CN110241072A - A kind of Embryo Production refrigerating process - Google Patents
A kind of Embryo Production refrigerating process Download PDFInfo
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- CN110241072A CN110241072A CN201910606151.4A CN201910606151A CN110241072A CN 110241072 A CN110241072 A CN 110241072A CN 201910606151 A CN201910606151 A CN 201910606151A CN 110241072 A CN110241072 A CN 110241072A
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- embryo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
-
- 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
Abstract
The invention discloses a kind of Embryo Production refrigerating process, comprising the following steps: (1) ovary for acquiring meat sheep used as donor is taken back 35-40 DEG C under the conditions of in 1-4h with physiological saline to laboratory, obtain egg mother cell and be placed in and save in liquid；(2) egg mother cell is washed 3-5 times with mature liquid, cultivates 25-30h；(3) sperm of ram is handled；(4) sperm is moved by sperm, by the egg mother cell for containing step (2) in sperm and handling；(5) it is fertilized after 15-20h, dissolves cumulus cell 1-2min with 0.2% hyaluronidase, then Voatex whirlpool shakes, and removes cumulus cell；(6) fertilized eggs are moved into granular cell co-culture system, changes the culture solution of half volume every 48h, collects embryo after cultivating 150-170h；(7) the freeze proof processing of embryo: (8) save；The present invention solves the problems, such as that current embryo quality source is insufficient and is of great significance that Refrigeration Technique can be reduced the cooling preservation effect that embryo is improved to physical injury caused by embryo.
Description
Technical field
The present invention relates to the technical field of embryo transfer, in particular to a kind of Embryo Production refrigerating process.
Background technique
Embryo transfer, also known as fertilized eggs inhibit, and refer to the body early embryo of jenny, or by vitro fertilization and its
The embryo that his mode obtains migrates in jenny body, is allowed to continue to the technology that development is new individual, the essence of embryo transfer
It is to produce the donor of embryo and breed the process that the receptor of embryo raises up seed jointly, female defect individual can be given full play to
Fertility, and the breeding cycle for substantially reducing donor itself, while increasing the quantity that donor all one's life raises up seed.
Using embryo transfer, the excellent female livestock breed potentiality of hereditary capacity can be developed, it is faster to expand breeding drove, it adopts
Excellent dam can be made to eliminate the interminable gestational period with embryo transfer, embryo take out after soon can again heat, breeding and
Fertilization, so as to generate more offspring within a certain period of time.
If can also induce it in an oestrus in delivery ratio nature situation to dam internal injection promoting sexual gland hormone
More ovums obtain multiple embryos and are used for transplanting, in addition, being also family since embryo can be transported with long-term preservation and remote way
The foundation of gene pool is raiseeed, introduction and exchange of variety source, and reduction transmission etc. provide better condition.
Embryo cryopreservation is that embryo and freezing liquid are fitted into cryovial, by making embryo with quick two groups of coolings placement at a slow speed
A kind of static method got off and can saved in liquid nitrogen, can be by the preferable Embryo freezing preservation of remaining quality, after
Free period or artificial cycle are implanted into uterine cavity after thawing, the chance that increase is become pregnant, and can be used for a super ovulation
Embryo's quantity that period obtains is more, high-quality, once cannot all transplant, can by extra Embryo freezing preservation, thus
Can reasonable restriction tire number, reduce multifetation rate, and can provide for embryo transfer failure or miscarriage dam
Tree-remover meeting again, while embryo transfer can be cancelled when ovarian hyperstimulation syndrome sign occur in superstimulated cycles, it will
Embryo carries out freezen protective, and the non-stimulated period carries out frozen embryo transfer behind.The parent of mutton sheep embryo transfer technology is " to borrow
The tire of the abdomen bosom breeding ewe of inferior strain ewe " carrys out fast-propagation breeding ewe.
The method of existing technology production embryo is complicated, and the survival rate of embryo is low；Existing embryo is in freezen protective timeliness
Fruit is poor, and embryo viability is lower when transplanting again after thawing, and lack has efficient, easy to operate Embryo freezing preservation at this stage
Method.
Summary of the invention
Invention is designed to provide a kind of Embryo Production refrigerating process, solves the problems in background technique.
