CN114214269A - Porcine embryo culture solution and application - Google Patents
Porcine embryo culture solution and application Download PDFInfo
- Publication number
- CN114214269A CN114214269A CN202111553715.6A CN202111553715A CN114214269A CN 114214269 A CN114214269 A CN 114214269A CN 202111553715 A CN202111553715 A CN 202111553715A CN 114214269 A CN114214269 A CN 114214269A
- Authority
- CN
- China
- Prior art keywords
- culture solution
- pig
- culture
- embryo
- reduced glutathione
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000001161 mammalian embryo Anatomy 0.000 title claims abstract description 50
- 238000000338 in vitro Methods 0.000 claims abstract description 36
- 108010024636 Glutathione Proteins 0.000 claims abstract description 27
- 241001122767 Theaceae Species 0.000 claims abstract description 27
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims abstract description 27
- 150000008442 polyphenolic compounds Chemical class 0.000 claims abstract description 27
- 235000013824 polyphenols Nutrition 0.000 claims abstract description 27
- 210000001733 follicular fluid Anatomy 0.000 claims abstract description 21
- 210000002257 embryonic structure Anatomy 0.000 claims abstract description 16
- 239000012530 fluid Substances 0.000 claims abstract description 4
- 239000012531 culture fluid Substances 0.000 claims description 11
- 230000004720 fertilization Effects 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 3
- 238000000034 method Methods 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 238000012136 culture method Methods 0.000 claims description 2
- 210000002459 blastocyst Anatomy 0.000 abstract description 14
- 238000011161 development Methods 0.000 abstract description 14
- 210000004027 cell Anatomy 0.000 abstract description 11
- 238000004519 manufacturing process Methods 0.000 abstract description 9
- 238000003776 cleavage reaction Methods 0.000 abstract description 7
- 230000007017 scission Effects 0.000 abstract description 7
- 230000001737 promoting effect Effects 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 47
- 230000018109 developmental process Effects 0.000 description 12
- 230000000052 comparative effect Effects 0.000 description 11
- 210000000287 oocyte Anatomy 0.000 description 10
- 229910002092 carbon dioxide Inorganic materials 0.000 description 7
- 210000001672 ovary Anatomy 0.000 description 7
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 6
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 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 description 3
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 3
- 239000007995 HEPES buffer Substances 0.000 description 3
- 229930003268 Vitamin C Natural products 0.000 description 3
- 239000001569 carbon dioxide Substances 0.000 description 3
- 235000013601 eggs Nutrition 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 235000019154 vitamin C Nutrition 0.000 description 3
- 239000011718 vitamin C Substances 0.000 description 3
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 235000006708 antioxidants Nutrition 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
- 210000001771 cumulus cell Anatomy 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000002468 fat body Anatomy 0.000 description 2
- 239000002480 mineral oil Substances 0.000 description 2
- 235000010446 mineral oil Nutrition 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 210000000582 semen Anatomy 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 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 description 1
- 108010003272 Hyaluronate lyase Proteins 0.000 description 1
- 102000001974 Hyaluronidases Human genes 0.000 description 1
- LPHGQDQBBGAPDZ-UHFFFAOYSA-N Isocaffeine Natural products CN1C(=O)N(C)C(=O)C2=C1N(C)C=N2 LPHGQDQBBGAPDZ-UHFFFAOYSA-N 0.000 description 1
- 239000004201 L-cysteine Substances 0.000 description 1
- 235000013878 L-cysteine Nutrition 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229960001948 caffeine Drugs 0.000 description 1
- VJEONQKOZGKCAK-UHFFFAOYSA-N caffeine Natural products CN1C(=O)N(C)C(=O)C2=C1C=CN2C VJEONQKOZGKCAK-UHFFFAOYSA-N 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229960002773 hyaluronidase Drugs 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000001331 nose Anatomy 0.000 description 1
- 210000004508 polar body Anatomy 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000011218 segmentation Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 210000004340 zona pellucida Anatomy 0.000 description 1
Classifications
-
- 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
-
- 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
-
- 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
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Gynecology & Obstetrics (AREA)
- Biotechnology (AREA)
- Reproductive Health (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Developmental Biology & Embryology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a porcine embryo culture solution. The culture solution is PZM-3 culture solution containing the following components in concentration: 1-8 g/L of reduced glutathione, 10-50 mg/L of tea polyphenol and 8-15 v/v% of pig follicular fluid. The culture solution is prepared by adding reduced glutathione, tea polyphenol and pig follicle fluid with proper concentration into the conventional PZM-3 culture solution. The combination of the reduced glutathione and the tea polyphenol can be compounded and synergized, so that the effect of the culture solution for promoting the development of pig embryos is better than that of singly using the reduced glutathione or the tea polyphenol. And the use of the pig follicular fluid further improves the efficacy of the culture solution. The culture solution provided by the invention can effectively promote the development of in vitro pig embryo culture, improve the 4-cell cleavage rate and blastocyst rate of the in vitro pig embryo culture, and the total cell number of the blastocyst, and has wide application prospect in vitro production of pig embryo.
