CN107034176A - A kind of nutrient solution and its cultural method for improving in vitro culture porcine oocytes developmental potentiality - Google Patents
A kind of nutrient solution and its cultural method for improving in vitro culture porcine oocytes developmental potentiality Download PDFInfo
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Abstract
The invention discloses a kind of nutrient solution and cultural method for improving in-vitro maturity of porcine oocytes quality and developmental potentiality；The nutrient solution includes following components：Forskolin 3~7 μm of ol/L, Roscovitine 3~7 μm of ol/L, TCM 199 8~12 g/L, NaHCO31~4g/L, 0.4~0.7g/L of D glucose, 0.05~0.15g/L of Sodium Pyruvate, 0.05~0.1g/L of penicillin sodium salt, 0.03~0.07g/L of streptomycin sulfate, 1~4g/L of polyvinyl alcohol, the μ g/mL of luteotropin 0.3~0.7, follicle-stimulating hormone 0.3~0.7 μ g/mL, 8~12ng/mL of EGF, L 0.3~0.7mmol/L of cysteine, liquor folliculi 8~12%；The nutrient solution can improve the cleavage rates of porcine oocytes, blastaea number and blastomere number, effectively improve the developmental potentiality of the porcine oocytes of in vitro culture, with larger application prospect.
Description
Technical field
The invention belongs to Embryo engineering technology field, in particular it relates to which a kind of improve the development of in vitro culture porcine oocytes
The nutrient solution and its cultural method of potentiality.
Background technology
Mammal ovocyte Vitro Culture Techniques（In vitro maturation, IVM）It is the important of embryo engineering
Part, refers to the immature oocyte that will be taken out in ovarian follicle and passes through In-vitro maturation, be developed to subtrahend second division
Mid-term, can carry out technology that is in vitro fertilization and splitting into embryo.Pig is biological as a kind of common pattern, to its egg mother cell body
Outer ripe research starts from late 1980s, by development for many years, though larger progress is achieved, in vitro
Ripe porcine oocytes, its ripe quality and developmental potentiality still have larger gap compared with vivo.Therefore, exploitation improves ovum mother
The nutrient solution of cell development potentiality, improves In-vitro maturation system, it is still necessary to which ours keeps punching.
The content of the invention
The technical problems to be solved by the invention are ripe quality when overcoming Porcine Oocytes In Vitro culture in the prior art
There is provided a kind of in-vitro maturity of porcine oocytes nutrient solution and cultural method, the culture with the defect and deficiency of developmental potentiality difference
Liquid can effectively improve the developmental potentiality of the porcine oocytes of in vitro culture, and prepare simply, and cultural method is simple and easy to do, has
Larger application prospect.
It is an object of the invention to provide a kind of in-vitro maturity of porcine oocytes accelerator.
Another object of the present invention is to provide the in-vitro maturity of porcine oocytes nutrient solution added with above-mentioned accelerator.
Another object of the present invention is to provide improves in-vitro maturity of porcine oocytes quality and development using above-mentioned nutrient solution
The cultural method of potentiality.
The above-mentioned purpose of the present invention is to give realization by the following technical programs：
A kind of in-vitro maturity of porcine oocytes accelerator, the accelerator is by 3~7 μm of ol/L forskolins and 3~7 μm of ol/L
Roscovitine is constituted.
The forskolin of the present invention（Forskolin）It is a kind of adenyl cyclase activator, by stimulating adenylate to be cyclized
Enzyme generates a large amount of cAMP, and cAMP delay oocyte nuclear maturations make oocyte cytoplasm fully be grown, finally reach and carry
The effect of high Oocyte Development potentiality, clinic is used for suppressing tumour growth, while also being obtained extensively in supplementary reproduction field
Pay attention to.
Roscovitine is a kind of adenine derivative, has high efficiency and spy when acting on cyclin kinase
The opposite sex, can with the cell cycle dependent kinases such as selective depression cdc2, cdk2 and cdk5 activity, can effectively suppress pig, ox,
The recovery of the species Oocyte Meiosis such as horse and goat.
