An embryo culture method for improving the in vitro blastocyst rate of cattle

Abstract

The present invention relates to the field of embryo engineering technology, and specifically relates to an embryo culture method for improving the in vitro blastocyst rate of cattle, which comprises the steps of: adding a set volume of maturation medium into each well of a four-well plate; placing the cumulus-oocyte complexes collected from cattle ovaries into the maturation medium for culture; adding the treated cattle semen into the fertilization medium containing the cumulus-oocyte complexes, and performing fertilization in an incubator; making a three-dimensional columnar culture droplet in the culture well to achieve the culture purpose of enabling the embryos to be in close contact, and increasing the paracrine between the embryos; and transferring the fertilized eggs into the three-dimensional columnar culture droplet for culture. The purpose of the embryo culture method for improving the in vitro blastocyst rate of cattle is to solve the problem of the relatively low blastocyst rate of cattle embryos cultured in vitro.

Description

Technical Field

[0001] The present invention relates to the technical field of embryo engineering, and particularly relates to an embryo culture method for improving the in vitro blastocyst rate of cattle. Background Art

[0002] As an important part of agriculture, animal husbandry is of great significance for ensuring national food security and promoting agricultural modernization. Applying embryo technology for genetic improvement of dairy cattle breeds and breeding of breeding cattle has become a powerful tool for dairy cattle breeding in dairy powerhouses. The low efficiency of in vitro embryo production is the key obstacle that prevents large-scale in vitro embryo production in China at present, which to a certain extent restricts the high-quality development of animal husbandry in China.

[0003] In vitro embryo production involves multiple links such as in vitro oocyte maturation, in vitro fertilization, and in vitro embryo culture. Among them, the low in vitro embryo blastocyst rate is the biggest obstacle restricting the application of in vitro embryo technology in China. The traditional methods of oil-covered maturation, microdrop fertilization, and planar embryo culture may inhibit the action of hormones, prolong the in vitro treatment time, and cause uneven distribution of embryos in the culture medium, affecting the interaction and signal transmission between embryos, thereby restricting the development of in vitro embryos and resulting in a decrease in the blastocyst rate.

[0004] Therefore, the inventor provides an embryo culture method for improving the in vitro blastocyst rate of cattle. Summary of the Invention

[0005] (1) Technical Problems to be Solved

[0006] The embodiment of the present invention provides an embryo culture method for improving the in vitro blastocyst rate of cattle, which solves the technical problem of the low blastocyst rate in in vitro culture of cattle embryos.

[0007] (2) Technical Solutions

[0008] The present invention provides an embryo culture method for improving the in vitro blastocyst rate of cattle, comprising the following steps:

[0009] S100. Add a set volume of maturation medium into each well of a four-well plate, and take it out for standby after equilibration in an incubator for a first set time;

[0010] S200. Place the cumulus-oocyte complexes collected from bovine ovaries into the maturation medium processed in step S100, and add them into each well of the four-well plate respectively, and then culture them in an incubator for a second set time;

[0011] S300. Add a set volume of fertilization medium to each well of the four-well plate in step S200 to obtain a fertilization medium containing cumulus-oocyte complexes, and then add the processed bovine semen to the fertilization medium containing cumulus-oocyte complexes and perform fertilization in an incubator for a third set time to obtain fertilized eggs;

[0012] S400. Add the first part of the in vitro culture medium to each well of the four-well plate to make small drops, then add mineral oil to each well, and then add the second part of the in vitro culture medium to form a three-dimensional columnar culture medium drop;

[0013] S500. Transfer the fertilized eggs obtained in step S300 into the three-dimensional columnar culture medium drop for culture, and perform half-volume medium replacement at intervals of a fourth set time until the fertilized eggs are cultured in vitro to the blastocyst stage.

[0014] Further, adding a set volume of maturation medium to each well of the four-well plate and taking it out for standby after equilibration in an incubator for a first set time specifically includes:

[0015] Add 500 - 750 μL of maturation medium to each well of the four-well plate, and do not drop mineral oil on the surface of the maturation medium, and equilibrate in an incubator at 38.5 °C, 5% CO2 and saturated humidity for at least 2 h.

