NO20120303A1 - Feed composition for fish for use in preventing or reducing symptoms related to cardiovascular disease, HSMB - Google Patents

Abstract

Summary A feed composition is described which is suitable for feeding fish to prevent or reduce symptoms related to cardiovascular and musculoskeletal disease (HSMB) in fish.

Description

FIELD OF THE INVENTION

The present invention relates to a pre-composition for fish comprising conventional feed ingredients such as proteins, lipids, vitamins, carbohydrates and minerals, a use of the pre-composition and a method for feeding fish susceptible to a heart and skeletal muscle inflammation infection.

BACKGROUND OF THE INVENTION

Heart and skeletal muscle inflammatory disease (HSMB) is a serious infectious disease in Atlantic salmon with a high potential for transmission from infected to healthy fish. Since the first occurrence in 1999, an increasing number of outbreaks of the disease have been recorded. Thus, after recording 68 outbreaks in 2003 and 54 in 2004, the number has increased every year. In 2007, the disease was diagnosed at 162 seawater facilities along the Norwegian coast and 144 in 2008 (Figure 1). These outbreaks cause great losses to the Norwegian salmon industry since the facilities that are attacked have a large number of fish (more than 90%) suffering from heart damage months after the disease event, which significantly reduces their growth potential. Most outbreaks are reported in January, June, July and October, in fish that have been transferred to seawater. The disease has a major economic impact on the Norwegian salmon farming industry, and a single fish farming company reported losses of up to NOK 10 million per year due to the disease 5 to 9 months later. However, there are indications that HSMB is also relevant in species other than those belonging to the salmonids.

Clinical signs of HSMB are typically reduced appetite and abnormal swimming behaviour. There are no obvious external signs, but at autopsy the heart appears pale, soft and limp. HSMB produces inflammation in the heart that results in the death of heart cells. These damages already appear in the early stages of the disease and can continue for several months. Mortality increases with increasing degree of heart inflammation. The inflammation and cell death can spread to other organs and tissues such as red skeletal muscle and liver, causing further serious damage to these organs.

The infectious nature of HSMB has been documented, as the disease can be induced in healthy fish by injecting homogenized tissue from fish diagnosed with HSMB, and further transferred to healthy cohabiting fish (Kongtorp et al., 2004. Heart and skeletal muscle inflammation in Atlantic salmon , Salmo salar L: a new infectious disease. Journal of Fish Disease: Vol 27(6), (p). 351-358). HSMB is caused by a virus, which has been isolated (Eliassen et al., 2004. Isolation of heart and skeletal muscleinflammation virus (HSMIV) from Salmon. The 6th International Symposium on Vimses of Lower Vertebrates, Poster 19). However, since the virus has not been fully characterized yet, vaccines against HSMB have not been developed to date. In addition, current challenge models of the disease in laboratories only reproduce the histopathology observed in the field, not the mortality. Thus, there is a clear need for alternative treatments and methods to reduce the negative effects of this serious infection. One purpose of the present invention is therefore to find new strategies and treatments that can be used to positively influence the development of the disease in HSMB-infected fish, and likewise in fish that are susceptible to infection.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a pre-composition for fish to prevent and/or treat heart and skeletal muscle inflammatory disease comprising conventional pre-ingredients such as proteins, lipids, vitamins, carbohydrates and minerals, characterized in that more than 20% of the total fatty acids are n-3 fatty acids.

In a preferred embodiment, more than 23% of the total fatty acids are n-3 fatty acids, and more preferably more than 24% of the total fatty acids are n-3 fatty acids.

In a currently preferred embodiment, the content of eicosapentaenoic acid 20:5 n-3 (EPA) is more than approx. 7% of the total fatty acids. More preferably, the content of eicosapentaenoic acid 20:5 n-3 (EPA) is more than about 8% of the total fatty acids, more preferably 9%, more preferably more than approx. 10%, such as approx. 10.2%. A preferred embodiment contains amounts of EPA as indicated in Table 1.

In a preferred embodiment, the protein:lipid ratio (w/w) in the lining is higher than 2, preferably higher than 2.7. Preferably, the proteins comprise more than 50% (based on weight) and that the lipids comprise less than 20% (based on weight) of the preformulation. More preferably, the proteins comprise more than 52% (based on weight) and the lipids comprise approx. 18% (based on weight) of the preformulation.

A preferred embodiment relates to a pre-composition for salmon fish where the composition has a reduced content of lipids and an increased content of proteins compared to commercial pre-compositions used for salmon fish of the same age, size and/or developmental stage.

In a further embodiment, the content of n-6 fatty acids is less than 11% of the total fatty acids, preferably in the range 7-11%, more preferably approx. 10%.

In a preferred embodiment, the content of n-3 fatty acids is more than 23% and the level of n-6 fatty acids is less than 11% of the total fatty acids.

