CN110628709A - Culture solution and culture method for improving in-vitro maturation quality of porcine oocytes - Google Patents
Culture solution and culture method for improving in-vitro maturation quality of porcine oocytes Download PDFInfo
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- 210000000287 oocyte Anatomy 0.000 title claims abstract description 97
- 238000000338 in vitro Methods 0.000 title claims abstract description 47
- 230000035800 maturation Effects 0.000 title claims abstract description 42
- 238000012136 culture method Methods 0.000 title claims abstract description 12
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims abstract description 22
- 210000001733 follicular fluid Anatomy 0.000 claims abstract description 13
- 229940054269 sodium pyruvate Drugs 0.000 claims abstract description 11
- PWKSKIMOESPYIA-UHFFFAOYSA-N 2-acetamido-3-sulfanylpropanoic acid Chemical compound CC(=O)NC(CS)C(O)=O PWKSKIMOESPYIA-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229930182555 Penicillin Natural products 0.000 claims abstract description 7
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims abstract description 7
- 229940049954 penicillin Drugs 0.000 claims abstract description 7
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 88
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 29
- 239000007995 HEPES buffer Substances 0.000 claims description 29
- JXSVIVRDWWRQRT-UYDOISQJSA-N asiatic acid Chemical compound C1[C@@H](O)[C@H](O)[C@@](C)(CO)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CC[C@@H](C)[C@H](C)[C@H]5C4=CC[C@@H]3[C@]21C JXSVIVRDWWRQRT-UYDOISQJSA-N 0.000 claims description 26
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- CLXOLTFMHAXJST-UHFFFAOYSA-N esculentic acid Natural products C12CC=C3C4CC(C)(C(O)=O)CCC4(C(O)=O)CCC3(C)C1(C)CCC1C2(C)CCC(O)C1(CO)C CLXOLTFMHAXJST-UHFFFAOYSA-N 0.000 claims description 26
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 24
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 22
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- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 20
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 claims description 11
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 claims description 11
- 235000020778 linoleic acid Nutrition 0.000 claims description 11
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- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 5
- 239000001110 calcium chloride Substances 0.000 claims description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 5
- 239000002480 mineral oil Substances 0.000 claims description 5
- 235000010446 mineral oil Nutrition 0.000 claims description 5
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- 239000011780 sodium chloride Substances 0.000 claims description 5
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- 239000001103 potassium chloride Substances 0.000 claims description 4
- 235000011164 potassium chloride Nutrition 0.000 claims description 4
- ZWQVYZXPYSYPJD-RYUDHWBXSA-N Glu-Gly-Phe Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 ZWQVYZXPYSYPJD-RYUDHWBXSA-N 0.000 claims description 3
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
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- 238000003825 pressing Methods 0.000 claims description 3
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 3
- NGSFWBMYFKHRBD-DKWTVANSSA-M sodium;(2s)-2-hydroxypropanoate Chemical compound [Na+].C[C@H](O)C([O-])=O NGSFWBMYFKHRBD-DKWTVANSSA-M 0.000 claims description 3
- 229960005322 streptomycin Drugs 0.000 claims description 3
- 210000004681 ovum Anatomy 0.000 claims description 2
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- 238000011161 development Methods 0.000 abstract description 4
- 229940000207 selenious acid Drugs 0.000 abstract description 3
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- XDFNWJDGWJVGGN-UHFFFAOYSA-N 2-(2,7-dichloro-3,6-dihydroxy-9h-xanthen-9-yl)benzoic acid Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC(Cl)=C(O)C=C2OC2=CC(O)=C(Cl)C=C21 XDFNWJDGWJVGGN-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 2
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- 238000011160 research Methods 0.000 description 2
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- 244000146462 Centella asiatica Species 0.000 description 1
- 235000004032 Centella asiatica Nutrition 0.000 description 1
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- VJEONQKOZGKCAK-UHFFFAOYSA-N caffeine Natural products CN1C(=O)N(C)C(=O)C2=C1C=CN2C VJEONQKOZGKCAK-UHFFFAOYSA-N 0.000 description 1
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- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0608—Germ cells
- C12N5/0609—Oocytes, oogonia
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/31—Pituitary sex hormones, e.g. follicle-stimulating hormone [FSH], luteinising hormone [LH]; Chorionic gonadotropins
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- Health & Medical Sciences (AREA)
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- Bioinformatics & Cheminformatics (AREA)
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- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Developmental Biology & Embryology (AREA)
- Microbiology (AREA)
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Abstract
The invention discloses a culture solution and a culture method for improving in-vitro maturation quality of porcine oocytes, wherein the culture solution comprises the following components: m199 culture solution, 10ng/mL EGF, 10. mu.M linoleic acid, 0.6mM L-cysteine, 0.91mM sodium pyruvate, 10% follicular fluid, 10IU/mL LH, 10IU/mL FSH and 100IU/mL penicillin. According to the invention, the selenious acid is added into the in vitro maturation culture solution of the porcine oocyte for the first time, so that the maturation rate and the quality of the oocyte can be effectively improved, the development rate and the embryo quality of parthenogenetic embryo and in vitro fertilization embryo can be promoted, and the problem of low in quality of the in vitro maturation and in vitro culture efficiency at present is effectively solved.
