AU2020100372A4 - Staining agent and staining method for juvenile mugil cephalus, and application of alizarin red in staining and labeling of juvenile mugil cephalus - Google Patents
Staining agent and staining method for juvenile mugil cephalus, and application of alizarin red in staining and labeling of juvenile mugil cephalus Download PDFInfo
- Publication number
- AU2020100372A4 AU2020100372A4 AU2020100372A AU2020100372A AU2020100372A4 AU 2020100372 A4 AU2020100372 A4 AU 2020100372A4 AU 2020100372 A AU2020100372 A AU 2020100372A AU 2020100372 A AU2020100372 A AU 2020100372A AU 2020100372 A4 AU2020100372 A4 AU 2020100372A4
- Authority
- AU
- Australia
- Prior art keywords
- juvenile
- staining
- mugil cephalus
- labeling
- aqueous solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
- 241000212850 Mugil cephalus Species 0.000 title claims abstract description 98
- 230000000366 juvenile effect Effects 0.000 title claims abstract description 96
- 238000010186 staining Methods 0.000 title claims abstract description 81
- 238000002372 labelling Methods 0.000 title claims abstract description 68
- 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 52
- 238000007447 staining method Methods 0.000 title claims abstract description 7
- 238000005470 impregnation Methods 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 13
- 239000007864 aqueous solution Substances 0.000 claims description 40
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 39
- 241000251468 Actinopterygii Species 0.000 claims description 28
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 27
- 235000002639 sodium chloride Nutrition 0.000 claims description 26
- 239000011780 sodium chloride Substances 0.000 claims description 26
- 238000009395 breeding Methods 0.000 claims description 23
- 230000001488 breeding effect Effects 0.000 claims description 23
- 239000003795 chemical substances by application Substances 0.000 claims description 13
- 238000002791 soaking Methods 0.000 claims description 10
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 8
- 229910052760 oxygen Inorganic materials 0.000 claims description 8
- 239000001301 oxygen Substances 0.000 claims description 8
- 230000009467 reduction Effects 0.000 claims description 5
- 230000000694 effects Effects 0.000 abstract description 28
- 230000008569 process Effects 0.000 abstract description 6
- 230000035755 proliferation Effects 0.000 abstract description 6
- 230000014759 maintenance of location Effects 0.000 abstract description 5
- 230000004083 survival effect Effects 0.000 abstract description 3
- 238000001917 fluorescence detection Methods 0.000 abstract description 2
- 238000012544 monitoring process Methods 0.000 abstract description 2
- 239000007850 fluorescent dye Substances 0.000 description 16
- 239000012452 mother liquor Substances 0.000 description 15
- JKYKXTRKURYNGW-UHFFFAOYSA-N 3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid Chemical compound O=C1C2=CC=CC=C2C(=O)C2=C1C(O)=C(O)C(S(O)(=O)=O)=C2 JKYKXTRKURYNGW-UHFFFAOYSA-N 0.000 description 12
- 239000000243 solution Substances 0.000 description 11
- 238000001215 fluorescent labelling Methods 0.000 description 10
- 239000000975 dye Substances 0.000 description 9
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical group [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 8
- 230000002354 daily effect Effects 0.000 description 6
- 238000010586 diagram Methods 0.000 description 6
- 239000013505 freshwater Substances 0.000 description 6
- 230000006698 induction Effects 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 4
- 230000002842 otolith Effects 0.000 description 4
- 210000001265 otolithic membrane Anatomy 0.000 description 4
- 230000001737 promoting effect Effects 0.000 description 4
- 235000017557 sodium bicarbonate Nutrition 0.000 description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- 238000005273 aeration Methods 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000035515 penetration Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000001044 red dye Substances 0.000 description 2
- 239000013535 sea water Substances 0.000 description 2
- 241001247278 Acanthopagrus schlegelii Species 0.000 description 1
- 241000269782 Mugil Species 0.000 description 1
- 241000269777 Mugilidae Species 0.000 description 1
- 241000134214 Mugiliformes Species 0.000 description 1
- 241000269979 Paralichthys olivaceus Species 0.000 description 1
- 241000700608 Sagitta Species 0.000 description 1
- 241001441745 Sepia esculenta Species 0.000 description 1
- 241001053214 Spinibarbus sinensis Species 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000013441 quality evaluation Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 235000019553 satiation Nutrition 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/90—Sorting, grading, counting or marking live aquatic animals, e.g. sex determination
- A01K61/95—Sorting, grading, counting or marking live aquatic animals, e.g. sex determination specially adapted for fish
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B1/00—Dyes with anthracene nucleus not condensed with any other ring
- C09B1/02—Hydroxy-anthraquinones; Ethers or esters thereof
- C09B1/06—Preparation from starting materials already containing the anthracene nucleus
