KR101207278B1 - Feed additives using Skullcap and Feed for raising fishes including the same - Google Patents

Abstract

The present invention relates to a feed additive using gold and farmed fish feed including the same. The present invention first put 100g of golden dried root with 1,000ml of distilled water in a celadon jar and put it in a stainless steel pot containing water and boil it in a water bath for 3 hours to extract about 800ml of sample solution. The activity of the extracted sample stock is analyzed. In addition, the sample stock solution is added to the concentration of 0.05% of the commercial commercial feed to prepare a culture feed, it is administered to the test for 8 weeks and at the same time, the control is to consume only the commercial feed for the commercial under the same conditions. As a result, according to the present invention, the cultured fish ingested the cultured feed increased the growth and immune activity, and hepatotoxicity due to hematologic physiological changes and administration compared to the cultured fish ingested the general commercial combination (EP) feed In addition, it can be seen that there is an advantage of providing a disease prevention effect for Ed Ward disease, a fish disease bacterium.

Description

Feed additives using Skullcap and Feed for raising fishes including the same}

The present invention relates to aquaculture fish feed, in particular, the sample extract extracted using the golden (skullcap) is added to a predetermined concentration in a commercial commercial formulation (EP) feed, and then orally administered to the cultured fish, general commercial formulation feed The present invention relates to a feed additive and a farmed fish feed using gold, which can prevent disease against fish disease bacteria compared to farmed fish ingested only.

Recently, the production of farmed fish has increased dramatically. In addition, large-scale and commercialization is proceeding rapidly, especially in the land tank farming industry. As such, domestic fish farming production is increasing per unit area. On the other hand, the rearing environment is gradually deteriorating, and the main issue of strengthening food stability for farmed fishes, along with various problems that directly or indirectly affect fish farming, such as year-round occurrence of mixed infectious diseases, increased marine pollution, increased feed costs, and lower fish prices Is emerging. In particular, suspicion and distrust of fish residues caused by misuse and abuse of antibiotics for fisheries has led to slow consumption of fish.

Therefore, it is required to develop natural herbal medicines that not only show bactericidal activity against pathogens such as bacteria, parasites, and fungi, but also enhance the natural anti-pathology by enhancing the inherent immune function of fish. Natural herbal medicines are natural plant resources, such as herbal medicines used in the human body, there is no concern about environmental pollution, and can produce high quality fish with good meat quality. This can generate high returns economically for aquaculture fishers.

Currently, research on natural substances that can improve physiological functions, improve meat quality and promote growth along with prevention of diseases of farmed fish is ongoing. For example, a combination of herbal medicines erbosan, plant extract aloe and green tea, wolfberry, ginseng, schisandra chinensis, sesame, cinnamon, sweet ginseng, lettuce, citrus fermentation solution, wormwood, yeonkyo, trifolium vinegar, manta mushroom, baejaja, palm cactus Extracts such as, ume, etc. have an antimicrobial effect against fish pathogens and have been reported to be effective in preventing disease when orally administered to farmed fish.

However, the efficacy of the herbal medicines from the above studies is mostly confirmed in the laboratory, and there are very few studies confirmed directly on the farm.

Therefore, an object of the present invention is to solve the above problems, to prepare a new natural herbal medicine with antimicrobial activity effect, parasite insecticidal effect, aquatic bacteria antifungal effect against fish disease bacteria, and directly orally administered to cultured fish at the farm This is to directly evaluate the nonspecific immune response of aquaculture fish and the resistance and growth of fish against fish pathogenic bacteria.

According to a feature of the present invention for achieving the above object, a first step of putting the golden dry root with distilled water in a first container; And a second step of preparing the feed additive by extracting the sample stock solution by placing the first container in a second container containing water and using a bath.

The first container of the first step is to mix 100g of the golden dry root and 1000ml of distilled water.

In the second step, the bath time is 3 hours, and the sample stock solution is 800 ml.

Preferably, the first container is a celadon jar, and the second container is a stainless steel pot.

And when tested in vitro (in vitro), if the feed additive contains 10% gold, the minimum growth inhibitory concentration (MIC) is the same as the minimum antibiotic growth concentration (MIC) of norfloxacin.

In addition, when tested in vitro, if the feed additive contains 5% of gold, there is a pesticidal insect against the parasites Skutika insects.

