CN109943522A - A kind of mammalian in vitro fertilization Embryo Culture method - Google Patents
A kind of mammalian in vitro fertilization Embryo Culture method Download PDFInfo
- Publication number
- CN109943522A CN109943522A CN201910153674.8A CN201910153674A CN109943522A CN 109943522 A CN109943522 A CN 109943522A CN 201910153674 A CN201910153674 A CN 201910153674A CN 109943522 A CN109943522 A CN 109943522A
- Authority
- CN
- China
- Prior art keywords
- sodium
- culture
- culture solution
- embryo
- chloride
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a kind of mammalian in vitro fertilization Embryo Culture methods.The method of the present invention includes the following steps: acquisition and the maturation in vitro of egg mother cell；It is in vitro fertilization；In vitro culture and preservation.The time of fertilization in vitro, mature COCs is cultivated in present invention fertilization culture solution, in In vitro culture, the granular cell around embryo is clean with stripping ovum needle removal, it is put into embryo medium and cultivates, it can the liquid nitrogen frozen preservation in freezing liquid with embryo after culture.Many technical effects excellent as used in the description are presented in the method for the present invention.
Description
Technical field
The invention belongs to technical field of animal reproduction, are related to a kind of technology bred for agricultural-animal and veterinary, specifically relate to
And a kind of method of IVF Embryos culture, in addition, the related culture solution the invention further relates to IVF Embryos culture exists
Application in IVF Embryos culture.Further, the invention further relates to the related trainings for using the IVF Embryos culture
The method of nutrient solution progress IVF Embryos culture.In particular, the method for IVF Embryos culture of the present invention is with excellent
Technical effect.
Background technique
(In Vitro Fertilization) or (external fertilization) in vitro fertilization refer to that lactation is dynamic
The sperm and ovum of object complete the technology of fertilization process, English abbreviation IVF in the environment of manual control in vitro.Due to it with
Embryo transfer technology (ET) is inseparable, and referred to as IVF-ET.In biology, after In vitro Fertilization Embryo Transfer to parent
The animal of acquisition claims test tube animal (test-tube animal).The success of this technology is in the 1950s, at nearest 20 years
It quickly grows, has reached its maturity and become an important and conventional animal reproduction biotechnology.
Technology in vitro fertilization has animals' reproduction mechanism study, husbandry sector, medicine and animals on the brink of extinction protection etc. important
Meaning.Such as make experimental material with mouse, rat or rabbit, technology in vitro fertilization can be used for studying mammalian gamete occur,
Fertilization and early embryo development mechanism.In livestock animal breeding, technology in vitro fertilization provides cheap and high for Embryo Production
The means of effect shorten the livestock reproduction period to excellent variety resource is made full use of, and accelerating breed improvement speed etc. has important valence
Value.In the mankind, IVF-ET technology is the certain infertilities for the treatment of and one of the important measures for overcoming sex-kink disease.Technology in vitro fertilization
Or the indispensable component parts of modern biotechnologies such as mammal embryo transplanting, clone, transgenosis and sexual control.
With the development of modern agriculture science and technology, in order to more make full use of the breeding potential of breeding mother, accelerate genetic breeding into
Journey, the efficient breeding new technology of application becomes inevitable in production practice.Ovum pick-up (Ovum pick UP, OPU) and external
Fertilization technique (In Vitro Fertilization, IVF) is the embryo engineering that 1980s fast development is got up
New technology, the two, which combines, can get the specific embryo of a large amount of Genetic lineages, so as to shorten the generation inteval.Currently, both skills
The farmer that art has become the animal husbandry developed countries such as American-European and Oceania is the important breeding for expanding breeding mother group and using
Technology.However, blastocyst rate in vitro fertilization is lower using conventional Embryo Culture system (CR1aa and SOF liquid), and
For embryo quality also far away from internal embryo, the pregnancy rate after leading to embryo transplantation acceptor is low, therefore how to improve blastocyst rate
And embryo quality becomes the emphasis of IVF Embryos production and the focus of research.
Early in 1878, German Scnenk just using rabbit and cavy as material, start to explore mammal it is external by
Smart technology.But it is in vitro fertilization after Chinese American Zhang Minjue and Austin have found sperm microcytotoxicity phenomenon respectively until nineteen fifty-one
Technology just obtains breakthrough.Technology in vitro fertilization is by the maturation in vitro of egg mother cell, the In-vitro Capacitation of sperm, fertilization
The influence of many aspects such as the vitro culture conditions of ovum.
The in vitro culture of embryo is a key link of IVF technology, is also oocyte in vitro maturation and in vitro fertilization
The embodiment and inspection of technology final effect.After fertilization in vitro, fertilized eggs need to be undergone into blastaea growth course it is a series of
Important variation, activation, densification and the formation blastaea of formation, First cleavage, embryonic gene group including zygote.This
In the process, the variation of external environment will lead to gene expression and change, to influence the normal development and quality of embryo.Mesh
Before, the in vitro culture research of Mammalian Embryo, which is concentrated mainly on, improves culture solution ingredient to meet different developmental phases
Embryotrophy demand.Based on (Rosenkrans, C.F., Jr.and N.L.First, the Effect of such as Rosenkrans
freeamino acids and vitamins on cleavage and developmental rate of bovine
Zygotesin vitro.J Anim Sci, 1994.72(2): p.434-7) the Charles Rosenkrans 1(CR1 developed)
(Tervit, H.R., D.G.Whittingham, the and L.E.Rowson, Successful such as culture solution and Tervit
Culture invitro of sheep and cattle ova.J Reprod Fertil, 1972.30(3): p.493-7) open
The synthesis Oviductal Fluid (Synthetic Oviductal Fluid, SOF) of hair, is continuously improved gradually formed two kinds for many years
Cultivating system.According to (Sagirkaya, H., et al., Developmental the potential of such as Hakan Sagirkaya
bovineoocytes cultured in different maturation and culture conditions.Anim
ReprodSci, 2007.101(3-4): p.225-40) and (Somfai, T., et al., the Development of such as Somfai
bovineembryos cultured in CR1aa and IVD101media using different oxygen
Tensions andculture systems.Acta Vet Hung, 2010.58(4): p.465-74) research achievement shows
CR1aa culture solution has preferable effect for Embryo Culture, the Embryo Culture that can be widely applied to；Thompson, J.G. etc.
(Thompson, J.G., et al., Effect of inhibitors and uncouplers of oxidative
phosphorylationduring compaction and blastulation of bovine embryos cultured
In vitro.JReprod Fertil, 2000.118(1): p.47-55) and Jean M.Feugang etc. (Feugang, J.M.,
O.Camargo-Rodriguez,and E.Memili,Culture systems for bovine embryos.Livestock
Science, 2009.121(2-3): result of study p.141-149) shows that SOF culture solution is also that one kind is suitable for embryo's training
Feeding cultivating system.(Zhang Zhiping, An Zhixing, rust, Zhang Yong, the northwest the optimization agriculture and forestry science and technology of Embryo Culture system such as Zhang Zhiping
College journal, 2006.34) and (Sang Guojun, egg mother cell and external embryo culture technique the study .2008) result of study such as Sang Guojun
It also shows, the embryo in vitro culture that the CR1aa and SOF culture solution by optimization is adapted to obtains good culture effect.
Mammalian Embryo development is a hight coordinate and the process that accurately adjusts.During evolution, gametid is gradually
A series of molecule cascade network is formd, to guarantee that embryonic development period is systematically carried out.In growth course, embryo's is interior
Outer active oxygen radical (Reactive Oxygen Species, ROS) and the balance of antioxidant play early embryonic development
Decisive role.
Most biochemical reaction generates ROS, in the cell, it is outer have important role, a part of ROS plays signal
The effect of molecule, but most of ROS are harmful to body.Brooker, R.J. etc. (Brooker, R.J., Genetics:
Analysis and principles(4th ed.) .McGraw-Hill Science, 2011) report, ROS can cause carefully
Born of the same parents' DNA damage, the oxidation of unsaturated fatty acid, the oxidation of Amino Acids in Proteins or even the inactivation that certain enzymes can be caused.One
As for, ROS exists in the form of four kinds, and wherein H2O2 oxidation is stronger, is the main factor for causing oxidative damage.
A large number of studies show that glutathione (GSH) be in the form of nonprotein existing for a kind of antioxidant, can remove
A variety of free radicals: ultra-oxygen anion free radical, hydroxyl radical free radical, hydrogen peroxide, hypochlorous acid and rouge oxygen radical, and can tie up
Hold intraor extracellular redox equilibrium.Intraor extracellular environment GSH and ROS level is two weights influenced during development of fertilized ova
Want factor.Early in 2000, (de Matos, D.G.and C.C.Furnus, the The importance such as de Matos
Ofhaving high glutathione(GSH) level after bovine in vitro maturation on
embryodevelopment effect of beta-mercaptoethanol,cysteine
Andcystine.Theriogenology, 2000.53(3): p.761-71) once by being added during Embryo Culture in vitro
Beta -mercaptoethanol, cysteine plus cystine improve blastocyst rate.
Although technology in vitro fertilization can be successfully applied to many mammals, led since blastocyst rate in vitro fertilization is low
High production cost, the low efficiency for causing IVF Embryos limit extensive use of the technology in fast-propagation practice.Cause
This, how reducing cost and improving IVF Embryo Production efficiency and embryo quality becomes urgent problem to be solved.
Currently, mainly using CR1aa and SOF liquid as embryo in-vitro culture solution in technical system in vitro fertilization, and in this base
It is improved on plinth, blastocyst rate has different degrees of raising, blastocyst rate average out to 30%-40%.For blastaea
Quality can be assessed by blastomere sum, ICM cell number/total cell number ratio, apoptosis rate.Blastomere is total
The number difference in the stages according to locating for blastaea and it is different, S.Iwasaki etc. (Iwasaki, S.and T.Nakahara,
Cellnumber and incidence of chromosomal anomalies in bovine
blastocystsfertilized in vitro followed by culture in vitro or in vivo in
Rabbitoviducts.Theriogenology, 1990.33(3): p.669-75) the early blastocyst total cell number obtained is average
It is 15.8% or so for 44, ICM cell number/blastaea total cell number ratio；(Watson, A.J., the et such as Andrew J.Watson
al.,Impact of bovine oocyte maturation media on oocyte transcript levels,
Blastocyst development, cell number, and apoptosis.Biol Reprod, 2000.62(2):
P.355-64) statistics blastomere apoptosis rate is about 7.7%-13%.
