CN108865982A - A kind of frozen cattle semens X, y sperm separation method - Google Patents
A kind of frozen cattle semens X, y sperm separation method Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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Abstract
The invention belongs to frozen cattle semens production technical fields, and in particular to a kind of frozen cattle semens X, y sperm separation method, including configuration Staining liquid and dilution, each ingredient and its final concentration of in Staining liquid：Hepes：9.520g/L MgCl2·6H2O：0.08g/L, NaCl：5.518g/L KCl：0.224g/L, Na2HPO4：0.04g/L, NaHCO3：0.84g/L, Napy：0.22g/L, glucose：0.90g/L, Nalatctate：3.61ml/L BSA：3.0g/L, gentamicin：2.5ml/L；Ox is freezed and freezes essence defrosting, is centrifuged after defrosting, supernatant is abandoned, calculates precipitating sperm quantity；To precipitate sperm to be diluted to concentration with Staining liquid is 1 × 108A sperm/ml, is then added fluorescent dye, and uniformly mixed be placed in water-bath stands, X, y sperm are separated with flow cytometer after water-bath, X or y sperm are placed in refrigerator and balanced, is centrifuged after balance, supernatant is abandoned, precipitating X or y sperm are collected, freezen protective into X or y sperm plus after dilution.This method separative efficiency is high, and sexual control accuracy rate is up to 92% or more.
Description
Technical field
The invention belongs to frozen cattle semens production technical fields, and in particular to a kind of frozen cattle semens X, y sperm separation method.
Background technique
In the breeding production of ox, ox life will be greatly improved with the other ox offspring of foreseeability by selectively breeding
The speed and efficiency of production are inseminated to cow with X or y sperm, the gender of offspring can be determined before fertilization, thus significantly
The sex ratio for changing offspring, achievees the purpose that sexual control.Currently, only flow cytometer spermatozoa partition method is acknowledged as
It is most scientific most reliable, while being also most efficient Gender preselection.Flow cytometric sorting mammal X, y sperm are roots
According to mammal X sperm DNA content ratio Y sperm more than principle, pass through resolved fluorometric difference after fluorescent staining for the two point
It opens, sexual control is achieved the purpose that after fertilization.
But use separation means used by flow cytometer as described in CN1667118A for X, y sperm at present
" antibiotic mixed solution is added in the male animal original sperm of acquisition, constant temperature saves in 24 hours, dilute with the sperm without yolk
Fluorescent dye will be added and be incubated for dyeing in water-bath by releasing liquid after former semen dilution, pigment is added after the water bath is over,
Using X or y sperm needed for flow cytometric sorting under constant temperature, collected with the test tube for being preinstalled with the sperm acceptable solution containing yolk
X sperm and y sperm after separation, isolated sperm can be directly used for artificial insemination or inseminatio externalis after centrifugal concentrating,
Or freezen protective after low-temperature balance." original sperm used by this kind of method is fresh essence, and require fresh precision 800,000,000 sperms/
Ml or more and vigor >=0.6 need to introduce living body ox or introduce embryo if necessary to introduce external required X or y sperm
Tire either introduces sex control embryo, and such method required cost is higher, is unable to satisfy the demand of domestic market.
Summary of the invention
Place in view of the deficiency of the prior art, the object of the invention is that providing a kind of easy to operate, expense
With method that is low and being applicable to freeze essence control separation X, y sperm.
