CN103039433B - Casein anti-freezing agent for cryopreservation of donkey semen and preparation method thereof - Google Patents

Abstract

The invention provides a casein anti-freezing agent for cryopreservation of donkey semen and a preparation method thereof. Each 100 milliliters of anti-freezing agent contains the following components: 1.0 to 3.0 grams of casein, 2.0 to 3.0 grams of glucose, 1.0 to 2.0 grams of lactose, 3.0 to 6.0 grams of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) BUFFER, 2.0 to 3.0 milliliters of glycerol, 2 to 4 milliliters of yolk, 450 to 550 thousand international units (IU) of penicillin, 4.5 to 5.5 grams of gentamicin, 20 to 30 milligrams of amphotericin B, and the balance of HANK solution. The casein anti-freezing agent solves the problems of long reproductive cycle of female animals, low artificial insemination rate, complex production operation of frozen donkey semen and high difficulty in the prior art.

Description

A kind of casein antifreeze for the freezing preservation of donkey testicular fluid and preparation method thereof

Technical field

The present invention relates to biological technical field, refer to especially a kind of casein antifreeze for the freezing preservation of donkey testicular fluid and preparation method thereof.

Background technology

Semen cryopreservation combines with technology of artificial insemination, in Modern Animal Husbandry is produced, is bringing into play huge effect.It is also to excavate the important means of outstanding kind of jack ass reproductive potential that seminal fluid preservation simultaneously utilizes technology; Adopt frozen semen can improve kind of a jack ass utilization ratio, reduce a raising number for kind of jack ass; And excellent naughty bad owing to selecting, kind jack ass productivity and hereditary quality that input is frozen essence production are good, have accelerated donkey genetic improvement speed; Technology of artificial insemination also can reduce generation and the propagation of genital diseases, has indirectly improved the conception rate of jenny ass; Frozen semen convenient transportation, can make not to be subject to regional limits with joining jenny ass, has equaled to extend utilizing the time limit and using region of kind jack ass.In addition, world's majority state, has all set up in imminent danger or rare animal seminal fluid gene pool at present, with long-term a large amount of preservation of form of frozen semen, to take at any time in the future.No. 662 dispatch bulletin of Ministry of Agriculture's on June 2nd, 2006, announces that 138 livestock and poultry species such as Dezhou donkey are national Genetic Resources of Domestic Animal protection kind.But because donkey is the reasons such as seasonal oestrus animal, the female livestock breed cycle is long, artificial insemination conception rate is low, both at home and abroad donkey testicular fluid is preserved with Refrigeration Technique and do not carried out systematic research always, also do not obtain the breakthrough of significance.

Present stage, main this friendship mode that adopts of donkey breeding, indivedual good herding improvement persons of technology also do not dilute the seminal fluid of extraction (fresh essence) or low power dilution (1: 2～4) sometimes, give immediately the jenny ass semen deposition of oestrusing, it is more obvious that it is applicable to jenny ass seasonal oestrus, and a fairly large number of area.Compared with this friendship, efficiency can improve 5-10 doubly.But because China's donkey kind is more, culture zone is distributed more widely, how in mountain area or the region that has inconvenient traffic, and feeding manner is in the majority mainly with casual household, and jenny ass distributes and do not concentrate, and the seminal fluid technology of artificial insemination popularizing area of fresh essence or low power dilution is less.In addition, because the external holding time of the fresh essence of donkey is extremely short, after several hours, vigor is close to and exhausts, and this makes the fresh smart technology of artificial insemination of donkey be subject to the very big restriction in time, place.Therefore, longer in the urgent need to the holding time in producing, the donkey production colony of carrying more convenient high-quality jack ass seminal fluid and improve the whole society of donkey breeding.

