CN104830755A - Novel, simple, convenient and low-energy-consumption maturation culture method for pig embryo in centrifugal tube - Google Patents
Novel, simple, convenient and low-energy-consumption maturation culture method for pig embryo in centrifugal tube Download PDFInfo
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- CN104830755A CN104830755A CN201510251865.XA CN201510251865A CN104830755A CN 104830755 A CN104830755 A CN 104830755A CN 201510251865 A CN201510251865 A CN 201510251865A CN 104830755 A CN104830755 A CN 104830755A
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Abstract
The invention discloses a novel, simple, convenient and low-energy-consumption maturation culture method for a pig embryo in a centrifugal tube. The novel, simple, convenient and low-energy-consumption maturation culture method comprises the following operation steps of (1) collecting a common commercial pig ovary, placing the common commercial pig ovary into normal saline, and carrying the common commercial pig ovary back to a laboratory within 5 hours; (2) screening oocytes; (3) placing the common commercial pig ovary into a centrifugal tube containing an oocyte maturation solution, and maturating by using a 3-D shaking table under the culture condition of 39 DEG C for 45 hours; and (4) after maturating for 45 hours, electrically activating the oocytes which contain first polar bodies and are uniform in cytoplasm, culturing in a culture solution composed of an improved pig zygote culture solution and cytochalasin for 3 hours after the activation operation is ended, and then, culturing in the improved pig zygote culture solution for 7 days. The pig embryo cultured by using the method for culturing in the centrifugal tube has the blastula developmental rate of more than 50% which is up to the domestic leading technological level, so that the lossless transportation of the embryo can be realized within the national scale, and the technical foundation is laid for national transportation of a pig somatic cell cloned embryo and cloning technology industrialization.
Description
technical field:
The present invention relates to a kind of Pig embryos centrifuge tube maturation culture method of novel, easy, low consumption, belong to technical field of cell biology.
background technology:
Tradition embryo culture mode mainly contains two kinds: cultivate to drip and cultivate and culture plate cultivation, but these two kinds of modes limit the long distance transportation of embryo, and because embryo culture generally needs to use import to cultivate consumptive material, further increase the cost of porcine somatic cell cloning embryos, limit porcine somatic cell cloning embryos technology commercial application at home.Centrifuge culture embryo is used in the present invention's research instead, the blastocyst rate of Pig embryos is more than 50%, solve the problem of embryo culture and long distance transportation, reach domestic leading technology level, for technical foundation has been established in porcine somatic cell cloning embryos embryo not damaged transport in China, and further for the application of porcine somatic cell cloning embryos technology industrialization at home plays a role in promoting.
summary of the invention:
Technical problem to be solved by this invention is: overcome the deficiencies in the prior art, a kind of Pig embryos centrifuge tube maturation culture method of novel, easy, low consumption is provided, solve the problem of embryo culture and long distance transportation, produce for embryo's industrialization and realize nationwide transport and established technical foundation.
The technical scheme that the present invention takes for technical solution problem is:
A Pig embryos centrifuge culture method for novel, easy, low consumption, comprise the collection method and in-vitro maturation culture method etc. of porcine oocytes, its concrete operation step is as follows:
1. collect general goods pig ovary from slaughterhouse, be placed in 0.9% physiological saline of 30 ~ 37 DEG C, in 5 h, transport laboratory back, and with normal saline flushing 3 ~ 5 times;
2. extracting diameter on Ovarian surface with 10 ml syringes of band No. 12 syringe needles is the ovocyte of 2 ~ 6 ml ovarian follicles, then under stereomicroscope with embryo pipettor draw with more than 2 layers of cumulus cell, the uniform ovocyte of kytoplasm, in egg-cleaning liquid, wash 3 times, then wash 2 times in ripe liquid;
3. the ovocyte of select is put into the centrifuge tube containing oocyte maturation liquid, with ripe 45 h of 3-D shaking table, culture condition is 39 DEG C;
4. after ripe 45h, with the Unidasa removing cumulus cell of 0.1 ~ 0.3%, select containing first polar body, the uniform ovocyte of kytoplasm, electro activation is carried out with ECM2001 electro' asion instrument, activate after cultivating 3h in the complete nutrient solution improveing pig zygote nutrient solution and cytochalasin composition, then cultivate 7 days in improvement pig zygote nutrient solution.
