WO2007061314A2

WO2007061314A2 - Use of fatty acid analogues

Info

Publication number: WO2007061314A2
Application number: PCT/NO2006/000424
Authority: WO
Inventors: Kjell-Arne Rorvik; Magny Thomassen; Rolf Berge
Original assignee: Berge Biomed As; Thia Medica As
Priority date: 2005-11-23
Filing date: 2006-11-22
Publication date: 2007-05-31
Also published as: EP1959751A2; NO20055541D0; NO20082546L; NO20055541L; WO2007061314A3

Abstract

Use of a fish feed comprising a compound represented by the general formula R'- COO-(CH2)2n+1-X-R', (I) or (II), which comprise non β-oxidizable fatty acids, for feeding fish during time periods where the requirements for energy of said fish is increased, in order to improve the health of said fish.

Description

Use of fatty acid analogues

The present application pertains to use of a feed comprising

(1) a compound represented by the general formula R"-COO-(CH2)2n+i-X-R\ wherein X is a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group or a SO₂ group; n is an integer of 0 to 11 ; and R' is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, wherein the main chain of said R' contains from 13 to 23 carbon atoms and optionally one or more heterogroups selected from the group comprising an oxygen atom, a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group and a SO₂ group; and R" is a hydrogen atom or an alkyl group containing from 1 to 4 carbon atoms; or

(2) a compound represented by the general formula (I),

wherein R1 , R2, and R3 represent i) a hydrogen atom; or ii) a group having the formula CO-R in which R is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, and the main chain of said R contains from 1 to 25 carbon atoms; or iii) a group having the formula CO-(CH₂)₂n+i-X-R', wherein X is a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group or a SO₂ group; n is an integer of 0 to 11 ; and R' is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, wherein the main chain of said R' contains from 13 to 23 carbon atoms and optionally one or more heterogroups selected from the group comprising an oxygen atom, a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group and a SO₂ group; iv) an entity selected from the group comprising -PO₃CH₂CHNH_SCOOH (serine), PO₃CH₂CH₂NH₃ (ethanolamine), P0₃CH₂CH₂N(CH₃)₃ (choline), PO₃CH₂CHOHCH₂OH (glycerol) and PO₃(CHOH)₆ (inositol); wherein R1 , R2, and R3 are chosen independently from i), ii), iii), or iv), but at least one of R1 , R2, or R3 is defined by iii); or

(3) a compound represented by the general formula (II),

R3

wherein A1 , A2 and A3 are chosen independently and represent an oxygen atom, a sulphur atom or an N-R4 group in which R4 is a hydrogen atom or a linear or branched alkyl group, saturated or unsaturated, optionally substituted, containing from 1 to 5 carbon atoms; wherein R1, R2, and R3 represent i) a hydrogen atom or a linear or branched alkyl group, saturated or unsaturated, optionally substituted, containing from 1 to 23 carbon atoms; or ii) a group having the formula CO-R in which R is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, and the main chain of said R contains from 1 to 25 carbon atoms; or iii) a group having the formula CO-(CH₂Wi-X-R'. wherein X is a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group or a SO₂ group; n is an integer of 0 to 11 ; and R' is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, wherein the main chain of said R' contains from 13 to 23 carbon atoms and optionally one or more heterogroups selected from the group comprising an oxygen atom, a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group and a SO₂ group; iv) an entity selected from the group comprising -PO₃CH₂CHNH₃COOH (serine), PO₃CH₂CH₂NH₃ (ethanolamine), PO₃CH₂CH₂N(CHs)₃ (choline), PO₃CH₂CHOHCH₂OH (glycerol) and PO₃(CHOH)₆ (inositol); wherein R1 , R2, and R3 are chosen independently from i), ii), iii), or iv), but at least one of R1 , R2, or R3 is defined by iii); or

(4) a salt, prodrug or complex of said compound as defined by (1), (2), or (3).

Infectious pancreatic necrosis (IPN) is a widespread disease in fish in most part of the world, and has been and still is a great problem in the Norwegian fish farming industry. In 1995 the financial loss caused by IPN alone was approximately 350-400 million NOK. In mid Norway 76% of the farms were hit by the disease in 2000. Since 1970 it has been ongoing work on vaccination against IPN, and a vaccine was finally introduced in 1995. Vaccine companies report good results in laboratories, but among fish farmers there are only 12 % that consider the IPN vaccine as an effective attempt to reduce IPN outbreaks. Hence, the efficacy of the vaccine in post smolts is uncertain.

The IPN virus (IPNV) is virulent to salmonids such as Atlantic salmon, rainbow trout (Oncorhynchus mykiss L.) and brown trout (Salmo trutta L.), but also a number of other species (Mortensen et al. 1999; Bruno 2004). In Atlantic salmon IPN occurs in the freshwater period, but in recent years disease outbreaks have increased after sea transfer. Experiments with smolt transferred to seawater in spring (1+) versus smolt transferred in autumn (0+) and outbreak of IPN have shown that there is a much higher mortality among 1+ smolt compared to 0+ smolt. Because of the low effect of the IPN vaccines experiments have been conducted to reduce the natural outbreak of IPN in 1+ smolt after sea transfer. Jarp et al. (1994) showed that stress related transportation affected negatively the frequency of IPN outbreaks after sea transfer. In an experiment by Leonardi et al. (2003) with dietary additives to improve the immune system rainbow trout given feed added nucleotides were shown to have a lower IPN mortality than fish fed the control diet.

Seasonal variations in energy stores, growth and feed utilization have been documented in both 1+ and 0+ farmed salmon during the grow out phase (Mørkøre and Rørvik 2001). To manage the transfer phase from freshwater to seawater the salmon go through a smoltification process, a complex and energy demanding process. It involves changes in morphology, and in the lipid metabolism. After sea transfer smolts have reduced energy stores seen as low levels of glycogen in liver and muscles, and reduced amount of fat in the abdominal cavity and in muscle (Staurnes et al. 1992). Experiments have showed that smolt have a lower content of fat compared to salmon that remain in freshwater (Usher et al. 1991). Salmon smolt transferred to seawater during spring also have been seen to reduce the energy stores even further in the weeks after transfer (Masøval et al. 1994).

Recently the present inventors have documented that Atlantic salmon survival during a natural outbreak of furunculosis, caused by Aeromonas salmonicida subsp. Salmonicida, was strongly and synergistically affected by dietary contents of iron and very long-chain polyunsaturated fatty acids of the n-3 family (EPA/DHA), where positive effects of high dietary levels of EPA/DHA were enhanced when combined with low levels of iron (Rørvik et al. 2003). Based on cumulative mortalities in the different experimental groups, relative percent survival (RPS) for the high EPA/DHA- low iron group was 70% during an outbreak of furunculosis and 96% during an outbreak of cold-water vibriosis, caused by Vibrio salmonicida.

Preventive actions against diseases are a necessary part of the aquaculture industry. A very important part of this is work dedicated to understand basic physiological processes in order to strengthen the fish and its natural resistance against diseases. Previous research by the inventors concerning fat content of diet and resistance to bacterial diseases (Rørvik et al. 2003) indicated that feed optimisation is of importance. The inventors wanted to investigate whether feed optimisation had significant effects also in relation to viral diseases such as IPN. Thus they conducted a feeding experiment on salmon smolt before and after release to seawater and natural exposure to IPN infection, testing several variables, among them the fat content of the diet before and/or after seawater release, the oxygen saturation level, IPN vaccination, and supplementation with medium fatty acid triacylglycerols (MCT), or supplementation with a fatty acid analogue, tetradecylthioacetic acid (TTA). To their surprise they found that the only factor tested to have a significant effect was the supplementation with TTA after release of the smolt to seawater. Not only was this effect unexpected, it was also quite strong, leading to a 70 % increase in survival.

