CN1226378A - Technology for crossbreeding sheep in 'tubes' - Google Patents
Technology for crossbreeding sheep in 'tubes' Download PDFInfo
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- CN1226378A CN1226378A CN 99103293 CN99103293A CN1226378A CN 1226378 A CN1226378 A CN 1226378A CN 99103293 CN99103293 CN 99103293 CN 99103293 A CN99103293 A CN 99103293A CN 1226378 A CN1226378 A CN 1226378A
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Abstract
A test-tube hybridization technique for sheep includes such technological steps as over-ovulation of young ewe, collecting the oocytes from its ovarium, external maturation, culturing in culture liquid, external fertilization with the spermia taken from ram, external culture of oosperm, transplanting it into uterus of the ewe as acceptor to obtain the first-generation hybridized sheep, and obtaining the second-generation and the third-generation hybridized sheep by same process while their inheritable characters are fixed by transverse hybridization. Its advantages are shorter period and low cost.
Description
The invention belongs to biotechnology, particularly the propagation method of animal.
" test tube domestic animal " technology, (In Vitro Fertilization IVF), is meant the ovum and the sperm ripe and fertilization under conditions in vitro that make domestic animal by manual operation, and can grows the technology for body early embryo also to be domestic animal technology in vitro fertilization.What this technology was achieved success the earliest domestic animal is in nineteen eighty-two, and (Bio.Reprod. 27:147-158) handles Niu Jingzi with hypertonic solution to the Brackett of the U.S. etc., has obtained the first calf in vitro fertilization in the world.But the capacitation method repeatability that they adopted is very poor, comprises that themselves also fails to obtain the result of repetition.Present patent application person in 1984 (rising sun is done) and Japanese scholar Hua Tianzhang, utilize chemicalses such as Calcium ionophore Ionophore A23187 and caffeine to induce goat, sheep and ox capacitation all to succeed first, and utilize the method cultivate the first carcass in the world be fertilized outward goat (Bull Nippon Vet.and Zootech.Coll., 33:219-221).This method was achieved success in the experiment of multiple domestic animals such as sheep, ox and pig in multinational laboratories such as Japan, Britain, the U.S., China and Russia afterwards, and progressively developed into comparative maturity on this basis, the biological high-technology that can develop.
The applicant presided over country's " 863 " high-tech program project " ox Study on Technology in vitro fertilization and exploitation " from 1987, obtained major progress through " the Seventh Five-Year Plan " basic research, " eight or five " application study and " 95 " pilot scale developmental research.Cultivated in 1989 domestic first tire, first batch of " test tube sheep " (1989, University of the Inner Mongol's journal, 20:564-569) and " tube cattle " (1990, the Chinese science circular, 21:1666-1667).Creatively proposed simultaneously to utilize the batch production of animal for slaughter ovary egg mother cell to produce the technology path of " in-vitro embryos ", ox process for industrializing technology in vitro fertilization has been proposed, proposed to utilize the lamb ovum to carry out the new ideas of " crossbreeding in vitro ", new technology.
The objective of the invention is: new ideas, the new technology of utilizing " crossbreeding in vitro ", can species hybriding on the reproductive cell level continuous, goat, thereby quick, low cost is cultivated high yield, new quality variety, and the cycle of cultivation new varieties will shorten more than 1/2 compared with conventional breeding.
Content of the present invention: in animal breeding, still adopt traditional breeding technique at present mostly.The shortcoming of this technology maximum is exactly that breeding cycle is long, often needs the time of more than ten years even decades.We utilize children dam in age can obtain the characteristics of good superovulation effect, and technology in vitro fertilization is applied to the breeding of sheep, shorten breeding cycle, and this research method does not still have report at present in the world.
(1) utilizing FSH-LH to carry out superovulation to 2-3 monthly age ewe lamb handles, reclaims egg mother cell with operation method from the ovary surface.After the super row of lamb handled, every lamb can obtain the ovum more than 50 pieces, has given full play to the breeding potential of ewe, and the fertility after this ewe grown up is without any damage.
(2) egg mother cell that reclaims is cultivated maturation in the maturation in vitro culture fluid after, carry out in vitro fertilization with the stud ram sperm of other kinds.
(3) ivf zygote is cultured to 2-cell-blastula embryo in the ectogenesis culture systems, moves into acceptor ewe oviduct or intrauterine.
(4) obtain the first familiar generation lamb after acceptor sheep gestation, the lambing.
(5) F1 godmother lamb 2-3 utilizes the method for superovulation to obtain egg mother cell once more after the monthly age, is used for carrying out in vitro fertilization with the ram sperm of the identical strain of its male parent.
(6) after in vitro fertilization, can obtain embryo in vitro fertilization again.
(7) embryo in vitro fertilization is moved into acceptor sheep intrauterine, after gestation, obtain F2 for lamb.
(8) F2 godmother lamb 2-3 handles the recovery egg mother cell through superovulation after the monthly age, carries out in vitro fertilization once more.
(9) embryo who obtains after in vitro fertilization can be used for embryo transplantation again.
