In vitro embryo culture medium and method for preparing the same

Abstract

The application provides an in-vitro embryo culture solution and a preparation method thereof. The IVM solution of the in-vitro embryo culture solution comprises CaCl2, KCl, KH2PO4, glycine, HEPES sodium salt, lactic acid, MgSO4, NaHCO3, myo-inositol, glucose, essential amino acids, non-essential amino acids, retinoic acid, taurine and hormones. When the in-vitro embryo culture solution is used for mammals such as cattle, the IVF embryo production efficiency and embryo quality of the cattle can be improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of embryo technology, in particular to an in vitro embryo culture solution and a preparation method thereof. BACKGROUND

[0002] In Vitro Fertilization, also known as external fertilization, refers to a technique of allowing sperm and oocytes of mammals to complete the fertilization process in an artificially controlled in vitro environment, and its English abbreviation is IVF. Because this technology is closely related to embryo transfer technology (ET), it is also referred to as IVF-ET. In the field of biology, animals obtained by in vitro fertilization and embryo transfer to the mother are called test-tube animals. This technology was successful in the 1950s, and has developed rapidly in the past 20 years. Today, it has become an important and routine animal breeding biotechnology.

[0003] With the continuous development of modern agricultural technology, in order to more fully tap the reproductive potential of good breed cows and speed up the process of genetic breeding, it has become an inevitable trend to apply efficient new reproductive technologies in production practice. Ovum pick UP (OPU) and in vitro fertilization technology (IVF) are new embryo engineering technologies that developed rapidly in the 1980s. The combination of these two technologies can obtain a large number of embryos with clear genetic pedigree, thereby shortening the generation interval. At present, these two technologies have become an important reproductive means for farmers in developed countries such as Europe and Oceania to expand good breed cow populations. However, when using conventional bovine embryo culture systems (CRlaa and SOF liquid), the blastocyst development rate of bovine in vitro fertilization is low, and the embryo quality is far inferior to in vivo embryos, resulting in a low pregnancy rate after embryo transfer to the recipient. Therefore, how to improve the blastocyst development rate and embryo quality has become the focus and core of in vitro fertilization embryo production research.

[0004] As early as 1878, the German Schenk began to explore the in vitro fertilization technology of mammals using rabbits and guinea pigs as research materials. However, it was not until 1951 that breakthroughs were made in in vitro fertilization technology when Zhang Minjue, a Chinese American, and Austin discovered the phenomenon of sperm in vitro capacitation. The in vitro fertilization technology of cattle is influenced by many factors, including the in vitro maturation of oocytes, the in vitro capacitation of sperm, and the in vitro culture environment of zygotes.

[0005] In vitro culture of embryos is one of the key steps of IVF technology, and is also the embodiment and test of the final success of in vitro maturation of oocytes and in vitro fertilization technology. After in vitro fertilization is completed, the zygote will undergo a series of important changes in the process of developing into a blastocyst, including zygote formation, first cleavage, activation of the embryonic genome, compaction, and blastocyst formation. During this process, changes in the external environment will cause changes in gene expression, which will affect the normal development and quality of the embryo. At present, the in vitro culture of mammalian early embryos mainly focuses on improving the composition of the culture medium to meet the nutritional needs of embryos at different stages of development. Mammalian early embryonic development is a highly coordinated and precisely regulated process. During evolution, gametes have gradually formed a series of molecular cascade networks to ensure that the embryonic development cycle proceeds systematically. During development, the balance between internal and external reactive oxygen species (ROS) and antioxidants plays a decisive role in early embryonic development.

[0006] Most biochemical reactions produce reactive oxygen species (ROS), which play an important role both inside and outside cells. Some of these ROS act as signaling molecules, but most are harmful to the body. Brooker, R.J., et al. (Brooker, R.J., Genetics: analysis and principles (4th ed.). McGraw-Hill Science, 2011) reported that ROS can cause DNA damage in cells, oxidation of unsaturated fatty acids, oxidation of amino acids in proteins, and even inactivation of certain enzymes. In general, ROS exists in four forms, of which hydrogen peroxide (H2O2) is the most important factor in causing oxidative damage due to its strong oxidizing effect.

[0007] Although in vitro fertilization technology has been successfully applied to many mammals, the low blastocyst rate of in vitro fertilization has resulted in high production costs and low efficiency of in vitro fertilized embryos, which has largely limited the widespread application of this technology in the practice of rapid expansion of cattle. Therefore, how to reduce costs and improve the production efficiency and embryo quality of bovine in vitro fertilized embryos has become a problem to be solved. SUMMARY

[0008] Based on the technical problems existing in the prior art, the present application provides an in vitro embryo culture medium and preparation method which can improve the production efficiency and embryo quality of IVF embryos, and when used for mammals such as cattle, it can improve the production efficiency and embryo quality of IVF embryos of cattle.

[0009] The specific technical solutions of the present application are as follows:

[0010] 1.An in vitro maturation medium (IVM medium) comprising CaCl 2, KCl, KH 2PO 4, glycine, HEPES sodium salt, lactic acid, MgSO 4, NaHCO 3, myo-inositol, glucose, essential amino acids, non-essential amino acids, retinoic acid, taurine, and hormones.

[0011] 2.The in vitro maturation medium (IVM medium) of claim 1, wherein the hormones comprise FSH, LH, estradiol, EGF, and melatonin.

[0012] 3.The in vitro maturation medium (IVM medium) of claim 2, wherein the concentration of CaCl 2 is 1.5-2.5 mmol / L, the concentration of KCl is 5-7 mmol / L, the concentration of KH 2PO 4 is 0.5-1.5 mmol / L, the concentration of glycine is 1-10 mmol / L, the concentration of HEPES sodium salt is 9-15 mmol / L, the concentration of L-lactic acid is 5-12 mmol / L, the concentration of MgSO 4 is 0.3-0.8 mmol / L, the concentration of NaHCO 3 is 20-30 mmol / L, the concentration of myo-inositol is 0.5-5 mmol / L, the concentration of glucose is 1.5-10 mmol / L, the essential amino acids are added at 0.5-5 ml per 100 ml of IVM medium, the non-essential amino acids are added at 0.5-5 ml per 100 ml of IVM medium, the concentration of retinoic acid is 5-15 mmol / L, the concentration of taurine is 0.05-0.3 mmol / L, the concentration of FSH is 0.5-5 μg / mL, the concentration of LH is 10-20 μg / mL, the concentration of estradiol is 0.5-5 μg / mL, the concentration of EGF is 30-60 ng / mL, and the concentration of melatonin is 1×10 -6 -1×10 -5 mmol / L.

