CN107916249A - Improve the nutrient solution and cultural method of the development quality of bovine somatic cells clone's embryo and embryo in vitro fertilization - Google Patents
Improve the nutrient solution and cultural method of the development quality of bovine somatic cells clone's embryo and embryo in vitro fertilization Download PDFInfo
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Abstract
The present invention provides a kind of nutrient solution and cultural method of the development quality for improving bovine somatic cells clone's embryo and embryo in vitro fertilization, nutrient solution includes the BME of volume fraction 2%, the glutamine of MEM, 1mmol/L of volume fraction 1%, the BSA of 8mg/mL, the gentamicin of 50 μ g/mL and the cephalosporin of 50 μ g/mL, ox embryo is cultivated in vitro to after 96h, adds the KSR of volume fraction 5%；Bovine somatic cells clone's embryo is carried out using above-mentioned nutrient solution and embryo in vitro culture in vitro fertilization, the results show blastocyst rate and quality are all remarkably higher than control group, can efficient acquisition bovine somatic cells clone embryos or IVF Embryos with the present invention.
Description
Technical field
The invention belongs to livestock embryo engineering field, and in particular to the training of a kind of bovine somatic cells clone's embryo and embryo in vitro fertilization
Nutrient solution and cultural method.
Background technology
Livestock embryo in vitro culture is a technology with huge research and commercial value, may be used on moving outside embryoid body
Plant, animal gene editor breeding and the amplification of excellent domestic animal kind etc..But so far the extracorporeal embryo development quality of ox relative to
The internal embryo of ox is not still high, and the blastaea for being mainly reflected in acquisition is few, poor morphology, the cell number of blastaea are few, apoptotic cell
Situations such as number is more, pregnancy rate is low, pregnant time increases, giant embryo occurs, and birth Sex Ratio is unbalance, therefore, significantly restrict
The extensive use of this technology.
Knockout serum replacement (KSR) are a kind of serum-free nutrient materials of principal component, in the mankind
During with Mouse Embryonic Stem Cell Culture, embryonic stem cell can be made to maintain kilter, and promote stem cell to be bred.
The content of the invention
It is an object of the invention to provide a kind of training for the development quality for improving bovine somatic cells clone's embryo and embryo in vitro fertilization
Nutrient solution and cultural method, it is possible to increase bovine somatic cells clone the developmental rate and development quality of embryo and ox embryo in vitro fertilization.
To reach above-mentioned purpose, present invention employs following technical scheme：
A kind of nutrient solution for the development quality for improving bovine somatic cells clone's embryo and embryo in vitro fertilization, the nutrient solution include
MSOFaa liquid, the mSOFaa liquid include SOF (synthesis Oviductal Fluid) basal mediums and additive, SOF basal mediums
Including the sodium lactate that volume fraction is 0.28~0.285% and the Sodium Pyruvate of 30~35mg/L, additive includes 4~8mg/
The BSA of mL.
Preferably, KCl, 0.162g of NaCl, 0.0534g of 0.6294g are specifically included per 100mL SOF basal mediums
KH2PO4, 0.0172g CaCl2·2H2O, the MgCl of 0.00996g2·6H2O, the NaHCO of 0.2106g3, 0.003~
The sodium lactate of the Sodium Pyruvate of 0.0035g and 28~28.5 μ L；The additive specifically includes BME (Basal Eagle
Medium), MEM (Minimum Essential Medium), glutamine, BSA (bovine serum albumin(BSA)), gentamicin and head
P0-357, wherein, the volume fraction that the volume fraction of BME is 2%, MEM is 1%, and the concentration of glutamine is 1mmol/L, BSA
Concentration be 4~8mg/mL, the concentration of gentamicin is 50 μ g/mL, and the concentration of cephalosporin is 50 μ g/mL.
A kind of cultural method for the development quality for improving bovine somatic cells clone's embryo and embryo in vitro fertilization, comprises the following steps：
By Niu Kelong reconstituted embryos, ox embryo in vitro fertilization or (embryonic development period can be with certain embryonic development period
For example, 2- cell stages are to any one embryonic development period between blastula stage) be placed in from the ox embryo obtained in vitro
Cultivated in above-mentioned mSOFaa liquid；Ox clone reconstituted embryo, ox embryo culture in vitro fertilization to 78~100h, alternatively, the ox
Embryo has been developed to after 2- cell stages, then adds serum substitute into mSOFaa liquid, then proceedes to cultivate,
The volume fraction of the serum substitute added in mSOFaa liquid is 1~10%.
