CN109182253A - A method of improving oocyte in vitro maturation quality and efficiency - Google Patents
A method of improving oocyte in vitro maturation quality and efficiency Download PDFInfo
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Abstract
The present invention provides a kind of methods for improving oocyte in vitro maturation quality and efficiency, the method includes at least replacing a maturation in vitro liquid in maturation in vitro, and are further included in maturation in vitro liquid and add a certain amount of FGF.The egg mother cell of method preparation of the invention has the nuclear maturation efficiency significantly improved and cell cytoplasm at heat efficiency, MII phase egg mother cell is up to 95%, and egg mother cell ratio, glutathione and the ATP level of cortical granule and mitochondria normal distribution are all remarkably higher than normal mature group；A certain amount of FGF is added in mature liquid in vitro, can further improve the mature quality and efficiency of egg mother cell.For the egg mother cell of method preparation of the invention after in vitro fertilization, efficiency in vitro fertilization is significantly higher than normal mature group, is presented as higher cleavage rates, blastocyst rate and more blastomere numbers, has great application value.
Description
Technical field
The present invention relates to technical field of bioengineering, and in particular to the method for Mammalian Oocytes in Vitro Maturation efficiency
Background technique
Oocyte in vitro maturation (IVM) is the key link and technology in vitro fertilization of livestock embryo biotechnology
(IVF) important component.However compared with vivo environment, existing egg mother cell IVM system is still not perfect, and then causes
The quality of IVM egg mother cell is lower.A large number of studies show that in the method for the prior art IVM egg mother cell quality, such as be fertilized
Produce surviving of son ability after ability, developmental potency, embryo transfer, substantially lower than egg mother cell (Shi JM etc., J of cylinder mature
Pineal Res,2009；47(4):318-323；Lonergan P etc., Annu Rev Anim Biosci, 2016；4:255-
268).The lower quality of IVM egg mother cell and developmental potentiality have become the bottleneck of limitation embryo biotechnology efficiency, it would be highly desirable to
It solves.
In vivo under environment, the growth of livestock Oocytes and maturation are all in hypothalamic pituitary gonadal axis secreting hormone
Accuracy controlling carry out, these hormones include hypophysis follicle-stimulating hormone (FSH), luteotropin (LH), estrogen (E2) etc..In ox
In oestrous cycle, begins to ramp up within the concentration of FSH 12 days or so before heat, peak value occurs within 1~2 day before heat, and LH's is dense
Then the 12h after heat steeply rises and reaches peak value, later in reduced levels to degree.And E2Concentration then a few days ago opened in heat
Begin to increase, heat reaches peak value after starting 12h, then begins to decline, and second peak value of appearance in the 5th~9 day after heat.By
This is as it can be seen that the hormonal readiness of above-mentioned regulation oocyte maturation process is dynamic change during egg mother cell cylinder mature
's.
Currently, egg mother cell IVM system is prepared by imitating domestic animal vivo environment, generally all by basal liquid
(M199), serum (FBS) and reproductive hormone etc. are constituted, and reproductive hormone includes FSH, LH, E2, wherein the effect of FSH can promote ovum
Mound Cell expansions, cytoplasmic maturation etc., and rate of ovine oocyte maturation, rate of fertilization and developmental rate can be improved in LH.Bovine oocyte IVM
Program is usual are as follows: will be put into IVM liquid from the cumulus oocyte complex (COCs) that Ovarian surface obtains, in 38.5 DEG C, 5%
CO2Maturation for 24 hours, is not any change in this period IVM system in incubator.
Due to the method for currently used bovine oocyte in vitro maturation, can not be provided most for oocyte in vitro maturation
Good hormonal milieu, therefore oocyte maturation quality and efficiency will certainly be had an impact.
Summary of the invention
It is an object of the invention to for oocyte maturation quality and effect caused by method for in-vitro maturity in the prior art
The lower problem of rate provides a kind of method that can effectively improve oocyte in vitro maturation quality and efficiency.
In order to solve the above technical problems, the present invention adopts the following technical scheme:
In a first aspect, the present invention provides a kind of method for improving oocyte in vitro maturation quality and efficiency, feature
It is, the method includes at least replacing a maturation in vitro liquid in maturation in vitro.
In method of the invention, the egg mother cell is preferably bovine oocyte.
