CN101709290A - Simple, economic and efficient method for in-vitro maturity of porcine oocytes - Google Patents
Simple, economic and efficient method for in-vitro maturity of porcine oocytes Download PDFInfo
- Publication number
- CN101709290A CN101709290A CN200910185795A CN200910185795A CN101709290A CN 101709290 A CN101709290 A CN 101709290A CN 200910185795 A CN200910185795 A CN 200910185795A CN 200910185795 A CN200910185795 A CN 200910185795A CN 101709290 A CN101709290 A CN 101709290A
- Authority
- CN
- China
- Prior art keywords
- vitro
- ovocyte
- ovary
- porcine oocytes
- oocytes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The invention relates to a simple, economic and efficient method for the in-vitro maturity of porcine oocytes, which comprises the steps of collecting an ovary, collecting oocytes from the ovary, sieving the oocytes and maturing and culturing in vitro. The method is characterized in that the entire process of maturing and culturing the oocytes in vitro is carried out in a manual lung gas expiration environment, 40 of the sieved ovarian cumulus-oocyte complexes are in one hole on average and are transplanted into a four-hole plate which is transplanted into an inflation bag, a bag opening is sealed by a sealing machine, respiratory gas exchange is carried out by testing personnel, the gas of the lung is blown into the inflation bag by an air blowing needle, air in the inflation bag is exhausted completely, the inflation bag is filled with the gas of the lung, sealed, put into a constant temperature tank with the temperature of 38 DEG C and cultured, and maturing and culturing are carried out for 42-44h.
Description
Technical field
The invention belongs to the cytobiology field, particularly a kind of simple, economic, in-vitro maturity of porcine oocytes novel method efficiently.
Background technology
Chinese patent application 200810218336.X discloses " a kind of porcine oocytes mature method in vitro culture ", comprise and gather the ovary step, oocyte collection step from ovary, ovocyte screening step and ripe culturing step, it is characterized in that, before the ovocyte acquisition step, the ovary of collection is deposited in the container that is loaded with physiological saline and is preserved, and temperature remains on 32～34 ℃ in the container; Contain 1～10 in the egg-cleaning liquid that ovocyte screening step adopts and comprise collection ovary step, oocyte collection step from ovary, ovocyte screening step and ripe culturing step, it is characterized in that, before the ovocyte acquisition step, the ovary of gathering is deposited in the container that is loaded with physiological saline and is preserved, and temperature remains on 32～34 ℃ in the container; The amphotericin B that contains 1～10 μ g/mL in the egg-cleaning liquid that ovocyte screening step adopts, the serum of 2%～20%v/v; The TCM-199[1X that contains 50～90%v/v in the maturation culture solution that the oocyte maturation culturing step adopts] basal liquid, the PMSG (pregnant mare serum gonadotrop(h)in (PMSG)) of the hCG of 5～25IU/mL (human chorionic gonadotrophin) and 5～20IU/mL.
This patent application has the following disadvantages.At first, before the ovocyte collection, when ovary is gathered ovary placed in the insulation can 2h that contains 32～34 ℃ of physiological saline and transport the laboratory back, but do not add penicillin G and two kinds of microbiotic of Vetstrep in the physiological saline, make the bacterial contamination ovocyte easily.The second, ovocyte uses vacuum pump that pressure source is provided when gathering, depend on the ovarian follicle of 3～6mm on the machine negative pressure-pumping ovary, but vacuum pump costs an arm and a leg, the cost height, and carry also inconveniently, be unfavorable for production line and field work.The 3rd, the egg-cleaning liquid composition is too many, and the cost height is unfavorable for on-the-spot preparation, and IVM liquid washing ovocyte of no use before ripe the cultivation, easily egg-cleaning liquid is mixed in the IVM drop, causes ripe weak effect.The 4th, this patent application is the same with other prior aries, and it also is to adopt industrial mixed gas (5%CO that oocyte maturation is cultivated
2+ 95% air), expensive, the difficult of industrial mixed gas price, be unfavorable in the production line or use of large-scale production during field work.Because the gas of existing maturation in vitro porcine oocytes all need be bought the gas mixture of Production in Chemical Plant, such as: 5%CO
2, 95% air or 5%CO
2, 2%～8%O
2, 93%～87%N
2Gaseous environment.These two kinds of gaseous environments all be unable to do without business-like bottled CO
2Or N
2, and need be by the special-purpose CO of costliness
2Incubator is regulated and could be realized, so the cost height; In addition, dress CO
2Or N
2Big, the quality of steel cylinder volume heavy, is unfavorable for transportation and moves, and there is certain risk in the very big handling process of pressure that industry mixes in the gas cylinder; The industry gas mixture can only be confined to use in the laboratory, and can not be in the production line or carry out the maturation in vitro of porcine oocytes during field work on a large scale.
