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DIGESTIBILITY, FEEDING VALUE AND LIMITING AMINOftCIDS IN HIGH-FIBRE AND FIBRE-REDUCED SUNFLOWER CAKES FED TO TILAPIA {OREOCHROMIS NJLOTICUS) BY JOYCE GICHIKU MAINA B.Sc. The University of Nairobi, 1981. M.Sc, The University of Nairobi, 1992, A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE FACULTY OF GRADUATE STUDIES (FACULTY OF AGRICULTURE) We accept this thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA FEBRUARY 2001. (g) Joyce Gichiku Maina, 2001

Transcript of joyce gichiku maina - Open Collections

DIGESTIBILITY, FEEDING VALUE AND LIMITING AMINOftCIDS IN HIGH-FIBRE AND FIBRE-REDUCED SUNFLOWER CAKES FED TO TILAPIA {OREOCHROMIS NJLOTICUS)

B Y

JOYCE GICHIKU M A I N A

B.Sc. The University of Nairobi, 1981. M . S c , The University of Nairobi, 1992,

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN

THE F A C U L T Y OF GRADUATE STUDIES (FACULTY OF AGRICULTURE)

We accept this thesis as conforming to the required standard.

THE UNIVERSITY OF BRITISH COLUMBIA FEBRUARY 2001.

(g) Joyce Gichiku Maina, 2001

In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.

Department of ffrCuL-7Y frf AQAldyLTUje/H-. S ^ i B v C e

The University of British Columbia Vancouver, Canada

Date Fg&%Uff*~V g P p /

DE-6 (2/88)

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Abstract

Four experiments were conducted at the University of Nairobi, in Kenya, to determine

the effect of reducing the amount of fibre in sunflower cake on nutrient digestibility and

feed utilization in tilapia (O. niloticus), and to compare this low-fibre cake with a

commercially available high-fibre sunflower cake. The extent to which protein from a

high-fibre and a fibre-reduced sunflower cake could replace fishmeal protein in tilapia

diets, and the effects of supplementing diets made from a low-fibre sunflower cake with

amino acids lysine, methionine, and threonine on growth, feed intake, and feed utilization

were also investigated. Also of interest was to compare digestibility and feeding value of

Kenyan omena fishmeal with that of Low-Temperature (LT) anchovy fishmeal.

Tilapia (O. niloticus) fingerlings were used in all the experiments. Water

temperatures and dissolved oxygen concentrations were maintained above 26 °C and 5.5

mg/litre respectively. Dehulling of sunflower seeds was done using a manual dehuller.

Crude fibre levels in the dehulled cakes were all below 15% (DM basis).

Protein from the low-fibre and high-fibre sunflower cakes was well digested by

tilapia. The apparent digestibilities of protein in the sunflower cakes and the fishmeals

were not significantly different. Reduction of fibre in sunflower cake had no effect on

the digestibility of protein. Digestibility of energy in the sunflower cakes was low.

Apparent digestibility coefficient for energy (ADC-E) and digestible energy

concentration (DE) were higher in the low-fibre sunflower cake than in the high fibre

cake, but the differences were only significant for DE. There were no differences in the

apparent digestibilities of protein, energy and organic matter between omena and

anchovy fishmeals.

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In Experiment 2, the feeding value of a high-fibre and a low-fibre sunflower cake, omena

and anchovy fishmeals was evaluated at two dietary protein levels (20% and 30%).

There was no significant interaction between protein level and protein source. Fish fed at

the 30% protein level gained more weight and had better feed conversion efficiency

(FCE) than those fed at the 20% level. There were no significant differences in weight

gain between fish fed diets based on anchovy and omena fishmeals and the low-fibre

sunflower cake. Fish fed diets based on the high-fibre cake gained significantly (P <

0.05) less weight than those fed diets based on anchovy fishmeal.

The low-fibre and high-fibre sunflower cakes were tested over a wide range of

dietary inclusion in Experiment 3, each supplying 30%, 60%, and 80% of the dietary

protein. The extent to which body fatty acids in tilapia reflect dietary fatty acids was also

investigated. The low-fibre and high-fibre sunflower cakes could comprise up to 60%

and 30% of the dietary protein respectively without compromising the performance of the

fish. The inclusion of higher levels of the cakes in the diets caused a depression in feed

intake, which resulted in lower weight gains of the fish fed these diets compared to those

fed the control diet. Body fatty acid composition closely reflected dietary fatty acid

composition.

In Experiment 4, a basal diet in which a fibre-reduced sunflower cake provided

80% of the dietary protein was supplemented with amino acids lysine, methionine and

threonine. The levels of these amino acids in the basal diet were 1.17%, 0.75% and

1.05% for lysine, methionine and threonine respectively, while the stipulated

requirements (NRC, 1993) are 1.54%, 0.8% and 1.2% respectively. There was a trend to

improved growth rate and FCE in fish fed diets supplemented with lysine and threonine,

i i i

but the improvement did not attain statistical significance. Methionine, added alone or

together with threonine did not elicit any response in fish.

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TABLE OF CONTENTS

Abstract ii Table of contents v List of Tables viii List of Appendices ix Acknowledgements x

Chapter 1: General introduction 1 1.2 References 5 Chapter 2: Review of Literature 6 2.1 Aquaculture in Kenya 6 2.2 Fish species cultured 9 2.3 Dietary protein and amino acids requirement in fish 10 2.3.1 Search for new sources of protein 16 2.4 Sunflower (Helianthus annuus) 26 2.4.1 Taxonomy and origin of the domesticated sunflower 26 2.4.2 Morphology of the sunflower plant 27 2.4.3 Chemical and physical composition of sunflower seeds 27 2.4.4 Sunflower seed oil 28 2.4.5 Sunflower seed proteins 29 2.4.6 Composition of sunflower hulls 29 2.4.7 Anti-nutritive factors in sunflower seeds 31 2.4.8 Processing methods 32 2.4.9 Sunflower meal 32 2.5 Use of sunflower meal in animal feeds 36 2.5.1 Ruminants 36 2.5.2 Non-ruminants 37 2.5.2.1 Sunflower meal in swine diets 37 2.5.2.2 Sunflower meal in poultry diets 39 2.5.2.2.1 Broilers 39 2.5.2.2.2 Sunflower meal in Layer diets 40 2.6 Use of sunflower meal in fish diets 40 2.7 References 45

Chapter 3: Experiment 1: Digestibility of nutrients and energy in wheat bran, high-fibre and fibre-reduced sunflower cakes, anchovy fishmeal and omena fishmeal by Oreochromis niloticus 56

3.0 Abstract 56 3.1 Introduction and objectives 57 3.2 Materials and methods 60 3.2.1 Sunflower cakes 60 3.2.1.1 Fibre-reduced sunflower cake 60

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3.2.1.2 High-fibre sunflower cake 60 3.2.2 Ingredients other than sunflower cakes 60 3.2.3 Chemical analyses 61 3.2.4 Experimental diets 61 3.2.5 Supply and maintenance of fish 63 3.2.6 Fecal collection 64 3.2.7 Digestibility assessment 64 3.2.8 Data collection and analytical procedures 66 3.2.9 Statistical analyses 66 3.3 Results and discussion 67 3.3.1 Chemical composition of the reference diet, test

diets and test ingredients. 67 3.3.2 Fish performance 67 3.3.3 Apparent digestibility of nutrients in test ingredients 70 3.3.4 Apparent digestibility coefficient for protein (ADC-P) 70 3.3.5 Apparent digestibility coefficient for energy (ADC-E) and

digestible energy concentration (DE) in test ingredients 75 3.3.6 Apparent digestibility coefficient for organic matter (ADC-OM) 78 3.4 Conclusions 79 3.5 References 80

Chapter 4: Experiment 2: The feeding value and protein quality in high-fibre and fibre-reduced sunflower cakes and Kenya's "omena" fishmeal for tilapia (Oreochromis niloticus) 83 4.0. Abstract 83 4.1 Introduction and objectives 85 4.2 Materials and methods 85 4.2.1 Experimental diets and design 87 4.2.2 Fish sampling 90 4.2.3 Data collection and analytical procedures 91 4.2.4 Chemical analyses 91 4.2.5 Statistical analysis 92 4.3 Results and discussion 93 4.3.1 Chemical composition of the diets 93 4.3.2 Fish performance, PER, and PPV 97 4.3.3 Effect of diets on whole body composition 106 4.4 Conclusions 110 4.5 References 113

Chapter 5: Experiment 3: Partial replacement of fishmeal with high-fibre and low-fibre sunflower cakes in diets for tilapia (O. niloticus): Effect on fish performance and whole body fatty acids. 117

5.0 Abstract 117 5.1 Introduction and objectives 119

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5.2 Materials and methods 120 5.2.1 Experimental diets and design 120 5.2.2 Fish sampling 122 5.2.3 Data collection and analytical procedures 123 5.2.4 Chemical analyses 123 5.2.5 Statistical analysis 124 5.3 Results and discussion 125 5.3.1 Chemical composition of the diets 125 5.3.2 Fish performance PER, PPV, body and fatty acid composition 127 5.4 Conclusions 142 5.5 References 144 Chapter 6: Experiment 4. Evaluation of the most limiting amino acids in diets based on sunflower cake fed to tilapia (O. niloticus). 148

6.0 Abstract 148 6.1 Introduction and objectives 149 6.2 Materials and methods 151 6.2.1 Experimental diets and design 151 6.2.2 Fish sampling 153 6.2.3 Data collection and analytical procedures 153 6.2.4 Chemical analyses 153 6.2.5 Statistical analyses 154 6.3 Results and discussion 155 6.3.1 Chemical compositions of the diets 155 6.3.2 Fish performance 156 6.4 Conclusions 166 6.2 References 167

Chapter 7: General discussion, conclusions and recommendations 170 7.1 References 176

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LIST OF T A B L E S

Table # 2.1 Fish production in Kenya 8 2.2 Some fibre components of sunflower seed hulls and other

agricultural residues 30 2.3 Proximate compositions of Kenyan sunflower seed cakes 34 2.4 Average amino acid compositions of sunflower meal, soybean meal,

cottonseed meal and rapeseed (or canola) meal. 35 3.1 Compositions of the diets used in Experiment 1 62 3.2 Compositions of the ingredients used in Experiment 1 68 3.3 Performance of O. niloticus after 50 days of feeding on the

experimental diets 69 3;'4 Apparent digestibility coefficients (ADCs) and apparent digestible

energy (ADE) values of the reference and test diets 71 3.5 Apparent digestibility coefficients (ADCs) and digestible energy

values for the fibre-reduced and high-fibre sunflower cakes, omena fishmeal, anchovy fishmeal and wheat bran 72

4.1 Chemical compositions of the ingredients 88 4.2 Compositions of the diets used in Experiment 2 89 4.3 Amino acid compositions of the test diets 95 4.4 Effect of protein level on fish performance 98 4.5 Effect of source of protein on fish performance 99 4.6 Performance of O. niloticus fed diets containing high-fibre and fibre-

reduced sunflower cakes, and LT. anchovy and omena fishmeals for 78 days. 100

4.7 Effect of feeding diets based on high-fibre and fibre-reduced sunflower seed cakes, LT. anchovy and omena fishmeals on whole body composition of O. niloticus after 78 days 107

5.1 Compositions of the diets used in Experiment 3 121 5.2 Amino acid compositions of diets used in Experiment 3 126 5.3 Percentages of fatty acid in the diets 128 5.4 Fatty acid compositions of corn oil, sunflower oil and herring oil. 129 5.5 Fish performance in relation to diet treatment after 70 days 130 5.6 Effect of protein source and level of sunflower cake on fish performance 131 5.7 Percentages of body proximate constituents viz., moisture, protein fat and

ash (Air-dry basis) at 70 days in relation to diet treatments. 132 5.8 Percentages of fatty acid levels in the whole body of fish in relation to

diet treatment. 137

5.9 Effect of type of sunflower cake and level in the diet on percentages of whole body fatty acids 138

6.1 Compositions and chemical analyses of diets used in Experiment 4 152

6.2 Determined Amino acid compositions of the diets used in Experiment 4 (DM basis) 157

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6.3 Determined amino acid compositions of the diets used in Experiment 4 (% of dietary protein)

6.4 Performance of fish (absolute weight, weight gains, specific growth rates (SGR), feed intake, and feed conversion efficiency) in relation to diet treatment

LIST OF APPENDIXES

Appendix # 1 Kabete water quality parameters assessed at the start of the study

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Acknowledgments

This work was made possible by many people to whom I am greatly indebted. To Dr. Beames, my thesis supervisor, I say a big thank you for going beyond the call of duty to ensure that the work was completed. Though you retired from active teaching more than three years ago, you always found the time to go through my work patiently, meticulously and thoroughly, and were always ready for discussions. Special thanks to members of my committee, Dr. Higgs, Dr. Mbugua, Dr. Iwama and Dr. Kisia for going through all those drafts, and providing very valuable ideas. I am also grateful to Gay Huchelega for proof reading the thesis, valuable comments, and for being a very genuine friend. Thanks also to Mike Pitt for the concern you showed to all of us who were on the CIDA program.

My family deserves special mention. To my husband, Julius Maina, I could not have done it without you. Thank you for taking very good care of our daughter during the years I have been away. Nobody would have done a better job. I am very grateful for your love, encouragement and support. I greatly appreciated all those long distance calls; emails, and letters which helped me maintain my sanity in a distant land. To my daughter, Beatrice, I wish to apologize for the years that I could not be physically present with you. I am very grateful that you understood, and survived without me. To my parents, Ester and Lasidslas Mwangi, thank you for sowing the seed, and believing that I could do it. I am also very appreciative to my brothers and sisters, for your concern for me, and for taking care of mum and dad.

I owe special gratitude to all my friends. Gracias for making U B C and Vancouver a home away from home, and for welcoming me into your homes and your lives. I am immensely grateful to the Gichane's, Kamabu's, Tidyebwa's, Grace Wangu, Muthoni and Mugo Kimari, The Njenga's, Kangethe's, Wanjau's, Kamande's, Emmah Kishindo, Charles Ochieng, John Agak, Lucy Karanja, Abba Hammond, Rebeccca Biegon, and many others. Thank you for your support. I also wish to acknowledge my friend, Dr. Lucy Kabuage. I am grateful for your fellowship and prayers.

To Giles, Sylvia, and Siva, thank you for your help in the lab. To Dr. Thompson, and Joyce Tom, I appreciated the interest you showed in my work, and for spurring me on to the finish line. To Rachel Njoroge and Thomas Njau, who helped with the experiments, thank you for your patience and dedication. I also wish to thank all the people who helped with the data analyses, C. Matere, Dr. Wanjau, and Dr. Charagu -God bless you all.

Lastly, I wish to acknowledge some organizations that contributed greatly to the accomplishment of this work. I am grateful to the Canadian International Development Agency (CIDA) and Rockefeller Foundation for funding the work, and the University of Nairobi, for giving me the opportunity.

It has been a long journey. I am grateful to each and everyone of you who helped along the way.

Chapter 1

General Introduction

Nile tilapia (O. niloticus) has become increasingly important as an inexpensive source of

dietary protein in many countries. Tilapia culture is widespread in Africa and Asia on

account of the fast growth, adaptability to a wide range of culture conditions and high

consumer acceptability of this genus of fish. Nile tilapias, like all fish, require energy,

protein, lipids, vitamins and minerals in their diets. In the wild these nutrients may be

provided by the natural feed in the ponds. However, as fish biomass increases, e.g. in

aquaculture, the provision of artificial feeds becomes essential.

The characteristic diet of tilapia in the wild is a mixture of plant matter and

detritus of plant origin. Blue green algae, diatoms, macrophytes and amorphous detritus

are all common natural constituents of an adult tilapia diet (Bowen, 1990). Tilapia

possess morphological and physiological adaptation mechanisms for utilization of these

dietary components. Pharyngeal teeth break food particles into smaller units for easier

peristaltic mixing and increased exposure to digestive enzymes. Gastric acid secreted to

an unusually low pH lyses prokaryotic and eucaryotic cell walls to expose the cytoplasm

to intestinal enzymes. This ability of tilapia to digest high-fibre materials has not been

fully exploited in the development of tilapia diets for intensive culture.

The protein component of most commercial fish diets generally includes a large

proportion of fishmeal, usually 30-50% of the diet. Fishmeal is expensive and therefore

considerable effort has gone into research to evaluate new protein sources to totally or

partially replace the fishmeal. Soybean meal has an acceptable amino acid profile for the

growth of most fish species, and therefore has been widely used to partially replace

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fishmeal (Lovell, 1991). Soybeans, however, are not suitable as a replacement for

fishmeal in some countries because they would have to be imported. In Kenya, for

example, in 1990 and 1991 more than five million tonnes of soybean meal were imported

for the Animal Feed Industry (Dept. of Animal Production, 1992). Thus there is a need to

evaluate the more readily available locally produced sources of protein.

Sunflower seed cake is an inexpensive and common oil-processing byproduct in

many countries. In Kenya, sunflower farming was revitalized after a period of decline

when the government decided to decontrol consumer prices for edible oils, fats, and

animal feeds. Despite the ready availability of sunflower seed cake, its very high crude

fibre content limits its use in animal feeds, especially for monogastric species, which

account for 70% of the total feed produced. The crude fibre level of sunflower seed

cakes in Kenya ranges from 24.1% to 40.2% (Jacob, 1993). Complete industrial

dehulling of sunflower seeds has not been achieved (Tranchino et al, 1984; Cargill,

1980). Partial dehulling of the seed (10 - 12% removal) is common in the oil-seed

industry. In the present study, one of the primary objectives was to determine the effect

of fibre reduction of sunflower seed cake on the utilization of this protein product by fish.

A second objective was to evaluate Kenya's "omena" fishmeal, made from

Rastrineobola argentea. This is a small cyprinid fish endemic to Lake Victoria, Lake

Kyoga and Lake Nabugabo in Uganda. It is locally known as "omena" in Kenya,

"dagaa" in Tanzania and "mukene" in Uganda (Manyala et el., 1992; Wandera, 1992). It

has a short life span of 1-2 years and its total length rarely exceeds 100 mm (Wanink,

1989). Prior to 1960, R.. argentea was of little economic importance in Kenya, forming

an insignificant proportion of fish landed from Lake Victoria (Chitamwemba, 1992;

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FAO, 1992). Catches of this fish have undergone explosive changes in the last 15 - 20

years (Manyala et al., 1992). It has become very important commercially, especially in

the animal feed industry. Prior to 1991, Kenya relied heavily on imported fishmeal,

mainly herring meal from Denmark. In 1992, Kenya adopted a Structural Adjustment

Program as recommended by the PMF. As a result of this, the value of the Kenya shilling

fell sharply against the major currencies of the world and importation of goods became

very expensive. Feed manufacturers turned to omena fishmeal to replace imported

fishmeal. According to the Kenya Bureau of Statistics, before 1991 the country imported

more than 350,000 metric tonnes of fishmeal every year. This figure fell to 800 metric

tonnes in 1991.

Currently, omena fishmeal is widely used in the animal feeds industry. Despite

this widespread use, no studies have been done to assess its quality and feeding value.

The Kenya Bureau of Standards (KBS), which sets standards for consumer products, has

not set any specifications for omena fishmeal due to lack of scientific research data.

Hence there is need to assess the quality and feeding value of omena fishmeal relative to

the imported fishmeals.

Specific objectives of this study were:

a) To determine the effect of reducing fibre content in sunflower cake on the

apparent digestibility of protein, energy, and organic matter using tilapia (O.

niloticus) as the test animal.

b) To compare the nutritional values and protein qualities of diets based on high-

fibre and low-fibre sunflower cakes, omena and anchovy fishmeals, when fed to

tilapia (O. niloticus) at two levels of dietary protein.

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c) To establish the highest level at which high-fibre and low-fibre sunflower cakes

could replace fishmeal in diets of tilapia (0. niloticus) without affecting growth,

and to evaluate the effect of substituting sunflower cake for fishmeal on whole

body fatty acid composition.

d) To determine the most limiting amino acids in diets based on sunflower cake fed

to O. niloticus, and to evaluate the effect of supplementing these diets with the

fore-going amino acids on fish performance.

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1.2 References

Bowen, S.H., 1982. Feeding, digestion and growth - Qualitative considerations In: The biology and culture of tilapia. R.S.V. Pullin and R.H. Lowe Mclonell (Eds.) I C L A R M , Manila Philipines.

Cargill Inc., 1980. Industry News: America's native oilseed crop rediscovered. Journal of American Oil Chemists Society, 57: 264 - 268

Chitamwemba, D B F . , 1992. The fishery of Rastrineobola argentea in Southern Sector of Lake Victoria. In: The Lake Victoria dagaa 7?. argentea. Report of the first meeting of the working group on the Lake Victoria R. argentea, 9 - 1 1 Dec. 1991, Kisumu, Kenya. UNDP/FAO Regional Project for Inland Fisheries Planning. IFIP

FAO., 1992. Report of the sixth session of the CLFA subcommittee for the development & management of the fisheries of Lake Victoria, 10-13 February, 1992. FAO Fish Reports.

Jacob, J.P., 1993. The feeding value of Kenyan sorghum, sunflower seed cake, and sesame seed cake for poultry. Ph.D. Thesis, The University of British Columbia.

Lovell, T., 1991. Nutrition of aquaculture species. J. Anim. Science, 69: 4193 - 4200

Manyala, J.O., Nyawade, C O . , and Rabour, C O . , 1992. The Dagaa (Rastrineobola argentea Pellegrin) Fishery in the Kenyan Waters of Lake Victoria: A natural review and proposal for future research. In: The Lake Victoria dagaa (R. argentea). Report of the first meeting of the working group on L. Victoria R. argentea. 9 - 1 1 December, 1991, Kisumu, Kenya. UNDP/FAO Regional Project for Inland Fisheries Planning IFIP.

Tranchino, L. , Melle, F., and Sodini G., 1984. Almost complete dehulling of high oil sunflower seed. Journal of American Oil Chemists Society., 61: 1261 - 1265.

Wandera, S B . , 1992. A study of R. argentea in Ugandan lakes. In: The Lake Victoria Dagaa. Report of the first meeting of the working group on Lake Victoria R. argentea 9 -11 December, 1991, Kisumu, Kenya. UNDP/FAO Regional Project for Inland Fisheries Planning IFIP.

Wanink, J. H . , 1989. The Ecology and the Fisheries of dagaa (R. argentea). In: Fish Stock and Fisheries in Lake Victoria. A handbook to the Hest/TAFIRI & FAO/DANTDA regional seminar, Mwanza, January - February 1989. Report of the Ftaplocromis Ecology Survey Team, (HEST) and the Tanzanian Fisheries Research Institute (TAFIRI) no. 53, Leiden, The Netherlands, RUL.

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Chapter 2

Review of literature

2.1 Aquaculture in Kenya

Development of aquaculture in Sub-Saharan Africa is relatively recent. Most of the

aquaculture systems were introduced in the last 35 years. Trout farming in high altitude

areas was first introduced in South Africa in 1859, and in Kenya in 1910. According to

FAO statistics (Coche et al. 1994), total aquaculture production in Africa in 1990 was

14,700 metric tonnes, which was equivalent to 0.5% of world aquaculture production.

The estimated value of this production was US$ 25 million. Nigeria, Ivory Coast,

Zambia, and Kenya were among the largest producers. More than 30 indigenous and

exotic species are cultured in the region. Tilapia, particularly Oreochromis niloticus are

the major species cultured, but other species like Clarias gariepinus (catfish) and

Cyrpinus carpio (carp) are also important. Vincke (1995) lists three production systems

practiced in Sub-Saharan Africa, viz., extensive, semi-intensive and intensive systems.

The extensive system is the oldest, where aquaculture is mainly rural and directed

to satisfying nutritional needs of the family. Small and large-scale commercial farmers

prefer the semi-intensive system where aquaculture is integrated with raising farm

animals. This system is becoming increasingly important in the development of

aquaculture in the region. Intensive farming has not been fully developed (Coche et al.,

1994). There are only a few private commercial farms in Kenya, Malawi, Nigeria,

Zambia and Zimbabwe. At the Continental level, there are various constraints to fish

farming, such as a lack of a good national data bank, a lack of good statistical production

data, scarcity of public funds and a lack of good co-ordination between researchers and

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producers (Coche et al., 1994). There are also social and technological constraints such

as an inaccessibility to credit for small- scale fish farmers, an excessively low

technological level of the farmers and a shortage of various key feed ingredients because

of competition for food for humans and other animal species such as poultry and swine.

Kenya has 10,000 square kilometers of inland lakes and 6500 km of coastline.

Eighty percent of the fish landings are from fresh water lakes, and 19% from marine

sources . Aquaculture contributes only 0.5% of the total fish production (Table 2.1).

About 86% of the fish from inland waters come from Lake Victoria, while 6% comes

from Lake Turkana. Other lakes and Rivers contribute 8%. The main species in the wild

catch are Lates niloticus (Nile perch), Rastrineobola argentea (Omena), Oreochromis

niloticus (Nile tilapia), Cyprinus carpio (Common carp) and Micro salmoides (black

bass).

The history of fish farming in Kenya dates back to 1910 during the colonial era.

European settlers, unfamiliar with Kenyan indigenous fish, imported trout (Onchorhycus

mykiss and Salmo trutta), black bass, and common carp. The fish were stocked into

various rivers for sport fishing. Black bass was also stocked into Lake Naivasha.

Government involvement in fisheries started around 1926, with allocation of funds for the

care of trout and trout fishing. From 1926 to 1937, the fisheries program was

administered by the Game Department. In 1954, a separate department for fisheries was

formed and a trout hatchery established at Kiganjo. After the Second World War, the

government of Kenya started showing an interest in raising indigenous fish, particularly

tilapia, as a potential food crop for the rural population.

Table 2.1: Fish Production in Kenya (metric tonnes)

Year Aquaculture Inland Marine Total capture capture Production

1987 310 124,096 6,875 131,281

1989 530 131,000 14,566 146,096

1993 1,014 167,510 14,966 183,490

1994 1,114 173,500 28,249 202,863

1995 1,083 154,164 38,541 193,788

Source: Fisheries Department, Kenya (1995).

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A program for stocking dams and ponds was started in Western Kenya and in the sixties,

a campaign "eat more fish" was launched and quickly spread to various parts of Kenya,

including the non-fish-eating communities in Central Province (Ochieng, 1994).

Currently, fish farming is mostly practiced as part of other farming activities. At

the national level, the contribution of fish farming to fish production is insignificant, but

it has an important effect on nutrition and income at the farmer level. Besides, the main

sources of fish, which have traditionally been the fresh-water lakes, particularly Lake

Victoria, are having problems with water hyacinth, pollution, over-fishing, and the

disappearance of some species from the catches. Consequently, the gap between the

national fish requirement and production can only be met through aquaculture.

2.2 Fish species cultured

According to Balarin (1985), the warm-water species currently cultured in Kenya are

tilapias (O. niloticus, O. mossambicus, T. rendali and T. zilli), common and mirror carp

(Cyprinus carpio), and black bass (Micropterus salmoides). Rainbow trout

(Onchorhynchus mykiss) and to a lesser extent, brown trout (Salmo trutta) are cultured in

high-altitude cold-water areas. Marine shrimp (Penaeus indicus and P. monodon) are

cultured at the coast. There is a wide range of culture practices. Small family fish farms

consist of earthen ponds (130 m2 to 1000 m2), stocked with tilapia (Western Kenya), or

tilapia and carp (Central Kenya) (Ochieng, 1994). Water may be stagnant or flowing

through. On-farm organic fertilizers may be applied at varying rates. Productivity is in

the order of 500 - 2000 kg/ha/year (Ochieng 1994), with the fish being consumed by the

house-hold. Tilapia fry are produced at the Department of Fisheries in Sagana, and at the

Lake Basin Development Authority production centers. Trout farming is done on

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commercial farms at the slopes of Mt. Kenya. Trout farming requires clean, clear, cold

(10-18 °C) water flowing in large quantities; this restricts its practice in Kenya. In

addition, investment and operating costs are high.

2.3 Dietary Protein and Amino Acid Requirements of fish

Dietary protein quality and quantity are major factors that influence fish performance.

Protein is the most expensive component of fish diets. Most studies done on protein

requirement in fish have been designed to maximize growth; hence growth rate has been

the main criterion used to determine requirement (Siddiqui et al, 1988; Wang et al,

1985; Santiago et al, 1982; De Silva and Perera 1985). On small farms, such as the

family farms in Kenya, where the fixed costs are low and feed costs would be the major

components of the variable costs, a more suitable measure of diet quality would be feed

conversion ratio.

Protein requirement as a percentage of diet is higher for fish than for most

terrestrial animals. Some researchers have explained this by relating requirement to the

feeding habits of fish, pointing out that most fish are carnivorous and hence the high

protein requirement (Cowey, 1975 and Watanabe et al, 1979). However, high protein

requirements are also a characteristic of omnivorous and herbivorous fish such as

common carp, tilapia and grass carp. Not much difference has been noted between the

requirements of these latter fish and the carnivorous fish. A plausible explanation may be

that a portion of the ingested protein is catabolized for energy. In fish, both lipids and

proteins are readily available energy sources, while the value of carbohydrates as an

energy source varies among species. It has been shown that tilapia (O. niloticus) (Popma,

1982) and channel catfish (Wilson and Poe, 1985), which are warm water herbivorous

10

and omnivorous fish respectively, digest over 70% of the gross energy in non-cooked

starch, while rainbow trout, which are cold water carnivorous fish, digest less than 50%

(Cho and Slinger 1979). Another reason for the higher dietary protein concentration is

that fish have lower dietary energy requirement because they exert relatively less energy

to maintain position, do not maintain a constant body temperature, and excrete most of

their nitrogenous waste as ammonia.

Wilson and Halver (1986), stated that fish do not have a requirement for protein per

se, but rather require amino acids that are usually obtained from the diet by digestion of

protein. All fish require the 10 indispensable amino acids that are required by other

animals (Cowey, 1994), i.e. arginine, histidine, isoleucine, leucine, lysine, methionine,

phenylalanine, threonine, tryptophan, and valine. Tyrosine is a non-essential amino acid

that has a sparing effect on phenylalanine, while cystine has been shown to spare

methionine in fish diets (Page, 1978). Maintaining an optimum amino acid balance is

essential for optimal fish growth. Excess methionine has a depressing effect on growth,

which may be due to its inhibitory effect on the absorption of neutral amino acids

(Ingham and Arme, 1977). Similarly, a growth reduction has been observed when the

ratio between leucine and isoleucine is increased (Nose, 1979). Antagonism between

branched chain amino acids has been reported in mammals (May et al, 1991; Hargrove

et al, 1988; Calvert et al, 1982), and in fish (Hughes et al, 1984).

