CN103013908A - New method of in vitro fertilization for mixed semens of bovine and sheep - Google Patents
New method of in vitro fertilization for mixed semens of bovine and sheep Download PDFInfo
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- CN103013908A CN103013908A CN2013100060629A CN201310006062A CN103013908A CN 103013908 A CN103013908 A CN 103013908A CN 2013100060629 A CN2013100060629 A CN 2013100060629A CN 201310006062 A CN201310006062 A CN 201310006062A CN 103013908 A CN103013908 A CN 103013908A
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Abstract
The invention discloses a new method of in vitro fertilization for mixed semens of bovine and sheep, belonging to the technical field of animal propagation. The new method comprises the following steps: washing COCs (Cumulus Oocyte Complexes) respectively with bovine oocyte follicular fluid and bovine oocyte maturation fluid, then placing the COCs into the bovine oocyte maturation fluid for maturation culturing, washing in BO-AB fluid, and placing in sperm drip in which raw bovine and sheep semens for culturing; and taking off granule cells with SOF fluid, and placing ovum with taken-off granule cells into the SOF fluid for culturing. The new method has the advantages that raw bovine and sheep semens are mixed so as to conduct in vitro fertilization on the bovine ovum by utilizing fertilization biology principle of reproductive isolation of uncrossed fertilization of heterogeneity animals; through the plug flow action of sheep sperms, bovine sperms needed in being lower than normal fertilization is promoted to be fertilized with ovum, thereby achieving the purpose of producing a bovine embryo.
Description
Technical field
The invention belongs to the animal reproduction technical field, specifically belong to a kind of ox and mix sheep seminal fluid novel method in vitro fertilization.
Background technology
Mammalian Fertilization is that sperm and ovum are the process of a zygote at the female reproductive tract endomixis, at present the conventional technology that refers to homozoic sperm and ovum are finished fertilization process in external manually operated environment in vitro fertilization.Mammiferous fertilization has specificity between kind, that is to say that most Mammalss can not carry out being fertilized between xenogenesis.
In order to seek a kind of minimizing Niu Jingzi usage quantity, but can guarantee to be subjected to extract Iuality, can be with same ox sperm quantity when producing cow frozen semen, but can produce the method for more product, carried out the research of ox mixing sheep seminal fluid method in vitro fertilization.
Summary of the invention
The object of the present invention is to provide a kind of ox to mix sheep seminal fluid novel method in vitro fertilization, the method utilizes heterogenous animal not intersect the fertilization Biological Principles of reproduction isolation of fertilization, and the sheep essence is mixed into Niu Jingzhong, carries out the processing in vitro fertilization of ox ovum; By the effect of sheep sperm plug-flow, promote to be lower than the required Niu Jingzi of normal fertilization and ovum fertilization, reach the purpose of making the ox embryo, realize minimizing Niu Jingzi usage quantity but can guarantee to be subjected to extract Iuality, when making the ox embryo, can reduce simultaneously the ox sperm quantity by adding the sheep sperm, produce the purpose of more product.
The objective of the invention is to be achieved through the following technical solutions: a kind of ox mixes sheep seminal fluid novel method in vitro fertilization, and it is characterized in that: it comprises the steps:
(1), reagent preparation:
(1) the preparation bovine oocyte is picked up ovum liquid:
H-M199+10%FBS
4 ℃ of preservations, time limit of service are one month;
(2) the ripe liquid of configuration bovine oocyte:
M199+1μg/mlE
2+0.1IU/mlFSH+1IU/mlLH+10%FBS
4 ℃ of preservations, time limit of service are one month;
(3) preparation ox liquid BO in vitro fertilization liquid:
Use ultrapure water to dissolve, 4 ℃ of preservations, time limit of service is one month;