In a preferred embodiment, the ratio of n-3 to n-6 fatty acids is higher than 1.6, preferably more than 2.2, more preferably between 2.3 and 3.4.

In a preferred embodiment, a proportion of said lipids is South American fish oil. Preferably, the pre-composition contains less than 0.5%, preferably 0.2% based on the weight of the composition of added vegetable oil, and that the oil is preferably added as a hydrolyzate.

In a preferred embodiment, the composition comprises hydrolysed phospholipids, preferably approx. 0.1-0.2% by weight of the diet, more preferably more than 0.2% by weight of the composition.

In a preferred embodiment, the composition comprises krill meal, preferably approx. 5% by weight, more preferably more than 5% by weight of the diet.

In a preferred embodiment, the feed comprises nucleotides and/or manna-oligosaccharides and/or anti-inflammatory compounds such as flavonoids.

A preferred embodiment relates to a pre-composition, where said proteins make up more than 50% (based on weight) of the composition, where the lipids make up less than 20% (based on weight), where more than 20% of the total fatty acids are n-3 fatty acids . More preferably, the protein makes up more than 52% (based on weight) of the composition, and where the lipids make up less than 18% (based on weight), preferably 18% of the diet, where more than 23% of the total fatty acids are n-3 fatty acids.

A preferred embodiment relates to a pre-composition essentially as stated for anti-HSMB diet 1 or for anti-HSMB diet 2 in table 1 or table 2.

A second aspect of the present invention relates to a pre-composition as indicated above for the production of a pharmaceutical composition and/or nutritional composition and/or functional for the prophylaxis and/or treatment of cardio- and skeletal muscle inflammatory disease.

A preferred embodiment relates to preventing or reducing symptoms related to heart and skeletal muscle inflammation disease in fish.

In a preferred embodiment, said fish is a salmon fish, flatfish or another fish species suitable for aquaculture, preferably the species Atlantic salmon (Salmo salar).

In a preferred embodiment, the application is for effective recovery after heart and skeletal muscle inflammation infection, and/or reduction of the risk of reinfection.

A third aspect of the present invention relates to a method for feeding fish susceptible to a cardiomyositis infection, characterized in that a composition as indicated is transferred to the fish for a period before challenge with an infection, during the infection and after the fish has been infected.

Preferably, the composition is administered for a period of at least 8 weeks pre-infection, more preferably 10-16 weeks, most preferably 16-20 weeks and 8-20 weeks post-infection.

A fourth aspect of the present invention relates to a pre-composition for fish comprising conventional pre-ingredients such as proteins, lipids, vitamins, carbohydrates and minerals, characterized in that more than 20% of the total fatty acids are n-3 fatty acids.

Preferably, more than 23% of the total fatty acids are n-3 fatty acids.

Preferably, more than 24% of the total fatty acids are n-3 fatty acids.

More preferably, the content of eicosapentaenoic acid 20:5 n-3 (EPA) is more than about 7% of the total fatty acids.

More preferably, the content of eicosapentaenoic acid 20:5 n-3 (EPA) is more than about 8% of the total fatty acids, more preferably more than 9%, more preferably more than approx. 10%, such as approx. 10.2%.

Preferably, the protein:lipid ratio (wt/wt) in the feed is higher than 2, preferably higher than 2.7, or said proteins comprise more than 50% (based on weight) and the lipids comprise less than 20% (based on weight ) of the pre-composition, more preferably that said proteins make up more than 52% (based on weight) and the lipids make up approx. 18% (based on weight) of the preformulation.

In a preferred embodiment, the content of n-6 fatty acids is less than 11% of the total fatty acids, preferably in the range 7-11%, more preferably approx. 10%.

In a preferred embodiment, the content of n-3 fatty acids is more than 23% and the level of n-6 fatty acids is less than 11% of total fatty acids.

In a preferred embodiment, the ratio of n-3 to n-6 fatty acids is higher than 1.6, preferably more than 2.2, more preferably between 2.3 and 3.4.

In a preferred embodiment, a proportion of said lipids is South American fish oil.

Preferably, the content of added vegetable oils is less than 0.5%, preferably 0.2% based on the weight of the composition, and that the oil is preferably added as a hydrolyzate.

Preferably, the composition comprises hydrolysed phospholipids, preferably approx. 0.1-0.2% by weight of the diet, more preferably more than 0.2% by weight of the diet.

Preferably, the composition comprises krill meal, preferably approx. 5% by weight, more preferably more than 5% by weight of the diet.

Preferably, the feed further comprises nucleotides and/or manna-oligosaccharides and/or anti-inflammatory compounds such as flavonoids.

Preferably, the proteins make up more than 50% (based on weight) of the composition, where the lipids make up less than 20% (based on weight), where more than 20% of the total fatty acids are n-3 fatty acids, and more preferably the proteins make up more than 52% (based on weight) of the composition, and where the lipids constitute less than 18% (based on weight), preferably 18% of the diet, where more than 23% of the total fatty acids are n-3 fatty acids.