Preferably, the serum substitute is selected from KSR (KnockOutTMSerum Replacement)。
Preferably, the cultural method specifically includes following steps：The above-mentioned mSOFaa liquid of 500 μ L is added to culture vessel
Liquid level is covered with paraffin oil after in (such as four orifice plates), 30~40 oxen are then cloned into reconstituted embryo or ox embryo in vitro fertilization is inserted
In the culture vessel, then in 38.5 DEG C, 5%CO2, 7%O2And cultivated under the conditions of saturated humidity, when cultivating to 96h
KSR is added into the culture vessel, the volume fraction of the KSR added in mSOFaa liquid is 5%, then proceedes to culture to the 8th
My god.
Ox clone's reconstituted embryo is by the way that ox donorcells is moved into non-nucleus egg mother cell and completes to merge and activates
Obtained from.
The ox embryo in vitro fertilization be by will be fertilized in Niu Jingzi and ox the ovum in vitro environment of manual control and
Arrive.
The present invention has the beneficial effect that：
The present invention proposes a kind of mSOFaa formula of liquid of improvement, it is by controlling sodium lactate, Sodium Pyruvate and BSA etc. to close
Key component, has more preferable culture effect, Ke Yiti compared to general commercial embryo medium (SOFaa, KSOMaa etc.)
The outer development quality of high bovine embryo.
Further, the present invention is (special by increasing the serum substitute that volume fraction is 1~10% in mSOFaa liquid
It is KSR), significantly improve development rate, quality of blastocysts and the blastomere number of ox embryo.
Further, KSR (is about exactly to be fertilized embryo or reconstituted embryo in culture bar after embryonic development to 2- cell stages
78~100h or so is developed under part) add in cultivating system, so as to play KSR as the positive of embryotrophy prime factor
Effect, efficient acquisition bovine somatic cells clone embryos or IVF Embryos.
Certain time of the cultural method of the present invention after Embryo Culture starts adds a certain amount of serum substitute
Into nutrient solution, not only efficient bovine somatic cells clone embryos or IVF Embryos can be obtained, but also can significantly improve
The development rate of somatic cell clone embryo and IVF Embryos, quality of blastocysts and blastomere number, can be greatly reduced preparation
The production cost of embryo.
Embodiment
Elaborate with reference to embodiment to the present invention.
(1) nutrient solution of the development quality of bovine somatic cells clone's embryo and embryo in vitro fertilization is improved
The nutrient solution that the present invention uses is the culture medium based on SOF liquid for mSOFaa liquid, also include BME, MEM,
Glutamine, BSA, gentamicin and cephalosporin, and also it is included in the component (KSR) that can be in addition added in incubation.
Developed in view of produced in vitro embryo (somatic cell clone embryo and IVF Embryos) in embryo's densification stage
Slow, it is few to result in the cell number of blastaea, and it is substantially weaker to increase associated signal paths for cell in growth course.And in embryo
After adding KSR this nutriment in vitro culture liquid, the developmental rate of the embryo of produced in vitro is improved, quality of blastocysts and
Blastaea number cell number is obviously improved.
(2) source of reagent or preparation
Trypsase, EDTA, gentamicin, cephalosporin, inorganic salts, paraffin oil (M8410) are sigma Products,
DMEM/F12 (Dulbecco's Modified Eagle Medium) fluid nutrient medium (Nutrient Mixture F-12),
Superfine hyclone (FBS) is Gibco Products, KnockOutTMSerum Replacement (KSR) are Thermo
Fisher products (article No. A3181501).KSOMaa is Millipore Products, SOFaa Caisson
Laboratories Products.It is sigma Products that other are not specified.
A, oocyte in vitro maturation culture solution
Maturation culture solution is that 2.2mg/mL NaHCO are added in TCM199 liquid3、0.075IU/mL HMG、1μg/mL 17β-
E2, 0.33mM Sodium Pyruvates, 2mM L-Glutamines and the gentamicin of 50 μ g/mL and the cephalosporin of 50 μ g/mL.
