Composition for treating mucus on fish gills

Abstract

A composition for treating mucus on the gills of fish for the therapeutic or prophylactic treatment of amoebic gill disease in fish, wherein the composition comprises extruded fish feed supplemented with arginine; the fish feed comprises protein, binder, fat, vitamins and minerals; the total arginine content of the fish feed is at least 3.0% (wt / wt) of the total feed weight.

Description

[0001] This application is a divisional application of patent application filed on August 1, 2016, with application number 201680048190.1 and titled "Composition for treating mucus on fish gills".

[0002] This invention relates to compositions for treating mucus on fish gills. More specifically, this invention relates to compositions that increase the viscosity of mucus on fish gills. The invention also relates to compositions that increase the polysaccharide content of said mucus. Mucus treatment is a therapeutic or preventative treatment for amoebic gill disease in fish. Amoebic gill disease is caused by marine amoebas, specifically by *Paramoeba perurans*. The fish can be salmonid fish such as Atlantic salmon (*Salmo salar*) or rainbow trout (*Onchorhynchus mykiss*).

[0003] Amoebic gill disease (AGD) is one of the biggest challenges facing the global marine salmonid aquaculture industry. It has been found in a variety of fish species, both farmed and wild, including carp, halibut, ayu, mackerel, and roundfin. It was first reported in Australia in 1984, and subsequently found on the west coast of the United States, Ireland (1995), Scotland and Norway after 2006, and Chile (2007). Outbreaks typically occur in late summer to early winter when water temperatures are above 10°C; however, recently it has garnered year-round attention.

[0004] AGD is caused by *Paramoeba perurans*, formerly *Neoparamoeba perurans*, a free-living and conditionally parasitic amoeba that can be fatal if left untreated. It is estimated to cause up to 20% of total production costs in terms of treatment, reduced fish growth, and fish mortality. *P. perurans* is a marine amoeba. *P. perurans* is an extracellular parasite belonging to the phylum *Flabellinea*.

[0005] Risk factors include high salinity, warmer water temperatures, high stocking density of fish, suspended organic matter in the water, and early gill damage.

[0006] Clinically, AGD leads to anorexia (reduced food intake), respiratory distress, open gill covers, and lethargy. Generally, an increased number of white to gray, mucus-like patches can be seen on the gill surface. The presence of amoebas is usually associated with excessive mucus production in the gills. Microscopically, the disease is characterized by epithelial hyperplasia (an increase in the number of epithelial cells) and fusions of palisade plates with mucus metaplasia. As the disease progresses, inflammatory cells (neutrophils and macrophages) aggregate in the edematous areas of the lesion. Sometimes, eosinophilic granular cells are seen in the blood vessels surrounding the filocartilage. In later stages of the disease, epithelial hypertrophy and epithelial stratification appear on the lesion surface, accompanied by mucus cell infiltration, decreased chloride cell count, and the formation of interpalisade vesicles that may contain amoebas.

[0007] Diagnosis is made by microscopic examination of fresh gill sections or paraffin-embedded fixed gill tissue and / or by specific PCR tests for P. perurans.

[0008] Paramoeba pemaquidensis is considered a predilection for AGD (aggregate disease) caused by P. perurans. It is usually found on the gills alongside P. perurans as part of a mixed infection. The behavior of amoebas towards unfavorable or toxic substances is thought to be similar only within the same family or group of amoebas. Amoebas possess a similar mechanism in which they curl up and retract their filopodia in adverse environments.

[0009] Despite over 30 years of research, no vaccine or drug has been approved for the treatment of AGD. Current treatments include a freshwater bath (2-3 hours at <4 ppt salinity) or a hydrogen peroxide bath (1000-1400 ppm H2O2 for 20-30 minutes). Hydrogen peroxide baths are generally not used above 15-16°C and should be used with caution at temperatures between 12 / 13°C and 15 / 16°C.

