Dr. Subhendu Datta Sr. Scientist CIFE, Kolkata Centre e-mail: subhdatta@yahoo.

com Introduction: The micronutrients, as the name suggests, are those components of the diet, which, although essential, are required in only relatively small quantities. If these are absent, or insufficient, they give rise to specific deficiency diseases, which often have particular clinical and histopathological features. To many workers the term ‘micronutrient’ means trace elements e.g. Fe, Mn, Cu, Co, Zn, Mo, Se etc. Where as Halver et al. (2002) in the latest edition of “Fish Nutrition” included only vitamins under micronutrients (Chapter 8, article, 8.4.p 464). However, I will describe here both trace minerals and vitamins because these are two groups of unrelated inorganic and organic compounds required only in small amounts. But one must remember that they may be required at higher levels at times of fast growth or reproduction. They may also be required at significantly increased levels in high-energy diets. Trace minerals: All forms of aquatic animals required inorganic elements or minerals for their normal life processes. Unlike most terrestrial animals, fish have the ability to absorb some inorganic elements not only from their diets but also from their external environment in both fresh water and seawater. Many essential elements are required in such small quantities that it is difficult to formulate diets and maintain an environment that is low in minerals to demonstrate a mineral deficiency. It is well recognized that all living organisms contain most naturally occurring elements in the periodic table. At present, 29 of the 90 naturally occurring elements are known to be essential for animal life. The greater proportion of living matter consists of six basic structural elements – C, H, N, O, P an S. These elements, found at high concentrations, are required in gram amounts. In addition, five macroelements, Ca, Mg, Na, K and Cl (as chloride), are also required in gram quantities. The remaining elements occur in the body at much lower concentrations (milligrams or micrograms per kilogram body wt.). Initial difficulties in the 1

accurate determination of low levels of many of these elements inevitably led to their description as “trace elements”. Fifteen trace elements are considered to be essential in animals. Among these the physiological role of deficiency of iron, manganese, copper, zinc, selenium, iodine, cobalt, chromium, molybdenum and fluorine is well recognized. Although deficiencies of nickel, vanadium, silicon and arsenic have been demonstrated in an ultraclean environment, with the exception of silicon, the physiological function of these trace elements has not been clearly demonstrated. Other elements, including cadmium, lead, bromine and tin, have also been claimed to be essential, but their essentiality remains to be confirmed. Most of these trace elements have been detected in fish tissues; however, the essentiality of only a few of these elements has been demonstrated. In general, the major functions of essential elements in the body include the formation of skeletal structure, maintenance of colloidal systems (osmotic pressure, viscosity, diffusion) and regulation of acid-base equilibrium. They are important components of hormones, enzymes and activators of enzymes. A fixed number of specific trace elements (Fe, Mn, Cu, Co, Zn, Mo, Se etc.) are firmly associated with specific protein in metalloenzymes, which produce a unique catalytic function (Table 1). Certain minerals, such as Ca, Mg and Mn, are of particular significance as enzyme activators. A nonmetal, iodine, is necessary for the biosynthesis of thyroid hormones, which in turn greatly affect development and metabolism in all vertebrates. Some biologically important compounds contain trace mineral as an inherent part of their structure, e.g., haemoglobin (Fe) and vitamin B12 (Co). Role and requirements of Fe, Mn, Zn, Cu, Se, Mo and I in fish are presented in tabular form in Table 1-3. Chromium (III) is required for normal carbohydrate and lipid metabolism. The biological function of Cr is closely related to that of insulin and chromium supplementation in carp diet improved the glucose utilization.

Vitamins: Vitamins are organic molecules that act as cofactors or substrates in some metabolic reactions. They are generally required in small quantities in the diet, and if present in adequate amounts, may result in nutrition-related diseases, poor growth or increased susceptibility to infections. The vitamin requirements of majority of the species of fish in culture have not been determined. As a result, the data obtained from studies of salmonids, carp or catfish are usually applied to other species. As can be observed from Table 4, measured vitamin requirements of different species vary considerably. If data from a different species are used, one of the three 2

outcomes may occur. The first is that too great a level of vitamin may be included in the diet, which at least increases the cost of the feed.

