CN113801840A - Operating fluid for improving mouse in-vitro fertilization efficiency and using method thereof - Google Patents
Operating fluid for improving mouse in-vitro fertilization efficiency and using method thereof Download PDFInfo
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- 230000004720 fertilization Effects 0.000 title claims abstract description 101
- 238000000338 in vitro Methods 0.000 title claims abstract description 48
- 238000000034 method Methods 0.000 title claims abstract description 34
- 239000012530 fluid Substances 0.000 title claims abstract description 19
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 35
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 25
- 239000011259 mixed solution Substances 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 11
- 239000012894 fetal calf serum Substances 0.000 claims abstract description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 8
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims abstract description 7
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims abstract description 6
- CYDQOEWLBCCFJZ-UHFFFAOYSA-N 4-(4-fluorophenyl)oxane-4-carboxylic acid Chemical compound C=1C=C(F)C=CC=1C1(C(=O)O)CCOCC1 CYDQOEWLBCCFJZ-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 5
- 229940005581 sodium lactate Drugs 0.000 claims abstract description 5
- 239000001540 sodium lactate Substances 0.000 claims abstract description 5
- 235000011088 sodium lactate Nutrition 0.000 claims abstract description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 4
- 239000007836 KH2PO4 Substances 0.000 claims abstract description 4
- 239000008103 glucose Substances 0.000 claims abstract description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 4
- 239000001103 potassium chloride Substances 0.000 claims abstract description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims abstract description 4
- 239000011780 sodium chloride Substances 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000000839 emulsion Substances 0.000 claims abstract description 3
- 229940054269 sodium pyruvate Drugs 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 31
- 239000012091 fetal bovine serum Substances 0.000 claims description 20
- 210000002257 embryonic structure Anatomy 0.000 claims description 17
- 238000005406 washing Methods 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 12
- 210000000582 semen Anatomy 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 11
- 230000008010 sperm capacitation Effects 0.000 claims description 10
- 239000005662 Paraffin oil Substances 0.000 claims description 7
- 201000010063 epididymitis Diseases 0.000 claims description 7
- 102000011022 Chorionic Gonadotropin Human genes 0.000 claims description 6
- 108010062540 Chorionic Gonadotropin Proteins 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 239000012224 working solution Substances 0.000 claims description 6
- 210000000683 abdominal cavity Anatomy 0.000 claims description 5
- 102000006771 Gonadotropins Human genes 0.000 claims description 4
- 108010086677 Gonadotropins Proteins 0.000 claims description 4
- 239000002622 gonadotropin Substances 0.000 claims description 4
- 229940084986 human chorionic gonadotropin Drugs 0.000 claims description 4
- 210000002966 serum Anatomy 0.000 claims description 4
- 241000700159 Rattus Species 0.000 claims description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 3
- 244000309466 calf Species 0.000 claims 1
- 230000001605 fetal effect Effects 0.000 claims 1
- 241000699666 Mus <mouse, genus> Species 0.000 abstract description 27
- 241000699670 Mus sp. Species 0.000 abstract description 14
- 230000000052 comparative effect Effects 0.000 description 10
- 210000000287 oocyte Anatomy 0.000 description 8
- 235000013601 eggs Nutrition 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
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- 210000003101 oviduct Anatomy 0.000 description 4
- 241000283690 Bos taurus Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 210000002459 blastocyst Anatomy 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
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- 210000000918 epididymis Anatomy 0.000 description 3
- 244000144972 livestock Species 0.000 description 3
- 210000001672 ovary Anatomy 0.000 description 3
- 210000004681 ovum Anatomy 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 210000004291 uterus Anatomy 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000012449 Kunming mouse Methods 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 230000003187 abdominal effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000013020 embryo development Effects 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
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- 210000004303 peritoneum Anatomy 0.000 description 2
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- 238000002054 transplantation Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
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- 238000003975 animal breeding Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