Research finds that the greatest problem that in-vitro maturity of porcine oocytes is present is exactly the ripe disunity of caryoplasm, this
Two kinds of the medicines Forskolin and Roscovitine selected are invented, all there is the effect for suppressing Nuclear maturity, single high concentration addition
Though there can be certain delay effect to Nuclear maturity, unknowable influence can be also caused to egg mother cell simultaneously.The present invention passes through
Forskolin and Trichostatin A are carried out into each single medicine, a variety of concentration of combination drug and addition incubation time respectively to attempt
Afterwards, Forskolin and Roscovitine composite reagents are found in certain concentration and there is significant collaboration to increase under the addition time
Effect, can effectively improve the ectogenesis potentiality of egg mother cell, greatly promote embryo's yield, it is to avoid what single drug was brought lacks
Fall into.
It is latent with development that the in-vitro maturity of porcine oocytes accelerator of the present invention can improve in-vitro maturity of porcine oocytes quality
Power, therefore, the accelerator can be added in in-vitro maturity of porcine oocytes nutrient solution.
A kind of in-vitro maturity of porcine oocytes nutrient solution, it is characterised in that the nutrient solution includes following components：Forskolin
3~7 μm of ol/L, Roscovitine 3~7 μm of ol/L, TCM-1998~12g/L, NaHCO3 1~4g/L, D-Glucose 0.4
~0.7 g/L, the g/L of Sodium Pyruvate 0.05~0.15, the g/L of penicillin sodium salt 0.05~0.1, streptomycin sulfate 0.03~
0.07 g/L, the g/L of polyvinyl alcohol 1~4, the μ g/mL of luteotropin 0.3~0.7, the μ g/mL of follicle-stimulating hormone 0.3~0.7, epidermis
The ng/mL of growth factor 8~12, the mmol/L of Cys 0.3~0.7, liquor folliculi 8~12%
Preferably, the nutrient solution includes following components：Forskolin 4~6 μm of ol/L of 4~6 μm of ol/L, Roscovitine,
TCM-199 9~11 g/L, NaHCO32~3 g/L, D-Glucose 0.5~0.6 g/L, 0.08~0.12g/L of Sodium Pyruvate,
0.06~0.09g/L of penicillin sodium salt, 0.04~0.06g/L of streptomycin sulfate, the g/L of polyvinyl alcohol 1~3, luteotropin
0.4~0.6 μ g/mL, follicle-stimulating hormone 0.4~0.6 μ g/mL, the ng/mL of EGF 9~11, Cys 0.4~
0.6mmol/L, liquor folliculi 9~11%.
More preferably, the nutrient solution includes following components：Forskolin 5 μm of ol/L of 5 μm of ol/L, Roscovitine,
TCM-199 9.87 g/L, NaHCO3 2.2 g/L, the g/L of D-Glucose 0.5496, the g/L of Sodium Pyruvate 0.1, penicillin sodium salt
0.075 g/L, the g/L of streptomycin sulfate 0.05, the g/L of polyvinyl alcohol 1, luteotropin 0.5 μ g/mL, the μ of follicle-stimulating hormone 0.5
G/mL, the ng/mL of EGF 10, the mmol/L of Cys 0.57, liquor folliculi 10%.
Above-mentioned in-vitro maturity of porcine oocytes nutrient solution is in in-vitro maturity of porcine oocytes quality and developmental potentiality is improved
Application also in the scope of the present invention.
It is a kind of to improve in-vitro maturity of porcine oocytes quality and the method for developmental potentiality, it regard any of the above-described nutrient solution as training
Nutrient solution A, porcine oocytes are cultivated to be transferred to after 22~24h in nutrient solution B in nutrient solution A and continue to cultivate 22~24h；
The nutrient solution B includes following components：TCM-199 8~12 g/L, NaHCO31~4 g/L, D-Glucose 0.4~0.7
G/L, the g/L of Sodium Pyruvate 0.05~0.15, the g/L of penicillin sodium salt 0.05~0.1, the g/ of streptomycin sulfate 0.03~0.07
L, the g/L of polyvinyl alcohol 1~4, the ng/mL of EGF 9~11, liquor folliculi 9~11%.
Preferably, the nutrient solution A or/and nutrient solution B need to be placed in CO after preparing2Incubator balance is stayed overnight.
Preferably, the CO2Condition in incubator is 38.6 °C, 5%CO2, relative humidity 100%.
Above-mentioned cultural method can effectively improve the developmental potentiality of the porcine oocytes of in vitro culture, and the pig ovum of in vitro culture
Mother cell is the important raw and processed materials of porcine clone embryos, therefore application of the above-mentioned cultural method in porcine clone embryos culture is also at this
In invention protection domain.
