Application of 10-HDA in in-vitro culture of ovarian granular cells

Abstract

The invention provides an application of 10-HDA (10-histone deacetylase) in in-vitro culture of ovarian granular cells. The invention provides a new application of 10-HDA, and particularly relates to an application of 10-HDA in preparation of an ovarian granular cell in-vitro culture agent. The 10-HDA can obviously reduce the increase of the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) of H2O2-induced granulosa cells, and relieve the reduction of the activity of glutathione (GSH) and superoxide dismutase (SOD) of H2O2-induced granulosa cells. By adopting 10-HDA, the oxidative stress state of the primary granular cells of the dairy cow can be effectively improved, and the problem that in the prior art, the oxidative stress of the primary granular cells of the dairy cow lacks an effective method for preventing the oxidative stress of the primary granular cells of the dairy cow is solved.

Description

Technical Field

[0001] The present invention belongs to the field of animal reproduction, and specifically relates to the application of 10-HDA in the in vitro culture of ovarian granulosa cells. Background Art

[0002] The development of dairy cow follicles depends on the synergistic action among somatic cells, granulosa cells and theca cells within the follicles. Among them, ovarian granulosa cells, as the main cell population within the follicles, play an important role in follicular development regulation, oocyte nutritional support and reproductive endocrine regulation by virtue of their heterogeneity in structural differentiation and function. The dynamic changes of granulosa cells are significantly correlated with the follicular development process. The development of granulosa cells precedes that of oocytes, and it affects the fertility of dairy cows by influencing the fate of oocytes. In addition, granulosa cells have high metabolic activity and are sensitive to reactive oxygen species (ROS), and are prone to becoming the main targets attacked by ROS, which not only affects the function and structure of granulosa cells, but may also seriously affect the follicular development and ovulation of dairy cows by promoting apoptosis and inhibiting steroid hormone synthesis.

[0003] 10-Hydroxy-2-decenoic acid (10-HDA), also known as royal jelly acid, is one of the main active components of royal jelly and is also a key marker for evaluating the quality of royal jelly. 10-HDA belongs to unsaturated medium-chain fatty acids, with a molecular weight of 186.25 g / mol and a chemical formula of C 10 H 18 O3, including having a hydroxyl (-OH) functional group at the 10th position of the carbon chain and forming a cis double bond (C=C) at the 2nd position. This unique structure endows it with special physical and chemical properties. Research shows that 10-HDA has various biological activities such as anti-inflammatory, antioxidant, anti-tumor, anti-aging and immunomodulatory effects. However, there are few reports on the research of 10-HDA regulating the antioxidant capacity of dairy cow ovarian granulosa cells. Summary of the Invention

[0004] The purpose of the present invention is to provide a new use of 10-HDA. Another purpose of the present invention is also to provide a method for enhancing the antioxidant capacity of primary ovarian granulosa cells of dairy cows using 10-HDA.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] The application of 10-HDA in the preparation of an in vitro culture medium for ovarian granulosa cells.

[0007] For the application as described above, preferably, the in vitro culture medium is a reagent for enhancing the antioxidant capacity of ovarian granulosa cells, improving the oxidative stress performance of ovarian granulosa cells, and used for alleviating the reduction of GSH activity and SOD activity of granulosa cells induced by H2O2.

[0008] The application as described above, preferably, the ovarian granulosa cells are primary ovarian granulosa cells of dairy cows, ovarian granulosa cells of beef cattle, ovarian granulosa cells of water buffalo, ovarian granulosa cells of pigs, ovarian granulosa cells of mice, and ovarian granulosa cells of sheep.

[0009] An in vitro culture medium for ovarian granulosa cells, the in vitro culture medium contains 10-HDA. Further, the concentration of 10-HDA is 1-100 μM.

[0010] An in vitro culture medium for primary ovarian granulosa cells of dairy cows, the in vitro culture medium contains 10-HDA. Further, the concentration of 10-HDA is 1-100 μM.

[0011] Further, the in vitro culture medium further includes DMEN / HIGH GLUCOSE medium with a volume ratio of 89%, 10% FBS, and 1% penicillin-streptomycin.

[0012] A method for enhancing the antioxidant capacity of primary ovarian granulosa cells of dairy cows using 10-HDA, which includes adding 10-HDA to the culture medium for culturing primary ovarian granulosa cells of dairy cows.

