Method for improving ovum in-vitro maturation quality and blastocyst quality and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for improving the in-vitro maturation quality of ovum and the blastocyst quality and application of the ovum and blastocyst quality. The invention discloses beta-sodium glycerophosphate pentahydrate. According to the preparation, at least one of the retinol binding protein and the sodium beta-glycerophosphate pentahydrate is added to improve the cleavage rate and blastocyst rate of an in-vitro embryo, the oxidation resistance of an oocyte and the like, so that the maturation quality of the in-vitro oocyte and the development potential of the in-vitro embryo are remarkably improved, and the application prospect is wide.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for improving the quality of in vitro maturation of oocytes and blastocyst and application thereof. BACKGROUND

[0002] Retinoic acid (RA) is a metabolite of vitamin A (retinol), which plays a key role in the body, especially in cell differentiation, embryonic development, immune function and visual process. In the field of in vitro embryo culture, the production process of in vitro embryo starts from the collection of oocytes, mainly including three stages of in vitro maturation of oocytes, in vitro fertilization and in vitro culture of fertilized eggs. Related studies show that the addition of retinoic acid during in vitro maturation of oocytes can promote oocyte maturation, improve embryonic development potential, regulate intercellular gap communication, regulate intracellular antioxidant status and anti-apoptosis, and affect the expression of related genes.

[0003] Although retinoic acid has certain application value in in vitro embryo culture, its use still has many limitations in actual production, such as the difficulty in accurately determining the concentration of directly added retinoic acid, the significant differences in tolerance, demand and cis-trans stereoisomer requirements of oocytes in different culture states, for example, 5 nM of 9-cis retinoic acid can improve the maturation rate of buffalo oocytes, 10 nM of all-trans retinoic acid can significantly improve the maturation and survival rate of goat oocytes, but 500 nM of retinoic acid can inhibit the maturation of pig and cattle oocytes, and even produce cytotoxicity. In addition, retinoic acid and its derivatives have high teratogenicity, although they have a short half-life, in theory, the addition time can be set to reduce the risk of teratogenicity, but it will be difficult to mass-produce before safety is ensured.

[0004] Therefore, it is of great significance to explore a direct addition of retinoic acid alternative and to explore compounds that can produce synergies with it for efficient in vitro embryo culture of livestock. SUMMARY

[0005] The present application aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, the present application provides a method for improving the quality of in vitro maturation of oocytes and blastocyst and application thereof. The preparation of the present application adds at least one of retinol binding protein and sodium beta-glycerophosphate pentahydrate to improve the cleavage rate and blastocyst rate of in vitro embryos, the antioxidant capacity of oocytes, and thus significantly improve the maturation quality of in vitro oocytes and the development potential of in vitro embryos, and has a wide application prospect.

[0006] In a first aspect, the present application provides a preparation for in vitro embryo production. According to an embodiment of the present application, the preparation contains at least one of the following components: retinol binding protein; sodium beta-glycerophosphate pentahydrate. According to the preparation of the embodiment of the present application, at least one of the retinol binding protein and the sodium beta-glycerophosphate pentahydrate is added to promote in vitro maturation of oocytes, improve the maturation quality of in vitro oocytes and the development potential of in vitro embryos, and provide an effective new strategy for in vitro embryo production of livestock, animal genetic improvement, and assisted reproductive technology, which has a wide application prospect.

[0007] According to an embodiment of the present application, the preparation described above can also have the following additional technical features: According to an embodiment of the present application, the retinol binding protein includes one or more of retinol binding protein 1, retinol binding protein 2, and retinol binding protein 4.

[0008] According to an embodiment of the present application, the concentration of the retinol binding protein is 0.1-200 ng / mL.

[0009] According to an embodiment of the present application, the concentration of the sodium beta-glycerophosphate pentahydrate is 0.1-100 mM.

[0010] According to an embodiment of the present application, the preparation contains the retinol binding protein and the sodium beta-glycerophosphate pentahydrate, the concentration of the retinol binding protein is 0.1-200 ng / mL, and the concentration of the sodium beta-glycerophosphate pentahydrate is 0.1-100 mM.

[0011] According to an embodiment of the present application, the preparation is used in at least one of the stages of in vitro maturation of oocytes, in vitro fertilization, or in vitro culture of embryos.

[0012] According to an embodiment of the present application, the preparation is used in the in vitro maturation stage of oocytes, and the use time is from the GV stage to the MII stage.

[0013] According to an embodiment of the present application, the preparation further includes one or more of inorganic salts, amino acids, vitamins, sugars, follicle stimulating hormone, luteinizing hormone, estradiol, fetal bovine serum, pH indicator, and antibiotics.

[0014] According to an embodiment of the present application, the preparation further includes 0.04-2.5 mg / mL of inorganic salts, 0.05-2.0 mM of amino acids, 0.5-2.0 mM of vitamins, 0.5-10 mM of sugars, 0.02-0.1 IU / mL of follicle stimulating hormone, 0.02-0.1 IU / mL of luteinizing hormone, 1.0-2.0 ug / mL of estradiol, 5-10% of fetal bovine serum, and 95-105 IU / mL of antibiotics.

[0015] According to an embodiment of the present application, the oocyte or the in vitro embryo is derived from a non-human mammal.

[0016] According to an embodiment of the present application, the mammal comprises one or more of non-human primates, bovine, ovine, porcine, equine and mouse.

[0017] In a second aspect of the present application, the present application provides a use of retinol binding protein, sodium beta-glycerophosphate pentahydrate or the preparation for in vitro embryo production of the first aspect in in vitro embryo production.

[0018] It is understood by those skilled in the art that the features and advantages described above for the preparation for in vitro embryo production also apply to the use, which will not be repeated here.

[0019] According to an embodiment of the present application, the use described above can further have the following additional technical features: According to an embodiment of the present application, the retinol binding protein, sodium beta-glycerophosphate pentahydrate or the preparation for in vitro embryo production of the first aspect has at least one of the following uses: improving the maturation quality of in vitro oocyte; improving the development potential of in vitro embryo; wherein the improving the development potential of in vitro embryo comprises improving the development efficiency and / or development quality of in vitro embryo.

[0020] According to an embodiment of the present application, the retinol binding protein, sodium beta-glycerophosphate pentahydrate or the preparation for in vitro embryo production of the first aspect has at least one of the following uses: improving the glutathione level in in vitro oocyte; reducing the level of reactive oxygen species in in vitro oocyte; improving the cleavage rate of in vitro embryo; improving the blastocyst rate of in vitro embryo; improving the inner cell mass cell number of in vitro embryo.

