fluid.
Embodiment 2.A kind of Tibetan mastiff sperm freezing diluent, this diluent are based on 1 liquid, under aseptic condition, get 1 liquid 35mL, join calf serum 30mL, yolk 25mL, glycerine 10mL, penicillin 1000IU/mL mix, and the osmotic pressure of this diluent is 310mosmo/L, the pH value is 6.5; Wherein each component of 1 liquid and weight are: calcium chloride 0.20g, Repone K 0.18g, magnesium chloride 0.22g, sodium-chlor 5.0g, SODIUM PHOSPHATE, MONOBASIC 0.3g, sodium bicarbonate 1.38g, Sodium.alpha.-ketopropionate 0.03g, add distilled water and be settled to 1L and form, and heating for dissolving, with this area ordinary method filter-sterilized, reserved filtrate is 1 liquid.Described diluent is splendid to the Tibetan mastiff sperm freezing preservation effect.During use, carry out the equality of temperature dilution in 1: 2 ratio and Tibetan mastiff seminal fluid.
Embodiment 3.A kind of Tibetan mastiff sperm freezing diluent, this diluent are based on 1 liquid, under aseptic condition, get 1 liquid 55mL, join calf serum 20mL, yolk 20mL, glycerine 5mL, penicillin 1000IU/mL mix, and the osmotic pressure of this diluent is 330mosmo/L, the pH value is 7; Wherein each component of 1 liquid and weight are: calcium chloride 0.50g, Repone K 0.36g, magnesium chloride 0.45g, sodium-chlor 3.5g, SODIUM PHOSPHATE, MONOBASIC 0.55g, sodium bicarbonate 3.2g, Sodium.alpha.-ketopropionate 0.036g, add distilled water and be settled to 1L and form, and heating for dissolving, with this area ordinary method filter-sterilized, reserved filtrate is 1 liquid.Described diluent is splendid to the Tibetan mastiff sperm freezing preservation effect.During use, carry out the equality of temperature dilution in 1: 2 ratio and Tibetan mastiff seminal fluid.
Claims (2)
1. Tibetan mastiff sperm freezing diluent, it is characterized in that: this diluent is based on 1 liquid, get 1 liquid, 35～55mL, join calf serum 20～30mL, yolk 20～25mL, glycerine 5～10mL, penicillin 1000IU/mL mix, and the osmotic pressure of this diluent is 300～330mosmo/L, and the pH value is 6.5～7.0; Wherein each component of 1 liquid and weight are: calcium chloride 0.20～0.50g, Repone K 0.18～0.36g, magnesium chloride 0.22～0.45g, sodium-chlor 3.5～5.85g, SODIUM PHOSPHATE, MONOBASIC 0.30～0.55g, sodium bicarbonate 1.38～3.20g, Sodium.alpha.-ketopropionate 0.025～0.036g, add distilled water and be settled to 1L and form.
2. Tibetan mastiff sperm freezing diluent according to claim 1, it is characterized in that: this diluent is based on 1 liquid, get 1 liquid 50mL, join calf serum 25mL, yolk 20mL, glycerine 5mL, penicillin 1000IU/mL mix, and the osmotic pressure of this diluent is 325mosmo/L, and the pH value is 6.8; Wherein each component of 1 liquid and weight are: calcium chloride 0.30g, Repone K 0.25g, magnesium chloride 0.32g, sodium-chlor 5.85g, SODIUM PHOSPHATE, MONOBASIC 0.45g, sodium bicarbonate 2.38g, Sodium.alpha.-ketopropionate 0.025g, add distilled water and be settled to 1L and form.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101609863A CN101353643B (en) | 2008-09-17 | 2008-09-17 | Tibetan mastiff sperm freezing diluent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101609863A CN101353643B (en) | 2008-09-17 | 2008-09-17 | Tibetan mastiff sperm freezing diluent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101353643A CN101353643A (en) | 2009-01-28 |
| CN101353643B true CN101353643B (en) | 2010-08-11 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN2008101609863A Expired - Fee Related CN101353643B (en) | 2008-09-17 | 2008-09-17 | Tibetan mastiff sperm freezing diluent |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109644983A (en) * | 2018-12-17 | 2019-04-19 | 浙江万里学院 | Boar spermatozoa low temperature dilution based on Shelled Turtle Trionyx Sinensis hibernation serum |
| CN115362961B (en) * | 2022-04-14 | 2023-12-08 | 中国水产科学研究院北戴河中心实验站 | Artificial insemination method of Songjiang perch and application thereof |
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Granted publication date: 20100811 Termination date: 20120917 |