[0013] 4.An in vitro fertilization medium (IVF medium) comprising CaCl 2, KCl, KH 2PO 4, glycine, HEPES sodium salt, lactic acid, MgSO 4, NaHCO 3, NaCl, glucose, and non-essential amino acids.

[0014] Optionally, the concentrations of CaCl2, KCl, KH2PO4, glycine, HEPES sodium salt, L-lactic acid, MgSO4, NaHCO3, NaCl, and non-essential amino acids are added to 0.5-5 ml per 100 ml IVF solution. The concentrations of CaCl2, KCl, KH2PO4, glycine, HEPES sodium salt, L-lactic acid, MgSO4, NaHCO3, NaCl, and L-lactic acid are 10-80 mmol / L.

[0015] 5. An embryo culture medium 1 (IVC1 solution) comprising CaCl2, KCl, KH2PO4, glycine, sodium HEPES, lactic acid, MgSO4, NaHCO3, NaCl, inositol, EDTA, non-essential amino acids and taurine;

[0016] Optionally, the concentrations of CaCl2, KCl, KH2PO4, glycine, HEPES sodium salt, L-lactic acid, MgSO4, NaHCO3, NaCl, and taurine are 1.5-2.5 mmol / L, 5-7 mmol / L, 0.5-1.5 mmol / L, 1-10 mmol / L, 9-15 mmol / L, 5-12 mmol / L, 0.3-1 mmol / L, 20-30 mmol / L, 0.5-80 mmol / L, 2-5 mmol / L, 0.005-0.02 mmol / L, 0.5-5 ml of non-essential amino acids per 100 ml of IVC1 solution, and 0.05-0.15 mmol / L.

[0017] 6. An embryo culture medium 2 (IVC2 solution) comprising CaCl2, KCl, KH2PO4, glycine, sodium HEPES, lactic acid, MgSO4, NaHCO3, NaCl, inositol, essential amino acids and non-essential amino acids;

[0018] Optionally, the concentration of CaCl2 is 1.5-2.5 mmol / L, the concentration of KCl is 5-7 mmol / L, the concentration of KH2PO4 is 0.5-1.5 mmol / L, the concentration of glycine is 1-10 mmol / L, the concentration of HEPES sodium salt is 9-15 mmol / L, the concentration of L-lactic acid is 5-12 mmol / L, the concentration of MgSO4 is 0.3-1 mmol / L, the concentration of NaHCO3 is 20-30 mmol / L, the concentration of NaCl is 0.5-80 mmol / L, the concentration of myo-inositol is 2-5 mmol / L, the essential amino acids are added at 0.5-5 ml per 100 ml of IVC2 liquid, and the non-essential amino acids are added at 0.5-5 ml per 100 ml of IVC2 liquid.

[0019] 7. An in vitro embryo culture solution, comprising the in vitro oocyte maturation culture solution (IVM solution) according to any one of items 1-3; and / or

[0020] the fertilization culture solution (IVF solution) according to item 4; and / or

[0021] the embryo culture solution 1 (IVC1 solution) according to item 5; and / or

[0022] the embryo culture solution 2 (IVC2 solution) according to item 6.

[0023] 8. The in vitro embryo culture solution according to item 7, comprising the in vitro oocyte maturation culture solution (IVM solution) according to any one of items 1-3, the fertilization culture solution (IVF solution) according to item 4, the embryo culture solution 1 (IVC1 solution) according to item 5, and the embryo culture solution 2 (IVC2 solution) according to item 6.

[0024] 9. A method for preparing an in vitro embryo culture solution, comprising:

[0025] dissolving the components in the in vitro oocyte maturation culture solution according to any one of items 1-3 to obtain the in vitro oocyte maturation culture solution;

[0026] dissolving the components in the fertilization culture solution (IVF solution) according to item 4 to obtain the fertilization culture solution;

[0027] dissolving the components in the embryo culture solution 1 (IVC1 solution) according to item 5 to obtain the embryo culture solution 1 (IVC1 solution);

[0028] dissolving the components in the embryo culture solution 2 (IVC2 solution) according to item 6 to obtain the embryo culture solution 2 (IVC2 solution).

[0029] 10. Use of retinoic acid in the preparation of an in vitro oocyte maturation medium (IVM medium); optionally, the concentration of the retinoic acid is 5-15 mmol / L.

[0030] Effects of the Invention

[0031] The in vitro embryo culture medium described in the present application can improve the fertilization effect, and the cleavage rate and blastocyst rate are statistically significantly higher than those of the control group, thereby improving the embryo quality, reducing the cost of in vitro production of embryos, providing an experimental basis for the application of IVF technology in practice, and greatly accelerating the genetic breeding process. DETAILED DESCRIPTION

[0032] The present application will be described in detail below with reference to the described embodiments. Although specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0033] It should be noted that certain terms are used in the specification and claims to refer to particular components. It will be understood by those skilled in the art that the same component can be referred to by different names. The present specification and claims do not distinguish components based on the difference in names, but rather on the difference in function. As used throughout the specification and claims, "comprising" or "including" is an open term, which should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment for implementing the present application, but the description is for the purpose of illustrating the general principles of the specification, and is not intended to limit the scope of the present application. The scope of protection of the present application is defined by the appended claims.

[0034] The present application provides an in vitro oocyte maturation medium (IVM medium) comprising CaCl2, KCl, KH2PO4, glycine, HEPES sodium salt, lactic acid, MgSO4, NaHCO3, inositol, glucose, essential amino acids, non-essential amino acids, retinoic acid, taurine and hormones.

