JP5024985B2 - Fish labeling and labeling methods - Google Patents

Description

  The present invention relates to a fish labeling agent and a method for labeling fish using the labeling agent. Specifically, the present invention relates to a novel fish labeling agent and a method for labeling fish by staining a hard tissue of a fish body using the labeling agent. More specifically, the present invention relates to a labeling agent made of a material that has been confirmed to be safe for the human body, and a method for labeling fish safely and inexpensively using the labeling agent.

  In recent years, with the dramatic improvement of seed production technology, the number of marine larvae produced for the purpose of release nationwide reached 37 species and 94 million in 2002. Among them, red sea bream is one of the target species representing cultivated fishery. In 2002, 1,955,000 fry were released nationwide. In addition, flounder is a fish species that represents cultivated fishery together with red sea bream. In recent years, the number of juveniles produced and the number of released tails are the largest among the fishes intended for release. In 2002, 25.9 million nationwide. The tail is released. Oniokoze is a promising target species for cultivated fishery. In 2002, 640,000 fry were released throughout the country.

  The fish labeling methods used in these discharge surveys have been conventionally applied to in vitro labeling methods such as attaching various tagging tags such as anchor tags, phlegm excision, phlegm removal, and branding, and alizarin complexone (hereinafter “ ALC ”), oxytetracycline hydrochloride (hereinafter“ OTC ”), alizarin red S (hereinafter“ ARS ”) and other otolith fluorescent staining methods, and in-vivo labeling methods by subcutaneous injection such as ink and illustrator fluorescent tags. It is used.

  As a problem of fish labeling technology, in vitro labeling methods require a long time to put on, the tag slips off, the rehabilitation of the salmon after excision or removal, the regeneration of the brand mark, etc., making it difficult to identify the label It has been pointed out. On the other hand, in the in-body labeling method, since labeling agents such as ALC and ARS are expensive, a problem of economic efficiency when a large amount is attached to a fry is pointed out. Note that OTC is a veterinary drug prescribed by the Pharmaceutical Affairs Law, and uses other than approved usage are subject to criminal penalties, and therefore cannot be used for actual release tests.

  In recent years, public interest in food safety has increased, but ALC and ARS cannot be clearly stated as drugs that have been confirmed to be safe for the human body. Further, in the future, any labeling method is highly likely to be a safety concern for the human body in that a foreign object is attached to the fish body. Therefore, in the future, it will be necessary to develop a labeling technology that takes this into consideration.

  In the conventional in vivo labeling method, ALC, which is a quantitative reagent for fluoride ions, is frequently used as a labeling agent. When ALC is taken into the fish body, it forms calcium and chelate and deposits on hard tissues such as otoliths. Since the deposited ALC is confirmed as fluorescence by observing with a fluorescence microscope, it can be easily determined whether or not the investigated fish is a released fish. However, as described above, since ALC is concerned with safety and is quite expensive, it is highly preferable if a safer and cheaper labeling agent can be developed for the human body.

  According to the in vivo labeling method, it is possible to label a large amount of fertilized eggs and hatched larvae (larvae just after hatching from fertilized eggs), which is difficult with in vitro labeling methods, and has been confirmed by ALC. Thus, the persistence after labeling is high, and the labeling rate can be increased to 100%. Furthermore, when distinguishing several release groups in a release study, the release of fish requires a multiplicity of fluorescent rings in the otolith, so fluorescent labeling technology in the fertilized egg or hatchling larva stage is indispensable. The labeling method is compatible with this point. In addition, the label | marker in a fertilized egg thru | or hatched larva stage can make the tag unit price per individual low compared with the label | marker in the juvenile stage.

In view of such a situation, the present inventors aimed at the development of a fish internal labeling method capable of solving all the above-mentioned problems, and first investigated patent documents in order to obtain technical information.
Looking at the patent publications, several inventions have been filed and disclosed regarding fish labeling technology, but most of them are inventions related to the improvement of the in vitro labeling method, and there are almost no inventions related to the in vivo labeling method. Slightly, Patent Document 1 discloses a method of labeling otoliths of released fish, but this labeling method changes the water temperature and water quality of fish hatchery ponds to bar code on fish otoliths. It is an invention for forming a sign having a pattern, and is not information to be used as a reference for the problem of the present invention. Patent Document 2 discloses a method of measuring the concentration distribution of strontium in a cross-section passing through the otolith growth base point of ayu captured in a river and discriminating whether it is natural or released. However, this method has nothing to do with the problem of the present invention. Thus, the patent publication does not disclose any information that serves as a reference for solving the problems of the present invention.
Japanese Patent Laid-Open No. 2003-9701 JP 2001-275512 A

  In view of the above situation, it is a first object of the present invention to provide a novel fish labeling agent that has been confirmed to be safe for the human body and a method for safely labeling fish using the labeling agent. . Another object of the present invention is to provide a method for labeling fish safely and inexpensively using an inexpensive labeling agent and the labeling agent that have been confirmed to be safe for the human body.

  Of the present invention for solving the above-mentioned problems, the invention described in claims 1 and 2 is a fish labeling agent comprising a cochineal dye or a drug containing this as an active ingredient.

  The invention described in claim 2 is a fish labeling agent comprising a rack dye or a drug containing the same as an active ingredient.

  The invention described in claim 3 is a fish labeling agent comprising a sicon pigment or a drug containing the same as an active ingredient.

