The chemical composition of the feed gives its potential

nutritive value and hence, in the assessment of quality of a

feed the proximate principles are first determined. The Weende

proximate analysis system for analyzing animal feed includes the

determination of moisture (dry matter) ether extract (crude fat)

crude protein, ash, crude fibre and nitrogen free extract (NFE)

Ahamed Ali, S

Paulraj, R

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 1 PROXIMAT%COMPOSITION ANALYSIS OF FEEDS* 1 INTRODUCTION The chemical composition of the feed gives its potential nutritive value and hence, in the assessment of quality of a feed the proximate principles are first determined. The Weende proximate analysis system for analyzing animal feed includes the determination of moisture (dry matter) ether extract (crude fat) crude protein, ash, crude fibre and nitrogen free extract (NFE). 7, COLLECTING AND LABELLING SAMPLES FOR ANALYSIS The method used to collect samples for chemical analysis affects the values of the parameters obtained. The aim is to take a sample of the material which represents the total and to analyse it in such a way that it represents the material which is consumed by the animal or to sample other material which affects the objectives of the experiment. Sampling of bagged ingredients is done with a spear probe. The probe is inserted diagonally and as horizontally as possible, from one corner of the bag to the other. In lots of 1-10 bags, all bags are sampled. In larger lots, 10 percent of all bags are sampled. Materials received in bulk are sampled using a scoop, according to the size of the consignment. For smaller than 10 tonnes consignments, two samples per tonne are taken. Larger consignments, up to 100 tonnes, require one sample per tonne or one sample for every two tonnes depending on the size of the consignment. oil cakes and other coarse materials are sampled by random selection of pieces from different parts of the entire consignment. * prepared, by R. Paul Raj and Syed Ahamed'Ali, Centre of Advanced Studies in Mariculture, Central Marine Fisheries Research institute, cochin-18. 2.1 Grinding and sub-sampling Samples taken In the above manner should be pooled, thoroughly mixed, and ground to pass through 1 mm screen in Wiley Mill (large samples may be ground through hammer mill (3/8" screen), sub sampled, regrbund through 4 mm screen, sub sampled and ground %hrough 1 mm screen). Combine all the materials left in mills With the ground portion before subsampling; take precautions to nix ground samples well before subsampllng in a waring blender. Place sample in airtight containers. If possible preserve all samples high in moisture content by freezing. 2.2 Method of submitting samples for chemical analysis At the time a sample is collected, a tag is attached. The pejreoa collecting the sample should fill in the project number, experiment number, date taken, a brief description of the sample and the name of the person doing the sampling. 2«3 Laboratory sample numbers At the time the sample comes to the laboratory, it is given a laboratory sample inumber. 3 DETERMINATION OP DRV MATTER 3.1 Principle The moiature of the sample is lost by volatilization caused by heat. The amount of material left after the. removal of the moisture is the dry matter. 3.2 Apparatuet (a) oven, 105*C (b) Covered aluminium dishes, 50 mm diameter (c) Dessicators 3.3 Procedure (a) Wash the dishes with a detergent. Dry the dishes In 105"c oven overnight. Place in dessioator, cool, and weigh. Handle dishes with metal tongs. (b) Weigh by different 2.0 g. of sample Into a weighed dish, place it In 105*C oven overnight. Remove dishes, put cover on top and place in dessicator and cool. Remove from dessicator and weigh as quickly as possible. 