CN103461323A - Method used for in vitro time-delay preservation of oocyte biological activity - Google Patents
Method used for in vitro time-delay preservation of oocyte biological activity Download PDFInfo
- Publication number
- CN103461323A CN103461323A CN201310286127XA CN201310286127A CN103461323A CN 103461323 A CN103461323 A CN 103461323A CN 201310286127X A CN201310286127X A CN 201310286127XA CN 201310286127 A CN201310286127 A CN 201310286127A CN 103461323 A CN103461323 A CN 103461323A
- Authority
- CN
- China
- Prior art keywords
- mother cell
- tcm199
- egg mother
- beaker
- time delay
- 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
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a method used for in vitro time-delay preservation of oocyte biological activity, and belongs to the field of biotechnology. The method comprises following steps: oocytes are washed with an oocyte collecting liquid and a TCM199 mixed liquid respectively, and are added into the TCM199 mixed liquid at a ratio of 1.5 to 3.5ml per 100 oocytes; and the mixture is preserved for 6 to 8h at a temperature of 30 to 37 DEG C and in saturation humidity. The TCM199 mixed liquid comprises 11.01g/L of M199, 4 to 5g/L of HEPES and 0.3 to 0.4g/L of NaHCO3, and the pH value of the TCM199 mixed liquid is 7.2 to 7.3. The method is capable of realizing in vitro time-delay preservation of oocytes for 6 to 8h; it is possible to regulate and control the initial time of oocyte maturation by the method; and maturing rate, cleavage rate and blastocyst rate of oocytes will not be influenced.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of bioactive method of external time delay preservation egg mother cell that can be used for.
Background technology
In recent years, due to developing rapidly of Embryo engineering technology research, scientists is devoted to study maturation in vitro and the technology in vitro fertilization that the animal for slaughter ovary obtains egg mother cell, to explore the approach in the needed a large amount of body early embryos of embryo transplantation source.Yet still there is puzzlement in obtaining of egg mother cell source now, as all butchered at dead of night due to most animal-slaughtering in fixed place field, by normal operating and the ripe required time of cultivating, calculate, this will cause the maturation in vitro of egg mother cell and in vitro fertilization, and the subsequent operation of Related Experimental Study all will be carried out at dead of night, the labour intensity that this has increased experiment operator undoubtedly, be unfavorable for carrying out of normal experimental study.Therefore, be necessary after obtaining the egg mother cell source, the initial time that gimmick etc. regulates and controls Oocyte Development is preserved in the time delay by external egg mother cell, with this, realizes meeting the controllability of the working time of subsequent experimental.
Summary of the invention
The problem existed for prior art, the object of the invention is to design a kind of technical scheme that the bioactive method of egg mother cell is preserved in external time delay that can be used for is provided, not only the initial time of Oocyte Development be can regulate and control by the method, and maturing rate, spilting of an egg rate and the blastaea rate of egg mother cell can not be affected.
A kind of described bioactive method of external time delay preservation egg mother cell that can be used for, it is characterized in that egg mother cell after adopting ovum liquid and TCM199 mixed liquor and cleaning respectively, with the ratio of 1.5～3.5ml/100 piece, egg mother cell is put into to the TCM199 mixed liquor, preserve 6～8 hours under 30～37 ℃ of temperature, saturated humidity, in above-mentioned TCM199 mixed liquor, contain M199 11.01g/L, HEPES4～5g/L, NaHCO
30.3～0.4 g/L, PH is 7.2～7.3.
A kind of described bioactive method of external time delay preservation egg mother cell that can be used for, is characterized in that egg mother cell is put into to the TCM199 mixed liquor in the ratio of 2～3ml/100 piece.
A kind of described bioactive method of external time delay preservation egg mother cell that can be used for, is characterized in that in the TCM199 mixed liquor containing M199 11.01g/L, HEPES4.2～4.8g/L, NaHCO
30.32～0.38g/L.