[0021] In a third aspect of the present application, the present application provides a method for in vitro embryo production. According to an embodiment of the present application, the method comprises: culturing oocyte or sperm cell or zygote in a culture system containing retinol binding protein and / or sodium beta-glycerophosphate pentahydrate; wherein the oocyte or sperm cell or zygote is derived from a non-human mammal; and the culture system is the preparation for in vitro embryo production of the first aspect. According to the method of the embodiment of the present application, by co-culturing the oocyte or sperm cell or zygote with the aforementioned preparation containing retinol binding protein and / or sodium beta-glycerophosphate pentahydrate, precise regulation and support can be provided in the whole stage of in vitro embryo production, thereby comprehensively improving the efficiency and quality of in vitro embryo production.

[0022] According to an embodiment of the present application, the method described above can further have the following additional technical features: According to embodiments of the present application, the non-human mammal comprises one or more of a non-human primate, a cow, a sheep, a pig, a horse, and a mouse.

[0023] Additional aspects and advantages of the present application will be made apparent from the following description. DETAILED DESCRIPTION

[0024] Embodiments of the present application are described in detail below. The embodiments described below are examples only and are not intended to limit the present application.

[0025] It should be noted that the terms "first", "second" are used only for descriptive purposes and are not to be construed as indicating or implying relative importance or a quantity of the specified technical features. Thus, features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0026] The endpoints of the ranges and any values described herein are not limited to the precise values stated. The ranges and values should be construed to be approximations that allow for significant variation. Within the range and values, endpoints are included. Other values not explicitly mentioned can be included from the endpoints by simple combination.

[0027] In this document, the terms "comprise" or "comprising" are open-ended, that is, they mean including, but not limited to, what is recited.

[0028] In this document, the terms "optionally", "optional" or "option" generally mean that the subsequently described event or circumstance can or can not occur, and the description includes situations where the event or circumstance occurs and situations where it does not.

[0029] Terms and definitions In this document, the term "retinol binding protein" refers to a class of proteins that can specifically bind to retinol (alcohol form of vitamin A), common retinol binding proteins include RBP1, RBP2 and RBP4, etc. In the embodiments of the present application, RBP4 is taken as an example, which is additionally added to the basic in vitro maturation solution for related experiments.

[0030] In this document, the term "sodium beta-glycerophosphate pentahydrate" refers to a biologically active endogenous metabolite, its chemical formula is C3H 17 Na2O 11 P.

[0031] In this context, the term "0.1% PVP-PBS" refers to a 0.1% (mass / volume ratio) Polyvinylpyrrolidone (PVP) solution in Phosphate-Buffered Saline (PBS), which is mainly used for washing embryos during immunofluorescence staining or oocytes during GSH / ROS detection in embodiments of the present application.

[0032] In this context, the term "0.5% Triton X-100 (PBS)" refers to a Phosphate-Buffered Saline (PBS) solution containing 0.5% (volume percentage) Triton X-100, which is mainly used for permeabilizing oocytes or embryos in embodiments of the present application, making their cell membranes permeable for subsequent processing.

[0033] In this context, the term "DCFH-DA working solution" refers to a working solution of DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) dissolved in serum-free culture medium, where DCFH-DA is a commonly used fluorescent probe for detecting intracellular reactive oxygen species (ROS) levels.

[0034] In this context, the term "Glutathione (GSH) level" refers to the content of Glutathione (GSH) in cells, which is an important intracellular antioxidant that maintains the redox balance in cells by reacting with reactive oxygen species (ROS), protecting cells from oxidative stress damage. The level of GSH reflects the antioxidant capacity of cells, and a higher GSH level usually means that cells have stronger antioxidant capacity and better physiological status.

[0035] In this context, the term "Reactive Oxygen Species (ROS) level" refers to the content of Reactive Oxygen Species (ROS) in cells, which plays an important role in cell signaling, apoptosis and immune response. However, excessive ROS can cause cellular oxidative stress, damaging cell structure and function. Therefore, the level of ROS reflects the oxidative stress state of cells, and a lower ROS level usually means that cells are in a healthier redox balance state.

[0036] In this context, the term "Basic in vitro maturation medium" refers to the basic culture medium used for in vitro maturation of oocytes, i.e. oocyte in vitro maturation culture medium, which contains basic nutrients necessary for oocyte growth and maturation, such as amino acids, vitamins, minerals, energy sources, buffers and antibiotics.

[0037] In the present document, the term "ICM" refers to the Inner Cell Mass, which is a group of cells in early embryos (such as blastocysts) with developmental totipotency, capable of differentiating into all tissues of the embryo, and thus the ICM cell number is an important indicator for evaluating the developmental potential of embryos, and a higher ICM cell number means that the embryo has stronger developmental potential.

[0038] Formulation for in vitro embryo production The present application provides a formulation for in vitro embryo production. According to an embodiment of the present application, the formulation contains at least one of the following components: retinol binding protein; sodium beta-glycerophosphate pentahydrate. The formulation according to the embodiment of the present application, by adding at least one of retinol binding protein and sodium beta-glycerophosphate pentahydrate, can promote in vitro maturation of oocytes, improve the maturation quality of in vitro oocytes and the developmental potential of in vitro embryos, and provide an effective new strategy for in vitro embryo production of livestock, animal genetic improvement, and assisted reproductive technology, and has a wide application prospect.

[0039] According to an embodiment of the present application, the retinol binding protein includes one or more of retinol binding protein 1, retinol binding protein 2 and retinol binding protein 4. Thus, these retinol binding proteins have similar functional and structural properties, and by providing a selection of multiple retinol binding proteins, different retinol binding proteins can be selected according to different experimental purposes, different types of oocytes or embryos and application requirements, providing flexibility for further optimization of the present application for in vitro embryo production.

[0040] It should be noted that in the embodiments of the present application, although only the functional properties of retinol binding protein 4 are verified, retinol binding protein 4 and other retinol binding proteins are a class of proteins with similar structural characteristics and metabolic functions, and adding them to the formulation for in vitro embryo production will have similar technical effects, and therefore all retinol binding proteins (including but not limited to RBP4, RBP1 and RBP2) should belong to the protection scope of the present application.

[0041] It should be noted that the formulation contains at least one of retinol binding protein and sodium beta-glycerophosphate pentahydrate, which mainly includes several cases: single addition of two components (retinol binding protein and sodium beta-glycerophosphate pentahydrate), simultaneous addition of two components (retinol binding protein and sodium beta-glycerophosphate pentahydrate), all of which belong to the protection scope of the present application.

