FARMING OF TILAPIA

Breeding Plans, Mass Seed Production and Aquaculture Techniques

ii

FARMING OF TILAPIA
Breeding Plans, Mass Seed Production and Aquaculture Techniques

M.G. Hussain
BSc Fisheries (Hons), MSc Aquaculture & Management, PhD Aquaculture Genetics (Stirling, UK)

Director, Research & Planning

Bangladesh Fisheries Research Institute

Mymensingh 2201, Bangladesh

iii

FARMING OF TILAPIA
Breeding Plans, Mass Seed Production and Aquaculture Techniques
First Edition 2004 M.G. Hussain 2004 Hussain, M.G. 2004. Farming of tilapia: Breeding plans, mass seed production and aquaculture techniques. 149 p.

Published by Habiba Akter Hussain 55 Kristawpur, Mymensingh 2200 Bangladesh

ISBN 984-32-1839-6

Cover and other photos by M.G. Hussain Printed by Momin Offset Press, Dhaka, Bangladesh

Price: Taka 300 (Three Hundred) Outside Bangladesh: US$ 15

iv

Dedication

The author dedicates this book to his family, especially to his wife Habiba Akter Hussain and sons Sazzad Hussain and Ali Hussain without whose love, patience, kindness and encouragement this book could never have been completed

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Contents
List of figures List of tables Preface Acknowledgements Chapter 1 1.1 1.2 1.3 Chapter 2 2.1 2.2 2.3 2.4 Chapter 3 3.1 3.2 3.3 3.4 Chapter 4 4.1 Introduction Importance and potential of tilapia species in aquaculture Tilapia species introduction in Bangladesh Performance and potentials of the GIFT strain of Oreochromis niloticus General and reproductive biology of tilapia Taxonomic classification Generic groups of tilapias General biology of Nile tilapia Breeding and reproductive biology of Nile tilapia x xiv xv xvii 1 1 2 3 9 9 9 10 11

Brood stock replacement and breeding plans for 18 tilapia hatchery stocks Brood stock management to avoid genetic stock deterioration Brood stock replacement techniques Breeding plan and genetic stock improvement of tilapia Maximizing the effective population size (Ne) Ploidy manipulation and production of all sterile, female and male population Genomic status and determination of sex
vii

18 19 20 30 31 31

4.2 4.3

4.4 4.5 Chapter 5 5.1 5.2 5.3 5.4 Chapter 6

Production of genetically induced all sterile population 4.2.1 Induction of polyploidy Production of genetically induced all female population 4.3.1 Induction of meiotic gynogenesis 4.3.2 Induction of mitotic gynogenesis Production of genetically induced all male population Protocols for chromosome karyopyting Body colour inheritance and development of purebred strains of red tilapia Inheritance of body colour in commercially available strains Importance and problems associated with the present stocks Mechanisms of progeny testing to develop purebred strains of red tilapia Maintenance of purebred brood stock for seed production in the hatchery Development and operation of mixed sex commercial tilapia seed production systems Mixed sex tilapia seed production in ponds Mixed sex tilapia seed production in concrete tanks Mixed sex tilapia seed production in hapas Mixed sex tilapia seed production in rice fields Development and operation of monosex commercial tilapia seed production systems Sex reversal technique for the production of monosex fish fry
viii

32 32 38 38 39 42 42 45 45 46 48 51 53

6.1 6.2 6.3 6.4 Chapter 7 7.1

53 56 59 61 63 63

7.2

All male monosex seed production through inversion of sexes in tilapia 7.2.1 Hatchery design and operation of monosex seed production systems 7.2.2 Production of YY males and operation of monosex all male seed production system Development and operation of semi-intensive tilapia culture systems Tilapia culture in seasonal ditches and ponds Tilapia culture in rice fields Polyculture of tilapia with carps Tilapia culture in pens Tilapia culture in ponds under commercial farming management Development and operation of intensive tilapia culture systems The suitability of tilapia for intensive culture Tilapia culture in cages Tilapia culture in tanks and raceways 9.3.1 Tilapia culture in tanks 9.3.2 Tilapia culture in raceways Diseases and parasites of tilapia and their control measures Marketing of tilapia Strategies and prospects of frontier development of tilapia aquaculture

63 66 78 86 86 87 90 91 93 102 102 103 107 108 110 113 121 126 130 134 144

Chapter 8 8.1 8.2 8.3 8.4 8.5 Chapter 9 9.1 9.2 9.3

Chapter 10 Chapter 11 Chapter 12 Glossary References Index

ix

List of figures
1 2 3 4 5 Mozambique tilapia, Oreochromis mossambicus Nile tilapia, Oreochromis niloticus Red tilapia strain Genetically Improved Farmed Tilapia (GIFT) strain a. Genital papilla of male Oreochromis niloticus having two opening; the urogenital opening, where the milt and urine are excreted and the anus, for the discharge of fecal waste. b. Genital papilla of female Oreochromis niloticus having three openings; the anus, the urethra for urine passing and the oviduct, where egg passes through Histological section of an ovary shows various stages of development at peak maturation of female Oreochromis niloticus Histological section of a testis shows various stages of development at peak maturation of male Oreochromis niloticus A simple tilapia egg incubation system having a. plastic water bottles and b. medium type of trays connected to the recirculating system Floy tagging underneath the scale below the dorsal fin and above the lateral line of tilapia Plastic numbered tags with nylon thread PIT tagging into the visceral cavity of tilapia
x

4 4 5 5 13

16

16

25

9 10 11

28 28 29

12 13 14 15

A schematic diagram of inducing polyploids in O. niloticus using pressure, heat and cold shocks 1 1.5 L vessel capacity pressure apparatus Thermostatically regulated 50 L capacity water bath A schematic diagram of inducing two types of gynogenesis (meiotic and mitotic) in Oreochromis niloticus using pressure, heat and cold shocks Metaphase chromosome of Oreochromis niloticus. a. haploid (n = 22), b. diploid (2n = 44), c. triploid (3n = 66), d. aneuploid metaphase (hyperhaploid or hypodiploid) Purebred red tilapia Impure blotched type tilapia of red phenotype Tilapia fry holding hapas Low cost tilapia breeding and fry rearing tanks Tilapia egg incubation and hatching system model Typical modern monosex tilapia seed production hatchery system in Thailand Tilapia breeding hapas in pond Gathering tilapia breeders at regular intervals for egg collection purpose in the breeding hapa Plastic vowels placed in a bamboo frame for separating the collected fertilized eggs having different colours (based on different age groups)

33 35 35 38

16

44

17 18 19 20 21 22 23 24 25

52 52 57 57 68 68 73 73 74

xi

26 27 28 29 30 31 32

A series of round bottom plastic jars and flat trays for incubating the fertilized eggs/hatched fry with yolk sac Separate flat trays where hatched larvae are kept until their yolk sac resorption stage is over The protocol for preparation and application of hormone mixed feeds for sex reversal of Nile tilapia fry Automatic hormone feed mixing machine The technique of application of hormone mixed feeds to the first feeding fry in the transitory hapas Manual counting of tilapia fry a. Hapas can be installed in the pond and fixed and tied to nylon ropes and bamboo poles for feeding hormone mixed feeds; b. Hapas can be installed and fixed with RCC frame made over the pond for feeding hormone mixed feeds The protocol for feeding of hormone mixed feeds The technique of application of hormone mixed feeds to the early fry in the nursery hapas The protocol for sex identification in tilapia fry The protocol of producing all male monosex population through the indirect method of sex reversal The monk in a pond A typical layout of a fish arm View of a commercial fish farm

74 75 75 79 79 80 80 81 81 82 82 85 100 100 101

33 34 35 36 37 38 39

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40 41 42 43 44 45 46 47 48 49 50 51

Simple paddle wheel type of aerators set in the ponds for aeration of water to add more oxygen The floating rafts with net cages for intensive tilapia culture The cemented tanks for intensive tilapia culture Race ways for intensive tilapia culture Abdominal dropsy in tilapia Protozoan parasite Chilodonella sp. Protozoan parasite Trichodina sp. Fish lice Argulas sp. Washing of harvested live tilapias in the holding tank with the inflowing cool water before marketing Tilapias in the retailing fish market for the consumers Tilapia is a fish of the decade Tilapia is a good food fish

101 106 109 111 116 117 118 119 123 123 129 129

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List of tables
1 Serum calcium concentration and steroid hormone levels in mature female and male O. niloticus. All values are mean SE estimated from an equal number (n=10) of fish in each category Inbreeding resulting from some matings between closely related individuals Correlation between effective population size (Ne) and rate of inbreeding in a hatchery Polyploidy induction in various Oreochromis spp. using pressure, heat and cold shocks Gynogenesis induction in various Oreochromis spp. using pressure and heat shocks Formulated feed for feeding tilapia fry in rearing hapas and nursery ponds Formulated feed for feeding tilapia under semi-intensive system in the grow out ponds Available data on tilapia cage culture in different countries Available data on tilapia culture in tanks in different countries 17

2 3 4 5 6 7 8 9

19 30 37 41 61 98 107 110 112

10 Available data on tilapia culture in raceways in different countries

xiv

Preface
In the context of declining trends both in inland and marine capture fisheries, aquaculture is the most promising option for increasing fish production. In addition to earning profits, aquaculture can improve the livelihoods and nutrition of the resource-poor rural people in the region. In fact, promotion of aquaculture of Indian and Chinese major carp and shrimp species has taken place for many years in the developing countries of this part of Asia, like Bangladesh, India, Nepal, Pakistan and Sri Lanka. Although production was promisingly increased especially for carps through adoption of improved technologies, but these could not be diversified out of freshwater areas. On the other hand, mass involvement of rural people in carp and shrimp culture was found difficult due to their limited water resources and financial incapability in many cases. Like Thailand and Vietnam, recently farming of riverine catfish (Pangasius sutchi) has dramatically increased in Bangladesh. However, feed crisis and low market prices have severely damaged the progress of farming of this fish in the country. On the other hand, improved extensive shrimp culture is in collapse due to disease outbreaks in recent times. Under such conditions, a large number of commercial catfish and shrimp producers are looking for alternative species to culture in their farms to maximize the production. Of the available farmed species, tilapias are among the best candidates to overcome the situation due to their desirable characteristics like ease of seed production, high yield, disease resistance and efficiency of growing well with organic and agricultural wastes and low cost feeds. In spite of the promising future of tilapia farming in Bangladesh, it took a long time to realize the fact, due to some negative attitudes of the respective organization(s) and decision makers. Although the best tilapia farming species like Nile tilapia (Oreochronis niloticus) was introduced in this country in 1974, but it was not clear that the species is highly potential and productive for suitable water bodies until the Bangladesh Fisheries Research Institute (BFRI) discovered the truth through introduction of GIFT strain (1994) and subsequently conducted intensive research and developed the super strain of GIFT. Tilapia farming is gaining popularity day by day in Bangladesh and a number of entrepreneurs have already initiated its hatchery development
xv

for commercial mixed and monosex seed production and farming in different parts of the country. It has been felt that very little readily available information on farming practices of tilapia is available in Bangladesh and elsewhere in this region. Therefore, an attempt has been made to prepare a comprehensive handbook and publish it initially in English for the national and international readers. This book highlights the importance of tilapia species in aquaculture, the performance of the GIFT strain of Oreochromis niloticus, general and reproductive biology of tilapia, brood stock replacement and development of outbred and genetically improved hatchery stocks of tilapia in the Chapter 1, Chapter 2 and Chapter 3. Simple classical biotechnological tools to develop genetically induced all sterile, female and male populations in tilapia and production of purebred red tilapia strains are detailed in Chapter 4 and Chapter 5 . In Chapter 6 and Chapter 7, special emphasis has been given to simple techniques for design and operation of mixed and monosex mass seed production systems. Detailed development and operation of semi-intensive systems, intensive systems of tilapia culture, disease and parasites of tilapia and their control measures and marketing of tilapia are presented respectively in Chapter 8, Chapter 9, Chapter 10 and Chapter 11. All these chapters are designed for progressive fish farmers and entrepreneurs. Finally in Chapter 12, a note on future strategies and prospects of frontier development of tilapia aquaculture is highlighted. I have tried my best to invest my knowledge on the subject in compiling the best information on tilapia farming in this book, which I believe will be useful as a guide to hatchery operators, entrepreneurs, progressive farmers, researchers and planners developing programs for simple breeding, stock improvement, mass seed production and various aquaculture techniques of the fish in Bangladesh and elsewhere in Asia where tilapias are being used for promotion of aquaculture. M.G. Hussain
xvi

Acknowledgements
The author wishes to acknowledge the kindness of all of his good friends and colleagues, who have provided information, materials and photographs for use in this book. These persons are: Dr. R.S.V. Pullin, Dr. M.V. Gupta, Dr. A.E. Eknath, Dr. R.A. Dunham, Dr. M.M. Dey, Dr. G. C. Mair, Dr. R.W. Ponzoni, Drs. J. Janssen, Prof. B.J. McAndrew, Dr. M. Karim, Dr. A. Wahab, Dr. R.I. Sarder, Dr. M.J. Alam, Dr. A.H.M. Kohinoor and Mr. S. Islam. Dr. E. Hoq assisted and formulated the overall design of the book. Special thanks are due to Dr. M.A. Mazid, Director General of Bangladesh Fisheries Research Institute for his kindness to allow me for using many materials from the institute library and stations including his valuable suggestions and encouragement. Dr. Nuanmanee Pongthana, the former Director, National Aquaculture Genetic Research Institute, Thailand guided me to collect information and design of monosex tilapia hatchery from private tilapia hatchery entrepreneurs of several provinces in Thailand. She also provided a number of her diagrams and photographs for this document. I am also indebted to Mr. Yong Kim Thai, Managing Director of PKPS Farm Mart, Selangor, Malaysia for his kind permission to use some of his farm photographs in this book. All the assistance and support from the tilapia hatchery and farming entrepreneurs of Bangladesh viz. NIRIBILI Group, Shubra Hatchery Group, Bismillah Hatchery Group, Muktagacha Fisheries, Poultry and Dairy Farms Ltd. and Riliance Aqua Ltd. are greatly acknowledged. I owe my greatest debt to my lovely wife Habiba Akhter Hussain (Koli) for her continuous support and encouragement until the completion of this book. Above all my thanks go to Dr. David J. Penman, Fish Genetics and Reproduction Research Group, Institute of Aquaculture, Stirling University, Scotland, who provided a wealth of information and critically reviewed and improved the first draft of the manuscript.
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Farming of Tilapia

1
1.1

Introduction
IMPORTANCE AND POTENTIAL OF TILAPIA SPECIES IN AQUACULTURE

Tilapias are a group of Cichlid fish native to African countries. In the early days of the 20th century, tilapias were wild fish in the great lakes and rivers of that continent. In the central African countries, farming of tilapias in ponds was introduced after Second World War. After that the tilapia species were spread over most of the tropical and sub-tropical countries of the world. In recent years, commercial farming of several species of tilapia has become a common practice in aquaculture throughout several regions of the world such as China, SE Asia, Africa, USA and Latin America/Caribbean (Vannuccini 1998). According to FAO, the total world production of tilapias (wild and aquaculture) has increased from 37, 500 mt in 1950 to 1, 265, 800 mt in 2000. While widespread introductions have provided the mechanism for expansion of tilapia culture, effective management of reproduction is the primary factor that has been instrumental in the realization of their aquaculture potential in the later half of the 20th century (Shelton 2002). Although the important natural tilapia genetic resources are in Africa, the major aquaculture industries at present are in Asia. About 989,899 mt tilapia were produced in Asia in 1999 of which 62.6% came from China. Other countries like, Thailand (151,647 mt), Philippines (99,724 mt), Indonesia (86,930 mt) and Sri Lanka (31, 450 mt) are the major tilapia producing countries in Asia (Guerrero 2002). A total of about 70 species of tilapia have been so far listed as native to Africa (Anon 1984). Only a few species are suitable and popular for farming in ponds and other culture systems, which include Nile tilapia (Oreochromis niloticus), Blue tilapia (O. aureus), Mozambique tilapia (O. mossambicus), three spotted tilapia (O. andersonii), longfin tilapia (O. macrochir), Galilee tilapia (Sarotherodon galilaeus), blackchin tilapia (S.

Farming of Tilapia

melanotheron) and redbelly tilapia (Tilapia zillii). There are also some genetically improved strains such as Genetically Improved Farmed Tilapia (GIFT), red tilapia strains and hybrids. Pullin (1983) compared the attributes of various species with culture potential; he suggested concentrating research efforts on the Blue tilapia and Nile tilapia. While the former is still used to produce hybrids, it has been effectively left behind as the Nile tilapia (O. niloticus) has taken the lead as the principal species (cited by Shelton 2002). Above all, O. niloticus has been recognized as the prime domesticated species for farming in a wide range of aquaculture systems from simple waste-fed fishponds to intensive culture systems (ICLARM 1991). In the First International Symposium on Tilapia in Aquaculture (May 1983, Nazareth, Israel) Drs. Liao and Chen concluded that Tilapia is no longer an African fish but an International fish. It is believed that in the future it may become the most important fin fish in the world. Vannuccini (1998) stated Tilapia, once considered a low value fish, only suitable for the ethnic market, has in recent times gained wider consumer acceptance and is now considered an attractive menu item in chain restaurants. Tilapia has also been described as the important aquaculture species of the 21st century, which being grown on commercial farms in 100 countries of the world from extensive to super-intensive. It remains to be seen whether the food fish of the 21st century will surpass production of the carps in aquaculture during the new millennium (Fitzsimmons 2000).

1.2

TILAPIA SPECIES INTRODUCTION IN BANGLADESH

Among tilapias, Mozambique tilapia, O. mossambicus (Figure 1) was the first species, which was introduced into Bangladesh from Thailand in 1954. The fish did not flourish and proved to be a pest due to its early maturation and prolific breeding habits in the ponds. As a result, producers
2

Farming of Tilapia

and consumers regarded the fish as nuisance fish. During the 1970s a renewed interest in tilapia culture developed in some Asian countries including Bangladesh with the introduction of Nile tilapia, O. niloticus (Figure 2). Overall performance of Nile tilapia and other fast growing tilapias have proved that they are no longer pests but have come to be known as aquatic chicken. In 1974, the Chitralada strain of Nile tilapia, a promising farmed species, was introduced into Bangladesh from Thailand through UNICEF. The Bangladesh Fisheries Research Institute (BFRI, formerly FRI) initiated the second introduction of the fish in this country, also from Thailand, in 1987. Meanwhile, a red mutant tilapia (Figure 3), a hybrid between albino O. mossambicus x O. niloticus, was developed in Taiwan and introduced into Thailand. In 1988 Drs. M.G. Hussain and S. Dewan brought a batch of this red strain of tilapia to Bangladesh from the Asian Institute of Technology (AIT), Bangkok, Thailand. Under the Dissemination and Evaluation of Genetically Improved Tilapia in Asia (DEGITA) project of WorldFish Center (Formerly ICLARM), another promising Genetically Improved Farmed Tilapia (GIFT) strain (Figure 4), a synthetic strain of O. niloticus, was introduced in July 1994 from the Philippines.

1.3 PERFORMANCE AND POTENTIAL OF THE GIFT STRAIN OF OREOCHROMIS NILOTICUS

The GIFT strain was developed by the International Center for Living Aquatic Resources Management (ICLARM) through several generations of selection from a base population involving eight different strains of Nile tilapia, O. niloticus (Eknath et al. 1993). In on-station trials, the synthetic GIFT strain was reported to show on an average 60% faster growth and 50% better survival at harvest than the most commonly farmed strain in the Philippines, the 'Israel strain (Eknath 1992). For evaluating this strain in other countries of Asia, a research project was initiated in Bangladesh, China, Philippines, Thailand and Vietnam under the auspices of a WorldFish Center's project entitled "Dissemination and Evaluation of
3

Farming of Tilapia

the Genetically Improved Tilapia in Asia (DEGITA, Asian Development Bank (ADB) Technical Assistant Grant Project: RETA No. 5558)".

Fig. 1 Mozambique tilapia, Oreochromis mossambicus.

Fig 2 Nile tilapia, Oreochromis niloticus.

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Fig. 3 Red tilapia strain.

Fig. 4 Genetically Improved Farmed Tilapia (GIFT) strain.

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1.3.1 Growth and production performance

In Bangladesh, comparative growth and production potential of GIFT and existing Nile tilapia strains (O. niloticus) was evaluated both under onstation and on-farm conditions. Comparative production performance of GIFT and existing O. niloticus strains was assessed in five test environments (i.e. nursery systems, cisterns, hapas, net cages and growout ponds) and six multi-locational sites (i.e. Trishal, Chandina, Manikganj, Paikgacha, Jessore Sadar and Mithapukur) respectively under on-station and on-farm conditions (Hussain et al. 2000). In on-station ponds, the mean final weights of GIFT and existing strains were 134.42.4 g and 85.32.4 g, respectively. The average gross production of GIFT and existing strains were estimated at 4,411 and 2,966 kg/ha/6 months, respectively. Total yield of the GIFT was significantly higher (57% more; P<0.05) than that of the existing strain. The growth performance expressed as least square mean (LSM) values of body weight at harvest of individual strains across different environments revealed that GIFT was consistently better performing than the existing strain. In multi-locational on-farm trials in ponds, at harvest, the average production of the GIFT strain per unit area in the on-farm conditions was also found to be significantly (P<0.05) higher (52%) than that of the existing local strain. In other DEGETA countries viz. China, Indonesia, Thailand and Vietnam, where there are longer histories of tilapia farming, greater climatic variation, and therefore, the possibility of both natural and artificial selection of local strains to their environments, in on-station conditions, the GIFT strain appears to be about 10-15% superior to local strains in terms of growth (Dey 1996). On the other hand, in on-farm conditions, the GIFT strain gave about 15 to 25% higher yield than the other strains like 1978 introduced, 1988 introduced and hybrid strain of China, 15 to 20% higher yield than that of the Chitralada strain of Thailand; and 18 to 40% higher yield in comparison to the local strains of Vietnam.

