Method for improving the efficiency of bovine in vitro embryo preparation

Abstract

The present invention provides a method for improving the efficiency of bovine in vitro embryo preparation, comprising the steps of in vitro collection of bovine oocytes, in vitro maturation and culture of cumulus-oocyte complexes, in vitro fertilization of bovine oocytes, and in vitro development and culture. The culture medium used in the in vitro development and culture contains 0.01-70 μM C 35 H 49 F2N7O4·CF3CO2H (MM102). By selecting the optimal bovine IVF system under in vitro conditions and placing the blastomeres in development medium supplemented with varying concentrations of MM102 after 48 hours of IVF development, the results showed that MM102 promoted the in vitro development of bovine zygotes, resulting in a high blastocyst rate. This method can be used to improve the efficiency of bovine IVF embryo preparation. This invention can provide a theoretical basis for research on livestock IVF technology and can be further applied to livestock breeding and genetic conservation.

Description

Technical Field

[0001] The present invention relates to the fields of cell biology and molecular biology, and in particular to a method for improving the efficiency of bovine in vitro embryo preparation. Background Art

[0002] Cattle IVF technology includes the in vitro collection and maturation of oocytes, in vitro capacitation of sperm and in vitro fertilization of sperm and eggs, and in vitro culture of fertilized eggs. In 1977, Iritane and Niwa obtained the world's first in vitro fertilized cow. my country's in vitro fertilization research began in the late 1980s. Based on the research of predecessors, my country has made rapid progress. In 1989, Academician Xu Rigan and others successfully bred my country's first in vitro fertilized cow. Since then, research teams such as Fan Biqin, Lu Kehuan, and Shi Deshun have also successively obtained in vitro fertilized cattle. So far, due to the widespread use of in vitro embryo production technology, the offspring of most domestic animals have been successfully obtained. Although cattle are one of the earliest animals studied in in vitro fertilization of livestock, their production efficiency is not satisfactory.

[0003] Mixed lineage leukemia (MLL1) is an H3K4 methyltransferase. Inhibiting the activity of MLL1 is a new therapeutic strategy for acute leukemia caused by MLL1 fusion protein. MLL1 is only active when WDR / MLL protein interacts. 35 H 49 F2N7O4·CF3CO2H) is an inhibitor of the WDR / MLL protein interaction. When leukemia cells express the MLL1-AF9 fusion gene, it inhibits MLL1 methyltransferase activity and MLL1-induced expression of the HoxA9 and Meis-1 genes. Currently, MM102 has not been studied for improving embryonic development. Summary of the Invention

[0004] The purpose of this invention is to provide a method for improving the efficiency of bovine in vitro embryo preparation. By adding MM102 during embryonic development after in vitro fertilization, the effect of MM102 on bovine embryo development is explored, providing a reference for in vitro fertilization of livestock and improving blastocyst rates.

[0005] In order to achieve the purpose of the present invention, the present invention provides a method for improving the efficiency of bovine in vitro embryo preparation, including the steps of in vitro collection of bovine oocytes, in vitro maturation and culture of cumulus ovary-oocyte complexes, in vitro fertilization of bovine oocytes, and in vitro development and culture.

[0006] The culture medium (IVC-2 medium) used for in vitro development culture contains 0.01-70 μM C 35 H 49F2N7O4·CF3CO2H. Preferably, the IVC-2 solution contains 30μM, 50μM or 70μM C 35 H 49 F2N7O4·CF3CO2H, more preferably 50μM.

[0007] According to the aforementioned method, when the IVC-2 solution contained 0 μM MM102, the blastocyst rate was 24.33%.

[0008] According to the aforementioned method, when the IVC-2 solution contained 30 μM MM102, the blastocyst rate was 25.47%.

[0009] In the aforementioned method, when the IVC-2 solution contained 50 μM MM102, the blastocyst rate was 32.61%, which was higher than that of other systems.

