JP6351016B2 - Infectious disease preventive agent for salmon fry for release - Google Patents

Description

  The present invention relates to an infectious disease preventive agent for salmon fry for release, and in particular, contains one or more components selected from the group consisting of oregano oil, carvacrol, para-cymene, linseed oil, cottonseed oil and linolenic acid. One or more selected from the group consisting of oregano oil, carvacrol, para-cymene, gamma-terpinene, linseed oil, cottonseed oil, linolenic acid and linoleic acid, The present invention relates to an infectious disease preventive agent for preventing trichodinosis of salmon fry for release containing ingredients, and a method for preventing infectious diseases of salmon fry for release using these infectious agent preventive agents.

  Salmon is a food fish that is widely popular. Salmon is a breeding project for securing stable fishery resources. In the salmon breeding project, artificial eggs are collected from the parent fish that have returned to Haha River, fertilized with sperm, and then hatched. After hatching and raising fry until it reaches a certain size, it is released into the river.

  On the other hand, both Trichodina and Ikutiobodo are eukaryotic unicellular parasites (protozoa), which parasitize the body surface of salmon juveniles and cause trichodinosis and ichthyobodosis. Salmon juveniles with trichodinosis or ichthyobodosis cause damage or necrosis of the body surface tissue, eventually leading to drowning. Moreover, even if it does not lead to drowning, it will be damaged by the parasite by these parasite parasites and lose its ability to adapt to seawater. In recent years, salmon hatchery has suffered health problems or massive drowning due to trichodinosis or ichthyobodosis, which has become a problem.

  In this regard, Patent Document 1 discloses a ichiobod control agent for salmon and trout containing tea extract, and Patent Document 2 discloses a composition for preventing / treating trichodinosis for fish containing matrine, Each is disclosed.

JP 2006-219478 A JP 2006-28072 A

  However, the ichytobodo control agent described in Patent Document 1 is used by immersing fish in breeding water in which the control agent is dissolved, and in the breeding site of salmon fry that is usually used for pouring, the control agent. It takes time to use. In addition, the composition for preventing / treating trichodinosis described in Patent Document 2 only shows an anthelmintic effect against white spot worms and neobenedenia and an infection prevention effect against white spotted worms in red sea bream and flounder. Has not been shown to prevent trichodinosis or ichthyobodosis. Accordingly, there is a need for an infectious disease preventive agent that can effectively prevent trichodinosis and ichthyobodosis in fry salmon by a simple method such as oral administration.

  The present invention has been made in order to solve such problems, and can effectively prevent trichodinosis and ichthyobodosis by a simple method such as oral administration. It is an object of the present invention to provide a preventive agent for infectious diseases and a method for preventing infectious diseases of salmon juveniles using the same.

  As a result of intensive studies, the inventors have added one or more components selected from the group consisting of oregano oil, carvacrol, para-cymene, linseed oil, cottonseed oil and linolenic acid to the feed and fed, I found that Ikutibodo's infestation of salmon fry can be prevented. Moreover, by adding one or more components selected from the group consisting of oregano oil, carvacrol, para-cymene, gamma-terpinene, linseed oil, cottonseed oil, linolenic acid and linoleic acid to the feed, the salmon fry I found that Trichodina parasitism can be prevented. Accordingly, the following inventions have been completed based on these findings.

(1) The infectious disease preventive agent for preventing ichthyovodosis of salmon fry for release according to the present invention is at least one selected from the group consisting of oregano oil, carvacrol, para-cymene, linseed oil, cottonseed oil and linolenic acid Contains ingredients.

(2) Infectious disease preventive agent for preventing trichodinosis of salmon fry for release according to the present invention is a group consisting of oregano oil, carvacrol, para-cymene, gamma-terpinene, linseed oil, cottonseed oil, linolenic acid and linoleic acid 1 or more components selected from.

(3) In the infectious disease preventive agent of (1), it is preferable that the component is used by adding it to the feed at the following dosage or addition concentration;
Oregano oil; dosage 27 mg / day / kg body weight or less, added concentration 0.09 weight% or less,
Carvacrol; dose of 15.91 mg / day / kg body weight or less, added concentration of 0.053% by weight or less,
Para-cymene; dose 4.23 mg / day / kg body weight or less, additive concentration 0.014% by weight or less,
Linseed oil; dosage 270 mg / day / kg body weight or more, addition concentration 0.9 weight% or more,
Cottonseed oil; dosage 270 mg / day / kg body weight or more, addition concentration 0.9 weight% or more,
Linolenic acid; dosage 156.6 mg / day / kg body weight or more, added concentration 0.5 weight% or more,
Linoleic acid; dosage 40.5 mg / day / kg body weight or more, addition concentration 0.1 weight% or more.

(4) In the infectious disease preventive agent of (2), it is preferable that the above components are used by adding to the feed at the following dosage or addition concentration;
Oregano oil; dosage less than 27 mg / day / kg body weight, added concentration less than 0.09% by weight,
Carvacrol; dose of less than 15.91 mg / day / kg body weight, added concentration of 0.053% by weight or less,
Para-cymene; dose 4.23 mg / day / kg body weight less than 0.014% by weight added,
Gamma-terpinene; dose 2.85 mg / day / kg body weight or less, added concentration less than 0.010% by weight,
Linseed oil; dosage greater than 270 mg / day / kg body weight, added concentration greater than 0.9% by weight,
Cottonseed oil; dosage less than 810 mg / day / kg body weight, added concentration less than 2.7% by weight,
Linolenic acid; dose greater than 156.6 mg / day / kg body weight, added concentration greater than 0.5% by weight,
Linoleic acid; dose greater than 40.5 mg / day / kg body weight, added concentration greater than 0.1% by weight.

(5) The infectious disease preventive agent according to the present invention preferably contains oregano oil and linseed oil, and linseed oil is preferably used as an excipient for oregano oil.

(6) The method for preventing infection of a salmon fry for discharge according to the present invention comprises a step of administering the infection preventive agent according to any one of (1) to (5) to a salmon fry for discharge.

(7) The method of (6) preferably includes a step of administering the infectious disease preventive agent to a salmon fry for release that is not infected with trichodinosis or ichthyobodosis for 7 days or more.

  According to the infectious disease preventive agent and the method for preventing infectious diseases according to the present invention, trichodinosis or ichthyobodosis of salmon fry without adversely affecting the growth and various physiological functions of fry by a simple method such as oral administration. Can be effectively prevented. In addition, according to the infectious disease preventive agent and the method for preventing infectious diseases according to the present invention, the adverse effects on the growth and various physiological functions of salmon fry caused by trichodina and ichthyobodo are significantly reduced to produce healthy salmon fry. can do.

It is a bar graph which shows the parasitic amount of Trichodina in the salmon fry reared by feeding the feed which added the herb essential oil (mint oil, eucalyptus oil, and oregano oil). It is a bar graph which shows the parasitic amount of an ichthyodo in the salmon fry reared with the feed which added the herb essential oil (mint oil, eucalyptus oil, and oregano oil). It is a bar graph which shows the cumulative number of moribund tails of the salmon fry reared with the feed which added the herb essential oil (mint oil, eucalyptus oil, and oregano oil). It is a figure which shows the content component and its content rate of oregano oil. It is a line graph which shows the parasitic amount of Trichodina in the salmon fry reared by feeding the feed which added oregano oil and an oregano oil containing component (carbacrol, para-cymene, gamma-terpinene, and cineol). It is a line graph which shows the parasitic amount of an ichthyodo in the salmon juvenile which was fed with the feed which added oregano oil and an oregano oil containing component (carbacrol, para-cymene, gamma-terpinene, and cineol). It is a bar graph which shows the cumulative number of moribund tails of a salmon fry reared by feeding a feed supplemented with oregano oil and oregano oil-containing components (carbacrol, para-cymene, gamma-terpinene and cineol). It is a bar graph which shows the parasitic amount of Trichodina in the salmon juvenile which was fed with the feed which added the oil for food and feed addition (fish oil, cottonseed oil, and flaxseed oil). It is a bar graph which shows the parasitic amount of Trichodina in the salmon juvenile which was fed with the feed which added the oil for food and feed addition (fish oil, cottonseed oil, and flaxseed oil). It is a bar graph which shows the cumulative number of moribund tails of the salmon fry reared with the feed which added the oil for food and feed addition (fish oil, cottonseed oil, and flaxseed oil). It is a figure which shows the fatty acid composition of a linseed oil. It is a bar graph which shows the parasitism amount of a trichodina and an ichthyobodo, and the cumulative number of moribund tails in the salmon juvenile fed with the feed which added linoleic acid and linolenic acid. Salmon juveniles (Group 1) that had not been fed while being fed with feed supplemented with oregano oil and flaxseed oil before the infection test (hereinafter referred to as “pre-administration rearing”), and pre-administration rearing It is a bar graph which shows the parasitic amount of Trichodina in the salmon fry which performed for 7 days (group 2) and 14 days (group 3). Ichtiobodo parasitism in salmon larvae (Group 1) that had not been pre-administered prior to infection testing, and salmon larvae that had been pre-administered for 7 days (Group 2) and 14 days (Group 3) It is a bar graph which shows quantity. Prior to the infection test, the number of salmon juveniles that had not been pre-administered (Group 1) and the number of salmon fry that had been pre-administered for 7 days (Group 2) and 14 days (Group 3) It is a bar graph to show. It is a bar graph which shows body weight and various physiological functions in the salmon juvenile which was fed with the feed which added oregano oil and flaxseed oil, and was not infected with the protozoa. It is a bar graph which shows the body weight and various physiological functions in the salmon fry after the infection test, which was fed and fed with oregano oil and flaxseed oil.

