A culture solution for improving the blastocyst rate and quality of bovine embryos, application thereof and in vitro culture method

Abstract

The application discloses a culture solution for improving the blastocyst rate and quality of bovine embryos, application and an in-vitro culture method, and relates to the technical field of animal embryo engineering. The application provides a culture solution for in-vitro culture of bovine embryos, which comprises a first culture solution and a second culture solution with different osmotic pressure concentrations. By controlling the osmotic pressure concentrations of the first culture solution and the second culture solution in a suitable range, and by using the first culture solution in the early stage of embryo development and the second culture solution in the late stage of embryo development, the in-vitro culture system can better simulate the environment in the oviduct in the early stage of embryo development and better simulate the environment in the uterus in the late stage of embryo development, so that the osmotic pressure stress of the embryo in the development process is reduced, and the blastocyst rate and the blastocyst quality of the bovine embryo are improved. Compared with a traditional culture system with constant osmotic pressure, the application significantly improves the blastocyst rate by sequential regulation of the osmotic pressure, the total cell number of the obtained blastocysts is more, and the apoptosis index is lower.

Description

Technical Field

[0001] This invention relates to the field of animal embryo engineering technology, and in particular to a culture medium for improving the blastocyst rate and quality of bovine embryos, its application, and an in vitro culture method. Background Technology

[0002] In vitro embryo production (IVF) technology in cattle is a key technology for accelerating the genetic improvement and propagation of superior dairy and beef cattle breeds. This technology mainly includes three core steps: in vitro oocyte maturation, in vitro fertilization, and in vitro embryo culture. Among these, in vitro embryo culture is the bottleneck to the technology's success. Currently, the blastocyst rate of bovine IVF embryos is typically only around 30%-40%, far lower than that of in vivo embryos, which severely limits the efficiency of the technology's industrial application.

[0003] The suitability of the in vitro culture environment is a key factor determining whether an embryo can successfully develop into a blastocyst. However, blastocysts cultured using conventional culture media and methods do not exhibit ideal developmental quality and blastocyst rate. Therefore, there is an urgent need in the field for a new in vitro culture method that can effectively improve the blastocyst rate and quality of bovine embryos. Summary of the Invention

[0004] The main objective of this invention is to propose a culture medium for improving the blastocyst rate and quality of bovine embryos, its application, and an in vitro culture method, aiming to solve the problem that the development quality and blastocyst rate of blastocysts cultured using existing bovine embryo in vitro culture methods are not ideal.

[0005] To achieve the above objectives, this invention provides a culture medium for improving the blastocyst rate and quality of bovine embryos, comprising:

[0006] A first culture medium, comprising a basal culture medium and a first osmotic pressure regulator, is used for culturing bovine embryos in the early stages of development, specifically the zygote to morula stage; and... The second culture medium includes a basal culture medium and a second osmotic pressure regulator. The second culture medium is used for culturing bovine embryos in the later stages of development, which is the stage from morula to blastocyst. The first culture medium has an osmotic concentration of 280-320 mOsm / kg, the second culture medium has an osmotic concentration of 250-290 mOsm / kg, and the first culture medium has a higher osmotic concentration than the second culture medium.

[0007] In one embodiment, the osmotic concentration of the first culture medium is 290~310 mOsm / kg, and the osmotic concentration of the second culture medium is 260~280 mOsm / kg.

[0008] In one embodiment, the basal culture medium includes synthetic fallopian tube fluid and a supplement, wherein the supplement includes at least one of serum, bovine serum albumin, amino acids, vitamins, and antibiotics.

[0009] In one embodiment, the first osmotic pressure regulator includes sodium chloride, and the second osmotic pressure regulator includes sodium chloride.

[0010] The present invention also proposes the application of a culture medium, as described in the foregoing technical solution, for improving the blastocyst rate and quality of bovine embryos in bovine breeding.