Table 1. Essential Metalloenzymes in aquatic Animals Trace Enzymes elements Fe Succinate dehydrogenase Cytochromes (a,b,c) Catalase Cu Cytochrome oxidase Lysyl oxidase Ceruloplasmin (ferroxidase) Superoxide dismutase Zn Carbonic anhydrase Alcohol dehydrogenase Carboxypeptidases Alkaline phosphatase Polymerases Collegenase Mn Pyruvate carboxylase Superoxide dismutase Mo Glycosylaminotranferases Xanthine dehydrogenase Sulfite oxidase Aldehyde oxidase Se Glutathione peroxidase Type I and III deiodinases Function Aerobic oxidation of carbohydrates Electron transfer Protection against H2O2 Terminal oxidase Lysine oxidation Iron utilization, copper trasport
Dismutation of the superoxide free radical (O2.-)

CO2 formation Alcohol metabolisms Protein digestion Hydrolysis of phosphate esters Synthesis of RNA and DNA chains Wound healing Pyruvate metabolisms
Dismutation of the superoxide free radical (O2.-)

Proteoglycan synthesis Purine metabolism Sulfite oxidation Purine metabolism Removal of H2O2 Conversion of thyroxide to the active form.

Table 2. Mineral requirements of certain finfisha Species Rainbow trout Atlantic salmon Pacific salmon Tilapia Channel catfish Common carp Japanese eel Red sea bream
a

Fe (mg) Rb 30-60 R R 30 150 170 R

Cu (mg) 3 5 R 3.5 5 3 R R

Mn (mg) 13 10 R 12 2.4 13 R R

Zn (mg) 15-30 37-67 R 20 20 15-30 R R

I (µg) 1.1 R 0.6-1.1 R 1.1 R R R

Se (mg) 0.15-0.3 R R R 0.25 R R R

Amount per kg feed. Requirements were determined using purified or semi-purified diets. Factors affecting the bioavilability of these elements and nutrient interactions must be considered when formulating fish feeds. b Essential in the diet but the quantitative requirement was not reported.

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Table 3. Mineral deficiency signs reported in certain fin fish Minerals Fe Deficiency Signs (fish species)a Reduced growth and poor feed conversion (6), hypochromic microcytic anemia (2,3,7-9), low hematocrit and hemoglobin levels (1,3,6), reduced plasma Fe and Fe transferring saturation (3,6) Reduced growth (1,3,6,7), anorexia (6,7), short-body dwarfism (1), cataracts (1,7), fin erosion (1,7), skin erosion (7), reduced body zinc (3), bone zinc (1,6) and bone Ca (6) concentrations, low serum Zn level (6), mortality (1,7). Reduced growth (1,7,11), loss of equilibrium (11), short-body dwarfism (1,7), cataracts (1,7), high mortality (3, 11), reduced bone (2,3) and body (3) Mn concentration (2,3), poor hatchability of eggs (1,2,3), abnormal tail growth (1). Reduced growth (7), cataracts (7), reduced liver Cu/Zn-superoxide dismutase (3) and heart cytochrome c oxidase activity (3,6) Reduced growth (6,7), anemia (7), cataracts (7), mascular dystrophy (3), exudative diathesis (1), reduced glutathione peroxidase activity (1,3,6) Thyroid hyperplasia (1,2,5)

Zn

Mn

Cu Se I
a

Key to fish species: 1. Rainbow trout (Oncorhynchus mykiss); 2. Brook trout (Salvelinus fontinalis); 3. Atlantic salmon (Salmo salar); 5. Chinook salmon (Oncorhynchus tshawytscha); 6. Channel catfish (Ictalurus punctatus); 7. Common carp (Cyprinus carpio); 8. Japanese eel (Anguilla japonica); 9. Red sea bream (Chrysophrys major); 11. Mozambique tilapia (Oreochromis mossambica).

Table 4. Vitamin requirements (mg/kg dry diet basis) for growtha Vitamins Trout Salmon Carp Channel catfish 1-3 9 3 25-50 14 R R Sea bream R R 5-6 R R R R 300-900 R N R
1000-2000 IU ?