Description
Technical Field
The invention relates to a culture solution and a culture method for improving the quality of porcine oocytes, in particular to a culture solution and a culture method for improving the in-vitro maturation quality of porcine oocytes, and belongs to the field of application of animal breeding technology.
Background
Animal embryo engineering is rapidly developed at present, and requirements on quality and quantity of oocytes are gradually improved. Since the number of oocytes matured in vivo and the method of retrieval are limited, in vitro maturation of oocytes becomes an important source for the in vitro production of embryos and plays an important role in the field of animal reproduction. In vitro maturation of oocytes recovered from slaughterhouses is therefore the most cost effective means. The quality of the oocyte is of great significance to fertilization, early embryo development and embryo transfer, and how to improve the quality of the oocyte becomes a current research hotspot.
Asiatic acid, also known as asiatic acid, is a pentacyclic triterpene isolated from the juice of centella asiatica leaves, which has proven to have few side effects in numerous applications and is widely used in medicine. According to the previous literature reports, three functionally related structures are: three hydroxyl groups, one unsaturated bond and one carboxyl group. In vivo and in vitro experiments, the asiatic acid has proved many pharmacological activities, and its biological actions mainly include antioxidation, anti-apoptosis and anti-aging. Currently, no research indicates whether the quality of oocytes cultured in vitro can be improved by using the asiatic acid, so that the addition of the asiatic acid into the in vitro culture solution for improving the quality of the oocytes has important practical significance for producing high-quality oocytes and promoting the development of early embryos.
Disclosure of Invention
The present invention has been made to solve the above problems, and an object of the present invention is to provide a culture solution and a culture method for improving the quality of in vitro maturation of porcine oocytes.
The invention realizes the aim through the following technical scheme, and provides a culture solution for improving the in vitro maturation quality of porcine oocytes, which comprises the following components: m199 culture solution, 10ng/mL EGF, linoleic acid, 0.6mM L-cysteine, 0.91mM sodium pyruvate, 10% follicular fluid, 10IU/mL LH, 10IU/mL FSH and 100IU/mL penicillin.
Preferably, the follicular fluid is prepared by the following steps:
(1) precipitating the liquid extracted from 3-8mm follicle for 1h, and collecting the supernatant;
(2) centrifuging the supernatant at 8000rpm for 15min, removing precipitate, and repeating the above steps for 3 times;
(3) finally, the liquid was filtered through a 0.22 μm filter and dispensed as 1mL per tube and stored at-20 ℃.
Preferably, the M199 culture solution, EGF, asiatic acid, L-cysteine, sodium pyruvate, penicillin are all purchased from Sigma, and the LH and FSH are purchased from Ningbo second hormone plant, and the asiatic acid is sold under the reference 546712.
Preferably, the culture solution preparation process comprises the following steps:
(1) 97.74mg of fine linoleic acid was weighed out and dissolved in 2mL of DMSO and filtered through a 0.22 μm filter to prepare a 100mM concentrated solution of fine linoleic acid;
(2) preparing the culture solution of claim 1 into a basic culture solution, filtering with a 0.22 μm filter, and storing at 4 deg.C;
(3) diluting the 100mM concentrated solution of the asiatic acid prepared in the step (1) into 10 mu M working solution of the asiatic acid by using the basic culture solution prepared in the step (2);
(4) adding the 10. mu.M of the working solution of linoleic acid prepared in step (3) to the basic culture solution according to claim 1 to prepare a culture solution.
Preferably, the DMSO is purchased from Sigma company.