[0011] This invention also proposes an in vitro culture method for improving the blastocyst rate of bovine embryos, wherein the in vitro culture is performed using the culture medium described above for improving the blastocyst rate and quality of bovine embryos, and the culture medium for improving the blastocyst rate and quality of bovine embryos comprises: A first culture medium, comprising a basal culture medium and a first osmotic pressure regulator, is used for culturing bovine embryos in the early stages of development, specifically the zygote to morula stage; and... The second culture medium includes a basal culture medium and a second osmotic pressure regulator. The second culture medium is used for culturing bovine embryos in the later stages of development, which is the stage from morula to blastocyst. The first culture medium has an osmotic concentration of 280-320 mOsm / kg, the second culture medium has an osmotic concentration of 250-290 mOsm / kg, and the first culture medium has a higher osmotic concentration than the second culture medium. The in vitro culture method includes the following steps: Zygotes or early embryos after in vitro fertilization are provided and cultured in a first culture medium to obtain embryos cultured in the first culture medium; The embryos cultured in the first culture medium are transferred to the second culture medium for further culture until they form blastocysts.

[0012] In one embodiment, the zygote or early embryo after in vitro fertilization is cultured in the first culture medium for a period of 1 to 3-5 days after fertilization.

[0013] In one embodiment, the embryo cultured in the first culture medium is cultured in the second culture medium for 3-5 days to 6-7 days after fertilization.

[0014] The present invention provides a culture medium for in vitro culture of bovine embryos, comprising a first culture medium and a second culture medium with different osmotic concentrations. By controlling the osmotic concentrations of the first and second culture media within an appropriate range, and using the first culture medium in the early stages of embryonic development and the second culture medium in the later stages, the in vivo environment within the fallopian tube can be better simulated in the early stages of embryonic development, and the in vivo environment within the uterus can be better simulated in the later stages of embryonic development. This reduces osmotic stress on the embryo during development and improves the blastocyst rate and quality of bovine embryos. Compared with culture media using traditional constant osmotic pressure culture systems, the present invention significantly improves the blastocyst rate of bovine embryos through sequential osmotic pressure regulation, achieving a blastocyst rate as high as 49.1%, and obtaining blastocysts with a higher total cell count and a lower apoptosis index. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0016] Figure 1 This is a flowchart of the bovine embryo in vitro culture method provided in Embodiment 1 of the present invention; Figure 2 This is a bar chart comparing the blastocyst development rates of Example 1 and Comparative Example 1 of the present invention, wherein, " "" indicates a significant difference between groups, P<0.05.

[0017] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] It should be noted that if the embodiments of the present invention involve directional indicators (such as up, down, left, right, front, back, etc.), the directional indicators are only used to explain the relative positional relationship and movement of the components in a specific posture. If the specific posture changes, the directional indicators will also change accordingly.

[0020] Furthermore, if the embodiments of this invention involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the use of "and / or" or "and / or" throughout the text includes three parallel solutions. For example, "A and / or B" includes solution A, solution B, or a solution where both A and B are satisfied simultaneously. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0021] In vitro embryo production technology in cattle is a key technology for accelerating the genetic improvement and propagation of superior dairy and beef cattle breeds. This technology mainly includes three core steps: in vitro oocyte maturation, in vitro fertilization, and in vitro embryo culture. Among these, in vitro embryo culture is the bottleneck to the technology's success. Currently, the blastocyst rate of bovine in vitro fertilization embryos is typically around 30%-40%, far lower than that of in vivo embryos, which severely limits the efficiency of the technology's industrial application.

[0022] The suitability of the in vitro culture environment is a key factor determining whether an embryo can successfully develop into a blastocyst. Current techniques often employ culture media with constant composition and concentration, such as single-component SOFaa, CR1aa, or G1 / G2 sequential culture media. However, the developmental environment of an embryo in vivo is dynamic. Fertilization and early cleavage occur in the oviduct, where the osmotic pressure is relatively high; while development from morula to blastocyst occurs in the uterus, where the osmotic pressure of uterine fluid is relatively low. A constant in vitro culture environment cannot simulate this physiological dynamic change, potentially leading to osmotic stress in the embryo, affecting its normal metabolism and gene expression, and ultimately causing developmental arrest. Therefore, blastocysts cultured using conventional media and methods do not exhibit ideal developmental quality and blastocyst development rates. There is an urgent need in this field for a new in vitro culture method that can better simulate the in vivo environment and effectively improve the blastocyst rate and quality of bovine embryos.