Process of the present invention it is preferred maturation in vitro liquid of every 4-20 hours replacement, it is highly preferred that every 8-16 hours more
A maturation in vitro liquid is changed, most preferably, maturation in vitro liquid of every 8-12 hours replacement.
In terms of In-vitro maturation 24 hours, replacing maturation in vitro liquid for every eight hours indicates that after culture starts, the 8th is small
When, the 16th hour replacement maturation in vitro liquid；The maturation in vitro liquid of replacement in every 12 hours indicates the 12nd hour after culture starts
Replace a maturation in vitro liquid.
In method of the invention, replaceable all mature liquid when maturation in vitro liquid is replaced；It in some embodiments, can be more
Shift to few 50%, 60%, 70%, 80% or 90% mature liquid.
In method of the invention, the component of maturation in vitro liquid includes: TCM199 basic culture solution, 0.01IU/mL FSH, and 10
μ g/mL heparin, 40ng/mL IGF, 50ng/mL EGF, 0.01IU/mL LH, 1 μ g/mL E2And 10%FBS.
In method of the invention, maturation in vitro liquid is not limited to said components and content, in the prior art other packets
Maturation in vitro liquid containing basic culture solution and reproductive hormone, may also apply to the present invention.
Further, in method of the invention, FGF (fibroblast growth factor) is further comprised in maturation in vitro liquid,
The concentration of the FGF is 10-30ng/mL；Preferably, the concentration of the FGF is 20ng/mL.
Second aspect, the present invention provides the egg mother cells by maturation in vitro prepared by the above method.
Further, the egg mother cell of the maturation in vitro is bovine oocyte.
Using egg mother cell prepared by method of the invention, relative to the method for not replacing maturation in vitro liquid, MII phase ovum
The ratio of mother cell significantly improves, and the level of the ratio of cortical granule and mitochondria normal distribution, glutathione and ATP is significant
It improves, indicates that the egg mother cell of method preparation of the invention has higher mature quality and efficiency.
The third aspect, the present invention provides the egg mother cell of prepared maturation in vitro be fertilized in vitro in application.It adopts
It is fertilized after (IVF) in vitro with egg mother cell prepared by method of the invention, relative to the method for not replacing maturation in vitro liquid,
Cleavage rates, blastocyst rate and blastomere number are significantly increased, and indicate the egg mother cell and fertilization archiblast of method preparation of the invention
Amount is higher, and practical application value is bigger.
The present invention by Oocyte Maturation Process replace maturation in vitro liquid, overcome in the method for the prior art because
There is oocyte in vitro maturation quality and inefficiency caused by half-life period in reproductive hormone, to provide one kind
Easy to operate, repeatability is high, the method for significantly improving oocyte maturation quality and efficiency.
The egg mother cell prepared using method of the invention has the nuclear maturation efficiency significantly improved and cell cytoplasm
At heat efficiency, MII phase egg mother cell is up to 95%, egg mother cell ratio, the gluathione of cortical granule and mitochondria normal distribution
Peptide and ATP level are all remarkably higher than normal mature group；A certain amount of FGF is added in culture solution in vitro, can further improve
The mature quality and efficiency of egg mother cell.
For the egg mother cell of method preparation of the invention after in vitro fertilization, efficiency in vitro fertilization is significantly higher than normal mature
Group is presented as higher cleavage rates, blastocyst rate and more blastomere numbers；A certain amount of FGF is added in culture solution in vitro,
It can further improve the efficiency of Oocytes in Vitro Fertilization, there is great practical application value.
Detailed description of the invention
Cortical granule distribution situation in Fig. 1 egg mother cell.In figure A, B, C, D be respectively annular spread, it is discontinuously arranged,
Cytoplasm is uniformly distributed and discharges completely, and wherein A is normal distribution.Scale=20 μm.
Fig. 2 egg mother cell Mitochondria distribution situation.A, B, C are respectively annular spread, are uniformly distributed and divide extremely in figure
Cloth, wherein A, B are normal distribution.Scale=20 μm.
Fig. 3 .COCs maturation photo.A:20ng/mL FGF+12h changes liquid group, B: normal mature group.Amplification factor: 200x.
Specific embodiment
The present invention is discussed in detail below with reference to embodiment, and advantages of the present invention will be with description and apparent.Ying Li
Solution, the scope of protection of present invention are not limited by the specific embodiment, and specific embodiment provided by the invention is only
It is exemplary, any restrictions are not constituted to the scope of the present invention, the description of those skilled in the art's reference specification is to the present invention
Specific embodiment modify or some technical features can be equivalently replaced, these are not necessarily to the improvement of creative work
It should also be fallen within the protection scope of the appended claims of the present invention with replacement.