In addition, when tested in vitro, if the feed additive contains more than 5% of gold provides an antifungal effect on aquatic bacteria.

According to another feature of the invention, the feed additive prepared by the above method is added to a general commercial formulation (EP) feed, but adjusts the final concentration of the feed additive in the general commercial formulation (EP) feed to 0.05% To produce aquaculture feed. Such aquaculture feed can prevent Edward disease, a major fish disease bacterium, when administered to farmed fish for 8 weeks.

According to another feature of the present invention, the method comprising the steps of administering to aquaculture fish and a commercial commercial formulation (EP) feed prepared by the above method; Comparing the growth, hemochemical, immunological, pathological and anti-history for each cultured fish ingested the aquaculture feed and general commercial formulation (EP) feed, wherein the aquaculture feed is the final concentration of the feed additive Is adjusted to 0.05% and compared to the cultured fish ingested with the cultured fish and the general commercial combination (EP) feed while being administered to the cultured fish for 8 weeks.

In the present invention, the feedstock is prepared by extracting the sample stock solution using gold, and is added to the general commercial formulation (EP) while controlling the concentration to a predetermined concentration, and then administering the fish to the fish for a certain administration period, wherein the commercial formulation ( EP) We compare the characteristics of the farmed fish with the feed.

As a result, aquaculture fish that consumed 0.05% of cultured fish for 8 weeks showed better growth rate than fish that consumed commercially marketed compounded (EP) food. Not only do not induce, the lysozyme activity and phagocytic activity is increased to increase the immune activity of cultured fish ingested aquaculture feed, there is an effect that the defense ability against fish disease bacteria is improved.

In particular, Edward disease, a major bacterial disease that causes large mortality in flounder farming, provides disease prevention effects for a certain period of time, resulting in a high survival rate of farmed fish.

As a result, natural aquatic extracts are manufactured to produce farmed feeds, thereby reducing concerns about environmental pollution as well as producing high quality fish with high quality, which is expected to generate high profits.

1 is a graph of the parasite insecticidal effect on the Scutika insects of the sample stock solution containing gold at a predetermined concentration according to an embodiment of the present invention
Figure 2 is an antifungal effect graph for aquatic bacteria by concentration of sample stock solution according to an embodiment of the present invention
Figure 3 is a graph comparing the growth of the test and control in accordance with an embodiment of the present invention
4 is a graph showing the change in phagocytic activity in the immunological index analysis of the test and control in accordance with an embodiment of the present invention
Figure 5 shows liver tissues of control and liver tissues at 4 and 12 weeks after administration of 1% aquaculture feed, and liver and kidney tissues at 4 and 8 weeks after administration of 0.05% aquaculture feed.
6A and 6B are graphs showing cumulative mortality according to fish disease bacterial attack in the test plots treated with 0.05% aquaculture feed for 4 and 8 weeks.

Extract the sample stock solution using gold according to an embodiment of the present invention, the extracted sample stock solution is added to a general commercial formulation (EP) feed to a predetermined concentration so that it is ingested by the farmed fish, and then to the fish Various features, etc. will be described with reference to the accompanying drawings.

The cultured fish to be described in the present embodiment will be described by taking the flounder as an example, will be referred to as 'test sphere'. Therefore, in the present embodiment, aquaculture fish is used as a flounder or a test mix. The test spheres were absorbed while changing the sample stock solution extracted according to the present invention in a general commercial compounding (EP) feed to the final dosage concentration (0.05%, 0.1%, 1%) by administration period (4, 8, 12 weeks) Administered. The feed at this time will be referred to as 'food feed'. On the other hand, there is a 'control' as a target to be compared with the test, the control was administered only a commercial feed formula. This is referred to as 'mixed feed'.

The test zone was an average of 90 g of flounder, with 200 animals per tank for each final dose. Of course, if it is difficult to accommodate all 200 animals in one tank, a plurality of tanks can be used, in which case the experimental conditions applied to the tank should be the same. During the experiment, the water temperature of the bath is maintained at the natural water temperature from 13 ℃ to 24 ℃.

In addition, the natural herbal medicine used in the present invention was used in vitro (gold) excellent in antimicrobial activity, insecticidal effect, antifungal efficacy, and extracted the sample stock solution in the method of preparing a sample solution to be described below.

