CN108103011B - A kind of bovine oocyte in vitro maturation culture solution and cultural method - Google Patents

A kind of bovine oocyte in vitro maturation culture solution and cultural method Download PDF

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CN108103011B
CN108103011B CN201810134698.4A CN201810134698A CN108103011B CN 108103011 B CN108103011 B CN 108103011B CN 201810134698 A CN201810134698 A CN 201810134698A CN 108103011 B CN108103011 B CN 108103011B
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culture solution
oocyte
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bovine
vitro maturation
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CN108103011A (en
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张景程
张涌
马晓楠
张敏
王勇胜
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Northwest A&F University
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N2500/10—Metals; Metal chelators
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/31—Pituitary sex hormones, e.g. follicle-stimulating hormone [FSH], luteinising hormone [LH]; Chorionic gonadotropins
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    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • C12N2501/392—Sexual steroids

Abstract

The present invention provides a kind of bovine oocyte in vitro maturation culture solution and cultural method, maturation culture solution includes 10% fetal calf serum and 1% ITS-G, and the CXCL12 of the bFGF and 50ng/mL of EGF, 2.2mg/mL of the HMG containing 0.075IU/mL, 17 beta estradiols of 1 μ g/mL, 10ng/mL.Bovine oocyte in vitro maturation culture is carried out using above-mentioned culture solution, mature quality, developmental rate of in-vitro fertilization embryo and the body-cell neucleus transplanting embryo development rate and quality of blastocysts of bovine oocyte in vitro culture can be improved.

Description

A kind of bovine oocyte in vitro maturation culture solution and cultural method
Technical field
The present invention relates to livestock embryo field of engineering technology more particularly to a kind of bovine oocyte in vitro maturation culture solution, And the method using the culture solution culture bovine oocyte.
Background technique
In vitro maturation is in vitro fertilization, body-cell neucleus transplanting, animal embryo produced in vitro and transgenosis The basis of the Embryo engineering technologies such as clone and key link, the mature quality of egg mother cell are directly determined in fertilization or nuclear transfer The potentiality of development of embryo afterwards.Oocyte in vitro maturation is not only by ovarian cycle, animal varieties, follicle size, hormone, serum Equal many factors influence, and closely related with the program of oocyte in vitro maturation and method and culture solution ingredient, although ovum Mother cell maturation in vitro technology makes considerable progress, but Oocytes Maturation In vitro is significantly lower than body in developmental potency at present Interior mature oocyte.
Chemokine CXCL12 is also known as stromal cell derived factor-1 (SDF-1), is the cell factor of small molecule, belongs to become Change factor protein family.In-vitro maturation liquid often adds cytokine profiles (FSH, LH, estradiol, HCG) and reaches at present Promote the maturation in vitro purpose of animal oocyte.
Summary of the invention
The present invention is to improve the mature quality of bovine oocyte in vitro culture, provides a kind of bovine oocyte in vitro maturation training Nutrient solution and cultural method.
In order to achieve the above objectives, the invention adopts the following technical scheme:
A kind of bovine oocyte in vitro maturation culture solution, the In-vitro maturation liquid include the tire of volume fraction 5~20% Cow's serum, volume fraction 0.5~2% ITS-G (transferrins of insulin and 2.8~11.0mg/L containing 5~20mg/L), The HMG of 0.05~0.1IU/mL, 17 beta estradiols of 0.5~1.5 μ g/mL, the EGF of 5~10ng/mL, 1.1~3.3mg/mL The CXCL12 of bFGF and 10~100ng/mL.
Preferably, the In-vitro maturation liquid includes the ITS- of the fetal calf serum of volume fraction 10%, volume fraction 1% G (transferrins of insulin and 5.5mg/L containing 10mg/L), the HMG of 0.075IU/mL, 1 μ g/mL 17 beta estradiols, The CXCL12 of the bFGF and 50ng/mL of EGF, 2.2mg/mL of 10ng/mL.
Preferably, the In-vitro maturation liquid by no Hepes M199 basic culture solution and be added to the basis culture The fetal calf serum, ITS-G, HMG, 17 beta estradiols, EGF, bFGF and CXCL12 composition in liquid.
A kind of bovine oocyte in vitro maturation cultural method, comprising the following steps:
Bovine oocyte is put into oocyte in vitro maturation culture solution, it is small to be subsequently placed in culture 20~22 in incubator When, that is, obtain mature egg mother cell (M2 phase)；The oocyte in vitro maturation culture solution, that is, above-mentioned bovine oocyte is external Maturation culture solution.
