CN103039433A - Casein anti-freezing agent for cryopreservation of donkey semen and preparation method thereof - Google Patents
Casein anti-freezing agent for cryopreservation of donkey semen and preparation method thereof Download PDFInfo
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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
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, is bringing into play huge effect in Modern Animal Husbandry production.It also is to excavate the important means of outstanding kind of jack ass reproductive potential that simultaneously seminal fluid preservation utilizes technology; Adopt frozen semen can improve kind of a jack ass utilization ratio, reduce the raising number of kind of jack ass; And excellent naughty bad owing to selecting, kind jack ass productivity and hereditary quality that input is frozen smart 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; The frozen semen convenient transportation can make with joining jenny ass and not be subjected to regional limits, has equaled to prolong utilizing the time limit and using the region of kind jack ass.In addition, world's majority state has all been set up in imminent danger or rare animal seminal fluid gene pool at present, with long-term a large amount of preservation of the form of frozen semen, in order to take at any time in the future.No. 662 dispatch bulletin of Ministry of Agriculture's on June 2nd, 2006,138 livestock and poultry species such as declaration Dezhou donkey are national Genetic Resources of Domestic Animal protection kind.But owing to donkey is the reasons such as seasonal oestrus animal, the female livestock breed cycle is long, the artificial insemination conception rate is low, donkey testicular fluid is preserved with Refrigeration Technique do not carried out systematic research always both at home and abroad, also do not obtain the breakthrough of significance.
Present stage, main this friendship mode that adopts of donkey breeding, the herding improvement person that indivedual technology are good does not also dilute the seminal fluid (bright smart) of extraction or low power dilution (1: 2～4) sometimes, gives immediately the jenny ass semen deposition of oestrusing, it is more obvious that it is applicable to the jenny ass seasonal oestrus, and a fairly large number of area.Compare with this friendship, efficient can improve 5-10 doubly.But because China's donkey kind is more, the culture zone is distributed more widely, and how in the mountain area or the zone that has inconvenient traffic, and feeding manner is in the majority mainly with the casual household, and jenny ass distributes and do not concentrate, and the seminal fluid technology of artificial insemination popularizing area of bright essence or low power dilution is less.In addition, because the bright smart external holding time of donkey is extremely short, vigor is close to and exhausts after several hours, and this is so that the bright smart technology of artificial insemination of donkey is subject to the very big restriction in time, place.Therefore, donkey breeding longer in the urgent need to the holding time in producing, carry the donkey production colony that more convenient high-quality jack ass seminal fluid is improved the whole society.
Seminal fluid is preserved and is divided into normal temperature (15 ℃～25 ℃) preservation, three kinds of methods of low temperature preservation (0 ℃～5 ℃) and freezing (79 ℃～-196 ℃) preservation.The research of normal temperature Techniques of preserving is carried out the earliest, and is relatively ripe, but at normal temperatures, growth of microorganism can affect semen quality rapidly on the one hand, and sperm viability descended very fast when normal temperature was preserved on the other hand.The relatively freezing preservation of low temperature Techniques of preserving is simple to operation, and cost is low, but the 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 the dry ice through specially treated, can preserve this species germ plasm resource several years even many decades, conscientiously brings into play the genetic resources of herd sire.But because the biological property of different male animal sperms is different, also there is very large difference in its controlled-rate freezing.Such as the freezing Techniques of preserving comparative maturity of ox seminal fluid, especially ox semen thin tube method program is produced and is popularized; But the 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 is higher because of fat content, dehydration is difficulty relatively, the problem such as sperm exists ultra microstructure to sustain damage (such as sperm membrane, acrosome and sperm nucleus alveolation, damage or break) behind the freeze-thaw, the lost of life and fertilization rate be low in female reproductive tract, thereby Cryopreservation of Boar Semen is also always under test.At present both at home and abroad about the research of the freezing preservation of donkey testicular fluid seldom, 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 has obvious difference between different donkey individualities, and donkey sperm itself hits responsively especially to low temperature, in the freeze-thaw process, very easily damage, thereby 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; At last, the each average ejaculatory amount of jack ass reaches 70ml, and density is rarer, and the electrolyte ingredient in the seminal plasma can promote sperm early ageing.Thereby the frozen semen production operation degree of donkey is complicated, and difficulty is large, is seriously restricting the application of frozen semen.
