5. ESSENTIAL NUTRIENTS - VITAMINS

5.1 Definition and classification

Vitamins are a heterogeneous group of organic compounds essential for the growth and maintenance of animal life. The majority of vitamins are not synthesized by the animal body or at a rate sufficient to meet the animals needs. They are distinct from the major food nutrients (proteins, lipids, and carbohydrates) in that they are not chemically related to one another, are present in very small quantities within animal and plant foodstuffs, and are required by the animal body in trace amounts. Approximately 15 vitamins have been isolated from biological materials; their essentiality depending on the animal species, the growth rate of the animal, feed composition, and the bacterial synthesizing capacity of the gastro-intestinal tract of the animal. In general, all animals display distinct morphological and physiological deficiency signs when individual vitamins are absent from the diet.

Vitamins may be classified into two broad groups, depending on their solubility; the water-soluble vitamins and the fat-soluble vitamins (Table 9)

As their name suggests, the fat-soluble vitamins are absorbed from the gastrointestinal tract in the presence of fat and can be stored within the fat reserves of the body whenever dietary intake exceeds metabolic demands; storage increasing with dietary intake to the extent that a toxic condition (hypervitaminosis) may be produced. By contrast, the water-soluble vitamins are not stored in appreciable quantities in the animal body; body stores being rapidly depleted in the absence of regular dietary water-soluble vitamin sources. Water-soluble vitamins toxicities are therefore unlikely.

5.2 Water-soluble vitamins

5.2.1 Thiamine

Structure:

Biological function: Thiamine, in the form of its di-phosphate ester (thiamine pyrophosphate, TPP), functions as a coenzyme in carbohydrate metabolism. In particular, TPP is involved in the oxidative decarboxylation (ie. CO₂ removal) of pyruvic acid and alpha-ketoglutaric acid to acetylcoenzyme A and succinyl coenzyme A respectively, and as an activator of the enzyme transketolase which is involved in the oxidation of glucose via the pentose phosphate pathway. Thiamine is therefore intimately involved in carbohydrate metabolism.

Dietary sources: Rich dietary sources of thiamine include: dried brewers yeast (100–50 mg/kg); wheat middlings, wheat mill run, rice bran, rice polishings (50–10 mg/kg); dried torula yeast, groundnut (peanut) meal, wheat bran, oats, barley, dried fish solubles, cottonseed meal, soybean meal, linseed meal, dried distillers solubles, broad beans, lima beans, dried delactose whey (10–5 mg/kg). Other rich dietary sources include glandular meals (liver/kidney), green leafy crops, and the outer coat or germ of cereals.

5.2.2 Riboflavin

Structure:

Biological function: Riboflavin, as a constituent of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), functions as a coenzyme for many enzyme oxidases and reductases, and therefore plays an important role in energy metabolism; FMN and FAD facilitating the enzymatic breakdown of energy-yielding nutrients such as fatty acids, amino acids and pyruvic acid. In addition, riboflavin is particularly important for the respiration of poorly vascularised tissues such as the cornea of the eye, and in conjunction with pyridoxine (vitamin B6) for the conversion of tryptophan to nicotinic acid. Riboflavin is therefore essential for the metabolism of carbohydrates, fats and proteins.

Dietary sources: Rich dietary sources of riboflavin include: dried torula yeast, dried brewers yeast, liver and lung meal, dried delactose whey (50–30 mg/kg); chicken egg white, dried skim milk, dried distillers solubles, safflower seed meal, dried fish solubles, alfalfa meal (30–10 mg/kg); poultry by-product meal, fish meal, meat meal, meat and bone meal, groundnut meal, rapeseed meal (10–5 mg/kg). Other rich dietary sources include green vegetables and to a lesser extent germinated cereal grains.

5.2.3 Pyridoxine

Structure:

Pyridoxal	Pyridoxol	Pyridoxamine

All three forms of pyridoxine are readily interchangeable in animal tissues and have equal activity.

Biological function: Pyridoxine, in the form of its phosphate ester (pyridoxal phosphate), functions as a coenzyme in nearly all reactions involved in the non-oxidative degradation of amino acids, which include transaminations, deaminations, decarboxylations, and sulfhydrations. Pyridoxine therefore plays a vital role in protein metabolism. Pyridoxal phosphate is also required for the metabolic breakdown of tryptophan to nicotinic acid, the synthesis of haemoglobin, acetyl coenzyme A and messenger RNA, and for the metabolism of carbohydrates by facilitating the release of glycogen from muscle and liver.

