In-vitro embryo culture solution and preparation method thereof

Abstract

The invention 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, inositol, glucose, essential amino acid, non-essential amino acid, retinoic acid, taurine and hormones, and when the in-vitro embryo culture solution is used as mammals such as cows, the in-vitro embryo culture solution can be used for preparing the in-vitro embryo culture solution. And the IVF embryo production efficiency and embryo quality of the cattle can be improved.

Description

Technical Field

[0001] This application relates to the field of embryo technology, and particularly relates to an in vitro embryo culture solution and a preparation method thereof. Background Art

[0002] In Vitro Fertilization, also known as external fertilization, refers to the technology that enables sperm and eggs of mammals to complete the fertilization process in an artificially controlled in vitro environment. Its English abbreviation is IVF. Since this technology is closely related to the embryo transfer technology (ET), it is also simply abbreviated as IVF-ET. In the biological field, an animal obtained by in vitro fertilization and embryo transfer into the mother body is called a test-tube animal. This technology was successfully achieved in the 1950s and has developed rapidly in the past 20 years. Now it has become an important and conventional animal reproduction biotechnology.

[0003] With the continuous development of modern agricultural technology, in order to more fully explore the reproductive potential of high-quality dairy cows and accelerate 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 (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 pedigrees, thereby shortening the generation interval. At present, these two technologies have become important reproductive means adopted by farmers in developed livestock countries such as Europe, America, and Oceania to expand the population of high-quality dairy cows. However, when using the conventional bovine embryo culture system (CRlaa and SOF solutions), the blastocyst development rate of bovine in vitro fertilization is relatively low, and the embryo quality is also far inferior to that of 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 fertilized embryo production.

[0004] As early as 1878, the German Scnenk began to explore the in vitro fertilization technology of mammals using rabbits and guinea pigs as research materials. However, it wasn't until 1951 that the Chinese-American Min Chueh Chang and Austin respectively discovered the phenomenon of sperm capacitation in vitro that the in vitro fertilization technology made a breakthrough. The in vitro fertilization technology of cows is affected by multiple aspects, including the in vitro maturation of oocytes, the in vitro capacitation of sperm, and the in vitro culture environment of fertilized eggs.

[0005] In vitro culture of embryos is one of the key steps in IVF technology and also reflects and tests the ultimate effectiveness of in vitro maturation and in vitro fertilization of oocytes. After in vitro fertilization is completed, a series of crucial changes will occur during the development of the fertilized egg into a blastocyst, including the formation of the zygote, the first cleavage, the activation of the embryonic genome, compaction, and the formation of the blastocyst. During this process, changes in the external environment can cause changes in gene expression, thereby affecting the normal development and quality of the embryo. Currently, the research on in vitro culture of early mammalian embryos mainly focuses on improving the composition of the culture medium to meet the nutritional needs of embryos at different developmental stages. The development of early mammalian embryos is a highly coordinated and precisely regulated process. During evolution, gametes have gradually formed a series of molecular cascade networks to ensure the systematic progress of the embryonic development cycle. During development, the balance between intracellular and extracellular 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 important roles both intracellularly and extracellularly. Some of these ROS act as signaling molecules, but most ROS 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. Generally, ROS exist in four forms, and among them, hydrogen peroxide (H 2 O 2 ) has a stronger oxidizing effect and is the main factor causing oxidative damage.

[0007] Although in vitro fertilization technology has been successfully applied to many mammals, however, due to the low blastocyst rate of in vitro fertilization, the production cost of in vitro fertilized embryos remains high and the efficiency is low, which greatly limits the wide application of this technology in the practice of rapid bovine propagation. Therefore, how to reduce costs and improve the production efficiency and embryo quality of bovine in vitro fertilized embryos has become an urgent problem to be solved. Summary of the Invention

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

[0009] The specific technical solution of the present application is as follows:

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

[0011] 2. The in vitro oocyte maturation culture medium (IVM medium) according to item 1, wherein the hormones comprise FSH, LH, estradiol, EGF and melatonin.

[0012] 3. The in vitro oocyte maturation culture medium (IVM medium) according to item 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 2 PO 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 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. A fertilization culture medium (IVF medium), which comprises CaCl 2 , KCl, KH 2 PO 4 , glycine, HEPES sodium salt, lactic acid, MgSO 4 , NaHCO 3 , NaCl, glucose and non-essential amino acids;

[0014] Optionally, the concentration of CaCl 2 is 1.5 - 2.5 mmol / L, the concentration of KCl is 5 - 7 mmol / L, KH 2 PO 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, MgSO 4 is 0.3 - 1 mmol / L, NaHCO 3 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 non-essential amino acids are added at 0.5 - 5 ml per 100 ml of IVF solution.

