CN105123575A - Method for continuous observation of Tiretrack eel embryonic development - Google Patents
Method for continuous observation of Tiretrack eel embryonic development Download PDFInfo
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- CN105123575A CN105123575A CN201510448308.7A CN201510448308A CN105123575A CN 105123575 A CN105123575 A CN 105123575A CN 201510448308 A CN201510448308 A CN 201510448308A CN 105123575 A CN105123575 A CN 105123575A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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Abstract
The invention belongs to the technical field of aquatic animal breeding biology and discloses a method for continuous observation of Tiretrack eel embryonic development. A tiretrack eel oosperm is processed by trypsin solution and then continuous microscopic observation is conducted; the method further comprises steps of preparation of the trypsin solution, acquisition and processing of the tiretrack eel oosperm, continuous microscopic observation of the oosperm and daily management. The concentration of the trypsin solution is 1 to 3g/L; and 3 to 5ml is filled into each culture dish; after the tiretrack eel oosperm is processed by the trypsin solution, the external film of the oosperm is thinned and becomes transparent and then continuous microscopic observation can be achieved; clear view can be provided during the observation and reliable results can be achieved; compared with the same batches of oosperms yet to be processed by the trypsin solution, each development phase can be clearly observed during the embryonic development; and fish embryonic development research level is raised to a high new phase due to the formation of radial oosperm holes features of red artery and venous blood circulation system, so the method has huge research and reference value.
Description
Technical field
The invention belongs to aquatic livestock reproductive biology technical field, be specifically related to a kind of method that sustainability observes large mastacembelus aculeatus embryonic development.
Background technology
Large mastacembelus aculeatus (Mastacembelusarmatus) is under the jurisdiction of Perciformes, Mastacembeloidei, mastacembelus aculeatus section in classification, and mastacembelus aculeatus belongs to.Be commonly called as and receive cone, coupdepoing, thick fiber crops are cut, capsicum fish, weapons fish.Adult fish length 30 ~ 50 centimetres, body weight 200 ~ 500 grams, maximum individuality can reach 2.5kg, and meat is carefully solid, fragrant and sweet good to eat, nutritious.Mainly be distributed in each water system on the south the Changjiang river, particularly abundant with fluviatic wild resources in ground such as Guangdong, Guangxi, Fujian.After the eighties in 20th century, because cruel fishing is excessively captured and rivers environmental pollution, the rivers wild resource of large mastacembelus aculeatus is seriously damaged, resource exhaustion, one of province wild water animals having been listed in focused protection such as Guangdong, Fujian, Guizhou.At present; although the research of existing large mastacembelus aculeatus Embryonic aspect; but because fertilized egg chorion is thick; generally can only be confined to regular microscopic photography and use the process of Photoshop software again; fail to realize continuation microexamination; be difficult to obtain clearly that embryonic development image is to ensure correct observed result, thus extreme influence large mastacembelus aculeatus reproductive biology progress, is unfavorable for that large mastacembelus aculeatus large-scale artificial propagation technique is studied.
Summary of the invention
The object of this invention is to provide a kind of method that sustainability observes large mastacembelus aculeatus embryonic development.
For achieving the above object, the present invention by the following technical solutions:
Sustainability observes a method for large mastacembelus aculeatus embryonic development, is to carry out continuation microexamination with trypsin solution to after large mastacembelus aculeatus fertilized egg process.
Further, the concentration of described trypsin solution is 1 ~ 3g/L.
Further, described tryptic specific activity is 200 ~ 300U/mg.
Further, the consumption of described trypsin solution is consumption is that each culture dish adds 3 ~ 5mL.
Further, described trypsin solution is specially large mastacembelus aculeatus fertilized egg process: add trypsin solution toward the culture dish that large mastacembelus aculeatus fertilized egg is housed, culture dish 5 ~ 10s is shaken every 1min, when in culture dish 1/3 ~ 1/2 fertilized egg beginning become bright, namely pour out trypsin solution, and clean 3 ~ 5 times to stop tryptic effect with distilled water.