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- 230000007547 defect Effects 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 230000008143 early embryonic development Effects 0.000 description 1
- 210000002308 embryonic cell Anatomy 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 239000003777 experimental drug Substances 0.000 description 1
- 210000001733 follicular fluid Anatomy 0.000 description 1
- 230000006543 gametophyte development Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 208000000509 infertility Diseases 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 231100000535 infertility Toxicity 0.000 description 1
- BTNMPGBKDVTSJY-UHFFFAOYSA-N keto-phenylpyruvic acid Chemical compound OC(=O)C(=O)CC1=CC=CC=C1 BTNMPGBKDVTSJY-UHFFFAOYSA-N 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000003739 neck Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000004508 polar body Anatomy 0.000 description 1
- 239000003761 preservation solution Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
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- 238000004904 shortening Methods 0.000 description 1
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- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
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
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0604—Whole embryos; Culture medium therefor
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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
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
-
- 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
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
- C12N2500/14—Calcium; Ca chelators; Calcitonin
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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
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
- C12N2500/16—Magnesium; Mg chelators
-
- 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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- 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
- C12N2500/34—Sugars
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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
- C12N2501/998—Proteins not provided for elsewhere
Abstract
The invention discloses an operating fluid for improving the in vitro fertilization efficiency of a mouse and a using method thereof, belonging to the technical field of in vitro fertilization of mice; the formula of the operating fluid comprises HTF mixed solution and fetal calf serum; the formula of HTF mixed solution comprises NaCl 0.6mg/ml, KCl 0.35mg/ml and KH2PO40.05mg/ml、MgSO4.7H2O0.05 mg/ml, glucose 0.5mg/ml, NaHCO32.1mg/ml、CaCl2.2H20.6mg/ml of O, 0.037mg/ml of sodium pyruvate, 3.42 mu.l/ml of 60% sodium lactate water emulsion and 5-8mg/ml of bovine serum albumin. The invention also provides a specific use method for carrying out mouse in-vitro fertilization operation by using the operation liquid, and the fertilization efficiency and the 2-cell embryo rate of the mouse in-vitro fertilization can be obviously improved by using the operation liquid and the method.
Description
Technical Field
The invention belongs to the technical field of mouse in-vitro fertilization, and particularly relates to an operating fluid for improving mouse in-vitro fertilization efficiency and a using method thereof.
Background
In vitro fertilization refers to a technique in which sperm and eggs of a mammal complete a fertilization process in an environment artificially controlled in vitro, and is abbreviated as IVF in English. Because it is inseparable from the embryo transfer technique (ET), also referred to as IVF-ET for short. In biology, an animal obtained after transferring an in vitro fertilized embryo to a mother is called a test-tube animal. The technology is successful in the 50 s of the 20 th century, develops rapidly in the last 20 years, and is mature day by day to become an important and conventional animal breeding biotechnology.
The in vitro fertilization technology has important significance for animal reproductive mechanism research, livestock production, medicine, endangered animal protection and the like. For example, using mouse, rat or rabbit as experimental material, the in vitro fertilization technique can be used to study the gametogenesis, fertilization and early embryonic development mechanism of mammals. In the livestock breed improvement, the in vitro fertilization technology provides a cheap and efficient means for embryo production, and has important values for fully utilizing excellent breed resources, shortening the breeding cycle of livestock, accelerating the breed improvement speed and the like. In humans, IVF-ET technology is one of the important measures to treat certain infertility and to overcome sexual linked diseases. The in vitro fertilization technology is an indispensable component of modern biotechnology such as mammalian embryo transplantation, cloning, transgenosis, sex control and the like, and how to improve the success rate (efficiency) of in vitro fertilization is one of the important factors influencing the development of the technology at present.
