CN1232231C - Livestock sex control method by using sperm separation and embryo sex identification as basis - Google Patents
Livestock sex control method by using sperm separation and embryo sex identification as basis Download PDFInfo
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- CN1232231C CN1232231C CN 03109426 CN03109426A CN1232231C CN 1232231 C CN1232231 C CN 1232231C CN 03109426 CN03109426 CN 03109426 CN 03109426 A CN03109426 A CN 03109426A CN 1232231 C CN1232231 C CN 1232231C
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Abstract
The present invention relates to a livestock sex control method on the basis of sperm separation and fetus sex identification, which comprises the steps of the separation and freezing preservation of sperms, the production of a sex control fetus and the identification of the sex of the fetus, wherein the separation and freezing preservation of sperms comprises the collection of fresh sperms, the treatment of sperms before separation, the separation of sperms and the freezing preservation, and the production of a sex control fetus and the identification of the sex of the fetus comprise the production of the fetus(a vivo or test tube technique), the incision of the fetus and the identification of the sex of the fetus by a PCR method. The present invention has the following advantages that the accurate rate of the separation of the sperms reaches more than 90%, the vigor of the sperms is maintained to be more than 50%, and the accurate rate of the identification of the sex of fetuses is 100%.
Description
Technical field
The invention belongs to a kind of domestic animal sex-control method, particularly a kind of domestic animal sex-control method that is accredited as the basis with sperm separation, embryo gender.
Background technology
The sex of mammal (containing domestic animal) is decided by the x and y sperm in the time of fertilization seminal fluid.X sperm and ovum are in conjunction with producing the maternal instinct individuality, and y sperm then causes the generation of male.Because the ratio of X sperm and y sperm is suitable, the male and female that therefore produce among the offspring respectively account for 50% approximately in normal livestock semen.Manage for actual industry, if can separate X sperm and y sperm, just can select the sex of domestic animal according to producing needs, require to improve the natality of male calf as far as possible such as the beef cattle beginner, the milch cow operator thinks that then many Rhizoma Anemarrhenae calf can obtain bigger economic benefit.
Since the popularization and application of the sixties in last century technology of artificial insemination, and the trial of the embryo transfer technology that begins of the seventies, numerous research worker begin to carry out that sperm separates and the research and inquirement of embryo gender evaluation.The isolating basis of sperm is a difference of utilizing aspects such as physics and chemistry between X sperm and the y sperm, physiology, gene, wait such as weight, surface charge, pH value and antigenicity and to design various experimental models, comprising the sedimentation method, density, electrophoresis method, antigen-antibody method etc.But above-mentioned all methods are because the accurate rate variance of separated sperm or do not have effect fully, reasons such as infringement to motility of sperm, to last century the nineties therefore negated also just can't be applied to production practices basically by the experimental result of most of research worker as a practical technique.In addition, embryo's sex identification also is an analogue.
Summary of the invention
The purpose of this invention is to provide a kind of domestic animal sex-control method that is accredited as the basis with sperm separation, embryo gender, it has overcome above-mentioned defective, accuracy rate based on the fluidic cell partition method of the minute differences of X sperm and y sperm dna content is 90%, motility of sperm based on artificial insemination is 50%, has reached the industry application level; Based on the embryo gender authenticate technology accuracy rate of y sperm specific DNA segment near 100%.
The present invention may further comprise the steps with the domestic animal sex-control method that sperm separates, embryo gender is accredited as the basis:
(1) sperm separates and freezing preservation
A, from tame carcass, take 1 milliliter of fresh semen, use phosphate buffer PBS-0.05%PVP, centrifuge washing 2 times, 2000 change, and 5 minutes, with described phosphate buffer sperm concentration are transferred to 100 * 10 then
6Individual/milliliter, every milliliter of fresh semen can be modulated the dilution seminal fluid of the above-mentioned concentration of 10-15 milliliter.
