CN110066764A

CN110066764A - Promote the method for ox embryo in vitro culture oocyte in vitro maturation

Info

Publication number: CN110066764A
Application number: CN201910421475.0A
Authority: CN
Inventors: 王小武; 郭晶; 王娜; 郝少强; 赵明礼; 张月桥; 杨尚斌; 何伟; 马毅; 郭春明
Original assignee: Tianjin Li Mu Ding Feng Biotechnology Co Ltd; Tianjin Zhongke Boya Biological Breeding Research Co Ltd; Tianjin Bo Yu Li Husbandry Technology Co Ltd
Current assignee: Tianjin Boyu Limu Technology Co ltd; Tianjin Zhongke Boya Biological Breeding Research Co ltd
Priority date: 2019-05-21
Filing date: 2019-05-21
Publication date: 2019-07-30

Abstract

The present invention relates to the methods for promoting ox embryo in vitro culture oocyte in vitro maturation.Wherein the method for ox IVF Embryos culture includes the following steps: the acquisition and maturation in vitro of (1) egg mother cell, when carrying out maturation in vitro operation, it will acquire in vitro or living body acquisition gained COCs is washed 1 time in oocyte maturation culture solution, it is then transferred in new maturation culture solution and cultivates 22-24h, condition of culture is 38.8 DEG C, 5.5-6.5%CO2, saturated humidity；(2) in vitro fertilization；(3) In vitro culture and preservation, when preservation, can be placed in freezen protective in liquid nitrogen.Particularly, oocyte in vitro maturation rate can effectively improve using the method for the present invention mature liquid especially therein.

Description

Promote the method for ox embryo in vitro culture oocyte in vitro maturation

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 ox IVF Embryos culture, in addition, the invention further relates to the correlation cultures of ox IVF Embryos culture Application of the liquid in ox IVF Embryos culture.Further, the invention further relates to use the ox IVF Embryos culture Related culture solution carry out the culture of ox IVF Embryos method.In particular, the side of ox IVF Embryos culture of the present invention Method has excellent technical effect, such as can obtain higher culture efficiency in the method for low cost.In particular, the present invention relates to And a kind of from Niu Jinhang living body or from acquisition body egg mother cell and then carry out in vitro fertilization and Embryo Culture method.Particularly It is that the present invention relates to a kind of and promotes in ox embryo in vitro incubation the method for oocyte in vitro maturation.

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.

Embryo production in vitro (IVP) technology, in particular, for example ovum pick-up-embryo production in vitro (OPU-IVP) technology are Excellent individual egg mother cell is acquired by living body, through maturation in vitro (IVM), (IVF) in vitro fertilization, In vitro culture (IVC) Deng a large amount of embryo qualities can be quickly obtained under in vitro conditions.Compared to internal embryo, production efficiency is up to 4~8 times (Blondin P.Logistics of large scale commercial IVF embryo production.Reprod Fertil Dev.2016Jan；29 (1): 32-36), it can effectively promote excellent female livestock breed potentiality.Currently, external ox OPU-IVP Technology has reached higher level (Merton JS, de Roos AP, Koenen EP, Roelen BA, Vos PL, Mullaart E,Knijn HM.Bovine OPU-derived oocytes can be matured in vitro for 16-28h with similar developmental capacity.Reprod Domest Anim.2012Dec；47(6):1037-42； Oliveira LH,Sanches CP,Seddon AS,Veras MB,Lima FA,Monteiro PLJ Jr,Wiltbank MC,Sartori R.Short communication:Follicle superstimulation before ovum pick- up for in vitro embryo production in Holstein cows.J Dairy Sci.2016Nov；99 (11):9307-9312；Cavalieri FLB,Morotti F,Seneda MM,Colombo AHB,Andreazzi MA, Emanuelli IP,Rigolon LP.Improvement of bovine in vitro embryo production by ovarian follicular wave synchronization prior to ovum pick- up.Theriogenology.2018Sep 1；117:57-60), commercial applications have been realized in some areas.But China is in the skill The deficiencies of art field is lower there are still efficiency limits its use.

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.

As the development of modern agriculture science and technology accelerates genetic breeding to more make full use of the breeding potential of breeding cow Process, the efficient breeding new technology of application becomes inevitable in production practice.Ovum pick-up (Ovum pick UP, OPU) and body Outer fertilization technique (In Vitro Fertilization, IVF) is embryo's work that 1980s fast development is got up Journey 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 The farmer that technology has become the animal husbandry developed countries such as American-European and Oceania is to expand breeding cow group and use important Reproduction technique.However, using conventional ox Embryo Culture system (CR1aa and SOF liquid), ox blastocyst rate in vitro fertilization compared with It is low, and embryo quality, also far away from internal embryo, the pregnancy rate after leading to embryo transplantation acceptor is low, therefore how to improve blastaea Developmental rate and embryo quality become 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.Ox technology in vitro fertilization by the maturation in vitro of egg mother cell, sperm In-vitro Capacitation, by The influence of many aspects such as the vitro culture conditions of smart 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, Effect of free such as Rosenkrans amino acids and vitamins on cleavage and developmental rate of bovine zygotes In vitro.J Anim Sci, 1994.72 (2): p.434-7) Charles Rosenkrans 1 (CR1) culture solution developed With (Tervit, H.R., D.G.Whittingham, and L.E.Rowson, Successful the culture in such as Tervit Vitro of sheep and cattle ova.J Reprod Fertil, 1972.30 (3): the synthesis p.493-7) developed is defeated Oviduct liquid (Synthetic Oviductal Fluid, SOF), is continuously improved gradually formed two kinds of cultivating systems for many years.According to (Sagirkaya, H., et al., Developmental potential of bovine such as Hakan Sagirkaya oocytes cultured in different maturation and culture conditions.Anim Reprod P.225-40) and (Somfai, T., et al., Development of bovine such as Somfai Sci, 2007.101 (3-4): embryos cultured in CR1aa and IVD101media using different oxygen tensions and Culture systems.Acta Vet Hung, 2010.58 (4): p.465-74) research achievement shows that CR1aa culture solution is used There is preferable effect in ox Embryo Culture, can be widely applied to the Embryo Culture of ox；Thompson, J.G. etc. (Thompson, J.G.,et al.,Effect of inhibitors and uncouplers of oxidative phosphorylation during compaction and blastulation of bovine embryos cultured in vitro.J Reprod Fertil, 2000.118 (1): p.47-55) and (Feugang, J.M., the O.Camargo- such as Jean M.Feugang 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 a kind of training for being suitable for ox Embryo Culture The system of supporting.(Zhang Zhiping, An Zhixing, rust, Zhang Yong, the optimization Xibei Univ. of Agricultural & Forest Science & Technology of ox Embryo Culture system such as Zhang Zhiping Journal, 2006.34) and (Sang Guojun, bovine oocyte and external embryo culture technique the study .2008) result of study such as Sang Guojun It has been shown that, CR1aa the and SOF culture solution by optimization are adapted to the embryo in vitro culture of ox, obtain 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 of such as de Matos having high glutathione(GSH)level after bovine in vitro maturation on embryo development effect of beta-mercaptoethanol,cysteine and Cystine.Theriogenology, 2000.53 (3): p.761-71) once by adding β-mercapto during Embryo Culture in vitro Base ethyl alcohol, 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 the practice of ox fast-propagation.Cause This, how reducing cost and improving ox 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 ox technical system in vitro fertilization, and herein On the basis of improve, blastocyst rate has different degrees of raising, blastocyst rate average out to 30%-40%.For capsule Idioplasm amount can be assessed by blastomere sum, ICM cell number/total cell number ratio, apoptosis rate.Blastomere The difference in sum stage according to locating for blastaea and different, (Iwasaki, S.and T.Nakahara, the Cell such as S.Iwasaki number and incidence of chromosomal anomalies in bovine blastocysts fertilized in vitro followed by culture in vitro or in vivo in rabbit Oviducts.Theriogenology, 1990.33 (3): p.669-75) the ox early blastocyst total cell number average out to 44 obtained, ICM cell number/blastaea total cell number ratio is 15.8% or so；Andrew J.Watson etc. (Watson, A.J., et 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 ox blastomere apoptosis rate is about 7.7%-13%.