The invention is realized in this way a kind of Embryo Production refrigerating process, the production refrigerating process the following steps are included:
(1) use physiological saline at 35-40 DEG C in 1-4h in the ovary for acquiring the eupraxic meat sheep used as donor of reproductive system
Under the conditions of take back laboratory, with suction method obtain egg mother cell be placed in save liquid in；
(2) egg mother cell is washed 3-5 times with mature liquid, cultivates 25-30h；
(3) sperm that will be provided with the eupraxic ram of reproductive system is handled；
(4) sperm of step (3) processing is moved by sperm, by the egg mother cell for containing step (2) in sperm and handling；
(5) it is fertilized after 15-20h, dissolves cumulus cell 1-2min with 0.2% hyaluronidase, then Voatex whirlpool shakes
It swings, removes cumulus cell；
(6) fertilized eggs by step (5) processing move into granular cell co-culture system, change half volume every 48h
Culture solution collects embryo after cultivating 150-170h；
(7) the freeze proof processing of embryo: anti-freezing processing first is carried out to embryo with anti-freezing liquid, then embryo is packed into tubule sealing
It is placed at 4 DEG C, then gradually decreases extremely -7 DEG C of temperature according to the speed of 2 DEG C/min, carry out plant ice after osmotic equilibrium 4min, so
Osmotic equilibrium 4min again afterwards gradually decreases temperature to -35 DEG C then according to the speed of 0.4 DEG C/min, then puts into Liquid nitrogen storage；
(8) it saves: when embryo is cooled to -35 DEG C by tubule by freezen protective in liquid nitrogen is taken out and put into frigorimeter,
Liquid nitrogen is supplemented weekly, replaces liquid nitrogen every year.
A further technical solution of the present invention is: the preservation liquid in the step (1) is that PBS liquid adds polyvinyl alcohol
0.4mg/mL, sheep blood serum albumin 3mg/mL.
A further technical solution of the present invention is: oocyte maturation liquid is to cultivate base in M199 in the step (2)
Be added in liquid: 8~12 μ g/mL follicle-stimulating hormone, 8~12 μ g/mL luteotropins, 0.5~1.5 μ g/mL estrogen, 0.05~
0.15mmol/L β mercaptoethanol and 10~20% heat ewe serum.
A further technical solution of the present invention is: liquefacient duration in the step (3): the essence after taking fresh or defrosting
Liquid is diluted to the spermatozoa diluent of 4 × 106~6 × 106/mL.
A further technical solution of the present invention is: by the preparation of sperm in the step (4): adding in fallopian tubal Synthesis liquid
Enter: 0.1~0.2 μm of ol/L penicillamine, 0.1~0.2 μm of ol/L hypotaurine, 5~10 μ g/mL heparin and 2~10% heats are female
Sheep blood serum.
A further technical solution of the present invention is: culture solution is that basis liquid M199 adds 10% tire sheep in the step (6)
Serum；Granular cell co-culture system preparation method: it after having acquired egg mother cell, takes remaining containing granular cell in ovoscopy plate
Liquor folliculi 1mL, add 2mL0.2% hyaluronic acid enzymic digestion 3-5min, with equivalent culture solution terminate digestion after, 200g centrifugation
5min, precipitating are suspended again with 5mL culture solution, and 200g is centrifuged 5min, and final precipitating is suspended with culture solution, will be hanged with pipettor
Supernatant liquid is added in culture drop, and ultimate density is 106/mL.
A further technical solution of the present invention is: the anti-freezing liquid in the step (7) mainly includes following material composition:
Ethylene glycol (EG) solution that concentration is 20%, dimethyl sulfoxide (DMSO) solution that concentration is 20%, concentration are the sucrose of 0.5M
Solution.
A further technical solution of the present invention is: plant ice described in step (7) is used in the tweezers folder being pre-chilled in liquid nitrogen
Live in tubule 12s.
Beneficial effects of the present invention: the present invention, which solves the problems, such as that current embryo quality source is insufficient, has important meaning
Justice shortens the generation inteval of excellent variety breeding, improves the genetic improvement speed and production efficiency of drove；Freezing skill of the invention
Art can be reduced the cooling preservation effect that embryo is improved to physical injury caused by embryo, and it is active higher after thawing.