Tilapia, compared to other species of fish, require relatively low concentrations of

dietary crude protein (NRC 1993). There have been several attempts to determine protein

requirements, and a wide range of dietary protein levels has been proposed. One of the

major problems with the stated requirements (Jauncey, 1982; Cruz and Laudencia, 1977;

11

Davies and Stickney, 1978), is that diets used to determine them were not formulated to

meet the requirements of essential amino acids, which were not quantified until recently

(Santiago and Lovell, 1988). Scott et al. (1982) stipulated that amino acid requirements

might differ depending on the balance (excesses and deficiencies) of amino acids > in the

diet. It therefore follows that diets formulated to determine protein requirements must

first and foremost meet the requirement for amino acids. Lack of knowledge of amino

acid requirements may explain some of the variation in the stated requirements. There

are only three studies that have investigated the requirements of some tilapia species for

some or all of the essential amino acids. Jackson and Capper (1982) studied the

requirements of O. mossambicus for lysine, methionine and arginine, while Jauncey et al.

(1983) studied the essential amino acid requirement of the same species based on the

amino acid analysis of fish flesh protein. Santiago and Lovell (1988) studied the

requirements of O. niloticus fry (15 to 87 mg) for 10 essential amino acids using casein

/gelatin diets. The latter study represents the most extensive and complete study on the

essential amino acid requirements of tilapia. The values reported were significantly

higher than those reported by Jauncey et al. (1983) for O. mossambicus. Santiago and

Lovell (1988) postulated that essential amino acid requirements of the two related species

differ considerably. Research in this area encounters methodological problems such as

lower growth rates when fish are fed diets based on synthetic amino acids (Mazid et al,

1978; Yamada et al, 1982). The reason for this has not been established.

A further confounding factor is that the minimum requirement for crude protein

varies with the rearing system used. Clark et al. (1990) observed no significant

differences in weight gain or FCR among tilapia grown in outdoor seawater pools and fed

12

diets containing 20, 25, and 30% crude protein. The authors postulated that fish on the

lowest level of dietary protein maintained a good growth rate by feeding on algae present

in the pools.

A wide range of estimates of the optimal dietary crude protein concentration for

tilapia has been reported. Winfree and Stickney (1981) reported that 56% crude protein

promoted maximum weight gain in tilapia (O. niloticus) weighing 2.5 g. Shiau and

Huang (1989) reported 24% crude protein as the optimum crude protein concentration for

tilapia (O. niloticus x 0. aureus) weighing 2.9 g. Fish in the latter study were maintained

at a salinity of 32 -34 ppt. Water salinity has been observed to influence protein

requirement, being lower at full salinity than in fresh water (Shiau and Huang, 1989;

Clark, 1990).

Luquet (1991) reviewed several studies, and recommended 30-35% crude protein

as the optimum for tilapia. In making this recommendation, the author relied on studies

that utilized good protein sources such as fish meal and casein. The quality of dietary

protein affects protein requirements. Both fishmeal and casein have good amino acid

profiles and good protein digestibility and are considered to be high quality protein

sources. The quality of fishmeal, however, may vary depending on the species of fish,

processing method and freshness of the raw material used (Anderson, 1996). In most of

the published studies, the type of fishmeal used has not been specified. McCallum and

Higgs (1989) reported that low-temperature dried herring meal had a slightly reduced

protein quality compared to freeze-dried herring meal, whereas the high-temperature-

dried meal had a dramatically reduced protein quality.

13

Estimates of optimal dietary protein levels have typically been in the range of 35 -

40% for tilapia weighing less than 5 g (Mazid et al, 1979; Jauncey, 1982; Siddiqui et al.,

1988). The crude protein requirement for tilapia is inversely related to their size.

Winfree and Stickney (1981), observed that maximum weight gain in tilapia (O. aureus),

weighing 2.5 g, was realized when diets contained 56% crude protein, while in fish

weighing 7.5 g, 34% crude protein was adequate. Siddiqui et al. (1988) observed that the

optimal dietary protein level for Nile tilapia (O. niloticus) fry weighing 0.8 g was 40%,

while the corresponding level for fish weighing 40 g was 30%. Twibell and Brown

(1998), determined that the crude protein requirement for tilapia with an initial weight of

21 g was 28%). In the latter study, the protein content of the diet was increased by

increasing the level of soybean meal. The diet containing the highest level of crude

protein (34%) contained 35% soybean meal. Soybean meal contains anti-nutritive

factors, the content of which may vary depending on the processing method. It is not

clear whether the higher level of soybean meal in the high-protein diets may have caused

the depressed growth rates observed at the higher protein levels.

Thus, the stated requirements for protein show a wide variation, reflecting the

different environmental conditions in which the studies were done. Fish factors such as

size, and stocking density also affect the requirements. Similarly, dietary factors such as

protein quality, the ingredients used and the way they were processed would all affect

requirements.

The stated values for optimal dietary protein level have been estimated from

growth response curves. There are various problems when the requirements are

estimated in this way. Growth is a non-specific response, and it is affected by many

14

factors such as temperature, water quality, biomass density and water flow rates.

Furthermore, different growth rates were attained for the "optimal" diet in the various

experiments, and, in some of the studies, the growth rates were quite low for the "optimal

diet", indicating that the environmental or dietary factors or both were not really optimal.

The other major problem in stating protein requirements for tilapia is that there is

very little information available on the digestibility of feedstuffs. Digestible energy (DE)

concentration is an important factor affecting protein requirement (Scott et al., 1982). If

the DE content of the diet is low, most of the protein will be catabolized for provision of

energy. Unfortunately, data on the digestible energy content of major dietary components

generally used in tilapia diets are inadequate, and this has hindered the expression of

protein requirements in relation to digestible energy content of the diet. Protein to energy

ratio is important in determining the requirement of protein. A low protein to energy

ratio will lead to slow growth, while a ratio that is too high would lead to catabolism of

proteins for provision of energy. Cisse (1996) observed that in tilapia S. melanotheron a

protein:energy ratio of 70mg protein.kcal"1 was optimal. Values higher or lower than this

resulted in poorer growth performance. In determining protein requirements, some

authors have treated energy and protein as independent variables, although their effects

are not independent. Protein can be catabolized for energy, and energy is used in the

synthesis of half of the 21 amino acids used in growth and metabolism (Bowen et al,

1995). Furthermore, feed intake is regulated by the available energy content of the diet.

The other reason for the different values reported by various authors would be in

the source of protein used. Low-temperature (LT) high-quality fishmeal would contain a

favorable amino acid profile for the growth of most fish (Anderson, 1996). In contrast,

15

most plant proteins may be limiting in one of more of the indispensable amino acids. It is

therefore unlikely that diets with LT fish meal as the main source of protein would result

in the same optimal dietary protein estimate as diets composed entirely of plant

feedstuffs.

2.3.1 Search for new sources of protein

Tilapia are the third largest group of farmed fish species after carp and salmonids (FAO,

1997). Nile tilapia was the sixth most cultured fish species in the world in 1995, with a

total production of 473,641 m.t, and an average annual increase in production of 12% per

annum since 1986. Between 1984 and 1997, the global production of farmed tilapia

increased more than three-fold i.e., from 186,544 tonnes to 659,000 tonnes, and in 1995,

it represented more than 4.48% of the total farmed fish, with a value of US$ 925 million

(Tacon, 1997).

Feed represents the highest operating cost in intensive fish aquaculture, with

protein being the most expensive dietary component. Traditionally, fish diets have been

based on fishmeals as the main protein sources due to their high protein content, good

amino acid profile, and excellent supply of essential fatty acids, minerals and vitamins of

high digestibilities. Fishmeal is also the single most expensive ingredient in aquaculture

feeds (Tacon, 1993). A reduction in world production of fishmeal, coupled with

increased demand and competition with terrestrial domestic animals for the limited

supply, has further increased fishmeal prices. Many developing countries are unable to

afford fish meal for inclusion in feed for fish and other domestic animals. Furthermore,

high dependence on fishmeal would make fish prices high and less competitive compared

to other meats. For that reason, considerable research has been done to evaluate new

16

protein sources. El-Sayed (1999) reviewed the alternative protein sources tested as

replacements for fishmeal in tilapia diets.

Fishery by-products have shown promising results. One such product is fish

silage which is prepared from fish or fish-processing wastes. Feeding experiments have

indicated that fish silage can replace fishmeal in tilapia diets. The nutritional value of

silage depends on the source of the fish species, and on the care taken during silage

preparation (Fagbrenro and Jauncey, 1994). In particular, good knowledge is needed

regarding the chemical changes that occur during the digestion and storage of silage. The

nutritional quality of fish silage can be improved by limiting the extent to which proteins

are hydrolyzed to polypeptides and free amino acids. Termination of the ensiling process

after 3 - 7 days was shown to result in improved weight gain, protein efficiency ratio,

biological value and net protein utilization when these products were fed to mink (Screde,

1981), calves (Offer and Hussain, 1987) and salmonids (Lall, 1991). Fish silage that has

been acid or enzymatically digested, is a viscous liquid that is difficult to transport, store,

or feed to animals. It has a low solid and high moisture content which makes it difficult

to dry. Carbohydrates, cereals, crop residues and by-products have been used as filler

materials, making it possible to dry the silage in conventional driers. In tilapia, fish

silage can successfully replace fishmeal in diets. Lapie and Bigueras (1992) fed Nile

tilapia fish silage preserved in formic acid, and blended with fishmeal in a 1:1 ratio, and

observed that growth rate was similar to that of the fish fed on the fishmeal control diet.

When silage to fishmeal ratio was increased to 3:1, growth was significantly reduced,

presumably due to the high acidity of the diet, which may have depressed the appetite of

the fish. Formaldehyde formed in the ensiling process inhibits protein hydrolysis (Haard

17

et al., 1985; Hussain and Offer, 1987), but may be toxic to some animals at high

concentration. Fagbenro and Jauncey (1993) found that fermented fish silage blended

with soybean meal, hydrolyzed feather meal, or meat and bone meal in a ratio of 1:1,

could replace 75% of the fish meal in diets for Nile tilapia, with no significant differences

in weight gain, or hemoglobin and hematocrit levels in the fish.

Terrestrial animal by-products have been used successfully in tilapia feeds.

Poultry by-product meal, hydrolyzed feather meal, blood meal, and meat and bone meal

have high protein contents. Unfortunately, most of these protein sources are deficient in

one or more of the essential amino acids, particularly lysine, isoleucine, and methionine

(Tacon and Jackson, 1985). When the limiting amino acids are supplemented, the diet

quality is improved. Tacon et al. (1983) found that hexane-extracted meat and bone meal

alone, or mixed with blood meal, in the ratio of 4:1, and supplemented with methionine,

successfully replaced 50% of fish meal protein in diets fed to Nile tilapia fry. When

blood meal was used alone, the results were still comparable to those of the control. This

was contrary to a later study by El-Sayed (1998), who observed significantly reduced

growth rates and feed efficiencies when fish meal was replaced by blood meal. The

differences between the two studies could be due to the fact that methionine, which is

deficient in blood meal was supplemented in the first but not the second study.

Hydrolyzed feather meal has been used as a protein source for tilapia with

contradictory results. In studies by Tacon et al. (1983), Viola and Zohar (1984), and

Davies et al. (1989) with O. niloticus, O. mossambicus, and all male tilapia hybrids

respectively, fish fed on diets based on hydrolyzed feather meal exhibited poor

performance, presumably due to poor digestibility and low levels of lysine in the meal.

18

On the contrary, Falaye (1982), Bishop et al. (1995) and Gaber (1996) observed that

hydrolyzed feather meal could replace between 40% and 66% of the fishmeal in tilapia

diets.

Chicken offal silage has also been tested (Belal et al, 1995) in O. niloticus

fingerlings weighing 10.8 g. The authors only tested the range of 0-20% inclusion level

and found that it could replace fishmeal up to the 20% level. Additional studies should

have been conducted to determine the highest level to which chicken offal silage could

replace fishmeal.

Animal manures have also been used as protein sources for tilapia. Alhadrami

and Yousif (1994) reported that camel and cow manures could be successfully

incorporated in tilapia diets at 10% and 20% levels, respectively. It is not clear, however,

whether the increased growth of the fish was the result of direct consumption of the

manures, or whether the manures increased natural food productivity in the ponds.

Plant protein sources have also received considerable attention as full or partial

replacements for fishmeal. Among them, soybean meal has been the most widely used.

It has a high protein content and a good essential amino acid profile, but is limiting in

lysine, and the sulfur amino acids, methionine and cystine. Raw and under-heated

soybeans contain proteins that inactivate the digestive enzymes trypsin and chymotrypsin,

and cause agglutination of red blood cells in-vitro (Scott et al., 1982). Heat treatment

inactivates these proteins, making soybean meal a major protein source in diets of many

fish species.

In tilapia (O. niloticus), studies to evaluate the potential of soybean meal to

wholly or partially replace fishmeal have yielded varying results. In most of the studies

19

conducted, soybean meal could replace between 67% and 100% of the fishmeal,

depending on fish species and size, dietary protein level, source of the soybean meal,

processing methods and the culture system used. Studies done also indicate that there are

no added benefits in supplementing diets based on soybean meal with the assumed

limiting amino acids. For instance, in studies conducted by Tacon et al. (1983) and

Jackson et al. (1982) pre-pressed solvent extracted meal, with or without methionine

supplementation, was found to successfully replace 75% of the fishmeal in diets fed to

tilapia (0. niloticus) and (0. mossambicus), respectively. Also, Shiau et al. (1989), found

that 67% of the fishmeal in diets for hybrid tilapia (O. niloticus x O. aureus) could be

replaced by soybean meal at a low dietary protein level (24%). Despite the low protein

level and the high content of soybean meal in the diets, addition of methionine did not

improve fish performance. Similarly, Viola et al. (1988) did not observe any

improvement in the growth of hybrid tilapia (O. niloticus x O. aureus) fed diets in which

50% of the protein originated from soybean meal, supplemented with the amino acids

lysine and methionine versus soybean meal alone.

The lack of response to amino acid supplementation in the quoted studies is

surprising considering that most of the soybean meal used was heat treated to inactivate

anti-nutritive factors. The Maillard reaction between reducing sugars and amino acids,

particularly lysine, results in linkages that are not hydrolyzed by digestive enzymes.

These amino acids become unavailable to the fish, even though they are chemically

present. It would therefore be expected that supplementing such diets with amino acids

such as lysine would improve fish performance, but this is contrary to what has been

observed. Viola et a/.(1988) postulated that tilapia are able to utilize lysine from heat-

20

treated soybean meal because of their stronger gastric digestion compared to that of most

other fish. Also, it can be postulated that the lysine and methionine requirements of

tilapia are lower than those generally assumed.

Minerals, rather than amino acids may be the factors that limit the efficient

utilization of soybean meal in tilapia. Ogino et al. (1979) found that availability of

phosphorus from bone and from plant protein sources was low in carp, which are

stomachless, but in contrast, tilapia could effectively utilize mineral phosphorus, but were

also unable to utilize phytin phosphorus. Thus, replacement of animal proteins with plant

proteins creates a phosphorus deficiency in tilapia, which should be balanced by dietary

inclusion of a suitable mineral supplement.

The extent to which soybean meal protein can replace fishmeal protein in tilapia

diets is affected by dietary protein level. Davis and Stickney (1978) found that at a low

dietary protein level (15%), replacement of fishmeal protein with soybean meal protein

caused growth depression, while at a high protein level (36%), soybean meal protein

could totally replace fish meal protein in the diets without any significant decline in fish

performance. This was contrary to the observations of Shiau et al. (1989), who reported

that at 24% dietary protein, soybean meal protein could effectively replace 67% of the

fishmeal protein in diets of tilapia (O. niloticus x O. aureus). When the diets contained

32% protein, replacing 30% of the fishmeal protein with soybean meal protein

significantly decreased fish growth and FCR. The differences in findings between the

two studies may have been caused by the type of soybean meal, and the way it had been

processed. In the studies by Shiau et al. (1989), the anti-nutritive factors in soybean meal

may not have been completely destroyed, thus causing the reduced growth observed

21

when soybean was added at high levels in the diet. Indeed, El Sayed (1999) postulated

that the differences in findings between the two studies may have been related to the

quality and processing of the soybean meal, fish species, size and culture systems used.

The potential of cotton seed cake as a replacement for fishmeal has been studied

in many fish species. Cotton seed cake is one of the most available plant protein sources

in the world. It is relatively cheap, has a protein content ranging from 26% to 54%, air-

dry basis), and a reasonably good amino acid profile for a plant protein. However, it has

relatively low levels of lysine, cystine and methionine and contains a phenolic anti-

nutritive compound, gossypol which is toxic to many animal species and also binds to

lysine, reducing its availability (Jauncey and Ross, 1982). Its effect on fish is species

specific. In rainbow trout, Herman (1970) reported that 0.03% free gossypol was toxic,

while Dorsa et al. (1982) found that channel catfish could tolerate up to 0.09% free

gossypol without any reduction in growth. In tilapia (O. aureus), Robinson et al. (1984)

reported that a free dietary gossypol content of 0.2% had no adverse effect on fish

performance.

Various authors have studied the use of cotton seed cake as a protein supplement

for tilapia, with inconsistent results. Ofojekwu and Ejike (1984) found that O. niloticus

fed diets with cotton seed cake grew at slower rates than fish fed the fishmeal control

diet. Similarly, Abdel-Fattah and El-Sayed. (1990), working with O. niloticus

fingerlings, observed that fish fed on diets where 65% to 80% of the protein originated

from cotton seed cake had poor weight gains compared to fish fed on the fishmeal control

diet. Supplemention of the diets containing cotton seed cake with lysine did not improve

fish performance. In a later study, Abdul-Aziz et al. (1999), using Nile tilapia

22

fingerlings, found that fish fed on diets in which 25% of the protein was from cotton seed

cake had lower growth rates compared to those fed on the control diet based on fishmeal.

In contrast to the above findings, Jackson et al. (1982) successfully used diets in which

50% of the fishmeal protein was replaced with cottonseed meal. There were differences

in the way the various studies were done, which may account for the differences in the

results. In the study by Ofojekwu and Ejike (1984) the diets contained unconventional

feed ingredients, which may have affected the results. Gari, defined as grated cassava

partially fermented, dried under the sun, and dried to 13.5% moisture level, was one of

the ingredients used. Similarly, the cellulose used in the study was prepared by soaking

filter paper in hot water and extracting it with 18% KOH for 24 hours. The growth rates

obtained for fish of initial weight 3.71 g ranged from 0.08 % to 0.38%, while growth

rates of fish with an initial weight of 45 g were 0.23 to 1.05%. These growth rates were

low even for fish fed the control diet, indicating that there were other factors that affected

the fish negatively. Abdel-Fattah and El-Sayed. (1990) used a control diet where wheat-

bran was incorporated at a level of 60%. Recent studies have shown that the digestibility

of protein and energy in wheat bran is low in tilapia (Popma, 1982; Anderson et al.,

1991). The use of such high levels of wheat bran in the control diet, therefore, would

have negatively influenced the digestibility of the energy and protein. In the studies by

Jackson et al. (1982), the fish on the control diets also performed poorly, making it

difficult to draw any firm conclusions from the findings.

Other oilseed by-products that have been tested in diets for tilapia have included

groundnut, sunflower meal, canola meal, rapeseed meal, sesame meal, copra, macadamia

nuts, palm kernel and defatted cocoa cake (Jackson et al., 1982; Pereira and Pezzato,

23

1999; Higgs et al, 1990; Davies et al, 1990; Hossain et al, 1992; Guerrero, 1995;

Balogun and Fagbenro, 1995; Omoregie and Ogbemudia, 1993; Fagbenro, 1988). All

may have good potential as protein sources for tilapia, but further work needs to be done

on their utilization.

Leguminous and cereal plants and their by-products have been tested as partial

replacements for fishmeal in tilapia diets. Leucaena leaf meal (LLM, 30% CP) from the

plant Leucaena leucocephala is a potential protein source. The plant is drought resistant

and the leaves have a high protein content. It is widely used as an animal feed,

particularly for ruminants. However, the presence of the toxic amino acid mimosine

limits its use in diets for monogastric animals. It is also low in the essential amino acids,

arginine, threonine, isoleucine, histidine and methionine (Lim and Dominy, 1991).

Several studies have been undertaken to assess the potential of using leucaena leaf

meal as a protein source in tilapia diets, with varying results. Salaro et al. (1995)

observed that Leucaena seed meal could comprise only 20% of the dietary protein in O.

niloticus fry weighing 0.5 g. In larger fish, Santiago et al. (1988), noted that the fish

performed poorly when leucaena leaf meal exceeded 40% of the diet.

Mimosine in leucaena can be degraded to a relatively less-toxic form, through

various processing methods, thereby increasing its nutritive value. Wee and Wang

(1987) found that fish fed diets with leucaena leaf meal that had been soaked in tap-water

for 48 hours and sun-dried for 12 hours had better growth rates than those fed the diets in

which the leaf protein had only been sun-dried. The soaked leucaena leaf meal could

supply 25%) of the total protein in the diet. In studies by Osman et al. (1996), the best

performance was found for tilapia that were fed diets containing leaves that had been

24

dried for 48 hours or autoclaved for 15 minutes, compared to the leaves that had been

treated with sodium hydroxide or incubated with rumen liquor.

In conclusion, leucaena leaf protein can be used successfully as a protein source

in tilapia diets. It can supply between 20% and 40% of the protein depending on the fish

size and the processing method used to detoxify the mimosine. Despite the fact that

mimosine can be completely or partially destroyed, the nutritive value of the leaf protein

appears to be limited by other factors.

Single cell proteins (SCP) such as unicellular algae, bacteria, cyanobacteria, and

yeast have received a lot of attention in tilapia culture. Of particular interest has been the

the biosynthesis and utilization of SCP by tilapia within intensive and semi-intensive

farming systems. The production of single-cell proteins is simple, inexpensive and an

effective way of producing natural fish food. Chamberlain and Hopkins (1994) reported

that spraying a source of carbon such as wheat bran or cellulose on the surface of pond

water with continuous aeration, at the optimum carbon:nitrogen ratio (15:1), would

increase bacterial growth. Bacteria that are produced consume the carbon as an energy

source and reduce ammonia concentration through nitrification. Fish may feed on the

bacteria so produced directly, or they can be harvested and used commercially as a

protein source. Viola and Zohar (1984) found that a commercial single cell protein diet

(Pruteen, 70% CP) could replace 50% of the fishmeal protein in diets fed to tilapia hybrid

(O. niloticus x O. aureus).

25

2.4 Sunflower (Helianthus annuus)

2.4.1 Taxonomy and origin of the domesticated sunflower

Commercial sunflower (Helianthus annuus) belongs to the compositae family. All

species of Helianthus are native to the Americas, where archeological evidence reveals

that wild sunflower was used by American Indians as a source of food, and also in

medicine and ceremonies (Heiser, 1955). Sunflower seed meal was mixed with flour to

make bread. Oil from the seeds was used to season food and anoint hair, and it also

served as a base for pigments. Further, sunflower was used as a medicine to cure

rattlesnake bites and for other remedies (Heiser, 1976). After the discovery of the

Americas, sunflower was introduced to Europe, where it spread eastward and northward,

eventually reaching Russia in the middle of the eighteenth century (Zukovsky, 1950). By

the beginning of the twentieth century, sunflower became a major edible oil crop in

Russia. Russian plant breeders devoted much effort to the improvement of sunflower

productivity and disease resistance in cultivated plants. They increased the oil content of

the seeds from 28% in the 1920's to 43% by 1935, and to 49% in 1955. Presently, some

varieties have oil contents that exeed 50%. Cultivated sunflower was re-introduced to

North America by the immigrants from Europe around 1875. Currently, Russia is the

leading producer of the crop followed by France, USA, China and Spain (Putt, 1997).

Eighty percent of the monetary value of sunflower is derived from the oil. The meal is

the main by-product after oil extraction, and it contains 30 - 45% protein.

European settlers introduced sunflower into Kenya in the nineteenth century. It

was cultivated as an export crop for bird feed (Zulberti, 989). When independence was

attained in 1963, it was grown as a cash crop in the high-potential areas of the country.

26

Production of the crop declined steadily due to the fact that monetary returns from it were

much lower than those from other cash crops such as wheat and corn, which could be

grown in the same areas. Production has recently been revitalized, however, following

government decontrol of consumer prices of edible oils and fats and animal feeds.

Sunflower farming is also moving from high-potential to marginal areas where few

alternative crops can be grown.

2.4.2 Morphology of the sunflower plant

The most striking feature of the sunflower plant is the head inflorescence which carries

the seeds. The floral head consists of individual small flowers which are congested and

attached on a single horizontal plane to simulate a large individual flower. The whole

intricate arrangement of the head and structure of the flowers is believed to be an

adaptation to improve the efficiency of pollination by insects and by other means (Heiser,

1976). Each flower has a single ovary containing one seed that ripens into the fruit or

achene. The achene consists of a seed (kernel), and adhering pericarp (hull).

2.4.3 Chemical and physical composition of sunflower seeds

Two different types of sunflower cultivars are cultivated, viz., the oilseed varieties that

have an oil content of 40 to 51%, and the low oil seed varieties in which the oil content

varies between 21 and 32% (Earles et al, 1968). The high-oil seed cultivars are black-

seeded and have thin hulls, which adhere to the kernel tightly. Edible oil is the main

product of the oil-seed cultivars, with the meal being an important by-product. In addition

to high oil content, high-oil seed cultivars generally have low hull content and smaller

size compared to the low-oil cultivars. The low-oil seed varieties, referred to as

"confectionary" sunflowers, have large stripped seeds, and relatively thick hulls which

27

remain loosely attached to the kernel (Vaughan, 1970; Park et al., 1997) They are mainly

used in snack, confectionary, bakery and bird food markets. Within each type of cultivar,

the composition of the seeds varies with location, year of planting, type of soil and

cultural practices. The oil content of the high-oil cultivars compares favorably with that

of other oilseeds (Unilever, 1976). Due to extensive selection for high oil content,

sunflower seeds have a higher oil content than found in most other oil seeds except

peanuts.

2.4.4 Sunflower seed oil

Intensive selection has been done in the sunflower plant for high oil content (Senkoylu

and Dale, 1999). The oil content was increased from 28% to over 50% during a period of

seven decades (Zatari, 1989). In the seeds of any one cultivar, the oil content may vary

depending on geographical location, environmental temperature, planting season and

other cultural practices (Zatari, 1989). The fatty acid composition of sunflower oil is

characterized by a low level of linolenic acid (NRC, 1993), and for that reason, the oil has

excellent storage qualities. It has also has a lower level of palmitic acid than soybean oil,

and a higher level of linoleic acid. Sunflower oil is considered to be a highly desirable oil

for human consumption because of its light color, bland flavor, high smoke point, and

high level of linoleic acid.

The fatty acid composition of sunflower oil is affected by the environmental

temperatures that occur during growth of the plant (Canvin, 1965). Seeds produced in

cool climates contain 70% or more linoleic acid, while those produced in hot climates

may contain as little as 25% (Unger and Thompson, 1982). The use of breeding to

modify the fatty acid composition of sunflower oil has received little attention and, for

28

that reason, the fatty acid composition of the oil from seeds of different cultivars within

similar environments is quite uniform (Kharchenko and Borodulina, 1976). Sunflower oil

has high levels of oleic and linoleic acids.

2.4.5 Sunflower seed proteins

The protein content of the sunflower kernel ranges from 9% to 24 %, and depends on

variety, climate, soil and cultivation conditions (Dorrell and Vick, 1997). Selection of

sunflower for a high oil content has resulted in an attendant decrease in protein content

because protein and oil contents are negatively correlated with one another.

Environmental temperatures also affect protein quality and content. In studies by

Canvin (1965), the protein content of the seeds increased from 14% to 20% as the mean

environmental temperatures increased from 10°C to 26°C. Under optimal processing and

dehulling conditions, the protein quality of sunflower meal is equivalent to that of

soybean meal. However, when processing conditions are harsh, or when excess heat is

used to desolventize the meal, some decline in the biological value of the protein occurs

due to destruction of lysine, arginine and tryptophan (Clandinin, 1958). The amino acid

composition of sunflower meal and soybean meal is shown in Table 2.4. Sunflower meal

protein is relatively deficient in lysine, but rich in sulfur amino acids when compared

with soybean meal protein.

2.4.6 Composition of sunflower hulls

Sunflower hulls are the outer covering of the sunflower seed. They make up 22% to 30%

of the total weight of the seed. Cellulose, lignin and hemicellulose comprise 74% to 90%

of the total components (Table 2.2), and are highly indigestible by animals. Lipids,

proteins and minerals make up the rest. Generally, sunflower hulls contain more lignin

29

-v

Table 2.2: Some fibre components of sunflower seed hulls and other agricultural residues (Earles etal, 1968)

% (Air-dry basis) and range

Material Lignin Pentosans Cellulose

Oat hulls 17 39 36 Wheat straw 18 (15-21) 30 (27-32) 33 (29-37) Sugarcane bagasse 19 (16-22) 30 (27-32) 33 (30-37) Corncobs 14(8-17) 41 (31-45) 32 (22-39) Sunflower hulls 27 (25-30) 27 (25-31) 30 (29-32)

30

and less pentosans and cellulose compared to other agricultural residue materials. They

can be utilized as a roughage source, but they are low in nutrient content, poorly digested

and highly unpalatable (Park et al., 1997). Hulls derived from oil-seed processing may

be of slightly higher quality and contain more protein and fat than the larger

confectionery hulls.

2.4.7 Anti-nutritive factors in sunflower seeds

Unlike most other oilseed meals, anti-nutritive factors are not a major problem in

sunflower. The seeds, however, contain arginase and trypsin inhibitors which are heat

labile and easily inactivated (Roy and Bhat, 1974). The potential use of sunflower

protein isolates for human food is limited by the presence of phenolic compounds in the

seed. During protein extraction in alkaline medium, chlorogenic acid and other phenolic

compounds are oxidized to o-quinones and form covalent linkages with proteins giving

dark-green or brown products (Sosulski and McCleary, 1972).

While chlorogenic acid is not considered to be toxic, Delic et al. (1975) found that

a 2% inclusion in the feed of mice resulted in a depressed feed intake, and a reduced

weight gain. Methionine and choline chloride partially offset these effects. Both

genotype of the seeds and environmental conditions during seed maturation have a direct

effect on the concentration of chlorogenic acids in the seed. Dorrell, (1976) analyzed 38

inbred lines and found that the concentration of chlorogenic acid ranged from 1.4% to

4%. Early seeding and warm temperatures during seed maturation favored higher levels

of chlorogenic acid. Eliminating or reducing the amount of chlorogenic acid in the seed

through genetic selection is difficult because oil and chlorogenic acid content are

positively correlated.

31

2.4.8 Processing methods

The method used to process sunflower seeds is one of the most important factors that

determines the composition of the meal. The seeds may or may not be dehulled prior to

oil extraction, depending primarily on the design of the processing plant. In the older

plants, dehulling equipment is not available, and the seeds are crushed whole. Modern

processing plants dehull from 40 to 75% of the achenes, but even a very efficient

dehulling system can only remove a maximum of 90% of the hulls from the seeds. The

hulls are discarded as trash or used as fuel for plant operations (Dorrell and Vick, 1997).