CN103898046B(Chinese Patent Application No. 201410073635.4) it discloses one kind and is exclusively used in embryo in vitro fertilization
The culture solution of tire, the culture formula of liquid are as follows: NaCl 109.5mM, KCl 3.1mM, NaHCO3 26.2mM, MgCl26H2O
0.8mM, KH2PO31.19mM, Sodium Pyruvate 0.4mM, glucose 1.5mM, galactonic acid calcium 5mM, 10v/v% tire serum, L- paddy
Glutamine 1mM, 2v/v% essential amino acid, 1v/v% nonessential amino acid and glutathione 3mM, are prepared with water；It is described required
Amino acid is the aqueous solution prepared after following amino acid mixes in proportion, wherein each amino acid content are as follows: L- R-gene
6.32g/L, one water object 2.1g/L of l-cysteine dihydrochloride 1.564g/L, L- histidine monohydrochloride, l-Isoleucine 2.625g/L,
L-Leu 2.62g/L, L lysine HCL 3.625g/L, L-Methionine 0.755g/L, L-phenylalanine 1.65g/L, L-
Threonine 2.38g/L, L-Trp 0.51g/L, l-tyrosine 1.8g/L and Valine 2.34g/L；The nonessential amino
Acid is the aqueous solution prepared after following amino acid mixes in proportion, wherein each amino acid content are as follows: l-Alanine 0.89g/L, L-
One water object 1.5g/L of asparagine, L-Aspartic acid 1.33g/L, Pidolidone 1.47g/L, glycine 0.75g/L, L- dried meat ammonia
Sour 1.15g/L and Serine 1.05g/L.IVF Embryos are placed in above-mentioned culture solution and carry out in vitro culture, it is believed that knot
Fruit, which shows, is substantially better than the control group for being not added with GSH, improves blastocyst rate and embryo quality, reduces produced in vitro embryo's
Cost is applied to practice for IVF technology and provides experiment basis, can greatly accelerate genetic breeding process.
Other bibliography:
Chen great Yuan fertilization biology .2003, Beijing: Science Press；
Fang Junshun embryo IVC technical research " Chinese excellent MA theses full-text database agricultural science and technology volume "
.2007, (the 4th phase)；
" Chinese excellent MA theses are complete by the influence of Cao Haiqing cumulus cell and condition of culture to embryo production in vitro efficiency
Literary database agricultural science and technology volume " .2008, (the 9th phase)；
I.H.KIM et al.Effect of exogenous glutathione on the in
Vitrofertilization of bovine oocytes.Theriogenology.1999, Vol.52(3).
However, the method for having the culture of the improved IVF Embryos of performance is still expected in this field, such as still expect
The method for being improved IVF Embryos culture efficiency, such as have the IVF Embryos culture with excellent cost advantage
Method.
Summary of the invention
The purpose of the present invention is to provide a kind of methods of the IVF Embryos culture of performance improvement, especially expect to mention
High IVF Embryo Production efficiency and embryo quality.More specifically, the present invention needs to provide one kind and is exclusively used in reach above-mentioned purpose
The related culture solution of IVF Embryos and and using the culture solution carry out IVF Embryos culture method.The present inventor
It has been had now surprisingly been found that, excellent technical effect is presented using the method for the present invention, the present invention is consequently found that and be accomplished.
For this purpose, first aspect present invention provides a kind of method of IVF Embryos culture comprising following steps:
(1) acquisition of egg mother cell and maturation in vitro
It takes ovary to be contained in the insulation barrel for adding dual anti-physiological saline, under the conditions of 30-33 DEG C, transports laboratory in 3h back；Extract table
The ovarian follicle of face 2-8mm collects precipitating, and the egg mother cell at least containing the cumulus cell package that haves three layers is picked under stereomicroscope
COCs(is that is, cumulus oocytes complesxes), it is washed 2 times in egg-cleaning liquid, removes undesired impurities；
Gained COCs is washed 1 time in oocyte maturation culture solution, is then transferred in new maturation culture solution and cultivates 22-
For 24 hours, condition of culture is 38.8 DEG C, 5.5-6.5%CO2, saturated humidity；
(2) in vitro fertilization
Mature COCs is washed 1 time in fertilization culture solution, is then transferred in fertilization culture solution, is put into incubator, it is spare；
A jelly fine tube is taken from liquid nitrogen, 37 DEG C of water-baths are thawed；Tubule both ends are cut off in sterile working, and sperm injection is made to fill essence
Liquid is prepared in the 15mL centrifuge tube of culture solution, and 328 × g is centrifuged 2 times, and each 5min abandons supernatant after centrifugation；By 300 μ L sperm systems
Above-mentioned centrifuge tube is added in standby culture solution, and Sperm pellets are resuspended, and sperm suspension appropriate is taken to carry out sperm count；
The sperm suspension for calculating volume is added in the fertilization culture drop for filling egg mother cell, and culture plate is put into culture
Case, makes smart ovum be incubated for 16-20h altogether, and condition of culture is 38.8 DEG C, 5.5-6.5%CO2, saturated humidity；
(3) In vitro culture and preservation
It is after end of operation in vitro fertilization, the granular cell around embryo is clean with stripping ovum needle removal, it is put into embryo medium
Culture is denoted as the 1st day of Embryo Culture at this time, and condition of culture is 38.8 DEG C, 6%O2,88%N2, saturated humidity, records within the 3rd day
Cleavage rates；7th day record blastocyst rate, until (it is hatched blastocyst number divided by hundred obtained by blastaea number to statistics hatching rate at the 9th day
Score), and carry out Quality Identification；
It can be washed 3 times in saving liquid with embryo, freezing liquid is transferred to after balancing 10min in equilibrium liquid, by 5 sections of dress liquid methods
It is packed into embryo, marks, places into programmed cooling instrument after being down to -35 DEG C according to the speed of 0.5 DEG C/min, by embryo's tubule
It takes out rapidly, is placed in freezen protective in liquid nitrogen.
According to method of the first aspect of the present invention, wherein in step (1), described plus dual anti-physiological saline is comprising penicillin
The physiological saline of 400IU/mL, 400 μ g/mL of streptomysin.
According to method of the first aspect of the present invention, wherein in step (1), the egg-cleaning liquid is that be added to 3mg/mL serum white
The BY basic culture solution of albumen.
The preparation method of egg-cleaning liquid or other culture solutions of the invention is that those skilled in the art are easy to accomplish, such as
For egg-cleaning liquid, usually addition seralbumin is corresponding to it in preparatory prepared BY basic culture solution of the present invention
It is required that concentration, this preparation method is also that those skilled in the art are common.
According to method of the first aspect of the present invention, wherein in step (1), the maturation culture solution is to be added to 100mL/
The BY basic culture solution of LFBS, 10 μ g/mL FSH, 10 μ g/mL LH, 1 μ g/mL E2,20ng/mL EGF.
In the present invention, EGF --- epidermal growth factor, FSH --- follicular stimulating hormone, FBS --- tire serum, E2 ---
Estradiol, LH --- interstitialcellstimulating hormone (ICSH).
According to method of the first aspect of the present invention, wherein in step (2), the fertilization culture solution is comprising 112.0mM chlorine
Change sodium, 4.02mM potassium chloride, the calcium chloride dihydrate of 2.25mM, 0.52mM magnesium chloride hexahydrate, 0.83mM potassium dihydrogen phosphate,
37.0mM sodium bicarbonate, 1.25mM Sodium Pyruvate, 10 μ g/ml heparin, 4mg/ml seralbumin (BSA), 100U/ml mould
The aqueous solution of element, 100 μ g/ml streptomysins.
According to method of the first aspect of the present invention, wherein in step (2), it is to include that the sperm, which prepares culture solution,
112.0mM sodium chloride, 4.02mM potassium chloride, the calcium chloride dihydrate of 2.25mM, 0.52mM magnesium chloride hexahydrate, 0.83mM phosphoric acid
Potassium dihydrogen, 37.0mM sodium bicarbonate, 1.25mM Sodium Pyruvate, 10 μ g/ml heparin, 4mg/ml seralbumin (BSA), 10mM coffee
Coffee is because of, the aqueous solution of 100U/ml penicillin, 100 μ g/ml streptomysins.
According to method of the first aspect of the present invention, wherein in step (3), the embryo medium includes: 109.5mM chlorination
Sodium, 3.1mM potassium chloride, 26.2mM sodium bicarbonate, 0.8mM magnesium chloride hexahydrate, 1.19mM potassium dihydrogen phosphate, 0.4mM Sodium Pyruvate,
1.5mM glucose, 5mM galactonic acid calcium, 2.5v/v% tire serum (FBS), L-Glutamine 1mM, 2v/v% essential amino acid,
1v/v% nonessential amino acid, the glutathione of 3mM, sodium citrate 0.04w/v%, maltose 0.02w/v% aqueous solution；Institute
Stating essential amino acid is that following amino acid ratio by weight is added: L- R-gene 6.32g, l-cysteine dihydrochloride
One water object 2.1g of 1.564g, L- histidine monohydrochloride, l-Isoleucine 2.625g, L-Leu 2.62g, L lysine HCL
3.625g, L-Methionine 0.755g, L-phenylalanine 1.65g, L-threonine 2.38g, L-Trp 0.51g, l-tyrosine
1.8g and Valine 2.34g, the nonessential amino acid are that following amino acid ratio by weight is added: l-Alanine 0.89g,
One water object 1.5g of L- asparagine, L-Aspartic acid 1.33g, Pidolidone 1.47g, glycine 0.75g, L-PROLINE
1.15g and Serine 1.05g.
