CN101341863B - Injection method for single-bird egg - Google Patents
Injection method for single-bird egg Download PDFInfo
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- CN101341863B CN101341863B CN 200810013123 CN200810013123A CN101341863B CN 101341863 B CN101341863 B CN 101341863B CN 200810013123 CN200810013123 CN 200810013123 CN 200810013123 A CN200810013123 A CN 200810013123A CN 101341863 B CN101341863 B CN 101341863B
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Abstract
The invention relates to a method for injecting to a single bird egg; the operation steps are as follows: the bird egg is positioned at a position with preset direction under incubation temperature; an opening is selected by a position on the egg shell which is to be injected to be disinfected by alcohol; a disinfected solid pin is used to form small openings in the same size on the opening of the egg shell and the outer shell membrane and the inner shell membrane below the opening; the bird egg is slightly rotated, so that the position in the egg to be injected aims to the small openings on the egg shell; a disinfected disposable injection needle is extended into the small openings; materials are injected into the egg by the disposable injection needle; the temperature of the egg is reduced in the environment 1 DEG C to 10 DEG C lower than the incubation temperature; the disposable injection needle is collected; the bird egg rotates for some angles within a longitudinal section, so that the crossing of the injection needle stays from the opening of the egg shell; melted paraffin is used to seal the opening; the paraffin is solidified under the incubation temperature; the bird eggis put to the original place to be incubated. The method has the advantages of low cost, fast operation, single-egg rejection, small simulation and damage, no pollution, fixed point and quantitative injection, and direct view.
Description
Technical field
The present invention relates to a kind of method, particularly relate to a kind of method, be particularly suitable for each phase injection of embryo of the small-sized bird ovum of altricial young bird under the nature hatching state to single bird ovum fixed point, calibrated shot to the injection of bird ovum.
Background technology
Birds are oviparous animal, parent is left in its embryonic development, helps using modern biology technology such as transgenic technology, embryonic stem cell technology, technology of artificial insemination, animal cloning technology and labelled with radioisotope tracer technique that its ontogeny is studied.
Compare with relatively large ovum such as chicken, duck, geese, it is thin that small-sized bird ovum such as lonchura striata, parrot, canary have a chorion, easy opening, characteristics such as the content volume is little.Injection success rate is subject to the influence of factors such as temperature, injected material volume, mechanical damage, want effectively to finish fixed point, calibrated shot under the prerequisite that guarantees higher hatching rate and survival rate, the method for fixed point, calibrated shot material in the bird ovum that a cover is suitable is particularly crucial.
Traditional single ovum injecting method uses medical injector or microsyringe usually, injects under the sidelight irradiation.This method is the injection position inaccuracy not only, and injection depth is wayward, and is big to the damage of bird ovum, and after the injection, pin internal memory dose is big, the waste injection mass.In addition, entry needle is recycled usually, causes cross-infection easily, reduces hatching rate, the survival rate of bird ovum.Another weak point of conventional method is, fails to solve well middle-size and small-size bird ovum fixed point, calibrated shot problem, brings very big inconvenience to correlative study.Chinese patent 02105130.5 and 99815436.9 method relatively are fit to the industrial mass injection of the big-and-middle-sized precocity nestling ovum (as chicken, duck etc.) of artificial incubation, apparatus expensive not only, and can't stop cross-infection problem in the injection process; The altricial nestling ovum in position and hatching process of bird ovum is hatched the problem towards position consistency naturally in the time of also can't solving simultaneously injection.(because of the two must be in advance be adjusted to the blunt end air chamber upwards with the bird ovum towards the position, utilize entry needle to inject then through the air chamber of bird ovum blunt end) in addition, at small-sized bird ovum, as small-sized bird ovum such as lonchura striata ovum, spun gold bird ovum, parrot bird ovum, can increase because of the content volume in the ovum behind the material that is injected into certain volume, and the phenomenon that content (comprising injection mass) overflows from the chorion upper shed appears, be difficult for finishing calibrated shot, also be difficult for sealing, also cause environmental pollution easily for the isotope-labeled material of injection.If under the opening part chorion important structure is arranged, easily causes the damage of this important structure during opening.The above problem, other relevant patents are all unresolved.
Summary of the invention
The objective of the invention is to,, provide cost lower for addressing the above problem, pollution-free, a kind of method that can improve hatching rate and survival rate to the injection of bird ovum.
The present invention relates to a kind of method to single bird ovum fixed point, calibrated shot, its operating procedure is as follows:
1, the bird ovum is positioned under incubation temperature predetermined towards the position;
2, the other 1-2mm of desire injection position selects aperture position in the place and disinfects in alcohol on chorion;
3, with the solid needle of sterilization the aperture position on the chorion with and following periostracum, inner shell membrane on make an equal big or small little opening simultaneously, diameter is less than 2mm.
