CN103013908B - 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
- Publication number
- CN103013908B CN103013908B CN201310006062.9A CN201310006062A CN103013908B CN 103013908 B CN103013908 B CN 103013908B CN 201310006062 A CN201310006062 A CN 201310006062A CN 103013908 B CN103013908 B CN 103013908B
- Authority
- CN
- China
- Prior art keywords
- liquid
- vitro fertilization
- preparation
- bovine
- preservations
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
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 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 female reproductive tract endomixis, and at present conventional in vitro fertilization referring to completes the technology of fertilization process in manually operated environment in vitro homozoic sperm and ovum.Mammiferous fertilization has specificity between kind, that is to say that most Mammalss can not carry out different interbreed.
In order to find a kind of minimizing Niu Jingzi usage quantity, but can guarantee to be subject 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 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, promotion is lower than the required Niu Jingzi of normal fertilization and ovum fertilization, reach the object of making ox embryo, realized minimizing Niu Jingzi usage quantity but can guarantee to be subject to extract Iuality, when making ox embryo, can reduce ox sperm quantity by adding sheep sperm simultaneously, produce the object of more product.
The object 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, it is characterized in that: it comprises the steps:
(1), reagent preparation:
(1) preparation bovine oocyte is picked up ovum liquid:
H-M199+10%FBS
4 ℃ of preservations, time limit of service is 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 is 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 subject to seminal fluid:
Use BO liquid to dissolve, now with the current;
(5) preparation BO-B is subject to seminal fluid:
Use BO liquid to dissolve, now with the current;
(6) preparation BO-AB is subject to seminal fluid:
Equivalent BO-A mixes with BO-B, 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:
| 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 is one month;
(2), in vitro fertilization:
(1) maturation of ox ovum is cultivated:
The fresh ox ovary of 1. butchering within 3h rinses 3 times and is immersed in clean physiological saline by 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 10cm culture dish, picks under the microscope ovarian cumulus-ovocyte complex body COCs;
2. bovine oocyte is picked up to the ripe liquid of ovum liquid and bovine oocyte and be placed on 38.5 ℃, in 5% CO2gas incubator more than balance 1h, then ovarian cumulus-ovocyte complex body the COCs sorting out being picked up to ovum liquid with the good bovine oocyte of balance washes one time, with the ripe liquid of bovine oocyte, wash after twice again, get one or four orifice plates, every hole first adds the ripe liquid of 500 μ l bovine oocytes, add again 500 μ l paraffin oils, according to the number of every hole 50-120 piece, put into the ripe liquid of bovine oocyte, at 38.5 ℃, in 5% CO2gas incubator, carry out maturation and cultivate 22h;
(2) in vitro fertilization:
1. get two, 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 liquid BO-A in vitro fertilization liquid configuring and BO-B liquid are filled in new 50ml beaker with 0.2 μ m filter respectively, all put into 37.5 ℃ of thermostat water bath preheatings;
2. get ox, the former essence mixing of sheep, add the ox liquid BO-A in vitro fertilization liquid of 5ml preheating, after gently mixed semen being mixed, the centrifugal 5min of 2000rpm/min room temperature, abandons supernatant; The ox liquid BO-A in vitro fertilization liquid that adds again 5ml preheating, after mixing gently, the centrifugal 5min of 2000rpm/min, discards after supernatant, and with the ox liquid BO-AB in vitro fertilization liquid of preheating, cattle and sheep being mixed to 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 35mm ware drips, and adds paraffin oil to put into 38.5 ℃, in 5% CO2gas incubator after carrying out;
4. open microscope warming plate and rise to 37.5 ℃, sort out under the microscope the ox ovum of ripe 22h, in the ox liquid BO-AB in vitro fertilization of preheating liquid, wash 3 times, in inciting somebody to action 3., ready-made sperm drips and takes out from incubator, each sperm is added dropwise to 10 pieces of ovum, 38.5 ℃, in 5% CO2gas incubator, cultivate 6h;
(3) ectogenesis of zygote is cultivated:
After fertilization 6h, with the ox nutrient solution IVC in vitro fertilization SOF liquid of preheating in incubator, take off granulosa cell, then, the ovum of taking off granulosa cell is put into every hole of preheating containing 500 μ l ox nutrient solution IVC in vitro fertilization SOF liquid, in four orifice plates of upper cover 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 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 lower than the required Niu Jingzi of normal fertilization and ovum fertilization, reach the object of making ox embryo, realized minimizing Niu Jingzi usage quantity, but can guarantee to be subject to extract Iuality, when producing ox embryo, can reduce ox sperm count by adding sheep sperm simultaneously, but can guarantee that ox ovum fertilization produces the object 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; these described 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.In embodiment, M-199 buys the GIBICO company in the U.S., and article No. is 11150; H-M199 buys the GIBICO company in the U.S., and article No. is 12340, and other reagent not particularly pointing out is commercially available prod.
