CN105695398A - O-acetyl-L-carnitine hydrochloride containing buffalo oocyte in-vitro maturation liquid and culture method - Google Patents
O-acetyl-L-carnitine hydrochloride containing buffalo oocyte in-vitro maturation liquid and culture method Download PDFInfo
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- CN105695398A CN105695398A CN201610259386.7A CN201610259386A CN105695398A CN 105695398 A CN105695398 A CN 105695398A CN 201610259386 A CN201610259386 A CN 201610259386A CN 105695398 A CN105695398 A CN 105695398A
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Abstract
The invention discloses O-acetyl-L-carnitine hydrochloride containing buffalo oocyte in-vitro maturation liquid. A corresponding culture method is further established by an inventor accordingly. Experiments indicate that O-acetyl-L-carnitine hydrochloride is applied to buffalo oocyte in-vitro maturation, compared with buffalo oocyte in-vitro maturation liquid to which no O-acetyl-L-carnitine hydrochloride is added, buffalo ovum in-vitro maturation quality and the development capacity of subsequent embryos can be remarkably improved, O-acetyl-L-carnitine hydrochloride is low in cost, and the O-acetyl-L-carnitine hydrochloride containing buffalo oocyte in-vitro maturation liquid can be applied and popularized in buffalo buffalo in-vitro production conveniently.
Description
Technical field
The invention belongs to oocyte IVM technical field, particularly relate to a kind of buffalo oocytes maturation in vitro liquid containing O-acetyl-l-carnitine hydrochlorate and cultural method。
Background technology
Development along with embryo biotechnology, the ovum in source in opposite bank, utilizes in vitro ovary to obtain immature ovum, through In-vitro maturation, substantial amounts of mature egg can be obtained, carry out research and the production application of subsequent in vitro fertilization, somatic cell clone, embryonic stem cell etc.。And currently buffalo oocytes maturation in vitro is inefficient, limit the bionic research of buffalo embryo and application。
Maturation of ovum and subsequent embryo grow that final embryo is attached to plant successfully, and this process needs significantly high energy。In vitro culture liquid adds carbohydrate ovum full maturity and fetal development are played very crucial effect, but recent studies have shown that the beta oxidation of fatty acid is another very important energy source in maturation of ovum and subsequent embryo are grown。On power supply characteristic, ATP produced by fatty acid metabolism is several times of carbohydrate, and ATP produced by carbohydrate is utilized limited by ovum and embryo。Although the ovum fat content of various animals is not quite similar, but research shows, even if in Mouse Eggs maturation in vitro, by adding lipometabolic inhibitor, Mouse Eggs can being suppressed ripe and subsequent embryo is grown, result prompting lipid metabolism plays vital effect on oocyte and subsequent embryo developmental capacity。
Fatty acid is mainly composed of triglyceride, with drop-wise by fat drop around form be present in Cytoplasm, resolve into the fatty acid of different length and different saturation through lipase, then fatty acid enter mitochondrion, carry out beta oxidation。
The fatty acid carrier of unique activation on VBT mitochondrial membrane, its major function is to carry, transport the fatty acid of activation, enters and carries out beta oxidation and tricarboxylic acid cycle reaction in mitochondrion, and the various metabolic activities for body provide energy。Follicle adds VBT before the chamber of mice and pig and can improve the subsequent development ability of ovum, but also studies have reported that, maturation of ovum adds VBT, improves the division rate of embryo, but the formation of follow-up blastaea is not had significant impact。
The VBT of acetyl group is the derivant of VBT, when in case of need, ALC naturally can be converted into VBT。Having been reported that and show that ALC has more antioxidative ability than VBT, therefore ALC is likely to there is more effective bioavailability than VBT。
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of buffalo oocytes maturation in vitro liquid containing O-acetyl-l-carnitine hydrochlorate and cultural method, to improve the follow-up developmental capacity of Babalus bubalis L. maturation in vitro ovum and embryo quality。
For solving above-mentioned technical problem, the present invention by the following technical solutions:
Buffalo oocytes maturation in vitro liquid containing O-acetyl-l-carnitine hydrochlorate。
The concentration of O-acetyl-l-carnitine hydrochlorate is 2.5-5.0mM。