For reference, the golden cap (skullcap) is dried roots peeled off the skin of the perennial perennial golden (scutellaria baicalensis) belonging to the Lamiaceae. Gold is a herb that is classified as a food, and it is known to be less toxic and effective in protecting the liver. The main ingredient is a flavonoid compound, which is separated from about 30 species. The pharmacological action of gold is known to be bile excretion, acceleration, antibacterial action, anti-inflammatory action, antioxidant action.

First, a manufacturing method of extracting a sample stock solution using gold will be described.

100 g of golden dry root, known as a natural herb, is added to the celadon jar with 1,000 ml of distilled water. The reason why celadon jars are used is to prevent the destruction of various components contained in gold when preparing the sample stock solution. Thus, instead of celadon jars, jars or containers made of different materials may be used.

Next, the celadon jar is placed in a stainless steel pot containing water, and extracted by cutting off in a bath for 3 hours using a gas fire.

Therefore, distilled water containing the golden dry root is finally made into 800 ml, which is used as a sample stock solution.

On the other hand, when the sample stock solution is extracted, it is preferable to measure the activity state of the sample stock solution in vitro. This is because it is difficult to clinically measure the effect of the sample stock using fish. On the other hand, after measuring the active effects on various fish pathogens, parasites, aquatic bacteria, etc. in vitro, the clinical effects on fish and the like can be easily measured and demonstrated with active sample stocks.

Activity on the sample stock solution can be proved by measuring the following three effects.

1. Antimicrobial Activity (MIC) Effects of Sample Solution on In Vitro Fish Bacteria

2. Insecticidal effect of sample stock on in vitro Scutika insects

3. Antifungal effect on sample in vitro fungi (aquatic bacteria).

The three effects will be described separately. In order to measure the effect, the above-described method should be diluted with the medium or water of the experiment to be tested by the method described above, and mixed with the same amount with bacteria, parasites and aquatic bacteria growing in a tube or '96 well plate '. do.

First is the in vitro antimicrobial activity (MIC) effect on the sample stock solution. The MIC indicates the minimum growth inhibition concentration.

In order to measure the MIC effect, first, the final concentration of the sample stock is adjusted to 10 to 50%. The concentration is adjusted by dilution with distilled water or the medium to be tested. For example, when the final concentration is adjusted to 50%, the mixture may be diluted with 100 ml of sample stock solution and 100 ml of water.

In addition, the antibacterial activity strain is a representative fish disease bacterium isolated from the flounder kidney ' Edwardella ' , a gram-negative causative agent causing Edward disease. tarda ( G3) 'and' Streptococcus , a gram-positive bacterium that causes streptococcosis iniae (2-24) '.

The antimicrobial activity test is to adjust each fish disease bacteria to 10 ⁸ CFU / ㎖ to dilute the sample stock with medium and mix the components of the sample stock with 100 μl (final concentration is 20% of gold) + 100 μl of bacteria. The actual concentration is then sample stock containing 10% gold. After incubation at 25 ° C., 0.2 mg of p-idonitrotetazolium violet was added to investigate the minimum growth inhibitory concentration (MIC) in an environment in which red color due to the production of INT formazan was not developed.

As a result, Table 1 below shows that the minimum growth inhibitory concentration (MIC) is equal to 0.125mg / ml, the MIC concentration of norfloxacin (nor) antimicrobial used as a control. have. That is, the sample stock containing 10% of gold is the MIC of the fish disease bacteria is the same as the MIC of norfloxacin, the representative antibiotic.

[Table 1]

Second is the pesticidal effect of sample stock solution on in vitro Scutika insects.

For this purpose, the parasites are separated by subcultured from the flounder at 15 ° C. on a flounder cell line 15 ° C., and 100 µl of the medium containing Scutica insects and 100 µl of the sample stock solution are mixed. Then, 100 μl of the mixed medium and 100 μl of the sample stock solution were observed at 10 ° C. incubator at 10 ° C. for 10 minutes to observe the activity change and denaturation (ie, degree of death, movement of the carcass, and cytoplasmic destruction).

The observation result is shown in FIG. 1 is a graph of the parasite insecticidal effect on the Scutika insects of the sample stock solution containing gold at a predetermined concentration according to an embodiment of the present invention.

Looking at the measured insecticidal effect on the Scutika insects of Figure 1 it can be seen that if the sample contains more than 5% of the gold in the pesticidal effect against Scutika insects. In contrast, when gold is not used at all (control), it can be seen that there is almost no insecticidal effect on the Scutika insects. In other words, Scutika is not dead at all.

