Application of dihydromyricetin or its derivatives in the preparation of products for improving the quality of in vitro embryos

Abstract

The present invention belongs to the field of in vitro embryo technology and relates to the technology of in vitro cultivation of pre-implantation embryos of mammals. The present invention provides the use of dihydromyricetin or its derivatives in the preparation of products for improving the quality and developmental ability of in vitro embryos, and products for improving the developmental ability of in vitro embryos, the use of dihydromyricetin or its derivatives in improving the quality of in vitro embryos, and a method for improving the developmental ability of in vitro embryos. The present invention discovers that dihydromyricetin or its derivatives can increase the blastocyst rate of in vitro embryo culture, reduce the content of cellular reactive oxygen species, increase the content of cellular glutathione, and promote the expression of oxidative stress genes, thereby effectively improving the quality and developmental ability of in vitro embryos.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro embryo technology, relates to the technology of in vitro culture of mammalian pre-implantation embryos, and specifically relates to the application of dihydromyricetin or its derivatives in the preparation of products for improving the quality and developmental ability of in vitro embryos. Background Art

[0002] In vitro embryo production is a key link in embryo engineering technology and a key technology for animal husbandry, human assisted reproductive technology, and research on the mechanism of pre-implantation embryo development [1]. In vitro embryo production mainly includes in vitro fertilization and in vitro embryo culture. In vitro embryo culture is the process of culturing activated oocytes into blastocysts in vitro [2]. The activation of oocytes is divided into three types: spontaneous activation (common in animals that perform parthenogenesis and aging oocytes), sperm activation (the combination of sperm and egg in sexually reproducing animals under natural conditions), and artificial activation. By artificially simulating the activation mechanism of oocytes under natural conditions, oocytes in the MII phase can also be activated in vitro through various physical and chemical stimuli, which is the artificial parthenogenetic activation of oocytes. Parthenogenetically activated eggs can start cleavage and develop under certain culture conditions, going through the 2-cell, 4-cell, 8-cell, 16-cell, morula stages, and finally developing into blastocysts. Successfully developing into blastocysts is a very crucial step. Blastocysts have tightly arranged trophoblasts and loosely arranged inner cell masses, and generally form blastocysts on the 5th to 7th day after oocyte activation. Only embryos with good quality can develop into blastocysts, and using blastocysts for transplantation can better improve the embryo implantation rate. Currently, the in vitro blastocyst rate is generally low. Taking pigs as an example, the in vitro blastocyst rate is generally only about 30%, while the in vivo environment can reach 80%. In recent years, some scholars have continuously improved problems such as embryo transplantation rate, cryopreserved embryo recovery rate, and embryo survival rate by changing conditions.

[0003] During in vitro embryo production, since oocytes contain a large amount of lipids, and these lipids are particularly sensitive to reactive oxygen species and are more easily interfered by reactive oxygen species in the in vitro environment compared to the in vivo environment. Oocytes in a state of oxidative stress are greatly affected in terms of their quality and developmental potential, manifested as depletion of the follicle pool, an increase in oocyte aneuploidy, a low oocyte maturation rate, and significant decreases in fertilization rate, cleavage rate, and blastocyst rate, resulting in a decrease in the success rate of embryo transplantation, or causing serious problems such as recurrent miscarriage and fetal chromosomal aberration.

[0004] To solve these problems, the prior art adds substances such as oviduct fluid and melatonin to the culture medium of in vitro embryos [3]. However, the side effects of these substances are unclear, the mechanism is not obvious, and the price is expensive, with certain limitations. There is an urgent need to find new drugs to improve the composition of in vitro embryo culture media to enhance the developmental ability and quality of in vitro embryos.

[0005] [1] Kruip T, Bevers M M, Kemp B. Environment of oocyte and embryo determines health of IVP offspring[J]. Theriogenology, 2000, 53(2): 611-618.

[0006] [2] Wang Na, Li Jiao, Wang Xiaowu, et al. Factors affecting the efficiency of bovine in vitro embryo production[J]. Today's Animal Husbandry and Veterinary Medicine, 2018, 34(11): 4.

[0007] [3] Liu Zeyu, Sun Zheng. Research progress on melatonin metabolism patterns[J]. Life Sciences, 2017, 29(2): 6. Summary of the Invention

[0008] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides the use of dihydromyricetin and / or its derivatives in the preparation of products for improving the quality and developmental ability of in vitro embryos. Dihydromyricetin ((2R,3R)-3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-2,3-dihydrochromen-4-one) is a flavonoid compound extracted from natural plants and has the following chemical structural formula:

[0009]

[0010] The applicant surprisingly found that this component can increase the blastocyst rate, reduce the content of cellular reactive oxygen species (ROS), increase the content of cellular glutathione (GSH), and promote the expression of oxidative stress genes in early pre-implantation embryos, thereby improving the quality and developmental ability of in vitro embryo culture and being beneficial to in vitro embryo culture.