The present invention is achieved through the following technical solutions above-mentioned purpose：
A kind of frozen cattle semens X, y sperm separation method, include the following steps：
Step (1) configures Staining liquid and dilution
1.1) Staining liquid
Each ingredient and its final concentration of in Staining liquid：Hepes：9.520g/L MgCl2·6H2O：0.08g/L,
NaCl：5.518g/L KCl：0.224g/L, Na2HPO4：0.04g/L, NaHCO3：0.84g/L, Napy：0.22g/L, grape
Sugar：0.90g/L, Nalatctate：3.61ml/L BSA：3.0g/L, gentamicin： 2.5ml/L；
1.2) dilution
Dilution includes 796 parts of working solutions, 204 parts of yolk and final concentration of 0.007467ml/ml gentamicin, end
Concentration is the antibiotic Taylor rhzomorph and cillimycin of 0.005633ml/ml, each ingredient and its final concentration in the working solution
For：Tris 30.44g/L, citric acid 17.21g/L, fructose 12.65g/L；
Step (2), which freezes ox, freezes essence defrosting, is centrifuged after defrosting, abandons supernatant, calculates precipitating sperm quantity；
It is 1 × 10 that the resulting precipitating sperm of step (2) is diluted to concentration with Staining liquid by step (3)8A sperm/
Then fluorescent dye is added in ml, uniformly mixed be placed in water-bath stands, and makes X, y sperm point with flow cytometer after water-bath
From X or y sperm are placed in refrigerator and balanced, is centrifuged after balance, supernatant is abandoned, precipitating X or y sperm is collected, to X or y sperm
In plus dilution after freezen protective.
Preferably, above-mentioned frozen cattle semens X, y sperm separation method, the configuration method of Staining liquid is in step 1.1)：
The ultrapure water of 700-800ml is poured into beaker, stirring is opened, is sequentially added into Hepes, MgCl2·6H2O、
NaCl、KCl、Na2HPO4、NaHCO3, after Napy, glucose, Nalatctate and BSA shake up, PH is adjusted using NaOH solution
Value is transferred to volumetric flask to 7.4, by the solution in beaker, and addition ultrapure water is settled to 1000ml, and sealer shakes up；By volumetric flask
In liquid standing filter afterwards for 24 hours, filtrate is placed in reagent bottle, for use；It is final concentration of being added before into reagent bottle
The gentamicin of 2.5ml/L.
Preferably, above-mentioned frozen cattle semens X, y sperm separation method, in step 1.2), configuration dilution includes the following steps：
(1) configuration work liquid
The configuration method of working solution is that 700-800ml ultrapure water is added in beaker, opens stirring, and sequentially add
Tris, citric acid and fructose adjust PH to 6.80 using NaOH solution after dissolution, the solution in beaker are transferred to volumetric flask,
Addition ultrapure water is settled to 1000ml, filters after mixing, abandons filter residue, filtrate is saved backup in 4 DEG C of conditions,
(2) dilution is configured
It weighs 796ml working solution to be placed in 1000ml volumetric flask, and 204ml yolk is added into volumetric flask, put after shaking up
It sets 24 hours, three times by solution centrifugal filtration in volumetric flask, is then separately added into antibiosis by final concentration of 0.005633ml/ml
Gentamicin is added in plain finished product Taylor rhzomorph and cillimycin, final concentration of 0.007467ml/ml, is uniformly mixed, obtains dilute
Release liquid.
Preferably, above-mentioned frozen cattle semens X, y sperm separation method, in step (2), it is that vigor is big that essence is frozen in the ox freezing
Essence is frozen in freezing under 0.35 Liquid nitrogen storage.
Preferably, above-mentioned frozen cattle semens X, y sperm separation method, in step (2), the thawing condition is to freeze ox
Freeze essence and is placed in 38 DEG C of water-bath the 10s that thaws.
Preferably, above-mentioned frozen cattle semens X, y sperm separation method, in step (2), the centrifugally operated is to freeze ox
Freeze after essence is thawed and cuts the non-tampon end for the tubule that essence is frozen in splendid attire ox freezing, it is from tampon end that the jelly essence after defrosting is smart with pushing pin
Liquid is pushed into centrifuge tube, is centrifuged 10 minutes under the conditions of 18 DEG C, 850G, supernatant is abandoned after centrifugation.
Preferably, above-mentioned frozen cattle semens X, y sperm separation method, in step (3), the fluorescent dye is H33342.
Preferably, above-mentioned frozen cattle semens X, y sperm separation method, in step (3), the bath temperature is 34 DEG C, water-bath
Time is 48 minutes.
