Application of phenethyl caffeate in preparation of product for promoting in-vitro embryonic development

Abstract

The invention discloses application of caffeic acid phenethyl ester in preparation of products for promoting embryonic development, belongs to the technical field of in-vitro embryos, and relates to an in-vitro embryo cultivation technology before implantation of mammals. The invention discloses the application of the caffeic acid phenethyl ester in preparation of the medicine for improving the in-vitro embryonic developmental ability for the first time, the correlation between the caffeic acid phenethyl ester and the in-vitro embryonic developmental ability is not reported before, and the application range of the caffeic acid phenethyl ester is expanded. The caffeic acid phenethyl ester has the effects of improving the blastocyst rate of in-vitro embryo culture, improving the number of blastocyst cells of in-vitro embryo culture, protecting the mitochondrial function of in-vitro embryo cells, improving the oxidation resistance of the in-vitro embryo cells and reducing endoplasmic reticulum stress response, autophagy and cell apoptosis of in-vitro embryos; therefore, the development quality and development capability of in-vitro embryos are effectively improved.

Description

Technical Field The invention belongs to the field of in vitro embryo technology, relates to in vitro cultivation technology of mammal pre-implantation embryos, and in particular to the application of caffeic acid phenethyl ester in the preparation of a product for promoting in vitro embryo development. Background Art The increasing use of advanced reproductive technologies such as in vitro fertilization, embryo production, animal cloning and transgenics requires high-quality embryos. [1] In vitro embryo production (IVEP) is able to produce large numbers of viable embryos and can have applications ranging from the treatment of human reproductive disorders to the preservation of gametes in animals with high genetic conservation value, thus contributing to the understanding of early embryonic development in mammals. However, it is well known that IVEP technology has various disadvantages, such as developmental arrest during in vitro embryo development and low offspring survival rate. [2],[3] , mammalian embryos developed in vitro have lower developmental competence than embryos developed in vivo [4] Oocytes and embryos cultured in vitro are more susceptible to external harmful physical and chemical factors (such as high oxygen, temperature, toxins, etc.) due to the lack of protection of the natural antioxidant system in the in vivo environment. Caffeic acid phenethyl ester, one of the chemical components of propolis, is a phenolic compound with antioxidant, antiviral, anti-inflammatory, anti-cancer and immunomodulatory properties. It also has the functions of lowering blood sugar, protecting the heart and inhibiting gastric secretion. [57 However, there is no information on the use of caffeic acid phenethyl ester in the preparation of products for improving the quality and developmental capacity of in vitro embryos.

