CN101310729A - Animal origin free flocks and herds frozen semen diluent and production method of flocks and herds frozen semen - Google Patents
Animal origin free flocks and herds frozen semen diluent and production method of flocks and herds frozen semen Download PDFInfo
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Abstract
A cattle and sheep non-animal-derived frozen semen diluent comprises at least one or the combination of the following ingredients: trehalose, propylene glycol, soybean lecithin, soluble soybean protein, nitrogen-trishydroxymethyl aminoethanesulfonic acid and dodecanoyl sodium sulfate. A production method of a cattle and sheet frozen semen by using the cattle and sheep non-animal-derived frozen semen diluent fully mixes the cattle or sheet semen and the dilutent, lowers the temperature and carries out the preservation. Compared with the prior art, the animal semen dilutent of the invention eliminates the animal-derived ingredients; the preservation method is more practical, the technical effects are better, thus thoroughly eliminating the potential risks of spreading various epidemic diseases between the animals and the people by the animal-derived ingredients in the dilutent. The cattle and sheep non-animal-derived frozen semen diluent can promote the popularization of animal artificial insemination technology to the utmost extent and improve the labor productivity and the economic benefits, thus having great economic and social values.
Description
Technical field:
The present invention relates to biology and chemical science, provide a kind of especially with the cattle and sheep semen diluent of animal origin-free composition preparation and the method for utilizing this kind diluent making ox and sheep freezing sperm.
Background technology:
In the prior art, the composition of the various semen diluents of animal composition is not also broken away from the development degree of leaving the animal origin composition fully, and the store method of semen diluent mainly also rests on the level of matching while using or freezing preservation.Under above-mentioned this state, certainly existing the animal origin composition that passes through in the diluent is propagated various eqpidemic diseases between the animals and human beings class potential risk.The store method of too simple single semen diluent has limited the popularization of animal technology of artificial insemination to a great extent, has increased the amount of labour of prepared and diluted liquid, has wasted semen diluent, has influenced the raising of labor productivity.
Therefore, people (for example: cattle and sheep) semen diluent and utilize this kind diluent (for example: the cattle and sheep) method of frozen semen to make animal expect to obtain a kind of animal with the preparation of animal origin-free composition.
Summary of the invention:
The objective of the invention is to from non-animal origin composition, filter out the semen diluent of active substance preparation animal, stop the potential danger that traditional diluent is propagated eqpidemic disease; And improved the store method of traditional semen diluent, prolonged the holding time of semen diluent, simplified the using method of semen diluent, filled up prior art blank in this regard.
The smart diluent of animal origin free flocks and herds frozen of the present invention is characterized in that: include one of following composition or its combination in the described diluent at least: trehalose, propylene glycol, soybean lecithin, soluble soy protein matter, nitrogen-trihydroxy methyl aminoethane sulphonic acid, lauroyl sodium sulfate.
In the preferred version, in described diluent, also include one of following composition or its combination: lactose, fructose, ethylene glycol, ethylenediaminetetraacetic acid, tri methylol amino methane;
Further preferred version: in described diluent, also include one of following composition or its combination: cholesterol, erythritol, vinegar chromic acid, Phosphatidylserine, soybean isoflavone;
In described diluent, also include one of the motility of sperm that is used to improve after thawing, the following composition that prolongs the sperm post-thaw survival time or its combination: Cloprostenol, vitamin B
12, caffeine, vitamin E sodium selenite injection, sucrose, phosphate buffer, nitrogen-trihydroxy methyl aminoethane sulphonic acid (TES), vitamin B1, V
E, PG, ethylenediaminetetraacetic acid (EDTA), tri methylol amino methane (Tris), phosphate buffer (the no calf serum BSA of PBS. composition), cysteine, mannitol, penicillin, streptomycin.
