Oocyte culture method and application thereof

Abstract

The invention relates to the technical field of tissue culture, in particular to an oocyte culture method and application thereof. The invention provides a culture method of oocytes. The culture method comprises the following steps: S10, providing oogonocytes; s20, carrying out hanging drop culture on the oogonium to obtain a recombinant ovary; and S30, carrying out suspension culture on the recombinant ovary to obtain the oocyte. According to the technical scheme provided by the invention, after the oogonium is cultured into the recombinant ovary through hanging drop culture, suspension culture is carried out, so that the oogonium in the recombinant ovary is differentiated into the oocyte, and the hanging drop culture and the suspension culture can reduce the situation of adherent growth of the oogonium or the oocyte; natural arrangement and interaction of the cells in vivo are simulated, so that a better in-vitro growth and differentiation environment is provided for the cells; moreover, relatively frequent digestion passage for contact inhibition between the cells is not needed in the hanging drop culture and suspension culture processes, so that the damage to the cells is smaller.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue culture, and in particular to an oocyte culture method and application thereof. Background Art

[0002] As the main component of Chinese residents' daily meat consumption, pork has a huge demand in the domestic market. At the same time, China's pig supply fluctuates greatly. In order to maintain the Chinese people's demand for pork, this problem must be solved by expanding the scale of pig production and improving pig production efficiency. At present, China's pig breeding work is still widely adopted due to the constraints of various factors such as funds and technology. With the development of the social market economy, pork products produced by traditional pig breeding technology cannot meet market demand, so it is urgent to develop faster and more accurate pig breeding technology.

[0003] The reproductive life of sows depends on the number of primordial follicles in reserve. Abnormal follicle development can lead to egg failure and induce reproductive failure in sows. However, the differentiation and development of female germ cells are all carried out in the body, and it is difficult to artificially regulate the differentiation and development process.

[0004] At present, the application of in vitro breeding technology for domestic pigs mainly includes semen cryopreservation and transportation, and in vitro fertilization, while the research on in vitro maturation of pig oocytes is still shallow, and there is a lack of in vitro cultivation technology for pig oocytes. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for culturing oocytes and an application thereof, aiming to provide a method for culturing oocytes in vitro.

[0006] To achieve the above object, the present invention provides a method for culturing oocytes, comprising the following steps:

[0007] S10, provide oogonia;

[0008] S20, culturing the oogonia in hanging drops to obtain a reconstructed ovary;

[0009] S30, suspending and culturing the recombinant ovary to obtain oocytes.

[0010] Optionally, in step S20, the hanging drop culture time is 2 to 4 days; and / or,

[0011] In step S30, the suspension culture time is 4 to 8 weeks.

[0012] Optionally, step S10 includes:

[0013] S11, taking out the fetal pig from the uterus of the pregnant sow, selecting the female fetal pig, and taking the reproductive ridge thereof;

[0014] S12, remove the cortical area in the reproductive ridge and mince the ovarian tissue blocks;

[0015] S13. Digest the ovarian tissue blocks, centrifuge and discard the supernatant, terminate the digestion, centrifuge and discard the supernatant to obtain oogonia.

[0016] Optionally, in step S13, the digestion time is 20 to 30 minutes.

[0017] Optionally, the digestion-stopping medium includes glucose, amino acids, vitamins, zinc, putrescine, hypoxanthine, and thymidine.

[0018] Optionally, in step S13, the digestion solution includes a collagenase IV solution, wherein the concentration of collagenase IV is 1.5-2.5 mM.

[0019] Optionally, step S20 includes:

[0020] S21, adding oogonia to the polymerization culture medium and mixing to obtain oogonia culture fluid;

[0021] S22. Culture the oogonia culture medium in batches by hanging drops to obtain reconstructed ovaries.

[0022] Optionally, in step S22, the concentration of oogonia in the oogonia culture medium is 1.5×10 6 ~1.8×10 6 Pieces / mL.

[0023] Optionally, the polymerization medium comprises ethanolamine, glutathione, ascorbic acid, insulin, transferrin, lipids, bovine serum albumin, fetal bovine serum, non-essential amino acids, sodium pyruvate, penicillin-streptomycin, 2-mercaptoethanol and Y-27632 dihydrochloride.