Donkey is equus, is widely distributed, spread all over the 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 is upright, and the neck trunk is in conjunction with good.It is the small-sized donkey kind of improvement and as a desirable kind of interspecific cross.Therefore need the freezing preservation research of carrying out Dezhou donkey testicular fluid in producing badly.
The antifreeze composition that in the semen freezing process, is absolutely necessary.In refrigerating process, the effect of antifreeze is mainly manifested in the following aspects: replenish sperm nutrition 1.; 2. minimizing human semen viscosity; 3. reducing foreign matter affects sperm; 4. relax metabolite to the damaging effect of sperm; 5. in refrigerating process, the protection sperm strengthens sperm to resistance and the oxidation resistance of low temperature, reduces the freezing injury of sperm.At present, in the manufacturing process of frozen semen, it is can Inhibit sperm movable usually to select, and reduces energy loss, prolongs the antifreeze of sperm life.The yolk of the multiplex chicken of antifreeze of ox, sheep, horse, Cryopreservation of Boar Semen has all been obtained good controlled-rate freezing.Yolk can reduce sperm in the extent of damage that suffers cold shock in 4 ℃ of temperature-fall periods on the one hand; The yolk low price is drawn materials conveniently on the other hand.But, be not in the yolk all the components all useful to sperm, but breathe such as yolk middle-high density lipoprotein Inhibit sperm, reduce sperm motility rate; The second, yolk belongs to animal derived material, wherein may carry multiple pathogenic microorganisms, can cause the bio-safety problem.As causing 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 size of yolk, content what etc. quality standard be difficult to unifiedly, be used for freezing the smart quality that smart product is frozen in not only impact of producing, and be difficult to realize standardization, standardization.
Summary of the invention
The present invention proposes a kind of casein antifreeze for the freezing preservation of donkey testicular fluid, solved in the prior art female livestock breed cycle long, the artificial insemination conception rate is low, the frozen semen production operation degree of donkey is complicated, the problem that difficulty is large.
Technical scheme of the present invention is achieved in that
A kind of casein antifreeze for the freezing preservation of donkey testicular fluid comprises following component in every 100mL antifreeze:
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 comprises following component in every 100mL antifreeze:
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 for the preparation of the method for 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 by weighing each component, and is for subsequent use;
2) the above-mentioned casein that takes by weighing, glucose, lactose, HEPES BUFFER are become basal liquid with the HANK solution allocation;
3) add the penicillin, gentamicin, the amphotericin B that take by weighing in the step 1) in the above-mentioned basal liquid that is configured to and be configured to I liquid;
4) be 7.1～7.8 with the whole pH of described I liquid, then carry out filtration sterilization, it is for subsequent use to put into 3～5 ℃ of refrigerators
5) get described I liquid before the use, to wherein adding the yolk that takes by weighing in the step 1), be configured to II liquid, namely get 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 by weighing.
Owing to adopted technique scheme, a kind of casein antifreeze for the freezing preservation of donkey testicular fluid and preparation method thereof, comprise following component in every 100mL antifreeze: 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 the prior art female livestock breed cycle, the artificial insemination conception rate is low, the frozen semen production operation degree of donkey is complicated, the problem that difficulty is large.
Concrete experiment:
Taking of yolk
Egg should derive from the chicken house without epidemic disease, and the egg that uses must be fresh, complete, clean, with 75% cotton ball soaked in alcohol eggshell surface is carried out disinfection first before taking yolk, after alcohol volatilization to the greatest extent with the complete yolk of egg white separator taking-up; Also can knock a crackle open in egg waist midline, with one minute two halves of egg, the method for utilizing two eggshells alternately to topple over is removed egg white, stays yolk; Then the syringe with sterilization passes vitellinae membrana extraction yolk.
The preparation method of donkey frozen semen antifreeze
Take by weighing 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, it is dissolved in the 50mL HANK solution, after even with 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, and then the filter membrane with 0.22 μ m carries out filtration sterilization, and it is for subsequent use to put into 4 ℃ of refrigerators.
Carry out donkey testicular fluid freezing before, get 360mL I liquid and add 7.2-14.4ml yolk, seal mouth with sealed membrane after, the mixing that turns upside down is in 4 ℃ of refrigerator overnight, layering.Second day sucks the top layer foam, and the dilution middle level is poured in the beaker, discards lower sediment.Dilution centrifugal 3 times through 4000rpm/min, each 30min, dilution top layer impurity and the bottom removed after centrifugal precipitate, get the middle level and pour in saline bottle or the blue mouth bottle, add 6-9mL glycerine, after stirring with magnetic stirring apparatus, be configured to II liquid, place 4 ℃ of refrigerators stand-by.After I liquid prepares, can in 4 ℃ of refrigerators, deposit about 10 days.II liquid matching while using, the resting period should not surpass 24h in 4 ℃ of refrigerators.
