ANNEXES

ANNEX A
TERMS OF REFERENCE FOR THE 1986 TWO-MONTHS MISSION OF THE FISH NUTRITIONIST

1. Preparation of a general Research Plan for the ‘Fish (and crustacean) Feed and Nutrition Programme’ of FRI, to be carried out in the Freshwater Aquaculture Research Station at Mymensingh. This Programme is expected to contain the following projects:

   • nationwide survey of ingredients, including costs and availability;
   • analysis of ingredients;
   • feed technology, including formation, packaging, and application;
   • diet testing;

   • nutritional studies.

2. Preparation of detailed workplans for each of the projects for the year to come, and discussion of these workplans with the relevant researchers and their supervisor. The workplans should specify objectives, justification, methodology, expected output, as well as suggestions for co-operation with other research groups in the Bangladesh Agricultural University (BAU, Mymensingh) or elsewhere.

3. Preparation of specifications and cost estimates for equipment and materials to be purchased for implementation of the complete Research Plan. Equipment should include a laboratory-type feed mill (processing line). Very special and/or expensive equipment for the feed laboratory should be considered in relation to availability at BAU or elsewhere. A purchase plan has to be drawn, including phasing of purchases, alternative brands (if any), suppliers, and costs, based on the administrative rules for purchase in Bangladesh.

4. Preparation of a proposal for later consultancy visits of the Fish Nutritionist to Bangladesh, including his activities in the fields of:

   • monitoring of research results;
   • preparation/adjustment of follow-up projects;

   • training.

5. Reporting; a full technical report is to be submitted within two weeks after the end of each contract period; format will be discussed with the teamleader.

ANNEX B
LIST OF ADDITIONAL EQUIPMENT NEEDED FOR THE NUTRITION LAB AT FARS

*) FOB Seattle, USA

ANNEX C
ELEMENTS OF THE GENERAL FISH NUTRITION AND FEED TECHNOLOGY RESEARCH AND DEVELOPMENT PLAN FOR FRI

1. Finfish applied nutrition research

   1. Protein/energy requirements;
   2. Fatty acid needs and sources;
   3. Vitamin needs and supplements;
   4. Mineral levels;
   5. Fiber and inhibiter restraints;

   6. Nutrition and fish health.

2. Crustacean applied nutrition research

   1. Protein/energy needs and sources;
   2. Fatty acid needs and sources;
   3. Toxicants and inhibitors in feeds;

   4. Limiting vitamins and sources.

3. Feed technology for finfish and crustaceans

   1. Least cost formulations for:

      1. Complete feeds;
      2. Supplemental feeds;
      3. Fry feeds;

      4. Larval feeds.

   2. Nutrient stability during processing of:

      1. Wet diets;
      2. Semi-moist diets;
      3. Cold rolled diets;
      4. Cold compressed pellets;
      5. Steam-treated compressed pellets;
      6. Crumbles (from 4 and 5);
      7. Flake feeds;

      8. Micro-pelletized feeds.

   3. Fish feed manufacturing techniques:

      1. Raw material reprocessing;
      2. Wet ingredient mixture feeds;
      3. Semi-moist extruded feeds;
      4. Dravo-rolled pellets;
      5. Cold compressed pellets;
      6. Steam-treated compressed pellets;
      7. Crumbles and top dressing;
      8. Medicated feeds;
      9. Flake feeds;

      10. Micro-pelletized larval feeds.

4. Quality control on feeds

   1. Agricultural commodities;
   2. By-products;
   3. Pre-and post-manufactured formulae;
   4. Packaging and shipping;
   5. Feeding techniques;
   6. Water stability and nutrient loss;
   7. Storage nutrient loss.

ANNEX D
HOW TO DEVELOP A RESEARCH PROJECT

ANNEX E
FISHERIES RESEARCH INSTITUTE - RESEARCH PROJECT PROFORMA

ANNEX F
FIVE YEAR GENERAL PLAN FOR FISH FEED AND NUTRITION RESEARCH

Md. Abdul Khaleque & Nurunnahar Begum

1 Introduction

Bangladesh is one of the richest countries in its inland water resources. But fish production has, however, never attained to a satisfactory level, even though some efforts were made. In spite of the existence of such water resources the daily per capita consumption of animal protein is only 7.9 g, although total animal protein requirement is 25.0 g. This is mainly due to inefficient knowledge regarding fish feed formulation, resulting in low fish production. Artificial balanced feeding is an effective measure to increase fish production to many folds. In this situation, it is quite beneficial to explore new types of fish feed from the indigenous raw materials which can easily and cheaply be supplied to fishes for boosting up production by increasing their conversion efficiency so as to enable independent fish feeding. Fortunately our country shows great promise having vast resources of indigenous raw materials. If a balanced fish feed is prepared from the indigenous ingredients and fed to fishes correctly, it may bring a revolutionary change in fish production. In view of the above, a critical study relating to the fabrication of high quality fish feed from the well spread indigenous raw materials is indispensable.