(10) after embryo transplantation, can obtain F3 for lamb.F3 fixes its genetic character for lamb by the method for traversed by, reaches the purpose of genetic breeding.
Advantage of the present invention and good effect:
1. adopt " in vitro " crossbreeding technology that sheep is carried out breeding and can greatly shorten breeding cycle.In the conventional breeding method of sheep, the filial generation ewe just can be used for breeding after common 2 years old, so breeding cycle is very long at present, and cultivating a kind often needs the more than ten years, even the time of decades.Adopt this technology, promptly can be used for breeding, and so just greatly shortened the generation interval in the breeding process in filial generation ewe 2-3 month.In lamb ovary Oocyte in Vitro fertilization technology, after lamb carried out superovulation and handle, can be recovered to the egg mother cell more than 50 pieces, this quantity is far above super row's effect of the 10 pieces of left and right sides egg mother cells of ewe of growing up, and the fecundity after lamb grown up does not have any damage.This result has not only shifted to an earlier date the breeding time of ewe, and has greatly improved the reproductive potential of ewe, and the ovary egg mother cell is fully utilized, for breeding process provides more sufficient genetic resources.The ovary egg mother cell is after culture in vitro maturation, processing in vitro fertilization, ovum more than 50% can be grown for supplying the 2-4 cell stage embryo of embryo transplantation, wherein 25% embryo can pregnant lambing after transplanting, and these results of study provide technical assurance for this The Application of Technology.
2. adopt " in the formula pipe " crossbreeding technology sheep to be carried out breeding can reduce the breeding cost.Adopt the generation interval that has significantly shortened sheep after this technology, the feeding and management time that is used in the breeding sheep shortens greatly, thereby has reduced the feeding and management expense of breeding process significantly.Simultaneously because this technology can significantly increase the quantity of filial generation lamb again when shortening breeding time, can provide more sufficient genetic resources for breeding process, thereby avoided the situation of breeding process generation of neutrons lamb lazy weight, otherwise also need drop into great amount of manpower and material resources and financial resources and carry out artificial breeding, both increase the breeding cost, further prolonged breeding time again.
In sum, adopt this new technical method of " in vitro " crossbreeding technology to sheep carry out breeding be a kind ofly save time, laborsaving, effective effective way.
Accompanying drawing is a process chart of the present invention.
Embodiment:
Present embodiment carries out at Ih Ju League, Inner Mongolia Autonomous Region En Gebei sheep stud, to the Cashmere Goat cross-breeding.Select the cashmere quality good, but lower this down producing goat of Alba of velour yield is as female parent; The selection cashmere is fair, but the higher Gai County down producing goat of velour yield is a male parent.Breeding objective is to cultivate the new down producing goat kind of velour yield height, cashmere quality better.
This ewe of Alba that grows up is carried out superovulation with FSH+LH handle, artificial insemination is carried out with Gai County ram seminal fluid in the back of oestrusing.Adopt operation method in the ewe oviduct, to reclaim 2 ~ 8 cell stage embryos after the artificial insemination about 70 hours, then these embryos are moved in the acceptor ewe oviduct of synchronization of estrus.Acceptor ewe can be given birth to a fairly large number of F1 for lamb after gestation.
F1 godmother lamb to the 2-3 monthly age utilizes the method for FSH+LH to carry out superovulation, adopts operation method to reclaim egg mother cell in 15-24 hour in LH injection back.Adopting ovum liquid is the PBS+BSA+10iu/ml heparin, draws egg mother cell with syringe from the development follicle of ovary surface.The egg mother cell that has complete cumulus cell that is recovered to is incubated at 39 ℃, 5%CO
2Incubator in, maturation culture solution is TCM199+HCG+FCS+E
2, ripe incubation time is 20 hours.With picking up from the medium centrifugal washed twice of the fresh semen of Gai County ram with the BO+2mM caffeine, handled 1 minute with 0.5 μ M Ionophore A23187 then, add in the insemination droplet that contains egg mother cell and carry out processing in vitro fertilization.Insemination is handled the back and will be continued to cultivate in the ovum immigration growth culture systems in 6-8 hour, and growing culture fluid is the M199+FCS+ Sodium Pyruvate.Ectogenesis is moved in the acceptor sheep oviduct to the embryo of 2-8 cell stage, acceptor ewe after gestation, give birth to F2 for lamb.
The F2 godmother lamb at 2-3 monthly age is carried out processing in vitro fertilization as stated above once more, obtain to move in the acceptor ewe oviduct behind the embryo in vitro fertilization, after gestation, give birth to F3 for lamb.
F3 can carry out traversed by to fix its hereditary capacity for lamb development to the manhood (2 years old), reaches the preliminary purpose of breeding.
