METHOD OF IMPROVING MEAT PIGMENTATION IN AN AQUATIC ANIMAL AND ANIMAL FEED COMPOSITION

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to methods of improving meat pigmentation of a carotenoid in an aquatic animal using animal feed comprising vitamin D or a vitamin D metabolite, preferably 25- hydroxy vitamin D3 (25-OH D3).

Background of the invention

Color is an important characteristic of food. Since color is closely associated with expectations, the addition of color to food is a way for food producers to meet customer expectations. Some markets require high pigmentation levels of animal skin and/or meat. Especially for aquatic animals markets often demand highly coloured meat products, such as shrimps and fish meat. Salmon flesh, for example, is generally orange to red, although there are some examples of white- fleshed wild salmon. The natural color of salmon results from carotenoid pigments, largely astaxanthin and canthaxanthin in the flesh.

Carotenoids are widely used as a dietary supplement, feed supplements and food colorants for salmon, crabs, shrimp and other animal meat products and the cost of these carotenoids represents a large financial strain on the producers. Although carotenoids generally have good heat and pressure stability, they can be degraded by light, low pH, oxygen and enzymes. Therefore, storage conditions can be detrimental on pigment concentration, for example canthaxanthin. This has caused the need of increased quantities of pigments being added to the animal's diet to compensate for the degrading effects of processing and storage, increasing the financial strain on producers.

Therefore it is of great economic importance to supply a way to improve meat pigmentation of a carotenoid in an aquatic animal. Surprisingly, the inventors of the present invention discovered that vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), can be used in feed to improve meat pigmentation of a carotenoid in an aquatic animal. Such a solution is of great interest of aquaculture farmers. SUMMARY OF THE INVENTION

Accordingly, the present invention provides a method for improving meat pigmentation of a carotenoid in an aquatic animal comprising administering to the animal an animal feed or animal feed additive comprising vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3),

Brief description of the figures

Figure 1. Effect of Vitamin D3 and 25-OH D3 on pigmentation in the fillet of trouts; Figure 1 shows the increase in redeness of the fillet with increasing vitamin D. HyD achieves higher redness levels than D3.

DEFINITIONS

Aquatic Animal: The term “animal” or “aquatic animal” refers to all kinds of fish and shrimp, including but not limited to crustaceans, such as shrimps and prawns, and including but not limited to fish, such as amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sauger, sea bass, seabream, shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleye and whitefish.

Animal feed: The term “animal feed” refers to any compound, preparation, or mixture suitable for, or intended for intake by an animal. Animal feed for an aquatic animal typically comprises concentrates as well as vitamins, minerals, enzymes, direct fed microbial, amino acids and/or other feed ingredients (such as in a premix) whereas animal feed for ruminants generally comprises forage (including roughage and silage) and may further comprise concentrates as well as vitamins, minerals, enzymes direct fed microbial, amino acid and/or other feed ingredients (such as in a premix).

Feed Additive: The term feed additive according to the invention refers to a formulation comprising vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), as active ingredient intended for intake by an aquatic animal.

Feed Premix: The incorporation of the composition of feed additives as exemplified herein above to animal feeds, for example fish feeds, is in practice carried out using a concentrate or a premix. A premix designates a preferably uniform mixture of one or more microingredients with diluent and/or carrier. Premixes are used to facilitate uniform dispersion of micro-ingredients in a larger mix. A premix according to the invention can be added to feed ingredients as solids (for example as water soluble powder) or liquids.

Concentrates: The term “concentrates” means feed with high protein and energy concentrations, such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (either whole or prepared by crushing, milling, etc. from e.g. corn, oats, rye, barley, wheat), oilseed press cake (e.g. from cottonseed, safflower, sunflower, soybean (such as soybean meal), rapeseed/canola, peanut or groundnut), palm kernel cake, yeast derived material and distillers grains (such as wet distillers grains (WDS) and dried distillers grains with solubles (DDGS)).

Meat: The term “meat” according to the present invention refers to the flesh of an animal and is not limited to a mammal or bird, but includes the flesh of quatic animals.

Vitamin D: “Vitamin D” means either Vitamin D2, Vitamin D3 or a combination. Vitamin D3 is preferred.