Step 3. in, described oocyte maturation liquid is the NaHCO of the hydroxyethyl piperazine second thiosulfonic acid Hepes+5-15mM of the lutropin LH+5.9g/L of follitropin FSH+1 μ g/ml of liquor folliculi+1 μ g/ml of 10%
3the vitamin E of the vitamin b6 usp BT+20 μMs of Streptomycin sulphate+2 ~ 8 mM of penicillin+50 μ g/ml of halfcystine L-Cysteine+75 μ g/ml of the epithelical cell growth factor EGF+ 0.57mM of+10 ~ 20 ng/ml.
Step 4. in, the electro activation parameter of described ECM2001 electro' asion instrument is 150 ~ 170v/mm, 50 μ s, 2 pulses.
Through the Pig embryos that centrifuge tube maturation culture method is cultivated out, blastocyst rate is more than 50%.
testing data of the present invention is as follows:
One, experiment material and method:
The collection of 1.1 porcine oocytes and maturation in vitro:
Collect general goods pig ovary from slaughterhouse, in 0.9% physiological saline 5 h being placed in 30 ~ 37 DEG C, transport laboratory back, and with normal saline flushing 3 ~ 5 times.The ovocyte of diameter 2 ~ 6 ml ovarian follicle on Ovarian surface is extracted with 10 ml syringes of band No. 12 syringe needles, under stereomicroscope with embryo pipettor draw with more than 2 layers of cumulus cell, the uniform ovocyte of kytoplasm (COCs), clean 3 times in egg-cleaning liquid, then clean 2 times in ripe liquid.
1.2 Pig embryos maturation culture method contrast experiments:
1, the ovocyte of select moves into the ripe liquid (TCM-199 of ripe drop mTCM199 at incubator balance at least 4 h, add 10% liquor folliculi, 0.57 mM halfcystine, 10-20ng/ml Urogastron, 75 μ g/ml penicillin, 50 μ g/ml Streptomycin sulphates, 1 μ g/ml follitropin FSH, 1 μ g/ml lutropin LH) in, cover with embryo's level mineral oil, ripe 45 h, incubator condition is: the air of 5 % CO2, maximal phase to saturated humidity, 39 DEG C.
2, the ovocyte of select puts into the NaHCO of the hydroxyethyl piperazine second thiosulfonic acid Hepes+5-15mM of the lutropin LH+5.9g/L of follitropin FSH+1 μ g/ml of liquor folliculi+1 μ g/ml containing ripe liquid mTCM199(10%
3the vitamin E of the vitamin b6 usp BT+20 μMs of Streptomycin sulphate+2 ~ 8 mM of penicillin+50 μ g/ml of halfcystine L-Cysteine+75 μ g/ml of the epithelical cell growth factor EGF+0.57mM of+10 ~ 20 ng/ml) centrifuge tube in, with ripe 45 h of 3-D shaking table, culture condition is 39 DEG C.
The 3-D shaking table of commercially available suitable size is positioned in thermostat container by this experiment, and the centrifuge tube that ovocyte is housed then is put on shaking table, forms the 3-D shaking table maturation culture system of ovocyte.
Two, test design: compare two kinds of different embryo culture modes and volume of culture to the impact of the female activation embryo development rate of orphan.
Two kinds of embryo's training methods: culture plate is cultivated and centrifuge culture.The embryo medium adding 1ml, 1.5ml, 2ml respectively in centrifuge tube for the cultivation of the female activation embryo of orphan.