Dietary sources: Rich dietary sources of pyridoxine include: dried brewers yeast, dried torula yeast, dried delactose whey (50–30 mg/kg); dried fish solubles (30–20 mg/kg); wheat mill run, sunflower seed meal (20–10 mg/kg); wheat middlings, groundnut meal, dried distillers solubles, rapeseed meal, meat and bone meal, fish meal, corn, alfalfa meal, cottonseed meal, rice, sorghum, soybean meal (10–5 mg/kg).

5.2.4 Pantothenic acid

Structure:

Biological function: Pantothenic acid, in the form of 3 phospho-adenosine-5-diphospho-pantotheine (commonly known as acetyl coenzyme A), functions as a coenzyme which plays a central role in all acetylation reactions (ie. reactions involving the formation or transfer of a 2-carbon acetyl group). Since carbohydrates, fats and proteins are first converted to acetyl coenzyme A before they are oxidized in the krebs or tricarboxylic acid cycle, pantothenic acid is therefore essential for the release of energy from the major food nutrients. Acetyl coenzyme A is also involved in the synthesis of fatty acids, cholesterol, steroids, haemoglobin, and in the acetylation of choline. Pantothenic acid is therefore a key substance in carbohydrate, fat and protein metabolism.

Dietary sources: Rich dietary sources of pantothenic acid include: dried brewers yeast, dried torula yeast (130–100 mg/kg); dried delactose whey (100-75 mg/kg); dried fish solubles, whole hens egg (75–50 mg/kg); rice polishings, groundnut meal, sunflower seed meal, wheat bran, safflower meal, dried skim milk, alfalfâ meal, dried cane molasses (50–25 mg/kg); rice bran, wheat middlings, wheat mill run, dried distillers solubles, fish meal, soybean meal, linseed meal, sorghum, maize, cottonseed meal, poultry by-product meal, oats (25–10 mg/kg). Other rich dietary sources include the glandular meals (liver/ kidney) and green leafy crops.

5.2.5 Nicotinic acid

Structure:

Nicotinic acid	Nicotinamide

Biological function: Nicotinic acid, as a constituent of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), functions as a coenzyme in enzyme systems that provide a mechanism for electron transfer in metabolic processes (ie. hydrogen removal andtransport). The metabolism of NAD and NADP is therefore closely linked with that of FAD and FMN; both vitamins playing a central role in tissue oxidation and therefore essential for the release of energy from carbohydrates, fats and proteins. In addition, NAD and NADP also play an important role in the synthesis of fatty acids and cholesterol, respectively.

Dietary sources: Rich dietary sources of nicotinic acid include: rice polishings, dried torula yeast, dried brewers yeast, rice bran (600–300 mg/kg); wheat bran, dried fish solubles, sunflower seed meal, groundnut meal, rapeseed meal, liver and lung meal, dried distillers solubles, wheat mill run (300–100 mg/kg); fish meal, wheat middlings, safflower seed meal, corn gluten meal, meat and bone meal, meat meal, dried brewers grains, poultry by-product meal, sorghum, alfalfa meal, barley grain, dried cane molasses, rice mill run (100–40 mg/kg). Other rich sources include green leafy vegetables.

5.2.6 Biotin

Structure:

Biological function: Biotin functions as a coenzyme in those tissue reactions involving the transfer of carbon dioxide from one compound to another (ie. carboxylation reactions). For example, as a component of the enzymes pyruvate carboxylase and acetyl coenzyme A carboxylase, biotin is responsible for the conversion of pyruvic acid to oxaloacetic acid (an intermediate in gluconeogenesis and the krebs cycle), and for the conversion of acetyl coenzyme A to malonyl coenzyme A (the latter required for the synthesis of long chain fatty acids). Biotin therefore plays a key role in carbohydrate and fat metabolism. Biotin is also reported to be involved in purine and protein synthesis, certain deamination reactions, and in the urea cycle; however, the precise role of biotin in most of these actions is still unclear.

Dietary sources: Rich dietary sources of biotin include: dried brewers yeast, dried torula yeast, dried distillers solubles, rapeseed meal, safflower seed meal, sunflower seed meal (2-1 mg/kg); whole hens egg, rice polishings, dried brewers grains, liver and lung meal, rice bran, dried delactose whey, cottonseed meal (1–0.5 mg/kg); groundnut meal, soybean meal, dried skim milk, alfalfa meal, oats, sorghum, dried blood meal, dried fish solubles, fish meal, wheat bran, wheat mill run (0.5-0.2 mg/kg). Other rich sources of biotin include the legumes and green vegetables.

5.2.7 Folic acid

Structure:

Biological function: Folic acid, in the form of tetrahydrofolic acid, functions as a coenzyme for those reactions effecting the transfer of one-carbon units (ie. formyl, methyl, formate and hydroxymethyl units) from one compound to another. For example, tetrahydrofolic acid is involved in the synthesis of haemoglobin, glycine, methionine, choline, thymine (pyrimidine) and purines, and in the metabolism of phenylalanine, tyrosine and histidine.