Compared with prior art, the invention has the advantages that：
（1）The invention provides a kind of in-vitro maturity of porcine oocytes accelerator.
（2）The invention provides a kind of nutrient solution for improving in-vitro maturity of porcine oocytes quality and developmental potentiality.
（3）The invention provides a kind of cultural method for improving in-vitro maturity of porcine oocytes quality and developmental potentiality.
（4）The present invention accelerator, nutrient solution and cultural method can improve the cleavage rates of porcine oocytes, blastaea number and
Blastomere number, effectively improves the developmental potentiality of the porcine oocytes of in vitro culture, and prepares simply, easy to operate, has
Larger application prospect.
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment, but embodiment does not do any type of to the present invention
Limit.Unless stated otherwise, the reagent of the invention used, method and apparatus is the art conventional reagent, methods and apparatus.
Unless stated otherwise, following examples agents useful for same and material are purchased in market.
The Porcine Oocytes In Vitro culture of embodiment 1
1st, solution and nutrient solution are prepared
（1）Forskolin（Forskolin）：1 mg Forskolin are dissolved with 2.44 mL DMSO, final concentration of 1 mM storage is obtained
Standby liquid, is placed in -20 DEG C of preservations；DdH is used again2O is diluted to 10 μM of working solution, and 4 DEG C save backup.
（2）Core inhibitor Roscovitine：1 mg Roscovitin are dissolved with 2.83 ml DMSO, final concentration of 1 is obtained
MM storing solution, is placed in -20 DEG C of preservations；DdH is used again2O is diluted to 10 μM of working solution, and 4 DEG C save backup.
（3）0~22 h nutrient solutions：Take 9.87 g TCM-199,2.2 g NaHCO3, 0.5496 g D-Glucoses, 0.1
G Sodium Pyruvates, 0.075 g penicillin sodium salts, 0.05 g streptomycin sulfates, 1 g polyvinyl alcohol is dissolved in 1 L ddH2In O, fill
Divide after mixing, add 0.5 μ g/mL LH, 0.5 μ g/mL FSH, 10 ng/mL EGF, 0.57 mM Cys, 10%
PFF；Fully mix again, suction filtration is degerming, is placed in CO2Incubator balance is stayed overnight.
（4）22~44 h nutrient solutions：Take 9.87 g TCM-199,2.2 g NaHCO3, 0.5496 g D-Glucoses, 0.1
G Sodium Pyruvates, 0.075 g penicillin sodium salts, 0.05 g streptomycin sulfates, 1 g polyvinyl alcohol is dissolved in 1 L ddH2In O, fill
Divide after mixing, add 10 ng/mL EGF and 10% PFF, suction filtration is degerming, is placed in CO2Incubator is balanced.
（5）Egg mother cell operates liquid：By 9.5 g TCM-199,0.05 g NaHCO3,1.755 g NaCl, 3.0
GBSA, 0.75 g Hepes, 0.06 g streptomysins, 0.05 g penicillin is dissolved in 1 L ddH2In O, after fully mixing, pH is measured,
Suction filtration is degerming, packing, is placed in 4 DEG C of refrigerators and saves backup.
（6）Merge liquid：Take 0.0203 g MgCl2-6H2O, 0.147 gCaCl2-2H2O, 54.66 g mannitol, 0.119
G Hepes are dissolved in 1 L ddH2In O, suction filtration is degerming, packing, is placed in -20 DEG C of refrigerators and saves backup.
（7）Embryo medium：Take 6.312 g NaCl, 0.746 g KCl, 0.048 g KH2PO4, 0.098 g MgSO4-
7H2O, 2.106 g NaHCO3, 3.0 g BSA, 0.1460 g Glus, 0.546g hypotaurine, 0.05 g celebrates big mould
Element, 0.022g Sodium Pyruvates, 20 mL essential amino acids, 10 mL nonessential amino acid sequentially add ddH2In O, treat that medicine is complete
After dissolving, pH is measured, suction filtration is degerming, packing is placed in 4 DEG C of refrigerators and saved backup.