[0042] According to an embodiment of the present application, the concentration of the retinol binding protein is 0.1-200 ng / mL. Thereby, by controlling the concentration of the retinol binding protein, the optimal effect of the preparation of the present application in regulating oocyte maturation, in vitro fertilization or in vitro culture of embryos is further ensured; exemplarily, the concentration of the retinol binding protein is 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 30 ng / mL, 50 ng / mL, 100 ng / mL, 120 ng / mL, 150 ng / mL, 180 ng / mL, 200 ng / mL, preferably 1-50 ng / mL, more preferably 20 ng / mL.

[0043] According to a preferred embodiment of the present application, the concentration of the retinol binding protein 4 is 0.1-200 ng / mL. Thereby, by controlling the concentration of the retinol binding protein 4, the optimal effect of the preparation of the present application in regulating oocyte maturation, in vitro fertilization or in vitro culture of embryos is further ensured; exemplarily, the concentration of the retinol binding protein 4 is 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 30 ng / mL, 50 ng / mL, 100 ng / mL, 120 ng / mL, 150 ng / mL, 180 ng / mL, 200 ng / mL, preferably 1-50 ng / mL, more preferably 20 ng / mL.

[0044] According to an embodiment of the present application, the concentration of the sodium beta-glycerophosphate pentahydrate is 0.1-100 mM. Thereby, by controlling the concentration of the sodium beta-glycerophosphate pentahydrate, the optimal effect of the preparation of the present application in regulating oocyte maturation, in vitro fertilization or in vitro culture of embryos is further ensured; exemplarily, the concentration of the sodium beta-glycerophosphate pentahydrate is 0.1 mM, 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, preferably 0.2-4 mM, more preferably 1 mM.

[0045] According to an embodiment of the present application, the preparation contains retinol binding protein and sodium beta-glycerophosphate pentahydrate, the concentration of the retinol binding protein is 0.1-200 ng / mL, and the concentration of the sodium beta-glycerophosphate pentahydrate is 0.1-100 mM. Thus, when the preparation contains the retinol binding protein and the sodium beta-glycerophosphate pentahydrate, the concentration of the two components when added synergistically is further optimized so that the two components are synergistically matched, and thus exert the best effect (better synergistic effect) in regulating oocyte maturation, in vitro fertilization or in vitro culture of embryos. Exemplarily, the concentration of the retinol binding protein is 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 30 ng / mL, 50 ng / mL, 100 ng / mL, 120 ng / mL, 150 ng / mL, 180 ng / mL, 200 ng / mL, preferably 1-50 ng / mL, and more preferably 20 ng / mL; and the concentration of the sodium beta-glycerophosphate pentahydrate is 0.1 mM, 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, preferably 0.2-4 mM, and more preferably 1 mM.

[0046] According to a preferred embodiment of the present application, the preparation contains retinol binding protein 4 and sodium beta-glycerophosphate pentahydrate, the concentration of the retinol binding protein 4 is 0.1-200 ng / mL, and the concentration of the sodium beta-glycerophosphate pentahydrate is 0.1-100 mM. Thus, when the preparation contains the retinol binding protein 4 and the sodium beta-glycerophosphate pentahydrate, the concentration of the two components when added synergistically is further optimized so that the two components are synergistically matched, and thus exert the best effect (better synergistic effect) in regulating oocyte maturation, in vitro fertilization or in vitro culture of embryos. Exemplarily, the concentration of the retinol binding protein 4 is 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 30 ng / mL, 50 ng / mL, 100 ng / mL, 120 ng / mL, 150 ng / mL, 180 ng / mL, 200 ng / mL, preferably 1-50 ng / mL, and more preferably 20 ng / mL; and the concentration of the sodium beta-glycerophosphate pentahydrate is 0.1 mM, 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, preferably 0.2-4 mM, and more preferably 1 mM.

[0047] According to an embodiment of the present application, the preparation is used in at least one stage of oocyte in vitro maturation, in vitro fertilization or in vitro culture of embryos.

[0048] According to an embodiment of the present application, the preparation is used for the in vitro maturation stage of oocytes, and the use time is from the GV stage to the MII stage.

[0049] Therefore, the preparation can be added at each key stage of in vitro maturation, in vitro fertilization and in vitro culture of embryos; it should be noted that the preparation of the present application can be used in the whole production process of in vitro embryos, mainly including three stages of in vitro maturation of oocytes, in vitro fertilization and in vitro culture of fertilized eggs.

[0050] According to an embodiment of the present application, the preparation further comprises one or more of inorganic salts, amino acids, vitamins, sugars, follicle stimulating hormone, luteinizing hormone, estradiol, fetal bovine serum, pH indicator and antibiotics. Therefore, the presence of these substances provides basic conditions for the growth and development of oocytes / in vitro embryos, and together with retinol binding protein and / or sodium beta-glycerophosphate, they form a complete nutrient system, which provides an ideal growth environment for the in vitro maturation of oocytes / in vitro embryos, further improves the maturation efficiency and quality of oocytes, and ensures that they exhibit better development potential in the subsequent development process of embryos; for example, in an embodiment of the present application, the basic in vitro maturation solution comprises Medium 199 basic medium, follicle stimulating hormone, luteinizing hormone, estradiol, fetal bovine serum, penicillin and streptomycin, wherein the Medium 199 basic medium is a classic cell culture medium suitable for various cell types, and the medium mainly comprises (1) inorganic salts: sodium salt, potassium salt, calcium salt and magnesium salt, etc.; (2) amino acids: various essential and non-essential amino acids, such as alanine, aspartic acid, glutamic acid, lysine, tryptophan, etc.; (3) vitamins: water-soluble vitamins (such as vitamin B group) and fat-soluble vitamins (such as vitamin A, vitamin D, etc.); (4) sugars: glucose (5) pH indicator: phenol red (6) others: trace elements.

[0051] According to an embodiment of the present application, the preparation further comprises 0.04-2.5 mg / mL inorganic salt, 0.05-2.0 mM amino acid, 0.5-2.0 mM vitamin, 0.5-10 mM sugar, 0.02-0.1 IU / mL follicle stimulating hormone, 0.02-0.1 IU / mL luteinizing hormone, 1.0-2.0 ug / mL estradiol, 5-10% fetal bovine serum, 95-105 IU / mL antibiotic. Therefore, the presence of these substances provides basic conditions for the growth and development of oocytes.

[0052] It should be noted that the specific types, concentrations and combination modes of other components in the preparation described in the present application except for retinol binding protein, sodium beta-glycerophosphate pentahydrate can be adjusted appropriately according to the source of oocytes / embryos, the development stage and the experimental requirements, and the scheme for reasonably optimizing or equivalently replacing the components of the preparation for improving the quality of oocytes in vitro maturation and the quality of blastocysts after in vitro fertilization should all belong to the protection scope of the present application.