Thiamin (B1) 10-12 10-15 2-3 Riboflavin (B2) 20-30 20-25 7-10 Pyridoxine (B6) 10-15 15-20 5-10 Pantothenate 40-50 40-50 30-40 Niacin 120-150 150-200 30-50 Folacin 6-10 6-10 N Cyanocobalamin R 0.015-0.02 N (B12) myo-Inositol 200-300 300-400 200-300 R Choline 2000-4000 3000 1500-2000 R Biotin (Vit H) 1-1.2 1-1.5 1-1.5 R Ascorbate (C) 100-150 100-150 30-50 60 2000-2500 IU 2000-2500 IU 1000-2000 IU 1000-2000 IU A 2400 IU 2400 IU N 500 – 1000 IU D E 30 30 80-100 30 K 10 10 R R a R. required, but level not known; N, no requirement shown; ?, unknown.

? ?

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unnecessarily, or at the most can result in vitaminosis, a disease induced by excess amounts of vitamin in the diet. Vitaminosis is usually caused by excess amounts of fat-soluble vitamins, which are difficult for an animal to excrete. If insufficient vitamin is included in the diet, a nutritional disease or poor growth performance is observed. The third alternative is that the right amount of vitamin is included. Most nutritionist hope for the last outcome. Determining vitamin requirements in fish, as in other animals, is difficult, since microorganisms in the gut produce many vitamins. Depending upon the activity of an animal’s gut microflora, the requirement for dietary vitamins can vary. It is likely that all fish require all vitamins, but whether it is necessary to include them in formulated diet is a different matter. A species such as carp can obtain many nutrients from decaying organic matter which it will consume in addition to any artificial diet in all but the most sterile of conditions, and so the requirements for vitamins in supplementary feeds for this species are less than for a species which has a carnivorous feeding habit and relies entirely upon the components of an artificial diet for its vitamin intake. Similarly, animals held in tanks have less opportunity to consume natural sources of vitamins than those held in ponds and so require greater levels of vitamins in any artificial feed they may be given. Vitamins can be divided into two groups, the fat-soluble vitamins, A, D, E and K and the water-soluble vitamins, which are the B group, C and some specific cofactors. The major vitamins, their active forms and physiological functions are listed below: Thiamine (Vitamin B1): The active form of thiamine is thiamine pyrophosphate. Thiamine pyrophosphate is a coenzyme for reactions, which are involved in carbohydrate metabolisms. These reactions are the decarboxylation of α-keto acids and the formation and degradation of αketols. Magnesium is also required as cofactor. Of particular importance is the role of thiamine in the conversion of pyruvate to acetyl CoA. Ribloflavin (Vitamin B2): Riboflavin is a yellow-pigmented molecule, which in the body forms a component of the molecule flavin adenine dinucleotide (FAD). FAD is a cofactor in a number of oxidation-reduction reactions and acts as an energy currency similar to ATP. It is particularly important in the degradation of pyruvate, fatty acids and amino acids and in the process of electron transport. Nicotinic acid (Niacin): It is a component of two high-energy molecules – nicotinamide adenibe dinucleotide (NAD) and nicotinamide adenibe dinucleotide phosphate (NADP). These two, like 5

FAD, are important in a number of oxidation and reduction reactions that occur within cells, e.g. reactions involved in the citric acid cycle. Nicotinic acid can be synthesized by most of the animal from the amino acid tryptophan. However, tryptophan, is only present in very small amounts in most animal meals and supplementation of the diet with nicotinic acid is therefore advisable. Pantothenic acid (Coenzyme A): It is the coenzyme of acetyl, acyl or propionic CoA. This molecule serves as a carrier of various carbohydrate groups and is involved in reactions in fatty acid oxidation, fatty acid synthesis, pyruvate oxidation and acetylations. The presence of coenzyme A within a cell is fundamental to the transfer of energy throughout the various reactions. Pyridoxine (Vitamin B6): Pyridoxine as pyridoxal phosphate acts as an important coenzyme in transamination reactions thereby plays an important role in protein metabolisms. This vitamin plays an important role in fish metabolism, because major sources of energy in fish metabolism occur from the degradation of amino acids. Carnivorous fish have stringent requirements for pyredoxine in the diet and stores are rapidly exhausted. Biotin: Biotin acts to facilitate CO2 transfer in reactions which require the addition of CO2 to another molecule. Biotin is made by the intestinal bacteria and a deficiency in this vitamin is not readily produced by withholding the vitamin from the diet. A glycoprotein component of egg white called avidin binds biotin in the gut in such away that it can not be absorbed by the intestinal mucosa and thus can be used to induce biotin deficiency. Heat denatures avidin, allowing it to be digested and so any biotin complexed can be absorbed. Clearly care must be taken if including raw eggs into fish feeds. Folic acid: Folic acid is a cofactor in the transfer of single carbon entities (e.g. methyl group, CH3) to the other molecules in the same way that biotin is a carrier for CO2. The folic acid molecule is composed of three separate parts called pterodine, p-amino benzoic acid and glutamic acid. In some organisms, folic acid can be synthesized if p-amino benzoic acid is provided in the diet.