B, SOF solution and mSOFaa solution
It is prepared by SOF solution：By the KH of KCl, 0.162g of NaCl, 0.0534g of the 0.6294g of weighing2PO4、0.0172g
CaCl2·2H2O, the MgCl of 0.00996g2·6H2O, the NaHCO of 0.2106g3, 0.0033g Sodium Pyruvate, and 28.24 μ
The sodium lactate of L is added in 98mL water, 1mM NaOH tune pH to 7.2~7.4, deionized water constant volume to 100mL.
MSOFaa solution：The nutrient solution based on SOF solution, also includes BME, the volume fraction of volume fraction 2%
The glutamine of 1% MEM, 1mmol/L, the BSA of 8mg/mL, the cephalosporin of the gentamicin of 50 μ g/mL and 50 μ g/mL；
And the KSR (do not add KSR or add KSR after a certain period of time in culture) of volume fraction 0~10%
C, BO liquid
BO liquid is the nutrient solution based on SOF solution, also include the BSA of 48mg/mL, 50 μ g/mL gentamicin,
Cephalosporin, the caffeine of 0.1942mg/mL and the heparin sodium of 0.05mg/mL of 50 μ g/mL.
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.
(3) preparation method 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 gather and cloned from Yang Ling sections member from liquid nitrogen
Limited company cattle farm, in January, 2014 in March, 2016) thaw in 38 DEG C, add the DMEM/F12 cell culture fluids of 0.8mL
Centrifugation, abandons supernatant, adds cell culture fluid to be resuspended, take 3mL cell suspending liquids to be inoculated in the culture dish of diameter 6cm, be placed in CO2Training
Support in case and cultivated under the conditions of 38.5 DEG C.
When bovine fetal fibroblast reaches 80% and converges, nutrient solution is abandoned in suction, with no Ca2+、Mg2+PBS rinse it is thin
Born of the same parents, add trypsase and EDTA mixture slaking liquid, vitellophag.Cell is observed under inverted microscope, treats most cells
Bounce back, be rounded, space between cells expand when, with containing 10% hyclone DMEM/F12 cell culture fluids terminate digestion, use liquid relief
After device piping and druming, it is collected by centrifugation, suspends, by 1:3 ratio is inoculated in 24 orifice plates, is put into CO2Cultivated in incubator.Carry out body cell
Clone embryo to make a few days ago, nutrient solution is replaced with the DMEM/F12 containing 0.5% hyclone.
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), will
Ovary is placed in 20~25 DEG C of the physiological saline that vacuum flask includes penicillin and streptomycin, transports laboratory within 5h back.Ovary is transported back
Afterwards, the connective tissue, fat and the fallopian tubal of attachment of Ovarian surface are wiped out with sterilizing scissors, three are cleaned in sterile saline
It is secondary, the egg mother cell in Ovarian surface 2~8mm ovarian follicles is extracted with the 10mL syringes equipped with 12G syringe needles, is put into 6cm glass dishes
In, cumulus oocytes complesxes (cumulus-oocyte complexes, COCs) are collected under stereomicroscope.Collect
Cleaned three times in the PBS containing 10% hyclone afterwards.The normal COCs of oocyte morphology is selected to be trained for maturation in vitro
Support.
The COCs of selection is washed twice in maturation culture solution, is then moved into the 3cm plates equipped with 3mL maturation culture solutions
(being balanced one hour in incubator in advance), in 38.5 DEG C, 5%CO2, 20~22h is cultivated under the conditions of saturated humidity.Will culture maturation
COCs cleaned 3 times with PBS liquid, be put into containing 0.1% hyaluronidase without Ca2+、Mg2+1~2min is digested in PBS, is used in combination
1000mL liquid-transfering guns are blown and beaten repeatedly, to remove the cumulus cell of egg mother cell external diffusion.After piping and druming is clean, washed in PBS 3 times,
Then under entity stereomicroscope, egg mother cell is stirred with foreign body needle, the egg mother cell for selecting polar body is stand-by.
C. the structure of bovine somatic cells clone embryo
Stoning：
Before stoning egg mother cell first containing 7.5 μ g/mL cytochalasin Bs, 10%FBS PBS in be incubated 15min.Aobvious
Under Micromanipulators, first polar body and surrounding part ooecium matter are drawn with the stoning pipe that internal diameter is 20 μm.