[0023] In view of this, the present invention proposes a culture medium for improving the blastocyst rate and quality of bovine embryos, comprising: A first culture medium, comprising a basal culture medium and a first osmotic pressure regulator, is used for culturing bovine embryos in the early stages of development, specifically the zygote to morula stage; and... The second culture medium includes a basal culture medium and a second osmotic pressure regulator. The second culture medium is used for culturing bovine embryos in the later stages of development, which is the stage from morula to blastocyst. The first culture medium has an osmotic concentration of 280-320 mOsm / kg, the second culture medium has an osmotic concentration of 250-290 mOsm / kg, and the first culture medium has a higher osmotic concentration than the second culture medium.

[0024] The present invention provides a culture medium for in vitro culture of bovine embryos, comprising a first culture medium and a second culture medium with different osmotic concentrations. By controlling the osmotic concentrations of the first and second culture media within an appropriate range, and using the first culture medium in the early stages of embryonic development and the second culture medium in the later stages, the in vivo environment within the fallopian tube can be better simulated in the early stages of embryonic development, and the in vivo environment within the uterus can be better simulated in the later stages of embryonic development. This reduces osmotic stress on the embryo during development and improves the blastocyst rate and quality of bovine embryos. Compared with culture media using traditional constant osmotic pressure culture systems, the present invention significantly improves the blastocyst rate of bovine embryos through sequential osmotic pressure regulation, achieving a blastocyst rate as high as 49.1%, and obtaining blastocysts with a higher total cell count and a lower apoptosis index.

[0025] To further enhance the effect of the sequential culture system on blastocyst development, in this embodiment of the invention, the osmotic concentration of the first culture medium is set to 290-310 mOsm / kg, and the osmotic concentration of the second culture medium is set to 260-280 mOsm / kg. This results in better improvement of blastocyst quality, a higher number of cells after culture, and a lower apoptosis rate.

[0026] In embodiments of the present invention, the basal culture medium includes synthetic oviduct fluid (SOF) and supplements, wherein the supplements include at least one selected from serum, bovine serum albumin, amino acids, vitamins, and antibiotics. In one embodiment of the present invention, a first culture medium is prepared using modified SOFaa (essential and non-essential amino acids added to the basal SOF) in combination with bovine serum albumin, and a second culture medium is prepared using modified SOFaa in combination with fetal bovine serum.

[0027] In embodiments of the present invention, the first osmotic pressure regulator includes sodium chloride, and the second osmotic pressure regulator includes sodium chloride. The osmotic pressure concentration range of the first culture medium / second culture medium can be controlled by adjusting the concentration of sodium chloride (NaCl) in the first culture medium / second culture medium.

[0028] This invention proposes the application of a culture medium, as described in the foregoing technical solution, for improving the blastocyst rate and quality of bovine embryos in bovine breeding. Applying the provided culture medium to bovine breeding can increase the blastocyst rate and quality of bovine embryos, thereby improving the overall quality of bovine breeding.

[0029] This invention proposes an in vitro culture method for improving the blastocyst rate of bovine embryos. The method utilizes a culture medium specifically designed to improve the blastocyst rate and quality of bovine embryos for in vitro culture. This culture medium comprises: A first culture medium, comprising a basal culture medium and a first osmotic pressure regulator, is used for culturing bovine embryos in the early stages of development, specifically the zygote to morula stage; and... The second culture medium includes a basal culture medium and a second osmotic pressure regulator. The second culture medium is used for culturing bovine embryos in the later stages of development, which is the stage from morula to blastocyst. The first culture medium has an osmotic concentration of 280-320 mOsm / kg, the second culture medium has an osmotic concentration of 250-290 mOsm / kg, and the first culture medium has a higher osmotic concentration than the second culture medium. The in vitro culture method includes the following steps: Zygotes or early embryos after in vitro fertilization are provided and cultured in a first culture medium to obtain embryos cultured in the first culture medium; The embryos cultured in the first culture medium are transferred to the second culture medium for further culture until they form blastocysts.