Third, the antifungal effect on the in vitro fungi (aquatic bacteria) of the sample stock solution.

It targets aquatic bacteria (Saprolegnia sp.) Isolated from eel, and aquatic mycelia of about 8 mm diameter are planted in the bottom of a 15 ml test tube. At this time, the bottom of the test tubing includes a medium prepared by adding the sample stock solution to SDB (Sabouraud Dextrose Broth) by concentration. Then incubate for one week at 15 ℃ to examine the development of aquatic bacteria.

A graph of growth inhibition effect on aquatic bacteria by concentration of gold as a result of the investigation is shown in FIG. 2.

2, it can be seen that there is antifungal activity when the sample stock contains 5% or more of gold. That is, 5% or more indicates that aquatic bacteria can hardly grow. On the other hand, when gold is not used at all (control), it can be seen that aquatic bacteria are grown more than about 30 mm.

By the method described above it can be confirmed that the sample stock solution is active.

Subsequently, the aquaculture feed prepared by adding the sample stock solution to the blended feed is orally administered to the aquaculture fish being bred by the above-described breeding method to look at the analysis result of the measurement index. At this time, the aquaculture feed absorbs the sample stock solution into the blended feed, and adjusts the final dose of the sample stock solution to 0.05%, 0.1%, 1%, and the state of the farmed fish by administration period (4 weeks / 8 weeks / 12 weeks). Was analyzed. At this time, the final dosage concentration of 0.05%, 0.1%, 1% means that the sample stock solution containing gold in 0.5kg, 1ml, 10ml of 1kg of the general commercial formulation (EP) feed. Hereinafter, the cultured feed having the concentration of 0.05%, 0.1%, and 1% of the sample stock solution will be referred to as 0.05% farmed feed, 0.1% farmed feed, and 1% farmed feed, respectively.

The analysis was carried out for the following items 1 to 5 for each test and control, and the results will be examined.

1. growth analysis,

2. hemochemical analysis;

3. immunological analysis,

4. histopathological analysis,

5. Antimorbidity analysis.

For each of the above items, each data was extracted for each test and control, and the characteristics of the test and control were compared.

Analysis method

1. Growth Analysis

The growth rate was randomly selected from each of the test and control groups for each administration period, and the average value of 10 rats was determined by measuring the hepatosomatic index (HSI: liver weight / weight * 100).

The resulting graph of growth is shown in FIG. 3. Figure 3a is a graph comparing the growth of the control group intake of 0.1% and 1% aquaculture feed and the control compound intake of general compound feed, Figure 3b is the control group intake of 0.05% aquaculture feed and control compound intake of general compound feed This is a graph comparing the growth of.

Referring first to Figure 3a, between 4 weeks and 8 weeks, the test group intake of 1% aquaculture feed showed better growth than the control group ingesting 0.1% aquaculture feed and the control group ingesting only a general compound feed. At week 12, the Interval Index did not show any difference.

On the other hand, when the concentration is adjusted to consume 0.05% aquaculture feed, it can be seen that there is a difference between the test group and the liver weight index from the 8th week as compared to the control group as shown in FIG. 3b.

Therefore, when looking at the growth of the flounder, it can be seen that the aquaculture feed to which the sample feed solution is added improves the growth rate compared to the general commercial feed.

However, during the growth test, the water temperature was maintained at 18 ° C until the 5th week, but gradually dropped to 12 ° C, and the flounder growth was slowing down overall.

2. Blood Chemical Analysis

The hemochemical index is to investigate the physiological phenomena and side effects of farmed fishes by aquaculture feed with sample stock.

To this end, test specimens are collected from the nasal vessels with a heparin-treated syringe at necropsy, and the plasma obtained by centrifugation (12,000 rpm, 10 minutes, 4 ° C) of the collected blood is stored at -80 ° C. Blood biochemical changes were measured using FujiDri-Chem3000 within 3 days.

The measurement results are shown in the following [Table 2] and [Table 3].

[Table 2]

[Table 3]

Looking at the [Table 2] and [Table 3], the hemochemical index is 0.05%, 0.1%, 1% compared to the test group and the control group ingested the feed can be seen that there is no difference in numerical value. In other words, no physiological changes occur in the flounder, which is a farmed fish that consumed aquaculture feed.

