CN107365738A - A kind of milk cow and the preparation method of ox xenogenesis IVF Embryos - Google Patents
A kind of milk cow and the preparation method of ox xenogenesis IVF Embryos Download PDFInfo
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- CN107365738A CN107365738A CN201710667052.8A CN201710667052A CN107365738A CN 107365738 A CN107365738 A CN 107365738A CN 201710667052 A CN201710667052 A CN 201710667052A CN 107365738 A CN107365738 A CN 107365738A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0604—Whole embryos; Culture medium therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2517/00—Cells related to new breeds of animals
- C12N2517/10—Conditioning of cells for in vitro fecondation or nuclear transfer
Abstract
The invention discloses a kind of milk cow and the preparation method of ox IVF Embryos.The milk cow ovary discarded in method using slaughterhouse, the physiological saline gradient wash by temperature increment formula, extract egg mother cell therein and carry out In-vitro maturation, obtain the egg mother cell of the In-vitro maturation of high quality；Inserted after the defrosting of high yield cow sperm in fertilization liquid after gently being blown and beaten with liquid-transfering gun, turn upside down several times, floated in incubator, progress is in vitro fertilization, prepares high yield cow and ox IVF Embryos.The ovary that the present invention is discarded using slaughterhouse obtains egg mother cell, improves the utilization rate of cow resource.And by the improvement of ins and outs, obtain preferable effect.The utilization rate of excellent sire can be increased substantially using the present invention, excellent sire is obtained substantial amounts of offspring, so as to expand its effect in drove genetic improvement.
Description
Technical field
The invention belongs to ox breeding field, and in particular to milk cow and the preparation method of ox xenogenesis IVF Embryos.
Background technology
Technology in vitro fertilization refers to that the sperm of mammal and ovum are completed to be fertilized in the environment of manual control in vitro
The technology of journey.The frozen semen that can make full use of the discarded ovary in slaughterhouse and high-quality milk cow using external fertilization method carries out embryo
Tire produced in vitro, not only cost is cheap, and effect stability, it is not necessary to superfecundation and Estrus synchronization are carried out to cow,
Greatly reduce workload, reduce production cost, while increase substantially the utilization rate of excellent sire, obtain excellent sire
Substantial amounts of offspring, so as to expand its effect in drove genetic improvement.
But in actual production, raising dairy cattle cost is higher, and the mortality of milk cow is relatively low, even if superseded milk cow is also old
Weak invalid, therefore, the milk cow that slaughterhouse is butchered is mostly old milk cow or sick milk cow, has had a strong impact on effect in vitro fertilization
Fruit.
The content of the invention
It is milk cow embryo it is an object of the invention to establish the preparation method of a kind of milk cow and ox xenogenesis IVF Embryos
The application of tire biotechnology provides rational technology path, gives full play to the genetic potential of high yield and high quality milk cow.
Problem be present to solve prior art, the present invention provides following technical scheme：
A kind of milk cow and the preparation method of ox xenogenesis IVF Embryos, its step are：
(1) the ox ovary that slaughterhouse gathers is put into and added in 25 DEG C of dual anti-sterile salines in constant temperature preservation 7h
Laboratory is taken back, is washed successively with 30 DEG C, 35 DEG C of sterile salines, it is standby in 2 rearmounted 39 DEG C of water-baths of washing respectively, 10
Milliliter disposable syringe suction method obtains cumulus oocytes complesxes (COCs)；
(2) cumulus oocytes complesxes picked (COCs) are contained with what 2h in advance was preheated in 39 DEG C of water-baths
The DPBS buffer solutions of 5% hyclone (FBS) are washed 3 times, then the maturation in vitro preheated with 2h in advance in 39 DEG C of incubators is trained
Nutrient solution is placed in CO after washing 3 times2In-vitro maturation is carried out in incubator, using five orifice plate cultures, cultivates concentration：100-150
