In-vitro maturation culture medium and in-vitro maturation culture method of porcine oocytes

Abstract

The invention provides an in-vitro maturation culture medium of porcine oocytes and an in-vitro maturation culture method of the porcine oocytes. The cell culture medium comprises the following components: fibroblast growth factors, glucose, cysteine, sodium pyruvate, epidermal growth factors, polyvinyl alcohol, pregnant horse serum gonadotropin, human chorionic gonadotropin, follicle-stimulating hormone, recombinant porcine interleukin-6, keratinocyte growth factors and the balance of a TCM199 culture medium. The in-vitro maturation culture method of the porcine oocytes comprises the step of adding the porcine oocytes into the in-vitro maturation culture medium for culture. The in-vitro maturation culture medium is stable and controllable in component, the maturation rate and quality of oocytes are remarkably improved, the storage time of the oocytes is prolonged, and the in-vitro maturation culture medium is an efficient and standardized culture medium.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an in vitro maturation culture medium and an in vitro maturation culture method for pig oocytes. Background Art

[0002] Mammalian oocytes provide most of the maternal genetic material for embryonic development, such as cytoplasmic components, organelles, and cell membranes. Oocytes must undergo chromosome division and cytoplasmic division to reach maturity. The maturation process of oocytes is a prerequisite and necessary physiological event that determines the success of fertilization, the formation of fertilized eggs, and the normal development of embryos. With the application and development of in vitro fertilization, nuclear transplantation, and transgenic animal production technology, the demand for mature oocytes is increasing. Due to the small number of mature oocytes in vivo and the limited access to them. Therefore, the technology of in vitro culture of mature oocytes has developed rapidly. However, the quality of mature oocytes cultured in vitro and the subsequent developmental capacity of embryos are far inferior to those of mature oocytes in vivo. The in vitro culture medium for porcine oocytes may lack certain essential nutrients, growth factors, or hormones, resulting in a maturation rate that is usually less than 80%. Therefore, there is an urgent need to optimize the formulation of the in vitro culture medium for porcine oocytes.

[0003] Porcine follicular fluid (PFF) is the fluid inside the follicle, which provides the necessary nutrients and environment for the maturation and development of the oocyte. Specifically, it has the following functions: (1) Providing nutrient supply: The follicular fluid contains a variety of nutrients, such as amino acids, vitamins, minerals and metabolic intermediates, which can directly provide nutrients to the oocyte; (2) Promoting oocyte maturation: The hormones (such as estradiol, progesterone, gonadotropin, etc.) and growth factors contained in the follicular fluid are essential for the maturation of the oocyte; (3) Playing the role of intercellular communication: The cytokines and signaling molecules in the follicular fluid can simulate the communication between the oocyte and the granulosa cell, which is very important for the maturation of the oocyte and the subsequent embryonic development; (4) Regulating pH and osmotic pressure: The follicular fluid can help regulate the pH of the culture medium. and osmotic pressure, making it closer to the in vivo environment, which is beneficial to the survival and function of oocytes; (5) Providing antioxidant protection: The follicular fluid contains antioxidants that can help oocytes resist oxidative stress and protect them from damage by free radicals; (6) Promoting metabolic activity: The metabolites in the follicular fluid can promote the metabolic activity of oocytes and maintain their normal physiology; (7) Improving the culture environment: Adding follicular fluid can improve the composition of the culture medium, making it closer to the growth environment of oocytes in vivo, thereby improving the maturation rate of oocytes and the potential for embryonic development.

[0004] However, there are many problems with the use of follicular fluid in the in vitro culture of porcine oocytes: (1) Batch-to-batch differences: The composition of follicular fluid varies from season to season and from individual to individual, resulting in poor reproducibility and unstable experimental results of cultured oocytes; (2) Contamination risk: Follicular fluid contains bacteria, viruses or other harmful substances and needs to be strictly processed before it can be used for in vitro culture of oocytes; (3) Complex composition: The composition of follicular fluid is very complex, and the specific mechanism of action of some of the components is still unclear, which poses a challenge to the standardization of the culture medium. At the same time, serum in oocyte culture medium will also limit the standardized production of culture medium due to batch-to-batch differences, viral and bacterial contamination, ethical and cost issues, and unclear composition.

[0005] Therefore, in order to overcome the above-mentioned problems existing in the existing culture medium, there is an urgent need to develop a new type of oocyte in vitro maturation culture medium with high efficiency and standardized formulation to provide a more stable and controllable in vitro culture environment for porcine oocytes that is conducive to improving the maturation rate.