[0016] Further, placing the cumulus-oocyte complexes collected from bovine ovaries in the maturation medium processed in step S100, adding them to each well of the four-well plate respectively, and then culturing them in an incubator for a second set time specifically includes:

[0017] Collect cumulus-oocyte complexes from follicles with a diameter of 2 - 8 mm on the bovine ovaries, place the cumulus-oocyte complexes in the maturation medium processed in step S100, add them to each well of the four-well plate respectively, and then culture them in the incubator at 38.5 °C, 5% CO2 and saturated humidity for 22 - 24 h.

[0018] Further, in step S300, the preparation of the semen includes: thawing bovine frozen straw semen in a water bath and then adding washing semen, and preparing semen after centrifugal precipitation; and / or,

[0019] The processing conditions for fertilization in the incubator for a third set time are: fertilize in the incubator at 38.5 °C, 5% CO2 and saturated humidity for 8 - 18 h.

[0020] Further, after thawing bovine frozen straw semen in a water bath, add washing semen, after centrifugal precipitation, adjust the sperm density with fertilization medium as semen; among them, the sperm density is 2×106 ~5×10 6 cells / mL.

[0021] Furthermore, after the cumulus-oocyte complex is subjected to fertilization treatment, it is digested with hyaluronidase to remove cumulus granulosa cells, obtaining a fertilized egg.

[0022] Furthermore, 300 - 500 μL of fertilization medium is added to each well of the four-well plate in step S200.

[0023] Furthermore, the concentration of the hyaluronidase is 1 g / L.

[0024] Furthermore, the digestion time is 1 - 2 min.

[0025] Furthermore, the volume ratio of the in vitro culture medium in the first part to the in vitro culture medium in the second part is 1:(10 - 20).

[0026] Furthermore, the fourth set time is 48 h.

[0027] (3) Beneficial effects

[0028] In summary, in the embryo culture stage of the present invention, a three-dimensional columnar culture drop is used to reduce the bottom area of the culture drop, enabling the embryos to come into close contact, generating an effective paracrine effect, sharing cytokines and growth factors, activating certain common signaling pathways, helping to reduce apoptosis in embryos, assisting the embryos to resist stress and damage, thereby achieving the purpose of increasing the quantity and quality of blastocysts. Brief description of the drawings

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the embodiments of the present invention will be briefly introduced below. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0030] Figure 1 is a schematic flowchart of an embryo culture method for improving the in vitro blastocyst rate of cows provided by an embodiment of the present invention;

[0031] Figure 2 is a SEM image of the morphology of a blastocyst on the seventh day cultured by an embryo culture method for improving the in vitro blastocyst rate of cows provided by an embodiment of the present invention;

[0032] Figure 3 is a SEM image of the morphology of a blastocyst on the seventh day cultured by a traditional embryo culture method for the in vitro blastocyst rate of cows. Detailed implementation manners

[0033] The following further describes the embodiments of the present invention in detail in conjunction with the accompanying drawings and embodiments. The detailed descriptions and drawings of the following embodiments are used to exemplarily illustrate the principles of the present invention, but cannot be used to limit the scope of the present invention, that is, the present invention is not limited to the described embodiments.

[0034] It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other. The following will describe this application in detail with reference to the accompanying drawings and embodiments.

[0035] How to optimize the systems of in vitro maturation, in vitro fertilization, and in vitro embryo culture, reduce the impact on reproductive hormones, shorten the residence time of fertilized eggs outside the incubator, increase the paracrine effect among embryos, and better support the development of in vitro embryos is of great significance for enhancing the overall competitiveness of China's animal husbandry, promoting the transformation and upgrading of animal husbandry, and realizing the sustainable development of animal husbandry. At the same time, this also helps to improve the economic benefits of animal husbandry, increase farmers' income, and make positive contributions to the modernization of China's agriculture and rural areas. In the future, we should increase scientific research investment, strengthen technological innovation, break through the key technical bottlenecks of in vitro embryo production, and promote the high-quality development of China's animal husbandry.