Note core and electro' asion：
When noting core, a diameter of 15~20 μm of bovine fetal fibroblast injection non-nucleus egg mother cell oolemma is selected
Under.Reconstituted embryo after note core is merged using the method for microelectrode.Before fusion, reconstituted embryo pre-equilibration in electro' asion liquid
3min, electro' asion carry out under 150 times of micromanipulation instrument.Two " Z " font microelectrode top end diameters for mixing operation
For 15 μm, rear end is connected on micromanipulation instrument, the donor, recipient cell after birth contact surface and the line of two electrodes of reconstituted embryo is hung down
Directly, fusion parameters：Voltage 32V, 20 μ s of pulse duration, 2 subpulse interval 10ms.1h observes fusion feelings under the microscope after fusion
Condition.
D, the activation of bovine somatic cells clone reconstituted embryo
1~2h after electro' asion, the bovine somatic cells clone's reconstituted embryo for selecting successful fusion pass through ionomycin
(Ionomycin) activation for combining 6-DMAP is handled into line activating：It is molten in the mSOFaa containing 2~5 μm of ol/L ionomycins first
Incubation at room temperature 4min in liquid (being free of KSR), then in the mSOFaa solution (being free of KSR) of the 6-DMAP containing 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
Treatment group：Four orifice plates add the mSOFaa solution without KSR of 500 μ L per hole, are also covered with mSOFaa solution
500 μ L paraffin oils, paraffin oil is in advance in CO2Balance at least 2h in incubator, bovine somatic cells clone reconstituted embryo at line activating
After reason, it is transferred in above-mentioned four orifice plate.35~40 pieces of bovine somatic cells clone's reconstituted embryos are placed per hole, in 38.5 DEG C, 5%CO2、
7%O2And cultivated under the conditions of saturated humidity, add KSR after starting the 96h of culture.Correspondingly, control group is trained with mSOFaa solution
Support (not adding KSR), observe and record developmental state.
Compared with control group, the KSR processing of 5% volume fraction is added when 96h, bovine somatic cells clone can be significantly improved
The developmental rate and quality of blastocysts of embryo, can efficient produced in vitro bovine somatic cells clone embryos.It is embodied in：
1) developmental rate and quality of bovine somatic cells clone embryos
As can be seen from Table 1, somatic cell clone embryo is cultivated by adding the mSOFaa of 5% volume fraction KSR, can shown
Write the developmental rate and quality for improving blastaea.
The KSR of 1. different volumes fraction of table is developed by bovine somatic cells clone embryos and the influence of quality of blastocysts
Number in bracket is developmental rate, the number before bracket for statistics total number of embryos；2- cell stages embryo and the hair of blastaea
Rate statistics is educated respectively in restructuring embryo culture 72h and 192h (the 8th day)；In same row, the Superscript letters of data are different to represent poor
Different significantly (P<0.05, Chi-square Test)；Blastaea(1-3)Refer to the 8th day total blastaea number obtained and total blastocyst rate；Blastaea (1-2)
Then refer to reach International Embryo transplanting association's grade 1 and the high quality of 2 morphologic criterias, recommend to carry out further transplanting culture
Blastaea
2) result of the test of the cell number of bovine somatic cells cloned blastocysts
As can be seen from Table 2, after 5%KSR processing, the total number of cells of bovine somatic cells clone embryos (blastaea), inner cell mass
(ICM) cell number significantly improves.
The cell number analysis of 2. bovine somatic cells cloned blastocysts of table
| Blastaea number | Blastomere sum | TE cell numbers | ICM cell numbers | |
| Control group | 37 | 95.62±5.32b | 69.36±3.56 | 25.97±3.47b |
| Treatment group | 37 | 110.45±7.46a | 76.32±6.72 | 35.14±4.26a |
The data in same row, Superscript letters are different to represent significant difference (P<0.05)
3) with it has been reported that cultivating system compared with (add 5%KSR after when same culture 96 is small), test result indicates that
(for example, table 3 and table 4), mSOFaa solution proposed by the present invention, regardless of whether adding KSR, cultivates developmental rate and the development of embryo
Quality all significantly improves.