[0010] Various feed and bathing chemotherapeutic treatments were tested to more effectively remove amoebic gill disease from the gills. Oral supplementation with levamisole or dextran had no significant effect on mortality levels. In commensal studies, oral administration of L-cysteine ​​ethyl ester (LCEE) two weeks prior to attack significantly delayed the progression of AGD-related gill pathology (Roberts SD, Powell MD. 2005. Oral L-cysteine ​​ethyl ester (LCEE) reduces amoebic gill disease (AGD) in Atlantic salmon Salmosalar. Dis Aquat Org, 66(1):21-28). LCEE was found to reduce mucus viscosity in Ontario salmon. Freshwater treatment is also thought to reduce mucus viscosity by breaking down mucus and helping it detach from the skin (Roberts SD. 2004. Improving the treatment of amoebic gill disease in salmonids with soft freshwater and the mucolytic drug L-cysteine ​​ethyl ester. PhD thesis, University of Tasmania, Launceston). Both freshwater treatment and LCEE treatment produce positive clinical effects on AGD, with the common feature being a reduction in mucus viscosity.

[0011] Feed administration of the antiprotozoan drug bis(dichlorophen) at 25 mg / kg (no maximum residue limit (MRL) has been established in any food animal species) showed a delayed and reduced intensity of AGD-related damage. In in vitro bath treatments, administration of 10 mg / L of ionophores: salinomycin, lasaloxic acid, and maduramycin, respectively, significantly reduced amoebae numbers. However, when tested in feed treatments, the ionophores reduced the percentage of damaged palps in fish fed with P. perurans only 7 days post-attack. No difference was observed at 14 and 21 days post-attack.

[0012] A thin layer of mucus is found on the gills and skin of fish, serving as the first physical defense barrier against waterborne pathogens. It also functions in respiration, ion and osmotic regulation, reproduction, communication, excretion, and disease resistance. The protective function of mucus is a result of a combination of mechanical and biochemical properties. Mucus is primarily secreted by mucinous cells of the epithelium. Besides capturing and detaching pathogens, mucus contains a wide range of substances that can act against pathogens. Mucus is mainly composed of water and glycoproteins. However, many components of mucus have been described, including some innate immune components such as lectins, penetrantin, lysozyme, proteolytic enzymes, alkaline phosphatase, C-reactive protein, complement and antimicrobial peptides, and immunoglobulins.

[0013] Recently, changes in protein abundance in gill mucus have been described in salmon infected with *P. perurans*. This is supported by histological observations of AGD-associated gill lesions, specifically reduced attachment of amoebae to epithelial regions rich in mucus cells. Later stages of AGD lesion development are characterized by the variable aggregation of superficial epithelial cells with squamous structures and mucus cells at the lesion surface, which may indicate defensive strategies designed to exclude and / or disrupt or isolate amoebae from suspected tissue. Palisade fusion reduces the total available surface area for amoebae colonization. Enzymes and / or other substances secreted by mucus cells can affect the aggregation and attachment of amoebas to these areas (Adams MB, Ellard K, Nowak BF. 2004. Gross pathology and its relationship with histopathology of amoebic gill disease (AGD) in farmed Atlantic salmon, Salmo salar LJ Fish Dis, 27(3):151-61; Adams MB, Nowak BF. 2003. Amoebic gill disease: sequential pathology in cultured Atlantic salmon, Salmo salar LJ Fish Dis, 26(10):601-614.).

[0014] Patent document EP 1234508 discloses the use of L-arginine, alone or in combination with ibuprofen, for the prophylactic treatment of coccidiosis in poultry. The causative microorganism of coccidiosis is several species of Eimeria spp. Eimeria spp. is an intracellular parasite belonging to the phylum Sporozoa or subphylum Atopycium. Eimeria spp. invade the epithelial cells and related glands of the digestive tract.

[0015] The purpose of this invention is to remedy or reduce at least one disadvantage of the prior art, or at least to provide a useful alternative to the prior art.

[0016] The objective is achieved by the features described below and detailed in the following claims.

[0017] This invention is defined by the independent patent claims. The dependent claims define advantageous embodiments of the invention.

[0018] The results show that adding dietary arginine to fish feed at a level 3 percent higher than the total level improves the survival of fish infected with AGD. This is important for the global marine salmon farming industry because an effective diet can help minimize AGD-related costs by reducing mortality, maintaining growth rates, and perhaps reducing the frequency of bathing events.