  Moreover, the invention described in claim 4 is a method in which a fertilized egg of a target fish is immersed in a labeling solution in which the labeling agent according to any one of claims 1 to 3 is dissolved, and the hard tissue of the fertilized egg or its hatched larvae It is a method of labeling fish by staining.

  The invention described in claim 5 is the labeling method according to claim 4, wherein the concentration of the labeling solution is 4000 to 8000 mg / L when the labeling agent according to claim 1 is used. When the labeling agent described in 2 is used, it is about 4000 mg / L. When the labeling agent according to claim 3 is used, it is about 0.2 mg / L, and the immersion time is about 24 hours, respectively. Is the method.

  The invention described in claim 6 is the method for labeling fish according to claim 4 or 5, wherein the fish to be labeled is either red sea bream, oniochoze or flounder.

  Further, the invention described in claim 7 is a method of labeling fish by immersing a fry of a fish to be labeled in a labeling solution in which the labeling agent according to claim 1 or 3 is dissolved and staining the hard tissue thereof. is there.

  Further, the invention described in claim 8 is the labeling method according to claim 7, wherein the target fish for labeling is oniochoze or trough puffer and a labeling solution in which the cochineal dye is dissolved at a concentration of about 16000 mg / L is used. This is a method for labeling fry of Oniochoze or tiger puffer fish by staining otoliths among the hard tissues.

  The invention described in claim 9 is the labeling method according to claim 7, wherein a labeling solution in which a cochineal dye is dissolved at a concentration of about 2000 mg / L is used, and spines, scales, It is a method of labeling fish by staining either the lower skull or soft stripes.

  The invention described in claim 10 is the method for labeling fish according to claim 9, wherein the fish to be labeled is a larvae of red sea bream, oniokoze, flounder, or tiger puffer fish.

  Further, the invention described in claim 11 is the labeling method according to claim 7, wherein the labeling fish is red sea bream or onyokoze, and a labeling solution in which a sycon dye is dissolved at a concentration of about 0.5 mg / L is used. This is a method for labeling red sea bream or oniokoze fry by staining otoliths or scales in the hard tissues of the fry.

  Since the fish labeling agents according to the present invention are made of materials that are officially recognized as food additives and herbal medicines, they are safe in terms of food safety even if they are used for the labeling of released fish. There is no fear of mischief. In addition, since the fish labeling methods according to the present invention are both safe for the human body and suitable for use in large-scale labeling, it is possible to reduce the cost required for fish body labeling.

  In addition, the method for labeling fish according to the present invention can sufficiently meet the requirement for multiplicity of the otolith fluorescent ring of the released fish because the target fish can be labeled at the time of fertilized eggs or hatched larvae.

  In particular, in the case of a fish labeling agent comprising a sicon dye or a drug containing this as an active ingredient in the present invention, when labeling a fertilized egg using this, the labeling cost can be kept lower than when using ALC. . In addition, fish labeling agents consisting of sicon pigments or drugs containing them as active ingredients are useful for labeling fertilized eggs or hatched larvae, and for labeling fry when red sea bream or oniokoze are targeted. Therefore, it is very convenient.

  Regarding the labeling agent for fish comprising the cochineal dye or a drug containing this as an active ingredient in the present invention, when labeling hard tissue other than otoliths of fry, the labeling cost can be kept lower than when using ALC. it can. That is, a fish labeling agent comprising a cochineal pigment or a drug containing this as an active ingredient can stain scales, spines, the lower skull, etc., not only for otolith labeling, when juveniles are to be labeled. Thus, it is possible to easily distinguish whether or not the fish is a labeling target fish with these staining conditions. Moreover, it is effective at a concentration of 2000 mg / L that can be safely immersed in any fish species, and scales, thorns, corals, etc. can be sufficiently stained even if the concentration of the labeling solution is kept low, so that the labeling cost is reduced by ALC. It can be kept lower than when using. Thus, the labeling method in which the fry is immersed in a solution (labeling solution) of a labeling agent comprising a cochineal dye or a drug containing this as an active ingredient is a very useful labeling method when the target is not an otolith.

  If the otolith is not targeted, scales can be easily collected from the outside, and therefore sampling is easy when discriminating by scales. Oniochoze and tiger pufferfish do not have scales, but the scorpionfish usually collects spines that have been excised before they are put on the market, and trough puffer can collect the thorns present on the skin, so both can be sampled easily. .

  In addition, a fish labeling agent comprising a cochineal dye or a drug containing the same as an active ingredient is very useful for labeling fry otoliths. For example, it has been confirmed that, for Oniochoze and tiger puffer, labeling of otoliths in the hard tissue is possible. As described above, the fish labeling agent comprising a cochineal dye or a drug containing the same as an active ingredient in the present invention is useful for labeling fertilized eggs or hatched larvae and also for labeling fry. Great results are expected as an agent.