Description
Technical Field
The invention belongs to the technical field of pig breeding, and particularly relates to a pig embryo culture solution and application thereof.
Background
In recent years, in vitro embryo production has been widely used for producing animals with economic value, and the development of animal husbandry has been promoted by the application of In Vitro Fertilization (IVF), single sperm injection (ICSI), embryo transfer, embryo segmentation, and embryonic stem cell engineering. With the development of modern reproduction biotechnology and the gradual improvement of embryo in vitro production technology, the production of in vitro embryos by using in vitro fertilization technology has a potential wide application prospect. In vitro production of livestock embryos is an effective way to rapidly expand the number of elite populations. Although live offspring have been obtained by in vitro fertilization techniques, the early embryo after in vitro fertilization has a low in vitro developmental rate, which severely limits the application of in vitro embryo production.
Pigs and omnivorous mammals have fat bodies, short limbs, long noses and mouths, short fat bodies and limbs, warm nature, strong adaptability and quick reproduction. With the rapid development of the fields of social economy, animal husbandry and the like, more and better animal embryos need to be prepared in vitro urgently, however, in the actual production process, the embryo retardation of in vitro development is particularly serious in a certain stage, a large amount of resources are wasted, and the embryo retardation of a pig is serious in a 4-cell stage in the in vitro development process.
The in vitro culture solution of the embryo currently used in the pig includes modified Whitten culture solution, NCSU23/NCSU37, Beltsville Embryo Culture Medium (BECM) culture solution, PZM series (PZM-3/PZM-4/PZM-5) culture solution and the like. However, after embryo transplantation, the pregnancy rate and the litter average litter size of the embryos cultured in vitro are very low, and the number of cells developed to the blastocyst is also obviously lower than that of in vivo embryos, so that a culture medium capable of promoting the in vitro development of the porcine embryos is still required to be developed to improve the in vitro production efficiency of the porcine embryos at the early stage.
Disclosure of Invention
Based on the above, the invention aims to provide a pig embryo culture solution, which can effectively promote the development of pig in-vitro culture embryos, and improve the 4-cell cleavage rate and blastocyst rate of the pig in-vitro culture embryos and the total cell number of the blastocysts.
The specific technical scheme is as follows:
a porcine embryo culture solution, which is PZM-3 culture solution comprising the following concentration components: 1-8 g/L of reduced glutathione, 10-50 mg/L of tea polyphenol and 8-15 v/v% of pig follicular fluid.
In some of these embodiments, the culture fluid is a PZM-3 culture fluid comprising the following concentration components: 1.5-5 g/L of reduced glutathione, 20-30 mg/L of tea polyphenol and 10-15 v/v% of pig follicular fluid.
In some of these embodiments, the culture fluid is a PZM-3 culture fluid comprising the following concentration components: 2g/L of reduced glutathione, 30mg/L of tea polyphenol and 12 v/v% of pig follicular fluid.
In some embodiments, the mass ratio of reduced glutathione to tea polyphenol in the culture solution is 200: (2-3).
In some embodiments, the mass ratio of reduced glutathione to tea polyphenol in the culture solution is 200: 3.
the invention also provides a preparation method of the pig embryo culture solution, which comprises the following steps: adding reduced glutathione, tea polyphenol and pig follicle fluid into PZM-3 culture solution according to the using amount, and filtering and sterilizing to obtain the product;
the invention also provides application of the porcine embryo culture solution in porcine embryo in-vitro culture.
The invention also provides a pig embryo in vitro culture method, which comprises the following steps: placing in the above porcine embryo culture solution at 38.5 deg.C and 5% CO2The cultivation was carried out under 100% humidity.
The culture solution capable of promoting in-vitro culture of the porcine embryo is obtained by optimizing the inventor, and reduced glutathione, tea polyphenol and porcine follicular fluid with proper concentration are added into the existing PZM-3 culture solution. The combination of the reduced glutathione and the tea polyphenol can be compounded and synergized, so that the effect of the culture solution for promoting the development of pig embryos is better than that of singly using the reduced glutathione or the tea polyphenol. And the use of the pig follicular fluid further improves the efficacy of the culture solution. The culture solution provided by the invention can effectively promote the development of in vitro pig embryo culture, improve the 4-cell cleavage rate and blastocyst rate of the in vitro pig embryo culture, and the total cell number of the blastocyst, and has wide application prospect in vitro production of pig embryo.
