CN1667118A - Method for separating XY germ cell of mammal - Google Patents
Method for separating XY germ cell of mammal Download PDFInfo
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- CN1667118A CN1667118A CN 200410060793 CN200410060793A CN1667118A CN 1667118 A CN1667118 A CN 1667118A CN 200410060793 CN200410060793 CN 200410060793 CN 200410060793 A CN200410060793 A CN 200410060793A CN 1667118 A CN1667118 A CN 1667118A
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Abstract
This invention is a method of separating mammal XY sperm. Fluorescence colorant Hoechst33342 is used to color sperm DNA and then they are separated by flow cytometry according to theory of DNA content of X sperm is more than Y sperm of mammal. Preservation method, sperm diluent, sheath liquor and acceptance liquor used for separating sperm before sperm separating is improved to confirm improve the separation accuracy rate and activity after separation at the same time of ensuring separating XY sperm efficiency. It is fit for big scale commercialization separating generation of XY sperm of scalper, buffalo, goat and other mammal. It can be bonded to artificial insemination and external insemination and other animal breeding new techniques, it greatly improved the efficiency of animal husbandry production.
Description
Technical field
The present invention relates to a kind of method of separating Mammals X sperm and y sperm.
Background technology
In livestock industry is produced, breed selectively and have other livestock and poultry offspring of foreseeability and can improve speed and the efficient that livestock industry is produced greatly.Inseminate to dam with X or y sperm, before fertilization, just can determine offspring's sex, thereby change offspring's sex ratio significantly, reach sex-controlled purpose.With the milk cow is example, and the milk cow farmer wishes to breed more heifer from the core group of its good quality and high output, to increase or to upgrade its milk cows, just can reach this purpose by sex control.Equally, the beef cattle farmer then wishes to breed more bullock, because the quality of the speed of growth of ox and meat is gender-related, the bull speed of growth is faster than cow, and it is higher to castrate the price of bull meat.Aspect varieties breeding, if the accuracy rate of giving birth to the offspring by sex control, breed the speed of drove quantity and character improvement more than 90% will be than the raising greatly of asexuality control, thereby can save a large amount of time, energy and expense.
Mammals XY sperm isolation technique is effective sex-control method, if can in advance the X sperm be separated with y sperm, then by artificial insemination or in vitro fertilization, just can obtain other offspring of foreseeability.Since nineteen twenty-five Lush reported first is separated the sperm of rabbit, many scientific workers have done unremitting effort and trial to the research of separated sperm, characteristics such as this density, form, size, vigor, surface charge and immunogenicity comprising the foundation sperm, invented various methods and attempted decisive other X sperm and Y essence are separated, but these methods all there are poor reliability, repeatability is low and efficient is not high problem.So far, it is the most reliable to have only flow cytometer sperm partition method to be acknowledged as science, also is simultaneously sperm separation method the most efficiently.It is the principle of Duoing than y sperm according to the dna content of Mammals X sperm that flow cytometer separates Mammals XY sperm, by differentiating the fluorescence difference the two is separated after the fluorescent dye, reaches sex-controlled purpose after the fertilization.
At present, the weak point of flow cytometer separated sperm technology is that the efficient of separated sperm is also lower, cost is higher, containing lecithality etc. in the spermatozoa diluent influences the material of chromospermism, because of XY sperm after the fluorescent dye is differentiated rate variance and then has been influenced the purity of separated sperm, and the loss of sperm after separating vigor is big, influences its fertility.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of isolating method of mammalian sperm that is applicable to, when guarantee separating XY sperm efficient, improves the isolating accuracy rate of sperm and separates the vigor of sperm afterwards, and be applicable to and commercially produce.
The present invention solves the problems of the technologies described above with following technical scheme:
(1), in the former seminal fluid of gathering of male animal, adds the microbiotic mixing solutions, constant temperature preservation down in 24 hours.
(1), in the former seminal fluid of gathering of male animal, adds the microbiotic mixing solutions, constant temperature preservation down in 24 hours.
(2), add fluorescent dye and in water-bath, hatch dyeing after with former semen dilution, water-bath finishes the back and adds pigment with the spermatozoa diluent that does not contain yolk.
(3), under constant temperature, utilize flow cytometer to separate required X or y sperm.
(4), collect X sperm and y sperm after separating, separate the seminal fluid that obtains and after centrifugal concentrating, can be directly used in artificial insemination or in vitro fertilization with test tube that the sperm that contains yolk receives liquid is housed in advance, or freezing preservation after the low-temperature balance.