Summary of the invention
Technical problem to be solved by this invention is, a kind of simple, economic, method for in-vitro maturity of porcine oocytes efficiently is provided, solves used industrial mixed gas cost height in the present method for in-vitro maturity of porcine oocytes, difficult, be unfavorable in the production line or the problem of scale operation during field work.
Technical scheme of the present invention is, a kind of method for in-vitro maturity of porcine oocytes, comprise and gather the ovary step, oocyte collection step from ovary, the screening step of ovocyte and maturation in vitro culturing step, it is characterized in that the whole process that oocyte in vitro maturation is cultivated is all carried out in artificial exhalation lung qi environment, promptly adopt this gas phase of human body lung qi to realize the maturation in vitro ovocyte.
Described method for in-vitro maturity of porcine oocytes is characterized in that, described collection ovary step may further comprise the steps, and gets pig ovary from the slaughterhouse, puts into the thermos cup that fills penicillin G and 28～35 ℃ of physiological saline of Vetstrep, transports the laboratory in the 2h back.
Described method for in-vitro maturity of porcine oocytes, it is characterized in that, describedly may further comprise the steps from ovary oocyte collection step, ovarian follicle with 3～6mm on the asepsis injector suction ovary, inject the centrifuge tube that places 38 ℃ of thermostat water baths with extracting liquid, make the fit natural subsidence of ovarian cumulus ovocyte.
Described method for in-vitro maturity of porcine oocytes, it is characterized in that, the screening step of described ovocyte may further comprise the steps, liquor folliculi sedimentation 13-17min abandons supernatant liquor, then throw out is diluted with egg-cleaning liquid and the piping and druming mixing, under stereoscopic microscope, choose the ovarian cumulus parcel more than 2 layers, the uniform cumulus cell ovocyte of densification and kytoplasm complex body is through egg-cleaning liquid washing 3 times, again with IVM nutrient solution washing 3 times.
Described method for in-vitro maturity of porcine oocytes, it is characterized in that, the outer mature oocyte step of described pulmonary gas may further comprise the steps, ovarian cumulus-ovocyte complex body with screening, average 40 ovarian cumulus-hole of ovocyte complex body, move in four orifice plates, four orifice plates are moved in the air bag, sealing machine is sealed sack, and testing crew carries out the breathing gas exchange, with blowing needle pulmonary gases is blown in the air bag, air in the air bag has been arranged, air bag is blown full lung qi and seal, place 38 ℃ thermostat container to cultivate this air bag, the ripe 42-44h that cultivates.
Compared with prior art, the present invention places the ovary of gathering and adds antibiotic thermos cup and preserve, easy to carry, high insulating effect and can prevent that bacterium is to the pollution of ovocyte when the suction liquor folliculi; The composition of screening ovocyte egg-cleaning liquid is simple, cheap, be easy to preparation, and can obtain good washing effect; Particularly the whole process that oocyte in vitro maturation is cultivated among the present invention is all carried out in artificial exhalation lung qi environment, not only need not industrial gas mixture, economic security, simple to operate, and ripe effect and later stage ovocyte developmental potency are very high, the first polar body rate of discharge can reach 70% after the 42-44h maturation in vitro is cultivated, and blastocyst rate is 39% after the lonely female activation.If using the lung qi mature oocyte to alleviate in the production line or during field work, the herding staff do not have CO at that time
2The emergency situation of incubator; In the economically underdeveloped area,, be difficult to buy expensive CO for the teaching unit of being engaged in the reproduction biotechnology research
2Incubator, at this moment, lung qi can be with its economy, advantage substitutes industrial gas mixture easily.The present invention is a kind of simple, economic, novel method efficiently.