[0024] The present invention also proposes an application of the above-mentioned oocyte in pig breeding.

[0025] In the technical solution provided by the present invention, after the oogonia are cultured into recombinant ovaries through hanging drop culture, suspension culture is performed to allow the oogonia in the recombinant ovaries to differentiate into oocytes. The hanging drop culture and suspension culture can reduce the situation of oogonia or oocytes adhering to the wall, thereby simulating the natural arrangement and interaction of cells in the body, thereby providing a better in vitro growth and differentiation environment for cells; and, during the hanging drop culture and suspension culture, there is no need to perform relatively frequent digestion and passage for contact inhibition between cells, which causes less damage to the cells; in addition, the 3D culture that simulates the in vivo environment can also provide a model for exploring cell-to-cell interactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 These are white light images of reconstructed ovarian tissues under a 10x microscope after 1 week, 4 weeks, and 8 weeks of in vitro culture in one embodiment of the present invention;

[0028] Figure 2 This is a Hoechst immunofluorescence staining image of a recombinant ovarian tissue section after 8 weeks of in vitro culture in one embodiment of the present invention;

[0029] Figure 3 This is an MVH immunofluorescence staining image of a recombinant ovarian tissue section after 8 weeks of in vitro culture in one embodiment of the present invention;

[0030] Figure 4 This is a γH2AX immunofluorescence staining image of a recombinant ovarian tissue section after 8 weeks of in vitro culture in one embodiment of the present invention;

[0031] Figure 5 This is an immunofluorescence staining image of Hoechst / MVH / γH2AX superimposed on the recombinant ovarian tissue slice after 8 weeks of in vitro culture in one embodiment of the present invention.

[0032] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0033] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0034] It should be noted that, in the embodiments, those without specifying specific conditions are carried out according to normal conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer of reagents or instruments used are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, and "A and / or B" is taken as an example, including schemes A, B, or A and B that meet the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of the technical schemes is contradictory or cannot be achieved, it should be considered that the combination of such technical schemes does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work, all belong to the scope of protection of the present invention.

[0035] At present, the application of in vitro breeding technology for domestic pigs mainly includes semen cryopreservation and transportation, and in vitro fertilization, while the research on in vitro maturation of pig oocytes is still shallow, and there is a lack of in vitro cultivation technology for pig oocytes.

[0036] In view of this, if Figures 1 to 5 As shown, the present invention provides a method for culturing oocytes, comprising the following steps:

[0037] S10, provide oogonia;

[0038] S20, culturing the oogonia in hanging drops to obtain a reconstructed ovary;

[0039] S30, suspending and culturing the recombinant ovary to obtain oocytes.

[0040] It should be noted that the hanging drop culture in step S20 refers to a culture method in which the cell culture fluid is placed on the surface of a coverslip to form a hanging drop using surface tension, the tissue or organ explant is inoculated into the culture fluid, and then the coverslip is flipped so that the explant and the culture fluid are suspended under the coverslip, and then placed on a concave slide and placed in an incubator for culture; the suspension culture in step S30 refers to a tissue culture method in which single cells or small cell clusters are cultured in a liquid culture medium that is constantly stirred or shaken.

[0041] In the technical solution provided by the present invention, after the oogonia are cultured into recombinant ovaries by hanging drop culture, suspension culture is performed to differentiate the oogonia in the recombinant ovaries into oocytes. Hanging drop culture and suspension culture can reduce the situation of oogonia or oocytes adhering to the wall, thereby simulating the natural arrangement and interaction of cells in the body, thereby providing cells with a better environment for in vitro growth and differentiation; and, during the hanging drop culture and suspension culture process, it is not necessary to perform relatively frequent digestion and passage for contact inhibition between cells, which causes less damage to cells; in addition, 3D culture simulating the in vivo environment can also provide a model for exploring cell-to-cell interactions. It can be understood that in step S30, the suspension culture can be cultured by stirring or shaking, or by placing the material in an anti-adherence culture container for culture; specifically, in one embodiment of the present invention, in step S30, an anti-adherence 96-well plate is used to suspend and culture the recombinant ovary.

