CN112725263A - Porcine oocyte in-vitro maturation culture solution with polyspermy inhibition effect and preparation method and application thereof - Google Patents
Porcine oocyte in-vitro maturation culture solution with polyspermy inhibition effect and preparation method and application thereof Download PDFInfo
- Publication number
- CN112725263A CN112725263A CN202110114495.0A CN202110114495A CN112725263A CN 112725263 A CN112725263 A CN 112725263A CN 202110114495 A CN202110114495 A CN 202110114495A CN 112725263 A CN112725263 A CN 112725263A
- Authority
- CN
- China
- Prior art keywords
- porcine
- culture solution
- vitro maturation
- oocyte
- streptomycin
- 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.)
- Granted
Links
Images
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
- C12N5/0608—Germ cells
- C12N5/0609—Oocytes, oogonia
-
- 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
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- 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
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- 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
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Developmental Biology & Embryology (AREA)
- Microbiology (AREA)
- Reproductive Health (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Gynecology & Obstetrics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a porcine oocyte in-vitro maturation culture solution with the function of inhibiting polyspermia fertilization, a preparation method and application thereof, and belongs to the technical field of animal husbandry. In order to effectively inhibit the occurrence of polyspermia fertilization and ensure the development quality of later embryos. The culture solution for in vitro maturation of the porcine oocyte in vitro maturation with the effect of inhibiting the polyspermia fertilization is calculated by taking TCM199 as a reference, and is 0.6mmol/L cysteine, 0.91mmol/L sodium pyruvate, 10ng/mL EGF, 10% porcine follicular fluid, 10IU/mL FSH, 10IU/mL LH, 1% streptomycin, 10% streptomycin‑11‑10‑5mol/L melatonin. The porcine oocyte in-vitro maturation culture solution containing melatonin improves the in-vitro maturation quality of porcine oocytes by promoting the maturation of porcine oocytes cytoplasm, thereby inhibiting the polyspermia fertilization effect of the oocytes in the in-vitro fertilization process and ensuring the later-stage embryo development quality.
Description
Technical Field
The invention belongs to the technical field of livestock raising, and particularly relates to a porcine oocyte in-vitro maturation culture solution with a function of inhibiting polyspermia fertilization, and a preparation method and application thereof.
Background
The In Vitro Fertilization (IVF) technology of pigs can preserve the genetic resources of excellent breeding pigs in China, expand and breed excellent varieties, shorten breeding time, promote genetic improvement and support the preparation of disease model pigs and organ donor pigs. Polyspermia is an abnormal fertilization mode, which means that more than one sperm enters the oocyte cytoplasm, resulting in the formation of polyploid zygotes and abnormal embryonic development. In the process of pig in-vitro fertilization, the incidence rate of polyspermia fertilization is particularly prominent and can reach about 80 percent, and the method is a main obstacle for producing high-quality in-vitro embryos. Effectively inhibiting the occurrence rate of polyspermia fertilization and ensuring the development quality of later embryos, which is also one of the important contents of the research in the current in vitro fertilization technology.
Disclosure of Invention
The invention aims to effectively inhibit the occurrence of polyspermy and guarantee the development quality of later-stage embryos, and provides a porcine oocyte in-vitro maturation culture solution with the function of inhibiting polyspermy, which consists of the following components: TCM199, cysteine, sodium pyruvate, porcine follicular fluid, FSH, LH, streptomycin and melatonin.
Further defined, the in vitro maturation culture solution of the porcine oocytes is based on TCM199, and comprises 0.6mmol/L cysteine, 0.91mmol/L sodium pyruvate, 10ng/mLEGF, 10% volume fraction porcine follicular fluid, 10IU/mL FSH, 10IU/mLLH, 1% mass fraction streptomycin and 10% mass fraction streptomycin-11-10-5mol/L melatonin.
Further defined, the in vitro maturation culture solution of the porcine oocytes is based on TCM199, and comprises 0.6mmol/L cysteine, 0.91mmol/L sodium pyruvate, 10ng/mLEGF, 10% volume fraction porcine follicular fluid, 10IU/mL FSH, 10IU/mLLH, 1% mass fraction streptomycin and 10% mass fraction streptomycin-11mol/L melatonin.
