CN101129333A - Method of preparing nano slow release agent for simplifying animal FSH superovulation - Google Patents
Method of preparing nano slow release agent for simplifying animal FSH superovulation Download PDFInfo
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- CN101129333A CN101129333A CNA2007101319989A CN200710131998A CN101129333A CN 101129333 A CN101129333 A CN 101129333A CN A2007101319989 A CNA2007101319989 A CN A2007101319989A CN 200710131998 A CN200710131998 A CN 200710131998A CN 101129333 A CN101129333 A CN 101129333A
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Abstract
The invention discloses a making method of nanometer slow-release agent to simplify animal FSH superovulation in the embryo engineering technical domain, which comprises the following steps: 1) dissolving 0. 5%-3% fatty acid in the alkaline condition for 8-12h to make vesicle; 2) adjusting the grain size of vesicle and surface load; adding cationic surfactant in the vesicle liquid with weight rate of vesicle liquid and cationic surfactant at 1: 0. 05-0. 1; 3) adopting phosphoric acid buffer to adjust the grain size and surface load into 0. 05%-1. 5% vesicle slow-release agent. The invention simplifies the operation of superovulation without special request for instrument, which has strong repeatability.
Description
Technical field
The invention belongs to the embryo engineering technical field, particularly control the technical field of animal FSH superovulation.
Background technology
Contain 200,000～400,000 oocytes on mammal when birth ovary, but in the whole life process of animal 99% follicular atresia, have only 1% the ripe and ovulation of follicular development.In order to obtain more oocyte or embryo, certain phase in the animal estrus cycle, handle by exogenous hormone, improve promoting sexual gland hormone concentration in the blood, reduce the locking rate of development follicle, increase the quantity of early stage follicular development to mature follicle, stimulate the oocyte that temporarily is in dormant state to enter the ripe period of development, discharge ripe back, can obtain thus than oocyte much more under the spontaneous ovulation situation, and this process just is called superovulation (being called for short super row).
Super row provides ovum and embryonic origin, this technology to be widely used in the livestock reproduction for embryo transfer, clone and transgenic animal production etc., and up to now, super row is a kind of effective ways that obtain neat in a large number, fine ovum or embryo.Premium animal is surpassed row, can make the superior progeny number increase several times even tens times, and then can accelerate the paces of improved seeds expanding propagation and animal breeding.
Super at present row is mainly used in animals such as cattle, sheep, rabbit, mice, and conventional method has two kinds usually: 1. reduction or elimination ovary are primarily aimed at follicle stimulating hormone (FSH) to the negative feedback reaction that promoting sexual gland hormone discharges; 2. increase exogenous hormone (to use FSH or PMSG), improve promoting sexual gland hormone concentration in the blood.Aborning, extensively adopt second method, generally adopt super row's method of FSH or the super row's method of PMSG.The ovary functional recovery was very fast after application FSH surpassed row's processing, and number of eggs ovulated is higher, but FSH half-life weak point (2-4 hour) need carry out the continuous several times injection to donor, relatively be tired of and lock, and workload is big; And surpass row with PMSG, though only need intramuscular injection once, less to the stimulation of donor, easy to operate; But the half-life of PMSG is grown (more than 8 hours), is difficult in vivo being eliminated, and often makes the large follicle of not ovulation residue in ovary and secrete estrogen, and duration of estrus prolongs, influence fertilization and recovery embryo's quality.Sometimes even form follicular cyst, prolong the convalescent period of ovary.When improving number of eggs ovulated by increase PMSG dosage, above-mentioned phenomenon is particularly serious.Have experimental result to show: the super row's method of FSH is obtaining to be significantly higher than the PMSG method on Niu Keyong ovum number and the super row's effect stability, and phenomenons such as the ovarian cyst of cattle and continuous estrus can not appear in the super row of FSH, the super row of FSH effect is better than PMSG, so in the superovulation of big domestic animal, use the most general still FSH.
Therefore, how to bring into play to the full extent under the prerequisite of PMSG, FSH effect, reduce or eliminate exploration that their shortcoming carries out as far as possible and improve and have important practical value.People such as Yamamoto once were dissolved in F SH and used among the PVP, had both improved number of eggs ovulated, had increased transplantation embryo's quantity again.
Summary of the invention
The purpose of this invention is to provide a kind of method of preparing nano slow release agent, adopt prepared nano slow release agent can simplify FSH superovulation method, for production of embryo transfer, clone, transgenic animal etc. provides neat in a large number, fine oocyte or embryo.