  The present invention is a fish labeling agent comprising a cochineal dye or a rack dye or a drug containing these as an active ingredient. Furthermore, the present invention is a method for labeling fish by dyeing hard tissues of fish using these labeling agents. Specifically, the fertilized egg or the hatched larvae hard tissue is stained by immersing the fertilized egg of the target fish for about 24 hours in a labeling solution prepared by dissolving these labeling agents in filtered seawater or the like. It is preferable to adopt a labeling method. When a labeling agent comprising a cochineal dye or a drug containing this as an active ingredient is used, the target fish is soaked in a labeling solution in which it is dissolved in filtered seawater for about 24 hours, and its hard tissue (especially otoliths). , Scales, thorns, lower skull, soft stripes, etc.) can also be used to label the fry. This method is particularly useful for larvae of Oniochoze or tiger puffer fish when labeling otoliths, and is particularly useful for red sea bream, scallops, flounder, and tiger puffers when targeting hard tissues other than otoliths.

  In addition, the present invention is a fish labeling agent comprising a sicon pigment or a drug containing the same as an active ingredient. Furthermore, the present invention is a method for labeling fish by dyeing the hard tissue of fish using these labeling agents. Specifically, the fertilized egg or the hatched larvae hard tissue is stained by immersing the fertilized egg of the target fish for about 24 hours in a labeling solution prepared by dissolving these labeling agents in filtered seawater or the like. It is preferable to adopt a labeling method. In addition, when using a labeling agent comprising a sicon pigment or a drug containing this as an active ingredient, the target fry is immersed in a labeling solution in which it is dissolved in filtered seawater for about 24 hours, and its hard tissue (especially otolith or It is also possible to take a method of labeling fry by staining scales. This method is particularly useful when the target to be labeled is a red sea bream or a scallop.

  The present inventors speculated that fluorescent labeling on otoliths and the like is possible due to the chemical structure of the labeling agent used, and focused on the anthraquinone ring structure possessed by ALC and ARS. In addition, the labeling agent developed in the present invention is intended for a material that is certified as a food or food additive from the viewpoint of food safety, and a food or food additive that contains a chemical substance having an anthraquinone ring. Searched and examined as targets. As a result, it has been estimated that the cochineal dye and the same rack dye, which are colorant preparations, are useful.

  The inventors confirmed the above estimation by testing. That is, as shown in Test Example 1 to be described later, for the purpose of selecting a material suitable for a labeling agent, there are 29 kinds of commercially available dyes that are recognized as food additives. For a total of 30 pigment materials including shikonin, materials other than the silicone pigment are dissolved in filtered seawater to a concentration of 2000 mg / L, and the silicone pigment is dissolved in filtered seawater to a concentration of 50 mg / L. Then, after immersing red sea bream hatchlings in each solution for 24 hours, the staining condition of otoliths was observed. As a result, the red sea bream hatchling larvae survived and the otolith fluorescent staining was recognized only in the cochineal pigment and the rack pigment among the 30 pigment materials. In addition, although the larvae died (perhaps the concentration of the labeling solution was probably too high), the sicon dye was added to the material under consideration because fluorescent staining was observed in the otoliths. .

  That is, the above preliminary test confirmed that among the anthraquinones, food additives “cochineal dye” and “lac dye” commercially available as anthraquinone dyes are suitable as fish labeling agents. In addition, it was estimated that “sicon dye”, which is a naphthoquinone pigment among naphthoquinones and marketed as a Chinese medicine, is also useful as a fish labeling agent. Therefore, the present inventors decided to further test and examine the usefulness as a fish labeling agent for three materials obtained by adding a chicone pigment to a cochineal pigment and a rack pigment.

  Cochineal pigments belong to anthraquinone pigments and are food additives (coloring agents) extracted from the dried body of scale insects, which are mainly composed of carminic acid. Cochineal pigments are recognized as food safe by a committee that formulates a food standard plan jointly formed by the United Nations Food and Agriculture Organization (FAO) and the World Health Organization (WHO). Etc. are used.

  A rack pigment belongs to an anthraquinone-based pigment, and is a food additive (colorant) extracted from a resinous substance secreted by a scale insect, the scale insect, and mainly contains laccaic acid. In Japan, it is used for rice cakes, confectionery, noodles, etc.

  Sikon pigment is a naphthoquinone pigment extracted from the root of the plant "Murasaki" and is commercially available as a Chinese medicine. Its main components are shikonin, acetylshikonin, isobutylshikonin and the like. Shikonin is naturally present in a very small amount, and most of it exists as an ester compound such as acetylshikonin and isobutylshikonin. In addition to being used as a dye for purple fabrics that have long been known as a noble color, sicon pigments have anti-inflammatory and antitumor activities, and are used as herbal medicines for antipyretic / detoxification, acne treatment, burn treatment, etc. Used for purposes. It is also used as a lipstick pigment. In addition, the sycon pigment | dye in this invention contains what was made to make the cultured cell by biotechnology and refine | purified.

  As the fish labeling agent according to the present invention, not only cochineal pigments, lac pigments and sicon pigments but also agents containing these as active ingredients are useful. Examples of drugs containing these dyes as active ingredients include dye preparations and reagent preparations. For example, as for the cochineal dye, a dye preparation is commercially available under the trade name of each manufacturer, and a reagent preparation is commercially available under the trade name of “Cochineal” or “Cochineal dye”. The same applies to rack dyes and silicon dyes, and reagent preparations are commercially available in addition to dye preparations.