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[0007] Kleczka A,Kubina R,Dzik R,et al.Caffeic Acid Phenethyl Ester(CAPE)Induced Apoptosis in Serous Ovarian Cancer OV7 Cells by Deregulation of BCL2 / BAX Genes[J].Molecules(Basel,Switzerland),2020,25(15). Summary of the invention The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes the use of caffeic acid phenethyl ester in the preparation of a product for promoting in vitro embryo development, which can effectively improve the quality and development ability of in vitro embryo culture and is conducive to in vitro embryo culture. The first aspect of the present invention provides the use of caffeic acid phenethyl ester in preparing a product for improving in vitro embryo development. According to a preferred embodiment of the present invention, at least the following beneficial effects are achieved: The present invention discloses for the first time the application of caffeic acid phenethyl ester in the preparation of a drug for improving the in vitro embryo development ability. Previously, there has been no report that caffeic acid phenethyl ester is related to the in vitro embryo development ability, which expands the application scope of caffeic acid phenethyl ester. Caffeic acid phenethyl ester can significantly improve the quality and proliferation ability of pig embryos by increasing the blastocyst rate, the total cell number of blastocysts and the cell proliferation level and the expression level of embryo development-related genes (BMP15, PCNA, OCT4, CCNB1, CDK2, CDK1, SOX2, Nanog) in early preimplantation embryos. The component reduces the accumulation of reactive oxygen, increases the content of glutathione, increases the expression level of Nrf2 protein and the expression level of antioxidant-related genes (SOD1, SOD2, GPX, CAT, PRDX2) to improve the antioxidant level of embryos. This ingredient improves mitochondrial function by increasing mitochondrial membrane potential, regulating mitochondrial biogenesis, increasing ATP content, and increasing the expression level of mitochondrial function-related genes (PGC1α, TFAM, TFB1M, TFB2M, NRF1, NRF2, SIRT1, ATP5B). This ingredient reduces the protein expression level of the endoplasmic reticulum stress marker GRP78 and the expression level of endoplasmic reticulum stress-related genes (CHOP, uXBP1, sXBP1, GRP78, ATF4, ATF6), reduces the protein expression level of the autophagy-related protein LC3B and the expression level of autophagy-related genes (LC3B,, P62), reduces the cell apoptosis rate and the expression level of pro-apoptotic genes (CASP3, JNK, BAX), and increases the expression level of the anti-apoptotic gene BCL2, showing its role in alleviating endoplasmic reticulum stress response, autophagy and cell apoptosis, thereby improving the quality of in vitro embryo culture and developmental ability in many aspects, which is beneficial to in vitro embryo culture. Caffeic acid phenethyl ester can be used to prepare pharmaceutical compositions, culture media or kits for promoting embryonic development, and has good industrial application prospects. Caffeic acid phenethyl ester is a phenolic compound extracted from propolis, which has the following chemical structure: According to some embodiments of the present invention, the promoting of in vitro embryo development includes at least one of improving embryo quality or improving embryo development ability. According to some embodiments of the present invention, the improvement of in vitro embryo development ability includes at least one of increasing the embryo blastocyst rate, increasing the total cell number of embryo blastocyst, enhancing the embryo's antioxidant capacity, increasing the embryo's mitochondrial abundance, increasing the embryo's mitochondrial membrane potential level, reducing the embryo's endoplasmic reticulum stress level, and reducing the embryo's autophagy and cell apoptosis levels. According to some embodiments of the present invention, the product is a kit, a culture medium or a drug. According to some specific embodiments of the present invention, such as by detecting / calculating the blastocyst rate, the total number of blastocyst cells, the EdU positive cell rate, the content of ROS and GSH, the expression of Nrf2 protein, the abundance of mitochondria, the level of mitochondrial membrane potential, the content of ATP, the expression of PGC1α+β protein, the expression of endoplasmic reticulum stress protein GRP78, the expression of autophagy-related LC3B protein, the apoptosis rate, and the embryonic development-related genes (BMP15, PCNA, OCT4, CCNB1, CDK2, CDK1, SOX2, Nanog), the antioxidant-related genes (S Caffeic acid phenethyl ester improved the quality and developmental ability of in vitro embryos, as demonstrated by the gene expression levels of genes related to mitochondrial function (PGC1α, TFAM, TFB1M, TFB2M, NRF1, NRF2, SIRT1, ATP5B), anti-apoptotic gene BCL2, endoplasmic reticulum stress-related genes (CHOP, uXBP1, sXBP1, GRP78, ATF4, ATF6), autophagy-related genes (LC3B, P62), and pro-apoptotic genes (CASP3, JNK, BAX). According to some embodiments of the present invention, the quality and developmental capacity of in vitro embryos are evaluated by detecting / calculating the blastocyst rate, the total number of blastocyst cells and the expression levels of related genes of the in vitro embryos. According to some embodiments of the present invention, the quality and developmental ability of in vitro embryos are evaluated by detecting / calculating the proliferation ability of in vitro embryonic cells and the expression levels of embryonic development-related genes (BMP15, PCNA, OCT4, CCNB1, CDK2, CDK1, SOX2, Nanog), wherein the proliferation ability of in vitro embryonic cells can be evaluated by detecting / calculating the EdU positive cell rate and the total number of cells in the in vitro embryo. Cell proliferation detection is a commonly used biological experimental method, which mainly reflects the growth, cycle and vitality of cells. The incorporation of 5-ethynyl-2'-deoxyuridine (EdU) can directly detect DNA synthesis in cells and reflect the S phase of the cell cycle. EdU is a new thymine deoxynucleotide analog that can be incorporated into newly synthesized DNA instead of thymidine during DNA synthesis. The acetylene group coupled to EdU can react with the azide coupled to the fluorescent probe through the catalysis of monovalent copper ions to form a stable triazole ring, which is referred to as the click reaction. The newly synthesized DNA can be labeled with the corresponding fluorescent probe. The proliferating cells can be detected using appropriate fluorescence detection equipment, and the proliferation capacity of the embryo can be evaluated by calculating the EdU-positive cell rate (the ratio of the number of EdU-positive cells to the total number of cells). According to some embodiments of the present invention, the quality and developmental ability of in vitro embryos are evaluated by detecting / calculating the content of ROS and GSH in in vitro embryonic cells, the expression of Nrf2 protein, and the expression level of antioxidant-related genes (SOD1, SOD2, GPX, CAT, PRDX2). When the redox balance in the body is disturbed and the free radicals produced exceed the antioxidant scavenging capacity of the body, it will lead to the accumulation of reactive oxygen species (ROS), reactive nitrogen species (RNS), etc. in cells and organs, causing oxidative damage. ROS is an inevitable byproduct in the cell process and plays an important role in cell signal transduction. ROS is the most direct evaluation index for detecting the content of oxidative free radicals, GSH is one of the most commonly used non-enzymatic antioxidant substances, and Nrf2 is involved in the induction and expression of a series of genes encoding cell protection proteins. Its protein can be translocated to the nucleus and combined with antioxidant response elements, and can also be used as an evaluation index to judge the antioxidant capacity of cells, thereby reflecting the quality and developmental ability of embryos. According to some embodiments of the present invention, the quality and developmental capacity of in vitro embryos are evaluated by detecting / calculating the mitochondrial abundance, mitochondrial membrane potential level, ATP content, expression of PGC1α+β protein and expression level of mitochondrial function-related genes (PGC1α, TFAM, TFB1M, TFB2M, NRF1, NRF2, SIRT1, ATP5B) of in vitro embryonic cells. Mitochondria are not only the main producers of ROS, but also the main place for cell energy production, playing a key role in the regulation of cell growth, information transmission and apoptosis. Oxidative phosphorylation in mitochondria provides fuel for processes such as gamete formation, fertilization and early embryonic development, and the balance of mitochondria is crucial to the successful reproduction of mammals. Mitochondria are the main place for animal and plant cells to generate ATP, and are important organelles that promote cell energy conversion and participate in cell apoptosis. When generating energy, mitochondria store electrochemical potential energy in the inner membrane of mitochondria. On both sides of the inner membrane, if the concentration of protons and other ions is asymmetrically distributed, the mitochondrial membrane potential will be formed. PGC-1α and PGC-1β are the master regulators of mitochondrial biogenesis and