[0053] According to an embodiment of the present application, the oocyte or the in vitro embryo is derived from a non-human mammal. Thus, the preparation for in vitro embryo production of the present application is suitable for oocytes or in vitro embryos derived from various non-human mammals.

[0054] According to an embodiment of the present application, the mammal includes one or more of non-human primates, cows, sheep, pigs, horses and mice.

[0055] It should be noted that the application range of the preparation for in vitro embryo production of the present application is not only limited to the above-mentioned several non-human mammals, but also suitable for all other mammals, such as buffalos, sheep, goats, donkeys, camels, rats, rabbits, guinea pigs, hamsters, ferrets, cats, dogs, etc., which should all belong to the protection scope of the present application.

[0056] Application The present application proposes the application of retinol binding protein, sodium beta-glycerophosphate pentahydrate or the aforementioned preparation for in vitro embryo production in in vitro embryo production.

[0057] It can be understood by those skilled in the art that the features and advantages described above for the preparation for in vitro embryo production are also applicable to the application, which will not be described here again.

[0058] According to an embodiment of the present application, the retinol binding protein, sodium beta-glycerophosphate pentahydrate or the aforementioned preparation for in vitro embryo production has at least one of the following uses: improving the maturation quality of in vitro oocytes; improving the development potential of in vitro embryos; wherein the improving the development potential of in vitro embryos includes improving the development efficiency and / or development quality of in vitro embryos.

[0059] According to an embodiment of the present application, the retinol binding protein, sodium beta-glycerophosphate pentahydrate or the aforementioned preparation for in vitro embryo production has at least one of the following uses: improving the glutathione level in in vitro oocytes; reducing the level of reactive oxygen species in in vitro oocytes; improving the cleavage rate of in vitro embryos; improving the blastocyst rate of in vitro embryos; improving the inner cell mass cell number of blastocysts of in vitro embryos.

[0060] According to an embodiment of the present application, the retinol binding protein includes one or more of retinol binding protein 1, retinol binding protein 2 and retinol binding protein 4.

[0061] It should be noted that although the application examples only verify the application of retinol binding protein and sodium β-glycerophosphate pentahydrate in improving the maturation quality of oocytes in vitro, it can be understood that oocyte in vitro maturation is the core initial link in the whole process of in vitro embryo production, and its quality directly determines the potential of subsequent fertilization, embryo development and successful pregnancy, which is a decisive stage of in vitro embryo production. Therefore, the application of retinol binding protein and sodium β-glycerophosphate pentahydrate in the whole process of in vitro embryo production is universal, mainly including three stages of in vitro maturation of oocytes, in vitro fertilization and in vitro culture of fertilized eggs. In addition, in the process of in vitro embryo production, high oxygen environment, insufficient antioxidant system, accumulation of metabolic by-products, lack of intercellular support and stress caused by experimental operation will stimulate oxidative stress in oocytes, sperm and fertilized eggs. Among them, oxidative stress (manifested as ROS accumulation, GSH depletion, etc.) is a key limiting factor in the process of oocyte in vitro maturation, in vitro fertilization and in vitro culture of embryos. No matter which stage is stimulated by oxidative stress, it will interfere with the physiological function of reproductive cells (oocytes, sperm) and early embryos through multiple molecular mechanisms, ultimately affecting the embryo development potential, and its influence is continuous. The cascade damage from oocyte maturation to embryo development has been confirmed in multiple species, including but not limited to humans, mice, cows, sheep, etc., which is highly conserved in mammals. Therefore, those skilled in the art can understand that retinol binding protein, sodium β-glycerophosphate pentahydrate or the aforementioned preparation for in vitro embryo production has application potential in the whole process of in vitro embryo production, and should belong to the protection scope of the present application.

[0062] Method for in vitro embryo production The present application provides a method for in vitro embryo production. According to an embodiment of the present application, the method comprises: culturing oocyte or sperm cell or fertilized egg in a culture system containing retinol binding protein and / or sodium β-glycerophosphate pentahydrate; wherein the oocyte or sperm cell or fertilized egg is derived from a non-human mammal; and the culture system is the aforementioned preparation for in vitro embryo production. According to the method of the embodiment of the present application, by co-culturing oocyte or sperm cell or fertilized egg with the aforementioned preparation containing retinol binding protein and / or sodium β-glycerophosphate pentahydrate, precise regulation and support can be provided in the whole stage of in vitro embryo production, thereby comprehensively improving the efficiency and quality of in vitro embryo production.

[0063] According to an embodiment of the present application, the non-human mammal includes one or more of non-human primates, cows, sheep, pigs, horses and mice. Therefore, the method for in vitro embryo production of the present application is suitable for oocytes derived from a variety of non-human mammals.

[0064] It should be noted that the scope of application of the method of the present application includes many non-human mammals, such as non-human primates, cows, sheep, pigs, horses, mice, buffalos, sheep, goats, donkeys, camels, rats, rabbits, guinea pigs, hamsters, ferrets, cats, dogs, etc., which should all belong to the protection scope of the present application.

[0065] The scheme of the present application will be explained below in combination with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If the specific technology or condition is not specified in the examples, it is carried out according to the technology or condition described in the literature in the art or according to the product instruction. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained by market purchase.

[0066] Example 1: Application of combined administration of RBP4 and sodium beta-glycerophosphate pentahydrate in improving the quality of bovine oocytes in vitro maturation and the quality of blastocysts after in vitro fertilization 1. Experimental grouping Control group: normal in vitro fertilization, using basic in vitro maturation solution (addition amount is 500 μL / well).

[0067] Treatment group 1: normal in vitro fertilization, adding 20 ng / mL of retinol binding protein 4 (RBP4, purchased from R&D systems, item number 3378-LC-050) on the basis of using basic in vitro maturation solution (addition amount is 500 μL / well).

[0068] Treatment group 2: normal in vitro fertilization, adding 1 mM of sodium beta-glycerophosphate pentahydrate (purchased from MedChemExpress, item number HY-D0886) on the basis of using basic in vitro maturation solution (addition amount is 500 μL / well).

[0069] Treatment group 3: normal in vitro fertilization, combined addition of 15 ng / mL of RBP4 and 1 mM of sodium beta-glycerophosphate pentahydrate on the basis of using basic in vitro maturation solution (addition amount is 500 μL / well).