Dehulling has many advantages. First, it reduces the movement of unnecessary mass

through the system, and second, it reduces wear and tear in the expeller. Third, it reduces

the wax content of the oil and lastly, it reduces the fibre content of the meal (Dorrell,

1976). Three basic methods are available for extraction of sunflower oil, namely;

mechanical screw press, direct solvent extraction, and a combination of screw-press and

solvent extraction.

2.4.9 Sunflower meal

Sunflower is grown for the oil, but the meal left behind after oil extraction is a valuable

and nutritious by-product. The chemical composition of the meal compares favourably

with that of most other vegetable-type meals. Exceptions are the higher fibre and ash

contents of sunflower meal, which reduce its metabolizable energy. Approximately 8.3

million tons of sunflower meal were produced world-wide in 1990/1991, making

sunflower meal the fourth largest source of oil seed meal following soybean, cottonseed

and rapeseed meals (Dorrell and Vick, 1997). Almost all sunflower meal that is marketed

comes from the processing of the black-hulled oilseed type sunflower. The chemical

32

composition of sunflower meal depends on the variety of the seed, the processing

method, and the degree of dehulling or decortication (Earles et al, 1968; Ravindran and

Blair, 1992). The oil content of the meal varies with the type and efficiency of the oil

extraction process. If seeds are completely dehulled before oil extraction, a meal with a

protein content in excess of 4 0 % can be achieved with a solvent extraction system, and

37%) protein with a mechanical extraction system. In contrast, if no dehulling occurs, the

meal contains only 2 8 % protein after oil extraction by either method. Intermediate

dehulling results in a sunflower meal with about 34%> protein. Sixty to sixty five percent

of the sunflower meal produced in the USA and Canada is this type. The reminder is in

the 28%) protein category. In Kenya, where the present project was done, the crude

protein content of sunflower meal is typically in the range of 2 5 % to 30 %. The protein

and fibre contents of the sunflower meal are inversely related. The fibre content has a

negative impact on nutrient availability and the digestible energy level (Villamide and

San Juan, 1998). Kondra et al, (1974) found that feeding a high- fibre diet (19 .6% fibre

DM basis) to chicken layers and broilers resulted in significant reduction in food

consumption and body weight gain, but had no effect on FCR during the growing period.

The levels of proximate constituents in some Kenyan sunflower seed cakes are

given in Table 2.3. There was considerable variation among the samples, which was

mainly caused by differences in processing methods (Jacob, 1993). The levels of crude

fibre were high, and ranged from 2 4 . 1 % to 40.2%o. Crude protein ranged from 24 .9% to

37.6%. The amino acid compositions of sunflower meal, soybean meal, cottonseed meal

and rapeseed (or canola) meal are presented in table 2.4.

33

Table 2.3: Proximate compositions of Kenyan sunflower seed cakes (adapted from Jacob, 1993). A l l samples were produced by the expeller process.

% ( D M basis)

M i l l Sample no. % D M 1 C P E E C F Ash N F E

*A 1 94.1 30.8 16.6 28.4 7.5 16.7 A 2 97.2 24.9 12.7 33.5 7.0 21.9 A 3 96.9 25.8 13.1 29.0 8.7 23.4 A 4 96.9 25.0 13.7 32.1 7.8 21.4 B 1 93.1 32.4 12.2 24.7 9.2 21.4 B 2 96.0 37.6 8.0 24.8 8.9 20.7 B 3 96.8 33.4 10.3 24.1 10.3 21.9 C 1 91.0 29.9 14.4 35.7 5.5 14.4 C 2 96.4 28.9 12.6 29.7 7.0 21.9 D 1 93.2 33.5 12.4 25.8 6.8 19.7 D 2 93.5 29.8 14.9 33.8 6.6 14.9 D 3 94.4 32.2 12.0 33.1 5.9 16.8 D 4 94.3 32.7 12.0 32.8 5.9 16.6 E 1 95.5 25.3 11.0 40.2 5.1 18.3 E 2 93.9 32.5 13.0 27.4 7.8 19.4 E 4 93.6 34.0 11.6 24.9 7.3 22.2 F 1 95.6 27.0 12.3 33.1 9.4 18.2 Misc . 1 92.6 27.5 14.5 29.6 6.3 22.1 Misc. 2 93.8 26.0 11.4 38.3 4.6 19.7 Mean 94.6 29.8 12.5 30.8 7.2 19.7 SD 1.7 3.7 1.8 4.6 1.5 2.6 M a x 97.2 37.6 16.6 40.2 10.3 23.4 M i n 91.0 24.9 8.0 24.1 4.6 14.4

'DM- Dry matter, CP-Crude protein, EE- Ether extract, CF-Crude fibre, NFE- Nitrogen-free extract

* A , B , etc. refer to mills from which the samples were obtained.

34

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Sunflower meal is lower in lysine content than noted in soybean, cottonseed, and

rapeseed meals (Senkoylu and Dale, 1999). Methionine content is higher in sunflower

cake than in soybean meal, but equal to that of cottonseed and rapeseed meals. The

average digestibility of amino acids in sunflower meal is less than in soybean meal, but

higher than in canola and cottonseed meals. The average amino acid digestibility

coefficients for the meals are 91%, 89%, 84% and 77% in soybean, sunflower, canola and

cottonseed meals respectively (Table 2.4)).

The amino acid profile is affected by processing temperatures. Lower availability

of lysine, arginine, and tryptophan has been reported for sunflower products produced

using high processing temperatures (Rhone Poulenc, 1993; Feedstuffs, 1998). In poultry,

Villamide and San Juan (1998) observed that lysine had the lowest digestibility of the

essential amino, while Green and Kiener (1989), working with pigs, found no significant

differences in true amino acid digestibility between partially and completely dehulled

sunflower meals with crude protein contents of 31% and 36 %, respectively.

2.5 Use of sunflower meal in animal feeds

2.5.1 Ruminants

The high protein and high fibre contents of sunflower meal makes it ideal for use in

ruminant diets. Park et al. (1981) and Erickson et al. (1984) fed high-producing dairy

cattle diets containing dehulled sunflower meal during early lactation and found that milk

production and fat and protein contents were the same as for cows fed diets containing

soybean meal. Similarly, sunflower meal is well utilized by calves and growing heifers

(Schingoethe, 1981)

36

2.5.2 Non-ruminants

The use of sunflower meal as a replacement for soybean meal in poultry and swine diets

is limited by its high fibre content and low lysine availability. The degree to which each

of the above limitations contribute to the poor performance of non-ruminants fed diets

containing sunflower meal is controversial. Studies with swine and poultry have shown

that the high fibre content of sunflower meal reduced performance even with adequate

lysine supplementation. The specific mechanisms of this reduction in performance have

not been well elucidated. Fibres are not well digested by non-ruminants and hence

fibrous feedstuffs act as diluents of nutrients and add bulkiness to feeds. Animals usually

respond to the diluting effects of fibres by increasing feed intake, and they may be able to

consume nutrients at rates comparable to those of controls. This compensatory increase in

feed consumption may be partially or totally hindered by physical limitations in the

ability of the gut to distend. In these cases, feed consumption may not be increased at all

or not enough to satisfy nutrient requirements, and in more severe cases, feed intake may

be reduced to an extent that growth is impaired. Moreover, many fibrous feeds have poor

palatability that may reduce feed intake and growth.

2.5.2.1 Sunflower meal in swine diets

Lysine is the first-limiting amino acid in swine diets containing sunflower meal. Reports

on the growth and feed intake responses of swine to lysine supplementation of diets

containing sunflower meal are contradictory. Seerly et al. (1974) evaluated sunflower

meal as a replacement for soybean meal in diets of growing swine. Average daily weight

gain and feed consumption were depressed when sunflower meal replaced 50 to 100% of

37

soybean meal protein in the diet. Lysine levels in the diets containing sunflower meal

were less than the minimum requirement recommended by the National Research Council

(NRC, 1993) for growing swine. When the same diets were supplemented with synthetic

lysine up to requirement, feed consumption increased and weight gains were comparable

to those of the control. The improvement in performance observed suggests that lysine

deficiency was the main factor contributing to the poor performance of the animals, and

that the adverse effects of fibre were minimal. In contrast, Moser et al. (1985) reported a

reduction in weight gain, and an increase in feed consumption when they fed diets

containing 30% sunflower meal to growing-finishing swine, despite supplementing the

diets with lysine. The authors attributed the reduction in performance to the high fibre

content of the sunflower meal (20.5%> crude fibre). They speculated that the increased

feed consumption response was an attempt by the animals to satisfy their nutrient

requirements, and that the pigs were unable to consume enough feed for normal growth

because of its high level of fibre. It is reasonable to conclude from the data of the above-

mentioned studies that, when lysine level is adequate in the diet, adjustment in feed

intake to satisfy energy requirement could be impaired, at least partially by the bulkiness

of the diets containing sunflower cake. The sunflower meal used by Seerley et al. (1974)

contained only 3% crude fibre, a value comparable to that of soybean meal. Hence,

energy level and consequently feed consumption were not significantly affected by

replacement of soybean meal with sunflower meal. On the other hand, in the studies by

Moser et al. (1985), the sunflower meal had a high fibre content (20.5%).

38

2.5.2.2 Sunflower meal in poultry diets

2.5.2.2.1 Broilers

High fibre content limits the use of sunflower meal in broiler diets. The dehulled meal

contains at least 11% to 12% fibre, which is quite high compared to dehulled soybean

meal that contains 3% crude fibre (NRC, 1994). This characteristic of sunflower meal

may lead to bulky diets which could be a problem for young chicks due to the limited

capacity of their digestive system. The density of the diet is of prime concern in terms of

nutrient intake and the resultant growth rates. Pelleting has been shown to improve the

utilization of sunflower meal in diets for poultry (Waldroup et al., 1970; Nir et al., 1994).

An important factor to be considered when sunflower meal is included at high

levels in diets for broilers is the energy status of the diet. Large amounts of dietary fibre

reduce the available energy density of the diet. The metabolisable energy value of the

meal depends on the effectiveness of the dehulling process. If sunflower meal is

incorporated into diets at high levels, the nutrient and energy densities of the resulting

diets may be significantly diluted and consequently growth is retarded. The addition of

fat has been used to improve energy densities in broiler feeds. Zatari and Sell (1990), for

example, found that adding 6% of an animal-vegetable fat blend to a broiler diet

containing 20% sunflower meal containing 33%> crude protein and 18% crude fibre

improved weight gain and feed conversion ratio.

Lysine has been observed to be the first limiting amino acid in SFM diets fed to

poultry. Supplementing lysine to these diets however, has given mixed results,

depending on the other ingredients in such diets and their total lysine content.

39

2.5.2.2.2 Sunflower meal in layer diets

Egg-type chicks are more tolerant to fibrous feeds than broilers because of their slower

growth rates and greater capacity to adjust feed intake according to energy needs.

McNaughton and Deaton (1981) reported that sunflower meal could be included at up to

30% in layers diets without adversely affecting body weight, egg production or egg

weight. Birds responded to decreased energy in the diets by increasing feed intake.

Similar observations were made by Deaton et al. (1979) using a high-fibre SFM (36%

crude protein, and 24% crude fibre). At high dietary levels of inclusion of sunflower

cake (30%>), FCR was significantly decreased, but egg production, egg weight and shell

strength were not affected. The gizzards and intestines of the birds were enlarged. These

findings suggest that layers tend to consume more feed to maintain the same rate of egg

production when the diet contains high levels of SFM. The pelleting of such diets has

been shown by several workers to increase the feed intake of layers (Waldroup et al.,

1970; Nir era/., 1994; Jensen, 1998).

2.6 Use of sunflower meal in fish diets

There is little information on the utilization of sunflower meal by fish. The few

studies available have mainly been done with rainbow trout (Salmo gairdneri) and the

results are not consistent. This is mainly due to differences in experimental methodology,

and differences in the quality of the sunflower meal between studies. The maximum

level of sunflower meal that can be included in fish diets without affecting growth rate or

feed efficiency depends on a number of factors, These include the composition of the

remainder of the diet and the nutritive value of the sunflower meal itself. Some of the

40

reported studies do not make reference to the crude fibre content, which is the main factor

limiting the use of sunflower meal in fish diets.

Tacon et al. (1984) fed rainbow trout diets containing 0 to 37% of solvent

extracted sunflower meal with a fibre content of 24.7% (AD basis) and found no

difference in performance between fish fed the diet with the highest level of sunflower

meal and those fed the soybean meal control diet. Similarly, Morales et al. (1993) also

evaluated sunflower meal as a protein source for rainbow trout. The sunflower meal used

in their study was solvent extracted, with a fibre content of 16.5%. They found that

sunflower meal could replace all the soybean meal in the diet, and could be included at a

level up to 40%). In the study by Morales et al. (1993), rainbow trout fed on the soybean

and sunflower meal diets had better feed intake than those fed on the fishmeal control

diet, which had a reduced feed intake and performed poorly. These results suggest that

the fishmeal used in the latter study was of poor quality. Scott and co-workers (1982)

also noted that fish fed diets based on sunflower meal (17.5% fibre) had better growth

than those fed on the soybean meal control diet. The soybean meal used in the study was

solvent extracted and had undergone the normal degree of heat processing associated

with the solvent extraction process, but the level of trypsin inhibitors was still quite high,

indicating that the treatment did not completely destroy the inhibitor, which may account

for the poor growth of the fish fed these diets.

The results of studies on rainbow trout in which diets based on sunflower meal

have been supplemented with amino acids are not consistent. Sanz et al. (1994) fed

rainbow trout diets containing 36% sunflower meal (16.4% crude fibre), and observed

that without amino acid supplementation the fish had lower feed intake, growth and feed

41

efficiency compared to those fed a fishmeal control diet. Addition of the amino acids

lysine, leucine and methionine improved the growth rate to a level comparable with those

fed the fishmeal control diet. Contrary to the above findings, Tacon et al. (1984),

however, did not observe any beneficial effects on growth and feed utilization when

rainbow trout were fed diets containing 36% sunflower meal and 0.2%> supplemental

methionine. This may be due to the fact that the sunflower meal had high levels of sulfur

amino acids. Indeed it may have been more worthwhile to supplement the diets

containing sunflower meal with lysine, which was very low in these diets (1.1% of diet),

compared to the fishmeal control diet (5.9%> of diet).

In conclusion, the studies available so far indicate that rainbow trout can utilize

sunflower meal at levels up to 40% in the diet. It is possible that a higher level than this

could be used if a good source of digestible energy is also included in the diet. However,

at higher levels, it may be necessary to supplement the diets with amino acids. The

question of whether or not supplementing diets with synthetic amino acids is of any

benefit has not been adequately resolved. Further work needs to be done to establish the

limiting amino acids.

There are only limited studies on the use of sunflower seed cake in tilapia diets.

Abdul-Aziz et al. (1999), working with O. niloticus fingerlings with an initial weight of

19.4 g, replaced 25% to 50%> of soybean meal with sunflower cake. The digestibilities of

protein and organic matter were significantly lower in the diets that contained the

sunflower cake compared to the soybean meal control diet. Among the two diets based on

sunflower cake, the digestibility of protein and organic matter was lower in the diet where

sunflower cake replaced 50% of the soybean meal protein than in the diet where the

42

replacement level was 25%. Sintayehu et al. (1996) evaluated the digestibility of high-

fibre sunflower cake by tilapia (O. niloticus), and observed that the apparent digestibility

of crude protein was higher in soybean than in cotton seed cake and sunflower cake. The

digestibility of organic matter and gross energy was lower in sunflower meal than in

soybean meal and cotton seed cake. In the growth trial, sunflower cake protein replaced

32% of the fishmeal protein with no adverse effects on fish performance.

Supplementation of the diets containing sunflower cake with lysine and methionine did

not improve fish performance. Fish used in the study by Sintayehu and co-workers

(1996) had an initial weight of 90 g., and the diet containing sunflower cake also had

30% fishmeal, which would have adequately supplied all of the lysine needed by fish of

that size. Jackson et al. (1982) fed tilapia (O. mossambicus) (initial weight 13 g) diets

where 75% of the protein originated from sunflower cake and observed that there were no

differences in fish performance between fish fed the diet with the high level of sunflower

cake, and those fed the fishmeal control diet. The results of this experiment, however,

should be interpreted with caution, because the growth rates that were obtained for fish in

this study were well below optimal (0.9% to 1.25%). Furthermore, fish fed the positive

control diet had a specific growth rate of 1.09% per day, indicating that there were other

factors that may have adversely affected fish in this trial. Growth rates for tilapia of

similar size and fed on a positive control diet have typically been in the range of 1.7% to

over 2% (El Sayed, 1998; Abdel-Fattah and El-Sayed, 1990)

In summary, the high fibre content of sunflower cake limits its broad use in fish

diets. In other animal species e.g., swine and poultry, the low lysine content of sunflower

cake has also been identified as a factor limiting its use, whereas in fish, the response to

43

dietary lysine supplementation has not always been consistent. Further work is certainly

needed on this topic. The high fibre content of sunflower cake could be a strong

hindrance to its extensive use in fish diets because fibres are highly indigestible by most

fish species. Dietary fibres also act as diluents, and they may reduce diet palatability, as

well as mineral bioavailability.

44

2.7 References

Abdel-Fattah, M., and El-Sayed, 1990. Long term evaluation of cotton seed meal as a protein source for Nile Tilapia, Oreochromis niloticus (Linn). Aquaculture, 84: 315-320.

Abdul-Aziz, G.M., El-Nady, M.A, Shalaby, AS., and Mahmoud, S.H., 1999. Partial substitution of soybean meal protein by different protein sources in diets for Nile tilapia fingerlings. Bulletin of Faculty of Agriculture, University of Cairo, Vol. 50: Issue 2:

Anderson, J., Capper, B.S., and Bromage, N.R., 1991. Measurement and prediction of digestible energy value in feedstuffs for the herbivorous fish tilapia (Oreochromis niloticus Linn). British Journal of Nutrition, 66: 37- 48.

Anderson, J.S., 1996. Dietary protein quality and quantity for Atlantic Salmon (Salmo salar) reared in sea water. PhD. thesis, The University of British Columbia. 156 pp.

Balarin, J.D., 1985. National reviews for aquaculture development in Africa. 7. Kenya. FAO fish circ, 770: (7) 96pp.

Balogun, A.M., and Fagbenro, OA., 1995. Use of macadamia press-cake as a protein feedstuff in practical diets for tilapia, (Oreochromis niloticus) (L). Aquaculture and Research, 26: (6) 371-377.

Belal, I.E. H., Al-Owaifeir, A , and Al-Dosari, M., 1995. Replacing fishmeal with chicken offal silage in commercial Oreochromis niloticus (L) feed. Aquaculture Research, 26: 855 - 858.

Bishop, CD. , and Angus, R.A., and Watts, A., 1995. The use of feather meal as a replacement for fishmeal in the diet of Oreochromis niloticus fry. Bio-Resource Technology, 54: 291-295.

Bowen, S.H., Lutz, E.V., and Ahlgren, M.O., 1995. Dietary protein and energy determinants of food quality. Ecology, Vol 76: (3) 899-907.

Calvert, C C , Klasing, K . C , and Austic, R.E., 1982. Involvement of food intake and amino acid catabolism in the branched-chain amino acids. Antagonism in chicks. Journal of Nutrition, 112: (4) 627-635.

Canvin, D.T., 1965. The effect of temperature on the oil content and fatty acid composition of the oils from several oilseed crops. Canadian Journal of Botany, 43: 63-69.

Chamberlain, G.N., and Hopkins, J.S., 1994. Reducing waste use and feed cost in intensive ponds. World aquaculture, 25: (3) 29 - 32.

45

Cho, C.V., and Slinger, S.J., 1979. Apparent digestibility measurements in feedstuffs for rainbow trout. In: Finfish Nutrition and Fish-feed Technology. J.E. Halver and K. Tiews, (Eds.) Vol. 2 239-247

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55

Chapter 3

Experiment 1: Digestibility of nutrients and energy in wheat bran, high-

fibre and fibre-reduced sunflower cakes, anchovy fishmeal and omena

fishmeal by Oreochromis niloticus

3.0 Abstract

The apparent digestibility of protein, organic matter, and energy of high-fibre and fibre-

reduced sunflower cake, wheat bran, omena fishmeal and anchovy fishmeal was

investigated in tilapia (O. niloticus) fingerlings. A reference diet in which anchovy fish

meal and soybean meal were the main sources of protein was formulated. Test diets were

made by combining 70% of the reference diet with 30% of each of the following: wheat

bran, high-fibre and fibre-reduced sunflower cakes, anchovy and omena fishmeals. Each

diet was provided to 3 tanks offish, each with 12 fish. Water temperature was maintained

at 26°C, and dissolved oxygen concentration above 5.5 mg/litre. Digestibility of the diets,

nutrients and energy was done by the indirect method using chromic oxide as a marker.

Fecal collection was done by stripping.

Among the fishmeals and the sunflower cakes, apparent protein digestibility

coefficients were similar. Protein digestibility was lower (P < 0.05) in wheat bran than in

the other ingredients. Apparent digestibility coefficients for energy and organic matter,

and the digestible energy concentrations were significantly higher for the fishmeals (P <

0.05) than the plant protein ingredients. There were no significant differences in the

digestibility of energy and organic matter between anchovy fishmeal and omena

fishmeal, and this was also true for the digestible energy concentrations (P > 0.05). The

dehulling of sunflower cake increased the digestibility of energy by 12% relative to the

undehulled meal.

56

3.1 Introduction and objectives

In order to formulate practical diets for feeding fish, we need to know the nutrient

compositions of the feedstuffs that are potential ingredients for inclusion into such diets.

We also need to know the biological availability of the nutrients and energy in each of the

ingredients for the species under consideration. The diets for intensive fish culture are

formulated where possible on the basis of literature values for digestible energy and

protein in the feedstuffs. For some warm water fish such as tilapia, however,

comprehensive tables showing the availability of nutrients and energy from various

ingredients are not available. Consequently, diets for tilapia are formulated using

published data extrapolated from other fish species such as carp (Cyprinus carpio), and

catfish (Clarias gariepinus). There are differences in the ability of tilapia, carp and

catfish to digest proteins, fats and carbohydrates (Degani and Revach, 1991). Tilapia are

herbivorous fish and they digest carbohydrates efficiently (Anderson et al., 1984; Viola

and Arieli, 1983). Tilapia have also been shown to digest animal proteins better than carp

(Degan et al, 1991). Carp are omnivorous, and under natural conditions, they find their

feeds from benthic sources. By contrast, tilapia feed at all trophic levels, with the result

that the natural diet of tilapia contains a higher percentage of plant life. On the other

hand, tilpia cope less efficiently with fat than carp and catfish (Degani and Revach,

1991).

Methods for determining digestibility coefficients involve either direct or indirect

measurements of the amounts of nutrients ingested, and subsequently excreted. The

direct method involves measuring all the feed consumed by the fish, and all the resulting

excreta (Smith,1971; Smith et al, 1980). In studies by Smith et al, (1980), individual

57

1

fish were confined in metabolic chambers and force-fed a measured amount of feed. The

excrements were quantitatively collected and analyzed for nutrient content. This method

was very stressful to the fish, and likely compromised feed utilization.

The indirect method involves the use of an indigestible marker (Maynard et al,

1979). Chromic oxide, crude fibre, polyethylene and acid-insoluble ash have all been

used as markers. In using markers, several assumptions are made. For example, it is

assumed that the marker is inert, indigestible, does not stream ahead of the intestinal

contents, and is not preferentially retained at some level in the gastrointestinal tract.

The relative concentration of the marker is also observed to increase in the intestinal

contents as the ingesta passes along the tract and the nutrients are absorbed.

In digestibility studies in fish by direct or indirect methods, several problems are

encountered in the collection of feces from water. One problem is the likelihood of

leaching of soluble fecal compounds and the fragmentation of the feces with the

dispersion of fine particulate matter into the water. Besides, it is difficult to separate

feces from the water and to avoid contamination of the feces with the uneaten feed. In

view of the above, several methods of collecting samples of feces where a marker has

been used have been devised such as intestinal dissection, (Smith and Lovell, 1971),

stripping (Nose, 1960), anal suction (Windell et al, 1978) and the use of special

collection tanks (Cho and slinger, 1979; Cho et al. 1982; Talbot, 1985). In the present

study, chromic oxide was used as a marker and feces were collected by stripping. Cho

and Slinger (1979), Cho et al. (1982) and Talbot (1985) showed that the results of

digestibility studies obtained by intestinal dissection, stripping and special collection

58

tanks were not significantly different for digestibility of dry matter, crude protein and

lipids.

The main objective of this experiment was to determine the effect of reducing

fibre content in sunflower cake on the apparent digestibility of protein, energy and

organic matter using tilapia (0. niloticus) as the test animal. Also, it was of interest to

compare the digestibility of protein, energy and organic matter of Kenyan omena

fishmeal with that of prime quality anchovy fishmeal for the reasons provided previously.

A growth study was also undertaken to assess whether all of the diets were acceptable to

the fish.

59

3.2 Materials and Methods

3.2.1. Fibre-reduced sunflower cake

Hybrid sunflower seeds (Kenya Fedha) were purchased from a commercial trader (Rift

Valley Products, Nakuru, Kenya). The seeds were partly dehulled using a manually-

operated Cecoco dehuller (Ibaraki, Osaka 567 Japan) which incorporated a dehuller and a

sorting machine. Throughput was 25 kg/hr to achieve a 75% dehulling efficiency.

Efficiency depended on the seed type and moisture content. Seeds with low moisture

content were easier to dehull than those with a high moisture content, and for that reason,

all seeds were dried to less than 10% moisture before dehulling. The oil content of the

partly dehulled seeds was reduced by a commercial screw press oil extractor (Gold Feeds,

Nairobi, Kenya).

3.2.1.2 High-fibre sunflower cake

The high-fibre sunflower cake used in this experiment and all other experiments reported

in this thesis was a commercial cake that is marketed as prime quality sunflower cake. It

is processed by Rift Valley Products, Nakuru, Kenya, for Unga Feed Millers in Nairobi,

the largest feed manufacturers in the country. The fibre content is considerably lower

than in other cakes available in the country and it fetches a premium price. It was

processed from the same variety of sunflower as the low-fibre sunflower cake.

3.2.2 Ingredients other than sunflower cakes.

Omena fishmeal was purchased from Tamfeeds, (Nairobi, Kenya). It was made from the

cyprinid fish, Rastrineobola argentea. The anchovy meal was a high quality Chilean LT.

meal purchased from Moore Clark, B.C., Canada. This fishmeal was purchased in Canada

because, at the outset of the study, it was not possible to obtain prime quality herring

60

meal elsewhere to act as a positive control. Herring meal became available in Kenya

later in the project, and was used in Experiments 3 and 4. The oc-cellulose and cornstarch

were bought from ICN, Canada, and chromic oxide from Fisher Scientific (Ontario,

Canada). Soybean meal and wheat bran were bought from Sigma Feeds, (Nairobi,

Kenya).

3.2.3 Chemical analyses

All ingredients and the experimental diets were analyzed in duplicate for dry matter, ash,

crude fibre, fat and protein according to standard procedures (AOAC, 1984). Gross

energy was determined by adiabatic bomb calorimeter at the Kenya Industrial Research

Development Institute, Nairobi, Kenya. Acid detergent fibre (ADF) and neutral detergent

fibre (NDF) contents were determined by the method of Waldern (1971), using an

Ankom technoloanalyzer (Ankom Technology, 140 Turk Hill Park, Fairport, N Y 14450).

The chromic oxide contents in the diets and feces were determined by the acid digestion

method of Farukawa and Tsukahara (1966). Water quality parameters (Appendix 1) (pH,

colour, turbidity, and the levels of iron, manganese, calcium, magnesium, sodium,

potassium, aluminium, chlorides, fluorides, nitrates, nitrites, ammonia, total nitrogen,

sulphate, orthophosphates, total hardness, total alkalinity, total suspended solids, free

carbon dioxide, total dissolved solids, dissolved oxygen and residual chlorine were

analysed at the Ministry of Water Development, Water Quality and Pollution Control

laboratory in Nairobi.

3.2.4 Experimental diets

Six diets, whose compositions are shown in Table 3.1, were formulated. A reference diet

based on prime quality anchovy fishmeal (Moore Clark, BC, Canada), soybean meal,

61

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the main sources of protein while wheat bran was used as an inexpensive binder to ensure

good water durability of the pellets.

Test diets were formulated by combining 70% of the reference diet, with 30% each of the

test ingredients (high-fibre and fibre-reduced sunflower cakes, "omena" fishmeal,

anchovy fishmeal, and wheat bran). Chromic oxide (0.5%) was used as an indigestible

marker, and was added at the expense of cellulose. Al l diets were pelleted at the

Naivasha Station of the Kenya Agricultural Research Institute using an Ottevanger

pelleting machine (Ottevanger Machine Fabrieken B.V, 2750 A A Moekapelle, Holland).

Three tanks, each containing 12 fish, were used for each diet. The fish were fed to

satiation 3 times daily at 8.30 a.m., 12.30 p.m. and 4.30 p.m.

3.2.5 Supply and maintenance of fish

Male O. niloticus fingerlings weighing 59g ± 5.4 g (+ sd) were purchased from Baobab

Fish Farm (Bamburi Nature Trail, Mombasa) and transferred to the University of Nairobi

for this experiment. They were acclimated to laboratory conditions for a period of three

weeks. During the acclimation period they were fed on commercial feed (Baobab tilapia

pellets) for a period of one week, and then the reference diet for another 2 weeks before

the onset of the digestibility trial. After the acclimation period, they were weighed in

groups of 12 fish selected at random and allocated to the experimental circular tanks.

Eighteen tanks with a diameter of one metre and filled with water to a depth of 0.26

metres were used at the start of the experiment. Water level was adjusted as the fish

biomass increased to maintain a stocking density below 0.1 kilograms of fish per litre of

63

water. The chemical parameters of the well water, which was used in all experiments are shown

in Appendix 1. A Sweetwater T M Regenerative blower was used for aeration. Each tank was

fitted with an AS8-1 (3 inches) diffuser. Water temperatures and dissolved oxygen concentration

in the tanks was maintained at 26°C ± 2°C and above 5.5 mg/liter, respectively. Water was

completely exchanged in each tank every 48 hours. The fish were kept under a natural

photoperiod (Nairobi, Kenya, 1° 16' S, 36° 48' E).

Duration of the growth trial was 50 days, but feces collection continued for 3 months.

All fish that died were replaced by others of similar weight from a reserve tank to ensure that

there were enough fish to provide the required amount of the fecal samples. Fish were starved

24 hours before handling.

