CN105519521A - Cryopreservation liquid for horse semen and preparation method of cryopreservation liquid - Google Patents
Cryopreservation liquid for horse semen and preparation method of cryopreservation liquid Download PDFInfo
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- CN105519521A CN105519521A CN201610084180.5A CN201610084180A CN105519521A CN 105519521 A CN105519521 A CN 105519521A CN 201610084180 A CN201610084180 A CN 201610084180A CN 105519521 A CN105519521 A CN 105519521A
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- 239000007788 liquid Substances 0.000 title claims abstract description 190
- 210000000582 semen Anatomy 0.000 title claims abstract description 119
- 241000283073 Equus caballus Species 0.000 title claims abstract description 105
- 238000005138 cryopreservation Methods 0.000 title claims abstract description 76
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims abstract description 73
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 67
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims abstract description 56
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- 235000020183 skimmed milk Nutrition 0.000 claims abstract description 45
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims abstract description 38
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims abstract description 38
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 38
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- 239000008103 glucose Substances 0.000 claims abstract description 30
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims abstract description 29
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- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a cryopreservation liquid for horse semen. 100 mL of the cryopreservation liquid for horse semen is prepared from 2.00-2.40 g of glucose, 3.40-4.00 g of lactose, 0.60-1.00 g of trehalose, 0.061-0.092 g of glutathione, 0.50-0.70 g of sodium citrate, 30-50 mL of skimmed milk, 2.5-4.5 mL of egg yolk, 50,000-100,000 IU of penicillin, 50,000-100,000 IU of streptomycin, 3.5-5.5 mL of glycerin and double distilled water. The invention further discloses a preparation method of the cryopreservation liquid. The cryopreservation liquid for horse semen can effectively increase the survival rate and semen activity of the horse semen during cryopreservation. The unfrozen semen has high physiological indexes, has the stable and reliable fecundation effect after field application and can be applied to production. The preparation method comprises few steps and is simple and easy to operate, and the preparation condition is mild and easy to control.
Description
Technical field
The present invention relates to a kind of horse semen cryopreservation liquid and compound method thereof, belong to animal semen freezen protective technical field.
Background technology
Semen cryopreservation is one of horse human assistance propagation technique means.Nineteen fifty, glycerine is applied to horse semen cryopreservation as antifreeze by Smith and Polge the earliest.Nineteen fifty-seven, Barker and Gandier utilizes horse epididymis frozen semen to carry out breeding to become pregnant the earliest.China just started to carry out the research about horse semen cryopreservation as far back as late 1950s, and achieved good achievement.The eighties in 20th century, along with the cultivar registration tissues such as Arab, quarter horse, U.S.'s dapple allow the young coltfoal registration using frozen semen breeding birth, horse semen cryopreservation technology is rapidly developed.Horse semen freezing can expand stallion breeding scope; reduce the propagation of horse genital diseases; protect excellent genetic resources; can cross between race and carry out mating; to semen freezing and the artificial insemination supporting technology of improving horse, the development of Ma Ye, Local Excellent horse kind are preserved, adds the aspects such as flying horse breed improvement and all have great importance.
Freezen protective liquid is the protectant of sperm, has conclusive impact to the effect of semen freezing.At present, the Refrigeration Technique of domestic horse seminal fluid is also under test, and main cause is: in (1) horse semen freezing process, freezing one thaw mechanism, Sperm lesion mechanism, external environment condition change still imperfectly understands all many-sides such as the Changing Pattern of the impact of sperm, the inner component of sperm and molecular mechanisms; (2) composition of a freezing thaw routine, dilution and thawing solution in practical operation is inconsistent, and preparation composition, method are complicated, is not easy to extemporaneous preparation in production and uses; (3) vigor of frozen semen is also difficult to reach artificial insemination requirement.Above problem seriously constrains the application of horse frozen semen in horse breeding and merchandized handling.Therefore, study a kind of horse semen cryopreservation liquid, effectively can improve the survival rate in horse spermatozoa cryopreservation process and vigor, be convenient to a freezing thaw routine operation and use, seem particularly necessary.
