CN103076283A - Method for observing developmental process of salmonidae roes - Google Patents
Method for observing developmental process of salmonidae roes Download PDFInfo
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- CN103076283A CN103076283A CN2013100175266A CN201310017526A CN103076283A CN 103076283 A CN103076283 A CN 103076283A CN 2013100175266 A CN2013100175266 A CN 2013100175266A CN 201310017526 A CN201310017526 A CN 201310017526A CN 103076283 A CN103076283 A CN 103076283A
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Abstract
The invention discloses a method for observing the developmental process of salmonidae roes, relates to a method for observing the developmental process of roes and aims to solve the problems of difficulty in observing details during cell division and unclear microimaging in an existing method for observing the developmental process of the salmonidae roes. The method for observing the developmental process of the salmonidae roes, which is disclosed by the invention, is executed according to the following steps: 1, preparing, i.e. preparing roe stationary liquid and placing the roe stationary liquid into a refrigerator for storage; 2, sampling, i.e. sampling salmonidae fertilized eggs, placing the salmonidae fertilized eggs into sampling tubes with the roe stationary liquid and placing the sampling tubes into the refrigerator for storage; and 3, development observing, i.e. taking out the roes from the stationary liquid, placing the roes on a glass slide, slightly peeling off roe membranes by a dissecting needle, placing the obtained roes under an anatomical lens to observe, shooting, and according to different labels and different developmental phases, which are marked on the corresponding sampling tubes, totally recording the integral developmental process of salmonidae fish from the moment that the salmonidae fish is fertilized to the moment that the membranes are ruptured. The method is suitable for the fields of observation and research application of the developmental process of the salmonidae fertilized eggs.
Description
Technical field
The present invention relates to the observation procedure of fish-egg growth course.
Background technology
Salmon fishes have fine and tender taste, nutritious, edible part large, without thorn between flesh, be easy to the characteristics such as processing, the most important thing is that protein content is high in the muscle, therefore at present and contain a large amount of unsaturated fatty acids, become gradually China and even in the world one of important high-quality protein and source of nutriment.Along with China's expanding economy, people's daily life level improves constantly, and the traditional fish species such as green grass or young crops, grass, silver carp, flathead to be can not satisfying people's consumer need fully, so the high-quality protein fish culture such as China's salmon fishes already develops very rapid.
Because the demand in market strengthens, country has also strengthened the R﹠D intensity of the aspects such as physiology, biochemistry and Developmental Biology to salmon fishes.China mainly from traditional temperate water fisheses such as green grass or young crops, grass, silver carp, flathead, because these fish ovum ovum footpaths are little and egg membrane is thin, therefore generally adopts microscopically directly observation and manual method of drawing for the observational study of fish ovum growth course.Along with the progress of science and technology, the exploitation of high-tech product is taken pictures with digital camera gradually in research subsequently and has been substituted intuitive and the accuracy that manual drawing has so improved research greatly.But along with continually developing of the salmon fishes such as China rainbow trout, the research of also growing to fish ovum has brought new challenge, mainly consisted of by albumen because the salmon fishes major part all is the ovum of its product of cold water fishes, have that the ovum footpath is large, egg membrane is thick, a direct observational characteristic of microscope.The research that early stage scholar grows Salmonoid fish spawn mainly contains 3 kinds: trypsin digestion transparent processing method, transparent liquid processing method and 5% formaldehyde are fixedly peeled off the method that the egg membrane microscopically is taken pictures.Although method has realized the observation of salmon flying fish egg development process to a certain extent in above 3, but also all have deficiency separately, mainly as follows: trypsase must be processed the live body ovum, and is thinner because of egg membrane after processing, observe the concrete Growth phrase of fish-egg although can see through egg membrane, but cost is higher, needs to process immediately, and the time is nervous (processing untimely albuminous degeneration can't observe) relatively, operation easier is also larger, egg membrane is very easily broken, and the egg membrane reflective is strong, and micro-imaging is unintelligible; Although transparent liquid can make the egg membrane sex change and increase transparency, but the thickness of egg membrane does not change, the cell division phase details is difficult to observe, therefore transparent liquid can only be when roughly determining to grow the instrument of phase, lack accuracy but be used for the further investigation that fish-egg grows; 5% formaldehyde is fixedly peeled off the method that the egg membrane microscopically is taken pictures, although the characteristic that has possessed processing ease, can observe at any time after fixing, but have formaldehyde fixedly idiosome and the livetin of fish-egg be white, therefore without colour contrast, the sharpness of photomicrograph is inadequate when the observation cell division phase.