Vitamin D metabolite: "Vitamin D metabolite" means any metabolite of Vitamin D as for example 25-hydroxy vitamin D3, 1 ,25-dihydroxy vitamin D3 or 24,25-dihydroxy vitamin D3.

25-OH D: “25-OH D” or “25-hydroxyvitamin D” refers to any form of 25-hydroxyvitamin D (i.e. either 25-OH D2 or 25-OH D3, or mixes thereof). 25-OH D3 specifically refers to 25-hydroxyvitamin D3 (referred to HyD and also known as calcifediol, calcidiol, 25-hydroxycholecalciferol or 25- hydroxyvitamin D ₃ ); 25-OH D2 specifically refers to 25-hydroxyvitamin D2. In a preferred embodiment, 25-hydroxyvitamin D is 25-hydroxyvitamin D3.

DETAILED DESCRIPTION OF THE INVENTION

Methods of improving meat pigmentation

It has been surprisingly found that supplementing an animal feed with vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), results in a significant benefit of improving meat pigmentation of a carotenoid in an aquatic animal, compared to an animal feed without the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3),.

Thus, the invention relates to a method of improving skin pigmentation of a carotenoid in an aquatic animal comprising administering to the animal an animal feed or animal feed additive comprising vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3),.

In the present invention, the improvement is compared to an animal feed or animal feed additive wherein the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), is not present (herein referred to as the negative control) or present in standard levels (DSM’s Optimum Vitamin Nutrition guidelines).

Preferably, the one or more parameters such as redness in the fillet of fish is increased by at least 0.1 %, such as by at leas 0.5%, at least 1.0%, at least 1.5%, at least 2.0%, at least 2.5%, at least 3.0%, at least 3.5%, at least 4.0%, at least 4.5%, at least 5.0%, at least 5.5%, at least 6.0%, at least 6.5%, at least 7.0%, at least 7.5% or at least 8.0% compared to fish fed a diet with a standard level of vitamin D or a vitamin D metabolite.

In the present invention, the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), may be dosed at a range from 50ug to 1500ug per kg feed, preferably 100ug to 1500ug per kg feed, preferably 150ug to 1500ug per kg feed, more preferably 150ug to 1250ug per kg feed, more preferably 150ug to 1000ug per kg feed.

In another embodyment, the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), may be dosed at a level of at least 80, preferably 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 mg per kg feed.

In another embodyment, the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), may be dosed at a level of 80-1000, 150-1000, 200-1000, 250-1000, 300- 1000, 350-1000, 400-1000, 450-1000 500-1000, 550-1000, 600-1000, 650-1000, 700-1000, 750- 1000 or 800-1000 mg per kg feed.

In the present invention, the animal is an aquatic animal. The aquatic animal may be selected from the group consisting of crustaceans, shrimps, prawns, fish, amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sauger, sea bass, seabream, shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleye and whitefish.

In the present invention, the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), may be fed to the animal from birth until slaughter. Preferably, the the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), is fed to the animal on a daily basis from birth until slaughter. More Preferably, the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), is fed to the animal on a daily basis for at least 10 days, such as at least 15 days or at least 20 days (where the days can be continuous or non- continuous) during the life span of the animal. Further preferably, the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), is fed to the animal for 10-20 days followed by a non-treatment period of 5-10 days, and this cycle is repeated during the life span of the animal.

In the present invention, the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), may be fed to aquatic animals during life span of the animal. The vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), can be fed to all types of fish, including cold-water fish and shrimp. Some examples are turbot, halibut, yellow tail salmon, trout, bream, bass and tuna. The feed is particularly suitable for feeding salmonids, including Atlantic salmon (Salmo salar), other salmon species and trout, and non-salmonids such as cod, sea bass, sea bream and eel. It is suitable for feeding salmon, trout, bream and/or bass in the fresh water (FW) phase and the in the sea water (SW) phase and in the period after hatching and until slaughter and in all stages, such as fry, fingerlings, parr, smolts and adult fish.

In the present invention, examples of the carotenoid include but are not limited to betacarotene, canthaxanthin, astaxanthin, lutein and mixture thereof.