[0035] In the present application, neither the essential amino acids nor the non-essential amino acids comprise the above-mentioned glycine.

[0036] In the present application, the essential amino acids and non-essential amino acids can use the commonly used essential amino acids and non-essential amino acids in the art, for example, the essential amino acids and non-essential amino acids can be commercially available amino acids, for example, can be purchased from Sigma, etc.

[0037] In some embodiments, the hormones comprise FSH, LH, estradiol, EGF, and melatonin.

[0038] FSH refers to follicle-stimulating hormone, LH refers to luteinizing hormone, and EGF refers to epidermal growth factor.

[0039] In some embodiments, the concentration of CaCl2 is 1.5-2.5 mmol / L, the concentration of KCl is 5-7 mmol / L, the concentration of KH2PO4 is 0.5-1.5 mmol / L, the concentration of glycine is 1-10 mmol / L, the concentration of HEPES sodium salt is 9-15 mmol / L, the concentration of L-lactic acid is 5-12 mmol / L, the concentration of MgSO4 is 0.3-0.8 mmol / L, the concentration of NaHCO3 is 20-30 mmol / L, the concentration of inositol is 0.5-5 mmol / L, the concentration of glucose is 1.5-10 mmol / L, the concentration of essential amino acids is 0.5-5 ml per 100 ml of IVM solution, the concentration of non-essential amino acids is 0.5-5 ml per 100 ml of IVM solution, the concentration of retinoic acid is 5-15 mmol / L, the concentration of taurine is 0.05-0.3 mmol / L, the concentration of FSH is 0.5-5 μg / mL, the concentration of LH is 10-20 μg / mL, the concentration of estradiol is 0.5-5 μg / mL, the concentration of EGF is 30-60 ng / mL, and the concentration of melatonin is 1 x 10 -6 -1 x 10 - 5 mmol / L.

[0040] For example, the concentration of CaCl2 can be 1.5 mmol / L, 1.6 mmol / L, 1.7 mmol / L, 1.8 mmol / L, 1.9 mmol / L, 2.0 mmol / L, 2.1 mmol / L, 2.2 mmol / L, 2.3 mmol / L, 2.4 mmol / L, 2.5 mmol / L, etc.

[0041] The concentration of KCl can be 5 mmol / L, 5.1 mmol / L, 5.2 mmol / L, 5.3 mmol / L, 5.4 mmol / L, 5.5 mmol / L, 5.6 mmol / L, 5.7 mmol / L, 5.8 mmol / L, 5.9 mmol / L, 6 mmol / L, 6.1 mmol / L, 6.2 mmol / L, 6.3 mmol / L, 6.4 mmol / L, 6.5 mmol / L, 6.6 mmol / L, 6.7 mmol / L, 6.8 mmol / L, 6.9 mmol / L, 7 mmol / L, etc.

[0042] The concentration of KH2PO4 can be 0.5 mmol / L, 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L, 0.9 mmol / L, 1.0 mmol / L, 1.1 mmol / L, 1.2 mmol / L, 1.3 mmol / L, 1.4 mmol / L, 1.5 mmol / L, etc.

[0043] The concentration of glycine can be 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, etc.

[0044] The concentration of HEPES sodium salt can be 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, etc.

[0045] The concentration of L-lactic acid can be 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, etc.

[0046] The concentration of MgSO4 can be 0.3 mmol / L, 0.4 mmol / L, 0.5 mmol / L, 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L, etc.

[0047] The concentration of NaHCO3 can be 20 mmol / L, 21 mmol / L, 22 mmol / L, 23 mmol / L, 24 mmol / L, 25 mmol / L, 26 mmol / L, 27 mmol / L, 28 mmol / L, 29 mmol / L, 30 mmol / L, etc.

[0048] The concentration of myo-inositol can be 0.5 mmol / L, 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L, 0.9 mmol / L, 1 mmol / L, 1.5 mmol / L, 2 mmol / L, 2.5 mmol / L, 3 mmol / L, 3.5 mmol / L, 4 mmol / L, 4.5 mmol / L, 5 mmol / L, etc.

[0049] The concentration of glucose can be 1.5 mmol / L, 1.6 mmol / L, 1.7 mmol / L, 1.8 mmol / L, 1.9 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, etc.

[0050] The essential amino acid can be added 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, etc. per 100 ml of IVM liquid.

[0051] The non-essential amino acid can be added 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, etc. per 100 ml of IVM liquid.

[0052] The concentration of retinoic acid can be 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, etc.

[0053] The concentration of taurine can be 0.05 mmol / L, 0.06 mmol / L, 0.07 mmol / L, 0.08 mmol / L, 0.09 mmol / L, 0.1 mmol / L, 0.11 mmol / L, 0.12 mmol / L, 0.13 mmol / L, 0.14 mmol / L, 0.15 mmol / L, 0.16 mmol / L, 0.17 mmol / L, 0.18 mmol / L, 0.19 mmol / L, 0.2 mmol / L, 0.21 mmol / L, 0.22 mmol / L, 0.23 mmol / L, 0.24 mmol / L, 0.25 mmol / L, 0.26 mmol / L, 0.27 mmol / L, 0.28 mmol / L, 0.29 mmol / L, 0.3 mmol / L, etc.

[0054] The concentration of FSH can be 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, etc.

[0055] The concentration of LH can be 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, 20 μg / mL, etc.

[0056] The concentration of estradiol can be 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, etc.

[0057] The concentration of EGF is 30 ng / mL, 31 ng / mL, 32 ng / mL, 33 ng / mL, 34 ng / mL, 35 ng / mL, 36 ng / mL, 37 ng / mL, 38 ng / mL, 39 ng / mL, 40 ng / mL, 41 ng / mL, 42 ng / mL, 43 ng / mL, 44 ng / mL, 45 ng / mL, 46 ng / mL, 47 ng / mL, 48 ng / mL, 49 ng / mL, 50 ng / mL, 51 ng / mL, 52 ng / mL, 53 ng / mL, 54 ng / mL, 55 ng / mL, 56 ng / mL, 57 ng / mL, 58 ng / mL, 59 ng / mL, 60 ng / mL, etc.