[0015] 5. An embryo culture medium 1 (IVC1 solution), which contains CaCl 2 , KCl, KH 2 PO 4 , glycine, HEPES sodium salt, lactic acid, MgSO 4 , NaHCO 3 , NaCl, inositol, EDTA, non-essential amino acids and taurine;

[0016] Optionally, the concentration of CaCl 2 is 1.5 - 2.5 mmol / L, the concentration of KCl is 5 - 7 mmol / L, KH 2 PO 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, MgSO 4 is 0.3 - 1 mmol / L, NaHCO 3 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 concentration of EDTA is 0.005 - 0.02 mmol / L, 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.

[0017] 6. An embryo culture medium 2 (IVC2 solution), which contains CaCl 2 , KCl, KH 2 PO 4 , glycine, HEPES sodium salt, lactic acid, MgSO 4 , NaHCO 3, NaCl, inositol, essential amino acids and non-essential amino acids;

[0018] Optionally, CaCl 2 has a concentration of 1.5 - 2.5 mmol / L, KCl has a concentration of 5 - 7 mmol / L, KH 2 PO 4 has a concentration of 0.5 - 1.5 mmol / L, glycine has a concentration of 1 - 10 mmol / L, sodium HEPES has a concentration of 9 - 15 mmol / L, L-lactic acid has a concentration of 5 - 12 mmol / L, MgSO 4 has a concentration of 0.3 - 1 mmol / L, NaHCO 3 has a concentration of 20 - 30 mmol / L, NaCl has a concentration of 0.5 - 80 mmol / L, inositol has a concentration of 2 - 5 mmol / L. The essential amino acids are added at 0.5 - 5 ml per 100 ml of IVC2 solution, and the non-essential amino acids are added at 0.5 - 5 ml per 100 ml of IVC2 solution.

[0019] 7. An in vitro embryo culture medium, which comprises the in vitro oocyte maturation culture medium (IVM medium) described in any one of items 1 - 3; and / or

[0020] the fertilization culture medium (IVF medium) described in item 4; and / or

[0021] the embryo culture medium 1 (IVC1 medium) described in item 5; and / or

[0022] the embryo culture medium 2 (IVC2 medium) described in item 6.

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

[0024] 9. A method for preparing an in vitro embryo culture medium, which includes:

[0025] Dissolving the components in the in vitro oocyte maturation culture medium described in any one of items 1 - 3 to obtain the in vitro oocyte maturation culture medium;

[0026] Dissolving the components in the fertilization culture medium (IVF medium) described in item 4 to obtain the fertilization culture medium;

[0027] Dissolving the components of the embryo culture medium 1 (IVC1 medium) described in item 5 to obtain the embryo culture medium 1 (IVC1 medium);

[0028] The components of the embryo culture medium 2 (IVC2 medium) described in item 6 are dissolved to obtain the embryo culture medium 2 (IVC2 medium).

[0029] 10. Use of retinoic acid in the preparation of an in vitro oocyte maturation culture 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 this application can improve the fertilization effect. At the same time, the cleavage rate and blastocyst rate are statistically analyzed, both of which are significantly higher than those of the control group, improving the embryo quality, reducing the cost of in vitro embryo production, providing an experimental basis for the application of IVF technology in practice, and greatly accelerating the genetic breeding process. Specific embodiments

[0032] The following describes the present application in detail in combination with 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 specific components. Those skilled in the art should understand that technicians may use different terms to refer to the same component. The specification and claims do not use the difference in terms as a way to distinguish components, but use the difference in the functions of components as the criterion for distinction. As used throughout the specification and claims, the terms "comprising" or "including" are open-ended terms and 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 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 shall be determined by the scope defined by the appended claims.

[0034] This application provides an in vitro oocyte maturation culture medium (IVM medium), which contains CaCl 2 , KCl, KH 2 PO 4 , glycine, HEPES sodium salt, lactic acid, MgSO 4 , NaHCO 3 , inositol, glucose, essential amino acids, non-essential amino acids, retinoic acid, taurine and hormones.

[0035] In this application, neither the essential amino acids nor the non-essential amino acids include the glycine described above.

