CN109628382B - A kind of culture solution and cultural method of the development quality improving ox embryo in vitro fertilization - Google Patents
A kind of culture solution and cultural method of the development quality improving ox embryo in vitro fertilization Download PDFInfo
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Abstract
It include following additive: Insulin-Transferrin-selenium additive, beta -mercaptoethanol, hypotaurine, 1- oleoyl-sn- glycerol-3-phosphate sodium salt and rhodioside in culture solution the invention discloses a kind of culture solution of development quality for improving ox embryo in vitro fertilization.And disclose the cultural method that the development quality of ox embryo in vitro fertilization is improved using the culture solution.The developmental rate and development quality of ox IVF Embryos are effectively improved using culture solution of the present invention and cultural method.
Description
Technical field
The present invention relates to livestock embryo engineering field, more particularly to a kind of development for improving ox embryo in vitro fertilization
The culture solution and cultural method of quality.
Background technique
Livestock embryo in vitro culture is the technology with potential researching value and commercial value, be can be widely applied to
Transplanting, animal gene editor breeding and the amplification of excellent domestic animal kind etc. outside embryoid body.But the embryo that in vitro culture obtains so far
Tire often not as good as the embryo of normal development in vivo in quality and quantity, develop in embryo's densification stage by IVF Embryos
It is slow, cause embryo's cleavage rates to be gradually reduced, the cell number for forming blastaea is few, and then damages embryo quality, influences early embryo
Tire ectogenesis significantly restricts the extensive use of this technology.
Therefore, how to improve ox embryonic development rate in vitro fertilization and development quality is asking for those skilled in the art's urgent need to resolve
Topic.
Summary of the invention
In view of this, ox embryonic development in vitro fertilization can be effectively improved the present invention provides a kind of culture solution and cultural method
Rate and development quality.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of culture solution for the development quality improving ox embryo in vitro fertilization, includes following additive: insulin-in culture solution
Transferrins-selenium additive, beta -mercaptoethanol, hypotaurine, 1- oleoyl-sn- glycerol-3-phosphate sodium salt and rhodioside.
Insulin-Transferrin-selenium additive ITS-G can preferably maintain the Motility and proliferation of cell；β-mercapto
Base ethyl alcohol can promote cell Proliferation, slow down cell ageing rate, conducive to the growth of cell；Hypotaurine can neutralize cytotoxin second
The final substance of aldehyde-peroxidating cascade reaction, to limit the illeffects of ROS；1- oleoyl-sn- glycerol-3-phosphate sodium energy
Enough promote cell Proliferation；Rhodioside, which has, improves cell viability, improves cellular morphology, reduces karyon agglutination, anti-cell oxidation
Stress protective effect.
Five kinds of additives are added in culture solution, can effectively reduce the oxidative damage of ox embryo in vitro fertilization, and it is external to improve ox
Embryonic development rate of being fertilized and development quality.
Preferably, Insulin-Transferrin-selenium additive volume fraction is 0.1-2%, and beta -mercaptoethanol concentration is 10-
100 μM, hypotaurine concentration is 1-10mM, and 1- oleoyl-SN- glycerol-3-phosphate sodium salt concentration is 0.1-1 μ g/mL, root of kirilow rhodiola
Glycosides concentration is 0.01-0.2mM.
Preferably, culture solution further includes SOF basal medium, essential amino acid, nonessential amino acid and no fatty acids ox
Seralbumin EFAF-BSA.
Preferably, it is necessary to which amino acid volume fraction is 0.1-2%, and nonessential amino acid volume fraction is 0.1-2%, no rouge
Fat acid bovine serum albumin(BSA) concentration is 1-10mg/mL.
It can further improve ox embryo by control essential amino acid, nonessential amino acid and no fatty acids bovine serum albumin(BSA)
Tire ectogenesis quality.
