Culture medium for improving in-vitro culture efficiency and quality of pig embryo and application of culture medium

Abstract

The invention relates to the technical field of animal cell culture, in particular to a culture medium for improving in-vitro culture efficiency and quality of pig embryos and application of the culture medium. The invention provides a new application of taurine and a taurine-containing pig embryo in-vitro culture medium, the in-vitro culture medium can significantly improve the in-vitro culture efficiency of the pig embryo and the quality of a pig blastocyst, can significantly prolong the in-vitro culture time of the pig embryo to 35 days, can improve the pluripotent state of the pig embryo, and has a good application prospect. Differentiation of epiderm and hypoderm of a pig embryo can be promoted, and differentiation and development of an embryo pedigree as well as development of nerves and a heart can be improved. The taurine-containing pig embryo in-vitro culture medium provided by the invention is a novel efficient in-vitro culture medium, can promote high-quality development of a pig blastocyst, and can be used for later culture of the pig embryo. In conclusion, the invention provides the culture medium for in-vitro long-term culture of the pig embryo, and also develops a method for improving the development efficiency and the development quality of the in-vitro cultured pig embryo.

Description

Technical Field

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

[0002] In vitro culture of mammalian embryos is a crucial biotechnology. It 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, proteins, and osmotic pressure regulators. The mouse embryo is a commonly used embryonic model for establishing mammalian in vitro culture systems and has laid 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 culture technology of embryos of large livestock such as pigs, cattle, and sheep lags significantly behind that of mice, humans, and primates. The reasons are: first, the quality of in vitro cultured embryos is poor; second, it cannot support long-term in vitro culture. Currently, PZM-3 is the most widely used culture system for in vitro embryo cultivation in pigs. It is designed based on inorganic substances and energy substrates in the pig oviduct. Although the composition of PZM-3 culture medium is clear, the efficiency of embryos cultured therein to develop into blastocysts is low, the quality of blastocysts is poor, and they can only be cultured to the mid-blastocyst stage. Therefore, the goal of the present invention is to develop a new and efficient in vitro culture medium that can be used for late-stage embryo culture and can also promote the high-quality development of pig blastocysts. Summary of the Invention

[0005] The purpose of the present invention is to provide a culture medium and application thereof for improving the efficiency and quality of in vitro culture of pig embryos, so as 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 taurine 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 promoting 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 promoting 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, the development includes one or more of neural development, cardiac development and placental development.

[0023] The invention provides a porcine embryo in vitro culture medium, which comprises an N2B27 culture medium and taurine; 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, ActivinA, Y-27632, PS48, Glucose, ITS, Arg, GlutaMAX, FBS, KSR and lipids.

[0024] Preferably, the N2B27 culture medium further comprises antibiotics.

[0025] Preferably, the antibiotics include one of penicillin, streptomycin, penicillin-streptomycin combination and gentamicin.

[0026] 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-40ng / mL, the concentration of 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 Arg is 1mM-3mM, the concentration of GlutaMAX is 1.5mM-5mM, the volume percentage of FBS is 3%-15%, the volume percentage of KSR is 5%-30%, the lipid addition ratio is 1:200-1:800, the concentration of antibiotics is 40-60IU / mL, and the concentration of taurine is 0.5mM-5mM.

[0027] Preferably, the volume ratio of the DMEM / F12 culture medium to the Neurobasal culture medium is 1:1.

[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 concentration, amino acids, and vitamins of DMEM with the various components of F-12, but does not contain 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 maintains 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) is a chemically defined, serum-free supplement based on Bottenstein's N-1 formulation. Supplied as a 100X concentrate, N-2 Supplement can be used with Neurobasal medium supplemented with growth factors (such as bFGF and EGF). It can also be used with Dulbecco's Membrane Ingredient (DMEM). This product is available from Thermo Fisher Scientific.

[0031] B-27 Supplement (Vitamin A Depleted, B-27) 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 an important role in cell growth, differentiation, and organogenesis. Retinoic acid is required for nearly 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 (RARα-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 cells, then recruits and phosphorylates the type I activin receptor (ActRI). ActivinA primarily signals through SMAD2 / 3 proteins to regulate various 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. This product was purchased from Peprotech.

