Oocyte in-vitro culture solution and culture method

Abstract

The invention discloses an oocyte in-vitro culture solution and a culture method. The culture solution comprises a basal culture medium, procyanidine B2, coenzyme Q10, BAPTA-AM, alpha-lipoic acid and sodium pyruvate. According to the method, the in-vitro maturation efficiency and quality of the oocytes can be remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to an oocyte in vitro culture medium and a culture method. Background Art

[0002] In the field of oocyte in vitro culture, optimizing the culture system to improve oocyte maturation quality and subsequent developmental potential has always been a key research challenge. While existing culture media can meet basic nutritional requirements, they lack targeted antioxidant and metabolic regulatory components. Oocytes are susceptible to damage from reactive oxygen species (ROS) during in vitro culture. The lack or irrational ratio of antioxidants in conventional culture media makes it difficult to effectively control the accumulation of intracellular ROS, which in turn affects mitochondrial function and chromosomal stability. Furthermore, the lack of energy metabolism regulatory substances makes oocytes inefficient in ATP production in vitro, making it impossible to maintain normal meiotic progression.

[0003] Therefore, it is necessary to design a technical solution that can overcome the above-mentioned defects. Summary of the Invention

[0004] One object of the present invention is to provide an oocyte in vitro culture medium and culture method, which can significantly improve the efficiency and quality of oocyte in vitro maturation.

[0005] To achieve these objectives and other advantages of the present invention, according to one aspect of the present invention, the present invention provides an oocyte in vitro culture medium, comprising a basal culture medium, procyanidin B2, coenzyme Q10, BAPTA-AM, α-lipoic acid and sodium pyruvate.

[0006] Furthermore, the concentration of the proanthocyanidin B2 is 3-7 μg / mL, the concentration of the coenzyme Q10 is 5-15 μM, the concentration of the BAPTA-AM is 0.1-1.0 μM, the concentration of the α-lipoic acid is 0.5-2.0 mM, and the concentration of the sodium pyruvate is 5-20 mM.

[0007] Furthermore, the basal culture medium is TCM-199 culture medium.

[0008] According to another aspect of the present invention, an in vitro oocyte culture method is also provided, comprising: S1: placing GV stage oocytes in a basal culture medium containing proanthocyanidin B2, coenzyme Q10, BAPTA-AM, α-lipoic acid and sodium pyruvate, and culturing at an oxygen concentration of 4.5%-5.5% for 18 hours; S2: transferring the oocytes treated in step S1 to an environment with an oxygen concentration of 18%-22% and continuing to culture for 24 hours; wherein, throughout steps S1 and S2, a planar oscillation with a frequency of 1-2 Hz and a duration of 8-12 seconds is applied once every 120 minutes; the intracellular reactive oxygen level is monitored in real time using a dichlorodihydrofluorescein diethyl ester probe, and when the fluorescence intensity is ≥150 relative fluorescence units, 1 μg / mL proanthocyanidin B2 is added to the culture medium.

[0009] Furthermore, the establishment of the hypoxic environment in S1 is achieved by using a light-controlled oxygen consumption hydrogel system, which is composed of a thermosensitive poly (N-isopropylacrylamide) hydrogel loaded with hemoglobin-manganese dioxide nanoparticles. When the culture is started, 808nm near-infrared light is applied for 60 seconds to trigger hemoglobin to release oxygen molecules, causing the local oxygen concentration to drop from 21% to 4.5%-5.5% within 120 seconds, and maintaining a fluctuation range of ≤±0.3%.

[0010] Furthermore, in S2, the oocytes were moved into the lower layer of a double-layer gas diffusion chamber, a 20% O2 / 5% CO2 mixture was introduced into the upper layer, and a 5% O2 / 5% CO2 mixture was initially introduced into the lower layer, and the oxygen concentration was linearly increased to 20% within 60±5 minutes through the microporous ceramic partition.

