CN110331129A - A kind of the embryo transfer liquid and its application method of ox - Google Patents
A kind of the embryo transfer liquid and its application method of ox Download PDFInfo
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses the embryo transfer liquid and its application method of a kind of ox; the embryo transfer liquid of ox includes SOF basal medium, further includes following additive: 4- hydroxyethyl piperazineethanesulfonic acid, sodium bicarbonate, Insulin-Transferrin-selenium additive, beta -mercaptoethanol, hypotaurine, 1- oleoyl-sn- glycerol-3-phosphate sodium salt, L-citrulline and rhodioside.The pregnancy rate after ox embryo transfer can be effectively improved using embryo transfer liquid of the invention.
Description
Technical field
The present invention relates to livestock embryo field of engineering technology, the embryo transfer liquid of more particularly to a kind of ox and its
Application method.
Background technique
Animal embryo transfer is the technology with potential researching value and commercial value, can be widely applied to external embryo
Tire transplanting, animal gene editor breeding and the amplification of excellent domestic animal kind etc..But due to laboratory position, farm's cowshed limit
System and actual demand generally require the transport of certain distance in transplanting embryo, embryo's exposure for a long time in air,
Rich oxygen content in variation of the meeting due to pH and air generates damage to embryo, influences the development of embryo after the implantation.
Therefore it provides a kind of embryo transfer liquid of ox, reducing embryo, brought damage is this field during transportation
The problem of technical staff's urgent need to resolve.
Summary of the invention
In view of this, can reduce embryo the present invention provides the embryo transfer liquid and its application method of a kind of ox and transport
Damage brought by during defeated.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of embryo transfer liquid of ox, including SOF basal medium, further include following additive: 4- hydroxyethyl piperazine second
Sulfonic acid, sodium bicarbonate, Insulin-Transferrin-selenium additive, beta -mercaptoethanol, hypotaurine, 1- oleoyl-sn- glycerol-
3- sodium ascorbyl phosphate, L-citrulline and rhodioside.
4- hydroxyethyl piperazineethanesulfonic acid and sodium bicarbonate are used to maintain the pH value of embryo transfer liquid；Insulin-Transferrin-
Selenium additive (ITS-G) can preferably maintain the Motility and proliferation of cell；Beta -mercaptoethanol can promote cell Proliferation, subtract
Slow cell ageing rate, resists oxidative damage caused by oxygen environment, conducive to the growth of cell；Hypotaurine can neutralize cell toxicant
The final substance of plain acetaldehyde-peroxidating cascade reaction, to limit the illeffects of ROS；1- oleoyl-sn- glycerol-3-phosphate
Sodium can promote cell Proliferation；Rhodioside, which has, improves cell viability, improves cellular morphology, reduces karyon agglutination, anti-cell
The protective effect of oxidative stress.
Eight kinds of additives are added in embryo transfer liquid, can effectively reduce soda acid damage and the oxidative damage of ox embryo, mention
The developmental rate and development quality of embryo after high ox transplanting.
Further, the 4- hydroxyethyl piperazineethanesulfonic acid concentration is 1-3mg/mL, and sodium bicarbonate concentration is 0.1-0.2mg/
ML, Insulin-Transferrin-selenium additive volume fraction are 0.1-2%, and beta -mercaptoethanol concentration is 10-200 μM, sub- ox sulphur
Acid concentration is 1-10mM, and 1- oleoyl-sn- glycerol-3-phosphate sodium salt concentration is 0.1-1 μ g/mL, L-citrulline concentration are as follows:
0.01-0.03mg/mL, rhodioside concentration are 0.01-0.2mM.
Further, the embryo transfer liquid further includes that essential amino acid, nonessential amino acid and no fatty acids ox blood are pure
Albumen.
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, 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 1-10mg/mL.
