CN1654636A - Method for producing sex controllable in vitro embryo of buffalo - Google Patents
Method for producing sex controllable in vitro embryo of buffalo Download PDFInfo
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- CN1654636A CN1654636A CN 200510031159 CN200510031159A CN1654636A CN 1654636 A CN1654636 A CN 1654636A CN 200510031159 CN200510031159 CN 200510031159 CN 200510031159 A CN200510031159 A CN 200510031159A CN 1654636 A CN1654636 A CN 1654636A
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Abstract
The method of producing extracorporeal buffalo embryo in controlled sex includes recovering buffalo egg in butchery or from living buffalo body, extracorporeal culturing to mature, external fertilization with fresh or frozen X or Y sperm of fine breed bull in a flow cytometer; and recovering embryo of the fertilized egg via external culture to blastula stage for transplanting or further freeze maintaining. The embryo is used in generating posterity of the expected sex. The present invention has the advantages of industrial embryo production in controlled sex and raised propagation speed of high yield milk buffalo.
Description
Technical field
The invention belongs to technical field of bioengineering, particularly the sex-controlled method of animal.
Background technology
The sex of mammalian subject sperm during with ovum fertilization by different karyomit(e) 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 genome of zygote is combined into XX and then grows for female; Combine with ovum if carry the sperm of Y chromosome, the genome of zygote is combined into XY and then grows for male.With isolating X or y sperm give dam artificial insemination or with the Oocyte in Vitro fertilization, before fertilization, just can predict the offspring sex, thereby reach sex-controlled purpose.
Utilizing the sperm isolation technique to produce the animal offspring with in vitro fertilization combining, is at the biological company limited (ABC) of Britain Camb unfolded the earliest.Cran etc. (1993) are the people who carries out this research the earliest.Embryo transfer to 9 receptor cow that they produce the separated sperm after fertilization, wherein 4 pregnancies, and give birth to 6 (3 cow calves and 3 bull calf oxen) other calves of preselection, the sex accuracy rate is 100%.Nineteen ninety-five, Cran etc. have carried out second research again, promptly produce the experiment of male calf with isolating y sperm.Transplant behind 106 pieces of embryo cryopreservations that they will be undertaken obtaining behind the IVF by isolating y sperm, following 41 calves of common property, wherein 37 is the bull calf ox, male ratio is 90%.
Except the research of the separated sperm IVF of ox, the somebody has carried out the research of the separated sperm IVF of pig, by successfully having given birth to piglet after the embryo transfer.But carry out in vitro fertilizationly with the separated sperm of buffalo, yet there are no report at present both at home and abroad.
Buffalo is that the southern buffalo milk of China has very high nutritive value, but the present situation of just present China milk buffalo sees that its quantity and milk yield also do not satisfy the demand in market far away.And by the Embryo Production technology of the producing controlled sex embryo milk ox head number that can increase sharply, thereby improving the selection intensity of milk buffalo, this is very great to development meaning of accelerating milk buffalo industry.
One of the inventor of this patent doctor Lu Kehuan, professor, be international well-known scientist, once studied the two calves of the first test tube in the successful world in Ireland, during " the Seventh Five-Year Plan " and " eight or five ", once presided over country's " 863 " major project " ox research and development in vitro fertilization " subject study in 1988.Every technical indicator of this problem is all reached advanced world standards, and studies maximum " tube cattle " group of successfully national quantity, totally 228.This problem obtained the Guangxi scientific-technical progress first prize in 1997, calendar year 2001 is obtained national science and technology progress second prize.Another inventor doctor Zhang Ming of he and this patent in addition, professor once was engaged in the research in vitro fertilization of ox separated sperm in the XY Co.,Ltd of the U.S. together.
The objective of the invention is to utilize sperm isolation technique this animal reproduction new technology that combines with technology in vitro fertilization to produce other buffalo embryo of foreseeability, to reach control breeding milk buffalo offspring's sex.Also provide technical support simultaneously for breeding milk buffalo embryo batch production, sex production.The maximum characteristics of producing this method of sex controllable in vitro embryo of buffalo be efficient, save time, laborsaving and cost saving, so this method is to accelerate high yield milk buffalo reproductive speed one of effective technical means the most at present.
Summary of the invention
Utilize flow cytometer to separate this at present international most advanced and effective sperm isolation technique of XY sperm in conjunction with in vitro fertilization, other buffalo embryo of mass production foreseeability, to satisfy the demand of market to controlled sex buffalo embryo, thereby reach the speed of breeding of quickening breeding milk buffalo, promote the development of buffalo milk industry, this technological method is the first in the world.Particular content is as follows:
(1) the buffalo ovary is reclaimed in the slaughterhouse, places stroke-physiological saline solution, takes back the laboratory with vacuum flask.From the ovary surface extraction ovocyte that cleans up (or obtain buffalo ovocyte by ovum pick-up), ovocyte placed collect liquid, under stereoscopic microscope, choose ovocyte, with collecting liquid washing 2～3 times, more once with the maturation culture solution washing.
