A culture medium for bovine cloned embryos and its application

Abstract

The present invention relates to the technical field of bovine cloned embryo culture, and discloses a culture medium for bovine cloned embryos and its application. The culture medium is prepared with water, and includes the following substances and their contents: 6.81 g / L sodium chloride, 0.23 g / L potassium chloride, 2.2 g / L sodium bicarbonate, 0.04 g / L sodium pyruvate, 0.76 g / L calcium galactate, 0.05 v / v% double antibody, 2 v / v% essential amino acids, 1 v / v% non-essential amino acids, 0.146 g / L glutamine, 4 g / L bovine serum albumin and NAD + 10~100μmol / L. This culture medium can significantly increase the blastocyst rate and pregnancy rate of bovine cloned embryos, reduce the apoptosis rate and reactive oxygen level of bovine cloned embryos, and significantly improve the quality of bovine cloned embryos, which is conducive to optimizing the bovine somatic cell cloning operation system and reducing the cost of bovine cloned embryos from production to application.

Description

Technical Field

[0001] The invention belongs to the technical field of bovine cloned embryo culture, and in particular relates to a bovine cloned embryo culture solution and application thereof. Background Art

[0002] As a large agricultural animal, the development of cattle cloning technology has always attracted much attention. When different types of somatic cells, such as fibroblasts, cumulus cells, muscle cells, and mammary epithelial cells, are used as donor cells, cloned animals are obtained. At present, compared with the in vivo embryos of cattle, the development quality of cloned embryos of cattle is still not high, which is mainly reflected in the small number of blastocysts obtained, poor morphology, small number of cells in blastocysts, large number of apoptotic cells, low pregnancy rate, increased pregnancy time, giant fetuses, imbalanced sex ratio at birth, and high cost. The above reasons have seriously restricted the practical application of cloned embryo technology.

[0003] Therefore, the existing technology still needs to be improved and developed. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a culture medium for bovine cloned embryos and its application, wherein the culture medium can significantly reduce the reactive oxygen species (ROS) content and apoptosis level of bovine cloned embryos, improve the blastocyst rate and embryo quality of bovine cloned embryos, thereby reducing the cost of in vitro production of bovine cloned embryos.

[0005] The technical solution of the present invention is as follows:

[0006] In a first aspect, a culture medium for bovine cloned embryos is provided, wherein the culture medium is prepared with water and comprises the following substances and their contents: 6.81 g / L sodium chloride, 0.23 g / L potassium chloride, 2.2 g / L sodium bicarbonate, 0.04 g / L sodium pyruvate, 0.76 g / L calcium galactate, 0.05 v / v% bispecific antibody, 2 v / v% essential amino acids, 1 v / v% non-essential amino acids, 0.146 g / L glutamine, 4 g / L bovine serum albumin and NAD + 10~100μmol / L;

[0007] The dual antibody is prepared in water, and the substances and their contents are: penicillin 10000U / mL and streptomycin 10000μg / L;

[0008] Wherein, the essential amino acids are prepared with water, and the substances and their contents are as follows: L-arginine hydrochloride 6.32 g / L, L-cystine 1.20 g / L, L-histidine hydrochloride monohydrate 2.10 g / L, L-isoleucine 2.62 g / L, L-leucine 2.62 g / L, lysine hydrochloride 3.625 g / L, L-methionine 0.755 g / L, L-phenylalanine 1.65 g / L, L-threonine 2.38 g / L, L-tryptophan 0.51 g / L, L-tyrosine 1.80 g / L, L-valine 2.34 g / L;

[0009] The non-essential amino acids are prepared with water, and include the following substances and their contents: L-alanine 0.89 g / L, L-asparagine monohydrate 1.5 g / L, L-aspartic acid 1.33 g / L, L-glutamic acid 1.47 g / L, glycine 0.75 g / L, L-proline 1.15 g / L and L-serine 1.05 g / L.

[0010] The preferred technical solution is prepared with water, and the substances and their contents are as follows: sodium chloride 6.81g / L, potassium chloride 0.23g / L, sodium bicarbonate 2.2g / L, sodium pyruvate 0.04g / L, calcium galactate 0.76g / L, double antibody 0.05v / v%, essential amino acids 2v / v%, non-essential amino acids 1v / v%, glutamine 0.146g / L, bovine serum albumin 4g / L and NAD + 50μmol / L.