  In general, the otolith is the most suitable site for labeling fish, because the most suitable site for observing the presence or absence of fluorescent staining in a hard tissue of a fish is the otolith. Is widely used. Otoliths, also known as auditory sand, are granular calcareous materials in the vestibule of the inner ear of the fish body that serve to maintain a sense of balance. Even in fertilized eggs, otoliths appear soon after the formation of otocysts in the embryo. However, in the method for labeling fish according to the present invention, particularly when a fry fish is labeled using a cochineal dye solution, the fluorescent staining site is not limited to the otolith, but a hard tissue other than the otolith, such as a spine ( In particular, dorsal fin second spine, heels (especially the lower skull), scales, soft stripes, etc. can be easily stained, and the fluorescence status can be confirmed by a fluorescence microscope.

  In the present invention, a method of injecting the labeling agent into the fish body can also be adopted. For example, it has been confirmed that otolith labeling is possible by injecting 0.1 mL of physiological saline in which 20 mg of cochineal dye is dissolved into one onioze with a total length of 68 mm. That is, a labeling solution in which a labeling agent is dissolved in physiological saline or the like is injected into the peritoneal cavity of the fish to be labeled at a rate of 0.1 mL / tail. Anesthesia may be performed by immersing fish in sand-filtered seawater in which 0.03% 2-phenoxyethanol is dissolved before injection. The injection is performed under a stereomicroscope, and after fixing the right thoracic fin of the fish with tweezers, the labeling solution is injected into the abdominal cavity from the base of the right pleural cavity using a syringe. When the otolith of the fish injected with the labeling solution is observed with a fluorescence microscope, it can be confirmed that it is a fish with a label because it emits fluorescence, as in the case of the immersion method.

  In the otolith labeling of fertilized eggs in the present invention, the required concentration of the labeling solution varies depending on the labeling agent used and the type of fish to be labeled. That is, as shown in Test Examples 2 to 4 below, in the case where a cochineal dye or a drug containing this as a main component is used as a labeling agent, the concentration of the cochineal dye is 2000 to 8000 mg / L for a red sea bream fertilized egg. Is effective for 2000-16000 mg / L and for flounder fertilized eggs, 4000-16000 mg / L. Therefore, in the case of using a labeling agent comprising a cochineal dye or a drug containing this as an active ingredient, it can be widely used for fertilized eggs in general if the concentration of the cochineal dye is adjusted to “4000 to 8000 mg / L”.

  In addition, when a rack dye or a drug containing this as an active ingredient is used as a labeling agent, the concentration of the rack dye is 1000 to 4000 mg / L for a red sea bream fertilized egg, 1000 to 16000 mg / L for a fertilized onyokoze egg, and a flatfish fertilized egg. Then, it is effective to set it to about 4000 mg / L. Therefore, when a labeling agent comprising a lac dye solution or a drug containing this as an active ingredient is used, it can be widely used for fertilized eggs in general if the concentration of the lac dye is adjusted to about “4000 mg / L”. In the present invention, “about 4000 mg / L” refers to a concentration in a range generally accepted as a fish labeling solution, mainly 4000 mg / L, for example, a concentration of 3900 to 4100 mg / L.

  In addition, when using a sicon dye or a drug containing this as an active ingredient as a labeling agent, the concentration of the sicon dye is 0.1 to 0.2 mg / L for red sea bream fertilized eggs and 0.5 to 2 mg for oniochoze fertilized eggs. For L / F, flounder fertilized eggs, it is effective to be about 0.2 mg / L. Therefore, when using a labeling agent comprising a sycon dye or a drug containing this as an active ingredient, it can be widely used for fertilized eggs in general if the concentration of the sicon dye is adjusted to about “0.2 mg / L”.

  In the labeling of the hard tissue of the fry in the present invention, the required concentration of the labeling solution varies depending on the labeling agent used, the type of the target fish to be labeled, and the labeling site. That is, as shown in Test Examples 5 and 6 to be described later, in the case of using a cochineal dye or a drug containing this as a main component as a labeling agent, the concentration of the cochineal dye is about 16000 mg / L in the otolith labeling of fry of Oniokoze, It is effective to set it to 8000 to 16000 mg / L for a hard tissue label other than the otolith of Oniokose fry, and to 8000 to 16000 mg / L for a label of otolith, thorn, and soft streak of tiger puffer fish. In addition, when using a labeling agent comprising a cochineal dye or a drug containing the same as an active ingredient, adjusting the concentration of the cochineal dye to about “2000 mg / L” can be safely used for fry in general such as red sea bream, oniokoze, flounder, and tiger pufferfish. At the same time, hard tissues other than otolith can be sufficiently stained.

  Further, in the labeling of fry hard tissue in the present invention, when a sykon pigment or a drug containing this as a main component is used, otoliths, scales, or dorsal spines of red sea bream larvae or larvae larvae with a concentration of about 0.5 mg / L can do.

  According to the fish labeling agent and the labeling method using the same according to the present invention, the labeling effect on the hard tissue of fish can be maintained for a long period of time. For example, after immersing an onyokoze with a total length of 65 mm in a 16000 mg / L solution of cochineal pigment, labeling it, and then continuing the breeding after returning to fresh seawater, after 6 months, otoliths, dorsal spines, lower armpits Fluorescent staining has been observed for every part of the skull.

  The target fish of the fish labeling agent and the labeling method according to the present invention is not limited to red sea bream, onyokoze, flounder, and tiger puffer, and it has been confirmed that it can be applied to many fishes such as rockfish, medaka, goldfish. For example, in the case of labeling fry otoliths using a cochineal dye solution based on the present invention, it has been confirmed that the staining effect is observed when the concentration of the labeling solution is about 8000 mg / L in the rockfish, medaka and goldfish. Yes.