Thus the use of the feed according to the present invention is for feeding fish during time periods where the requirements for energy of said fish is increased, in order to improve the health of said fish.

DETAILED DESCRIPTION OF THE INVENTION

The Invention will now be described in detail with references to the following figures:

Figure 1

Cumulative IPN mortality in smolt, from week 8 to week 13 after sea transfer, fed the same level of dietary fat. S2 is the unsupplemented control whereas S3 and S4 are the diets supplemented with TTA or MCT, respectively

Figure 2

Relationship between fat content in the muscle (a) and osmotic stress (b) six weeks after sea transfer, and mortality (c) during a natural outbreak of IPN in week 8-13 after sea transfer among farmed 1+ smolt fed the same dietary level of fat (29%). S2 is the unsupplemented control whereas S3 and S4 are diets supplemented with TTA or MCT, respectively.

Figure 3

Beta-oxidation expressed as nmol palmitoylCoA oxidised/μg protein (a) and fat content in the muscle (b) for farmed 1+ smolt fed control diet or a diet supplemented with TTA during an six weeks period after sea transfer.

Compounds of the present application

TTA is a non β-oxidizable fatty acid analogue, belonging to a group of compounds comprising: (1) a compound represented by the general formula R"-COO-(CH₂)₂n₊i-X-R\ wherein X is a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group or a SO₂ group; n is an integer of 0 to 11 ; and R' is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, wherein the main chain of said R' contains from 13 to 23 carbon atoms and optionally one or more heterogroups selected from the group comprising an oxygen atom, a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group and a SO₂ group; and R" is a hydrogen atom or an alkyl group containing from 1 to 4 carbon atoms; or

(2) a compound represented by the general formula (I),

wherein R1 , R2, and R3 represent i) a hydrogen atom; or ii) a group having the formula CO-R in which R is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, and the main chain of said R contains from 1 to 25 carbon atoms; or iii) a group having the formula CO-(CH₂)_2n+i-X-R', wherein X is a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group or a SO₂ group; n is an integer of O to 11 ; and R' is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, wherein the main chain of said R' contains from 13 to 23 carbon atoms and optionally one or more heterogroups selected from the group comprising an oxygen atom, a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group and a SO₂ group; iv) an entity selected from the group comprising -PO₃CH₂CHNH₃COOH (serine), PO₃CH₂CH₂NH₃ (ethanolamine), PO₃CH₂CH₂N(CHs)₃ (choline), PO₃CH₂CHOHCH₂OH (glycerol) and PO₃(CHOH)₆ (inositol); wherein R1 , R2, and R3 are chosen independently from i), ii), iii), or iv), but at least one of R1 , R2, or R3 is defined by iii); or

(3) a compound represented by the general formula (II),

wherein A1 , A2 and A3 are chosen independently and represent an oxygen atom, a sulphur atom or an N-R4 group in which R4 is a hydrogen atom or a linear or branched alkyl group, saturated or unsaturated, optionally substituted, containing from 1 to 5 carbon atoms; wherein R1 , R2, and R3 represent i) a hydrogen atom or a linear or branched alkyl group, saturated or unsaturated, optionally substituted, containing from 1 to 23 carbon atoms; or ii) a group having the formula CO-R in which R is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, and the main chain of said R contains from 1 to 25 carbon atoms; or iii) a group having the formula CO-(CH₂)_2n+i-X-R', wherein X is a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group or a SO₂ group; n is an integer of 0 to 11 ; and R' is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, wherein the main chain of said R' contains from 13 to 23 carbon atoms and optionally one or more heterogroups selected from the group comprising an oxygen atom, a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group and a SO₂ group; iv) an entity selected from the group comprising -PO₃CH₂CHNH₃COOH (serine), PO₃CH₂CH₂NH₃ (ethanolamine), P0₃CH₂CH₂N(CH₃)₃ (choline), PO₃CH₂CHOHCH₂OH (glycerol) and PO₃(CHOH)₆ (inositol); wherein R1 , R2, and R3 are chosen independently from i), ii), iii), or iv), but at least one of R1 , R2, or R3 is defined by iii); or

(4) a salt, prodrug or complex of said compound as defined by (1), (2), or (3). In a preferred embodiment of a compound according to the invention at least one of R1 , R2 or R3 is an alkyl.

In a preferred embodiment of a compound according to the invention at least one of R1 , R2 or R3 is an alkene.

In a preferred embodiment of a compound according to the invention at least one of R1 , R2 or R3 is an alkyne.

In a preferred embodiment of a compound according to the invention at least one of R1 , R2 or R3 is tetradecylthioacetic acid.

In a preferred embodiment of a compound according to the invention at least one of R1 , R2 or R3 is tetradecylselenoacetic acid.

In a preferred embodiment of a compound according to the invention X is a sulphur or selenium atom.

Preferred embodiments of the compounds according to the invention are tetradecylthioacetic acid (TTA), tetradecylselenoacetic acid and 3-Thia-15- heptadecyne.

In a preferred embodiment of a compound according to the invention n is 0 or 1.

In a preferred embodiment of a compound according to the invention said compound is a phospholipid, wherein said phospholipid is selected from the group comprising phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl glycerol, diphosphatidyl glycerol.

In a preferred embodiment of a compound according to the invention said compound is a triacylglycerol, most preferably comprising tetradecylthioacetic acid (TTA).

In a preferred embodiment of a compound according to the invention said compound is a diacylglycerol.

In a preferred embodiment of a compound according to the invention said compound is a monoacylglycerol. In a preferred embodiment of a compound according to the invention said compound is a non β-oxidizable fatty acid.

In a preferred embodiment of a compound according to the invention said compound is the phosphatidyl choline derivative 1 ,2-ditetradecylthioacetoyl-s/7-glycero-3- phosphocholine.

In a preferred embodiment of a compound according to the invention said compound is the phosphatidyl ethanolamine derivative i ^-ditetradecylthioacetoyl-sn-glycero-S- phosphoethanolamine.

In a preferred embodiment of a compound according to formula (II) A1 and A3 both represent an oxygen atom, while A2 represent a sulphur atom or an N-R4 group in which R4 is a hydrogen atom or a linear or branched alkyl group, saturated or unsaturated, optionally substituted, containing from 1 to 5 carbon atoms.

The compounds according to the invention are analogues of naturally occurring compounds, and as such are recognized by the same systems which process the natural compounds, including the enzymes that β- and in some cases ω- oxidize natural long chain fatty acids. The analogues differ from their naturally occurring counterparts in that they cannot be completely oxidized in this manner.