[0036] In the present application, for essential amino acids and non-essential amino acids, commonly used essential amino acids and non-essential amino acids in the art can be used. For example, the essential amino acids and non-essential amino acids can be commercially available amino acids, such as those that can be purchased from Sigma and the like.

[0037] In some embodiments, the hormones include 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 CaCl 2 is 1.5 - 2.5 mmol / L, the concentration of KCl is 5 - 7 mmol / L, the concentration of KH 2 PO 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 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 solution, the non-essential amino acids are added at 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 1×10 -6 -1×10 - 5 mmol / L.

[0040] For example, the concentration of CaCl 2 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] KH 2 PO 4 The concentration of 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] MgSO 4 The concentration of 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] NaHCO 3 The concentration of 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 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 acids 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 IVM solution.

[0051] The non-essential amino acids 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 IVM solution.

[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 medium (IVM medium) described in this application can help the oocytes recover after meiosis and reduce the occurrence of oxidative stress, thereby improving the oocyte maturation effect.

[0060] The present application provides a fertilization culture medium (IVF solution), which contains CaCl 2 , KCl, KH 2 PO 4 , glycine, HEPES sodium salt, lactic acid, MgSO 4 , NaHCO 3 , NaCl, glucose and non-essential amino acids.

[0061] In the present application, the non-essential amino acids do not include the above-mentioned glycine.

[0062] In some embodiments, 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 2 PO 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 - 1 mmol / L, the concentration of NaHCO 3 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 the IVF solution.

[0063] For example, the concentration of CaCl 2 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] KH 2 PO 4The concentration 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] MgSO 4 The concentration 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] NaHCO 3 The concentration 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 acids 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 binding of oocytes and sperm, and promote the fertilization process.

[0075] The present application provides an embryo culture medium 1 (IVC1 solution), which contains CaCl 2 , KCl, KH 2 PO 4 , glycine, HEPES sodium salt, lactic acid, MgSO 4 , NaHCO 3 , NaCl, 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 CaCl 2 is 1.5 - 2.5 mmol / L, the concentration of KCl is 5 - 7 mmol / L, the concentration of KH 2 PO 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 - 1 mmol / L, the concentration of NaHCO 3 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 concentration of EDTA is 0.005 - 0.02 mmol / L, the non-essential amino acids are added in an amount of 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, CaCl 2The concentration 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] KH 2 PO 4 The concentration 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] MgSO 4 The concentration 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] NaHCO 3 The concentration of NaHCO 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 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, etc.

[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, etc.

[0089] The non-essential amino acids 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 IVC1 solution.

[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, etc.

[0091] The IVC1 solution described in this application can provide a suitable living environment for fertilized eggs and contains components that support the development of early embryo cleavage stages. For example, it contains an appropriate amount of non-essential amino acids, etc., to meet the metabolic needs of cleavage-stage embryos.

[0092] This application provides an embryo culture solution 2 (IVC2 solution), which contains CaCl 2 , KCl, KH 2 PO 4 , glycine, HEPES sodium salt, lactic acid, MgSO 4 , NaHCO 3 , NaCl, inositol, essential amino acids and non-essential amino acids.

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

[0094] In some embodiments, 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 2 PO 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, MgSO4 The concentration of [substance] is 0.3 - 1 mmol / L, and the concentration of NaHCO 3 is 20 - 30 mmol / L, the concentration of NaCl is 0.5 - 80 mmol / L, the concentration of inositol is 2 - 5 mmol / L. For essential amino acids, 0.5 - 5 ml is added to every 100 ml of IVC2 solution, and for non-essential amino acids, 0.5 - 5 ml is added to every 100 ml of IVC2 solution.

[0095] For example, the concentration of CaCl 2 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] KH 2 PO 4 The concentration 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] MgSO 4 The concentration of it 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] NaHCO 3 The concentration of it 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 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, etc.

[0105] The essential amino acids can be added in amounts such as 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 IVC2 solution.

[0106] The non-essential amino acids can be added in amounts such as 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 IVC2 solution.

[0107] The IVC2 solution described in this application can meet the requirements for the rapid proliferation and differentiation of blastocyst cells. It contains all kinds of nutritional components such as amino acids to support the further development of the embryo to form structures such as blastocoel, inner cell mass, and trophoblast cells.