Preferably, every 100mL SOF basal medium includes 26.168mg CaCl2·2H2O、9.999mg
C6H5Na3O7·2H2O, 2.923mg glutamine, 37.218mg MgSO4·7H2O, 49.904mg inositol, 1.000mg it is phenol red,
53.378mg KCl、16.195mg KH2PO4, 4.402mg Sodium Pyruvate, 210.025mg NaHCO3, 629.399mg NaCl and
0.0757mg sodium lactate.
It preferably, further include 12 μ g/mL of 12 μ g/mL of gentamicin and cephalosporin in culture solution.
A kind of cultural method for the development quality improving ox embryo in vitro fertilization, is placed in above-mentioned culture solution for ox embryo in vitro fertilization
In cultivated.
Preferably, culture solution is preheated at least 2 hours, training then is added in the embryo in vitro fertilization of ox sperm ovum binding 16-22h
In nutrient solution, CO2It is cultivated 7-8 days in incubator.
Insulin-Transferrin-selenium additive, beta -mercaptoethanol, hypotaurine, 1- oleoyl-sn- glycerol-3-phosphate
Sodium salt and rhodioside before embryonic development (ox sperm ovum binding 16-22h) are added in cultivating system, can give full play to it
As the positive effect of Embryo Culture environmental effectors, ox IVF Embryos are expeditiously obtained, it is external to significantly improve ox
Fertilized embryo developmental rate, blastaea number and blastomere number, are greatly reduced the production cost for preparing embryo.
Preferably, Insulin-Transferrin-selenium additive, beta -mercaptoethanol, hypotaurine, 1- oleoyl-sn- glycerol-
3- sodium ascorbyl phosphate and rhodioside make an addition in culture solution in preheating.
Preferably, ox embryo in vitro fertilization is in 38.5 DEG C, 5%CO2, cultivate under saturated humidity.
It can be seen via above technical scheme that compared with prior art, turning iron added with insulin-in culture solution of the present invention
Albumen-selenium additive, beta -mercaptoethanol, hypotaurine, 1- oleoyl-sn- glycerol-3-phosphate sodium salt and rhodioside, can have
Effect improves the developmental rate and development quality of ox IVF Embryos.
Specific embodiment
The following is a clear and complete description of the technical scheme in the embodiments of the invention, it is clear that described embodiment
Only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1
(1) source of reagent and preparation
Trypsase, EDTA, gentamicin, cephalosporin, inorganic salts, paraffin oil (M8410) are sigma Products；
The M199 basic culture solution of no Hepes is Thermo Products；
Superfine fetal calf serum (FBS) is Gibco Products；
Essential amino acid is Thermo Fisher Products (article No. 1831512)；
Nonessential amino acid is Thermo Fisher Products (article No. 1958909)；
No fatty acids bovine serum albumin(BSA) EFAF-BSA is sigma Products (article No. A6003-5G)；
Insulin-Transferrin-selenium additive ITS-G is Thermo Fisher Products (article No. 1865342, pancreas islet
Plain 1g/L, transferrins 0.55g/L, selenium 0.00067g/L)；
Beta -mercaptoethanol is Thermo Fisher Products (article No. 1922445)；
Hypotaurine is sigma Products (article No. H1384)；
1- oleoyl-sn- glycerol-3-phosphate sodium is Enzo Life Science Products (article No. LP100-0025)；
Rhodioside is sigma Products (article No. SMB00072-1MG)；
SOF basal medium is voluntarily to configure；
It is sigma Products that other are not specified.
A. oocyte in vitro maturation culture solution
10% (v/v) fetal calf serum, 1% (v/v) ITS-G, 0.075IU/ are added in the M199 basic culture solution of no Hepes
ML HMG, 1 μ g/mL, 17 beta estradiol, 10ng/mL EGF, 12ng/mLbFGF and 50ng/mL CXCL12.
B.SOF solution, SOFaa solution, mSOFaa solution
SOF solution: 26.168mg CaCl2·2H2O、9.999mg C6H5Na3O7·2H2O, 2.923mg glutamine,
37.218mg MgSO4·7H2O, 49.904mg inositol, 1.000mg be phenol red, 53.378mg KCl, 16.195mg KH2PO4、
4.402mg Sodium Pyruvate, 210.025mg NaHCO3, 629.399mg NaCl, 0.0757mg sodium lactate, deionized water constant volume arrives
100mL, 1mM NaOH tune pH to 7.2-7.4.
