CN104585163A - Buffalo oocyte vitrification pretreating method - Google Patents
Buffalo oocyte vitrification pretreating method Download PDFInfo
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Abstract
The invention discloses a buffalo oocyte vitrification pretreating method. A buffalo oocyte is pretreated by applying certain concentration of cytochalasin B before being subjected to vitrification. In vitro tests prove that the cytochalasin B has a remarkable protecting function on the vitrification of the mature buffalo oocyte, so that the damage to a cytoskeleton after the oocyte is cryopreserved is reduced, and the survival rate and the developmental rate of the cryopreserved oocyte are increased.
Description
Technical field
The invention belongs to buffalo mature oocyte vitrificated cryopreserration field, particularly relate to a kind of buffalo oocytes glass freezing preprocess method.
Background technology
Buffalo is the domestic animal variety source of south China preciousness, and one of domestic animal of most potentiality to be exploited and Development volue is thought by food and agricultural organization of united state.The protein of buffalo milk, fat, micro-equal size are all higher than black-and-white flower milk, therefore its high-end dairy produce is more competitive.But the low large obstacle becoming restriction buffalo development of dairy industry of the reproduction rate of China buffalo, the biological self reproducing technology being badly in need of application of advanced improves, but the research and development of these technology application needs a large amount of egg mother cells.Buffalo oocytes is carried out freezen protective; fundamentally can solve the technical barrier that buffalo ovum source can not get ensureing; and contribute to setting up breeding buffalo ovum and offer an opportunity with conservation genetics resource and for species genetic improvement, obtain larger social effect and economic benefit.
Within 1985, Rall uses vitrifying freeze process successfully to save the embryo of mouse first, and after this glass freezing constantly improves as a kind of technology of new development, and the egg mother cell of Some Species obtains effective preservation successively.Though through studying for many years, still there is many obstacles in vitrification in preservation egg mother cell.Egg mother cell, to the susceptibility of low temperature, causes the change of structure and physicochemical property after its glass freezing, the especially damage of cytoskeleton.The growth of egg mother cell, maturation and fertilization need the participation of cytoskeleton, glass freezing easily causes the depolymerization of microtubule and the entanglement distribution of microfilament, thus causing the exception of spindle and chromosome structure and the change of cytoskeletal protein, this has a strong impact on the survival rate after egg mother cell freeze thawing, developmental rate.Therefore, how to reduce the freezing damage to egg mother cell and just become the study hotspot of egg mother cell and embryo cryopreservation.
Have document to show, cytochalasin B (being called for short CB), as Cytoskeleton Stabilizer, makes cell more high resilience.Use cytochalasin B pretreatment can reduce the damage of microfilament and microtubule to a certain extent before freezing, improve the stability of cytoskeleton in oocyte vitrification process.Once report confirmation 7.5 μ g/mL CB was had to have obvious effect porcine oocytes is freezing, but about the report of CB action effect in buffalo oocytes glass freezing process is little, only several sections of its results of report are also usually contradiction, the result obtained also is verified further, and its working concentration is all test according to the gradient freezing at porcine oocytes.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of buffalo oocytes glass freezing preprocess method, to reduce the damage of cytoskeleton after egg mother cell freeze thawing, and then improves survival rate and the developmental rate of egg mother cell after freeze thawing.
For solving the problems of the technologies described above, the present invention by the following technical solutions: buffalo oocytes glass freezing preprocess method is that the cytochalasin B of 4-12 μ g/mL is in incubator process by concentration before buffalo oocytes carries out glass freezing.
Above-mentioned buffalo oocytes glass freezing preprocess method, before buffalo oocytes carries out glass freezing, adds the cytochalasin B that concentration is 8 μ g/mL, is then placed in incubator process 30min in ripe liquid.
Cause damage for current glass freezing to buffalo oocytes thus cause the problem that egg mother cell survival rate and developmental rate reduce, inventor applies certain density cytochalasin B and carries out pretreatment before buffalo oocytes carries out glass freezing.In vitro test proves, the glass freezing of cytochalasin B to buffalo mature oocyte has the protective effect of significance, reduces the damage of cytoskeleton after egg mother cell freeze thawing, improves survival rate and the developmental rate of egg mother cell after freeze thawing.Accompanying drawing explanation
Fig. 1 is buffalo oocytes developmental capacity cytological map after the pretreatment of variable concentrations cytochalasin B.