According to method of the first aspect of the present invention, wherein in step (1), the BY basic culture solution is comprising following component
Aqueous solution: 180~220mg/L of calcium chloride, nine 0.70~0.75mg/L of water ferric nitrate, 380~420mg/L of potassium chloride, sulfuric acid
90~100mg/L of magnesium, 6500~7000mg/L of sodium chloride, 130~150mg/L of sodium dihydrogen phosphate-water, sodium bicarbonate 2000~
2500mg/L, 40~60mg/L of sodium acetate, 20~30mg/L of l-Alanine, 60~80mg/L of L-arginine hydrochloride, L- asparagus fern
25~35mg/L of propylhomoserin, 0.10~0.12mg/L of L-cysteine hydrochloride monohydrate, l-cysteine dihydrochloride 20~
30mg/L, 70~80mg/L of Pidolidone, 40~60mg/L of glycine, 20~25mg/L of L-Histidine hydrochloride monohydrate,
8~12mg/L of L- hydroxyproline, 15~25mg/L of l-Isoleucine, 50~70mg/L of L-Leu, L lysine HCL 60
~80mg/L, 10~20mg/L of L-Methionine, 20~30mg/L of L-phenylalanine, 30~50mg/L of L-PROLINE, Serine
20~30mg/L, 25~35mg/L of L-threonine, 8~12mg/L of L-Trp, 55~60mg/ of l-tyrosine disodium dihydrate
L, 20~30mg/L of Valine, 0.04~0.06mg/L of ascorbic acid, 0.008~0.012mg/L of α-D- tocopherol phosphate,
0.008~0.012mg/L of biotin, 0.08~0.12mg/L of ostelin, 0.008~0.012mg/L of D-VB5 calcium, choline chloride
0.4~0.6mg/L, 0.008~0.012mg/L of folic acid, 0.04~0.06mg/L of inositol, three hydration Menadione Sodium Bisulfites
0.015~0.025mg/L, 0.02~0.03mg/L of niacin, 0.02~0.03mg/L of niacinamide, p- aminobenzoic acid 0.04~
0.06mg/L, 0.04~0.06mg/L of pyridoxine hydrochloride, 0.008~0.012mg/L of riboflavin, thiamine hydrochloride 0.008~
0.012mg/L, 0.1~0.2mg/L of retinyl acetate, 8~12mg/L of adenine sulfate, 0.15~0.25mg/L of adenine,
0.8~1.2mg/L of Adenosine Triphosphate Disodium, 0.15~0.25mg/L of cholesterol, 0.4~0.6mg/L of 2-deoxy-D-ribose, D-
800~1200mg/L of glucose, 0.04~0.06mg/L of glutathione, 0.25~0.35mg/L of guanine hydrochloride, hypoxanthine
0.3~0.4mg/L of sodium, 0.4~0.6mg/L of ribose, thymosin beta 10 .25~0.35mg/L, 4~6mg/L of Tween 80, uracil
0.25~0.35mg/L, 0.3~0.4mg/L of xanthine sodium, phenol red 8~12mg/L.
According to method of the first aspect of the present invention, wherein in step (1), the BY basic culture solution is comprising following component
Aqueous solution: calcium chloride 200mg/L, nine water ferric nitrate 0.72mg/L, potassium chloride 400mg/L, magnesium sulfate 97.7mg/L, sodium chloride
6800mg/L, sodium dihydrogen phosphate-water 140mg/L, sodium bicarbonate 2200mg/L, sodium acetate 50mg/L, l-Alanine 25mg/L,
L-arginine hydrochloride 70mg/L, L-Aspartic acid 30mg/L, L-cysteine hydrochloride monohydrate 0.11mg/L, L- Guang ammonia
Sour dihydrochloride 26mg/L, Pidolidone 75mg/L, glycine 50mg/L, L-Histidine hydrochloride monohydrate 21.88mg/L,
L- hydroxyproline 10mg/L, l-Isoleucine 20mg/L, L-Leu 60mg/L, L lysine HCL 70mg/L, L- egg ammonia
Sour 15mg/L, L-phenylalanine 25mg/L, L-PROLINE 40mg/L, Serine 25mg/L, L-threonine 30mg/L, L- color ammonia
Sour 10mg/L, l-tyrosine disodium dihydrate 57.66mg/L, Valine 25mg/L, ascorbic acid 0.05mg/L, α-D- are raw
Educate phenol phosphoesterase 30 .01mg/L, biotin 0.01mg/L, ostelin 0.1mg/L, D-VB5 calcium 0.01mg/L, choline chloride
0.5mg/L, folic acid 0.01mg/L, inositol 0.05mg/L, three hydration Menadione Sodium Bisulfite 0.019mg/L, niacin
0.025mg/L, niacinamide 0.025mg/L, p- aminobenzoic acid 0.05mg/L, pyridoxine hydrochloride 0.05mg/L, riboflavin
0.01mg/L, thiamine hydrochloride 0.01mg/L, retinyl acetate 0.14mg/L, adenine sulfate 10mg/L, adenine
0.2mg/L, Adenosine Triphosphate Disodium 1mg/L, cholesterol 0.2mg/L, 2-deoxy-D-ribose 0.5mg/L, D-Glucose
1000mg/L, glutathione 0.05mg/L, guanine hydrochloride 0.3mg/L, hypoxanthine sodium 0.354mg/L, ribose 0.5mg/L,
Thymosin beta 10 .3mg/L, Tween 80 5mg/L, uracil 0.3mg/L, xanthine sodium 0.34mg/L, phenol red 10mg/L.
It according to method of the first aspect of the present invention, further include selenous acid in the BY basic culture solution wherein in step (1)
Sodium, concentration are 0.2~0.3mg/L, such as 0.25mg/L；And/or in the BY basic culture solution further include copper sulphate,
Concentration is calculated as 0.05~0.1mg/L, such as 0.075mg/L with anhydride.
In the present invention, such as in step (3), preservation liquid, equilibrium liquid, freezing liquid used in embryo etc. are this fields
It is well known and can be easy to obtain from commercially available approach, such as freezing liquid can be Vitrolife company, Sweden at home
The FreezeKit Cleave of sale.
Further, second aspect of the present invention provides a kind of egg-cleaning liquid, is that be added to 3mg/mL sero-abluminous
BY basic culture solution.
Egg-cleaning liquid according to a second aspect of the present invention, wherein the BY basic culture solution is water-soluble comprising following component
Liquid: 180~220mg/L of calcium chloride, nine 0.70~0.75mg/L of water ferric nitrate, 380~420mg/L of potassium chloride, magnesium sulfate 90~
100mg/L, 6500~7000mg/L of sodium chloride, 130~150mg/L of sodium dihydrogen phosphate-water, 2000~2500mg/ of sodium bicarbonate
L, 40~60mg/L of sodium acetate, 20~30mg/L of l-Alanine, 60~80mg/L of L-arginine hydrochloride, L-Aspartic acid 25~
35mg/L, 0.10~0.12mg/L of L-cysteine hydrochloride monohydrate, 20~30mg/L of l-cysteine dihydrochloride, L- paddy
70~80mg/L of propylhomoserin, 40~60mg/L of glycine, 20~25mg/L of L-Histidine hydrochloride monohydrate, L- hydroxyproline 8
~12mg/L, 15~25mg/L of l-Isoleucine, 50~70mg/L of L-Leu, 60~80mg/L of L lysine HCL, L-
10~20mg/L of methionine, 20~30mg/L of L-phenylalanine, 30~50mg/L of L-PROLINE, 20~30mg/L of Serine,
25~35mg/L of L-threonine, 8~12mg/L of L-Trp, 55~60mg/L of l-tyrosine disodium dihydrate, Valine
20~30mg/L, 0.04~0.06mg/L of ascorbic acid, 0.008~0.012mg/L of α-D- tocopherol phosphate, biotin
0.008~0.012mg/L, 0.08~0.12mg/L of ostelin, 0.008~0.012mg/L of D-VB5 calcium, choline chloride 0.4~
0.6mg/L, 0.008~0.012mg/L of folic acid, 0.04~0.06mg/L of inositol, three hydration Menadione Sodium Bisulfites 0.015~
0.025mg/L, 0.02~0.03mg/L of niacin, 0.02~0.03mg/L of niacinamide, 0.04~0.06mg/L of p- aminobenzoic acid,
0.04~0.06mg/L of pyridoxine hydrochloride, 0.008~0.012mg/L of riboflavin, 0.008~0.012mg/L of thiamine hydrochloride, dimension
Raw 0.1~0.2mg/L of element A acetic acid esters, 8~12mg/L of adenine sulfate, 0.15~0.25mg/L of adenine, adenosine triphosphate two
0.8~1.2mg/L of sodium, 0.15~0.25mg/L of cholesterol, 0.4~0.6mg/L of 2-deoxy-D-ribose, D-Glucose 800~
1200mg/L, 0.04~0.06mg/L of glutathione, 0.25~0.35mg/L of guanine hydrochloride, hypoxanthine sodium 0.3~
0.4mg/L, 0.4~0.6mg/L of ribose, thymosin beta 10 .25~0.35mg/L, 4~6mg/L of Tween 80, uracil 0.25~
0.35mg/L, 0.3~0.4mg/L of xanthine sodium, phenol red 8~12mg/L.
Egg-cleaning liquid according to a second aspect of the present invention, wherein the BY basic culture solution is water-soluble comprising following component
Liquid: calcium chloride 200mg/L, nine water ferric nitrate 0.72mg/L, potassium chloride 400mg/L, magnesium sulfate 97.7mg/L, sodium chloride
6800mg/L, sodium dihydrogen phosphate-water 140mg/L, sodium bicarbonate 2200mg/L, sodium acetate 50mg/L, l-Alanine 25mg/L,
L-arginine hydrochloride 70mg/L, L-Aspartic acid 30mg/L, L-cysteine hydrochloride monohydrate 0.11mg/L, L- Guang ammonia
Sour dihydrochloride 26mg/L, Pidolidone 75mg/L, glycine 50mg/L, L-Histidine hydrochloride monohydrate 21.88mg/L,
L- hydroxyproline 10mg/L, l-Isoleucine 20mg/L, L-Leu 60mg/L, L lysine HCL 70mg/L, L- egg ammonia
Sour 15mg/L, L-phenylalanine 25mg/L, L-PROLINE 40mg/L, Serine 25mg/L, L-threonine 30mg/L, L- color ammonia
Sour 10mg/L, l-tyrosine disodium dihydrate 57.66mg/L, Valine 25mg/L, ascorbic acid 0.05mg/L, α-D- are raw
Educate phenol phosphoesterase 30 .01mg/L, biotin 0.01mg/L, ostelin 0.1mg/L, D-VB5 calcium 0.01mg/L, choline chloride
0.5mg/L, folic acid 0.01mg/L, inositol 0.05mg/L, three hydration Menadione Sodium Bisulfite 0.019mg/L, niacin
0.025mg/L, niacinamide 0.025mg/L, p- aminobenzoic acid 0.05mg/L, pyridoxine hydrochloride 0.05mg/L, riboflavin
0.01mg/L, thiamine hydrochloride 0.01mg/L, retinyl acetate 0.14mg/L, adenine sulfate 10mg/L, adenine
0.2mg/L, Adenosine Triphosphate Disodium 1mg/L, cholesterol 0.2mg/L, 2-deoxy-D-ribose 0.5mg/L, D-Glucose
1000mg/L, glutathione 0.05mg/L, guanine hydrochloride 0.3mg/L, hypoxanthine sodium 0.354mg/L, ribose 0.5mg/L,
Thymosin beta 10 .3mg/L, Tween 80 5mg/L, uracil 0.3mg/L, xanthine sodium 0.34mg/L, phenol red 10mg/L.