- C09B1/12—Dyes containing sulfonic acid groups
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B67/00—Influencing the physical, e.g. the dyeing or printing properties of dyestuffs without chemical reactions, e.g. by treating with solvents grinding or grinding assistants, coating of pigments or dyes; Process features in the making of dyestuff preparations; Dyestuff preparations of a special physical nature, e.g. tablets, films
- C09B67/0071—Process features in the making of dyestuff preparations; Dehydrating agents; Dispersing agents; Dustfree compositions
- C09B67/0083—Solutions of dyes
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1011—Condensed systems
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Marine Sciences & Fisheries (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Farming Of Fish And Shellfish (AREA)
Abstract
The present invention provides a staining agent and staining method for juvenile Mugil
cephalus, and application of alizarin red in staining and labelling of the juvenile Mugil cephalus,
5 belonging to the technical field of tracking and monitoring of proliferation and release. Alizarin
red has high solubility and a good labeling effect. After impregnation staining of the juvenile
Mugil cephalus, good labeling is achieved on both scales and fin spines, and the total labeling
rate in fluorescence detection of all samples reaches 100%. After temporary culture for 40 d, the
labeling retention rate is 100%, and thus the labeling retention effect is good. In addition,
J staining and labeling the juvenile Mugil cephalus by employing alizarin red is simple in
operation, can obtain a high-quality labeling effect, and can ensure the survival rate of the
juvenile Mugil cephalus in the labeling process.
Description
The present invention relates to the technical field of tracking and monitoring of proliferation and release, and in particular to a staining agent and staining method for juvenile Mugil cephalus, and application of alizarin red in staining and labelling of the juvenile Mugil cephalus.
J Mugil cephalus belongs to the genus Mugil of family Mugilidae under Mugiliformes. It is an eurythermic and euryhalic fish that is commonly seen in South-East coastal areas of China, can live in fresh water, brackish water and saline water, prefers to inhabit coastal nearshore, bays and river estuaries, grows rapidly, has a large body and thick meat, and a delicious taste. It is one of the main economic fishes cultured in brackish water in the south coast of China, and is also an important species in fishery production all long.
Proliferation and release is recognized as one of the more direct and effective means for conserving aquatic biological resources at home and abroad. It has achieved relatively good results in aspects of promoting the recovery of fishery population resources, improving the ecological environment of water areas, promoting the protection of endangered species and biodiversity, promoting the increase of fishermen's income and increasing fishery efficiency. As an important basic technical means for evaluating an effect of proliferation and release, a marking/labeling technology is also a key technology for improving the accuracy of evaluating the effect of proliferation and release and promoting the progress of the proliferation and release work.
Traditional marking/labeling techniques such as external marking (using various types of signboards) and external labeling (pterygiophore excision and branding) have a great damage to a marking object and a high mortality rate, and signboard-detachment problems are existed for the hanged signboard marks, while for pterygiophore excision, it may be difficult to identify the labeled fish after regeneration due to the strong recovery ability of the labeled fish. Internal marks such as miniature line code marks, passive radar integrated marks and embedded fluorescent marks have less influence on an object to be labeled, a wide application range, a high group discriminability, but expensive equipment. As a potentially effective and low-cost labeling method, fluorescent impregnation-staining labeling has an obvious effect in forming labels on a hard bone tissue of the object to be labeled by using a fluorescent dye, and has been successfully tested in technologies of labeling various release species such as Acanthopagrus schlegelii, Paralichthysolivaceus, Spinibarbussinensis, Sepia esculenta and the like.
For the selection of labeling materials and labeling methods, it is usually necessary to conduct comparison and screening from many aspects such as the labeling efficiency, safety, J retention rate of the labeling effect, etc., and meanwhile factors such as operation popularity and labeling cost should also be considered comprehensively.
Due to the different sensitivities of different objects to be labeled to dyes, the manner of impregnation-staining labeling, the type and concentration selection of dyes are also different. At present, there is no patent report on fluorescent impregnation-staining labeling of Mugil cephalus at home and abroad. Therefore, it is urgent to provide a fluorescent impregnation-staining labeling material and method suitable for releasing species of Mugil cephalus.