2 Literature review

Biochemical research into the nutritional requirements of the indigenous variety of fish is relatively a new approach in Bangladesh. Little work on the nutritional requirements of major carps, magur and nilotica has been done in Bangladesh. The literature thus reviewed here obviously includes studies conducted mainly in other countries with different species.

The art of preparing fish feed from the natural feed stuffs has been reviewed by a few scientists (Schaeperclaus, 1933; Wood, 1952; Huet, 1960). They are of the opinion that fish meal, frozen animal products and insects are the backbone of production ration in hatcheries. Swingle (1958) and Prother (1958) cultured catfish and minnows with a food containing 35% soyabean meal, 25% peanut meal, 15 % fish meal and 15 % distiller saleable and found the production quite satisfactory. Halver (1980) reported the nutritional requirements of cultivated warmwater fish species. According to him, requirements for the ten essential amino acids and sparing effects of several others are reported for only a few species of fish, held under specific environmental conditions.

Harris (1980) described a detailed review about the nature of different feed stuffs that are being presently used for preparing formulated fish feed. He discussed the international feed recording of data on feed composition. Calculation used in summerization of feed composition data, energy feeds, non-chemical characteristics of energy feeds, quality in energy feeds, protein supplements, vitamin and mineral supplements and other additives.

The technology of fish feed formulation has been discussed by various authors (Hastings, 1980; Hardy, 1980; Chow, 1980; Hastings & Higgs, 1980; Pigott, 1980; Lovell, 1980).

Sen et al. (1978) reported a level of 26 % carbohydrate as optimum for the growth of carp. Cruz & Laudencia (1976) suggested the protein requirement for Clarias batrachus (magur) as 35.69 – 37.72 %. The body weight gain of Tilapia zillii was improved with the increase of the caloric value of the diet, suggesting that lipids are more effective than carbohydrates (Teshima et al., 1978). Rifai (1979) reported that the aquatic plant Lemna minor was very effective on the growth rate of U. nilotica in different levels of stocking density (5, 15 and 45 fish per floating cage), being a high energy feed. Mazid et al. (1985a) found a level of 38 % protein as optimum for the growth of Labeo rohita. Maximum growth was obtained at a protein:energy ratio of 122. Coconut oil was found superior to soybean and cod liver oils in sustaining growth of fish.

Mazid et al. (1985b) found that the growth of Clarias batrachus increased proportionately with the increase of protein content upto the highest level of dietary inclusion. However, a level of 40 % protein obtained from indigenous materials in the diet was suggested as optimum for the fingerlings raised in a mini-pond.

3 Objectives of the proposed work

The proposed investigations will be designed:

• to prepare a check-list of the cheaply available indigenous raw materials in respect to availability and cost;
• to formulate a low-cost balanced fish feed to minimise the cost of fish production for maximum yield;
• to prepare fish feeds for culturable species of Bangladesh at their different life stages and differentenvironmental conditions;
• to find out the specific nutrient requirements for carp, both major and chinese, catfishes (especially Clarias batrachus) and tilapia (Sarotherodon nilotica);
• to study the growth of the stated species at their different life stages in response to the formulated diets incorporated with indigenous raw materials;
• to study shelf life of the formulated feed and to provide storage guidelines.

4 Methodology

4.1 Ingredient survey

The proposed survey should be conducted at 6 representative districts, namely Mymensingh, Dhaka, Rajshahi, Cox's Bazar, Barisal and Jessore. Area of the survey will be extended later on. Information and samples will be collected after every two months.

4.2 Chemical analysis

Proximate composition of raw materials and the finished diet will be analyzed in the FARS nutrition laboratory and in the laboratory of the Dept. of Fisheries Technology, BAU. Proximate composition includes protein, moisture, ash, fat, lipid, carbohydrate, salt, phosphorus, and calcium.

4.3 Selection of species

Feeding trials will be conducted with the major carps (Catla, Rohu, Mrigal and Calbasu), chinese carps (grass carp, silver carp, black carp and bighead), catfish (magur) and tilapia (O. nilotica). Feeding trials should be done in 3 stages, namely fry (4 – 12 mm), food fish and brood fish.

4.4 Fish feed manufacturing

• Physical inspection. Preliminary physical inspection of raw materials to be used as ingredient. Physical inspection includes:
  • evidence of wetting; mold growth confirms water damage;
  • presence of scrap metals, stones, dirt or other non-biological contaminants;

  • presence of insects.