Claims (3)
1, sheep crossbreeding technology in vitro is characterized in that taking the following step;
(1) utilize FSH-LH to carry out superovulation to 2-3 monthly age ewe lamb and handle, reclaim egg mother cell with operation method from the ovary surface, after the super row of lamb handled, every lamb can obtain the ovum more than 50 pieces;
(2) egg mother cell that reclaims is cultivated maturation in the maturation in vitro culture fluid after, carry out in vitro fertilization with the stud ram sperm of other kinds;
(3) ivf zygote is cultured to 2-cell-blastula embryo in ectogenesis cultivation property system, moves into acceptor sheep oviduct or intrauterine;
(4) obtain the first familiar generation lamb after acceptor sheep gestation, the lambing;
(5) F1 godmother lamb 2-3 utilizes the method for superovulation to obtain egg mother cell once more after the monthly age, is used for carrying out in vitro fertilization with the ram sperm of the identical strain of its male parent;
(6) after in vitro fertilization, can obtain embryo in vitro fertilization again;
(7) embryo in vitro fertilization is moved into acceptor sheep intrauterine, after gestation, obtain F2 for lamb;
(8) the F2 godmother lamb 2-3 monthly age is reclaimed egg mother cell after superovulation is handled, and carries out in vitro fertilization once more;
(9) embryo who obtains after in vitro fertilization can be used for embryo transplantation again;
(10) move through the embryo and can obtain F3 for lamb after growing; F3 fixes its genetic character for lamb by the method for traversed by, reaches the genetic breeding purpose.
2, sheep as claimed in claim 1 crossbreeding technology in vitro is characterized in that utilizing in the step 1 FSH-LH to carry out superovulation, adopts operation method to reclaim egg mother cell in 15-24 hour in LH injection back, and adopting ovum liquid is the PBS+BSA+10IU/ml heparin.
3, sheep as claimed in claim 1 crossbreeding technology in vitro is characterized in that in the step 2 egg mother cell that reclaims being incubated at 39 ℃, 5%CO
2Culture fluid in, maturation culture solution is TCM199+HCG+FCS+E
2, ripe incubation time is 20 hours, the stud ram sperm was handled 1 minute with 0.5 μ MIOnophoreA23187 then with the medium centrifugal washed twice of BO+2mM caffeine, adding contain carry out in the fertility of oocytes droplet in vitro fertilization.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99103293 CN1226378A (en) | 1999-04-01 | 1999-04-01 | Technology for crossbreeding sheep in 'tubes' |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 99103293 CN1226378A (en) | 1999-04-01 | 1999-04-01 | Technology for crossbreeding sheep in 'tubes' |
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| CN1226378A true CN1226378A (en) | 1999-08-25 |
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| CN 99103293 Pending CN1226378A (en) | 1999-04-01 | 1999-04-01 | Technology for crossbreeding sheep in 'tubes' |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1323583C (en) * | 2002-06-24 | 2007-07-04 | 健康及生物科学促进中心 | Methods for developing animal models |
| CN101120670B (en) * | 2007-08-02 | 2010-10-06 | 姜俊青 | Method for cultivating meat and fine hair goat |
| CN103275930A (en) * | 2013-06-20 | 2013-09-04 | 江苏皓康生物医药科技有限公司 | Oocyte in-vitro maturity nutrient solution and preparation method thereof |
| CN108669002A (en) * | 2018-05-23 | 2018-10-19 | 马鞍山市金农牧业有限公司 | A kind of double cross method improving quality of mutton and yield |
| CN111406711A (en) * | 2020-05-20 | 2020-07-14 | 山西农业大学 | Artificial propagation-hybridization combined breeding method for meat goats |
| CN111596073A (en) * | 2020-06-04 | 2020-08-28 | 昆明天沃生物科技有限公司 | Method for detecting early pregnancy of sheep |
| CN114931137A (en) * | 2022-05-30 | 2022-08-23 | 内蒙古农业大学 | Sperm survival increasing liquid and sheep artificial hybridization breeding method |
-
1999
- 1999-04-01 CN CN 99103293 patent/CN1226378A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1323583C (en) * | 2002-06-24 | 2007-07-04 | 健康及生物科学促进中心 | Methods for developing animal models |
| CN101120670B (en) * | 2007-08-02 | 2010-10-06 | 姜俊青 | Method for cultivating meat and fine hair goat |
| CN103275930A (en) * | 2013-06-20 | 2013-09-04 | 江苏皓康生物医药科技有限公司 | Oocyte in-vitro maturity nutrient solution and preparation method thereof |
| CN103275930B (en) * | 2013-06-20 | 2014-07-09 | 江苏皓康生物医药科技有限公司 | Oocyte in-vitro maturity nutrient solution and preparation method thereof |
| CN108669002A (en) * | 2018-05-23 | 2018-10-19 | 马鞍山市金农牧业有限公司 | A kind of double cross method improving quality of mutton and yield |
| CN111406711A (en) * | 2020-05-20 | 2020-07-14 | 山西农业大学 | Artificial propagation-hybridization combined breeding method for meat goats |
| CN111596073A (en) * | 2020-06-04 | 2020-08-28 | 昆明天沃生物科技有限公司 | Method for detecting early pregnancy of sheep |
| CN114931137A (en) * | 2022-05-30 | 2022-08-23 | 内蒙古农业大学 | Sperm survival increasing liquid and sheep artificial hybridization breeding method |
| CN114931137B (en) * | 2022-05-30 | 2023-09-08 | 内蒙古农业大学 | Sperm activating solution and sheep artificial hybridization breeding method |
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