CN1732983A - Freeze method for preserving separated sperm of bull - Google Patents
Freeze method for preserving separated sperm of bull Download PDFInfo
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- CN1732983A CN1732983A CNA2005100977883A CN200510097788A CN1732983A CN 1732983 A CN1732983 A CN 1732983A CN A2005100977883 A CNA2005100977883 A CN A2005100977883A CN 200510097788 A CN200510097788 A CN 200510097788A CN 1732983 A CN1732983 A CN 1732983A
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Abstract
Disclosed is a freeze method for preserving separated sperm of bull, wherein the TRIS is employed as the cushioning solution system, which mainly comprises A solution and B solution, particularly, components beneficial for keeping vitality of separated sperm. The process comprises receiving separated sperm, centrifugal concentration, equalizing and programmed freezing.
Description
Technical field
The invention belongs to technical field of bioengineering, particularly the method for cryobiology.
Background technology
The sex of mammalian subject sperm and ovum in conjunction with the time of fertilization by different chromosomes redistribute and make up determine.The hereditary basis of sex is the phenotype that depends on spermatid, if taking X-bearing sperm combines with ovum, the chromosome set of germ cell is combined into XX, and the offspring then grows for female; Combine with ovum if carry the sperm of Y chromosome, the chromosome set of germ cell is combined into XY, and the offspring then grows for male.With isolating X or y sperm give the female animal artificial insemination or with the Oocyte in Vitro fertilization, before fertilization, just can predict the offspring sex, thereby reach sex-controlled purpose.With the milch cow is example, and the milch 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, by just reaching this purpose with the insemination of X sperm.Equally, the stocker farmer wishes that then because the quality of the speed of growth of cattle and meat is gender-related, the bull speed of growth is faster than cow by breeding more stot with y sperm insemination, 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 controll, 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.
At present, the key technology that the flow cytometer method is separated bull sperm solves, and has given birth to other animal offspring of foreseeability by technology such as artificial insemination and external fertilization.Yet, this achievement will be expected large-scale Industry Promotion, the isolating sperm of institute must can long preservation and long-distance transport, can conveniently use in any remote cattle farm or pastoral area, and the freezing preservation technology of sperm is exactly the auxiliary key of promoting separated sperm.The objective of the invention is by freezing preservation separated sperm, be implemented in-196 ℃ medium-term and long-term the preservation or remote transportation of liquid nitrogen, make that separated sperm can be widely used in the middle of.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of freezing and storing method that is applicable to bull separation x and y sperm, when guaranteeing that the isolating motility of sperm of process sex can satisfy artificial insemination and external fertilization, realize that separated sperm preserves or remote transportation in that-196 ℃ liquid nitrogen is medium-term and long-term, and be applicable to and commercially produce and promote.
The present invention solves the problems of the technologies described above with following technical scheme:
A) collect sperm
According to present sophisticated flow cytometer separated sperm technology to the sperm of the gathering separation of dyeing, the test tube that receives separated sperm adds the A liquid of 1～5ml in advance, become the solution that contains X or y sperm after receiving separated sperm, it finally contains 2%～6% yolk.
For the sperm that separates is maintained vigour, contain the yolk of 2.0～3.0% TRIS (Tris), single water citric acid of 1.0～2.0%, 0.8～1.2% fructose and 15.0～25.0% in the A liquid.
B) low-temperature balance
The separation seminal fluid that step a) is finally obtained moves on to 5 ℃ of balances, equilibration time 2～6 hours;
C) centrifugal concentrating
Centrifuge is pre-chilled to 5 ℃, with the separation seminal fluid after the balance in the step b) 3000～5000 rev/mins (under 288～800g) speed centrifugal 20 minutes; After the centrifugal end, supernatant discarded stays 100～200 microlitre sperms precipitation; Converge separating the sperm precipitation that obtains each batch in same the bull 4 hours;
D) glycerol balance
5 ℃ of constant temperature B liquid in advance mix with the seminal fluid precipitation that step c) obtains according to volume 1:1, shake up also balance 10～15 minutes.