[0019] More specifically, a first aspect of the invention relates to a composition for treating fish gill mucus for therapeutic or preventative treatment of amoebic gill disease in fish, wherein the composition comprises an extruded fish feed supplemented with arginine; the fish feed comprises protein, binder, fat, vitamins and minerals; and the total arginine content of the fish feed is at least 3.0% (wt / wt) of the total weight of the feed.

[0020] Fish feed can be prepared by extrusion, in which the extruded clumps are cooked, and the extrudate is porous to absorb and retain a large amount of added liquid fat. The final fish feed can have a total fat content of less than 25% of the total weight of the fish feed, or it can be more than 25%, such as 30%, 35%, and even 40%. Starch from wheat and other vegetable raw materials such as broad beans is used as a binder to maintain the shape and integrity of the fish feed. Other binders may also be used.

[0021] Treatment of mucus may include increasing its viscosity. Treatment of mucus may include increasing its polysaccharide content.

[0022] Amoebic gill disease can be caused by marine amoebas. Fish that can be salmonids, such as Atlantic salmon or rainbow trout, may be infected. Amoebic gill disease can be caused by infection with at least one of the amoebas, *Paramoeba perurans* (also known as *Neoparamoeba perurans*) and *Paramoeba pemaquidensis* (also known as *Neoparamoeba pemaquidensis*).

[0023] The fish feed can be used for the preventive and / or therapeutic treatment of amoebic gill disease in fish. The fish feed can be used for the preventive and / or therapeutic treatment of fish infections caused by marine amoebas. The fish feed can be used for the preventive and / or therapeutic treatment of infection with at least one of the amoebas *Paramoeba perurans* (also known as *Neoparamoeba perurans*) and *Paramoeba pemaquidensis* (also known as *Neoparamoeba pemaquidensis*).

[0024] Preventative and / or therapeutic treatment can be initiated by feeding salmonids a diet supplemented with arginine for 6 weeks after they have been transferred from freshwater to seawater. This fish feed can be used for preventative and / or therapeutic treatment of infection with at least one of the following: *Paramoeba perurans* (also known as *Neoparamoeba perurans*) and *Paramoeba pemaquidensis* (also known as *Neoparamoeba pemaquidensis*).

[0025] The application of arginine in treating mucus on fish gills for the treatment or prevention of amoebic gill disease in fish is also described. Arginine can be added to fish feed in an amount sufficient to increase the total arginine content of the feed to at least 3% (wt / wt) of the total feed weight.

[0026] Amoebic infection can be caused by at least one of Paramoeba perurans (also known as Neoparamoeba perurans) and Paramoeba pemaquidensis (also known as Neoparamoeba pemaquidensis). The fish can be salmonids. The fish feed of this invention can be started after the fish have been transferred from freshwater to seawater. Alternatively, the fish feed of this invention can be started 6 weeks after the fish have been transferred from freshwater to seawater.

[0027] The following describes an example of a preferred implementation scheme.

[0028] Figure 1-3 This shows the survival of Atlantic salmon after being attacked with the amoeba Paramoeba perurans in different studies;

[0029] Figure 4 The results showed that Paramoeba pemaquidensis survived in vitro after being incubated with fish mucus for 72 hours.

[0030] Figure 5 This indicates the viscosity of Atlantic salmon mucus;

[0031] Figure 6This indicates the concentration of lysozyme in the mucus of Atlantic salmon;

[0032] Figure 7 This indicates the polysaccharide concentration in Atlantic salmon mucus;

[0033] Figure 8 This indicates the concentration of lysozyme in the mucus of Atlantic salmon;

[0034] Figure 9 The results showed that Paramoeba pemaquidensis survived in vitro after being incubated with fish mucus for 48 hours.

[0035] Figure 10 The study showed that Paramoeba perurans survived in vitro after being incubated with fish mucus for 48 hours.

[0036] Example 1

[0037] The test was conducted for 15 days using Atlantic salmon (Ontario salmon (S. salar)) in 250L tanks containing brine with a salinity of 35 ppt and a temperature of 16°C. Each tank contained 30 fish, with an average weight of 121g at the start of the test, and each diet was used for two tanks.