Further, described continuation microexamination is specially: fertilized egg is placed on stereomicroscope and carries out Continuous Observation, carries out microscopic photography in each spilting of an egg and the artis of each developmental stage to the key character of fertilized egg.
Further, the method comprises the following steps:
(1) preparation of trypsin solution: weigh 0.2g trypsase in container bottle, add 100mL distilled water, do not stop after adding a cover to rock, allow trypsase fully dissolve, be made into the trypsin solution of 2g/L, be placed on constant temperature laboratory stand-by;
(2) acquisition of large mastacembelus aculeatus fertilized egg and process: large mastacembelus aculeatus parent obtains fertilized egg through artificial induced spawning, fertilized egg is evenly distributed in culture dish, the fertilized egg quantity of culture dish controls at 25 ~ 35, from obtain fertilized egg to add trypsin solution and complete in 3min, then trypsin solution is added toward culture dish, culture dish 5 ~ 10s is shaken every 1min, when in culture dish 1/3 ~ 1/2 fertilized egg beginning become bright, namely trypsin solution is poured out, and clean 3 ~ 5 times to stop tryptic effect with distilled water, add trypsin solution from fertilized egg to complete to terminating in 7 ~ 8min,
(3) the lasting microexamination of fertilized egg and daily management: the fertilized egg after trypsin treatment is placed on stereomicroscope and carries out Continuous Observation, the artis of each spilting of an egg and each developmental stage carries out microscopic photography to the key character of fertilized egg, and records the corresponding time; Fertilized egg changes water 2 times every day, each quantity of exchanged water 1/3.
The present invention has following beneficial effect:
Adopt method of the present invention to observe large mastacembelus aculeatus embryonic development, after utilizing trypsase to process large mastacembelus aculeatus fertilized egg, make fertilized egg adventitia thinning, transparent, can carry out continuation microexamination, observation process gets a clear view, reliable results.Compared with studying with forefathers, there is bigger difference in its development time, duration and development characteristics; With without trypsin treatment with compared with batch fertilized egg, in embryo development procedure, each developmental stage feature is more clear, especially radial Fertilization Hole occurs, the features such as the formation of red artery and vein circulatory system, fish embryo is grown research level and bring up to the new stage, there is huge research and reference value.The present invention is that the observation of large mastacembelus aculeatus embryonic development continuation provides effective approaches and methods, ensure that the correctness of observed result, greatly can promote large mastacembelus aculeatus reproductive biology aspect research.
Accompanying drawing explanation
Fig. 1 is the fertilized egg of experimental group,
Fig. 2 is the muscle effects phase of experimental group,
Fig. 3 and Fig. 4 is fertilized egg period without same batch of fertilized egg (control group) of trypsin treatment and muscle effects phase respectively.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further:
embodiment 1
The process of large mastacembelus aculeatus embryonic development is observed according to following steps:
The preparation of 1 experiment condition
Experiment material comprises the fertilized egg, trypsase, distilled water, culture dish etc. that obtain through the large mastacembelus aculeatus parent of artificial induced spawning, and the binocular stereo microscope, computer, camera head etc. that can carry out photomicrography are a set of; Experimental implementation and observation process are all carried out in constant temperature laboratory, and indoor temperature control is at 26 ± 0.5 DEG C;
The preparation of 2 trypsin solutions
Weigh 0.2g trypsase (specific activity is 250U/mg) and in container bottle, add 100mL distilled water, do not stop after adding a cover to rock, allow trypsase fully dissolve, be made into the trypsin solution of 2g/L, be placed on constant temperature laboratory stand-by;
The acquisition of 3 large mastacembelus aculeatus fertilized eggs and process
