CN111548987B - Pig oocyte in-vitro maturation culture solution and application thereof - Google Patents
Pig oocyte in-vitro maturation culture solution and application thereof Download PDFInfo
- Publication number
- CN111548987B CN111548987B CN202010407723.9A CN202010407723A CN111548987B CN 111548987 B CN111548987 B CN 111548987B CN 202010407723 A CN202010407723 A CN 202010407723A CN 111548987 B CN111548987 B CN 111548987B
- Authority
- CN
- China
- Prior art keywords
- concentration
- culture solution
- mass concentration
- growth factor
- volume fraction
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
- C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/35—Polyols, e.g. glycerin, inositol
-
- 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
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- 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
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/235—Leukemia inhibitory factor [LIF]
Abstract
The invention discloses a culture solution for in vitro maturation of porcine oocytes. The culture solution comprises insulin transferrin selenium solution ITS, fibroblast growth factor FGF, leukemia inhibitory factor LIF and inositol. When the culture solution is applied to oocyte in-vitro maturation culture, serum and follicular fluid do not need to be added, and the culture solution is safer, more stable and more efficient.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a porcine oocyte in-vitro maturation culture solution and application thereof.
Background
With the development of embryo transfer, frozen semen technology and clone transgenic technology, the demand of oocytes is increasing. Because the number of oocytes or embryos obtained by utilizing the porcine superovulation technology is extremely limited, the requirement of a large number of high-quality oocytes cannot be met, and the operation is complex and the cost is high, the in vitro maturation culture is very important.
The in vitro maturation of the pig oocyte refers to the purpose of directly collecting the waste ovary from a slaughterhouse, taking out the immature oocyte and simulating the in vivo maturation environment in vitro to achieve maturation. In the existing mature culture system, hormones, serums, growth factors or other small molecular substances are generally added into a basic culture solution to achieve the optimal in vitro culture condition. The basic culture medium commonly used for pig oocytes comprises NCSU-23 and M199, wherein M199 is a commercial product and can be directly purchased. These chemically synthesized media have appropriate osmotic pressure, energy substances, amino acids, vitamins, trace elements and buffered pH values to meet the most basic requirements for in vitro culture of oocytes. Other additives are selectively added to the culture system in each laboratory. Wherein, the serum contains rich nutrient substances, and the addition amount of the serum in a culture system is 10 percent. However, it is difficult to exclude the interfering effects of growth factors in serum when one wants to observe the effect of a growth factor on oocytes due to its uncertainty of composition. The alternative product KSR to serum has been studied and is available directly. In addition, the follicular fluid of the pig can provide a development microenvironment for oocyte maturation, and the addition amount of the follicular fluid in a culture system is generally 10%. Can provide the nutritional factors such as hormone, vitamin, growth factor and the like required by the growth of the oocyte, but has the disadvantages. First, because follicular fluid is usually obtained by directly extracting large follicles from ovaries collected from slaughterhouses, the quality of ovaries varies from batch to batch, resulting in different quality of follicular fluid obtained. And many components in the follicular fluid are changed along with the starting state of the growth and development of the oocyte, and play a positive or negative regulating role in the maturation of the oocyte. Secondly, the pig follicular fluid is a potential disease infection source, and particularly, the use of the follicular fluid from slaughter houses has great potential safety hazard under the normal state of African swine fever. Even if no African swine fever virus was detected before use, it may not be detected due to too low a concentration. Once the embryo is transferred to the recipient sow, the virus proliferates in the body, resulting in infection of the recipient pig or surrounding herds, and the loss will be enormous.
Disclosure of Invention
The invention aims to provide a porcine oocyte in-vitro maturation culture solution to solve the problems.
According to one aspect of the invention, a porcine oocyte in vitro maturation culture solution is provided, which comprises insulin transferrin selenium solution ITS, fibroblast growth factor FGF, leukemia inhibitory factor LIF and inositol.
In certain embodiments, the volume fraction of insulin transferrin selenium solution ITS in the culture solution is 0.1-1.5%, the mass concentration of fibroblast growth factor FGF is 10-80ng/mL, the mass concentration of leukemia inhibitory factor LIF is 10-40ng/mL, and the mass concentration of inositol in the culture solution is 1-10 μ g/mL.
In certain embodiments, the culture fluid comprises: based on a basic culture solution, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of a serum substitute is 5%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, the mass concentration of epidermal growth factor EGF is 10ng/mL, the volume fraction of insulin transferrin selenium solution ITS is 0.1-1.5%, the mass concentration of fibroblast growth factor FGF is 10-80ng/mL, the mass concentration of leukemia inhibitory factor LIF is 10-40ng/mL, and the mass concentration of inositol is 1-10 mug/mL.
