CN101948799A - Method for improving developmental capacity of sheep mature oocytes after vitrification - Google Patents
Method for improving developmental capacity of sheep mature oocytes after vitrification Download PDFInfo
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- CN101948799A CN101948799A CN 201010270432 CN201010270432A CN101948799A CN 101948799 A CN101948799 A CN 101948799A CN 201010270432 CN201010270432 CN 201010270432 CN 201010270432 A CN201010270432 A CN 201010270432A CN 101948799 A CN101948799 A CN 101948799A
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Abstract
The invention relates to a method for improving the developmental capacity of sheep mature oocytes after vitrification refrigeration, belonging to the technical field of biology. The method comprises the steps of micro-injection fertilization of sheep mature oocytes and activation of fertilized eggs, wherein the activation of the fertilized eggs adopts a direct current pulse-chemical activation-direct current pulse mode, and experiments prove that the embryonic developmental capacity of the vitrified sheep mature oocytes subject to is greatly improved after the vitrified sheep mature oocytes is subject to intracytoplasmic sperm injection and is activated by the activation method of the invention. Compared with the conventional external fertilization method, the cleavage rate and the blastocyst rate are respectively increased to 13.7% and 6.8%. At the same time, by comparing the chromosome ploidy of embryos subject to different treatments, the result indicates that the proportion of normal-ploidy embryos (71.5%) obtained by the ECE method is apparently higher than that (45.4%) obtained by the common chemical activation method and no obvious difference exists compared with the external fertilization groups.
Description
Technical field
The present invention relates to the animal embryo biological technical field, particularly relate to the method for the growth that improves sheep oocyte, the application is referred to as Electric-Chemical-Electric, the ECE method.
Background technology
The freezing preservation of ovocyte is not only significant for the preservation that is on the verge of species, and can provide a large amount of available materials for fundamental research.In recent years, vitrifying freeze process is preserved the effect that ovocyte has obtained to be better than the traditional slow cold method.Many reports utilize vitrifying freeze process to preserve the ovocyte of different animals, and the offspring who also secures good health in some reports.But the success ratio of sheep oocyte glass freezing is still lower at present, and with respect to fresh ovocyte, the ovocyte developmental potency behind the glass freezing is very low.There is report to show that the glass freezing process can cause multiple damage to the animal ovocyte, for example release in advance of the destruction of cytoskeleton, cortical granule and the Ultrastructural variation of zona pellucida.These variations have seriously hindered normal fertilization process, make sperm not enter ovocyte and be fertilized, adopt microinjection can improve the ratio that sperm enters ovocyte, but the ovocyte developmental potency behind the glass freezing is very low, need extra stimulation could start its normal development, be used for Activation of Oocyte at present and use the chemokinesis method more, but behind the chemokinesis, be easy to shine into lonely female activation embryo, can not form normal diploid embryo.
Summary of the invention
Defective at above-mentioned field, the invention provides a kind of method of the developmental potency of sheep oocyte behind the glass freezing that improved, its lonely female activation embryo ratio reduces, and normal diploid embryo ratio improves greatly, compare with the external fertilization method of routine simultaneously, its spilting of an egg rate and blastaea rate bring up to 13.7% and 6.8% respectively.
A kind of method that improves developmental potency behind the sheep mature oocyte glass freezing, comprise the microinjection fertilization of sheep mature oocyte and the activation step of zygote, it is characterized in that: the activation of described zygote comprise the steps: 1. with the sheep mature oocyte after the microinjection (2.4kv/cm 10us) stimulates 1 time down in DC pulse; 2. put into the SOF nutrient solution 5 minutes that has added 5mMionomycin; 3. in the SOF nutrient solution 3 hours; 4. moved in the SOF nutrient solution added 1.9mM6-dimethylaminopurine (6-DMAP) 1.5 hours; 5. (2.4kv/cm 10us) stimulates 1 time again with DC pulse.
Described SOF nutrient solution composition is as follows: 107.63mM NaCl+7.16mM KCl+1.19mM KH
2PO
4+ 1.51mMMgSO
4+ 1.78mM CaCl
2.2H
2O+25.00mM NaHCO
3+ 5.35mM Sodium.alpha.-hydroxypropionate+7.27mM Sodium.alpha.-ketopropionate+0.20mM glutamine+20.0 μ L/mL indispensable amino acid mixed solutions+10.0 μ L/mL non-essential amino acid mixed solution+0.34mM Trisodium Citrate+2.77mM inositol+100IU/mL penicillin+100IU/mL Streptomycin sulphate+0.2mg/mL are phenol red.
The microinjection of described sheep mature oocyte fertilization is to utilize the micrurgy instrument that single sheep sperm is injected directly into sheep mature oocyte behind the glass freezing.