[0013] Further, the preferred concentration of 10-HDA is 1-100 μM.

[0014] The beneficial effects of the present invention are as follows:

[0015] The present invention provides a new use of 10-HDA, for use in the preparation of an in vitro culture medium for ovarian granulosa cells, for enhancing the antioxidant capacity of ovarian granulosa cells.

[0016] The present invention also provides a method for enhancing the antioxidant capacity of primary ovarian granulosa cells of dairy cows using 10-HDA. By exogenous addition of 10-HDA, it can significantly reduce the reactive oxygen species level of primary ovarian granulosa cells of dairy cows at the normal level, up-regulate the GSH activity, and thus enhance the antioxidant defense ability of granulosa cells.

[0017] The present invention pretreats with different concentrations of 10-HDA for 24 h, and then combines with H2O2 stimulation for 6 h. At the same time, a NAC positive control group is set. It is found that 10-HDA can significantly reduce the increase in the MDA level of granulosa cells induced by H2O2, and relieve the decrease in the GSH activity and SOD activity of granulosa cells induced by H2O2. The present invention determines that 10-HDA can improve the oxidative stress state of primary ovarian granulosa cells of dairy cows, and solves the problem in the prior art that there is a lack of effective prevention methods for the oxidative stress of primary ovarian granulosa cells of dairy cows. Description of the Drawings

[0018] Figure 1Fluorescence intensity images of ROS in primary ovarian granulosa cells of dairy cows under normal conditions after treatment with different concentrations of 10-HDA.

[0019] Figure 2 Quantitative analysis of the average fluorescence intensity of ROS in primary ovarian granulosa cells of dairy cows under normal conditions after treatment with different concentrations of 10-HDA.

[0020] Figure 3 GSH activity in primary ovarian granulosa cells of dairy cows under normal conditions after treatment with different concentrations of 10-HDA.

[0021] Figure 4 Cell viability of primary ovarian granulosa cells of dairy cows after treatment with different concentrations of H2O2.

[0022] Figure 5 Fluorescence intensity images of ROS in primary ovarian granulosa cells of dairy cows after treatment with different concentrations of H2O2.

[0023] Figure 6 Quantitative analysis of the average fluorescence intensity of ROS in primary ovarian granulosa cells of dairy cows after treatment with different concentrations of H2O2.

[0024] Figure 7 Fluorescence intensity images of ROS in primary ovarian granulosa cells of dairy cows after treatment with different concentrations of 10-HDA, H2O2, and NAC.

[0025] Figure 8 Quantitative analysis of the average fluorescence intensity of ROS in primary ovarian granulosa cells of dairy cows after treatment with different concentrations of 10-HDA, H2O2, and NAC.

[0026] Figure 9 MDA level in primary ovarian granulosa cells of dairy cows after treatment with different concentrations of 10-HDA, H2O2, and NAC.

[0027] Figure 10 GSH activity in primary ovarian granulosa cells of dairy cows after treatment with different concentrations of 10-HDA, H2O2, and NAC.

[0028] Figure 11 SOD activity in primary ovarian granulosa cells of dairy cows after treatment with different concentrations of 10-HDA, H2O2, and NAC. Specific implementation mode

[0029] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Modifications or substitutions made to the present invention without departing from the spirit and essence of the present invention all fall within the scope of the present invention.

[0030] Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art. Unless otherwise specified, the reagents used in the examples are of analytical grade or above.

[0031] Example 1

[0032] A method for enhancing the antioxidant capacity of primary ovarian granulosa cells in dairy cows using 10-HDA, comprising the following steps:

[0033] S1: First, under in vitro conditions, primary ovarian granulosa cells of dairy cows are cultured. Healthy dairy cow ovarian samples are collected from a local slaughterhouse in Wuhan, Hubei Province. After collection, the ovaries are immediately placed in PBS containing 1% penicillin-streptomycin solution preheated to 37°C and transported to the laboratory within 2 h. The ovarian surface is disinfected and washed with 75% ethanol. Granulosa cells and follicular fluid are aspirated from the follicles using a 25-gauge needle. The obtained cell suspension is centrifuged at 1500 r / min for 10 min to separate the granulosa cells and follicular fluid. After centrifugation, the supernatant is discarded, and the precipitated granulosa cells are retained. Subsequently, the granulosa cells are washed twice with DMEM / HIGH medium containing 1% penicillin-streptomycin solution, and a cell culture medium composed of DMEN / HIGH GLUCOSE medium with a final concentration of 89%, 10% FBS, and 1% penicillin-streptomycin is added. The cells are cultured in a cell culture incubator at 38.5°C and 5% CO2. When the cell confluence reaches 70%, the fresh medium is replaced for 10-HDA treatment to carry out subsequent experiments.