Farming of Tilapia

1.3.2 The suitability of GIFT strain for seed production and aquaculture
The GIFT strain has, meanwhile, proved to be very suitable fish for aquaculture in Bangladesh and other DEGITA countries. The desirable characteristics of this genetically improved strain are as follows: High yielding Excellent breeder Efficient converter of organic and agricultural wastes in to high quality protein Resistant to disease Very hardy Tolerant to over crowding conditions Able to grow in either fresh or brackish water

The GIFT strain of O. niloticus is being presently widely used for mono sex seed production in a large number of tilapia hatcheries in Thailand (Dr. Nuanmanee Pongthana, personal communication).

1.3.3 Further genetic selection and development of superior strain

BFRI has been maintaining the gene pool of pure GIFT strain since its introduction and has enhanced the stock by introduction of new generations from the Philippines in 1996. Subsequently, further stock improvement has also been initiated using selective breeding technique. Evaluation of growth performance was carried out through comparative trials between selected and non-selected average control (existing stock of GIFT) groups. A 5.0 % genetic gain in growth performance was attained by the F1 generation over the non-selected control group. The F2, F3 and F4 selected generations attained 2.9%, 12.5% and 6.9% cumulative weight gain, respectively, over three generations. The average gain per generation across four generations of selection for growth performance in weight was estimated 6.8%. The weight gain values of 4th generation of
7

Farming of Tilapia

the selected group showed 27.3% superiority over the non-selected GIFT control. Due to excellent performances for growth and other relevant traits (survival, fecundity and disease resistance) the new strain can be termed as Super Strain of GIFT (ie. BFRI Super GIFT). The new super strain is gaining popularity day by day in Bangladesh and a number of entrepreneurs have already initiated its hatchery development for commercial mixed and monosex seed production and farming in several regions of the country. To date, tilapias have yet to be farmed widely in Bangladesh, because interest in tilapia farming is growing due to its success in other Asian countries and increasing consumer acceptance (ADB 2004).

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General and reproductive biology of tilapia

TAXONOMIC CLASSIFICATION

2.1

Tilapias are predominantly freshwater finfishes often some species characterized with nest building and surface or mouth brooding habits. The taxonomic classification of the Nile tilapia is given below: Phylum: Subphylum: Class: Order: Family: Genus: Species: Common name: Chordata Vertebrata Osteichthyes Perciformes Cichlidae Oreochromis Oreochromis niloticus Linnaeus Nile tilapia

2.2

GENERIC GROUPS OF TILAPIAS

The fishery scientists and taxonomists change the generic names of tilapias from time to time on the basis of research on their various characters but mainly by spawning and breeding behaviors. Dr. E. Trewavas, a Senior Tilapia Taxonomist of British Museum (Natural History) made a thorough review of natural history of African cichlid species in her book Tilapiine Species and showed the basis for distinguishing Tilapia, Sarotherodon and Oreochromis as genera. According to Trewavas (1983) the three main distinct generic groups of tilapias are as below: Genus Tilapia: Includes those species which are substrate spawners and do not keep the eggs in the mouth for incubation, e.g. T. zillii.
9

Farming of Tilapia

Genus Sarotherodon: Includes those species which are mostly paternal mouth brooders and sometimes eggs and hatched larvae are brooded by both parents, e.g. S. galilaeus, S. melanotheron. Genus Oreochromis: Includes those species which are exclusively maternal mouth brooders. In this group the males construct and defend a mating territory in an arena with other males in adjacent terrirories, and females come to find spawning partners, e.g. O. niloticus, O. aureus, O. mossambicus.

2.3

GENERAL BIOLOGY OF NILE TILAPIA

2.3.1 Distinguishing characters

Body elongate and deep, covered with moderately large scales. Scales in lateral line series are 30-34; usually these scales remain around 31-33. The numbers of gill-rakers are 20 26, which are available just on the lower part of the first arch. Number of dorsal spines are 17. Unlike other cichlid fish, the body colour of the males is more attractive than females. Caudal fin covered with narrow vertical stripes, the upper margin of dorsal fin black or gray. Both sexes at breeding time show red flush on the belly and lower flanks, which is brighter in male. In particular, the dorsal and caudal fin margins of the males become bright red.

2.3.2 Tolerance to ecological conditions

The Nile tilapia, is a very hardy fish and can thrive a wide range of aquatic ecological conditions from purely freshwater to brackish or semi saline waters. The suitable ranges of water quality parameters under which the fish can survive well are:
10

Water temperatures 12 to 35 oC; pH 6.5 8.0;

Farming of Tilapia

Dissolve oxygen 2.0 8.0 mg/l; Salinity 3 - 25 ppt.

2.3.3 Growth
In tropical pond waters under semi-intensive culture management, the Nile tilapia can grow to 150 250 g in 4 to 6 months, 500 to 800 g in 10 to 12 months and 2 3 kg in 2 years. In the great lakes of Africa, several scientists (Worthington and Ricardo 1936; Lowe-McConnell 1958) recorded maximum sizes of 61- 64 cm, weights 4-7 kg.

2.3.4 Food
O. niloticus is capable of using a wide range of food materials from tiny plankton (phytoplanton and zooplankton) to macrophytes. It grows well on artificial feeds. The young fry are omnivorous, actively pursuing copepods, hydracarines and various insects, both aquatic larvae and terrestrial insects that fall on the water (Trewavas 1983).

2. 4

BREEDING AND REPRODUCTIVE BIOLOGY OF NILE TILAPIA

2.4.1 Breeding habit and natural spawning (a) Sexual dimorphism

Sexual dimorphism is common in Nile tilapia. The males are larger than females. It is reported that the males become 20 30% heavier in weight in comparison to the females (Hussain and Kohinoor 2003).

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Farming of Tilapia

(b) Morphology of sexual organs

The genital papilla of male is larger than that of the females and has 2 openings (Velasco 2003a); the urogenital opening, where the milt and urine are excreted and the anus, for the discharge of fecal waste (Figure 5a). The female has a flatter and shorter papilla with 3 openings; the anus, the urethra (for excretion of urine) and the oviduct, where eggs pass through (Figure 5b).

(c) Sexual maturation of brood fish

The Nile tilapia sexually matures at the age of 3 months, when it attains a body weight of around 40 60 g (Hussain 1989). In Bangladesh, it breeds from February to November when water temperatures remain around 22 30 oC.

(d) Nest building for natural breeding

For natural breeding males build nests on the substratum in shallow regions of stagnant water bodies/ ponds to attract the females for spawning. The nest has a circular shape with a depression from 0.2 to 0.3 meter in diameter and 0.1 to 0.3 meter deep. It is also recorded that the nest is about twice the length of the male making it (Hussain 1989).

(e) Mating and spawning activity

In most of the tilapia species, prespawning courtship behavior between males and females is observed. They choose partners of more or less same age group. In particularly the males are more active to exhibit their secondary sexual characteristics (i.e. erection of fins and body colour patterns) to attract the females. When a mating partnership is finally settled, the selected female is allowed to enter to the nest. The female spawns in the nest after a short mating ritual. During the spawning the female shows the movement of touching the bottom of the nest and the
12

Farming of Tilapia

erected conical genital papilla fully descends to deposit the ovulated ova (unfertilized eggs) in batches (30 60 eggs/batch). It is reported that the females subsequent to ovulation attract the partner males through releasing sexual pheromones (Velasco 2003a). The male ejects milt over the eggs to effect fertilization immediately after deposition of each batch of eggs by the female (Chen 1976). In view of mouth brooding the female picks up the fertilized eggs in to her mouth. Ova deposition and fertilization activities occur over a period of up to 2 3 hours.

Fig. 5 a. Genital papilla of male Oreochromis niloticus having two opening; the urogenital opening, where the milt and urine are excreted and the anus, for the discharge of fecal waste. b. Genital papilla of female Oreochromis niloticus having three openings; the anus, the urethra for urine passing and the oviduct, where egg passes through.

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Farming of Tilapia

(f) Fecundity
The colour of ripe and fertilized eggs is pale yellow orange; they are ovoid in shape and the size ranges between 1.0-2.0 mm x 1.5-3.0 mm in diameter, and 2.3 2.8 mm in length. The fecundity fluctuates widely from a few hundred to several thousand eggs, depending on the size and age of the female. It is reported that as the weight of GIFT strain O. niloticus increases to a range of 180 498 g, the number of eggs decreases. It means that absolute fecundity in this species is inversely correlated with the weight of sexually mature females (Velasco 2003a).

(g) Mouth brooding and development of fertilized eggs and embryos

The Nile tilapia is maternal mouth brooder, so the female immediately takes the fertilized eggs into her mouth for incubation. Hatching of eggs take place after 7090 hours in the mouth at 281oC and the female holds the hatched larvae and gives parental care until the swim up stage, which might need another 610 days. In O. niloticus embryonic development there are five easily observed developmental stages (Hussain 1992): Morula stage: Pigmentation stage: Hatching stage: Yolk sac resorption stage: First feeding stage: 6-8 hrs. after fertilization (a.f.) 45-50 hrs. a.f. 70-90 hrs. a.f. 6-10 days a.f. 12 14 days a.f.

The weight of first feeding tiny fry of Nile tilapia is about 0.01g and after this stage they are able to take natural or artificial feeds and move easily in the surrounding waters and do not need any parental care.

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2.4.2 Process of gonad development

In Nile tilapia development of gonadal products (eggs and sperms) is a short process. In the female fish, early egg development starts with the oogonia, then primary oocytes which give rise to secondary oocytes. During the pre-ovulatory period, high-energy yolk (vitellogenin) deposition occurs in the growing oocytes. The secondary oocytes complete such yolk deposition by the process of vitellogenesis and remain as mature oocytes in the ovary for a variable period of time until their final maturation or ovulation. A histological section of an ovary shows various stages of development at peak maturation of female O. niloticus (Figure 6). The process of sperm development starts in the males with the spermatogonia, spermatogonia develop to primary spermatocytes, each of which gives rise to secondary spermatocytes and then to spermatozoa or mature sperm. Various stages of germ cell development are visible in testicular section at maturation (Figure 7).

2.4.3 Endocrine hormone profiles

In the process of sexual maturation and gonad development in the fish including tilapia, the most vital role is played by the endocrine hormones like vitellogenin, 17-oestradiol, testosterone, 11-ketotestosterone etc. Development of secondary sexual characteristics such as bright and shiny body colour, bluish fins with reddish margins, prominent urogenital papilla etc. are also controlled by these sex steroid hormones. Normally in female fish egg yolk is derived from a precursor of lipophosphoprotein-calcium complex called vitellogenin, which is synthesized by the liver, released into the blood and finally sequestered by the oocytes by means of a receptor-mediated endocytotic process (Tyler, Sumpter and Bromage 1987). The yolk precursor is believed to be synthesized during the vitellogenesis phase of ovarian growth under the influence of oestrogenic control (ie. vitellogenin production is normally stimulated by 17-oestradiol hormone). On the other hand, testosterone
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Fig. 6 Histological section of an ovary shows various stages of development at peak maturation of female Oreochromis niloticus.

Fig. 7 Histological section of a testis shows various stages of development at peak maturation of male Oreochromis niloticus.

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hormone acts as precursor in oestrogen synthesis. Ultimately, vitellogenin and all other sex steroid hormones have interlinked functions to regulate oocyte maturation. Similarly in male fish sex steroid hormones also regulate more or less the sexual maturation and development of testicular products (ie. semen and sperm). Serum calcium concentration (index of vitellogenin) and sex steroid hormone levels in sexually matured female and male O. niloticus have been determined respectively by using the atomic absorption spectrophotometry and radioimmunoassay techniques (Hussain et al. 1995) and the data are presented in Table 1.
Table 1. Serum calcium concentration and steroid hormone levels in mature female and male O. niloticus. All values are mean SE estimated from an equal number (n=10) of fish in each category. Sexes Female Male Serum Ca++level -1 (mg 100 ml ) 34.286.3 16.460.5 Testosterone (ng ml-1) 39.077.1 82.0717.2 11-ketotestosterone (ng ml-1) 0.0 37.4512.5 17--oestradiol (ng ml-1) 0.012.542.3 0.0

Hussain et al. (1995)

2.4.4 Sex ratio

In normal O. niloticus brood stock and fish larger than 20-30 g, examining their urogenital papilla can easily differentiate sex. In small fish (3-5 g size) where manual sexing is not possible, an alternative aceto-carmine squash technique can be used (see Chapter 7). In this species the overall sex ratio does not generally differ from the expected 1:1 ratio.

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Brood stock replacement and breeding plans for tilapia hatchery stocks
BROOD STOCK MANAGEMENT TO AVOID GENETIC STOCK DETERIORATION

3.1

As it is an established fact that the main drawback of all the existing commercial tilapia strains is their precocious maturation and short cycle habit of reproduction in pond conditions, which ultimately leads the subsequent generations to become stunted and undesirable. The most detrimental problem associated with the growing population, as they rapidly become poor genetic material due to inbreeding of stocks for poor brood stock management. A short generation time and essentially uncontrolled reproduction make tilapia susceptible to inbreeding. On the other hand, in a small-scale seed production system of tilapia inbreeding will be common because of the mating of close relatives as in most cases female and male breeders are chosen from the finite population. Such uncontrolled inbreeding often leads to the loss of genetic variability and other deleterious consequences known as inbreeding depression. Inbreeding depression makes a stock having the characteristics of reduced growth rate, loss of fecundity, increased incidence of deformities, poor survival etc. Inbreeding tends to increase the homozygosity across all loci fixing some alleles while others are lost (Mair 1999). Such genetic stock deterioration will undoubtedly be a critical problem for tilapia seed production. Therefore, a good brood stock management practice in a hatchery or seed production system of tilapia is essential to avoid inbreeding and other related genetic stock deterioration aspects.

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Table 2. Inbreeding resulting from some matings between closely related individuals (Dr. Raul Ponzoni, personal communication). Mating between breeding stocks Full sibs Parent offspring Half sibs Uncle-niece First cousins Unrelated Inbreeding rate at 1st generation (%) 25.0 25.0 12.5 12.5 6.5 0.0

3.2

BROOD STOCK REPLACEMENT TECHNIQUES

Use of a high effective population size (ie. Ne) supported by standard reference stocks/strains will be the main principle of the tilapia breeding plan in a hatchery. The mating protocol should be devised in such a way that will reduce the chances of declining genetic variation between pairs. A tilapia hatchery operator should maintain at least 500 brood fish (more or less equal number of females and males) per generation as a standard population size for breeding. For brood stock replacement a simple protocol should be developed and maintained to ensure that each pair of breeders will contribute only once to the next generation. The steps of the protocol is furnished below: Required numbers of founder stock need to be collected from a known source (either from BFRI or from its regional stations/sub-stations) and reared in the holding tanks/ponds until maturity. Brood stock should be transferred and kept separately by sex in two transitory hapas (size 8 m x 2.5m x 1 m). Before stocking at least 80 to 100 breeding hapas (size 1 x 1 x 1 m) need to be set in a pond. A pair of female and male breeders (1:1 ratio) are stocked in each breeding hapa.

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It is necessary to check the mouth of all the stocked fish in each hapa 10-14 days interval to collect the fertilized eggs/larvae with yolk sac in view of their artificial incubation. Fertilized eggs/larvae with yolk sac that have been collected from the mouth of each female fish will be kept separately and incubated family wise in plastic jars. Immediately after hatching, the larvae are shifted to a series of trays and kept until their yolk sac resorption stage i.e. first feeding fry stage (in farmers condition, if the egg/larvae incubation system is not available, in that case, mouth checking is not necessary, alternatively fry produced in the hapas can be collected at fortnightly interval by scooping them from the inside or lifting up the bottom). Subsequently, at least 200 fry from each progeny family will be transferred to separate nursery hapas (size 1 x 1 x 1 m). Fry need to be grown up to 30 40 g size in the same hapa by reducing their numbers 40 50 fingerlings/m3. From each progeny group at least 5 females and 5 males should be selected as reference stock by manual sexing and shifted them for brood stock replacement. This will be done to ensure the equal contribution of each breeding pair to brood stock replacement as well as to maximize the effective population size (Ne).

3.3

BREEDING PLAN AND GENETIC STOCK IMPROVEMENT OF TILAPIA

In view of enhancing productivity of aquaculture species including tilapia, a number of advanced genetic techniques are presently available viz. chromosome manipulation, hybridization, cross breeding, sex reversal (including YY-male technology), gene transfer and selective breeding. None of these techniques are suitable for continuous improvement of desired traits of a population except selective breeding. Selective breeding is a long-term continuous strategy to improve the production performance and quality of tilapia (WorldFish Center 2004). In a well-designed
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selective breeding program the pedigree of brood fish can be monitored to increase the accuracy of selection and to restrict inbreeding. Through adopting brood stock replacement program as described above, the tilapia hatchery operators have a chance to collect and maintain superior brood stock and mate them accordingly. If the selective breeding technique can be added with brood stock replacement program, then it will be wonderful to improve a stock with desirable traits and high genetic variation. In this way, genetically superior individuals can be developed per generation, heritability and genetic variability of all traits can be increased to a maximum level and inbreeding depression can be kept to a minimum (Hussain and Mazid 2001). Such superior and outbred stocks may be of benefit to breeding and aquaculture by increasing growth rate, fecundity, survival and disease resistance of commercially important tilapia species/strains.

3.3.1 Development of the base population

It is worthy to mention here that a breeding population has every chance of attaining genetic gain every generation if they are produced through a planned selective breeding. The success of this phenomenon is dependant on the base population, which needs to have high additive genetic variation. In the process of selective breeding, the rate of selection response, therefore, depends on the additive genetic variation present in the breeding population and the intensity of selection (WorldFish Center 2004). The base population requires a large effective population size with a large number of breeders, which might need to develop or maintain huge tilapia reference stocks. The rationale of this stock development/maintenance is to form a heterogeneous, outbred and broad genetic base in the population at the beginning of the selection program.

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3.3.2 The selection methods

In tilapia breeding program among three selection methods, viz. (a) Mass or individual selection; (b) Family selection and (c) Combined selection, any one can be followed for genetic stock improvement. Mass selection as an initial method of choice is described below:

(a) Mass selection

This is a relatively simple selection strategy where breeding candidates are selected on the basis of individual performance. Mass selection is normally used to improve phenotypic traits (e.g. growth, shape and colour) that are recorded on the living fish. Several authors found this to be a useful selection strategy for genetic improvement of commercially important fish stocks. Schom and Baily (1986) reported that mass selection of brood stock in subsequent generations improves the chances of genetic gains through the accumulation of favorable alleles/traits with high genetic variability in the population. According to Bentsen et al. (1996) an assumed heritability for body weight of about 0.3 and assumed coefficient of variation of 30%, as shown in many fish species, the genetic gain in the progeny should be about 15 to 17% per generation compared to the mean of parent generation. Eknath et al. (1998) showed about 15% additive genetic gain in the GIFT strain though five generations of selection. Hussain et al. (2002) reported that in silver barb the average gain per generation across two generations of selection for growth performance in weight was 7.2%. Similar performance was also attained by Super Strain of GIFT (derived from pure GIFT strain through several generations of genetic selection) in Bangladesh, where the weight gain values of fifth generation of the selected group showed 31.2% superiority (average genetic gain per generation across four generations of selection was 6.0%) over the non-selected control group. The most important step to be considered by the hatchery operators in mass selection strategy is that in every generation at least 5-10% bestselected breeders should be collected and used for breeding program for
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the production of next generation. Individual selection of the breeders is followed based on empirical assessment of best size and weight, good growth and healthy appearance (Hussain and Mazid 2001). It is also reported that when using a mass selection method, after several generations the accumulation of inbreeding can be a major problem. The selected breeding candidates can become more related to each other. If large families are naturally produced or stocked in the test environment, the wide genetic variation and common non-genetic effects (e.g. maternal and age effects) may easily result in a disproportionately high contribution of individuals from just a few parents to the next generation, and a consequent accumulation of inbreeding. Such inbreeding reduces the genetic variation and the potential for further genetic improvement in the breeding population (WorldFish Center 2004). In this situation either family selection strategy or combined selection strategy can be followed as described in the manual entitled Gift technology manual: An aid to tilapia selective breeding published by WorldFish Center (2004).

3.3.3 Initiate breeding and testing programs (a) Selection, sexing and maintenance of brood stock
During the month March/April, when the Nile tilapia brood stock derived as base population become sexually mature, the best females and males are chosen (weighing between 100-150 gm each) and kept separately by sex in two transitory hapas (size 8 m x 2.5m x 1 m) set in a pond until they are used for the planned selective breeding program. During this transitory period, the brood stock are maintained by proper feeding.

(b) Setting of breeding hapas and stocking of breeders

At least 80 to 100 breeding hapas (size 1 x 1 x 1 m) are prepared and set in breeding ponds and fixed and tied to nylon ropes and bamboo poles. A pair of female and male breeders (1:1 ratio) is stocked in each breeding
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hapa. Artificial feeds having 35% crude protein can be fed @ 3 - 4% per estimated weight of biomass 2-3 times daily.

(c) Collection of fertilized eggs/larvae

The female fish normally holds the fertilized eggs in her mouth for natural incubation. So, it is necessary to check the mouth of all the stocked females in each hapa twice a week to collect the fertilized eggs/larvae with yolk sac in view of their artificial incubation.