(4) preparation BO-A is subjected to seminal fluid:
Use BO liquid to dissolve, now with the current;
(5) preparation BO-B is subjected to seminal fluid:
Use BO liquid to dissolve, now with the current;
(6) preparation BO-AB is subjected to seminal fluid:
Equivalent BO-A mixes with BO-B, and is now with the current;
(7) preparation ox nutrient solution Stock in vitro fertilization A liquid:
| Title | Concentration |
| Nacl | 997.6mM |
| Kcl | 71.63mM |
| KH 2PO 4 | 11.90mM |
| Mgcl 2·6H 20 | 4.919mM |
| Na-lactate | 0.616 (V/V)% |
| Glucose | 14.99mM |
[0026]Use ultrapure water to dissolve, 4 ℃ of preservations, time limit of service is one month;
(8) preparation ox nutrient solution Stock in vitro fertilization B liquid:
| Title | Concentration |
| NaHCO 3 | 250.0mM |
Use ultrapure water to dissolve, 4 ℃ of preservations, time limit of service is one month;
(9) preparation ox nutrient solution Stock in vitro fertilization C liquid:
| Title | Concentration |
| Na Pyrucate | 32.72mM |
Use ultrapure water to dissolve, 4 ℃ of preservations, time limit of service is one month;
(10) preparation ox nutrient solution Stock in vitro fertilization D liquid:
| Title | Concentration |
| Cacl 2·2H 2O | 171.4mM |
Use ultrapure water to dissolve, 4 ℃ of preservations, time limit of service is one month;
(11) preparation ox nutrient solution Stock in vitro fertilization X liquid:
| Title | Concentration |
| L-Glutamine | 99.90mM |
Use ultrapure water to dissolve, 4 ℃ of preservations, time limit of service is one month;
(12) preparation ox nutrient solution Stock in vitro fertilization Y liquid:
| Title | Concentration |
| Sodium citrate | 34.00mM |
Use ultrapure water to dissolve, 4 ℃ of preservations, time limit of service is one month;
(13) preparation ox nutrient solution Stock in vitro fertilization Z liquid:
| Title | Concentration |
| myo-Inositol | 277.0mM |
Use ultrapure water to dissolve, 4 ℃ of preservations, time limit of service is one month;
(14) preparation ox nutrient solution IVC in vitro fertilization SOF liquid:
| The SOF total liquid volume | 10ml |
| stockA | 1ml |
| stockB | 1ml |
| stockC | 100μl |
| stockD | 100μl |
| stockX | 100μl |
| stockY | 100μl |
| stockZ | 100μl |
| NEAA | 100μl |
| EAA | 200μl |
| BSA | 0.08g |
| P/S | 10μl |
| Phenol red | 10μl |
| Ultrapure water | 7.18ml |
4 ℃ of preservations, time limit of service are one month;
(2), in vitro fertilization:
(1) maturation of ox ovum is cultivated:
The fresh ox ovary of 1. butchering within the 3h washes 3 times and is immersed in the clean physiological saline with stroke-physiological saline solution, with 20ml syringe 17
#Syringe needle extracts diameter at the ovarian follicle of 2-8mm, and the liquor folliculi after gathering is placed in the 10cm culture dish, picks ovarian cumulus-ovocyte complex body COCs at microscopically;
2. bovine oocyte is picked up the ripe liquid of ovum liquid and bovine oocyte and be placed on 38.5 ℃, in 5% the CO2gas incubator more than the balance 1h, then the ovarian cumulus sorted out-ovocyte complex body COCs is picked up ovum liquid with the good bovine oocyte of balance and washes one time, after washing twice with the ripe liquid of bovine oocyte again, get one or four orifice plates, every hole adds first the ripe liquid of 500 μ l bovine oocytes, add again 500 μ l paraffin oils, put into the ripe liquid of bovine oocyte according to every hole 50-120 piece number, at 38.5 ℃, carry out maturation in 5% CO2gas incubator and cultivate 22h;
(2) in vitro fertilization:
1. get two in 50ml beaker, be labeled as respectively A and B:A preparation ox liquid BO-A in vitro fertilization liquid, B preparation ox liquid BO-B in vitro fertilization liquid; The ox that configures liquid BO-A in vitro fertilization liquid and BO-B liquid are filled in the new 50ml beaker with 0.2 μ m filter respectively, all put into 37.5 ℃ of thermostat water bath preheatings;
2. get ox, former smart mixing of sheep, add the ox liquid BO-A in vitro fertilization liquid of 5ml preheating, gently with behind the mixed semen mixing, the centrifugal 5min of 2000rpm/min room temperature abandons supernatant; The ox liquid BO-A in vitro fertilization liquid that adds again the 5ml preheating, gently behind the mixing, the centrifugal 5min of 2000rpm/min, discard supernatant after, with the ox of preheating liquid BO-AB in vitro fertilization liquid cattle and sheep being mixed the sperm concentration dilution is 1,000 ten thousand/ml;
