CN110305836A - A kind of ox is in vitro fertilization by sperm and its application method - Google Patents
A kind of ox is in vitro fertilization by sperm and its application method Download PDFInfo
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Abstract
It is in vitro fertilization by sperm and its application method that the invention discloses a kind of oxen; included the SOF basal medium without inositol by sperm, further includes following additive: L-arginine, L-Aspartic acid, l-carnitine, adrenaline, penicillamine, hypotaurine, heparin.Using of the invention by sperm, the development quality of bovine embryo outer rate of fertilization and fertilized embryo can effectively ensure that.
Description
Technical field
The present invention relates to livestock embryo field of engineering technology, more particularly to a kind of ox is in vitro fertilization by sperm
And its application method.
Background technique
Livestock embryo in vitro culture is the technology with potential researching value and commercial value, can be widely applied to embryo
Transplanting, animal gene editor breeding and the amplification of excellent domestic animal kind etc. outside carcass.But the embryo that in vitro culture obtains so far
Often not as good as the embryo of normal development in vivo in quality and quantity, in ox is in vitro fertilization, freezing is often used to protect
The sperm deposited, which results in it is in vitro fertilization when sperm unstable quality, concentration is low, cause embryo fertilization rate unstable, formed
The fertilized eggs of protokaryon are few, and then influence pronucleus and the spilting of an egg, influence body early embryo ectogenesis, significantly restrict this technology
It is widely applied.
Therefore, it is in vitro fertilization by sperm and to establish a set of stable application method be those skilled in the art how to improve ox
The problem of member's urgent need to resolve.
Summary of the invention
In view of this, that the present invention provides a kind of oxen is in vitro fertilization by sperm and its application method, promote body early embryo
Ectogenesis.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of ox is in vitro fertilization by sperm, and the SOF basal medium including being free of inositol, further includes following additive:
L-arginine, L-Aspartic acid, l-carnitine, adrenaline, penicillamine, hypotaurine, heparin.
Heparin is spermatoblast chemistry energy substance living, L-arginine, L-Aspartic acid, adrenaline, hypotaurine and L-
The external vigor of the enough preferable stimulation spermatoblasts of carnitine；Penicillamine can promote insemination, and hypotaurine and l-carnitine can slow down
The final substance of cytotoxin acetaldehyde-peroxidating cascade reaction, to limit the illeffects of ROS；L-arginine is also improved
Cell viability improves the protective effect of cellular morphology.
Seven kinds of additives are added to by that in sperm, can effectively improve ox sperm motility, improve ox it is in vitro fertilization when be fertilized embryo
The development quality of Pronucleus formation rate and fertilization embryo.
Further, the concentration of the L-arginine is 10-100mg/L, and the concentration of L-Aspartic acid is 1-10mg/L, L- meat
Alkali concentration is 10-100 μM, and Adrenaline Concentration is 0.2-20 μM, and mould amine concentration is 2-200 μM, and hypotaurine concentration is 1-
100 μM, heparin concentration 5-20mg/L.
Further, described by sperm is further included essential amino acid, nonessential amino acid and no fatty acids bovine serum albumin(BSA).
Further, the essential amino acid volume fraction is 0.1-2%, and nonessential amino acid volume fraction is 0.1-2%,
No fatty acids bovine serum albumin(BSA) concentration is 1-100mg/mL.
It can further improve ox embryo by control essential amino acid, nonessential amino acid and no fatty acids bovine serum albumin(BSA)
Tire ectogenesis quality.
Further, SOF basal medium described in every 100mL includes 26.168mg CaCl2·2H2O、9.999mg
C6H5Na3O7·2H2O, 2.923mg glutamine, 37.218mg MgSO4·7H2O, 1.000mg is phenol red, 53.378mg KCl,
16.195mg KH2PO4, 4.402mg Sodium Pyruvate, 210.025mgNaHCO3, 629.399mgNaCl and 0.0757mg sodium lactate.
Further, described by sperm is further included 12 μ g/mL gentamicins and 12 μ g/mL cephalosporins.
Further, a kind of ox application method in vitro fertilization by sperm, is placed in right for bovine oocyte after maturation and wants
It asks and is co-cultured by sperm with sperm described in any one of 1-6.
Further, a kind of ox application method in vitro fertilization by sperm will be preheated to few 2h by sperm, then by ox essence
Son is placed on ox ovum by sperm, in CO28-10h is combined in incubator；Then embryo medium is added in fertilized bovine oocytes again
In, CO2N2It is cultivated in incubator, the embryo of the spilting of an egg is picked out in the 3rd after embryo medium culture day, continues culture to 7-8
It, the discarding of the non-spilting of an egg.
