CN109112100A - The collection method of the small cumulus oocyte complex for having chamber of one boar - Google Patents
The collection method of the small cumulus oocyte complex for having chamber of one boar Download PDFInfo
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- CN109112100A CN109112100A CN201811027903.3A CN201811027903A CN109112100A CN 109112100 A CN109112100 A CN 109112100A CN 201811027903 A CN201811027903 A CN 201811027903A CN 109112100 A CN109112100 A CN 109112100A
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- 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
- C12N5/0608—Germ cells
- C12N5/0609—Oocytes, oogonia
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Abstract
The invention discloses the collection methods of the small cumulus oocyte complex for having chamber of a boar, using the ovary of pig as raw material, it is drawn by ovary acquisition, equipment disinfection, ovarian follicle, oocytes collection, the effect for collecting egg mother cell is best, recovery efficiency highest, it can make full use of existing resource in hand, simply, the liquor folliculi and cumulus oocyte complex of economic, the efficient recycling small antral follicles of pig, for providing material abundant about the development of research and the animal husbandry of the Oocyte Development in the small antral follicles of pig from now on.
Description
Technical field
The present invention relates to cell biology, the collections of the small cumulus oocyte complex for having chamber of a specially boar
Method.
Technical background
Nowadays while, with animal embryo engineering technology fast development, animal husbandry, aquaculture have also been greatly facilitated
And biomedical progress.Using animal embryo function engineering technology can improve domestic animal kind, cultivate transgenosis new varieties,
Prepare human disease model, production pharmaceutical protein, research embryonic development mechanism etc..And pig is embryo biologist and geneticist
One of the important model animal of research, because the physiology of pig and anatomical structure and the mankind have very big similarity.
Currently, how simply, efficiently to collect egg mother cell in pig ovary is to carry out subsequent in vitro mature technology
(IVM), modem animals embryo's biology skill such as technology (IVF) in vitro fertilization, somatic cell clone technique (SCNT), lonely female activation (PA)
The essential link of art is also to support the essential guarantee of above-mentioned technology, especially with respect to the small antral follicles liquid of pig and ovum
The collection for steeping cumulus oocyte complex in liquid is then very difficult, because the invention is a kind of simple, it is economical, conveniently, efficient ovum
Mother cell collection method just seems particularly significant.
Summary of the invention
It is an object of the invention to: the collection method of the small cumulus oocyte complex for having chamber of a boar is provided, simply,
Economical, conveniently, efficiently, the effect for collecting egg mother cell is good, and recovery efficiency is high, for from now on about the ovum in the small antral follicles of pig
The research of mother cell development and the development of animal husbandry provide material abundant.
To achieve the goals above, the invention provides the following technical scheme:
The collection method of the small cumulus oocyte complex for having chamber of one boar, comprising the following steps:
S1: ovary acquisition: the ovary of pig is chosen from slaughterhouse, and ovary is deposited in and is loaded with benzyl penicillin and sulfate chain
In the vacuum cup of the physiological saline of two kinds of antibiotic of mycin, and temperature is maintained at 28~35 DEG C in vacuum cup, and experiment is taken back in 2h
Room；
S2: equipment disinfection: in the lab, equipment required by next step is all carried out disinfection；
S3: ovarian follicle draw: using 5ml syringe needle cooperation 5ml asepsis injector syringe suction ovary on 0.5~
The ovarian follicle of 2mm, and the liquor folliculi of extraction and egg mother cell are slowly injected into the 15ml point bottom centrifugation for being placed in 38 DEG C of thermostat water baths
Guan Zhong makes cumulus oocyte complex (Cumulus-Oocyte complexes, COCs) natural subsidence；
S4: oocytes collection: the liquor folliculi of previous step natural subsidence and egg mother cell be diluted to egg-cleaning liquid suitable
Suitable concentration, then the egg mother cell in liquid is collected by Stereo microscope, so that it is female thin to complete ovarian cumulus ovum in the small antral follicles of pig
The collection of born of the same parents' complex.
Preferably, the syringe needle is strong scholar's board disposable sterilized injector syringe needle.
Preferably, the equipment to carry out disinfection in the S2 step, including 5ml syringe needle, 5ml asepsis injector needle
Cylinder, 15ml conical centrifuge tube.
Preferably, the egg-cleaning liquid in the S4 step the preparation method comprises the following steps: first with 100 mL deionized water dissolving 1g
PVA (Polyvinyl alcohol, Sigma), is completely dissolved to PVA, weighs 9.55g DPBS powder again after cooling
(Dulbecco ' s phosphate buffered saline, Gibco, 758054, no calcium and magnesium), adds deionized water, make dissolution,
It is settled to 1L, is not stirred；Then masking foil on high pressure front cover, it is cooling after high pressure sterilization, with the ratio of 1mL/L add it is phenol red and
It is dual anti-；Finally in super-clean bench, using 0.22 μm of filter, packing is into 50mL centrifuge tube after degerming, 4 DEG C of preservations, before use
It is preheated under the conditions of 38.5 DEG C.