In the preferred version, the various compositions that comprise in the smart dilution of described animal origin free flocks and herds frozen and content thereof can be (the various material mass/volumes of the following stated are relative mass/volume ratio relation): fructose: sucrose: lactose: trehalose: ethylenediamine tetra-acetic acid (EDTA): tri methylol amino methane (Tris): nitrogen-trihydroxy methyl aminoethane sulphonic acid (TES): chromic acetate: cysteine=(0.5g～1.4g): (1.5g～5g): (2g～5g): (0.3g～1g): (1g～2.5g): (1.5g～4g): (0.001g～0.002g): (0.001g～0.01g): (0.01g～0.2g);
Phosphate buffer (PBS. does not have calf serum BSA composition): V
ESodium selenite injection solution: phosphate buffer: ethylene glycol: propylene glycol: red Ji sugar alcohol: mannitol: cholesterol=(1ml～3ml): (0.1ml～1ml): (1ml～6ml): (1ml～5ml): (1.5ml～7ml): (0.1ml～1.5ml): (0.1ml～1.5ml): (0.1ml～1ml).
It specifically can be (the various material mass/volumes of the following stated are relative mass/volume ratio relation) that the preparation of the smart diluent of animal origin free flocks and herds frozen of the present invention requires:
The first step takes by weighing fructose 0.85g, sucrose 3.10g, lactose 3.75g, trehalose 0.58g, ethylenediaminetetraacetic acid (EDTA) 1.80g, tri methylol amino methane (Tris) 2.45g, nitrogen-trihydroxy methyl aminoethane sulphonic acid (TES) 0.015g, chromium acetate 0.003g, phosphate buffer (the no calf serum BSA of PBS. composition) 2.0ml, phosphate buffer 3.0ml, cysteine 0.05g, ethylene glycol or propylene glycol or the combination of the two 6.0ml, red Ji sugar alcohol 0.50ml, mannitol 0.50ml, cholesterol 0.30ml altogether with electronic balance; After adding DDW 3.0ml above-mentioned substance is fully mixed, cooling immediately adds DDW again after the heated and boiled, makes the volume before original material keeps heated and boiled;
In second step, behind the abundant mixing of above-mentioned substance, in the mixed liquor that obtains by said process, add following material again: Phosphatidylserine 2.18g, soluble soy protein 2.00g, soybean lecithin 0.25g, V
B10.18g, V
B120.09g, caffeine 0.01g, Cloprostenol 0.013g, lauroyl sodium sulfate 0.50ml, penicillin 100,000 units, streptomycin 0.1g;
The 3rd step, above-mentioned substance is added the back fully mix, the lucifuge environment of putting into 5 ℃～10 ℃ then left standstill more than 20 hours, got its supernatant, discarding the insoluble composition of container bottom, is that 2 ℃～8 ℃, the condition lower seal of air-out, lucifuge are preserved in temperature.
The Main Ingredients and Appearance of the smart diluent of animal origin free flocks and herds frozen of the present invention is multiple medicines such as sugared source, buffer agent, alcohols cryoprotective agent, lecithin matter, soluble soy protein matter, vitamin B group, Cloprostenol, caffeine, nitrogen-trihydroxy methyl aminoethane sulphonic acid and antibiotics.Wherein necessary composition is: trehalose, lactose, fructose, propylene glycol, ethylene glycol, ethylenediaminetetraacetic acid, tri methylol amino methane, lauroyl sodium sulfate, soybean lecithin, soluble soy protein matter, nitrogen-compositions such as trihydroxy methyl methylamino ethane sulfonic acid
Be with former other domestic diluent institute difference, in diluent, used compositions such as trehalose, propylene glycol, cholesterol, erythritol, soybean lecithin, soluble soy protein matter, nitrogen-trihydroxy methyl aminoethane sulphonic acid, lauroyl sodium sulfate, vinegar chromic acid, Phosphatidylserine, soybean isoflavone first.
And Cloprostenol, vitamin B
12, microcomponent such as caffeine, vitamin E sodium selenite injection and sucrose, phosphate buffer use, played further the improved motility of sperm after thawing, the effect that has prolonged the time-to-live after sperm thaws.
The interpolation of compositions such as ethylene glycol, propylene glycol, cholesterol, soybean lecithin, Phosphatidylserine, soybean isoflavone has improved the freexing tolerance energy of sperm; reduced the destructiveness of sperm acrosome in refrigerating process; obviously alleviated with the toxic and side effects of glycerol to sperm, finally the conception rate of joining ewe is frozen in raising has played very positive effect.