[0011] Furthermore, the intracellular calcium oscillation frequency was monitored in real time using a Cal-520 AM probe. When the frequency was > 0.8 times / min, 0.75 ± 0.25 mM sodium pyruvate was injected into the microfluidic chip within 3 seconds, while a frequency of 20 ± 1 kHz and an acoustic intensity of 0.5 ± 0.1 W / cm 2 A directional ultrasound pulse of 2 seconds was applied; 2 hours after the end of oxygenation, the ATP concentration was detected to be ≥7.5 pmol / oocyte, which was considered to be a valid conversion.

[0012] Furthermore, the planar oscillator is integrated with a piezoelectric sensor array to monitor the shear force of the culture medium in real time and dynamically regulate it; when the shear force is less than 0.4 dyne / cm 2 When the amplitude is increased to 1.0 mm, when the shear force is greater than 0.8 dyne / cm 2 The frequency was reduced to 1 Hz to maintain the shear force of the fluid on the oocyte surface at 0.5-0.7 dyne / cm 2 within the physiological range.

[0013] Furthermore, GV stage oocytes were placed in a pretreatment solution containing 0.1 mM dichlorodihydrofluorescein diethyl ester probe and 0.5% Pluronic F-127, incubated at 37°C and 5% CO2 for 30 minutes, and then washed and transferred to the culture system.

[0014] Furthermore, the fluorescence intensity threshold was set at 135 RFU for 0-18 h of culture, and at 165 RFU for 18-42 h of culture; when the real-time fluorescence intensity was ≥ the current stage threshold but <200 RFU, 1 μg / mL proanthocyanidin B2 was added to the culture medium, and when the real-time fluorescence intensity was ≥200 RFU, 2 μg / mL proanthocyanidin B2 was added.

[0015] The present invention has at least the following beneficial effects: The oocyte in vitro culture medium and culture method provided by the present invention significantly improve the efficiency and quality of oocyte maturation in vitro through the synergistic effect of multiple components and dynamic culture regulation. Proanthocyanidin B2, coenzyme Q10, and α-lipoic acid in the culture medium form a composite antioxidant system that can simultaneously reduce intracellular ROS levels, enhance mitochondrial membrane potential and ATP production efficiency, and significantly improve the MII stage maturation rate compared to single antioxidants. BAPTA-AM and sodium pyruvate synergistically regulate calcium oscillation frequency and glycolysis metabolism, reducing the abnormal distribution rate of cortical granules and downregulating the expression of endoplasmic reticulum stress markers. Dynamic oxygen concentration regulation combined with planar oscillation simulates the in vivo fallopian tube microenvironment, significantly improving the blastocyst formation rate, and achieving a high effective conversion rate of ATP concentration. Real-time ROS monitoring and dynamic proanthocyanidin supplementation can control the intracellular fluorescence intensity below a reasonable range, avoid oxidative stress damage, and provide high-quality oocytes for subsequent embryonic development.

[0016] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. DETAILED DESCRIPTION

[0017] The present invention is described in further detail below so that those skilled in the art can implement the invention with reference to the description.

[0018] It should be understood that terms such as "having," "comprising," and "including" used in the embodiments of this application do not exclude the presence or addition of one or more other elements or combinations thereof. All directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of this application are intended only to explain the relative positional relationships and movement of components in a specific posture. If the specific posture changes, the directional indications will also change accordingly. When an element is referred to as being "fixed to" or "disposed on" another element, it can be directly on the other element or there may be an intervening element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element through an intervening element. References to "first," "second," etc. in the embodiments of this application are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, features designated as "first" or "second" may explicitly or implicitly include at least one of such features.

[0019] It should be noted that the technical solutions between the various embodiments of the present application can be combined with each other, but it must be based on the fact that ordinary technicians in this field can implement it. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0020] The embodiments of the present application provide an oocyte in vitro culture medium, comprising a basal culture medium, procyanidin B2, coenzyme Q10, BAPTA-AM, α-lipoic acid, and sodium pyruvate.