Seminal fluid is preserved and is divided into normal temperature (15 DEG C～25 DEG C) preservation, three kinds of methods of low temperature preservation (0 DEG C～5 DEG C) and freezing (79 DEG C～-196 DEG C) preservation.The research of normal temperature Techniques of preserving is carried out the earliest, relatively ripe, but at normal temperatures, growth of microorganism can affect rapidly semen quality on the one hand, and when normal temperature is preserved on the other hand, sperm viability declines very fast.The relatively freezing preservation of low temperature Techniques of preserving is simple to operation, and cost is low, but low temperature Techniques of preserving needs to provide nutrition can protect again impermeability cryoprotective agent and the dilution of sperm.Freezing preservation refers to seminal fluid is placed in liquid nitrogen or dry ice through specially treated, can preserve this even many decades of species germ plasm resource several years, conscientiously brings into play the genetic resources of herd sire.But due to the biological property difference of different male animal sperms, also there is very large difference in its controlled-rate freezing.As the freezing Techniques of preserving comparative maturity of ox seminal fluid, especially ox semen thin tube method program is produced universal; But sheep sperm freezes the problems such as rear ubiquity non-return rate is unstable, conception rate is low, repetitive rate is low; And the sperm of pig because of fat content higher, dewater more difficult, after freeze-thaw sperm exist ultra microstructure sustain damage (as sperm membrane, acrosome and sperm nucleus alveolation, damage or break), in female reproductive tract the lost of life and the problem such as fertilization rate is low, thereby Cryopreservation of Boar Semen is also always under test.Little about the research of the freezing preservation of donkey testicular fluid both at home and abroad at present, main cause is: the reproduction rate that is on the one hand donkey is low, and the pregnancy duration is long; The second, because its semen quality between different donkey individualities has obvious difference, and donkey sperm itself low temperature is hit responsive especially, very easily damage in freeze-thaw process, thus refrigeration and freezing after seminal fluid greatly reduced conception rate; The 3rd, because donkey is long estrus time, inaccurate its pregnancy rate that causes of test-tube timing is low; Finally, the each average ejaculatory amount of jack ass reaches 70ml, and density is rarer, and electrolyte ingredient in seminal plasma can promote sperm early ageing.Thereby, the frozen semen production operation degree complexity of donkey, difficulty is large, is seriously restricting the application of frozen semen.

Donkey is equus, is widely distributed, spread all over Asia, non-, draw each continent.Dezhou donkey, is one of China's five large good donkey kinds, and physique is tall and big, and structure is well-balanced, good looking appearance, and build Founder, neck trunk is in conjunction with good.The small-sized donkey kind of improvement and a desirable kind as interspecific cross.Therefore in producing, need the freezing preservation research of carrying out Dezhou donkey testicular fluid badly.

The antifreeze composition that is absolutely necessary in semen freezing process.In refrigerating process, the effect of antifreeze is mainly manifested in the following aspects: supplement sperm nutrition 1.; 2. reduce human semen viscosity; 3. reducing foreign matter affects sperm; 4. relax the damaging effect of metabolite to sperm; 5. in refrigerating process, protection sperm, strengthens resistance and the oxidation resistance of sperm to low temperature, reduces the freezing injury of sperm.At present, in the manufacturing process of frozen semen, usually selecting can Inhibit sperm activity, reduces energy loss, extends the antifreeze of sperm life.The yolk of the multiplex chicken of antifreeze of ox, sheep, horse, Cryopreservation of Boar Semen, has all obtained good controlled-rate freezing.Yolk can reduce sperm in the extent of damage that suffers cold shock in 4 DEG C of temperature-fall periods on the one hand; Yolk low price on the other hand, draws materials conveniently.But not in yolk, all the components is all useful to sperm, can breathe by Inhibit sperm as yolk middle-high density lipoprotein, reduce sperm motility rate; The second, yolk belongs to animal derived material, wherein may carry multiple pathogenic microorganisms, can cause bio-safety problem.As can be caused acceptor jenny ass RTI by artificial insemination, affect conception rate, pathogenic microorganism also can affect motility rate and the holding time of seminal fluid; Derive from the other hand different time, different physiological statuss, the yolk of Different Individual, the quality standard such as the size of yolk, content are how many is difficult to unified, produces the quality that not only smart product is frozen in impact, and be difficult to realize standardization, standardization for freezing essence.