The compounds according to the invention may be non β-oxidizable fatty acid analogues, as represented by the formula R¹¹CCO-(CH₂WrX-R'- However, said compounds may also be more complex structures derived from one or more of said non β-oxidizable fatty acid analogues, as represented by the general formulas (I) or (II). These compounds are analogues of naturally occurring mono-, di-, and triacylglycerols, or phospholipids including phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl glycerol, and diphosphatidyl glycerol. Said compounds may also comprise a substitution in the glycerol backbone, as shown in formula (II). Said substitution of the oxygen(s) is achieved by replacing the oxygen(s) with sulphur or a nitrogen containing group. This may block hydrolysis before uptake by the intestines, thus increasing the bioavailability of the compounds. The above complex structures derived from one or more of said non β-oxidizable fatty acid analogues have their effect because the fatty acid analogues they comprise are not capable of being fully β-oxidized. Said complex structures may have an effect as complete structures, and as naturally resulting degradation products comprising the fatty acid analogues. Because the compounds are not able to be fully β-oxidized, they will build up, and this triggers an increase in the β- oxidation of naturally occurring fatty acids. Many of the effects of the compounds according to the invention are due to this increase in β-oxidation.

During β-oxidation, a fatty acid is enzymatically oxidized cleaved between carbons 2 and 3 (when counting from the carboxylic end of the fatty acid), resulting in the removal of the two carbon atoms on either side of the oxidation site as acetic acid. This step is then repeated on the now two carbons shorter fatty acid, and repeated again until the fatty acid is fully oxidized, β-oxidation is the usual way in which the majority of fatty acids are catabolized in vivo. The β-oxidation blocking by the compounds according to the invention is achieved by the insertion of a non- oxidizable group in the X position in the formula of the present invention. Because the mechanism for β-oxidation is well known, X is defined as S, O, SO, SO₂, CH₂ or Se. Anyone skilled in the art would assume, without an inventive step, that these compounds would all block β-oxidation in the same manner.

In addition, the compounds may contain more than one block, i.e. in addition to X, R' may optionally comprise one or more heterogroups selected from the group comprising an oxygen atom, a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group and a SO₂ group. As an example, one may insert two or three sulphurs as X to induce a change in the degradation of the fatty acid and thus a modulated effect. Multiple sulphur atoms would also modulate the polarity and stability somewhat. From a pharmacological viewpoint it is generally desirable to be able to present a spectrum of compounds rather than just one single compound to avoid or counteract problems with resistance.

In addition to the identity of X, its position is also an issue. The distance of X from the carboxylic end of the fatty acid is defined by how many CH₂ groups are positioned between X and the carboxylic end of the fatty acid, which is defined by (CH₂)_2n+i, where n is an integer of O to 11. Thus there are an odd number of CH₂ groups, that is; the position of X relative to the carboxyl group is such that X eventually blocks β- oxidation. The range of n is chosen to include all variations of the fatty acid analogue which has the desired biological effect. Since β-oxidation in theory can work on infinitely long molecules, n could be infinite, but in practice this is not so. The fatty acids which normally undergo β-oxidation are usually 14 to 24 carbon atoms long, and this length is therefore most ideal for undergoing enzymatic β-oxidation. The ranges of n and R' are thus given so that the fatty acid analogues will cover this range. (Likewise, option ii) of formulas (I) and (II) and define R to have 1 to 25 carbon groups, and option i) of formula (II) define the alkyl group to contain from 1 to 23 carbon atoms, to be analogous to naturally occurring compounds.) The total number of carbon atoms in the fatty acid backbone is preferably between 8 and 30, most preferably between 12 and 26. This size range is also desirable for the uptake and transport through cell membranes of the fatty acid analogues of the present invention.

Although all fatty acid analoges with an odd positioning of the β-oxidation blocker X away from the carboxylic end block β-oxidation, the extent of their biological effect may be variable. This is due to the difference in biological degradation time of the various compounds. The inventors have done experiments to show the effect of moving X further from the carboxylic fatty acid end. In these experiments the activity (in nmol/min/mg/protein) of mitochondrial β-oxidation in the liver of fatty acid analogues was measured with sulphur in the 3, 5 and 7 positions relative to the carboxyl end. The activities were 0,81 for sulphur in the 3^rd position, 0,61 for sulphur in the 5^th position, 0,58 for sulphur in the 7^th position, and 0,47 for palmitic acid, the non β-oxidation blocking control. This shows, as expected, that β-oxidation is indeed blocked by fatty acid analogues with varying positioning of the block, and that the effect thereof is lessened the further away from the carboxylic end the block is positioned at, because it takes the β-oxidation longer to reach the block so more of the fatty acid analogue is degraded by then. However, as the decline is great for going from the 3^rd to 5^th position, but small going from the 5^th to 7^th position, it is reasonable to assume that this decline will continue to be less as one moves out the chain, and thus that it will be very far out indeed before no effect (compared to the control) is seen at all.

Thus, it is reasonable to include as compounds of the present invention, fatty acid analogues and other compounds represented by the general formulas (I) and (II), (which comprise said fatty acid analogue(s),) which block β-oxidation at different distances from the carboxylic end of the analogues, as the compounds of the present invention all do indeed block β-oxidation, even if the effect thereof can be modulated. This modulation will after all differ under varying conditions; in different tissues, with varying dosages, and by changing the fatty acid analogue so that it is not so easily broken down, as will be described next. Thus it is reasonable to include in the formula all distances of the β-oxidation blocker from the carboxylic end of the fatty acid analogue which are biologically relevant.

Although fatty acid analogues as described with a block in the X position cannot undergo β-oxidation, they may still undergo ω-oxidation. This is a much less common and slower biological process, which oxidizes the fatty acid not from the carboxylic end, but rather from the methyl/hydrophobic head group, here termed R'. In this pathway the carbon atom at the ω-end of the fatty acid is hydroxylated by a member of the cytochrome P450 enzyme family. This hydroxylated fatty acid is then converted into an aldehyde by an alcohol dehydrogenase, and subsequently this aldehyde is converted into a carboxyl group by an aldehyde dehydrogenase. As a consequence, the final product of the pathway is a dicarboxylic fatty acid, which can be degraded further by oxidation from the ω-end.

Oxidation from the ω-end ("ω-oxidation") is believed to be the main pathway for degradation of the fatty acid analogues as described with a block in the X position. Experiments were thus performed where R' was changed to block ω-oxidation, by introducing a triple bond at the methyl end of the fatty acid analogue. This resulted in the fatty acid analogue 3-thia-15-heptadecyn, which when tested showed the expected result: a substantially increased degradation time in vivo. This is important for the use of the fatty acid analogues in pharmaceutical preparation, as it may potentiate the effects of the β-oxidizable fatty acid analogues by further slowing down their breakdown.

Again, as with the blocking of β-oxidation, it is routine to find other fatty acid analogues witch would block ω-oxidation in exactly the same manner, based upon knowledge of how ω-oxidation occurs. A double bond will for instance have the exact same effect as the triple bond did, and it is therefore included in the definition of the methyl/hydrophobic head group end of the molecule, here termed R', that it may be saturated or unsaturated. A branch may also block oxidation, so R' is defined as linear or branched.

In order to block ω-oxidation by the insertion of a substitute in R', said R' may be substituted in one or several positions with heterogroups selected from the group comprising an oxygen atom, a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group and a SO₂ group. R' may also be substituted with one or more compounds selected from the group comprising fluoride, chloride, hydroxy, Ci- C₄ alkoxy, Ci-C₄ alkylthio, C₂-C₅ acyloxy or Ci-C₄ alkyl.

Thus the compounds according to the present invention are either fatty acids analogous to naturally occurring fatty acids, which are not capable of being β- oxidized, or naturally occurring lipids comprising said fatty acid analogues. In vivo, the fatty acid analogues show a strong preference for being incorporated into phospholipids. In some cases it is indeed advantageous to mimic nature and incorporate the fatty acid analogues in naturally occurring lipids, such as mono-, di-, and triglycerides and phospholipids. This changes the absorption of the compounds (when comparing fatty acids to fatty acids incorporated in larger lipid structures) and may increase the bioavailability or stability.

