CN111778204B - Oocyte in-vitro maturation culture solution additive and application thereof - Google Patents
Oocyte in-vitro maturation culture solution additive and application thereof Download PDFInfo
- Publication number
- CN111778204B CN111778204B CN202010531982.2A CN202010531982A CN111778204B CN 111778204 B CN111778204 B CN 111778204B CN 202010531982 A CN202010531982 A CN 202010531982A CN 111778204 B CN111778204 B CN 111778204B
- Authority
- CN
- China
- Prior art keywords
- oocyte
- culture solution
- ceruloplasmin
- rate
- vitro maturation
- 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.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0608—Germ cells
- C12N5/0609—Oocytes, oogonia
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Developmental Biology & Embryology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an additive of an oocyte in-vitro maturation culture solution, which comprises ceruloplasmin. Therefore, the first polar body discharge rate of the oocyte can be obviously improved, the ROS content of the oocyte can be obviously reduced, the embryo blastocyst rate after parthenogenetic activation can be obviously improved, and the maturation rate and the quality of the oocyte can be greatly improved.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an oocyte in-vitro maturation culture solution additive and application thereof.
Background
The in vitro fertilization embryo, the clone embryo, the parthenogenetic embryo and the transgenic embryo of the mammal are widely applied to the technical fields of embryo engineering such as genetic improvement, the research of early development mechanism of the embryo, epigenetic regulation and the like, the in vitro culture quality of the oocyte is a key factor for determining the development of the embryo, but the maturation efficiency and the quality of the in vitro culture of the oocyte are not greatly improved so far. Reactive Oxygen Species (ROS) is an important factor affecting the in vitro culture of oocytes. ROS are a product of biological aerobic metabolism and include oxygen ions, superoxide ions, hydroxyl radicals, hydrogen peroxide, and the like. In equilibrium, ROS play a beneficial role as signaling molecules in physiological processes such as hormonal signaling, intracellular redox regulation, and embryonic development. However, in vitro culture is a static environment, without the exchange of nutrients and metabolites, and is prone to cause the accumulation of ROS, thereby altering their function and impairing cell survival. Therefore, ROS negatively affect oocyte viability, gene expression, protein synthesis and molecular signaling, affecting oocyte maturation and developmental competence.
Disclosure of Invention
The invention aims to provide an oocyte in-vitro maturation culture solution additive and application thereof, so as to solve the problems.
According to one aspect of the present invention, there is provided an additive for a culture solution for in vitro maturation of oocytes, the additive comprising ceruloplasmin. Therefore, the first polar body discharge rate of the oocyte can be obviously improved, the ROS content of the oocyte can be obviously reduced, the embryo blastocyst rate after parthenogenetic activation can be obviously improved, and the maturation rate and the quality of the oocyte can be greatly improved.
In certain embodiments, the chalcocyanin concentration of the oocyte in vitro maturation medium additive is 1.25-10 μ g/ml. Therefore, the maturative rate of the oocytes cultured by the ceruloplasmin additive in the concentration range is higher, and the quality is better.
In certain embodiments, the chalcocyanin concentration of the oocyte in vitro maturation medium additive is 2.5 μ g/ml. Therefore, the oocytes cultured in vitro with the addition of ceruloplasmin at a concentration of 2.5. mu.g/ml had the highest maturation rate and the best quality.
According to another aspect of the present invention, there is provided a culture solution for in vitro maturation of oocytes, the culture solution comprising ceruloplasmin; or 1.25-10 μ g/ml ceruloplasmin; or 2.5. mu.g/ml ceruloplasmin is included. Therefore, the oocyte in-vitro maturation culture solution containing ceruloplasmin/or 1.25-10 mu g/ml ceruloplasmin/or 2.5 mu g/ml ceruloplasmin is used, so that the oocyte in-vitro maturation rate and the in-vitro maturation quality can be greatly improved.