[0021] For example, the basal culture medium used is TCM-199 medium, a classic animal cell culture medium manufactured by Gibco. It contains L-glutamine, sodium bicarbonate, and other ingredients. It should be dissolved in triple-distilled water according to the manufacturer's instructions and supplemented with 10% fetal bovine serum and a double-stranded antibody. Proanthocyanidin B2 (PCB2) is used as an antioxidant at concentrations of 3, 5, and 7 μg / mL. Its polyphenolic structure, extracted from grape seeds, can scavenge intracellular reactive oxygen species. Coenzyme Q10 is used at concentrations of 5, 10, and 15 μM, as a component of the mitochondrial electron transport chain involved in ATP synthesis. BAPTA-AM is used as a calcium chelator at concentrations of 0.1, 0.5, and 1.0 μM. Upon entry into cells, it is hydrolyzed to BAPTA-bound calcium ions. α-Lipoic acid is used at concentrations of 0.5, 1.0, and 2.0 mM to regenerate glutathione and enhance antioxidant capacity. Sodium pyruvate is used at concentrations of 5, 10, and 20 mM to provide energy to cells through the tricarboxylic acid cycle. During preparation, each component must be sterilized by filtration using a 0.22 μm filter membrane and added to the basal culture medium in sequence.

[0022] In this example, the above components were mixed at optimized concentrations and used for in vitro culture of sheep oocytes. The culture medium provided a suitable microenvironment for oocytes through synergistic effects of antioxidant and metabolic regulation, thereby improving maturation efficiency.

[0023] In another embodiment, the in vitro oocyte culture method includes: S1: placing the GV stage oocytes in a basic culture medium containing antioxidants and culturing them at an oxygen concentration of 4.5%-5.5% for 18 hours; S2: transferring to an oxygen concentration environment of 18%-22% and continuing to culture for 24 hours, with planar oscillation once every 120 minutes throughout the process, real-time monitoring of ROS and supplementation of proanthocyanidin B2.

[0024] For example, GV-stage oocytes were obtained by puncturing 2-4 mm follicles of adult sheep, and intact cumulus cells were selected to encapsulate the cells. The S1 hypoxic environment was controlled by a Thermo Fisher gas incubator, with oxygen concentrations set to 4.5%, 5.0%, and 5.5%, simulating the in vivo environment of the ampulla of the fallopian tube. The S2 normoxia environment was adjusted to approximately 20% using an ESCO incubator, simulating the oxygen concentration in the isthmus of the fallopian tube. Planar oscillation was performed using a Qsonica oscillator at a frequency of 1-2 Hz and a duration of 8-12 seconds, with oscillation every 2 hours to simulate tubal peristalsis. After labeling with a dichlorodihydrofluorescein diethyl ester probe (0.1 mM), the sample was monitored in real time using a Nikon fluorescence microscope. When the fluorescence intensity reached ≥150 RFU, 1 μg / mL of proanthocyanidin B2 was added using an Eppendorf pipette.

[0025] In this embodiment, hypoxic culture is first used to promote nuclear maturation, and then normoxic culture is used to synchronize cytoplasmic maturation. Combining oscillation and dynamic antioxidant regulation can reduce oxidative stress damage and improve the synchronization of oocyte maturation.

[0026] In another embodiment, the hypoxic environment in S1 is established by a light-controlled oxygen consumption hydrogel system, which is composed of a thermosensitive hydrogel loaded with hemoglobin-manganese dioxide nanoparticles. When the culture is started, 808nm near-infrared light is used to trigger a decrease in oxygen concentration.

[0027] For example, during hydrogel preparation, hemoglobin-manganese dioxide nanoparticles were synthesized by coprecipitation and then loaded into a poly (N-isopropylacrylamide) (PNIPAM) hydrogel to form 5 mm diameter hydrogel beads. During culture, the hydrogel beads were placed at the bottom of a culture dish, and oocytes were seeded in culture medium above. Irradiation with a Thorlabs near-infrared laser (808 nm) for 60 seconds triggered the release of oxygen from the nanoparticles, reducing the local oxygen concentration from 21% to 4.5%-5.5% within 120 seconds. Real-time monitoring was performed using a PreSens oxygen electrode, and a PID feedback system maintained the oxygen concentration fluctuation within ±0.3%.

[0028] In this embodiment, the precise establishment and maintenance of a hypoxic environment is achieved through light control, avoiding the lag of traditional gas incubators and providing a stable hypoxic microenvironment for the early development of oocytes.