In certain embodiments, the culture fluid comprises: based on a basic culture solution, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of a serum substitute is 5%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, the mass concentration of epidermal growth factor EGF is 10ng/mL, the volume fraction of insulin transferrin selenium solution ITS is 0.5%, the mass concentration of fibroblast growth factor FGF is 40ng/mL, the mass concentration of leukemia inhibitory factor LIF is 20ng/mL, and the mass concentration of inositol is 5 mug/mL.
In certain embodiments, the culture fluid comprises: based on a basic culture solution, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of a serum substitute is 5%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, the mass concentration of epidermal growth factor EGF is 10ng/mL, the volume fraction of insulin transferrin selenium solution ITS is 0.1%, the mass concentration of fibroblast growth factor FGF is 10ng/mL, the mass concentration of leukemia inhibitory factor LIF is 10ng/mL, and the mass concentration of inositol is 1 mug/mL.
In certain embodiments, the culture fluid comprises: based on a basic culture solution, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of a serum substitute is 5%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, the mass concentration of epidermal growth factor EGF is 10ng/mL, the volume fraction of insulin transferrin selenium solution ITS is 1.5%, the mass concentration of fibroblast growth factor FGF is 80ng/mL, the mass concentration of leukemia inhibitory factor LIF is 40ng/mL, and the mass concentration of inositol is 10 mug/mL.
In certain embodiments, the culture fluid comprises: based on a basic culture solution, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of a serum substitute is 5%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, the mass concentration of epidermal growth factor EGF is 10ng/mL, the volume fraction of insulin transferrin selenium solution ITS is 0.8%, the mass concentration of fibroblast growth factor FGF is 50ng/mL, the mass concentration of leukemia inhibitory factor LIF is 25ng/mL, and the mass concentration of inositol is 8 mug/mL.
In certain embodiments, the basal medium is any one of TCM-199, NCSU-37, NCSU-23, Ham's F10, CRI-aa.
According to another aspect of the invention, the application of the in vitro maturation culture solution of the porcine oocytes in the porcine somatic cell cloning technology is provided.
In certain embodiments, a porcine oocyte in vitro maturation medium for use in a somatic cloning technique includes: insulin transferrin selenium solution ITS with volume fraction of 0.1-1.5%, fibroblast growth factor FGF with mass concentration of 10-80ng/mL, leukemia inhibitory factor LIF with mass concentration of 10-40ng/mL, and inositol with mass concentration of 1-10 mug/mL.
In certain embodiments, a porcine oocyte in vitro maturation medium for use in a somatic cloning technique includes: based on a basic culture solution, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of a serum substitute is 5%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, the mass concentration of epidermal growth factor EGF is 10ng/mL, the volume fraction of insulin transferrin selenium solution ITS is 0.1-1.5%, the mass concentration of fibroblast growth factor FGF is 10-80ng/mL, the mass concentration of leukemia inhibitory factor LIF is 10-40ng/mL, and the mass concentration of inositol is 1-10 mug/mL.
In certain embodiments, a porcine oocyte in vitro maturation medium for use in a somatic cloning technique includes: based on a basic culture solution, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of a serum substitute is 5%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, the mass concentration of epidermal growth factor EGF is 10ng/mL, the volume fraction of insulin transferrin selenium solution ITS is 0.5%, the mass concentration of fibroblast growth factor FGF is 40ng/mL, the mass concentration of leukemia inhibitory factor LIF is 20ng/mL, and the mass concentration of inositol is 5 mug/mL.
In certain embodiments, a porcine oocyte in vitro maturation medium for use in a somatic cloning technique includes: based on a basic culture solution, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of a serum substitute is 5%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, the mass concentration of epidermal growth factor EGF is 10ng/mL, the volume fraction of insulin transferrin selenium solution ITS is 0.1%, the mass concentration of fibroblast growth factor FGF is 10ng/mL, the mass concentration of leukemia inhibitory factor LIF is 10ng/mL, and the mass concentration of inositol is 1 mug/mL.
In certain embodiments, a porcine oocyte in vitro maturation medium for use in a somatic cloning technique includes: based on a basic culture solution, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of a serum substitute is 5%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, the mass concentration of epidermal growth factor EGF is 10ng/mL, the volume fraction of insulin transferrin selenium solution ITS is 1.5%, the mass concentration of fibroblast growth factor FGF is 80ng/mL, the mass concentration of leukemia inhibitory factor LIF is 40ng/mL, and the mass concentration of inositol is 10 mug/mL.