3. the sperm of using the seminal fluid of 2. handling well to be 100 μ l in the 35mm ware drips, and carries out rear adding paraffin oil and puts into 38.5 ℃, in 5% CO2gas incubator;
4. open the microscope warming plate and rise to 37.5 ℃, sort out the ox ovum of ripe 22h at microscopically, in the ox of preheating liquid BO-AB in vitro fertilization liquid, wash 3 times, ready-made sperm drips and takes out from incubator in inciting somebody to action 3., each sperm is added dropwise to 10 pieces of ovum, 38.5 ℃, cultivate 6h in 5% CO2gas incubator;
(3) ectogenesis of zygote is cultivated:
Behind the fertilization 6h, take off granulosa cell with the ox of preheating in incubator nutrient solution IVC in vitro fertilization SOF liquid, then, every hole of the ovum of taking off granulosa cell being put into preheating contains 500 μ l oxen nutrient solution IVC in vitro fertilization SOF liquid, in four orifice plates of loam cake 500 μ l paraffin oils, 38.5 ℃, 5% CO2gas incubator continues to cultivate
Advantage of the present invention and beneficial effect are: it is to utilize heterogenous animal not intersect the fertilization Biological Principles of reproduction isolation of fertilization, and the sheep essence is mixed into Niu Jingzhong, carries out the processing in vitro fertilization of ox ovum; Promote to be lower than the required Niu Jingzi of normal fertilization and ovum fertilization by the effect of sheep sperm plug-flow, reach the purpose of making the ox embryo, realized minimizing Niu Jingzi usage quantity, but can guarantee to be subjected to extract Iuality, when producing the ox embryo, can reduce simultaneously the ox sperm count by adding the sheep sperm, but can guarantee that the ox ovum fertilization produces the purpose of more product.
Embodiment
Below in conjunction with embodiment the present invention is described; the scheme of embodiment described here; do not limit the present invention; one of skill in the art can make improvements and change according to spirit of the present invention; described these improvement and variation all should be considered as in protection scope of the present invention, and scope of the present invention and essence are limited by claim.M-199 buys the GIBICO company in the U.S. among the embodiment, and article No. is 11150; H-M199 buys the GIBICO company in the U.S., and article No. is 12340, and other reagent that does not particularly point out is the commercially available prod.
Being explained as follows of the english abbreviation letter that occurs among the present invention:
The FBS-foetal calf serum; FSH-pituitary follicular stimulating hormone; The LH-lutropin;
Glucose-glucose; Na Pyrucate-Sodium.alpha.-ketopropionate; The SA-bovine serum albumin;
The stock-stock solution; The NEAA-non-essential amino acid; The EAA-indispensable amino acid;
The P/S-penicillin/streptomycin; The Na-lactate-Sodium.alpha.-hydroxypropionate; The L-Glutamine-L-glutamine; Sodium citrate-Trisodium Citrate; The myo-Inositol-inositol; SOF-synthesizes Oviductal Fluid;
Embodiment 1:
1, the fresh ox ovary within the 3h of slaughterhouse collection, washes 3 times and be immersed in the clean physiological saline with stroke-physiological saline solution; With 20ml syringe 17
#Syringe needle extracts diameter at the ovarian follicle of 2-8mm, and the liquor folliculi after gathering is placed in the 10cm culture dish, picks ovarian cumulus-ovocyte complex body COCs at microscopically; The COCs that sorts out is placed in the egg-cleaning liquid of balance 1h in 38.5 ℃ the CO2gas incubator and washes one time, wash twice in the ripe liquid, put into every hole according to the number in 50 pieces in every hole four orifice plates that contain 500 μ l maturation culture solution loam cakes, 500 μ l paraffin oils are arranged, 38.5 ℃, carry out maturation in 5% CO2gas incubator and cultivate 22h;
