CN106614217B - Method for labeling large yellow croaker with alizarin red - Google Patents
Method for labeling large yellow croaker with alizarin red Download PDFInfo
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- CN106614217B CN106614217B CN201610823069.3A CN201610823069A CN106614217B CN 106614217 B CN106614217 B CN 106614217B CN 201610823069 A CN201610823069 A CN 201610823069A CN 106614217 B CN106614217 B CN 106614217B
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- large yellow
- alizarin red
- yellow croaker
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- 241001596950 Larimichthys crocea Species 0.000 title claims abstract description 52
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000002372 labelling Methods 0.000 title claims abstract description 27
- 238000004043 dyeing Methods 0.000 claims abstract description 28
- 239000013535 sea water Substances 0.000 claims abstract description 28
- 238000002791 soaking Methods 0.000 claims abstract description 15
- 239000012452 mother liquor Substances 0.000 claims abstract description 12
- 239000002244 precipitate Substances 0.000 claims abstract description 10
- 150000003839 salts Chemical class 0.000 claims abstract description 9
- 238000007865 diluting Methods 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 19
- 229920000742 Cotton Polymers 0.000 claims description 9
- 238000005273 aeration Methods 0.000 claims description 6
- 230000001376 precipitating effect Effects 0.000 claims description 6
- 239000008399 tap water Substances 0.000 claims description 5
- 235000020679 tap water Nutrition 0.000 claims description 5
- 239000004575 stone Substances 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 2
- 239000012895 dilution Substances 0.000 claims description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims 1
- 235000017557 sodium bicarbonate Nutrition 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 9
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 abstract description 3
- 229910001424 calcium ion Inorganic materials 0.000 abstract description 3
- 238000004090 dissolution Methods 0.000 abstract description 3
- 241000251468 Actinopterygii Species 0.000 description 18
- 239000000975 dye Substances 0.000 description 12
- 241000356847 Otolithes Species 0.000 description 5
- XMEVHPAGJVLHIG-FMZCEJRJSA-N chembl454950 Chemical compound [Cl-].C1=CC=C2[C@](O)(C)[C@H]3C[C@H]4[C@H]([NH+](C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O XMEVHPAGJVLHIG-FMZCEJRJSA-N 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 230000002842 otolith Effects 0.000 description 5
- 210000001265 otolithic membrane Anatomy 0.000 description 5
- 230000003578 releasing effect Effects 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 229960004989 tetracycline hydrochloride Drugs 0.000 description 5
- 210000002816 gill Anatomy 0.000 description 4
- 230000000366 juvenile effect Effects 0.000 description 4
- 210000003097 mucus Anatomy 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 239000013049 sediment Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 238000010668 complexation reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000001215 fluorescent labelling Methods 0.000 description 2
- 230000001418 larval effect Effects 0.000 description 2
- 229960002378 oftasceine Drugs 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 235000006550 Liquidambar Nutrition 0.000 description 1
- 241000208682 Liquidambar Species 0.000 description 1
- 241001550339 Lutjanus erythropterus Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000001044 red dye Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 229910052712 strontium Inorganic materials 0.000 description 1
- CIOAGBVUUVVLOB-UHFFFAOYSA-N strontium atom Chemical compound [Sr] CIOAGBVUUVVLOB-UHFFFAOYSA-N 0.000 description 1
- 229940047908 strontium chloride hexahydrate Drugs 0.000 description 1
- AMGRXJSJSONEEG-UHFFFAOYSA-L strontium dichloride hexahydrate Chemical compound O.O.O.O.O.O.Cl[Sr]Cl AMGRXJSJSONEEG-UHFFFAOYSA-L 0.000 description 1
- 238000009941 weaving Methods 0.000 description 1
Classifications
-
- 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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- Farming Of Fish And Shellfish (AREA)
Abstract
The invention relates to a method for labeling large yellow croaker with alizarin red, which comprises the following steps: (1) preparing alizarin red stock mother liquor, diluting the mother liquor to an application concentration, and adjusting the salinity to more than 15 per thousand by using refined salt and the like to obtain artificial seawater dye liquor; (2) soaking the large yellow croaker fry to be dyed in the dyeing solution for dyeing; and after dyeing is finished, transferring the fry into natural seawater for rinsing to obtain the alizarin red-labeled large yellow croaker. The method disclosed by the invention avoids the influence of calcium ions on the dissolution of alizarin red and the generation of precipitate, improves the effective concentration and the dip-dyeing effect of alizarin red, and solves the problem that suspended precipitate is easy to block gill parts of large yellow croakers to cause mass death.