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

[0007] In view of the problems of low oocyte maturation rate, poor quality and unstable experimental repetition in the existing in vitro maturation culture technology of porcine oocytes, the present invention provides an in vitro maturation culture medium and an in vitro maturation culture method for porcine oocytes. The components of the in vitro maturation culture medium are stable and controllable, and the oocyte maturation rate is significantly improved. The in vitro maturation culture medium is a new type of oocyte in vitro maturation culture medium with high efficiency and standardized preparation.

[0008] To solve the above problems, this application provides the following technical solutions: In a first aspect, the present application provides an in vitro maturation medium for porcine oocytes, comprising the following components: Fibroblast growth factor, glucose, cysteine, sodium pyruvate, epidermal growth factor, polyvinyl alcohol, pregnant mare serum gonadotropin, human chorionic gonadotropin, follicle-stimulating hormone, recombinant porcine interleukin-6, keratinocyte growth factor, and the remainder is TCM199 culture medium.

[0009] Optionally, the in vitro maturation culture medium for porcine oocytes further comprises: double antibodies, which are penicillin and streptomycin.

[0010] Experiments have found that the three ingredients, Fibroblast Growth Factor (FGF), recombinant porcine interleukin-6 (IL-6) and keratinocyte growth factor (KGF-2), work together to promote the differentiation of porcine skin stem cells. On this basis, it was further found that the above three ingredients can effectively replace the essential follicular fluid (PFF) and serum in the existing formula, and form a new type of oocyte in vitro maturation medium with high efficiency and standardized formulation by combining with other ingredients in the above culture medium. Therefore, the composition of the culture medium is stable and the quality is controllable, effectively avoiding the batch differences, contamination risks and difficulty in standardization caused by follicular fluid (PFF) and serum.

[0011] Optionally, the in vitro maturation medium for porcine oocytes comprises the following components: 0.5~10 ng / ml fibroblast growth factor, 1~10 mM glucose, 0.1~1 mM cysteine, 0.1~3 mM sodium pyruvate, 1~10 ng / mL epidermal growth factor, 5~15% polyvinyl alcohol, 50~150 IU / mL pregnant mare serum gonadotropin, 50~150 IU / mL human chorionic gonadotropin, 200~300 IU / mL follicle-stimulating hormone, 1~25 ng / ml recombinant porcine interleukin-6, 0.5~5 ng / ml keratinocyte growth factor and 0.1~5% double antibody, and the remainder is TCM199 medium.

[0012] Preferably, the in vitro maturation medium for porcine oocytes comprises the following components at the following concentrations: 0.5 ng / ml fibroblast growth factor, 3.05 mM glucose, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 2.5 ng / mL epidermal growth factor, 10% polyvinyl alcohol, 100 IU / mL pregnant mare serum gonadotropin, 100 IU / mL human chorionic gonadotropin, 250 IU / mL follicle-stimulating hormone and 1% double antibody, 2.5 ng / ml recombinant porcine interleukin-6, 2.5 ng / ml keratinocyte growth factor, and the remainder is TCM199 medium.

[0013] Experiments have shown that the above-mentioned in vitro maturation medium for porcine oocytes has the highest maturation rate and the most optimized scheme. The maturation rate of porcine oocytes cultured in this medium is the highest, reaching more than 80%, and the oocytes are of good quality, with a high blastocyst rate and good stability. They can be stored at 2-8°C for one month without inactivation.

[0014] In a second aspect, the present application also provides an in vitro maturation culture method for porcine oocytes. The in vitro maturation culture method comprises: adding porcine oocytes to the aforementioned in vitro maturation culture medium for culture.

[0015] Optionally, the in vitro maturation culture method of porcine oocytes comprises the following steps: S1. Add the aforementioned porcine oocyte in vitro maturation medium into the well plate, seal it with mineral oil, and place it in a 38.5°C, 5% CO2, fully saturated humidity incubator for 2-4 h. S2. In vitro separation of porcine oocyte-follicle complexes (COCs): extract follicular fluid from fresh follicles with a diameter of 2-6 mm into a centrifuge tube, and naturally settle in a 37°C water bath for 10 min. After discarding the supernatant, wash the cell pellet with TL-HEPES operating solution, and then settle in a warm bath for 10 min. Repeat at least 3 times, and then select porcine oocyte-follicle complexes (COCs) with uniform cytoplasm and 3-5 layers of granulosa cells under a microscope; S3. In vitro culture of porcine oocyte-follicle complexes: The porcine oocyte-follicle complexes selected in the previous step were washed with TL-HEPES, and then 40-50 porcine oocyte-follicle complexes from each group were randomly selected and transferred to the well plate balanced in S1, and cultured for 44 h to the MII stage to obtain mature porcine oocytes.

