Culture solution for recovering quality of porcine oocytes by adding uridine and application of culture solution

Abstract

The invention relates to a culture solution for recovering the quality of porcine oocytes by adding uridine. The culture solution comprises a basic culture solution and uridine, the basic culture solution contains 10% of pig follicular fluid, 0.57 mM of cysteine, 0.5 IU / mL of LH, 0.5 IU / mL of FSH, 10 ng / mL of epidermal growth factors, 1% of a double-antibody TCM 199 culture medium and the balance of water; the addition amount of uridine is 100 [mu] M of the basic culture solution. According to the method disclosed by the invention, metabolite uridine (Urd) required by a human body is used for respectively rescuing in-vitro cultured H2O2 induced oxidative stress porcine oocytes and delayed cultured aged porcine oocytes, so that the quality of the oocytes is improved.

Description

Technical Field

[0001] The invention relates to the technical field of reproductive biology, and in particular to a culture solution for restoring the quality of pig oocytes by adding uridine and an application thereof. Background Art

[0002] High-quality oocytes are a prerequisite for successful fertilization and subsequent embryonic development, and are the material basis for the beginning of life. In most mammals, female reproductive aging is characterized by a significant decline in the number and quality of follicles and oocytes. Studies have shown that female fertility begins to decline in the early 30s and declines faster after the age of 35, accompanied by a sharp increase in the incidence of infertility, miscarriage, embryonic lethality, and congenital birth defects. Although assisted reproductive technology can compensate for suboptimal reproductive outcomes in humans to a certain extent, the success rate of assisted reproductive technology also decreases with age. Therefore, for older women, low-quality oocytes are a common and difficult to overcome problem and a major cause of poor reproductive outcomes. Despite the importance of this issue, strategies to maintain oocyte quality with aging have not been well explored.

[0003] Oocyte maturation is a cell cycle transition process stimulated by external signals, including two levels: oocyte nuclear maturation and cytoplasmic maturation. Oocyte nuclear maturation is the process from the early stage of the first meiotic division (Metaphase I, MI) to the metaphase of the second meiotic division (Metaphase II, MII) to the discharge of the first polar body (PBE), also known as meiotic maturation. This process occurs under the induction of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) or spontaneously without follicle microenvironment stimulation, and ultimately enables oocytes to acquire the ability to recover from meiosis during in vitro culture and produce haploid eggs with fertilization function. Oocyte cytoplasmic maturation is the process of accumulating and storing maternal factors, preparing for later oocyte fertilization and early embryonic development. Most cytoplasmic maturation events are not visible under conventional low-power microscopes, and mainly occur in changes in the distribution of ultrastructural morphology such as mitochondria and cortical granules.

[0004] Uridine (Urd) is a compound belonging to the nucleoside group, which is composed of uracil and ribose (furanose) ring, which are connected by a β-N1-glycosidic bond. At the same time, Urd, as a biologically active molecule, has potential anti-aging effects. In addition, Urd also plays an important role in the synthesis of glycogen.

[0005] We cultured porcine COCs (cumulus oocyte complexes) in vitro and subjected oocytes to oxidative stress by adding a strong oxidant, H202, which affected the quality of oocytes. The quality of oocytes can be restored by adding an appropriate concentration of Urd. Based on this, we also simulated the natural aging of porcine oocytes by prolonging the in vitro culture time, thus obtaining aged oocytes, and then added a certain amount of Urd for rescue. By testing functional indicators such as the first polar body extrusion of oocytes, oocyte fragmentation, and oocyte apoptosis, it was found for the first time that the addition of Urd in vitro had a good rescue effect on oocyte quality.

[0006] Therefore, the prior art needs to be further improved. Summary of the invention

[0007] The present invention aims to solve the above problems and provides a culture medium and application thereof for restoring the quality of pig oocytes by adding uridine. Specifically, the technical solution provided by the present application is as follows:

[0008] In the first aspect, the present invention provides a culture medium for adding uridine to restore the quality of pig oocytes, including a basic culture medium and uridine; the basic culture medium contains 10% pig follicular fluid, 0.57mM cysteine, 0.5IU / mL LH, 0.5IU / mL FSH, 10ng / mL epidermal growth factor, 1% double double antibody TCM 199 culture medium, and the rest is water; the amount of uridine added is 100μM of the basic culture medium.