In certain embodiments, the oocyte in vitro maturation medium includes: basic solution, 0.6mM cysteine, 0.91mM sodium pyruvate, 10ng/mL epidermal growth factor, 10% volume fraction fetal bovine serum, 10% volume fraction follicular fluid, 10IU/mL pregnant mare serum gonadotropin, 10IU/mL human chorionic gonadotropin and ceruloplasmin. Therefore, the culture solution can greatly improve the in vitro maturation rate and the in vitro maturation quality of the oocyte.
In certain embodiments, the oocyte in vitro maturation medium includes: basic solution, 0.6mM cysteine, 0.91mM sodium pyruvate, 10ng/mL epidermal growth factor, 10% volume fraction fetal bovine serum, 10% volume fraction follicular fluid, 10IU/mL pregnant mare serum gonadotropin, 10IU/mL human chorionic gonadotropin and 1.25-10 mug/mL ceruloplasmin. Therefore, the culture solution can greatly improve the in vitro maturation rate and the in vitro maturation quality of the oocyte.
In certain embodiments, the oocyte in vitro maturation medium includes: basic solution, 0.6mM cysteine, 0.91mM sodium pyruvate, 10ng/mL epidermal growth factor, 10% volume fraction fetal bovine serum, 10% volume fraction follicular fluid, 10IU/mL pregnant mare serum gonadotropin and 10IU/mL human chorionic gonadotropin, 2.5 mug/mL ceruloplasmin. Therefore, the culture solution has the best effect on improving the in vitro maturation rate and the in vitro maturation quality of the oocytes.
According to another aspect of the invention, the application of the oocyte in-vitro maturation culture solution additive in the somatic cell cloning technology is provided. Therefore, the in vitro maturation rate and quality of the oocyte can be greatly improved, and the success rate and quality of the somatic cell cloned embryo can be improved when the oocyte cloning method is applied to a somatic cell cloning technology.
In certain embodiments, the oocyte in vitro maturation medium additive used in the somatic cloning technology is 1.25-10 μ g/ml ceruloplasmin. Therefore, the in vitro maturation rate and quality of the oocyte can be greatly improved, and the success rate and quality of the somatic cell cloned embryo can be improved when the oocyte in vitro maturation rate and quality are further applied to a somatic cell cloning technology.
In certain embodiments, the oocyte in vitro maturation medium additive applied to the somatic cloning technique is 2.5 μ g/ml ceruloplasmin. Therefore, the in vitro maturation rate and quality of the oocyte can be improved with the best effect, and the success rate and quality of the somatic cell cloned embryo can be improved when the oocyte in vitro maturation rate and quality are further applied to a somatic cell cloning technology.
The invention has the beneficial effects that:
1. the ceruloplasmin is added into the oocyte maturation culture solution, so that the first polar body discharge rate of the oocyte can be obviously improved, the ROS content of the oocyte can be obviously reduced, the embryo blastocyst rate after parthenogenetic activation can be obviously improved, and the maturation rate and the quality of the oocyte can be greatly improved.
2. The maturative culture solution for the oocytes containing the ceruloplasmin can obviously improve the first polar body discharge rate of the oocytes, obviously reduce the ROS content of the oocytes and obviously improve the blastocyst rate of embryos after parthenogenetic activation, so that the maturative rate and the quality of the oocytes can be greatly improved.
3. The ceruloplasmin is applied to the somatic cell cloning technology and is used as an additive of an oocyte maturation culture solution, the first polar body discharge rate of the oocyte can be obviously improved, the ROS content of the oocyte can be obviously reduced, the embryo blastocyst rate after parthenogenesis activation can be obviously improved, the maturation rate and the quality of the oocyte can be greatly improved, and the ceruloplasmin is further applied to the somatic cell cloning technology and can improve the success rate and the quality of the embryo cloned by the somatic cell.