(d) Development of incubation system and hatching of fertilized eggs/larvae

A simple tilapia egg incubation system can be developed using 2.0 liter capacity empty plastic coca cola or drinking water bottles and medium type plastic trays (30 x 25 x 8 cm). The bottles and trays need to be set over the designed concrete platform and connected to the recirculating system, where fresh water (281 oC) comes directly from a header tank by gravity (Figure 8). Fertilized eggs/larvae with yolk sac that have been collected from the mouth of each female fish are incubated separately in this system. Immediately after hatching, the larvae are shifted to a series of trays and kept separately until their yolk sac resorption stage i.e. first feeding fry stage. Note: If there is no facility for artificial hatching and incubation system (s), in that case, it is wise not to remove the fertilized eggs from the mouth of the female breeder(s). Rather these eggs should be allowed to hatch and incubate in the mouth of the female(s). In that case only first feeding or advanced fry can be collected by using a scoop net for their separate nursing in the other series of hapas.

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Fig. 8a A simple tilapia egg incubation system having plastic water bottles.

Fig. 8b A simple tilapia egg incubation system having medium type of trays.

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(e) Nursing and rearing of fry/fingerlings

At least 200 fry from each progeny family will be transferred to a series of separate nursery hapas (size 1 x 1 x 1 m) for rearing for 21 24 days. To reduce environmental differences between families, all nursery hapas should be installed in the same pond. Fry need to be grown up to 8 10 g (taggable size) in the same hapas or another series of similar size of hapas by reducing their numbers to 80 100 individuals/m3 and reared for another 40 - 60 days.

(f) Record keeping/data collection

The tilapia hatchery manager/operator must maintain a record book and keep all the necessary family wise data of the hapa breeding and fry/fingerling rearing. He should note the body weight and length data of individual male and female stocked in each hapa including the date of eggs/larvae collection. During incubation, he should record the number of eggs/larvae per jar/tray and their survival. During fry/fingerling rearing, the number of fry stocked in each nursery hapa, and the fingerlings shifted to rearing hapas should also be recorded. For producing sufficient number of fingerlings and standardizing the rearing conditions of all full-sib families such records are essential.

(g) Tagging of fingerlings

. Before stocking tilapia fingerlings for any desired communal testing, tagging with suitable materials is essential. Several types of tagging materials are available in the market presently but not all are found suitable. Floy fingerling tags with different colours have been found to be convenient for external tagging to uniquely identify each one of the large numbers of experimental fish (WorldFish Center 2004). The Floy tag having two plastic protection discs, which are fixed through its thread
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either side with the needle carefully inserted underneath the scale below the dorsal fin and above the lateral line (Figure 9). Another type of flat, comparatively longer shaped plastic numbered tags with nylon thread (Figure10) can be used by fixing them at the dorsal fin or tail region of the tilapia fingerlings. For tilapia the most convenient is digital tagging by Passive Integrated Transponder (PIT) tags (Figure 11). The capsulated PIT tags are implanted within the visceral cavity of the fish, which provide a safe, secured and permanent identification of experimental fish (both for fingerlings and breeders). An electronic scanner is used to read out the digital number at the time of sampling and harvesting.

(h) Testing in common farm environments

Communal testing of fish in common farm environment might be essential for evaluation of growth and other performance traits as well as estimating the breeding values for any desired selection strategy(s) in each generation. After tagging all the representative number of fingerlings from each family should be shifted to a holding tank or hapa. The earthen ponds, cemented tanks, cages and rice fields as common farm environments should be made ready for communal stocking of all full-sib groups. A few equal numbers of tagged fingerlings per full-sib group can be stocked in the selected environments for a full grow-out period (3 5 months). Stocking density needs to be calculated as per recommended numbers for each test environments. Before stocking, the body weight, standard length and tag number of the fingerlings should be recorded. Throughout the entire grow-out period, the fish should be fed with formulated feeds as shown in Table 7 @ 3% per estimated body weight. Feeding intensity should be at least 2 times daily. Every 30 days, sampling of growing fish should be made to check the growth and adjust the feeding rate.

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Fig. 9 Floy tagging underneath the scale below the dorsal fin and above the lateral line of tilapia.

Fig. 10 Plastic numbered tag with nylon thread.

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Fig. 11 PIT tagging into the visceral cavity of tilapia.

Fish can be harvested when they attain the weight about 100 150 g. All the test fish should be captured as carefully as possible and the individual tag numbers, sex, sexual maturation condition, body weight, standard length, body width, body depth etc. should be recorded.

3.3.4 Continue selection program(s) to produce next generation(s)

For the production of the next generation, the very best individuals in terms of individual weight for mass selection/breeding values for other selection strategies should be used. But a compromise needs to be maintained between selection intensity and effective population size (WorldFish Center 2004). Closely related individuals (i.e. full-sibs, halfsibs, parent-offspring, uncle-nice and cousins) mating should always be avoided to prevent inbreeding depression in further genetic improvement program of tilapia.

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3.4 MAXIMIZING THE EFFECTIVE POPULATION SIZE (NE)

Ne should be maximized to minimize the loss of genetic variation and inbreeding. In a well designed breeding program, Ne will be made by collecting very few but equal numbers of individuals from each progeny group (ie. individuals produced by each pair of breeders) from maximum number of broodstock and maintaining a 1:1 sex ratio (Mair 1999). In a well planned breeding program, the effective population size (Ne) should be maintained >50 up to 1000 to keep the estimated level of inbreeding less than 1% per generation. In a breeding population where random mating is done, Ne can be calculated by using the following formula: Ne = 4 x F x M / (F + M) where F = number of female & M = number of males Effective population size is inversely related to the rate of inbreeding (F), which is shown as: F = 1/2Ne It is worthy to mention here that the rate of inbreeding depends on the skewed sex ratio. The rate of inbreeding per generation in the range of effective population size (Ne) 50 to 1000 using the above formula is furnished in Table 3.
Table 3. Correlation between effective population size (Ne) and rate of inbreeding in a hatchery. Effective population size (Ne) (Sex ratio 1:1) 50 100 250 500 750 1000 Rate of inbreeding (F value) per generation 1% 0.5% 0.2% 0.1% 0.07% 0.05%

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Ploidy manipulation and production of all sterile, female and male population
GENOMIC STATUS AND DETERMINATION OF SEX

4.1

Like other finfish, the gametes (i.e. sperms and eggs) are produced by the male and female O. niloticus. A single gamete can be termed as genome (i.e. having a haploid set of chromosomes). At fertilization of mature egg and sperm, the two haploid genomes or gametes form a zygote and this complement is termed as diploid. Each genome in this species of tilapia contains 22 chromosomes, therefore, a zygote has a diploid genomic status having a total of 44 chromosomes. O. niloticus has an XX/XY sex determining system. Here, each haploid set of chromosomes in the egg contains a single X sex chromosome and in the sperm it contains either an X or a Y sex chromosome. In this case, the offspring that are produced from the fertilized eggs having XX (homogametic) set of sex chromosomes become females, while those are produced having XY (heterogametic) set of sex chromosomes become males. As the mechanism of sex determination is known in Nile tilapia, the methods of controlling sex have become easier. Ploidy or chromosome manipulation has become popular research in this fish since 1980s for the generation of genetically induced sterile and mono-sex (either all female or all male) populations. Various techniques have been developed so far to interfere with normal functioning of the metaphase spindle apparatus during cell division in eggs using several causal agents, such as physical shocks (temperature and hydrostatic pressure), chemicals (i.e. endomitotics) and anesthetics (i.e. nitrous oxide and freon). As a result,
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individuals with differing genomic status can be produced in a population as below: Polyploids (triploid and tetraploid) Gynogenetics (both meiotic and mitotic gynogenetics) Androgenetics (embryonic development with chromosomes)

paternal

4.2

PRODUCTION OF GENETICALLY INDUCED ALL STERILE POPULATION

Genetically induced sterile populations can be produced by the induction of polyploidy (triploidy and tetraploidy) in the developing eggs of Nile tilapia. Such sterility in both female and male fish can be of benefit to aquaculture, where control of reproduction and population is desirable.

4.2.1 Induction of polyploidy

There are two types of polyploids, i) triploids and ii) tetraploids. Triploids are produced directly by blocking of second polar body extrusion during second meiotic division shortly after fertilization of fish eggs using various physical shocks and chemical treatments. Triploid individuals are expected to be functionally and endocrinologically sterile due to their meiotic inhibition of gametogenesis and lack of essential steroid hormone levels to support gonadal growth (Hussain 1998). Tetraploid individuals are generated by the disruption of the first mitotic division shortly prior to formation of cleavage furrow of developing fertilized eggs using several agents both physical and chemical. The production of tetraploids might have tremendous impact, because of promising future of large-scale production of genetically induced sterile fish from mating of normal diploid and viable tetraploid individuals. But until now no viable tetraploids in tilapia have produced except the

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embryos. A schematic diagram of inducing polyploids in O. niloticus using pressure, heat and cold shocks is shown in Figure 12.

Fig. 12 A schematic diagram of inducing polyploids in O. niloticus using pressure, heat and cold shocks (Hussain 1996).

Methods for triploidy and tetraploidy induction (a) Collection and maintenance of brood stock
Sufficient numbers (>100 pairs) of purebred O. niloticus brood stock including improved GIFT strain are to be collected from a known source and attention to be given for their special maintenance in ponds or tanks and feeding with protein rich artificial feeds. For artificial breeding in view of chromosome manipulation works, sexually mature fish are maintained under at least 12-h photoperiod and transferred into a series of 120 L glass aquaria provided a recirculated, aerated and temperature controlled (281 oC) water supply in a wet laboratory. A single male and female are accommodated in each aquarium but are kept separate by a sheet of Perspex. In these aquaria the fish need to be fed with commercial pellets (at least 40% protein) at the rate of 2-3% body weight per day.
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(b) Collection and preservation of sperm

Milt of sexually mature male O. niloticus is collected by manual stripping. The stripping is done by applying gentle downward pressure with the thumb and index fingers from just below the pectoral fins up to the genital opening of the fish. The urine is first ejected and the genital papilla dried with a paper towel and the milt is sucked into a micro-pipette by capillary attraction when it is placed at the opening of the urethra. Milt contaminated with urine or water is always eliminated. Before any milt is used for fertilization, motility of sperm is always examined under microscope. For short storage undiluted milt is held at 4 oC in a refrigerator can be used to fertilize eggs until 3-4 days.

(c) Egg collection and artificial fertilization

Under experimental conditions sexually mature female O. niloticus spawn at approximately 14-20 day intervals. Readiness of females to spawn is ascertained by examining the degree of swelling of the urogenital papilla and by the pre-spawning behaviour of the fish. The ripe female is removed from the aquarium and the ovulated eggs are obtained by manual stripping. The eggs are collected in a clean and sterile Petri dish. To avoid the female prematurely releasing her eggs, she can be held in a scope net for up to 2 hrs, while the experiment is being prepared. Fertilization is carried out in vitro by mixing 0.1-0.2 ml pre-collected dry sperm per batch of eggs (ca. 100-200 eggs) followed by the addition of 10-20 ml of 281 o C water. After that fertilized eggs are left in the Petri dish for 2-3 min. for water hardening before using for further treatments or transfer to the incubation system.

(d) Application of shock treatments

For applying physical shock treatments (pressure, heat and cold shocks) to induce both triploidy and tetraploidy, both 1 1.5 L vessel capacity pressure apparatus (Figure 13) and thermostatically regulated 50 L capacity water bath (Figure 14) are used.
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Fig. 13 1 1.5 L vessel capacity pressure apparatus.

Fig. 14 Thermostatically regulated 50 L capacity water bath.

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For pressure shocking the fertilised eggs, the vessel and hydraulic pump reservoir are first filled with 281 oC clean water. Eggs are held in individual uncapped vials and, after the vessel is sealed and purged of air, the pressure release valve is closed, pressure was applied gradually by a manually operated hydraulic pump. The time is taken to raise the pressure level from ambient to 8,000 9,000 psi typically in the region of 30 sec with the passage from 8000 10,000 psi taking a further 10 sec. Pressure is released by gradually opening the valve and so the pressure dropped typically over 30 sec (9,000 0 psi). After the pressure treatment, the eggs are removed from the vials and transferred directly to incubating jars. For thermal (heat and cold) shocking the fertilized eggs, the water bath (temp. range 0 to 100 oC capable of maintaining 0.1 oC) needs to be filled with clean water and allowed to heat the water up to required temperature. The optimal pressure, heat and cold shock parameters for the induction of triploidy in O. niloticus (Hussain et al., 1991) are as follows: Pressure shock: 8000 psi, 2 min duration to be applied 9 min after fertilization (a.f.). Heat shock: 41 oC, 3.5 min duration to be applied 5 min a.f. Cold shock: 9 oC, 30 min duration to be applied 7 min a.f.

The optimal pressure and heat shock parameters to suppress the first cleavage or mitotic events of cell division in the fertilized eggs in the Nile tilapia (Hussain et al. 1993) can be used for the induction of tetraploidy are as below: Pressure shock: 9000 psi, 2 min duration to be applied 40-50 min a.f. Heat shock: : 41 oC, 3.5 duration to be applied 27.5-30 min a.f.

The induction of triploidy and tetraploidy has already been carried out in various Oreochromis spp. is shown in Table 4.

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(e) Incubation of eggs

Fertilized, treated and controlled eggs are identically incubated in a series of round bottom plastic jars (750 1500 ml capacity) connected to the warm water (281 oC ) recirculating system. Additionally, the water is sterilized through a UV sterilization unit (ca. 62,000 Wsec/cm2) and there should be a provision so as to ensure gentle movement of the developing eggs at all times (Rana, 1986). The survival rate in treated and control groups is checked at three development stages, namely: morula, pigmentation and hatching stages (see section 2.2.3). The numbers of normal and deformed fry at hatch are also need to be recorded.
Table 4. Polyploidy induction in various Oreochromis spp. using pressure, heat and cold shocks.
Species Oreochromis niloticus Oreochromis niloticus Oreochromis niloticus Oreochromis aureus Oreochromis niloticus Oreochromis aureus Causal agents PS HS CS CS PS + CS CS Intensity level 8000 psi for 3.5 min o 41 C for 3.5 min 9 C for 30 min 11 C for 60 min 7000 psi for 7 min + o 7.5 C o 11 C for 60 min
o o

Induction widow 9 min a.f. 5 min a.f. 7 min a.f. 15 min a.f. 57-60 a.f. 92 min a.f.

Ploidy status Triploidy Triploidy Triploidy Triploidy Tetraploidy Tetraploidy

Authors Hussain et al. (1991)) Hussain et al. (1991)) Hussain et al. (1991)) Valenti (1975)) Myers (1986) Don & Avatalion (1988)

(f) Determination of ploidy status

The ploidy status of all treatment and control groups can be determined by chromosome preparation of sub-samples of new hatched or 1 day old larvae (Hussain and McAndrew 1994). For detailed protocol of chromosome karyotyping see section 4.5 of this chapter. The triploid and teraploid metaphases in O. niloticus are composed of three (3n=66 chromosomes) and four (4n=88 chromosomes) sets of genomes respectively.
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4.3

PRODUCTION OF GENETICALLY INDUCED ALL FEMALE POPULATION

Genetically induced all female populations in fish can be produced by artificial diploidization of the maternal chromosome complement either by retention of the second polar body or inhibition of first cleavage using physical and chemical treatments. So two types of gynogenetic individuals can thus be generated through the induction of meiotic or mitotic events of fertilized eggs. A schematic diagram of inducing two types of gynogenesis (meiotic and mitotic) in O. niloticus using pressure, heat and cold shocks is shown in Figure 15.

Fig. 15 A schematic diagram of inducing two types of gynogenesis (meiotic and mitotic) in O. niloticus using pressure, heat and cold shocks (Hussain 1996).

4.3.1 Induction of meiotic gynogenesis

In the process of meiotic gynogenesis, eggs are fertilized with UV irradiated sperm and then are exposed to a variety of physical shock or chemical treatments, which suppress the anaphase stages of second meiotic division by disruption of metaphase spindles. As a result, embryonic development proceeds with the inheritance of only maternal
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chromosome set(s). At present there are few direct applications of meiotic gynogens in aquaculture because the fish partly or mostly are inbred and have reduced variability compared to normal diploids. It has commonly suggested that meiotic gynogentic induction coupled with sex inversion such that functional XX males could be produced (Nagy 1987; Thorgaard and Allen 1987; Pongathana et al. 1995). Such sex-reversed males are thought to be useful in cross breeding experiments to produce all outbred monosex female population, where the growth rate of females is superior to males.

4.3.2 Induction of mitotic gynogenesis

In this process, putative gynogenetic progeny derive by the artificial diploidization of the maternal chromosome complement due to prevention of mitotic cleavage. The main rationale of mitotic gynogenesis induction in fish has been its potential for generating rapidly inbred lines. Han et al. (1991) suggested that using meiotic gynogenetic diploids, even when reproduction is repeated for several generations would never produce homozygous inbred lines. Therefore, induction of diploid gynogenesis by inhibition of first cleavage at mitotic division of a zygote might be more promising method for producing inbred lines, which will be homozygous at every gene locus (Chourrout 1984; Streisinger et al. 1981; Hussain et al. 1994). Despite the first generation of mitotic gynogenetics have limitations to use them directly for culture but they are potential and valuable as completely homozygous brood stock to produce second generation of clonal lines in fish including tilapia (Hussain et al. 1998).

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Methods for induction of meiotic and mitotic gynogenesis (a) Source of brood stock, their maintenance, sperm/egg collection and fertilization
The true breeding O. niloticus brood stock collection, their maintenance, stripping of sperm/egg collection and fertilization protocols are described in section 3.1.1 of this chapter.

(b) Ultraviolet irradiation of sperm

Milt samples required for UV treatment are checked for motility and irradiated with an ultraviolet lamp set using a radiometer (Ultra-Violet Products Inc.). Irradiation is carried out in a 5 cm diameter Petri dish at 4 o C to give a dose of 300-310 W/cm2 for 2 min with a sperm concentration of 2.5x107 ml-1, 2.05 ml of modified Cortlands solution (Hussain et al. 1993).

(c) Application of shock treatments

The same protocols for the application of both pressure and thermal shocks treatments can be followed as described in the section 4.2.1. All the treatment batches of eggs are fertilized by mixing 0.4 0.5 ml diluted (with modified Cortlands solution) UV irradiated sperm, un-irradiated sperm from the same pool is used to fertilize a portion of eggs as a control. After fertilization, when not being submitted for shock treatments, eggs are at all times incubated at 281 oC. All treatment batches of eggs except the UV control are exposed to elevated hydrostatic pressure and heat shocks. The optimal second polar body retaining pressure and heat shock parameters (Hussain et al. 1991) can be applied for the induction of meiotic gynogenesis as follows:
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Farming of Tilapia

Pressure shock: 8000 psi, 2 min duration to be applied 9 min after fertilization (a.f.). Heat shock: 41 oC, 3.5 min duration to be applied 5 min a.f.

To interfere with the first mitosis for the induction of mitotic gynogenesis the recommended optimal parameters of pressure and heat shocks (Hussain et al. 1993) can be used as below: Pressure shock: 9000 psi, 2 min duration to be applied 40-50 min a.f. Heat shock: 41 oC, 3.5 min duration to be applied 27.5-30 min a.f.

Table 5 shows a brief review of suppression of meiotic and mitotic events of cell division in the fertilised eggs to produce meiotic and mitotic gynogenetics in various Oreochromis spp. Egg incubation and checking of survival rates of embryos at various developmental stages can be done as described in the protocols in section 2.2.3. and 3.1.1. Determination of ploidy status can be performed as explained in the protocols in section 4.5.
Table 5. Gynogenesis induction in various Oreochromis spp. using pressure and heat shocks.
Species Oreochromis niloticus Oreochromis niloticus Oreochromis mossambicus Oreochromis niloticus Oreochromis niloticus Oreochromis niloticus Causal agents PS HS HS PS HS HS Intensity level 8000 psi for 3.5 min o 41 C for 3.5 min 41.7 C for 3 min 9000 psi for 2 min o 41 C for 3.5 min 41 C for 3.5 min
o o

Induction widow 9 min a.f. 5 min a.f. 32-54 min a.f. 40-50 min a.f. 27.5-30 min a.f. 25-35 min a.f.

Ploidy status Meiotic gynogenetic Meiotic gynogenetic Meiotic gynogenetic Mitotic gynogenetic Mitotic gynogenetic Mitotic gynogenetic

Authors Hussain et al. (1991) Mair (1988); Hussain et al. (1991) Varadaraj & Pandian (1990) Hussain et al. (1993) Hussain et al. (1993) Mair (1988)

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4.4

PRODUCTION OF GENETICALLY INDUCED ALL MALE POPULATION

The induction of androgenesis is the alternative method of producing genetically induced all male population in tilapia and other selected fish species to replace hormonal sex reversal. Another possible application of genetically induced males lies in recovering genotypes from cryopreserved sperm, which is important as egg and embryo cryopreservation has not yet been succeeded. Androgenesis is a genome manipulation technique, the reverse to gynogenesis, which involves a genetically inactivated egg fertilised with normal sperm. The resulting embryo develops with entirely paternal chromosomal inheritance without any contribution from the maternal chromosomes. The eggs can be inactivated successfully by gamma or x-rays including UV irradiation. The first androgenetic diploids were produced by the suppression of first cleavage of inactivated eggs in salmonids (Parsons and Thorgaard, 1985; May et al. 1988; Thorgaard et al. 1990) and later in Nile tilapia (J.M. Myers personal communication). For the commercial production and application of genetically induced males need further research.

