JP2547371B2 - Feeding method for crustacean, fish feed, and fish feed - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a crustacean and fish feed for administration to shrimps, crabs, and fish. Here, prawns are prawns (Penaeus japonicus) and bull shrimp (P
enaeus monodon), Black shrimp (Penaeus chinensis)
, And Kuruma prawns such as banana shrimp (Penaeus morguiensis) and other seawater and freshwater shrimp. Crabs include all seawater and freshwater crabs, and fish are seawater and freshwater. Includes sex fish.

[0002]

2. Description of the Related Art In recent years,
Cultivation of crustaceans and fish is actively carried out, but due to the frequent occurrence of infectious diseases, economic loss due to death or growth delay is extremely large, which is a problem in the aquaculture industry.
The infectious diseases include infectious diseases caused by Vibrio spp. And other bacteria, and infectious diseases caused by baculovirus and other viruses.

At present, various antibacterial agents such as antibiotics and sulfa drugs are used for the prevention and treatment of these diseases.
The effect is not always sufficient, and in recent years, antibacterial agents such as antibiotics have a tendency to be restricted as much as possible due to their residual effects in the living body and the appearance of drug-resistant bacteria, which have an adverse effect on the human body.

The present invention has been made in view of the above-mentioned circumstances, and the purpose thereof is to activate the biological defense function of crustaceans and fish by feeding, enhance the biological function and the immune function, and exert a weight gain effect. The present invention provides a feed for fish, fish feed, and fish feed.

[0005]

Means for Solving the Problems In order to achieve the above-mentioned object, the crustacean and fish feed of the present invention is characterized by adding peptidoglycan derived from Gram-positive bacteria, yeasts and fungi to the feed. . Further, the feeding method of the fish feed of the present invention, the relationship between the daily dose of peptidoglycan to fish and its proper administration period is set by a preliminary test, and the peptidoglycan-containing feed is appropriately fed at a ratio corresponding to the above dose. Administration period It is characterized by daily administration.

[0006]

[Function] Peptidoglycan (hereinafter, abbreviated as PG) is taken up in the body to enhance phagocytic activity of blood cells.
It enhances the defense function of the body, thereby enhancing immunity and weight gain. Further, PG has a relationship between the dose for fish and its proper administration period, and the effect is diminished even if it is continuously administered in excess.

[0007]

EXAMPLES The present invention will be described in detail below with reference to specific test examples. Test Example 1 Test method: Kuruma prawns having an average body weight of 25 g were divided into 4 groups of 30 fish each, and PG was added to the feed in the present groups 1, 2 and 3
Is a daily dose per 1 kg of body weight of shrimp.
It was added so as to be 05, 0.2 and 1.0 mg and given every other day. On the day without PG administration of the present invention group and P on the control group
The same feed without G was fed. After breeding for 7 days in this way, 8 days later, blood was collected from the shrimp heart and
10 ⁷ latex beads (0.81 micron diameter) were mixed per 1 ml of blood in M medium, and after 45 minutes, the number of beads taken up in 100 cells of blood cells and the total number of beads taken up in 100 cells of blood cells were examined. It was

Results: The number of blood cells incorporating latex beads per 100 blood cells in the prawns of the present invention and the control was shown in FIG. Average of 3 in the control area
8.2 blood cells had incorporated latex beads, while 0.05 mg group had an average of 59.0 cells, 0.2 mg.
The group had an average of 62.5 cells and the 1.0 mg group had an average of 53.0 cells, and in any PG-administered group, more blood cells were phagocytosed with the latex beads than in the control group.

The total number of incorporated latex beads per 100 blood cells is shown in FIG. Compared to the average of 134 beads taken up in the control, 0.0
Average of 372 in 5 mg group, 450 in 0.2 mg group
The number of beads was 1.03 mg, and the number of 1.0 mg groups was 313, and more beads were taken up in blood cells in each of the present invention groups than in the control group. From the above, it became clear that the phagocytic activity of blood cells was enhanced by administering PG to the prawns. Moreover, it was suggested from FIGS. 1 and 2 that there is an optimum dose when PG is administered.