Dietary sources: Rich dietary sources of folic acid include: dried torula yeast, dried brewers yeast, dried brewers grains (10–5 mg/kg); alfalfa meal, full-fat soybeans, liver and lung meal, wheat germ meal, rapeseed meal, rice bran, linseed meal, sunflower seed meal, cottonseed meal, whole hens egg, dried distillers solubles, wheat bran, wheat mill run, safflower seed meal, dried delactose whey (5–1 mg/kg). Other rich sources include mushrooms, fruits (lemons, strawberries, bananas) and dark green leafy vegetables.

5.2.8 Cyanocobalamin

Structure:

Biological function: Cyanocobalamin, in the form of cobamide coenzymes, is required for normal red blood cell formation and the maintenance of nerve tissue. Although the precise role of cyanocobalamin in these processes is still unclear, its metabolism is closely linked with that of folic acid in that they are both involved in the metabolism of single carbon units. Physiological processes in which cobamide coenzymes have been found to be involved include: the synthesis of nucleic acid (through its role in the synthesis of thymine and deoxyribose), the recycling of tetrahydrofolic acid, the mainentance of glutathione activity ( carbohydrate metabolism), the conversion of methylmalonyl coenzyme A to succinyl coenzyme A (fat metabolism), and in the methylation of homocysteine to methionine (amino acid metabolism).

Dietary sources: Rich dietary sources of cyanocobalamin include: animal by-products, liver, kidney, fish meals, meat and bone meal, condensed fish solubles, poultry by-product meal (1–0.1 mg/kg).

5.2.9 Inositol

Structure:

Nine possible stereoisomers, of which only myo-inositol is biologically active.

Biological function: Myo-inositol, as a constituent of inositol phospholipids, is an important structural component of skeletal, heart and brain tissue. Although the physiological role of myo-inositol is still unclear, it is believed to play an important role in the growth of liver and bone marrow cells, liver lipid (cholesterol) transport, and in the synthesis of RNA. No coenzyme function has so far been ascribed to myo-inositol.

Dietary sources: Rich dietary sources of myo-inositol include animal tissues (skeletal, brain, heart, liver), dried brewers yeast and fish meal. In plant tissues myo-inositol exists in a phosphorylated form as phytic acid (inositol hexaphosphate); although phytic acid is considered to be an anti-nutritional factor for most monogastric animals by interfering with mineral absorption, rich dietary sources include cereal grains and legumes.

5.2.10 Choline

Structure:

Biological function: Choline is an essential component of phospholipids and acetylcholine, and as such plays a vital role in the maintenance of cell structure and the transmission of nerve impulses respectively. Choline also acts as a methyl donor in trans-methylation reactions (ie. synthesis of methionine) and in the form of the phospholipid lecithin plays an important role in the transport of lipid within the body. No coenzyme functions have so far been ascribed to choline.

Dietary sources: Rich dietary sources of choline include: rapeseed meal, poultry by-product meal, shrimp meal, liver and lung meal, dried fish solubles (7000–6000 mg/kg); dried distillers solubles, dried brewers yeast, sunflower seed meal, dried delactose whey (6000–4000 mg/kg); brown fish meal, dried torula yeast, wheat germ meal, white fish meal, safflower seed meal, cottonseed meal, soybean meal, meat meal, meat and bone meal, groundnut meal (4000–2000 mg/kg); whole hens egg, wheat bran, dried brewers grains, wheat middlings, linseed meal, sesame meal, alfalfa meal, barley, rice bran, rice polishings, wheat mill run, oats (2000–1000 mg/kg).

5.2.11 Ascorbic acid

Structure:

	⇌
L-ascorbic acid (reduced form)		Dehydro-L-ascorbic acid (oxidized form)

Biological function: Ascorbic acid and its oxidation product dehydro-L-ascorbic acid act as physiological antioxidants by facilitating hydrogen transport within the animal cell. Ascorbic acid is also required for numerous hydroxylation reactions within the body, including the hydroxylation of tryptophan, tyrosine, lysine, phenylalanine and proline. Of the above mentioned hydroxylation reactions probably the most important is the formation of hydroxyproline from proline; both amino acids being important constituents of collagen, mucopolysaccharides and chondroitin sulphate (ie. intracellular substances that bind bone cells, blood capillary cells, and connective tissue cells). Ascorbic acid therefore plays a vital role in maintaining the integrity of connective tissue, blood vessels, bone tissue and wound tissue. Ascorbic acid is also required for the conversion of folic acid into its metabolically active form of tetrahydrofolic acid, for the conversion of tryptophan to serotonin, and for the synthesis of steroid hormones by the adrenal cortex.