Reagent and instrument: removing specified otherwise, and all experiment reagents of the present invention are purchased from Sigma company (U.S.), and milk cow is frozen
Essence is purchased from Beijing Milk Cow Center.
The acquisition of 1 egg mother cell of embodiment and method for in-vitro maturity
1. acquisition and the maturation in vitro of egg mother cell
The ovary obtained from slaughterhouse, is put into 37 DEG C of physiological saline and transports to laboratory in 2h.By vacuum pump, extraction is straight
Cumulus oocyte complex (COCs) of the diameter in 2-8mm ovarian follicle detects under the microscope after the washing of IVM liquid, then will
COCs is put into IVM liquid in 38.5 DEG C, 5%CO2Incubator in cultivate for 24 hours.Wherein, IVM liquid ingredient is as follows: the basis TCM199
Culture solution+0.01IUmL-1FSH+10μg·mL-1Heparin+40ngmL-1IGF+50ng·mL-1EGF+0.01IU·mL-1LH
+1μg·mL-1E2+ 10%FBS.
2. oocyte nuclear maturation counts
Each group egg mother cell IVM for 24 hours after, using hyaluronidase remove cumulus cell, select MII phase egg mother cell, into
And count MII phase egg mother cell ratio.
3. oocyte cytoplasm maturation counts
Each group MII phase egg mother cell is chosen, cortical granule, Distribution of mitochondria and glutathione, ATP horizontal analysis are carried out.
Cortical granule analyzes program: egg mother cell is digested with pronase (5mg/mL), after removing zona-free, in DBPS
It is cleaned 3 times in solution, then room temperature is fixed on 30min in 4% paraformaldehyde, it is washed 3 times with the DBPS confining liquid containing 3%BSA,
It is placed in the penetrating processing 5min of room temperature in 0.1%Triton X-100 again, confining liquid is washed 3 times, each 5min, in 20 μ g/mL
Incubation 30min is protected from light for 38.5 DEG C in FITC-LCA dyestuff, and DBPS solution washs 3 times.It is burnt aobvious by copolymerization after egg mother cell tabletting
Micro mirror photograph, cortical granule is divided into: annular spread, discontinuously arranged, cytoplasm are uniformly distributed and discharge completely, middle ring
Shape is distributed as normal distribution (Fig. 1).
Mitochondria analyzes program: egg mother cell dyes 30min using 12.5 μM of MitoTracker RED CMXRos, and
It is washed twice afterwards using DPBS, each 5min, and then carries out tabletting, (TE2000- is then observed under laser confocal microscope
U,Nikon,Japan).The distribution of mitochondria is divided into: edge distribution, cytoplasm distribution, cytoplasm Distributed parts missing, wherein edge point
Cloth and cytoplasm are distributed as normal distribution (Fig. 2).
Glutathione level analysis: GSH level intracellular uses Fluorescence Intensity Assays, and egg mother cell first uses 10mM GSH to contaminate
Expect (CellTracker Blue CMF2HC；Invitrogen, Carlsbad, CA, USA) dyeing 10min, then use DPBS
2 times, each 5min are washed, is observed after tabletting.Using Image J software (NIH, USA) to egg mother cell GSH fluorescence intensity
It is analyzed, GSH concentration is indicated using fluorescence intensity.
ATP horizontal analysis: bioluminescent assay kit (11699709001, ATP Bioluminescence are used
Assay Kit HS II, Roche) detection ATP content.It is blown and beaten with pipettor so that ovum rupture, release ATP.First toward ELISA Plate
100 μ L ATP reaction solutions of middle addition are added 20 μ L samples after standing 5min, are measured in microplate reader after mixing, and record is each
A sample microplate reader reading.While being measured, by specification operating method prepares standard curve using standard sample.Mark
Directrix curve gradient is 0,1.0,2.0,3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0pmol.Measurement redeterminates mark every time
Directrix curve value, while setting blank control.According to the numerical value of measurement, standard curve is established.Each sample microplate reader reading is put into
It converts in standard curve, divided by egg mother cell content in sample, i.e. ATP content in acquisition sample.