In a preferred embodiment, the composition is essentially as stated for anti-HSMB diet 1 or for anti-HSMB diet 2 in table 1 or table 2.

A fifth aspect of the present invention relates to a pre-composition for fish comprising conventional pre-ingredients such as proteins, lipids, vitamins, carbohydrates and minerals, characterized in that the protein:lipid ratio (weight/weight) in the feed is higher than 2, preferably higher than 2.7 to prevent or reduce symptoms related to heart and skeletal muscle inflammation disease in fish.

Preferably, the protein:lipid ratio (w/w) of the feed is greater than 2.7.

Preferably, the protein comprises more than 50% (based on weight) of the diet, preferably more than 52% and that the lipids comprise less than 20% (based on weight), preferably 28% of the diet.

Preferably, more than 20% of the total fatty acids are n-3 fatty acids, preferably more than 23%, more preferably 24%.

Preferably, the composition comprises more than 23% n-3 fatty acids and less than II% n-6 FA of the total fatty acids.

Preferably, the content of eicosapentaenoic acid 20:5 n-3 is more than 7% of the total fatty acids, preferably more than 9%, more preferably 10.2%.

It must be recognized that properties according to the invention described above can be combined in any combination without deviating from the framework of the invention.

DESCRIPTION OF THE INVENTION.

Embodiments of the invention will now be described, using examples with references to the accompanying figures, where: Figure 2 shows modeled cumulative probability of myocardial score between for at 10, 12, 14 and 16 weeks. The graph shows the probability that the myocardial score is equal to or less than what is read on the x-axis. Figure 3 shows the model coefficient (± 95% confidence intervals) of the myocardial score model compared between liners at 10, 12, 14 and 16 weeks post-challenge. This means that if the 95% confidence intervals do not overlap the red dashed lines indicating the coefficient for the control diet for a particular week, then the difference can be considered significant at the 5% level. Furthermore, if the joins are compared across the subplots, weeks that do not have 95% confidence intervals that cross the zero line (not drawn) can be considered significantly different from 10 weeks. Figure 4 shows modeled cumulative epicardial score probabilities for each at 10, 12, 14 and 16 weeks. The figures give the probability that the epicardial score is equal to or lower than what is read on the x-axis. Although the differences were not significant between the different diets on epicardial score, there is a clear trend that epicardial score is improved when fed with anti-HSMB1 or 2. Figure 5 shows the model coefficients (± 95% confidence intervals) of the epicardial score model comparable between for at 10, 12, 14 and 16 weeks postal challenge.

This means that if the 95% confidence interval does not overlap the red dashed line indicating the coefficient for the control diet for a particular week, then the difference can be considered significant at the 5% level. Furthermore, if the control plots are compared across subplots, weeks that do not have 95% confidence intervals that cross the zero line (not drawn) can be considered significantly different from 10 weeks.

Figure 6 shows the modulated cumulative probabilities for liver scores between linings within the sampling weeks. The graph shows the probability that the score is equal to or less than what is read on the x-axis. Figure 7 shows model coefficients (± 95% confidence intervals) of the liver score model by comparing between for at 10, 12, 14 and 16 weeks post-challenge. This means that if the 95% confidence interval does not overlap the red dashed line indicating the coefficient for the control diet for a particular week, then the difference can be considered significant at the 5% level. Furthermore, if the control plots are compared across the subplots, weeks that do not have 95% confidence intervals that cross the zero line (not drawn) can be considered significantly different from 10 weeks. Figure 8 shows the mean weight gain and 95% confidence interval during the 8-week feeding period before the challenge. Figure 9 shows the level of EPA in heart tissue of Atlantic salmon 8,10, 12 g 16 weeks after challenge.

EXPERIMENTAL SECTION

Experiment.

The dietary effect of HSMB was tested in a feeding experiment with infected Atlantic salmon (Salmo salar). Salmon smolts used for experiments were all fed the same commercial diet for a period of 10 weeks during the acclimatization period. All the fish were in good condition and showed good and quick adaptation to seawater.

Lining experiment:

The experiment consisted of two phases. During the first part, an 8-week pre-challenge period was carried out, which was carried out at a feeding trial facility. After this period, the fish were transferred to a disease challenge facility and the fish were acclimatized for 2 weeks. After this period, the second part of the study included a challenge, a post-challenge, and lasted 16 weeks (that is, 16 weeks post-challenge).

A total of 945 Atlantic salmon Salmo salar, pit-tagged, with an average initial growth of 10 g, seawater adapted were distributed in 9 tanks (1.0 x 1.0 m, 50 cm depth; 500 liter volume) at the feeding trial facility. Three diets were randomly allocated among the 9 tanks (3 tanks per diet) and fed to the fish for a period of 8 weeks. The amount of feed offered during the 8-week period was based on measured or estimated biomass in the tanks. The daily feeding ratio that was offered varied between 0.8-1.1% fish body weight per day. The trial feeds provided were adjusted throughout the trial to complicate fish growth. The fish were kept in seawater at ambient temperature and salinity and diurnal photoperiod, 24:0 (light (L): dark (D)). Water temperature and salinity were measured every day in a representative tank.