[0011] In a first aspect of the present invention, there is provided the use of dihydromyricetin and / or its derivatives in the preparation of products for improving the quality of in vitro embryos. According to some embodiments of the present invention, there is provided the use of dihydromyricetin and / or its derivatives in the preparation of products for improving the developmental ability of in vitro embryos.

[0012] According to some specific embodiments of the present invention, as demonstrated by detecting / calculating the blastocyst rate of in vitro embryos, the content of ROS in cells, the content of GSH in cells, and the expression level of oxidative stress genes, dihydromyricetin and / or its derivatives improve the quality and developmental ability of in vitro embryos.

[0013] According to some embodiments of the present invention, the quality and developmental ability of in vitro embryos are evaluated by detecting / calculating the blastocyst rate of in vitro embryos.

[0014] According to some embodiments of the present invention, the quality and developmental ability of in vitro embryos are evaluated by detecting / calculating the ROS content and GSH content of in vitro embryo cells. ROS refers to the general term for substances composed of oxygen, containing oxygen and being chemically active in the body or natural environment; in vitro embryo cells are more susceptible to ROS interference in the in vitro environment compared to the in vivo environment. An increase in the ROS content in cells will cause damage to lipids, proteins, and DNA, leading to cell death. GSH is a tripeptide containing a γ-amide bond and a sulfhydryl group, which participates in various intracellular processes, including protecting cells from oxidative stress and regulating intracellular redox metabolism, and can help maintain normal immune system function. The ROS and GSH content levels of embryo cells are two important factors affecting the in vitro embryo development process.

[0015] According to some embodiments of the present invention, the quality and developmental ability of in vitro embryos are evaluated by detecting / calculating the expression level of oxidative stress genes in in vitro embryo cells. According to some specific embodiments of the present invention, the oxidative stress genes include the SOD1 gene, the SOD2 gene, and the Sirt1 gene. SOD protein is an antioxidant enzyme present in mammalian bodies, which can dismutate oxygen free radicals into hydrogen peroxide (H2O2), and H2O2 is further scavenged under the catalysis of catalase and glutathione peroxidase, thereby protecting cells from damage. There are three isoenzymes of SOD protein in mammalian bodies, namely SOD1 (copper-zinc superoxide dismutase) located in the cytoplasm, SOD2 (manganese superoxide dismutase) located in the mitochondria, and extracellular SOD3 located in the extracellular fluid. Sirt1 protein is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, which removes acetyl groups from various proteins. The SOD1 gene, the SOD2 gene, and the Sirt1 gene are genes encoding the enzyme proteins SOD1, SOD2, and Sirt1 respectively, and the level of their expression reflects the ability of cells to resist oxidative stress.

[0016] According to some embodiments of the present invention, improving the developmental ability of in vitro embryos includes increasing the blastocyst rate of in vitro embryos, reducing the ROS content of in vitro embryo cells and / or increasing the GSH content of in vitro embryo cells, and increasing the expression level of oxidative stress genes in in vitro embryo cells.

[0017] In some embodiments, improving the developmental ability of in vitro embryos is to increase the blastocyst rate of in vitro embryos. Preferably, as verified by the in vitro embryo blastocyst rate test, compared with the case of not applying the product of the present invention, applying the product of the present invention increases the blastocyst rate of in vitro embryos by 2% - 90%.

[0018] Specifically, in some embodiments of the present invention, compared with the situation where the product of the present invention is not applied, applying the product of the present invention increases the in vitro embryo blastocyst rate by 45%-85%. In some specific embodiments of the present invention, applying the product of the present invention increases the in vitro embryo blastocyst rate by at least about 82%. In some other embodiments of the present invention, applying the product of the present invention increases the in vitro embryo blastocyst rate to 25%-65%; preferably, applying the product of the present invention increases the in vitro embryo blastocyst rate to 40-60%, and more preferably, at least to about 57%.

[0019] In some embodiments, the improvement of the in vitro embryo development ability is to reduce the ROS content in in vitro embryo cells. Preferably, as verified by the test of the ROS content in in vitro embryo cells, compared with the situation where the product of the present invention is not applied, applying the product of the present invention reduces the ROS content in in vitro embryo cells by 10%-30%.

