TUDCA-added porcine oocyte culture solution as well as preparation method and application of TUDCA-added porcine oocyte culture solution

Abstract

The invention discloses a TUDCA-added porcine oocyte culture solution as well as a preparation method and application of the TUDCA-added porcine oocyte culture solution. The TUDCA-added pig oocyte culture solution comprises TUDCA and a basic culture solution, wherein the basic culture solution comprises a TCM-199 culture medium containing a 1% Pen Strep solution, an epidermal growth factor, insulin, pyruvic acid, cysteine, pig follicular fluid, PMSG and hCG; the addition amount of the TUDCA is 0.5 mmol / L of the basic culture solution. The TUDCA-added pig oocyte culture solution can effectively repair heat stress-induced pig oocyte damage, significantly improve the in-vitro mature ovum rate of heat stress-induced pig oocytes, and improve the oocyte quality.

Description

Technical Field

[0001] The present invention belongs to the technical field of oocyte culture, and specifically relates to a porcine oocyte culture solution supplemented with TUDCA, a preparation method thereof, and an application thereof. Background Art

[0002] Constant body temperature is very important for the normal physiological functions of organisms. Animals usually suffer from heat stress in hot weather or environments, especially when the temperature is extremely high and the exposure time is long. Heat stress has a negative impact on human health, animal growth, and livestock production [1] . Heat stress is one of the most stressful and costly events in the life of livestock, having a negative impact on animal health, productivity, and product quality. Pigs, poultry, and ruminants are susceptible to heat stress due to their high metabolic rate, high basal metabolic heat production, fast growth rate, and high production level [2,3] . Heat stress mainly impairs oocyte quality and embryonic development by inducing mitochondrial dysfunction, oxidative stress, and apoptosis in oocytes and granulosa cells or cumulus cells. Studies have also shown that under high-temperature stress, sheep oocytes arrest at the germinal vesicle breakdown (GVBD) stage, with abnormal chromatin structure [4] . More importantly, high-temperature stress has an adverse effect on mitochondrial function, inducing the production of reactive oxygen species (ROS) and oxidative stress, reducing antioxidant capacity, and leading to cell DNA damage and apoptosis [5] .

[0003] Tauroursodeoxycholic acid (TUDCA) is a white crystalline powder, odorless, bitter, easily soluble in water and methanol, insoluble in low-polarity organic solvents, and its aqueous solution is acidic. It is the most hydrophilic bile acid [6] . Studies have shown that hydrophilic bile acids (especially TUDCA) have anti-apoptotic effects and are therefore considered potential tools for treating neurodegenerative diseases [7] . Recent studies have shown that TUDCA also plays an important role in anti-aging and extending the lifespan of the body. TUDCA promotes protein folding activity by targeting HSP90, reduces the accumulation of unfolded proteins in cells, and improves protein homeostasis, thereby delaying aging [8] . In addition, TUDCA treatment has been proven to reduce endoplasmic reticulum stress in various tissues [9] . TUDCA can prevent endoplasmic reticulum stress-induced apoptosis and thereby improve mouse brain and vascular functions

[10] . TUDCA increases fetal weight and improves uterine artery function by inhibiting endoplasmic reticulum stress in the placenta of aged rats

[11] . The above studies show that TUDCA plays an important role in improving the body's metabolic function, preventing endoplasmic reticulum stress, and anti-aging. However, whether TUDCA can relieve the effect on oocytes under HS remains unknown.

[0004] Therefore, the present invention provides a new culture medium composed of TUDCA and porcine oocyte culture medium, which is used to improve the maturation rate of porcine oocytes induced by heat stress and improve the quality of oocytes. Summary of the Invention

[0005] The object of the present invention is to provide a porcine oocyte culture medium added with TUDCA.

[0006] Another object of the present invention is to provide a preparation method of a porcine oocyte culture medium added with TUDCA.

[0007] Another object of the present invention is to provide the application of a porcine oocyte culture medium added with TUDCA in promoting the maturation of porcine oocytes induced by heat stress.