Accordingly, the invention also relates to a method of improving meat pigmentation of an aquatic animal comprising administering to the animal an animal feed or animal feed additive comprising vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), and one or more carotenoids such as beta-carotene, canthaxanthin, astaxanthin, lutein and mixture thereof.

Formulation

The vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), of the present invention may be formulated as a composition for improving meat pigmentation of a carotenoid in an aquatic animal, which is also the present invention intents to cover. The vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), of the present invention may be formulated as a liquid or a solid.

For a solid formulation, the composition of the present invention may be for example a granule, powder or agglomerate. The formulating agent may comprise a salt (organic or inorganic zinc, sodium, potassium or calcium salts such as e.g. such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or a sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol). The formulating agent may comprise, for example, 40-50 % by weight of wood fibers, 8-10 % by weight of stearin, 4-5 % by weight of curcuma powder, 4-5 % by weight of rosemary powder, 22-28 % by weight of limestone, 1-3 % by weight of a gum, such as gum Arabic, 5-50 % by weight of sugar and/or starch and 5-15 % by weight of water.

For a liquid formulation, the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), embedded in an oil carrier. The oil carrier may comprise an oil is selected from the group consisting of fish oil, microbial oil and/or one or more vegetable oil(s) (such as e.g. rape seed oil, soy oil and camelina oil). In another aspect, the oil may be a source of eicosapentaenoic acid ("EPA") and/or docosahexaenoic acid ("DHA"). Thus the composition of the present invention may a liquid composition comprising the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), of the present invention and one or more formulating agents. The liquid formulation may be sprayed onto the feed after it has been pelleted or may be added to drinking water given to the animals.

Feed or Aquaculture feed or Animal Feed or Animal Feed Additives

The term “Feed” or “Aquaculture feed” or “aquatic feed” or “animal feed” refers to any compound, preparation, or mixture suitable for, or intended for intake by aquatic animals and decapod crustacean.

The vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), of the present invention may also be formulated as animal feed or animal feed additive for improving meat pigmentation of a carotenoid in an animal, which is also the present invention intents to cover.

Animal feed compositions or diets have a relatively high content of protein. Fish diets can be characterised as indicated in column 4 of Table B of WO 2001/058275. Furthermore, such fish diets usually have a crude fat content of 200-310 g/kg and can be characterized by a pellet size (diameter) in a range from 0.5 to 16 mm pellet size (diameter) roughly.

An animal feed composition according to the present invention may have a crude protein content of between 50 and 800 g/kg, and furthermore comprises one or more vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3),s as described herein. Furthermore, or in the alternative (to the crude protein content indicated above), the animal feed composition of the present invention may have a content of metabolisable energy of 10-30 MJ/kg; and/or a content of calcium of 0.1-200 g/kg; and/or a content of available phosphorus of 0.1- 200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or a content of methionine plus cysteine of 0.1-150 g/kg; and/or a content of lysine of 0.5-50 g/kg.

Particularly, the content of metabolisable energy, crude protein, calcium, phosphorus, methionine, methionine plus cysteine, and/or lysine may be within any one of ranges 2, 3, 4 or 5 in Table B of WO 2001/058275 (R. 2-5). Metabolisable energy can be calculated on the basis of the NRC publication Nutrient requirements.

The nitrogen content is determined by the Kjeldahl method (A.O.A.C., 1984, Official Methods of Analysis 14th ed., Association of Official Analytical Chemists, Washington DC) and crude protein is calculated as nitrogen (N) multiplied by a factor 6.25 (i.e. Crude protein (g/kg)= N (g/kg) x 6.25).

The dietary content of calcium, available phosphorus and amino acids in complete animal diets is calculated on the basis of feed tables such as Veevoedertabel 1997, gegevens over chemische samenstelling, verteerbaarheid en voederwaarde van voedermiddelen, Central Veevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

The animal feed composition of the present invention may contain at least one vegetable protein as defined above.

The animal feed composition of the present invention may also contain animal protein, such as Meat and Bone Meal, Feather meal, and/or Fish Meal, typically in an amount of 0-25%. The animal feed composition of the present invention may also comprise Dried Distillers Grains with Solubles (DDGS), typically in amounts of 0-30%.

Preferaly, the animal feed composition of the present invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybean meal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or 0-20% whey.