Further, SOF basal medium described in every 100mL includes 26.168mg CaCl2·2H2O、9.999mg
C6H5Na3O7·2H2O, 2.923mg glutamine, 37.218mg MgSO4·7H2O, 49.904mg inositol, 1.000mg it is phenol red,
53.378mg KCl、16.195mg KH2PO4, 4.402mg Sodium Pyruvate, 629.399mg NaCl and 0.0757mg sodium lactate.
Further, the embryo transfer liquid further includes 12 μ g/mL gentamicins and 12 μ g/mL cephalosporins.
Further, the application method of the embryo transfer liquid of a kind of ox, bovine IVF embryo or cloned blastocysts are placed in
It is transported and is transplanted in the embryo transfer liquid.
Further, embryo transfer liquid is preheated to by the application method of the embryo transfer liquid of a kind of ox under the conditions of 38.5 DEG C
Not a half hour, then by ox, 6-7 days clone embryos are added after 6-7 days embryos or chemokinesis in vitro fertilization after in vitro fertilization
In embryo transfer liquid, and sucked in sterile straw under body formula mirror with syringe；Straw is put into and is preheated to 38.5 DEG C in advance
In portable incubator, cowshed is taken to；Loaded on the transplanting rifle to sterilize after straw is taken out, plastic packaging film is put on, the embryo of ox is carried out
Tire transplanting.
Insulin-Transferrin-selenium additive, beta -mercaptoethanol, hypotaurine, 1- oleoyl-sn- glycerol-3-phosphate
Sodium salt and rhodioside are added in embryo transfer liquid, can give full play to it as the positive of Embryo Culture environmental effectors
Effect, efficiently reduces high oxygen and low carbon dioxide in environment and gives embryo's bring side effect, significantly improve ox embryo transfer
The production cost for preparing embryo is greatly reduced in pregnancy rate afterwards；And it without using serum, avoids and potentially results in giant embryo disease
Risk.
It can be seen via above technical scheme that compared with prior art, the present disclosure provides a kind of embryo of ox shiftings
Liquid and its application method are planted, adds Insulin-Transferrin-selenium additive, beta -mercaptoethanol, Asia in embryo transfer liquid of the present invention
Taurine, 1- oleoyl-sn- glycerol-3-phosphate sodium salt, L-citrulline and rhodioside, after ox embryo transfer can be effectively improved
Pregnancy rate.
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
4- hydroxyethyl piperazineethanesulfonic acid, gentamicin, cephalosporin, inorganic salts, paraffin oil (M8410) are sigma company
Product；
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 prepare；
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, embryo transfer liquid
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, 629.399mg NaCl, 0.0757mg sodium lactate, deionized water constant volume to 100mL, 1mM NaOH tune
PH to 7.2-7.4.
SOFaa solution: using SOF solution as basic culture solution, essential amino acid, the volume point of volume fraction 1% are added
The nonessential amino acid of number 1%, 8mg/mL no fatty acids bovine serum albumin(BSA), 12g/mL gentamicin and 12 μ g/mL cephalos are mould
Element.
MSOFaa solution: using SOFaa solution as basic culture solution, addition volume fraction is 1% when preheating ITS-G,
55 μM of beta -mercaptoethanols, 2.5mM hypotaurine, 0.4 μ g/mL 1- oleoyl-sn- glycerol-3-phosphate sodium salt and the red scape of 0.1mM
Its glycosides, the L-citrulline of 0.02mg/mL, the hyaluronic acid of 0.5mg/mL.
By sperm
It is using the SOF solution without inositol as basic culture solution by sperm, also includes 48mg/mL no fatty acids ox blood
Pure albumen, 12 μ g/mL gentamicins, 12 μ g/mL cephalosporins, 63 μ g/mL L-arginines, 6 μ g/mL L-Aspartic acids,
50 μM of l-carnitine, 2mM adrenaline, 20mM penicillamine, 10mM hypotaurine, 10 μ g/mL heparin.