[0036] Figure 1 is a schematic flowchart of an embryo culture method for improving the in vitro blastocyst rate of cattle provided by an embodiment of the present invention. As Figure 1 shown, the method may include the following steps:

[0037] S100. Add a set volume of maturation medium to each well of a four-well plate, and take it out for standby after equilibration in an incubator for a first set time.

[0038] Specifically, add a set volume of maturation medium to each well of a four-well plate, and equilibrate it in an incubator at 38.5°C, 5% CO2, and saturated humidity for at least 2 h. There is no limitation on the specific type selection of the maturation medium, and it can be purchased through products on the market. The maturation medium can specifically adopt the BO-IVM oocyte maturation medium of Huayuexing. Further, no mineral oil is dropped on the surface of the maturation medium to reduce the impact of mineral oil on reproductive hormones such as estrogen.

[0039] S200. Place the cumulus-oocyte complexes collected from bovine ovaries into the maturation medium processed in step S100, and add them to each well of the four-well plate respectively, and then culture them in an incubator for a second set time.

[0040] Specifically, collect the cumulus-oocyte complexes in follicles with a diameter of 2 - 8 mm on bovine ovaries, place the cumulus-oocyte complexes in an in vitro maturation medium, and culture them in an incubator at 38.5°C, 5% CO2, and saturated humidity for 22 - 24 h.

[0041] S300. Add a set volume of fertilization medium to each well of the four-well plate in step S200 to obtain a fertilization medium containing cumulus-oocyte complexes. Then, add the processed bovine semen to the fertilization medium containing cumulus-oocyte complexes and perform fertilization in an incubator for a third set time to obtain fertilized eggs.

[0042] Specifically, S300 may include the following steps:

[0043] S301. Thaw the frozen bovine straw semen in a water bath and then add washing semen. After centrifuging and precipitating multiple times, it is used as semen.

[0044] S302. Aspirate a set volume of semen and add it to the fertilization medium containing cumulus-oocyte complexes, and perform fertilization in an incubator at 38.5 °C, 5% CO2, and saturated humidity for 8 - 18 h.

[0045] Among them, in step S301, after sperm precipitation, the sperm density is adjusted with fertilization medium to be used as semen.

[0046] In step S302, after in vitro fertilization of the cumulus-oocyte complexes, they are digested with hyaluronidase to remove cumulus granulosa cells. Among them, add 300 - 500 μL (i.e., large drop) of fertilization medium to each well of the four-well plate in step S200, which is convenient for fertilization treatment and shortens the residence time of oocytes and sperm outside the incubator. The concentration of hyaluronidase is 1 g / L. Specifically, 0.1 g of hyaluronidase and 100 ml of TCM199 solvent can be mixed and prepared, and the digestion time is 1 - 2 min.

[0047] S400. Add the first part of the in vitro culture medium to each well of the four-well plate to make small drops. Then, add mineral oil to each well, and then add the second part of the in vitro culture medium to form a three-dimensional columnar culture medium drop.

[0048] Specifically, by controlling the dripping amount of the culture medium, mainly use a white pipette tip to first drip 5 - 10 μL of small drops of the culture medium, add mineral oil to achieve the purpose of fixing the bottom area and preventing the evaporation of the culture medium. Further, the volume ratio of the first part of the in vitro culture medium to the second part of the in vitro culture medium is 1:(10 - 20). It should be noted here that the components of the first part of the in vitro culture medium and the second part of the in vitro culture medium are exactly the same, only the added amounts are different. The entire IVC culture drop is a 100 μL three-dimensional columnar liquid drop. The purpose of designing it as a three-dimensional columnar liquid drop is to ensure the close contact between embryos to produce beneficial paracrine effects, thereby increasing the number and quality of blastocysts. It should be noted here that the specific volumes of the first part and the second part of the in vitro culture medium are not limited and can be dripped according to the actual situation. There is no limit to the specific selection of the in vitro culture medium, and it can be purchased through products on the market. Specifically, the maturation medium can adopt the BO series culture medium of Huayuehang.