3.4 Calculation Dry matter (54) (Wt. of dish + wt. of dried sample) - wt. of dish X 100 X 100 Wt. of sample before drying m Wt. of dry sample X 100 Wt. of sample before drying Moisture content (%) Wt. of fresh sample - wt. of dry sample Wt. of fresh.sample 4 DETERMIHATION OP ASH 4.1 Principle The sample is ignited at 600°C to burn off all organic material. The inorganic material which does not volatilize at that temperature is called ash. 4.2 Apparatus (a) Muffle furnace (b) Silica crucibles (c) Dessicator, with magnesium perchlorate dessicant 4 4.3 Procedure (a) place clean crucibles in a muffle furnace at 600"C for one hour. Transfer crucibles from furnace to a dessicator and cool to room temperature. Weigh as quickly as possible to prevent moisture absorption, use metal tongs to move the crucibles after they are ashed or dried. (b) Weigh by difference 2.0 g. of sample into tared silica crucibles. Place in a muffle furnace and hold the temperature at 600*C for 6 h. (c) Transfer the crucibles to a dessicator and cool to room temperature. When cool, weigh the crucibles as quicldy as possible to prevent moisture absorption. (d) save the ash sample if mineral determinations are to be made. 4.4 Calculation Ash (%) on partial dry or as fed basis Wt. of ash - X 100 Wt. of sample Adjusting to dry basis ash % on as fed sample . X 100 dry matter % of as fed sample 5 ACID SOLUBLE AND INSOLUBLE ASH 5.1 Apparatus and reagents (a) Hydrochloric acid (1-2.5 v/v) (b) Filter paper, ashless, and (c) Dishes, porcelain 5.2 Procedure use the residue obtained from the ash determination. Boll with 25 ml HC;# taking care to avoid spattering, filter through ashless filter paper, and wash with hot water until acid-free. Place filter paper and residue Into a dry, tared porcelain dish and place In a muffle furnace at 600*C for 2 h. or until carbon-free. 5.3 Calculation Acid Insoluble ash (°/.) wt. of acid-treated ash = — , X 100 wt. of sample 6 DETERMINATION OF CRUDE FIBRE. 6.1 Principle A moisture-free and ether extracted sample Is digested first with a weak acid solution, then a weak base solution. The organic residue is collected in a filter crucible. The loss of weight on ingition is called crude fibre. 6.2 Apparatus (a) Beakers, 600 ml tall-sided (b) Round-bottom flask condenser unit (c) Buchner flasks, 1 litre (d) Buchner funnels, Hartley 3 section pattern (e) Crucibles, silica with porous base, and (f) Rubber cones to fit above. 6.3 Reagents (a) Sulphuric acid solution (0.25 N) (b) Sodium hydroxide solution (0.313 N) (c) Antifoam reagent (Octyl alcohol) (d) Ethyl alcohol (e) Hydrochloric acid, 1% v/v •6 6.4 Procedure Weigh about 2 g. of the dried, fat-free sample into a 600 ml beaker. Add 200 ml of hot sulphuric acid, place the beaker under the condenser, and bring to boiling within 1 min. Boil gently for exactly 30 min., using distilled water to maintain volume and to wash down particles adhering to the sides, use antifoam if necessary. Filter through Whatman No.541 paper in a auchner funnel, using suction, and wash well with boiling water. Transfer residue back to beaker and add. 200 ml hot sodium hydroxide solution. Replace under the con-denser and again bring to boiling within 1 min. After boiling for exactly 30 min., filter through porous crucible and wash with boiling water; 1% hydrochloric acid and then again with boiling water. Mash twige with alcohol or acetone, dry over-night at 100°C, cool, and weigh. Ash at 500°C for 3 h,, cool, and weigh. Calculate the weight of fibre by difference. 6.5 Calculation Crude fibre {% of fat-free DM) (Wt. of crucible + _ (fit. of crucible + dried residue) ~ ash residue) X 100 (Vft. of sample) 7 DETERMINATION OF CRUDE FAT (Soxhlet Method) 7.1 Principle Ether is continuously volatilized, then condensed and allowed to pass through the sample, extracting ether soluble materials. The extract is collected in flask. When the process is completed, the ether is distilled and collected in another container and the remaining crude fat is dried and weighed. 