Methods and animal feed compositions for improving meat pigmentation in aquatic animals

Abstract

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

Description

Background of the Invention Technical Field

[0002] The present invention relates to a method for improving carotenoid meat pigmentation in aquatic animals using an animal feed comprising vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OHD3). Background Art

[0003] Color is an important property of food. Since color is closely related to expectations, adding color to food is one way for food manufacturers to meet customer expectations. Some markets require animal skin and / or flesh with high pigmentation levels. Especially for aquatic animals, the market often requires highly pigmented meat products, such as shrimp and fish. For example, salmon meat is typically orange to red, although some examples of white-fleshed wild salmon exist. The natural color of salmon comes from carotenoid pigments, primarily astaxanthin and canthaxanthin, which are present in the fish meat.

[0004] Carotenoids are widely used as dietary supplements, feed supplements and food colorants for salmon, crabs, shrimps and other animal meat products, and the cost of these carotenoids represents a huge economic pressure for producers. Although carotenoids generally have good heat and pressure stability, they can be degraded by light, low pH, oxygen and enzymes. Therefore, storage conditions may be harmful to pigment concentrations (such as canthaxanthin). This has led to the need to increase the amount of the pigment added to the animal diet, to compensate for the degradation effects of processing and storage, thereby increasing the economic pressure of producers.

[0005] Therefore, providing a way to improve carotenoid meat pigmentation in aquatic animals is of great economic significance. Surprisingly, the inventors of the present invention have found that vitamin D or vitamin D metabolites, preferably 25-hydroxyvitamin D3 (25-OH D3), can be used in feed to improve carotenoid meat pigmentation in aquatic animals. This solution has aroused great interest among aquaculture farmers. Summary of the invention

[0006] Accordingly, the present invention provides a method for improving carotenoid meat pigmentation in an aquatic animal, the method comprising administering to the animal an animal feed or an animal feed additive comprising vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3). BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1. Effect of Vitamin D3 and 25-OH D3 on pigmentation in trout fillets: Figure 1 shows that the redness of the fillets increased with increasing Vitamin D. HyD achieved higher redness levels than D3.

[0008] definition

[0009] Aquatic Animals: The term "animal" or "aquatic animals" refers to all species of fish and shrimp, including but not limited to crustaceans such as shrimp and prawns; and includes but is not limited to fish such as bluegill, arapaima, barbel, sea bass, flathead, bocachico, bream, bullhead, giant pacu, carp, catfish, catlarva, milkfish, char, cichlid, cobia, cod, crappie, gilthead sea bream, drum, eel, goby, goldfish, threadfin, grouper, guapote, halibut fish, java, rohu, wrasse, loach, mackerel, milkfish, silver perch, mudfish, mullet, paco, pearlspot, silverside, perch, pike, pomfret, roach, salmon, long-fin catfish, bigeye amberjack, sea bass, sea bream, shiner, sleeper, snakehead, bream, snook, sole, bluefish, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, white trout, glass pike perch, and whitefish.

[0010] Animal feed: The term "animal feed" means any compound, preparation or mixture suitable or intended for ingestion by an animal. Animal feed for aquatic animals typically comprises concentrates as well as vitamins, minerals, enzymes, direct-fed microorganisms, amino acids and / or other feed ingredients (e.g. in a premix), while animal feed for ruminants typically comprises forage (including roughage and silage) and may also comprise concentrates as well as vitamins, minerals, enzymes, direct-fed microorganisms, amino acids and / or other feed ingredients (e.g. in a premix).

[0011] Feed additive: The term "feed additive" according to the present invention refers to a preparation comprising vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3), as active ingredient intended for ingestion by aquatic animals.

[0012] Feed premix: The incorporation of a feed additive composition as exemplified above into an animal feed, e.g. fish feed, is in practice carried out using a concentrate or a premix. A premix represents a preferably homogeneous mixture of one or more microingredients with a diluent and / or a carrier. A premix serves to facilitate the uniform dispersion of the microingredients in a larger mixture. The premix according to the invention can be added to the feed ingredients as a solid (e.g. as a water-soluble powder) or as a liquid.

[0013] Concentrates: The term "concentrates" means feeds with high protein and energy concentrations, such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (whole corn, oats, rye, barley, wheat, or made from, for example, corn, oats, rye, barley, wheat by crushing, milling, etc.), oilseed press cakes (e.g., from cottonseed, safflower, sunflower, soybean (e.g., soybean meal), rapeseed / canola, peanuts), palm kernel cake, yeast-derived materials, and distillers grains (e.g., wet distillers grains (WDS) and dried distillers grains with solubles (DDGS)).

[0014] Meat: The term "meat" according to the present invention refers to the meat of animals, and is not limited to mammals or birds, but includes the meat of aquatic animals.

[0015] Vitamin D: "Vitamin D" refers to vitamin D2, vitamin D3 or a combination. Vitamin D3 is preferred.

