CN110438068A - A method of improving river type In Vitro Fertilization in Water Buffalo embryo development rate - Google Patents
A method of improving river type In Vitro Fertilization in Water Buffalo embryo development rate Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 37
- 230000004720 fertilization Effects 0.000 title claims abstract description 25
- 230000013020 embryo development Effects 0.000 title claims abstract description 18
- 241000030939 Bubalus bubalis Species 0.000 title claims abstract description 10
- 210000001733 follicular fluid Anatomy 0.000 claims abstract description 72
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims abstract description 49
- 230000035800 maturation Effects 0.000 claims abstract description 41
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims abstract description 25
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- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims abstract description 25
- 239000004472 Lysine Substances 0.000 claims abstract description 25
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims abstract description 25
- 235000004279 alanine Nutrition 0.000 claims abstract description 25
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- 239000004471 Glycine Substances 0.000 claims abstract description 24
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- CYDQOEWLBCCFJZ-UHFFFAOYSA-N 4-(4-fluorophenyl)oxane-4-carboxylic acid Chemical compound C=1C=C(F)C=CC=1C1(C(=O)O)CCOCC1 CYDQOEWLBCCFJZ-UHFFFAOYSA-N 0.000 claims description 5
- 229920000089 Cyclic olefin copolymer Polymers 0.000 claims description 5
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 claims description 5
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- 229930182555 Penicillin Natural products 0.000 claims description 5
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 5
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 claims description 5
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- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 3
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 3
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 3
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- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 2
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- AQGDXJQRVOCUQX-UHFFFAOYSA-N N.[S] Chemical compound N.[S] AQGDXJQRVOCUQX-UHFFFAOYSA-N 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0604—Whole embryos; Culture medium therefor
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
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- 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
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Abstract
The present invention relates to buffalo embryos to develop technical field, in particular to a kind of method for improving river type In Vitro Fertilization in Water Buffalo embryo development rate, the present invention, which mainly studies, improves buffalo oocytes maturation culture solution, in low concentration liquor folliculi (1%-4%), still it can reach the division rate and blastocyst rate for improving buffalo fertilized eggs, to improve the effect of buffalo extracorporeal embryo development rate and production efficiency；In formula, in order to activate the activity of egg mother cell, applicant is dissolved in TCM199 solution the influence that can significantly improve liquor folliculi with liquor folliculi mixing chelating again to embryo development rate is promoted using cysteine, methionine, alanine, lysine, tryptophan and glycine by numerous studies discovery.
Description
[technical field]
The present invention relates to buffalo embryos to develop technical field, in particular to a kind of raising river type In Vitro Fertilization in Water Buffalo embryo
The method of developmental rate.
[background technique]
Embryo Culture technology excavates the breeding potential of domestic animal, fast-propagation and plasm resource protection are with important
Value.The cause that In vitro culture and embryo in vitro save is influenced to be known as very much, wherein oocyte in vitro maturation, in vitro by
Essence, body early embryo in vitro culture and embryo cryopreservation are four key links during embryo production in vitro.It is existing very much
Maturation in vitro effect is less desirable always in Vitro Culture Techniques, is difficult the technology and embryo transfer, ovum pick-up (OPU)
Technology combines, and is applied to production practices, promotes and applies；In the prior art also about utilization liquor folliculi to oocyte maturation
The research of buffalo oocytes effect in vitro fertilization, for example, " different cultivars bovine follicular fluid is to water for the paper that applicant oneself delivers
The influence of IVF of Oocyte in Bovine effect " (Chinese animal and veterinary the 4th phase of volume 37 in 2010)；Just to different product in this article
Research of the ox ovarian follicle of kind to buffalo oocytes effect in vitro fertilization, this article indicate the optimum amount of liquor folliculi in 5%-
10%, and point out that 5% and 10% liquor folliculi of addition has obviously the embryonic development after slaughterhouse collection Oocytes in Vitro Fertilization
Facilitation, excessive concentration or the too low development that can all inhibit In Vitro Fertilization in Water Buffalo embryo, but in actual operation, liquor folliculi
It is big due to extracting difficulty, it is often not readily available, for this purpose, applicant studies how to improve formula and behaviour on the basis of original research
Make method, enable liquor folliculi under low concentration: 1%-4% achievees the purpose that promote In Vitro Fertilization in Water Buffalo embryonic development.
[summary of the invention]
In view of above content, it is necessary to a kind of method for improving river type In Vitro Fertilization in Water Buffalo embryo development rate is provided, it should
Method can improve mature liquid, make liquor folliculi under low concentration: 1%-4%, which remains to reach, improves In Vitro Fertilization in Water Buffalo embryo
The purpose of tire developmental rate.
In order to achieve the above objectives, the technical scheme adopted by the invention is that:
A kind of maturation culture solution improving river type In Vitro Fertilization in Water Buffalo embryo development rate, the maturation culture solution includes matter
Measure the liquor folliculi that percentage is 1%-4%.
Further, the liquor folliculi is the liquor folliculi handled by amino acid chelate.
Further, the method for the amino acid chelate processing are as follows: by cysteine, methionine, alanine, rely ammonia
Acid, tryptophan and glycine are dissolved in TCM199 solution, make final concentration of 500-700 μm of ol/L, the first sulphur ammonia of cysteine
The final concentration of 300-500 μm of ol/L of acid, the final concentration of 100-300 μm of ol/L of alanine, lysine final concentration of 100-
300 μm of ol/L, the final concentration of 50-100 μm of ol/L of tryptophan, glycine final concentration of 50-150 μm of ol/L；It is pressed after mixing
It is mixed for 1:1 with the liquor folliculi directly acquired according to mass ratio, shaken cultivation under the conditions of 39 DEG C.
Further, the maturation culture solution includes the mature liquid A of ovum in vitro culture being adopted for slaughterhouse and/or for living
Body adopts the mature liquid B of ovum in vitro culture.
Further, mature liquid A's the preparation method is as follows: basal liquid of 50mL is taken then to add into CM basal liquid
The FSH-LH storing liquid of 200 μ L, the E2 storing liquid of 45 μ L, the EGF liquid of 100 μ L, the cysteamine storing liquid of 50 μ L, then add ovarian follicle
Liquid makes the final concentration of 1%-4% of liquor folliculi.