Drawings
FIG. 1 is a fluorescence plot of the effect of control treatment on oocyte ROS levels;
FIG. 2 is a fluorescence plot of the effect of 1.25. mu.g/ml of ceruloplasmin treatment on oocyte ROS levels;
FIG. 3 is a fluorescence plot of the effect of 2.5. mu.g/ml of ceruloplasmin treatment on oocyte ROS levels;
FIG. 4 is a fluorescence plot of the effect of 5. mu.g/ml of ceruloplasmin treatment on oocyte ROS levels;
FIG. 5 is a fluorescence plot of the effect of 10. mu.g/ml ceruloplasmin treatment on oocyte ROS levels;
FIG. 6 is a graph showing the results of different concentrations of ceruloplasmin treatment on the ROS level in oocytes.
Detailed Description
The invention is described in further detail below with reference to the accompanying drawings.
1. Oocyte Collection and maturation culture
Ovaries of young sows were harvested from a slaughterhouse, returned to the laboratory in 32 ℃ saline, washed 3 times with saline supplemented with antibiotics, 2-6mm follicles were aspirated by a 10mL syringe equipped with an 18G needle, the follicular fluid was collected in a 50mL conical centrifuge tube, the supernatant was discarded after natural sedimentation for 10min, the sediment was resuspended in DPBS containing 0.05% (wt/vol) PVA, Cumulus oophorus-oocyte complexes (COCs) that wrapped more than 3 layers of Cumulus cells and homogenized cytoplasm were collected under a stereomicroscope, and after three washes in maturation medium, the COCs were transferred to four-well Nunc dishes containing 500 μ L of maturation medium and equilibrated overnight in an incubator at 39 ℃, 5% CO2, saturated humidity. 50 COCs are put into a culture dish with one hole, the temperature of the culture dish is 39 ℃, and 5 percent CO is added2And carrying out maturation culture for 44h in an incubator with saturated humidity. Mixing the mature cultured COCs with 0.1% hyaluronidase, repeatedly sucking and spitting out cumulus cells by using a pipette, selecting the oocytes under a stereoscopic microscope, wherein the oocytes with obvious perivitelline gaps, no impurities, uniform cytoplasm and obviously discharged first polar bodies are mature oocytes, and the oocytes without perivitelline systems and cytoplasm divergence are regarded as dead oocytes.
The mature culture solution is as follows: TCM-199(Gibco) base solution, added with 0.6mM cysteine, 0.91mM sodium pyruvate, 10ng/mL epidermal growth factor, 10% volume fraction fetal bovine serum, 10% volume fraction follicular fluid, 10IU/mL pregnant horse serum gonadotropin (PMSG), and 10IU/mL human chorionic gonadotropin (hCG).
Mature cultures of COCs are divided into five groups: control group (mature culture without adding ceruloplasmin), 1.25. mu.g/ml group (mature culture with 1.25. mu.g/ml ceruloplasmin added), 2.5. mu.g/ml group (mature culture with 2.5. mu.g/ml ceruloplasmin added), 5. mu.g/ml group (mature culture with 5. mu.g/ml ceruloplasmin added), 10. mu.g/ml group (mature culture with 10. mu.g/ml ceruloplasmin added). After the maturation culture for 44h, the discharge rate of the first polar body of the group cells of 2.5 mu g/ml is obviously improved (P is less than 0.05), and the maturation rate of the oocyte can be obviously improved (P is less than 0.05) by adding the maturation culture solution of Ceruloplastin (CP) of 2.5 mu g/ml, and the results are shown in the table 1.
TABLE 1 influence of ceruloplasmin on oocyte maturation
| Group of | Number of eggs cultured | Mortality of oocytes | First pole body discharge rate |
| Control group | 725 | 126(17.38％) | 441(60.83％)a |
| 1.25 μ g/ml group | 530 | 95(20.00％) | 338(63.77％)ab |
| 2.5 μ g/ml group | 589 | 87(14.77％) | 389(66.04％)b |
| 5 μ g/ml group | 559 | 88(18.60) | 351(62.79％)ab |
| 10 μ g/ml group | 599 | 111(21.65％) | 361(60.27％)a |
Remarking: data from 5 replicates were statistically analyzed and different lower case letters in the same column indicated significant differences (P < 0.05), as follows.