CN103898046B (Chinese Patent Application No. 201410073635.4) discloses one kind, and to be exclusively used in ox in vitro fertilization The culture solution of embryo, the culture formula of liquid are as follows: NaCl 109.5mM, KCl 3.1mM, NaHCO3 26.2mM, MgCl2 6H2O 0.8mM, KH2PO31.19mM, Sodium Pyruvate 0.4mM, glucose 1.5mM, galactonic acid calcium 5mM, 10v/v% tire ox blood Clearly, L-Glutamine 1mM, 2v/v% essential amino acid, 1v/v% nonessential amino acid and glutathione 3mM, are prepared with water；Institute Stating essential amino acid is the aqueous solution prepared after following amino acid mixes in proportion, wherein each amino acid content are as follows: L- hydrochloric acid essence Propylhomoserin 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；It is described 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 One water object 1.5g/L of 0.89g/L, L- asparagine, L-Aspartic acid 1.33g/L, Pidolidone 1.47g/L, glycine 0.75g/L, L-PROLINE 1.15g/L and Serine 1.05g/L.Ox IVF Embryos are placed in above-mentioned culture solution and are carried out In vitro culture, it is believed that be substantially better than the control group for being not added with GSH as the result is shown, improve blastocyst rate and embryo quality, drop The cost of low produced in vitro embryo, for ox IVF technology be applied to practice experiment basis being provided, can greatly accelerate genetic breeding into Journey.

Chinese Patent Application No. 2018105767643 discloses a kind of method of IVF of Oocyte in Bovine Embryo Culture It is incorporated herein by reference with the full content of culture medium used, the document.Other bibliography: Chen great Yuan fertilization biology Learn .2003, Beijing: Science Press；Fang Junshun bovine embryo vitro produces technical research, and " Chinese excellent MA theses are complete Literary database agricultural science and technology volume " .2007, (the 4th phase)；Cao Haiqing cumulus cell and condition of culture imitate ox embryo production in vitro Influence " Chinese excellent MA theses full-text database agricultural science and technology volume " .2008 of rate, (the 9th phase)；I.H.KIM et al.Effect of exogenous glutathione on the in vitro fertilization of bovine oocytes.Theriogenology.1999,Vol.52(3)。

However, the method for having the culture of the improved ox IVF Embryos of performance is still expected in this field, especially expect There is the method for promoting oocyte in vitro maturation in ox embryo in vitro incubation.

Summary of the invention

The purpose of the present invention is to provide a kind of methods of the ox IVF Embryos culture of performance improvement, especially expect A kind of improved method for promoting oocyte in vitro maturation in ox embryo in vitro incubation is provided.The present inventor has gone out Excellent technical effect is presented using the method for the present invention for the discovery of people's will material, and the present invention is consequently found that and be accomplished.

For this purpose, first aspect present invention provides a kind of maturation culture solution, be added in BY basic culture solution as Lower ingredient: the FBS of 100mL/L, the FSH of 10 μ g/mL, the LH of 10 μ g/mL, 1 μ g/mL E2,20ng/mL EGF.

Maturation culture solution according to a first aspect of the present invention, wherein the BY basic culture solution is a kind of basic culture solution, It includes the aqueous solutions of following component: 180~220mg/L of calcium chloride, nine 0.70~0.75mg/L of water ferric nitrate, potassium chloride 380 ~420mg/L, 90~100mg/L of magnesium sulfate, 6500~7000mg/L of sodium chloride, 130~150mg/L of sodium dihydrogen phosphate-water, 2000~2500mg/L of sodium bicarbonate, 40~60mg/L of sodium acetate, 20~30mg/L of l-Alanine, L-arginine hydrochloride 60~ 80mg/L, 25~35mg/L of L-Aspartic acid, 0.10~0.12mg/L of L-cysteine hydrochloride monohydrate, l-cysteine two 20~30mg/L of hydrochloride, 70~80mg/L of Pidolidone, 40~60mg/L of glycine, L-Histidine hydrochloride monohydrate 20 8~12mg/L of~25mg/L, L- hydroxyproline, 15~25mg/L of l-Isoleucine, 50~70mg/L of L-Leu, L-lysine 60~80mg/L of hydrochloride, 10~20mg/L of L-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, l-tyrosine disodium dihydrate 55~60mg/L, 20~30mg/L of Valine, 0.04~0.06mg/L of ascorbic acid, α-D- tocopherol phosphate 0.008~ 0.012mg/L, 0.008~0.012mg/L of biotin, 0.08~0.12mg/L of ostelin, 0.008~0.012mg/ of D-VB5 calcium L, 0.4~0.6mg/L of choline chloride, 0.008~0.012mg/L of folic acid, 0.04~0.06mg/L of inositol, three hydration menadiones are sub- 0.015~0.025mg/L of sodium bisulfate, 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, adenine 0.15~ 0.25mg/L, 0.8~1.2mg/L of Adenosine Triphosphate Disodium, 0.15~0.25mg/L of cholesterol, 2-deoxy-D-ribose 0.4~ 0.6mg/L, 800~1200mg/L of D-Glucose, 0.04~0.06mg/L of glutathione, 0.25~0.35mg/ of guanine hydrochloride L, 0.3~0.4mg/L of hypoxanthine sodium, 0.4~0.6mg/L of ribose, thymosin beta 10 .25~0.35mg/L, 4~6mg/ of Tween 80 L, 0.25~0.35mg/L of uracil, 0.3~0.4mg/L of xanthine sodium, phenol red 8~12mg/L.

Maturation culture solution according to a first 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 first 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.Maturation culture solution according to a first aspect of the present invention, wherein the BY base It further include copper sulphate in plinth culture solution, concentration is calculated as 0.05~0.1mg/L, such as 0.075mg/L with anhydride.

Maturation culture solution according to a first aspect of the present invention, wherein also added 10~20mmol/L (such as 15mmol/L) 4- hydroxyethyl piperazineethanesulfonic acid.

In the present invention, some abbreviations have following meaning: EGF --- epidermal growth factor, FSH --- ovarian follicle stimulation Element, FBS --- fetal calf serum, E2 --- estradiol, LH --- interstitialcellstimulating hormone (ICSH), HEPES --- 4- hydroxyethyl piperazine second sulphur Acid.

The maturation culture solution of any embodiment according to a first aspect of the present invention is added in BY basic culture solution Following ingredient: 50~100 μ g/mL (such as 75 μ g/mL) Sodium Hyaluronate, 100~200 μ g/mL (such as 150 μ g/mL) acid iodide Potassium, the 4- hydroxyethyl piperazineethanesulfonic acid of 10~20mmol/L (such as 15mmol/L), the FBS of 100mL/L, the FSH of 2 μ g/mL, 10 The EGF of E2,20ng/mL of the LH of μ g/mL, 1 μ g/mL.It has been unexpectedly discovered that micro when being added in maturation culture solution The dosage (reducing to about 1/5 when not adding both this) of FSH, and mature training can be greatly reduced when Sodium Hyaluronate and Potassiumiodate Feeding efficiency can't reduce, and have quite high maturing rate.It is high in view of the cost of FSH, and Sodium Hyaluronate and Potassiumiodate Source is easy to get, is cheap, therefore this this field that is replaced by realizes that the target of efficient ox IVF Embryos culture provides It may.

