CN110484494A - A method of improving Porcine In Vitro Maturation Oocytes quality - Google Patents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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Abstract
The invention discloses a kind of methods for improving Porcine In Vitro Maturation Oocytes quality, pre-process pig GV phase egg mother cell using methyl-B-cyclodextrin before glass freezing.Preferably, the concentration of methyl-B-cyclodextrin is 3~8mg/mL, and the pretreated time is 5~6.5min.The present invention directlys adopt methyl-B-cyclodextrin and handles immature cumulus oocytes complesxes, then glass freezing is carried out again, In-vitro maturation is carried out after defrosting again, it was found that adding exogenous cholesterol before glass freezing enhances the quality of the porcine oocytes of maturation in vitro after GV phase glass freezing, be conducive to the long-term preservation of porcine oocytes.
Description
Technical field
The present invention relates to a kind of methods for improving Porcine In Vitro Maturation Oocytes quality.
Background technique
Animal oocyte is widely used in during embryo production in vitro, nuclear transfer and gene pool preservation etc., therefore, research
Personnel produce sizable interest to the multiple fields of egg mother cell freezen protective.In past 20 years, porcine oocytes
Preservation be constantly subjected to hinder.In recent years, the trial of multiple freezen protective porcine oocytes has been carried out, it is as a result different.Pig
The successful freezen protective of egg mother cell is of great significance to the protection of genetic resources and assisted reproductive technology.Although pig ovum is female thin
Born of the same parents are highly sensitive to low temperature, but the development of vitrification improves result in recent years.Somfai researches show that from cold
Freeze in the GV phase egg mother cell saved and successfully produces piglet.The research of Gajda then shows the MII phase egg mother cell from freezen protective
It is middle successfully to produce piglet.Although glass freezing GV phase porcine oocytes can produce offspring, the speed of embryonic development is still
Very low Appeltant etc..Optimize glass freezing process therefore, it is necessary to keep punching.
During the freezen protective of mammal ovocyte, a significant points of freezen protective damage are cytoplasm
Lipid in film, especially film.Reducing temperature causes lipid to undergo the transformation (lipid phase transition) from liquid crystalline phase to gel phase, this
It can lead to film integrality reduction and cell death.In this respect, Membrane cholesterol: phosphatide (C:P) ratio is in process of cryopreservation
The important determinant of membrane fluidity and stability.Film (high cholesterol film: phosphatide ratio) with high cholesterol concentration is lower
At a temperature of more mobility, therefore to freeze it is insensitive.
Summary of the invention
The technical problem to be solved by the present invention is to overcome survival rate after the freezing of porcine oocytes in the prior art is lower scarce
It falls into, a kind of method improving Porcine In Vitro Maturation Oocytes quality is provided.
In order to solve the above-mentioned technical problems, the present invention provides the following technical solutions:
A method of improving Porcine In Vitro Maturation Oocytes quality, methyl-B-cyclodextrin is used before glass freezing
Pre-process pig GV phase egg mother cell.
Further, the concentration of methyl-B-cyclodextrin is 3~8mg/mL.Preferably, the concentration of methyl-B-cyclodextrin is
5mg/mL.
Further, the pretreated time is 5~6.5min.
The beneficial effects obtained by the present invention are as follows being: the present invention directlys adopt methyl-B-cyclodextrin and handles immature ovarian cumulus-
Then oocyte complex carries out glass freezing again, carry out In-vitro maturation after defrosting again, finds in glass freezing
Exogenous cholesterol is added before and enhances the quality of the porcine oocytes of maturation in vitro after GV phase glass freezing, is conducive to
The long-term preservation of porcine oocytes.
Specific embodiment
Hereinafter, preferred embodiments of the present invention will be described, it should be understood that preferred embodiment described herein is only used
In the description and interpretation present invention, it is not intended to limit the present invention.