[0016] Vitamin D metabolite: "Vitamin D metabolite" means any metabolite of vitamin D, such as, for example, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3 or 24,25-dihydroxyvitamin D3.

[0017] 25-OH D: "25-OH D" or "25-hydroxyvitamin D" refers to any form of 25-hydroxyvitamin D (i.e., 25-OH D2 or 25-OH D3, or a mixture thereof). 25-OH D3 specifically refers to 25-hydroxyvitamin D3 (referred to as HyD, and also referred to as calcifediol, calcidiol, 25-hydroxycholecalciferol, or 25-hydroxyvitamin D3); 25-OH D2 specifically refers to 25-hydroxyvitamin D2. In a preferred embodiment, 25-hydroxyvitamin D is 25-hydroxyvitamin D3. DETAILED DESCRIPTION

[0018] Methods to improve meat pigmentation

[0019] It has surprisingly been found that supplementation of animal feed with vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3), results in significant benefits in improving carotenoid meat pigmentation in aquatic animals compared to animal feed without vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3).

[0020] Therefore, the present invention relates to a method for improving carotenoid skin pigmentation in an aquatic animal, said method comprising administering to said animal an animal feed or an animal feed additive comprising vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OHD3).

[0021] In the present invention, the improvement is compared to an animal feed or animal feed additive in which vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3), is absent (referred to herein as a negative control) or is present at standard levels (DSM's Optimum Vitamin Nutrition guidelines).

[0022] Preferably, one or more parameters, e.g. redness in the fillet of the fish, is increased by at least 0.1%, e.g. at least 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.

[0023] In the present invention, vitamin D or vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3), can be added in a dosage range of 50ug to 1500ug per kg of feed, preferably 100ug to 1500ug per kg of feed, preferably 150ug to 1500ug per kg of feed, more preferably 150ug to 1250ug per kg of feed, more preferably 150ug to 1000ug per kg of feed.

[0024] In another embodiment, vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OHD3) may be dosed at a level of at least 80 mg, preferably 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg per kg of feed.

[0025] In another embodiment, vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OHD3), may be dosed at a level of 80-1000 mg, 150-1000 mg, 200-1000 mg, 250-1000 mg, 300-1000 mg, 350-1000 mg, 400-1000 mg, 450-1000 500-1000 mg, 550-1000 mg, 600-1000 mg, 650-1000 mg, 700-1000 mg, 750-1000 mg or 800-1000 mg per kg of feed.

[0026] In the present invention, the animal is an aquatic animal. The aquatic animal can be selected from the group consisting of crustaceans, prawns, shrimps, fish, green snapper, arapaima, barbel, sea bass, flat trevally, bocachico, sea bream, bighead carp, bighead carp, carp, catfish, catla barb, milkfish, char, cichlid, cobia, cod, crappie, gilthead sea bream, drum, eel, goby, goldfish, threadfin, grouper, guapote, halibut, java, rohu, Wrasse, loach, mackerel, milkfish, silver perch, mudfish, mullet, paco, pearlspot, silverside, perch, pike, pomfret, roach, salmon, long-finned catfish, bigeye amberjack, sea bass, sea bream, shiner, sleeper, snakehead, bream, snook, sole, bluefish, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, white trout, glass pike and whitefish.

[0027] In the present invention, vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3), can be fed to animals from birth until slaughter. Preferably, vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3) is fed to animals every day from birth to slaughter. More preferably, vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3), is fed to the animal every day for at least 10 days, such as at least 15 days or at least 20 days during the life of the animal (wherein the number of days may be continuous or discontinuous). Further preferably, vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin 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 of the animal.

[0028] In the present invention, vitamin D or vitamin D metabolites, preferably 25-hydroxyvitamin D3 (25-OH D3) can be fed to the aquatic animal during its lifespan. Vitamin D or vitamin D metabolites, preferably 25-hydroxyvitamin D3 (25-OH D3), can be fed to all types of fish, including cold water fish and shrimp. Some examples are turbot, halibut, yellowtail salmon, trout, bream, sea bass and tuna. The feed is particularly suitable for feeding salmonids, including Atlantic salmon (Ontario salmon), 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 sea bass in the freshwater (FW) stage and the seawater (SW) stage and in the period from hatching to slaughter and in all stages (such as fry, fingerlings, young salmon, smolt and adult fish).

[0029] In the present invention, examples of carotenoids include, but are not limited to, β-carotene, canthaxanthin, astaxanthin, lutein, and mixtures thereof.

[0030] Therefore, the present invention also relates to a method for improving meat pigmentation of aquatic animals, the method comprising administering to the animal an animal feed or an animal feed additive, the animal feed or animal feed additive comprising vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3), and one or more carotenoids, such as β-carotene, canthaxanthin, astaxanthin, lutein and mixtures thereof.

