The requirement of essential amino acids (EAA) are

generally estimated on the basis of the following parameters:

(1) The weight gain in the feeding experiments using the test

diets (amino acid-diets) whose protein sources were replaced

entirely or partly by the mixture of amino acids; or (2) The

daily increase of each EAA in the fish bodies when they were

fed on the diets containing proteins with high biological values

Kanazawa, Akio

CMFRI Digital Repository

^PM^BlQMfflM^SMQMQM^HiQH •/' f r \ Phe ^ His ' f'^ , • % -i0P? ': i*P P^ ie* « COOH ^4& k i ^ <|A jg : m «? up. Ey^^ftni^^fflB^ ^ B^"rijiBMSSMt^ Br QlTiJ^B I^B •• >'Vi :'f{ij;j..V^;. i>_V'" ^ ..--.* CMFRI SPECIAL PUBLICATION Number 8 MANUAL OF RESEARCH METHODS FOR FISH AND SHELLFISH NUTRITION Issued on the occasion of the Workshop on METHODOLOGY FOR FISH AND SHELLFISH NUTRITION organised by The Centre of Advanced Studies in Mariculture, Central Marine Fisheries Research Institute, held at Cochin from 11 -16 January 1982 Published by: E. G. SILAS Director Central Marine Fisheries Research Institute COCHIN " " " " • • • f f c f PREFACE The Centre of Advanced Studies in Mariculture established at the Central Marine Fisheries Research Institute has been conducting '.Jorkshops in Research Methodologies on specialised disciplines with a view to enhance the competence of the scienti-fic workers specialising in researches connected with mariculture. The main emphasis in mariculture research has been directed towards the development of economically viable culture techniques for culturable species of fish and shellfish, with a view to augmenting the fish and shellfish production of the country. In order to develop low-cost technologies the essential operational inputs have to be rationally utilized. + It has been well established that feeding constitutes the major cost of production, often exceeding 50 per cent of the » operating costs in intensive aquaculture operations. Two main factors affecting the cost of feeding are composition of the diet and efficiency of feed conversion. In order to develop least-cost formula diets of high conversion efficiency, knowledge of the nutritional requirements of the different species during the different phases of the life cycle and the nutritive value of the complex feed ingredients available in the country to the candidate species is a prerequisite. The existing information on the nutritional requirements of cultivated species of fish and shellfish in India, is meagre and recently research has been intensified in this area. If researches on this field could be carried ut using standardised experimental procedures, the data obtained on the nutritional requirements ot the different species could be stored in a fish and shellfish nutrition data batik, from where data could be disseminated to the users such as feed manufacturers, farmers, extension workers and research workers as and when required. It. is also necessary that the data collected on the chemical composition of the feed ingredients and their nutritive value tor the species should be based un standard chemical methods and experimental procedures so that the data could be stored in ii the dafca batik which eventually could become a National Fish Seed information. yCentpe.. i , r .'i;.u .,To.iUBd?fftake studies on the above lines, especially by the technicians and .research workers entering afresh into the field/ the,need of praptiqal guides describing the research techniques and methods, planning of investigations, collection of data and their interpretation peed not be emphasized. Keeping this in Y£»w<,.;the, .present manual, on Research Methods in Fish and Shellfish Nutrition is issued by the centre<of Advanced studies in Mari-culture on the occasion of the Workshop on Methodology of Fish and Shellfish Nutrition. Drf Akio Kanajawa, Professor of Nutritional Chemistry, University of Kagpsh,iniat| Japan and Consultant in Fish and Shell-fish Nutrition at the CAS in Mariculture, has been kind enough to cooperate with the scientists of CAS in Mariculture of the Central Marine Fisheries Research institute in the preparation of t£i» Bvanua,!. There are chapters in this manual covering various methods on(composition analysis of feeds, including growth inhibitors and toxins; deterrainetlpn of digestibility coefficient; protein evaluation; bioenergetics; determination of essential amino acid requirements using radioisotope method; research, test diets for fishes and prawns; feed formulation methods; experimental design, etc. Methods of preparation of mlcroparfciculate diets, phytoplankton and zppplankton culture methods, etc, are also included to facilitate larval nutrition studies. , Many of the methods given in the manual have been standardized for fish and shellfish nutrition studies in India and abroad. , The users can also gain maximum benefit by suitable modifications of other methods which are given as guidelines. I would like to thank ail the scientific and technical staff especially Shri s. Ahamed All, Dr. K. Alagarswami, Shri D.C.V. Ea'sterson, Shri C.P., Gopina^han, Shri T. Jacob, Shri M.S. Muthu,,Dr. R. Paul Raj, Dr. A.G. ponniah and ill / in:, p. vedavyasa Rao who have rendered assistance