Preferably, the bovine oocyte is selected from GV phase egg mother cell.
Preferably, the oocyte in vitro maturation culture solution preheats 1~3 hour before being put into bovine oocyte.
Preferably, described to preheat and be incubated at 38.5 DEG C, 5%CO2And it is carried out in the incubator of saturated humidity.
Preferably, 30~50 pieces of bovine oocyte ovum are put into the oocyte in vitro maturation culture solution of every 400~600 μ L Mound complex (COCs).
The beneficial effects of the present invention are embodied in:
Bovine oocyte in vitro maturation culture solution proposed by the present invention, by added CXCL12, ITS, (insulin turns Ferritin), the components such as EGF and bFGF, the body early embryo of ox IVF Embryos and body-cell neucleus transplanting embryo can be effectively improved Developmental rate and quality have achieved the purpose that the mature quality for effectively improving bovine oocyte in vitro culture.It is proposed by the present invention at Ripe cultural method is easy to operate, no replacement is required culture solution, so that cultivating the Oocytes Maturation In vitro obtained in developmental potency On significantly increase.
Further, by being preheated (balance) in culture environment to maturation culture solution, maturation culture can be improved Effect.
Specific embodiment
It elaborates below with reference to embodiment to the present invention.
Through experiments, it was found that CXCL12 high abundance in bovine follicular fluid exists, therefore speculate that the factor (CXCL12) may be for Oocyte maturation has facilitation.Therefore the present invention improves In-vitro maturation liquid by components such as addition CXCL12, with Improve the mature quality of bovine oocyte in vitro culture.
(1) bovine oocyte in vitro maturation culture solution
Bovine oocyte in vitro maturation culture solution includes the basis the M199 culture of no Hepes (4- hydroxyethyl piperazineethanesulfonic acid) Liquid and the bFGF being added in the basic culture solution (basic fibroblast growth factor), EGF (epithelical cell growth factor), 17 beta estradiols, HMG (people's menopause menotropins), fetal calf serum, ITS and CXCL12.
In one embodiment of the invention, based on volumn concentration, bovine oocyte in vitro maturation culture solution contains 10% Fetal calf serum and 1% ITS-G (source of people), and 17 beta estradiols, the 10ng/mL of the HMG containing 0.075IU/mL, 1 μ g/mL EGF (source of people), 2.2mg/mL bFGF (source of people) and 50ng/mL CXCL12 (source of people).
(2) reagent source and preparation
The M199 basic culture solution of no Hepes is bought (Thermo company) from commercialization channel, superfine fetal calf serum (FBS) For Gibco Products, ITS-G is that (ITS-G is the ITS dissolved with sodium selenite to Thermo Products, and ITS-G includes Two kinds of albumen: insulin (Insulin) and transferrins (Transferrin)), it is that sigma company produces that other are not specified Product.
A. bovine oocyte in vitro maturation culture solution
1) the tire ox of 10% (v/v) maturation culture solution (addition CXCL12): is added in the M199 basic culture solution of no Hepes Serum, the ITS-G (Insulin:10mg/L, Transferrin:5.5mg/L) of 1% (v/v), the HMG of 0.075IU/mL, 1 μ g/ The CXCL12 of 17 beta estradiols of mL, the bFGF and 50ng/mL of EGF, 2.2mg/mL of 10ng/mL.
2) tire of 10% (v/v) maturation culture solution (not adding CXCL12): is added in the M199 basic culture solution of no Hepes Cow's serum, the ITS-G (Insulin:10mg/L, Transferrin:5.5mg/L) of 1% (v/v), the HMG of 0.075IU/mL, 1 μ The bFGF of 17 beta estradiols of g/mL, the EGF and 2.2mg/mL of 10ng/mL.
B.SOF solution and mSOFaa solution
SOF solution preparation: by the KH of KCl, 0.162g of NaCl, 0.0534g of 0.6294g2PO4, 0.0172g CaCl2·2H2O, the MgCl of 0.00996g2·6H2O, the NaHCO of 0.2106g3, 0.0033g Sodium Pyruvate and 28.24 μ L Sodium lactate is added in 98mL water, 1mM NaOH tune pH to 7.2~7.4, deionized water constant volume to 100mL.
MSOFaa solution: also including BME, the volume fraction 1% of volume fraction 2% using SOF solution as basic liquid The BSA (bovine serum albumin(BSA)) of the glutamine of MEM, 1mmol/L, 8mg/mL.