In certain embodiments, a porcine oocyte in vitro maturation medium for use in a somatic cloning technique includes: based on a basic culture solution, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of a serum substitute is 5%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, the mass concentration of epidermal growth factor EGF is 10ng/mL, the volume fraction of insulin transferrin selenium solution ITS is 0.8%, the mass concentration of fibroblast growth factor FGF is 50ng/mL, the mass concentration of leukemia inhibitory factor LIF is 25ng/mL, and the mass concentration of inositol is 8 mug/mL.
The invention has the beneficial effects that:
1. conventional porcine oocyte maturation medium will be supplemented with 10% PFF. Many studies have shown that the addition of follicular fluid to the culture medium is beneficial for in vitro maturation of porcine oocytes. Algriny considers follicular fluid as a source of growth factors, gonadotropins, meiosis activating sterols, all of which have been shown to be involved in nuclear maturation of porcine oocytes. However, as the follicular fluid is directly derived from ovaries collected in slaughterhouses, the collection method comprises the following steps: extracting follicular fluid with diameter of 6-8mm from ovary with 12-gauge needle, centrifuging, collecting supernatant, filtering with 0.22 μm filter membrane for sterilization, and freezing at-20 deg.C for storage. Whereas African swine fever viruses are approximately 0.175-0.215 μm in diameter, effective virus filtration is not possible using this method. In the normal state of the African swine fever, the use of follicular fluid has a great safety hazard. The scheme disclosed by the invention can achieve a considerable or even better culture effect without adding follicular fluid, thereby being safer.
2. The composition of serum and follicular fluid is complex and unstable, with the quality of follicular fluid being more unstable. The follicular fluid is usually obtained by directly extracting large follicles from ovaries collected from slaughterhouses, and the quality of ovaries varies from batch to batch, resulting in different quality of follicular fluid obtained. And many components in the follicular fluid are changed along with the starting state of the growth and development of the oocyte, and play a positive or negative regulating role in the maturation of the oocyte. In the scheme disclosed by the invention, serum FBS and follicular fluid PFF do not need to be added. Therefore, the influence of serum FBS and follicular fluid on the culture system can be reduced. The oocyte in-vitro maturation system can be safer, more stable, more standardized and more efficient. Moreover, in the process of in vitro culture of the oocyte, the maturation rate is higher than that of the traditional culture solution added with the follicular fluid. After parthenogenetic activation and somatic cell nuclear transfer are carried out on the obtained mature oocytes, higher cleavage rate and blastocyst rate can be obtained through in vitro culture.
3. The substances added into the culture solution have definite components and stable properties, can be accurately quantified, and have extremely important significance for mature culture and research of in vitro oocytes and application of the oocytes in a somatic cell cloning technology.
Drawings
FIG. 1 is a graph showing the total number of cells in blastocysts of nuclear transfer pigs, which had developed up to day 6, in control group culture broths under a fluorescent microscope;
FIG. 2 is a graph showing the total number of cells in the blastocyst of a nuclear transfer pig, which had developed up to day 6, observed in the culture medium of the experimental group under a fluorescent microscope.
Detailed Description
Reagents for the experimental studies were purchased from SIGMA, Inc. without specific instructions. Oocyte maturation and embryo culture consumables are products of NUNC company. The egg washing liquid is DPBS + PVA liquid, the operation liquid is H-NCSU-23 without calcium, the oocyte culture liquid is TCM-199 as basic culture liquid, and the embryo culture liquid is PZM-3.
Example one
First, preparation method of oocyte maturation culture solution
Formula of control group culture solution (oocyte maturation culture solution adopted in the prior art): based on TCM-199 basic culture solution, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of serum FBS is 10%, the volume fraction of follicular fluid PFF is 10%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, and the mass concentration of epidermal growth factor EGF is 10 ng/mL.
The formula of the culture solution of the experimental group is as follows: based on TCM-199 basic culture solution, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of serum substitute is 5%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, the mass concentration of epidermal growth factor EGF is 10ng/mL, the volume fraction of insulin transferrin selenium solution ITS is 0.5%, the mass concentration of fibroblast growth factor FGF is 40ng/mL, the mass concentration of LIF is 20ng/mL, and the mass concentration of inositol is 5 mug/mL.
In the following steps, at least three parallel group tests are designed for the experimental group and the control group, and data statistical analysis is carried out.