during the preparation of this manual. Thanks are also due to Shri Johnson, Librarian and shri Kambadkar, Technical Assistant, Central Marine Fisheries Research institute, for the help rendered bythtra in printing this manual. (E.G. Silas) Director, CMPRI, Sub-Project Coordinator, Centre of Advanced studies in Maricultuce CHAPTER IX DETERMINATION OF ESSENTIAL AMINO ACID REQUIREMENT OP PRAWN (Radioisotopic tracer method) 1 INTRODUCTION The requirement of essential amino acids (EAA) are generally estimated on the basis of the following parameters: (1) The weight gain in the feeding experiments using the test diets (amino acid-diets) whose protein sources were replaced entirely or partly by the mixture of amino acids; or (2) The daily increase of each EAA in the fish bodies when they were fed on the diets containing proteins with high biological values. In the case of the prawn, however, the amino acid-diets are known to attain only poor growth as also observed in the carp. This makes it difficult for us to estimate the EAA requirements of prawn by the feeding trials. Therefore, to define the amino acids essential for the growth of the prawn, radio-isotopic methods are used. 2 PROCEDURE n 2.1 Injection of 14 Acetate to the prawn Two speciments of the prawn, about 6.0g in total body weight, are each injected with 15 uCl of 14 sodium acetate dissolved in 1.6yul of aqueous solution containing carrier sodium acetate (0.1%). The radioactive acetate is injected into the muscle between the carapace and the second abdominal segment, and then the prawns are maintained on the fresh meat of short-necked clam in the aquarium (30 1) at 25°C. 2-2 Fractionation of the prawn n Six days after injection of 14 acetate, the whole body of prawns is fractionated into 4 fractions by the method of * Prepared by Aklo.Kanazawa, Professor of Nutritional Chemistry, University of Kagoshima, Japan. 07 SCHNEIDER; that is, the pravms are minced, homogenized in an ice-cold trichloroacetic acid (TCA), and sej>arated into the TCA-soluble compound fraction and the residue. The residua is then extracted with organic solvents such as ethanol-water (4:1), ethanol, and ethanol-ether (3:1) to give the lipid fraction. The defatted residue is further separated into the protein and nucleic acid fractions by using a hot TCA solution. 2.3 separation and Quantification of individual amino acids The protein fraction is oxidized with performic acid as described by HIRS and than hydrolized in a sealed test tube with 5.7N hydrochloric acid by the method of MOORE and STEIN. The performic acid oxidation converts cysteine and cystine to cystelc acid and methionine to the corresponding sulphone. The amino acids of protein hydrolysate are separated and quanti-fied by the method of MOORE et al. using column chromatography on Amberlite IR-120 (type II, Rohm and Hass co.) with sodium citrate buffers. Tryptophan is also quantified and separated in the same manner after hydrolyzing the protein fraction in 5N sodium hydroxide solution as mentioned by 03!>SHLEGEIi et al. 2.4 Measurements of Radioactivity The radioactivity of lipid, TCA-soluble compound, and nucleic acid fractions from the prawns and individual amino acids from the protein fraction is measured wth a Beckman Liquid scintillation Counter LS-230 using a dioxane solution of ppo (0.6;) and naphthalene (11.2/i) as a scintillation cocktail. The radioactivity of protein fraction is determined in the similar manner after hydrolysis with hydrochloric acid. Distribution of radioactivity in the fractions obtained from the prawns 6 days after injection of acetate (30 iiCi) Fraction TCA-soluble compounds Lipids Nucleic acids Proteins Whole body of prawns sea Water Total radioactivity recovered Total radioactivity recovered (yuci ) 0.39 0.73 0.03 0.15 1.30 25.80 27.10 Incorporation of radioactivity into the individual amino acids of protein fraction in the prawns 6 days after injection of acetate Amino acids Aspartic acid Serine Glutamic acid Proline Glycine Alanine Cysteic acid Tyrosine Threonine Specific activity cpm/yumol) 203 102 196 13S 132 187 159 1.2 1.9 Amino acids Valine Methionine Isoleucine Leucine Phenylalanine Lysine :iistidine Arginine Tryptophan Specific activity (cpm/ ^ umol) 0.1 2.5 0.7 0.1 0.1 0.2 0.7 1.2 0.4 39 Radioactivity is unambigously incorporated into aspartic acid, serine, glutamic acid, proline, glycine, alanine, and cysteic acid. These amino acids are suspected to be unnecessary for the prawn. On the other hand, little or no radioactivity is incorporated into valine, methionine, isoleucine, leucine, phenylalanine, lysine, histidine, arginine, threonine, end tryptophan. These 10 amino acids are not synthesized de novo and are probably essential for the growth of the prawn. Tyrosine is thoughfto be formed from the ingested phenylalanine. 3 REFERENCE 1. Manual Book of Experiments, Laboratory of Fisheries Chemistry, University of Kagoshima, Japan. 1980-81. 

Determination of essential amino acid requirement of prawn

(radioisotopic tracer method)

http://eprints.cmfri.org.in/3269/1/Spl_8-11.pdf

Determination of essential amino acid requirement of prawn (radioisotopic tracer method)

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