[0009] Furthermore, the preparation method of the culture solution is:

[0010] Select fresh ovaries with bright appearance, no corpus luteum, no obvious abnormalities in development, and many plump, translucent pink antral follicles, place them in a thermos bottle containing 3% double antibody and 37°C sterile saline, and transport them back to the laboratory within 2 hours; then wash the ovaries with pre-sterilized and preheated 3% double antibody and 37°C sterile saline until there is no obvious blood color, transfer the ovaries to new 3% double antibody and 37°C sterile saline, use a disposable 10mL syringe to extract follicular fluid from follicles with a diameter of 2-6mm to 50ml L centrifuge tube (warmed in a 38°C water bath throughout the process), naturally settle in a 38°C water bath for 10 minutes, use a 20mL syringe to extract the slightly yellowish translucent supernatant liquid, i.e., the porcine follicular fluid, and transfer the porcine follicular fluid to a 50mL centrifuge tube, and centrifuge it in a high-speed centrifuge (5000rpm, 30min). After centrifugation, avoid the precipitate containing granulosa cells at the bottom of the centrifuge tube, take the supernatant porcine follicular fluid, filter the porcine follicular fluid after centrifugation with a 0.22μm filter membrane, divide it into portions, and store it at -20°C.

[0011] Based on the above technical scheme, the method for preparing porcine follicular fluid is as follows: select healthy follicles of 2 to 6 mm, extract follicular fluid, centrifuge at 5000 rpm for 30 min, collect the supernatant, divide it into 1.5 mL centrifuge tubes at 1 mL / tube, and freeze it at -20°C for later use.

[0012] In the second aspect, based on the same invention, the present invention also provides an application of a culture medium with uridine added to restore the quality of pig oocytes, and the application method of the culture medium is as follows:

[0013] S1: In vitro separation of porcine COCs: Select fresh ovaries with bright appearance, no corpus luteum, no obvious abnormalities in development, and many plump, translucent pink antral follicles, and place them in a thermos bottle containing 3% double antibody and 37°C sterile saline. Wash the ovaries with pre-sterilized and preheated 3% double antibody and 37°C sterile saline until there is no obvious blood color. Transfer the ovaries to new 3% double antibody and 37°C sterile saline. Use a disposable 10mL syringe to extract follicular fluid from follicles with a diameter of 2-6mm into a 550mL centrifuge tube, and let it settle naturally in a 38°C water bath for 10 minutes. Discard the supernatant, wash the cell pellet with TL-HEPES operating solution, and then let it settle in a warm bath for 10 minutes. Repeat twice. Then, use an egg picking needle under a stereo microscope to select COCs with uniform cytoplasm and 3-5 layers of granulosa cells;

[0014] S2: In vitro culture of porcine COCs: artificially prepare simulated aging porcine COCs, transfer the porcine COCs into the culture medium as described in claim 1, and continue to culture until the MII stage.

[0015] Furthermore, in S2, there are two preparation method models for artificially preparing simulated aging pig COCs: a H2O2 oxidative stress model and an in vitro delayed culture aging model.

[0016] Specifically, the specific method steps of the H2O2 oxidative stress model are: wash the selected COCs three times in TL-HEPES operating solution, wash them once with basal culture medium, and then randomly select 40 to 50 COCs / group and transfer them to a 4-well plate, wherein the 4-well plate already contains one well of basal culture medium that has been equilibrated for 2-4 hours in advance and two wells of H2O2 group culture medium, 500 μL culture medium in each well, and culture them in a 38.5°C, 5% CO2, fully saturated humidity incubator for 30 minutes, so as to construct an in vitro porcine oocyte oxidative stress model.

[0017] Specifically, the specific method steps of the in vitro delayed culture aging model are: wash the selected COCs three times in TL-HEPES operating solution, wash once with basic culture medium, randomly select 40 to 50 COCs / group and transfer them to a 4-well plate, wherein the 4-well plate already contains basic culture medium that has been equilibrated for 2-4 hours in advance, 500 μL culture medium per well, and culture for 44 to 46 hours, so as to construct an in vitro delayed culture aging model.