Cyanocobalamin (Vitamin B12): It is essential for normal maturation and development, required for the synthesis of choline and the metabolisms of single carbon fragments. The requirement for 6

vitamin B12 in the diet is only as a trace, and is difficult to induce a dietary deficiency because the microorganisms in the gut synthesize vitamin B12. The usual problem with vitamin B12 deficiency occurs because a carrier mucoglycoprotein called intrinsic factor is lacking in the gut. The absence of intrinsic factor means that the vitamin B12 present in the intestinal contents will not be absorbed by the animal and deficiency results. Ascorbic acid (Vitamin C): Ascorbic acid is required in the diet of some species of fish. The first recognized function of ascorbic acid is its role in hydroxylating the proline to hydroxyproline for use in cartilage synthesis. However, it acts as a strong reducing agent in a number of other reactions. It is involved in carnitine synthesis and in the detoxification of pesticides and other toxicants in process involving cytochrome P450. It is a highly labile vitamin easily destroyed by cooking or lengthy or improper storage of food. It is usually added to five- to 10 fold excess to allow for degradation during storage and to provide some shelf-life to the feed. Inositol: Inositol has no known cofactor activity but as an important component of phospholipids, is important in the synthesis of membranes in cells. Choline: Choline also has no known coenzymes function but acts as an important methyl donor in a number of metabolic reactions, such as the production of acetylcholine from acetyl CoA. Animals can synthesize choline if there is adequate methionine in their diet. Inclusion of choline into fish diets alleviates some of the requirement for the methionine which is present in low amounts in most protein sources. Retinoic acid (Vitamin A): Vitamin A is a fat soluble vitamin which is important as a component of the protein rhodopsin, a light absorbing pigment found in the retina of the eye. Two forms of vitamin A are known. Vitamin A1 is common in marine fishes, whilst vitamin A2 is common in freshwater fishes. The two molecules are very similar; the only difference being that there is an additional double bond present in the carbon ring structure of vitamin A2. Vitamin A is also important in cells other than the retina since deficiency causes damage in epithelial, bony and connective tissues. However, its exact function is not known. β-Carotene is often used as a dietary source of vitamin A since it is composed of two vitamin A molecules, which are readily separated by hydrolysis.

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Vitamin D (Calciferol): The vitamin D group of compounds consists of several molecules, although the most important are vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Of these it appears that fish are only able to use vitamin D3. Vitamin D is a precursor to 1, 25dihydroxycholecalciferol, a hormone important in regulating calcium and phosphate levels in the serum. Vitamin D synthesis occurs in most animals by ultraviolet radiation of 7dehydrocholesterol. Whilst this reaction is likely to occur in many fishes, the shallow penetration of UV irradiation through water is likely to lead to a requirement for vitamin D in the diet of fish. Vitamin E (Tocopherol): It acts as an antioxidant, particularly protecting polyunsaturated fatty acids. High dietary levels of polyunsaturated fatty acids increase the requirement for dietary vitamin E. An interaction exists between vitamin E and selenium, a metallic antioxidant; vitamin E requirements are greater in selenium-depleted fish. Vitamin K: Vitamin K is a group of several compounds called Phylloquinone or menaquinone, which are isolated from plant or animal tissue respectively. A third compound, menadione is a synthetic compound that has greater vitamin K activity than either of the naturally occurring substances. Vitamin K is important in the synthesis of prothrombin, a protein, which is important in blood clotting. All animals require Vitamin K, including fish, for the normal blood clotting. Vitamin requirements for fish As stated earlier, the requirement for vitamins by different species of fish varies greatly according to their usual feeding habit and capacity to synthesize them. The known requirements of those fish that have been investigated are shown in Table 4. The observed difference in vitamin requirements in Table 4 may be due to the variation between experimental regimes, but more likely to be the result of variations between species. It is therefore very difficult to determine a generally recommended level of vitamin supplementation that will be satisfactory for all fish species. Inclusion of vitamins in diets is complicated further since most of them are highly labile molecules and are readily destroyed during processing. It is easy to overcome this problem for the water-soluble vitamins by adding excess amounts since any excess amount consumed is readily excreted. However, in the case of fat-soluble vitamins, excess amounts accumulate in the body and cause vitaminosis or vitamin poisoning. Adding large amounts of vitamins, even those that are water soluble, has the additional disadvantage of the extra expense, which is undesirable. The common practice of basing inclusion of vitamins on known 8