For the separated sperm behind the freeze-thaw is maintained vigour, contain 3.0～3.5% TRIS (Tris), single water citric acid of 1.0～2.0%, 1.2～1.4% fructose, 15.0～25.0% yolk and 10.0～14.0% glycerol in the B liquid.
E) tubule packing
The good bull of labelling number, date, sperm type (X or Y) move into 5 ℃ of constant temperature pre-coolings on the tubule of 0.25ml; The seminal fluid branch that step d) is obtained installs in the 0.25ml tubule and seals;
F) programmed cooling
Be ready to the program frigorimeter, inject liquid nitrogen and initial temperature is set in 5 ℃, move into the seminal fluid tubule that step e) obtained.The opening program cooling is freezing, cools to-10 ℃ with 6 ℃/minute speed, and constant temperature stopped 2 minutes, and then lowers the temperature with 6 ℃/minute speed, when reaching-80 ℃ of terminations, tubule is dropped into the liquid nitrogen long preservation.
Characteristics of the present invention
Main feature of the present invention is:
(1) the inventive method adopts TRIS as buffer solution system, mainly comprises A liquid and B liquid two parts, and the two complements each other, and has added the composition that helps keeping the separated sperm vigor especially, is specially adapted for separating the freezing preservation of bull sperm.
When (2) passing through conventional method frozen fresh collecting semen, all be earlier seminal fluid to be diluted in the past, then carry out freezing again with the diluent that contains lecithality; In the present invention, when separating X or y sperm with flow cytometer, sperm is subjected to high dilution, thereby centrifugal concentrated seminal fluid earlier, and then adds glycerinated B liquid and continue freezing.
The specific embodiment
Embodiment one: the separated sperm of freezing He Sitan bull
A) collect sperm: the flow cytometer method is separated the He Sitan bull sperm routinely, and the test tube that receives separated sperm adds the A liquid that contains 20% yolk of 2ml in advance, accepts that cumulative volume is 10ml behind the separated sperm, finally contains 4.0% yolk;
The prescription of A liquid is:
| Reagent chemicals | Quality or volume |
| TRIS (Tris) | 2.792g |
| Single water citric acid | 1.360g |
| Fructose | 1.000g |
| Tylosin 100 μ g/ml (storage liquid) | 0.480m1 |
| Lincoln-spectinomycin 300/600 μ g/ml (storage liquid) | 0.480ml |
| Gentamycin 500 μ g/ml (storage liquid) | 0.640ml |
| Yolk | 20.000ml |
Add deionized water to 100ml.
B) low-temperature balance: the solution that step a) is obtained moves on to 5 ℃ of balances, equilibration time 4 hours;
C) centrifugal concentrate: after balance finishes, the solution that step b) obtains under 5 ℃, 3000 rev/mins speed centrifugal 20 minutes; Centrifugal end is supernatant discarded afterwards, stays the precipitation of 100～200 microlitres, mixing;
D) glycerol balance: 5 ℃ of homothermic B liquid mixes balance 15 minutes according to volume 1: 1 with the sperm precipitation that step c) obtains;
The prescription of B liquid:
| Reagent chemicals | Quality or volume |
| TRIS (Tris) | 3.146g |
| Single water citric acid | 1.744g |
| Fructose | 1.293g |
| Tylosin 100 μ g/ml (storage liquid) | 0.011ml |
| Lincoln-spectinomycin 300/600g/ml (storage liquid) | 0.011ml |
| Gentamycin 500 μ g/ml (storage liquid) | 0.011ml |
| Yolk | 20.310ml |
| Glycerol | 12.000ml |
Add deionized water to 100ml.
E). the tubule packing: the good bull of labelling number, date, sperm type (X or Y) on the tubule of 0.25ml move into 5 ℃ of constant temperature; The seminal fluid branch that step d) is obtained installs in the 0.25ml tubule and seals;
F) programmed cooling: with the straw semen cooling that step e) obtained, when reaching-80 ℃ of terminations, tubule is dropped into the liquid nitrogen long preservation with the program frigorimeter.