Second, collection of oocytes
The pig ovary is collected to Guangzhou Baiyun slaughter house, tissues such as oviduct and the like are removed by scissors, then the ovary is placed in physiological saline with 37 ℃ containing antibiotics, and the physiological saline is transported back to a laboratory in a thermos bottle for 3 hours. After 3-5 washes with saline containing antibiotics, follicles of 2mm to 6mm in diameter were aspirated by a 10mL syringe equipped with an 18G needle. A self-made ovum picking needle is used for picking up Cumulus-oocyte complexes (COCs) with complete cytoplasm and more than 3 layers of Cumulus cells. Washing with ovum-washing solution for 3 times, washing with mature culture solution for 2 times, and adding into mature culture solution (divided into control group and experimental group) which has been balanced in carbon dioxide incubator for more than 4 hr. Mature culturing for 42-44 h in an incubator with 39 ℃, 5% CO2 and saturated humidity.
Thirdly, obtaining mature oocyte with polar body
Adding matured oocytes for 42-44 h into a pointed-bottom centrifuge tube containing 1mg/ml hyaluronidase, and repeatedly blowing and sucking peripheral cumulus cells by a pipette. And (3) cleaning the oocyte by using the operating liquid, picking out the oocyte with uniform cytoplasm and a mature mark discharged from the first polar body by using a round-head glass needle under a stereoscopic microscope, and placing the oocyte into the operating liquid drop for later use.
Fourth, parthenogenetic activation of mature oocytes
The selected mature oocytes are firstly kept still for 2min in fusion activating solution (0.25mol/L Mannitol, 0.1mmol/L CaCl 2.2H2O, 0.1mmol/L Mg-Cl 2.6H2O, 0.5mmol/L HEPES and 0.01% PVA (w/v)), and then are moved to a fusion tank for parthenogenetic activation. After activation, the cells were treated with a CO-activating solution containing 5. mu.g/mL CB + 10. mu.g/mL CHX for 4 hours and cultured in embryo culture PZM-3 at 39 ℃ in a 5% CO2 incubator saturated with humidity for 6 days. As the parthenogenetic activation embryo, the influence of sperms, fertilization processes and nuclear transfer operation processes on the quality of the embryo is eliminated, so that the development quality of the parthenogenetic activation embryo is less in operation steps and single in influence condition relative to the in-vitro sperm embryo and nuclear transfer operation embryo, the parthenogenetic activation embryo is only related to the quality of the oocyte, and the problem of the maturation quality of the oocyte can be relatively measured through the development rate of the parthenogenetic activation embryo.
Five, cloned embryo production of mature oocytes
Preparation of donor cells: collecting pig ear samples, washing with physiological saline containing double antibody, storing in DMEM culture solution containing double antibody, and taking back to the laboratory with ice box. And (4) establishing a fibroblast line by the ear sample according to a conventional somatic cell line establishing method, and freezing and storing. The donor cells are revived and cultured 1-2 weeks before nuclear transfer operation. Fibroblasts passaged to 3-6 passages were cultured to 100% confluence, after 2 days of contact inhibition, conventionally digested, washed by centrifugation, and finally resuspended in a working solution to serve as a nuclear donor.
Removing kernels and injecting kernels: constructs cells were cloned using blind enucleation method embryos (reconstituted embryos): the oocyte was fixed with a fixed needle in a micromanipulator, the enucleated needle was moved to the polar body at 5 o' clock position, the polar body and nearby 1/3 cytoplasm inside the oocyte were removed with the enucleated needle and the somatic cell was injected into the zona pellucida so that it was in close proximity to the cytoplasm.
Fusion and activation: the well-constructed reconstructed embryos are transferred into a fusion activating solution (0.25mol/L Mannitol, 0.1mmol/L CaCl 2.2H 2O, 0.1mmol/L Mg-Cl 2.6H 2O, 0.5mmol/L HEPES, 0.01% PVA (w/v)) in batches for balancing for 2min, after being washed for 3 times by the fusion activating solution, 15-20 reconstructed embryos in each batch are placed into a fusion tank which is fully paved with the fusion activating solution, the reconstructed embryos are stirred by a drawn very thin solid glass needle, the contact surface of a donor cell membrane and a receptor egg is parallel to an electrode, direct current electric pulses are applied to induce fusion and activate simultaneously, then the embryo culture solution PZM-3 is washed for 3 times, and the reconstructed embryos are immediately transferred into an auxiliary activating solution containing 5 mu g/mL +10 mu g/mL CHX for treatment for 4H.
In vitro culture: the reconstructed embryos judged to have fused are washed 3 times with the embryo culture solution PZM-3 and then placed in the PZM-3 culture solution which is balanced in a carbon dioxide incubator for more than 4 hours. Culturing at 39 deg.C in 5% CO2 saturated humidity incubator for 168 h.