Summary of the invention

The present invention proposes a kind of casein antifreeze for the freezing preservation of donkey testicular fluid, has solved in prior art that the female livestock breed cycle is long, artificial insemination conception rate is low, the frozen semen production operation degree complexity of donkey, the problem that difficulty is large.

Technical scheme of the present invention is achieved in that

For a casein antifreeze for the freezing preservation of donkey testicular fluid, every 100mL antifreeze comprises following component:

Casein 1.0～3.0g, glucose 2.0～3.0g, lactose 1.0～2.0g, HEPES BUFFER3.0～6.0g, glycerine 2.0～3.0mL, yolk 2～4mL, penicillin 45～550,000 IU, gentamicin 4.5～5.5g, amphotericin B 20～30mg, surplus is HANK solution.

As preferred technical scheme, a kind of casein antifreeze for the freezing preservation of donkey testicular fluid, every 100mL antifreeze comprises following component:

Casein 2.0g, glucose 2.5g, lactose 1.5g, HEPES BUFFER 5.0g, glycerine 2.5mL, yolk 3mL, penicillin 500,000 IU, gentamicin 5g, amphotericin B 25mg, surplus is HANK solution.

The present invention proposes another method for the preparation of the casein antifreeze of the freezing preservation of donkey testicular fluid, and it comprises step:

1) according to comprising casein 1.0～3.0g, glucose 2.0～3.0g, lactose 1.0～2.0g, HEPES BUFFER 3.0～6.0g, glycerine 2.0～3.0mL, yolk 2～4mL, penicillin 45～550,000 IU, gentamicin 4.5～5.5g, amphotericin B 20～30mg in every 100mL antifreeze, surplus is that HANK solution takes each component, for subsequent use;

2) the above-mentioned casein taking, glucose, lactose, HEPES BUFFER are become to basal liquid by HANK solution allocation;

3) be configured to I liquid to adding the penicillin, gentamicin, the amphotericin B that take in step 1) in the above-mentioned basal liquid being configured to;

4) be 7.1～7.8 by the whole pH of described I liquid, then carry out filtration sterilization, put into 3～5 DEG C of refrigerators for subsequent use

5) before use, get described I liquid, add wherein the yolk taking in step 1), be configured to II liquid, obtain the described casein antifreeze for the freezing preservation of donkey testicular fluid.

As preferred technical scheme, according to comprising casein 2.0g, glucose 2.5g, lactose 1.5g, HEPES BUFFER 5.0g, glycerine 2.5mL, yolk 3mL, penicillin 500,000 IU, gentamicin 5g, amphotericin B 25mg in every 100mL antifreeze, it is for subsequent use that surplus is that HANK solution takes.

Owing to having adopted technique scheme, a kind of casein antifreeze for the freezing preservation of donkey testicular fluid and preparation method thereof, every 100mL antifreeze comprises following component: casein 1.0～3.0g, glucose 2.0～3.0g, lactose 1.0～2.0g, HEPES BUFFER 3.0～6.0g, glycerine 2.0～3.0mL, yolk 2～4mL, penicillin 45～550,000 IU, gentamicin 4.5～5.5g, amphotericin B 20～30mg, surplus is HANK solution, it is long to have solved in prior art the female livestock breed cycle, artificial insemination conception rate is low, the frozen semen production operation degree complexity of donkey, the problem that difficulty is large.

Specific experiment:

Taking of yolk

Egg should derive from the chicken house without epidemic disease, and the egg that uses must be fresh, complete, clean, first with 75% cotton ball soaked in alcohol, eggshell surface is carried out disinfection before taking yolk, after alcohol volatilization is use up, take out complete yolk with egg white separator; Also can knock a crackle open in egg waist midline, by one point of two halves of egg, the method for utilizing two eggshells alternately to topple over, removes egg white, leaves yolk; Then extract yolk with the syringe of sterilizing through vitellinae membrana.

