Embryo culture method for improving bovine in-vitro blastocyst rate

Abstract

The invention relates to the technical field of embryo engineering, in particular to an embryo culture method for improving the bovine in-vitro blastocyst rate, which comprises the following steps: adding a maturation solution with a set volume into each hole in a four-hole plate; putting the collected cumulus-oocyte complex on the bovine ovary into a maturation solution, and culturing the cumulus-oocyte complex; adding the treated bovine semen into a fertilization liquid containing a cumulus-oocyte complex, and carrying out fertilization in an incubator; three-dimensional cylindrical culture liquid drops are manufactured in the culture holes, so that the culture purpose of tight contact between embryos is achieved, and paracrine between the embryos is increased; and transferring the fertilized ovum into a three-dimensional cylindrical culture liquid drop for culturing. The embryo culture method for improving the bovine in-vitro blastocyst rate aims at solving the problem that the bovine embryo in-vitro culture blastocyst rate is low.

Description

Technical Field

[0001] The invention relates to the technical field of embryo engineering, and in particular to an embryo culture method for improving the rate of bovine in vitro blastocysts. Background Art

[0002] As an important part of agriculture, animal husbandry is of great significance to ensuring national food security and promoting agricultural modernization. The application of embryo technology for genetic improvement of dairy cow breeds and breeding of breeding cattle has become a powerful tool for dairy cow breeding in dairy powerhouses. The low efficiency of in vitro embryo production is the key obstacle to my country's inability to carry out in vitro embryo production on a large scale, which to a certain extent restricts the high-quality development of my country's animal husbandry.

[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 blastocyst rate of in vitro embryos is the biggest obstacle to the application of in vitro embryo technology in my country. Traditional oil-covered maturation, droplet fertilization and flat embryo culture methods may inhibit hormone effects, prolong in vitro treatment time, and cause uneven distribution of embryos in the culture medium, affecting the interaction and signal transmission between embryos, thereby limiting the development of in vitro embryos and reducing the blastocyst rate.

[0004] Therefore, the inventors provide an embryo culture method for improving the bovine in vitro blastocyst rate. Summary of the invention

[0005] (1) Technical issues to be solved

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

[0007] (2) Technical solution

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

[0009] S100, adding a set volume of maturation solution to each well of the four-well plate, and equilibrating the wells in the incubator for a first set time before taking them out for later use;

[0010] S200, placing the collected cumulus-oocyte complex on the bovine ovary in the maturation solution treated in step S100, and adding the complex to each well in the four-well plate, and then culturing the complex in an incubator for a second set time;

[0011] S300, adding a set volume of fertilization solution to each well in the four-well plate in step S200 to obtain a fertilization solution containing the cumulus ovum-oocyte complex, then adding the treated bovine semen to the fertilization solution containing the cumulus ovum-oocyte complex, and fertilizing in an incubator for a third set time to obtain a fertilized egg;

[0012] S400, adding the first part of the in vitro culture solution to each well of the four-well plate to make small drops, then adding mineral oil to each well, and then adding the second part of the in vitro culture solution to form three-dimensional columnar culture solution drops;

[0013] S500, transferring the fertilized egg obtained in step S300 into the three-dimensional columnar culture droplet for culture, and replacing the medium by half at a fourth set time interval until the fertilized egg is cultured in vitro to the blastocyst stage.

[0014] Furthermore, a set volume of maturation solution is added to each well of the four-well plate, and the solution is taken out for standby after being balanced in an incubator for a first set time, specifically:

[0015] 500-750 μL of maturation solution was added to each well of the four-well plate without dropping mineral oil on the surface of the maturation solution, and the plate was equilibrated in an incubator at 38.5° C., 5% CO 2 and saturated humidity for at least 2 hours.

[0016] Further, the cumulus-oocyte complex collected from the bovine ovary is placed in the maturation solution treated in step S100, and is added to each well of the four-well plate, and then cultured in an incubator for a second set time, specifically:

[0017] The cumulus-oocyte complexes in the follicles with a diameter of 2 to 8 mm are collected from the bovine ovaries, the cumulus-oocyte complexes are placed in the maturation solution treated in step S100, and are added to each well of a four-well plate respectively, and then cultured in the incubator at 38.5° C., 5% CO2 and saturated humidity for 22 to 24 hours.