As an example, one could make a complex by including a fatty acid(s) which are not capable of being β-oxidized into a triacylglycerol. Such compounds are encompassed by formulas (I) and (II). If such a triacylglycerol was taken orally, for instance in an animal feed product, it would probably be transported like any triacylglycerol, from the small intestine in chylomicrons and from the liver in the blood in lipoproteins to be stored in the adipose tissue or used by muscles, heart or the liver, by hydrolyzes of the triacylglycerol into glycerol and 3 free fatty acids. The free fatty acids would at this point be the parent compound of the present invention, and not a complex anymore.

Yet other possible glycerophospholipid derivatives of the fatty acids of the present invention includes, but are not limited to, phosphatidyl cholines, phosphatidyl ethanolamines, phosphatidyl inositols, phosphatidyl serines and phosphatidyl glycerols.

Another esterification of fatty acids found in vivo which could be easily used to make a complex for a compound of the present invention would be to make the alcohol or polyalcohol corresponding to the fatty acid, for example one could make a sphingolipid derivative such as ceramide or sphingomyelin by making the corresponding amino alcohol. Like the glycerophospholipid complexes, such complexes would be very water insoluble and less hydrophilic. These kinds of hydrophobic complexes of the present invention would pass easier through biological membranes. Other possibilities of polar complexes of the present invention may be, but are not limited to, lysophospholipids, phosphatidic acis, alkoxy compounds, glycerocarbohydrates, gangliosiedes, and cerebrosides.

Although there can be large structural differences between different compounds of the invention, the biological functions of all compounds are expected to be similar because they all block β-oxidation in the same manner. This inability of the lipid analogues to be β-oxidized (and in some cases, ω-oxidized,) causes the analogues to build up in the mitochondria, which triggers the β-oxidation of the in vivo naturally occurring fatty acids, which in turn leads to many of the biological effects of the fatty acid analogues of the present invention. (Berge RK et al. (2002) Curr Opin Lipidol 13(3):295-304)

Preparation and characterisation of the compounds according to the invention

A) The synthesis of 3-substituted fatty acid analogues:

The compounds used according to the present invention wherein the substituent X_/=3 is a sulphur atom or selenium atom may be prepared according to the following general procedure:

X is a sulphur atom:

The thio-substituted compound used according to the present invention may be prepared by the general procedure indicated below:

Base Alkyl-Hal + HS-CH₂COOR ===> Alkyl-S-CH2-COOR

The sulphur-compound, namely, tetradecylthioaceticacid (TTA), (CHs-(CH₂) 13-S- CH₂-COOH was prepared as shown in EP-345.038.

X is a selenium atom: the seleno-substituted compound used according to the present invention may be prepared by the following general procedure

1. Alkyl-Hal + KSeCN =-> Alkyl-SeCN...

2. Alkyl-SeCN + BH₄ ^" => Alkyl-Se^"

3. Alkyl-Se^" + O₂ => Alkyl-Se-Se-Alkyl

This compound was purified by carefully crystallisation from ethanol or methanol.

BH₄ ^"

4. Alkyl-Se-Se-Alkyl => 2 Alkyl-Se^"

5. Alkyl-Se- + Hal-CH₂_COOH => Alkyl-Se-CH₂ - COOH

The final compound, e.g. when alkyl is tetradecyl,

(CH3-(CH₂)₁3-Se-CH₂-COOH (tetradecylselinioacetic acid (TSA)) can be purified by crystallisation from diethyl ether and hexane.

B) Synthesis of non-β-oxidizable fatty acid analogous comprising a carbon- carbon triple binding:

The synthesis of a compound in accordance with the present invention is representatively elaborated with reference to the synthesis of the 3-Thia-15- heptadecyne:

KOH (2,76 g, 49,0 mmol) was dissolved in methanol (30 ml), and thioglycolic acid (2,04 g, 22,1 mmol) in methanol (25 ml) was added drop by drop. After 10 minutes the 14-Bromo-2-tetradecyne (5,5 g, 20,1 mmol) was carefully added drop by drop, and the mixture was heated to 50 ⁰C overnight. The mixture was cooled to 0°C, and 30 ml HCI was added (pH = 1). The precipitate was filtered and washed with water (2x). The solid material was dissolved in chloroform (100 ml) and washed with water (1x), dried (MgSO₄) and the solvent was evaporated off. The yield of the compound 14-Bromo-2-tetradecyne was 4,4 g (77 %).

The synthesis of the above non β-oxidizable fatty acid analogues were given as examples, not to illustrate the synthesis of all compounds of formula (I) or (II). Other compounds in accordance with the present invention can be synthesised as indicated in applicant patent applications PCT/NO99/00135 and NO 20001123. Any of the fatty acid analogues described can be inserted into triacylglycerols or other fats in accordance with the invention by means well known in the art. Toxicity study of TTA

A 28 days toxicity study in dogs according to GLP guidelines has been performed by Corning Hazleton (Europe), England. Oral administration of TTA at dose levels up to 500 mg/kg/day was generally well tolerated. Some lipid related parameters were lowered in the animals given high dosages. This is consistent with the pharmacological activity of TTA.

The dose level of 500 mg/kg/day also elicited body weight loss. There was no evidence of toxicity at dose levels of 50 or 500 mg/day/kg.

Tests for mutagenic activity have been performed by Covance Laboratories Limited, England. It was concluded that TTA and TSA did not induce mutations in strains of Salmonella typhimurium and Escherichia coli. Furthermore, TTA was not mutagenic when tested in mouse lymphoma cells and L5178Y.

The concentration of the compounds tested in S. typhimurium and E. coli 3-1000 mg/plate (TTA) 2-5000 mg/plate (TSA). In mouse lymphoma cells, L5178Y, the concentration was 2,5 - 50 mg/ml.

TSA and TSA were found not to be mutagenic in these tests. TSA and TTA have been tested for chromosomal aberrations in cultured Chinese hamster ovary cells and no aberrations were induced by the doses tested (12-140 mg/ml).

The compounds of the present invention are therefore potentially useful as pharmaceutical or nutritional compounds in this respect.

Administration of the compounds according to the present invention.

As a general proposition, the total effective amount of the compounds according to the invention administered in the feed will preferably be in the range of about 1 mg/kg/day to 2g/kg/day of total body weight. A dose of 5 - 500 mg/kg/day is preferable, while a dose of 50-100 mg/kg/day is most preferable.

As a percentage of the total feed, the amount of compounds according to the invention present in the feed should be between 0.005 % and 5 % of the total weight of the feed, preferably between 0.05 % and 1 %, more preferably between 0.1 % and 0.5 %, most preferably 0.5 % of the total weight of the feed.

The feed may contain any amount of fat common in fish feed. But the feed should preferably be optimized by being energy rich, that is, it should preferably comprise fairly high levels of fat, at least 15 % by weight, preferably 15 % - 45 %, more preferably 20 % - 40 % fat by weight. The exact preference will of course vary with different fish species. However, a high fat content is not required for the compounds according to the invention to work, but it will most likely enhance their effect.