6. A culture method for improving the in vitro maturation quality of porcine oocytes comprises the following steps:
(1) collecting the pig ovaries which are just slaughtered from a slaughterhouse, putting the pig ovaries into physiological saline water with double antibodies and the temperature of 37 ℃, and sending the pig ovaries to a laboratory within 2 hours by workers; the culture solution prepared in claim 3 was placed at 38.5 ℃ in 5% CO2Balancing in a carbon dioxide incubator with 95% of air and saturated humidity for 3 hours in advance;
(2) washing ovary with 0.9% physiological saline at 37 deg.C for 2-3 times, temporarily storing the washed ovary in water bath at 37 deg.C, selecting 10mL syringe with 12-gauge needle, pressing ovary by hand to generate certain negative pressure, and puncturing follicle of 3-8mm from one side of ovary;
(3) storing the extracted follicular fluid into a 10mL centrifugal tube, automatically precipitating for 10min, sucking the supernatant by using a pasteur tube and discarding, and adding an HEPES buffer solution into the centrifugal tube to repeatedly wash the follicular fluid precipitate for 3 times;
(4) placing the cleaned follicular fluid precipitate and HEPES buffer solution into a culture dish, picking up oocytes with two to three layers of cumulus ova, uniform cytoplasm and good morphology by using a mouth suction tube under a stereoscopic microscope, placing the oocytes into a four-hole plate, adding 600 mu L of culture solution balanced in advance in the step (1) into each hole, covering the surface of the culture solution with 450 mu L of mineral oil, placing the four-hole plate into a culture dish, placing the four-hole plate into a container with 38.5 ℃ and 5% CO2Culturing in a carbon dioxide incubator with 95% air and saturated humidity for 42-44 h.
Preferably, the HEPES buffer comprises the following components: 1L of high Voltage ddH2O, 6.6633g/L sodium chloride, 0.2386g/L potassium chloride, 0.1680g/L sodium bicarbonate, 0.0408g/L sodium dihydrogen phosphate, 1.868mL/L sodium lactate, 0.1017g/L magnesium chloride, 0.022g/L sodium pyruvate, 2.38g/L HEPES,0.065g/L streptomycin, 0.294g/L calcium chloride, 1 g/LPVA.
Preferably, the HEPES buffer solution is preheated by a 38.5 ℃ water bath before use, and the pH of the HEPES buffer solution is 7.2-7.4, and the osmotic pressure is 270-290 mOsm.
Preferably, the formulated HEPES buffer is filtered through a 0.22 μm filter and stored at 4 ℃.
Preferably, the number of oocytes in each well of the four-well plate is 100-110.
The invention has the beneficial effects that: according to the invention, the selenious acid is added into the in vitro maturation culture solution of the porcine oocyte for the first time, so that the maturation rate and the quality of the oocyte can be effectively improved, the development rate and the embryo quality of parthenogenetic embryo and in vitro fertilization embryo can be promoted, and the problem of low in quality of the in vitro maturation and in vitro culture efficiency at present is effectively solved.
Drawings
FIG. 1 is a schematic representation of the detection of ROS and GSH levels in oocytes of the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 preparation of in vitro maturation Medium containing Fine Iminic acid
A culture solution for improving the in vitro maturation quality of porcine oocytes, comprising the following components: m199 culture solution, 10ng/mL EGF, linoleic acid, 0.6mM L-cysteine, 0.91mM sodium pyruvate, 10% follicular fluid, 10IU/mL LH, 10IU/mL FSH and 100IU/mL penicillin.
The M199 culture solution, EGF, asiatic acid, L-cysteine, sodium pyruvate, penicillin were purchased from Sigma, and the LH and FSH were purchased from Ningbo second hormone plant, and the asiatic acid was purchased under the reference number 546712.
The preparation process of the culture solution comprises the following steps:
(1) 97.74mg of fine linoleic acid was weighed out and dissolved in 2mL of DMSO and filtered through a 0.22 μm filter to prepare a 100mM concentrated solution of fine linoleic acid;
(2) preparing the culture solution of claim 1 into a basic culture solution, filtering with a 0.22 μm filter, and storing at 4 deg.C;
(3) diluting the 100mM concentrated solution of the asiatic acid prepared in the step (1) into 10 mu M working solution of the asiatic acid by using the basic culture solution prepared in the step (2);
(4) adding the 10. mu.M of the working solution of linoleic acid prepared in step (3) to the basic culture solution according to claim 1 to prepare a culture solution.