Summary of the invention
For solving the deficiencies in the prior art, the object of the present invention is to provide a kind of horse semen cryopreservation liquid, effectively can improve the survival rate of horse seminal fluid in frozen process and sperm viability, the preparation method of this horse semen cryopreservation liquid is simple and easy to realize.
In order to realize above-mentioned target, the present invention adopts following technical scheme:
A kind of horse semen cryopreservation liquid is mixed with 100mL horse semen cryopreservation liquid by glucose 2.00g ~ 2.40g, lactose 3.40g ~ 4.00g, trehalose 0.60g ~ 1.00g, glutathione 0.061 ~ 0.092g, sodium citrate 0.50g ~ 0.70g, skim milk 30mL ~ 50mL, yolk liquid 2.5 ~ 4.5mL, penicillin 50,000 ~ 100,000 IU, streptomycin 50,000 ~ 100,000 IU, glycerine 3.5 ~ 5.5mL and distilled water.
Further, aforementioned horse semen cryopreservation liquid is mixed with 100mL horse semen cryopreservation liquid by glucose 2.20g, lactose 3.70g, trehalose 0.80g, glutathione 0.077g, sodium citrate 0.60g, skim milk 40mL, yolk liquid 3.5mL, penicillin 100,000 IU, streptomycin 100,000 IU, glycerine 4.5mL and distilled water.
A compound method for horse semen cryopreservation liquid, comprises the following steps: (1) preparation skim milk; (2) basal liquid I is prepared; (3) freezen protective liquid is prepared.
The compound method of aforementioned horse semen cryopreservation liquid, specifically comprises the following steps:
(1) skim milk is prepared: after getting the filtration of fresh mare's milk, 100 DEG C of water-bath sterilization 10min, after slow cooling to room temperature, be placed in 4 DEG C of refrigerations (can fully cool, lipid is made to float on upper strata), centrifugal segregation upper strata lipid and lower sediment, obtain skim milk subsequently, and its holding time is generally no more than three days;
(2) basal liquid I is prepared: by measuring glucose, lactose, trehalose, glutathione, sodium citrate, skim milk, yolk liquid, penicillin and streptomycin, first glucose, lactose, trehalose, glutathione and sodium citrate are put into sterile chamber, add distilled water to dissolve, be settled to 100mL, add skim milk, yolk liquid, penicillin and streptomycin subsequently, namely stirring at room temperature obtains basal liquid I;
(3) prepare freezen protective liquid: get basal liquid I96.5mL ~ 94.5mL and glycerine 3.5 ~ 5.5mL, in basal liquid I, add glycerine stir 20min ~ 40min, obtain freezen protective liquid.
The compound method of aforementioned horse semen cryopreservation liquid, in step (1), is centrifugally: with the centrifugal 20 ~ 30min of 2000 ~ 3000r/min rotating speed.
The compound method of aforementioned horse semen cryopreservation liquid, specifically comprises the following steps:
(1) prepare skim milk: extracting degreasing milk powder adds in distilled water, stir, being mixed with concentration is 0.1g/mL solution, obtains skim milk;
(2) basal liquid I is prepared: by measuring glucose, lactose, trehalose, glutathione, sodium citrate, skim milk, yolk liquid, penicillin and streptomycin, first glucose, lactose, trehalose, glutathione and sodium citrate are put into sterile chamber, add distilled water to dissolve, be settled to 100mL, add skim milk, yolk liquid, penicillin and streptomycin subsequently, namely stirring at room temperature obtains basal liquid I;
(3) prepare freezen protective liquid: get basal liquid I96.5mL ~ 94.5mL and glycerine 3.5 ~ 5.5mL, in basal liquid I, add glycerine stir 20min ~ 40min, obtain freezen protective liquid.
The compound method of aforementioned horse semen cryopreservation liquid, in step (2), yolk liquid is obtained by following steps: get Fresh Egg, after removing egg white, being transferred to by yolk on filter paper rolls so that the chalaza of sticking together on membrane of yolk of removing residue egg white and white, then membrane of yolk thorn thoroughly, yolk liquid is collected with in ice-cooled container.