2nd, Porcine Oocytes In Vitro culture
（1）Individually add Forskolin：By forskolin（Forskolin）Added in 0~22 h nutrient solutions, make its final concentration
For 5 μM；Porcine oocytes are cultivated after 24h in above-mentioned 0~22 h nutrient solutions added with Forskolin, it is transferred to 22~
Continue to cultivate 24h in 44 h nutrient solutions, carry out follow-up lonely female activation experiment.
（2）Individually add Roscovitine：Core inhibitor Roscovitine is added in 0~22 h nutrient solutions, made
Its final concentration of 5 μM；Porcine oocytes are cultivated after 24h in above-mentioned 0~22 h nutrient solutions added with Roscovitine,
It is transferred in 22~44 h nutrient solutions and continues to cultivate 24h, carries out follow-up lonely female activation experiment.
（3）Forskolin+Roscovitine is added simultaneously：By forskolin（Forskolin）With core inhibitor
Roscovitine is added separately in 0~22 h nutrient solutions, and it is 5 μM to make Forskolin and Roscovitine final concentrations；
Porcine oocytes are cultivated after 24h in above-mentioned 0~22 h nutrient solutions added with Forskolin and Roscovitine, are transferred to
Continue to cultivate 24h into 22~44 h nutrient solutions, carry out follow-up lonely female activation experiment.
（4）Positive controls：Porcine oocytes culture is trained in 0~22 h nutrient solutions containing 100nm or so DMSO
Support after 24h, be transferred in no DMSO 22~44 h nutrient solutions and continue to cultivate 24h, carry out follow-up lonely female activation experiment.
（5）Negative control group：Porcine oocytes culture is cultivated after 24h in normal 0~22 h nutrient solutions, is transferred to just
Continue to cultivate 24h in 22~44 normal h nutrient solutions, carry out follow-up lonely female activation experiment.
3rd, result
As a result as shown in table 1, find that pig ovum is female by investigating the form natural rate of interest, cleavage rates and blastocyst rate of porcine oocytes
Developmental potentiality after cell is cultivated in the nutrient solution for only individually adding Forskolin is not so good as control group, and significant difference on the contrary；
Developmental potentiality of the porcine oocytes in the nutrient solution for only individually adding Roscovitine after culture is with control group without significance difference
It is different；Cellular morphology of the porcine oocytes in the nutrient solution for adding 5 μM of Forskolin and 5 μM of Roscovitine after culture
Natural rate of interest is relatively compareed with control group without significant difference, but cleavage rates and blastocyst rate with control group significant difference, especially blastocyst rate
Group is greatly improved.
The developmental potentiality of porcine oocytes after the Forskolin of table 1 processing（Lonely female activation）
| Group | Oocyte number | Form natural rate of interest（%） | Cleavage rates（%） | Blastocyst rate（%） |
| Experimental group Forskolin | 380 | 85.4 (324/390) | 66.2(214/324)a | 17.5(37/214)a |
| Experimental group Roscovitine | 290 | 83.4 (242/290) | 44.6(108/242)a | 18.5(20/108)a |
| Accelerator group Forskolin+ Roscovitine | 318 | 86.5 (275/318) | 84.4(232/275) | 59.9(138/232)a |
| Positive controls | 320 | 91.9(294/320) | 79.6(234/294) | 26.1(61/234)b |
| Negative control group | 300 | 89.3(268/300) | 78.7(211/268) | 27.5(58/211)b |
Note：Same column letter is different represent differences have conspicuousness (P＜ 0.05), there was no significant difference for no letter expression (P＞ 0.05).
The Porcine Oocytes In Vitro culture of embodiment 2
1st, solution and nutrient solution are prepared
It is same as Example 1.
2nd, Porcine Oocytes In Vitro culture
（1）By forskolin（Forskolin）It is added separately in 0~22 h nutrient solutions, makes with core inhibitor Roscovitine
Final concentration of 7 μM of final concentration of 3 μM of Forskolin, Roscovitine；Porcine oocytes are added with above-mentioned
Cultivated in Forskolin and Roscovitine 0~22 h nutrient solutions after 24h, be transferred in 22~44 h nutrient solutions and continue to train
24h is supported, follow-up lonely female activation experiment is carried out.
（2）Control group is set, it is same as Example 1.
