CN101418282A - Pig bagged freezing semen preservation liquor and preservation method thereof - Google Patents
Pig bagged freezing semen preservation liquor and preservation method thereof Download PDFInfo
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Abstract
The invention discloses a liquid for preserving bagged frozen pig semen and a method for preserving the same. The preservation liquid consists of liquid A and liquid B, wherein the liquid A contains 1 to 10 grams of glucose, 1 to 10 grams of lactose, 1 to 8 grams of sodium glutamate, 1 to 8 grams of polyvinylpyrrolidone, 100 milliliters of distilled water, 100 to 200 thousand of international units of antibiotic, 0.02 to 0.1 gram of vitamin B12, 0.02 to 0.1 gram of caffeine sodium benzoate, and 10 to 30 milliliters of egg yolk; and the liquid B contains 50 to 100 milliliters of the liquid A, 2 to 10 milliliters of glycerol, and 2 to 10 milliliters of N-N-dimethylformamide. The method for preserving the semen comprises the following steps: concentrating the semen after sampling the sperm, and diluting and refrigerating the liquid A with the concentrated semen proportionally and isothermally; diluting the mixture by using the liquid B for the second time, and subpackaging the semen into bags; and preserving the bagged semen in liquid nitrogen. The method is effective in maintaining the vitality of the sperms, has simple and convenient use and low cost, and can meet the needs of artificial insemination of the frozen pig semen.
Description
One, technical field:
The present invention relates to the animal reproduction field, relate to the store method that a kind of seminal fluid is preserved liquid and seminal fluid, particularly relate to the store method of a kind of pig bagged freezing semen preservation liquor and seminal fluid.
Two, technical background:
Along with the development need of pig industry, the artificial insemination of pig normal temperature preservation seminal fluid can not have been satisfied the development of modern pig industry, and the frozen semen artificial insemination technology of pig has become problem demanding prompt solution in the domestic and international Swine Production.
The pig frozen semen is preserved with the normal temperature seminal fluid and is compared, frozen semen can make excellent genes obtain indefinite preservation (as the conservation of resources of local pig kind), prolong boar working life, be not subjected to this boar death, damage, semen collection interval or breed sterile restriction, help using artificially the herd boar of high performance level and the insemination of sows of plurality, obtain bigger genetic progress; Make boar select not to be subjected to the restriction of time or geographical position difference, can introduce external high-performance boar and freeze the smart live body boar that replaces, can save the foreign exchange expenditure; Help the perfect of artificial insemination system, the boar frozen semen can begin a large amount of supplies fully after progeny testing or disease detection, for stable genetic progress and strict Biosafety system construction provide Additional Protection.Consider that from health perspectives freezing essence provides time enough for disease detection, freeze essence and also can when outbreak of disease, utilize large-scale artificial insemination station that stable seminal fluid supply guarantee is provided.
At present, the boar semen method is mainly contained particle frozen semen, tubule frozen semen and three kinds of methods of ampoule frozen semen.The granular size of particle frozen semen generally about 0.1ml, when particle is excessive nexine and outer seminal fluid be subjected to temperature too inhomogeneous, effect is relatively poor; If it is particle is too small, then bothersome also not too convenient again when freezing.The tubule type of tubule frozen semen has two kinds on 0.25ml type and 0.5ml type, and as satisfying the semen deposition dosage of 1 sow, the tubule of 0.5ml is wanted more than 10 at least, otherwise, can't satisfy the total amount requirement of sperm; There are problems such as manufacture craft is numerous and diverse, cost is higher, ampoule easily breaks in the method for ampoule frozen semen.
The patent No. is the preservation liquid that the file of 200410013364.X discloses the pig seminal fluid under a kind of normal temperature, and this preservation liquid raw material is compositions such as glucose, bovine serum albumin, Repone K, Trisodium Citrate, ethylenediamine tetraacetic acid (EDTA), sodium bicarbonate, microbiotic and water.This invention to a certain extent can high outstanding herd boar the seminal fluid utilization ratio, but the shelf time of seminal fluid is shorter, generally has only about 7 days, can not prolonged preservation.