A kind of described bioactive method of external time delay preservation egg mother cell that can be used for, it is characterized in that the egg mother cell after cleaning is put into to the first sterilizing beaker that the TCM199 mixed liquor is housed, again by the first sterilizing beaker outer wall parcel sterile gauze, the first sterilizing beaker that then will be surrounded by sterile gauze is put into the second sterilizing beaker that fills ultra-pure water, then the beaker mouth of the second sterilizing beaker is sealed with preservative film, finally the second sterilizing beaker is placed under 30～37 ℃ of temperature, saturated humidity and preserves 6～8 hours.
A kind of described bioactive method of external time delay preservation egg mother cell that can be used for, is characterized in that the beaker mouth of the height of ultra-pure water lower than the first sterilizing beaker.
The reagent that the present invention adopts is available reagent, and wherein PMSG and HCG are purchased from Ningbo hormone products factory, and the TCM199 pulvis is purchased from day water Pharmaceutical Co., Ltd, and other reagent is all purchased from Sigma company.
A kind of above-mentioned bioactive method of external time delay preservation egg mother cell that can be used for, not only time delay is preserved egg mother cell 6～8 hours in vitro, and can regulate and control the initial time that oocyte maturation is grown by the method, and can not affect maturing rate, spilting of an egg rate and the blastaea rate of egg mother cell.
The accompanying drawing explanation
Fig. 1 is the signal ideograph that egg mother cell is preserved in external time delay;
Fig. 2 is the schematic diagram (100X) when after time delay preservation egg mother cell, maturation in vitro is cultivated 42～44h;
Fig. 3 removes the schematic diagram of cumulus cell after time delay preservation Oocytes in Vitro Fertilization 6～8h;
Fig. 4 is the early embryonic development schematic diagram after Oocytes in Vitro Fertilization is preserved in time delay.
In figure: 1-the first sterilizing beaker; The 2-sterile gauze; 3-the second sterilizing beaker; The 4-preservative film.
Embodiment
Further illustrate the present invention below in conjunction with specific embodiment.
Embodiment: the egg mother cell time delay is preserved after ripening and is cultivated and conventional ripe comparison of cultivating immediately
1) collection of egg mother cell
Gather replacement gilt ovary in 1～2 years old age by slaughter house, put into and add two 30～37 ℃ of anti-sterile salines, send laboratory in 2h back to.Use the 10ml syringe, No. 22 syringe needle extracts egg mother cell from the ovarian follicle of diameter 3～6 mm, sorts out under the microscope containing cumulus cell at 3 layers and above and wrap up fine and close ovarian cumulus-egg mother cell complex (COCs), for maturation, cultivates.
2) oocyte in vitro maturation
Experimental design
(1) control group: COCs is after adopting ovum liquid and maturation culture solution (NCSU-37 medium) and cleaning respectively, the 100 μ l that put into pre-equilibration 3～6h be take the ripe drop that the NCSU-37 medium is the maturation in vitro basic culture solution, every 20～25 pieces, in 39 ℃, 5%CO
2, 5% O
2, cultivate 42～44h under saturated humidity, front 20-22h adds dbcAMP, pig follicle liquid (PFF), PMSG+HCG hormone combinations (CG) and mercaptoethanol (β-ME) in culture fluid, rear 22～24h cultivates without hormone.
(2) experimental group: COCs is after adopting ovum liquid and TCM199 mixed liquor and cleaning respectively 3 times, ratio with 1.5～3.5ml/100 piece (being preferably 2～3ml/100 piece) is put into egg mother cell in the first sterilizing beaker 1 that the TCM199 mixed liquor is housed, again by the first sterilizing beaker 1 outer wall parcel sterile gauze 2, the first sterilizing beaker 1 that then will be surrounded by sterile gauze 2 is put into the second sterilizing beaker 3 that fills ultra-pure water, wherein the height of ultra-pure water is lower than the beaker mouth of the first sterilizing beaker 1, then the beaker mouth of the second sterilizing beaker 3 is sealed with preservative film 4, finally the second sterilizing beaker 3 is placed in to 30～37 ℃ of temperature, preserve 6～8h under saturated humidity, be preferably and be placed in 35 ℃ of temperature, preserve 6～8h(as shown in Figure 1 under saturated humidity).