Dietary sources: Rich dietary sources of ascorbic acid include: citrus fruits, green leafy vegetables, fresh insects, and to a lesser extent the glandular meals (liver/kidney). For example, the ascorbic acid content (expressed as mg ascorbic acid per 100g fresh product) of a few of the above listed food items can be approximated as follows; blackcurrants - 200, green peppers - 91, cauliflower - 64, watercress - 60, strawberries - 60, green cabbage - 53, oranges/lemons - 50, potatoes - 8–30, raw liver - 30, raw kidney - 12.

5.3 Fat-soluble vitamins

5.3.1 Retinol

Vitamin A exists only in animal tissues, and is present either in the form of retinol (vitamin A1: mammals and marine fish) or 3,4-dehydroretinol (vitamin A2: freshwater fish). However, a vitamin A precursor is found in plant tissues in the form of the carotenoid pigments (ie. beta carotene). Once ingested by the animal, these plant pigments can be converted into active vitamin A; the conversion efficiency depending on the animal species and the isomeric form of the carotenoid ingested, with ‘trans’ isomers having the highest biological activity.

Biological function: Vitamin A is required for normal vision; in the retina of the eye vitamin A is combined with a specific protein (opsin) to form a visual pigment, which in turn functions in the reception and transmission of light from the eye to the brain. In addition, vitamin A is required for the maintenance of the mucous secreting epithelial tissues of the reproductive tract, skin, bone and gastro-intestinal tract. Its role in protecting mucous membranes and developing bone tissue is thought to be due to its participation in the formation of mucopolysaccharides. However, the precise role of vitamin A in epithelial cell metabolism remains to be fully elucidated. Vitamin A is also believed to be required for the release of proteolytic enzymes from lysosomes.

Dietary sources: Rich dietary sources of retinol include fish liver oils (ie. halibut liver oil - 9000 μg/g, cod liver oil - 180 μg/g) and animal liver meals (25–100 μg/g). Within plant foodstuffs rich sources of vitamin A (expressed as retinol equivalents μg/g fresh weight) include old carrots - 20, spinach - 10, and watercress - 5. The amounts of vitamin A or retinol in feed stuffs are often quoted in international units (i.u). One i.u. of vitamin A is equal to 0.344 μg retinol or 0.6 μg beta carotene.

5.3.2 Cholecalciferol

Structure:

The major source of vitamin D in nature is cholecalciferol (vitamin D₃). Like vitamin A, cholecalciferol exists only in animal tissues. In most terrestrial animals cholecalciferol is produced in the skin through the UV irradiation of the provitamin 7-dehydrocholesterol.

Biological function: Cholecalciferol plays an essential role in calcium and phosphorus metabolism in animals. In particular, cholecalciferol is required for the absorption of calcium from the gastro-intestinal tract and for the calcification of growing bone tissue. Before cholecalciferol can perform these metabolic functions it is first converted to 25-hydroxycalciferol (25-HCC) in the liver, which in turn is converted to the physiological active form 1,25-dihydroxycalciferol (1,25-DHCC) in the kidney. It is 1,25-DHCC which acts on the respective target tissues and is responsible for the synthesis of the calciumbinding protein in the intestinal epithelial cells. Additional functions which have been ascribed to 1,25-DHCC include: the conversion of organic phosphorus to inorganic phosphorus in bone, the resorption of phosphate and amino acids from the kidney tubules, the maintenance of blood calcium level, and the deposition and oxidation of citrate in bone.

Dietary sources: Rich dietary sources of cholecalciferol include fish liver oils (cod liver oil 2–10 μg/g), animal liver meals and oils, and fish meal. As with vitamin A, the amounts of vitamin D in feedstuffs is often quoted in international units (i.u.). The i.u. has the potency of 0.025 μg of cholecalciferol and is equal to one British Standard Unit (BSI) or 1.3 Association of Official Analytical Chemists USA (AOAC) units.

5.3.3 Tocopherol

Structure:

When R₁, R₂, R₃ are all CH₃ groups, the tocopherol isomer is D-alpha-tocopherol. Of the eight naturally occuring isomers of tocopherol, the alpha isomer is the most widely distributed and has the greatest vitamin activity.

Biological function: Tocopherols act as lipid-soluble extracellular and intracellular antioxidants within the animal body. In particular, the tocopherols protect the highly unsaturated fatty acids present in cellular and subcellular membranes, and other reactive compounds (ie. vitamins A and C) from oxidative damage by acting as free radical traps. It has also been suggested that the tocopherols play an important role in cellular respiration and in the biosynthesis of DNA and coenzyme Q.

