CN107574144B - Carnosic acid-added porcine oocyte in-vitro maturation culture solution and application thereof - Google Patents
Carnosic acid-added porcine oocyte in-vitro maturation culture solution and application thereof Download PDFInfo
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- CN107574144B CN107574144B CN201710985604.XA CN201710985604A CN107574144B CN 107574144 B CN107574144 B CN 107574144B CN 201710985604 A CN201710985604 A CN 201710985604A CN 107574144 B CN107574144 B CN 107574144B
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Abstract
The invention relates to the technical field of cell culture, in particular to a carnosic acid-added oocyte in-vitro maturation culture solution and application thereof. The in vitro maturation culture solution is characterized in that the components comprise carnosic acid; the concentration range of carnosic acid is 1-20 μ M. According to the invention, carnosic acid is added into the mature culture solution of the porcine oocytes, and the synergistic effect of other components is matched, so that the mature quality of the porcine oocytes is improved, and the early development of porcine embryos is promoted well. The result shows that the carnosic acid with proper concentration can effectively improve the maturation quality of the oocyte, thereby improving the in vitro development potential of the porcine oocyte. And the in vitro maturation culture solution is simple to prepare, convenient to operate and has a wide application prospect.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a carnosic acid-added 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. The in vitro maturation of the oocyte is regulated by a plurality of factors, the regulation mechanism is relatively complex, and the research on the oocyte has important significance for the theory of the egg maturation mechanism and the actual production of the embryo. Since the number of oocytes or embryos obtained by using the porcine superovulation technique 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 technique 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. As the porcine oocytes contain a relatively large amount of fat drops, the porcine oocytes are sensitive to temperature and are relatively difficult to operate in vitro. At present, the rate of in vitro embryo development to blastocyst of in vitro matured eggs is lower compared to in vivo produced embryos. In response to these problems, researchers have conducted constant research on the maturation of porcine oocytes and hope to improve the quality of oocytes matured in vitro. Therefore, optimizing the oocyte in vitro maturation culture condition has important significance for improving the in vitro embryo production efficiency.
Disclosure of Invention
The invention provides a carnosic acid-added oocyte in-vitro maturation culture solution and application thereof.
The invention provides a carnosic acid-added oocyte in-vitro maturation culture solution, which comprises the components of carnosic acid. Preferably, the carnosic acid concentration is 1-20 μ M.
Further preferably, the carnosic acid concentration is in the range of 2-10. mu.M, most preferably 2-5. mu.M.
According to the invention, the carnosic acid is added into the oocyte in-vitro maturation culture solution, so that the oocyte maturation quality is improved, and the embryo development is promoted well.
Preferably, the components also comprise conventional culture solution of oocyte, glutamine, gentamicin, pregnant mare serum gonadotropin, human chorionic gonadotropin, pig follicular fluid and serum;
preferably, the volume ratio of the conventional oocyte culture solution is 70-80%, the concentration of glutamine is 0.02-0.06g/L, the concentration of gentamicin is 0.1-0.2g/L, the concentration of pregnant mare serum gonadotropin is 20000-25000U/L, the concentration of human chorionic gonadotropin is 10000-15000U/L, the volume ratio of follicular fluid concentration is 8-12%, and the volume ratio of serum concentration is 8-12%.
Regarding the conventional oocyte culture solution, preferably, the conventional oocyte culture solution is one of Medium199, DMEM, TCM. Further preferably Medium 199.
With respect to serum, preferably, the serum is one of calf serum, fetal bovine serum or neonatal bovine serum.
The invention provides a relatively preferable oocyte in-vitro maturation culture solution, which comprises the following formula: medium199 is 75-80% by volume, glutamine is 0.02-0.06g/L, gentamicin concentration is 0.12-0.16g/L, pregnant mare serum gonadotropin concentration is 20000-25000U/L, human chorionic gonadotropin concentration is 10000-15000U/L, pig follicular fluid concentration is 8-12% by volume, serum concentration is 8-12% by volume, and carnosic acid concentration is 2-5 μ M.
The in vitro maturation culture solution provided by the invention can improve the antioxidant capacity of the oocyte, and the synergistic effect of the active ingredients can effectively improve the maturation quality of the oocyte, so that the in vitro development potential of the oocyte is improved. And the in vitro maturation culture solution is simple to prepare, convenient to operate and has a wide application prospect.
The invention also provides application of the in vitro maturation culture solution in culturing oocytes of animals.
The application of culturing the oocyte by using the oocyte in-vitro maturation liquid comprises the step of placing the oocyte in the carnosic acid-added oocyte in-vitro maturation culture liquid for culturing.