3.2.6 Fecal collection

Feces were collected by stripping, (Nose, 1960), once a fortnight during the growth trial (50

days), and thereafter, once every week for the next 56 days to the end of the trial. Fish were fed

at 8.30 a.m and 10.30 a.m and fecal collection was done approximately 5 to 7 hours after the first

feeding. Al l fish in a tank were first anaesthetized with MS 222 and blotted dry. Urine was then

pressed out by gentle pressure applied along the lateral line towards the vent. Pressure was then

applied on the region between the anal fin and the vent and the expressed feces collected into

aluminium dishes. Feces from each tank were dried immediately after collection at 60°C for a

period of 24 hours. They were then labeled and and frozen (-5°C) until the end of the trial, when

all feces from each tank were pooled. Fecal samples were analyzed for moisture, chromic oxide,

protein, gross energy and ash.

i

64

3.2.7 Digestibility assessment

Apparent digestibility coefficients of the test diets and test ingredients were calculated by the

indicator method of difference as described by Maynard et al. (1979). Apparent digestibility

coefficients (%) for each of the 6 test diets and test ingredients were determined for crude

protein, gross energy and organic matter.

The apparent digestibility of each nutrient was calculated as follows;

100 [ 1 - marker cone, in diet X nutrient cone, in feces] marker cone, in feces nutrient cone, in diet

Digestible energy concentration in the diet was calculated as follows;

Gross energy in diet - [fecal energy concentration X marker cone, in diet] marker cone, in feces

Apparent digestibility coefficient for a nutrient in the test ingredient was calculated from the

digestibility coefficient for the reference and the test diets according to the following equation of

Forster (1999).

ADCNingr = {(a + b) * ADCNcom - (a)*ADCN ref}/b

ADCNingr = apparent digestibility coefficient of a nutrient in test ingredient

A D C N ref - apparent digestibility coefficient of a nutrient n the reference diet.

a = nutrient contribution of reference diet to nutrient content of combined diet,

= (% nutrient in reference diet) * (100 - i)

b = nutrient contribution of test ingredient to nutrient content of combined diet,

= (% nutrient in test ingredient)*i

i = percent of test ingredient in the combined diet.

ADCNcom = Apparent digestibility coefficient of a nutrient in the diet consisting of a

combination of the reference diet and the test ingredient

65

3.2.8 Data collection and analytical procedures

Fish growth and performance was assessed by the following parameters; absolute weight, weight

gain, specific growth rate, feed intake and feed conversion ratio. Specific growth rate (% per day

was calculated as follows: 100(ln final wt (g) - ln initial wt (g))/number of experimental days.

Feed conversion ratio was calculated as ingested feed (g)/wet weight gain.

3.2.9 Statistical analyses

Data on final weight of fish, weight gain, specific growth, feed consumption, feed conversion

ratio and digestibility of various nutrients and energy in the diets were analyzed using PROC.

G L M of the SAS statistical package. Means were compared using Tukeys's test with the level of

significance set at P < 0.05. In analyzing data on final weight, weight gain, and feed

consumption, an analysis of covariance was done with the initial weight of the 12 fish as the

covariate, to control for the differences in the initial weight of the fish. The statistical model

used in the analyses of various fish performance parameters and digestibility was;

Yij = p + Ai + Bj + eij

where, p = overall mean

A = effect of diet

e = random error

66

3.3 Results and discussion

3.3.1. Chemical composition of the reference diet, test diets and test ingredients

The results of proximate analyses of the reference and test diets are presented in Table 3.1, while

those of the test ingredients are presented on Table 3.2. Dehulling sunflower seeds considerably

reduced the fibre content and increased the percentage of protein in the cake. Dehulled

sunflower seeds are difficult to press using the conventional screw press machines and

consequently the fibre-reduced cake had a slightly higher percentage of lipid than the high-fibre

cakes. Gross energy was also higher in the fibre-reduced cake than noted in the high-fibre cake

when the results were calculated on a dry weight basis. The dehulled cake had a dry matter

content of 92%. Crude protein, fibre, fat and ash contents (DM basis) were 44.6%, 12%, 11%,

and 9.8% respectively. Gross energy content was 5.01 kcal/g., while acid detergent fibre and

neutral detergent fibre were 14% and 26%, respectively. The high-fibre sunflower cake had a

dry matter content of 92% and contained the following levels of nutrients and energy (DM

basis); crude protein, 30%; crude fibre, 29%; crude fat, 9.7%; gross energy, 5.00 kcal/g; ADF,

24.5%; NDF, 48.9%.

Gross energy contents in the diets did not vary appreciably, ranging from 4807 kcal/kg to

4954 kcal/kg ( D M basis), while crude protein levels were in the range of 31.1% to 44.6% (DM

basis). Crude fibre, ADF and NDF values were highest in the diets containing high-fibre

sunflower cake, while ash content was highest in the diets that contained both fishmeals.

3.3.2. Fish performance

Final weights, weight gains, specific growth rates, feed intakes, feed efficiencies and percent

mortalities are presented in Table 3.3. For the 50 days of the feeding trial, feed intake and feed

67

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inclusion of high-fibre sunflower cake at the 30% level in the diet did not depress feed

intake. Mortality was highest for the fish fed the diet with a high level of anchovy

fishmeal.

The specific growth rates observed were 0.8%, 0.75%, 0.73%, 0.72%, 0.69% and

0.64% for fish fed the reference diet, and diets based on omena fishmeal, fibre-reduced

sunflower cake, wheat bran, anchovy fishmeal, and high-fibre sunflower cake. The

growth rate of fish fed on the high-fibre cake was significantly less than that found for the

fish fed the reference diet. This could be due to the low digestible energy content of the

diet based on the high-fibre cake. The reason that fish performance was assessed was

mainly to determine whether all diets were acceptable to the fish, and whether each

would support positive nitrogen balance. There were no significant differences between

groups in feed intake indicating that all diets were equally acceptable.

3.3.3 Apparent digestibility of nutrients in test ingredients.

The apparent digestibilities of energy, protein and organic matter of the reference diet and

test diets are presented in Table 3.4. The digestibility coefficients were subsequently

used to calculate the digestibility of nutrients and energy in the test ingredients. In regard

to the latter, the digestibility of nutrients and energy was determined according to the

equation of Forster (1999) considering that each of the test diets consisted of 70 % of the

reference diet and 30%> of the test ingredient.

3.3.4 Apparent digestibility coefficient for protein (ADC-P)

The apparent digestibility coefficients for protein (ADC-P) in the test ingredients are

listed in Table 3.5. Anchovy and omena fishmeals each had an ADC-P of 90%. The

70

Table 3.4: Apparent digestibility coefficients (ADCs) and apparent digestible energy (ADE) values of the reference and test diets (70% reference diet, 30% test material).

Protein Energy DE Organic matter ADC ADC (kcal/kg) ADC (%) (%) (DM basis) (%)

Reference 92.8 78.5 3840 76.2 Ref-Fibre reduced SFC1 91.3 67.1 3341 63.1 Ref-High fibre SFC 90.9 63.7 3101 61.6 Ref-Omena fishmeal 91.4 78.4 3771 75.8 Ref-Anchovy 91.5 80.8 3889 73.5 Ref -wheat bran 89.8 66.2 3228 60.2

'SFC Sunflower Seed Cake ADC Apparent digestibility coefficient DE Digestible energy

71

Table 3.5: Apparent digestibility coefficients (ADCs) and digestible energy values for the fibre-reduced and high-fibre sunflower cakes, omena fishmeal, anchovy fishmeal, and wheat bran.

Protein Energy DE Organic matter ADC ADC (kcal/kg) ADC (%) (%) (DM basis) (%)

Test Ingredient Fibre-reduced SFC1 88.60" High-fibre cake 85.60a

Omena Fish Meal 89.70a

Anchovy Fish meal , 90.00a

Wheat bran 75.20b

SEM 1.90

42.10b 2203b 37.20b

29.70b 1401° 30.80b

78.203 3624a 74.70" 86.00a 4003a 76.50a

37.10b 1787bc 30.30b

2.65 132.7 2.57

A D C Apparent digestibility coefficient D E Digestible energy 1 SFC Sunflower cake Means (n = 3) within a column with a common superscript are not significantly different (P>0.05)

72

fibre-reduced and high-fibre sunflower cakes had ADC-P values of 89% and 86%

respectively, which were not significantly different. Differences in protein digestibility

between the fishmeals and the sunflower cakes were not significant (P > 0.05). Wheat

bran had an apparent protein digestibility coefficient of 75%, which was significantly

lower (P < 0.05) than those found for the other test ingredients.

The results of this study are in agreement with the findings of other authors,

despite the fact that different methodologies were used in the various studies. For

example, with respect to fishmeal, Watanabe et al. (1996), observed an apparent protein

digestibility of 92% for white fishmeal and a local fishmeal fed to tilapia (O. niloticus).

Further, Hanley (1987) reported an ADC-P value of 87%> for a fishmeal made from

different species of fish fed to O. niloticus. Watanabe and co-workers (1996) used

digestibility chambers (as described by Cho et al, 1982) for collection of feces, while

Hanley (1987) collected feces by intestinal dissection. There is limited information on

the digestibility of protein in sunflower cake by tilapia (O. niloticus). Sintayehu et al.

(1996) determined the protein digestibility of sunflower cake that had a crude fibre

content of 29.4% (AD basis) using HCL-insoluble ash as the indigestible marker. The

fish were hand fed, and the feces were collected by the sedimentation technique. The

apparent protein digestibility coefficient of sunflower cake in that study was 89.8%,

which was not appreciably different from the values of 86% and 89% observed in the

current study for the high-fibre and the fibre-reduced cakes, respectively.

In studies with carp by Eid and Matty (1989), true protein digestibilities of two

sunflower cakes (compositions not defined) were reported as 65%> and 68%. These

values were much lower than those obtained for tilapia in the current study. In the studies

73

by Eid and Matty (1989), an in-vitro digestion method was used, where the protein

sources were incubated in-vitro with carp intestinal extract, and then the digestibility of

the various nutrients was determined. The method used by Eid and Matty (1989) would

give true digestibility values, which should be higher than the apparent values.

Differences between the two studies may be due to the different methods used to assess

digestibility and possibly to differences between the two species in utilization of plant

proteins. Differences in digestive capabilities between tilapia and carp have been

reported by Degani and Revach (1991), who compared the digestive capabilities of

tilapia, carp, and catfish, and by Watanabe et al. (1996), who compared protein

digestibility in rainbow trout, carp, tilapia, and ayu (Plecoglossus altivelis). The studies

by Degani and Revach (1991) showed that tilapia digest protein from animal sources

better than carp, while Watanabe et al. (1996) observed that the digestibility of protein

from plant protein sources by carp and tilapia was similar. It therefore follows that the

observed differences in protein digestibility for sunflower cake between the current study

and that of Eid and Matty (1989) may have been due to the different methodologies used

in the two studies.

Commercial feed binders are expensive, and wheat and wheat products are often

used to increase pellet stability in water. Wheat bran is an inexpensive product that could

be used as a binder in tilapia pellets. Information on the digestibility of protein in wheat

bran for tilapia species is limited. Popma (1982) determined that the apparent protein

digestibility of wheat bran in tilapia was 71%, which was close to the value of 75%

observed in the present study. It is not clear why the digestibility of protein in wheat bran

was lower than that of the other ingredients assessed in this study, particulary the high-

74

fibre and fibre-reduced sunflower cake. Popma (1982) attributed the low digestibility of

wheat bran protein to the feeding habits of tilapia, pointing out that tilapia repeatedly pick

up and expel pelleted feeds before swallowing. In this process, the pellet disintegrates

which may lead to selective feeding on individual components of the diet. The author

observed selective feeding in coarsely-textured and less palatable diets (coffee pulp,

wheat bran and alfalfa meal). Furthermore, for the above mentioned diets, Popma (1982)

found that the concentration of chromic oxide in the diets was more than twice the

concentration in the faeces, which would indicate a negative digestibility. The author

concluded that chromic oxide was not suitable for determination of digestibility in such

feeds. In contrast to the above argument, the digestibility of protein in the high-fibre

sunflower cake used in the current study was not significantly different from that of

fishmeal, despite being "coarsely" textured. Furthermore, in the study by Popma (1982),

raw corn which had the same texture as wheat bran had an apparent protein digestibility

of 84%, which was not significantly different from that of the fishmeal used in that study.

This may indicate that there was another factor that affected the digestibility of protein in

the wheat bran which needs further investigation.

3.3.5 Apparent digestibility coefficient for energy (ADC-E) and digestible energy

concentration (DE) in test ingredients

The results of the apparent digestibility coefficients for energy (ADC-E) and digestible

energy (DE) concentrations of the test ingredients are listed in Table 3.5. Anchovy

fishmeal had an ADC-E of 86%, while omena fishmeal had an ADC-E of 78%>, which

were not significantly different.

75

The A D C - E values for the two fishmeals in this study are comparable to those

reported by other authors for tilapia species. Hanley (1987) reported an A D C - E of 87%,

and a digestible energy concentration of 3283 kcal/kg D M for a fishmeal made from

different species of fish, while Anderson et al. (1991) noted a digestible energy content of

3876 kcal/kg D M . The type of fishmeal used in the latter study was not defined. In a

study with carp, Hossain and Jauncey (1989) reported an apparent energy digestibility

coefficient of 80% for fish meal (type not denned), which is closer to the value of 78%

observed for omena fish meal in this study, but lower than that of 86% for anchovy fish

meal. The A D C - E of fishmeal is affected by many factors, including the source,

composition and freshness of the fish, and the processing temperatures involved in the

production of the meal (Anderson, 1996). Fish lipids are high in polyunsaturated fatty

acids, which are susceptible to oxidation, during processing. Fligh temperatures cause

lipid peroxidation, resulting in reduced digestibility (Davadasan et al, 1985). Opstvedt

(1974), working with poultry, observed that oxidation of lipids in fishmeal reduced the

energy value of the meal.

The digestibility of energy in fibre-reduced sunflower cake has not been reported

previously for tilapia or carp. A D C - E values in the high-fibre and fibre-reduced

sunflower cakes in the present study were 30% and 42%, respectively, while the

digestible energy contents were 1401 kcal/kg D M and 2203 kcal/kg D M , respectively.

However, these preceding differences in the A D C - E coefficients were not significant (P <

0.05), although the difference in energy value, when expressed as D E concentration was

significant (P < 0.05). The lack of a significant difference in the energy digestibility

coefficients may have been caused by the large random variation in values observed for

76

the fibre-reduced sunflower, high-fibre cake, and wheat bran. Despite the reduction in

fibre content, the fibre-reduced sunflower cake had a low A D C - E compared to the

fishmeals. Some authors have reported low digestibility of carbohydrates in sunflower

cake. In rainbow trout, Sanz et al. (1994) found a digestibility of 40% for carbohydrates

(NFE plus fibre) in sunflower cake compared to 50% for soybean meal carbohydrates.

Bendi and Spandorf (1953) also found low (26%) digestibility of sunflower cake

carbohydrates in carp. It was not possible to assess the digestibility of all nutrients in the

present experiment. It is plausible that the digestibility of sunflower carbohydrates in

tilapia is also low, and that this accounted for the observed low values for digestible

energy content.

The high-fibre sunflower cake was markedly higher in fibre content as measured

by the different fibre analysis methods (crude fibre, ADF and NDF). Anderson et al.

(1991) and Kirchgessner et al. (1986) stipulated that the digestibility of energy in plant

protein sources is determined by the amount of cell wall fractions in the respective

feedstuffs. In studies by Anderson et al. (1991), NDF was well correlated (inversely)

with the D E content in feedstuffs fed to tilapia (O. niloticus), while Kirchgessner et al.

(1986) related energy digestibility to the ADF content of feedstuffs.

Sintayehu et al. (1996), reported an apparent digestibility coefficient for energy of

49%, and a D E content of 2298 kcal/kg D M in high-fibre sunflower cake, while

Anderson et al. (1991) observed a D E value of 867 kcal/kg D M . The crude fibre contents

of the sunflower cake in the studies refered to were 26%-29%. In the study by Anderson

and Associates (1991), ADF and NDF values for the sunflower cake were 31% and 40%,

respectively, while in the study by Sintayehu et al. (1996) these values were not stated.

77

The apparent digestibility coefficient for energy of 30%, and digestible energy content of

1401 kcal/kg D M observed in the present study for the high-fibre sunflower cake are

closer to the values observed by Anderson et al. (1991). The higher D E concentration in

the present study could be attributed to the higher crude lipid content of the high-fibre

sunflower cake which was 9% while in the studies by Anderson et al. (1991) it was only

2%.

3 . 3 . 6 Apparent digestibility coefficient for organic matter (ADC-OM)

The trend in apparent organic matter digestibility was similar to that observed for energy

digestibility. The fishmeals had the highest organic matter digestibility. Among the two

sunflower cakes, organic matter digestibility was higher in the fibre-reduced cake than in

the high-fibre cake, but the differences were not significant. The high-fibre cake and

wheat bran had a similar A D C - O M of 31 and 30%, respectively. Kirchgessner et al.

(1986) reported that, in general, ingredients with higher protein content had higher

digestibility values for organic matter and energy in tilapia. The authors also noted a

negative correlation between ADF content and the digestibility of organic matter and

energy. In the present study, anchovy and omena fishmeals had higher organic matter

digestibility than the sunflower cakes and wheat bran, both of which were high in ADF.

ADF in the fibre-reduced sunflower cake and wheat bran, was lower than in the high-

fibre sunflower cake, but, despite this, the differences in organic matter digestibility

between the three ingredients were not significant

78

3.4 Conclusions

The results from this study indicate that the protein from sunflower cake, regardless of its

fibre content, is well digested by tilapia (O. niloticus) at a level not markedly different

from that of high quality fishmeal. Similarly, the digestibility of protein in the locally

produced omena fishmeal was equal to that of the high-quality anchovy fishmeal.

Reducing the fibre content in sunflower cake increased the digestibility of nutrients and

energy, thereby increasing its feeding value. The greatest effect of fibre reduction was

observed in the digestibility of energy, with the value increasing from 30% to 42%.

The digestibility of omena fishmeal in tilapia had not been studied before. This

study showed that digestibility of nutrients in omena fishmeal was similar to that of

anchovy fishmeal. Compared to fishmeal and soybean meal, sunflower cake is

inexpensive and the digestibility values suggest that the fibre-reduced sunflower cake

could be used at high levels of replacement in tilapia diets, with little need for adjustment

in dietary crude protein level. Additional lipid may be necessary to adjust the D E

concentrations, but increasing voluntary feed intake by using appetite enhancers could at

least partially counteract the dilution in dietary DE. This is an area that needs further

research. The results suggest that it may be possible to use omena fishmeal, which is

locally available, to replace the imported expensive fishmeals, in diets for tilapia and

perhaps other fish species.

Al l the plant sources tested" had high levels of crude fibre. They were

incorporated at a level of 30% in the diets. The subject of the additional studies described

in this thesis was to investigate the effect of increasing the levels of these ingredients in

the diets on tilapia performance.

79

3.5 References

Anderson, J., Capper, B.S., and Bromage, N R . , 1991. Measurement and prediction of digestible energy value in feedstuffs for the herbivorous fish tilapia (Oreochromis niloticus Linn). British Journal of Nutrition, 66: 37 - 48.

Anderson, J., Jackson, A.J., Matty, A.J. , and Capper, B.S., 1984. Effects of dietary carbohydrates and fibre on the tilapia (O. niloticus). Aquaculture, 37: 303 - 314.

Anderson, J.S., 1996. Dietary protein quality and quantity for Atlantic Salmon (Salmo salar) reared in sea water. PhD thesis, The University of British Columbia. 156 pp

A O A C , 1984. Association of Official Analytical Chemists. Official methods of analysis. Animal Feed Section. 1141 pp

Bendi, A. and Spandorf, A , 1953. The activity of digestion enzymes of the carp. Bamidgeh, 5: 116-130.

Cho, C.Y., and Slinger S.J., 1979. Apparent digestibility measurements in feedstuffs for rainbow trout. In: Finfish Nutrition and Fish Food Technology. Proceedings of a World symposium. Hamburg, 20 - 23 June 1978.

Cho, C.Y., Slinger, S.J., and Bayley H.S., 1982. Bioenergetics of Salmonid fishes: Energy intake, expenditure, and productivity. Comp. Biochem. Physiol., 73B: 2 5 - 4 1 .

Davadasan, K. , Nair, P.G.V., and Antony, P.D., 1985. Effect of oxidation of dietary fish lipids on the quality of proteins in the diet. Fishery Technology. Society of Fisheries Technologists, (India) 22: (1) 70-73.

Degani, G., and Revach, A., 1991. Digestive capabilities of three commensal fish species: carp (Cyprinus carpio), L.tilapia (Oreochromis aureus x O. niloticus) and African catfish (Clarias gariepinus). Aquaculture and Fisheries M anagement, 22: 397 - 403.

Degani, G., Viola, S., and Yehuda, Y. , 1997. Digestibility of protein and carbohydrates in feed ingredients for adult tilapia (Oreochromis aureus x O. niloticus). The Israeli Journal of Aquaculture - Bamidgeh, 49: (3) 115 - 123.

Eid, A.E. , and Matty, A.J., 1989. A simple in-vitro method for measuring protein digestibility. Aquaculture, 77: 111-119.

Farukawa, A., and Tsukahara, H. , 1966. Acid digestion method for the determination of chromic oxide as an index substance in the study of digestibility of fish feed. Bull. Jpn. Soc. Sci. Fish., 32: 502 - 504.

80

Forster, I., 1999. A note on the method of calculating digestibility coefficients of nutrients provided by single ingredients to feeds of aquatic animals. Aquaculture Nutrition. Vol 5, no. 2. 143 - 145.

Hanley, F., 1987. The digestibility of feedstuff's and the effects of selectivity on digestibility determinations in tilapia (Oreochromis niloticus). Aquaculture, 66: 163 — 179.

Hossain, M.A . , and Jauncey, K. , 1989. Studies on the protein, energy, and amino acid digestibility of fishmeal, mustard oil cake, linseed and sesame meal for common carp (Cyprinus carpio L ) . Aquaculture, 83: 59-72 .

Kirchgessner, M . , Kurzinger, H. , and Scwarz, F. J. 1986. Digestibility of crude nutrients in different feeds and estimation of their energy content for carp (Cyprinus carpio L.). Aquaculture, 58: 185 - 194.

Maynard, L .A . , Loosli, J.K., Hintz, H.F., and Warner, R.G., 1979. Animal Nutrition. 7 t h

Edition. McGraw Hill. 602pp.

Nose, T., 1960. Digestion of food protein by goldfish (Carassius auratus) and rainbow trout (Salmo gairdneri). Bull. Freshwater Fish Res. Lab., 10: (1) 12 - 22.

Opstvedt, J., 1974. Influence of lipids on the nutritive value of fishmeal. Effects of fat addition to diets high in fishmeal to fatty acid composition and flavor in broiler meat. Acta. Agric. Scand., 24: 62 - 67.

Popma, JT., 1982. Digestibility of selected feedstuffs and naturally occurring algae by tilapia. Ph.D. Thesis, Auburn University, Alabama. 80pp.

Sanz, A., Morales, A.E. , De LaHiguera, M . , and Cardenete, G , 1994. Sunflower meal compared with soybean meal as partial substitutes for fish meal in rainbow trout (Oncorhyncus mykiss) diets: protein and energy utilization. Aquaculture, 128: 287-300.

Shiau, S.Y., and Chen, M.J. , 1993. Carbohydrates utilization by tilapia (Oreochromis niloticus XO. aureus) as influenced by different chromium sources. Journal of Nutrition, 123: 1747- 1753.

Shiau, S.Y., and Liang, H.S., 1995. Carbohydrates utilization and digestibility by tilapia, (Oreochromis niloticus x O. aureus), are affected by chromic oxide inclusion in the diet. Journal of Nutrition, 125: 976 - 982.

Sintayehu, A. , Mathies, E., Mayer, Burgdorff, K - H . , RosenowH., and Gunther, K-D. , 1996. Apparent digestibilities and growth experiments with tilapia (Oreochromis niloticus) fed soybean meal, cottonseed meal, and sunflower seed meal. Journal of Applied Ichthyology, 13: (2) 125 - 130.

81

Smith, B.W., and R.T. Lovell., 1971. Digestibility of nutrients in semi purified rations by channel catfish in stainless troughs. Proc. Annu. Conf. Southeast Asia Association of Game Fish. Commun., 25: 425 - 459.

Smith, R.R., 1971. A method for determination of digestibility and metabolizable energy of feedstuffs for finfish. Prog, fish Cult., 33: 132-134.

Smith, R.R., Peterson, M.C. , and Allred, A.C. , 1980. The effect of leaching on apparent digestion coefficients in determining digestibility and metabolizable energy of feedstuffs for salmonids. Prog. Fish - Cult., 42: 195 - 199.

Talbot, C , 1985. Laboratory methods in fish feeding and nutrition studies. In: Fish Energetics, New Perspectives. P. Tyler, P. Calow, and H . Croom (Eds), John Hopkins Univ. Press. Baltimore, M D . 349 pp

Viola, S., and Arieli, Y . , 1983. Nutrition studies with tilapia hybrids. The effect of oil supplements to practical diets for intensive culture. Bamidgeh, 35: 44 - 52.

Waldern, D.E., 1971. A rapid micro digestion procedure for neutral and acid detergent fibre. Canadian Journal of Animal Science, 51: 67-69 .

Watanabe, T., Takeuchi, T., Satoh, S., and Kiron, V. , (1996). Digestible crude protein in various feedstuffs determined with four freshwater fish species. Fisheries Science, 62: (2) 278 - 282.

Windell, J.T., Foltz J.W., and Sarokon J.A., 1978. Methods of fecal collection and nutrient leaching in digestibility studies. Prog. Fish-Cult., 40: (2) 51 - 55.

82

Chapter 4: Experiment 2: The feeding value and protein quality in high-fibre and fibre-reduced sunflower cakes and Kenya's "omena" fishmeal for tilapia {Oreochromis niloticus)

4.0 Abstract

This study was undertaken to assess the nutritive values of some locally available protein

sources in Kenya, as replacements for anchovy fishmeal in tilapia diets. The test protein

sources included were omena fishmeal made from Rastrineobola argentea, anchovy

fishmeal, as well as fibre-reduced and high-fibre sunflower cakes. The four protein

sources were each tested at two protein concentrations. O. niloticus fingerlings with an

initial weight of 16 g. were used for the study. Water temperature was maintained above

26°C throughout the trial and dissolved oxygen concentration in the tanks was maintained

above 5.5 mg/litre. Eight experimental diets, four based on fishmeal and four on

sunflower cake were formulated. In the diets based on the anchovy and omena fishmeals,

the fishmeals provided most of the the dietary protein, while in diets based on the

sunflower cakes, 50% of the protein was provided by each of the cakes while the

remaining 50% was provided mainly by anchovy fishmeal. Each diet contained one of

two levels of protein, viz., approximately 20% (low-protein) and 30%> (high-protein).

Further, each diet was fed to triplicate groups of fish for 78 days.

Diets based on the fibre-reduced cake had higher levels of all amino acids than the

ones based on the high-fibre cake. Lysine and threonine concentrations were lower in

diets based on the sunflower cakes than the ones based on the fishmeals. Dietary protein

level had a significant effect on growth rate and weight gain. Fish fed diets with 20%

protein gained less weight and had higher feed:gain ratios than those fed diets with 30%

protein. The source of protein had a significant effect on weight gain. Fish fed diets based

83

on anchovy fishmeal had higher weight gains than those fed diets based on the high-fibre

sunflower cake. Reducing the fibre content of sunflower cake improved growth rate and

weight gain, but the improvements in both of the parameters were not significant. Diets

based on omena fishmeal had lower protein concentrations than noted for the other diets

due to an error during mixing. Despite this, the growth rates and weight gains of fish fed

these diets were not significantly different from those of fish fed the anchovy fishmeal

diets.

84

4.1. Introduction and objectives

Tilapia (Oreochromis niloticus) are herbivorous fish that possess morphological and

physiological adaptations for utilization of diets high in fibre content. This aspect of its

feeding habits has not been fully exploited in commercial aquaculture. Most formulated

feeds for tilapia resemble those for omnivorous fish in that they contain significant levels

of animal proteins (Hughes and Handwerker, 1993). Much research has been done to

evaluate new protein sources to partially or wholly replace fishmeal in diets for all types

of fish. Among the plant protein sources, soybean meal has been used widely because of

its good amino acid profile, which, as the main protein source, supports the growth of

most fish species (Tacon et al., 1984; Wilson and Poe, 1985; Shiau et al., 1987; Viola and

Arieli, 1983). Soybeans, however, are not suitable for growing in many countries; hence

the need to evaluate other plant proteins sources.

Sunflower cake contains little or no known anti-nutritional factors. Sunflower is

cultivated extensively due to its adaptability to a wide range of climatic and soil

conditions (Ravindran and Blair, 1992). Its seeds are inexpensive to process, and the

cake remaining after oil extraction is used as a protein supplement in animal diets (Daghir

et al., 1980). The crude protein content of the cake ranges from 25 to 45% (air-dry basis)

depending on the extent of dehulling and the efficiency of the oil extraction process. The

crude fibre level in the cake generally varies between 14% and 39% (air-dry basis)

(Villamide and San Juan, 1998). Protein concentration in sunflower cake is inversely

proportional to the fibre content. Kenyan sunflower cakes contain 25% to 40% crude

protein, and 24% to 40% crude fibre (air-dry basis) (Jacobs, 1998). Differences in these

85

components among the various cakes tested were caused mainly by differences in seed

types and in the processing methods used.

The potential use of sunflower cake in fish diets is limited by its high fibre

content. Crude fibre not only has no known dietary value for fish, but it also dilutes

digestible nutrient densities, thus increasing the release of polluting wastes into the

environment. In view of the above, a fibre-reduced, high-fat sunflower cake was tested in

the present experiment as a replacement for fishmeal in tilapia feeds. A high-fibre

sunflower cake was also tested to determine the extent to which dietary fibre level would

influence fish performance. In addition to the sunflower cakes, Kenya's omena fishmeal

was also evaluated as a source of protein. From the first experiment (Chapter 3), it was

established that the digestibility of energy and nutrients in omena fishmeal produced

values comparable to those obtained with LT Anchovy meal. Furthermore, it was also

demonstrated that, at low levels of dietary inclusion (30% of the diet), there were no

significant differences in fish performance between fish fed the omena fishmeal diets and

those fed anchovy meal diets. In the present experiment, the feeding value of omena

fishmeal, and low-fibre and high-fibre sunflower cakes were evaluated at a higher level

of inclusion (providing 50% of the dietary protein), and over a longer period than in

Experiment 1 (Chapter 3).

The objectives of this study were to compare the nutritional values and protein

qualities of diets based on high-fibre and low-fibre sunflower cakes, and omena and

anchovy fishmeals when fed to tilapia (O. niloticus) at each of two levels of dietary

protein.

86

4.2 Materials and Methods

4.2.1 Experimental diets and design

The fibre-reduced cake used in this experiment was prepared as described in Experiment

1, Chapter 3. The high-fibre cake was from the same batch as used in Experiment 1.