function, inducing mitochondrial biogenesis by activating NRF1 / NRF2. Therefore, they can be used as indicators of mitochondrial function along with mitochondrial abundance, mitochondrial membrane potential, and ATP content. According to some embodiments of the present invention, the quality and developmental capacity of in vitro embryos are evaluated by detecting / calculating the expression level of the endoplasmic reticulum stress protein GRP78 of in vitro embryonic cells and the expression level of endoplasmic reticulum stress-related genes (CHOP, uXBP1, sXBP1, GRP78, ATF4, ATF6). The endoplasmic reticulum and mitochondria are two important organelles with high dynamics in mammalian cells. The processing, modification and folding of proteins in the endoplasmic reticulum have a decisive influence on cells. High levels of endoplasmic reticulum stress can cause cell damage, so cells initiate an unfolded protein response (UPR) to alleviate endoplasmic reticulum stress caused by adverse external stimuli. Glucose-regulated protein 78 (GRP78) forces unfolded proteins to refold or be degraded by cellular degradation mechanisms, and is a marker protein that can respond to endoplasmic reticulum stress in cells. According to some embodiments of the present invention, the quality and developmental capacity of in vitro embryos are evaluated by detecting / calculating the expression of autophagosome protein LC3B in in vitro embryonic cells, the apoptosis rate, and the expression levels of autophagy-related genes (LC3B, P62), pro-apoptotic genes (CASP3, JNK, BAX), and anti-apoptotic gene BCL2. Animal cell death is generally divided into type I (apoptosis), type II (autophagy), or necrosis. Calcium ion signals are regulated by mitochondria, and the accumulation of calcium ions in mitochondria can lead to cell apoptosis. When cells undergo apoptosis, the double-stranded breaks of chromosome DNA produce a large number of sticky 3'-OH ends. Under the action of terminal deoxyribonucleotide transferase (TdT), the derivatives formed by deoxyribonucleotides and fluorescein, peroxidase, alkaline phosphatase or biotin can be labeled to the 3'-end of DNA, so that apoptotic cells can be detected (TUNEL method), and apoptosis can be evaluated according to the percentage of apoptotic cell nuclei. Autophagy is a process in which eukaryotic cells selectively degrade and recycle misfolded, damaged or excess cytoplasmic products, and is induced under various stress conditions such as endoplasmic reticulum stress. LC3B is an RNA-binding protein that can engulf cytokines and rapidly degrade mRNA. Its protein expression level can be used as an assessment of cellular autophagy. According to some embodiments of the present invention, the improvement of in vitro embryo development ability includes improving the blastocyst rate, the total number of blastocyst cells, the EdU positive cell rate, the GSH content, the expression of Nrf2 protein, the mitochondrial abundance, the mitochondrial membrane potential level, the ATP content, the expression of PGC1α+β protein, embryo development related genes (BMP15, PCNA, OCT4, CCNB1, CDK2, CDK1, SOX2, Nanog), antioxidant related genes (SOD1, SOD2, GPX, CAT, PRDX2), mitochondrial function related genes (PAGE), and the like. It also decreased the expression levels of ER stress protein GRP78, autophagy-related LC3B protein, cell apoptosis rate, as well as the expression levels of ER stress-related genes (CHOP, uXBP1, sXBP1, GRP78, ATF4, ATF6), autophagy-related genes (LC3B, P62), and pro-apoptosis genes (CASP3, JNK, BAX). In some embodiments, the improvement of in vitro embryo development ability is to improve the in vitro embryo blastocyst rate. Preferably, as verified by the in vitro embryo blastocyst rate test, the administration of the product of the present invention increases the in vitro embryo blastocyst rate by 11.43%-28.81% relative to the case where the product of the present invention is not administered. In some embodiments, the improvement of in vitro embryo development ability is to increase the total cell number. Preferably, as verified by the total cell number test, the administration of the product of the present invention increases the total cell number of in vitro embryos by 20.65%-35.78% relative to the case where the product of the present invention is not administered. In some embodiments, the improvement of in vitro embryo development ability is to increase the EdU positive cell rate. Preferably, as verified by the EdU positive cell rate test, the administration of the product of the present invention increases the in vitro embryo EdU positive cell rate by 16.82%-48.70% relative to the case where the product of the present invention is not administered. In certain embodiments, the improvement of in vitro embryo development ability is to reduce the oxidative stress level of in vitro embryo cells. Preferably, as verified by in vitro embryo cell ROS and GSH tests, relative to the situation where the product of the present invention is not used, the use of the product of the present invention reduces the ROS level of in vitro embryo cells by 1.26%-5.57% and increases the GSH level by 13.56%-37.48%. In some embodiments, the improving the in vitro embryo development ability is to improve the antioxidant level of in vitro embryo cells. Preferably, as verified by the in vitro embryo cell Nrf2 immunofluorescence test, the application of the product of the present invention increases the Nrf2 level of in vitro embryo cells by 7.49%-13.07% relative to the case where the product of the present invention is not applied. In some embodiments, the improvement of in vitro embryo development ability is to improve the mitochondrial abundance of in vitro embryonic cells. Preferably, as verified by the in vitro embryonic cell mitochondrial abundance test, relative to the situation where the product of the present invention is not applied, the application of the product of the present invention increases the mitochondrial abundance of in vitro embryonic cells by 30.18%-37.33%. In some embodiments, the improvement of in vitro embryo development ability is to improve the mitochondrial membrane potential of in vitro embryonic cells. Preferably, as verified by the in vitro embryonic cell mitochondrial membrane potential test, relative to the situation where the product of the present invention is not applied, the application of the product of the present invention increases the mitochondrial membrane potential of in vitro embryonic cells by 13.64%-24.44%. In some embodiments, the improving in vitro embryo development ability is to improve the in vitro embryonic cell ATP content. Preferably, as verified by the in vitro embryonic cell ATP content test, the application of the product of the present invention increases the in vitro embryonic cell ATP content by 61.11%-79.58% relative to the situation where the product of the present invention is not applied. In some embodiments, the improvement of in vitro embryo development ability is to increase the expression of in vitro embryo PGC1α+β protein. Preferably, as verified by the test of the expression of in vitro embryo PGC1α+β protein, the application of the product of the present invention increases the expression of in vitro embryo PGC1α+β protein by 3.77%-9.14% relative to the case where the product of the present invention is not applied. In some embodiments, the improvement of in vitro embryo development ability is to reduce the expression of in vitro embryo GRP78 protein. Preferably, as verified by the in vitro embryo GRP78 protein expression test, the application of the product of the present invention reduces the expression of in vitro embryo GRP78 protein by 7.53%-13.64% relative to the case where the product of the present invention is not applied. In some embodiments, the improvement of in vitro embryo development ability is to reduce the expression of in vitro embryo LC3B protein. Preferably, as verified by the test of the expression of in vitro embryo LC3B protein, the application of the product of the present invention reduces the expression of in vitro embryo LC3B protein by 3.89%-11.80% relative to the case where the product of the present invention is not applied. In some embodiments, the improvement of in vitro embryo development ability is to reduce the in vitro embryo cell apoptosis rate. Preferably, as verified by the in vitro embryo cell apoptosis rate test, the application of the product of the present invention reduces the in vitro embryo cell apoptosis rate by 54.74%-65.73% relative to the situation where the product of the present invention is not applied. In the context of the numerical value of the effect of improving the in vitro embryo development ability, the numerical value of "improved" used is calculated by the following method: [(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). The numerical value of "reduced" is calculated by the following method: [(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). According to some embodiments of the present invention, there is provided a use of caffeic acid phenethyl ester in preparing a product. According to some embodiments of the present invention, there is provided the use of caffeic acid phenethyl ester in a product for improving the blastocyst rate of in vitro embryos. According to some embodiments of the present invention, there is provided a use of caffeic acid phenethyl ester in preparing a product for increasing the total cell number of in