Embodiment
The preparation of 1.1 related solutions
Glass freezing and the minimal medium of defrosting solution are the TCM199 with the HEPES buffering of 20%FBS.In base
The CLC of various concentration (0,0.5,5 and 10mg/mL) is supplemented in basal culture medium.Balance solution is by 7.5% ethylene glycol (EG), 7.5%
Dimethyl sulfoxide (DMSO) and containing 20%FBS TCM199 composition.Vitrification solution is by containing 15%EG, 15%DMSO, 20%
The TCM199 of FBS, 0.4M sucrose is formed.Defrosting I liquid: 0.5m sucrose, defrosting II liquid are added in the TCM199 containing 20%FBS
It is made of the 0.25M sucrose in the TCM199 containing 20%FBS.Incubating Solution is the TCM199 containing 10%FBS.
TCM 199 (containing 10%FBS) is supplemented with 3.05mM D-Glucose, 0.91mM Sodium Pyruvate, 25.07mM bicarbonate
Sodium, 0.1% polyvinyl alcohol, 75ug/ml penicillin, 50 μ g/ml streptomysins, 10IU/ml hCG, 10IU/ml eCG and 10ng/ml
EGF is used for oocyte maturation culture.ZP solvent soln: using deionized water dissolving 0.06g sodium dihydrogen phosphate, be diluted to 100ml,
The phosphate sodium dihydrogen buffer solution for preparing 5mM is adjusted to pH 2.5.Lock solution for Western blotting contains 5g defatted milk
Powder is dissolved in 100mL Tris buffered saline tween (10 × TBS), is saved using preceding in 4 DEG C.
Capacitation liquid is referring to Garc í a etc.: 96mM NaCl, 4.7mM KCl, 0.4mM MgSO4、0.3mM NaH2PO 4、
5.5mM glucose, 1mM Sodium Pyruvate, 21.6mM sodium lactate, 0.5mM CaCl2、10mM NaHCO3、20mM HEPES(pH
And 3mg/ml BSA 7.45).Embryo culture medium (NCSU23): 0.6355g NaCl, 0.2105g NaHCO3、0.0356g KCl、
0.0162g KH2PO4、0.0142g MgSO4、0.025g CaCl2·2H2O、0.100g Glucose、0.0146g
Glutamine、0.0875g Taurine、0.0545g Hypotaurine、0.0065g Penicilin G sodium
Salt, 0.005g Streptomycin sulfate are dissolved in 100ml deionized water (to be opened by North Carolina State University 23
Culture medium (NCSU-23) composition is sent out, 0.4%BSA is supplemented).
The collection of 1.2 cumulus oocyte compounds
Sow ovary used in this test is taken from Yanji slaughterhouse.Ovary is immediately placed in after taking out out of sow body
Laboratory is transported back in vacuum flask equipped with 38 DEG C or so 0.9% physiological saline.For separating cumulus oocyte compound
(COCs) postmortem program carries out on the glass slide heater platform for be set as 38.5 DEG C.After blotting salt water, selection is each
The ovarian follicle of 3-6mm in ovary, and liquor folliculi is individually sucked out using the 10ml disposable syringe with 18G syringe needle, then gently
It is transferred in sterile 50ml pipe.After being incubated for 30-60 minutes in 38 DEG C of water-baths, with pasteur pipet draw bottom of the tube COCs and
Other precipitatings, are then transferred into TCM cleaning solution (at 38,5 DEG C).Hereafter, selection display is consistent, equal under inverted microscope
Even dark color and the high quality COCs surrounded by three layers or more cumulus granulosa cells.
1.3 incubate the GV phase egg mother cell of culture together with the CLC for nitrobenzoxadiazole (NBD) label that NBD is marked
It educates
Cumulus cell is removed with 0.1% (w/v) hyaluronidase.For the GV phase of cholesterol detection and non-glass freezing
The combination of egg mother cell, by egg mother cell be supplemented with 20% (v/v) FBS and 20%22-N- (oxa- -1 7- nitro benzo -2-,
3- diazole -4- base) amino -23,24 diine -5- indoles -3b- alcohol label cholesterol (NBD-CLC) TCM199 in be incubated for it is 1 small
When.NBD-CLC is prepared as described in Horvath and Seidel etc..After 1 hour, by egg mother cell in DPBS and 0.1% (w/v) BSA
Middle washing, fixed (4% paraformaldehyde fixative), and use the Nikon Eclipse with 11001v2 blue color filter
Epifluorescence E800 microscope photographing COCs and egg mother cell.