2, get 2 in 50ml beaker, be labeled as respectively A, B:A preparation BO-A liquid 40ml, B preparation BO-B liquid 6ml, the BO-A liquid, the BO-B liquid that configure are filled in the new beaker, and put into 37.5 ℃ of thermostat water bath preheatings;
3, get Niu Yuanjing 1,000 ten thousand and put into the 15ml centrifuge tube, add the BO-A liquid 5ml of preheating, gently behind the mixing, the centrifugal 5min of 2000rpm/min room temperature abandons supernatant with sperm; Add again 5ml BO-A liquid gently behind the mixing, the centrifugal 5min of 2000rpm/min, discard supernatant after, add 1ml BO-AB liquid, be 1,000 ten thousand/ml with the dilution of ox sperm concentration, be pure ox group;
4, each 5,000,000 is put into the 15ml centrifuge tube and mixes to get ox, the former essence of sheep, adds the BO-A liquid 5ml of preheating, and gently with behind the mixed semen mixing, the centrifugal 5min of 2000rpm/min room temperature abandons supernatant; Add again 5ml BO-A liquid, gently behind the mixing, the centrifugal 5min of 2000rpm/min; After discarding supernatant, add 1ml BO-AB liquid, it is 1,000 ten thousand/ml that cattle and sheep are mixed the sperm concentration dilution, is ox: the mixed smart group of sheep 1:1;
5, get Niu Yuanjing 1,000,000, former smart 900 contingency of sheep rise puts into the 15ml centrifuge tube, adds the BO-A liquid 6ml of preheating, and gently with behind the mixed semen mixing, the centrifugal 5min of 2000rpm/min room temperature abandons supernatant; Add again 6ml BO-A liquid, gently behind the mixing, the centrifugal 5min of 2000rpm/min; After discarding supernatant, add 1ml BO-AB liquid, it is 1,000 ten thousand/ml that cattle and sheep are mixed the sperm concentration dilution, is ox: the mixed smart group of sheep 1:9;
6, the seminal fluid of handling well with step 3-5 is 100 μ l in the 35mm ware sperm drips, and carries out rear adding paraffin oil, puts into 38.5 ℃, in 5% CO2gas incubator;
7, open the microscope warming plate and rise to 37.5 ℃, the ox ovum after microscopically is sorted out ripe 22h is washed 3 times in the BO-AB of preheating mixed solution, takes out the ready-made sperm of step 6 and drips, and each sperm is added dropwise to 10 pieces of ovum, 38.5 ℃, 5%CO
2Cultivate 6h in the incubator;
8, be subjected to precision processing 6h after, zygote is taken out from incubator, IVC SOF liquid with preheating in the incubator is taken off its granulosa cell, then every hole of the zygote of handling well being put into preheating contains four orifice plates of 500 μ l IVC SOF liquid loam cakes, 500 μ l paraffin oils, 38.5 ℃, 5% CO2gas incubator continues to cultivate;
9, statistics spilting of an egg rate, blastaea rate:
Ectogenesis is after 48 hours (with ovocyte put into sperm drip begin to calculate) observe spilting of an egg situation:
The embryo of spilting of an egg rate=normal spilting of an egg/for the ovum number of being fertilized (being M II ovum after taking off granulosa cell)
Being fertilized added 4% serum in 5 days afterwards, cultivated after 2 days again and added up the hatching rate;
The embryo number of blastaea rate=blastaea number/normal spilting of an egg;
Three different breeding oxens to be gathered its seminal fluid carry out pure ox, ox in the below's table: mixed smart, the ox of sheep 1:1: the mixed smart statistics in vitro fertilization of sheep 1:9:
Can find out from their result: cattle and sheep mixed smart during for 1:1 and spilting of an egg rate and the blastaea rate of pure ox essence do not have significant difference, cattle and sheep are mixed smart in dropping to the also normally spilting of an egg of 1:9, just the spilting of an egg rate than the smart and mixed smart 1:1 of pure ox is slightly low, and the blastaea rate does not have significant difference.
Can draw the following conclusions thus: when making the ox embryo, reduce simultaneously the ox sperm quantity by adding the sheep sperm, adopt between mammiferous xenogenesis nonfertilization to carry out the ox ovum fertilization, be subjected under the prerequisite of extract Iuality guaranteeing, reach the ox ovum fertilization and produce more ox embryo.
