CN101347458A - Sheep X/Y sperm separation freezing sperm as well as preparation method and use thereof - Google Patents
Sheep X/Y sperm separation freezing sperm as well as preparation method and use thereof Download PDFInfo
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- CN101347458A CN101347458A CNA2008101353522A CN200810135352A CN101347458A CN 101347458 A CN101347458 A CN 101347458A CN A2008101353522 A CNA2008101353522 A CN A2008101353522A CN 200810135352 A CN200810135352 A CN 200810135352A CN 101347458 A CN101347458 A CN 101347458A
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Abstract
The invention relates to sheep X/Y sperm-separated frozen semen, the production method and application thereof. The sheep X/Y sperm-separated frozen semen provided by the invention contains a frozen balance solution A, a frozen balance solution B including 12 percent glycerin cryoprotectant and a sheep X or Y sperm-separated frozen semen. The invention applies the difference of DNA content of sheep X and Y sperms and separates X and Y sperms through flow cytometry so as to manufacture the sex-control frozen semen and the frozen storage condition is 20 to 30min treatment under -90 to 120 DEG C. The frozen semen provided by the invention controls the lamb sex through artificial insemination or embryo transplantation. With high separation efficiency and sex control accuracy up to 90 percent, the frozen semen is suitable for industrialization production and culture, thus greatly increasing the culturing effect.
Description
Technical field
The present invention relates to a kind of domestic animal X/Y sperm isolation technics, particularly a kind of sheep X/Y sperm separation freezing sperm and production method and application.
Background technology
Frozen semen, artificial fertilization and external fertilization technology based on domestic animal are widely used in producing to breed with biology of reproduction studying.Under normal circumstances, the filial generation of sheep (sheep and goat) is bred male and female and is respectively accounted for 50%, but in production reality, in order to improve the breed effect of sheep (sheep and goat), need breed ewe lamb (sheep, Goat Breeding and expansion group) or public lambs (production of commodity Carnis caprae seu ovis, the production of commodity cashmere) for specific strain, so just need control the sex of children of sheep (sheep and goat) by separating the X/Y sperm more.In addition, the sperm after the separation needs freezing preservation usually before insemination, and the difference of each species, its requirement for pH value, freezing buffer and freezing processing method is also different, and this has proposed higher requirement to freezing conditions and buffer thereof.
CN1232231C discloses the isolating method of a kind of domestic animal X/Y sperm, but this method need be used phosphate buffer solution washing seminal fluid, separates by the fluidic cell seperator, and its speed is 3000/second.Yet, when using this method to separate the X/Y sperm of sheep, its separating rate, separate that accuracy rate, buffer are used, the aspects such as motility of sperm after the freezing preservation still improve a lot the space.
CN101112392A discloses dosage livestock semen of a kind of end and production method thereof, this method is handled multiple livestock semen by the freezing balance liquid B (Tris-B) that uses freezing balance liquid A (Tris-A) and added glycerol, by the research of multiple animal semen physiological property, find the fertilization facilitation effect between specific heterogenous animal sperm.This method need be added the seminal fluid of xenogenesis domestic animal, and using the conception rate of the goat+sheep sperm freezing liquid of this method is 51%, in the space that still has lifting.
Summary of the invention
The purpose of this invention is to provide sheep X/Y sperm separation freezing sperm and production thereof and methods and applications, sheep X/Y sperm can be separated, and realize industrialization production and application, thus the sex of children of control sheep.
The invention provides a kind of sheep X/Y sperm separation freezing sperm, comprise freezing balance liquid A, freezing balance liquid B and sheep X or y sperm sperm separation freezing, its content is as follows:
Freezing balance liquid A Tris-A 50%
Freezing balance liquid B contains the Tris-B 50% of 12% glycerol cryoprotective agent, wherein:
Sheep X or y sperm sperm separation freezing concentration are 50-200 ten thousand sperms/0.25mL.
Above-mentioned freezing balance liquid A, pH 6.8, and is composed of the following components:
Tris Tris 291.7mM
Citric acid 95.1mM
Fructose 70.2mM
Yolk 20%
Ultra-pure water;
Freezing balance liquid B, pH 6.8, and is composed of the following components:
Tris Tris 291.7mM
Citric acid 95.1mM
Fructose 70.2mM
Yolk 20%
Glycerol 12%
Ultra-pure water.