After the 8-week period, the fish were weighed and measured, the pit-mark was recorded and transferred to the disease challenge facility. As soon as it reached the challenge facility, the fish were allocated to 18 tanks (2 laboratories x 9 tanks, i.e. 6 tanks per diet) where the same diet history as during the previous 8 weeks was kept. The fish were allowed to acclimatise to the tanks at the challenge facility for 2 weeks and then challenged with HSMB. The fish were anesthetized before challenge to HSMB virus. HSMB virus was isolated from diseased fish during a clinical outbreak of HSMB. All fish were challenged by intramuscular injection (0.1 ml on each side near the lateral line) of virus collected from cell culture supernatant of HSMB virus. The general condition of the fish was recorded daily and any mortality was summed on a daily basis (no mortality was expected from the challenge). All other mortality due to other conditions, which occurred during the course of the experiment, was recorded.

Diets:

Fish were either fed a control diet or the two different experimental diets anti-HSMB 1 and anti-HSMB 2 in accordance with the invention. The pellet size of all diets was 3.5 mm. The diets included conventional ingredients that are usually found in fish feed such as proteins, lipids, carbohydrates, vitamins, minerals. The compositions of the diets were as follows (Table 1):

Sampling:

Samples were taken of the fish at regular intervals after 8, 10, 12, 14 and 16 weeks, and histological samples of heart muscle and liver were analyzed with regard to histopathological changes as described below. A total of 360 fish were analyzed from the feeding experiment.

Further processing and analysis of the samples:

The histopathology of the heart and liver was performed by a professional veterinary service. The samples were fixed in 10% neutral phosphate-buffered formalin. Formalin-fixed specimens were separated for histological examination using standard paraffin box techniques and stained with hematoxylin and eosin (H & E).

Analyzed parameters:

1. Heart inflammation:

The histological changes have been evaluated on the basis of a predetermined categorization of changes in the heart. The focus was on cardiac inflammation: Endothelial changes in the boundary between the compact layer and the spongy layer in the heart were important parameters in the characterization. In early phases, swelling and proliferation were important, followed by infiltration with mononuclear inflammatory cells -Infiltration of inflammatory cells in the compact layer, focal or diffuse in appearance -infiltration of inflammatory cells in the spongy layer, focal or diffuse in appearance accompanied by degeneration and necrosis of myocytes, and swelling of the endothelial cells lining the spongy muscle fibres. Scores were available from 8, 10, 12, 14 and 16 weeks post challenge. Scores from 8 weeks were omitted from the analysis since only a total of 5 fish were examined (2 from one diet and 3 from another). Histological changes in the epicardium and myocardium were graded according to a non-continuous score graded from 0 to 4 (0 indicates no pathology, normal tissue; 4 indicates intense inflammation). A description of each of the scores for epicardium and myocardium is included in Table 3.

2. Liver inflammation:

Liver histopathology was studied with changes related to micro and macro vesicular steatosis. This is characterized by varying degrees of vacuole formation in the cytoplasm of the hepatocytes. Grading of scores followed a non-continuous score from 0 to 5, where 5 is the score indicating the highest degree of steatosis in the liver (fatty liver). A description of each of the grading scores is as follows.

Score 0: Individual formation of vacuoles in the cytoplasm, involving less than 10% of the hepatocytes and including less than 25% of the area of the individual hepatocytes.

Score 1: Individual formation of vacuoles in the cytoplasm, involving less than 25% of the hepatocytes, and including less than 25% of the area of the individual hepatocytes.

Score 2: Individual formation of vacuoles in the cytoplasm, involving less than 50% of the hepatocytes, and including less than 50% of the area of the individual hepatocytes.

Score 3: Individual formation of vacuoles in the cytoplasm, involving less than 75% of the hepatocytes, and including less than 75% of the area of the individual hepatocytes.

Score 4: Individual formation of vacuoles in the cytoplasm, involving less than 90% of the hepatocytes, and including less than 80% of the area of the individual hepatocytes.

Score 5: Individual formation of vacuoles in the cytoplasm, involving less than 90% of the hepatocytes, and including less than 80% of the area of the individual hepatocytes.

Statistical analyses:

All preparation of data and simulation output analyzes was performed with "R language"

(R: A Language and Environment for Statistical Computing, R Development Core Team, R Foundation for Statistical Computing, Vienna, Austria, 2009, ISBN 3-900051-07-0, http:// www. R- project. org). Individual weight gain data were analyzed using a linear mixed-effects model (multilevel model) fitted between the response and predictor by allowing the intercepts to vary from tank to tank to account for tank-level correlations and variability. The model was a mixed-effects linear model estimated with an Imer function in the Ime4 package. The treatment estimates were based on posterior simulation (n=2500) with 95% confidence intervals as absolute and proportional to the reference level (control diet).