[0058] The concentration of melatonin can be 1 × 10 -6 mmol / L, 2 × 10 -6 mmol / L, 3 × 10 -6 mmol / L, 4 × 10 -6 mmol / L, 5 × 10 -6 mmol / L, 6 × 10 -6 mmol / L, 7 × 10 -6 mmol / L, 8 × 10 -6 mmol / L, 9 × 10 -6 mmol / L, 1 × 10 - 5 mmol / L, etc.

[0059] The in vitro oocyte maturation culture solution (IVM solution) described in the present application can help oocytes to recover after meiosis, reduce the occurrence of oxidative stress, and thus improve the oocyte maturation effect.

[0060] The present application provides a fertilization culture solution (IVF solution) comprising CaCl2, KCl, KH2PO4, glycine, HEPES sodium salt, lactic acid, MgSO4, NaHCO3, NaCl, glucose and non-essential amino acids.

[0061] In the present application, the non-essential amino acid does not include glycine as described above.

[0062] In some embodiments, the concentration of CaCl2 is 1.5-2.5 mmol / L, the concentration of KCl is 5-7 mmol / L, the concentration of KH2PO4 is 0.5-1.5 mmol / L, the concentration of glycine is 1-10 mmol / L, the concentration of HEPES sodium salt is 9-15 mmol / L, the concentration of L-lactic acid is 5-12 mmol / L, the concentration of MgSO4 is 0.3-1 mmol / L, the concentration of NaHCO3 is 20-30 mmol / L, the concentration of NaCl is 10-80 mmol / L, the concentration of glucose is 0.5-2 mmol / L, and the non-essential amino acid is 0.5-5 ml per 100 ml of IVF solution.

[0063] For example, the concentration of CaCl2 can be 1.5 mmol / L, 1.6 mmol / L, 1.7 mmol / L, 1.8 mmol / L, 1.9 mmol / L, 2.0 mmol / L, 2.1 mmol / L, 2.2 mmol / L, 2.3 mmol / L, 2.4 mmol / L, 2.5 mmol / L, etc.

[0064] The concentration of KCl can be 5 mmol / L, 5.1 mmol / L, 5.2 mmol / L, 5.3 mmol / L, 5.4 mmol / L, 5.5 mmol / L, 5.6 mmol / L, 5.7 mmol / L, 5.8 mmol / L, 5.9 mmol / L, 6 mmol / L, 6.1 mmol / L, 6.2 mmol / L, 6.3 mmol / L, 6.4 mmol / L, 6.5 mmol / L, 6.6 mmol / L, 6.7 mmol / L, 6.8 mmol / L, 6.9 mmol / L, 7 mmol / L, etc.

[0065] The concentration of KH2PO4 can be 0.5 mmol / L, 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L, 0.9 mmol / L, 1.0 mmol / L, 1.1 mmol / L, 1.2 mmol / L, 1.3 mmol / L, 1.4 mmol / L, 1.5 mmol / L, etc.

[0066] The concentration of glycine can be 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, etc.

[0067] The concentration of HEPES sodium salt can be 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, etc.

[0068] The concentration of L-lactic acid can be 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, etc.

[0069] The concentration of MgSO4 can be 0.3 mmol / L, 0.4 mmol / L, 0.5 mmol / L, 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L, 0.9 mmol / L, 1 mmol / L, etc.

[0070] The concentration of NaHCO3 can be 20 mmol / L, 21 mmol / L, 22 mmol / L, 23 mmol / L, 24 mmol / L, 25 mmol / L, 26 mmol / L, 27 mmol / L, 28 mmol / L, 29 mmol / L, 30 mmol / L, etc.

[0071] The concentration of NaCl can be 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, 50 mmol / L, 55 mmol / L, 60 mmol / L, 65 mmol / L, 70 mmol / L, 75 mmol / L, 80 mmol / L, etc.

[0072] The concentration of glucose can be 0.5 mmol / L, 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L, 0.9 mmol / L, 1.0 mmol / L, 1.1 mmol / L, 1.2 mmol / L, 1.3 mmol / L, 1.4 mmol / L, 1.5 mmol / L, 1.6 mmol / L, 1.7 mmol / L, 1.8 mmol / L, 1.9 mmol / L, 2 mmol / L, etc.

[0073] The non-essential amino acid can be added in an amount of 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, etc. per 100 ml of IVF solution.

[0074] The IVF solution described in the present application can simulate the in vivo environment, complete sperm capacitation and maintain sperm motility, provide an environment for the combination of oocytes and sperm, and promote the fertilization process.

[0075] The present application provides an embryo culture solution 1 (IVC1 solution) comprising CaCl2, KCl, KH2PO4, glycine, HEPES sodium salt, lactic acid, MgSO4, NaHCO3, NaCl, myo-inositol, EDTA, non-essential amino acids, and taurine.

[0076] As described above, the non-essential amino acids do not include glycine.

[0077] In some embodiments, the concentration of CaCl2 is 1.5-2.5 mmol / L, the concentration of KCl is 5-7 mmol / L, the concentration of KH2PO4 is 0.5-1.5 mmol / L, the concentration of glycine is 1-10 mmol / L, the concentration of HEPES sodium salt is 9-15 mmol / L, the concentration of L-lactic acid is 5-12 mmol / L, the concentration of MgSO4 is 0.3-1 mmol / L, the concentration of NaHCO3 is 20-30 mmol / L, the concentration of NaCl is 0.5-80 mmol / L, the concentration of myo-inositol is 2-5 mmol / L, the concentration of EDTA is 0.005-0.02 mmol / L, the non-essential amino acids are added at 0.5-5 ml per 100 ml of IVF solution, and the concentration of taurine is 0.05-0.15 mmol / L.

[0078] For example, the concentration of CaCl2 can be 1.5 mmol / L, 1.6 mmol / L, 1.7 mmol / L, 1.8 mmol / L, 1.9 mmol / L, 2.0 mmol / L, 2.1 mmol / L, 2.2 mmol / L, 2.3 mmol / L, 2.4 mmol / L, 2.5 mmol / L, etc.