Specifically, the culture is carried out according to the following steps:
placing the oocyte in the balanced oocyte in-vitro maturation culture solution, and then placing the oocyte in CO2And (3) mature culturing for 42-44 hours in an environment with the volume concentration of 4-6% to obtain the culture medium.
Preferably, the balance is in particular: equilibrating the oocyte in vitro maturation medium for at least 3 hours.
Preferably, the oocyte is a porcine oocyte and the temperature of the maturation culture is 38-39 ℃.
Preferably, the oocytes are Cumulus Oocyte Complexes (COCs).
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The pig ovaries were supplied from the slaughterhouse, placed in a 37 ℃ thermos bottle in 0.9% saline and transported back to the laboratory within 1 h.
Serum was bovine serum, purchased from Gibico, pregnant mare serum gonadotropin for injection and human chorionic gonadotropin for injection were purchased from Ningbo second hormone plant, and other materials were purchased from Sigma.
Carnosic acid storage solution: 10mg of carnosic acid powder was first dissolved in 0.15ml of DMSO to a final concentration of 0.2M and stored in a refrigerator at-20 ℃ in the dark.
Physiological saline: 9g of NaCl was dissolved in 1L of sterile water and sterilized by autoclaving at high temperature.
PBS egg wash: 1G of PVA, 9.55G of PBS powder, 0.066G of penicillin G sodium and 0.05G of streptomycin are dissolved in sterile water, the volume is determined to be 1L, and the mixture is sterilized.
IVM mature liquid: the volume ratio of the M199 concentration is 78%, Glutamine is 0.04g/L, Gentamicin-stock is 0.146g/L, pregnant mare serum gonadotropin concentration is 20000U/L, human chorionic gonadotropin concentration is 10000U/L, pig follicular fluid is 10%, and bovine serum is 10%. The osmotic pressure is adjusted to about 280mOsm, and the pH is adjusted to 7.2-7.4. Filtering with 0.22 filter, packaging, and storing at-20 deg.C.
HAE hyaluronidase solution: 20mg of hyaluronidase was dissolved in 20mL of PBS, filtered through a 0.22 filter and dispensed, and stored at-20 ℃ until use.
T2 in vitro procedure fluid: NaHCO 230.168mg/mL, 0.22mg/mL of pyruvic acids (Na Salt), 3.6mg/mL of HEPES (Na Salt), 2.63mg/mL of HEPES (acid), 10% of M199(10 ×), 0.1461mg/mL of L-Glutamine, 2% of FBS, osmotic pressure adjusted to about 280mOsm, pH adjusted to 7.2-7.4.0.22, filter, subpackaged, and stored at-20 ℃ for later use.
P/A electric activation liquid: mannitol 0.25M, CACl2·2H2O 0.1mM，MgCl2·6H2O0.1 mM, HEPES (Na salt)0.5mM, PVA 0.1 g/L. Filtering with 0.22 filter, packaging, and storing at-20 deg.C.
PZM-3 in vitro embryo development solution: NaCl 108mM, NaHCO325.07mM，KCl 100mM，KH2PO40.35mM，MgSO40.4mM，Ca-Lactate·5H2O 2mM，Na-Pyvate 0.2mM，Myo-inositol 2.78mM，PhenolRed 10mM，L-Glutamine 1mM，Hypotaurine 5mM，EAA(50×)2％，NEAA(100 ×), 0.05mg/mL Gentamicin, 3g/L BSA, adjusting osmotic pressure to about 280mOsm, adjusting pH to 7.2-7.4.0.22, filtering with a filter, packaging, and storing at-20 deg.C for use.
CC auxiliary activating liquid: dimethylsulfoxide was used to lyse cytochalasin B and cycloheximide into 5. mu.L/mL and 1mg/mL stock solutions, respectively. To 1mL of PZM-3, 5. mu.L of cytochalasin B and 10. mu.L of cycloheximide stock solution were added to a working solution concentration.
Example 1
This example provides a carnosic acid-supplemented oocyte in vitro maturation medium (CA2) having the following formulation: medium199 is 78%, glutamine is 0.04g/L, gentamicin concentration is 0.146g/L, pregnant mare serum gonadotropin concentration is 20000U/L, human chorionic gonadotropin concentration is 10000U/L, pig follicular fluid concentration is 10%, serum concentration is 10% and carnosic acid concentration is 2 μ M.
Example 2
This example provides a carnosic acid-supplemented oocyte in vitro maturation medium (CA5) that differs from example 1 only in that the carnosic acid concentration is 5. mu.M.
Example 3
This example provides a carnosic acid-supplemented oocyte in vitro maturation medium (CA1) that differs from example 1 only in that the carnosic acid concentration is 1. mu.M.
Example 4
This example provides a carnosic acid-supplemented oocyte in vitro maturation medium (CA10) that differs from example 1 only in that the carnosic acid concentration is 10. mu.M.