The preparation method of donkey frozen semen antifreeze

Take casein 1.0-3.0g, glucose 2.0-3.0g, lactose 1.0-2.0g, HEPES BUFFER 3.0-6.0g, glycerine 2.0-3.0mL, yolk 2-4mL with electronic analytical balance, be dissolved in 50mL HANK solution, after even by magnetic stirrer, add HANK solution and become basal liquid to 100mL.In basal liquid, add penicillin 500,000 IU, gentamicin 5g, amphotericin B 25mg, be configured to I liquid; Adjusting pH with accurate pH meter is 7.1-7.8, then carries out filtration sterilization with the filter membrane of 0.22 μ m, puts into 4 DEG C of refrigerators for subsequent use.

Carry out donkey testicular fluid freezing before, get 360mL I liquid and add 7.2-14.4ml yolk, seal after mouth with sealed membrane, turn upside down and mix, in 4 DEG C of refrigerator overnight, layering.Second day, suck top layer foam, dilution middle level is poured in beaker, discard lower sediment.Dilution centrifugal 3 times through 4000rpm/min, each 30min, removes dilution top layer impurity and bottom precipitation after centrifugal, get middle level and pour in saline bottle or blue mouthful of bottle, add 6-9mL glycerine, after stirring with magnetic stirring apparatus, be configured to II liquid, be placed in 4 DEG C of refrigerators stand-by.After I liquid prepares, can in 4 DEG C of refrigerators, deposit approximately 10 days.II liquid matching while using, in 4 DEG C of refrigerators, the resting period should not exceed 24h.

Experimental example describes with the freezing preservation of Dezhou donkey testicular fluid.

The preparation of antifreeze

Take casein 3.0g, glucose 2.0g, lactose 1.5g, HEPESBUFFER 3.0g, glycerine 2.0mL, yolk 2mL with electronic analytical balance, be dissolved in 50mL HANK solution, after even by magnetic stirrer, add HANK solution and become basal liquid to 100mL.In basal liquid, add penicillin 500,000 IU, gentamicin 5g, amphotericin B 25mg, be configured to I liquid; Adjusting pH with accurate pH meter is 7.5, then carries out filtration sterilization with the filter membrane of 0.22 μ m, puts into 4 DEG C of refrigerators for subsequent use.

Carry out donkey testicular fluid freezing before, get 360mL I liquid and add 8ml yolk, seal after mouth with sealed membrane, turn upside down and mix, in 4 DEG C of refrigerator overnight, layering.Second day, suck top layer foam, dilution middle level is poured in beaker, discard lower sediment.Dilution centrifugal 3 times through 4000rpm/min, each 30min, removes dilution top layer impurity and bottom precipitation after centrifugal, get middle level and pour in saline bottle or blue mouthful of bottle, add 6mL glycerine, after stirring with magnetic stirring apparatus, be configured to II liquid, be placed in 4 DEG C of refrigerators stand-by.

The collection of seminal fluid

Utilize artificial vagina method to gather jack ass seminal fluid, take out the gel-shaped sexual accessory gland thing in institute's semen collection liquid, after filtering with 8 layers of sterile gauze, microscopy on the phase contrast microscope of being furnished with 38 DEG C of thermostats immediately, evaluation sperm viability, and by sperm concentration instrument measurement sperm concentration.The seminal fluid gathering is milky or canescence, free from extraneous odour, and sperm morphology is normal, density >=100,000,000/ml, vigor >=0.7, can be used for making Straw Frozen Semen.

Semen freezing

In former seminal fluid, add I liquid in the ratio of 1:1, then place 10min at 22 DEG C, divide with 600 × g/, centrifugal 15min, removes supernatant, and the atherosclerotic seminal fluid in each centrifuge tube is finally diluted with dilution II liquid.Sperm dilution is undertaken by two-step method: first add 1/4 of final amount of dilution, afterwards, first at room temperature place 10min through the seminal fluid of preliminarily diluted, then add remaining II liquid dilution.The final seminal fluid having diluted moves in a large container, carries out packaging operation with tubule all-in-one, and every tubule sperm total amount is 7,500 ten thousand, and volume is 0.5mL.Straw semen after packing is put into opaque plastic casing of uncapping, then plastic casing is put into the refrigerator balance 3-4h of 4 DEG C.