The present invention includes following steps:
1) preparation vesicle: be that 0.5%～3% fatty acid dissolves under alkali condition with concentration, after 8～12 hours, promptly make vesicle;
2) adjust vesicle particle diameter and surface charge: add cationic surfactant in vesicle liquid, wherein, the weight ratio of the cationic surfactant of vesicle liquid and interpolation is: 1: 0.05～0.1;
3) preparation vesicle slow releasing agent: adopting phosphate buffer that the vesicle of particle diameter and surface charge adjustment is diluted to concentration is 0.05%～1.5% vesicle slow releasing agent.
The slow-releasing system that the present invention makes is vesicle (vesicle), and vesicle is a kind of amphiphile, amphiphilic molecule assembly, and it is made up of two-layer amphiphile, amphiphilic molecule, hydrophilic base of outer amphiphile, amphiphilic molecule outwardly, hydrophilic base of internal layer amphiphile, amphiphilic molecule is then towards vesicle inside.Vesicle is structurally similar to cell membrane, and chemical property is stable, inexpensive.Recent research shows, can make the release of medicine have targeting, long-lasting and slow-releasing with vesicle as pharmaceutical carrier.
It is simple, convenient to adopt the made slow-releasing system of the present invention to carry out the operating process of superovulation, need not special instrument requirement, only needs to grasp the operation of general superovulation, and repeatable strong.
Nanoscale slow-releasing system-the vesicle of the present invention's preparation is as the FSH carrier, in the disposable injection donor of the FSH total amount body with the injection of need continuous several times, it is slowly discharged in vivo, to reach identical super row's effect with continuous several times injection FSH, and then simplified the super scheduling preface of FSH, reached purpose such as save time, laborsaving.
Preparation is during vesicle, and fatty acid of the present invention can be in palmitic acid or oleic acid or the myristic acid any one.
During the preparation vesicle, the pH value of described alkali condition is 7～9.
Described cationic surfactant is any one in cetyl trimethyl ammonium bromide or Dodecyl trimethyl ammonium chloride or the dodecyl triethyl group ammonium bromide.
The specific embodiment
This enforcement is example with the mice, the concrete operations step:
1, reagent is prepared:
Follicle stimulating hormone (FSH) and lutropin (LH) are all available from Ningbo City, Zhejiang Province hormone two factories; Self-control vesicle liquid; Du Shi phosphate buffer (PBS); 0.9% normal saline
2, equipment:
The 1ml syringe; Cotton balls; Dactylethrae
3, operating procedure
3.1 the selection of female Mus
Selected for 6～7 ages in week, the cleaning level Kunming white mice of body weight 25g～30g.It is raised under the control environment of 5 ℃ in ambient temperature 25 soil.
3.2 superovulation
3.2.1 synthesis nano slow-releasing system:
Before mice is carried out superovulation, prepare vesicle liquid earlier:
At first be that 0.5%～3% fatty acid (palmitic acid or oleic acid or myristic acid) is 7～9 times dissolvings and vibration at pH value, after 8～12 hours, promptly make vesicle liquid concentration.
In vesicle liquid, add cationic surfactant (cetyl trimethyl ammonium bromide or Dodecyl trimethyl ammonium chloride or dodecyl triethyl group ammonium bromide), with regulation and control vesicle particle diameter and surface charge.Wherein, the weight ratio of the cationic surfactant of vesicle liquid and interpolation is: 1: 0.05～0.1.
Adopt phosphate buffer (PBS) by 2～10 multiples dilution vesicle, promptly make concentration and be 0.05%～1.5% vesicle slow releasing agent.
With the nano slow release agent for preparing follicule-stimulating hormone (FSH) (FSH) is diluted to 10IU/ml, and at room temperature leaves standstill after 1 hour and re-use, lutropin (LH) gets final product with 0.9% physiological saline solution.
3.2.2 operation:
It is the nano slow release agent of 10IU/ml that first day 20:00 gives injected in mice follicule-stimulating hormone (FSH) (FSH) concentration, and injected in mice dosage is 1ml/.
The 3rd day 20:00 LH injection diluent, injected dose 1ml/ are only.
The characteristics of the method, promptly FSH is disposable injection, has simplified injecting program.