[0031] preparation

[0032] Vitamin D or vitamin D metabolites of the present invention, preferably 25-hydroxyvitamin D3 (25-OH D3) can be formulated into a composition for improving carotenoid meat pigmentation in aquatic animals, which composition is also intended to be covered by the present invention. Vitamin D or vitamin D metabolites of the present invention, preferably 25-hydroxyvitamin D3 (25-OH D3) can be formulated into a liquid or a solid.

[0033] For solid preparations, the composition of the present invention can be, for example, granules, powders or agglomerates. The formulation reagents can include salts (organic or inorganic zinc, sodium, potassium or calcium salts, 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 sugar or sugar derivatives (such as sucrose, dextrin, glucose, lactose, sorbitol). The formulation agent may include, for example, 40-50 wt% wood fiber, 8-10 wt% stearin, 4-5 wt% turmeric powder, 4-5 wt% rosemary powder, 22-28 wt% limestone, 1-3 wt% gum (e.g., gum arabic), 5-50 wt% sugar and / or starch, and 5-15 wt% water.

[0034] For liquid formulations, the vitamin D or vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OHD3), is embedded in an oil carrier. The oil carrier may comprise an oil selected from the group consisting of fish oil, microbial lipids and / or one or more vegetable oils (e.g., rapeseed oil, soybean oil, and camelina seed oil). In another aspect, the oil may be a source of eicosapentaenoic acid ("EPA") and / or docosahexaenoic acid ("DHA"). Therefore, the composition of the present invention may be a liquid composition comprising vitamin D or a vitamin D metabolite of the present invention, preferably 25-hydroxyvitamin D3 (25-OH D3), and one or more formulation agents. The liquid formulation may be sprayed onto the feed after it has been granulated, or may be added to the drinking water given to the animal.

[0035] Feed or aquaculture feed or animal feed or animal feed additives

[0036] The term "feed" or "aquaculture feed" or "aquafeed" or "animal feed" means any compound, preparation or mixture suitable or intended for ingestion by aquatic animals and decapod crustaceans.

[0037] The vitamin D or vitamin D metabolite of the present invention, preferably 25-hydroxyvitamin D3 (25-OH D3), can also be formulated into animal feed or animal feed additives for improving carotenoid meat pigmentation in animals, which are also intended to be covered by the present invention.

[0038] The animal feed composition or diet has a relatively high protein content. The fish diet may be characterized as shown in column 4 of Table B of WO 2001 / 058275. In addition, such fish diets typically have a crude fat content of 200-310 g / kg and may be characterized by a pellet size (diameter) in the range of about 0.5 mm to 16 mm pellet size (diameter).

[0039] The animal feed composition according to the present invention may have a crude protein content of between 50 g / kg and 800 g / kg and further comprise one or more vitamin D or vitamin D metabolites, preferably 25-hydroxyvitamin D3 (25-OH D3), as described herein.

[0040] In addition, or as an alternative (relative to the crude protein content mentioned above), the animal feed composition of the present invention may have a metabolizable energy content of 10-30 MJ / kg; and / or a calcium content of 0.1-200 g / kg; and / or an available phosphorus content of 0.1-200 g / kg; and / or a methionine content of 0.1-100 g / kg; and / or a methionine plus cysteine ​​content of 0.1-150 g / kg; and / or a lysine content of 0.5-50 g / kg.

[0041] Specifically, the content of metabolizable energy, crude protein, calcium, phosphorus, methionine, methionine plus cysteine ​​and / or lysine may be within any of ranges 2, 3, 4 or 5 (range 2-5) in Table B of WO 2001 / 058275. Metabolizable energy may be calculated based on the NRC publication "Nutrient requirements".

[0042] The nitrogen content was determined by the Kjeldahl method (AOAC, 1984, Official Methods of Analysis, 14th Edition, Association of Official Analytical Chemists, Washington DC), and the crude protein was calculated as nitrogen (N) multiplied by a factor of 6.25 (ie, crude protein (g / kg) = N (g / kg) x 6.25).

[0043] The dietary content of calcium, available phosphorus and amino acids in fully fed animal diets is calculated based on feed tables, e.g. Veevoedertabel 1997, gegevens over chemische samenstelling, verteerbaarheid envoederwaarde van voedermiddelen, Central Veevoederbureau, Runderweg 6, 8219pk Lelystad. ISBN 90-72839-13-7.

[0044] The animal feed composition of the invention may contain at least one vegetable protein as defined above.

[0045] 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 contain distillers dried grains with solubles (DDGS), typically in an amount of 0-30%.

[0046] Preferably, the animal feed composition of the present invention contains 0-80% corn; 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.

[0047] Preferably, the animal feed of the present invention comprises vegetable protein. The protein content of the vegetable protein is at least 10% (w / w), 20% (w / w), 30% (w / w), 40% (w / w), 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w) or 90% (w / w).