Further, the L-arginine, L-Aspartic acid, l-carnitine, adrenaline, penicillamine and hypotaurine, pre-
It is made an addition to before heat in the SOF basal medium without inositol, by not covering paraffin oil on sperm.
L-arginine, L-Aspartic acid, l-carnitine, adrenaline, penicillamine, hypotaurine, heparin are added by sperm,
It can be given full play to as sperm motility and the positive effect of environment culture acting factor, it is in vitro fertilization expeditiously to obtain ox
Ovum significantly improves ox developmental rate of in-vitro fertilization embryo, blastaea number and blastomere number, is greatly reduced and prepares being produced into for embryo
This.
Further, ox embryo in vitro fertilization is in 38.5 DEG C, 5%CO2It is cultivated under saturated humidity.
It can be seen via above technical scheme that compared with prior art, the present disclosure provides a kind of ox is in vitro fertilization
By sperm and its application method, the present invention by be added in sperm L-arginine, L-Aspartic acid, l-carnitine, adrenaline,
Penicillamine, hypotaurine, heparin can effectively ensure that the development quality of bovine embryo outer rate of fertilization and fertilized embryo.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1
(1) source of reagent and preparation
Gentamicin, cephalosporin, inorganic salts, L-arginine, L-Aspartic acid, l-carnitine, adrenaline, penicillamine,
Hypotaurine, heparin, paraffin oil (M8410) are sigma Products；
The M199 basic culture solution of no Hepes is Thermo Products；
Superfine fetal calf serum (FBS) is Gibco Products；
Essential amino acid is Thermo Fisher Products (article No. 1831512)；
Nonessential amino acid is Thermo Fisher Products (article No. 1958909)；
No fatty acids bovine serum albumin(BSA) EFAF-BSA is sigma Products (article No. A6003-5G)；
Insulin-Transferrin-selenium additive ITS-G is Thermo Fisher Products (article No. 1865342, pancreas islet
Plain 1g/L, transferrins 0.55g/L, selenium 0.00067g/L)；
Beta -mercaptoethanol is Thermo Fisher Products (article No. 1922445)；
Hypotaurine is sigma Products (article No. H1384)；
1- oleoyl-sn- glycerol-3-phosphate sodium is Enzo Life Science Products (article No. LP100-0025)；
Rhodioside is sigma Products (article No. SMB00072-1MG)；
SOF basal medium is voluntarily to configure；
It is sigma Products that other are not specified.
A. oocyte in vitro maturation culture solution
10% (v/v) fetal calf serum, 1% (v/v) ITS-G, 0.075IU/ are added in the M199 basic culture solution of no Hepes
ML HMG, 1 μ g/mL, 17 beta estradiol, 10ng/mL EGF, 12ng/mL bFGF and 50ng/mL CXCL12.
B.SOF solution, SOFaa solution, mSOFaa solution
SOF solution: 26.168mg CaCl2·2H2O、9.999mg C6H5Na3O7·2H2O, 2.923mg glutamine,
37.218mg MgSO4·7H2O, 49.904mg inositol, 1.000mg be phenol red, 53.378mg KCl, 16.195mg KH2PO4、
4.402mg Sodium Pyruvate, 210.025mgNaHCO3, 629.399mg NaCl, 0.0757mg sodium lactate, deionized water constant volume arrives
100mL, 1mMNaOH tune pH to 7.2-7.4.
SOFaa solution: using SOF solution as basic culture solution, essential amino acid, the volume point of volume fraction 1% are added
The nonessential amino acid of number 1%, 6mg/mL no fatty acids bovine serum albumin(BSA), 12g/mL gentamicin and 12 μ g/mL cephalos are mould
Element.
MSOFaa solution (embryo medium): using SOFaa solution as basic culture solution, addition volume fraction is when preheating
1% ITS-G, 55 μM of beta -mercaptoethanols, 2.5mM hypotaurine, 0.4 μ g/mL1- oleoyl-SN- glycerol-3-phosphate sodium salt and
0.1mM rhodioside, the hyaluronic acid of 0.5mg/100mL, the L-citrulline of 0.02mg/mL.
C. by sperm (BO liquid)
BO liquid is using the SOFaa solution without inositol as basic culture solution, also includes 63 μ g/mL L-arginines, 6 μ
G/mL L-Aspartic acid, 50 μM of l-carnitine, 2mM adrenaline, 20mM penicillamine, 10mM hypotaurine, 10 μ g/mL heparin.