3rd, result
As a result as shown in table 4, find that pig ovum is female by investigating the form natural rate of interest, cleavage rates and blastocyst rate of porcine oocytes
Cell add 3 μM of Forskolin and 7 μM of Roscovitine nutrient solution in cultivate after cellular morphology natural rate of interest with
Control group is carried significantly without significant difference, but cleavage rates and blastocyst rate with control group significant difference, especially blastocyst rate compared with control group
It is high.
The developmental potentiality of porcine oocytes after Forskolin and 7 μM of Roscovitine processing of table 43 μM（It is lonely female sharp
It is living）
| Group | Oocyte number | Form natural rate of interest（%） | Cleavage rates（%） | Blastocyst rate（%） |
| Accelerator group | 310 | 85.6 (272/318) | 85(231/272)a | 54.3(125/231)a |
| Positive controls | 320 | 91.9(294/320) | 79.6(234/294) | 26.1(61/234)b |
| Negative control group | 300 | 89.3(268/300) | 78.7(211/268) | 27.5(58/211)b |
Note：Same column letter is different represent differences have conspicuousness (P＜ 0.05), there was no significant difference for no letter expression (P＞ 0.05).
The Porcine Oocytes In Vitro culture of embodiment 3
1st, solution and nutrient solution are prepared
It is same as Example 1.
2nd, Porcine Oocytes In Vitro culture
（1）By forskolin（Forskolin）It is added separately in 0~22 h nutrient solutions, makes with core inhibitor Roscovitine
Final concentration of 3 μM of final concentration of 7 μM of Forskolin, Roscovitine；Porcine oocytes are added with above-mentioned
Cultivated in Forskolin and Roscovitine 0~22 h nutrient solutions after 24h, be transferred in 22~44 h nutrient solutions and continue to train
24h is supported, follow-up lonely female activation experiment is carried out.
（2）Control group is set, it is same as Example 1.
3rd, result
As a result as shown in table 5, find that pig ovum is female by investigating the form natural rate of interest, cleavage rates and blastocyst rate of porcine oocytes
Cell add 7 μM of Forskolin and 3 μM of Roscovitine nutrient solution in cultivate after cellular morphology natural rate of interest with
Control group is carried significantly without significant difference, but cleavage rates and blastocyst rate with control group significant difference, especially blastocyst rate compared with control group
It is high.
The developmental potentiality of porcine oocytes after Forskolin and 3 μM of Roscovitine processing of table 57 μM（It is lonely female sharp
It is living）
| Group | Oocyte number | Form natural rate of interest（%） | Cleavage rates（%） | Blastocyst rate（%） |
| Accelerator group | 315 | 86.3 (271/315) | 86.1(207/271)a | 51.2(106/207)a |
| Positive controls | 320 | 91.9(294/320) | 79.6(234/294) | 26.1(61/234)b |
| Negative control group | 300 | 89.3(268/300) | 78.7(211/268) | 27.5(58/211)b |
Note：Same column letter is different represent differences have conspicuousness (P＜ 0.05), there was no significant difference for no letter expression (P＞ 0.05)
The Porcine Oocytes In Vitro culture of embodiment 4
1st, solution and nutrient solution are prepared
It is same as Example 1.
2nd, Porcine Oocytes In Vitro culture
（1）By forskolin（Forskolin）It is added separately in 0~22 h nutrient solutions, makes with core inhibitor Roscovitine
Final concentration of 8 μM of final concentration of 2 μM of Forskolin, Roscovitine；Porcine oocytes are added with above-mentioned
Cultivated in Forskolin and Roscovitine 0~22 h nutrient solutions after 24h, be transferred in 22~44 h nutrient solutions and continue to train
24h is supported, follow-up lonely female activation experiment is carried out.
（2）Control group is set, it is same as Example 1.
3rd, result
As a result as shown in table 6, find that pig ovum is female by investigating the form natural rate of interest, cleavage rates and blastocyst rate of porcine oocytes
Cellular morphology natural rate of interest, ovum of the cell in the nutrient solution for adding 2 μM of Forskolin and 8 μM of Roscovitine after culture
Rate and blastocyst rate are split with control group without significant difference.