The DMSO was purchased from Sigma.
The culture solution is used within three days after preparation.
Example 2 maturation of porcine oocytes
A culture method for improving the in vitro maturation quality of porcine oocytes comprises the following steps:
(1) collecting the pig ovaries which are just slaughtered from a slaughterhouse, putting the pig ovaries into physiological saline water with double antibodies and the temperature of 37 ℃, and sending the pig ovaries to a laboratory within 2 hours by workers; placing the culture solution prepared in the step (4) at 38.5 deg.C and 5% CO2The test group is obtained by balancing in a carbon dioxide incubator with 95% air and saturated humidity for 3 hours in advance, 10 mu M of the asiatic acid is replaced by DMSO with the same concentration in a control group, and other test conditions are the same;
(2) washing ovary with 0.9% physiological saline at 37 deg.C for 2-3 times, temporarily storing the washed ovary in water bath at 37 deg.C, selecting 10mL syringe with 12-gauge needle, pressing ovary by hand to generate certain negative pressure, and puncturing follicle of 3-8mm from one side of ovary;
(3) storing the extracted follicular fluid into a 10mL centrifugal tube, automatically precipitating for 10min, sucking the supernatant by using a pasteur tube and discarding the supernatant, and adding an HEPES buffer solution into the centrifugal tube to repeatedly wash the follicles for 3 times;
(4) putting the cleaned follicle and HEPES buffer solution into a culture dish, picking up oocytes with two to three layers of cumulus with uniform cytoplasm and intact morphology by using a mouth suction tube under a stereoscopic microscope, putting the oocytes into a four-hole plate, adding 600 mu L of the culture solution of the experimental group balanced in advance in the step (1) into each hole of the two holes, namely the culture solution containing 10 mu M of the linoleic acid, adding 600 mu L of the culture solution of the control group balanced in advance in the step (1) into each hole of the other two holes, namely the culture solution containing 10 mu M of DMSO, covering 450 mu L of mineral oil on the surface of the culture solution of each hole, putting the four-hole plate into a culture dish at 38.5 ℃ and 5% CO2Culturing in a carbon dioxide incubator with 95% air and saturated humidity for 42-44 h.
The HEPES buffer solution comprises the following components: 1L of high Voltage ddH2O, 6.6633g/L sodium chloride, 0.2386g/L potassium chloride, 0.1680g/L sodium bicarbonate, 0.0408g/L sodium dihydrogen phosphate, 1.868mL/L sodium lactate, 0.1017g/L magnesium chloride, 0.022g/L sodium pyruvate, 2.38g/L HEPES,0.065g/L streptomycin, 0.294g/L calcium chloride and 1 g/LPVA.
The HEPES buffer solution is preheated by a 38.5 ℃ water bath before use, and the pH value of the HEPES buffer solution is 7.2-7.4, and the osmotic pressure is 270-290 mOsm.
The prepared HEPES buffer solution is filtered by a 0.22-micron filter membrane and is stored at the temperature of 4 ℃.
The number of oocytes in each well of the four-well plate is 100-110.
The experimental results show that: after maturation culture for 44h, the average oocyte nucleus maturation rate in the control group and the experimental group is: 76.85 percent and 80.96 percent, compared with the control group, the nucleus maturation rate is increased after the addition of the asiatic acid, and the increase of the experimental group is 4.11 percent; the average number of oocytes in both groups was 18.92% and 14.59%, and the experimental group showed a 4.33% decrease compared to the control group. (for details see table one)
TABLE-analysis of the Effect of Asiatic acid on the maturation and mortality of oocyte nuclei
Note: the numbers in the same column and different letters show significant difference (P < 0.05)
Experimental results prove that the in vitro maturation culture solution containing 10 mu M of the asiatic acid prepared in example 1 can effectively improve the maturation rate of the porcine oocytes and reduce the death rate.