The compound method of aforementioned horse semen cryopreservation liquid, in step (2), stirring at room temperature 20min ~ 45min obtains basal liquid I.
The application of a kind of horse semen cryopreservation liquid in horse semen cryopreservation.
In the application of aforementioned horse semen cryopreservation liquid in horse semen cryopreservation, horse seminal fluid cryopreservation method comprises the following steps: get horse seminal fluid to be saved, add basal liquid I to dilute, the volume ratio of horse seminal fluid and basal liquid I is 1:1 ~ 2, wherein, basal liquid I is by glucose, lactose, trehalose, glutathione, sodium citrate, skim milk, yolk liquid, penicillin, streptomycin and distilled water formulated, subsequently at ambient temperature, with 600 × g centrifugal force, 8 ~ 12min, abandoning supernatant, being diluted to sperm concentration with freezen protective liquid is again 5 × 10
7~ 1 × 10
8individual/mL, after mixing, loads in 0.5mL tubule, and slow cooling to 4 DEG C balance 90 ~ 120min, can make glycerine infiltrate in sperm body, produce freeze proof protective effect, after the stifling 8 ~ 10min in 4 ~ 6cm place (preferred distance liquid nitrogen aspect 4cm eminence), drop in liquid nitrogen and preserve on distance liquid nitrogen surface again.Controlling the rate of temperature fall of straw semen in the present invention by adjusting stifling height and time, fumigating apart from too high or too low, overlong time or too short, sperm all can be made to catch a cold strike, cause irreversible dead sperm consequence.
In the present invention, after seminal fluid adopts Pseudopyloric metaplasia semen collection, filter the jellies such as accessory sex glands with 4 layers of sterile gauze, carry out routine examination after semen collection, finally get sperm viability more than 0.60, density " in " seminal fluid for freezen protective.Course of defrosting is: from liquid nitrogen, take out semen thin tube, immerses rapidly water-bath and thaws, and shake is to melting gently.Thaw point is: 37 DEG C, 30s.
Easily oxidative damage is there is in sperm in refrigerating process, oxidative damage is active chalcogen (ReactiveOxygenSpecies, ROS) generation and the unbalanced result of removing activity: under normal circumstances, polyphenoils in injection seminal fluid refining (SP) has the potentiality removing ROS, but seminal fluid is in process of cryopreservation, seminal fluid centrifugal segregation refining, makes sperm lose the protective effect of refining antioxidant, causes sperm to be subject to oxidative attacks; Refrigerating process can produce ROS by induced sperm again in addition.Wherein defectiveness sperm and Necrospermia are the main sources of ROS between pool period.ROS, by oxidation unsaturated fatty acid, destroys plasma membrane lipid bimolecular structure, causes spermoblast film functional disorder and interior damage.
Carbohydrate is the element of semen cryopreservation liquid, both can provide energy for sperm metabolism, is again important cryoprotector.Trehalose, be the nonreducing sugar be made up of two glucose molecules, character is highly stable.Trehalose can contain Ca effectively
2+flowing, slow down the instantaneous variation of intraor extracellular osmotic pressure, Cell protection film is not destroyed.It can also be combined by the hydrogen on sperm membrane, forms a kind of protective layer, effectively can stop the outflow of the inner active ingredient of sperm when sperm membrane breaks.In addition, the change of moisture in trehalose controllable cell and between solution, in freeze-thaw process, Cell protection is from damage.Physical configuration and the chemical property of the defense mechanism of trehalose and its crystal structure, solution are closely related.When sperm freezing, trehalose can strong Bound Water Molecules, own Bound moisture together with membrane lipid or itself play the function replacing film Bound moisture, keep moistening in cell, prevent cell from causing the loss of nutrient and the damage of cell because of dehydration, there is the characteristic of stabilizing cell membrane and protein structure.
The penetrating sperm membrane of glycerine very capable, can concentrate or in conjunction with moisture in born of the same parents, reduce the osmotic pressure of salinity and freezing liquid in solution.But glycerine has certain toxic action to sperm.Harm being reduced to minimum for reaching protective effect simultaneously, in freezen protective formula of liquid of the present invention, reducing glycerol concentration.Time in 100mL freezen protective liquid containing glycerine 3.5 ~ 5.5mL, after a freezing course of defrosting, sperm viability is the highest.