Histopathological scores were analyzed using multilevel ordered categorical logistic regression because the data are multinominal. The model was written in (BUGS language (Lunn, D.J., Thomas, A., Best, N., and Spiegelhalter, D. (2000) WinBUGS - a Bayesian modeling framework; concepts, structure, and extensibility. Statistics and Computing, 10: 325-337) and adapted with JAGS (JAGS= Just another Gibbs Sampler, Plummer, M. (2009) JAGS Version 1.03 manual, http:// wwwfis. iarc. fr/- martyn/ software/ iags/).

Fuzzy non-informative uniform priors [0,100] were given for the variance parameters, and fuzzy non-informative normal priors (0,1,0E+4) for all other parameters. 25,000 "run-in" simulations were used to fit Markov Chain Monte Carlo (MCMC) consecutive 2,500 runs that were used for interference. Three rows were run in parallel, i.e. there was a total of 7500 simulations for interference. These were thinned so that only every tenth simulation was saved to reduce the size of stored objects and to reduce the effects of autocorrelation. In sum, the posterior density is based on 750 draws from the posterior probability distribution. Convergence of the MCMC simulation was assessed with the so-called Gelman-Rubin convergence diagnostic (Gelman, A- and Rubin, D.B. (1992) Inference from iterative simulation using multiple sequences, Statistical Science, 7, 457-511).

Results and conclusion:

The results from the feeding experiments show that it is possible to reduce the impact of HSMB disease through the diet. By using specific dietary formulations, it is possible to reduce both heart damage and liver steatosis during disease outbreaks.

The main changes observed between the diet groups were in the myocardium, while there were no clear differences between the diets observed in the epicardium.

With respect to the myocardium, at 10 weeks postchallenge no significant differences in myocardial scores between the diets could be observed since the 95% confidence intervals crossed the dashed line (Figure 3a). As the disease progressed, scores appeared to become more severe for the control litter at 14 and 16 weeks compared to 10 and 12 weeks (negative coefficients for the control litter in Fig 3). At 12 weeks postchallenge, the myocardium started to show a trend towards differences between diets and 99% of the fish fed with diet anti-HSMB 1 had myocardial scores equal to or lower than 1, and for diet control and anti-HSMB 2 around 96% of the fish had score equal to or lower than 1 (Figure 2b); the statistical analyzes showed that diet anti-HSMB 1 had higher coefficients resulting in better (lower scores) than the control feed at week 12, however, at this point it can only be considered as a trend since 95%

coefficient intervals slightly crossed the dotted line (Figure 3b). At week 14

postchallenge, 62% of fish fed the control diet had myocardial scores equal to or lower than 1, compared to 86% on diet anti-HSMB 2 and 96% on diet anti-HSMB 1 (Figure 2c). At week 16 postchallenge, 94% of fish fed the anti-HSMB 1 diet had myocardial scores equal to or less than 1 compared to 85% and 51% on the anti-HSMB 2 and control diets, respectively (Figure 2d). The differences between diets anti-HSMB 1 and control at 14 and 16 weeks were statistically significant since higher coefficients result in significantly better (lower) myocardial scores observed with for anti-HSMB 1 compared to control (Figure 3c). Similar positive effects, but much milder and less certain, were observed with anti-HSMB 2, however not statistically significant (Figure 3d). With respect to the epicardium, at 10 weeks post-challenge for all for, 91-92% of the epicardial scores are equal to or lower than 1, and about 40% of the scores were 0 for all for (figure 4a). At 12 weeks post-challenge, anti-HSMB 2 fed fish were slightly less likely (81%) to have epicardial scores equal to or lower than 1 compared to 90% for diet control and 92% for anti-HSMB 1 diet, although these differences were not significant (Figure 4b). At week 14 postchallenge, there was a trend for anti-HSMB 1 with a higher probability of 85% to have epicardial scores equal to or lower than 1 compared to 69% and 63% probability for the anti-HSMB 2 and control diets, respectively ( figure 4). At week 16 postchallenge, it appeared that the anti-HSMB 1 and anti-HSMB 2 diets had higher probabilities (90% and 85%, respectively) of epicardial scores equal to or lower than 1 than controls (74%). Although this cannot be claimed as significant, a clear trend emerges for anti-HSMB 1 in improving the pathological status of the fish (figure 4d).

Thus, clear effects of dietary treatments on the inflammatory status of the fish were observed from 12 weeks onwards (figures 3-4), where in particular the diet anti-HSMB 1 resulted in a reduced inflammation on the heart as indicated by a higher probability of having low histopathological score compared to the control diet. Even more marked differences were observed later in the feeding experiment after 14 and 16 weeks of feeding with anti-HSMB diets 1 and 2 compared to the control group. This means that fish fed anti-HSMB diet 1 had significantly less damage compared to the control diet.