Consisting of of this buffalo oocytes maturation in vitro liquid: solute O-acetyl-l-carnitine hydrochlorate is 2.5mM, and solvent is containing NaHCO326.2mM, Hepes5mM, 10%ECS, 3%BFF, 0.5 μ g/mlFSH, 5 μ g/mlLH, the TCM-199 culture fluid of 10 μ g/mlCysteine。
The above-mentioned buffalo oocytes maturation in vitro liquid containing O-acetyl-l-carnitine hydrochlorate is used to carry out the cultural method of buffalo oocytes maturation in vitro。
Above-mentioned cultural method; undertaken by following operation: pick out the oocyte with intact cumuli oophori cell envelope, first wash 2 times with egg-cleaning liquid, then wash 2 times with ripe liquid; it is then placed in the buffalo oocytes maturation in vitro liquid containing O-acetyl-l-carnitine hydrochlorate, in the CO of 38.5 DEG C, 5%2Environment in carry out Invitro maturation 22-24h。
For the current inefficient problem of Babalus bubalis L. Oocytes Maturation, inventor have developed a kind of buffalo oocytes maturation in vitro liquid containing O-acetyl-l-carnitine hydrochlorate。Accordingly, the corresponding cultural method that inventor also sets up。Test shows; O-acetyl-l-carnitine hydrochlorate is applied to buffalo oocytes maturation in vitro; compare the Babalus bubalis L. Oocytes Maturation liquid not adding O-acetyl-l-carnitine hydrochlorate; the developmental capacity of Babalus bubalis L. maturation in vitro Oocytes Maturation quality and subsequent embryo can be significantly improved; and O-acetyl-l-carnitine hydrochlorate cost is low, it is simple to popularization and application in buffalo embryo produced in vitro。
Up to the present, can O-acetyl-l-carnitine hydrochlorate be used for buffalo embryo and improve buffalo oocytes maturation in vitro quality and have not been reported。Feature and the function of O-acetyl-l-carnitine hydrochlorate is closed in inventor's tying, obtains the present invention through further investigation。Wherein, the main Physiological Function of L-carnitine is: 1. transhipment fatty acid enters mitochondrion and carries out B-oxidation at this, to promote that fat combustion provides energy, so having blood fat reducing, improving the effect such as Human Stamina and fat-reducing。2. cell is protected: excessive acyl coenzyme A got rid of and external prevent it from damaging cell。3. lactic acid accumulation is prevented。Producing a large amount of lactic acid after vigorous exercise, L-carnitine can reduce lactic acid growing amount, improves exercise tolerance。Meanwhile, it also has matter energies such as promoting carbohydrate to utilize, and improves the effect such as sperm number and vigor。Except having the function identical with L-carnitine, acetyl-L-carnitine also has following function: neuroprotective and neural promotion functions, improves immunologic function。
Detailed description of the invention
Embodiment 1
The preparation buffalo oocytes maturation in vitro liquid containing O-acetyl-l-carnitine hydrochlorate: first prepare the storage solutions of 50mMO-acetyl-l-carnitine hydrochlorate; with the EP pipe subpackage of 200 μ l; each 110 μ l are (with without 10%ECS; 3%BFF; 0.5 μ g/mlFSH; 5 μ g/mlLH, 10 these somatomedin of μ g/mlCysteine but containing NaHCO326.2mM, the TCM-199 solution dilution of Hepes5mM), the used time dilutes 20 times, namely takes storing in the buffalo oocytes maturation in vitro working solution that liquid joins 950 μ l of 50 μ l。
Concrete maturation in vitro formula of liquid is as follows
The formula of the table 1 buffalo oocytes maturation in vitro liquid containing O-acetyl-l-carnitine hydrochlorate
Cultural method: extract the follicle of 2-6mm with the 10ml syringe with No. 12 syringe needles from vitro buffalo ovaries, pick out the oocyte with intact cumuli oophori cell envelope under common Stereo microscope, first washs 2 times with egg-cleaning liquid, then washs 2 times with ripe liquid；Buffalo oocytes In-vitro maturation liquid is placed in the glass culture dish of 25mm × 10mm, puts into 1ml maturation liquid, uncovered mineral oil, put ovum 70-80, in the CO of 38.5 DEG C, 5%2Environment in carry out Invitro maturation 22-24h。
Obtain the oocyte of Babalus bubalis L. maturation in vitro according to aforementioned cultural method, select the assessment that Cytoplasm is uniform, discharge first housing oocyte carries out ovum subsequent development potential。The Ionomycin of 5 μm processes 5min, 4h is acted on again in the 6-DMAP of 2.5mM, put into and cultivate containing in Babalus bubalis L. granular cell monolayer co-culture system, 20 μ l every, put into 15-20h embryo for every to cultivate, every 48h half amount changes liquid, and after lonely female activation, 2d observes embryo's division rate, and 7d observes Blastocyst formation rate。