7.2 Apparatus and reagents (a) Soxhlet extraction apparatus (b) Extraction thimbles (c) Petroleum ether (b.p. 40-60°C) or ethyl ether 7 7.3 procedure Weigh by difference 2 to 3 g. of the dried sample (residue from dry matter determination can be used). Place the thimble inside the soxhlet apparatus. Connect a dry pre-weighed solvent flask beneath the apparatus and' add the required quantity of solvent and connect to condenser. Adjust the heating rate to give a condensation rate of 2 to 3 drops and extract for 16 h. On completion, remove the thimble and reclaim ether using the apparatus. Complete the removal of ether on a boiling water bath and dry flask at 105°C for 30 minutes. Cool in dessicator and weigh. 7.4 Calculation Wt. of fat 100 crude fat (% of Dry matter) - - <• X — Wt. of sample 1 8 DETERMINATION OF FREE FATTY ACIDS 8.1 Apparatus and reagents (a) Ethyl alcohol (b) Phenolphthalein (154 solution in alcohol) (c) Sodium hydroxide (0.25N) (d) Stoppered flasks, 250 ml. 8.2 procedure Weigh oil or fat into a stoppered flask and add 50 ml. alcohol previously neutralised by adding sufficient 0.25N sodium hydroxide to give faint pinkish colour with phenolphthalein (2 ml.). Titrate with sodium hydroxide and vigorously shake until a permanent faint pink colour appears. 8.3 Calculation Free fatty acids % (as oleic acid) g. oil or fat « • » X volume of 0.25N NaOH used in titration 7,05. . . 8 9 DETERMINATION OF CRUDE PROTEIN (Kjeldahl Method) 9.1 Apparatus (a) Macro kjeldahl digestion and distillation units or micro kjeldahl apparatus (b) Kjeldahl flasks (500 ml. capacity or larger), and (c) Conical flasks, 250 ml. 9.2 Reagents (a) Sulphuric acid (98%), nitrogen free, (b) Potassium sulphate, reagent grade, (c) Mercuric oxide, reagent grade, (d) paraffin wax, (e) Sodium hydroxide, 40% solution (f) Sodium sulphide, 4% solution (g) Pumice chips, (h) Boric acid/indicator solution. Add 5 ml of indicator solution (0.1% methyl red and 9.2% bromocresol green in alcohol) to 1 litre saturated boric acid solution, (1) Hydrochloric acid standard solution (0.1N) 9.3 Procedure Accurately weigh 1 g. of sample into a digestion flask. Add 10 g. potassium sulphate, 0.7 g. mercuric oxide and 20 ml sulphuric acid. Heat the flask gently at an inclined angle until frothing subsides and then boil until the solution clears. Con-tinue boiling for an additional half hour. If the frothing is excessive, a small amount of paraffin was may be added. On cooling, add about 90 ml. distilled water, reccol, add 25 ml. sulphide solution, and mix. Add a small piece of boiling chip to prevent bumping and 80 ml. of sodium hydroxide solution while tilting the flask so that two layers are formed. Connect rapidly to the condenser unit, heat, and collect distilled ammonia in 50 ml. boric acid/indicator solution. Collect 50 ml of distillate, on completion of distillation, remove the receiver (wash condenser tip) and titrate against standard acid solution. !» 9.4 Calculate Nitrogen content of aample 1%) fad. acid x normality of atendard acid) . -II ,1, , , . , , ..„,,)„., ;„„ i.rfrn, X 0 . 0 X 4 X 100 wt. of aample (g) crude protein oontent (%) • nitrogen oontent X 6.25 1 0 DE1TRMIHATI0N OF WIJROOW fUES KXBttCT • " • - • • ' • • ' , JOfl Procedura Nitrogen fro* extract (WR> of a food la determined by difference after the analyaee nawa BOM ootapleted for aeh, exuda flora, crude fat and crude protein. 10.2 ffaicalat^n MTt (%) on dry Jaaa*t m 100% - (% aah on dry baala + % crude fibre on dry baala + % crude fat on dry baala * X protein on dry baala) 11 REr*?EMCES 1. AOAC - 1965 Offlolal Nethoda of Analyala of the Aaaoelatlon of Official Agricultural Chamlete. 1965, 10th Edition Published by the Aaaoelatlon of Official Agricul-tural Chamiata, Kaahlngton, D.C. 2. enow, K.tf, Quality control in flan feed manufacturing. 1 9 8 0 In Pish feed mchnology, Aquaculture Development and Coordination Programme, FAO ADCP/Rep/80/11 p, 369-385. 3. Harria, L.E. Nutrition reeearcn technique* for domeatlc 1970 ' and wild anlmala. volume 1. Utah state Unlvaralty, Logan, Utah. 

Proximate composition analysis of feeds

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Proximate composition analysis of feeds

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