An objective of the present invention is to provide a staining agent and staining method for J juvenile Mugil cephalus, and application of alizarin red in staining and labelling of the juvenile Mugil cephalus, where the alizarin red can achieve a good labeling effect when applicationd for staining and labelling of the juvenile Mugil cephalus.
In order to realize the objective of the present invention, the present invention provides the following technical solutions.
The present invention provides application of alizarin red of formula I in staining and labeling of juvenile Mugil cephalus;
S OH Na
0 OH
Formula I.
The present invention provides a staining agent for juvenile Mugil cephalus. The staining agent includes an aqueous solution of alizarin red and an aqueous solution of sea salt; the mass concentration of alizarin red in the aqueous solution of alizarin red is 150-450 mg/L; the percentage mass content of a sea salt in the aqueous solution of sea salt is 30%-40%; and the aqueous solution of alizarin red and the aqueous solution of sea salt are separately packaged.
Preferably, the pH value of the aqueous solution of alizarin red is 7.0-8.0.
The present invention provides a method for staining juvenile Mugil cephalus based on the staining agent recited in the aforementioned solution, including the following steps:
J 1) from 10-15 days before staining, gradually reducing the salinity of the juvenile Mugil cephalus breeding water to 0, wherein the mass fraction of daily salinity reduction is 2%o-3%o;
2) after feed deprivation of the juvenile Mugil cephalus is conducted for 1 day, putting and soaking the juvenile Mugil cephalus into an aqueous solution of sea salt at a density of 4-6 fishes/L for 8-12 min; and
3) after soaking in the aqueous solution of sea salt, putting the soaked juvenile Mugil cephalus into an aqueous solution of alizarin red at a density of 3.5-4 fishes/L for impregnation staining for 10-14 h to obtain a labeled juvenile Mugil cephalus.
Preferably, the staining method further includes feeding feedstuff to the juvenile Mugil cephalus twice a day 10-15 days before staining, where the mass fraction of the feeding amount at each time of feeding accounts for 3-5% of the weight of the juvenile Mugil cephalus.
Preferably, the initial body length of the juvenile Mugil cephalus is 2-4 cm.
Preferably, during the soaking in the step 2) the dissolved oxygen amount in the aqueous solution of sea salt is maintained > 5.00 mg/L.
Preferably, during the impregnation staining in the step 3) the dissolved oxygen in the aqueous solution of alizarin red is maintained > 5.00 mg/L.
Preferably, after the impregnation staining in the step 3), the method further comprises putting the labeled juvenile Mugil cephalus into water for 1-3 h.
The beneficial effects of the present invention: the present invention provides the application of alizarin red in the staining and labeling of juvenile Mugil cephalus. Alizarin red has high solubility and a good labeling effect. After impregnation staining of the juvenile Mugil cephalus, good labeling is achieved on both scales and fin spines, and the total labeling rate in fluorescence detection of all samples reaches 100%. After temporary culture for 40 d, the labeling retention rate is 100%, and thus the labeling retention effect is good. In addition, staining and labeling the juvenile Mugil cephalus by employing alizarin red is simple in operation, can obtain a high-quality labeling effect, and can ensure the survival rate of the juvenile Mugil cephalus in the labeling process.
FIG. 1 shows a body length measurement result of juvenile Mugil cephalus that has been labeled with an ARS fluorescent dye and cultured for 0-40 days;
FIG. 2 shows a weight measurement result of juvenile Mugil cephalus that has been labeled with an ARS fluorescent dye and cultured for 0-40 days;
FIG. 3 shows the labeling effects of ARS at different concentrations in staining pterygiophores, fin spines, scales and otoliths of juvenile Mugil cephalus;
FIG. 4 is an observation diagram of the fluorescent labeling effect on the scales of juvenile Mugil cephalus;
FIG. 5 is an observation diagram of the fluorescent labeling effect on the fin spines of juvenile Mugil cephalus; and
FIG. 6 is an observation diagram of the fluorescent labeling effect on the pterygiophores of juvenile Mugil cephalus.
The present invention provides application of alizarin red of formula I in staining and labeling of juvenile Mugil cephalus; during the application, the alizarin red is applicationd as a fluorescent labeling dye; the alizarin red (ARS) is orange yellow or yellow brown powder, is easily soluble in water, and has a molecular formula of C4 H7NaO 7S-H20; and the alizarin red has high solubility and a good labeling effect; o O Na'
Formula I.