[0048] In the present invention, the vegetable protein can be derived from vegetable protein sources such as legumes and cereals, for example, materials from the Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae and Poaceae, such as soy flour, lupin flour, rapeseed flour, and combinations thereof.

[0049] The vegetable protein source can be material from one or more plants of the Leguminosae family, such as soybeans, lupines, peas or beans. The vegetable protein source can also be material from one or more plants of the Chenopodiaceae family, such as beets, sugar beets, spinach or quinoa. Other examples of vegetable protein sources are rapeseed and cabbage. Soybeans are a preferred vegetable protein source. Other examples of vegetable protein sources are cereals, such as barley, wheat, rye, oats, maize (corn), rice and sorghum.

[0050] The diet is usually prepared as a pelleted feed or an extruded feed. Typically, the ground feed ingredients are mixed and sufficient amounts of necessary vitamins and minerals are added according to the instructions for the species in question. Vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3) can be added as a solid or liquid preparation. For example, for pelleted feeds, the (liquid or solid) preparation can also be added before or during the feed ingredient step. Typically, the liquid preparation comprises vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3) of the present invention, optionally with an oil carrier, and is added after the granulation or extrusion step, for example by spraying the liquid preparation onto the pellets. The vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3) can also be incorporated into a feed additive or premix.

[0051] In the present invention, the animal feed composition may further comprise one or more additional enzymes, microorganisms, vitamins, minerals, amino acids and / or other feed ingredients.

[0052] Preferably, the composition comprises vitamin D or a vitamin D metabolite of the present invention, preferably 25-hydroxyvitamin D3 (25-OH D3), one or more formulation agents and one or more components selected from the list consisting of: one or more additional enzymes; one or more microorganisms; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients.

[0053] The final concentration of vitamin D or vitamin D metabolites, preferably 25-hydroxyvitamin D3 (25-OH D3), in the animal feed composition of the present invention may be in the range of 80 mg-1000 mg per kg of animal feed, for example 200 mg to 900 mg, 300 mg to 800 mg, 400 mg to 800 mg, 400 mg to 1000 mg, 500 mg to 600 mg per kg of animal feed, or any combination of these intervals.

[0054] In another embodiment, vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OHD3) may be dosed at a level of at least 80 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg per kg of feed.

[0055] In another embodiment, vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OHD3), may be dosed at a level of 80-1000 mg, 200-1000 mg, 250-1000 mg, 300-1000 mg, 350-1000 mg, 400-1000 mg, 450-1000 500-1000 mg, 550-1000 mg, 600-1000 mg, 650-1000 mg, 700-1000 mg, 750-1000 mg, 800-1000 mg, 850-1000 mg, 900-1000 mg, or 950-1000 mg per kg of feed.

[0056] Premix

[0057] In the present invention, the composition, animal feed or animal feed additive may comprise a premix comprising, for example, vitamins, minerals, enzymes, amino acids, preservatives, antibiotics, other feed ingredients or any combination thereof that is mixed into the animal feed.

[0058] Amino Acids

[0059] The composition, animal feed or animal feed additive of the present invention may further comprise one or more amino acids. Examples of amino acids include, but are not limited to, lysine, alanine, beta-alanine, threonine, methionine and tryptophan.

[0060] Vitamins and minerals

[0061] In the present invention, the composition, animal feed or animal feed additive may comprise 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 comprise one or more minerals, such as one or more trace minerals and / or one or more macro minerals.

[0062] Typically fat-soluble and water-soluble vitamins and trace minerals form part of a so-called premix intended for addition to feed, whereas macrominerals are usually added to the feed separately.

[0063] Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, such as vitamin K3.

[0064] Non-limiting examples of water-soluble vitamins include vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid, and pantothenate, such as calcium D-pantothenate.

[0065] Non-limiting examples of trace minerals include boron, cobalt, chlorine, chromium, copper, fluorine, iodine, iron, manganese, molybdenum, selenium, and zinc.

[0066] Non-limiting examples of macrominerals include calcium, magnesium, potassium, and sodium.

[0067] The nutritional requirements of these components (exemplified by poultry and piglets / pigs) are listed in Table A of WO 2001 / 058275. The nutritional requirements mean that these components should be provided in the diet at the concentrations specified.

[0068] Alternatively, 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 refers to one or more of the following: one or two, three, or four, etc. up to all thirteen, or up to all fifteen individual components. More specifically, this at least one individual component is contained in the composition, animal feed or animal feed additive of the present invention in such an amount as to provide an intra-feed concentration within the ranges shown in the fourth, fifth or sixth column of Table A.

[0069] Other feed ingredients

[0070] The composition, animal feed or animal feed additive of the present invention may further comprise colorants, growth promoting additives and aromatic compounds / flavorings, polyunsaturated fatty acids (PUFA); reactive oxygen species generating substances, antimicrobial peptides and antifungal polypeptides.