[0049] It should be noted here that since mineral oil has been added, when adding the second part of the in vitro culture medium, it needs to be pushed into the original liquid drop, otherwise a new small liquid drop will be formed and cannot fuse with the original liquid drop; secondly, during the process of transferring the plate, it is also necessary to pay attention to being stable because what is formed is a three-dimensional columnar liquid drop, and if the movement amplitude is too large, it is very easy for the three-dimensional columnar liquid drop to disperse and form small round drops.

[0050] S500. Transfer the fertilized eggs obtained by the treatment in step S300 into the three-dimensional columnar culture medium drop for culture, and change the liquid by half at intervals of the fourth set time until the fertilized eggs are cultured in vitro to the blastocyst stage. Specifically, the number of oocytes added to the three-dimensional columnar culture medium drop is generally 15 - 20, and the fourth set time is 48 h.

[0051] Example 1

[0052] The specific process of embryo culture is divided into four groups: A, B, C, and D. Among them, group A uses traditional flat large-drop culture, group B does not cover mineral oil at the oocyte maturation stage on the basis of group A, group C uses large-drop fertilization solution at the in vitro fertilization stage on the basis of group B; group D uses three-dimensional columnar culture drops at the embryo culture stage on the basis of group C.

[0053] The culture process of group A is as follows:

[0054] 1) First, add 750 μL of maturation medium to each well in the four-well plate, cover mineral oil on the surface of the maturation medium, and equilibrate in an incubator at 38.5 °C, 5% CO2 and saturated humidity for 2 h;

[0055] 2) Collect cumulus-oocyte complexes (COCs) from follicles with a diameter of 2-8 mm on bovine ovaries, place the COCs in the treated maturation medium, and culture them in an incubator at 38.5 °C, 5% CO2 and saturated humidity for 22 h;

[0056] 3) After thawing bovine frozen straw semen in a 37 °C water bath, add 3.5 mL of washing semen, centrifuge at 1800 rpm for 5 min, and centrifuge twice; adjust the sperm density of the sperm pellet to 5×10 6 cells / mL with fertilization medium as semen;

[0057] 4) In a 35 mm dish, make 50 μL drops (7 drops can be made in each dish), cover with mineral oil and equilibrate in an incubator at 38.5 °C, 5% CO2 and saturated humidity for 2 h. Pipette 50 μL of semen into 50 μL of semen containing 15 bovine oocytes, and fertilize in an incubator at 38.5 °C, 5% CO2 and saturated humidity for 16 h;

[0058] 5) After in vitro fertilization of cumulus-oocyte complexes, digest with 1 g / L hyaluronidase for 1 min to remove granulosa cells;

[0059] 6) In the IVC (in vitro culture) stage, add 500 μL of IVC medium to a four-well plate, cover with mineral oil and equilibrate in an incubator at 38.5 °C, 5% CO2 and saturated humidity for 2 h.

[0060] 7) Transfer the fertilized eggs to a large drop of embryo culture medium for culture, change half of the medium every 48 h, and culture the fertilized eggs in vitro to the blastocyst stage.

[0061] The culture process of group B is as follows:

[0062] 1) First, add 750 μL of maturation medium to each well in a four-well plate, do not cover the surface of the maturation medium with mineral oil, and equilibrate in an incubator at 38.5 °C, 5% CO2 and saturated humidity for 2 h;

[0063] 2) Collect cumulus-oocyte complexes (COCs) from follicles with a diameter of 2-8 mm on bovine ovaries, place the COCs in the treated maturation medium, and culture them in an incubator at 38.5 °C, 5% CO2 and saturated humidity for 22 h;