[0016] The culture conditions were as follows: 38.5°C, 5% CO2 incubator for 44 h to the MII stage.

[0017] Optionally, the in vitro maturation culture method of pig oocytes further comprises: S4, determination of the cumulus diffusion rate, maturation rate, cleavage rate and blastocyst rate of the oocytes.

[0018] Furthermore, step S4 is: photographing the oocytes 44 hours after culture and maturation under a microscope and calculating the cumulus diffusion rate; digesting the mature oocytes with 0.1% hyaluronidase, selecting naked eggs, observing and counting the number of polar bodies discharged under a stereoscope to calculate the maturation rate; and calculating the cleavage rate and blastocyst rate after parthenogenetic activation of the mature oocytes.

[0019] The present invention has the following beneficial effects: The invention provides an in vitro maturation culture medium for porcine oocytes and an in vitro maturation culture method. The components of the in vitro maturation culture medium are stable and controllable, and the maturation rate of oocytes is significantly improved. The maturation rate can be as high as 80% or more, and the oocytes have good quality and a high blastocyst rate. The oocytes can be stored at 2-8°C for one month. The in vitro maturation culture medium is an efficient and standardized culture medium with a high repetition rate, stable and controllable results, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the statistical graph of oocyte maturation rate of experimental groups 1-12 and comparative example 1; Figure 2 The COCs cumulus diffusion rate and statistical graph of Example 3; Figure 3 The oocyte maturation rate and statistical graph of Example 3; Figure 4 The cleavage diagram and cleavage rate statistical diagram of the oocyte after parthenogenetic activation of Example 3; Figure 5 The blastocyst diagram and blastocyst rate statistical diagram of the oocyte parthenogenetic activation in Example 3; Figure 6 This is the blastocyst nuclear staining and blastocyst nuclear statistics diagram after the oocyte parthenogenetic activation in Example 3. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the 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. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0022] Preparation of test reagents and materials of the present invention: 1. Culture medium (1) Porcine follicular fluid (PFF) Select healthy porcine follicles of 2-6 mm with bright appearance, no corpus luteum, no obvious developmental abnormalities, and many plump and translucent pink antral follicles. Extract follicular fluid and centrifuge at 1,600 × g for 30 min at 4°C. Collect the supernatant in a 1.5 mL centrifuge tube and store at -20°C for later use.

[0023] (2) Control group oocyte in vitro maturation medium 10% porcine follicular fluid, 0.57 mM cysteine ​​(Sigma), 10% serum (Gibco), 0.91 mM sodium pyruvate (Sigma), 3.05 mM glucose (Sigma), 10% polyvinyl alcohol (Sigma), 10 ng / mL epidermal growth factor (Peprotech), 10 IU / mL pregnant mare serum gonadotropin (Ningbo Estrogen Factory), 10 IU / mL human chorionic gonadotropin (Ningbo Estrogen Factory), 25 IU / mL follicle-stimulating hormone (Sigma) and 1% double antibody (penicillin with a working concentration of 100 U / ml and streptomycin with a working concentration of 0.1 mg / ml, Bio-Time) were added to TCM199 (Gibco) and filtered through a 0.22 μm filter membrane before use.

[0024] (3) PZM-3 embryo culture medium composition table:

[0025] 2. Other reagents (1) TL-HEPES operating solution: CaCl2·2H2O (Sigma), 0.15 g, KCl (Sigma), 0.118 g, MgCl2·6H2O (Sigma), 0.05 g, NaCl (Sigma), 3.33 g, NaHCO3 (Sigma), 0.084 g, KH2PO4 (Sigma), 0.0232 g, HEPES (Sigma), 1.19 g, sodium lactate (Sigma), 0.56 g, sodium pyruvate (Sigma), 0.011 g, polyvinyl alcohol (Sigma), 0.5 g, phenol red (Sigma), 0.005 g, NaOH (Sigma), adjust the pH to 7.2-7.4, add 1% double antibody, dilute to 500 mL with ultrapure water, filter with 0.22 μm filter membrane and dispense into 50 mL Store the centrifuge tube at 4°C for later use.