Dietary sources: Rich dietary sources of tocopherol include; alfalfa meal, wheat germ meal (125–100 mg/kg); whole chicken egg, rice polishings (100–75 mg/kg), rice bran, wheat middlings (75–50 mg/kg); dried brewers grains, dried distillers solubles, barley grain, full fat soybean meal, maize grain, wheat mill run (50–25 mg/kg); corn gluten meal, wheat bran, rye grain, sorghum, fish meal, oats, sunflower seed meal, cottonseed meal (25–10 mg/kg). Other rich sources include all vegetable oils and green leafy crops.

5.3.4 Phylloquinone

Structure:

	Phylloquinone (vitamin K,)
	Multiprenylmanaquinone (vitamin K2)
	Menadione (vitamin K3)

Compounds possessing vitamin K activity are all derivatives of naphthoquinone; the two naturally occuring forms being phylloquinone (synthesized by green leafy plants) and multiprenylmenaquinones (synthesized by micro-organisms). However, the simplest and most potent form of vitamin K is a synthetic derivative, menadione (vitamin K₃).

Biological function: Vitamin K is required for the maintenance of normal blood coagulation by facilitating the production and/or release of various plasma proteins required for blood coagulation, including; prothrombin, proconvertin, plasma thromboplastin, and the Stuart-Prower factor. Although it has been suggested that vitamin K may play a role in electron transport and oxidative phosphorylation in micro-organisms, these functions remain to be confirmed in higher animals.

Dietary sources: Rich dietary sources of vitamin K include alfalfa meal (9 mg/kg), fish meal (2 mg/kg), liver meal, and green leafy vegetables (spinach, kale, cabbage, pine neddles, nettles).

5.4 Dietary vitamin requirements

The dietary vitamin requirements of fish and shrimp have been determined by feeding graded levels of each vitamin within a purified or semi-purified test diet under controlled laboratory conditions; dietary requirement being taken at ‘break-point’ on the basis of the observed growth response, feed efficiency or vitamin tissue concentration (for review see ADCP, 1980; Castell et al., 1986; Cho, Cowey and Watanabe, 1985; Halver, 1985; Kanazawa, 1983; Koenig, 1981; NRC, 1983; and Robinson and Wilson, 1985).

Despite an obvious physiological need by most fish and shrimp for the fifteen or so vitamins mentioned, under practical farming conditions the quantitative dietary vitamin requirements will depend upon a number of important factors, including:

• The feeding behaviour of the fish or shrimp species cultured. For example, species such as shrimp which consume their food slowly over a period of hours require higher dietary vitamin levels so as to counteract the progressive loss of water-soluble vitamins through leaching.

• The vitamin synthesizing capacity of the gut microflora of the fish or shrimp species cultured. For example, a well developed gut microflora is capable of synthesizing most B vitamins, pantothenic acid, biotin, choline, inositol and vitamin K, which in turn may become available to the animal, thereby reducing the dietary requirement. This may be particularly true for pond reared herbivorous or omnivorous fish and shrimp species.

• The intended culture system to be used (ie. intensive, semi-intensive or extensive) and availability of natural food organisms within the water body. For example, no beneficial effect of dietary vitamin supplementation was observed with tilapia or carp either in fertilized ponds or cages (within the pond) at stocking densities of 2/m² and 100/m³ respectively (ca. 400g fish; S. Viola, personal communication, Ashrat, Israel, 1985). Here, the important factor is the natural fertility of the water body and the total biomass of the fish or shrimp species stocked; the importance of dietary vitamin supplementation increasing with increasing stocking density and decresing natural food availability per animal stocked. Natural pond food organisms therefore represent a potential source of dietary vitamins for pond cultured aquaculture species.

• The size and growth rate of the fish or shrimp species cultured (ie. daily vitamin requirement per unit of body weight decreasing with increasing animal size and decreasing growth rate).

• The nutrient content of the diet used. For example, the dietary requirement for tocopherol, thiamine and pyridoxine has been shown to increase with increasing dietary concentrations of polyunsaturated fatty acids, carbohydrate and protein, respectively.

• The manufacturing process to be used for the production of the ration. For example, so as to counteract the destruction of the heat labile vitamins during feed manufacture, dry heat or steam pelleted feeds require higher dietary vitamin fortification than cold or wet pelleting processes.

• The physico-chemical characteristics of the water body and physiological condition of the fish or shrimp species cultured. For example, the negative effects of pollution, disease, body wounds, and stress on fish have been found to be reduced in-part by dietary supplementation with ascorbic acid over and above that normally required by a healthy ‘non-stressed’ animal.