The present invention also provides the production method of above-mentioned sheep X/Y sperm separation freezing sperm, comprises the steps:
1) gathers fresh semen 2-5mL (hundred million of total sperm count 25-50) from the ram of health, bacterial multiplication adds antibiotic (tylosin Tylosin:0.103mg/mL in the seminal fluid in order to suppress, gentamycin Gentamicin:0.508mg/mL, woods can-miramycin Linco-spectin:0.6mg/mL), be incubated under 18-20 ℃ of condition, every part of fresh semen can be used under this condition 10-12 hour;
2) use sperm concentration-vigor instrument to detect the concentration and the vigor of fresh semen, have only vigor to can be used for separating treatment at the sample more than 80%, amount to volume according to seminal concentration and get the centrifugal removal refining of 400,000,000 sperms, add 10 μ g/mL Hochest 33342 and in 24 ℃ of tepidariums, dyeed 20 minutes;
3) sheep X/Y sperm separated DNA content difference is set at 3.2-3.4%, set fluidic cell seperator parameter (first phase region is just got the former seminal fluid necrospermia of 80%/the second phase region rate 10%=X/Y unisexuality sperm separation of sperm jet flow sum and got rate 45-47%) according to sheep X/Y sperm DNA content difference, separate x and y sperm by the fluidic cell seperator, make sheep X/Y sperm separating rate be controlled at 5000-7000/second, X or y sperm separation purity reach 85-95%;
4) show according to separated sperm accumulative total, learnt from else's experience isolating X or y sperm by 1-1.1 hundred million, change/5 minutes centrifugal treating with 2000 behind the following machine, detect separated sperm concentration with microscope-computer sperm scale of notation, be diluted to 2 times of final design concentration with freezing balance liquid A, under 4-5 ℃ of condition, place more than 90 minutes;
5) after the freezing balance liquid A balance, add the freezing balance liquid B that contains 12% glycerol cryoprotective agent of equivalent, 4 ℃ of following balances 30 minutes;
6) separated X that under 4 ℃ of conditions step 5) is obtained or the Y seminal fluid cryovial of packing into is made and is contained sheep X/Y sperm separation freezing sperm.
7) because the effect difference of frozen solution, the sheep X/Y sperm separation freezing sperm that further step 6) is made is inserted in-90 ℃ the nitrogen vapor and was handled 20-30 minute; When temperature drops to-120 ℃, preserving in the cryovial input liquid nitrogen.
Above-mentioned sheep X/Y sperm separation freezing sperm is used for conventional artificial insemination or operation artificial insemination method, and every receptor once can be imported 50-200 very much from sperm, needs orientation to breed public lamb or ewe lamb according to production; Perhaps, by sheep X/Y sperm separation freezing sperm productivity control embryo, carry out the speed of breeding that embryo transfer is accelerated defect individual again.
The present invention uses the difference of sheep X, y sperm dna content, separates x and y sperm by the fluidic cell mode, makes the sex controll frozen semen then, controls the sex of sheep lambing in the artificial insemination mode according to producing needs.The inventive method does not need to add buffer before sperm separates, its separating rate reaches 5000-7000/second, separation efficiency significantly improves, resulting separated sperm product vigor height, test-tube conception rate height (can reach more than 74%), the sex controll accuracy rate of lambing can reach more than 90%.Product of the present invention and method are applicable to industrialization production and breed, can increase substantially the breed effect of sheep.
The specific embodiment
Below in conjunction with specific embodiments method of the present invention is done more detailed description.It will be appreciated by those skilled in the art that following embodiment all is used for the present invention's scope required for protection is carried out the description of exemplary, summarize the relative scope of each parameter of the present invention, thereby it can not be interpreted as a kind of concrete restriction of the present invention with this.