[0034] Y-27632, 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 well-characterized 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. Its effectiveness is proven, significantly increasing human embryonic stem cell viability and enhancing their clonogenic capacity without affecting their pluripotency. This product was purchased from Thermo Fisher.

[0035] PS48 is a signaling pathway activator. It is 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 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 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 acts as an antioxidant in culture media. This product was purchased from Thermo Fisher Scientific.

[0038] Arg (L-arginine or arginine) is a substrate for nitric oxide synthase (eNOS) to produce nitric oxide (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 secondary energy source, particularly during rapid cell division. It also participates in the formation of purine and pyrimidine nucleotides, amino sugars, glutathione, L-glutamate, and other amino acids, and is involved in protein synthesis and glucose production. This product was purchased from Thermo Fisher Scientific.

[0040] KSR, a serum replacement, offers the advantages of clear, controllable composition and improved batch stability. Using KSR instead of traditional serum during the embryo implantation phase not only maintains nutrient supply in the culture system but also reduces exogenous interference factors, providing a more optimal microenvironmental support for embryonic development. This product was purchased from Thermo Fisher.

[0041] Fetal bovine serum (FBS) provides essential nutrients and growth factors for cell maintenance and growth and was purchased from Thermo Fisher.

[0042] Lipids: Chemically defined lipid concentrates are concentrated fat emulsions and were purchased from Thermo Fisher Scientific.

[0043] 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.

[0044] Antibiotics: can be used to prevent bacterial contamination of embryo culture, this product was purchased from Thermo Fisher.

[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 promoting 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] The present invention provides an in vitro culture method for pig embryos, which comprises the step of using the above-mentioned in vitro culture medium for culture starting from the 9th day after parthenogenetic activation.

[0054] The present invention provides a method for promoting the developmental potential of pig embryos cultured in vitro, which comprises the step of using the above-mentioned pig embryo in vitro culture medium for culture starting on the 9th day after parthenogenetic activation.

[0055] The present invention discloses the following technical effects:

[0056] The present invention provides a new application of taurine and an in vitro culture medium for pig embryos containing taurine. The in vitro culture medium can significantly improve the in vitro culture efficiency of pig embryos (embryo survival rate is 10% at D35) and the quality of pig blastocysts, can significantly increase the in vitro culture time of pig embryos, extending it to 35 days, can improve the pluripotency of pig embryos, can promote the differentiation of the epiblast and hypoblast of pig embryos, increase the differentiation and development of embryonic lineages, and the development of nerves and heart. The in vitro culture medium for pig embryos containing taurine provided by the present invention is a new and efficient in vitro culture medium that can promote the high-quality development of pig blastocysts and can be used for the late culture of pig embryos. In summary, the present invention provides a culture medium for long-term in vitro culture of pig embryos, and also develops a method for improving the development efficiency and development quality of in vitro cultured pig embryos. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] 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.

[0058] Figure 1 The effects of different concentrations of taurine on pig embryo development; A shows the developmental status of embryos at D9-D12 under different concentrations of taurine, with a scale bar of 100 μm; B shows the changes in embryo diameter at D9-D12 under different concentrations of taurine; C shows the statistical graph of embryo diameter at D12 under different concentrations of taurine treatment; D shows the statistical graph of embryo survival rate at D12 under different concentrations of taurine treatment;

[0059] Figure 2 The effect of adding 2.5mM taurine at different stages on embryonic development; A shows the status of embryos added with 2.5mM taurine at different stages until D10; B shows the statistical graph of embryo diameter at different stages until D10; C shows the statistical graph of embryo survival rate at different stages until D10;

[0060] Figure 3 This is a bright field image of the embryos in the experimental group with taurine added and cultured for D35; A is a bright field image of the embryos in the parthenogenetic activation group with 2.5 mM taurine added continuously starting from D9 and cultured for D35; B is a statistical graph of the in vitro delayed culture time; C is a statistical graph of the survival rate of the embryos in the experimental and control groups cultured for D35; D is a statistical graph of the survival rate curves of the control and experimental groups; E is the immunofluorescence results of the control and experimental groups; F is a statistical graph of the SOX expression levels in the control and experimental groups; G is a statistical graph of the SOX expression levels in the control and experimental groups;