requirements of other species generally means using data from rainbow trout for cold-water fish, and from catfish or carp for warm water fish. This strategy probably provides a compromise between determining the exact requirement experimentally and adding vitamins to excess. Deficiency syndromes of different vitamins are mentioned below in Table 5.

Table 5. Vitamin deficiency syndromes Vitamins
Thiamin Riboflavin

Deficiency signs in salmonids, ictalurids, cyprinids etc.
Poor appetite, muscle atrophy, convulsions, instability and loss of equilibrium, edema, poor growth Corneal vascularization, cloudy lens, hemorrhagic eyes, photophobia, dim vision, incoordination, abnormal pigmentation of iris, striated constrictions of abdominal wall, dark coloration, poor appetite, anemia, poor growth. Nervous disorders, epileptiform fits, hyperirritability, ataxia, anemia, loss of appetite, edema of peritoneal cavity, colourless serous fluid, rapid postmortem rigor mortus, rapid and gasping breathing, flexing of opercles. Clubbed gills, prostration, loss of appetite, necrosis and scarring, cellular atrophy, gill exudates, sluggishness, poor growth Loss of appetite, lesions in colon, jerky or difficult motion, weakness, edema of stomach and colon, muscle spasm while resting, poor growth Poor growth, lethargy, fragility of caudal fin, dark colouration, macrocytic anemia Poor appetite, low hemoglobin, fragmentation of erythrocytes, macrocytic anemia Poor growth, distended stomach, increased gastric empting time, skin lesions Poor growth, poor food conversion, hemorrhagic kidney and intestine Loss of appetite, lesions in colon, skin colouration, muscle atrophy, spastic convulsions, fragmentation of erythrocytes, skin lesions, poor growth Scoliosis, lordosis, impaired collagen formation, altered cartilage, eye lesions, hemorrhagic skin, liver, kidney, intestine and muscle No abnormal indication in growth, appetite, mortality Impaired growth, exophthalmos, eye lens displacement, edema, ascites, depigmentation, corneal thining and expansion, degeneration of retina Poor growth, tetany of white skeletal muscle, impaired calcium homeostasis Reduced survival, poor growth, anemia, ascites, immature erythrocytes, variable-sized erythrocytes, erythrocyte fragility and fragmentation, nutritional muscular dystrophy, elevated body water Prolonged blood clotting, anemia, lipid peroxidation, reduced hematocrit

Pyridoxine

Pantothenic acid Nicotinic acid Folic acid Cyanocobalamin Inositol Choline Biotin Ascorbic acid
p-Amino benzoic acid

A D E

K

Dietary sources of vitamins: Vitamins are contained in the fresh products from which diets are made. However, processing method and time can destroy these compounds, a problem not easily resolved. For example, cooking destroys vitamin C, whilst another vitamin, thiamin, is protected by heat. Heat-treating fishmeal destroys the thiaminase, which breaks down thiamine. Processes such as solvent extraction of oil seeds remove the fat-soluble vitamins and choline. Inositol, on 9

the other hand, is common in seeds but is largely present in a form, which is indigestible to fish. Considerable work is being undertaken to develop methods of protecting vitamins included in animal feeds, particularly vitamin C. The use of techniques, which physically protect (encapsulation) or chemically protect active sites (e.g. esterification of vitamin C) is proving to be successful in delivering accurate doses of vitamins to fish.