Preferably, the animal feed of the present invention comprises vegetable proteins. The protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (w/w).

In the present invention, the vegetable proteins may be derived from vegetable protein sources, such as legumes and cereals, for example, materials from plants of the families Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy bean meal, lupin meal, rapeseed meal, and combinations thereof. The vegetable protein source may be material from one or more plants of the family Fabaceae, e.g., soybean, lupine, pea, or bean. The vegetable protein source may also be material from one or more plants of the family Chenopodiaceae, e.g. beet, sugar beet, spinach or quinoa. Other examples of vegetable protein sources are rapeseed, and cabbage. Soybean is a preferred vegetable protein source. Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, and sorghum.

Diets are usually manufactured as pelleted or extruded feed. Typically, the milled feed-stuffs are mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications for the species in question. Vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), can be added as a solid or liquid formulation. For example, for pelleted feed the (liquid or solid) preparation may also be added before or during the feed ingredient step. Typically, a liquid preparation comprises the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), of the present invention optionally with an oil carrier, and is added after the pelleting or extruding step, such as by spraying the liquid formulation onto the pellets. The vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), may also be incorporated in a feed additive or premix.

In the present invention, the animal feed composition may further comprise one or more additional enzymes, microbes, vitamins, minerals, amino acids, and/or other feed ingredients.

Preferably, the composition comprises vitamin D or a vitamin D metabolite, preferably 25- hydroxy vitamin D3 (25-OH D3),s of the present invention, one or more formulating agents and one or more components selected from the list consisting of: one or more additional enzymes; one or more microbes; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients.

The final concentration of vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), in the animal feed compositon of the present invention may be within the range of 80 mg - 1000 mg per kg animal feed, such as 200 to 900 mg, 300 to 800 mg, 400 to 800 mg, 400 to 1000 mg, 500 to 600 mg per kg animal feed, or any combination of these intervals.

In another embodyment, the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), may be dosed at a level of at least 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 mg per kg feed.

In another embodyment, the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), may be dosed at a level of 80-1000, 200-1000, 250-1000, 300-1000, 350- 1000, 400-1000, 450-1000 500-1000, 550-1000, 600-1000, 650-1000, 700-1000, 750-1000, 800-

1000, 850-1000, 900-1000, or 950-1000 mg per kg feed.

Premix

In the present invention, the composition, animal feed or animal feed additive may include a premix, comprising e.g. vitamins, minerals, enzymes, amino acids, preservatives, antibiotics, other feed ingredients or any combination thereof which are mixed into the animal feed.

Amino Acids the composition, animal feed or animal feed additive of the present invention may further comprise one or more amino acids. Examples of the amino acids include but are not limited to lysine, alanine, beta-alanine, threonine, methionine and tryptophan.

Vitamins and Minerals

In the present invention, the composition, animal feed or animal feed additive may include one or more vitamins, such as one or more fat-soluble vitamins and/or one or more water-soluble vitamins. Optionally, the composition, animal feed or animal feed additive of the present invention may include one or more minerals, such as one or more trace minerals and/or one or more macro minerals.

Usually fat- and water-soluble vitamins, as well as trace minerals form part of a so-called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed.

Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, e.g., vitamin K3.

Non-limiting examples of water-soluble vitamins include vitamin B12, biotin and choline, vitamin B1 , vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g., Ca-D-panthothenate.

Non-limiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium and zinc.

Non-limiting examples of macro minerals include calcium, magnesium, potassium and sodium.

The nutritional requirements of these components (exemplified with poultry and piglets/pigs) are listed in Table A of WO 2001/058275. Nutritional requirement means that these components should be provided in the diet in the concentrations indicated. In the alternative, the composition, animal feed or animal feed additive of the present invention comprises at least one of the individual components specified in Table A of WO 01/58275. At least one means either of, one or more of, one, or two, or three, or four and so forth up to all thirteen, or up to all fifteen individual components. More specifically, this at least one individual component is included in the composition, animal feed or animal feed additive of the present invention in such an amount as to provide an in-feed-concentration within the range indicated in column four, or column five, or column six of Table A.