Further defined, the in vitro maturation culture solution of the porcine oocytes is based on TCM199, and comprises 0.6mmol/L cysteine, 0.91mmol/L sodium pyruvate, 10ng/mLEGF, 10% volume fraction porcine follicular fluid, 10IU/mL FSH, 10IU/mLLH, 1% mass fraction streptomycin and 10% mass fraction streptomycin-8mol/L melatonin.
Further defined, the porcine oocyte in vitro maturation culture solution is based on TCM199, and comprises 0.6mmol/L cysteine, 0.91mmol/L sodium pyruvate, 10ng/mL EGF, 10% volume fraction porcine follicular fluid, 10IU/mL FSH, 10IU/mL LH, and,The mass fraction of the streptomycin is 1 percent and 10 percent-5mol/L melatonin.
The invention also provides a preparation method of the in vitro maturation culture solution for the porcine oocytes, which comprises the following specific steps:
(1) obtaining the pig follicular fluid: extracting liquid from the follicle of the pig ovary, centrifuging the liquid for 30min at 1500rpm at 4 ℃ for 3-4 times, retaining the supernatant, filtering the supernatant with a 0.45 μm filter to remove impurities, and filtering with a 0.22 μm filter to obtain the required follicle liquid of the pig;
(2) obtaining culture solution of TCM-199, cysteine, sodium pyruvate, EGF, FSH, LH, streptomycin and melatonin;
(3) and (3) mixing the culture solution obtained in the step (2) with the porcine follicular fluid obtained in the step (1) to obtain the porcine oocyte in-vitro maturation culture solution.
The invention also provides application of the porcine oocyte in-vitro maturation culture solution in preparing a reagent for inhibiting polyspermia fertilization.
The invention also provides application of the porcine oocyte in-vitro maturation culture solution in preparing a medicament for preventing polyspermia.
The invention also provides application of the porcine oocyte in-vitro maturation culture solution in preparing a medicament for improving the developmental capacity of in-vitro fertilized embryos.
Has the advantages that: experiments prove that the melatonin-added oocyte maturation culture solution group has no significant difference in oocyte nucleus maturation rate, but indexes for reacting oocyte cytoplasm maturation such as GSH, ROS, BMP15 and GDF9 have significant difference with an oocyte maturation culture solution without melatonin, and therefore, the melatonin is remarkably improved in oocyte cytoplasm maturation. The oocyte in vitro fertilization result shows that: the number and the percentage of double pronuclei generated by the oocyte maturation culture solution group added with the melatonin are obviously higher than those of the oocyte maturation culture solution not added with the melatonin, and the number and the percentage of multiple pronuclei generated by the oocyte maturation culture solution group added with the melatonin are obviously lower than those of the oocyte maturation culture solution not added with the melatonin. The blastocyst rate and the total number of blastocyst cells of the oocyte maturation culture solution group added with the melatonin are obviously higher than those of the oocyte maturation culture solution not added with the melatonin. The above results show that melatonin improves the quality of in vitro maturation of porcine oocytes by promoting the cytoplasmic maturation of porcine oocytes, thereby inhibiting the polyspermia fertilization of oocytes during in vitro fertilization and improving the developmental capacity of in vitro fertilized embryos.
Drawings
FIG. 1 is a graph showing the effect of in vitro maturation medium for porcine oocytes on GSH, ROS, BMP15, GDF9 in porcine oocytes;
FIG. 2 is a graph showing the effect of in vitro maturation culture medium of porcine oocytes on GSH, ROS, BMP15, GDF9 in porcine oocytes, wherein A is the effect on GSH, the abscissa is the group, and the ordinate is the fluorescence intensity of GSH; panel B is the effect on ROS, with the abscissa being the group and the ordinate being the ROS fluorescence intensity; panel C shows the effect on BMP15, group on the abscissa and BMP15 fluorescence intensity on the ordinate; panel D is the effect on GDF9, group on abscissa and fluorescence intensity of GDF9 on ordinate.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The experimental reagents were all purchased commercially.
Example 1.