Sixthly, observation of in vitro development of reconstructed embryo
The number of cleavage and blastocysts were recorded at 48h and 144h of embryo culture. Meanwhile, the blastocyst is stained to record the number of blastocyst cells, because the number of blastocyst cells is a necessary condition for reflecting the quality of blastocyst. The specific method comprises the following steps: and taking out the blastocyst at the 6d, washing the blastocyst in DPBS containing 3.7 percent paraformaldehyde for 2 times, fixing the blastocyst for 10min, transferring the fixed blastocyst into DPBS containing Hoechst33342 of 10 microgram/ml, dyeing the fixed blastocyst in a dark place for 5min, washing the fixed blastocyst with the operating solution for 3 times, transferring the fixed blastocyst onto a glass slide, and slightly pressing the glass slide. The photographs were then observed under uv excitation under an Olympus fluorescence microscope and the cells counted.
Seventh, data processing
And (3) performing t-test statistical analysis on the oocyte maturation rate, the cleavage rate and the blastocyst rate by using SPSS16.0 statistical software, wherein the data are represented by the mean value +/-standard error, and the difference is obvious when P is less than 0.05.
Eighth, analysis of results
8.1 Effect of control and Experimental formulas on oocyte maturation Rate
| Group of | Total number of oocytes cultured | Number of mature oocytes | Maturation Rate (%) |
| Control group | 1273 | 790 | 62.79a±0.32 |
| Experimental group | 1211 | 885 | 74.37b±0.13 |
Remarking: the representations with different superscript letters in the same column are significantly different (P < 0.05), as follows.
8.2 parthenogenetic activation efficiency of mature eggs in control group formula and experimental group formula
| Group of | Total number of parthenogenetic embryos cultured | Number of cleavage | Number of blastula | Cleavage Rate (%) | Percentage of blastocyst (%) |
| Control group | 195 | 170 | 100 | 84.24a±0.51 | 45.37a±0.10 |
| Experimental group | 260 | 246 | 140 | 91.52b±0.49 | 49.68b±0.67 |
8.3 mature egg cloning embryo development efficiency of control group formula and experimental group formula
In conclusion, the results of repeated experiments show that the in vitro maturation rate of the porcine oocytes can be remarkably improved by the experimental group formula in the first embodiment. Parthenogenetic activation is carried out on the obtained mature oocytes, the cleavage rate and the blastocyst rate are higher than those of a control group, and the difference is obvious. The cleavage rate and blastocyst rate of the nuclear transfer embryo are both higher than those of the control group, the cell number is also better than that of the control group, and the difference is obvious. Therefore, the formula of the experimental group can be completely used for in-vitro maturation culture of the porcine oocytes, the culture solution system is safer, more stable and more efficient, and the obtained mature oocytes have better development potential.
Example two
First, preparation method of oocyte maturation culture solution
Formula of control group culture solution (oocyte maturation culture solution adopted in the prior art): based on TCM-199 basic culture solution, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of serum FBS is 10%, the volume fraction of follicular fluid PFF is 10%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, and the mass concentration of epidermal growth factor EGF is 10 ng/mL.
The formula of the culture solution of the experimental group is as follows: based on TCM-199 basic culture solution, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of serum substitute is 5%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, the mass concentration of epidermal growth factor EGF is 10ng/mL, the volume fraction of insulin transferrin selenium solution ITS is 0.1%, the mass concentration of fibroblast growth factor FGF is 10ng/mL, the mass concentration of LIF is 10ng/mL, and the mass concentration of inositol is 1 mug/mL.
In the following steps, at least three parallel group tests are designed for the experimental group and the control group, and data statistical analysis is carried out.
The other steps refer to steps two to seven in the first embodiment.
Eighth, analysis of results
8.1 Effect of control and Experimental formulas on oocyte maturation Rate
8.2 parthenogenetic activation efficiency of mature eggs in control group formula and experimental group formula
| Group of | Total number of parthenogenetic embryos cultured | Number of cleavage | Number of blastula | Cleavage Rate (%) | Percentage of blastocyst (%) |
| Control group | 202 | 178 | 108 | 85.62a±0.61 | 48.24a±0.19 |
| Experimental group | 210 | 200 | 115 | 93.12b±0.38 | 52.11b±0.52 |
8.3 mature egg cloning embryo development efficiency of control group formula and experimental group formula
In conclusion, the results of repeated experiments show that the experimental group formula in the second embodiment can obviously improve the in vitro maturation rate of the porcine oocytes. Parthenogenetic activation is carried out on the obtained mature oocytes, the cleavage rate and the blastocyst rate are higher than those of a control group, and the difference is obvious. The cleavage rate and blastocyst rate of the nuclear transfer embryo are both higher than those of the control group, the cell number is also better than that of the control group, and the difference is obvious. Therefore, the formula of the experimental group can be completely used for in-vitro maturation culture of the porcine oocytes, the culture solution system is safer, more stable and more efficient, and the obtained mature oocytes have better development potential.