[0070] Among them, the preparation method of the basic in vitro maturation solution is as follows: Medium 199 (purchased from Gibco, item number 11150059) as the basic solvent, add 0.02 IU / mL Folltropin, 0.02 IU / mL luteinizing hormone (purchased from Ningbo Second Hormone), 1 μg / mL estradiol (purchased from Sigma, item number E2257), 10% fetal bovine serum (purchased from Mei5bio, item number MF195-plus-10), 1% penicillin-streptomycin mixture (purchased from Gibco, item number 15140122).

[0071] 2. Oocyte collection and in vitro maturation First, the cow ovary was incubated at 32~35°C physiological saline and sent to the laboratory, and the visible follicle on the surface of the ovary was extracted by a 20 mL syringe to obtain follicular fluid containing oocytes; Then, the COCs containing more than 3 layers of cumulus cells were obtained by screening under a microscope, and washed twice with oocyte washing solution (add 500 μL), and then washed twice with pre-equilibrated basic oocyte in vitro maturation solution for 3 h; Then, the washed COCs were placed in a four-well plate basic in vitro maturation solution (500 μL volume of maturation solution / well) pre-equilibrated in a CO2 incubator for more than 2 h, about 50 oocytes per well, the culture conditions were set as 5% CO2 in air, temperature 38.5°C, saturated humidity, culture time 24 h, and mature COCs were obtained.

[0072] Among them, the washed COCs need to be counted, and the total number of oocytes in each group is recorded, that is, the number of oocytes.

[0073] The preparation method of the oocyte washing solution is as follows: DPBS (purchased from Sigma, item number 14190144) as the basic solvent, add 0.7 mg / mL heparin sodium (purchased from Sigma, item number H3149), 1 mg / mL BSA (purchased from Sigma, item number A6003), 1% penicillin-streptomycin mixture (purchased from Gibco, item number 15140122).

[0074] 3. In vitro fertilization After maturation of the COCs, most cumulus cells were removed by gently pipetting, and then the COCs were transferred into the fertilization medium (500 μL) equilibrated in a CO2 incubator for more than 2 h to obtain the fertilization preparation (50 mature COCs per well). Frozen bovine sperm (purchased from Beijing Dairy Center) was thawed in a 38°C water bath for 30 s, and then centrifuged (320 xg, 5 min) in a 15 mL centrifuge tube containing washing medium (purchased from Beijing Dairy Center). The supernatant was discarded, and the sperm were resuspended in 3 mL of washing medium and centrifuged again. The sperm were resuspended in the fertilization medium, and the sperm activity was detected. Then, 75 μL of the sperm suspension was added to the fertilization preparation, and in vitro fertilization was performed for 18 h at 38.5°C, 5% CO2, and 95% air.

[0075] The fertilization medium was prepared as follows: The base solvent was embryo culture water (purchased from Sigma, W1503), to which 20 μg / mL of sodium heparin (purchased from Sigma, H3149), 6 mg / mL of BSA (purchased from Sigma, A6003), 37.00 mM of sodium bicarbonate (purchased from Sigma, S5761), 1.25 mM of sodium pyruvate (purchased from Sigma, P2256), 112 mM of sodium chloride (purchased from Sigma, S5886), 4.02 mM of potassium chloride (purchased from Sigma, P5405), 0.954 mM of sodium phosphate monobasic (purchased from Sigma, S5011), 0.52 mM of magnesium chloride hexahydrate (purchased from Sigma, M2393), 2.25 mM of calcium chloride dihydrate (purchased from Sigma, C7902), 13.98 mM of glucose (purchased from Sigma, G6152), 0.1 mg / mL of phenol red (purchased from Sigma, P0290), and 1% of penicillin-streptomycin (purchased from Gibco, 15140122) were added.

[0076] 4. In vitro culture and counting After fertilization, the zygotes were removed, and all the residual cumulus cells and sperm were removed. The zygotes were washed three times with in vitro embryo development medium (400 μL) and then cultured in a four-well plate (500 μL of in vitro embryo development medium per well) at 38.5°C, 5% CO2, 90% N2, and saturated humidity for 7-8 days. Then, the cell counting was performed, and the cleavage rate and the 8-day blastocyst rate were calculated. The cleavage rate = (the number of cleaved oocytes / the total number of zygotes) * 100%, and the 8-day blastocyst rate = (the number of embryos developed to the blastocyst stage / the total number of zygotes) * 100%.

[0077] The preparation method of the in vitro embryo development solution is as follows: Water suitable for embryo culture (purchased from Sigma, item number W1503) as a base solvent, 26.31 mM sodium bicarbonate (purchased from Sigma, item number S5761), 2.97 mM myo-inositol (purchased from Sigma, item number I7508), 1.06 mM L-glutamine (purchased from Sigma, item number G3126), 2% essential amino acids (purchased from Sigma, item number B6766), 1% non-essential amino acids (purchased from Sigma, item number M7145), 107.64 mM sodium chloride (purchased from Sigma, item number S5886), 7.18 mM potassium chloride (purchased from Sigma, item number P5405), 1.19 mM potassium phosphate monobasic (purchased from Sigma, item number P5655), 0.73 mM magnesium sulfate (purchased from Sigma, item number M2643), 0.06% sodium lactate (purchased from Sigma, item number L7900), 1% penicillin-streptomycin mixture (purchased from Gibco, item number 15140122), 0.33 mM sodium pyruvate (purchased from Sigma, item number P2256), 1.71 mM calcium chloride dihydrate (purchased from Sigma, item number C7902), 3 mg / mL bovine serum albumin (purchased from Sigma, item number A6003), 0.1 mg / mL phenol red (purchased from Sigma, item number P0290).

[0078] 5. Number of inner cell mass cells The blastocyst was collected and washed 3 times with 0.1% PVP-PBS, and then fixed in 4% paraformaldehyde for 30 min; then treated with 0.5% TritonX-100 (PBS) at room temperature for 30 min, and washed 3 times with 0.1% PVP-PBS; then blocked with 0.1% TritonX-100 + 0.1% BSA (PBS) for 30 min, and washed 3 times with 0.1% PVP-PBS to obtain the treated blastocyst; then the treated blastocyst was incubated with the primary antibody at 4°C overnight, washed 3 times with 0.1% PVP-PBS after incubation, and incubated with the secondary antibody at room temperature in the dark, washed 3 times with 0.1% PVP-PBS after incubation, and finally stained the cell nucleus with DAPI, mounted after incubation in the dark for 5-10 min; then observed using a fluorescence microscope, and counted the cells using image analysis software ImageJ to obtain the number of inner cell mass (ICM) in the blastocyst; wherein the primary antibody was Anti-SOX2 (ebioscience-14-9811-82); Anti-CDX2 (BioGenex-MU392A-UC). The secondary antibody was Alexa Fluor 488 (Goat anti-mouse) (Invitrogen, -A-11029); Alexa Fluor 594 (Goat anti-rat) (Abcam-ab150160).