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

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

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

[0111] the embryo culture medium 2 (IVC2 solution) described in any one of the above. In some embodiments, it comprises the in vitro oocyte maturation culture medium (IVM solution) described in any one of the above; the fertilization culture medium (IVF solution) described in any one of the above; the embryo culture medium 1 (IVC1 solution) described in any one of the above; and the embryo culture medium 2 (IVC2 solution) described in any one of the above.

[0112] In this application, the in vitro embryo culture medium can be used for in vitro embryo culture of mammals, and the mammals can be, for example, cattle, etc.

[0113] This application provides the above in vitro embryo culture medium, in which IVM significantly improves the quality of in vitro matured oocytes, reduces the oxidative stress level of bovine oocytes, and IVF, IVC1, and IVC2 reduce production costs through self-configuration while further increasing the blastocyst formation rate and blastocyst quality after in vitro fertilization, greatly improving the efficiency of bovine in vitro embryo production.

[0114] This application provides a method for preparing an in vitro embryo culture medium, which includes:

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

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

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

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

[0119] In the present application, for the above in vitro embryo culture medium, conventional solvents in the art can be used to dissolve the above components. For example, the solvent can be water or the like.

[0120] The culture medium is prepared by the method described in the present application, and its preparation process is simple, which can greatly improve the production efficiency of bovine in vitro embryos.

[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] During the growth process of some oocytes, retinoic acid can stagnate at the prophase of the first meiotic division, activate relevant signaling pathways, promote the oocytes to resume meiosis and develop towards the mature stage; and it can regulate the distribution and function of various organelles in the oocytes, promote the synthesis and distribution of substances such as cortical granules to be more reasonable, and prepare for fertilization and subsequent embryo development. In addition, retinoic acid enhances the response ability of oocytes to activation signals after fertilization by regulating gene expression, and improves their potential to develop into high-quality embryos.

[0124] Examples

[0125] This application generally and / or specifically describes the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, that is, weight percentage. For reagents or instruments whose manufacturers are not indicated, they are all conventional reagent products that can be obtained through commercial purchase. Among them, the sources of the experimental supplies used in this application are as follows: CaCl2, product number: C3881-500G, purchased from: Sigma; KCl, product number: P-5405-250G, purchased from: Sigma; KH2PO4, product number: P5379-500G, purchased from: Sigma; Glycine, product number: G7126-100G, purchased from: Sigma; HEPES NA SALT, product number: H3784-100G, purchased from: Sigma; L-Lactate, product number: L7022-50G, purchased from: Sigma; MgSO4, product number: M2643-500G, purchased from: Sigma; NaHCO3, product number: S5761-500G, purchased from: Sigma; NaCl, product number: S-9625-500G, purchased from: Sigma; Inositol, product number: I7508-50G, purchased from: Sigma; EDTA, product number: E6635-100G, purchased from: Sigma; Glucose, product number: G6152-500G, purchased from: Sigma; EAAS (essential amino acids), product number: B6766-100ml, purchased from: Sigma; NEAAS (non-essential amino acids), product number: M7145-100ml, purchased from: Sigma; Retinoic acid, product number: R2625-50mg, purchased from: Sigma; Taurine, product number: T8691-25G, purchased from: Sigma; FSH, purchased from: Ningbo Hormone Factory No. 2; LH, purchased from: Ningbo Hormone Factory No. 2; Estradiol, product number: E2758-250mg, purchased from: Sigma; EGF, product number: E9644-0.2mg, purchased from: Sigma; Melatonin, product number: M5250-250mg, purchased from: Sigma.

[0126] Example 1 Preparation of IVM Solution

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

[0128] Table 1

[0129]

[0130]

[0131] Step 2: Prepare the hormones according to Table 2

[0132] Table 2

[0133]

[0134] Step 3: Mix the base solution in Table 1 and the hormones in Table 2 to obtain the IVM solution.

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

[0136] The method in Example 2-5 is the same as that in Example 1, except that the amounts of some components are different, as shown in Table 3 for details.

[0137] Comparative Examples 1-4

[0138] The methods in Comparative Examples 1-4 are the same as that in Example 1, except that the amounts of some components are different, as shown in Table 3 for details.

[0139] Table 3

[0140] 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] The IVF solution is obtained by adding each component according to the concentrations in Table 4.

[0143] Table 4

[0144]

[0145]

[0146] Comparative Examples 5-7

[0147] The methods in Comparative Examples 5-7 are the same as that in Example 6, except that the added amounts of some components are different, as shown in Table 5 for details.

[0148] Table 5

[0149] 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] The IVC1 solution is obtained by adding each component according to the concentrations in Table 6.