4, rotating the bird ovum gently makes the interior desire of ovum injection position aim at the little opening on the chorion;
5, stretch into an injection needles of having sterilized by little opening;
6, by injection needles injection mass in ovum;
7, ovum was lower than under the incubation temperature 1-10 ℃ environment cooling less than 10 minutes;
8, regain injection needles;
9, make the bird ovum in vertical section, rotate 5-45 °, make the entry needle road junction depart from opening on the chorion;
10, with the paraffin sealing opening that melts;
11, treat that under incubation temperature paraffin solidifies;
12, the bird ovum is replaced hatching.
The invention has the advantages that:
1. cost is lower
This method is with traditional physiology instrument---and glass microelectrode connects during use on liquid-transfering gun as injection tool, and is therefore, with low cost.
2. swift to operate
Under the nature hatching state, the major axis of bird ovum generally is horizontal level, and the blunt end air chamber is positioned at the side.Must not pass through air chamber during this method injection, ovum under position and natural hatching state towards position consistency, the ovum that takes out from Bird's Nest directly can be injected according to this method, does not need other to adjust its blunt end position by spended time again, thereby has fundamentally saved the time.Simultaneously, avoided the influence of the change of environment in the long-time put procedure, be more suitable for ovum, especially be fit to small-sized altricial nestling ovum by close bird hatching by close bird hatching to embryonic development and hatching rate and survival rate.
3. single ovum injection
Bird ovum embryo in embryo development procedure is changed the relative motion state into by relative static conditions gradually.The embryo in mid-term becomes irregular rotation or swimming (naked eyes are high-visible under transmitted light) usually in ovum.Single ovum injection can be at different concrete condition jet injections, and specific aim is stronger.Be more suitable for ovum by the hatching of close bird, and by the small-sized altricial nestling ovum of close bird hatching, the injection of especially rare bird ovum.
4. stimulate little, the damage little
One of bird ovum hatching rate after the influence injection and principal element of survival rate are exactly the degree of injury of injecting the ovum that causes.Big stimulation or damage often reduce hatching rate and survival rate significantly.The entry needle that this method is used is glass microelectrode, and needle tip is thin, and diameter is lower than 100 microns, even is lower than 20 microns, the mechanical damage when having reduced inserting needle and injection; Opening required on eggshell is little, not only is convenient to sealing, and also little to the mechanical damage of ovum; Paraffin sealing is solid, is difficult for being fallen to cause the accidental mechanical damage by close unfirmly closing.In addition, before sealing, the bird ovum is turned an angle, so that the important biomolecule structure, leave opening as blood vessel, blastodisc etc., and the non-important structure that makes a tool trophism such as egg white then with the sealing of sealers such as paraffin, has been avoided stimulation and the damage of sealer to important structure in the face of opening.In addition, relatively more responsive to variations in temperature because the bird ovum is little, shell is thin, the most of processing of this method carried out under incubation temperature, reduced stimulation and the influence of variations in temperature to ovum.These can reduce stimulation and mechanical damage to ovum on the whole, thereby have improved hatching rate and survival rate.
5. pollution-free
This method uses injection needles as injection tool, can avoid cross-infection; The opening of traditional method on eggshell is big, use marine glue and sealer to seal usually, and this method opening is very little, as joint filling material, nontoxic, free from extraneous odour seals firmly with paraffin, isolation is good, is difficult for that nature comes off or is fallen by close unfirmly closing, has avoided pollutant to enter in the ovum.Therefore, this method can obtain higher hatching rate and survival rate (seeing Table).In addition, handle injection back cooling in advance, has avoided content and injection mass to overflow from opening, thereby avoided the pollution to the ovum external environment.
The fixed point, calibrated shot
Want to carry out quantitative analysis in later stage research, fixed point, calibrated shot are particularly crucial.When utilizing this law injection, entry needle need not pass through air chamber, and entry needle has scale.Therefore, the degree of depth that entry needle enters in the ovum is controlled easily, can realize accurate fixed-point injection.The glass microelectrode that this method is used links to each other with liquid-transfering gun, can realize quantitative sampling, calibrated shot.This law must be to utilize the principle of expanding with heat and contract with cold to reduce the temperature of object in the ovum to the processing of lowering the temperature of bird ovum after finishing injecting step, the interior material volume of ovum is reduced, thereby avoid the interior material of ovum to overflow, also can make the drop Hui Na that overflows, thereby guarantee calibrated shot.