[0034] S2: Adjust the density of primary ovarian granulosa cells of dairy cows to 1×10 6 cells / mL, seeded into a 6-well cell culture plate. When the cell density reaches 70%, cell culture media containing 1, 10, and 100 μM 10-HDA at the final concentration are added respectively and treated for 24 h. Then, the cell culture medium is removed and diluted DCFH-DA working solution is added. DCFH-DA (2,7-dichlorofluorescein diacetate) is diluted with serum-free medium to a final concentration of 5 μM as the DCFH-DA working solution. After incubating at 37°C in the dark for 30 min, the cells are washed 3 times with serum-free medium to remove the DCFH-DA probe that has not entered the cells. Fluorescence inverted microscopy is used to observe and collect images, and finally Image J 1.8.0 software is used to analyze the average fluorescence intensity.

[0035] Among them, the preparation of 10-HDA with different concentrations was carried out in a laminar flow hood. A pipette was used to aspirate 10.74 μL of dimethyl sulfoxide (DMSO) to dissolve 2 mg of 10-HDA with a molecular weight of 186.25 g / mol, obtaining a concentrated stock solution with a concentration of 1 M. Then, it was successively diluted with DMSO to 1 mM, 10 mM, and 100 mM 10-HDA, which were dispensed into PCR tubes. The lids of the PCR tubes were wrapped with sealing film and stored in a -20 °C refrigerator.

[0036] The results are as Figure 1 and Figure 2 shown. Among them, Figure 1 the ROS fluorescence intensity in granulosa cells was measured using a fluorescence inverted microscope, Figure 2 and the average fluorescence intensity was quantitatively analyzed and plotted using Image J. From Figure 1 and Figure 2 it can be seen that adding 100 μM 10-HDA can significantly reduce the ROS level in granulosa cells (P < 0.001).

[0037] S3: After treating primary ovarian granulosa cells of dairy cows with 10-HDA at different concentrations for 24 h, a GSH detection kit (Nanjing Jiancheng) was used to detect the level of glutathione (GSH) in primary ovarian granulosa cells of dairy cows at normal levels, and a microplate reader was used to measure the GSH activity.

[0038] Adjust the density of primary ovarian granulosa cells of dairy cows to 1×10 6 cells / mL, and seed them into a 6-well cell culture plate. When the cell density reaches about 70%, cell culture media with final concentrations of 1, 10, and 100 μM 10-HDA are added respectively and cultured for 24 h. After adding 0.2 mL of RIPA lysis buffer to each well, the cells are lysed on ice for 30 min, and then the lysed cells are collected, centrifuged at 10000 g / min at 4 °C for 10 min, and the supernatant is collected to remove the lower precipitate. Then, the protein concentration in the supernatant is measured using a BCA protein concentration assay kit. Take 100 μL of the homogenized supernatant, add 100 μL of reagent one in the GSH kit and mix well, centrifuge at 3500 r / min for 10 min, remove the supernatant and perform a color reaction. At the same time, set standard wells (standards provided by the kit), sample wells (100 μL of the treated sample is added), and blank wells (blank control with nothing added) in a 96-well plate. Subsequently, different reagents are added successively according to the GSH detection kit instructions. Finally, the absorbance is measured at 405 nm using a microplate reader, and the GSH activity is calculated.

[0039] From Figure 3 it can be seen that adding 100 μM 10-HDA can significantly increase the GSH level in granulosa cells (P < 0.05).

[0040] S4: Add H2O2 at different concentrations to evaluate its effects on the cell viability and the content of reactive oxygen species (ROS) in primary bovine ovarian granulosa cells, determine the optimal treatment concentration and time of H2O2, use a CCK-8 assay kit (Dojindo, Japan) and combine it with a microplate reader to measure cell viability; use a ROS assay kit (Beyotime, Shanghai) and a fluorescence inverted microscope to detect the content of ROS.