Preferably, above-mentioned frozen cattle semens X, y sperm separation method in steps (3), X or y sperm are placed in refrigerator and balanced,
Temperature in refrigerator is controlled at 4 DEG C, and centrifuging temperature control is at 4 DEG C when being centrifuged after balance, and centrifugal force controlled is in 850G, centrifugation
Between control after 20min, centrifugation use diluted, dilution be placed on 4 DEG C under the conditions of save.
Preferably, above-mentioned frozen cattle semens X, y sperm separation method, in step (2), the calculating precipitates contained sperm count
Method used by measuring is blood cell counting plate counting method.
Preferably, above-mentioned frozen cattle semens X, y sperm separation method, the blender used in step 1.1) when stirring is magnetic force
Blender.
Preferably, above-mentioned frozen cattle semens X, y sperm separation method, NaOH used in step 1.1) is that mass fraction is
15% NaOH solution.
The Chinese name of Hepes described in present specification：4- hydroxyethyl piperazineethanesulfonic acid；N- (2- ethoxy) piperazine
Piperazine-N'-2- ethane sulfonic acid, the Chinese name of Napy are：The Chinese name of Sodium Pyruvate, Nalatctate is：Sodium lactate.
The Chinese name of BSA described in present specification is bovine serum albumin(BSA)；
Antibiotic Taylor described in present specification refers to Taylor's rhzomorph, and the configuration method of antibiotic Taylor is will
0.76g tartaric acid Taylor rhzomorph (Tylosin tartrate) and 40ml2.9% (mass fraction, similarly hereinafter) sodium citrate mix
Uniformly obtain antibiotic Taylor；
Antibiotic Lincoln described in present specification refers to cillimycin, and the configuration method of antibiotic Lincoln is：With strong
Miromycin (Spectinomycin dihydrochloride pentahydrate): cillimycin (Lincomycin
Hydrochloride): the ratio of 2.9% sodium citrate is that mixed liquor made of 4g: 2g: 40ml configuration is antibiotic Lincoln.
Compared with prior art, frozen cattle semens X of the present invention, y sperm separation method have following innovative point：
Frozen cattle semens X provided by the invention, y sperm separation method, are made by reasonably configuring Staining liquid and dilution
Separation can also be realized using flow cytometer by freezing essence, and existing flow cytometer is only limitted to fresh essence and requires fresh precision 8
Hundred million sperms/ml and vigor is required to be more than or equal to 0.6, the only two doses of freezings of frozen cattle semens X provided by the present invention, y sperm separation method
Freeze essence (to freeze precision >=80,000,000 sperm/ml, freeze smart specification：0.25ml/ agent) required sperm can be met.
And the sperm motility after separating reaches 0.3-0.36, sexual control rate >=90% reaches as high as 99%, can satisfy body completely
Condition required for outer fertilization.
Specific embodiment
In order to enable those skilled in the art to better understand the solution of the present invention, With reference to embodiment to this
Invention is described in further detail.
Embodiment 1
A kind of frozen cattle semens X, y sperm separation method, include the following steps：
Step (1) configures Staining liquid and dilution
1.1) Staining liquid
Each ingredient and its final concentration of in Staining liquid：Hepes：9.520g/L MgCl2·6H2O：0.08g/L,
NaCl：5.518g/L KCl：0.224g/L, Na2HPO4：0.04g/L, NaHCO3：0.84g/L, Napy：0.22g/L, grape
Sugar：0.90g/L, Nalatctate：3.61ml/L BSA：3.0g/L, gentamicin：2.5ml/L；
The configuration method of Staining liquid is：
The ultrapure water of 700-800ml is poured into beaker, opens stirring (using magnetic stirrer), respectively successively
Hepes, MgCl is added2·6H2O、NaCl、KCl、Na2HPO4、NaHCO3, Napy, glucose, Nalatctate and BSA shake up
Afterwards, pH value is adjusted to 7.4 using NaOH solution, addition ultrapure water is settled to 1000ml volumetric flask, and sealer shakes up；By volumetric flask
In liquid stand for 24 hours after, liquid in volumetric flask is placed in 200 mu of filters and is filtered, filtrate is placed in reagent bottle, according to