Detailed Description
In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It is to be understood that the experimental procedures in the following examples, where specific conditions are not noted, are generally in accordance with conventional conditions, or with conditions recommended by the manufacturer. The various reagents used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Example 1
This example is a porcine embryo culture solution, which is a PZM-3 culture solution comprising the following concentration components: 2g/L of reduced glutathione, 30mg/L of tea polyphenol and 12 v/v% of pig follicular fluid.
Example 2
This example is a porcine embryo culture solution, which is a PZM-3 culture solution comprising the following concentration components: 5g/L of reduced glutathione, 25mg/L of tea polyphenol and 10 v/v% of pig follicular fluid.
Example 3
This example is a porcine embryo culture solution, which is a PZM-3 culture solution comprising the following concentration components: 3g/L of reduced glutathione, 20mg/L of tea polyphenol and 15 v/v% of pig follicular fluid.
Comparative example 1
The comparative example is a porcine embryo culture solution, which is a PZM-3 culture solution comprising the following concentration components: 2g/L of reduced glutathione, 30mg/L of vitamin C and 12 v/v% of pig follicular fluid.
The culture solution of this comparative example is different from that of example 1 in that another common antioxidant, vitamin C, is used instead of the tea polyphenol component.
Comparative example 2
The comparative example is a porcine embryo culture solution, which is a PZM-3 culture solution comprising the following concentration components: reduced glutathione 2g/L and tea polyphenol 30 mg/L.
The culture solution of this comparative example was different from that of example 1 in that no follicular fluid was added.
The preparation method of the porcine embryo culture solution described in examples 1-3 and comparative examples 1-2 is as follows: adding the components into PZM-3 culture solution according to the formula usage amount, and filtering and sterilizing to obtain the PZM-3 culture solution.
The culture solutions of examples 1-3 and comparative examples 1-2 were used to culture porcine embryos formed by in vitro fertilization, specifically as follows:
(1) collecting the pig ovaries which are just slaughtered from a slaughterhouse, putting the pig ovaries into physiological saline water with the temperature of 37 ℃ and containing antibiotics, and sending the pig ovaries to a laboratory within 2 hours;
(2) washing ovary with 0.9% physiological saline at 37 deg.C for 3 times, placing the washed ovary in water bath at 37 deg.C, selecting 10mL syringe with 12-gauge needle, pressing ovary with hand to generate certain negative pressure, and puncturing and extracting follicle of 3-8mm from one side of ovary;
(3) storing the extracted follicular fluid into a 10mL centrifuge tube, standing for precipitation for 10min, sucking the supernatant by using a Pasteur tube, discarding, and adding HEPES buffer solution into the centrifuge 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 oocytes with three layers of cumulus, uniform cytoplasm and good morphology by using a mouth suction tube under a stereomicroscope, placing the oocytes into a four-hole plate, adding 600 mu L PZM-3 culture solution into each hole, covering the surface of the culture solution with 450 mu L mineral oil, placing the four-hole plate into a culture dish, placing the four-hole plate at 38.5 ℃ and 5% CO2And culturing for 42-44h in a carbon dioxide incubator with saturated humidity.
(5) Sucking the oocyte in the step (4) out of the culture medium, putting the oocyte into 1mg/ml hyaluronidase, gently blowing and beating the oocyte by using a pipette gun, observing the oocyte under a microscope until all cumulus cells are removed, and picking the oocyte by using a suction tube;
(6) eluting cumulus cells for 3 times by using HEPES buffer solution, washing off granular cells, and picking up dead oocytes;
(7) transferring the cumulus oophorus cells in the step (6) into operating fluid microdroplets to be cleaned for 3 times, and picking up the oocytes which are discharged with the first polar body, complete zona pellucida, clear perivitelline space, complete shape and uniform cytoplasm;
(8) sperm concentration was diluted 1X 10 with mTBM6Per mL to obtain diluted semen;
(9) 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% CO2Balancing in a carbon dioxide incubator with saturated humidity for 2 hours in advance;
(10) the oocytes obtained in step (7) were placed in each mTBM droplet and 20. mu.L of diluted semen obtained in step (8) was added to each droplet at 38.5 ℃ in 5% CO2And incubating the fertilized eggs in a carbon dioxide incubator with saturated humidity for 6 hours, and transferring the fertilized eggs to the culture solution and PZM-3 culture solution described in examples 1-3 and comparative examples 1-2 for culture.