The developmental potentiality of porcine oocytes after Forskolin and 8 μM of Roscovitine processing of table 62 μM（It is lonely female sharp
It is living）
| Group | Oocyte number | Form natural rate of interest（%） | Cleavage rates（%） | Blastocyst rate（%） |
| Accelerator group | 320 | 86.4(276/320) | 81.1(223/276) | 30.3(67/223) |
| Positive controls | 320 | 91.9(294/320) | 79.6(234/294) | 26.1(61/234) |
| Negative control group | 300 | 89.3(268/300) | 78.7(211/268) | 27.5(58/211) |
Note：Same column letter is different represent differences have conspicuousness (P＜ 0.05), there was no significant difference for no letter expression (P＞ 0.05)
The Porcine Oocytes In Vitro culture of embodiment 5
1st, solution and nutrient solution are prepared
It is same as Example 1.
2nd, Porcine Oocytes In Vitro culture
（1）By forskolin（Forskolin）It is added separately in 0~22 h nutrient solutions, makes with core inhibitor Roscovitine
Final concentration of 2 μM of final concentration of 8 μM of Forskolin, Roscovitine；Porcine oocytes are added with above-mentioned
Cultivated in Forskolin and Roscovitine 0~22 h nutrient solutions after 24h, be transferred in 22~44 h nutrient solutions and continue to train
24h is supported, follow-up lonely female activation experiment is carried out.
（2）Control group is set, it is same as Example 1.
3rd, result
As a result as shown in table 7, find that pig ovum is female by investigating the form natural rate of interest, cleavage rates and blastocyst rate of porcine oocytes
Cellular morphology natural rate of interest, ovum of the cell in the nutrient solution for adding 8 μM of Forskolin and 2 μM of Roscovitine after culture
Rate and blastocyst rate are split with control group without significant difference.
The developmental potentiality of porcine oocytes after Forskolin and 2 μM of Roscovitine processing of table 78 μM（It is lonely female sharp
It is living）
| Group | Oocyte number | Form natural rate of interest（%） | Cleavage rates（%） | Blastocyst rate（%） |
| Accelerator group | 306 | 84.5 (258/306) | 76.3(196/258) | 28.4(56/196) |
| Positive controls | 320 | 91.9(294/320) | 79.6(234/294) | 26.1(61/234) |
| Negative control group | 300 | 89.3(268/300) | 78.7(211/268) | 27.5(58/211) |
Note：Same column letter is different represent differences have conspicuousness (P＜ 0.05), there was no significant difference for no letter expression (P＞ 0.05)
The Porcine Oocytes In Vitro culture of embodiment 6
1st, solution and nutrient solution are prepared
It is same as Example 1.
2nd, Porcine Oocytes In Vitro culture
By forskolin（Forskolin）It is added separately in 0~22 h nutrient solutions, makes with core inhibitor Roscovitine
Forskolin and Roscovitine final concentrations are 5 μM；By porcine oocytes it is above-mentioned added with Forskolin and
Roscovitine 0~22 h nutrient solutions, the time as described in table 8 is cultivated respectively.
3rd, result
As a result as shown in table 8, when porcine oocytes are in 0~22 h nutrient solutions added with Forskolin and Roscovitine
Cultivate after 22~24h, be transferred in 22~44 h nutrient solutions and continue to cultivate after 22~24h, its developmental potentiality is different compared with other
The developmental potentiality of incubation time is good.
Influence of the different incubation times of table 8 to porcine oocytes developmental potentiality
The Porcine Oocytes In Vitro culture of embodiment 7
1st, solution and nutrient solution are prepared
（1）Forskolin（Forskolin）：It is same as Example 1；
（2）Core inhibitor Roscovitine：It is same as Example 1；
（3）0~22 h nutrient solutions：Take 8 g TCM-199,4 g NaHCO3, 0.4 g D-Glucoses, 0.15 g Sodium Pyruvates,
0.05 g penicillin sodium salts, 0.07 g streptomycin sulfates, 1 g polyvinyl alcohol is dissolved in 1 L ddH2In O, after fully mixing, plus
Enter 0.7 μ g/mL LH, 0.3 μ g/mL FSH, 12 ng/mL EGF, 0.3 mM Cys, 12% PFF；It is fully mixed again
Even, suction filtration is degerming, is placed in CO2Incubator balance is stayed overnight.
（4）22~44 h nutrient solutions：Take 12 g TCM-199,1 g NaHCO3, 0.7 g D-Glucoses, 0.05 g acetone
Sour sodium, 0.1 g penicillin sodium salts, 0.03 g streptomycin sulfates, 4 g polyvinyl alcohol are dissolved in 1 L ddH2In O, fully mix
Afterwards, 9 ng/mL EGF and 8% PFF are added, suction filtration is degerming, is placed in CO2Incubator is balanced.