Table 3.5%KSR is to the development of bovine somatic cells clone embryos and quality of blastocysts under the different ox Embryo Culture systems
Influence
The data in same row, Superscript letters are different to represent significant difference (P<0.05)
Influences of the table 4.5%KSR to the bovine somatic cells cloned blastocysts cell number under different ox Embryo Culture systems
| Blastaea number | Blastomere sum | TE cell numbers | ICM cell numbers | |
| mSOFaa | 31 | 110.01±8.25a | 76.32±5.54a | 35.14±4.26a |
| KSOMaa | 22 | 98.75±5.32b | 71.36±6.68b | 26.97±3.47b |
| SOFaa | 27 | 105.23±9.78b | 74.01±7.69a | 31.42±3.99a |
The data in same row, Superscript letters are different to represent significant difference (P<0.05)
(4) preparation method 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 (acquisition time in January, 2014 in March, 2016), will
Ovary is placed in 20~25 DEG C of the physiological saline that vacuum flask includes penicillin and streptomycin, transports laboratory within 5h back.Ovary is transported back
Afterwards, the connective tissue, fat and the fallopian tubal of attachment of Ovarian surface are wiped out with sterilizing scissors, three are cleaned in sterile saline
It is secondary, the egg mother cell in Ovarian surface 2~8mm ovarian follicles is extracted with the 10mL syringes equipped with 12G syringe needles, is put into 6cm glass dishes
In under stereomicroscope collect cumulus oocytes complesxes (cumulus-oocyte complexes, COCs).After collection
Cleaned three times in the PBS containing 10% hyclone.The selection normal COCs of oocyte morphology is used for In-vitro maturation.
The COCs of selection is washed twice in maturation culture solution, is then moved into the 3cm plates equipped with 3mL maturation culture solutions
(being balanced one hour in incubator in advance), in 38.5 DEG C, 5%CO2, 20~22h is cultivated under the conditions of saturated humidity.Will culture maturation
COCs blown and beaten repeatedly with 1000mL liquid-transfering guns, to remove the cumulus cell of egg mother cell external diffusion.Piping and druming to around COCs only
During four to five layers of cumulus cell of remaining tight, washed 2 times in by sperm (BO liquid), the free ovarian cumulus that wash clean is blown down is thin
Born of the same parents, then the egg mother cell of four to five layers of cumulus cell of tight is moved to be placed into balance more than 2h in incubator in advance
It is fertilized in drop.
B, the defrosting of sperm, purifies, fertilization
Essence (Xi'an milk cow center, in October, 2013 collection) will be frozen after liquid nitrogen container taking-up, be positioned over 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 levels, then the sperm after defrosting is placed on 45%Percoll liquid levels,
1500rpm centrifuges 20min, about 200 μ L sperm of careful suction foot, and adds containing egg mother cell in by sperm (BO liquid),
Putting back in incubator makes smart ovum be combined 16~22h.
C, the culture of fertilized embryo
Treatment group：After fertilization, remaining cumulus cell, sperm around ovum are blown away with mouth suction pipe or pipette tips.Such as
Fruit can not remove totally, can be removed in hyaluronidase solution.In the mSOFaa solution without KSR (on liquid level also
Covered with 500 μ L paraffin oils, and in advance in CO2At least 2h is balanced in incubator) in culture 72h (40 pieces of embryos in vitro fertilization), choose
Go out the fertilization embryo of the non-spilting of an egg, put back to incubator and continue to cultivate, KSR is added after starting the 96h of culture, continue culture to the 8th day
(192h).Correspondingly, control group with mSOFaa hydroponics (not adding KSR), is observed and records developmental state.
Compared with control group, the KSR processing of 5% volume fraction is added, the development of IVF Embryos can be significantly improved
Rate and quality of blastocysts, can efficient produced in vitro ox IVF Embryos.It is embodied in：
1) developmental rate and quality of ox IVF Embryos
As can be seen from Table 5, IVF Embryos are cultivated by adding the mSOFaa of 5% volume fraction KSR, can be notable
Improve the developmental rate and quality of blastaea.
The KSR of 5. different volumes fraction of table is to fertilized embryo development and the influence of quality of blastocysts outside ox
Number in bracket is developmental rate, the number before bracket for statistics total number of embryos；2- cell stages embryo and the hair of blastaea
Educate rate statistics difference fertilization embryo culture 72h and 192h (the 8th day) in vitro；In same row, the Superscript letters difference table of data
Show significant difference (P<0.05, Chi-square Test)；Blastaea(1-3)Refer to the 8th day total blastaea number obtained and total blastocyst rate；Capsule
Embryo(1-2)Then refer to reach International Embryo transplanting association's grade 1 and the high quality of 2 morphologic criterias, recommendation is further transplanted
The blastaea of culture
2) result of the test of the cell number of bovine IVF embryo
As can be seen from Table 6, add mSOFaa start culture 96 it is small when add 5%KSR processing, ox embryo in vitro fertilization
The total number of cells of tire (blastaea) significantly improves.