Test Example 2 Test Method: Juvenile prawns (PL30, average weight 0.01 g) were put in two 500-liter water tanks of running water type 20.
Each of them was housed 0, and 60 days after the start of the test, each was transferred to a 5-ton aquarium and bred for a total of 95 days. The present invention group was fed with a feed supplemented with PG in an amount of 0.2 mg per 1 kg of body weight of shrimp for 1 week, and for the next 1 week, a method of feeding a PG-free feed was repeated until the end of the test. . The control group was always fed with PG-free feed. Then, in order to confirm the effect of PG, the following items were examined. (1) Shrimp survival rate 60, 75, 95 days after the start of the test (2) Shrimp weight 60, 75, 95 days after the start of the test (3) 60 days, 75 after the start of the test Protective effect against experimental infection in shrimp on day 95, the experimental infection of the test item (3), Vibrio sp. 1.7-2.
30 shrimp on the 67th day and the 95th day after the start of PG administration were immersed in 7 × 10 ⁷ cells / ml of suspended seawater for 1 hour each, and then the survival rate for 10 days was examined.

Results: (1) Table 1 shows the survival rate of the prawns of the present invention group and the control group during the test period.

[Table 1] Note) The numbers in parentheses indicate the number of surviving fish / the number of test fish.

As described above, the survival rate at the end of the test was 58.5% in the present invention group, whereas it was 30.5% in the control group.
%, And the survival rate of the former was excellent. In addition, when a bacterium was tested for dead shrimp, a bacterium of the genus Vibrio was isolated from a part of the present invention and most of the control. Therefore, it is considered that the reason why the survival rate of the present invention group was higher than that of the control group was that PG promoted the biological defense function and protected the bacterial infection due to Vibrio etc.

(2) Table 2 shows the average body weight of the prawns of the present invention group and the control group during the test period.

[Table 2] Note) Numbers are mean ± standard deviation. Thus, the average weight at the end of the test was 2.
While it was 58 g, the control group was 2.00 g,
The former was growing well.

(3) Changes in the survival rate after experimental infection in the prawns of the present invention group and the control group are shown in FIGS. 3 and 4. That is, the prawns on the 67th day after the start of the test
brio sp. The survival rate after 1 hour of immersion in the bacterial solution was 86.7% after 1 day and 76.7% after 2 days, and no mortality was observed thereafter. Control area is infected 1
56.7% after day, 30% after 2 days, 26.7 after 3 days
%, And the final survival rate was 26.7% (see FIG. 3).

95 days after the start of the test
rio sp. The survival rate after immersion in the bacterial solution for 1 hour was 86.7% after 3 days in the present invention group and 80% after 4 days, and the final survival rate was 80%. 7 days after infection in the control area
The rate was 3.3%, 56.7% after 2 days, 33.3% after 3 days, and the final survival rate was 33.3%. (See Figure 4). As described above, the survival rate after experimental infection was significantly high (p <0.01) in all of the present invention plots. Regarding this cause, it is considered that PG enhanced the biological defense function centered on the phagocytic activity of the blood cells of Shrimp prawn.

Test Example 3 Test method: A fine feed containing PG (PG 5 mg / Kg feed) was daily administered to larvae of the zoea, mysis and post-lava (PL15) stages of the shrimp to examine the survival rate and zoea stage. The rate of transformation from to the Mitsis stage was investigated.
The dose of the fine feed was 0.16 mg / day in the zoea stage, 0.2 mg / day in the mesis stage, and 0.24 mg / day in the post-rava stage per larva. The daily dose per larva of PG calculated from the dose of feed was 5 × 10 ^-7 mg in the Zoea period and 6.25 × 10 ^{-7 in the} Mesis period.
mg, 7.5 × 10 ⁻⁷ mg in the post-lava period.
This dose is about 0.1 to 0.2 mg as the daily dose of PG per 1 kg of body weight of the larva.

Results: Table 3 shows the rate of transformation of the shrimp of the present invention group and the control group from the zoea stage to the mesis stage and the survival rate from the zoea stage to the post-lava stage (PL15).