(2) preparation method of ox IVF Embryos
A. the maturation culture of egg mother cell
Ox ovary picks up from three bridge animal-slaughtering in fixed place field of Xi'an City, Shanxi Province (acquisition time in January, 2015 in December, 2017),
Ovary is placed in vacuum flask to include in 20-25 DEG C of physiological saline of 200U/ml penicillin, 0.2mg/ml streptomysin, within 5h
Transport laboratory back.After ovary is transported back, the connective tissue, fat and the fallopian tubal of attachment of Ovarian surface are wiped out with sterilizing scissors,
It is cleaned in sterile saline three times, it is female to extract the ovum in Ovarian surface 2-8mm ovarian follicle with the 10mL syringe equipped with 12G syringe needle
Cell is put into 6cm glass dish, and cumulus oocytes complesxes (cumulus-oocyte is collected under stereomicroscope
Complexes, COCs)；It is cleaned three times in the PBS containing 10% fetal calf serum after collection.Select oocyte morphology normal
COCs is used for In-vitro maturation (being denoted as total oocyte number).
The COCs of selection is washed twice in oocyte in vitro maturation culture solution, is then moved into equipped with 3mL egg mother cell
In the 3cm plate of In-vitro maturation liquid (being balanced one hour in 38.5 DEG C of incubators in advance), in 38.5 DEG C, 5%CO2, saturation
20-22h is cultivated under damp condition.The mature COCs of culture is blown and beaten repeatedly with 1000mL liquid-transfering gun, to remove outside egg mother cell
The cumulus cell of diffusion.Piping and druming to around COCs only four to five layers of cumulus cell of remaining tight when, by sperm (BO
Liquid) in wash 2 times, the free cumulus cell that wash clean is blown down, then by the egg mother cell of four to five layers of cumulus cell of tight
It moves in fertilization drop (being placed into balance 2h or more in 38.5 DEG C of incubators in advance).
B. defrosting, purifying, the fertilization of sperm
Essence (Xi'an milk cow center, the acquisition of in September, 2015) will be frozen after liquid nitrogen container taking-up, be placed in 37 DEG C of water-bath solutions
Freeze, Percoll layering liquid is done in centrifuge tube.2mL 90%Percoll liquid is placed in centrifugation bottom of the tube, 2mL 45%
Percoll liquid is carefully added on 90%Percoll liquid level, then the sperm after defrosting is placed on 45%Percoll liquid level,
1500rpm is centrifuged 20min, careful suction foot about 100 μ L sperm be added to containing egg mother cell by sperm, putting back to culture
Make sperm ovum binding 10h in case.
C. the culture of fertilized embryo
Processing group: adding mSOFaa solution in four orifice plates, and every 500 μ L of hole covers 500 μ L paraffin oils on liquid level, in advance
In 38.5 DEG C, 5%CO2, balance at least 2h in saturated humidity incubator.
Blow away remaining cumulus cell and sperm around ovum with mouth suction pipe or pipette tips after fertilization, obtain assume by
The ox embryo in vitro fertilization of essence, is placed in the mSOFaa solution of four orifice plates, 40 pieces or so of every hole, 38.5 DEG C, 5%CO2, 7%N2Saturation
72h is cultivated in humidified incubator, chooses the fertilization embryo of the non-spilting of an egg, is put back to incubator and is continued culture to the 8th day (192h).
The SOF hydroponics of heparin of the control group 1 containing 10ug/ml are fertilized.
It observes and records the discharge of each group polar body and Pronucleus formation situation, test result is as shown in table 1.
Table 1
Table 1 the result shows that, use it is of the invention by sperm carry out ox it is in vitro fertilization, the embryo of obtained normal procaryosis
Number is significantly more than general BO by sperm.
Each group developmental state is observed and recorded, test result is as shown in table 2.
Table 2
A, b subscript difference indicates significant, and conspicuousness is detected using Chi-square Test；AB grades of blastaeas are portable blastaea, C grades
Not recommend to transplant blastaea, blastaea ABC classification is to transplant the quality of blastocysts standards of grading of association according to International Embryo to evaluate.
Table 2 the result shows that, using it is of the invention by sperm carry out ox it is in vitro fertilization, obtained portable embryo's number is significant
More than general BO by sperm.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Claims (10)
1. a kind of ox is in vitro fertilization by sperm, the SOF basal medium including being free of inositol, which is characterized in that the fertilization
Liquid further includes following additive: L-arginine, L-Aspartic acid, l-carnitine, adrenaline, penicillamine, hypotaurine, heparin.