  Hereinafter, the infectious disease preventive agent for salmon fry for discharge and the method for preventing the infectious disease of salmon fry for discharge according to the present invention will be described in detail.

  In the present invention, “salmon fry for release” refers to salmon fry reared for the purpose of releasing into the natural environment such as rivers and the sea in the future. Here, “salmon” according to the present invention refers to a fish belonging to the family Salmonid. The “fry” according to the present invention is a broad concept including larva and may be used interchangeably with “larf and fry”.

  The infectious disease preventive agent for preventing ichthyobodosis of salmon fry for release according to the present invention contains one or more components selected from the group consisting of oregano oil, carvacrol, para-cymene, linseed oil, cottonseed oil and linolenic acid To do. Further, the infectious disease preventive agent for preventing trichodinosis of salmon fry for release according to the present invention is from the group consisting of oregano oil, carvacrol, para-cymene, gamma-terpinene, linseed oil, cottonseed oil, linolenic acid and linoleic acid. Contains one or more selected components.

Here, oregano oil is a kind of essential oil (volatile oil produced by plants) extracted from the above-ground part (mainly leaves) of the family Lamiaceae oregano ( Origanum vulgare ). Used for. The “oregano oil” according to the present invention can be a commercially available product, or can be extracted from the above-ground part of oregano by a conventional method such as a steam distillation method.

Flaxseed oil is extracted from the seeds of the genus Linum usitatissimum and is generally used for edible, feed, or paint solvents. The “linseed oil” according to the present invention can be a commercially available product or can be extracted from flax seeds by a conventional method such as pressing.

Cottonseed oil is extracted from the seeds of cotton ( Gossypium plant) and is generally used for food. The “cotton seed oil” according to the present invention can be a commercially available product or can be extracted from cotton seeds by a conventional method such as pressing.

  In the present invention, “carbacrol”, “para-cymene”, “gamma-terpinene”, “linoleic acid”, and “linolenic acid” may be chemically synthesized or may be obtained from natural products. In the case of obtaining from natural products, natural products containing these components or extracts thereof may be used as they are, or these components may be further extracted, separated and purified from the natural products or extracts thereof. Good.

  For example, natural products containing “carbachlor” and extracts thereof include perilla plants such as oregano, thyme, medusa, lynx, rosaceae, cypress plants such as hiba, and essential oils thereof. In addition, examples of natural products containing “para-cymene” and extracts thereof include plants such as oregano, thyme, cumin, and lemon, and essential oils thereof. Examples of natural products containing gamma-terpinene and extracts thereof include plants such as oregano, tea tree, thyme, mandarin, lemon, cedar and hiba, and essential oils thereof. Examples of natural products containing linolenic acid and extracts thereof include flax, sesame, rapeseed and soybean seeds and oils extracted from them. In addition, examples of natural products containing linoleic acid and extracts thereof include flax, grape, cotton and soybean seeds, corn germ and oil extracted from them.

  As a method for extracting from a natural product, a general method can be used, and examples thereof include a solvent extraction method using water, an organic solvent, or a mixture of water and an organic solvent. Examples of the solvent extraction method include a method of immersing the natural product, a method of immersing and stirring while heating (room temperature to the boiling point of the solvent). The obtained extract can be used as it is or after the extract is concentrated or dried after filtering and centrifuging to remove solids, if necessary.

  Examples of the organic solvent include lower alcohols such as methanol, ethanol, propanol and butanol, esters such as ethyl acetate, polyhydric alcohols such as ethylene glycol, butylene glycol, propylene glycol, 1,3-butylene alcohol and glycerin, Examples thereof include ethers such as diethyl ether and petroleum ether, polar solvents such as acetone and acetic acid, and hydrocarbon solvents such as benzene, hexane and xylene. These solvents may be used alone or in appropriate combination of two or more.

  As the separation / purification method, a general method can be used, and examples thereof include adsorption / desorption using activated carbon, silica gel, a polymer carrier, column chromatography, liquid-liquid extraction, fractional precipitation, and the like. be able to.

  Examples of the method for administering the agent for preventing infection according to the present invention include oral administration. Oral administration, for example, only the infection prevention agent according to the present invention may be directly orally administered to juvenile fish, and orally administered by adding the infection prevention agent according to the present invention to the feed and feeding the feed. Alternatively, it may be added orally to the breeding water.

  In the infectious disease preventive agent for preventing ichthyododosis of salmon fry for release according to the present invention, the dosage of the above components or the concentration of addition to the feed is not particularly limited. For example, Example 1 (1) described later As shown in FIG. 4, it was found that “oregano oil” has a parasite prevention effect or a mortality suppression effect of ichthiobod at a dose of 27 mg / day / kg body weight or less or an addition concentration to the feed of 0.09% by weight or less. In order to further enhance the parasitic prevention effect of ikutiovo, the dosage is preferably less than 13.5 mg / day / kg body weight or less than 0.045% by weight added to the feed, and the dosage is 5.4 mg / day / kg body weight. More preferably, the concentration is 0.018% by weight or less. In addition, as shown in Example 2 (1) described later, for “linseed oil” and “cotton seed oil”, both doses of 270 mg / day / kg body weight or more or an addition concentration to the feed of 0.9% by weight or more were used. , It has been found that the parasitic prevention effect of iktiovod can be obtained.

  Further, in the infectious disease preventive agent for preventing trichodinosis of salmon fry for release according to the present invention, the dosage of the above components or the concentration of addition to the feed is not particularly limited, but for example, Example 1 described later As shown in (1), for "Oregano oil", it is known that the parasitic prevention effect of Trichodina can be obtained at a dose less than 27 mg / day / kg body weight or less than 0.09% by weight added to the feed. . In addition, as shown in Example 2 (1) described later, for “linseed oil”, the dose of 270 mg / day / kg body weight or greater than 0.9% by weight added to the feed, the effect of preventing trichodina parasitism In order to further enhance the parasitic prevention effect of Trichodina, a dose of 540 mg / day / kg body weight or higher or a concentration added to feed of 1.8% by weight or higher is preferable. Further, as shown in Example 2 (1) described later, with “cotton seed oil”, the parasitic prevention effect of Trichodina can be obtained at a dose of less than 810 mg / day / kg body weight or less than 2.7% by weight added to the feed. In order to further enhance the parasitic prevention effect of Trichodina, a dose of 270 mg / day / kg body weight or more and less than 810 mg / day / kg body weight or an addition concentration to the feed of 0.9 wt% or more and 2.7 wt% % Is preferred.

  Here, as shown in FIG. 4, “carbacrol”, “para-cymene” and “gamma-terpinene” are contained in oregano oil at 58.92 wt%, 15.65 wt% and 10.57 wt%, respectively. . Further, as shown in FIG. 11, “linolenic acid” and “linoleic acid” are contained in linseed oil at 58 wt% and 15 wt%, respectively. Therefore, by multiplying the appropriate dosage of the above oregano oil and linseed oil and the concentration added to the feed by these content ratios, “carbacrol”, “para-cymene”, “gamma-terpinene”, “linolenic acid” ”And“ linoleic acid ”can be determined at suitable dosages and concentrations added to the feed. That is, specifically:

<< Infectious disease preventive agent for preventing ichthyobodosis of salmon fry for release according to the present invention >>
<Carvacrol>
Dose; 27 × 58.92% by weight = 15.99084 = 15.91 mg / day / kg body weight or less, preferably 13.5 × 58.92% by weight = 7.995542 = 7.96 mg / day / kg body weight or less More preferably, 5.4 × 58.92% by weight = 3.118168 = 3.18 mg / day / kg body weight or less.
Concentration added: 0.09 × 58.92 wt% = 0.053028 = 0.053% wt% or less, preferably 0.045 × 58.92 wt% = 0.026514 = 0.027 wt% or less, more preferably 0.018 × 58.92 wt% = 0.01006056 = 0.011 wt% or less.
<Para-cymene>
Dose: 27 × 15.65 wt% = 4.2255 = 4.23 mg / day / kg body weight or less, preferably 13.5 × 15.65 wt% = 2.121275 = 2.11 mg / day / kg body weight More preferably, 5.4 × 15.65 wt% = 0.8451 = 0.85 mg / day / kg body weight or less.
Addition concentration: 0.09 × 15.65 wt% = 0.14085 = 0.014 wt% or less, preferably 0.045 × 15.65 wt% = 0.704425 = 0.007 wt% or less, more preferably 0.018 x 15.65 wt% = 0.002817 = 0.003 wt% or less.
<Linolenic acid>
Dose: 270 × 58 wt% = 156.6 = 156.6 mg / day / kg body weight or more.
Addition concentration: 0.9 × 58 wt% = 0.522 = 0.5 wt% or more.
<Linoleic acid>
Dose amount: 270 × 15% by weight = 40.5 = 40.5 mg / day / kg body weight or more.
Addition concentration: 0.9 × 15 wt% = 0.135 = 0.1 wt% or more.