[0071] Examples of colorants are carotenoids, such as beta-carotene, canthaxanthin, astaxanthin and lutein.

[0072] Examples of stabilizers (e.g., acidulants) are organic acids. Examples of these are benzoic acid ( DSM Nutritional Products), formic acid, butyric acid, fumaric acid and propionic acid.

[0073] Examples of aromatic compounds / flavoring agents are cresols, anethole, decalactone, undecalactone and / or dodecalactone, ionones, irones, gingerol, piperidine, propylidene phatalide, butylidene phatalide, capsaicin and tannins.

[0074] Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid.

[0075] Examples of active oxygen-generating substances are chemical substances such as perborate, persulfate or percarbonate; and enzymes such as oxidase, oxygenase or synthetase.

[0076] Examples of antimicrobial peptides (AMPs) are CAP18, Lincomycin A, Tributyrin, Protegrin-1, Thanatin, Defensins, Lactoferrin, Lactoferrin and Ovispirin (e.g. Novispirin) (Robert Lehrer, 2000), Plectasin and Statins, including compounds and polypeptides disclosed in WO 03 / 044049 and WO 03 / 048148, and variants or fragments thereof that retain antimicrobial activity.

[0077] Examples of antifungal polypeptides (AFPs) are Aspergillus giganteus peptides and Aspergillus niger peptides, and variants and fragments thereof which retain antifungal activity, as disclosed in WO 94 / 01459 and WO 02 / 090384.

[0078] Use of vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3)

[0079] In another aspect, the present invention relates to the use of a composition, an animal feed or an animal feed additive for improving carotenoid meat pigmentation in aquatic animals, wherein the composition, animal feed or animal feed additive comprises vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3).

[0080] In the present invention, vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3), may be dosed at a level of 80-1000 mg, 200-1000 mg, 250-1000 mg, 300-1000 mg, 350-1000 mg, 400-1000 mg, 450-1000 500-1000 mg, 550-1000 mg, 600-1000 mg, 650-1000 mg, 700-1000 mg, 750-1000 mg, 800-1000 mg, 850-1000 mg, 900-1000 mg, or 950-1000 mg per kg of feed.

[0081] In another embodiment, vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OHD3) may be dosed at a level of at least 80 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg per kg of feed.

[0082] In the present invention, the aquatic animal can be selected from the group consisting of: fish or decapod crustaceans. In a particularly preferred embodiment of the present invention, the aquatic animal can be selected from the group consisting of: crustaceans, prawns, shrimps, fish, green snapper, arapaima, barbel, perch, flat trevally, bocachico, porgy, bighead carp, bighead carp, carp, catfish, catla barb, milkfish, char, cichlid, cobia, cod, crappie, gilthead sea bream, drum, eel, goby, goldfish, silk foot fish, grouper, guapote, halibut, Java fish, va), rohu, wrasse, loach, mackerel, milkfish, silver perch, mudfish, mullet, paco, pearlspot, silverside, perch, pike, pomfret, roach, salmon, long-fin catfish, bigeye amberjack, sea bass, sea bream, shiner, sleeper, snakehead, bream, snook, sole, bluefish, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, white trout, glass pike perch, and whitefish.

[0083] The salmon may be of the family Salmonidae or the subfamily Salmoninae. In one embodiment, the salmon is selected from the group consisting of: Salmo, Oncorhynchus, and Salvenis. In another embodiment, the Salmon is selected from the group consisting of: Atlantic salmon (Salmo salar) and brown salmon (Salmo trutta). In another embodiment, the Salmon 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 other embodiments, the salmonid is selected from the group consisting of Arctic char (Salvelinus alpinus), brook trout (Salvelinus fontinalis), and lake trout (Salvelinus namaycush). The sea bream may be gilthead sea bream (Sparus aurata), and the sea bass may be European sea bass (Dicentrarchus labrax).