The present invention provides a staining agent for juvenile Mugil cephalus, where the staining agent includes an aqueous solution of alizarin red and an aqueous solution of sea salt that are packaged separately; the mass concentration of the alizarin red in the aqueous solution of alizarin red is 150-450 mg/L, preferably 200-400 mg/L, and more preferably 250-350 mg/L; the percentage mass content of a sea salt in the aqueous solution of sea salt is 30%-40%, and preferably 35%, where the salinity of 35% will not bring any additional stress damage to the J object to be labeled while ensuring the permeation induction effect, thereby avoiding safety risks arising therefrom; and the aqueous solution of alizarin red and the aqueous solution of sea salt are separately packaged. In the present invention, the pH value of the aqueous solution of alizarin red is preferably 7.0-8.0, and more preferably 7.5-7.8, and the aforementioned pH value ranges are suitable for survival of juvenile Mugil cephalus.
In the specific implementation process of the present invention, the aqueous solution of alizarin red is preferably obtained by diluting an alizarin red mother liquor; the alizarin red mother liquor includes water and the alizarin red; the mass concentration of the alizarin red in the alizarin red mother liquor is preferably 1,800-2,200 mg/L, and more preferably 2,000 mg/L; the pH value of the alizarin red mother liquor is preferably 7.0-8.0, and more preferably 7.5-7.8; the reagent for adjusting the pH value of the alizarin red mother liquor is preferably sodium bicarbonate; the present invention has no special limitation on the water in the alizarin red mother liquor, and the juvenile Mugil cephalus breeding water, fresh water or distilled water all can be adopted; and the dilution process is realized by adding water into the alizarin red mother liquor.
The present invention provides a method for staining juvenile Mugil cephalus based on the staining agent recited in the aforementioned solution, including the following steps:
1) from 10-15 days before staining, gradually reducing the salinity of the juvenile Mugil cephalus breeding water to 0, wherein the mass fraction of daily salinity reduction is 2%o-3%o;
2) after feed deprivation of the juvenile Mugil cephalus is conducted for 1 day, putting and soaking the juvenile Mugil cephalus into an aqueous solution of sea salt at a density of 4-6 fishes/L for 8-12 min; and
3) after soaking in the aqueous solution of sea salt, putting the soaked juvenile Mugil cephalus into an aqueous solution of alizarin red at a density of 3.5-4 fishes/L for impregnation staining for 10-14 h to obtain a labeled juvenile Mugil cephalus.
In the present invention, from 10-15 days before staining, the salinity of juvenile Mugil cephalus breeding water is gradually reduced to 0, where the mass fraction of daily salinity reduction is 2%o-3%o, and preferably 2.5%o; preferably, it further includes feeding feedstuff to the J juvenile Mugil cephalus from 10-15 days before staining, twice a day, and preferably once in the morning and once in the evening, where the mass fraction of the feeding amount at each time of feeding accounts for 3-5% of the weight of the juvenile Mugil cephalus; the body length (initial body length) of the juvenile Mugil cephalus before the treatment is preferably 2-4 cm, and more preferably 3 cm.
In the present invention, feed deprivation of the juvenile Mugil cephalus is conducted for 1 day, and then the juvenile Mugil cephalus are put into the aqueous solution of sea salt at a density of 4-6 fishes/L for 8-12 min, and preferably 10 min, where the function of feed deprivation is to reduce stress and metabolism, thereby avoiding fish death caapplicationd by the stress and metabolism; It is preferable that the amount of dissolved oxygen in the aqueous J solution of sea salt is maintained > 5.00 mg/L during the soaking process; and the density of the juvenile Mugil cephalus is preferably 5 fishes/L.
In the present invention, the juvenile Mugil cephalus is soaked in the aqueous solution of sea salt, and the effect of the soaking is to carry out penetration induction before the operation of impregnation-staining labeling; surface cells of the object to be labeled are rapidly dehydrated by changing the salinity of a water body in which the juvenile Mugil cephalus is located at a high intensity in a short time; and then impregnation-staining is carried out, so that the absorption of dye by the juvenile Mugil cephalus can be enhanced, thereby enhancing the labeling effect.
After soaked in the aqueous solution of sea salt, the soaked juvenile Mugil cephalus are put into the aqueous solution of alizarin red at a density of 3.5-4 fishes/L for impregnation staining for 10-14 h, and preferably 12 h, so as to obtain labeled juvenile Mugil cephalus; the amount of dissolved oxygen in the aqueous solution of alizarin red is preferably > 5.00 mg/L during the impregnation staining; and the density of the juvenile Mugil cephalus is preferably 3.7 fishes/L.
In the present invention, after the impregnation staining, it preferably further includes putting the labeled juvenile Mugil cephalus into water for 1-3 h, preferably 2 h, so as to remove the residual dye (the alizarin red).
The technical solutions in the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are merely a part rather than all of the embodiments of the present invention. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
J Example 1
The experimental Mugil cephalus to be tested as provided in this example had an initial body length of 3.08 0.07 cm, were temporarily cultured in a 500 L breeding bucket for 10 days at a temporary culture density of 1.4 fishes/L. The whole experimental process was carried out in a workshop. Sea water was taken from open seas and then subjected to rough filtration through a sand filter before entering the workshop. Fresh water was taken from mountain spring water, and then filtered through a mesh bag before entering the workshop to remove larger particulate impurities from the water. A total of 500 juvenile Mugil cephalus (0.2 fishes/L) were transferred to a 5 m3 square culture pond (250 x 250 cm, with a water depth of 80 cm) in a seedling workshop for temporary culture for 10 days. During the temporary culture, commercial grain fish J feed was fed twice a day until satiation.
During the initial stage of the temporary culture, the salinity was 15.0 2.0 %o, and the salinity of the culture pond was reduced by continuously pouring fresh water into the culture pond every day until all the sea water is replaced by fresh water, where the daily salinity reduction was 2%o-3%o. During the breeding, the water temperature was 21.7-23.2°C, the dissolved oxygen was 5.16-5.42 mg/L, the pH was 7.5-8.3, the photoperiod was 14 L/OD, and the water is replaced daily during the breeding at the water replacement quantity of 50%.
Alizarin red was dissolved in filtered breeding water (fresh water) to prepare a 2,000 mg/L mother liquor for later application; and during the preparation of the mother liquor, the pH value of the solution was adjusted by adding sodium bicarbonate (molecular formula: NaHCO3) to keep it at 6.5-8.0.
The mother liquor was subjected to shading and aeration for 12 h, and then the pH value of the solution was adjusted by using sodium bicarbonate again, so that the pH value of the solution was finally stabilized at 7.5-7.8.
A temporary culture bucket with a capacity of 30 L was applicationd as a impregnation staining vessel A, and was added with 22 L of a 35%o saline solution prepared from clean cultivation water and sea salt; 110 juvenile Mugil cephalus that had been subjected to feed deprivation for one day were randomly placed into the bucket and soaked for 10 min to carry out penetration induction while remaining an aeration state.
A temporary culture bucket with a capacity of 60 L was applicationd as a impregnation staining vessel B, and added with filtered breeding water, 30 L of a 150 mg/L of J impregnation-staining labeling solution was formulated from the mother liquor, with the pH being adjusted to 7.6 and the water temperature being natural (23 2.3°C). 110 juvenile Mugil cephalus were randomly placed in each impregnation-staining bucket that was continuously inflated by an air pump, so as to conduct impregnation-staining for 12 h.
After the impregnation staining was finished, the labeled fishes were put into fresh breeding water for 2 h to remove the residual dye and complete the labeling operation.
Example 2
The temporary culture of juvenile Mugil cephalus, infiltration induction and preparation of the mother liquor of the alizarin red dye liquor provided in this example were as those shown in the above example 1.
A temporary culture bucket with a capacity of 60 L was applicationd as a impregnation staining vessel B, and added with filtered breeding water, 30 L of a 300 mg/L of impregnation-staining labeling solution was formulated from the mother liquor, with the pH being adjusted to 7.6 and the water temperature being natural (23 2.3°C). 110 juvenile Mugil cephalus were randomly placed in each impregnation-staining bucket that was continuously inflated by an air pump, so as to conduct impregnation-staining for 12 h.
Example 3
The temporary culture of juvenile Mugil cephalus, infiltration induction and preparation of the mother liquor of the alizarin red dye liquor provided in this example were as those shown in the above example 1.
A temporary culture bucket with a capacity of 60 L was applicationd as a impregnation staining vessel B, and added with filtered breeding water, 30 L of a 450 mg/L of impregnation-staining labeling solution was formulated from the mother liquor, with the pH being adjusted to 7.6 and the water temperature being natural (23 2.3°C). 110 juvenile Mugil cephalus were randomly placed in each impregnation-staining bucket that was continuously inflated by an air pump, so as to conduct impregnation-staining for 12 h.
Comparative Example 1
The temporary culture of juvenile Mugil cephalus and infiltration induction provided in this example were as those shown in the above example 1.