Egg-cleaning liquid according to a second aspect of the present invention, wherein further include sodium selenite in the BY basic culture solution, it is dense
Degree is 0.2~0.3mg/L, such as 0.25mg/L；And/or in the BY basic culture solution further include copper sulphate, concentration is with nothing
Water object is calculated as 0.05~0.1mg/L, such as 0.075mg/L.
Further, third aspect present invention provides a kind of maturation culture solution, is to be added to 100mL/L FBS, 10 μ
The BY basic culture solution of g/mL FSH, 10 μ g/mL LH, 1 μ g/mL E2,20ng/mL EGF.
Maturation culture solution according to a third aspect of the present invention, wherein the BY basic culture solution is the water comprising following component
Solution: 180~220mg/L of calcium chloride, nine 0.70~0.75mg/L of water ferric nitrate, 380~420mg/L of potassium chloride, magnesium sulfate 90
~100mg/L, 6500~7000mg/L of sodium chloride, 130~150mg/L of sodium dihydrogen phosphate-water, sodium bicarbonate 2000~
2500mg/L, 40~60mg/L of sodium acetate, 20~30mg/L of l-Alanine, 60~80mg/L of L-arginine hydrochloride, L- asparagus fern
25~35mg/L of propylhomoserin, 0.10~0.12mg/L of L-cysteine hydrochloride monohydrate, l-cysteine dihydrochloride 20~
30mg/L, 70~80mg/L of Pidolidone, 40~60mg/L of glycine, 20~25mg/L of L-Histidine hydrochloride monohydrate,
8~12mg/L of L- hydroxyproline, 15~25mg/L of l-Isoleucine, 50~70mg/L of L-Leu, L lysine HCL 60
~80mg/L, 10~20mg/L of L-Methionine, 20~30mg/L of L-phenylalanine, 30~50mg/L of L-PROLINE, Serine
20~30mg/L, 25~35mg/L of L-threonine, 8~12mg/L of L-Trp, 55~60mg/ of l-tyrosine disodium dihydrate
L, 20~30mg/L of Valine, 0.04~0.06mg/L of ascorbic acid, 0.008~0.012mg/L of α-D- tocopherol phosphate,
0.008~0.012mg/L of biotin, 0.08~0.12mg/L of ostelin, 0.008~0.012mg/L of D-VB5 calcium, choline chloride
0.4~0.6mg/L, 0.008~0.012mg/L of folic acid, 0.04~0.06mg/L of inositol, three hydration Menadione Sodium Bisulfites
0.015~0.025mg/L, 0.02~0.03mg/L of niacin, 0.02~0.03mg/L of niacinamide, p- aminobenzoic acid 0.04~
0.06mg/L, 0.04~0.06mg/L of pyridoxine hydrochloride, 0.008~0.012mg/L of riboflavin, thiamine hydrochloride 0.008~
0.012mg/L, 0.1~0.2mg/L of retinyl acetate, 8~12mg/L of adenine sulfate, 0.15~0.25mg/L of adenine,
0.8~1.2mg/L of Adenosine Triphosphate Disodium, 0.15~0.25mg/L of cholesterol, 0.4~0.6mg/L of 2-deoxy-D-ribose, D-
800~1200mg/L of glucose, 0.04~0.06mg/L of glutathione, 0.25~0.35mg/L of guanine hydrochloride, hypoxanthine
0.3~0.4mg/L of sodium, 0.4~0.6mg/L of ribose, thymosin beta 10 .25~0.35mg/L, 4~6mg/L of Tween 80, uracil
0.25~0.35mg/L, 0.3~0.4mg/L of xanthine sodium, phenol red 8~12mg/L.
Maturation culture solution according to a third aspect of the present invention, wherein the BY basic culture solution is the water comprising following component
Solution: calcium chloride 200mg/L, nine water ferric nitrate 0.72mg/L, potassium chloride 400mg/L, magnesium sulfate 97.7mg/L, sodium chloride
6800mg/L, sodium dihydrogen phosphate-water 140mg/L, sodium bicarbonate 2200mg/L, sodium acetate 50mg/L, l-Alanine 25mg/L,
L-arginine hydrochloride 70mg/L, L-Aspartic acid 30mg/L, L-cysteine hydrochloride monohydrate 0.11mg/L, L- Guang ammonia
Sour dihydrochloride 26mg/L, Pidolidone 75mg/L, glycine 50mg/L, L-Histidine hydrochloride monohydrate 21.88mg/L,
L- hydroxyproline 10mg/L, l-Isoleucine 20mg/L, L-Leu 60mg/L, L lysine HCL 70mg/L, L- egg ammonia
Sour 15mg/L, L-phenylalanine 25mg/L, L-PROLINE 40mg/L, Serine 25mg/L, L-threonine 30mg/L, L- color ammonia
Sour 10mg/L, l-tyrosine disodium dihydrate 57.66mg/L, Valine 25mg/L, ascorbic acid 0.05mg/L, α-D- are raw
Educate phenol phosphoesterase 30 .01mg/L, biotin 0.01mg/L, ostelin 0.1mg/L, D-VB5 calcium 0.01mg/L, choline chloride
0.5mg/L, folic acid 0.01mg/L, inositol 0.05mg/L, three hydration Menadione Sodium Bisulfite 0.019mg/L, niacin
0.025mg/L, niacinamide 0.025mg/L, p- aminobenzoic acid 0.05mg/L, pyridoxine hydrochloride 0.05mg/L, riboflavin
0.01mg/L, thiamine hydrochloride 0.01mg/L, retinyl acetate 0.14mg/L, adenine sulfate 10mg/L, adenine
0.2mg/L, Adenosine Triphosphate Disodium 1mg/L, cholesterol 0.2mg/L, 2-deoxy-D-ribose 0.5mg/L, D-Glucose
1000mg/L, glutathione 0.05mg/L, guanine hydrochloride 0.3mg/L, hypoxanthine sodium 0.354mg/L, ribose 0.5mg/L,
Thymosin beta 10 .3mg/L, Tween 80 5mg/L, uracil 0.3mg/L, xanthine sodium 0.34mg/L, phenol red 10mg/L.
Maturation culture solution according to a third aspect of the present invention, wherein further include sodium selenite in the BY basic culture solution,
Its concentration is 0.2~0.3mg/L, such as 0.25mg/L；It and/or in the BY basic culture solution further include copper sulphate, concentration
0.05~0.1mg/L, such as 0.075mg/L are calculated as with anhydride.
Any technical characteristic possessed by any embodiment of either side or the either side of the present invention is equally applicable
Any embodiment of other any embodiments or other either sides, as long as they will not be conflicting, certainly mutual
Between where applicable, if necessary can individual features be made with appropriate modification.Make to various aspects of the present invention with feature into one below
The description of step.
All documents recited in the present invention, their full content are incorporated herein by reference, and if these are literary
When offering expressed meaning and the inconsistent present invention, it is subject to statement of the invention.In addition, the various terms that use of the present invention and
Phrase has that well known to a person skilled in the art general senses, nonetheless, the present invention remain desirable at this to these terms and
Phrase is described in more detail and explains, the term and phrase referred to is if any inconsistent with common art-recognized meanings, with institute's table of the present invention
Subject to the meaning stated.
The tire serum that the present invention uses readily can buy its from market and standardize commercial form, such as can be from various
Bought at agent Gibco company Australia tire serum (article No.: 10099141), New Zealand's tire serum (article No.: 10091148),
North America tire serum (article No.: 16000044), Mexico's tire serum (article No.: 10437028) etc., the valence of these commercialization tire serum
Lattice 500ml is mostly at 8000 yuan or more, and tire serum is cost main contributions when in IVF Embryos culture solution of the present invention
Person.In the test of the context of the invention, if not otherwise specified, tire serum used is Australia tire serum (goods of Gibco company
Number: 10099141).
Detailed description of the invention
It, below will be to attached drawing needed in embodiment description in order to illustrate more clearly of the technical solution of the application
It is briefly described, it should be apparent that, the drawings in the following description are only some examples of the present application, general for this field
For logical technical staff, without creative efforts, it is also possible to obtain other drawings based on these drawings.
A kind of process for mammalian in vitro fertilization Embryo Culture method that Fig. 1 is proposed by the embodiment of the present application is illustrated
Figure.
Specific embodiment
The present invention can be further described by the following examples, however, the scope of the present invention and unlimited
In following embodiments.One of skill in the art, can be with it is understood that under the premise of without departing substantially from the spirit and scope of the present invention
Various change and modification are carried out to the present invention.The present invention carries out the material and test method arrived used in test general
And/or specific description.Although to realize the present invention many materials and operating method used in purpose be it is known in the art that
But the present invention is still described in this detail as much as possible.Unless otherwise specified, technological means used in embodiment is ability
Conventional means known to field technique personnel, raw materials used is commercial goods.
In specific test of the invention, if not otherwise indicated, details are as follows for the related reagent used:
Add dual anti-physiological saline: the physiological saline comprising penicillin 400IU/mL, 400 μ g/mL of streptomysin.
Egg-cleaning liquid: it is added to the sero-abluminous BY basic culture solution of 3mg/mL.
Maturation culture solution: 100mL/L FBS, 10 μ g/mL FSH, 10 μ g/mL LH, 1 μ g/mL E2,20ng/ are added to
The BY basic culture solution of mLEGF.Wherein, EGF --- epidermal growth factor, FSH --- follicular stimulating hormone, FBS --- tire serum,
E2 --- estradiol, LH --- interstitialcellstimulating hormone (ICSH).