Proceed to afterwards in maturation culture solution, with the identical cultivation of (1) scheme.Above-mentioned TCM199 mixed liquor is formed by the distilled water configuration, and it contains M199 11.01g/L, HEPES4～5g/L, NaHCO
30.3～0.4 g/L, PH is 7.2～7.3, is preferably and contains M199 11.01g/L, HEPES4.2～4.8g/L, NaHCO
30.32～0.38 g/L.
3) in vitro fertilization
Gather the bright seminal fluid of healthy boar, wash 2 times with the PGM-tac solution (peptide research institute Japan) of improvement, adjusting sperm concentration is 2～4 * 10
6individual/ml, equivalent joins the improvement PGM-tac culture fluid in vitro fertilization of 50 μ l pre-equilibration 12～13h, makes 100 μ l fertilization drops.Mature oocyte after the PGM-tac solution washing is moved into to the PGM-tac solution fertilization drop of improvement, every 20～25 pieces, in 39 ℃, 5%CO
2, 5% O
2, under saturated humidity more than smart ovum co-incubation 6～8h.
4) fertilized egg is cultivated
After essence ovum acting in conjunction, will suppose that fertilized egg removes ovarian cumulus by washing in immigration PZM-5 Development culture liquid in 39 ℃, 5%CO in the TALP-HEPES washing lotion
2, cultivate under saturated humidity.
5) result
(1) result (in Table 1) of In vitro maturation is preserved in time delay
The ovocyte in-vitro standard: the cumulus cell that egg mother cell is wrapped with evenly spreads, and discharges first polar body after cultivating 42～44h.
3 experiments are carried out in this test altogether, gather totally 1990 pieces of effective COCs, and wherein experimental group is 960 pieces, obtains 76.25% maturing rate; 1030 pieces of control groups, obtain 78.08% maturing rate, can find out that experimental group and control group are without significant difference (p > 0.05).(see figure 2)
(2) Oocytes in Vitro Fertilization effect (in Table 2) is preserved in time delay
Standard in vitro fertilization: after the effect of incubating altogether, ovum discharges second polar body or cultivates 48h and grow to 2 born of the same parents' phases.
3 experiments are carried out in this test altogether, gather totally 1506 pieces of effective COCs, and wherein the experimental group egg mother cell is totally 720 pieces, and spilting of an egg number is 396 pieces, and obtaining spilting of an egg rate is 55.00%; Totally 786 pieces of control group egg mother cells, spilting of an egg number is 441 pieces, and spilting of an egg rate is 55.87%, and experimental group and control group difference is (p > 0.05) not significantly.
(3) influential effect (in Table 3) of Oocytes in Vitro Fertilization embryonic development is preserved in time delay
The embryonic development criterion: the blastaea developmental rate of 5-8d is grown in rear cultivation in vitro fertilization.
The experimental group development of fertilized ova is cultured to totally 48 pieces of blastula embryos, and blastocyst rate is 12.12%; 56 pieces of control groups, blastocyst rate is 12.23%, experimental group and control group fertilized egg embryo and grow difference not significantly (p > 0.05).(Fig. 3, Fig. 4)
Table 1 TCM199(is containing HEPES) postpone to preserve the impact that egg mother cell is grown maturation
Table 2 TCM199(is containing HEPES) postpone to preserve the impact of egg mother cell on effect in vitro fertilization
Table 3 TCM199(is containing HEPES) postpone to preserve the impact of egg mother cell on early embryonic development
Claims (5)
1. one kind can be used for the bioactive method of external time delay preservation egg mother cell, it is characterized in that egg mother cell after adopting ovum liquid and TCM199 mixed liquor and cleaning respectively, with the ratio of 1.5～3.5ml/100 piece, egg mother cell is put into to the TCM199 mixed liquor, preserve 6～8 hours under 30～37 ℃ of temperature, saturated humidity, in above-mentioned TCM199 mixed liquor, contain M199 11.01g/L, HEPES4～5g/L, NaHCO
30.3～0.4 g/L, PH is 7.2～7.3.