Present status of research on Indian major carps: While vitamins are vital for growth and metabolism their requirement depends on the maturity stage, environmental conditions, dietary nutrient interactions etc. Vitamin requirements except that of vitamin-C have not been adequately studied in Indian major carps. All the three species of major carps lack L-Glucono-γlactone oxidase, the terminal enzyme for the conversion of glucose to ascorbic acid (Mukhopadhyay et al., 1996). This indicates that these species are unable to synthesize ascorbic acid de novo and exogenous supply along with feed is mandatory. The requirements are based on the minimum dietary level that support maximum growth and/or maximum tissue storage and prevent deficiency signs. Exact requirement level is difficult to assess because the vitamin is highly susceptible to storage loss. However, when dietary supply was 1000 mg/kg feed; growth, feed efficiency, tissue storage and vertebral collagen content were found to be optimum for Labeo rohita fry and fingerlings. Jain et al. (1994) showed that diet supplemented with 1 mg chromium/100 g enhanced growth of Labeo rohita by about 72%. However, excess dietary chromium (4 mg/100 g) proved to be deleterious. Devraj and Seenappa (1991) incorporated a mineral premix to a diet and observed increased growth compared to fish fed diets, which were not supplemented with minerals.

References:
1. Mukhopadhyay, P.K., Nandi, S., Hassan, M.A., Dey, A. and Sarkar, S. (1996). Effects of dietary deficiency and supplementation of ascorbic acid on growth performances, vertebral collagen, tissue vitamin and enzyme status in the carp Labeo rohita. In: Hamed, M.S. and Karup, B.M. (Eds.). Technological Advancement of Fisheries, Cochin University of Science and Technology publication.pp.101-107. Jain, K.K., Sinha, A., Srivastava, P.P., Berendra, D.K. (1994). Chromium: an efficient growth enhancer in Indian major carp Labeo rohita. J. Aqua. Trop. 9: 49-54. Devraj, K.V. and Seenappa, D. (1991). Studies on nutritional requirements and feed formulations for cultivable carps. Final Research Report 1987-1991. Inland Fisheries Division, University of Agricultural Science, Hebbal, Bengalore.pp185.

2. 3.

(Go ahead......) 10

There are several methods of estimation for each trace element and vitamin, describing those methods is beyond the scope of this article and in terms will be irrelevant also. Only few methods/principles are described below. Estimation of Vitamin-C in feed: Titrimetric method: Principle: Vitamin-C is ascorbic acid chemically. Ascorbic acid is a reducing agent and it is determined by its reaction with 2,6 –dichlorophenol. The dye, which is blue in alkaline solution and red in the acidic solution is reduced to colourless form in the pH range 1-3.5. Reagents: 1. Ascorbic acid: Dissolve 100 mg of l-ascorbic acid in 3% HPO3 and make up the volume to 1 litre (1 ml = 0.1 mg of ascorbic acid). 2. Metaphosphoric acid (HPO3): prepare 3% solution with glass distilled water. 3. Dye solution: Dissolve 50 mg of sodium salt of 2,6-dichlorophenol (indophenol) in 150 ml of a solution containing 42 g of sodium bicarbonate. Make it to 200 ml with water and store in a refrigerator. It should be standardized every day. Standardization: To 5 ml of standard ascorbic acid in a conical flask add 5 ml of HPO3. Titrate this solution with dye solution taken in a microburette to pink colour end point, which persists for 15 seconds. Dye factor = mg of ascorbic acid per ml of dye = 0.5/titre Preparation of sample: Take 10 g of solid food and blend it with 3 % HPO3 to make total volume 100 ml. Filter or centrifuge this material. Procedure: Take 5-10 ml of sample prepared in 3% HPO3 and titrate with dye solution to a pink end point which should persist for 15 seconds. Repeat this titration to get more accurate result. The volume of dye used may be 3-5 ml. If SO2 is present in the sample it will interfere in the titration. To eliminate the interference take 10 ml filtrate in a test tube and add 1 ml of 40% formaldehyde and 0.1 ml of HCl. Lay it aside for 10 minutes and titrate as before. Calculation: Titre x dye factor x volume made up x 100 mg of ascorbic acid in 100 g = Volume of extract taken for titration x weight of sample 11