Be fertilized culture solution: comprising 112.0mM sodium chloride, 4.02mM potassium chloride, 2.25mM calcium chloride dihydrate,
0.52mM magnesium chloride hexahydrate, 0.83mM potassium dihydrogen phosphate, 37.0mM sodium bicarbonate, 1.25mM Sodium Pyruvate, 10 μ g/ml heparin,
4mg/ml seralbumin (BSA), 100U/ml penicillin, 100 μ g/ml streptomysins aqueous solution.
Sperm prepares culture solution: comprising 112.0mM sodium chloride, 4.02mM potassium chloride, 2.25mM calcium chloride dihydrate,
0.52mM magnesium chloride hexahydrate, 0.83mM potassium dihydrogen phosphate, 37.0mM sodium bicarbonate, 1.25mM Sodium Pyruvate, 10 μ g/ml heparin,
4mg/ml seralbumin (BSA), 10mM caffeine, 100U/ml penicillin, 100 μ g/ml streptomysins aqueous solution.
Embryo medium: include: 109.5mM sodium chloride, 3.1mM potassium chloride, 26.2mM sodium bicarbonate, six water chlorine of 0.8mM
Change magnesium, 1.19mM potassium dihydrogen phosphate, 0.4mM Sodium Pyruvate, 1.5mM glucose, 5mM galactonic acid calcium, 2.5v/v% tire serum
(FBS), L-Glutamine 1mM, 2v/v% essential amino acid, 1v/v% nonessential amino acid, the glutathione of 3mM, citric acid
The aqueous solution of sodium 0.04w/v%, maltose 0.02w/v%；The essential amino acid is that following amino acid ratio by weight is added:
L- R-gene 6.32g, one water object 2.1g of l-cysteine dihydrochloride 1.564g, L- histidine monohydrochloride, l-Isoleucine
2.625g, L-Leu 2.62g, L lysine HCL 3.625g, L-Methionine 0.755g, L-phenylalanine 1.65g, L- Soviet Union
Propylhomoserin 2.38g, L-Trp 0.51g, l-tyrosine 1.8g and Valine 2.34g, the nonessential amino acid are following ammonia
Base acid ratio by weight is added: one water object 1.5g of l-Alanine 0.89g, L- asparagine, L-Aspartic acid 1.33g, L- paddy
Propylhomoserin 1.47g, glycine 0.75g, L-PROLINE 1.15g and Serine 1.05g.
BY basic culture solution is the aqueous solution comprising following component: calcium chloride 200mg/L, nine water ferric nitrate 0.72mg/L,
Potassium chloride 400mg/L, magnesium sulfate 97.7mg/L, sodium chloride 6800mg/L, sodium dihydrogen phosphate-water 140mg/L, sodium bicarbonate
2200mg/L, sodium acetate 50mg/L, l-Alanine 25mg/L, L-arginine hydrochloride 70mg/L, L-Aspartic acid 30mg/L, L-
Cysteine hydrochloride monohydrate 0.11mg/L, l-cysteine dihydrochloride 26mg/L, Pidolidone 75mg/L, glycine
50mg/L, L-Histidine hydrochloride monohydrate 21.88mg/L, L- hydroxyproline 10mg/L, l-Isoleucine 20mg/L, L- are bright
Propylhomoserin 60mg/L, L lysine HCL 70mg/L, L-Methionine 15mg/L, L-phenylalanine 25mg/L, L-PROLINE 40mg/
L, Serine 25mg/L, L-threonine 30mg/L, L-Trp 10mg/L, l-tyrosine disodium dihydrate 57.66mg/L,
Valine 25mg/L, ascorbic acid 0.05mg/L, α-D- tocopherol phosphate 0.01mg/L, biotin 0.01mg/L, ossification
Alcohol 0.1mg/L, D-VB5 calcium 0.01mg/L, choline chloride 0.5mg/L, folic acid 0.01mg/L, inositol 0.05mg/L, three hydration first
Naphthoquinones sodium hydrogensulfite 0.019mg/L, niacin 0.025mg/L, niacinamide 0.025mg/L, p- aminobenzoic acid 0.05mg/L, salt
Sour Benadon 0.05mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L, retinyl acetate 0.14mg/L, sulfuric acid
Adenine 10mg/L, adenine 0.2mg/L, Adenosine Triphosphate Disodium 1mg/L, cholesterol 0.2mg/L, 2-deoxy-D-ribose
0.5mg/L, D-Glucose 1000mg/L, glutathione 0.05mg/L, guanine hydrochloride 0.3mg/L, hypoxanthine sodium
0.354mg/L, ribose 0.5mg/L, thymosin beta 10 .3mg/L, Tween 80 5mg/L, uracil 0.3mg/L, xanthine sodium 0.34mg/
L, phenol red 10mg/L.
It takes ovary to be contained in the insulation barrel for adding dual anti-physiological saline, under the conditions of 30-33 DEG C, transports laboratory in 3h back；It takes out
The ovarian follicle of surface 2-8mm is taken, precipitating is collected, the ovum picked under stereomicroscope at least containing the cumulus cell package that haves three layers is female thin
Born of the same parents COCs(is that is, cumulus oocytes complesxes), it is washed 2 times in egg-cleaning liquid, removes undesired impurities；
Gained COCs is washed 1 time in oocyte maturation culture solution, is then transferred in new maturation culture solution and cultivates 22-
(practical operation 24 hours) for 24 hours, condition of culture are 38.8 DEG C, 5.5-6.5%CO2, saturated humidity；
Mature COCs is washed 1 time in fertilization culture solution, is then transferred in fertilization culture solution, is put into incubator, it is spare；
A jelly fine tube is taken from liquid nitrogen, 37 DEG C of water-baths are thawed；Tubule both ends are cut off in sterile working, and sperm injection is made to fill essence
Liquid is prepared in the 15mL centrifuge tube of culture solution, and 328 × g is centrifuged 2 times, and each 5min abandons supernatant after centrifugation；By 300 μ L sperm systems
Above-mentioned centrifuge tube is added in standby culture solution, and Sperm pellets are resuspended, and sperm suspension appropriate is taken to carry out sperm count；
The sperm suspension for calculating volume is added in the fertilization culture drop for filling egg mother cell, and culture plate is put into culture
Case makes smart ovum be incubated for 16-20h(practical operation altogether 18 hours), condition of culture is 38.8 DEG C, 5.5-6.5%CO2, saturated humidity；
It is after end of operation in vitro fertilization, the granular cell around embryo is clean with stripping ovum needle removal, it is put into embryo medium
Culture is denoted as the 1st day of Embryo Culture at this time, and condition of culture is 38.8 DEG C, 6%O2,88%N2, saturated humidity, records within the 3rd day
Cleavage rates；7th day record blastocyst rate, until (it is hatched blastocyst number divided by hundred obtained by blastaea number to statistics hatching rate at the 9th day
Score), and carry out Quality Identification；
It can be washed 3 times in saving liquid with embryo, freezing liquid is transferred to after balancing 10min in equilibrium liquid, by 5 sections of dress liquid methods
It is packed into embryo, marks, places into programmed cooling instrument after being down to -35 DEG C according to the speed of 0.5 DEG C/min, by embryo's tubule
It takes out rapidly, is placed in freezen protective in liquid nitrogen.
1. choosing the 7th day blastaea of in vitro culture, the fixed 20min of 2% paraformaldehyde is used.
2. washed twice using the phosphate buffer (PBS-BSA) containing 0.5%BSA, be put into permeabilization liquid (50 μ l Triton,
5 μ l Tween 80s and 9.945ml PBS) in, it is placed at room temperature for 30min.
3. handling 20min using 2M hydrochloric acid room temperature, 10min then is handled using the Tris-HCl room temperature of 100mM, makes CDX2
Albumen can be with an anti-binding.
4. using PBS-BSA cleaning three times, by blastaea be put into confining liquid (1ml lowlenthal serum, 5 μ l Tween 80s and
In 8.995mlPBS), room temperature closes 1h, is then transferred to 4 DEG C of refrigerator closings overnight.
5. discarding confining liquid, CDX2 primary antibody is diluted with confining liquid by 1:200, is incubated at room temperature 2h, is discarded primary antibody dilution, is used
PBS-BSA is cleaned 3 times, each 5min.
6.caspase-3 primary antibody (being purchased from Cell Signaling Technology company) is dilute by 1:200 with confining liquid
It releases, is incubated at room temperature 2h, discards primary antibody dilution, cleaned 3 times with PBS-BSA, each 5min.
7. with confining liquid by 1:200 dilution CDX2 specificity secondary antibody (being purchased from Sigma company) under the conditions of being protected from light, at room temperature
Avoid light place 1h.Secondary antibody diluent is discarded under the conditions of being protected from light, and is cleaned 3 times with PBS, each 5min.
8. (being purchased from confining liquid by 1:200 dilution caspase-3 specificity secondary antibody under the conditions of being protected from light
LifeTechnologies company), avoid light place 1h at room temperature.Secondary antibody diluent is discarded under the conditions of being protected from light, and is cleaned 3 times with PBS,
Each 5min.
9. 33342 dye liquor of Hochest that 10 μ g/mL are added contaminates nucleus, room temperature acts on 5min, under fluorescence microscope
It observes and takes pictures.
10. experiment is in triplicate, 10 blastaeas are randomly selected every time, calculate apoptosis rate, ICM cell number/total cell number is come
Assess quality of blastocysts.
Data statistical approach: experimental data is analyzed using the ANOVA program in statistical software SAS V8, Duncan '
The significance of difference between smultiple-range method of inspection determination processing thinks significant difference as p < 0.05.
In the present invention, cleavage rates=fertilization spilting of an egg number/fertilized eggs number.In the present invention, blastocyst rate=blastaea number/spilting of an egg
Embryo number.
The present embodiment 1 the step of in (1), through In-vitro maturation after, observed under inverted microscope, by ovum mother
Cell have first polar body release, keep secreting sticky matrix between cumulus cell, cellular layer significantly expands, during cell with ovum is
The heart, substantially radial diffusion person around is determined as maturation, records mature oocyte number, calculates maturing rate.
The present embodiment 1 tests Chinese Yellow (kind is used as a servant in Nanyang).As a result, cleavage rates 88.7%, morula rate are
63.7%, blastocyst rate 51.2%, apoptosis rate 5.1%；In addition, hatching rate was up to 72.3% at the 9th day.Egg mother cell body
The maturing rate of outer maturation is up to 75.6%.
