CN105519521B - A kind of horse semen cryopreservation liquid and its preparation method - Google Patents
A kind of horse semen cryopreservation liquid and its preparation method Download PDFInfo
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- CN105519521B CN105519521B CN201610084180.5A CN201610084180A CN105519521B CN 105519521 B CN105519521 B CN 105519521B CN 201610084180 A CN201610084180 A CN 201610084180A CN 105519521 B CN105519521 B CN 105519521B
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- 241000283073 Equus caballus Species 0.000 title claims abstract description 102
- 210000000582 semen Anatomy 0.000 title claims abstract description 83
- 238000005138 cryopreservation Methods 0.000 title claims abstract description 69
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 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 72
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 65
- 235000013336 milk Nutrition 0.000 claims abstract description 50
- 239000008267 milk Substances 0.000 claims abstract description 50
- 210000004080 milk Anatomy 0.000 claims abstract description 50
- 210000002969 egg yolk Anatomy 0.000 claims abstract description 48
- 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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- 239000012153 distilled water Substances 0.000 claims abstract description 20
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 28
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 20
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- OQUFOZNPBIIJTN-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;sodium Chemical compound [Na].OC(=O)CC(O)(C(O)=O)CC(O)=O OQUFOZNPBIIJTN-UHFFFAOYSA-N 0.000 claims 1
- 238000001816 cooling Methods 0.000 claims 1
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- 230000008014 freezing Effects 0.000 abstract description 24
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- 239000003642 reactive oxygen metabolite Substances 0.000 description 1
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- 235000020183 skimmed milk Nutrition 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 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
- -1 sulfhydryl compound Chemical class 0.000 description 1
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
Landscapes
- 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 kind of horse semen cryopreservation liquid, 100mL horse semen cryopreservation liquid is configured to by glucose 2.00g~2.40g, lactose 3.40g~4.00g, trehalose 0.60g~1.00g, 0.061~0.092g of glutathione, sodium citrate 0.50g~0.70g, defatted milk 30mL~50mL, 2.5~4.5mL of yolk liquid, the IU of penicillin 50,000~100,000, the IU of streptomysin 50,000~100,000,3.5~5.5mL of glycerine and distilled water.Also disclose the preparation method of the freezen protective liquid.Horse semen cryopreservation liquid provided by the invention can effectively improve survival rate and sperm motility of the horse sperm during freezing.Sperm physical signs is high after defrosting, and field application fertilization effect stability is reliable, available for producing.Preparation method step is few, simple to operation, and preparation condition is mildly easy to control.
Description
Technical field
The present invention relates to a kind of horse semen cryopreservation liquid and its preparation methods, belong to animal semen freezen protective technology neck
Domain.
Background technology
Semen cryopreservation is one of horse human assistance reproduction technique means.Nineteen fifty, Smith and Polge earliest will be sweet
Oil is applied to horse semen cryopreservation as antifreeze.Nineteen fifty-seven, Barker and Gandier utilize horse epididymis frozen semen earliest
Breeding is carried out to become pregnant.China began to carry out the research in relation to horse semen cryopreservation, and obtain early in late 1950s
Preferable achievement.In the 1980s, as the cultivar registrations tissues such as Arab, quarter horse, U.S.'s dapple allow to use
The young coltfoal registration of frozen semen breeding birth so that horse semen cryopreservation technology is rapidly developed.Horse freezing of semen can expand
Big stallion breeding range, reduces the propagation of horse genital diseases, protects excellent genetic resources, can be handed over across between race
Match, to improving the freezing of semen of horse and artificial insemination supporting technology, development, Local Excellent horse kind for Ma Ye are preserved, added
Flying horse breed improvement etc. all has great importance.
Freezen protective liquid is the protective agent of sperm, has conclusive influence to the effect of freezing of semen.At present, domestic horse essence
The Refrigeration Technique of liquid also in experimental stage, main reason is that：(1) a defrosting mechanism, essence are freezed during horse freezing of semen
Sub- micromechanism of damage, influence of the external environment variation to sperm, the changing rule of component and molecular mechanism etc. are all inside sperm
Various aspects are not fully understood；(2) composition of one thaw routine of freezing in practical operation, dilution and thawing solution is inconsistent,
It is complicated to prepare ingredient, method, is not easy to extemporaneous preparation in production and uses；(3) vigor of frozen semen is also difficult to reach and manually award
Essence requirement.Problem above seriously constrains application of the horse frozen semen in horse breeding and merchandized handling.Therefore, research is a kind of
Horse semen cryopreservation liquid can effectively improve survival rate and vigor during horse spermatozoa cryopreservation, convenient for freezing a defrosting journey
Sequence operates with, it appears particularly necessary.