In the present embodiment, He Sitan bull J03001, PT01003 from the Guangxi University pasture have been carried out 11 times respectively in successive two middle of the month separated freezing experiment, had straight-line sperm motility rate after thawing and be respectively 0.41 ± 0.026 and 0.39 ± 0.019.
Embodiment two: the separated sperm of freezing Babalus bubalis L.
A) collect sperm: the flow cytometer method is separated the Babalus bubalis L. sperm routinely, and the test tube that receives separated sperm adds the A liquid that contains 22% yolk of 1.0ml in advance, accepts that cumulative volume is 10ml behind the separated sperm, finally contains 2.2% yolk;
The prescription of A liquid is:
| Reagent chemicals | Quality or volume |
| TRIS (Tris) | 2.562g |
| Single water citric acid | 1.579g |
| Fructose | 1.100g |
| Tylosin 100 μ g/ml (storage liquid) | 0.480ml |
| Lincoln-spectinomycin 300/600 μ g/ml (storage liquid) | 0.480ml |
| Gentamycin 500 μ g/ml (storage liquid) | 0.640ml |
| Yolk | 22ml |
Add deionized water to 100ml.
B) low-temperature balance: the solution that step a) is obtained moves on to 5 ℃ of balances, equilibration time 4 hours;
C) centrifugal concentrate: after balance finishes, the solution that step b) obtains under 5 ℃, 3000 rev/mins speed centrifugal 20 minutes; Centrifugal end is supernatant discarded afterwards, stays the precipitation of 100～200 microlitres, mixing;
D) glycerol balance: 5 ℃ of homothermic B liquid mixes balance 15 minutes according to volume 1: 1 with the sperm precipitation that step c) obtains;
The prescription of B liquid:
| Reagent chemicals | Quality or volume |
| TRIS (Tris) | 3.225g |
| Single water citric acid | 1.900g |
| Fructose | 1.357g |
| Tylosin 100 μ g/ml (storage liquid) | 0.011ml |
| Lincoln-spectinomycin 300/600g/ml (storage liquid) | 0.011ml |
| Gentamycin 500 μ g/ml (storage liquid) | 0.011ml |
| Yolk | 22.470ml |
| Glycerol | 11.000ml |
Add deionized water to 100ml.
E) tubule packing: the good bull of labelling number, date, sperm type (X or Y) on the tubule of 0.25ml move into 5 ℃ of constant temperature; The seminal fluid branch that step d) is obtained installs in the 0.25ml tubule and seals;
F) programmed cooling: with the straw semen cooling that step e) obtained, when reaching-80 ℃ of terminations, tubule is dropped into the liquid nitrogen long preservation with the program frigorimeter.
In the present embodiment, separate freezing experiment to having carried out 7 times respectively in successive two middle of the month with 9 times, have straight-line sperm motility rate after thawing and be respectively 0.35 ± 0.035 and 0.36 ± 0.022 from the seminal fluid of the Murrah ML811 at Guangxi livestock and poultry species improvement station and Buddhist nun's interior edema cattle NL440.
Claims (8)
1, a kind of freezing and storing method that is applicable to bull separation x and y sperm, when the separated sperm vigor of the freezing preservation of assurance process can satisfy artificial insemination and external fertilization, realize that separated sperm is in medium-term and long-term preservation of-196 ℃ liquid nitrogen or remote transportation, and be applicable to and commercially produce and promote, it is characterized in that:
A) sperm is collected:
Separate the x and y sperm technology to the sperm of the gathering separation of dyeing according to present sophisticated flow cytometer, the test tube that receives separated sperm adds the A liquid of 1～5ml in advance, become the solution that contains X or y sperm after receiving separated sperm, it finally contains 2%～6% yolk.