Trehalose is when mammalian sperm deep-bed drying, and only having could available protecting sperm with glycerine conbined usage.Therefore, when the trehalose in freezen protective liquid and glycerine acting in conjunction, be more suitable for protecting the cellular morphology of horse sperm and cell function etc., the formation of ice crystal in block cell, effectively counteracting glycerine is to the toxic action of sperm.In addition, the double action of trehalose is very beneficial for the integrality protecting equine sperm cell film, can stand the maturation relevant with sperm viability and fertility and change.Trehalose and glycerine, in conjunction with acting in conjunction, finally play best protective effect to horse sperm.
Glutathione (GSH) is non-protein sulfhydryl compound important in mammalian cell; it is as the agent of peroxidating-reductase system; sperm can be protected in time to exempt from the injury of oxidation and peroxidization termination product; make sperm lipid membrane structures keep stable, finally improve semen quality.
Reagent: glycerine (Japan), benzylpenicillin sodium for injection (HARBIN PHARMACEUTICAL GROUP CO., LTD. General Pharm. Factory), streptomycin sulphate for injection (Dalian Merro Pharmaceutical Co., Ltd.), skim milk powder (Britain), glucose (Sigma), lactose (Sigma), trehalose (Sigma).
Instrument: TDZ5-WS type centrifuge (Changsha Xiang Zhi centrifuge Instrument Ltd.), 400 × stereomicroscope (Olympus), high-temperature high-pressure sterilizing chamber (Shanghai Boxun Industrial Co., Ltd.).
Have detected different formulations horse semen cryopreservation liquid respectively and carry out freezen protective, on the impact of sperm quality after freeze thawing.Specific experiment process is as follows:
Choose 5-15 year blood horse 2, Mongolian horse 2, weekly collecting semen 3 times, vigor more than 0.6 carries out freezen protective.2 weeks totally 24 groups of samples.Use 4 different formulations freezen protective respectively, wherein, be adopt horse semen cryopreservation liquid of the present invention from dosing 1 group and 2 groups, INRA82 and INRA96 is commercially available prod (French IMV (card Soviet Union) company).Thaw point is 37 DEG C, 30s.Freezing and storing method is: get horse seminal fluid to be saved, add basal liquid I to dilute, the volume ratio of horse seminal fluid and basal liquid I is 1:1, wherein, basal liquid I is formulated by glucose, lactose, trehalose, glutathione, sodium citrate, skim milk, yolk liquid, penicillin, streptomycin and distilled water, subsequently at ambient temperature, with 600 × g centrifugal force 10min, abandoning supernatant, then to be diluted to sperm concentration with freezen protective liquid be 1 × 10
8individual/mL, after mixing, loads in 0.5mL tubule, slow cooling to 4 DEG C balance 100min, then on distance liquid nitrogen surface after the stifling 8min in 4cm place, drops in liquid nitrogen and preserve.
Concrete detection method is: the mensuration of the sperm after thawing being carried out indices, kinematic parameter, abnormal rate, the bacterial population of more each experimental group freezing front and back sperm.Detect with CASA (Computer-aidedspermanalysis) compater assisted sperm analysis after different formulations freeze thawing, each sample detection 3 repetition.
(1) CASA detects sperm concentration, and each detection at least reaches the sperm of 5 visuals field and more than 200;
(2) a Samples detection 3 times, will observe the visual field of more than 15, the sperm of more than 600 altogether.
Rate of teratosperm is surveyed by Ji's nurse Sa decoration method.I.e. smear-dry-fix-wash-dry-dye-wash-dry-microscopy.At least observe 200 sperms under 400 power microscopes, repeat 2 times.Bacterial population plain agar cultivation.Put in 37 DEG C of insulating boxs to cultivate after 48 hours and take out, clump count in counting plate.Often organize sample and get two flags, two plates, average.