The glass freezing of 1.4 GV phase porcine oocytes
Glass freezing balancing processing step carries out under the microscope at 38.5 DEG C.COC is placed in balance solution first
In 5 minutes, be then transferred into vitrification solution, be incubated for 30s after, egg mother cell is loaded on OPS and puts into liquid nitrogen (LN2)
In.COCs is transferred to vitrification solution should be less than 90 seconds until total exposure duration that they put into liquid nitrogen.
The defrosting of 1.5 GV phase porcine oocytes and maturation culture
Vitrified egg mother cell is placed in thawing solution I 5 minutes, is then transferred in thawing solution II 5 points
Clock.Then, egg mother cell is washed three times in 0.1%PBS-PVA and TCM199 containing 10%FBS, is then transferred to ovum
Mother cell maturation medium, in 5%CO2It is cultivated 46-48 hours in (38.5 DEG C, 95% maximum saturation humidity environment) incubator.
The maturing rate and survival rate of 1.6 assessment egg mother cells
After cultivating 46 hours in vitro, with 0.1% hyaluronic acid enzymatic treatment egg mother cell until the most of ovarian cumulus of removing is thin
Born of the same parents.Oocyte maturation is measured under microscope (400 ×), such as the discharge of first polar body.
The measurement of 1.7 egg mother cell mitochondrial membrane potentials
After removing cumulus cell, the MII egg mother cell of maturation in vitro is washed in PBS buffer solution, it is thin to be subsequently placed in 1ml
In the mixture of born of the same parents' culture medium (mature liquid) and 1ml JC-1 dyeing working solution (being configured by kit specification).Dyeing is trained
Feeding base is sufficiently mixed, then in 37 DEG C of incubator (5%CO2, 95% maximum saturation humidity environment) in be incubated for 20 minutes.In absorption
Clearly, it is then washed twice in the JC-1 dye solution of pre-cooling.Finally, by the egg mother cell of dyeing and 2ml cell culture medium
Mixing, is observed in dark surrounds under fluorescence microscope.
1.8 promote the measurement of apoptogene mRNA expression
After egg mother cell IVM is handled 46 hours and removed cumulus cell, useSuperTotal RNA
Extraction Kit (Promega, Shanghai, China) cracks egg mother cell.Then DNAse I incubation buffer is added
To eliminate any contaminating genomic DNA, and washed repeatedly using RNA washing buffer until obtaining high-purity RNA.It extracts
RNA is according to the Prime Script of TaKaRa companyTMRT Master Mix kit specification (catalog number (Cat.No.): RR036A) cDNA
Reverse transcription.Measure the cDNA concentration of sample.CDNA is used as to the template of amplification target gene, and expands products therefrom.
The dissolution of 1.9 egg mother cell oolemmas
Sample containing 200 to 300 MII egg mother cells is washed three times in 0.1%PVA-PBS, is then transferred to
In 0.6ml centrifuge tube.After removing excess buffer, 20 μ l ZP solvent solns are added into each pipe to dissolve oolemma, then
It is incubated for 90 minutes in 70 DEG C of water-baths.When observing under the microscope, confirmation oolemma is completely dissolved.Collect sample and with 5000
× g is centrifuged 5 minutes.Then by supernatant equal part and be stored in -20 DEG C until analysis.
1.10 sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (kit, P0012A)
15%SDS-PAGE separating gel is prepared indoors.Protein example is mixed with sample sample-loading buffer, then
It is denaturalized 5 minutes at 99 DEG C.After being cooled to room temperature, 20-25 μ l protein example is loaded into each hole of gel.Electrophoresis exists
It carries out under 100V, and stops as Bromophenol Blue dye Front distance gel bottom about 2cm.