[0061] Figure 4 Morphological changes of embryos obtained by in vitro delayed culture system; A shows the occurrence of hematopoiesis during culture, and the blue arrow indicates the hematopoietic part; B shows the occurrence of primitive streak during culture; C shows the occurrence of blastoderm during culture, and the blue arrow indicates the blastoderm structure; D shows the result of immunofluorescence detection; E shows the result of immunofluorescence detection

[0062] Figure 5 Embryonic cardiomyocyte differentiation occurs in an in vitro delayed culture system;

[0063] Figure 6 The images show beating cardiomyocytes in the embryo during culture; A is the result of immunofluorescence detection; B is the result of immunofluorescence detection;

[0064] Figure 7 These are the immunofluorescence detection results of D33 embryos in the in vitro delayed culture system; A is the immunofluorescence detection result; B is the immunofluorescence detection result, and the scale bar is 100 μm. DETAILED DESCRIPTION

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0070] Example 1

[0071] 1. Experimental Procedure

[0072] 1. Pig fertilized eggs and in vitro culture

[0073] Pig ovaries collected from slaughterhouses were transported to the laboratory at 37°C. The tissues were washed with 37°C 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 and diluted in a 100mm dish. Oocytes with intact COCs were selected using a glass needle under a microscope and transferred to oocyte maturation medium and cultured at 39°C and 5% CO2. After 42 hours of culture, the granulosa cells of the oocytes were removed by digestion with 0.1% hyaluronidase. Mature oocytes with the first polar body were selected under a microscope for subsequent experiments, and the data were recorded.

[0074] 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 in the fusion tank and apply a DC electric shock twice at 1.2V / cm with a pulse time of 30us. Transfer the oocytes to PZM-3 that has been equilibrated for at least 12 hours and rinse them three to four times. Then, transfer the oocytes to 500μL of PZM-3 and culture in an incubator at 38.5°C, 5% CO2, and saturated humidity. Observe and record the embryonic cleavage rate after 48 hours, and the embryonic rate on day 6 of development.

[0075] 2. Continuous Culture of Pig Embryos in Vitro

[0076] Select well-developed blastocysts from day 6.5 of PZM-3 culture (parthenogenetic activation) and transfer them to N2B27+Taurine medium (Table 2) that has been equilibrated for at least 4 hours. Rinse 3-4 times. Then, transfer the embryos to 500 μL of N2B27+Taurine medium and culture in an incubator. Change half of the medium every 24 hours, using N2B27 medium (Table 1) as a control. Take photos to record embryonic morphological changes.

[0077] Table 1 Components and concentrations of N2B27 culture medium

[0078] Element Concentration / volume percentage / addition ratio DMEM / F12: Neurobasal 1：1 N-2 1％ B-27 2％ RA 5nM ActivinA 20 ng / mL Y27632 10 μM PS48 5μM Glucose 2mg / mL lipids 1:500 ITS 1× Arg 1.69mM GlutaMAX 3.75mM FBS 10％ KSR 20％ Antibiotics (penicillin-streptomycin) 60 IU / mL

[0079] Table 2 Components and concentrations of N2B27+Taurine medium

[0080] Element Concentration / volume percentage / addition ratio DMEM / F12: Neurobasal 1：1 N-2 1％ B-27 2％ RA 5nM ActivinA 20 ng / mL Y27632 10 μM PS48 5μM Glucose 2mg / mL lipids 1:500 ITS 1× Arg 1.69mM GlutaMAX 3.75mM FBS 10％ KSR 20％ Antibiotics (penicillin-streptomycin) 60 IU / mL Taurine 0mM, 2.5mM, 5mM and 10mM

[0081] 3. Immunofluorescence detection of pig embryos

[0082] (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.

[0083] (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.

[0084] (3) Blocking: Transfer the sample into 500 μL of blocking solution and block for more than 1 hour.

[0085] (4) Primary antibody binding: Dilute the primary antibody (manufacturer: Peprotech, catalog number: 740013T) 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.

[0086] (5) Wash the sample in the cleaning solution three times, each time for 5 minutes.

[0087] (6) Secondary antibody binding: Dilute the secondary antibody (manufacturer: Invitrogen, catalog number: A21202) with cleaning solution in a certain ratio as required, add the sample to 400 μL of secondary antibody dilution solution, and incubate at room temperature in the dark for 1-2 h.