1. In vitro maturation of pig oocytes:
composition of In Vitro Maturation (IVM) culture solution of porcine oocytes: the IVM culture solution adopts TCM-199 as a basic culture solution, and is added with 0.6mmol/L cysteine, 0.91mmol/L sodium pyruvate, 10ng/mL EGF, 10% pig follicular fluid by volume fraction, 10IU/mL FSH (follicle stimulating hormone), 10IU/mL LH (luteinizing hormone) by mass fraction of 1% streptomycin and melatonin 10-11mol/L。
2. The preparation method of the porcine oocyte culture solution comprises the following steps:
(1) obtaining the pig follicular fluid: extracting liquid from the follicle of the pig ovary, centrifuging the liquid for 30min at 1500rpm at 4 ℃ for 3-4 times, retaining the supernatant, filtering the supernatant with a 0.45 μm filter to remove impurities, and filtering with a 0.22 μm filter to obtain the required follicle liquid of the pig;
(2) obtaining culture solution of TCM-199, cysteine, sodium pyruvate, EGF, FSH, LH, streptomycin and melatonin;
(3) and (3) mixing the culture solution obtained in the step (2) with the porcine follicular fluid obtained in the step (1) to obtain the porcine oocyte in-vitro maturation culture solution.
3. The obtained pig ovaries are placed in normal saline at 32 ℃ and transported back to a laboratory. Cumulus Oocyte Complexes (COCs) with a diameter of 3-6 mm were extracted with a 18-gauge needle. Transferring COCs to In Vitro Maturation (IVM), culturing oocyte in IVM culture medium for 21h, transferring to IVM culture medium without FSH and LH, and culturing for 21h at 38.5 deg.C and 5% CO2Culturing in saturated humidity environment to obtain MII stage oocyte.
Example 2.
1. Composition of In Vitro Maturation (IVM) culture solution of porcine oocytes: the IVM culture solution adopts TCM-199 as a basic culture solution, and is added with 0.6mmol/L cysteine, 0.91mmol/L sodium pyruvate, 10ng/mL EGF, 10% pig follicular fluid in volume fraction, 10IU/mL FSH, 10IU/mL LH in mass fraction of 1% streptomycin and 10% melatonin-8mol/L。
2. The preparation method of the porcine oocyte culture solution comprises the following steps:
(1) obtaining the pig follicular fluid: extracting liquid from the follicle of the pig ovary, centrifuging the liquid for 30min at 1500rpm at 4 ℃ for 3-4 times, retaining the supernatant, filtering the supernatant with a 0.45 μm filter to remove impurities, and filtering with a 0.22 μm filter to obtain the required follicle liquid of the pig;
(2) obtaining culture solution of TCM-199, cysteine, sodium pyruvate, EGF, FSH, LH, streptomycin and melatonin;
(3) and (3) mixing the culture solution obtained in the step (2) with the porcine follicular fluid obtained in the step (1) to obtain the porcine oocyte in-vitro maturation culture solution.
3. The obtained pig ovaries are placed in normal saline at 32 ℃ and transported back to a laboratory. Cumulus Oocyte Complexes (COCs) with a diameter of 3-6 mm were extracted with a 18-gauge needle. The COCs were transferred to In Vitro Maturation (IVM) and the oocytes were cultured in IVM medium for 21h, thenTransferring to IVM culture medium without FSH and LH, culturing for 21 hr at 38.5 deg.C and 5% CO2Culturing in saturated humidity environment to obtain MII stage oocyte.
Example 3.
1. Composition of In Vitro Maturation (IVM) culture solution of porcine oocytes: IVM culture solution adopts TCM-199 as basic culture solution: adding 0.6mmol/L cysteine, 0.91mmol/L sodium pyruvate, 10ng/mL EGF, 10% volume fraction pig follicular fluid, 10IU/mL FSH, 10IU/mL LH, 1% mass fraction streptomycin and 10% melatonin-5mol/L。
2. The preparation method of the porcine oocyte culture solution comprises the following steps:
(1) obtaining the pig follicular fluid: extracting liquid from the follicle of the pig ovary, centrifuging the liquid for 30min at 1500rpm at 4 ℃ for 3-4 times, retaining the supernatant, filtering the supernatant with a 0.45 μm filter to remove impurities, and filtering with a 0.22 μm filter to obtain the required follicle liquid of the pig;
(2) obtaining culture solution of TCM-199, cysteine, sodium pyruvate, EGF, FSH, LH, streptomycin and melatonin;
(3) and (3) mixing the culture solution obtained in the step (2) with the porcine follicular fluid obtained in the step (1) to obtain the porcine oocyte in-vitro maturation culture solution.
