CN106834216A - A kind of in vitro culture liquid and cultural method for the lonely female activation embryo of pig - Google Patents
A kind of in vitro culture liquid and cultural method for the lonely female activation embryo of pig Download PDFInfo
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Abstract
The invention discloses a kind of in vitro culture liquid (mPZM 3) and cultural method for the lonely female activation embryo of pig, belong to animal embryo in vitro culture field, the raw material of the in vitro culture liquid (mPZM 3) includes the basic culture solutions of mPZM 3 and five water lactic acid calcium and Sodium Pyruvate, and the concentration of five water lactic acid calcium is 0.606g/L in the in vitro culture liquid, the concentration of Sodium Pyruvate is 0.022g/L；The in vitro culture liquid solvent for use is hydrogen rich water, and wherein H2Content be 1.2~1.6ppm；The cultural method that the in vitro culture liquid is used for the lonely female activation embryo of pig is：First the lonely female activation embryo of pig is washed and 3~5h for the treatment of wherein with the in vitro culture liquid containing 5 μm of ol/L cytochalasin Bs, then is washed with the in vitro culture liquid for being not added with cytochalasin B and is cultivated 168h wherein, to embryonic development into blastaea.The present invention can guarantee that preferably for the lonely female activation embryo of pig provides energy and developing environment, so as to improve its Embryo Culture quality and effect.
Description
Technical field
The present invention relates to animal embryo in vitro culture field, a kind of external training for the lonely female activation embryo of pig is specifically related to
Nutrient solution and cultural method.
Background technology
The in vitro culture of animal embryo is the important step in embryo biotechnology.In vitro under condition of culture, early embryo
Tire tends not to complete the development overall process from embryonated egg to blastaea, and is parked on certain specific developmental stage, this phenomenon quilt
Referred to as vitro Development of Embryos blocks (in vitro block of embryonic development), thus has a strong impact on morning
Phase vitro Development of Embryos quality, such as external blastocyst rate of pig are typically only 20% or so, inner cell mass quantity at 30 or so,
Cause the birth rate of offspring to decline, greatly hinder research application of the pig in agricultural, bioengineering and medical science.
The in vitro culture of animal embryo with three aspect factor it is relevant, i.e., embryo's species, condition of culture and nutrient solution into
Point, the improvement of wherein nutrient solution composition is the basis and key link place of Vitro Culture Techniques, directly influences in vitro culture
Effect and the quality of produced in vitro embryo.
Lonely female activation embryo be by activation of oocytes after, maturation division can be completed, and thus to mitosis
Cross, start new individual generation.Research to the lonely female activation of mammal, will deepen many heavy to embryo's generation, nuclear transfer etc.
The understanding of broad theory problem.The conservation of wildlife, transgenic animals are made and biological development mechanism by the success of lonely female activation
Further investigation etc. also there is important theory and realistic meaning.
At present, it is existing for the in vitro culture liquid and cultural method of the lonely female activation embryo of pig it cannot be guaranteed that swashing for egg mother cell
Stabilization living, it is impossible to the energy preferably for needed for cell metabolism is provided, and be not enough to obtain Embryo Culture quality and effect higher.
The content of the invention
1. the technical problem to be solved
The technical problem to be solved in the present invention is a kind of promotion vitro Development of Embryos of offer for the lonely female activation embryo of pig
In vitro culture liquid and cultural method.
2. technical scheme
To solve the above problems, the present invention is adopted the following technical scheme that：
A kind of in vitro culture liquid for cultivating the lonely female activation embryo of pig, the raw material of the in vitro culture liquid includes mPZM-3
The concentration of five water lactic acid calcium is 0.606g/ in embryo medium and five water lactic acid calcium and Sodium Pyruvate, and the in vitro culture liquid
L, the concentration of Sodium Pyruvate is 0.022g/L；The in vitro culture liquid solvent for use is hydrogen rich water, and wherein H2Content be 1.2
~1.6ppm.
Further, the raw material of the mPZM-3 embryo mediums includes mPZM-3 basic culture solutions, Glu, Asia
Taurine, Cys, penicillin, streptomycin sulphate and bovine serum albumin(BSA), and their proportioning is 1L：0.146g：
0.546g：0.069g：100000IU：70000IU：3g.