Three, summary of the invention:
The technical problem to be solved in the present invention: the store method of a kind of pig bagged freezing semen preservation liquor and seminal fluid is provided, and this method can keep the vigor of sperm, simple, the convenience of use effectively, and is with low cost, can satisfy the needs of pig frozen semen artificial insemination.
Technical scheme of the present invention is:
Pig bagged freezing semen preservation liquor, described preservation liquid is made up of A liquid and B liquid, A liquid preparation: take by weighing glucose 1～10 gram, lactose 1～10 gram, Sodium Glutamate 1～8 gram, polyvinylpyrrolidone 1～8 gram, add successively in 100 ml distilled waters, it is dissolved fully, add microbiotic 10～20 million international units again, filter, add vitamins B then with biofilter
120.02 10～30 milliliters of～0.1 grams, caffeine and sodium benzoate 0.02～0.1 gram, yolk fully obtain A liquid behind the mixing; B liquid preparation: get 50～100 milliliters of A liquid, add 2～10 milliliters of glycerine, 2～10 milliliters of N-N-dimethyl formamides, fully obtain B liquid behind the mixing.
Described A liquid composition is: glucose 1～3 gram, lactose 1～3 gram, Sodium Glutamate 1～2 gram, polyvinylpyrrolidone 1～2 gram, microbiotic 10～15 million international units, vitamins B
120.02 10～20 milliliters of～0.04 grams, caffeine and sodium benzoate 0.02～0.04 gram, yolk; Described B liquid composition is: 75～95 milliliters of A liquid, 2～6 milliliters of glycerine, 2～6 milliliters of N-N-dimethyl formamides.
Described A liquid composition is: glucose 3～7 grams, lactose 1～3 gram, Sodium Glutamate 2～6 grams, polyvinylpyrrolidone 2～5 grams, microbiotic 15～20 million international units, vitamins B
120.04 10～20 milliliters of～0.08 grams, caffeine and sodium benzoate 0.04～0.08 gram, yolk; Described B liquid composition is: 70～100 milliliters of A liquid, 4～8 milliliters of glycerine, 4～8 milliliters of N-N-dimethyl formamides.
Described microbiotic is gentamicin sulphate, penicillin or Streptomycin sulphate.
The pH value of described A liquid and B liquid is 6.5～7.5, osmotic pressure is 300～500mmol/L.
A kind of pig bagged freezing semen preservation liquor is preserved the method for seminal fluid, and this method may further comprise the steps:
(1) semen collection and concentrated: boar is carried out semen collection, seminal fluid centrifugation, collect lower floor's liquid, control sperm concentration more than 1,000,000,000/ml, preservation, standby;
(2) cooling: the ratio in A liquid and concentrated seminal fluid is the ratio of 1:0.5～2, and with the dilution of seminal fluid isothermal, refrigeration was cooled to 1～4 ℃ through 1～2 hour then under 18～35 ℃;
(3) balance: cryopreserved semen is carried out the dilution second time with B liquid, the proportioning 1:0.5 of B liquid and cryopreserved semen～2, dilution back seminal fluid was preserved 1～2 hour down at 1～4 ℃;
(4) packing: seminal fluid is distributed into bag;
(5) freezing: that packed seminal fluid is preserved in liquid nitrogen.
Described seminal fluid be distributed into the bag be with seminal fluid with sterile bag by the packing of every bag of 5ml seminal fluid.
The described preservation in liquid nitrogen is that packed seminal fluid was fumigated 5～10 minutes at distance liquid nitrogen surface 2～5cm place earlier, drops into freezing preservation in the liquid nitrogen then.
Positive beneficial effect of the present invention:
(1) in the pig bagged freezing semen preservation liquor prescription of the present invention, glucose and lactose are mainly sperm nutrition and energy are provided, and have protection sperm, cryophylactic effect; Sodium Glutamate is mainly seminal fluid shock absorption is provided; Yolk and polyvinylpyrrolidone are mainly by the effect of anti-cryopreserving sperm; Vitamins B
12With caffeine and sodium benzoate the minimizing rate of teratosperm is arranged, improve the function of motility of sperm; Microbiotic such as gentamicin sulphate mainly are the effects of sterilization, bacteria growing inhibiting, and each composition can produce good synergism after adopting suitable mixed, helps the preservation of pig seminal fluid.