In the prior art, for example, chinese granted patent CN112322579B provides a culture solution for cattle in vitro fertilization and a method for improving cattle in vitro fertilization, in which a cumulus oophorus-oocyte complex is washed in a fertilization culture solution 1 time and then transferred to the fertilization culture solution for standby; then taking a frozen semen thin tube, unfreezing in a water bath, injecting the semen into a centrifugal tube containing a semen preparation culture solution, centrifuging and removing the supernatant; adding 300 mu L of semen preparation culture solution into the centrifuge tube, re-suspending the sperm precipitate, and taking proper sperm suspension to limit the formula of the fertilization culture solution. The patent also provides a preparation method of the cumulus oophorus-oocyte complex, wherein the cumulus oophorus-oocyte complex is collected from a cow ovary and is put into a transportation culture solution for temporary storage; then putting the oocyte into a mature culture solution for washing for 1 time, and finally putting the oocyte into a new mature culture solution for culturing for 22 to 24 hours, and defining the specific formulas of the transport culture solution and the mature culture solution. The patent also provides a method for culturing and preserving embryos in vitro, after the operation of in vitro fertilization is finished, granular cells around the embryos are removed cleanly by using an egg stripping needle, the embryos are placed into an embryo culture solution for culture, and the cleavage rate is recorded on day 3; recording the blastocyst rate on the 7 th day, counting the blastocyst hatchability by the 9 th day, and performing quality identification; washing the available embryo in preservation solution for 3 times, balancing in balancing solution for 10min, transferring into freezing solution, loading into embryo according to 5-stage liquid loading method, marking, cooling to-35 deg.C, rapidly taking out embryo tubule, and placing into liquid nitrogen for freezing preservation; the specific method of embryo culture solution is defined.
For another example, the chinese patent application CN111849872A provides a method for improving the in vitro fertilization effect of thawed pig sperm, wherein the frozen semen is first taken out and then thawed, and the formula of the freezing base solution for semen freezing is limited; then washing and centrifuging thawed semen, washing and centrifuging precipitate with mTBM capacitation solution for 1 time, adding mTBM capacitation solution into the precipitate, resuspending, and adjusting semen concentration to 4-9 × 106Putting the seeds/mL into an incubator for capacitation for 35-45min to obtain capacitation semen, and limiting the formula of mTBM capacitation liquid; then extracting follicular fluid from the sow ovary, picking out a cumulus-oocyte complex, taking out the oocyte after culturing, putting the oocyte into a fertilization operation fluid for cleaning, picking out a mature oocyte with a polar body, cleaning the mature oocyte in a mTBM capacitation fluid, and putting the mature oocyte into a well-balanced mTBM fertilization droplet for standby preservation; finally adding capacitation semen into each fertilization droplet, putting the fertilization droplets into an incubator for culture, washing the sperms attached to the oocytes in embryo culture solution, then putting the fertilization droplets into the embryo culture solution for culture, and finishing the in vitro fertilization culture.
However, the above methods for improving the in vitro fertilization effect are still general in fertilization effect and are not suitable for the in vitro fertilization operation process of mice.
For example, although it is disclosed in the document "influence of different culture solutions on the in vitro fertilization rate and early embryo in vitro development of Kunming mice" (Wu Chang Li et al, proceedings of south China university of agriculture, vol.27, No. 1, p. 1, page 107-109) that FSH (fetal bovine serum) with different concentrations (0.1, 0.5, 1.0IU/ml) can affect the in vitro fertilization rate and 2-cell embryo rate of mice, the formula of other culture solutions and equilibrium solutions is not disclosed, and the fertilization rate obtained by the disclosed in vitro fertilization method is 69.21% -78.06%, and the fertilization effect is still general.
Disclosure of Invention
In view of the defects of the prior art, the invention provides an operating fluid for improving the efficiency of mouse in vitro fertilization and a using method thereof, and particularly the operating fluid is realized by the following technology.