[Table 3]

Compared to car shrimp and the like, bull shrimp
The survival rate in the larval period is extremely low, and is said to be usually around 20%. However, as shown in Table 3, it was revealed that administration of PG increases the survival rate and the transformation rate.

Test Example 4 Test Method: Ayu having an average body weight of about 58 g were divided into two ponds according to the test section shown in Table 4 and subjected to the test. The test fish were bred for 1 month in a 2 × 1 × 1 m concrete pond. The water temperature during the breeding period is about 20-23 ° C.
Met. In the present invention group, PG was added to the basic feed in an external proportion and molded into pellet crumble, and then the test feed was fed so that the dose of PG was 0.01 to 1.0 mg per 1 kg of fish body weight per day. did. The control group was fed with PG-free feed.

Results: After feeding the PG-added feed for 1 month, blood was collected from the tail blood vessels of 10 ayu from each group, and the blood plasma obtained by the chick feeding was 2 × 10 ⁷ cells / ml of rabbit red blood cells at 20 ° C. After obtaining the ratio of hemolysis in 90 minutes,
The reciprocal of the plasma volume for hemolyzing half the amount was determined as the ACH ₅₀ value. The results are shown in Table 4 and FIG. In addition, AC
H ₅₀ values were expressed as mean ± standard error.

[0021]

[Table 4] Note) There is a significant 5% level difference between different signs by DANCAN test.

As shown in Table 4 and FIG. 5, the ACH ₅₀ values of all the present invention plots tended to be higher than those of the control plots,
It was confirmed that the ACH ₅₀ value of the plasma of ayu increases by the administration of the PG-added feed. However, the value was highest when 0.1 mg of PG was administered and tended to be lower in the 1.0 mg group than in the 0.1 mg group. Have been suggested to exist. The ACH ₅₀ value represents the ability to attack antigens by the complement alternative pathway, but in recent years it has been reported that the receptor for C3, which is one of the complements, may be present in phagocytes and peripheral blood lymphocytes (Matsuyama et al. 1991 Spring Meeting of the Japanese Society of Fish Diseases, Nishimura et al., Nisui, 57 (12), 22
19 (1991)), it is considered that complement also has the ability to activate phagocytic cells and peripheral blood lymphocytes, and plays an important role as a biological defense function of fish. In addition, since the ACH ₅₀ value of fish is significantly higher than that of mammals, it is considered that fish have a higher rate of dependence on alternative pathways which are said to be phylogenetically older than the classical pathway (Yano). Etc., Nisshi, 54 (6), 10
49 (1988)). In this test, it was confirmed that the administration of PG improves the biological defense ability of fish.

Test Example 5 Test Method: Frying rainbow trout juveniles having an average body weight of about 0.1 g immediately after ascending were subjected to the test according to the test classification shown in Table 5 by dividing 800 fish in each section into four aquariums. The test fish were bred for 4 weeks in a 60-liter glass water tank. The water temperature during the breeding period was 15 to 16 ° C. In the present invention group, after adding PG to the basic feed in an outer percentage, it was molded into a diameter of 0.3 to 0.5 mm, and the dose of PG was 0.01 to 0.1 mg per 1 kg of fish body weight per day. I was paid to become. The control group was fed a PG-free diet.

Results: After feeding PG-added feed for 4 weeks, 40 rainbow trouts in each ward were picked up and 2.5 × 1
Vibrio a of 0 ^{5 to} 2.5 × 10 ⁶ CFU / ml
It was dipped in a physiological saline solution in which nguillarem was suspended for 30 minutes, and the survival rate for 21 days was measured. The non-infected area was immersed in physiological saline free of bacteria for 30 minutes. The results are shown in Table 5 and FIG. 6 (results of 2.5 × 10 ⁵ CFU / ml), and FIG. 7 (results of 2.5 × 10 ⁶ CFU / ml).
It was shown to.