C.BO liquid
BO liquid is using mSOFaa solution as basic liquid, and every 100mL mSOFaa solution adds 0.05g heparin sodium.
D. electro' asion liquid
Electro' asion liquid includes: 0.3mol/L mannitol, 0.05mol/L calcium chloride, 0.1mol/L magnesium sulfate, 0.27mol/L The BSA of histidine and 1mg/mL.
E. cell dissociation buffer
0.02g EDTA and 0.25g trypsase is weighed, 100mL PBS is added, is stirred continuously to abundant and dissolves, 0.22 μm After membrane filtration, the packing of 4mL/ pipe, -20 DEG C are saved backup.
(3) preparation of bovine somatic cells clone embryos
A. the culture of bovine fetal fibroblast
The bovine fetal fibroblast in 2~5 generations of a pipe holstein cow is taken (to acquire and clone from Yang Ling section member from liquid nitrogen Limited liability company cattle farm, acquisition time: in January, 2014 in March, 2016) it thaws in 38 DEG C, add the DMEM/F12 of 0.8mL thin Born of the same parents' culture solution, centrifugation abandon supernatant, add cell culture fluid to be resuspended, 3mL cell suspending liquid is taken to be inoculated in the culture dish of diameter 6cm, It is placed in CO2It is cultivated under the conditions of 38.5 DEG C in incubator.
When bovine fetal fibroblast reaches 80% and converges, inhales and abandon culture solution, with no Ca2+、Mg2+PBS rinse it is thin The cell dissociation buffer that trypsase is mixed with EDTA, vitellophag is added in born of the same parents.Cell is observed under inverted microscope, to most When number cell bounces back, is rounded, space between cells expands, digestion is terminated with the DMEM/F12 cell culture fluid containing 10% fetal calf serum, It after being blown and beaten with pipettor, is collected by centrifugation, is suspended with the DMEM/F12 cell culture fluid containing 10% fetal calf serum, in the ratio of 1:3 24 orifice plates are inoculated in, CO is put into2Culture (guaranteeing cell within ten generations) in incubator.Before carrying out the production of somatic cell clone embryo Two days, culture solution is replaced with the DMEM/F12 containing 0.5% fetal calf serum.
B. the maturation culture of egg mother cell
Ox ovary picks up from three bridge animal-slaughtering in fixed place field of Xi'an City, Shanxi Province (acquisition time: in January, 2014 in March, 2016), Ovary is placed in vacuum flask to include in 20~25 DEG C of physiological saline of penicillin and streptomycin, transports laboratory within 5h back.Ovary is transported back Afterwards, the connective tissue, fat and the fallopian tubal of attachment that Ovarian surface is wiped out with sterilizing scissors, clean three in sterile saline It is secondary, the egg mother cell (GV phase) in 2~8mm of Ovarian surface ovarian follicle is extracted with the 10mL syringe equipped with 12G syringe needle, is put into 6cm In glass dish, under stereomicroscope collect cumulus oocytes complesxes (cumulus-oocyte complexes, COCs).It is cleaned three times in the PBS containing 10% fetal calf serum after collection.The normal COCs of oocyte morphology is selected to be used for body Outer maturation culture.
Processing group: the COCs (30) of selection is washed twice in maturation culture solution (addition CXCL12), is then moved into every (balanced one hour in incubator in advance) in four orifice plates of the hole equipped with 500 μ L maturation culture solutions (addition CXCL12), 38.5 DEG C, 5%CO2, 20~22h is cultivated under the conditions of saturated humidity.The mature COCs of culture is cleaned 3 times with PBS liquid, is put into containing 0.1% thoroughly Bright matter acid enzyme without Ca2+、Mg2+1~2min is digested in PBS, and is blown and beaten repeatedly with 1000mL liquid-transfering gun, to remove egg mother cell The cumulus cell of external diffusion.After piping and druming is clean, is washed in PBS 3 times, then under entity stereomicroscope, ovum is stirred with foreign body needle Mother cell, the egg mother cell for selecting polar body are stand-by.