Piece/500 μ L；39 DEG C of condition of culture, 5%CO2, saturated humidity；Incubation time 22-24h；
In-vitro maturation formula of liquid：TCM-199 (Gibco)+10%FBS+10 μ L/mL dual anti-(i.e. 100IU/mL)+
The μ g/mL estradiol (E2) of 10IU/mL human chorionic gonadotrophins (HMG)+1；
(3) milk cow frozen semen is handled with BO liquid floating method：From liquid nitrogen container take straw frozen semen be put into 30 in 37 DEG C of water-baths~
40s quick-thawings, with tubule is cut off after 75% alcohol disinfecting, sperm is put into the centrifuge tube containing 1mLBO liquid, uses liquid-transfering gun
After gently blowing and beating, after turning upside down several times, it is placed in CO2gas incubator and stands 20-30min, sperm is floated, after floating
By the sperm centrifugal treating of floating, 1500r/min, 5min, with BO, liquid again centrifugal treating twice, 1500r/min, 5min；
The formula of BO liquid：6.55mg/mL sodium chloride+0.3mg/mL potassium chloride+0.249mg/mL calcium chloride+0.0497mg/
ML magnesium chloride+0.0869mg/mL sodium dihydrogen phosphate+3.104mg/mL sodium acid carbonate+2.5mg/mL glucose+0.055mg/mL third
Ketone acid sodium+0.065mg/mL penicillin+0.05mg/mL streptomycin sulphates；BO, the formula of liquid：BO liquid+5mM caffeines+10mg/
ML liquaemins+3mg/mLBSA；
(4) egg mother cell in step (2) after maturation in vitro is placed in after cumulus cell is sloughed in 2% hyaluronic acid enzymic digestion
Liquid BO in vitro fertilization, middle balance 30min；
(5) sperm handled well in step (3) is added in the droplet in vitro fertilization containing egg mother cell in step (4), essence
Final concentration of the 3 × 10 of son5Individual/mL；
(6) after 6h in vitro fertilization, gently blow and beat embryonated egg with pipettor, remove the sperm for floating over embryonated egg surface, with carrying
The IVF Embryos nutrient solution that preceding 2h is preheated in 39 DEG C of incubators is placed in nutrient solution after washing three times and cultivated；Culture
Concentration：10-15 embryonated egg is cultivated in 50 μ L culture drop；24h half, which is measured, changes liquid, and blastaea can be obtained after 7d；
Nutrient solution is CRlaa：114.7mM NaCl、3.1mM KCl、26.2mM NaHCO3, 0.4mM Sodium Pyruvates,
The calcium of 5.0mM lactic acid half, 3mg/mL BSA, 1.0mM.
Milk cow of the present invention is simon Simmental Cattle Chromosome.
Technical scheme provided by the invention has the characteristics that：
(1) ovary can preserve 7 hours and not influence the COC obtained under the conditions of 25 DEG C with constant temperatureSQuality, improve people
The utilization rate of power resource, reduce the waste in human resources；
(2) ovary is handled：Carrying out washing treatment is carried out using the physiological saline of temperature increment.It is placed under the conditions of 25 DEG C of physiological saline
Ovary, washed successively with 30 DEG C, 35 DEG C of sterile salines after taking back laboratory, the ovary after washing is placed in 39 DEG C of water-baths
Middle preservation, acquisition contain COCSLiquor folliculi be placed in 39 DEG C of water-baths constant temperature and preserve, COCSAll the time 39 are placed in during picking
In DEG C thermal station, influence of the change to oocyte in vitro maturation of ambient temperature is avoided, ensure that the matter of egg mother cell
Amount and maturation in vitro efficiency；
(3) thaw after seminal fluid, in BO liquid enter incubator in float before gently blown and beaten with pipettor or gently on
Lower reverse seminal fluid-BO for several times mixed liquor, avoids spermatium being present, substantially increases efficiency in vitro fertilization, embryonated egg cleavage rates
Compared with control group height.
Milk bovine oocyte is replaced using the egg mother cell of ox in the present invention, and by the improvement of ins and outs,
Obtain preferable effect.
Brief description of the drawings
Fig. 1 is the egg mother cell of after fertilization in the step of embodiment 1 (6) that Hoechst 33342 is dyed；
Fig. 2 is blastaea and hatched blastocyst；
Fig. 3 is the different spermatiums presented of Sperm treatment method and single sperm after thawing；
Embodiment
By combination accompanying drawing described further below it will be further appreciated that the features and advantages of the invention.The implementation provided
Example is only explanation to the inventive method, remaining content without limiting the invention in any way announcement.