The cell number analysis of 6. bovine IVF embryo of table
| Blastaea number | Blastomere sum | TE cell numbers | ICM cell numbers | |
| Control group | 49 | 117.58±6.39b | 77.79±4.56 | 39.47±6.01 |
| Treatment group | 50 | 127.81±3.25a | 83.32±5.02 | 44.14±8.35 |
The data in same row, Superscript letters are different to represent significant difference (P<0.05)
3) with it has been reported that cultivating system compared with (add 5%KSR after when same culture 96 is small), test result indicates that
(for example, table 7 and table 8), mSOFaa solution proposed by the present invention, regardless of whether adding KSR, cultivates the developmental rate and quality of embryo
All significantly improve.
Table 7.5%KSR is to IVF Embryos development and the influence of quality of blastocysts under different ox Embryo Culture systems
The data in same row, Superscript letters are different to represent significant difference (P<0.05)
Influences of the table 8.5%KSR to the bovine IVF embryo cell number under different ox Embryo Culture systems
| Blastaea number | Blastomere sum | TE cell numbers | ICM cell numbers | |
| mSOFaa | 37 | 129.70±6.59a | 78.47±4.68a | 51.58±3.19a |
| KSOMaa | 23 | 101.75±8.00b | 73.31±7.99b | 27.68±4.63b |
| SOFaa | 33 | 115.23±7.18b | 74.01±7.10a | 41.22±2.83a |
The data in same row, Superscript letters are different to represent significant difference (P<0.05) .
Claims (10)
- A kind of 1. nutrient solution for the development quality for improving bovine somatic cells clone's embryo and embryo in vitro fertilization, it is characterised in that：The culture Liquid includes mSOFaa liquid, and the mSOFaa liquid includes SOF basal mediums and additive, and SOF basal mediums include volume Fraction is 0.28~0.285% sodium lactate and the Sodium Pyruvate of 30~35mg/L, and additive includes the BSA of 4~8mg/mL.
- 2. a kind of nutrient solution for the development quality for improving bovine somatic cells clone's embryo and embryo in vitro fertilization according to claim 1, It is characterized in that：Include the KH of KCl, 0.162g of NaCl, 0.0534g of 0.6294g per 100mL SOF basal mediums2PO4、 The CaCl of 0.0172g2·2H2O, the MgCl of 0.00996g2·6H2O, the NaHCO of 0.2106g3, 0.003~0.0035g acetone The sodium lactate of sour sodium and 28~28.5 μ L；The additive includes BME, MEM, glutamine, BSA, gentamicin and cephalo Mycin, wherein, the volume fraction that the volume fraction of BME is 2%, MEM is 1%, and the concentration of glutamine is 1mmol/L, BSA's Concentration is 4~8mg/mL, and the concentration of gentamicin is 50 μ g/mL, and the concentration of cephalosporin is 50 μ g/mL.
- A kind of 3. culture of development quality for improving bovine somatic cells clone's embryo and embryo in vitro fertilization according to claim 1 or claim 2 Liquid, it is characterised in that：The additive further includes the serum substitute that volume fraction is 1~10%.
- 4. a kind of nutrient solution for the development quality for improving bovine somatic cells clone's embryo and embryo in vitro fertilization according to claim 3, It is characterized in that：The serum substitute is selected from KSR.
- 5. a kind of nutrient solution for the development quality for improving bovine somatic cells clone's embryo and embryo in vitro fertilization according to claim 3, It is characterized in that：The serum substitute is added in cultivating system after Embryo Culture to 2- cell stages.
- A kind of 6. cultural method for the development quality for improving bovine somatic cells clone's embryo and embryo in vitro fertilization, it is characterised in that：The training Foster method comprises the following steps：By Niu Kelong reconstituted embryos, ox embryo in vitro fertilization or being put from the ox embryo obtained in vitro in certain embryonic development period Cultivated in mSOFaa liquid；Ox clone reconstituted embryo, ox embryo culture in vitro fertilization to 78~100h, alternatively, the ox embryo Tire, which has been developed in the backward mSOFaa liquid of 2- cell stages, adds serum substitute, then proceedes to cultivate, in mSOFaa liquid The volume fraction of the serum substitute of addition is 1~10%.