After the former seminal fluid of male animal had added the microbiotic mixing solutions, the temperature that constant temperature is preserved was 22.5～27.5 ℃.
The composition of semen diluent and content are:
| Composition | Restrain/200 milliliters |
| Sodium chloride and potassium chloride sodium hydrogen phosphate sodium acid carbonate magnesium chloride hexahydrate Sodium Pyruvate glucose sodium lactate HEPES bovine serum albumin(BSA) gentamicin sulphate | 1.00～1.20 0.04～0.05 0.005～0.010 0.15～0.18 0.01～0.02 0.04～0.05 0.15～0.20 0.70～0.75 milliliters/200 milliliter 1.5～2.0 0.10～0.80 0.005～0.010 |
The seminal fluid of dilution adds pigment FD﹠amp after adding fluorescent dye Hoechst33342 water-bath dyeing; The mass percent of C#40 is 0.0025%～0.005%.
Under 10 ℃～25 ℃ constant temperature, separate the XY sperm with flow cytometer.
Separating used sheath fluid composition and content is:
| Composition | Restrain/1000 milliliters |
| TRIS citric acid fructose sodium-chlor penicillin streptomycin | ????22.00～25.00 ????10.00～13.00 ????8.00～10.00 ????0.80～1.00 ????0.05～0.29 ????0.05～0.25 |
Sperm receives the liquid composition and content is:
| Composition | Restrain/100 milliliters |
| TRIS citric acid fructose tylosin lincomycin spectinomycin gentamicin | ????3.00～4.50 ????1.50～2.50 ????1.00～1.50 ????0.01～0.02 ????0.03～0.05 ????0.06～0.08 ????0.05～0.08 |
Use adds 10%～30% fresh yolk before according to volume percent.
Compare with present sperm isolation technique, the invention has the advantages that:
(1), adopt method of the present invention can effectively improve the resolving power of XY sperm to staining of sperm.
Existing sperm isolation technique is before sperm separates, utilization contains the diluent of lecithality former semen dilution is preserved, its maximum defective is that proteic substance such as yolk has influenced sperm to the homogeneity that fluorescence dye Hoechst33342 absorbs, and is unfavorable for the resolution of flow cytometer to X sperm and y sperm.And the present invention preserves the direct constant temperature of fresh former seminal fluid, and adds the growth that mixes microbiotic inhibition bacterium, and the material of protection motility of sperm effect has been avoided the influence of materials such as yolk to the staining of sperm homogeneity simultaneously in making full use of refining.The present invention utilizes improved Luo Shi liquid, the sperm dilution of carrying out one batch every 2 hours is dyeed, and dyeing finishes to separate immediately, shortens the timed interval of dyeing between separating, when effectively guaranteeing motility of sperm, make it to obtain evenly painted, be beneficial to the resolution of X sperm and y sperm.
(2), adopt method separated sperm of the present invention can effectively keep motility of sperm.
Existing sperm isolation technique is when staining of sperm, and the extension rate of sperm is bigger, and the protection material in the refining is by high dilution, and provide protection descends, and motility of sperm is made a big impact.The present invention is diluted to 1.0～1.5 hundred million/milliliter according to different types of animal with sperm when carrying out staining of sperm, add fluorescent dye and hatch dyeing in water-bath.Water-bath finishes the back and adds pigment, removes dead sperm, and only the sperm alive that cell membrane is complete separates.The present invention also adds the sperm that contains lecithality and receives liquid, the effect that has the protection sperm membrane and keep motility of sperm in the receiving tube of separated sperm.
(3) in existing sperm separation method, sepn process is generally carried out under 4 ℃ in room temperature or constant temperature; The present invention is undertaken by flow cytometer in the isolating whole process at sperm, is included in flow cytometer sample introduction test tube, sample introduction capillary passages and receives liquid in vitro, all keeps constant temperature under high slightly temperature.The quick loss of motility of sperm and the quick breeding of bacterium in the time of can avoiding like this under comparatively high temps separating, the cold strike that can avoid the rapid variation of temperature that sperm is caused again.
Technology of the present invention and artificial insemination and animal reproduction new technology such as in vitro fertilization combined is applied to livestock industry, can greatly improve the production efficiency of livestock industry.And technology of the present invention goes for the separation of XY sperm among the human seminal fluid equally, suffers from gender-related disease in order to avoid some as far as possible.
