JP3013871B2 - Method for activating immune activity of marine cultured fish - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for activating immunological activity of marine cultured fish. More specifically, the present invention relates to a method for stimulating the immunoreactivity of marine cultured fish using the principle of stimulating the immunoreactivity of promoting the production of glutathione peroxidase by providing a fish feed containing selenium.

[0002]

2. Description of the Related Art Aerobic organisms have effectively utilized molecular oxygen for metabolism and have acquired a function of protecting against oxygen damage. The strong chemical reactivity of oxygen can be used as a powerful energy source, but involves the formation of reaction intermediates that are dangerous for living organisms. In general, such active oxygen that is toxic to living organisms includes superoxide (O 2 O).
2) and hydrogen peroxide (H2O2). Unsaturated fatty acids undergo peroxidation by these active oxygens to produce lipid peroxides.

Heretofore, fish feed has often used, as a raw material thereof, an animal or animal raw material having a higher nutritional value than a plant raw material, such as fish meal and meat powder. Lipid peroxide is also attached to this fish meal, and it is considered that the lipid peroxide has an adverse effect on intercellular communication in a living body. At present, antioxidants such as tocopherol and ethoxyquin are added to feed containing fish meal in order to prevent the production of lipid peroxide. However, these are only antioxidants and are ineffective against the produced lipid peroxide. However, a substance that is effective against the produced lipid peroxide has not yet been developed.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a means which is effective even for lipid peroxide produced. An object of the present invention is to provide a method for stimulating immune activity of marine farmed fish, which stimulates immune activity by promoting the production of glutathione peroxidase by providing a fish feed containing selenium.

[0005]

Under these circumstances, the present inventor has searched for a substance which is also effective against the produced lipid peroxide, and converts the lipid peroxide into non-toxic hydroxyperoxide. Focusing on glutathione peroxidase, which is known as an enzyme having an action, the present inventors have conducted research on conditions under which glutathione peroxidase is produced and reduced in cost, and finally completed the present invention. One of the important components of this glutathione peroxidase is selenium (hereinafter sometimes referred to as “Se”).

[0006] In the United States, it is necessary to add selenium as a trace element to livestock feed. The average selenium content of grass in Japan is 0.02-0.03p
pm, and the selenium content in the roughage is very low.
Therefore, feed yeasts with high selenium content and high safety have been developed. Bread yeast Saccharomyces cerevis containing more Se than ordinary yeast
e-yeast, which is commercially available under the name TOYOKOBO (registered trademark, Asahi Kasei Kogyo Co., Ltd.). Selenium has been added to livestock feed as a trace element as being effective for livestock. However, there is no report that it is equally effective for fish.

[0007] Therefore, the present inventor has fed selenium-containing feed for fish farming, and raised marine farmed fish in a state where glutathione peroxidase is easily produced in vivo. As a result, it was confirmed by measuring the immunoreactivity of marine cultured fish that glutathione peroxidase detoxifies activated oxygen which is toxic to the living body by activating its activity in the living body. Accordingly, the present invention is a result activating the immune activity using the principle of immunostimulating
A immunostimulatory activation method marine farmed fish using the principle of immunostimulatory say.

[0008] The present invention promotes the production of glutathione peroxidase in the living body of marine farmed fish by adding selenium to the raw material for fish farming to form a feed for selenium-containing fish farming and breeding marine farmed fish. is there. Contains a large amount of selenium as selenium to be added to the above fish feed ingredients
Use the yeast that you are .

That is, in the present invention, a selenium-containing fish feed is preferably used by mixing yeast containing more selenium than ordinary yeast with ordinary fish feed. The above-mentioned TOYOKOBO (registered trademark) was used as a yeast containing more selenium than a normal yeast. Selenium in yeast is thought to be present in the organic selenium selenomethionine.

When used as livestock feed, inorganic selenium hardly migrates in selenium transfer in milk, whereas organic selenium transfer is good. As a result,
The serum selenium and blood glutathione peroxidase activity values of the larvae that ingested such milk remained high, and the palatability for livestock was good, and when administered to livestock, a clear gain-improving effect was observed. It is reported that yeast containing more selenium than ordinary yeast is preferably used as a selenium source.

The feed for fish farming of the present invention contains ordinary fish meal.
Any feed may be used as long as selenium is added to the feed for farmed fish . The usual fishmeal-containing feed ingredients for adding selenium <br/> fish feed, fishmeal, Antarctic krill meal,
Shrimp powder, fish soluble powder, feather meal,
Animal raw materials such as blood meal, bone meal, gelatin: defatted soybeans, yeast, defatted rice bran, corn gluten meal, alfalfa meal, flour, wheat germ, 、, chlorella, spirulina, seaweed powder and other plant raw materials; vitamins; minerals ;
Flavors; pigments; and other swelling agents can be used, and can be appropriately selected and used depending on the target fish species.