EXAMPLE III
First, preparation method of oocyte maturation culture solution
Formula of control group culture solution (oocyte maturation culture solution adopted in the prior art): based on TCM-199 basic culture solution, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of serum FBS is 10%, the volume fraction of follicular fluid PFF is 10%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, and the mass concentration of epidermal growth factor EGF is 10 ng/mL.
The formula of the culture solution of the experimental group is as follows: based on TCM-199 basic culture solution, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of serum substitute is 5%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, the mass concentration of epidermal growth factor EGF is 10ng/mL, the volume fraction of insulin transferrin selenium solution ITS is 0.8%, the mass concentration of fibroblast growth factor FGF is 50ng/mL, the mass concentration of LIF is 25ng/mL, and the mass concentration of inositol is 8 mug/mL.
In the following steps, at least three parallel group tests are designed for the experimental group and the control group, and data statistical analysis is carried out.
The other steps refer to steps two to seven in the first embodiment.
Eighth, analysis of results
8.1 Effect of control and Experimental formulas on oocyte maturation Rate
8.2 parthenogenetic activation efficiency of mature eggs in control group formula and experimental group formula
| Group of | Total number of parthenogenetic embryos cultured | Number of cleavage | Number of blastula | Cleavage Rate (%) | Percentage of blastocyst (%) |
| Control group | 201 | 175 | 103 | 84.92a±0.46 | 47.99a±0.31 |
| Experimental group | 208 | 199 | 118 | 95.02b±0.27 | 55.32b±0.19 |
8.3 mature egg cloning embryo development efficiency of control group formula and experimental group formula
In conclusion, the results of repeated experiments show that the in vitro maturation rate of the porcine oocytes can be remarkably improved by the formula of the experimental group in the third embodiment. Parthenogenetic activation is carried out on the obtained mature oocytes, the cleavage rate and the blastocyst rate are higher than those of a control group, and the difference is obvious. The cleavage rate and blastocyst rate of the nuclear transfer embryo are both higher than those of the control group, the cell number is also better than that of the control group, and the difference is obvious. Therefore, the formula of the experimental group can be completely used for in-vitro maturation culture of the porcine oocytes, the culture solution system is safer, more stable and more efficient, and the obtained mature oocytes have better development potential.
Example four
First, preparation method of oocyte maturation culture solution
Formula of control group culture solution (oocyte maturation culture solution adopted in the prior art): based on TCM-199 basic culture solution, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of serum FBS is 10%, the volume fraction of follicular fluid PFF is 10%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, and the mass concentration of epidermal growth factor EGF is 10 ng/mL.
The formula of the culture solution of the experimental group is as follows: based on TCM-199 basic culture solution, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of serum substitute is 5%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, the mass concentration of epidermal growth factor EGF is 10ng/mL, the volume fraction of insulin transferrin selenium solution ITS is 1.5%, the mass concentration of fibroblast growth factor FGF is 80ng/mL, the mass concentration of LIF is 40ng/mL, and the mass concentration of inositol is 10 mug/mL.
In the following steps, at least three parallel group tests are designed for the experimental group and the control group, and data statistical analysis is carried out.
The other steps refer to steps two to seven in the first embodiment.
Eighth, analysis of results
8.1 Effect of control and Experimental formulas on oocyte maturation Rate
| Group of | Total number of oocytes cultured | Number of mature oocytes | Maturation Rate (%) |
| Control group | 1155 | 705 | 61.03a±0.82 |
| Experimental group | 1108 | 820 | 73.05b±0.29 |
8.2 parthenogenetic activation efficiency of mature eggs in control group formula and experimental group formula
| Group of | Total number of parthenogenetic embryos cultured | Number of cleavage | Number of blastula | Cleavage Rate (%) | Percentage of blastocyst (%) |
| Control group | 190 | 160 | 88 | 83.99a±0.21 | 45.88a±0.13 |
| Experimental group | 185 | 168 | 95 | 91.23b±0.72 | 50.12b±0.82 |
8.3 mature egg cloning embryo development efficiency of control group formula and experimental group formula
| Group of | Total number of cultured nuclear transfer embryos | Number of cleavage | Number of blastula | Cleavage Rate (%) | Percentage of blastocyst (%) | Total number of blastula |
| Control group | 502 | 377 | 91 | 75.12a±0.77 | 18.03a±0.28 | 41.29a±3.19 |
| Experimental group | 508 | 416 | 123 | 83.10b±0.91 | 24.39b±0.93 | 49.98b±1.29 |
In conclusion, the results of repeated experiments show that the in vitro maturation rate of the porcine oocytes can be remarkably improved by the formula of the experimental group of the fourth embodiment. Parthenogenetic activation is carried out on the obtained mature oocytes, the cleavage rate and the blastocyst rate are higher than those of a control group, and the difference is obvious. The cleavage rate and blastocyst rate of the nuclear transfer embryo are both higher than those of the control group, the cell number is also better than that of the control group, and the difference is obvious. Therefore, the formula of the experimental group can be completely used for in vitro maturation culture of the porcine oocytes, the culture solution system is safer, more stable and more efficient, and the obtained mature oocytes have better development potential.