SOFaa solution: using SOF solution as basic culture solution, essential amino acid, the volume point of volume fraction 1% are added
The nonessential amino acid of number 1%, 6mg/mL no fatty acids bovine serum albumin(BSA), 12g/mL gentamicin and 12 μ g/mL cephalos are mould
Element.
MSOFaa solution: using SOFaa solution as basic culture solution, addition volume fraction is 1% when preheating ITS-G,
55 μM of beta -mercaptoethanols, 2.5mM hypotaurine, 0.4 μ g/mL 1- oleoyl-SN- glycerol-3-phosphate sodium salt and the red scape of 0.1mM
Its glycosides.
C.BO liquid
BO liquid is using the SOF solution without inositol as basic culture solution, also includes 48mg/mL no fatty acids cow's serum
Albumin, 12 μ g/mL gentamicins, 12 μ g/mL cephalosporins, 0.1942mg/mL caffeine and 0.05mg/mL heparin sodium.
(2) 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, 2017 in December, 2017),
Ovary is placed in vacuum flask to include in 20-25 DEG C of physiological saline of 200U/ml penicillin, 0.2mg/ml streptomysin, within 5h
Transport laboratory back.After ovary is transported back, the connective tissue, fat and the fallopian tubal of attachment of Ovarian surface, In are wiped out with sterilizing scissors
It is cleaned in sterile saline three times, it is female to extract the ovum in Ovarian surface 2-8mm ovarian follicle with the 10mL syringe equipped with 12G syringe needle
Cell is put into 6cm glass dish, and cumulus oocytes complesxes (cumulus-oocyte is collected under stereomicroscope
complexes,COCs).It is cleaned three times in the PBS containing 10% fetal calf serum after collection.Select oocyte morphology normal
COCs is used for In-vitro maturation (being denoted as total oocyte number).
The COCs of selection is washed twice in oocyte in vitro maturation culture solution, is then moved into equipped with 3mL egg mother cell
In the 3cm plate of In-vitro maturation liquid (being balanced one hour in 38.5 DEG C of incubators in advance), in 38.5 DEG C, 5%CO2, saturation
20-22h is cultivated under damp condition.The mature COCs of culture is blown and beaten repeatedly with 1000mL liquid-transfering gun, to remove outside egg mother cell
The cumulus cell of diffusion.Piping and druming to around COCs only four to five layers of cumulus cell of remaining tight when, by sperm (BO
Liquid) in wash 2 times, the free cumulus cell that wash clean is blown down, then by the egg mother cell of four to five layers of cumulus cell of tight
It moves in fertilization drop (being placed into balance 2h or more in 38.5 DEG C of incubators in advance).
B. defrosting, purifying, the fertilization of sperm
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, 2mL45%Percoll
Liquid is carefully added on 90%Percoll liquid level, then the sperm after defrosting is placed on 45%Percoll liquid level, 1500rpm centrifugation
20min, about 200 μ L sperm of careful suction foot are added to containing egg mother cell and, by sperm (BO liquid), put back in incubator
Smart ovum is set to be combined 20h.
C. the culture of fertilized embryo
Processing group: adding mSOFaa solution in four orifice plates, and every 500 μ L of hole covers 500 μ L paraffin oils on liquid level, in advance
In 38.5 DEG C, 5%CO2, balance at least 2h in saturated humidity incubator.
Blow away remaining cumulus cell and sperm around ovum with mouth suction pipe or pipette tips after fertilization, obtain assume by
The ox embryo in vitro fertilization of essence, is placed in the mSOFaa solution of four orifice plates, 40 pieces or so of every hole, 38.5 DEG C, 5%CO2, saturated humidity
72h is cultivated in incubator, chooses the fertilization embryo of the non-spilting of an egg, is put back to incubator and is continued culture to the 8th day (192h).
The SOFaa hydroponics of control group 1.