Further, which is characterized in that the preparation method of the CM basal liquid is as follows:
(1) it configures Cm-Stock solution: weighing the TCM199 of 2.45~3.65g, the NaHCO of 0.18~0.30g3, 1.0~
The Sodium Pyruvate of 2.0mg, the sodium lactate that 35~45 μ L percentage compositions are 70%, the penicillin of 18~22mg, the chain of 17~27mg
Mycin, 9~13mg's is phenol red；And mentioned component is dissolved in the physiological saline of 200mL, it is uniformly mixed and obtains the Cm-
Stock solution；
(2) CM basal liquid is prepared: the step of fetal calf serum of 15mL-25mL is dissolved in 50mL (1) Cm-Stock solution
In obtain CM basal liquid；
The preparation method of the FSH-LH storing liquid is as follows: the follicular stimulating hormone of 120~150ug being taken to be dissolved in 10mL physiology
Salt water obtains follicle stimulating hormone solution, then weighs the interstitialcellstimulating hormone (ICSH) of 2.11~3.04mg and be dissolved in above-mentioned follicular stimulating hormone
After mixing in solution, after being distributed into 10 μ L/ pipes, FSH-LH storing liquid is obtained in -20 DEG C of preservations；
The preparation method of the E2 storing liquid is as follows: the ethyl alcohol for taking the estradiol of 2.01~2.43mg to be dissolved in 1mL mixes
Afterwards, it is distributed into 10 μ L/ pipe, obtains E2 storing liquid in -20 DEG C of preservations；
The preparation method of the EGF liquid is as follows: it is 10 that epithelical cell growth factor, which is configured to solutes content, with distilled water
The solution of~18 ng/mL obtains EGF liquid；
The preparation method of the cysteamine storing liquid is as follows: the cysteamine for weighing 7.0~7.5mg is dissolved in 10mL step
(2) in CM basal liquid, the 100 every pipes of μ L are packed as, obtain cysteamine storing liquid in -20 DEG C of preservations.
Further, the maturation liquid B is by as follows at being grouped as: 8% fetal calf serum, 10 μ g/mL follicular stimulating hormone,
15 μ g/mL interstitialcellstimulating hormone (ICSH)s, the estradiol of 5 μ g/mL, the Sodium Pyruvate of 0.9mmoL/L, 100 μm of ol/L cysteine,
The epicuticle growth factor of 35ng/mL, the liquor folliculi of 1%-4%, surplus are TCM199 solution.
A method of in vitro culture being carried out using culture solution, described method includes following steps:
(1) ovum in vitro culture is adopted in slaughterhouse: being collected buffalo ovaries, ovary excess tissue is removed, with containing dual anti-physiology salt
Water cleans 2-3 times, draws ovarian follicle with syringe, the ovum of uniform, the extracellular above granular cell that haves three layers of cytoplasm is chosen under stereoscope
Mother cell is washed 2~3 times, is placed in mature liquid A, in 39 DEG C, 5%CO2It is cultivated in saturated air humidified incubator；Culture
The cumulus cell around egg mother cell is blown off after 22-24h, suspends after then washing twice with new mature liquid A, droplet is made
After cover paraffin, in 39 DEG C, 5%CO2It is cultivated in saturated air humidified incubator；By buffalo sperm hatching after with egg mother cell into
Row fertilization, embryo in vitro culture；Record each embryonic development situation；The blastaea that collection is developed to 7d observes and records under the microscope
Blastomere sum；
(2) ovum pick-up in vitro culture: ovum pick-up is carried out to living body buffalo, egg mother cell is collected, is chosen under stereoscope
It selects cytoplasm uniformly and wraps up the COCs of one layer or more cumulus cell, in fetal calf serum and 20mmol/L Hepes containing 5%
It washes in TCMl99 liquid 2 times, then is cleaned 1 time with maturation liquid B, is transferred in the mature liquid B of 50 μ L/ drops, in 39 DEG C, 5%CO2, maximum
Maturation culture 22 in the incubator of relative humidity~for 24 hours；It is fertilized after culture with the buffalo sperm after hatching, embryo in vitro training
It supports, record each embryonic development situation；The blastaea that collection is developed to 7d observes and records blastomere sum under the microscope.
The invention has the following beneficial effects:
1, the main research of the present invention improves buffalo oocytes maturation culture solution, in low concentration liquor folliculi (1%-
4%) in, it still can reach the division rate and blastocyst rate for improving buffalo fertilized eggs, to improve buffalo extracorporeal embryo development rate
With the effect of production efficiency；In formula, in order to activate the activity of egg mother cell, applicant utilizes half by numerous studies discovery
Cystine, methionine, alanine, lysine, tryptophan and glycine are dissolved in TCM199 solution and mix again with liquor folliculi
Chelating can significantly improve influence of the liquor folliculi to embryo development rate, and Influencing Mechanism is still not clear at present, and applicant is considered certain
The synergistic effect of amino acid formula improves point of Some Circulating Factors and egg mother cell and follicle cell in liquor folliculi from serum
Secrete effect brought by factor isoreactivity substance.
[specific embodiment]
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, below to specific reality of the invention
The mode of applying is described in detail.In the following description, numerous specific details are set forth in order to facilitate a full understanding of the present invention.But
The invention can be embodied in many other ways as described herein, and those skilled in the art can be without prejudice to this hair
Similar improvement is done in the case where bright intension, therefore the present invention is not limited to the specific embodiments disclosed below.
Embodiment 1:
Adopt ovum extracorporeal culturing method in slaughterhouse:
1. In-vitro maturation:
Buffalo ovaries are collected from slaughterhouse, is placed in the thermo jug for including 30 DEG C of physiological saline and keeps the temperature, reality is sent within 2h
Test room, it is ensured that the temperature behind arrival laboratory in thermo jug is still best at 30 DEG C.Ovary is poured out, mesovarium and fallopian tubal are removed
Equal excess tissues, then with cleaning 2 times containing dual anti-physiological saline, extracting diameter with 10mL syringe after cleaning up is 2mm's
Ovarian follicle is slowly injected into the ovum mother for choosing uniform, the extracellular above granular cell that haves three layers of cytoplasm in plate rapidly under stereomicroscope
Cell washs 2 times, using plate culture, is placed in 39 DEG C, 5%CO in mature liquid A2It is cultivated in saturated air humidified incubator,
In, mature liquid A's the preparation method is as follows: take 50mL basal liquid then added into CM basal liquid 200 μ L FSH-LH storage
Liquid, the E2 storing liquid of 45 μ L, the EGF liquid of 100 μ L, the cysteamine storing liquid of 50 μ L, then liquor folliculi is added, keep the end of liquor folliculi dense
Degree is 1%.