And in the super scheduling preface of routine, need to give mice 4～6 FSH of injection (each 12 hours at interval, the total injection units of FSH is 10IU) continuously, operate loaded down with trivial details (seeing Table 1).
Through experiment confirm: adopt the super row of vesicle group and routine group effect to compare there was no significant difference, illustrate that slow releasing agent-vesicle liquid (being the nanoscale slow-releasing system) that the inventor adopts has had effect to the super row of mice, slow releasing agent has played the effect (seeing Table 2) of slow release to FSH.
The comparison of the different super scheduling prefaces of table 1
| Time | The 1st day 20:00 FSH | The 2nd day 8:00 FSH | The 2nd day 20:00 FSH | The 3rd day 8:00 FSH | The 3rd day 20:00 LH |
| The conventional group of vesicle group 10IUFSH+10IULH 10IUFSH+10IULH | 10IU 4IU | - 3IU | - 2IU | - 1IU | 10IU 10IU |
The effect of the different super scheduling prefaces of table 2 relatively
| The experiment group | Average uterus weight in wet base (g) | On average pick up ovum number (individual) |
| Vesicle group 10IU FSH+10IU LH matched group 10IU FSH+10IU LH | 0.1811 0.2050 | 12 13 |
Claims (4)
1. the method for preparing nano slow release agent of simplifying animal FSH superovulation is characterized in that may further comprise the steps:
1) preparation vesicle: be that 0.5%～3% fatty acid dissolves under alkali condition with concentration, after 8～12 hours, promptly make vesicle liquid;
2) adjust vesicle particle diameter and surface charge: add cationic surfactant in vesicle liquid, wherein, the weight ratio of the cationic surfactant of vesicle liquid and interpolation is: 1: 0.05～0.1;
3) preparation slow releasing agent: adopting phosphate buffer that vesicle is diluted to concentration is 0.05%～1.5% vesicle slow releasing agent.
2. according to the method for preparing nano slow release agent of the described simplifying animal FSH superovulation of claim 1, when it is characterized in that preparing vesicle, described fatty acid is any one in palmitic acid or oleic acid or the myristic acid.
3. according to the method for preparing nano slow release agent of the described simplifying animal FSH superovulation of claim 1, when it is characterized in that preparing vesicle, the pH value of described alkali condition is 7～9.
4. according to the method for preparing nano slow release agent of the described simplifying animal FSH superovulation of claim 1, it is characterized in that described cationic surfactant is any one in cetyl trimethyl ammonium bromide or Dodecyl trimethyl ammonium chloride or the dodecyl triethyl group ammonium bromide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101319989A CN101129333A (en) | 2007-09-18 | 2007-09-18 | Method of preparing nano slow release agent for simplifying animal FSH superovulation |
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| CNA2007101319989A CN101129333A (en) | 2007-09-18 | 2007-09-18 | Method of preparing nano slow release agent for simplifying animal FSH superovulation |
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| CNA2007101319989A Pending CN101129333A (en) | 2007-09-18 | 2007-09-18 | Method of preparing nano slow release agent for simplifying animal FSH superovulation |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107456295A (en) * | 2017-09-06 | 2017-12-12 | 黑龙江省农业科学院畜牧研究所 | A kind of new and effective sustained release Superovulation of sheep |
| CN111616830A (en) * | 2020-07-03 | 2020-09-04 | 宁夏大学 | Novel ICR mouse superovulation method |
| US11376220B2 (en) | 2017-06-30 | 2022-07-05 | Therio, LLC | Single-injection methods and formulations to induce and control multiple ovarian follicles in bovine, caprine, ovine, camelid and other female animals |
-
2007
- 2007-09-18 CN CNA2007101319989A patent/CN101129333A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11376220B2 (en) | 2017-06-30 | 2022-07-05 | Therio, LLC | Single-injection methods and formulations to induce and control multiple ovarian follicles in bovine, caprine, ovine, camelid and other female animals |
| US11964053B2 (en) | 2017-06-30 | 2024-04-23 | Therio, LLC | Single-injection methods and formulations to induce and control multiple ovarian follicles in bovine, caprine, ovine, camelid and other female animals |
| CN107456295A (en) * | 2017-09-06 | 2017-12-12 | 黑龙江省农业科学院畜牧研究所 | A kind of new and effective sustained release Superovulation of sheep |
| CN111616830A (en) * | 2020-07-03 | 2020-09-04 | 宁夏大学 | Novel ICR mouse superovulation method |
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Open date: 20080227 |