[0020] Specifically, in some embodiments of the present invention, compared with the situation where the product of the present invention is not applied, applying the product of the present invention reduces the ROS content in in vitro embryo cells by 15%-25%. In some specific embodiments of the present invention, applying the product of the present invention reduces the ROS content in in vitro embryo cells by at least about 21%.

[0021] In some embodiments, the improvement of the in vitro embryo development ability is to increase the GSH content in in vitro embryo cells. Preferably, as verified by the test of the GSH content in in vitro embryo cells, compared with the situation where the product of the present invention is not applied, applying the product of the present invention increases the GSH content in in vitro embryo cells by 10%-20%.

[0022] Specifically, in some embodiments of the present invention, compared with the situation where the product of the present invention is not applied, applying the product of the present invention increases the GSH content in in vitro embryo cells by 10%-15%. In some specific embodiments of the present invention, applying the product of the present invention increases the GSH content in in vitro embryo cells by at least about 13%.

[0023] In some embodiments, the improvement of the in vitro embryo development ability is to increase the expression level of oxidative stress genes in in vitro embryo cells. Preferably, as verified by the test of the expression level of oxidative stress genes in in vitro embryo cells, compared with the situation where the product of the present invention is not applied, applying the product of the present invention increases the expression level of oxidative stress genes in in vitro embryo cells by 50%-100%.

[0024] Specifically, in some embodiments of the present invention, compared with the situation where the product of the present invention is not applied, applying the product of the present invention increases the expression level of oxidative stress genes in in vitro embryo cells by 60%-90%. In some specific embodiments of the present invention, applying the product of the present invention increases the expression level of oxidative stress genes in in vitro embryo cells by at least about 88%.

[0025] In the context of the numerical values regarding the effect of increasing or decreasing the in vitro embryo development ability, the numerical values of "increased to" or "decreased to" used are the measured values in the case of applying the product of the present invention. In the context of the numerical value regarding the effect of increasing the in vitro embryo development ability, the numerical value of "increased by" is calculated as follows: [(measured value of applying the product of the present invention) - (measured value of not applying the product of the present invention)] / (measured value of not applying the product of the present invention). In the context of the numerical value regarding the effect of decreasing the in vitro embryo development ability, the numerical value of "decreased by" is calculated as follows: [(measured value of not applying the product of the present invention) - (measured value of applying the product of the present invention)] / (measured value of not applying the product of the present invention).

[0026] According to some embodiments of the present invention, there is provided the use of dihydromyricetin and / or its derivatives in the preparation of a product for increasing the in vitro embryo blastocyst rate.

[0027] According to some embodiments of the present invention, there is provided the use of dihydromyricetin and / or its derivatives in the preparation of a product for reducing the ROS content in in vitro embryo cells.

[0028] According to some embodiments of the present invention, there is provided the use of dihydromyricetin and / or its derivatives in the preparation of a product for increasing the GSH content in in vitro embryo cells.

[0029] According to some embodiments of the present invention, there is provided the use of dihydromyricetin and / or its derivatives in the preparation of a product for increasing the expression levels of SOD1 gene, SOD2 gene, and Sirt1 gene in in vitro embryo cells.

[0030] Preferably, the product includes a pharmaceutical composition, a culture medium, and a kit. Preferably, the in vitro embryo includes mammalian in vitro embryos. Preferably, the mammal includes but is not limited to humans, domestic animals, farm animals, zoo animals, sports animals, pet animals (such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, camels, bison, domestic cattle, dairy cows), primates (such as apes, monkeys, orangutans, and chimpanzees), canids (such as dogs and wolves), felids (such as cats, lions, and tigers), equids (such as horses, donkeys, and zebras), food animals (such as dairy cows, pigs, and sheep), ungulates (such as deer and giraffes), and rodents (such as mice, rats, hamsters, and guinea pigs). In a preferred embodiment, the mammal includes pigs.

[0031] Preferably, the mammalian in vitro embryo includes mammalian in vitro parthenogenetically activated embryos and mammalian in vitro fertilized embryos. Preferably, the mammalian in vitro embryo includes parthenogenetically activated oocytes, fertilized eggs, and 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst developed from parthenogenetically activated oocytes and / or fertilized eggs.

[0032] In a second aspect of the present invention, there is provided a product for improving the in vitro embryo development ability, which product comprises dihydromyricetin and / or its derivatives.

[0033] Preferably, the dihydromyricetin derivatives include esterified products, acylated products, metal complexes, and glycosylated products of dihydromyricetin.