【Embodiment 1】Simon Simmental Cattle Chromosome and the preparation method of ox xenogenesis IVF Embryos
(1) the ox ovary that slaughterhouse gathers is put into and added in 25 DEG C of dual anti-sterile salines in constant temperature preservation 7h
Laboratory is taken back, is washed successively with 30 DEG C, 35 DEG C of sterile salines, it is standby in 2 rearmounted 39 DEG C of water-baths of washing respectively, 10
Milliliter disposable syringe suction method obtains cumulus oocytes complesxes (COCs)；
(2) cumulus oocytes complesxes picked (COCs) are contained with what 2h in advance was preheated in 39 DEG C of water-baths
The DPBS buffer solutions of 5% hyclone (FBS) are washed 3 times, then the maturation in vitro preheated with 2h in advance in 39 DEG C of incubators is trained
Nutrient solution is placed in CO after washing 3 times2In-vitro maturation is carried out in incubator, using five orifice plate cultures, cultivates concentration：100-150
Piece/500 μ L；39 DEG C of condition of culture, 5%CO2, saturated humidity；Incubation time 22-24h；
In-vitro maturation formula of liquid：TCM-199 (Gibco)+10%FBS+10 μ L/mL dual anti-(i.e. 100IU/mL)+
The μ g/mL estradiol (E2) of 10IU/mL human chorionic gonadotrophins (HMG)+1；
(3) simon tal fibre cow frozen semen is handled with BO liquid floating method：Milk cow frozen semen is bought to be changed in Wuhan City milk cow
Good station, kind are simon tal fibre milk cow；The processing method of sperm：From liquid nitrogen container take straw frozen semen be put into 30 in 37 DEG C of water-baths~
40s quick-thawings, with tubule is cut off after 75% alcohol disinfecting, sperm is put into the centrifuge tube containing 1mLBO liquid, uses liquid-transfering gun
After gently blowing and beating, turn upside down 2-3 times, be placed in CO2gas incubator and stand 20-30min, sperm is floated, will after floating
The sperm centrifugal treating of floating, 1500r/min, 5min, with BO, liquid again centrifugal treating twice, 1500r/min, 5min；
The formula of BO liquid：6.55mg/mL sodium chloride+0.3mg/mL potassium chloride+0.249mg/mL calcium chloride+0.0497mg/
ML magnesium chloride+0.0869mg/mL sodium dihydrogen phosphate+3.104mg/mL sodium acid carbonate+2.5mg/mL glucose+0.055mg/mL third
Ketone acid sodium+0.065mg/mL penicillin+0.05mg/mL streptomycin sulphates；BO, the formula of liquid：BO liquid+5mM caffeines+10mg/
ML liquaemins+3mg/mLBSA；
(4) egg mother cell in step (2) after maturation in vitro is placed in after cumulus cell is sloughed in 2% hyaluronic acid enzymic digestion
Liquid BO in vitro fertilization, middle balance 30min；
(5) sperm handled well in step (3) is added in the droplet in vitro fertilization containing egg mother cell in step (4), essence
Final concentration of the 3 × 10 of son5Individual/mL；
(6) after 6h in vitro fertilization, gently blow and beat embryonated egg with pipettor, remove the sperm for floating over embryonated egg surface, with carrying
The IVF Embryos nutrient solution that preceding 2h is preheated in 39 DEG C of incubators is placed in nutrient solution after washing three times and cultivated；Culture
Concentration：10-15 embryonated egg is cultivated in 50 μ L culture drop；24h half, which is measured, changes liquid, and blastaea can be obtained after 7d；
Nutrient solution is CRlaa：114.7mM NaCl、3.1mM KCl、26.2mM NaHCO3, 0.4mM Sodium Pyruvates,
The calcium of 5.0mM lactic acid half, 3mg/mL BSA, 1.0mM.
【Embodiment 2】The dyeing identifications of Hoechst 33342
(1) egg mother cell of after fertilization in the step of section Example 1 (6) is taken to be washed 3 times with DPBS；
Cumulus cell is sloughed in (2) 2% hyaluronic acid enzymic digestions；
(3) lucifuges of Hoechst 33342 dyeing 5-7min；
(4) for inverted microscope observed under fluorescent light as shown in figure 1, left figure is the egg mother cell of after fertilization, right figure is in fluorescence
The dot to be lighted at visible 2 under the visual field, as by the male pronucleus dyed of Hoechst 33342 and female pronucleus, this shows xenogenesis
Sperm in vitro fertilization method is feasible.