SA temporary culture bucket with a capacity of 60 L was applicationd as an impregnation staining vessel B, and added with 30 L of filtered breeding water, the pH was adjusted to 7.6, and the water temperature was natural (23 2.3°C). 110 juvenile Mugil cephalus were randomly placed in each impregnation-staining bucket that was continuously inflated by an air pump, so as to conduct impregnation-staining for 12 h.
After the impregnation-staining test was completed, a growth test was carried out by comparing the labeled test juvenile Mugil cephalus from the above 3 examples (examples 1-3) with the juvenile Mugil cephalus of the same batch (comparative example 1). 90 juvenile fishes were randomly selected from the juvenile fishes (110 fishes) of each group that had been labeled with dyes of different concentrations and specifications for breeding experiments. Three replicate J treatments (n = 30) were set up for each concentration gradient group, and were respectively placed in three net cages (140 x 70 x 120 cm; where the water depth in the net cage was 80 cm, and the density of experimental fishes was 0.038 fishes/L). Continuous aeration was carried out throughout the breeding experiment. During the breeding, the water temperature was 21.7-23.2°C, the dissolved oxygen was 5.16-5.42 mg/L, the pH was 7.5-8.3, the photoperiod was 14 L/1OD, and the daily water replacement rate during the breeding accounted for 25% of the breeding water body.
The overall lengths and weights of all juvenile Mugil cephalus were measured with the data being accurate to 0.1 mm and 0.1 g respectively, and the breeding experiment lasted 40 days. At day 20 and day 40 of breeding, the body lengths and weights of experimental fishes in each group were determined.
After the breeding of the growth experiment was completed, 10 fishes were taken from each replicate treatment (a total of 30 fishes for each staining concentration) and dissected to remove sagittas, scales (5 scales were taken from each of the back, abdomen and tail of each fish, and 15 scales were obtained in total), fin spines and pterygiophores. The taken samples were cleaned, and then stored with protection from light. Detection and analysis were completed within 1 month.
The fluorescence labels were observed by a fluorescence microscope (Olympus BX51) equipped with a digital camera (Olympus DP70). Labeling quality evaluation was graded and quantified, and the grading standards were referred in the following table (Table 1) for the . The fluorescence labels of all samples were observed, and recorded to evaluate the staining quality of J fluorescence labeling. A sample was evaluated and recorded by two testers separately for the labeling quality, and if there is a deviation, a third tester will determine it. In this test, if the labeling quality was >2, then the labeling quality was considered good.
Table 1 Labeling Clarity Grades and Classification Standard
Labeling Clarity ClassificationStandard Grades
Grade 0 No label can be seen under the fluorescence microscope
Grade 1 Blurred labels can be seen under the fluorescence microscope
Grade 2 Easily-distinguishable labels can be seen under the fluorescence microscope
Grade 3 Bright labels can be seen under the fluorescence microscope.
Grade 4 Labels can be seen under natural transmission light
Grade 5 Clear labels can be seen under natural transmission light
Statistical analysis: SPSS and Excel were applicationd to process the relevant data.
One-way ANOVA was applicationd to analyze the significant difference level. The data was expressed by "mean standard deviation".
The body length determination results of juvenile Mugil cephalus after being labeled with the ARS fluorescent dye and cultured for 0-40 days were referred in FIG. 1; and the weight determination results of juvenile Mugil cephalus after being labeled with the ARS fluorescent dye and cultured for 0-40 days were referred in FIG. 2. As could be seen from FIGs. 1 and 2, there was no significant difference in body length and weight of juvenile Mugil cephalus of examples 1-3 compared with the control group (comparative example 1) (p > 0.05) during breeding on day 0, day 20 and day 40.
J The labeling effects of ARS at different concentrations in staining pterygiophores, fin spines, scales and otoliths of juvenile Mugil cephalus were referred in FIG. 3; the observation diagram of the fluorescent labeling effect on the scales of juvenile Mugil cephalus was referred in FIG. 4; the observation diagram of the fluorescent labeling effect on the fin spines of juvenile Mugil cephalus was referred in FIG. 5; and the observation diagram of the fluorescent labeling effect on the pterygiophores of juvenile Mugil cephalus was referred in FIG. 6.