Embodiment
In order to make the technical problem to be solved in the present invention, technical scheme and beneficial effect clearer, the present invention is described in further detail below in conjunction with embodiment.Should be appreciated that specific embodiment described herein only in order to explanation the present invention, and be not used in qualification the present invention.
Embodiment 1
1. reagent and nutrient solution are prepared
(1) egg-cleaning liquid preheating
Egg-cleaning liquid is placed 38 ℃ of thermostat container preheatings, use when selecting for ovocyte.
(2) balance oocyte in vitro maturation liquid (IVM liquid)
Every hole adds 400 μ l IVM liquid respectively in four orifice plates, makes it be paved with the bottom, covers 400 μ l paraffin oils (mineral oil) in fluid surface again, places 5%CO
2, in the incubator of 95% air, balance is 4 hours at least.
2. ovary collection
Get ovary from the slaughterhouse, put into the thermos cup of 28～35 ℃ of physiological saline that fill penicillin G and Vetstrep, transport the laboratory in the 2h back.
3. oocytes collection
During the suction ovarian follicle, ovarian follicle with 3～6mm on the 10ml asepsis injector suction ovary of being furnished with No. 18 syringe needles, slowly inject the 15ml conical centrifuge tube that places 38 ℃ of thermostat water baths with extracting liquid, make ovarian cumulus ovocyte zoarium (Cumulus-Oocyte complexes, COCs) natural subsidence.
4. ovocyte screening
Can see obvious limit about liquor folliculi sedimentation 15min, abandon supernatant liquor, to precipitate then with egg-cleaning liquid (DPBS-0.01%PVA) dilution and piping and druming mixing, under stereoscopic microscope, choose the ovarian cumulus parcel rapidly more than 2 layers, the uniform cumulus cell ovocyte of densification and kytoplasm complex body, after DPBS-0.01%PVA washing 3 times, again with IVM nutrient solution washing 3 times.
5. the outer mature oocyte of pulmonary gas
With the above-mentioned ovarian cumulus that screens-ovocyte complex body (COCs), average 40 ovarian cumulus-hole of ovocyte complex body (COCs), move in four orifice plates, then four orifice plates are moved in the air bag, the plastics film sealing machine is sealed sack, testing crew is air-breathing deeply and keep 10～20s to carry out utilizing the blowing needle that is equipped with strainer that pulmonary gases is blown in the air bag after the gaseous interchange, double air blowing exhaust again, air in the air bag has been arranged, and then sack is blown full lung qi and seal, last sack places 38 ℃ thermostat container to cultivate, and carries out lonely female activation behind the ripe cultivation 42-44h.
6. result
After the lung qi maturation in vitro was cultivated 42-44 hour, first polar body rate of discharge (maturing rate) reached 66%-70%.
The above is preferred embodiment of the present invention only, is not used in restriction the present invention, and all any modifications of being done within the spirit and principles in the present invention are equal to replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. method for in-vitro maturity of porcine oocytes, comprise and gather the ovary step, oocyte collection step from ovary, the screening step of ovocyte and maturation in vitro culturing step, it is characterized in that, the whole process that oocyte in vitro maturation is cultivated is all carried out in artificial exhalation lung qi environment, promptly adopts human lung's gas to realize the maturation in vitro ovocyte.