Three, test results and analysis:
Table 1: different group training method is on the impact of the female sharp embryo development rate of orphan
Can draw from Fig. 1 ~ Fig. 4 of table 1 and Figure of description: two kinds of different training methods are used for the lonely female activation embryo culture of pig, and result is without significant difference, and the impact of different nutrient solution volumes on embryo development rate there is not significant difference, as shown in table 1 and Fig. 1 yet.Result shows: the embryo of the lonely female activation of pig can cultivate with centrifuge tube, can replace expensive culture plate culture method completely.
In the present invention, centrifuge tube can replace culture plate method for the cultivation of embryo, a kind of embryo culture and mode of transport of novel, easy, low consumption, the developmental rate of the method pig blastocyst is more than 50%, reach domestic leading technology level, solve embryo and can realize the harmless transport in nationwide, the embryo for porcine somatic cell cloning embryos realizes nationwide transport and technical foundation has been established in clone technology industrialization.
accompanying drawing illustrates:
Fig. 1 be during the present invention tests culture plate training method on the impact of the female sharp embryo development rate of orphan;
Fig. 2 be during the present invention tests 1ml centrifuge culture on the impact of the female sharp embryo development rate of orphan;
Fig. 3 be during the present invention tests 1.5ml centrifuge culture on the impact of the female sharp embryo development rate of orphan;
Fig. 4 be during the present invention tests 2ml centrifuge culture on the impact of the female sharp embryo development rate of orphan.
embodiment:
Below in conjunction with specific embodiment, the present invention will be further explained:
Embodiment: a kind of Pig embryos centrifuge culture method of novel, easy, low consumption, comprise the collection method and in-vitro maturation culture method etc. of porcine oocytes, its concrete operation step is as follows:
1. collect general goods pig ovary from slaughterhouse, be placed in the 0.9 % physiological saline of 30 ~ 37 DEG C, in 5 h, transport laboratory back, and with normal saline flushing 3 ~ 5 times;
2. extracting diameter on Ovarian surface with 10 ml syringes of band No. 12 syringe needles is the ovocyte of 2 ~ 6 ml ovarian follicles, then under stereomicroscope with embryo pipettor draw with more than 2 layers of cumulus cell, the uniform ovocyte of kytoplasm, in egg-cleaning liquid, wash 3 times, then wash 2 times in ripe liquid;
3. the ovocyte of select is put into the centrifuge tube containing oocyte maturation liquid, with ripe 45 h of 3-D shaking table, culture condition is 39 DEG C;
4. after ripe 45h, with the Unidasa removing cumulus cell of 0.1 ~ 0.3%, select containing first polar body, the uniform ovocyte of kytoplasm, electro activation is carried out with ECM2001 electro' asion instrument, activate after cultivating 3h in the complete nutrient solution improveing pig zygote nutrient solution and cytochalasin composition, then cultivate 7 days in improvement pig zygote nutrient solution.
Step 3. in, described oocyte maturation liquid is the NaHCO of the hydroxyethyl piperazine second thiosulfonic acid Hepes+5-15mM of the lutropin LH+5.9g/L of follitropin FSH+1 μ g/ml of liquor folliculi+1 μ g/ml of 10%
3the vitamin E of the vitamin b6 usp BT+20 μMs of Streptomycin sulphate+2 ~ 8 mM of penicillin+50 μ g/ml of halfcystine L-Cysteine+75 μ g/ml of the epithelical cell growth factor EGF+ 0.57mM of+10 ~ 20 ng/ml.
Step 4. in, the electro activation parameter of described ECM2001 electro' asion instrument is 150 ~ 170v/mm, 50 μ s, 2 pulses.
Through the Pig embryos that centrifuge tube maturation culture method is cultivated out, blastocyst rate is more than 50%, reach domestic leading technology level, solve embryo and can realize the harmless transport in nationwide, the embryo for porcine somatic cell cloning embryos realizes nationwide transport and technical foundation has been established in clone technology industrialization.