[0079] 6. Glutathione (GSH) level detection First, the mature COCs obtained in step 2 were stripped of cumulus granulosa cells using a 1 mg / mL hyaluronic acid solution to obtain clean oocytes, and then the clean oocytes were incubated with 5 µM 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (fluorescent probe, labeling GSH) in a 37°C cell incubator for 20 min, and then washed 3 times with 0.1% PVP-PBS, and then observed and photographed under 405 nm laser excitation using a fluorescence microscope, and analyzed the GSH level using Image J software.

[0080] 7. Reactive oxygen species (ROS) level detection The working solution of the oxidation-sensitive fluorescent probe DCFH-DA was prepared by diluting DCFH-DA (final concentration = 10 μM) with Medium 199 (purchased from Gibco, item number 11150059) at a ratio of 1:1000; meanwhile, the mature COCs obtained in step 2 were cleaned by removing the cumulus cells using a 1 mg / mL hyaluronic acid solution, and then the cleaned oocytes were incubated with the 10 uM working solution of the oxidation-sensitive fluorescent probe DCFH-DA in a 37°C cell incubator for 20 min, followed by washing 3 times with 0.1% PVP-PBS, and then observed and photographed under a fluorescence microscope under 488 nm laser excitation, and the ROS level was analyzed using Image J software.

[0081] 8. Experimental results The results of the determination of the effects of RBP4 and sodium β-glycerophosphate pentahydrate on the development rate of bovine embryos in vitro are shown in Table 1, the results of the determination of the effects of RBP4 and sodium β-glycerophosphate pentahydrate on the number of inner cell mass cells in bovine embryos in vitro are shown in Table 2, and the results of the determination of the effects of RBP4 and sodium β-glycerophosphate pentahydrate on the quality of bovine mature oocytes in vitro are shown in Table 3.

[0082] Table 1. Results of the determination of the effects of RBP4 and sodium β-glycerophosphate pentahydrate on the development rate of bovine embryos in vitro

[0083] Among them, the different lowercase letters indicate significant differences P <0.05), and the same lowercase letters indicate no significant difference P >0.05) (mean ± standard deviation).

[0084] The results show that, compared with the cleavage rate of the control group (76.36 ± 6.53), the cleavage rates of the RBP4-only added group (treatment group 1, 84.28 ± 2.50), the sodium β-glycerophosphate pentahydrate-only added group (treatment group 2, 80.34 ± 3.75), and the RBP4 and sodium β-glycerophosphate pentahydrate combined added group (treatment group 3, 89.56 ± 3.19) are all significantly improved, and the RBP4 and sodium β-glycerophosphate pentahydrate combined added group (treatment group 3) has the most significant improvement effect. At the same time, the results of the detection of the 8-day blastocyst rate show that, compared with the blastocyst rate of the control group (32.73 ± 1.86), the blastocyst rates of the RBP4-only added group (treatment group 1), the sodium β-glycerophosphate pentahydrate-only added group (treatment group 2), and the RBP4 and sodium β-glycerophosphate pentahydrate combined added group (treatment group 3) are all significantly improved, and are 40.76 ± 2.41, 37.65 ± 3.19, and 49.23 ± 3.57, respectively, and the RBP4 and sodium β-glycerophosphate pentahydrate combined added group (treatment group 3) has the most significant improvement effect.

[0085] The above results show that the single or combined addition (synergistic effect) of RBP4 and sodium beta-glycerophosphate pentahydrate can significantly improve the in vitro development ability of bovine oocytes, specifically reflected in the significant increase of cleavage rate and blastocyst rate, and the effect is better when the two are used together.

[0086] Table 2 Determination results of RBP4 and sodium beta-glycerophosphate pentahydrate on bovine in vitro embryo inner cell mass cell number

[0087] Among them, the shoulder notes of different lowercase letters represent significant differences P <0.05), and the same lowercase letters represent no significant difference P >0.05) (mean ± standard deviation).

[0088] The results show that compared with the ICM cell number (35.83±6.28) of the control group, the ICM cell number of the RBP4 single addition group (treatment group 1, 57.39±8.29), the sodium beta-glycerophosphate pentahydrate single addition group (treatment group 2, 49.27±7.51), and the RBP4 and sodium beta-glycerophosphate pentahydrate combined addition group (treatment group 3, 61.95±5.45) can be significantly improved, and the ICM cell number of the RBP4 and sodium beta-glycerophosphate pentahydrate combined addition group (treatment group 3) is the most significant.

[0089] The above results show that the single or combined addition (synergistic effect) of RBP4 and sodium beta-glycerophosphate pentahydrate can significantly improve the in vitro development ability of bovine oocytes, specifically reflected in the significant increase of cleavage rate and blastocyst rate, and the effect is better when the two are used together.

[0090] Table 3 Determination results of RBP4 and sodium beta-glycerophosphate pentahydrate on bovine in vitro matured oocyte quality

[0091] Among them, the shoulder notes of different lowercase letters represent significant differences P <0.05), and the same lowercase letters represent no significant difference P >0.05) (mean ± standard deviation).

[0092] The results show that: compared with the GSH level (18.74±5.17) of the control group, the GSH level of the RBP4 single addition group (treatment group 1, 35.76±6.30), the sodium beta-glycerophosphate pentahydrate single addition group (treatment group 2, 27.38±5.79), and the RBP4 and sodium beta-glycerophosphate pentahydrate combined addition group (treatment group 3, 39.12±4.29) are all improved, among which the RBP4 and sodium beta-glycerophosphate pentahydrate combined addition group (treatment group 3) has the best improvement effect; at the same time, compared with the ROS level (58.39±7.45) of the control group, the ROS levels of the RBP4 single addition group (treatment group 1, 29.35±6.15), the sodium beta-glycerophosphate pentahydrate single addition group (treatment group 2, 35.93±7.13), and the RBP4 and sodium beta-glycerophosphate pentahydrate combined addition group (treatment group 3, 21.84±5.49) are all significantly reduced, among which the RBP4 and sodium beta-glycerophosphate pentahydrate combined addition group (treatment group 3) has the best reduction effect.

[0093] The above results show that: RBP4 and sodium beta-glycerophosphate pentahydrate single or combined addition (synergistic effect) can significantly improve the antioxidant capacity and redox balance of bovine oocytes, thereby improving the quality and development potential of bovine oocytes, and the effect is better when they are used together.