Summary of the invention
The present invention will solve the observation procedure that exists the cell division phase details to be difficult to observation and the unsharp problem of micro-imaging in the observation procedure of existing salmon flying fish egg development process and propose a kind of salmon flying fish egg development process.
The observation procedure of a kind of salmon flying fish egg development process among the present invention carries out according to the following steps:
One, early-stage preparations: at first prepare the observation outfit, comprising: landing-spoon, specification be the sampling pipe, pincet, dissecting needle, microslide of 5ml, with the anatomical lens of camera function; Then prepare the fish-egg immobile liquid, the picric acid that takes by weighing 20g ~ 50g is put into the beaker that fills 400ml ~ 600ml distilled water, after repeatedly stirring 20min ~ 40min with glass bar, the beaker mouth is tight with the sealed membrane envelope, be positioned in 3 ℃ ~ 5 ℃ the refrigerator, after placing 20h ~ 28h, with Filter paper filtering and get supernatant, measure this supernatant of 350ml ~ 450ml and put into another beaker, and in this beaker, add 80ml ~ 120ml formaldehyde and 16ml ~ 24ml glacial acetic acid, after mixing, in the container opaque and with cover of packing into, be positioned in 3 ℃ ~ 5 ℃ the refrigerator for subsequent use;
Two, sample collection: salmon fishes incubating oosperm water temperature is 7 ℃ ~ 9 ℃, needs 18d ~ 22d could hatch rupture of membranes, and concrete sampling interval is as follows: in after fertilization 0h ~ 3h, and every interval 15min once sampling; In after fertilization 3h ~ 8h, every 30min once sampling; In after fertilization 8h ~ 33h, every interval 1h once sampling; In after fertilization 33h ~ 81h, every interval 2h once sampling; In after fertilization 81h ~ 129h, every interval 6h once sampling; The every interval of after fertilization 129h ~ 215h 12h once sampling; After fertilization 215h is during to rupture of membranes, every interval 24h once sampling; Sample collection process: at first indicate different labels and different puberties at each sampling pipe, then in each sampling pipe, inject the fish-egg immobile liquid that 2ml ~ 4ml step 1 is prepared, getting 15 ~ 20 in embryonated egg during each sampling puts in the sampling pipe that the fish-egg immobile liquid is housed, after rocking 1min ~ 3min, sampling pipe is positioned in 3 ℃ ~ 5 ℃ the refrigerator, behind 20h ~ 28h the embryonated egg that gathers is changed the fish-egg immobile liquid of preparing in the step 1 of an equivalent, namely finish the sample collection of salmon fishes embryonated egg;
Three, grow observation: from immobile liquid, take out fish-egg and be placed on year fragmentation, the idiosome of fish-egg up, clamp fish-egg with tweezers, peel gently egg membrane off with dissecting needle, be placed under the anatomical lens and observe, and take pictures, according to the different labels and different puberties that indicate on each sampling pipe, whole process record salmon fishes by after fertilization 0h the whole growth course during to rupture of membranes.