Fig. 2 is the column statistical chart of variable concentrations cytochalasin B pretreatment on female activation effect impact lonely after the freeze thawing of buffalo mature oocyte, in figure: A: represent difference extremely remarkable, a, b: it is not remarkable that identical lowercase represents difference, different representative significant differences.
Fig. 3 is the microtubule of buffalo mature oocyte and chromosome is normal, the immunofluorescence dyeing figure of exception and disappearance after application the present invention, in figure: buffalo oocytes is under fluorescence microscope in finding spindle structure, chromosome morphology, microtubule is in green, and chromosome and polar body take on a red color; Wherein: A1-A3: normal spindle structure, Chromosomal arrangement is in equatorial plate; B1-B3: spindle disappearance and shrinkage, chromosome is normal; C1-C3: spindle and Chromosome spread; D1-D3: spindle is at random, rare, chromosome abnormality; E1-E3: spindle disappears, chromosome abnormality.
Fig. 4 is the immunofluorescence dyeing figure of normal, the exception of the microfilament of buffalo mature oocyte and disappearance after application the present invention, in figure: buffalo oocytes is under fluorescence microscope in finding microfilament form, and microfilament is in green, and chromosome and polar body take on a red color; Wherein: A1-A3: normal microfilament distribution; B1-B3: the punishment of microfilament polar body is loose; C1-C3: microfilament spatial abnormal feature cell periphery; D1-D3: microfilament reduces; E1-E3: microfilament lacks.
Fig. 5 be after application the present invention the tubulin of buffalo mature oocyte and actin at the expression column statistical chart of different disposal group, in figure: A, B, C: it is extremely remarkable that different capitalization represents difference, it is not remarkable that identical capitalization represents difference, a: represent difference not remarkable.
Fig. 6 is the electrophoretogram on cytoskeletal protein impact after buffalo mature oocyte glass freezing after application the present invention, in figure: 1: fresh control group; 2: fresh CB processed group; Freezing group of 3:CB process; 4: direct freezing group
Fig. 7 is the blastaea and the blastaea fluorescent staining figure that apply different disposal group of the present invention, and in figure: control is control group, CB+Cryotop is CB process freezing group of group, and Cryotop is direct freezing group of group.
Embodiment
One, test material
Test agents useful for same is except TCM-199 is purchased from except Gibco company, and other reagent are if no special instructions all purchased from Sigma chemical company.
The formula of major experimental reagent is as follows:
Ripe liquid: TCM-199+26.2mmol/L NaHCO
3+ 5mmol/L Hepes+10%ECS, 0.06g penicillin, 0.1g streptomycin.With 0.22 μm of membrane filtration sterilizing, 4 DEG C of preservations, add 3%BFF, 10 μ g/mL FSH, 10 μ g/mL LH, 1 μ g/mL E2,100 μm of ol/L cysteines, 1 μ g/m LMLT before using, at CO
2at least 1h is balanced in incubator.
2 μ g/mL cytochalasin B storage liquid: add 2.5mL DMSO and dissolve 5mg CB, fully dissolve mixing, filter, are dispensed into Eppendof pipe, often pipe 10 μ l ,-20 degree freezen protective.Dilute according to the ripe liquid of different ratios during use, can use.
Cryoprotective extender: TCM-199+5.96g/L Hepes+1.25g/L NaHCO3+0.056g/L Sodium Pyruvate;
Equilibrium liquid: cryoprotective extender+10% DMSO (DMSO)+10% ethylene glycol (EG)+20%FCS+0.1%PS;
Freezing liquid: cryoprotective extender+20%DMSO+20%EG+0.5mol/L Sucrose+20%FCS+0.1%PS;
Thawing solution: cryoprotective extender+0.5mol/L Sucrose+20%FCS+0.1%PS.
Prepare test solution and all filter with the miillpore filter of 0.02 μm.