[0026] (2) 6-(Dimethylamino)purine solution: 0.0068 g of 6-(Dimethylamino)purine (Sigma) powder was dissolved in 25 ml of TCM19 medium containing 4% BSA. After dissolution, the solution was filtered through 0.22 μm and stored at -20°C until use.

[0027] (3) Ionomycin solution: Dissolve 1 mg of ionomycin powder (Sigma) in 267 μL of DMSO, divide into aliquots, and store at -20°C until needed.

[0028] Example 1 In vitro maturation culture medium and in vitro maturation culture method for porcine oocytes 1. This embodiment provides a culture medium for in vitro maturation of porcine oocytes The in vitro maturation medium comprises the following components: 0.5 ng / mL fibroblast growth factor (Peprotech), 3.05 mM glucose, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 2.5 ng / mL epidermal growth factor, 10% polyvinyl alcohol, 2.5 ng / mL recombinant porcine interleukin-6 (Peprotech), 100 IU / mL pregnant mare serum gonadotropin, 100 IU / mL human chorionic gonadotropin, 2.5 ng / mL keratinocyte growth factor, 250 IU / mL follicle-stimulating hormone, 1% double antibody (penicillin and streptomycin mixture), and the remainder is TCM199 medium.

[0029] 2. The preparation method of the above-mentioned in vitro maturation medium of porcine oocytes is as follows: TCM199 medium was supplemented with 0.5 ng / mL fibroblast growth factor (Peprotech), 3.05 mM glucose, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 2.5 ng / mL epidermal growth factor, 10% polyvinyl alcohol, 2.5 ng / mL recombinant porcine interleukin-6 (Peprotech), 100 IU / mL pregnant mare serum gonadotropin, 100 IU / mL human chorionic gonadotropin, 2.5 ng / mL keratinocyte growth factor, 250 IU / mL follicle-stimulating hormone, and 1% double-antibody (penicillin and streptomycin), filtered through a 0.22 μm filter membrane, and stored at 4°C until use.

[0030] 3. This embodiment also provides a method for in vitro maturation and culture of porcine oocytes, the method comprising the following steps: The first step is the in vitro separation of porcine COCs: Fresh porcine ovaries with bright appearance, no corpus luteum, no obvious developmental abnormalities, and many plump and translucent pink antral follicles were selected from the slaughterhouse and placed in a thermos bottle containing 3% double-antibody 37℃ sterile saline and transported back to the laboratory within 2 hours. Follicular fluid of follicles with a diameter of 2-6 mm was extracted into a 50 mL centrifuge tube with a disposable 10 mL syringe, and naturally settled in a 37℃ water bath for 10 min, the supernatant was discarded, and the porcine COCs were washed with TL-HEPES operating solution for 10 min, and repeated 3 times. Then, porcine oocyte-follicle complexes (COCs) with uniform cytoplasm and 3-5 layers of granulosa cells were selected with a mouth pipette under a stereomicroscope.

[0031] Step 2: In vitro culture of porcine oocyte-follicle complexes (COCs): The porcine oocyte-follicle complexes (COCs) selected in the previous step were washed three times in TL-HEPES operating solution, and then washed once with the in vitro maturation medium of this example. 40-50 porcine oocyte-follicle complexes in each group were randomly selected and transferred to a 4-well plate that had been equilibrated for 2-4 h in advance. 500 μL of the in vitro maturation medium was added to each well, and the plates were cultured in a 38.5°C, 5% CO2 incubator for 44 h to the MII stage.

[0032] Subsequently, the maturity of the pig oocytes cultured in this example was measured by referring to the measurement method in Example 3.

[0033] Comparative Example 1 1. This embodiment provides a control group culture medium for porcine oocytes, comprising the following components: 10% PFF, 0.57 mM cysteine, 10% serum, 0.91 mM sodium pyruvate, 3.05 mM glucose, 10% polyvinyl alcohol, 10 ng / mL epidermal growth factor, 10 IU / mL pregnant mare serum gonadotropin, 10 IU / mL human chorionic gonadotropin, 25 IU / mL follicle-stimulating hormone and 1% double antibody, and the remainder is TCM199 culture medium.