<< Infectious disease preventive agent for preventing trichodinosis of salmon fry for release according to the present invention >>
<Carvacrol>
Dose; 27 x 58.92 wt% = 15.9084 = 15.91 mg / day / kg body weight.
Addition concentration: 0.09 × 58.92% by weight = 0.053028 = 0.053% by weight or less.
<Para-cymene>
Dose; 27 x 15.65 wt% = 4.2255 = 4.23 mg / day / kg body weight.
Addition concentration: 0.09 × 15.65 wt% = 0.014085 = 0.014 wt% or less.
<Gamma-Terpinene>
Dose amount: 27 × 10.57% by weight = 2.8539 = 2.85 mg / day / kg body weight or less.
Addition concentration: 0.09 × 10.57 wt% = 0.000513 = 0.010 wt%
<Linolenic acid>
Dose; 270 × 58 wt% = 156.6 = 156.6 mg / day / kg body weight, preferably 540 × 58 wt% = 313.2 mg / day / kg body weight or more.
Addition concentration: 0.9 × 58 wt% = 0.522 = 0.5 wt% or more, preferably 1.8 × 58 wt% = 1.04 = 1.0 wt% or more.
<Linoleic acid>
Dose: 270 × 15 wt% = 40.5 = 40.5 mg / day / kg body weight or more, preferably 540 × 15 wt% = 81 mg / day / kg body weight or more.
Addition concentration: 0.9 × 15 wt% = 0.135 = 0.1 wt% or more, preferably 1.8 × 15 wt% = 0.27 = 0.2 wt% or more.

  Here, since the suitable dosage amount of oregano oil or the addition concentration to the feed is a minimum amount, it is difficult to uniformly mix the feed with the feed when added to the feed. Therefore, in the infectious disease preventive agent according to the present invention, among the above components, it is preferable to contain oregano oil and linseed oil and to use linseed oil as an excipient for oregano oil. Since the suitable dosage of linseed oil or the concentration added to the feed is relatively large, it is possible to mix the flaxseed oil with oregano oil to increase the volume and add it to the feed, so that it can be mixed uniformly in the feed . Oregano oil is highly volatile and volatilizes and diminishes during storage or use, whereas flaxseed oil is less volatile. By using linseed oil as an excipient for oregano oil, Oil volatilization can also be suppressed.

  Next, the present invention provides a method for preventing infection of salmon fry for release. The method for preventing infection of a salmon fry for release according to the present invention comprises the step of administering the agent for preventing infection according to the present invention to a salmon fry for discharge. In the method for preventing an infectious disease according to the present invention, the description of the same or corresponding configuration as the above-described infectious disease preventing agent according to the present invention will be omitted.

  In the method for preventing infectious diseases according to the present invention, in order to enhance the preventive effect of trichodinosis and ichthyobodosis, the infectious disease preventive agent according to the present invention is applied to salmon juveniles that are not infected with trichodinosis and ichthyobodosis. Is preferably administered for 7 days or more.

  Hereinafter, the infectious disease preventive agent and method for preventing infectious diseases of salmon fry for release according to the present invention will be described based on each example. The technical scope of the present invention is not limited to the features shown by these examples.

<Experiment method>
In the following examples, the following experimental methods were used unless otherwise specified.
(1) Breeding of fry The fry was grouped into 100 or 500 fish in a 60 L acrylic tank, and was bred under a stream of spring water (water temperature 7.8-8.0 ° C.). The water exchange rate of the water tank was 2 times / hour. The feed used was an initial feed for fish (Altech K-2: Nisshin Marubeni Feed Co., Ltd.). Feeding was given daily in an amount of about 3% body weight / day, divided into three times a day. In each example, the same amount of feed was given to all groups.

(2) Calculation of dose The dose of feed additives (herb essential oil, edible / feed additive oil, or their components) is calculated from the concentration of addition and the amount of feed per day. It was expressed as a daily dose (mg / day / kg body weight) per body weight.

(3) Quantification of Trichodina Since the size of Trichodina is as large as about 100 μm, quantification of Trichodina was performed by microscopic observation. Specifically, first, live larvae were soaked in a 0.2% tricaine aqueous solution for 10 minutes, and all protozoa parasitic on the body surface were detached. Next, the fry was taken out from the tricaine aqueous solution, the entire amount of the remaining tricaine aqueous solution was put into a plankton calculating board, and the number of trichodina individuals existing in the liquid was counted using a stereoscopic microscope observation. The amount of trichodina parasitism was expressed as the number of trichodina individuals per g body weight of fry (individual / g body weight). In comparing the trichodina parasite amount between the groups, an average value was calculated for each group, and the test was performed by the one-way analysis of variance method and the multiple comparison test Tukey-Kramer method.

(4) Quantitative determination of ichthiobodo The size of ikutiovo is as small as 10 μm, and cannot be counted by microscopic observation. Therefore, quantification of ichthiobodo was performed by quantifying the DNA copy number of the ichthiobodo ribosomal RNA gene. In addition, it is known that there is a significant linear correlation between the number of DNA copies and the number of individuals of the ichthiobodo ribosome RNA gene (Mizuno et al., Presented at the 2014 Spring Meeting of the Japanese Fisheries Society).

Specifically, first, live larvae were soaked in a 0.2% tricaine aqueous solution for 10 minutes, and all protozoa parasitic on the body surface were detached. Next, the juvenile was taken out from the tricaine aqueous solution, and the remaining tricaine aqueous solution was centrifuged at 10,000 × g for 15 minutes at 4 ° C. to collect a precipitate, and DNA was extracted from the precipitate according to a conventional method. Using the extracted DNA as a template, specific primers (forward primer; 5'-GTCGTTGTTACGATGCC-3 '(SEQ ID NO: 1), reverse primer; 5'-GCTGTATCTCCTTCTCCC-3' (sequence) No. 2)) and real-time PCR was performed using a real-time PCR apparatus (model 7500: Applied Biosystems). The amount of ictivodo parasitism was expressed as the number of DNA copies (10 ⁻¹⁸ mol (amol) / g body weight) of the ichthiobod ribosomal RNA gene per g body weight of the fry. When comparing the parasitic amount of iktiovo between the groups, an average value was calculated for each group, and the test was performed by the one-way analysis of variance method and the multiple comparison test Tukey-Kramer method.

(5) Seawater transfer test The juvenile fish was put into a 60-liter aquarium filled with 40-liter artificial seawater and reared without feed at 8 ° C. for 48 hours with aeration, and the number of surviving juveniles was counted. The number of survivors after the seawater transfer test was expressed as a percentage of the number of juveniles introduced into the tank as the survival rate (%) after seawater transfer.

(6) Analysis of 鰓 Na ⁺ , K ⁺ -ATPase activity After anesthetizing the juvenile fish with 200 ppm phenoxyethanol, the leftmost outermost valve was collected and stored frozen at −80 ° C. until analysis. In the analysis, the left sputum sample was ground in SEI buffer, and the protein was quantified by the Lowry method, and then used as a sample. This sample was mixed with SEI buffer solution in which ATP was dissolved, and reacted at 37 ° C. for 30 minutes (enzyme reaction group). At the same time, ouabain, which is an inhibitor of Na ⁺ , K ⁺ -ATPase, was added and reacted in the same manner (enzyme reaction inhibition group). Subsequently, the amount of free phosphorus contained in these reaction solutions was measured, and the difference in the amount of free phosphorus between the enzyme reaction group and the enzyme reaction inhibition group was determined. The value of 鰓 Na ⁺ , K ⁺ -ATPase activity was expressed as the difference between the amount of cocoon protein and the amount of free phosphorus released per hour (μmol Pi / mg protein / h).

(7) Analysis of 鰓 Na ⁺ , K ⁺ -ATPase α subunit gene expression level After anesthetizing the juvenile fish with 200 ppm phenoxyethanol, the outermost phlegm valve on the right side was collected and RNase inactive agent (RNAlater; Ambion) until analysis. Company). In the analysis, total RNA was extracted from the right eyelid sample using ISOGEN (Wako Pure Chemical Industries, Ltd.), and then DNase I treatment was performed to remove genomic DNA, thereby preparing a sample. This sample was reverse transcribed using a random primer and reverse transcriptase (Superscript III; Invitrogen) to obtain cDNA. Using the obtained cDNA as a template, specific primers (forward primer; 5′-AGCTCTGCCCACTGGGGCT-3 ′ (SEQ ID NO: 3), reverse primer; 5) designed from the sequence of a known Na ⁺ , K ⁺ -ATPase α subunit gene Real-time PCR was performed using a '-ATTTGTTGGTGGAGTTTGA-3' (SEQ ID NO: 4)) and a real-time PCR apparatus (model 7500: Applied Biosystems). Using the same template and quantitative primers (forward primer; 5′-CCCAAGGCCAACAGAGAGAAA-3 ′ (SEQ ID NO: 5), reverse primer; 5′-GGCGGGGAACGTTTGAAGGT-3 ′ (SEQ ID NO: 6)), an internal control gene (β- The expression level of the actin gene) was also quantified. As the standard, a salmon Na ⁺ , K ⁺ -ATPase α subunit cDNA fragment obtained by cDNA cloning in advance was used.発 現 Na ⁺ , K ⁺ -ATPase α subunit gene expression level was expressed as a relative value relative to the expression level of the internal control gene.