[0029] In another embodiment, the oocyte in S2 is moved into a double-layer gas diffusion chamber, and the oxygen concentration is linearly increased by gas diffusion in the upper and lower layers.

[0030] For example, a Merck Millicell double-layer diffusion chamber was used, separated by a 1-5 μm microporous ceramic separator (CoorsTek). A 20% O2 / 5% CO2 mixture was introduced into the upper chamber, while a 5% O2 / 5% CO2 mixture was initially introduced into the lower chamber. Gases were supplied from Air Liquide cylinders, with flow controlled by a Brooks mass flowmeter. Oxygen concentration changes were monitored by an Oxford Optronix oxygen electrode. By adjusting the separator pore size and gas flow rate, the oxygen concentration in the lower chamber increased linearly from 5% to 20% over 55-65 minutes, mimicking the in vivo oxygen concentration gradient.

[0031] In this embodiment, the device achieves a slow increase in oxygen concentration through the gas diffusion effect, which is consistent with the physiological oxygen environment changes when the oocyte moves from the ampulla to the isthmus of the fallopian tube, thereby promoting cytoplasmic maturation.

[0032] In another embodiment, the calcium oscillation frequency is monitored using a Cal-520 AM probe during the culture process. When abnormal, sodium pyruvate is injected through a microfluidic chip and ultrasonic pulses are applied. The ATP concentration is then measured after oxygenation.

[0033] For example, Cal-520 AM probe (Invitrogen) was prepared with DMSO to a 0.5 μM working solution and incubated with oocytes at 37°C for 30 minutes. Calcium signals were recorded using a Nikon fluorescence microscope and analyzed using ImageJ software. When the frequency was > 0.8 times / min, 0.5-1.0 mM sodium pyruvate was injected within 3 seconds using an Elveflow microfluidic chip, while a Hielscher ultrasonic processor was used to apply 20 ± 1 kHz, 0.5 ± 0.1 W / cm 2 Two hours after the end of oxygenation, the ATP concentration was measured using the Promega Cell Titer-Glo kit. A value of 7.5 pmol / oocyte or higher was considered to be a valid conversion.

[0034] In this embodiment, the operation ensures that the oocyte maintains normal physiological functions during the oxygen concentration conversion process and enhances developmental potential through calcium signal regulation and energy metabolism intervention.

[0035] In another embodiment, the planar oscillator is integrated with a piezoelectric sensor array to monitor shear force in real time and dynamically control amplitude and frequency.

[0036] For example, the oscillator is modified from a Branson ultrasonic disruptor, with a PI Ceramic piezoelectric sensor array integrated at the bottom, with a spacing of 2 mm covering the culture area. The shear force is calculated as τ = μ × (dv / dy). When the shear force is detected to be less than 0.4 dyne / cm 2 When the PLC system increases the amplitude from 0.5mm to 1.0mm; >0.8dyne / cm 2 Reduce the frequency from 2 Hz to 1 Hz and maintain the shear force at 0.5-0.7 dyne / cm 2 This range is consistent with the physiological shear force in the fallopian tube, and dynamic regulation can be used to avoid cell membrane damage or insufficient material transport.

[0037] In this embodiment, the closed-loop control system provides a bionic fluid microenvironment for oocytes through real-time mechanical monitoring and feedback regulation, thereby optimizing the establishment of cell polarity and the distribution of cytoplasmic components.

[0038] In another embodiment, GV stage oocytes are incubated with a pretreatment solution containing DCFH-DA probe and Pluronic F-127 and then transferred into a culture system.

[0039] For example, the pretreatment solution consists of 0.1 mM DCFH-DA (Invitrogen) and 0.5% Pluronic F-127 (Sigma-Aldrich) dissolved in TCM-199 culture medium and filtered through a 0.22 μm filter. Oocytes are incubated in a 37°C, 5% CO2 incubator for 30 minutes, gently shaking the culture dish every 10 minutes. After incubation, the oocytes are washed three times with probe-free culture medium for 5 minutes each. Pluronic F-127 enhances the permeability of the probe cell membrane, allowing DCFH-DA to enter the cell and be hydrolyzed by esterases to form DCFH, which reacts with ROS to generate a fluorescent substance, facilitating subsequent real-time monitoring.