Further, second aspect of the present invention provides a kind of method of ox IVF Embryos culture comprising following step It is rapid:

(1) acquisition of egg mother cell and maturation in vitro

In vitro acquisition: taking slaughterhouse ovary to be contained in the insulation barrel for adding dual anti-physiological saline, under the conditions of 30.5-33 DEG C, Transport laboratory in 4h back；The ovarian follicle of surface 2-8mm is extracted, precipitating is collected, is picked under stereomicroscope at least containing the ovarian cumulus that haves three layers The egg mother cell COCs (that is, cumulus oocytes complesxes) of cell encapsulation is washed 2 times in egg-cleaning liquid, is removed extra miscellaneous Matter；

Living body acquisition: carrying out ovum pick-up to ox, gained liquor folliculi is picked under stereomicroscope at least containing 3 layers of ovarian cumulus The cumulus oocytes complesxes (COCs) of cell encapsulation are put into the maturation culture solution comprising HEPES 38.8 DEG C, transport in 3h Go back to laboratory；

It acquisition in vitro or living body acquisition gained COCs will wash 1 time, then turn in oocyte maturation culture solution above It moves on in new maturation culture solution and cultivates 22-24h, 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, contain sperm injection There is sperm to prepare in the 15mL centrifuge tube of culture solution, 328 × g is centrifuged 2 times, and each 5min abandons supernatant after centrifugation；By 300 μ L essences Liquid prepares culture solution and above-mentioned centrifuge tube is added, 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 Incubator, 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 Culture It is cultivated in liquid, is denoted as the 1st day of Embryo Culture at this time, condition of culture is 38.8 DEG C, 6%O2,88%N2, saturated humidity, the 3rd day Record cleavage rates；7th day record blastocyst rate, until (it is hatched blastocyst number divided by blastaea number institute to statistics hatching rate at the 9th day Obtain percentage), and carry out Quality Identification；

It can be washed 3 times in saving liquid with embryo, be transferred to freezing liquid after balancing 10min in equilibrium liquid, filled by 5 sections Liquid method 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 Tubule takes out rapidly, is placed in freezen protective in liquid nitrogen.

Method according to a second 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.

Method according to a second aspect of the present invention, wherein in step (1), the egg-cleaning liquid is to be added to 3mg/mL cow's serum The BY basic culture solution of albumin.

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, bovine serum albumin(BSA) is usually added in preparatory prepared BY basic culture solution of the present invention to its phase The concentration that should be required, this preparation method are also that those skilled in the art are common.

Method according to a second aspect of the present invention, wherein in step (1), the maturation culture solution such as first aspect present invention Described in any embodiment.

Method according to a second 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 bovine serum albumin(BSA) (BSA), 100U/ml mould The aqueous solution of element, 100 μ g/ml streptomysins.

Method according to a second 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 bovine serum albumin(BSA) (BSA), 10mM Caffeine, 100U/ml penicillin, 100 μ g/ml streptomysins aqueous solution.

Method according to a second 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, The required amino of 1.5mM glucose, 5mM galactonic acid calcium, 2.5v/v% fetal calf serum (FBS), L-Glutamine 1mM, 2v/v% Acid, 1v/v% nonessential amino acid, the glutathione of 3mM, sodium citrate 0.04w/v%, maltose 0.02w/v% it is water-soluble Liquid；The essential amino acid is that following amino acid ratio by weight is added: L- R-gene 6.32g, two hydrochloric acid of l-cysteine One water object 2.1g of salt 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.

In the present invention, such as in step (3), preservation liquid, equilibrium liquid, freezing liquid etc. used in embryo 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 of sale^TM Cleave。

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.

Follicular stimulating hormone is a kind of hormone of anterior pituitary basophil cell secretion, and ingredient is glycoprotein, because of discovery earliest Its stimulation to follicle maturity and obtain (English: Follicle-stimulating hormone, referred to as FSH, also known as Flitropin).The chemical structure of FSH is glycoprotein, by two Asia fostimon (Ford illiteracy) base peptide chains of a and b with covalent Key is combined into.

Detailed description of the invention

Fig. 1: influence of the mature liquid to ox COCs maturation effect

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.Such as FSH and LH used in the present invention can be easy Ground is bought from Beijing than Brunswick biotech company.In another example the fetal calf serum that the present invention uses can be purchased readily from market Its standardize commercial form, such as can be bought from various agents Gibco company Australia fetal calf serum (article No.: 10099141), New Zealand's fetal calf serum (article No.: 10091148), North America fetal calf serum (article No.: 16000044), Mexico's tire Cow's serum (article No.: 10437028) etc., in the test of the context of the invention, if not otherwise specified, fetal calf serum used is Australia fetal calf serum (article No.: 10099141) of Gibco company.

Embodiment 1: the cultural method of ox IVF Embryos (in vitro without selenium)

One, reagent

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 BY basic culture solution of 3mg/mL bovine serum albumin(BSA).

Maturation culture solution: 100mL/L FBS, 10 μ g/mL FSH, 10 μ g/mL LH, 1 μ g/mL E2,20ng/mL are added to The BY basic culture solution of EGF.

HEPES maturation culture solution: 15mmol/L HEPES, 100mL/L FBS, 10 μ g/mL FSH, 10 μ g/mL are added to The BY basic culture solution of LH, 1 μ g/mL E2,20ng/mL EGF.

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 bovine serum albumin(BSA) (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 bovine serum albumin(BSA) (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 ox blood (FBS), L-Glutamine 1mM, 2v/v% essential amino acid, 1v/v% nonessential amino acid, the glutathione of 3mM, citron clearly The aqueous solution of sour sodium 0.04w/v%, maltose 0.02w/v%；The essential amino acid is that following amino acid ratio by weight adds Add: 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.

Two, ox is in vitro fertilization and Embryo Culture:

Step (1), the acquisition of egg mother cell and maturation in vitro

It takes slaughterhouse ovary to be contained in the insulation barrel for adding dual anti-physiological saline, under the conditions of 30.5-33 DEG C, transports reality in 4h back Test room；The ovarian follicle of surface 2-8mm is extracted, precipitating is collected, is picked under stereomicroscope at least containing the cumulus cell package that haves three layers Egg mother cell COCs (that is, cumulus oocytes complesxes) 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 22-24h (practical operation 24 hours), condition of culture are 38.8 DEG C, 5.5-6.5%CO2, saturated humidity；

It is step (2), in vitro fertilization

The sperm suspension for calculating volume is added in the fertilization culture drop for filling egg mother cell, and culture plate is put into Incubator is incubated for smart ovum altogether 16-20h (practical operation 18 hours), and condition of culture is 38.8 DEG C, 5.5-6.5%CO2, saturation Humidity；

Step (3), In vitro culture and preservation

Three, the method for embryo's differential dyeing

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. blastaea three times using PBS-BSA cleaning, is put into confining liquid (1ml lowlenthal serum, 5 μ l Tween 80s and 8.995ml PBS in), 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 Life by 1:200 dilution caspase-3 specificity secondary antibody with confining liquid under the conditions of being protected from light Technologies company), avoid light place 1h at room temperature.Secondary antibody diluent is discarded under the conditions of being protected from light, and cleans 3 times with PBS, every time 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 ' s The significance of difference between multiple-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.

Four, maturation in vitro (IVM) effect --- the maturing rate of egg mother cell

The present embodiment 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.

Five, result

The present embodiment tests Chinese Cattle (Nanyang cattle uses as a servant kind).As a result, cleavage rates 88.7%, morula Rate is 63.7%, blastocyst rate 51.2%, apoptosis rate 5.1%；In addition, hatching rate was up to 72.3% at the 9th day.Ovum is female thin The maturing rate of born of the same parents' maturation in vitro is up to 75.6%.