After balance, tubule is piled up on freezing frame, while piling up, should be noted tubule placing direction, make tampon sealing section near operator.On every, put a label that is similar to tubule, a label for donkey, is convenient to identification.As there being the straw semen of different donkeys on same, separately pile up, between the donkey of two, to separate some distances, in order to avoid obscure.Drop to after approximately 4 DEG C until frigorimeter temperature, the tubule of piling up is moved in frigorimeter, according to pre-set cryogenic temperature curve, automatically complete refrigerating process.

Thawing and freezing rear motility rate of straw frozen semen detects

In advance by the water temp. heating to 38 in thermostat water bath DEG C, after the tubule of a superfreeze being taken out rapidly from liquid nitrogen with tweezers, drop in 38 DEG C of water-baths and rock about 30s, take out immediately until completely dissolved, dry the globule with gauze, cut and first cut off ultrasonic sealing end with tubule again, cut off again tampon end, seminal fluid in tubule is all placed in a small test tube, shake up, get one on slide, and covered, microscopy on the phase contrast microscope of being furnished with 38 DEG C of thermostats, the conventional quality standard of evaluation seminal fluid.

Semen quality qualification after thawing

1. acrosomal integnity detects.Adopt after the peanut agglutinin dyeing of FITC mark, detect the integrality of the perforatorium after freeze-thaw with fluorescence microscope.Get the straw semen smear after 30 μ L thaw, natural air drying afterwards.Smear after natural seasoning is soaked and is fixed for 10 minutes in the methyl alcohol of 20-22 DEG C.Then in each smear after air-dry, drip the dye liquor of the peanut agglutinin of the FITC mark that contains 100mg/mL, be placed on the 30min that dyes in the dark wet box of 37 DEG C.Afterwards, with PBS flushing, after air-dry smear, drip anti-fluorescence quenching, covered with after colourless nail polish mounting, checks the situation of perforatorium under fluorescence microscope.

2.DNA integrity detection.Utilize after acridine orange (AO) dyeing, detect the integrality of the sperm DNA after freeze-thaw with fluorescence microscope.With TNE buffer solution by the sperm dilution after thawing to suitable concentration, afterwards, the seminal fluid of getting after 200 μ L dilutions adds 400 μ L abstergents immediately.After 30s, add 1.2mL AO dyeing.After dyeing, get 15 μ L seminal fluid and be added drop-wise on slide, covered, at fluorescence microscopy Microscopic observation.Judge that according to sperm head color sperm DNA is complete or degraded.

3. mitochondria activity detects.After utilizing Rhodamine 123 (R123) and propidium iodide (PI) to dye, by the mitochondria of sperms activity after fluorescence microscope detection freeze-thaw.With PBS by the sperm dilution after thawing to suitable concentration, the seminal fluid of getting after 1mL dilution adds in 1.5mL centrifuge tube, adds wherein 3 μ L Rhodamine 123s.Centrifuge tube is placed on to 20min in the dark wet box of 37 DEG C, afterwards, adds 10 μ L propidium iodides, the dark wet box that is placed in 37 DEG C is hatched 10min.Afterwards, seminal fluid is through the centrifugal 6min of 600 × g.After centrifugal, remove supernatant, with the resuspended lower floor of 1mL PBS sperm agglomerate.Get the seminal fluid of 10 μ L after resuspended and drop on slide, then drip the anti-fluorescence quenching of 10 μ L.Slide is placed in to fluorescence microscopy Microscopic observation.According to sperm Show Color, judge mitochondria of sperms activity.

4. membrane integrity detects.Utilize hypotonic expansion experiment (HOST) to detect the integrality of plasmalemmae of sperms.The seminal fluid of getting after 50 μ L thaw adds in the hypotonic solution of 1mL.After mixing, be placed in 37 DEG C and hatch 60min, afterwards, get 15 μ L seminal fluid sample drops and be added on slide, add after cover glass, whether curling according to sperm tail, judge that whether plasmalemmae of sperms is complete.

Artificial insemination test

Artificial input after the thawing of semen of above-mentioned development is oestrused in jenny ass body, and joining pregnant rate is 55.7%, has reached fresh essence breeding effect.