Specific embodiment
A kind of Embryo Production refrigerating process, the production refrigerating process the following steps are included:
(1) use physiological saline at 35-40 DEG C in 1-4h in the ovary for acquiring the eupraxic meat sheep used as donor of reproductive system
Under the conditions of take back laboratory, with suction method obtain egg mother cell be placed in save liquid in；
(2) egg mother cell is washed 3-5 times with mature liquid, cultivates 25-30h；
(3) sperm that will be provided with the eupraxic ram of reproductive system is handled；
(4) sperm of step (3) processing is moved by sperm, by the egg mother cell for containing step (2) in sperm and handling；
(5) it is fertilized after 15-20h, dissolves cumulus cell 1-2min with 0.2% hyaluronidase, then Voatex whirlpool shakes
It swings, removes cumulus cell；
(6) fertilized eggs by step (5) processing move into granular cell co-culture system, change half volume every 48h
Culture solution collects embryo after cultivating 150-170h；
(7) the freeze proof processing of embryo: anti-freezing processing first is carried out to embryo with anti-freezing liquid, then embryo is packed into tubule sealing
It is placed at 4 DEG C, then gradually decreases extremely -7 DEG C of temperature according to the speed of 2 DEG C/min, carry out plant ice after osmotic equilibrium 4min, so
Osmotic equilibrium 4min again afterwards gradually decreases temperature to -35 DEG C then according to the speed of 0.4 DEG C/min, then puts into Liquid nitrogen storage；
(8) it saves: when embryo is cooled to -35 DEG C by tubule by freezen protective in liquid nitrogen is taken out and put into frigorimeter,
Liquid nitrogen is supplemented weekly, replaces liquid nitrogen every year.
Preservation liquid in the step (1) is that PBS liquid adds polyvinyl alcohol 0.4mg/mL, sheep blood serum albumin 3mg/mL.
Oocyte maturation liquid is to be added in M199 culture medium stoste in the step (2): 8~12 μ g/mL promote ovarian follicle
Element, 8~12 μ g/mL luteotropins, 0.5~1.5 μ g/mL estrogen, 0.05~0.15mmol/L β mercaptoethanol and 10~20%
Heat ewe serum.
Liquefacient duration in the step (3): take it is fresh or thaw after sperm, be diluted to 4 × 106~6 × 106/
The spermatozoa diluent of mL.
By the preparation of sperm in the step (4): it is added in fallopian tubal Synthesis liquid: 0.1~0.2 μm of ol/L penicillamine,
0.1~0.2 μm of ol/L hypotaurine, 5~10 μ g/mL heparin and 2~10% heat ewe serum.
In the step (6), culture solution is that basis liquid M199 adds 10% tire sheep blood serum；Granular cell co-culture system system
Preparation Method: after having acquired egg mother cell, the remaining liquor folliculi 1mL containing granular cell in ovoscopy plate is taken, adds 2mL0.2% saturating
Bright matter acid enzymic digestion 3-5min, after terminating digestion with equivalent culture solution, 200g is centrifuged 5min, and precipitating is hanged again with 5mL culture solution
Floating, 200g is centrifuged 5min, and final precipitating is suspended with culture solution, and suspension is added in culture drop with pipettor, final
Concentration is 106/mL.
Anti-freezing liquid in the step (7) mainly includes following material composition: the ethylene glycol (EG) that concentration is 20% is molten
Liquid, dimethyl sulfoxide (DMSO) solution that concentration is 20%, concentration are the sucrose solution of 0.5M.
Plant ice described in step (7) is used in the tweezers being pre-chilled in liquid nitrogen and clamps tubule 12s.