[0011] According to a preferred technical solution, the culture solution further comprises 50-150 μmol / L of sodium selenite.

[0012] According to a further preferred technical solution, the culture solution further comprises 100 μmol / L sodium selenite.

[0013] In a second aspect, a use of the culture medium as described in the first aspect in culturing bovine cloned embryos is provided.

[0014] A preferred technical solution is a method for applying the culture medium to bovine cloned embryo culture, comprising: placing the bovine cloned embryo in the culture medium and culturing in vitro under the conditions of 38.5°C, 5v / v% CO2, and 100% humidity.

[0015] In a further preferred technical solution, the in vitro culture time is 7 to 9 days.

[0016] Beneficial effect: The present invention provides a culture medium for bovine cloned embryos, wherein NAD is added to the embryo basic culture medium. + Furthermore, sodium selenite was added to the culture medium. 50 μmol / L NAD+ The culture medium can increase the blastocyst rate of bovine cloned embryos from 27.67% to 38.10%; the addition of 50μmol / L NAD + The culture medium containing 100 μmol / L sodium selenite can increase the blastocyst rate of bovine cloned embryos from 27.67% to 42.34%; thus, it can be seen that the culture medium of the present invention can significantly increase the blastocyst rate. At the same time, the ROS level and apoptosis of bovine cloned embryos cultured with the culture medium of the present invention were significantly reduced, and the pregnancy rate after embryo transplantation was significantly increased, further confirming the effectiveness of the culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a fluorescence graph of ROS content in bovine cloned embryos of the control group, experimental group 2, experimental group 5, and experimental group 7.

[0018] Figure 2 Different concentrations of NAD were added to the embryo basal culture medium + Histogram of blastocyst rate of cloned bovine embryos.

[0019] Figure 3 This is a bar graph of the blastocyst rate of bovine cloned embryos after adding different concentrations of sodium nitrite to the embryo basal culture medium.

[0020] Figure 4 50 μmol / L NAD was added to the embryo basal culture medium + and 100μmol / L sodium selenite.

[0021] Figure 5 It is a bar graph of apoptosis gene expression in bovine cloned embryos of the control group and experimental group 7.

[0022] Figure 6 It is a bar graph of the transplantation pregnancy rate of bovine cloned embryos in the control group and experimental group 7. DETAILED DESCRIPTION

[0023] The present invention provides a culture medium for bovine cloned embryos and application thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below.

[0024] The embodiment of the present invention provides a culture medium for cloned bovine embryos. The culture medium is prepared with water and includes the following substances and their contents: 6.81 g / L sodium chloride, 0.23 g / L potassium chloride, 2.2 g / L sodium bicarbonate, 0.04 g / L sodium pyruvate, 0.76 g / L calcium galactate, 0.05 v / v% double antibody, 2 v / v% essential amino acids, 1 v / v% non-essential amino acids, 0.146 g / L glutamine, 4 g / L bovine serum albumin and NAD +10~100μmol / L;

[0025] The dual antibody is prepared in water, and the substances and their contents are: penicillin 10000U / mL and streptomycin 10000μg / L;

[0026] Wherein, the essential amino acids are prepared with water, and the substances and their contents are as follows: L-arginine hydrochloride 6.32 g / L, L-cystine 1.20 g / L, L-histidine hydrochloride monohydrate 2.10 g / L, L-isoleucine 2.62 g / L, L-leucine 2.62 g / L, lysine hydrochloride 3.625 g / L, L-methionine 0.755 g / L, L-phenylalanine 1.65 g / L, L-threonine 2.38 g / L, L-tryptophan 0.51 g / L, L-tyrosine 1.80 g / L, L-valine 2.34 g / L;

[0027] The non-essential amino acids are prepared with water, and include the following substances and their contents: L-alanine 0.89 g / L, L-asparagine monohydrate 1.5 g / L, L-aspartic acid 1.33 g / L, L-glutamic acid 1.47 g / L, glycine 0.75 g / L, L-proline 1.15 g / L and L-serine 1.05 g / L.