[0041] Preparation of H2O2 at different concentrations: Use a pipette to aspirate 45.4 μL of 3% H2O2, then add 54.6 μL of phosphate buffer solution (PBS) to obtain 100 μL of H2O2 concentrated stock solution with a concentration of 400 mM. Then, dilute it with PBS to 200 mM, 100 mM, and 50 mM H2O2 in sequence, and aliquot them into PCR tubes. Wrap the lids of the PCR tubes with sealing film and store them in a 4°C refrigerator.

[0042] Adjust the density of primary bovine ovarian granulosa cells to 1×10 6 cells / mL, seed them into a 96-well cell culture plate. When the cell density reaches 70%, add cell culture media with final concentrations of 50, 100, 200, and 400 μM H2O2 to each well, with 6 replicates for each treatment. At the same time, set wells with only complete medium as blank controls. After 6 h of treatment, add 90 μL of serum-free medium and 10 μL of cell proliferation detection (CCK-8) solution to the cell wells and blank wells, then wrap them with tin foil to avoid the influence of light and incubate them in an incubator for 2 h. Subsequently, use a microplate reader to measure the absorbance of each well at a wavelength of 450 nm, and calculate the cell viability according to the instructions. Then, use the ROS assay kit instructions to measure the ROS content in cells treated with different concentrations of H2O2 (the measurement steps are the same as in S2).

[0043] The results are as Figures 4 - 6 shown. Among them, Figure 4 is to measure the cell viability in granulosa cells using a microplate reader, Figure 5 is to measure the ROS fluorescence intensity in granulosa cells using a fluorescence inverted microscope, Figure 6 is to quantitatively analyze the average fluorescence intensity using Image J and plot a graph. As Figure 4 can be seen, compared with the control group, the cell activities in the 100, 200, and 400 μM H2O2 treatment groups decreased by 23.81%, 60%, and 96.5% respectively (P<0.001); as Figure 5 and Figure 6 can be seen, the 100, 200, and 400 μM H2O2 treatments can significantly increase the ROS level in granulosa cells (P<0.05). Based on the above results, the treatment condition of 100 μM H2O2 for 6 h is selected for subsequent experiments.

[0044] S5: First, the primary ovarian granulosa cells of cows were pretreated with 1, 10, 100 μM 10-HDA for 24 h, and then stimulated with H2O2 for 6 h. Meanwhile, a positive control group of NAC (N-acetylcysteine) was set up. Subsequently, the ROS content in granulosa cells of different treatment groups was measured according to the instructions of the ROS kit (Shanghai Beyotime) (the measurement steps were the same as those in S2).

[0045] Preparation of NAC (positive control group): Use a pipette to aspirate 6.1 μL of PBS to dissolve 5 mg of NAC with a molecular weight of 163.2 g / mol to obtain a concentrated stock solution with a concentration of 5 M, and aliquot it into PCR tubes. Wrap the lids of the PCR tubes with sealing film and store them in a -20 °C refrigerator.

[0046] Adjust the density of primary ovarian granulosa cells of cows to 1×10 6 cells / mL, and seed them into a 6-well cell culture plate. When the cell density reaches 70%, cell culture media with final concentrations of 1, 10, and 100 μM 10-HDA are added respectively and cultured for 24 h. Then, stimulate with cell culture media containing 100 μM H2O2 with a final concentration for 6 h. At the same time, set up a CT group, an H2O2 group, and a NAC positive group. Among them, the control group (CT group) is not pretreated with 10-HDA and not treated with H2O2; the H2O2 group is only stimulated with cell culture media containing 100 μM H2O2 with a final concentration for 6 h; the positive group uses 5 mM NAC to replace 10-HDA, and other treatments are the same as those in the 10-HDA group. Subsequently, the ROS content in cells was measured using a ROS detection kit (Shanghai Beyotime).

[0047] The results are as Figure 7 and Figure 8 shown. Among them, Figure 7 is to measure the ROS fluorescence intensity in granulosa cells using a fluorescence inverted microscope, Figure 8 and Figure 7 is to quantitatively analyze the average fluorescence intensity and plot a graph using Image J. From Figure 8 and

[0048] it can be seen that compared with the group treated with H2O2 alone, pretreatment with 10-HDA can significantly reduce the increase in the ROS level of granulosa cells induced by H2O2 (P<0.001), and there is no significant difference in the alleviating effect compared with the positive control group of NAC.