• Biochemical analysis. “Weende” analysis for proximate composition of both raw material and finished diet. It includes:
  • moisture, ash, crude fibre, crude lipid, nitrogen free extract, non-protein nitrogen;
  • acid insoluble ash (for materials having high ash content);
  • salt;
  • calcium and phosphorus;
  • amino acid spectrum;
  • determination of toxic substances in the ingredients, like urease, gossypol, aflatoxins;

  • determination of total sugar using molasses.

• Formulation and manufacture of fish diet. Depending on the chemical composition and caloric values of the different ingredients dietary formulae will be prepared. Physical properties that are to be considered include:
  • size;
  • flavour and adour;
  • texture;
  • colour;
  • density (sinking rate);
  • dehydration capacity;
  • water stability.

  Diets in the form of meal, paste and cakes will be tried; however at the later stages pellets (soft) will be prepared. Preparation of flake diets may also be considered.

• Feeding trials. Feeding trials will be conducted in pond series, depending of the available facilities at FARS. Feeding will be done for each kind of fish separately. The following are to be recorded:
  • species of fish;
  • type of fish;
  • sex;
  • age;
  • number of animals in treatment;
  • physiological state;
  • percent of test ingredient in ration;
  • lenght of trial;
  • daily dry matter consumed.

  This information will be used for studying some of the following parameters depending on the importance and existing facilities in the laboratory:

  • measuring indexes of growth and food intake;
  • indexes of food utilization;
  • general relationship between food and growth;
  • growth efficiency;
  • body size, food and growth and different rations;
  • factors influencing the use of food for growth;
  • effect of temperature on food-growth relations;
  • effect of food quality on growth;

  • effect of crowding.

5 Equipment and chemicals needed

• Glass or metal dishes with lids (air tight)
• Force-draught oven, capable of maintaining 103°C ± 2°C
• Catalyst table (kjeltabs M)
• Sulphuric acid
• Sodium thiosulphate
• Boric acid
• Indicator Soln (screened methyl red)
• Sodium hydroxide
• Paraffin wax
• Volumetric flasks
• Pipettes
• Petroleum spirit
• Water bath
• Extraction apparatus, Quickfit plain-body (Bolton type) extractor Ex 7/23 and coil Ex13/26 or similar continuous extraction type
• Condenser, Quickfit Cx5/23 and adaptor Da37
• Round flat-bottomed flask, Quickfit FF250/35
• Anti-bumping granules
• Pestle and mortar
• Extraction thimbles, Whatman 30 × 80 mm, 33 × 113 mm
• Funnel, Pyrex 100
• Absorbent cotton wool
• Electric ovens (2), maintained at 103°C and 70°C
• Analytical balance
• Measuring cylinder
• Sodium hydroxide Soln
• Ethyl/methyl alcohol
• Diethyl ether
• Buchner funnel, I.D. 21 cm
• Buchner flask, 2 1
• Scintered glass crucibles
• Rubber cones
• Cotton circles (19 cm dia)
• Nickel dishes, 6 cm
• Stop watch
• Nomex or leather heat resistent gloves
• Muffle furnace
• Cetyl trimethyl ammonium bromide (CTAB)
• Decalin (decahydronaphthalene)
• Acetone
• Hexane
• Ammonium molybdate
• Ammonium vapadate
• Potassium dihydrogenphosphate
• Desiccator
• Spectrophotometer
• Benzyl alcohol
• Ferric aluminium indicator
• Potassium thiocyanate solution.

ANNEX G

A. INTRODUCTION

To minimize protein malnutrition prevailing in the country, the easiest and best way is to increase the per capita fish production through extensive action programmes for intensive fish culture in all types of culturable water bodies. To make this programme a success story, supplementary feeding with well-formulated feedstuffs (cheaper, but containing an optimum level of protein and energy) is a must. It is well established, that weight gain in case of fish production with supplementary feeds may be 3 to 4 times more than that of fish grown on natural feeds (Singh and Singh, 1975).

We have many kinds of by-products, natural (of plant and animal origin) as well as from domestic and industrial activities. These locally available indigenous raw materials may serve as good ingredients for the formulation and development of cheaper and as well as quality fish feed. To do this, it is obviously necessary to develop the technology for proper formulation and manufacturing of the feeds, assuring the optimum contents of indispensable amino-acids, minerals, vitamins, growth promoting substances and energy. Though in many developed countries quality supplemental fish feeds from locally available ingredients have been developed for better fish nutrition (Dabrowski & Kozak, 1976; Pal et al., 1977; Andrews, 1979; Reddy, 1979; Mazid et al., 1979; Tacon & Silva, 1983), in our country authentic and informative studies pertaining to the development of a suitable quality fish feed for intensive fish culture have not yet been undertaken.

Not only the quality feed alone, but also selection of species to be cultivated is an important aspect in any intensive fish culture programme. In many countries, a great deal of attention has been paid to mono-culture of catfish-species, and also to their introduction into composite fish culture with carps (Sidthimunka, 1973; Devraj, 1976). Recently, in Bangladesh, some pilot schemes on artificial propagation and rearing of catfish (particularly Clarias spp.) have been undertaken by the Directorate of Fisheries and the Fisheries Research Institute, because of their:

• capability of inhabiting different types of waters, like ponds, tanks, canals, ditches, paddy fields, and even marshy lands or drains having distress environmental conditions;
• omnivorous feeding habits;
• great market demand and high market value;
• fast growth rate;
• breeding tendency in confined waters;

• high therapeutic nutritional value.

It has been reported, that for economically successful culture operation of Clarias batrachus the importance should be given to the supply of adequate amounts of supplementary diets, prepared from indigenously available raw materials, particularly of plant origin (Mollah et al., 1973; Protap, 1979). Again, protein contents of feeds to be used should be considered as one of the most important factors to determine its quality to fulfil the nutritional requirements of catfish (Cruz et al., 1976; Protap et al., 1978; Mollah & Tan, 1982).

B. OBJECTIVES

Practically no feeds have been developed for the economically and nutritionally valuable catfish species Clarias batrachus, locally called ‘Magur’. Hence, the present study is proposed with the following objectives:

• to develop isocaloric fish feeds containing different levels of protein from available indigenous raw materials;
• to estimate the proximate chemical composition of feed ingredients to be used for the formulation and development of quality fish feed;
• to evaluate the quality of prepared feeds on the basis of growth performance of the catfish Clarias batrachus.

C. LITERATURE REVIEW

Mollah et al. (1973) conducted an experiment with 6 types of feed (five had a common base of vegetable origin), to determine a suitable feed for catfish fry (Heteropneustes fossilis). The feeds differed only in their animal protein content. Feed I (with meat offal and fish meal), feed II (with meat offal and blood meal) and feed III (with lysine-cum-methionine) gave the best results. Feeds consisting of only fish meal with no vegetable base resulted in weight reduction of the fry.

Cruz & Landencia (1976) studied the protein requirements of Clarias batrachus. For this purpose, C. batrachus fingerlings were fed with practical type diets, containing 20 – 45 % crude protein for 90 days. Results indicated, that rations containing 35.69 and 37.72 % crude protein gave optimum results in terms of gain in weight and feed conversion.

Protap et al. (1978) studied the effect of dietary protein levels on the proteolytic activity in the intestines of the air-breathing catfish Clarias batrachus, feeding 3 different diets containing 25, 50 and 75 % protein. It was found that in fish with 50 % protein diet protease activity was maximum as compared to the fish maintained on the other diets. This lead to the conclusion that the optimum protein requirement of this species may be somewhere in the vicinity of 50 %.

Andrews (1979) conducted an experiment on the effects of feeding-rate on growth, feed conversion and nutrient absorbtion of channel catfish (Ictalurus punctatus). Significant decrease in growth and feed conversion (feed/gain) rates was observed in treatments fed with 110 % of the control rates. Water quality monitoring indicated that this reduction in performance was not due to poorer water quality. Gains were not significantly affected by feeding 90 % of the control level, but were reduced in the 75 and 50 % treatments. Food efficiencies were improved by reduction of the feeding rate to 90 or 75 % of the control level, but were not further improved at the 50 % feeding level.

Rahaman et al. (1983) conducted an experiment to study the growth performance of catfish fry (Heteropneustes fossilis), reared in laboratory aquaria on 3 fish feeds prepared such indigenous materials as frog meal, mustard oil cake, rice bran, aroid leaves and salt, having 40, 30 and 20 % protein levels. They found that feed containing 40 % protein level was better for fish growth. They concluded that a good feed must contain 40 % or more of good quality protein.

Mazid et al. (1979) studied the growth response of Tilapia zillii fingerlings with 6 purified isocaloric casein diets within a range of 21 – 53 % crude protein to determine the optimum protein requirement. A protein-energy ratio of 81 appeared to be more efficiently utilized by the fish in terms of protein deposition and energy retention than diets with higher levels of crude protein. These results indicated that Tilapia zillii requires about 35 % protein in its diet for optimum growth.

D. METHODS

The proposed study would be conducted at FARS in colaboration with the Dept. of Fisheries Technology, Animal Nutrition and Biochemistry of BAU for a period of 3 months.

a) Design of the experiment

The collected catfish fry, either from natural sources or from laboratory hatched larvae, would be divided into 4 groups by random sampling for rearing in 12 glass aquaria having equal size of 0.90 × 0.35 × 0.35 m. Among the 4, three groups would be fed with 3 different supplemental feeds, A, B, and C, formulated with indigenous ingredients either alone or in mixed forms with 50, 40 and 30 % protein level respectively. In this study, the following ingredients would be used:

• frog waste meal;
• mustard oil cake;
• fish meal;
• blood meal;
• wheat bran;
• rice bran;
• soyabean oil;
• duckweeds and/or Azolla spp.;
• a high starch component.

Another group of fry with no supplemental feed will be treated as the negative control group D. Three replications for each treatment and also for the control would be made in order to get good comparable data. Statistically, the randomized block design (RBD) would be applied to study the significance of the developed feeds for the growth of the experimental fish. The proposed design may be summarized as follows:

b) Preparation of diet/feed

The selected ingredients as mentioned above will be used for the preparation of 3 different isocaloric fish feeds, denoted by A, B and c according to the treatment groups, by mixing the ingredients in such a manner so as to give crude protein values of 50, 40 and 30 % respectively. The amounts of ingredients needed to prepare 1 kg of feed should be calculated from their proximate chemical composition and adjusted in such a manner that all the feeds contain nearly the same amount of energy per kg of feed. The feed should be made into bite size pellets by adding starch solution or liquid from boiled rice and dried in an oven at 40° C for 2 days; or be extracted as pellets from a pelleting machine and stored, sealed and frozen until used. The pelleted feeds have many advantages over the conventional feeding system, since it keeps down wastage of feed to a minimum and further also provides a good way of effective checking food utilization by the cultured fish (Jeychandran & Paulraj, 1977).

c) Analytical procedures

All samples of fish feed ingredients would be analyzed either in duplicate or triplicate to determine the percentage of moisture, ash, crude protein, total lipid and crude fibre, following the procedures developed by the Association of Official Agricultural Chemists (AOAC, 1965). The moisture content would be determined by heating in an air oven at 150° C for about 4 hours until there is no further loss in weight. The crude protein would be determined by Kjeldahl method, crude fat by extraction with petroleum ether (60 – 80° C) in a Soxhelt extraction apparatus. Crude fibre is the portion of the total carbohydrate of a feed that is resistant to the acid and alkali treatment. The moisture and fat free sample would be boiled first with 1.25 % sulfuric acid for 30 minutes, filtered, and then with 1.25 % sodium hydroxide for 30 minutes and filtered. The residu would be dried and weighed. It would be then ashed and weighed. And crude fibre would be calculated. Ash content would be determined by ashing in a furnace at 600°C for several hours. The carbohydrate content (nitrogen free extract, Cullison 1975) was calculated by difference. The percentage of total energy would be calculated theoretically on the basis of the knowledge that 1 g each of metabolizable protein, fat and carbohydrate yield 4.0, 9.0 and 4.0 Kcal of energy respectively.

d) Diet plan

The diet plan for the feed treatments having 50, 40 and 30 % protein level would be made depending on the calculated proximate composition of protein of each ingredient. Here a diet plan of some selected feed ingredients (depending on the standard protein content) is given (Nutrient requirements of warm water fishes and shellfishes, 1983):

e) Stocking of fish
Under the proposed study, each of the aquaria will be stocked with 50 fish fry of about one month old, having an average weight of 1 g each.

f) Application of feeds
After acclimatization of the released fish in the aquaria the fry would be fed at a rate of 4 % body weight at a particular time, two or three times daily, 6 days per week.

g) Analysis of data
The data obtained would be analyzed statistically to see whether the effects of different supplemental feeds on the growth (in terms of length and weight of the experimental fish) are significant or not. Considering the ‘time’ as block, analysis of variance (ANOVA) would be done following the experimental design layed out earlier. To evaluate the best feed, among the feeds applied, for the growth and better nutrition of the experimental fish, comparison of the experimental feeds would be done following the Dunett's test and Duncans new multiple range test methods.

E. NEEDS

a) Materials

The following materials will be required to carry out the whole experiment as per schedule:

• 12 glass aquaria of 0.90 × 0.35 × 0.35 m each;

• a total of 660 (10 % mortality) fry of Clarias batrachus of about 1 g each.

b) Equipment

• Electrical balance;
• Thermosthatically controlled oven;
• Kjeldahl apparatus;
• Muffle furnace;
• Porcelain crucibles;
• Desiccator;
• Soxhelt apparatus;
• Heating units for fibre determinations;
• Glassware (volumetric flask, Erlenmeyer, beakers, burettes, pipettes);
• Homogenizer.

c) Chemicals

Different kinds of chemicals will be required to carry out the proposed experiment for complete proximate analysis, such as H2SO4, HCl, NaS203, NaOH, isopropanol, boric acid, mercury oxide, potassium sulphate, Ba(OH) 2, and some others.

d) Staff

• Technician/laboratory attendant: 3 manmonths;
• Labourers: 3 manmonths.

These personnel will work under the direct supervision of the project co-investigator throughout the study period. Their availability should be ascertained to the project investigator as per work schedule.

F. RELATION WITH OTHER PROJECTS

The work is strongly related with all other projects in the research programme on Fish Feed and Nutrition at FARS; the teamleader of that programme, M.A. Khaleque, will be co-investigator in this project.

G. PHASING OF WORK

The entire work would be completed in 3 months time.

Notes:

• The presently proposed experiment should be considered as the Phase I for subsequent experiments in various fields of nutritional investigations and feed technology. Research proposals of the consequent phases will be prepared just after the completion of the present work and in the framework of the overall 5 year research plan.
• If the nutrition laboratory of FARS will not be well equiped as per requirement before or on the start of the experiment, the laboratory facilities of concerned Depts. of BAU should be availed through mutual understanding.

H. REFERENCES

Anrews, J.W., 1979. Some effects of feeding rate on growth, feed conversion and nutrient absorbtion of channel catfish. Aquaculture 16(3): 169 – 175.

AOAC, 1965. Official methods of analysis. Association of Official Agricultural Chemists, 10th ed., Washington DC.

Cruz, C.M. & I.L. Landencia, 1976. Preliminary study on the protein requirements of Clarias batrachus. Fish. Res. J. Philipp. 1(2): 43 – 45.

Cullison, A., 1975. Feeds and feeding. Reston Publishing Co. Inc., Virginia, USA, pp 1 – 486.

Dabrowski, K. & B. Kozak, 1979. The ude of fish meal and soyabean meal as a protein source in the diet of grass carp fry. Aquaculture 18(2): 107–114.

Devraj, K.V., 1976. On the food of channel catfish stocked in farm ponds. Aquaculture 7(1): 27 – 32.

Jeyachandran, P. & S. Paulraj, 1977. Formulation of pelleted feed and feeding trials with common carp. J. Inland Fish. Soc. India, IX: 45 – 52.

Mazid, M.A., Y. Tanaka, T. Kataynama, M.A. Rahaman, K.L. Simpson & C.O. Chochester, 1979. Growth response of Tilapia zillii fingerlings fed isocaloric diets with variable protein level. Aquaculture 18: 115 – 122.

Mollah, F.H., M.K. Inamul Haque & A.K.M. Aminul Haque, 1973. An experiment on the feeding of fry of catfish (Heteropneustes fossilis). Indian J. Fish. 28(1): 35 – 42.

Mollah, M.F.A. & E.S.P. Tan, 1982. Effects of feeding of Magur (Clarias batrachus). Indian J. Fish. 28 (1 & 2): 1 – 7.

Munnet, S.K., 1978. An experiment on the feeding of Magur (Clarias batrachus). J. Inland Fish. Soc. India 11(2): 10 – 14.

Nutrient requirements of Warmwater Fishes and Shellfishes, 1983. National Academy Press, Washington DC: pp 71 – 94.

Pal, R.N., S.C. Pathak, D.N. Singh & P.V. Dehadrai, 1977. Efficacy of feeds in survival of spawn of Anabas testudineus (Bloch). J. Inland Fish. Soc. India IX: 165 – 167.

Protap, K.M., 1977. Studies on the enzymatic activities related to varied pattern of diets in the air-breathing catfish, Clarias batrachus Linn. Hydrobiologia 52 (2–3): 235 – 237.

Protap, K.M., V.D. Padmaker & K.B. Sudib, 1978. Studies on intestinal protease, isolation, purification and effect of dietary proteins on alkaline protease activity of the air-breathing fish Clarias batrachus. Hydrobiologia 57(1): 11 – 15.

Reddy, S.R., 1979. Growth rate and conversion efficiency of the air-breathing catfish, Heteropneustes fossilis, in relation to ration size. Aquaculture 18(1): 22 – 28.

Sidhimunka, A., 1973. Culture of catfish on Thailand farms. Fish Farm. Inst. 1: 81 – 83.

Singh, C.S. & K.P. Singh, 1975. Feeding experiments on Indian major carps on Tarai ponds. J. Inland Fish. Soc. India 7: 212 – 215.

Tacon, A.G.J. & S.S. De Silva, 1983. Mineral composition of some commercial fish feeds available in Europe. Aquaculture 31(1): 11 – 20.