4.IVF program
IVF program is carried out referring to the method for Brackett and Oliphant (1975), is slightly changed.Concrete operations are as follows: will
Freeze essence to be removed from liquid nitrogen, shakes freeze smart surface liquid nitrogen to volatilize rapidly, and then be put into 38 DEG C of water-baths and thaw.After defrosting
After sperm uses alcohol disinfecting, immigration is washed in the 15mL centrifuge tube of sperm containing 7mL, and 1500r/min washs 2 times, each 5min.
After centrifugation, the Sperm pellets after abandoning supernatant are added by sperm, adjustment adjustment sperm concentration to 5 × 106A/mL.By 20 μ L
Sperm and 80 μ L fertilization drop are mixed into 100 μ L fertilization drop, and (sperm final densities are 1 × 106A/mL), then it is put into CO2Incubator
(5%CO2, 38.5 DEG C, saturated humidity) balance 1.5h.Finally, egg mother cell is moved to fertilization drop (20~30 pieces/drop), it is put into
CO2Be fertilized 16~18h in incubator.After fertilization, it is put into mCR1aa and cultivates after fertilized eggs are washed.After 48h, ovum is recorded
Rate is split, and the embryo of the spilting of an egg is changed in later period liquid and is cultivated, changes a not good liquor (half measures), the 7th day blastaea of statistics every 48h later
Rate.
5. blastomere counts
Each group blastaea first removes oolemma (ZP) using the pronase enzymatic treatment 2-3min of 5mg/mL, then uses 10 μ
The Hoechst33342 of g/mL dyes 10min.Fluorescence microscope is used after dyeing blastaea tabletting, records blastomere number.
6. hormone concentration detects
Using radio immunoassay, in conjunction with FSH, LH, E2Detection kit (is purchased from Beijing North biotechnology research
Institute) measure FSH, LH, E in sample to be tested2Concentration.
7. statistical analysis
All biotinylated biomolecule recombinations of the present invention are at least more than three times.Data indicate with " mean+SD ", wherein
Percentage data is after arcsin transformation for analyzing.ANOVA significance analysis, p < 0.05 are carried out to data using SAS9.1
It is considered as significant difference.
The hormonal readiness of different time in 2 blank group of embodiment and COCs group IVM liquid
1. experimental design
Blank group is the IVM liquid without COCs in 38.5 DEG C, 5%CO2Incubator in cultivate for 24 hours.
COCs group is containing the IVM liquid on COCs in 38.5 DEG C, 5%CO2Incubator in cultivate for 24 hours.
Above-mentioned IVM liquid component is as follows: TCM199 basic culture solution+0.01IUmL-1FSH+10μg·mL-1Heparin+
40ng·mL-1IGF+50ng·mL-1EGF+0.01IU·mL-1LH+1μg·mL-1E2+ 10%FBS.
The 0th after IVM starts, 4,8,12,16,20, for 24 hours, take blank group and COCs group IVM liquid, -80 DEG C save,
For hormone test.
2. the hormonal readiness testing result of blank group and COCs group different time
Blank group hormonal readiness testing result is as shown in table 1,8h blank group FSH, LH, E2Level (5.75 ± 0.52mIU/
ML, 6.85 ± 0.59mIU/mL, 703.74 ± 63.45ng/ml) be substantially less than 0h blank group (9.93 ± 0.73mIU/mL, 9.72
±0.86mIU/mL,995.98±87.12ng/ml；P < 0.05), 16h blank group (3.38 ± 0.31mIU/mL, 4.87 ±
0.33mIU/mL,655.12±51.79ng/ml)FSH、LH、E2Level is substantially less than 8h blank group (p < 0.05).
Hormonal readiness detects in 1 blank group of table and COCs group IVM liquid
a,b,c,dSignificant difference (p < 0.05) is indicated with column data subscript difference, similarly hereinafter.
COCs group hormonal readiness testing result is as shown in table 2,8h COCs group FSH, LH, E2Level (7.98 ± 0.73mIU/
ML, 6.83 ± 0.52mIU/mL, 823.12 ± 80.98ng/ml) be substantially less than 0h COCs (9.75 ± 0.89mIU/mL, 9.56
± 0.84mIU/mL, 991.54 ± 92.78ng/ml；P < 0.05), 16h COCs group FSH, LH, E2Horizontal (5.42 ±
0.41mIU/mL, 4.29 ± 0.38mIU/mL, 781.32 ± 75.09ng/mL) it is substantially less than 8h COCs group (p < 0.05).