  A recent standard price of the labeling agent according to the present invention was examined. The ALC was about 1,000 yen / g, the cochineal dye was about 13 yen / g, the rack dye was about 9 yen / g, and the chicone dye was about 6000. It was confirmed that the product was sold in yen / g (by a commercially available pigment preparation). When a fertilized egg is labeled, the appropriate concentration of the labeling solution is 4000 to 8000 mg / L for the cochineal dye, about 4000 mg / L for the rack dye, and about 0.2 mg / L for the sicon dye as described above. Therefore, the cost of the labeling agent per liter of labeling solution is 52 to 104 yen for the cochineal dye, 36 yen for the rack dye, and about 1 yen for the chicone dye. When this is compared with ALC, since the appropriate concentration of ALC when labeling a fertilized egg is about 5 mg / L, the labeling cost per 1 L is 5 yen. Therefore, among the labeling agents developed this time, the labeling cost of the sicon dye is lower than that of ALC.

  In addition, when labeling the otoliths of juvenile Oniokose using a cochineal pigment, assuming that the release size is 50 mm in total length and accommodating 3000 fish per 1 kl of seawater, the preferred concentration of the labeling solution is ALC (1000 yen / g) For 50 mg / L and cochineal dye (13 yen / g), the cost per label is 17 yen for ALC and 69 yen for cochineal dye, so ALC is cheaper. . However, when labeling spines, scales, etc., the cochineal dye can be labeled at 1/8 concentration, that is, at a concentration of about 2000 mg / L, so the labeling cost is estimated at 8.7 yen / tail. In terms of cost, it is more effective than ALC.

  Moreover, when labeling red sea bream or scallop fry using a silicon pigment, the preferred concentration of the labeling solution is 50 mg / L for ALC (1000 yen / g), and 0.5 mg / L for the silicon pigment (6000 yen / g). Therefore, the cost of the labeling agent per liter of the labeling solution is 50 yen for ALC and 3 yen for the sicon dye, and the use of the sicon dye is more effective than ALC in terms of cost.

  Hereinafter, the present invention will be described in more detail with test examples. In addition, in Test Examples 1 to 4, the test fish were fertilized eggs of onyokoze that were naturally spawned by the parent fish cultivated at the Fisheries Research Center of Independent Administrative Institution, Hakatajima Cultivation Fishery Center, hatchling larvae of red sea bream and private aquaculture companies Red sea bream fertilized eggs and flounder fertilized eggs that were naturally laid by the cultured parent fish were used. In Test Example 5, Oniokose larvae produced in 2004 and 2005 at the Fisheries Research Center and Hakatajima Cultivation Fisheries Center, flounder fry produced in 2005, and produced in 2006 at the Doyajima Cultivation Fisheries Center Larvae and larvae produced in 2005 by a private farmer were used. In Test Example 6, red sea bream fry produced in 2006 at the Fisheries Research Center and Yajima Cultivation Fisheries Center and Oniokose fry produced in 2006 at the Hakuhojima Cultivation Technology Development Center were used.

  In the following test examples, commercially available pigment preparations were used. That is, “Carmine Red MK-40” (trade name: Kiriya Chemical Co., Ltd.) as the cochineal pigment, “Luckine Red R” (trade name: Kiriya Chemical Co., Ltd.) as the rack pigment, and “Sikonin” as the silicone pigment. (Trade name: Pinoa Co., Ltd.). Of course, the material of the labeling agent according to the present invention is not limited to these. The present inventors have used “SR Red KR” (trade name: Saneigen Co., Ltd.), “Cochineal dye” (trade name: Wako Pure Chemical Industries, Ltd.), “Cochineal” (trade name: Kanto Chemical Co., Ltd.) as cochineal dyes. Company), and “SR Red L” (trade name: Saneigen Co., Ltd.) and “Rack Dye” (trade name: Wako Pure Chemical Industries, Ltd.) It was confirmed that almost the same results as in the following test examples were obtained.

  In the following test examples, in Tables 1 to 11 summarizing the test results, “◯” indicates that the test fish survived or was labeled, and “×” indicates that the test fish was Indicates death or no labeling. In addition, “△” in the term of survival or survival indicates that a part of the tested individuals survived, and “△” in the term of fluorescence staining or staining does not clearly show the fluorescence. No, but not visible. In addition, "-" in a table | surface shows not having observed.

  In addition, when using ALC from the past, the time for immersing fish in the labeling solution is normally “24 hours”. Therefore, in each of the following test examples, the immersion time of the test fish in the labeling solution was tested for 24 hours. Yes. However, in the fish labeling method according to the present invention, the time for immersing the fish in the labeling solution is not limited to 24 hours, and may be arbitrarily set. For example, it can be shortened to about 6 to 12 hours. However, since it is necessary to increase the concentration of the labeling solution when the immersion time is shortened, a large amount of labeling agent is used, and it is undeniable that the labeling cost increases accordingly.