[0038] Before the start of the 65-day study phase, the fish were acclimatized to the new environment and fed a control diet for 5 weeks, followed by either a control diet or a test diet until the end of the trial. The control diet, also known as the control feed or Control 2, contained wheat, gluten, North Atlantic fishmeal, soy protein concentrate, rapeseed oil, North Atlantic fish oil, astaxanthin, vitamins, and minerals. The control diet was produced by press-cooking and contained 26.5% fat, 50.1% protein, and 5.7% water, and represented commercially available fish feed. The test diet, also known as the test feed or Control 2+A, had the same composition as Control 2 but with the addition of 1.0% arginine. Arginine was added as a dry powder to the dietary mixture, which was then cooked and press-cooked. On an as-is basis, the calculated arginine level in Control 2 was 2.61%.

[0039] Following the method described by Morrison RN, Crosbie PBB, Nowak BF. 2004 (The induction of laboratory-based amoebic gill disease revisited. J. Fish Dis, 27, 445–449), *P. perurans* were harvested from Atlantic salmon placed in infection tanks. After being fed an experimental diet for 4 weeks, the fish were attacked with a total dose of 500 cells per liter of *P. perurans* on a series of days (0, 8, 9, 10, 12, and 16 post-infection). For the attack, water circulation in all tanks was stopped, and the amoebas were added to each tank using a spray bottle containing an additional 7 liters of seawater to ensure even distribution of the amoebas within the tanks. Water flow was restored after 1.5–2 hours.

[0040] The experiment ended when the mortality rate in the control group reached 60%. The average fish weight at the end of the experiment was 192g. *P. perurans* was confirmed in the selected deceased individuals by qPCR and histological examination.

[0041] like Figure 1 As shown, fish fed the test diet control 2+A had a relative survival rate of 19% compared to fish fed the control diet. The relative survival rate was calculated as: (1 – (% mortality / % control mortality)) x 100.

[0042] Table 1 shows that the test diet effectively reduced mortality caused by AGD compared to fish fed the control diet.

[0043] Table 1: Summary of Mortality Rates at 35 Days Post-Infection

[0044]

[0045] Example 2

[0046] The test used Atlantic salmon (Ontario salmon) in 250L tanks for 144 days, with the tanks containing brine with a salinity of 135 ppt and a temperature of 16°C. Each tank contained 30 fish, with an average weight of 171g at the start of the test, and each diet was used in 3 tanks.

[0047] Fish were acclimatized to the new environment and fed a control diet for 4 weeks, followed by either a control diet or a test diet until the end of the trial. The control diet, also known as the control feed or Control 1, contained wheat, gluten, sunflower seed meal, North Atlantic fish meal, soybean protein concentrate, broad beans, rapeseed oil, North Atlantic fish oil, astaxanthin, vitamins, and minerals. The control diet was produced by press-cooking and contained 24.2% fat, 49.9% protein, 5.3% ash, and 6.3% water, and represented commercially available fish feed. The test diet, also known as the test feed or Control 1+A, had the same composition as Control 2 but with the addition of 0.58% arginine. Arginine was added as a dry powder to the dietary mixture, which was then cooked and press-cooked. Analysis showed that, based on the samples taken, Control 1 contained 2.92% arginine, while Control 1+A for the test group contained 3.24% arginine based on the samples taken.

[0048] Following the method described by Morrison et al., *P. perurans* were harvested from Atlantic salmon placed in infection tanks. After being fed an experimental diet for 4 weeks, the fish were attacked for 2 days with a total dose of 500 cells / L of *P. perurans*. Due to the low mortality observed during the attack, an additional dose of amoebae (50 *P. perurans* cells / L) was added on day 55 post-attack. For the attack, water circulation in all tanks was stopped, and amoebae were added to each tank using a spray bottle containing an additional 7L of seawater to ensure even distribution of the amoebae. Water flow was restored after 1.5–2 hours.

[0049] The experiment ended when the mortality rate in the control group reached 40%. The average fish weight at the end of the experiment was 391g. *P. perurans* was confirmed in the selected deceased individuals by qPCR and histology.

[0050] like Figure 2 As shown, compared to fish fed control diet 1, fish fed the test diet 1+A had a 19% relative survival rate.

[0051] Table 2 shows that the test diet effectively reduced mortality caused by AGD compared to fish fed the control diet 1.