vitro embryo blastocysts. According to some embodiments of the present invention, there is provided a use of caffeic acid phenethyl ester in preparing a product for improving the proliferation ability of in vitro embryonic cells. According to some specific embodiments of the present invention, there is provided the use of caffeic acid phenethyl ester in preparing a product for enhancing the antioxidant capacity of in vitro embryonic cells. According to some specific embodiments of the present invention, there is provided the use of caffeic acid phenethyl ester in preparing a product for improving the mitochondrial function of in vitro embryonic cells. According to some specific embodiments of the present invention, there is provided the use of caffeic acid phenethyl ester in preparing a product for inhibiting endoplasmic reticulum stress of embryonic cells in vitro. According to some specific embodiments of the present invention, there is provided the use of caffeic acid phenethyl ester in preparing a product for inhibiting autophagy of embryonic cells in vitro. According to some specific embodiments of the present invention, there is provided the use of caffeic acid phenethyl ester in preparing a product for inhibiting apoptosis of embryonic cells in vitro. According to some embodiments of the present invention, the product includes at least one of a pharmaceutical composition, a culture medium, and a kit. According to some embodiments of the present invention, the in vitro embryo includes a mammal in vitro embryo. 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, cattle, cows), primates (such as apes, monkeys, orangutans and chimpanzees), canines (such as dogs and wolves), felines (such as cats, lions and tigers), equines (such as horses, donkeys and zebras), edible animals (such as 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 a pig. According to some embodiments of the present invention, the mammalian in vitro embryo includes a mammalian in vitro parthenogenetic activated embryo, a mammalian in vitro fertilized embryo. Preferably, the mammalian in vitro embryo includes a parthenogenetic activated oocyte, a fertilized egg cell, and a 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst developed from the parthenogenetic activated oocyte and / or the fertilized egg cell. The second aspect of the present invention provides a product for promoting in vitro embryo development, wherein the raw material of the product includes caffeic acid phenethyl ester. According to some embodiments of the present invention, the product for improving the in vitro embryo development ability includes a drug, a culture medium or a kit. According to some embodiments of the present invention, the medicine also includes a pharmaceutically acceptable adjuvant. Preferably, the adjuvant includes a pharmaceutically acceptable diluent, excipient, carrier, adhesive, lubricant, suspending agent, coating agent, solubilizing agent. The medicine can be in various forms, such as liquid, semisolid and solid dosage forms. In other embodiments of the present invention, the form of the medicine 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 medicine is a liquid solution. Preferably, the culture medium also includes basal culture medium, inorganic salts, solvents, antibiotics, and nutritional functional factors that are beneficial to the development of in vitro embryonic cells. Preferably, the basal culture medium includes TCM-199 culture medium, PZM-5 culture medium, CR1-aa culture medium, Ham'S F10 culture medium, Ham'S F12, NCSU-37 culture medium, and NCSU-23 culture medium. Preferably, the inorganic salt includes sodium chloride, potassium chloride, potassium dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, sodium bicarbonate. Preferably, the antibiotics include gentamicin, penicillin, streptomycin, kanamycin. Preferably, the nutritional functional factors that are beneficial to the development of in vitro embryonic cells include hormones, energy substrates, amino acids, and growth factors. 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). According to some embodiments of the invention, the energy substrate comprises glucose, pyruvate, lactate. 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, and L-lysine hydrochloride. According to some embodiments of the present invention, the growth factor includes epidermal growth factor (EGF), insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF). The third aspect of the present invention provides the use of caffeic acid phenethyl ester in promoting porcine oocyte embryonic development in vitro. The fourth aspect of the present invention provides a method for promoting the development of porcine oocyte embryos in vitro, comprising the following steps: applying an effective amount of caffeic acid phenethyl ester to the in vitro embryos. The method can be used for non-therapeutic purposes. Specifically, the 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. According to some embodiments of the present invention, the method comprises the following steps: S1. Take mature oocytes for parthenogenesis activation; S2. The oocytes treated in step S1 are placed in an embryo culture medium containing caffeic acid phenethyl ester to form blastocysts. Preferably, the effective amount of caffeic acid phenethyl ester can be administered in the form of a product prepared according to the first embodiment of the present invention or the second embodiment of the present invention. Preferably, the caffeic acid phenethyl ester in S1 can be applied at any time point during the in vitro embryo development to the blastocyst stage, including after the oocyte is parthenogenetically activated, after sperm and egg fertilization, and at any time point during the development to the 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst stages. Preferably, the caffeic acid phenethyl ester in S1 can be administered once, but more preferably multiple times. According to some embodiments of the present invention, the caffeic acid phenethyl ester can be administered from once a day to once a week. Specifically, the caffeic acid phenethyl ester 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. According to some embodiments of the present invention, the method further comprises the step of preparing a mature oocyte. According to some embodiments of the present invention, the effective amount is 0.1 to 1 nM; preferably, the effective amount is 1 nM. The term "effective amount" as used in this specification and in the claims refers to the amount of the compound administered that is sufficient to provide the expected effect compared to the situation where the compound is not administered, and the amount is calculated based on the mass of the applied caffeic acid phenethyl ester and the volume of the applied liquid environment (e.g., culture medium); the expected effect includes significantly increasing the in vitro embryo blastocyst rate, and / or significantly increasing the total cell number of in vitro embryonic cells and the expression level of genes related to embryonic development, and / or significantly increasing the mitochondrial membrane potential of in vitro embryonic cells and the expression level of genes related to mitochondrial function, and / or significantly reducing the oxidative stress level of in vitro embryonic cells, and / or increasing the antioxidant level of in vitro embryonic cells and the expression level of related antioxidant genes, and / or significantly reducing the endoplasmic reticulum stress level of in vitro embryonic cells and the expression level of endoplasmic reticulum stress-related genes, and / or significantly reducing the autophagy and apoptosis level of in vitro embryonic cells and the expression level of related genes that promote autophagy and apoptosis, and / or increasing the expression level of related anti-apoptotic genes. The beneficial effects of the present invention are: The present invention discloses for the first time the application of caffeic acid phenethyl ester in the preparation of a drug for improving the in vitro embryo development ability. Previously, there has been no report that caffeic acid phenethyl ester is related to the in vitro embryo development ability, thus broadening the application scope of caffeic acid phenethyl ester. Caffeic acid phenethyl ester can effectively improve the development quality and development ability of in vitro embryos by increasing the blastocyst rate, increasing the total number of blastocyst cells, increasing cell proliferation ability, increasing antioxidant levels, enhancing mitochondrial function, and reducing the effects of endoplasmic reticulum stress response, autophagy and cell apoptosis. It can be used to prepare pharmaceutical compositions, culture media or kits for promoting embryo development, and has good industrial application prospects. Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS The above and / or additional aspects and advantages of the present invention will become apparent and easily understood by describing the embodiments in conjunction with the following drawings, in which: Figure 1The figures and bar graphs show the effects of different concentrations (0, 0.1, 1 and 20 nM) of caffeic acid phenethyl ester on the blastocyst formation rate at day 7 in pigs; data are expressed as mean ± standard error; n = 3 independent parallel experiments; significant differences are expressed as *P<0.05, ***P<0.001. Figure 2 The figure and bar graph show the effect of caffeic acid phenethyl ester on the total cell number of embryos in vitro; the data are expressed as mean ± standard error; n = 3 independent parallel experiments; significant differences are expressed as ***P < 0.001. Figure 3 The figure and bar graph show the effect of caffeic acid phenethyl ester on the proliferation ability of early embryos in vitro; the data are expressed as mean ± standard error; n = 3 independent parallel experiments; significant differences are expressed as **P < 0.01. Figure 4 The figure and bar graph show the effect of caffeic acid phenethyl ester on genes related to early in vitro embryonic development; data are expressed as mean ± standard error; n = 3 independent parallel experiments; significant differences are expressed as *P<0.05, **P<0.01, ***P<0.001. Figure 5 The figure and bar graph show the effect of caffeic acid phenethyl ester on the ROS and GSH content of early embryos in vitro; the data are expressed as mean ± standard error; n = 3 independent parallel experiments; significant differences are expressed as *P < 0.05. Figure 6 The figure and bar graph show the effect of caffeic acid phenethyl ester on the expression of Nrf2 protein in early embryos in vitro; the data are expressed as mean ± standard error; n = 3 independent parallel experiments; significant differences are expressed as ***P < 0.001. Figure 7 The figure and bar graph show the effect of caffeic acid phenethyl ester on antioxidant-related genes in early embryos in vitro; data are expressed as mean ± standard error; n = 3 independent parallel experiments; significant differences are expressed as *P < 0.05, **P < 0.01. Figure 8 The figure and bar graph show the effect of caffeic acid phenethyl ester on the abundance of mitochondria in early embryos in vitro; the data are expressed as mean ± standard error; n = 3 independent parallel experiments; significant differences are indicated as *P < 0.05. Fig. 9 The figure and bar graph show the effect of caffeic acid phenethyl ester on mitochondrial membrane potential of early embryos in vitro; data are expressed as mean ± standard error; n = 3 independent parallel experiments; significant differences are expressed as *P < 0.05. Fig.10The figure and bar graph show the effect of caffeic acid phenethyl ester on the expression of PGC1α+β protein in early in vitro embryos; data are expressed as mean ± standard error; n = 3 independent parallel experiments; significant differences are indicated as *P<0.05. Fig.11 The figure and bar graph show the effect of caffeic acid phenethyl ester on ATP content of early embryos in vitro; data are expressed as mean ± standard error; n = 3 independent parallel experiments; significant differences are expressed as *P < 0.05. Fig.12 The figure and bar graph show the effect of caffeic acid phenethyl ester on genes related to mitochondrial function of early embryos in vitro; data are expressed as mean ± standard error; n = 3 independent parallel experiments; significant differences are expressed as *P < 0.05, ***P < 0.001. Fig.13 The figure and bar graph show the effect of caffeic acid phenethyl ester on the expression level of GRP78 protein in early in vitro embryos measured by immunofluorescence staining; the data are expressed as mean ± standard error; n = 3 independent parallel experiments; significant differences are expressed as ***P < 0.001. Fig.14 The figure and bar graph show the effect of caffeic acid phenethyl ester on the expression level of GRP78 protein in early in vitro embryos measured by protein immunoblotting; data are expressed as mean ± standard error; n = 3 independent parallel experiments. Fig.15 The figure and bar graph show the effect of caffeic acid phenethyl ester on endoplasmic reticulum stress-related genes in early embryos in vitro; data are expressed as mean ± standard error; n = 3 independent parallel experiments; significant differences are expressed as *P < 0.05, **P < 0.01. Fig.16 The figure and bar graph show the effect of caffeic acid phenethyl ester on the expression level of LC3B protein in early embryos in vitro; the data are expressed as mean ± standard error; n = 3 independent parallel experiments; significant differences are expressed as **P < 0.01. Fig.17 The figure and bar graph show the effect of caffeic acid phenethyl ester on the apoptosis rate of early embryonic cells in vitro; the data are expressed as mean ± standard error; n = 3 independent parallel experiments; significant differences are expressed as ***P < 0.001. Fig.18 The figure and bar graph show the effect of caffeic acid phenethyl ester on autophagy-related genes and apoptosis-related genes in early embryos in vitro; data are expressed as mean ± standard error; n = 3 independent parallel experiments; significant differences are expressed as *P < 0.05, **P < 0.01. DETAILED DESCRIPTION The present invention is further described in detail below in conjunction with examples, but the embodiments 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 cannot be understood as limiting the present invention. Example 1 A method for promoting porcine oocyte embryonic development in vitro. A method for promoting porcine oocyte embryonic development in vitro comprises the following steps: (1) Oocyte collection and in vitro maturation (IVM) The ovaries of adolescent sows collected from slaughterhouses were washed with physiological saline preheated to 37.5°C. The follicle-oocyte complexes (COCs) in the ovaries were extracted with a syringe and placed in a preheated operating solution (TL-HEPES) in a 37.5°C water bath. When the COCs precipitated to the bottom, the supernatant was discarded to separate the cumulus-oocyte complexes from other impurities. TCM-199 maturation medium (Thermo Fisher Scientific, #11150-059, Waltham, MA, USA) supplemented with 10% porcine follicular fluid, 0.57mM L-cysteine, 20ng / mL epidermal growth factor, 1% penicillin-streptomycin (ThermoFisher Scientific, #15140122, Waltham, MA, USA), 0.2mM sodium pyruvate, 10IU / mL follicle stimulating hormone (Ningbo Hormone Plant No. 2) and 10IU / mL luteinizing hormone (Ningbo Hormone Plant No. 2) to make IVM medium. The cumulus-oocyte complex with good cumulus coating was selected, washed with TL-HEPES and IVM medium, and then immediately placed in IVM medium covered with mineral oil, and cultured in an incubator at 37°C, containing 5% carbon dioxide for 44-46h. (2) Oocyte parthenogenesis activation The cumulus-oocyte complex was placed in an operating solution containing 1% hyaluronidase and blown to separate the outer cumulus, and the oocyte containing the first polar body was selected. A 120V DC pulse lasting 60 μs was used in the activation solution to activate the oocyte for parthenogenesis. (3) In vitro culture of embryos The activated embryos were incubated for 3-4 hours in a mixture containing 4 mg / ml bovine serum albumin (BSA) and 7.5 mg / ml cytochalasin B to inhibit the extrusion of the dipolar body. Finally, the oocytes were transferred to an in vitro culture medium covered with mineral oil and cultured in a 5% CO2, 37°C saturated humidity incubator for 7 days. Both the activation solution and IVC medium (PZM-bicarbonate buffer supplemented with 4 mg / mL bovine serum albumin) need to be pre-equilibrated in the incubator in advance. The activation solution consists of 300 mM mannitol (containing 0.5 mM HEPES, 0.05 mM calcium chloride, 0.1 mM magnesium sulfate, and 0.01% polyvinyl alcohol. Caffeic acid phenethyl ester was dissolved to a higher concentration with dimethyl sulfoxide and then diluted with IVC to the specific concentration required for the experiment. As a control group, embryos were cultured in IVC medium without adding any other substances. Too low a concentration of the drug may make it difficult to maintain its effect on embryo quality, while too high a concentration may produce inhibitory behavior on the embryo. In order to determine the dose of caffeic acid phenethyl ester suitable for in vitro culture of early porcine embryos, we cultured embryos with different concentrations of CAPE (0, 0.1, 1, and 20 nM) and screened the optimal concentration based on the blastocyst formation rate. The results are as follows Figure 1 As shown. It can be seen that the blastocyst formation rate of the group with 1nM caffeic acid phenethyl ester added was significantly better than that of the other groups. Among them, the blastocyst rate of the control group was 28.97±1.48%, and the blastocyst rate of the group treated with 1nM caffeic acid phenethyl ester was 34.67±0.74%, which was significantly (*P<0.05) increased by 11.43%-28.81% compared with the control group. Example 2 Application of caffeic acid phenethyl ester in the preparation of a product for enhancing the proliferation ability of in vitro embryonic cells. Cell proliferation was assessed using the BeyoClick EdU-647 Cell Proliferation Assay Kit (Beyotime, Jiangsu, China). Blastocysts were removed from the IVC medium, rinsed with PBS-PVA, fixed with 3.7% paraformaldehyde in PBS-PVA for 30 min, and then permeabilized in 0.1% Triton X-100 for 30 min, all at room temperature. After permeabilization, blastocysts were washed four times and fixed in blocking buffer (1% BSA in PBS-PVA) for one hour, then promptly washed with PVA-PBS and incubated with BeyoClick supplement for one hour. Subsequently, blastocysts were incubated with 10 μg / mL Hoechst 33342 at 37.5°C in the dark for 15 min to label the nuclei. The number of EdU-positive cells and the