[0010] According to the aforementioned method, when the IVC-2 solution contained 70 μM MM102, the blastocyst rate was 29.65%.

[0011] In the aforementioned method, when the MM102 concentration was between 0 and 50 μM, the blastocyst rate gradually increased with the increase of MM102 concentration, and the blastocyst morphology was normal.

[0012] In the aforementioned method, when the MM102 concentration reached 70 μM, the blastocyst rate decreased, but the blastocyst morphology remained normal.

[0013] Immunofluorescence staining of pluripotency genes in blastocysts cultured under the above developmental conditions revealed no difference.

[0014] Preferably, the formula of the IVC-2 solution is:

[0015]

[0016] Wherein, the formula of the SOF liquid is:

[0017]

[0018] The formula of the storage solution A is:

[0019]

[0020] The formula of the storage solution B is:

[0021] 10 mL of water

[0022] NaHCO3 0.21g

[0023] The formula of the storage solution C is:

[0024] 10 mL of water

[0025] Sodium pyruvate 0.08g

[0026] The formula of the storage solution D is:

[0027] 10 mL of water

[0028] CaCl2·2H2O 0.262g

[0029] In the aforementioned method, the culture medium formula used for in vitro maturation culture of the cumulus-oocyte complex is:

[0030]

[0031] In the aforementioned method, in vitro fertilization is performed in solution A and solution B, wherein the formula of solution A is: in vitro fertilization stock solution + 10 mM caffeine, and the formula of solution B is: in vitro fertilization stock solution + 20 mg / mL bovine serum albumin + 1 IU / mL heparin;

[0032] Wherein, the formula of the in vitro fertilization storage solution is:

[0033]

[0034] The aforementioned method preferably uses a syringe extraction method to collect cumulus oocyte-oocyte complexes with uniform cytoplasm and multiple layers of intact granulosa cells, and then matures and cultures them in vitro for 22-24 hours.

[0035] The above-mentioned method uses frozen semen from a bull in the in vitro fertilization of bovine oocytes. The washed semen and the cumulus ovary-oocyte complex that has matured in vitro are placed in the fertilization solution. After fertilization, the granulosa cells are removed and the oocytes are cultured in IVC-1 solution for 48 hours. Then, they are transferred to IVC-2 solution and developed in vitro for 7-9 days, and the blastocyst rate is counted.

[0036] Wherein, the formula of the IVC-1 liquid is:

[0037]

[0038] In the present invention, the in vitro culture conditions are: 38.5°C, 5% CO2, and a saturated humidity incubator.

[0039] The present invention also provides an in vitro development culture medium IVC-2 for improving the efficiency of bovine in vitro embryo preparation, wherein the IVC-2 solution contains 0.01-70 μM C 35 H 49 F2N7O4·CF3CO2H. Preferably, the IVC-2 solution contains 30μM, 50μM or 70μM C 35 H 49 F2N7O4·CF3CO2H, more preferably 50μM.

[0040] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0041] This method uses an optimized bovine IVF system under in vitro conditions. After 48 hours of IVF development, the blastomeres are placed in a development medium supplemented with varying concentrations of MM102. Results show that MM102 promotes the in vitro development of bovine zygotes, resulting in a high blastocyst rate. This method can be used to improve the efficiency of bovine IVF embryo preparation. This invention provides a theoretical basis for research into livestock IVF technology and can be further applied to livestock breeding and genetic conservation. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The morphology of the blastocysts after in vitro fertilization on the 7th day under the culture system of MM102 at different concentrations in Example 3 of the present invention is shown.

[0043] Figure 2 These are the results of immunofluorescence staining of pluripotency genes in blastocysts cultured in Example 3 of the present invention when the IVC-2 solution contained 0 μM MM102.

[0044] Figure 3 The results of immunofluorescence staining of pluripotency genes in blastocysts cultured in Example 3 of the present invention when the IVC-2 solution contained 30 μM MM102.