Embodiment one:
A kind of Embryo Production refrigerating process, the production refrigerating process the following steps are included:
(1) will acquire the ovary of the eupraxic meat sheep used as donor of reproductive system in 2h with physiological saline 36 DEG C under the conditions of
Laboratory is taken back, egg mother cell is obtained with suction method and is placed in preservation liquid；
(2) egg mother cell is washed 3 times with mature liquid, cultivates 26h；
(3) sperm that will be provided with the eupraxic ram of reproductive system is handled；
(4) sperm of step (3) processing is moved by sperm, by the egg mother cell for containing step (2) in sperm and handling；
(5) it is fertilized after 18h, dissolves cumulus cell 1min with 0.2% hyaluronidase, then Voatex whirlpool shakes, and takes off
Except cumulus cell；
(6) fertilized eggs by step (5) processing move into granular cell co-culture system, change half volume every 48h
Culture solution collects embryo after cultivating 150h；
(7) the freeze proof processing of embryo: anti-freezing processing first is carried out to embryo with anti-freezing liquid, then embryo is packed into tubule sealing
It is placed at 4 DEG C, then gradually decreases extremely -7 DEG C of temperature according to the speed of 2 DEG C/min, carry out plant ice after osmotic equilibrium 4min, so
Osmotic equilibrium 4min again afterwards gradually decreases temperature to -35 DEG C then according to the speed of 0.4 DEG C/min, then puts into Liquid nitrogen storage；
(8) it saves: when embryo is cooled to -35 DEG C by tubule by freezen protective in liquid nitrogen is taken out and put into frigorimeter,
Liquid nitrogen is supplemented weekly, replaces liquid nitrogen every year.
Preservation liquid in the step (1) is that PBS liquid adds polyvinyl alcohol 0.4mg/mL, sheep blood serum albumin 3mg/mL.
Oocyte maturation liquid is to be added in M199 culture medium stoste in the step (2): 8~12 μ g/mL promote ovarian follicle
Element, 8~12 μ g/mL luteotropins, 0.5~1.5 μ g/mL estrogen, 0.05~0.15mmol/L β mercaptoethanol and 10~20%
Heat ewe serum.
Liquefacient duration in the step (3): take it is fresh or thaw after sperm, be diluted to 4 × 106~6 × 106/
The spermatozoa diluent of mL.
By the preparation of sperm in the step (4): it is added in fallopian tubal Synthesis liquid: 0.1~0.2 μm of ol/L penicillamine,
0.1~0.2 μm of ol/L hypotaurine, 5~10 μ g/mL heparin and 2~10% heat ewe serum.
In the step (6), culture solution is that basis liquid M199 adds 10% tire sheep blood serum；Granular cell co-culture system system
Preparation Method: after having acquired egg mother cell, the remaining liquor folliculi 1mL containing granular cell in ovoscopy plate is taken, adds 2mL0.2% saturating
Bright matter acid enzymic digestion 3-5min, after terminating digestion with equivalent culture solution, 200g is centrifuged 5min, and precipitating is hanged again with 5mL culture solution
Floating, 200g is centrifuged 5min, and final precipitating is suspended with culture solution, and suspension is added in culture drop with pipettor, final
Concentration is 106/mL.
Anti-freezing liquid in the step (7) mainly includes following material composition: the ethylene glycol (EG) that concentration is 20% is molten
Liquid, dimethyl sulfoxide (DMSO) solution that concentration is 20%, concentration are the sucrose solution of 0.5M.
Plant ice described in step (7) is used in the tweezers being pre-chilled in liquid nitrogen and clamps tubule 12s.
Embodiment two:
A kind of Embryo Production refrigerating process, the production refrigerating process the following steps are included:
(1) will acquire the ovary of the eupraxic meat sheep used as donor of reproductive system in 3h with physiological saline 38 DEG C under the conditions of
Laboratory is taken back, egg mother cell is obtained with suction method and is placed in preservation liquid；
(2) egg mother cell is washed 4 times with mature liquid, cultivates 28h；
(3) sperm that will be provided with the eupraxic ram of reproductive system is handled；
(4) sperm of step (3) processing is moved by sperm, by the egg mother cell for containing step (2) in sperm and handling；
(5) it is fertilized after 18h, dissolves cumulus cell 2min with 0.2% hyaluronidase, then Voatex whirlpool shakes, and takes off
Except cumulus cell；
(6) fertilized eggs by step (5) processing move into granular cell co-culture system, change half volume every 48h
Culture solution collects embryo after cultivating 160h；
(7) the freeze proof processing of embryo: anti-freezing processing first is carried out to embryo with anti-freezing liquid, then embryo is packed into tubule sealing
It is placed at 4 DEG C, then gradually decreases extremely -7 DEG C of temperature according to the speed of 2 DEG C/min, carry out plant ice after osmotic equilibrium 4min, so
Osmotic equilibrium 4min again afterwards gradually decreases temperature to -35 DEG C then according to the speed of 0.4 DEG C/min, then puts into Liquid nitrogen storage；
(8) it saves: when embryo is cooled to -35 DEG C by tubule by freezen protective in liquid nitrogen is taken out and put into frigorimeter,
Liquid nitrogen is supplemented weekly, replaces liquid nitrogen every year.
