The amount of heat, measured in calories, that is released

when a substance is completely oxidized in a bomb calorimeter

containing 25 to 30 atmospheres of oxygen, is called the gross

energy (GE) of the substance. A sample of the material to be

tested is weighed into a combustion capsule. The combustion

capsule is placed in an oxygen bomb containing 25 to 30 atmospheres

of oxygen. The oxygen bomb is covered with 2000 g of

water in an adiabatic calorimeter. After the bomb and calorimeter

have been adjusted to the same temperature, the sample is Ignited

with a fuse wire. The temperature rise is measured under adiabatic

conditions. From the hydrothermal equivalent of the

calorimeter the temperature rise minus some small corrections for

fuse wire oxidation and acid production, the caloric content of

the sample is calculated

Paulraj, R

Ponniah, A G

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^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 2 DETERMINATION OF GROSS ENERGY OF FEEDS* 1 Principle The amount of heat, measured in calories, that is released when a substance is completely oxidized in a bomb calorimeter containing 25 to 30 atmospheres of oxygen, is called the gross energy (GE) of the substance. A sample of the material to be tested is weighed into a combustion capsule. The combustion capsule is placed in an oxygen bomb containing 25 to 30 atmos-pheres of oxygen. The oxygen bomb is covered with 2000 g of water in an adiabatic calorimeter. After the bomb and calorimeter have been adjusted to the same temperature, the sample is Ignited with a fuse wire. The temperature rise is measured under adia-batic conditions. From the hydrothermal equivalent of the calorimeter the temperature rise minus some small corrections for fuse wire oxidation and acid production, the caloric content of the sample is calculated. 2 Apparatus (a) parr oxygen bomb calorimeter and accessories or equivalent (b) The calorimeter may be equipped with an automatic temperature controller. If the temperature controller is on the calorimeter; it will take less labour to run the analysis, but the controller is not necessary to obtain accurate results. (c) Solution or trip balance with capacity to 3000 g and accurate to 0.1 g. * Prepared by R. Paul Raj and A.G. Ponniah, Centre of Advanced Ptuoies in Mariculture, Central Marine Fisheries Research Tn :t.! t-ute, Cochln-18. 11 3 £2*2234! (a) standard sodium carbonate solution, equivalent to J. cal/ml (3.658g Na2 Co3 per litre) (b) Methyl orange indicator (0) Benzoic acid combustion tablets or primary standard grade crystals 4 Determining the hydrothermal equivalent of the bomb (a) Determine the hydrothermal'equivalent of the bomb, bucket and water by determining the temperature rise using the same procedure as outlined above, but with a sample of known caloric content (benaoic acid combus-tion tablet). Make at least four determinations and use the average value, once this value is determined, it should not change unless some parts of the bomb are replaced. (b) Dry the benzoic acid at 105*C overnight, cool in dessicator and weigh by difference from a covered weighing bottle one tablet or approximately a lg sample of dry calorific standard grade benzole acid crystals. Determine the temperature rise from the benzoic acid in the bomb as with other samples. Hydrothermal equivalent per degree (Cal) wt. of benzoic Calories Length Cal/cm acid v per gram of fuse v fuse v . x benzoic + wire x wire x ml N a2 °°» acid burned (final temp. - Initial temp.) 4.1 Examplet A 1.0622g sample of benzoic acid had a beat of combustion of 6319 cal per g. The corrected Initial temperature of the bomb waa 20.280'C and the final corrected temperature was 23.045'C. There were 4.8 cm of fuse wire burned with a caloric value of 2.3 cal per cm. There were 7.5 ml of Na2Co3 titrated (equivalent to 7.5 cal). 12 Hydrothermal equivalent calories per degree (1.0622 X 6319) + (4.8 X 2,3) + 7.5 • I , , , . 