Example 3 application of Asiatic acid to improvement of in vitro maturation quality of porcine oocytes
Parthenogenetic activation of oocytes
Parthenogenetic activation processes are as follows:
(1) preparing a cumulus delusion cell: respectively sucking the oocytes of the experimental group and the control group in example 2 out of the culture medium by using a 200 mu L pipette, putting the oocytes into 0.1% hyaluronidase, lightly blowing the oocytes by using the pipette, observing the oocytes under a microscope until all the oocytes are shed, and picking the oocytes out by using a mouth suction tube;
(2) cleaning: eluting cumulus cells for 2-3 times by using HEPES buffer solution, washing off granular cells, and picking up dead oocytes;
(3) selecting oocytes: transferring the cumulus cell cleaned in the step (2) from the microdroplet of the HEPES buffer solution to microdroplet of a working solution for cleaning for 2-3 times, and picking up the oocyte with the first polar body discharged and intact in morphology;
(4) parthenogenetic activation: adding an activating solution into an activation groove, carrying out primary pre-activation, keeping a detection resistor and an instrument intact, cleaning oocytes for 2-3 times by using the activating solution, arranging the cleaned oocytes in the middle of the activation groove, and carrying out electric shock twice on the conditions that the direct-current voltage is 100V/mm, the frequency is 0.1s, and the time is 60 mu s;
(5) treating cytochalasin: transferring activated oocyte into cytochalasin with concentration of 7.5 μ g/ml, washing for 2-3 times, adding into 38.5 deg.C and 5% CO2And a carbon dioxide incubator with 95% air and saturated humidity was equilibrated for three hours, and early embryo culture was performed.
(6) Early embryo culture: adding early culture solution into four-well plate, each well is 450 μ L, covered with 450 μ L mineral oil, and cultured at 38.5 deg.C under 5% CO2And (3) balancing in a carbon dioxide incubator with 95% of air and saturated humidity for three hours in advance, transferring the early embryos from cytochalasin to the early embryo culture solution, and adding 10% of serum into each hole until the fifth day of culture, and recording data in the process.
Wherein the preparation process of the operating fluid comprises the steps of adding sodium chloride into the prepared HEPES buffer solution, adjusting the osmotic pressure to 300-320mOsm, and storing at 4 ℃;
the activating solution comprises the following components, 300 mM of mannitol, 0.1 mM of calcium chloride, 0.05 mM of magnesium sulfate, 0.01% of PVA and 0.5 mM of HEPES.
The early embryo culture solution adopts a PZM-5 system.
The reagents and cytochalasin used in the activating solution were obtained from sigma.
The results show that: compared with the control group, after 10 mu M of the asiatic acid is added to the porcine oocytes during in vitro maturation, the cleavage rate of 4-cells of parthenogenetic embryos is improved by 18.13%, and the blastocyst rate is improved by 18.02%. (see Table II for details)
Table for analyzing influence of epidiylidene acid on parthenogenetic activation of oocytes
Note: numbers in the same column and different letters indicate significant differences (P < 0.05).
Experimental results prove that the growth rate of the parthenogenetic cells in the early embryo can be effectively improved by adding the asiatic acid into the porcine oocytes during in vitro maturation.
Second, in vitro fertilization of oocytes
The specific process of oocyte in vitro fertilization is as follows:
(1) preparing a cumulus delusion cell: respectively sucking the oocytes of the experimental group and the control group in example 2 out of the culture medium by using a 200 mu L pipette, putting the oocytes into 0.1% hyaluronidase, lightly blowing the oocytes by using the pipette, observing the oocytes under a microscope until the oocytes are completely removed, and picking the oocytes out by using a mouth suction tube;
(2) cleaning: eluting cumulus cells for 2-3 times by using HEPES buffer solution, washing off granular cells, and picking up dead oocytes;
(3) selecting oocytes: transferring the cumulus cell cleaned in the step (2) from the microdroplet of the HEPES buffer solution to microdroplet of a working solution for cleaning for 2-3 times, and picking up the oocyte with the first polar body discharged and intact in morphology;
(4) and (3) processing sperms: thawing frozen sperm in 37.5 deg.C water bath, transferring into 1.5mL centrifuge tube, resuspending sperm with 1mL mTBM, centrifuging at 700g for 3min, repeating for three times, and adjusting sperm concentration to 1 × 10 with mTBM for the last time6/mL；
(5) mTBM droplet preparation: mTBM was made into 60. mu.L droplets each, and the droplets were covered with mineral oil until the droplets were completely covered, and the droplets were placed in 38.5 ℃ 5% CO295% air, saturated humidityBalancing in a carbon dioxide incubator for 2 hours in advance;
(6) in vitro fertilization, 15-20 selected oocytes are put into each mTBM microdrop, 20 mu L of diluted semen is added into each microdrop, and the microdroplets are transferred into early embryo culture solution for early embryo culture after being incubated for 6h in a carbon dioxide incubator with 38.5 ℃, 5% CO2, 95% air and saturated humidity.