3. The obtained pig ovaries are placed in normal saline at 32 ℃ and transported back to a laboratory. Cumulus Oocyte Complexes (COCs) with a diameter of 3-6 mm were extracted with a 18-gauge needle. Transferring COCs to In Vitro Maturation (IVM), culturing oocyte in IVM culture medium for 21h, transferring to IVM culture medium without FSH and LH, and culturing for 21h at 38.5 deg.C and 5% CO2Culturing in saturated humidity environment to obtain MII stage oocyte.
Comparative example 1.
1. Composition of In Vitro Maturation (IVM) culture solution of porcine oocytes: IVM culture solution adopts TCM-199 as basic culture solution: adding 0.6mmol/L cysteine, 0.91mmol/L sodium pyruvate, 10ng/mL EGF, 10% volume fraction pig follicular fluid, 10IU/mLFSH, 10IU/mL LH and 1% mass fraction streptomycin.
2. The culture solution was prepared according to example 1, except that melatonin was not added.
3. The obtained pig ovaries are placed in normal saline at 32 ℃ and transported back to a laboratory. Cumulus Oocyte Complexes (COCs) with a diameter of 3-6 mm were extracted with a 18-gauge needle. Transferring COCs to In Vitro Maturation (IVM), culturing oocyte in IVM culture medium for 21h, transferring to IVM culture medium without FSH and LH, and culturing for 21h at 38.5 deg.C and 5% CO2Culturing in saturated humidity environment to obtain MII stage oocyte.
The effect was verified with the following experiment:
1. GSH levels and ROS levels in porcine oocytes were determined by staining with CellTracker Blue CMF2HC (4-chloromethyl-6.8-difluoro-7-hydroxyformanin, Invitrogen) and H2DCFDA (2 ', 7' -diflorofluorescein diacetate, Invitrogen), the former being Blue fluorescence and the latter green fluorescence, respectively. 10. mu. mol/L of H2DCFDA and CellTracker Blue CMF2HC were added to PBS, respectively, and the matured oocytes were placed therein and cultured in the dark for 30 min. Washed 3 times with PBS, placed in 4 μ L TALP-HEPES droplets, and fluorescence observed under a fluorescence microscope, and ROS and GSH fluorescence intensity were analyzed using Image J software.
2. Immunofluorescent staining of BMP15 (bone morphogenetic protein 15) and GDF9 (growth differentiation factor 9) in porcine oocytes
The oocytes of the groups of examples 1-3 and comparative example 1 were fixed in 4% paraformaldehyde at room temperature for 60min, washed 3 times in 1% PVA-PBS, permeabilized with 1% TritonX-100 at room temperature for 30min, and washed 5 times in 1% PVA-PBS. Blocking was performed in 2% BSA at 37 ℃ for 1 h. BMP15 and GDF9 antibody were used and incubated overnight at 4 ℃. Incubate for 1h in FITC-labeled goat anti-rabbit secondary antibody. After 3 washes, the slides were mounted and fluorescence was observed under a fluorescence microscope and analyzed for BMP15 and GDF9 fluorescence intensity using Image J software.
The results in Table 1 show that the groups of examples 1 to 3 and comparative example 1 had no significant difference in oocyte maturation rate, i.e., no significant difference in oocyte nucleus maturation.
TABLE 1 Effect of porcine oocyte in vitro maturation culture Medium on porcine oocyte in vitro maturation
The results in FIG. 1 and FIG. 2 show that the fluorescence intensities of GSH, BMP15 and GDF9, which are important markers for cytoplasmic maturation of oocytes in the groups of oocytes in examples 1-3, are significantly higher than those of oocytes in the group of comparative example 1, and the fluorescence intensity of reactive oxygen species ROS in oocytes in the groups of oocytes in examples 1-3 is significantly lower than that of oocytes in the group of comparative example 1. Thus, the oocytes of the groups of examples 1 to 3 were improved in quality by cytoplasmic maturation of porcine oocytes.