[0034] 2. The preparation method of the control group culture medium (10 mL system as an example) of this embodiment is as follows: TCM199 medium (Gibco) was supplemented with 10% PFF, 0.57 mM cysteine ​​(Sigma), 10% serum (Gibco), 0.91 mM sodium pyruvate (Sigma), 3.05 mM glucose (Sigma), 10% polyvinyl alcohol (Sigma), 10 ng / ml epidermal growth factor (Peprotech), 10 IU / ml pregnant mare serum gonadotropin (Ningbo Estrogen Factory), 10 IU / ml human chorionic gonadotropin (Ningbo Estrogen Factory), 25 IU / ml follicle-stimulating hormone (Sigma) and 1% double-antibody (Biyuntian) and filtered through a 0.22 μm filter membrane.

[0035] 3. The in vitro maturation culture method of the porcine oocytes of this comparative example 1: The washed porcine COCs were transferred to the pre-equilibrated control group culture medium provided in Comparative Example 1 and cultured in an incubator at 38.5°C and 5% CO2 for 44 h to obtain mature porcine oocytes for subsequent research. The operation method was referred to Example 1 as a control for Example 1.

[0036] Subsequently, the maturity of the pig oocytes cultured in this example was measured by referring to the measurement method in Example 3.

[0037] Example 3 Determination of the maturity of porcine oocytes This example performs the following tests on the porcine oocytes cultured and matured for 44 hours in Example 1 and Comparative Example 1: 1. Determination method (1) Statistics of porcine COCs cumulus diffusion rate; After 44 h of mature culture, the area of ​​each COC was statistically analyzed by ImageJ software according to the two-dimensional area method. 40-50 COCs / COCs were considered as one treatment group, and each treatment group had at least 3 independent repeated experiments.

[0038] (2) Statistics of porcine oocyte maturation rate After 44 h of maturation culture, porcine COCs were transferred to preheated 0.1% hyaluronidase, and cumulus cells were removed by blowing with a 200 μL pipette to obtain naked eggs. Naked eggs were transferred to preheated TL-HEPE operating solution by mouth pipette to terminate hyaluronidase digestion. Oocytes were rolled with a mouth pipette under a stereoscope, the number of polar bodies discharged was counted, and the maturation rate was calculated. 40-50 / COCs were a treatment group, and each treatment group had at least 3 independent repeated experiments.

[0039] (3) Statistics of the cleavage rate of porcine oocytes After 44 h of maturation culture, porcine COCs were transferred to preheated 0.1% hyaluronidase, and cumulus cells were removed by blowing with a 200 μL pipette to obtain naked eggs. The naked eggs were transferred to preheated TL-HEPE operating solution by mouth pipette to terminate hyaluronidase digestion. Under a stereoscope, oocytes with mature polar bodies were selected and transferred to preheated ionomycin for incubation for 5 min. After the incubation, they were washed three times with 4 h equilibrated (dimethylamino) purine solution and transferred to 6-(dimethylamino) purine solution for further incubation for 4 h. After the incubation, they were washed three times with 4 h equilibrated PZM-3, transferred to PZM-3 droplets and covered with paraffin oil, and placed in a 38.5°C, 5% CO2 incubator for 48 h to check their cleavage rate. 40-50 / MII stage was a treatment group, and each treatment group had at least 3 independent repeated experiments.

[0040] (4) Statistics of porcine oocyte blastocyst rate After 44 h of maturation culture, porcine COCs were transferred to preheated 0.1% hyaluronidase, and cumulus cells were removed by blowing with a 200 μL pipette to obtain naked eggs. The naked eggs were transferred to preheated TL-HEPE operating solution by mouth pipette to terminate hyaluronidase digestion. Under a stereoscope, oocytes with polar body maturity were selected and transferred to preheated ionomycin for incubation for 5 min. After the incubation, they were washed three times with 6-(dimethylamino)purine equilibrated for 4 h in advance, and then transferred to 6-(dimethylamino)purine for further incubation for 4 h. After the incubation, they were washed three times with PZM-3 equilibrated for 4 h, transferred to PZM-3 droplets and covered with paraffin oil, and placed in a 38.5℃, 5% CO2 incubator for 168 h to check their blastocyst rate. 20-30 / MII stage was a treatment group, and each treatment group had at least 3 independent repeated experiments.