Further, the mPZM-3 basic culture solutions include the solute of following concentration：Sodium chloride 6.312g/L, carbonic acid
Hydrogen sodium 2.119g/L, potassium chloride 0.746g/L, potassium dihydrogen phosphate 0.048g/L, magnesium sulfate 0.048g/L；Also include 2% it is required
Amino acid and 1% nonessential amino acid.
For the lonely female activation embryo of pig, the H of hydrogen rich water in the in vitro culture liquid2Concentration is preferable with 1.4ppm effects.
Present invention also offers the method that above-mentioned in vitro culture liquid is used to cultivate the lonely female activation embryo of pig, including following step
Suddenly：
(1) the lonely female activation embryo of pig is washed three times in containing 5 μm of in vitro culture liquid of ol/L cytochalasin Bs, moves into and contain 5
3~5h is cultivated in the in vitro culture liquid of μm ol/L cytochalasin Bs；
(2) embryo after above-mentioned treatment is washed three times with the nutrient solution without cytochalasin B, moves into and be free of cell relaxation
In the in vitro culture liquid of plain B, while covering 500 μ L mineral oil, 168h is cultivated, to the embryonic development into blastaea.
Preferably, the lonely female activation embryo of pig described in above-mentioned steps (1) is containing 5 μm of external trainings of ol/L cytochalasin Bs
The time that treatment is cultivated in nutrient solution is 4h.
Specifically, the culture density of the lonely female activation embryo of pig described in above-mentioned steps (2) is every 500 μ L in vitro cultures liquid training
40~60 pieces of embryos are supported, condition of culture is 38.5 DEG C, 5%CO2, saturated humidity.
3. beneficial effect
(1) pyruvic acid is necessary energy matter in cell metabolism, is the substrate that glycometabolism is produced, and is played in the medium
The effect of carbon source is substituted, cytotrophy metabolism is participated in.Similar also glucose, sodium lactate etc. is acted in cell culture, but
It is that its mechanism of action has differences for different animals.Especially effect of the glucose to body early embryo culture, has proven to grape
Sugar has significant inhibitory action to the embryonated egg of mouse, hamster, is the obstacle that embryo overcomes development to block, and pig also has similar showing
As.So the present invention uses pyruvic acid as the energy matter of cell culture.
(2) calcium ion is right to maintaining the stability of cell membrane, between permeability and cell being connected with important effect
It is morular deflation and blastaea formation and extended vital effect；In addition, the rising of calcium ion concentration is female thin ovum
The important inducement signal of born of the same parents' activation.And five water lactic acid calcium have solubility very high, the calcium ion of high concentration can be provided.
(3) solvent for use of the present invention is hydrogen rich water, and the hydrogen in solution can be generated with the oxygen radical direct reaction in cell
The nontoxic water that can be utilized by cell itself, so as to the oxidative damage to external blastaea has significant protective effect.
(4) cell pine is added in nutrient solution in vitro in the cultural method of the lonely female activation embryo of pig provided by the present invention
Relax element B, the formation of cytochalasin B interior micro-pipe capable of inhibiting cell and microfilament so that the cytoskeleton in egg mother cell relaxes, and increases
Add the flexibility of cell, be conducive to cell division；Cytochalasin B destroys the formation of spindle simultaneously, it is suppressed that polar body
Formation and chromosome discharge, it is ensured that chromosome G banding, being used cooperatively with other activator can improve the ovum of lonely female activation
The blastocyst rate of mother cell, increased the cell number in blastaea and reduces the reactive oxygen species in blastomere, so as to improve
The quality and quantity of blastaea.
In vitro culture liquid and its application process for cultivating the lonely female activation embryo of pig provided by the present invention, can guarantee that ovum
The activation stabilization of mother cell, the energy preferably for needed for cell metabolism is provided, and be conducive to improving Embryo Culture quality and effect.
Brief description of the drawings
Fig. 1 is blastomere nuclear staining result of the lonely female activation Embryo Culture of the pig of control group and hydrogen-rich group to 168h；
Fig. 2 is that the lonely female activation Embryo Culture of the pig of control group and hydrogen-rich group to the blastaea mitochondrial membrane potential of 168h is dyeed
As a result；
Fig. 3 is that the lonely female activation Embryo Culture of the pig of control group and hydrogen-rich group is in situ to the blastaea Pan-caspase of 168h
The result of fluorescent staining.
Specific embodiment
To make the present invention easier to understand, specific embodiment of the invention is further illustrated below.The following example
The standard laboratory practices of inventor are schematically illustrated, for example pattern of the invention, without that should invention is construed as being defined in
The scope of these embodiments.The present embodiment discloses the general level with those skilled in the art according to the present invention, and technical staff will
Understanding is following to be for illustration only, and various variations, modification and transformation can be carried out in no more than the scope of the present invention.It is wherein involved
And technology, be conventional laboratory techniques well known to those skilled in the art unless stated otherwise；If not specializing, below
The reagent and material used in embodiment are commercially obtained.
Embodiment
First, the preparation of porcine oocytes maturation culture solution
In-vitro maturation liquid is by TCM-199 add 10% (V/V) hyclone (Gibco, the U.S.), 10% (V/V) pig ovum
Bubble liquid (laboratory self-control), 10IU/mL pregnant mare serum gonadotrop(h)in (PMSG)s (hormone preparations Co., Ltd of Ningbo City the 3rd, Chinese),
10IU/mL human chorionic gonadotrophins (hormone preparations Co., Ltd of Ningbo City the 3rd, China), 10IU/mL it is dual anti-(Sigma,
The U.S.), the Cys (Sigma, the U.S.) of 0.1mg/mL, 10ng/mL EGFs (Sigma, the U.S.) mixing and
Into.
2nd, the acquisition of porcine oocytes and maturation in vitro
From slaughterhouse, collection pig ovary is placed in 37 DEG C of physiological saline (penicillin containing 1000IU/mL and streptomysin), and
Transport laboratory in 2h back.Cumulus oocyte complex (COCs) of the FD in 2~8mm is extracted with No. 18 needle applicators
In injection 15mL centrifuge tubes, supernatant is abandoned after precipitation 30min, the above granular cell and kytoplasm is uniform of having three layers is selected under the microscope
COCs, washed three times respectively with tyrode's solution and TCM-199 (Gibco, the U.S.) successively, be put into pre-temperature balance maturation in vitro training
44h is cultivated in nutrient solution.Condition of culture is 38.5 DEG C, 5%CO2, saturated humidity.
3rd, the preparation of lonely female activation liquid and embryo medium
Lonely female activation liquid：0.3mol/L mannitol, 0.05mmol/L calcium chloride and 0.1mmol/L magnesium chlorides.
In vitro culture liquid：Sodium chloride 6.312g/L, sodium acid carbonate 2.119g/L, potassium chloride 0.746g/L, potassium dihydrogen phosphate
0.048g/L, magnesium sulfate 0.048g/L, five water lactic acid calcium 0.606g/L, Sodium Pyruvate 0.022g/L, Glu 0.146g/
L, hypotaurine 0.546g/L, cysteine 0.069g/L, penicillin 10000IU/L, streptomysin 70000IU/L, required amino
Sour (50 ×) 2%, nonessential amino acid (100 ×) 1%, bovine serum albumin(BSA) 3g/L.Experimental group solvent for use is hydrogen rich water, its
Middle H2Content 1.4ppm (experimental group is alternatively referred to as hydrogen-rich group), control group solvent for use is distilled water.
4th, the lonely female activation of pig MII egg mother cells
Will cultivate 44h after COCs move into 0.1% hyaluronidase (Sigma, the U.S.) in liquid-transfering gun blow and beat 2min,
Choose that kytoplasm is uniform, form normal and the egg mother cell of discharge first polar body under the microscope, move into and wash 3 times in TCM-199,
It is placed in standby on 37 DEG C of thermostatic platforms.
The egg mother cell picked out balances 5min in liquid is activated, and immigration is covered with the 0.2mm activation slots of activation liquid and electricity
Extremely parallel discharge, is electrically activated, and activation parameter is 1.2kV/cm, 30 μ s, pulsatile once.
5th, the in vitro culture of the lonely female activation embryo of pig
Egg mother cell after activation is washed three times in the nutrient solution containing 5 μm of ol/L cytochalasin Bs (Sigma, the U.S.), and
3~5h is cultivated wherein, is then washed three times with the nutrient solution without cytochalasin B.
The in vitro culture liquid of hydrogen-rich group and control group has been configured in advance, has been respectively charged into two four orifice plates (NUNC, Denmark),
500 μ L are filled per hole.
Egg mother cell after activating and washing is divided into two parts, in moving into ready four orifice plate respectively, with 500 μ L ore deposits
Thing oil covering.Condition of culture is 38.5 DEG C, 5%CO2, saturated humidity.
6th, cleavage rates, blastocyst rate and the blastomere number of lonely female activation embryo are counted.
Embryo Culture 44h statistics cleavage rates after lonely female activation, 168h statistics blastocyst rates.With 5 μ g/mL's
The dyeing liquor lucifuges of Hoechst 33342 are incubated 10min, carry out nuclear targeting, are washed 3 times with TCM-199 afterwards, are put in slide
Upper compressing tablet, observes under inverted fluorescence microscope (IX71, OLYMPUS, Japan), and observation result in blastaea as shown in figure 1, own
The nucleus of cell shows blue-fluorescence after being dyeed through Hoechst33342, and then counts blastomere number.
7th, blastaea Mitochondria film potential (Δ Ψ m) level is detected
Using containing 10 μ g/mL JC-1 (5,5 ', 6,6 '-tetrachloro-1,1 ', 3,3 '-tetraethyl-
Imidacarbocyanine iodide) the PZM-3 solution in (the green skies, China) carries out mitochondrial membrane electricity to above-mentioned three groups of blastaeas
Position detection, dyeing condition is 37 DEG C, and lucifuge is incubated 20min, is then washed with TCM-199 3 times, uses laser confocal microscope
(TCS SP2, Leica, Germany) is detected.At blastomere maximum cross section, red and green is observed and recorded respectively
Fluorescence signal intensity.The ratio of result such as Fig. 2, red (RITC) and green (FITC) fluorescence intensity is Δ Ψ m.Every group of experiment
It is repeated 3 times, every time about 20 blastaeas.
8th, Pan-caspase Activity determinations in blastaea
Caspase is responsible for optionally cutting some protein, so as to cause Apoptosis.I.e. theirs is active stronger,
Apoptosis is faster.The Pan-caspase fluorescence in situ staining kits produced using Promega (U.S.) company are contaminated
Color.FITC-VAD-FMK is diluted to 10 μm of ol/L with TCM-199, blastaea is used after 37 DEG C of lucifuges incubation 15min in dyeing liquor
TCM-199 is washed 3 times, is then taken pictures in fluorescence microscopy Microscopic observation.Washed 3 times with TCM-199, be placed in the bat of fluorescence microscopy Microscopic observation
According to.Result such as Fig. 3, is quantified to fluorescence photo using the softwares of Image-Pro Plus 6.0 and is recorded fluorescence intensity level, knot
Fruit fluorescence average relative density is represented.Every group of experiment is repeated 3 times, every time about 20 blastaeas.
9th, result
1st, the statistics of cleavage rates, blastocyst rate and blastomere number.
The cleavage rates of control group and hydrogen-rich group, blastocyst rate and blastomere number result of calculation are as shown in table 1.
Influence of the table 1, hydrogen rich water to the lonely female activation vitro Development of Embryos effect of pig
Note：With column data shoulder mark, difference letter person represents significant difference (P ＜ 0.05).
As shown in Table 1, to 44h, the cleavage rates of hydrogen-rich group are significantly higher than control group to the ectogenesis of lonely female activation embryo.It is lonely female
To 168h, cell number is also significantly high during the blastocyst rate of hydrogen-rich group is significantly higher than control group, and blastaea for activation embryo ectogenesis
In control group (P ＜ 0.05).
In Fig. 1, cell quantity in hydrogen-rich group blastaea is apparently higher than control group.
2nd, mitochondrial membrane potential (the Δ Ψ m) level and Pan-caspase Activity determinations of blastaea
Δ Ψ m and Pan-caspase the activity statistics of control group and hydrogen-rich group are as shown in table 2.
Influence of the table 2, hydrogen rich water to the lonely female activation blastaea mitochondrial membrane potential of pig and Pan-caspase activity
| Group | ΔΨm | Pan-caspase activity |
| Control group | ||
| Hydrogen-rich group |
As shown in Table 2, the mitochondrial membrane potential (Δ Ψ m) of hydrogen-rich group blastaea is significantly higher than control group.And Pan-caspase
Activity is substantially less than control group (P ＜ 0.05).
In Fig. 2, the yellow or orange fluorescent intensity of hydrogen-rich group blastaea will be apparently higher than control groups.
In Fig. 3, the fluorescence intensity of control group blastaea is apparently higher than hydrogen-rich group.
Analyzed from Fig. 1, Fig. 2, Fig. 3 and the above results, effect of the hydrogen rich water to the lonely female activation In vitro culture of pig
With following influence：Hydrogen rich water can improve cell cleavage rates, improve blastaea incubation rate, and can lift the quantity of cell in blastaea；
The film potential of blastaea Mitochondria can also be strengthened；And Pan-caspase activity is significantly reduced in blastaea, be conducive to keeping cell
Vigor.
Those of ordinary skill in the art it should be appreciated that the embodiment of the above be intended merely to explanation the present invention,
And be not used as being limitation of the invention, as long as in spirit of the invention, the change to embodiment described above
Change, modification will all fall in scope of the presently claimed invention.
Claims (7)
1. a kind of in vitro culture liquid for the lonely female activation embryo of pig, it is characterised in that：The raw material of the in vitro culture liquid includes
The concentration of five water lactic acid calcium is in mPZM-3 embryo mediums and five water lactic acid calcium and Sodium Pyruvate, and the in vitro culture liquid
0.606g/L, the concentration of Sodium Pyruvate is 0.022g/L；The in vitro culture liquid solvent for use is hydrogen rich water, and wherein H2Contain
It is 1.2~1.6ppm to measure.
2. a kind of in vitro culture liquid for the lonely female activation embryo of pig according to claim 1, it is characterised in that described
The raw material of mPZM-3 embryo mediums includes mPZM-3 basic culture solutions, Glu, hypotaurine, Cys, green grass or young crops
Mycin, streptomycin sulphate and bovine serum albumin(BSA), and their proportioning is 1L：0.146g：0.546g：0.069g：
100000IU：70000IU：3g；The mPZM-3 embryo mediums solvent for use is hydrogen rich water, and wherein H2Content be
1.4ppm。
3. a kind of in vitro culture liquid for the lonely female activation embryo of pig according to claim 2, it is characterised in that described
MPZM-3 basic culture solutions include the solute of following concentration：Sodium chloride 6.312g/L, sodium acid carbonate 2.119g/L, potassium chloride
0.746g/L, potassium dihydrogen phosphate 0.048g/L, magnesium sulfate 0.048g/L；Also include 2% essential amino acid and 1% it is nonessential
Amino acid.
4. the in vitro culture liquid described in a kind of any one of claims 1 to 3 is used for the method for cultivating the lonely female activation embryo of pig, and it is special
Levy and be, comprise the following steps：
(1) the lonely female activation embryo of pig is washed three times in containing 5 μm of in vitro culture liquid of ol/L cytochalasin Bs, moves into and contain 5 μ
3~5h is cultivated in the in vitro culture liquid of mol/L cytochalasin Bs；
(2) embryo after above-mentioned treatment is washed three times with the nutrient solution without cytochalasin B, moves into and be free of cytochalasin B
In vitro culture liquid in cultivate 168h, to the embryonic development into blastaea.
5. a kind of cultural method according to claim 4, it is characterised in that the lonely female activation embryo of pig described in step (1)
The time that treatment is cultivated in containing 5 μm of in vitro culture liquid of ol/L cytochalasin Bs is 4h.
6. a kind of cultural method according to claim 4, it is characterised in that the lonely female activation embryo of pig described in step (2)
Culture density be every 500 μ L in vitro culture liquid 40~60 pieces of embryos of culture.
7. a kind of cultural method according to claim 4, it is characterised in that the condition of culture in step (2) is 38.5 DEG C,
5%CO2, saturated humidity.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110184233A (en) * | 2019-05-15 | 2019-08-30 | 湖北省农业科学院畜牧兽医研究所 | A kind of oocyte in vitro maturation culture solution additive and its application |
| CN110205284A (en) * | 2019-05-27 | 2019-09-06 | 中国农业大学 | L-PROLINE is improving the application in egg mother cell early embryonic development and oxidation resistance |
| CN110551682A (en) * | 2018-06-01 | 2019-12-10 | 深圳华大生命科学研究院 | Kits and methods for dissociation of animal embryos |
| CN111778204A (en) * | 2020-06-11 | 2020-10-16 | 温氏食品集团股份有限公司 | Oocyte in-vitro maturation culture solution additive and application thereof |
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| CN111778204B (en) * | 2020-06-11 | 2022-05-27 | 温氏食品集团股份有限公司 | Oocyte in-vitro maturation culture solution additive and application thereof |
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