In a complementary testing, referring to the method for the present embodiment 1, it is directed to Holstein cow (dairy breeds), west gate respectively Three kinds of Simmental Cattle Chromosome (beef breed), CHINESE BUFFALO (labour kind) oxen are tested, and as a result: cleavage rates are in 84~89% ranges Interior, morula rate in 62~65% ranges, blastocyst rate in 50~53% ranges, apoptosis rate is in 4~7% ranges Interior, the 9th day hatching rate is in 71~75% ranges；Holstein cow, Simmental, the ovum of three kinds of oxen of CHINESE BUFFALO are female The maturing rate of cells in vitro maturation is respectively 69.3%, 65.6%, 57.8%.

Embodiment 2: the cultural method of ox IVF Embryos (living body is without selenium)

One, reagent

Add dual anti-physiological saline, egg-cleaning liquid: same as Example 1.

Fertilization culture solution, sperm prepare culture solution, embryo medium, BY basic culture solution: same as Example 1.

Two, ox is in vitro fertilization and Embryo Culture:

Step (1), the acquisition of egg mother cell and maturation in vitro

Ovum pick-up is carried out to ox, gained liquor folliculi is picked under stereomicroscope at least containing 3 layers of cumulus cell package Cumulus oocytes complesxes (COCs), be put into the maturation culture solution comprising HEPES 38.8 DEG C, transport laboratory back in 3h；

It is step (2), in vitro fertilization

Step (3), In vitro culture and preservation

Three, the method for embryo's differential dyeing

Four, maturation in vitro (IVM) effect --- the maturing rate of egg mother cell

Five, result

The present embodiment tests Chinese Cattle (Nanyang cattle uses as a servant kind).As a result, cleavage rates 87.4%, morula Rate is 64.2%, blastocyst rate 51.8%, apoptosis rate 4.8%；In addition, hatching rate was up to 71.1% at the 9th day.Ovum is female thin The maturing rate of born of the same parents' maturation in vitro is up to 76.3%.

In a complementary testing, referring to the method for example 2 above, respectively for Holstein cow (dairy breeds), west Three kinds of door Simmental Cattle Chromosome (beef breed), CHINESE BUFFALO (labour kind) oxen are tested, and as a result: cleavage rates are in 83~89% models In enclosing, morula rate in 61~65% ranges, blastocyst rate in 50~54% ranges, apoptosis rate is in 4~6% ranges Interior, the 9th day hatching rate is in 72~75% ranges；Holstein cow, Simmental, the ovum of three kinds of oxen of CHINESE BUFFALO are female The maturing rate of cells in vitro maturation is respectively 68.8%, 66.3%, 58.3%.

Embodiment 3: the cultural method (in vitro selenium copper) of ox IVF Embryos

The main distinction of the present embodiment and example 1 above is that also additional addition 0.25mg/L is sub- in BY basic culture solution Sodium selenate and 0.075mg/L anhydrous cupric sulfate.

One, reagent

Add dual anti-physiological saline, egg-cleaning liquid: same as Example 1.

Fertilization culture solution, sperm prepare culture solution, embryo medium: same as Example 1.

BY basic culture solution: on the basis of the BY basic culture solution used in embodiment 1 is formulated, also supplement is added to 0.25mg/L sodium selenite and 0.075mg/L anhydrous cupric sulfate.

Two, ox is in vitro fertilization and Embryo Culture:

Step (1), the acquisition of egg mother cell and maturation in vitro

It is step (2), in vitro fertilization

Step (3), In vitro culture and preservation

Three, the method for embryo's differential dyeing

Four, maturation in vitro (IVM) effect --- the maturing rate of egg mother cell

Five, result

The present embodiment tests Chinese Cattle (Nanyang cattle uses as a servant kind).As a result, cleavage rates 88.2%, morula Rate is 64.2%, blastocyst rate 50.8%, apoptosis rate 5.6%；In addition, hatching rate was up to 73.4% at the 9th day.Ovum is female thin The maturing rate of born of the same parents' maturation in vitro 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 3 above, respectively for Holstein cow (dairy breeds), west Three kinds of door Simmental Cattle Chromosome (beef breed), CHINESE BUFFALO (labour kind) oxen are tested, and as a result: cleavage rates are in 84~90% models In enclosing, morula rate in 61~65% ranges, blastocyst rate in 50~54% ranges, apoptosis rate is in 3~6% ranges Interior, the 9th day hatching rate is in 71~76% ranges；Holstein cow, Simmental, the ovum of three kinds of oxen of CHINESE BUFFALO are female The maturing rate of cells in vitro maturation is respectively 84.5%, 79.5%, 73.2%, and the maturing rate relative to 1 method of embodiment is divided equally Do not increase about 14~15 percentage points.The above results, although 1 method of the embodiment of the present invention can obtain the knot being entirely satisfactory Fruit, however, having now surprisingly been found that is, it can when adding micro selenides and copper sulphate in BY basic culture solution of the present invention The maturing rate of oocyte in vitro maturation is dramatically increased, this is significantly especially no any teaching in prior art The addition of above-mentioned selenides and copper sulphate can dramatically increase the technical teaching of the maturing rate of oocyte in vitro maturation.

In a complementary testing, with reference to the present embodiment 3, 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), in four kinds of ox kinds, ovum It is almost the same with 3 result of embodiment in both cases to split rate, morula rate, blastocyst rate, four parameter of apoptosis rate, to four kinds of ox kinds Cleavage rates in 84~89% ranges, morula rate in 63~65% ranges, blastocyst rate is in 52~55% ranges Interior, apoptosis rate in 3~6% ranges, the 9th day hatching rate in 70~75% ranges, such as Chinese Cattle Cleavage rates be respectively 87.7% and 88.5%；But the maturing rate of oocyte in vitro maturation relative to 1 result of embodiment not See there is increase, in the case of two kinds to Chinese Cattle, Holstein cow, Simmental, CHINESE BUFFALO oocyte in vitro maturation Maturing rate be respectively 73~75%, 67~70%, 65~66%, 55~57%.This shows selenides in BY basal medium It will not influence cleavage rates, morula rate, blastocyst rate, four parameter of apoptosis rate whether with the addition of copper sulphate, but only while adding Add the maturing rate that oocyte in vitro maturation could be effectively improved when selenides and copper sulphate.

Embodiment 4: the cultural method (living body selenium copper) of ox IVF Embryos

The main distinction of the present embodiment and example 2 above is that also additional addition 0.25mg/L is sub- in BY basic culture solution Sodium selenate and 0.075mg/L anhydrous cupric sulfate.

One, reagent

Add dual anti-physiological saline, egg-cleaning liquid: same as Example 1.

Two, ox is in vitro fertilization and Embryo Culture:

Step (1), the acquisition of egg mother cell and maturation in vitro

It is step (2), in vitro fertilization

Step (3), In vitro culture and preservation

Three, the method for embryo's differential dyeing

Four, maturation in vitro (IVM) effect --- the maturing rate of egg mother cell

Five, result

The present embodiment tests Chinese Cattle (Nanyang cattle uses as a servant kind).As a result, cleavage rates 88.4%, morula Rate is 64.7%, blastocyst rate 50.3%, apoptosis rate 5.1%；In addition, hatching rate was up to 74.4% at the 9th day.Ovum is female thin The maturing rate of born of the same parents' maturation in vitro is up to 89.6%, about 13 percentage points of maturing rate increase relative to 2 method of embodiment.