total number of cells were analyzed using a fluorescent inverted microscope (Nikon) and ImageJ software. The proliferation rate was calculated as the ratio of EdU-positive cells to the total number of cells. Detection of relevant gene expression: According to the manufacturer's instructions, blastocysts on the 7th day were selected from the culture medium, and total RNA was extracted from 20 blastocysts using the Dynabeads mRNA DIRECT Purification Kit (Invitrogen). The first-strand cDNA was obtained by reverse transcription using a reverse transcription kit (TIANGEN, Beijing, China). After reverse transcription, the concentration of cDNA was determined to ensure that the quality of the obtained cDNA was sufficient for subsequent qRT-PCR experiments. According to the instructions, the qRT-PCR system was prepared using the KAPA SYBR FAST qpcr Master Mix (2×) Kit (Kapa Biosystems, USA); each system was 20 μL, containing 1 μL cDNA template and 1 μL each of forward and reverse primers. The qRT-PCR program included an initial denaturation at 93°C for 30 minutes, followed by 40 cycles; the conditions for each cycle were 95°C for 3 seconds, 60°C for 30 seconds, and 72°C for 20 seconds, and finally extended at 37°C for 30 seconds. The melting curve was analyzed before the experiment, and it was observed that the melting curve showed a single peak and was within a reasonable temperature range. Gene expression was analyzed by LightCycler96 using the 2-ΔΔCt method, with RN18S as the internal standard, and the annealing temperature for all reactions was 60°C. The relative mRNA levels of embryonic development-related factors BMP15, PCNA, OCT4, CCNB1, CDK2, CDK1, SOX2, and Nanog were detected. The results are as follows Figure 2 , 3 , 4. From Figure 2 It can be seen that the total number of embryonic cells in the control group was 47.2333±1.6433, and the total number of embryonic cells in the 1 nM caffeic acid phenethyl ester treatment group was 60.4286±1.4652, which was significantly (***P<0.001) increased by 20.65%-35.78% compared with the control group. Figure 3 It can be seen that the EdU positive cell rate in the control group was 26.3528±1.7709%, and the EdU positive cell rate in the 1 nM caffeic acid phenethyl ester treatment group was 34.7017±1.8543%, which was significantly (**P<0.01) increased by 16.82%-48.70% compared with the control group. Figure 4It can be seen that caffeic acid phenethyl ester treatment can significantly (*P<0.05, **P<0.01, ***P<0.001) increase the expression level of genes related to embryonic development, among which the gene expression levels of BMP15, PCNA, OCT4, CCNB1, CDK2, CDK1, SOX2, and Nanog are 1.56 times, 1.52 times, 1.06 times, 1.21 times, 1.31 times, 1.40 times, 1.72 times, and 1.75 times that of the control group, respectively. It can be found that the total number of cells in the blastocysts with 1nM caffeic acid phenethyl ester added is significantly more than that in the control group, and its proliferation ability is also significantly enhanced. This shows that caffeic acid phenethyl ester has a good application prospect in the preparation of products that enhance the proliferation ability of embryonic cells. Example 3 Application of caffeic acid phenethyl ester in the preparation of a product for enhancing the antioxidant capacity of embryos. (1) Measurement of ROS and glutathione (GSH) levels in embryos The washed 4-cell embryos were stained with 10 μM 2′, 7-dichlorodihydrofluorescein diacetate (DCFH, Beyotime, Shanghai, China) and 10 μM 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (CMF2HC, Invitrogen) to detect the levels of intracellular ROS and GSH. Specifically, the 4-cell embryos were removed from the culture medium and rinsed with PBS-PVA. Then, the 4-cell embryos were placed in 10 μM DCFH-DA and CMF2HC at 37.5°C for 30 minutes each, rinsed three times with PBS-PVA, and imaged with a fluorescence microscope (Nikon). The corresponding fluorescence intensity was analyzed and calculated using ImageJ software. (2) Immunofluorescence staining Day 7 blastocysts were removed from the culture medium, rinsed with PBS-PVA, and then fixed in PBS-PVA containing 3.7% paraformaldehyde for 30 minutes and permeabilized in 0.1% TritonX-100 for 30 minutes, all at room temperature. After permeabilization, the blastocysts were washed and fixed in blocking buffer (1% BSA in PBS-PVA) for one hour. After blocking, the blastocysts were incubated with anti-Nrf2 antibody (#ab31163, diluted 1:200; Abcam, Cambridge, MA, USA) at 4°C overnight. After washing the blastocysts with PBS-PVA, they were stained with goat anti-rabbit IgG (#ab150077, diluted 1:500; Abcam, Cambridge, MA, USA) at room temperature for 1 hour. Subsequently, the blastocysts were incubated with 10 μg / mL Hoechst 33342 at 37.5°C in the dark for 15 minutes to label the nuclei. A fluorescence inverted microscope (Nikon) and ImageJ software were used to analyze and calculate the corresponding fluorescence intensity. (3) Detection of related gene expression levels According to the method in Example 2, the mRNA expression levels of antioxidant-related factors SOD1, SOD2, GPX, CAT and PRDX2 were detected. The results are as follows Figure 5 , 6 , 7. From Figure 5 It can be seen that the ROS content of the control group was 1.0000±0.0101, the GSH content was 0.9999±0.0098, the ROS content of the 1nM caffeic acid phenethyl ester treatment group was 0.9657±0.01195, and the GSH content was 1.1466±0.0215. Compared with the control group, the ROS content of the 1nM caffeic acid phenethyl ester treatment group was significantly (*P<0.05) reduced by 1.26%-5.57%, and the GSH content was significantly (*P<0.05) increased by 13.56%-37.48%. Figure 6 It can be seen that the expression of Nrf2 protein in the control group was 1.0000±0.0168, and the expression of Nrf2 protein in the 1nM caffeic acid phenethyl ester treatment group was 1.0931±0.0187. Compared with the control group, the expression of Nrf2 protein in the 1nM caffeic acid phenethyl ester treatment group was significantly (***P<0.001) increased by 7.49%-13.07%. Figure 7 It can be seen that caffeic acid phenethyl ester treatment can significantly (*P<0.05, **P<0.01) increase the expression level of antioxidant-related genes, among which the gene expression levels of SOD1, SOD2, GPX, CAT, and PRDX2 are 1.43 times, 1.66 times, 1.24 times, 1.11 times, and 1.70 times that of the control group, respectively. It can be found that the antioxidant capacity of the blastocysts added with 1nM caffeic acid phenethyl ester is significantly stronger than that of the control group, indicating that caffeic acid phenethyl ester has a good application prospect in the preparation of products that enhance the antioxidant capacity of embryos. Example 4 Application of caffeic acid phenethyl ester in the preparation of a product for enhancing mitochondrial function of embryonic cells in vitro (1) Determination of mitochondrial abundance To measure the distribution of mitochondria, 4-cell embryos were placed in 200 nM MitoTracker Red CMXRos (C1049B; Beyotime, Shanghai, China) for 30 min, washed with PBS-PVA, and imaged using a fluorescent inverted microscope (Nikon). ImageJ software was used to analyze and calculate the corresponding fluorescence intensity. (2) Determination of mitochondrial membrane potential To measure mitochondrial membrane potential, 4-cell embryos were placed in 10 μM 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolylcarbocyanineiodide (JC-1.Invrogen) dye for 30 min, washed three times with PBS-PVA, and imaged using a fluorescence inverted microscope (Nikon). The corresponding fluorescence intensity was analyzed and calculated using ImageJ software. The average mitochondrial membrane potential of all 4-cell embryos was calculated as the ratio of red fluorescence intensity (J-aggregates) to green fluorescence intensity (J-monomers). (3) ATP content determination To evaluate mitochondrial function, we measured ATP levels in embryos using an ATP assay kit (S0026; Beyotime, Shanghai, China). Day 7 blastocysts were removed from the culture medium and rinsed with PBS-PVA. They were concentrated and lysed in cell lysis buffer, and the ATP standard solution was diluted to the appropriate concentration gradient with ATP standard diluent. Then 100 μL of ATP assay working solution was added to each well of a 96-well plate, and a standard curve was drawn using the fluorescence emission values ​​of a multi-function microplate. We added ATP assay lysate and ATP assay reagent to each assay well and measured luminescence using an automated microplate reader (BIO-TEK), all on ice. (4) Immunofluorescence staining Immunofluorescence staining was performed according to the method of (2) in Example 3, wherein the primary antibody was PGC1alpha+beta (#ab72230, diluted 1:200; Cambridge, MA, USA), the secondary antibody was goat anti-rabbit IgG (#ab150077, diluted 1:500; Abcam, Cambridge, MA, USA), and the rest of the methods were the same. (5) Detection of related gene expression levels According to the method in Example 2, the mRNA expression levels of mitochondrial function-related factors PGC1α, TFAM, TFB1M, TFB2M, NRF1, NRF2, SIRT1 and ATP5B were detected. The results are as follows Figure 8 , 9 , 10, 11, 12. From Figure 8 It can be seen that the mitochondrial abundance of the control group was 0.9999±0.0149, and the mitochondrial abundance of the 1nM caffeic acid phenethyl ester-treated group was 1.3211±0.0316. Compared with the control group, the mitochondrial