1.11 Western blotting
After SDS-PAGE, pvdf membrane (Millipore ISEQ00010) is cut into the size of PAGE gel, immerses first
5 minutes in alcohol, it is then immersed in film transfer buffer (P0021B) 3-5 minutes.It will be coagulated at 25V using half photosensitive transfer instrument
Protein transfer in glue was to pvdf membrane 90 minutes.
After Protein transfer, pvdf membrane is washed three times (every time with TBST buffer (Tris buffered saline, Tween-20)
10 minutes), it is then incubated in lock solution, vibrates 2 hours at room temperature.Hereafter, lock solution is removed, washs film with TBST
(10 minutes) three times.Then pvdf membrane is placed in culture dish, which contains the rabbit polyclonal antibody for ubiquitin protein
(AB1690) it solution (thinner ratio 1:2000) and is incubated for 12 hours at 4 DEG C.Topple over antibody and washs film three times (10 with TBST
Minute).Then film is incubated with 2 hours with rabbit igg peroxidase secondary antibody (A0545) (1:500 dilution) at room temperature, with
(10 minutes) three times are washed in TBST afterwards.Image J be used for quantify different proteins expression and each protein bars
The chemiluminescence intensity of band.The ubiquitination level of ZP albumen is standardized by the amount of GAPDH albumen.
1.12 in vitro fertilization and Embryo Cultures
Mature oocyte is moved into capacitation culture medium, each droplet puts 15 cells, is then injected again with liquid-transfering gun
The 1x10 of capacitation650 μ l of cell/ml sperm, be put into 38.5 DEG C, 5%CO2,95% maximum saturation humidity incubator in be fertilized
4-6 hours.Egg mother cell is washed 4 times in 0.1%PBS-PVA after 4-6 hours, at room temperature in 5% paraformaldehyde
Fix 30 minutes.Hereafter, egg mother cell is incubated with 15 minutes with 5 μ g/ml Hoechst 33342 in the dark, In
It washes twice in 0.1%PBS-PVA solution, is then observed at nu-425-600e fluorescence microscope (Leica, Germany)
(ultraviolet light): excitation wavelength: 330nm~400nm, launch wavelength: 425nm), oolemma is attached to each egg mother cell of determination
Sperm quantity.The egg mother cell being fertilized is transferred in embryo culture medium, 10 are put in each droplet, is put into 38.5 DEG C, 5%
CO2, 95% maximum saturation humidity incubator in cultivate.Spilting of an egg situation is checked after 48h, is then moved into new embryo culture medium
Continue culture to the 7th day.
1.13 statistical analysis
Experiment determines western blot band density in triplicate, using Image J software, uses ANOVA points of SPSS19
All data are analysed, the Multiple range test of Duncan is tested for comparing average value, and P < 0.05 is considered significant.
2 results
The assessment of 2.1 cholesterol fluorescence levels
The results show that the egg mother cell that non-glass freezing, unused NBD-CLC are incubated for does not have fluorescence；Non- glass freezing,
Hyperfluorescence is shown with the egg mother cell that NBD-CLC is incubated for；Glass freezing, with NBD-CLC be incubated for egg mother cell show it is weak
Fluorescence.
The maturation in vitro rate of 2.2 GV phase egg mother cells
Pig GV phase egg mother cell, glass are handled with the CLC (0.5,5 and 10mg/ml) of various concentration in vitrification solution
The oocyte in vitro maturation rate of change is slightly above non-freezing processing group, wherein the maturing rate of 5 and 10mg/ml CLC processing group is significant
Higher than 0 and 0.5mg/ml CLC vitrifying processing group (P < 0.05).