The present invention includes following advantage:
1, the inventive method adopts new prescription to make the fish-egg immobile liquid, and its term of validity generally about 1 year, possesses characteristics easy to use;
2, behind the fish-egg immobile liquid in use the inventive method, the film on the fish-egg is easy to peel off, and is conducive to observe;
3, the inventive method has adopted the fish-egg immobile liquid that new prescription is made, it has dyes orange-yellow effect with the idiosome of fish-egg, overcome greatly the drawback that adopts the formaldehyde method, made the growth course of observation salmon fishes embryonated egg become more convenient, more directly perceived and more accurate.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the combination in any between each embodiment.
Embodiment one: the observation procedure of a kind of salmon flying fish egg development process in the present embodiment carries out according to the following steps:
One, early-stage preparations: at first prepare the observation outfit, comprising: landing-spoon, specification be the sampling pipe, pincet, dissecting needle, microslide of 5ml, with the anatomical lens of camera function; Then prepare the fish-egg immobile liquid, the picric acid that takes by weighing 20g ~ 50g is put into the beaker that fills 400ml ~ 600ml distilled water, after repeatedly stirring 20min ~ 40min with glass bar, the beaker mouth is tight with the sealed membrane envelope, be positioned in 3 ℃ ~ 5 ℃ the refrigerator, after placing 20h ~ 28h, with Filter paper filtering and get supernatant, measure this supernatant of 350ml ~ 450ml and put into another beaker, and in this beaker, add 80ml ~ 120ml formaldehyde and 16ml ~ 24ml glacial acetic acid, after mixing, in the container opaque and with cover of packing into, be positioned in 3 ℃ ~ 5 ℃ the refrigerator for subsequent use;
Two, sample collection: salmon fishes incubating oosperm water temperature is 7 ℃ ~ 9 ℃, needs 18d ~ 22d could hatch rupture of membranes, and concrete sampling interval is as follows: in after fertilization 0h ~ 3h, and every interval 15min once sampling; In after fertilization 3h ~ 8h, every 30min once sampling; In after fertilization 8h ~ 33h, every interval 1h once sampling; In after fertilization 33h ~ 81h, every interval 2h once sampling; In after fertilization 81h ~ 129h, every interval 6h once sampling; The every interval of after fertilization 129h ~ 215h 12h once sampling; After fertilization 215h is during to rupture of membranes, every interval 24h once sampling; Sample collection process: at first indicate different labels and different puberties at each sampling pipe, then in each sampling pipe, inject the fish-egg immobile liquid that 2ml ~ 4ml step 1 is prepared, getting 15 ~ 20 in embryonated egg during each sampling puts in the sampling pipe that the fish-egg immobile liquid is housed, after rocking 1min ~ 3min, sampling pipe is positioned in 3 ℃ ~ 5 ℃ the refrigerator, behind 20h ~ 28h the embryonated egg that gathers is changed the fish-egg immobile liquid of preparing in the step 1 of an equivalent, namely finish the sample collection of salmon fishes embryonated egg;
Three, grow observation: from immobile liquid, take out fish-egg and be placed on year fragmentation, the idiosome of fish-egg up, clamp fish-egg with tweezers, peel gently egg membrane off with dissecting needle, be placed under the anatomical lens and observe, and take pictures, according to the different labels and different puberties that indicate on each sampling pipe, whole process record salmon fishes by after fertilization 0h the whole growth course during to rupture of membranes.
The present invention includes following advantage:
1, the inventive method adopts new prescription to make the fish-egg immobile liquid, and its term of validity generally about 1 year, possesses characteristics easy to use;
2, behind the fish-egg immobile liquid in use the inventive method, the egg membrane on the fish-egg is easy to peel off, and is conducive to observe;
3, the inventive method has adopted the fish-egg immobile liquid that new prescription is made, it has dyes orange-yellow effect with the idiosome of fish-egg, overcome greatly the drawback that adopts the formaldehyde method, made the growth course of observation salmon fishes embryonated egg become more convenient, more directly perceived and more accurate.
