CN101886059A - Culture solution used for embryo vitro production and method for bovine embryo vitro production - Google Patents
Culture solution used for embryo vitro production and method for bovine embryo vitro production Download PDFInfo
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- CN101886059A CN101886059A CN 201010224589 CN201010224589A CN101886059A CN 101886059 A CN101886059 A CN 101886059A CN 201010224589 CN201010224589 CN 201010224589 CN 201010224589 A CN201010224589 A CN 201010224589A CN 101886059 A CN101886059 A CN 101886059A
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Abstract
The invention relates to the technical field of biology and discloses culture solution used for embryo vitro production. The culture solution is TCM-199 culture solution comprising 5 to 10mmol /L of Hepes, 15 to 26.2mmol/L of NaHCO3, 30 to 50mu g/L of brain derived neurotrophic factor (BNDF) and 3 to 5 percent OCS, wherein the concentration of the BDNF in the culture solution is preferably 40mu g/L. The culture solution of the invention can obviously improve the ratio of blastocyst developed from bovine embryo body. The invention also provides a method for performing bovine embryo vitro production by using the culture solution.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of nutrient solution of embryo vitro product and method of cattle early embryo produced in vitro of being used for.
Background technology
Animal embryo is transplanted and to be meant the zygote of will take out or to grow body early embryo to the certain period from the reproduction pipeline of the dam of becoming pregnant (donor), be transplanted to certain position with dam (acceptor) the reproduction pipeline of donor dam synchronization of estrus, make its continued growth, growth, grow up to young animal's technology.Utilize embryo transfer can shorten the animal reproduction cycle, further excavate the breeding potential of animal, be a large amount of breedings of superior animals, race's continuity of rare animal provides effective solution, plays an important role in fields such as livestock industry and pharmacy industries.
Owing to collect the influence that zygote is subject to the animal reproductive cycle from the donor dam, embryo transfer now mostly adopts the animal body early embryo to transplant, and this just makes the vitro culture of animal body early embryo occupy critical role in embryo transfer.Animal body early embryo vitro culture be meant the ovocyte that will collect external after the artificial culture maturation, make oocyte fertilization or be activated by technology such as in vitro fertilization, nuclear transplantation and lonely female activation, move to then it is grown to the embryo engineering technology of blastula stage, this technology can not be subjected to the influence of animal reproductive cycle, carries out the produced in vitro of animal body early embryo on a large scale.
Embryo's vitro culture has 40 years of development history.At present, the embryo can be survived and grow under condition of in vitro culture, can gestation farrow after the transplanting, has had in embryo engineering and production practice widely and has used.Present external embryo's production efficiency is still very low, and embryo's quality is compared the also very big gap of existence with embryo in the body.The outer main problem that exists of bovine embryo is: conception rate is on the low side, the Gestation period prolongs, abortion ratio increases, the vitality power of calf differs, the mortality ratio of sex ratio deflection, perinatal period and introduction stage calf and abnormal rate increase etc.Its major cause is that the vitro culture system can't be simulated the dam internal milieu fully, and is stable not enough.In vivo, the embryo migrates to the uterus by uterine tube, and the embryo is exposed to dynamic environment.And the most embryo culture system that has grown up all is at the stable environment of for some time relative equilibrium, the embryo still is in a relative immobilized environment, also physics identical or envrionment conditions can't be simulated fully, the demand of body early embryo differentiation and development can't be adapted to female reproductive tract.Therefore, embryo's vitro culture system awaits further to optimize, improve.
Cultural method commonly used at present is that somatocyte is cultivated and two kinds of sequential culture altogether.Cultivate altogether be helper or somatocyte with the embryo in external cultivation, more preferably simulated embryo's developing environment in vivo, the interpolation of various somatic mixed culture and some somatomedins has all greatly promoted the further perfect of technology in vitro fertilization.This culture systems can promote embryo's growth, and high embryo quality is improved the rate of formation of blastaea.Yet a lot of uncertain factors are arranged in the co-culture system, and somatocyte may serve as the transmitting carrier of cause of disease, produce pollution the embryo.
Sequential culture be embryo according to vitro culture in different growth times to the different substances demand with metabolic different, prepare the nutrient solution of a series of heterogeneities, carrying out sequence changes, thereby prolong the embryo at external incubation time, increase the method for in-vitro screening chance, but this method need be changed multiple substratum, used substratum needs screening, troublesome poeration, and pollute easily.