Description of drawings:
The detected XY sperm DNA of flow cytometer contains spirogram among Fig. 1 the present invention
Separate the accuracy rate master drawing that the X sperm sample that obtains utilizes reanalysis method to detect among Fig. 2 the present invention
Separate the accuracy rate master drawing that the y sperm sample that obtains utilizes reanalysis method to detect among Fig. 3 the present invention
Embodiment
Embodiment 1
(1) the bull fresh semen that collects is added the microbiotic mixing solutions, place 22.5 ℃ of following constant temperature water baths to preserve.The antibiotic concentration that is added is as follows:
| Title | Concentration (mcg/ml) |
| Tylosin lincomycin spectinomycin gentamicin | ????100 ????300 ????600 ????500 |
(2), with former semen dilution to 1.5 hundred million/milliliter, add fluorescent dye Hoechst33342 to 40 mcg/ml, in 35 ℃ water-bath, dye and hatched 50 minutes.Water-bath finishes the back and adds pigment FD﹠amp; C#40 is to mass percent concentration 0.0025%.The composition of semen diluent and content are:
| Composition | Restrain/200 milliliters |
| Sodium chloride and potassium chloride sodium hydrogen phosphate sodium acid carbonate magnesium chloride hexahydrate Sodium Pyruvate glucose sodium lactate HEPES bovine serum albumin(BSA) gentamicin sulphate | 1.1036 0.0448 0.0080 0.1680 0.0160 0.0440 0.1800 0.7220 milliliters/200 milliliter 1.9040 0.6000 0.0050 |
The full name of HEPES is N-2-hydroxyethyl piperazine-N '-2-ethanesulfonic acid;
Add deionized water to 200 milliliter;
Regulate pH7.0 with 1 mol sodium hydroxide solution;
Membrane filtration degerming with 0.22 micron pore size.
(3), under 16 ℃ of constant temperature, the X sperm is separated with y sperm, separate used sheath fluid and receive the liquid composition and content is respectively with flow cytometer:
Sheath fluid:
| Composition | Restrain/1000 milliliters |
| TRIS citric acid fructose sodium-chlor penicillin streptomycin | ????23.88 ????11.63 ????8.55 ????0.8 ????0.29 ????0.25 |
The full name of TRIS is a Tutofusin tris;
Add deionized water to 1000 milliliter;
Hydrochloric acid with 5 mol is regulated pH6.8;
0.22 the membrane filtration of micron pore size.
Sperm receives liquid:
| Composition | Restrain/100 milliliters |
| TRIS citric acid fructose tylosin lincomycin spectinomycin gentamicin | ????3.773 ????1.838 ????1.351 ????0.010 ????0.030 ????0.060 ????0.050 |
Add deionized water to 100 milliliter;
Hydrochloric acid with 5 mol is regulated pH7.0;
0.22 the membrane filtration of micron pore size;
Per 16 milliliters add 4 milliliters of fresh yolk before using.
(4), the sperm that obtains of separated and collected is centrifugal concentrates, and detects sperm motility rate under the sperm image analysis system, the average motility rate of sperm that obtains in 10 revision tests is: (67 ± 1.6) %.
(5), separate X sperm and the y sperm sample obtain and dock with ultrasonic wave, add Hoechst33342 again and redye sperm, by flow cytometer weight analysis sperm DNA, utilize the gaussian curve approximation figure to determine the accuracy rate of separated sperm.The average accuracy rate of X sperm that obtains in 5 times revision test is 94% (as Fig. 2), and the average accuracy rate of y sperm is 89% (as Fig. 3).
Embodiment 2
The grand woods goat fresh semen that collects is added the microbiotic mixing solutions, place 25 ℃ of constant temperature water baths to preserve, the antibiotic concentration that is added is expressed as follows with mcg/ml: tylosin, lincomycin, spectinomycin, gentamicin are respectively 200,500,800 and 800.
With former semen dilution to 1.3 hundred million/milliliter, add fluorescent dye Hoechst33342 to 30 mcg/ml, in 33 ℃ water-bath, dye and hatched 60 minutes.Water-bath finishes the back and adds pigment FD﹠amp; C#40 is to mass percent concentration 0.003%.
In the semen diluent, the concentration of sodium-chlor, Repone K, Sodium phosphate dibasic, sodium bicarbonate, magnesium chloride hexahydrate, Sodium.alpha.-ketopropionate, glucose, HEPES, bovine serum albumin, gentamicin sulphate is respectively 1.1,0.04,0.005,0.15,0.01,0.04,0.15,1.5,0.1,0.005 with/200 milliliters of expressions of gram, and Sodium.alpha.-hydroxypropionate is 0.7 milliliter/200 milliliters.Add deionized water to 200 milliliter; Regulate pH7.2 with 1 mol sodium hydroxide solution; Membrane filtration with 0.22 micron pore size.