7. directly perceived
Traditional bird ovum injection is used metal needle injection usually, only depends on naked eyes to be difficult to observe and deposits dose and bubble in the syringe needle.And this law uses glass material as entry needle, and liquid level and bubble naked eyes in the entry needle are high-visible, directly perceived, convenient.
Embodiment
The present invention relates to a kind of method to single bird ovum fixed point, calibrated shot, its operating procedure is as follows:
A, the bird ovum is positioned under incubation temperature predetermined towards the position.Be about to the light source hole top that the bird ovum is positioned over desk-top candler, and make its with hatch naturally towards position consistency;
B, on chorion, desire the other 1-2mm of injection position place selection aperture position and disinfect in alcohol;
C, with the solid needle of sterilization the aperture position on the chorion with and following periostracum, inner shell membrane on make an equal big or small little opening simultaneously, diameter is less than 2mm;
D, rotate the bird ovum gently and make and desire injection position in the ovum and aim at the little opening on the chorion;
E, stretch into an injection needles of having sterilized by little opening;
F, by injection needles injection mass in ovum;
G, ovum was lower than under the incubation temperature 1-10 ℃ environment cooling less than 10 minutes.The control of this temperature and time can be decided according to the situations such as size of bird ovum.Onesize bird ovum is not advisable so that its injection back interior material of ovum does not overflow little opening.Cooling extent is big more, and the required time is short more; Cooling extent is more little, and the required time is long more;
H, withdrawal injection needles;
I, make the bird ovum in vertical section, rotate 5-45 °, make the entry needle road junction depart from opening on the chorion, and opening part does not have important structure;
The paraffin sealing opening that j, usefulness are melted;
K, treat that under incubation temperature paraffin solidifies;
L, with the hatching of replacing of bird ovum.
Described incubation temperature is meant with the bird ovum hatches corresponding to temperature range naturally.
The predetermined of described bird ovum comprises towards the position and the natural artificial incubation state corresponding to position towards the position under position and natural laying state under position or close naturally bird hatching state down, and preferably scheme is that the major axis that the blunt end air chamber of bird ovum is positioned at side or bird ovum is horizontal.During injection, entry needle is injected without air chamber.
Under the nature hatching state, various altricial nestling ovum such as lonchura striata ovum, canary ovum, Passeris montani saturati ovum, dove ovum, and the orientation great majority of precocies ovum such as the chicken in hatching naturally, duck, goose, quail are in level for major axis or the blunt end air chamber is positioned at the side.Therefore the present invention is particularly suitable for the injection of the bird ovum in the nature hatching, is particularly suitable for the injection of small-sized altricial nestling ovum (as lonchura striata ovum, canary ovum, Passeris montani saturati ovum, various dove ovum etc.) and the injection of rare bird ovum.
Before the injection, the bird ovum is positioned over the loophole place of desk-top candler, and the corresponding to position towards the position that is in and hatches naturally, make transmitted light from the irradiation of the below of bird ovum.The bird ovum is placed steadily like this, is difficult for rolling, and interior is clear and legible, is convenient to observe and free-hand operation.
Easy desk-top candler can be by the bulb in the ball bearing made using and a desk-top organization, in the central authorities of desk-top body a loophole is arranged, and bulb is positioned at the loophole below, and there is a pit loophole top.Ovum is put in the pit of loophole top during use.The desk-top candler of increasing work efficiency is found in Chinese patent 200520093870.4.
Described bird ovum is lived bird ovum, contains the embryo in blastodisc or any period, and the method that its developmental stage is judged is to utilize desk-top candler irradiation, distinguishes mutually according to the egg of embryonic development different times.
Described at the other 1-2mm of desire injection position place's selection aperture position (also need not having important structure under the chorion herein) on the chorion, to desire injection position aligning opening behind the opening again injects, be because the solid needle of opening is thicker, chorion is very thin, when opening, before injection, just damage the important structure of desiring the injection position place easily in advance, and then influence later stage embryonic development and hatching rate.And so handle, because of entry needle very thin, with the diameter of solid needle not at the same order of magnitude, therefore very little to desiring the damage that injection position and corresponding position thereof produce, the influence of mechanical damage to the embryonic development of ovum later stage, hatching rate, survival rate can be minimized like this.
Described little opening is positioned at the one side up of ovum, is for the diameter that is positioned at the equal size on chorion, periostracum, the inner shell membrane little opening less than 2mm, can be in any position, non-air chamber place.Opening is more little, and is more little to the damage of ovum, is convenient to sealing more, and sealer is more little, intense little to the thorn of ovum, more little to embryo's influence like this with the area that material in the ovum contacts during sealing, and is also more little to the influence of hatching rate, survival rate.
Injection position can be selected near the blood vessel, near the blastodisc or near the embryo, select to be arranged in the ovum position of the degree of depth arbitrarily as required.Also can choose around blastodisc, embryo, yolk, egg white, the blood vessel, in the blood vessel and location about.
Described injection needles is for drawing the glass microelectrode form with microelectrode puller, tip diameter is less than 500 microns, also can be less than 100 microns, even less than 20 microns.Can select the thin as far as possible glass microelectrode of tip diameter as required.The tip is thin more, and the mechanical damage to ovum during injection is more little.And glass microelectrode has scale from most advanced and sophisticated beginning label, can accurately control injection depth like this.In addition, glass transparent is injected more directly perceived.
The other end of described injection needles links to each other with the micropipette rifle by flexible pipe, has a certain amount of nontoxic non-injecting fluid in flexible pipe, as the paraffin wet goods.Between the desire injection mass in non-injecting fluid and entry needle and between non-injecting fluid and the liquid-transfering gun cavity is arranged.During operation, the tip portion of injection needles is inserted under the liquid level, drawn a certain amount of desire injection mass by the swabbing action of liquid-transfering gun.During injection,, material is injected in the ovum, realize the site-directed quantitative injection desiring to press the button of liquid-transfering gun in the injection structure (degree of depth is reference with the chorion, shown in the scale on the entry needle) in the needle point opening insertion from childhood ovum.
Described injection needles uses for the sterilization back.After the use, can pull up, change new entry needle again from hose end.Every is only used once, pollution-free, also can avoid cross-infection, thereby improves hatching rate and survival rate.
Described injection mass can comprise nucleic acid, nucleotide, antibiotic, vitamin, virus, vaccine, hormone, peptide, variant cell etc., and contain radioisotopic or, or any material that can be used in ovum, injecting by fluorescently-labeled above-mentioned substance.
Step a-f carries out under incubation temperature, can make in the ovum in the raw.The injection back increases because of ovum content volume, and pressure increases, when extracting entry needle out, ovum content (comprising injection mass) can overflow from the chorion opening, be difficult for finishing calibrated shot control, also be difficult for sealing, also cause environmental pollution easily for the isotope-labeled material of injection.And according to the principle of expanding with heat and contract with cold, by step g in advance cooling its ovum content volume is reduced, the level control that so can make opening part is under chorion, avoid the loss of overflowing of injection mass,, can well address the above problem so that seal, especially to small-sized bird ovum, this step is very crucial.
The purpose of step I is to destroy and stimulate the important structure at injection position place when avoiding sealing, and avoids destroying or the blocking-up source of nutrition.Make unessential structure such as egg white relative by this step, allow these unessential structures when sealing, directly contact, can reduce influence, thereby the ovum after helping handling recovers normal development as quickly as possible injection position and needle track with sealer with opening.
The present invention further comprises and the bird ovum after handling is replaced the Bird's Nest or the incubator box of promptly original residing close bird hatching, the step of further hatching into birdling.
Experiment and effect
In developmental neurobiology research
3H-TDR can be used for the newborn neuron in a certain period in the mark brain, also can be in the destiny of neuron in the later stage growth course of following the tracks of these marks behind the mark etc.
For the present invention is described, seven days lonchura striata ovum of embryonic development to the is injected according to the inventive method
3H-TDR has carried out experimental study, and is comparing result such as table one aspect hatching rate and the survival rate with the bird ovum that uses traditional microsyringe list ovum injecting method injection the same period and without the bird ovum of hatching naturally of injection treatment.Choose the health fertilization bird ovum and the hatching healthy close bird hatching suitable of suitable injection in the experiment, injection with the nurture condition
3The H-TDR amount is 5ul/ (1uci/ul), and injection position is the other 0.5-1mm of trunk, and degree of depth 2.5mm puts back to primitive pit immediately and continues hatching after the processing.
The result is grown in seven days lonchura striata ovum of table one embryonic development to the injection back
As can be seen from Table I, no matter the bird ovum that uses the inventive method to handle all is better than using the bird ovum of traditional microsyringe injection treatment on hatching rate still is survival rate, and close with bird ovum result without injection treatment.
Therefore, this method is the effective bird ovum site-directed quantitative injecting method that guarantees higher hatching rate and survival rate.Use the inventive method and can solve traditional bird ovum injecting method institute outstanding question in fields such as biology and medical science, can improve the efficient of these field Related Experimental Study greatly.
Claims (3)
1. one kind to single bird ovum injecting method, is characterised in that its operating procedure is as follows:
A, the bird ovum is positioned under incubation temperature predetermined towards the position;
B, on chorion, desire the other 1-2mm of injection position place selection aperture position and disinfect in alcohol;
C, with the solid needle of sterilization the aperture position on the chorion with and following periostracum, inner shell membrane on make an equal big or small little opening simultaneously, diameter is less than 2mm.
D, rotate the bird ovum gently and make and desire injection position in the ovum and aim at the little opening on the chorion;
E, stretch into an injection needles of having sterilized by little opening;
F, by injection needles injection mass in ovum;
G, ovum was lower than under the incubation temperature 1-10 ℃ environment cooling less than 10 minutes;
H, withdrawal injection needles;
I, make the bird ovum in vertical section, rotate 5-45 °, make the entry needle road junction depart from opening on the chorion;
The paraffin sealing opening that j, usefulness are melted;
K, treat that under incubation temperature paraffin solidifies;
L, with the hatching of replacing of bird ovum.
2. according to the method for the said a kind of bird ovum injection of claim 1, be characterised in that the bird ovum is positioned over the loophole place of desk-top candler, the bird ovum be that the major axis that the blunt end air chamber of bird ovum is positioned at side or bird ovum is horizontal towards the position, during injection, entry needle is injected without air chamber.
3. according to the method for the said a kind of bird ovum injection of claim 1, be characterised in that said injection needles is for drawing the glass microelectrode that forms with microelectrode puller.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200810013123 CN101341863B (en) | 2008-09-02 | 2008-09-02 | Injection method for single-bird egg |
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| CN 200810013123 CN101341863B (en) | 2008-09-02 | 2008-09-02 | Injection method for single-bird egg |
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| CN101341863A CN101341863A (en) | 2009-01-14 |
| CN101341863B true CN101341863B (en) | 2011-04-20 |
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| CN 200810013123 Expired - Fee Related CN101341863B (en) | 2008-09-02 | 2008-09-02 | Injection method for single-bird egg |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100186674A1 (en) * | 2009-01-26 | 2010-07-29 | E. I. Du Pont De Nemours And Company | Methods and compositions for treating fertilized avian eggs |
| ES2704066T3 (en) * | 2011-11-02 | 2019-03-14 | Zoetis Services Llc | Indexing system for an in ovo injection device and associated procedure |
| CN102499148B (en) * | 2011-11-02 | 2013-05-01 | 浙江大学舟山海洋研究中心 | Shrimp larvae microinjection method |
| CN102613109B (en) * | 2012-04-10 | 2014-05-07 | 刘红林 | Poultry egg windowing and sealing method |
| CN104082237B (en) * | 2014-05-04 | 2016-09-28 | 东北农业大学 | A kind of effective hatching of breeding eggs method prepared for transgenic poultry |
| CN107027694B (en) * | 2016-10-19 | 2019-10-01 | 中国水产科学研究院长江水产研究所 | A kind of device and method importing soft-shelled turtle ovum for exogenous inducer |
| CN113475425B (en) * | 2021-05-25 | 2023-04-11 | 攀枝花学院 | In ovo injection device for immunization or nutrition supply of early embryos of poultry animals |
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| US6286455B1 (en) * | 1998-01-12 | 2001-09-11 | Embrex, Inc. | Automated in ovo injection apparatus |
| CN1332603A (en) * | 1999-01-06 | 2002-01-23 | 恩布里克斯公司 | Concurrent in ovo injection and detection method and apparatus |
| US6601533B1 (en) * | 1997-01-27 | 2003-08-05 | Edward G. Bounds, Jr. | Egg injection apparatus and method |
| CN1989802A (en) * | 2002-01-09 | 2007-07-04 | 恩布里克斯公司 | Methods and apparatus for punching through egg shells with reduced force |
| CN101018481A (en) * | 2004-09-03 | 2007-08-15 | 恩布里克斯公司 | Methods for manipulating air cells within avian eggs |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US6601533B1 (en) * | 1997-01-27 | 2003-08-05 | Edward G. Bounds, Jr. | Egg injection apparatus and method |
| US6286455B1 (en) * | 1998-01-12 | 2001-09-11 | Embrex, Inc. | Automated in ovo injection apparatus |
| CN1332603A (en) * | 1999-01-06 | 2002-01-23 | 恩布里克斯公司 | Concurrent in ovo injection and detection method and apparatus |
| CN1989802A (en) * | 2002-01-09 | 2007-07-04 | 恩布里克斯公司 | Methods and apparatus for punching through egg shells with reduced force |
| CN101018481A (en) * | 2004-09-03 | 2007-08-15 | 恩布里克斯公司 | Methods for manipulating air cells within avian eggs |
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