Embodiment two: what present embodiment and embodiment one were different is that the picric acid that takes by weighing 25g ~ 45g in the step 1 is put into the beaker that fills 450ml ~ 550ml distilled water, repeatedly stirs 25min ~ 35min with glass bar.Other step and parameter are identical with embodiment one.
Embodiment three: what present embodiment and embodiment one were different is that the picric acid that takes by weighing 35g in the step 1 is put into the beaker that fills 500ml distilled water, repeatedly stirs 30min with glass bar.Other step and parameter are identical with embodiment one.
Embodiment four: present embodiment is different from one of embodiment one to three is in the step 1 that the beaker mouth is tight with the sealed membrane envelope, is positioned in 4 ℃ the refrigerator, places 24h.Other step and parameter are identical with embodiment one.
Embodiment five: what present embodiment was different from one of embodiment one to four is to measure this supernatant of 380ml ~ 420ml in the step 1 to put into another beaker, and in this beaker, add 90ml ~ 110ml formaldehyde and 18ml ~ 22ml glacial acetic acid, mix.Other step and parameter are identical with one of embodiment one to four.
Embodiment six: what present embodiment was different from one of embodiment one to four is to measure this supernatant of 400ml in the step 1 to put into another beaker, and adds 100ml formaldehyde and 20ml glacial acetic acid in this beaker, mixes.Other step and parameter are identical with one of embodiment one to four.
Embodiment seven: what present embodiment was different from one of embodiment one to six is to inject the fish-egg immobile liquid that 2.5ml ~ 3.5ml step 1 is prepared in the step 2 in each sampling pipe, gets 16 ~ 18 in embryonated egg at every turn when sampling and puts in the sampling pipe that the fish-egg immobile liquid is housed.Other step and parameter are identical with one of embodiment one to six.
Embodiment eight: what present embodiment was different from one of embodiment one to six is to inject the fish-egg immobile liquid that the 3ml step 1 is prepared in the step 2 in each sampling pipe, gets 17 in embryonated egg at every turn when sampling and puts in the sampling pipe that the fish-egg immobile liquid is housed.Other step and parameter are identical with one of embodiment one to six.
Embodiment nine: present embodiment is different from one of embodiment one to eight is in the step 2 sampling pipe to be positioned in 3.5 ℃ ~ 4.5 ℃ the refrigerator, behind 22h ~ 26h the embryonated egg that gathers is changed the fish-egg immobile liquid of preparing in the step 1 of an equivalent.Other step and parameter are identical with one of embodiment one to eight.
Embodiment ten: present embodiment is different from one of embodiment one to eight is in the step 2 sampling pipe to be positioned in 4 ℃ the refrigerator, behind the 24h embryonated egg that gathers is changed the fish-egg immobile liquid of preparing in the step 1 of an equivalent.Other step and parameter are identical with one of embodiment one to eight.