[0084] In a preferred embodiment of the present invention, the decapod crustacean may be a shrimp or a prawn. The shrimp may be selected from the group consisting of Pacific white shrimp (Penaeus vannamei or Litopenaeus vannamei), white-legged shrimp (Penaeus vannamei or Litopenaeus vannamei), black tiger shrimp (Penaeus monodon), Japanese prawn (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 indica) indicus or Indian white shrimp (Fenneropenaeusindicus), banana shrimp (Penaeus merguiensis or Fenneropenaeusmerguiensis), autumn shrimp (Metapenaeus spp.), yellowleg shrimp (Penaeuscaliforniensis or Farfantepenaeuscaliforniensis), brown shrimp (Penaeuscaliforniensis or Farfantepenaeuscaliforniensis), São Paulo shrimp (Penaeuspaulensis or Farfantepenaeuspaulensis), Carpas shrimp (Penaeuspaulensis or Farfantepenaeuspaulensis), red spot shrimp (Penaeusbrasiliensis or Farfantepenaeus brasiliensis), spotted pink shrimp (Penaeus erythrocephalus or Penaeus brasiliensis) and southern white shrimp (Penaeus schmitti).The prawn may be selected from the group consisting of: broad groove shrimp (Penaeus vannamei or Penaeus vannamei), giant tiger shrimp (Penaeus monodon), giant freshwater prawn (Macrobrachium rosenbergii), giant river prawn (Macrobrachium rosenbergii), Malaysian shrimp (Macrobrachium rosenbergii), Japanese shrimp (Penaeus japonicas or Marsupenaeus japonicas), Chinese shrimp (Penaeus chinensis or Fenneropenaeus chinensis), Indian shrimp (Penaeus indicus or Fenneropenaeus indicus), banana shrimp (Penaeus merguiensis or Fenneropenaeus merguiensis), Oriental river prawn (Macrobrachium nipponense)) and Monsoon river prawn (Macrobrachium malcolmsonii).

[0085] In the present invention, vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3), can be fed to animals from birth until slaughter. Preferably, vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3) is fed to animals every day from birth to slaughter. More preferably, vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3), is fed to the animal every day for at least 10 days, such as at least 15 days or at least 20 days (wherein the number of days may be continuous or discontinuous) during the life of the animal. In one embodiment, vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin 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 of the animal.

[0086] In the present invention, examples of carotenoids include, but are not limited to, β-carotene, canthaxanthin, astaxanthin, lutein, and mixtures thereof.

[0087] Therefore, the present invention also relates to the use of a composition, an animal feed or an animal feed additive for improving the pigmentation of meat of aquatic animals, wherein the composition, animal feed or animal feed additive comprises one or more vitamin D or vitamin D metabolites, preferably 25-hydroxyvitamin D3 (25-OH D3), and one or more carotenoids, such as β-carotene, canthaxanthin, astaxanthin, lutein and mixtures thereof.

[0088] The oil carrier can be fish oil, microbial lipids and / or one or more vegetable oils. The vegetable oil can be selected from the group consisting of rapeseed oil and soybean oil. An example of a microbial lipid according to the present 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 cis-5,8,11,14,17-eicosapentaenoic acid. This fatty acid is an ω-3 fatty acid of 20:5. "Docosahexaenoic acid" ["DHA"] is the common name for cis-4,7,10,13,16,19-docosahexaenoic acid. This fatty acid is an ω-3 fatty acid of 22:6. 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.

[0089] Incorporation of a feed additive composition containing vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3), into fish feed can be performed as described in Examples 1 and 2. The final concentration of vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3) in the feed can be determined by HPLC according to standard methods.

[0090] Incorporation of a feed additive composition containing vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3), into fish feed can alternatively be carried out by preparing a premix of vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3), and other suitable additives.

[0091] Example

[0092] Example 1: Preparation of compressed fish feed

[0093] The main raw materials are ground and mixed. Minor ingredients are then added to the mixer and a homogeneous mixture is conditioned by adding water and steam to the material in the preconditioner. This starts the cooking process in the starch fraction (binding component). The material is fed into a pelletizer. The material is forced through the die of the pelletizer and the ribbons are broken into pellets outside the die. The moisture content is low and no drying of the feed is required.

[0094] The additional oil comprising the fish feed composition according to the invention is then sprayed onto the surface of the pellets, but since the pellets are quite compact, the total lipid content rarely exceeds 24%. The added oil can be fish oil, microorganism / algae or vegetable oil, such as rapeseed oil or soybean oil, or a mixture of oils. After oiling, the pellets are cooled in a cooler and bagged. The final pressed fish feed contains 10ppm to 5000ppm of the composition as described in the invention.

[0095] Example 2: Method for preparing puffed fish feed

[0096] The main raw materials are ground and mixed. Minor ingredients (including the fish feed composition according to the present invention) are added to the mixer. A homogeneous mixture is adjusted by adding water and steam to the material in a preconditioner. Additional oil can also be added to the material at this stage. This starts the cooking process in the starch fraction (binding component). The material is fed into an extruder. The extruder can be a single screw or twin screw type. Due to the rotational movement of the material in the extruder, the material is further mixed. Additional oil, water and steam can be added to the material in the extruder. At the end of the extruder, the temperature of the material is above 100°C and the pressure is above ambient pressure. The material is forced through an opening in the extruder die plate. Due to the reduction in temperature and pressure, some of the moisture will evaporate immediately (flash) and the extruded material becomes porous. The ribbons are cut into pellets with a rotating knife. The moisture content is quite high (18-28%), and therefore the pellets are immediately dried in a dryer to a moisture content of about 10%.