[0094] Example 2: Application of RBP4 and sodium beta-glycerophosphate pentahydrate combined administration in improving the quality of sheep oocytes in vitro maturation and the quality of blastocysts after in vitro fertilization 1. Experimental grouping The experimental grouping is set as in Example 1.

[0095] 2. Oocyte collection and in vitro maturation First, the sheep ovary is incubated with 35℃ physiological saline and sent to the laboratory, and the follicle is pierced with a 20G needle syringe containing 5 mL oocyte aspiration solution to obtain follicular fluid containing oocytes; then the follicle-oocyte complex (COCs) containing more than 3 layers of cumulus cells is obtained by screening under a microscope, and washed 3 times with oocyte washing solution, and then washed 3 times with basic in vitro maturation solution; then the washed COCs are placed in a four-well plate basic in vitro maturation solution (600 μL volume of maturation solution per well, 300 μL mineral oil covering) pre-equilibrated in a CO2 incubator for more than 2 h, about 30~35 oocytes per well, the culture conditions are set as 5% CO2 in air, temperature 38.5℃, saturated humidity, and the culture time is 22~24 h, to obtain mature COCs.

[0096] The preparation method of the oocyte aspiration solution is as follows: 0.7 mg / mL heparin sodium (purchased from Sigma, H3149), 2% fetal bovine serum (purchased from MF195, plus-10), 1% penicillin-streptomycin mixture (purchased from Gibco, 15140122), DPBS (purchased from Gibco, 14190144) as a base solvent.

[0097] 3. In vitro fertilization After the maturation of COCs, most of the cumulus cells were removed by gently blowing, and then moved into the fertilization solution equilibrated in a CO2 incubator for more than 2 h, 300 μL of mineral oil was covered to obtain the fertilization preparation cell solution (30 mature COCs per well); and the frozen semen of sheep (donated by Tianjin Agricultural University) was taken out, thawed in a 38℃ water bath for 30 s, and then the thawed sperm was transferred to 600 μL of fertilization solution and incubated in the incubator for 30 min to make the sperm fully float; then 300~400 μL of supernatant was added to the fertilization preparation cell solution, and in vitro fertilization was performed for 20 h, and the fertilization conditions were set as 38.5℃, 5% CO2, 95% air.

[0098] Among them, the preparation method of the fertilization solution is the same as in Example 1.

[0099] 4. In vitro culture and counting After the fertilization was completed, the fertilized eggs were aspirated, all the residual cumulus cells and sperm on the surface were removed, and then washed with in vitro embryo development solution for 3 times, and then placed in a four-well plate for culture (600 μL of in vitro embryo development solution per well), and the culture conditions were set as 38.5℃, 5% CO2, 90% N2, saturated humidity, and cultured for 6~7 days; then the cell counting was performed, the cleavage rate and the 7-day blastocyst rate were counted, wherein the cleavage rate = (the number of cleaved eggs / the total number of fertilized eggs) * 100%, and the 7-day blastocyst rate = (the number of embryos developed to the blastocyst stage / the total number of fertilized eggs) * 100%.

[0100] 5. Inner cell mass cell number and cell proportion determination The blastocyst was collected and washed 3 times with 0.1% PVP-PBS, and then fixed in 4% paraformaldehyde for 30 min; then treated with 0.5% TritonX-100 (PBS) at room temperature for 30 min, and washed 3 times with 0.1% PVP-PBS; then blocked with 0.1% TritonX-100 + 0.1% BSA (PBS) for 30 min, and washed 3 times with 0.1% PVP-PBS to obtain the treated blastocyst; then the treated blastocyst was incubated with the primary antibody at 4°C overnight, after incubation, washed 3 times with 0.1% PVP-PBS, and incubated with the secondary antibody at room temperature in the dark, after incubation, washed 3 times with 0.1% PVP-PBS, and finally stained the cell nucleus with DAPI, and mounted after incubation in the dark for 5-10 min; then observed using a fluorescence microscope, and counted the cells using image analysis software ImageJ to obtain the number of inner cell mass (ICM) in the blastocyst, and the ratio of ICM / TE (ICM cell number and trophoblast cell number (Trophoblast Cell Number, TE)); wherein the primary antibody is Anti-SOX2 (purchased from ebioscience, item number 14-9811-82); Anti-CDX2 (purchased from BioGenex, item number MU392A-UC); the secondary antibody is Alexa Fluor 488 (Goat anti-mouse) (purchased from Invitrogen, item number A-11029); Alexa Fluor 594 (Goat anti-rat) (purchased from Abeam, item number ab150160).

[0101] 6. Glutathione (GSH) level detection The same as step 6 of Example 1.

[0102] 7. Reactive oxygen species (ROS) level detection The same as step 7 of Example 1.

[0103] 8. Experimental results The determination results of the effects of RBP4 and sodium β-glycerophosphate pentahydrate on the development rate of sheep embryos in vitro are shown in Table 4, the determination results of the effects of RBP4 and sodium β-glycerophosphate pentahydrate on the number of inner cell mass cells of sheep embryos in vitro are shown in Table 5, and the determination results of the effects of RBP4 and sodium β-glycerophosphate pentahydrate on the quality of sheep mature oocytes in vitro are shown in Table 6.

[0104] Table 4 Determination results of the effects of RBP4 and sodium β-glycerophosphate pentahydrate on the development rate of sheep embryos in vitro

[0105] Wherein, the shoulder notes different lowercase letters represent significant difference P <0.05), the same lowercase letters represent no significant difference P >0.05).

[0106] The results show that: compared with the control group cleavage rate (73.48±4.97), RBP4 alone added group (treatment group 1, 83.67±3.57), five water beta glycerol phosphate sodium alone added group (treatment group 2, 79.53±3.72), RBP4 and five water beta glycerol phosphate sodium combined addition group (treatment group 3, 85.67±4.34) cleavage rate were significantly improved, among them, RBP4 and five water beta glycerol phosphate sodium combined addition group (treatment group 3) cleavage rate of the most significant effect of improvement; the 7th day blastocyst rate detection results show that, compared with the control group blastocyst rate (35.87±4.12), RBP4 alone added group (treatment group 1), five water beta glycerol phosphate sodium alone added group (treatment group 2), RBP4 and five water beta glycerol phosphate sodium combined addition group (treatment group 3) blastocyst rate were significantly improved, respectively 42.17±3.11, 40.97±4.75, 49.25±3.72, among them, RBP4 and five water beta glycerol phosphate sodium combined addition group (treatment group 3) blastocyst rate of the most significant effect of improvement.