[0049] Adjust the density of primary ovarian granulosa cells of dairy cows to 1×10 6 cells / mL, seed them into a 6-well cell culture plate. When the cell density reaches 70%, add cell culture medium with a final concentration of 1, 10, and 100 μM 10-HDA, and culture for 24 h. Then, stimulate with cell culture medium containing a final concentration of 100 μM H2O2 for 6 h. At the same time, set up a CT group, an H2O2 group, and an NAC positive control group. After adding 0.2 mL of RIPA lysis buffer to each well, lyse the cells on ice for 30 min, collect the lysed cells, centrifuge at 12,000 r / min at 4°C for 10 min, collect the supernatant, and remove the lower precipitate. Then, measure the protein concentration using a BCA protein concentration assay kit and prepare the working solution for MDA detection. At the same time, set up a standard tube (the standard provided by the kit), a sample tube (0.1 mL of the treated sample is added), and a blank tube (a blank control with nothing added). Subsequently, add 0.2 mL of the MDA working solution to each tube, mix well by shaking, heat at 100°C for 15 min. Cool to room temperature in a water bath, centrifuge at 1000 g at room temperature for 10 min. Take 200 μL of the supernatant and add it to a 96-well plate, and then measure the absorbance at 532 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Finally, calculate the molar concentration of MDA according to the standard curve.

[0050] It can be seen from Figure 9 that compared with the group treated with H2O2 alone, pre-addition of 10-HDA can significantly reduce the increase in the MDA content of granulosa cells induced by H2O2 (P<0.001), and there is no significant difference in the alleviating effect compared with the NAC positive control group.

[0051] S7: First, pretreat primary ovarian granulosa cells of dairy cows with 1, 10, 100 μM 10-HDA for 24 h, and then stimulate with H2O2 for 6 h in combination, and set up an NAC positive control group at the same time. Use a GSH detection kit (Nanjing Jiancheng) to detect the GSH activity in primary ovarian granulosa cells of dairy cows, and use an ELISA reader to measure the GSH activity (the detection method is the same as S3).

[0052] It can be seen from Figure 10 that compared with the group treated with H2O2 alone, 10-HDA pretreatment can significantly alleviate the decrease in GSH activity of granulosa cells induced by H2O2 (P<0.001), and there is no significant difference in the alleviating effect compared with the NAC positive control group.

[0053] S8: First, pretreat primary ovarian granulosa cells of dairy cows with 1, 10, 100 μM 10-HDA for 24 h, and then stimulate with H2O2 for 6 h in combination, and set up an NAC positive control group at the same time. Use an SOD detection kit (Shanghai Beyotime) to detect the SOD activity in primary ovarian granulosa cells of dairy cows, and use an ELISA reader to measure the SOD activity.

[0054] Adjust the density of primary ovarian granulosa cells of dairy cows to 1×10 6 cells / mL, and seed them into a 6-well cell culture plate. When the cell density reaches 70%, add cell culture medium containing 1, 10, and 100 μM 10-HDA at the final concentration, and culture for 24 h. Then, stimulate with cell culture medium containing 100 μM H2O2 at the final concentration for 6 h. At the same time, set up a CT group, an H2O2 group, and a positive NAC group. After adding 0.2 mL of RIPA lysis buffer to each well and lysing on ice for 30 min, collect the lysed cells, centrifuge at 10000 g / min at 4°C for 10 min, collect the supernatant, and remove the lower precipitate. Then, measure the protein concentration using a BCA protein concentration assay kit. Set up standard wells (standards provided by the kit), sample wells (20 μL of the treated sample added), and blank wells (distilled water) in a 96-well plate. Subsequently, add different reagents in sequence according to the SOD detection kit instructions. Finally, measure the absorbance at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader and calculate the SOD activity.

[0055] It can be seen from Figure 11 that compared with the H2O2 treatment group alone, 10-HDA pretreatment can significantly alleviate the decrease in SOD activity in granulosa cells induced by H2O2 (P<0.001), and there is no significant difference in the alleviating effect compared with the positive control NAC group.