Through artificial induced spawning, large mastacembelus aculeatus parent obtains fertilized egg, fertilized egg is evenly distributed in 12 culture dishes, wherein experimental group 8, control group 4, the fertilized egg quantity of each culture dish controls at about 30, from obtain fertilized egg to add trypsin solution and complete in 3min, then 4mL trypsin solution is added toward each culture dish of experimental group, culture dish 5 ~ 10s is shaken gently every 1min, make fertilized egg and trypsin solution fully mixed, when in culture dish about 1/3 fertilized egg beginning become bright, namely trypsin solution is poured out, and clean 3 ~ 5 times to stop tryptic effect with distilled water, add trypsin solution from fertilized egg to complete to terminating in 7 ~ 8min,
The lasting microexamination of 4 fertilized eggs and daily management
Fertilized egg after trypsin treatment is placed on stereomicroscope and carries out Continuous Observation, the artis of each spilting of an egg and each developmental stage carries out microscopic photography to the key character of fertilized egg, meanwhile observe and take the fertilized egg of control group, and recording the corresponding time; Fertilized egg changes water 2 times every day, each quantity of exchanged water 1/3;
5 observed results
Under 26 ± 0.5 DEG C of conditions, large mastacembelus aculeatus fertilized egg is hatched whole process and is lasted 72.5h from artificial insemination to fry, respectively through 8 continuous developmental stage such as fertilized egg, blastodisc protuberance, the spilting of an egg, blastaea, primitive gut, neurula, organ generation and membranes, according to large mastacembelus aculeatus embryonic development order and metamorphosis situation, whole growth course is divided into 23 periods, as shown in table 1, according to each phase duration length of embryonic development and water temperature, total accumulated temperature that extrapolating whole embryo development procedure needs is 1885 ± 36.25 DEG C of h.
The embryo development procedure of the large mastacembelus aculeatus of table 1
| Puberty | Development time (h) | Duration (h) | Principal character |
| Fertilized egg | 0.0 | 1.0 | In oval, golden yellow, there is radial Fertilization Hole |
| Blastodisc swells | 1.0 | 0.3 | Hat shape protuberance is blastodisc |
| 2 cells | 1.3 | 0.15 | For the first time through splitting, in 2 blastomeres |
| 4 cells | 1.45 | 0.15 | For the second time through splitting, in 4 blastomeres |
| 8 cells | 1.6 | 0.2 | For the third time through splitting, in 8 blastomeres |
| 16 cells | 1.8 | 0.3 | Occur that latitude splits, in 16 blastomeres |
| 32 cells | 2.1 | 0.3 | Continuing the spilting of an egg is, in 32 blastomeres |
| Many cells | 2.4 | 0.7 | Continue the spilting of an egg, blastomere geometry level increases |
| Mulberry body | 3.1 | 0.7 | Continue the spilting of an egg, blastomere is mulberry fruit shape |
| Blastaea is early stage | 3.8 | 1.5 | Blastoderm is held high on yolk |
| Blastaea mid-term | 5.3 | 1.7 | Blastoderm height reduction |
| Blastaea late period | 7.0 | 3.0 | Blastoderm becomes flat, and cell is lower bag obviously |
| Primitive gut is early stage | 10.0 | 6.5 | Wrap under blastodisc to 1/3-1/2, occur germ ring |
| Primitive gut mid-term | 16.5 | 6.3 | Wrap under blastodisc to 1/2-2/3, occur embryonic shield |
| Primitive gut late period | 22.8 | 7.6 | Wrap under blastodisc to 2/3-3/4, occur blastopore |
| Neurula stage | 30.4 | 5.3 | Wrap under blastodisc to 2/3-3/4, embryonic shield continues to extend |
| Blastopore closes the phase | 35.7 | 6.0 | Blastopore is closed |
| The muscle segment apparition | 41.7 | 1.5 | 2-3 muscle segment is there is in the middle part of idiosome |
| The optic vesicle phase | 43.2 | 6.8 | There is former base in idiosome head |
| The tail tooth phase | 55.0 | 6.4 | Embryo's afterbody stretches out bud shape projection |
| The muscle effects phase | 61.4 | 4.0 | Muscle segment increases, and microtremor appears in idiosome |
| Heart beat period | 65.4 | 7.1 | Heart occurs, faintly beats, and occurs clear red flow |
| The membrane phase | 72.5 | — | Head embryophoric membrane is thinning, progressively expands, rupture of membranes and going out |
Fig. 1 is the fertilized egg of experimental group, and Fig. 2 is the muscle effects phase of experimental group, Fig. 3 and Fig. 4 is fertilized egg period without same batch of fertilized egg (control group) of trypsin treatment and muscle effects phase respectively.