Embryo transfer liquid: using SOFaa solution as basic culture solution, addition volume fraction is 1% when preheating ITS-G,
100 μM of beta -mercaptoethanols, 2.5mM hypotaurine, 0.4 μ g/mL 1- oleoyl-sn- glycerol-3-phosphate sodium salt and the red scape of 0.1mM
The L-citrulline of its glycosides, 0.02mg/mL, 4- hydroxyethyl piperazineethanesulfonic acid concentration are as follows: 2.38mg/mL, sodium bicarbonate concentration are as follows:
0.168mg/mL。
(2) application method of ox embryo transfer liquid
A. the maturation culture of egg mother cell
Ox ovary picks up from three bridge animal-slaughtering in fixed place field of Xi'an City, Shanxi Province (acquisition time in January, 2017 in December, 2017),
Ovary is placed in vacuum flask to include in 20-25 DEG C of physiological saline of 200U/ml penicillin, 0.2mg/ml streptomysin, within 5h
Transport laboratory back.After ovary is transported back, the connective tissue, fat and the fallopian tubal of attachment of Ovarian surface 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 3% fetal calf serum after collection.Select oocyte morphology normal
COCs is used for In-vitro maturation (being denoted as total oocyte number).
The COCs of selection is washed twice in oocyte in vitro maturation culture solution, is then moved into equipped with 3mL egg mother cell
In the 3cm plate of In-vitro maturation liquid (being balanced one hour in 38.5 DEG C of incubators in advance), in 38.5 DEG C, 5%CO2, saturation
20-22h is cultivated under damp condition.The mature COCs of culture is blown and beaten repeatedly with 1000mL liquid-transfering gun, to remove outside egg mother cell
The cumulus cell of diffusion.Piping and druming to around COCs only four to five layers of cumulus cell of remaining tight when, by sperm (BO
Liquid) in wash 2 times, the free cumulus cell that wash clean is blown down, then by the egg mother cell of four to five layers of cumulus cell of tight
It moves in fertilization drop (being placed into balance 2h or more in 38.5 DEG C of incubators in advance).
B. defrosting, purifying, the fertilization of sperm
Essence (Xi'an milk cow center, in October, 2013 acquisition) will be frozen after liquid nitrogen container taking-up, be placed in 37 DEG C of water-bath solutions
Freeze, Percoll layering liquid is done in centrifuge tube.2mL 90%Percoll liquid is placed in centrifugation bottom of the tube, 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 200 μ L sperm be added to containing egg mother cell by sperm, putting back to culture
Smart ovum is set to be combined 8h 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, saturated humidity
72h is cultivated in incubator, chooses the fertilization embryo of the non-spilting of an egg, is put back to incubator and is continued culture to the 7th day (168h), picks out capsule
Embryo carries out embryo transfer.
D. the transplanting of blastaea
Embryo transfer liquid at least half an hour is preheated under the conditions of 38.5 DEG C, then by ox 6-7 days in vitro fertilization it is external by
Smart embryo is added in embryo transfer liquid, and is sucked in sterile straw under body formula mirror with syringe, and straw is put into and is preheated in advance
Into 38.5 DEG C of portable incubator, cowshed is taken to.Loaded on the transplanting rifle to sterilize after straw is taken out, plastic packaging film is put on,
Carry out the embryo transfer of ox.
Control group 1 is transplanted with PBS solution.
Control group 2 is transplanted with mSOFaa solution.
Each group developmental state is observed and recorded, test result is as shown in table 1.
Table 1
A, b subscript difference indicates significant, and conspicuousness is detected using Chi-square Test；Each recipient cattle transplants 1-2 pieces of mulberries
Or blastaea.
Table 1 statistics indicate that, carry out embryo transfer using embryo transfer liquid of the invention, compare the Transplanted Embryonic being disclosed
Tire liquid, embryo transfer liquid of the invention, which achieves the developmental potency after embryo transfer, to be obviously improved, and illustrates this hair
Bright embryo transfer liquid can be good at reducing the environment side effect that embryo is subject to during the transplantation process.