Colorimetric method: Principle: The procedure is based on the decolourisation of 2,6-dichloro-phenol-indophenol with ascorbic from the extract and comparing it with standard ascorbic acid. Reagents: 1. Metaphosphoric acid HPO3: 2% solution 2. Dye solution: Weigh 100 mg of 2,6-dichlorophenol-indo-phenol and 84 g of sodium bicarbonate solution in water by heating to 90°C. Cool and make up the volume to 100 ml. Take 25 ml of this solution and dilute to 500 ml. 3. Ascorbic acid: Dissolve 100 mg of ascorbic acid in 100 ml of 2% HPO3. To 4 ml of this solution add 2% HPO3 to make 100 ml. [1ml of this solution = 40 µg of vitamin C]. Procedure: Prepare the samples as in volumetric method but using 2% HPO3. Blend 50-100 g sample with equal weight of HPO3 and make volume to 100 ml. Standard curve: Take 0.5, 1, 2, 3, 4 and 4.5 ml of ascorbic acid in different test tubes and make volume to 5 ml by adding 4.5, 4,3,2,1,0.5 ml of 2% HPO3. Add 10 ml of dye solution with a pipette. Shake and measure optical density (absorbance) of these solutions within 15-20 seconds at 518 nm setting the spectrophotometer at 100% transmission using 5 ml of 2% HPO3 as blank. Plot the absorbance against concentration. Now measure 1-4 ml of the extract and make to 5 ml with 2% HPO3 in a test tube. Add 10 ml of the dye solution and measure its absorbance. Calculation: ascorbic acid content x volume made x 100

mg of ascorbic acid per 100 g of sample = ml of solution taken x 100 x weight of sample Principle for the estimation of Riboflavin in feed: Riboflavin is extracted with dilute acids and after removing the interfering substances by treatment with KMnO4, it is determined in a Fluorimeter at 450-500 nm wavelength. The intensity of fluorescence is proportional to the concentration. Principle for the estimation of folic acid in feed: Feed material is treated with alkaline buffer to extract folic acid, which is oxidized with KMnO4 to form an amine. This is diazotised and then coupled with N-(1 naphthyl) ethylene diamine to form a coloured compound which is evaluated colorimetrically at 550 nm.

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Principle for the estimation of Thiamine in feed: Feed material is treated with dilute HCl to extract thiamine complex, which is then treated with phosphatase to liberate free thiamine. It is purified by passing through base-exchange silicate alkaline column to remove interfering compounds. The column is eluted with ferricyanide to oxidize thiamine to thiochrome, which is measured fluorometrically (For details method of these three estimations please check: Reference 1). Estimation of trace minerals in feed: Moisture, protein, fat, fibre, ash, calcium, phosphorus, salt and sugar are generally estimated in feed samples. Fish nutrition point of view, estimation of trace elements in fish feeds is not so common in India. However, in the field of aquatic pollution and toxicology, some of the trace elements e.g. Pb, Cd, Zn, Mn, Cu, As, Cr, Se are intensely studied and estimated. So if required these elements could also be studied at ease in feed samples also to judge the nutrient values, as the basic principles are same. Various volumetric, gravimetric and colorimetric/spectrophotometric methods are often used for analysis of various elements, while only atomic absorption spectrophotometric (AAS) method is reliable because of low concentration and chances of interferences from other elements. Detailed AAS method for all the trace minerals is also beyond the scope of this article. Only outline of AAS method for few trace minerals is given below. Sample preparation: Samples are homogenized using a sample mill, which should not give any metallic contamination. The regular type of sample mill normally provides contamination of iron from the cutting blade and zinc and copper from brass sieves. The sample mill consisting of hard plastic, for example as developed by Tecator, can be used for grinding. After grinding, the sample may be dried at 70°C before taking up weighing for digestion. Digestion of the sample: For nutrients other than N, the feed material can be digested in a diacid mixture or a triacid mixture or dry ashed and dissolved in acid. The diacid digestion is used for the determination of Ca, Mg, S, Fe, Mn, Zn and Cu. The triacid digestion is recommended only when P and K are to be estimated. Since H2SO4 can contribute some trace elements and heavy metals, diacid digestion is normally recommended for the estimation of trace minerals in feed samples prepared from plant materials. Wet digestion is normally not used for the estimation of B and Mo. Dry ashing is the preferred technique for these two minerals estimation. Diacid digestion: Take 1 g ground sample and placed it in 100 ml volumetric flask. To this, 10 ml of acid mixture is added (9:4 mixture of HNO3: HClO4) and the content of the flask is mixed by swirling. The flask is placed on low heat hot plate in a digestion chamber. Then the flask is 13