[0028] Specifically, the present invention adds NAD to the embryo basal culture medium. + Nicotinamide adenine dinucleotide (NAD) is also known as coenzyme I, NAD + is its oxidized state. + NAD is present in every cell and is involved in hundreds of metabolic processes. It is an important substance for maintaining the healthy balance of the body and keeping the body young. + It can significantly increase the blastocyst development rate, effectively improve the quality of cloned embryos, and increase the pregnancy rate of cloned embryo transplantation, thereby reducing the cost of cloned embryo production and optimizing the cloned embryo production system.

[0029] In one embodiment, the culture medium is prepared with water, and the substances and their contents are as follows: sodium chloride 6.81 g / L, potassium chloride 0.23 g / L, sodium bicarbonate 2.2 g / L, sodium pyruvate 0.04 g / L, calcium galactate 0.76 g / L, double antibody 0.05 v / v%, essential amino acids 2 v / v%, non-essential amino acids 1 v / v%, glutamine 0.146 g / L, bovine serum albumin 4 g / L and NAD + 50μmol / L.

[0030] In one embodiment, the culture medium further comprises 50-150 μmol / L of sodium selenite. Sodium selenite can enhance the antioxidant capacity of cells by improving glutathione peroxidase activity and selenoprotein content.

[0031] In a more specific embodiment, the culture solution further includes 100 μmol / L sodium selenite.

[0032] An embodiment of the present invention provides a use of the culture medium as described above in bovine cloned embryo culture.

[0033] In one embodiment, the method of applying the culture medium to bovine cloned embryo culture comprises: placing the bovine cloned embryo in the culture medium and culturing in vitro under the conditions of 38.5° C., 5 v / v% CO 2 , and 100% humidity.

[0034] In a more specific embodiment, the in vitro culture time is 7 to 9 days.

[0035] The present invention will be further described below by means of specific examples.

[0036] The solution formula used in the embodiment of the present invention is as follows:

[0037] Egg collection fluid: M199 culture medium + 2.2g / L sodium bicarbonate (NaHCO3) + 2.38g / L 4-hydroxyethylpiperazineethanesulfonic acid (Hepes) + 1v / v% fetal bovine serum (FBS) + 0.2w / v% heparin + 1v / v% double antibody.

[0038] Maturation culture medium: M199 medium + 10mmol / L 4-hydroxyethylpiperazineethanesulfonic acid (Hepes) + 10v / v% fetal bovine serum (FBS) + 0.01μg / mL follicle stimulating hormone (FSH) + 1μg / mL luteinizing hormone (LH) + 0.01μg / mL estradiol (E2).

[0039] Operating solution: M199 medium + 10v / v% fetal bovine serum (FBS) + 7.5μg / mL cytochalasin (CB).

[0040] Fusion solution: water + 0.27 mol / L mannitol + 0.1 mmol / L calcium chloride (CaCl2) + 0.1 mmol / L magnesium chloride (MgCl2) + 0.05 w / v% bovine serum albumin (BSA).

[0041] Embryo basal culture medium: water + 6.81g / L sodium chloride (NaCl) + 0.23g / L potassium chloride (KCl) + 2.2g / L sodium bicarbonate (NaHCO3) + 0.04g / L sodium pyruvate + 0.76g / L hemi-calcium lactate + 0.05v / v% double antibody + 2v / v% essential amino acid + 1v / v% non-essential amino acid + 0.146g / L glutamine (Glutamax) + 4g / L bovine serum albumin (BSA).

[0042] Washing solution: water + 6.81g / L sodium chloride (NaCl) + 0.23g / L potassium chloride (KCl) + 2.2g / L sodium bicarbonate (NaHCO3) + 0.04g / L sodium pyruvate + 0.76g / L hemi-calcium lactate + 0.05v / v% double antibody + 2v / v% essential amino acid + 1v / v% non-essential amino acid + 0.146g / L glutamine (Glutamax) + 3v / v% fetal bovine serum (FBS).

[0043] M199 culture medium was purchased from Gibco with catalog number 31100019.