[0052] Table 2: Summary of Mortality Rates at 74 Days Post-Infection

[0053]

[0054] Example 3

[0055] The test used Atlantic salmon (Ontario salmon) in 250L tanks for 144 days. The tanks contained brine with a salinity of 35 ppt and a temperature of 16°C. Each tank contained 30 fish, with an average weight of 179g at the start of the test. Each diet was used in 3 tanks.

[0056] Fish were acclimatized to the new environment and fed a control diet for 4 weeks, followed by either a control diet or a test diet until the end of the trial. The control diet, also known as the control feed or Control 2', contained wheat, gluten, sunflower seed meal, North Atlantic fish meal, soybean protein concentrate, broad beans, rapeseed oil, North Atlantic fish oil, astaxanthin, vitamins, and minerals. The control diet was produced by press-cooking and contained 24.3% fat, 47.7% protein, 5.6% ash, and 7.1% water, and represented commercially available fish feed. The test diet, also known as the test feed or Control 2'+A', had the same composition as the control 2' feed but with the addition of 0.58% arginine. Arginine was added as a dry powder to the dietary mixture, which was then cooked and press-cooked. Analysis showed that, based on the samples taken, the control 2' feed contained 2.75% arginine, while the control 2'+A' feed used in the test group contained 3.30% arginine, based on the samples taken.

[0057] Following the method described by Morrison et al., *P. perurans* were harvested from Atlantic salmon placed in infection tanks. After being fed an experimental diet for 4 weeks, the fish were attacked for 2 days with a total dose of 500 cells / L of *P. perurans*. Due to the low mortality observed during the attack, an additional dose of amoebae (50 *P. perurans* cells / L) was added on day 55 post-attack. For the attack, water circulation in all tanks was stopped, and amoebae were added to each tank using a spray bottle containing an additional 7L of seawater to ensure even distribution of the amoebae. Water flow was restored after 1.5–2 hours.

[0058] The experiment ended when the mortality rate in the control group reached 40%. The average fish weight at the end of the experiment was 422g. *P. perurans* was confirmed in the selected deceased individuals by qPCR and histological examination.

[0059] like Figure 3 As shown, fish fed the control 2' diet had a 36% relative survival percentage compared to fish fed the control 2' diet. Fish fed the control 2' diet had a significantly increased survival rate compared to fish fed the control 2' diet, with a significance level of 0.1% (log-rank, Mantel-Cox, P = 0.09).

[0060] Table 3 shows that, compared to fish fed the control diet 2', the test diet effectively reduced mortality caused by AGD.

[0061] Table 3: Summary of Mortality Rates at 74 Days Post-Infection

[0062]

[0063] Example 4

[0064] The test used Atlantic salmon (Ontario salmon) in tanks 1 meter in diameter for 37 days, with the tanks containing brine with a salinity of 32.9–34.0 ppt. The water temperature varied from 11.8 to 12.1°C. Each tank contained 40 fish, with an average weight of 132 g at the start of the test, and each diet was used in 3 tanks.

[0065] The control diet, also known as control feed or Control 1', contains wheat, gluten, sunflower seed meal, Scandinavian fish meal, soy protein concentrate, rapeseed oil, North Atlantic fish oil, astaxanthin, vitamins, and minerals. Produced by press-cooking, the control diet consists of 23.2% fat, 48.0% protein, 11.1% ash, and 4.9% water, and represents commercially available fish feed. The test diet, also known as test feed or Control 1'+A', has the same composition as Control 1' feed. Multiple batches of 12.5 kg Control 1' feed were top-coated with 1% arginine and mixed for 90 seconds in a commercial bread mixer, followed by the addition of 0.05% Scandinavian fish oil, and then mixed for another 30 seconds.

[0066] At the end of the experiment, the fish weighed 156g.

[0067] Protocol for culturing fish mucus samples

[0068] Mucus Sampling: Skin mucus was collected separately as follows: Each fish was placed on a plastic bag, the bag was carefully wrapped around the fish, and the fish was allowed to slide out of the bag. The mucus was immediately and rapidly frozen in liquid nitrogen and stored at -80°C until analysis. Skin mucus was collected instead of gill mucus because it is impossible to collect a sufficient volume of gill mucus from an individual fish for viscosity, lysozyme, and polysaccharide analysis. Literature reports that skin and gill mucus are similar in characteristics for the properties being analyzed, and that changes in skin mucus reflect changes in gill mucus.