3. In vitro fertilization of pig oocytes:
culturing the oocyte for 42h to obtain an MII stage oocyte, randomly placing the MII stage oocyte in an mTris-buffered medium (mTBM) droplet, and covering the mTBM droplet with mineral oil preheated in advance. Fresh semen transported to a laboratory within 2h from a local pig farm is washed twice by calcium-magnesium-free DPBS, centrifuged at 1000g for 2 minutes, and the precipitate obtained after centrifugation is resuspended by mTBM. After resuspension, the semen resuspension solution was added to mTBM droplets to achieve a sperm-egg ratio of 500: 1. Sperm viability > 80% is ensured before fertilization proceeds. mTBM droplet containing oocytes and sperm at 39 deg.C, 5% CO2Cultured for 5 h. After incubation for 5h, the oocytes were gently blown off of unattached, tight sperm on the zona pellucida. Gametes were washed with PZM-3 medium and subsequently placed in 500. mu.L four-well plates (50 gametes/well) at 39 ℃ with 5% CO2Under the conditions of (3), the cultivation was continued for 7 d.
The oocyte is cultured for 42h, and after in vitro fertilization for 10h, the formation of pronucleus is observed, wherein double pronucleus is in a normal development state, and single pronucleus and multiple pronucleus are in abnormal development. The results in Table 2 show that the proportion of normal double pronuclei produced by the groups of examples 1-3 is significantly higher than that of the group of comparative example 1, no significant difference exists among the groups on the aspect of producing abnormal single pronuclei, and the group of comparative example 1 on the aspect of producing abnormal multiple pronuclei is higher than that of the groups of examples 1-3.
TABLE 2 Effect of porcine oocytes cultured in vitro maturation Medium on polyspermia
Note that: different letters of a and b represent significant differences in the data from the same column (P < 0.05).
The cleavage rate of oocytes cultured by IVM of 42h groups of examples 1-3 and comparative example 1 was observed after 48h in vitro fertilization, and the blastocyst rate was observed after 168h, and the results in Table 3 show that there was no significant difference in cleavage rate between the groups of examples and comparative examples, but the groups of examples 1-3 were significantly higher than the group of comparative example 1 in blastocyst rate and total number of blastocysts.
TABLE 3 Effect of porcine oocytes on in vitro fertilization by porcine oocytes cultured in vitro maturation Medium
Note that: different letters of a and b represent significant differences in the data from the same column (P < 0.05).
The oocyte in vitro maturation results show that: although the oocyte maturation rate of the oocyte maturation culture medium group added with melatonin (namely, the groups of examples 1 to 3) has no significant difference, indexes for reacting oocyte cytoplasm maturation such as GSH, ROS, BMP15 and GDF9 have significant difference with the group of comparative example 1, which shows that the melatonin significantly improves oocyte cytoplasm maturation.
The oocyte in vitro fertilization result shows that: the melatonin-supplemented oocyte maturation medium group (i.e., examples 1-3) produced significantly higher numbers and percentages of pronuclei than the melatonin-unsupplemented oocyte maturation medium group (i.e., comparative example 1), and the melatonin-supplemented oocyte maturation medium group (i.e., examples 1-3) produced significantly lower numbers and percentages of pronuclei than the melatonin-unsupplemented oocyte maturation medium group (i.e., comparative example 1). The blastocyst rate and total blastocyst cells of the melatonin-supplemented oocyte maturation medium group (i.e., examples 1-3) were significantly higher than those of the melatonin-supplemented oocyte maturation medium group (i.e., comparative example 1).
The results show that the melatonin improves the in vitro maturation quality of the porcine oocytes by promoting the cytoplasmic maturation of the porcine oocytes, thereby inhibiting the polyspermia fertilization effect of the oocytes in the in vitro fertilization process and improving the developmental capacity of the in vitro fertilized embryos.
Claims (9)
1. The in vitro maturation culture solution for the porcine oocytes with the function of inhibiting the fertilization of polysperms is characterized by comprising the following components: TCM199, cysteine, sodium pyruvate, porcine follicular fluid, FSH, LH, streptomycin and melatonin.
2. The in vitro maturation culture solution of porcine oocytes according to claim 1, wherein the in vitro maturation culture solution of porcine oocytes comprises 0.6mmol/L cysteine, 0.91mmol/L sodium pyruvate, 10ng/mL EGF, 10% volume fraction porcine follicular fluid, 10IU/mL FSH, 10IU/mL LH, 1% mass fraction streptomycin and 10% mass fraction streptomycin based on TCM199-11-10-5mol/L melatonin.