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. The in vitro maturation culture solution for the porcine oocytes comprises the following components: based on basic culture solution TCM-199, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of serum substitute is 5%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, the mass concentration of epidermal growth factor EGF is 10ng/mL, the volume fraction of insulin transferrin selenium solution ITS is 0.1-1.5%, the mass concentration of fibroblast growth factor FGF is 10-40ng/mL, the mass concentration of leukemia inhibitory factor LIF is 10-40ng/mL, and the mass concentration of inositol is 1-10 mug/mL.
2. The culture solution according to claim 1, wherein the culture solution comprises the following components: based on basic culture solution TCM-199, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of serum substitute is 5%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, the mass concentration of epidermal growth factor EGF is 10ng/mL, the volume fraction of insulin transferrin selenium solution ITS is 0.5%, the mass concentration of fibroblast growth factor FGF is 40ng/mL, the mass concentration of leukemia inhibitory factor LIF is 20ng/mL, and the mass concentration of inositol is 5 mug/mL.
3. The culture solution according to claim 1, wherein the culture solution comprises the following components: based on basic culture solution TCM-199, the concentration of sodium bicarbonate is 1.99mmol/L, the concentration of sodium pyruvate is 0.91mmol/L, the volume fraction of serum substitute is 5%, the concentration of cysteine is 0.57mmol/L, the concentration of pregnant mare serum gonadotropin PMSG is 10IU/mL, the concentration of human chorionic gonadotropin HCG is 10IU/mL, the mass concentration of epidermal growth factor EGF is 10ng/mL, the volume fraction of insulin transferrin selenium solution ITS is 0.1%, the mass concentration of fibroblast growth factor FGF is 10ng/mL, the mass concentration of leukemia inhibitory factor LIF is 10ng/mL, and the mass concentration of inositol is 1 mug/mL.
4. The use of the porcine oocyte in vitro maturation culture medium of any one of claims 1 to 3 in porcine somatic cell cloning technology.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010407723.9A CN111548987B (en) | 2020-05-14 | 2020-05-14 | Pig oocyte in-vitro maturation culture solution and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010407723.9A CN111548987B (en) | 2020-05-14 | 2020-05-14 | Pig oocyte in-vitro maturation culture solution and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111548987A CN111548987A (en) | 2020-08-18 |
| CN111548987B true CN111548987B (en) | 2021-07-09 |
Family
ID=71996758
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010407723.9A Active CN111548987B (en) | 2020-05-14 | 2020-05-14 | Pig oocyte in-vitro maturation culture solution and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111548987B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140435A (en) * | 2010-11-22 | 2011-08-03 | 广西大学 | Method for improving in-vitro production efficiency of buffalo embryos |
| CN104651407A (en) * | 2015-02-06 | 2015-05-27 | 广东温氏食品集团股份有限公司 | Method of efficiently preparing porcine cloned embryo |
| KR20190052542A (en) * | 2017-11-08 | 2019-05-16 | 서울대학교산학협력단 | A follicular fluid replacement medium for in vitro Maturation of oocytes and The Use thereof |
| 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 |
| CN110709505A (en) * | 2017-04-12 | 2020-01-17 | 派乔易股份有限公司 | Cytokine-free adjuvants, in particular for in vitro fertilization or for cell culture media for follicles, male germ cells or embryos |
| 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 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170258495A1 (en) * | 2016-02-29 | 2017-09-14 | The Curators Of The University Of Missouri | Medium supplement to increase the efficiency of oocyte maturation and embryo culture in vitro |
-
2020
- 2020-05-14 CN CN202010407723.9A patent/CN111548987B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140435A (en) * | 2010-11-22 | 2011-08-03 | 广西大学 | Method for improving in-vitro production efficiency of buffalo embryos |
| CN104651407A (en) * | 2015-02-06 | 2015-05-27 | 广东温氏食品集团股份有限公司 | Method of efficiently preparing porcine cloned embryo |
