CN102676449A - Ghrelin-containing sheep embryo in-vitro culture solution and culture method thereof - Google Patents
Ghrelin-containing sheep embryo in-vitro culture solution and culture method thereof Download PDFInfo
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Abstract
The invention provides a ghrelin-containing sheep embryo in-vitro culture solution. The sheep embryo in-vitro culture solution takes a TCM199 culture solution as a matrix; and the matrix also contains 400-800mg/mL ghrelin, 0.15mg/mL glutamine, 100IU/mL penicillin, 100mug/mL streptomycin and 5-10mg/mL bovine serum albumin. In the embryo in-vitro culture solution provided by the invention, ghrelin is used as the endogenous ligand of a growth hormone secretagogue receptor, and has the functions of regulating the secretion of reproductive hormone, promoting the growth of ovarian follicle and ovulation, adjusting the ovarian function, participating in the early developmental regulation of embryo and the like. By applying ghrelin to the sheep embryo in-vitro culture, the ectogenesis of the embryo can be effectively promoted, thus the problem of low ectogenesis rate of the sheep early embryo at present is solved. The sheep embryo in-vitro culture solution provided by the invention has relatively low economic cost, and is convenient to popularize and use in production.
Description
(1) technical field
The present invention relates to the sheep embryo in-vitro culture solution of a kind of ghrelin of containing, and utilize this nutrient solution to carry out the outer cultured method of sheep embryoid body, belong to the livestock embryo field of engineering technology.
(2) background technology
Along with modern times breeding development of biology and embryo vitro produce the progressively perfect of technology, the sheep embryo vitro produces technology (in vitro fertilization or clone) has become the important channel of accelerating breed breeding and good variety population reproductive speed.Though obtained the offspring who lives through the embryo vitro technology of producing; But external embryo ubiquity in culturing process in vitro fertilization or that the clone obtains is grown the blocking-up phenomenon; The stasi that is fetal development behind the certain phase is also degenerated, and causes external embryo's blastocyst rate far below embryo in the body.In addition, compare with embryo in the body, the TCS of external embryo's blastaea and inner cell mass cell count obviously reduce, and after acceptor was gone in external embryo transfer, farrowing rate significantly was lower than embryo in the body.The outer culture system of present sheep embryoid body can not provide with body in the identical or close nutrient environment of body early embryo be one of key reason that causes the problems referred to above, therefore improve the embryo early stage culture system, set up the vitro culture liquid that can improve blastocyst rate and become embryo vitro and produce the breach that technology popularization is used.
Ghrelin is the specific endogenic ligand of the secretagogue receptor of from the stomach of mouse, purifying and identifying in 1999.Early stage research shows that the major function of ghrelin is through activating phosphatase lipase C and protein kinase C signal path, stimulates the prepituitary gland growth hormone releasing, the secretion of appetite stimulator, adjusting energy metabolism balance and promotion hydrochloric acid in gastric juice.But along with going deep into of research, more and more evidences shows that ghrelin is bringing into play important effect aspect genital regulating.In pig, sheep and people's hypothalamus and hypophysis, detected the expression of ghrelin.Ghrelin all expresses in the whole oestrus cycle at rat ovary, but the highest at the 1st day expression amount of dioestrus, the expression amount of proestrus is minimum, and the function of this periodicity expression pattern and corpus luteum adapts.And in the whole Gestation period, the expression amount of the early stage ovary ghrelin of gestation is higher, and later stage of pregnancy, expression amount was lower.Research also finds, the ghrelin gene is expressed in the uterine endometrium of non-pregnancy and deciduaization and placenta, and ghrelin temporarily regulates and control the dialogue between uterine endometrium and the embryo as the paracrine regulator of the deciduaization of endometrial stromal cell during embryo's implantation.
The Ghrelin gene efficiently expresses confirmation in reproductive systems such as ovary, uterus and placenta, its differentiation to embryo in the body, growth and implantation have important regulation.The relevant ghrelin of Shang Weijian is used for the livestock reproduction technical field, especially for the report of embryo's vitro culture but up to the present.