By SAS9.0 software, all data all adopt one-way analysis of variance method to process, wherein, and TM=total activity; PM=forward motile sperm percentage; VCL=mean curvilinear velocity (μm/s); VSL=mean linear speed; VAP=average path speed; ALH=sperm head side-sway amplitude; LIN=linearity, the linearity of curvilinear path, VSL/VCL; Tropism before STR=, the linearity in space average path, VSL/VAP; BCF=beat frequency (Hz).Experimental result is as shown in table 1.
As shown in Table 1, in the sample with a collection of fresh essence, after different dilution process, after freeze-thaw, routine index of semen evaluation result is variant, in 4 experimental group, from dosing 1 group and 2 groups of total activity average out to 68.75 and 64.14, higher than INRA group 2 groups, significant difference (P < 0.05).According to every detection of seminal fluid after thawing, semen cryopreservation liquid (from dosing) of the present invention is in the control of seminal fluid abnormal rate, bacterial population and on the basis of commercialization freezen protective liquid no significant difference, effectively improve the total activity of seminal fluid, forward motile sperm percentage, stability is best.
Also screening experiment is carried out to glutathione in the present invention.Specific experiment process is as follows: choose 5-15 year Mongolian horse 4, weekly collecting semen 3 times, vigor more than 0.6 carries out freezen protective.1 week totally 12 groups of sample.Often organize design 3 test gradient group (preparation 100mL freezen protective liquid, glutathione raw material addition is respectively 0.077g, 0.154g, 0.230g) and 1 control group (not adding glutathione).Thaw point is 37 DEG C, 30s.Concrete freezing and storing method is tested identical with different formulations horse semen cryopreservation liquid on the impact of sperm quality after freeze thawing with detection method.Test result is as shown in table 2.
As shown in Table 2, the 100mL freezen protective liquid obtained when glutathione raw material addition is 0.077g, for preserving horse seminal fluid, freeze essence thaw after the total activity of sperm, forward motile sperm percentage be all better than the reduced glutathione of other concentration, illustrate and effectively can improve the sperm viability of frozen semen by the 100mL freezen protective liquid that glutathione raw material addition obtains when being 0.077g.
In addition, adopt horse semen cryopreservation liquid of the present invention and freezing and storing method freezing after horse seminal fluid for artificial insemination, breeding 32 mares in feelings phase, wherein conceived 15, conception rate is 46.88%.
Table 1 different formulations horse semen cryopreservation liquid is on the impact of sperm quality after freeze thawing
Table 2 adds the impact of variable concentrations glutathione on horse semen cryopreservation effect
Note: represent significant difference (P < 0.05) with having different Superscript letters person in a line in table 1 and table 2, identical table differential is different not significantly (P > 0.05).
Usefulness of the present invention is: a kind of horse semen cryopreservation liquid provided by the invention, effectively can improve the survival rate of horse seminal fluid in frozen process and sperm viability.After thawing, sperm physical signs is high, effectively improves the total activity of seminal fluid, forward motile sperm percentage, and on-the-spot application is impregnated effect stability reliably, can be used for producing.The raw material of this horse semen cryopreservation liquid is easy to get, and cost is low, and preparation method's step is few, simple to operation, and preparation condition gentleness is easy to control.Adopt the freezing and storing method of horse semen cryopreservation liquid of the present invention, condition is easy to control, and easily realize, controlled-rate freezing is good.By adopt horse semen cryopreservation liquid of the present invention and freezing and storing method freezing after horse seminal fluid be used for artificial insemination, breeding 32 mares in feelings phase, wherein conceived 15, conception rate is 46.88%.
Embodiment
Below in conjunction with specific embodiment, the present invention is further introduced.
Embodiment 1
A kind of horse semen cryopreservation liquid, is mixed with 100mL horse semen cryopreservation liquid by glucose 2.00g, lactose 3.40g, trehalose 0.60g, glutathione 0.061g, sodium citrate 0.50g, skim milk 30mL, yolk liquid 2.5mL, penicillin 50,000 IU, streptomycin 50,000 IU, glycerine 3.5mL and distilled water.