(2) ovocyte is changed over to be added with serum, hormone and liquor folliculi and contain in TCM199 (tissue culture medium (TCM)) culture dish of Hepes (N-2-hydroxyethyl piperazine-N '-2-ethanesulfonic acid), put into incubator and carry out maturation and cultivate.
(3) will by Percoll (but piperazine liquid) gradient centrifugation the Necrospermia in the seminal fluid and the sperm of living be separated by the buffalo X or the y sperm of the isolating fresh or freeze-thaw of flow cytometer.
(4) ovocyte after the vitro culture maturation is sorted out from ripe liquid, dripped with the fertilization that made by seminal fluid with being subjected to change over to after the seminal fluid washing 2 times.After the sperm dilution X or y sperm joined and contain during fertility of oocytes drips, put into incubator and carry out cultivation in vitro fertilization.
(5) cultivation in vitro fertilization is after 6～8 hours, and the zygote washes clean with supposition places the ox granulosa cell monolayer culture liquid for preparing in advance, contain new-born calf serum then, puts into the incubator vitro culture 6～8 days.
(6) when after fertilization the 6th, the 7th and the 8th day, reclaim blastaea,, the embryo is transplanted if any suitable recipient cattle.Or well-developed embryo quality is carried out freezing preservation handle, place liquid nitrogen container to store then.
Compare with present external Embryo Production technology, the invention has the advantages that:
(1) adopt this kind method can make the Embryo Production sexization.Carry out in vitro fertilization because use selectively by isolating X of flow cytometer or y sperm at the time of fertilization, can control embryo's sex, promptly as to select the ovocyte of X sperm and the maturation in vitro embryo who is produced that is fertilized be female, and the embryo who selects the y sperm fertilization to be produced is male, transplants the offspring that just can give birth to desired sex with other embryo of these controllabilitys then and comes.
(2) can make the Embryo Production batch production by this technological approaches.Utilize the 30-40% of the ovary ovocyte that present method can reclaim from the slaughterhouse to develop into the embryo that portable is used.Controlled sex embryo is more than 1000 pieces in annual production.
(3) this method the breeding of buffalo of suckling that combine with other various modern Embryo Production technology can be improved the efficient of breeding of milk buffalo, and the breeding that increases sharply milk buffalo quantity improves milk cows quality.Because separating the accuracy rate of XY sperm at present is more than 90%, with the offspring more than 90% after the embryo transfer of X sperm fertilization generation is cow, change under the natural condition 50: 50 sex ratio significantly, therefore, using the sex control techniques to breed the efficient of milk cow will be than nearly one times of the raising of asexuality control, thereby can save nearly half time, energy and expense.
Embodiment
Specific embodiment:
Gather the buffalo ovary from the slaughterhouse, place 25 ℃ of physiological saline, take back the laboratory with vacuum flask.Cleaning ovary 3-4 time with aseptic physiological saline, with the syringe that is equipped with No. 8, is to extract ovocyte the 2-8mm ovarian follicle from the ovary surface diameter.Under stereoscopic microscope, chosen dense granule cell more than three layers, and the bright uniform ovocyte of tenuigenin of profile, with the Hepes buffered, be added with 5mmol/L NaHCO
3The oocytes collection liquid washed twice of the TCM199 of (sodium hydroxide) 3%OCS (bovine serum of oestrusing) again with ripe liquid washing once, changes over to and carries out the maturation cultivation in the culture dish.Maturation culture solution is Hepes buffered TCM199+5%OCS+0.1 μ g/mL FSH (follitropin)+3%BFF (an ox liquor folliculi).The ripe liquid of every culture dish is 1mL, puts into ovocyte 50-100 piece, 39 ℃, and 5%CO
2Cultivated 21～24 hours under the air of (carbonic acid gas) and the condition of maximal humidity.
Will be by the buffalo X or the y sperm of the isolating fresh or freeze-thaw of flow cytometer, seminal fluid is put into the centrifuge tube of 2mL90%:2mL45%Percoll gradient, centrifugal 20 minutes of 700xg, abandon supernatant, add 1mL and be subjected to seminal fluid 400xg centrifugal 5 minutes, remove the upper strata suspension, stay the seminal fluid of about 50 μ l.Estimate motility of sperm at microscopically, and calculate sperm count to calculate sperm concentration with blood counting chamber.Ovocyte is taken out from ripe culture dish, stand seminal fluid washing 2 times, changing over to during fertilization drips.Being subjected to seminal fluid is Tyrodes (the platform Luo Shi liquid) nutrient solution of revising, and is added with 10mg/mLBSA (bovine serum albumin), 40 μ l/mLPHE (penicillamine, taurine, suprarenin mixed solution), 5mM caffeine, 30 μ g/mL heparin.It is 20 μ l that fertilization is dripped, and puts into 15-20 piece of ovocyte for every.Sperm after the dilution is added to during fertilization drips, makes its ultimate density reach 2 * 10
6/ ml, at 39 ℃, 5%CO
2Air and the maximal humidity condition under cultivate 6-8h.