[0040] In this embodiment, the pretreatment step is carried out by fluorescent probe labeling to achieve real-time dynamic monitoring of intracellular ROS levels, providing a basis for the precise addition of antioxidants.

[0041] In another embodiment, different fluorescence intensity thresholds are set at different stages of the culture, and proanthocyanidin B2 is dynamically added according to the thresholds.

[0042] For example, the threshold was set at 135 RFU for 0-18 hours (GV-MI stage) and 165 RFU for 18-42 hours (MI-MII stage), monitored using an Olympus fluorescence microscope with MetaMorph software. When the fluorescence intensity was ≥135 RFU and <200 RFU, 1 μg / mL of proanthocyanidin B2 (prepared from a 10 mg / mL stock solution in DMSO) was added using a Gilson pipette; when the fluorescence intensity was ≥200 RFU, 2 μg / mL was added. This regulation was based on the oxidative stress tolerance of oocytes at different developmental stages to avoid either insufficient or excessive amounts of antioxidants.

[0043] In this embodiment, through staged threshold control and dynamic supplementation mechanism, the intracellular redox balance is accurately maintained, oxidative stress damage is reduced, and the quality of oocyte maturation is guaranteed.

[0044] The following is an explanation with specific examples.

[0045] Example 1: First, prepare the oocyte in vitro culture medium using Gibco TCM-199 medium as the base, dissolving 10g of powder in 1L of triple-distilled water. Add 10% fetal bovine serum and double-stranded antibodies, adjust the pH to 7.3, and filter the medium until ready for use. Add 5μg / mL proanthocyanidin B2, 10μM coenzyme Q10, 0.5μM BAPTA-AM, 1.0mM α-lipoic acid, and 10mM sodium pyruvate to the basal medium, mix thoroughly, filter, and aliquot. GV-stage oocytes were obtained from adult sheep 2-4mm follicles and incubated with a pretreatment solution containing 0.1mM DCFH-DA probe and 0.5% Pluronic F-127 at 37°C, 5% CO2 for 30 minutes. Wash and set aside.

[0046] Pretreated oocytes were placed in a culture dish containing a photo-controlled oxygen depletion hydrogel system composed of PNIPAM loaded with hemoglobin-manganese dioxide nanoparticles. The oocytes were irradiated with 808nm near-infrared light for 60 seconds, causing the oxygen concentration to decrease from 21% to 5.0% over 120 seconds and maintain a fluctuation of ≤±0.3%. Simultaneously, the oocytes were oscillated for 10 seconds every 120 minutes using a Qsonica planar oscillator at a 1Hz frequency and 0.5mm amplitude. Eighteen hours later, the oocytes were transferred to a Merck double-layer gas diffusion chamber. The lower chamber was initially aerated with 5% O2 / 5% CO2, while the upper chamber was aerated with 20% O2 / 5% CO2. The oxygen concentration was then linearly increased to 20% over 60 minutes via a 3μm microporous ceramic separator. ROS were monitored using an Olympus fluorescence microscope throughout the culture process. When the fluorescence intensity was ≥135 RFU from 0 to 18 hours or ≥165 RFU from 18 to 42 hours, 1 μg / mL proanthocyanidin B2 was added, and when it was ≥200 RFU, 2 μg / mL was added. Calcium oscillations were monitored using a Cal-520 AM probe. When the frequency was >0.8 times / min, 0.75 mM sodium pyruvate was injected and a 20 kHz, 0.5 W / cm 2 Ultrasonic pulse for 2 seconds.

[0047] After 42 hours of culture, the first polar body extrusion rate was observed to assess MII maturation rate. Blastocyst formation rate was calculated on day 7 after in vitro fertilization. Oocyte ATP concentration was measured 2 hours after the end of oxygenation, and a concentration ≥7.5 pmol / oocyte was considered effective. The entire process enhances the efficiency of in vitro oocyte maturation by combining TCM-199 basal medium with functional components at specific concentrations, light-controlled oxygen consumption, oxygen concentration gradient conversion in a gas diffusion chamber, shear force regulation by planar oscillating fluids, and real-time physiological signal intervention.