B, add 10 mcg/ml fluorescent dyes in seminal fluid, DNA dyeing is 30 minutes under 37 ℃ condition, uses phosphate buffer PBS-0.05%PVP then, centrifuge washing 2 times, and 2000 change 5 minutes;
C, again sperm concentration is transferred to 100 * 10
6Individual/milliliter, pack into then in the fluidic cell seperator, the speed of 3000 sperms is separated with each second, after X sperm separately and Y are skillful in being recovered, according to the sum of every 250～5,000,000 sperms, the 0.25 milliliter of freezing of semen pipe of packing into is made frozen semen according to conventional method, be stored in the liquid nitrogen, 1 milliliter of fresh semen can be produced X sperm after the above-mentioned separation or each 10-15 of frozen semen of y sperm props up;
D, production sex controll embryo: the sperm after separating is carried out in vitro fertilization and cultivation with the ratio of every 200 ovums, can produce 25-30, be used for embryo transfer through sex-controlled A-B grade embryo (blastaea);
(2), embryo gender is identified:
A, to use frozen embryo;
B, embryonic cell cutting: get the blastaea after thawing, add in 0.25% the protease at described phosphate buffer and to handle 2-3 minute, make embryo's zona pellucida softening, move into described phosphorus enzyme buffer liquid then and add in 0.3% the serum albumin solution, handle about embryo number 20-40 at every turn:
C, at microscopically, be 15 ° of angles with the cutter end, the special-purpose metal blade of embryo's cutting, or the glass fine needle downcuts nutritive layer cell 10-15 of the embryo, cell that downcuts and embryo's major part, embryo group cell, reference numeral respectively in comprising, embryo's major part is put into the embryo and is preserved liquid TCM199-10%FBS, at CO
2Kept 2-4 hour in the incubator;
D, the cell that downcuts is carried out female, malely Jian Ding not with the external synthetic technology PCR method of a kind of DNA, each the embryonic cell sample that cuts moves in the PCR reaction tube of 10 microlitre pure water, Rapid Cleaning is 3 times in the water, with 100 ℃ water bath processing 10 minutes, get 1 microlitre then and carry out the PCR reaction, with BOV97M (157bp, the specific gene part of the Y chromosome of a kind of male cattle) is male probe, is female sample probe with alpha-lactalbumin gene fragment (109bp);
The composition of PCR reactant liquor: 200 micromolar Deoxydization nucleotides, each 40 micromolar male and female sample probe template, the archaeal dna polymerase (1.25Iu Taq polymerase) of 1.25 ius, PCR buffer transfer to 50 microlitres with pure water with total liquid measure;
The condition of PCR reaction: 95 ℃ 1 minute, 55 ℃ 2 minutes, 72 ℃ 3 minutes, above-mentioned condition repeats 40 times;
The PCR response sample is checked: get reacted each sample, the sepharose electrophoresis with 3% 20 minutes has 157bp, and the embryo of 109bp biobelt is male, and it is female having only the embryo of 109bp band, and band does not slip up or lose for sample operation.
According to the check result of PCR, just can determine corresponding embryo's sex, as required the embryo is transplanted then or freezing preservation.This technical process needs 2-3 hour approximately, and the processing of sample can overlappingly be carried out, and embryo's sex identification accuracy rate is about 100%.
Said fluorescent dye is Hoechst 33342.
The present invention has the following advantages: the rate of accuracy reached of separated sperm is more than 90%, and motility of sperm remains on more than 50%, and embryo's sex identification accuracy rate is about 100%.
Description of drawings:
Accompanying drawing 1 is a process method flow chart of the present invention.
Accompanying drawing 2 is embryo's cutting illustraton of model and the actual cutting drawing of embryo.
Accompanying drawing 3 is that PCR figure is as a result confirmed in chromosome examination of the present invention.
The specific embodiment:
As shown in the figure: a kind of with the domestic animal sex-control method that sperm separates, embryo gender is accredited as the basis, may further comprise the steps:
(1) sperm separates and freezing preservation:
A, take 1 milliliter of fresh semen, use phosphate buffer PBS-0.05%PVP from normal breeding oxen, centrifuge washing 2 times, 2000 change, and 5 minutes, with described phosphate buffer sperm concentration are transferred to 100 * 10 then
6Individual/milliliter, every milliliter of fresh semen can be modulated the dilution seminal fluid of the above-mentioned concentration of 10-15 milliliter;
B, add 10 mcg/ml fluorescent dye Hoechst 33342 in sperm liquid, DNA dyeing is 30 minutes under 37 ℃ condition, uses phosphate buffer PBS-0.05%PVP centrifuge washing 2 times then, and 2000 change 5 minutes;