[0018] Furthermore, in step S300, the preparation of semen includes: thawing the frozen semen in a water bath, adding semen washing solution, and obtaining semen after centrifugal precipitation; and / or,

[0019] The treatment conditions for fertilization at the third set time in the incubator are: fertilization for 8 to 18 hours in the incubator at 38.5° C., 5% CO 2 and saturated humidity.

[0020] Furthermore, the frozen semen in a capillary tube of cattle was thawed in a water bath, and then a semen washing solution was added, and the semen was centrifuged and precipitated. After the sperm was precipitated, the sperm density was adjusted with fertilization solution and used as semen; wherein the sperm density was 2×106 ~5×10 6 Pieces / mL.

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

[0022] Furthermore, 300 to 500 μL of fertilization solution 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 to 2 minutes.

[0025] Furthermore, 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).

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

[0027] (3) Beneficial effects

[0028] In summary, the present invention uses three-dimensional columnar culture drops during the embryo culture stage to reduce the bottom area of ​​the culture droplets, allowing the embryos to achieve close contact, produce effective paracrine effects, share cytokines and growth factors, and activate certain common signaling pathways, which helps to reduce cell apoptosis in the embryo and help the embryo resist stress and damage, thereby achieving the purpose of improving the number 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 for use in the embodiments of the present invention are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0030] Figure 1 It is a schematic flow chart of an embryo culture method for improving the rate of bovine in vitro blastocysts provided by an embodiment of the present invention;

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

[0032] Figure 3 This is a SEM image of the morphology of the blastocyst on the seventh day of culture using the traditional bovine in vitro blastocyst embryo culture method. DETAILED DESCRIPTION

[0033] The following detailed description of the embodiments of the present invention is provided in conjunction with the accompanying drawings and examples. The following detailed description of the embodiments and the accompanying drawings are used to 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, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

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

[0036] Figure 1 FIG. 1 is a flow chart of a method for culturing an embryo to improve the rate of in vitro blastocysts in cattle provided by an embodiment of the present invention. Figure 1 As shown, the method may include the following steps:

[0037] S100, adding a set volume of maturation solution to each well of the four-well plate, and taking it out for use after equilibration in the incubator for a first set time.

[0038] Specifically, a set volume of maturation solution is added to each well of the four-well plate and equilibrated in an incubator at 38.5°C, 5% CO2 and saturated humidity for at least 2 hours. There is no limitation on the specific selection of maturation solution, which can be purchased from commercial products. Specifically, the maturation solution can be BO-IVM oocyte maturation solution from Huayuexing. Furthermore, mineral oil is not dripped onto the surface of the maturation solution to reduce the effect of mineral oil on reproductive hormones such as estrogen.

[0039] S200, placing the collected cumulus-oocyte complex on the bovine ovary in the maturation solution treated in step S100, and adding them to each well of the four-well plate respectively, and then culturing them in an incubator for a second set time.

[0040] Specifically, cumulus-oocyte complexes are collected from follicles with a diameter of 2 to 8 mm on bovine ovaries, placed in in vitro maturation fluid, and cultured in an incubator at 38.5° C., 5% CO 2 and saturated humidity for 22 to 24 hours.

[0041] S300, adding a set volume of fertilization solution to each well of the four-well plate in step S200 to obtain fertilization solution containing cumulus ovary-oocyte complex, then adding the treated bovine semen to the fertilization solution containing cumulus ovary-oocyte complex, and performing fertilization in an incubator for a third set time to obtain a fertilized egg.

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

[0043] S301, thawing the frozen semen in a water bath, adding a semen washing solution, and centrifuging and precipitating the semen several times as semen;

[0044] S302, a set volume of semen is drawn and added into the fertilization solution containing the cumulus-oocyte complex, and fertilization is carried out in an incubator at 38.5°C, 5% CO2 and saturated humidity for 8 to 18 hours.

[0045] Among them, in step S301, after the sperm is precipitated, the sperm density is adjusted with fertilization fluid to form semen.