[0044] The dual antibody was purchased from Gibco with the catalog number 15140-122. Specifically, the dual antibody was prepared with water, and the substances included and their contents were: penicillin 10000 U / mL and streptomycin 10000 μg / L.

[0045] Essential amino acids were purchased from Gibco with a catalog number of 11130-051. Specifically, the essential amino acids were prepared with water, and the substances included and their contents were: L-arginine hydrochloride 6.32 g / L, L-cystine 1.20 g / L, L-histidine hydrochloride monohydrate 2.10 g / L, L-isoleucine 2.62 g / L, L-leucine 2.62 g / L, lysine hydrochloride 3.625 g / L, L-methionine 0.755 g / L, L-phenylalanine 1.65 g / L, L-threonine 2.38 g / L, L-tryptophan 0.51 g / L, L-tyrosine 1.80 g / L, and L-valine 2.34 g / L.

[0046] Non-essential amino acids were purchased from Sigma with the catalog number M-7145; specifically, the non-essential amino acids were prepared with water, and the substances included and their contents were: L-alanine 0.89 g / L, L-asparagine monohydrate 1.5 g / L, L-aspartic acid 1.33 g / L, L-glutamic acid 1.47 g / L, glycine 0.75 g / L, L-proline 1.15 g / L and L-serine 1.05 g / L.

[0047] Example

[0048] 1. Collection of ovarian oocytes

[0049] The oocytes of this embodiment are derived from slaughtered female livestock. After the female livestock is slaughtered, its ovaries are removed as soon as possible and placed in sterile saline at 20-25°C. During the collection process and before egg retrieval, the ovaries are repeatedly rinsed to prevent bacterial contamination. A 10mL injection needle is used to extract visible follicles from the surface of the ovary and placed in a pre-configured egg collection solution. According to the appearance characteristics of the cumulus-oocyte complex (COCs), oocytes with uniform cytoplasm and more than 3-5 layers of complete and dense cumulus cells are selected under a stereomicroscope.

[0050] 2. Oocyte maturation culture

[0051] Add about 500 μL of maturation culture medium to each well of the four-well plate, and place it in 38.5℃, 5v / v% CO2 for 1~2h to prepare a culture drop. Add 50 COCs to the culture drop, and after culturing for 24h, observe the expansion state of cumulus cells and whether the first polar body (PB1) is discharged to judge the maturation of oocytes. Add 0.1w / v% hyaluronidase to the four-well plate containing COCs, blow with a pipette until the cumulus cells fall off, pick up the oocytes from which the cumulus cells have been removed under a stereomicroscope, and check the discharge of PB1 and the morphology of the oocytes. The oocytes that discharge PB1 are oocytes that have matured to the MII stage. All oocytes with good morphology are washed with maturation culture medium, transferred to new culture drops, and wait for enucleation.

[0052] 3. Somatic cell culture

[0053] Bovine fetal fibroblasts were used as donor cells. The bovine fetuses that were 40 to 50 days old were taken out, the trunks were cleaned, and cut into pieces about 1 mm. 3 After washing, inoculate in a culture dish and then move into a CO2 incubator. During the primary culture period, replace the culture medium every 48 hours. When the cell confluence reaches 80%~90%, subculture.

[0054] 4. Remove the core

[0055] Use the operating solution to prepare the operating drop. Pick the oocyte with PB1 and transfer it into the pre-prepared 20μL operating drop, with 20 to 30 oocytes in each drop. Use the bevel injection needle to gently pierce the zona pellucida, move the injection needle to the bottom of PB1 and gently suck up PB1, and then the nuclear material of the egg will also enter the injection pipette. Quickly withdraw the injection needle to complete the enucleation operation.

[0056] 5. Nuclear transplantation

[0057] Use an injection needle to absorb donor cells with good morphology, uniform cytoplasm and a diameter of about 12μm, and then insert them into the zona pellucida along the hole left when the nucleus is removed. The donor cells are embedded between the egg plasma membrane and the zona pellucida to complete the production of reconstructed embryos.

[0058] 6. Fusion

[0059] The reconstructed embryos were washed 3-5 times in the maturation culture medium, transferred to new maturation culture medium and cultured for 15-30 minutes before fusion. The fusion voltage was 1.25 kV / cm and the shock time was 25 μs. The fusion was observed about 30 minutes after the fusion operation. If there were still unfused embryos, a second fusion operation could be performed.