C, again sperm concentration is transferred to 100 * 10
6Individual/milliliter, pack into then in the fluidic cell seperator, the speed of 3000 sperms is separated with each second, X sperm positively charged separately and y sperm is electronegative be recovered respectively after, according to the sum of every 250～5,000,000 sperms, the 0.25 milliliter of freezing of semen pipe of packing into is made frozen semen according to conventional method, be stored in the liquid nitrogen, 1 milliliter of fresh semen can be produced X sperm after the above-mentioned separation or each 10-15 of frozen semen of y sperm props up;
D, production sex controll embryo: because embryo's sex is by the sperm type decision of the time of fertilization, therefore utilize X sperm or y sperm after separating directly to produce in-vitro embryos, enlarge the service efficiency of separated sperm, concrete grammar is that the sperm after separating is carried out external fertilization and cultivation with the ratio of every 200 ovums, can produce 25-30 through sex-controlled A-B grade embryo (blastaea), freezing preservation is used for embryo transfer;
(2), embryo gender is identified:
A, embryo gender are identified to use frozen embryo;
The cutting of B, embryonic cell: the blastaea of getting after thawing was handled 2-3 minute in described phosphate buffer adds 0.25% protease, make embryo's zona pellucida softening, move into described phosphate buffer then and add in 0.3% the serum albumin solution, handle about embryo number 20-40 at every turn;
C, at microscopically, be 15 ° of angles with the cutter end, the special-purpose metal blade of embryo's cutting downcuts nutritive layer cell 10-15 of embryo, cell that downcuts and embryo's major part, embryo group cell in comprising, the difference reference numeral, embryo's major part is put into the embryo and is preserved liquid TCM199-10%FBS, at CO
2Kept 2-4 hour in the incubator;
D, the cell that downcuts is carried out female, malely Jian Ding not with the external synthetic technology PCR method of a kind of DNA, each the embryonic cell sample that cuts moves in the PCR reaction tube of 10 microlitre pure water, Rapid Cleaning is 3 times in the water, with 100 ℃ water bath processing 10 minutes, gets 1 microlitre then and carries out the PCR reaction; With BOV97M (157bp, the specific gene part of the Y chromosome of a kind of male cattle) is male probe, is female sample probe with alpha-lactalbumin gene fragment (109bp);
The composition of PCR reactant liquor: 200 micromolar Deoxydization nucleotides, each 40 micromolar male and female sample probe template, the archaeal dna polymerase of 1.25 ius, PCR buffer transfer to 50 microlitres with pure water with total liquid measure;
The condition of PCR reaction: 95 ℃ 1 minute, 55 ℃ 2 minutes, 72 ℃ 3 minutes, above-mentioned condition repeats 40 times;
The PCR response sample is checked: get reacted each sample, the sepharose electrophoresis with 3% 20 minutes has 157bp, and the embryo of 109bp biobelt is male, and it is female having only the embryo of 109bp band, and band does not slip up or lose for sample operation.
According to the check result of PCR, just can determine corresponding embryo's sex, as required the embryo is transplanted then or freezing preservation.This technical process needs 2-3 hour approximately, and the processing of sample can overlappingly be carried out, and embryo's sex identification accuracy rate is about 100%.
Testing result:
Present technique has all been carried out concrete enforcement on laboratory and industry pilot scale level, and its result has been carried out the detection of different levels, and concrete grammar and result are as follows: (referring to Fig. 3)
1, the accuracy rate of separated sperm and fertilization property:
(1) the accuracy rate inspection of separated sperm (molecular hybridization in situ method)
Inspection sperm sum: 2000
X sample of sperm (hyperfluorescence) X sperm 93.6% y sperm 6.4%
Y sperm sample (hypofluorescence) y sperm 81.8% X sperm 18.2%
(2) utilize the calving inspection (micro-fertilization embryo) of separated sperm after fertilization
10 male 8/female 2 accuracys rate 80.0% of y sperm sample
(3) sex controll embryo's production and evaluation (inspection of PCR method)
The y sperm sample: external fertilization is handled 187 in ovum, obtains 62 of A-B blastaeas
(26.3%), wherein male embryo accounts for 86%
The X sample of sperm: external fertilization is handled 235 in ovum, obtains 79 of A-B blastaeas
(36.6%), wherein female embryo accounts for 91%
2, sex identification embryo's accuracy rate inspection:
Check 46 of embryo numbers, 24 of wherein male embryos (52.1%), 15 (conception rate 62.5%) of calving sum behind these embryo transfers: male 15 (100%), female 0 (0%).