[0042] Further, in step S20, the hanging drop culture time is 2 to 4 days; and / or, in step S30, the suspension culture time is 4 to 8 weeks. Through the hanging drop culture for 2 to 4 days, the oogonia can be cultured to form a recombinant ovarian tissue block, which is convenient for further culture; if the culture time exceeds 4 days, the recombinant ovarian tissue block is too large, and the gravity of the hanging drop overcomes the surface tension of the hanging drop, causing the hanging drop to fall from the culture surface; through the suspension culture for 4 to 8 weeks, the recombinant ovary matures, enters the meiotic stage, and then produces oocytes.

[0043] Further, step S10 includes:

[0044] S11, taking out the fetal pig from the uterus of the pregnant sow, selecting the female fetal pig, and taking the reproductive ridge thereof;

[0045] S12, remove the cortical area in the reproductive ridge and mince the ovarian tissue blocks;

[0046] S13. Digest the ovarian tissue blocks, centrifuge and discard the supernatant, terminate the digestion, centrifuge and discard the supernatant to obtain oogonia.

[0047] By selecting ovarian tissue from the cortical region and cutting and digesting it, oogonia with a high degree of dispersion can be obtained, so as to form reconstituted ovarian tissue of sufficient size in hanging drop culture.

[0048] It should be noted that the reproductive ridge of the female fetal pig can be divided into the cortical region and the medullary region. During the growth and development of the fetal pig, the ovaries grow from the outside to the inside of the cortical region to form the medullary region. Therefore, the degree of differentiation of the medullary region is higher, while the degree of differentiation of the cortical region is lower, and more oogonia are isolated from the cortical region.

[0049] Further, in step S13, the digestion time is 20 to 30 minutes. In this way, the cells in the ovarian tissue block can be connected and digested to form dispersed oogonia, so as to form multiple recombinant ovaries and increase the number of cultures in the same batch; if the digestion time is too short, the digestion is not thorough, and some oogonia maintain the original cell connection. During the hanging drop culture, the recombinant ovary is too large to cause the hanging drop to fall, and the recombinant ovary will differentiate into oocytes in advance; if the digestion time is too long, further digestion may digest the membrane proteins or receptors on the cell membrane, resulting in subsequent oogonia differentiation being blocked.

[0050] Furthermore, the culture medium for terminating digestion includes glucose, amino acids, vitamins, zinc, putrescine, hypoxanthine and thymidine. Such a configuration facilitates in vitro culture of oocytes; in addition, the addition of culture medium can dilute the residual digestive fluid and completely terminate digestion. Specifically, in one embodiment of the present invention, the culture medium for terminating digestion is DMEM / F12 culture medium, which includes glucose, amino acids and vitamins in DMEM culture medium, and other components in Ham's F-12 culture medium, including zinc, putrescine, hypoxanthine and thymidine.

[0051] Furthermore, in step S13, the digestion solution includes a collagenase IV solution, wherein the concentration of collagenase IV is 1.5 to 2.5 mM. By using the collagenase IV solution as the digestion solution, the low pancreatic activity of collagenase IV is utilized to reduce damage to membrane proteins and receptors, thereby avoiding failure to differentiate into oocytes due to damage to membrane proteins and receptors.

[0052] Further, step S20 includes: S21, adding oogonia to the polymerization culture medium and mixing, to obtain oogonia culture fluid; S22, culturing the oogonia culture fluid in batches to obtain reconstructed ovaries. It should be noted that the polymerization culture medium can promote the re-aggregation of dispersed oogonia to form reconstructed ovaries. Through batch hanging drop culture, oogonia from the same ovary can be digested and cultured to form multiple reconstructed ovarian tissues, thereby increasing the batches cultured simultaneously and improving the yield of oocytes.