[0018] Furthermore, the preparation method of the H2O2 group culture medium is as follows: first, 68 μL of 3% H2O2 is taken and made into a 30 mM concentrated stock using a basal culture medium. According to the volume requirement, the basal culture medium is used to dilute the working solution with a working concentration of 100 μM. For example, if a volume of 3 mL is required, 10 μL of the 30 mM concentrated stock is diluted with 3 mL of the Fresh group culture medium.

[0019] On the basis of the above technical scheme, the preparation method of the TL-HEPES operating solution is as follows: accurately weigh CaCl2·2H2O, 0.15g; KCl, 0.118g; MgCl2·6H2O, 0.05g; NaCl, 3.33g; NaHCO3, 0.084g; KH2PO4, 0.0232g; HEPES, 1.19g; sodium lactate, 428μL / 0.56g; sodium pyruvate, 0.011g; polyvinyl alcohol, 0.5g; phenol red, 0.005g; adjust the pH to 7.2-7.4 with NaOH, add 1% double antibody, make up to 500mL with ultrapure water, filter with 0.22μm filter membrane, and dispense into 50mL centrifuge tubes, and store at 4°C for later use.

[0020] The present invention has the following advantages / benefits:

[0021] The purpose of the present invention is to use metabolites required by the human body to save the quality of eggs. The method of the present invention uses uridine (Urd), a metabolite required by the human body, to save pig oocytes induced by oxidative stress in vitro cultured with H2O2 and pig oocytes aged by delayed culture, so as to improve the quality of oocytes.

[0022] Experimental data show that the aged pig oocytes obtained by the two artificial simulation methods can be rescued by the culture medium provided by the present invention with added uridine to restore the quality of pig oocytes, and the indicators such as oocyte maturation rate, oocyte fragmentation rate and oocyte apoptosis are all rescued. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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 prior art descriptions are briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0024] Figure 1 : Statistical graph of the first polar body extrusion rate of oocytes in different groups in the H2O2 oxidative stress model in Example 3;

[0025] Figure 2 : Statistical graph of the fragmentation rate of oocytes in different groups in the in vitro delayed culture aging model in Example 3;

[0026] Figure 3 : Statistical graph of the first polar body extrusion rate of oocytes in different groups in the H2O2 oxidative stress model in Example 4;

[0027] Figure 4 : Fragmentation diagrams of oocytes in different groups in the in vitro delayed culture aging model in Example 5;

[0028] Figure 5 : Statistical graph of the fragmentation rate of oocytes in different groups in the in vitro delayed culture aging model in Example 5;

[0029] Figure 6 : Typical images of apoptosis staining of oocytes in different groups in the in vitro delayed culture aging model in Example 6;

[0030] Figure 7 : Statistical graph of apoptosis rates of oocytes in different groups in the in vitro delayed culture aging model in Example 6. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following embodiments are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. The experimental methods used in the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the present invention, unless otherwise specified, can be obtained from commercial channels. In addition, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0032] Example 1: Preparation of relevant culture medium

[0033] (1) Porcine follicular fluid (pFF): Select healthy follicles of 2 to 6 mm, extract the follicular fluid, centrifuge at 5000 rpm for 30 min, collect the supernatant, divide into 1.5 mL centrifuge tubes at 1 mL / tube, and freeze at -20°C for later use. Avoid repeated freezing and thawing during use. To ensure the stability of the experiment, collect pFF multiple times, mix well, and divide according to the above method for later use.

[0034] The specific preparation method is as follows:

[0035] Fresh ovaries with bright appearance, no corpus luteum, no obvious abnormalities in development, and many plump, translucent pink ovarian follicles were selected from Qingdao Laixi Wanfu Meat Processing Factory, placed in a thermos bottle containing 3% double antibody and 37°C sterile saline, and transported to the laboratory within 2 hours. Then, the ovaries were washed with pre-sterilized and preheated 3% double antibody and 37°C sterile saline until there was no obvious blood color, and the ovaries were transferred to new 3% double antibody and 37°C sterile saline, and the follicular fluid of follicles with a diameter of 2 to 6 mm was extracted with a disposable 10 mL syringe into a 50 mL centrifuge tube (warmed in a 38°C water bath throughout the process), and naturally settled in a 38°C water bath for 10 min. The yellowish translucent supernatant liquid, i.e., porcine follicular fluid, was extracted with a 20 mL syringe, and the porcine follicular fluid was transferred to a 50 mL centrifuge tube and centrifuged in a high-speed centrifuge (5000 rpm, 30 min). After centrifugation, the precipitate containing granulosa cells at the bottom of the centrifuge tube was avoided, and the supernatant porcine follicular fluid was taken, and the porcine follicular fluid after centrifugation was filtered with a 0.22 μm filter membrane and packaged, and stored at -20°C.