Description
Technical Field
The invention belongs to the field of large yellow croaker labeling, and particularly relates to a method for labeling large yellow croakers with alizarin red.
Background
Marker recapture is the most common method for assessing fish proliferation and release effects, which requires tagging of fish prior to release to distinguish released from wild populations. The currently used labeling methods include physical labeling, biomolecular labeling, chemical labeling, and the like. In the methods, physical marking can cause certain damage to the fish body, and the method is only suitable for marking the fish body with larger specification, has higher marking cost, needs to be operated one by one, and is not suitable for large-scale marking and releasing. The procedure for detecting the biomolecular marker is too complicated. The chemical marker is mainly formed by soaking, injecting, feeding and other methods and can be stably kept at the gill cover, otolith and other parts. The soaking method is the most main chemical marking mode, can mark released groups in a large scale, has small damage to the fish, and is particularly suitable for marking larval and juvenile fish. Common chemiluminescent dyes include: tetracycline hydrochloride, calcein, alizarin red and an alizarin complexing indicator. These dyes can bind to calcium in the body to form stable chelates and deposit them on the hard group of aquatic organismsIn weaving, a fluorescent marker is finally formed. Tetracycline hydrochloride is an antibiotic, when the tetracycline hydrochloride is used for chemical labeling, the labeling effect is influenced when the tetracycline hydrochloride is used at a low concentration, the survival rate of a labeled target is reduced when the tetracycline hydrochloride is used at an excessive concentration, and the quality safety of aquatic products is threatened. The background fluorescence generated by calcein is strong, and the marking detection effect is poor. The alizarin red and alizarin complex indicator has good performance in fluorescent labeling application, has no obvious damage to a labeling target, has high labeling efficiency, obvious labeling effect and longer retention time, and is an ideal labeling method. It is noteworthy that both dyes are susceptible to Ca in seawater 2+ The isochelation produces a precipitate that affects the amount of dissolution in seawater. As reported in the study of Lutjanus erythropterus fluorescence labeling technology by Liquidambar brevifiliformis et al, the amount of alizarin red dissolved in seawater is only 30mg/L, and the dyeing concentration needs to reach 100-200 mg/L to form an obvious labeling color. This feature makes both of them less effective in marking marine life and suspended sediment may also block the gill of fish and cause death. In addition, the high use cost is also a big factor for limiting the labeling application of the alizarin complex indicator, and the labeling cost of the dye on 5cm juvenile fish reaches 1-2 yuan per tail, so that the application of the dye in releasing labeling is limited. Compared with the prior art, the application cost of alizarin red is lower, which is about 1/200 of alizarin red, and the alizarin red is particularly suitable for large-scale marking of fish.
The large yellow croaker is an important marine economic fish in China, and due to the decline of resources of wild large yellow croaker, the large yellow croaker becomes one of the most important proliferation and release fishes in China, and the release rate of the large yellow croaker in every part of the year reaches tens of millions of tails. The releasing specification of the large yellow croaker is about 5cm generally, and how to mark the larval large yellow croaker of the specification in a large scale becomes a great problem which needs to be solved urgently for evaluating the proliferation releasing effect. The pseudosciaena crocea belongs to the family of the stone-headed fishes, has large otolith, is easy to mark and detect, is very suitable for carrying out fluorescence marking by taking the otolith as a marker, but the existing marking schemes have some defects. For example, the invention patent of a method for marking proliferation and releasing of large yellow croakers, application No. 201410283245.X discloses a method for marking strontium in young large yellow croakers by adding strontium chloride hexahydrate in a nursery pond. The method has long marking time, and the identification of the mark needs to be detected by professional equipment after the otolith is ground, so the method has complex operation and poor practicability. In addition, patent "a method of marking juvenile fish of the family of Shihou", application No. 201410145230.7, discloses a method of marking juvenile fish of the family of Shihou with an alizarin complexation indicator, which is relatively expensive to mark and does not solve the problem that the alizarin complexation indicator is liable to precipitate in seawater. The large yellow croaker can secrete a large amount of mucus under the dye soaking stress state, and the mucus is combined with suspended sediments to form floccules, so that the cheek of the large yellow croaker is easily blocked and the large yellow croaker dies. The survival rate of the large yellow croaker marked in actual operation is less than 50 percent.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for labeling large yellow croakers with alizarin red, which avoids the influence of calcium ions on the dissolution of the alizarin red and the generation of precipitates by blending artificial seawater, improves the effective concentration and the dip-dyeing effect of the alizarin red, and solves the problem that suspended precipitates easily block gills of the large yellow croakers to cause a great amount of death.