abundance of the 1nM caffeic acid phenethyl ester-treated group was significantly (*P<0.05) increased by 30.18%-37.33%. Fig. 9It can be seen that the mitochondrial membrane potential of the control group was 0.9706±0.0302, and the mitochondrial membrane potential of the 1nM caffeic acid phenethyl ester treatment group was 1.1373±0.0329. Compared with the control group, the mitochondrial membrane potential of the 1nM caffeic acid phenethyl ester treatment group was significantly (*P<0.05) increased by 13.64%-24.44%. Fig.10 It can be seen that the expression of PGC1α+β protein in the control group was 0.9999±0.0172, and the expression of PGC1α+β protein in the 1nM caffeic acid phenethyl ester-treated group was 1.0554±0.0171. Compared with the control group, the expression of PGC1α+β protein in the 1nM caffeic acid phenethyl ester-treated group was significantly (*P<0.05) increased by 3.77%-9.14%. Fig.11 It can be seen that the ATP content of the control group was 1, and the ATP content of the 1nM caffeic acid phenethyl ester-treated group was 1.6111±0.1848. Compared with the control group, the ATP content of the 1nM caffeic acid phenethyl ester-treated group was significantly (*P<0.05) increased by 61.11%-79.58%. Fig.12 It can be seen that caffeic acid phenethyl ester treatment can significantly (*P<0.05, ***P<0.001) increase the expression level of genes related to mitochondrial function, among which the gene expression levels of PGC1α, TFAM, TFB1M, TFB2M, NRF1, NRF2, SIRT1, and ATP5B are 1.56 times, 1.20 times, 1.10 times, 1.22 times, 1.65 times, 2.31 times, 1.34 times, and 1.79 times that of the control group, respectively. It can be found that the mitochondrial function of the blastocysts added with 1nM caffeic acid phenethyl ester is significantly better than that of the control group, indicating that caffeic acid phenethyl ester has a good application prospect in the preparation of products that enhance the mitochondrial function of embryonic cells. Example 5 Application of caffeic acid phenethyl ester in the preparation of a product for inhibiting endoplasmic reticulum stress of embryonic cells in vitro (1) Immunofluorescence staining Immunofluorescence staining was performed according to the method of (2) in Example 3, wherein the primary antibody was GRP78 BIP antibody (#ab21685, diluted 1:200; Cambridge, MA, USA), the secondary antibody was rabbit anti-IgG (H+L) (#8889, diluted 1:500; Cell Signaling Technology, Danvers, MA, USA), and the rest of the methods were the same. (2) Detection of related gene expression levels According to the method in Example 2, the mRNA expression levels of endoplasmic reticulum stress-related factors CHOP, uXBP1, sXBP1, GRP78, ATF4 and ATF6 were detected. (3) Immunoblotting Embryos were collected and placed in SDS-PAGE sample loading buffer and heated at 95°C for 10 min. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with TBST containing 5% BSA for two hours at room temperature and incubated with GRP78 BIP primary antibody (#ab21685, diluted 1:1000; Abcam, Cambridge, MA, USA) at 4°C overnight. The membranes were washed three times with TBST for 10 minutes each and then incubated with anti-rabbit IgG, HRP-linked antibody (#7074, diluted 1:10000; Cell Signaling Technology, Danvers, MA, USA) for 1 hour at room temperature. The blots were visualized using the Azure Sapphire Biomolecular Imager (AZURE), and the band intensity values ​​were analyzed using ImageJ. The results are as follows Fig.13 , 14 , 15. From Fig.13 It can be seen that the GRP78 protein expression in the control group was 1.0000±0.0186, and the GRP78 protein expression in the 1nM caffeic acid phenethyl ester treatment group was 0.8936±0.0139. Compared with the control group, the GRP78 protein expression in the 1nM caffeic acid phenethyl ester treatment group was significantly (***P<0.001) reduced by 7.53%-13.64%. Fig.14 It can be seen that the gray value of the GRP78 protein immunoblotting results of the control group is 1.0000±0.1987, and the gray value of the GRP78 protein immunoblotting results of the 1nM caffeic acid phenethyl ester treatment group is 0.8603±0.0998. Compared with the control group, the GRP78 protein expression level of the 1nM caffeic acid phenethyl ester treatment group decreased by 3.99%-23.95%. Fig.15 It can be seen that caffeic acid phenethyl ester treatment can significantly (*P<0.05, **P<0.01) reduce the expression levels of genes related to endoplasmic reticulum stress, among which the gene expression levels of CHOP, uXBP1, sXBP1, GRP78, ATF4, and ATF6 are 0.65 times, 0.91 times, 0.81 times, 0.86 times, 0.74 times, and 0.86 times of the control group, respectively. It can be found that the endoplasmic reticulum stress response of the blastocysts added with 1nM caffeic acid phenethyl ester is significantly weaker than that of the control group, indicating that caffeic acid phenethyl ester has a good application prospect in the preparation of products for inhibiting endoplasmic reticulum stress of embryonic cells in vitro. Example 6 Application of caffeic acid phenethyl ester in the preparation of a product for inhibiting autophagy and apoptosis of embryonic cells in vitro. (1) Immunofluorescence staining Immunofluorescence staining was performed according to the method of (2) in Example 3, wherein the primary antibody was LC3B (#ab48394, diluted 1:200; Abcam, Cambridge, MA, USA), the secondary antibody was goat anti-rabbit IgG (#ab150077, diluted 1:500; Abcam, Cambridge, MA, USA), and the rest of the methods were the same. (2) TUNEL assay Day 7 blastocysts were rinsed with PBS-PVA and fixed in PBS-PVA containing 3.7% paraformaldehyde for 30 minutes, and then permeabilized in 0.1% Triton X-100 for 30 minutes, all at room temperature. After permeabilization, the blastocysts were washed and fixed in blocking buffer (1% BSA in PBS-PVA) for one hour. The blastocysts were then washed with PVA-PBS and incubated with fluorescein-linked dUTP and terminal deoxynucleotidyl transferase (provided in the In Situ Cell Death Detection Kit; Roche) in the dark at 37.5°C for one hour. Subsequently, the blastocysts were incubated with 10 μg / mL Hoechst 33342 in the dark at 37.5°C for 15 minutes to label the cell nuclei. The number of apoptotic nuclei and the total number of cells were analyzed using a fluorescence microscope (Nikon) and ImageJ software. Cell apoptosis was evaluated based on the percentage of apoptotic nuclei. (3) Detection of related gene expression levels According to the method in Example 2, the mRNA expression levels of autophagy and apoptosis-related factors LC3B, P62, CASP3, JNK, BAX and BCL2 were detected. The results are as follows Fig.16 , 17 , 18. From Fig.16 It can be seen that the expression of LC3B protein in the control group was 1.0000±0.1675, and the expression of LC3B protein in the 1nM caffeic acid phenethyl ester treatment group was 0.9207±0.0198. Compared with the control group, the expression of LC3B protein in the 1nM caffeic acid phenethyl ester treatment group was significantly (**P<0.01) reduced by 3.89%-11.80%. Fig.17 It can be seen that the TUNEL positive cell rate of the control group was 13.1129±0.7513%, and the TUNEL positive cell rate of the 1nM caffeic acid phenethyl ester treatment group was 5.1728±0.4220%. Compared with the control group, the TUNEL positive cell rate of the 1nM caffeic acid phenethyl ester treatment group was significantly (***P<0.001) reduced by 54.74%-65.73%. Fig.15It can be seen that caffeic acid phenethyl ester treatment can significantly (*P<0.05, **P<0.01) reduce the expression levels of autophagy-related genes and pro-apoptotic genes, and significantly (**P<0.01) increase the expression levels of anti-apoptotic genes, among which the gene expression levels of LC3B, P62, CASP3, JNK, BAX, and BCL2 were 0.39 times, 0.84 times, 0.80 times, 0.83 times, 0.69 times, and 1.11 times that of the control group, respectively. It can be found that the occurrence of cell autophagy and apoptosis in the blastocysts with 1nM caffeic acid phenethyl ester added was significantly less than that in the control group, indicating that caffeic acid phenethyl ester has a good application prospect in the preparation of products that inhibit autophagy and apoptosis of embryonic cells in vitro. The above statistical results are expressed as mean ± standard error (SEM). The t-test was used to compare two groups of data, and one-way analysis of variance (ANOVA, Tukey-Kramer test) was used to analyze three or more mean values. All statistical analyses were performed according to *P<0.05, **P<0.01 and ***P<0.001. All statistical analyses were performed using SPSS 22.0 version (IBM Corp., Chicago, IL, USA). In summary, the present invention proves that caffeic acid phenethyl ester can increase the blastocyst rate, increase the total number of blastocyst cells, improve cell proliferation ability, improve antioxidant level, enhance mitochondrial function, reduce the effects of endoplasmic reticulum stress response, autophagy and cell apoptosis, thereby effectively improving the development quality and development ability of in vitro embryos, and has good market application prospects in the preparation of products promoting embryo development. 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 according to the structures, features and principles described in the scope of the patent application of the present invention should be included in the scope of protection of the patent application of the present invention.