[0079] The concentration of KCl can be 5 mmol / L, 5.1 mmol / L, 5.2 mmol / L, 5.3 mmol / L, 5.4 mmol / L, 5.5 mmol / L, 5.6 mmol / L, 5.7 mmol / L, 5.8 mmol / L, 5.9 mmol / L, 6 mmol / L, 6.1 mmol / L, 6.2 mmol / L, 6.3 mmol / L, 6.4 mmol / L, 6.5 mmol / L, 6.6 mmol / L, 6.7 mmol / L, 6.8 mmol / L, 6.9 mmol / L, 7 mmol / L, etc.

[0080] The concentration of KH2PO4 can be 0.5 mmol / L, 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L, 0.9 mmol / L, 1.0 mmol / L, 1.1 mmol / L, 1.2 mmol / L, 1.3 mmol / L, 1.4 mmol / L, 1.5 mmol / L, etc.

[0081] The concentration of glycine can be 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, etc.

[0082] The concentration of HEPES sodium salt can be 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, etc.

[0083] The concentration of L-lactic acid can be 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, etc.

[0084] The concentration of MgSO4 can be 0.3 mmol / L, 0.4 mmol / L, 0.5 mmol / L, 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L, 0.9 mmol / L, 1 mmol / L, etc.

[0085] The concentration of NaHCO3 can be 20 mmol / L, 21 mmol / L, 22 mmol / L, 23 mmol / L, 24 mmol / L, 25 mmol / L, 26 mmol / L, 27 mmol / L, 28 mmol / L, 29 mmol / L, 30 mmol / L, etc.

[0086] The concentration of NaCl can be 0.5 mmol / L, 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L, 0.9 mmol / L, 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, 50 mmol / L, 55 mmol / L, 60 mmol / L, 65 mmol / L, 70 mmol / L, 75 mmol / L, 80 mmol / L, etc.

[0087] The concentration of myo-inositol can be 2 mmol / L, 2.1 mmol / L, 2.2 mmol / L, 2.3 mmol / L, 2.4 mmol / L, 2.5 mmol / L, 2.6 mmol / L, 2.7 mmol / L, 2.8 mmol / L, 2.9 mmol / L, 3 mmol / L, 3.1 mmol / L, 3.2 mmol / L, 3.3 mmol / L, 3.4 mmol / L, 3.5 mmol / L, 3.6 mmol / L, 3.7 mmol / L, 3.8 mmol / L, 3.9 mmol / L, 4 mmol / L, 4.1 mmol / L, 4.2 mmol / L, 4.3 mmol / L, 4.4 mmol / L, 4.5 mmol / L, 4.6 mmol / L, 4.7 mmol / L, 4.8 mmol / L, 4.9 mmol / L, 5 mmol / L, or the like.

[0088] The concentration of EDTA can be 0.005 mmol / L, 0.006 mmol / L, 0.006 mmol / L, 0.007 mmol / L, 0.008 mmol / L, 0.009 mmol / L, 0.01 mmol / L, 0.011 mmol / L, 0.012 mmol / L, 0.013 mmol / L, 0.014 mmol / L, 0.015 mmol / L, 0.016 mmol / L, 0.017 mmol / L, 0.018 mmol / L, 0.019 mmol / L, 0.02 mmol / L, or the like.

[0089] The non-essential amino acid can be added in an amount of 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, or the like per 100 ml of IVC1 liquid.

[0090] The concentration of taurine can be 0.05 mmol / L, 0.06 mmol / L, 0.07 mmol / L, 0.08 mmol / L, 0.09 mmol / L, 0.10 mmol / L, 0.11 mmol / L, 0.12 mmol / L, 0.13 mmol / L, 0.14 mmol / L, 0.15 mmol / L, or the like.

[0091] The IVC1 liquid described in the present application can provide a suitable living environment for fertilized eggs, and contains components that support the development of early embryonic cleavage stage, such as an appropriate amount of non-essential amino acids and the like, to meet the metabolic needs of cleavage stage embryos.

[0092] The application provides an embryo culture solution 2 (IVC2 solution) comprising CaCl2, KCl, KH2PO4, glycine, HEPES sodium salt, lactic acid, MgSO4, NaHCO3, NaCl, inositol, essential amino acids and non-essential amino acids.

[0093] Here, neither the non-essential amino acids nor the essential amino acids comprise glycine.

[0094] In some embodiments, the concentration of CaCl2 is 1.5-2.5 mmol / L, the concentration of KCl is 5-7 mmol / L, the concentration of KH2PO4 is 0.5-1.5 mmol / L, the concentration of glycine is 1-10 mmol / L, the concentration of HEPES sodium salt is 9-15 mmol / L, the concentration of L-lactic acid is 5-12 mmol / L, the concentration of MgSO4 is 0.3-1 mmol / L, the concentration of NaHCO3 is 20-30 mmol / L, the concentration of NaCl is 0.5-80 mmol / L, the concentration of inositol is 2-5 mmol / L, the essential amino acids are 0.5-5 ml per 100 ml of IVC2 solution, and the non-essential amino acids are 0.5-5 ml per 100 ml of IVC2 solution.

[0095] For example, the concentration of CaCl2 can be 1.5 mmol / L, 1.6 mmol / L, 1.7 mmol / L, 1.8 mmol / L, 1.9 mmol / L, 2.0 mmol / L, 2.1 mmol / L, 2.2 mmol / L, 2.3 mmol / L, 2.4 mmol / L, 2.5 mmol / L, etc.

[0096] The concentration of KCl can be 5 mmol / L, 5.1 mmol / L, 5.2 mmol / L, 5.3 mmol / L, 5.4 mmol / L, 5.5 mmol / L, 5.6 mmol / L, 5.7 mmol / L, 5.8 mmol / L, 5.9 mmol / L, 6 mmol / L, 6.1 mmol / L, 6.2 mmol / L, 6.3 mmol / L, 6.4 mmol / L, 6.5 mmol / L, 6.6 mmol / L, 6.7 mmol / L, 6.8 mmol / L, 6.9 mmol / L, 7 mmol / L, etc.