After fertilization 6～7h blows and beats the zygote of supposition repeatedly with suction pipe in fertilization is dripped, remove the part cumulus cell.The zygote of supposition is shifted out from fertilization is dripped, change in the co-culture system that contains the ox granulosa cell with vitro culture liquid washed twice and cultivate.Nutrient solution is the Sodium.alpha.-ketopropionate of Hepes buffered TCM199+10%NCS (new-born calf serum)+0.11mg/mL, whenever is added dropwise to nutrient solution 25 μ l, puts into 50 pieces of zygotes, at 39 ℃, and 5%CO
2, 5%O
2, 90%N
2And external cultivation 6～8 days under the maximal humidity condition, changed liquid once in per two days between incubation period, change 1/2 liquid at every turn.When after fertilization the 6th, the 7th and the 8th day, reclaim blastaea,, the embryo is transplanted if any suitable recipient cattle.Or well-developed embryo quality is carried out freezing preservation handle, place liquid nitrogen container to store then.
Adopt aforesaid method, in the process of the external Embryo Production of buffalo, the maturation in vitro rate of ovocyte is more than 70%, and the spilting of an egg rate of Oocyte in Vitro after fertilization is about 60%, and the developmental rate of blastaea is about 30%.
Claims (7)
1. produced in vitro buffalo sex control embryo technology is characterized in that the following step forms:
(1) reclaims the buffalo ovary from the slaughterhouse, place 20～25 ℃ of stroke-physiological saline solution, take back the laboratory with vacuum flask.From the ovary surface extraction ovocyte that cleans up (or obtain buffalo ovocyte by ovum pick-up), ovocyte placed collect liquid, under stereoscopic microscope, choose ovocyte, with collecting liquid washing 2～3 times, more once with the maturation culture solution washing.
(2) ovocyte is changed over to be added with serum, hormone and liquor folliculi and contain in the maturation culture solution of TCM199 (tissue culture medium (TCM)) of Hepes (N-2-hydroxyethyl piperazine-N '-2-ethanesulfonic acid), put into incubator and carry out maturation and cultivate.
(3) will by Percoll (but piperazine liquid) gradient centrifugation the Necrospermia in the seminal fluid and the sperm of living be separated by the buffalo X or the y sperm of the isolating fresh or freeze-thaw of flow cytometer.
(4) ovocyte after the vitro culture maturation is sorted out from ripe liquid, dripped with the fertilization that made by seminal fluid with being subjected to change over to after the seminal fluid washing 2 times.After the sperm dilution X or y sperm joined and contain during fertility of oocytes drips, put into incubator and carry out cultivation in vitro fertilization.
(5) cultivation in vitro fertilization is after 6～8 hours, and the zygote washes clean with supposition places the ox granulosa cell monolayer culture liquid for preparing in advance, contain NCS (new-born calf serum) then, puts into the incubator vitro culture 6～8 days.
(6) when after fertilization the 6th, the 7th and the 8th day, reclaim blastaea,, the embryo is transplanted if any suitable recipient cattle.Or well-developed embryo quality is carried out freezing preservation handle, place liquid nitrogen container to store then.
2. sex controllable in vitro embryo of buffalo production technology according to claim 1 is characterized in that the collection liquid in the step (1) is 20～30mM Hepes buffered, is added with 2～8mmol/L NaHCO
3The TCM199 of (sodium bicarbonate), 2～5%OCS (bovine serum of oestrusing).
3. produced in vitro buffalo sex control embryo technology according to claim 1, it is characterized in that maturation culture solution is 20～30mM Hepes buffered TCM199+3～8%OCS+0.1 μ g/mL FSH (follitropin)+2～5%BFF (ox liquor folliculi) in the step (2), incubation time is 21～24 hours, condition is 39 ℃ in the incubator, 5%CO
2(carbonic acid gas) air and maximum humidity.
4. produced in vitro buffalo sex control embryo technology according to claim 1, its feature is at X or the Y seminal fluid of the used buffalo seminal fluid of step (3) for the fresh or freezing preservation after separating through flow cytometer; The Percoll gradient centrifugation is that seminal fluid is put into 2mL90%: the centrifuge tube of 2mL 45%Percoll gradient, centrifugal 20 minutes of 700xg abandons supernatant, adds 1mL and is subjected to seminal fluid 400xg centrifugal 5 minutes.