If the feed is used as a supplementary feed in addition to another feed(s), the given daily amounts should of course be the total daily amounts in the total feed consumed, and the percentages of the compounds according to the invention and the percentages of fats should also be for the total amounts of feed. Thus if the feed is used as such a supplementary feed the percentages by weight of both compounds according to the invention and fats may greatly exceed the percentages given above, up to a feed comprised entirely of fats (therein included the compounds according to the invention).

If the feed is used as a supplementary feed in addition to another feed(s), it may be administered to the fish separately from said another feed(s), or at the same time, or mixed with said another feed(s), as an example any conventional feed can be sprayed or soaked with a mixture of fat (preferably in liquid form) and a compound according to the invention, to thus obtain a feed according to the present invention.

Definitions used in the present invention

Baseline intake

The term "baseline intake" when used in regard to energy intake, refers to the intake of energy from food that the fish experience during normal growth, that is, when the fish is not subject to any extraneous stresses, such as an abrupt change of/in the environment, exposure to disease etc.

Feed

The term feed is used to describe any food fed to animals, comprising any fodder or specialty or supplemental feed or feed additive. Any feed can be supplemented with the composition of the present invention, to attain the biological effects thereof, and this is intended to be included in the definition of feed.

Prodrug

The term "prodrug" is commonly used in the art to describe a medicament which has been modified so that it has no effect in vitro, but is activated in vivo. Usually this modification is the addition of some compound to the parent drug, which the natural mechanisms in the body will remove, yielding the now active (original) parent drug. Thus such a "prodrug" is, biologically speaking, identical in its effects to the parent compound and the inclusion of the word "prodrug" in the compound descriptions does not widen the scope of compounds included in the patent.

Example 1: Reduction of IPN mortality in 1+ Atlantic salmon smolt after sea transfer in spring by potentially energy enhancing additives

Materials and methods:

Fish and treatments in freshwater

Two different vaccines, two different levels of oxygen saturation (100 % in the inlet and 80% in the outlet or 170 % in the inlet and 120% in the outlet) and three different diets were administrated for 5,5 weeks (from April 2nd to May 11th 2004) to 1 +

Atlantic salmon smolt in duplicate tanks (2 m2) at AKVAFORSK research station at

Sunndalsøra on the west cost of Norway. Initial body weight was 38 grams and all fish were individually marked with pit tag to make salmon in seawater traceable to freshwater treatments. Totally 12 tanks and 800 smolt per tank were used with a mean water temperature of 9 ⁰C.

One third of the smolt was vaccinated with Norvax Compact 4 (Aeromonas salmonicida susp. Salmonicida, Vibrio salmonicida, Vibrio Anguillarum serovar O1 and 02) and two third with Norvax Compact 6 Aeromonas salmonicida susp. Salmonicida, Vibrio salmonicida, Vibrio Anguillarum serovar 01 and 02, Vibrio viscosus and a surface protein from IPNV). Intervet Norbio AS delivered both vaccines.

All diets (also in seawater) were manufactured by high-pressure moist extrusion by Ewos (Table 1). Eight tanks were given control feed (F1), two tanks were given control feed supplemented with 0.5% clofibrate (CIo) (F2) and the two last tanks were given control feed with addition of 0.5% tetradecylthioacetic acid (TTA) (F3). Among fish fed F1 four tanks were administrated the normal level of oxygen saturation (100% in the inlet) whereas four tanks got the high level of oxygen saturation (170% in the inlet). The last four tanks, which were given feed F2 or F3, all had the normal level of oxygen saturation. Throughout the experiment, the fish were fed with 120% of predicted growth (Austreng et al. 1987), assuming a feed:gain ratio of 1.0.

Table 1. Chemical content of feed (F1 , F2 and F3) used in freshwater.

Type of feed Fat (%) Protein (%) DM ¹ (%) Ash (%) NFE² (%) Additives

F1 22,8 48,1 92, 1 9,4 11 , 8 F2 22,7 48,8 91 , 3 9,4 10, 4 0,5 % CIo F3 24,6 48,8 93, 1 9,5 10, 3 0,5 % TTA

1 DM = dry matter 2 NFE = Nitrogen free extract

Prior to start of the freshwater period the Atlantic salmon were reared under continuous light (24L0D). To prepare for smoltification the photoperiod was changed from long to short (12L12D) on February 2nd and back to continuous light again on March 24th.

Fish and treatments in seawater

On May 11th the fish were transferred to AKVAFORSK research station at Averøy on the west cost of Norway. On arrival to the sea site 60 smolts from each tank in freshwater were stocked together in each of 12 net pens (5x5x5 m). Thus, 720 smolt per pen representing all freshwater treatments, totally 8840 fish. Mean body weight of the smolt by transfer was 61 grams. During the seawater period (terminated September 14th) four diets were fed to the salmon in a randomised block design of triplicate net pens. Seawater temperature at transfer was 9DC increasing to about 16DC in mid August with an average of 12⁰C.

The four different seawater diets were: S1 , a low fat control diet with 20% fat; S2, a high fat control diet with 29% fat; S3, the high fat control diet added 0.5% TTA and finally S4, the high fat control where 14.8% of the fat was substituted by MCT (Table 2).

Table 2. Chemical content of feed (S1 , S2, S3 and S4) used in seawater.

Type of feed Fat (%) Protein (%) DM¹ (%) Ash (%) NFE² (%) Addetives 51 19,8 47,2 92,4 9,3 16,2

52 29,2 48,0 94,7 9,9 7,6

53 30,1 48,4 95,3 9,9 6,8 0,5 % TTA

S4 29,3 48J 94,7 ^■ 10,0 6,7 4,3 % MCT

1 DM = Dry matter

2 NFE = Nitrogen free extracts

Evaluation of protection from IPN

To evaluate the effect of dietary supplementation in seawater cumulative mortalities were compared. In order to characterize this difference, the method for determination of relative percent survival (RPS), described by Amend (1981), was used. The control group was fish fed the high fat diet without supplementation. Relative percent survival for a given dietary group was estimated by the following equation:

RPS = 100% x (1- % mortality in a given dietary group / % mortality in the control group)

Sampling and analyses

All diets were analysed gravimetrically for dry matter (DM) after drying at 105⁰C (16 18 h), and ash (flame combustion followed by 3 4 h at 55O⁰C). The content of crude proteins was calculated as N*6.25 using the semi micro Kjeldahl method (Kjeltec Auto System, Tecator, Hδganas, Sweden), and crude lipid after diethyl ether extraction in a Fostec analyser (Tecator, Hδganas, Sweden) after HCI hydrolysis (Stoldt, 1952). Proportion of Nitrogen Free Extract (NFE) was estimated as: NFE = DM - (Fat + Protein + Ash).

Fish were randomly sampled for analysis of fat content in the muscle at the end of the freshwater period (10 fish per tank) and after six weeks in sea (60 fish per pen - five from each tank in freshwater). Plasma chloride was analysed in fish sampled for analyses of muscle fat six weeks after sea transfer. All fish were anaesthetised (MS 222, metacaine, 0.1 g L-1). Fat content in the muscle (Norwegian Quality Cut (NQC), NS9401 1994) was analysed according to Folch et al. (1957) and level of plasma chloride was essayed using a Radiometer CMT Chloride titrator (Copenhagen, Denmark). Plasma was separated by centrifugation of blood samples taken from the caudal vein using heparinized vacuum tubes.

Dead fish was recorded daily. Control for IPN was performed at Norwegian Veterinarian University of Norway.