Preferably, the physiological saline in the S1 step, preparation method includes the following steps:
(1), 1.3g penicillin (P3032) and 1g streptomysin (S1277) is weighed in 15mL Embryo Culture water, it is sufficiently molten
Be settled to 20mL after solution, reuse 0.22 μm of filter filtration sterilization, be prepared into concentration be 1000 × mother liquor, mother liquor is dispensed into
In the EP centrifuge tube of 800 μ L, 500 μ L of every pipe point are placed in -20 DEG C and save backup；
(2), 9g NaCl powder (Chinese medicines group) is weighed, 1L is settled to primary filtering Milli-Q water, then by liquid
It is dispensed into the vial of 300mL, every bottle of packing 250mL liquid, high pressure sterilization after packing is spare；
(3), it chooses in 1 centrifuge tube step (1) and dispenses the penicillin and streptomysin saved backup, then draw 500 μ L,
And be added in step (2) in the vial of the spare physiological saline containing 250mL, it mixes, contains benzyl penicillin and sulphur to be made
The mixed liquor of normal saline of sour two kinds of antibiotic of streptomysin.
The beneficial effects of the present invention are:
The collection method of the small cumulus oocyte complex for having chamber of a boar of the invention, collects the effect of egg mother cell most
Good, recovery efficiency highest can make full use of existing resource in hand, simple, economic, the efficient recycling small antral follicles of pig
Liquor folliculi and cumulus oocyte complex, for from now on about the research of the Oocyte Development in the small antral follicles of pig
And the development of animal husbandry provides material abundant.
Specific embodiment
In order to facilitate the understanding of those skilled in the art, the present invention is further illustrated combined with specific embodiments below.
Embodiment 1:
The collection method of the small cumulus oocyte complex for having chamber of one boar, comprising the following steps:
S1: ovary acquisition: the ovary of pig is chosen from slaughterhouse, and ovary is deposited in and is loaded with benzyl penicillin and sulfate chain
In the vacuum cup of the physiological saline of two kinds of antibiotic of mycin, and temperature is maintained at 28~35 DEG C in vacuum cup, and experiment is taken back in 2h
Room；
Wherein, physiological saline preparation method the following steps are included:
(1), 1.3g penicillin (P3032) and 1g streptomysin (S1277) is weighed in 15mL Embryo Culture water, it is sufficiently molten
Be settled to 20mL after solution, reuse 0.22 μm of filter filtration sterilization, be prepared into concentration be 1000 × mother liquor, mother liquor is dispensed into
In the EP centrifuge tube of 800 μ L, 500 μ L of every pipe point are placed in -20 DEG C and save backup；
(2), 9g NaCl powder (Chinese medicines group) is weighed, 1L is settled to primary filtering Milli-Q water, then by liquid
It is dispensed into the vial of 300mL, every bottle of packing 250mL liquid, high pressure sterilization after packing is spare；
(3), it chooses in 1 centrifuge tube step (1) and dispenses the penicillin and streptomysin saved backup, then draw 500 μ L,
And be added in step (2) in the vial of the spare physiological saline containing 250mL, it mixes, contains benzyl penicillin and sulphur to be made
The mixed liquor of normal saline of sour two kinds of antibiotic of streptomysin.
S2: equipment disinfection: in the lab, by equipment required by next step, including the disposable 5ml injection of strong scholar's board
Device syringe needle, 5ml asepsis injector syringe, 15ml conical centrifuge tube etc. all carry out disinfection.
S3: ovarian follicle draw: using 5ml syringe needle cooperation 5ml asepsis injector syringe suction ovary on 0.5~
The ovarian follicle of 2mm, and the liquor folliculi of extraction and egg mother cell are slowly injected into the 15ml point bottom centrifugation for being placed in 38 DEG C of thermostat water baths
Guan Zhong makes cumulus oocyte complex (Cumulus-Oocyte complexes, COCs) natural subsidence.