[0053] Furthermore, in step S22, the concentration of oogonia in the oogonia culture medium is 1.5×10 6 ~1.8×10 6 With this setting, the concentration of oogonia in the oogonia culture medium is optimal; if the concentration of oogonia is lower than 1.5×10 6 / mL, the oogonia concentration is too low, the reconstructed ovary is too small, resulting in slow development and a longer time required for differentiation into oocytes; if the oogonia concentration is higher than 1.8×10 6The concentration of oogonia in the oogonia culture medium is preferably 1.6×10 6 Pieces / mL.

[0054] Furthermore, the polymeric medium includes ethanolamine, glutathione, ascorbic acid, insulin, transferrin, lipids, bovine serum albumin, fetal bovine serum, non-essential amino acids, sodium pyruvate, penicillin-streptomycin, 2-mercaptoethanol and Y-27632 dihydrochloride. Such a setting can provide a stable culture environment for oogonia. Among them, fetal bovine serum (FBS) provides essential nutrients and growth factors for the maintenance and growth of in vitro cultured cells, which can meet the specific metabolic needs of cell culture; sodium pyruvate can be used as an alternative carbon source in cell culture; 2-mercaptoethanol and vitamin C are used as reducing agents in the cell culture process to prevent cell oxygen free radical poisoning; Y-27632 dihydrochloride is an ATP-competitive ROCK-Ⅰ and ROCK-Ⅱ inhibitor, which dissociates the induced apoptosis of cultured human embryonic stem cells and improves cloning efficiency.

[0055] The present invention also proposes an application of the above-mentioned oocyte in pig breeding. By adopting the above-mentioned oocyte culture method, the real growth of the ovary can be simulated to culture a large number of recombinant ovarian tissues, which are then differentiated to produce oocytes, which are applied to pig breeding and reduce the cost of oocyte culture. The application of the oocyte in pig breeding also has all the beneficial effects of the above-mentioned oocyte culture method, which will not be repeated here.

[0056] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0057] Example 1

[0058] Provided is a method for culturing an oocyte, comprising the following steps:

[0059] (1) In vitro isolation and identification of fetal pig genital ridges:

[0060] The uterus of the pregnant sow was retrieved from the slaughterhouse, transferred to the laboratory, rinsed with saline, and then sprayed with 75% alcohol for disinfection. The outer membrane of the uterus was peeled off, and the fetal pig wrapped in the amniotic membrane was sprayed with 75% alcohol, then divided into plastic bags and transferred to the intercellular transfer window. The amniotic membrane wrapped around the fetal pig was torn open on the intercellular operation table, and the fetal pig was transferred to a culture dish with a diameter of 10 cm with tweezers, and it was rinsed with PBS containing 1% penicillin and streptomycin. The abdominal skin and muscle layer near the hind legs of the fetal pig were clamped with tweezers, and the abdominal cavity of the fetal pig was cut open with tweezers. The underdeveloped liver and kidney of the fetal pig were opened, and the white and smooth fetal pig genital ridge structure could be observed. The genital ridge was transferred to a culture dish containing DMEM / F12 culture medium (Gibco) by gently holding the bottom of the fetal pig with tweezers. After observing the reproductive organs of the fetal pig to identify the sex, the genital ridge of the fetal pig was removed for further identification. The genital ridge of the female fetal pig was slender and twisted, while the genital ridge of the male fetal pig was smooth and rounded in the shape of a kidney, with red vascular-like seminiferous tubules visible on the surface.