Culture solution used in control group 2 adds 3mM glutathione on the basis of SOFaa solution.
Culture solution used in control group 3 only adds 55 μM of beta -mercaptoethanols on the basis of SOFaa solution.
Each group developmental state is observed and recorded, test result is as shown in table 1.
Table 1
A, b subscript difference indicates significant, and conspicuousness is detected using Chi-square Test；AB grades of blastaeas are portable blastaea, C grades
Not recommend to transplant blastaea, blastaea ABC classification is to transplant the quality of blastocysts standards of grading of association according to International Embryo to evaluate.It is right
The above description of the disclosed embodiments, enables those skilled in the art to implement or use the present invention.To these implementations
A variety of modifications of example will be readily apparent to those skilled in the art, and the general principles defined herein can
Without departing from the spirit or scope of the present invention, to realize in other embodiments.Therefore, the present invention will not be limited
It is formed on the embodiments shown herein, and is to fit to consistent with the principles and novel features disclosed in this article widest
Range.
Claims (8)
1. a kind of culture solution for the development quality for improving ox embryo in vitro fertilization, which is characterized in that the culture solution includes the basis SOF
Culture medium, essential amino acid, nonessential amino acid and no fatty acids bovine serum albumin(BSA)；It further include following add in the culture solution
Add agent: Insulin-Transferrin-selenium additive, beta -mercaptoethanol, hypotaurine, 1- oleoyl-sn- glycerol-3-phosphate sodium salt
And rhodioside；Insulin-Transferrin-selenium additive volume fraction is 0.1-2%, and beta -mercaptoethanol concentration is 10-100 μM,
Hypotaurine concentration is 1-10mM, and 1- oleoyl-SN- glycerol-3-phosphate sodium salt concentration is 0.1-1 μ g/mL, rhodioside concentration
For 0.01-0.2mM.
2. a kind of culture solution of development quality for improving ox embryo in vitro fertilization according to claim 1, which is characterized in that must
Needing amino acid volume fraction is 0.1-2%, and nonessential amino acid volume fraction is 0.1-2%, and no fatty acids bovine serum albumin(BSA) is dense
Degree is 1-10mg/mL.
3. a kind of culture solution of development quality for improving ox embryo in vitro fertilization according to claim 2, which is characterized in that every
100mL SOF basal medium includes 26.168mg CaCl2•2H2O、9.999mg C6H5Na3O7•2H2O, 2.923mg paddy ammonia
Amide, 37.218mg MgSO4•7H2O, 49.904mg inositol, 1.000mg be phenol red, 53.378mg KCl, 16.195mg
KH2PO4, 4.402mg Sodium Pyruvate, 210.025mg NaHCO3, 629.399mg NaCl and 0.0757mg sodium lactate.
4. a kind of culture solution of development quality for improving ox embryo in vitro fertilization according to claim 3, which is characterized in that institute
Stating in culture solution further includes 12 μ g/mL of 12 μ g/mL of gentamicin and cephalosporin.
5. a kind of cultural method for the development quality for improving ox embryo in vitro fertilization, which is characterized in that ox embryo in vitro fertilization to be placed in
It is cultivated in culture solution of any of claims 1-4.
6. a kind of cultural method of development quality for improving ox embryo in vitro fertilization according to claim 5, which is characterized in that
Culture solution is preheated at least 2 hours, then the embryo in vitro fertilization of ox sperm ovum binding 16-22h is added in culture solution, CO2Culture
It is cultivated 7-8 days in case.
7. a kind of cultural method of development quality for improving ox embryo in vitro fertilization according to claim 6, which is characterized in that
Insulin-Transferrin-selenium additive, beta -mercaptoethanol, hypotaurine, 1- oleoyl-sn- glycerol-3-phosphate sodium salt and red
Red-spotted stonecrop glycosides makes an addition in culture solution in preheating.
8. a kind of cultural method of development quality for improving ox embryo in vitro fertilization according to claim 7, which is characterized in that
Ox embryo in vitro fertilization is in 38.5 DEG C, 5%CO2, cultivate under saturated humidity.
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