Being explained as follows of the english abbreviation letter occurring in the present invention:
FBS-foetal calf serum; FSH-pituitary follicular stimulating hormone; LH-lutropin;
Glucose-glucose; Na Pyrucate-Sodium.alpha.-ketopropionate; SA-bovine serum albumin;
Stock-stock solution; NEAA-non-essential amino acid; EAA-indispensable amino acid;
P/S-penicillin/streptomycin; Na-lactate-Sodium.alpha.-hydroxypropionate; L-Glutamine-L-glutamine; Sodium citrate-Trisodium Citrate; Myo-Inositol-inositol; SOF-synthesizes Oviductal Fluid;
Embodiment 1:
1, the fresh ox ovary within 3h slaughterhouse being collected, rinses 3 times and is immersed in clean physiological saline by 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 10cm culture dish, picks under the microscope ovarian cumulus-ovocyte complex body COCs; The COCs sorting out is placed in the egg-cleaning liquid of balance 1h in the CO2gas incubator of 38.5 ℃ and washes one time, in ripe liquid, wash twice, according to the number in 50 pieces, every hole, put into four orifice plates of every Kong Youhan 500 μ l maturation culture solution upper cover 500 μ l paraffin oils, 38.5 ℃, in 5% CO2gas incubator, carry out maturation and cultivate 22h;
2, get 2,50ml 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 new beaker, and put into 37.5 ℃ of thermostat water bath preheatings;
3, get Niu Yuanjing 1,000 ten thousand and put into 15ml centrifuge tube, add the BO-A liquid 5ml of preheating, after sperm is mixed gently, the centrifugal 5min of 2000rpm/min room temperature, abandons supernatant; After adding 5ml BO-A liquid to mix gently, the centrifugal 5min of 2000rpm/min, discards after supernatant again, adds 1ml BO-AB liquid, by the dilution of ox sperm concentration, is 1,000 ten thousand/ml, is pure ox group;
4, each 5,000,000 is put into 15ml centrifuge tube and mixes to get ox, the former essence of sheep, adds the BO-A liquid 5ml of preheating, and after gently mixed semen being mixed, the centrifugal 5min of 2000rpm/min room temperature, abandons supernatant; Add again 5ml BO-A liquid, after mixing gently, the centrifugal 5min of 2000rpm/min; Discard after supernatant, add 1ml BO-AB liquid, it is 1,000 ten thousand/ml that cattle and sheep are mixed to sperm concentration dilution, is ox: mixed smart group of sheep 1:1;
5, get Niu Yuanjing 1,000,000, former smart 900 contingency of sheep rise puts into 15ml centrifuge tube, adds the BO-A liquid 6ml of preheating, and after gently mixed semen being mixed, the centrifugal 5min of 2000rpm/min room temperature, abandons supernatant; Add again 6ml BO-A liquid, after mixing gently, the centrifugal 5min of 2000rpm/min; Discard after supernatant, add 1ml BO-AB liquid, it is 1,000 ten thousand/ml that cattle and sheep are mixed to sperm concentration dilution, is ox: mixed smart group of sheep 1:9;
6, the sperm that the seminal fluid of handling well with step 3-5 is 100 μ l in 35mm ware drips, and after carrying out, adds paraffin oil, puts into 38.5 ℃, in 5% CO2gas incubator;
7, open microscope warming plate and rise to 37.5 ℃, sort out under the microscope the ox ovum after ripe 22h, in the BO-AB of preheating mixed solution, wash 3 times, take out the ready-made sperm of step 6 and drip, each sperm is added dropwise to 10 pieces of ovum, 38.5 ℃, 5%CO
2in incubator, cultivate 6h;
8, be subject to after precision processing 6h, zygote is taken out from incubator, with the IVC SOF liquid of preheating in incubator, take off its granulosa cell, then the zygote of handling well is put into every hole of preheating containing four orifice plates of 500 μ l IVC SOF liquid upper cover 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 (by ovocyte put into sperm drip start to calculate) observe spilting of an egg situation:
The ovum number (being M II ovum after de-granulosa cell) of the embryo of spilting of an egg rate=normal spilting of an egg/for being fertilized
Be fertilized and after 5 days, add 4% serum, then cultivate after 2 days and add up hatching rate;
The embryo number of blastaea rate=blastaea number/normal spilting of an egg;
In table, be three different breeding oxens to be gathered to its seminal fluid carry out pure ox, ox below: the mixed essence of sheep 1:1, ox: the mixed essence of sheep 1:9 statistics in vitro fertilization:
From their result, can find out: the mixed essence of cattle and sheep during for 1:1 and spilting of an egg rate and the blastaea rate of pure ox essence there is no significant difference, cattle and sheep are mixed smart in dropping to the also normally spilting of an egg of 1:9, just slightly low than the spilting of an egg rate of pure ox essence and mixed smart 1:1, blastaea rate does not have significant difference.