4.5

PROTOCOLS FOR CHROMOSOME KARYOPYTING

Hussain and McAndrew (1994) developed an improved technique for chromosome karyrotyping from embryonic and soft tissues of tilapia. The protocol for chromosome preparation from embryonic tissues is as follows: Embryonic tissues need to be collected from newly hatched or 1 dayold larvae of treatment groups. For each group (ca 100) 15 - 20 embryos are placed in a small Petri dish containing 8 - 10 ml of 0.002 - 0.005% colchicine solution (freshly prepared or stored for 4 - 6 hrs at 28oC).
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Tissues are obtained from the embryos in chilled 0.75% saline solution under a dissecting microscope by removing their heads and yolk sacs and putting these in distilled water (hypotonic solution) for 8 - 12 min. The tissues are then immersed in a fixative of 4:1 methanol - acetic acid at 4oC. After two changes the tissues are stored in the fixative for 30 - 90 days. To prepare the slides, the tissues are removed from the fixative and, later blotting out the excess fixative, placed in the cavity of a Perspex slide with two to three drops 60% glacial acetic acid and minced for 1 min. with a glass rod to allow sufficient dissociation of epithelial cells. After 15 - 20 min., three to four drops of cell suspension are dropped from a height of 30 - 40 cm onto a clean glass slide on a warmed hot plate (44 - 48oC) and withdrawn within 8 - 12 seconds leaving a fine and clean ring of cells using a single micro-hematocrit dropper. Slides are air dried and stained with freshly prepared 10% Giemsa stain (prepared in 0.01M phosphate buffer pH 7.0) for 15 - 20 min. The slides are rinsed in distilled water, air dried and mounted with DPX after 10 min. of Xylene wash.

The protocol for chromosome preparation from soft tissues is as follows: Soft tissues like gill epithelia and the soft edges of the caudal fin are collected from 25-30 day old (after hatching) fry. The are placed overnight (10-12 hours) in a plastic container with aerated 0.01-0.02% colchicine solution. The temperature of the solution is maintained 281 oC. Tissues are collected with fine scissors and forceps then transferred immediately to distilled water for 10-20 min before being fixed in 4:1 methanol-acetic acid (two changes) and stored at 4 oC up to 30 days. Slides are prepared according to the same technique described for chromosome karyotyping from embryonic tissues. Metaphase spreads of chromosomes are to be checked and chromosome number noted by observing the slides under x400 and x1000 (oil immersion) magnifications, respectively, with a compound microscope.
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Counting the chromosomes of as many karyotypes as possible per slide carries out the karyological examination. The karyotypes of O. niloticus consisting of 22 pairs with no morphologically distinct sex chromosomes. In fact only one pair large marker chromosomes are recognizable and remaining 21 being similar in size and acrocentric morphologically. The haploid, diploid and triploid metaphases, which are composed of respectively one (n=22 including one large marker chromosome), two (2n=44 including two large marker chromosomes) and three (3n=66 including three large marker chromosomes) sets of chromosomes are shown in Figure 16 a-c. Aneuploid metaphase (hyperhaploid or hypodiploid) is composed of more than 22 and less than 44 chromosomes in this species (Figure 16 d).

Fig. 16 Metaphase chromosome of Oreochromis niloticus. a. haploid (n = 22), b. diploid (2n = 44), c. triploid (3n = 66), d. aneuploid metaphase (hyperhaploid or hypodiploid) (Hussain 1995).

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Body colour inheritance and development of purebred strains of red tilapia

INHERITANCE OF BODY COLOUR IN COMMERCIALLY AVAILABLE STRAINS

5.1

After the discovery of Mendels theory of heredity or inheritance, many geneticists working with various plants and animals have since conducted an immense number of experimental works. Meanwhile, studies have also been made particularly to determine the genetics of body colour inheritance in a limited number of commercial and experimental populations of fishes including tilapias. Commercially available red tilapia strains are mostly hybrids and products of cross breeding involving as many as four different species in which O. mossambicus and O. niloticus are predominant (McAndrew et al. 1988). The Taiwanese red tilapia has been reported as a hybrid between albino O. mossambicus and O. niloticus by Kuo (1969, 1988) and Liao and Chang (1983). Initially the founder hybrid strain did not produce a high frequency of red fry, but after several years of continued selection and hybridization trials using F1 progeny, the proportion of red phenotypic fry was increased from 30% in 1969 to 80% in 1974. Later further genetic improvement of such red tilapia was made through cross breeding (Kuo and Tsay 1988). These crosses were made between phenotypic individuals such as red, white, brown and wild type: colour segregations occurred in all crosses. The pink phenotype was homozygous dominant, the red heterozygous and the wild type homozygous recessive. The red strain in the Philippines was introduced from Singapore in 1978 and the breeding characteristics of various crosses of different phenotypes
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(ie. pink, grey, black spotted and albino) were subsequently investigated (Galman et al. 1988). Among all red, pink and albinos, the pink phenotype seemed to be homozygous dominant. Among U.S. strains of hybrid red tilapias, the first one derived from cross breeding involving O. aureus, O. hornorum and O. mossambicus (Sipe 1979) and the second strain a red-gold colour mutant hybrid between O. hornorum and red O. mossambicus (Behrends et al. 1982). Observing several generations, the authors stated that red-gold colouration was dominant and controlled by two or three gene pairs in this strain. In Israel, the red/gold body colour of O. mossambicus has been determined and it was revealed that the mutant phenotype was inherited as a Mendelian recessive (Wohlfarth et al. 1990). Tave et al (1989) demonstrated that black body coloured fish were homozygous dominant, gold fish were homozygous recessive and bronze fish were heterozygotes. In an Egyptian strain of O. niloticus, the red body colour was inherited as an autosomal dominant trait in presence of wild type (McAndrew et al. 1988). Hussain (1994) also observed similar pattern of colour inheritance both in Egyptian and Thai red strains. His results demonstrated that red body colour in these two mutant strains is controlled by a single autosomal dominant R gene. The frequency of blotchy pattern in these strains further indicated that blotched phenotype are heterozygotes (Rr), which might be epistatic to the R gene and expressed only in its presence.

5.2

IMPORTANCE AND PROBLEMS ASSOCIATED WITH THE PRESENT STOCKS

Red tilapia strains have become increasingly popular to fish farmers and entrepreneurs for their characteristic body colour, fast growth, tasty flesh and high demand in the market. These strains are commercially cultured in many tropical and sub-tropical countries of the world such as Taiwan, the Philippines, Thailand, Malaysia, Indonesia, Israel, Guam, Greece, Brazil, Jamaica and USA. Although the Thai red strain was introduced into Bangladesh in 1988, extensive farming practice of this fish has not yet
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flourished. It is expected that like the GIFT strain of Nile tilapia, the red strain will also take a place in aquaculture soon due to its commercial importance and high demand in the international markets. Despite the commercial importance and development of several red tilapia strains in many regions of the world, one major problem of these mutant strains is that the majority of them do not breed true, including Thai red strain. Another problem associated with the appearance of varying proportions of blotched types (presence of black spots on the skin) of fish in each generation, which are not as valuable to the consumers as the pure red individuals. It will be difficult to maintain or improve the quality and development of pure breeding red populations of the present stocks until the mode of body colour inheritance is well understood by tilapia hatchery workers and researchers.

5.2.1 Genetic status of Thai red strain

The origin of the Thai red strain is less certain and its origins were discussed for the first time in a workshop on Tilapia Genetic Resources for Aquaculture held in Bangkok, Thailand in 1987. An Official of Thai Government informed the meeting that red tilapia was found in a pond in northern Thailand, where O. mossambicus was introduced from Malaysia in 1949. Thus, this fish was assumed to be a hybrid between O. mossambicus and O. niloticus. Electrophoretic analysis of Thai red tilapia samples showed that both O. mossmabicus and O. niloticus alleles were present (Pullin 1988; Prof. B.J. McAndrew and Dr. P. Sodsuk, pers. communication). It has been experimentally proved that the existing stocks of Thai red strain are a mixture of both homozygous RR that breed true, and heterozygous Rr individuals that do not (Hussain 1994). In this case, progeny testing is a valuable method for maintaining the production of all pure red progenies of Thai and other mutant strains. Red mutant brooders can be made as true breeders when they will be fixed
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as all homozygous at the R allele, to allow the undesirable r allele to be selected out.

5.3

MECHANISMS OF PROGENY TESTING TO DEVELOP PUREBRED STRAINS OF RED TILAPIA

5.3.1 Collection of red and wild brood stock

Both red and wild type pure O. niloticus stocks should be collected from a known source or from a tilapia reference collection maintained at the research institute(s). The brood stocks of these strains need to be maintained separately initially in the earthen ponds and subsequently in a recirculated water system ie. in mini tanks or glass aquaria. During all the phases of growing period of brood stocks, the fish should be fed with formulated or commercial feeds having at least 30% crude protein at recommended rates.

5.3.2 Fish breeding, stripping, fertilization and incubation of eggs

Fish breeding, stripping, egg fertilization and incubation protocols to be used for progeny production will be similar to section 4.2.1.

5.3.3 Parental cross breeding (a) Red x Red parental cross

48

A number of red females can be used to cross with red males. Stripped eggs of each red female need to be fertilized with freshly collected milt of red males and incubated separately. No females in these crosses are used more than once and males can be used several times with different females.

Farming of Tilapia

F1 progenies produced in these crosses need to be reared for few weeks in the nursery hapas until the fry develop distinguishable body colour. It is expected that body colour segregation of progenies of all these crosses will be all red. Thus it can be presumed that the parental red stocks will be either RR or Rr genotypes or combination of both. In these crosses it will be difficult to identity the true breeding parental stocks to develop purebred red strains for breeding purpose until the F1 progenies are used for sib crosses.

(b) Red x Wild type parental cross

A number of red females or males can be used to cross with wild type males or females. Stripped eggs of each female (red or wild) need to be fertilized with freshly collected milt of corresponding males (red or wild) and incubated separately. No females in these crosses are used more than once and males can be used several times with different females. F1 progenies produced in these crosses need to be reared for few weeks in the nursery hapas until the fry develop distinguishable body colour. It is expected that body colour segregation of F1 progenies of all these crosses will be either 100% red or 50% red plus 50% wild type. Thus it can be presumed that the parental red stocks will be either RR (where F1 progenies are 100% red) or Rr (where F1 progenies are expected 1 red:1 wild) genotypes or combination of both. From this test crosses, 100% red progeny producing parental stocks (RR genotype) can be isolated to develop purebred red strains (Figure 17) for commercial breeding purpose. .

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5.3.4 Sib cross breeding

A number of F1 red females can be used to cross with F1 red males. Stripped eggs of each red female need to be fertilized with freshly collected milt of red males and incubated separately. No females in these crosses are used more than once and males can be used several times with different females. F2 progenies produced in these crosses need to be reared for few weeks in the nursery hapas until the fry develop distinguishable body colour. It is expected that body colour segregation of F2 progenies of all these crosses will be either all red or 75% red plus 25% wild type. Thus it can be presumed that the parental red stocks will be either RR (where F2 progenies are 100% red) or Rr (where F2 progenies are expected 3 red:1 wild) genotypes or combination of both. From this test crosses, 100% red progeny producing F1 stocks (RR genotype) can be isolated to develop purebred red strains for commercial breeding purpose.

5.3.5 Scoring of progeny phenotypes

Progeny phenotypes in all the crosses can be categorized as red (including blotched type) and wild type (those normally pigmented and completely different from those of red phenotype) of the same strains. Only F1 or F2 progenies can be categorized into full red (approximately <10% body surface with melanophores) and blotched types (approximately >10% body surface with melanophores), although both types together are designated as red (McAndrew et al. 1988). To determine the observed ratio of colour segregation, the proportion of progeny phenotypes are calculated as: (No. of progeny of a given phenotype / Total no. of survivors) x 100

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5.4

MAINTENANCE OF PUREBRED BROOD STOCK FOR SEED PRODUCTION IN THE HATCHERY

For purebred red brood stock development, fingerlings (20 30 g in weight) of RR genotypes could be produced or collected and stocked at the rate of 3-4 fish/m2 in the small and medium type brood stock rearing earthen ponds ranging from 1000 1500 m2 with the depth of 1.0 to 1.5 m. During all the phases of growing period of brood stocks, the fish should be fed with formulated or commercial feeds having at least 30% crude protein @ 3-10% body weight. During 1st and 2nd week of rearing the fish can be fed @ 10%, during 3rd and 4th week @ 5% and during 5th and 6th week onwards @ 3% body weight. Care should be taken not to contaminate with wild type or impure red blotched type Rr genotypes (Figure 18) in the rearing ponds. Brood stock replacement and stock improvement protocols and monosex production techniques for red strains will be the same as described respectively in Chapter 3 and Chapter 7.

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Fig. 17 Purebred red tilapia strain.

Fig. 18 Impure blotched type tilapia of red phenotype.

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Development and operation of mixed sex commercial tilapia seed production systems
MIXED SEX TILAPIA SEED PRODUCTION IN PONDS

6.1

Mixed sex seed production through controlled natural spawning in small and medium earthen ponds is a common practice for tilapia breeders. This system consists of three basic components as follows: i) ii) iii) Brood stock collection and maintenance. Fry production through natural spawning. Rearing of fry in nursery ponds.

6.1.1 Brood stock collection and maintenance

For seed production, the brood stock should be collected from the regional stations and sub-stations of BFRI or any other reliable known sources, who are maintaining outbred and improved stocks of O. niloticus. For brood stock development, fingerlings (20 30 g in weight) could be collected and stocked at the rate of 3-4 fish/m2 in the small and medium type brood stock rearing earthen ponds ranging from 1000 1500 m2 with the depth of 1.0 to 1.5 m. During all the phases of growing period of brood stocks, the fish should be fed with formulated or commercial feeds having at least 30% crude protein @ 3-10% body weight. During 1st and 2nd week of rearing the fish can be fed @ 10%, during 3rd and 4th week @ 5% and during 5th and 6th week onwards @ 3% body weight. If formulated feeds are not available
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alternatively a mixture of 60% rice bran and 40% mustard oil cake can be given at 5 -10 % biomass 2 times a day. Feeding of brood fish during low temperature and rainy days should be avoided to minimize the loss of resources.

6.1.2 Fry production through natural spawning (a) Pond selection and preparation
One or two ponds having an area of 400 800 m2 with inside slope about 1:3 and average water depth of 1.0 meter need to be selected for the purpose of natural spawning. All the predatory fish and animals are to be completely eradicated by dewatering and drying of ponds before stocking of fish. If this is not possible, then ponds need to be poisoned by using rotenone @ 10 12 kg/ha.. Ponds should be limed @ 250 300 kg CaO /ha. Seven days after liming, manuring and fertilization of each pond should be made respectively with cattle dung @ 800 1000 kg/ha and Urea plus T.S.P (25 + 25 kg/ha).

(b) Stocking of brood fish Sexually matured breeders weighing 80 to 100 g each should be stocked @ 2-3 fish/m2 with a sex ratio of 1 male to 3 females.

(c) Feeding
Supplementary feeds with a mixture of 60% rice bran and 40% mustard oil cake or 75% rice barn or 25% fish meal can be given at 3-5% biomass 2 times a day.

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(d) Collection of early fry

Three to four weeks after stocking of brood stock, when early fry are found schooling near the shore of spawning ponds, the available fry should be collected regularly in the early morning with a fine-mesh seine net and transferred to holding hapas (Figure 19) set in the ponds prior to stocking in the nursery ponds.

6.1.3 Rearing of fry in nursery ponds

The size and preparation of nursery ponds should be more or less the same as brood stock ponds described above. Rearing of tilapia fry to stockable size can be made following the two stages technique as follows:

(a) Primary stage

In well prepared nursery ponds, the first feeding fry can be stocked @ 500 600 per m2,. Early fry can be fed with powdered feeds as a mixture of rice bran and mustard oilcake (1:1 ratio) at the rate of 12 15% initial body weight 3 4 times per day. The growing fry need to be reared for 21 days in these ponds to attain an average weight about 1 g each. At this stage, the available fry can be sold directly to the buyers or reared in another series of nurseries by reducing their stocking densities.

(b) Secondary stage

In view of producing better stockable size, advanced fry can be reared further by stocking @ 100 200 m2 in a series of well prepared nursery ponds for 40 60 days.
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Fry can be fed formulated feeds (Table 6) and feeding rate can be reduced to 8 10% biomass 2 4 times per day. The fingerlings are expected to attain the average body weight about 8 10 g each.

6.2

MIXED SEX TILAPIA SEED PRODUCTION IN CONCRETE TANKS

Concrete tanks are often useful for tilapia seed production. Tanks can be constructed at any places including the towns and cities, which do not need much surface land area.

6.2.1 Tilapia breeding in the tanks (c) Design of the tanks

Tilapia breeding tanks are generally simple and smaller in comparison to the fattening tanks normally used for intensive culture systems. Shape of the tanks can be either circular, square, rectangular or oval Rectangular tanks are suitable and the size of individual tank may vary from 2.0 10.0 m long; 2.0 4.0 m wide with a depth of 0.8 1.0 m. 4-6 m diameter circular tank is also most economic size and self cleaning for tilapia seed production. Low cost breeding and fry rearing tanks might not have access to water flowing or recirculation system but can be facilitated for irregular water flashing for cleaning the tanks once a week (Figure 20).

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Fig. 19 Tilapia fry holding hapas.

Fig. 20 Low cost tilapia breeding and fry rearing tanks.

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(b) Stocking of brood fish

Sexually matured breeders weighing 80 to 100 g each should be stocked in the breeding tanks @ 3-4 fish/m2 with a sex ratio of 1 male to 3 females. Breeders need to be replaced when they attain an average weight of 250 g or more.

(c) Feeding
Supplementary feeds with a mixture of 60% rice bran and 40% mustard oil cake or 75% rice barn or 25% fish meal can be given at 3-5% biomass 2 times a day. The females while mouth brooding, they do not actively take artificial feeds. In this case, reducing the feed may lessen water quality deterioration in the tanks.

(d) Early fry collection

At the 12th to 15th day from stocking of the breeders, using a fine meshed dip net the first schooling fry should be collected. Every alternative week the fry collection needs to be repeated. Fry collected from the breeding tanks need to be graded, counted and stocked in a series of nursery tanks.

6.2.2 Fry/fingerling rearing of in the nursery tanks

For one week, the stocking of fry will be 1000 1200 per m2 and feeding will be made with high protein powder feed (at least 35% crude protein) @ 15-20% of biomass 4-6 times per day. After a week, the density should be reduced to 500 600 per m2 and feeding rates will be 12-15% of biomass.

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After second week, the density will be 300-400 per m2 and feeding rates will be 10-12% of biomass. Fry will be ready to sell when they will be >1 g each. Advanced fry can be reared further by stocking 100 150 2 individuals/m by feeding formulated feeds (Table 6) for 40 60 days. The fry/fingerlings can be harvested in the tanks by netting or draining the water.

6.2.3 Overall maintenance of tilapia breeding and nursery tanks

Breeding tanks should be cleaned manually or by water flashing fortnightly. Water depth of the nursery tanks can be kept 0.6 - 0.8 meter. Partial water change in nursery tanks can be done depending on the water quality. For healthy growth of fry/fingerings, water in the tanks should not be too green or foul in odor.

6.3

MIXED SEX TILAPIA SEED PRODUCTION IN HAPAS

A commercial mixed sex seed production system in fine meshed hapas (net cages) can be operated easily for large-scale production of tilapia seed for aquaculture where monosex tilapia culture is not preferred. The marginal or small-scale farmers with one or two earthen ponds having an area of 1500 2000 m2 each can follow a simple and efficient method.

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6.3.1 Tilapia breeding in the hapas (a) Setting of breeding hapas

A series of breeding hapas having the size of 2 m x 1 m to 12 m x 3 m with depths of 1.5 m can be set in the pond(s) by fixing them to bamboo poles by nylon thread. At least 0.25 m of top side of the hapas should above the waterline. The hapas can be covered with fine meshed nets or kept uncovered.

(b) Stocking of brood fish

Sexually matured breeders weighing 80 to 100 g each should be stocked @ 2-3 fish/m2 with a sex ratio of 1 male to 3 females. The breeders need to be fed with supplementary feed consisting of 60% rice bran and 40% mustard oil cake or 75% rice barn or 25% fish meal. The feeds can be given at 3-5% biomass 2 times a day. About 10 15 days after stocking of breeders, schooling of tiny fry will be visible in each hapa.

6.3.2 Fry/fingerling rearing of in the nursery hapas

60

Prior to capture and transfer of fry from breeding hapas another series of nursery hapas having the size of 8 m x 2.5m x 0.8 m need to be set in the same pond(s) or other pond(s). Fry can be collected by using dip or push net or by lifting the hapas and transferred to nursery hapas. Breeders weighing more than 250 g should always be replaced with new batches. During the first week, the stocking of fry will be 1000 1500 per m2 and feeding will be made with high protein powder feed (at least 35% crude protein) @ 15-20% of biomass 4-6 times per day. During the second week, their density should be reduced to 500 700 per m2 and feeding rates will be 12-15% of biomass.

Farming of Tilapia

During third week, the density will be 300-400 per m2 and feeding rates will be 10-12% of biomass. Fry will be ready to sell when they will be >1 g each. In view of producing better stockable size, advanced fry can be 2 reared further by stocking 100 200 individuals/m in nylon hapas (8 m x 2.5m x 0.8 m) by feeding formulated feeds (Table 6) for 40 60 days.