[0097] The concentration of KH2PO4 can be 0.5 mmol / L, 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L, 0.9 mmol / L, 1.0 mmol / L, 1.1 mmol / L, 1.2 mmol / L, 1.3 mmol / L, 1.4 mmol / L, 1.5 mmol / L, etc.

[0098] The concentration of glycine can be 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, etc.

[0099] The concentration of HEPES sodium salt can be 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, etc.

[0100] The concentration of L-lactic acid can be 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, etc.

[0101] The concentration of MgSO4 can be 0.3 mmol / L, 0.4 mmol / L, 0.5 mmol / L, 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L, 0.9 mmol / L, 1 mmol / L, etc.

[0102] The concentration of NaHCO3 can be 20 mmol / L, 21 mmol / L, 22 mmol / L, 23 mmol / L, 24 mmol / L, 25 mmol / L, 26 mmol / L, 27 mmol / L, 28 mmol / L, 29 mmol / L, 30 mmol / L, etc.

[0103] The concentration of NaCl can be 0.5 mmol / L, 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L, 0.9 mmol / L, 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, 50 mmol / L, 55 mmol / L, 60 mmol / L, 65 mmol / L, 70 mmol / L, 75 mmol / L, 80 mmol / L, etc.

[0104] The concentration of myo-inositol can be 2 mmol / L, 2.1 mmol / L, 2.2 mmol / L, 2.3 mmol / L, 2.4 mmol / L, 2.5 mmol / L, 2.6 mmol / L, 2.7 mmol / L, 2.8 mmol / L, 2.9 mmol / L, 3 mmol / L, 3.1 mmol / L, 3.2 mmol / L, 3.3 mmol / L, 3.4 mmol / L, 3.5 mmol / L, 3.6 mmol / L, 3.7 mmol / L, 3.8 mmol / L, 3.9 mmol / L, 4 mmol / L, 4.1 mmol / L, 4.2 mmol / L, 4.3 mmol / L, 4.4 mmol / L, 4.5 mmol / L, 4.6 mmol / L, 4.7 mmol / L, 4.8 mmol / L, 4.9 mmol / L, 5 mmol / L, or the like.

[0105] The essential amino acid can be added at 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, or the like per 100 ml of IVC2 liquid.

[0106] The non-essential amino acid can be added at 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, or the like per 100 ml of IVC2 liquid.

[0107] The IVC2 liquid described in the present application can meet the needs of rapid proliferation and differentiation of blastomere cells, and contains all kinds of amino acids and other nutrients to support the further development of embryos to form structures such as blastocyst cavity, inner cell mass, and trophoblast cells.

[0108] The present application provides an in vitro embryo culture solution, which comprises the in vitro oocyte maturation culture solution (IVM liquid) described in any of the above; and / or

[0109] The fertilization culture solution (IVF liquid) described in any of the above; and / or

[0110] The embryo culture solution 1 (IVC1 liquid) described in any of the above; and / or

[0111] The embryo culture solution 2 (IVC2 liquid) described in any of the above. In some embodiments, the in vitro oocyte maturation culture solution (IVM liquid) described in any of the above; the fertilization culture solution (IVF liquid) described in any of the above; the embryo culture solution 1 (IVC1 liquid) described in any of the above; and the embryo culture solution 2 (IVC2 liquid) described in any of the above are comprised.

[0112] In the present application, the in vitro embryo culture medium can be used for in vitro embryo culture of mammals, such as cattle and the like.

[0113] The present application provides the above-mentioned in vitro embryo culture medium, wherein IVM significantly improves the quality of in vitro matured oocytes, reduces the oxidative stress level of bovine oocytes, IVF, IVC1, IVC2 reduces the production cost while further improving the blastocyst formation rate and blastocyst quality after in vitro fertilization, and greatly improves the efficiency of bovine in vitro embryo production.

[0114] The present application provides a method for preparing an in vitro embryo culture medium, comprising:

[0115] Dissolving the components in the in vitro oocyte maturation culture medium of any one of the above to obtain the in vitro oocyte maturation culture medium;

[0116] Dissolving the components in the fertilization culture medium (IVF medium) of any one of the above to obtain the fertilization culture medium;

[0117] Dissolving the components of the embryo culture medium 1 (IVC1 medium) of any one of the above to obtain the embryo culture medium 1 (IVC1 medium);

[0118] Dissolving the components of the embryo culture medium 2 (IVC2 medium) of any one of the above to obtain the embryo culture medium 2 (IVC2 medium).

[0119] In the present application, for the above-mentioned in vitro embryo culture medium, the above-mentioned components can be dissolved by a solvent commonly used in the art, for example, the solvent can be water and the like.

[0120] The method described in the present application for preparing the above-mentioned culture medium has a simple preparation process and can greatly improve the efficiency of bovine in vitro embryo production.

[0121] The present application provides the use of retinoic acid in the preparation of an in vitro oocyte maturation culture medium (IVM medium). In some embodiments, the concentration of the retinoic acid is 5-15 mmol / L.

[0122] For example, the concentration of retinoic acid can be 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, etc.

[0123] Retinoic acid can make part of the oocytes stop in prophase of the first meiosis, activate the relevant signal pathways, promote the oocytes to resume meiosis, and develop to the mature stage. In addition, retinoic acid can regulate the distribution and function of various organelles in oocytes, promote the synthesis and distribution of cortical granules and other substances, and make preparations for fertilization and subsequent embryonic development. Furthermore, retinoic acid can regulate gene expression, enhance the response ability of oocytes to post-fertilization activation signals, and improve the potential of oocytes to develop into high-quality embryos.