The omena fishmeal was prepared by sun-drying "omena" fish (Rastrineobola argentea),

and then grinding them in a Wiley mill.

Eight diets whose compositions are shown in Table 4.2 were formulated. LT

anchovy fishmeal, omena fishmeal, high-fibre and fibre-reduced sunflower cakes were

used as sources of protein. In the diets based on anchovy and omena fishmeals, the

fishmeals provided most of the dietary protein, while in the diets based on sunflower

cakes, only 5 0 % of the dietary protein was provided by the cake, while the remaining

5 0 % was provided by anchovy fishmeal, as shown in Table 4.2. Each diet contained one

of two levels of protein, approximately 2 0 % or 30%. At each protein level, the diets were

formulated to contain similar levels of digestible energy by varying the level of corn oil.

In calculating the digestible energy contents of the diets, the apparent digestibility

coefficients for energy that were used were as follows; anchovy fishmeal, 8 6 % (Anderson

et al, 1991; Hanley, 1987), omena fishmeal, 7 8 % (present study), low-fibre sunflower

cake 44%o (present study), and high-fibre cake, 3 0 % (present study). The digestible

energy concentrations of cornstarch and corn oil were taken as 2700 kcal/kg D M (NRC

1993, for channel catfish), and 8100 kcal/kg D M (Santiago and Reyes, 1993)

respectively.

87

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their prescribed diets by hand to satiation three times daily.

4.2.2 Fish sampling

O. niloticus fry with an average weight of 16 g were transferred to the experimental

facilities and managed as in Experiment 1. They were acclimated to the experimental

tanks for a period of two weeks before the onset of the experiment. At the end of the two

weeks, they were weighed in groups of 25 fish that were selected at random, and then

they were allocated to the experimental tanks as described in Experiment 1. Water

temperature was maintained at 26 ±1°C, and dissolved oxygen concentration was

maintained above 5.5 mg/1. The natural photoperiod was followed (Nairobi, Kenya, 1°

16' S, 36° 48' E).

The duration of the experiment was 78 days. The fish were weighed on day 0,

day 38, and at the conclusion of the trial. They were starved for 24 hours before each

weighing, and each fish was weighed individually. Sampling of the fish for

determination of whole body compositions was done at the end of the experiment and in

this regard, five fish representative of each group (tank) were selected for this purpose.

They were killed with an overdose of MS222, and frozen at -5°C in plastic bags until

analyzed approximately a month later. During analysis, all five fish from each tank were

chopped into small pieces and thoroughly minced in a blender. They were analyzed for

their content of dry matter, crude lipid, crude protein, and ash respectively, according to

standard procedures (AOAC, 1984).

90

4.2.3 Data collection and analytical procedures

Fish growth and performance were assessed by calculating the following parameters:

initial and final absolute weights, weight gain, specific growth rates (SGR, % day_1)

which were calculated as follows: 100 [(In final wt (g) - ln initial wt (g))/number of

experimental days], feed consumption (g/fish), and feed conversion ratio (feed

consumption, g/wet weight gain, g). The protein quality parameters that were assessed

included: protein efficiency ratio (PER: wet weight gain, g/protein consumption, g), and

productive protein value (PPV: 100*(gain in body protein/protein intake)).

4.2.4 Chemical analyses

All ingredients and feed samples were ground using a Wiley mill with a 1mm

sieve, and subsequently they were stored in sealed containers at room temperature. The

standard procedures (AOAC, 1984) were used to determine the various proximate

fractions. Al l analyses were carried out in duplicate. Calcium was determined by atomic

absorption spectroscopy at U B C (Perkin-Elmer, model 2380), while phosphorus was

determined colorimetrically using a Beckman Model Du-8B spectrophotometer at 450

nm wavelength. Samples for the analyses of calcium and phosphorous were digested by

wet ashing. Amino acid analyses of the feed samples were conducted at the University of

Alberta. Performic acid oxidation was done prior to hydrolysis, to oxidize cystine and

methionine to cysteic acid and methionine sulfone, respectively (AOAC, 1998). Sodium

metabisulfite was added to to neutralize the performic acid. Amino acids were released

from protein by hydrolysis with 6 N HCL. Hydrolysed samples were diluted with sodium

citrate buffer and pH adjusted to 2.2. Individual amino acids were quantified using a

91

HPLC. Tyrosine was destroyed by oxidation and tryptophan by hydrolysis, and they

could not be determined this way.

4.2.5 Statistical analyses

The data were analyzed using PROC G L M of the SAS Statistical Package (1985). The

means were compared using Tukey's test with level of significance set at P < 0.05. All

parameters were analyzed as a 4 x 2 factorial design (4 protein sources at 2 levels of

protein intake). Data on body composition parameters were analyzed by conducting

analyses of covariance with the fish weight as the covariate (Shearer 1994).

The statistical model employed in the analyses of the various fish performance

parameters was;

Y = n + Si + Pj + (SP)ij + e i j k

Where Y = Observation, e.g. weight gain, feed intake, growth rate, etc.

u = overall mean

Si = effect of the source of protein

Pj = effect of the protein level

(SP)ij = effect of interaction between source of protein and protein level

e;jk = error term

Means were compared using Tukey's multiple range test and the level of significance was

set at (P < 0.05). All data on body composition parameters were analyzed by conducting

analyses of covariance with the fish weight as the covariate (Shearer 1994).

92

4.3 Results and discussion

4.3.1 Chemical composition of the diets

The chemical compositions of the diets used in this experiment are presented in

Table 4.2. The low-protein diets were formulated to contain a D E concentration of 2800

kcal/kg and a protein content of about 20% (air-dry basis). The high-protein diets were

formulated to contain a D E concentration of 3000 kcal/kg, and a crude protein content of

30%> (air-dry basis). Calculated D E concentrations in low-protein diets ranged from 2751

kcal/kg ( D M basis) in the diet based on high-fibre sunflower cake (HFSC-20), to 2965

kcal/kg ( D M basis) in the diet based on anchovy fishmeal. The calculated D E

concentrations in the high-protein diets ranged from 2797 kcal/kg to 3077 kcal/kg (DM

basis). The lower D E concentrations of the diets based on the high-fibre sunflower cake

were lower than in the other diets due to an over-estimation of the D E concentration of

the cake during formulation of the diets. The D E concentrations of the sunflower cakes

and omena fishmeal were calculated based on the results of the digestibility study in

chapter 3. Later on, it became necessary to recalculate the digestibility coefficients for

energy and the digestible energy concentrations of the ingredients (Experiment 1, Chapter

3). The high-fibre sunflower cake had lower levels of digestible energy than had been

assumed during formulation of the diets. The low protein diets were formulated to

contain less D E concentration than the high-protein diets in order to minimize differences

in energy:protein ratios between the diets at the two protein levels. The stipulated D E

requirement for tilapia (O. niloticus) is 3000 kcal/kg D M (NRC, 1993). The calculated

D E concentrations in most of the diets (except the ones based on anchovy fishmeal) were

slightly below this level.

93

The determined crude protein levels in the diets based on omena fishmeal at both

high and low protein levels (O-20 and 0-3 0), were slightly lower than the values found

for the other diets. It is not clear why this situation arose. It may have been caused by an

error during the mixing of the diets. The fibre-reduced cake was rich in oil and

consequently diets made from this cake (LFSC-20 and LFSC-30) had a high oil content.

In diets based on the fishmeals, the ADF and NDF values reflected the amount of a-

cellulose added to the diets. Despite the fact that the high-fibre sunflower cake had

higher ADF and NDF contents than the fibre-reduced cake, this was not clearly reflected

in the diets at the low protein level because a-cellulose was added to the diet based on the

fibre-reduced cake, thus increasing its ADF and NDF contents.

In the diets where fishmeal was partially replaced by the sunflower cakes,

phosphorus was balanced by the addition of dicalcium phosphate. Extensive replacement

of animal proteins that contain high levels of phosphorus by plant proteins may result in

phosphorus deficiency (Viola et al, 1988). This is because plant proteins contain phytate

phosphorus which is not available to tilapia. Gur (1996) stated that a phosphorus level of

0.6%- 0.7%) of the diet (air-dry basis), supplied from dicalcium phosphate, animal by­

products, or a combination of both is adequate to meet the minimum demand of tilapia

for inorganic phosphorus. Most of the phosphorus in the diets based on sunflower cake

was supplied by anchovy fishmeal and dicalcium phosphate. Determined levels of

phosphorus in the diets were all above 1%> (DM basis).

The amino acid profiles of the diets are shown in Table 4.3. When the amino

acids were expressed as a percentage of the dietary protein, diets based on the tw

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fishmeals had a profile that was almost similar. The diets based on the two sunflower

cakes had lower levels of lysine and threonine than the diets made from the fishmeals.

Among the diets made from sunflower cake, the ones based on fibre-reduced

sunflower cake (FRSC-20 and FRSC-30) had higher levels of all the determined amino

acids than the ones based on the high-fibre cake (HFSC-20 and HFSC-30). Villamide

and San Juan (1998) evaluated three sunflower cakes containing different crude protein

levels, and observed that amino acids (%DM) decreased with decreasing protein content

in the cakes. Similarly, Green and Kiener (1989) compared high-fibre and partially

dehulled sunflower cakes that had crude protein contents of 31% and 36%> respectively,

and observed that dehulling increased the concentration (% DM) of amino acids in the

cake. The findings in the present experiment are consistent with these observations made

by Green and Kiener (1989).

When the dietary amino acids were expressed as a percentage of the diet, the diets

with a crude protein content of 20% did not meet tilapia requirements for most of the

essential amino acids except leucine and valine. It must be cautioned at this point that

strict comparisons between the requirements as stipulated in N R C (1993) may not be

appropriate because the N R C figures quoted are for juvenile tilapia weighing less than 1

gram. The fish used in this study had an initial weight of about 16 g. In fish, as in all

animals, protein and consequently amino acid requirements (as percentage of the diet)

decrease as the animal grows. At a protein level of 30%, diets based on omena fishmeal,

anchovy fishmeal, and fibre-reduced sunflower cake met all the essential amino acid

requirements for tilapia (NRC, 1993). The lysine levels were lower in the sunflower

96

cake diets than in the diets based on fishmeal at both protein levels. Lysine has been

identified as one of the limiting amino acids in sunflower cake (McGinnis et al, 1948;

Klain et al., 1956). Methionine and cystine levels in the fibre-reduced sunflower cake

were almost similar to the levels in the fishmeal diets. Jackson et al. (1982) observed that

sunflower cake had high levels of methionine and cystine compared to other plant

proteins.

4.3.2 Fish performance, PER and PPV

The effects of the dietary protein level and protein source on absolute weights,

weight gains, growth rates, and feed and protein utilizations after 78 days of feeding on

the various diets are shown in Tables 4.4 and 4.5, respectively. The effects of both

protein level and protein source on the various parameters are shown in Table 4.6. At

the start of the study, the mean weights of fish fish in all the groups were not significantly

different (P > 0.05). After 78 days, the fish fed the diets with high protein contents had

higher weight gains and growth rates (P < 0.05) than those fed the diets with low protein

contents (Table 4.6). The interaction between protein level and protein source was not

significant for any of the parameters (Table 4.6). The growth rates of the fish increased

in direct relation to the

dietary protein level. Feed intake was not significantly affected by dietary protein level,

but feed utilization was better for fish fed the high protein diets than the low protein diets.

Protein utilization (PER and PPV) decreased at the higher level of protein intake. The

analyzed protein content of the low-protein diets (DM basis) ranged from 20% to 23.6%,

while in the high protein diets, the range was 29% to 33.8% (DM basis). Protein intake

(feed intake x protein concentration) was therefore lower for fish receiving the

97

Table 4.4: Effect of protein level on fish performance

Protein level Low-protein High-protein SEM

Final weight (g/fish)1 51.40b 57.10" 0.96 Wt gain (g/fish)2 35.00b 40.40" 0.90 Specific growth rate (% per day)2 1.47b 1.58a 0.03 Feed intake (g/fish)2 76.703 75.30a 0.47 FCR (Feed:gain ratio)2 2.20a 1.87b 0.04 PER 2 2.16a 1.69b 0.02 PPV 2 39.44a 32.21b 0.36

Means with a different superscript for each factor in a row are significantly different (P < 0.05) 1 Means (n = 300) (Individual fish weights were used, 4 diets x 75 fish/diet) 2 Means (n = 12) PER = protein efficiency ratio PPV = productive protein value

98

Table 4.5: Effect of source of protein on fish performance.

OM ANC Fibre-reduced High-fibre SEM Diets fishmeal fishmeal sunfl. cake sunfl.cake

Final weight (g/fish)1 52.40b 57.70" 54.90ab 52.00b 1.34 Weight gain (g/fish)2 35.92ab 40.86" 38.73ab 35.39b 1.27 Sp. growth (% per day)2 1.48 1.59 1.57 1.47 0.04 Feed consumption (g/fish)2 77.10 FCR (feed: gain ratio)2 2.17

75.90 76.10 74.90 0.66 Feed consumption (g/fish)2 77.10 FCR (feed: gain ratio)2 2.17 1.87 1.98 2.12 0.07 PER 2 2.01 1.98 1.86 1.84 0.06 PPV 2 38.90" 37.00ab 35.14ab 32.30b 1.27

Means that do not have a superscript or share a common superscript letter for each factor within a row, are not significantly different (P > 0.05) 'Means (n = 150) (Individual fish weights used, 2 diets x 75 fish/diet) 2Means (n= 6)

OM - Omena ANC - Anchovy PER = protein efficiency ratio; PPV = productive protein value

99

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low-protein diets, compared to the ones ingesting the high-protein diets. In tilapia (O.

niloticus), Twibell and Brown (1998) and Bowen et al. (1995) observed that at low

protein intake, increasing the protein level of the low protein diets resulted in

proportional improvements in weight gain and feed conversion ratios up to 30% (DM

basis) level in the diet.

A wide range of estimates have been reported for the optimal dietary protein

content of tilapia feeds. Winfree and Stickney (1981) reported that 56% dietary crude

protein level promoted maximum weight gain of tilapia (0. aureus) weighing 2.5g, while

in those weighing 7.5 g., 34% dietary crude protein was adequate. Shiau and Huang

(1989) reported 24% crude protein as the optimum for tilapia (0.niloticus x O. aureus)

weighing 2.9 g., and fed on purified diets with dietary protein levels ranging from 0% to

56%. Weight gain was proportional to the dietary protein content up to a protein level of

24%. At higher protein levels, there was no increase in weight gain or protein gain.

Luquet (1991) reviewed several studies and recommended a protein content of 30 - 35%

as the optimum for tilapia. This recommendation was based on studies that utilized good

protein sources such as fishmeal and casein. N R C (1993) gives the protein requirement

as 30% of the diet ( D M basis). Estimates of optimal dietary crude protein concentrations

have typically been within the range of 30-35% for tilapia weighing less than 5 g (Mazid

et al., 1979; Jauncey, 1982; Siddiqui et al., 1988). The stated requirements for protein

show a wide variation, reflecting the different environmental conditions in which the

experiments were done, and the different fish and feed factors involved. There is

evidence that the optimal protein requirement for tilapia is inversely related to fish size.

In studies by Twibell and Brown (1998) using (O. niloticus x O. aureus) fingerlings with

101

an initial weight of 21 g., there was no improvement in growth at dietary protein levels

higher than 28%. The initial weight of the fish used in this study was 16 g., which was

close to the starting weight of the fish employed in the latter study.

The source of protein (omena fishmeal, anchovy fishmeal, fibre-reduced and

high-fibre sunflower cakes) had a significant effect on the final fish weights, weight

gains, and PPV values (Table 4.5), but not on growth rates, feed intakes, feed utilization

and PER values. Fish fed diets based on anchovy fishmeal and the fibre-reduced

sunflower cake had an 8% and 7% improvement in growth over those fed diets based on

the high-fibre sunflower cake, but the differences were not significant (P < 0.05).

Fish fed diets based on the high-fibre cake gained less weight (P < 0.05) over the

whole experiment than fish receiving the diets based on anchovy fishmeal (Table 4.5).

As noted above, the growth rate was also lower for these fish than for those fed diets

based on omena fishmeal and the fibre-reduced sunflower cake, but the differences were

not significant. Fish fed diets based on the high-fibre cake tended to have lower feed

intake, and a higher feed:gain ratio compared to those fed on the other three diets, but

again the differences were not significant.

Tilapia, like other fish, consume organoleptically acceptable diets in an attempt to

satisfy energy demands. The D E concentration in all the diets was almost similar, which

may explain the similarity in feed intake. Diets based on the high-fibre cake had a

slightly lower D E concentration compared to the other diets. Despite this, the fish fed on

these diets did not increase their feed intakes to compensate for the lower dietary D E

content. Residual hulls in the high-fibre sunflower cake diets (FTF-SC20 and FTF-SC30)

may have hindered this compensatory increase in feed intake. The percentages of high-

102

fibre sunflower cake were 36% and 54% for the low-protein and high-protein diets,

respectively. The weight gain of the fish fed the diets containing the high-fibre sunflower

cake was significantly lower than for the fish fed on the anchovy fishmeal-based diets.

This may be attributed to the lower digestibility of the diet, or the fact that essential

amino acids lysine, phenylalanine and threonine in that diet did not meet tilapia

requirements for these amino acids as stipulated in NRC (1993).

a-Cellulose was used as an inert filler in the diets based on omena and anchovy

fishmeal at both protein levels. Different components of dietary fibre vary in their

chemical and physiological properties and thus have different effects on physiological

functions. Studies on the utilization of fibre in fish have yielded varying results. In sea

bream, Morita et al. (1982), studied the effect of carboxymethylcellulose (CMC) on

carbohydrate utilization by adding 0 to 12% C M C to diets containing 10%, 20%, or 30%

dextrin. They noted that C M C supplementation improved weight gain and feed

conversion ratio. It was not clear from the study what the mechanism of improvement

was. It could be postulated that C M C , which is a water-soluble fibre, formed a highly

viscous solution, that slowed the flow of the digesta, leading to a lower gastric emptying

time, and thus more time for absorption of the nutrients in the intestine. Cellulose, unlike

C M C , is an insoluble fibre which has been reported to increase gastric emptying time in

rainbow trout (Hilton et al., 1983). In channel catfish (Ictalurus punctatus) reared on

purified diets, Dupree and Sneed (1966) reported growth improvement when 21% a-

cellulose was added to these diets. Indeed, N R C (1977) indicates that it is desirable to

have some fibre in semi-purified test-diets for catfish to add structural integrity to

pelleted diets. The fibre level however, should not exceed 8% of the diet.

103

Contrary to these findings, Anderson et al. (1984), working with tilapia (O.

niloticus), observed that growth was depressed when diets contained more than 10% a-

cellulose. In the study by Anderson and co-workers, a-cellulose was added at 10% to

40% of the diet at the expense of glucose, sucrose, dextrin or starch and no attempt was

made to balance the digestible energy content in the diets. The fish were also fed at 3%

of their body weight daily. Since cellulose is not digestible by fish, diets that contained

the cellulose had less digestible energy content than the diets that were based on

carbohydrates. In studies by Dioundick and Storm (1990) with tilapia (O. mossambicus),

the best growth rates and feed utilization values were obtained with diets containing 2%

to 5% supplemental fibre as a-cellulose. Fish fed on cellulose-free diets or on diets that

contained 10% a-cellulose demonstrated reduced growth. In that study, a-cellulose was

added at the expense of cornstarch, and the fish fed at 6% of their body weight twice a

day. Feed intake was higher for fish fed diets containing 7.5% and 10% a-cellulose

compared to those fed on diets containing 0, 2.5% and 5% a-cellulose. Despite the

higher feed intake, the fish fed on diets containing 10% a-cellulose had reduced growth

rate compared to those fed diets containing 2.5% and 5% a-cellulose.

Hence, the fish responded differently to the inclusion of dietary fibre in the

various studies quoted above. In most of the studies, increasing the cellulose levels in the

diet resulted in a reduction of digestible energy concentration. The other explanations for

the different observations are likely related to the amount of feed offered to the fish and

the number of feedings per day. As explained earlier, fish respond to low dietary energy

intake by increasing feed intake if given the opportunity. In studies by Hilton et al.

(1983) with trout, fish fed ad libitum with diets containing 10% and 20% a-cellulose,

104

were able to increase feed intake so that the nutrient levels consumed were similar to

those of the control fish (no cellulose added). On the other hand, when they were fed on

a restricted feeding regime (3% of body weight), feed intake was markedly less than that

of the control fish. The other factor that may have affected the utilization of fibre in the

experiments quoted above may have been the dissimilar fish sizes that were used.

Jackson et al. (1982), used fish sizes ranging from 13 g to 50 g to test utilization of

various plant proteins (copra, groundnut, soya, sunflower, rapeseed, cottonseed and

leucaena meals) in complete diets for O. mossamhicus, and showed that tilapia could

effectively utilize diets containing relatively high levels of fibre, compared to other fish.

In that study, fish fed diets containing 13% crude fibre (from natural sources) performed

as well as those fed the control diet.

The initial weight of the fish in the present study was about 16 g, while the final

average weight, after 78 days, was approximately 50g. The digestible energy

concentration was not appreciably different between the different diets, and the fish were

fed ad libitum. Feed intake did not vary significantly between fish fed the various diets.

Fish on the high-fibre sunflower cake diets showed a trend to a reduced feed intake. PER

was significantly affected by dietary protein level, but not by protein source (table 4.5).

Fish fed diets containing 20% protein had higher PERs than those fed on diets containing

30%> protein. Protein source did not significantly affect PER, although fish fed diets

based on the two fishmeals tended to have higher PER values. PER is a measure of

protein quality that is presented as a ratio of gain/protein intake, and is affected by the

level of protein in the diet, the digestibility of the protein, and the levels of essential

amino acids, particularly the first limiting amino acid. Within each protein level, dietary

105

were able to increase feed intake so that the nutrient levels consumed were similar to

those of the control fish (no cellulose added). On the other hand, when they were fed on

a restricted feeding regime (3% of body weight), feed intake was markedly less than that

of the control fish. The other factor that may have affected the utilization of fibre in the

experiments quoted above may have been the dissimilar fish sizes that were used.

Jackson et al. (1982), used fish sizes ranging from 13 g to 50 g to test utilization of

various plant proteins (copra, groundnut, soya, sunflower, rapeseed, cottonseed and

leucaena meals) in complete diets for O. mossambicus, and showed that tilapia could

effectively utilize diets containing relatively high levels of fibre, compared to other fish.

In that study, fish fed diets containing 13% crude fibre (from natural sources) performed

as well as those fed the control diet.

The initial weight of the fish in the present study was about 16 g, while the final

average weight, after 78 days, was approximately 50g. The digestible energy

concentration was not appreciably different between the different diets, and the fish were

fed ad libitum. Feed intake did not vary significantly between fish fed the various diets.

Fish on the high-fibre sunflower cake diets showed a trend to a reduced feed intake. PER

was significantly affected by dietary protein level, but not by protein source (table 4.5).

Fish fed diets containing 20% protein had higher PERs than those fed on diets containing

30% protein. Protein source did not significantly affect PER, although fish fed diets

based on the two fishmeals tended to have higher PER values. PER is a measure of

protein quality that is presented as a ratio of gain/protein intake, and is affected by the

level of protein in the diet, the digestibility of the protein, and the levels of essential

amino acids, particularly the first limiting amino acid. Within each protein level, dietary

105

protein content was not appreciably different between diets, while the digestibility of

protein in fishmeal and sunflower cakes was similar (Experiment 1, Chapter 3).

Consequently, the observed trends for PER for fish ingesting the diets containing the

various protein sources likely resulted from differences in amino acid levels (particularly

lysine) between the diets based on fishmeals and those based on sunflower cakes.

The trend observed for PPV in relation to diet treatment was similar to PER. Fish

fed the diets containing the 20% protein level had significantly higher PPV values than

those fed the diets containing 30% protein (Table 4.4). Protein source also had a

significant effect on PPV. Fish fed on the high-fibre sunflower cake diets had

significantly (P < 0.05) lower values than those fish fed on the diets based on the omena

fishmeal. PPV, like PER, is sensitive to dietary protein level. Diets based on omena

fishmeal had slightly lower protein levels compared to the other diets and this may have

contributed to the observed differences. The differences would also have resulted from

differences in the dietary levels of essential amino acids, particulary lysine, which was

low in the diets based on the high-fibre sunflower cake.

4.3.3 Effect of diets on whole body composition.

Dietary treatment had no significant effect on whole body proximate composition (Table

4.7). There was a trend for fish fed the diets with anchovy fishmeal at the 20%> and 30%>

dietary protein level to have higher moisture and lower lipid contents than fish fed the

other diets. Generally, fish fed diets high in fat showed a trend to higher lipid levels,

106

protein content was not appreciably different between diets, while the digestibility of

protein in fishmeal and sunflower cakes was similar (Experiment 1, Chapter 3).

Consequently, the observed trends for PER for fish ingesting the diets containing the

various protein sources likely resulted from differences in amino acid levels (particularly

lysine) between the diets based on fishmeals and those based on sunflower cakes.

The trend observed for PPV in relation to diet treatment was similar to PER. Fish

fed the diets containing the 20% protein level had significantly higher PPV values than

those fed the diets containing 30% protein (Table 4.4). Protein source also had a

significant effect on PPV. Fish fed on the high-fibre sunflower cake diets had

significantly (P < 0.05) lower values than those fish fed on the diets based on the omena

fishmeal. PPV, like PER, is sensitive to dietary protein level. Diets based on omena

fishmeal had slightly lower protein levels compared to the other diets and this may have

contributed to the observed differences. The differences would also have resulted from

differences in the dietary levels of essential amino acids, particulary lysine, which was

low in the diets based on the high-fibre sunflower cake.

4.3.3 Effect of diets on whole body composition.

Dietary treatment had no significant effect on whole body proximate composition (Table

4.7). There was a trend for fish fed the diets with anchovy fishmeal at the 20% and 30%

dietary protein level to have higher moisture and lower lipid contents than fish fed the

other diets. Generally, fish fed diets high in fat showed a trend to higher lipid levels,

106

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but the results were not significant. Protein and ash percentages were fairly constant

between fish fed on the different diets, and were not affected by the diets.

In his review on factors that affect composition of farmed fish, Shearer (1994)

stated that the levels of body protein are life cycle and size dependent. He further stated

that data on whole body proximate composition should be subjected to an analysis of

covariance to remove the effect of size. The average fish weights in the current study

ranged from 47.82 grams to 60.66 grams at the end of the experiment. Van der Meer.

(1995) observed that there were significant differences in whole body protein percentage

between fish weighing 5 grams and those weighing 50 grams, but the differences were

not significant between fish weighing 50 grams and 150 grams. It could be postulated

from the study above that there is a certain size where fish attain "chemical maturity" and

where further increases in size do not affect whole body protein composition.

Trends in whole body lipid concentration followed trends in dietary lipid content.

Fish fed high fat diets tended to accumulate more body lipid compared to ones fed on low

fat diets, regardless of the D E concentrations of the diets. The differences between

groups however, were not statistically significant. Shearer (1994) stated that whole body

lipid stores are more influenced by energy intake than by dietary lipid levels.

Recently, the utilization of dietary lipids by tilapia has been the subject of study

by various authors. De Silva et al. (1991) for example, observed that the addition of

lipids to diets of tilapia fry (mean weight, 1 g.), increased growth rate, and that the best

growth response was elicited with diets containing 18% lipid. They also observed that

whole body fat concentration was directly proportional to dietary lipid content. The diets

used in their study were isocaloric. Hanley (1991) on the other hand, using O. niloticus

108

fingerlings (average initial weight, 42 g) noted that increasing dietary lipid level above

9% did not result in any increase in growth. Also, the fish had significantly higher

carcass lipid concentrations than those fed the diets containing 5% fat.

The lack of fish growth response to an increase in dietary lipid content in the latter

study could be attributed to the large size of the fish used and the fact that the experiment

was carried out in outdoor ponds where there was substantial natural productivity. It

could also be postulated that tilapia can effectively utilize both fat and carbohydrates as

energy sources. In most of the studies quoted above, fat was added as a replacement for

carbohydrates in isocaloric diets. Chou and Shiau (1996) fed hybrid tilapia (O. niloticus

x O. aureus) on isocaloric and isonitrogenous diets containing 0% to 20% fat and

observed that fish fed diets with 5%, 10% and 15% lipid, had similar weight gains,

although whole body lipid concentrations increased as dietary lipid content was

increased. Fish that were fed high dietary lipid levels had higher percentages of body

lipid and lower percentages of body moisture than fish fed the low lipid diets.

An interesting point in lipid nutrition in tilapia is the fat distribution in the body.

Despite the fact that tilapia have a tendency to store much of the dietary fat, they have

been described as lean fish compared to carp (Viola et al., 1988). Viola et al. (1988) and

Hanley (1991) observed that 40% of the body fat in tilapia was distributed around the

viscera while the muscles contained only 8% of the total body fat. This observation is

opposite to the pattern of fat distribution in carp, where fat distribution between the

viscera and the muscle are almost equal.

109

4.4 Conclusions

The low protein diets were formulated to contain a CP level of approximately 20% (DM

basis) and a D E concentration of 2800 kcal/kg (DM), while the high protein diets were

formulated to contain a CP level of 30% (DM basis) and a D E concentration of

approximately 3000 kcal/kg D M . The D E concentrations in the diets based on the high-

fibre sunflower cake at both protein concentrations were lower than observed in the other

diets. This occured due to an over-estimation of the D E energy concentration in that cake

during the formulation of the diets. Diets based on the omena fishmeal at the 30% and

the 20% protein levels had a low determined protein concentration compared to the other

diets, which may have been caused by an error during the formulation or mixing of the

diets.

There was no significant interaction between protein level and protein source.

Protein level had a significant effect on weight gains and growth rates. Generally, fish

fed diets at the 20% protein level gained less weight and had higher feed:gain values

compared to the ones fed diets at the 30% protein level.

The source of protein had a significant effect on weight gain, but not on specific

growth rate, feed intake, or feed utilization. Fish fed diets based on anchovy fishmeal

had higher weight gains than those fed diets based on omena fishmeal, fibre-reduced cake

and the high-fibre cake respectively, but the differences were only significant relative to

the fish fed the diets based on the high-fibre sunflower cake. Results from this study

showed that omena fishmeal could supply all the protein in tilapia diets, while the fibre-

reduced sunflower cake could provide up to 50% of the dietary protein.

110

Fish fed diets based on the fibre-reduced cake had a mean weight gain of 38.75 g,

and a specific growth rate of 1.57% per day, while those fed on the high-fibre sunflower

cake had an average weight gain of 35.4 g and a specific growth rate of 1.47% per day.

Reducing the fibre content of sunflower cake improved weight gain and growth rate, but

the improvement was not significant in any of the parameters tested (P < 0.05). The

failure of the statistical test used to detect significant differences despite the relatively

large absolute differences may have been caused by the small sample size used in the

tests. Most of the parameters were tested using a mean of 3 replicates (n = 3), except for

the final fish weights where individual fish weights were used (n = 75). The effect of

sample size is illustrated in Table 4.5, where there was a significant difference in absolute

weights (n = 75) between fish fed the anchovy and the omena fishmeal diets, yet weight

gain (n = 3) was not significantly different, despite the fish having a similar initial weight.

Diets with a crude protein content of 20% did not meet tilapia requirements for

most of the essential amino acids except leucine and valine. At a protein level of 30%,

diets based on omena fishmeal and anchovy fishmeal met all the essential amino acid

requirements for tilapia (NRC, 1993). The diet based on the fibre-reduced sunflower

cake at this protein level had a slightly lower level of methionine, while the diet based on

the high-fibre cake had lower levels of lysine, methionine and threonine.

Lysine and threonine levels were low in diets based on the sunflower cakes,

compared to the fishmeal diets. Levels of the other essential amino acids in the fibre-

reduced sunflower cake were comparable to those in the fishmeal diets.

Ill

PER was significantly affected by dietary protein level, while PPV was affected

by both dietary protein level and protein source. Fish fed diets based on the high-fibre

sunflower cake had lower PPV values compared to those fed the omena fishmeal diets.

112

4.5 References Anderson, J., Capper, A J . , Matty, A.J., and Capper, B.S., 1984. Effects of dietary carbohydrate and fibre on the tilapia (Oreochromis niloticus - Linn). Aquaculture, 37: 303-314.

Anderson, J., Capper, B.S., and Bromage, N R . , 1991. Measurement and prediction of digestible energy values in feedstuffs for herbivorous fish tilapia (Oreochromis niloticus-Linn). British Journal of Nutrition, 66: 37-48.

AO A C , 1984. Association of Official Analytical Chemists. Official Methods of Analyses Animal Feeds section. 1141 pp.

A O AC, 1998. Official Methods of Analyses of AO A C International, 16 th Edition. AO A C Official Method 994.12. Amino acids in Feeds (CD ROM)

Bowen, S.H., Lutz, E .V. and Ahlgren, M.O., 1995. Dietary protein and energy as determinants of food quality. Ecology, vol 76: 3.

Chou, Ben-Shan., and Shiau, Shi-Yen., 1996. Optimal dietary lipid level for growth of juvenile hybrid tilapia, (Oreochromis niloticus X O. aureus). Aquaculture, 143: 185-195.

Daghir, N.J. , Ras, M.A . , and Wwayjan M . , 1980. Studies on the utilization of full fat sunflower seed in broiler rations. Poultry Science, 59: 2273-2278.

Desilva, Sena S., Rasanihi, M.G. , and Shim, K.F. , 1991. Interactions of varying dietary protein and lipid levels in young red tilapia. Evidence of protein sparing. Aquaculture, 95: 305-318.

Dioundick, O. B. , and Stom, D.L., 1990. Effects of dietary a cellulose levels on the juvenile tilapia, (O. mossambicus, Peters). Aquaculture, 91: 311-315.

Dupree, H.K., and Sneed, K .E . , 1966. Responses of channel catfish to different levels of major nutrients in purified diets. U.S. Bull of Sport Fish. Wildlife Technical paper 9: 21pp

Green, S., and Kiener, T., 1989. Digestibilities of nitrogen and amino acids in soya-bean, sunflower and meat and rapeseed meals measured with pigs and poultry. Anim. Prod., 48: 157-179.

Gur, N . , 1996. Minerals in tilapia nutrition - the protein phosphorus connection. In Proceedings of the intensive aquaculture workshop. Shefayim Israel March 9 t h - April 16 th 1996.

113

Hanley, F., 1987. The digestibility of feedstuffs and the effect of selectivity on digestibility determinations in tilapia (Oreochromis niloticus). Aquaculture, 66: 163-179.

Hanley, F., 1991. Effect of feeding supplementary diets containing varying levels of lipids on growth, food conversion and body composition of Nile tilapia (Oreochromis niloticus). Aquaculture, 93: 323-334.

Hilton, J.W., Atkinson, J.L., and Slinger, S.J., 1983. Effect of increased dietary fibre on the growth of rainbow trout Salmo gairdneri. Can. J. Fish Aquacult, Sci., 40: 81-85.

Hughes, S.G., and Handwerker, T.S., 1993. Formulating for tilapia: Al l vegetable protein feeds. Feed International, September, 1993. 55 -60 .

Jacob, J.P., 1993. The feeding value of Kenyan sorghum, sunflower seed cake, and sesame seed cake for poultry. Ph.D. Thesis, The University of British Columbia.

Jackson, A.J., Capper, B.S., and Matty, A.J., 1982. Evaluation of some plant proteins in complete diets for the tilapia (Oreochromis mossamhicus). Aquaculture, 27: 97-109.

Jauncey, K. , 1982. The effects of varying dietary protein level on the growth, food conversion, protein utilization and body composition of juvenile tilapias (Oreochromis mossamhicus). Aquaculture, 27: 43-54.

Klain, G.J., Hill , D . C , Branion, H.D., and Gray, J.A., 1956. The value of rapeseed oil meal and sunflower oil meal in chick starter rations. Poultry Science 35: 1315-1326.

Luquet, P., 1991. Tilapia Oreochromis sp. In: Handbook of nutrient requirements of finfish. R P Wilson (Ed). CRC Press. Boca Raton, Florida, USA. 196pp

Mazid, M.A . , Tanaka, Y. , Katayama, T., Rahaman, M.A. , Simpson, K . L . , and Chichester, C O . , 1979. Growth response of tilapia zilli fingerlings fed isocaloric diets with variable protein levels. Aquaculture, 18: 115-122.

McGinnis, J., Hsu, P.T., and Carver, J.S., 1948. Nutritional deficiencies of sunflower oil meal for chicks. Poultry Science, 27: 389-393.

Morita, K. , Furuichi, M . , and Yone, Y. , 1982. Effects of carboxymethylcellulose supplemented to dextrin containing diets on the growth and feed efficiency of red sea bream. Bull. Jap. Soc. Sci. Fish., 48: 1617-1620.

NRC, 1977. Nutrient Requirements of Warmwater Fish. In: Nutrient Requirements of Domestic Animals Series, National Academy Press, Washington, D . C , 78pp.

NRC, 1993. Nutrient Requirements. In: Nutrient Requirements of Fish. National Academy Press. Washington, D.C. pp 114.

114

Ravindran, V. , and Blair, R., 1992. Feed resources for poultry production in Asia and the Pacific. Plant protein sources. World's Poult Sci. J., 48: 205-231.

Santiago, C.B., and Reyes, S.O., 1993. Effects of dietary lipid source on the reproductive performance and tissue lipid levels of Nile tilapia (Oreochromis niloticus) (linnaeus) broodstock, J. Appl. Ichthyology, 9: 33-40.

SAS Users Guide: Statistics, Version 5 Edition. 1985. SAS Inst., Inc., Caary, NC.

Shiau, S.Y., and Huang, S.L., (1989) Optimal dietary protein level for hybrid tilapia (O. niloticus x O. aureus), reared in seawater, Aquaculture, 81: 119-

Shiau, S.Y., Kwok, C C , Chen, C I , Hong, H.T., and Hsieh, H.B., 1989. Effects of dietary fibre on the intestinal absorption of dextrin, blood sugar level and growth of tilapia (Oreochromis niloticus x O. aureus). J. Fish Biol., 34: 929-935.

Shiau, Shi-Yen., Chuang, Jan-Lung., and Sun, Chan-Lan , 1987. Inclusion of soybean meal in tilapia (Oreochromis niloticus x O. aureus) at two protein levels. Aqaculture, 65: 251-261.

Shearer, K .D. , 1994. Factors affecting proximate composition of cultured fishes with emphasis on salmonids. Aquaculture, 119: 63-88.

Shiau, S. Y. , and Huang, S.L., 1989. Optimal dietary protein level for hybrid tilapia (Oreochromis niloticus X O. aureus) reared in sea-water. Aquaculture, 81: 119-127.

Siddiqui, A.Q., Howlader, M.S., and Adam, A. A., 1988. Effect of dietary protein levels on growth, feed conversion, and protein utilization in fry and young Nile tilapias (Oreochromis niloticus). Aquaculture, 70: 63-73.

Tacon, A.G.J. , Webster, J.L., and Martinez, C.A., 1984. Use of solvent extracted sunflower seed meal in complete diets for fingerling trout (Salmo gairdneri). Aquaculture, 43: 381-389.

Twibell, R.G., and Brown P.B., 1998. Optimal dietary protein concentration for hybrid tilapia (Oreochromis niloticus x 0. aureus) fed an all plant diet. World Aquaculture Society, Vol. 29 1 9-16.

Van der Meer, M B . , 1995. The effect of dietary protein levels on growth, protein utilization, and body composition of Colossoma macropomum (Cuvier). Aquaculture Research, 26: 901-909.

Villamide, M.J . , and San Juan L.D. , 1998. Effect of chemical composition of sunflower seed meal on its true metabolizable energy and amino acid digestibility. Poultry Science, 77: 1884-1892.

115

Viola, S., and Arieli, Y . , 1983. Nutrition studies with tilapia. Replacement of fishmeal by soybean meal in feeds for intensive tilapia culture. Bamidgeh, 35. 1 9-17.

Viola, S., Arieli Y . , and Zohar, G., 1988. Animal protein free feeds for hybrid tilapia (Oreochromis niloticus x O. aureus) in intensive aquaculture. Aquaculture, 75 (1-2) 115-125.

Wilson, R.P. and Poe, W.E., 1985. Apparent digestible protein and energy coefficients of common feed ingredients for channel catfish. Prog. Fish Cult., 47: 154-158.

Winfree, R.D., and Stickney, R.R., 1981. Effects of dietary protein and energy on growth, feed conversion efficiency and body composition of tilapia aureus. Journal of Nutrition, 111: 1001-1012.

116

Chapter 5

Experiment 3: Partial replacement of fishmeal with high-fibre and low-

fibre sunflower cakes in diets for tilapia (O. niloticus): Effect on fish

performance and whole body fatty acids

5.0 Abstract

The objectives of the study were to determine the effects of replacing fishmeal with high-

fibre and low-fibre sunflower cakes (HFSC and LFSC) on fish performance, body

proximate composition, and whole body fatty acid, and to determine the upper limit of

sunflower cake inclusion that would not compromise fish performance.

Sex-reversed O. niloticus males with an initial weight of 15.65g + 0.95 (SD) were

used. They were stocked in circular tanks with a base circumference of 1 metre and filled

with water to a depth of 0.3 metres. Stocking density for the heaviest fish at the end of the

trial was 0.013 kg per litre of water. Water temperature was maintained at 27°C ± 2 °C

throughout the trial, and dissolved oxygen concentration in the tanks was above 5.5

mg/litre.

A control diet based on herring meal and soyabean meal was formulated. Six test

diets were formulated such that low fibre (LF) and high-fibre (FTF) sunflower cakes (SC)

contributed 30%, 60% and 80% of the dietary protein and the diets were designated as

LFSC-30, LFSC-60, LFSC-80, HFSC-30, FTFSC-60, and HFSC-80 respectively. The rest

of the protein in each diet was supplied by herring meal. Fish were fed on the

experimental diets for a period of 70 days.

At the end of this period, they were starved for 24 hours and weighed. Five fish

representing the average weight of each replicated group (n = 3 per diet treatment) were

117

frozen in plastic bags at -5 °C for determination of body composition and fatty acid

composition.

At 70 days, the absolute weights and weight gains were similar between fish fed

the control diet, and those fed the LFSC-30, LFSC-60, LFSC-80 and HFSC-30 diets.

Specific growth rates were 1.60%, 1.60%, 1.51%, and 1.32% for the control fish, and

those fed the LFSC-30, LFSC-60, and LFSC-80 diets, respectively, and 1.57%, 1.38%,

and 1.26% for the HFSC-30, HFSC-60, and HFSC-80 diets respectively. Feed intake

decreased with increasing levels of sunflower cake in the diet. Fish fed diets LFSC-80,

HFSC-30, HFSC-60 and HFSC-80 had significantly lower feed intakes than those fed the

control diet.

Protein efficiency ratio (PER) and productive protein value (PPV) did not differ

significantly between fish fed the control, LFSC-30 and HFSC-30 diets. Fatty acid levels

(%) in the whole body were significantly influenced by diet. Linoleic (18:2 co 6), oleic

(18:1 co 9), and palmitic (16:0) acids were the most abundant fatty acids in the diets and

in the fish bodies. Percentages of the long chain poly-unsaturated acids, of the co3 family

viz., docosahexaenoic (22:6 co 3) and eicosapentaenoic (20:5 co 3) acid, were low in the

diets and in the fish bodies.

The low-fibre sunflower cake could replace 60% of the dietary protein without

compromising fish performance. By contrast, the high fibre cake could only constitute

30% of the protein in the fishmeal/soybean control diet without adversely affecting fish

performance. At higher levels of inclusion, the fish did not consume enough feed to meet

their nutrient requirements, and hence their growth was reduced compared to those fed

the control diet.

118

5.1 Introduction and objectives

In the first experiment (Chapter 3), it was established that the digestibility of protein in

the high-fibre and low-fibre sunflower cake diets by tilapia (O. niloticus) was high, and

that despite the low level of lysine in these diets, the fish fed the diets where low-fibre

sunflower cake supplied 50% of the dietary protein performed as well as those fed diets

based on anchovy fishmeal as the major protein source. Digestibility of the energy in

low-fibre and high-fibre sunflower cakes was found to be low (Experiment 1, Chapter 3).

Consequently, the digestible energy levels in the diets based on sunflower cakes were

also low, and they were increased by the addition of corn-oil, which also served as a

source of linoleic acid. Kanazawa et al. (1980), and Tekeuchi et al. (1983), established

that the only essential fatty acid required in the diet of tilapia is linoleic acid (18:2 eo 6).

Moreover, it was shown that tilapia possess enzymes that desaturate and elongate fatty

acids of the co6 series to provide sufficient levels of the long chain unsaturated fatty acids

necessary for membrane fluidity and function. For that reason no attempt was made to

add these fatty acids to the diets.

There is evidence that suggests that consumption of fish containing high levels of

highly unsaturated ©3 fatty acids is favorable for human health (Higgs, 1986; Bates et al,

1989; Thais and Stahl, 1987). Generally, marine fish oils are characterised by low levels

of linoleic (18:2 oo 6) and linolenic acids (18:3 oo 3), and high levels of the long chain ©3

polyunsaturated fatty acids, with eicosapentaenoic (20:5 oo 3) and docosahexaenoic acid

(22:6 co 3) being the predominant oo 3 fatty acids (Hilditch and Williams, 1964; Yamada

and Hayashi, 1975). Many studies have been done to assess the effect of the fatty acid

composition of the diet on the fatty acid fatty acid composition of fish. Body fatty acid

119

composition to a large extent has been found to reflect the dietary fatty acid composition

(Toyomizu etal, 1963; Braekhan etal, 1971; Yu and Sinnhuber, 1972).

The objectives of this experiment were:

a) To establish the upper limit to which high-fibre and low-fibre sunflower cakes could

replace fishmeal in the diets of tilapia (O. niloticus) without affecting growth rate,

c) To evaluate the effect of substituting sunflower cake for fishmeal on whole body fatty

acid composition.

5.2 Materials and Methods

5.2.1 Experimental diets and design

Seven diets whose compositions are shown in Table 5.1 were formulated. They contained

between 2600 and 3297 kcal/kg of D E (DM basis), and approximately 31% crude protein

( D M basis). A low-fibre sunflower cake (CF = 10%) supplied 30, 60, or 80% of the

dietary protein in the first three test diets. A high fibre cake (CF = 24%) was used in a

similar manner for the other test diets. Danish herring meal and soybean meal were the

protein sources in the control diet. The low-fibre sunflower cake was processed from a

high-oil hybrid variety (Kenya Fedha) as explained in Experiment 1. Each diet was fed

to three groups of 17 fish, three times daily, for a period of 70 days. The D E

concentration of the diets was calculated as explained in Experiment 2, chapter 4. The

digestible energy concentration in herring meal was estimated as 4000 kcal/kg (DM

basis) (Degani et al, 1997). Digestible energy for wheat flour was estimated at 4169

kcal/kg ( D M basis) (Degani et al, 1997) and the digestibility coefficient for energy in

soybean meal was estimated as 70% (Popma, 1982).

120

Table 5.1: Compositions of the diets used in Experiment 3 (g/100 g air-dry).

Low-fibre SFC 1 High-fibre SFC Control % protein supplied by sunflower cake Diets

30 2 LFSC-30

60 LFSC-60

80 LFSC-80

30 3HFSC-30

60 HFSC-60

80 HFSC-80

0 Control

Diets Herring meal 28.4 15.8 7.5 28.3 16.3 7.3 31.5 SFC (low- fibre) 25.0 49.0 65.0 - - - -SFC (high- fibre) - - - 26.0 50.0 68.0 -Soybean meal - - - - - - 16.0 Corn starch 31.0 16.1 5.9 29.6 10.7 - 38.9 Whole wheat 8.0 8.0 8.0 8.0 8.0 8.0 8.0 Corn oil 3.5 5.9 7.7 4.0 10.0 11.3 1.5 Dicalcium phosphate 1.5 2.6 3.3 1.5 2.4 2.9 1.5 *Vit/min premix 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Iodized salt 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Ascorbic acid 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Choline chloride 0.1 0.1 0.1 0.1 0.1 0.1 0.1

Chemical composition (DM basis)4

DM 92.8 91.2 92.3 90.9 90.2 91.3 89.0 DE (kcal/kgDM) 3150 2974 2870 29871 2850 2600 3057 Crude protein (%) 30.0 31.8 31.4 30.8 31.0 30.0 32.6 Crude fat (%) 5.3 13.5 20.3 7.4 8.5 13.9 4.7 Crude fibre (%) 3.2 5.9 7.6 6.6 12.3 17.5 1.2 ADF (%) 4.7 14.5 17.4 8.1 15.0 20.0 3.3 NDF (%) 6.5 13.8 17.4 11.7 22.7 30.5 10.5 Calcium (%) 1.6 1.8 1.2 1.5 1.7 1.0 1.6 Phosphorus. (%) 1.4 1.5 1.1 1.4 1.5 1.2 1.4

'SFC Sunflower cake. 2LFSC Low-fibre sunflower cake 3HFSC High-fibre sunflower cake 4 All values were determined by analysis except for DE, which was estimated from published data (see text).

Vitamin/mineral premix contained the following per kg: Vitamin A, 6000 IU; Vitamin D 3 ) 600 IU; Vitamin E, 100 mg; Vitamin K 3 , 3 mg; Vitamin B,, 10 mg; Vitamin B 2 , 20 mg; niacin, 150 mg; D-pantothenic acid, 50 mg; Vitamin B 6 , 10 mg; Vitamin B 1 2 , 0.03 mg; folic acid, 4 mg; biotin, 0.8 mg; choline, 600 mg; Vitamin C, 600 mg; inositol, 300 mg; manganese, 192 mg; iron 51.2 mg; copper, 6.4 mg; zinc, 57.6 mg; selenium, 0.15 mg; traces of cobalt and iodine.

121

5.2.2 Fish sampling

O. niloticus sex-reversed males weighing 15.65g + 0.95 (at the start of the experiment)

were used for this trial. They were bought at Sagana Farm in Kenya (Sagana, Kenya) and

transported to the research facilities in Nairobi. They were acclimated to laboratory

conditions for a period of 2 weeks before the onset of the trial. At the end of the second

week, they were weighed in groups of 17 fish and these were randomly allocated to the

experimental tanks. Stocking density for the heaviest fish at the end of the trial was

maintained below 0.013 kg per litre of water. Each tank was fitted with an AS8-1 (3 inch)

diffuser. Dissolved oxygen concentration in the tanks was maintained above 5 mg/litre.

Water in the tanks was completely exchanged every 48 hours, or when the dissolved

oxygen concentration in the tanks fell below 5 mg/litre. Feed intake for each group of fish

was recorded daily, while water temperatures were taken 3 times a day. Water

temperature was maintained between 25°C and 28°C throughout the experimental period

by the use of thermostatically-controlled heaters. Fish were weighed 3 times - namely on

day 0, 32 and 70. Before weighing time, the fish were starved for 24 hours. At the end

of the experimental period, 5 fish representing the average weight of fish in the tank from

which they were taken were selected and killed with an overdose of MS-222. They were

frozen at -5 °C and stored in plastic bags pending analysis of moisture, protein, crude fat,

ash and fatty acids. Before analysis, the fish were partly thawed and chopped into small

pieces. Then they were homogenized in a food blender. The homogenized samples were

divided into two portions. One part was used for the analyses of moisture, protein and

ash at the University of Nairobi, while the other portion was used for fatty acid analyses

in Canada.

122

5.2.3 Data collection and analytical procedures

The following parameters were used to assess fish growth and performance: absolute

weights, weight gain, specific growth rate, feed intake, and feed conversion. Specific

growth rate (% per day) was calculated as: 100*(ln final wt (g) - ln (initial wt

(g))/number of experimental days. Feed conversion ratio was calculated as ingested dry

feed (g)/wet weight gain (g). PER was calculated as wet weight gain/ingested protein and

PPV as 100 (gain in body protein/protein intake).

5.2.4 Chemical analyses

All ingredients and diets were analyzed in duplicate for their contents (%) of dry matter,

ash, protein, lipid, fibre, calcium and phosphorus according to standard procedures

(AOAC, 1984). ADF and NDF were analyzed according to the method of Waldern

(1971), using an Ankom technoloanalyzer (Ankom Technology, 140 Turk Hill Park,

Fairport, N Y 14450).

Analyses of total lipids in fish and diets were done at the Department of Food

Science, U B C . Total lipids were extracted in 50 ml of chloroform:methanol mixture

(2:1) according to the procedure of Folch et al. (1957). The fatty acid compositions of

the diets and fish samples were measured after methylation of the samples by gas

chromatography (Shimadzu GC-17A). In methylation, 10 mg of the lipid material was

saponified with 2.5 ml of 0.5 N CH3 -OH-KOH. This was achieved by first neutralizing

with 0.4N H C L , and then adding 5 ml of boron trifluoride. The mixture was heated for

15 to 20 minutes to achieve complete methylation. The fatty acid methyl esters were

extracted 3 times with hexane and concentrated. In the analysis for fatty acids, a fused

silica capillary column (Omegawax 320, Supleco Park, Bellefonte, PA, USA) was used.

123

Temperature of injection was 150 °C. It was increased by 2 °C per minute to 170 °C. It

was then increased by 3 °C per minute to 210 °C, and maintained at that temperature for

9.5 minutes. The detector temperature was set at 220 °C. An auto injector was used, and

helium was used as the carrier gas at 1 ml per min. Peak areas were quantified using a

Shimadzu Class VP chromatography data system, Version 4.2, and by reference to an

internal standard (heptadecanoic acid, C 17:0).

Amino acid compositions were determined by H P L C after performic acid

oxidation (AOAC 1998) (See Chapter 4).

5.2.5 Statistical analyses

Data for absolute weights, weight gain, specific growth rate, feed intake, feed conversion,

body composition and fatty acid composition were subjected to statistical analyses using

PROC G L M of the Statistical Analysis Systems (SAS 1985). An analysis of covariance

was done on all the fish performance data using the initial weight of the fish as the

covariate. In the analyses of the body composition data, the final weight of the fish for

each group was used as the covariate. After the initial comparison of the seven diets as a

7 x 1 completely randomized design, the performance of fish fed on the low-fibre and

high-fibre sunflower cakes was compared using a 2 x 3 factorial design (2 sunflower

cakes x 3 levels of dietary inclusion). Treatment means were separated using Tukey's

multiple range test. The level of significance was set at P < 0.05.

124

5.3 R e s u l t s a n d d i s c u s s i o n

5.3.1 Chemical composition of the diets

The chemical compositions of the diets used in this experiment are presented in Table

5.1. The low digestible energy content in the HFSC-80 diet is consistent with the low

digestible energy level in that cake. Despite the addition of corn oil, the energy level of

this diet was still below the levels of the other diets. The D E concentration in most of the

diets based on sunflower cake was slightly below the 3000 kcal/kg (DM) stipulated by

N R C (1993) for tilapia (O. niloticus).

Crude protein percentages of the diets ranged from approximately 30% to 32.6%>

(DM basis). As in Experiment 2, crude fat levels (%) in the LFSC-60 and LFSC-80 diets

were high due to the high residual oil content in the low-fibre cake and the high amounts

of the cake needed to provide 80% of the dietary protein. Extraction of oil from dehulled

seeds using a conventional screw press is more difficult than from seeds that contain

husks, which explains why the low-fibre cake diets had consistently higher oil levels than

the diets based on the high-fibre cake. ADF and NDF percentages increased with

increasing levels of sunflower cake in the diets, and they were higher in diets based on

the high-fibre sunflower cake. Phosphorus was maintained above 0.6 %» (air-dry basis).

Dietary levels of essential amino acids are shown in Table 5.2. The lysine levels

in the diets containing the highest levels of the sunflower cakes were 1.34 % and 1.00%

for the LFSC-80 and HFSC-80 diets, respectively, which were lower than the stipulated

requirement for juvenile tilapia (O. niloticus) (Santiago and Lovell, 1988). Methionine

level was also low in the diets based on the high-fibre sunflower cake.

125

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0.96% (DM % ( D M basis), which were also lower than the stated requirement of 1.05%

of diet ( D M basis) (NRC, 1993). All the other diets met the requirements for all the

amino acids.

The percentages of fatty acids in the diets are presented in Table 5.3, and the fatty

acid compositions of corn oil and sunflower oil are shown in Table 5.4. The control diet,

LFSC-30 and HFSC-30 diets had high levels of palmitic acid (16:0) compared to the

other diets. These diets contained more herring meal, which has a higher level of that

fatty acid than corn oil and sunflower oil (Table 5.4). The fatty acid compositions of the

diets based on low-fibre sunflower cake reflected the fatty acid composition of sunflower

oil, which was high in these diets, while the fatty acid composition of the high-fibre cake

diets reflected the composition of both corn-oil and sunflower oil. Both sunflower oil and

corn oil have high levels of oleic (18:1) and linoleic acids (18:2 co 6) which were

reflected in the diets. Linolenic (18:3 co 3), eicosapentaenoic (20:5 co 3) and

docosahexaenoic acids (22:6 co 3) were all very low in the diets. The control diet had a

slightly higher level of docosahexaenoic acid than the other diets.

5.3.2 Fish performance, PER, PPV, body and fatty acids composition

Data on absolute weight, weight gain, specific growth rate, feed intake, and feed and

protein utilization are shown in Tables 5.5 and 5.6, while data on whole body proximate

composition are shown in Table 5.7. Specific growth rates were not significantly

different (P > 0.05) for fish fed the control diet, and those fed the LFSC-30, LFSC-60,

and HFSC-30 diets. Fish fed the HFSC-60, LFSC-80 and HFSC-80 diets had

127

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Fatty acid Corn oil Sunflower oil Herring oil

14:0 - - 6.4 16:0 10.9 5.9 12.7 16:1 - - 8.8 18:0 1.8 4.5 0.9 18:1 24.2 19.5 12.7 18:2 ©6 58.0 65.7 1.1 18:3 co 3 0.7 - 0.6 18:4 co 3 - 1.6 20:1 - - 10.7 20:4 © 6 - - 0.4 20:5 © 3 - - 8.1 22:1 - - 12.0 22:5 © 3 - - 0.8 22:6© 3 4.8

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% Protein from SFC 'Moisture 'Protein 'Fat 'Ash

Control 0 69.1 16.60 7.40 5.40 LFSC 30 68.0 17.00 8.30 5.10

60 68.7 16.20 7.90 5.40 80 68.4 16.30 8.50 5.30

HFSC 30 67.9 16.00 8.70 5.30 60 69.6 16.00 7.00 5.60 80 69.4 15.80 7.70 5.40

SEM 0.53 0.28 0.36 0.12 2NS NS NS NS

^eans (n = 3)

2 Not significant

132

significantly lower growth rates than those receiving the control diet. Weight gain was

significantly influenced by diet (P < 0.05). Generally, the fish fed on the diets made from

the low-fibre cake (LFSC), gained more weight than those fish fed the diets made from

the high-fibre cake (HFSC). Among the fish fed the diets based on the LFSC, the weight

gains for those fed the LFSC-30 and LFSC-60 diets were not significantly different from

that of the control fish (P > 0.05), while fish fed the LFSC-80 diet gained significantly

less weight (P < 0.05) than those fed the control diet. For fish fed the diets based on the

HFSC, only those fed the HFSC-30 diet had an average weight gain comparable to those

fed the control diet (P > 0.05). Generally, the weight gain for fish fed the HFSC-30 diet

was higher than that noted for fish receiving diets with higher levels of the HFSC, but the

differences were only significant relative to the fish fed the HFSC-80 diet.

Mean feed intakes for the fish fed the LFSC-30 and LFSC-60 diets were not

significantly different from that of the control fish. The feed intakes of fish fed the

LFSC-80 diet and all of the diets based on the HFSC were lower than observed for the

control fish. The type of cake (low-fibre and high-fibre), and the level of cake in the diet

(30% of protein, 60% of protein and 80% of protein) significantly affected feed intake (P

< 0.05) (Table 5.6). Generally, fish fed diets based on the LFSC had a higher feed intake

than those fed the HFSC diets. The interaction between the type of cake and the level of

cake in the diet was significant for feed intake. Feed intakes decreased as the level of

sunflower cake in the diets was increased (Table 5.6), but the decline was higher for fish

fed diets based on the HFSC. Differences in feed intake between fish fed the LFSC and

HFSC diets and between those fed the diets where sunflower cakes supplied 30%, 60%,

and 80% of the protein may have been caused by the amount of fibre in the diets.

133

Dietary fibre dilutes nutrient density and increases bulkiness in feeds. Fish respond to the

diluting effects of fibre by increasing feed intake, thereby consuming enough nutrients to

grow at a rate comparable to that of the control fish. This compensatory increase in feed

consumption may however be hindered by physical limitations in the ability of the gut to

extend, in which case feed consumption may not increase enough to satisfy nutrient

requirements. Tilapia have a relatively small stomach (Balarin, 1979), which may limit

the extent to which the gut can distend to compensate for the increased dietary fibre level.