2. Mature oocyte ROS level detection
The ROS level in oocytes was detected using the ROS detection kit (Sigma-Aldrich). After incubation of oocytes in serum-free medium containing 10 μ M DCFH-DA ((2,7-Dichlorodi-hydrofluorescein diacetate) for 1h without light, the oocytes were washed 1-2 times with serum-free medium, fluorescence signals were detected and photographed on a Leeka fluorescence microscope, and the relative ratio of fluorescence intensity of the oocytes was analyzed using ImageJ software.
3. Oocyte parthenogenetic activation, in vitro culture and blastocyst cell counting
Five groups of matured oocytes were transferred to an activation solution (0.25mM mannitol, 0.05mM MgCl2, 0.05mM CaCl2 and 0.5mM Hepes, 0.01% PVA (w/v)) for equilibration for 1min, then transferred to a chamber between two electrodes 0.5cm apart which had been filled with the activation solution, and the oocytes were activated for 80 μ s with electrical stimulation using a BLS fusion apparatus with 80kV/cm DC pulses. The activated oocytes were transferred to PZM-3 medium and cultured in 5% CO2, 5% O2, saturated humidity incubator for 7 days. Cleavage rate and blastocyst formation rate were measured at 48h and 168h, respectively. Selecting parthenogenetic embryo which has developed into blastocyst, fixing with 4% paraformaldehyde for 10min, staining in 10mg/L Hoechst33342 for 10min, tabletting, and observing under fluorescent microscope to record blastocyst cell number. The mature oocytes are cultured by adding 2.5 mu g/ml ceruloplasmin group in the mature culture solution, the blastocyst rate of the embryo after parthenogenetic activation is obviously higher than that of a control group (P is less than 0.05), and the results are shown in Table 2.
TABLE 2 development and quality of maturated oocyte parthenogenetic embryos cultured with ceruloplasmin-supplemented groups
What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept herein, and it is intended to cover all such modifications and variations as fall within the scope of the invention.
Claims (4)
1. An oocyte in vitro maturation culture solution, wherein the culture solution comprises: basic solution, 0.6mM cysteine, 0.91mM sodium pyruvate, 10ng/mL epidermal growth factor, 10% volume fraction fetal bovine serum, 10% volume fraction follicular fluid, 10IU/mL pregnant mare serum gonadotropin, 10IU/mL human chorionic gonadotropin and ceruloplasmin.
2. The culture solution of claim 1, wherein the culture solution comprises: basic solution, 0.6mM cysteine, 0.91mM sodium pyruvate, 10ng/mL epidermal growth factor, 10% volume fraction fetal bovine serum, 10% volume fraction follicular fluid, 10IU/mL pregnant mare serum gonadotropin, 10IU/mL human chorionic gonadotropin and 1.25-10 mug/mL ceruloplasmin.
3. The culture solution of claim 2, wherein the culture solution comprises: basic solution, 0.6mM cysteine, 0.91mM sodium pyruvate, 10ng/mL epidermal growth factor, 10% volume fraction fetal bovine serum, 10% volume fraction follicular fluid, 10IU/mL pregnant mare serum gonadotropin and 10IU/mL human chorionic gonadotropin, 2.5 mug/mL ceruloplasmin.