In order to verify beneficial effect of the present invention, carried out following experiment:
Experiment one: a kind of observation procedure of salmon flying fish egg development process carries out according to the following steps:
One, early-stage preparations: at first prepare the observation outfit, comprising: landing-spoon, specification be the sampling pipe, pincet, dissecting needle, microslide of 5ml, with the anatomical lens of camera function; Then prepare the fish-egg immobile liquid, the picric acid that takes by weighing 35g is put into the beaker that fills 500ml distilled water, repeatedly stir 30min with glass bar after, the beaker mouth is tight with sealed membrane envelope, be positioned in 4 ℃ the refrigerator, after placing 24h, with Filter paper filtering and get supernatant, measure this supernatant of 400ml and put into another beaker, and in this beaker, add 100ml ~ 120ml formaldehyde and 20ml glacial acetic acid, after mixing, in the container opaque and with cover of packing into, be positioned in 4 ℃ the refrigerator for subsequent use;
Two, sample collection: salmon fishes adopts salmon in this experiment, and its incubating oosperm water temperature is 7 ℃ ~ 9 ℃, needs 18d ~ 22d could hatch rupture of membranes, and concrete sampling interval is as follows: in after fertilization 0h ~ 3h, and every interval 15min once sampling; In after fertilization 3h ~ 8h, every 30min once sampling; In after fertilization 8h ~ 33h, every interval 1h once sampling; In after fertilization 33h ~ 81h, every interval 2h once sampling; In after fertilization 81h ~ 129h, every interval 6h once sampling; The every interval of after fertilization 129h ~ 215h 12h once sampling; After fertilization 215h is during to rupture of membranes, every interval 24h once sampling; Sample collection process: at first indicate different labels and different puberties at each sampling pipe, then in each sampling pipe, inject the fish-egg immobile liquid that the 3ml step 1 is prepared, getting 17 ~ 18 in embryonated egg during each sampling puts in the sampling pipe that the fish-egg immobile liquid is housed, after rocking 2min, sampling pipe is positioned in 4 ℃ the refrigerator, behind the 24h embryonated egg that gathers is changed the fish-egg immobile liquid of preparing in the step 1 of an equivalent, namely finish the sample collection of salmon embryonated egg;
Three, grow observation: from immobile liquid, take out fish-egg and be placed on year fragmentation, the idiosome of fish-egg up, clamp fish-egg with tweezers, peel gently egg membrane off with dissecting needle, be placed under the anatomical lens and observe, and take pictures, according to the different labels and different puberties that indicate on each sampling pipe, whole process record salmon by after fertilization 0h the whole growth course during to rupture of membranes.
The fish-egg immobile liquid that has adopted new prescription to make in this experiment, it has dyes orange-yellow effect with the idiosome of fish-egg, has overcome greatly the drawback that adopts the formaldehyde method, makes the growth course of observation salmon embryonated egg become more convenient, more directly perceived and more accurate.
Claims (10)
1. the observation procedure of a salmon flying fish egg development process is characterized in that it realizes by following steps:
One, early-stage preparations: at first prepare the observation outfit, comprising: landing-spoon, specification be the sampling pipe, pincet, dissecting needle, microslide of 5ml, with the anatomical lens of camera function; Then prepare the fish-egg immobile liquid, the picric acid that takes by weighing 20g ~ 50g is put into the beaker that fills 400ml ~ 600ml distilled water, after repeatedly stirring 20min ~ 40min with glass bar, the beaker mouth is tight with the sealed membrane envelope, be positioned in 3 ℃ ~ 5 ℃ the refrigerator, after placing 20h ~ 28h, with Filter paper filtering and get supernatant, measure this supernatant of 350ml ~ 450ml and put into another beaker, and in this beaker, add 80ml ~ 120ml formaldehyde and 16ml ~ 24ml glacial acetic acid, after mixing, in the container opaque and with cover of packing into, be positioned in 3 ℃ ~ 5 ℃ the refrigerator for subsequent use;
Two, sample collection: salmon fishes incubating oosperm water temperature is 7 ℃ ~ 9 ℃, needs 18d ~ 22d could hatch rupture of membranes, and concrete sampling interval is as follows: in after fertilization 0h ~ 3h, and every interval 15min once sampling; In after fertilization 3h ~ 8h, every 30min once sampling; In after fertilization 8h ~ 33h, every interval 1h once sampling; In after fertilization 33h ~ 81h, every interval 2h once sampling; In after fertilization 81h ~ 129h, every interval 6h once sampling; The every interval of after fertilization 129h ~ 215h 12h once sampling; After fertilization 215h is during to rupture of membranes, every interval 24h once sampling; Sample collection process: at first indicate different labels and different puberties at each sampling pipe, then in each sampling pipe, inject the fish-egg immobile liquid that 2ml ~ 4ml step 1 is prepared, getting 15 ~ 20 in embryonated egg during each sampling puts in the sampling pipe that the fish-egg immobile liquid is housed, after rocking 1min ~ 3min, sampling pipe is positioned in 3 ℃ ~ 5 ℃ the refrigerator, behind 20h ~ 28h the embryonated egg that gathers is changed the fish-egg immobile liquid of preparing in the step 1 of an equivalent, namely finish the sample collection of salmon fishes embryonated egg;
Three, grow observation: from immobile liquid, take out fish-egg and be placed on year fragmentation, the idiosome of fish-egg up, clamp fish-egg with tweezers, peel gently egg membrane off with dissecting needle, be placed under the anatomical lens and observe, and take pictures, according to the different labels and different puberties that indicate on each sampling pipe, the whole growth course when whole process records salmon fishes by after fertilization 0h ~ rupture of membranes.