The volume for the liquid being obtained by filtration in reagent bottle is added gentamicin and obtains Staining liquid, gentamicin in Staining liquid
Final concentration of 2.5ml/L；
1.2) dilution
Dilution includes 796 parts of working solutions, 204 parts of yolk and final concentration of 0.007467ml/ml gentamicin, end
Concentration is the antibiotic Taylor rhzomorph and cillimycin of 0.005633ml/ml, each ingredient and its final concentration in the working solution
For：Tris 30.44g/L, citric acid 17.21g/L, fructose 12.65g/L；
Specifically, configuration dilution includes the following steps：
Firstly, configuration work liquid
The configuration method of working solution is that 700-800ml ultrapure water is added in 1000ml beaker, opens stirring and (uses magnetic
The stirring of power blender), and Tris, citric acid and fructose are sequentially added, PH to 6.80 is adjusted using NaOH solution after dissolution, it will
Solution in beaker is transferred to volumetric flask, and addition ultrapure water is settled to 1000ml, filters after mixing, filter residue is abandoned, by filtrate
It is saved backup in 4 DEG C of conditions,
Then, dilution is configured
It weighs 796ml working solution to be placed in 1000ml volumetric flask, and 204ml yolk is added into volumetric flask, put after shaking up
It sets 24 hours, three times by solution centrifugal filtration in volumetric flask, is then separately added into antibiosis by final concentration of 0.005633ml/ml
Gentamicin is added in plain finished product Taylor rhzomorph and cillimycin, final concentration of 0.007467ml/ml, is uniformly mixed, obtains dilute
Release liquid.
Step (2), which freezes ox, freezes essence defrosting, is centrifuged after defrosting, supernatant is abandoned, using blood cell counting plate counting method meter
Calculate precipitating sperm quantity；It is that essence, solution are frozen in the freezing under Liquid nitrogen storage of the vigor more than or equal to 0.35 that essence is frozen in the ox freezing
Jelly condition is that ox freezing is frozen to essence to be placed in 38 DEG C of water-bath the 10s that thaws, and centrifugally operated is to freeze ox after jelly essence is thawed to cut
The non-tampon end that the tubule of essence is frozen in ox freezing is contained, is pushed into centrifuge tube with pushing pin from tampon end by the jelly essence sperm after defrosting,
It is centrifuged 10 minutes under the conditions of 18 DEG C, 850G, supernatant is abandoned after centrifugation；
It is 1 × 10 that the resulting precipitating sperm of step (2) is diluted to concentration with Staining liquid by step (3)8A sperm/
Then fluorescent dye is added in ml, uniformly mixed be placed in water-bath stands, and makes X, y sperm point with flow cytometer after water-bath
From X or y sperm are placed in refrigerator and balanced, is centrifuged after balance, supernatant is abandoned, precipitating X or y sperm is collected, to X or y sperm
In plus dilution after freezen protective.
Specifically, in step (3), the fluorescent dye is H33342.
Specifically, in step (3), the bath temperature is 34 DEG C, and water bath time is 48 minutes.
Specifically, in step (3), X or y sperm is placed in refrigerator and balanced, the temperature in refrigerator is controlled at 4 DEG C, balance
Centrifuging temperature control is at 4 DEG C when being centrifuged afterwards, and centrifugal force controlled is in 850G, and centrifugation time control is after 20min, centrifugation with dilution
Liquid dilution, dilution save under the conditions of being placed on 4 DEG C.
Specifically, NaOH used in step 1.1) is the NaOH solution that mass fraction is 15%.
Embodiment 2
Staining liquid and dilution used by embodiment 2 are identical as embodiment 1.
1. the defrosting of frozen cattle semens
Two doses of (the 0.25ml/ of common jelly essence for the ox that the ox number for taking 04 Japanese firm November in 2014 to produce is 41104818
Agent), it thaws 10 seconds in 38 DEG C of warm water, cuts off non-tampon end, shift 15ml sky centrifuge tube (a) onto from tampon end sperm with pushing pin
In, centrifuge tube (a) net weight M1=6.6746g.