Hormonal readiness detects in 2 blank group of table and COCs group IVM liquid
Embodiment 3 replaces influence of the maturation in vitro liquid to oocyte in vitro maturation and IVF efficiency
1. experimental design
Normal mature group: COCs is put into maturation in IVM liquid and for 24 hours, has no change liquid during maturing.
12h changes liquid group: COCs being put into maturation 12h in IVM liquid, then moves into and continues maturation in the IVM liquid newly prepared
12h。
8h changes liquid group: COCs is put into IVM liquid to the IVM mature, 8h, 16h immigration are newly prepared after IVM starts respectively
Continue in liquid mature.
Above-mentioned IVM liquid component is as follows: TCM199 basic culture solution+0.01IUmL-1FSH+10μg·mL-1Heparin+
40ng·mL-1IGF+50ng·mL-1EGF+0.01IU·mL-1LH+1μg·mL-1E2+ 10%FBS.
After maturation, the MII phase egg mother cell rate of three groups of egg mother cells is counted, it is female thin then to select each group MII phase ovum
Born of the same parents carry out cortical granule distribution, Distribution of mitochondria and glutathione level, ATP content detection.Finally, each group MII phase ovum is female
After cell IVF, statistics cleavage rates, blastocyst rate and blastomere number.
2.IVM changes influence of the liquid to oocyte nuclear maturation efficiency
IVM changes influence of the liquid to Nuclear maturity efficiency, and the results are shown in Table 3, and 8h changes liquid group (95.03 ± 7.28%) and 12h is changed
Liquid group (86.25 ± 6.73%) rate of ovine oocyte maturation is significantly higher than normal mature group (77.66 ± 6.82%；p<0.05)；
8h, which changes liquid group rate of ovine oocyte maturation and is also significantly greater than 12h, simultaneously changes liquid group.
3 IVM of table changes influence of the liquid to oocyte nuclear maturation efficiency
3.IVM changes influence of the liquid to oocyte cytoplasm at heat efficiency
As a result as shown in table 4 and table 5, according to table 4 the results show that 8h changes the cortical granule normal distribution rate (93.02 of liquid group
± 8.39%), mitochondria normal distribution rate (91.30 ± 8.12%) be significantly higher than 12h change liquid group (72.50 ± 6.12%,
80.95 ± 7.81%) and normal mature group (60.98 ± 5.73%, 66.67 ± 5.03%；p<0.05)；12h changes liquid group simultaneously
Also significantly greater than normal mature group.
4 IVM of table changes influence of the liquid to oocyte cytoplasm at heat efficiency
Shown according to table 5,8h change the glutathione level of liquid group, ATP it is horizontal (93.39 ± 8.21 fluorescence intensities, 1.16 ±
0.09pmol) it is significantly higher than 12h and changes liquid group (67.74 ± 6.37 fluorescence intensities, 0.98 ± 0.07pmol) and normal mature group
(44.12 ± 3.98 fluorescence intensities, 0.85 ± 0.04pmol；p<0.05)；12h changes liquid group and is also significantly greater than normal mature simultaneously
Group.
5 IVM of table changes influence of the liquid to oocyte cytoplasm at heat efficiency
4.IVM changes influence of the liquid to egg mother cell IVF efficiency
The results are shown in Table 6, and 8h changes the cleavage rates, blastocyst rate and blastomere number (95.65 ± 8.53%, 54.55 of liquid group
± 3.91%, 118.82 ± 10.71) be all remarkably higher than normal mature group (75.59 ± 5.38%, 32.29 ± 1.82%,
100.32±5.62；P < 0.05) and 12h change liquid group (85.12 ± 6.12%, 42.72 ± 2.17%, 109.18 ± 8.18；p<
0.05)；12h changes liquid group and is also significantly greater than normal mature group simultaneously.
6 IVM of table changes influence of the liquid to egg mother cell IVF efficiency
Embodiment 4 adds FGF and replaces influence of the maturation in vitro liquid to oocyte in vitro maturation
1. adding influence of the FGF to oocyte nuclear maturation
1) experimental design
10ng/mL, 20ng/mL, 30ng/ml FGF are added in IVM liquid respectively, COCs presses normal mature program maturation extremely
For 24 hours, and then influence of the addition FGF to oocyte nuclear maturation is detected.