Claims

1. Application of caffeic acid phenethyl ester in the preparation of products for promoting in vitro embryo development.

2. The use according to claim 1, characterized in that: The promoting of in vitro embryo development includes at least one of improving embryo quality or improving embryo development ability.

3. The use according to claim 2, characterized in that: The improving of in vitro embryo development ability includes at least one of improving embryo blastocyst rate, increasing total cell number of embryo blastocyst, enhancing embryo antioxidant capacity, improving embryo mitochondrial function, reducing embryo endoplasmic reticulum stress level, and reducing embryo autophagy and cell apoptosis level.

4. The use according to any one of claims 1 to 3, characterized in that: The product is a kit, a culture medium or a medicine.

5. The application of caffeic acid phenethyl ester in preparing products, characterized in that: The product has at least one of the following functions: (1) Improve the embryo blastocyst rate; (2) Increase the total cell number of embryonic blastocyst; (3) Enhance the antioxidant level of embryos; (5) Improve embryonic mitochondrial function; (6) inhibiting embryonic endoplasmic reticulum stress; (7) Inhibit embryonic cell autophagy and apoptosis.

6. A product for promoting in vitro embryo development, characterized in that: The raw materials of the product include caffeic acid phenethyl ester.

7. The product according to claim 6, characterized in that The product is a kit, a culture medium or a medicine.

8. Use of caffeic acid phenethyl ester in promoting porcine oocyte embryonic development in vitro.

9. A method for promoting porcine oocyte embryonic development in vitro, characterized in that: The method comprises the following steps: applying an effective amount of caffeic acid phenethyl ester to the in vitro embryo.

10. The method according to claim 9, characterized in that The effective amount is 0.1-1 nM; preferably, the effective amount is 1 nM.

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