It is evident from the above that the apparent ‘physiological’ vitamin requirements of fish and shrimp (ie. minimum dietary vitamin level that will support maximum growth, maximum vitamin tissue storage, or prevent deficiency signs) will differ markedly from the vitamin level required within a practical fish or shrimp ration. Sadly, scant information exists on the dietary vitamin requirements of fish or shrimp under practical semi-intensive or intensive farming conditions; the majority of studies having been conducted under controlled ‘in-door’ laboratory conditions and animals fed purified or semipurified diets manufactured using laboratory or hand operated feed processing equipment.

Despite these serious drawbacks, the known dietary vitamin requirements of fish and shrimp are summarised in Table 10. Unless otherwise indicated, the vitamin requirements presented represent the minimum dietary requirements for growth and the prevention of deficiency signs, and thus do not allow for dietary vitamin processing or storage losses.

¹ Basal diet contained 8.2 mg/kg riboflavin from the feed ingredients used

² R - dietary requirement shown, but quantitative requirements unknown

¹ NR - no dietary requirement demonstrated during the experiment

¹ No dietary requirement shown with fish fed a practical ration

² No dietary requirement shown with fish fed a practical ration which already contained 0.51 mg/kg biotinfrom the dietary ingredients used

¹ lm³ cages situated within earthen ponds, 400 fingerlings per cage

² 578 m² plastic lined ponds, 580 fish/pond

The above vitamin requirements represent the minimum dietary requirements for growth and the prevention of deficiency signs, and thus do not allow for dietary vitamin processing or storage losses.

5.5 Vitamin pathology

5.5.1 Vitamin deficiency

The following gross anatomical deficiency signs have been reported in fish and shrimp fed vitamin deficient diets under controlled laboratory conditions:

¹ 1-McLaren et al. (1947); 2-Phillips & Brockway (1957); 3-Halver (1957);4-Kitamura et al. (1967); 5-Poston et al. (1977); 6-Takeuchi, Takeuchi &Ogino (1980); 7-Hughes, Rumsey & Nickum (1981); 8-Woodward (1982); 9-Woodward(1985); 10-Dupree (1966); 11-Murai & Andrews (1978); 12-Aoe et al. (1967);13-Ogino (1967); 14-Yone (1975); 15-Arai, Nose & Hashimoto (1972); 16-Coates& Halver (1958); 17-Murai & Andrews (1979); 18-Wilson, Bowser & Poe (1983);19-Poston & Di Lorenzo (1973); 20-Aoe, Masuda and Takada (1967); 21-Andrews& Murai (1978); 22-Aoe et al. (1969); 23-Murai & Andrews (1978a); 24-Hashimoto,Arai & Nose (1970); 25-Smith, Brin & Halver (1974); 26-Jürss & Jonas (1981);27-Jürss (1978); 28-Hardy, Halver & Brannon (1979); 29-Ogino (1965); 30-Andrews& Murai (1979); 31-Adron, Knox & Cowey (1978); 32-Kissil et al. (1981); 33-Sakaguchi,Takeda & Tange (1969); 34-Agrawal & Mahajan (1983); 35-Walton,Cowey & Adron (1984a); 36-Pos n & McCartney (1974); 37-Poston (1976), 38-Castledineet al. (1978); 39-Poston & Page (1982); 40-Ogino et al. (1970);41-Robinson & Lovell (1978); 42-Lovell & Buston (1984); 43-John & Mahajan (1979);Phillips et al. (1963); 45-Kashiwada & Teshima (1966); 46-Kashiwada, Teshima& Kanazawa (1970); 47-Limsuwan & Lovell (1981); 48-Ketola (1976); 49-Ogino et al.(1970a); 50-Aoe & Masuda (1967); 51-Burtle (1981); 52-Kitamura et al. (1965);53-Hilton, Cho & Slinger (1978); 54-Sato, Yoshinaka & Ikeda (1978); 55-Poston(1967); 56-Halver, Ashley & Smith (1969); 57-LOvell (1973); 58-Andrews & Murai(1974); 59-LOvell & Lim (1978); 60-Wilson & Poe (1973); 61-Lim & Lovell (1978);62-Li & Lovell (1985); 63-Mahajan & Agrawal (1979); 64-Soliman, Jauncey &Roberts (1986); 65-Agrawal & Mahajan (1980); 66-Poston et al. (1977); 67-Aoeet al. (1968); 68-Barnett, Cho & Slinger (1979); 69-Letherland et al. (1980);70-Lovell & Li (1978); 71-Andrews, Murai & Page (1980); 72-Poston (19 ); 73-Poston(1976a); 74-Murai & Andrews (1977); 75-Woodall et al. (1964); 76-Poston(1965); 77-Poston, Combs & Leibovitz (1976), 78-Cowey et al. (1984); 79-Watanabeet al. (1970); 80-Watanabe & Takashima (1977); 81-Murai & Andrews (1974); 82-Lovell, Miyazaki & Rabegnator (1984); 83-Wilson, Bowser & Poe(1984); 84-Butthep, Sitasit & Boonyaratpalin (1985); 85-Poston & Wolfe (1985);86-Herman (1985); 87-Deshimaru & Kuroki (1979); 88-Kanazawa, Teshima & Tanaka(1976); 89-Kanazawa (1983); 90-Guary et al. (1976); 91-Lightner et al. (1979);92-Sandnes et al., (1984); 93-Soliman, Jauncey and Roberts (1986a); 94-Satoh,Takeuchi & Watanabe (1987)