Can draw the following conclusions thus: when making ox embryo, by adding sheep sperm, reduce ox sperm quantity simultaneously, adopt nonfertilization between mammiferous xenogenesis to carry out ox ovum fertilization, be subject under the prerequisite of extract Iuality guaranteeing, reach ox ovum fertilization and produce more ox embryo.
Claims (1)
1. ox mixes a sheep seminal fluid novel method in vitro fertilization, it is characterized in that: by following step, undertaken:
(1), reagent preparation:
(1) preparation bovine oocyte is picked up ovum liquid:
H-M199+10% foetal calf serum
4 ℃ of preservations, time limit of service is one month;
(2) the ripe liquid of configuration bovine oocyte:
M199+1 μ g/ml estradiol+0.1IU/ml pituitary follicular stimulating hormone+1IU/ml lutropin+10% foetal calf serum
4 ℃ of preservations, time limit of service is 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 subject to seminal fluid:
Use BO liquid to dissolve, now with the current;
(5) preparation BO-B is subject to seminal fluid:
Use BO liquid to dissolve, now with the current;
(6) preparation BO-AB is subject to seminal fluid:
Equivalent BO-A mixes with BO-B, 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 is one month;
(2), in vitro fertilization:
(1) maturation of ox ovum is cultivated:
The fresh ox ovary of 1. butchering within 3h rinses 3 times and is immersed in clean physiological saline by stroke-physiological saline solution, with 20ml syringe 17# syringe needle, extract diameter at the ovarian follicle of 2-8mm, liquor folliculi after gathering is placed in 10cm culture dish, picks under the microscope ovarian cumulus-ovocyte complex body COCs;
2. bovine oocyte is picked up to the ripe liquid of ovum liquid and bovine oocyte and be placed on 38.5 ℃, in 5% CO2gas incubator more than balance 1h, then ovarian cumulus-ovocyte complex body the COCs sorting out being picked up to ovum liquid with the good bovine oocyte of balance washes one time, with the ripe liquid of bovine oocyte, wash after twice again, get one or four orifice plates, every hole first adds the ripe liquid of 500 μ l bovine oocytes, add again 500 μ l paraffin oils, according to the number of every hole 50-120 piece, put into the ripe liquid of bovine oocyte, at 38.5 ℃, in 5% CO2gas incubator, carry out maturation and cultivate 22h;
(2) in vitro fertilization:
1. get two, 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 liquid BO-A in vitro fertilization liquid configuring and BO-B liquid are filled in new 50ml beaker with 0.2 μ m filter respectively, all put into 37.5 ℃ of thermostat water bath preheatings;
2. get ox, the former essence of sheep in 1:1 or the mixing of 1:9 ratio, add the ox liquid BO-A in vitro fertilization liquid of 5ml preheating, after gently mixed semen being mixed, the centrifugal 5min of 2000rpm/min room temperature, abandons supernatant; The ox liquid BO-A in vitro fertilization liquid that adds again 5ml preheating, after mixing gently, the centrifugal 5min of 2000rpm/min, discards after supernatant, and with the ox liquid BO-AB in vitro fertilization liquid of preheating, cattle and sheep being mixed to 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 35mm ware drips, and adds paraffin oil to put into 38.5 ℃, in 5% CO2gas incubator after carrying out;
4. open microscope warming plate and rise to 37.5 ℃, sort out under the microscope the ox ovum of ripe 22h, in the ox liquid BO-AB in vitro fertilization of preheating liquid, wash 3 times, in inciting somebody to action 3., ready-made sperm drips and takes out from incubator, each sperm is added dropwise to 10 pieces of ovum, 38.5 ℃, in 5% CO2gas incubator, cultivate 6h;
(3) ectogenesis of zygote is cultivated:
After fertilization 6h, with the ox nutrient solution IVC in vitro fertilization SOF liquid of preheating in incubator, take off granulosa cell, then, the ovum of taking off granulosa cell is put into every hole of preheating containing 500 μ l ox nutrient solution IVCSOF in vitro fertilization liquid, in four orifice plates of upper cover 500 μ l paraffin oils, 38.5 ℃, 5% CO2gas incubator continues to cultivate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310006062.9A CN103013908B (en) | 2013-01-08 | 2013-01-08 | New method of in vitro fertilization for mixed semens of bovine and sheep |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310006062.9A CN103013908B (en) | 2013-01-08 | 2013-01-08 | New method of in vitro fertilization for mixed semens of bovine and sheep |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103013908A CN103013908A (en) | 2013-04-03 |