（5）Egg mother cell operates liquid：It is same as Example 1；
（6）Merge liquid：It is same as Example 1；
（7）Embryo medium：It is same as Example 1；
2nd, Porcine Oocytes In Vitro culture
By forskolin（Forskolin）It is added separately in 0~22 h nutrient solutions, makes with core inhibitor Roscovitine
Forskolin and Roscovitine final concentrations are 5 μM；By porcine oocytes it is above-mentioned added with Forskolin and
Cultivated in Roscovitine 0~22 h nutrient solutions after 24h, be transferred in 22~44 h nutrient solutions and continue to cultivate 24h, carry out
Follow-up lonely female activation experiment.
3rd, result
Porcine oocytes cultivated in the nutrient solution added with 5 μM of Forskolin and 5 μM of Roscovitine after cell
The porcine oocytes of form natural rate of interest, cleavage rates and blastocyst rate than being cultivated without accelerator are high.
The Porcine Oocytes In Vitro culture of embodiment 8
1st, solution and nutrient solution are prepared
（1）Forskolin（Forskolin）：It is same as Example 1；
（2）Core inhibitor Roscovitine：It is same as Example 1；
（3）0~22 h nutrient solutions：Take 12 g TCM-199,1 g NaHCO3, 0.7 g D-Glucoses, 0.05 g Sodium Pyruvates,
0.1 g penicillin sodium salts, 0.03 g streptomycin sulfates, 4 g polyvinyl alcohol are dissolved in 1 L ddH2In O, after fully mixing, add
0.3 μ g/mL LH, 0.7 μ g/mL FSH, 8 ng/mL EGF, 0.7 mM Cys, 8% PFF；Fully mix, take out again
Bacterium is filtered out, CO is placed in2Incubator balance is stayed overnight.
（4）22~44 h nutrient solutions：Take 8 g TCM-199,4 g NaHCO3, 0.4 g D-Glucoses, 0.15 g acetone
Sour sodium, 0.05 g penicillin sodium salts, 0.07 g streptomycin sulfates, 1 g polyvinyl alcohol is dissolved in 1 L ddH2It is fully mixed in O
After even, 11 ng/mL EGF and 12% PFF are added, suction filtration is degerming, is placed in CO2Incubator is balanced.
（5）Egg mother cell operates liquid：It is same as Example 1；
（6）Merge liquid：It is same as Example 1；
（7）Embryo medium：It is same as Example 1；
2nd, Porcine Oocytes In Vitro culture
By forskolin（Forskolin）It is added separately in 0~22 h nutrient solutions, makes with core inhibitor Roscovitine
Forskolin and Roscovitine final concentrations are 5 μM；By porcine oocytes it is above-mentioned added with Forskolin and
Cultivated in Roscovitine 0~22 h nutrient solutions after 24h, be transferred in 22~44 h nutrient solutions and continue to cultivate 24h, carry out
Follow-up lonely female activation experiment.
3rd, result
Porcine oocytes cultivated in the nutrient solution added with 5 μM of Forskolin and 5 μM of Roscovitine after cell
The porcine oocytes of form natural rate of interest, cleavage rates and blastocyst rate than being cultivated without accelerator are high.
The forskolin of the present invention（Forskolin）With core inhibitor Roscovitine, its optimum combined collocation is passed through
After our each single medicine, a variety of concentration of combination drug and addition addition incubation times are attempted, it is determined that the pig of in vitro culture
Egg mother cell cultivates 22~24 h in the nutrient solution after addition concentration is 5 μM of Forskolin and 5 μM of Roscovitine,
The developmental potentiality of the porcine oocytes of in vitro culture can be effectively improved.The porcine oocytes of in vitro culture are the weights of porcine clone embryos
Want raw material, therefore improve Porcine Oocytes In Vitro to be trained heat efficiency significant for scientific research and production practices.This
The promotion formulation that there is provided is invented through the experimental verification such as multiple Cell culture invitro and follow-up lonely female activation, cleavage rates, blastaea number and
Blastomere number, which is better than, is not added with group, and the pig ovum that such a inhibitor combination that has been unequivocally established can effectively improve in vitro culture is female
The developmental potentiality of cell.