[0069] Mucus Preparation: Thaw all mucus samples and use them only once. Avoid refreezing and reuse, as the activity of substances or immune components in the mucus may be affected by freeze-thaw cycles. Use the mucus samples according to their viscosity. If the mucus sample is very viscous, briefly rotate it at 1000g for 1 minute to precipitate cells. Use the resulting supernatant for testing.

[0070] Incubation with Amoebas: All mucus samples were diluted 1:1 with cultured Paramoeba pemaquidensis amoebas. The amoebas were observed and their survival checked at 4–5 hours, 24 hours, 48 ​​hours, and 6–9 days. Stronger effects were typically observed in the mucus several days after exposure.

[0071] Viability staining: For viability staining, according to the procedure of Yokoyama et al. (Journal of Fish Diseases 1997, 20(4), 281-286), amoebas are stained with the fluorescent dyes propidium iodide (red for dead cells) and fluorescein diacetate (green for live cells), with an incubation time modified to only 5 minutes. Alternatively, amoebas are stained with neutral red, which stains lysosomes in live cells (Chazotte, 2010, Imaging: A Laboratory Manual) (ed. Yuste). CSHLPress). Count 100 cells per concentration or per individual fish mucus sample in triplicate.

[0072] After incubation for 72 hours in the mucus of fish harvested from the control group 1'+A', the survival rate of amoebas decreased from 96% to 92%. Figure 4 As shown.

[0073] Example 5

[0074] The test used Atlantic salmon (Ontario salmon) in tanks 1 meter in diameter for 34 days, with a salinity of 34.1-34.2 ppt. The water temperature varied from 11.5 to 11.8°C. Each tank contained 20 fish, with an average weight of 379 g at the start of the test, and each diet was used for one tank.

[0075] The control diet, also known as the control feed or Control 1", contains wheat, gluten, sunflower seed meal, North Atlantic fish meal, soy protein concentrate, broad beans, rapeseed oil, North Atlantic fish oil, astaxanthin, vitamins, and minerals. The control diet is produced by extrusion cooking and consists of 24.2% fat, 49.9% protein, 6.3% water, and 5.3% ash, and represents commercially available fish feed. The test diet, also known as the test feed or Control 1"+A, has the same composition as Control 1" but with the addition of 0.58% arginine. Arginine is added as a dry powder to the dietary mixture, which is then cooked and extruded. Based on the samples taken, the total arginine level analyzed in Control 1" was 2.92%, while that in the test diet Control 1"+A was 3.24% based on the samples taken.

[0076] At the end of the experiment, the fish weighed 470.5 g. Skin mucus was collected separately as follows: each fish was placed on a plastic bag, the bag was carefully wrapped around the fish, and the fish was allowed to slide out of the bag. The mucus was immediately and rapidly frozen in liquid nitrogen and stored at -80°C until analysis. Skin mucus was collected instead of gill mucus because it was impossible to collect a sufficient volume of gill mucus from an individual fish for viscosity, lysozyme, and polysaccharide analysis. Literature reports that skin and gill mucus are similar in characteristics for the properties being analyzed, and that changes in skin mucus reflect changes in gill mucus.

[0077] The viscosity of the mucus was analyzed using a Brookfield cone-plate DV3T rheometer. The mucus was centrifuged at 4000 rpm for 4 minutes, and the viscosity of 0.5 ml of clear, particle-free mucus was measured at 80 rpm and 12 °C.

[0078] Lysozyme activity was measured using a Varioskan Flash microplate reader. 250 μl of a suspension of *Micrococcus lysodeikticus* in 0.4 M sodium phosphate buffer (pH 5.8) was added to 5 μl of clear, particle-free mucus, and absorbance was monitored for 30 minutes. A decrease in absorbance of 0.001 μL per minute was taken as the unit of lysozyme activity.