Other feed ingredients the composition, animal feed or animal feed additive of the present invention may further comprise colouring agents, growth improving additives and aroma compounds/flavourings, polyunsaturated fatty acids (PLIFAs); reactive oxygen generating species, anti-microbial peptides and anti-fungal polypeptides.

Examples of the colouring agents are carotenoid such as beta-carotene, canthaxanthin, astaxanthin, and lutein.

Examples of the stabilizing agents (e.g. acidifiers) are organic acids. Examples of these are benzoic acid (VevoVitall®, DSM Nutritional Products), formic acid, butyric acid, fumaric acid and propionic acid.

Examples of the aroma compounds/flavourings are creosol, anethol, deca-, undeca-and/or dodeca-lactones, ionones, irone, gingerol, piperidine, propylidene phatalide, butylidene phatalide, capsaicin and tannin.

Examples of the polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid.

Examples of the reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a syntethase.

Examples of the antimicrobial peptides (AMP’s) are CAP18, Leucocin A, Tritrpticin, Protegrin- 1 , Thanatin, Defensin, Lactoferrin, Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000), Plectasins, and Statins, including the compounds and polypeptides disclosed in WO 03/044049 and WO 03/048148, as well as variants or fragments of the above that retain antimicrobial activity. Examples of the antifungal polypeptides (AFP’s) are the Aspergillus giganteus, and Aspergillus niger peptides, as well as variants and fragments thereof which retain antifungal activity, as disclosed in WO 94/01459 and WO 02/090384.

Use of vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3),

In another aspect, the invention relates to the use of a composition, an animal feed or an animal feed additive for improving meat pigmentation of a carotenoid in an aquatic animal wherein the composition, the animal feed or the animal feed additive comprises vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3),.

In the present invention, the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), may be dosed at a level of 80-1000, 200-1000, 250-1000, 300-1000, 350- 1000, 400-1000, 450-1000 500-1000, 550-1000, 600-1000, 650-1000, 700-1000, 750-1000, 800- 1000, 850-1000, 900-1000, or 950-1000 mg per kg feed.

In another embodyment, the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), may be dosed at a level of at least 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 mg per kg feed.

In the present invention, the aquatic animal may be selected from the group consisting of fish or decapod crustacean. In a particular preferred embodiment of the present invention, the aquatic animal may be selected from the group consisting of crustaceans, shrimps, prawns, fish, amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sauger, sea bass, seabream, shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleye and whitefish.

The salmon may be of the family Salmonidae and of the subfamily of Salmoninae. In one embodiment the salmon is selected from the group consisting of the genus Salmo, Oncorhynchus and Salvenis. In a further embodiment the genus Salmo is selected from the group consisting of Atlantic salmon (Salmo salar) and Brown trout (Salmo trutta). In yet an embodiment the genus Oncorhynchus is selected from the group consisting of Chinook salmon (Oncorhynchus tshawytsch), Rainbow trout (Oncorhynchus mykiss), Sockeye salmon (Oncorhynchus nerka) and Coho salmon (Oncorhynchus kisutch). In a further embodiment the genus Salvenis is selected from the group consisting of Arctic charr (Salvelinus al pin us), Brook trout (Salvelinus fontinalis) and Lake trout (Salvelinus namaycush). The sea bream may be gilt-head sea bream (Sparus aurata) wheras the sea bass may be European bass (Dicentrarchus labrax)