Under intensive culture conditions, and in the absence of natural food organisms, dietary vitamin deficiencies may arise through:

Feed processing and storage

Riboflavin: used in the form of a spray dried powder or a dry-dilution product, riboflavin is generally stable in dry multivitamin premixes. Processing losses of 26% have been reported for expanded pet foods (NRC, 1983). Feeds containing riboflavin should be protected from intensive light/ultraviolet radiation (liable to oxidation) and alkaline conditions.

Pantothenic acid: used in the form of calcium d-pantothenate (92% activity) or calcium dl-pantothenate (46% activity), pantothenic acid is generally stable in dry multivitamin premixes. Processing losses during pelleting or expansion have been reported to be as high as 10% (Slinger, Razzaque and Cho, 1979).

Niacin: used in the form of nicotinic acid or niacinamide and added as a drydilution product, niacin is generally stable in dry multivitamin premixes. Processing losses of 20% have been reported for expanded pet foods (NRC, 1983). Stability of niacin is good only if the feed is kept cool and dry.

Thiamine: used in the form of thiamine mononitrate (91.88% activity), thiamine is stable in dry multivitamin premixes that contain no added choline or trace elements. Thiamine is rapidly destroyed under alkaline conditions or in the presence of sulphide. Processing (pelleting/expansion) and storage (7 months, room temperature) losses have been reported to be 0–10% and 11–12%, respectively (Slinger, Razzaque and Cho, 1979).

Pyridoxine: used in the form of pyridoxine hydrochloride in dry-dilution, pyridoxine is stable in dry multivitamin premixes that contain no added trace elements. Prepared feeds that contain pyridoxine need protection from sunlight (UV), heat and moisture. Processing and storage (10 months) losses have been reported to be 7–10% (Slinger, Razzaque and Cho, 1979).

Biotin: used in the form of d-biotin in dry dilution, biotin is generally stable in dry multivitamin premixes. Processing losses is expanded petfoods have been reported to be 10% (NRC, 1983).

Folic acid: used in the crystalline form and in dry-dilution, folic acid can be lost during the storage of dry multivitamin premixes particularly at elevated temperatures (43% activity lost after 3 months at room temperature). Processing and storage losses have been reported to be 3–10% (Slinger, Razzaque and Cho, 1979). Folic acid is liable to oxidation on storage at elevated temperatures and on exposure to sunlight.

Vitamin B12: used in the crystalline form and in dry-dilution, vitamin B12 stability in multivitamin premixes is dependent on storage temperature; elevated temperatures reduce activity, particularly in the presence of mild acid conditions.

Choline: used as a 70% choline chloride solution or as a dry powder (25–60% activity choline chloride is stable in multivitamin premixes but can decrease the stability of other vitamins present. Relatively stable on processing and storage (NRC, 1983).

Vitamin C: used as L-ascorbic acid, ethylcelullose or fat coated (to improve stability) and generally not added to dry multivitamin premixes because of its poor stability. Readily oxidized in the presence of moisture, trace elements, elevated temperatures, light and oxidation products (rancid oils). Stability is dependent on form of product used and method of feed processing (Soliman, Jauncey and Roberts, 1987). For example, the effect of mixing (mash), water addition, cold pelleting and drying on the percent retention of L-ascorbic acid, the sodium salt of ascorbic acid, glyceride-coated L-ascorbic acid and the barium salt of L-ascorbic acid 2-sulphate (original dietary concentration 125 mg ascorbic acid/100g diet) have been reported to be 94.89%, 93.77%, 98.99% and 96.78% respectively (for mixing), 74.59%, 71.12%, 94.40% and 95.70% respectively (after water addition), 64.80%, 61.14%, 87.55% and 95.50% respectively (after cold pelleting), and 33.50%, 26.26% 58.10% and 94.70% respectively (after drying the moist pelleted feed; Soliman, Jauncey and Roberts, 1987). Processing and storage losses for practical fish feeds have been reported to be as high as 95% for uncoated L-ascorbic acid (Slinger, Razzaque and Cho, 1979; Sandnes and Utne, 1982). However, these difficulties may be overcome (in part) by using higher dietary fortification levels or by using protected forms of ascorbic acid such as ascorbic acid 2-sulphate and glyceridecoated ascorbic acid (Soliman, Jauncey and Roberts, 1987; Hilton, Cho and Slinger, 1977; Halver et al., 1975).