Control group: the COCs (30) of selection is washed twice in maturation culture solution (not adding CXCL12), is then moved into Every hole is equipped in four orifice plates of 500 μ L maturation culture solutions (not adding CXCL12) and (balances one hour in incubator in advance), 38.5 DEG C, 5%CO2, 20~22h is cultivated under the conditions of saturated humidity.The mature COCs of culture is cleaned 3 times with PBS liquid, is put into and contains 0.1% hyaluronidase without Ca2+、Mg2+1~2min is digested in PBS, and is blown and beaten repeatedly with 1000mL liquid-transfering gun, to remove ovum The cumulus cell of mother cell external diffusion.After piping and druming is clean, washed in PBS 3 times, then under entity stereomicroscope, with foreign body needle Egg mother cell is stirred, the egg mother cell for selecting polar body is stand-by.
C. the building of bovine somatic cells clone embryo
Stoning:
Before stoning, processing group and control group egg mother cell are respectively first containing 7.5 μ g/mL cytochalasin Bs, 10%FBS 15min is incubated in PBS.Under micromanipulation instrument, first polar body and surrounding part are drawn with the stoning pipe that internal diameter is 20 μm Ooecium matter.
Infuse core and electro' asion:
When infusing core, processing group and control that the bovine fetal fibroblast that diameter is 15~20 μm is injected separately into stoning are selected Under group egg mother cell oolemma.Reconstituted embryo after note core is merged using the method for microelectrode.Before fusion, reconstituted embryo melts in electricity It closes in liquid and pre-equilibrates 3min, electro' asion carries out under 150 times of micromanipulation instrument.Two " Z " fonts for mixing operation are micro- Electrode tip diameter is 15 μm, and rear end is connected on micromanipulation instrument, makes the donor, recipient cell after birth contact surface and two electricity of reconstituted embryo The line of pole is vertical, fusion parameters: voltage 32V, 20 μ s of pulse duration, 2 subpulse interval 10ms.1h is under the microscope after fusion Observation fusion situation.
D, the activation of bovine somatic cells clone reconstituted embryo
1~2h after electro' asion, the processing group and control group ox somatic cell clone reconstituted embryo for selecting successful fusion pass through The activation processing of ionomycin (Ionomycin) joint 6-DMAP is activated: containing 2~5 μm of ol/L ionomycins first 4min is incubated at room temperature in mSOFaa solution, then in the mSOFaa solution of the 6-DMAP Han 1~2mmol/L, in 38.5 DEG C, 5% CO2, cultivate 4h under the conditions of saturated humidity.
E, the in vitro culture of bovine somatic cells clone embryos
The mSOFaa solution of 500 μ L is added in the every hole of four orifice plates, is also covered with 500 μ L paraffin oils, paraffin on mSOFaa solution Oil is in advance in CO2At least 2h, processing group and control group ox somatic cell clone reconstituted embryo are balanced in incubator is carrying out activation processing Afterwards, it is transferred in above-mentioned four orifice plate.Every hole places 35~40 pieces of bovine somatic cells and clones reconstituted embryo, in 38.5 DEG C, 5%CO2, 7%O2 And cultivated under the conditions of saturated humidity, record developmental state.
Test result: compared with the control group, adding 50ng/mL CXCL12 in oocyte maturation culture solution, can be significant The developmental rate and quality of blastocysts of bovine somatic cells clone embryos are improved, it can efficient produced in vitro bovine somatic cells clone embryos.Specific table It is existing are as follows:
1) developmental rate and quality of bovine somatic cells clone embryos
As can be seen from Table 1, the oocyte in vitro maturation culture solution for adding 50ng/mL CXCL12, is remarkably improved ovum The developmental rate and quality of mother cell blastaea after nuclear transfer.
Egg mother cell of the table 1. after the oocyte in vitro maturation culture solution containing various concentration CXCL12 is mature is in core Developmental rate after transplanting
Note: the number in bracket is developmental rate, and the number before bracket is the total number of embryos of statistics；2- cell stage embryo and blastaea Developmental rate statistics is respectively in recombination embryo culture 72h and 192h (the 8th day)；In same row, the Superscript letters difference of data is indicated Significant difference (P < 0.05, Chi-square Test)；Blastaea(1-3)Refer to the total blastaea number and total blastocyst rate that the 8th day obtains；Capsule Embryo(1-2)Then refer to the high quality for having reached the International Embryo transplanting morphologic criteria of association's grade 1 and 2, recommendation is further transplanted The blastaea of culture
2) cell number of bovine somatic cells cloned blastocysts
As can be seen from Table 2, egg mother cell mature in the oocyte maturation culture solution of the CXCL12 containing 50ng/mL Total number of cells, inner cell mass (ICM) cell number of the embryo's (blastaea) obtained after nuclear transfer significantly improves.