Embodiment 1
The making of sheep X frozen semen and artificial insemination control lambing sex:
1) gathering 4mL, total sperm count from the Mongolian ram of a health is 4,000,000,000 fresh semen, bacterial multiplication adds antibiotic (tylosin Tylosin:0.103mg/mL in the seminal fluid in order to suppress, gentamycin Gentamicin:0.508mg/mL, woods can-miramycin Linco-spectin:0.6mg/mL), under 18-20 ℃ of condition, be incubated, use sperm concentration-vigor instrument to detect the concentration and the vigor of fresh semen, after testing, the motility of sperm of this seminal fluid is 85%, transports back laboratory and separates use 5 hours for the X/Y sperm.
2) amount to volume according to seminal concentration and get the centrifugal removal refining of 4 * 200,000,000 sperms (using), add 10 μ g/mL Hochest 33342 and in 24 ℃ of tepidariums, dyeed 20 minutes for 2 sperm seperators.
3) sheep X/Y sperm separated DNA content difference is set at 3.2-3.4%, set sperm streaming separator cell (the SX-MoFlo fluidic cell seperator of U.S. XY Co.,Ltd) parameter (first phase region is just got the former seminal fluid necrospermia of 80%/the second phase region rate 10%=X/Y unisexuality sperm separation of sperm jet flow sum and got rate 45-47%) according to sheep X/Y sperm DNA content difference, separate x and y sperm by the fluidic cell seperator, make sheep X/Y sperm separating rate be controlled at 5000-7000/second, X or y sperm separation purity reach 85-95%.
4) show according to separated sperm accumulative total, learnt from else's experience isolating X or y sperm by 1-1.1 hundred million, carried out centrifugal treating in/5 minutes with 2000 commentaries on classics behind the following machine, detect separated sperm concentration with microscope-computer sperm scale of notation, be diluted to 2 times of final design concentration with Tris-A (freezing balance liquid A), under 4-5 ℃ of condition, place more than 90 minutes; Wherein, above-mentioned freezing balance liquid A, pH 6.8, and is composed of the following components:
Tris Tris 291.7mM
Citric acid 95.1mM
Fructose 70.2mM
Yolk 20%
Ultra-pure water.
5) after the Tris-A balance, add the Tris-B that contains 12% glycerol cryoprotective agent (freezing balance liquid B) of equivalent, 4 ℃ of refrigerator balances 30 minutes; Wherein, freezing balance liquid B, pH 6.8, and is composed of the following components:
Tris Tris 291.7mM
Citric acid 95.1mM
Fructose 70.2mM
Yolk 20%
Glycerol 12%
Ultra-pure water.
6) isolating X or the Y seminal fluid 0.25mL cryovial of packing under 4 ℃ of conditions is made the sheep property control frozen semen that contains 2,000,000 sperms; This sample used 5 hours for 2 seperators, made 75 of sheep X control frozen semens.
7) because of the effect difference of frozen solution, the separation seminal fluid of the 0.25mL cryovial of will packing into is inserted in-90 ℃ the nitrogen vapor and was handled 20-30 minute; When temperature drops to-120 ℃, preserving in the seminal fluid pipe input liquid nitrogen.
This sheep X control frozen semen is with operation artificial insemination method (every receptor once can use 2,000,000 sperms), and 75 of ewes are handled in insemination, are impregnated 56,63 of lambings (7 of twins), and wherein ewe lamb is 60, conception rate 74.6%, property control accuracy rate is 95%.
Embodiment 2
The making of goat X frozen semen and artificial insemination, embryo transfer control lambing sex:
1) gathering 6mL, total sperm count altogether from the down producing goat ram of 2 health is 4,800,000,000 fresh semen, bacterial multiplication adds an amount of antibiotic (tylosin Tylosin:0.103mg/mL in the seminal fluid in order to suppress, gentamycin Gentamicin:0.508mg/mL, woods can-miramycin Linco-spectin:0.6mg/mL), under 18-20 ℃ of condition, be incubated.Use sperm concentration-vigor instrument to detect the concentration and the vigor of fresh semen, after testing, the vigor of this seminal fluid is 83%, transports laboratory back and separates use 6 hours for the X/Y sperm.
2) amount to volume according to seminal concentration and get the centrifugal removal refining of 4 * 200,000,000 sperms (using), add 10 μ g/mL Hochest 33342 and in 24 ℃ of tepidariums, dyeed 20 minutes for 2 sperm seperators.