Experimental example describes with the freezing preservation of Dezhou donkey testicular fluid.
The preparation of antifreeze
Take by weighing casein 3.0g, glucose 2.0g, lactose 1.5g, HEPESBUFFER 3.0g, glycerine 2.0mL, yolk 2mL with electronic analytical balance, it is dissolved in the 50mL HANK solution, after even with 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, and then the filter membrane with 0.22 μ m carries out filtration sterilization, and it is for subsequent use to put into 4 ℃ of refrigerators.
Carry out donkey testicular fluid freezing before, get 360mL I liquid and add 8ml yolk, seal mouth with sealed membrane after, the mixing that turns upside down is in 4 ℃ of refrigerator overnight, layering.Second day sucks the top layer foam, and the dilution middle level is poured in the beaker, discards lower sediment.Dilution centrifugal 3 times through 4000rpm/min, each 30min, dilution top layer impurity and the bottom removed after centrifugal precipitate, get the middle level and pour in saline bottle or the blue mouth bottle, add 6mL glycerine, after stirring with magnetic stirring apparatus, be configured to II liquid, place 4 ℃ of refrigerators stand-by.
The collection of seminal fluid
Utilize the artificial vagina method to gather the 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 ℃ of thermostats is evaluated sperm viability immediately, and measures sperm concentration with the sperm concentration instrument.The seminal fluid that gathers 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, in the ratio adding I liquid of 1:1, then place 10min at 22 ℃, divide with 600 * g/, centrifugal 15min removes supernatant, and the congee liquid semen in each centrifuge tube is diluted at last with dilution II liquid.Sperm dilution is undertaken by two-step method: add first 1/4 of final amount of dilution, afterwards, at room temperature place 10min first through the seminal fluid of preliminarily diluted, then add remaining II liquid dilution.The final seminal fluid that has diluted moves in the large container, carries out packaging operation with the tubule all-in-one, and every tubule sperm total amount is 7,500 ten thousand, and volume is 0.5mL.Straw semen after the packing is put into opaque plastic casing of uncapping, again plastic casing is put into 4 ℃ refrigerator balance 3-4h.
After balance is complete, tubule is piled up on freezing frame, should be noted the tubule placing direction when piling up, make tampon sealing section near the operator.Put a label that is similar to tubule on every, a donkey is convenient to identification with a label.As the straw semen of different donkeys is arranged on same, separately pile up, to separate some distances between the donkey of two, in order to avoid obscure.After the frigorimeter temperature drops to about 4 ℃, the tubule of piling up is moved in the frigorimeter, according to pre-set cryogenic temperature curve, automatically finish refrigerating process.
Thawing and freezing rear motility rate of straw frozen semen detects
In advance with the water temp. heating to 38 in the thermostat water bath ℃, after with tweezers the tubule of a superfreeze being taken out rapidly from liquid nitrogen, drop in 38 ℃ of water-baths and rock about 30s, take out immediately until completely dissolved, dry the globule with gauze, cut with tubule again and cut off first the ultrasonic sealing end, cut off again the tampon end, seminal fluid in the tubule is all placed in the small test tube, shake up, get one on slide, and covered, microscopy on the phase contrast microscope of being furnished with 38 ℃ of thermostats, the conventional quality standard of evaluation seminal fluid.
The rear semen quality that thaws is identified
1. acrosomal integnity detects.After adopting the peanut agglutinin dyeing of FITC mark, with the integrality of the perforatorium behind the fluorescence microscope detection freeze-thaw.Get the straw semen smear after 30 μ L thaw, afterwards natural air drying.Smear after the natural seasoning is soaked in 20-22 ℃ methyl alcohol and was fixed in 10 minutes.Then the smear after each is air-dry is dripped the dye liquor of the peanut agglutinin of the FITC mark contain 100mg/mL, is placed on the 30min that dyes in 37 ℃ the dark wet box.Afterwards, with the PBS flushing, after the air-dry smear, drip anti-fluorescence quenching, covered after colourless nail polish mounting, checks the situation of perforatorium under fluorescence microscope.