In a preferred embodiment of the present invention the decapod crustacean may be shrimp or prawn. The shrimp may be selected from the group consisting of Pacific white shrimp (Penaeus vannamei or Litopenaeus vannamei), Whiteleg shrimp (Penaeus vannamei or Litopenaeus vannamei), Black tiger shrimp (Penaeus monodori), Kuruma shrimp (Penaeus japonicas or Marsupenaeus japonicas), Western blue shrimp (Penaeus stylirostris or Litopenaeus stylirostris), blue shrimp (Penaeus stylirostris or Litopenaeus stylirostris), Chinese white shrimp (Penaeus chinensis or Fenneropenaeus chinensis), Oriental shrimp (Penaeus chinensis or Fenneropenaeus chinensis), Indian white shrimp (Penaeus indicus or Fenneropenaeus indicus), Banana shrimp (Penaeus merguiensis or Fenneropenaeus merguiensis), Akiami paste shrimp (Metapenaeus spp.), yellowleg shrimp (Penaeus californiensis or Farfantepenaeus californiensis), brown shrimp (Penaeus californiensis or Farfantepenaeus californiensis), Sao Paulo shrimp (Penaeus paulensis or Farfantepenaeus paulensis), Carpas shrimp (Penaeus paulensis or Farfantepenaeus paulensis), redspotted shrimp (Penaeus brasiliensis or Farfantepenaeus brasiliensis), spotted pink shrimp (Penaeus brasiliensis or Farfantepenaeus brasiliensis)ar\d southern white shrimp (Penaeus schmitti). The prawn may be selected from the group consisting of King prawn (Penaeus vannamei or Litopenaeus vannamei), Giant tiger prawn (Penaeus monodon), Giant Freshwater Prawn (Macrobrachium rosenbergii), Giant river prawn (Macrobrachium rosenbergii), Malaysian prawn (Macrobrachium rosenbergii), Kuruma prawn (Penaeus japonicas or Marsupenaeus japonicas), Fleshy prawn (Penaeus chinensis or Fenneropenaeus chinensis), Indian prawn (Penaeus indicus or Fenneropenaeus indicus), Banana prawn (Penaeus merguiensis or Fenneropenaeus merguiensis), Oriental river prawn (Macrobrachium nipponense) and Monsoon river prawn (Macrobrachium malcolmsonii).

In the present invention, the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), may be fed to the animal from birth until slaughter. Preferably, the the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), is fed to the animal on a daily basis from birth until slaughter. More Preferably, the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), is fed to the animal on a daily basis for at least 10 days, such as at least 15 days or at least 20 days (where the days can be continuous or non- continuous) during the life span of the animal. In one embodiment, the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), is fed to the animal for 10-20 days followed by a non-treatment period of 5-10 days, and this cycle is repeated during the life span of the animal.

In the present invention, examples of the carotenoid include but are not limited to betacarotene, canthaxanthin, astaxanthin, lutein and mixture thereof.

Accordingly, the invention also to the use of a composition, an animal feed or an animal feed additive for improving meat pigmentation of an aquatic animal wherein the composition, the animal feed or the animal feed additive comprises one or more vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), and one or more carotenoids such as beta-carotene, canthaxanthin, astaxanthin, lutein and mixture thereof.

Said oil carrier can be fish oil, microbial oil and/or one or more vegetable oil(s). The vegetable oil can be selected from the group consisting of rape seed oil and soy oil. An example of a microbial oil according to the invention is an oil from Schizochytrium. Preferably, the oil is a source of eicosapentaenoic acid ("EPA") and/or docosahexaenoic acid ("DHA"). "Eicosapentaenoic acid" ["EPA"] is the common name for eis-5, 8, 11 ,14, 17-eicosapentaenoic acid. This fatty acid is a 20:5 omega-3 fatty acid. "Docosahexaenoic acid" ["DHA"] is the common name for eis-4, 7, 10, 13, 16, 19-docosahexaenoic acid. This fatty acid is a 22:6 omega-3 fatty acid. A Preferred EPA/ARA ratio is between 0.4 and 25.0 and a preferred DHA/EPA ratio is between 0.1 and 15.0.

The incorporation of the feed additive composition containing the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), into fish feed may be performed as described in example 1 and 2. The final concentration of vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), in the feed may be determined by HPLC according to standard methods.

The incorporation of the feed additive composition containing the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25-OH D3), into fish feed may alternatively be carried out by preparing a premix of the vitamin D or a vitamin D metabolite, preferably 25-hydroxy vitamin D3 (25- OH D3), and other suitable additives.

EXAMPLES

Example 1 : Preparation of Pressed Fish Feed The main raw materials are ground and mixed. Microingredients are then added to the mixer and the homogenous mix is conditioned by adding water and steam to the mass in a preconditioner. This starts a cooking process in the starch fraction (the binding component). The mass is fed into a pellet mill. The mass is forced through the mill's die and the strings are broken into pellets on the outside of the die. The moisture content is low and drying of the feed is not necessary.