Evaluation result

The freeze-thaw method of applying antifreeze of the present invention and seminal fluid, its evaluation result is as follows:

1. after freeze-thaw, the motility rate of sperm reaches more than 40%;

2. the acrosomal integrity of sperm reaches more than 84%;

3. in every dose of tubule, effectively number of sperm is no less than 3,000 ten thousand;

4. there is active mitochondrial sperm to reach more than 13%;

5. the plasma membrane percentage of head rice of sperm reaches more than 38%.

Brief description of the drawings

In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, to the accompanying drawing of required use in embodiment or description of the Prior Art be briefly described below, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, do not paying under the prerequisite of creative work, can also obtain according to these accompanying drawings other accompanying drawing.

Fig. 1 is preparation method's of the present invention process flow diagram.

Embodiment

Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiment.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtaining under creative work prerequisite, belong to the scope of protection of the invention.

As shown in Figure 1, a kind of casein antifreeze for the freezing preservation of donkey testicular fluid, every 100mL antifreeze comprises following component:

Casein 1.0～3.0g, glucose 2.0～3.0g, lactose 1.0～2.0g, HEPES BUFFER3.0～6.0g, glycerine 2.0～3.0mL, yolk 2～4mL, penicillin 45～550,000 IU, gentamicin 4.5～5.5g, amphotericin B 20～30mg, surplus is HANK solution.

For a casein antifreeze for the freezing preservation of donkey testicular fluid, every 100mL antifreeze comprises following component:

Casein 2.0g, glucose 2.5g, lactose 1.5g, HEPES BUFFER 5.0g, glycerine 2.5mL, yolk 3mL, penicillin 500,000 IU, gentamicin 5g, amphotericin B 25mg, surplus is HANK solution.

For the preparation of a method for the casein antifreeze of the freezing preservation of donkey testicular fluid, comprise step:

1) according to comprising casein 1.0～3.0g, glucose 2.0～3.0g, lactose 1.0～2.0g, HEPES BUFFER 3.0～6.0g, glycerine 2.0～3.0mL, yolk 2～4mL, penicillin 45～550,000 IU, gentamicin 4.5～5.5g, amphotericin B 20～30mg in every 100mL antifreeze, surplus is that HANK solution takes each component, for subsequent use;

2) the above-mentioned casein taking, glucose, lactose, HEPES BUFFER are become to basal liquid by HANK solution allocation;

3) be configured to I liquid to adding the penicillin, gentamicin, the amphotericin B that take in step 1) in the above-mentioned basal liquid being configured to;

4) be 7.1～7.8 by the whole pH of described I liquid, then carry out filtration sterilization, put into 3～5 DEG C of refrigerators for subsequent use;

5) before use, get described I liquid, add wherein the yolk taking in step 1), be configured to II liquid, obtain the described casein antifreeze for the freezing preservation of donkey testicular fluid.

According to comprising casein 2.0g, glucose 2.5g, lactose 1.5g, HEPESBUFFER 5.0g, glycerine 2.5mL, yolk 3mL, penicillin 500,000 IU, gentamicin 5g, amphotericin B 25mg in every 100mL antifreeze, it is for subsequent use that surplus is that HANK solution takes.

Embodiment mono-

According to comprising casein 1.0g, glucose 2.0g, lactose 1.0g, HEPESBUFFER 3.0g, glycerine 2.0mL, yolk 2mL, penicillin 450,000 IU, gentamicin 4.5g, amphotericin B 20mg in every 100mL antifreeze, surplus is that HANK solution takes each component, for subsequent use;

2) the above-mentioned casein taking, glucose, lactose, HEPES BUFFER are become to basal liquid by HANK solution allocation;

3) be configured to I liquid to adding the penicillin, gentamicin, the amphotericin B that take in step 1) in the above-mentioned basal liquid being configured to;

4) be 7.1 by the whole pH of described I liquid, then carry out filtration sterilization, put into 3 DEG C of refrigerators for subsequent use;

5) before use, get described I liquid, add wherein the yolk taking in step 1), be configured to II liquid, obtain the described casein antifreeze for the freezing preservation of donkey testicular fluid.