(8) Analysis of plasma lysozyme activity The fry was anesthetized with 200 ppm phenoxyethanol, then the caudal portion was cut with a scalpel, and blood was collected in a hematocrit tube treated with heparin. The collected blood was subjected to centrifugation at 4 ° C. and 2000 × g for 15 minutes, and then the supernatant (plasma) was collected. On the other hand, Micrococcus lysodeikticus was suspended in 0.067M sodium phosphate buffer (pH 6.24) to a concentration of 0.15 mg / mL to prepare a bacterial suspension. 5 mL of plasma was added to 100 mL of the bacterial suspension, and the absorbance was measured at a wavelength of 540 nm every minute for 5 minutes, and the average value of the absorbance change per minute was calculated. The same absorbance measurement was performed using lysozyme standard (Sigma) instead of plasma, and the change in absorbance that 1 g of lysozyme standard changed per minute was defined as 1 Unit. Plasma lysozyme activity was expressed as Unit per minute per mL of plasma (Unit / mL / min).

(9) Analysis of plasma cortisol amount Plasma was collected by the method described in this experimental method (8). Steroids were extracted from plasma using diethyl ether. The amount of cortisol in the steroid was measured using a kit for measuring cortisol by enzyme immunoassay (EA65; Oxford Biomedical Research). The plasma cortisol amount was expressed as the amount per 1 mL of plasma (ng / mL).

(10) Analysis of heat shock protein 70 (Heat Shock Proteins; HSP70) gene expression level Liver was collected from juvenile fish, and cDNA was obtained from the liver sample according to the method described in this experimental method (7). Using the obtained cDNA as a template, specific primers designed from the sequence of a known HSP70 gene (forward primer; 5′-TTCTACACCTCCATCACC-3 ′ (SEQ ID NO: 7), reverse primer; 5′-GTGATGGTGATCTTGTTC-3 ′ (SEQ ID NO: 8)) and a real-time PCR apparatus (model 7500: Applied Biosystems) was used for real-time PCR. The expression level of the internal control gene (β-actin gene) was also quantified using the same template and the quantification primers of SEQ ID NOs: 5 and 6. As the standard, a salmon HSP70 cDNA fragment obtained by cDNA cloning in advance was used. The expression level of HSP70 gene in the liver was expressed as a relative value relative to the expression level of the internal control gene.

(11) Electrophysiological analysis of olfactory response The fry was immobilized using an anesthetic (FA100; DS Pharma Animal Health). The olfaction is stimulated by dropping 10 ^-5 M L-serine into the nasal cavity of fry at a flow rate of about 2.7 mL / min for about 1 minute and 30 seconds, and an electro-olfogram (EOG) is recorded. did. Similarly, EOG was recorded by dropping the water for breeding. The value of the olfactory response was expressed as a comparative relative value of the EOG response strength to breeding water / 10 to the EOG response strength to 10 ⁻⁵ M L-serine.

<Example 1> Examination of herb essential oil (1) Examination of type and concentration of herb essential oil [1-1] Pre-administration fry (average body weight 0.31 g, n = 30) is divided into 13 groups of 500 fish It was set as 1-13 groups. January 22 to February 14, 2014, while feeding each group with a feed supplemented with herbal essential oils (mint oil, eucalyptus oil and oregano oil) so as to have the addition concentrations and dosages shown in Table 1 Until about 3 weeks. On February 14, when protozoa parasitizing the fry of each group were observed or detected, no parasitism was observed.

[1-2] Infection test Subsequently, a basket basket with a side of 10 cm square was installed in each water tank in a form suspended from the top, and ikutibod (average number of parasites 0.17 amol / g body weight, n = 30) in the basket. Infection test by housing and rearing 50 larvae parasitized with Trichodina (average number of parasites: 107 individuals / g body weight, n = 30) each (average body weight: 0.50 g, n = 30) Went. The infection test was conducted for 4 weeks from February 15. The same feed as in Table 1 was used during the infection test. After completion of the infection test (March 15), 7 fry fish were randomly collected from each group, and trichodina and ikutiodo were quantified by the methods described in Experimental Methods (3) and (4). In addition, the cumulative number of juveniles moribund in each group by March 15 (cumulative moribund tails) was counted. The total amount of food fed to each group from January 22 to March 15 was 580 g. FIG. 1 shows the quantification results of Trichodina, FIG. 2 shows the quantification results of ichiobodo, and FIG. 3 shows the cumulative number of moribund tails of fry.

  As shown in FIG. 1, the parasitic amount (average value) of Trichodina was 2377 individuals / g body weight in the first group (control), whereas the second group (addition of mint oil 0.009% by weight) ) In 2190 individuals / g body weight, in group 3 (addition of mint oil 0.018 wt%), 2457 individuals / g body weight, in group 4 (addition of mint oil 0.045 wt%), 1291 individuals / g body weight, In group 5 (addition of mint oil 0.090% by weight), 2316 individuals / g body weight, in group 6 (addition of eucalyptus oil 0.009% by weight), 2493 individuals / g body weight, group 7 (0% eucalyptus oil added) 2412 individuals / g body weight in the case of .018 weight%), 1291 individuals / g body weight in the eighth group (0.045% by weight addition of eucalyptus oil), and 1874 in the ninth group (0.090% by weight addition of eucalyptus oil). Individual / g body weight, group 10 (Ole No. oil (0.009 wt% added) 112.7 individuals / g body weight, Group 11 (Oregano oil added 0.018 wt%) 5.317 individuals / g body weight, Group 12 (Oregano oil 0 0. 45% by weight) was 1132 individuals / g body weight, and the 13th group (0.090% by weight of oregano oil added) was 2011 individuals / g body weight.

  That is, in the group fed with a feed supplemented with 0.09% by weight of oregano oil, although there is no significant difference compared to the control, the parasitic amount of trichodina tends to be small, 0.009% by weight of oregano oil, In the group fed with the diet supplemented with 0.018 wt% and 0.045 wt%, the parasitic amount of Trichodina was remarkably small compared to the control (P <0.05). From this result, it became clear that oregano oil significantly prevented Trichodina parasitism on salmon fry. In addition, it has been clarified that if the concentration of oregano oil added to the feed is less than 0.09% by weight or the dose is less than 27 mg / day / kg, the effect of preventing trichodina parasitism can be obtained.

  Moreover, as shown in FIG. 2, the parasitic amount (average value) of ichthiobod was 42.76 amol / g body weight in the first group (control), whereas the second group (mint oil was 0.009). In the third group (addition of 0.018% by weight of mint oil), 691.9 amol / g body weight in the third group (addition of peppermint oil), and 61 in the fourth group (addition of mint oil 0.045% by weight). 20 amol / g body weight, 52.29 amol / g body weight in group 5 (addition of mint oil 0.090 wt%), 28.08 amol / g body weight in group 6 (addition of 0.009 wt% eucalyptus oil), In the seventh group (addition of 0.018 wt% eucalyptus oil), 20.06 amol / g body weight, in the eighth group (addition of 0.045 wt% eucalyptus oil), 123.5 amol / g body weight, the ninth group (eucalyptus oil) 0.0 0 wt%) was 8.523 amol / g body weight, Group 10 (adding 0.009 wt% oregano oil) was 5.406 amol / g body weight, and Group 11 (0.01 g wt. Oregano oil added) 5.578 amol / g body weight, 45.71 mol / g body weight in group 12 (adding 0.045% by weight of oregano oil), 20.26 amol / g body weight in group 13 (adding 0.090% by weight of oregano oil) Met.

  That is, in the group fed with the feed supplemented with 0.009% by weight, 0.018% by weight and 0.09% by weight of oregano oil, the parasitic amount of ichiobod was small compared to the control. In particular, in the group fed with a diet supplemented with 0.009% by weight and 0.018% by weight of oregano oil, the parasitic amount of ichthiobod was remarkably small compared to the control (P <0.05). From this result, it became clear that oregano oil markedly prevented the infestation of ichthyobodo on salmon fry. Further, in the group fed with the feed supplemented with 0.045% by weight of oregano oil, the parasitic amount of trichodina was similar to that of the control, but 0.009% by weight, 0.018% by weight of oregano oil and In the group fed with the feed supplemented with 0.09% by weight, the parasitic amount of ichthiobod was reduced compared to the control, and the feed suppression effect of larvae of juvenile fish described below was fed with feed supplemented with 0.045% by weight oregano oil. In view of the fact that the concentration of oregano oil added to the feed is 0.09% by weight or less, or the dose is 27 mg / day / kg or less, the effect of preventing ichtibodo parasitism is considered. Was thought to be obtained.