In a complementary testing, referring to the method for example 4 above, respectively for Holstein cow (dairy breeds), west Three kinds of door Simmental Cattle Chromosome (beef breed), CHINESE BUFFALO (labour kind) oxen are tested, and as a result: cleavage rates are in 85~90% models In enclosing, morula rate in 62~65% ranges, blastocyst rate in 51~54% ranges, apoptosis rate is in 4~6% ranges Interior, the 9th day hatching rate is in 71~75% ranges；Holstein cow, Simmental, the ovum of three kinds of oxen of CHINESE BUFFALO are female The maturing rate of cells in vitro maturation is respectively 85.2%, 79.1%, 74.32%, and the maturing rate relative to 2 method of embodiment is divided equally Do not increase about 14~15 percentage points.The above results, although 2 method of the embodiment of the present invention can obtain the knot being entirely satisfactory Fruit, however, having now surprisingly been found that is, it can when adding micro selenides and copper sulphate in BY basic culture solution of the present invention The maturing rate of oocyte in vitro maturation is dramatically increased, this is significantly especially no any teaching in prior art The addition of above-mentioned selenides and copper sulphate can dramatically increase the technical teaching of the maturing rate of oocyte in vitro maturation.

In a complementary testing, with reference to the present embodiment 4, 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), in four kinds of ox kinds, ovum It is almost the same with 4 result of embodiment in both cases to split rate, morula rate, blastocyst rate, four parameter of apoptosis rate, to four kinds of ox kinds Cleavage rates in 84~89% ranges, morula rate in 63~66% ranges, blastocyst rate is in 53~55% ranges Interior, apoptosis rate in 3~5% ranges, the 9th day hatching rate in 70~74% ranges, such as Chinese Cattle Cleavage rates be respectively 88.4% and 88.1%；But the maturing rate of oocyte in vitro maturation relative to 2 result of embodiment not See there is increase, in the case of two kinds to Chinese Cattle, Holstein cow, Simmental, CHINESE BUFFALO oocyte in vitro maturation Maturing rate be respectively 72~74%, 67~71%, 65~67%, 55~58%.This shows selenides in BY basal medium It will not influence cleavage rates, morula rate, blastocyst rate, four parameter of apoptosis rate whether with the addition of copper sulphate, but only while adding Add the maturing rate that oocyte in vitro maturation could be effectively improved when selenides and copper sulphate.

Embodiment 5: the cultural method of ox IVF Embryos (in vitro without selenium)

Referring to example 1 above and the method for the corresponding complementary testing in the embodiment 1, in BY basic culture solution not Selenium and copper are added, different is only that BY basic culture solution used uses the aqueous solution (basis BY of the present embodiment as following formula instead In culture solution, each composition is same as Example 1, is only that ratio varies slightly): calcium chloride 180mg/L, nine water ferric nitrates 0.75mg/L, potassium chloride 380mg/L, magnesium sulfate 100mg/L, sodium chloride 6500mg/L, sodium dihydrogen phosphate-water 150mg/L, carbon Sour hydrogen sodium 2000mg/L, sodium acetate 60mg/L, l-Alanine 20mg/L, L-arginine hydrochloride 80mg/L, L-Aspartic acid 25mg/L, L-cysteine hydrochloride monohydrate 0.12mg/L, l-cysteine dihydrochloride 20mg/L, Pidolidone 80mg/ L, glycine 40mg/L, L-Histidine hydrochloride monohydrate 25mg/L, L- hydroxyproline 8mg/L, l-Isoleucine 25mg/L, L-Leu 50mg/L, L lysine HCL 80mg/L, L-Methionine 10mg/L, L-phenylalanine 30mg/L, L-PROLINE 30mg/L, Serine 30mg/L, L-threonine 25mg/L, L-Trp 12mg/L, l-tyrosine disodium dihydrate 55mg/ L, Valine 30mg/L, ascorbic acid 0.04mg/L, α-D- tocopherol phosphate 0.012mg/L, biotin 0.008mg/L, Ostelin 0.12mg/L, D-VB5 calcium 0.008mg/L, choline chloride 0.6mg/L, folic acid 0.008mg/L, inositol 0.06mg/L, Three hydration Menadione Sodium Bisulfite 0.015mg/L, niacin 0.03mg/L, niacinamide 0.02mg/L, p- aminobenzoic acid 0.06mg/L, pyridoxine hydrochloride 0.04mg/L, riboflavin 0.012mg/L, thiamine hydrochloride 0.008mg/L, retinyl acetate 0.2mg/L, adenine sulfate 8mg/L, adenine 0.25mg/L, Adenosine Triphosphate Disodium 0.8mg/L, cholesterol 0.25mg/L, It is 2-deoxy-D-ribose 0.4mg/L, D-Glucose 1200mg/L, glutathione 0.04mg/L, guanine hydrochloride 0.35mg/L, secondary Xanthine sodium 0.3mg/L, ribose 0.6mg/L, thymosin beta 10 .25mg/L, Tween 80 6mg/L, uracil 0.25mg/L, xanthine Sodium 0.4mg/L, phenol red 8mg/L.

As a result: the result of the result of each test parameters and the corresponding complementary testing in example 1 above and the embodiment 1 Essentially identical, for example, to Chinese Cattle, cleavage rates 88.3%, morula rate be 64.2%, blastocyst rate 51.6%, apoptosis rate 5.3%, at the 9th day, hatching rate was up to 71.7%, and the maturing rate of oocyte in vitro maturation is up to 74.5%；To He Sitan The maturing rate of the oocyte in vitro maturation of ox, three kinds of Simmental, CHINESE BUFFALO oxen is respectively 68.8%, 65.9%, 57.3%.

Embodiment 6: the cultural method of ox IVF Embryos (living body is without selenium)

Referring to example 2 above and the method for the corresponding complementary testing in the embodiment 2, in BY basic culture solution not Selenium and copper are added, different is only that BY basic culture solution used uses the aqueous solution (basis BY of the present embodiment as following formula instead In culture solution, each composition is same as Example 2, is only that ratio varies slightly): calcium chloride 220mg/L, nine water ferric nitrates 0.70mg/L, potassium chloride 420mg/L, magnesium sulfate 90mg/L, sodium chloride 7000mg/L, sodium dihydrogen phosphate-water 130mg/L, carbonic acid Hydrogen sodium 2500mg/L, sodium acetate 40mg/L, l-Alanine 30mg/L, L-arginine hydrochloride 60mg/L, L-Aspartic acid 35mg/ L, L-cysteine hydrochloride monohydrate 0.10mg/L, l-cysteine dihydrochloride 30mg/L, Pidolidone 70mg/L, sweet ammonia Sour 60mg/L, L-Histidine hydrochloride monohydrate 20mg/L, L- hydroxyproline 12mg/L, l-Isoleucine 15mg/L, L- are bright Propylhomoserin 70mg/L, L lysine HCL 60mg/L, L-Methionine 20mg/L, L-phenylalanine 20mg/L, L-PROLINE 50mg/ L, Serine 20mg/L, L-threonine 35mg/L, L-Trp 8mg/L, l-tyrosine disodium dihydrate 60mg/L, L- figured silk fabrics Propylhomoserin 20mg/L, ascorbic acid 0.06mg/L, α-D- tocopherol phosphate 0.008mg/L, biotin 0.012mg/L, ostelin 0.08mg/L, D-VB5 calcium 0.012mg/L, choline chloride 0.4mg/L, folic acid 0.012mg/L, inositol 0.04mg/L, three hydrations Menadione Sodium Bisulfite 0.025mg/L, niacin 0.02mg/L, niacinamide 0.03mg/L, p- aminobenzoic acid 0.04mg/L, salt Sour Benadon 0.06mg/L, riboflavin 0.008mg/L, thiamine hydrochloride 0.012mg/L, retinyl acetate 0.1mg/L, sulphur Adenine 12mg/L, adenine 0.15mg/L, Adenosine Triphosphate Disodium 1.2mg/L, cholesterol 0.15mg/L, 2- deoxidation-D- Ribose 0.6mg/L, D-Glucose 800mg/L, glutathione 0.06mg/L, guanine hydrochloride 0.25mg/L, hypoxanthine sodium 0.4mg/L, ribose 0.4mg/L, thymosin beta 10 .35mg/L, Tween 80 4mg/L, uracil 0.35mg/L, xanthine sodium 0.3mg/ L, phenol red 12mg/L.