Invention content
To solve the deficiencies in the prior art, the purpose of the present invention is to provide a kind of horse semen cryopreservation liquid, Neng Gouyou
Effect improves survival rate and sperm motility of the horse sperm during freezing, and the preparation method of the horse semen cryopreservation liquid is simply easy
It realizes.
In order to realize above-mentioned target, the present invention adopts the following technical scheme that：
A kind of horse semen cryopreservation liquid is by glucose 2.00g~2.40g, lactose 3.40g~4.00g, trehalose
0.60g~1.00g, 0.061~0.092g of glutathione, sodium citrate 0.50g~0.70g, defatted milk 30mL~50mL, yolk
The IU of the IU of 2.5~4.5mL of liquid, penicillin 50,000~100,000, streptomysin 50,000~100,000,3.5~5.5mL of glycerine and distilled water are configured to
100mL horse semen cryopreservation liquid.
Further, aforementioned horse semen cryopreservation liquid, be by glucose 2.20g, lactose 3.70g, trehalose 0.80g,
Glutathione 0.077g, sodium citrate 0.60g, defatted milk 40mL, yolk liquid 3.5mL, 100,000 IU of penicillin, streptomysin 100,000
IU, glycerine 4.5mL and distilled water are configured to 100mL horse semen cryopreservation liquid.
A kind of preparation method of horse semen cryopreservation liquid, includes the following steps：(1) defatted milk is prepared；(2) basis is prepared
Liquid I；(3) freezen protective liquid is prepared.
The preparation method of aforementioned horse semen cryopreservation liquid, specifically includes following steps：
(1) defatted milk is prepared：After fresh mare's milk is taken to filter, 100 DEG C of water-bath sterilization 10min after slow cooling to room temperature, are put
(can be fully cooled, lipid is made to float on upper strata) is refrigerated in 4 DEG C, then centrifugation removal upper strata lipid and lower sediment,
Defatted milk is obtained, the holding time is usually no more than three days；
(2) basal liquid I is prepared：By measurement glucose, lactose, trehalose, glutathione, sodium citrate, defatted milk, yolk
Glucose, lactose, trehalose, glutathione and sodium citrate are first put into sterile chamber, add by liquid, penicillin and streptomysin
Enter distilled water dissolving, 100mL is settled to, then adds in defatted milk, yolk liquid, penicillin and streptomysin, is stirred at room temperature up to base
Plinth liquid I；
(3) freezen protective liquid is prepared：3.5~5.5mL of basal liquid I 96.5mL~94.5mL and glycerine is taken, in basal liquid I
Middle addition glycerine stirs 20min~40min to get freezen protective liquid.
The preparation method of aforementioned horse semen cryopreservation liquid in step (1), centrifuges and is：With 2000~3000r/min rotating speeds
Centrifuge 20~30min.
The preparation method of aforementioned horse semen cryopreservation liquid, specifically includes following steps：
(1) defatted milk is prepared：Extracting degreasing milk powder is added in distilled water, and stirring is configured to a concentration of 0.1g/mL solution, obtains
To defatted milk；
(2) basal liquid I is prepared：By measurement glucose, lactose, trehalose, glutathione, sodium citrate, defatted milk, yolk
Glucose, lactose, trehalose, glutathione and sodium citrate are first put into sterile chamber, add by liquid, penicillin and streptomysin
Enter distilled water dissolving, be settled to 100mL, then add in defatted milk, yolk liquid, penicillin and streptomysin, be stirred at room temperature up to base
Plinth liquid I；
(3) freezen protective liquid is prepared：3.5~5.5mL of basal liquid I 96.5mL~94.5mL and glycerine is taken, in basal liquid I
Middle addition glycerine stirs 20min~40min to get freezen protective liquid.