The cell number of 2. bovine somatic cells cloned blastocysts of table is analyzed
Note: the data in same row, Superscript letters difference indicate significant difference (P < 0.05)
(4) preparation of ox IVF Embryos
A. the maturation culture of egg mother cell
Ox ovary picks up from three bridge animal-slaughtering in fixed place field of Xi'an City, Shanxi Province, and ovary is placed in vacuum flask and includes penicillin and streptomycin In 20~25 DEG C of physiological saline, laboratory is transported within 5h back.After ovary is transported back, the connective of Ovarian surface is wiped out with sterilizing scissors The fallopian tubal of tissue, fat and attachment is cleaned three times in sterile saline, is taken out with the 10mL syringe equipped with 12G syringe needle The egg mother cell in Ovarian surface 2~8mm ovarian follicle is taken, is put into 6cm glass dish, it is female that ovarian cumulus-ovum is collected under stereomicroscope Cell conjugate (cumulus-oocyte complexes, COCs).Three are cleaned in the PBS containing 10% fetal calf serum after collection Time.The normal COCs of oocyte morphology is selected to be used for In-vitro maturation.
Processing group: the COCs (30) of selection is washed twice in maturation culture solution (addition CXCL12), is then moved into every (balanced one hour in incubator in advance) in four orifice plates of the hole equipped with 500 μ L maturation culture solutions (addition CXCL12), 38.5 DEG C, 5%CO2, 20~22h is cultivated under the conditions of saturated humidity.The mature COCs of culture is blown and beaten repeatedly with 1000mL liquid-transfering gun, to remove Remove the cumulus cell of egg mother cell external diffusion.Piping and druming to around COCs only four to five layers of cumulus cell of remaining tight when, It is washed 2 times in by sperm (BO liquid), the free cumulus cell that wash clean is blown down, then by four to five layers of cumulus cell of tight Egg mother cell move to be placed into advance in incubator balance 2h or more fertilization drop in.
Control group: the COCs (30) of selection is washed twice in maturation culture solution (not adding CXCL12), is then moved into Every hole is equipped in four orifice plates of 500 μ L maturation culture solutions (not adding CXCL12) and (balances one hour in incubator in advance), 38.5 DEG C, 5%CO2, 20~22h is cultivated under the conditions of saturated humidity.The mature COCs of culture is blown repeatedly with 1000mL liquid-transfering gun It beats, to remove the cumulus cell of egg mother cell external diffusion.It blows and beats to only four to five layers of ovarian cumulus of remaining tight around COCs When cell, washed 2 times in by sperm (BO liquid), the free cumulus cell that wash clean is blown down, then by four to five layers of tight The egg mother cell of cumulus cell moves in the fertilization drop for being placed into balance 2h or more in incubator in advance.
B, the defrosting of sperm purifies, fertilization
Essence (Xi'an milk cow center, in October, 2013 acquisition) will be frozen after liquid nitrogen container taking-up, be placed in 37 DEG C of water-bath solutions Freeze, Percoll layering liquid is done in centrifuge tube.2mL 90%Percoll liquid is placed in centrifugation bottom of the tube, 2mL 45% Percoll liquid is carefully added on 90%Percoll liquid level, then the sperm after defrosting is placed on 45%Percoll liquid level, 1500rpm is centrifuged 20min, and about 200 μ L sperm of careful suction foot is separately added into the fertilization of processing group and control group egg mother cell In liquid (BO liquid), putting back in incubator makes smart ovum be combined 16~22h.
C, the in vitro culture of fertilized embryo
After fertilization, remaining cumulus cell, the sperm around ovum are blown away with mouth suction pipe or pipette tips.If can not go Except clean, can be removed in hyaluronidase solution.MSOFaa solution is moved into afterwards (500 μ L stones are also covered on liquid level Wax oil, and in advance in CO2At least 2h is balanced in incubator) in culture (35~40 pieces of fertilized bovine oocytes are placed in every hole, 38.5 DEG C, 5%CO2, 7%O2And cultivated under the conditions of saturated humidity), observe and record developmental state.
Test result: compared with the control group, adding 50ng/mL CXCL12 in oocyte maturation culture solution, can be significant The developmental rate and quality of blastocysts of ox IVF Embryos are improved, it can efficient produced in vitro ox fertilized embryo.Specific manifestation are as follows:
1) developmental rate and quality of ox IVF Embryos
As can be seen from Table 3, egg mother cell mature in the oocyte maturation culture solution of the CXCL12 containing 50ng/mL It is significantly raised in the embryonic development potential that after fertilization obtains.