- 7. according to the method described in claim 6, it is characterized in that：The mSOFaa liquid includes SOF basal mediums and adds Adding thing, SOF basal mediums include the sodium lactate that volume fraction is 0.28~0.285% and the Sodium Pyruvate of 30~35mg/L, Additive includes the BSA of 4~8mg/mL.
- 8. according to the method described in claim 6, it is characterized in that：Include 0.6294g's per 100mL SOF basal mediums The KH of KCl, 0.162g of NaCl, 0.0534g2PO4, 0.0172g CaCl2·2H2O, the MgCl of 0.00996g2·6H2O、 The NaHCO of 0.2106g3, the Sodium Pyruvate of 0.003~0.0035g and the sodium lactate of 28~28.5 μ L；The additive includes BME, MEM, glutamine, BSA, gentamicin and cephalosporin, wherein, the volume fraction of BME is the volume fraction of 2%, MEM For 1%, the concentration of glutamine is 1mmol/L, and the concentration of BSA is 4~8mg/mL, and the concentration of gentamicin is 50 μ g/mL, head The concentration of p0-357 is 50 μ g/mL.
- 9. according to the method described in claim 6, it is characterized in that：The serum substitute is selected from KSR.
- 10. according to the method described in claim 6, it is characterized in that：The cultural method specifically includes following steps：By 500 μ L mSOFaa liquid is added into culture vessel, and 30~40 oxen then are cloned reconstituted embryo or ox embryo in vitro fertilization inserts the culture In container, then in 38.5 DEG C, 5%CO2, 7%O2And cultivated under the conditions of saturated humidity, cultivate to during 96h to described KSR is added in culture vessel, the volume fraction of the KSR added in mSOFaa liquid is 5%, then proceedes to culture to the 8th day.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628382A (en) * | 2019-01-18 | 2019-04-16 | 西北农林科技大学 | A kind of culture solution and cultural method of the development quality improving ox embryo in vitro fertilization |
| CN111593020A (en) * | 2020-06-17 | 2020-08-28 | 草原和牛投资有限公司 | Culture solution and culture method for bovine in-vitro fertilized embryo or bovine cloned embryo |
| CN111718892A (en) * | 2020-06-29 | 2020-09-29 | 内蒙古大学 | Culture solution and method for improving culture efficiency and quality of bovine in vitro embryos |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4348458B2 (en) * | 2004-03-17 | 2009-10-21 | 有限会社山口ティー・エル・オー | Serum-free early embryo culture |
| CN102618496A (en) * | 2012-03-23 | 2012-08-01 | 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心 | Method for enhancing ectogenesis of sheep oocyte |
| CN102703377A (en) * | 2012-06-08 | 2012-10-03 | 中国农业大学 | Method for improving blastocyst rate of in-vitro embryos of animals |
-
2017
- 2017-11-13 CN CN201711117369.0A patent/CN107916249A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4348458B2 (en) * | 2004-03-17 | 2009-10-21 | 有限会社山口ティー・エル・オー | Serum-free early embryo culture |
| CN102618496A (en) * | 2012-03-23 | 2012-08-01 | 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心 | Method for enhancing ectogenesis of sheep oocyte |
| CN102703377A (en) * | 2012-06-08 | 2012-10-03 | 中国农业大学 | Method for improving blastocyst rate of in-vitro embryos of animals |
Non-Patent Citations (3)
| Title |
|---|
| 刘东军 等: "碳水化合物对牛体外受精胚胎体外发育的影响", 《中国实验动物学报》 * |
| 张林 等: "牛-牛及山羊-牛克隆胚胎体外培养条件的优化", 《生物工程学报》 * |
| 赵晓娥 等: "牛性控精子体外受精及受精卵的体外培养", 《农业生物技术学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628382A (en) * | 2019-01-18 | 2019-04-16 | 西北农林科技大学 | A kind of culture solution and cultural method of the development quality improving ox embryo in vitro fertilization |
| CN111593020A (en) * | 2020-06-17 | 2020-08-28 | 草原和牛投资有限公司 | Culture solution and culture method for bovine in-vitro fertilized embryo or bovine cloned embryo |
| CN111718892A (en) * | 2020-06-29 | 2020-09-29 | 内蒙古大学 | Culture solution and method for improving culture efficiency and quality of bovine in vitro embryos |
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