3) sheep X/Y sperm separated DNA content difference is set at 3.2-3.4%, set sperm streaming separator cell (the SX-MoFlo fluidic cell seperator of U.S. XY Co.,Ltd) parameter (first phase region is just got the former seminal fluid necrospermia of 80%/the second phase region rate 10%=X/Y unisexuality sperm separation of sperm jet flow sum and got rate 45-47%) according to sheep XY sperm DNA content difference, separate x and y sperm by the fluidic cell seperator, make sheep X/Y sperm separating rate be controlled at 5000-7000/second, X or y sperm separation purity reach 85-95%.
4) get isolating X or y sperm by 1-1.1 hundred million, carried out centrifugal treating in/5 minutes with 2000 commentaries on classics behind the following machine, detect separated sperm concentration with microscope-computer sperm scale of notation, be diluted to 2 times of final design concentration with Tris-A (freezing balance liquid A), placement is more than 90 minutes under 4-5 ℃ of condition; Wherein, above-mentioned freezing balance liquid A, pH 6.8, and is composed of the following components:
Tris Tris 291.7mM
Citric acid 95.1mM
Fructose 70.2mM
Yolk 20%
Ultra-pure water.
5) after the Tris-A balance, add the Tris-B that contains 12% glycerol cryoprotective agent (freezing balance liquid B) of equivalent, 4 ℃ of refrigerator balances 30 minutes.
6) isolating X or the Y seminal fluid 0.25mL cryovial of packing under 4 ℃ of conditions is made the sheep property control frozen semen that contains 2,000,000 sperms; This sample used 6 hours for 2 seperators, made 82 of sheep X control frozen semens; Wherein, freezing balance liquid B, pH 6.8, and is composed of the following components:
Tris Tris 291.7mM
Citric acid 95.1mM
Fructose 70.2mM
Yolk 20%
Glycerol 12%
Ultra-pure water.
7) because of the effect difference of frozen solution, the separation seminal fluid of the 0.25mL cryovial of will packing into is inserted in-90 ℃ the nitrogen vapor and was handled 20-30 minute; When temperature drops to-120 ℃, preserving in the seminal fluid pipe input liquid nitrogen.
8) 40 property control frozen semens are with operation artificial insemination method (every receptor once can use 2,000,000 sperms), and 40 of ewes are handled in insemination, are impregnated 33,45 of lambings (12 of twins), and 42 of ewe lambs, conception rate 82.5%, property control accuracy rate is 93%.
9) other 42 property control frozen semens are with operation artificial insemination method (every receptor once can use 2 * 2,000,000 sperms), 21 of the she-goats that the superovulation of insemination process hormone is handled, obtain 165 pieces of embryos, these embryos transplant 128 pieces after selecting, 82 of lambings, wherein ewe lamb is 79, and property control accuracy rate is 96%.
Product of the present invention and production thereof, store method, its sperm does not need to add buffer before separating, its separating rate reaches 5000-7000/second, separation efficiency significantly improves, resulting product motility of sperm height, conception rate height (can reach more than 74%), the sex controll accuracy rate of lambing can reach more than 90%.The product and the method for wood invention are applicable to industrialization production and breed, thereby increase substantially the breed effect of sheep.
Claims (8)
1, a kind of sheep X/Y sperm separation freezing sperm comprises freezing balance liquid A, freezing balance liquid B and sheep X or y sperm sperm separation freezing, and its content is as follows:
Freezing balance liquid A Tris-A 50%
Freezing balance liquid B contains the Tris-B 50% of 12% glycerol cryoprotective agent, wherein:
Sheep X or y sperm sperm separation freezing concentration are 50-200 ten thousand sperms/0.25mL.
2, sheep X/Y sperm separation freezing sperm according to claim 1 is characterized in that: described freezing balance liquid A, and pH 6.8, and is composed of the following components:
Tris Tris 291.7mM
Citric acid 95.1mM
Fructose 70.2mM
Yolk 20%
Ultra-pure water;
And, described freezing balance liquid B, pH 6.8, and is composed of the following components:
Tris Tris 291.7mM
Citric acid 95.1mM
Fructose 70.2mM
Yolk 20%
Glycerol 12%
Ultra-pure water.