As a result: the result of the result of each test parameters and the corresponding complementary testing in example 2 above and the embodiment 2 Essentially identical, for example, to Chinese Cattle, cleavage rates 87.9%, morula rate be 64.6%, blastocyst rate 50.9%, apoptosis rate 5.1%, at the 9th day, hatching rate was up to 72.2%, and the maturing rate of oocyte in vitro maturation is up to 75.3%；To He Sitan The maturing rate of the oocyte in vitro maturation of ox, three kinds of Simmental, CHINESE BUFFALO oxen is respectively 69.5%, 65.3%, 57.8%.

Embodiment 7: the cultural method (in vitro selenium copper) of ox IVF Embryos

Referring to example 3 above and the method for the corresponding complementary testing in the embodiment 3, add in BY basic culture solution Add selenium and copper, different is only that BY basic culture solution used is to augment to add again on the basis of the BY basic culture solution of embodiment 5 Add sodium selenite 0.3mg/L, copper sulphate (in terms of anhydride) 0.05mg/L.

As a result: the result of the result of each test parameters and the corresponding complementary testing in example 3 above and the embodiment 3 Essentially identical, for example, to Chinese Cattle, cleavage rates 87.4%, morula rate be 64.9%, blastocyst rate 51.6%, apoptosis rate 5.4%, at the 9th day, hatching rate was up to 72.5%, and the maturing rate of oocyte in vitro maturation is up to 89.1%；To He Sitan The maturing rate of the oocyte in vitro maturation of ox, three kinds of Simmental, CHINESE BUFFALO oxen is respectively 85.6%, 80.2%, 73.6%.

Embodiment 8: the cultural method (living body selenium copper) of ox IVF Embryos

Referring to example 4 above and the method for the corresponding complementary testing in the embodiment 4, add in BY basic culture solution Add selenium and copper, different is only that BY basic culture solution used is to augment to add again on the basis of the BY basic culture solution of embodiment 6 Add sodium selenite 0.2mg/L, copper sulphate (in terms of anhydride) 0.1mg/L.

As a result: the result of the result of each test parameters and the corresponding complementary testing in example 4 above and the embodiment 4 Essentially identical, for example, to Chinese Cattle, cleavage rates 87.8%, morula rate be 65.5%, blastocyst rate 51.2%, apoptosis rate 4.4%, at the 9th day, hatching rate was up to 73.8%, and the maturing rate of oocyte in vitro maturation is up to 90.2%；To He Sitan The maturing rate of the oocyte in vitro maturation of ox, three kinds of Simmental, CHINESE BUFFALO oxen is respectively 84.7%, 80.5%, 73.8%.

Embodiment 11: the cultural method of ox IVF Embryos (in vitro without selenium)

The maturation culture solution that the present embodiment uses is changed to: be added to 75 μ g/mL Sodium Hyaluronates, 150 μ g/mL Potassiumiodates, The BY basic culture solution of 100mL/L FBS, 2 μ g/mL FSH, 10 μ g/mL LH, 1 μ g/mL E2,20ng/mL EGF；

The HEPES maturation culture solution that the present embodiment uses is changed to: being added to 75 μ g/mL Sodium Hyaluronates, 150 μ g/mL iodine Sour potassium, 15mmol/L HEPES, 100mL/L FBS, 2 μ g/mL FSH, 10 μ g/mL LH, 1 μ g/mL E2,20ng/mL EGF BY basic culture solution；

Remaining reagent reagent and operations method used in the present embodiment are with embodiment 1, such as the wherein BY Basic culture solution is same as Example 1.

As a result: the present embodiment tests Chinese Cattle (Nanyang cattle uses as a servant kind).As a result, cleavage rates 89.2%, mulberry Shen embryo rate is 63.1%, blastocyst rate 50.8%, apoptosis rate 5.4%；In addition, hatching rate was up to 73.7% at the 9th day.Ovum The maturing rate of mother cell maturation in vitro is up to 75.2%；Each result is substantially the same manner as Example 1.

In a complementary testing, referring to the method for the present embodiment 11, it is directed to Holstein cow (dairy breeds), west gate respectively Three kinds of Simmental Cattle Chromosome (beef breed), CHINESE BUFFALO (labour kind) oxen are tested, and as a result: cleavage rates are in 84~88% ranges Interior, morula rate in 63~65% ranges, blastocyst rate in 50~54% ranges, apoptosis rate is in 4~6% ranges Interior, the 9th day hatching rate is in 71~74% ranges；Holstein cow, Simmental, the ovum of three kinds of oxen of CHINESE BUFFALO are female The maturing rate of cells in vitro maturation is respectively 69.6%, 65.3%, 57.3%；Each result is substantially the same manner as Example 1.

Embodiment 12: the cultural method of ox IVF Embryos (living body is without selenium)

Remaining reagent reagent and operations method used in the present embodiment are with embodiment 2, such as the wherein BY Basic culture solution is same as Example 2.

As a result: the present embodiment tests Chinese Cattle (Nanyang cattle uses as a servant kind).As a result, cleavage rates 88.1%, mulberry Shen embryo rate is 63.8%, blastocyst rate 52.3%, apoptosis rate 5.1%；In addition, hatching rate was up to 70.8% at the 9th day.Ovum The maturing rate of mother cell maturation in vitro is up to 75.7%；Each result is substantially the same manner as Example 2.

In a complementary testing, referring to the method for example 12 above, respectively for Holstein cow (dairy breeds), west Three kinds of door Simmental Cattle Chromosome (beef breed), CHINESE BUFFALO (labour kind) oxen are tested, and as a result: cleavage rates are in 85~89% models In enclosing, morula rate in 60~64% ranges, blastocyst rate in 51~54% ranges, apoptosis rate is in 4~6% ranges Interior, the 9th day hatching rate is in 72~74% ranges；Holstein cow, Simmental, the ovum of three kinds of oxen of CHINESE BUFFALO are female The maturing rate of cells in vitro maturation is respectively 69.3%, 65.8%, 59.2%；Each result is substantially the same manner as Example 2.

Embodiment 13: the cultural method (in vitro selenium copper) of ox IVF Embryos

Remaining reagent reagent and operations method used in the present embodiment are with embodiment 3, such as the wherein BY Basic culture solution is same as Example 3.

As a result: the present embodiment tests Chinese Cattle (Nanyang cattle uses as a servant kind).As a result, cleavage rates 88.7%, mulberry Shen embryo rate is 63.6%, blastocyst rate 50.3%, apoptosis rate 5.2%；In addition, hatching rate was up to 73.8% at the 9th day.Ovum The maturing rate of mother cell maturation in vitro is up to 88.8%, relative to about 13 percentage points of maturing rate increase of 1 method of embodiment, with reality The result for applying 3 method of example is essentially identical.