From table 1 and Fig. 1 ~ 4, utilize trypsase to after large mastacembelus aculeatus fertilized egg process, make fertilized egg adventitia thinning, transparent, continuation microexamination can be carried out, get a clear view, reliable results, with without trypsin treatment with compared with batch fertilized egg, in embryo development procedure, each developmental stage feature is more clear, and especially radial Fertilization Hole occurs, the features such as the formation of red artery and vein circulatory system.
In prior art (see large mastacembelus aculeatus Embryonic, fresh water fishery, 2014(3): 101 ~ 104), with trypsase, large mastacembelus aculeatus fertilized egg is not processed, picture Photoshop process, the embryo development procedure observed does not have the present embodiment reliably clear.
embodiment 2
The process of large mastacembelus aculeatus embryonic development is observed according to following steps:
(1) preparation of trypsin solution: weigh 0.1g trypsase (specific activity is 200U/mg) in container bottle, add 100mL distilled water, do not stop after adding a cover to rock, allow trypsase fully dissolve, be made into the trypsin solution of 1g/L, be placed on constant temperature laboratory stand-by;
(2) acquisition of large mastacembelus aculeatus fertilized egg and process: large mastacembelus aculeatus parent obtains fertilized egg through artificial induced spawning, fertilized egg is evenly distributed in culture dish, the fertilized egg quantity of culture dish controls at about 30, from obtain fertilized egg to add trypsin solution and complete in 3min, then 3mL trypsin solution is added toward each culture dish, culture dish 5 ~ 10s is shaken gently every 1min, when in culture dish 1/2 fertilized egg beginning become bright, namely trypsin solution is poured out, and clean 5 times to stop tryptic effect with distilled water, add trypsin solution from fertilized egg to complete to terminating in 7 ~ 8min,
(3) the lasting microexamination of fertilized egg and daily management: the fertilized egg after trypsin treatment is placed on stereomicroscope and carries out Continuous Observation, the artis of each spilting of an egg and each developmental stage carries out microscopic photography to the key character of fertilized egg, and records the corresponding time; Fertilized egg changes water 2 times every day, each quantity of exchanged water 1/3.
embodiment 3
The process of large mastacembelus aculeatus embryonic development is observed according to following steps:
(1) preparation of trypsin solution: weigh 0.3g trypsase (specific activity is 300U/mg) in container bottle, add 100mL distilled water, do not stop after adding a cover to rock, allow trypsase fully dissolve, be made into the trypsin solution of 3g/L, be placed on constant temperature laboratory stand-by;
(2) acquisition of large mastacembelus aculeatus fertilized egg and process: large mastacembelus aculeatus parent obtains fertilized egg through artificial induced spawning, fertilized egg is evenly distributed in culture dish, the fertilized egg quantity of culture dish controls at about 30, from obtain fertilized egg to add trypsin solution and complete in 3min, then 5mL trypsin solution is added toward each culture dish, culture dish 5 ~ 10s is shaken gently every 1min, when in culture dish about 1/3 fertilized egg beginning become bright, namely trypsin solution is poured out, and clean 3 times to stop tryptic effect with distilled water, add trypsin solution from fertilized egg to complete to terminating in 7 ~ 8min,
(3) the lasting microexamination of fertilized egg and daily management: the fertilized egg after trypsin treatment is placed on stereomicroscope and carries out Continuous Observation, the artis of each spilting of an egg and each developmental stage carries out microscopic photography to the key character of fertilized egg, and records the corresponding time; Fertilized egg changes water 2 times every day, each quantity of exchanged water 1/3.
The above; be only the specific embodiment of the present invention, but protection scope of the present invention is not limited thereto, anyly belongs to those skilled in the art in the technical scope that the present invention discloses; the change that can expect easily or replacement, all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain of claim.