[0088] (7) Wash the sample in the cleaning solution three times, each time for 5 minutes.

[0089] (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.

[0090] (9) Sealing: Pressing: Add 30 μL of anti-fluorescence quencher to a clean glass slide, move the sample into the anti-fluorescence quencher drop, and place one end of a clean cover glass on the slide. Slowly tilt the cover glass until it is completely attached to the slide, and fix the cover glass with glycerol or nail polish. Observation under 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.

[0091] 2. Experimental results

[0092] To explore the optimal concentration of taurine, we cultured parthenogenetically activated blastocysts in an in vitro culture system until day 9 (D9), and added different concentrations of taurine, with the concentration gradient set as 0mM (control group), 2.5mM, 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 Figure A, the embryo morphology was observed to be the fullest in the experimental group supplemented with 2.5 mM taurine at D12. The embryo diameters of each group were further analyzed. Figure 1 As shown in B in . The results showed that the diameter of the control group embryos showed a downward trend during the culture process, which indicates that the control group did have the phenomenon of embryo shrinkage. The experimental groups that added 2.5mM and 5mM taurine were able to improve the phenomenon of embryo shrinkage, among which the experimental group that added 2.5mM taurine had the best effect. However, the higher the concentration of taurine added, the better. Although the embryos of the experimental group that added 10mM taurine were the fullest and had the largest diameter compared with the other groups when cultured to D10, the embryos showed the phenomenon of bursting. In order to more comprehensively evaluate the effect of taurine on embryos, this example further conducted a statistical analysis on the survival rate of each group of embryos. The statistical analysis results showed that when cultured to the 12th day (D12), the embryo survival rate of the group that added 2.5mM taurine was the highest (as shown in Figure 2). Figure 1 The diameter of the embryos in this group was the largest on D12 (as shown in D). Figure 1 The results were statistically significant (P < 0.05). Based on the above experimental results, it was concluded that the optimal concentration of taurine in the in vitro embryo culture system is 2.5 mM. The taurine concentration in the culture medium (2) used in subsequent experiments was 2.5 mM.

[0093] To explore the optimal time for adding taurine, a control group without taurine was set up, and 2.5mM taurine was added to the experimental group on the 6th day (D6), 7th day (D7), 8th day (D8), 9th day (D9) and 10th day (D10) of embryo culture (parthenogenetic activation). All groups were cultured from the 6th day (D6) and continued to the 12th day (D12) to observe and compare the effects of taurine addition at different times on embryo development. When cultured in vitro to the 12th day (D12), the morphology of the embryos in each group (such as Figure 2The results show that the embryos of the experimental group that added 2.5mM taurine on day 9 (D9) showed the most plump and best condition at day 12. Furthermore, the inventors calculated the embryo survival rate of each group (as shown in Figure 1). Figure 2 B in the figure) and embryo diameter (as shown in Figure 2 (As shown in C). In terms of survival rate, except for the experimental group supplemented with 2.5mM taurine on day 10 (D10), which showed no significant difference between the experimental group and the control group (P>0.05), all other experimental groups showed significant differences compared with the control group (P<0.05). More importantly, the experimental group supplemented with 2.5mM taurine on day 9 (D9) had the highest survival rate, and the difference from the control group was the most significant (P<0.05). In terms of embryo diameter, all experimental groups showed significant differences compared with the control group. Among them, the embryo diameter of the experimental group supplemented with 2.5mM taurine on day 9 (D9) was the largest, and the difference from the control group was the most significant. Conclusion: The optimal addition scheme for taurine in the in vitro embryo culture system is to continuously add it at a concentration of 2.5mM on day 9 of culture (D9, i.e., day 9 of parthenogenetic activation).