[0097] After the dryer, more oil containing the feed additive composition according to the present invention can be added to the feed by spraying the oil onto the surface of the feed or by immersing the feed in the oil. It is advantageous to add the oil to the feed in a closed container with an air pressure lower than the ambient pressure (vacuum coating) so that the porous feed pellets absorb more oil. Feeds containing more than 40% lipids can be produced in this way. After the coating machine, the feed is cooled and bagged. As mentioned above, the oil can be added at several places in the process and can be fish oil, microorganism / algae or vegetable oil, such as rapeseed oil or soybean oil, or a mixture of oils.

[0098] Fish require proteins, fats, minerals and vitamins to grow and stay healthy. The diet of carnivorous fish is particularly important. Initially in the farming of carnivorous fish, whole fish or ground fish were used to meet the nutritional requirements of farmed fish. Ground fish mixed with various types of dry raw materials such as fish meal and starch was called soft feed or semi-moist feed. As agriculture became industrialized, soft feed or semi-moist feed was replaced by pressed dry feed. This itself was gradually replaced by extruded dry feed.

[0099] Today, extruded feeds are almost universally used in the farming of a variety of fish species, such as various types of salmonids, cod, sea bass and sea bream.

[0100] The main source of protein in dried fish feed is fish meal of varying qualities. Other animal protein sources are also used in dried fish feed. Thus, it is known to use blood meal, bone meal, feather meal and other types of meal produced from other slaughterhouse wastes, such as chicken meal. These are generally cheaper than fish meal and fish oil. However, in some geographical areas, the use of such raw materials in the production of feed for animals and fish used to produce food has been banned.

[0101] It is also known to use vegetable proteins, such as wheat gluten, maize (corn) gluten, soy protein, lupin flour, pea flour, bean flour, rapeseed flour, sunflower seed meal and rice flour.

[0102] Example 3: Effects of Vitamin D3 and 25-OH D3 on the pigmentation of Carophyll Pink in trout fillets.

[0103] Materials and methods

[0104] The experiment was conducted with 270 trout. The trial was conducted for 8 weeks and had been designed with 6 treatments (see Table 1) and 3 replicates of 15 fish.

[0105] Feeding

[0106] The diet given to the fish was a standard feed to which the tested products were added. The feed met all the nutritional requirements relevant to the developmental stage of the fish.

[0107] Vitamin D3 was added by including the commercial product Rovimix D3-500 D3. 25-OH D3 was added by including the commercial product Rovimix HyD. Astaxanthin was added by including the commercial product arophyll Pink 10% CWS B-free.

[0108] Table 1. Treatments used in trout experiments.

[0109]

[0110]

[0111] *Vitamin and mineral premixes do not contain vitamin D3

[0112] **Rovimix D3-500 D3, Rovimix HyD and Carizo Pink 10% will be included in the paste (pre-extruded) at the expense of wheat

[0113] Table 2. Product inclusion levels for 45 kg feed

[0114]

[0115]

[0116] *80, 400 and 800 in D3-80 aso refer to the inclusion levels of 80ug, 400ug and 800ug respectively.

[0117] Experimental design

[0118] After eight weeks of feeding, fish fillet samples were collected.

[0119] According to L * a * b * The color space (also known as CIELAB) method measures the redness of the fillet digitally with a colorimeter on the surface of the fillet. D3-80 corresponds to the vitamin D level in the standard feed and can be used as a control.

[0120] result

[0121] The redness of the fillet increased with increasing vitamin D (see Figure 1). Significant improvements in vitamin D3 were observed at D3-800. Significant improvements in HyD were observed at higher HyD-400abd.

[0122] Example 4: Effects of Vitamin D3 and 25-OH D3 on the pigmentation of carnelian pink in trout fillets.

[0123] Materials and methods

[0124] The experiment was conducted with 270 trout. Six test diets were used. The trial was conducted for 8 weeks and 6 treatments (see Table 3) and 3 replicates of 15 fish have been designed.

[0125] Feeding

[0126] The diet given to the fish was a standard feed to which the tested products were added. The feed met all the nutritional requirements relevant to the developmental stage of the fish.

[0127] Vitamin D3 was added by including the commercial product Rovimix D3-500 D3. 25-OH D3 was added by including the commercial product Rovimix HyD. Astaxanthin was added by including the commercial product arophyll Pink 10% CWS B-free.

[0128] Table 3. Treatments used in trout experiments.

[0129]

[0130] *Vitamin and mineral premixes do not contain vitamin D3

[0131] **Rovimix D3-500 D3, Rovimix HyD and Carizo Pink 10% will be included in the paste (pre-extruded) at the expense of wheat

[0132] Table 4. Product inclusion levels for 45 kg feed

[0133]

[0134] *80, 400 and 800 in D3-80 aso refer to the inclusion levels of 80ug, 400ug and 800ug respectively.