Vitamin A: used as the acetate, palmitate or propionate ester, usually in beadlet form with vitamin D. Vitamin A is stable in dry multivitamin premixes. However, vitamin A is readily oxidised at elevated storage temperatures and in the presence of oxidation products (rancid oils). Processing losses of 20% have been reported for expanded pet foods (NRC, 1983). Similarly, six months storage at room temperature resulted in a 53% loss of vitamin A activity (NRC, 1983). Stability can be increased by suitable antioxidant protection and spraying on to the outside of the pellet in a lipid medium (NRC, 1983).

Vitamin D: used as vitamin D₃, and normally added in beadlet form with vitamin A or as a spray dried/drum dried powder. Stability is generally high.

Vitamin K: used in the form of a menadione salt (vitamin K₃), either as menadione sodium bisulphite (50% K₃ activity) or menadione sodium bisulphite complex (33% K₃ activity). Stability in multivitamin premixes is good in the absence of trace elements (Frye, 1978). Under processing conditions heat, moisture, alkaline pH and trace elements accelerate the destruction of menadione salts (NRC, 1983). Prepared feeds should be protected from sunlight to obviate further oxidative losses.

Vitamin E: used in the form of dl-alpha-tocopherol acetate, either spray dried or absorbed, vitamin E is stable in dry multivitamin premixes stored at below room temperature. Stability is increased when used in the acetate form, but is prone to oxidation on storage at high temperatures and in the presence of oxidation products (rancid oils).

Leaching of water soluble vitamins

In contrast to the fat soluble vitamins (A, D, E, K) the water soluble vitamins can readily be lost from the feed through leaching prior to ingestion by the fish or shrimp. In general, the smaller the feed particle size and the longer the feed remains uneaten in the water, the greater the loss of water soluble vitamins through leaching.

L-ascorbic acid (vitamin C) has been found to be particularly prone to loss through leaching. For example, despite the excessive losses of vitamin C which occur during feed preparation and storage, up to 50–70% of the residual vitamin C activity has been reported to be lost through leaching after a 10 second immersion period in water (1.18–2.36mm diam. pellet; Slinger, Razzaque and Cho, 1979). In the same study, these authors also reported a 5–20% loss in pantothenic acid, 0–27% loss in folic acid, 0–17% loss in thiamine, and a 3–13% loss in pyridoxine activity through leaching, after a 10 second water immersion period. Murai and Andrews (1975) reported a 50% loss in pantothenic acid after a 10 second immersion of a trout pellet originally containing 500 mg/kg pantothenic acid. Similarly, water stability tests with complete diets for penaeid shrimps reported water soluble vitamin losses of 97% (thiamine), 94% (pantothenic acid), 93% (pyridoxine), 90% (vitamin C), 86% (riboflavin), 50% (inositol), and 45% (choline) after a one hour immersion period in sea water (Cuzon, Hew and Cognie, 1982).

Deficiencies due to the presence of dietary anti-vitamin factors

Avidin: anti-biotin factor present in raw egg white. Readily destroyed by heat.

Thiaminase: heat labile anti-thiamine factor present in raw fish, shellfish, rice polishings, Indian mustard seed, mung bean (green gram), and linseed (liener, 1980). Dietary thiamine deficiency may be overcome by heat processing the raw material so as to deactivate the thiaminase, or by the use of supplemental dibenzoylthiamine (DBT) as a thiaminase resistant form of dietary thiamine.

Anti-vitamin A, E, D, B12: these anti-vitamin factors are reported to be present in raw soybeans. May be deactivated by heat treatment (Liener, 1980).

Anti-pyridoxine: anti-pyridoxine factor present in linseed meal; treatment as above.

Deficiencies due to dietary antibiotic addition

The use of feed antibiotics to treat disease outbreaks may destroy the vitamin synthesizing capacity of the gut microflora of fish, which in omnivorous/ herbivorous species may play an important role toward the vitamin requirements of the animal.

5.5.2 Vitamin toxicity

In contrast to the water soluble vitamins, fish and shrimp accumulate fat soluble vitamins under conditions where dietary intake exceeds metabolic demand. Under certain circumstances accumulation is such that a toxic condition (hypervitaminosis) may be produced. Although such a condition is unlikely to occur under practical farming conditions, hypervitaminosis has been experimentally induced in fish. Toxicity signs which have been reported include:

1 -Hilton (1983);

2 -Poston (1971);

3 -Halver (1980);

4 -Andrews, Murai & Page (1980)