B) low-temperature balance:
The separation seminal fluid that step a) is finally obtained moves on to 5 ℃ of balances, equilibration time 2～6 hours;
C) centrifugal concentrating:
Separation seminal fluid in the step b) after the balance 5 ℃, 3000～5000 rev/mins (under 288～800g) speed centrifugal 20 minutes; After the centrifugal end, supernatant discarded stays 100～200 microlitre sperms precipitation; Converge separating the sperm precipitation that obtains each batch in same the bull 4 hours;
D) glycerol balance:
5 ℃ of constant temperature B liquid in advance mix with the seminal fluid precipitation that step c) obtains according to volume 1: 1, shake up also balance 10～15 minutes.
E) tubule packing:
The good bull of labelling number, date, sperm type (X or Y) move into 5 ℃ of constant temperature pre-coolings on the tubule of 0.25ml; The seminal fluid branch that step d) is obtained installs in the 0.25ml tubule and seals;
F) programmed cooling:
Be ready to the program frigorimeter, inject liquid nitrogen and initial temperature is set in 5 ℃, move into the seminal fluid tubule that step e) obtained.The opening program cooling is freezing, cools to-10 ℃ with 6 ℃/minute speed, and constant temperature stopped 2 minutes, and then lowers the temperature with 6 ℃/minute speed, when reaching-80 ℃ of terminations, tubule is dropped into the liquid nitrogen long preservation.
2, the freezing and storing method of separated sperm of bull as claimed in claim 1 is characterized in that the frozen soln system comprises A liquid and B liquid two parts composition.
3, the freezing and storing method of separated sperm of bull as claimed in claim 1 is characterized in that receiving the test tube of separated sperm or the A liquid that centrifuge tube is equipped with 1～5ml in advance;
4, the freezing and storing method of separated sperm of bull as claimed in claim 1 or 2 is characterized in that containing in the A liquid TRIS (Tris), single water citric acid, fructose and yolk.
5,, it is characterized in that the content of TRIS in the A liquid (Tris), single water citric acid, fructose and yolk is respectively 2.0～3.0%, 1.0～2.0%, 0.8～1.2% and 15.0～25.0% as the freezing and storing method of claim 1 or 4 described separated sperm of bull.
6, the freezing and storing method of separated sperm of bull as claimed in claim 1, it is characterized in that separating seminal fluid and (centrifugally under 288～800g) speed concentrate 20 minutes at 5 ℃, 3000～5000 rev/mins, centrifugal end back supernatant discarded stays 100～200 microlitre sperms precipitation;
7, the freezing and storing method of separated sperm of bull as claimed in claim 1 or 2 is characterized in that containing in the B liquid TRIS (Tris), single water citric acid, fructose, yolk and glycerol;
8,, it is characterized in that the content of the TRIS (Tris) in the B liquid, single water citric acid, fructose, yolk and glycerol is respectively 3.0～3.5%, 1.0～2.0%, 1.2～1.4%, 15.0～25.0% and 10.0～14.0% as the freezing and storing method of claim 1 or 7 described separated sperm of bull.