3. The in vitro maturation culture solution of porcine oocytes according to claim 1, wherein the in vitro maturation culture solution of porcine oocytes comprises 0.6mmol/L cysteine, 0.91mmol/L sodium pyruvate, 10ng/mL EGF, 10% volume fraction porcine follicular fluid, 10IU/mL FSH, 10IU/mL LH, 1% mass fraction streptomycin and 10% mass fraction streptomycin based on TCM199- 11mol/L melatonin.
4. The in vitro maturation culture medium of porcine oocytes according to claim 1, wherein the in vitro maturation culture medium of porcine oocytes comprises 0.6mmol/L cysteine, 0.91mmol/L sodium pyruvate, 10ng/mL EGF, 10% volume fraction porcine follicular fluid, 10IU/mL FSH, 10IU/mL LH,The mass fraction of the streptomycin is 1 percent and 10 percent- 8mol/L melatonin.
5. The in vitro maturation culture solution of porcine oocytes according to claim 1, wherein the in vitro maturation culture solution of porcine oocytes comprises 0.6mmol/L cysteine, 0.91mmol/L sodium pyruvate, 10ng/mL EGF, 10% volume fraction porcine follicular fluid, 10IU/mL FSH, 10IU/mL LH, 1% mass fraction streptomycin and 10% mass fraction streptomycin based on TCM199- 5mol/L melatonin.
6. The method for preparing the in vitro maturation culture medium of porcine oocytes according to any one of claims 1 to 5, comprising the following steps:
(1) obtaining the pig follicular fluid: extracting liquid from the follicle of the pig ovary, centrifuging the liquid for 30min at 1500rpm at 4 ℃ for 3-4 times, retaining the supernatant, filtering the supernatant with a 0.45 μm filter to remove impurities, and filtering with a 0.22 μm filter to obtain the required follicle liquid of the pig;
(2) obtaining culture solution of TCM-199, cysteine, sodium pyruvate, EGF, FSH, LH, streptomycin and melatonin;
(3) and (3) mixing the culture solution obtained in the step (2) with the porcine follicular fluid obtained in the step (1) to obtain the porcine oocyte in-vitro maturation culture solution.
7. Use of the culture solution for in vitro maturation of porcine oocytes according to any one of claims 1 to 5 for the preparation of a reagent for inhibiting polyspermy.
8. Use of the porcine oocyte in vitro maturation culture medium of any one of claims 1 to 5 for the preparation of a medicament for the prevention of polyspermy.
9. Use of the porcine oocyte in vitro maturation culture medium of any one of claims 1 to 5 in the preparation of a medicament for increasing the developmental competence of an in vitro fertilized embryo.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110114495.0A CN112725263B (en) | 2021-01-26 | 2021-01-26 | Porcine oocyte in-vitro maturation culture solution with polyspermy inhibition effect and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110114495.0A CN112725263B (en) | 2021-01-26 | 2021-01-26 | Porcine oocyte in-vitro maturation culture solution with polyspermy inhibition effect and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112725263A true CN112725263A (en) | 2021-04-30 |
| CN112725263B CN112725263B (en) | 2022-04-29 |
Family
ID=75594249
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110114495.0A Active CN112725263B (en) | 2021-01-26 | 2021-01-26 | Porcine oocyte in-vitro maturation culture solution with polyspermy inhibition effect and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112725263B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113834936A (en) * | 2021-08-20 | 2021-12-24 | 李竞宇 | Application of growth differentiation factor 9 in predicting embryonic development potential |
| CN115161266A (en) * | 2022-06-23 | 2022-10-11 | 东北农业大学 | Application of tannic acid in inhibiting polyspermia fertilization of porcine oocytes |
| CN117511854A (en) * | 2024-01-05 | 2024-02-06 | 金宝医学科技(深圳)有限公司 | Immature oocyte culture solution and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628709A (en) * | 2019-10-22 | 2019-12-31 | 吉林大学 | Culture solution and culture method for improving in-vitro maturation quality of porcine oocytes |
-
2021
- 2021-01-26 CN CN202110114495.0A patent/CN112725263B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628709A (en) * | 2019-10-22 | 2019-12-31 | 吉林大学 | Culture solution and culture method for improving in-vitro maturation quality of porcine oocytes |