Result shows, adds 2.5mMO-acetyl-l-carnitine hydrochlorate in buffalo oocytes maturation in vitro liquid, and after chemokinesis, the division rate of 2d 79.31% is significantly higher than without group 64.38%, improves 14.93% than matched group。The Blastocyst formation rate of 7d is 26.45%, is significantly higher than matched group 13.70%, improves 12.75% (referring to table 2) than matched group
Table 2
| Group | Embryo number | Division number (%) | Blastaea number (%) |
| 0 | 73 | 47(64.38)b | 10(13.70)b |
| 2.5mM | 87 | 69(79.31)a | 23(26.45)a |
| 5mM | 68 | 42(61.76)b | 7(10.29)b |
Note: with column of figure difference letter representation significant difference (P < 0.05)。
As a result, it was confirmed that after the outer mature egg of buffalo oocytes maturation in vitro liquid adding the O-acetyl-l-carnitine hydrochlorate of 2.5mM, buffalo oocytes subsequent embryo developmental rate can be improved。
Embodiment 2
The qualification of concrete buffalo oocytes maturation in vitro and subsequent embryo developmental capacity is with embodiment 1。
The blastaea formed of 7d after lonely female activation is carried out the total counting number of inner cell mass, first blastaea is put into lucifuge dyeing 5min in the Hoechst33342 of 10mg/ml, place into and PBS washes 2～3 times, then carry out tabletting。Under common fluorescence microscope, the visual field of 200 times is observed, and the bright spot of visual field exhibits blue fluorescence is counted。
Result shows, the blastaea obtained carries out inner cell mass sum and is counted as 85.64, is significantly higher than matched group 56.40, high 29.24 inner cell mass sums, illustrates that the quality of blastocysts that embodiment 2 obtains is higher than matched group。(referring to table 3) buffalo oocytes maturation in vitro liquid containing O-acetyl-l-carnitine hydrochlorate can improve the inner cell mass sum of Babalus bubalis L. blastaea。
Table 3
| Group | Embryo number | Inner cell mass number |
| 0 | 10 | 56.40b |
| 2.5 | 11 | 85.64a |
| 5 | 6 | 79.17ab |
Note: with column of figure difference letter representation significant difference (P < 0.05)。
Claims (5)
1. the buffalo oocytes maturation in vitro liquid containing O-acetyl-l-carnitine hydrochlorate。
2. the buffalo oocytes maturation in vitro liquid containing O-acetyl-l-carnitine hydrochlorate according to claim 1, it is characterised in that: the concentration of described O-acetyl-l-carnitine hydrochlorate is 2.5-5.0mM。
3. the buffalo oocytes maturation in vitro liquid containing O-acetyl-l-carnitine hydrochlorate according to claim 2, it is characterised in that consisting of of this buffalo oocytes maturation in vitro liquid: solute O-acetyl-l-carnitine hydrochlorate is 2.5mM, and solvent is containing NaHCO326.2mM, Hepes5mM, 10%ECS, 3%BFF, 0.5 μ g/mlFSH, 5 μ g/mlLH, the TCM-199 culture fluid of 10 μ g/mlCysteine。
4. use described in claim 1 the buffalo oocytes maturation in vitro liquid containing O-acetyl-l-carnitine hydrochlorate to carry out the cultural method of buffalo oocytes maturation in vitro。
5. cultural method according to claim 4; it is characterized in that being undertaken by following operation: pick out the oocyte with intact cumuli oophori cell envelope; first wash 2 times with egg-cleaning liquid; wash 2 times with the ripe liquid without O-acetyl-l-carnitine hydrochlorate again; it is then placed in the buffalo oocytes maturation in vitro liquid containing O-acetyl-l-carnitine hydrochlorate, in the CO of 38.5 DEG C, 5%2Environment in carry out Invitro maturation 22-24h。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110547290A (en) * | 2019-08-26 | 2019-12-10 | 南京农业大学 | vitrification freezing method of porcine oocytes |
| KR102158946B1 (en) | 2019-12-27 | 2020-09-23 | 서울대학교산학협력단 | Peroxisome dynamics in oocytes affirmed through phytanic acid treatment |
| CN113755429A (en) * | 2021-09-29 | 2021-12-07 | 中国科学院苏州生物医学工程技术研究所 | Composition for preparing cleavage culture solution, cleavage culture solution and preparation method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110547290A (en) * | 2019-08-26 | 2019-12-10 | 南京农业大学 | vitrification freezing method of porcine oocytes |
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| KR102158946B1 (en) | 2019-12-27 | 2020-09-23 | 서울대학교산학협력단 | Peroxisome dynamics in oocytes affirmed through phytanic acid treatment |
| CN113755429A (en) * | 2021-09-29 | 2021-12-07 | 中国科学院苏州生物医学工程技术研究所 | Composition for preparing cleavage culture solution, cleavage culture solution and preparation method |
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