The preparation method of aforementioned horse semen cryopreservation liquid, in step (2), yolk liquid is obtained by following steps：It takes new
Fresh hen egg after removing egg white, yolk is transferred on filter paper to roll and is sticked together to remove remaining egg white and white in yolk
Chalaza on film, then pierces membrane of yolk, and yolk liquid is collected into the container being cooled with ice.
The preparation method of aforementioned horse semen cryopreservation liquid in step (2), is stirred at room temperature 20min~45min and obtains basis
Liquid I.
A kind of application of horse semen cryopreservation liquid in horse semen cryopreservation.
In application of the aforementioned horse semen cryopreservation liquid in horse semen cryopreservation, horse seminal fluid cryopreservation method includes
Following steps：Horse sperm to be saved is taken, basal liquid I is added in and is diluted, the volume ratio of horse sperm and basal liquid I are 1:1~
2, wherein, basal liquid I be by glucose, lactose, trehalose, glutathione, sodium citrate, defatted milk, yolk liquid, penicillin,
Streptomysin and distilled water are formulated, and then at ambient temperature, with 600 × g, 8~12min of centrifugal force, are discarded supernatant
Liquid, then it is 5 × 10 to be diluted to sperm concentration with freezen protective liquid7~1 × 108A/mL after mixing, is packed into 0.5mL tubules
In, slow cooling can be such that glycerine penetrates into sperm body, generate freeze proof protective effect to 4 DEG C of 90~120min of balance；Again away from
After 8~10min stifling at 4~6cm on liquid nitrogen surface (preferred distance liquid nitrogen level 4cm eminences), put into liquid nitrogen and preserve.This hair
The rate of temperature fall of straw semen is controlled in bright by adjusting stifling height and time, is fumigated apart from too high or too low, time mistake
It is long or too short, sperm can all be made to be cooled strike, cause irreversible dead sperm consequence.
In the present invention, after sperm uses Pseudopyloric metaplasia semen collection, the jellies such as accessory sex gland, semen collection are filtered with 4 layers of sterile gauze
After carry out routine examination, finally take sperm motility more than 0.60, density " in " sperm be used for freezen protective.Course of defrosting
For：Semen thin tube is taken out from liquid nitrogen, it is rapid to immerse water-bath defrosting, it gently shakes to thawing.Thaw point is：37℃、30s.
Oxidative damage easily occurs in refrigerating process for sperm, and oxidative damage is active chalcogen (Reactive Oxygen
Species, ROS) generate and the unbalanced result of removing activity：Under normal circumstances, it projects anti-oxidant in sperm refining (SP)
Substance has the potentiality for removing ROS, but sperm, in process of cryopreservation, sperm centrifugation eliminates refining, loses sperm
The protective effect of refining antioxidant causes sperm easily by oxidative attacks；In addition refrigerating process can induce sperm and generates ROS again.
The main source of ROS during wherein defective sperm and Necrospermia are freezings.ROS can be destroyed by aoxidizing unrighted acid
Plasma membrane lipid bimolecular structure causes spermatoblast film dysfunction and internal structure to be damaged.
Carbohydrate is the element of semen cryopreservation liquid, energy can be both provided for sperm metabolism, and be important
Cryoprotector.Trehalose, is the nonreducing sugar being made of two glucose molecules, and property is highly stable.Trehalose energy
It is enough effectively to contain Ca2+Flowing, slow down the instantaneous variation of intraor extracellular osmotic pressure, cell membrane protected not to be destroyed.It may be used also
To be combined with the hydrogen on sperm membrane, a kind of protective layer is formed, can effectively stop inside sperm in the case of sperm membrane fracture
The outflow of active ingredient.In addition, the variation of the controllable intracellular moisture between solution of trehalose, during freeze-thaw
It protects cells from damaging.The protection mechanism of trehalose and its crystal structure, the physical configuration of solution and the close phase of chemical characteristic
It closes.In sperm freezing, trehalose can be co-owned with reference to water or itself risen with membrane lipid with strong Bound Water Moleculess
To the function for replacing film combination water, intracellular moistening is kept, prevents cell from causing the loss of nutrient and the damage of cell due to dehydration
Wound has the characteristic of stabilizing cell membrane and protein structure.