In a complementary testing, referring to the method for example 13 above, respectively for Holstein cow (dairy breeds), west Three kinds of door Simmental Cattle Chromosome (beef breed), CHINESE BUFFALO (labour kind) oxen are tested, and as a result: cleavage rates are in 85~90% models In enclosing, morula rate in 61~64% ranges, blastocyst rate in 51~55% ranges, apoptosis rate is in 4~6% ranges Interior, the 9th day hatching rate is in 72~76% ranges；Holstein cow, Simmental, the ovum of three kinds of oxen of CHINESE BUFFALO are female The maturing rate of cells in vitro maturation is respectively 84.8%, 79.1%, 72.8%, and the maturing rate relative to 1 method of embodiment is divided equally Do not increase about 14~15 percentage points, the result relative to 3 method of embodiment is essentially identical.

The above results show in maturation culture solution or HEPES maturation culture solution, supplement add micro Sodium Hyaluronate and Potassiumiodate, and greatly reduce FSH concentration (reduce to 1/5), acquired results and largely using FSH and be not used Sodium Hyaluronate and Result when Potassiumiodate is suitable, is conducive to greatly reduce production cost (the usually every 1mg of commercially available FSH in view of FSH dosage is greatly reduced Price up to 200 yuan or more, and city's price of 50~100 yuan/g of Sodium Hyaluronate is very easy to find, and Potassiumiodate is more just Preferably), therefore this is significantly in production practice；In particular, without the above-mentioned hyaluronic acid of any teaching in prior art The addition of sodium and Potassiumiodate is able to maintain the maturing rate of oocyte in vitro maturation and greatly reduces the technology religion of the dosage of FSH It leads.

In a complementary testing, with reference to the present embodiment 13, do not added in maturation culture solution or HEPES maturation culture solution Sodium Hyaluronate (and the Potassiumiodate for only augmenting corresponding amount) does not add Potassiumiodate (and only augmenting corresponding amount Sodium Hyaluronate) When, in four kinds of ox kinds, cleavage rates, morula rate, blastocyst rate, four parameter of apoptosis rate in both cases with 3 result of embodiment It is almost the same, to the cleavage rates of four kinds of ox kinds in 85~89% ranges, morula rate is in 62~64% ranges, blastaea Rate in 52~56% ranges, apoptosis rate in 3~6% ranges, the 9th day hatching rate be in 70~74% ranges It is interior, such as the cleavage rates of Chinese Cattle be respectively 87.1% and 88.9%；But the maturing rate of oocyte in vitro maturation It there are no increase relative to 11 result of embodiment, to Chinese Cattle, Holstein cow, Simmental, China Water in the case of two kinds The maturing rate of the oocyte in vitro maturation of ox is respectively 72~74%, 67~71%, 63~66%, 55~58%.This shows It will not influence cleavage rates, mulberries whether the addition of Sodium Hyaluronate and Potassiumiodate in maturation culture solution or HEPES maturation culture solution Embryo rate, blastocyst rate, four parameter of apoptosis rate, but only while when adding Sodium Hyaluronate and Potassiumiodate could effectively improve ovum Greatly reduce the dosage of FSH while the maturing rate of mother cell maturation in vitro.

Embodiment 14: the cultural method (living body selenium copper) of ox IVF Embryos

Remaining reagent reagent and operations method used in the present embodiment are with embodiment 4, such as the wherein BY Basic culture solution is same as Example 4.

As a result: the present embodiment tests Chinese Cattle (Nanyang cattle uses as a servant kind).As a result, cleavage rates 88.7%, mulberry Shen embryo rate is 65.3%, blastocyst rate 49.8%, apoptosis rate 5.3%；In addition, hatching rate was up to 74.1% at the 9th day.Ovum The maturing rate of mother cell maturation in vitro is up to 89.3%, relative to about 13 percentage points of maturing rate increase of 2 method of embodiment, with reality The result for applying 4 method of example is essentially identical.

Fig. 1 shows the micrograph of influence of the mature liquid of the present embodiment 14 to ox COCs maturation effect, and A is to use this in figure Influence of the embodiment maturation liquid to ox COCs maturation effect；As contrasting, B is that the mature liquid of embodiment 2 is mature to ox COCs in figure The influence of effect, wherein maturity is schemed the ox COCs ovarian cumulus diffusion of A culture solution culture and is glued not as good as the figure A of embodiment 14 for display Even degree is higher than figure B culture solution；C is the micrograph for the egg mother cell that first polar body is discharged in the present embodiment 14 in figure, and D is this in figure The egg mother cell of first polar body is not discharged for embodiment 14.

In a complementary testing, referring to the method for example 14 above, respectively for Holstein cow (dairy breeds), west Three kinds of door Simmental Cattle Chromosome (beef breed), CHINESE BUFFALO (labour kind) oxen are tested, and as a result: cleavage rates are in 85~91% models In enclosing, morula rate in 62~64% ranges, blastocyst rate in 51~55% ranges, apoptosis rate is in 3~6% ranges Interior, the 9th day hatching rate is in 71~74% ranges；Holstein cow, Simmental, the ovum of three kinds of oxen of CHINESE BUFFALO are female The maturing rate of cells in vitro maturation is respectively 85.7%, 79.4%, 74.5%, and the maturing rate relative to 2 method of embodiment is divided equally Do not increase about 14~15 percentage points, the result relative to 4 method of embodiment is essentially identical.

In a complementary testing, with reference to the present embodiment 14, do not added in maturation culture solution or HEPES maturation culture solution Sodium Hyaluronate (and the Potassiumiodate for only augmenting corresponding amount) does not add Potassiumiodate and (and only augments the hyaluronic acid of corresponding amount Sodium) when, in four kinds of ox kinds, cleavage rates, morula rate, blastocyst rate, four parameter of apoptosis rate in both cases with 4 knot of embodiment Fruit is almost the same, to the cleavage rates of four kinds of ox kinds in 85~88% ranges, morula rate is in 63~65% ranges, capsule Embryo rate in 52~55% ranges, apoptosis rate in 3~6% ranges, the 9th day hatching rate be in 71~74% ranges It is interior, such as the cleavage rates of Chinese Cattle be respectively 88.9% and 88.5%；But the maturing rate of oocyte in vitro maturation It there are no increase relative to 12 result of embodiment, to Chinese Cattle, Holstein cow, Simmental, China Water in the case of two kinds The maturing rate of the oocyte in vitro maturation of ox is respectively 70~74%, 67~70%, 64~67%, 56~59%.

Embodiment 15: the cultural method of ox IVF Embryos (in vitro without selenium)

Referring to example 11 above and the method for the corresponding complementary testing in the embodiment 11, the basis BY culture used Liquid is changed to (not adding selenium and copper) identical as the formula of embodiment 5, and is added in maturation culture solution and HEPES maturation culture solution The Sodium Hyaluronate and Potassiumiodate of corresponding amount and reduce FSH phase application amount.

As a result: the result of the result of each test parameters and the corresponding complementary testing in example 5 above and the embodiment 5 Essentially identical, for example, to Chinese Cattle, cleavage rates 88.6%, morula rate be 63.7%, blastocyst rate 50.8%, apoptosis rate 5.1%, at the 9th day, hatching rate was up to 71.1%, and the maturing rate of oocyte in vitro maturation is up to 75.1%；To He Sitan The maturing rate of the oocyte in vitro maturation of ox, three kinds of Simmental, CHINESE BUFFALO oxen is respectively 68.5%, 66.4%, 57.7%.

Embodiment 16: the cultural method of ox IVF Embryos (living body is without selenium)

Referring to example 12 above and the method for the corresponding complementary testing in the embodiment 12, the basis BY culture used Liquid is changed to (not adding selenium and copper) identical as the formula of embodiment 6, and is added in maturation culture solution and HEPES maturation culture solution The Sodium Hyaluronate and Potassiumiodate of corresponding amount and reduce FSH phase application amount.