3, the production method of sheep X/Y sperm separation freezing sperm according to claim 2 comprises the steps:
1) gathers ram fresh semen 2-5mL (hundred million of total sperm count 25-50), add antibiotic, under 18-20 ℃ of condition, be incubated;
2) concentration and the vigor of detection fresh semen have only vigor to can be used for separating treatment at the sample more than 80%, amount to volume according to seminal concentration and get the centrifugal removal refining of 400,000,000 sperms, add 10 μ g/mL Hochest33342 and dye 20 minutes in 24 ℃ of tepidariums;
3) sheep X/Y sperm separated DNA content difference is set at 3.2-3.4%, set fluidic cell seperator parameter according to sheep X/Y sperm DNA content difference, separate x and y sperm by the fluidic cell seperator, make sheep X/Y sperm separating rate be controlled at 5000-7000/second;
4) get isolating X or y sperm 1-1.1 hundred million, change with 2000 behind the following machine and carried out centrifugal treating in/5 minutes, be diluted to 2 times of final design concentration with freezing balance liquid A then, placement is more than 90 minutes under 4-5 ℃ of condition;
5) after the freezing balance liquid A balance, add the freezing balance liquid B that contains 12% glycerol cryoprotective agent of equivalent, 4 ℃ of following balances 30 minutes;
6) separating X or the Y seminal fluid cryovial of under 4 ℃ of conditions, packing into, make sheep X/Y sperm separation freezing sperm.
4, according to the described production method of claim 3, it is characterized in that: described antibiotic is tylosin 0.103mg/mL, gentamycin: 0.508mg/mL, Linco-spectin:0.6 mg/mL.
5, according to the described production method of claim 3, it is characterized in that: described sperm seperator parameter is that the former seminal fluid necrospermia of 80%/the second phase region rate 10%=X/Y unisexuality sperm that first phase region is just got sperm jet flow sum separates and to get rate 45-47%.
6, according to the described production method of claim 3, further comprise the steps: the sheep X/Y sperm separation freezing sperm that step 6) the is made 0.25mL cryovial of packing into, insert in-90 ℃ the nitrogen vapor and handled 20-30 minute; When temperature drops to-120 ℃, preserving in the cryovial input liquid nitrogen.
7, be used for artificial insemination according to the described sheep X/Y sperm separation freezing sperm of claim 2 or productivity control embryo carries out embryo transfer again.
8, sheep X/Y sperm separation freezing sperm according to claim 6 is used for artificial insemination or productivity control embryo carries out embryo transfer again, it is characterized in that: described sheep X/Y sperm separation freezing artificial insemination dosage is 50-200 ten thousand sperms/only or inferior.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN104705290A (en) * | 2015-03-19 | 2015-06-17 | 新疆天山畜牧生物工程股份有限公司 | Sheep frozen semen dilution and process used for preparing sheep straw frozen semens |
| CN106472484A (en) * | 2016-10-31 | 2017-03-08 | 内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 | Containing glutathione and catalatic sheep frozen semen preparation method |
| CN108103012A (en) * | 2017-12-14 | 2018-06-01 | 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 production method of mouse X/Y spermatozoa isolation sperm and application |
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| CN100551382C (en) * | 2006-07-26 | 2009-10-21 | 李喜和 | A kind of low dosage livestock semen and production method and application |
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2008
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| 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 |
| CN103222458B (en) * | 2013-04-08 | 2015-01-14 | 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 |
| CN104705290A (en) * | 2015-03-19 | 2015-06-17 | 新疆天山畜牧生物工程股份有限公司 | Sheep frozen semen dilution and process used for preparing sheep straw frozen semens |
| CN104705290B (en) * | 2015-03-19 | 2016-06-15 | 新疆天山畜牧生物工程股份有限公司 | Sheep extender of freezing semen and the manufacture craft for sheep straw frozen semen |
| CN106472484A (en) * | 2016-10-31 | 2017-03-08 | 内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 | Containing glutathione and catalatic sheep frozen semen preparation method |
| CN108103012A (en) * | 2017-12-14 | 2018-06-01 | 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 production method of mouse X/Y spermatozoa isolation sperm and application |
| CN108103012B (en) * | 2017-12-14 | 2021-04-02 | 内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 | Production method and application of mouse X/Y sperm separation semen |
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