heated at higher temperature until the production of red NO2 fumes ceases. The contents are further evaporated until the volume is reduced to about 3 to 5 ml but not to dryness. The completion of digestion is confirmed when the liquid become colourless. After cooling the flask, add 20 ml of deionized water. Volume is made up with deionized water and the solution is filtered through Whatman No.1 filter paper. Aliquots of this solution are used for the determination of Fe, Mn, Zn and Cu. If the sample is high in fats/oils, pre-digestion using 25 ml HNO3/g sample is recommended to avoid explosion. Triacid digestion is carried out using mixture of HNO3:H2SO4:HClO4 in the ratio of 9:4:1. The sample digestion is carried out as described under the diacid digestion.

Determination of Fe, Mn, Zn and Cu by AAS in fish feed: Principle: The AAS is based on the principle that atoms of metallic elements (Fe, Mn, Zn and Cu), which normally remain in ground state under flame conditions absorb energy when subjected to radiations of specific wavelength. The absorption of radiation is proportional to the concentration of atoms of that element. The absorption of radiation by the atoms is independent of the wavelength of absorption and temperature of the atoms. The AAS technique is very specific because only the atoms of a particular element can absorb radiation of their own characteristics wavelength; therefore, the spectral interferences rarely occur in AAS. The use of AAS is not only restricted to aqueous solutions, but also to non-aqueous solutions. The AAS instrument has the ability to calibrate and compute concentration using absorbance data from linear and nonlinear curves. Sample preparation: 1 g oven dried feed samples are digested using diacid mixture (as described above) and made up to 100 ml using deionized water on cooling. Preparation of standard stock solution: Iron: Dissolve 1 g of pure iron wire in 50 ml of (1+1) analytical grade HNO3. Dilute to 1 litre with deionized water. Manganese: Dissolve 1 g of manganese metal in a minimum volume of (1+1) HNO3. Dilute to 1 litre with 1% (v/v) HCl. Zinc: Dissolve 0.5 g of zinc metal in a minimum volume of (1+1) HCl and dilute to 1 litre with 1% (v/v) HCl. Copper: Dissolve 1 g of copper metal in minimum volume of (1+1) HNO3. Dilute to 1 litre with 1% (v/v) HCl. 14

Determination: The determination of Fe, Mn, Zn, Cu is done by using AAS with the following specifications for mono element hollow cathode lamp.

Specifications Lamp current (mA) Wave length (A°) Linear range (ppm) Silt width (A°) Integration time (sec.)

Fe 30 2483 0-5 2 2

Mn 20 2795 0-2 2 2

Zn 6* 2139 0-1 7 2

Cu 15 3248 0-5 7 2

*For determination of Zn, instead of hollow cathode lamps, Electrodeless Discharge Lamp (EDL) is used. EDL will
provide improved sensitivity and lower detection limits than hollow cathod lamp.

References:
1. Chopra, S.L. and Kanwar, J.S. (1991). Analytical Agricultural Chemistry (Fourth Edition). Kalyani Publishers, New Delhi, 488 pp. 2. De Silva, S.S. and Anderson, T.A. (1995). Fish Nutrition in Aquaculture. Chapman & Hall. London. 319 pp. 3. FAO/UNDP (1980). Compilation of lectures on ‘Fish Feed Technology’ held at College of Fisheries, University of Washington, from 9 Oct – 15 Dec, 1978. 395 pp. 4. Halver, J.E. and Hardy, R.W. (2002). Fish Nutrition. Academic Press, London. 824pp. 5. John, G. and Ninawane, A.S. (2000). Aquaculture: Feed and Health. Biotech Consortium India Ltd., New Delhi.187 pp. 6. Maiti, S. K. (2001). Handbook of methods in environmental studies.Vol-1. ABD publishers, Jaipur, India. 307 pp. 7. Tandon, H.L.S. (1993). Methods of Analysis of soils, plants, waters and fertilizers. FDCO, New Delhi.143 pp.

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