[0064] 3) After thawing bovine frozen straw semen in a 37 °C water bath, add 3.5 mL of washing semen, centrifuge at 1800 rpm for 5 min, and centrifuge twice; adjust the sperm density of the sperm pellet to 5×10 6 cells / mL with fertilization medium as semen;

[0065] 4) In a 35-mm Petri dish, make 50-μL drops (7 drops can be made per dish), cover with mineral oil, and equilibrate in an incubator at 38.5 °C, 5% CO2, and saturated humidity for 2 h. Aspirate 50 μL of semen and add it to 50 μL of semen containing 15 bovine oocytes, and fertilize in an incubator at 38.5 °C, 5% CO2, and saturated humidity for 16 h;

[0066] 5) After in vitro fertilization of the cumulus-oocyte complex, digest with 1 g / L hyaluronidase for 1 min to remove granulosa cells;

[0067] 6) In the IVC (in vitro culture) stage, add 500 μL of IVC solution to a four-well plate, cover with mineral oil, and equilibrate in an incubator at 38.5 °C, 5% CO2, and saturated humidity for 2 h.

[0068] 7) Transfer the fertilized eggs to a large drop of embryo culture medium for culture, change half of the medium every 48 h, and culture the fertilized eggs in vitro until the blastocyst stage.

[0069] Table 1 Effects of covering and not covering mineral oil on embryo development in the IVM mode

[0070]

[0071] The data in Table 1 above show that not covering the IVM culture medium with mineral oil during the IVM stage can increase the blastocyst rate. During the in vitro maturation (IVM) stage, E2 (estradiol) added to the culture medium is a lipophilic substance, which means it is easily soluble in oil substances. Mineral oil, as a substance commonly used as a solvent or protective agent, is hydrophobic and can therefore be compatible with E2. Based on the lipophilic nature of E2, we can reasonably speculate that when mineral oil is covered above the IVM solution, E2 may be absorbed by the mineral oil. This is because lipophilic substances generally tend to dissolve in an oil environment. However, the specific absorption efficiency and degree may be affected by various factors, including the concentration of E2, the properties of the mineral oil, and the specific composition of the IVM solution.

[0072] The culture process of Group C is as follows:

[0073] 1) First, add 750 μL of maturation solution to each well of a four-well plate, do not cover the surface of the maturation solution with mineral oil, and equilibrate in an incubator at 38.5 °C, 5% CO2, and saturated humidity for 2 h;

[0074] 2) Collect cumulus-oocyte complexes (COCs) from follicles with a diameter of 2-8 mm on the bovine ovary, place the COCs in the treated maturation solution, and culture in an incubator at 38.5 °C, 5% CO2, and saturated humidity for 22 h;

[0075] 3) After thawing bovine frozen straw semen in a 37 °C water bath, 3.5 mL of washing semen was added, and centrifugation was performed at 1800 rpm for 5 min twice; the sperm precipitate was adjusted to a sperm density of 5×10 6 cells / mL with fertilization medium as semen;

[0076] 4) Add 400 μL of fertilization medium to each well in a four-well plate, equilibrate for 2 h in an incubator at 38.5 °C, 5% CO2 and saturated humidity, aspirate 60 μL of semen and add it to 400 μL of semen containing 50 bovine oocytes, and fertilize for 16 h in an incubator at 38.5 °C, 5% CO2 and saturated humidity;

[0077] 5) After in vitro fertilization of the cumulus-oocyte complex, digest with hyaluronidase at a concentration of 1 g / L for 1 min to remove granulosa cells;

[0078] 6) In the IVC (in vitro culture) stage, add 500 μL of IVC medium to a four-well plate, cover with mineral oil, and equilibrate for 2 h in an incubator at 38.5 °C, 5% CO2 and saturated humidity.

[0079] 7) Transfer the fertilized eggs to a large drop of embryo culture medium for culture, change half of the medium every 48 h, and culture the fertilized eggs in vitro to the blastocyst stage.