Two, test method
Vitrification method reference literature (Liang Y Y, et al.In vitro development of vitrifiedbuffalo oocytes following parthenogenetic activation and intracytoplasmic sperminjection] .Theriogenology, 2011,75 (9): 1652-1660), concrete operation step is as follows:
(1) maturation in vitro of egg mother cell: after obtaining ovary from slaughter house, Nanning, with being added with antibiotic brine 2-3 time, to remove the bloodstain adhered to, the unnecessary tissue of periphery is cut off again with scissors, with the egg mother cell that the 10ml syringe extraction Ovarian surface diameter of No. 12 syringe needles is in 2 ~ 6mm ovarian follicle, 3 times are washed with ripe liquid, at 39 DEG C, 5%CO
2in-vitro maturation 22 ~ 24h is carried out with in the incubator of maximum saturation humidity.
(2) pretreatment of cytochalasin B: by the egg mother cell after step (1) maturation culture, in ripe liquid, add 0.1% hyaluronidase blow and beat the ovarian cumulus layer removed around gently, select the mature oocyte that discharge first polar body, kytoplasm are even, full.Before egg mother cell carries out glass freezing, use the cytochalasin B of variable concentrations (0,4,8,12 μ g/mL) at incubator process 30min.
(3) freezing step: the egg mother cell that step (2) is disposed is carried out freezing.Time freezing, room temperature is adjusted to 22-24 DEG C.First, mature oocyte is first moved into 5min in the cryoprotective extender (TCM199-Hepes buffer solution) containing 20%FBS, move into 1min in freezing equilibrium liquid (TCM199-Hepes+20%FBS+10% dimethyl sulfoxide (DMSO)+10% ethylene glycol) subsequently, observe egg mother cell substantially to permeate fully, move in freezing liquid (TCM199-Hepes+20%FBS+20% dimethyl sulfoxide (DMSO)+20% ethylene glycol+0.5M sucrose), transfer in 30s in cryovial, drop into liquid nitrogen rapidly.Each operation oocyte number is 5, institute carrying liqs volume <1 μ L.Thawing of egg mother cell: the egg mother cell after freezing, at Liquid nitrogen storage 1-2 days, thaws immediately.Thawing solution is shifted to an earlier date 2 hours in incubator preheating.Course of defrosting is all carried out on the heating plate of 37.Taken out from liquid nitrogen by cryovial, immersed rapidly in thawing solution by the cryovial containing egg mother cell end, because of the effect of being heated, egg mother cell is discharged in thawing solution very soon.At thawing solution balance 5min, moved into 5min in the cryoprotective extender containing 20%FBS, with the cryoprotective extender washing 4-5 time containing 20%FBS, finally moved in the cryoprotective extender containing 20%FBS, at 39 DEG C, 5%CO
2with recover 1h in the incubator of maximum saturation humidity after carry out subsequent experimental.
(4) lonely female activation: the egg mother cell after freeze/thaw recovery is washed 2 times fast at ionomycin working solution drop, the culture fluid be all placed in containing 5 μm of ol/L ionomycins activates process 5min, wash the culture fluid moved into again containing 2mmol/l 6-DMAP after 3 times through culture fluid and cultivate 4h, wash 3 times finally by culture fluid and move into granular cell individual layer droplet afterwards, in 39 DEG C, 5%CO
2with Dual culture in the incubator of maximum saturation humidity.Change culture fluid every 2 days half amounts, check division rate after cultivating 48h, 7-8d records blastocyst rate.This step is used for screening the concentration for the treatment of of optimum cytochalasin B.
(5) immunofluorescence dyeing: after obtaining optimum cytochalasin B concentration for the treatment of from step (4), carry out step (3) method freezing, egg mother cell after freeze thawing recovery fixes 30min (or 4 spend night) under 4% paraformaldehyde room temperature, subsequently by fixing egg mother cell cleaning solution cleaning 3-4 time (moving sap cavity to blow and beat gently), often all over 3-4min.Put into permeable membrane liquid after cell washing, 37 DEG C of permeable membranes spend the night (10 ~ 16h).The egg mother cell that permeable membrane completes is blown and beaten clean gently with liquid-transfering gun in cleaning solution, is generally 3-4 time, often all over 3-4min.Proceed to confining liquid afterwards and close 1 ~ 1.5h (or 4 spend night) in 37 DEG C.