Influence of 2.3 CLC to the mitochondrial membrane potential after the porcine oocytes of GV phase glass freezing are mature
The red fluorescence intensity of 5mg/ml CLC processing group egg mother cell is significantly higher than in glass freezing processing group
0.5mg/ml (P < 0.05), 10mg/ml and 0mg/ml CLC group, although fluorescence is significantly lower than shown in unverified control.Control
The mitochondrial membrane that most weak fluorescence signal shows these egg mother cells in group is led to mitochondria activity by damage to a certain degree
It reduces.5mg/ml CLC processing group also shows that red green fluorescence (P < 0.05) more stronger than other two processing groups.In short, this
A bit the result shows that 5mg/ml CLC processing provides the protection of the highest level to egg mother cell damage in During Vitrification in vitro, because
This these cell is able to maintain that the mitochondria activity of highest level.
2.4 promote the mRNA expression of apoptogene after the porcine oocytes of GV phase glass freezing are mature
The expression of apoptotic gene amount of glass freezing processing group egg mother cell is all remarkably higher than non-glass freezing processing group,
Wherein, Caspase3, Caspase8 and the Caspase9 gene expression of 5 and 10mg/ml CLC processing group egg mother cell are significantly low
In 0 and 0.5mg/ml CLC processing group (P < 0.01).
Influence of 2.5 CLC to the ZP proteins ubiquitin after the porcine oocytes of GV phase glass freezing are mature
Western blot is the results show that three kinds of ZP albumen of 61kDa (ZP1), 80kDa (ZP2) and 106kDa (ZP3) exist
It experienced different degrees of proteins ubiquitin in maturation.With 0.5mg/ml CLC, 10mg/ml CLC and glass freezing pair
It is compared according to group, the total ZP proteins ubiquitin level of egg mother cell of 5mg/ml CLC processing is higher, and especially ZP3 albumen is especially aobvious
It writes.These results indicate that these ZP albumen experienced proteins ubiquitinization modification during the maturation in vitro of porcine oocytes.
Influence of 2.6 CLC to porcine oocytes embryonic development
This shows that the associated value of sperm and egg mother cell is significantly higher than vitrifying in 0.5,5 and 10mg/mL CLC processing group
Frozen control group (P < 0.01).The blastocyst rate of glass freezing processing group egg mother cell is substantially less than non-vitrifying processing group, glass
The blastocyst rate that CLC processing group egg mother cell is added in glass refrigerating process is slightly above un-added.
Influence of 3.1 CLC to egg mother cell survival and maturing rate and embryonic development
The display of this result of study, the survival rate of vitrifying processing group egg mother cell are below the control group not freezed, wherein
The maturing rate of 5mg/mL and 10mg/mLC LC vitrifying processing group egg mother cell is slightly above non-freezing processing group.However vitrifying
The embryo development rate of processing group egg mother cell is substantially less than non-vitrifying processing group, this shows that vitrifying GV ovum can be improved in CLC
The maturing rate of mother cell, but embryo development rate cannot be improved.
Influence of 3.2 CLC to egg mother cell mitochondrial membrane potential
Porcine oocytes are especially sensitive to low temperature.Facts proved that contacting their bases of 15 DEG C or more of low temperature in a short time
Originally survival rate is lost.High lipid content is considered as one of the main reason for they is to low-temperature sensitive in porcine oocytes.Its
His reason, such as injury of mitochondria, may will affect survival or the apoptosis of glass freezing porcine oocytes.
Mitochondria is main cellular relevant to energy supply and organelle migration, therefore, injury of mitochondria or line
The variation of plastochondria distribution may generate strong influence to the development of egg mother cell.Jc-1 is widely used in the detection of mitochondria Δ Ψ m,
Mitochondria Δ Ψ m can be accumulated in mitochondrial matrix and be formed aggregation by monomer, and can be contaminated under fluorescence microscope
At red.The red ratio with green fluorescence intensity is generally used to indicate that the functional status of mitochondria.