Concrete compound method comprises the following steps:
(1) prepare skim milk: after getting the filtration of fresh mare's milk, 100 DEG C of water-bath sterilization 10min, after slow cooling to room temperature, are placed in 4 DEG C of refrigerations, subsequently with the centrifugal 30min of 2000r/min rotating speed, remove upper strata lipid and lower sediment, obtain skim milk;
(2) basal liquid I is prepared: by measuring glucose, lactose, trehalose, glutathione, sodium citrate, skim milk, yolk liquid, penicillin and streptomycin, first glucose, lactose, trehalose, glutathione and sodium citrate are put into sterile chamber, add distilled water to dissolve, be settled to 100mL, add skim milk, yolk liquid, penicillin and streptomycin subsequently, namely stirring at room temperature 20min obtains basal liquid I;
(3) prepare freezen protective liquid: get basal liquid I96.5mL and glycerine 3.5mL, in basal liquid I, add glycerine stir 20min, obtain freezen protective liquid.
Wherein, yolk liquid is obtained by following steps: get Fresh Egg, and after removing egg white, then membrane of yolk thorn thoroughly, yolk liquid is collected with in ice-cooled container.
Embodiment 2
A kind of horse semen cryopreservation liquid, is mixed with 100mL horse semen cryopreservation liquid by glucose 2.40g, lactose 4.00g, trehalose 1.00g, glutathione 0.092g, sodium citrate 0.70g, skim milk 50mL, yolk liquid 4.5mL, penicillin 60,000 IU, streptomycin 80,000 IU, glycerine 5.5mL and distilled water.
The compound method of aforementioned horse semen cryopreservation liquid, specifically comprises the following steps:
(1) prepare skim milk: extracting degreasing milk powder adds in distilled water, stir, being mixed with concentration is 0.1g/mL solution, obtains skim milk;
(2) basal liquid I is prepared: by measuring glucose, lactose, trehalose, glutathione, sodium citrate, skim milk, yolk liquid, penicillin and streptomycin, first glucose, lactose, trehalose, glutathione and sodium citrate are put into sterile chamber, add distilled water to dissolve, be settled to 100mL, add skim milk, yolk liquid, penicillin and streptomycin subsequently, namely stirring at room temperature 45min obtains basal liquid I;
(3) prepare freezen protective liquid: get basal liquid I94.5mL and glycerine 5.5mL, in basal liquid I, add glycerine stir 40min, obtain freezen protective liquid.
Wherein, yolk liquid is obtained by following steps: get Fresh Egg, after removing egg white, is transferred to by yolk on filter paper and rolls so that the chalaza of sticking together on membrane of yolk of removing residue egg white and white, then membrane of yolk thorn thoroughly, yolk liquid is collected with in ice-cooled container.
Embodiment 3
Horse semen cryopreservation liquid, is mixed with 100mL horse semen cryopreservation liquid by glucose 2.20g, lactose 3.70g, trehalose 0.80g, glutathione 0.077g, sodium citrate 0.60g, skim milk 40mL, yolk liquid 3.5mL, penicillin 100,000 IU, streptomycin 100,000 IU, glycerine 4.5mL and distilled water.
The compound method of horse semen cryopreservation liquid, specifically comprises the following steps:
(1) prepare skim milk: after getting the filtration of fresh mare's milk, 100 DEG C of water-bath sterilization 10min, after slow cooling to room temperature, are placed in 4 DEG C of refrigerations, subsequently with the centrifugal 20min of 3000r/min rotating speed, remove upper strata lipid and lower sediment, obtain skim milk;
(2) basal liquid I is prepared: by measuring glucose, lactose, trehalose, glutathione, sodium citrate, skim milk, yolk liquid, penicillin and streptomycin, first glucose, lactose, trehalose, glutathione and sodium citrate are put into sterile chamber, add distilled water to dissolve, be settled to 100mL, add skim milk, yolk liquid, penicillin and streptomycin subsequently, namely stirring at room temperature 30min obtains basal liquid I;
(3) prepare freezen protective liquid: get basal liquid I95.5mL and glycerine 4.5mL, in basal liquid I, add glycerine stir 30min, obtain freezen protective liquid.