The results show surprising and clear potential for these dietary compositions in modulating and treating HSMB disease in Atlantic salmon.

Fish suffering from HSMB disease have been described to have changes in the liver. Thus, the liver is yellowish, shows an increased fat content (commonly known as hepatic steatosis) and sometimes necrotic foci have been observed in which the cells are vacuolated and pyknotic. Feeding new dietary compositions to HSMB-infected Atlantic salmon surprisingly affected the pathological status of the liver (Figures 6-7).

At 10 weeks post-challenge, fish fed anti-HSMB 1 and anti-HSMB 2 diets had a clearly higher probability of having lower scores (that is, scores less than or equal to 1)

(79% and 72% respectively) compared to 16% for the control diet fish (Figure 6a). The coefficients for liners with anti-HSMB 1 and anti-HSMB 2 were significantly better (lower score - less steatosis) at 10 weeks (Figure 6a). At 14 weeks postchallenge, anti-HSMB 1 were also more likely (74%) to have a lower score (ie, indicating a lower degree of hepatic steatosis) compared to 40% for anti-HSMB 2 and to 23% for controls . These differences were significantly lower for diet anti-HSMB 1, while a trend emerged for diet anti-HSMB 2 compared to control (figure 7c).

Thus, clear effects of anti-HSMB 1 diets on the liver status of the fish were observed at 10 and 14 weeks (figures 6-7), where in particular the anti-HSMB 1 diet resulted in a reduced hepatic steatosis indicated by a higher probability of having low histopathological score compared to the control diet. These effects were particularly observed at 10 and 14 weeks post-challenge.

In addition, clear significant differences in weight gain were observed between prechallenge diets (Figure 8). After 8 week feeding period before challenge.

The exact mechanism(s) behind this surprising effect in reducing cardiac inflammation in HSMB-infected fish with the new dietary compositions in accordance with the present invention are not yet clear. A high content of n-3 fatty acids, especially of the fatty acid eicosapentaenoic acid (EPA20:5n-3) is beneficial. Furthermore, a reduced content of lipids combined with an increased content of protein, compared to conventional pre-compositions used for the growth of the same species, appeared to be beneficial for the modulation of HMSI diseases.

Figure 9 shows that the level of the fatty acid eicosapentaenoic acid (EPA 20:5n-3) accumulates in heart tissue, and without being bound by mechanism, it is assumed that this increased level of EPO in the right place in the fish body, i.e. in the heart tissue, is beneficial for HSMB - infected fish.

Furthermore, in a preferred embodiment in accordance with the present invention, South American fish oil containing approximately twice as much EPA as Nordic Atlantic fish oil is used to increase the content of EPA in the anti-HSMB diets.

Furthermore, a reduction in the content of n-6 fatty acids, which are typically found in vegetable oils, appeared to be useful in modulating the HSMB disease. The result also indicates that a reduced n-3/n-6 ratio so that this is in the range 2.3 - 3.4 can be beneficial.

The liver of HSMB-infected fish is affected by the disease as described above. This can have an effect on lipid digestibility in the fish. In a preferred embodiment of the present invention, vegetable oils are added to the diets in a hydrolyzed form. It also seemed beneficial to include hydrolyzed phospholipids in the diet.

It may also be helpful to add krill meal and/or krill oil to the diet. Preferably, the diet may further comprise nucleotides, anti-inflammatory compounds and/or mannaoligosaccharides (MOS).

It is assumed that the pre-composition in accordance with the present invention is also relevant for teleost species other than salmonids which can be affected by HSMB.

It should be realized that the properties of the present invention as described above can be modified without deviating from the scope of the invention.

Definitions of terms and abbreviations:

"FA" is the abbreviation used for "fatty acids"

"n-3 fatty acids" a family of unsaturated fatty acids which have in common a final carbon-carbon double bond in the n-3 position, which is the third bond from the methyl end of the fatty acid. These fatty acids are also known as omega 3 fatty acids.

"n-6 fatty acids" are a family of unsaturated fatty acids that have in common a final carbon-carbon double bond in the n-6 position, which is the sixth bond from the methyl end of the fatty acid. These fatty acids are also known as omega 6 fatty acids.

The term "n-3/n-6 ratio" means the ratio between fatty acids belonging to the n-3 family and those belonging to the n-6 family in the composition or diet.

"Protein/lipid ratio" indicates the ratio of proteins to lipids in the composition or diet (each related to the weight of the diet).

"HSMB" is the abbreviation used for "heart and skeletal muscle inflammatory disease".

The term "conventional feed ingredients" refers to ingredients that are usually part of a fish feed such as proteins, lipids, carbohydrates, vitamins, minerals, etc. The ingredients may be derived from marine, vegetable, animal by-products, and/or from any other relevant source and can be used in any suitable combination.