[0060] 7. Activation of fusion embryos

[0061] The activation method used is the combined use of ionomycin and cycloheximide (CHX). During the operation, the fused embryos are first placed in a washing solution with an additional 5μmol / L ionomycin, and cultured in an incubator for 5 minutes; then, the embryos are washed in the washing solution, repeated twice, and then the embryos are placed in a washing solution with an additional 10μg / mL CHX for 5~6 hours; finally, after the embryos are fully washed in the washing solution, they are transferred to different culture solutions for culture. The different culture solutions are as follows:

[0062] Control group: embryo basal culture medium;

[0063] Experimental group 1: embryo basal culture medium + 10μmol / L NAD + ;

[0064] Experimental group 2: embryo basal culture medium + 50μmol / L NAD + ;

[0065] Experimental group 3: embryo basal culture medium + 100 μmol / L NAD + ;

[0066] Experimental group 4: embryo basal culture medium + 50 μmol / L sodium selenite;

[0067] Experimental group 5: embryo basal culture medium + 100 μmol / L sodium selenite;

[0068] Experimental group 6: embryo basal culture medium + 150 μmol / L sodium selenite;

[0069] Experimental group 7: embryo basal culture medium + 50μmol / L NAD + +100μmol / L sodium selenite.

[0070] 8. Detection of ROS levels in cloned embryos

[0071] The ROS level of cloned embryos at the 1-cell stage was detected using the Reactive Oxygen Species Detection Kit from Solebo. The procedure was performed according to the instructions of the kit, and images were collected using a laser confocal microscope. The fluorescence intensity was analyzed using Image J software. The results are shown in the figure below. Figure 1 shown.

[0072] 9. Embryo culture and blastocyst quality assessment

[0073] Add the culture medium of the control group and experimental groups 1 to 7 to the four-well plate, and cover with paraffin oil. After the fusion embryos are activated and washed, place the embryos in the four-well plate and culture them at 38.5℃, 5v / v%CO2 and saturated humidity. Culture 50 embryos in each well. Replace the corresponding culture medium every 72h, culture until the 7th day, and count the blastocyst rate.

[0074] Different concentrations of NAD were added to embryo basal culture medium + The statistical results of blastocyst rate are shown in Table 1 and Figure 2 As shown in Table 1 and Figure 2 It can be seen that compared with the control group, the experimental group 2 added 50μmol / L NAD in the embryo basal culture medium. + The blastocyst rate of cloned bovine embryos was significantly improved.

[0075] Table 1 Different concentrations of NAD added to embryo basal culture medium + Blastocyst rate

[0076]

[0077] The statistical results of the blastocyst rate in the embryo basal culture medium supplemented with sodium selenite are shown in Table 2 and Figure 3 As shown in Table 2 and Figure 3 It can be seen that compared with the control group, the blastocyst rate of bovine cloned embryos in experimental group 5 was significantly improved after adding 100 μmol / L sodium selenite to the embryo basal culture medium.

[0078] Table 2 Blastocyst rate of embryo basal culture medium with different concentrations of sodium selenite added

[0079]

[0080] NAD is added to embryo basal culture medium + The statistical results of blastocyst rate of sodium selenite are shown in Table 3 and Figure 4 As shown in Table 3 and Figure 4 It can be seen that compared with the control group, the experimental group 7 added 50μmol / L NAD in the embryo basal culture medium + and 100μmol / L sodium selenite, the blastocyst rate of bovine cloned embryos was significantly increased, and the difference was significant.

[0081] Table 3 Sodium selenite and NAD added to embryo culture medium + Blastocyst rate

[0082]

[0083] 10. Timed fluorescence quantitative PCR detection of apoptosis-related genes Bcl-2 and Caspase-3 mRNA expression levels

[0084] After the blastocysts were cleaned, 15 blastocysts were taken from each experimental group 7 and the control group, and lysed (purchased from Nanjing Novozyme Biotechnology Co., Ltd., catalog number: R711-01) and centrifuged. RNA from the blastocysts of each group was extracted using a kit and reverse transcribed to synthesize cDNA. The kit was used to complete gene amplification using a timed quantitative PCR instrument. ―ΔΔCt The relative expression of each gene was calculated by Figure 5 As shown. Figure 5 It can be seen that compared with the control group, the addition of NAD + and sodium selenite can significantly reduce the ROS content in bovine cloned embryos, and it can be seen that NAD + and sodium selenite can effectively reduce the ROS content in bovine cloned embryos and improve embryo quality.

[0085] 11. Pregnancy rate statistics

[0086] The pregnancy status of the cows was examined by B-ultrasound on the 45th, 90th and 180th day after transplantation. The results are shown in Table 4 and Figure 6 As shown in Table 4 and Figure 6 It can be seen that compared with the control group, the experimental group 7 added 50μmol / L NAD in the embryo basal culture medium + The pregnancy rate of bovine cloned embryos was significantly increased after addition of 100 μmol / L sodium selenite, P < 0.05.

[0087] Table 4 Pregnancy rate after transplantation of cloned bovine embryos

[0088]

[0089] In summary, the present invention provides a culture medium for bovine cloned embryos, wherein the culture medium is supplemented with NAD based on the embryo basal culture medium. + ; Furthermore, sodium selenite is added to the culture medium. The culture medium can significantly improve the blastocyst rate and pregnancy rate of bovine cloned embryos, reduce the apoptosis rate and ROS level of bovine cloned embryos, and significantly improve the quality of bovine cloned embryos, which is conducive to optimizing the bovine somatic cell cloning operation system and reducing the cost of bovine cloned embryos from production to application.

[0090] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A culture medium for bovine cloned embryos, characterized in that: The culture medium is prepared with water, and the substances and their contents are as follows: sodium chloride 6.81g / L, potassium chloride 0.23g / L, sodium bicarbonate 2.2g / L, sodium pyruvate 0.04g / L, calcium galactate 0.76g / L, double antibody 0.05v / v%, essential amino acids 2v / v%, non-essential amino acids 1v / v%, glutamine 0.146g / L, bovine serum albumin 4g / L, NAD + 10~100μmol / L and sodium selenite 50~150μmol / L; The dual antibody is prepared in water, and the substances and their contents are: penicillin 10000U / mL and streptomycin 10000μg / L; Wherein, the essential amino acids are prepared with water, and the substances and their contents are as follows: L-arginine hydrochloride 6.32 g / L, L-cystine 1.20 g / L, L-histidine hydrochloride monohydrate 2.10 g / L, L-isoleucine 2.62 g / L, L-leucine 2.62 g / L, lysine hydrochloride 3.625 g / L, L-methionine 0.755 g / L, L-phenylalanine 1.65 g / L, L-threonine 2.38 g / L, L-tryptophan 0.51 g / L, L-tyrosine 1.80 g / L, L-valine 2.34 g / L; The non-essential amino acids are prepared with water, and include the following substances and their contents: L-alanine 0.89 g / L, L-asparagine monohydrate 1.5 g / L, L-aspartic acid 1.33 g / L, L-glutamic acid 1.47 g / L, glycine 0.75 g / L, L-proline 1.15 g / L and L-serine 1.05 g / L.

2. The culture solution according to claim 1, characterized in that The culture medium is prepared with water, and the substances and their contents are as follows: sodium chloride 6.81g / L, potassium chloride 0.23g / L, sodium bicarbonate 2.2g / L, sodium pyruvate 0.04g / L, calcium galactate 0.76g / L, double antibody 0.05v / v%, essential amino acids 2v / v%, non-essential amino acids 1v / v%, glutamine 0.146g / L, bovine serum albumin 4g / L, NAD + 50μmol / L and sodium selenite 100μmol / L.

3. Use of the culture solution as claimed in any one of claims 1 to 2 in culturing bovine cloned embryos.

4. The use according to claim 3, characterized in that: The method of applying the culture medium to bovine cloned embryo culture comprises: placing the bovine cloned embryo in the culture medium and culturing in vitro under the conditions of 38.5°C, 5v / v% CO2 and 100% humidity.

5. The use according to claim 3, characterized in that: The in vitro culture time is 7 to 9 days.

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