Mitochondrial membrane potential Δ Ψ m is easy impaired during cryo-conservation and defrosting.When mitochondrial transmembrane potentials are low,
Entire cytoplasm should generate green fluorescence.The result shows that the egg mother cell mitochondrial membrane potential that glass freezing thaws is not lower than
The egg mother cell that glass freezing thaws.The result is consistent with the result of Dai etc..Compared to other glass freezing processing groups,
The mitochondria Δ Ψ m of 5mg/ml CLC processing egg mother cell is significantly increased, and illustrates that exogenous CLC, can be with after infiltrating cells matter
The opposite egg mother cell mitochondrial membrane damage for reducing cold-induction, so that mitochondria be made to maintain normal activity.
Influence of 3.3 CLC to apoptogene mRNA expression is promoted
Vallorani et al. has found that glass freezing can pass through by freezing research glass freezing porcine oocytes
Activation caspase activity, mitochondrial membrane potential are lost and are induced cell apoptosis.The MII phase porcine oocytes of glass freezing
It is characterized in that developmental potency reduction, Elisa et al related with the activation of apoptosis pathway.Huang etc. points out addition antioxidant,
Oxidative damage can be reduced and reduce apoptosis rate.
In the exogenous apoptosis pathway that death receptor mediates, death ligand and its receptor knot merga pass self-catalysis are activated
Caspase 8, and self-catalysis is further through cutting and activation Caspase-3 or 7 stimulate Apoptosis.It withers as exogenous
The promoter of approach is died, caspase 8 plays an important role in exogenous apoptosis.After glass freezing, the activity of caspase 8
It greatly increases.When application 8 inhibitor of caspase, the not only activity of caspase 8, the activity of caspase 3 also by
Inhibit.These are the result shows that the exogenous apoptosis pathway that death receptor mediates promotes the generation of Apoptosis after glass freezing.
We analyze the mRNA expression for promoting apoptogene in mature oocyte, the results show that glass freezing
The expression of the Oocyte Apoptosis gene of processing group is all remarkably higher than non-glass freezing processing group.Glass freezing processing group
In, gene expression is substantially less than other several groups (P < 0.01) after 5 and 10mg/ml CLC processing.These are observation indicate that glass
Change and add the adjustable apoptosis process of CLC in refrigerating process, improves the damage that porcine oocytes induce freezen protective
Resistance.The result is similar to the result of study of Elisa etc..
Influence of 3.4 CLC to ZP proteins ubiquitin
The Western blot analysis of porcine oocytes shows, 61kDa (ZP1), 80kDa (ZP2), the three of 106kDa (ZP3)
Kind ZP albumen experienced different degrees of proteins ubiquitin when handling in response to CLC.In 5mg/ml CLC processing group, ZP egg
White total ubiquitination level is significantly higher than to be observed in other groups, and in these albumen, ZP3 shows highest level
Ubiquitination.Our result indicate that the exogenous CLC penetrated into oolemma before glass freezing protects this
A little tissues.Then, the egg mother cell of defrosting can carry out normal ubiquitination in cultivation stage.
The frostbite for observing egg mother cell helps to assess glass including the damage to oolemma, DNA and aneuploid embryos
The quality of glassization freezing egg mother cell.Oolemma is as the mass exchange channel between egg mother cell and internal environment, in sperm-
It plays an important role in egg fusion and embryonic development.It is reported that glass freezing enhances the hardness of oolemma, increase repeatedly
Sperm fertilization, and reduce rate of fertilization in vitro fertilization.
Oolemma around mammal ovocyte works in the various aspects of fertilization.Previous studies show (1)
Proteasome is present in the acrosome surface of acrosome and mammalian sperm, and (2) ubiquitin protein is present in mammalian oocyte
In film.The ZP albumen of ubiquitination is hydrolyzed by the relevant proteasome of sperm, and participates in the transparent carrying substrates of Sperm penetration.Egg mother cell
Glass freezing induce relevant to the secondary structure of protein and carbohydrate residue Biochemical changes.