Claims (1)
1. an ox mixes sheep seminal fluid novel method in vitro fertilization, it is characterized in that: undertaken by following step:
(1), reagent preparation:
(1) the preparation bovine oocyte is picked up ovum liquid:
H-M199+10%FBS
4 ℃ of preservations, time limit of service are one month;
(2) the ripe liquid of configuration bovine oocyte:
M199+1μg/mlE
2+0.1IU/mlFSH+1IU/mlLH+10%FBS
4 ℃ of preservations, time limit of service are one month;
(3) preparation ox liquid BO in vitro fertilization liquid:
Use ultrapure water to dissolve, 4 ℃ of preservations, time limit of service is one month;
(4) preparation BO-A is subjected to seminal fluid:
Use BO liquid to dissolve, now with the current;
(5) preparation BO-B is subjected to seminal fluid:
Use BO liquid to dissolve, now with the current;
(6) preparation BO-AB is subjected to seminal fluid:
Equivalent BO-A mixes with BO-B, and is now with the current;
(7) preparation ox nutrient solution Stock in vitro fertilization A liquid:
Use ultrapure water to dissolve, 4 ℃ of preservations, time limit of service is one month;
(8) preparation ox nutrient solution Stock in vitro fertilization B liquid:
Use ultrapure water to dissolve, 4 ℃ of preservations, time limit of service is one month;
(9) preparation ox nutrient solution Stock in vitro fertilization C liquid:
Use ultrapure water to dissolve, 4 ℃ of preservations, time limit of service is one month;
(10) preparation ox nutrient solution Stock in vitro fertilization D liquid:
Use ultrapure water to dissolve, 4 ℃ of preservations, time limit of service is one month;
(11) preparation ox nutrient solution Stock in vitro fertilization X liquid:
Use ultrapure water to dissolve, 4 ℃ of preservations, time limit of service is one month;
(12) preparation ox nutrient solution Stock in vitro fertilization Y liquid:
Use ultrapure water to dissolve, 4 ℃ of preservations, time limit of service is one month;
(13) preparation ox nutrient solution Stock in vitro fertilization Z liquid:
Use ultrapure water to dissolve, 4 ℃ of preservations, time limit of service is one month;
(14) preparation ox nutrient solution IVC in vitro fertilization SOF liquid:
4 ℃ of preservations, time limit of service are one month;
(2), in vitro fertilization:
(1) maturation of ox ovum is cultivated:
The fresh ox ovary of 1. butchering within the 3h washes 3 times and is immersed in the clean physiological saline with stroke-physiological saline solution, with 20ml syringe 17
#Syringe needle extracts diameter at the ovarian follicle of 2-8mm, and the liquor folliculi after gathering is placed in the 10cm culture dish, picks ovarian cumulus-ovocyte complex body COCs at microscopically;
2. bovine oocyte is picked up the ripe liquid of ovum liquid and bovine oocyte and be placed on 38.5 ℃, in 5% the CO2gas incubator more than the balance 1h, then the ovarian cumulus sorted out-ovocyte complex body COCs is picked up ovum liquid with the good bovine oocyte of balance and washes one time, after washing twice with the ripe liquid of bovine oocyte again, get one or four orifice plates, every hole adds first the ripe liquid of 500 μ l bovine oocytes, add again 500 μ l paraffin oils, put into the ripe liquid of bovine oocyte according to every hole 50-120 piece number, at 38.5 ℃, carry out maturation in 5% CO2gas incubator and cultivate 22h;
(2) in vitro fertilization:
1. get two in 50ml beaker, be labeled as respectively A and B:A preparation ox liquid BO-A in vitro fertilization liquid, B preparation ox liquid BO-B in vitro fertilization liquid; The ox that configures liquid BO-A in vitro fertilization liquid and BO-B liquid are filled in the new 50ml beaker with 0.2 μ m filter respectively, all put into 37.5 ℃ of thermostat water bath preheatings;
2. get ox, former smart mixing of sheep, add the ox liquid BO-A in vitro fertilization liquid of 5ml preheating, gently with behind the mixed semen mixing, the centrifugal 5min of 2000rpm/min room temperature abandons supernatant; The ox liquid BO-A in vitro fertilization liquid that adds again the 5ml preheating, gently behind the mixing, the centrifugal 5min of 2000rpm/min, discard supernatant after, with the ox of preheating liquid BO-AB in vitro fertilization liquid cattle and sheep being mixed the sperm concentration dilution is 1,000 ten thousand/ml;
3. the sperm of using the seminal fluid of 2. handling well to be 100 μ l in the 35mm ware drips, and carries out rear adding paraffin oil and puts into 38.5 ℃, in 5% CO2gas incubator;
4. open the microscope warming plate and rise to 37.5 ℃, sort out the ox ovum of ripe 22h at microscopically, in the ox of preheating liquid BO-AB in vitro fertilization liquid, wash 3 times, ready-made sperm drips and takes out from incubator in inciting somebody to action 3., each sperm is added dropwise to 10 pieces of ovum, 38.5 ℃, cultivate 6h in 5% CO2gas incubator;
(3) ectogenesis of zygote is cultivated:
Behind the fertilization 6h, take off granulosa cell with the ox of preheating in incubator nutrient solution IVC in vitro fertilization SOF liquid, then, every hole of the ovum of taking off granulosa cell being put into preheating contains 500 μ l oxen nutrient solution IVC in vitro fertilization SOF liquid, in four orifice plates of loam cake 500 μ l paraffin oils, 38.5 ℃, 5% CO2gas incubator continues to cultivate.