Wherein, in the selection of syringe needle and injector syringe in this step, early period is gradually groped,
Main method are as follows: in the lab, with the small antral follicles (diameter 0.5-2mm) of pig for experimental material, have chosen 4 kinds of models
Strong scholar's board disposable sterilized injector syringe needle (4 kinds of syringe needle models are respectively 1ml, 2.5ml, 5ml, 10ml), in order to
Facilitate exclude 4 kinds of models syringe needle in operation brought by difference, we uniformly use 5ml to be good for scholar's syringe
The syringe needle of above-mentioned 4 kinds of models is connected respectively on the syringe of 5ml syringe by syringe；Then, using the injection of every kind of model
Device respectively aspirates ovarian follicle of 100 specifications between 0.5-2mm, and collected liquor folliculi is diluted to suitable concentration with egg-cleaning liquid,
Collected cumulus oocyte complex (COCs) is picked out by Stereo microscope again.Through the above steps, we carry out
Multiple test operation, and the par of this 4 kinds of different size syringe needles recycling egg mother cells has been counted, it finally obtains following
As a result: the use of the oocyte number that 1ml syringe needle is drawn being 56 pieces；The ovum drawn using 2.5ml syringe needle
Mother cell number is 48 pieces；It the use of the oocyte number that 5ml syringe needle is drawn is 75 pieces；Use 10ml syringe needle
The oocyte number drawn is 54 pieces.By the way that the quantity of the drawn egg mother cell of syringe needle of this above 4 kinds of models is compared
Compared with, it has been found that the syringe needle of these four models can recycle egg mother cell, but collect ovum mother with 5ml syringe needle
The effect of cell is best, recovery efficiency highest.
S4: oocytes collection: the liquor folliculi of previous step natural subsidence and egg mother cell be diluted to egg-cleaning liquid suitable
Suitable concentration, then the egg mother cell in liquid is collected by Stereo microscope, so that it is female thin to complete ovarian cumulus ovum in the small antral follicles of pig
The collection of born of the same parents' complex.
Wherein, egg-cleaning liquid the preparation method comprises the following steps: first use 100mL deionized water dissolving 1g PVA (Polyvinyl
Alcohol, Sigma), it is completely dissolved to PVA, weighs 9.55g DPBS powder (Dulbecco ' s phosphate again after cooling
Buffered saline, Gibco, 758054, no calcium and magnesium), add deionized water, makes to dissolve, be settled to 1L, not stir；Then
Masking foil on high pressure front cover, it is cooling after high pressure sterilization, added with the ratio of 1mL/L phenol red and dual anti-；Finally in super-clean bench,
Using 0.22 μm of filter, packing is into 50 mL centrifuge tubes after degerming, 4 DEG C of preservations, using preheating under the conditions of first 38.5 DEG C.
Above-mentioned is that invention is exemplarily described, it is clear that present invention specific implementation is not subject to the restrictions described above,
As long as using this insubstantial improvement that the inventive concept and technical scheme of the present invention carry out, or the not improved structure by invention
Think and technical solution directly applies to other occasions, it is within the scope of the present invention.
Claims (5)
1. the collection method of the small cumulus oocyte complex for having chamber of a boar, which comprises the following steps:
S1: ovary acquisition: the ovary of pig is chosen from slaughterhouse, and ovary is deposited in and is loaded with benzyl penicillin and streptomycin sulphate
In the vacuum cup of the physiological saline of two kinds of antibiotic, and temperature is maintained at 28~35 DEG C in vacuum cup, and laboratory is taken back in 2h；
S2: equipment disinfection: in the lab, equipment required by next step is all carried out disinfection；
S3: ovarian follicle is drawn: using 0.5~2mm's on 5ml syringe needle cooperation 5ml asepsis injector syringe suction ovary
Ovarian follicle, and the liquor folliculi of extraction and egg mother cell are slowly injected into the 15ml conical centrifuge tube for being placed in 38 DEG C of thermostat water baths,
Make cumulus oocyte complex (Cumulus-Oocyte complexes, COCs) natural subsidence；
S4: oocytes collection: it is suitable for dense for being diluted to the liquor folliculi of previous step natural subsidence and egg mother cell with egg-cleaning liquid
Degree, then the egg mother cell in liquid is collected by Stereo microscope, so that it is multiple to complete cumulus oocyte in the small antral follicles of pig
Fit collection.
2. the collection method of the small cumulus oocyte complex for having chamber of a boar according to claim 1, feature exist
In the syringe needle is strong scholar's board disposable sterilized injector syringe needle.
3. the collection method of the small cumulus oocyte complex for having chamber of a boar according to claim 1, feature exist
In, the equipment to carry out disinfection in the S2 step, including 5ml syringe needle, 5ml asepsis injector syringe, 15ml point bottom from
Heart pipe.