The nutrient solution composition of each culture systems is a part the most key in the whole culturing process, and it has determined the animal body early embryo rate of growing.Simple basic culture solutions such as TCM-199, CZB, KSOM can not be simulated the environment of growing in the embryoid body fully at present, and necessary nutrition of early embryonic development and non-nutritious matter can not be provided fully, greatly reduce the efficient that fetal development becomes blastaea thus.With regard to the cattle early embryo produced in vitro, these problems are particularly outstanding, greatly restricting Developing of Animal Industry.
Summary of the invention
In view of this, an object of the present invention is to provide a kind of nutrient solution that embryo vitro produces that is used for, described nutrient solution can significantly promote the growth of external embryo to blastaea, further improves the external Embryo Production efficient of ox thereby improve blastocyst rate.
In order to realize the foregoing invention purpose, the present invention adopts following technical scheme:
The nutrient solution of embryo vitro product that is used for of the present invention is for comprising 5-10mmol/L Hepes, 15-26.2mmol/L NaHCO
3, the BDNF of 30～50 μ g/L and the TCM-199 basic culture solution of 3-5%OCS.
BDNF, Brain Derived Neurotrophic Factor (Brain derived neurotrophic factor) mainly is present in brain and peripheral tissues, and the differentiation and the neuronal survival of neurocyte had vital role.Studies show that recently these factors also are expressed in some non-neural system, as cardiovascular, immune, internal secretion, reproductive system, and the function of these systems is regulated in participation.
In the specific embodiment of the present invention, find that BDNF all has expression in the body early embryo of the ovocyte of different developmental phases and embryo and different methods production, BDNF has promoter action to early embryonic development.Cultivate body early embryo with the nutrient solution that contains BDNF,, significantly improve the growth of the external embryo of ox to blastaea when BDNF concentration during between 30～50 μ g/L.Those skilled in the art can be applied to the embryo vitro product of other Mammals such as horse, sheep etc. according to general knowledge.Same, described nutrient solution also can improve remarkable promotion grows, and improves the blastocyst rate of body early embryo.
As preferably, described to be used for the nutrient solution BDNF concentration that embryo vitro produces be 40 μ g/L.
Another object of the present invention provides the method that a kind of bovine embryo vitro produces, and is included in to contain 5-10mmol/L Hepes, 15-26.2mmol/L NaHCO
3, the BDNF of 30～50 μ g/L and 3-5%OCS the TCM-199 nutrient solution cultivate body early embryo, the periodic replacement nutrient solution obtains the step of blastula embryo.
As preferably, described nutrient solution is for containing 5mmol/L Hepes, 26.2mmol/LNaHCO
3, the BDNF of 40 μ g/L and the TCM-199 nutrient solution of 3%OCS.
The body early embryo of described body early embryo for producing by in vitro fertilization, lonely female activation or nuclear transplantation.
In vitro fertilization, English abbreviates IVF as, is meant that mammiferous sperm and ovum finish the technology of fertilization process in external manually operated environment.
Lonely female activation is meant that M II phase ovocyte stimulates without sperm, and by artificial stimulations such as chemistry or physics simulation fertilization processes, recovers and finishes second meiotic division, the spilting of an egg takes place also form body early embryo.
Nuclear transplantation is meant donorcells nuclear is moved in the non-nucleus egg mother cell, makes the latter can be activated, divide and develop into body early embryo without sexual processes such as sperm penetrate.
As preferably, be replaced by fresh nutrient solution of the present invention every 48h in the described method.
The present invention is applied to embryo's vitro culture field with BNDF, has obtained the embryo in-vitro culture solution even more ideal, that developmental rate is higher.Utilize nutrient solution vitro culture ox embryo of the present invention, can significantly improve the blastocyst rate of body early embryo, perfect existing culture systems has improved the quality and the efficient of external Embryo Production greatly.
Embodiment:
The invention discloses a kind of nutrient solution of embryo vitro product and utilize this nutrient solution to carry out the method that bovine embryo vitro produces, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as being included in the present invention.Nutrient solution of the present invention and method are described by preferred embodiment, the related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.