| CN110709505A (en) * | 2017-04-12 | 2020-01-17 | 派乔易股份有限公司 | Cytokine-free adjuvants, in particular for in vitro fertilization or for cell culture media for follicles, male germ cells or embryos |
| KR20190052542A (en) * | 2017-11-08 | 2019-05-16 | 서울대학교산학협력단 | A follicular fluid replacement medium for in vitro Maturation of oocytes and The Use thereof |
| 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 |
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| 体外卵母细胞的抗氧化作用研究进展;刘宇等;《黑龙江畜牧兽医》;20180131(第1期);第64页右栏第1段 * |
| 生长因子对猪卵母细胞成熟及孤雌胚胎发育的影响;张曼玲等;《内蒙古大学学报(自然科学版)》;20190731;第50卷(第4期);第397页最后一段，第399页第1段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111548987A (en) | 2020-08-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Paramio et al. | Recent advances in in vitro embryo production in small ruminants | |
| Fair et al. | Bovine oocyte diameter in relation to maturational competence and transcriptional activity | |
| Lee et al. | Synergistic effect of alanine and glycine on bovine embryos cultured in a chemically defined medium and amino acid uptake by in vitro-produced bovine morulae and blastocysts | |
| Hall et al. | Early embryonic development, assisted reproductive technologies, and pluripotent stem cell biology in domestic mammals | |
| Ito et al. | Effect of follicular fluid collected from various diameter follicles on the progression of nuclear maturation and developmental competence of pig oocytes | |
| Głabowski et al. | Growth factors effects on preimplantation development of mouse embryos exposed to tumor necrosis factor alpha | |
| CN110951678B (en) | Culture solution for promoting in-vitro maturation of porcine oocytes | |
| Gutnisky et al. | Evaluation of the Cryotech Vitrification Kit for bovine embryos | |
| CN110760473B (en) | Porcine oocyte in-vitro maturation culture solution and preparation method and application thereof | |
| Yong et al. | Production of a transgenic piglet by a sperm injection technique in which no chemical or physical treatments were used for oocytes or sperm | |
| Cao et al. | Effects of chemically defined medium on early development of porcine embryos derived from parthenogenetic activation and cloning | |
| Kharche et al. | Effect of somatic cells co-culture on cleavage and development of in-vitro fertilized caprine embryos | |
| Das et al. | Supplementation of insulin–transferrin–selenium to embryo culture medium improves the in vitro development of pig embryos | |
| Aghaz et al. | Enhanced in vitro developmental competence of sheep embryos following sericin supplementation of the in vitro maturation and in vitro culture media | |
| Romar et al. | Effects of oviductal and cumulus cells on in vitro fertilization and embryo development of porcine oocytes fertilized with epididymal spermatozoa | |
| CN111548987B (en) | Pig oocyte in-vitro maturation culture solution and application thereof | |
| KR20190052542A (en) | A follicular fluid replacement medium for in vitro Maturation of oocytes and The Use thereof | |
| CN110577928A (en) | Oocyte in-vitro maturation culture solution and application thereof | |
| WO2019245278A1 (en) | Medium composition for culturing porcine pluripotent stem cells | |
| Yang et al. | Improving in vitro development of cloned bovine embryos with hybrid (Holstein–Chinese Yellow) recipient oocytes recovered by ovum pick up | |
| Li et al. | Synergetic effects of epidermal growth factor and estradiol on cytoplasmic maturation of porcine oocytes | |
| Kobayashi et al. | Blastocyst production by in vitro maturation and development of porcine oocytes in defined media following intracytoplasmic sperm injection | |
| CN114410573A (en) | Oocyte in-vitro maturation culture solution additive and application thereof | |
| CN109136172B (en) | Mouse sperm in-vitro culture kit for improving blastocyst rate and use method thereof | |
| Lee et al. | Effect of glycine and various osmolarities of culture medium on in vitro development of parthenogenesis and somatic cell nuclear transfer embryos in pigs |
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 | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230110 Address after: One of Shop 202, No. 16, Peixian East Road, Dongyong Town, Nansha District, Guangzhou City, Guangdong Province, 510000 Patentee after: Guangdong Zhongxin Seed Technology Co.,Ltd. Address before: 527300 9 East Causeway North Road, Xinxing County new town, Yunfu, Guangdong Patentee before: WENS FOODSTUFF GROUP Co.,Ltd. |
|
| TR01 | Transfer of patent right |