  Finally, as shown in FIG. 3, the cumulative number of moribund tails of fry was 353 in the first group (control), whereas it was 223 in the second group (addition of minced oil 0.009% by weight). Tail, 201 in the third group (addition of 0.018% by weight of mint oil), 180 in the fourth group (addition of mint oil of 0.045% by weight), fifth group (0.090% by weight of mint oil) 188 fish in the 6th group (0.009% by weight added eucalyptus oil), 92 fish in the 7th group (added 0.018% by weight eucalyptus oil), and 8th group (0% eucalyptus oil added). 245 fish in 0.045 wt%), 180 fish in group 9 (0.090 wt% eucalyptus oil added), 3 fish in group 10 (0.009 wt% oregano oil added), group 11 ( Oregano oil added at 0.018% by weight), 1 fish, group 12 (Oregano 0.045 wt% added) In 58 fish, were 13th group (oregano oil added 0.090% by weight) in 178 tails.

  That is, in the group fed with diets supplemented with mint oil, eucalyptus oil and oregano oil, the cumulative number of moribund tails of fry decreased compared to the control. In particular, in the group fed with a diet supplemented with 0.009% by weight and 0.018% by weight of oregano oil, the cumulative number of moribund tails of fry was significantly small. From these results, it became clear that oregano oil significantly suppresses the death of salmon fry by preventing infestation of ichthyodobo and trichodina on the salmon fry. In addition, it became clear that when the concentration of oregano oil added to the feed is less than 0.045% by weight or the dose is less than 13.5 mg / day / kg, the effect of suppressing the moribund death of fry is increased. .

(2) Examination of active ingredients in oregano oil [2-1] Analysis of contained ingredients Oregano oil, which was found to be effective in preventing ictivodo and trichodina parasitism, was subjected to gas chromatography mass spectrometry (GC / MS). About the main peak of the obtained mass spectrum, it searched with the fragrance | flavor library and identified the contained component. Moreover, the content rate of each containing component was calculated | required with the area percentage of the obtained total ion chromatogram. The result is shown in FIG.

Sample preparation: 90 μL of oregano oil was dissolved in 1500 μL of acetone.
GC / MS equipment; GCMS-QP2010SE
Column; Rtx-5 ms (length 30 m, inner diameter 0.25 mm, film thickness 0.25 μm)
Sample injection volume: 0.5 μL
Vaporization chamber; temperature 250 ° C, split ratio 50: 1
Gas type; helium gas pressure; 37.1 kPa (linear velocity: 32.4 cm / sec)
Column temperature: The temperature was raised from 50 ° C. at 3 ° C./min, and held at 300 ° C. for 5 minutes.
Ion source temperature: 200 ° C
Interface temperature: 250 ° C
Measurement; measured in scan mode from 40 to 400 m / z

  As shown in FIG. 4, the oregano oil contains carvacrol 58.92 wt%, para-cymene 15.65 wt%, gamma-terpinene 10.57 wt% and eucalyptol (cineole) 0.8. It was revealed that the content was 06% by weight. Then, the preventive effect of parasitosis of ikuthiobod and trichodina was examined for carvacrol, para-cymene, gamma-terpinene and cineol (hereinafter collectively referred to as “oregano oil-containing components”).

[2-2] Pre-administration Specifically, first, larvae that are not parasitized at all (average body weight 1.80 g, n = 30) were divided into 6 groups of 100 fish each to be the first to sixth groups. . In each group, oregano oil, carvacrol (reagent special grade; Wako Pure Chemical Industries, Ltd.), para-cymene (reagent special grade; Wako Pure Chemical Industries, Ltd.), gamma-terpinene so that the addition concentrations and dosages shown in Table 2 are obtained. (Reagent Special Grade; Wako Pure Chemical Industries, Ltd.) and Cineol (Reagent Special Grade; Wako Pure Chemical Industries, Ltd.) were fed for 2 weeks from March 21 to April 4, 2014 while feeding the feed. In addition, the addition concentration and dosage of each oregano oil-containing component in the feed correspond to the case where the addition concentration of oregano oil is 0.018% by weight.

[2-3] Infection test Subsequently, an infection test was performed for 4 weeks from April 5 to May 2 by the method described in Example 1 (1) [1-2]. The same feed as in Table 2 was used during the infection test. At the start of the infection test, it was confirmed beforehand that no protozoa were parasitic on the fry of each group. On the 14th day (April 18) and 28th day (May 2) from the start of the infection test, 10 larvae were randomly collected from each group, and the methods described in the experimental methods (3) and (4) Was used to quantify trichodina and ichthiobodo. In addition, for each group, the cumulative number of moribund tails of fry until May 2 was counted. The total feed amount for each group from March 21 to May 2 was 160 g. FIG. 5 shows the quantification results of Trichodina, FIG. 6 shows the quantification results of ichiobodo, and FIG. 7 shows the cumulative number of moribund tails of fry.

  As shown in FIG. 5, the parasitic amount (average value) of Trichodina on the 28th day was 2581 individuals / g body weight in the first group (control), whereas the second group (0. 485.8 individuals / g body weight in 018 wt%), 777.7 individuals / g body weight in group 3 (added 0.010 wt% carvacrol), and group 4 (0.0028 wt% para-cymene). Added) is 732.8 individuals / g body weight, in the fifth group (added 0.0019% by weight of gamma-terpinene) is 839.1 individuals / g body weight, and in the sixth group (added 0.000010% by weight of cineol) is 2635 Individual / g body weight.

  That is, in the group fed with the diet supplemented with cineol, the parasitic amount of trichodina was comparable to the control, whereas the diet supplemented with oregano oil, carvacrol, para-cymene and gamma-terpinene. In the group fed with No. 3, the amount of parasitism of Trichodina was remarkably small compared to the control (P <0.05). The results revealed that oregano oil, carvacrol, para-cymene and gamma-terpinene prevent trichodina infestation on salmon fry.

  Moreover, as shown in FIG. 6, the parasite amount (average value) of ichthiobod on the 28th day was 1.659 amol / g body weight in the first group (control), whereas the second group (oregano oil) In the third group (added 0.010% by weight of carvacrol), 0.9216 amol / g body weight in the third group (added 0.010% by weight of carvacrol), and 0.0028% in the fourth group (para-cymene). %) Was 0.7398 amol / g body weight, the fifth group (gamma-terpinene was added 0.0019 wt%) 3.294 amol / g body weight, and the sixth group (cineole was added 0.000010 wt%) was 2. It was 420 amol / g body weight.

  That is, in the group fed with the feed supplemented with gamma-terpinene and cineol, the parasitic amount of ichthiobod was larger than that of the control, whereas the diet supplemented with oregano oil, carvacrol and para-cymene was fed. In the group, the amount of ikutiodo parasite was significantly smaller than the control (P <0.05). The results revealed that oregano oil, carvacrol and para-cymene prevent ichthyodod infestation on salmon fry.

  Finally, as shown in FIG. 7, the cumulative number of moribund tails of fry was 45 in the first group (control), whereas it was 7 in the second group (added 0.018 wt% oregano oil). Tail, 14 in 3rd group (0.010 wt% carvacrol added), 24 in 4th group (added 0.0028 wt% para-cymene), 5th group (0.0019 gamma-terpinene) 40 in the weight% addition) and 43 in the sixth group (addition of 0.000010 wt% cineol).

  That is, in the group fed with the diet supplemented with cineole, the cumulative number of moribund tails of juveniles was comparable to the control, whereas oregano oil, carvacrol, para-cymene and gamma-terpinene were added. In the group fed with feed, the cumulative number of moribund tails of fry decreased compared to the control. From this result, it became clear that oregano oil, carvacrol, para-cymene and gamma-terpinene suppress the death of salmon fry by suppressing the infestation of ichthyodobo and trichodina to the salmon fry.

<Example 2> Examination of edible / feed additive oil (1) Examination of type and concentration of edible / feed additive oil [1-1] Pre-administration fry (average weight 0.31 g, n = 30) 500 The tails were divided into 10 groups and designated as groups 1 to 10. January 22, 2014 while feeding each group with feed supplemented with edible oil (cotton seed oil and flaxseed oil) and feed additive feed oil (fish oil) so as to have the addition concentrations and dosages shown in Table 3 -Raised for about 3 weeks until 14 February. On February 14, when protozoa parasitizing the fry of each group were observed or detected, no parasitism was observed.

[1-2] Infection test Subsequently, an infection test was performed by the method described in Example 1 (1) [1-2]. However, the same feed as in Table 3 was used during the infection test. FIG. 8 shows the quantification results of Trichodina, FIG. 9 shows the quantification results of ichiobodo, and FIG. 10 shows the cumulative number of moribund tails of fry.

  As shown in FIG. 8, the parasitic amount (average value) of Trichodina was 2377 individuals / g body weight in the first group (control), whereas the second group (0.9% by weight of fish oil was added) In 821 individuals / g body weight, 687.3 individuals / g body weight in the third group (with 1.8% fish oil added), 967.5 individuals / g body weight in the fourth group (with 2.7% fish oil added) In the fifth group (0.9% by weight of cottonseed oil added), 1071 individuals / g body weight, in the sixth group (added 1.8% by weight of cottonseed oil) 53.85 individuals / g body weight, and in the seventh group (2% cottonseed oil added) 7 wt%), 2493 individuals / g body weight, 8th group (0.9% linseed oil added) 3229 individuals / g body weight, 9th group (1.8% linseed oil added) 2 0.076 individuals / g body weight, in group 10 (2.7% by weight of linseed oil added), 1.000 individuals / g body weight Was Tsu.