According to the present invention, the described nutrient solution of embryo vitro product that is used for is for containing 5mmol/LHepes, 26.2mmol/L NaHCO
3, the BDNF of 40 μ g/L and the TCM-199 nutrient solution of 3%OCS.
In the experiment of BDNF to the early embryonic development influence, with two kinds of body early embryos in vitro fertilization and lonely female active mode production, in the nutrient solution of cattle early embryo produced in vitro of the present invention, cultivate, when BDNF concentration is 30～50 μ g/L, compare with nutrient solution that is not added with BDNF and the nutrient solution that adds the BDNF of other concentration, the blastaea rate significantly improves.
Preferably, when BDNF concentration is 40 μ g/L in the described nutrient solution, can make the blastaea rate of the body early embryo of producing in mode in vitro fertilization be increased to 51.22%, be increased to 58.41% with the blastaea rate of the body early embryo of the female active mode production of orphan.
The present invention also provides a kind of method of cattle early embryo produced in vitro on the other hand, is included in to contain 5-10mmol/L Hepes, 15-26.2mmol/L NaHCO
3, the BDNF of 30～50 μ g/L and 3-5%OCS the TCM-199 nutrient solution cultivate body early embryo, change nutrient solution every 48h, obtain the step of blastula embryo.
The mode of production of described body early embryo comprises in vitro fertilization, lonely female activation or nuclear transplantation.
During the ovocyte normal fertilization, sperm and ovum plasma membrane directly merge or cause Ca in the ovum by lip-deep receptor protein
2+The instantaneous rising of concentration, cause thus comprise that cortical granule (CG) discharges, in the tenuigenin pH change, source of parents mRNA a series of activating reactions such as replenish, finally cause formation, the spilting of an egg of DNA synthetic initial sum of protokaryon.
Nuclear transplantation, Oocyte in Vitro fertilization and lonely female activation are the important channels that the embryo engineering research field obtains the embryo.The lonely female activation of ovocyte is a kind of mode of artificial activation's ovum, by electricimpulse, Ionomycin, ethanol, Calcium ionophore, cycloheximide and 1,4,5-InsP3 (IP3) etc. can cause Activation of Oocyte, make MII phase ovocyte finish early embryonic development, obtain the parthenogenetic embryo tire without fertilization process.
Lonely female activation with in vitro fertilization all be to be starting point with MII phase ovocyte, can both obtain body early embryo, but lonely female activation and the essential difference of existence in vitro fertilization; The former manually activates ovocyte, and the latter is the activation ovocyte; Lonely female activation embryo is by suppressing the second polar body discharging, make itself and original ovocyte nuclear fusion constitute diplontic embryo, and embryo in vitro fertilization is merged the embryo obtained by female and male gametophyte, lonely female activation and in vitro fertilizationly be two relatively independent technology but close contact is arranged.
Nuclear transplantation is meant donorcells nuclear is moved in the non-nucleus egg mother cell, makes the latter can be activated, divide and develop into body early embryo without sexual processes such as sperm penetrate.
Below in conjunction with embodiment, further set forth the present invention.
Embodiment 1: the detection of body early embryo BDNF mRNA expression level
Detect BDNF with fluorescent quantitative PCR technique and produce expression level in the body early embryo (embryo in vitro fertilization, lonely female activation, nuclear transfer embryo), the results are shown in Table 1 and table 2 at different developmental phases ovocyte and embryo and different methods.
Table 1 different developmental phases bovine oocyte and embryo BDNF mRNA expression level in vitro fertilization
Annotate: different capitalizations are represented difference extremely significantly (P＜0.01)
BDNF mRNA expression level among the dissimilar embryos of table 2 ox
Annotate: different capitalizations are represented difference extremely significantly (P＜0.01)
Above result shows, BDNF all has expression in the body early embryo of the ovocyte of different developmental phases and embryo and different methods production, and BDNF has promoter action to early embryonic development.
Embodiment 2: detect the expression of bdnf protein in the embryo with immunofluorescence technique
Detect the expression of bdnf protein in the embryo with immunofluorescence technique, show that the bdnf protein signal mainly concentrates on the ectoderm of blastaea.
Embodiment 3: the collection of ovocyte and maturation in vitro
Ox ovary with collect in the slaughterhouse places and contains K
+, Ca
2+And Mg
2+35～37 ℃ of physiological saline vacuum flask in, deliver to the laboratory in the 4h, clean with 37 ℃ physiological saline.Extracting diameter with the 10mL syringe that has No. 12 syringe needles then is ovocyte in 2～6mm ovarian follicle.Under stereomicroscope, pick out tenuigenin evenly and ovocyte complete or the fine and close cumulus cell of part is arranged, clean twice with egg-cleaning liquid after, put into the glass dish that contains the 1.5mL maturation culture solution (30 * 10mm), at 38.5 ℃, 5%CO
2With cultivate 22～24h in the incubator of saturated humidity, obtain sophisticated ovocyte.
Embodiment 4: production body early embryo in vitro fertilization
To freeze smart in 37 ℃ of water-baths, thaw after, get the round bottom test tube bottom that the 0.25mL seminal fluid carefully places the Tyrode ' s that contains the 1.5mL improvement to be subjected to seminal fluid, after 15min suspends, sperm swim-up alive, with upper strata liquid sucking-off, centrifuge washing reaches to concentrate and once removes supernatant, stays about 30 μ L standby then.In culture dish, do the fertilization droplet of 30 μ l, cover, put into 38.5 ℃, 5%CO with paraffin oil
2With balance 1h at least in the incubator of maximum saturation humidity.Remove the mature oocyte cumulus cell on every side that surrounds embodiment 1 preparation with tubule machinery, wash ovocyte 2～3 times with fresh egg-cleaning liquid.Ovocyte after the washing is put into droplet, and 15～25 ovocytes of every dropping add the sperm suspension of 2～5 μ L again, make that sperm concentration is 1.0 * 10
6～1.5 * 10
6Individual/mL.The culture dish that will contain smart ovum is put back to 38.5 ℃, 5%CO
2Cultivate 24h in the incubator, obtain body early embryo.
Embodiment 5: body early embryo is produced in lonely female activation
The maturation in vitro of getting embodiment 3 preparation is cultivated the ovocyte behind 20～22h, blows and beats the cumulus cell that removes around the ovocyte repeatedly with suction pipe.Ovocyte is handled 5min in the nutrient solution that contains 5 μ mol/L ionomycins, wash 3 times with nutrient solution afterwards, move into again to contain and cultivate 3h in the 2mmol/L 6-dimethylamino-purine nutrient solution, obtain body early embryo.
Embodiment 6: nutrient solution of the present invention is to the early embryonic development influence of production in vitro fertilization
With TCM-199+5mmol/L Hepes+26.2mmol/L NaHCO
3+ 3%OCS is embryo's basal liquid, to the BDNF that wherein adds different concns, and at 38.5 ℃, 5%CO
2Under the condition body early embryo of embodiment 4 productions in vitro fertilization is cultivated, nutrient solution is changed once every 48h, checks division rate behind the cultivation 48h, and the blastaea rate that record is grown behind the 7d the results are shown in Table 3.(the hundreds of proportions by subtraction of the spilting of an egg=spilting of an egg number/oocyte number %; % is counted in the hundreds of proportions by subtraction of blastaea number=blastaea number/spilting of an egg; Hatch hundreds of proportions by subtraction=hatching number/spilting of an egg and count %)
Table 3 different concns BDNF is to ox embryo's in vitro fertilization influence
Annotate: subscript is represented and is not added the BDNF group and compare significant difference: a:P＜0.05, b:P＜0.01
Learn by table 3, contain 5-10mmol/L Hepes, 15-26.2mmol/L NaHCO
3, the BDNF of 30～50 μ g/L and 3-5%OCS the TCM-199 nutrient solution can significantly improve embryo's in vitro fertilization blastaea rate, preferably BDNF concentration is 40 μ g/L in the nutrient solution.
Embodiment 7: the early embryonic development influence that nutrient solution of the present invention is produced the female activation of orphan
With reference to method among the embodiment 6, the body early embryo that 5 lonely female activation are produced to embodiment is cultivated, and the results are shown in Table 4.