The principal character of cattle and sheep straw frozen semen diluent is the composition that has replaced animal origins such as yolk in traditional diluent, milk, Mel, the cattle and sheep of being developed are frozen do not contained the mentioned kind material in the composition of smart diluent, but the result of use that is used to make straw frozen semen does not have significant difference with the result of use that original tradition is joined.
Because the stock solution of the straw frozen semen diluent of the animal origin-free of being invented is concentrated solution, osmotic pressure is higher, in addition because the concentration of antibiotics quasi drugs after dilution that contains in the concentrated solution is Mlc, and the concentration in stock solution is bacteriocidal concentration, thereby strong guarantee the smart diluent that freezes of the animal origin-free prepared do not have the source of disease microorganism and exist, under lower refrigerated storage temperature, can accomplish long preservation.
The manufacture method of ox and sheep freezing sperm of the present invention is fully mixed sperm and the diluent of cattle or sheep, lowers the temperature then and preserves; It is characterized in that: include one of following composition or its combination in the described diluent at least: trehalose, propylene glycol, soybean lecithin, soluble soy protein matter, nitrogen-trihydroxy methyl aminoethane sulphonic acid, lauroyl sodium sulfate.
In the preferred version, in described diluent, also include one of following composition or its combination:
Lactose, fructose, ethylene glycol, ethylenediaminetetraacetic acid, tri methylol amino methane; Cholesterol, erythritol, vinegar chromic acid, Phosphatidylserine, soybean isoflavone; Cloprostenol, vitamin B
12, caffeine, vitamin E sodium selenite injection, sucrose, phosphate buffer; Nitrogen-trihydroxy methyl aminoethane sulphonic acid (TES), vitamin B1, V
E, PG, ethylenediaminetetraacetic acid (EDTA), tri methylol amino methane (Tris), phosphate buffer (the no calf serum BSA of PBS. composition), cysteine, mannitol, penicillin, streptomycin.
The manufacturing process of animal frozen semen of the present invention is successively:
The first step, described diluent are with semen dilution and mix homogeneously, and extension rate is 1: 1～1: 20, and dilution is that the temperature range requirement of seminal fluid and diluent is 30 ℃～35 ℃;
Second step, will lower the temperature until 12～18 ℃ with the cooling rate of 1.5 ℃～2.0 ℃/min with the good seminal fluid of diluted; To dilute good seminal fluid then and be cooled to 8 ℃～10 ℃ with the speed of 0.5～1.5 ℃/min; To dilute again good seminal fluid since 8 ℃～10 ℃ speed with 0.1～1 ℃/min be cooled to 0 ℃～4 ℃ and under this temperature balance 0.5 hour～2 hours;
In the 3rd step, behind the freezing 3min～8min of cooling under-100 ℃ ± 5 ℃ the condition, the container that will hold the animal frozen semen then immerses the liquid nitrogen intermediate package or/and preserve.
Preferred version: in the manufacturing process of described animal frozen semen, lower the temperature after 12～18 ℃ with the cooling rate of 1.5 ℃～2.0 ℃/min to diluting good seminal fluid, putting it in the low temperature cabinet of prior modulated good temperature (this moment, the temperature of filling machine and any instrument that contact with seminal fluid all equated with the Current Temperatures that dilutes the seminal fluid of getting well) carries out isothermal fill, printing, and puts on the tubule frame; When will dilute good animal semen be cooled to 0 ℃～4 ℃ and under the isothermy after 0.5 hour～2 hours balances, straw semen after the balance is put into temperature buffer case isothermal with it, be transplanted on after the casing sealing lower the temperature in-100 ℃ ± 5 ℃ the slot type frigorimeter freezing;
In practical operation, to diluting good animal semen behind the freezing 3min～8min of cooling under-100 ℃ ± 5 ℃ the condition, animal semen after freezing taken out together with its packing sampling immerse liquid nitrogen 5 second～0.5 hour, again it is taken out from liquid nitrogen in the warm water that drops into 38 ℃～40 ℃ and thaw, just the animal semen after freezing is all immersed liquid nitrogen intermediate package and preservation together with its packing after the passed examination at 38 ℃ of following microscopies.