Preservation liquid in the step (1) is that PBS liquid adds polyvinyl alcohol 0.4mg/mL, sheep blood serum albumin 3mg/mL.
Oocyte maturation liquid is to be added in M199 culture medium stoste in the step (2): 8~12 μ g/mL promote ovarian follicle
Element, 8~12 μ g/mL luteotropins, 0.5~1.5 μ g/mL estrogen, 0.05~0.15mmol/L β mercaptoethanol and 10~20%
Heat ewe serum.
Liquefacient duration in the step (3): take it is fresh or thaw after sperm, be diluted to 4 × 106~6 × 106/
The spermatozoa diluent of mL.
By the preparation of sperm in the step (4): it is added in fallopian tubal Synthesis liquid: 0.1~0.2 μm of ol/L penicillamine,
0.1~0.2 μm of ol/L hypotaurine, 5~10 μ g/mL heparin and 2~10% heat ewe serum.
In the step (6), culture solution is that basis liquid M199 adds 10% tire sheep blood serum；Granular cell co-culture system system
Preparation Method: after having acquired egg mother cell, the remaining liquor folliculi 1mL containing granular cell in ovoscopy plate is taken, adds 2mL0.2% saturating
Bright matter acid enzymic digestion 3-5min, after terminating digestion with equivalent culture solution, 200g is centrifuged 5min, and precipitating is hanged again with 5mL culture solution
Floating, 200g is centrifuged 5min, and final precipitating is suspended with culture solution, and suspension is added in culture drop with pipettor, final
Concentration is 106/mL.
Anti-freezing liquid in the step (7) mainly includes following material composition: the ethylene glycol (EG) that concentration is 20% is molten
Liquid, dimethyl sulfoxide (DMSO) solution that concentration is 20%, concentration are the sucrose solution of 0.5M.
Plant ice described in step (7) is used in the tweezers being pre-chilled in liquid nitrogen and clamps tubule 12s.
The present invention solves the problems, such as that current embryo quality source is insufficient and is of great significance, and shortens excellent variety breeding
Generation inteval, improve the genetic improvement speed and production efficiency of drove；Refrigeration Technique of the invention can be reduced to be caused to embryo
Physical injury, improve the cooling preservation effect of embryo, and its activity is higher after thawing.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (8)
1. a kind of Embryo Production refrigerating process, it is characterised in that: the production refrigerating process the following steps are included:
(1) use physiological saline in 35-40 DEG C of condition in 1-4h in the ovary for acquiring the eupraxic meat sheep used as donor of reproductive system
Under take back laboratory, with suction method obtain egg mother cell be placed in save liquid in；
(2) egg mother cell is washed 3-5 times with mature liquid, cultivates 25-30h；
(3) sperm that will be provided with the eupraxic ram of reproductive system is handled；
(4) sperm of step (3) processing is moved by sperm, by the egg mother cell for containing step (2) in sperm and handling；
(5) it is fertilized after 15-20h, dissolves cumulus cell 1-2min with 0.2% hyaluronidase, then Voatex whirlpool shakes, and takes off
Except cumulus cell；
(6) fertilized eggs by step (5) processing move into granular cell co-culture system, change the culture of half volume every 48h
Liquid collects embryo after cultivating 150-170h；
(7) the freeze proof processing of embryo: anti-freezing processing first is carried out to embryo with anti-freezing liquid, then embryo is packed into tubule and seals postposition
At 4 DEG C, extremely -7 DEG C of temperature then are gradually decreased according to the speed of 2 DEG C/min, carries out plant ice after osmotic equilibrium 4min, then again
Osmotic equilibrium 4min gradually decreases temperature to -35 DEG C then according to the speed of 0.4 DEG C/min, then puts into Liquid nitrogen storage；
(8) it saves: when embryo is cooled to -35 DEG C by tubule by being taken out in frigorimeter and putting into freezen protective in liquid nitrogen, weekly
Liquid nitrogen is supplemented, replaces liquid nitrogen every year.