Regarding how the above-mentioned toyocobo acts on marine farmed fish, since this toyocobo is abundantly present in the form of organic selenomethionine in which Se is substituted for S of methionine, administration of toyocobo is performed. Then, it is easily considered that glutathione peroxidase is easily generated. FIG. 1 schematically shows the function of selenium in detoxifying lipid peroxide.

Using the above-mentioned TOYOKOBO as a selenium source,
Investigations were made on fish breeding tests to examine the effects of animal and selenium contents in feed on rearing performance, and it was confirmed that humoral immunity increased when Se was added to feed and fed. That is, by mixing fish meal containing a large amount of lipid peroxide and ordinary fish meal, the effect of a feed containing a large amount of Se on test fish (marine cultured fish) was evaluated by an immune reaction. As the test fish, any fish species can be used as long as it is a marine cultured fish that can be bred with a feed for aquaculture containing fish meal. Therefore, the fish to be cultured according to the present invention may be any fish species such as trout, bream, hamachi and the like that can be cultured in seawater. Listonell as immunogen
a. Anguillarum formalin-killed bacteria were used to evaluate antibody production after immunization, leukocyte chemotaxis, phagocytosis, and the like. As a result, when Se was added to feed and fed, humoral immunity increased. Do you get it.

As a means for adding the above-mentioned toyo-cobo to the feed material, it may be added at an appropriate time during the production process of the feed for fish farming. The concentration of selenium added to feed ingredients
At least, the effect of the addition is not obtained, and when it is too large, the breeding results are inferior. The feed for fish farming of the present invention can be in the form of, for example, powder, granules, pellets, crumbles or any other suitable shape.

[0015]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail with reference to embodiments. The present invention is not limited by these examples. Real
The basic composition (Free) of the feed used in the examples is based on fish meal.
Selenium (SE), vitamins (VE, VC)
Is not included. Selenium (SE) in the feed is celeomethioni
Containing 1000 ppm as selenium
(Toyo Kobo) as 1 pp
m with Toyo Kobo at a concentration of 0.1%
It is. Vitamin E (VE) and Vitamin C (VC)
The amount is the amount normally used in food. In the examples,
The growing environment water used artificial seawater. Selenium in artificial seawater
(SE) is not included. 3 kinds of feed (SE + VE
+ VC, VE + VC, Free) for 2 weeks,
Immunize and sample 1, 3, 5 weeks after immunization. So
During the period, feed any one of the three types
Gave. Feeding method that feed is given until it is not eaten
So the exact amount is not measured, but usually 2% of body weight
It is supposed to eat much.

Example 1 [Materials and Methods] (1) Test fish Aged salmon with an average body weight of 85 g were used to form dried pellets while performing aeration using a flowing water-type circular water tank containing artificial seawater. They were fed once a day, bred and used for experiments. The breeding water temperature during the test was set at 15 ° C. One sampling consisted of 5 fish. (2) Trial strain Lis isolated from marine ayu, distributed in November 1992 from the Aquarium Pathology Laboratory, Faculty of Agriculture, Kochi University
tonella anguillarum V-110
Nine strains were used which were subcultured and stored in a 1/3 concentration brain heart infusion agar high-layer medium.

(3) Preparation of standard formalin-inactivated cell suspension A test strain was prepared at 25 ° C. for 24 hours in 5 ml of dry broth medium.
After pre-cultivation for an hour, main culture was performed at 25 ° C. for 48 hours in 500 ml of a dry broth medium. Next, formalin was added to the medium at a final concentration of 0.3%, left at room temperature for 48 hours, and then collected by centrifugation at 5,000 rpm for 20 minutes. In addition, the collected cells are used for PB
After washing three times with S, the same solution was used to adjust the wet bacterial weight to 100 mg / ml, and this was subjected to experiments as standard formalin-inactivated cells.

(4) Immunization 0.1 ml of formalin-inactivated bacterial cells was placed in the abdomen of the test fish.
Immunized by inoculation. According to the same method, PB
S was inoculated as a control. (5) Collection of serum Eight samples were taken for each sampling from the immunized and control groups before immunization, 1, 3, and 5 weeks after immunization, and blood was collected by cardiac puncture. The obtained blood was left overnight at 4 ° C., and centrifuged at 4,000 × g for 3 minutes to collect serum. The obtained serum was stored frozen at -80 ° C and thawed before use.

(6) Measurement of agglutination antibody titer of serum The agglutination antibody titer was measured by a microtiter method. (7) Kidney cell suspension The kidney was excised from coho salmon in the immunized zone and the control zone after blood collection, immersed in MEM medium (Nissui), and minced with dissecting scissors. After standing for 10 minutes to precipitate red blood cells,
The supernatant was aspirated with a micropipette, transferred to another sampling tube, and washed three times with MEM medium by centrifugation. The cell concentration at the time of use in the experiment was such that dead cells and red blood cells were removed by eosin-Y,
It was adjusted to × 10 8 cells / ml.