The penetrating sperm membrane of glycerine it is very capable, can concentrate or with reference to intracellular moisture, reduce salinity and freezing in solution
The osmotic pressure of liquid.But glycerine has sperm certain toxic action.To reach protective effect while harm being reduced to minimum, this hair
Glycerol concentration is reduced in bright freezen protective formula of liquid.When containing 3.5~5.5mL of glycerine in 100mL freezen protective liquid, pass through
Freeze sperm motility highest after a course of defrosting.
For trehalose in mammalian sperm deep-bed drying, being only used in combination with glycerine could effective protection sperm.
Therefore, when the trehalose in freezen protective liquid is with glycerine collective effect, more suitable for protecting the cellular morphology and cell of horse sperm
Toxic action of the glycerine to sperm is effectively offset in function etc., the formation of ice crystal in block cell.In addition, the dual work of trehalose
With the integrality for being very beneficial for protecting equine sperm cell film, the ripe change related with sperm motility and fertility can be subjected to
Change.Both trehalose and glycerine combine collective effect, and best protective effect is finally played to horse sperm.
Glutathione (GSH) is non-protein sulfhydryl compound important in mammalian cell, it as peroxidating-also
The agent of protoenzyme system can protect sperm from oxidation and the injury of peroxidization termination product, make sperm adipose membrane in time
Structure remained stable finally improves semen quality.
Reagent：Glycerine (Japan), benzylpenicillin sodium for injection (HARBIN PHARMACEUTICAL GROUP CO., LTD. General Pharm. Factory), streptomycin sulphate for injection are (big
The even big pharmaceutical factory of Metro), skimmed milk power (Britain), glucose (Sigma), lactose (Sigma), trehalose (Sigma).
Instrument：TDZ5-WS types centrifuge (Changsha Xiang Zhi centrifuges Instrument Ltd.), 400 × stereomicroscope
(Olympus), high-temperature high-pressure sterilizing chamber (Shanghai Boxun Industrial Co., Ltd.).
Different formulations horse semen cryopreservation liquid is had detected respectively and carries out freezen protective, to the shadow of sperm quality after freeze thawing
It rings.Specific experiment process is as follows：
5-15 Sui blood horse 2 of selection, Mongolian horse 2, weekly collecting semen 3 times, more than 0.6 vigor carry out freezing guarantor
It deposits.2 weeks totally 24 groups of samples.Respectively with 4 different formulations freezen protectives, wherein, 1 group and 2 groups of autogamy liquid is using the present invention
Horse semen cryopreservation liquid, INRA82 and INRA96 are commercial product (French IMV (card Soviet Union) company).Thaw point is 37
℃,30s.Freezing and storing method is：Horse sperm to be saved is taken, basal liquid I is added in and is diluted, horse sperm is with basal liquid I's
Volume ratio is 1:1, wherein, basal liquid I is by glucose, lactose, trehalose, glutathione, sodium citrate, defatted milk, yolk
Liquid, penicillin, streptomysin and distilled water are formulated, and then at ambient temperature, with 600 × g centrifugal force 10min, are abandoned
Supernatant is removed, then it is 1 × 10 to be diluted to sperm concentration with freezen protective liquid8A/mL after mixing, is packed into 0.5mL tubules
In, slow cooling is to 4 DEG C of balance 100min, then after 8min is fumigated at 4cm on liquid nitrogen surface, puts into liquid nitrogen and preserve.
Specifically detection method is：Sperm after defrosting is carried out to the measure of indices, before and after more each experimental group freezing
Kinematic parameter, abnormal rate, the bacterial population of sperm.CASA (Computer-aided sperm are used after different formulations freeze thawing
Analysis) compater assisted sperm analysis is detected, each 3 repetitions of sample detection.
(1) CASA detects sperm concentration, and detection every time is at least up to 5 visuals field and the sperm of 200 or more；
(2) portion Samples detection 3 times will observe the visual field of 15 or more, the sperm of 600 or more in total.
Rate of teratosperm is surveyed with Ji's nurse Sa decoration method.That is smear-dry-fixation-wash-dry-dyes-water
Wash-dry-microscopy.200 sperms are at least observed under 400 power microscopes, are repeated 2 times.Bacterial population plain agar cultivation.
It puts culture in 37 DEG C of insulating boxs to take out after 48 hours, counts clump count in plate.Every group of sample takes two flags, two plates, makes even
Mean value.