2. being centrifuged, centrifuge tube (a) is placed in a centrifuge and is centrifuged, centrifugal condition is：18 DEG C, 850G, 10 minutes.
Supernatant in centrifuge tube (a) is removed after centrifugation, quality is M after weighing2=6.7578g.Precipitate sperm gross weight M=M2-M1=
6.7578g-6.6747g=0.0831g precipitating sperm concentration ρ is 1.04g/ml, sperm volume=M/ ρ=0.0831g/ is precipitated
1.04 (g/ml) × 1000 μ l=80 μ l take 10 μ l to be put into the Staining of 90 μ l, are uniformly mixed from precipitating sperm, then
10 μ l are taken to be added in 290 μ l formalin in above-mentioned mixed liquor from the Staining liquid containing precipitating sperm, from above-mentioned
It takes 10 μ l to instill blood cell counting plate in 300 μ l formalin mixed liquors and counts sperm count under the microscope, pass through extension rate
Calculating precipitating sperm count is 22,510,000.
3. dyeing
To 225-80=135 μ l sperm Staining liquid is added in centrifuge tube (a), add H33342 fluorescent dye=
0.2251 × 15/2=1.68 μ l.Mixed liquid is put into the loading pipe of 5ml, the water-bath 48min in 34 DEG C of water-baths.
Upper machine (being put into flow cytometer loading area) separates and collects required sexed sperm in national standard control separation condition after water-bath.This
Place's separation is X, and the X sperm of collection is collected with the 50ml specification centrifuge tube for being placed with 1ml dilution and covers centrifuge tube lid juxtaposition
Two hours are balanced in 4 DEG C of refrigerators, are shown according to the data on flow cytometer, collecting X sperm count is 3,000,000.
The computation rule of " 225-80=135 μ l " described in above-mentioned " 3. dyeing " generally will in subsequent operation
Sperm concentration is tuned into 1 × 108A/ml, the solution that 22,510,000 precipitating sperm corresponding volumes are 225 microlitres, the precipitating being centrifuged
Sperm volume is 80 microlitres, so the volume for needing to be added Staining liquid is 225-80=135 μ l.
The X sperm obtained to collection surveys its sperm motility, and the detection method of sperm motility is that the sperm obtained drops to load
It on slide, is observed using phase contrast microscope, and microscope is connected with display screen, the ratio that the good sperm of range estimation calculating state accounts for
Rate.Sperm motility after calculating gained separation reaches 0.36 (i.e. 36%).
4. being centrifuged and saving
Sperm after balance is centrifuged 20 minutes under 4 DEG C, 850G centrifugal condition, supernatant is abandoned, by 60 microlitres of dilution/100
Ten thousand sperms add dilution, and amount=300 × 60/100=180 microlitres of dilution of added dilution is subsequently placed in 4 DEG C of ice
Case saves backup.
Embodiment 3
Staining liquid and dilution used by embodiment 3 are identical as embodiment 1.
1. Niu Putong freezes the defrosting of essence
Two doses of (the 0.25ml/ of common jelly essence for the ox that the ox number for taking 23 Japanese firm November in 2015 to produce is 41110294
Agent), it thaws 10 seconds in 38 DEG C of warm water, cuts off non-tampon end, shift 15ml sky centrifuge tube (b) onto from tampon end sperm with pushing pin
In, centrifuge tube (b) net weight M1=6.6815g；
2. being centrifuged
Centrifugation, centrifuge tube (b) is placed in a centrifuge and is centrifuged, centrifugal condition is：18 DEG C, 850G, 10 minutes.It abandons
Supernatant.Weight M after weighing2=6.7501g.Calculate precipitation volume=(6.7501-6.6815)/1.04 × 1000 μ l=66 μ
l.It takes 10 μ l to be put into the Staining liquid of 90 μ l from precipitating sperm, mixes, then take 10 μ l to be added to from the mixed liquor
In 290 μ l formalin, 10 μ l is taken to instill blood cell counting plate under the microscope from above-mentioned 300 μ l formalin mixed liquor
Number sperm count, calculating sperm count in precipitating finally by extension rate is 24,430,000.