[0036] (2) Prepare the raw materials: Uridine powder (Sigma, U3003) tightly sealed and stored in a dry container, 3% H2O2 (Sigma), and sterile water.

[0037] (3) Prepare the fresh culture medium (the fresh culture medium is the basal culture medium, which will be used in the following for the convenience of describing the examples): containing 10% pFF, 0.57 mM cysteine ​​(Sigma), 0.5 IU / mL LH (Biyuntian), 0.5 IU / mL FSH (Biyuntian), 10 ng / mL epidermal growth factor (Epidermal growth factor, EGF, Biyuntian), and 1% double-antibody TCM199 culture medium (Sigma), and filter it with a 0.22 μm filter membrane before use.

[0038] The specific preparation method is as follows:

[0039] Weigh 0.007 g of cysteine ​​crystals, dissolve in 5 mL of TCM199 medium, take 500 μL, add to 8.5 mL of TCM199 medium, add 1 mL of porcine follicular fluid, 5 μL of LH concentrated stock solution, 10 ng / mL epidermal growth factor, 10 μL of FSH concentrated stock solution, 100 μL of double antibody, and filter with a 0.22 μm filter membrane.

[0040] Among them, concentrated stock solution refers to concentrated reserve solution, which is as follows:

[0041] Each bottle of LH powder contains 200 IU, which can be diluted with 200 μL TCM199 culture medium and stored at -20°C to obtain LH concentrated stock solution. Each bottle of FSH powder contains 200 IU, which can be diluted with 200 μL TCM199 culture medium and stored at -20°C to obtain FSH concentrated stock solution.

[0042] (4) Prepare H2O2 medium: First, take 68 μL of 3% H2O2 and use basal medium to make a 30 mM stock solution. According to the volume required, use the Fresh medium to dilute it to a working solution with a working concentration of 100 μM. For example, if a volume of 3 mL is required, 10 μL of the 30 mM stock solution can be diluted with 3 mL of the Fresh medium. (This medium is not required for the in vitro delayed culture aging model)

[0043] (5) Preparation of Urd group culture medium: Weigh 0.1 g of uridine powder and use 13.6 mL of sterile water to prepare a 30 mM concentrated stock. As above, finally, according to the volume required, just dilute the concentrated stock with Fresh group culture medium to a 100 μM working solution.

[0044] (6) TL-HEPES operating solution: accurately weigh CaCl2·2H2O (Sigma), 0.15 g; KCl, 0.118 g; MgCl2·6H2O (Sigma), 0.05 g; NaCl, 3.33 g; NaHCO3, 0.084 g; KH2PO4, 0.0232 g; HEPES, 1.19 g; sodium lactate (Na Lactic acid), 428 μL / 0.56 g; sodium pyruvate (Na-Pyruvate), 0.011 g; polyvinyl alcohol (PVA), 0.5 g; phenol red (Phenol red), 0.005 g; adjust the pH to 7.2-7.4 with NaOH, add 1% double antibody, make up to 500 mL with ultrapure water, filter with 0.22 μm filter membrane, dispense into 50 mL centrifuge tubes, and store at 4°C for later use.

[0045] (7) Preparation and collection of MII oocyte culture medium (MM culture medium): TCMl99 powder (Invitrogen), 1 bag, accurately weigh NaHCO3, 0.05 g; HEPES, 0.75 g; NaCl, 1.855 g; PVA, 1.0 g, dilute to 1 L with ultrapure water, filter through 0.22 μm filter membrane, divide into portions and store at 4°C until use, add 1% double antibody when using.