Statistical analyses All statistical analyses were performed using the SAS software package (SAS Institute Inc. 1990). Data were analysed using one-way analyses of variance (ANOVA) or simple and multiple regression analyses. Diet in freshwater, type of vaccine, level of oxygen saturation, dietary content of fat in seawater and high fat diet in seawater were used as treatment. Experimental units were tanks for treatments in freshwater and net pens in seawater. Proportion of the total variation explained by the model is expressed by R2 and calculated as the marginal contribution of the mean square of the parameter (type I sum of squares). Significant differences among variables within treatments were indicated by least-square means comparison. Person's product moment correlations were used to describe the relationship between variables. The level of significance is chosen at P±O.05, and the results are presented as mean ± s.e.m. (standard error of the mean).

Results of experiment 1 :

Fish mortality

Eight weeks after sea transfer the mortality began to increase. IPN was confirmed to be the cause of death. After week 13 unspecific mortality were registered. Mean cumulative mortality from week eight to week thirteen was 5,3 %.

Neither feed given in freshwater, level of oxygen saturation, type of vaccine nor dietary level of fat in seawater significantly affected IPN mortality in seawater. Type of high fat diets (S2-S4) given in seawater, however, revealed a significant effect (Fig.1). Throughout week 8 to week 13 the dietary effect increased, explaining 45% to 76% of observed variation in mortality becoming significant in week 10. After 14 weeks in sea the explanation decreased again coinciding with the observation of unspecific mortality in the last period of the outbreak. Highest mortality was found for fish fed the high fat control diet (S2; 7,8 %), whereas lowest mortality was observed among those fed the high fat diet added TTA (S3; 2,3 %) (Fig. 2c). The relative percent survival (RPS) for fish fed the TTA diet was 70 % compared to the control group without supplementation. Dietary supplementation of TTA revealed significantly reduced mortality compared to both the control group and the group supplemented MCT (S4; 6.4%).

Fat content in muscle

Mean fat content in the muscle of pre-smolt at the start of the freshwater rearing was 2.4% ± 0.1. At the end of the freshwater period a significant difference in fat content in the fish muscle was observed. Experimental diet in freshwater was the only treatment significantly affecting the fat content. Smolt fed control diet (F1) had significantly higher fat content (3.2 % + 0.1) than fish fed either control diet added TTA (2.6 % ± 0.3) or control diet added clofibrat (2.5 % ± 0.4). Overall mean fat content at sea transfer was 3.0%± 0.4.

After six weeks in seawater type of feed given in freshwater was the only treatment revealing a significant effect on the fat content. Smolt fed control diet added TTA in freshwater (F3) had significantly lower fat content in the muscle (3.3 % ± 0.2) than those fed control diet added clofibrate (4.0 % ± 0.2), but not significantly different from smolt fed the control diet (3.7 % ± 0.1). No significant effect of the high fat diets in seawater was observed. However, as in freshwater numerically lowest fat content was observed in the TTA group in sea. Hence, just prior to the start of the IPN outbreak among fish given the same content of dietary fat (29%) a trend (p=0.17) to reduced fat in the muscle was observed in smolt fed diet supplemented with TTA (Fig.2a).

Plasma chloride

Six weeks after sea transfer the high fat diets in sea revealed an overall almost significant (p=0.06) effect on the levels of plasma chloride in the fish. Fish fed the high fat control diet S2 (149 mmol L-1) had significantly elevated levels of chloride compared to fish in the TTA group (143 mmol L-1) and almost significantly so (p=0.08) compared to the MCT group (145 mmol L-1) (Fig. 2b)

Relationship between levels of muscle fat, plasma chloride and IPN mortality A not significant positive correlation between levels of fat in the muscle and plasma chloride (r=0.82), between plasma chloride and IPN mortality (r=0.95), and between muscle fat and IPN mortality (r=0.96) was registered. The same dietary ranking was observed in all three variables (Fig. 2a, b, c).

IPN vaccine

During the natural outbreak of IPN in the present study no effect of the IPN vaccine alone or dietary interactions was observed. This relates well with the low percentage of fish farmers (12%) that consider the IPN vaccine as an effective attempt to reduce IPN outbreaks (FHL & VESO 2003). The apparent contradiction between the good results obtained in controlled challenge tests by the vaccine companies and the rather uncertain efficacy observed in commercial post smolt is a great problem for the farming industry. This makes, however, alternative strategies in the struggle for reduced IPN mortality even more important.

Example 2: Increase in β-oxidation in muscle of salmon fed a diet containing TTA during the critical period following sea transfer.

Fish and treatments

Example 2 is part of a larger ongoing experiment with Atlantic salmon 1+ smolt hatched in January 2005 (Marine Harvest, Slørdal). On May 19^tn the fish were transferred to AKVAFORSK seawater research station at Averøy on the west coast of Norway. On arrival to the sea site the fish were distributed in net pens with about 1100 fish in each. Mean body weight of the smolt was 104 grams at transfer. The part of the experiment described in this experiment focuses on two different diets fed in triplicates. Seawater temperature at transfer was 9.2°C increasing to 12.3⁰C at the end of the six weeks period with an average of 10.5⁰C. The two different diets were: control diet and a diet added 0.5% TTA (Table 3), which is the same amount as in the feed fed in study 1. The fish were fed for six-nine weeks, straight after transfer to seawater, until sampling for beta-oxidation and fat content in muscle, respectively.

Table 3. Chemical content of the diets in the present experiment 2.

Type of feed Fat (%) Protein (%) DM¹ (%) Ash (%) NFE² (%) Additives

Control 28.1 45.6 92.6 8.7 10.1

TTA-diet 27.2 47.2 94.2 9.0 10.9 0.5% TTA

Samplinq and analyses

Fish were randomly sampled for analysis of beta-oxidation in muscle after six weeks in sea. A very small piece of white muscle was sampled from ten fish in each group. Muscle was then homogenized and the mitochondria isolated using a mitochondria isolation kit (MITO-ISO1 , Sigma-Aldrich, USA). The mitochondrial beta-oxidation was analysed in accordance to Lazarow (1981), with some small modifications. The main modification is that the reaction is started by transferring the tubes to room temperature for 30 minutes instead of 11 minutes in water bath held at 37⁰C. This modification is done because the method is designed for warm-blooded animals, while fish is a cold-blooded animal. Further; the amount of enzymes added to the tubes with reaction mixture was 5, 7.5 and 10 μl. To evaluate the enzyme activity in the result chapter we used the average value of these three concentrations. The protein analysis in the mitochondria samples was measured using Total Protein Kit, micro Lowry, Peterson's modification (Lowry, Rosebrough, Farr & Randall, 1951 ; Peterson, 1977). Fat content in muscle was analysed on samples of 10 fish (NQC) as described under example 1.

Statistical analyses

All statistical analyses were performed using the SAS software package (SAS

Institute Inc. 1990) as described for example 1. Diets were used as treatments, while net pens were used as experimental units. The level of significance is chosen at pθ.05, and the results are presented as lsmeans s.e.m. (standard error of the mean).

Results of experiment 2:

Mitochondrial beta-oxidation

After six weeks of feeding the activity of muscle mitochondrial beta-oxidation was found to be significantly higher (p=0.04; R2=0.93) in fish given feed added 0.5% TTA

(12.3 ± 1.4 nmol palmitoylCoA oxidised/μg protein) than in fish given the control diet

(3.6 ± 1.1 nmol palmitoylCoA oxidized/μg protein).