Under 10 ℃ of constant temperature, the X sperm is separated with y sperm with flow cytometer.Separating used sheath fluid composition is: the concentration of TRIS, citric acid, fructose, sodium-chlor, penicillin, Streptomycin sulphate is respectively with/1000 milliliters of expressions of gram: 22.00,10.0,10.0,1.0,0.05,0.05; Add deionized water to 1000 milliliter; Hydrochloric acid with 5 mol is regulated pH6.8; Membrane filtration with 0.22 micron pore size.
The concentration of separating used sperm reception liquid composition in back and content: TRIS, citric acid, fructose, tylosin, lincomycin, spectinomycin, gentamicin is respectively 3.0,1.5,1.0,0.015,0.030,0.060,0.070 with/100 milliliters of expressions of gram; Add deionized water to 100 milliliter; Hydrochloric acid with 5 mol is regulated pH7.0; 0.22 the membrane filtration of micron pore size, per 12 milliliters receive the use afterwards of 4 milliliters of fresh yolk of liquid adding.
The sperm that separated and collected obtains is centrifugal to be concentrated, and detects sperm motility rate under the sperm image analysis system, and the average motility rate of goat separated sperm that obtains in 10 revision tests is: (73 ± 4.8) %.
Separate the X sperm and the y sperm sample that obtain and dock, add Hoechst33342 again and redye sperm,, utilize the gaussian curve approximation figure to determine the accuracy rate of separated sperm by flow cytometer weight analysis sperm DNA with ultrasonic wave.The average accuracy rate of separation goat X sperm that obtains in 5 times revision test is 96%, and the average accuracy rate of y sperm is 92%.
Embodiment 3
The danish landrace boar fresh semen that collects is added the microbiotic mixing solutions, place 27.5 ℃ of constant temperature water baths to preserve, the antibiotic concentration that is added is as follows: the concentration of tylosin, lincomycin, spectinomycin, gentamicin represents to be respectively 180,400,700,700 with mcg/ml;
With semen dilution to 1.0 hundred million/milliliter, add fluorescent dye Hoechst33342 to 30 mcg/ml, in 37 ℃ water-bath, dye and hatched 45 minutes.Water-bath finishes the back and adds pigment FD﹠amp; C#40 is to mass percent concentration 0.0035%.
In the semen diluent, the concentration of sodium-chlor, Repone K, Sodium phosphate dibasic, sodium bicarbonate, magnesium chloride hexahydrate, Sodium.alpha.-ketopropionate, glucose, HEPES, bovine serum albumin, gentamicin sulphate is respectively 1.2,0.05,0.01,0.18,0.02,0.05,0.20,2.0,0.8,0.01 with/200 milliliters of expressions of gram, and Sodium.alpha.-hydroxypropionate is 0.75 milliliter/200 milliliters.Add deionized water to 200 milliliter; Regulate pH7.0 with 1 mol sodium hydroxide solution, with the membrane filtration of 0.22 micron pore size.
Under 25 ℃ of constant temperature, the X sperm is separated with y sperm with flow cytometer; Separating used sheath fluid composition is: the concentration of TRIS, citric acid, fructose, sodium-chlor, penicillin, Streptomycin sulphate is respectively with/1000 milliliters of expressions of gram: 25.00,12.0,9.0,0.9,0.2,0.15.Add deionized water to 1000 milliliter; Hydrochloric acid with 5 mol is regulated pH6.8; Membrane filtration with 0.22 micron pore size.
Separate back used reception liquid composition and content: the concentration of TRIS, citric acid, fructose, tylosin, lincomycin, spectinomycin, gentamicin is respectively 4.5,2.5,1.5,0.02,0.050,0.080,0.080 with/100 milliliters of expressions of gram; Add deionized water to 100 milliliter; Hydrochloric acid with 5 mol is regulated pH7.0; 0.22 the membrane filtration of micron pore size; After adding 1.8 milliliters of fresh yolk, use per 12 milliliters of reception liquid.
The sperm that separated and collected obtains is centrifugal to be concentrated, and detects sperm motility rate under the sperm image analysis system, and the average motility rate of sperm that obtains in 10 revision tests is: (60 ± 5.5) %.
Separate the X sperm and the y sperm sample that obtain and dock, add Hoechst33342 again and redye sperm,, utilize the gaussian curve approximation figure to determine the accuracy rate of separated sperm by flow cytometer weight analysis sperm DNA with ultrasonic wave.The average accuracy rate of X sperm that obtains in 5 times revision test is 90%, and the average accuracy rate of y sperm is 86%.