Wherein, yolk liquid is obtained by following steps: get Fresh Egg, after removing egg white, is transferred to by yolk on filter paper and rolls so that the chalaza of sticking together on membrane of yolk of removing residue egg white and white, then membrane of yolk thorn thoroughly, yolk liquid is collected with in ice-cooled container.
Embodiment 4 ~ 6 is the horse semen cryopreservation liquid embody rule in horse semen cryopreservation embodiment 1 ~ 3 obtained.
Embodiment 4
The application of horse semen cryopreservation liquid in horse semen cryopreservation of embodiment 1 ~ 3, concrete store method comprises the following steps: get horse seminal fluid to be saved, add basal liquid I to dilute, the volume ratio of horse seminal fluid and basal liquid I is 1:1.5, wherein, basal liquid I is formulated by glucose, lactose, trehalose, glutathione, sodium citrate, skim milk, yolk liquid, penicillin, streptomycin and distilled water, subsequently at ambient temperature, with 600 × g centrifugal force 8min, abandoning supernatant, then to be diluted to sperm concentration with freezen protective liquid be 5 × 10
7individual/mL, after mixing, loads in 0.5mL tubule, slow cooling to 4 DEG C balance 90min; After the stifling 10min in 5cm place, drop in liquid nitrogen and preserve on distance liquid nitrogen surface again.
Embodiment 5
The application of horse semen cryopreservation liquid in horse semen cryopreservation of embodiment 1 ~ 3, concrete store method comprises the following steps: get horse seminal fluid to be saved, add basal liquid I to dilute, the volume ratio of horse seminal fluid and basal liquid I is 1:2, wherein, basal liquid I is formulated by glucose, lactose, trehalose, glutathione, sodium citrate, skim milk, yolk liquid, penicillin, streptomycin and distilled water, subsequently at ambient temperature, with 600 × g centrifugal force 12min, abandoning supernatant, then to be diluted to sperm concentration with freezen protective liquid be 8 × 10
7individual/mL, after mixing, loads in 0.5mL tubule, slow cooling to 4 DEG C balance 120min; After the stifling 9min in 6cm place, drop in liquid nitrogen and preserve on distance liquid nitrogen surface again.
Embodiment 6
The application of horse semen cryopreservation liquid in horse semen cryopreservation of embodiment 1 ~ 3, concrete store method comprises the following steps: get horse seminal fluid to be saved, add basal liquid I to dilute, the volume ratio of horse seminal fluid and basal liquid I is 1:1, wherein, basal liquid I is formulated by glucose, lactose, trehalose, glutathione, sodium citrate, skim milk, yolk liquid, penicillin, streptomycin and distilled water, subsequently at ambient temperature, with 600 × g centrifugal force 10min, abandoning supernatant, then to be diluted to sperm concentration with freezen protective liquid be 1 × 10
8individual/mL, after mixing, loads in 0.5mL tubule, slow cooling to 4 DEG C balance 100min; After the stifling 8min in 4cm place, drop in liquid nitrogen and preserve on distance liquid nitrogen surface again.
Claims (10)
1. a horse semen cryopreservation liquid, is characterized in that: be mixed with 100mL horse semen cryopreservation liquid by glucose 2.00g ~ 2.40g, lactose 3.40g ~ 4.00g, trehalose 0.60g ~ 1.00g, glutathione 0.061 ~ 0.092g, sodium citrate 0.50g ~ 0.70g, skim milk 30mL ~ 50mL, yolk liquid 2.5 ~ 4.5mL, penicillin 50,000 ~ 100,000 IU, streptomycin 50,000 ~ 100,000 IU, glycerine 3.5 ~ 5.5mL and distilled water.
2. horse semen cryopreservation liquid according to claim 1, is characterized in that: be mixed with 100mL horse semen cryopreservation liquid by glucose 2.20g, lactose 3.70g, trehalose 0.80g, glutathione 0.077g, sodium citrate 0.60g, skim milk 40mL, yolk liquid 3.5mL, penicillin 100,000 IU, streptomycin 100,000 IU, glycerine 4.5mL and distilled water.