An operating fluid for improving the in vitro fertilization efficiency of a mouse comprises HTF mixed liquor and fetal calf serum;
the formula of the HTF mixed solution comprises 0.6mg/ml of NaCl, 0.35mg/ml of KCl and KH2PO4 0.05mg/ml、MgSO4.7H2O0.05 mg/ml, glucose 0.5mg/ml, NaHCO3 2.1mg/ml、CaCl2.2H20.6mg/ml of O, 0.037mg/ml of sodium pyruvate, 3.42 mu.l/ml of 60% sodium lactate water emulsion and 5-8mg/ml of bovine serum albumin.
The invention provides the operating fluid, which comprises HTF mixed solution and Fetal Bovine Serum (FBS) prepared by adopting a special formula. By combining the two, the applicant of the present invention has discovered that the efficiency (success rate) of mouse in vitro fertilization can be greatly improved.
Preferably, the fetal bovine serum is used in an amount of 60-80 μ l/ml based on the volume of the HTF mixture.
More preferably, the fetal bovine serum is used in an amount of 75 μ l/ml based on the volume of the HTF mixture.
The invention also provides a use method of the operating fluid for improving the mouse in-vitro fertilization efficiency, which comprises the following steps:
s1, super-discharging and balancing: injecting Pregnant Mare Serum Gonadotropin (PMSG) into the abdominal cavity of a female mouse on the third day before the female mouse is sacrificed, wherein the injection amount is 5-10 IU/mouse; injecting Human Chorionic Gonadotropin (HCG) into abdominal cavity of female mouse after 44-48h, wherein the injection amount is 5-10IU per mouse; the HTF mixture was taken in 5% CO the day before the male rats were sacrificed semen2The culture system is balanced overnight, and simultaneously, the HTF mixed solution and fetal calf serum are uniformly mixed in 5 percent CO2Balancing overnight under a culture system to prepare HTF mixed liquor and operating liquor;
s2, sperm capacitation: sacrifice the male mice of step S1 and obtain the epididymal tail, transfer epididymal tail semen into 500. mu.l of the HTF mixture of step S1, cover with paraffin oil, and add 5% CO2Capacitation under a culture system is 40-60 min;
s3, making a fertilization disc and taking eggs: taking 200 mul of the operation liquid 100-200 of the step S1 as fertilization drops, taking 2-5 fertilization drops as fertilization discs, and covering with paraffin oil; killing the female mouse of the step S1 for detecting the pessary, taking out an egg mass, and transferring the egg mass into a fertilization drop of the fertilization disc; each of the fertilization drops contains 3-5 egg masses;
s4, fertilization: adding 5-10 μ l of the product of step S2 to each of the fertilization drops obtained in step S3, respectively, in 5% CO2Culturing for 4-6h under a culture system;
s5, embryo washing and culturing: picking out the embryos in the fertilization tray cultured in step S4, picking out the fertilization embryos (under a microscope), washing the embryos with the culture solution several times, and transferring the embryos to the culture wells for culture and embryo washing.
The applicant of the present invention found that sperm of a male mouse can be capacitated by using a normal HTF mixed solution balancing solution, and fertilized by using an HTF mixed solution containing Fetal Bovine Serum (FBS), and finally the fertilized eggs are obtained after culturing for 4-6 hours. Compared with the method provided by the existing method for influencing the in vitro fertilization rate and early embryo in vitro development of Kunming mice by different culture solutions, the in vitro fertilization rate and the 2-cell embryo rate of the mice obtained by the special preparation method are obviously improved.
Preferably, the energy obtaining time of step S2 is 55 min.
Preferably, the amount of the operation solution used as the fertilization drop in step S3 is 150 ul.
Preferably, in step S4, the product of step S2 is added in an amount of 8 μ l at 5% CO2Culturing for 5.5h under the culture system.