Fat content in muscle

After 9 weeks of feeding fish fed the diet added TTA had significant lower (p=0.03,

R2=O.94 %) fat content in muscle (2.95 ± 0.05) than fish fed control diet (3.35 ± 0.05

%).

Consequently, as suggested in our discussions of the results from experiment 1 , a relationship between mitochondrial beta-oxidation and fat content in muscle was observed (Fig. 3 a, b). Fish fed TTA had significantly higher mitochondrial beta- oxidation and significantly lower fat content in muscle than fish fed the control diet.

Conclusion

The described examples (1 and 2) demonstrates that dietary addition of TTA significantly increased survival (RPS=70%) during a natural outbreak of IPN after sea transfer of 1⁺ salmon. It is suggested that TTA has resulted in a re-allocation of dietary fatty acids from storage to energy producing oxidation due to the increased mitochondrial beta-oxidation capacity and decreased level of fat in muscle found in smolt fed TTA in example 2. The increased access to energy explain the observation of significantly lower levels of plasma chloride and hence reduced osmotic stress in smolt fed TTA compared to the control six weeks after sea transfer, two weeks prior to the outbreak of the disease. During this critical, but temporary, high metabolic energy-requiring period of 1⁺ smolt after sea transfer dietary addition of TTA increase significantly the ability of salmon smolt to resist IPN.

The scope of the invention

Based upon the experiments detailed herein, the inventors believe that the energy requirement of fish during critical and stressful periods is higher than can be obtained through the energy content in traditional feed, reducing growth and feed conversion, and acting as a predisposing factor for outbreak of diseases. This increase in the requirements for energy of fish can be detected by the reduction in body lipid/energy stores and/or as the need of energy intake above the baseline intake.

Timing of the natural outbreak of IPN eight weeks after sea transfer in the present study is in accordance with earlier observations by Bowden et al. (2002). They also observed that IPN was associated with mortality in salmon smolt about eight weeks after sea transfer, especially in Norway and Shetland. However, such outbreaks may occur as early as 2-4 weeks after sea transfer and until the smolt have completely adapted to their new environment, at 16-20 weeks, but usually occurs within 4 to 14 weeks from sea transfer.

The major unexpected finding in this experiment was the observed high reduction in IPN mortality in Atlantic salmon fed diet added TTA (S3) compared to the high fat control diet (S2). The experiment revealed a cumulative mortality as low as 2.3 % in the TTA groups compared to 7.8 % in the control. This is equivalent to a relative percent survival (RPS) of 70 %, which is over two times higher than that reported by use of glucan nucleotides as dietary additives (Johnsen 2002). Addition of TTA also showed a trend to lower mortality than dietary MCT (p=0.08).

Adding a compound according to the invention to feed during the critical and temporary high metabolic energy-requiring period of 1+ smolt after sea transfer thus increase the ability of salmon smolt to resist IPN virus infection. However, there is no reason to believe that this effect is limited to salmon smolt or IPN virus infections. Compounds according to the present invention should have a beneficial effect on any farmed fish during any temporary high metabolic energy-requiring period. The compounds according to the present invention are known to increase β-oxidation, and may thus help to supply extra energy in any time periods where the requirements for energy of said fish is increased.

Such critical temporary high energy-requiring periods may comprise any time periods where the fish is subjected to unusual stress. This comprises time periods when fish is subjected to infections, comprising bacterial, viral or fungal infections, including but not limited to IPN infections, furunculosis, Pancreas dieseas (PD), HSMB (heart and skeletal muscle infection) and CMS (Cardiac Myopathy Syndrome). During such time periods the fish is in need of extra energy to fight off the infection, to lessen both the symptoms and/or mortality caused thereby.

These critical temporary time periods where the requirements for energy of said fish is increased also comprises transfer of fish to a new environment, including sea- transfer of fish, as well as transferral of hatchery reared fish to fresh or brackish water, or transferral from indoor tanks to the outdoors, or from one outdoor environment to a different environment, during any time of the year. Following transferral to a new environment the fish must undergo a process of adaptation, whereupon the fish must expend energy, and thus the fish may need more energy than it can utilize from normal feed. If such extra energy is not provided, the fish may be more susceptible to disease, and to reduced growth and health in general. In some cases the need for extra energy does not follow immediately after the transferral to a new environment, for instance the first spring spent in the sea after fall release of smolt from hatchery is a very critical time period, where the fish is susceptible to infections, in particular PD infections. A feed in accordance with the invention would then be of great benefit to the health of the fish, providing extra energy needed to avoid or lessen the effects of infection.

Thus these temporary time periods where the requirements for energy of said fish is increased also comprises time periods when the fish is not transferred to a new environment, but rather when their environment is changing. An example would be a particularly cold winter, especially with water temperatures at 5°C or lower, or very sudden changes in the environment that would cause the fish undue stress. During such time periods, a feed according to the invention would be highly beneficial.

Since the events leading to temporary high energy-requiring periods may differ greatly, the amount of time for which a feed in accordance with the present invention should be used will also vary greatly. But as such events can be predicted, it should be feasible for those in the field of fish-farming to establish when and for how long the feeds according to the present application should be applied. However, this is supposed to be a feed for temporary use only, not for general use. The applicant has previously described the use of the compounds according to the present invention in PCT patent applications NO05/00271 and NO05/00272, for feeds for general use. These feeds for general use were not for keeping fish healthy during critical temporary high energy-requiring periods, as according to the present invention, but rather for improving the fatty acid profile of the fish and products there from, and thus preventing or treating various non-infectious diseases. Thus the use of the compounds according to the present invention for keeping fish healthy during critical temporary high energy-requiring periods is both new and inventive compared to the prior art.

Salmon is not the only type of farmed fish that is subject to critical temporary high energy-requiring periods where said fish is in need of exceeding the energy intake above the baseline intake. Any farmed fish could benefit from a feed according to the present invention tailored to their needs during such time periods. The compounds according to the present invention have a basic effect on β-oxidation, and this should not be any different in other fish species. Thus the invention could be used for any farmed fish species, although preferred species comprise cod, charr, halibut and catfish, more preferred species comprise salmon and trout, and the most preferred species comprise Atlantic salmon, Coho salmon, rainbow trout and brown trout.

References

• Amend D. F. (1981) Potency testing of fish vaccines. Developments in Biological Standardization 49, 447-454

• AKVAFORSK AS, (2000) Metode for bestemmelse av klorid. Dokumentkode: 091004.

• Bowden T.J., Smail D.A. & Ellis A.E. (2002) Development of a reproducible infectious pancreatic necrosis virus challenge model for Atlantic salmon (Salmo salaή. Journal of Fish Diseases 25, 555-563.

• Bratberg B. (1978) Histologisk teknikk. Manual for histolabs product WD4. Manual for histolab 1002.

• FHL & VESO (2003) IPN in salmonids a review. 115s. www.veso.no

• Folch J., Lees M. & Sloana Stanley G. H. (1957) Simple method for isolation and purification of total lipids from animal tissues. Journal of Biology and Chemistry 226, 497 - 507.

• Jarp J., Gjevre G., Olsen A.B. & Bruheim T. (1994) Risk factors for furunculosis, infectious pancreatic necrosis and mortality in post-smolt of Atlantic salmon (Salmo salaή. Journal of Fish Diseases 18, 67-78.