[0080] Table 2 Effects of fertilization systems on embryo development

[0081]

[0082] The data in Table 2 above show that further at the fertilization stage, fertilization using a large drop in a well plate (generally 300 - 500 μL, specifically 400 μL in this example) can improve the blastocyst rate compared to a small drop (50 - 100 μL). This phenomenon may be attributed to the fact that the large droplet can reduce the in vitro operation time, thereby shortening the time it stays outside the incubator and reducing the adverse effects of light; at the same time, the large droplet provides a larger operation space, which helps to reduce the damage to sperm and eggs during the operation; providing more nutrient supply is beneficial to the early development of fertilized eggs; lower sperm and egg densities help to avoid over-activation and density-dependent problems.

[0083] The culture process of group D is as follows:

[0084] 1) First, add 750 μL of maturation medium to each well in a four-well plate, do not cover the surface of the maturation medium with mineral oil, and equilibrate for 2 h in an incubator at 38.5 °C, 5% CO2 and saturated humidity;

[0085] 2) Collect cumulus-oocyte complexes (COCs) from follicles with a diameter of 6 mm on bovine ovaries, place the COCs in the treated maturation medium, and culture them in an incubator at 38.5 °C, 5% CO2 and saturated humidity for 22 h;

[0086] 3) After thawing bovine frozen straw semen in a 37 °C water bath, add 3.5 mL of washing semen, centrifuge at 1800 rpm for 5 min, and centrifuge twice; adjust the sperm density of the sperm precipitate to 5×10 6 cells / mL with fertilization medium as semen;

[0087] 4) Add 400 μL of fertilization medium to each well of a four-well plate, equilibrate it in an incubator at 38.5 °C, 5% CO2 and saturated humidity for 2 h, aspirate 60 μL of semen and add it to 400 μL of semen containing 50 bovine oocytes, and fertilize it in an incubator at 38.5 °C, 5% CO2 and saturated humidity for 16 h;

[0088] 5) After in vitro fertilization of cumulus-oocyte complexes, digest with hyaluronidase at a concentration of 1 g / L for 1 min to remove granulosa cells;

[0089] 6) In the IVC (in vitro culture) stage, first add 5 μL of IVC culture medium to each well of a four-well plate with a 10 μL pipette to make small drops with a small area, then add 1 mL of mineral oil to each well, and then add IVC culture medium. The whole IVC culture drop is a 100 μL three-dimensional columnar culture medium drop. The purpose is that the close contact between embryos may produce beneficial paracrine effects, thereby increasing the number and quality of blastocysts;

[0090] 7) Transfer the fertilized eggs into a 100 μL culture drop of bovine in vitro fertilized embryo culture medium for culture, change half of the medium every 48 h, and culture the fertilized eggs in vitro until the blastocyst stage.

[0091] Table 3 Effects of different drop culture modes in the IVC stage on embryo development

[0092]

[0093] The data in Table 3 above show that on the basis of the results in Tables 1 and 2, further adopting a three-dimensional columnar culture system can increase the blastocyst formation rate, which can reach 33.5%. This phenomenon may be attributed to the environment closer to natural physiological conditions provided by the three-dimensional columnar drop culture technology, which makes the contact between embryos closer and increases the paracrine between embryos; it creates more favorable conditions for the early development of embryos, thereby promoting the development and quality improvement of embryos.

[0094] The experimental results show thatFigure 2 The embryos cultured by three-dimensional columnar drops in Group D shown have significantly higher quality and quantity of blastocysts on the seventh day than Figure 3 the conventional planar large-drop culture in Group A shown.

[0095] It should be clear that each embodiment in this specification is described in a progressive manner. For the same or similar parts between each embodiment, reference can be made to each other. The key point of each embodiment is to illustrate the differences from other embodiments. The present invention is not limited to the specific steps and structures described above and shown in the figures. And, for the sake of brevity, the detailed description of known method technologies is omitted here.

[0096] The above are only the embodiments of the present application and do not limit the present application. For those skilled in the art, various changes and modifications can be made to the present application without departing from the scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included within the scope of the claims of the present application.