5. produced in vitro buffalo sex control embryo technology according to claim 1; it is characterized in that the seminal fluid that is subjected in the step (4) is improved Tyrodes (platform Luo Shi liquid) nutrient solution; be added with 5～15mg/mLBSA (bovine serum albumin), 20～50 μ l/mLPHE (penicillamine, taurine, suprarenin mixed solution), 2～7mM caffeine, 30～60 μ g/mL heparin; it is 15～30 μ l that size is dripped in fertilization; put into 15～20 pieces of ovocytes for every; sperm is joined during fertilization drips, make sperm concentration reach 2 * 10
6/ mL, culture condition is identical with 3, and incubation time is 6～8 hours.
6. produced in vitro buffalo sex control embryo technology according to claim 1, it is characterized in that the nutrient solution in the step (5) is the Sodium.alpha.-ketopropionate of 20～30mM Hepes buffered TCM199+5～15%NCS+0.05～0.15mg/mL, and with the ox granulosa cell as individual layer, at 39 ℃, 5%CO
2, 5%O
2External cultivation is 6～8 days under (oxygen) and the maximal humidity condition, changes liquid once in per two days between incubation period, changes 1/2 liquid at every turn.
7. produced in vitro buffalo sex control embryo technology according to claim 1, recovery after fertilization the 6th, the 7th and the 8th day blastaea of it is characterized in that indication in the step (6) are meant that the embryo with segmentation cavity and inner cell mass comprises early stage blastaea, blastaea, expands and expanded blastaea.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100432219C (en) * | 2006-07-28 | 2008-11-12 | 浙江大学 | Cow ovum cell in vitro ripening culturing liquid containing tea polyphenol and its culturing method |
| CN101974482A (en) * | 2010-11-05 | 2011-02-16 | 大连雪龙产业集团有限公司 | Artificial culture method of black cattle embryo |
| CN102258004A (en) * | 2010-05-27 | 2011-11-30 | 中国科学院动物研究所 | Method for preserving/culturing oocytes in vitro to inhibit oocytes from germinal vesicle breakdown |
| 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 |
| CN103215220A (en) * | 2013-03-22 | 2013-07-24 | 四川省草原科学研究院 | Method for producing dzo gender controllable in vitro embryos |
| CN104312972A (en) * | 2014-10-20 | 2015-01-28 | 广西大学 | Percoll continuous density gradient separation method for high/low-activity sperms of water buffalos |
| CN106635963A (en) * | 2016-04-25 | 2017-05-10 | 广西大学 | Method suitable for metabonomic buffalo embryo culture and embryo development potential detection |
| CN112961824A (en) * | 2015-07-14 | 2021-06-15 | 吉纳斯公司 | Cryopreservation of ungulate embryos |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1226377A (en) * | 1999-04-01 | 1999-08-25 | 旭日干 | Process for industrializing technology of 'tube cattle' |
| CN1304443A (en) * | 1999-06-30 | 2001-07-18 | 黄禹锡 | Method for producing clened cows |
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2005
- 2005-01-12 CN CNB2005100311590A patent/CN100334205C/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100432219C (en) * | 2006-07-28 | 2008-11-12 | 浙江大学 | Cow ovum cell in vitro ripening culturing liquid containing tea polyphenol and its culturing method |
| CN102258004A (en) * | 2010-05-27 | 2011-11-30 | 中国科学院动物研究所 | Method for preserving/culturing oocytes in vitro to inhibit oocytes from germinal vesicle breakdown |
| CN102258004B (en) * | 2010-05-27 | 2013-07-17 | 中国科学院动物研究所 | Method for preserving/culturing oocytes in vitro to inhibit oocytes from germinal vesicle breakdown |
| CN101974482A (en) * | 2010-11-05 | 2011-02-16 | 大连雪龙产业集团有限公司 | Artificial culture method of black cattle embryo |
| 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 |
| CN103215220A (en) * | 2013-03-22 | 2013-07-24 | 四川省草原科学研究院 | Method for producing dzo gender controllable in vitro embryos |
| CN104312972A (en) * | 2014-10-20 | 2015-01-28 | 广西大学 | Percoll continuous density gradient separation method for high/low-activity sperms of water buffalos |
| CN112961824A (en) * | 2015-07-14 | 2021-06-15 | 吉纳斯公司 | Cryopreservation of ungulate embryos |
| CN106635963A (en) * | 2016-04-25 | 2017-05-10 | 广西大学 | Method suitable for metabonomic buffalo embryo culture and embryo development potential detection |
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| CN100334205C (en) | 2007-08-29 |
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