In the process of prepared and diluted liquid, used medicine is the analytical pure chemical reagent, and used dispensing instrument must and require no any disinfectant composition residual through thorough disinfection;
In terms of existing technologies, (for example: the composition composition of semen diluent cattle and sheep) has been broken away from the animal origin composition to animal of the present invention, and the store method of semen diluent has also broken away from the level of original matching while using or simple freezing preservation within a short period of time simultaneously.Thereby thoroughly eliminated the potential risk of between the animals and human beings class, propagating various eqpidemic diseases by the animal origin composition in the diluent.The store method of more practical, the better semen diluent of technique effect will promote the popularization of animal technology of artificial insemination to a great extent, reduces the amount of labour of prepared and diluted liquid, has reduced the waste of semen diluent, has improved labor productivity.It has huge economy and social value.
The specific embodiment:
Embodiment 1
1) configuration animal semen diluent:
Take by weighing fructose 0.85 gram with electronic balance, sucrose 3.10 grams, lactose 3.75 grams, trehalose 0.58 gram, ethylenediaminetetraacetic acid 1.80 grams, tri methylol amino methane 2.45 grams, nitrogen-trihydroxy methyl aminoethane sulphonic acid 0.015 gram, chromium acetate 0.003 gram, phosphate buffer (no calf serum composition) liquid 2.0ml, V
ESodium selenite injection solution 0.30ml, phosphate buffer 3.0ml cysteine 0.05 gram, ethylene glycol 2.50ml, propylene glycol 3.50ml, red Ji sugar alcohol 0.50ml, mannitol 0.50ml, cholesterol 0.30ml adds DDW 3.0ml.Above material fully mixes, cooling immediately after the heated and boiled, add DDW again, make the volume before original material keeps heated and boiled, firmly stir, use distilled water and the abundant mixing of original liquid, add following material again: Phosphatidylserine 2.18 grams, soluble soy protein 2.00 grams, soybean lecithin 0.25 gram, soybean isoflavone 0.10 gram, vitamin
B10.18 gram, vitamin
B120.09 gram, caffeine 0.05 gram, Cloprostenol 0.013 gram, SLS0.50ml, penicillin 100,000 units, streptomycin 0.1 gram.Right to use above-mentioned substance fully mixes in volumetric flask, dry powder sample drug substances is sticked on the inwall of container, add stirrer, fully stirred 1 hour with agitator, the lucifuge environment of putting into 5 ℃～10 ℃ then left standstill more than 20 hours, get its supernatant, with 5000 rev/mins rotating speed centrifugal 20 minutes, discard the insoluble composition of container bottom, put into 2 ℃～8 ℃ brown container, air-out, sealing is preserved.
In the process of prepared and diluted liquid, used medicine is the analytical pure chemical reagent, and used dispensing instrument must and require no any disinfectant composition residual through thorough disinfection;
2) seminal fluid is handled:
With the animal of just having gathered (for example: cattle and sheep) seminal fluid, measure ejaculate volume with syringe, by microscopy qualified after, use diluent isothermal (temperature is 30 ℃～35 ℃) to dilute immediately with it, extension rate is 1: 1～1: 20, and the reuse syringe is slowly taken out repeatedly and pushed away for several times until seminal fluid and diluent mix homogeneously behind the adding diluent.
The seminal fluid that dilution is good is lowered the temperature with 1.5 ℃～2.0 ℃/minute cooling rate, when the temperature of the good seminal fluid of dilution drops to 15 ℃, the low temperature cabinet (this moment, the temperature of filling machine and any instrument that contacts with seminal fluid was 15 ℃) of putting into prior modulated good temperature carries out fill, printing, and puts on the tubule frame.Adjust the low-temperature operation cabinet, make the seminal fluid in the cabinet be cooled to 8 ℃～10 ℃, be cooled to 0 from 8 ℃～10 ℃ speed again with 0.5/ clock with 1 ℃/minute speed, ℃～4 ℃ of balances 0.5 hour～2 hours.