Claims

1. An embryo culture method for improving the in vitro blastocyst rate of cattle, characterized in that, The method includes the following steps: S100: Add a set volume of maturation medium into each well of a four-well plate, and after equilibration in an incubator for a first set time, take it out for standby; S200: Place the cumulus-oocyte complexes collected from bovine ovaries into the maturation medium processed in step S100, and add them into each well of the four-well plate respectively, then culture in an incubator for a second set time; S300: Add a set volume of fertilization medium into each well of the four-well plate in step S200 to obtain a fertilization medium containing cumulus-oocyte complexes, then add the processed bovine semen into the fertilization medium containing cumulus-oocyte complexes, and perform fertilization in an incubator for a third set time to obtain fertilized eggs; S400: Add the first part of in vitro culture medium into each well of the four-well plate to make droplets, then add mineral oil into each well, and then add the second part of in vitro culture medium to form a three-dimensional columnar culture medium droplet; the volume ratio of the first part of in vitro culture medium to the second part of in vitro culture medium is 1:(10 - 20); S500: Transfer the fertilized eggs obtained by processing in step S300 into the three-dimensional columnar culture medium droplet for culture, and change half of the medium at intervals of a fourth set time until the fertilized eggs are cultured in vitro to the blastocyst stage.

2. The embryo culture method for improving the in vitro blastocyst rate of cattle according to claim 1, characterized in that, The step of adding a set volume of maturation medium into each well of the four-well plate and after equilibration in an incubator for a first set time, taking it out for standby, specifically is: Add 500 - 750 μL of maturation medium into each well of the four-well plate, and do not drop mineral oil on the surface of the maturation medium, and equilibrate in an incubator at 38.5 °C, 5% CO2 and saturated humidity for at least 2 h.

3. The embryo culture method for improving the in vitro blastocyst rate of cattle according to claim 1, characterized in that, The step of placing the cumulus-oocyte complexes collected from bovine ovaries into the maturation medium processed in step S100, adding them into each well of the four-well plate respectively, and then culturing in an incubator for a second set time, specifically is: Collect the cumulus-oocyte complexes in follicles with a diameter of 2 - 8 mm on the bovine ovaries, place the cumulus-oocyte complexes into the maturation medium processed in step S100, add them into each well of the four-well plate respectively, and then culture in the incubator at 38.5 °C, 5% CO2 and saturated humidity for 22 - 24 h.

4. The embryo culture method for improving the in vitro blastocyst rate of cattle according to claim 1, characterized in that, In step S300, the preparation of the semen includes: thawing bovine frozen straw semen in a water bath and adding washing semen, and obtaining semen after centrifugal precipitation; and / or, The processing conditions for performing fertilization in the incubator for a third set time are: fertilize in the incubator at 38.5 °C, 5% CO2 and saturated humidity for 8 - 18 h.

5. The embryo culture method for improving the in vitro blastocyst rate of cattle according to claim 4, characterized in that, After thawing bovine frozen straw semen in a water bath, washing semen was added. After centrifugation and precipitation, the sperm precipitate was adjusted to a sperm density with fertilization fluid to obtain semen; among them, the sperm density was 2×10 6 ~5×10 6 cells / mL.

6. The embryo culture method for improving the in vitro blastocyst rate of cattle according to claim 1, characterized in that, After the cumulus-oocyte complexes are subjected to fertilization treatment, digest them with hyaluronidase to remove cumulus granulosa cells to obtain fertilized eggs.

7. The embryo culture method for improving the in vitro blastocyst rate of cattle according to claim 6, characterized in that, The concentration of the hyaluronidase is 1 g / L.

8. The embryo culture method for improving the in vitro blastocyst rate of cattle according to claim 1, characterized in that, Add 300 - 500 μL of fertilization medium into each well of the four-well plate in step S200.

9. The embryo culture method for improving the in vitro blastocyst rate of cattle according to claim 1, characterized in that The fourth set time is 48 h.

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