[0152] Table 6

[0153]

[0154]

[0155] Comparative Example 8

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

[0157] Table 7

[0158] 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 Solution in Example 8

[0160] Add each component according to the concentrations in Table 8 to obtain IVC2 solution.

[0161] Table 8

[0162]

[0163]

[0164] Comparative Example 9

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

[0166] Table 9

[0167] 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] Collect oocytes by follicular puncture from Holstein heifer ranches: Under the pressure of a vacuum pump, select follicles with a diameter of 2 - 8 mm for egg collection through follicular puncture technology. After egg collection, collect a small amount of in vivo egg collection fluid to rinse the egg collection needle and catheter, and select cumulus-oocyte complexes (COCs) under a stereomicroscope. Transfer the COCs to IVM solution after washing in 3 mL of WASH solution (manufacturer: stroebech media, batch number: S23WASB05), and send them to the laboratory within 3 hours. Open the lid of the cryotube and place it in an incubator under the conditions of: 38.8 °C, 6% CO 2 , 100% humidity for 21 - 23 hours.

[0171] 2. In vitro fertilization

[0172] Transfer the COCs from the IVM solution to a 500 μL IVF drop for washing once, and then transfer them to a 90 μL IVF drop to wait for fertilization. Place 15 COCs in each drop. Thaw the semen in a 35.5 °C water bath, centrifuge it at 328 g for 5 minutes using 4 ml of semen washing solution; discard the supernatant, add 3 ml of semen washing solution again and centrifuge for 5 minutes, discard the supernatant, and adjust the sperm concentration using the semen washing solution to make the sperm concentration approximately 2×10 6 cells / ml. Take 50 μL of semen and mix it with the fertilization drop containing COCs to form a 100 μL droplet. Incubate the sperm and eggs together at 38.8 °C, 6% CO 2 gas, 100% humidity for 18 hours.

[0173] 3. Embryo in vitro culture

[0174] Before in vitro culture, prepare balanced IVC1 culture medium one night in advance. 18 hours after in vitro fertilization, use an embryo stripping gun to aspirate the fertilized eggs multiple times until the cumulus is completely stripped. Subsequently, wash the fertilized eggs in IVC1 solution and then transfer them to the pre-balanced IVC1 culture medium. Place the treated fertilized eggs in an incubator at 38.8 °C, 6.0% CO 2 , 7.0% O 2 with saturated humidity for 56 hours of culture.

[0175] At 56 hours (day 3) after fertilization, transfer to the pre-balanced IVC2 solution and continue the culture. Perform embryo grading and cryopreservation on day 7, and count the cleavage rate and blastocyst rate. The results are shown in Table 10. Among them, the cleavage rate is observed morphologically, and the fertilized eggs that cleave are counted in the cleavage rate; early blastocysts, blastocysts, expanded blastocysts, and hatched blastocysts are all counted in the blastocyst rate.

[0176] Table 10

[0177]

[0178] The above are only the preferred embodiments of the present application and do not limit the present application in other forms. Any person skilled in the art may use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present application without departing from the technical solution content of the present application still fall within the protection scope of the technical solution of the present application.

Claims

1. An in vitro oocyte maturation culture medium (IVM solution), 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.

2. The in vitro oocyte maturation medium (IVM medium) according to claim 1, wherein the hormones comprise FSH, LH, estradiol, EGF and melatonin.

3. in vitro oocyte maturation culture fluid (IVM liquid) according to claim 2, wherein CaCl concentration is that the concentration of 1.5-2.5mmol / L, KCl is 5-7mmol / L, KH PO concentration is that the concentration of 0.5-1.5mmol / L, glycine is that the concentration of 1-10mmol / L, HEPES sodium salt is that the concentration of 9-15mmol / L, L-lactic acid is 5-12mmol / L, MgSO concentration is 0.3-0.8mmol / L, NaHCO concentration is that the concentration of 20-30mmol / L, inositol is that the concentration of 0.5-5mmol / L, glucose is that the concentration of 1.5-10mmol / L, essential amino acids are every 100ml 0.5-5 ml of IVM solution was added, 0.5-5 ml of non-essential amino acids were added to every 100 ml of IVM solution, the concentration of retinoic acid was 5-15 mmol / L, the concentration of taurine was 0.05-0.3 mmol / L, the concentration of FSH was 0.5-5 μg / mL, the concentration of LH was 10-20 μg / mL, the concentration of estradiol was 0.5-5 μg / mL, the concentration of EGF was 30-60 ng / mL and the concentration of melatonin was 1×10 -6 -1×10 -5 mmol / L.