Claims (4)
1. a Pig embryos centrifuge tube maturation culture method for novel, easy, low consumption, comprise collection method and the in-vitro maturation culture method of porcine oocytes, its concrete operation step is as follows:
1. collect general goods pig ovary from slaughterhouse, be placed in the 0.9 % physiological saline of 30 ~ 37 DEG C, in 5 h, transport laboratory back, and with normal saline flushing 3 ~ 5 times;
2. extracting diameter on Ovarian surface with 10 ml syringes of band No. 12 syringe needles is the ovocyte of 2 ~ 6 ml ovarian follicles, then under stereomicroscope with embryo pipettor draw with more than 2 layers of cumulus cell, the uniform ovocyte of kytoplasm, in egg-cleaning liquid, wash 3 times, then wash 2 times in ripe liquid;
3. the ovocyte of select is put into the centrifuge tube containing oocyte maturation liquid, with ripe 45 h of 3-D shaking table, culture condition is 39 DEG C;
4. after ripe 45h, with the Unidasa removing cumulus cell of 0.1 ~ 0.3%, select containing first polar body, the uniform ovocyte of kytoplasm, electro activation is carried out with ECM2001 electro' asion instrument, activate after cultivating 3h in the complete nutrient solution improveing pig zygote nutrient solution and cytochalasin composition, then cultivate 7 days in improvement pig zygote nutrient solution.
2. Pig embryos centrifuge tube maturation culture method according to claim 1, it is characterized in that: step 3. in, described oocyte maturation liquid is the NaHCO of the hydroxyethyl piperazine second thiosulfonic acid Hepes+5-15mM of the lutropin LH+5.9g/L of follitropin FSH+1 μ g/ml of liquor folliculi+1 μ g/ml of 10%
3the vitamin E of the vitamin b6 usp BT+20 μMs of Streptomycin sulphate+2 ~ 8 mM of penicillin+50 μ g/ml of halfcystine L-Cysteine+75 μ g/ml of the epithelical cell growth factor EGF+ 0.57mM of+10 ~ 20 ng/ml.
3. Pig embryos centrifuge tube maturation culture method according to claim 1, is characterized in that: step 4. in, the electro activation parameter of described ECM2001 electro' asion instrument is 150 ~ 170v/mm, 50 μ s, 2 pulses.
4. Pig embryos centrifuge tube maturation culture method according to claim 1, is characterized in that: the Pig embryos cultivating out through centrifuge tube maturation culture method, blastocyst rate is more than 50%.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104073465A (en) * | 2014-07-04 | 2014-10-01 | 广东温氏食品集团股份有限公司 | Long-distance transportation method for oocytes |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101857878A (en) * | 2010-04-08 | 2010-10-13 | 广西大学 | Bama miniature pig somatic cell cloning method |
| CN104073465A (en) * | 2014-07-04 | 2014-10-01 | 广东温氏食品集团股份有限公司 | Long-distance transportation method for oocytes |
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- 2015-05-18 CN CN201510251865.XA patent/CN104830755A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101857878A (en) * | 2010-04-08 | 2010-10-13 | 广西大学 | Bama miniature pig somatic cell cloning method |
| CN104073465A (en) * | 2014-07-04 | 2014-10-01 | 广东温氏食品集团股份有限公司 | Long-distance transportation method for oocytes |
Non-Patent Citations (4)
| Title |
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| JANG-WON LEE等: "Optimization of Parthenogenetic Activation Protocol in Porcine", 《MOLECULAR REPRODUCTION AND DEVELOPMENT》 * |
| 刘希: "维生素C和E及电脉冲刺激对犬卵母细胞体外成熟影响的研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 吴国权等: "L-肉碱对猪卵母细胞体外成熟及孤雌激活早期胚胎发育能力的影响", 《中国畜牧兽医学会动物繁殖学分会第十五届学术研讨会论文集》 * |
| 曹胜兰等: "电激活对猪卵母细胞孤雌发育的影响", 《育种繁殖》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104073465A (en) * | 2014-07-04 | 2014-10-01 | 广东温氏食品集团股份有限公司 | Long-distance transportation method for oocytes |
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