[0124] EMBODIMENT

[0125] The materials used in the experiment and the experimental methods are generally and / or specifically described in the present application. In the following examples, % means wt%, i.e., weight percentage, unless otherwise specified. The reagents or instruments used are not specified by the manufacturer, and are all conventional reagent products that can be obtained commercially. Among them, the experimental supplies used in the present application are from: CaCl2, item number: C3881-500G, purchased from Sigma; KCl, item number: P-5405-250G, purchased from Sigma; KH2PO4, item number: P5379-500G, purchased from Sigma; Glycine, item number: G7126-100G, purchased from Sigma; HEPES NA SALT, item number: H3784-100G, purchased from Sigma; L-Lactate, item number: L7022-50G, purchased from Sigma; MgSO4, item number: M2643-500G, purchased from Sigma; NaHCO3, item number: S5761-500G, purchased from Sigma; NaCl, item number: S-9625-500G, purchased from Sigma; Myo-inositol, item number: I7508-50G, purchased from Sigma; EDTA, item number: E6635-100G, purchased from Sigma; Glucose, item number: G6152-500G, purchased from Sigma; EAAS (essential amino acids), item number: B6766-100ml, purchased from Sigma; NEAAS (non-essential amino acids), item number: M7145-100ml, purchased from Sigma; Retinoic acid, item number: R2625-50mg, purchased from Sigma; Taurine, item number: T8691-25G, purchased from Sigma; FSH, purchased from Ningbo Hormone Factory No. 2; LH, purchased from Ningbo Hormone Factory No. 2; Estradiol, item number: E2758-250mg, purchased from Sigma; EGF, item number: E9644-0.2mg, purchased from Sigma; Melatonin, item number: M5250-250mg, purchased from Sigma.

[0126] EMBODIMENT

[0127] Step one: configure the base solution according to Table 1

[0128] Table 1

[0129]

[0130]

[0131] Step two: configure the hormone according to Table 2

[0132] Table 2

[0133]

[0134] Step three: mix the base solution in Table 1 and the hormone in Table 2 to obtain the IVM solution.

[0135] Preparation of IVM solution in Example 2-5

[0136] Example 2-5 is the same as Example 1, except that the amount of some components is different, as shown in Table 3.

[0137] Comparative Example 1-4

[0138] Comparative Example 1-4 is the same as Example 1, except that the amount of some components is different, as shown in Table 3.

[0139] Table 3

[0140] Myo-inositol (mmol / l) EDTA (mmol / l) Retinoic acid (mmol / l) Example 1 2.8 0 10 Example 2 2 0 10 Example 3 4 0 10 Example 4 2.8 0 5 Example 5 2.8 0 15 Comparative Example 1 0 0 10 Comparative Example 2 2.8 0.5 10 Comparative Example 3 10 0 10 Comparative Example 4 2.8 0 0.3

[0141] Preparation of IVF solution in Example 6

[0142] Add each component according to the concentration in Table 4 to obtain the IVF solution.

[0143] Table 4

[0144]

[0145]

[0146] Comparative Example 5-7

[0147] Comparative Example 5-7 is the same as Example 6, except that the amount of some components is different, as shown in Table 5.

[0148] Table 5

[0149] Myo-inositol (mmol / l) EDTA (mmol / l) Retinoic acid (mmol / l) Example 6 0 0 0 Comparative Example 5 0 0 0.5 Comparative Example 6 0 0.5 0 Comparative Example 7 0.5 0 0

[0150] Preparation of IVC1 solution in Example 7

[0151] Add each component according to the concentration in Table 6 to obtain the IVC1 solution.

[0152] Table 6

[0153]

[0154]

[0155] Comparative Example 8

[0156] Comparative Example 8 is the same as the method of Example 7, except that the amounts of some ingredients are different, as shown in Table 7.

[0157] Table 7

[0158] Myo-inositol (mmol / l) EDTA (mmol / l) Retinoic acid (mmol / l) Example 7 2.769145 0.01266 0 Comparative Example 8 2.769145 0 0.5

[0159] Preparation of IVC2 liquid of Example 8

[0160] The ingredients were added according to the concentrations in Table 8 to obtain the IVC2 liquid.

[0161] Table 8

[0162]

[0163]

[0164] Comparative Example 9

[0165] Comparative Example 9 is the same as the method of Example 8, except that the amounts of some ingredients are different, as shown in Table 9.

[0166] Table 9

[0167] Myo-inositol (mmol / l) EDTA (mmol / l) Retinoic acid (mmol / l) Example 8 2.769145 0 0 Comparative Example 9 2.769145 0 0.5

[0168] Experimental Example:

[0169] 1. Collection and in vitro maturation of oocytes

[0170] Collection of oocytes from live young Holstein dairy cows: Under the pressure of a vacuum pump, oocytes were collected by follicle aspiration technique, and follicles with a diameter of 2-8 mm were selected for oocyte collection. After oocyte collection was completed, a small amount of live oocyte collection fluid was collected for flushing the oocyte collection needle and catheter, and under a stereomicroscope, cumulus-oocyte complexes (COCs) were selected, washed in 3 mL of WASH fluid (manufacturer: stroebech media, batch number: S23WASB05), and transferred to IVM fluid, and sent to the laboratory within 3 h, the frozen tube cap was opened, and placed in an incubator with conditions of 38.8°C, 6% CO2, and 100% humidity for 21-23 h.

[0171] 2. In vitro fertilization

[0172] COCs were transferred from the IVM solution to a 500 μL IVF drop for washing once, then transferred to a 90 μL IVF drop for fertilization, with 15 COCs per drop. The semen was thawed in a 35.5°C water bath, and centrifuged at 328g for 5 min using 4 ml of washing buffer. The supernatant was discarded, and 3 ml of washing buffer was added again, centrifuged for 5 min, and the supernatant was discarded again. The sperm concentration was adjusted using the washing buffer to approximately 2 × 10⁻⁶ sperm. 6 / ml. Take 50μL of semen and mix it with a fertilized droplet containing COCs to form a 100μL droplet. Incubate the sperm and egg together at 38.8℃, 6% CO2 gas, and 100% humidity for 18h.

[0173] 3. In vitro embryo culture

[0174] Before in vitro culture, a balanced IVC1 culture medium was prepared overnight. Eighteen hours after in vitro fertilization, fertilized eggs were repeatedly aspirated using an embryo stripper until the cumulus oophorus was completely removed. The fertilized eggs were then washed in IVC1 medium and transferred to the pre-balanced IVC1 culture medium. The treated fertilized eggs were then cultured in a cell culture incubator at 38.8°C, 6.0% CO2, and 7.0% O2 with saturated humidity for 56 hours.