  That is, in the group fed with feed containing 0.9% and 1.8% by weight of cottonseed oil and 1.8% and 2.7% by weight of linseed oil, the parasitic amount of Trichodina was higher than that of the control. It was small. In particular, in the group fed with 1.8% by weight of cottonseed oil and 1.8% and 2.7% by weight of linseed oil, the parasitic amount of Trichodina was significantly smaller than that of the control (P <0.05). The results revealed that cottonseed oil and linseed oil prevent trichodina infestation on salmon fry. In addition, when the concentration of cottonseed oil added to the feed is less than 2.7% by weight, or when the dose is less than 810 mg / day / kg body weight, and the concentration of flaxseed oil added to the feed is from 0.9% by weight It has been found that the preventive effect of Trichodina parasitism can be obtained when the dose is larger or larger than 270 mg / day / kg.

  Further, as shown in FIG. 9, the parasitic amount (average value) of ikuthiobod was 42.76 amol / g body weight in the first group (control), whereas the second group (0.9 weight of fish oil). 24.83 amol / g body weight in Group 3 (with 1.8% by weight of fish oil added), 20.56 amol / g body weight with Group 3 (with 1.8% by weight of fish oil added), and 17.38 amol / g in Group 4 (with 2.7% by weight of fish oil added). g body weight, 9.788 amol / g body weight in group 5 (0.9% cotton seed oil added), 28.94 amol / g body weight in group 6 (1.8% cotton seed oil added), group 7 (cotton seed oil) 2.7 wt%), 2.203 amol / g body weight, Group 8 (0.9% by weight linseed oil added) 2.579 amol / g body weight, Group 9 (1.8% by weight linseed oil) Added) 2.105 amol / g body weight, group 10 ( AsaHitoshiabura was 8.024amol / g body weight in a 2.7 wt% addition).

  That is, in the group fed with a feed supplemented with 1.8% by weight of cottonseed oil, there was no significant difference compared to the control, but the parasitic amount of ichthiobod tended to be small, and 0.9% by weight of cottonseed oil and 2. In the group fed with 7% by weight and a diet supplemented with 0.9%, 1.8% and 2.7% by weight of linseed oil, the parasitic amount of ikutiobodo was smaller than that of the control. In particular, in the group fed with 2.7% by weight of cottonseed oil and 0.9%, 1.8% and 2.7% by weight of linseed oil, the amount of ichytobodo infestation was higher than that of the control. It was significantly smaller (P <0.05). The results revealed that cottonseed oil and linseed oil prevent the infestation of ichthyobodo on salmon fry. As for cottonseed oil, there was no significant difference in the parasitic amount of ikutiobodo in the group fed with 1.8% by weight of feed compared to control, but 0.9% and 2.7% by weight of feed were added. In the group fed with cucumber, the parasitism of ikutiobodo decreased compared to the control, and the mortality control effect of juvenile fish, which will be described later, was significantly obtained even in the group fed with 1.8% by weight of cottonseed oil. In view of the above, it was considered that if the concentration of cottonseed oil added to the feed was 0.9% by weight or higher, or the dose was higher than 270 mg / day / kg, the effect of preventing ichytobodo parasitism was obtained. In addition, for flaxseed oil, it is clear that the effect of preventing parasitism of ikuthiobod can be obtained if the concentration added to the feed is 0.9% by weight or more, or the dose is more than 270 mg / day / kg. It was.

  Finally, as shown in FIG. 10, the cumulative number of moribund tails of fry was 353 in the first group (control), compared with 93 in the second group (0.9% by weight of fish oil added). 73 in the third group (1.8% by weight of fish oil added), 70 in the fourth group (2.7% by weight of fish oil added), 22 in the fifth group (0.9% by weight of cottonseed oil added) 5 tails in group 6 (1.8% by weight cottonseed oil added), 61 tails in group 7 (2.7% by weight cottonseed oil added), 8th group (0.9% by weight flaxseed oil added) The number of fish was 91, 9 in group 9 (1.8% by weight of linseed oil added), and 4 in group 10 (2.7% by weight of linseed oil added).

  That is, in the group fed with a feed supplemented with fish oil, cottonseed oil and flaxseed oil, the cumulative number of moribund tails of fry decreased compared to the control. In particular, in the group fed with a diet supplemented with 1.8% by weight of cottonseed oil and 1.8% and 2.7% by weight of flaxseed oil, the cumulative number of moribund tails of fry was remarkably small. These results revealed that cottonseed oil and linseed oil significantly suppressed moribund of salmon larvae by preventing infestation of ichthyobodo and trichodina to salmon larvae. In addition, when the concentration of cottonseed oil added to the feed is 0.9 wt% or more and less than 2.7 wt%, or the dose is 270 mg / day / kg or more and less than 810 mg / day / kg body weight, and linseed oil It has been clarified that the effect of suppressing the moribund of salmon fry is enhanced when the concentration added to the feed is greater than 0.9% by weight or the dose is greater than 270 mg / day / kg body weight.

(2) Examination of active ingredients in linseed oil [2-1] Analysis of contained components Fig. 11 shows the fatty acid composition of linseed oil that was found to be effective in preventing parasitism of ikutiodo and trichodina (Kanto Chemical Co., Ltd. Reagent Business Division) Provided by Reagent Technology Department). As shown in FIG. 11, it became clear that the linseed oil contains 58% by weight of linolenic acid and 15% by weight of linoleic acid. Then, the preventive effect of ichinobodo and trichodina parasitism was examined for linoleic acid and linolenic acid.

[2-2] Pre-administration Specifically, first, fry (average weight 0.31 g, n = 30) was divided into 4 groups of 500 fish each to be the first to fourth groups. While feeding each group with a feed supplemented with linoleic acid (reagent special grade; Wako Pure Chemical Industries) and linolenic acid (reagent special grade; Wako Pure Chemical Industries) so as to achieve the addition concentrations and dosages shown in Table 4. The animals were bred for about 3 weeks from January 22 to February 14, 2014. On February 14, when protozoa parasitizing the fry of each group were observed or detected, no parasitism was observed.

[2-3] Infection test Subsequently, an infection test was performed by the method described in Example 1 (1) [1-2]. However, the same feed as in Table 4 was used during the infection test. The result is shown in FIG.

  As shown in the graph on the left side of FIG. 12, the parasitic amount (average value) of Trichodina was 2377 individuals / g body weight in the first group (control), whereas the second group (in which linoleic acid was 0.1%). Nine weight percent) was 198.7 individuals / g body weight, group 3 (0.9% by weight of linolenic acid) was 0 individuals / g body weight, and group 4 (linoleic acid and linolenic acid were each 0.9 weight) % Addition) was about 69.73 individuals / g body weight. That is, in the group fed with the diet supplemented with linoleic acid and linolenic acid, the parasitic amount of Trichodina was remarkably small compared to the control (P <0.05). From this result, it was revealed that linoleic acid and linolenic acid prevent trichodina infestation on salmon fry.

  In addition, as shown in the central graph of FIG. 12, the parasitic amount (average value) of ichthiobod was 42.76 amol / g body weight in the first group (control), whereas the second group (linoleic acid). 129.0 amol / g body weight in Group 3 (0.9% by weight of linolenic acid added), 6.302 amol / g body weight in Group 3 (0.9% by weight of linolenic acid), and Group 4 (linoleic acid and linolenic acid 0% each). It was 44.32 amol / g body weight. That is, in the group fed with the diet supplemented with linolenic acid, the amount of parasitic ichytobodo was significantly smaller than that of the control (P <0.05). From this result, it was revealed that linolenic acid prevents infestation of ichthiobodo on salmon fry.

  Finally, as shown in the graph on the right side of FIG. 12, the cumulative number of moribund tails of fry was 353 in the first group (control), whereas the second group (0.9% by weight of linoleic acid) (Added), 1 fish in the third group (0.9% by weight of linolenic acid), and 10 fish in the 4th group (0.9% by weight of linoleic acid and linolenic acid added). That is, in the group fed with a diet supplemented with linoleic acid and linolenic acid, the cumulative number of moribund tails of fry was significantly smaller than that of the control. From these results, it has been clarified that linoleic acid and linolenic acid remarkably suppress the death of salmon fry by preventing infestation of ichthyodobo and trichodina to the salmon fry.

<Example 3> Examination of administration period (1) Pre-administration Fry fish (average body weight 1.80 g, n = 30) in which no protozoa parasitize are divided into 12 groups of 100 fish each to be the first to 12th groups. . The 12 groups were divided into groups of 4 groups, and groups 1 to 3 were obtained. Each group of groups 2 and 3 was reared while being fed with a feed supplemented with oregano oil and linseed oil before the infection test (hereinafter referred to as “pre-administration rearing”). Table 5 shows the concentrations and dosages of oregano oil and linseed oil added to the feed. Pre-administration breeding was carried out for 7 days (from March 21 to March 28, 2014) for Group 2 and 14 days (from March 21 to April 4, 2014) for Group 3. Group 1 was not bred in advance.