[0079] The amount of polysaccharide was measured using a Varioskan Flash microplate reader. 25 μl of clear, particle-free mucus was mixed with 60 μl of water containing 2.5% phenol and 150 μl of concentrated sulfuric acid, and then incubated at 100 °C for 20 minutes. After cooling to room temperature, the absorbance was measured, and the concentration was calculated based on a glucose-containing standard.

[0080] Figure 5 The viscosity of the mucus was shown at 80 revolutions per minute (rpm). The mucus in the test group fed with control 1”+A” feed was significantly more viscous than that in the control group fed with control 1” feed (P<0.0001, unpaired t-test).

[0081] The mucus composition in the test group fed with control 1"+A" feed was significantly different from that in the control group fed with control 1" feed. The lysozyme concentration in the mucus of the test group fed with control 1"+A" feed was significantly higher than that of the control group fed with control 1" feed (P = 0.0005, unpaired t-test). Figure 6 As shown, the polysaccharide concentration in the test group fed with control 1"+A" diet was significantly higher ( Figure 7 ).

[0082] Example 6

[0083] The test used Atlantic salmon (Ontario salmon) in tanks with a diameter of 1 meter for 41 days, with the tanks containing 33.6-34.6 ppt of brine. The water temperature ranged from 11.9 to 12.3°C. Each tank contained 30 fish, with an average weight of 322 g at the start of the test, and each diet was used for two tanks.

[0084] The control diet, also known as the control feed or Control 2", contains wheat, gluten, North Atlantic fishmeal, soy protein concentrate, broad beans, rapeseed oil, North Atlantic fish oil, sunflower seed meal, astaxanthin, vitamins, and minerals. The control diet is produced by extrusion cooking and contains 25.8% fat, 45.0% protein, 7.3% water, and 5.7% ash, and represents commercially available fish feed. The test diet, also known as the test feed or Control 2"+A, has the same composition as the Control 2" feed, but with 0.86% arginine added as dry powder to the dietary mixture before extrusion. Based on the samples taken, the total arginine level analyzed in the Control 2" feed was 2.63%, while that in the Test Diet Control 2"+A was 3.14% based on the samples taken.

[0085] At the end of the experiment, the fish weighed 545g. Skin mucus was collected separately as described in Example 4.

[0086] As described in Example 4, the lysozyme activity in the sampled mucus was measured. Figure 8 The test group fed with control 2”+A” showed a higher concentration of lysozyme in the mucus than the control group fed with control 2”.

[0087] Incubation with amoebas: All mucus samples were diluted 1:1 with cultured Paramoeba pemaquidensis or Paramoeba perurans amoebas. The amoebas were observed and their survival checked at 4–5 hours, 24 hours, 48 ​​hours, and 72 hours.

[0088] Viability staining was performed as described in Example 4. In mucus collected from feeds fed to control 2”+A”, the survival rate of P. pemaquidensis decreased significantly from 97.8% to 96.8% after 48 hours of incubation (P = 0.011, unpaired t-test), and the survival rate of P. perurans decreased significantly from 95.9% to 91.2% after 48 hours of incubation (P = 0.004, unpaired t-test).

[0089] In addition, in the mucus collected from the control 2”+A” diet, the survival rate of P. pemaquidensis decreased from 96.6% to 91.8% after 72 hours of incubation, and the survival rate of P. perurans decreased significantly from 92.1% to 90.2% after 72 hours of incubation.

[0090] It should be noted that the above embodiments illustrate, rather than limit, the invention, and those skilled in the art can devise many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference marks placed in parentheses shall not constitute a limitation of the claims. The use of the verb "comprising" and its variations does not exclude the presence of elements or steps other than those shown in the claims. The article "a" or "an" preceding an element does not exclude the presence of multiple such elements.

[0091] The fact that certain measures are referenced in different subsidiary claims does not indicate that a combination of these measures cannot yield a benefit.

Claims

1. Use of the composition in the preparation of a feed for increasing the polysaccharide content in the mucus of fish skin and / or gills, wherein the composition comprises an extruded fish feed supplemented with arginine; the fish feed comprises protein, binder, fat, vitamins and minerals; and the total arginine content of the fish feed is at least 3.0% by weight of the total feed weight.

2. The use as described in claim 1, wherein the fish is a salmonid fish.

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