[0041] (5) Statistics of the number of porcine oocyte nuclei After parthenogenetic activation, porcine oocytes were cultured in PZM3 for 168 h, and blastocysts were selected with a mouth pipette under a stereoscope, incubated with Hoechst 33342 (1:1000, Bio-Tech) for 25 min, washed three times, and immediately photographed with a fluorescence microscope to count the number of blastocyst nuclei in each group. 10-20 blastocysts per treatment group, and each treatment group had at least 3 independent replicates.

[0042] 2. Test results and analysis (1) If Figure 2As shown, the cumulus diffusion area of ​​comparative example 1 is 1.03±0.04, and the cumulus diffusion area of ​​embodiment 1 is 2.13±0.14. It can be seen that compared with the control group culture medium of comparative example 1, the optimized culture medium significantly increases the expansion area of ​​COCs.

[0043] (2) If Figure 3 As shown, the maturation rate of comparative example 1 is 58.87±2.46%, and the maturation rate of embodiment 1 is 81.24±1.69%. Compared with the control group culture medium of comparative example 1, the optimized in vitro maturation culture medium of embodiment 1 significantly improves the maturation rate of pig oocytes.

[0044] (3) If Figure 4 As shown, the cleavage rate of comparative example 1 is 54.43±2.77%, and the maturation rate of embodiment 1 is 85.63±2.56%. Compared with the control group culture medium of comparative example 1, the optimized in vitro maturation culture medium of embodiment 1 significantly improves the cleavage rate of porcine oocytes.

[0045] (4) If Figure 5 As shown, the blastocyst rate of Comparative Example 1 is 15.20±1.33%, and the blastocyst rate of Example 1 is 35.01±1.50%. It can be seen that compared with the control group culture medium of Comparative Example 1, the optimized in vitro maturation culture of Example 1 significantly improves the blastocyst rate of pig oocytes.

[0046] (5) If Figure 6 As shown, the number of blastocyst nuclei in Comparative Example 1 is 32.34±1.22, and the number of blastocyst nuclei in Example 1 is 49.03±1.96. It can be seen that compared with the control group culture medium of Comparative Example 1, the optimized in vitro maturation culture of Example 1 significantly increases the number of blastocyst nuclei in oocytes.

[0047] Example 4 Optimization experiment of the in vitro maturation medium of porcine oocytes of the present invention 1. Experimental group settings In order to optimize the in vitro maturation culture medium of the porcine oocytes of the present invention, the following experimental groups 1 to 12 were set, specifically as follows: Experimental group 1: Prepare 10 ml of basal culture medium, which includes: 3.05 mM glucose, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 2.5 ng / mL epidermal growth factor, 10% polyvinyl alcohol, 100 IU / mL pregnant mare serum gonadotropin, 100 IU / mL human chorionic gonadotropin, 250 IU / mL follicle-stimulating hormone and 1% penicillin-streptomycin, and the rest is TCM199 culture medium; transfer fibroblast growth factor (FGF) and mix evenly with the above basal culture medium to make the final concentration of FGF 0.1 ng / mL to prepare porcine oocyte in vitro maturation medium; transfer the washed porcine COCs to the porcine oocyte in vitro maturation medium (containing 0.1 ng / mL FGF) equilibrated in advance for this experimental group, and culture in an incubator at 38.5℃, 5% CO2 for 44 h to obtain mature porcine oocytes for subsequent research.

[0048] The difference between the in vitro maturation medium of experimental groups 2 to 4 and experimental group 1 is that fibroblast growth factor (FGF) with a final concentration of 0.5 ng / mL, 5 ng / mL, and 10 ng / mL was added respectively, and the other components were consistent with those in Example 1. The culture method was similar to that in Example 1, and the only difference was the in vitro maturation medium used.

[0049] The difference between the in vitro maturation medium of experimental groups 5 to 8 and experimental group 1 is that recombinant porcine interleukin-6 (IL-6) with a final concentration of 0.5 ng / mL, 2.5 ng / mL, 25 ng / mL and 100 ng / mL was added, and the other components were consistent with those in Example 1. The culture method was the same as in Example 1, and the only difference was the in vitro maturation medium used.

[0050] The in vitro maturation medium of experimental groups 9 to 12 differed from that of experimental group 1 in that keratinocyte growth factor (KGF-2) was added at final concentrations of 0.5 ng / mL, 2.5 ng / mL, 25 ng / mL, and 100 ng / mL.