[0034] Preferably, the product for improving the in vitro embryo development ability comprises a pharmaceutical composition, a culture medium, and a kit.

[0035] Preferably, the drug further comprises a pharmaceutically acceptable excipient. Preferably, the excipient includes a pharmaceutically acceptable diluent, excipient, carrier, binder, lubricant, suspending agent, coating agent, solubilizer. The drug can be in various forms, such as liquid, semi-solid, and solid dosage forms. In some other embodiments of the present invention, the form of the drug includes a liquid solution (such as an injectable and infusible solution), a spray, a dispersion, or a suspension. In some specific embodiments of the present invention, the drug is a liquid solution.

[0036] Preferably, the culture medium further comprises a basal medium, inorganic salts, a solvent, antibiotics, and nutritional functional factors beneficial to the development of in vitro embryo cells.

[0037] Preferably, the basal medium includes TCM-199 medium, PZM-5 medium, CR1-aa medium, Ham's F10 medium, Ham's F12, NCSU-37 medium, NCSU-23 medium.

[0038] Preferably, the inorganic salts include sodium chloride, potassium chloride, potassium dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, and sodium bicarbonate.

[0039] Preferably, the antibiotics include gentamicin, penicillin, streptomycin, and kanamycin.

[0040] Preferably, the nutritional functional factors beneficial to the development of in vitro embryo cells include hormones, energy substrates, amino acids, and growth factors.

[0041] According to some embodiments of the present invention, the hormones include follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), pregnant mare serum gonadotropin (PMSG), and estradiol (E2).

[0042] According to some embodiments of the present invention, the energy substrates include glucose, pyruvate, and lactate.

[0043] According to some embodiments of the present invention, the amino acids include essential amino acids and non-essential amino acids. According to some specific embodiments of the present invention, the amino acids include L-cysteine, L-isoleucine, L-leucine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-arginine hydrochloride, L-cystine dihydrochloride, L-histidine hydrochloride monohydrate, L-lysine hydrochloride.

[0044] According to some embodiments of the present invention, the growth factors include epidermal growth factor (EGF), insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF).

[0045] In a third aspect of the present invention, there is provided the use of dihydromyricetin and / or its derivatives in improving the quality of in vitro embryos. Preferably, there is provided the use of dihydromyricetin and / or its derivatives in improving the developmental ability of in vitro embryos.

[0046] According to some embodiments of the present invention, there is provided the use of dihydromyricetin and / or its derivatives in increasing the blastocyst rate of in vitro embryos.

[0047] According to some embodiments of the present invention, there is provided the use of dihydromyricetin and / or its derivatives in reducing the ROS content in in vitro embryo cells.

[0048] According to some embodiments of the present invention, there is provided the use of dihydromyricetin and / or its derivatives in increasing the GSH content in in vitro embryo cells.

[0049] According to some embodiments of the present invention, there is provided the use of dihydromyricetin and / or its derivatives in increasing the expression level of oxidative stress genes in in vitro embryo cells. According to some specific embodiments of the present invention, the oxidative stress genes include SOD1 gene, SOD2 gene, and Sirt1 gene.

[0050] Preferably, the in vitro embryos include mammalian in vitro embryos. Preferably, the in vitro embryos include mammalian in vitro parthenogenetically activated embryos. More preferably, the in vitro embryos include porcine in vitro parthenogenetically activated embryos. More preferably, the in vitro embryos include porcine in vitro parthenogenetically activated oocytes.

[0051] In a fourth aspect of the present invention, there is provided a method for improving the developmental ability of in vitro embryos, which comprises the following steps:

[0052] Applying an effective amount of dihydromyricetin and / or its derivatives to the in vitro embryos.

[0053] This method can be used for non-therapeutic purposes. Specifically, this method can be used for scientific research, such as in vitro cell culture research, in vitro embryo culture research, and pre-implantation embryo development mechanism research, etc.

[0054] According to some embodiments of the present invention, the method comprises the following steps:

[0055] S1. Applying an effective amount of dihydromyricetin and / or its derivatives to in vitro embryos;

[0056] S2. Culturing the in vitro embryos until the blastocyst stage.

[0057] Preferably, the effective amount of dihydromyricetin and / or its derivatives can be administered in the form of the product obtained in the embodiments of the first aspect of the present invention or the product of the embodiments of the second aspect.

[0058] Preferably, the dihydromyricetin and / or its derivatives in S1 can be applied at any time point during the development of in vitro embryos to the blastocyst stage, including any time point after parthenogenetic activation of oocytes, after fertilization of sperm and eggs, and at the 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst stages.