Reduced feed intake may also be due to the presence of phenolic compounds in

sunflower cake. The most important of these compounds is chlorogenic acid (Sabir et al,

1974). Sunflower cake contains 1% to 3% of chlorogenic acid (Sosulski and McCleary,

1972), which has been shown to reduce feed intake and weight gain in rats (Liener,

1980). In fish, no deleterious effect of chlorogenic acid has been reported. The

concentration of the acid is highest in the hulls (Bau et al, 1983), which may explain why

the reduction in feed intake of the fish in the current study was more prominent when

they ingested diets containing the high-fibre sunflower cake. In trout, Stickney (1996)

observed that feed intake was depressed when sunflower protein concentrate was fed at

35% of the diet. The depressed feed intake was attributed to low palatability of the

protein concentrate. Tacon et al (1984) also noted that rainbow trout fed diets containing

36% sunflower cake (25% fibre) had reduced feed intake compared to those fed the

control diet. In tilapia, Jackson et al. (1982) fed diets where sunflower cake provided

75% of the protein and observed a growth rate similar to that of fish fed the control diet

based on fishmeal. The sunflower cake used in their study had a fibre content of 14%,

which is comparable to the low-fibre sunflower cake used in the present study. However,

134

the growth rates of tilapia attained in the study by Jackson et al. (1982) were low, even

for those fed the control diet, indicating that there could have been other factors affecting

fish performance. In the present experiment, feed intake was reduced in fish fed diets

containing the highest level of sunflower cake, especially those containing HFSC.

The type of sunflower cake (low-fibre and high-fibre) in the diets also affected

weight gain and PPV, but not specific growth rate, FCR or PER. In this regard, fish fed

diets based on the low-fibre cake gained more weight than those fed the high-fibre cake

diets (Table 5.6). Although the type of cake had no significant effect on PER, the level of

cake in the diet did significantly affect values for PER (Table 5.6). Fish fed diets where

sunflower cake provided 30% of the protein had significantly higher PER values than

those fed diets where sunflower cake provided 60% and 80% of the protein. In a review

of factors affecting dietary protein utilization, Steffens (1981) lists fish species, fish size,

environmental and feed factors as some of the main factors that determine protein

utilization. In the current experiment, the same source of fish was used and they were

maintained under similar environmental conditions, regardless of diet treatment. Thus

the only difference in the current study was in diet formulations. Some of the dietary

factors that could have contributed to the observed differences were; digestibility, levels

of essential amino acids, lysine to total protein ratio, digestible energy concentration and

palatability of the diets. In relation to these, the digestibility of protein in the low-fibre

and high-fibre cakes was high (Experiment 1, Chapter 3), and the determined levels of

essential amino acids did not differ markedly among the diets, except for lysine, which

decreased as the level of sunflower cake in the diet was raised. The other probable cause

of the observed differences was the dietary D E concentration which was lower in the

135

diets containing high levels of sunflower cake (LFSC-80, HFSC-60 and HFSC-80) than

the other diets. Therefore, it is plausible that some of the dietary protein in the latter diets

could have been used for energy production.

PPV (protein gain/protein intake) is a measure of protein quality that takes into

account elaboration of new tissue protein in relation to dietary protein intake. It is an

indicator of the amount of dietary protein required to gain a unit weight of protein in fish

(Steffens, 1981), and it is affected by the same factors that influence PER. The type of

sunflower cake and the level of sunflower cake in the diets significantly affected PPV.

Fish fed diets based on low-fibre sunflower cake had higher PPV values than those fed

diets based on the high-fibre sunflower cake. As with PER, PPV values decreased with

increasing levels of sunflower cake in the diet. Fish fed diets where sunflower cake

provided 30% of the protein had significantly higher PPV values compared to those fed

diets where sunflower cake provided 60% and 80% of the dietary protein.

Diet did not have a significant effect on the whole body proximate composition of

the fish (Table 5.7). Morever, there were no specific trends in the contents (%) of

moisture, lipid, protein or ash in relation to diet treatment. In salmonids, body lipid

content is influenced more by energy intake, rather than lipid intake (Shearer, 1994),

whereas in tilapia, De Silva et al. (1991), Hanley (1991) and Chou and Shiau. (1996)

observed that fish fed high-fat diets had more body fat than those fed low-fat diets. In the

current experiment, fat deposition did not show any particular pattern.

Fatty acid levels in the whole body of fish are presented in Tables 5.8 and 5.9.

Whole body fatty acid composition closely resembled dietary fatty acid composition

(Table 5.8).

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Main effects

Type of sunflower cake Percentage protein from SFC ! L F S C 2FfFSC S E M 30 60 80 S E M

Fatty acid 3 2 2 a b 12:0 2.92 3.40 0.24 2.23b 4.05a 3 2 2 a b 0.29

14:0 4.18 6.26 0.93 4.63 7.23 3.80 1.14 16:0 20.19 20.88 1.08 24.22 18.59 18.80 1.33 18:0 6.16 7.62 1.62 6.48 8.95 5.25 1.99 16:1 2.01 2.59 0.29 2.69 2.76 1.44 0.35 18:1 31.39 31.97 0.34 31.74ab 2997b 33.33a 0.41 18:2 30.93a 24.48b 1.60 25.22 25.69 32.21 1.96 18:3 0.31 0.37 0.01 0.36 0.35 0.29 0.017 20:4 0.48 0.54 0.05 0.33b 0.48ab 0.71a 0.06 20:5 0.06 0.21 0.10 0.14 0.25 0.013 0.13 22:6 1.36b 1.68a 0.05 1.95a 1.66b 0.94° 0.06

Means that do not have a superscript or share a common superscript letter for the same factor in a row within a main effect are not significantly different ( P > 0.05) T J S C Low fibre sunflower cake 2 HFSC High fibre sunflower cake 3Means (n = 6) 4Means (n = 4)

138

Palmitic (16:0), oleic (18:1 co 9) and linoleic (18:2 co 6) acids were the most abundant in

both the diets and fish. The levels of these fatty acids in the diets ranged from 16.0% to

31.0% for palmitic acid (16:0), 25.5% to 41.9% for oleic acid (18:1 co 9), and 23% to 45.0

% for linoleic acid (18:2 co 6). Palmitic acid (16:0) was the most abundant fatty acid in

the control diet, which was reflected in fish fed this diet. Indeed the control fish had a

significantly higher content of palmitic acid (16:0) than fish fed diets with high levels of

sunflower cake (LFSC-60, LFSC-80 and HFSC-80). The higher level of 16:0 in the

control diet was due to the higher percentage of herring fishmeal, which has a higher

content of palmitic acid (16:0) than present in sunflower oil and corn oil (Table 5.4).

Oleic acid (18:1 co 9) was the most abundant of the mono-unsaturated fatty acids

in both the diets and the fish. Fish fed the control diet had significantly lower levels of

this fatty acid than those fed diets based on the two sunflower cakes. There was a trend

to increasing levels of oleic acid (18:1 co 9) in fish fed diets with high levels of sunflower

cake, especially the high-fibre cake. Diets based on sunflower cake had high levels of

corn oil and sunflower oil, while the control diet was rich in herring oil from the herring

fishmeal. Corn oil and sunflower oil have higher levels of oleic acid (18:1 co 9) than

herring oil (Table 5.4).

Linoleic acid (18:2 co 6) was the most abundant fatty acid in diets based on

sunflower cake. The level of this fatty acid was also significantly higher in fish fed diet

with a high level of sunflower cake (LFSC-60, LFSC-80 and HFSC-80) than in those fed

the control diet. It is also worth noting that the levels of linoleic acid (18:2 co 6) were

much lower in the fish bodies than those determined in the diets. In tilapia (O. niloticus),

139

desaturation and elongation enzymes efficiently convert C 18 P U F A to longer chain

P U F A (Kanazawa et al, 1980; Olsen et al, 1990; Tekeuchi et al, 1983). It is plausible

that some of the dietary linoleic acid was converted into the long chain highly unsaturated

fatty acids, especially arachidonic acid (20:4 co 6), which was not detected the diets, and

yet was present in the fish.

The fish had low levels of eicosapentaenoic acid (20:5 co 3) and docosahexaenoic

acid (22:6 co3). This may have been caused by several factors. First, the fish were frozen

at -5 °C for a period of about one year before they were analyzed for fatty acids. The

highly unsaturated fatty acids are more susceptible to oxidation and may have undergone

some degree of oxidation during storage, leading to the low observed values. This

notwithstanding, levels of these fatty acids are generally low in fresh-water fish compared

to marine fish. Ackman (1967) and Hilditch and Williams (1964) noted that fatty acids

of fish could be altered by manipulating temperatures. At low temperatures, there was an

increase in the long chain highly unsaturated fatty acids which are necessary for

membrane fluidity at these temperatures. It is possible that the high temperatures

prevailing during the experimental period may also have contributed to the low levels of

these (20:5 co 3 and 22:6 co 3) fatty acids observed. Besides, the diet had very low levels

of the 20:5 co 3 and 22:6 co 3 fatty acids, as well as 18:3 co 3 precusor needed to make

20:5 co 3 and 22:6 co 3 fatty acids. This was reflected in the fish bodies. The addition of

fish oils to fish diets has been shown to increase the body and tissue contents of co3

unsaturated fatty acids in a number of fish species. This observation has been made in

various species such as rainbow trout (Oncorhynchus mykiss) (Yu et al, 1977), channel

catfish (Ictalurus punctatus) (Stickney and Andrews, 1972), grass carp

140

(Ctenopharyngodon idella) (Tekeuchi et al., 1991) and hybrid tilapia (Oreochromis

niloticus x Oreochromis aureus) (Chou and Shiau, 1999). In tilapia, recent studies (Chou

and Shiau, 1999) have shown significant increases in the percentages of ©-3 fatty acids in

the muscle and liver by feeding diets supplemented with cod liver oil. In the same study,

it was also observed that eicosapentaenoic acid (20:5 co 3) is not well retained by tilapia.

The levels of docosahexaenoic acid (22:6 co 3) were not appreciably different

from those determined by Huang et al. (1998) in the muscles of hybrid tilapia (O.

niloticus x O. aureus) fed diets fortified with soy oil. The levels of docosahexaenoic acid

reported in the above study were 2.1% for the fish fed a lipid-free diet (0.02% lipid) and

3.8% for fish fed diets containing soy oil.

In addition to dietary lipid composition and level, the fatty acid composition of

fish is affected by other factors such as temperature, fish size, section analyzed, sex, and

physiological status (Kinsella et al, 1977; Stephens, 1997). In the present experiment, all

these factors were similar for all treatments. The only difference between treatments was

in the final weights but this was taken into account when analyzing the fatty acids

composition data by analysis of covariance, with the final weight of the fish as the

covariate. Whole body percentages of fatty acids did not differ appreciably between the

two types of sunflower cakes mainly because of the similarity in fatty acid composition

of sunflower oil and corn oil. Diets based on the LFSC had higher level of linoleic (18:2

co 6) acid than those made from the HFSC. This was due to the higher content of residual

sunflower oil in the LFSC. Sunflower oil contains approximately 66% linoleic acid

(Table 5.4).

141

5.4 Conclusions

Al l diets contained adequate levels of essential amino acids, except for lysine and

threonine, which were low in the diets containing high levels of sunflower cakes. There

was little variation in dietary fatty acid percentages. The long chain polyunsaturated fatty

acids, eicosapentaenoic and docosahexaenoic were not detected in most of the diets.

Generally, the fish performed well on all diets even when they contained high

levels of both the low-fibre and the high-fibre sunflower cakes. Absolute weights, weight

gains, feed intakes and PPV values were higher for fish fed diets based on the low-fibre

cake than those fed diets containing the high-fibre cake. Feed intake decreased with

increasing levels of sunflower cake in the diet, but the decline was higher for fish fed

diets based on the high-fibre cake. The reduced feed intake was reflected in the growth

rates of fish, which also decreased with increasing levels of sunflower. The low-fibre

cake could comprise up to 60% of the dietary protein without compromising the

performance of the fish. At higher dietary inclusion levels, feed intake was depressed,

leading to a lower growth rate than that of the control fish. The high-fibre sunflower cake

could supply up to 30% of the dietary protein with the same performance as that obtained

with fish fed the control diet despite a reduction in feed intake. The inclusion of higher

levels of the cake in the diet led to a drastic reduction in feed intake which in turn

resulted in lower weight gains of the fish fed these diets, compared to those fed the

control diet.

The level of sunflower cake in the diet had a significant effect on most of the

parameters assessed except for whole body proximate composition. Growth rates and

feed intakes of the fish decreased with increasing levels of the sunflower cakes in the

diets (Table 5.6). FCR values were better for fish fed diets where sunflower cake

142

contributed 30% of the protein than for those fed diets where it contributed 80% of the

protein. PER and PPV values were significantly lower for fish fed diets containing a high

level of the sunflower cake (60% and 80%), than those with a low level of sunflower cake

(30%)

Whole body fatty acid percentages were significantly influenced by diet.

Palmitic, oleic and linoleic acids were the most abundant fatty acids both in the diets and

the fish. Eicosapentaenoic and docosahexaenoic acids were low in the fish reflecting the

low level in the diets and perhaps other factors.

143

5.5 References Ackman, R.G., 1967. Characteristics of the fatty acid composition and biochemistry of some fresh-water fish oils and lipids in comparison to marine oils and lipids. Comp. Biochem. Physiol., 22: 907-922.

A O A C , 1984. Association of Official Analytical Chemists. Official methods of analysis. Animal Feed Section

Balarin J.D., 1979. Tilapia; A guide to their biology and culture in Africa JP . Hatton (Ed). University of Stirling. 174pp.

Bates, D., Cartlidge, N . , French, J.M., Jackson, M.J. , Nightingale, S., Shaw, D A , Smith, S., Woo, E., Hawkins, S.A., Millar, J.H.D., Berlin, J., Conroy, D M . , Gill, S.K., Sidey, M . , Smith, A.D. , Thompson, R.H.S., Zilka, K., Gale, M . , and Sinclair, H . M . , 1989. A double-blinded controlled trial of long chain n-3 polyunsaturated fatty acids in the treatment of multiple sclerosis. J. Neurol. Neurosurg. Psychiatr., 52: 18-22.

Bau, H . M . , Mohtadi-Nia, D.J., Mejean, L. , and Debry, G., 1983. Preparation of coloress sunflower protein products: Effect of processing on physiological and nutritional properties. Journal of American Oil Chemists Society, 60 (6) 1141-1146.

Braekhan, O R . , Lamberstein, G., and Andresen, J., 1971. Influence of dietary fat on the fatty acid patterns of muscle and liver lipids in rainbow trout. SKR. Fiskeridir, 5 (8) 1-12.

Chou, B.S., and Shiau, Shi Yen, 1999. Both n-6 and n-3 fatty acids are required for Maximal Growth of juvenile Hybrid Tilapia. North American Journal of Aquaculture, 61: 13-20.

Chou, B.S., and Shiau, Shi-Yen, 1996. Optimal dietary lipid level for growth of juvenile hybrid (Oreochromis niloticus x Oreochromis aureus). Aquaculture, 143: 185-195.

De-Silva, S.S., Rosanthi M . , Gunasekera and Shim, K.F. , 1991. Interaction of varying dietary protein and lipid levels in young red tilapia. Evidence of protein sparing. Aquaculture, 95: 305-318.

Degani, G., Viola, S., and Yehuda, Y. , 1997. Apparent digestibility of proteins and carbohydrates in feed ingredients for adult hybrid tilapia (O. niloticus x O. aureus). Israeli Journal of Aquaculture Bamidgeh, 49: 3 115-123.

Folch, J., Lee, M . , and Sloane-Stanely G.H., 1957. A simple method for isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry, 226: 497-509.

Hanley, F., 1991. Effects of feeding supplementary diets containing varying levels of lipid on growth, feed conversion and body composition of Nile tilapia (Oreochromis niloticus). Aquaculture, 93: 323-334.

144

Higgs, G.A., 1986. The role of eicosanoids in inflammation. Prog. Lipid Res., 25: 555-561.

Hilditch, T. P., and Williams, P.N., 1964. The chemical constitution of natural fats. 4 t h

Edition. Chapman and Hill (Eds). London.

Huang, Chen-Huei., Huang, Ming-Chi., and Hou, Ping-Chun, 1998. Effect of dietary fatty acid composition and lipid peroxidation in sarcoplasmic reticulum of hybrid tilapia, (Oreochromis niloticus x O. aureus). Comp. Biochem. Physiol., PartB 120: 331-336.

Jackson, A.J. , Capper, B.S., and Matty, A.J., 1982. Evaluation of some plant proteins in complete diets for tilapia (O. mossambicus). Aquaculture, 27: 97 - 109.

Kanazawa, A , Teshima, S. I., Sakamoto, M . and Awal, M.A. , 1980. Requirements of tilapia zilli for essential fatty acids. Bull. Jap. Soc. Sci. Fish., 46: 1353-1356.

Kinsella, J.E., Shimp, J.L., Mai J., and Weihrauch, J., 1977. Fatty acid content and composition of freshwater finfish. Journal of American Oil Chemists Society. 424-429

Liener, L .E . , 1980. Toxic constituents of plant feedstuffs. Academic Press London and New York. 502.

NRC, National Research Council, 1993. Nutrient Requirements of Fish. National Academy Press, Washington, D.C. 144 pp.

Olsen, F.E., Henderson, R.J., and McAndrew, B.J., 1990. The conversion of linoleic acid to longer chain polyunsaturated fatty acids by Tilapia (Oreochromis nilotica) in vivo. Fish Physiology and Biochemistry, Vol. 8: (3) 261-270.

Popma T.J., 1982. Digestibilities of selected feedstuffs and naturally occurring algae by tilapia. Ph.D. Dissertation, Auburn University, Alabama.

Sabir, M . A., Sosulski, F.W., and Kernan, J.A., 1974. Phenolic constituents in sunflower flour. J. Agric. Food Chem., 22: 572-574.

Santiago, C.B., and Lovell, R.T., 1988. Amino acid requirements for growth of Nile tilapia. Journal of Nutrition, 118: 1540-1546.

Santiago, C.B., and Reyes, OS . , 1993. Effect of dietary lipid source on reproductive performance and tissue lipid levels of Nile tilapia (Oreochromis niloticus (L.)) broodstock. J. Appl. Ichthyol, 9: 33-40.

SAS Institute, 1985. General Linear Models Procedure. SAS Institute, Cary, North Carolina.

145

Shearer, K .D. , 1994. Factors affecting the proximate composition of cultured fishes with emphasis on salmonids. Aquaculture, 119: 63 -88.

Sosulski, F. W., and McCleary, C. W., 1972. Diffusion extraction of chlorogenic acid from sunflower kernels. Journal of Food Science, Vol. 37: 253-257.

Steffens, W., 1981. Protein utlization by rainbow trout {Salmo gairdneri) and carp (Cyprinus carpio). A brief review. Aquaculture, 23: 337-345.

Stephens, W., 1997. Effects of variation in essential fatty acids in fish feeds on nutritive value of freshwater fish for humans. Aquaculture, 151, 97-119.

Stickney, R.R., and J.W. Andrews, 1972. Effects of dietary lipid on growth, food conversion, lipid and fatty acid composition of channel catfish. Journal of Nutrition, 102: 249-258.

Stickney, R.R., 1996. The effects of substituting selected oilseed protein concentrate for fishmeal in rainbow trout (Salmo gairdneri) and Carp (Cyprinus Carpio). A brief review. Aquaculture, 23: 337 - 345.

Tacon, A.G.J. , Webster, J.L., and Martinez, C.A., 1984. Use of solvent extracted sunflower seed meal in complete diets for fingerling rainbow trout (Salmo gairdneri Richardson). Aquaculture, 43: (4) 381-389.

Tekeuchi, T., Watanabe, K. , Yong, W.Y., and Watanabe, T., 1991. Essential fatty acids of grass carp (Cteno-pharyngodon idella). Nippon Suisan Gakkaishi, 57: 467-473.

Tekeuchi, T., Sitoh, S., and Watanabe T., 1983. Requirements of tilapia nilotica for essential fatty acids. Bull. Jap. Soc. Sci. Fish., 49: 1127 - 1134.

Thais, F., and Stahl, R.A.K. , 1987. Effect of dietary fish oil on renal function in immune mediated glomerular injury. In: W.E.M. Lands (Ed.) Proceedings of A O A C short course on polyunsaturated fatty acids and eicosanoids. American Oil Chemists Society., Champaign, Illinois, pp 123-126.

Toyomizu, M . , Kawasaki, K. , and Tomiyasu, Y. , 1963. Effect of dietary oil on fatty acids composition of rainbow trout oil. Bull. Jap. Soc. Sci. Fish., 29: 957 - 961.

Viola, S., Arieli, Y . , and Zohar, G., 1988. Animal protein free feeds for hybrid tilapia (O. niloticus xO. aureus) in intensive culture. Aquaculture, 75: 115 - 125.

Viola, S., Mokady, S., Behar, D., and Cogan,U, 1988. Effects of polyunsaturated fatty acids in feeds of tilapia and carp. Body composition and fatty acid profiles at different environmental temperatures. Aquaculture, 75: 127-137.

146

Waldern, D.E., 1971. A rapid micro digestion procedure for neutral and acid detergent fibre. Canadian Journal of Animal Science, 51: 67-69 .

Yamada, M . and Hayashi, K. , 1975. Fatty acids composition of lipids from 22 species of fish and molluscs. Bull. Jap. Soc. Sci. Fish., 41: 1143-1152.

Yu, T C , and Sinnhuber, R.O., 1972. Effect of linolenic acid and docosahexaenoic acid on growth and fatty acid composition of rainbow trout. Lipids, 7: 450-454.

Yu, T C , Sinnhuber, R.O., and Putnam, G.B., 1977. Effect of dietary lipid on fatty acid composition of body lipid in rainbow trout (Salmo gairdneri). Lipids, 12: 495- 499.

147

Chapter 6

Experiment 4: Evaluation of the most limiting amino acids in diets

based on sunflower cake fed to tilapia (Oreochromis niloticus)

6.0 Abstract

The objective of this study was to determine the effects of supplementing diets based on

sunflower cake with lysine, threonine and methionine on the performance of tilapia (O.

niloticus). A basal diet in which a fibre-reduced sunflower cake provided 80% of the

dietary protein was formulated. The levels of lysine, threonine and methionine in the

basal diet expressed as a percent of the diet (DM basis) were 1.17%, 1.05% and 0.75%»

respectively, while the stipulated requiments (NRC, 1993) are 1.54%, 1.2% and 0.8%

respectively. The amino acids were added to the basal diet singly or in various

combinations. A positive control diet based on herring fishmeal and soybean meal was

also formulated. All diets were isonitrogenous and isocaloric and were fed to triplicate

groups of fish with an initial weight of 24 g + 0.59 (+ Sd). Further, all groups were held

at 27 °C for a period of 39 days. There was a trend to improved growth rate in fish fed

diets supplemented with lysine and threonine, but the improvement was not significant.

There was no response to methionine added alone or together with threonine, but fish fed

diets in which methionine and lysine were added together had a 12% increase in growth

rate over those fed the basal diet, while those fed diets in which threonine was added

together with lysine had a 10%> increase. There was a trend to improved FCR with the

addition of lysine, threonine, lysine and methionine, lysine and threonine, and lysine,

methionine and threonine. Growth rates and FCR's for the fish supplemented with the

above amino acids were not significantly different from those of fish fed the basal diet,

148

but the values observed were also not significantly different from those obtained with the

positive control fish.

6.1 Introduction and objectives.

Feed accounts for about 50 % of the total cost of production in an intensive fish farm,

with protein being the most expensive dietary component (El-Sayed, 1999). Fishmeal

has traditionally been the most widely used protein source for many cultured fish species

(Tacon, 1993). Due to its high cost, many attempts have been made to reduce its

proportion in fish diets (Fagbenro and Jauncey, 1994; Mansour, 1998; El-Sayed, 1998).

Many protein sources have been tested as complete or partial replacements for fishmeal.

Jackson et al. (1982) demonstrated that certain plant protein sources could be used to

meet much of the protein requirements of tilapia (O. mossambicus). In their study, one of

the limiting factors when high dietary inclusion levels of plant proteins were tested

related to a deficiency of certain essential amino acids, particularly lysine and

methionine. Information on limiting amino acids in plant protein sources for tilapia

species is inadequate, making it difficult to use free amino acids to supplement

comparatively poor protein sources.

Sunflower seeds are important sources of edible oils and protein for inclusion in

animal feeds. The meal resulting from the oil extraction process is a valuable source of

protein, and has been tested in many animal diets. In studies with pigs and poultry, Green

and Kiener (1989) found lysine to be the most limiting amino acid. In their study, the

highest growth response was obtained when lysine and methionine were supplemented in

diets based on grain and sunflower cake. In ducks, Attia et al. (1998) found lysine to be

the most limiting amino acid in diets containing sunflower cake, while in broilers,

149

Mohme et al. ( 1 9 9 7 ) established that partly-dehulled and solvent-extracted sunflower

meal supplemented with lysine and methionine could be included up to 3 0 % in broiler

diets without compromising their performance. In pigs, Jorgensen and Sauer ( 1 9 8 2 )

reported that the apparent availability of lysine in sunflower cake was 7 1 % , while the

digestibilities of methionine and threonine in the same cake were 8 1 % and 69%o,

respectively.

In fish, studies on supplementation of diets based on sunflower meal with

crystalline amino acids have yielded conflicting results. In the European eel, (Anguila

anguila), Hinguera et al. ( 1 9 9 9 ) noted that the inclusion of sunflower meal as the only

source of dietary protein resulted in poor growth, and that growth could be improved

when sunflower meal was mixed with fishmeal or supplemented with essential amino

acids. Contrary to these findings, Sanz et al. ( 1 9 9 4 ) found no improvement in dietary

protein utilization of rainbow trout when diets that contained 3 9 % sunflower meal were

supplemented with lysine, leucine, and methionine. Similarly, Tacon et al. ( 1 9 8 4 ) , did

not observe any beneficial effects on growth and feed utilization when rainbow trout were

fed diets containing 3 6 % sunflower meal, supplemented with 0 . 2 % methionine. The

goals of this experiment were to:

1. determine the most limiting amino acids in diets based on sunflower cake fed

to O. niloticus.

2. evaluate the effect of these diets with the foregoing limiting amino acids on

fish performance.

150

6.2 Materials and Methods

6.2.1 Experimental diets and design

The sunflower used in this study was dehulled using a manual "Cecolo" dehuller (Ibraki,

Osaka, 567, Japan) as described in Experiment 1, and the oil was extracted using a

laboratory Komet screw press (model 80B/2Q FDR, Germany).

The compositions of the basal, control, and the test diets are shown in Table 6.1.

Test diets were supplemented with the synthetic amino acids, L-lysine H C L , D L -

methionine, and L-threonine singly or in various combinations. Herring meal and

soybean meal were the main protein sources in the control diet. The calculated level of

methionine in the control diet was considered to be low, and it was added to bring it up to

0.8% of the dry diet. It was not possible to determine the amino acid contents of the diets

before commencement of the trial. These were calculated from the values of Scott et al.

(1982). L-lysine H C L , and DL-methionine, and L-threonine were added to the diets at

levels which were estimated to be approximately 10% higher than the respective

requirements of tilapia as stated by N R C (1993). The diets were as follows:

Diet 1. Basal diet with no amino acid supplementation

Diet 2. Basal diet + lysine (1.7% total in diet D M basis)

Diet 3. Basal diet + methionine (0.9% total in diet D M basis)

Diet 4. Basal diet + threonine (1.25% total in diet D M basis)

Diet 5. Basal diet + lysine and methionine (1.7% and 0.9% of diet D M basis)

respectively.

Diet 6. Basal diet + lysine and threonine (1.7% and 1.25%) of diet Dm basis) respectively.

151

Table 6.1: Compositions and chemical analyses of diets used in Experiment 4 (air-dry basis).

Diets Amino acids

1 Basal

2 ] L

3 M

4 Th

5 L+M

6 L+T

7 M+Th

8 L,M,T

9 Control

Sunflower cake 61.0 61.0 61.0 61.0 61.0 61.0 61.0 61.0 Soybean cake - - - - - - - - 30.0 Herring meal 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 24.0 Corn starch 7.4 6.2 6.7 7.0 5.5 5.9 6.29 5.2 29.8 Wheat 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 Corn oil 8.6 9.0 8.9 8.7 9.2 9.1 9.0 9.3 0.5 Iodized salt 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Dicalcium phospate 2.8 2.8 2.8 2.8 2.8 2.8 2.8 2.8 2.1 2Vit/mineral premix 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.3 Ascorbic acid 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.2 Choline chloride 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 L-lysine HCL - 0.8 - - 0.8 0.8 - 0.8 -DL-methionine - - 0.5 - 0.5 - 0.5 0.5 0.3 Threonine - - - 0.2 - 0.2 0.2 0.2 -

Chemical analyses (DM basis3

Dry matter 92.7 92.09 92.05 92.04 92.1 92.1 92.07 92.14 90.75 4 DE (kcal/kg D M 2951 2951 2958 2949 2949 2951 2954 2949 3182 Protein, % 33.0 33.1 33.4 33.4 33.1 32.9 33.3 33.1 33.9 Crude fat, 19.6 20.2 20.0 19.9 20.4 20.3 20.1 20.5 4.7 Crude fibre, % 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 2.1 Calcium, % 1.2 1.3 1.2 1.3 1.3 1.3 1.3 1.3 1.3 Phosphorus, % 1.6 1.6 1.7 1.6 1.7 1.7 1.6 1.6 1.5

1 L = lysine, M = methionine, T = threonine. (For diet description, see text). 2 Composition of vitamin/mineral premix is as described in Table 5.1 (composition of diets used in Experiment 3). 3 All values were determined by analysis, except D E concentrations which were calculated as described in Chapter 4, Experiment 2.

152

Diet 7. Basal diet + methionine and threonine (0.9% and 1.25% of diet Dm basis)

respectively.

Diet 8. Basal diet + lysine, methionine and threonine (1.7%, 0.9%, and 1.25% of diet Dm

basis) respectively.

Diet 9. Positive control diet.

6.2.2 Fish sampling

Sex-reversed tilapia (O. niloticus) male fingerlings weighing 23.9 + 0.60g were used.

They were bought from the Sagana Government Fisheries Farm, in Sagana, Kenya, and

transported to the University of Nairobi. They were acclimated to laboratory conditions

for a period of two weeks before the onset of the trial. Thereafter, they were weighed in

groups of 16 fish that were selected at random, allocated to the experimental tanks, and

managed as in Experiment 3. Fish were weighed at the beginning of the trial and 39 days

later. They were starved for a period of 24 hours before each weighing. Water

temperatures in each tank were maintained between 25 °C and 28 °C, and dissolved

Dissolved oxygen concentration in the tanks was maintained above 5.5 mg/L during the

experimental period.

6.2.3 Data collection and analytical procedures

Fish growth and performance were assessed by absolute weights, weight gain, specific

growth rate, feed intake, and feed conversion ratio. Specific growth rates, feed intakes

and feed conversion ratios were determined as described in Experiment 3.

6.2.4 Chemical analyses

The ingredients and diets were analyzed in duplicate for percentages of dry matter, ash,

protein, lipid, calcium and phosphorus according to standard procedures (AOAC, 1984).

153

Dietary amino acid compositions were determined by H P L C after performic acid

oxidation (see Chapter 4)

6.2.5 Statistical analyses

Data on absolute weight, weight gain, specific growth rates, feed intake and feed

utilization were subjected to statistical analysis using PROC G L M of the SAS statistical

analysis systems (SAS, 1985). The model for a 9 x 1 CRD was used. An analysis of

covariance was done using the initial weights as the covariate. The covariate was not

significant in any of the parameters. Treatment means were compared using Tukey's

multiple range test, with the level of significance set at P < 0.05.

154

6.3 Results and Discussion

6.3.1 Chemical composition of the diets

The low-fibre sunflower cake used in this experiment had a dry matter content of 93%,

while crude protein, crude fibre, fat and ash contents were 42%>, 14%>, 15%>. and 7.5%

( D M basis) respectively. All diets were formulated to contain 30% protein (as fed), and

in the test diets, 80% of the protein originated from dehulled sunflower cake, and 20%

from LT herring meal.

The chemical compositions of the diets are presented in Table 6.1. The

determined protein levels in the diets were similar in each case. Due to the low digestible

energy content of sunflower cake, corn oil was used to increase the energy level in the

diets. This resulted in the crude fat levels in the diets based on sunflower cake being

higher than in the control diet. The crude protein content of the diets was approximately

30%o (AD basis). Estimates of the protein requirements of young tilapia range from 25%

to 35%> (AD basis) depending mainly on the protein source and fish size (Santiago et al,

1982; Siddiqui et al, 1988). In the current experiment, the estimated energy to protein

ratio in the diets based on sunflower cake was 9.0 kcal of digestible energy per gram of

protein (113 mg protein/kcal), while in the control diet it was 9.4 kcal of digestible

energy per gram (107 mg per kcal). Kubaryk (1980) reported that an energy to protein

ratio of 8 - 10 kcal digestible energy per gram of protein (100 - 125 mg protein/kcal) was

adequate for young tilapia fingerlings, while El-Sayed and Teshima (1992) reported an

optimum protein to energy ratio of 103 mg of protein per kcal DE. The calcium content

of the diets was approximately 1.3%, while the total phosphorus content was

approximately 1.6% for the diets based on sunflower cake and 1.5% for the control diet

155

(AD basis). In tilapia, the calcium requirement is largely met by absorption of calcium

through the gills and skin, while phosphorus has to be supplied in the diet. Tilapia has

been reported to require 0.6 to 0.7% phosphorus in the diet (AD basis) (Viola and Arieli,

1988), which can be met using animal by-products, dicalcium phosphate or a

combination of both.

The quality of protein is influenced by its amino acid profile. The amino acid

profiles of the diets expressed as a percentage of the diet, and as a percentage of the

dietary protein, are presented in Tables 6.2 and 6.3 respectively. The basal diet met or

exceeded the requirements for most of the essential amino acids except lysine,

methionine and threonine. The stipulated requirements for these amino acids are 1.54%,

0.8%, and 1.12% of the dry diet respectively (NRC, 1993) while their levels in the basal

diet were 1.17, 0.75 and 1.05% (DM basis) respectively. Diet 2 was deficient in

methionine and threonine, while diets 3, 4, 5, 6 and 7 were deficient in lysine and

threonine, lysine and methionine, threonine, methionine, and lysine, respectively. Due to

budgetary constraints, it was not possible to determine tryptophan and tyrosine levels in

all of the diets. This was only done for the basal and control diets. Determined amino

acid levels were not appreciably different among the different diets.

6.3.2 F i sh performance

Data on fish performance are presented in Table 6.4. The specific growth rate for fish fed

the positive control diet was higher than the values found for fish fed the other diets, but

the differences were only significant (P< 0.05) relative to those fed the basal diet, the

basal + methionine, and the basal diet + methionine and threonine. Fish fed the positive

control diet had a growth rate of 2.28% per day, followed by those fed the diet

156

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supplemented with both lysine and methionine (diet 5) which had a growth rate of 2.17%

per day, while those fed the diet supplemented with lysine and threonine (diet 6) had a

growth rate of 2.14% per day. Fish fed the diet supplemented with lysine, methionine

and threonine (diet 8) had a growth rate of 2.08%>. There were no significant differences

(P > 0.05) in growth rates between fish fed the basal diet and those fed diets

supplemented with amino acids. However, the addition of lysine and methionine (diet 5)

to the basal diet improved growth rate by approximately 12%, while the addition of lysine

and threonine (diet 6), and lysine, methionine and threonine (diet 8) improved growth rate

by 10%) and 7% respectively. Lysine, added alone improved the growth rate of tilapia by

approximately 6% over that noted for fish fed the basal diet. Although the improvement

in growth rate for fish fed the above diets was not significant relative to those ingesting

the basal diet, the growth rates attained were also not significantly different from that of

fish fed the the positive control diet (P > 0.05). Fish fed the basal diet alone or

supplemented with methionine had the lowest growth rates of 1.94%, and 1.96% per day,

and as indicated above, these values were lower than those observed for the fish fed the

control diet (P < 0. 05).

There were no significant differences in feed intake between fish fed the various

diets, but there was a trend to improved feed conversion ratios for the fish fed the diets

supplemented with amino acids compared to those fed the basal diet. FCR values of fish

fed diets 2, 4, 5, 6, 7, and 8 were not significantly different from that of fish fed the

control diet.

Lysine has been identified as the first limiting amino acid in sunflower cake

(Senkoylu and Dale, 1999). Besides its effect on growth, it has been shown to chemically

160

enhance feed intake together with glutamic, aspartic, citric and malic acids (Adams et al.,

1988). In the current experiment, there was no evidence of higher feed intake in fish fed

diets supplemented with lysine, but there was a trend to improved FCR in fish fed diets

where lysine was added alone (diet 2) or together with methionine (diet 5), threonine (diet

6) or both methionine and threonine (diet 8). The values for FCR of fish fed these diets

were not significantly different from that of the control fish (P > 0.05).

Data on the response of tilapia to dietary lysine supplementation are inconsistent.

In studies by Sintayehu et al. (1996) with sunflower cake, the addition of lysine and

methionine did not enhance fish performance. The initial weight of the fish used in the

above study was 93 g, which may explain the lack of response. Furthermore, the lysine

level in the basal diet was 2.3% (air-dry basis), which was higher than the stipulated

requirement (NRC, 1993; Jackson and Capper, 1982).

The lysine to protein ratios in the diets used in the current experiment are shown

in Table 6.3. In the basal diet and in diets 3 (basal + methionine) and 4 (basal +

threonine), the ratio was below 3.9%, while in diet 7 (basal + methionine and threonine),

the ratio was 4.53%. The ratio was above 5.5%> in all the other diets. Viola et al. (1994)

found that in hybrid tilapia weighing 80, 190 and 203 g respectively, supplementing diets

with lysine where the lysine to protein ratio was above 4.9%, did not enhance fish

performance, whereas lysine supplementation of diets where the ratio was below 4.4%o

improved growth rate. In the present experiment, the lysine to protein ratios in the basal

diet and diets which were not supplemented with lysine were below 4.4%. There was a

trend to increased growth rate and weight gain in fish fed diets supplemented with lysine,

to a level where they were not significantly different from those of fish fed the control

161

diet (P > 0.05). The lack of significant differences in fish performance over fish fed the

basal diet may have been due to the small sample sizes (n = 3) used to test for statistical

significance.

Supplemention of the basal diet with methionine alone did not have any effect on

fish performance. Methionine levels (% of protein) in the control diet and the other diets

supplemented with this amino acid were 3.07%, 3.83%, 3.38%, 3.49% and 4.25% in the

control diet, diet 3 (basal + methionine), diet 5 (basal +lysine and methionine), diet 7

(basal + methionine and lysine) and diet 8 (basal + lysine, methionine and threonine),

respectively. The lack of response was presumably because the methionine level in the

basal diet was adequate for fish growth. It is also plausible that diet 3 (basal +

methionine) and diet 8 (basal + lysine, methionine and threonine) contained excessive

levels of this amino acid. As explained earlier in the text, it was not possible to determine

amino acid composition of the diets before the onset of the experiment. One of the signs

of amino acid imbalances in animals is depression in feed intake (Harper et al., 1970;

D'Mello, 1994). There were no significant differences in feed intake between fish fed

diet 3 and diet 8 and those fed the other diets, indicating that the levels of methionine in

these diets were not sufficiently excessive to create an amino acid imbalance.

Different responses have been reported in tilapia fed diets supplemented with

methionine. In studies by El-Dahhar and El-Shazly (1993) with tilapia (O.niloticus)

(initial weight 16 g), the addition of methionine and lysine to diets where soybean meal

and cotton seed cake supplied 100%» of the dietary protein improved growth and FCR

slightly, but the values obtained were still lower than those attained with the fish fed the

fishmeal control diet. Shiau et al. (1987) observed a significant increase in weight gain

162

of tilapia when they were fed diets in which fishmeal and soybean meal supplied 70%

and 30% of the protein, supplemented with methionine. The methionine level in the

unsupplemented diets in the above study was 0.84% of diet (DM basis), and the average

initial weight of the fish was 1.24 g. Teshima and Kanazawa (1988) did not observe any

improvement in growth rate in tilapia fry weighing about 0.4g, when diets in which

soybean protein was the sole protein source, were supplemented with crystalline

methionine. However, when methionine was added as methionine-enriched soybean

plastein, the growth rate of tilapia was improved significantly to a level comparable to

that of fish fed the fishmeal control diet. The authors postulated that methionine in

soybean plastein is more effectively utilized by tilapia fry than crystalline methionine.

The growth responses of tilapia to methionine addition to diets varied in the studies

quoted above. The variation may have been caused by the different sizes of fish used in

the studies. It is also worth noting that in all of the studies quoted above, soybean meal

provided part or all of the dietary protein. Sunflower cake contains a higher level of

methionine than soybean meal (Jackson et al, 1982; NRC, 1993; Scott et al, (1982).

Santiago and Lovell (1988) reported that the dietary methionine requirement of juvenile

tilapia was 0.8% in diets containing 0.15% cystine, while Jackson and Capper (1982)

observed that the minimum methionine requirement for tilapia (O. mossamhicus) (1.7 g.)

was below 0.53% of the diet (DM basis), when the cystine level was 0.74%. The diets

used by Jackson and Capper (1982) had a high level of cystine, and the low methionine

requirement observed may indicate a high level of methionine sparing. In tilapia (O.

mossamhicus) weighing approximately 12.5g., Jackson et al (1982) reported that the

minimum requirement for methionine was 0.7%> of diet (DM basis), in the presense of

163

0.45% cystine ( D M basis). The methionine and cystine levels in the basal diet used in the

current experiment were 0.75% and 0.66%> of the diet (DM basis). The methionine level

was lower than that recommended by Santiago and Lovell. (1988), but it was higher than

the value reported by Jackson and Capper, (1982) and Jackson et al. (1982). The lack of

response was presumably because the methionine level in the basal diet was adequate,

especially in the in the presense of the high levels of cystine in the diets based on

sunflower cake (> 0.45%).

Methionine added with lysine elicited a positive response with respect to the

growth rate and FCR of fish in the current study. The values observed for growth rate

and FCR were not significantly different from those obtained with fish fed the basal diet,

but nevertheless they were higher than those observed when methionine or lysine were

added alone. It is also possible that methionine may have been marginally deficient, but

it was not the first limiting amino acid.

The threonine level in the basal diet was 1.05 % of the diet, while the stated

requirement for juvenile tilapia (O. niloticus) (Santiago and Lovell, 1988) is 1.12%

(DM). The basal diet may have been marginally deficient in this amino acid. Adding

threonine to the basal diet improved growth rate by approximately 6%, while adding it

together with methionine and lysine (diet 8), improved the growth rate by 7 %. The

growth rates were not significantly different from that of fish fed the basal diet (P > 0.05),

but they were also not significantly less than that of fish fed the control diet.

In chicken broilers fed diets containing sunflower meal as the only protein source,

threonine was identified as the second most limiting amino acid in high protein diets (37-

43%) CP) (DM), while lysine was the first limiting amino acid. Information on the dietary

164

threonine requirement of tilapia species has only been reported in one study (Santiago

and Lovell, 1988), for juvenile fish weighing less than 1 g. In the current study, it is

plausible that lack of response to dietary threonine supplementation may have been due to

the fact that lysine may also have been inadequate.

165

6.4 Conclusions

There was no response in growth rate or FCR in fish fed diets supplemented with

methionine alone, but there was some improvement when it was added together with

lysine, to a level that was not significantly different from that of fish fed the control diet.

N R C (1993) recommends a methionine level of 0.8% in diets containing 0.16% cystine

(DM basis), while Jackson and Capper (1982) determined the optimum methionine level

in the diet as 0.53% in diets with a cystine level of 0.73% of diet (DM basis). The

methionine level in the unsupplemented diets in the current study,was 0.75%, while the

cystine level was 0.66% of the diet. The level of sulfur amino acids in the diets was

higher than that recommended by Jackson and Capper (1982). In addition, the high level

of cystine in the diets may have spared methionine for growth, leading to a lower

requirement than stipulated by N R C (1993). It is therefore likely that methionine was not

the first limiting amino acid in the diets fed to tilapia in the current study.

Threonine added alone or together with lysine to the basal diet improved growth

rate and FCR of tilapia, but the values attained were not significantly different from those

obtained for fish fed the basal diet. There was very little response in weight gain or FCR

in fish fed diets supplemented with both threonine and methionine, suggesting that they

were not the first limiting amino acids.

From the observations presented above, it it is probable that lysine was the most

limiting amino acid in the basal diet, and possibly threonine may have been marginally

deficient in the diet as well.

166

6.5 References

Adams, M.A . , Johnsen, P.B., and Zhou, Hongi - Qi, 1988. Chemical enhancement of feeding for a herbivorous fish, tilapia zilli. Aquaculture, 72: (1-2) 95-107.

A O A C , 1984. Association of Official Analytical Chemists. Official methods of analyses. Animal Feed Section.

Attia, Y . A . , El-Deek, A . A , and Osman, M . , 1998. Evaluation of sunflower meal a a feedstuff in diets for ducks. Archiv. Fur Geflugelkunde, 62: (6) 273 - 282.

D'Mello, J.P.F., 1994. Amino acid imbalances, antagonisms, and toxicities. In: Amino acids in Farm Animal Nutrition. LP.D'Mello (Ed.) 418 pp.

El-Dahhar, A. A., and El-Shazly, K. , 1993. Effect of essential amino acids (methionine and lysine) and treated oil in fish diet on growth performance and feed utilization of Nile tilapia, Tilapia nilotica (L). Aquaculture and Fisheries Management, 24: 731-739.

El Sayed, A . F . M . , 1999. Alternative dietary protein sources for farmed tilapia, Oreochromis spp. Aquaculture, 179: 149-168.

El-Sayed, A .F .M. , 1998. Total replacement of fishmeal with animal protein sources in Nile tilapia (Oreochromis niloticus (L.) feeds. Aquaculture Research, 29 (4) 275-280.

El-Sayed, A . F . M . , and Teshima, S., 1992. Protein and energy requirements of Nile tilapia (Oreochromis niloticus) fry•. Aquaculture, 103: 55-63.

Fagbenro, O.A., and Jauncey, K. , 1994. Chemical and nutritional quality of dried fermented silage and their nutritive value for tilapia (Oreochromis niloticus). Animal Feed Sci. Technol., 45: (2) 167-176.

Green, S., and Kiener, T., 1989. Digestibilites of nitrogen and amino acids in soya bean, sunflower, meat and rapeseed meals measured with pigs and poultry. Animal Production, 48: (1) 157- 179.

Harper, A.E. , Benevenga, N.J., and Wohlhueter, R .M. , 1970. Effects of ingestion of disproportionate amounts of amino acids. Physiological Reviews, 50: 428-558.

Hinguera, D.L. , Akharbach, M . , Hidalgo, H , Peragon, M C , Lupanez, J., and Garcia Gallago M . , 1999. Liver and white muscle turn-over rates in the European eel (Anguila Anguila) effects of dietary protein quality. Aquaculture, 179: (1 - 4) 203 - 216.

Jackson, A.J., and Capper, B.S., 1982. Investigations into the requirement of the tilapia (Oreochromis mossambicus) for dietary methionine, lysine and arginine in semi synthetic diets. Aquaculture, 29: 289 - 297.

167

Jackson, A.J. , Capper, B.S., and Matty, A.J. , 1982. Evaluation of some plant proteins in complete diets for tilapia (O. mossamhicus). Aquaculture, 27: 97 - 109.

Jorgensen, H. , and Sauer, C.N., 1982. Amino acid availabilities in soybean meal, sunflower meal, fishmeal and meat and bone meal fed to growing pigs. Journal of Animal Science, 58: 926-934.

Kubaryk, J.M., 1980. Effects of diet, feeding schedule, and sex on food consumption, growth and retention of protein and energy by tilapia. Ph.D. dissertation, Auburn University, Auburn, A L .

Mansour, C.R., 1998. Nutrient requirements of red tilapia fingerlings. MSc. Thesis, Faculty of Science, University of Alexandria, Egypt 121 pp.

Mohme, H. , Toska, M . , and Gunther, K.D. , 1997. Sunflower seed meal as a protein source in nutrition of broilers. Fett-Lipid, 99: (3) 78-80.

National Research Council, 1993. Nutrient Requirements of Fish. National Academy Press, Washington, D . C , 144pp.

Santiago, C.B., Aldaba, M.B . , and Laron, M.A. , 1982. Dietary crude protein requirements of tilapia nilotica fry. Kalikasan, Phil. J. Biol., 11: 61 - 71.

Santiago, C.B., and Lovell, R.T., 1988. Amino acid requirements for growth of Nile tilapia. Journal of Nutrition, 111: 46-52

Sanz, A., Morales, A.E. , De La Higuera M . , and Carenete, G., 1994. Sunflower meal compared with soybean meal as a partial substitute for fishmeal in rainbow trout (Orchorhynchus mykiss) diets. Protein and energy utilization. Aquaculture, 128: 287 -289.

SAS Users Guide: Statistics, Version 5 t h Edition. 1985. SAS Inst., Inc., Caary, NC.

Scott, M L . , Nesheim, M . C and Young, R.J., 1982. Nutrition of the chicken. 3 r d Edition, M L . Scott and Associates, Ithaca, New York.

Senkoylu, N . , and Dale N . , 1999. Sunflower in poultry diets. A review. World's Poultry Science Journal Vol. , 55: 153 - 174.

Shiau, Shi-Yen, Chuang, Jan-Lung, and Sun, Chan-Lan., 1987. Inclusion of soybean meal in tilapia (Oreochromis niloticus x O. aureus) diets at two protein levels. Aquaculture, 65: 251-261.

Siddiqui, A.Q., Howlander, M.S., and Adams, A. A., 1988. Effects of dietary protein levels on growth, feed conversion and protein utilization in fry and young Nile tilapia (Oreochromis niloticus). Aquaculture, 70: 63 - 7 3 .

168

Sintayehu, A. , Mathies, E. , Mayer- Burgdorff, K . H . , Rosenow, H. , and Guenter, K .D. , 1996. Apparent digestibility and growth experiments with tilapia (Oreochromis niloticus), fed soybean meal, cotton seed meal and sunflower seed meal. Journal of Applied Ichthyology, 12: (2) 125 - 130.

Tacon, A.G.J. , Webster, J.L., and Martinez, C.A., 1984. Use of solvent extracted sunflower seed meal in complete diets for fingerling rainbow trout (Salmo gairdneri Richardson). Aquaculture, 43: (4) 381-389.

Tacon, A.G.J. , 1993. Feed ingredients for warm water fish. Fishmeal and other processed feedstuffs, FAO Fish. Circ , No. 856, FAO, Rome, Italy, 64pp.

Teshima, S., and Kanazawa, A., 1988. Nutritive value of methionine enriched plastein for Oreochromis niloticus fry. 2 n d Intl. Symp. on Tilapia in Aquaculture, 16 -20 March 1987. Bangkok, Thailand. 393 - 399.

Viola, S., Angeoni, H. , Gur, N . , and Lahav, E., 1994. Growth performance, protein and energy balances of hybrid tilapia fed two levels of lysine at three levels of protein. Israeli Journal of Aquaculture/ Bamidgeh, 46: (4) 212 - 222.

Viola, S., Arieli, Y . , and Zohar, G., 1988. Animal protein free feeds for hybrid tilapia (Oreochromis niloticus x O. aureus) in intensive culture. Aquaculture, 75: 115- 125.

169

Chapter 7

7.0 General discussion, conclusions and recommendations.

Sunflower cake, a by-product of the oil extraction industry is an inexpensive protein

source with potential for use in fish diets. In addition to its high protein content, it also

has a high fibre level, which reduces its digestible energy concentration. Fish respond to

low-energy diets by increasing their feed intake in an attempt to meet their energy

requirements. However, in some cases, this adjustment in feed intake is hindered by the

bulkiness of the diet. This is especially true for tilapia, which have a small stomach size

(Balarin, 1979). Based on this information from the literature, four experiments were

designed to investigate the effect of reducing the amount of fibre in sunflower cake on

nutrient digestibility and feed utilization, and to compare this fibre-reduced cake with the

commercially available high-fibre cake. The extent to which protein from both the high-

fibre and the fibre-reduced cake could replace fishmeal protein in tilapia diets was

investigated. The effects of supplementing diets made from a fibre-reduced sunflower

cake with the amino acids lysine, methionine and threonine singly or in combination on

growth, feed intake and FCR were also investigated.

Protein from both high-fibre and fibre-reduced sunflower cake was well digested

by tilapia. ADC-P of protein was not appreciably different between the two types of

cakes. A D C - E of energy improved with fibre-reduction from 30% in the high-fibre cake

to 42% in the fibre-reduced sunflower cake. Despite the low fibre content of the fibre-

reduced cake (11% Air-dry basis), the digestibility of energy was still poor compared to

the fishmeals. In rainbow trout, Sanz et al. (1994) found poor (40%) digestibility of

carbohydrates (NFE and fibre) in sunflower cake compared to soybean meal

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carbohydrates (50%). Similarly, in carp, Bendi and Spandorf (1953) observed very low

digestibility of carbohydrates in sunflower cake (26%). It was not possible to determine

the digestibility of all nutrients in this study. It is plausible that the digestibility of

carbohydrates in sunflower cake by tilapia (O. niloticus) may be low, which could have

contributed to the observed results. In this study, the digestible energy density of the

diets was increased by the addition of corn oil. Research has shown that adult tilapia

(over lOOg) have a limited ability to use high amounts of fat in the diet (Degani et al.,

1997). Al l the fish used in this study were below 90 g. There was no evidence of poor

utilization of oil.

The fibre-reduced cakes used in all of the experiments had higher levels of crude

protein and all of the amino acids that were measured than the high-fibre cake.

Sunflower cake is low in lysine and consequently diets based on this cake were relatively

low in lysine. They were also low in threonine.

When protein from both the fibre-reduced sunflower cake and the high-fibre cake

replaced 50% of the fishmeal protein, the weight gain of the fish receiving the fibre-

reduced cake was not significantly different from that of those fed the anchovy fishmeal

diets. Fish receiving the fibre-reduced cake also had a trend towards a higher growth

rate, feed intake, and a better FCR than those on the high-fibre diets. From Experiment

2, it was established that the protein from the fibre-reduced sunflower cake could

effectively replace up to 50% of the fishmeal without compromising the performance of

the fish.

In Experiment 3, the fibre-reduced and high-fibre sunflower cakes were tested

over a wide range of dietary inclusion, i.e., each supplied 30%, 60% and 80% of the

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dietary protein. It was found that the low-fibre cake could supply up to 60% of the

dietary protein without compromising fish performance. Feed intake and weight gain

were reduced when the cake was incorporated into the diet at higher levels. The high-

fibre sunflower cake could satisfactorily supply only 30% of the dietary protein. Higher

levels of this cake caused a reduction in feed intake and weight gain.

Experiment 4 was designed to investigate the effect of supplementing diets

containing high levels of the low-fibre sunflower cake with the amino acids, lysine,

methionine, and threonine. The levels of these amino acids were found to be low in diets

containing high levels of sunflower cake in Experiment 3. Lysine levels were low in

diets containing high levels (80%>) of sunflower cake. Besides its direct effect on growth,

lysine has been shown to enhance feed intake in tilapia (O. niloticus) (Adams et al.,

1988). There was a trend to improved weight gain and FCR with dietary lysine

supplementation, so that the values obtained were not significantly different from those of

the control fish.

Methionine, added singly had no effect, but there was a trend to improved growth

rate and FCR when it was added together with lysine, to a level comparable to that of the

control fish. Methionine and cystine levels in the unsupplemented diets were 0.75%> and

0.66% ( D M basis). N R C (1993) recommends a methionine level of 0.8% in diets with a

cystine level of 0.16% (DM basis), based on a study with juvenile tilapia (O. niloticus)

weighing less than 1 g. It is plausible that with the larger size of fish used in the current

study, and the higher level of cystine in the diets decreased the requirement tilapia for

methionine. Moreover, it appears that methionine was not the first limiting dietary amino

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acid for the fish in this study. This may account for the observed trend of improved

growth rate and FCR when lysine was added together with methionine.

Threonine, added alone or together with lysine improved growth rate and FCR by 6% and

10% respectively, but there was no response when it was added together with methionine.

It is not clear why this situation arose. The threonine level in the basal diet was 1.07 %

(DM basis), while the stipulated requirement (NRC, 1993) is 1.12% of the diet (DM

basis). There is only one study on the threonine requirement of tilapia (O. niloticus)

(Santiago and Lovell, 1988), which was done with tilapia fry weighing less than 1 g. It is

plausible that the threonine level in the diet was adequate for the fish size used in the

present study, or that threonine was not the first limiting amino acid in the diets.

The fatty acid compositions of the diets were determined and compared with the

whole body fatty acid compositions of the fish. Earlier studies (Kanazawa et al., 1980;

Tekeuchi et al., 1983) established that the only essential fatty acid required by tilapia is

linoleic acid (18:2 co 6), and that they possess the enzymes that are required to desaturate

and elongate fatty acids of the co 6 and co 3 series to make the long chain highly

unsaturated fatty acids viz., 20: 4 co 6, 20: 5 co 3 and 22: 6 co3 of nutritional significance.

For that reason, many formulated diets for tilapia do not contain a source of the long

chain poly-unsaturated fatty acids. Recently, Chou and Shau ( 1999) observed significant

increases in the co 3 fatty acids in tilapia muscle and liver by feeding cod liver oil. In this

study, the fatty acid composition of the fish reflected the dietary fatty acid composition.

In view of the recent studies that have shown the beneficial effect of the long chain co 3

fatty acids for humans, it may be worthwhile to consider adding these fatty acids to the

tilapia diets to improve the nutritional quality of the flesh.

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One of the primary objectives of this work was to evaluate locally produced raw

materials (high-fibre sunflower cake, fibre-reduced sunflower cake and omena fishmeal)

as replacements for imported fishmeal in the diets of tilapia (O. niloticus). Omena

fishmeal, made from a cyprinid fish Rastrineobola argentea is used widely in the animal

feed industry in Kenya, but there is no documented information on its quality and feeding

value. In this study, the digestibility of nutrients and energy and the feeding value of

Omena fishmeal were compared to that of prime quality anchovy meal at two levels of

protein intake. Apparent digestibility coefficients for protein, energy and organic matter

in omena fishmeal were comparable to those of anchovy fishmeal. In Experiment 2, the

determined CP level for the diets based on omena fishmeal at both protein levels were

slightly lower than the calculated (expected) values, which may have been caused by an

error during the mixing of the diets. Despite this, there were no significant differences in

the growth rate and FCR in fish fed diets based on the two fishmeals. Generally, omena

fishmeal did show some promising results. There is a need for a more detailed study of

this fishmeal, especially the processing methods that are cost effective and that produce a

high quality fishmeal.

The fish did well on most of the ingredients tested. Reducing fibre in sunflower

cake improved the growth rate and FCR of the fish to levels that were not significantly

different from those of the control fish. In Kenya, labour costs as a percentage of the

total cost of production are relatively low, and the feed cost is the major component of the

variable costs. FCR is therefore a useful measure of feed quality. Replacing high- cost

herring meal and soybean meal with low cost ingredients such as omena fishmeal, fibre-

reduced sunflower cake and high-fibre sunflower cake would reduce the importation of

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expensive dietary ingredients, and this in turn, will reduce the cost of tilapia culture

Kenya

175

7.1 References

Adams, M.A . , Johnsen, P.B., and Zhou, Hongi - Qi, 1988. Chemical enhancement of feeding for a herbivorous fish, tilapia zilli. Aquaculture, 72: (1-2) 95-107.

Balarin, J.D., 1979. Biological characteristics of tilapia In: Tilapia, A guide to their biology and culture in Africa. J.D. Balarin and J.P Hatton (Eds.), University of Stirling, U.K. 174pp.

Bendi, A. , and Spandorf, A., 1953. The activity of digestion enzymes of carp. Bamidgeh, 5: 116-130.

Chou, B.S., and Shiau, Shi-Yen., 1996. Optimal dietary lipid level for the growth of juvenile hybrid (Oreochromis niloticus x Oreochromis aureus). Aquaculture, 143: 185-195.

Degani, G., Viola, S., Yehuda, Y. , 1997. Apparent digestibility of carbohydrates in feed ingredients for adult tilapia (O. aureus x O. niloticus). Israel Journal of Aquaculture/Bamidgeh, 49: 115-123.

Kanazawa, A., Teshima, S. I , Sakamoto, M . and Awal, M.A. , 1980. Requirements of tilapia zilli for essential fatty acids. Bull. Jap. Soc. Sci. Fish., 46: 1353-1356.

N R C (National Research Council) 1993. Nutrient requirements for fish. National Academy Press. Washington, D.C., U.S.A. 114pp.

Santiago, C.B., and Lovell, R.T., 1988. Amino acid requirements for growth of Nile tilapia. Journal of Nutrition, 118: 1540-1546.

Sanz, A., Morales, A.E. , HigueraM., Cardenete, G., 1994. Sunflower meal compared with soybean meal as partial substitute for fishmeal in rainbow trout (Onchorhyncus mykiss) diets: protein and energy utilization. Aquaculture, 128: 287-300.

Tekeuchi, T., Sitoh, S., and Watanabe T., 1983. Requirements of tilapia nilotica for essential fatty acids. Bull. Jpn. Soc. Sci. Fish., 49: 1127 - 1134.

176

i

Appendix 1. Kabete water quality parameters.

Parameters Unit of measurement Results

pH pH scale 7.2

Color mg pt/1 Less than 5

Turbidity N . T . U 7

Manganese mg/1 Less than 0.1

Calcium mg/1 16.8

Magnesium mg/1 4.86

Sodium mg/1 30

Potassium mg/1 8.8

Aluminium mg/1 -

Chlorides mg/1 29

Fluoride mg/1 0.2

Nitrates mg/1 -

Nitrites mg/1 Less than 0.01

Ammonia mg/1 -

Total nitrogen mg/1 -

Sulfates mg/1 5.33

Orthophospate mg/1 Less than 0.01

Total suspended solids mg/1 -

Free carbon dioxide mg/1 12

Total dissolved solids mg/1 210

Residual chlorine mg/1 Less than 0.1

177