Claims (2)
1, a kind of with the domestic animal sex-control method that sperm separates, embryo gender is accredited as the basis, it is characterized in that: may further comprise the steps:
(1) sperm separates and freezing preservation:
A, take 1 milliliter of fresh semen from tame carcass, with phosphate buffer PBS-0.05%PVP centrifuge washing 2 times, 2000 change, and 5 minutes, with described phosphate buffer sperm concentration are transferred to 100 * 10 then
6Individual/milliliter, every milliliter of fresh semen can be modulated the dilution seminal fluid of the above-mentioned concentration of 10-15 milliliter;
B, add 10 mcg/ml fluorescent dyes in seminal fluid, DNA dyeing is 30 minutes under 37 ℃ condition, uses phosphate buffer PBS-0.05%PVP centrifuge washing 2 times then, and 2000 change 5 minutes;
C, again sperm concentration is transferred to 100 * 10
6Individual/milliliter, pack into then in the fluidic cell seperator, the speed of 3000 sperms is separated with each second, after X sperm separately and y sperm are recovered, according to the sum of ten thousand sperms of every 250-500, the 0.25 milliliter of freezing of semen pipe of packing into is made frozen semen, be stored in the liquid nitrogen, 1 milliliter of fresh livestock semen is produced X sperm after the above-mentioned separation or each 10-15 of frozen semen of y sperm props up;
D, production sex controll embryo: the sperm after separating is carried out external fertilization and cultivation with the ratio of every 200 ovums, produce 25-30 through sex-controlled A-B grade embryo, freezing preservation is used for embryo transfer;
(2) embryo gender is identified:
A, the above-mentioned frozen embryo of use;
The cutting of B, embryonic cell: get embryo after thawing and add in 0.25% the protease at described phosphate buffer and handled 2-3 minute, make embryo's zona pellucida softening, move into described phosphate buffer then and add in 0.3% the serum albumin solution, handle about embryo number 20-40 at every turn;
C, at microscopically, downcut nutritive layer cell 10-15 of embryo with metal blade or glass fine needle, the cell of cutting-out and embryo's major part, embryo group cell in comprising, the difference reference numeral, embryo's major part is put into the embryo and is preserved liquid TCM199-10%FBS, at CO
2Kept 2-4 hour in the incubator;
D, the cell that downcuts is carried out female, malely Jian Ding not with the external synthetic technology PCR method of a kind of DNA, each the embryonic cell sample that cuts moves in the PCR reaction tube of 10 microlitre pure water, Rapid Cleaning is 3 times in the water, with 100 ℃ water bath processing 10 minutes, get 1 microlitre then and carry out the PCR reaction, with BOV97M is male probe, is female sample probe with the alpha-lactalbumin gene fragment;
The composition of PCR reactant liquor: 200 micromolar Deoxydization nucleotides, each 40 micromolar male and female sample probe template, the archaeal dna polymerase of 1.25 ius, PCR buffer transfer to 50 microlitres with pure water with total liquid measure;
The condition of PCR reaction: 95 ℃ 1 minute, 55 ℃ 2 minutes, 72 ℃ 3 minutes, above-mentioned condition repeats 40 times;
The PCR response sample is checked: get reacted each sample with 3% sepharose electrophoresis 20 minutes, have 157bp, the embryo of 109bp biobelt is male, and it is female having only the embryo of 109bp band, and band does not slip up or lose for sample operation.
2, according to claim 1 a kind of with the domestic animal sex-control method that sperm separates, embryo gender is accredited as the basis, it is characterized in that: said fluorescent dye is Hoechst33342.
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| CN 03109426 CN1232231C (en) | 2003-04-10 | 2003-04-10 | Livestock sex control method by using sperm separation and embryo sex identification as basis |
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| CN 03109426 CN1232231C (en) | 2003-04-10 | 2003-04-10 | Livestock sex control method by using sperm separation and embryo sex identification as basis |
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| CN1232231C true CN1232231C (en) | 2005-12-21 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7713687B2 (en) | 2000-11-29 | 2010-05-11 | Xy, Inc. | System to separate frozen-thawed spermatozoa into x-chromosome bearing and y-chromosome bearing populations |
| US7758811B2 (en) | 2003-03-28 | 2010-07-20 | Inguran, Llc | System for analyzing particles using multiple flow cytometry units |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2408939C (en) | 2000-05-09 | 2011-11-08 | Xy, Inc. | High purity x-chromosome bearing and y-chromosome bearing populations of spermatozoa |
| US7169548B2 (en) | 2002-09-13 | 2007-01-30 | Xy, Inc. | Sperm cell processing and preservation systems |
| CN100478675C (en) * | 2005-02-07 | 2009-04-15 | 北京大学 | DNA sex verification of scrod and fish fertilized egg |
| CN101322489B (en) * | 2008-08-01 | 2011-11-30 | 内蒙古蒙牛繁育生物技术有限公司 | Deer X/Y sperm separation freezing sperm and producing method thereof and use |
| CN103858821B (en) * | 2014-03-25 | 2015-08-12 | 西北农林科技大学 | A kind of feeding method and nourishing additive agent increasing milch goat X sperm ratio |
| JP2018143102A (en) * | 2017-03-01 | 2018-09-20 | 山下 直樹 | Human y-bearing sperm selection method |
-
2003
- 2003-04-10 CN CN 03109426 patent/CN1232231C/en not_active Expired - Lifetime
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7713687B2 (en) | 2000-11-29 | 2010-05-11 | Xy, Inc. | System to separate frozen-thawed spermatozoa into x-chromosome bearing and y-chromosome bearing populations |
| US7758811B2 (en) | 2003-03-28 | 2010-07-20 | Inguran, Llc | System for analyzing particles using multiple flow cytometry units |
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| CN1536361A (en) | 2004-10-13 |
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