3. dyeing
To 244-66=178 μ l sperm Staining liquid is added in centrifuge tube (b), add H33342 fluorescent dye=
0.2443 × 15/2=1.8 μ l.Mixed liquid is put into the loading pipe of 5ml the water-bath 48min in 34 DEG C of water-baths.Water
Upper machine (being put into flow cytometer loading area) separates and collects required gender essence under national standard control separation condition parameter after bath
Son.What is separated herein is y sperm.It collects y sperm to be collected with the 50ml specification centrifuge tube for being placed with 1ml dilution, and covers centrifugation
Pipe lid is placed in 4 DEG C of refrigerators and balances two hours, is shown according to the data on flow cytometer, and collecting y sperm number is 3,800,000.
The y sperm obtained to collection surveys its sperm motility, and the detection method of sperm motility is that the sperm obtained drops to load
It on slide, is observed using phase contrast microscope, and microscope is connected with display screen, the ratio that the good sperm of range estimation calculating state accounts for
Rate.Sperm motility after calculating gained separation reaches 0.3 (i.e. 30%).
4. being centrifuged and saving
Y sperm after balance is centrifuged 20 minutes under 4 DEG C, 850G centrifugal condition, abandon supernatant, by 60 microlitres of dilutions/
1000000 sperms add dilution, and amount=3.8 × 60=228 microlitres of dilution of added dilution is subsequently placed in 4 DEG C of refrigerators
It saves backup.
Design scheme provided by the present invention is described in detail above.Specific case used herein is to this
The embodiment of invention is expounded, method and its core of the invention that the above embodiments are only used to help understand
Thought is thought.It should be pointed out that for those skilled in the art, before not departing from the principle of the invention
It puts, can be with several improvements and modifications are made to the present invention, these improvement and modification also fall into the guarantor of the claims in the present invention
It protects in range.
Claims (10)
1. a kind of frozen cattle semens X, y sperm separation method, which is characterized in that include the following steps：
Step (1) configures Staining liquid and dilution
1.1) Staining liquid
Each ingredient and its final concentration of in Staining liquid：Hepes：9.520g/L MgCl2·6H2O：0.08g/L, NaCl：
5.518g/L KCl：0.224g/L, Na2HPO4：0.04g/L, NaHCO3：0.84g/L, Napy：0.22g/L, glucose：
0.90g/L, Nalatctate：3.61ml/L BSA：3.0g/L, gentamicin：2.5ml/L；
1.2) dilution
Dilution includes 796 parts of working solutions, 204 parts of yolk and final concentration of 0.007467ml/ml gentamicin, final concentration
For the antibiotic Taylor rhzomorph and cillimycin of 0.005633ml/ml, each ingredient and its final concentration of in the working solution：
Tris 30.44g/L, citric acid 17.21g/L, fructose 12.65g/L；
Step (2), which freezes ox, freezes essence defrosting, is centrifuged after defrosting, abandons supernatant, calculates precipitating sperm quantity；
It is 1 × 10 that the resulting precipitating sperm of step (2) is diluted to concentration with Staining liquid by step (3)8A sperm/ml, then
Fluorescent dye is added, uniformly mixed be placed in water-bath stands, and separates X, y sperm with flow cytometer after water-bath, by X or Y
Sperm is placed in refrigerator and balances, and is centrifuged after balance, abandons supernatant, collects precipitating X or y sperm, and dilution is added into X or y sperm
Freezen protective afterwards.
2. frozen cattle semens X according to claim 1, y sperm separation method, which is characterized in that Staining in step 1.1)
The configuration method of liquid is：
The ultrapure water of 700-800ml is poured into beaker, stirs, is sequentially added into Hepes, MgCl2·6H2O、NaCl、KCl、
Na2HPO4、NaHCO3, after Napy, glucose, Nalatctate and BSA shake up, pH value is adjusted to 7.4 using NaOH solution, will
Solution in beaker is transferred to volumetric flask, and addition ultrapure water is settled to 1000ml, and sealer shakes up；Liquid in volumetric flask is stood
It filters afterwards for 24 hours, filtrate is placed in reagent bottle, for use；
Using the preceding gentamicin that final concentration of 2.5ml/L is added into reagent bottle.