In a complementary testing, referring to the method for example 1 above, it is directed to He Sitan (dairy products kind), west gate tower respectively
That (meat kind), three kinds of China Water (labour kind) are tested, and as a result: cleavage rates are in 84~89% ranges, morula
Rate in 62~65% ranges, blastocyst rate in 50~53% ranges, apoptosis rate is in 4~7% ranges, the 9th day capsule
Embryo hatching rate is in 71~75% ranges；The maturation of He Sitan, Simmental, three kinds of China Water of oocyte in vitro maturation
Rate is respectively 69.3%, 65.6%, 57.8%.
The main distinction of the present embodiment 2 and example 1 above is the also additional addition 0.25mg/L in BY basic culture solution
Sodium selenite and 0.075mg/L anhydrous cupric sulfate.
In specific test of the invention, if not otherwise indicated, details are as follows for the related reagent used:
Add dual anti-physiological saline: the physiological saline comprising penicillin 400IU/mL, 400 μ g/mL of streptomysin.
Egg-cleaning liquid: it is added to the sero-abluminous BY basic culture solution of 3mg/mL.
Maturation culture solution: 100mL/L FBS, 10 μ g/mL FSH, 10 μ g/mL LH, 1 μ g/mL E2,20ng/ are added to
The BY basic culture solution of mLEGF.Wherein, EGF --- epidermal growth factor, FSH --- follicular stimulating hormone, FBS --- tire serum,
E2 --- estradiol, LH --- interstitialcellstimulating hormone (ICSH).
Be fertilized culture solution: comprising 112.0mM sodium chloride, 4.02mM potassium chloride, 2.25mM calcium chloride dihydrate,
0.52mM magnesium chloride hexahydrate, 0.83mM potassium dihydrogen phosphate, 37.0mM sodium bicarbonate, 1.25mM Sodium Pyruvate, 10 μ g/ml heparin,
4mg/ml seralbumin (BSA), 100U/ml penicillin, 100 μ g/ml streptomysins aqueous solution.
Sperm prepares culture solution: comprising 112.0mM sodium chloride, 4.02mM potassium chloride, 2.25mM calcium chloride dihydrate,
0.52mM magnesium chloride hexahydrate, 0.83mM potassium dihydrogen phosphate, 37.0mM sodium bicarbonate, 1.25mM Sodium Pyruvate, 10 μ g/ml heparin,
4mg/ml seralbumin (BSA), 10mM caffeine, 100U/ml penicillin, 100 μ g/ml streptomysins aqueous solution.
Embryo medium: include: 109.5mM sodium chloride, 3.1mM potassium chloride, 26.2mM sodium bicarbonate, six water chlorine of 0.8mM
Change magnesium, 1.19mM potassium dihydrogen phosphate, 0.4mM Sodium Pyruvate, 1.5mM glucose, 5mM galactonic acid calcium, 10v/v% tire serum
(FBS), L-Glutamine 1mM, 2v/v% essential amino acid, 1v/v% nonessential amino acid, the glutathione of 3mM, citric acid
The aqueous solution of sodium 0.04w/v%, maltose 0.02w/v%；The essential amino acid is that following amino acid ratio by weight is added:
L- R-gene 6.32g, one water object 2.1g of l-cysteine dihydrochloride 1.564g, L- histidine monohydrochloride, l-Isoleucine
2.625g, L-Leu 2.62g, L lysine HCL 3.625g, L-Methionine 0.755g, L-phenylalanine 1.65g, L- Soviet Union
Propylhomoserin 2.38g, L-Trp 0.51g, l-tyrosine 1.8g and Valine 2.34g, the nonessential amino acid are following ammonia
Base acid ratio by weight is added: one water object 1.5g of l-Alanine 0.89g, L- asparagine, L-Aspartic acid 1.33g, L- paddy
Propylhomoserin 1.47g, glycine 0.75g, L-PROLINE 1.15g and Serine 1.05g.
BY basic culture solution is the aqueous solution comprising following component: calcium chloride 200mg/L, nine water ferric nitrate 0.72mg/L,
Potassium chloride 400mg/L, magnesium sulfate 97.7mg/L, sodium chloride 6800mg/L, sodium dihydrogen phosphate-water 140mg/L, sodium bicarbonate
2200mg/L, sodium acetate 50mg/L, l-Alanine 25mg/L, L-arginine hydrochloride 70mg/L, L-Aspartic acid 30mg/L, L-
Cysteine hydrochloride monohydrate 0.11mg/L, l-cysteine dihydrochloride 26mg/L, Pidolidone 75mg/L, glycine
50mg/L, L-Histidine hydrochloride monohydrate 21.88mg/L, L- hydroxyproline 10mg/L, l-Isoleucine 20mg/L, L- are bright
Propylhomoserin 60mg/L, L lysine HCL 70mg/L, L-Methionine 15mg/L, L-phenylalanine 25mg/L, L-PROLINE 40mg/
L, Serine 25mg/L, L-threonine 30mg/L, L-Trp 10mg/L, l-tyrosine disodium dihydrate 57.66mg/L,
Valine 25mg/L, ascorbic acid 0.05mg/L, α-D- tocopherol phosphate 0.01mg/L, biotin 0.01mg/L, ossification
Alcohol 0.1mg/L, D-VB5 calcium 0.01mg/L, choline chloride 0.5mg/L, folic acid 0.01mg/L, inositol 0.05mg/L, three hydration first
Naphthoquinones sodium hydrogensulfite 0.019mg/L, niacin 0.025mg/L, niacinamide 0.025mg/L, p- aminobenzoic acid 0.05mg/L, salt
Sour Benadon 0.05mg/L, riboflavin 0.01mg/L, thiamine hydrochloride 0.01mg/L, retinyl acetate 0.14mg/L, sulfuric acid
Adenine 10mg/L, adenine 0.2mg/L, Adenosine Triphosphate Disodium 1mg/L, cholesterol 0.2mg/L, 2-deoxy-D-ribose
0.5mg/L, D-Glucose 1000mg/L, glutathione 0.05mg/L, guanine hydrochloride 0.3mg/L, hypoxanthine sodium
0.354mg/L, ribose 0.5mg/L, thymosin beta 10 .3mg/L, Tween 80 5mg/L, uracil 0.3mg/L, xanthine sodium 0.34mg/
L, phenol red 10mg/L, 0.25mg/L sodium selenite and 0.075mg/L anhydrous cupric sulfate.
It takes ovary to be contained in the insulation barrel for adding dual anti-physiological saline, under the conditions of 30-33 DEG C, transports laboratory in 3h back；It takes out
The ovarian follicle of surface 2-8mm is taken, precipitating is collected, the ovum picked under stereomicroscope at least containing the cumulus cell package that haves three layers is female thin
Born of the same parents COCs(is that is, cumulus oocytes complesxes), it is washed 2 times in egg-cleaning liquid, removes undesired impurities；
Gained COCs is washed 1 time in oocyte maturation culture solution, is then transferred in new maturation culture solution and cultivates 22-
(practical operation 24 hours) for 24 hours, condition of culture are 38.8 DEG C, 5.5-6.5%CO2, saturated humidity；
Mature COCs is washed 1 time in fertilization culture solution, is then transferred in fertilization culture solution, is put into incubator, it is spare；
A jelly fine tube is taken from liquid nitrogen, 37 DEG C of water-baths are thawed；Tubule both ends are cut off in sterile working, and sperm injection is made to fill essence
Liquid is prepared in the 15mL centrifuge tube of culture solution, and 328 × g is centrifuged 2 times, and each 5min abandons supernatant after centrifugation；By 300 μ L sperm systems
Above-mentioned centrifuge tube is added in standby culture solution, and Sperm pellets are resuspended, and sperm suspension appropriate is taken to carry out sperm count；
The sperm suspension for calculating volume is added in the fertilization culture drop for filling egg mother cell, and culture plate is put into culture
Case makes smart ovum be incubated for 16-20h(practical operation altogether 18 hours), condition of culture is 38.8 DEG C, 5.5-6.5%CO2, saturated humidity；
It is after end of operation in vitro fertilization, the granular cell around embryo is clean with stripping ovum needle removal, it is put into embryo medium
Culture is denoted as the 1st day of Embryo Culture at this time, and condition of culture is 38.8 DEG C, 6%O2,88%N2, saturated humidity, records within the 3rd day
Cleavage rates；7th day record blastocyst rate, until (it is hatched blastocyst number divided by hundred obtained by blastaea number to statistics hatching rate at the 9th day
Score), and carry out Quality Identification；
It can be washed 3 times in saving liquid with embryo, freezing liquid is transferred to after balancing 10min in equilibrium liquid, by 5 sections of dress liquid methods
It is packed into embryo, marks, places into programmed cooling instrument after being down to -35 DEG C according to the speed of 0.5 DEG C/min, by embryo's tubule
It takes out rapidly, is placed in freezen protective in liquid nitrogen.
1. choosing the 7th day blastaea of in vitro culture, the fixed 20min of 2% paraformaldehyde is used.
2. washed twice using the phosphate buffer (PBS-BSA) containing 0.5%BSA, be put into permeabilization liquid (50 μ l Triton,
5 μ l Tween 80s and 9.945ml PBS) in, it is placed at room temperature for 30min.
3. handling 20min using 2M hydrochloric acid room temperature, 10min then is handled using the Tris-HCl room temperature of 100mM, makes CDX2
Albumen can be with an anti-binding.
4. using PBS-BSA cleaning three times, by blastaea be put into confining liquid (1ml lowlenthal serum, 5 μ l Tween 80s and
In 8.995mlPBS), room temperature closes 1h, is then transferred to 4 DEG C of refrigerator closings overnight.
5. discarding confining liquid, CDX2 primary antibody is diluted with confining liquid by 1:200, is incubated at room temperature 2h, is discarded primary antibody dilution, is used
PBS-BSA is cleaned 3 times, each 5min.
6.caspase-3 primary antibody (being purchased from Cell Signaling Technology company) is dilute by 1:200 with confining liquid
It releases, is incubated at room temperature 2h, discards primary antibody dilution, cleaned 3 times with PBS-BSA, each 5min.