3. Immunological Analysis

Immunological index analysis measures lysozyme activity, phagocytic activity, and immunogenic activity (IL-1β) in the blood.

Serum lysozyme activity is a turbidimetric assay method modified from the method of Ellis (1990). This method is used to measure the amount of micrococcus lysodeikticus (Sigma) degradation using a 96 well plate at 37 ° C, 0 min, 5 min and 10 min at absorbance 600 nm. One unit of lysozyme is defined as the amount of sample that reduces the absorbance 0.001 / min. Tissue lysozyme activity was homogenized with tissue (0.1 g) in PBS (pH 6.2), centrifuged (12,000 rpm, 10 minutes), and the supernatant was calibrated to OD 1.0 at absorbance 595 nm and analyzed by the turbidimetric assay method. .

In addition, the blood lysozyme activity values measured by the above method for each test group ingesting 0.05%, 0.1% and 1% aquaculture feed and the control group ingesting the general compound feed are shown in [Table 4] and [Table 5].

[Table 4]

[Table 4] analyzes the changes in lysozyme of the flounder by administering 0.1% and 1% aquaculture feed for each administration period of 0 weeks, 4 weeks, 8 weeks, 12 weeks. In this study, there was little difference in the changes of blood lysozyme activity in the olive flounder by 0.1% and 1% aquaculture.

[Table 5]

Table 5 shows the change in lysozyme of the olive flounder by administering 0.05% aquaculture feeds for each administration period of 0, 4, 8, and 12 weeks.

In the fourth week, the test group showed a difference in lysozyme activity change compared to the control group, but there was no significant difference. However, the lysozyme activity of the body was different from the control group at 4 weeks, and the lysozyme activity of the spleen was found to be different from the control at 8 weeks. The greater activity of the lysozyme activity compared to the control means that the lysozyme activity increased the nonspecific immune response of the flounder fed aquaculture. In other words, the ability to defend against bacterial attacks on the flounder improved.

In the meantime, we look at changes in phagocytic activity in immunological index analysis. This is shown in FIG. 4. Figure 4 is a graph comparing the change of phagocytic activity of the flounder by administering the control and 0.05% aquaculture feed to the flounder for 4 weeks, 8 weeks, and 12 weeks. From this, it can be seen that at 4 weeks and 8 weeks the test cells increased phagocytic activity compared to the control. Increasing the phagocytic activity is an increase in the immune activity of the flounder means that the defense ability against bacterial attack is improved. At week 12, however, there was no significant difference between the test and control groups.

To measure immunogene activity (IL-1β), spleen and body tissues are extracted for each test zone, and then total RNA is separated using trizol. Then cDNA is made using a cDNA synthesis kit (Invitrogen). Real-time gene analysis is then used to measure gene changes in IL-1b against the b-actin gene. The measurement results do not provide experimental data, but the test group was analyzed to be less affected than the control group.

4. Histopathological Analysis

Histopathological analysis was performed by taking liver and kidney tissues for each period and fixing them in 10% neutral buffered formalin to make tissue sections. Hematoxyline and Eosin (H & E) staining were observed as optical microscopes.

The observations are shown in FIG.

5A shows three liver tissues. In turn, the normal liver tissue of the flounder, the liver tissue of the test group fed 1% aquaculture feed at 4 weeks, and the liver tissue of the test group fed 1% aquaculture feed at 8 weeks, respectively.

The results show that the first picture is normal liver tissue, but non-nausea necrosis (part B) was observed in the liver tissues of 1% aquaculture fed at 4 weeks in the second picture, and at 8 weeks in the third picture. Necrosis (C part) was clearly observed in the liver tissues of the 1% aquaculture diet, which was clearly distinguished from the normal area around the blood vessels. In other words, it can be expected that if the concentration of the sample stock extracting gold from aquaculture feed is 1% or more, it causes stress of necrosis and fat degeneration of liver cells of the olive flounder.

On the other hand, Figure 5b is a comparison of the liver and kidney tissue and control of the test when the concentration of the sample stock solution to 0.05% in aquaculture feed. This suggests that degeneration does not occur in the liver tissue, and that no problem occurs in the kidney tissue.

As a result, it can be inferred that necrosis and fat degeneration are caused by the administration of aquaculture with 1% or more of the sample extract of gold based on this, and the degree of denaturation and necrosis increases in proportion to the concentration of the sample solution. Can be.