In mammals, by ubiquitination in Process of oogenesis, sperm-receptor is dropped ZP albumen in fertilization process
Solution, and the relevant sperm-receptor albumen of proteasome degradation ZP of sperm carrying is had proven in mammals.Therefore, we
It is proposed that egg mother cell undergoes ubiquitination during maturation culture, and the ZP3 of ubiquitination may serve as the receptor of sperm.Although
It is observed that the ZP albumen to ubiquitination is hydrolyzed by the relevant proteasome of sperm and participated in Boar spermatozoa and penetrates oolemma, but do not have
Egg mother cell low-temperature resistance is assigned about report of the exogenous cholesterol in conjunction with oolemma and its during acting on slow freezing.
It is presumed that the cholesterol amount used has a significant impact these processes, therefore we intend to conduct further research, it is intended to
Develop the implementation procedure of egg mother cell freezen protective.
Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention
Within protection scope.
Claims (4)
1. a kind of method for improving Porcine In Vitro Maturation Oocytes quality, which is characterized in that use methyl-before glass freezing
Beta-cyclodextrin pre-processes pig GV phase egg mother cell.
2. improving the method for Porcine In Vitro Maturation Oocytes quality as described in claim 1, which is characterized in that methyl-β-ring
The concentration of dextrin is 3~8mg/mL.
3. improving the method for Porcine In Vitro Maturation Oocytes quality as described in claim 1, which is characterized in that methyl-β-ring
The concentration of dextrin is 5mg/mL.
4. the method as described in any one of claims 1-3 for improving Porcine In Vitro Maturation Oocytes quality, which is characterized in that pre-
The time of processing is 5~6.5min.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588011A (en) * | 2018-05-08 | 2018-09-28 | 中国农业科学院北京畜牧兽医研究所 | A method of improving glass freezing Oocytes in Vitro Fertilization ability |
| CN108841781A (en) * | 2018-06-13 | 2018-11-20 | 延边大学 | Crocin is improving the application in porcine oocytes oxidation resistance and cleavage rates |
| CN110923330A (en) * | 2019-11-29 | 2020-03-27 | 河北医科大学第一医院 | Method for evaluating frozen oocyte quality by micro RNA |
| CN115152746A (en) * | 2022-07-28 | 2022-10-11 | 河北大学 | New application of all-methyl cyclodextrin |
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|---|---|---|---|---|
| CN108588011A (en) * | 2018-05-08 | 2018-09-28 | 中国农业科学院北京畜牧兽医研究所 | A method of improving glass freezing Oocytes in Vitro Fertilization ability |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588011A (en) * | 2018-05-08 | 2018-09-28 | 中国农业科学院北京畜牧兽医研究所 | A method of improving glass freezing Oocytes in Vitro Fertilization ability |
Non-Patent Citations (2)
| Title |
|---|
| HIROYUKI WATANABE ET AL.: "Variation of cholesterol contents in porcine cumulus-oocyte complexes is a key factor in regulation of fertilizing capacity.", 《THERIOGENOLOGY.》 * |
| XUAN CHEN ET AL.: "Addition of cholesterol loaded cyclodextrin prior to GV-phase vitrification improves the quality of mature porcine oocytes in vitro.", 《CRYOBIOLOGY.》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588011A (en) * | 2018-05-08 | 2018-09-28 | 中国农业科学院北京畜牧兽医研究所 | A method of improving glass freezing Oocytes in Vitro Fertilization ability |
| CN108588011B (en) * | 2018-05-08 | 2022-04-05 | 中国农业科学院北京畜牧兽医研究所 | Method for improving in vitro fertilization capability of vitrified frozen oocyte |
| CN108841781A (en) * | 2018-06-13 | 2018-11-20 | 延边大学 | Crocin is improving the application in porcine oocytes oxidation resistance and cleavage rates |
| CN110923330A (en) * | 2019-11-29 | 2020-03-27 | 河北医科大学第一医院 | Method for evaluating frozen oocyte quality by micro RNA |
| CN115152746A (en) * | 2022-07-28 | 2022-10-11 | 河北大学 | New application of all-methyl cyclodextrin |
| CN115152746B (en) * | 2022-07-28 | 2024-05-17 | 河北大学 | New application of full-methyl cyclodextrin |
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