7. with confining liquid by 1:200 dilution CDX2 specificity secondary antibody (being purchased from Sigma company) under the conditions of being protected from light, at room temperature
Avoid light place 1h.Secondary antibody diluent is discarded under the conditions of being protected from light, and is cleaned 3 times with PBS, each 5min.
8. (being purchased from confining liquid by 1:200 dilution caspase-3 specificity secondary antibody under the conditions of being protected from light
LifeTechnologies company), avoid light place 1h at room temperature.Secondary antibody diluent is discarded under the conditions of being protected from light, and is cleaned 3 times with PBS,
Each 5min.
9. 33342 dye liquor of Hochest that 10 μ g/mL are added contaminates nucleus, room temperature acts on 5min, under fluorescence microscope
It observes and takes pictures.
10. experiment is in triplicate, 10 blastaeas are randomly selected every time, calculate apoptosis rate, ICM cell number/total cell number is come
Assess quality of blastocysts.
Data statistical approach: experimental data is analyzed using the ANOVA program in statistical software SAS V8, Duncan '
The significance of difference between smultiple-range method of inspection determination processing thinks significant difference as p < 0.05.
In the present invention, cleavage rates=fertilization spilting of an egg number/fertilized eggs number.In the present invention, blastocyst rate=blastaea number/spilting of an egg
Embryo number.
The present embodiment 2 the step of in (1), through In-vitro maturation after, observed under inverted microscope, by ovum mother
Cell have first polar body release, keep secreting sticky matrix between cumulus cell, cellular layer significantly expands, during cell with ovum is
The heart, substantially radial diffusion person around is determined as maturation, records mature oocyte number, calculates maturing rate.
The present embodiment 2 tests Chinese Yellow (kind is used as a servant in Nanyang).As a result, cleavage rates 88.2%, morula rate are
64.2%, blastocyst rate 50.8%, apoptosis rate 5.6%；In addition, hatching rate was up to 73.4% at the 9th day.Egg mother cell body
The maturing rate of outer maturation is up to 88.3%, about 13 percentage points of maturing rate increase relative to 1 method of embodiment.
In a complementary testing, referring to the method for example 2 above, it is directed to He Sitan (dairy products kind), west gate tower respectively
That (meat kind), three kinds of China Water (labour kind) are tested, and as a result: cleavage rates are in 84~90% ranges, morula
Rate in 61~65% ranges, blastocyst rate in 50~54% ranges, apoptosis rate is in 3~6% ranges, the 9th day capsule
Embryo hatching rate is in 71~76% ranges；The maturation of He Sitan, Simmental, three kinds of China Water of oocyte in vitro maturation
Rate is respectively 84.5%, 79.5%, 73.2%, and the maturing rate relative to 1 method of embodiment increases separately about 14~15 hundred
Branch.The above results, although 1 method of the embodiment of the present invention can obtain it is being entirely satisfactory as a result, however, unexpected
It is the discovery that, egg mother cell body can be dramatically increased when adding micro selenides and copper sulphate in BY basic culture solution of the present invention
The maturing rate of outer maturation, this is the significantly above-mentioned selenides of especially no any teaching in prior art and copper sulphate
Addition can dramatically increase oocyte in vitro maturation maturing rate technical teaching.
In a complementary testing, with reference to the present embodiment 2, does not add sodium selenite in BY basal medium and (and only increase
Mend the copper sulphate of corresponding amount) or when not adding copper sulphate (and the sodium selenite for only augmenting corresponding amount),
Four it is various in, cleavage rates, morula rate, blastocyst rate, four parameter of apoptosis rate in both cases with 2 result base of embodiment
This is consistent, to four various cleavage rates in 84~89% ranges, morula rate in 63~65% ranges, blastocyst rate it is equal
In 52~55% ranges, apoptosis rate in 3~6% ranges, the 9th day hatching rate in 70~75% ranges, example
It is respectively such as 87.7% and 88.5% for the cleavage rates of Chinese Yellow；But the maturing rate of oocyte in vitro maturation is relative to reality
Applying 1 result of example there are no increase, in the case of two kinds to Chinese Yellow, He Sitan, Simmental, China Water Oocyte in Vitro at
Ripe maturing rate is respectively 73~75%, 67~70%, 65~66%, 55~57%.This shows selenizing in BY basal medium
The addition of object and copper sulphate or not it will not influence cleavage rates, morula rate, blastocyst rate, four parameter of apoptosis rate, but only simultaneously
The maturing rate of oocyte in vitro maturation could be effectively improved when addition selenides and copper sulphate.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a kind of method of IVF Embryos culture comprising following steps:
(1) acquisition of egg mother cell and maturation in vitro
It takes ovary to be contained in the insulation barrel for adding dual anti-physiological saline, under the conditions of 30-33 DEG C, transports laboratory in 3h back；Extract table
The ovarian follicle of face 2-8mm collects precipitating, and the egg mother cell at least containing the cumulus cell package that haves three layers is picked under stereomicroscope
COCs(is that is, cumulus oocytes complesxes), it is washed 2 times in egg-cleaning liquid, removes undesired impurities；
Gained COCs is washed 1 time in oocyte maturation culture solution, is then transferred in new maturation culture solution and cultivates 22-
For 24 hours, condition of culture is 38.8 DEG C, 5.5-6.5%CO2, saturated humidity；
(2) in vitro fertilization
Mature COCs is washed 1 time in fertilization culture solution, is then transferred in fertilization culture solution, is put into incubator, it is spare；
A jelly fine tube is taken from liquid nitrogen, 37 DEG C of water-baths are thawed；Tubule both ends are cut off in sterile working, and sperm injection is made to fill essence
Liquid is prepared in the 15mL centrifuge tube of culture solution, and 328 × g is centrifuged 2 times, and each 5min abandons supernatant after centrifugation；By 300 μ L sperm systems
Above-mentioned centrifuge tube is added in standby culture solution, and Sperm pellets are resuspended, and sperm suspension appropriate is taken to carry out sperm count；
The sperm suspension for calculating volume is added in the fertilization culture drop for filling egg mother cell, and culture plate is put into culture
Case, makes smart ovum be incubated for 16-20h altogether, and condition of culture is 38.8 DEG C, 5.5-6.5%CO2, saturated humidity；
(3) In vitro culture and preservation
It is after end of operation in vitro fertilization, the granular cell around embryo is clean with stripping ovum needle removal, it is put into embryo medium
Culture is denoted as the 1st day of Embryo Culture at this time, and condition of culture is 38.8 DEG C, 6%O2,88%N2, saturated humidity, records within the 3rd day
Cleavage rates；7th day record blastocyst rate, until (it is hatched blastocyst number divided by hundred obtained by blastaea number to statistics hatching rate at the 9th day
Score), and carry out Quality Identification；
It can be washed 3 times in saving liquid with embryo, freezing liquid is transferred to after balancing 10min in equilibrium liquid, by 5 sections of dress liquid methods
It is packed into embryo, marks, places into programmed cooling instrument after being down to -35 DEG C according to the speed of 0.5 DEG C/min, by embryo's tubule
It takes out rapidly, is placed in freezen protective in liquid nitrogen.
2. the method according to claim 1, wherein in step (1), described plus dual anti-physiological saline is comprising penicillin 400IU/
The physiological saline of mL, 400 μ g/mL of streptomysin.
3. the method according to claim 1, wherein in step (1), the egg-cleaning liquid is that be added to 3mg/mL sero-abluminous
BY basic culture solution.
4. the method according to claim 1, wherein in step (1), the maturation culture solution is to be added to 100mL/L FBS, 10 μ
The BY basic culture solution of g/mL FSH, 10 μ g/mL LH, 1 μ g/mL E2,20ng/mL EGF.
5. the method according to claim 1, wherein in step (2), the fertilization culture solution be comprising 112.0mM sodium chloride,
4.02mM potassium chloride, the calcium chloride dihydrate of 2.25mM, 0.52mM magnesium chloride hexahydrate, 0.83mM potassium dihydrogen phosphate, 37.0mM
Sodium bicarbonate, 1.25mM Sodium Pyruvate, 10 μ g/ml heparin, 4mg/ml seralbumin (BSA), 100U/ml penicillin, 100 μ
The aqueous solution of g/ml streptomysin.
6. the method according to claim 1, wherein in step (2), it is comprising 112.0mM chlorination that the sperm, which prepares culture solution,
Sodium, 4.02mM potassium chloride, the calcium chloride dihydrate of 2.25mM, 0.52mM magnesium chloride hexahydrate, 0.83mM potassium dihydrogen phosphate,
37.0mM sodium bicarbonate, 1.25mM Sodium Pyruvate, 10 μ g/ml heparin, 4mg/ml seralbumin (BSA), 10mM caffeine,
The aqueous solution of 100U/ml penicillin, 100 μ g/ml streptomysins.
7. the method according to claim 1, wherein in step (3), the embryo medium includes: 109.5mM sodium chloride,
3.1mM potassium chloride, 26.2mM sodium bicarbonate, 0.8mM magnesium chloride hexahydrate, 1.19mM potassium dihydrogen phosphate, 0.4mM Sodium Pyruvate,
1.5mM glucose, 5mM galactonic acid calcium, 2.5v/v% tire serum (FBS), L-Glutamine 1mM, 2v/v% essential amino acid,
1v/v% nonessential amino acid, the glutathione of 3mM, sodium citrate 0.04w/v%, maltose 0.02w/v% aqueous solution；Institute
Stating essential amino acid is that following amino acid ratio by weight is added: L- R-gene 6.32g, l-cysteine dihydrochloride
One water object 2.1g of 1.564g, L- histidine monohydrochloride, l-Isoleucine 2.625g, L-Leu 2.62g, L lysine HCL
3.625g, L-Methionine 0.755g, L-phenylalanine 1.65g, L-threonine 2.38g, L-Trp 0.51g, l-tyrosine
1.8g and Valine 2.34g, the nonessential amino acid are that following amino acid ratio by weight is added: l-Alanine 0.89g,
One water object 1.5g of L- asparagine, L-Aspartic acid 1.33g, Pidolidone 1.47g, glycine 0.75g, L-PROLINE
1.15g and Serine 1.05g.