[0059] Preferably, the dihydromyricetin and / or its derivatives in S1 can be administered once, but more preferably multiple times. According to some embodiments of the present invention, the dihydromyricetin and / or its derivatives can be administered from once a day to once a week. Specifically, the dihydromyricetin and / or its derivatives can be administered once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once a week.

[0060] Preferably, the effective amount in S1 is 1-10 μmol / L, preferably 2.5-7.5 μmol / L, more preferably 4.5-6.5 μmol / L. As used in this specification and in the claims, the term "effective amount" refers to the amount of the compound sufficient to provide the desired effect compared to the case where the compound is not administered, and this amount is calculated based on the mass of the dihydromyricetin and / or its derivatives applied and the volume of the liquid environment (such as the culture medium) applied; the desired effects include significantly increasing the in vitro embryo blastocyst rate, and / or significantly reducing the ROS content in in vitro embryo cells, and / or significantly increasing the GSH content in in vitro embryo cells, and / or significantly increasing the expression level of oxidative stress genes in in vitro embryo cells, and the oxidative stress genes include SOD1 gene, SOD2 gene, and Sirt1 gene. Significantly increasing the in vitro embryo blastocyst rate means that the in vitro embryo blastocyst rate is increased by 2%-90% compared to the case where the compound is not administered, preferably increased by 45%-85%, more preferably increased by at least about 82%. Significantly reducing the ROS content in in vitro embryo cells means that the ROS content in in vitro embryo cells is reduced by 10%-30% compared to the case where the compound is not administered, preferably reduced by 15%-25%, more preferably reduced by at least about 21%. Significantly increasing the GSH content in in vitro embryo cells means that the GSH content in in vitro embryo cells is increased by 10%-20% compared to the case where the compound is not administered, preferably increased by 10%-15%, more preferably increased by at least about 13%. Significantly increasing the expression level of oxidative stress genes in in vitro embryo cells means that the expression level of oxidative stress genes in in vitro embryo cells is increased by 50%-100% compared to the case where the compound is not administered, more preferably increased by 60%-90%, more preferably increased by at least about 88%.

[0061] Preferably, the culture temperature in S2 is 36-40 °C and the culture time is 40-50 h.

[0062] The beneficial effects of the present invention are:

[0063] The present invention discloses for the first time the application of dihydromyricetin or its derivatives in the preparation of drugs for improving the in vitro embryo development ability. There has been no previous report on the correlation between dihydromyricetin or its derivatives and the in vitro embryo development ability, which expands the application scope of dihydromyricetin or its derivatives.

[0064] Dihydromyricetin or its derivatives can effectively improve the in vitro development ability and embryo quality of embryos by increasing the blastocyst rate, reducing the ROS content in cells, increasing the GSH content in cells, and increasing the expression level of oxidative stress genes in cells.

[0065] Dihydromyricetin is a natural flavonoid extract, which is non-toxic and has no side effects on mammalian cells. It has a wide source and is easy to obtain. Adding dihydromyricetin or its derivatives to the culture medium is simple to operate, easy to control, and has a significant effect.

[0066] Dihydromyricetin or its derivatives can be used to prepare pharmaceutical compositions, culture media or kits for promoting embryonic development, etc., and have good industrial application prospects. Description of the Drawings

[0067] The following examples will be described in conjunction with the accompanying drawings, which will make the above and / or additional aspects and advantages of the present invention obvious and easy to understand, where:

[0068] Figure 1 Shows the effect of different concentrations of dihydromyricetin on the blastocyst rate of in vitro embryos in Example 2; shows the blastocyst rates on the 6th and 7th days of culture with different concentrations of dihydromyricetin; the data are expressed as mean ± standard error; n = 5 independent parallel experiments; the significant difference is expressed as: ***P < 0.001.

[0069] Figure 2A Shows the effect of dihydromyricetin on the relative content of reactive oxygen species (ROS) in 4-cell in vitro embryos in Example 3; the ROS fluorescence intensity of the control group was set to 1.0, and the relative ROS fluorescence intensity of the experimental group embryos relative to the control group was shown; the data are expressed as mean ± standard error; n = 4 independent parallel experiments; the significant difference is expressed as: ***P < 0.001.

[0070] Figure 2B Shows the effect of dihydromyricetin on the relative content of glutathione (GSH) in 4-cell in vitro embryos in Example 3. The GSH fluorescence intensity of the control group was set to 1.0, and the relative GSH fluorescence intensity of the experimental group embryos relative to the control group was shown; the data are expressed as mean ± standard error; n = 4 independent parallel experiments; the significant difference is expressed as: ***P < 0.001.