Compared with the prior art, the invention has the advantages that: the invention provides a working solution for mouse in-vitro fertilization and a using method thereof, and by adopting the working solution and the method, the fertilization efficiency and the 2-cell embryo rate of the mouse in-vitro fertilization can be obviously improved.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be understood 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: mouse in vitro fertilization efficiency verification experiment
1. Laboratory animal
Donor: selecting female mice C57 with normal age of 4 weeks and male mice C57 with age of 10 weeks, and purchasing from Witongli Hua (KM strain mice can also be selected);
2. experimental equipment
Microshearing, ophthalmic scissors, ophthalmic forceps, fat clip, forceps, 1ml sterile syringe, transplantation needle, alcohol lamp, mouth grinder, magnetic stirrer, mouth suction tube, rubber tube, air filter, medical cotton, pipette gun and gun head, centrifuge tube, 100mm culture dish, 35mm culture dish, CO2An incubator.
3. Experimental drugs:
raw materials of HTF mixed solution: NaCl, KCl, KH2PO4，MgSO4.7H2O，Glucose，NaHCO3，CaCl2.2H2O, naporuvate (phenylpyruvate), Sodium lactate (Sodium lactate), BSA (bovine serum albumin).
FBS (fetal bovine serum), PMSG (pregnant mare serum gonadotropin), HCG (human chorionic gonadotropin), M2 operating fluid, KSOM embryo culture fluid, mineral oil (paraffin oil), 70% medical alcohol and the like.
The medicines are all obtained by conventional purchase in the market.
4. Experimental procedure
S1, super-arranging and balancing
Injecting PMSG (pregnant mare serum gonadotropin) into abdominal cavity of female mouse at 17 pm on the first day, wherein the injection amount is 10IU per mouse; 46h later, the female mouse was injected with HCG (human chorionic gonadotropin) in an amount of 10 IU/mouse, and HTF mixture was prepared at 17 PM on the day of injection and in 5% CO2Culture System (CO)2Incubator, same below) overnight equilibration;
simultaneously adding Fetal Bovine Serum (FBS) into HTF mixed solution, mixing well to obtain operation solution, and adding 5% CO2Balancing overnight under a culture system to prepare HTF mixed liquor and operating liquor; adding 75 mul fetal calf serum into each 1ml HTF mixed solution;
s2 sperm capacitation
Killing the male mice in the step S1 by breaking the necks, taking out epididymis tails on two sides of the male mice, and sucking 0.5ml of HTF mixed solution to place the HTF mixed solution in a flat dish; clamping oviduct at both ends with forceps under stereomicroscope, cutting off the fracture of about 1/3 depth in the middle of epididymis, squeezing sperm out of epididymis tail with forceps, transferring into 500 μ l of HTF mixed liquid drop prepared in step S1, covering with paraffin oil, and adding 5% CO2Capacitation is 55min under a culture system;
s3, preparation of fertilization plate
Taking 150 mul of the operation liquid in the step S1 as fertilization drops, taking 4 fertilization drops as fertilization discs, and covering with paraffin oil; killing female mice subjected to cervical dislocation in the step S1 for detecting pessaries as required, wiping abdominal hair with alcohol, transversely cutting a 1-2cm opening above the vagina by about 0.5cm, holding two legs of the mice with the left hand, tearing the epidermis of the mice upwards with the right hand, exposing the peritoneum, transversely cutting an opening of the peritoneum at the same position, expanding wounds obliquely upwards at two sides, exposing abdominal organs of the mice, picking up intestinal tracts upwards, finding Y-shaped uterus, sequentially finding fallopian tubes and ovaries upwards along the uterus, clamping and picking up the uterus, cleaning the paranoid, cutting out the fallopian tubes, placing the fallopian tubes in M2 operating fluid, tearing under a stereoscopic microscope, taking out the ova and transferring the ova into fertilization drops, wherein each fertilization drop contains 5 ova;
s4, fertilization
Adding 8 μ l of the product of step S2 to each of the fertilization drops obtained in step S3, respectively, in 5% CO2Culturing for 5.5h under a culture system;
s5, embryo washing and culturing
Picking out the embryos in the fertilization discs cultured in the step S4, picking out the fertilization embryos under a microscope, washing the fertilization embryos for a plurality of times by KSOM embryo culture solution, and transferring the fertilization embryos to culture wells for embryo washing.