8. the method according to claim 1, wherein in step (1), the BY basic culture solution is water-soluble comprising following component
Liquid: 180~220mg/L of calcium chloride, nine 0.70~0.75mg/L of water ferric nitrate, 380~420mg/L of potassium chloride, magnesium sulfate 90~
100mg/L, 6500~7000mg/L of sodium chloride, 130~150mg/L of sodium dihydrogen phosphate-water, 2000~2500mg/ of sodium bicarbonate
L, 40~60mg/L of sodium acetate, 20~30mg/L of l-Alanine, 60~80mg/L of L-arginine hydrochloride, L-Aspartic acid 25~
35mg/L, 0.10~0.12mg/L of L-cysteine hydrochloride monohydrate, 20~30mg/L of l-cysteine dihydrochloride, L- paddy
70~80mg/L of propylhomoserin, 40~60mg/L of glycine, 20~25mg/L of L-Histidine hydrochloride monohydrate, L- hydroxyproline 8
~12mg/L, 15~25mg/L of l-Isoleucine, 50~70mg/L of L-Leu, 60~80mg/L of L lysine HCL, L-
10~20mg/L of methionine, 20~30mg/L of L-phenylalanine, 30~50mg/L of L-PROLINE, 20~30mg/L of Serine,
25~35mg/L of L-threonine, 8~12mg/L of L-Trp, 55~60mg/L of l-tyrosine disodium dihydrate, Valine
20~30mg/L, 0.04~0.06mg/L of ascorbic acid, 0.008~0.012mg/L of α-D- tocopherol phosphate, biotin
0.008~0.012mg/L, 0.08~0.12mg/L of ostelin, 0.008~0.012mg/L of D-VB5 calcium, choline chloride 0.4~
0.6mg/L, 0.008~0.012mg/L of folic acid, 0.04~0.06mg/L of inositol, three hydration Menadione Sodium Bisulfites 0.015~
0.025mg/L, 0.02~0.03mg/L of niacin, 0.02~0.03mg/L of niacinamide, 0.04~0.06mg/L of p- aminobenzoic acid,
0.04~0.06mg/L of pyridoxine hydrochloride, 0.008~0.012mg/L of riboflavin, 0.008~0.012mg/L of thiamine hydrochloride, dimension
Raw 0.1~0.2mg/L of element A acetic acid esters, 8~12mg/L of adenine sulfate, 0.15~0.25mg/L of adenine, adenosine triphosphate two
0.8~1.2mg/L of sodium, 0.15~0.25mg/L of cholesterol, 0.4~0.6mg/L of 2-deoxy-D-ribose, D-Glucose 800~
1200mg/L, 0.04~0.06mg/L of glutathione, 0.25~0.35mg/L of guanine hydrochloride, hypoxanthine sodium 0.3~
0.4mg/L, 0.4~0.6mg/L of ribose, thymosin beta 10 .25~0.35mg/L, 4~6mg/L of Tween 80, uracil 0.25~
0.35mg/L, 0.3~0.4mg/L of xanthine sodium, phenol red 8~12mg/L.
9. the method according to claim 1, wherein in step (1), the BY basic culture solution is water-soluble comprising following component
Liquid: calcium chloride 200mg/L, nine water ferric nitrate 0.72mg/L, potassium chloride 400mg/L, magnesium sulfate 97.7mg/L, sodium chloride
6800mg/L, sodium dihydrogen phosphate-water 140mg/L, sodium bicarbonate 2200mg/L, sodium acetate 50mg/L, l-Alanine 25mg/L,
L-arginine hydrochloride 70mg/L, L-Aspartic acid 30mg/L, L-cysteine hydrochloride monohydrate 0.11mg/L, L- Guang ammonia
Sour dihydrochloride 26mg/L, Pidolidone 75mg/L, glycine 50mg/L, L-Histidine hydrochloride monohydrate 21.88mg/L,
L- hydroxyproline 10mg/L, l-Isoleucine 20mg/L, L-Leu 60mg/L, L lysine HCL 70mg/L, L- egg ammonia
Sour 15mg/L, L-phenylalanine 25mg/L, L-PROLINE 40mg/L, Serine 25mg/L, L-threonine 30mg/L, L- color ammonia
Sour 10mg/L, l-tyrosine disodium dihydrate 57.66mg/L, Valine 25mg/L, ascorbic acid 0.05mg/L, α-D- are raw
Educate phenol phosphoesterase 30 .01mg/L, biotin 0.01mg/L, ostelin 0.1mg/L, D-VB5 calcium 0.01mg/L, choline chloride
0.5mg/L, folic acid 0.01mg/L, inositol 0.05mg/L, three hydration Menadione Sodium Bisulfite 0.019mg/L, niacin
0.025mg/L, niacinamide 0.025mg/L, p- aminobenzoic acid 0.05mg/L, pyridoxine hydrochloride 0.05mg/L, riboflavin
0.01mg/L, thiamine hydrochloride 0.01mg/L, retinyl acetate 0.14mg/L, adenine sulfate 10mg/L, adenine
0.2mg/L, Adenosine Triphosphate Disodium 1mg/L, cholesterol 0.2mg/L, 2-deoxy-D-ribose 0.5mg/L, D-Glucose
1000mg/L, glutathione 0.05mg/L, guanine hydrochloride 0.3mg/L, hypoxanthine sodium 0.354mg/L, ribose 0.5mg/L,
Thymosin beta 10 .3mg/L, Tween 80 5mg/L, uracil 0.3mg/L, xanthine sodium 0.34mg/L, phenol red 10mg/L.
10. the method according to claim 1 further includes sodium selenite in the BY basic culture solution wherein in step (1),
Concentration is 0.2~0.3mg/L, such as 0.25mg/L；And/or further include copper sulphate in the BY basic culture solution, concentration with
Anhydride is calculated as 0.05~0.1mg/L, such as 0.075mg/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910153674.8A CN109943522A (en) | 2019-03-01 | 2019-03-01 | A kind of mammalian in vitro fertilization Embryo Culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910153674.8A CN109943522A (en) | 2019-03-01 | 2019-03-01 | A kind of mammalian in vitro fertilization Embryo Culture method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109943522A true CN109943522A (en) | 2019-06-28 |
Family
ID=67007032
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910153674.8A Withdrawn CN109943522A (en) | 2019-03-01 | 2019-03-01 | A kind of mammalian in vitro fertilization Embryo Culture method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109943522A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110669724A (en) * | 2019-11-08 | 2020-01-10 | 广州达瑞生殖技术有限公司 | Saccharum embryo culture solution and preparation method thereof |
| CN111406711A (en) * | 2020-05-20 | 2020-07-14 | 山西农业大学 | Artificial propagation-hybridization combined breeding method for meat goats |
| CN112675155A (en) * | 2021-03-15 | 2021-04-20 | 南京鼓楼医院 | Application of mevalonate metabolic intermediate product in preparation of medicine for improving aged ovarian oocyte quality and meiosis process |
| CN118222489A (en) * | 2024-05-22 | 2024-06-21 | 广州市新甡命科技有限公司 | Embryo culture solution and culture method for improving embryo health |
-
2019
- 2019-03-01 CN CN201910153674.8A patent/CN109943522A/en not_active Withdrawn
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110669724A (en) * | 2019-11-08 | 2020-01-10 | 广州达瑞生殖技术有限公司 | Saccharum embryo culture solution and preparation method thereof |
| CN111406711A (en) * | 2020-05-20 | 2020-07-14 | 山西农业大学 | Artificial propagation-hybridization combined breeding method for meat goats |
| CN112675155A (en) * | 2021-03-15 | 2021-04-20 | 南京鼓楼医院 | Application of mevalonate metabolic intermediate product in preparation of medicine for improving aged ovarian oocyte quality and meiosis process |
| CN118222489A (en) * | 2024-05-22 | 2024-06-21 | 广州市新甡命科技有限公司 | Embryo culture solution and culture method for improving embryo health |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109943522A (en) | A kind of mammalian in vitro fertilization Embryo Culture method | |
| CN110066763A (en) | Promote the method for ox embryo in vitro culture development of fertilized ova | |
| Thompson | Comparison between in vivo-derived and in vitro-produced pre-elongation embryos from domestic ruminants | |
| Merton et al. | Cysteamine supplementation during in vitro maturation of slaughterhouse-and opu-derived bovine oocytes improves embryonic development without affecting cryotolerance, pregnancy rate, and calf characteristics | |
| US20020131957A1 (en) | Cryopreservation of sperm | |
| CN107142239B (en) | Method for improving culture efficiency of bovine in vitro fertilization embryos | |
| CN111254109B (en) | Method for in vitro fertilization and embryo culture of bovine oocytes and transport culture solution | |
| Mito et al. | Birth of piglets from in vitro–produced porcine blastocysts vitrified and warmed in a chemically defined medium | |
| CN107034173A (en) | The method of ox IVF Embryos culture and the nutrient solution used | |
| CN113201484B (en) | Method for improving cryopreservation and thawing of bovine in vitro fertilization blastocysts | |
| CN110066764A (en) | Promote the method for ox embryo in vitro culture oocyte in vitro maturation | |
| CN112501114B (en) | Method for improving cattle in-vitro fertilization efficiency | |
| CN108753689A (en) | The method of ox IVF Embryos culture | |
| CN108728401A (en) | The method of IVF of Oocyte in Bovine Embryo Culture and culture medium used | |
| CN108728404A (en) | Ox live body or the method for in vitro oocyte collection IVF Embryos culture | |
| Gardner | Blastocyst culture: toward single embryo transfers | |
| CN112980778B (en) | Method for culturing and cryopreserving bovine in-vitro fertilization embryos | |
| Herrid et al. | Optimization of a vitrification protocol for hatched blastocysts from the dromedary camel (Camelus dromedarius) | |
| CN112322579B (en) | Culture solution for cattle in-vitro fertilization and method for improving cattle in-vitro fertilization | |
| CN112914784B (en) | Bovine embryo segmentation method | |
| CN113151160B (en) | Method for improving cattle in-vitro fertilization efficiency and used freezing medium and thawing medium | |
| US11208622B2 (en) | Processing and use of reproductive tract fluids to improve the in vitro production of mammalian embryos | |
| EP3598895A1 (en) | An in vitro method for freezing mammalian embryos | |
| CN111280163B (en) | Egg extracting liquid for egg taking of live cattle and preparation method and application thereof | |
| RU2823596C1 (en) | Method for producing in vitro cattle embryos |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20190628 |
|
| WW01 | Invention patent application withdrawn after publication |