By SAS9.0 softwares, all data are handled using one-way analysis of variance method, wherein, TM=always lives
Power；PM=forward motile sperm percentage；VCL=mean curvilinear velocities (μm/s)；VSL=mean linear speed；VAP=is averaged
Path velocity；ALH=sperm head side-sway amplitudes；LIN=linearity, the linearity of curvilinear path, VSL/VCL；Before STR=to
Property, the linearity in space average path, VSL/VAP；BCF=beat frequencies (Hz).Experimental result is as shown in table 1.
As shown in Table 1, in the sample of the fresh essence of same a batch, after different dilution processing, sperm after freeze-thaw
Index evaluation result is variant, and in 4 test groups, 1 group and 2 groups total activity average out to 68.75 and 64.14 of autogamy liquid are higher than
2 groups of INRA groups, significant difference (P ＜ 0.05).It is detected according to the every of sperm after defrosting, semen cryopreservation liquid of the invention
(autogamy liquid) in the control of sperm abnormal rate, bacterial population on the basis of commercialization freezen protective liquid no significant difference, effectively
Total activity, the forward motile sperm percentage of sperm are improved, stability is best.
Screening experiment also has been carried out to glutathione in the present invention.Specific experiment process is as follows：Choose 5-15 Sui Mongolian horse 4
, collecting semen 3 times weekly, more than 0.6 vigor carries out freezen protective.1 week totally 12 groups of sample.3 experiment gradients of every group of design
Group (preparing 100mL freezen protective liquid, glutathione raw material addition is respectively 0.077g, 0.154g, 0.230g) and 1 control
Group (does not add glutathione).Thaw point is 37 DEG C, 30s.Specific freezing and storing method and detection method and different formulations horse
Semen cryopreservation liquid is identical to the influence experiment of sperm quality after freeze thawing.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 protecting
Horse sperm is deposited, total activity, the forward motile sperm percentage for freezing sperm after essence is thawed are superior to the reduced form paddy Guang of other concentration
Sweet peptide illustrates the 100mL freezen protective liquid obtained when glutathione raw material addition is 0.077g, can effectively improve freezing essence
The sperm motility of liquid.
In addition, the horse sperm after horse semen cryopreservation liquid using the present invention and freezing and storing method freezing is for artificial
Insemination, 32 mares of breeding in a feelings phase, wherein pregnancy 15, conception rate 46.88%.
Influence of the 1 different formulations horse semen cryopreservation liquid of table to sperm quality after freeze thawing
Table 2 adds influence of the various concentration glutathione to horse semen cryopreservation effect
Note：With in a line there are different Superscript letters persons to represent significant difference (P ＜ 0.05), identical expression in Tables 1 and 2
Difference is not notable (P ＞ 0.05).
The invention has the beneficial effects that：A kind of horse semen cryopreservation liquid provided by the invention, can effectively improve horse
Survival rate and sperm motility of the sperm during freezing.Sperm physical signs is high after defrosting, effectively increases total work of sperm
Power, forward motile sperm percentage, field application fertilization effect stability is reliable, available for producing.The horse semen cryopreservation liquid
Raw material be easy to get, at low cost, preparation method step is few, simple to operation, and preparation condition is mildly easy to control.Using horse of the present invention essence
The freezing and storing method of liquid freezen protective liquid, condition is easily controllable, easy to implement, and controlled-rate freezing is good.The present invention will be used
Horse semen cryopreservation liquid and freezing and storing method freezing after horse sperm for artificial insemination, breeding 32 in a feelings phase
Mare, wherein pregnancy 15, conception rate 46.88%.
Specific embodiment
The present invention is further introduced below in conjunction with specific embodiment.
Embodiment 1
A kind of horse semen cryopreservation liquid, by glucose 2.00g, lactose 3.40g, trehalose 0.60g, glutathione
0.061g, sodium citrate 0.50g, defatted milk 30mL, yolk liquid 2.5mL, 50,000 IU of penicillin, 50,000 IU of streptomysin, glycerine 3.5mL
100mL horse semen cryopreservation liquid is configured to distilled water.