Claims (6)
1. a method of separating Mammals XY sperm according to the principle that the dna content of Mammals X sperm is Duoed than y sperm, uses flow cytometer to separate by after the fluorescent dye, it is characterized in that:
(1), in the former seminal fluid of gathering of male animal, adds the microbiotic mixing solutions, constant temperature preservation down in 24 hours.
(2), add fluorescent dye and in water-bath, hatch dyeing after with the spermatozoa diluent that does not contain yolk former semen dilution.Water-bath finishes the back and adds pigment.
(3), under constant temperature, utilize flow cytometer to separate required X or y sperm.
(4), collect X sperm and y sperm after separating, separate the seminal fluid that obtains and after centrifugal concentrating, can be used for artificial insemination or in vitro fertilization with test tube that the sperm that contains yolk receives liquid is housed in advance, or freezing preservation after the low-temperature balance.
2. the method for separation as claimed in claim 1 Mammals XY sperm, it is characterized in that the former seminal fluid of male animal has added the microbiotic mixing solutions after, the temperature that constant temperature is preserved is 22.5～27.5 ℃.
3. the method for separation Mammals XY sperm as claimed in claim 1 or 2 is characterized in that the composition of semen diluent and content are:
One-tenth decigram/200 milliliters
Sodium-chlor 1.0～1.2
Repone K 0.04～0.05
Sodium phosphate dibasic 0.005～0.010
Sodium bicarbonate 0.15～0.18
Magnesium chloride hexahydrate 0.01～0.02
Sodium.alpha.-ketopropionate 0.04～0.05
Glucose 0.15～0.20
0.70～0.75 milliliter/200 milliliters of Sodium.alpha.-hydroxypropionates
HEPES????????????????????????????????1.5～2.0
Bovine serum albumin 0.1～0.8
Gentamicin sulphate 0.005～0.010
4. the method for separation Mammals XY sperm as claimed in claim 1 is characterized in that utilizing under 10 ℃～25 ℃ constant temperature flow cytometer to separate X or y sperm.
5. the method for separation Mammals XY sperm as claimed in claim 4, used sheath fluid composition and content is when it is characterized in that utilizing flow cytometer to separate X or y sperm:
One-tenth decigram/1000 milliliters
TRIS????????????????????????????22.00～25.00
Citric acid 10.00～13.00
Fructose 8.00～10.00
Sodium-chlor 0.80～1.00
Penicillin 0.05～0.29
Streptomycin sulphate 0.05～0.25
6. as the methods of claim 4 or 5 described separation Mammals XY sperms, it is characterized in that receiving liquid composition and content and be:
One-tenth decigram/100 milliliters
TRIS????????????????????????????3.00～4.50
Citric acid 1.50～2.50
Fructose 1.00～1.50
Tylosin 0.010.02
Lincomycin 0.03～0.05
Spectinomycin 0.06～0.08
Gentamicin 0.05～0.08
Use adds 10%～30% fresh yolk before according to volume percent.
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| CN 200410060793 CN1284849C (en) | 2004-08-31 | 2004-08-31 | Method for separating XY germ cell of mammal |
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| CN1667118A true CN1667118A (en) | 2005-09-14 |
| CN1284849C CN1284849C (en) | 2006-11-15 |
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| CN101793641A (en) * | 2010-04-14 | 2010-08-04 | 内蒙古蒙牛繁育生物技术股份有限公司 | Fluorescent staining method for separating dairy cow sperms |
| CN102907417A (en) * | 2012-10-22 | 2013-02-06 | 西北农林科技大学 | Freezing diluent formula for improving quality of Holstein bull frozen-thawed X-spermatozoa |
| CN103222458A (en) * | 2013-04-08 | 2013-07-31 | 陕西石羊集团蒲城畜牧发展有限公司 | Swine gender-controlling sperm and preparation method and application thereof |
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| CN102907417B (en) * | 2012-10-22 | 2014-01-29 | 西北农林科技大学 | Freezing diluent formula for improving quality of Holstein bull frozen-thawed X-spermatozoa |
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| CN109370981A (en) * | 2018-11-07 | 2019-02-22 | 临泉县欣达生态羊业有限公司 | The X Gender preselection of Anhui White Goats |
| CN114085809A (en) * | 2021-11-30 | 2022-02-25 | 吉林大学 | Method for separating X sperm and Y sperm |
| CN114085809B (en) * | 2021-11-30 | 2023-08-25 | 吉林大学 | Method for separating X sperm and Y sperm |
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