(8) Measurement of chemotaxis of phagocytic cells Performed according to the Boyden method. In the lower chamber of the chemotaxis chamber, a 2-fold dilution of the immunized and control sera was brought to a total volume of 0.2 ml. And a filter was fixed thereon. Then, 0.2 ml of an immune or control coho salmon kidney cell suspension, which had been diluted twice with MEM medium, was injected into the upper chamber, and the upper chamber was closed with parafilm to prevent drying. Further, after leaving the chemotaxis chamber in a thermostat at 20 ° C. for 90 minutes, the filter was taken out, and Giemsa staining was performed on the kidney cells leached on the lower surface thereof. The number of transformed cells was measured.

(9) Test for phagocytic cells of leukocytes In a sampling tube, 90 ul of a standard kidney cell suspension of an immunological group and a control group are mixed with 10 ul of a latex solution diluted 1,000 times from a stock solution at 20 ° C. for 60 minutes. Cultured. After the culture, the cells collected by centrifugation at 3,000 rpm for 3 minutes were suspended in fetal calf serum, and then spread on a slide glass. The cell smear thus prepared was subjected to Giemsa staining, and the phagocytic activity of phagocytic cells in the kidney [phagocytosis rate: Sly
Phagocytosis of 500 phagocytic cells on Douglas
Ratio (%)] was measured.

[Results] Listonella anguil after immunization of test fish
The antibody titer against larum is as shown in FIG.
In the group containing organic selenium, the antibody titer gradually started to increase from 3 weeks after immunization, and showed an agglutinated antibody titer of 2048 times after 5 weeks from immunization, and the antibody rise was clearly more rapid than in the other groups. Was done. FIG. 3 shows the change in the migration ability of the non-immune fish-derived kidney cells by the immune serum for each week (the number of migrated cells is indicated by the total per 10 visual fields by microscopic observation). No significant difference was observed in the leukocyte chemotaxis. FIG.
Shows changes in the phagocytosis rate of immune fish-derived kidney cells. As for the phagocytic ability of leukocytes, phagocytic activity was observed in a section to which toyocobo was slightly added. From the above results, it was found that humoral immunity increased when Se was added to the feed and fed after immunization.

In this test, the effect of serum on opsonin was not examined, but it can be easily expected that this effect would be observed. In addition, since administration of Se resulted in such a high increase in the agglutinating antibody titer, vaccines for bacterial diseases and viral diseases and feed containing a certain concentration of organic Se were fed as a set. This proved that a high vaccine effect was obtained.

[0024]

Industrial Applicability The present invention can provide a marine cultured fish having activated immunological activity and a method for activating immunological activity of marine cultured fish. Provided is a marine cultured fish in which immune activity is activated by providing selenium-containing fish feed for facilitating the production of glutathione peroxidase, and a method for stimulating immunity of marine cultured fish using the principle of immunostimulation. it can.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing the function of selenium in detoxifying lipid peroxide.

FIG. 2 Listonella anguillaru
1 is a graph showing the change in agglutinating antibody titer in coho salmon serum after intraperitoneal immunization of m- formalin-inactivated bacterial cells.

FIG. 3 is a graph showing changes in migration ability of non-immune fish-derived kidney cells by immune serum each week.

FIG. 4 is a graph showing a change in the phagocytosis rate for immune fish-derived kidney cells.

Continued on the front page (72) Inventor Masahiro Izumida 559-6 Kitanocho, Hachioji-shi, Tokyo Nippon Suisan Co., Ltd. Central Research Laboratory (72) Inventor Tatsuyoshi Hirata 559-6 Kitanocho, Hachioji-shi, Tokyo Nippon Suisan Co., Ltd. (56) References JP-A-61-146155 (JP, A) JP-A-4-237468 (JP, A) JP-A-4-40888 (JP, A) U.S. Pat. "Aquaculture" Special Issue "Additives" (Vol. 347) Midori Shobo Co., Ltd. Issued January 5, 1992, pp. 46-51 "Fish Nutrition and Feed" Revised Edition Koseisei Koseikaku Heisei Apr. 10, 1987, p. 244, "Utient Requirements of Fish," Natl. Research Research Council, 1993, pp. 20-21, 26, 63. (58) Fields investigated (Int. Cl. ⁷ , DB name) A01K 61/00 A23K 1/16-1/18

Claims (2)

(57) [Claims] (1) The selenium is contained more than ordinary yeast.
Gluta in vivo by raising marine farmed fish with selenium-containing fish feed mixed with yeast
Promotes the production of thione peroxidase
For stimulating the immunity of marine cultured fish . 2. A fish culture wherein the usual fish feed contains fish meal.
2. The method for activating immunological activity of a marine cultured fish according to claim 1, which is a feed for use .