[0046] Example 2: In vitro separation of porcine COCs

[0047] Fresh ovaries with bright appearance, no corpus luteum, no obvious abnormalities in development, and many, plump, and translucent pink antral follicles were selected from Qingdao Laixi Wanfu Meat Processing Plant, placed in a thermos bottle containing 3% double antibody and 37°C sterile saline, and transported back to the laboratory within 2 hours. Then, the ovaries were washed with pre-sterilized and preheated 3% double antibody and 37°C sterile saline until there was no obvious blood color, and the ovaries were transferred to new 3% double antibody and 37°C sterile saline. A disposable 10mL syringe was used to extract follicular fluid from follicles with a diameter of 2 to 6mm into a 50mL centrifuge tube (warmed in a 38°C water bath throughout the process), and the follicles were naturally precipitated in a 38°C water bath for 10 minutes, the supernatant was discarded, and the cell pellet was washed with TL-HEPES operating solution, and then warmed and precipitated for 10 minutes. This was repeated twice, and then COC with uniform cytoplasm and 3 to 5 layers of granulosa cells were selected with an egg picking needle under a stereomicroscope. s .

[0048] Example 3: In vitro culture of porcine COCs

[0049] 3.1 Model 1: H2O2 oxidative stress model:

[0050] The selected COCs were washed three times in TL-HEPES operating solution, and then washed once with Fresh group culture medium. Then, 40-50 COCs / group were randomly selected and transferred to a 4-well plate, in which one well of Fresh group culture medium and two wells of H2O2 group culture medium were equilibrated for 2-4 hours in advance. Each well was filled with 500 μL culture medium and cultured in a 38.5°C, 5% CO2, fully saturated humidity incubator for 30 minutes to construct an in vitro porcine oocyte oxidative stress model.

[0051] Then the COCs in the Fresh group were transferred to the new Fresh group culture medium, recorded as the Fresh group, and the COCs in the H2O2 group were transferred to the Fresh group culture medium without or with Urd, recorded as the H2O2 group and the Urd group, and cultured for 44 to 46 hours until the MII stage. It is worth noting that the three groups of cells need to be transferred synchronously to exclude the influence of mechanical damage on oocyte maturation during the transfer process.

[0052] 3.2 Model 2: In vitro delayed culture aging model:

[0053] The selected COCs were washed three times in TL-HEPES operating solution, and then washed once in the fresh group culture medium. 40-50 COCs / group were randomly selected and transferred to a 4-well plate, in which the 4-well plate contained the fresh group culture medium that had been equilibrated for 2-4 hours in advance, with 500 μL culture medium in each well. After culturing for 44-46 hours, it was recorded as the fresh group, and the relevant indicators of the fresh group could be detected.

[0054] At the same time, the COCs in the remaining wells were transferred to the culture medium of the Fresh group without or with Urd using glass tubes, recorded as the Aged group and the Urd group, and cultured in a 38.5°C, 5% CO2, fully saturated humidity incubator for 48 hours, and relevant indicators were detected.

[0055] 3.3 Uridine concentration screening

[0056] 3.3.1 Model 1: H2O2 oxidative stress model

[0057] Compared with the control group and the H2O2 treatment group, three concentrations of uridine rescue group were set to screen the optimal concentration. We added 100μM, 150μM and 200μM uridine to the basal culture medium and calculated the extrusion rate of the first polar body of oocytes. The experimental results are shown in Figure 2. Figure 1 The experiment showed that 100 μM uridine in the basal medium had the best rescue effect on senescence caused by H2O2, and 100 μM uridine was used in subsequent experiments.

[0058] 3.3.2 Model 2: In vitro delayed culture aging model

[0059] Compared with the control group and the aging group, three concentrations of uridine rescue group were set to screen the optimal concentration. We added 50μM, 100μM and 200μM uridine to the basal culture medium and calculated the oocyte fragmentation rate. The experimental results are as follows Figure 2 The experiment showed that 100 μM uridine in the basal culture medium had the best rescue effect on senescence caused by delayed culture in vitro.

[0060] Example 4: Oocyte maturation rate statistics

[0061] In the oxidative stress model, the COCs of the Fresh group, H2O2 group, and Urd group were transferred to preheated 1% hyaluronidase and pipetted 50 to 70 times with a 200 μL pipette to remove the cumulus cells and obtain naked eggs. The digested oocytes were transferred to the preheated MM culture medium using a mouth pipette to terminate the digestion of hyaluronidase. Then the oocytes were transferred to the new preheated MM culture medium as quickly as possible to further reduce the damage of the remaining hyaluronidase to the oocytes. The oocytes were rolled with an egg picking needle under a stereoscope to observe and count the number of PBE discharged to calculate the oocyte maturation rate (see Figure 3 ).