【Embodiment 3】The hatching of blastaea
(1) laboratory will be taken back in physiological saline (containing dual anti-) in the ox ovary 3h of collection, ovum is obtained with suction method
Mound-oocyte complex (COCs)；
(2) 22-24h is cultivated after cumulus oocytes complesxes (COCs) are washed；
(3) simon Simmental Cattle Chromosome seminal fluid is handled with BO liquid floating method；
(4) egg mother cell in step (2) after maturation sloughs cumulus cell through digestion；
(5) sperm in step (3) is moved into by seminal fluid, and the egg mother cell in step (4) is contained in by seminal fluid；
(6) it is fertilized after 6-8h, is cultivated after scrubbed, 24h half, which is measured, changes liquid；
(7) after cultivating 7d, it was observed that blastaea, 24h half, which is measured, changes liquid, as a result as shown in Fig. 2 left figure be blastaea, right figure for
Continue that transparent desmorrhexis occurs during culture, embryo, which therefrom expands, carrys out i.e. hatched blastocyst.
【Embodiment 4】Temperature increment method washs the contrast experiment of ovary
Experimental design：It is the same that the step of embodiment 1 (1), is divided into 3 groups of subsequent steps, the 1st group：25 DEG C of constant temperature physiology salt washings
Inserted after washing ovary in 39 DEG C of water-baths；2nd group：(25 DEG C, 30 DEG C, 35 DEG C, 39 DEG C) washing ovum of physiological saline temperature increment method
Inserted after nest in 39 DEG C of water-baths；3rd group：Directly inserted with 39 DEG C of preservations with brine ovary in 39 DEG C of water-baths.
As a result such as table 1：Temperature increment formula washs ovary, certain cushioning effect is played to egg mother cell, to the ovum of acquisition
Mother cell quality influences notable, acquisition A levels COCSRatio it is more directly high with 25 DEG C and 39 DEG C of brine ovaries, have aobvious
Difference (89.4%vs64.5%vs61.6%) is write, oocyte maturation rate is also high, significant difference (79.5%vs51.6%
Vs48.2%), but cleavage rates there were significant differences.
The temperature increment method of table 1 washs the contrast experiment of ovary
Note：The different lowercase letter indication differences significantly (P with after column data<0.05).
【Embodiment 5】The Comparative result experiment that Sperm treatment method is different after defrosting
Experimental design：The step of embodiment 1 (3), is divided into 2 groups, and as step, the 1st group is control group for it：Sperm thaws
Directly directly insert in by seminal fluid floating in incubator afterwards；2nd group is experimental group：Sperm is inserted after thawing in fertilization liquid
Gently blown and beaten with liquid-transfering gun after being floated in incubator.
As a result such as table 2：After gently blowing and beating sperm with pipettor in experimental group, embryonated egg cleavage rates are high, compared with control group difference
Significantly (88.7%vs54.5%).
The different Comparative result experiment of Sperm treatment method after table 2 thaws
Note：The different lowercase letter indication differences significantly (P with after column data<0.05).
Fig. 3 left figures are shown：Sperm is inserted after thawing in fertilization liquid, and part sperm exists in the form of spermatium, plus essence
The characteristic that son is easily united, the availability of sperm is greatly reduced, but after liquid-transfering gun is gently blown and beaten, the essence in spermatium
Son is released, and the ratio of single sperm increases substantially (as shown in Fig. 3 right figures), substantially increases the ratio of available sperm
Example, reduce the waste of sperm, improve rate of fertilization.
Although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of changes, modification can be carried out to these embodiments, replace without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (1)
1. a kind of milk cow and the preparation method of ox xenogenesis IVF Embryos, its step are：
（1）The ox ovary that slaughterhouse gathers is put into add in 25 DEG C of dual anti-sterile salines and taken back in constant temperature preservation 7h
Laboratory, washed successively with 30 DEG C, 35 DEG C of sterile salines, it is standby in 2 rearmounted 37 DEG C of water-baths of washing respectively, 10 milliliters
Disposable syringe suction method obtains cumulus oocytes complesxes（COCs）；
（2）The cumulus oocytes complesxes that will be picked（COCs）Contain 5% tire with what 2h in advance was preheated in 39 DEG C of water-baths