Additional oil including a fish feed composition according to the present invention is then sprayed onto the surface of pellets, but as the pellets are rather compact, the total lipid content rarely exceeds 24 %. The added oil may be fish oil, microbial/algal or vegetable oils, for example rape seed oil or soy oil, or a mixture of oils. After oil coating, the pellets are cooled in a cooler and bagged. The final pressed fish feed contains 10 to 5000 ppm of the composition as described in the invention.

Example 2: Method for Preparation of Extruded Fish Feed

The main raw materials are ground and mixed. Micro ingredients incl. a fish feed composition according to the invention are added to the mixer. The homogenous mix is conditioned by adding water and steam to the mass in a preconditioner. Additional oil may also be added to the mass at this stage. This starts a cooking process in the starch fraction (the binding component). The mass is fed into an extruder. The extruder may be of the single screw or the twin-screw type. Due to the rotational movement of the mass in the extruder, the mass is further mixed. Additional oil, water and steam may be added to the mass in the extruder. At the end of the extruder, the mass has a temperature above 100 °C and a pressure above ambient pressure. The mass is forced through the openings in the extruder's die plate. Due to the relief in temperature and pressure, some of the moisture will evaporate immediately (flash off) and the extruded mass becomes porous. The strings are cut into pellets by a rotating knife. The water content is rather high (18-28 %) and the pellets are therefore immediately dried to approximately 10 % water content in a dryer.

After the dryer, more oil including a feed additive composition according to the invention may be added to the feed by spraying oil onto the surface of the feed, or by dipping the feed in oil. It is advantageous to add the oil to the feed in a closed vessel where the air pressure is below ambient (vacuum coating) so that the porous feed pellets absorb more oil. Feed containing more than 40 % lipid may be produced this way. After the coater, the feed is cooled and bagged. Oil may be added at several places in the process as explained above, and may be fish oil, microbial/algal or vegetable oils, by example rape seed oil or soy oil, or a mixture of oils. Fish need protein, fat, minerals and vitamins in order to grow and to be in good health. The diet of carnivorous fish is particularly important. Originally in the farming of carnivorous fish, whole fish or ground fish were used to meet the nutritional requirements of the farmed fish. Ground fish mixed with dry raw materials of various kinds, such as fish meal and starch, was termed soft or semimoist feed. As farming became industrialized, soft or semi-moist feed was replaced by pressed dry feed. This was itself gradually replaced by extruded dry feed.

Today, extruded feed is nearly universal in the farming of a number of fish species such as various types of salmonid, cod, sea bass and sea bream.

The dominant protein source in dry feed for fish has been fish meal of different qualities. Other animal protein sources are also used for dry fish feed. Thus, it is known to use blood meal, bone meal, feather meal and other types of meal produced from other slaughterhouse waste, for example chicken meal. These are typically cheaper than fish meal and fish oil. However, in some geographic regions, there has been a prohibition against using such raw materials in the production of feeds for food-producing animals and fish.

It is also known to use vegetable protein such as wheat gluten, maize (corn) gluten, soya protein, lupin meal, pea meal, bean meal, rape meal, sunflower meal and rice flour.

Example 3: Effect of Vitamin D3 and 25-OH D3 on pigmentation of Carophyll Pink in the fillet of trouts.

Material & Methods

An experiment has been conducted with 270 trouts. The trial was conducted during 8 weeks and has been designed with six treatments (see tablel) and 3 replicates of 15 fish.

Feeding

The diet given to the fish was a standard feed with the addition of the products that were tested. The feed met all nutritional requirements in relation to the developmental stage of the fish.

Vitamin D3 is added by inclusion of the commercial product Rovimix D3-500 D3. 25-OH D3 is added by inclusion of the commercial product Rovimix HyD. Astaxanthin is added by inclusion of the commercial product Carophyll Pink 10% CWS B-free.

TABLE 1. Treatments used in the experiment on trout.

* Vitamin and Mineral premix does not contain vitamin D3

** Rovimix D3-500 D3, Rovimix HyD and Carophyll pink 10% will be included at expenses of wheat in the mash (pre-extrusion) TABLE 2. Product inclusion level for 45 kg of feed

The 80, 400 and 800 in D3-80 aso. Refer to inclusion levels of 80, 400 and 800 ug respectively.