2. a kind of bioactive method of external time delay preservation egg mother cell that can be used for as claimed in claim 1, is characterized in that egg mother cell is put into to the TCM199 mixed liquor in the ratio of 2～3ml/100 piece.
3. a kind of bioactive method of external time delay preservation egg mother cell that can be used for as claimed in claim 1, is characterized in that in the TCM199 mixed liquor containing M199 11.01g/L, HEPES4.2～4.8g/L, NaHCO
30.32～0.38g/L.
4. as claimed in claim 1ly a kind ofly can be used for external time delay and preserve the bioactive method of egg mother cell, it is characterized in that the egg mother cell after cleaning is put into to the first sterilizing beaker (1) that the TCM199 mixed liquor is housed, again by the first sterilizing beaker (1) outer wall parcel sterile gauze (2), the first sterilizing beaker (1) that then will be surrounded by sterile gauze (2) is put into the second sterilizing beaker (3) that fills ultra-pure water, then by the beaker of the second sterilizing beaker (3) for mouth preservative film (4) seal, finally the second sterilizing beaker (3) is placed in to 30～37 ℃ of temperature, under saturated humidity, preserve 6～8 hours.
5. a kind of bioactive method of external time delay preservation egg mother cell that can be used for as claimed in claim 4, is characterized in that the beaker mouth of the height of ultra-pure water lower than the first sterilizing beaker (1).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310286127.XA CN103461323B (en) | 2013-07-09 | 2013-07-09 | One can be used for external time delay and preserves the bioactive method of egg mother cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310286127.XA CN103461323B (en) | 2013-07-09 | 2013-07-09 | One can be used for external time delay and preserves the bioactive method of egg mother cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103461323A true CN103461323A (en) | 2013-12-25 |
| CN103461323B CN103461323B (en) | 2015-10-28 |
Family
ID=49786566
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310286127.XA Active CN103461323B (en) | 2013-07-09 | 2013-07-09 | One can be used for external time delay and preserves the bioactive method of egg mother cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103461323B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110760473A (en) * | 2019-11-29 | 2020-02-07 | 东北农业大学 | Porcine oocyte in-vitro maturation culture solution and preparation method and application thereof |
| CN114854674A (en) * | 2022-06-20 | 2022-08-05 | 中国农业大学 | Application of dbcAMP in preparation of primordial follicle in-vitro activator |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101220346A (en) * | 2008-02-03 | 2008-07-16 | 西北农林科技大学 | Biological cell freezing antifreeze agent |
| CN102258005A (en) * | 2010-05-27 | 2011-11-30 | 中国科学院动物研究所 | Method for maintaining meiotic arrest of oocytes by storing/culturing oocytes in vitro |
| WO2012047678A2 (en) * | 2010-09-27 | 2012-04-12 | Auxogyn, Inc. | Apparatus, method, and system for the automated imaging and evaluation of embryos, oocytes, and stem cells |
| CN103013908A (en) * | 2013-01-08 | 2013-04-03 | 内蒙古赛科星繁育生物技术股份有限公司 | New method of in vitro fertilization for mixed semens of bovine and sheep |
-
2013
- 2013-07-09 CN CN201310286127.XA patent/CN103461323B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101220346A (en) * | 2008-02-03 | 2008-07-16 | 西北农林科技大学 | Biological cell freezing antifreeze agent |
| CN102258005A (en) * | 2010-05-27 | 2011-11-30 | 中国科学院动物研究所 | Method for maintaining meiotic arrest of oocytes by storing/culturing oocytes in vitro |
| WO2012047678A2 (en) * | 2010-09-27 | 2012-04-12 | Auxogyn, Inc. | Apparatus, method, and system for the automated imaging and evaluation of embryos, oocytes, and stem cells |
| CN103013908A (en) * | 2013-01-08 | 2013-04-03 | 内蒙古赛科星繁育生物技术股份有限公司 | New method of in vitro fertilization for mixed semens of bovine and sheep |
Non-Patent Citations (2)
| Title |
|---|
| 卞桂华 等: "水牛卵母细胞玻璃化冷冻保存", 《中国兽医学报》, vol. 29, no. 1, 31 January 2009 (2009-01-31) * |