[0025]

[Table 5]

As shown in Table 5 and FIGS. 6 and 7, 2.5
× 10 ⁵ GFU / ml attack and 2.5 × 10 ⁶ CFU / ml
In both cases of ml attack, the survival rates of all the inventive plots were significantly superior to those of the control plots. As a result of the activation of the immune function of juvenile rainbow trout by the administration of PG,
It is considered that it has acquired the disease resistance against Vibrio anguillarum. In this test, it was confirmed that the administration of PG improves the biological defense ability of fish. Since the survival rate in the non-infected area was 100%, the cause of death of the fish during the infection test was the V used for the attack.
It is considered to be an infection caused by ibrio anguillarum.

Test Example 6 Test method: Eels having an average body weight of 200 g were subjected to the test according to the test categories shown in Table 6 with 30 fish in each group. The test fish were bred for 1 month in a 2 × 1 × 1 m concrete pond. In the present invention group, PG was added to the basic feed in an external proportion, and once every two days, the dose of PG was 0.1% per 1 kg of fish body weight per day.
The test feed was fed so as to be 2 to 1.0 mg. still,
The control group was fed with PG-free feed.

Results: Edwardsiella t of 1 × 10 ⁵ CFU per eel after feeding PG-added feed for 1 month
Arda was inoculated intraperitoneally and the survival rate for 20 days was examined. The results are shown in Table 6 and FIG.

[0029]

[Table 6] Note) In the table, the number in parentheses indicates a significant difference from the control.

As shown in Table 6 and FIG. 8, the survival rate of the control group was 53.3%, whereas that of the group of the present invention to which 0.2 mg of PG was administered was 90.0%. it was high.
It is considered that the administration of PG resulted in the activation of the immune function of the eel, resulting in the acquisition of anti-pathogenicity against Edwardsilella tarda. However, PG is 1.
The survival rate of the 0 mg-administered group was 73.3%, and there was no significant difference from the control group, suggesting that there is an optimum dose when PG is administered. In this test, it was confirmed that the administration of PG improves the biological defense ability of fish. In addition, the cause of the death of the eel was all Edw based on the results of the bacterial and histopathological examinations.
It was determined to be due to infection with Ardsilella tarda.

Test Example 7 (Long-term continuous administration test of PG) Test method: According to the test classification shown in Table 7, rainbow trout having an average body weight of about 20 g was divided into two ponds, each of which had 160 fish, and used for the test. The test fish were bred for 24 weeks in a circulation type 1.5t FRP water tank equipped with an ultraviolet sterilizer. The water temperature was adjusted to 17 ° C during the breeding period. In the present invention group, PG was externally added to the basic feed and molded into pellets having a diameter of 3.2 mm, and then the dose of PG was adjusted to 0.
The test feed was fed so as to be 004 mg to 4 mg.
The control group was fed with PG-free feed.

Results: After feeding the PG-added feed, blood was collected from the tail blood vessels of 10 rainbow trouts in each group every 4 weeks, and the separated plasma was 2 × 10 ⁷ cells / ml of rabbit red blood cells at 20 ° C. The proportion of hemolysis in 90 minutes was determined, and the reciprocal of the plasma volume for hemolyzing half the amount was determined as the ACH ₅₀ value. The results are shown in Table 7.

[0033]

[Table 7] Note) The numbers in the table are ACH ₅₀ values (units / ml) (mean ± standard error). Note) There is a significant difference of 5% level between different signs in the vertical line by DANCAN test.

As shown in Table 7, in the 4th week, 0.
The 4 mg dose was 0.004 m in the 8th week.
g, 0.04mg, and 4mg administration group is the control group ACH
_The value was higher than ₅₀ . However, the 0.4 mg and 4 mg dose groups were 0.004 m
The activity was lower than that in the control group at 16th week and 0.04mg administration group at 20th week, respectively. This indicates that PG has an appropriate dose or administration period, and the effect is diminished even if excessive administration is continued. From the above test results, the ACH ₅₀ value of rainbow trout plasma is increased by administration of PG, and PG has an appropriate dose and administration period. When 0.4 mg is administered daily, it is 4 weeks, and It was found that 8 weeks or 12 weeks is appropriate when 0.004 mg to 0.04 mg is administered. It is considered that this tendency also appears in the fish species to be cultivated having the same biological defense mechanism (red sea bream, yellowtail, eel, sweetfish, carp, etc.).