2. method for in-vitro maturity of porcine oocytes as claimed in claim 1, it is characterized in that described collection ovary step may further comprise the steps, and gets pig ovary from the slaughterhouse, put into the vacuum flask of 28～35 ℃ of physiological saline that fill penicillin G and Vetstrep, transport the laboratory in the 2h back.
3. method for in-vitro maturity of porcine oocytes as claimed in claim 1, it is characterized in that, describedly may further comprise the steps from ovary oocyte collection step, ovarian follicle with 3～6mm on the asepsis injector suction ovary, inject the Centrifuge Cup that places 38 ℃ of thermostat water baths with extracting liquid, make the fit natural subsidence of ovarian cumulus ovocyte.
4. method for in-vitro maturity of porcine oocytes as claimed in claim 1, it is characterized in that, the screening step of described ovocyte may further comprise the steps, liquor folliculi sedimentation 13-17min abandons supernatant liquor, then throw out is diluted with egg-cleaning liquid and the piping and druming mixing, under stereoscopic microscope, choose the ovarian cumulus parcel more than 2 layers, the uniform cumulus cell ovocyte of densification and kytoplasm complex body is through egg-cleaning liquid washing 3 times, again with IVM nutrient solution washing 3 times.
5. method for in-vitro maturity of porcine oocytes as claimed in claim 1, it is characterized in that, the outer mature oocyte step of described pulmonary gas may further comprise the steps, with the ovarian cumulus-ovocyte complex body that screens, average 40 ovarian cumulus-hole of ovocyte complex body, move in four orifice plates, then four orifice plates are moved in the air bag, sealing machine is sealed sack, testing crew dark air-breathing and keep 10～20s to carry out gaseous interchange after, utilize blowing needle that pulmonary gases is blown in the air bag, air in the air bag has been arranged in double air blowing exhaust again, and then sack is blown full lung qi and seals, last sack places 38 ℃ thermostat container to cultivate, and carries out lonely female activation behind the ripe cultivation 42-44h.
6. method for in-vitro maturity of porcine oocytes as claimed in claim 5 is characterized in that, the first polar body rate of discharge reaches 70% behind lung qi maturation in vitro porcine oocytes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910185795A CN101709290B (en) | 2009-12-03 | 2009-12-03 | Simple, economic and efficient method for in-vitro maturity of porcine oocytes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910185795A CN101709290B (en) | 2009-12-03 | 2009-12-03 | Simple, economic and efficient method for in-vitro maturity of porcine oocytes |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101709290A true CN101709290A (en) | 2010-05-19 |
| CN101709290B CN101709290B (en) | 2012-10-03 |
Family
ID=42402095
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910185795A Expired - Fee Related CN101709290B (en) | 2009-12-03 | 2009-12-03 | Simple, economic and efficient method for in-vitro maturity of porcine oocytes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101709290B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154198A (en) * | 2010-12-16 | 2011-08-17 | 西北农林科技大学 | Simple in-vitro maturation culture method for oocytes |
| CN102268404A (en) * | 2011-07-20 | 2011-12-07 | 安徽农业大学 | Method for separating pig cumulus stem cells |
| CN103289891A (en) * | 2013-04-23 | 2013-09-11 | 青海省畜牧兽医科学院 | Fast bovine oocyte collector |
| CN104928236A (en) * | 2015-07-06 | 2015-09-23 | 中国农业科学院特产研究所 | Nutrient solution and culturing method for maturation of oocyte in vitro |
| CN106226133A (en) * | 2016-07-01 | 2016-12-14 | 内蒙古农业大学 | Comprise the preparation method of the novel C OCs composite construction tissue of multiple complex |
| CN109112100A (en) * | 2018-09-04 | 2019-01-01 | 安徽农业大学 | The collection method of the small cumulus oocyte complex for having chamber of one boar |
