CN101709290B - 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
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- CN101709290B CN101709290B CN200910185795A CN200910185795A CN101709290B CN 101709290 B CN101709290 B CN 101709290B CN 200910185795 A CN200910185795 A CN 200910185795A CN 200910185795 A CN200910185795 A CN 200910185795A CN 101709290 B CN101709290 B CN 101709290B
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- 210000004072 lung Anatomy 0.000 claims abstract description 13
- 230000002611 ovarian Effects 0.000 claims abstract description 13
- 238000012258 culturing Methods 0.000 claims abstract description 8
- 238000007664 blowing Methods 0.000 claims abstract description 6
- 238000007789 sealing Methods 0.000 claims abstract description 4
- 238000012360 testing method Methods 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 20
- 239000007789 gas Substances 0.000 claims description 17
- 230000035800 maturation Effects 0.000 claims description 14
- 238000004140 cleaning Methods 0.000 claims description 12
- 238000012216 screening Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 230000002685 pulmonary effect Effects 0.000 claims description 6
- 210000002394 ovarian follicle Anatomy 0.000 claims description 5
- 210000001733 follicular fluid Anatomy 0.000 claims description 4
- 235000015097 nutrients Nutrition 0.000 claims description 4
- 229940056360 penicillin g Drugs 0.000 claims description 4
- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 claims description 4
- 230000032258 transport Effects 0.000 claims description 4
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- 238000004500 asepsis Methods 0.000 claims description 3
- 210000001771 cumulus cell Anatomy 0.000 claims description 3
- 238000000280 densification Methods 0.000 claims description 3
- 210000004508 polar body Anatomy 0.000 claims description 3
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- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 1
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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
One 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 saline water 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 is characterized in that, before the ovocyte acquisition step; The ovary of gathering is deposited in the container that is loaded with saline water 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; TCM-199 [1X] basal liquid that contains 50～90%v/v in the maturation culture solution that the oocyte maturation culturing step adopts, the PMSG (pregnant mare serum gonadotrop(h)in (PMSG)) of the hCG of 5～25IU/mL (pregnancy urine extract) and 5～20IU/mL.
It is not enough below this patented claim exists.At first; Before the ovocyte collection; When ovary is gathered ovary placed in the insulation can 2h that contains 32～34 ℃ of saline water and transport the laboratory back, but do not add penicillin G and two kinds of microbiotic of Vetstrep in the saline water, 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, and cost is high, and carries also inconveniently, is unfavorable for production line and field work.The 3rd, the egg-cleaning liquid composition is too many, and cost is high, 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 patented claim 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), industrial mixed gas expensive, difficult, 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 expensive dedicated CO
2Incubator is regulated and could be realized, so cost is high; 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 comprises and gathers 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 manual work 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 saline water 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 injects the centrifuge tube that places 38 ℃ of thermostat water baths with extracting liquid, makes the fit natural subsidence of ovarian cumulus ovocyte.
Described method for in-vitro maturity of porcine oocytes is characterized in that, the screening step of described ovocyte may further comprise the steps; Liquor folliculi sedimentation 13-17min abandons supernatant, 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 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 of screening, average 40 ovarian cumulus-hole of ovocyte complex body move in four orifice plates; Four orifice plates are moved in the air bag, and 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, has arranged the air bag air, air bag is blown full lung qi and seals; 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; The whole process that particularly oocyte in vitro maturation is cultivated among the present invention is all carried out in manual work exhalation lung qi environment; Not only need not industrial gas mixture; Economic security, simple to operate, and also 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
Clearer for technical problem, technical scheme and beneficial effect that the present invention will be solved, below in conjunction with embodiment the present invention is done further explain.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 supplying ovocyte to select.
(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 Yellow Protopet 2As (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 saline water 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 fit (Cumulus-Oocyte complexes, COCs) natural subsidence.
4. ovocyte screening
Can see obvious limit about liquor folliculi sedimentation 15min; Abandon supernatant; 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, and testing crew is air-breathing deeply and keep 10～20s to carry out utilizing the blowing needle that is equipped with strainer pulmonary gases to be blown in the air bag double air blowing exhaust again after the gaseous interchange; Arranged the air bag air; And then sack is blown full lung qi and seal, last sack places 38 ℃ thermostat container to cultivate, and ripely carries out lonely female activation after cultivating 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 merely preferred embodiment of the present invention, is not used in restriction the present invention, and all any modifications of within spirit of the present invention and principle, being done are equal to replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (2)
1. a method for in-vitro maturity of porcine oocytes comprises and gathers the ovary step, oocyte collection step from ovary, and the screening step of ovocyte and maturation in vitro culturing step is characterized in that:
(1) described collection ovary step may further comprise the steps, and gets pig ovary from the slaughterhouse, puts into the vacuum flask of 28～35 ℃ of saline water that fill penicillin G and Vetstrep, transports the laboratory in the 2h back;
(2) describedly may further comprise the steps from ovary oocyte collection step, the ovarian follicle with 3～6mm on the asepsis injector suction ovary injects the Centrifuge Cup that places 38 ℃ of thermostat water baths with extracting liquid, makes the fit natural subsidence of ovarian cumulus ovocyte;
(3) the screening step of described ovocyte may further comprise the steps; Liquor folliculi sedimentation 13-17min abandons supernatant, 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;
(4) the outer mature oocyte step of described pulmonary gas may further comprise the steps, with the ovarian cumulus that screens-ovocyte complex body, and 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, double air blowing exhaust again; Arranged the air bag air; And then sack is blown full lung qi and seal, last sack places 38 ℃ thermostat container to cultivate, and ripely carries out lonely female activation after cultivating 42-44h.
2. method for in-vitro maturity of porcine oocytes as claimed in claim 1 is characterized in that, the first polar body rate of discharge reaches 70% behind lung qi maturation in vitro porcine oocytes.
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|---|---|---|---|
| CN200910185795A CN101709290B (en) | 2009-12-03 | 2009-12-03 | Simple, economic and efficient method for in-vitro maturity of porcine oocytes |
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| 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 |
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| CN101709290B true CN101709290B (en) | 2012-10-03 |
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Families Citing this family (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 |
| CN102268404B (en) * | 2011-07-20 | 2012-07-11 | 安徽农业大学 | Method for separating pig cumulus stem cells |
| CN103289891B (en) * | 2013-04-23 | 2014-11-26 | 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 |
| 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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1810967A (en) * | 2006-02-22 | 2006-08-02 | 李宁 | Production process of somatic cell clone pig |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1810967A (en) * | 2006-02-22 | 2006-08-02 | 李宁 | Production process of somatic cell clone pig |
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