2. prepared by granular cell single layer:
The egg mother cell surrounding cumulus cells of mature 22h are gently blown off, remain in the granular cell in mature liquid A with newly
Twice of centrifuge washing of mature liquid A after suspend, control cell density 2 × 106/ mL or so is made of the plastic culture dish of 35mm
The droplet of 30 μ L covers paraffin oil, 39 DEG C, 5%CO2Cultivated in saturated air humidified incubator it is spare, culture three days after change CM
A CM basal liquid is changed after basal liquid every other day.
3. in vitro fertilization:
The defrosting of water frozen cattle semens is placed on the liquid about 30min that climbs, it during this period will be around the buffalo oocytes of mature 22h
Cumulus cell gently blow and beat, be placed in by sperm, 10 pieces of ovums of every droplet, about 30min be incubated for, after climbing, washing
Sperm is added by sperm droplet, controls sperm concentration 1 × 106Fertilization disk is placed in incubator and is incubated for by/mL.
4. embryo in vitro culture:
The sperm piping and druming for being attached to fertilized eggs surface is fallen after fertilization 18h, is transferred to after being cleaned 2 times in embryo medium
In preprepared monolayer cultivation disk, in 39 DEG C, 5%CO2It is cultivated in saturated air humidified incubator, records each rank of embryo
Section developmental state.
5. blastaea total cell number counts:
The blastaea for being developed to 7d is collected, after cleaning 2 times in PBS buffer solution, is placed in diluted 10 μ of dehydrated alcohol
Mol/mL Hoechst33342 dye liquor is protected from light stained over night in room temperature, and taking-up is cleaned 2 times in PBS buffer solution, and paraffin-is all
Intellectual circle's mounting observes and records blastomere total number under fluorescence microscope.
Liquor folliculi used in the present embodiment maturation liquid A is the liquor folliculi handled by amino acid chelate, wherein amino acid
The method of chelating processing are as follows: cysteine, methionine, alanine, lysine, tryptophan and glycine are dissolved in TCM199
In solution, keep the final concentration of 500 μm of ol/L, final concentration of 300 μm of ol/L of methionine, the end of alanine of cysteine dense
Degree is 100 μm of ol/L, final concentration of 100 μm of ol/L of lysine, final concentration of 50 μm of ol/L of tryptophan, the end of glycine are dense
Degree is 50 μm of ol/L；It is mixed for 1:1 with the liquor folliculi directly acquired after mixing according to mass ratio, shaken cultivation under the conditions of 39 DEG C
1h。
In the present embodiment, used CM basal liquid, FSH-LH storing liquid, E2 storing liquid, EGF liquid and cysteamine storing liquid
The preparation method is as follows:
(1) preparation method of CM basal liquid is as follows:
1. configuration Cm-Stock solution: weighing the TCM199 of 2.45g, the NaHCO of 0.18g3, the Sodium Pyruvate of 1.0mg,
The sodium lactate that 35 μ L percentage compositions are 70%, the penicillin of 18mg, the streptomysin of 17mg, 9mg's is phenol red；And it is mentioned component is molten
Solution is uniformly mixed in the physiological saline of 200mL and obtains the Cm-Stock solution；
2. preparing CM basal liquid: being obtained in the step of fetal calf serum of 15mL is dissolved in 50mL (1) Cm-Stock solution
CM basal liquid；
(2) preparation method of FSH-LH storing liquid is as follows:
It takes the follicular stimulating hormone of 120ug to be dissolved in 10mL physiological saline and obtains follicle stimulating hormone solution, then weigh 2.11mg
Interstitialcellstimulating hormone (ICSH) be dissolved in above-mentioned follicular stimulating hormone solution mix after, after being distributed into 10 μ L/ pipes, -20 DEG C save
Obtain FSH-LH storing liquid；
(3) preparation method of E2 storing liquid is as follows:
After taking the estradiol of 2.01mg to be dissolved in the ethyl alcohol mixing of 1mL, it is distributed into 10 μ L/ pipe, obtains E2 in -20 DEG C of preservations
Storing liquid；
(4) preparation method of EGF liquid is as follows:
Epithelical cell growth factor is configured to the solution that solutes content is 10ng/mL with distilled water and obtains EGF liquid；
(5) preparation method of cysteamine storing liquid is as follows:
The cysteamine for weighing 7.0mg is dissolved in the CM basal liquid of 10mL step (2), the 100 every pipes of μ L is packed as, -20
DEG C preservation obtains cysteamine storing liquid.
Embodiment 2:
1. In-vitro maturation:
Buffalo ovaries are collected from slaughterhouse, is placed in the thermo jug for including 39 DEG C of physiological saline and keeps the temperature, reality is sent within 3h
Test room, it is ensured that the temperature behind arrival laboratory in thermo jug is still best at 33 DEG C.Ovary is poured out, mesovarium and fallopian tubal are removed
Equal excess tissues, then cleaned 3 times with containing dual anti-physiological saline.Extracting diameter with 10mL syringe after cleaning up is 6mm's
Ovarian follicle is slowly injected into the ovum mother for choosing uniform, the extracellular above granular cell that haves three layers of cytoplasm in plate rapidly under stereomicroscope
Cell washs 3 times, using plate culture, is placed in 39 DEG C, 5%CO in mature liquid A2It is cultivated in saturated air humidified incubator,
In, mature liquid A's the preparation method is as follows: take 50mL basal liquid then added into CM basal liquid 200 μ L FSH-LH storage
Liquid, the E2 storing liquid of 45 μ L, the EGF liquid of 100 μ L, the cysteamine storing liquid of 50 μ L, then liquor folliculi is added, keep the end of liquor folliculi dense
Degree is 4%.
2. prepared by granular cell single layer:
Mature egg mother cell surrounding cumulus cells for 24 hours are gently blown off, remain in the granular cell in mature liquid A with newly
Twice of centrifuge washing of mature liquid A after suspend, control cell density 2 × 106/ mL or so is made of the plastic culture dish of 35mm
The droplet of 30 μ L covers paraffin oil, in 39 DEG C, 5%CO2Cultivated in saturated air humidified incubator it is spare, culture three days after change
A CM basal liquid is changed after CM basal liquid every other day.
3. in vitro fertilization:
The defrosting of water frozen cattle semens is placed on the liquid about 30min that climbs, it during this period will be around mature buffalo oocytes for 24 hours
Cumulus cell gently blow and beat, be placed in by sperm, 15 pieces of ovums of every droplet, about 30min be incubated for, after climbing, washing
Sperm is added by sperm droplet, controls sperm concentration 1.5 × 106Fertilization disk is placed in incubator and is incubated for by/mL.