Specific preparation method includes the following steps：
(1) defatted milk is prepared：After fresh mare's milk is taken to filter, 100 DEG C of water-bath sterilization 10min after slow cooling to room temperature, are put
In 4 DEG C of refrigerations, 30min is then centrifuged with 2000r/min rotating speeds, upper strata lipid and lower sediment is removed, obtains defatted milk；
(2) basal liquid I is prepared：By measurement glucose, lactose, trehalose, glutathione, sodium citrate, defatted milk, yolk
Glucose, lactose, trehalose, glutathione and sodium citrate are first put into sterile chamber, add by liquid, penicillin and streptomysin
Enter distilled water dissolving, be settled to 100mL, then add in defatted milk, yolk liquid, penicillin and streptomysin, 20min is stirred at room temperature i.e.
Obtain basal liquid I；
(3) freezen protective liquid is prepared：Basal liquid I 96.5mL and glycerine 3.5mL are taken, glycerine stirring is added in basal liquid I
20min is to get freezen protective liquid.
Wherein, yolk liquid is obtained by following steps：Fresh Egg is taken, after removing egg white, then membrane of yolk is pierced, ovum
Yellow liquor is collected into the container being cooled with ice.
Embodiment 2
A kind of horse semen cryopreservation liquid, by glucose 2.40g, lactose 4.00g, trehalose 1.00g, glutathione
0.092g, sodium citrate 0.70g, defatted milk 50mL, yolk liquid 4.5mL, 60,000 IU of penicillin, 80,000 IU of streptomysin, glycerine 5.5mL
100mL horse semen cryopreservation liquid is configured to distilled water.
The preparation method of aforementioned horse semen cryopreservation liquid, specifically includes following steps：
(1) defatted milk is prepared：Extracting degreasing milk powder is added in distilled water, and stirring is configured to a concentration of 0.1g/mL solution, obtains
To defatted milk；
(2) basal liquid I is prepared：By measurement glucose, lactose, trehalose, glutathione, sodium citrate, defatted milk, yolk
Glucose, lactose, trehalose, glutathione and sodium citrate are first put into sterile chamber, add by liquid, penicillin and streptomysin
Enter distilled water dissolving, be settled to 100mL, then add in defatted milk, yolk liquid, penicillin and streptomysin, 45min is stirred at room temperature i.e.
Obtain basal liquid I；
(3) freezen protective liquid is prepared：Basal liquid I 94.5mL and glycerine 5.5mL are taken, glycerine stirring is added in basal liquid I
40min is to get freezen protective liquid.
Wherein, yolk liquid is obtained by following steps：Fresh Egg is taken, after removing egg white, yolk is transferred to filter paper
Upper rolling then pierces membrane of yolk, yolk liquid is received to remove the chalaza sticked together on membrane of yolk of remaining egg white and white
Collect in the container being cooled with ice.
Embodiment 3
Horse semen cryopreservation liquid, by glucose 2.20g, lactose 3.70g, trehalose 0.80g, glutathione 0.077g,
Sodium citrate 0.60g, defatted milk 40mL, yolk liquid 3.5mL, 100,000 IU of penicillin, 100,000 IU of streptomysin, glycerine 4.5mL and double
It steams water and is configured to 100mL horse semen cryopreservation liquid.
The preparation method of horse semen cryopreservation liquid, specifically includes following steps：
(1) defatted milk is prepared：After fresh mare's milk is taken to filter, 100 DEG C of water-bath sterilization 10min after slow cooling to room temperature, are put
In 4 DEG C of refrigerations, 20min is then centrifuged with 3000r/min rotating speeds, upper strata lipid and lower sediment is removed, obtains defatted milk；
(2) basal liquid I is prepared：By measurement glucose, lactose, trehalose, glutathione, sodium citrate, defatted milk, yolk
Glucose, lactose, trehalose, glutathione and sodium citrate are first put into sterile chamber, add by liquid, penicillin and streptomysin
Enter distilled water dissolving, be settled to 100mL, then add in defatted milk, yolk liquid, penicillin and streptomysin, 30min is stirred at room temperature i.e.
Obtain basal liquid I；
(3) freezen protective liquid is prepared：Basal liquid I 95.5mL and glycerine 4.5mL are taken, glycerine stirring is added in basal liquid I
30min is to get freezen protective liquid.