The invention relates to a method for labeling large yellow croaker with alizarin red, which comprises the following steps:
(1) preparing alizarin red stock mother liquor, diluting the mother liquor to an application concentration, and adjusting the salinity to be more than 15 per thousand to obtain artificial seawater dye liquor;
(2) soaking the large yellow croaker fry to be dyed in the dyeing solution for dyeing; and after dyeing is finished, transferring the fry into natural seawater for rinsing to obtain the alizarin red marked large yellow croaker.
And (2) strongly aerating for 12h by using air stones when preparing alizarin red stock mother liquor in the step (1).
In the step (1), the concentration is 100-300 mg/L, and tap water with aeration time of more than 48 hours is adopted for dilution.
In the step (1), iodine-free refined salt and MgSO are adopted 4 、MgCl 2 And KCl regulates salinity. The addition amount is as follows: adding iodine-free refined salt 12.3g and MgSO 2 to each liter of dye liquor 4 1.0g，MgCl 2 1.6g，KCl 0.3g。
Aerating after the artificial seawater obtained in the step (1), and adopting pneumatic filter cotton for adsorption and sedimentationPrecipitation (possible precipitate formed by adsorption of small amount of calcium ions carried in tap water and refined salt), and application of NaHCO 3 Adjusting the pH value of the solution to 7.8-8.2.
And (3) the large yellow croaker fries to be dyed in the step (2) are adapted to low-salt seawater with the salinity of 15 per mill for more than 48 hours in advance (the adaptability to the low-salt artificial seawater is enhanced).
And (3) dyeing in the step (2) is carried out in a culture pond or a dyeing barrel.
In the step (2), the soaking density of the large yellow croaker to be dyed is 1.5-2.5 tails/L (the total length is about 5 cm), and the soaking time is 24 hours.
Continuously inflating during the soaking period, adsorbing precipitate (small amount of precipitate and fish body secretion mucus brought into seawater from body surface of Pseudosciaena crocea) with pneumatic filter cotton, and adding NaHCO 3 And adjusting the pH value of the solution to be 7.8-8.2.
And (3) filtering the dyeing solution obtained after dyeing in the step (2) for repeated use.
Advantageous effects
(1) The method adopts a mode of modulating artificial seawater, improves the effective concentration and dyeing effect of alizarin red dye solution, has the marking rate of 100 percent, and can still clearly detect otolith coloring after 90 days of dip dyeing. The detection mode is simple, the marked fry can be distinguished by naked eye observation in the early stage (within about 50 days), and the distinguishing accuracy can be improved by means of fluorescent microscope observation in the later stage;
(2) the artificial seawater avoids the generation of suspended sediments, prevents large amount of large yellow croakers from dying due to blocking gill of large yellow croakers caused by floccules formed by combination of the suspended sediments and mucus secreted by the large yellow croakers, and keeps the survival rate of the large yellow croakers above 95% after marking for 48 hours;
(3) the influence of a small amount of precipitate possibly generated in the dip dyeing process on the fry is further reduced by virtue of the filtering action of the pneumatic filter cotton;
(4) the method avoids the problems that the methods such as in-vitro tag hanging and the like have large damage to the fish body, are not suitable for marking small-size fish and are easy to lose;
(5) the method has extremely low marking cost, the marking cost of each fish is less than 0.1 yuan, and the method is suitable for large-scale marking of large yellow croaker release;
(6) the large yellow croaker has strong tolerance to salinity change, the large yellow croaker is directly transferred from natural seawater to artificial seawater with the salinity of 15 per mill, the survival of the large yellow croaker is not obviously influenced, the use amount of refined salt and the like can be reduced by properly reducing the dip-dyeing salinity, and the cost is further saved.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
(1) Preparing 2000mg/L alizarin red stock mother liquor by using distilled water, and fully dissolving alizarin red by using air stone strong aeration for 12 hours during preparation; diluting the mother liquor to 200mg/L by tap water after 48 hours of aeration; using iodine-free refined salt (commercially available), MgSO 4 、MgCl 2 Adjusting salinity to 15 ‰ with KCl to obtain artificial seawater dye solution (per liter dye solution contains iodine-free refined salt 12.3g and MgSO 4 1.0g，MgCl 2 1.6g, KCl 0.3 g). Aerating the dye solution for 12h, adsorbing and precipitating with pneumatic filter cotton (2 per cubic meter of water), and adding NaHCO 3 The solution was adjusted to pH 8.2.