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| CNB2005100977883A CN100359003C (en) | 2005-08-29 | 2005-08-29 | Freeze method for preserving separated sperm of bull |
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Cited By (12)
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| WO2010045758A1 (en) * | 2008-10-22 | 2010-04-29 | Li Xihe | Dairy cow x/y sex-control frozen mixed semen and production method thereof |
| CN101810163A (en) * | 2010-05-10 | 2010-08-25 | 广西大学 | Method for freezing semen of Macaca fascicularis |
| US8251887B2 (en) | 2009-01-24 | 2012-08-28 | Xihe Li | Reproductive technology of low dose semen production and in vitro/in vitro fertilization in domestic animals |
| CN103125448A (en) * | 2013-03-22 | 2013-06-05 | 四川省草原科学研究院 | Method for cultivating gender-controlled dzo by hybridizing common cattle and yak |
| US8486618B2 (en) | 2002-08-01 | 2013-07-16 | Xy, Llc | Heterogeneous inseminate system |
| US8512224B2 (en) | 2009-01-24 | 2013-08-20 | Xy, Llc | Method of producing an inseminate |
| CN103493800A (en) * | 2013-09-29 | 2014-01-08 | 大连金弘基种畜有限公司 | Freezing preservation method for separated semen |
| US9474591B2 (en) | 2010-06-09 | 2016-10-25 | Xy, Llc | Heterogeneous inseminate system |
| CN106106433A (en) * | 2016-06-22 | 2016-11-16 | 湖北省畜禽育种中心 | A kind of Binglangjiang waterbuffalo freezing of semen process |
| CN108575989A (en) * | 2018-07-05 | 2018-09-28 | 河南省鼎元种牛育种有限公司 | A kind obstinacy control sperm Cryopreservation |
| CN108865982A (en) * | 2018-07-05 | 2018-11-23 | 河南省鼎元种牛育种有限公司 | A kind of frozen cattle semens X, y sperm separation method |
| CN110811914A (en) * | 2019-12-09 | 2020-02-21 | 中南百草原集团有限公司 | Method for extracting sperm from strong cattle |
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| CN100383237C (en) * | 2004-03-12 | 2008-04-23 | 中国科学院昆明动物研究所 | Low-temperature cryopreservation method for semen of gayals |
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2005
- 2005-08-29 CN CNB2005100977883A patent/CN100359003C/en not_active Expired - Fee Related
Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US8486618B2 (en) | 2002-08-01 | 2013-07-16 | Xy, Llc | Heterogeneous inseminate system |
| WO2010045758A1 (en) * | 2008-10-22 | 2010-04-29 | Li Xihe | Dairy cow x/y sex-control frozen mixed semen and production method thereof |
| US8512224B2 (en) | 2009-01-24 | 2013-08-20 | Xy, Llc | Method of producing an inseminate |
| US8251887B2 (en) | 2009-01-24 | 2012-08-28 | Xihe Li | Reproductive technology of low dose semen production and in vitro/in vitro fertilization in domestic animals |
| CN101810163A (en) * | 2010-05-10 | 2010-08-25 | 广西大学 | Method for freezing semen of Macaca fascicularis |
| US9474591B2 (en) | 2010-06-09 | 2016-10-25 | Xy, Llc | Heterogeneous inseminate system |
| US10492896B2 (en) | 2010-06-09 | 2019-12-03 | Xy, Llc | Heterogeneous inseminate system |
| US11364104B2 (en) | 2010-06-09 | 2022-06-21 | Xy, Llc | Heterogeneous inseminate system |
| CN103125448A (en) * | 2013-03-22 | 2013-06-05 | 四川省草原科学研究院 | Method for cultivating gender-controlled dzo by hybridizing common cattle and yak |
| CN103493800A (en) * | 2013-09-29 | 2014-01-08 | 大连金弘基种畜有限公司 | Freezing preservation method for separated semen |
| CN103493800B (en) * | 2013-09-29 | 2015-09-16 | 大连金弘基种畜有限公司 | Be separated the freezing method of seminal fluid |
| CN106106433A (en) * | 2016-06-22 | 2016-11-16 | 湖北省畜禽育种中心 | A kind of Binglangjiang waterbuffalo freezing of semen process |
| CN106106433B (en) * | 2016-06-22 | 2019-04-16 | 湖北省畜禽育种中心 | A kind of Binglangjiang waterbuffalo freezing of semen process |
| CN108575989A (en) * | 2018-07-05 | 2018-09-28 | 河南省鼎元种牛育种有限公司 | A kind obstinacy control sperm Cryopreservation |
| CN108865982A (en) * | 2018-07-05 | 2018-11-23 | 河南省鼎元种牛育种有限公司 | A kind of frozen cattle semens X, y sperm separation method |
| CN110811914A (en) * | 2019-12-09 | 2020-02-21 | 中南百草原集团有限公司 | Method for extracting sperm from strong cattle |
| CN110811914B (en) * | 2019-12-09 | 2022-01-11 | 中南百草原集团有限公司 | Method for extracting sperm from strong cattle |
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