Wherein, yolk liquid is obtained by following steps：Fresh Egg is taken, after removing egg white, yolk is transferred to filter paper
Upper rolling then pierces membrane of yolk, yolk liquid is received to remove the chalaza sticked together on membrane of yolk of remaining egg white and white
Collect in the container being cooled with ice.
Embodiment 4~6 is tool of the horse semen cryopreservation liquid for obtaining Examples 1 to 3 in horse semen cryopreservation
Body application.
Embodiment 4
Application of the horse semen cryopreservation liquid of Examples 1 to 3 in horse semen cryopreservation, specific store method include
Following steps：Horse sperm to be saved is taken, basal liquid I is added in and is diluted, the volume ratio of horse sperm and basal liquid I are 1:1.5
Wherein, basal liquid I is by glucose, lactose, trehalose, glutathione, sodium citrate, defatted milk, yolk liquid, penicillin, chain
Mycin and distilled water are formulated, and then at ambient temperature, with 600 × g centrifugal force 8min, discard supernatant liquid, then use
It is 5 × 10 that freezen protective liquid, which is diluted to sperm concentration,7A/mL after mixing, is fitted into 0.5mL tubules, slow cooling to 4
DEG C balance 90min；Again after 10min is fumigated at 5cm on liquid nitrogen surface, put into liquid nitrogen and preserve.
Embodiment 5
Application of the horse semen cryopreservation liquid of Examples 1 to 3 in horse semen cryopreservation, specific store method include
Following steps：Horse sperm to be saved is taken, basal liquid I is added in and is diluted, the volume ratio of horse sperm and basal liquid I are 1:2,
In, basal liquid I is by glucose, lactose, trehalose, glutathione, sodium citrate, defatted milk, yolk liquid, penicillin, strepto-
Element and distilled water are formulated, and then at ambient temperature, with 600 × g centrifugal force 12min, discard supernatant liquid, then with cold
It is 8 × 10 to freeze preservation liquid to be diluted to sperm concentration7A/mL after mixing, is fitted into 0.5mL tubules, and slow cooling is to 4 DEG C
Balance 120min；Again after 9min is fumigated at 6cm on liquid nitrogen surface, put into liquid nitrogen and preserve.
Embodiment 6
Application of the horse semen cryopreservation liquid of Examples 1 to 3 in horse semen cryopreservation, specific store method include
Following steps：Horse sperm to be saved is taken, basal liquid I is added in and is diluted, the volume ratio of horse sperm and basal liquid I are 1:1,
In, basal liquid I is by glucose, lactose, trehalose, glutathione, sodium citrate, defatted milk, yolk liquid, penicillin, strepto-
Element and distilled water are formulated, and then at ambient temperature, with 600 × g centrifugal force 10min, discard supernatant liquid, then with cold
It is 1 × 10 to freeze preservation liquid to be diluted to sperm concentration8A/mL after mixing, is fitted into 0.5mL tubules, and slow cooling is to 4 DEG C
Balance 100min；Again after 8min is fumigated at 4cm on liquid nitrogen surface, put into liquid nitrogen and preserve.
Claims (8)
1. a kind of horse semen cryopreservation liquid, it is characterised in that：By glucose 2.00g~2.40g, lactose 3.40g~4.00g,
Trehalose 0.60g~1.00g, 0.061~0.092g of glutathione, sodium citrate 0.50g~0.70g, defatted milk 30mL~
The IU of the IU of 50mL, 2.5~4.5mL of yolk liquid, penicillin 50,000~100,000, streptomysin 50,000~100,000,4.5~5.5mL of glycerine and double
It steams water and is configured to 100mL horse semen cryopreservation liquid.
2. horse semen cryopreservation liquid according to claim 1, it is characterised in that：By glucose 2.20g, lactose 3.70g,
Trehalose 0.80g, glutathione 0.077g, sodium citrate 0.60g, defatted milk 40mL, yolk liquid 3.5mL, 100,000 IU of penicillin,
100,000 IU of streptomysin, glycerine 4.5mL and distilled water are configured to 100mL horse semen cryopreservation liquid.