2. a kind of Embryo Production refrigerating process according to claim 1, it is characterised in that: the preservation in the step (1)
Liquid is that PBS liquid adds polyvinyl alcohol 0.4mg/mL, sheep blood serum albumin 3mg/mL.
3. a kind of Embryo Production refrigerating process according to claim 1, it is characterised in that: ovum is female thin in the step (2)
Born of the same parents' maturation liquid be added in M199 culture medium stoste: 8~12 μ g/mL follicle-stimulating hormone, 8~12 μ g/mL luteotropins, 0.5~
1.5 μ g/mL estrogen, 0.05~0.15mmol/L β mercaptoethanol and 10~20% heat ewe serum.
4. a kind of Embryo Production refrigerating process according to claim 1, it is characterised in that: in the step (3) at sperm
Reason: the sperm after taking fresh or defrosting is diluted to the spermatozoa diluent of 4 × 106~6 × 106/mL.
5. a kind of Embryo Production refrigerating process according to claim 1, it is characterised in that: by sperm in the step (4)
Preparation: be added in fallopian tubal Synthesis liquid: 0.1~0.2 μm of ol/L penicillamine, 0.1~0.2 μm of ol/L hypotaurine, 5~10
μ g/mL heparin and 2~10% heat ewe serum.
6. a kind of Embryo Production refrigerating process according to claim 1, it is characterised in that: in the step (6), culture solution
10% tire sheep blood serum is added for basic liquid M199；Granular cell co-culture system preparation method: after having acquired egg mother cell, inspection is taken
The remaining liquor folliculi 1mL containing granular cell in ovum plate, adds 2mL0.2% hyaluronic acid enzymic digestion 3-5min, with equivalent culture
After liquid terminates digestion, 200g is centrifuged 5min, and precipitating is suspended again with 5mL culture solution, and 200g is centrifuged 5min, final precipitating training
Nutrient solution suspends, and suspension is added in culture drop with pipettor, ultimate density is 106/mL.
7. a kind of Embryo Production refrigerating process according to claim 1, it is characterised in that: resisting in the step (7) is cold
Freezing liquid mainly includes following material composition: ethylene glycol (EG) solution that concentration is 20%, the dimethyl sulfoxide that concentration is 20%
(DMSO) solution, concentration are the sucrose solution of 0.5M.
8. a kind of Embryo Production refrigerating process according to claim 1, it is characterised in that: plant ice described in step (7)
It is used in the tweezers being pre-chilled in liquid nitrogen and clamps tubule 12s.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0340934A1 (en) * | 1988-04-14 | 1989-11-08 | University College Dublin | In vitro culture of bovine embryos |
| CN102703378A (en) * | 2011-12-06 | 2012-10-03 | 中国农业科学院兰州畜牧与兽药研究所 | Method for producing yak embryo in vitro |
| CN107232179A (en) * | 2016-03-29 | 2017-10-10 | 中国农业大学 | A kind of semen diluent for improving seminal fluid low temperature/freezen protective quality and its application |
| CN109006805A (en) * | 2018-09-17 | 2018-12-18 | 内蒙古草原乌骨羊生物科技有限公司 | A kind of Embryo Production refrigerating process |
-
2019
- 2019-07-05 CN CN201910606151.4A patent/CN110241072A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0340934A1 (en) * | 1988-04-14 | 1989-11-08 | University College Dublin | In vitro culture of bovine embryos |
| CN102703378A (en) * | 2011-12-06 | 2012-10-03 | 中国农业科学院兰州畜牧与兽药研究所 | Method for producing yak embryo in vitro |
| CN107232179A (en) * | 2016-03-29 | 2017-10-10 | 中国农业大学 | A kind of semen diluent for improving seminal fluid low temperature/freezen protective quality and its application |
| CN109006805A (en) * | 2018-09-17 | 2018-12-18 | 内蒙古草原乌骨羊生物科技有限公司 | A kind of Embryo Production refrigerating process |
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Application publication date: 20190917 |