4. Use of an oocyte in vitro maturation medium according to any one of claims 1 to 3 in porcine somatic cloning techniques, which do not involve diagnosis and treatment of disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010531982.2A CN111778204B (en) | 2020-06-11 | 2020-06-11 | Oocyte in-vitro maturation culture solution additive and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010531982.2A CN111778204B (en) | 2020-06-11 | 2020-06-11 | Oocyte in-vitro maturation culture solution additive and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111778204A CN111778204A (en) | 2020-10-16 |
| CN111778204B true CN111778204B (en) | 2022-05-27 |
Family
ID=72757422
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010531982.2A Active CN111778204B (en) | 2020-06-11 | 2020-06-11 | Oocyte in-vitro maturation culture solution additive and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111778204B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113151159B (en) * | 2021-05-06 | 2023-09-26 | 内蒙古大学 | Oocyte in-vitro maturation culture solution additive and application thereof |
| CN113444683B (en) * | 2021-07-12 | 2022-11-04 | 华南农业大学 | Additive for improving oocyte in-vitro maturation quality, culture method and application |
| CN113583942A (en) * | 2021-07-12 | 2021-11-02 | 华南农业大学 | Additive for promoting oocyte in-vitro maturation and application thereof |
| CN114934011A (en) * | 2022-05-07 | 2022-08-23 | 南京优而生物科技发展有限公司 | In-vitro culture method for high-quality culture of oocytes |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2093164C1 (en) * | 1993-07-01 | 1997-10-20 | Башкирский государственный медицинский институт | Method of treatment of syringomyelia |
| WO2007080442A2 (en) * | 2006-01-09 | 2007-07-19 | Mcgill University | Method to determine state of a cell exchanging metabolites with a fluid medium by analyzing the metabolites in the fluid medium |
| CN101225373A (en) * | 2008-01-23 | 2008-07-23 | 北京锦绣大地农业股份有限公司 | Bovine oocyte in vitro maturation serum-free medium |
| WO2011057411A1 (en) * | 2009-11-12 | 2011-05-19 | Universite Laval | Ovarian markers of oocyte competency and uses thereof |
| CN106834216A (en) * | 2017-02-20 | 2017-06-13 | 上海市农业科学院 | A kind of in vitro culture liquid and cultural method for the lonely female activation embryo of pig |
| CN109792984A (en) * | 2019-02-01 | 2019-05-24 | 北京健坤禾润科技有限公司 | It is a kind of for the cell cryopreservation culture medium of cell injuring model and its application |
-
2020
- 2020-06-11 CN CN202010531982.2A patent/CN111778204B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2093164C1 (en) * | 1993-07-01 | 1997-10-20 | Башкирский государственный медицинский институт | Method of treatment of syringomyelia |
| WO2007080442A2 (en) * | 2006-01-09 | 2007-07-19 | Mcgill University | Method to determine state of a cell exchanging metabolites with a fluid medium by analyzing the metabolites in the fluid medium |
| CN101225373A (en) * | 2008-01-23 | 2008-07-23 | 北京锦绣大地农业股份有限公司 | Bovine oocyte in vitro maturation serum-free medium |
| WO2011057411A1 (en) * | 2009-11-12 | 2011-05-19 | Universite Laval | Ovarian markers of oocyte competency and uses thereof |
| CN106834216A (en) * | 2017-02-20 | 2017-06-13 | 上海市农业科学院 | A kind of in vitro culture liquid and cultural method for the lonely female activation embryo of pig |
| CN109792984A (en) * | 2019-02-01 | 2019-05-24 | 北京健坤禾润科技有限公司 | It is a kind of for the cell cryopreservation culture medium of cell injuring model and its application |
Non-Patent Citations (6)
| Title |
|---|
| Effects of copper sulphate concentrations during in vitro maturation of bovine oocytes;S.J.Picco等;《Theriogenology》;20120115;第77卷(第2期);第373页摘要，第377页右栏第3.3节，第380页左栏第1段 * |
| S.J.Picco等.Effects of copper sulphate concentrations during in vitro maturation of bovine oocytes.《Theriogenology》.2012,第77卷(第2期),373-381. * |