(2) method of the present invention adopt the 5ml aseptic plastic bag to the pig seminal fluid carry out packing, freezing, thaw and operation such as semen deposition, have the advantages that every bag of pig seminal fluid can satisfy 1 sow semen deposition needs, entire operation is simple, convenient, avoided secondary pollution, with low cost, can satisfy the needs of pig frozen semen artificial insemination.
(3) pig bagged freezing semen of utilization the inventive method preservation shows, the seminal fluid that this method is preserved can just be usually used in the artificial insemination of pig, and the pig seminal fluid artificial insemination effect that the pregnancy rate of pig, average nest farrowing number average and normal temperature are preserved is suitable, can normally use.
(4) pig bagged freezing semen of the inventive method preservation can significantly improve the seminal fluid utilization ratio of outstanding herd boar in the medium-term and long-term preservation of liquid nitrogen, reduces production costs, and can obtain bigger genetic progress, helps the perfect of swine artificial insemination system.
Four, embodiment:
Embodiment one:
(1) preparation of pig bagged freezing semen preservation liquor
A liquid preparation: take by weighing glucose 10 grams, lactose 1 gram, Sodium Glutamate 8 grams, polyvinylpyrrolidone 8 grams, be dissolved in 100 ml distilled waters, adding is dissolved it fully successively again, adds gentamicin sulphate 10 million international units again, filter with biofilter, add vitamins B then
120.02 10 milliliters of grams, caffeine and sodium benzoate 0.02 gram, yolk are A liquid behind the mixing fully.
B liquid preparation: get 50 milliliters of A liquid, add 2 milliliters of glycerine, 2 milliliters of N-N-dimethyl formamides, fully obtain B liquid behind the mixing.
Require above-mentioned agents useful for same to be analytical pure, the pH value of A liquid and B liquid is 6.5～7.5, osmotic pressure is 300～500mmol/L, and acid, alkali are all had certain surge capability.
(2) utilize the method for the preservation pig seminal fluid of above-mentioned freezing semen preservation liquor, may further comprise the steps:
1. semen collection and seminal fluid concentrate: adopt hand grip that 8 boars are carried out semen collection, the seminal fluid gathered with centrifugal 10 minutes of 800 rev/mins in whizzer, is collected the sperm part of lower floor after the layering, sperm concentration be controlled at 1,000,000,000/more than the ml.Seminal fluid is carried out the quality inspection, guarantee motility of sperm more than 0.6, abnormal rate is preserved, standby in 20%;
2. cooling: with spissated seminal fluid, the ratio that in A liquid and concentrated seminal fluid is 1:0.5 can slowly join A liquid in the seminal fluid 20 ℃ of following isothermals dilutions, is cooled to 1～2 ℃ at cold compartment of refrigerator through 1～2 hour then.
3. balance: will be cooled to 1～4 ℃ seminal fluid, be that the ratio of 1:0.5 is carried out the dilution second time under 1～2 ℃ in B liquid and concentrated seminal fluid, B liquid slowly can be joined in the seminal fluid, deposited 1～2 hour at 1～2 ℃.
4. packing: by every bag of 5ml packing, promptly the consumption branch by every pig 5ml installs the seminal fluid that balance is good with aseptic plastic bag.
5. freezing: the seminal fluid that branch is installed was fumigated 5 minutes at distance liquid nitrogen surface 2cm place earlier, directly put in the liquid nitrogen freezingly then, and itself and liquid nitrogen temperature are consistent (promptly-196 ℃), preserved in liquid nitrogen then always.
6. semen quality inspection after thawing and thawing: before using the refrigerated seminal fluid is put in 38～40 ℃ of warm water, thawed for 10～20 seconds; Carry out the semen quality inspection then, motility of sperm more than 0.4, abnormal rate below 20%, acrosomal integrity be qualified more than 60%.
Embodiment two:
(1) preparation of pig bagged freezing semen preservation liquor is with embodiment one difference:
A liquid composition: glucose 1 gram, lactose 1 gram, Sodium Glutamate 1 gram, polyvinylpyrrolidone 1 gram, 100 milliliters of distilled water, gentamicin sulphate 20 million international units, vitamins B
120.02 10 milliliters of grams, caffeine and sodium benzoate 0.02 gram, yolk.