[0046] In step S302, after in vitro fertilization, cumulus-oocyte complex is digested with hyaluronidase to remove cumulus granulosa cells. Wherein, 300~500 μL (i.e., large drops) of fertilization solution are added in each hole in the four-well plate of step S200, fertilization process is convenient, and the residence time of oocyte and sperm outside incubator is shortened. The concentration of hyaluronidase is 1g / L, and specifically 0.1g hyaluronidase and 100ml TCM199 solvent can be used for mixed preparation, and digestion time is 1~2min.

[0047] S400, adding the first part of the in vitro culture solution to each well of the four-well plate to make small droplets, then adding mineral oil to each well, and then adding the second part of the in vitro culture solution to form three-dimensional columnar culture solution droplets.

[0048] Specifically, by controlling the amount of culture solution dripped in, a small drop of 5 to 10 μL of culture solution is first dripped in with a white gun tip, and mineral oil is added to achieve the purpose of fixing the bottom area and preventing the evaporation of the culture solution. Furthermore, the volume ratio of the in vitro culture solution of the first part and the in vitro culture solution of the second part is 1: (10 to 20). It should be noted here that the components of the in vitro culture solution of the first part and the in vitro culture solution of the second part are exactly the same, except for the different amounts added. The entire IVC culture drop is a 100 μL three-dimensional columnar droplet, which is designed to be a three-dimensional columnar droplet to ensure close contact between embryos to produce beneficial paracrine effects, thereby improving the number and quality of blastocysts. It should be noted here that the specific volume of the in vitro culture solution of the first and second parts is not limited, and can be dripped according to actual conditions. There is no limitation on the specific selection of the in vitro culture solution, which can be purchased through products on the market. The mature solution can specifically use the BO series culture solution of Huayuexing.

[0049] It should be noted here that since mineral oil has been added, the second part of in vitro culture medium must be added to the original droplet, otherwise a new small droplet will be formed that cannot merge with the original droplet; secondly, attention should be paid to stability during the plate transfer process, because three-dimensional columnar droplets are formed, and too large a movement can easily cause the three-dimensional columnar droplets to disperse and form small round droplets.

[0050] S500, transferring the fertilized eggs obtained in step S300 into a three-dimensional columnar culture droplet for culture, and replacing the medium by half at a fourth set time interval until the fertilized eggs are cultured in vitro to the blastocyst stage. Specifically, the number of oocytes added to the three-dimensional columnar culture droplet is generally 15 to 20, and the fourth set time is 48 hours.

[0051] Example 1

[0052] The specific process of embryo culture was divided into four groups: A, B, C, and D. Group A adopted traditional flat large drop culture; Group B was based on Group A but did not cover the oocyte with mineral oil during the oocyte maturation stage; Group C was based on Group B but used large drops of fertilization solution during the in vitro fertilization stage; Group D was based on Group C but used three-dimensional columnar culture drops during the embryo culture stage.

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

[0054] 1) First, add 750 μL of maturation solution to each well of a four-well plate, 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;

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

[0056] 3) After the frozen bovine semen was thawed in a 37°C water bath, 3.5 mL of semen washing solution was added and centrifuged at 1800 rpm for 5 min, twice; the sperm sediment was adjusted to a sperm density of 5×10 6 pcs / mL, as semen;

[0057] 4) In a 35 mm small dish, make 50 μL drops (7 drops per dish), cover with mineral oil and equilibrate in an incubator at 38.5°C, 5% CO2 and saturated humidity for 2 hours, draw 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 hours;

[0058] 5) After in vitro fertilization, the cumulus-oocyte complex was digested with hyaluronidase at a concentration of 1 g / L for 1 min to remove granulosa cells;

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

[0060] 7) The fertilized eggs were transferred into large drops of embryo culture medium for culture, and half of the medium was replaced every 48 hours. The fertilized eggs were cultured in vitro to the blastocyst stage.

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

[0062] 1) First, add 750 μL of maturation solution to each well of a four-well plate, without covering 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 hours;

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

[0064] 3) After the frozen bovine semen was thawed in a 37°C water bath, 3.5 mL of semen washing solution was added and centrifuged at 1800 rpm for 5 min, twice; the sperm sediment was adjusted to a sperm density of 5×10 6 pcs / mL, as semen;

[0065] 4) In a 35 mm small dish, make 50 μL drops (7 drops per dish), cover with mineral oil and equilibrate in an incubator at 38.5°C, 5% CO2 and saturated humidity for 2 hours, draw 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 hours;

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

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

[0068] 7) The fertilized eggs were transferred into large drops of embryo culture medium for culture, and half of the medium was replaced every 48 hours. The fertilized eggs were cultured in vitro to the blastocyst stage.