5. Anti History

This is to investigate the preventive effect of bacterial diseases. To this end, E. tarda GY-01 strain (0.99 × 10 ⁶⁾ , which is the major fish disease bacterium, was applied to 10 rats at each time of administration of cultured feed for each 4, 8 and 12 weeks. 2.8 × 10 ⁶ CFU / fish) and S. iniae FT5228 strain (1.15 × 10 ⁶ to 12.7 × 10 ⁶ CFU / fish) were injected and compared for survival after 2 weeks.

First, in Table 6, 0.1% and 1% aquaculture feeds were administered, and the relative survival rate with the control group was examined at 4, 8, and 12 weeks.

TABLE 6

As shown in [Table 6], the test group administered 0.1% aquaculture feed was slightly increased compared to the control group, and the test group administered 1% aquaculture feed did not show a big difference from the control group.

On the other hand, Table 7 shows the relative survival rate of the control group at the 4th, 8th, and 12th week after the administration of 0.05% aquaculture feed.

[Table 7]

[Table 7], the test group administered with 0.05% aquaculture feed showed the highest relative survival rate at 4 and 8 weeks for ED disease compared to the control group, and there was no significant difference with the control group at 12 weeks. have. This suggests that the treatment group treated with 0.05% aquaculture feed selectively prevented disease for up to 8 weeks for Edward disease.

On the other hand, through the confirmation of the cumulative mortality shown in Figures 6a and 6b with respect to relative survival rate it can be confirmed that the disease prevention effect is high up to 4 weeks and 8 weeks only for the Ed Ward disease.

That is, Figures 6a and 6b is a graph showing the cumulative mortality caused by the attack of fish disease bacteria in the test group administered 0.05% aquaculture feed. Specifically, in FIG. 6a, two fish disease bacteria, E. tarda and S. iniae, were attacked in the state of administering to the flounder for 4 weeks, and in FIG. 6b, the flounder was administered to the flounder for 8 weeks. In this state, two fish disease bacteria, E. tarda and S. iniae, were attacked.

First, as shown in Figure 6a, the control group (S. iniae) and the control group (HwangGum-S. Iniae) administered the aquaculture feed for streptococcus bacteria showed high mortality, whereas for Ed Ward disease HwangGum-E. Tarda showed lower cumulative mortality than control (E. tarda).

Similarly, in FIG. 6B, the experimental group (HwangGum-E. Tarda) administered the aquaculture feed showed a low cumulative mortality rate only for Ed Ward disease, thereby preventing disease.

As described above, in the above experimental results, when the sample extract from which gold was generally extracted was added to a commercial commercial feed and administered to aquaculture fish at 0.05% concentration for 8 weeks, the control group ingested only a commercial commercial feed. Compared with the growth rate, it can be seen that the defense ability against bacteria due to increased immune activity is improved. In addition, the disease prevention effect of Edward disease, which is a fish disease bacterium, was found to have a higher survival rate than the control group.

The scope of the present invention is not limited to the embodiments described above, but is defined by the claims, and various changes and modifications can be made by those skilled in the art within the scope of the claims. It is self evident.

Claims (10)

A first step of putting 100 g of golden dry root and 1000 ml of distilled water into a first container;
A feed additive using a gold, characterized in that it comprises a second step of preparing a feed additive by extracting the sample stock solution of 800ml by placing the first container in a second container containing water for 3 hours in a hot bath . delete delete The method of claim 1,
The first container is a celadon jar, the second container is a feed additive using gold, characterized in that the stainless steel pot. delete delete delete The feed additive of claim 1 is added to a general commercial formulation (EP) feed,
Aquaculture feed, characterized in that the final concentration of the feed additive to occupy in the general commercial formulation (EP) feed prepared by adjusting to 0.05% by weight. delete Administering to the farmed fish the cultured feed prepared using the 8 and commercial commercial combination (EP) feed; And
Comparing the growth, hemochemical, immunological, pathological, and anti-history for each cultured fish ingested the cultured feed and general commercial formulation (EP) feed,
The cultured feed was adjusted to a final concentration of 0.05% by weight of the feed additive to the cultured fish for 12 weeks while ingesting the fish and the commercial commercial combination (EP) feed at 4 weeks, 8 weeks, 12 weeks A method of evaluating farmed feed, characterized in that compared with farmed fish.

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