The mTBM comprises the following components: 113mM sodium chloride, 3mM potassium chloride, 7.5mM calcium chloride, 5mM sodium pyruvate, 11mM glucose, 20mM Tris, 1mM caffeine, 0.57mM L-cysteine and 0.1 w/v% BSA.
The fertilized eggs obtained from each group of the culture medium were examined for 4-cell cleavage rate, blastocyst rate and total blastocyst cell count, and the results were as follows:
the results are shown in table 1 below:
TABLE 1 test results
From the results in table 1, it can be seen that, compared with the PZM-3 culture solution group, in vitro culture of porcine embryos using the culture solution provided by the present invention (examples 1 to 3), the cleavage rate and blastocyst rate of porcine embryos 4 cells can be effectively increased, and the total number of blastocyst cells can be increased. The PZM-3 culture solution containing reduced glutathione, tea polyphenol and pig follicular fluid can better promote the in vitro development ability of pig embryos.
Compared with the example 1, in the comparative example 1, another common antioxidant vitamin C is used for replacing a tea polyphenol component to be compounded with reduced glutathione, and the result shows that the 4-cell cleavage rate, the blastocyst rate and the total number of blastocyst cells of the in vitro cultured pig embryo are all reduced, so that the compound synergistic effect of the reduced glutathione and the tea polyphenol can be generated, and the development of the pig embryo can be promoted.
Compared with the example 1, the culture solution in the comparative example 2 is not added with the porcine follicular fluid, so that the effect of promoting the in vitro development of the porcine embryo is reduced to a certain extent, and the addition of the porcine follicular fluid is beneficial to improving the cleavage rate and blastocyst rate of the porcine embryo 4 cell.
The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the scope of the present description should be considered as being described in the present specification.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (8)
1. A porcine embryo culture solution is PZM-3 culture solution which comprises the following components in concentration: 1-8 g/L of reduced glutathione, 10-50 mg/L of tea polyphenol and 8-15 v/v% of pig follicular fluid.
2. The porcine embryo culture fluid of claim 1, wherein the culture fluid is PZM-3 culture fluid comprising the following concentration components: 1.5-5 g/L of reduced glutathione, 20-30 mg/L of tea polyphenol and 10-15 v/v% of pig follicular fluid.
3. The porcine embryo culture fluid of claim 2, wherein the culture fluid is PZM-3 culture fluid comprising the following concentration components: 2g/L of reduced glutathione, 30mg/L of tea polyphenol and 12 v/v% of pig follicular fluid.
4. The porcine embryo culture solution of claim 1, wherein the mass ratio of the reduced glutathione to the tea polyphenol is 200: (2-3).
5. The porcine embryo culture solution of claim 4, wherein the mass ratio of the reduced glutathione to the tea polyphenol is 200: 3.
6. the method for preparing a porcine embryo culture solution according to any of claims 1 to 5, comprising the steps of: adding reduced glutathione, tea polyphenol and pig follicle fluid into PZM-3 culture solution according to the using amount, and filtering and sterilizing to obtain the product.
