Composition and method for increasing number of inner cell mass cells of in vitro embryo and use

Abstract

Disclosed in the present invention are a composition and method for increasing the number and proportion of inner cell mass cells of an in vitro embryo and use. According to the present invention, in view of the problems of in vitro fertilized embryos such as low blastocyst rate during early development, low inner cell mass cell number in the blastocyst stage, low proportion of the inner cell mass cells, and poor subsequent embryonic development potential, by adding one or two of regulators folic acid and laxiflorin B into the culture medium for early embryo in vitro culturing, the blastocyst rate, the number and proportion of the inner cell mass cells in the blastocyst, the survival rate of blastocysts after extended culturing, and the diameter of blastocysts are significantly improved, effectively improving the developmental potential of in vitro embryos. In addition, the present invention also effectively improves the ratio of in vitro fertilized embryos that can be subsequently transplanted. The present invention is low in cost, safe, and efficient, provides a new strategy for in vitro embryo production of livestock and development of human-assisted reproductive technology, and exhibits broad application prospects.

Description

Compositions, methods, and applications for increasing the number of cells in the inner cell mass of in vitro embryos.

[0001] Cross-reference to related applications

[0002] This application claims priority to patent application No. CN202411570128.1, filed on November 6, 2024, entitled "Composition, Method and Application for Increasing the Number of Cells in the Inner Cell Mass of an In Vitro Embryo", the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This invention belongs to the field of in vitro fertilization technology, and in particular relates to compositions, methods and applications for increasing the number and proportion of cells in the inner cell mass of in vitro embryos. Background Technology

[0004] In vitro fertilization (IVF) has been widely used to rapidly expand the breeding stock of high-quality livestock and in human assisted reproduction. However, the pregnancy rate after embryo transfer from IVF is much lower than that of in vivo fertilized embryos, indicating that the developmental potential of IVF embryos is lower than that of in vivo embryos. The developmental potential of IVF embryos is crucial to the success of IVF embryo transfer and has always been a challenge in IVF technology. Embryo developmental potential is closely related to the first cell differentiation (i.e., differentiation into the 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 assessing the developmental potential of blastocysts. Reports have indicated that the in vitro culture environment affects the number and proportion of inner cell mass cells; the ICM / TE ratio of blastocysts after IVF in mice and cattle is 14%-23% lower than that of in vivo blastocysts, which is also a significant factor determining the lower developmental potential of IVF embryos.

[0005] In recent years, researchers have established an in vitro extended culture system for livestock blastocysts to assess the subsequent developmental potential of in vitro fertilized embryos. Compared to methods using embryo transfer to determine embryo developmental potential, the in vitro extended culture system for blastocysts significantly saves time and money. Furthermore, it can serve as a model for studying pre- and post-implantation developmental mechanisms, allowing for rapid, high-throughput assessment of 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 and diameter of blastocysts in in vitro extended culture.

[0006] To increase the number and proportion of cells in the inner cell mass (ICM) of in vitro embryos, current techniques primarily utilize the addition of secretory factors from the pre-implantation embryo or fallopian tube to regulate in vitro embryo development. For example, BMP, belonging to the TGF-β superfamily, promotes blastocyst formation. BMP signaling, by regulating the expression of pluripotent and lineage-specific genes, promotes ICM proliferation and inhibits TE differentiation. EGF, a growth factor, promotes cell proliferation and differentiation; adding EGF during in vitro embryo culture can increase the blastocyst rate. Although some progress has been made in recent years in improving in vitro embryo development by adding secretory factors, the effect on increasing the number and proportion of cells in the ICM is limited. Therefore, there is an urgent need to establish more effective methods to increase the number and proportion of cells in the ICM to further improve in vitro embryo technology. Summary of the Invention

[0007] To address at least some of the technical problems in the prior art, this invention significantly improves the blastocyst rate, increases the number of cells in the inner cell mass and the proportion of cells in the inner cell mass, improves the survival rate and diameter of blastocysts in extended culture, and effectively enhances the developmental potential of in vitro embryos by adding one or both of folic acid and phytohemagglutinin B to the culture medium for early embryo in vitro culture. Specifically, this invention includes the following:

[0008] In a first aspect, the present invention provides a composition for increasing the number and percentage of cells in the inner cell mass of an in vitro embryo, comprising a regulator selected from one or both of folic acid and phytoestrogens B.

[0009] In some embodiments, the composition for increasing the number and percentage of cells in the in vitro embryonic inner cell mass according to the present invention comprises any one of (a)-(c):

[0010] (a) Folic acid;

[0011] (b) succinate B;

[0012] (c) Folic acid and phytoestrogens B, wherein the concentration ratio of folic acid to phytoestrogens B is (0.001-100):1.

[0013] In some embodiments, the composition according to the invention for increasing the number and percentage of cells in the in vitro embryonic inner cell mass, wherein the amount of folic acid enables the working concentration of folic acid during in vitro culture to reach 0.01-10 μM; and the amount of phytoestrogens B enables the working concentration of phytoestrogens B during in vitro culture to reach 0.1-10 μM.

[0014] In some embodiments, the composition according to the invention for increasing the number and percentage of cells in the inner cell mass of an in vitro embryo is wherein the in vitro embryo is derived from a mammalian in vitro fertilized embryo.

[0015] In some embodiments, the composition according to the invention for increasing the number and percentage of cells in the in vitro embryonic inner cell mass further includes a basal culture medium.

[0016] A second aspect of the present invention provides a method for increasing the number and percentage 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.

[0017] In some embodiments, the method for increasing the number and proportion of cells in the inner cell mass of an in vitro embryo according to the present invention includes increasing the number of cells in the inner cell mass of the blastocyst and the proportion of cells in the inner cell mass.

[0018] A third aspect of the present invention provides a method for improving the development of in vitro fertilized embryos, comprising the step of contacting the embryo with the composition according to the present invention in vitro.

[0019] In some embodiments, the method for improving in vitro fertilized embryo development according to the present invention includes improving cleavage rate and improving blastocyst development rate.

[0020] In some embodiments, the method for improving in vitro fertilized embryo development according to the present invention further includes increasing the survival rate and diameter of blastocysts in extended in vitro culture.

[0021] A fourth aspect of the present invention provides the application of the compositions according to the present invention, or the methods according to the present invention for increasing the number and percentage of cells in the inner cell mass of an in vitro embryo, or the methods according to the present invention for improving the development of in vitro fertilized embryos in livestock genetic improvement, breeding, in vitro embryo production, or human assisted reproduction.

[0022] This invention addresses the problems of low inner cell mass cell count, low blastocyst rate, and low developmental potential of embryos in the early developmental stages of in vitro fertilized embryos (IVF). By adding one or both of folic acid and phytohexidine B to the culture medium during early embryo in vitro culture, it significantly improves the blastocyst rate, the number and proportion of cells in the inner cell mass of blastocysts, the survival rate and diameter of blastocysts in extended culture, and effectively enhances the developmental potential of in vitro embryos. Furthermore, this invention effectively increases the rate of IVF embryos usable for subsequent embryo transfer, and is low-cost, safe, and efficient, providing a new strategy for the development of in vitro embryo production in livestock and assisted reproductive technology in humans, with broad application prospects. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] In this article, the term "embryo" refers to the early stage of development from a fertilized egg. Since development is a continuous process, the term "embryo" in this article includes different forms of embryos generated at any developmental process or stage, generally referring to pre-implantation embryos, such as 2-cell embryos, 4-cell embryos, 8-cell embryos, morula, blastocysts, etc.

[0027] In this article, the term "in vitro" refers to events that occur in artificial environments, such as in test tubes or reaction vessels, in cell cultures, in petri dishes, etc., rather than in living organisms (e.g., animals, plants, or microorganisms).

[0028] In this article, the term "folic acid" or "vitamin B9," abbreviated as "FA," refers to a water-soluble vitamin. Folic acid is crucial for early embryonic development, serving as a methyl donor in the single-carbon cycle and directly participating in transcriptional regulation of signaling pathways by stimulating folic acid receptors via the receptor pathway.

[0029] In this article, the term "floridin B" refers to the chemical formula C. 20 H 24 O5 is a traditional Chinese medicine compound with CAS number 165337-71-3 and has the following structural formula:

[0030] In this article, the term "working concentration," also known as "use concentration," refers to the initial concentration at which the regulator is brought into contact with the embryo for effective treatment. The unit of concentration is not limited and can be, for example, μM, ng / ml, μg / ml, mg / ml, etc.

[0031] Composition

[0032] A first aspect of the present invention provides a composition for increasing the number and percentage of cells in the inner cell mass of an in vitro embryo, comprising one or both of a regulator selected from folic acid and phytoestrogens B. In this invention, folic acid and phytoestrogens B interact to produce a synergistic effect; the combined use of the two is more effective than their individual addition. The form of the composition of the present invention is not particularly limited and may be a solid, such as a dry powder, or a liquid, such as a solution.

[0033] In some embodiments, the modifier in the composition of the present invention can be a single modifier or a combination of two modifiers, meaning it contains no components other than unavoidable impurities besides the modifier. The modifiers in the composition of the present invention can exist in a mixed form or in individual forms. When used, the individually present modifiers can be pre-mixed before use, or each modifier can be used individually, simultaneously, or sequentially.

[0034] In some embodiments, the compositions of the present invention comprise the above-described combination of regulators, and further comprise other components. The composition or type of these other components is not limited and can be freely selected as needed. Such other components can be any known ingredients, particularly embryo culture-related reagents and compositions, such as culture media, culture solutions, or additives thereof.

[0035] In some embodiments, the composition of the present invention is a combination of folic acid and phytohemagglutinin B. Preferably, the concentration ratio of folic acid to phytohemagglutinin 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 phytohemagglutinin B can be achieved, significantly improving the efficacy.

[0036] In some embodiments, the amount of folic acid in the present invention enables a working concentration of 0.01-10 μM for use. The concentration or amount of folic acid in the composition is not limited as long as it can achieve the above range for use. When the composition is a solution and is used as an additive, 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 the above working concentration. When the composition is a solution and is used directly as a culture medium, the concentration of folic acid is generally substantially 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.

[0037] In some embodiments, the amount of phytoestrogens B of the present invention enables a working concentration of phytoestrogens B during in vitro culture of 0.1-10 μM. The concentration or amount of phytoestrogens B in the composition is not limited as long as it achieves the above range during use. When the composition is a solution and is used as an additive, the concentration of phytoestrogens 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 achieve the above working concentration. When the composition is a solution and is used directly as a culture medium, the concentration of phytoestrogens B is generally substantially equivalent to the working concentration. The working concentration of phytoestrogens 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.

[0038] In some embodiments, the in vitro embryo includes mammalian in vitro fertilization embryos. The mammals include, but are not limited to, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, pigs, and humans. In some embodiments, the animals include transgenic animals, genetically engineered animals, or clones.

[0039] Methods to increase the number and percentage of cells in the inner cell mass of in vitro embryos

[0040] A second aspect of the present invention provides a method for increasing the number and percentage of cells in the inner cell mass of an in vitro embryo, comprising the step of contacting the embryo in vitro with the composition according to the first aspect of the present invention.

[0041] In this invention, the increase includes increasing the number of cells in the inner cell mass of the blastocyst and the percentage of cells in the inner cell mass.

[0042] Methods to improve in vitro fertilization embryo development

[0043] A third aspect of the present invention provides a method for improving the development of in vitro fertilized embryos, comprising the step of contacting the embryo with the composition according to the first aspect of the present invention in vitro.

[0044] In this invention, the improvements include increasing the cleavage rate and increasing the blastocyst development rate.

[0045] In this invention, the improvement further includes increasing the survival rate and diameter of blastocysts in in vitro extended culture.

[0046] application

[0047] A fourth aspect of the present invention provides the application of the composition according to the first aspect of the present invention, or the method according to the second aspect of the present invention for increasing the number and percentage of cells in the inner cell mass of an in vitro embryo, or the method according to the third aspect of the present invention for improving the development of in vitro fertilized eggs or in vitro embryos in livestock genetic improvement, breeding, in vitro embryo production, or human assisted reproduction.

[0048] In some embodiments, the genetic improvement and propagation of improved varieties of the present invention include genetic improvement or propagation relying on in vitro embryo production technology.

[0049] Example 1

[0050] This embodiment demonstrates the application of combined folic acid and phytohexidine B in increasing the number and percentage of cells in the inner cell mass of bovine in vitro embryos.

[0051] 1. Experimental Design and Methods

[0052] (1) Experimental Grouping

[0053] Control group: Conventional in vitro development group;

[0054] Treatment group 1: Folic acid (2 μM) administered alone;

[0055] Treatment group 2: The group treated with phytosalicylate B (1 μM) alone;

[0056] Treatment group 3: Combined administration of folic acid (1 μM) and phytosalicylate B (0.5 μM).

[0057] The control group consisted of the basic in vitro developmental solution, while treatment groups 1-3 consisted of the basic in vitro developmental solution with the addition of different regulators or a combination of both.

[0058] (2) In vitro maturation of bovine oocytes and embryonic development

[0059] a) Oocyte collection and in vitro maturation of oocytes

[0060] Bovine ovaries were obtained from the slaughterhouse, follicular fluid was extracted, and cumulus-oocyte complexes were sorted out under a stereomicroscope. The complexes were washed three times in maturation solution, and the last 50 cumulus-oocyte complexes were incubated in 500 μL of bovine in vitro maturation culture medium and cultured in vitro for 21 h at 38.5℃ and saturated humidity in a 6% CO2 incubator.

[0061] b) In vitro fertilization

[0062] After maturation, the cells are gently blown away to remove large granulosa cells. The mature cumulus-oocyte complex is then transferred into a pre-balanced fertilization medium. Bovine frozen semen is thawed and washed twice with semen washing solution, then resuspended in fertilization medium. Sperm counts are performed, and 75 μL of the resuspended sperm is added to the fertilization medium containing the oocytes, resulting in a final sperm concentration of 1 × 10⁻⁶. 6 Sperm / mL, in vitro fertilization 18h.

[0063] c) In vitro culture

[0064] All granulosa cells and cumulus cells were removed from the fertilized eggs. The cleaned fertilized eggs were washed three times in in vitro embryo development medium and then cultured in four-well plates: approximately 50 fertilized eggs were placed in each well, and 500 μL of in vitro embryo development medium was added. 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 (on day 8 of embryonic development) were recorded. Blastocysts were collected, fixed, and subjected to immunofluorescence staining for cell counting.

[0065] (3) In vitro extended culture of embryos

[0066] Three days prior to incubation, four-well plates were incubated with ultrapure low-melting-point agarose, with the culture medium changed daily. The culture medium was prepared using Neurobasal:DMEM / F12 at a 1:1 ratio, with the addition of glutamine substitute, non-essential amino acids, N2 additive, and B27 additive. Day 8 blastocysts were transferred to four-well plates containing agarose for further culture. Culture conditions were 38.5℃, 5% CO2, and saturated humidity.

[0067] (4) Count the number of inner cell mass cells and the percentage of inner cell mass cells.

[0068] Blastocysts were collected, washed three times with 0.1% PVP-PBS, fixed in 4% paraformaldehyde for 30 minutes, treated with 0.5% Triton X-100 (PBS) at room temperature for 30 minutes, washed three times with 0.1% PVP-PBS, blocked with 0.1% Triton X-100 + 0.1% BSA (PBS) for 30 minutes, washed three times with 0.1% PVP-PBS, and incubated overnight at 4°C with primary antibody. After washing three times with 0.1% PVP-PBS, the cells were 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 the slides were mounted after incubation in the dark for 5-10 minutes.

[0069] 2. Experimental Results

[0070] The experimental results, including the in vitro embryo development rate, are shown in Table 1, and the inner cell mass cell count is shown in Table 2. Table 1 data shows that compared with the control group (70.2±3.2%), the cleavage rates of the folic acid alone (80.6±2.5%), the folic acid B alone (75.6±2.6%), and the combined folic acid and folic acid B group (90.2±1.9%) were significantly higher than the control group (p<0.05). Treatment group 3, i.e., the combined folic acid and folic acid B group, showed the most significant increase in cleavage rate. The blastocyst rate on day 8 was also measured. Table 1 data shows that compared with the control group (30.5±3.6%), the blastocyst rates of all three treatment groups were significantly higher, at 41.1±5.2%, 36.2±3.1%, and 50.4±4.3%, respectively, indicating that the combined use of folic acid and folic acid B was the most effective.

[0071] Table 1. Effects of folic acid and thiobutanol B on bovine in vitro embryonic development rate.

[0072] Note: Different lowercase letters above the table indicate significant differences (P<0.05), while the same lowercase letters indicate no significant differences (P>0.05).

[0073] The number of cells in the inner cell mass (ICM) and the proportion of ICM cells determine the developmental potential of the embryo. Table 2 shows that compared with the control group (36.0±16.3 cells in the blastocyst ICM), the folic acid group (57.2±28.5), the phytoestrogens B group (52.1±20.7), and the combination of folic acid and phytoestrogens B (63.5±26.7) all significantly increased the number of ICM cells in the blastocyst. Treatment group 3, the combination of folic acid and phytoestrogens B, showed the best effect in increasing the number of ICM cells in the blastocyst. Not only did they increase the number of ICM cells, but all three treatments also significantly increased the ICM / TE ratio. Compared with the control group's ICM / TE ratio of 36.2±12.5%, the ICM / TE ratios in 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 fructose B on cell number and percentage in the inner cell mass of bovine in vitro embryos

[0075] Note: Different lowercase letters above the table indicate significant differences (P<0.05), while the same lowercase letters indicate no significant differences (P>0.05).

[0076] Table 3 shows that, after 7 days of in vitro culture, the survival rate of blastocysts cultured with folic acid, phytoestrogens B, and a combination of folic acid and phytoestrogens B was 17.5±5.7% in the control group, 37.2±3.1% and 32.9±7.1% respectively when folic acid and phytoestrogens B were added alone, and 51.3±4.3% when folic acid and phytoestrogens B were used in combination. Statistical analysis of blastocyst diameter revealed that the diameter of embryos in the control group was 723.4±8.9 μm, while the diameters in the folic acid group, phytoestrogens B group, and the combined folic acid and phytoestrogens B group were 878.9±9.3 μm, 830.8±11.4 μm, and 1032.8±14.2 μm respectively, all significantly higher than the control group. Furthermore, the combined folic acid and phytoestrogens B group showed significantly higher diameters than the groups treated with folic acid and phytoestrogens B alone.

[0077] Table 3. Effects of folic acid and thiophene B on the survival rate and blastocyst diameter of bovine in vitro embryos in extended culture.

[0078] Note: Different lowercase letters above the table indicate significant differences (P<0.05), while the same lowercase letters indicate no significant differences (P>0.05).

[0079] Example 2

[0080] This example demonstrates the application of combined folic acid and phytohexidine B in increasing the number and percentage of cells in the inner cell mass of sheep in vitro embryos.

[0081] 1. Experimental Design and Methods

[0082] (1) Experimental Grouping

[0083] Control group: Conventional in vitro development group;

[0084] Treatment group 1: Folic acid (1 μM) administered alone;

[0085] Treatment group 2: The group treated with phytosalicylate B (1 μM) alone;

[0086] Treatment group 3: Combined administration of folic acid (0.5 μM) and phytohexidine B (0.5 μM).

[0087] The control group consisted of the basic in vitro developmental solution, while treatment groups 1-3 consisted of the basic in vitro developmental solution with the addition of different regulators or a combination of both.

[0088] (2) In vitro maturation of sheep oocytes and embryonic development

[0089] a) Oocyte collection and in vitro maturation of oocytes

[0090] Collected sheep oocytes were washed three times with oocyte washing solution, then washed three times with basal oocyte maturation solution. They were then transferred to in vitro maturation culture medium and cultured in a 38.5℃, 5% CO2 incubator for 22-24 hours. The cells were cultured in four-well plates with 600 μL of in vitro maturation culture medium per well, covered with 300 μL of mineral oil, and containing 30-35 sheep cumulus-oocyte complexes per well.

[0091] b) In vitro fertilization

[0092] Mature cocci (COCs) were placed in 0.5% hyaluronic acid and gently and repeatedly pipetted to remove most of the cumulus cells. The sperm were then transferred to a pre-balanced fertilization medium. The frozen sperm were removed and thawed in a 38°C water bath. The thawed sperm were then transferred to 600 μL of sperm suspension medium and incubated in an incubator for 30 minutes to allow the sperm to float. The supernatant was then aspirated and added to a plate containing mature oocytes for in vitro fertilization (IVF) for 20 hours. Fertilization conditions included saturated humidity, 38.5°C, 5% CO2, and 95% air.

[0093] c) In vitro culture

[0094] After fertilization, the fertilized eggs were aspirated, and all residual cumulus cells and sperm were removed from the surface. After washing three times in in vitro embryonic development medium, the eggs were cultured in four-well plates: 600 μL of in vitro embryonic development medium was added to each well. 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 (day 7 of embryonic development) were recorded. Blastocysts were collected, fixed, and subjected to immunofluorescence staining for cell counting.

[0095] (3) Count the number of inner cell mass cells and the percentage of inner cell mass cells.

[0096] Blastocysts were collected, washed three times with 0.1% PVP-PBS, fixed in 4% paraformaldehyde for 30 minutes, treated with 0.5% Triton X-100 (PBS) at room temperature for 30 minutes, washed three times with 0.1% PVP-PBS, blocked with 0.1% Triton X-100 + 0.1% BSA (PBS) for 30 minutes, washed three times with 0.1% PVP-PBS, and incubated overnight at 4°C with primary antibody. After washing three times with 0.1% PVP-PBS, the cells were 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 the cells were mounted after incubation in the dark for 5-10 minutes.

[0097] 2. Experimental Results

[0098] Table 4 shows the experimental results. Compared with the control group (72.4±2.7%), the folic acid group and the phytoestrogens B group did not affect the in vitro cleavage rate of sheep. However, the combination of folic acid and phytoestrogens B (85.6±2.3%) significantly improved the cleavage rate of sheep. All three treatment groups significantly improved the blastocyst rate of sheep in vitro, 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.

[0099] As shown in Table 5, folic acid or phytoestrogens B alone had no significant effect on the number of ICM cells and the ICM / TE ratio in sheep blastocysts. However, the combination of folic acid and phytoestrogens B significantly increased the number of ICM cells and the ICM / TE ratio to 43.6±17.1 and 52.0±16.0%, respectively.

[0100] Table 4. Effects of folic acid and thioglycoside B on in vitro embryonic development rate in sheep.

[0101] Note: Different lowercase letters above the table indicate significant differences (P<0.05), while the same lowercase letters indicate no significant differences (P>0.05).

[0102] Table 5. Effects of folic acid and thioglycoside B on the number and percentage of cells in the inner cell mass of sheep in vitro embryos.

[0103] Note: Different lowercase letters above the table indicate significant differences (P<0.05), while the same lowercase letters indicate no significant differences (P>0.05).

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate 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 percentage of cells in the inner cell mass of an in vitro embryo, characterized in that, Including regulators selected from one or both of folic acid and phytoestrogens B.

2. The composition according to claim 1 for increasing the number and percentage of cells in the inner cell mass of an in vitro embryo, characterized in that, The composition comprises any one of (a)-(c): (a) Folic acid; (b) succinate B; (c) Folic acid and phytoestrogens B, wherein the concentration ratio of folic acid to phytoestrogens B is (0.001-100):

1.

3. The composition according to claim 1 for increasing the number and percentage of cells in the inner cell mass of an in vitro embryo, characterized in that, The amount of folic acid is sufficient to achieve a working concentration of 0.01-10 μM during in vitro culture; the amount of phytoestrogens B is sufficient to achieve a working concentration of phytoestrogens B during in vitro culture.

4. The composition according to claim 1 for increasing the number and percentage of cells in the inner cell mass of an in vitro embryo, characterized in that, The in vitro embryos are derived from mammalian in vitro fertilization embryos.

5. The composition according to claim 1 for increasing the number and percentage of cells in the inner cell mass of an in vitro embryo, characterized in that, This further includes the basic culture medium.

6. A method for increasing the number and percentage of cells in the inner cell mass of an in vitro embryo, characterized in that, Includes the step of contacting an embryo with the composition according to any one of claims 1-5 in vitro.

7. The method for increasing the number and percentage of cells in the inner cell mass of an in vitro embryo according to claim 6, characterized in that, The increase includes increasing the number of cells in the inner cell mass of the blastocyst and the percentage of cells in the inner cell mass.

8. A method for improving the development of in vitro fertilized embryos, characterized in that, Includes the step of contacting an embryo with the composition according to any one of claims 1-5 in vitro.

9. The method for improving in vitro fertilized embryo development according to claim 8, characterized in that, The improvements include increasing cleavage rate and increasing blastocyst development rate.

10. The method for improving in vitro fertilized embryo development according to claim 8, characterized in that, The improvements include increasing the survival rate of blastocysts in in vitro extended culture and / or blastocyst diameter.

11. The composition according to any one of claims 1-5, or the method for increasing the number and percentage of cells in the inner cell mass of an in vitro embryo according to claim 6 or 7, or the method for improving the development of an in vitro fertilized embryo according to claim 8, 9 or 10, and its application in the fields of livestock genetic improvement, breed propagation, in vitro embryo production or human assisted reproduction.

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