Example 2
In the experimental procedure of this example, the difference from example 1 is that the amount of the fetal calf serum is: to 1ml of HTF mixture, 60. mu.l of fetal bovine serum was added.
Example 3
In the experimental procedure of this example, the difference from example 1 is that the amount of the fetal calf serum is: to 1ml of HTF mixture, 80. mu.l of fetal bovine serum was added.
Comparative example 1
In the experimental procedure of this comparative example, the difference from example 1 is that the operating fluid of step S1 was prepared using the following amounts of fetal bovine serum: 90. mu.l fetal bovine serum was added to each 1ml HTF mixture. The other steps are unchanged.
Comparative example 2
In the experimental procedure of this comparative example, the difference from example 1 is that the operating fluid of step S1 was prepared using the following amounts of fetal bovine serum: 55. mu.l fetal bovine serum was added to each 1ml HTF mixture. The other steps are unchanged.
Comparative example 3
In the experimental procedure of this comparative example, the difference from example 1 was that the operation liquid was replaced with the HTF mixed liquid in step S3. The other steps are unchanged.
Embryo development observation and verification
In the in vitro fertilization methods used in the above examples and comparative examples, the number of two cells in a normal developmental state was observed and counted on the 5 th day of culture, and then the number of four cells, eight cells, and blastocysts were observed, so that the corresponding fertilization rate and embryonic cell development rate (embryo rate) were calculated, and the IVF effect of the methods used in the above examples and comparative examples was judged.
Fertilization rate ═ 100% (number of two-cell embryos/total number of fertilized embryos)%
The specific test results are shown in table 1 below:
TABLE 1 observations of embryonic development
As can be seen from table 1 above, the fertilization rates were all 90% or more by the methods of examples 1 to 3, and the fertilization rate of example 1 reached 93%. By adopting the method of the comparative example 1, although the fertilization rate reaches 92%, the amount of the fetal calf serum used is relatively large, which indicates that the improvement of the mouse in-vitro fertilization efficiency is not obvious when too much fetal calf serum is used; with the methods of comparative examples 2, 3, the fertilization rates were not more than 68% at the highest, both significantly lower than the in vitro fertilization rates obtained with the methods of examples 1-3. Therefore, the fertilization efficiency and success rate of mouse in vitro fertilization can be obviously improved only by combining the HTF mixed solution and fetal calf serum by adopting the method disclosed by the invention.
Claims (7)
1. An operating fluid for improving the in vitro fertilization efficiency of a mouse is characterized in that the formula comprises HTF mixed solution and fetal calf serum;
the formula of the HTF mixed solution comprises 0.6mg/ml of NaCl, 0.35mg/ml of KCl and KH2PO4 0.05mg/ml、MgSO4.7H2O0.05 mg/ml, glucose 0.5mg/ml, NaHCO3 2.1mg/ml、CaCl2.2H20.6mg/ml of O, 0.037mg/ml of sodium pyruvate, 3.42 mu.l/ml of 60% sodium lactate water emulsion and 5-8mg/ml of bovine serum albumin.
2. The operating fluid for improving the efficiency of in vitro fertilization of a mouse according to claim 1, wherein the fetal bovine serum is used in an amount of 60 to 80 μ l/ml based on the volume of the HTF mixed solution.
3. The working solution for improving the efficiency of in vitro fertilization of a mouse according to claim 2, wherein the fetal bovine serum is used in an amount of 75 μ l/ml based on the volume of the HTF mixture.