Embodiment bis-

According to comprising casein 2.0g, glucose 2.5g, lactose 1.5g, HEPESBUFFER 5.0g, glycerine 2.5mL, yolk 3mL, penicillin 500,000 IU, gentamicin 5g, amphotericin B 25mg in every 100mL antifreeze, surplus is that HANK solution takes each component, for subsequent use;

2) the above-mentioned casein taking, glucose, lactose, HEPES BUFFER are become to basal liquid by HANK solution allocation;

3) be configured to I liquid to adding the penicillin, gentamicin, the amphotericin B that take in step 1) in the above-mentioned basal liquid being configured to;

4) be 7.5 by the whole pH of described I liquid, then carry out filtration sterilization, put into 4 DEG C of refrigerators for subsequent use;

5) before use, get described I liquid, add wherein the yolk taking in step 1), be configured to II liquid, obtain the described casein antifreeze for the freezing preservation of donkey testicular fluid.

Embodiment tri-

According to comprising casein 3.0g, glucose 3.0g, lactose 2.0g, HEPESBUFFER 6.0g, glycerine 3.0mL, yolk 4mL, penicillin 550,000 IU, gentamicin 5.5g, amphotericin B 30mg in every 100mL antifreeze, surplus is that HANK solution takes each component, for subsequent use;

2) the above-mentioned casein taking, glucose, lactose, HEPES BUFFER are become to basal liquid by HANK solution allocation;

3) be configured to I liquid to adding the penicillin, gentamicin, the amphotericin B that take in step 1) in the above-mentioned basal liquid being configured to;

4) be 7.8 by the whole pH of described I liquid, then carry out filtration sterilization, put into 5 DEG C of refrigerators for subsequent use;

5) before use, get described I liquid, add wherein the yolk taking in step 1), be configured to II liquid, obtain the described casein antifreeze for the freezing preservation of donkey testicular fluid.

The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any amendment of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.

Claims (1)

1. for the preparation of a method for the casein antifreeze of the freezing preservation of donkey testicular fluid, it is characterized in that, comprise step:

1) taking of yolk

Egg should derive from the chicken house without epidemic disease, and the egg that uses must be fresh, complete, clean, first with 75% cotton ball soaked in alcohol, eggshell surface is carried out disinfection before taking yolk, after alcohol volatilization is use up, take out complete yolk with egg white separator; Also can knock a crackle open in egg waist midline, by one point of two halves of egg, the method for utilizing two eggshells alternately to topple over, removes egg white, leaves yolk; Then extract yolk with the syringe of sterilizing through vitellinae membrana;

2) preparation of donkey frozen semen antifreeze

Take casein 1.0-3.0g, glucose 2.0-3.0g, lactose 1.0-2.0g, HEPES BUFFER3.0-6.0g, glycerine 2.0-3.0mL, yolk 2-4mL with electronic analytical balance, be dissolved in 50mL HANK solution, after even by magnetic stirrer, add HANK solution and become basal liquid to 100mL; In basal liquid, add penicillin 500,000 IU, gentamicin 5g, amphotericin B 25mg, be configured to I liquid; Adjusting pH with accurate pH meter is 7.1-7.8, then carries out filtration sterilization with the filter membrane of 0.22 μ m, puts into 4 DEG C of refrigerators for subsequent use;

Carry out donkey testicular fluid freezing before, get 360mL I liquid and add 7.2-14.4ml yolk, seal after mouth with sealed membrane, turn upside down and mix, in 4 DEG C of refrigerator overnight, layering; Second day, suck top layer foam, dilution middle level is poured in beaker, discard lower sediment.Dilution centrifugal 3 times through 4000rpm/min, each 30min, removes dilution top layer impurity and bottom precipitation after centrifugal, get middle level and pour in saline bottle or blue mouthful of bottle, add 6-9mL glycerine, after stirring with magnetic stirring apparatus, be configured to II liquid, be placed in 4 DEG C of refrigerators stand-by; After I liquid prepares, can in 4 DEG C of refrigerators, deposit approximately 10 days; II liquid matching while using, in 4 DEG C of refrigerators, the resting period should not exceed 24h.