The microinjection of described sheep mature oocyte fertilization comprises the steps: that (1) is with the granulosa cell removal of 0.2% Unidasa with the sheep mature oocyte of survival, (2) ovocyte is moved in the micrurgy drop again, the micrurgy drop is the SOF nutrient solution that has added 25mM Hepes and 10% serum, (3) microscopically with single sperm injection in the sheep mature oocyte.
The activation of zygote of the present invention adopts the mode of DC pulse-chemokinesis-DC pulse to activate, and normal diploid embryo is significantly improved, and lonely female activation embryo reduces.After handling through present method simultaneously, improved the developmental potency behind the sheep oocyte intracytoplasmic sperm injection behind the glass freezing, compared with the external fertilization method of routine, spilting of an egg rate and blastaea rate have improved 13.7% and 6.8% respectively.
Embodiment
The present invention is described in further detail below in conjunction with embodiment.
1. key instrument equipment
Micrurgy instrument, CO
2Incubator, constant temperature platform, view volume microscope, thermostat container, super clean bench.
2. the sheep oocyte behind the glass freezing is prepared
Select the sheep mature oocyte of survival at microscopically, slough granulosa cell, under stereoscopic microscope, select to have ovocyte first polar body, the kytoplasm uniformity, place the HCO that contains 10% serum with 0.2% Unidasa
3Stand-by in-199 liquid.
3. microinjection
The microinjection of mature oocyte is to carry out on the micrurgy instrument.10 pieces of mature oocytes of sloughing granulosa cell are put into an end of operating drop, the operation drop is made up of the SOF nutrient solution that adds 25mMHepes and 10% serum, hold one piece of ovocyte with holding the ovum pin, adjust the position of polar body, make its first polar body be positioned at the position of 12 on clock.In dripping, the sperm operation sucks 1 sperm with entry needle, entry needle is moved into during ovocyte operation drips, and shift sperm onto the entry needle tip, entry needle penetrates the ovocyte deep from 3 positions, and a small amount of ovocyte matter of resorption is injected ovocyte with sperm again, entry needle is withdrawn from from ovocyte gently, after the injection, the one side that ovocyte is moved into drop finishes up to microinjection, places incubator to preserve 1h the ovocyte after the injection.
4. zygote is activated
Injecting the laggard line activating that finishes when all ovocytes handles.The activation treatment process is: 1. (2.4kv/cm 10us) stimulates once down in DC pulse with the sheep mature oocyte after the microinjection; 2. put into the SOF nutrient solution 5 minutes that has added 5mM ionomycin; 3. in the SOF nutrient solution 3 hours; 4. moved in the SOF nutrient solution added 1.9mM 6-dimethylaminopurine (6-DMAP) 1.5 hours; 5. (2.4kv/cm 10us) stimulates 1 time again with DC pulse.
5. fetal development
After the activation, the embryo cultivates, and cultural method is: zygote was cultivated 7 days in the SOF nutrient solution that has added 10% serum.Changed liquid in per 2 days half.Observe the fetal development situation, statistics spilting of an egg rate and blastaea rate.
From year October in January, 2008 to 2009, after the ECE method is handled, fetal development ability behind the sheep oocyte intracytoplasmic sperm injection behind the glass freezing improves greatly, compares with the external fertilization method of routine, and spilting of an egg rate and blastaea rate bring up to 13.7% and 6.8% respectively.Simultaneously, the ploidy that has compared the embryo after the different treatment, found that, after utilizing the ECE method to handle, normal ploidy embryo's ratio (71.5%) is apparently higher than common chemokinesis method (45.4%), and with in vitro fertilization group do not have notable difference, this explanation, present method helps the normal development of embryo behind the sheep oocyte intracytoplasmic sperm injection behind the glass freezing, and experimental result is as follows:
The developmental state of sheep mature oocyte under different fertilization methods behind table one glass freezing
Annotate: 1. the chemokinesis method is: in the SOF of 5mM ionomycin nutrient solution 5 minutes earlier, moved into then in the SOF nutrient solution that has added 1.9mM6-dimethylaminopurine (6-DMAP) 1.5 hours.
2. no extra activation is handled in conventional in vitro fertilization
3. identical subscript represents that difference does not show blue, and different subscripts are represented significant difference, have statistical significance.