2. the observation procedure of a kind of salmon flying fish egg development process as claimed in claim 1 is characterized in that the picric acid that takes by weighing 25g ~ 45g in the step 1 puts into the beaker that fills 450ml ~ 550ml distilled water, repeatedly stirs 25min ~ 35min with glass bar.
3. the observation procedure of a kind of salmon flying fish egg development process as claimed in claim 1 is characterized in that the picric acid that takes by weighing 35g in the step 1 puts into the beaker that fills 500ml distilled water, repeatedly stirs 30min with glass bar.
4. such as the observation procedure of each described a kind of salmon flying fish egg development process in the claims 1 to 3, it is characterized in that in the step 1 beaker mouth is tight with the sealed membrane envelope, be positioned in 4 ℃ the refrigerator, place 24h.
5. the observation procedure of a kind of salmon flying fish egg development process as claimed in claim 1, it is characterized in that measuring in the step 1 this supernatant of 380ml ~ 420ml and put into another beaker, and in this beaker, add 90ml ~ 110ml formaldehyde and 18ml ~ 22ml glacial acetic acid, mix.
6. the observation procedure of a kind of salmon flying fish egg development process as claimed in claim 1 is characterized in that measuring in the step 1 this supernatant of 400ml and puts into another beaker, and adds 100ml formaldehyde and 20ml glacial acetic acid in this beaker, mixes.
7. such as the observation procedure of each described a kind of salmon flying fish egg development process in the claim 1,2,3,5 and 6, it is characterized in that in each sampling pipe, injecting the fish-egg immobile liquid that 2.5ml ~ 3.5ml step 1 is prepared in the step 2, get 16 ~ 18 in embryonated egg at every turn when sampling and put in the sampling pipe that the fish-egg immobile liquid is housed.
8. such as the observation procedure of each described a kind of salmon flying fish egg development process in the claim 1,2,3,5 and 6, it is characterized in that in each sampling pipe, injecting the fish-egg immobile liquid that the 3ml step 1 is prepared in the step 2, get 17 in embryonated egg at every turn when sampling and put in the sampling pipe that the fish-egg immobile liquid is housed.
9. the observation procedure of a kind of salmon flying fish egg development process as claimed in claim 7, be characterised in that in the step 2 in the refrigerator that sampling pipe is positioned over 3.5 ℃ ~ 4.5 ℃, behind 22h ~ 26h the embryonated egg that gathers changed the fish-egg immobile liquid of preparing in the step 1 of an equivalent.
10. the observation procedure of a kind of salmon flying fish egg development process as claimed in claim 7 is characterised in that in the step 2 in the refrigerator that sampling pipe is positioned over 4 ℃, behind the 24h embryonated egg that gathers is changed the fish-egg immobile liquid of preparing in the step 1 of an equivalent.
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| CN108918520A (en) * | 2018-07-09 | 2018-11-30 | 长沙健金电子技术有限公司 | A kind of devices and methods therefor of fish egg cell screening |
| CN112167124A (en) * | 2020-10-24 | 2021-01-05 | 武汉市农业科学院 | Method for improving hatching rate of mandarin fish fertilized eggs and uniformity of seeds |
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Application publication date: 20130501 |