[0071] Figure 3 Shows the effect of dihydromyricetin on the expression levels of oxidative stress genes in blastocyst cells in Example 4; the expression levels of SOD1 gene, SOD2 gene, and Sirt1 gene in the control group were set to 1.0, and the relative expression levels of SOD1 gene, SOD2 gene, and Sirt1 gene in the experimental group blastocyst cells relative to the control group were shown; the data are expressed as mean ± standard error; n = 4 independent parallel experiments. Detailed Embodiments

[0072] The present invention will be further described in detail below in conjunction with embodiments, but the implementation manners of the present invention are not limited thereto. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention and should not be construed as a limitation of the present invention.

[0073] Pretreatment of material samples

[0074] 1. Collection of porcine oocytes:

[0075] Ovaries from prepubertal gilts were collected from a slaughterhouse and transported to the laboratory in 0.9% saline at 37°C. Cumulus-oocyte complexes (COCs) and follicular fluid in follicles with a diameter of 3 - 6 mm in the ovaries were aspirated with a syringe (18-gauge needle) and placed in a shaking tube. The tube was left standing in a 38°C water bath for 15 min until the COCs settled to the bottom of the tube, and the supernatant was discarded. An appropriate amount of 199 medium containing HEPES (GIBCO, 12340030) with 0.1% (w / v) bovine serum albumin (BSA, SIGMA, A3311) was added, and the precipitate was washed repeatedly 3 times (15 min, 5 min, 3 min). The washed precipitate was aspirated with a Pasteur pipette and transferred to a 90 mm × 20 mm culture dish. Under a stereomicroscope (Nikon, SMZ745), COCs were aspirated with a glass pipette, and the cumulus should be wrapped in three or more layers.

[0076] 2. In vitro maturation of porcine oocytes:

[0077] COCs were cultured in a medium containing the following components. The culture plates were placed in an incubator at 38°C, 5% CO2, and maximum saturated humidity for 45 h. The medium included: 199 medium (GIBCO, 11150-059), 0.6 mmol / L cysteine (SIGMA, C7477), 10 ng / mL epidermal growth factor (SIGMA, E4127), 10 IU / mL follicle-stimulating hormone (Ningbo No. 2 Hormone Factory), 10 IU / mL luteinizing hormone (Ningbo No. 2 Hormone Factory), 1% penicillin-streptomycin (GIBCO, 15140-122), 0.1 g / L sodium pyruvate (SIGMA, P4562), and 10% porcine follicular fluid.

[0078] 3. Parthenogenetic activation of porcine oocytes:

[0079] After the oocytes matured, the cumulus oocyte complexes were repeatedly pipetted with hyaluronidase to remove granulosa cells until the zona pellucida was completely transparent, and denuded oocytes were obtained. The denuded oocytes were transferred to a 297 mmol / L (mM) mannitol solution (pH 7.2, SIGMA, M9546), which contained 0.1 mM CaCl2 (SIGMA, C7902), 0.05 mM MgSO4 (SIGMA, M1880), 0.01% (w / v) polyvinyl alcohol (PVA, SIGMA, P8136), and 0.5 mM HEPES buffer (SIGMA, H6147). The denuded oocytes were parthenogenetically activated by applying direct current pulses with an electric field strength of 1.2 kV / cm for 60 μs each time, for a total of two times.

[0080] Example 1: Applying dihydromyricetin to porcine parthenogenetically activated embryos and in vitro embryo culture:

[0081] 1. The parthenogenetically activated oocytes were cultured in a porcine embryo in vitro culture medium (containing 7.5 mg / mL cytochalasin B) for 3 h. Among them, the porcine embryo in vitro culture medium consisted of the following components: 108 mmol / L (mM) sodium chloride (sigma, S5886), 10 mM potassium chloride (sigma, P5405), 0.35 mM potassium dihydrogen phosphate (sigma, P5655), 0.4 mM magnesium sulfate (sigma, M2643), 25 mM sodium bicarbonate (sigma, S5761), 0.2 mM sodium pyruvate (sigma, P5280), 2.0 mM calcium lactate (sigma, C8356), 2.0 mM glutathione (sigma, G8540), 5.0 mM taurine (sigma, H1384), 20 mL / L essential amino acids (sigma, B6766), 10 mL / L non-essential amino acids (sigma, M7145), 25 mg / mL gentamicin (sigma, B6766), 4 mg / mL bovine serum albumin (sigma, A8806), and added sterilized ultrapure water to make up to 1 L.