<< Test Example 1 >>
<Preliminary test for selecting materials suitable for fish labeling>
For the purpose of selecting materials suitable for the labeling agent, the following tests were conducted on a total of 30 types of materials, including 29 types of commercially available dyes designated as food additives and silicon dyes marketed as traditional Chinese medicines.
(1) Test method Among the test materials, materials other than the sicon pigment were each dissolved in filtered seawater so as to have a concentration of 2000 mg / L, and used as a labeling solution. In addition, the sicon dye was dissolved in filtered seawater so as to have a concentration of 50 mg / L to obtain a labeling solution. Next, after immersing red sea bream hatchlings in each labeling solution for 24 hours, the staining state of otoliths was observed.
(2) Test results The test results are as shown in Table 1.
(3) Consideration The larval larvae survived and fluorescent staining of otoliths was observed only in Cochineal pigment (Carmine Red MK-40) and Lac pigment (Luckine Red R) among 29 pigment materials. It was. In addition, the sicon pigment (Shikonin) did not survive the red sea bream larvae, but fluorescent staining of otolith was observed. It is presumed that the larvae of the red sea bream died because the concentration of the cocoon pigment was too high. Therefore, this test confirmed that cochineal dyes and rack dyes are useful materials as labeling agents. It was also inferred that sicon dye is a useful material as a labeling agent.

<< Test Example 2 >>
<Practical application test of three kinds of labeling materials using red sea bream fertilized eggs>
About the possibility of labeling of red sea bream fertilized eggs and the conditions of use of three kinds of materials, which are a combination of cochineal dyes and lac dyes that have been recognized as useful as otolith labeling agents, as well as Chinese dyes Tested.
(1) Test method The above-mentioned three kinds of materials were dissolved in filtered seawater so as to have the respective concentrations shown in Table 2, and red sea bream fertilized eggs in which formation of otocysts was confirmed in each solution were immersed for 24 hours. . Fertilized eggs or hatched larvae were collected after a predetermined immersion time, including those that became larvae by hatching during immersion, and the presence or absence of fluorescence of the otolith was observed under a fluorescence microscope.
(2) Test results The test results are as shown in Table 2.
(3) Discussion i. In the cochineal dye solution, fluorescence to the otolith was confirmed at a concentration of 1000 mg / L or more. No deaths occurred at any concentration. The highest fluorescence was at a concentration of 8000 mg / L.
B. In the rack dye solution, fluorescence to the otolith was observed at a concentration of 500 to 4000 mg / L, but death occurred at a concentration of 8000 mg / L. The fluorescence was strong at a concentration of 2000 to 400 mg / L.
C. In the silicon dye solution, death occurred at a concentration of 0.4 mg / L or more. In addition, strong fluorescence to the otolith was confirmed at a concentration of 0.1 mg / L or more.

(4) Comprehensive evaluation The above test results are summarized as follows.
A. As a result of immersing red sea bream fertilized eggs in a solution containing 500 to 8000 mg / L of cochineal pigment and lac pigment for 24 hours, normal hatching can be achieved at a concentration of 8000 mg / L or less for cochineal pigment and 4000 mg / L or less for rack pigment. Admitted. Fluorescence on otoliths was confirmed in all test individuals at a concentration of 2000 mg / L or more for the cochineal dye and 1000 mg / L or more for the rack dye.
B. With the sicon pigment, as a result of being immersed in a solution having a concentration of 0.1 to 10 mg / L for 24 hours, hatching was observed in all test individuals at a concentration of 0.1 to 0.3 mg / L. Fluorescence was also observed.

<< Test Example 3 >>
<Practical application test of three kinds of labeling materials using fertilized eggs
Regarding the use of three kinds of materials, which have been recognized as useful as otolith markers, cochineal dyes and lac dyes, plus sicon dyes used as traditional Chinese medicines, the possibility of labeling fertilized eggs of onyokoze on a practical scale Tested.
(1) Test method The above-mentioned three kinds of materials were dissolved in filtered seawater so as to have the respective concentrations shown in Table 3, and onyokoze fertilized eggs in which the formation of otocysts was confirmed in each solution were immersed for 24 hours. . Fertilized eggs or hatched larvae were collected after a predetermined immersion time, including those that became larvae by hatching during immersion, and the presence or absence of fluorescence of the otolith was observed under a fluorescence microscope.
(2) Test results The test results are as shown in Table 3.
(3) Discussion i. In the cochineal dye solution, fluorescence to the otolith was confirmed at a concentration of 1000 mg / L or more. No deaths occurred at any concentration. The intensity of fluorescence was 4000 mg / L or more.
B. In the rack dye solution, fluorescence to the otolith was confirmed at a concentration of 500 mg / L or more. No deaths occurred at any concentration. The strong fluorescence was 2000 mg / L or more.
C. In the silicone dye solution, death occurred at a concentration of 5 mg / L or more, but death was not observed at a concentration of 2 mg / L or less, and strong fluorescence to otoliths was confirmed at all remaining concentrations.

(4) Comprehensive evaluation The above test results are summarized as follows.
A. Onyokoze fertilized eggs were immersed in a solution containing 250 to 16000 mg / L of cochineal pigment and lac pigment for 24 hours. As a result, normal hatching was observed at a concentration of 16000 mg / L or less using any pigment. It was. Fluorescence on otoliths was confirmed for all test specimens at a concentration of 2000 mg / L or more for cochineal color and 1000 mg / L or more for lac dye.
B. In the case of the sicon dye, as a result of being immersed in a solution having a concentration of 0.1 to 50 mg / L for 24 hours, hatching of all test individuals was observed in the range of 0.1 to 2 mg / L, and fluorescence to the otolith was also observed. Confirmed for all specimens.