Wherein the mTBM comprises 113mM sodium chloride, 3mM potassium chloride, 7.5mM calcium chloride, 5mM sodium pyruvate, 11 mM glucose, 20 mM Tris, 1mM caffeine, 0.57 mM L-cysteine, and 0.1% wt/vol BSA.
Reagents used in the formulation of mTBM were purchased from sigma.
The results show that: compared with the control group, after 10 mu M of the linoleic acid is added into the porcine oocyte during in vitro maturation, the cleavage rate of the early embryo 4-cell completing in vitro fertilization is improved by 3.82 percent, the morula rate is improved by 4.42 percent, and the blastocyst rate is improved by 5.66 percent. (details are shown in table three)
Table for analyzing influence of epitricylidene acid on in vitro fertilization of porcine oocyte
Note: numbers in the same column and different letters indicate significant differences (P < 0.05).
Experimental results prove that the addition of the asiatic acid to the porcine oocytes during in vitro maturation can effectively improve the development rate of in vitro fertilization early embryos.
Third, ROS and GSH level detection of oocyte
Intracellular ROS and GSH levels were measured using 10. mu.M 2', 7' -dichlorodihydrofluorescein and 10. mu.M 4-chloromethyl-6, 8-difluoroo-7-hydroxyomycin, respectively.
The specific process for detecting the ROS and GSH levels of the oocyte is as follows:
(1) preparing a cumulus delusion cell: respectively sucking the oocytes of the experimental group and the control group in example 2 out of the culture medium by using a 200 mu L pipette, putting the oocytes into 0.1% hyaluronidase, lightly blowing the oocytes by using the pipette, observing the oocytes under a microscope until the oocytes are completely removed, and picking the oocytes out by using a mouth suction tube;
(2) cleaning: eluting cumulus cells for 2-3 times by using HEPES buffer solution, washing off granular cells, and picking up dead oocytes;
(3) selecting oocytes: transferring the cumulus cell cleaned in the step (2) from the microdroplet of the HEPES buffer solution to microdroplet of a working solution for cleaning for 2-3 times, and picking up the oocyte with the first polar body discharged and intact in morphology;
(4) incubating 10. mu.M of 2', 7' -dichlorodihydrofluorescein and 10. mu.M of 4-chloromethyl6, 8-difluoroo-7-hydroxycoumarin, respectively, with each set of oocytes of step (3) for 20min at 37 ℃ in the dark;
(5) after incubation, the oocytes of each group were washed 3 times with 0.1% (wt/vol) PBS/PVA, placed in 10. mu.l drops of 0.1% (wt/vol) PBS/PVA, fluorescence signals were captured with a fluorescence microscope and photographed, and the fluorescence intensity of the oocytes was analyzed with Image J software.
Wherein, the ROS and GSH kits are purchased from Invitrogen company.
The results show that: after 10 μ M of linoleic acid was added to the porcine oocytes during in vitro maturation, the ROS levels decreased and GSH levels increased in the oocytes compared to the control group. (for details see Table four)
TABLE OF EFFECT OF TETRA-FINIA ON ROS AND GSH LEVELS IN PIG OVERCARE
Note: numbers in the same column and different letters indicate significant differences (P < 0.05).
Experimental results prove that the oocyte quality can be effectively improved by adding the selenious acid in the in-vitro maturation period.
The results can prove that the addition of the asiatic acid in the in vitro maturation culture solution can obviously improve the maturation rate and the quality of the oocytes, effectively promote the parthenogenetic activation and the cleavage rate and the blastocyst rate of the early embryo after in vitro fertilization, improve the quality of the blastocyst, and have important potential application value in the aspect of improving the quality of the porcine oocytes.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (10)
1. A culture solution for improving the in vitro maturation quality of porcine oocytes, which comprises the following components: m199 culture solution, 10ng/mL EGF, linoleic acid, 0.6mM L-cysteine, 0.91mM sodium pyruvate, 10% follicular fluid, 10IU/mL LH, 10IU/mL FSH and 100IU/mL penicillin.