7. The use of a porcine embryo culture fluid according to any of claims 1-5 for the in vitro culture of porcine embryos.
8. A pig embryo in vitro culture method is characterized by comprising the following steps: placing porcine embryo formed by in vitro fertilization or porcine cloned embryo in porcine embryo culture solution of any of claims 1-5 at 38.5 deg.C and 5% CO2The cultivation was carried out under 100% humidity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111553715.6A CN114214269A (en) | 2021-12-17 | 2021-12-17 | Porcine embryo culture solution and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111553715.6A CN114214269A (en) | 2021-12-17 | 2021-12-17 | Porcine embryo culture solution and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114214269A true CN114214269A (en) | 2022-03-22 |
Family
ID=80703844
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111553715.6A Pending CN114214269A (en) | 2021-12-17 | 2021-12-17 | Porcine embryo culture solution and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114214269A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000030441A1 (en) * | 1998-11-24 | 2000-06-02 | Bresagen Limited | Cryopreservation of oocytes and embryos and methods for producing animals involving the same |
| CN1904039A (en) * | 2006-07-28 | 2007-01-31 | 浙江大学 | Ox embryo in vitro culturing liquid containing tea polyphenol and its culturing method |
| CN106676136A (en) * | 2016-12-30 | 2017-05-17 | 中国农业科学院北京畜牧兽医研究所 | Method for improving pig cell nucleus transplantation efficiency |
| CN112877279A (en) * | 2019-12-11 | 2021-06-01 | 上海厚超生物科技股份有限公司 | Culture solution and culture method for rat embryonic stem cells and light source controllable cell culture box |
| CN113444683A (en) * | 2021-07-12 | 2021-09-28 | 华南农业大学 | Additive for improving oocyte in-vitro maturation quality, culture method and application |
-
2021
- 2021-12-17 CN CN202111553715.6A patent/CN114214269A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000030441A1 (en) * | 1998-11-24 | 2000-06-02 | Bresagen Limited | Cryopreservation of oocytes and embryos and methods for producing animals involving the same |
| CN1904039A (en) * | 2006-07-28 | 2007-01-31 | 浙江大学 | Ox embryo in vitro culturing liquid containing tea polyphenol and its culturing method |
| CN106676136A (en) * | 2016-12-30 | 2017-05-17 | 中国农业科学院北京畜牧兽医研究所 | Method for improving pig cell nucleus transplantation efficiency |
| CN112877279A (en) * | 2019-12-11 | 2021-06-01 | 上海厚超生物科技股份有限公司 | Culture solution and culture method for rat embryonic stem cells and light source controllable cell culture box |
| CN113444683A (en) * | 2021-07-12 | 2021-09-28 | 华南农业大学 | Additive for improving oocyte in-vitro maturation quality, culture method and application |
Non-Patent Citations (2)
| Title |
|---|
| 叶小芳;陈静波;吕雪峰;陈世斌;黄俊成;: "卵母细胞老化对绵羊胚胎早期发育及胞质谷胱甘肽含量的影响", 中国细胞生物学学报, no. 03 * |
| 宋雪梅;许惠艳;郝朝亮;梁世忠;卢晟盛;卢克焕;: "胰岛素和胚胎培养密度对猪孤雌胚早期体外发育的影响", 西南农业学报, no. 06 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Moore et al. | A 100-Year Review: Reproductive technologies in dairy science | |
| Barceló‐Fimbres et al. | Effects of fetal calf serum, phenazine ethosulfate and either glucose or fructose during in vitro culture of bovine embryos on embryonic development after cryopreservation | |
| CN107034173B (en) | Method for culturing bovine in vitro fertilized embryo and culture solution used in method | |
| CN107142239B (en) | Method for improving culture efficiency of bovine in vitro fertilization embryos | |
| CN103898046B (en) | Ox IVF Embryos nutrient solution and cultural method | |
| WO2016206086A1 (en) | Pig oocyte in vitro maturation culture solution and preparation method and culture method thereof | |
| CN111254109B (en) | Method for in vitro fertilization and embryo culture of bovine oocytes and transport culture solution | |
| CN100334205C (en) | Method for producing sex controllable in vitro embryo of buffalo | |
| CN107365738B (en) | Method for preparing cow and cattle xenogenesis in-vitro fertilization embryo | |
| CN110846272A (en) | Oocyte culture solution and in-vitro culture method for improving oocyte quality | |
| KR100666595B1 (en) | Compositions of sequential synthetic culture media for in vitro maturation and fertilization of human oocyte and culture methods using thereof | |
| An et al. | Significant heparin effect on bovine embryo development during sexed in vitro fertilization | |
| CN108707578B (en) | Pig monose embryo in vitro culture method | |
| CN112980778B (en) | Method for culturing and cryopreserving bovine in-vitro fertilization embryos | |
| CN107980765B (en) | Semen diluent for improving low-temperature preservation quality of donkey semen and preparation method and application thereof | |
| Binyameen et al. | Recent trends in bovine reproductive biotechnologies. | |
| CN110305836B (en) | Fertilization liquid for cattle in vitro fertilization and use method thereof | |
| CN114214269A (en) | Porcine embryo culture solution and application | |
| Poniedziałek-Kempny et al. | Piglets produced by transfer of embryos obtained by in vitro fertilization of oocytes matured in vitro with thymosin: a case report. | |
| CN103013908A (en) | New method of in vitro fertilization for mixed semens of bovine and sheep | |
| CN107043743B (en) | In-vitro maturation method of canine oocytes | |
| CN112914784B (en) | Bovine embryo segmentation method | |
| CN103215220A (en) | Method for producing dzo gender controllable in vitro embryos | |
| CN114410573A (en) | Oocyte in-vitro maturation culture solution additive and application thereof | |
| CN113767896A (en) | Bovine in vitro fertilization embryo vitrification freezing method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20220322 |
|
| WD01 | Invention patent application deemed withdrawn after publication |