Composition, method and application for increasing the number of cells in the inner cell mass of in vitro embryos

Abstract

The present invention discloses a composition, method and application for increasing the number of cells and the proportion of cells in the inner cell mass of in vitro embryos. The present invention aims at the problems of low blastocyst rate, small number of cells in the inner cell mass at the blastocyst stage, low proportion of cells in the inner cell mass, low potential for subsequent embryonic development, etc. in the early development process of in vitro fertilized embryos. By adding one or two regulators of folic acid and sedum B to the culture medium of early embryo in vitro culture, the blastocyst rate, the number of cells in the inner cell mass of the blastocyst and the proportion of the number of cells in the inner cell mass, as well as the survival rate and blastocyst diameter of the delayed culture of the blastocyst are significantly improved, and the potential for in vitro embryonic development is effectively improved. In addition, the present invention also effectively improves the ratio of embryos that can be subsequently transplanted by in vitro fertilized embryos, is low in cost, safe and efficient, and provides a new strategy for the development of in vitro embryo production and assisted reproductive technology for livestock, with broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro fertilization, and in particular relates to a composition, method and application for increasing the number of cells and the cell ratio of inner cell mass of in vitro embryos. Background Art

[0002] In vitro fertilization has been widely used to improve the rapid expansion of high-quality breeding stock and in the field of human assisted reproduction. However, the pregnancy rate after embryo transplantation obtained by in vitro fertilization is much lower than that of embryos fertilized in vivo, indicating that the developmental potential of in vitro embryos is lower than that of in vivo embryos. The developmental potential of in vitro embryos is the key to determining the success of in vitro embryo transplantation, and it has always been the difficulty of in vitro embryo technology. The embryonic developmental potential is closely related to the first cell differentiation (i.e., differentiation into inner cell mass (ICM) and trophoblast (TE)). Since the inner cell mass and trophoblast cells will develop into the fetus and placenta respectively, the number and proportion of inner cell mass cells are important indicators for evaluating the developmental potential of blastocysts. It has been reported that the in vitro culture environment affects the number and proportion of inner cell mass cells. The ICM / TE ratio of blastocysts after in vitro fertilization of mice and cattle is 14%-23% lower than that of blastocysts in vivo, which is also an important factor determining the low developmental potential of in vitro embryos.

[0003] In recent years, researchers have established a delayed in vitro culture system for livestock blastocysts to evaluate the subsequent developmental potential of in vitro fertilized embryos. Compared with the method of using embryo transfer to determine the developmental potential of embryos, the delayed in vitro culture system for blastocysts greatly saves time and financial costs; in addition, the delayed in vitro culture system for blastocysts can also be used as a model to study the developmental mechanism of embryos before and after implantation. This system can quickly and high-throughput evaluate the developmental potential of in vitro fertilized embryos. Studies have shown that the number and proportion of cells in the inner cell mass of the blastocyst are closely related to the survival rate of the delayed in vitro culture of the blastocyst and the diameter of the blastocyst.

[0004] In order to increase the number and proportion of inner cell masses of in vitro embryos, the existing technology mainly uses the addition of secretory factors of pre-implantation embryos or fallopian tubes to regulate the development of in vitro embryos. For example, BMP, which belongs to the TGF-β superfamily, can promote blastocyst formation. BMP signals promote ICM proliferation and inhibit TE differentiation by regulating the expression of pluripotency and lineage-specific genes; EGF is a growth factor that promotes cell proliferation and differentiation. The addition of EGF during in vitro embryo culture can increase the blastocyst rate. Although in recent years, people have made certain progress in adding secretory factors to improve in vitro embryo development, the effect of increasing the number and proportion of cells in the inner cell mass of in vitro embryos is limited. Therefore, it is urgent to establish a more effective method to increase the number and proportion of cells in the inner cell mass of in vitro embryos in order to further improve the level of in vitro embryo technology. Summary of the invention

[0005] In order to solve at least some of the technical problems in the above-mentioned prior art, the present invention significantly improves the blastocyst rate, the number of cells in the inner cell mass of the blastocyst and the proportion of the number of cells in the inner cell mass, the survival rate and the diameter of the blastocyst in delayed culture, and effectively improves the in vitro embryo development potential by adding one or two regulators of folic acid and sedum B to the culture medium of early embryos cultured in vitro. Specifically, the present invention includes the following contents.