[0094] Bright field photos of embryonic development in the control group and experimental group are shown in the figure below. Figure 3 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 3 As shown in A in the figure, the embryos in the experimental group were more plump 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, and there was a significant difference between the two groups (P < 0.05). In terms of embryo survival rate, the embryo survival curves of the control group and the experimental group are shown in Figure 2. Figure 3 As shown in D. The data showed that the survival time of the embryos in the control group could only be maintained for about 11 days ( Figure 3 B), and the survival rate of the experimental group was much higher than that of the control group. Figure 3 On the 10th day of culture (D10), embryo samples from the control group and the experimental group were selected for immunofluorescence detection. The test results are as follows: Figure 3 As shown in E. The expression level of SOX2 in the embryos of the experimental group was significantly higher than that in the control group, and there was a significant difference compared with the control group (P < 0.05) (as shown in Figure 3 F in Figure 3 G). Immunofluorescence detection of upper and lower embryos. The results are as follows. Figure 3The results showed that compared with the control group, the experimental group had a higher expression of the mesoderm marker gene SOX2, with significant differences between the two groups. There was also a lower expression of the marker gene CDX2, indicating that N2B27+Taurine culture medium can promote the differentiation of embryonic germ layers.

[0095] After culturing for D16, the embryos were observed and the results showed that the embryonic disc appeared in the experimental group with taurine addition ( Figure 4 B in), blood cell formation ( Figure 4 A) and primitive streak-like structures ( Figure 4 C). Further in-depth research was conducted on parthenogenetic embryo samples that showed hematopoiesis during in vitro culture. The results of immunofluorescence staining are shown in Figure 4. Figure 4 As shown in D, the specific expression of RUNX1 gene was detected, which may form the yolk sac structure. At the same time, the immunofluorescence results also detected the specific expression of PAX3 gene (as shown in Figure 4 E) in the figure may form somites and somitic cavity structures.

[0096] When parthenogenetically activated embryos were cultured continuously until day 33, it was found that two embryo samples showed regular and sustained autonomous contraction rhythms, and it was preliminarily determined that they had myocardial activity characteristics ( Figure 5 ).

[0097] To further verify the differentiation characteristics of the embryos, immunofluorescence staining was used to detect the above samples. The results showed that the embryo samples specifically expressed the myocardial development marker genes GATA4 and NKX2.5 (such as Figure 6 A and B in the figure indicate that the embryos have undergone directed differentiation toward the cardiomyocyte lineage.

[0098] Immunofluorescence detection was performed on embryos cultured in vitro to day 33 (D33), and the specific expression of SOX2, SOX17, and TFAP2C was detected ( Figure 7 The results confirmed that this culture system effectively supports the differentiation of the three germ layers of the embryo.

[0099] Immunofluorescence detection was performed on embryos cultured in vitro to day 33 (D33), and the specific expression of BLIMP1 and TFAP2C genes was detected ( Figure 7 B), which indicates that this culture system may effectively support PGCs specification.

[0100] 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. The use of taurine 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 promoting 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 promoting 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 The development includes one or more of neural development, heart development and blastoderm development.

3. A porcine embryo in vitro culture medium, characterized in that: The porcine embryo in vitro culture medium includes N2B27 culture medium and taurine; the N2B27 culture medium uses DMEM / F12 culture medium and Neurobasal culture medium as a basic culture medium, and also includes N-2, B-27, RA, ActivinA, Y-27632, PS48, Glucose, ITS, Arg, GlutaMAX, FBS, KSR and lipids.

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-40ng / mL, the concentration of Y-27632 is 5μM-15μM, and 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 Arg is 1mM-3mM, the concentration of GlutaMAX is 1.5mM-5mM, the volume percentage of FBS is 3%-15%, the volume percentage of KSR is 5%-30%, the lipid addition ratio is 1:200-1:800, the concentration of antibiotics is 40-60IU / mL, and the concentration of taurine is 0.5mM-5mM.

7. The porcine embryo in vitro culture medium according to claim 5, characterized in that The volume ratio of the DMEM / F12 culture medium to the Neurobasal culture medium is 1:

1.

8. The porcine embryo in vitro culture medium according to any one of claims 3 to 7 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 promoting 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.

9. A method for culturing pig embryos in vitro, characterized in that: The in vitro culture method comprises the step of culturing the pig embryo in vitro culture medium according to any one of claims 3 to 7 starting from the 9th day of parthenogenetic activation.

10. A method for promoting the developmental potential of porcine embryos cultured in vitro, characterized in that: The method comprises the step of culturing the pig embryo in vitro culture medium according to any one of claims 3 to 7 starting from the 9th day of parthenogenetic activation.

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