Comparing the labeling qualities of examples 1-3, there was a very significant difference of the 300 mg/L concentration group and the 450 mg/L concentration group (with better labeling qualities) compared with the 150 mg/L concentration group (p < 0.01), and there was no significant difference between the 300 mg/L concentration group and the 450 mg/L concentration J group (p > 0.05). For the overall labeling effect, fin spines > pterygiophores > scales > otoliths. When the ARS concentration reached the level of 300 mg/L or above, the fluorescent staining labeling of the scales, pterygiophores and fin spines could reach the level that was visible to naked eyes, and the staining effect on the otoliths had a labeling effect < 3 under the conditions of each of the three treatment groups.
From the above content, it can be seen that by using the labeling dye and labeling method disclosed by the present invention, there is no significant difference in the body length and weight of juvenile Mugil cephalus compared with the control group after 40 days (p > 0.05). Labeling juvenile Mugil cephalus by ARS impregnation staining can achieve better effects, and is a good object to be selected for fluorescent labeling of juvenile Mugil cephalus, where the formation of labels that can be seen by naked eyes under natural light on fin spines by ARS, provides convenience for direct observation under field conditions, and can greatly improve the detection efficiency. By regarding the lowest impregnation staining concentration, the best effect and the avoidance of impregnation staining risks as principles, in view of the above, the fluorescent labeling dye ARS at a labeling concentration of 350 mg/L is recommended as the optimum labeling condition. Meanwhile, the raw materials applicationd for the labeling are stable in source, easy to obtain, simple and convenient for labeling and high in success rate.
The above description is only preferred embodiments of the present invention. It should be pointed out that, for those of ordinary skills in the art, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be considered as falling into the claimed scope of the present invention.
Claims (5)
1. Application of alizarin red of formula I in staining and labeling of juvenile Mugil cephalus;
0o Na 4
0 OH
Formula I.
2. A staining agent for juvenile Mugil cephalus, wherein the staining agent comprises an aqueous solution of alizarin red and an aqueous solution of sea salt; the mass concentration of alizarin red in the aqueous solution of alizarin red is 150-450 mg/L; the percentage mass content J of a sea salt in the aqueous solution of sea salt is 30%-40%; and the aqueous solution of alizarin red and the aqueous solution of sea salt are separately packaged.
3. The staining agent according to claim 2, wherein the pH value of the aqueous solution of alizarin red is 7.0-8.0.
4. A method for staining juvenile Mugil cephalus based on the staining agent according to claim 2 or 3, comprising the following steps:
1) from 10-15 days before staining, gradually reducing the salinity of the juvenile Mugil cephalus breeding water to 0, wherein the mass fraction of daily salinity reduction is 2%o-3%o;
2) after feed deprivation of the juvenile Mugil cephalus is conducted for 1 day, putting and soaking the juvenile Mugil cephalus into an aqueous solution of sea salt at a density of 4-6 fishes/L for 8-12 min; and
3) after soaking in the aqueous solution of sea salt, putting the soaked juvenile Mugil cephalus into an aqueous solution of alizarin red at a density of 3.5-4 fishes/L for impregnation staining for 10-14 h to obtain a labeled juvenile Mugil cephalus.
5. The staining method according to claim 4, further comprising feeding feedstuff to the juvenile Mugil cephalus twice a day 10-15 days before staining, wherein the mass fraction of the feeding amount at each time of feeding accounts for 3-5% of the weight of the juvenile Mugil cephalus; wherein the initial body length of the juvenile Mugil cephalus is 2-4 cm; wherein during the soaking in the step 2) the dissolved oxygen amount in the aqueous solution of sea salt is maintained > 5.00 mg/L; wherein during the impregnation staining in the step 3) the dissolved oxygen in the aqueous solution of alizarin red is maintained > 5.00 mg/L; wherein after the impregnation staining in the step 3), the method further comprises putting the labeled juvenile Mugil cephalus into water for 1-3 h.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911407011.0A CN111165417B (en) | 2019-12-31 | 2019-12-31 | Mullet juvenile fish coloring agent, coloring method and application of alizarin red in mullet juvenile fish coloring marker |
| CN201911407011.0 | 2019-12-31 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2020100372A4 true AU2020100372A4 (en) | 2020-09-10 |
Family
ID=70620256