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| CN201310006062.9A CN103013908B (en) | 2013-01-08 | 2013-01-08 | New method of in vitro fertilization for mixed semens of bovine and sheep |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103392670A (en) * | 2013-08-10 | 2013-11-20 | 内蒙古赛科星繁育生物技术股份有限公司 | Method for evaluating sperm intracorporal conception rate of breeding bull according to extracorporal fertilization rate of breeding bull |
| CN103461323A (en) * | 2013-07-09 | 2013-12-25 | 浙江省农业科学院 | Method used for in vitro time-delay preservation of oocyte biological activity |
| CN108588007A (en) * | 2018-04-10 | 2018-09-28 | 内蒙古大学 | The method for improving ox embryo in vitro preparation efficiency |
| CN110747160A (en) * | 2019-11-27 | 2020-02-04 | 浙江大学 | High-survival-rate sheep fertilized egg culture method for extra-embryonic-body culture |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1163110A (en) * | 1997-02-19 | 1997-10-29 | 郝易风 | Preservation and utilization of mixed bull and goat sperm |
| CN1675354A (en) * | 2002-06-17 | 2005-09-28 | 哥本哈根政府医院,位于海莱乌 | In vitro fertilisation |
| CN101962626A (en) * | 2010-09-20 | 2011-02-02 | 北京奶牛中心 | Calf in vitro embryo culture solution |
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2013
- 2013-01-08 CN CN201310006062.9A patent/CN103013908B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1163110A (en) * | 1997-02-19 | 1997-10-29 | 郝易风 | Preservation and utilization of mixed bull and goat sperm |
| CN1675354A (en) * | 2002-06-17 | 2005-09-28 | 哥本哈根政府医院,位于海莱乌 | In vitro fertilisation |
| CN101962626A (en) * | 2010-09-20 | 2011-02-02 | 北京奶牛中心 | Calf in vitro embryo culture solution |
Non-Patent Citations (1)
| Title |
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| 郝易风: "牛兔混合冻精配种实验", 《甘肃畜牧兽医》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103461323A (en) * | 2013-07-09 | 2013-12-25 | 浙江省农业科学院 | Method used for in vitro time-delay preservation of oocyte biological activity |
| CN103461323B (en) * | 2013-07-09 | 2015-10-28 | 浙江省农业科学院 | One can be used for external time delay and preserves the bioactive method of egg mother cell |
| CN103392670A (en) * | 2013-08-10 | 2013-11-20 | 内蒙古赛科星繁育生物技术股份有限公司 | Method for evaluating sperm intracorporal conception rate of breeding bull according to extracorporal fertilization rate of breeding bull |
| CN108588007A (en) * | 2018-04-10 | 2018-09-28 | 内蒙古大学 | The method for improving ox embryo in vitro preparation efficiency |
| CN110747160A (en) * | 2019-11-27 | 2020-02-04 | 浙江大学 | High-survival-rate sheep fertilized egg culture method for extra-embryonic-body culture |
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| CN103013908B (en) | 2014-03-12 |
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| C56 | Change in the name or address of the patentee |
Owner name: INNER MONGOLIA SKXING REPRODUCTIVE BIOTECHNOLOGY ( Free format text: FORMER NAME: INNER MONGOLIA SEXING REPRODUCTIVE BIOTECHNOLOGY CO., LTD. |
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| CP03 | Change of name, title or address |
Address after: 011517 the Inner Mongolia Autonomous Region Helingeer County of Hohhot city Shengle economic Park gathering service area management committee building 4 New South Building Room 4422 Patentee after: Inner Mongolia saikexing reproductive biotechnology (Group) Limited by Share Ltd Address before: Shengle Economic Zone Hohhot city 011517 the Inner Mongolia Autonomous Region Avenue, six floor six growth of Mengniu Patentee before: Saikexing Reproductive Biotechnology CO., Ltd. |