Experimental Design

After eight weeks of feeding fillet samples were taken. Redness of the fillet was measured digitally on the surface of the fish fillet with a chromameter according to the L*a*b* color space (also referred to as Cl ELAB) method. D3-80 corresponds to the Vitamin D levels in standard feed and can be used as a control. Results

Redeness of the fillet is increased with increasing vitamin D (see Figure 1). Significant improvements are observed for Vitamin D3 at D3-800. Significant improvements are observed for HyD at HyD-400 abd higher.

Example 4: Effect of Vitamin D3 and 25-OH D3 on pigmentation of Carophyll Pink in the fillet of trouts.

Material & Methods

An experiment has been conducted with 270 trouts. 6 test diets were used. The trial was conducted during 8 weeks and has been designed with six treatments (see table 3) and 3 replicates of 15 fish.

Feeding

The diet given to the fish was a standard feed with the addition of the products that were tested. The feed met all nutritional requirements in relation to the developmental stage of the fish. Vitamin D3 is added by inclusion of the commercial product Rovimix D3-500 D3. 25-OH D3 is added by inclusion of the commercial product Rovimix HyD. Astaxanthin is added by inclusion of the commercial product Carophyll Pink 10% CWS B-free.

TABLE 3. Treatments used in the experiment on trout.

* Vitamin and Mineral premix does not contain vitamin D3

** Rovimix D3-500 D3, Rovimix HyD and Carophyll pink 10% will be included at expenses of wheat in the mash (pre-extrusion) TABLE 4. Product inclusion level for 45 kg of feed

The 80, 400 and 800 in D3-80 aso. Refer to inclusion levels of 80, 400 and 800 ug respectively.

Experimental Design

After eight weeks of feeding fillet samples were taken. 10 fish were sampled from each tank, 30 fish per group. Fillets were removed and the standard ‘Norwegian Quality Cut’ measured in three locations for fillet colour (redness (a*) using a chromameter (CR-400, Minolta).

Redness of the fillet was measured digitally on the surface of the fish fillet with a chromameter according to the L*a*b* color space (also referred to as Cl ELAB) method. D3-80 corresponds to the Vitamin D levels in standard feed and can be used as a control. Results

Redeness of the fillet is increased with increasing vitamin D (see Figure 2). Significant improvements are observed for increased levels of Vitamin D3. Significant improvements are observed for increased levels of HyD. Example 5: Effect of Vitamin D3 and 25-OH D3 on pigmentation of Carophyll Pink in the fillet of trouts.

Material & Methods

An experiment has been conducted with 480 trouts. 6 test diets were used. The trial was conducted during 12 weeks and has been designed with six treatments (see table 5) and 4 replicates of 20 fish. Feeding

The diet given to the fish was a standard feed with the addition of the products that were tested. The feed met all nutritional requirements in relation to the developmental stage of the fish.

Vitamin D3 is added by inclusion of the commercial product Rovimix D3-500 D3. 25-OH D3 is added by inclusion of the commercial product Rovimix HyD. Astaxanthin is added by inclusion of the commercial product Carophyll Pink 10% CWS B-free.

TABLE 5. Treatments used in the experiment on trout.

* Vitamin and Mineral premix does not contain vitamin D3

** Rovimix D3-500 D3, Rovimix HyD and Carophyll pink 10% will be included at expenses of wheat in the mash (pre-extrusion)

TABLE 6. Product inclusion level for 45 kg of feed

The 80, 400 and 800 in D3-80 aso. Refer to inclusion levels of 80, 400 and 800 ug respectively.

Experimental Design

After eight weeks of feeding fillet samples were taken. 10 fish were sampled from each tank, 40 fish per group. Fillets were removed and the standard ‘Norwegian Quality Cut’ measured in three locations for fillet colour (redness (a*) using a chromameter (Minolta). Redness of the fillet was measured digitally on the surface of the fish fillet with a chromameter according to the L*a*b* color space (also referred to as Cl ELAB) method. D3-80 corresponds to the Vitamin D levels in standard feed and can be used as a control.

Results Redeness of the fillet is increased with increasing vitamin D (see Figure 3). Significant improvements are observed for increased levels of Vitamin D3. Significant improvements are observed for increased levels of HyD.