[0069] Table 1 Effects of IVM model with or without oil covering on embryonic development

[0070]

[0071] The data in Table 1 above indicate 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 fat-soluble substance, which means that it is easily dissolved in oil substances. Mineral oil, a substance commonly used as a solvent or protective agent, is hydrophobic and therefore compatible with E2. Based on the fat-soluble nature of E2, we can reasonably speculate that E2 may be absorbed by the mineral oil when covering the IVM fluid. This is because fat-soluble substances generally tend to dissolve in oil environments. However, the specific absorption efficiency and degree may be affected by many factors, including the concentration of E2, the properties of the mineral oil, and the specific composition of the IVM fluid.

[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, without covering 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 hours;

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

[0075] 3) After the frozen bovine semen was thawed in a 37°C water bath, 3.5 mL of semen washing solution was added and centrifuged at 1800 rpm for 5 min, twice; the sperm sediment was adjusted to a sperm density of 5×10 6 pcs / mL, as semen;

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

[0077] 5) After in vitro fertilization, the cumulus-oocyte complex was digested 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 500ul of IVC solution to the four-well plate, cover with mineral oil and equilibrate in an incubator at 38.5°C, 5% CO2 and saturated humidity for 2 hours.

[0079] 7) The fertilized eggs were transferred into large drops of embryo culture medium for culture, and half of the medium was replaced every 48 hours. The fertilized eggs were cultured in vitro to the blastocyst stage.

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

[0081]

[0082] The data in Table 2 above show that, further in the fertilization stage, the use of large droplets (generally 300-500 μL, 400 μL is specifically selected in this embodiment) for fertilization can improve the blastocyst rate compared with small droplets (50-100 μL). This phenomenon may be attributed to the fact that large droplets can reduce the in vitro operation time, thereby shortening the time they stay outside the incubator and reducing the adverse effects of light; at the same time, large droplets provide a larger operating space, which helps to reduce 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 density helps to avoid over-activation and density dependence problems.

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

[0084] 1) First, add 750 μL of maturation solution to each well of a four-well plate, without covering 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 hours;

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

[0086] 3) After the frozen bovine semen was thawed in a 37°C water bath, 3.5 mL of semen washing solution was added and centrifuged at 1800 rpm for 5 min, twice; the sperm sediment was adjusted to a sperm density of 5×10 6 pcs / mL, as semen;

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

[0088] 5) After in vitro fertilization, the cumulus-oocyte complex was digested 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 use a 10μL pipette to add 5μL IVC culture medium to each well of a four-well plate to make a small droplet, then add 1mL of mineral oil to each well, and then add IVC culture medium. The entire IVC culture droplet is a 100μL three-dimensional columnar culture droplet. The purpose is that close contact between embryos may produce beneficial paracrine effects, thereby increasing the number and quality of blastocysts;

[0090] 7) The fertilized eggs were transferred into 100 μL of bovine in vitro fertilization embryo culture medium and cultured. Half of the medium was replaced every 48 hours. The fertilized eggs were cultured in vitro to the blastocyst stage.

[0091] Table 3 Effects of different droplet culture modes on embryonic development at the IVC stage

[0092]

[0093] The data in Table 3 above show that, based on the results in Tables 1 and 2, the blastocyst formation rate can be increased to 33.5% by further adopting a three-dimensional columnar culture system. This phenomenon may be attributed to the environment provided by the three-dimensional columnar droplet culture technology that is closer to natural physiological conditions, making the contact between embryos closer and increasing the paracrine secretion between embryos; creating more favorable conditions for the early development of embryos, thereby promoting the development and quality of embryos.

[0094] The experimental results show that Figure 2 The quality and quantity of embryos cultured in the three-dimensional column drop in group D were significantly higher than those in group B on day 7. Figure 3 Group A shown is a conventional planar macrodroplet culture.

[0095] It should be clear that the various embodiments in this specification are described in a progressive manner, and the same or similar parts between the various embodiments can be referred to each other, and each embodiment focuses on the differences from other embodiments. The present invention is not limited to the specific steps and structures described above and shown in the figures. In addition, for the sake of brevity, a detailed description of known methods and technologies is omitted here.