Claims (10)
1. a kind of in-vitro maturity of porcine oocytes accelerator, it is characterised in that the accelerator by 3~7 μm of ol/L forskolins with
3~7 μm of ol/L Roscovitine compositions.
2. application of the accelerator described in claim 1 in in-vitro maturity of porcine oocytes nutrient solution is prepared.
3. a kind of in-vitro maturity of porcine oocytes nutrient solution, it is characterised in that the nutrient solution includes following components：Forskolin 3
~7 μm of ol/L, Roscovitine 3~7 μm of ol/L, TCM-199 8~12g/L, NaHCO3 1~4g/L, D-Glucose 0.4
~0.7 g/L, the g/L of Sodium Pyruvate 0.05~0.15, the g/L of penicillin sodium salt 0.05~0.1, streptomycin sulfate 0.03~
0.07 g/L, the g/L of polyvinyl alcohol 1~4, the μ g/mL of luteotropin 0.3~0.7, the μ g/mL of follicle-stimulating hormone 0.3~0.7, epidermis
The ng/mL of growth factor 8~12, the mmol/L of Cys 0.3~0.7, liquor folliculi 8~12%.
4. nutrient solution according to claim 3, it is characterised in that the nutrient solution includes following components：Forskolin 4~6
μm ol/L, Roscovitine 4~6 μm of ol/L, TCM-199 9~11 g/L, NaHCO32~3 g/L, D-Glucose 0.5
~0.6 g/L, 0.08~0.12g/L of Sodium Pyruvate, 0.06~0.09g/L of penicillin sodium salt, streptomycin sulfate 0.04~
0.06g/L, the g/L of polyvinyl alcohol 1~3, the μ g/mL of luteotropin 0.4~0.6, the μ g/mL of follicle-stimulating hormone 0.4~0.6, epidermal growth
The factor 9~11 ng/mL, 0.4~0.6mmol/L of Cys, liquor folliculi 9~11%.
5. nutrient solution according to claim 3, it is characterised in that the nutrient solution includes following components：The μ of forskolin 5
Mol/L, Roscovitine 5 μm of ol/L, TCM-199 9.87 g/L, NaHCO3 2.2 g/L, the g/ of D-Glucose 0.5496
L, the g/L of Sodium Pyruvate 0.1, the g/L of penicillin sodium salt 0.075, the g/L of streptomycin sulfate 0.05, the g/L of polyvinyl alcohol 1, promote yellow
The μ g/mL of voxel 0.5, follicle-stimulating hormone 0.5 μ g/mL, the ng/mL of EGF 10, the mmol/L of Cys 0.57, ovum
Steep liquid 10%.
6. any nutrient solution of claim 3~5 is improving in-vitro maturity of porcine oocytes quality and answering in developmental potentiality
With.
7. a kind of method for improving in-vitro maturity of porcine oocytes quality and developmental potentiality, it is characterised in that by claim 3~
5 any described nutrient solutions are transferred to nutrient solution as nutrient solution A after porcine oocytes are cultivated into 22~24h in nutrient solution A
Continue to cultivate 22~24h in B；
The nutrient solution B includes following components：TCM-199 8~12 g/L, NaHCO31~4 g/L, D-Glucose 0.4~0.7
G/L, the g/L of Sodium Pyruvate 0.05~0.15, the g/L of penicillin sodium salt 0.05~0.1, the g/ of streptomycin sulfate 0.03~0.07
L, the g/L of polyvinyl alcohol 1~4, the ng/mL of EGF 9~11, liquor folliculi 9~11%.
8. the method according to right will go 7, it is characterised in that the nutrient solution A or/and nutrient solution B need to put after preparing
In CO2Incubator balance is stayed overnight.
9. method according to claim 8, it is characterised in that the CO2Condition in incubator is 38.6 °C, 5%CO2,
Relative humidity 100%.
10. application of any described method of claim 7~9 in porcine clone embryos culture.
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| CN110577928A (en) * | 2019-10-25 | 2019-12-17 | 东北农业大学 | Oocyte in-vitro maturation culture solution and application thereof |
| CN110628709A (en) * | 2019-10-22 | 2019-12-31 | 吉林大学 | Culture solution and culture method for improving in-vitro maturation quality of porcine oocytes |
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