Cow's serum（FBS）DPBS buffer solutions wash 3 times, then the In-vitro maturation liquid preheated with 2h in advance in 39 DEG C of incubators washes
CO is placed in after washing 3 times2In-vitro maturation is carried out in incubator, using five orifice plate cultures, cultivates concentration：100-150 pieces/500 μ
L；39 DEG C of condition of culture, 5% CO2, saturated humidity；Incubation time 22-24h；
In-vitro maturation formula of liquid：TCM-199 (Gibco)+10%FBS+10 μ L/mL are dual anti-（That is 100IU/mL）+10IU/mL
Human chorionic gonadotrophin（HMG）+ 1 μ g/mL estradiol（E2）；
（3）Milk cow frozen semen is handled with BO liquid floating method：Straw frozen semen is taken to be put into 30 ~ 40s in 37 DEG C of water-baths from liquid nitrogen container fast
Speed is thawed, and with tubule is cut off after 75% alcohol disinfecting, sperm is put into the centrifuge tube containing 1mLBO liquid, gently blown with liquid-transfering gun
After beating, after turning upside down several times, it is placed in CO2gas incubator and stands 20-30min, sperm is floated, will be floated after floating
Sperm centrifugal treating, 1500r/min, 5min, use BO,Liquid again centrifugal treating twice, 1500r/min, 5min；
The formula of BO liquid：6.55mg/mL sodium chloride+0.3mg/mL potassium chloride+0.249mg/mL calcium chloride+0.0497mg/mL chlorine
Change magnesium+0.0869mg/mL sodium dihydrogen phosphate+3.104mg/mL sodium acid carbonate+2.5mg/mL glucose+0.055mg/mL pyruvic acid
Sodium+0.065mg/mL penicillin+0.05mg/mL streptomycin sulphates；BO,The formula of liquid：The mg/mL livers of BO liquid+5mM caffeines+10
Plain sodium+3mg/mLBSA；
（4）Step（2）Egg mother cell after middle maturation in vitro is placed in vitro after cumulus cell is sloughed in 2% hyaluronic acid enzymic digestion
By seminal fluid BO,Middle balance 30min；
（5）Step（3）In the sperm handled well add and contain step（4）In the droplet in vitro fertilization of middle egg mother cell, sperm
Final concentration of 3 × 105Individual/mL；
（6）After 6h in vitro fertilization, embryonated egg is gently blown and beaten with pipettor, the sperm for floating over embryonated egg surface is removed, with 2h in advance
The IVF Embryos nutrient solution preheated in 39 DEG C of incubators is placed in nutrient solution after washing three times and cultivated；Cultivate dense
Degree：10-15 embryonated egg is cultivated in 50uL culture drop；24h half, which is measured, changes liquid, and blastaea can be obtained after 7d；
Nutrient solution is CRlaa：114.7mM NaCl、3.1 mM KCl、26.2 mM NaHCO3, 0.4 mM Sodium Pyruvates, 5.0
The calcium of mM lactic acid half, 3mg/mL BSA, 1.0 mM；
Described milk cow is simon Simmental Cattle Chromosome.
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| CN108148800A (en) * | 2018-01-29 | 2018-06-12 | 浙江省医学科学院 | A kind of sperm microcytotoxicity liquid, for kit in vitro fertilization and mammalian in vitro fertilization method |
| CN108424935A (en) * | 2018-03-22 | 2018-08-21 | 湖北省农业科学院畜牧兽医研究所 | A kind of preparation method of high yield cow blastomere reconstruct embryo |
| CN108642001A (en) * | 2018-05-08 | 2018-10-12 | 中国农业科学院北京畜牧兽医研究所 | A method of it improving obstinacy control and freezes essence ability in vitro fertilization |
| CN110684720A (en) * | 2019-10-14 | 2020-01-14 | 湖北省农业科学院畜牧兽医研究所 | Method for obtaining male pronucleus by taking oocyte among heterogeneous animals as medium |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108148800A (en) * | 2018-01-29 | 2018-06-12 | 浙江省医学科学院 | A kind of sperm microcytotoxicity liquid, for kit in vitro fertilization and mammalian in vitro fertilization method |
| CN108424935A (en) * | 2018-03-22 | 2018-08-21 | 湖北省农业科学院畜牧兽医研究所 | A kind of preparation method of high yield cow blastomere reconstruct embryo |
| CN108642001A (en) * | 2018-05-08 | 2018-10-12 | 中国农业科学院北京畜牧兽医研究所 | A method of it improving obstinacy control and freezes essence ability in vitro fertilization |
| CN108642001B (en) * | 2018-05-08 | 2022-09-02 | 中国农业科学院北京畜牧兽医研究所 | Method for improving bovine sexual control frozen semen in vitro fertilization capability |
| CN110684720A (en) * | 2019-10-14 | 2020-01-14 | 湖北省农业科学院畜牧兽医研究所 | Method for obtaining male pronucleus by taking oocyte among heterogeneous animals as medium |
| CN110684720B (en) * | 2019-10-14 | 2021-07-20 | 湖北省农业科学院畜牧兽医研究所 | Method for obtaining male pronucleus by taking oocyte among heterogeneous animals as medium |
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