4. A fertilization medium (IVF medium), comprising CaCl2, KCl, KH2PO4, glycine, HEPES sodium salt, lactic acid, MgSO4, NaHCO3, NaCl, glucose and non-essential amino acids; Optionally, the concentration of CaCl2 is 1.5-2.5mmol / L, the concentration of KCl is 5-7mmol / L, the concentration of KH2PO4 is 0.5-1.5mmol / L, the concentration of glycine is 1-10mmol / L, the concentration of HEPES sodium salt is 9-15mmol / L, the concentration of L-lactic acid is 5-12mmol / L, the concentration of MgSO4 is 0.3-1mmol / L, the concentration of NaHCO3 is 20-30mmol / L, the concentration of NaCl is 10-80mmol / L, the concentration of glucose is 0.5-2mmol / L, and 0.5-5ml of non-essential amino acids is added to every 100ml of IVF fluid.

5. An embryo culture medium 1 (IVC1 solution), comprising CaCl2, KCl, KH2PO4, glycine, HEPES sodium salt, lactic acid, MgSO4, NaHCO3, NaCl, inositol, EDTA, non-essential amino acids and taurine; Optionally, the concentration of CaCl2 is 1.5-2.5mmol / L, the concentration of KCl is 5-7mmol / L, the concentration of KH2PO4 is 0.5-1.5mmol / L, the concentration of glycine is 1-10mmol / L, the concentration of HEPES sodium salt is 9-15mmol / L, the concentration of L-lactic acid is 5-12mmol / L, the concentration of MgSO4 is 0.3-1mmol / L, the concentration of NaHCO3 is 20-30mmol / L, the concentration of NaCl is 0.5-80mmol / L, the concentration of inositol is 2-5mmol / L, the concentration of EDTA is 0.005-0.02mmol / L, 0.5-5ml of non-essential amino acids is added to every 100ml of IVC1 solution, and the concentration of taurine is 0.05-0.15mmol / L.

6. An embryo culture medium 2 (IVC2 medium), comprising CaCl2, KCl, KH2PO4, glycine, HEPES sodium salt, lactic acid, MgSO4, NaHCO3, NaCl, inositol, essential amino acids and non-essential amino acids; Optionally, the concentration of CaCl2 is 1.5-2.5mmol / L, the concentration of KCl is 5-7mmol / L, the concentration of KH2PO4 is 0.5-1.5mmol / L, the concentration of glycine is 1-10mmol / L, the concentration of HEPES sodium salt is 9-15mmol / L, the concentration of L-lactic acid is 5-12mmol / L, the concentration of MgSO4 is 0.3-1mmol / L, the concentration of NaHCO3 is 20-30mmol / L, the concentration of NaCl is 0.5-80mmol / L, the concentration of inositol is 2-5mmol / L, 0.5-5ml of essential amino acids is added to every 100ml of IVC2 solution, and 0.5-5ml of non-essential amino acids is added to every 100ml of IVC2 solution.

7. An in vitro embryo culture medium, comprising the in vitro oocyte maturation culture medium (IVM medium) according to any one of claims 1 to 3; and / or The fertilization medium (IVF medium) according to claim 4; and / or The embryo culture medium 1 (IVC1 solution) according to claim 5; and / or The embryo culture medium 2 (IVC2 medium) according to claim 6.

8. The in vitro embryo culture medium according to claim 7, comprising the in vitro oocyte maturation culture medium (IVM solution) according to any one of claims 1 to 3, the fertilization culture medium (IVF solution) according to claim 4, the embryo culture medium 1 (IVC1 solution) according to claim 5, and the embryo culture medium 2 (IVC2 solution) according to claim 6.

9. A method for preparing an in vitro embryo culture medium, comprising: Dissolving the components in the in vitro oocyte maturation culture medium according to any one of claims 1 to 3 to obtain the in vitro oocyte maturation culture medium; Dissolving the components in the fertilization culture medium (IVF solution) according to claim 4 to obtain the fertilization culture medium; Dissolving the components of the embryo culture solution 1 (IVC1 solution) according to claim 5 to obtain the embryo culture solution 1 (IVC1 solution); The embryo culture solution 2 (IVC2 solution) is obtained by dissolving the components of the embryo culture solution 2 (IVC2 solution) according to claim 6.

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

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