3. the preparation method of horse semen cryopreservation liquid as claimed in claim 1 or 2, it is characterised in that：Include the following steps：
(1) defatted milk is prepared：After fresh mare's milk is taken to filter, 100 DEG C of water-bath sterilization 10min after slow cooling to room temperature, are placed in 4
DEG C refrigeration, then centrifugation removal upper strata lipid and lower sediment, obtains defatted milk；
(2) basal liquid I is prepared：By measure glucose, lactose, trehalose, glutathione, sodium citrate, defatted milk, yolk liquid,
Glucose, lactose, trehalose, glutathione and sodium citrate are first put into sterile chamber by penicillin and streptomysin, are added in double
Water dissolution is steamed, is settled to 100mL, then adds in defatted milk, yolk liquid, penicillin and streptomysin, is stirred at room temperature up to basal liquid
I；
(3) freezen protective liquid is prepared：4.5~5.5mL of basal liquid I 95.5mL~94.5mL and glycerine is taken, is added in basal liquid I
Enter glycerine and stir 20min~40min to get 100mL freezen protective liquid.
4. the preparation method of horse semen cryopreservation liquid according to claim 3, it is characterised in that：In the step (1),
It centrifuges and is：20~30min is centrifuged with 2000~3000r/min rotating speeds.
5. the preparation method of horse semen cryopreservation liquid according to claim 3, it is characterised in that：Include the following steps：
(1) defatted milk is prepared：Extracting degreasing milk powder is added in distilled water, and stirring is configured to a concentration of 0.1g/mL solution, is taken off
Fat milk；
(2) basal liquid I is prepared：By measure glucose, lactose, trehalose, glutathione, sodium citrate, defatted milk, yolk liquid,
Glucose, lactose, trehalose, glutathione and sodium citrate are first put into sterile chamber by penicillin and streptomysin, are added in double
Water dissolution is steamed, is settled to 100mL, then adds in defatted milk, yolk liquid, penicillin and streptomysin, is stirred at room temperature up to basal liquid
I；
(3) freezen protective liquid is prepared：4.5~5.5mL of basal liquid I 95.5mL~94.5mL and glycerine is taken, is added in basal liquid I
Enter glycerine and stir 20min~40min to get freezen protective liquid.
6. the preparation method of the horse semen cryopreservation liquid according to claim 3 or 5, it is characterised in that：The step (2)
In, yolk liquid is obtained by following steps：Fresh Egg is taken, after removing egg white, membrane of yolk is pierced, yolk liquid, which is collected into, uses ice
In the container of cooling.
7. the preparation method of the horse semen cryopreservation liquid according to claim 3 or 5, it is characterised in that：The step (2)
In, 20min~45min is stirred at room temperature and obtains basal liquid I.
8. application of the horse semen cryopreservation liquid as claimed in claim 1 or 2 in horse semen cryopreservation, feature exist
In：Horse seminal fluid cryopreservation method includes the following steps：Horse sperm to be saved is taken, basal liquid I is added in and is diluted, horse sperm
Volume ratio with basal liquid I is 1:1~2, wherein, basal liquid I is by glucose, lactose, trehalose, glutathione, citric acid
Sodium, defatted milk, yolk liquid, penicillin, streptomysin and distilled water are formulated；Then at ambient temperature, it is centrifuged with 600 × g
Power centrifuges 8~12min, discards supernatant liquid, then it is 5 × 10 to be diluted to sperm concentration with freezen protective liquid7~1 × 108A/mL is mixed
After closing uniformly, is fitted into 0.5mL tubules, slow cooling balances 90~120min, then on liquid nitrogen surface at 4~6cm to 4 DEG C
After stifling 8~10min, put into liquid nitrogen and preserve.
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| CN103858858A (en) * | 2012-12-11 | 2014-06-18 | 中国农业大学 | Horse semen dilution and its preparation method |
| CN103120155B (en) * | 2013-02-05 | 2014-07-16 | 新疆维吾尔自治区畜牧科学院畜牧科学研究所 | Cryopreservation solution of horse sperms and freezing method thereof |
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| CN103783032A (en) * | 2013-11-21 | 2014-05-14 | 青岛德瑞骏发生物科技有限公司 | Equus animal frozen semen diluting liquid and preparation method thereof |
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| 马冷冻精液氧自由基的产生和清除系统;段洪云 等;《中国畜牧兽医》;20130220;第40卷(第2期);第133-136页 * |
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