[0056] S9: Analyze the detected data. All results are expressed as mean ± standard error (Mean±SEM). The data are plotted using Graphpad Prism 8.0 software. Each group has at least 3 biological replicates. The average fluorescence intensity is analyzed using Image J1.8.0 software. Student's t-test is used for significant analysis between two groups, and one-way analysis of variance (ANOVA) is used for significant analysis among multiple groups. * indicates significant difference compared with the control group (P<0.05), and ** and *** both indicate extremely significant differences compared with the control group (P<0.01, P<0.001). # indicates significant difference between the specified treatment groups (P<0.05). ## and ### both indicate extremely significant differences between the specified treatment groups (P<0.01, P<0.001).

[0057] In summary, adding 10-HDA can reduce the ROS content in granulosa cells during normal culture and increase the activity of GSH. Moreover, adding 10-HDA can significantly reduce the increase in MDA and ROS content in granulosa cells induced by H2O2, and alleviate the decrease in the antioxidant enzyme activities of GSH and SOD induced by H2O2. The alleviating effect has no significant difference compared with the positive control NAC group.

[0058] ROS can cause oxidative stress in granulosa cells, thereby activating the MAPK and JNK pathways to inhibit granulosa cell proliferation. In addition, ROS can inhibit Akt phosphorylation, reduce downstream mTOR activity, reduce the synthesis of pro-proliferation proteins, and promote β-catenin degradation to inhibit the expression of granulosa cell proliferation-related genes. It can be seen that excessive ROS can inhibit granulosa cell proliferation and promote granulosa cell apoptosis through multiple pathways, thereby affecting the follicular development and ovulation of dairy cows.

[0059] MDA is the end product of lipid peroxidation and is often used to assess oxidative stress levels. Its increased concentration reflects damage to cell membrane lipids, suggesting that cells have suffered oxidative damage. High MDA levels may also inhibit steroidogenic enzymes (such as aromatase), reduce estrogen synthesis, and affect follicular development.

[0060] GSH is the most important non-enzymatic antioxidant in cells. It directly removes reactive oxygen species (ROS) and protects cells from oxidative damage through the cycle of reduced state (GSH) and oxidized state (GSSG). GSH maintains the activity of steroidogenic enzymes (such as aromatase) by protecting mitochondrial function. The maturation of oocytes requires granulosa cells to provide antioxidant factors such as GSH to prevent growth arrest caused by oxidative stress. Therefore, by adding 10-HDA, the content of ROS and MDA can be reduced, and increasing GSH is beneficial to promoting the development of dairy cow follicles and the maturation of oocytes.

Claims

1. Application of 10-HDA in the preparation of an in vitro culture medium for ovarian granulosa cells.

2. The application according to claim 1, characterized in that The in vitro culture medium is a reagent for enhancing the antioxidant capacity of ovarian granulosa cells, improving the oxidative stress performance of ovarian granulosa cells, and alleviating the decrease in the activities of GSH and SOD in granulosa cells induced by H2O2.

3. The application according to claim 1, wherein The granulosa cells are primary ovarian granulosa cells of cows, beef cattle, buffaloes, pigs, mice, and sheep.

4. An in vitro culture medium for ovarian granulosa cells, characterized in that, The in vitro culture medium contains 10-HDA.

5. The in vitro culture agent for ovarian granulosa cells according to claim 4, wherein, The concentration of 10-HDA is 1 - 100 μM.

6. A primary ovarian granulosa cell in vitro culture medium for dairy cows, characterized in that, The in vitro culture medium contains 10-HDA.

7. The primary bovine ovarian granulosa cell in vitro culture agent according to claim 6, characterized in that, The concentration of 10-HDA is 1 - 100 μM.

8. The primary ovarian granulosa cell in vitro culture medium for dairy cows according to claim 6, wherein The in vitro culture medium further includes a DMEN / HIGH GLUCOSE medium at a volume ratio of 89%, 10% FBS, and 1% penicillin-streptomycin.

9. A method for enhancing the antioxidant capacity of primary ovarian granulosa cells of dairy cows using 10-HDA, characterized in that, It includes culturing by adding 10-HDA to the culture medium for primary ovarian granulosa cells of cows.

10. The method according to claim 9, wherein The added concentration of 10-HDA is 1 - 100 μM.

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