<< Test Example 4 >>
<Practical application test of three kinds of labeling materials using flounder fertilized eggs>
About the possibility of labeling of flounder fertilized eggs and the conditions of use on three scales of three kinds of materials that add the chicon pigment used as a Chinese medicine to the cochineal pigment and the rack pigment that have been recognized as useful as otolith markers Tested.
(1) Test method The above three kinds of materials were dissolved in filtered seawater so as to have respective concentrations shown in Table 4, and flounder fertilized eggs in which formation of otocysts was confirmed in the embryos were immersed in the respective solutions for 24 hours. . Fertilized eggs or hatched larvae were collected after a predetermined immersion time, including those that became larvae by hatching during immersion, and the presence or absence of fluorescence of the otolith was observed under a fluorescence microscope.
(2) Test results The test results are as shown in Table 4.
(3) Discussion i. In the cochineal dye solution, fluorescence to the otolith was confirmed at a concentration of 2000 mg / L or more. No deaths occurred at any concentration. The intensity of fluorescence was 4000 mg / L or more.
B. In the rack dye solution, the fluorescence to the otolith was confirmed at a concentration of 2000 mg / L or more. No death occurred at a concentration of 4000 mg / L or less. The fluorescence was strong at a concentration of 4000 mg / L or more.
C. With the sicon dye solution, death occurred at a concentration of 1 mg / L or more, but hatching was observed at a concentration of 0.5 mg / L or less. The fluorescence to the otolith was confirmed at a concentration of 0.1 to 0.2 mg / L. Strong fluorescence was confirmed at a concentration of 0.2 mg / L.

(4) Comprehensive evaluation The above test results are summarized as follows.
A. As a result of immersing the flounder fertilized eggs in a solution containing 500 to 16000 mg / L of cochineal pigment and lac pigment for 24 hours, the cochineal pigment is normal at a concentration of 16000 mg / L or less and the rack pigment is 4000 mg / L or less. Hatching was observed. The fluorescence to the otolith was confirmed at a concentration of 4000 mg / L or more for the cochineal dye and 4000 mg / L or more for the rack dye.
B. As for the sicon dye, it was immersed in a solution of 0.1 to 5 mg / L for 24 hours. In the concentration range of 2 mg / L, hatching survival of all test individuals was observed, and otolith fluorescence was observed for all test individuals at 0.2 mg / L.

<< Test Example 5 >>
<Laboratory test of various fry using cochineal dye>
(1) Test method Cochineal pigments are dissolved in seawater in a water tank so as to have concentrations of 2000, 4000, 8000, and 16000 mg / L, respectively, and oniokoze, red sea bream, Japanese flounder, and trough fish with a total length of 14 to 100 mm. 10 fry were soaked for 24 hours. A water tank having a capacity of 1 to 30 L was used according to the size of the fry. After immersion, all test fish were moved into fresh seawater and their survival status was confirmed. In addition, some individuals are sampled and otoliths and hard tissues other than otoliths are collected, and the presence or absence (staining degree) of fluorescent labels on otoliths is observed under a fluorescence microscope, soaking with the growth of fry Concentration and labeling status were investigated. Furthermore, some individuals were continuously raised to observe the remaining state of the label on the hard tissue.
(2) Test results Test results are shown in Tables 5 and 6 (Otstone staining), Tables 7 and 8 (Hard tissue staining other than otoliths), and Table 9 (Survaf fry survival and staining). ).
(3) Discussion i. In the larvae of 100 mm in length, the onyokoze was confirmed to survive the test fish at any concentration. For all fish species, clear fluorescence staining of otoliths was confirmed at a concentration of 16000 mg / L. However, red sea bream and flounder did not survive at a concentration of 16000 mg / L (Table 5).
B. It was confirmed that even onionkoze with a total length of less than 100 mm survived all test fish at a concentration of 16000 mg / L, and that otoliths could be fluorescently labeled (Table 6).
C. In the trough puffer fish with a total length of 47 mm, the survival of the test fish was confirmed at any concentration. In addition, clear fluorescence to the otolith was confirmed at 8000 mg / L or more. Even at a concentration of 4000 mg / L or less, fluorescence was not invisible (Table 9).

D. Regarding the staining of hard tissues other than otoliths, at the concentration of 2000 mg / L, the dorsal spine and the lower skull (Table 7 and Table 8) are observed in Oniokoze, scales (Table 7) in Japanese flounder, and in red sea bream. It was confirmed that dorsal spines and scales (Table 7) and skin thorns and soft stripes (Table 9) were stained with trough fish. As shown in Tables 5 and 9, 2000 mg / L is a concentration at which any fish species can be safely immersed, and the labeling method for immersing fry in a labeling solution containing about 2000 mg / L of cochineal pigment as an active ingredient is as follows. It is considered to be a useful labeling method when the target is not an otolith.