2. The culture solution for improving the in vitro maturation quality of porcine oocytes according to claim 1, wherein the follicular fluid is obtained by a method comprising:
precipitating the liquid extracted from 3-8mm follicle for 1h, and collecting the supernatant;
centrifuging the supernatant at 8000rpm for 15min, removing precipitate, and repeating the above steps for 3 times;
finally, the liquid was filtered through a 0.22 μm filter and dispensed as 1mL per tube and stored at-20 ℃.
3. The culture solution of claim 1, wherein the M199 culture solution, EGF, asiatic acid, L-cysteine, sodium pyruvate, and penicillin are all purchased from Sigma, and the LH and FSH are purchased from Ningbo second hormone plant, and the asiatic acid is available under the reference 546712.
4. The culture solution for improving the in vitro maturation quality of the porcine oocytes according to claim 1, wherein the preparation process of the culture solution comprises the following steps:
(1) 97.74mg of fine linoleic acid was weighed out and dissolved in 2mL of DMSO and filtered through a 0.22 μm filter to prepare a 100mM concentrated solution of fine linoleic acid;
(2) preparing the culture solution of claim 1 into a basic culture solution, filtering with a 0.22 μm filter, and storing at 4 deg.C;
(3) diluting the 100mM concentrated solution of the asiatic acid prepared in the step (1) into 10 mu M working solution of the asiatic acid by using the basic culture solution prepared in the step (2);
(4) adding the 10. mu.M of the working solution of linoleic acid prepared in step (3) to the basic culture solution according to claim 1 to prepare a culture solution.
5. The culture solution for improving the in vitro maturation quality of porcine oocytes according to claim 4, wherein the DMSO is purchased from Sigma.
6. A culture method for improving in vitro maturation quality of porcine oocytes is characterized by comprising the following steps:
(1) collecting the pig ovaries which are just slaughtered from a slaughterhouse, putting the pig ovaries into physiological saline water with double antibodies and the temperature of 37 ℃, and sending the pig ovaries to a laboratory within 2 hours by workers; the culture solution prepared in claim 3 was placed at 38.5 ℃ in 5% CO295% air, saturated humidity carbon dioxide incubator in advance balance 3h；
(2) Washing ovary with 0.9% physiological saline at 37 deg.C for 2-3 times, temporarily storing the washed ovary in water bath at 37 deg.C, selecting 10mL syringe with 12-gauge needle, pressing ovary by hand to generate certain negative pressure, and puncturing follicle of 3-8mm from one side of ovary;
(3) storing the extracted follicular fluid into a 10mL centrifugal tube, automatically precipitating for 10min, sucking the supernatant by using a pasteur tube and discarding, and adding an HEPES buffer solution into the centrifugal tube to repeatedly wash the follicular fluid precipitate for 3 times;
(4) placing the cleaned follicular fluid precipitate and HEPES buffer solution into a culture dish, picking up oocytes with two to three layers of cumulus ova, uniform cytoplasm and good morphology by using a mouth suction tube under a stereoscopic microscope, placing the oocytes into a four-hole plate, adding 600 mu L of culture solution balanced in advance in the step (1) into each hole, covering the surface of the culture solution with 450 mu L of mineral oil, placing the four-hole plate into a culture dish, placing the four-hole plate into a container with 38.5 ℃ and 5% CO2Culturing in a carbon dioxide incubator with 95% air and saturated humidity for 42-44 h.
7. The culture method for improving the in vitro maturation quality of the porcine oocytes according to claim 6, wherein the HEPES buffer comprises the following components: 1L of high Voltage ddH2O, 6.6633g/L sodium chloride, 0.2386g/L potassium chloride, 0.1680g/L sodium bicarbonate, 0.0408g/L sodium dihydrogen phosphate, 1.868mL/L sodium lactate, 0.1017g/L magnesium chloride, 0.022g/L sodium pyruvate, 2.38g/L HEPES,0.065g/L streptomycin, 0.294g/L calcium chloride, 1 g/LPVA.
8. The method as claimed in claim 6, wherein the HEPES buffer solution is preheated in a 38.5 ℃ water bath before use, and the pH of the HEPES buffer solution is 7.2-7.4, and the osmotic pressure is 270-290 mOsm.
9. The culture method for improving the in vitro maturation quality of porcine oocytes according to claim 6, wherein the HEPES buffer prepared is filtered through a 0.22 μm filter and stored at 4 ℃.
10. The culture method for improving in vitro maturation quality of porcine oocytes according to claim 6, wherein the number of oocytes in each well of the four-well plate is 100-110.
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