[0048] Control group: Using TCM-199 basal medium without antioxidants and functional components, and static culture at a constant oxygen concentration of 20%, the MII maturation rate was 45.68% (n=122), the blastocyst formation rate was 46.2%, and the oocyte ATP concentration was 5.2±0.8 pmol / oocyte after 42 hours of culture. The intracellular ROS fluorescence intensity averaged 210 RFU at the end of culture, indicating that the oocyte maturation efficiency and subsequent developmental potential were low under basal culture conditions, and the level of oxidative stress was high.

[0049] Comparative Example 1: When only 5 μg / mL proanthocyanidin B2 was added to the culture medium, without other functional components such as coenzyme Q10 and BAPTA-AM, and static culture was adopted at a constant 5% oxygen concentration without a dynamic supplementation mechanism, the MII stage maturation rate increased to 49.8% (n=115), the blastocyst formation rate was 52.3%, the ATP concentration was 6.1±0.5 pmol / oocyte, and the intracellular ROS fluorescence intensity averaged 185 RFU at the end of culture, a decrease of 12% compared with the control group. However, due to the lack of dynamic supplementation, the ROS peak still reached 230 RFU, indicating that although a single antioxidant can improve oxidative stress, it lacks the synergistic effect of metabolic regulatory components, and the improvement in maturation efficiency is limited.

[0050] Comparative Example 2: All functional components were retained in the culture medium, but the light-controlled oxygen consumption and the oxygen concentration gradient conversion in the gas diffusion chamber were eliminated. 20% oxygen concentration was used throughout the culture process. The MII stage maturation rate was 50.2% (n=118), the blastocyst formation rate was 55.7%, and the ATP concentration was 6.5±0.7 pmol / oocyte. JC-1 staining showed that the mitochondrial membrane potential was 15% lower than that in Example 1, confirming that the lack of the hypoxia to normoxia gradient conversion affects mitochondrial function, thereby resulting in insufficient improvement in the developmental potential of oocytes.

[0051] Comparative Example 3: When 5 μg / mL proanthocyanidin B2 was added to the culture medium at one time, the real-time ROS monitoring and dynamic supplementation mechanism was canceled, and sodium pyruvate injection and ultrasound intervention were not performed when calcium oscillation was abnormal, the MII stage maturation rate was 53.1% (n=110), and the blastocyst formation rate was 58.9%. However, the abnormal cortical granule distribution rate increased by 11% to 22% compared with Example 1. Although the average ROS fluorescence intensity at the end of culture was 170 RFU, it peaked at 215 RFU during the 18-42 hour period, indicating that the lack of real-time physiological signal intervention can lead to uncontrolled stage-by-stage oxidative stress and affect the quality of cytoplasmic maturation.

[0052] Summarize: Compared with the above-mentioned control group and comparative example, Example 1, by synergistically adding 5 μg / mL proanthocyanidin B2, 10 μM coenzyme Q10 and other functional components to the TCM-199 basal culture medium, combined with dynamic culture of light-controlled oxygen consumption (5.0% oxygen concentration) to gas diffusion chamber oxygen concentration gradient conversion (linearly increased to 20%), and dynamic supplementation of proanthocyanidin B2 based on the ROS fluorescence intensity staged threshold (0-18 hours 135RFU, 18-42 hours 165RFU) and intervention of abnormal calcium oscillation, the MII stage maturation rate was increased to 58-62%, the blastocyst formation rate reached 68-72%, the oocyte ATP concentration was ≥7.5 pmol / oocyte, and the intracellular ROS fluorescence intensity was effectively controlled below 165RFU. Data show that the synergistic effect of multi-component antioxidant and metabolic regulation, dynamic culture simulating the in vivo oxygen concentration gradient, and precise intervention based on real-time physiological signals can systematically improve the efficiency of oocyte in vitro maturation and subsequent embryonic development potential, which has significant advantages over single component addition or static culture conditions.

[0053] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. An oocyte in vitro culture medium, characterized in that: Includes basal culture medium, proanthocyanidin B2, coenzyme Q10, BAPTA-AM, α-lipoic acid and sodium pyruvate.