Comparative example 1
This comparative example provides a culture solution (blank control) for in vitro maturation of oocytes, differing from example 1 only in that the composition does not include carnosic acid.
Test example 1
The purpose of the test is as follows: verifying the effect of the in vitro maturation culture solution of the oocyte added with the carnosic acid;
test subjects: porcine oocytes;
and (3) test operation:
1. oocyte collection and in vitro maturation culture
Extraction of immature oocytes was performed in the embryo laboratory. Evacuating the follicle with the diameter of 3-8mm by connecting a vacuum pump with an acquisition tube, and pouring the follicle fluid containing the ovum into a 50ml centrifuge tube; precipitating for 30min, removing supernatant, washing with ovum-washing solution for 2 times at an interval of 15 min; adding ovum-washing solution in the 3 rd time, shaking up gently, pouring into a culture dish of 100mm, and picking out Cumulus Oocyte Complexes (COCs) by using a self-drawn glass Pasteur tube under a stereoscopic microscope; washing the picked COCs in the pre-balanced oocyte maturation solution for three times, putting the washed COCs into a 24-pore plate for oocyte maturation, adding 500 mu L of the in-vitro maturation culture solution prepared in the examples 1-4 and the comparative example 1 into each hole of the 24-pore plate, covering the upper layer with 300 mu L of mineral oil, and putting 75 COCs into each hole; ripened for 42 hours in a5 percent CO2 culture box at 38.5 ℃. Three replicates were used as parallel groups.
2. Preparation of oocytes
Transferring Cumulus Oocyte Complexes (COCs) cultured for 42h into a 1.5ml centrifuge tube filled with 500 mul of hyaluronidase solution, blowing and beating the Cumulus Oocyte Complexes (COCs) for about 65 times by a 200 mul pipette gun, and removing granular cells; washing the oocyte in vitro operating fluid for three times, and transferring the oocyte in vitro operating fluid into 50 mu l of operating fluid microdroplets coated with mineral oil; oocytes with the first polar body in the space between eggs were picked up in microdroplets and counted. After discarding oocytes with diffuse cytoplasm, incomplete cytoplasm, unclear perivitelline space and unclear cell membrane, the number of remaining oocytes was counted.
3. Parthenogenetic activation of oocytes
The fusion tank was washed 3 times with the fusion solution, 75 μ l of the fusion solution was added to the fusion tank, and the oocytes were first equilibrated in the fusion solution for 1min and then placed in the fusion tank to which the fusion solution was added. Two direct current pulses of 80 mus at an interval of 100ms and a voltage of 1.5kV/cm are applied to activate the oocytes in a CF-150/B fusion instrument, the reconstructed embryos are picked up and washed 3 times by PZM-3, and the washed reconstructed embryos are placed in an auxiliary activating solution for treatment for 4 h.
4. Embryo culture
Removing oocytes from the helper activation solution, removing dead eggs, washing with PZM-3 solutionTransferring into pre-balanced culture solution PZM-3 after 3 times, and culturing in low-oxygen culture environment (low-oxygen incubator or sealed culture by filling low-oxygen mixed gas distribution and sealing bag). Culturing with micro-drop or four-well plate under gas phase condition of 7% O2、88％N2And 5% CO2The culture temperature of the mixed gas of (3) was 38.5 ℃ and the humidity was 100%. Cleavage rate and blastocyst formation rate were observed and recorded at 48 and 168 hours of activation culture, respectively.
5. Blastocyst cell count
Collecting blastocysts formed by culturing embryos for 168 hours, fixing with 4% formaldehyde for 30min, breaking membranes with 1% Triton-100, staining with 0.3mM DAPI (stained nuclei) for 20min, performing conventional tabletting treatment on the blastocysts, exciting with ultraviolet light by a fluorescence microscope, and collecting images to count the number of blastocyst cells.
6. Data processing
DPS7.0 statistical software is used for analyzing the maturation rate of the oocytes of each group, the cleavage rate after parthenogenetic activation, the blastocyst formation rate and the number of blastocytes by adopting one-factor variance, and the difference P <0.05 has statistical significance.
Test results and analysis:
effect of carnosic acid on in vitro maturation of porcine oocytes and early development of parthenogenetic embryos
The influence of different doses of carnosic acid on the in vitro development effect of early embryos after in vitro fertilization of pigs in examples 1-5 was observed by using the comparative example 1 as a blank control group and different doses of carnosic acid as test groups, and the results are shown in table 1. The results prove that carnosic acid does not affect the maturation rate and the cleavage rate of porcine oocytes, but can improve the blastocyst rate and the number of blastocysts of embryo development after parthenogenetic activation, wherein the blastocyst rate and the number of blastocysts of CA2, CA5 and CA10 groups are obviously improved, the blastocyst rate of CA2 group is higher than that of other groups, and the number of blastocysts of CA5 group is higher than that of other groups, which indicates that the effect of promoting the early development of porcine embryos by adding carnosic acid with the concentration of 2-5 mu M in IVM is optimal.