2.DNA integrity detection.After utilizing acridine orange (AO) dyeing, with the integrality of the sperm DNA behind the fluorescence microscope detection freeze-thaw.Sperm dilution after will thawing with the TNE buffer solution is to suitable concentration, and afterwards, the seminal fluid of getting after the 200 μ L dilution adds 400 μ L abstergents immediately.Behind the 30s, add 1.2mL AO dyeing.After the dyeing, get 15 μ L seminal fluid and be added drop-wise on the slide, covered is at the fluorescence microscopy Microscopic observation.Judge that according to the sperm head color sperm DNA is complete or degraded.
3. mitochondria activity detects.After utilizing Rhodamine 123 (R123) and propidium iodide (PI) to dye, active with the mitochondria of sperms behind the fluorescence microscope detection freeze-thaw.Sperm dilution after will thawing with PBS is to suitable concentration, and the seminal fluid of getting after the 1mL dilution adds in the 1.5mL centrifuge tube, to wherein adding 3 μ L Rhodamine 123s.Centrifuge tube is placed on 20min in 37 ℃ the dark wet box, afterwards, adds 10 μ L propidium iodides, place 37 ℃ dark wet box to hatch 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 the slide, drip again the anti-fluorescence quenching of 10 μ L.Slide is placed the fluorescence microscopy Microscopic observation.According to the sperm Show Color, judge that mitochondria of sperms is active.
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.Behind the mixing, place 37 ℃ to hatch 60min, afterwards, get 15 μ L seminal fluid sample drops and be added on the slide, add cover glass after, whether curling according to sperm tail, judge whether plasmalemmae of sperms complete.
The artificial insemination test
With artificial the input in the jenny ass body of oestrusing after the thawing of semen of above-mentioned development, joining pregnant rate is 55.7%, has reached bright smart breeding effect.
Evaluation result
Use the freeze-thaw method of antifreeze of the present invention and seminal fluid, its evaluation result is as follows:
1. the motility rate of sperm reaches more than 40% behind the freeze-thaw;
2. the acrosomal integrity of sperm reaches more than 84%;
In every dose of tubule effectively number of sperm be 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%.
Description of drawings
In order to be illustrated more clearly in the embodiment of the invention or technical scheme of the prior art, the below will do to introduce simply to the accompanying drawing of required use in embodiment or the description of the Prior Art, apparently, accompanying drawing in the following describes only is some embodiments of the present invention, for those of ordinary skills, under the prerequisite of not paying 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 invention, the technical scheme in the embodiment of the invention is clearly and completely described, obviously, described embodiment only is the present invention's part embodiment, rather than whole embodiment.Based on the embodiment among the present invention, those of ordinary skills belong to the scope of protection of the invention not making the every other embodiment that obtains under the creative work prerequisite.
As shown in Figure 1, a kind of casein antifreeze for the freezing preservation of donkey testicular fluid comprises following component in every 100mL antifreeze:
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.
A kind of casein antifreeze for the freezing preservation of donkey testicular fluid comprises following component in every 100mL antifreeze:
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.
A kind of method of the casein antifreeze for the preparation of the freezing preservation of donkey testicular fluid 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 by weighing each component, and is for subsequent use;
2) the above-mentioned casein that takes by weighing, glucose, lactose, HEPES BUFFER are become basal liquid with the HANK solution allocation;
3) add the penicillin, gentamicin, the amphotericin B that take by weighing in the step 1) in the above-mentioned basal liquid that is configured to and be configured to I liquid;
4) be 7.1～7.8 with the whole pH of described I liquid, then carry out filtration sterilization, it is for subsequent use to put into 3～5 ℃ of refrigerators;
5) get described I liquid before the use, to wherein adding the yolk that takes by weighing in the step 1), be configured to II liquid, namely get 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 by weighing.
Embodiment one
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 by weighing each component, and is for subsequent use;
2) the above-mentioned casein that takes by weighing, glucose, lactose, HEPES BUFFER are become basal liquid with the HANK solution allocation;
3) add the penicillin, gentamicin, the amphotericin B that take by weighing in the step 1) in the above-mentioned basal liquid that is configured to and be configured to I liquid;
4) be 7.1 with the whole pH of described I liquid, then carry out filtration sterilization, it is for subsequent use to put into 3 ℃ of refrigerators;
5) get described I liquid before the use, to wherein adding the yolk that takes by weighing in the step 1), be configured to II liquid, namely get described casein antifreeze for the freezing preservation of donkey testicular fluid.