Microtubule dyes: the anti alpha tubulin mouse monoclonal antibody that the egg mother cell closed and 1:100 dilute is hatched jointly, 4 DEG C spend the night (10 ~ 16h), after one process resistant, through cleaning solution cleaning 3-4 time, often all over 3-4min, be placed in 1:80 dilution: two anti-FITC-mountain sheep anti-mouse igg room temperature lucifuge process 1 ~ 1.5h, after two process resistant terminate, cleaning 3-4 time, often all over 3-4min.After two process resistant terminate, carry out nuclear staining, cell being put into concentration is 10 μ g/mL propidium iodides (PI), room temperature lucifuge dyeing 5min, after cleaning solution cleaning 3 ~ 4 times, self-contained compressing tablet, namely on clean slide, two regions are marked with the marker of black, a circle medical ventolin is coated in area peripheral edge, cell after washing is placed in the middle of region, lack carrying liqs as far as possible, in case during compressing tablet outside overflow sheet, covered, under stereomicroscope, cover glass is pressed gently with the rifle head of 1mL, note making the slight pressurized of cell, in case excessively press and make clasmatosis.The micropipe aggregation of buffalo mature oocyte is at polar body and chromosome place, and the microtubule at chromosome place forms spindle state, and due to shooting angle, chromosome has two states: one is arranged in a linear, and spindle is symmetrically distributed in chromosome both sides; Another kind is arrangement in plate-like, and spindle is embedded in wherein, and the microtubule at polar body place is without particular state.
Microfilament dyes: the egg mother cell closed is placed in 1 μ g/mL FITC-Phalloidin room temperature (or 37 DEG C) lucifuge dyeing 1-1.5h.Carry out core dye and compressing tablet afterwards, step and dye microtubule time the same.The microfilament of buffalo mature oocyte is mainly enriched in polar body place, without particular state.
(6) Western-blotting Western blotting: after obtaining optimum cytochalasin B concentration for the treatment of from step (4), carry out step (3) method freezing, egg mother cell after freeze thawing recovery carries out Western-blotting Western blotting, is studied cytoskeleton main component tubulin and actin content change.
The collection of a protein sample
The egg mother cell of each group is washed 3 times with PBS respectively, and with fine needle, egg mother cell sucking-off is loaded EP pipe (often pipe 100 egg mother cells) of sterilizing in advance, brief centrifugation removes unnecessary PBS.Often pipe adds 10 μ L RIPA and 1% protease inhibitors, utilizes liquid nitrogen multigelation 3-5 time, middle at interval 5min on ice, and the abundant cracking of its albumen is separated out.After cracking, add 2 μ L 5 × albumen sample-loading buffers, sealed by EP pipe, in boiling water, boil 10min with sealing compound, 4 degree of centrifugal 10min of 12000rpm get supernatant loading EP pipe afterwards, preserve or namely use for-80 °.
The preparation of b SDS-PAGE gel and loading
After in a certain order electrophoresis tank being assembled, separation gel solution is joined between glass plate slowly, after the corresponding scale of glue plate, add 75% alcohol, to avoid producing bubble and flattening separation gel.After gelling to be separated is solid, add concentrated glue, insert comb.After gelling is solid, pulls out comb gently, adds the SDS electrophoretic buffer prepared.The light and slow successively afterwards 4 × albumen sample-loading buffer adding equivalent, albumen marker, sample 1, sample 2, sample 3, sample 4 and 4 × sample-loading buffer.
C electrophoresis
The complete cover lid of application of sample, carries out electrophoresis.First regulation voltage is 80V, and electric current is 10mA, and sample protein is concentrated, and then regulation voltage is to 120V, electric current 20mA, protein isolate, and powered-down when band arrives bottom, stops electrophoresis.
D transferring film
Glue after electrophoresis taken out and cuts unnecessary part, putting into the transferring film immersion bubble of now joining.The pvdf membrane onesize with glue sheared in advance is soaked 2min in formalin, then formalin and pvdf membrane is poured in transferring film liquid, mixing, put into simultaneously 2 pieces with the thick filter paper of the equal size of glue.Glue, pvdf membrane and thick filter paper soak 15min in transferring film liquid.Put into paper-glue-film-paper from top to bottom at transferring film folder, should flatten between every layer and drain bubble, then putting into electric turn trough transferring film.Transferring film condition is: 25V, 0.1mA, 30min.