2) interpretation of result
The results are shown in Table 7, in egg mother cell IVM liquid add FGF after, 10ng/ml FGF group, 20ng/ml FGF group,
30ng/ml FGF group Nuclear maturity rate (77.27 ± 7.38%, 77.78 ± 5.63%, 78.57 ± 7.38%) and normal mature group
There was no significant difference (75.00 ± 4.37%；p>0.05).
7 FGF of table adds the influence to oocyte nuclear maturation
2. adding FGF and changing influence of the liquid to oocyte nuclear maturation, cytoplasmic maturation and IVF efficiency
1) experimental design
Normal mature group: COCs is put into maturation in IVM liquid and has no during maturing to for 24 hours and changes liquid.
8h changes liquid group: COCs is put into IVM liquid it is mature, and after maturation starts the 8th, 16h moves into newly prepare respectively
IVM liquid in continue maturation to for 24 hours.
FGF+12h changes liquid group (addition FGF simultaneously change liquid group): COCs is put into the IVM liquid for be added to 20ng/mLFGF at
It is ripe, and continue maturation to for 24 hours in the IVM liquid for being added to 20ng/mLFGF that 12h immigration is newly prepared after IVM starts.
After maturation, the MII phase egg mother cell rate of three groups of egg mother cells is counted, it is female thin then to select each group MII phase ovum
Born of the same parents carry out cortical granule distribution, ATP content detection.Finally, counting cleavage rates, blastocyst rate after each group MII phase egg mother cell IVF
And blastomere number.
2) it adds FGF and changes influence of the liquid to oocyte nuclear maturation and cytoplasmic maturation
As shown in table 8 and Fig. 3, adds FGF and change the MII phase egg mother cell rate (95.16 ± 7.43%) of liquid group, ovum mother carefully
Born of the same parents' cortical granule normal distribution ratio (93.02 ± 8.28%) and ATP content (111.74 ± 10.83pmol) are all remarkably higher than just
Often maturation group (74.81 ± 6.34%, 63.33 ± 4.28%, 0.81 ± 0.04；P < 0.05), and liquid group is changed without significant area with 8h
Not (93.85 ± 8.23%, 92.11 ± 7.35%, 109.82 ± 8.08；p>0.05).
Table 8 adds FGF and 12h changes influence of the liquid to oocyte nuclear maturation and cytoplasmic maturation
Pass through comparison FGF and change liquid group (20ng/ml FGF+12h changes liquid group) and 12h changes liquid group, it has been found that is female in ovum
In cell mature process, FGF is added while changing liquid, can further improve the quality of oocyte maturation, MII phase egg mother cell
Rate, cortical granule normal distribution percentage, ATP content dramatically increase (p < 0.05).
3) it adds FGF and changes influence of the liquid to egg mother cell IVF efficiency
As shown in table 9, add FGF and change the cleavage rates (93.94 ± 9.24%) of liquid group, blastocyst rate (53.23 ±
4.92%), blastomere number (115.04 ± 10.13) be all remarkably higher than normal mature group (73.02 ± 5.92%, 32.61 ±
3.02%, 98.25 ± 7.15；P < 0.05), and with 8h change liquid group there was no significant difference (94.18 ± 7.31%, 53.13 ±
4.24%, 113.81 ± 9.38；p>0.05).
9 IVM of table changes influence of the liquid to egg mother cell IVF efficiency
Claims (10)
1. a kind of method of oocyte in vitro maturation, which is characterized in that the method includes in maturation in vitro at least
The step of replacing a maturation in vitro liquid.
2. the method as described in claim 1, which is characterized in that the egg mother cell is bovine oocyte.
3. the method as described in claim 1, which is characterized in that maturation in vitro liquid of every 4-20 hours replacement.
4. method as claimed in claim 3, which is characterized in that maturation in vitro liquid of every 8-12 hours replacement.
5. method according to any of claims 1-4, which is characterized in that described maturation in vitro liquid of replacement is that replacement is complete
The maturation in vitro liquid in portion.
6. the method according to claim 1 to 5, which is characterized in that also include 10-30ng/ in the maturation in vitro liquid
The FGF of mL.
7. method as claimed in claim 6, which is characterized in that the concentration of the FGF is 20ng/mL.
8. the egg mother cell of the maturation in vitro of method preparation according to claim 1-7.
9. the egg mother cell of maturation in vitro as claimed in claim 8, which is characterized in that the egg mother cell is that ox ovum is female thin
Born of the same parents.
10. the egg mother cell of maturation in vitro described in claim 8 or 9 be fertilized in vitro in application.
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