2434 small calories (23.045 - 20.280) 5 procedure (a) Weigh by difference approximately l.Og of sample and place in a clean, empty combustion capsule. Samples may be pelleted, but this is usually not necessary. (b) Attach a 10 cm length of fuse wire between the electrodes of the bomb (oxygen)and set the combustion capsule with sample in place in the loop electrode. Adjust the fuse wire so that it touches the sample. (c) Place about 1 ml of water in the bomb cylinder and swirl it around to wet the sides. This is not necessary if the bomb is still wet from a proceeding determination. (d) Assemble the bomb, tighten the screw cap, close the pressure release valve and fill with oxygen to 25 atmospheres gauge pressure. Place the bucket (oval) in the calorimeter, set the bomb in the bucket, and attach the clip terminal. (e) Weigh 2000g distilled water on the solution or trip balance (use a 2000 ml volumetric flask to hold the water) and carefully pour into the calorimeter bucket. The water temperature must be within the range of the calorimeter thermometers. (f) Close the cover, lower the thermometer and start the water circulating motor. Remove the cap from the jacket cover and fill the cover with water until it runs out of the drain hose. v (g) Adjust the temperature of the water in the outer jacket to approximately equal that of the calorimeter by adding hot or cold water, and allow one minute to 13 M attain equilibrium. Then carefully adjust the temperature to be exactly equal and check the ca^ori- , meter teniperature at one minute intervals for three minutes. \ (h) Read arid record the initial teniperature to the nearest. 0.0002* and Ignite the sample. Turn in hot or cold, water to keep the jacket temperature equivalent to the calorimeter temperatures during the period of rise. (1) Campar< 3 and adjust the teniperature of the outer jacket to the Inner bucket of the calorimeter tempera-ture frequently and carefully to insure adiabatic condition or that1 the1 temperatures are equal. Read and record the final temperature after the same temperature is observed in three successive one minute intervals. (j) Raise the thermometers. Open the calorimeter, take the bomb from the calorimeter bucket, release the residual pressure of the bomb and open. Carefully remove the remaining pieces of fuse wire from the electrodes; straighten and measure the combined total length in centimeters. The calories of wire burned can be determined with the measuring scale that is supplied witij the wire. (k) Rinse all inner bomb surfaces with a stream of neutral distilled water and collect all washings in a clean beaker. Titrate the washings with the standard sodium carbonate solution using methyl orange indicator to determine the amount of acid formed from the incidental oxidation of nitrogen and sulphur compounds. A correction is made to take care of the heat liberated in the formation of the acid. : (1) Correct the initial and final temperatures from the calibration curve supplied with the thermometer. 14 6 Calculations •:,.i ate (oai/g) on as fed basis fir,il temp.-1 Hydrotherraal [Length of Call Initial temp.I equivalent of -fuse wire X perl- Na, 1 J bomb (burned cml ca-l  ml   se 2 (weight of sample) 6.1.1 Example A 1.0214 g sample of feed (as fed basis) was used, the initial temperature waa 23.13'C and the final temperature was zs.as'c. The hydrothermal equivalent of the boob is 2412 cal per degree C. There was 7.0 cm of fuse wire burned with a correction of 2.3 cal per cm of wire and 6.0 ml of Ma2 Co3 titrated (equivalent of 6.0 cal). (25.25 - 23.13) 2412 - (7.0 X 2.3) - 6.0 GE ( -al/g) = -1.0214 g sample on as fed basis = 3804 cal/g or 3804 K cal/kg 2 Adjusting to dry basis; GE(KcalAg) on as fed sample GE (Kcal/kg) =» X 100 dry matter % of as fed sample References i. Harris, L.E. Nutrition Research Techniques 1 9 , 0 Volume 1 Utah State University* Logan, Utah. 2. parr instrument oxygen bomb calorlmetry and combustion Company 1966 methods. Technical Manual Ho.130. parr instrument Company, Hollas, Illinois. 

Determination of gross energy of feeds

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Determination of gross energy of feeds

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