[0051] The dosages of the three core components of the porcine oocyte in vitro maturation medium prepared in the above experimental groups 1 to 12 are shown in Table 1 below: Table 1 Differences in components of various examples Group Fibroblast growth factor (FGF) (ng / mL) Recombinant porcine interleukin-6 (IL-6) ng / mL Keratinocyte growth factor (KGF-2) ng / mL Experimental Group 1 0.1 0 0 Experimental Group 2 0.5 0 0 Experimental Group 3 5 0 0 Experimental Group 4 10 0 0 Experimental Group 5 0 0.5 0 Experimental Group 6 0 2.5 0 Experimental Group 7 0 25 0 Experimental Group 8 0 100 0 Experimental Group 9 0 0 0.5 Experimental Group 10 0 0 2.5 Experimental Group 11 0 0 25 Experimental Group 12 0 0 100 2. Test methods The maturation rates of mature porcine oocytes cultured in the culture medium of Examples 1-12 and Comparative Example 1 for 44 hours were statistically analyzed, and 7 groups of repeated experiments were performed.

[0052] The method for determining the maturity rate is as follows: refer to the method in Example 3.

[0053] 3. Test results and analysis The statistical results are shown in Figure 1 and Table 2. From the results of the univariate analysis of the maturation rate of oocytes in experimental groups 1 to 12, it can be seen that FGF has the best effect on the in vitro maturation medium for porcine oocytes at a concentration of 0.5 ng / mL, IL-6 has the best effect on the in vitro maturation medium for porcine oocytes at 2.5 ng / mL, and KGF-2 has the best effect on the in vitro maturation medium for porcine oocytes at 2.5 ng / mL. The three core components at the above three optimal concentrations can best improve the maturation rate of oocytes.

[0054] Table 2 Oocyte maturation rate (%) Group Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Comparative Example 1 66.67 63.64 60.87 59.57 62.50 56.62 61.64 Experimental Group 1 56.52 55.56 50.00 51.11 48.33 53.90 52.57 Experimental Group 2 70.00 76.67 75.00 74.05 75.56 78.82 75.02 Experimental Group 3 70.00 73.33 70.26 72.82 73.79 75.38 72.60 Experimental Group 4 70.20 70.87 69.30 68.57 71.12 65.00 69.18 Experimental Group 5 63.33 68.71 60.00 63.90 66.67 68.48 65.18 Experimental Group 6 80.00 75.00 71.85 74.05 76.19 75.56 75.44 Experimental Group 7 70.81 72.82 73.79 65.38 70.20 70.87 70.65 Experimental Group 8 68.82 60.00 62.00 68.48 75.49 70.67 67.58 Experimental Group 9 69.30 68.57 62.12 61.21 68.42 67.24 66.14 Experimental Group 10 71.62 78.75 70.59 70.83 78.75 72.22 73.79 Experimental Group 11 66.47 68.75 70.59 70.83 71.21 70.00 69.64 Experimental Group 12 66.00 64.00 67.24 65.98 66.84 60.43 65.08 Example 5 Storage stability experiment of porcine oocyte maturation culture medium 1. Experimental methods 0.5 ng / ml FGF, 3.05 mM glucose, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 2.5 ng / ml epidermal growth factor, 10% polyvinyl alcohol, 2.5 ng / ml IL-6, 100 IU / ml pregnant mare serum gonadotropin, 100 IU / ml human chorionic gonadotropin, 2.5 ng / ml keratinocyte growth factor, 250 IU / ml follicle-stimulating hormone and 1% double antibody were added to TCM199, and the culture medium for porcine oocyte maturation was prepared after filtration through a 0.22 μm filter membrane. The culture medium was stored at 4°C for 0 day, 1 week, 2 weeks, 3 weeks and 4 weeks, respectively, and then used for in vitro culture of porcine oocytes.

[0055] According to the method of Example 1, the washed porcine COCs were transferred to the pre-balanced optimized porcine oocyte in vitro maturation medium, and cultured in an incubator at 38.5°C, 5% CO2 for 44 h to obtain mature porcine oocytes, and the oocyte maturation rate was measured and counted according to the determination method of Example 3.

[0056] Three replicates were set up according to the above method, and the coefficient of variation of the three replicates was determined.

[0057] 2. Experimental results and analysis The results are shown in Table 3. After 4 weeks of storage, the maturation rate of oocytes cultured in the culture medium is still maintained at more than 80%, which shows that the storage stability of the porcine oocyte in vitro maturation culture medium is strong, and its activity remains basically unchanged after 4 weeks of storage. In addition, the coefficient of variation of 3 batches of repeated experiments shows that the results of the porcine oocyte in vitro maturation culture medium are highly consistent and stable.