| CN110628709A (en) * | 2019-10-22 | 2019-12-31 | 吉林大学 | Culture solution and culture method for improving in-vitro maturation quality of porcine oocytes |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100404675C (en) * | 2006-02-22 | 2008-07-23 | 李宁 | Production process of somatic cell clone pig |
-
2009
- 2009-12-03 CN CN200910185795A patent/CN101709290B/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154198A (en) * | 2010-12-16 | 2011-08-17 | 西北农林科技大学 | Simple in-vitro maturation culture method for oocytes |
| CN102268404A (en) * | 2011-07-20 | 2011-12-07 | 安徽农业大学 | Method for separating pig cumulus stem cells |
| CN102268404B (en) * | 2011-07-20 | 2012-07-11 | 安徽农业大学 | Method for separating pig cumulus stem cells |
| CN103289891A (en) * | 2013-04-23 | 2013-09-11 | 青海省畜牧兽医科学院 | Fast bovine oocyte collector |
| CN104928236A (en) * | 2015-07-06 | 2015-09-23 | 中国农业科学院特产研究所 | Nutrient solution and culturing method for maturation of oocyte in vitro |
| CN106226133A (en) * | 2016-07-01 | 2016-12-14 | 内蒙古农业大学 | Comprise the preparation method of the novel C OCs composite construction tissue of multiple complex |
| CN106226133B (en) * | 2016-07-01 | 2019-05-10 | 内蒙古农业大学 | The preparation method of novel C OCs composite construction tissue comprising multiple complexs |
| CN109112100A (en) * | 2018-09-04 | 2019-01-01 | 安徽农业大学 | The collection method of the small cumulus oocyte complex for having chamber of one boar |
| CN110628709A (en) * | 2019-10-22 | 2019-12-31 | 吉林大学 | Culture solution and culture method for improving in-vitro maturation quality of porcine oocytes |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101709290B (en) | 2012-10-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101709290B (en) | Simple, economic and efficient method for in-vitro maturity of porcine oocytes | |
| CN102154197B (en) | Baby hamster kidney (BHK)-21 cells obtained by high-density suspension culture in low-serum and serum-free culture medium and preparation method thereof | |
| CN101965394B (en) | Method and device for cell culture in the open continuous mode | |
| CN107354129B (en) | A kind of Fibroblast cell-culture method in autologous skin source | |
| CN102296029A (en) | perfusion bioreactor system | |
| CN104560868B (en) | A kind of primary isolated culture method of fat stem cell | |
| CN102304476B (en) | Three-dimensional cell dynamic culture reactor | |
| CN103849602B (en) | A kind of bull testis clone and establishment method thereof and application | |
| CN102239784A (en) | Microorganism production process | |
| CN102071137A (en) | Device and method for preparing stem cells through continuous perfusion bioreactor/tank (bag) system | |
| CN202369595U (en) | Cell culture flask | |
| CN107384879B (en) | A kind of fermentation manufacturing technique of recombinant yeast pichia pastoris bacterium production liver detoxification enzyme | |
| CN208857315U (en) | A kind of plant cell and organ Liquid Culture bag | |
| CN208562334U (en) | A kind of Bioexperiment observation incubator | |
| CN105385608A (en) | Lentinus edodes liquid strain submerged fermentation technology | |
| CN105420073A (en) | Anaerobe culture bag and method for culturing anaerobe by using anaerobe culture bag | |
| CN103146597B (en) | Method for preparing photosynthetic bacteria liquid | |
| CN105288608A (en) | Porcine circovirus 2 type inactivated vaccine continuous harvest and suspension cultivation production method | |
| CN108424855A (en) | A kind of plant cell, organ liquid culturing apparatus | |
| CN205408770U (en) | Hothouse plants cultivation device | |
| CN86102421A (en) | A kind of liquid culture method of edible fungi | |
| CN209481660U (en) | A kind of microalgae carbon sequestration is at mine coproduction integrated apparatus | |
| CN208857314U (en) | A kind of plant cell, organ liquid culturing apparatus | |
| CN203999636U (en) | A kind of zooblast adherent culture device | |
| CN110923162A (en) | Separation and identification of termite intestinal actinomycetes and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20121003 Termination date: 20151203 |
|
| EXPY | Termination of patent right or utility model |