3) freezing of semen:
Straw semen after the balance is put into temperature buffer case isothermal with it, after the casing sealing, anti-casing is transplanted in-100 ℃ ± 5 ℃ the slot type frigorimeter, take out the tubule frame of having piled up seminal fluid, putting into groove lowers the temperature freezing, rotation by the groove internal fan, make the interior each side of groove potential drop temperature more even, after freezing 3 minutes～8 minutes, straw frozen semen is taken out several doses immerse liquid nitrogen, from liquid nitrogen, take out in the warm water that drops into 38 ℃～40 ℃ again and thaw, after the passed examination remaining seminal fluid is all immersed liquid nitrogen intermediate package and preservation at 38 ℃ of following microscopies.
4) semen thawing:
Straw frozen semen is put into 38 ℃～40 ℃ warm water rapidly, just presented to the straw frozen semen surface and taken out immediately when transparent, under 25 ℃, carry out semen deposition.Vigor after straw frozen semen thaws reaches 54.88%, acrosomal integrity reaches 66.56%, the time that has the insemination ability after straw frozen semen thaws reached more than 18 hours, was experimental subject with Liaoning down producing goat, and the back semen deposition of thawing reaches more than 87% the non-return rate of ewe.
Embodiment 2:
1) configuration animal semen diluent:
Take by weighing fructose 0.85 gram with electronic balance, sucrose 3.10 grams, lactose 3.75 grams, trehalose 0.58 gram, ethylenediaminetetraacetic acid 1.80 grams, tri methylol amino methane 2.45 grams, nitrogen-trihydroxy methyl aminoethane sulphonic acid 0.015 gram, chromium acetate 0.003 gram, phosphate buffer (no calf serum composition) 2.0ml, phosphate buffer 3.0ml cysteine 0.05 gram, ethylene glycol 6.00ml, red Ji sugar alcohol 0.50ml, mannitol 0.50ml, cholesterol 0.30ml adds DDW 3.0ml.Above material fully mixes, cooling immediately after the heated and boiled, add DDW again, make the volume before original material keeps heated and boiled, firmly stir, use distilled water and the abundant mixing of original liquid, add following material again: Phosphatidylserine 2.18 grams, soluble soy protein 2.00 grams, soybean lecithin 0.25 gram, vitamin
B10.18 gram, vitamin
B120.09 gram, caffeine 0.01 gram,, penicillin 100,000 units, streptomycin 0.1 gram.Right to use above-mentioned substance fully mixes in volumetric flask, dry powder sample drug substances is sticked on the inwall of container, add stirrer, fully stirred 1 hour with agitator, the lucifuge environment of putting into 5 ℃～10 ℃ then left standstill more than 20 hours, get its supernatant, with 5000 rev/mins rotating speed centrifugal 20 minutes, discard the insoluble composition of container bottom, put into 2 ℃～8 ℃ brown container, air-out, sealing is preserved.
In the process of prepared and diluted liquid, used medicine is the analytical pure chemical reagent, and used dispensing instrument must and require no any disinfectant composition residual through thorough disinfection;
2) seminal fluid is handled:
With the seminal fluid of just having gathered, measure ejaculate volume with syringe, by microscopy qualified after, use diluent isothermal (temperature is 30 ℃～35 ℃) to dilute immediately with it, extension rate is 1: 1～1: 20, and the reuse syringe is slowly taken out repeatedly and pushed away for several times until seminal fluid and diluent mix homogeneously behind the adding diluent.
The seminal fluid that dilution is good is lowered the temperature with 1.5 ℃～2.0 ℃/minute cooling rate, when the temperature of the good seminal fluid of dilution drops to 15 ℃, the low temperature cabinet (this moment, the temperature of filling machine and any instrument that contacts with seminal fluid was 15 ℃) of putting into prior modulated good temperature carries out fill, printing, and puts on the tubule frame.Adjust the low-temperature operation cabinet, make the seminal fluid in the cabinet be cooled to 8 ℃～10 ℃, be cooled to 0 from 8 ℃～10 ℃ speed again with 0.5/ clock with 1 ℃/minute speed, ℃～4 ℃ of balances 0.5 hour～2 hours.
3) freezing of semen:
Straw semen after the balance is put into temperature buffer case isothermal with it, after the casing sealing, anti-casing is transplanted in-100 ℃ ± 5 ℃ the slot type frigorimeter, take out the tubule frame of having piled up seminal fluid, putting into groove lowers the temperature freezing, rotation by the groove internal fan, make the interior each side of groove potential drop temperature more even, after freezing 3 minutes～8 minutes, straw frozen semen is taken out several doses immerse liquid nitrogen, from liquid nitrogen, take out in the warm water that drops into 38 ℃～40 ℃ again and thaw, after the passed examination remaining seminal fluid is all immersed liquid nitrogen intermediate package and preservation at 38 ℃ of following microscopies.
4) semen thawing:
Straw frozen semen is put into 38 ℃～40 ℃ warm water rapidly, just presented to the straw frozen semen surface and taken out immediately when transparent, under 25 ℃, carry out semen deposition.
Vigor after straw frozen semen thaws reaches 48.95%, acrosomal integrity reaches 60.50%, the time that has the insemination ability after straw frozen semen thaws reached more than 11 hours, was experimental subject with Liaoning down producing goat, and the back semen deposition of thawing reaches more than 75% the non-return rate of ewe.
Claims (10)
1, the smart diluent of animal origin free flocks and herds frozen is characterized in that: include one of following composition or its combination in the described diluent at least: trehalose, propylene glycol, soybean lecithin, soluble soy protein matter, nitrogen-trihydroxy methyl aminoethane sulphonic acid, lauroyl sodium sulfate.
2, according to the smart diluent of the described animal origin free flocks and herds frozen of claim 1, it is characterized in that: in described diluent, also include one of following composition or its combination: lactose, fructose, ethylene glycol, ethylenediaminetetraacetic acid, tri methylol amino methane.
3, according to the smart diluent of the described animal origin free flocks and herds frozen of claim 2, it is characterized in that:
In described diluent, also include one of following composition or its combination: cholesterol, erythritol, vinegar chromic acid, Phosphatidylserine, soybean isoflavone.
4, according to the smart diluent of the described animal origin free flocks and herds frozen of claim 3, it is characterized in that:
In described diluent, also include one of the motility of sperm that is used to improve after thawing, the following composition that prolongs the sperm post-thaw survival time or its combination: Cloprostenol, vitamin B
12, caffeine, vitamin E sodium selenite injection, sucrose, phosphate buffer, nitrogen-trihydroxy methyl aminoethane sulphonic acid, vitamin B1, V
E, PG, ethylenediaminetetraacetic acid, tri methylol amino methane, cysteine, mannitol, penicillin, streptomycin.
5, according to the smart diluent of the described animal origin free flocks and herds frozen of one of claim 1～4, it is characterized in that: it includes at least a in the following composition, and various compositions and content thereof are specially:
Fructose: sucrose: lactose: trehalose: ethylenediaminetetraacetic acid: tri methylol amino methane: nitrogen-trihydroxy methyl aminoethane sulphonic acid: chromium acetate: cysteine=(0.5g～1.4g): (1.5g～5g): (2g～5g): (0.3g～1g): (1g～2.5g): (1.5g～4g): (0.001g～0.002g): (0.001g～0.01g): (0.01g～0.2g);
Phosphate buffer: V
ESodium selenite injection solution: phosphate buffer: ethylene glycol: propylene glycol: red Ji sugar alcohol: mannitol: cholesterol=(1ml～3ml): (0.1ml～1ml): (1ml～6ml): (1ml～5ml): (1.5ml～7ml): (0.1ml～1.5ml): (0.1ml～1.5ml): (0.1ml～1ml).