E closes
Transferring film takes out pvdf membrane after stopping, and closed to spend the night with 5% skim milk powder solution room temperature shaker being hatched 1h or 4 degree.
F primary antibodie is hatched
Take out the pvdf membrane after closing, put into TBST, shaking table concussion washing 4 times, each 10min, finally washes 1 time with TBS.Dilute primary antibodie (1:1000) with TBS, hatch washed pvdf membrane, 37 DEG C of effect 2-3h.
G bis-is anti-hatches
Take out pvdf membrane, with TBST shaking table concussion washing 4 times, each 10min, finally washes 1 time with TBS.Add two anti-(1:200) diluted with TBS, 37 DEG C of effect 2h
H develops the color
Two anti-hatch after, wash pvdf membrane 4 times with TBST.Mixed in proportion by the reagent of colour developing box, mixed liquor lucifuge and pvdf membrane hatch 10min, carry out exposure image in BIO-RAD imaging system.According to the exposure status adjustment time for exposure, select satisfied film.
(7) detection of potentiality of development:
After obtaining optimum cytochalasin B concentration for the treatment of from step (4), it is freezing to carry out step (3) method, and the egg mother cell after freeze thawing carries out intracytoplasmic sperm injection, detects its subsequent development potential.
Sperm prepares: from liquid nitrogen, take out a pipe seminal fluid, be placed in rapidly 37 degree of water-baths and be about 40s, dry the globule on spermaduct wall with paper handkerchief.Seminal fluid blowout in pipe is placed in the EP pipe of 1.5mL, put into the centrifuge tube of 15mL with 200 μ L pipettor sucking-offs, add PBS centrifuge washing, 1500 leave heart 5min.Remove supernatant, add a certain amount of PBS, prevent seminal fluid thickness.
The intracytoplasmic sperm injection of buffalo oocytes carries out under being inverted micromanipulation instrument at Nkion.Pre-prepd locking pin (internal diameter is 20 1 30um) and injection group (internal diameter is 8 one 9um) are arranged on micromanipulation instrument.With 60x culture dish lid as operation board, do from top to down 6 drip, above two be pancreatin, rinse entry needle and locking pin; Middle two is 7%PVP operation liquid (about 10uL), places sperm and cleans sperm and use; Two long is below operation liquid (containing 7.5 μ g/mL CB, about 20uL), puts egg mother cell to be injected.During micromanipulation, first locking pin and injection group are carried out rinse, the entry needle PVP moved to containing sperm operates in liquid subsequently, selects the measured sperm of matter to suck injection-tube openend from afterbody, operate in liquid at another PVP and sperm is carried out rinse, finally move on in the operation liquid containing egg mother cell.Now fix egg mother cell with locking pin, the polar body of egg mother cell is towards the position being equivalent to hour hands " 12 " point or " 6 " and putting, and the position puncture oolemma that entry needle is put in " 3 ", the direction of putting towards " 9 " is deep, the a small amount of cytoplasm of first resorption, then sperm is injected kytoplasm.Subsequently, by entry needle withdrawing from slowly, and egg mother cell is discharged from stationary pipes.
The chemokinesis process of egg mother cell after injection: the complete egg mother cell of injection is washed 3 times in culture fluid, is placed in incubator and recovers 30min, carry out chemokinesis subsequently.First use 5 μm of ol/L ionomycin process 5min, cultivate 3h at embryo medium afterwards, then choose the egg mother cell of discharging second polar body, with 2m,mol,L/6 mono-DMAP process 3.5h, finally move into granular cell layer droplet Dual culture.
(8) buffalo blastaea dyeing
Being collected in by step (7) each group blastaea is equipped with in the EP pipe of 4% paraformaldehyde, fixing 30min, egg-cleaning liquid washing 4-5 time.Dilute dyestuff Hoechst33342 by a certain percentage with PBS, make its working concentration be 10 μ g/ml.The blastaea fixed is placed in dyestuff, lucifuge effect 5min, with PBS washing 4-5 time, removes unnecessary dyestuff, carry out compressing tablet subsequently, at fluorescence microscopy Microscopic observation, statistics blastomere number.