[0062] We found that compared with the Fresh group, the oocyte maturation rate in the H2O2 group was reduced, which was restored after the addition of Urd.

[0063] Example 5: Statistics of oocyte fragmentation rate

[0064] In the delayed culture aging model, excellent fresh oocytes present a spherical structure with uniform and translucent cytoplasm surrounded by a uniform zona pellucida, while aged in vitro cultured oocytes often have loose and fragmented cytoplasm. We transferred COCs from the Fresh, Aged, and Urd groups into preheated 1% hyaluronidase and pipetted them 50 to 70 times with a 200 μL pipette to remove the cumulus cells and obtain naked eggs. Transfer them to 1× PBS, roll the oocytes with an egg picking needle under a stereoscope, observe and count the number of fragmented oocytes, and use this to calculate the oocyte fragmentation rate (see Figure 4 , 5 ).

[0065] We found that the fragmentation rate in the Aged group was increased compared with the Fresh group, and was reduced after the addition of Urd rescue.

[0066] Example 6: Detection of oocyte apoptosis using Annexin V-FITC apoptosis detection kit

[0067] The COCs of the Fresh, Aged, and Urd groups were transferred to preheated 1% hyaluronidase, and pipetted 50 to 70 times with a 200 μL pipette to remove the cumulus cells and obtain naked eggs. Then, the naked eggs were transferred to preheated 0.1% PVA-PBS with a mouth pipette, washed 3 times, and then transferred to 100 μL Annexin V-FITC binding solution, and 3 μL Annexin V-FITC was added. The mixture was gently mixed and incubated at room temperature (20-25°C) in the dark for 30 minutes. The cells can be resuspended 2-3 times during the incubation process to improve the staining effect. After the incubation, the fluorescence microscope was used to take pictures immediately, and the apoptosis rate of oocytes in each group was calculated (see Figure 6 , 7 ).

[0068] We found that compared with the Fresh group, the apoptosis rate in the Aged group was increased, which was reduced after the addition of Urd.

[0069] The present invention successfully utilizes the form of adding Urd in vitro to restore the phenomenon of decreased quality of pig oocytes caused by H2O2 treatment and in vitro delayed culture.

[0070] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A culture medium for restoring the quality of porcine oocytes by adding uridine, characterized in that: It includes basic culture fluid and uridine; the basic culture fluid contains 10% porcine follicular fluid, 0.57mM cysteine, 0.5IU / mL LH, 0.5IU / mL FSH, 10ng / mL epidermal growth factor, 1% double antibody TCM 199 culture medium, and the rest is water; the amount of uridine added is 100μM of the basic culture fluid.

2. A culture medium for restoring the quality of pig oocytes by adding uridine according to claim 1, characterized in that: The preparation method of the culture solution is: Select fresh ovaries with bright appearance, no corpus luteum, no obvious abnormalities in development, and many plump, translucent pink antral follicles, place them in a thermos bottle containing 3% double antibody and 37°C sterile saline, and transport them back to the laboratory within 2 hours; Then, the ovaries were washed with pre-sterilized and preheated 3% double antibody and 37°C sterile saline until there was no obvious blood color, and the ovaries were transferred to new 3% double antibody and 37°C sterile saline, and the follicular fluid of follicles with a diameter of 2 to 6 mm was extracted with a disposable 10 mL syringe into a 50 mL centrifuge tube (warmed in a 38°C water bath throughout the process), and naturally settled in a 38°C water bath for 10 min. The yellowish translucent supernatant liquid, i.e., porcine follicular fluid, was extracted with a 20 mL syringe, and the porcine follicular fluid was transferred to a 50 mL centrifuge tube and centrifuged in a high-speed centrifuge (5000 rpm, 30 min). After centrifugation, the precipitate containing granulosa cells at the bottom of the centrifuge tube was avoided, and the supernatant porcine follicular fluid was taken, and the porcine follicular fluid after centrifugation was filtered with a 0.22 μm filter membrane and packaged, and stored at -20°C.