[0107] The above results show that: RBP4 and five water beta glycerol phosphate sodium single or combined (play a synergistic effect), can significantly improve the in vitro development ability of sheep oocyte, specific embodiment for cleavage rate and blastocyst rate were significantly improved, among them, the effect is better when the two are used together.

[0108] Table 5 RBP4, five water beta glycerol phosphate sodium on the effect of sheep in vitro embryo inner cell mass cell number determination results

[0109] Wherein, the shoulder notes different lowercase letters represent significant difference P <0.05), the same lowercase letters represent no significant difference P >0.05).

[0110] The results show that: compared with the control group ICM cell number (30.27±7.56), RBP4 alone added group (treatment group 1, 31.45±6.98), five water beta glycerol phosphate sodium alone added group (treatment group 2, 32.17±7.34) on the effect of sheep blastocyst ICM cell number is not significant, but RBP4 and five water beta glycerol phosphate sodium combined addition group (treatment group 3) ICM cell number (42.31±6.14) significantly improved.

[0111] The above results show that the single or combined addition (synergistic effect) of RBP4 and sodium beta-glycerophosphate pentahydrate can improve the quality and development potential of sheep embryo development to a certain extent, and the effect is more significant when the two are used together.

[0112] Table 6 Determination results of the effect of RBP4 and sodium beta-glycerophosphate pentahydrate on the quality of sheep in vitro matured oocytes

[0113] Among them, the shoulder notes different lowercase letters represent significant differences P <0.05), and the same lowercase letters represent no significant difference P >0.05).

[0114] The results show that compared with the control group GSH level (25.31±4.76), the cleavage rate of RBP4 single addition group (treatment group 1, 29.13±3.34), sodium beta-glycerophosphate pentahydrate single addition group (treatment group 2, 31.05±4.01), RBP4 and sodium beta-glycerophosphate pentahydrate combined addition group (treatment group 3, 33.67±3.19) were significantly improved, and the RBP4 and sodium beta-glycerophosphate pentahydrate combined addition group (treatment group 3) was the best. At the same time, compared with the control group ROS level (52.78±5.98), the ROS level of RBP4 single addition group (treatment group 1, 32.45±4.22), sodium beta-glycerophosphate pentahydrate single addition group (treatment group 2, 34.19±4.02) and RBP4 and sodium beta-glycerophosphate pentahydrate combined addition group (treatment group 3, 25.17±3.67) were significantly reduced, and the RBP4 and sodium beta-glycerophosphate pentahydrate combined addition group (treatment group 3) was the best.

[0115] The above results show that the single or combined addition (synergistic effect) of RBP4 and sodium beta-glycerophosphate pentahydrate can significantly improve the antioxidant capacity and redox balance of sheep oocytes, thereby improving the quality and development potential of sheep oocytes, and the effect is better when the two are used together.

[0116] In the description of the specification, the description using the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" etc. means that the particular feature, structure, material or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. Descriptive expressions of the above terms in the specification do not necessarily refer to the same embodiment or example. Also, the particular feature, structure, material or characteristic described can be combined in any suitable manner in one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples without contradiction.

[0117] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and the person skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A formulation for in vitro embryo production, characterized in that, The formulation contains at least one of the following components: Retinol-binding protein; Sodium β-glycerophosphate pentahydrate.

2. The formulation according to claim 1, characterized in that, The retinol-binding protein includes one or more of retinol-binding protein 1, retinol-binding protein 2, and retinol-binding protein 4.

3. The formulation according to claim 1, characterized in that, The concentration of the retinol-binding protein is 0.1~200 ng / mL; Optionally, the concentration of the sodium β-glycerophosphate pentahydrate is 0.1~100 mM; Optionally, the formulation contains retinol-binding protein and sodium β-glycerophosphate pentahydrate, wherein the concentration of retinol-binding protein is 0.1~200 ng / mL and the concentration of sodium β-glycerophosphate pentahydrate is 0.1~100 mM.

4. The formulation according to any one of claims 1 to 3, characterized in that, The formulation is used in at least one stage of oocyte in vitro maturation, in vitro fertilization, or in vitro embryo culture. Optionally, the formulation is used for the in vitro maturation stage of oocytes, and the application time is from the GV stage to the MII stage; Optionally, the formulation further includes one or more of the following: inorganic salts, amino acids, vitamins, sugars, follicle-stimulating hormone, luteinizing hormone, estradiol, fetal bovine serum, pH indicators, and antibiotics. Optionally, the formulation further includes 0.04–2.5 mg / mL of inorganic salts, 0.05–2.0 mM of amino acids, 0.5–2.0 mM of vitamins, 0.5–10 mM of carbohydrates, 0.02–0.1 IU / mL of follicle-stimulating hormone, 0.02–0.1 IU / mL of luteinizing hormone, 1.0–2.0 μg / mL of estradiol, 5–10% fetal bovine serum, and 95–105 IU / mL of antibiotics; Optionally, the oocyte or in vitro embryo is derived from a non-human mammal; Optionally, the mammals include one or more of non-human primates, cattle, sheep, pigs, horses, and mice.

5. The use of retinol-binding protein, sodium β-glycerophosphate pentahydrate, or any of the formulations for in vitro embryo production according to claims 1 to 4 in in vitro embryo production.

6. The application according to claim 5, characterized in that, The retinol-binding protein, sodium β-glycerophosphate pentahydrate, or the formulation for in vitro embryo production according to any one of claims 1 to 4 has at least one of the following uses: Improve the quality of in vitro oocyte maturation; Enhance the developmental potential of in vitro embryos; in, Enhancing the developmental potential of in vitro embryos includes improving the developmental efficiency and / or quality of in vitro embryos.

7. The application according to claim 5, characterized in that, The retinol-binding protein, sodium β-glycerophosphate pentahydrate, or the formulation for in vitro embryo production according to any one of claims 1 to 4 has at least one of the following uses: Increase glutathione levels in in vitro oocytes; Reduce reactive oxygen species levels in in vitro oocytes; Improve the cleavage rate of in vitro embryos; Increase the blastocyst rate of in vitro embryos; Increase the number of cells in the inner cell mass of the blastocyst in vitro.

8. A method for in vitro embryo production, characterized in that, The method includes: Oocytes, sperm cells, or fertilized eggs are cultured in vitro in a culture system containing retinol-binding protein and / or sodium β-glycerophosphate pentahydrate. The oocytes, sperm cells, or fertilized eggs mentioned herein are all derived from non-human mammals; The culture system is the formulation for in vitro embryo production as described in any one of claims 1 to 4.

9. The method according to claim 8, characterized in that, The non-human mammals include one or more of the following: non-human primates, cattle, sheep, pigs, horses, and mice.

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