Three, result of the test
1. the pretreatment of variable concentrations cytochalasin B is on the impact of buffalo mature oocyte glass freezing effect
The cytochalasin B pretreatment of variable concentrations is added on the impact of buffalo mature oocyte glass freezing effect as depicted in figs. 1 and 2 in ripe liquid, CB pretreatment concentration is respectively 0 μ g/mL, 4 μ g/mL, 8 μ g/mL and 12 μ g/mL, and each group processing time is 30min.Survival rate as can be seen from Figure 1 in experimental group after egg mother cell freeze thawing and subsequent development potential are all well below control group.Survival rate between experimental group is without larger difference, but the cleavage rates of 8 μ g/mL groups is significantly higher than other three processed group (P<0.01), and blastocyst rate is also significantly higher than 0 μ g/mL group (P<0.05), and with 4 μ g/mL groups and 12 μ g/mL group differences not significantly (P<0.01).0 μ g/mL, between 4 μ g/mL and 12 μ g/mL tri-groups in blastocyst rate and there was no significant difference (P<0.01).Therefore buffalo mature oocyte glass freezing effect can be improved from whole structure 8 μ g/mLCB pretreatment.
2. cytochalasin B pretreatment is on the impact of buffalo mature oocyte glass freezing posterior microtubule
As shown in Figure 3, through fluorescence microscope after egg mother cell immunofluorescence dyeing, the egg mother cell in the stage of ripeness, its microtubule is symmetrically distributed in chromosome both sides (Fig. 3 A3) with the form of spindle microtubule, because viewing angle is different, aligned or the plate-like of chromosome, when in plate-like, spindle is embedded in wherein (Fig. 3 B3).The impact of cytochalasin B pretreatment on buffalo mature oocyte glass freezing posterior microtubule is as shown in table 1, compared with control group, glass freezing all causes spindle and chromosome normal distribution rate significantly to decline (70.57%vs 38.66%, 77.03%vs42.11%, P<0.01), abnormal spindle, also have remarkable rising (P<0.05) without the egg mother cell ratio of spindle and abnormal chromosome.But the pretreatment through 8 μ g/mL CB before egg mother cell is freezing can significantly improve spindle and chromosomal natural rate of interest (P<0.05), and the egg mother cell ratio (P<0.05) significantly reduced without spindle and abnormal chromosome, shows that the pretreatment of 8 μ g/mL CB produces significantly impact to spindle form and Chromosomal arrangement
Table 1
In table 1, the different lowercase alphabet of same column numerical value subscript shows significant difference (p<0.05), and different capitalization represents that difference extremely significantly (P<0.01).Following table is same.
3. cytochalasin B pretreatment is on the impact of microfilament after buffalo mature oocyte glass freezing
As shown in Figure 4, through fluorescence microscope after egg mother cell immunofluorescence dyeing, the egg mother cell in the stage of ripeness, its microfilament is mainly enriched in polar body place (Fig. 4 A3).Cytochalasin B pretreatment is as shown in table 2 on the impact of microfilament after buffalo mature oocyte glass freezing, after glass freezing, the natural rate of interest of egg mother cell microfilament is 43.54%, compared with 81.47% of control group, difference extremely significantly (P<0.01), illustrate that the freezing microfilament normal distribution rate that causes declines, but the natural rate of interest (P<0.05) of microfilament can be significantly improved before egg mother cell is freezing through the pretreatment of 8 μ g/mL CB, show that the pretreatment of 8 μ g/mL CB creates obvious impact to microfilament form.
Table 2
From table 1 and table 2, the natural rate of interest difference of spindle, chromosome and microfilament between control group and fresh group of CB process not significantly (P>0.05), illustrates that CB does not have an impact to cytoskeleton.
4. cytochalasin B pretreatment is on the impact of cytoskeletal protein after buffalo mature oocyte glass freezing
Carry out WB analysis to the egg mother cell of experimental group and control group, result is as Fig. 5 and 6.Known, compared with control group, freezing rear egg mother cell tubulin amount significantly declines (p<0.01), and the content of actin is substantially unaffected.Can reduce the decline of buffalo mature oocyte freeze thawing posterior microtubule protein content before freezing to a certain extent after 8 μ g/ml CB pretreatment, but the content of actin do not have significant difference in freezing front and back.