(2) Infection test Subsequently, for each of Groups 1 to 3, an infection test for 4 weeks was performed by the method described in Example 1 (1) [1-2]. However, the same feed as in Table 5 was used during the infection test. At the start of each group's infection test, it was confirmed in advance that no protozoa were parasitic on the fry. On the 14th day from the start of the infection test, 10 juvenile fish were sampled from each group, and trichodina and ikuthiobod were quantified by the method described in Experimental Methods (3) and (4). In addition, on the 28th day after the start of the infection test, the cumulative number of moribund tails of fry was obtained. The total amount of food fed to each group during the breeding period (a period in which the pre-administration breeding and the breeding in the infection test were combined) was 120 g in Group 1, 140 g in Group 2, and 160 g in Group 3. FIG. 13 shows the quantification results of Trichodina, FIG. 14 shows the quantification results of ichiobodo, and FIG.

  As shown in FIG. 13, the parasitic amount (average value) of Trichodina in group 1 was 595.1 individuals / g body weight in the first group (control), while the second group (added oregano oil was added) ) Was 471.1 individuals / g body weight, the third group (added linseed oil) was 333.6 individuals / g body weight, and the fourth group (added oregano oil and linseed oil) was 383.0 individuals / g body weight. It was. Further, the parasitic amount (average value) of trichodina in group 2 was 646.4 individuals / g body weight in the fifth group (control), whereas it was 265.0 in the sixth group (added with oregano oil). Individuals / g body weight, 210.5 individuals / g body weight in Group 7 (with linseed oil added), and 218.6 individuals / g body weight in Group 8 (with oregano oil and linseed oil added). Further, the parasitic amount (average value) of trichodina in group 3 was 444.6 individuals / g body weight in the ninth group (control), whereas it was 70.78 in the tenth group (added with oregano oil). Individual / g body weight was 54.96 individuals / g body weight in Group 11 (with linseed oil added) and 39.48 individuals / g body weight in Group 12 (with oregano oil and linseed oil added).

  That is, in Group 1, no significant decrease in the amount of Trichodina parasite was found compared to the control, whereas in Groups 2 and 3, the Trichodina parasite amount was significantly smaller than the control ( P <0.05). From these results, it became clear that oregano oil and linseed oil are effective for prevention while not effective in treating trichodinosis in salmon fry. In addition, it has been clarified that when oregano oil and / or flaxseed oil is administered to salmon juveniles not infected with trichodinosis for 7 days or more, the effect of preventing trichodina parasitism is enhanced.

  Further, as shown in FIG. 14, the parasitic amount (average value) of ichthiobod in group 1 was 7.085 amol / g body weight in the first group (control), whereas the second group (oregano oil was added). In addition, it was 5.294 amol / g body weight, in Group 3 (with linseed oil added) 3.864 amol / g body weight, and in Group 4 (with oregano oil and linseed oil added) 4.019 amol / g body weight. Further, the parasitic amount (average value) of ichthiobod in group 2 was 6.555 amol / g body weight in the fifth group (control), whereas it was 0.5298 amol / in the sixth group (added with oregano oil). g body weight, 0.9756 amol / g body weight in Group 7 (with linseed oil added) and 0.6411 amol / g body weight in Group 8 (with oregano oil and linseed oil added). Further, the parasitic amount (average value) of ichthiobod in group 3 was 5.247 amol / g body weight in the ninth group (control), whereas it was 0.2757 amol / in the tenth group (added with oregano oil). The weight was 0.1986 amol / g body weight in Group 11 (with linseed oil added) and 0.3271 amol / g body weight in Group 12 (with oregano oil and linseed oil added).

  That is, in Group 1, no significant decrease in the amount of ichotibodo was observed compared to the control, whereas in Groups 2 and 3, the amount of ikutiodo was significantly smaller than that of the control ( P <0.05). From these results, it became clear that oregano oil and linseed oil are effective for prevention while not effective in treating ichthyobodosis in salmon fry. In addition, it has been clarified that when oregano oil and / or flaxseed oil is administered to salmon fry not infected with ichthyobodosis for 7 days or more, the effect of preventing ichthyodobo infestation is enhanced.

  Finally, as shown in FIG. 15, the cumulative number of moribund tails of group 1 fry was 44 in the first group (control), compared with 46 in the second group (added with oregano oil), There were 40 fish in the third group (with linseed oil added) and 38 fish in the fourth group (with oregano oil and linseed oil added). In addition, the cumulative number of moribund tails of fry in Group 2 was 40 in the 5th group (control), while 22 in the 6th group (added with oregano oil), 7th group (added flaxseed oil) ) And 17 fish in Group 8 (added with oregano oil and linseed oil). In addition, the cumulative number of moribund tails of group 3 fry was 45 in the 9th group (control), but 7 in the 10th group (added with oregano oil) and 11th group (added linseed oil) ) And 5 in the 12th group (added with oregano oil and linseed oil).

  That is, in Group 1, there was no significant reduction in the number of juveniles killed by larvae compared to controls, whereas in Groups 2 and 3, the number of juveniles culled by larvae was significantly smaller than in controls. It was. From these results, it became clear that oregano oil and flaxseed oil suppress moribund of fry by preventing rather than treating trichodinosis and ichthyobodosis. In addition, it has been clarified that when oregano oil and / or flaxseed oil is administered to juvenile fish that are not infected with trichodinosis or ichthyobodosis for 7 days or more, the effect of suppressing the moribund death of the juvenile fish is enhanced.

<Example 4> Safety verification of oregano oil and linseed oil (1) Comparison with non-infected fish Young fish (average body weight 1.80 g, n = 30) that are not parasitized at all are divided into 4 groups of 100 fish each. Divided into groups 1 to 4. Each group was bred for 6 weeks from March 21 to May 2, 2014 while feeding with oregano oil and flaxseed oil in the same manner as Groups 1 to 4 of Group 1 in Table 5. Thereafter, the body weight of each group of fry was measured, and by the method described in Experimental Methods (5) to (11), a seawater transfer test (30 fish for each group), analysis of 鰓 Na ⁺ , K ⁺ -ATPase activity, Na ⁺ , K ⁺ -ATPase α subunit gene expression level analysis, plasma lysozyme activity analysis, plasma cortisol level analysis, HSP70 gene expression level analysis, and electrophysiological analysis of olfactory response were used. The result is shown in FIG. After the breeding period, when protozoa parasitizing the fry of each group was observed or detected, no parasitism was observed. Moreover, there was no moribund in all groups.

  As shown in FIG. 16, the weight (average value) of the fry was 3.334 g in the first group (control), whereas it was 3.297 g in the second group (added with oregano oil). It was 3.415 g in the group (added linseed oil) and 3.407 g in the fourth group (added oregano oil and linseed oil). That is, the weight of the fry of the group fed with the feed supplemented with oregano oil and linseed oil was similar to the control (P> 0.05: one-way analysis of variance). From this result, it became clear that oregano oil and flaxseed oil do not affect the growth of salmon fry.

  Survival rates after the seawater transfer test are the first group (control), the second group (added with oregano oil), the third group (added linseed oil) and the fourth group (added with oregano oil and linseed oil) ) Was 100%. From these results, it became clear that oregano oil and linseed oil do not affect the ability of salmon fry to adapt to seawater.

In addition, the Na ⁺ , K ⁺ -ATPase activity of sputum was 1.482 μmol Pi / mg protein / h in the first group (control), whereas that in the second group (added with oregano oil) was 1. 871 μmol Pi / mg protein / h, 1.420 μmol Pi / mg protein / h in Group 3 (added linseed oil), 1.450 μmol Pi / mg protein / h in Group 4 (added oregano oil and linseed oil) Met. That is, the 鰓 Na ⁺ , K ⁺ -ATPase activity of the group fed with the feed supplemented with oregano oil and linseed oil was similar to the control (P> 0.05: one-way analysis of variance). From this result, it became clear that oregano oil and linseed oil do not affect the osmotic pressure regulation function of salmon fry.

Moreover, the expression level of Na ⁺ , K ⁺ -ATPase α subunit gene in sputum was 17.37 in the first group (control), whereas 8.011 in the second group (added with oregano oil). The third group (with linseed oil added) was 7.180, and the fourth group (with oregano oil and linseed oil added) was 12.43. That is, no significant difference was observed in the expression level of 鰓 Na ⁺ , K ⁺ -ATPase α subunit gene between the control and the group fed with the diet supplemented with oregano oil and linseed oil (P> 0.05: One-way analysis of variance). From these results, it became clear that oregano oil and linseed oil do not affect the expression level of salmon Na ⁺ , K ⁺ -ATPase α subunit genes in juvenile salmon.

  The plasma lysozyme activity was 11.34 Unit / mL / min in the first group (control), whereas it was 5.544 Unit / mL / min in the second group (added with oregano oil), the third group. In (addition of linseed oil), it was 4.536 Unit / mL / min, and in the fourth group (added with oregano oil and linseed oil), it was 7.518 Unit / mL / min. That is, there was no significant difference in plasma lysozyme activity between the control and the group fed with oregano oil and linseed oil (P> 0.05: one-way analysis of variance). From this result, it became clear that oregano oil and linseed oil do not affect the immune function of fry.