Non-Patent Citations (6)
| Title |
|---|
| LING YANG, ET AL.: "Effect of Melatonin on the In Vitro Maturation of Porcine Oocytes, Development of Parthenogenetically Activated Embryos, and Expression of Genes Related to the Oocyte Developmental Capability", 《ANIMALS》 * |
| 冯怀亮 著: "《哺乳动物胚胎工程》", 31 July 1994, 吉林科学技术出版社 * |
| 周珍辉 主编: "《动物细胞培养技术》", 31 August 2006, 中国环境科学出版社 * |
| 徐仙 编著: "《生殖医学中心临床实践》", 31 May 2012, 阳光出版社 * |
| 黄晶晶 等人: "褪黑素不同添加量对猪***体外成熟和胚胎发育的影响", 《2015***业科技大会暨2015年学术年会》 * |
| 黄晶晶 等人: "褪黑素对猪***体外成熟和孤雌胚发育影响的研究", 《繁殖生理》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113834936A (en) * | 2021-08-20 | 2021-12-24 | 李竞宇 | Application of growth differentiation factor 9 in predicting embryonic development potential |
| CN115161266A (en) * | 2022-06-23 | 2022-10-11 | 东北农业大学 | Application of tannic acid in inhibiting polyspermia fertilization of porcine oocytes |
| CN117511854A (en) * | 2024-01-05 | 2024-02-06 | 金宝医学科技(深圳)有限公司 | Immature oocyte culture solution and preparation method thereof |
| CN117511854B (en) * | 2024-01-05 | 2024-03-19 | 金宝医学科技(深圳)有限公司 | Immature oocyte culture solution and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112725263B (en) | 2022-04-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112725263B (en) | Porcine oocyte in-vitro maturation culture solution with polyspermy inhibition effect and preparation method and application thereof | |
| Wongsrikeao et al. | Effect of the removal of cumulus cells on the nuclear maturation, fertilization and development of porcine oocytes | |
| Lai et al. | Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes | |
| US6872569B2 (en) | In vitro production of haploid germ cells | |
| CN110760473B (en) | Porcine oocyte in-vitro maturation culture solution and preparation method and application thereof | |
| Arat et al. | Effect of growth factors on oocyte maturation and allocations of inner cell mass and trophectoderm cells of cloned bovine embryos | |
| CN107365738B (en) | Method for preparing cow and cattle xenogenesis in-vitro fertilization embryo | |
| Dos Santos-Neto et al. | Cumulus cells during in vitro fertilization and oocyte vitrification in sheep: Remove, maintain or add? | |
| Feng et al. | Fertilization and early embryology: Effect of different co-culture systems in early human embryo development | |
| CN110684723B (en) | Porcine oocyte in-vitro maturation culture solution and preparation method and application thereof | |
| CN110577928A (en) | Oocyte in-vitro maturation culture solution and application thereof | |
| Chandra et al. | Effect of growth factors (epidermal growth factor, platelet derived growth factor, and insulin-like growth factor-1) on buffalo (Bubalus bubalis) embryos produced in vitro | |
| WO2021258423A1 (en) | Serum-free in vitro maturation culture solution for bovine oocytes and oocyte culture method | |
| CN107043743B (en) | In-vitro maturation method of canine oocytes | |
| Freitas et al. | In vitro embryo production in small ruminants | |
| CN114410573A (en) | Oocyte in-vitro maturation culture solution additive and application thereof | |
| CN114276984B (en) | Method for transdifferentiating female germ stem cells into functional sperm and application | |
| RU2410063C1 (en) | Method for extracorporeal cultivating bovine oocytes | |
| CN102499788B (en) | Application of SRY (sex determining region of the Y) antibody | |
| CN113151159A (en) | Oocyte in-vitro maturation culture solution additive and application thereof | |
| Mardenli et al. | Effects of Various Levels of Luteinizing Hormone and Caprine Follicular Fluid on In Vitro Embryo Production of Shami Goat | |
| CN115161266A (en) | Application of tannic acid in inhibiting polyspermia fertilization of porcine oocytes | |
| US20200399587A1 (en) | Culture medium for improving development of bovine in vitro fertilized embryos and cloned embryos | |
| Thomas et al. | Effects of cumulus cells on culture of bovine embryos derived from oocytes matured and fertilized in vitro | |
| EP1238584A1 (en) | Method for producing livestock individuals from cells of established cell line |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |