CN107574144A - A kind of in-vitro maturity of porcine oocytes nutrient solution for adding carnosic acid and its application - Google Patents
A kind of in-vitro maturity of porcine oocytes nutrient solution for adding carnosic acid and its application Download PDFInfo
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Abstract
The present invention relates to technical field of cell culture, and in particular to a kind of oocyte in vitro maturation culture solution for adding carnosic acid and its application.The In-vitro maturation liquid, it is characterised in that component includes carnosic acid；The carnosic acid concentration range is 1 20 μM.The present invention coordinates the synergy of other components, improves porcine oocytes maturation quality, there is good facilitation effect to Pig embryos early development by adding carnosic acid in porcine oocytes maturation culture solution.As a result show, the suitable carnosic acid of concentration can effectively improve oocyte maturation quality, so as to lift porcine oocytes in vitro potentiality.It is easy to operate and the In-vitro maturation liquid is prepared simply, there is larger application prospect.
Description
Technical field
The present invention relates to technical field of cell culture, and in particular to a kind of oocyte in vitro maturation for adding carnosic acid
Nutrient solution and its application.
Background technology
With embryo transfer, the development of frozen semen technology, cloned, transgenic technology, egg mother cell demand is increasingly
Greatly.Oocyte in vitro maturation is regulated and controled by many factors, and regulatory mechanism is more complicated, and though it is studied for ovum into
Theoretical or embryo's actual production of ripe mechanism, all has great importance.By being obtained using the super ovulation techniques of pig
Egg mother cell or embryo's quantity be extremely limited, can not meet the needs of a large amount of high-quality egg mother cell, and operate
Complexity, cost are higher, so maturation in vitro technology is just particularly important.In-vitro maturity of porcine oocytes refers to straight from slaughterhouse
The discarded ovary of collection is connect, after taking out immature egg mother cell, simulates cylinder mature environment in vitro to reach ripe purpose.
Because porcine oocytes contain relatively great amount of fat drop, more sensitive to temperature, manipulation in vitro difficulty is relatively bigger.Body at present
The egg mother cell of outer maturation culture is compared with the embryo produced in vivo, the ratio of the extracorporeal embryo development of maturation in vitro ovum to blastaea
Lowly.For these problems, each researcher is constantly studied the maturation of porcine oocytes, it is desirable to improves constantly body
The quality of outer mature oocyte.Therefore, In vitro maturation condition is optimized to improving embryo production in vitro efficiency
It is significant.
The content of the invention
The present invention provides a kind of oocyte in vitro maturation culture solution for adding carnosic acid and its application.
First purpose of the present invention, there is provided a kind of oocyte in vitro maturation culture solution for adding carnosic acid, its group
Dividing includes carnosic acid.Preferably, the carnosic acid concentration is 1-20 μM.
It is further preferred that 2-10 μM of carnosic acid concentration range, is optimal with 2-5 μM.
The present invention improves oocyte maturation matter by adding carnosic acid in oocyte in vitro maturation culture solution
Amount, there is good facilitation effect to embryonic development.
Preferably, component also includes egg mother cell cellar culture liquid, glutamine, gentamicin, pregnant mare serum rush sexual gland
Hormone, human chorionic gonadotrophin, pig follicle liquid, serum；
Preferably, the egg mother cell cellar culture liquid volume ratio is 70-80%, and the glutamine concentration is 0.02-
0.06g/L, the gentamicin concentration are 0.1-0.2g/L, and the pregnant mare serum gonadotrop(h)in (PMSG) concentration is 20000-
25000U/L, the human chorionic gonadotrophin concentration are 10000-15000U/L, and the liquor folliculi concentration volume ratio is 8-
12%, the serum-concentration volume ratio is 8-12%.
On egg mother cell cellar culture liquid, it is preferable that the egg mother cell cellar culture liquid is Medium199, DMEM,
One kind in TCM.More preferably Medium199.
On serum, it is preferable that the serum is calf serum, one kind in hyclone or NBCS.
The present invention provides a kind of more preferable oocyte in vitro maturation culture solution, and formula is as follows：Medium199 volumes
Than promoting sexual gland for 75-80%, glutamine 0.02-0.06g/L, gentamicin concentration 0.12-0.16g/L, pregnant mare serum
Hormone concentration is 20000-25000U/L, and human chorionic gonadotrophin concentration is 10000-15000U/L, pig follicle liquid concentration
Volume ratio is 8-12%, and serum-concentration volume ratio is 8-12%, and carnosic acid concentration is 2-5 μM.
In-vitro maturation liquid provided by the invention can improve egg mother cell oxidation resistance, the synergy of active component
Oocyte maturation quality can be effectively improved, so as to lift Oocyte in Vitro developmental potentiality, is applying body of the present invention
During outer maturation culture solution culture egg mother cell, the stress damage that external environment is brought can be effectively reduced, promotes egg mother cell
Cytoplasmic maturation, improve the lonely female activation of egg mother cell and the Embryo viability of after fertilization of maturation in vitro.And this in vitro into
Ripe nutrient solution is prepared simply, easy to operate, has larger application prospect.
The present invention also provides application of the above-mentioned In-vitro maturation liquid in the egg mother cell of culture animal.
A kind of application using above-mentioned oocyte in vitro maturation liquid culture egg mother cell, including egg mother cell is placed in this
Cultivated in the oocyte in vitro maturation culture solution of the described addition carnosic acid of invention.
Specifically, according to following steps culture：
By egg mother cell be put into balance after the oocyte in vitro maturation culture solution in, after be placed in CO2Volumetric concentration
Maturation culture 42~44 hours, are produced in 4%-6% environment.
Preferably, the balance is specially：The oocyte in vitro maturation culture solution is balanced at least 3 hours.
Preferably, the egg mother cell is porcine oocytes, and the temperature of the maturation culture is 38-39 DEG C.
Preferably, the egg mother cell is cumulus oocyte complex (COCs).
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
Pig ovary is provided by slaughterhouse, is positioned in 37 DEG C of vacuum flask of 0.9% salt solution, is transported laboratory in 1h back.
Serum is cow's serum, purchased from Gibico companies, injection pregnant mare serum gonadotrop(h)in (PMSG) and injection human chorionic
Promoting sexual gland hormone is purchased from Ningbo the second hormone factory, and other raw materials are purchased from Sigma companies.
Carnosic acid storing liquid：Take 10mg carnosic acid powder to be first dissolved in 0.15ml DMSO, final concentration of 0.2M, keep away
Light is stored in -20 DEG C of refrigerators.
Physiological saline：9g NaCl are taken to be dissolved in 1L sterilized waters, autoclave sterilization.
PBS egg-cleaning liquids：Take 1g PVA, 9.55g PBS powder, 0.066g penicillin G sodium, 0.05g
Streptomycin is dissolved in sterilized water, is settled to 1L, sterilizing.
IVM maturation liquid：M199 concentration volume ratio is 78%, Glutamine 0.04g/L, Gentamicin-stock
0.146g/L, pregnant mare serum gonadotrop(h)in (PMSG) concentration are 20000U/L, and human chorionic gonadotrophin concentration is 10000U/L, pig
Liquor folliculi 10%, cow's serum 10%.Osmotic pressure is adjusted to 280mOsm or so, and pH is adjusted to 7.2-7.4.Divide after the filtering of 0.22 filter
Dress, -20 DEG C store for future use.
HAE hyaluronidase solutions：20mg hyaluronidases are dissolved in 20mL PBS, are dispensed after the filtering of 0.22 filter, -20 DEG C
Store for future use.
T2 manipulation in vitro liquid：NaHCO30.168mg/mL, pyruvic acids (Na Salt) 0.22mg/mL, HEPES
(Na Salt) 3.6mg/mL, HEPES (Acid) 2.63mg/mL, M199 (10 ×) 10%, L-Glutamine 0.1461mg/
ML, FBS 2%.Osmotic pressure is adjusted to 280mOsm or so, and pH is adjusted to 7.2-7.4.Dispensed after the filtering of 0.22 filter, -20 DEG C of storages are standby
With.
P/A electrically activates liquid：Mannitol 0.25M, CACl2·2H2O 0.1mM, MgCl2·6H2O 0.1mM, HEPES
(Na Salt) 0.5mM, PVA 0.1g/L.Dispensed after the filtering of 0.22 filter, -20 DEG C store for future use.
PZM-3 extracorporeal embryo development liquid：NaCl 108mM, NaHCO325.07mM, KCl 100mM, KH2PO40.35mM,
MgSO40.4mM, Ca-Lactate5H2O 2mM, Na-Pyvate 0.2mM, Myo-inositol 2.78mM, Phenol
Red 10mM, L-Glutamine 1mM, Hypotaurine 5mM, EAA (50 ×) 2%, NEAA (100 ×) 1%,
Gentamicin 0.05mg/mL, BSA 3g/L.Osmotic pressure is adjusted to 280mOsm or so, and pH is adjusted to 7.2-7.4.0.22 filter mistake
Dispensed after filter, -20 DEG C store for future use.
CC Assisted Activation liquid：Dissolved respectively using dimethyl sulfoxide (DMSO) cytochalasin B and cycloheximide be 5 μ L/mL and
1mg/mL storing liquid.The storing liquid of 5 μ L cytochalasin Bs and 10 μ L cycloheximides is added in 1mL PZM-3, it is dense to working solution
Degree.
Embodiment 1
The present embodiment provides a kind of oocyte in vitro maturation culture solution (CA2) for adding carnosic acid, and formula is as follows：
Medium199 volume ratios are 78%, and glutamine 0.04g/L, gentamicin concentration 0.146g/L, pregnant mare serum promote sexual gland
Hormone concentration is 20000U/L, and human chorionic gonadotrophin concentration is 10000U/L, and pig follicle liquid concentration volume ratio is 10%,
Serum-concentration volume ratio is 10%, and carnosic acid concentration is 2 μM.
Embodiment 2
The present embodiment provides a kind of oocyte in vitro maturation culture solution (CA5) for adding carnosic acid, the area with implementing 1
It is not only that, carnosic acid concentration is 5 μM.
Embodiment 3
The present embodiment provides a kind of oocyte in vitro maturation culture solution (CA1) for adding carnosic acid, the area with implementing 1
It is not only that, carnosic acid concentration is 1 μM.
Embodiment 4
The present embodiment provides a kind of oocyte in vitro maturation culture solution (CA10) for adding carnosic acid, with implementing 1
Differ only in, carnosic acid concentration is 10 μM.
Comparative example 1
This comparative example provides a kind of oocyte in vitro maturation culture solution (blank control group), and the difference with embodiment 1 is only
It is, component does not include carnosic acid.
Test example 1
Test objective：The effect of the oocyte in vitro maturation culture solution of checking addition carnosic acid；
Subjects：Porcine oocytes；
Test operation：
1. collection and the In-vitro maturation of egg mother cell
The extraction of immature oocyte is carried out in embryo experiments room.It is straight that 3-8mm is evacuated using vavuum pump connection collection tube
The ovarian follicle in footpath, the liquor folliculi containing ovum is poured into 50ml centrifuge tubes；Supernatant is abandoned after precipitation 30min, is washed 2 times with egg-cleaning liquid,
Per minor tick 15min；Gently shake up, pour into right amount in 100mm culture dishes, under stereomicroscope after 3rd addition egg-cleaning liquid
Cumulus oocyte complex (COCs) is sorted out with the glass Pasteur pipe voluntarily drawn；The COCs sorted out is crossed in pre-equilibration
Wash after three times and be put into 24 orifice plates for oocyte maturation in oocyte maturation liquid, add 500 μ L in 24 orifice plates per hole
In-vitro maturation liquid prepared by embodiment 1-4, comparative example 1, upper strata cover 300 μ l mineral oil, and 75 pieces of COCs are put into per hole；
38.5 DEG C, ripe 42h in 5%CO2 incubators.In triplicate, as parallel group.
2. the preparation of egg mother cell
The cumulus oocyte complex (COCs) for cultivating 42h is moved into the 1.5ml equipped with 500 μ l hyaluronidase solutions
In centrifuge tube, 200 μ l liquid-transfering guns are blown and beaten 65 times or so, remove granular cell；After washing three times during Oocyte in Vitro operates liquid
Move into the operation liquid droplet that 50 μ l cover mineral oil；Choose the egg mother cell and statistics for thering is ovum gap to have first polar body in droplet
Quantity.Discard kytoplasm disperse, after the egg mother cell that kytoplasm is imperfect, perivitelline is unclear and after birth is unclear, count remaining ovum
Mother cell quantity.
3. the lonely female activation of egg mother cell
Integration slot is washed with fusion liquid 3 times, is added 75 μ l and is merged liquid in integration slot, egg mother cell is first put down in liquid is merged
Weigh 1min, is then placed in the integration slot that with the addition of fusion liquid.With two 80 μ s are applied in CF-150/B fusion instruments, it is spaced
100ms, voltage 1.5kV/cm electric pulse activation egg mother cell, pick reconstructed embryo and are washed 3 times, after washing with PZM-3
Reconstructed embryo be placed in Assisted Activation liquid and handle 4h.
4. Embryo Culture
Egg mother cell is taken out from Assisted Activation liquid, removes dead ovum, pre-balance is transferred to after washing 3 times with PZM-3 solution
In good nutrient solution PZM-3, it is put under hypoxemia culture environment that (hypoxemia incubator is filled with the closed training of hypoxemia mixing distribution envelope
Support) culture.Using droplet or four orifice plate cultures, gas phase condition is containing 7%O2, 88%N2And 5%CO2Mixed gas, culture
Temperature is 38.5 DEG C, humidity 100%.In activation culture cleavage rates and blastaea shape are observed and recorded respectively within the 48th and 168 hour
Into rate.
5. blastomere number counts
168 hours blastaeas formed of Embryo Culture are collected, after 4% formaldehyde fixes 30min, are broken using 1%Triton-100
With dyeing 20min in 0.3mM DAPI (dye nucleus) after film, Conventional compression processing, fluorescence microscope then are carried out to blastaea
With ultraviolet excitation, blastomere number is counted after gathering image.
6. data processing
Using DPS7.0 statistical softwares, to the cleavage rates after each group oocyte maturation rate, and lonely female activation, blastaea shape
One-way analysis of variance, P are used into rate and blastomere number<0.05 is that difference has statistical significance.
Test results and analysis：
Influence of the carnosic acid to in-vitro maturity of porcine oocytes and zona-free oocytes early development
It is blank control group with comparative example 1, using the carnosic acid for adding various dose as test group, observes embodiment respectively
Influence of the carnosic acid of 1-5 various doses to pig in-vitro fertilization body early embryo ectogenesis effect, the results are shown in Table 1.As a result demonstrate,prove
Real, carnosic acid does not influence the maturing rate and cleavage rates of porcine oocytes, but can improve the capsule of embryonic development after lonely female activation
Embryo rate and blastomere number, the wherein blastocyst rate of CA2, CA5 and CA10 group and blastomere number are obviously improved, and the capsule of CA2 groups
Embryo rate is higher than other groups, and CA5 group blastomeres number is higher than other groups, illustrated, the carnosic acid pair of 2-5 μM of concentration is added in IVM
The facilitation effect of Pig embryos early development is optimal.
The various concentrations carnosic acid of table 1 is to oocyte in vitro maturation and the influence of parthenogenetic development
Note：Same perpendicular field mark notes the different notable P of lowercase letter indication difference<0.05.
Although above the present invention is made to retouch in detail with general explanation, embodiment and experiment
State, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, are belonged to claimed
Scope.
Claims (10)
1. a kind of in-vitro maturity of porcine oocytes nutrient solution for adding carnosic acid, it is characterised in that active component includes rat-tail
Oxalic acid.
2. In-vitro maturation liquid according to claim 1, it is characterised in that the concentration range of the carnosic acid be 1-
20μM。
3. In-vitro maturation liquid according to claim 1 or 2, it is characterised in that also cultivated including egg mother cell basis
Liquid, glutamine, gentamicin, pregnant mare serum gonadotrop(h)in (PMSG), human chorionic gonadotrophin, liquor folliculi and serum.
4. In-vitro maturation liquid according to claim 3, it is characterised in that the oocyte in vitro maturation culture solution
In, the concentration volume ratio of the egg mother cell basic culture solution is 70-80%, and the concentration of the glutamine is 0.02-
0.06g/L, the concentration of the gentamicin is 0.1-0.2g/L, and the concentration of the pregnant mare serum gonadotrop(h)in (PMSG) is 20000-
25000U/L, the concentration of the human chorionic gonadotrophin are 10000-15000U/L, the concentration volume ratio of the liquor folliculi
For 8-12%, the concentration volume ratio of the serum is 8-12%.
5. the In-vitro maturation liquid according to claim 3 or 4, it is characterised in that the egg mother cell basic culture solution
For Medium199, one kind in DMEM, TCM.
6. according to the In-vitro maturation liquid described in claim any one of 3-5, it is characterised in that the serum is small ox blood
Clearly, one kind in hyclone or NBCS.
7. according to the In-vitro maturation liquid described in claim any one of 3-6, it is characterised in that formula is as follows：
Medium199 volume ratios are 75-80%, glutamine 0.02-0.06g/L, gentamicin concentration 0.12-0.16g/
L, pregnant mare serum gonadotrop(h)in (PMSG) concentration are 20000-25000U/L, and human chorionic gonadotrophin concentration is 10000-
15000U/L, pig follicle liquid concentration volume ratio are 8-12%, and serum-concentration volume ratio is 8-12%, and carnosic acid concentration is 2-5 μ
M。
8. the application of the in-vitro maturity of porcine oocytes nutrient solution culture egg mother cell described in claim any one of 1-7 is utilized,
Characterized in that,
By egg mother cell be put into balance after the oocyte in vitro maturation culture solution in, after be placed in CO2Volumetric concentration 4%-
Maturation culture 42~44 hours, are produced in 6% environment.
9. application according to claim 8, it is characterised in that the balance is specially：By the Oocyte in Vitro into
Ripe nutrient solution balances at least 3 hours.
10. application according to claim 8 or claim 9, it is characterised in that the temperature of the maturation culture is 38-39 DEG C.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108753699A (en) * | 2018-06-19 | 2018-11-06 | 青岛农业大学 | A kind of rescue method that zearalenone endangers porcine oocytes in vitro |
| CN113583942A (en) * | 2021-07-12 | 2021-11-02 | 华南农业大学 | Additive for promoting oocyte in-vitro maturation and application thereof |
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| CN105104890A (en) * | 2015-09-25 | 2015-12-02 | 四川农业大学 | Composition for promoting meiotic resumption of oocytes of sows and application of composition |
| CN106831403A (en) * | 2017-01-18 | 2017-06-13 | 河南佳尚农业科技发展有限公司 | It is a kind of that fat-soluble and water soluble antioxidant method is extracted from rosemary |
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| CN106831403A (en) * | 2017-01-18 | 2017-06-13 | 河南佳尚农业科技发展有限公司 | It is a kind of that fat-soluble and water soluble antioxidant method is extracted from rosemary |
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Cited By (3)
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|---|---|---|---|---|
| CN108753699A (en) * | 2018-06-19 | 2018-11-06 | 青岛农业大学 | A kind of rescue method that zearalenone endangers porcine oocytes in vitro |
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| CN113583942A (en) * | 2021-07-12 | 2021-11-02 | 华南农业大学 | Additive for promoting oocyte in-vitro maturation and application thereof |
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