[0096] The above are only embodiments of the present application and are not limited to the present application. For those skilled in the art, the present application may have various changes and variations without departing from the scope of the present invention. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A method for culturing embryos for improving the rate of bovine in vitro blastocysts, characterized in that: The method comprises the following steps: S100, adding a set volume of maturation solution to each well of the four-well plate, and equilibrating the wells in the incubator for a first set time before taking them out for later use; S200, placing the collected cumulus-oocyte complex on the bovine ovary in the maturation solution treated in step S100, and adding the complex to each well in the four-well plate, and then culturing it in an incubator for a second set time; S300, adding a set volume of fertilization solution to each well in the four-well plate in step S200 to obtain a fertilization solution containing the cumulus ovum-oocyte complex, then adding the treated bovine semen to the fertilization solution containing the cumulus ovum-oocyte complex, and fertilizing in an incubator for a third set time to obtain a fertilized egg; S400, adding the first part of the in vitro culture solution to each well of the four-well plate to make small drops, then adding mineral oil to each well, and then adding the second part of the in vitro culture solution to form three-dimensional columnar culture solution drops; S500, transferring the fertilized egg obtained in step S300 into the three-dimensional columnar culture droplet for culture, and replacing the medium by half at a fourth set time interval until the fertilized egg is cultured in vitro to the blastocyst stage.

2. The method for culturing embryos for improving the rate of bovine in vitro blastocysts according to claim 1, characterized in that: The method of adding a set volume of maturation solution to each well of the four-well plate and taking it out for standby after balancing it in the incubator for a first set time is as follows: 500-750 μL of maturation solution was added to each well of the four-well plate without dropping mineral oil on the surface of the maturation solution, and the plate was equilibrated in an incubator at 38.5° C., 5% CO 2 and saturated humidity for at least 2 hours.

3. The method for culturing embryos for improving the rate of bovine in vitro blastocysts according to claim 1, characterized in that: The cumulus-oocyte complex collected from the bovine ovary is placed in the maturation solution treated in step S100, and is added to each well of the four-well plate, and then cultured in an incubator for a second set time, specifically: The cumulus-oocyte complexes in the follicles with a diameter of 2 to 8 mm are collected from the bovine ovaries, the cumulus-oocyte complexes are placed in the maturation solution treated in step S100, and are added to each well of a four-well plate respectively, and then cultured in the incubator at 38.5° C., 5% CO2 and saturated humidity for 22 to 24 hours.

4. The method for culturing embryos for improving the rate of bovine in vitro blastocysts according to claim 1, characterized in that: In step S300, the preparation of semen includes: thawing the frozen semen in a water bath, adding semen washing solution, and centrifuging to obtain semen; and / or, The treatment conditions for fertilization at the third set time in the incubator are: fertilization for 8 to 18 hours in the incubator at 38.5° C., 5% CO 2 and saturated humidity.

5. The method for culturing embryos for improving the rate of bovine in vitro blastocysts according to claim 4, characterized in that: After the frozen semen in a capillary tube of cattle was thawed in a water bath, the semen washing solution was added, and the sperm was centrifuged and precipitated. After the sperm was precipitated, the sperm density was adjusted with fertilization solution and used as semen; among them, the sperm density was 2×10 6 ~5×10 6 Pieces / mL.

6. The method for culturing embryos for improving the rate of bovine in vitro blastocysts according to claim 1, characterized in that: After the cumulus-oocyte complex is fertilized, it is digested with hyaluronidase to remove the cumulus granulosa cells to obtain a fertilized egg.

7. The method for culturing embryos for improving the rate of bovine in vitro blastocysts according to claim 6, characterized in that: The concentration of the hyaluronidase is 1 g / L.

8. The method for culturing embryos for improving the rate of bovine in vitro blastocysts according to claim 1, characterized in that: 300-500 μL of fertilization solution is added to each well of the four-well plate in step S200.

9. The method for culturing embryos for improving the rate of bovine in vitro blastocysts according to claim 1, characterized in that: 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).

10. The method for culturing embryos for improving the rate of bovine in vitro blastocysts according to claim 1, characterized in that: The fourth set time is 48 hours.

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