[0045] Figure 4 The results of immunofluorescence staining of pluripotency genes in blastocysts cultured in Example 3 of the present invention when the IVC-2 solution contained 50 μM MM102.

[0046] Figure 5 These are the results of immunofluorescence staining of pluripotency genes in blastocysts cultured in Example 3 of the present invention when the IVC-2 solution contained 70 μM MM102.

[0047] Figure 1-Figure 5 The scale bar in the figure is 100 μm. DETAILED DESCRIPTION

[0048] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0049] Example 1 In vitro collection and in vitro maturation culture of bovine oocytes

[0050] Ovaries from freshly slaughtered cattle were collected from the slaughterhouse, stored in a clean, insulated bucket filled with 0.9% sterile saline, and transported to the laboratory within 2-4 hours. Upon arrival, the ovaries were washed at least three times in 0.9% sterile saline at 20-25°C. Oocytes were collected from the bovine ovaries using a syringe aspiration method, extracting follicles 2-7 mm in diameter from the ovarian surface. Cumulus-oocyte complexes (COCs) with uniform cytoplasm and multiple layers of intact granulosa cells were collected from the aspirated follicular fluid under a stereomicroscope. After washing three times with in vitro maturation medium, the COCs were quickly collected and cultured in maturation medium for in vitro maturation. All COCs were cultured in four-well plates using 700 μL of in vitro maturation medium per well, with 300 μL of paraffin oil overlayed on the surface of the medium. Culture conditions were 50 COCs per well in a 38.5°C, 5% CO2, and saturated humidity incubator.

[0051] The formula of the in vitro maturation culture medium is:

[0052]

[0053] Example 2 In vitro fertilization of bovine oocytes

[0054] After the oocytes have been matured in vitro for 22-24 hours, they are fertilized in vitro. Thaw the purchased bull semen in a 15mL sterile centrifuge tube containing 8mL of in vitro fertilization solution A at a ratio of 100:1 between the number of eggs and the number of semen tubes. Centrifuge at 37℃, 4000 rpm for 5 minutes. Wash twice, remove the supernatant, and slowly add 1-2mL of in vitro fertilization culture medium to the surface of the sperm sediment. Float it in a 37℃ water bath for 3-5 minutes, and draw the turbid part of the upper layer into a 1.5mL centrifuge tube. Add an equal volume of in vitro fertilization solution B, mix well, and add fertilization drops. The sperm concentration of each fertilization drop is 1×10 6 -5×10 6 After in vitro maturation, COCs were washed three times with IVF medium A+B (a mixture of A and B in equal volumes), and 15-20 were transferred into the fertilization drop. The culture dish was placed in an incubator for in vitro fertilization. The culture system consisted of 100 μL of IVF medium per drop, with paraffin oil layered on the surface of the liquid layer. The culture conditions were a 38.5°C, 5% CO2, and saturated humidity incubator.

[0055] Among them, the formula of IVF solution A is: IVF storage solution + 10mM caffeine, and the formula of IVF solution B is: IVF storage solution + 20mg / mL bovine serum albumin + 1IU / mL heparin.

[0056] The formula of the in vitro fertilization storage solution is:

[0057]

[0058]

[0059] Example 3 In vitro development and culture of bovine fertilized eggs

[0060] After 6 hours of in vitro fertilization, the zygotes were transferred to in vitro development medium. The zygotes were gently pipetted repeatedly with a 100 μL pipette to remove granulosa cells and sperm adhered to the zygotes, and then washed three times with IVC-1 solution. The zygotes were transferred to individual drops of in vitro development IVC-1 solution, 20 per drop, and the culture dishes were placed in an incubator for 48 hours of development. The culture system consisted of 50 μL of in vitro development medium per drop, with paraffin oil layered on the surface. The eggs were equilibrated in a CO2 incubator for at least 5 hours before in vitro development. After 48 hours of in vitro culture, the number of cleavages was counted, and the cleavage embryos were washed three times with IVC-2 solution containing different concentrations of MM102 and placed in the corresponding drop of development IVC-2 solution for further culture. The blastocyst rate was calculated on days 7-9 (Table 1). Culture conditions were 38.5°C, 5% CO2, and a saturated humidity in an incubator.

[0061] Wherein, the formula of the IVC-1 liquid is:

[0062]

[0063] The formula of the IVC-2 liquid is:

[0064]

[0065] Wherein, the formula of the SOF liquid is:

[0066]

[0067] The formula of the storage solution A is:

[0068]

[0069] The formula of the storage solution B is:

[0070] 10 mL of water

[0071] NaHCO3 0.21g

[0072] The formula of the storage solution C is:

[0073] 10 mL of water

[0074] Sodium pyruvate 0.08g

[0075] The formula of the storage solution D is:

[0076] 10 mL of water

[0077] CaCl2·2H2O 0.262g

[0078] In the aforementioned method, the culture medium formula used for in vitro maturation culture of the cumulus-oocyte complex is:

[0079]

[0080] Table 1 Blastocyst development rate under different concentrations of MM102 culture system

[0081]

[0082]

[0083] The morphology of blastocysts after in vitro fertilization on day 7 under different concentrations of MM102 culture system Figure 1 When the MM102 concentration was between 0 and 50 μM, the blastocyst rate gradually increased with the increase of MM102 concentration, and the blastocyst morphology was normal. When the MM102 concentration reached 70 μM, the blastocyst rate decreased, but the blastocyst morphology remained normal.

[0084] The blastocysts cultured in IVC-2 solution containing 0μM, 30μM, 50μM, and 70μM MM102 were subjected to immunofluorescence staining of pluripotency genes. The results are shown in Table 1. Figure 2-Figure 5 .from Figure 2-Figure 5 It can be seen that the addition of different concentrations of MM102 has no significant effect on the expression of pluripotent genes in bovine blastocysts.

[0085] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for improving the efficiency of bovine in vitro embryo preparation, characterized in that: The method includes the steps of in vitro collection of bovine oocytes, in vitro maturation and culture of cumulus oocyte-oocyte complexes, in vitro fertilization of bovine oocytes, and in vitro development and culture; The culture medium used for in vitro development culture contained 50 μM C 35 H 49 F2N7O4·CF3CO2H; The culture medium used for in vitro development culture, IVC-2 medium, has the following formula: Wherein, the formula of the SOF liquid is: The formula of the storage solution A is: The formula of the storage solution B is: 10 mL of water NaHCO3 0.21g The formula of the storage solution C is: 10 mL of water Sodium pyruvate 0.08g The formula of the storage solution D is: 10 mL of water CaCl2·2H2O 0.262g; The culture medium formula used for in vitro maturation of cumulus-oocyte complexes is: In vitro fertilization was performed in solution A and solution B, where the formula of solution A was: in vitro fertilization stock solution + 10 mM caffeine, and the formula of solution B was: in vitro fertilization stock solution + 20 mg / mL bovine serum albumin + 1 IU / mL heparin; Wherein, the formula of the in vitro fertilization storage solution is: In in vitro fertilization of bovine oocytes, frozen semen from bulls is used. Washed semen and in vitro matured cumulus oocyte-oocyte complexes are placed in fertilization fluid. After fertilization, granulosa cells are removed and oocytes are cultured in IVC-1 fluid for 48 hours. They are then transferred to IVC-2 fluid and allowed to develop in vitro for 7-9 days. The blastocyst rate is then calculated. Wherein, the formula of the IVC-1 liquid is:

2. The method according to claim 1, characterized in that The collected cumulus oocyte complex was matured and cultured in vitro for 22-24 hours.

3. The method according to claim 1 or 2, characterized in that The in vitro culture conditions were: 38.5°C, 5% CO2, and saturated humidity incubator.

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