[0082] 2. The medium was removed, and a porcine embryo in vitro culture medium supplemented with dihydromyricetin (Sigma, SML0295) (adding dihydromyricetin to the medium formula in step 1) was applied to the parthenogenetically activated oocytes. Three experimental groups were set up, adding 2.5, 5, and 10 μmol / L (μM) dihydromyricetin respectively; one control group was set up, and a porcine embryo in vitro culture medium without dihydromyricetin was applied to the parthenogenetically activated oocytes.

[0083] 3. The oocytes were cultured in an incubator at 38.5 °C, 5% CO2, and saturated humidity for 7 days, and the medium did not need to be changed during this period.

[0084] Example 2 Application of Dihydromyricetin in Improving the Blastocyst Rate of In Vitro Embryos

[0085] Testing the blastocyst rate of porcine parthenogenetically activated embryos in vitro: The oocytes in Example 1 were cultured in vitro for 6 days and 7 days, and the blastocyst rates of the control group and 3 experimental groups were observed respectively. The blastocyst rate = (number of blastocysts formed / number of all cultured early parthenogenetic embryos) × 100%.

[0086] The results are as Figure 1 shown. Compared with the control group (blastocyst rate on day 6: 27.23% ± 1.22%; on day 7: 37.74% ± 3.16%), adding 2.5 μmol / L (μM) and 5 μM of dihydromyricetin to the porcine embryo in vitro culture medium can both improve the blastocyst rates of porcine parthenogenetically activated embryos on day 6 and day 7. When the concentration of dihydromyricetin is 5 μM, the blastocyst rate on day 6 is 49.62% ± 4.37%, which is 82.23% higher than that of the control group; on day 7: 56.53% ± 1.75%, which is 49.79% higher than that of the control group; both are significantly higher than the control group (P < 0.001); when the concentration is 2.5 μM, the blastocyst rate on day 6 is 27.82% ± 4.41%, which is 2.17% higher than that of the control group; on day 7: 40.61% ± 6.81%, which is 7.60% higher than that of the control group; however, when the concentration of dihydromyricetin is 10 μM, the blastocyst rate is lower than that of the control group (blastocyst rate on day 6: 19.10% ± 5.59%; on day 7: 25.29% ± 8.77%).

[0087] Adding dihydromyricetin at a specific concentration (2.5 - 5 μM) to the culture medium helps to promote the smooth development of parthenogenetically activated oocytes to blastocysts. Whether the oocytes can develop smoothly to blastocysts after activation is the key to in vitro embryo culture. Only early embryos with good developmental ability and quality can develop to blastocysts, and subsequent in vivo transplantation using blastocysts can improve the embryo implantation rate. It can be seen that dihydromyricetin can improve the developmental ability and quality of early embryos before implantation, laying a good foundation for embryo implantation, and also indicating that dihydromyricetin has good application prospects in the preparation of products for improving the blastocyst rate of in vitro embryos.

[0088] Example 3 Application of Dihydromyricetin in Reducing Reactive Oxygen Species (ROS) in In Vitro Embryo Cells and Increasing Cellular Glutathione (GSH)

[0089] Testing the relative contents of ROS and GSH in porcine parthenogenetically activated embryo cells

[0090] The oocytes of the experimental group (the addition amount of dihydromyricetin in Example 1 was 5 μM) and the control group (without adding dihydromyricetin in Example 1) were cultured in vitro until the 4-cell stage of in vitro parthenogenetic embryos. For the early embryonic cells of the experimental group and the control group, 30 oocytes were taken respectively and stained with 2’,7’-dichlorodihydrofluorescein diacetate (DCHF-DA) (Beyotime, S0033S) for ROS, and stained with 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (invitrogen, C12881) for GSH. They were placed on a hot stage for 30 min, then washed 3 times with 0.1% polyvinyl alcohol-phosphate buffer solution, and then photographed under a fluorescence microscope. For ROS detection, the excitation wavelength was 460 nm, and for GSH detection, the excitation wavelength was 370 nm. The fluorescence images were analyzed with Image J software.