Table 4 different concns BDNF is to the lonely female activation embryo's of ox influence
Annotate: subscript is represented and is not added the BDNF group and compare significant difference: a:P＜0.05
Learn by table 4, contain 5-10mmol/L Hepes, 15-26.2mmol/L NaHCO
3, the BDNF of 30～50 μ g/L and 3-5%OCS the TCM-199 nutrient solution can significantly improve lonely female activation embryo's blastaea rate, preferably BDNF concentration is 40 μ g/L in the nutrient solution.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (8)
1. one kind is used for the nutrient solution that embryo vitro produces, and it is characterized in that described nutrient solution is for containing 5-10mmol/L Hepes, 15-26.2mmol/L NaHCO
3, the BDNF of 30～50 μ g/L and the TCM-199 nutrient solution of 3-5%OCS.
2. according to the described nutrient solution of claim 1, it is characterized in that described nutrient solution is for containing 5mmol/LHepes, 26.2mmol/L NaHCO
3, the BDNF of 40 μ g/L and the TCM-199 nutrient solution of 3%OCS.
3. according to claim 1 or 2 described nutrient solutions, it is characterized in that described embryo is a cattle early embryo.
4. the method that bovine embryo vitro produces is characterized in that, is included in to contain 5-10mmol/LHepes, 15-26.2mmol/L NaHCO
3, the BDNF of 30～50 μ g/L and 3-5%OCS the TCM-199 nutrient solution in cultivate cattle early embryo, the periodic replacement nutrient solution obtains the step of blastula embryo.
5. according to the described method of claim 4, it is characterized in that described nutrient solution is for containing 5mmol/LHepes, 26.2mmol/L NaHCO
3, the BDNF of 40 μ g/L and the TCM-199 nutrient solution of 3%OCS.
6. according to claim 4 or 5 described methods, it is characterized in that described body early embryo is the body early embryo by production in vitro fertilization.
7. according to claim 4 or 5 described methods, it is characterized in that the body early embryo of described body early embryo for producing by lonely female activation.
8. according to claim 4 or 5 described methods, it is characterized in that described body early embryo is the body early embryo of producing by nuclear transplantation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102499788A (en) * | 2011-11-10 | 2012-06-20 | 周虚 | Application of SRY (sex determining region of the Y) antibody |
| CN110331129A (en) * | 2019-06-24 | 2019-10-15 | 西北农林科技大学 | A kind of the embryo transfer liquid and its application method of ox |
| CN113846053A (en) * | 2021-07-27 | 2021-12-28 | 中山大学孙逸仙纪念医院 | Method for treating contaminated embryos |
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| CN1904039A (en) * | 2006-07-28 | 2007-01-31 | 浙江大学 | Ox embryo in vitro culturing liquid containing tea polyphenol and its culturing method |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1904039A (en) * | 2006-07-28 | 2007-01-31 | 浙江大学 | Ox embryo in vitro culturing liquid containing tea polyphenol and its culturing method |
Non-Patent Citations (4)
| Title |
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| 《Animal》 20081231 K.L.Yi et al The mRNA expression of brain-derived neurotrophic factor in oocytes and embryos and its effects on the development of early embryos in cattle 1786-1794 1-8 第2卷, 第12期 2 * |
| 《中国兽医学报》 20080531 易康乐 等 NGF对牛体外受精和孤雄激活早期胚胎发育的影响 588-591 1-8 第28卷, 第5期 2 * |
| 《中国博士学位论文全文数据库 农业科技辑》 20081115 易康乐 Figla和BDNF对猪和牛卵母细胞及早期胚胎生长发育的影响 D050-25 1-8 , 第11期 2 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102499788A (en) * | 2011-11-10 | 2012-06-20 | 周虚 | Application of SRY (sex determining region of the Y) antibody |
| CN102499788B (en) * | 2011-11-10 | 2014-12-10 | 周虚 | Application of SRY (sex determining region of the Y) antibody |
| CN110331129A (en) * | 2019-06-24 | 2019-10-15 | 西北农林科技大学 | A kind of the embryo transfer liquid and its application method of ox |
| CN113846053A (en) * | 2021-07-27 | 2021-12-28 | 中山大学孙逸仙纪念医院 | Method for treating contaminated embryos |
| CN113846053B (en) * | 2021-07-27 | 2023-08-29 | 中山大学孙逸仙纪念医院 | Method for treating contaminated embryo |
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Application publication date: 20101117 |