Test Example 8 (Effect of Extrusion Processing Reappearing on PG) Test Method: According to the test classification shown in Table 8, rainbow trout having an average body weight of about 20 g are divided into two aquariums, and 160 fish in each section are subjected to the test. did. The test fish were bred for 4 weeks in a circulation type 1.5 t FRP water tank equipped with an ultraviolet sterilizer. The water temperature during the breeding period was adjusted to 17 ° C. In the present invention group, PG was externally added to the basic feed and molded into 3.2 mm diameter pellets and about 4 mm diameter expansion pellets, respectively, and then the projected amount of PG was 0 per 1 kg of fish weight per day. The test feed was fed so that the amount would be 0.04 mg. In addition,
A PG-free feed was fed to the control plot.

Results: Four weeks after feeding the PG-added feed, blood was collected from 10 rainbow trout tail blood vessels in each section, and the blood plasma obtained after separation was 2 × 10 ⁷ cells / ml of rabbit red blood cells at 20 ° C. The rate of hemolysis per minute was determined, and the reciprocal of the plasma volume at which half the amount was hemolyzed was determined as the ACH ₅₀ value. Table 8 shows the results.

[0037]

[Table 8] Note) Numbers in the table are mean ± standard error.

As shown in Table 8, also in the expansion pellets, it was confirmed that the ACH ₅₀ value was increased by the addition of PG, which was comparable to the usual pellets. It is known that expansion pellets have higher energy utilization than normal pellets by converting the starch quality in the feed raw material into α, and thus are superior in growth.
Further, since the feed is less pulverized and the feed is less soluble in water, the contamination of aquaculture water is significantly reduced. It is also possible to feed saltwater fish such as yellowtail, eels, etc., which have not been eaten pellets in the past. For the above reasons, it is said to be a new form of feed that is becoming popular in recent years. However, generally, when the feed is subjected to the extrusion process, severe physical conditions such as a high pressure of about 10 atm and a high temperature of 100 ° C. or higher are added, so that the transformation of the raw material becomes a problem. This test revealed that PG can improve the biological defense function of fish without being affected by the severe physical conditions of such extrusion processing. This means that PG can be used not only for normal pellets and mash feeds, but also for expansion pellets, and thus in the shellfish and fish diets where various feed forms are required depending on the target organism, It shows that PG is more widely available.

As described above, the present invention feeds shrimp, crab crustaceans and fish to enhance the biological functions and immune functions of crustaceans and fish, and exerts a weight gain effect.

Further, according to the feeding method of the present invention, the biological function and the immune function of fish can be achieved by an appropriate standard, and at the same time, there is an advantage in actual operation that the economical effect thereof is high.

[Brief description of drawings]

FIG. 1 is a graph showing the phagocytic activity of blood cells of prawns.

FIG. 2 is a graph showing the phagocytic activity of blood cells of prawns.

FIG. 3 is a graph showing the survival rate of Shrimp after infection with Vibrio esculenta.

FIG. 4 is a graph showing the survival rate of Shrimp after infection with the Vibrio test.

FIG. 5 is a graph showing the ACH ₅₀ value of the plasma of sweetfish which was administered with PG-added feed for 1 month.

FIG. 6: Vibrio anguillarum (2.5 × 10 ⁵ CFU /
(ml) is a graph showing the survival rate of PG-administered rainbow trout after challenge.

FIG. 7: Vibrio anguillarum (2.5 × 10 ⁶ CFU /
(ml) is a graph showing the survival rate of PG-administered rainbow trout after challenge.

[Figure 8] Edwardsiella tarda (1 x 10 ⁵ CFU / m
It is a graph which shows the survival rate of the PG administration eel after attacking by 1).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location A61K 35/74 A61K 35/74 B 38/00 37/02

Claims (2)

(57) [Claims] 1. A feed for crustaceans and fish, comprising peptidoglycan derived from Gram-positive bacteria, yeasts and fungi. 2. A relationship between the daily dose of peptidoglycan to fish and its appropriate administration period is set by a preliminary test, and peptidoglycan-containing feed is administered daily at the appropriate administration period at a rate corresponding to the above dose. Feeding method for fish feed.