| 姜国诚 等: "猪卵母细胞的室温（20℃）保存与成熟培养", 《畜牧兽医学报》, vol. 23, no. 4, 28 August 1992 (1992-08-28) * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110760473A (en) * | 2019-11-29 | 2020-02-07 | 东北农业大学 | Porcine oocyte in-vitro maturation culture solution and preparation method and application thereof |
| CN110760473B (en) * | 2019-11-29 | 2020-12-18 | 东北农业大学 | Porcine oocyte in-vitro maturation culture solution and preparation method and application thereof |
| CN114854674A (en) * | 2022-06-20 | 2022-08-05 | 中国农业大学 | Application of dbcAMP in preparation of primordial follicle in-vitro activator |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103461323B (en) | 2015-10-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104312971A (en) | Method for promoting in-vitro buffalo oocyte maturation | |
| Nomura et al. | Sperm cryopreservation protocols for the large-scale fertilization of Japanese eel using a combination of large-volume straws and low sperm dilution ratio | |
| CN102140435A (en) | Method for improving in-vitro production efficiency of buffalo embryos | |
| Song et al. | The future potential of cryopreservation for assisted reproduction | |
| CN103898046A (en) | Cow in-vitro fertilization embryo culture fluid and culture method thereof | |
| CN103184189A (en) | Cultivation method of cross-bred wagy | |
| CN103898048A (en) | In vitro maturation culture method of denuded oocytes of mice | |
| CN104130973A (en) | In-vitro maturating method for sheep oocyte, pretreatment solution and kit | |
| CN104789523A (en) | Simple, effective and low-consumption porcine oocyte in vitro mature cloning and culturing method | |
| Yavas et al. | Effect of different thawing rates on motility and fertilizing capacity of cryopreserved grass carp (Ctenopharyngodon idella) sperm | |
| Menéndez-Blanco et al. | Effect of vitrification of in vitro matured prepubertal goat oocytes on embryo development after parthenogenic activation and intracytoplasmic sperm injection | |
| CN103074294A (en) | Pig embryo culture solution capable of improving ectogenetic efficiency of pig embryo and preparation method thereof | |
| CN103461323B (en) | One can be used for external time delay and preserves the bioactive method of egg mother cell | |
| Gil et al. | Brief coincubation of gametes in porcine in vitro fertilization: role of sperm: oocyte ratio and post-coincubation medium | |
| CN101962626B (en) | Calf in vitro embryo culture solution | |
| CN104513807A (en) | Method for separating cells from blood and cultivating the cells and method for cloning non-human animal | |
| Strand et al. | 10 Culturing and biobanking of amphibian cell lines for conservation applications | |
| Douvres et al. | In vitro cultivation of Ostertagia ostertagi, the medium stomach worm of cattle. I. Development from infective larvae to egg-laying adults | |
| CN103039433A (en) | Casein anti-freezing agent for cryopreservation of donkey semen and preparation method thereof | |
| CN113767896B (en) | Bovine in vitro fertilization embryo vitrification freezing method | |
| CN105532583A (en) | Living body breeding method for entomopathogenic nematodes | |
| CN107460162A (en) | A kind of method for improving lamb extracorporeal embryo development ability | |
| CN103710300A (en) | In-vitro culture solution for culturing swine parthenogenetic activated embryos and swine in-vitro fertilized embryos | |
| CN105255823A (en) | Method and culture solution for lamb oocyte in-vitro maturation culture | |
| CN110684720B (en) | Method for obtaining male pronucleus by taking oocyte among heterogeneous animals as medium |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent of invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Pan Jianzhi Inventor after: Zhu Zhiwei Inventor after: Chen Xiaoyu Inventor after: Yu Fuxian Inventor after: Huang Jing Inventor after: Hu Xiaorui Inventor before: Pan Jianzhi Inventor before: Zhu Zhiwei |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: PAN JIANZHI ZHU ZHIWEI TO: PAN JIANZHI ZHU ZHIWEI CHEN XIAOYU YU FUXIAN HUANG JING HU XIAORUI |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |