Nutrients present in the feedstuffs are not completely

available to the animal body. Large portions of the nutrients

are excreted In the faeces because of being not digested In the

alimentary tract. Therefore, the digestibility of the feedstuff

is defined as the portion of a feed or nutrient of feed which is

not recovered In faeces, i.e., the portion which has been absorbed

by the animal. When the digestibility is expressed in

percentage it is known as digestibility coefficient. Digestibility

coefficients are calculated for dry matter, crude protein, crude

fibre, ether extract and nitrogen-free extract. Digestibility

of gross energy present in the food can also be determined. The

digestibility coefficients normally determined are the apparent

digestibility coefficients since the nutrients found in the

faeces contain small proportion of nutrients from the previously

utilized food In the form of mucosal debris, unspent enzymes ate

Easterson, D C V

Paulraj, R

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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 9 DETERMINATION OF DIGESTIBILITY COEFFICIENT 1 INTRODUCTION Nutrlants prasant In the feedstuffs are not completely available to tha animal body. Large portions of the nutrients are excreted In tha faeces because of being not digested In tha alimentary tract. Therefore, the digestibility of tha feedstuff is defined as tha portion of a feed or nutrient of feed which is not recovered In faeces, i.e., tha portion which has been absorb-ed by the animal. When the digestibility is expressed in percentage it is known as digestibility coefficient. Digestibility coefficients are calculated for dry matter, crude protein, crude fibre, ether extract and nitrogen-free extract. Digestibility of gross energy present in the food can also be determined. The digestibility coefficients normally determined are the apparent digestibility coefficients since the nutrients found in the faeces contain small proportion of nutrients from the previously utilized food In tha form of mucosal debris, unspent enzymes ate. 2 DIRECT FAECES COLLECTION METHOD 2.1 Apparatus and reagents required (a) Specially designed aquarium tanks for collection of faeces (b) Aerators (c) Feeding trays (d) polythene tubes (e) Porcelain crucibles (f) Hot air oven (g) Centrifuge (h) Single pan balance (i) Equipment and reagents required for protein analysis (j) Equipment and reagents required for fat analysis * Prepared by R. Paul Raj and D.C.v. Easterson. Central Marine Fisheries Research Institute, Cochin-18. (k) Equipment and reagents for crude fibre analysis (1) Equipment and reagents for determination of energy (m) Experimental animals (fishes or crustaceans) 2.2 Procedure (a) Take three specially designed aquarium tanks of identical size, with provision for continuous collection of faeces, pill them with water and maintain the level, volume and temperature of water similar in all the aquaria. The water used in all the aquaria should be of the same source and salinity level. Aerate the aquaria well to maintain oxygen levels near saturation in all the tanks. (b) introduce 10 healthy experimental animals of the same species, age, size group, etc in each of the t a n k s . ' , - . • • (c) During the pre-experimantal feeding, the animals are fed on the feed or feedstuff in question for a period of atleast 6 days to remove the effects of previous feeds. (The number of pre-experimental feeding days can be adjusted depending on the gastric evacuation time). (d) At tho end of six days the feeding trial can be started. Feed the three groups of experimental animals with a known quantity of feed, weighed accurately to O.lg in a feeding tray and allow the animals to feed for about 12 hours overnight. The feeding time can be increased or decreased i based on the. actual time taken for consumption by the experimental animals. (e) carefully remove the feading trays from the aquarium tanks and collect the left-over food by centrifuging or filtering. Dry the left-over food in an oven at 105*0. 77 (f) Collect the faeces at 2 hourly intervals using a polythene tube by siphoning out the faecal pellets if there is no specially designed aquaria for collecting faeces, centrifuge or filter the faeces and dry in an oven at 105'c (g) Continue the feeding, and collection of left-over food and faeces for a period of 10 days. (h) After drying, the representative samples are kept separately in polythene bags. (i) At the end of the digestibility trial all the faeoeB samples of each aquarium tanks are composited and analysed for the dry matter, crude protein, crude fat, crude fibre, NFE and energy contents. 2.3 Calculation I-F Apparent digestibility coefficient of nutrients » Da • — » I where I = nutrient intake and F = faecal nutrient A I-(P-Fk) True digestibility coefficient » TO • — • I I where A » absorbed nutrient; I = nutrient intake; F = faecal nutrient and Fk * metabolic nutrient excreted with the faeces. 3 CHROMIC OXIDE INDICATOR METHOD A breakthrough in digestibility studies of nutrients was the use of inert materials, and in particular the use of chromium oxide (Cr,o3). Chromium oxide mixed with prepared diets and measured in the faeces provides a general comparison of the overall digesti-bility of a feed expressed as: rsCr,o, in faftces Percent digestibility - l^n 78 • When the percentage of a nutrient in the feed and faeces is analysed as well as the corresponding percentage of the indicator substance the digestibility percentage can be calculated by the formula: Apparent digestibility coefficient %Cr,0, in feed % nutrient in faeces „ 1.00- — x %Cr203 in faeces % nutrient in feed Caution: The ohromic oxide for commercial use has to be purified for removing the toxic chromium compounds. The toxic chromium compounds can be removed by repeated washing in N HC1 using glass fibre filter (Whatman GFC) and borosilicate glassware. After repeatedly treating with hydrochloric acid the powder is washed, thoroughly in distilled water, dried at 120°C for 24 hours and stored. Thus, treated Cr.O. is mixed in a definite percentage to the feed. 4 DETERMINATION OP CHROMIC OXIDE IN FAECES 4.1 TITRIMETRIC METHOD 4.1.1 Principle The well mixed faecal matter containing organic waste and chromic oxide marker is digested with nitric acid to remove organic matter. The resultant solution is suitable for estimation of minerals in addition to chromium. On the addition of perchloric acid the chromium oxide is oxidised to dichromate which is estimated by adding an excess of ferrous ammonium sulphate and titrating the mixture. 4.1.2 Reagents (a) Hydrochloric acid (b) Nitric acid, Analar (c) perchloric acid, 60% (d) Sulphuric acid (6N-167 ml of Analar sulphuric acid diluted to 1 litre in distilled water) !j£',:. ; i i- ' i i i r.o qixi'o ti ban '33i:.ri-:iiuK (i,t,incii..i:^. SVO'R'1:-^ ' t e y ^ ^ b t ^ ^ d ^ c r i r o n ^ t e ' ' f&t'i7 H)'; stindardV'"" n-,-;>ip tsriJ ixJrm ^ifAWiMip fiiLt/a-.&Jof. •' X.'.; t>"5x> previous^y^hejjiei^ tg_. ^ j^C^j^o&edj and., s tored in a' dess ica tor in 1 l i t r e of 2 JJ sulphuric acid. Check the normality. t>3:io£.:-J2 ^AssoivaiJabout^iog-'e&rjtoas'-aniiHonittit sulphate CPeSO^fWH^'g-.'^'O'^H S ; ' I i t re of°2» lsulphuric : .acJLdi ;:.Ch©ck£ £h6 HOrffl«^it^J#«e^^af ^ £ t > r e tfse. • j j". (g) Indicator solution: Dissolve O.lg of N-Phen^iahthrariiiic Scid: ifl* 2 ml of 5% sodium carbonate and dilute to 100 ml with distilled water. i — 4.1.3 Procedure (a) Weigh about 50-100 mg of faeces containing--1-3 mg of Cr 2 0 3 i n to a 100 ml conical f lask or a kjeldahl f lask . *->-•••<•-(b) Add 5 ml of cone n i t r i c acid f SlisJtftM'SoiiinuteB,?Jtaii ' ' ttie oohtenW-ge'tttiy in a hot p l a t e for about half an hour in a fume cup board (Alternat ively samples can be digested in i i c t s ^ I ^ ^ a ^ fcJ(jft{.ec system, i f ava i l ab l e ) . Addi-•;3Xi-f-;*'i:ifai»i!lf£icid-'may Tae 'added to prevent the content becoming dry . (d) Cobi 'atid* ffdd 3 ml of 60-J perchloric" acid. Heat' v,;•.. -. -tfay'keVplrig insld'e • fune cupboard rfith p l a s t i c •'--fgct'eerc"''uTVtil no more fumes evolves and a l l n i t r i c ";' af&ld hasvbeen removed. o:U ': • • . ( _ • - ' (e> ifAcl-wpBti,um ds present-t . ie so lu t ion becomes b r i l l i a n t golden in colour. Cool and wash the d iges t i n to a 50 ml volumetric flask with d i s t i l l e d water. so (f) To this add 5 ml of 6N-H2S04, 5 ml of 0.1 H ferrous ammonium sulphate and a drop of indiacatoc Titrate the mixture in a 10 ml burette with stand-ard o.l N potassium dichromate until the green colour of the solution turns to slate-grey, which turns to cherry colour on standing. 4.1.4 Calculation If ' t* ml of 0.1 M potassium dichromate solution is required, then the dichromate in the digest has reached with 5 't1 ml of 0.1 fe.rous ammonium sulphate. 1 :al of 0.1 N dichromate is equivalent to 2.53 mg of Cr2o~. Thus 2.53 (5-'t') mg of Cr,03 can be present in the digest. 4.2 SPECTROPH0T0METRIC METHOD 4.2.1 Reagents As in the titrimetric method 4.2.2 Equipment spectrophotometer 4.2.3 procedure Follow procedures 'a' to 'd' as given in titrimetric method. (e) Cool and wash the digest into a 100 ml volumetric flask and make up the volume by adding distilled water. (£) Mix well and after allowing a minimum of 5 minutes measure the optical density at 350 m/ti in a spectrophotometer. The percent chromic oxide is rutd from a standard curve where ' V is the optical density at 350 myu and «X' the Cr-O, content of the sample in mg/100 r.il. «* i \, 9«f*n«l. ft. ISd.* HetHBll*'ftw MMMnont of efeflt 1978 production in frashwaters. 3rt •ditto*. IBP Handbook Ko. 3. «ac»—U •ciantific publications, QKfosfl. -f, JWR^WM*, A . ' sad on th« acid dlgsation matfcod. fetf «w Ht ItHtthttaca dstermination atf «h*o*iC ox$4t M~«* . Xf« indax aub«tenc« in tM> Mmdy of dia»atlbmty in IMh t—d. p^fr . 2SJ2.' foe. 3£i- £ i f5" M ! IWWWfe .' >. *H*»y, L.C, and jr.. Clip. I w w t . a9 i-*H • jjli 'IffHI' • • • ' ' ' - ' • i t tb' : Wap e^m, I . D . P . Micro«n*ly»i» in Mrttal IIMM 4th edition. J. «o« * OwrefctU, 

Determination of digestibility coefficient

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Determination of digestibility coefficient

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