3. The culture medium for restoring the quality of pig oocytes by adding uridine according to claim 1, characterized in that: The method for preparing the porcine follicular fluid is as follows: selecting healthy follicles of 2 to 6 mm, extracting the follicular fluid, centrifuging at 5000 rpm for 30 min, collecting the supernatant, and dispensing 1 mL / tube into 1.5 mL centrifuge tubes, freezing them at -20°C for later use.

4. An application of a culture medium for restoring the quality of pig oocytes by adding uridine, characterized in that: The application mode of the culture solution is as follows: S1: In vitro separation of porcine COCs: Select fresh ovaries with bright appearance, no corpus luteum, no obvious abnormalities in development, and many plump, translucent pink antral follicles, and place them in a thermos bottle containing 3% double antibody and 37°C sterile saline. Wash the ovaries with pre-sterilized and preheated 3% double antibody and 37°C sterile saline until there is no obvious blood color. Transfer the ovaries to new 3% double antibody and 37°C sterile saline. Use a disposable 10mL syringe to extract follicular fluid from follicles with a diameter of 2-6mm into a 50mL centrifuge tube, and let it settle naturally in a 38°C water bath for 10 minutes. Discard the supernatant, wash the cell pellet with TL-HEPES operating solution, and then let it settle in a warm bath for 10 minutes. Repeat twice. Then, use an egg picking needle under a stereo microscope to select COCs with uniform cytoplasm and 3-5 layers of granulosa cells; S2: In vitro culture of porcine COCs: artificially prepare simulated aging porcine COCs, transfer the porcine COCs into the culture medium as described in claim 1, and continue to culture until the MII stage.

5. The use of a culture medium for restoring the quality of pig oocytes by adding uridine according to claim 4, characterized in that: In S2, there are two preparation methods for artificially preparing simulated aging pig COCs: a H2O2 oxidative stress model and an in vitro delayed culture aging model.

6. The use of a culture medium for restoring the quality of pig oocytes by adding uridine according to claim 5, characterized in that: The specific method and steps of the H2O2 oxidative stress model are as follows: the selected COCs are washed three times in TL-HEPES operating solution, washed once with basal culture medium, and then 40 to 50 COCs / group are randomly selected and transferred to a 4-well plate, wherein the 4-well plate contains a basal culture medium in one well that has been equilibrated for 2-4 hours in advance and H2O2 group culture medium in two wells, 500 μL culture medium in each well, and cultured in a 38.5°C, 5% CO2, fully saturated humidity incubator for 30 minutes, thereby constructing an in vitro porcine oocyte oxidative stress model.

7. The use of a culture medium for restoring the quality of pig oocytes by adding uridine according to claim 5, characterized in that: The specific method and steps of the in vitro delayed culture aging model are as follows: the selected COCs are washed three times in TL-HEPES operating solution, washed once with basic culture medium, 40 to 50 COCs are randomly selected per group and transferred to a 4-well plate, wherein the 4-well plate contains a basic culture medium that has been equilibrated for 2 to 4 hours in advance, 500 μL of culture medium per well, and cultured for 44 to 46 hours, thereby constructing an in vitro delayed culture aging model.

8. The use of a culture medium for restoring the quality of pig oocytes by adding uridine according to claim 6, characterized in that: The preparation method of the H2O2 group culture medium is as follows: first, 68 μL of 3% H2O2 is drawn and made into a 30 mM concentrated stock using a basic culture medium. According to the volume requirement, the basic culture medium is used to dilute the working solution into a working concentration of 100 μM.

9. The use of a culture medium for restoring the quality of pig oocytes by adding uridine according to any one of claims 6 or 7, characterized in that: The preparation method of the TL-HEPES operating solution is as follows: accurately weigh CaCl2·2H2O, 0.15g; KCl, 0.118g; MgCl2·6H2O, 0.05g; NaCl, 3.33g; NaHCO3, 0.084g; KH2PO4, 0.0232g; HEPES, 1.19g; sodium lactate, 428μL / 0.56g; sodium pyruvate, 0.011g; Polyvinyl alcohol, 0.5 g; phenol red, 0.005 g; adjust the pH to 7.2-7.4 with NaOH, add 1% double antibody, make up to 500 mL with ultrapure water, filter with 0.22 μm filter membrane, dispense into 50 mL centrifuge tubes, and store at 4°C for later use.

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