Claims (7)
1. sustainability observes a method for large mastacembelus aculeatus embryonic development, it is characterized in that, carries out continuation microexamination with trypsin solution to after large mastacembelus aculeatus fertilized egg process.
2. sustainability according to claim 1 observes the method for large mastacembelus aculeatus embryonic development, it is characterized in that, the concentration of described trypsin solution is 1 ~ 3g/L.
3. sustainability according to claim 1 observes the method for large mastacembelus aculeatus embryonic development, it is characterized in that, described tryptic specific activity is 200 ~ 300U/mg.
4. sustainability according to claim 1 observes the method for large mastacembelus aculeatus embryonic development, it is characterized in that, the consumption of described trypsin solution is that each culture dish adds 3 ~ 5mL.
5. sustainability according to claim 1 observes the method for large mastacembelus aculeatus embryonic development, it is characterized in that, described trypsin solution is specially large mastacembelus aculeatus fertilized egg process: add trypsin solution toward the culture dish that large mastacembelus aculeatus fertilized egg is housed, culture dish 5 ~ 10s is shaken every 1min, when in culture dish 1/3 ~ 1/2 fertilized egg beginning become bright, namely pour out trypsin solution, and clean 3 ~ 5 times to stop tryptic effect with distilled water.
6. sustainability according to claim 1 observes the method for large mastacembelus aculeatus embryonic development, it is characterized in that, described continuation microexamination is specially: fertilized egg is placed on stereomicroscope and carries out Continuous Observation, carries out microscopic photography in each spilting of an egg and the artis of each developmental stage to the key character of fertilized egg.
7. sustainability according to claim 1 observes the method for large mastacembelus aculeatus embryonic development, it is characterized in that, comprises the following steps:
(1) preparation of trypsin solution: weigh 0.2g trypsase in container bottle, add 100mL distilled water, do not stop after adding a cover to rock, allow trypsase fully dissolve, be made into the trypsin solution of 2g/L, be placed on constant temperature laboratory stand-by;
(2) acquisition of large mastacembelus aculeatus fertilized egg and process: large mastacembelus aculeatus parent obtains fertilized egg through artificial induced spawning, fertilized egg is evenly distributed in culture dish, the fertilized egg quantity of culture dish controls at 25 ~ 35, from obtain fertilized egg to add trypsin solution and complete in 3min, then trypsin solution is added toward culture dish, culture dish 5 ~ 10s is shaken every 1min, when in culture dish 1/3 ~ 1/2 fertilized egg beginning become bright, namely trypsin solution is poured out, and clean 3 ~ 5 times to stop tryptic effect with distilled water, add trypsin solution from fertilized egg to complete to terminating in 7 ~ 8min,
(3) the lasting microexamination of fertilized egg and daily management: the fertilized egg after trypsin treatment is placed on stereomicroscope and carries out Continuous Observation, the artis of each spilting of an egg and each developmental stage carries out microscopic photography to the key character of fertilized egg, and records the corresponding time; Fertilized egg changes water 2 times every day, each quantity of exchanged water 1/3.
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| CN108651337A (en) * | 2018-05-15 | 2018-10-16 | 江西省水产科学研究所 | A kind of production Drifting egg fish early stage different developmental phases form microscopy observation method |
| CN113498748A (en) * | 2021-06-04 | 2021-10-15 | 广东海洋大学 | Method for calculating survival rate of drifting roes according to survival rates of roes at different stages of development |
| CN114561343A (en) * | 2022-02-18 | 2022-05-31 | 河南师范大学 | Batch preparation method of membrane-removed living fish eggs |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113498748A (en) * | 2021-06-04 | 2021-10-15 | 广东海洋大学 | Method for calculating survival rate of drifting roes according to survival rates of roes at different stages of development |
| CN113498748B (en) * | 2021-06-04 | 2022-09-27 | 广东海洋大学 | Method for calculating survival rate of drifting roes according to survival rates of roes at different stages of development |
| CN114561343A (en) * | 2022-02-18 | 2022-05-31 | 河南师范大学 | Batch preparation method of membrane-removed living fish eggs |
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