The term "nucleotides" refers to molecules comprising a nitrogen base (purine or pyrimidine base), a five-carbon sugar, and 1 to 3 phosphate groups.

Claims (51)

1. Pre-composition for fish to prevent and/or treat heart and skeletal muscle inflammation disease comprising conventional pre-ingredients such as proteins, lipids, vitamins, carbohydrates and minerals, characterized in that more than 20% of the total fatty acids are n-3 fatty acids. 2. Precomposition in accordance with claim 1, characterized in that preferably more than 23% of the total fatty acids are n-3 fatty acids. 3. Precomposition in accordance with claim 1, characterized in that preferably more than 24% of the total fatty acids are n-3 fatty acids. 4. Precomposition in accordance with one of the preceding claims, characterized in that the content of eicosapentaenoic acid 20:5 n-3 (EPA) is more than approx. 7% of the total fatty acids. 5. Pre-composition in accordance with claim 4, characterized in that the content of eicosapentaenoic acid 20:5 n-3 (EPA) is more than approx. 8% of the total fatty acids, more preferably 9%, more preferably more than approx. 10%, such as approx. 10.2%. 6. Pre-composition in accordance with one of the preceding claims, characterized in that the protein: lipid ratio (weight/weight) in the feed is higher than 2, preferably higher than 2.7. 7. Pre-composition in accordance with claim 6, characterized in that the proteins make up more than 50% (based on weight) and that the lipids make up less than 20% (based on weight) of the pre-composition. 8. Pre-composition in accordance with claim 6, characterized in that the proteins make up more than 52% (based on weight) and the lipids make up approx. 18% (based on weight) of the preformulation. 9. Pre-composition in accordance with one of claims 1 or 2, characterized in that the composition is for salmon and that the composition has a reduced content of lipids and an increased content of proteins compared to commercial pre-compositions used for salmon of the same age, size and/or stage of development . 10. Precomposition in accordance with one of the preceding claims, characterized in that the content of n-6 fatty acids is less than 11% of the total fatty acids, preferably in the range 7-11%, more preferably approx. 10%. 11. Pre-composition in accordance with claim 10, characterized in that the content of n-3 fatty acids is more than 23% and the level of n-6 fatty acids is less than 11% of the total fatty acids. 12. Precomposition according to one of the preceding claims, characterized in that the ratio of n-3 to n-6 fatty acids is higher than 1.6, preferably more than 2.2, more preferably between 2.3 and 3.4. 13. Precomposition in accordance with one of the preceding claims, characterized in that a proportion of said lipids is South American fish oil. 14. Pre-composition in accordance with one of the preceding claims, characterized in that the content of added vegetable oil is less than 0.5%, preferably 0.2% based on the weight of the composition, and that the oil is preferably added as a hydrolyzate. 15. Pre-composition in accordance with one of the preceding claims, characterized in that the composition comprises hydrolysed phospholipids, preferably approx. 0.1-0.2% by weight of the diet, more preferably more than 0.2% by weight of the diet. 16. Pre-composition in accordance with one of the preceding claims, characterized in that said composition comprises krill meal, preferably approx. 5% by weight, more preferably more than 5% by weight of the diet. 17. Pre-composition in accordance with one of the preceding claims, characterized in that the pre-form further comprises nucleotides and/or manna-oligosaccharides and/or anti-inflammatory compounds such as flavonoids. 18. Precomposition in accordance with one of the preceding claims, characterized in that said proteins make up more than 50% (based on weight) of the composition, where the lipids make up less than 20% (based on weight), where more than 20% of the total fatty acids are n-3 fatty acids. 19. Pre-composition according to claim 18, characterized in that the protein makes up more than 52% (based on weight) of the composition, and where the lipids make up less than 18% (based on weight), preferably 18% of the diet, where more than 23% of the total fatty acids are n-3 fatty acids. 20. Pre-composition in accordance with one of the preceding claims, characterized in that the composition is essentially as stated for anti-HSMB diet 1 or for anti-HSMB diet 2 in table 1 or table 2. 21. Use of the precomposition in accordance with one of the preceding claims 1-20 for the production of a pharmaceutical composition and/or nutritional composition and/or functional for the prophylaxis and/or treatment of heart and skeletal muscle inflammatory disease. 22. Use of the pre-composition in accordance with claim 20, to prevent or reduce symptoms related to heart and skeletal muscle inflammation disease in fish. 23. Application in accordance with claim 20, where said fish is a salmon fish, flatfish or another fish species suitable for aquaculture, preferably the species Atlantic salmon (Salmo salar). 24. Use in accordance with claim 20, for effective recovery after heart and skeletal muscle inflammation infection, and/or reduction of risk of reinfection. 25. Method for feeding fish susceptible to a heart and skeletal muscle inflammation infection, characterized in that a composition in accordance with one of claims 1-20 is fed to the fish for a period before challenge with an infection, during the infection and after the fish has been infected. 