[0006] In a first aspect, the present invention provides a composition for increasing the number and cell ratio of inner cell mass of embryos in vitro, comprising one or two regulators selected from folic acid and phytoserotonin B.

[0007] In certain embodiments, according to the composition for increasing the number and cell proportion of inner cell mass of an in vitro embryo according to the present invention, the composition comprises any one of (a)-(c):

[0008] (a) Folic acid;

[0009] (b) phytosinolide B;

[0010] (c) folic acid and phytosporin B, wherein the concentration ratio of folic acid to phytosporin B is (0.001-100):1.

[0011] In certain embodiments, according to the composition for increasing the number and cell ratio of inner cell mass of embryos in vitro, the amount of folic acid is such that the working concentration of folic acid during in vitro culture reaches 0.01-10 μM; the amount of phytosphingomyelin B is such that the working concentration of phytosphingomyelin B during in vitro culture reaches 0.1-10 μM.

[0012] In certain embodiments, according to the composition for increasing the number and proportion of cells in the inner cell mass of an in vitro embryo according to the present invention, the in vitro embryo is derived from a mammalian in vitro fertilized embryo.

[0013] In certain embodiments, the composition for increasing the number and proportion of cells in the inner cell mass of an in vitro embryo according to the present invention further comprises a basal culture medium.

[0014] The second aspect of the present invention provides a method for increasing the number and proportion of cells in the inner cell mass of an in vitro embryo, comprising the step of contacting the embryo with the composition according to the present invention in vitro.

[0015] In certain embodiments, according to the method for increasing the number and proportion of inner cell mass of an in vitro embryo according to the present invention, the increase comprises increasing the number of inner cell mass cells and the proportion of inner cell mass cells in the blastocyst.

[0016] The third aspect of the present invention provides a method for improving the development of in vitro fertilized embryos, comprising the step of contacting the embryos with the composition according to the present invention in vitro.

[0017] In certain embodiments, according to the method for improving in vitro fertilization embryo development of the present invention, the improvement includes increasing the cleavage rate and increasing the blastocyst development rate.

[0018] In certain embodiments, according to the method for improving in vitro fertilization embryo development of the present invention, the improvement further comprises increasing the survival rate and diameter of the blastocyst during delayed in vitro culture of the blastocyst.

[0019] In a fourth aspect, the present invention provides a composition according to the present invention, or a method according to the present invention for increasing the number and cell ratio of inner cell mass of an in vitro embryo, or a method according to the present invention for improving the development of an in vitro fertilized embryo, for use in animal genetic improvement, breeding of improved species, in vitro embryo production or assisted reproduction.

[0020] The present invention aims at the problems of few inner cell mass cells, low blastocyst rate, and low potential for subsequent embryonic development in the early development process of in vitro fertilized embryos. By adding one or two regulators of folic acid and sedum B to the culture medium of early embryo in vitro culture, the blastocyst rate, the number of cells in the inner cell mass of the blastocyst and the proportion of the number of cells in the inner cell mass, the survival rate and the diameter of the blastocyst in delayed culture are significantly improved, and the potential for in vitro embryonic development is effectively improved. In addition, the present invention effectively improves the ratio of in vitro fertilized embryos that can be used for subsequent embryo transplantation, and is low in cost, safe, and efficient, providing a new strategy for the development of in vitro embryo production and assisted reproductive technology for livestock, and has broad application prospects. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0024] Herein, the term "embryo" refers to an early stage larvae developed from a fertilized egg. Since development is a continuous process, the embryo herein includes larvae of different forms generated in any developmental process or stage, generally referring to pre-implantation embryos or pre-implantation embryos, such as 2-cell embryos, 4-cell embryos, 8-cell embryos, morulae, blastocysts, etc.