  In addition, the plasma cortisol amount was 2.202 ng / mL in the first group (control), whereas 2.142 ng / mL in the second group (added with oregano oil), the third group (linseed oil was added). Addition) was 2.415 ng / mL, and the fourth group (added with oregano oil and linseed oil) was 2.681 ng / mL. That is, the plasma cortisol amount of the group fed with the feed supplemented with oregano oil and linseed oil was comparable to the control (P> 0.05: one-way analysis of variance). From this result, it became clear that oregano oil and flaxseed oil do not affect the stress response of fry.

  Also, the HSP70 gene expression level in the liver was 2428 in the first group (control), 1402 in the second group (added with oregano oil), 2371 in the third group (added linseed oil), In the fourth group (added with oregano oil and linseed oil), it was 3404. That is, there was no significant difference in liver HSP70 gene expression level between the control and the group fed with oregano oil and linseed oil (P> 0.05: one-way analysis of variance). From this result, it became clear that oregano oil and flaxseed oil do not affect the stress response of fry.

  Finally, the value of the olfactory response was 4.838 in the first group (control), whereas it was 4.504 in the second group (added with oregano oil), and the third group (added linseed oil) Was 4.271, and the fourth group (added with oregano oil and linseed oil) was 4.320. That is, the value of the olfactory response of the group fed with the diet supplemented with oregano oil and linseed oil was comparable to the control (P> 0.05: one-way analysis of variance). The results revealed that oregano oil and flaxseed oil did not affect the olfactory response of fry.

  From the results of Example 4 (1) above, it has been clarified that oregano essential oil and linseed oil do not adversely affect the growth and various physiological functions of fry.

(2) Comparison after infection test Upon completion of the infection test of Example 3 (2), fry were collected from each group of Group 3 at random, the body weight was measured, and described in Experimental Methods (5) to (9) Seawater transfer test (30 fish for each group), analysis of Na ⁺ , K ⁺ -ATPase activity, analysis of Na ⁺ , K ⁺ -ATPase α subunit gene expression level, analysis of plasma lysozyme activity and plasma cortisol level We used for analysis. The result is shown in FIG.

  As shown in FIG. 17, the weight (average value) of the fry was 2.799 g in the ninth group (control), whereas it was 3.199 g in the tenth group (added with oregano oil). It was 3.314 g in the group (with linseed oil added) and 3.313 g in the twelfth group (with oregano oil and linseed oil added). That is, in the group fed with a diet supplemented with oregano oil and linseed oil, the weight of the fry was larger than that of the control (P <0.05: one-way analysis of variance and multiple comparison test Tukey-Kramer method). This is because growth was suppressed by the infestation of trichodina and ichthyobodo in the control, whereas in the group fed with the diet supplemented with oregano oil and flaxseed oil, the infestation of fryfish was prevented. It is thought that growth restraint has also been reduced. The results showed that oregano oil and linseed oil alleviated the adverse effects on the growth of salmon larvae caused by trichodina and ichthyobodo parasitism.

  Further, the survival rate after the seawater transfer test was 0% in the ninth group (control), whereas it was 80 in the tenth group (added with oregano oil) and the eleventh group (added linseed oil). %, And in Group 12 (added oregano oil and linseed oil), it was 83.33%. That is, in the group fed with the feed supplemented with oregano oil and linseed oil, the survival rate was remarkably large compared to the control. This is because, in the control, the larvae of the larvae were damaged due to the infestation of Trichodina and Ikutibodo, and the ability to adapt to seawater was lost. As a result of the prevention of parasitism, the damage to the coral is reduced and the seawater adaptability is thought to be maintained. From these results, it became clear that oregano oil and flaxseed oil alleviate the adverse effects on the seawater adaptability of salmon larvae caused by trichodina and ichthyobodo infestation.

In addition, the Na ⁺ , K ⁺ -ATPase activity of sputum was 0.376 μmol Pi / mg protein / h in the 9th group (control), whereas 1% in the 10th group (added with oregano oil). 073 μmol Pi / mg protein / h, 1.156 μmol Pi / mg protein / h for Group 11 (with flaxseed oil added), 1.134 μmol Pi / mg protein / h for Group 12 (added with oregano oil and flaxseed oil) Met. That is, in the group fed with the diet supplemented with oregano oil and linseed oil, the 鰓 Na ⁺ , K ⁺ -ATPase activity was significantly higher than that of the control (P <0.05: one-way analysis of variance and multiple analysis). Comparative test Tukey-Kramer method). This is because, in the control, the larvae of the larvae were damaged due to infestation of trichodina and ichthyobodo, and the ability to actively excrete Na ⁺ from the culm was reduced, whereas diets supplemented with oregano oil and flaxseed oil were fed. In the group, as a result of preventing the infestation of trichodina and ichthiobodo, it is considered that the damage to the soot was reduced and the ability to actively discharge Na ⁺ from the soot was maintained. From these results, it became clear that oregano oil and linseed oil alleviate the adverse effects on the osmotic pressure regulating function of salmon juvenile salmon caused by trichodina and ichthyobodo infestation.

In addition, the expression level of Na ⁺ , K ⁺ -ATPase α subunit gene in sputum was 2.123 in the ninth group (control), whereas it was 5.731 in the tenth group (added with oregano oil). The 11th group (with linseed oil added) was 5.009, and the 12th group (with oregano oil and linseed oil added) was 5.849. That is, in the group fed with a diet supplemented with oregano oil and linseed oil, the expression level of 鰓 Na ⁺ , K ⁺ -ATPase α subunit gene was remarkably large compared to the control (P <0.05: unity Configurational analysis of variance and multiple comparison test Tukey-Kramer method). This is because, in the control, the expression level of the gene decreased due to damage to the fry of the larvae caused by the infestation of Trichodina or Iktiobodo, whereas in the group fed with oregano oil and linseed oil, As a result of the prevention of iktibodo parasitism, it is considered that the damage of the moth was reduced and the expression level of the gene was maintained. These results indicate that oregano oil and linseed oil alleviate the adverse effects of trichodina and ichthyobodo on the ability of salmon fry to adapt to seawater.

  Plasma lysozyme activity was 0.607 Unit / mL / min in Group 9 (control), compared with 4.667 Unit / mL / min in Group 10 (added with oregano oil), Group 11. In (addition of linseed oil), it was 6.474 Unit / mL / min, and in Group 12 (added with oregano oil and linseed oil), it was 5.517 Unit / mL / min. That is, in the group fed with the diet supplemented with oregano oil and linseed oil, the plasma lysozyme activity was significantly higher than that of the control (P <0.05: one-way ANOVA and multiple comparison test Tukey-Kramer). Law). This is because, in the control, the amount of lysozyme or its activity decreased due to the infestation of trichodina or ichthiobod, whereas in the group fed with the diet supplemented with oregano oil and linseed oil, the infestation of trichodina and ikuthiobod was prevented. It is considered that the amount of lysozyme or its activity was also maintained. From these results, it became clear that oregano oil and linseed oil alleviate the adverse effects on the immune function of salmon juveniles caused by infestation of trichodina and ichthyobodo.

  The plasma cortisol amount was 1.137 ng / mL in the 9th group (control), whereas it was 2.763 ng / mL in the 10th group (added with oregano oil), and the 11th group (flaxseed oil was added). Addition) was 3.147 ng / mL, and Group 12 (added oregano oil and linseed oil) was 3.036 ng / mL. That is, in the group fed with the diet supplemented with oregano oil and linseed oil, the amount of plasma cortisol was remarkably large compared to the control (P <0.05: one-way analysis of variance and multiple comparison test Tukey-Kramer). Law). This is because, in the control group, the increase in the amount of cortisol associated with the stress response did not occur because the fry was weakened by infestation with trichodina and ichthiobodo, whereas in the group fed with oregano oil and flaxseed oil, As a result of the prevention of trichodina and ichthiobodo parasitism, it is considered that the increase in cortisol amount accompanying the stress response was also maintained. These results indicate that oregano oil and linseed oil alleviate the adverse effects on the stress response ability of salmon larvae caused by trichodina and ichthyobodo parasitism.

  From the results of Example 4 (2) above, it has been clarified that oregano oil and linseed oil significantly reduce the adverse effects on salmon fry growth and various physiological functions caused by trichodina and ichthyobodo parasitism.

Claims (7)

An infectious disease preventive agent containing oregano oil for preventing ichthyobodosis of salmon fry for release. An infectious disease preventive agent containing oregano oil for preventing trichodinosis of salmon fry for release. The infectious disease preventive agent according to claim 1, wherein oregano oil is used by being added to feed at a dose of 27 mg / day / kg body weight or less or an addition concentration of 0.09% by weight or less . The infectious disease preventive agent according to claim 2, wherein oregano oil is used by being added to the feed at a dose of 27 mg / day / kg body weight or less or an additive concentration of less than 0.09% by weight .   The infectious disease preventive agent according to any one of claims 1 to 4, comprising oregano oil and linseed oil, and using linseed oil as an excipient for oregano oil.   A method for preventing an infectious disease of a salmon fry for discharge, which comprises a step of administering the infectious disease preventive agent according to any one of claims 1 to 5 to the salmon fry for discharge.   The method of Claim 6 which has the process of administering the said infectious disease preventive agent to the salmon juvenile for release which is not infected with the trichodinosis and ichthyobodosis for 7 days or more.