26. Method in accordance with claim 25, characterized in that the composition is administered for a period of at least 8 weeks pre-infection, more preferably 10-16 weeks, most preferably 16-20 weeks and 8-20 weeks post-infection. 27. Pre-composition for fish comprising conventional pre-ingredients such as proteins, lipids, vitamins, carbohydrates and minerals, characterized in that more than 20% of the total fatty acids are n-3 fatty acids. 28. Precomposition in accordance with claim 27, characterized in that preferably more than 23% of the total fatty acids are n-3 fatty acids. 29. Precomposition in accordance with claim 28, characterized in that preferably more than 24% of the total fatty acids are n-3 fatty acids. 30. Precomposition according to one of claims 27-29, characterized in that the content of eicosapentaenoic acid 20:5 n-3 (EPA) is more than approx. 7% of the total fatty acids. 31. Precomposition in accordance with claim 30, characterized in that the content of eicosapentaenoic acid 20:5 n-3 (EPA) is more than approx. 8% of the total fatty acids, more preferably more than 9%, more preferably more than approx. 10%, such as approx. 10.2%. 32. Pre-composition according to one of claims 27-31, characterized in that the protein:lipid ratio (weight/weight) in the feed is higher than 2, preferably higher than 2.7. 33. Pre-composition in accordance with claim 32, characterized in that said proteins make up more than 50% (based on weight) and the lipids make up less than 20% (based on weight) of the pre-composition. 34. Pre-composition in accordance with claim 33, characterized in that said proteins make up more than 52% (based on weight) and the lipids make up approx. 18% (based on weight) of the preformulation. 35. Precomposition according to one of claims 27-34, characterized in that the content of n-6 fatty acids is less than 11% of the total fatty acids, preferably in the range 7-11%, more preferably approx. 10%. 36. Precomposition in accordance with claim 35, characterized in that the content of n-3 fatty acids is more than 23% and the level of n-6 fatty acids is less than 11% of total fatty acids. 37. Precomposition according to one of claims 27-36, characterized in that the ratio of n-3 to n-6 fatty acids is higher than 1.6, preferably more than 2.2, more preferably between 2.3 and 3.4. 38. Precomposition in accordance with one of claims 27-37, characterized in that a proportion of said lipids is South American fish oil. 39. Pre-composition in accordance with one of claims 27-38, characterized in that the content of added vegetable oils is less than 0.5%, preferably 0.2% based on the weight of the composition, and that the oil is preferably added as a hydrolyzate. 40. Pre-composition in accordance with one of claims 27-39, characterized in that the composition comprises hydrolysed phospholipids, preferably approx. 0.1-0.2% by weight of the diet, more preferably more than 0.2% by weight of the diet. 41. Pre-composition in accordance with one of claims 27-40, characterized in that the composition comprises krill meal, preferably approx. 5% by weight, more preferably more than 5% by weight of the diet. 42. Pre-composition in accordance with one of claims 27-41, characterized in that the pre-form further comprises nucleotides and/or manna-oligosaccharides and/or anti-inflammatory compounds such as flavonoids. 43. Pre-composition according to one of claims 27-42, characterized in that the proteins make up more than 50% (based on weight) of the composition, where the lipids make up less than 20% (based on weight), where more than 20% of the total fatty acids are n-3 fatty acids. 44. Pre-composition according to claim 43, characterized in that the proteins make up more than 52% (based on weight) of the composition, and where the lipids make up less than 18% (based on weight), preferably 18% of the diet, where more than 23% of the total fatty acids are n-3 fatty acids. 45. Pre-composition in accordance with one of the preceding claims 27-44, characterized in that the composition is essentially as stated for anti-HSMB diet 1 or for anti-HSMB diet 2 in table 1 or table 2. 46. Pre-composition for fish comprising conventional pre-ingredients such as proteins, lipids, vitamins, carbohydrates and minerals, characterized in that the protein:lipid ratio (weight/weight) in the feed is higher than 2, preferably higher than 2.7 in order to prevent or reduce symptoms related to cardiomyopathy in fish. 47. Pre-composition in accordance with claim 46, characterized in that the protein:lipid ratio (weight/weight) in the feed is higher than 2.7. 48. Precomposition in accordance with claim 46, characterized in that the protein constitutes more than 50% (based on weight) of the diet, preferably more than 52% and that the lipids constitute less than 20% (based on weight), preferably 28% of the diet. 49. Precomposition in accordance with claim 46, characterized in that more than 20% of the total fatty acids are n-3 fatty acids, preferably more than 23%, more preferably 24%. 50. Pre-composition in accordance with claim 46, characterized in that the composition comprises more than 23% n-3 fatty acids and less than 11% n-6 FA of the total fatty acids. 51. Precomposition in accordance with claim 46 or 49, characterized in that the content of eicosapentaenoic acid 20:5 n-3 is more than 7% of the total fatty acids, preferably more than 9%, more preferably 10.2%.