| The effect of copper supplementation on in vitro maturation of porcine cumulus-oocyte complexes and subsequent developmental competence after parthenogenetic activation;HyerinChoi等;《Theriogenology》;20210401;第164卷;84-92 * |
| 猪卵母细胞体外成熟培养的研究进展;周荣等;《广东畜牧兽医科技》;20170418(第02期);9-11、25 * |
| 硼对雏鸵鸟下丘脑—垂体—卵巢轴的作用机理研究;王岩;《中国博士学位论文全文数据库（电子期刊）农业科技辑》;20090215(第2期);D050-15 * |
| 精液中性粒细胞弹性蛋白酶的研究进展;冯瑞祥等;《中华男科学杂志》;20111120(第11期);68-73 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111778204A (en) | 2020-10-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111778204B (en) | Oocyte in-vitro maturation culture solution additive and application thereof | |
| Thouas et al. | The ‘GO’system–a novel method of microculture for in vitro development of mouse zygotes to the blastocyst stage | |
| Brinster | Oxidation of pyruvate and glucose by oocytes of the mouse and rhesus monkey | |
| CN103898046B (en) | Ox IVF Embryos nutrient solution and cultural method | |
| Lee et al. | Pig oocytes with a large perivitelline space matured in vitro show greater developmental competence after parthenogenesis and somatic cell nuclear transfer | |
| JP2008534000A (en) | Adjuvant for embryo and / or cell manipulation medium | |
| CN103710299A (en) | In-vitro culture solution for culturing swine parthenogenetic activated embryos and swine in-vitro fertilized embryos | |
| CN108504625A (en) | A kind of l cell and application thereof | |
| Senatore et al. | Improved in vitro development of OPU-derived bovine (Bos taurus) embryos by group culture with agarose-embedded helper embryos | |
| Kwak et al. | The new system of shorter porcine oocyte in vitro maturation (18 hours) using≥ 8 mm follicles derived from cumulus-oocyte complexes | |
| CN106834216B (en) | In-vitro culture solution and culture method for swine parthenogenetic activation embryos | |
| CN113215087A (en) | Method for improving in-vitro maturation development rate of porcine oocytes by adopting agomelatine | |
| Puglisi et al. | In vitro fertilisation with frozen–thawed bovine sperm sexed by flow cytometry and validated for accuracy by real-time PCR | |
| Yao et al. | Melatonin promotes the development of sheep transgenic cloned embryos by protecting donor and recipient cells | |
| CN113151159B (en) | Oocyte in-vitro maturation culture solution additive and application thereof | |
| CN103525759B (en) | Application of cordycepin in in-vitro maturation culture of oocytes of small pig follicles | |
| Lee et al. | In vitro oocyte maturation in a medium containing reduced sodium chloride improves the developmental competence of pig oocytes after parthenogenesis and somatic cell nuclear transfer | |
| Faerge et al. | The effect of FF-MAS on porcine cumulus–oocyte complex maturation, fertilization and pronucleus formation in vitro | |
| Kobayashi et al. | Blastocyst production by in vitro maturation and development of porcine oocytes in defined media following intracytoplasmic sperm injection | |
| Kim et al. | Embryotrophic effects of ethylenediaminetetraacetic acid and hemoglobin on in vitro porcine embryos development | |
| CN114410573A (en) | Oocyte in-vitro maturation culture solution additive and application thereof | |
| Sugimoto et al. | Growth and development of rabbit oocytes in vitro: effect of fetal bovine serum concentration on culture medium | |
| Varga et al. | Parthenogenetic development of in vitro matured porcine oocytes treated with chemical agents | |
| Koçyiğit et al. | The effect of macromolecule and growth factor combinations on in vitrodevelopment of bovine embryos | |
| Fujihira et al. | Developmental capacity of Antarctic minke whale (Balaenoptera bonaerensis) vitrified oocytes following in vitro maturation, and parthenogenetic activation or intracytoplasmic sperm injection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |