Porcine embryo in-vitro culture medium and application thereof

Abstract

The invention relates to the technical field of animal cell culture, in particular to a porcine embryo in-vitro culture medium and application thereof. The invention provides a novel application of betaine and a betaine-containing pig embryo in-vitro culture medium. According to the in-vitro culture medium, the in-vitro culture efficiency of the pig embryo and the quality of the pig blastocyst can be remarkably improved, the in-vitro culture time of the pig embryo can be prolonged to the 35th day, and differentiation of the epiderm and the hypoderm of the pig embryo, embryo elongation, protointestinal movement and development of nerves and the heart can be promoted. Therefore, the betaine-containing pig embryo in-vitro culture medium provided by the invention is a novel efficient in-vitro culture medium and can promote high-quality development of the pig blastocyst.

Description

Technical Field

[0001] The present invention relates to the technical field of animal cell culture, in particular to an in vitro culture medium for pig embryos and application thereof. Background Art

[0002] In vitro culture of mammalian embryos is a crucial biotechnology that simulates the maternal uterine environment in a laboratory setting, providing the necessary support for embryonic growth and development. This technology is crucial for animal husbandry, including breeding, disease modeling, drug screening, and research into embryonic development mechanisms.

[0003] When culturing embryos in vitro, the composition of the culture medium is crucial for successful embryo culture. Optimizing each component of the culture medium is crucial for efficient embryo production in vitro. The main additives in the culture medium include water, inorganic salts, amino acids, energy substrates, antioxidants, and proteins. The mouse embryo is a commonly used embryonic model for establishing mammalian in vitro culture systems, laying the foundation for the development of in vitro culture systems for higher mammals. In vitro culture of mouse embryos dates back to the 1980s, using Eagle's basal medium to culture embryos from E3.5 to E5.0. Morris et al. developed a new culture system using CMRL1066 as the basal medium. In 2014, Bedzhov et al. introduced a novel culture method, the now commonly used in vitro culture systems IVC1 and IVC2. Using Advanced DMEM / F12 as the basal medium, they characterized the formation of the anterior-posterior axis and egg cylinder morphogenesis in mouse embryos in vitro.

[0004] At present, the in vitro embryo culture technology of large livestock such as pigs, cattle, and sheep lags significantly behind that of mice, humans, and primates. First, the quality of in vitro cultured embryos is poor, and second, it cannot support long-term in vitro culture. Currently, PZM-3 medium is the most widely used culture system for pig in vitro embryo cultivation. It is designed based on inorganic substances and energy substrates in the pig oviduct. Although the composition of PZM-3 medium is clear, the efficiency of embryos cultured with it to develop into blastocysts is low, the blastocyst quality is poor, and they can only be cultured to the mid-blastocyst stage. Summary of the Invention

[0005] The purpose of the present invention is to provide a porcine embryo in vitro culture medium and its application to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides the use of betaine in any one or more of the following:

[0008] (1) Application in prolonging the in vitro culture time of pig embryos;

[0009] (2) Application in improving the pluripotency of porcine embryos;

[0010] (3) Application in promoting the differentiation of porcine embryonic germ layers;

[0011] (4) Application in increasing porcine embryonic lineage differentiation and development;

[0012] (5) Application in improving the developmental potential of in vitro cultured porcine embryos;

[0013] (6) Application in improving the efficiency of in vitro culture of porcine embryos;

[0014] (7) Application in increasing the diameter of pig embryos;

[0015] (8) Application in the preparation of a culture medium for prolonging the in vitro culture time of pig embryos;

[0016] (9) Application in the preparation of a culture medium for improving the pluripotency of pig embryos;

[0017] (10) Application in the preparation of a culture medium for promoting differentiation of porcine embryonic germ layers;

[0018] (11) Application in the preparation of a culture medium for increasing porcine embryonic lineage differentiation and development;

[0019] (12) Application in the preparation of a culture medium for improving the developmental potential of in vitro cultured porcine embryos;

[0020] (13) Application in the preparation of a culture medium for improving the efficiency of in vitro culture of porcine embryos;

[0021] (14) Application in the preparation of a culture medium for increasing the diameter of pig embryos.

[0022] Preferably, said development comprises neural development and / or cardiac development.

[0023] Further preferably, the improving the efficiency of in vitro culture of pig embryos includes improving the hatching rate of pig blastocysts and / or improving the survival rate of pig embryos.

[0024] The invention provides a porcine embryo in vitro culture medium, which comprises an N2B27 culture medium and betaine; the N2B27 culture medium uses a DMEM / F12 culture medium and a Neurobasal culture medium as a basal culture medium, and further comprises N-2, B-27, RA, Activin A, a ROCK signaling pathway inhibitor Y-27632, PS48, Glucose, ITS, arginine, GlutaMAX, lipids and FBS.

[0025] Preferably, the N2B27 culture medium further comprises antibiotics.

[0026] Preferably, the antibiotics include one of penicillin, streptomycin, penicillin-streptomycin combination, and gentamicin.

[0027] Preferably, the volume percentage of N-2 in the porcine embryo in vitro culture medium is 1%, the volume percentage of B-27 is 2%, the concentration of RA is 0.5nM-5nM, the concentration of ActivinA is 10ng / mL-20ng / mL, the concentration of ROCK signaling pathway inhibitor Y-27632 is 5μm-15μM, the concentration of PS48 is 1μM-10μM, the concentration of Glucose is 0.1mg / mL-3mg / mL, the concentration of ITS is 1×-10×, the concentration of arginine is 0.5nM-8nM, the concentration of GlutaMAX is 1.5nM-5nM, the concentration of lipid is 1:200-1:800, the volume percentage of FBS is 3%-15%, the concentration of betaine is 2mM-10mM, and the concentration of antibiotics is 40-60IU / mL.

[0028] Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F-12) is a widely used basal culture medium suitable for growing a variety of mammalian cells. This medium combines the high glucose, amino acids, and vitamins of DMEM with the various components of F-12, but without proteins, lipids, or growth factors. This product was purchased from Gibco.

[0029] Neurobasal medium is a basic medium developed to meet the specialized needs of neural cell culture. With the addition of B-27 Supplement (N-2 Supplement or G-5 Supplement), Neurobasal medium can maintain long-term neural cell growth and normal phenotype, and can maintain high-purity neuronal cultures without the need for an astrocyte feeder layer. This product is purchased from Gibco.

[0030] N-2 Supplement (N-2), based on Bottenstein's N-1 formulation, is a chemically defined, serum-free supplement recommended for supporting the growth of primary neuroblastoma cells and postmitotic neurons derived from the peripheral and central nervous systems. N-2 Supplement is supplied as a 100X concentrate and can be used with Neurobasal medium supplemented with growth factors such as bFGF and EGF. It can also be used with DMEM and is available from Thermo Fisher Scientific.

[0031] B-27 Supplement (B-27, Vitamin A Depleted) is a customized version of the original B-27 Supplement with Vitamin A (retinyl acetate) removed. B-27 Supplement (Vitamin A Depleted) is a serum-free supplement ideal for culturing neural progenitor and neural stem cells, whether in suspension neurospheres or adherent monolayers, without inducing differentiation. Supplied in a 50× liquid format, B-27 Supplement (Vitamin A Depleted) is suitable for use with Neurobasal Medium or Neurobasal-A Medium for culturing neuronal cells without the need for an astrocyte feeder layer. This product is available from ThermoFisher.

[0032] RA (retinoic acid) is a metabolite of vitamin A that plays a vital role in cell growth, differentiation, and organogenesis. Retinoic acid is required for virtually all essential physiological processes and functions, as it participates in the transcriptional regulation of over 530 different genes. Retinoic acid exerts its effects by acting as an activating ligand for nuclear retinoic acid receptors (RARα-γ), which form heterodimers with retinoic acid X receptors (RXRα-γ). This product was purchased from Thermo Fisher Scientific.

[0033] ActivinA is a multifunctional cytokine and a member of the TGF-β superfamily. ActivinA first binds to the type II activin receptor (ActIIRA or ActRIIB) on the surface of these cells, then recruits and phosphorylates the type I activin receptor (ActRI). ActivinA primarily signals through SMAD2 / 3 proteins to regulate a variety of functions, including inflammation, fibrosis, and tumorigenesis. It is a member of the transforming growth factor-β (TGF-β) superfamily and consists of two inhibin β subunits linked by a disulfide bond. ActivinA is widely expressed in various tissues and cells and was purchased from Peprotech.

[0034] A ROCK signaling pathway inhibitor. Rho, a member of the small G protein family, is primarily involved in cytoskeletal organization and gene expression regulation. Rho kinase (ROCK) is its most characteristic downstream effector, regulating numerous cellular functions, such as cell migration, division, apoptosis, adhesion, and phagocytosis. Y-27632 is commonly used in human embryonic stem cell culture. Y-27632 is a reliable and effective treatment that significantly improves the viability of human embryonic stem cells and enhances their clonogenic capacity without affecting their pluripotency. This product was purchased from Thermo Fisher.

[0035] PS48 is a signaling pathway activator and a PDK1 (phosphoinositide-dependent protein kinase 1) activator that binds to the HM / PIF binding pocket rather than the ATP binding site. PS48 is one of the few true allosteric compounds that targets a regulatory binding site on the protein kinase catalytic domain that is not adjacent to or overlapping with the ATP binding site. This product was purchased from MCE.

[0036] Glucose is a common, naturally occurring sugar involved in processes such as energy production, glycosylation, and the formation of glycans that provide cellular structure. Glucose is used as a cell culture supplement and in a variety of cellular processes and molecular biology applications. This product was purchased from MCE.

[0037] An important substance contained in the ITS is insulin, which promotes glucose and amino acid uptake, lipogenesis, intracellular transport, and protein and nucleic acid synthesis. Transferrin, an iron carrier, also helps reduce toxic levels of oxygen free radicals and peroxides. Selenium (provided as sodium selenite) is a cofactor for glutathione peroxidase and other proteins and serves as an antioxidant in the culture medium. This product was purchased from Thermo Fisher Scientific.

[0038] L-Arginine, or Arg (arginine), is a substrate for endothelial nitric oxide synthase (eNOS) to produce NO. L-Arginine is transported to vascular smooth muscle cells via a family of cationic amino acid transporters, where it is metabolized into nitric oxide (NO), polyamines, or L-proline. This product was purchased from Thermo Fisher Scientific.

[0039] GlutaMAX (L-glutamine) is an important amino acid supplement commonly added to mammalian cell culture media. L-glutamine serves as a supplemental energy source, particularly during rapid cell division. It is also involved in the formation of purine and pyrimidine nucleotides, amino sugars, glutathione, L-glutamate, and other amino acids, and participates in protein synthesis and glucose production. This product was purchased from Thermo Fisher Scientific.

[0040] Fetal bovine serum (FBS) provides essential nutrients and growth factors for cell maintenance and growth and was purchased from Thermo Fisher.

[0041] Lipids: Chemically defined lipid concentrates are concentrated fat emulsions and were purchased from Thermo Fisher Scientific.

[0042] Taurine is a sulfur-containing amino acid and an organic osmotic agent involved in cell volume regulation, playing a role in regulating intracellular free calcium concentration. This product was purchased from MCE.

[0043] Antibiotics: can be used to prevent bacterial contamination of embryo culture, this product was purchased from Thermo Fisher.

[0044] Betaine (betaine) is a naturally occurring compound found in many foods and is an active methyl group donor that maintains normal DNA methylation patterns. Betaine is found in a wide range of plants, animals, microorganisms, and dietary sources, including seafood, spinach, and wheat bran. Betaine also acts as an osmolyte, maintaining cellular water and ion balance in birds by preventing dehydration and osmotic inactivation, thereby improving their ability to withstand heat stress. It helps maintain protective osmotic activity, particularly in heat-stressed birds. Betaine promotes the resistance of various intestinal microorganisms to osmotic changes, thereby improving microbial fermentation activity. This product was purchased from MCE.

[0045] The present invention provides the use of the above-mentioned porcine embryo in vitro culture medium in any one or more of the following:

[0046] (1) Application in prolonging the in vitro culture time of pig embryos;

[0047] (2) Application in improving the pluripotency of porcine embryos;

[0048] (3) Application in promoting the differentiation of porcine embryonic germ layers;

[0049] (4) Application in increasing porcine embryonic lineage differentiation and development;

[0050] (5) Application in improving the developmental potential of in vitro cultured porcine embryos;

[0051] (6) Application in improving the efficiency of in vitro culture of porcine embryos;

[0052] (7) Application in increasing the diameter of pig embryos.

[0053] Further preferably, the development includes neural development and / or cardiac development.

[0054] Further preferably, the improving the efficiency of in vitro culture of pig embryos includes improving the hatching rate of pig blastocysts and / or improving the survival rate of pig embryos.

[0055] The present invention provides an in vitro culture method for pig embryos, comprising the steps of starting culture with the N2B27 culture medium on the 6th day after parthenogenetic activation; and culturing with the pig embryo in vitro culture medium on the 8.5th to 13.5th day.

[0056] The present invention provides a method for improving the developmental potential of in vitro cultured pig embryos. The in vitro culture method comprises the steps of starting culture with the N2B27 culture medium on the 6th day after parthenogenetic activation; and culturing with the pig embryo in vitro culture medium on the 8.5th to 13.5th day.

[0057] The present invention discloses the following technical effects:

[0058] The present invention provides a new application of betaine and an in vitro culture medium for pig embryos containing betaine. The in vitro culture medium can significantly improve the in vitro culture efficiency of pig embryos and the quality (diameter and hatchability) of pig blastocysts, and can extend the in vitro culture time of pig embryos to 35 days. It can promote the differentiation of the epiblast and hypoblast of pig embryos, embryonic elongation, gastrulation, and the development of nerves and the heart. Thus, the in vitro culture medium for pig embryos containing betaine provided by the present invention is a new and efficient in vitro culture medium that can promote the high-quality development of pig blastocysts. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 paying any creative work.

[0060] Figure 1 The effects of different betaine addition concentrations on embryo morphology;

[0061] Figure 2 These are bright field photos of embryonic development in the control and experimental groups; Control is the control group, and Betaine is the experimental group;

[0062] Figure 3 The results of the culture duration survey are shown in Table 2. Among them, Control is the control group and Betaine is the experimental group;

[0063] Figure 4 The results of the pig embryo diameter survey are shown in Figure 2. Control is the control group, and Betaine is the experimental group.

[0064] Figure 5 The statistical results of pig embryo hatching rate; Control is the control group and Betaine is the experimental group;

[0065] Figure 6 This is the result of a pig embryo survival survey; Control is the control group, and Betaine is the experimental group;

[0066] Figure 7The results of embryonic immunofluorescence detection of upper and lower embryos are shown in the figure; Control is the control group and Betaine is the experimental group;

[0067] Figure 8 Figure 1 shows the statistical results of embryonic cell counts on day 11 (A) and the percentage of SOX17-positive cells and SOX2-positive cells in the experimental group on day 11 (B); Control is the control group and Betaine is the experimental group;

[0068] Figure 9 The embryo shows beating myocardial cells;

[0069] Figure 10 Are the results of immunofluorescence detection; A is the expression of SOX17 gene and GATA4 gene; B is the expression of GATA4 gene and NESTIN gene; Control is the control group, and Betaine is the experimental group. DETAILED DESCRIPTION

[0070] 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0071] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0072] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0073] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0074] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0075] Unless otherwise specified, the agents used in the culture medium and culture method of the present invention are all commercially available products, and the methods adopted in the present invention are all methods well known to those skilled in the art.

[0076] Example 1

[0077] 1. Experimental Procedure

[0078] 1. Pig fertilized eggs and in vitro culture

[0079] Pig ovaries collected from slaughterhouses were transported to the laboratory at 37°C. The tissues were washed with 37°C physiological saline containing antibiotics. Follicular fluid was extracted from the follicles with a syringe and placed in a 39°C incubator for 10 minutes to obtain a follicular fluid precipitate. The precipitate was washed twice with HEPES (4-hydroxyethylpiperazineethanesulfonic acid) and diluted in a 100mm dish. Oocytes with intact COCs were selected with a glass needle under a microscope and transferred to oocyte maturation medium and cultured at 39°C and 5% CO2. After culturing for 4 hours, the granulosa cells of the oocytes were digested and removed with 0.1% hyaluronidase. Mature oocytes with the first polar body were selected under a microscope for subsequent experiments, and the data were recorded.

[0080] Preheat the activation solution in advance, clean the fusion tank, and rinse it three times with activation solution. First, transfer the digested oocytes to a 30mm dish containing activation solution, allowing the eggs to sink to the bottom of the activation solution to acclimate to the liquid environment. Then, arrange the oocytes in parallel between the electrodes of the fusion tank and apply a direct current shock twice at 1.2V / cm with a pulse time of 30us. Transfer the oocytes to PZM-3 medium that has been equilibrated for more than 12 hours and rinse them three to four times. Then, transfer the oocytes to 500μL of PZM-3 medium and place them in an incubator at 38.5°C, 5% CO2, and saturated humidity for incubation. Observe and record the embryonic cleavage rate after 48 hours, and observe and record the embryonic rate on days 5.5-6 of development.

[0081] 2. Continuous Culture of Pig Embryos in Vitro

[0082] Choose to be cultivated to the 6th day in PZM-3 culture medium, select the blastocyst with good development state, transfer it to the N2B27 culture medium (table 1) of more than 4h of balance, rinse 3-4 times.Then at the 8.5th day, embryo is moved to the porcine embryo in vitro culture medium (table 2) containing 500 μL, is placed in incubator and is cultivated, and the present embodiment is when the 8.5th day, embryo is moved to the porcine embryo in vitro culture medium containing 500 μL.Every 24h, half of the culture medium is changed, and is recorded as experimental group or N2B27+Betaine group, and the process of only adopting N2B27 culture medium to cultivate is control group (Control), and takes pictures and records the morphological changes of embryo.

[0083] Table 1 Components and concentrations of N2B27 culture medium

[0084] Element Concentration / volume percentage / addition ratio DMEM / F12: Neurobasal 1：1 N-2 1％ B-27 2％ RA 5nM ActivinA 20ng / ml Y27632 10μm GlutaMAX 3.75mM Glucose 2mg / ml PS48 5μm Arg 1.69mM lipids 1:500 ITS 1× FBS 10％ Antibiotics (penicillin-streptomycin) 60 IU / mL

[0085] Table 2 Components and concentrations of porcine embryo in vitro culture medium

[0086] Element Concentration / volume percentage / addition ratio DMEM / F12: Neurobasal 1：1 N-2 1％ B-27 2％ RA 5nM ActivinA 20ng / ml Y27632 10μm GlutaMAX 3.75mM Glucose 2mg / ml PS48 5μm Arg 1.69mM lipids 1:500 ITS 1× FBS 10％ Antibiotics (penicillin-streptomycin) 60 IU / mL Betaine 0mM, 5mM, or 10mM

[0087] 3. Immunofluorescence detection of pig embryos

[0088] (1) Fixation: Add 500 μL of fixative solution to a 24-well plate. Place the sample (no more than 40 eggs or embryos) in the fixative solution and fix at room temperature for 30-60 minutes. Alternatively, the sample can be fixed and stored at 4°C for up to 8 hours. Wash three times with 500 μL of wash solution, shaking on a shaker for 5 minutes each time.

[0089] (2) Permeabilization: Transfer the sample into 500 μL of permeabilization solution and incubate overnight at 4°C (8-10 hours). When detecting membrane proteins, the permeabilization time can be appropriately reduced or the membrane can be omitted depending on the positional relationship between the specific binding site of the protein and the antibody and the membrane.

[0090] (3) Blocking: Transfer the sample into 500 μL of blocking solution and block for more than 1 hour.

[0091] (4) Primary antibody binding: dilute the primary antibody (purchased from Peprotech) with blocking buffer according to the reference ratio in the instructions, add the sample to 200 μL of primary antibody dilution buffer, and incubate at 4°C overnight.

[0092] (5) Wash the sample in the cleaning solution three times, each time for 5 minutes.

[0093] (6) Secondary antibody binding: Dilute the secondary antibody (purchased from Peprotech) with a certain ratio in the cleaning solution as required, add the sample to 400 μL of the secondary antibody dilution solution, and incubate at room temperature in the dark for 1-2 h.

[0094] (7) Wash the sample in the cleaning solution three times, each time for 5 minutes.

[0095] (8) Nuclear staining: Transfer the sample into the diluted Hoechest 33342 solution and incubate in the dark for 10 minutes. Wash in the cleaning solution three times, 5 minutes each time.

[0096] (9) Sealing: Pressing: Add 30 μL of anti-fluorescence quencher to a clean glass slide, move the sample into the anti-fluorescence quencher drop, stick one end of a clean cover glass on the slide, slowly tilt it until it is all stuck on the slide, and fix the slide with glycerol or nail polish.

[0097] (10) Observation on the microscope: Turn on the Nikon 80i fluorescence microscope system, find the sample under non-fluorescence excitation conditions, draw a circle, adjust the excitation fluorescence, observe the results under different fluorescence excitation conditions as needed, take photos, and save the results.

[0098] 2. Experimental results

[0099] To explore the optimal concentration of betaine, we cultured parthenogenetic activated blastocysts in an in vitro culture system to day 8.5 (D8.5), and added different concentrations of betaine, with the concentration gradient set to 0mM (control group, Control), 5mM and 10mM, and continued to culture until day 12 (D12). The morphology of the embryos in each experimental group from day 9 (D9) to day 12 (D12) was as follows: Figure 1 As shown in the table, observation of embryo morphology revealed that at D12, the embryos in the experimental group supplemented with 5 mM betaine were the most plump and in the best condition. Based on these experimental results, it was concluded that the optimal concentration of betaine for in vitro embryo culture is 5 mM. Subsequent experiments used porcine embryo in vitro culture medium containing 5 mM betaine (other cultures were the same as in Table 2) as the experimental group.

[0100] Bright field photos of embryonic development in the control group and experimental group are shown in the figure below. Figure 2 The results showed that the pig blastocysts that developed to the 6th day after parthenogenetic activation were cultured in vitro in the control group and the experimental group. By comparing the embryonic status of the control group and the experimental group ( Figure 2 As shown in the figure, the embryos in the experimental group were fuller and in better condition than those in the control group. Further analysis of the culture time showed that the culture period of the experimental group was significantly longer than that of the control group ( Figure 3 The culture period could be extended to more than 35 days, and there was a significant difference between the two groups (P < 0.05).

[0101] Embryo diameter statistics Figure 4As shown, on the 8th day of embryonic development, the embryo diameter of the control group was lower than that of the experimental group (269.80±15.25μm vs 289.03±11.65μm), and there was no significant difference between the two groups; on the 9th day of embryonic development, the embryo diameter of the control group was lower than that of the experimental group (386.90±27.13μm vs 459.92±7.36μm), and there was no significant difference between the two groups; on the 10th day of embryonic development, the embryo diameter of the control group was lower than that of the experimental group (392.83±25.14μm vs 468.75±20.3μm), and there was no significant difference between the two groups; after the 12th day of embryonic development, the embryo diameter of the experimental group was significantly higher than that of the control group; thus, it can be seen that the culture medium provided by the present invention can prolong the time of in vitro embryo culture of late embryos, which can be extended to more than 35 days. Currently, many different culture media for early embryo development can culture embryos of various species to the blastocyst stage. However, these embryos typically require a connection to the maternal uterus to survive, resulting in very limited survival and significant difficulty in later-stage culture. The present invention overcomes these challenges by optimizing the composition and concentration of the culture media, providing a culture medium suitable for later-stage embryo culture in vitro.

[0102] The statistical results of blastocyst hatching rate are as follows Figure 5 As shown in the data, when the embryos developed to the 6th day, the blastocyst hatching rate of the experimental group was higher than that of the control group (25.80±9.8% vs 13.96±6.96%); when the blastocysts developed to the 7th day, the blastocyst hatching rate of the experimental group was significantly increased compared with the control group (33.8±7.8% vs 21.00±6.00%, P<0.05).

[0103] Embryo survival rate statistics are as follows Figure 6 As shown in the figure, when the embryos of the control group and the experimental group (N2B27+Betaine group) developed to day 7-35, the embryo survival rate of the Betaine group was higher than that of the control group, and there was a significant difference.

[0104] The results of embryonic immunofluorescence detection of upper and lower embryos are as follows Figure 7 As shown, compared with the control group, the experimental group expressed the mesoderm marker gene SOX2 and the hypoblast marker gene SOX17, while the control group did not express SOX2 and SOX17, indicating that N2B27+Betaine culture medium can promote the differentiation of embryonic germ layers.

[0105] The results of embryonic cell count on day 11 are as follows Figure 8 As shown in A, the number of cells in the control group was (388.6±51.4), and the number of cells in the experimental group was (543±66). The number of cells in the experimental group was significantly higher than that in the control group. The statistical results of the proportion of SOX2 and SOX17 cells in the experimental group are shown in Figure 8As shown in Figure B, the proportion of SOX17-positive cells in the experimental group was higher than that of SOX2-positive cells on day 11. This shows that adding betaine to the culture system can significantly promote embryonic development.