I. BUDGET

J. ALLOCATIONS REQUESTED

K. RECOVERY OF COSTS (INCOME)

ANNEX H

A. INTRODUCTION

In Bangladesh, culture of major carps is more popular that of other fish since they grow faster and their food conversion ratio is high. The primary concern in fish culture is to increase fish production per unit of culture space. Supplementary feeding with artificial diets is an effective measure to increase fish production. In our country supplementary feeding with mustard oil cake, fish meal, rice bran, wheat bran, etc. in different ratios is practiced. But there is lack of knowledge on the rate of digestion and absorption of the dietary components (protein, fat, carbohydrate, etc.). To render the production economical, supplemental diets must be formulated in accordance with digestability of food. There is not much information regarding digestability of food by the major carps.

The principal diet component for growth is protein and this has been given priority in nutritional requirement studies. Protein quality and quantity markedly effect the growth response of fish. Its high cost also effects the economics of fish farming. Gerking (1955) suggested that a large part of caloric requirements of fish is derived from protein. The ingredients that go intothe formulation of the diet must be related to the physiological needs of the particular fish being fed. Moreover, the nutritional value of a diet is determined ultimately by the ability of the fish to digest and absorb it. Hence, it is proposed to study digestability of protein by Indian carps. To start with, digestability of protein by mrigal (Cirrhina mrigala), fed with diets containing 30, 40 and 50 % protein would be studied.

B. OBJECTIVES

• To develop a technique to measure digestability co-efficients in mrigal;
• To measure digestability co-efficients for 3 protein level feeds for mrigal;
• To calculate the relative merits of 3 protein levels fed to mrigal.

C. LITERATURE REVIEW

Nose (1960) collected samples of rectal contents for digestability measurement, by manually stripping the fish and gently squeezing out the faecal matter from the rectum. Windell et al. (1978) collected faeces, either by applying suction to the anus or by dissecting the fish. While Ogino et al. (1973) collected faeces by passing effluent water from a fish tank through a filtration column, Choubert et al. (1979) used mechanically rotating screens to filter faecal material.

Law et al. (1985) worked on the digestability of carpet grass and napier grass by grass carp and observed that only 20.92 and 16.45 % dry matter respectively was digested, while 86.7 % of the protein released from the grass was absorbed by the species. However, with pelleted feed, digestability of dry matter and protein was 82.85 and 90.24 % respectively.

Wannigama et al. (1985) worked on the protein digestability by Sarotherodon nilotica of 4 diets containing 19, 20, 25 and 29 % crude protein and observed that digestability decreased with decreasing dietary crude protein levels, except in the case of diet containing 19 % crude protein, which did not contain only rice bran. The other diets contained rice bran, which was of poor quality.

Sena (1985) carried out digestability experiments on Sarotherodon nilotica fry at different salinities, using Cr203 as a marker at the rate of 3 %, and observed that dry matter and protein digestability varied from day to day, indicating a possible rhythmicity in digestability.

Cho et al. (1985) found the apparent digestability co-efficients of protein ingredients in diets fed to rainbow trout, as blood meal 87 %, fish meal 92 %, and soyabean meal 96 %.

D. METHODS

The digestability co-efficient will be measured by the indirect method, using chromic oxide (Cr203) as a marker. Experiments with mrigal will be conducted with 3 protein level feeds (30, 40 and 50 % crude protein), made from fish meal, blood meal, oil seed cake, rica bran, wheat bran, etc., as detailed below. Each trial will have 3 replications. The diet formulation will be as follows:

Nine aquaria will be prepared for stocking the fish. Each aquarium will be stocked with 10 mrigal, each weighing 100 g, and the fish will be acclimatized for one week. In the week-long experiment force feeding will be done twice a day at the rate of 2 % of the body weight. Feeding will be given at 08.00 and 14.00 hours. During feeding 0.5 % chromic oxide will be included in the feed as marker. Faeces will be collected by stripping each fish on the 3rd, 5th, and 7th day of this week-long experiment, two hours after morning feeding. The stripped material will be collected in weighing boats, then the pooled sample of faces from each fish will be placed in an oven at 105°C overnight for drying to constant weight.

The dried faeces from the oven will be weighed and ground in a mortar. The chromic oxide content of the faeces and of the diets will be determined by using the wet-acid oxidation method. Crude protein both in diet and faeces will be measured by the Kjeldahl method. Apparent digestability co-efficients will be calculated by using the formula of Maynard & Loosli (1956) employing chromic oxide as the dietary marker and dietary component (protein, carbohydrate, fat) as nutrient:

Significance of results will be analyzed by ANOVA and simple student T-test for significance between mean values obtained from the chemical analysis and calculated apparent digestability co-efficients.

E. NEEDS

1. Facilities
   • 90 fish each weighing 100 g;

   • 9 aquaria.

2. Equipment
   • Tecator Kjeltee system 1003, including distillation unit and digestion block unit;
   • Alluminium foil;
   • Weighing boats;
   • Safety bulb pipettes, 5 ml and 30 ml;
   • Burette 25 ml (0.05 gradation);
   • 100 ml volumatric flask;
   • Gas heating mantles;
   • Goggles;
   • Grinder;
   • Mortar & pestle;
   • Gloves;

   • Spectrophotometer set at 350 μm, with cuvettes.

3. Chemicals and reagents
   • Kjettale catalyst tablets (BDH Ltd., Dorset);
   • Sodium thiosulphate (330 g/l of Na2S203, 5 H2O);
   • Boric acid (saturated solution);
   • 0.2 N hydrochloric acid;
   • BDH 4.5 indicator;
   • Concentrated nitric acid;
   • Perchloric acid;

   • Chromic oxide.

4. Labour

   • 3.5 manmonths.

F. RELATION WITH OTHER PROJECTS

The work is stronly related with other projects in the research programme on fish feed and nutrition at FARS; the teamleader of that programme, M.A. Khaleque, will be co-investigator in this project.

G. PHASING OF WORK (barchart of activities)

The entire work will be completed within one month; it is expected that similar experiments will follow.

H. REFERENCES

AOAC, 1980. Official methods of analysis of the Association of Official Agricultural Chemists, 13th ed. Washington DC: 1018 pp.

Cho, C.Y., C.B. Cowey & T. Watanable, 1985. Methodological approaches to research and development. Fin Fish Nutrition in Asia, IDRC-233e: 35 pp.

Choubert, G., J. Dela Nowe & P. Luguet, 1979. Continuous quantitative automatic collector for fish faeces. Progr. Fish Cult. 41: 64 – 67.

De Silva & S. Gena, 1985. Evaluation of the use of internal and external markers in digestability studies. Fin Fish Nutrition in Asia, IDRC-233e: 96 – 101.

Gerking, S.D., 1955. Influence of feeding on body composition and protein metabolism of bluegill sunfish. Physiol. Zool. 28: 267 – 282.

Law, A.T., S.H. Cheah & K.J. Ang, 1985. An evaluation of the apparent digestability of some locally available plants and a pelleted feed in three finfish species, Malaysia. Fin Fish Nutrition in Asia, IDRC-233e: 90 – 95.

Maynard, L.A. & J.K. Loosli, 1956. Animal nutrition. 4th ed. McGraw Hill, New York: 484 pp.

Nose, T., 1960. On the digestion of food protein by goldfish and rainbow trout. Bull. Freshw. Fish. Res. Lab. 10: 23 – 28.

Ogino, C., J. Kakino & M.S. Chen, 1973. Determination of metabolic faecal nitrogen and endogenous nitrogen excretion of carp. Bull. Japan. Soc. Scient. Fish. 39: 519 – 523.

Suhenda, N. & R. Djajadiredja, 1985. Determination of the optimum level of vitamin premix for the diet of common carp (Cyprinus carpio L) fingerlings. Fin Fish Nutrition in Asia, IDRC-233e: 130 – 135.

Wannigama, N.D., D.E.M. Weerakoon & Muthukumarana, 1985. Cage culture of S. niloticus in Sri Lanka: effect of stocking density and dietary crude protein levels on growth. Fin Fish Nutrition in Asia, IDRC-233e: 113–117.

Windell, J.T., J.W. Foltz & J.A. Sarokon, 1978. Methods of fecal collection and nutrient leaching in digestability studies. Progr. Fish Cult. 40: 51 – 55.

I. BUDGET

J. ALLOCATIONS REQUESTED

K. RECOVERY OF COSTS (INCOME)

ANNEX I
TARGET SPECIES FOR NUTRITION AND DIET DEVELOPMENT IN BANGLADESH

Fish diet research priorities

ANNEX J
BINDERS FOR DIETS

1. Protein - protein (glue-like).
2. Protein + salt (2 – 3 %), 5 minutes blend.
3. Protein - CHO (minimum 10 – 30 % flour).
4. Protein - dehydration (hot air (70 C) or air-dried).
5. Protein polymerization - plastein reaction.
6. CMC (carboxymethylcellulose or other derivates).
7. Bentonite (clay).
8. Cement - < 3%.
9. Lignin sulforate.
10. Agar or other gums.
11. Starches - commercial alpha starch and others
12. Gelatin - 20 > 100 bloom.
13. Pectine - guarans.
14. Cooking - minimum 5 minutes, and less than 10 minutes.