4. The method of using the working solution for improving the efficiency of in vitro fertilization of a mouse according to claim 1, comprising the steps of:
s1, injecting pregnant mare serum gonadotropin into the abdominal cavity of a female mouse on the third day before the female mouse is sacrificed, wherein the injection amount is 5-10 IU/female mouse; injecting Human Chorionic Gonadotropin (HCG) into abdominal cavity of female mouse after 44-48h, wherein the injection amount is 5-10 IU/mouse; the HTF mixture was taken in 5% CO the day before the male rats were sacrificed semen2Balancing overnight under culture system, and simultaneously taking HTF mixed solution and fetal calf bloodMixing the obtained mixture at 5% CO2Balancing overnight under a culture system to prepare HTF mixed liquor and operating liquor;
s2, killing the male mouse in the step S1, obtaining the epididymal tail, transferring the epididymal tail semen into 500 mu l of the HTF mixed solution in the step S1, covering with paraffin oil, and adding 5% CO2Capacitation under a culture system is 40-60 min;
s3, taking 200 mul of the operation liquid of the step S1 as fertilization drops, taking 2-5 fertilization drops as fertilization discs, and covering with paraffin oil; killing the female mouse of the step S1 for detecting the pessary, taking out an egg mass, and transferring the egg mass into a fertilization drop of the fertilization disc; each of the fertilization drops contains 3-5 egg masses;
s4, adding 5-10 μ l of the product of step S2 into each fertilization drop obtained in step S3, respectively, in 5% CO2Culturing for 4-6h under a culture system;
s5, picking out the embryos in the fertilization disc cultured in the step S4, picking out the fertilization embryos, washing the fertilization embryos for a plurality of times by using the culture solution, and transferring the fertilization embryos to the culture hole for culture and embryo washing.
5. The method for using the operating fluid for improving the in vitro fertilization of the mouse according to claim 4, wherein the capacitation time of step S2 is 55 min.
6. The method of using the working solution for improving mouse in vitro fertilization according to claim 4, wherein the amount of the working solution used as the fertilization drop in step S3 is 150 ul.
7. The method of claim 4, wherein the product of step S2 is added in an amount of 8 μ l at 5% CO at step S42Culturing for 5.5h under the culture system.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001017160A (en) * | 1999-07-07 | 2001-01-23 | Fuso Pharmaceutical Industries Ltd | Medium composition for in vitro fertilization |
| CN105647853A (en) * | 2016-03-01 | 2016-06-08 | 中国农业大学 | Method for improving development quality of in vitro fertilization female embryos after implantation |
| CN109628380A (en) * | 2018-12-25 | 2019-04-16 | 深圳韦拓生物科技有限公司 | A kind of people's liquid in vitro fertilization and preparation method thereof |
-
2021
- 2021-08-26 CN CN202110989996.3A patent/CN113801840A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001017160A (en) * | 1999-07-07 | 2001-01-23 | Fuso Pharmaceutical Industries Ltd | Medium composition for in vitro fertilization |
| CN105647853A (en) * | 2016-03-01 | 2016-06-08 | 中国农业大学 | Method for improving development quality of in vitro fertilization female embryos after implantation |
| CN109628380A (en) * | 2018-12-25 | 2019-04-16 | 深圳韦拓生物科技有限公司 | A kind of people's liquid in vitro fertilization and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| 华月琴等: "精子孵育时间及培养液成分对小鼠体外受精和胚胎发育的影响", 《苏州大学学报(医学版)》, vol. 29, no. 4, pages 648 * |
| 姚瀛等: "激素对小鼠胚胎体外发育的影响", 《现代农业科技》, no. 3, pages 319 - 321 * |
| 苏忆非: "小鼠受精卵模拟微重力差异基因的验证及Ndufa4对其体外发育的影响", 《中国优秀硕士学位论文全文数据库 基础科学辑》, no. 11, pages 006 - 19 * |
| 郎伍营: "小鼠体外受精条件优化及活性氧对体外受精卵发育的影响", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》, no. 2, pages 3 * |
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