[0025] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a petri dish, etc., rather than in an organism (eg, an animal, plant, or microorganism).

[0026] In this article, the term "folic acid" or "vitamin B9", abbreviated as "FA", is a water-soluble vitamin. Folic acid is essential for early embryonic development and can act as a methyl donor to participate in the one-carbon cycle; it can also directly participate in the transcriptional regulation of signaling pathways through receptor pathways by stimulating folate receptors.

[0027] Herein, the term "spatholobus B" refers to 20 H 24 O5 is a Chinese herbal compound with a CAS number of 165337-71-3 and a structural formula as shown below:

[0028] .

[0029] Herein, the term "working concentration" is also referred to as "use concentration", which refers to the initial concentration when the regulator is brought into contact with the embryo and effectively treated. The concentration unit is not limited, and can be, for example, μM, ng / ml, μg / ml, mg / ml, etc.

[0030] Composition

[0031] The first aspect of the present invention provides a composition for increasing the number of cells and the proportion of cells in the inner cell mass of an in vitro embryo, which comprises a regulator selected from one or both of folic acid and phytosporin B. In the present invention, folic acid and phytosporin B interact with each other to produce a synergistic effect, and the combined use of the two is more effective than adding them separately. The form of the composition of the present invention is not particularly limited, and can be a solid such as a dry powder, or a liquid such as a solution.

[0032] In certain embodiments, the regulator in the composition of the present invention may be a single regulator or a combination of two regulators, i.e., it does not contain any components other than the regulators and unavoidable impurities. The regulators in the composition of the present invention may exist in a mixed form or in the form of two regulators existing alone. When used, the regulators existing alone may be pre-mixed and then used, or each regulator may be used alone simultaneously or separately in sequence.

[0033] In certain embodiments, the composition of the present invention comprises the above-mentioned combination of regulators, and further comprises other ingredients in addition. The composition or type of other ingredients is not limited and can be freely selected as needed. Such other ingredients can select any known ingredients, particularly reagents and compositions related to embryo culture, such as culture medium, culture fluid or its additives, etc.

[0034] In certain embodiments, the composition of the present invention is a combination of folic acid and phytosporin B. Preferably, the concentration ratio of folic acid to phytosporin B is (0.001-100):1, for example: 0.001:1, 0.005:1, 0.01:1, 0.05:1, 0.1:1, 0.5:1, 1:1, 2:1, 4:1, 8:1, 16:1, 32:1, 64:1, 100:1, etc. Within the above range, effective synergy between folic acid and phytosporin B can be achieved, greatly improving the effect.

[0035] In certain embodiments, the amount of folic acid of the present invention can make the working concentration of folic acid reach 0.01-10 μM when used. As long as the folic acid can reach the above range when used, the concentration or amount of folic acid in the composition is not limited. When the composition is a solution and used as an added component, the concentration of folic acid in the composition can be higher than the working concentration, so that when the regulator combination is added to the embryo culture medium, it can be diluted to reach the above working concentration. When the composition is a solution and used directly as a culture medium, the concentration of folic acid is generally equivalent to the working concentration. The working concentration of folic acid is preferably 0.1-5 μM, for example 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 μM.

[0036] In certain embodiments, the amount of phytosphingomyelin B of the present invention can make the working concentration of phytosphingomyelin B reach 0.1-10 μM during in vitro culture. As long as phytosphingomyelin B can reach the above range when used, the concentration or amount of phytosphingomyelin B in the composition is not limited. When the composition is a solution and used as an added ingredient, the concentration of phytosphingomyelin B in the composition can be higher than the working concentration, so that when the composition is added to the embryo culture medium, it can be diluted to reach the above working concentration. When the composition is a solution and is directly used as a culture medium, the concentration of phytosphingomyelin B is usually substantially equivalent to the working concentration. The working concentration of phytosphingomyelin B is preferably 0.2-5 μM, for example 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 μM.

[0037] In certain embodiments, the in vitro embryo includes a mammalian in vitro fertilized embryo. The mammal includes but is not limited to mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, pigs, etc. In certain embodiments, the animal includes a transgenic animal, a genetically engineered animal or a clone, etc.

[0038] Method for increasing the number and proportion of cells in the inner cell mass of embryos in vitro

[0039] The second aspect of the present invention provides a method for increasing the number and cell ratio of inner cell mass of an in vitro embryo, comprising the step of contacting the embryo with the composition according to the first aspect of the present invention in vitro.

[0040] In the present invention, the increase includes increasing the number of cells and the proportion of cells in the inner cell mass of the blastocyst.

[0041] Methods to improve embryo development in vitro fertilization

[0042] The third aspect of the present invention provides a method for improving the development of in vitro fertilized embryos, comprising the step of contacting the embryos with the composition according to the first aspect of the present invention in vitro.

[0043] In the present invention, the improvement includes increasing the cleavage rate and increasing the blastocyst development rate.

[0044] In the present invention, the improvement further includes increasing the survival rate and diameter of the blastocyst during delayed in vitro culture of the blastocyst.

[0045] application

[0046] The fourth aspect of the present invention provides the use of the composition according to the first aspect of the present invention, or the method for increasing the number and cell proportion of inner cell mass of in vitro embryos according to the second aspect of the present invention, or the method for improving the development of in vitro fertilized eggs or in vitro embryos according to the third aspect of the present invention in animal genetic improvement, breeding expansion, in vitro embryo production or assisted reproduction.

[0047] In certain embodiments, the genetic improvement and enrichment of the present invention include genetic improvement or enrichment relying on in vitro embryo production technology.

[0048] Example 1

[0049] This example shows the use of combined administration of folic acid and phytosinolide B in increasing the number and cell proportion of inner cell mass of bovine embryos in vitro.

[0050] 1. Experimental Design and Methods

[0051] (1) Experimental groups

[0052] Control group: conventional in vitro development group;

[0053] Treatment group 1: folic acid (2 μM) alone administration group;

[0054] Treatment group 2: phytosinolide B (1 μM) alone administration group;

[0055] Treatment group 3: the group to which folic acid (1 μM) and phytosinolide B (0.5 μM) were administered together.

[0056] The control group was a basic in vitro development fluid, and the treatment groups 1-3 were basic in vitro development fluid with different regulators added or a combination of the two.

[0057] (2) Bovine oocyte in vitro maturation and embryo development

[0058] a) Oocyte collection and in vitro oocyte maturation

[0059] Bovine ovaries were obtained from the slaughterhouse, and follicular fluid was extracted. Cumulus-oocyte complexes were sorted out under a stereoscope and washed three times in maturation medium. Finally, 50 cumulus-oocyte complexes were incubated in 500 μL bovine in vitro maturation medium and cultured in vitro for 21 h in a 6% CO2 incubator at 38.5℃ and saturated humidity.

[0060] b) In vitro fertilization

[0061] After maturation, gently blow to remove large pieces of granulosa cells, and then transfer the mature cumulus oocyte complex into the balanced fertilization solution. Thaw the frozen bovine semen, wash the sperm twice with sperm washing solution, and resuspend it in fertilization solution. Count the sperm, then take 75 μL of the resuspended sperm and add it to the fertilization solution that has been placed in the oocyte. The final sperm concentration is 1×10 6 Sperm / mL, in vitro fertilization 18 h.

[0062] c) In vitro culture

[0063] All granulosa cells and cumulus cells outside the fertilized eggs were removed, and the clean fertilized eggs were washed three times in in vitro embryo development fluid and cultured in a four-well plate: about 50 fertilized eggs were placed in each well, and 500 μL in vitro embryo development fluid was added. The culture conditions were 38.5℃, 5% CO2, 90% N2, and saturated humidity for 7-8 days. During this period, the cleavage rate and blastocyst development rate of the embryos were counted (embryo development day 8), and the blastocysts were collected and fixed, immunofluorescence staining, and cell counting was performed.