[0061] (2) In vitro construction of recombinant ovaries: The removed fetal pig ovaries were transferred to FBS for washing and then transferred to MⅡ culture medium. The blood clots of the fetal pig ovaries were removed under a stereoscope. Since the germ cells migrate from the cortical area to the medullary area of ​​the ovaries as they develop, the cortical and medullary areas of the fetal pig ovaries were separated and cultured separately. The separated fetal pig ovaries were divided into 3 mm2 sections using cell forceps and a 1 mL syringe needle under a stereoscope. 3 Use cell forceps to transfer the small pieces to a 1.5ml centrifuge tube, add 1mL collagenase IV (Sigma), and place in a 37℃ constant temperature incubator for digestion for 20min. Use a pipette to disperse it into a single cell suspension. Use a 40μm cell sieve to filter the cell suspension, transfer the cell suspension to a centrifuge and centrifuge at 1500rpm for 3min. After discarding collagenase IV, add 1mL DMEM / F12 culture medium to terminate digestion, blow and mix well, and then aspirate 200μL of single cell suspension. After counting with a hemocytometer, move it to a centrifuge and centrifuge at 1500rpm for 3min. Discard DMEM / F12, add 1ml Advanced MEM and count the cells. After counting, adjust the cell density to maintain the cell density at 1.6×10 6 / mL. Use a pipette to transfer the cell suspension to the lid of a 6-cm diameter culture dish. The volume of each droplet is 30 μL, so that the cell volume in each droplet is maintained at 5×10 4 The lid of the culture dish was quickly turned over to keep the hanging drop as round as possible, and then the lid was placed on the culture dish with 3 mL of Alpha-MEM medium, and it was smoothly transferred to a 37°C, 5% CO2 incubator. Depending on the gestational age of the fetus, the cells were cultured at a constant temperature for 2-4 days to allow the cells to reassemble into recombinant ovaries.

[0062] (3) In vitro culture of recombinant ovaries and in vitro follicle development: 200 μL of the prepared recombinant ovary suspension medium was added to an anti-adhesive 96-well plate. The recombinant ovary suspension medium included: AdvancedMEM (Gibco), 10% FBS, 2mM L-glutamine, 150mM vitamin C, 0.1% penicillin-streptomycin, and 55 μM 2-mercaptoethanol. The 96-well plate containing the recombinant ovary suspension medium was transferred to a constant temperature incubator for 10 minutes. The recombinant ovaries were transferred to the 96-well plate using a mouth pipette under a stereoscope. The 96-well plate was then placed in a 37°C, 5% CO2 incubator for culture. The medium was changed every other day. After 8 weeks of continuous culture, oocytes were obtained from the recombinant ovaries.

[0063] Differentiation detection experiment of oocytes in reconstructed ovaries:

[0064] After one week, four weeks and eight weeks of culture, the partially reconstructed ovarian tissue cultured in vitro in Example 1 was collected, and the excess water was removed and transferred to the embedding box. The location of the tissue was marked with a marker for subsequent slicing. The frozen section embedding agent OCT was slowly added along one side of the embedding box to avoid bubbles. The embedding box containing the sample and the embedding agent was transferred to a -80°C refrigerator. After freezing for 24 hours, the packaged sample was taken out of the embedding box, and the sample was trimmed to a suitable size with a scalpel and fixed on the operating table. The frozen microtome was adjusted to -20°C and the slice thickness was 7 μm for slicing. The sample was cut into 7 μm thick sheets and transferred to a slide treated with an anti-slice. The slices were air-dried at room temperature for about 1 hour, transferred to a wash cup, and washed twice with 1×PBS for 3 minutes each time. The slices were washed 3 times with TBST buffer for 3 minutes each time to remove the OCT embedding agent. After washing, reconstructed ovarian slices were obtained.

[0065] The reconstructed ovarian slices were placed under a 10x microscope for observation, and white light images of the reconstructed ovarian tissues under a 10x microscope were obtained after 1 week, 4 weeks, and 8 weeks of in vitro culture, as shown in FIG. Figure 1 shown.

[0066] Transfer the reconstituted ovarian slices to a wet box, add 50 μL of 4% paraformaldehyde to each slice, cover with sealing film and fix for 10 minutes. After fixation, remove the sealing film, transfer the slices to a washing cup, and wash with TBST buffer 3 times, 3 minutes each time; block: block in a wet box at room temperature for 30 minutes, the blocking solution is: 1% BSA and 0.1% Triton X-100 in PBS; discard the blocking solution, add 50 μL of 0.5% primary antibody (Rb MVH, MsγH2AX) diluted with blocking solution and incubate in a wet box at 4°C overnight; the next day, rewarm at room temperature for 30 minutes, wash with TBST buffer 3 times, 3 minutes each time. Add 50 μL of 0.5% secondary antibody (Rb555, Ms 488) diluted with blocking solution, incubate at 37°C for 1 hour; wash with TBST 3 times, 3 minutes each time; Hoechst stain cell nuclei for 4 minutes, rinse with PBS several times, and seal the slices with fluorescent anti-quencher.