Egg mother cell of the table 3. after the oocyte in vitro maturation culture solution containing various concentration CXCL12 is mature by Developmental rate after essence
Note: the number in bracket is developmental rate, and the number before bracket is the total number of embryos of statistics；2- cell stage embryo and blastaea Developmental rate statistics is fertilized embryo culture 72h and 192h (the 8th day) in vitro respectively；In same row, the Superscript letters of data are different It indicates significant difference (P < 0.05, Chi-square Test)；Blastaea(1-3)Refer to the total blastaea number and total blastocyst rate that the 8th day obtains；Capsule Embryo(1-2)Then refer to the high quality for having reached the International Embryo transplanting morphologic criteria of association's grade 1 and 2, recommendation is further transplanted The blastaea of culture
2) cell number of bovine IVF embryo
The cell number of 4. bovine IVF embryo of table is analyzed
Note: the data in same row, Superscript letters difference indicate significant difference (P < 0.05)
As can be seen from Table 4, egg mother cell mature in the oocyte maturation culture solution of the CXCL12 containing 50ng/mL It is significantly improved in the total number of cells for embryo's (blastaea) that after fertilization obtains.
In addition, with it has been reported that cultivating system compared with (for example, being basic liquid with M199, add 10% FBS, 1 μ g/ The FSH of the Estradiol-17 β, 1IU/mL of mL, the maturation culture solution of the LH of 5IU/mL), the experimental results showed that, the present invention proposes Bovine oocyte in vitro maturation culture solution, regardless of whether addition CXCL12, the quality of bovine oocyte after maturation can be improved.
In short, bovine oocyte in vitro maturation culture solution proposed by the present invention include the bFGF for making an addition to basic culture solution, The components such as EGF, ITS and CXCL12.Egg mother cell is put into the oocyte in vitro maturation culture solution of preheating, is subsequently placed in CO2Maturation culture 20~22 hours in the environment that volumetric concentration is 5%, that is, obtain mature egg mother cell.It is experimentally confirmed that this hair Bright In-vitro maturation liquid and in-vitro maturation culture method can be improved mature quality, the ox of bovine oocyte in vitro culture Developmental rate of in-vitro fertilization embryo and body-cell neucleus transplanting embryo (somatic cell clone embryo) developmental rate and quality of blastocysts.

Claims (3)

1. a kind of bovine oocyte in vitro maturation culture solution, it is characterised in that: the In-vitro maturation liquid by volume fraction 5~ 20% fetal calf serum, the insulin of 5~20mg/L, the transferrins of 2.8~11.0mg/L, 0.05~0.1IU/mL HMG, 17 beta estradiols of 0.5~1.5 μ g/mL, the EGF of 5~10ng/mL, the bFGF of 1.1~3.3mg/mL and 10~100ng/mL CXCL12, and the composition of the M199 basic culture solution without Hepes.
2. a kind of bovine oocyte in vitro maturation culture solution according to claim 1, it is characterised in that: the maturation in vitro training Nutrient solution is by the fetal calf serum of volume fraction 10%, the insulin of 10mg/L, the transferrins of 5.5mg/L, 0.075IU/mL HMG, 17 beta estradiols of 1 μ g/mL, 10ng/mL EGF, 2.2mg/mL bFGF and 50ng/mL CXCL12, Yi Jiwu The M199 basic culture solution of Hepes forms.
3. a kind of bovine oocyte in vitro maturation cultural method, it is characterised in that: the following steps are included:
GV phase bovine oocyte is put into the oocyte in vitro maturation culture solution of preheating 1~3 hour, incubator is subsequently placed in Middle culture 20~22 hours, that is, obtain mature egg mother cell；It is described to preheat and be incubated at 38.5 DEG C, 5%CO2And saturated humidity Incubator in carry out；30~50 pieces of bovine oocytes are put into the oocyte in vitro maturation culture solution of every 400~600 μ L； The oocyte in vitro maturation culture solution by the fetal calf serum of volume fraction 5~20%, the insulin of 5~20mg/L, 2.8~ The transferrins of 11.0mg/L, the HMG of 0.05~0.1IU/mL, 17 beta estradiols of 0.5~1.5 μ g/mL, 5~10ng/mL The CXCL12 of EGF, the bFGF of 1.1~3.3mg/mL and 10~100ng/mL, and the M199 basic culture solution group without Hepes At.
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