4. embryo in vitro culture:
The sperm piping and druming for being attached to fertilized eggs surface is fallen after fertilization 18h, is transferred to after being cleaned 3 times in embryo medium
In preprepared monolayer cultivation disk, in 39 DEG C, 5%CO2It is cultivated in saturated air humidified incubator, records each rank of embryo
Section developmental state.
5. blastaea total cell number counts:
The blastaea for being developed to 7d is collected, after cleaning 3 times in PBS buffer solution, is placed in diluted 10 μ of dehydrated alcohol
Mol/mL Hoechst33342 dye liquor is protected from light stained over night in room temperature, and taking-up is cleaned 3 times in PBS buffer solution, and paraffin-is all
Intellectual circle's mounting observes and records blastomere total number under fluorescence microscope.
Liquor folliculi used in the present embodiment maturation liquid A is the liquor folliculi handled by amino acid chelate, wherein amino acid
The method of chelating processing are as follows: cysteine, methionine, alanine, lysine, tryptophan and glycine are dissolved in TCM199
In solution, keep the final concentration of 700 μm of ol/L, final concentration of 500 μm of ol/L of methionine, the end of alanine of cysteine dense
Degree is the end of 300 μm of ol/L, final concentration of 300 μm of ol/L of lysine, final concentration of 100 μm of ol/L of tryptophan, glycine
Concentration is 150 μm of ol/L；It mixes for 1:1 with the liquor folliculi directly acquired after mixing according to mass ratio, is vibrated under the conditions of 39 DEG C
Cultivate 2h.
In the present embodiment, used CM basal liquid, FSH-LH storing liquid, E2 storing liquid, EGF liquid and cysteamine storing liquid
The preparation method is as follows:
(1) preparation method of CM basal liquid is as follows:
1. configuration Cm-Stock solution: weighing the TCM199 of 3.65g, the NaHCO of 0.30g3, the Sodium Pyruvate of 2.0mg,
The sodium lactate that 45 μ L percentage compositions are 70%, the penicillin of 22mg, the streptomysin of 27mg, 13mg's is phenol red；And by mentioned component
It is dissolved in the physiological saline of 200mL, is uniformly mixed and obtains the Cm-Stock solution；
2. preparing CM basal liquid: being obtained in the step of fetal calf serum of 25mL is dissolved in 50mL (1) Cm-Stock solution
CM basal liquid；
(2) preparation method of FSH-LH storing liquid is as follows:
It takes the follicular stimulating hormone of 150ug to be dissolved in 10mL physiological saline and obtains follicle stimulating hormone solution, then weigh 3.04mg
Interstitialcellstimulating hormone (ICSH) be dissolved in above-mentioned follicular stimulating hormone solution mix after, after being distributed into 10 μ L/ pipes, -20 DEG C save
Obtain FSH-LH storing liquid；
(3) preparation method of E2 storing liquid is as follows:
After taking the estradiol of 2.43mg to be dissolved in the ethyl alcohol mixing of 1mL, it is distributed into 10 μ L/ pipe, obtains E2 in -20 DEG C of preservations
Storing liquid；
(4) preparation method of EGF liquid is as follows:
Epithelical cell growth factor is configured to the solution that solutes content is 18ng/mL with distilled water and obtains EGF liquid；
(5) preparation method of cysteamine storing liquid is as follows:
The cysteamine for weighing 7.5mg is dissolved in the CM basal liquid of 10mL step (2), the 100 every pipes of μ L is packed as, -20
DEG C preservation obtains cysteamine storing liquid.
Embodiment 3:
1. In-vitro maturation:
Buffalo ovaries are collected from slaughterhouse, is placed in the thermo jug for including 33 DEG C of physiological saline and keeps the temperature, be sent within 2.5h
Laboratory, it is ensured that the temperature behind arrival laboratory in thermo jug is still best at 32 DEG C.Ovary is poured out, mesovarium and defeated ovum are removed
The excess tissues such as pipe, then cleaned 3 times with containing dual anti-physiological saline.Extracting diameter with 10mL syringe after cleaning up is 5mm
Ovarian follicle, be slowly injected into the ovum for choosing uniform, the extracellular above granular cell that haves three layers of cytoplasm in plate rapidly under stereomicroscope
Mother cell washs 3 times, using plate culture, is placed in 39 DEG C, 5%CO in mature liquid A2It is cultivated in saturated air humidified incubator,
Wherein, mature liquid A's the preparation method is as follows: take 50mL basal liquid then added into CM basal liquid 200 μ L FSH-LH storage
Liquid storage, the E2 storing liquid of 45 μ L, the EGF liquid of 100 μ L, the cysteamine storing liquid of 50 μ L, then liquor folliculi is added, make the end of liquor folliculi
Concentration is 3%.
2. prepared by granular cell single layer:
The egg mother cell surrounding cumulus cells of mature 23h are gently blown off, remain in the granular cell in mature liquid A with newly
Twice of centrifuge washing of mature liquid A after suspend, control cell density 2 × 106/ mL or so is made of the plastic culture dish of 35mm
The droplet of 30 μ L covers paraffin oil, 39 DEG C, 5%CO2Cultivated in saturated air humidified incubator it is spare, culture three days after change CM
A CM basal liquid is changed after basal liquid every other day.
3. in vitro fertilization:
The defrosting of water frozen cattle semens is placed on the liquid about 30min that climbs, it during this period will be around the buffalo oocytes of mature 23h
Cumulus cell gently blow and beat, be placed in by sperm, 12 pieces of ovums of every droplet, about 30min be incubated for, after climbing, washing
Sperm is added by sperm droplet, controls sperm concentration 1.2 × 106Fertilization disk is placed in incubator and is incubated for by/mL.
4. embryo in vitro culture:
The sperm piping and druming for being attached to fertilized eggs surface is fallen after fertilization 18h, is transferred to after being cleaned 3 times in embryo medium
In preprepared monolayer cultivation disk, in 39 DEG C, 5%CO2It is cultivated in saturated air humidified incubator, records each rank of embryo
Section developmental state.
5. blastaea total cell number counts:
The blastaea for being developed to 7d is collected, after cleaning 2 times in PBS buffer solution, is placed in diluted 10 μ of dehydrated alcohol
Mol/mL Hoechst33342 dye liquor is protected from light stained over night in room temperature, and taking-up is cleaned 3 times in PBS buffer solution, and paraffin-is all
Intellectual circle's mounting observes and records blastomere total number under fluorescence microscope.
Liquor folliculi used in the present embodiment maturation liquid A is the liquor folliculi handled by amino acid chelate, wherein amino acid
The method of chelating processing are as follows: cysteine, methionine, alanine, lysine, tryptophan and glycine are dissolved in TCM199
In solution, keep the final concentration of 600 μm of ol/L, final concentration of 400 μm of ol/L of methionine, the end of alanine of cysteine dense
Degree is 200 μm of ol/L, final concentration of 200 μm of ol/L of lysine, final concentration of 80 μm of ol/L of tryptophan, the end of glycine are dense
Degree is 100 μm of ol/L；It is mixed for 1:1 with the liquor folliculi directly acquired after mixing according to mass ratio, training is vibrated under the conditions of 39 DEG C
Support 1.5h.
In the present embodiment, used CM basal liquid, FSH-LH storing liquid, E2 storing liquid, EGF liquid and cysteamine storing liquid
The preparation method is as follows:
(1) preparation method of CM basal liquid is as follows:
1. configuration Cm-Stock solution: weighing the TCM199 of 2.95g, the NaHCO of 0.20g3, the Sodium Pyruvate of 1.5mg,
The sodium lactate that 40 μ L percentage compositions are 70%, the penicillin of 20mg, the streptomysin of 22mg, 11mg's is phenol red；And by mentioned component
It is dissolved in the physiological saline of 200mL, is uniformly mixed and obtains the Cm-Stock solution；
2. preparing CM basal liquid: being obtained in the step of fetal calf serum of 20mL is dissolved in 50mL (1) Cm-Stock solution
CM basal liquid；
(2) preparation method of FSH-LH storing liquid is as follows:
It takes the follicular stimulating hormone of 130ug to be dissolved in 10mL physiological saline and obtains follicle stimulating hormone solution, then weigh 2.88mg
Interstitialcellstimulating hormone (ICSH) be dissolved in above-mentioned follicular stimulating hormone solution mix after, after being distributed into 10 μ L/ pipes, -20 DEG C save
Obtain FSH-LH storing liquid；
(3) preparation method of E2 storing liquid is as follows:
After taking the estradiol of 2.21mg to be dissolved in the ethyl alcohol mixing of 1mL, it is distributed into 10 μ L/ pipe, obtains E2 in -20 DEG C of preservations
Storing liquid；
(4) preparation method of EGF liquid is as follows:
Epithelical cell growth factor is configured to the solution that solutes content is 14ng/mL with distilled water and obtains EGF liquid；
(5) preparation method of cysteamine storing liquid is as follows:
The cysteamine for weighing 7.2mg is dissolved in the CM basal liquid of 10mL step (2), the 100 every pipes of μ L is packed as, -20
DEG C preservation obtains cysteamine storing liquid.
Embodiment 4:
Ovum pick-up extracorporeal culturing method
1. ovum pick-up:
Ovum pick-up instrument is the Hs2000B type ultrasonic wave imager of Honda company, Japan production, vagina sectoring probe
(Aloka), 19G is installed on probe to adopt ovum needle (long 60cm), vacuum pump adopts ovum needle and equipped with adopting ovum liquid with silica gel catheter connection
50mL test tube, test tube, which is then placed in thermostat, is kept for 38 DEG C.Baoding is carried out to donor buffalo, operator's proficiency per rectum is held
Fixed ovary will be equipped with the probe for adopting ovum needle on the other hand and slowly be inserted at fornix vagina and adopt ovum side, and ovary is drawn and is close to
In probe distal end, ovary image and ovarian follicle position at this moment can be shown from B ultrasound display, ovum pin puncture ovarian follicle is adopted in promotion, with
The negative pressure of 40mm mercury column or so adopt ovum, after 2 visual ovarian follicles of every absorption, rushes Xian Guandao with adopting ovum liquid immediately.Recycle ovarian follicle
Liquid.After having adopted 2 donors, sends recovered liquid back to laboratory and collect egg mother cell.
2. the maturation in vitro of egg mother cell:
The recovered liquid that living body is collected is poured into embryo's filter cup, is cleaned 2 times with the DPBS containing 3%FBS, under stereoscope
Collect living body oocyte collection；And the liquor folliculi that syringe extracts directly selects cytoplasm uniformly under stereoscope and wraps up one layer
The COCs of the above cumulus cell is washed 2 times in the TCMl99 liquid of the Hepes containing 5%FBS and 20mmol/L, then with mature liquid B liquid
It washes 1 time, is transferred in the mature liquid B of 50 μ L/ drops in 39 DEG C, 5%CO2, maturation culture 22h in the incubator of maximum relative humidity,
Wherein, mature liquid B is by as follows at being grouped as: 8% fetal calf serum, 10 μ g/mL follicular stimulating hormone, that 15 μ g/mL promote corpus luteum is raw
Cheng Su, the estradiol of 5 μ g/mL, the Sodium Pyruvate of 0.9mmoL/L, the cysteine of 100 μm of ol/L, 35ng/mL epicuticle
Growth factor, 1% liquor folliculi, surplus are TCM199 solution.
3. external fertilization method: same as Example 1；
4. embryo in vitro culture observation method: same as Example 1.
Liquor folliculi used in the present embodiment maturation liquid B is the liquor folliculi handled by amino acid chelate, wherein amino acid
The method of chelating processing are as follows: cysteine, methionine, alanine, lysine, tryptophan and glycine are dissolved in TCM199
In solution, keep the final concentration of 500 μm of ol/L, final concentration of 300 μm of ol/L of methionine, the end of alanine of cysteine dense
Degree is 100 μm of ol/L, final concentration of 100 μm of ol/L of lysine, final concentration of 50 μm of ol/L of tryptophan, the end of glycine are dense
Degree is 50 μm of ol/L；It is mixed for 1:1 with the liquor folliculi directly acquired after mixing according to mass ratio, shaken cultivation under the conditions of 39 DEG C
2h。
Embodiment 5:
1. ovum pick-up:
Ovum pick-up instrument is the Hs2000B type ultrasonic wave imager of Honda company, Japan production, vagina sectoring probe
(Aloka), 19G is installed on probe to adopt ovum needle (long 60cm), vacuum pump adopts ovum needle and equipped with adopting ovum liquid with silica gel catheter connection
50mL test tube, test tube, which is then placed in thermostat, is kept for 38 DEG C.Baoding is carried out to donor buffalo, operator's proficiency per rectum is held
Fixed ovary will be equipped with the probe for adopting ovum needle on the other hand and slowly be inserted at fornix vagina and adopt ovum side, and ovary is drawn and is close to
In probe distal end, ovary image and ovarian follicle position at this moment can be shown from B ultrasound display, ovum pin puncture ovarian follicle is adopted in promotion, with
The negative pressure of 40mm mercury column or so adopt ovum, after 3 visual ovarian follicles of every absorption, rushes Xian Guandao with adopting ovum liquid immediately.Recycle ovarian follicle
Liquid.After having adopted 3 donors, sends recovered liquid back to laboratory and collect egg mother cell.
3. the maturation in vitro of egg mother cell:
The recovered liquid that living body is collected is poured into embryo's filter cup, is cleaned 3 times with the DPBS containing 3%FBS, under stereoscope
Collect living body oocyte collection；And the liquor folliculi that syringe extracts directly selects cytoplasm uniformly under stereoscope and wraps up one layer
The COCs of the above cumulus cell is washed 2 times in the TCMl99 liquid of the Hepes containing 5%FBS and 20mmol/L, then with mature liquid B liquid
It washes 1 time, is transferred in the mature liquid B of 50 μ L/ drops in 39 DEG C, 5%CO2, in the incubator of maximum relative humidity maturation culture for 24 hours,
Wherein, mature liquid B is by as follows at being grouped as: 8% fetal calf serum, 10 μ g/mL follicular stimulating hormone, that 15 μ g/mL promote corpus luteum is raw
Cheng Su, the estradiol of 5 μ g/mL, the Sodium Pyruvate of 0.9mmoL/L, the cysteine of 100 μm of ol/L, 35ng/mL epicuticle
Growth factor, 4% liquor folliculi, surplus are TCM199 solution.
4. external fertilization method:
It is same as Example 2；
5. embryo in vitro culture observation method:
It is same as Example 2.
Liquor folliculi used in the present embodiment maturation liquid B is the liquor folliculi handled by amino acid chelate, wherein amino acid
The method of chelating processing are as follows: cysteine, methionine, alanine, lysine, tryptophan and glycine are dissolved in TCM199
In solution, keep the final concentration of 700 μm of ol/L, final concentration of 500 μm of ol/L of methionine, the end of alanine of cysteine dense
Degree is the end of 300 μm of ol/L, final concentration of 300 μm of ol/L of lysine, final concentration of 100 μm of ol/L of tryptophan, glycine
Concentration is 150 μm of ol/L；It mixes for 1:1 with the liquor folliculi directly acquired after mixing according to mass ratio, is vibrated under the conditions of 39 DEG C
Cultivate 3h.
Embodiment 6:
1. ovum pick-up:
Ovum pick-up instrument is the Hs2000B type ultrasonic wave imager of Honda company, Japan production, vagina sectoring probe
(Aloka), 19G is installed on probe to adopt ovum needle (long 60cm), vacuum pump adopts ovum needle and equipped with adopting ovum liquid with silica gel catheter connection
50mL test tube, test tube, which is then placed in thermostat, is kept for 38 DEG C.Baoding is carried out to donor buffalo, operator's proficiency per rectum is held
Fixed ovary will be equipped with the probe for adopting ovum needle on the other hand and slowly be inserted at fornix vagina and adopt ovum side, and ovary is drawn and is close to
In probe distal end, ovary image and ovarian follicle position at this moment can be shown from B ultrasound display, ovum pin puncture ovarian follicle is adopted in promotion, with
The negative pressure of 40mm mercury column or so adopt ovum, after 3 visual ovarian follicles of every absorption, rushes Xian Guandao with adopting ovum liquid immediately.Recycle ovarian follicle
Liquid.After having adopted 2 donors, sends recovered liquid back to laboratory and collect egg mother cell.
6. the maturation in vitro of egg mother cell:
The recovered liquid that living body is collected is poured into embryo's filter cup, is cleaned 3 times with the DPBS containing 3%FBS, under stereoscope
Collect living body oocyte collection；And the liquor folliculi that syringe extracts directly selects cytoplasm uniformly under stereoscope and wraps up one layer
The COCs of the above cumulus cell is washed 2 times in the TCMl99 liquid of the Hepes containing 5%FBS and 20mmol/L, then with mature liquid B liquid
It washes 1 time, is transferred in the mature liquid B of 50 μ L/ drops in 39 DEG C, 5%CO2, maturation culture 23h in the incubator of maximum relative humidity,
Wherein, mature liquid B is by as follows at being grouped as: 8% fetal calf serum, 10 μ g/mL follicular stimulating hormone, that 15 μ g/mL promote corpus luteum is raw
Cheng Su, the estradiol of 5 μ g/mL, the Sodium Pyruvate of 0.9mmoL/L, the cysteine of 100 μm of ol/L, 35ng/mL epicuticle
Growth factor, 3% liquor folliculi, surplus are TCM199 solution.
4. external fertilization method:
It is same as Example 3；
5. embryo in vitro culture observation method:
It is same as Example 3.
Liquor folliculi used in the present embodiment maturation liquid B is the liquor folliculi handled by amino acid chelate, wherein amino acid
The method of chelating processing are as follows: cysteine, methionine, alanine, lysine, tryptophan and glycine are dissolved in TCM199
In solution, keep the final concentration of 600 μm of ol/L, final concentration of 400 μm of ol/L of methionine, the end of alanine of cysteine dense
Degree is 200 μm of ol/L, final concentration of 200 μm of ol/L of lysine, final concentration of 70 μm of ol/L of tryptophan, the end of glycine are dense
Degree is 100 μm of ol/L；It is mixed for 1:1 with the liquor folliculi directly acquired after mixing according to mass ratio, training is vibrated under the conditions of 39 DEG C
Support 2.5h.
Adopt the verifying of ovum In-vitro maturation in slaughterhouse:
Control group 1:
Other laboratory facilities and embodiment 1 of this control group are completely the same, and uniquely the difference is that, liquor folliculi is without ammonia
The liquor folliculi of base acid chelating processing.
Control group 2:
Other laboratory facilities and embodiment 1 of this control group are completely the same, it is unique unlike, the amino acid that liquor folliculi is
In chelating treatment process, amino acid chelate liquid is free of methionine and alanine；That is the method for amino acid chelate processing are as follows: will be partly
Cystine, lysine, tryptophan and glycine are dissolved in TCM199 solution, make cysteine final concentration of 500 μm of ol/L,
Final concentration of 100 μm of ol/L of lysine, final concentration of 50 μm of ol/L of tryptophan, glycine final concentration of 50 μm of ol/L；
It is mixed for 1:1 with the liquor folliculi directly acquired after mixing according to mass ratio, shaken cultivation 1h under the conditions of 39 DEG C.
Control group 3:
Other laboratory facilities and embodiment 1 of this control group are completely the same, uniquely the difference is that, do not use liquor folliculi, and
It is substituted using amino acid chelate liquid, that is, uses 1% amino acid chelate liquid, it may be assumed that the method for amino acid chelate processing are as follows: by half Guang
Propylhomoserin, methionine, alanine, lysine, tryptophan and glycine are dissolved in TCM199 solution, keep the end of cysteine dense
Degree is 500 μm of ol/L, final concentration of 300 μm of ol/L of methionine, final concentration of 100 μm of ol/L of alanine, lysine
Final concentration of 100 μm of ol/L, final concentration of 50 μm of ol/L of tryptophan, glycine final concentration of 50 μm of ol/L.
Control group 4:
Other laboratory facilities and embodiment 1 of this control group are completely the same, uniquely the difference is that, make without using liquor folliculi
With tire ox blood.
Buffalo oocytes are handled according to the method for embodiment 1-3 and control group 1-4, is fertilized later, develops research,
When embryonic development is to 7d, blastaea is collected, after cleaning in PBS buffer solution, carries out being protected from light stained over night, take out slow in PBS
It is cleaned in fliud flushing, paraffin-vaseline mounting observes and records blastomere total number under fluorescence microscope.
Wherein, the calculation formula of embryo's division rate are as follows:
Embryo's division rate=division number/total fertilized eggs number；
Blastocyst rate=blastaea number/total fertilization；Specific experiment the results are shown in Table 1:
Table 1
As seen from the above table, the egg cell division rate of embodiment 1-3 and blastocyst rate are all larger than control group 1-4, illustrate, the application
Amino acid chelate liquid and liquor folliculi synergistic effect can improve the development rate of buffalo embryo.
Ovum pick-up In-vitro maturation
Control group A:
Other laboratory facilities and embodiment 4 of this control group are completely the same, and uniquely the difference is that, liquor folliculi is without ammonia
The liquor folliculi of base acid chelating processing.
Control group B:
Other laboratory facilities and embodiment 4 of this control group are completely the same, it is unique unlike, the amino acid that liquor folliculi is
In chelating treatment process, amino acid chelate liquid is free of tryptophan and glycine；That is the method for amino acid chelate processing are as follows: by half Guang
Propylhomoserin, methionine, alanine and lysine are dissolved in TCM199 solution, make cysteine final concentration of 500 μm of ol/L,
Final concentration of 300 μm of ol/L of methionine, final concentration of 100 μm of ol/L of alanine, lysine final concentration of 100 μ
mol/L；It is mixed for 1:1 with the liquor folliculi directly acquired after mixing according to mass ratio, shaken cultivation 2h under the conditions of 39 DEG C.
Control group C:
Other laboratory facilities and embodiment 4 of this control group are completely the same, uniquely the difference is that, do not use liquor folliculi, and
It is substituted using amino acid chelate liquid, that is, uses 1% amino acid chelate liquid, it may be assumed that the method for amino acid chelate processing are as follows: by half Guang
Propylhomoserin, methionine, alanine, lysine, tryptophan and glycine are dissolved in TCM199 solution, keep the end of cysteine dense
Degree is 500 μm of ol/L, final concentration of 300 μm of ol/L of methionine, final concentration of 100 μm of ol/L of alanine, lysine
Final concentration of 100 μm of ol/L, final concentration of 50 μm of ol/L of tryptophan, glycine final concentration of 50 μm of ol/L.
Control group D:
Other laboratory facilities and embodiment 4 of this control group are completely the same, uniquely the difference is that, make without using liquor folliculi
With tire ox blood.
Buffalo oocytes are handled according to the method for embodiment 1-3 and control group 1-4, is fertilized later, develops research,
When embryonic development is to 7d, blastaea is collected, after cleaning in PBS buffer solution, carries out being protected from light stained over night, take out slow in PBS
It is cleaned in fliud flushing, paraffin-vaseline mounting observes and records blastomere total number under fluorescence microscope.
Wherein, the calculation formula of embryo's division rate are as follows:
Embryo's division rate=division number/total fertilized eggs number；
Blastocyst rate=blastaea number/total fertilization；Specific experiment the results are shown in Table 2:
Table 2
| Test | It repeats | Cultivate ovum number | Divide ovum number | Division rate | Blastocyst rate | Blastaea total cell number/ |
| Embodiment 4 | 10 | 82 | 77 | 93.90 | 54.88 | 45 |
| Embodiment 5 | 10 | 80 | 74 | 92.50 | 56.25 | 45 |
| Embodiment 6 | 10 | 86 | 79 | 91.86 | 60.47 | 52 |
| Control group A | 10 | 79 | 49 | 62.03 | 34.18 | 27 |
| Control group B | 10 | 91 | 55 | 60.44 | 31.87 | 29 |
| Control group C | 10 | 96 | 56 | 58.33 | 30.21 | 29 |
| Control group D | 10 | 94 | 56 | 59.57 | 30.85 | 29 |
As seen from the above table, the egg cell division rate of embodiment 4-6 and blastocyst rate are all larger than control group A-D, illustrate, the application
Amino acid chelate liquid and liquor folliculi synergistic effect can improve the development rate of buffalo embryo.
In conclusion the maturation culture solution of the application can reach under liquor folliculi low concentration and mention after improvement
The effect of high buffalo embryo developmental rate, and take the two ways of ovum and live egg-fetching to be applicable in for butchering.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitation of the scope of the invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art,
Without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection model of the invention
It encloses.Therefore, protection scope of the present invention should be determined by the appended claims.
Claims (8)
1. a kind of maturation culture solution for improving river type In Vitro Fertilization in Water Buffalo embryo development rate, which is characterized in that the mature training
Nutrient solution includes the liquor folliculi that mass percent is 1%-4%.
2. maturation culture solution according to claim 1, which is characterized in that the liquor folliculi is to handle by amino acid chelate
Liquor folliculi.
3. maturation culture solution according to claim 2, which is characterized in that the method for the amino acid chelate processing are as follows: will
Cysteine, methionine, alanine, lysine, tryptophan and glycine are dissolved in TCM199 solution, make cysteine
Final concentration of 500-700 μm of ol/L, the final concentration of 300-500 μm of ol/L of methionine, alanine final concentration of 100-300
The end of μm ol/L, the final concentration of 100-300 μm of ol/L of lysine, the final concentration of 50-100 μm of ol/L of tryptophan, glycine
Concentration is 50-150 μm of ol/L；It mixes for 1:1 with the liquor folliculi directly acquired after mixing according to mass ratio, shakes under the conditions of 39 DEG C
Swing culture.
4. maturation culture solution according to claim 1, which is characterized in that the maturation culture solution includes adopting for slaughterhouse
The mature liquid A of ovum in vitro culture and/or mature liquid B for ovum pick-up in vitro culture.
5. maturation culture solution according to claim 4, which is characterized in that the maturation liquid A's the preparation method is as follows: taking
Then the basal liquid of 50mL adds the FSH-LH storing liquid of 200 μ L, the E2 storing liquid of 45 μ L, the EGF of 100 μ L into CM basal liquid
Liquid, the cysteamine storing liquid of 50 μ L, then liquor folliculi is added, make the final concentration of 1%-4% of liquor folliculi.
6. maturation culture solution according to claim 5, which is characterized in that
The preparation method of the CM basal liquid is as follows:
(1) it configures Cm-Stock solution: weighing the TCM199 of 2.45~3.65g, the NaHCO of 0.18~0.30g3, 1.0~2.0mg
Sodium Pyruvate, 35~45 μ L percentage compositions be 70% sodium lactate, the penicillin of 18~22mg, the streptomysin of 17~27mg, 9
~13mg's is phenol red；And mentioned component is dissolved in the physiological saline of 200mL, it is uniformly mixed that obtain the Cm-Stock molten
Liquid；
(2) CM basal liquid is prepared: in the step of fetal calf serum of 15mL-25mL is dissolved in 50mL (1) Cm-Stock solution
To CM basal liquid；
The preparation method of the FSH-LH storing liquid is as follows: the follicular stimulating hormone of 120~150ug being taken to be dissolved in 10mL physiological saline
Follicle stimulating hormone solution is obtained, then weighs the interstitialcellstimulating hormone (ICSH) of 2.11~3.04mg and is dissolved in above-mentioned follicular stimulating hormone solution
After middle mixing, after being distributed into 10 μ L/ pipes, FSH-LH storing liquid is obtained in -20 DEG C of preservations；
The preparation method of the E2 storing liquid is as follows: after the ethyl alcohol for taking the estradiol of 2.01~2.43mg to be dissolved in 1mL mixes, point
10 μ L/ pipe is dressed up, obtains E2 storing liquid in -20 DEG C of preservations；
The preparation method of the EGF liquid is as follows: with distilled water by epithelical cell growth factor be configured to solutes content be 10~
The solution of 18ng/mL obtains EGF liquid；
The preparation method of the cysteamine storing liquid is as follows: the cysteamine for weighing 7.0~7.5mg is dissolved in 10mL step (2)
In CM basal liquid, the 100 every pipes of μ L are packed as, obtain cysteamine storing liquid in -20 DEG C of preservations.
7. maturation culture solution according to claim 4, which is characterized in that the maturation liquid B is by as follows at being grouped as: 8%
Fetal calf serum, the follicular stimulating hormone of 10 μ g/mL, 15 μ g/mL interstitialcellstimulating hormone (ICSH)s, the estradiol of 5 μ g/mL, 0.9mmoL/L
Sodium Pyruvate, the cysteine of 100 μm of ol/L, the epicuticle growth factor of 35ng/mL, the liquor folliculi of 1%-4%, surplus are
TCM199 solution.
8. a kind of method for carrying out in vitro culture using claim 1-7 any one culture solution, which is characterized in that the method
Include the following steps:
(1) ovum in vitro culture is adopted in slaughterhouse: being collected buffalo ovaries, is removed ovary excess tissue, with clear containing dual anti-physiological saline
It washes 2-3 times, draws ovarian follicle with syringe, the ovum that uniform, the extracellular above granular cell that haves three layers of cytoplasm is chosen under stereoscope is female thin
Born of the same parents wash 2~3 times, are placed in mature liquid A, in 39 DEG C, 5%CO2It is cultivated in saturated air humidified incubator；Cultivate 22-24h
The cumulus cell around egg mother cell is blown off afterwards, suspends after then washing twice with new mature liquid A, is covered after droplet is made
Paraffin, in 39 DEG C, 5%CO2It is cultivated in saturated air humidified incubator；Will buffalo sperm hatching after with egg mother cell carry out by
Essence, embryo in vitro culture；Record each embryonic development situation；The blastaea that collection is developed to 7d observes and records blastaea under the microscope
Total number of cells；
(2) ovum pick-up in vitro culture: ovum pick-up is carried out to living body buffalo, egg mother cell is collected, selects born of the same parents under stereoscope
Matter is uniform and wraps up the COCs of one layer or more cumulus cell, in the TCMl99 of fetal calf serum and 20mmol/L Hepes containing 5%
It washes in liquid 2 times, then is cleaned 1 time with maturation liquid B, is transferred in the mature liquid B of 50 μ L/ drops, in 39 DEG C, 5%CO2, maximum relatively wet
Maturation culture 22 in the incubator of degree~for 24 hours；It is fertilized after culture with the buffalo sperm after hatching, embryo in vitro culture, note
Record each embryonic development situation；The blastaea that collection is developed to 7d observes and records blastomere sum under the microscope.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113583944A (en) * | 2021-09-15 | 2021-11-02 | 广西壮族自治区水牛研究所 | Application of activated Wnt/beta-catenin signal path in improving production efficiency of buffalo in vitro embryos |
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| CN105624100A (en) * | 2016-04-06 | 2016-06-01 | 云南中科胚胎工程生物技术有限公司 | Buffalo oocyte in-vitro maturation (IVM) culture method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105624100A (en) * | 2016-04-06 | 2016-06-01 | 云南中科胚胎工程生物技术有限公司 | Buffalo oocyte in-vitro maturation (IVM) culture method |
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| ABD ELLAH等: "Ovarian Follicular Fluid Constituents in Relation to Stage of Estrus Cycle and Size of the Follicle in Buffalo", 《VETERINARY WORLD》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113583944A (en) * | 2021-09-15 | 2021-11-02 | 广西壮族自治区水牛研究所 | Application of activated Wnt/beta-catenin signal path in improving production efficiency of buffalo in vitro embryos |
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