[0030] In this invention, a first culture medium and a second culture medium with different osmotic concentrations are used for in vitro culture. By controlling the osmotic concentrations of the first and second culture media within a suitable range, and using the first culture medium in the early stages of embryonic development and the second culture medium in the later stages of bovine embryonic development, the in vivo environment within the fallopian tube can be better simulated in the early stages of embryonic development, and the in vivo environment within the uterus can be better simulated in the later stages of embryonic development. This reduces osmotic stress on the embryo during development and improves the blastocyst rate and quality of bovine embryos. Compared with traditional culture methods using a constant osmotic pressure system, this invention significantly improves the blastocyst rate (up to 49.1%) through sequential osmotic pressure regulation, resulting in a higher total number of cells in the obtained blastocysts and a lower apoptosis index. Furthermore, the in vitro culture method provided by this invention is simple to operate, easy to promote, and requires no complex equipment. It only adds the sequential use of culture media with different concentrations to the existing embryo culture process, resulting in minimal cost increase and making it suitable for industrial application.

[0031] It should be noted that in this patent, "early embryo after in vitro fertilization" refers to the embryo after fertilization and the embryo in the early morula stage.

[0032] In an embodiment of the present invention, the zygote obtained after in vitro fertilization is obtained through the following steps: S10. Superovulation (SOV) was performed on the cows, and oocytes were collected from the ovaries. Cumulus-Oocyte Complexes (COCs) in the follicular fluid were identified under a stereomicroscope. COCs with intact cumulus cell layers and uniform cytoplasm were selected, washed with DPBS (Dulbecco's Phosphate-Buffered Saline), and then cultured in maturation medium. Matured oocytes were obtained by culturing in an incubator at 38.9℃ and 5% CO2 for 24 h. S20. Take the high-quality bull semen frozen from the liquid nitrogen tank, thaw it, and wash it with semen washing solution. After washing, apply 1×10 ml of the solution. 6 A density of one effective sperm was placed in a 50 μL fertilization microdroplet containing mature oocytes and co-cultured for 18 h. S30. After fertilization, the fertilized eggs are washed with a granulosa cell removal needle to remove granulosa cells and sperm from the surface of the fertilized eggs. Then, they are washed with DPBS solution to obtain zygotes.

[0033] In step S10, the present invention uses a conventional in vitro maturation procedure for bovine oocytes to obtain zygotes. Specifically, the cow is first subjected to superovulation treatment, and then follicular fluid containing oocytes is collected from the ovary by surgical or non-surgical methods (such as ovarian puncture). The collected follicular fluid is then placed in a culture dish and observed under a stereomicroscope to identify COCs (co-occurring cells) encapsulating multiple layers of cumulus cells. The COCs are then gently washed with DPBS to remove blood, impurities, or residual follicular fluid. After washing, the COCs are transferred to a maturation culture medium for culture, allowing the immature oocytes (in the prophase I of meiosis, GV phase) to develop to the MII phase (metaphase II of meiosis), which is a mature oocyte capable of fertilization.

[0034] In steps S20 and S30, a standard bovine in vitro fertilization (IVF) process is used. Specifically, the frozen semen is first thawed, then washed with semen washing solution, resuspended, and the effective sperm density (effective sperm refers to sperm that simultaneously meet the criteria of being viable, motile, and morphologically normal) is adjusted to 1×10⁻⁶. 6 Add fertilization microdroplets containing mature oocytes to a fertilization incubator at a ratio of 1 / mL, then transfer to an incubator for co-culture to fertilize; after fertilization, wash the fertilized eggs to obtain naked zygotes.

[0035] In an embodiment of the present invention, the zygote or early embryo after in vitro fertilization is cultured in the first culture medium for a period of time from day 1 to day 3 to 5 after fertilization, covering the developmental stages from zygote to early / mid-term morula.

[0036] The morula stage includes the early morula stage, the mid-morula stage, and the late morula stage. In the early morula stage, the cell count is approximately 16-20, and the cells are not yet fully compacted. In the mid-morula stage, the cells are tightly adhered with blurred boundaries. In the late morula stage, the blastocoel is about to appear, transitioning to the blastocyst stage. Specifically, when providing zygotes after in vitro fertilization, they are usually transferred to a second culture medium for further culture when the cell count reaches 16. However, in practice, due to the different developmental rates of individual embryos, some cells may have already developed to the mid-morula stage. Transferring them to a second culture medium at this point can also improve the blastocyst rate and quality of bovine embryos.

[0037] In an embodiment of the present invention, the embryo cultured in the first culture medium is cultured in the second culture medium for 3-5 days to 6-7 days after fertilization, covering the developmental stages from early / mid-term morula to blastocyst.

[0038] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0039] reagents The composition of the basic SOF is shown in Table 1.

[0040] Table 1 Composition of basic SOFs

[0041] Modified SOFaa: Using a basic SOF solution as the basal culture medium, 1% (v / v) of essential amino acids (Gibco's commercial MEM EAA100X concentrate), 1% (v / v) of non-essential amino acids (Gibco's commercial MEM NEAA100X concentrate) and 1 mM of glutamine are added.

[0042] Example 1 An in vitro culture method for improving the blastocyst rate of bovine embryos includes the following steps: 1. Preparation of culture medium: 1) Preparation of the first culture medium: Modified SOFaa is used as the basic culture medium, with 3 mg / mL bovine serum albumin (BSA) added, and the osmotic pressure concentration of the first culture medium is adjusted to 300±5 mOsm / kg by adding NaCl. 2) Preparation of the second culture medium: Modified SOFaa is used as the basic culture medium, 10% fetal bovine blood (FBS) is added, and the osmotic pressure concentration of the second culture medium is adjusted to 275±5 mOsm / kg by adding NaCl; 3) Equilibrate the first and second culture media in an incubator at 38.9℃, 5% CO2, and saturated humidity for at least 4 hours.

[0043] 2. Embryo Acquisition and Culture: 1) such as Figure 1 As shown, cows were subjected to superovulation treatment, and oocytes were collected from the ovaries. Cumulus-oocyte complexes (COCs) in the follicular fluid were identified under a stereomicroscope. COCs with intact cumulus cell layers and homogeneous cytoplasm were selected, washed with DPBS, and cultured in maturation medium (BO-IVM maturation medium). Matured oocytes were obtained by maturation culture at 38.9℃ and 5% CO2 for 24 hours.

[0044] 2) Remove high-quality bull semen from the liquid nitrogen tank, thaw it, and wash it with semen washing solution (BO-IVF semen washing solution from IVF Bioscience). After washing, apply 1×10 ml of the solution. 6 A density of 1 effective sperm was placed in 50 μL of fertilization microdroplets containing mature oocytes (covered with paraffin oil) and co-cultured in an incubator at 38.9°C and 5% CO2 for 18 h.

[0045] 3) After fertilization, the fertilized eggs are washed with a granulosa cell removal needle to remove the granulosa cells and sperm on the surface of the fertilized eggs, and then washed with DPBS solution to obtain zygotes.

[0046] 3. In vitro culture: like Figure 1 As shown, the zygotes were cultured in the first culture medium with an osmotic pressure of 300 mOsm / kg until the 4th day after fertilization. On the 4th day, the embryos that had developed into early morula were transferred to the second culture medium with an osmotic pressure of 275 mOsm / kg and cultured for another 3 days to form blastocysts.

[0047] Example 2 The difference compared to Example 1 is that the osmotic concentration of the first culture medium is 295±5 mOsm / kg.

[0048] Example 3 The difference compared to Example 1 is that the osmotic concentration of the first culture medium is 310±5 mOsm / kg.

[0049] Example 4 The difference compared to Example 1 is that the osmotic concentration of the second culture medium is 265±5 mOsm / kg.

[0050] Example 5 The difference compared to Example 1 is that the osmotic concentration of the second culture medium is 285±5 mOsm / kg.

[0051] Comparative Example 1 Compared to Example 1, the difference lies in the use of a single culture medium for 7 days. The preparation steps of the culture medium are as follows: The modified SOFaa was used as the base culture medium, with 10% FBS added, and the osmotic pressure concentration of the culture medium was adjusted to 290±5 mOsm / kg by adding NaCl.

[0052] Performance testing To verify whether altering osmotic pressure affects blastocyst development rate and quality, the blastocyst rate of Example 1 and Comparative Example 1 was calculated on day 7 post-fertilization. On day 7, the total number of cells and the number of apoptotic cells in the blastocysts were counted using Hoechst 33342 and Propidium Iodide (PI) dual-fluorescence staining. The results of the blastocyst development rate test are shown in Table 2 and... Figure 2 As shown in Table 3, the test results for blastocyst quality are presented.

[0053] Table 2 shows the comparison of blastocyst rates between Example 1 and Comparative Example 1.

[0054] Table 3 shows the comparison results of blastocyst quality between Example 1 and Comparative Example 1.

[0055] Combining Table 2, Table 3 and Figure 1 The test results show that, compared with the traditional constant osmotic pressure culture of Comparative Example 1, the osmotic pressure sequential culture method used in Example 1 can not only significantly improve the blastocyst formation rate of bovine embryos, but also effectively improve the quality of blastocysts, and can culture more cells with a lower apoptosis rate. This indicates that the blastocysts cultured in Example 1 have a higher potential for implantation and pregnancy.

[0056] To verify the effect of different osmotic pressure combinations on blastocyst rate, blastocysts were cultured using the methods in Examples 1-5. Approximately 150 zygotes were used in each group, and the blastocyst rate on day 7 was calculated. The test results are shown in Table 4.

[0057] Table 4. Blastocyst rate test results for Examples 1-5

[0058] As can be seen from Table 4, the highest blastocyst development rate of 49.2% was achieved when the osmotic pressure of the first culture medium was 300 mOsm / kg and the osmotic pressure of the second culture medium was 275 mOsm / kg (Example 1), which was significantly better than other combinations.

[0059] The above description is merely an exemplary embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention specification and drawings under the technical concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A culture medium for improving the blastocyst rate and quality of bovine embryos, characterized in that, include: A first culture medium, comprising a basal culture medium and a first osmotic pressure regulator, is used for culturing bovine embryos in the early stages of development, specifically the zygote to morula stage; and... The second culture medium includes a basal culture medium and a second osmotic pressure regulator. The second culture medium is used for culturing bovine embryos in the later stages of development, which is the stage from morula to blastocyst. The first culture medium has an osmotic concentration of 280-320 mOsm / kg, the second culture medium has an osmotic concentration of 250-290 mOsm / kg, and the first culture medium has a higher osmotic concentration than the second culture medium.

2. The culture medium for improving the blastocyst rate and quality of bovine embryos as described in claim 1, characterized in that, The osmotic concentration of the first culture medium is 290~310 mOsm / kg, and the osmotic concentration of the second culture medium is 260~280 mOsm / kg.

3. The culture medium for improving the blastocyst rate and quality of bovine embryos as described in claim 1, characterized in that, The basic culture medium includes synthetic fallopian tube fluid and supplements, wherein the supplements include at least one of serum, bovine serum albumin, amino acids, vitamins and antibiotics.

4. The culture medium for improving the blastocyst rate and quality of bovine embryos as described in claim 1, characterized in that, The first osmotic pressure regulator includes sodium chloride, and the second osmotic pressure regulator includes sodium chloride.

5. The application of a culture medium as described in any one of 1 to 4 for improving the blastocyst rate and quality of bovine embryos in bovine breeding.

6. An in vitro culture method for improving the blastocyst rate and quality of bovine embryos, characterized in that, The culture medium for improving the blastocyst rate and quality of bovine embryos, as described in any one of claims 1 to 4, is used for in vitro culture, and the in vitro culture method includes the following steps: Zygotes or early embryos after in vitro fertilization are provided and cultured in a first culture medium to obtain embryos cultured in the first culture medium; The embryos cultured in the first culture medium are transferred to the second culture medium for further culture until they form blastocysts.

7. The in vitro culture method for improving the blastocyst rate and quality of bovine embryos as described in claim 6, characterized in that, The zygote or early embryo after in vitro fertilization is cultured in the first culture medium for a period of time from day 1 to day 3-5 after fertilization.

8. The in vitro culture method for improving the blastocyst rate and quality of bovine embryos as described in claim 6, characterized in that, The embryos cultured in the first culture medium are cultured in the second culture medium from day 3 to day 5 to day 6 to day 7 after fertilization.

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