2. The oocyte in vitro culture medium according to claim 2, wherein The concentration of the proanthocyanidin B2 is 3-7 μg / mL, the concentration of the coenzyme Q10 is 5-15 μM, the concentration of the BAPTA-AM is 0.1-1.0 μM, the concentration of the α-lipoic acid is 0.5-2.0 mM, and the concentration of the sodium pyruvate is 5-20 mM.

3. The oocyte in vitro culture medium according to claim 2, wherein The basic culture medium is TCM-199 culture medium.

4. A method for culturing oocytes in vitro, characterized in that: include: S1: GV stage oocytes were placed in a basal medium containing proanthocyanidin B2, coenzyme Q10, BAPTA-AM, α-lipoic acid, and sodium pyruvate and cultured at an oxygen concentration of 4.5%-5.5% for 18 hours; S2: The oocytes treated in step S1 are transferred to an environment with an oxygen concentration of 18%-22% and cultured for 24 hours; During steps S1 and S2, a planar oscillation with a frequency of 1-2 Hz and a duration of 8-12 seconds was applied once every 120 minutes. The intracellular reactive oxygen species level was monitored in real time using a dichlorodihydrofluorescein diethyl ester probe. When the fluorescence intensity was ≥150 relative fluorescence units, 1 μg / mL proanthocyanidin B2 was added to the culture medium.

5. The method according to claim 4, characterized in that The establishment of the hypoxic environment in S1 is achieved by using a light-controlled oxygen consumption hydrogel system, which is composed of a thermosensitive poly (N-isopropylacrylamide) hydrogel loaded with hemoglobin-manganese dioxide nanoparticles; At the start of the culture, 808nm near-infrared light was applied for 60 seconds to trigger hemoglobin to release oxygen molecules, causing the local oxygen concentration to drop from 21% to 4.5%-5.5% within 120 seconds, and maintaining a fluctuation range of ≤±0.3%.

6. The method according to claim 4, characterized in that In the S2, the oocytes were moved into the lower layer of a double-layer gas diffusion chamber, a 20% O2 / 5% CO2 mixture was introduced into the upper layer, and a 5% O2 / 5% CO2 mixture was initially introduced into the lower layer, and the oxygen concentration was linearly increased to 20% within 60±5 minutes through a microporous ceramic partition.

7. The method according to claim 6, characterized in that The intracellular calcium oscillation frequency was monitored in real time using a Cal-520 AM probe. When the frequency was > 0.8 times / min, 0.75 ± 0.25 mM sodium pyruvate was injected into the microfluidic chip within 3 seconds, while a frequency of 20 ± 1 kHz and an acoustic intensity of 0.5 ± 0.1 W / cm were applied. 2 Directed ultrasound pulses for 2 seconds; Two hours after the end of oxygenation, the ATP concentration was ≥7.5 pmol / oocyte and the conversion was considered effective.

8. The method according to claim 4, characterized in that The planar oscillator integrates a piezoelectric sensor array to monitor the shear force of the culture medium in real time and dynamically regulate it; When shear force is less than 0.4 dyne / cm 2 When the amplitude is increased to 1.0 mm, when the shear force is greater than 0.8 dyne / cm 2 The frequency was reduced to 1 Hz to maintain the shear force of the fluid on the oocyte surface at 0.5-0.7 dyne / cm 2 within the physiological range.

9. The method according to claim 4, characterized in that GV stage oocytes were placed in a pretreatment solution containing 0.1 mM dichlorodihydrofluorescein diethyl ester probe and 0.5% Pluronic F-127, incubated at 37°C and 5% CO2 for 30 minutes, and then washed and transferred to the culture system.

10. The method according to claim 9, characterized in that The fluorescence intensity threshold was set at 135 RFU for 0-18 h of culture and 165 RFU for 18-42 h of culture; When the real-time fluorescence intensity was ≥ the current stage threshold but <200 RFU, 1 μg / mL proanthocyanidin B2 was added to the culture medium; when the real-time fluorescence intensity was ≥200 RFU, 2 μg / mL proanthocyanidin B2 was added.