• Johnsen M. (2002) Kampen mot IPN. / Dybden 18(2). www.skretting.no

• Lazarow P. B. (1981). Assay of peroxisomal y-oxidation of fatty acids. Methods in Enzymology 72, 315-319.

• Leonardi M., Sandino A.M. & Klempau A. (2003) Effect of a nucleotide- enriched diet on the immune system, plasma Cortisol levels and resistance to infectious pancreatic necrosis (IPN) in juvenile rainbow trout (Oncorhyncus mykiss). Bulletin of EAFP 23(2), 52-59.

• Lowry O.H., Rosebrough N.J., Farr A.L & Randall R.J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193 (1), 265-275.

• Mann CK. & Yoe J. H. (1957) Spectrophotometric determination of magnesium with 1 -Azo-2-hydroxy-3-(2.4-dimethylcarboxanilido)-naphtalene- 1 ^'-(2- hydroxybenzene). Analytica Chimica Acta 16(2), 155- 160.

• Mortensen S. H., Hjeltnes B., Rødseth O. M., Krogsrud J. & Christie, K.E. (1999)lnfectious pancreatic necrosis virus, serotye N1 , isolated from Norwegian halibut (Hippoglossus hippoglossus), turbot (Scopthalamus maximus) and scallops (Pecten maximus). Bulletin of European Association; Fish Pathology 10, 42-43. • Mørkøre, T. and Rørvik, K-A. (2001) Seasonal variations in growth, feed utilisation, and product quality of farmed Atlantic salmon transferred to seawater as 0+smolt and 1+smolt. Aquaculture, 199: 145-157

• Peterson G. L. (1977) Simplification of protein assay method of Lowry et a/. - which is more generally applicable. Analytical Biochemistry 83 (2), 346-356.

• Rørvik K.-A. & Thommassen M.S. (2004) Effekt av økt energitilgang pa IPN hos laks etter utsett i sjø. Havbruk - produksjon av akvatiske organismer. Programkonferanse 23. - 24. mars 2004, Gardermoen.

• Røsjø C, Nordrum S., OHi JJ. , Krogdahl A., Ruyter B. & Holm H. (2000) Lipid digestibility and metabolism in Atlantic salmon (Salmo salaή fed medium- chain triglycerides. Aquaculture 190, 65-76.

• SAS institute Inc. (1990) SAS/STAT User's Guide. Version 6. 4th ed. SAS Institute Inc., Cary, NC 956 pp.

• Staurnes M., Sigholt T. & Reite O. B. (1992) Reproduksjon og livssyklus. In: K. Døving & E. Reimers, Fiskens Fysiologi pp. 277-328. John Grieg Forlag AS, Stavanger. ISBN 82-533-0268-1.

• Stoldt, W., 1952. Vorschlag zur Vereinheitlichung der Fettbestimmung in Lebensmitteln.

• Usher M. L., Talbot C. & Eddy F.B. (1991) Effects of transfer to seawater on growth and feeding in Atlantic salmon smolt (Salmo salaή. Aquaculture 94, 309-326.

Claims

1. Use of a feed comprising

(1) a compound represented by the general formula R"-COO-(CH₂)2n+i-X-R\ wherein X is a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group or a SO₂ group; n is an integer of 0 to 11 ; and R' is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, wherein the main chain of said R' contains from 13 to 23 carbon atoms and optionally one or more heterogroups selected from the group comprising an oxygen atom, a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group and a SO₂ group; and R" is a hydrogen atom or an alkyl group containing from 1 to 4 carbon atoms; or

(2) a compound represented by the general formula (I),

wherein R1 , R2, and R3 represent i) a hydrogen atom; or ii) a group having the formula CO-R in which R is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, and the main chain of said R contains from 1 to 25 carbon atoms; or iii) a group having the formula CO-(CH₂)_2n+i-X-R\ wherein X is a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group or a SO₂ group; n is an integer of 0 to 11 ; and R' is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, wherein the main chain of said R' contains from 13 to 23 carbon atoms and optionally one or more heterogroups selected from the group comprising an oxygen atom, a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group and a SO₂ group; iv) an entity selected from the group comprising -PO₃CH₂CHNH₃COOH (serine), PO₃CH₂CH₂NH₃ (ethanolamine), P0₃CH₂CH₂N(CH₃)₃ (choline), PO₃CH₂CHOHCH₂OH (glycerol) and PO₃(CHOH)₆ (inositol); wherein R1 , R2, and R3 are chosen independently from i), ii), iii), or iv), but at least one of R1 , R2, or R3 is defined by iii); or

(3) a compound represented by the general formula (II),

wherein A1 , A2 and A3 are chosen independently and represent an oxygen atom, a sulphur atom or an N-R4 group in which R4 is a hydrogen atom or a linear or branched alkyl group, saturated or unsaturated, optionally substituted, containing from 1 to 5 carbon atoms; wherein R1 , R2, and R3 represent i) a hydrogen atom or a linear or branched alkyl group, saturated or unsaturated, optionally substituted, containing from 1 to 23 carbon atoms; or ii) a group having the formula CO-R in which R is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, and the main chain of said R contains from 1 to 25 carbon atoms; or iii) a group having the formula CO-(CH₂)_2n+i-X-R', wherein X is a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group or a SO₂ group; n is an integer of 0 to 11 ; and R' is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, wherein the main chain of said R' contains from 13 to 23 carbon atoms and optionally one or more heterogroups selected from the group comprising an oxygen atom, a sulphur atom, a selenium atom, an oxygen atom, a CH₂ group, a SO group and a SO₂ group; iv) an entity selected from the group comprising -PO₃CH₂CHNH₃COOH (serine), PO₃CH₂CH₂NH₃ (ethanolamine), PO₃CH₂CH₂N(CHs)₃ (choline), PO₃CH₂CHOHCH₂OH (glycerol) and PO₃(CHOH)₆ (inositol); wherein R1 , R2, and R3 are chosen independently from i), ii), iii), or iv), but at least one of R1 , R2, or R3 is defined by iii); or

(4) a salt, prodrug or complex of said compound as defined by (1), (2), or (3)

for feeding fish during time periods where the requirements for energy of said fish is increased, in order to improve the health of said fish.

2. Use of a feed in accordance with claim 1 , where the increase in requirements for energy of said fish is seen by the reduction in body lipid/energy stores and/or as the need of energy intake above the baseline intake.

3. Use of a feed in accordance with claim 1 , where said fish comprise hatchery reared Atlantic salmon, Coho salmon, rainbow trout, brown trout and/or Cod.

4. Use of a feed in accordance with claim 1 , where the improvement of the health of said fish comprise reduced mortality.

5. Use of a feed in accordance with claim 1 , where the improvement of the health of said fish comprise reduced mortality from infections.

6. Use of a feed in accordance with claim 5, where the improvement of the health of said fish comprise reduced mortality from viral infections.

7. Use of a feed in accordance with claim 6, where said reduced mortality from virus infections is reduced mortality from IPN outbreaks.

8. Use of a feed in accordance with claim 6, where said IPN outbreaks occurs after sea transfer of smolt.

9. Use of a feed in accordance with claim 1 , where the time periods where the requirements for energy of said fish is increased as seen by reduction in body energy stores comprise

• the weeks after release of smolt from hatchery in the spring (1⁺), while the smolt is adapting to the new environment, and/or

• winters with water temperatures at 5⁰C or lower, and/or the first spring spent in the sea after fall release of smolt from hatchery in the autumn (O⁺).