B liquid composition: 60 milliliters of A liquid, 2 milliliters of glycerine, 2 milliliters of N-N-dimethyl formamides.
(2) utilize the method for the preservation pig seminal fluid of above-mentioned freezing semen preservation liquor, may further comprise the steps:
1. semen collection and seminal fluid is concentrated: with embodiment one.
2. lower the temperature: the ratio that in A liquid and concentrated seminal fluid is 1:1 is diluted at 25 ℃ of isothermals, is cooled to 4 ℃ at refrigerator through 1 hour then.
3. balance: will be cooled to 4 ℃ seminal fluid, and 4 ℃ of dilutions for the second time down, under 4 ℃, deposit 1 hour then in the ratio of B liquid and seminal fluid 1:1.
Other packing, freezing, the quality inspection of seminal fluid afterwards of thawing and thaw: with embodiment one.
Embodiment three:
(1) preparation of pig bagged freezing semen preservation liquor is with embodiment one, and difference is:
A liquid composition: glucose 3 grams, lactose 10 grams, Sodium Glutamate 5 grams, polyvinylpyrrolidone 3 grams, 100 milliliters of distilled water, gentamicin sulphate 15 million international units, vitamins B
120.03 30 milliliters of grams, caffeine and sodium benzoate 0.04 gram, yolk.
B liquid composition: 75 milliliters of A liquid, 5 milliliters of glycerine, 4 milliliters of N-N-dimethyl formamides.
(2) utilize the store method of pig bagged freezing semen preservation liquor, may further comprise the steps:
1. semen collection and seminal fluid is concentrated: with embodiment one.
2. cooling: the ratio in A liquid and concentrated seminal fluid 1:1.5 is diluted at 20 ℃ of isothermals, is cooled to 1 ℃ at refrigerator through 2 hours then.
3. balance: will be cooled to 1 ℃ seminal fluid, and under 1 ℃, carry out the dilution second time, and deposit 2 hours at 1 ℃ then in the ratio of B liquid and concentrated seminal fluid 1:1.5.
Other packing, freezing, the quality inspection of seminal fluid afterwards of thawing and thaw: with embodiment one.
Embodiment four:
(1) preparation of pig bagged freezing semen preservation liquor is with embodiment one, and difference is:
A liquid composition: glucose 1.5 grams, lactose 2 grams, Sodium Glutamate 1.5 grams, polyvinylpyrrolidone 1 gram, 100 milliliters of distilled water, penicillin 15 million international units, vitamins B
120.03 15 milliliters of grams, caffeine and sodium benzoate 0.04 gram, yolk.
B liquid composition: 80 milliliters of A liquid, 3 milliliters of glycerine, 3 milliliters of N-N-dimethyl formamides.
(2) utilize the store method of pig bagged freezing semen preservation liquor, may further comprise the steps:
1. semen collection and seminal fluid is concentrated: with embodiment one.
2. cooling: the ratio 20 ℃ of isothermal dilutions at normal temperatures in A liquid and concentrated seminal fluid 1:1.8 were cooled to 2 ℃ at cold compartment of refrigerator through 1.5 hours then.
3. balance: will be cooled to 2 ℃ seminal fluid, and 2 ℃ of dilutions for the second time down, deposit 1.5 hours at 2 ℃ then in the ratio of B liquid and concentrated seminal fluid 1:1.8.
Other packing, freezing, the quality inspection of seminal fluid afterwards of thawing and thaw: with embodiment one.
Embodiment five:
The preparation of pig bagged freezing semen preservation liquor is with embodiment one, and difference is:
A liquid composition: glucose 5 grams, lactose 7 grams, Sodium Glutamate 4 grams, polyvinylpyrrolidone 5 grams, 100 milliliters of distilled water, streptomycin 20 million international units, vitamins B
120.06 16 milliliters of grams, caffeine and sodium benzoate 0.03 gram, yolk.
B liquid composition: 60 milliliters of A liquid, 8 milliliters of glycerine, 7 milliliters of N-N-dimethyl formamides.
The store method of pig bagged freezing semen preservation liquor with embodiment one, no longer repeats.
Embodiment six:
The preparation of pig bagged freezing semen preservation liquor is with embodiment one, and difference is:
A liquid composition: glucose 7 grams, lactose 3 grams, Sodium Glutamate 2 grams, polyvinylpyrrolidone 4 grams, 100 milliliters of distilled water, gentamicin sulphate 10 million international units, vitamins B
120.04 10 milliliters of grams, caffeine and sodium benzoate 0.03 gram, yolk.
B liquid composition: 90 milliliters of A liquid, 4 milliliters of glycerine, 9 milliliters of N-N-dimethyl formamides.
The store method of pig bagged freezing semen preservation liquor with embodiment one, no longer repeats.
Embodiment seven:
The preparation of pig bagged freezing semen preservation liquor is with embodiment one, and difference is:
A liquid composition: glucose 3 grams, lactose 2 grams, Sodium Glutamate 4 grams, polyvinylpyrrolidone 5 grams, 100 milliliters of distilled water, gentamicin sulphate 15 million international units, vitamins B
120.03 15 milliliters of grams, caffeine and sodium benzoate 0.07 gram, yolk.
B liquid composition: 65 milliliters of A liquid, 8 milliliters of glycerine, 8 milliliters of N-N-dimethyl formamides.
The store method of pig bagged freezing semen preservation liquor with embodiment one, no longer repeats.
Embodiment eight:
The preparation of pig bagged freezing semen preservation liquor is with embodiment one, and difference is:
A liquid composition: glucose 4 grams, lactose 2 grams, Sodium Glutamate 3 grams, polyvinylpyrrolidone 4 grams, 100 milliliters of distilled water, gentamicin sulphate 20 million international units, vitamins B
120.08 18 milliliters of grams, caffeine and sodium benzoate 0.07 gram, yolk.
B liquid composition: 95 milliliters of A liquid, 5 milliliters of glycerine, 5 milliliters of N-N-dimethyl formamides.
The store method of pig bagged freezing semen preservation liquor with embodiment one, no longer repeats.
Embodiment nine:
The preparation of pig bagged freezing semen preservation liquor is with embodiment one, and difference is:
A liquid composition: glucose 6 grams, lactose 2 grams, Sodium Glutamate 5 grams, polyvinylpyrrolidone 3 grams, 100 ml distilled waters, gentamicin sulphate 15 million international units, vitamins B
120.07 20 milliliters of grams, caffeine and sodium benzoate 0.06 gram, yolk.
B liquid composition: 80 milliliters of A liquid, 7 milliliters of glycerine, 4 milliliters of N-N-dimethyl formamides.
The store method of pig bagged freezing semen preservation liquor with embodiment one, no longer repeats.
Embodiment ten:
The preparation of pig bagged freezing semen preservation liquor is with embodiment one, and difference is:
A liquid preparation: glucose 5 grams, lactose 1 gram, Sodium Glutamate 6 grams, polyvinylpyrrolidone 2 grams, 100 milliliters of distilled water, gentamicin sulphate 12 million international units, vitamins B
120.06 13 milliliters of grams, caffeine and sodium benzoate 0.04 gram, yolk.
B liquid composition: 70 milliliters of A liquid, 5 milliliters of glycerine, 3 milliliters of N-N-dimethyl formamides.
The store method of pig bagged freezing semen preservation liquor with embodiment one, no longer repeats.
Embodiment 11:
The preparation of pig bagged freezing semen preservation liquor is with embodiment one, and difference is:
A liquid composition: glucose 1 gram, lactose 1.5 grams, Sodium Glutamate 2 grams, polyvinylpyrrolidone 2 grams, 100 milliliters of distilled water, gentamicin sulphate 10 million international units, vitamins B
120.02 15 milliliters of grams, caffeine and sodium benzoate 0.03 gram, yolk.
B liquid composition: 80 milliliters of A liquid, 3 milliliters of glycerine, 4 milliliters of N-N-dimethyl formamides.
The store method of pig bagged freezing semen preservation liquor with embodiment one, no longer repeats.
Embodiment 12:
The preparation of pig bagged freezing semen preservation liquor is with embodiment one, and difference is:
A liquid composition: glucose 2.5 grams, lactose 1 gram, Sodium Glutamate 1.5 grams, polyvinylpyrrolidone 2 grams, 100 milliliters of distilled water, gentamicin sulphate 20 million international units, vitamins B
120.06 13 milliliters of grams, caffeine and sodium benzoate 0.04 gram, yolk.
B liquid composition: 80 milliliters of A liquid, 4 milliliters of glycerine, 3 milliliters of N-N-dimethyl formamides.
The store method of pig bagged freezing semen preservation liquor with embodiment one, no longer repeats.
Embodiment 13:
The preparation of pig bagged freezing semen preservation liquor is with embodiment one, and difference is:
A liquid composition: glucose 3 grams, lactose 2 grams, Sodium Glutamate 6 grams, polyvinylpyrrolidone 2 grams, 100 milliliters of distilled water, gentamicin sulphate 10 million international units, vitamins B
120.06 13 milliliters of grams, caffeine and sodium benzoate 0.04 gram, yolk.
B liquid composition: 70 milliliters of A liquid, 5 milliliters of glycerine, 4 milliliters of N-N-dimethyl formamides.
The store method of pig bagged freezing semen preservation liquor with embodiment one, no longer repeats.
Embodiment 14: on same pig farm, adopt identical boar to use the artificial insemination of pig bagged freezing semen of the present invention and the test-tube comparison of pig seminal fluid that normal temperature is preserved
(1) the test-tube utilization of pig bagged freezing semen of the present invention
1. the oestrus evaluation of sow: when pushing its back or people when riding over the sow back, sow plants oneself, and shows as ear simultaneously and holds up, and the back of the body arches, and tail is holded up, and trembles, and the stimulation sensitivity to vulva and flank portion is considered as oestrusing, and does semen deposition and prepares.
2. the processing of seminal fluid before the semen deposition: getting the freezing pig bagged freezing semen of depositing a year of the inventive method is 5ml for one bag, thaws, and can directly put in 38～40 ℃ of warm water, thaws for 10～20 seconds; Carry out the semen quality inspection then, require motility of sperm all more than 0.4, more than 60%, preserve liquid (as EDTA, GuelPh, BTS, Zorpva, Reading etc.) with porcine semen at normal temperature and dilute in disposable swine artificial insemination semen deposition bottle in the ratio of 1:10 by qualified sperm with interior, acrosomal integrity for abnormal rate 20%.
3. semen deposition: adopt the disposable nothing sterilization of pig inseminating syringe that the sow that oestruses is carried out semen deposition, do semen deposition for the first time after oestrusing in 6～12 hours, at interval semen deposition for the second time in 12 hours.
4. result: 2～5 years old brood sow of semen deposition is 26 altogether.Wherein 23 brood sow childbirths, 1 miscarriage, 2 are not pregnant, feelings occur returning.Pregnancy rate is 88.4%, 23 brood sow childbirth, and average nest litter size is 9.3.
(2) artificial insemination of the pig seminal fluid of employing normal temperature preservation
1. the oestrus evaluation of sow, the same;
2. the processing of seminal fluid before the semen deposition: gather the pig fresh semen, seminal fluid is carried out the quality inspection, preserving liquid (as EDTA, GuelPh, BTS, Zorpva, Reading etc.) with porcine semen at normal temperature installed by 5,000,000,000/60ml/ part branch, preserve down at normal temperature (17～18 ℃), (vigor can not be lower than 0.6) can be used for semen deposition in 4 days.
3. semen deposition:, adopt the disposable nothing sterilization of pig inseminating syringe that the sow that oestruses is carried out semen deposition with the pig seminal fluid that branch installs.Do semen deposition for the first time after oestrusing in 6～12 hours, at interval semen deposition for the second time in 12 hours.
4. result: 2～5 years old brood sow of semen deposition is 143 altogether.Wherein 132 brood sow childbirths, 3 miscarriages, 8 are not pregnant, feelings occur returning.Pregnancy rate is 92.3%, and average nest litter size is 9.5.
The tabulation contrast:
| The breeding mode | Breeding sow number (head) | Pregnancy rate (%) | Average nest litter size (head) |
| The pig seminal fluid artificial insemination of adopting normal temperature to preserve | 143 | 92.3 | 9.5 |
| Pig bagged freezing semen artificial insemination of the present invention | 26 | 88.4 | 9.3 |
By as can be seen above-mentioned, artificial insemination of pig seminal fluid and the pig bagged freezing semen artificial insemination of the present invention of adopting normal temperature to preserve are approaching at aspects such as pregnancy rate, average nest litter sizes.
Claims (8)
1. pig bagged freezing semen preservation liquor, it is characterized in that: described preservation liquid is made up of A liquid and B liquid,
A liquid preparation: take by weighing glucose 1～10 gram, lactose 1～10 gram, Sodium Glutamate 1～8 gram, polyvinylpyrrolidone 1～8 gram, add successively in 100 ml distilled waters, it is dissolved fully, add microbiotic 10～20 million international units again, filter with biofilter, add vitamins B then
120.02 10～30 milliliters of～0.1 grams, caffeine and sodium benzoate 0.02～0.1 gram, yolk fully obtain A liquid behind the mixing;
B liquid preparation: get 50～100 milliliters of A liquid, add 2～10 milliliters of glycerine, 2～10 milliliters of N-N-dimethyl formamides, fully obtain B liquid behind the mixing.
2. pig bagged freezing semen preservation liquor according to claim 1, it is characterized in that: described A liquid composition is: glucose 1～3 gram, lactose 1～3 gram, Sodium Glutamate 1～2 gram, polyvinylpyrrolidone 1～2 gram, microbiotic 10～15 million international units, vitamins B
120.02 10～20 milliliters of～0.04 grams, caffeine and sodium benzoate 0.02～0.04 gram, yolk; Described B liquid composition is: 75～95 milliliters of A liquid, 2～6 milliliters of glycerine, 2～6 milliliters of N-N-dimethyl formamides.
3. pig bagged freezing semen preservation liquor according to claim 1 is characterized in that: described A liquid composition is: glucose 3～7 grams, lactose 1～3 gram, Sodium Glutamate 2～6 grams, polyvinylpyrrolidone 2～5 grams, microbiotic 15～20 million international units, vitamins B
120.04 10～20 milliliters of～0.08 grams, caffeine and sodium benzoate 0.04～0.08 gram, yolk; Described B liquid composition is: 70～100 milliliters of A liquid, 4～8 milliliters of glycerine, 4～8 milliliters of N-N-dimethyl formamides.
4. pig bagged freezing semen preservation liquor according to claim 1 is characterized in that: described microbiotic is gentamicin sulphate, penicillin or Streptomycin sulphate.
5. pig bagged freezing semen preservation liquor according to claim 1 is characterized in that: the pH value of described A liquid and B liquid is 6.5～7.5, osmotic pressure is 300～500mmol/L.
6. method of preserving seminal fluid with the described pig bagged freezing semen preservation liquor of claim 1 is characterized in that this method may further comprise the steps:
(1) semen collection and concentrated: boar is carried out semen collection, seminal fluid centrifugation, collect isolating lower floor liquid, control sperm concentration more than 1,000,000,000/ml, preservation, standby;
(2) cooling: the ratio in A liquid and concentrated seminal fluid is the ratio of 1:0.5～2, and with the dilution of seminal fluid isothermal, refrigeration was cooled to 1～4 ℃ through 1～2 hour then under 18～35 ℃;
(3) balance: cryopreserved semen is carried out the dilution second time with B liquid, the proportioning 1:0.5 of B liquid and cryopreserved semen～2, dilution back seminal fluid was preserved 1～2 hour down at 1～4 ℃;
(4) packing: seminal fluid is distributed into bag;
(5) freezing: that packed seminal fluid is preserved in liquid nitrogen.
7. the method for preservation seminal fluid according to claim 6 is characterized in that: described seminal fluid be distributed into the bag be with seminal fluid with sterile bag by the packing of every bag of 5ml seminal fluid.
8. the method for preservation seminal fluid according to claim 6 is characterized in that: the described preservation in liquid nitrogen is that packed seminal fluid was fumigated 5～10 minutes at distance liquid nitrogen surface 2～5cm place earlier, drops into freezing preservation in the liquid nitrogen then.
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| CN103262837A (en) * | 2013-05-20 | 2013-08-28 | 北京市农林科学院 | Livestock seminal fluid cryoprotectant and application thereof |
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