(3) summary of the invention
The present invention promptly is in order to solve the demand that sheep embryo in-vitro culture solution in the prior art can not satisfy external early embryo development; Cause the lower deficiency of early embryo development rate, a kind of sheep embryo in-vitro culture solution that contains ghrelin and cultural method thereof that improves external early embryo development rate function that have is provided.
The technical scheme that the present invention adopts is:
A kind of sheep embryo in-vitro culture solution that contains ghrelin; Said sheep embryo in-vitro culture solution is a matrix with the TCM199 nutrient solution, also contains the ghrelin of 400～800ng/mL, the Stimulina of 0.15mg/mL, the penicillium mould of 100IU/mL, the Streptomycin sulphate of 100 μ g/mL and the bovine serum albumin of 5～10mg/mL in the said matrix.
Ghrelin is a peptide species class biologically active factors, belongs to biochemical reagents commonly used, and existing commodity selling.Used ghrelin is purchased from Sigma company by seminar among the present invention.
The TCM199 nutrient solution can adopt commercial commodity for this area routine is used for the nutrient solution of embryo culture, and used TCM199 nutrient solution is available from Sigma company among the present invention.
Preferably, the every 1000mL of said sheep embryo in-vitro culture solution forms as follows: ghrelin 400～800mg, and Stimulina 150mg, penicillium mould 100000IU, Streptomycin sulphate 100000 μ g, serum albumin 5～10g, the TCM199 nutrient solution complements to 1000mL.
Said sheep embryo do for oneself sheep or goat.
Preferably, in the said sheep embryo in-vitro culture solution, ghrelin content is 500～600ng/mL, and bovine serum albumin content is 8mg/mL.
The invention still further relates to said sheep embryo in-vitro culture solution carries out vitro culture to the sheep embryo the method for using; Said method is after the early stage sheep embryo with after fertilization washes with the TCM199 nutrient solution repeatedly; Put into the sheep embryo in-vitro culture solution of the said ghrelin of containing, in 38～39 ℃, 5% CO
2Environment in, carry out embryo's vitro culture.
Said early stage sheep embryo by the sheep ovocyte through maturation in vitro cultivate, acquisition in vitro fertilization.
Said sheep in-vitro maturation culture method for oocyte is following: the sheep ovocyte that from the ovary that the slaughterhouse obtains, extracts with syringe, with maturation culture solution at 38～39 ℃, 5% CO
2Environment in cultivated 20～24 hours, obtain sophisticated sheep ovocyte; Said maturation culture solution is a matrix with conventional nutrient solution; Also containing the FSH (follicular stimulating hormone) of 10 μ g/mL, the LH (lutropin) of 10 μ g/mL, the penicillium mould of 100IU/mL, Streptomycin sulphate and the volumn concentration of 100 μ g/mL in the said matrix is 8%～20% serum, and said conventional nutrient solution is one or more the mixture in TCM199, DMEM, F12, the MEM nutrient solution; Described serum is a kind of in calf serum, foetal calf serum or the NBCS.
Said sheep Oocyte in Vitro fertilization method is following: sophisticated sheep ovocyte and sperm are together put into the fertilization nutrient solution, at 38～39 ℃, 5% CO
2Environment in cultivated 18～24 hours, be fertilized, obtain early stage sheep embryo; Said fertilization nutrient solution is the TCM199 nutrient solution that contains 2% sheep blood serum.
Embryo in-vitro culture solution of the present invention is that the blastaea rate is lower during according to present embryo's vitro culture, can not satisfy the needs of applying aborning, develops in conjunction with the biological function of ghrelin.Ghrelin has the secretion of regulation and control reproductive hormone as the endogenic ligand of secretagogue receptor; Promote ovarian follicular growth, ovulation; Regulate ovarian function; Participate in functions such as early embryo development regulation and control.Therefore ghrelin is applied to sheep embryo vitro culture, can effectively promote embryo's ectogenesis, has solved the low problem of present sheep body early embryo ectogenesis rate.Its Financial cost is lower, is convenient to apply aborning.
(4) embodiment
Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: preparation contains the sheep embryo in-vitro culture solution of ghrelin
Accurately take by weighing ghrelin (Sigma company) 60 mg; TCM199 nutrient solution with 20 mL dissolves earlier; The penicillium mould that in nutrient solution, adds 15mg Stimulina, 800 mg bovine serum albumins and 10000 IU then, the Streptomycin sulphate of 10000 μ g, gentle agitation is even; Add the TCM199 nutrient solution again to 100mL, once more stirring and evenly mixing.After the microfiltration membrane suction filtration sterilization with 0.22 μ M, store down at 4 ℃, subsequent use.
Embodiment 2: the nutrient solution that contains ghrelin is used for goat embryo vitro culture
The goat ovocyte is ripe the cultivation in the Streptomycin sulphate of the penicillium mould of the LH of the FSH that contains 10 μ g/mL, 10 μ g/mL, 100IU/mL, 100 μ g/mL and TCM199 nutrient solution that volumn concentration is 10% serum, carries out in vitro fertilization then.The body early embryo of after fertilization continues to cultivate 7 days respectively in the sheep embryo in-vitro culture solution that contains ghrelin of TCM199 nutrient solution (control group) that does not contain ghrelin and embodiment 1 preparation.Cultivate the 2nd day, the body early embryo division rate in the control group was 64.9%, and the body early embryo division rate in the sheep embryo in-vitro culture solution that contains ghrelin of embodiment 1 preparation is 78.3%, improves 13.4% than control group.Cultivate the 7th day, body early embryo developmental rate blastula stage in the control group was 24.0%, and body early embryo developmental rate blastula stage in the sheep embryo in-vitro culture solution that contains ghrelin of embodiment 1 preparation is 33.7%, improves 9.7% than control group.The result confirms that the sheep embryo in-vitro culture solution vitro culture goat embryo who contains ghrelin can effectively improve the early embryonic development rate of after fertilization.
Embodiment 3: the nutrient solution that contains ghrelin is used for the sheep embryo vitro culture
Sheep oocyte is ripe the cultivation in the Streptomycin sulphate of the penicillium mould of the LH of the FSH that contains 10 μ g/mL, 10 μ g/mL, 100IU/mL, 100 μ g/mL and TCM199 nutrient solution that volumn concentration is 10% serum, carries out in vitro fertilization then.The body early embryo of after fertilization continues to cultivate 7 days respectively in the sheep embryo in-vitro culture solution that contains ghrelin of TCM199 nutrient solution (control group) that does not contain ghrelin and embodiment 1 preparation.Cultivate the 2nd day, the body early embryo division rate in the control group was 62.7%, and the body early embryo division rate in the sheep embryo in-vitro culture solution that contains ghrelin of embodiment 1 preparation is 76.4%, improves 13.7% than control group.Cultivate the 7th day, body early embryo developmental rate blastula stage in the control group was 25.3%, and body early embryo developmental rate blastula stage in the sheep embryo in-vitro culture solution that contains ghrelin of embodiment 1 preparation is 35.4%, improves 10.1% than control group.The result confirms that the sheep embryo in-vitro culture solution vitro culture sheep embryo that contains ghrelin can effectively improve the early embryonic development rate of after fertilization.
Claims (7)
1. sheep embryo in-vitro culture solution that contains ghrelin; It is characterized in that said sheep embryo in-vitro culture solution is a matrix with the TCM199 nutrient solution, also contain the ghrelin of 400～800ng/mL, the Stimulina of 0.15mg/mL, the penicillium mould of 100IU/mL, the Streptomycin sulphate of 100 μ g/mL and the bovine serum albumin of 5～10mg/mL in the said matrix.
2. sheep embryo in-vitro culture solution as claimed in claim 1; It is characterized in that the every 1000mL of said sheep embryo in-vitro culture solution forms as follows: ghrelin 400～800mg; Stimulina 150mg, penicillium mould 100000IU, Streptomycin sulphate 100000 μ g; Serum albumin 5～10g, the TCM199 nutrient solution complements to 1000mL.
3. according to claim 1 or claim 2 sheep embryo in-vitro culture solution is characterized in that in the said sheep embryo in-vitro culture solution that ghrelin content is 500～600ng/mL, and bovine serum albumin content is 8mg/mL.
4. use sheep embryo in-vitro culture solution according to claim 1 or claim 2 carries out vitro culture to the sheep embryo method; Said method is after the early stage sheep embryo with after fertilization washes with the TCM199 nutrient solution repeatedly; Put into the sheep embryo in-vitro culture solution of the said ghrelin of containing, in 38～39 ℃, 5% CO
2Environment in, carry out embryo's vitro culture.
5. method as claimed in claim 4, it is characterized in that said early stage sheep embryo by the sheep ovocyte through maturation in vitro cultivate, acquisition in vitro fertilization.
6. method as claimed in claim 5 is characterized in that said sheep in-vitro maturation culture method for oocyte is following: the sheep ovocyte that from the ovary that the slaughterhouse obtains, extracts with syringe, with maturation culture solution at 38～39 ℃, 5% CO
2Environment in cultivated 20～24 hours, obtain sophisticated sheep ovocyte; Said maturation culture solution is a matrix with conventional nutrient solution; Also containing the FSH of 10 μ g/mL, the LH of 10 μ g/mL, the penicillium mould of 100IU/mL, Streptomycin sulphate and the volumn concentration of 100 μ g/mL in the said matrix is 8%～20% serum, and said conventional nutrient solution is one or more the mixture in TCM199, DMEM, F12, the MEM nutrient solution; Described serum is a kind of in calf serum, foetal calf serum or the NBCS.
7. method as claimed in claim 5 is characterized in that said sheep Oocyte in Vitro fertilization method is following: sophisticated sheep ovocyte and sperm are together put into the fertilization nutrient solution, at 38～39 ℃, 5% CO
2Environment in cultivated 18～24 hours, be fertilized, obtain early stage sheep embryo; Said fertilization nutrient solution is the TCM199 nutrient solution that contains 2% sheep blood serum.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628707A (en) * | 2019-11-01 | 2019-12-31 | 浙江大学 | Culture method for improving in vitro survival rate of mammalian embryo |
| CN110747160A (en) * | 2019-11-27 | 2020-02-04 | 浙江大学 | High-survival-rate sheep fertilized egg culture method for extra-embryonic-body culture |
| CN110777111A (en) * | 2019-10-28 | 2020-02-11 | 浙江大学 | Hu sheep embryo in-vitro culture solution containing ginsenoside |
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| CN1634985A (en) * | 2003-12-26 | 2005-07-06 | 李宁 | Pig Ghrelin derivative, its encoding gene and application |
| WO2010061030A1 (en) * | 2008-11-25 | 2010-06-03 | Universidade De Santiago De Compostela | Isolation of multipotent hypophysary cells and in vitro differentiation thereof |
| WO2010088735A1 (en) * | 2009-02-05 | 2010-08-12 | Regenertech Pty Ltd | Method of producing progenitor cells from differentiated cells |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1634985A (en) * | 2003-12-26 | 2005-07-06 | 李宁 | Pig Ghrelin derivative, its encoding gene and application |
| WO2010061030A1 (en) * | 2008-11-25 | 2010-06-03 | Universidade De Santiago De Compostela | Isolation of multipotent hypophysary cells and in vitro differentiation thereof |
| WO2010088735A1 (en) * | 2009-02-05 | 2010-08-12 | Regenertech Pty Ltd | Method of producing progenitor cells from differentiated cells |
Non-Patent Citations (1)
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| 谢体三等: "Ghrelin对水牛体外受精和孤雌激活胚胎体外发育的影响", 《中国兽医学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110777111A (en) * | 2019-10-28 | 2020-02-11 | 浙江大学 | Hu sheep embryo in-vitro culture solution containing ginsenoside |
| CN110628707A (en) * | 2019-11-01 | 2019-12-31 | 浙江大学 | Culture method for improving in vitro survival rate of mammalian embryo |
| CN111534480A (en) * | 2019-11-01 | 2020-08-14 | 浙江大学 | In-vitro culture method of mammalian embryo |
| CN111534480B (en) * | 2019-11-01 | 2021-12-21 | 广州华珍生物科技有限公司 | In-vitro culture method of mammalian embryo |
| CN110747160A (en) * | 2019-11-27 | 2020-02-04 | 浙江大学 | High-survival-rate sheep fertilized egg culture method for extra-embryonic-body culture |
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