As a result: the result of the result of each test parameters and the corresponding complementary testing in example 6 above and the embodiment 6 Essentially identical, for example, to Chinese Cattle, cleavage rates 88.4%, morula rate be 64.1%, blastocyst rate 50.2%, apoptosis rate 5.5%, at the 9th day, hatching rate was up to 72.8%, and the maturing rate of oocyte in vitro maturation is up to 75.0%；To He Sitan The maturing rate of the oocyte in vitro maturation of ox, three kinds of Simmental, CHINESE BUFFALO oxen is respectively 69.1%, 65.8%, 57.2%.

Embodiment 17: the cultural method (in vitro selenium copper) of ox IVF Embryos

Referring to example 13 above and the method for the corresponding complementary testing in the embodiment 13, the basis BY culture used Liquid is changed to (supplement addition sodium selenite 0.3mg/L, copper sulphate (by anhydride in terms of) 0.05mg/ identical as the formula of embodiment 7 L), the Sodium Hyaluronate of corresponding amount is added and in maturation culture solution and HEPES maturation culture solution and Potassiumiodate and reduces FSH Phase application amount.

As a result: the result of the result of each test parameters and the corresponding complementary testing in example 7 above and the embodiment 7 Essentially identical, for example, to Chinese Cattle, cleavage rates 86.8%, morula rate be 64.5%, blastocyst rate 51.1%, apoptosis rate 5.2%, at the 9th day, hatching rate was up to 72.1%, and the maturing rate of oocyte in vitro maturation is up to 89.5%；To He Sitan The maturing rate of the oocyte in vitro maturation of ox, three kinds of Simmental, CHINESE BUFFALO oxen is respectively 85.3%, 80.8%, 72.8%.

Embodiment 18: the cultural method (living body selenium copper) of ox IVF Embryos

Referring to example 14 above and the method for the corresponding complementary testing in the embodiment 14, the basis BY culture used Liquid is changed to (supplement addition sodium selenite 0.2mg/L, copper sulphate (in terms of anhydride) 0.1mg/L) identical as the formula of embodiment 8, And the Sodium Hyaluronate of corresponding amount is added in maturation culture solution and HEPES maturation culture solution and Potassiumiodate and reduces FSH phase Application amount.

As a result: the result of the result of each test parameters and the corresponding complementary testing in example 8 above and the embodiment 8 Essentially identical, for example, to Chinese Cattle, cleavage rates 88.2%, morula rate be 65.2%, blastocyst rate 51.7%, apoptosis rate 4.6%, at the 9th day, hatching rate was up to 74.2%, and the maturing rate of oocyte in vitro maturation is up to 90.4%；To He Sitan The maturing rate of the oocyte in vitro maturation of ox, three kinds of Simmental, CHINESE BUFFALO oxen is respectively 84.3%, 80.1%, 74.2%.

Although above the present invention is described in detail with a general description of the specific embodiments, at this On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore, These modifications or improvements without departing from theon the basis of the spirit of the present invention are fallen within the scope of the claimed invention.

Claims

1. the method for N IVF Embryos culture comprising following steps:

(1) acquisition of egg mother cell and maturation in vitro

In vitro acquisition: slaughterhouse ovary is taken to be contained in the insulation barrel for adding dual anti-physiological saline, under the conditions of 30.5-33 DEG C, in 4h Transport laboratory back；The ovarian follicle of surface 2-8mm is extracted, precipitating is collected, is picked under stereomicroscope at least containing the cumulus cell that haves three layers Egg mother cell COCs, that is, cumulus oocytes complesxes of package wash 2 times in egg-cleaning liquid, remove undesired impurities；

Living body acquisition: carrying out ovum pick-up to ox, gained liquor folliculi is picked under stereomicroscope at least containing 3 layers of cumulus cell The cumulus oocytes complesxes of package are put into the maturation culture solution comprising HEPES 38.8 DEG C, transport laboratory back in 3h；

It acquisition in vitro or living body acquisition gained COCs will wash 1 time, be then transferred in oocyte maturation culture solution above 22-24h is cultivated in new maturation culture solution, condition of culture is 38.8 DEG C, 5.5-6.5%CO2, saturated humidity；

(2) in vitro fertilization

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 counting hatching rate at the 9th day, and carries 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 the physiological saline comprising penicillin 400IU/mL, 400 μ g/mL of streptomysin；And/or

The egg-cleaning liquid is the BY basic culture solution for being added to 3mg/mL bovine serum albumin(BSA).

3. the method according to claim 1, wherein in step (1), any one of the maturation culture solution such as claim 5~10 institute It states.

4. the method according to claim 1, wherein

It is described fertilization culture solution be 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 bovine serum albumin(BSA) (BSA), 100U/ml penicillin, 100 μ g/ml streptomysins aqueous solution；

It is that the calcium chloride two comprising 112.0mM sodium chloride, 4.02mM potassium chloride, 2.25mM is hydrated that the sperm, which prepares culture solution, Object, 0.52mM magnesium chloride hexahydrate, 0.83mM potassium dihydrogen phosphate, 37.0mM sodium bicarbonate, 1.25mM Sodium Pyruvate, 10 μ g/ml livers Element, 4mg/ml bovine serum albumin(BSA) (BSA), 10mM caffeine, 100U/ml penicillin, 100 μ g/ml streptomysins aqueous solution； And/or

The embryo medium includes: 109.5mM sodium chloride, 3.1mM potassium chloride, 26.2mM sodium bicarbonate, six water chlorination of 0.8mM Magnesium, 1.19mM potassium dihydrogen phosphate, 0.4mM Sodium Pyruvate, 1.5mM glucose, 5mM galactonic acid calcium, 2.5v/v% fetal calf 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.

5. a kind of maturation culture solution is to be added to following ingredient in BY basic culture solution: the FBS of 100mL/L, 10 μ g/mL FSH, the LH of 10 μ g/mL, 1 μ g/mL E2,20ng/mL EGF；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.

6. maturation culture solution according to claim 5, wherein the BY basic culture solution is the aqueous solution comprising following component: chlorine Change calcium 200mg/L, nine water ferric nitrate 0.72mg/L, potassium chloride 400mg/L, magnesium sulfate 97.7mg/L, sodium chloride 6800mg/L, one Water sodium dihydrogen phosphate 140mg/L, sodium bicarbonate 2200mg/L, sodium acetate 50mg/L, l-Alanine 25mg/L, L-arginine hydrochloric acid Salt 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-Leu 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, 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, salt Sour guanine 0.3mg/L, hypoxanthine sodium 0.354mg/L, ribose 0.5mg/L, thymosin beta 10 .3mg/L, Tween 80 5mg/L, urine Pyrimidine 0.3mg/L, xanthine sodium 0.34mg/L, phenol red 10mg/L.

7. maturation culture solution according to claim 5 is to be added to following ingredient in BY basic culture solution: 50~100 μ g/ ML (such as 75 μ g/mL) Sodium Hyaluronate, 100~200 μ g/mL (such as 150 μ g/mL) Potassiumiodate, 10~20mmol/L (such as The FBS of 4- hydroxyethyl piperazineethanesulfonic acid, 100mL/L 15mmol/L), the FSH of 2 μ g/mL, the LH of 10 μ g/mL, 1 μ g/mL The EGF of E2,20ng/mL.

8. maturation culture solution according to claim 5, wherein further including sodium selenite in the BY basic culture solution, concentration is 0.2~0.3mg/L, such as 0.25mg/L.

9. maturation culture solution according to claim 5, wherein further including copper sulphate in the BY basic culture solution, concentration is with nothing Water object is calculated as 0.05~0.1mg/L, such as 0.075mg/L.

10. maturation culture solution according to claim 5, wherein also added the 4- hydroxyl of 10~20mmol/L (such as 15mmol/L) Ethyl piperazidine ethanesulfonic acid.