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2020100372A Ceased AU2020100372A4 (en) | 2019-12-31 | 2020-03-12 | Staining agent and staining method for juvenile mugil cephalus, and application of alizarin red in staining and labeling of juvenile mugil cephalus |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN111165417B (en) |
| AU (1) | AU2020100372A4 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852408A (en) * | 2020-12-30 | 2021-05-28 | 西藏自治区农牧科学院水产科学研究所 | Labeling reagent and labeling method for lasalosijiri fish |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102696516B (en) * | 2012-06-08 | 2013-12-04 | 上海海洋大学 | Low-salinity pomfret aquiculture method |
| CN106614217B (en) * | 2016-09-13 | 2022-09-30 | 江苏省海洋水产研究所 | Method for labeling large yellow croaker with alizarin red |
| 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 |
-
2019
- 2019-12-31 CN CN201911407011.0A patent/CN111165417B/en not_active Expired - Fee Related
-
2020
- 2020-03-12 AU AU2020100372A patent/AU2020100372A4/en not_active Ceased
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852408A (en) * | 2020-12-30 | 2021-05-28 | 西藏自治区农牧科学院水产科学研究所 | Labeling reagent and labeling method for lasalosijiri fish |
| CN112852408B (en) * | 2020-12-30 | 2024-01-23 | 西藏自治区农牧科学院水产科学研究所 | Marking reagent and marking method for Lassa nojirimi |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111165417A (en) | 2020-05-19 |
| CN111165417B (en) | 2022-02-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Joint et al. | Production of picoplankton and small nanoplankton in the Celtic Sea | |
| Gruber et al. | Biological materials for the study of age and growth in a tropical marine elasmobranch, the lemon shark, Negaprion brevirostris (Poey) | |
| Kraeuter et al. | Oyster growth analysis: a comparison of methods | |
| Strohmeier et al. | Flow reduction, seston depletion, meat content and distribution of diarrhetic shellfish toxins in a long-line blue mussel (Mytilus edulis) farm | |
| Branstrator et al. | Zooplankton dynamics in Lake Victoria | |
| Fritz et al. | Fall and winter movements and activity level of the mummichog, Fundulus heteroclitus, in a tidal creek | |
| Bir et al. | The effects of different water quality parameters on zooplankton distribution in major river systems of Sundarbans Mangrove | |
| Gowing et al. | Feeding ecology of the copepod Lucicutia aff. L. grandis near the lower interface of the Arabian Sea oxygen minimum zone | |
| AU2020100372A4 (en) | Staining agent and staining method for juvenile mugil cephalus, and application of alizarin red in staining and labeling of juvenile mugil cephalus | |
| CN108770755A (en) | A kind of labeling method suitable for black porgy juvenile fish | |
| Strohmeier et al. | Response of Mytilus edulis to enhanced phytoplankton availability by controlled upwelling in an oligotrophic fjord | |
| CN107300546B (en) | Calcein marking method for sepiolite maindroni and detection means thereof | |
| Taib et al. | Density, recruitment and growth performance of Asian green mussel (Perna viridis) in Marudu Bay, Northeast Malaysian Borneo, three years after a massive mortality event. | |
| Weinstein et al. | Exchange of marked juvenile spots between adjacent tidal creeks in the York River estuary, Virginia | |
| Webb et al. | Individual growth rates and movement of juvenile white shrimp (Litopenaeus setiferus) in a tidal marsh nursery | |
| CN103875573B (en) | A kind of method that indicates Shishou section juvenile fish | |
| Braley | Mariculture potential of introduced oysters Saccostrea cucullata tuberculata and Crassostrea echinata, and a histoogical study of reproduction of C. echinata | |
| Friedman et al. | Variation in abundance of blacklip pearl oyster (Pinctada margaritifera Linne.) spat from inshore and offshore reefs in Solomon Islands | |
| Bhattacharyya et al. | Effect of physico-chemical variables on the growth and condition index of the rock oyster, Saccostrea cucullata(Born) in the Sundarbans, India | |
| CN108064773A (en) | A kind of method that saline-alkali water raises and train colored perch juvenile fish | |
| Ajdari et al. | Lobster Fishery and aquaculture development in the North coast of Gulf of Oman: with emphasis on spiny lobster Panulirus homarus | |
| Hossain et al. | Selection of freshwater pearl mussel species for mantle transplantation in Bangladesh | |
| Anam et al. | Morphological and meristic characters of six rabbitfish species (Family: Siganidae) in Kenya | |
| Griffiths | The reproductive season and larval development of the barnacle Tetraclita serrata Darwin | |
| Heasman | The influence of oceanographic conditions and culture methods on the dynamics of mussel farming in Saldanha Bay, South Africa |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FGI | Letters patent sealed or granted (innovation patent) | ||
| MK22 | Patent ceased section 143a(d), or expired - non payment of renewal fee or expiry |