[0008] The culture medium described in the present invention includes a basal culture medium and TUDCA.

[0009] The addition amount of TUDCA described in the present invention is 0.1-1 mmol / L of the basal culture medium.

[0010] Preferably, the addition amount of TUDCA described in the present invention is 0.3-0.5 mmol / L of the basal culture medium.

[0011] More preferably, the addition amount of TUDCA described in the present invention is 0.5 mmol / L of the basal culture medium.

[0012] The basal culture medium described in the present invention includes TCM-199 medium containing 1-5% Pen Strep solution, 5-20 ng / mL epidermal growth factor, 3-9 μg / mL insulin, 0.1-0.6 mmol / L pyruvate, 0.2-0.8 mmol / L cysteine, 5-20% porcine follicular fluid, 5-20 IU / mL PMSG, and 5-20 IU / mL hCG.

[0013] Preferably, the basal culture medium described in the present invention includes TCM-199 medium containing 1-3% Pen Strep solution, 10-15 ng / mL epidermal growth factor, 5-7 μg / mL insulin, 0.2-0.4 mmol / L pyruvate, 0.4-0.6 mmol / L cysteine, 10-15% porcine follicular fluid, 10-15 IU / mL PMSG, and 10-15 IU / mL hCG.

[0014] Further preferably, the basal culture medium of the present invention comprises TCM-199 medium containing 1% Pen Strep solution, 10 ng / mL epidermal growth factor, 5 μg / mL insulin, 0.2 mmol / L pyruvate, 0.6 mmol / L cysteine, 10% porcine follicular fluid, 10 IU / mL PMSG, and 10 IU / mL hCG.

[0015] The preparation method of the porcine oocyte culture medium supplemented with TUDCA of the present invention is as follows: 0.09994 - 0.49970 g of TUDCA powder is dissolved in 10 mL of embryo water to prepare a TUDCA embryo aqueous solution with a concentration of 20 - 100 mmol / L. 3 - 7 μL of the TUDCA embryo aqueous solution is taken and added to 500 - 700 μL of the basal culture medium and shaken well to obtain a culture medium containing 0.1 - 1 mmol / L TUDCA.

[0016] Preferably, the preparation method of the porcine oocyte culture medium supplemented with TUDCA of the present invention is as follows: 0.24985 g of TUDCA powder is dissolved in 10 mL of embryo water to prepare a TUDCA embryo aqueous solution with a concentration of 50 mmol / L. 5.0 μL of the TUDCA embryo aqueous solution is taken and added to 500 μL of the basal culture medium and shaken well to obtain a culture medium containing 0.5 mmol / L TUDCA.

[0017] Application of the porcine oocyte culture medium supplemented with TUDCA of the present invention in promoting the maturation of porcine oocytes induced by heat stress.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The porcine oocyte culture medium supplemented with TUDCA of the present invention can promote oocyte maturation. Compared with the heat stress group, the average oocyte maturation rate of the group supplemented with 0.5 mM TUDCA is 52.07%, which is significantly higher than the average maturation rate of the heat stress group (HS) of 44.08%. The expansion of most cumulus cells is also significantly improved.

[0020] 2. The present invention examines the addition amount of TUDCA. The results show that compared with the groups supplemented with 0.1 mM, 0.3 mM, and 1 mM TUDCA, the group supplemented with 0.5 mM TUDCA has the highest average oocyte maturation rate and the best cumulus cell expansion effect. Therefore, the addition amount of TUDCA is selected as 0.5 mM.

[0021] 3. The porcine oocyte culture medium supplemented with TUDCA of the present invention can alleviate the effects of heat stress on the endoplasmic reticulum and Golgi apparatus of oocytes. The fluorescence staining detection results show that heat stress significantly reduces the red signals in the endoplasmic reticulum and Golgi apparatus of oocytes, while the supplementation of TUDCA can significantly enhance them.

[0022] 4. The porcine oocyte culture medium supplemented with TUDCA according to the present invention can inhibit excessive reactive oxygen species in porcine oocytes under heat stress. From the results of fluorescence staining detection, it can be seen that heat stress significantly increases the green signal of reactive oxygen species in the cytoplasm of oocytes, while supplementation with TUDCA can significantly weaken it.

[0023] 5. The porcine oocyte culture medium supplemented with TUDCA according to the present invention can restore the glutathione level in porcine oocytes decreased under heat stress. From the results of fluorescence staining detection, it can be seen that heat stress significantly reduces the green signal of glutathione in the cytoplasm of oocytes, while supplementation with TUDCA can significantly enhance it.

[0024] 6. The porcine oocyte culture medium supplemented with TUDCA according to the present invention can restore the mitochondrial membrane potential level in porcine oocytes decreased under heat stress. From the results of fluorescence staining detection, it can be seen that heat stress significantly reduces the red signal of mitochondrial membrane potential in oocytes, while supplementation with TUDCA can significantly enhance it. Description of the Drawings

[0025] Figure 1 Effect of TUDCA on meiosis of porcine oocytes under high-temperature stress (wherein, A is the result of morphological observation under a microscope; B is the relationship between each treatment method and the maturation rate);

[0026] Figure 2 Effect of TUDCA on the distribution of endoplasmic reticulum and Golgi apparatus in porcine oocytes under high-temperature stress (wherein, A is the fluorescence staining result of endoplasmic reticulum distribution under a microscope; B is the relationship between each treatment method and the fluorescence intensity of the red signal of endoplasmic reticulum; C is the fluorescence staining result of Golgi apparatus distribution under a microscope; D is the relationship between each treatment method and the fluorescence intensity of the red signal of Golgi apparatus);

[0027] Figure 3 Effect of TUDCA on the reactive oxygen species level in porcine oocytes under high-temperature stress (wherein, A is the fluorescence staining result of reactive oxygen species level under a microscope; B is the relationship between each treatment method and the fluorescence intensity of reactive oxygen species signal);

[0028] Figure 4 Effect of TUDCA on the glutathione level in porcine oocytes under high-temperature stress (wherein, A is the fluorescence staining result of glutathione level under a microscope; B is the relationship between each treatment method and the fluorescence intensity of glutathione signal);

[0029] Figure 5 Effect of TUDCA on the mitochondrial membrane potential level in porcine oocytes under high-temperature stress (wherein, A is the fluorescence staining result of mitochondrial membrane potential level under a microscope; B is the relationship between each treatment method and the fluorescence intensity of TMRE signal). Detailed Embodiments

[0030] The technical solution of the present invention will be further specifically described below through specific embodiments.

[0031] Formulation of porcine oocyte culture medium supplemented with TUDCA in Example 1

[0032] TCM-199 medium with 0.5 mmol / L TUDCA, 1% pen strep solution, 10 ng / mL epidermal growth factor, 5 μg / mL insulin, 0.2 mmol / L pyruvate, 0.6 mmol / L cysteine, 10% porcine follicular fluid, 10 IU / mL PMSG, 10 IU / mL hCG.

[0033] Formulation of porcine oocyte culture medium supplemented with TUDCA in Example 2

[0034] TCM-199 medium with 1 mmol / L TUDCA, 5% pen strep solution, 20 ng / mL epidermal growth factor, 9 μg / mL insulin, 0.6 mmol / L pyruvate, 0.8 mmol / L cysteine, 20% porcine follicular fluid, 20 IU / mL PMSG, 20 IU / mL hCG.

[0035] Formulation of porcine oocyte culture medium supplemented with TUDCA in Example 3

[0036] TCM-199 medium with 0.1 mmol / L TUDCA, 1% pen strep solution, 5 ng / mL epidermal growth factor, 3 μg / mL insulin, 0.1 mmol / L pyruvate, 0.2 mmol / L cysteine, 5% porcine follicular fluid, 5 IU / mL PMSG, 5 IU / mL hCG.

[0037] Formulation of porcine oocyte culture medium supplemented with TUDCA in Example 4

[0038] TCM-199 medium with 0.3 mmol / L TUDCA, 3% pen strep solution, 15 ng / mL epidermal growth factor, 7 μg / mL insulin, 0.4 mmol / L pyruvate, 0.4 mmol / L cysteine, 15% porcine follicular fluid, 15 IU / mL PMSG, 15 IU / mL hCG.