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 (8)
1. a kind of embryo transfer liquid of ox, including SOF basal medium, which is characterized in that the embryo transfer liquid further include with
Lower additive: 4- hydroxyethyl piperazineethanesulfonic acid, sodium bicarbonate, Insulin-Transferrin-selenium additive, beta -mercaptoethanol, sub- ox
Sulfonic acid, 1- oleoyl-sn- glycerol-3-phosphate sodium salt, L-citrulline and rhodioside.
2. a kind of embryo transfer liquid of ox according to claim 1, which is characterized in that the 4- hydroxyethyl piperazineethanesulfonic acid
Concentration is 1-3mg/mL, and sodium bicarbonate concentration is 0.1-0.2mg/mL, and Insulin-Transferrin-selenium additive volume fraction is
0.1-2%, beta -mercaptoethanol concentration are 10-200 μM, and hypotaurine concentration is 1-10mM, 1- oleoyl-sn- glycerol-3-phosphate
Sodium salt concentration is 0.1-1 μ g/mL, L-citrulline concentration are as follows: 0.01-0.03mg/mL, rhodioside concentration are 0.01-0.2mM.
3. a kind of embryo transfer liquid of ox according to claim 1 or 2, which is characterized in that the embryo transfer liquid also wraps
Include essential amino acid, nonessential amino acid and no fatty acids bovine serum albumin(BSA).
4. a kind of embryo transfer liquid of ox according to claim 3, which is characterized in that the essential amino acid volume fraction
For 0.1-2%, nonessential amino acid volume fraction is 0.1-2%, and no fatty acids bovine serum albumin(BSA) concentration is 1-10mg/mL.
5. a kind of embryo transfer liquid of ox according to claim 4, which is characterized in that the culture of the basis SOF described in every 100mL
Base includes 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 acetone
Sour sodium, 629.399mg NaCl and 0.0757mg sodium lactate.
6. a kind of embryo transfer liquid of ox according to claim 5, which is characterized in that the embryo transfer liquid further includes 12
μ g/mL gentamicin and 12 μ g/mL cephalosporins.
7. a kind of application method of the embryo transfer liquid of ox, which is characterized in that set bovine IVF embryo or cloned blastocysts
It is transported and is transplanted in embryo transfer liquid of any of claims 1-6.
8. a kind of application method of the embryo transfer liquid of ox according to claim 7, which is characterized in that by embryo transfer liquid
It is preheated to not a half hour under the conditions of 38.5 DEG C, then by ox latter 6-7 days embryos or chemokinesis in vitro fertilization in vitro fertilization
6-7 days clone embryos are added in embryo transfer liquid afterwards, and are sucked in sterile straw under body formula mirror with syringe；Straw is put
Enter in the portable incubator for being preheated to 38.5 DEG C in advance, takes cowshed to；Loaded on the transplanting rifle to sterilize after straw is taken out, cover
Upper plastic packaging film carries out the embryo transfer of ox.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101886059A (en) * | 2010-07-06 | 2010-11-17 | 周虚 | Culture solution used for embryo vitro production and method for bovine embryo vitro production |
| 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 |
-
2019
- 2019-06-24 CN CN201910551211.7A patent/CN110331129A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101886059A (en) * | 2010-07-06 | 2010-11-17 | 周虚 | Culture solution used for embryo vitro production and method for bovine embryo vitro production |
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| Y.M.ELHASSAN等: "AMINO ACID CONCENTRATIONS IN FLUIDS FROM THE BOVINE OVIDUCT AND UTERUS AND IN KSOM-BASED CULTURE MEDIA", 《THERIOGENOLOGY》 * |
| 安民等: "法国的牛胚胎移植", 《国外畜牧学(草食家畜)》 * |
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