[0106] On the 35th day of in vitro culture, beating of cardiomyocytes was observed in the embryos ( Figure 9 ), and immunofluorescence detection was performed, and it was found that the myocardial development marker genes GATA4 and NKX2.5 were specifically expressed in the embryo samples (such as Figure 10 ), indicating that the embryos have undergone directed differentiation into the cardiomyocyte lineage.

[0107] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of betaine in any one or more of the following: (1) Application in prolonging the in vitro culture time of pig embryos; (2) Application in improving the pluripotency of porcine embryos; (3) Application in promoting the differentiation of porcine embryonic germ layers; (4) Application in increasing porcine embryonic lineage differentiation and development; (5) Application in improving the developmental potential of in vitro cultured porcine embryos; (6) Application in improving the efficiency of in vitro culture of porcine embryos; (7) Application in increasing the diameter of pig embryos; (8) Application in the preparation of a culture medium for prolonging the in vitro culture time of pig embryos; (9) Application in the preparation of a culture medium for improving the pluripotency of pig embryos; (10) Application in the preparation of a culture medium for promoting differentiation of porcine embryonic germ layers; (11) Application in the preparation of a culture medium for increasing porcine embryonic lineage differentiation and development; (12) Application in the preparation of a culture medium for improving the developmental potential of in vitro cultured porcine embryos; (13) Application in the preparation of a culture medium for improving the efficiency of in vitro culture of porcine embryos; (14) Application in the preparation of a culture medium for increasing the diameter of pig embryos.

2. The use according to claim 1, characterized in that Said development comprises neural development and / or cardiac development.

3. A porcine embryo in vitro culture medium, characterized in that: The porcine embryo in vitro culture medium includes N2B27 culture medium and betaine; the N2B27 culture medium uses DMEM / F12 culture medium and Neurobasal culture medium as a basal culture medium, and also includes N-2, B-27, RA, ActivinA, ROCK signaling pathway inhibitor Y-27632, PS48, Glucose, ITS, arginine, GlutaMAX, lipids and FBS.

4. The porcine embryo in vitro culture medium according to claim 3, characterized in that The N2B27 medium also includes antibiotics.

5. The porcine embryo in vitro culture medium according to claim 4, characterized in that The antibiotics include one of penicillin, streptomycin, penicillin-streptomycin combination antibiotic and gentamicin.

6. The porcine embryo in vitro culture medium according to claim 5, characterized in that The volume percentage of N-2 in the porcine embryo in vitro culture medium is 1%, the volume percentage of B-27 is 2%, the concentration of RA is 0.5nM-5nM, the concentration of ActivinA is 10ng / mL-20ng / mL, the concentration of ROCK signaling pathway inhibitor Y-27632 is 5μm-15μm, the concentration of PS48 is 1μM-10μM, the concentration of Glucose is 0.1mg / mL-3mg / mL, the concentration of ITS is 1×-10×, the concentration of arginine is 0.5nM-8nM, the concentration of GlutaMAX is 1.5nM-5nM, the concentration of lipid is 1:200-1:800, the volume percentage of FBS is 3%-15%, the concentration of betaine is 2mM-10mM, and the concentration of antibiotics is 40-60IU / mL.

7. The porcine embryo in vitro culture medium according to any one of claims 3 to 6 is used in any one or more of the following: (1) Application in prolonging the in vitro culture time of pig embryos; (2) Application in improving the pluripotency of porcine embryos; (3) Application in promoting the differentiation of porcine embryonic germ layers; (4) Application in increasing porcine embryonic lineage differentiation and development; (5) Application in improving the developmental potential of in vitro cultured porcine embryos; (6) Application in improving the efficiency of in vitro culture of porcine embryos; (7) Application in increasing the diameter of pig embryos.

8. A method for culturing pig embryos in vitro, characterized in that: The in vitro culture method comprises the steps of using the N2B27 culture medium described in claim 3 for culture starting on the 6th day of parthenogenetic activation; and using the pig embryo in vitro culture medium described in claim 3 for culture from the 8.5th day to the 13.5th day.

9. A method for improving the developmental potential of in vitro cultured pig embryos, characterized in that: The method comprises the steps of using the N2B27 culture medium described in claim 3 to culture starting from the 6th day of parthenogenetic activation; and using the pig embryo in vitro culture medium described in claim 3 to culture from the 8.5th day to the 13.5th day.

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