3. frozen cattle semens X according to claim 1, y sperm separation method, which is characterized in that in step 1.2), configuration dilution
Liquid includes the following steps：
(1) configuration work liquid
The configuration method of working solution is that 700-800ml ultrapure water is added in beaker, stirring, and sequentially adds Tris, citric acid
And fructose, PH to 6.80 is adjusted using NaOH solution after dissolution, the solution in beaker is transferred to volumetric flask, addition ultrapure water is fixed
Hold to 1000ml, filter after mixing, abandons filter residue, filtrate is saved backup in 4 DEG C of conditions,
(2) dilution is configured
It weighs 796ml working solution to be placed in 1000ml volumetric flask, and 204ml yolk is added into volumetric flask, place 24 after shaking up
Hour, three times by solution centrifugal filtration in volumetric flask, antibiotic finished product then is separately added by final concentration of 0.005633ml/ml
Gentamicin is added in Taylor's rhzomorph and cillimycin, final concentration of 0.007467ml/ml, is uniformly mixed, obtains dilution.
4. frozen cattle semens X according to claim 1, y sperm separation method, which is characterized in that in step (2), the ox
It is that essence is frozen in the freezing under Liquid nitrogen storage of the vigor more than or equal to 0.35 that essence is frozen in freezing.
5. frozen cattle semens X according to claim 1, y sperm separation method, which is characterized in that in step (2), the solution
Jelly condition is that ox is freezed to jelly essence to be placed in 38 DEG C of water-bath the 10s that thaws.
6. ox according to claim 1 freezes X, y sperm separation method, which is characterized in that in step (2), it is described from
Heart operation cuts the non-tampon end for containing the tubule that essence is frozen in ox freezing to freeze ox to freeze after essence is thawed, will from tampon end with pushing pin
Jelly essence sperm after defrosting is pushed into centrifuge tube, is centrifuged 10 minutes under the conditions of 18 DEG C, 850G, supernatant is abandoned after centrifugation.
7. ox according to claim 1 freezes X, y sperm separation method, which is characterized in that in step (3), described is glimmering
Photoinitiator dye is H33342.
8. ox according to claim 1 freezes X, y sperm separation method, which is characterized in that in step (3), the water
Bath temperature is 34 DEG C, and water bath time is 48 minutes.
9. ox according to claim 1 freezes X, y sperm separation method, which is characterized in that in step (3), by X or Y essence
Son is placed in refrigerator and balances, and temperature in refrigerator is controlled at 4 DEG C, and centrifuging temperature control is at 4 DEG C when being centrifuged after balance, centrifugal force control
System saves under the conditions of using diluted, dilution to be placed on 4 DEG C after 20min, centrifugation in 850G, centrifugation time control.
10. frozen cattle semens X according to claim 1, y sperm separation method, which is characterized in that in step (2), the meter
Calculating method used by precipitating contained sperm quantity is blood cell counting plate counting method.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109554338A (en) * | 2018-12-24 | 2019-04-02 | 内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 | The application of sperm flow cytometer separative efficiency is improved using percoll |
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| CN1732983A (en) * | 2005-08-29 | 2006-02-15 | 广西大学 | Freeze method for preserving separated sperm of bull |
| CN101322489A (en) * | 2008-08-01 | 2008-12-17 | 内蒙古蒙牛乳业（集团）股份有限公司 | Deer X/Y sperm separation freezing sperm and producing method thereof and use |
| CN103525760A (en) * | 2013-09-29 | 2014-01-22 | 大连金弘基种畜有限公司 | Separation method of sexed semen |
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| CN1732983A (en) * | 2005-08-29 | 2006-02-15 | 广西大学 | Freeze method for preserving separated sperm of bull |
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| CN109554338A (en) * | 2018-12-24 | 2019-04-02 | 内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 | The application of sperm flow cytometer separative efficiency is improved using percoll |
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