(2) Dyeing at 6m 3 Is carried out in the culture pond. Soaking 12000 average total length of Pseudosciaena crocea fry (about 5cm, pre-adapted in seawater containing alizarin red with salinity of 15 ‰ for 48 hr) in the artificial seawater for 24 hr, continuously aerating, adsorbing and precipitating with pneumatic filter cotton, and adding NaHCO 3 Adjusting the pH value of the solution to be 7.8-8.2; and after dyeing is finished, transferring the fry into natural seawater for rinsing for 24 hours, and removing superficial color on the body surface to obtain the alizarin red marked large yellow croaker.
By observation, the survival rate of the large yellow croaker after marking for 48 hours is 98 percent, and the marking rate reaches 100 percent.
Example 2
(1) Preparing 2000mg/L alizarin red stock mother liquor by using distilled water, and fully dissolving alizarin red by using air stone strong aeration for 12 hours during preparation; respectively diluting the mother liquor to 100 mg/L, 200mg/L and 300mg/L by using tap water after 48h of aeration in 3 300L dyeing barrels; adding iodine-free refined salt 12.3g and MgSO 4 to each liter of diluent 4 1.0g，MgCl 2 1.6g and KCl 0.3g to obtain artificial seawater dye solution with salinity of 15%, aerating for 12h, adsorbing and precipitating with 1 pneumatic filter cotton per barrel, and precipitating with NaHCO 3 The solution was adjusted to pH 8.2.
(2) Soaking and dyeing 500 large yellow croaker fries with total length of 4.68 + -0.82 cm in each barrel for 24 hr, continuously inflating during soaking, adsorbing with pneumatic filter cotton, precipitating, and adding NaHCO 3 Adjusting the pH value of the solution to be 7.8-8.2; and after dyeing is finished, transferring the fry into natural seawater for rinsing for 24 hours to remove the superficial color of the body surface.
The large yellow croaker dyeing marking effect is as follows:
Claims (6)
1. a method for labeling large yellow croaker with alizarin red comprises the following steps:
(1) preparing alizarin red stock mother liquor, diluting the mother liquor to an application concentration, and adjusting the salinity to be more than 15 per thousand to obtain artificial seawater dye liquor;
in the step (1), iodine-free refined salt and MgSO (MgSO) are adopted 4 、MgCl 2 And KCl to regulate salinity;
aerating for 12h after the artificial seawater dye solution is obtained in the step (1), and adsorbing and precipitating by adopting pneumatic filter cotton;
(2) soaking the large yellow croaker fry to be dyed in the dyeing solution for dyeing; after dyeing is finished, transferring the fry into natural seawater for rinsing to obtain alizarin red marked large yellow croaker;
the fry of the large yellow croaker to be dyed in the step (2) is adapted in advance in low-salt seawater with the salinity of 15 per mill for more than 48 hours;
and continuously inflating during the soaking period, adopting pneumatic filter cotton to adsorb and precipitate, and adjusting the pH value by using a NaHCO3 solution to be kept between 7.8 and 8.2.
2. The method for labeling large yellow croaker with alizarin red according to claim 1, wherein: and (2) strongly aerating for 12 hours by using air stones when preparing alizarin red stock mother liquor in the step (1).
3. The method for labeling large yellow croaker with alizarin red according to claim 1, wherein: in the step (1), the concentration is 100-300 mg/L, and tap water with aeration time of more than 48 hours is adopted for dilution.
4. The method for labeling large yellow croaker with alizarin red according to claim 1, wherein: NaHCO is used after the artificial seawater dye solution is obtained in the step (1) 3 Adjusting the pH value of the solution to 7.8-8.2.
5. The method for labeling large yellow croaker with alizarin red according to claim 1, wherein: the soaking density of the large yellow croaker to be dyed in the step (2) is 1.5-2.5 tails per liter, and the soaking time is 24 hours.
6. The method for labeling large yellow croaker with alizarin red according to claim 1, wherein: and (3) filtering the dyeing solution obtained after dyeing in the step (2) for repeated use.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610823069.3A CN106614217B (en) | 2016-09-13 | 2016-09-13 | Method for labeling large yellow croaker with alizarin red |
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| CN201610823069.3A CN106614217B (en) | 2016-09-13 | 2016-09-13 | Method for labeling large yellow croaker with alizarin red |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107279018B (en) * | 2017-07-04 | 2020-10-30 | 浙江海洋大学 | Traceable label for large yellow croaker |
| CN108770755A (en) * | 2018-04-08 | 2018-11-09 | 中国水产科学研究院南海水产研究所 | A kind of labeling method suitable for black porgy juvenile fish |
| CN111165417B (en) * | 2019-12-31 | 2022-02-08 | 中国水产科学研究院南海水产研究所 | Mullet juvenile fish coloring agent, coloring method and application of alizarin red in mullet juvenile fish coloring marker |
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