[0064] (3) Delayed in vitro culture of embryos

[0065] Three days in advance, incubate the four-well plate with ultrapure low melting point agarose and change the culture medium every day. The culture medium is prepared by Neurobasal: DMEM / F12 = 1:1, and glutamine substitute, non-essential amino acids, N2 additive, B27 additive, etc. are added. The blastocysts on the eighth day are transferred to the four-well plate with agarose for further culture. The culture conditions are 38.5℃, 5% CO2, and saturated humidity.

[0066] (4) Count the number of inner cell mass cells and the proportion of inner cell mass cells

[0067] The blastocysts were collected, washed three times with 0.1% PVP-PBS, fixed in 4% paraformaldehyde for 30 minutes, treated with 0.5% TritonX-100 (PBS) at room temperature for 30 minutes, washed three times with 0.1% PVP-PBS, blocked with 0.1% TritonX-100 + 0.1% BSA (PBS) for 30 minutes, washed three times with 0.1% PVP-PBS, and incubated with primary antibody at 4°C overnight. Washed three times with 0.1% PVP-PBS, incubated with secondary antibody at room temperature in the dark, and washed three times with 0.1% PVP-PBS. The nuclei were stained with DAPI and sealed after incubation in the dark for 5-10 minutes.

[0068] 2. Experimental Results

[0069] Experimental results The statistics of in vitro embryo development rate are shown in Table 1, and the number of cells in the inner cell mass is shown in Table 2. The data in Table 1 show that compared with the cleavage rate of the control group (70.2±3.2%), the cleavage rates of the folic acid alone group (80.6±2.5%), the sedative B alone group (75.6±2.6%), and the folic acid and sedative B combined group (90.2±1.9%) were significantly higher than those of the control group (p<0.05). The cleavage rate of treatment group 3, i.e., the combined addition of folic acid and sedative B, was the most significantly improved. The blastocyst rate on day 8 was detected. The data in Table 1 show that compared with the blastocyst rate of the control group (30.5±3.6%), the blastocyst rates of the three treatment groups were significantly increased, namely 41.1±5.2%, 36.2±3.1%, and 50.4±4.3%, respectively. The combined use of folic acid and sedative B had the best effect.

[0070] Table 1 Effects of folic acid and phytosinolide B on the development rate of bovine embryos in vitro

[0071]

[0072] Note: In the table, those with different lowercase letters indicate significant differences (P<0.05), and those with the same lowercase letters indicate no significant differences (P>0.05).

[0073] The number of cells in the inner cell mass and the proportion of cells in the inner cell mass determine the potential for embryonic development. The data in Table 2 show that compared with the number of cells in the inner cell mass of the blastocyst in the control group (36.0±16.3), the folic acid group (57.2±28.5), the phytosphingomyelin B group (52.1±20.7), and the combination group of folic acid and phytosphingomyelin B (63.5±26.7) can all significantly increase the number of cells in the inner cell mass of the blastocyst. Treatment group 3, i.e., the combination group of folic acid and phytosphingomyelin B, has the best effect on increasing the number of cells in the inner cell mass of the blastocyst. Not only did the number of ICM cells increase, but the three treatment groups could also significantly increase the ICM / TE ratio. Compared with the ICM / TE ratio of 36.2±12.5% ​​in the control group, the ICM / TE ratios of the three treatment groups were 45.1±14.5%, 43.2±15.7%, and 57.2±22.5%, respectively.

[0074] Table 2 Effects of folic acid and sclerotin B on the number and proportion of cells in the inner cell mass of bovine embryos in vitro

[0075]

[0076] Note: In the table, those with different lowercase letters indicate significant differences (P<0.05), and those with the same lowercase letters indicate no significant differences (P>0.05).

[0077] The data in Table 3 show that the blastocysts cultured with folic acid, phytosporin B, and folic acid and phytosporin B in combination had an embryo survival rate of 17.5±5.7% in the control group after 7 days in vitro extension, and the survival rates of 37.2±3.1% and 32.9±7.1% when folic acid and phytosporin B were added alone, and 51.3±4.3% when folic acid and phytosporin B were used together. Statistics of the blastocyst diameter showed that the embryo diameter of the control group was 723.4±8.9 μm, and the diameters of the folic acid group, phytosporin B group, and folic acid and phytosporin B combined group were 878.9±9.3 μm, 830.8±11.4 μm, and 1032.8±14.2 μm, respectively, which were significantly higher than those of the control group, and the folic acid and phytosporin B combined group was significantly higher than the folic acid and phytosporin B group alone.

[0078] Table 3 Effects of folic acid and sclerotin B on the survival rate and blastocyst diameter of bovine in vitro embryos in delayed culture

[0079]

[0080] Note: In the table, those with different lowercase letters indicate significant differences (P<0.05), and those with the same lowercase letters indicate no significant differences (P>0.05).

[0081] Example 2

[0082] This example shows the use of combined administration of folic acid and phytosinolide B in increasing the number and proportion of cells in the inner cell mass of sheep embryos in vitro.

[0083] 1. Experimental Design and Methods

[0084] (1) Experimental groups

[0085] Control group: conventional in vitro development group;

[0086] Treatment group 1: folic acid (1 μM) alone administration group;

[0087] Treatment group 2: phytosinolide B (1 μM) alone administration group;

[0088] Treatment group 3: the group to which folic acid (0.5 μM) and phytosinolide B (0.5 μM) were administered together.

[0089] The control group was a basic in vitro development fluid, and the treatment groups 1-3 were basic in vitro development fluid with different regulators added or a combination of the two.

[0090] (2) Sheep oocyte in vitro maturation and embryo development

[0091] a) Oocyte collection and in vitro oocyte maturation

[0092] The collected sheep eggs were washed three times with egg washing solution, then washed three times with oocyte in vitro maturation basic solution, transferred to in vitro maturation culture medium, and in vitro maturation cultured for 22-24 h in a 5% CO2 incubator at 38.5℃ saturated humidity. Four-well plate culture, 600 μL in vitro maturation culture medium per well, 300 μL mineral oil covering, 30-35 sheep cumulus oocyte complexes per well.

[0093] b) In vitro fertilization

[0094] Place the mature COCs in 0.5% hyaluronic acid, gently blow and repeatedly remove most of the cumulus cells, and transfer them into the balanced fertilization solution. Take out the frozen sperm, thaw it in a 38℃ water bath, and then transfer the thawed sperm to 600 μL sperm upstream solution and incubate it in an incubator for 30 minutes to allow the sperm to fully float. After that, aspirate the supernatant and add it to the plate with mature eggs for in vitro fertilization for 20 hours. Fertilization conditions are saturated humidity, 38.5℃, 5% CO2, and 95% air.

[0095] c) In vitro culture

[0096] After fertilization, the fertilized eggs were aspirated to remove all residual cumulus cells and sperm on the surface. After washing three times in in vitro embryo development medium, they were cultured in a four-well plate: 600 μL in vitro embryo development medium was added to each well. The culture conditions were 38.5℃, 5% CO2, 90% N2, and saturated humidity for 6-7 days. During this period, the cleavage rate and blastocyst development rate of the embryos were counted (embryo development day 7), and the blastocysts were collected and fixed, immunofluorescence staining, and cell counting was performed.

[0097] (3) Count the number of inner cell mass cells and the proportion of inner cell mass cells

[0098] The blastocysts were collected, washed three times with 0.1% PVP-PBS, fixed in 4% paraformaldehyde for 30 minutes, treated with 0.5% TritonX-100 (PBS) at room temperature for 30 minutes, washed three times with 0.1% PVP-PBS, blocked with 0.1% TritonX-100 + 0.1% BSA (PBS) for 30 minutes, washed three times with 0.1% PVP-PBS, and incubated with primary antibody at 4°C overnight. Washed three times with 0.1% PVP-PBS, incubated with secondary antibody at room temperature in the dark, and washed three times with 0.1% PVP-PBS. The nuclei were stained with DAPI and sealed after incubation in the dark for 5-10 minutes.

[0099] 2. Experimental Results

[0100] The experimental results are shown in Table 4. Compared with the cleavage rate of the control group (72.4±2.7%), the folic acid group and the sedum B group did not affect the in vitro cleavage rate of sheep, but the combination of folic acid and sedum B group (85.6±2.3) could significantly increase the cleavage rate of sheep in vitro embryos. The three treatment groups can significantly increase the blastocyst rate of sheep in vitro embryos, increasing the day 7 blastocyst rate of the control group (33.9±1.9%) to 43.7±3.1%, 37.5±2.5%, and 47.9±3.6%, respectively.

[0101] As shown in Table 5, the addition of folic acid or phytosuccinate B alone had no significant effect on the ICM cell number and ICM / TE ratio of sheep blastocysts, but the combination of folic acid and phytosuccinate B could significantly increase the ICM cell number and ICM / TE ratio, which were 43.6±17.1 and 52.0±16.0%, respectively.

[0102] Table 4 Effects of folic acid and phytosinolide B on the in vitro embryo development rate of sheep

[0103]

[0104] Note: In the table, those with different lowercase letters indicate significant differences (P<0.05), and those with the same lowercase letters indicate no significant differences (P>0.05).

[0105] Table 5 Effects of folic acid and phytoserotin B on the number and proportion of cells in the inner cell mass of sheep embryos in vitro

[0106]

[0107] Note: In the table, those with different lowercase letters indicate significant differences (P<0.05), and those with the same lowercase letters indicate no significant differences (P>0.05).

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition for increasing the number and cell ratio of inner cell mass of embryos in vitro, characterized in that: It comprises folic acid, phytosporin B and basic embryo culture medium. The amount of folic acid can make the working concentration of folic acid reach 0.01-10 μM during in vitro culture; the amount of phytosporin B can make the working concentration of phytosporin B reach 0.1-10 μM during in vitro culture.

2. The composition for increasing the number and cell ratio of inner cell mass of in vitro embryos according to claim 1, characterized in that: The in vitro embryo source is a mammal in vitro fertilized embryo, and the mammal is a cow or a sheep.

3. A method for increasing the number and cell proportion of inner cell mass of embryos in vitro, characterized in that: The method comprises the step of contacting an embryo in vitro with the composition according to claim 1 or 2, wherein the in vitro embryo source is a mammal in vitro fertilized embryo, and the mammal is a cow or a sheep.

4. The method for increasing the number and cell proportion of inner cell mass of embryos in vitro according to claim 3, characterized in that: The increase includes increasing the number of cells in the inner cell mass of the blastocyst and the proportion of cells in the inner cell mass.

5. A method for improving in vitro fertilization embryo development, characterized in that: The method comprises the step of contacting an embryo with the composition according to claim 1 or 2 in vitro, wherein the in vitro fertilized embryo is derived from a mammal, and the mammal is a cow or a sheep.

6. The method for improving in vitro fertilization embryo development according to claim 5, characterized in that: The improvement includes increasing the cleavage rate and increasing the blastocyst development rate.

7. The method for improving in vitro fertilization embryo development according to claim 5, characterized in that: The improvement includes increasing the survival rate and / or diameter of blastocysts during delayed in vitro culture of blastocysts.

8. Use of the composition according to claim 1 or 2, or the method for increasing the number and cell proportion of inner cell mass of in vitro embryos according to claim 3 or 4, or the method for improving in vitro fertilization embryo development according to any one of claims 5 to 7 in the field of genetic improvement, breeding expansion or in vitro embryo production of cattle or sheep.

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