[0039] Apply the formulations of Examples 1-4 to the following preparation method

[0040] Preparation method of porcine oocyte culture medium supplemented with TUDCA in Example 5

[0041] Dissolve 0.24985 g of TUDCA powder in 10 mL of embryo water to prepare a TUDCA embryo aqueous solution with a concentration of 50 mmol / L. Take 5.0 μL of the TUDCA embryo aqueous solution and add it to 500 μL of the basal culture medium, and shake well to obtain a culture medium containing 0.5 mmol / L TUDCA.

[0042] Preparation method of porcine oocyte culture medium added with TUDCA in Example 6

[0043] Dissolve 0.49970 g of TUDCA powder in 10 mL of embryo water to prepare a TUDCA embryo aqueous solution with a concentration of 100 mmol / L. Take 7.0 μL of the TUDCA embryo aqueous solution and add it to 700 μL of the basal culture medium, and shake well to obtain a culture medium containing 1.0 mmol / L TUDCA.

[0044] Preparation method of porcine oocyte culture medium added with TUDCA in Example 7

[0045] Dissolve 0.09994 g of TUDCA powder in 10 mL of embryo water to prepare a TUDCA embryo aqueous solution with a concentration of 20 mmol / L. Take 3.0 μL of the TUDCA embryo aqueous solution and add it to 600 μL of the basal culture medium, and shake well to obtain a culture medium containing 0.1 mmol / L TUDCA.

[0046] To verify the effectiveness of the present invention, the invention team conducted a series of tests, which are specifically as follows:

[0047] 1. Materials

[0048] 1.1 Animals

[0049] After collecting porcine ovaries at the slaughterhouse, place them in a thermos flask containing sterile normal saline with 1 mL of pen strep solution and transport them back to the laboratory within 1 hour. Rinse the collected ovaries 3 times with pre-warmed normal saline to remove impurities and blood, and use scissors to remove fat blocks, connective tissues, fallopian tubes, etc. on the surface of the ovaries, and then rinse the trimmed ovaries again with normal saline. Place the treated ovaries in a beaker of normal saline containing 1 mL of pen strep solution (10,000 IU / mL) and place it on a constant temperature table at 37 °C for standby. Then use a 10 mL syringe (equipped with a 12-gauge needle) to aspirate the follicular contents on the surface of the ovaries with a diameter of 2 - 8 mm.

[0050] 1.2 Drugs and reagents

[0051] Tauroursodeoxycholic acid (TUDCA) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; the reactive oxygen species test kit was purchased from Nanjing Jiancheng Bioengineering Institute, the mitochondrial membrane potential, endoplasmic reticulum red fluorescence detection and Golgi apparatus red fluorescence detection kits were purchased from Shanghai Beyotime Biotechnology Co., Ltd., and the glutathione detection kit was purchased from Invitrogen Life Technologies Co., Ltd.

[0052] 2. Test methods

[0053] 2.1 Experimental method steps

[0054] 0.24985 g of TUDCA powder was dissolved in 10 mL of embryo water to a concentration of 50 mM. 5.0 μL of TUDCA was added to 500 μL of the basic culture medium and shaken well (final concentration 0.5 mM). After the culture medium was pre-equilibrated with culture drops in a 24-well plate, oocytes with intact and dense cumulus cell masses were placed in an incubator at 38.5 °C, 5% CO2, and 100% humidity for in vitro maturation culture.

[0055] 2.2 Heat stress treatment

[0056] Porcine oocytes at the germinal vesicle (GV) stage were cultured at 41.5 °C for 24 hours for heat stress treatment, and then cultured at 38.5 °C until the specific developmental stage for subsequent analysis.

[0057] 2.2 Analytical methods

[0058] (1) Oocyte maturation rate

[0059] After in vitro culture for about 43 h, the in vitro cultured oocytes were aspirated and added to a petri dish containing 0.1% hyaluronidase, and pipetted to detach the granulosa cells around the oocytes for about 3 min. All the digested oocytes were aspirated with a pipette, added to a pre-prepared operation liquid dish and washed 3 times. The stereomicroscope was adjusted for field of view and interpupillary distance for observation, and oocytes that had extruded the first polar body were selected (the oocytes that had extruded the first polar body were regarded as mature oocytes). The selected mature oocytes were placed in a 200 μL operation liquid drop, covered with paraffin oil, and the experiment was continued.

[0060] (2) Oocyte reactive oxygen species level

[0061] The reactive oxygen species (ROS) level in oocytes was measured using a ROS assay kit (E004-1-1, njjcbio, China), and the operation and detection were carried out strictly according to the instructions of each kit. The specific operation was as follows: For the 0.5 mM TUDCA treatment group, heat stress group (HS), and control group, 5-8 mature oocytes were selected from each group and transferred to DPBS-0.1% PVA solution for washing, and then the oocytes were transferred into a DCFH-DA staining drop (dye concentration was 10 μM) and incubated in a 37 °C dark incubator for 30 min. Then, they were washed 3 times with DPBS-0.1% PVA solution for 5 min each time, and placed under an inverted fluorescence microscope for observation and photography. The fluorescence values of the oocytes were processed using Image-J software, and the average fluorescence intensity was calculated as Mean = IntDen / Area.

[0062] (3) Detection of glutathione level in oocytes

[0063] For the 0.5 mM TUDCA treatment group, heat stress group (HS), and control group, 8-15 mature oocytes were selected from each group, transferred to DPBS-0.1% PVA solution and washed three times to remove the maturation medium. Subsequently, the oocytes were transferred into Cell Trackergreen staining solution (dye concentration 10 μM) and incubated in the dark at 37 °C for 30 minutes. After incubation, they were washed 3 times with DPBS-0.1% PVA solution for 5 minutes each time to remove the Cell Tracker green dye. The samples were placed under a confocal microscope, and the fluorescent dye was excited with a specific wavelength, and photos were taken with the same parameters set. The fluorescent images of the oocytes were processed using Image-J software, and the average fluorescence intensity was calculated (Mean = IntDen / Area).

[0064] (4) Detection of mitochondrial membrane potential in oocytes

[0065] For the 0.5 mM TUDCA treatment group, heat stress group (HS), and control group, 8-15 mature oocytes were selected from each group, transferred to DPBS-0.1% PVA solution and washed three times to remove the maturation medium. Subsequently, the oocytes were transferred into TMRE staining solution (dye concentration 10 μM) and incubated in the dark at 37 °C for 30 minutes. After incubation, they were washed 3 times with DPBS-0.1% PVA solution for 5 minutes each time to remove the TMRE dye. The samples were placed under a confocal microscope, and the fluorescent dye was excited with a specific wavelength, and photos were taken with the same parameters set. The fluorescent images of the oocytes were processed using Image-J software, and the average fluorescence intensity was calculated (Mean = IntDen / Area).

[0066] (5) Detection of Golgi apparatus distribution in oocytes

[0067] For the 0.5 mM TUDCA treatment group, heat stress group (HS), and control group, 8 - 15 mature oocytes were selected and transferred to DPBS - 0.1% PVA solution for washing three times to remove the maturation medium. Subsequently, the oocytes were transferred into Golgi - Tracker Red staining solution (added to the Golgi - Tracker Red dilution at a ratio of 1:100) and incubated at 37°C in the dark for 30 minutes. After incubation, they were washed 3 times with DPBS - 0.1% PVA solution for 5 minutes each time to remove the Golgi - Tracker Red dye. The samples were placed under a confocal microscope, and the fluorescent dye was excited with a specific wavelength, and photos were taken with the same parameters set. The fluorescent images of the oocytes were processed using Image - J software, and the average fluorescence intensity was calculated (Mean = IntDen / Area).

[0068] (6) Detection of endoplasmic reticulum distribution in oocytes

[0069] For the 0.5 mM TUDCA treatment group, heat stress group (HS), and control group, 8 - 15 mature oocytes were selected and transferred to DPBS - 0.1% PVA solution for washing three times to remove the maturation medium. Subsequently, the oocytes were transferred into ER - Tracker Red staining solution (added to the ER - Tracker Red dilution at a ratio of 1:100) and incubated at 37°C in the dark for 30 minutes. After incubation, they were washed 3 times with DPBS - 0.1% PVA solution for 5 minutes each time to remove the ER - Tracker Red dye. The samples were placed under a confocal microscope, and the fluorescent dye was excited with a specific wavelength, and photos were taken with the same parameters set. The fluorescent images of the oocytes were processed using Image - J software, and the average fluorescence intensity was calculated (Mean = IntDen / Area).

[0070] 3 Results

[0071] 3.1 TUDCA can restore meiotic failure of porcine oocytes under heat stress

[0072] The results are shown in Figure 1 . As Figure 1 shown in A, in the control group, the cumulus cells of most COCs were fully expanded, with a large and uniform expansion area. In the high - temperature stress group, the expansion ability of the cumulus cells was significantly weakened, with partial or complete non - expansion. As Figure 1As can be seen from B, the average maturation rate without addition in the control group was 66.31%; the average maturation rate in the heat stress group (HS) was 44.08%, the average maturation rate with the addition of 0.1 mM TUDCA was 30.50%; the average maturation rate with the addition of 0.3 mM TUDCA was 32.10%; the average maturation rate with the addition of 0.5 mM TUDCA was 52.07%, and the average maturation rate with the addition of 1.0 mM TUDCA was 47.09%. Compared with the heat stress group, the maturation rate increased by 7.99% with the addition of 0.5 mM TUDCA, promoting the maturation of oocytes. Compared with the heat stress group, the expansion of most cumulus cells was also significantly improved. Considering the above, 0.5 mM TUDCA treatment was selected as the experimental group for subsequent experiments to continue the research.

[0073] 3.2 Effects of TUDCA on the distribution of endoplasmic reticulum and Golgi apparatus in porcine oocytes under heat stress

[0074] The results are shown in Figure 2 . It can be seen from Figure 2 that heat stress significantly reduced the red signals in the endoplasmic reticulum and Golgi apparatus of oocytes, while supplementing TUDCA could improve this phenomenon. Compared with the control group, the fluorescence intensity of the red signal in the endoplasmic reticulum and the fluorescence intensity of the red signal in the Golgi apparatus of oocytes in the heat stress group were significantly reduced.

[0075] 3.3 Effects of TUDCA on the level of reactive oxygen species in porcine oocytes under heat stress

[0076] The results are shown in Figure 3 . It can be seen from Figure 3 that heat stress significantly increased the green signal of reactive oxygen species in the cytoplasm of oocytes, while supplementing TUDCA could alleviate this phenomenon. Compared with the control group, the fluorescence intensity of the reactive oxygen species signal in heat-stressed oocytes was significantly increased.

[0077] 3.4 Effects of TUDCA on the detection of glutathione level in porcine oocytes under heat stress

[0078] The results are shown in Figure 4 . It can be seen from Figure 4 that heat stress significantly reduced the green signal of glutathione in the cytoplasm of oocytes, while supplementing TUDCA could alleviate this phenomenon. Compared with the control group, the fluorescence intensity of the glutathione signal in heat-stressed oocytes was significantly reduced.

[0079] 3.5 Effects of TUDCA on the mitochondrial membrane potential level in porcine oocytes under heat stress

[0080] The results are shown in Figure 5 . It can be seen from Figure 5It can be seen that heat stress significantly reduces the level of mitochondrial membrane potential in the cytoplasm of oocytes, and the supplementation of TUDCA can alleviate this phenomenon. Compared with the control group, the fluorescence intensity of the red signal of mitochondrial membrane potential in heat-stressed oocytes is significantly reduced.

[0081] Although the present invention has been described in detail above with general descriptions, specific embodiments and experiments, some modifications or improvements can be made on the basis of the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

[0082] References

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[0086] [4] Gharibzadeh Z, Riasi A, Ostadhosseini S, Hosseini SM, Hajian M, Nasr-Esfahani MH. Effects of heat shock during the early stage of oocyte maturation on the meiotic progression, subsequent embryonic development and gene expression in ovine[J]. Zygote, 2015, 23(4): 573-582.

[0087] [5] Nabenishi H, Ohta H, Nishimoto T, Morita T, Ashizawa K, Tsuzuki Y. The effects of cysteine addition during in vitro maturation on the developmental competence, ROS, GSH and apoptosis level of bovine oocytes exposed to heat stress[J]. Zygote, 2012, 20(3): 249-259.

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Claims

1. A porcine oocyte culture medium supplemented with TUDCA, characterized in that, The culture medium described above includes a basal culture medium and TUDCA.

2. The porcine oocyte culture medium supplemented with TUDCA according to claim 1, wherein, The addition amount of TUDCA is 0.1 - 1 mmol / L of the basal culture medium.

3. The porcine oocyte culture medium supplemented with TUDCA according to claim 2, wherein The addition amount of TUDCA is 0.3 - 0.5 mmol / L of the basal culture medium.

4. The porcine oocyte culture medium supplemented with TUDCA according to claim 3, characterized in that, The addition amount of TUDCA is 0.5 mmol / L of the basal culture medium.

5. The porcine oocyte culture medium supplemented with TUDCA according to claim 1, wherein The basal culture medium includes TCM - 199 medium containing 1 - 5% Pen Strep solution, 5 - 20 ng / mL epidermal growth factor, 3 - 9 μg / mL insulin, 0.1 - 0.6 mmol / L pyruvate, 0.2 - 0.8 mmol / L cysteine, 5 - 20% porcine follicular fluid, 5 - 20 IU / mL PMSG, and 5 - 20 IU / mL hCG.

6. The porcine oocyte culture medium supplemented with TUDCA according to claim 5, characterized in that, The basal culture medium includes TCM - 199 medium containing 1 - 3% Pen Strep solution, 10 - 15 ng / mL epidermal growth factor, 5 - 7 μg / mL insulin, 0.2 - 0.4 mmol / L pyruvate, 0.4 - 0.6 mmol / L cysteine, 10 - 15% porcine follicular fluid, 10 - 15 IU / mL PMSG, and 10 - 15 IU / mL hCG.

7. The porcine oocyte culture medium supplemented with TUDCA according to claim 6, characterized in that, The basal culture medium includes TCM - 199 medium containing 1% Pen Strep solution, 10 ng / mL epidermal growth factor, 5 μg / mL insulin, 0.2 mmol / L pyruvate, 0.6 mmol / L cysteine, 10% porcine follicular fluid, 10 IU / mL PMSG, and 10 IU / mL hCG.

8. A method for preparing a porcine oocyte culture medium supplemented with TUDCA according to any one of claims 1-7, characterized in that, The preparation method of the culture medium is as follows: Dissolve 0.09994 - 0.49970 g of TUDCA powder in 10 mL of embryo water to prepare a TUDCA embryo aqueous solution with a concentration of 20 - 100 mmol / L. Take 3 - 7 μL of the TUDCA embryo aqueous solution and add it to 500 - 700 μL of the basal culture medium, and shake well to obtain a culture medium containing 0.1 - 1 mmol / L of TUDCA.

9. The preparation method of the porcine oocyte culture solution added with TUDCA according to claim 8, characterized in that, The preparation method of the culture medium is as follows: Dissolve 0.24985 g of TUDCA powder in 10 mL of embryo water to prepare a TUDCA embryo aqueous solution with a concentration of 50 mmol / L. Take 5.0 μL of the TUDCA embryo aqueous solution and add it to 500 μL of the basal culture medium, and shake well to obtain a culture medium containing 0.5 mmol / L of TUDCA.

10. Use of the porcine oocyte culture medium supplemented with TUDCA according to any one of claims 1 - 7 in promoting the maturation of porcine oocytes induced by heat stress.

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