2. a kind of ox according to claim 1 is in vitro fertilization by sperm, which is characterized in that the concentration of the L-arginine
For 10-100mg/L, the concentration of L-Aspartic acid is 1-10mg/L, and l-carnitine concentration is 10-100 μM, and Adrenaline Concentration is
0.2-20 μM, mould amine concentration is 2-200 μM, and hypotaurine concentration is 1-100 μM, heparin concentration 5-20mg/L.
3. a kind of ox according to claim 1 or 2 is in vitro fertilization by sperm, which is characterized in that described also to be wrapped by sperm
Include essential amino acid, nonessential amino acid and no fatty acids bovine serum albumin(BSA).
4. a kind of ox according to claim 3 is in vitro fertilization by sperm, which is characterized in that the essential amino acid volume
Score is 0.1-2%, and nonessential amino acid volume fraction is 0.1-2%, and no fatty acids bovine serum albumin(BSA) concentration is 1-
100mg/mL。
5. a kind of ox according to claim 4 is in vitro fertilization by sperm, which is characterized in that the basis SOF described in every 100mL
Culture medium includes 26.168mg CaCl2·2H2O、9.999mg C6H5Na3O7·2H2O, 2.923mg glutamine, 37.218mg
MgSO4·7H2O, 1.000mg is phenol red, 53.378mg KCl, 16.195mg KH2PO4, 4.402mg Sodium Pyruvate, 210.025mg
NaHCO3, 629.399mgNaCl and 0.0757mg sodium lactate.
6. a kind of ox according to claim 5 is in vitro fertilization by sperm, which is characterized in that described by sperm is further included 12
μ g/mL gentamicin and 12 μ g/mL cephalosporins.
7. a kind of ox application method in vitro fertilization by sperm, which is characterized in that bovine oocyte after maturation is placed in right
It is required that being co-cultured by sperm with sperm described in any one of 1-6.
8. a kind of ox application method in vitro fertilization by sperm according to claim 7, which is characterized in that will be by sperm
At least 2h is preheated, then Niu Jingzi and ox ovum are placed on by sperm, in CO28-10h is combined in incubator；Then again by ox
Fertilized eggs are added in embryo medium, CO2N2It is cultivated in incubator, the spilting of an egg is picked out in the 3rd after embryo medium culture day
Embryo continues to cultivate to the 7-8 days, the discarding of the non-spilting of an egg.
9. a kind of ox application method in vitro fertilization by sperm according to claim 8, which is characterized in that the L- essence
Propylhomoserin, L-Aspartic acid, l-carnitine, adrenaline, penicillamine and hypotaurine make an addition to the SOF without inositol before preheating
In basal medium, by not covering paraffin oil on sperm.
10. a kind of ox application method in vitro fertilization by sperm according to claim 9, which is characterized in that ox is external
Embryo be fertilized in 38.5 DEG C, 5%CO2It is cultivated under saturated humidity.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111534479A (en) * | 2020-05-09 | 2020-08-14 | 西北农林科技大学 | Culture solution for improving development quality of bovine in-vitro fertilized embryo and cloned embryo |
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|---|---|---|---|---|
| CN102296047A (en) * | 2011-08-13 | 2011-12-28 | 新疆农垦科学院 | In vitro embryo production method by utilization of lamb superovulation oocytes |
| CN109628382A (en) * | 2019-01-18 | 2019-04-16 | 西北农林科技大学 | A kind of culture solution and cultural method of the development quality improving ox embryo in vitro fertilization |
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2019
- 2019-06-24 CN CN201910551341.0A patent/CN110305836B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102296047A (en) * | 2011-08-13 | 2011-12-28 | 新疆农垦科学院 | In vitro embryo production method by utilization of lamb superovulation oocytes |
| CN109628382A (en) * | 2019-01-18 | 2019-04-16 | 西北农林科技大学 | A kind of culture solution and cultural method of the development quality improving ox embryo in vitro fertilization |
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| Title |
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| GONCALVES 等: ""Heparin and penicillamine–hypotaurine–epinephrine (PHE) solution during bovine in vitro fertilization procedures impair the quality of spermatozoa but improve normal oocyte fecundation and early embryonic development"", 《IN VITRO CELL.DEV.BIOL.—ANIMAL》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111534479A (en) * | 2020-05-09 | 2020-08-14 | 西北农林科技大学 | Culture solution for improving development quality of bovine in-vitro fertilized embryo and cloned embryo |
| WO2021227225A1 (en) * | 2020-05-09 | 2021-11-18 | 西北农林科技大学 | Culture solution for improving developmental quality of bovine in-vitro fertilized embryos and cloned embryos |
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