[0067] The fluorescent anti-quencher sealant was placed under a fluorescence microscope for observation to obtain an immunofluorescence staining image, such as Figures 2 to 5 shown.

[0068] like Figure 1 As shown, comparing the microscope images of the reconstructed ovarian tissue after one week of culture and four weeks of culture, the volume of the reconstructed ovary was significantly reduced, and the volume of the reconstructed ovary increased slightly with the extension of the culture time.

[0069] The reduction in the volume of the reconstructed ovary may be due to the re-generation of cell connections between the dispersed cells after hanging drop culture, thus forming a compact reconstructed ovarian tissue mass; while the slight increase in the volume of the reconstructed ovary indicates that the cells in the reconstructed ovary grow and develop normally.

[0070] See also Figures 2 to 5 , Figure 3 The MVH antibody (red fluorescence) in the Figure 4 The γH2AX antibody (green fluorescence) in the Figure 3 and Figure 4 , indicating that meiosis exists in the fluorescence anti-quencher sealant.

[0071] Figure 2 The Hoechst antibody (blue fluorescence) in the Figure 2 , Figure 3 and Figure 4 By superposition, we can get Figure 5 ,like Figure 5As shown in the red frame, the red fluorescence is located around the blue fluorescence, indicating that the MVH antibody is bound to the area around the Hoechst antibody binding site, and the green fluorescence overlaps with the blue fluorescence, indicating that the γH2AX antibody is bound to the Hoechst antibody binding site, and the Hoechst antibody binding site is located in the cell nucleus, indicating that the MVH antibody is bound to the cytoplasm and the γH2AX antibody is bound to the cell nucleus, which is consistent with the theoretical staining situation.

[0072] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A method for culturing oocytes, characterized in that: The following steps are involved: S10, provide oogonia; S20, culturing the oogonia in hanging drops to obtain a reconstructed ovary; S30, suspending and culturing the recombinant ovary to obtain oocytes.

2. The method for culturing oocytes according to claim 1, wherein: In step S20, the hanging drop culture time is 2 to 4 days; and / or, In step S30, the suspension culture time is 4 to 8 weeks.

3. The method for culturing oocytes according to claim 1, wherein: Step S10 includes: S11, taking out the fetal pig from the uterus of the pregnant sow, selecting the female fetal pig, and taking the reproductive ridge thereof; S12, remove the cortical area in the reproductive ridge and mince the ovarian tissue blocks; S13. Digest the ovarian tissue blocks, centrifuge and discard the supernatant, terminate the digestion, centrifuge and discard the supernatant to obtain oogonia.

4. The method for culturing oocytes according to claim 3, wherein: In step S13, the digestion time is 20 to 30 minutes.

5. The method for culturing oocytes according to claim 3, wherein: The digestion-stopping medium includes glucose, amino acids, vitamins, zinc, putrescine, hypoxanthine, and thymidine.

6. The method for culturing oocytes according to claim 3, wherein: In step S13, the digestion solution includes a collagenase IV solution, wherein the concentration of collagenase IV is 1.5-2.5 mM.

7. The method for culturing oocytes according to claim 1, wherein: Step S20 includes: S21, adding oogonia to the polymerization culture medium and mixing to obtain oogonia culture fluid; S22. Culture the oogonia culture medium in batches by hanging drops to obtain reconstructed ovaries.

8. The method for culturing oocytes according to claim 7, characterized in that: In step S22, the concentration of oogonia in the oogonia culture medium is 1.5×10 6 ~1.8×10 6 Pieces / mL.

9. The method for culturing oocytes according to claim 7, wherein: The polymerization medium includes ethanolamine, glutathione, ascorbic acid, insulin, transferrin, lipids, bovine serum albumin, fetal bovine serum, non-essential amino acids, sodium pyruvate, penicillin-streptomycin, 2-mercaptoethanol and Y-27632 dihydrochloride.

10. Use of the oocyte according to any one of claims 1 to 9 in pig breeding.

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