5. cytochalasin B pretreatment is on the impact of potentiality of development after the freeze thawing of buffalo mature oocyte.
Observe through the impact of cytochalasin B pretreatment on potentiality of development after the freeze thawing of buffalo mature oocyte, this experiment is chosen 8 μ g/mL cytochalasin Bs and is carried out pretreatment, carries out intracytoplasmic sperm injection, the results are shown in Table 3 to the egg mother cell of experimental group and control group.The freezing discharge rate of egg mother cell second polar body, cleavage rates, 8-cell rate and the blastocyst rate of all causing declines, significant difference (43.88%vs.68.04% compared with control group, 62.71%vs 87.50%, 34.90%vs 77.43%, 10.21%vs 30.21%).Compared with freezing group, although carry out discharge rate and cleavage rates (P>0.05) that CB pretreatment fails to have improved egg mother cell second polar body before freezing, significantly improve 8-cell rate and blastocyst rate (P<0.05).Freezing front and back blastomere number difference is not significantly (P>0.05).Fig. 7 is shown in the blastaea that control group and experimental group are formed and blastaea fluorescent staining.
Table 3
Four, conclusion
Buffalo oocytes volume is large, washiness, lipid rich, and to freezing very responsive, refrigerating process easily causes the change of cytoskeleton physicochemical property and integrality.For this reason, during research, vitrificated cryopreserration is carried out to buffalo oocytes, first In-vitro maturation, then use cytochalasin B pretreatment, finally carry out vitrificated cryopreserration.The present invention adopts concentration to be that the cytochalasin B of 8 μ g/mL is at incubator process 30min, the refrigerating effect of buffalo mature oocyte can be significantly improved during freezen protective, the damage of cytoskeleton whether is decreased subsequently with the use of the method validation cytochalasin B of immunofluorescence dyeing, result shows that cytoskeleton improves really, whether also change with WB technical identification cytoskeletal protein afterwards, equally also draw corresponding result.Therefore, the pretreatment adopting 8 μ g/mL cytochalasin Bs really can stabilized cell skeleton, improves refrigerating effect.Because buffalo oocytes is generally applied in actual production by vitro fertilization or intracytoplasmic sperm injection technology, so the egg mother cell after freeze thawing is carried out intracytoplasmic sperm injection by the present invention, find and do not carry out approximately improving 10% compared with cytochalasin B pretreated group, this is a very large improvement in freezing, the damage caused because freezing is many-sided, refrigerating effect can not be changed a lot by improvement on the one hand, but this improvement of 10% is enough to skeleton damage to drop to minimum.
In a word, the present invention improves significantly to the preservation effect of buffalo oocytes glass freezing, and experiment proves, adds cytochalasin B and carries out pretreatment, really can play facilitation to buffalo oocytes Cord blood, guide direction for improving buffalo oocytes refrigerating effect or thinking is provided.Meanwhile, research institute of the present invention does not use in the research of cytochalasin B with checking means are former.
Claims (2)
1. a buffalo oocytes glass freezing preprocess method, to is characterized in that before buffalo oocytes carries out glass freezing by concentration being that the cytochalasin B of 4-12 μ g/mL is in incubator process.
2. buffalo oocytes glass freezing preprocess method according to claim 1, it is characterized in that before buffalo oocytes carries out glass freezing, in ripe liquid, add the cytochalasin B that concentration is 8 μ g/mL, be then placed in incubator process 30min.
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| CN108707578A (en) * | 2018-05-07 | 2018-10-26 | 湖北省农业科学院畜牧兽医研究所 | One boar list placenta in vitro culture method |
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Non-Patent Citations (1)
| Title |
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| 卞桂华等: "细胞松弛素B（CB）对水牛卵母细胞玻璃化冷冻保存的影响", 《黑龙江畜牧兽医》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108707578A (en) * | 2018-05-07 | 2018-10-26 | 湖北省农业科学院畜牧兽医研究所 | One boar list placenta in vitro culture method |
| CN108707578B (en) * | 2018-05-07 | 2021-01-22 | 湖北省农业科学院畜牧兽医研究所 | Pig monose embryo in vitro culture method |
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