| CN103013908B true CN103013908B (en) | 2014-03-12 |
Family
ID=47963025
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310006062.9A Active CN103013908B (en) | 2013-01-08 | 2013-01-08 | New method of in vitro fertilization for mixed semens of bovine and sheep |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103013908B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103392670B (en) * | 2013-08-10 | 2015-06-10 | 内蒙古赛科星繁育生物技术（集团）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 |
| CN110747160B (en) * | 2019-11-27 | 2020-10-30 | 浙江大学 | High-survival-rate sheep fertilized egg culture method for extra-embryonic-body culture |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1058147C (en) * | 1997-02-19 | 2000-11-08 | 郝易风 | Preservation and utilization of mixed bull and goat sperm |
| EP1539932A1 (en) * | 2002-06-17 | 2005-06-15 | Kobenhavns Amts Sygehus, Herlev | In vitro fertilisation |
| CN101962626B (en) * | 2010-09-20 | 2012-06-27 | 北京奶牛中心 | Calf in vitro embryo culture solution |
-
2013
- 2013-01-08 CN CN201310006062.9A patent/CN103013908B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103013908A (en) | 2013-04-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103898046B (en) | Ox IVF Embryos nutrient solution and cultural method | |
| CN102899286B (en) | Application of C-type natriuretic peptide to promotion on in vitro maturation of bovine oocyte | |
| CN102812950B (en) | Chicken semen diluent as well as preparation method and application thereof | |
| CN108103011B (en) | A kind of bovine oocyte in vitro maturation culture solution and cultural method | |
| CN102296047B (en) | In vitro embryo production method by utilization of lamb superovulation oocytes | |
| CN103013908B (en) | New method of in vitro fertilization for mixed semens of bovine and sheep | |
| WO2016206086A1 (en) | Pig oocyte in vitro maturation culture solution and preparation method and culture method thereof | |
| CN107460161B (en) | Culture medium for promoting in-vitro maturation of immature oocyte and application thereof | |
| CN103184189A (en) | Cultivation method of cross-bred wagy | |
| CN102140435A (en) | Method for improving in-vitro production efficiency of buffalo embryos | |
| CN104130973A (en) | In-vitro maturating method for sheep oocyte, pretreatment solution and kit | |
| CN110066763A (en) | Promote the method for ox embryo in vitro culture development of fertilized ova | |
| CN104928236A (en) | Nutrient solution and culturing method for maturation of oocyte in vitro | |
| CN103710299A (en) | In-vitro culture solution for culturing swine parthenogenetic activated embryos and swine in-vitro fertilized embryos | |
| CN102703377A (en) | Method for improving blastocyst rate of in-vitro embryos of animals | |
| CN105838668A (en) | In-vitro maturation culture solution for small follicle oocytes and application thereof | |
| CN105624100A (en) | Buffalo oocyte in-vitro maturation (IVM) culture method | |
| CN102618496A (en) | Method for enhancing ectogenesis of sheep oocyte | |
| CN104513807A (en) | Method for separating cells from blood and cultivating the cells and method for cloning non-human animal | |
| CN107916249A (en) | Improve the nutrient solution and cultural method of the development quality of bovine somatic cells clone's embryo and embryo in vitro fertilization | |
| CN100404675C (en) | Production process of somatic cell clone pig | |
| CN103392670B (en) | Method for evaluating sperm intracorporal conception rate of breeding bull according to extracorporal fertilization rate of breeding bull | |
| CN107460162B (en) | Method for improving lamb in-vitro embryo development capability | |
| CN101843530A (en) | Method for fertilizing oocytes in vitro of young animals twice | |
| CN110055212A (en) | A method of embryo is produced using Ovisammon Linnaeus tissue sperm in vitro |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| 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. |
|
| 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. |