6, according to the smart diluent of the described animal origin free flocks and herds frozen of claim 5, it is characterized in that: the preparation of described diluent requires specifically:
The first step takes by weighing fructose 0.85g, sucrose 3.10g, lactose 3.75g, trehalose 0.58g, ethylenediaminetetraacetic acid 1.80g, tri methylol amino methane 2.45g, nitrogen-trihydroxy methyl aminoethane sulphonic acid 0.015g, chromium acetate 0.003g, phosphate buffer 2.0ml, phosphate buffer 3.0ml, cysteine 0.05g, ethylene glycol or propylene glycol or the combination of the two 6.0ml, red Ji sugar alcohol 0.50ml, mannitol 0.50ml, cholesterol 0.30ml altogether with electronic balance; After adding DDW 3.0ml above-mentioned substance is fully mixed, cooling immediately adds DDW again after the heated and boiled, makes the volume before original material keeps heated and boiled;
In second step, behind the abundant mixing of above-mentioned substance, in the mixed liquor that obtains by said process, add following material again: Phosphatidylserine 2.18g, soluble soy protein 2.00g, soybean lecithin 0.25g, V
B10.18g, V
B120.09g, caffeine 0.01g, penicillin 100,000 units, streptomycin 0.1g;
The 3rd step, above-mentioned substance is added the back fully mix, the lucifuge environment of putting into 5 ℃～10 ℃ then left standstill more than 20 hours, got its supernatant, discarding the insoluble composition of container bottom, is that 2 ℃～8 ℃, the condition lower seal of air-out, lucifuge are preserved in temperature.
7, the manufacture method of ox and sheep freezing sperm is fully mixed sperm and the diluent of cattle or sheep, lowers the temperature then and preserves; It is characterized in that: include one of following composition or its combination in the described diluent at least: trehalose, propylene glycol, soybean lecithin, soluble soy protein matter, nitrogen-trihydroxy methyl aminoethane sulphonic acid, lauroyl sodium sulfate.
8, according to the manufacture method of the described ox and sheep freezing sperm of claim 7, it is characterized in that:
In described diluent, also include one of following composition or its combination:
Lactose, fructose, ethylene glycol, ethylenediaminetetraacetic acid, tri methylol amino methane; Cholesterol, erythritol, vinegar chromic acid, Phosphatidylserine, soybean isoflavone; Cloprostenol, vitamin B
12, caffeine, vitamin E sodium selenite injection, sucrose, phosphate buffer; Nitrogen-trihydroxy methyl aminoethane sulphonic acid, vitamin B1, V
E, PG, ethylenediaminetetraacetic acid, tri methylol amino methane, cysteine, mannitol, penicillin, streptomycin.
9, according to the manufacture method of the described ox and sheep freezing sperm of claim 8, it is characterized in that: the manufacturing process of described animal frozen semen is successively:
The first step, described diluent are with semen dilution and mix homogeneously, and extension rate is 1: 1～1: 20, and dilution is that the temperature range requirement of seminal fluid and diluent is 30 ℃～35 ℃;
Second step, will lower the temperature until 12～18 ℃ with the cooling rate of 1.5 ℃～2.0 ℃/min with the good seminal fluid of diluted; To dilute good seminal fluid then and be cooled to 8 ℃～10 ℃ with the speed of 0.5～1.5 ℃/min; To dilute again good seminal fluid since 8 ℃～10 ℃ speed with 0.1～1 ℃/min be cooled to 0 ℃～4 ℃ and under this temperature balance 0.5 hour～2 hours;
In the 3rd step, behind the freezing 3min～8min of cooling under-100 ℃ ± 5 ℃ the condition, the container that will hold the animal frozen semen then immerses the liquid nitrogen intermediate package or/and preserve.
10, according to the manufacture method of the described ox and sheep freezing sperm of claim 9, it is characterized in that: in the manufacturing process of described animal frozen semen:
Lower the temperature after 12～18 ℃ with the cooling rate of 1.5 ℃～2.0 ℃/min to diluting good seminal fluid, put it in the low temperature cabinet of modulated good temperature in advance and carry out isothermal fill, printing, and put on the tubule frame;
When will dilute good animal semen be cooled to 0 ℃～4 ℃ and under the isothermy after 0.5 hour～2 hours balances, straw semen after the balance is put into temperature buffer case isothermal with it, be transplanted on after the casing sealing lower the temperature in-100 ℃ ± 5 ℃ the slot type frigorimeter freezing.
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| CN102812950A (en) * | 2012-09-07 | 2012-12-12 | 江苏省家禽科学研究所 | Chicken semen diluent as well as preparation method and application thereof |
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