TABLE 1 Effect of different concentrations of carnosic acid on oocyte maturation and parthenogenesis in vitro
Note: the same column is labeled with different lower case letters indicating a significant difference P < 0.05.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. An in vitro maturation culture solution for porcine oocytes is characterized by consisting of carnosic acid, oocyte basic culture solution, glutamine, gentamicin, pregnant mare serum gonadotropin, human chorionic gonadotropin, follicular fluid and serum; the concentration range of carnosic acid is 1-20 μ M.
2. The in vitro maturation culture solution of claim 1, wherein in said oocyte in vitro maturation culture solution, the volume ratio of the concentration of said oocyte basic culture solution is 70-80%, the concentration of said glutamine is 0.02-0.06g/L, the concentration of said gentamicin is 0.1-0.2g/L, the concentration of said pregnant mare serum gonadotropin is 20000-25000U/L, the concentration of said human chorionic gonadotropin is 10000-15000U/L, the volume ratio of the concentration of said follicular fluid is 8-12%, and the volume ratio of the concentration of said serum is 8-12%.
3. The in vitro maturation culture Medium according to claim 1 or 2, wherein said oocyte basic culture Medium is one of Medium199, DMEM, TCM.
4. The in vitro maturation culture medium of claim 1 or 2, wherein the serum is one of calf serum, fetal bovine serum or neonatal bovine serum.
5. The in vitro maturation culture medium of claim 3, wherein the serum is one of calf serum, fetal bovine serum or neonatal bovine serum.
6. The in vitro maturation culture medium according to any one of claims 1, 2 and 5, wherein the formulation is as follows:
medium199 is 75-80% by volume, glutamine is 0.02-0.06g/L, gentamicin concentration is 0.12-0.16g/L, pregnant mare serum gonadotropin concentration is 20000-25000U/L, human chorionic gonadotropin concentration is 10000-15000U/L, pig follicular fluid concentration is 8-12% by volume, serum concentration is 8-12% by volume, and carnosic acid concentration is 2-5 μ M.
7. The in vitro maturation culture solution of claim 3, wherein the formulation is as follows:
medium199 is 75-80% by volume, glutamine is 0.02-0.06g/L, gentamicin concentration is 0.12-0.16g/L, pregnant mare serum gonadotropin concentration is 20000-25000U/L, human chorionic gonadotropin concentration is 10000-15000U/L, pig follicular fluid concentration is 8-12% by volume, serum concentration is 8-12% by volume, and carnosic acid concentration is 2-5 μ M.
8. Use of the in vitro maturation culture medium for oocytes of swine oocytes according to any one of claims 1 to 7, wherein oocytes are placed in the in vitro maturation culture medium for oocytes after equilibration and then placed in CO2And (3) mature culturing for 42-44 hours in an environment with the volume concentration of 4-6% to obtain the culture medium.
9. Use according to claim 8, wherein the balancing is in particular: equilibrating the oocyte in vitro maturation medium for at least 3 hours.
10. Use according to claim 8 or 9, wherein the temperature of the maturation culture is 38-39 ℃.
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| CN101429492B (en) * | 2008-12-12 | 2010-10-06 | 深圳华大基因科技有限公司 | Mature method for in vitro culture of porcine oocyte |
| CN106831403A (en) * | 2017-01-18 | 2017-06-13 | 河南佳尚农业科技发展有限公司 | It is a kind of that fat-soluble and water soluble antioxidant method is extracted from rosemary |
| CN105104890B (en) * | 2015-09-25 | 2018-11-16 | 四川农业大学 | It is a kind of promote sow Oocyte Meiosis restore composition and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101429492B (en) * | 2008-12-12 | 2010-10-06 | 深圳华大基因科技有限公司 | Mature method for in vitro culture of porcine oocyte |
| CN105104890B (en) * | 2015-09-25 | 2018-11-16 | 四川农业大学 | It is a kind of promote sow Oocyte Meiosis restore composition and application |
| CN106831403A (en) * | 2017-01-18 | 2017-06-13 | 河南佳尚农业科技发展有限公司 | It is a kind of that fat-soluble and water soluble antioxidant method is extracted from rosemary |
Non-Patent Citations (1)
| Title |
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| Antioxidant effect of rosemary (Rosmarinus officinalis;Mahdi Khodaei Motlagh等;《Cryobiology》;20141231;第69卷(第2期);全文 * |
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