[0058] Table 3 Oocyte maturation rate (%) Group Repeat 1 Repeat 2 Repeat 3 Coefficient of variation 0 Days 85.05 88.87 84.45 2.78% 1 week 84.46 86.79 85.65 1.36% 2 weeks 84.98 85.78 83.56 1.33% 3 weeks 85.65 82.72 83.85 1.75% 4 weeks 81.46 83.37 83.45 1.36% 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. An in vitro maturation medium for porcine oocytes, characterized in that: Includes the following ingredients: Fibroblast growth factor, glucose, cysteine, sodium pyruvate, epidermal growth factor, polyvinyl alcohol, pregnant mare serum gonadotropin, human chorionic gonadotropin, follicle-stimulating hormone, recombinant porcine interleukin-6, keratinocyte growth factor, and the remainder is TCM199 culture medium.

2. The in vitro maturation medium for porcine oocytes according to claim 1, characterized in that: Also includes: double antibody.

3. The in vitro maturation medium for porcine oocytes according to claim 1, characterized in that: include: 0.5~10ng / ml fibroblast growth factor, 1~10mM glucose, 0.1~1mM cysteine, 0.1~3mM sodium pyruvate, 1~10ng / mL epidermal growth factor, 5~15% polyvinyl alcohol, 50~150 IU / mL pregnant mare serum gonadotropin, 50~150 IU / mL human chorionic gonadotropin, 200~300 IU / mL follicle-stimulating hormone, 1~25ng / ml recombinant porcine interleukin-6, 0.5~5ng / ml keratinocyte growth factor and 0.1~5% double antibody, the remainder is TCM199 culture medium.

4. The in vitro maturation medium for porcine oocytes according to claim 3, characterized in that: Includes the following concentrations of ingredients: 0.5 ng / ml fibroblast growth factor, 3.05 mM glucose, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 2.5 ng / mL epidermal growth factor, 10% polyvinyl alcohol, 100 IU / mL pregnant mare serum gonadotropin, 100 IU / mL human chorionic gonadotropin, 250 IU / mL follicle-stimulating hormone and 1% double antibody, 2.5 ng / ml recombinant porcine interleukin-6, 2.5 ng / ml keratinocyte growth factor, and the remainder is TCM199 medium.

5. A method for in vitro maturation and culture of porcine oocytes, characterized in that: The in vitro maturation culture method comprises: adding pig oocytes to the in vitro maturation culture medium according to any one of claims 1 to 4 for culture.

6. The in vitro maturation culture method of porcine oocytes according to claim 5, characterized in that: The following steps are involved: S1. Add the in vitro maturation medium of porcine oocytes as described in any one of claims 1 to 4 into the well plate, seal it with mineral oil, and place it in a 38.5°C, 5% CO2, fully saturated humidity incubator for 2 to 4 hours in advance; S2. Isolation of porcine oocyte-follicle complex in vitro: extract follicular fluid from fresh porcine follicles with a diameter of 2-6 mm into a centrifuge tube, let it settle naturally in a 37°C water bath for 10 min, discard the supernatant, wash the cell pellet with TL-HEPES operating solution, let it settle in a warm bath for another 10 min, repeat at least 3 times, and then select porcine oocyte-follicle complexes with uniform cytoplasm and 3-5 layers of granulosa cells under a microscope; S3. In vitro culture of porcine oocyte-follicle complexes: The porcine oocyte-follicle complexes selected in the previous step were washed with TL-HEPES, and then 40-50 porcine oocyte-follicle complexes from each group were randomly selected and transferred to the well plate balanced in S1, and cultured for 44 h to the MII stage to obtain mature oocytes.

7. The in vitro maturation and culture method of porcine oocytes according to claim 5, characterized in that: Also includes: S4. Determination of cumulus diffusion rate, maturation rate, cleavage rate and blastocyst rate of oocytes.

8. The in vitro maturation and culture method of porcine oocytes according to claim 5, characterized in that: The step S4 is: taking photos of oocytes 44 hours after culture and maturation under a microscope and calculating the cumulus diffusion rate; digesting mature oocytes with 0.1% hyaluronidase, selecting naked eggs, observing and counting the number of polar bodies discharged under a stereoscope to calculate the maturation rate; and calculating the cleavage rate and blastocyst rate after parthenogenetic activation of mature oocytes.

Images