[0091] The results are shown in Figure 2, where Figure 2A shows the effect of dihydromyricetin on the relative content of ROS in 4-cell in vitro embryos. The ROS content in the 4-cell of the dihydromyricetin treatment group (5 μM) was 0.8 times that of the control group, which was 20% lower than that of the control group. It can be seen that adding 5 μM dihydromyricetin to the culture medium can significantly reduce the ROS content in in vitro embryonic cells; Figure 2B shows the effect of dihydromyricetin on the relative content of GSH in 4-cell in vitro embryos. The GSH content in the 4-cell of the dihydromyricetin treatment group (5 μM) was 1.2 times that of the control group, which was 12% higher than that of the control group. It can be seen that adding 5 μM dihydromyricetin to the culture medium can significantly increase the GSH content in in vitro embryonic cells. It shows that dihydromyricetin has good application prospects in the preparation of products for reducing the ROS content and / or increasing the GSH content in in vitro embryonic cells.

[0092] Example 4 Application of dihydromyricetin in increasing the expression level of oxidative stress genes in in vitro embryonic cells

[0093] Test on the expression levels of oxidative stress genes (SOD1 gene, SOD2 gene, Sirt1 gene) in porcine blastocyst cells:

[0094] The oocytes of the experimental group (the addition amount of dihydromyricetin in Example 1 was 5 μM) and the control group (without adding dihydromyricetin in Example 1) were cultured in vitro to the blastocyst stage of porcine early parthenogenetic embryos (7 days after in vitro culture of oocytes), and the blastocyst cells were collected and lysed in 50 μL of lysis buffer. According to the manufacturer's instructions, the mRNA in blastocyst cells was extracted using a kit (DynaBeads mRNA Direct Kit) on ice, and cDNA was synthesized by reverse transcription of mRNA. Then, real-time fluorescence quantitative PCR was performed using a kit (KAPA FAST) according to the instructions, and qPCR reaction was carried out using a CFXConnect Optics Module PCR instrument. The PCR reaction system was 20 μL of cDNA, 1 μL of upstream primer, 1 μL of downstream primer, 10 μL of SYBR Green, and 7 μL of ddH2O. The names and sequences of each primer are shown in Table 1, where the GAPDH gene was used as an internal reference gene for reference.

[0095] Table 1 Names and sequences of each primer for qPCR reaction

[0096]

[0097] The results are as Figure 3 shown. The expressions of SOD1 gene, SOD2 gene, and Sirt1 gene in blastocyst cells of the dihydromyricetin treatment group (5 μM) were 1.66 times, 1.87 times, and 1.55 times that of the control group, respectively. It can be seen that adding 5 μM of dihydromyricetin to the culture medium can increase the expression level of oxidative stress genes in in vitro embryo cells, and at the same time, it also indicates that dihydromyricetin has good application prospects in the preparation of products for increasing the expression level of oxidative stress genes in in vitro embryo cells.

[0098] Data statistics: In Examples 2 - 4, SPSS software was used for statistical analysis, and the student t-test was used to compare two groups of data. One-way analysis of variance (ANOVA) was used to analyze three or more means.

[0099] Example 5 In vitro embryo culture medium

[0100] An in vitro embryo culture medium, which includes 5 μM of dihydromyricetin, and also includes a basal medium, inorganic salts, a solvent, antibiotics, and nutritional functional factors beneficial to the development of in vitro embryo cells.

[0101] In summary, the present invention demonstrates that dihydromyricetin or its derivatives can increase the blastocyst rate of in vitro embryo culture, reduce the intracellular ROS content, increase the intracellular GSH content, and promote the expression of oxidative stress genes, thereby effectively improving the quality and developmental ability of in vitro embryos. Dihydromyricetin or its derivatives have good market application prospects in the preparation of products for improving the quality and developmental ability of in vitro embryos.

[0102] The above-described embodiments are only preferred embodiments of the present invention and are not intended to limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made to the structure, features, and principles described in the scope of the patent application of the present invention should be included within the protection scope of the present invention's patent application.

Claims

1. Use of an effective amount of dihydromyricetin in the preparation of a product for improving the in vitro embryo development ability, wherein the effective amount is 2.5 - 5 μmol / L.

2. The application according to claim 1, characterized in that, The improvement of the in vitro embryo development ability is to increase the blastocyst rate of in vitro embryos.

3. The application according to claim 1, wherein The improvement of the in vitro embryo development ability is to reduce the content of reactive oxygen species in in vitro embryo cells and / or increase the content of glutathione in in vitro embryo cells.

4. The application according to claim 1, characterized in that The improvement of the in vitro embryo development ability is to increase the expression level of oxidative stress genes in in vitro embryo cells.

5. The application according to claim 1, characterized in that The product for improving the in vitro embryo development ability includes a pharmaceutical composition, a culture medium, or a kit.

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