3. the compound method of horse semen cryopreservation liquid as claimed in claim 1 or 2, is characterized in that: comprise the following steps: (1) preparation skim milk; (2) basal liquid I is prepared; (3) freezen protective liquid is prepared.
4. the compound method of horse semen cryopreservation liquid according to claim 3, is characterized in that: comprise the following steps:
(1) prepare skim milk: after getting the filtration of fresh mare's milk, 100 DEG C of water-bath sterilization 10min, after slow cooling to room temperature, be placed in 4 DEG C of refrigerations, centrifugal segregation upper strata lipid and lower sediment, obtain skim milk subsequently;
(2) basal liquid I is prepared: by measuring glucose, lactose, trehalose, glutathione, sodium citrate, skim milk, yolk liquid, penicillin and streptomycin, first glucose, lactose, trehalose, glutathione and sodium citrate are put into sterile chamber, add distilled water to dissolve, be settled to 100mL, add skim milk, yolk liquid, penicillin and streptomycin subsequently, namely stirring at room temperature obtains basal liquid I;
(3) prepare freezen protective liquid: get basal liquid I96.5mL ~ 94.5mL and glycerine 3.5 ~ 5.5mL, in basal liquid I, add glycerine stir 20min ~ 40min, obtain 100mL freezen protective liquid.
5. the compound method of horse semen cryopreservation liquid according to claim 4, is characterized in that: in described step (1), is centrifugally: with the centrifugal 20 ~ 30min of 2000 ~ 3000r/min rotating speed.
6. the compound method of horse semen cryopreservation liquid according to claim 3, is characterized in that: comprise the following steps:
(1) prepare skim milk: extracting degreasing milk powder adds in distilled water, stir, being mixed with concentration is 0.1g/mL solution, obtains skim milk;
(2) basal liquid I is prepared: by measuring glucose, lactose, trehalose, glutathione, sodium citrate, skim milk, yolk liquid, penicillin and streptomycin, first glucose, lactose, trehalose, glutathione and sodium citrate are put into sterile chamber, add distilled water to dissolve, be settled to 100mL, add skim milk, yolk liquid, penicillin and streptomycin subsequently, namely stirring at room temperature obtains basal liquid I;
(3) prepare freezen protective liquid: get basal liquid I96.5mL ~ 94.5mL and glycerine 3.5 ~ 5.5mL, in basal liquid I, add glycerine stir 20min ~ 40min, obtain freezen protective liquid.
7. the compound method of the horse semen cryopreservation liquid according to claim 4 or 6, it is characterized in that: in described step (2), yolk liquid is obtained by following steps: get Fresh Egg, after removing egg white, membrane of yolk thorn thoroughly, yolk liquid is collected with in ice-cooled container.
8. the compound method of the horse semen cryopreservation liquid according to claim 4 or 6, is characterized in that: in described step (2), stirring at room temperature 20min ~ 45min obtains basal liquid I.
9. the application of horse semen cryopreservation liquid in horse semen cryopreservation as claimed in claim 1 or 2.
10. the application of horse semen cryopreservation liquid according to claim 9 in horse semen cryopreservation, it is characterized in that: horse seminal fluid cryopreservation method comprises the following steps: get horse seminal fluid to be saved, add basal liquid I to dilute, the volume ratio of horse seminal fluid and basal liquid I is 1:1 ~ 2, wherein, basal liquid I is formulated by glucose, lactose, trehalose, glutathione, sodium citrate, skim milk, yolk liquid, penicillin, streptomycin and distilled water; Subsequently at ambient temperature, with 600 × g centrifugal force, 8 ~ 12min, abandoning supernatant, then to be diluted to sperm concentration with freezen protective liquid be 5 × 10
7~ 1 × 10
8individual/mL, after mixing, loads in 0.5mL tubule, slow cooling to 4 DEG C balance 90 ~ 120min, then on distance liquid nitrogen surface after the stifling 8 ~ 10min in 4 ~ 6cm place, drops in liquid nitrogen and preserve.
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| NL2036490B1 (en) * | 2023-12-12 | 2024-08-09 | Northwest Agriculture & Forestry Univ | Freezing diluent of east friensian milk sheep semen and application thereof |
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