[0135] Experimental design

[0136] After eight weeks of feeding, fish fillet samples were collected. Ten fish were sampled from each tank, with 30 fish per group. The fillets were removed and the fillet color (redness (a*)) of the standard 'Norwegian Quality Cut' was measured at three locations using a colorimeter (CR-400, Minolta).

[0137] According to L * a * b * The color space (also known as CIELAB) method measures the redness of the fillet digitally with a colorimeter on the surface of the fillet. D3-80 corresponds to the vitamin D level in the standard feed and can be used as a control.

[0138] result

[0139] The redness of the fillet increased with increasing vitamin D (see Figure 2). Significant improvements were observed for increasing vitamin D3 levels. Significant improvements were observed for increasing HyD levels.

[0140] Example 5: Effects of Vitamin D3 and 25-OH D3 on the pigmentation of carnelian pink in trout fillets.

[0141] Materials and methods

[0142] The experiment was conducted with 480 trout. Six test diets were used. The trial was conducted for 12 weeks and had been designed with 6 treatments (see Table 5) and 4 replicates of 20 fish.

[0143] Feeding

[0144] The diet given to the fish was a standard feed to which the tested products were added. The feed met all the nutritional requirements relevant to the developmental stage of the fish.

[0145] Vitamin D3 was added by including the commercial product Rovimix D3-500 D3. 25-OH D3 was added by including the commercial product Rovimix HyD. Astaxanthin was added by including the commercial product arophyll Pink 10% CWS B-free.

[0146] Table 5. Treatments used in trout experiments.

[0147]

[0148]

[0149] *Vitamin and mineral premixes do not contain vitamin D3

[0150] **Rovimix D3-500 D3, Rovimix HyD and Carizo Pink 10% will be included in the paste (pre-extruded) at the expense of wheat

[0151] Table 6. Product inclusion levels for 45 kg feed

[0152]

[0153] *80, 400 and 800 in D3-80 aso refer to the inclusion levels of 80ug, 400ug and 800ug respectively.

[0154] Experimental design

[0155] After eight weeks of feeding, fish fillet samples were collected. Ten fish were sampled from each tank, 40 fish per group. The fillets were removed and the fillet color (redness (a*)) of a standard 'Norwegian Quality Cut' was measured at three locations using a colorimeter (Minolta).

[0156] According to L * a * b * The color space (also known as CIELAB) method measures the redness of the fillet digitally with a colorimeter on the surface of the fillet. D3-80 corresponds to the vitamin D level in the standard feed and can be used as a control.

[0157] result

[0158] The redness of the fillet increased with increasing vitamin D (see Figure 3). Significant improvements were observed for increasing vitamin D3 levels. Significant improvements were observed for increasing HyD levels.

Claims

1. A method for improving meat pigmentation in an aquatic animal, the method comprising administering to the animal an animal feed or an animal feed additive comprising vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3), and one or more carotenoids.

2. The method according to claim 1, wherein the carotenoid is β-carotene, astaxanthin, canthaxanthin, lutein and / or a mixture thereof.

3. The method according to any one of claims 1 to 2, wherein the vitamin D or vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3), is in a liquid formulation.

4. The method according to any one of claims 1 to 2, wherein the liquid formulation comprises vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3), in powder form.

5. The method according to any one of claims 1 to 4, wherein the vitamin D or vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3), is embedded in an oil carrier.

6. The method of claim 5, wherein the oil carrier is selected from the group consisting of fish oil, microbial oil and / or one or more vegetable oils.

7. A method according to any one of claims 5 to 6, wherein the liquid formulation is sprayed onto the feed after it has been pelletized or extruded.

8. The method according to any one of claims 1 to 7, wherein the animal feed comprises vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3), in an amount of at least 80 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg per kg of feed.

9. A composition comprising vitamin D or a vitamin D metabolite, preferably 25-hydroxyvitamin D3 (25-OH D3), and one or more carotenoids as active ingredients, wherein the composition is intended to be ingested by an aquatic animal, and wherein the concentration of the active ingredient added to the final feed of the animal is in the range of 80 ug to 1500 ug per kg feed, preferably 100 ug to 1500 ug per kg feed, preferably 150 ug to 1500 ug per kg feed, more preferably 150 ug to 1250 ug per kg feed, more preferably 150 ug to 1000 ug per kg feed.

10. The composition of claim 9, wherein the active ingredient is embedded in an oil carrier.

11. The composition according to any one of claims 9 or 10, wherein the composition is a feed premix or an aquaculture feed.

12. The composition according to claim 11, wherein the aquaculture feed is a coated feed pellet.

13. A composition according to claim 12, wherein the active ingredient is present in the coating of the coated feed pellets.

14. The composition of claim 13, wherein the coating comprises a coating oil and the active ingredient.

15. The composition according to any one of claims 9 to 14, wherein the carotenoid is β-carotene, astaxanthin, canthaxanthin, lutein and / or mixtures thereof.

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