[0175] On day 3 (56 hours after fertilization), embryos were transferred to pre-balanced IVC2 medium for further culture. On day 7, embryos were graded and frozen, and the cleavage rate and blastocyst rate were calculated. The results are shown in Table 10. The cleavage rate was determined by morphological observation, and cleavage of fertilized eggs was included in the cleavage rate. Early blastocysts, blastocysts, expanded blastocysts, and hatched blastocysts were all included in the blastocyst rate.

[0176] Table 10

[0177]

[0178] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. An in vitro embryo culture solution for a mammal, comprising an in vitro oocyte maturation culture solution (IVM solution); a fertilization culture solution (IVF solution); an embryo culture solution 1 (IVC1 solution); and an embryo culture solution 2 (IVC2 solution); the in vitro oocyte maturation culture solution (IVM solution) comprises CaCl2, KCl, KH2PO4, glycine, HEPES sodium salt, lactic acid, MgSO4, NaHCO3, myo-inositol, glucose, essential amino acids, non-essential amino acids, retinoic acid, taurine, and hormones comprising FSH, LH, estradiol, EGF, and melatonin; concentration of 1.5-2.5 mmol / L, a concentration of KCl of 5-7 mmol / L, a concentration of KH2PO4 of 0.5-1.5 mmol / L, a concentration of glycine of 1-10 mmol / L, a concentration of HEPES sodium salt of 9-15 mmol / L, a concentration of L-lactic acid of 5-12 mmol / L, a concentration of MgSO4 of 0.3-0.8 mmol / L, a concentration of NaHCO3 of 20-30 mmol / L, a concentration of myo-inositol of 0.5-5 mmol / L, a concentration of glucose of 1.5-10 mmol / L, essential amino acids of 0.5-5 ml per 100 ml of IVM liquid, non-essential amino acids of 0.5-5 ml per 100 ml of IVM liquid, a concentration of retinoic acid of 5-15 mmol / L, a concentration of taurine of 0.05-0.3 mmol / L, a concentration of FSH of 0.5-5 μg / mL, a concentration of LH of 10-20 μg / mL, a concentration of estradiol of 0.5-5 μg / mL, a concentration of EGF of 30-60 ng / mL, and a concentration of melatonin of 1×10 -6 - 1×10 -5 mmol / L. the fertilization culture solution (IVF solution) comprises CaCl2, KCl, KH2PO4, glycine, HEPES sodium salt, lactic acid, MgSO4, NaHCO3, NaCl, glucose, and non-essential amino acids, wherein the concentration of CaCl2 is 1.5-2.5 mmol / L, the concentration of KCl is 5-7 mmol / L, the concentration of KH2PO4 is 0.5-1.5 mmol / L, the concentration of glycine is 1-10 mmol / L, the concentration of HEPES sodium salt is 9-15 mmol / L, the concentration of L-lactic acid is 5-12 mmol / L, the concentration of MgSO4 is 0.3-1 mmol / L, the concentration of NaHCO3 is 20-30 mmol / L, the concentration of NaCl is 10-80 mmol / L, the concentration of glucose is 0.5-2 mmol / L, and the non-essential amino acids are added at 0.5-5 ml per 100 ml of IVF solution; the embryo culture solution 1 (IVC1 solution) comprises CaCl2, KCl, KH2PO4, glycine, HEPES sodium salt, lactic acid, MgSO4, NaHCO3, NaCl, myo-inositol, EDTA, non-essential amino acids, and taurine; wherein the concentration of CaCl2 is 1.5-2.5 mmol / L, the concentration of KCl is 5-7 mmol / L, the concentration of KH2PO4 is 0.5-1.5 mmol / L, the concentration of glycine is 1-10 mmol / L, the concentration of HEPES sodium salt is 9-15 mmol / L, the concentration of L-lactic acid is 5-12 mmol / L, the concentration of MgSO4 is 0.3-1 mmol / L, the concentration of NaHCO3 is 20-30 mmol / L, the concentration of NaCl is 0.5-80 mmol / L, the concentration of myo-inositol is 2-5 mmol / L, the concentration of EDTA is 0.005-0.02 mmol / L, the non-essential amino acids are added at 0.5-5 ml per 100 ml of IVC1 solution, and the concentration of taurine is 0.05-0.15 mmol / L; the embryo culture solution 2 (IVC2 solution) comprises CaCl2, KCl, KH2PO4, glycine, HEPES sodium salt, lactic acid, MgSO4, NaHCO3, NaCl, myo-inositol, essential amino acids, and non-essential amino acids; The concentration of CaCl2 is 1.5-2.5 mmol / L, the concentration of KCl is 5-7 mmol / L, the concentration of KH2PO4 is 0.5-1.5 mmol / L, the concentration of glycine is 1-10 mmol / L, the concentration of HEPES sodium salt is 9-15 mmol / L, the concentration of L-lactic acid is 5-12 mmol / L, the concentration of MgSO4 is 0.3-1 mmol / L, the concentration of NaHCO3 is 20-30 mmol / L, the concentration of NaCl is 0.5-80 mmol / L, the concentration of myo-inositol is 2-5 mmol / L, the necessary amino acid is 0.5-5 ml per 100 ml IVC2 liquid, and the non-essential amino acid is 0.5-5 ml per 100 ml IVC2 liquid. The non-essential amino acid is not glycine.

2. A method for preparing an in vitro embryo culture solution, comprising: dissolving the components in the in vitro oocyte maturation culture solution (IVM solution) of claim 1 to obtain the in vitro oocyte maturation culture solution (IVM solution); dissolving the components in the fertilization culture solution (IVF solution) of claim 1 to obtain the fertilization culture solution (IVF solution); dissolving the components in the embryo culture solution 1 (IVC1 solution) of claim 1 to obtain the embryo culture solution 1 (IVC1 solution); dissolving the components in the embryo culture solution 2 (IVC2 solution) of claim 1 to obtain the embryo culture solution 2 (IVC2 solution).

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