E. After continuous breeding of a 65 mm long onyokoze soaked in a 16,000 mg / L cochineal dye solution for 24 hours, fluorescence was observed from any of the otolith, dorsal spine, and lower skull after 6 months. It was done. As shown in Tables 5 and 6, the staining condition of the otolith varies depending on the concentration of the labeling solution. However, as shown in Table 7 and Table 8, when the dorsal spine and the lower heel bone, which are hard tissues other than otoliths, are immersed in a labeling solution having a concentration of 2000 mg / L, the labeling cost is lower than that of ALC. Fluorescence is observed in Therefore, it is expected that the fluorescence will remain even after 6 months have passed, as when immersed in a 16000 mg / L labeling solution. As described later, in the Japanese flounder and red sea bream, fluorescent labeling has been confirmed from dorsal spines and scales after 7 months of larvae immersed in a labeling solution of 2000 mg / L.
F. Further, when flounder and red sea bream having a total length of 100 mm soaked in a 4000 mg / L cochineal pigment solution were continuously bred, scales (flatfish, red sea bream), dorsal spines (red sea bream), spider soft stripes even after 7 months had passed. Fluorescence was observed in (flounder). As shown in Table 7, since the fluorescent label is observed especially in the scale even after being immersed in the labeling solution of 2000 mg / L, according to the labeling method of the present invention, the fluorescent label is Even at a concentration at which the labeling cost is cheaper than ALC, it is expected to remain for 7 months or more.
G. As described above, since the fluorescent label can remain for a long period of time, it can be labeled at a lower cost than the conventional labeling method in which the fluorescent label is immersed in the ALC solution. Is understood.

<< Test Example 6 >>
<Labeling test of juvenile red sea bream using sicon pigment>
(1) Test method In the seawater put in the aquarium, the concentration of sicon pigment is 0.1, 0.2, 0.5, 1.0, 2.0, 5.0 and 10.0 mg / L. Each of them was dissolved, and 10 red bream fish with a total length of 78 mm and 10 mm long fry fish were immersed for 24 hours. As the water tank, a red sea bream with a capacity of 10 L was used, and an onyokoze with a capacity of 1 L was used. After immersion, all test fish were moved into fresh seawater and the survival situation was confirmed. In addition, some individuals were sampled to collect otoliths and other hard tissues, and the presence or absence (staining degree) of fluorescent labels on otoliths and the like was observed under a fluorescence microscope.
(2) Test results The test results are as shown in Table 10 (survival and staining properties of red sea bream fry) and Table 11 (survival and staining properties of Oniokose).
(3) Discussion i. In both fry, all individuals survived at a concentration of 0.5 mg / L or less, and some individuals survived even at 1.0 mg / L.
B. In red sea bream otoliths, fluorescent staining was confirmed at a concentration of 0.5 to 1.0 mg / L. In other hard tissues, staining was confirmed with scales and dorsal spines at a concentration of 0.5 to 1.0 mg / L. At 5 mg / L, the dorsal spine staining was weak (Table 10).
C. Fluorescence staining was confirmed at a concentration of 0.1 to 1.0 mg / L in the otolith of scallops and spinal cords (Table 11).
D. Through this test, it was confirmed that Shikon pigment is also useful as a labeling agent for juvenile red sea bream and larvae. The preferred concentration of the labeling solution is 0.5 mg / L for red sea bream larvae and 0.1 to 0.5 mg / L for Oniokose larvae.

As described above in detail, the present invention provides a novel fish labeling agent that has been confirmed to be safe for the human body. The present invention also provides a method for safely labeling fish using the labeling agent. Furthermore, the present invention provides an inexpensive labeling agent that has been confirmed to be safe for the human body and a method for safely labeling a large amount of fish using the labeling agent. Thus, the present invention is an invention that greatly contributes to the improvement of cultivated fishery technology.

Claims (11)

A fish labeling agent comprising a cochineal dye or a drug containing the same as an active ingredient. A fish labeling agent comprising a lac dye or a drug containing the same as an active ingredient. A fish labeling agent comprising a sicon dye or a drug containing the same as an active ingredient. A method for labeling fish by immersing a fertilized egg of a fish to be labeled in a labeling solution in which the labeling agent according to any one of claims 1 to 3 is dissolved, and staining the hard tissue of the fertilized egg or its hatched larvae. In the labeling method according to claim 4, the concentration of the labeling solution is set to 4000 to 8000 mg / L when the labeling agent according to claim 1 is used, and when the labeling agent according to claim 2 is used. A method of labeling fish at 4000 mg / L, when using the labeling agent according to claim 3, 0.2 mg / L, and immersion time of about 24 hours each. 6. The labeling method according to claim 4 or 5, wherein the fish to be labeled is any one of red sea bream, oniokoze or flounder. A method for labeling fish by immersing a fry of a target fish in a labeling solution in which the labeling agent according to claim 1 or 3 is dissolved, and staining the hard tissue. The labeling method according to claim 7, wherein the target fish for labeling is oniochoze or tiger puffer fish, and the otolith is stained by staining the otolith in the hard tissue of the fry using a labeling solution in which the cochineal pigment is dissolved at a concentration of 16000 mg / L. Or a method for labeling fry of trough puffer fish. 8. The labeling method according to claim 7, wherein the spine, the scale, the lower rib or the soft streak of the hard tissue of the fry is stained using a labeling solution in which a cochineal pigment is dissolved at a concentration of 2000 mg / L. How to mark fish by. The labeling method according to claim 9, wherein the fish to be labeled is a fry of any one of red sea bream, oniokoze, flounder, or trough puffer fish. 8. The labeling method according to claim 7, wherein the target fish is red sea bream or onyokoze, and the otolith or the scale is stained in the hard tissue of the fry using a labeling solution in which the pigment is dissolved at a concentration of 0.5 mg / L. How to label red sea bream or oniokoze fry.