Embodiment two
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 by weighing each component, and is for subsequent use;
2) the above-mentioned casein that takes by weighing, glucose, lactose, HEPES BUFFER are become basal liquid with the HANK solution allocation;
3) add the penicillin, gentamicin, the amphotericin B that take by weighing in the step 1) in the above-mentioned basal liquid that is configured to and be configured to I liquid;
4) be 7.5 with the whole pH of described I liquid, then carry out filtration sterilization, it is for subsequent use to put into 4 ℃ of refrigerators;
5) get described I liquid before the use, to wherein adding the yolk that takes by weighing in the step 1), be configured to II liquid, namely get described casein antifreeze for the freezing preservation of donkey testicular fluid.
Embodiment three
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 by weighing each component, and is for subsequent use;
2) the above-mentioned casein that takes by weighing, glucose, lactose, HEPES BUFFER are become basal liquid with the HANK solution allocation;
3) add the penicillin, gentamicin, the amphotericin B that take by weighing in the step 1) in the above-mentioned basal liquid that is configured to and be configured to I liquid;
4) be 7.8 with the whole pH of described I liquid, then carry out filtration sterilization, it is for subsequent use to put into 5 ℃ of refrigerators;
5) get described I liquid before the use, to wherein adding the yolk that takes by weighing in the step 1), be configured to II liquid, namely get described casein antifreeze for the freezing preservation of donkey testicular fluid.
The above only is preferred embodiment of the present invention, and is in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (4)
1. a casein antifreeze that is used for the freezing preservation of donkey testicular fluid is characterized in that, comprises following component in every 100mL antifreeze:
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.
2. a kind of casein antifreeze for the freezing preservation of donkey testicular fluid as claimed in claim 1 is characterized in that, comprises following component in every 100mL antifreeze:
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.
3. a method for preparing the casein antifreeze for the freezing preservation of donkey testicular fluid as claimed in claim 1 is characterized in that, 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 by weighing each component, and is for subsequent use;
2) the above-mentioned casein that takes by weighing, glucose, lactose, HEPES BUFFER are become basal liquid with the HANK solution allocation;
3) add the penicillin, gentamicin, the amphotericin B that take by weighing in the step 1) in the above-mentioned basal liquid that is configured to and be configured to I liquid;
4) be 7.1～7.8 with the whole pH of described I liquid, then carry out filtration sterilization, it is for subsequent use to put into 3～5 ℃ of refrigerators;
5) get described I liquid before the use, to wherein adding the yolk that takes by weighing in the step 1), be configured to II liquid, namely get described casein antifreeze for the freezing preservation of donkey testicular fluid.
4. the method for a kind of casein antifreeze for the preparation of the freezing preservation of donkey testicular fluid as claimed in claim 3, it is characterized in that: 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 by weighing.
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| CN201210529076.4A CN103039433B (en) | 2012-12-10 | 2012-12-10 | Casein anti-freezing agent for cryopreservation of donkey semen and preparation method thereof |
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| CN103548812A (en) * | 2013-10-30 | 2014-02-05 | 西北农林科技大学 | Normal-temperature and low-temperature preservation diluents of milk goat seminal fluid |
| CN105519519A (en) * | 2015-08-31 | 2016-04-27 | 新疆畜牧科学院畜牧研究所 | Donkey seminal fluid refrigeration preservation method |
| CN107183013A (en) * | 2017-07-14 | 2017-09-22 | 天津农学院 | A kind of method of donkey testicular fluid Cord blood |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103548812A (en) * | 2013-10-30 | 2014-02-05 | 西北农林科技大学 | Normal-temperature and low-temperature preservation diluents of milk goat seminal fluid |
| CN103548812B (en) * | 2013-10-30 | 2015-05-13 | 西北农林科技大学 | Normal-temperature and low-temperature preservation diluents of milk goat seminal fluid |
| CN105519519A (en) * | 2015-08-31 | 2016-04-27 | 新疆畜牧科学院畜牧研究所 | Donkey seminal fluid refrigeration preservation method |
| CN105519519B (en) * | 2015-08-31 | 2018-10-30 | 新疆畜牧科学院畜牧研究所 | A kind of donkey testicular fluid freezing and storing method |
| CN107183013A (en) * | 2017-07-14 | 2017-09-22 | 天津农学院 | A kind of method of donkey testicular fluid Cord blood |
| CN107183013B (en) * | 2017-07-14 | 2021-01-19 | 天津农学院 | Method for preserving donkey semen at low temperature |
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