4. the collection method of the small cumulus oocyte complex for having chamber of a boar according to claim 1, feature exist
In, egg-cleaning liquid in the S4 step the preparation method comprises the following steps: using 100mL deionized water dissolving 1g PVA (Polyvinyl first
Alcohol, Sigma), it is completely dissolved to PVA, weighs 9.55g DPBS powder (Dulbecco ' s phosphate again after cooling
Buffered saline, Gibco, 758054, no calcium and magnesium), add deionized water, makes to dissolve, be settled to 1L, not stir；Then high
Masking foil on front cover is pressed, cooling after high pressure sterilization is added phenol red and dual anti-with the ratio of 1mL/L；Finally in super-clean bench, make
With 0.22 μm of filter, packing is into 50mL centrifuge tube after degerming, 4 DEG C of preservations, using preheating under the conditions of first 38.5 DEG C.
5. the collection method of the small cumulus oocyte complex for having chamber of a boar according to claim 1, feature exist
In physiological saline in the S1 step, preparation method includes the following steps:
(1), 1.3g penicillin (P3032) and 1g streptomysin (S1277) is weighed in 15mL Embryo Culture water, it is fixed after completely dissolution
Hold to 20mL, reuse 0.22 μm of filter filtration sterilization, be prepared into concentration be 1000 × mother liquor, mother liquor is dispensed into 800 μ L
EP centrifuge tube in, every pipe point 500 μ L are placed in -20 DEG C and save backup；
(2), 9g NaCl powder (Chinese medicines group) is weighed, 1L is settled to primary filtering Milli-Q water, then by liquid subpackage
Into the vial of 300mL, every bottle of packing 250mL liquid, high pressure sterilization after packing is spare；
(3), it chooses in 1 centrifuge tube step (1) and dispenses the penicillin and streptomysin saved backup, then draw 500 μ L, and add
It is added in step (2) in the vial of the spare physiological saline containing 250mL, mixes, contain benzyl penicillin and sulfate chain to be made
The mixed liquor of normal saline of two kinds of antibiotic of mycin.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811027903.3A CN109112100A (en) | 2018-09-04 | 2018-09-04 | The collection method of the small cumulus oocyte complex for having chamber of one boar |
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| CN201811027903.3A CN109112100A (en) | 2018-09-04 | 2018-09-04 | The collection method of the small cumulus oocyte complex for having chamber of one boar |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6271436B1 (en) * | 1996-10-11 | 2001-08-07 | The Texas A & M University System | Cells and methods for the generation of transgenic pigs |
| JP2004173635A (en) * | 2002-11-28 | 2004-06-24 | National Agriculture & Bio-Oriented Research Organization | Method for culturing oocyte and method for growing oocyte |
| CN101709290A (en) * | 2009-12-03 | 2010-05-19 | 安徽农业大学 | Simple, economic and efficient method for in-vitro maturity of porcine oocytes |
| CN102258004A (en) * | 2010-05-27 | 2011-11-30 | 中国科学院动物研究所 | Method for preserving/culturing oocytes in vitro to inhibit oocytes from germinal vesicle breakdown |
| CN103525759A (en) * | 2013-10-06 | 2014-01-22 | 吉林大学 | Application of cordycepin in in-vitro maturation culture of oocytes of small pig follicles |
-
2018
- 2018-09-04 CN CN201811027903.3A patent/CN109112100A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6271436B1 (en) * | 1996-10-11 | 2001-08-07 | The Texas A & M University System | Cells and methods for the generation of transgenic pigs |
| JP2004173635A (en) * | 2002-11-28 | 2004-06-24 | National Agriculture & Bio-Oriented Research Organization | Method for culturing oocyte and method for growing oocyte |
| CN101709290A (en) * | 2009-12-03 | 2010-05-19 | 安徽农业大学 | Simple, economic and efficient method for in-vitro maturity of porcine oocytes |
| CN102258004A (en) * | 2010-05-27 | 2011-11-30 | 中国科学院动物研究所 | Method for preserving/culturing oocytes in vitro to inhibit oocytes from germinal vesicle breakdown |
| CN103525759A (en) * | 2013-10-06 | 2014-01-22 | 吉林大学 | Application of cordycepin in in-vitro maturation culture of oocytes of small pig follicles |
Non-Patent Citations (4)
| Title |
|---|
| SILVA BR等: "Dose-dependent effects of frutalin on in vitro maturation and fertilization of pig oocytes", 《ANIMAL REPRODUCTION SCIENCE》 * |
| 吴光明: "猪卵母细胞体外成熟的研究", 《华南师范大学学报(自然科学版)》 * |
| 张坤等: "季节对猪体外成熟卵母细胞的发育能力的影响", 《自然科学进展》 * |
| 陈祥等: "猪卵巢卵母细胞采集方法的比较研究", 《畜牧兽医科技信息》 * |
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Application publication date: 20190101 |