The moiety of table two SOF nutrient solution
| Medicine | Concentration |
| NaCL | 107.63mM |
| KCL | 7.16mM |
| KH 2PO 4 | 1.19mM |
| MgSO4 | 1.51mM |
| CaCL 2.2H 2O | 1.78mM |
| NaHCO3 | 25.00mM |
| Sodium.alpha.-hydroxypropionate | 5.35mM |
| Sodium.alpha.-ketopropionate | 7.27mM |
| Glutamine | 0.20mM |
| Indispensable amino acid | 20.0μL/mL |
| Non-essential amino acid | 10.0μL/mL |
| Trisodium Citrate | 0.34mM |
| Inositol | 2.77mM |
| Two anti- | 20.0μL/mL |
| Phenol red | 0.2mg/mL |
Indispensable amino acid: MEM indispensable amino acid mixed solution (available from Sigma company)
Non-essential amino acid: MEM non-essential amino acid mixed solution (available from Sigma company).
Claims (4)
1. method that improves developmental potency behind the sheep mature oocyte glass freezing, comprise the microinjection fertilization of sheep mature oocyte and the activation step of zygote, it is characterized in that: the activation of described zygote comprise the steps: 1. with the sheep mature oocyte after the microinjection at 2.4kv/cm, stimulate under the DC pulse of 10us 1 time; 2. put into the SOF nutrient solution 5 minutes that has added 5mMionomycin; 3. in the SOF nutrient solution 3 hours; 4. moved in the SOF nutrient solution added 1.9mM6-dimethylaminopurine 1.5 hours; 5. use 2.4kv/cm, the DC pulse of 10us stimulates 1 time again.
2. method according to claim 1, described SOF nutrient solution composition is as follows: 107.63mM NaCl+7.16mM KCl+1.19mM KH
2PO
4+ 1.51mM MgSO
4+ 1.78mM CaCl
2.2H
2O+25.00mM NaHCO
3+ 5.35mM Sodium.alpha.-hydroxypropionate+7.27mM Sodium.alpha.-ketopropionate+0.20mM glutamine+20.0 μ L/mL indispensable amino acid mixed solutions+10.0 μ L/mL non-essential amino acid mixed solution+0.34mM Trisodium Citrate+2.77mM inositol+100IU/mL penicillin+100IU/mL Streptomycin sulphate+0.2mg/mL are phenol red.
3. method according to claim 1, the microinjection of described sheep mature oocyte fertilization are to utilize the micrurgy instrument that single sheep sperm is injected directly into sheep mature oocyte behind the glass freezing.
4. method according to claim 3, the microinjection of described sheep mature oocyte fertilization comprises the steps: that (1) is with the granulosa cell removal of 0.2% Unidasa with the sheep mature oocyte of survival, (2) ovocyte is moved in the micrurgy drop again, the micrurgy drop is the SOF nutrient solution that has added 25mM Hepes and 10% serum, (3) microscopically with single sperm injection in the sheep mature oocyte.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106900698A (en) * | 2017-03-08 | 2017-06-30 | 吉林大学 | The vitrification ultra-low temperature store method of taxus chinensis in northeast suspension cell |
| CN107201343A (en) * | 2017-07-14 | 2017-09-26 | 阜阳师范学院 | A kind of goat cells clone embryos nutrient solution and cultural method |
| CN108728403A (en) * | 2018-06-05 | 2018-11-02 | 瑞柏生物（中国）股份有限公司 | A kind of spilting of an egg culture solution and preparation method thereof |
| CN110628705A (en) * | 2019-10-22 | 2019-12-31 | 山东畜牧兽医职业学院 | Donkey embryo flushing fluid and application method thereof |
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| CN1304443A (en) * | 1999-06-30 | 2001-07-18 | 黄禹锡 | Method for producing clened cows |
| CN101260383A (en) * | 2008-04-17 | 2008-09-10 | 北京锦绣大地农业股份有限公司 | Single-semen injection activation method |
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| CN1304443A (en) * | 1999-06-30 | 2001-07-18 | 黄禹锡 | Method for producing clened cows |
| CN101260383A (en) * | 2008-04-17 | 2008-09-10 | 北京锦绣大地农业股份有限公司 | Single-semen injection activation method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106900698A (en) * | 2017-03-08 | 2017-06-30 | 吉林大学 | The vitrification ultra-low temperature store method of taxus chinensis in northeast suspension cell |
| CN107201343A (en) * | 2017-07-14 | 2017-09-26 | 阜阳师范学院 | A kind of goat cells clone embryos nutrient solution and cultural method |
| CN107201343B (en) * | 2017-07-14 | 2021-05-11 | 阜阳师范学院 | Goat cell clone embryo culture solution and culture method |
| CN108728403A (en) * | 2018-06-05 | 2018-11-02 | 瑞柏生物（中国）股份有限公司 | A kind of spilting of an egg culture solution and preparation method thereof |
| CN110628705A (en) * | 2019-10-22 | 2019-12-31 | 山东畜牧兽医职业学院 | Donkey embryo flushing fluid and application method thereof |
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