Table 6. Formulated feed for feeding tilapia fry in rearing hapas and nursery ponds (Hoq et al. 2003) . Proportion (%) Fish meal 28.00 Mustard oilcake 20.00 Rice polish 37.00 Wheat bran 10.00 Molasses 5.00 Total 100 Cost per kg feed: US$ 0.25 (Taka 15); FCR: 2.0 Feed ingredients Crude protein (%) 16.80 7.20 4.50 1.50 30.00

6.4

MIXED SEX TILAPIA SEED PRODUCTION IN RICE FIELDS

Rice fields are the ideal place for rearing tilapia fry either at the time of rice cultivation or after rice harvesting. Land can be prepared by using lime CaO @ 250 300 kg /ha; Seven days after liming, manuring and fertilization of rice plot should be made respectively with cattle dung @ 800 1000 kg/ha and Urea plus T.S.P (25 + 25 kg/ha). Immediately after sowing rice seedlings, mixed sex tilapia fry weighing 1 2 g can be stocked @ 0.1 0.2 million/ha for 30 40 days. Water depth in the rice plot should be at least 20 25 cm during fry rearing period. Fry can be fed rice polish and feeding rate should be 5 8% per estimated body weight of biomass for 2 - 3 times per day.
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The fingerlings are expected to attain the average body weight about 8 10 g each at end of rearing period. Enough care should be taken to protect any incidental escaping of fry/fingerlings from the rice fields to the surrounding canals/water bodies.

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Development and operation of monosex commercial tilapia seed production systems

SEX REVERSAL TECHNIQUE FOR THE PRODUCTION OF MONOSEX FISH FRY

7.1

Sex reversal is a technique of changing of sexes from one sex to another in fish by administering synthetic steroid hormones before and/or during the period of sexual differentiation. In this technique, the first feeding fry are treated with male hormones or androgens (ie. 17-methyl testosterone), which develops testes and male sexual characteristics at maturity and on the other hand, treatment with female hormones or oestrogens (17oestradiol) produces individuals with ovaries and female characteristics in fish. The choice of conversion of sexes (either all males or all females) depends on growth performance characteristics of individual sexes of fish species. For instance, in tilapia males grow faster than females, masculinization using androgen hormones (Shelton et al. 1978; Guerrero 1979; Guerrero and Guerrero 1988) and in case of salmonids and cyprinids, where females grow faster than males, feminization using oestrogen hormones (Shelton 1987; Bye and Lincoln 1986) have become a popular practice. The use of monosex populations also eliminates reproduction during grow out in the case of tilapias.

7.2

ALL MALE MONOSEX SEED PRODUCTION THROUGH INVERSION OF SEXES IN TILAPIA

Despite the popularity of tilapia species in worldwide aquaculture, the main drawback of all the existing commercial strains is their precocious
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maturation in tropical and sub-tropical climatic conditions. This leads to prolific breeding and over-crowding in grow-out systems, resulting in undesirable stunting and low yields of harvestable size fish. In many countries, where the acceptable market is 150 g or more and tilapias are normally grown in mixed sex culture this has become a critical problem (Guerrero 1982). In order to overcome this serious problem, since 1960 several methods have been proposed and developed to reduce and eliminate uncontrolled reproduction in grow-out systems. The main goal of all these methods was to produce monosex populations of tilapias by manual separation of sexes, interspecific hybridization and masculinization using hormones and genetic manipulation techniques. Hand or manual sexing of tilapia by examining urogenital papilla is a simple technique but it is time consuming, laborious, wasteful and sometimes unreliable at the small fish (<10 g). Interspecific hybridization of tilapias to produce all male hybrids was demonstrated first by Hickling (1960), using female O. mossambicus and male O. hornorum. After that many others also came forward to initiate similar interspecific hybridization between more supposedly homogametic species involving crosses of O. mossambicus x O. niloticus (Kuo 1969; Majumdar and McAndrew 1983); O. niloticus x O. aureus (Pruginin et al. 1975; Hulata et al. 1983). However, many of these crosses did not produce the predicted 100% male offspring. The results of Majumdar and McAndrew (1983) indicate that the mechanism responsible for sex determination in hybrids is indeed variable and complicated and that a number of different alleles of different strength are operating in the tilapias as a whole. On the other hand, genetic impurity of existing tilapia strains and careless maintenance of brood stocks in poor farming conditions made more problematic the method of hybridization for mass seed production of all male tilapia population. As interspecific hybridization and genetically induced all male seed production in tilapia have proved difficult in practice, direct masculinization of tilapias using hormones is the most common method
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for monosex male production (Shelton et al. 1978; Guerrero 1979; Guerrero and Guerrero 1988). The results of these authors suggest that functional sex-reversal using hormones can lead to the production of all male monosex population in tilapia for aquaculture. Monosexing has been the key that has facilitated the development of tilapia culture in the global food fish arena (Shelton 2002). In this process, male steroids is administered to first feeding tilapia fry so that the undifferentiated gonadal tissue of genetic females develops into testicular tissue, producing individuals that grow and function reproductively as males. Generally the primal gonadal tissue starts to differentiate into ovarian tissue with 34 weeks after hatching in O. niloticus (Velasco 2003b). Some concern has been raised that consumption of steroid-treated tilapia (ie. sex-reversed male tilapia using hormones) might be harmful for human health. But there is no evidence for any human health hazard (Green and Teichert-Coddington 2000). Recent studies have demonstrated that exogenous steroids are rapidly cleared from tissue after the end of treatment: no residual can be detected within one month of the termination of monosexing treatment (Rothbard et al. 1990; Green and TeichertCoddington 2000). In Asia, Philippines and Thailand are the two leading countries where commercial mass seed production of all male Nile tilapia using androgen hormones is a popular practice.

Advantages of all male seed production through sex-reversal

Technique is simple and economic. It does not need sophisticated laboratory and equipment. A technician or an experienced farmer can run monosex seed production hatchery and nursery systems. Overall input costs are not very high. Reliability of 98 100% male seed production. Ensures high production and high net profit.

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7.2.1 Hatchery design and operation of monosex seed production systems

Before initiating the construction of commercial monosex tilapia seed production systems, the following factors need to be considered for site selection: Site should be flood free and nearby tilapia grow out farms. Enough water supply source either from underground or other surface water source like rivers, canals or reservoirs. Recommended soil type for necessary pond construction. Road access for all weather for supply of products and services. Stable electricity. Market access of products.

(a) Design and construction of a hatchery complex

Major components and requirements of a tilapia hatchery to facilitate egg incubation, hatching and larvae nursing are as follows: Hatchery shed should be made by iron angles covered with CI sheets. Cemented hatchery floor. Egg incubating jars and trays with stands. Recirculating water system. Water supply lines (internal and external). Water pump (submersible pump). Overhead water tank of 10,000 20,000 liter capacity. Feed preparation and store room. Transitory nursing tanks. Nursery ponds for hormone treatment. Aeration systems.

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(b) Model of tilapia egg incubation system

Tilapia egg incubation and hatching system model is shown in Figure 21 and essential components are listed below: i. ii. iii. iv. v. vi. vii. Header tank to hold enough fresh water to supply the required quantity to incubation jars and trays. Incubation jars to hold and hatch the fertilized eggs. Nursing trays to hold and nurse the newly hatched larvae. Filter chamber where used waters from incubation jars and trays are drained and filtered. Bioball chamber through which incoming water is passed from filter for purification. Oyster shells chamber has the similar function to further purify water passing from other chambers (filter and bioball). Storage tank to hold purified water and back to the head tank through the submersible water pump.

(c) Hatchery shed and cemented floor construction

For constructing a medium size tilapia hatchery, the following components are essential: Hatchery shed structure: 86.25 m2 (11.5 m x 7 m). Cemented floor: 77 m2 (11 m x 7 m). Beams and column. MS angle truss. CI sheet roofing.

(d) Setting of incubation jars and trays

The incubation jars and trays with the following capacities and numbers need to be set with plastic containers and holding stands properly inside the hatchery (Figure 22):
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Fig. 21 Tilapia egg incubation and hatching system model (Courtesy: Dr. Nuanmane Pongthana).

Fig. 22 Typical modern monosex tilapia seed production hatchery system in Thailand.

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Number of 2 liter capacity jars: 30. Number of trays, 28 x 43 x 10 cm each: 60 (50% for jars plus 50% to use as nursing trays). Number of plastic container for holding the trays, 30 x 120 x 20 cm: 30. Steel/iron/wooden stands: 15 (50% for jars plus 50% for nursing trays).

(e) Construction of breeding ponds

At least 3 breeding ponds having an area of o.4 ha each can be constructed following a well-planned engineering design with the depth of 1.5 2 m. Ponds should be rectangular size with two bypasses for water supply and evacuation. The average water depth of each pond should be around 1.5 m.

(f) Making and setting of breeding hapas

Polyetheline or fine meshed nylon netting materials need to be procured from local market and rectangular size (40 m x 3 m with depths of 1.5 m) hapas are to be made by a tailor. About 8 10 hapas are installed in each pond (Figure 23) in the following ways: Hapas should be checked carefully before installation whether any hole is reaming or not. Hapas can be set by using bamboo/wooden or steel bar poles by tying the top and bottom corners into the poles staked into the pond bottom. Hapas need to be set 0.5 1m apart to allow water circulation. The top edge of the hapas should be 50 60 cm above the water surface. It is necessary to stretch the hapas as much as possible to prevent the folding by winds and perform the well setting.

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(g) Selection and stocking of brood stock in the breeding hapas

Stocking density of breeders varies with the size of hapas. Recommended densities ranges from 4 6 fish/m2. True breeding and improved strain(s) of Nile tilapia (eg. Super Strain of GIFT) weighing 100 150 g each either collected from known sources or already reared in separate ponds in the hatchery (section 4.1.1) can be stocked. Sex ratio should be maintained 1:3 (i.e. 1 male to 3 females). Breeders should be almost the same size at stocking, which will minimize the biting occurrence leading to skin lesions that can cause stress and even mortality of females. If the size of male is not comparable to female, clipping the premaxilla of the male needs to be done (Velasco, 2003b). Breeders >1-1.5 years old and >300 g in weight should be replaced by the new batches. Formulated or commercial feeds having 30% crude protein can be fed @ 3-4% per estimated weight of biomass 2 times daily.

(h) Collection of fertilized eggs from the fish mouth

Each sexually mature female normally liberates ovulated eggs on average at 2-3 week intervals under tropical pond conditions and subsequently the male partner in the breeding hapa fertilizes the eggs. The female fish holds the fertilized eggs in her mouth for natural incubation. So, it is necessary to check the mouth of all the stocked females in each hapa twice or at least once a week to collect the eggs as described below: The breeders are checked by gathering them at a place in the hapa using a 4 m long bamboo pole transversely run at the level of water surface from anterior end to other end to concentrate the brood fish at the posterior end of hapa. (Figure 24). For lifting the hapa and collection of fertilized eggs/near to hatch larvae or yolk sac fry at least 2-3 persons are needed. Each brood fish is taken by using scoop net and checked carefully to see if her mouth is holding fertilized eggs or yolk sac fry. For grading the different age groups of fertilized eggs or near to hatch larvae or yolk sac fry that are collected from mouth of the

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females are separated by their different colors (4 colors are normally identified viz. Whitish, pale yellow, deep yellow and reddish tiny fry with yolk sac) and can be kept in separate plastic bowls placed in a steel, iron or bamboo frame (Figure 25). Collected products can be disinfected with 5-7 ppt solution of saline water for 8-10 minutes at ambient temperature. After that the eggs/larvae/yolk sac fry are shifted to the hatchery for incubation.

(i) Incubation of collected eggs and larvae in the jars/trays

Fertilized eggs/hatched fry with yolk sac, that have been collected from the mouth of female breeders, are incubated in a series of round bottom plastic jars and flat trays connected to the recirculating system (Figure 26), where fresh water (281 oC) come directly from header tank by gravity. Immediately after hatching, the larvae are deposited at the basement of the attached tray and they are transferred to the series of separate trays (Figure 27) and kept until their yolk sac resorption stage is over i.e. first feeding fry stage. Each batch of fertilized eggs needs on an average about 10 12 days to complete the cycle of development (both embryonic and larval stages). At least 3 batches of first feeding fry per month can be produced at water temperatures between 27 - 30 oC. Fertilized eggs/hatched larvae can be stocked for incubation in each jar/tray by keeping the numbers given below: Estimated number of eggs in each jar: 25,000. Hatching period: 65 72 hours. Estimated number of hatched fry per tray: 24,000. Estimated total first feeding fry per batch: 7,20,000 (24,000 x 30 trays). Estimated total first feeding fry production per month: Approx. 2.2 million (7,20,000 x 3 batches).

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(j) Preparation, storage and application of androgen hormone mixed feeds

The protocol for preparation and application of hormone mixed feeds for sex reversal of Nile tilapia fry is shown as flow diagram in Figure 28 and briefly explained below:

Direct feed preparation of hormone mixed feeds

Hormone dose: 50 mg of 17- Methyl testosterone (MT) is dissolved in 100 ml Ethyl Alcohol (95%). MT solution is mixed with 1 kg powdered feed (mixture of 50% normal feed plus 50% fish meal) for 10 to 15 minutes. The treated feed is left to dry. Hormone mixed dry feeds are stored at room temperature or fridge at 4oC for maximum 7 days

Preparation of hormone mixed feeds using stock solution

Alternatively a stock solution can be prepared for 100 kg feeds and stored in refrigerator for few weeks for subsequent use (Velasco, 2003b): 5 g of 17- Methyl testosterone (MT) is dissolved in 1 liter Ethyl Alcohol (95%). 1 kg of finely sieved feeds (formulated feed as mentioned in Table 6) is placed in a clean dry mixing bowl. From prepared stock solution, 10 ml is further diluted to 100 ml of ethanol and shaken well. The solution is gradually poured into the feeds and mixed for 10 to 15 minutes. The treated feed is left to dry. Excess hormone mixed feeds can be stored at 4oC for a week.

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Fig. 23 Tilapia breeding hapas in pond.

Fig. 24 Gathering tilapia breeders at regular intervals for egg collection purpose in the breeding hapa.

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Fig. 25 Plastic vowels placed in a bamboo frame for separating the collected fertilized eggs having different colours (based on different age groups).

Fig. 26 A series of round bottom plastic jars and flat trays for incubating the fertilized eggs/hatched fry with yolk sac.

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Fig. 27 Separate flat trays where hatched larvae are kept until their yolk sac resorption stage is over.

Fig. 28 The protocol for preparation and application of hormone mixed feeds for sex reversal of Nile tilapia fry (Courtesy: Dr. Nuanmane Pongthana).

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For commercial operation automatic feed mixing machine can be used (Figure 29). For the safety of the workers involved in hormone feed preparation should use hand gloves and face mask.

Feeding of hormone mixed feeds to the first feeding fry in transitory tanks
The system for feeding of hormone mixed feeds to the first feeding fry in transitory tanks (Figure 30) is briefly explained below: Size of tank: 17 m x 3 m x 0.75 m. Number of tanks: 4. Covered with fine meshed netting materials. Hapa size: 8 m x 2.5 m x 0.6 m. Number of required hapas in each tank: 2. Number of fry in each hapa: 1,50,000. Water level in the tank: 60 cm. Feeding hormone treated feed is initiated in these hapas. Feeding rate: At satiation level. Feeding intensity: 4-6 times daily. Duration of feeding: 3 days. Water quality of the tanks need to be maintained by regular exchange of fresh cool water (temperature 24 27oC). At the end of transitory period fry are shifted to nursery feeding hapas by estimating their numbers (Figure 31).

Feeding of hormone mixed feeds to the early fry in nursery hapas

At least one pond having an area of 0.4 ha can be constructed following a well-planned engineering design for setting the nursery hapas. Ponds should be rectangular size with two bypasses for water supply and evacuation. The depth of pond should be 1.5 - 2 m. Hapas can be installed in the pond and fixed and tied to nylon ropes and bamboo poles like
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breeding hapas or well designed hapa holding frame can be made over the pond using RCC construction as shown in Figure 32a and 32b. The protocol for feeding of hormone mixed feeds is shown in a flow diagram (Figure 33) and technique of application of feeds to the early fry in the nursery hapas is shown in Figure 34. The well designed fry feeding system for the production of all male monosex fry is summarized below: Hapa size: 8 x 2.5 m x 0.75 m Number of required hapas: 25 hapas Stocking density of fry in each hapa: 100, 000 Total number of stocked fry : Apprx. 2.5 million Feeding rate: 15-30% per estimated body weight Feeding intensity: 4-6 times daily Duration of feeding hormone mixed feeds: 18 -21 days Monosex all male fry production: Apprx. 2 million per month Water quality of the nursery hapa holding ponds need to be maintained by regular exchange of fresh cool water (temperature 24 27oC)

Monosex fry storage and rearing for sale

It is expected that after 21-24 days of feeding hormone mixed feeds to the fry 95 - 98% will be sex reversed male. At this stage the fry will be reared by feeding normal feeds (Table 6) until selling in separate nursery hapas in an earthen pond and their maintenance will be as below: Hapa size: 8 m x 2.5 m x 0.8 m. Storing: Monosex fry of 21 24 days old. 3 Stocking density: 400 600 individuals/m . Nursery feeding rate: 8 - 10 % per estimated body weight. Rearing period: 1- 2 weeks.

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(k) Sex identification technique in tilapia fry

In tilapia fry/fingerlings larger than 20 30 g, sex can easily be identified manually by examining their urogenital papilla. But in case of early fry smaller than 2 g, where the manual sexing is not useful, an aceto-carmine squash technique is used as described by Guerrero and Shelton (1974). A protocol for sex identification in tilapia fry is shown in a flow diagram (Figure 35). A sub-sample of fry are killed and dissected using a sharp pointed surgical scissor. The tiny thread-like gonad that lies along the anterodorsal abdominal cavity is removed using fine forceps. The collected gonad is placed in a glass slide and a drop of acetocarmine stain is added on the gonad. The gonad is lightly squashed with a cover slip. Then the gonads are examined under the microscope, the male gonad is composed of fine granular like structure of spermatogonia and the female is characterized with the structure of circular oogonia.

The technique of aceto-carmine stain preparation is as follows: Carmine (granular stain): 45% Acetic acid: Boil for 2 4 minutes, cool and filter. 0.5 g. 100 ml.

7.2.2 Production of YY males and operation of monosex all male seed production system
Direct hormonal masculinization might not appear to be a viable technique in tilapia and might have adverse environmental impacts or consumer reaction in near future, in that case the indirect method of producing monosex all males (ie. YY males) by combining both sex-reversal and/or genetic manipulation of the sex determining system will once be the alternative choice of the commercial seed producers.
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Fig. 29 Automatic hormone feed mixing machine.

Fig. 30 The technique of application of hormone mixed feeds to the first feeding fry in the transitory hapas.

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Fig. 31 Manual counting of tilapia fry.

Fig. 32a Hapas can be installed in the pond and fixed and tied to nylon ropes and bamboo poles for feeding hormone mixed feeds.

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Fig. 32b Hapas can be installed and fixed with RCC frame made over the pond for feeding hormone mixed feeds.

Fig. 33 The protocol for feeding of hormone mixed feeds (Courtesy: Dr. Nuanmane Pongthana).

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Fig. 34 The technique of application of hormone mixed feeds to the early fry in the nursery hapas.

Fig. 35 The protocol for sex identification in tilapia fry (Courtesy: Dr. Nuanmane Pongthana).

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(a) Breeding and fry production for hormone treatment

The protocols for breeding, egg collection, incubation and hatching of fertilized eggs are same as described in the section 5.2.1 of this chapter.

(b) Preparation, storage and application of estrogen hormone mixed feeds

The protocols for preparation, storage and application of estrogen hormone mixed feeds are more or less the same as the protocol of androgen hormone. In this case 17-oestrodiol hormone is used. Feed preparation technique is summarized below: Hormone dose: 50-100 mg of 17-oestrodiol is dissolve in 95% Ethyl Alcohol. Hormone solution is mixed with 1 kg powdered feed (mixture of 50% normal feed plus 50% fish meal). Hormone mixed dry feeds are stored at room temperature or fridge at 4oC for maximum 7 days.

(c) Protocols for production of all male monosex population using YY males
It needs to go up to at least three generations to produce all male monosex population through this indirect method of sex reversal (Figure 36). The essential steps of the protocol are as follows:

Production of F1 generation
The first feeding fry are fed with estrogen hormone mixed feeds at the rate of 15-30% body weight 4 6 times daily for at least 21 days in a series of nursery hapas. It is expected that the sex reversed progeny will be about
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100% female having XX and XY genotypes. The XY genotypic females can be termed as neofemales and need to be identified by progeny testing.

Production of F2 generation
Sexually matured neofemales (XY genotype) can be crossed with normal female (XX genotype) to produce F2 generation of progeny. The genotype ration of the produced generation are expected to be 1XX females: 2 XY males: 1 YY males (75% males and 25% females). Among males, YY genotypic males need to be identified by further progeny testing at their maturity.

Production of F3 generation
YY genotypic males can be crossed with normal females (XX genotype) to produce F3 generation of all males (XY genotypes). Commercial production system of all male monosex population using YY males can be established and operated same as monosex seed production using androgen hormones. In this case, precautions must be taken for breeding, progeny testing and identifying carefully the YY genotypic males and preserved them separately in the system. True breeding tilapia strains and highly experienced technician(s) are prerequisite for running such system for commercial seed production.

All male monosex fry rearing and sale

About 100% monosex male fry produced by using YY males can be reared in the nursery hapas with normal feeding as per recommended rates for 25 30 days. Fry will be ready to sell when they will be >1-2 g each.

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Fig. 36 The protocol of producing all male monosex population through the indirect method of sex reversal.

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Development and operation of semi-intensive tilapia culture systems

TILAPIA CULTURE IN SEASONAL DITCHES AND PONDS

8.1

Because of the seasonal river flow and monsoon rains, 34% of the country is considered to be wetlands that remain under water for at least 6 months of the year. The country has millions of small ponds, seasonal water fieldditches; borrow pits, flooded paddy fields etc. that all have potential for producing increased yields of fish through managed aquaculture practices. The seasonal closed water areas particularly in the form of small natural depressions, hilly creeks, dead river lagoons, shallow marshy wetlands, road side ditches, backyard impoundments and ponds, where average water depth remains 80 100 cm, are highly suitable for mixed or monosex farming of improved strain (s) of tilapia.

8.1.1 Pond selection and preparation

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Seasonal pond is preferred having an area 0.04 0.08 ha (ie. 10 20 decimal) with average depth of 0.8 1.25 m. Selected ponds should be dried. Dykes should be repaired and free of unwanted vegetation. Liming of pond bottom with 250 300 kg CaO or CaCo3 per ha. Cattle dung or poultry manure can be applied on the bottom @ 2000 3000 kg per ha. Ponds need to be filled with fresh water and water level can be remained at optimum. Pond fertilization with urea and triple super phosphate (TSP) @ 50 kg in 1:1 ratio can be used before stocking of tilapia fingerlings.

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8.1.2 Stocking of fry and pond management

Mixed or monosex tilapia fry of 10 15 g weight can be stocked in the prepared ditches or ponds @ 15000 - 20000 per ha. Stocked fish need to be fed with rice polish @ 3 5% per estimated weight of biomass. In well-managed ponds fish can be fed with formulated feeds as shown in Table 7. To enhance the status of natural food in pond water, regular fertilization can be made at 15 days intervals with cattle dung or poultry manure @ 800 1000 kg per hectare. If the ponds are stocked with mixed sex tilapia, undesirable populations of tiny fry will be seen within 3 months of stocking. In that case as many as possible of the fry must be removed by repeated netting (using fine mesh seine net) at 15 days intervals to avoid the problem of overpopulation. Every month sampling of growing fish should be made to check the growth and adjust the feeding rate.

8.1.3 Fish harvesting and estimation of production

After 4 6 months of grow out when the fish have the average weight of 150 -200 g, harvesting of fish can be made by repeated netting or drying out the ponds. Under semi-intensive culture system, yields of 2500 3000 kg fish per ha can be obtained per crop.

8.2 TILAPIA CULTURE IN RICE FIELDS

Rice fields either irrigated or rain fed is the suitable plot, which can be used for tilapia fry rearing and culture. Due to application of adequate
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quantity of fertilizers and manures in the rice fields, the available aquatic condition become rich enough in natural food to support fish growth. So, in most cases supplementary feeding to growing fry and fish is not a prerequisite.

8.2.1 Selection of land

Land should not be flood prone. High land should always be avoided. Soil texture should be clay-loamy. Land should have irrigation facility.

8.2.2 Preparation of land

Dyke around the land should be constructed at the height of 25 30 cm. A small ditch (1 m deep) needs to be constructed at the lower part of the land. The ditch size should be 3 4% of the rice plots. Water depth in the rice plot should be at least 20 25 cm during culture period. Manures and fertilizers can be used as per recommended doses of rice plot preparation.

8.2.3 Stocking of fish

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15 days after transplantation of rice seedlings, when water depth of the plot remains around 20 cm, the plot will be ready to stock tilapia fry. About 4000 5000 tilapia fry (either mixed or monosex) having a weight of 10 - 15 g each can be stocked per hectare. Improved strain of Nile tilapia (eg. Super strain of GIFT) should be considered for stocking in the rice fields for better production.

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8.2.4 Overall management

Optimum level of water in the rice plots should always be kept during culture period. If necessary irrigation should be made at regular basis. Dykes should be protected from rat and crab nesting. Bamboo screens can be used at some places of the dykes to reduce the excess water level during heavy rainy season. Special care should be taken that growing tilapia or naturally produced fry (in case of mixed sex stocking) cannot escape from the rice plots to nearby water bodies. During culture period rice plots can be fertilized with chemical fertilizers at recommended doses. Normally feeding of fish is not required in the rice plots, but farmer can apply rice polish @ 2 3% per estimated weight of fish 5 days a week in the ditch area. Use of pesticides is prohibited in the rice plots during fish culture. Care should be taken to protect the incoming source of pesticide mixed water from nearby plots. During extreme hot weather, water hyacinth can be spread over the ditches to protect the growing fish.

8.2.5 Fish harvesting and estimation of production

Fish can only be harvested after sawing the rice from the plots. Either netting or complete dewatering the rice plots, all the fish can be captured and yield can be estimated. It is recorded that about 300 350 kg of tilapia can be produced along with rice per ha of land per crop (Hussain and Koohinoor 2003).

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8.3 POLYCULTURE OF TILAPIA WITH CARPS

In a Southeast Asian country like Bangladesh, polyculture of carp species in homestead perennial ponds is a traditional practice but the available small or medium seasonal water bodies also hold tremendous potential for culturing improved tilapia strain(s) along with various desirable carp species, involving low inputs in terms of labour and costs and ensuring high production within a short period (4-6 months) of time (Hussain et al. 2000). Such kind of fish farming operation cannot only increase intake of animal protein for rural people but can also generate income and employment opportunities (Hussain et al. 2000). To initiate polyculture of tilapia with carp species in seasonal ponds and such type of water bodies the steps as mentioned below can be followed:

8.3.1 Pond selection and preparation

In this case, pond selection and preparation procedures will also be same as section 8.1.1.

8.3.2 Stocking of fish and pond management

Mixed or monosex tilapia fry/fingerlings along with short cycle carp species viz. silver barb, common/mirror carp and silver carp can be stocked in the prepared ponds. The species combination should be genetically improved tilapia strain 60%; silver barb 20%; common/mirror carp 10% and silver carp 10% or improved tilapia strain 20%; silver barb 60%; common/mirror carp 10% and silver carp 10%. The average weight of fry/fingerlings should be 10 15 g each and stocking density should be maintained 20000 fish per ha. The fish can be fed with formulated feeds as shown in Table 7. To enhance the status of natural food in pond water, regular fertilization can be made at 15 days intervals with cattle dung or poultry manure @ 800 1000 kg per hectare.

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If the ponds are stocked with mixed sex tilapia, undesirable populations of tiny fry will be marked within 3 months of stocking, in that case as many as possible of the fry must be removed by repeated netting (using fine mesh seine net) at 15 days intervals to avoid the problem of over population. Every month sampling of growing fish should be made to check the growth and adjust the feeding rate.

8.3.3 Fish harvesting and estimation of production

Within 4 6 months of farming when the tilapia and silver barb will attain the average weight of 200 g; common/mirror carp and silver carp attain the average weight of 500 600 g, harvesting of fish can be made by repeated netting or drying out the ponds. Under semi-intensive culture system, a yield of 3000 3500 kg fish per ha can be obtained per crop.

8.4

TILAPIA CULTURE IN PENS

When a part of open water bodies like dead river basins, small streams, canals, hilly creeks etc. is encircled or blocked by using bamboo screens or nylon nets, it can be termed as a pen. Tilapia can be cultured in these types of suitable pens comparatively keeping moderate or high density with or without feeding.

8.4.1 Site selection and setting the pens

Large or small open water bodies which are not being used for fish culture can be selected. Selected water bodies can be encircled or screened either by bamboo fencing or net made by knotless polyethylene net materials with a mesh between 10 15 mm.
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The pen fencing should be fixed with strong bamboo or wooden poles having the enough height above the level of water to withstand wind pressure and water current, if any. Water depth of the pens should be 1 3 m.

8.4.2 Stocking of fish

All monosex tilapia fingerlings (10 15 g size) need to be stocked to avoid the risk of contamination and undesirable reproduction in the water bodies. Fingerlings should be collected from a known source or a hatchery where pure breeds of Nile tilapia or its improved strain(s) are maintained. Stocking density can be maintained @ 30 50 fish/m3.

8.4.3 Feeding
Fish should be fed with formulated feeds as shown in Table 7 @ 3% per estimated body weight. Feeding intensity should be at least 2 times daily. Every 30 days sampling of growing fish should be made to check the growth and adjust the feeding rate.

If the stocking density remains >30 fish/ m3 and the water bodies seem to be productive, in that fish might not need artificial feeding.

8.4.4 Harvesting and estimation of production

Fish can be harvested when they attain the weight about 150 250 g, which might take 4-5 months. It is roughly estimated that under a good management averagely 5-8 kg/ m3 can be harvested.

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8.5

TILAPIA CULTURE IN PONDS UNDER COMMERCIAL FARMING MANAGEMENT

8.5.1 Construction of Commercial Tilapia Farms (a) Site Selection

For site selection of tilapia farms, the important factors to be considered are as below: Soils: Soils with more than 20% clay are essential to reduce the loss of water by seepage; soils containing gravel or sand layers or rock strata formations should be avoided. A neutral pH (6.5 7.5) of soil is desirable and acid sulphate soil needs to be avoided. Water: Any aquaculture operation generally needs a good quantity and good quality of water. Water source might be from rivers, irrigation canals, reservoirs and shallow or deep tube wells (ie. underground). Any sort of water that will be used for fish farms must not be contaminated with toxic chemicals such pesticides, herbicides, heavy metals and organic matter. Water loss in a pond through evaporation is a common fact in both tropical and sub-tropical climates. It is roughly estimated that evaporation of water can reach on an average 2.5 cm (about 1 inch) per day, which might require an inflow of water at least 3 liters per second/ha (Huet 1979). Topography: Pond construction lay out should be made on the basis of favorable topographical conditions. A slightly depression type of land is ideal where simple dykes can be constructed easily and economically to hold adequate water depth.

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If the ground is completely flat type, completely new ponds need to be constructed. In such case, ponds can be made both with outlet and inlet by pass facilities for supply and evacuation of water.

(b) Pond Design

After finalization of site selection, the farm area should be marked as per engineering design. A well-designed farm might consist various types of ponds having all essential components, which include: Pond depth: Depth of a pond should be kept on the basis of type of fishes as well as pattern of culture systems. Generally, average depth for tilapia farm might be as below: a. Nursery pond for early fry: b. Rearing pond for advanced fry/fingerlings: c. Grow-out ponds: 08 to 1.0 m 1.0 m to 1.25 1.5 to 2.0 m

The dike construction: Building a solid and seepage protected earthen wall or dike is one of the most important task of pond construction to keep enough water for planned fish production. Manual laborers, bulldozers, mechanical excavators etc. can be used for digging ponds and transporting soils for constructing dikes. The following principles can be followed to construct an ideal dike: Width of the dike at the top should be equal to its height but should be never less than 1 m wide. The dike must be some 30-40 cm above the surface of water for small ponds and 50-60 cm for large ponds. The slope for outside angle of the dike should be 1:1 to 1:1.5 and on the inside slope about 1:2. For small pond it can be reduced to 1:1.

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When dike construction is complete, it can be covered with the topsoil and turf or soft grasses. Draining installation (the monk): In a pond the monk is a part of dike, which regulates the required level of water as well as draining of water at the time of urgency and harvesting period of fish in a pond (Figure 37). The emptying device consists of a horizontal channel or drainage pipe running the full length of the foot of the dike, and also a vertical branch or so called monk, quadrangular in shape 40-60 cm wide (also depends on the area of the pond) open on one side of front. The monk is normally built in concrete and should reach at least 30-40 cm above the level of water. Two or three parallel groves are made using U shaped iron rod or cutting the concrete for making the placing of screen and wooden boards (20-30 cm high and 4-6 cm thick).

(c) Arrangement of ponds and layout of a farm

Most ideal arrangement of ponds of a farm should include one or two or multiple series of parallel ponds having two bypasses. Each pond should have its own independent supply and evacuation facilities (Figure 38).

8.5.2 Operation and Management of Commercial Tilapia Farms (a) Area of farms
Area of commercial tilapia farms can vary from 3 to 25 ha, which depends on production target and management system. (Figure 39). Typical farm areas might be as follows: Small farm: Medium farm: Large farm: 3 5 ha 7 10 ha 15 25 ha
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(b) Grow out pond preparation: liming and fertilization

Liming of pond bottom should be with 250 300 kg CaO or CaCo3 per ha. Cattle dung or poultry manure can be applied on the bottom @ 2000 3000 kg per ha. Ponds need to be filled with fresh water and water level can be remained at optimum. Pond fertilization with urea and triple super phosphate (TSP) @ 50 kg in 1:1 ratio can be used before stocking of tilapia fingerlings.

(c) Stocking of fry and post stocking management

Mixed or monosex tilapia fry of 10 15 g weight can be stocked in the well prepared ponds @ 25000 30000 per ha. Fish should be fed with formulated feeds as shown in Table 7 @ 3% per estimated body weight. Feeding intensity should be at least 2 times daily. To enhance the status of natural food in pond water, regular fertilization can be made at 15 days intervals with cattle dung or poultry manure @ 800 1000 kg per hectare. If the ponds are stocked with mixed sex tilapia, undesirable populations of tiny fry will be marked within 3 months of stocking. In that case as many fry as possible must be removed by repeated netting (using fine mesh seine net) at 15 days intervals to avoid the problem of over population. Every month sampling of growing fish should be made to check the growth and adjust the feeding rate.

(d) Water quality monitoring

Water quality in tilapia farm should be monitored every week using HACH kit or other electronic equipments.
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For good fish growth of tilapias, desirable water quality parameters are: pH: Dissolved Oxygen: Free Carbon Dioxide: Total Alkalinity: Total Hardness: Temperature: Salinity: 6.5 to 9.0 3.0 to 8.0 mg/L 5.0 to 12 mg/L 10.0 to 100.0 mg/L 50.0 to 250.0 mg/L 25.0 to 30.0 oC 1.0 to 15.00 ppt

(e) Water supply and aeration

A regular water supply (twice a week) needs to be available in the grow out ponds just to fill up the level, to compensate for evaporation.

(f) Culture period

120 180 days.

(g) Fish harvesting and estimation of production

Within 4 6 months of farming when the fish having the average weight of 200 g, harvesting of fish can be made by repeated netting or drying out the ponds. Under such type of semi-intensive culture system, yield of 4000 6000 kg fish per ha can be obtained per crop.

8.5.3 Option for commercial tilapia farming in ponds under the management of high stoking density
In commercial farms tilapia can be cultured even under high stocking density provided simple paddle wheel type of aerators are set in the ponds
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(at least two aerators in 1 ha pond) for aeration of water to add more oxygen (Figure 40). Under such type of farming management, the grow out pond preparation; liming and fertilization procedure will be same as above. Stocking density of advanced fry/fingerlings should be maintained @ 80,000 100,000 per ha. In that case input cost will be high due to running aerators (the cost the machine and electric bills) but production of marketable size fish can reach up to 15,000 20,000 kg per ha per crop.

8.5.4 Option for commercial tilapia farming in brackish water ponds

As tilapia can tolerate salinities up to 25 ppt with gradual acclimation, so the fish can nicely be cultured in brackish water ponds with a salinity range of 10 15 ppt without any stress. Farm areas close to the sea and exposed to strong waves should be avoided. A range of at least 0.6 meter between mean high tide and mean low tide is necessary to ensure adequate tidal water flow (Guerrero 1997). Brackish water enclosures are extremely suitable for tilapia culture either by extensive or semi-intensive type of management. Alternate cropping of tilapia in shrimp farms has recently been introduced in the coastal belt of Bangladesh as a means of reducing the risk of shrimp disease outbreak. In brackish water farms, under semiintensive culture management, a yield of 3000 4000 kg fish per ha can be obtained per crop.
Table 7. Formulated feed for feeding tilapia under semi-intensive system in the grow out ponds (Hoq et al. 2003). Proportion (%) Fish meal 20.00 Mustard oilcake 15.00 Rice polish 40.00 Wheat bran 20.00 Molasses 5.00 Total 100 Cost per kg feed: US$ 0.21 (Taka 13); FCR: 2.0 Feed ingredients Crude protein (%) 12.00 5.40 4.90 2.90 25.00

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8.5.5 Overall management of commercial tilapia farms

Like aquaculture of other commercially important fish species, commercial tilapia farming is capital intensive. The main inputs needed are good water, quality seeds, feeds and fertilizers. The operation of such types of farming is also labor intensive, where regular maintenance of the ponds and farms requires various types of manpower but mostly labor. Therefore, good capital investment is a prerequisite for obtaining maximum profits from commercial fish farming and the overall management options as below can be considered: A commercial farm should have the opportunity to produce the required quantity of quality fry (both mixed and monosex) in the available nursery ponds or procure such fry from reliable hatcheries Before stocking, fry should be grown up to a suitable size (at least 10 15 g). If the fry are taken from other sources, they should be kept for few days in a series of tanks or small ponds for prophylactic treatment. In high density stocking ponds, partial harvesting of grow out fish (about 150 g weight each) can be followed to reduce the load of biomass per unit area of ponds.

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Fig. 37 The monk in a pond

Fig. 38 A typical layout of a fish farm (Bromage et al. 1992).

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Fig. 39 View of a commercial fish farm (Courtesy: Dr. M.J. Alam).

Fig. 40 Simple paddle wheel type of aerators set in the ponds for aeration of water to add more oxygen.

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Development and operation of intensive tilapia culture systems

THE SUITABILITY OF TILAPIA FOR INTENSIVE CULTURE

9.1

It is mentioned in the first chapter of this book that commercially important tilapia species or strains have some special characteristics, viz. ease of seed production, resistance to infectious diseases and poor water quality, tolerance to wide range of environmental conditions, efficiency to convert organic and agricultural wastes in to high quality protein and ability to grow well at high stocking and overcrowding situations, which make them feasible for farming under various culture systems. Among the desirable cultivable finfishes, tilapias are the most suitable candidates for intensive culture in cages and raceways due to their amenability to intensification. Bangladesh has more water resources per capita than most other countries of the world and demand of fish for consumption and export is very high, therefore, the aquaculture industry is booming day by day. The time is not very far off when intensive farming of commercially important fish species particularly tilapias will take the place in aquaculture due to limited land and other resources. Many entrepreneurs might come forward in near future to initiate developing the high input and high cost systems for tilapia farming in this country. Because, for the international and domestic markets, tilapia has tremendous prospects to be considered as a number one protein food item. In view of that an attempt has been made to narrate briefly the available techniques of tilapia culture in the cages, tanks and raceways.

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9.2

TILAPIA CULTURE IN CAGES

Among intensive culture methods, cage culture is one by which the farmer can stock a large number of fish, control over feeding, minimize unwanted reproduction, harvest easily and obtain maximum profit from per unit area. It permits the more intensive exploitation of a water system with a low capital expenditure. The method can be utilized minimal infrastructural requirements and the ease of management lends the system to intensification (Balarin and Haller 1982). Various materials viz. simple bamboo poles and nylon nets, plastic and steel materials etc., can be used to construct cages for tilapia culture. Very large operations have been developed in the Philippines and Indonesia based on cage culture of respectively tilapias and common carps that provide jobs for thousands as members of cooperatives or employees of companies and food for local consumption and for international trade (Fitzsimmons, 1997). Cage culture of tilapia has also been attempted commercially or experimentally in other countries viz. Brazil, China, Cote dIvoire, El Salvador, Guatemala, Israel, Indonesia, Philippines, Puerto Rico, Niger, Sri Lanka and USA. In Bangladesh, Bangladesh Fisheries Development Corporation (BFDC) was the pioneer to initiate experimental cage culture of Nile tilapia in Kaptai Lake, Ragamati some time during 80s but no production data is available. Subsequently CARE, Bangladesh conducted grow-out trials of GIFT strain in cages at Meghna river lagoon area near Munshiganj during 90s (Hussain et al. 2000). Even, meanwhile, CARE implemented a CAGES project for more than 5 years with limited success as potential livelihood option in different places of Bangladesh. Recently a number of private entrepreneurs have initiated tilapia cage culture at Meghna river canals near Chandpur. As public water bodies like reservoirs, river lagoons, lakes, irrigation canals, deep borrow pits, estuaries, coastal bays including perennial natural depressions, and village ponds etc. are suitable for cage culture, therefore, small or large scale operation of tilapia culture in cages in these or other selected suitable water bodies has a bright future to play an important role in aquaculture.
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9.2.1 Site selection

Dead rivers, lagoons, deepwater lakes, protected irrigation canals, creeks, deep ponds, borrow pits, closed coastal bays can be selected. Optimum water depth of a water body should be 4 10 m. The site should be free from remains of trees and other debris. The sites should be out of the reach of heavy flood, high tides and cyclones etc.

9.2.2 Construction and setting of net cages (a) Essential materials for constructing a floating net cage
Bamboo pieces Styrofoam blocks Empty Drums Nylon nets with a mesh size of 15 25 mm Wood pieces Plastic ropes Iron nails

(b) Construction of the raft

Bamboo pieces need to be arranged to make a square raft and drums should be fixed and set underneath of the raft so that drums can float the raft.

(c) Making the net cage

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Net materials (mesh size 15 25 mm) should be cut and fixed by sewing the ends with plastic ropes to form a square net. Various sizes of net can be considered viz. 1 x 1 x1 m; 2 x 2 x 2.0 m; 4 x 4 x 2.5 m; 6 x 6 x 2.5 m; 8 x 8 x 2.5 m.

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(d) Setting the net cage with the raft

The net can be attached to the floating raft by binding the net to bamboo poles fixed at equal distances to the raft. Weights (2 kg) need to be fixed and hung around the bottom of the net to stretch the net into a square. The floating rafts with net cages can be set in one or two series to form a battery of cages for large-scale operation (Figure 41). A wooden or bamboo platform can be constructed along the series of rafts to allow feeding trolleys, sampling equipment/buckets and harvesting nets etc. to be carried. The rafts with net cages finally need to be anchored strongly to the shore or bottom.

(e) Stocking of fish

Mixed or monosex tilapia fingerlings weighing 20 30 g can be procured for stocking in the net cages. Stocking density can be maintained at 200 400 individuals/m3.

(f) Feeds and feeding

Fish should be fed with formulated feeds as shown in Table 7 @ 3% per estimated body weight. Feeding intensity should be at least 2 times daily. Every 2 weeks sampling of growing fish should be made to check the growth and adjust the feeding rate.

(g) Culture duration

Duration of tilapia culture in cages should range between 90 150 days.
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(h) Harvesting of fish and expected production Fish weighing on an average about 200 g can be harvested by using dip or push net or by lifting the cages and transferred to bamboo baskets or plastic buckets. Under such intensive system of cage culture expected tilapia production will be 30 70 kg/m3 (averagely 50 kg/m3)..

The tilapia cage culture information and production data of different countries are summarized in Table 8.

Fig. 41 The floating rafts with net cages for intensive tilapia culture (Courtesy: Dr. Nuanmane Pongthana).

9.2.3 Overall management of tilapia culture in floating cages

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Water quality and fish health in the cages needs to be monitored regularly. Dead and sick fish should always be removed from the cages.

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If the growing fish do not take feed particularly in rainy or cloudy days, it is better to stop feeding until the next day. If the stocked fish show any attempt of jumping out, in that case lids made of fine mesh nets can be used over each net cage. At every harvest, the nets need to taken out from the raft to remove the algae scum and sun dried before using further. Security of the cages needs to be ensured to avoid poaching and escapement of fish during windy or stormy weather as well as during monsoon months.

Table 8. Available data on tilapia cage culture in different countries (Revised after Balarin
and Haller 1982) Countries Bangladesh China Philippines Cote dIvoire Niger Nigeria Puerto Rico USA USA Species O.niloticus O.niloticus Hybrid (O. mossambicus x O.niloticus) O.niloticus O.niloticus S.galilaeus O.niloticus T.zillii 0..aureus O.aureus O.aureus no./m 350 300400 200 215488 264 256 300500 286857 487
3

Duration (days) 120 96 103 92 210 171 70 156 87

Production 3 kg/m 30.68 100.6 63.0 35-76 68.64 17.43 17-23 35-94.30 66.5-85

FCR 1.4:1 1.9 1.9 2.63.7:1 2.353.63:1 12.2:1 1.21.7:1 2.24:1

Reference Hussain et al. (2000) Guerrero (1997) Guerrero (1997) Coche (1975) Mikolasek et al. (1997) Ita (1976) Jordan & Pagan (1973) Pagan (1970) Suwanasart (1971)

9.3 TILAPIA CULTURE IN TANKS AND RACEWAYS

Intensive culture of tilapias like salmonids in the recirculating or flow through tanks/raceways is one of the preferred techniques for large-scale commercial production. These practices ensure high yield per unit area and/or unit volume of water. Important processes in such systems are the removal of solids and dissolved metabolites. High water exchange is
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needed for this purpose. In water-limited areas, intensive tank and raceway culture requires water treatment and recirculation (Cole et al. 1997). In tanks and raceways tilapias are cultured under crowded conditions. These systems are especially attractive in areas that can recover the effluent water from these farm operations to use for field crop irrigation and can be used as good source of fertilizers (Fitzsimmons 1997). Experimental or active rearing of tilapia in tanks has been reported from a number of countries (Table 9) and use of raceways reported from a few countries (Table 10) of the world.

9.3.1 Tilapia culture in tanks (a) Site selection

Before initiating the construction of rearing or growing tanks for commercial tilapia production systems, the following factors need to be considered for site selection: Site should be flood free and nearby tilapia hatcheries. Enough water supply source either from underground or other surface water source like rivers, canals or reservoirs. Road access for all weather for supply of products and services. Stable electricity. Market access of products.

(b) Design and characteristics of the tanks

Cemented tanks are suitable for intensive tilapia culture, generally wider than raceways (Figure 42). Shape of the tanks can be circular, square, rectangular or oval. Rectangular size of individual tank may vary from 10.0 50.0 m long; 8.0 20.0 m wide with a depth of 0.8 1.0 m. 10 m diameter circular tank (10 t unit) is considered as the most economic size and self-cleaning for tilapia production

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An intensive tilapia culture tank is required to have a demand feeder, a low flow to velocity ratio, so that water can be made useful to maintain a high water quality, maximize the requirement of oxygen of growing fish and provide rapid expulsion of solid wastes and faecal materials through the central drain (Balarin and Haller 1982). The cost and construction complexity of tanks should be low and the system should have access to water recirculation and aeration. Tanks should be adaptable to fry rearing, on growing and fattening of desired numbers of fish.

Fig. 42 The cemented tanks for intensive tilapia culture (Courtesy: Mr. Y. K. Thai).

(c) Stocking of fish

For fattening tilapia fingerlings weighing 25 -50 g can be stocked @ 200 250 fish/m3 in the tanks in order to achieve the enhanced individual growth to produce larger fish.

(d) Feeds and feeding

Fish in the grow out tanks can be fed with formulated feeds (Table 7) @ 3% per estimated body weight 3 - 4 times daily. Every 2 weeks, sampling of growing fish should be made to check the growth and adjust the feeding rate.
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Table 9. Available data on tilapia culture in tanks in different countries (Revised after Balarin
and Haller 1982). Countries Beligium Kenya Malaysia Scotland Taiwan USA Species O. niloticus O. niloticus Male red tilapia O. niloticus x O.aureus O. niloticus x O.aureus hybrid O.aureus no./m 100800 200250 23 90 200250 12.5
3

Duration (days) 170-200 150 120 150 150 125

Production 3 kg/m >30 50 30 32.4 112 16-24

FCR 2:1 2:1 1.9 1.5:1

Reference Melard & Philippart (1980) Balarin & Haller (1979) Guerrero 1989 McAndrew (cited by Balarin & Haller 1982) Sports (1983) Lauenstein (1978)

(e) Duration of culture

Fattening of large fish: 120 150 days.

(f) Harvesting of fish and expected production

Fish weighing 200 250 g can be harvested by using dip or push net or by dewatering the tanks. Expected production will be around 50 kg/m3.

9.3.2 Tilapia culture in raceways (a) Site selection

Site selection criteria for commercial tilapia culture in raceways under intensive system are similar like tank culture. In both cases, enough water supplies either from underground or other surface water source should be ensured.

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(b) Design and characteristics of the raceways

Raceways are rectangular in shape having both inlet and outlet facilities. Construction can be made either by concrete or earth, in most cases cemented raceways are used for fry/fingerling rearing and fattening of large fish. Size of individual raceway may vary from 15 30 m long; 2.5 3.0 m wide with a depth of 0.75 1.0 m. Most ideal arrangement of raceways should include single or double or multiple series of parallel ponds. Each raceway should have its own independent supply and evacuation facilities (Figure 43). Continuous flow of water supply need to be ensured for each series of raceways and excess water should be out through the outlet drains fitted with fish protecting screens. Recirculation system to reuse the available water can minimize the input costs of raceway operation without harming the production of tilapia. In many ways raceways are easier to manage and maintain than tanks. Although the capital expenditure will be greater than in some other installations but the maintenance costs will be lower (Stevenson 1980). Raceways can be used both for fry rearing and fattening large fish.

Fig. 43 The raceways for intensive tilapia culture.

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(c) Stocking of fish

Tilapia fingerlings weighing 25 -30 g can be stocked @ 150 300 fish/m3 in the raceways for fattening purpose.

(d) Duration of culture

Fattening of large fish: 120 150 days.

(e) Feeds and feeding

Fish in the grow out tanks can be fed with formulated feeds (Table 7) @ 3% per estimated body weight 3 - 4 times daily. Every 2 weeks, sampling of growing fish should be made to check the growth and adjust the feeding rate.

(f) Harvesting of fish and expected production

By using dip or push net or by dewatering the raceways, fish weighing 200 250 g can be harvested. Expected production will be 40 - 60 kg/m3.

Table 10. Available data on tilapia culture in raceways in different countries (Revised after
Balarin and Haller 1982) Countries Hawaii Kenya Kenya USA USA Species O. mossambicus O. niloticus O. niloticus O. aureus O. aureus no./m 10002800 1650 1000 6502500 100400
3

Duration (daya 90 40-50 40-50 60 150

Production 3 kg/m 5.6 9.5 35.6 16-64 35-150

FCR 1.5:1

Reference Uchida & King (1962) Balarin & Haller (1979) Balarin & Haller (1979) Lauenstein (1978) Lauenstein (1978)

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10 Diseases and parasites of tilapia and their control measures

10.1 OCCURRENCE OF DISEASES IN FISH AND THEIR COMMON SYMPTOMS
Diseases and parasites often become problematic when animals are crowded or reared closely in confined places. High production levels and population densities in fish farming ponds might have a chance of increasing the probability of a variety of diseases. These diseases can be acute, marked by heavy losses in fish population, even sufficient to kill the entire crop. In intensive fish farming practice, fish diseases may occur frequently, but when water quality is good and supplementing of artificial feed is adequate, it become less probability to develop sickness and diseases, So, precaution is the better way of disease control. In this regard for preventing unnecessary death and losses of fish population occurred by epidemic infectious diseases, proper management and care of fishponds are a must. Observing their abnormal physiological conditions, which are expressed by some symptoms as follows, can easily identify infected fish in ponds:

10.1.1 Changes in behavior

Fish in good health is only seen during the time of feeding or playing but when the fish is found always gasping together at the surface or near the incoming water supply, there should be doubt about diseases. The presence of parasites may cause the fish to try dislodging them on vegetation. Infected fish also shows other symptoms, such as loss of balance, erratically swimming, discomfort, etc.
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10.1.2 Abnormal physical signs

Fish without any infection become physically clean and fresh. But when they become sick, some abnormal physical sign ultimately develop, for instance- excessive mucus over the skin; inflamed abdomen; swollen and pale gills; bulging and blind eyes; hemorrhagic areas on the head, body and fins; cysts in the skin, muscles or internal organs and lesions on the several part of the body, etc.

10.1.3 Failure to feed

Under good water quality, healthy fish normally feed when food is provided in water. But sickness in fish may cause to stop feeding themselves. Certain parasitic infections act slowly and may cause emaciation long before death. A gaunt belly, a tight skin over the head, a long thin body is the sign that the fish have not been feeding well. Many of the diseases affecting fish have similar symptoms. So, it is necessary to identify these diseases accurately, which is the first step towards control. After that before beginning any treatment some other essential steps are to be considered like a) knowing the exact volume of water, b) knowing about the life history of fishes, c) knowing about the content of toxicity of the chemicals in relation to fish species. The above information should be known before the chemicals are applied. Because susceptibility of fish to chemicals varies with the species and age of the fish and with the volume of water. Using any drug or chemical without following the suggested levels is worthless and in some cases become dangerous to fish health. Treatment must be effective in controlling the suspected parasites and diseases in fish farming ponds. Otherwise, it will usually mean that during this time a lot of money, valuable energy and a large number of fish might be unnecessarily lost.

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10.2 THE MOST COMMON DISEASES OF TILAPIA AND THEIR TREATMENT

The most common diseases and parasites of tilapia and their possible treatments are briefly described below:

10.2.1 Bacterial Diseases (a) Myxobacterial infections

Among the diseases caused by bacteria, the most common myxobacterial infection is fin rot. Fish are infected by the disease in a pond, when they are roughly handled, transferred or become wounded or they are reared in crowded conditions. The rot starts first at the dorsal or caudal fins, then spreads over the body surface, which becomes necrotic and sloughs off gradually. Fish show discomfort, listlessness and fatigue. Treatment: Several types of chemicals can be used for the treatment of fin rot infection of fish in ponds, such as Copper sulphate (0.5 to 1.0 ppm) or Malachite green (0.05 to 0.1 ppm) or Formalin (25 ppm). Any one of the chemicals should be applied at every alternative day for 3-4 times to control the disease. Dip or Bath treatment: The infected fish are treated by dipping them in the solution of Copper sulphate (250 ppm) for one minute or Formalin (250 ppm) for one hour.

(b) Abdominal dropsy

The disease is caused by accumulation of dropsy fluid in the abdominal cavity of carp, tilapia and other susceptible species of fishes (Figure 44). The abdominal dropsy in fish probably arises from the action of both bacteria and viruses. It is also believed that the disease might be caused primarily by the bacterium Aeromonas punctata and secondarily by
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viruses. Diseased fish exhibit extreme inflammation of abdomen, erratically swimming and discomfortness. At high water temperature, the infected fish generally start to die. Treatment: The infected fish can be treated by applying antibiotic Oxytetracycline or Streptomycin or Chlorampheniocol at the rate of 1 mg per 100 g of fish. Among the surgical treatment, the fluid of the abdomen can be taken out by a hypodermic needle with syringe. The best preventive measure against the dropsy is to liming and drying out the ponds at every alternative year before stocking.

Fig. 44 Abdominal dropsy in tilapia.

10.2.2 Parasitic Diseases (a) Chilodonella sp.

This is a protozoan parasite, belonging to the genus Chilodonella, mostly attached to the gills and skin of the fish, becoming dangerous by growing in number within a very short period and sufficient to kill even plenty of large fish. The body of the parasite is dorsoventrally flattened, heart shaped, the dorsal side is swollen and the ventral side is partially ciliated (Figure 45). Attachment of the parasite is associated with body lesions, pale colour of the gills, discomfort, erratic swimming, a tendency to stay
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near the incoming water supply and occurrence of a higher rate of fish mortality. Chilodonella sp. is generally active to cause disease in tilapia fry fingerlings at the temperature below 20 OC. Chilodonelliasis is a common parasitic disease in tilapia fry and fingerlings during the cold season (Hussain 1988).

Fig. 45 Protozoan parasite Chilodonella sp. (Sarig 1971).

Treatment: The pond should be treated by Potassium permanganate (3-5 ppm), Formalin (25 ppm), Methylene blue (3 ppm), Malachite green (0.10.15 ppm). Any one of these chemicals should be applied every alternative day for 2-3 times. Dip or Bath treatment: The infected fish may be treated by dipping them in solution of Potassium permanganate (10 ppm) for one hour. For prevention of the disease, every alternative year ponds should be disinfected by liming just before releasing the fish.

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(b) Trichodina sp.

This protozoan parasite belongs to the genus Trichodina (Figure 46). The infections caused by this type of parasite occur at the site of gills and skin, sometimes dangerous to breakdown the normal physiological conditions of the pond fishes, causing a serious anemia to fry and fingerling fish, finally leading to death. The infected fish exhibit irregular white patches, frayed fins, become sluggish and fail to feed. Trichodiniasis is also a common parasitic disease in tilapia fry and fingerlings during the winter season (Hussain 1988).

Fig. 46 Protozoan parasite Trichodina sp. (Hoffman and Meyer 1974).

Treatment: Treatment in ponds can be carried out by applying Malachite green (0.1-0.15 ppm), Formalin (25 ppm) or Sodium Chloride (200 ppm) for only one time. Dip or Bath treatment: The diseased fish can be bathed by Malachite green (1.25-5.0 ppm) or Formalin (250 ppm) for half an hour.

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(c) Myxobolus sp.

These protozoan parasites belong to the genus Myxobolus, which cause infections in gills of susceptible species of fishes. White cysts are appeared in the muscles and below the tissues. Diseased fish develop a thickened epithelium and excessive mucus on the gill regions. Treatment: No treatment is yet been carried out by the chemicals to control the disease. Necessary precautions can be taken for disinfections by liming and drying of ponds.

(d) Argulus sp.

The argulus or fish lice are copepods, and adhere to the body of the fish by means of its suckers and extremities. The parasite has a flattened, often pinkish or reddish disc like body, but lives on the surface of the fish or in the mouth of gill cavity (Figure 47). Infected fish show discomfort, erratic swimming and in the case of a serious infection the fish may stop feeding.

Fig. 47 Fish lice Argulas sp. (Sarig 1971)

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Treatment: For pond treatment Mesoten or Dylox may be applied at the rate of 0.25 ppm once in a week for at least 4 weeks. A preventive measure is to dry out ponds at every alternative year. Dip or Batch treatment: The infected fish can be treated by dipping in the solution of Potassium permanganate (10 ppm) or Sodium chloride (5%) for half an hour.

(e) Lernaea sp.

The lernaea or anchor parasite is sometime dangerous to fry and fingerling fish and can cause their early death by leading to an anemic physiological condition. Their attachment is associated with hemorrhagic reactions in all surfaces of the body, which lead to secondary fungus and bacterial infections. The parasite resembles a shaft of a small barb inserted into the flesh of the fish. The parasite cannot be easily removed because of the anchor. The infected fish show discomfort, often rubbing their body surface against the bottoms, grass stems, etc. Treatment: The pond should be treated by Mesoten or Dylox (0.15-0.25 ppm), Potassium permanganate (3-5 ppm), Malathion (0.5-1.0 ppm) or Sodium chloride (1.5%). Any one of these chemicals may be applied once a week for at least 4 weeks. Dip or Bath treatment: The infected fish may be bathed in the solution of Potassium permanganate (10 ppm) or Sodium chloride (5%).

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11 Marketing of tilapia
11.1 DEMAND OF TILAPIA IN THE DOMESTIC MARKETS
Tilapias are becoming popular fish recently among the traders due to their suitability for selling any size (fingerling to adult) in the domestic market of Bangladesh and other South-east Asian countries. In this part of the world, there are multi various purchasers of different income levels, who might prefer different size grade tilapia. It is obvious that rich people always prefer large size and poor people due to their financial incapability go for medium and smaller size for the cheaper price. Consumers of this country normally used to like indigenous carps, shrimp, catfish and other small species as food fish but due to their unavailability and extremely high price in the domestic market, they are bound to purchase tilapias and Chinese carp species.

11.1.1

Post harvest handling of tilapia

In many developing countries of South-east Asia, tilapias are marketed fresh or frozen. So, it is essential to handle the fish carefully at harvest to ascertain their freshness and quality for good market price. The harvested fish need to be kept alive for washing in the holding tanks with the inflowing cold water before their shifting and processing for the market (Figure 48). Many consumers prefer live tilapia like catfish. Pick up vans having fiberglass tanks can be used for live fish transportation. Live tilapias can also be shifted in plastic bags with oxygenated water like fish fry/fingerling transportation. Crushed ice is used for processing fresh tilapias for market. In this case, fish are kept with ice (ice to fish ratio 1:3) in bamboo baskets having inner
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side rapped with polythene sheet or Styrofoam boxes for transportation to local markets or distant places.

11.1.2

Domestic marketing systems

Like other fishes, in Bangladesh tilapias are sold in the domestic markets following the distribution channel of fish trade as below: Primary Market: Rural market, where the brokers are engaged to sell the fish to the small and medium traders by collecting them from the village ponds/farms. Secondary Market: Market nearby the administrative unit headquarters (ie. Thana Headquarters) operated by the commission agents and fish are sold and packet for shifting to urban markets. Higher Secondary Market: Market at the towns and cities also operated by commission agents and fish are sold to second distributors. Final Consumers Market: The second distributors sell the fish to retailers to make them available in the retailing markets for the consumers (Figure 49).

11.1.3

Present status of domestic wholesale and retail market conditions

The overall fish landing facilities and wholesale markets are not well established all over Bangladesh. Private fish traders are the dominant groups who control the major landing centers in the rural and urban areas. These centers are unhygienic and poor; there is no particular auction and packing sheds, no adequate drainage and cleaning facilities. Ministry of Local Government through its municipalities controls only the wholesale and retail markets in most of the cities and towns. Conditions of municipal markets are not also at the mark for controlling hygienic aspects of fish.

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Fig. 48 Washing of harvested live tilapias in the holding tank with the inflowing cool water before marketing (Courtesy: Mr. Y. K. Thai).

Fig. 49. Tilapias in the retailing fish market for the consumers (Courtesy: Mr. Y. K. Thai).

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11. 2 DEMAND OF TILAPIA IN THE INTERNATIONAL MARKETS

In most of the chain restaurants, tilapias are recently considered as attractive menu items, which were once accepted as low value fish, only suitable for ethnic markets and their traditional markets have been in Africa and Asia for a long time. But recently tilapias have also obtained consumer recognition in the USA and to some extend in Europe and around the world (Vannuccini 1998). These have become third preferred fish like channel catfish after shrimp and salmon in the United States. Accurate data on global trade for tilapia are not readily available. It is reported that the major exporting countries are Taiwan, Thailand, Philippines, Indonesia, Israel, Singapore, Costa Rica, Colombia, Jamaica, Venezuela and Ecuador. Israel and Taiwan are the two countries from where majority of tilapias are being exported in the global markets. Israel alone exports more than 60% of total supply to the markets of USA. Bangladesh could be in the same line to export tilapias like shrimp to the world markets. In developed countries, tilapia fillet might be the choice of consumers. There is a growing demand of tilapia fillets produced from large sized fish (0.6 to 1 kg) in Japan, USA and Canada. Particularly in Japan, the fillets of the Nile and red tilapias are used for raw fish gourmet dishes as sashimi and excellent substitutes for the high priced red sea bream (Guerrero 1997). Large or medium sized red or blue (Nile tilapia) strains can either be filleted or packet in icebox freshly for export marketing. Price of colored tilapia is 1.5 times higher than normal colored tilapias. Many entrepreneurs are coming forward to Bangladesh to initiate commercial tilapia farming and invest money for filleting tilapias for export. In view of this, for market diversification both in the country and abroad the following points need to be considered:
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Tilapia farming should be expanded and bank credits need to be provided to the interested farmers/entrepreneurs. As the fish producers ie. farmers get less price at the farm gate, so they are not getting enough profit from tilapia culture, therefore,

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Government should fix the farm gate price of the fish like rice and other food grains. Ancillary market facilities like road networks, concrete sheds, washing, packing and preservation facilities, holding tanks for keeping live fish and nice display facilities need to be created. Tilapia processing industries, in particularly filleting, ice packing and canning industries should be encouraged and supported. It is obvious that international regulations need to be strictly followed for processing of tilapias for export. Appropriate policies and strategies need to be formulated and adopted by the Government for commercial farming of tilapias and their export.

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12 Strategies and prospects of frontier development of tilapia aquaculture

12.1 LEARNING FROM THE EXPERIENCE OF OTHER TILAPIA PRODUCING COUNTRIES
Tilapia farming has become an important source of additional income and a cheap source of protein for rural communities in the Philippines (Fermin 1985). The steady expansion of tilapia industry has benefited small-scale hatchery operators, grow-out farmers, cage operators and merchants (Smith et al. 1985). According to Guerrero (1994), the Philippines government selected tilapia for development because of its potential to benefit resource poor farmers as well as commercial growers. Researchers developed the technology and overcame the marketing constraints. Tilapia technology was successfully adapted and extended to small-scale farmers and large commercial producers. The Philippines has recently become one of the largest producers of tilapia in the Asia (Guerrero 1994). A number of Asian countries like Thailand, Indonesia, and Taiwan are presently in the line of developing tilapia aquaculture industry. Bangladesh and other countries of this region with appropriate water resources could learn from Philippine and Asian tilapia-producing countries experience and promote tilapia production.

12.2 STRATEGIES FOR TILAPIA AQUCULTURE

Tilapia has great potential in Bangladesh as an alternative and additional species of farmed fish. In view of taking tilapia as one of the important and potential fish species, the following developmental areas and strategies are identified for necessary consideration:
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Among the South East Asian countries, Bangladesh in particular abounds with hundreds and thousands of seasonal water bodies (>0.1 m ha) in the form of ditches, shallow ponds, road side canals, barrow pits etc. which retain water for 4-6 months, where carp species can not be cultured. No doubt, these water bodies have tremendous potential for aquaculture of fish species with short life cycle and characteristics of faster growth rate and require low input support (Hussain et al. 2000). In such cases, tilapia can be a promising candidate for aquaculture in the suitable seasonal water bodies. Recently, adequate feed crisis and low market price severely damaged the exotic riverine catfish (Panagsius sp.) farming in the country, therefore, a large number of commercial catfish producers have found tilapia as an alternative species to culture in their farms to maximize the production. Success of such attempts will encourage the entrepreneurs to come forward for initiating commercial tilapia farming in freshwater ponds and other suitable water bodies. In brackish water ponds (0.14 m ha) of the country, where improve extensive shrimp culture is in collapse due to disease outbreak, commercial farming of tilapia will be an alternative. Similar aquaculture activities can further be expanded in the suitable brackish water polders and enclosures (0.87 m ha). In Kaptai reservoir (0.07 m ha) and other similar water bodies commercial cage culture of monosex tilapia could flourish to boost fish production. It is presumed that tilapia farming will largely be expanded both in small and commercial scales, so there will be an extreme need for huge number of quality seeds (both mixed sex and monosex). In that case, development and operation of commercial tilapia hatcheries will be essential throughout the country. Commercial seed production activities both in public and private hatcheries will enable to create additional employment opportunities for a large group of unemployed people.
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12.3 DIVERSIFIED AND FRONTIER DEVELOPMENT OF TILAPIA AQUACULTURE

In the recent past all over the Asia, the inland and marine capture fisheries have registered a gradual decline due to deterioration of aquatic environments and over fishing. Thus aquaculture is seen as the most promising option of filling the pending void in the aquatic food supply. Among the available farmed fish species, the tilapias are among the best candidates for culture because of their desirable qualities (Guerrero 2002). In spite of bright and promising future of tilapia farming in the developing countries of this part of Asia like Bangladesh, India, Nepal, Pakistan and Sri Lanka, until recently the Governments of these nations have not yet taken it seriously. Since many years enormous efforts and investments have been made to promote aquaculture of Indian and Chinese major carp and shrimp species particularly in Bangladesh and India. Although per unit area of production was promisingly increased especially for carps through adoption of improved technologies these could not be diversified except into freshwater ponds or related water bodies. On the other hand, mass involvement of rural people in carp and shrimp culture was found difficult due to their limited water resources and financial incapability in many cases. Diversified and frontier aquaculture development with short cycled fish species like tilapias can overcome all the above-mentioned situations and have access to all existing aquatic ecosystems, which will certainly benefit the nation like ours in providing income for small-scale farmers and large producers and helping the country to alleviate shortfalls in fish production. Therefore, it can confidently be presumed today that tilapia is a novel and excellent species (Figure 50) for future aquaculture in all the suitable aquatic ecosystems in Bangladesh and elsewhere in South East Asia. Broadly it can be said Tilapia is a good food fish (Figure 51) for you and all others, it is a good cash crop for all the stake holders mainly the poor farmers living in Asia and other developing countries of the world.
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Fig. 50 Tilapia is a fish of the decade

Fig. 51 Tilapia is a good food fish (Courtesy: Mr. Y. K. Thai).

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Glossary
Additive genetic variance: The proportion of the total phenotypic variance that depends on the additive effects of the genes. Alleles: Members of a pair of different hereditary factors that may occupy a given locus on a specific chromosome and that segregate in formation of gametes. Alternative form of gene. Androgenesis: Opposite to gynogenesis, which involves fertilization of eggs with inactivated maternal nucleus, and prevents any contribution of the female genome to the embryo. As a result, embryonic development proceeds with the inheritance of only paternal chromosome sets. Breed: Group of animals having a common origin and identifying characters that distinguish them as belonging to a breeding group. Breeding population: A group fish to be used for planned breeding. Breeding value: The genetic value of a fish or a population, in terms of its or their ability to transmit certain distinguished features that separate them from other such group. Broodstock: Parent (Female and Male) fish cultivated to provide eggs/milt or fry. Chromosomes: Darkly staining bodies in cell nuclei, which carry the heredity material. They occur in pairs in somatic cells with the number of pairs and morphology being characteristic of the species. Chromosome set: A group of chromosomes representing a genome, consisting of one representative from each of the pairs characteristic of the somatic cells in a diploid species. Combined selection: Combination of both within-family and betweenfamily selection to increase the accuracy of the estimates of breeding values, and thereby achieve a greater selection response. Crossbred: An animal produced by crossing two or more pure breeds, strains or lines. Crossbred individuals are from intraspecific matings. Crossbreeding: Mating systems in which hereditary material from two or more pure breeds, strains or lines is combined.
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Diploid: Cells with two members of each pair of chromosomes. This is termed the 2n condition and is characteristic of most fish species. Effective population size (Ne): The ideal population size is infinitely large, ensuring no loss of genetic variation which is a function of the total number of breeding individuals, the sex ratio, the mating system employed, and the variance of family size in the production of fish that are used to produce the next generation. Family selection: Selection of the best male and best female fish from a family, such type of selection based on deviations from average family performance. Fecundity: Number of eggs produced; sometimes expressed as number per fish, sometimes per kg of fish. Gene: The classical term of the basic unit of heredity. Genotype: Genetic makeup of a fish; that portion which is inherited. The complete genetic make up is also referred to as the genome. Gonad: Fish organ in which either eggs or sperm are produced; generally termed as ovary in female or testis in male. Gynogenesis: Gynogenesis involves fertilization of eggs with inactivated sperm, and prevents any contribution of the male genome to the embryo. As a result, embryonic development proceeds with the inheritance of only maternal chromosome sets. Hapa: Fine meshed rectangular or square structure on which fertilized eggs or larvae are deposited after artificial spawning and hatching. Haploid: Having one complete set of chromosomes (see also Diploid); most gametes are haploid so that when fertilization occurs the diploid condition is restored. Heritability: The proportionate amount of additive genetic variance: h2 = VA/Vp Inbreeding: A system of mating in which mates are more closely related than average individuals of the population to which they belong.

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Inbreeding depression: Decreased performance in growth rate, fecundity, survival, etc. and an increased percentage of deformed/abnormal fish that occur due to inbreeding. Karyotype: The somatic chromosomal complement of an individual or species. The term is often used for photomicrographs of the metaphase chromosomes arranged in a standard sequence. Mass selection: Individual selection or empirical selection of the best male and female individuals from a population. Meiotic gynogenesis: Gynogenetic diploids poduced by the suppression of the second meiotic cell division of fertilized eggs. Mitotic gynogenesis: Gynogenetic diploids produced by the inhibition of the first mitotic cell division of fertilized eggs. Morula: An embryo that consists of a cluster of cleaving blastomeres. Monosex population: Production of only female or male fish through genetic manipulation or sex inversion. Pedigree: A fishs family tree. Phenotype: Physical appearance or characteristics of an organism. Phenotypic variance: The variance that is observed or measured for a particular aspect of the phenotype in a population. Polyploidy: A state where a cell or an individual contains three or more sets of chromosomes: e.g. triploid (3n) or tetraploid (4n). Progeny: Offspring of any generation. Selection: A breeding program in which the breeder chooses which fish will be the next generations broodstock, based on some predetermined criteria. Sex reversal: Modification of sex using hormonal manipulation. Sexual maturation: Condition of female and male individuals having ripe gonadal materials (eggs and sperm). Triploidy: Individuals having 3 sets of chromosomes (4n). Tetraploidy: Individuals having 4 sets of chromosomes (4n). Vitellogenesis: The formation of yolk in the developing oocyte.
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Vitellogenin: A protein synthesized in the liver of sexually maturing females and incorporated into the yolk spheres of the developing oocyte. XX chromosome: Homozygous pair of sex chromosomes; produces in tilapia a female. XY chromosome: Heterozygous pair of sex chromosomes; produces in tilapia a male. YY male production: Indirect method of producing monosex all males (having YY genomic status) by combining both sex-reversal and/or genetic manipulation technique of the sex determining system in tilapia.

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References
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Hussain, M.G., A. Chatterji, B.J. McAndrew and R. Johnstone. 1991. Triploidy induction in Nile tilapia, Oreochromis niloticus L. using pressure, heat and cold shocks. Theor. Appl. Genet., 81:6-12. Hussain, M.G. 1992. Genetic manipulation studies in Oreochromis niloticus L. Ph.D. Thesis, University of Stirling, Scotland, UK. Hussain, M.G., D.J. Penman, B.J. McAndrew and R. Johnstone. 1993. Suppression of first cleavage in the Nile tilapia, Oreochromis niloticus L. a comparison of relative effective of pressure and heat shocks. Aquaculture, 111:263-270. Hussain, M.G., D.J. McAndrew, D. Penman and P. Sodsuk. 1994. Estimating gene centromere recombination frequencies in gynogenetic diploids of Oreochromis niloticus L., using allozyme, skin colour and a putative sex determining locus (SDL-2). p. 502509 In: A.R. Beaumont (Editor), Genetics and evolution of aquatic organisms. Chapman and Hall, London. Hussain, M.G. 1994. Genetics of body colour inheritance in Thai and Egyptian red tilapia strains. Asian Fisheries Science, 7:215-224. Hussain, M.G. and B.J. McAndrew. 1994. An improved technique for chromosome karyotyping from embryonic and soft tissues of tilapia and salmonids. Asian Fisheries Science, 7:187-190. Hussain, M.G. 1995. Suppression of meiotic and mitotic cell divisions in Nile tilapia. Asian Fisheries Science, 8: 133-142. Hussain, M.G., G.P.S. Rao, N.M. Humayun, C.F. Randal, D.J. Penman, D. Kime, N.R. Bromage, J.M. Myers and B.J. MCandrew. 1995. Comparative performance of growth, biochemical composition and endocrine profiles in diploid and triploid tilapia Oreochromis niloticus L. Aquaculture, 138: 87-97. Hussain, M.G. 1996. Advances in chromosome engineering research in fish: review of methods, achievements and applications. Asian Fisheries Science, 9:45-60. Hussain, M.G. 1998. Manipulation of chromosomes in fish: review of various techniques and their implications in aquaculture. Bangladesh J. Fish. Res., 2(1):99-108. Hussain, M.G., D.J. Penman and B.J. McAndrew. 1998. Production of heterozygous and homozygous clones in Nile tilapia. Aquaculture International, 6:197-205.
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Hussain, M.G., A.H.M Kohinoor, M.S. Islam, S.C., Mahata, M.Z Ali. M. Tanu, M.A Hossain and M.A Mazid. 2000. Genetic evaluation of GIFT and existing strains of Nile tilapia, Oreochromis niloticus, under on-station and on-farm conditions of Bangladesh. Asian Fish. Science, 13(2):117-126. Hussain, M.G., A.H.M Kohinoor, M.S. Islam, M.A Hossain, M.M. Dey and M.A Mazid. 2000. Growth and production performances of GIFT strain of Nile tilapia, Oreochromis niloticus L., in ponds and cages under different farming conditions in Bangladesh. J. Aqua. Trop., 15:273-280. Hussain, M.G. and M.A. Mazid. 2001. Genetic improvement and conservation of carp species in Bangladesh. Bangladesh Fisheries Research Institute and International Center for Living Aquatic Resources Management. 74p. Hussain, M.G., M.S. Islam, M.A. Hossain, M.J. Wahid, A.H.M. Kohinoor, M.M. Dey and M.A. Mazid. 2002. Stock improvement of silver barb (Barbodes gonionotus Bleeker) through several generations of genetic selection. Aquaculture, 204::469-480. Hussain, M.G. and A.H.M. Kohinoor. 2003. Breeding, monosex male tilapia seed production and culture technologies of BFRI super tilapia. Extension manual No. 25. Bangladesh Fisheries Research Institute, Mymensingh. Ita, E.O. 1976. Approaches to the evaluation of fishery reserves in the development and managements of Inland Fisheries. Rome, FAO, CIFA/72/514, 18p. Jordan, D.T. and F.A. Pagan. 1973. Developments in cage culture of Tilapia aurea in a rock-quarry pond in Puerto Rico. Comm. 10th Ann. Meet. Ass. Islands Mar. Lab. Carib, 59pp. Kuo, H. 1969. Notes on the hybridization of tilapia. JCRR Fish. Ser. No. 8, p.116-117. Kuo, H. 1988. Progress in genetic improvement of red hybrid tilapia in Taiwan. p. 219-221. In: R.S.V. Pullin, T. Bhukaswan, K. Tonguthai and J.L. Maclean (Editors), The Second Int. Symp. on Tilapia in Aquaculture. ICLARM Conference Proceedings 15, Department of Fisheries, Bangkok, Thailand and International Center for Aquatic Resources Management, Manila, Philippines.
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Kuo, H. and T.T. Tsay. 1988. Study on the genetic improvement of red tilapia-crossbreeding of red tilapia and its growth. Bull. Taiwanese Fish. Res. Inst., 44:151-165. Lauenstein, P.C. 1978. Intensive culture of tilapia with geothermally heated water. In: R.O. Smitherman, W.L. Shelton and J.H. Grover (Editors), Culture of exotic fishes, Symp. Proc. Fish Culture Selection, American Fisheries Soc., Auburn, Alabama: 82-85. Liao, I.C. and I.C. Chang. 1983. Studies of the feasibility of red tilapia culture in saline water. p. 524-543. In: L. Fishelson and Z. Yaron (Edirors), Proc. Int. Symp. on Tilapia in Aquaculture, 8-13 May, 1983, Nazareth, Israel. Lowe-McConnell, R.H. 1958. Observation on the biology of Tilapia nilotica Linne (Pisces: Cicklidae) in East African waters. Revue Zool. Bot. afr., 55:353-363. Mair, G.C. 1988. Studies on sex determining mechanisms in Oreochromis species. Ph.D. Thesis, University of Wales, UK. Mair, G.C. 1999. Genetics of broodstock management: Basic principles and practices. AARM Newsletter 4(4):4-6. May, B., K.J. Henely, C.C. Krueger and P.S. Gloss. 1988. Androgenesis as a mechanism for chromosome set manipulation in brook trout (Salvelinus fontinalis). Aquaculture, 75:57-70. McAndrew, B.J., F.R. Rubanol, R.J. Roberts, A.M. Bullock and I.M. McEwen. 1988. The genetics and histology of red, blond and associated colour variations in Oreochromis niloticus. Genetica, 76:127-137. Melard, Ch. and J.C. Philippart. 1980. Intensive culture of Sarotherodon niloticus in Belgium. EIFAC Symp. on New Dev. in Util. of Heated Effluents and of Recirc. Systems for Ints. Aquacult. (EIFAC/80/Symp,-E/11), 11:28pp. Mikolasek, O., J. Lazard, M. Alhassane, P. Parrel and I. Ali. 1997. Biotechnical management of small-scale tilapia production units in floating cages in Niger river (Niger) p. 348-356. In: K. Fitzsimmons (Editor), Proceedings of the Fourth International Symposium on Tilapia in Aquaculture, 9-12 November, 1997. Walt Disney World, Orlando, Florida, USA. Myers, J.M. 1986. Tetraploid induction in Oreochromis spp. Aquaculture, 57:281-287.
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Pagan, F.A. 1970. Cage culture in tilapia. FAO Fish Cult. Bull, 2(1):6. Parsons, J.E. and G.H. Thorgaard. 1985. Production of androgenetic diploid rainbow trout. Journal of Heredity, 76:177-181. Pruginin, Y., S. Rothbard, G. Wohlfarth, A. Halvey, R. Moav and G. Hulata. 1975. All male broods of Tilapia nilotica x T. aurea hybrids. Aquaculture 6:11-21. Pullin, R.S.V. 1983. Choice of tilapia species for aquaculture. p. 64-74. In: L. Fishelson and Z. Yaron (Editors), Proceedings of the International Symposium on Tialpia in Aquaculture. Tel Aviv University, Israel. Pullin, R.S.V. (Editor). 1988. Tilapia genetic resources for aquaculture. ICLARM Conference Proceedings 16. 108p. Rothbard, S., Y. Zohar, N. Zmora, B. Vevavi-Sivan, B. Moab and Y. Yaron. 1990. Clearance of 17 -ethyltestosterone from muscle of sex-inversed tilapia hybrids treated for growth enhancement with two doses of the androgen. Aquaculture, 89:365-376. Sarig, S. 1971. The prevention and treatment of diseases of warm water fishes under subtropical conditions, with special emphasis on intensive fish farming. In. S.F. Snieszko and H.R. Axerlord (Editors), Diseases of Fishes. T.F.H. Publications, Inc. Ltd. USA. Shelton, W.L., K.D. Hopkins, and G.L. Jensen. 1978. Use of hormones to produce monosex tilapia for aquaculture. p. 10-33. In: R.O. Smitherman, W.L. Shelton and J.H. Grover (Editors), Proc. Symp. Culture of Exotic Fishes. Fish Culture Section. American Fisheries Society, Auburn, Alabama, USA. .Shelton, W.L. 1987. Genetic manipulations-sex control of exotic fish for stocking. p. 175-197. In: K. Tiews (Editor), Proceedings of World Symposium on Selection, Hybridization and Genetic Engineering in Aquaculture, Bordeaux, 27-30 June, 1986, Vol.II. Heeneman, Berlin. Shelton, W.L. 2002. Tilapia culture in the 21st century. p. 1-19. In: R.D. Guerrero III and M.R. Gurrero-del Castillo (Editors), Tilapia farming in the 21st century. Proceedings of the International Forum on Tilapia Farming in the 21st Century (Tilapia Forum 2002), Philippines Fisheries Association, Inc, Los Benos, Laguna, Philippines.
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Sipe, M. 1979. Golden perch. Commer. Fish Farmer Aquaculture News, 5(5):56 Smith, I.R., E.B. Torres and E.D. Tan. 1985. Philippines Tilapia Economics. ICLARM Conference Proceedings 12. 261p. Sports. D. 1983. High density tilapia culture in Taiwan. Aquacult. Magazine. 9(6): 21-23. Streisinger, G.C., C. Walker, N. Dower, D. Knauber and F. Singer. 1981. Production of clones of homozygous diploid zebra fish (Brachyndanio rerio), Nature 291: 293-296. Stevenson, J.P. 1980. Trout farming manual. Fishing News Books Limited, Farnham, Surrey, England. 186p. Suwanasart, P. 1971. Effects of feeding mesh size and stocking size on the growth of Tilapia aurea in cages. Ann. Rep. Int. Centr. Aquacult. Agric. Exp. St., 1971, Auburn Univ., Alabama:71-79. Tave, D., M. Rezk and O. Smitherman. 1989. Genetics of body colour in T. mossambica. J. World Aquacult. Soc., 20:214-222. Thorgaard, G.H., P.D. Scheerer, W.K. Hersberger and J.M. Myers. 1990. Androgenetic rainbow trout produced using sperm from tetraploid males show improved survival. Aquaculture, 85:215-221. Trewavas, E. 1983. Tilapiine fishes of the genera Sarotheroden, Oreochromis and Danakilia. British Museum (Natural History), Cromwell Road, London. 583 p. Tyler, C., J. Sumpter and N.R. Bromage, 1987. Vittellogenin uptake by cultured oocytes of rainbow trout. Gen. Comp. Endocrinol., 66:920. Uchida, R.N. and J.E. King. 1962. Tank culture of tilapia. US Fish Wildl. Serv. Fish. Bull., 62:21-52. Valenti, R.J. 1975. Induced polyploidy in Tilapia aurea (Steindachner) by means of temperature shock treatments. J. Fish Biol., 7:519-528. Vannuccini, S.1998. Western world the focus of new tilapia market. INFOFISH International, 4:20-24. Varadaraj, K. and T.J. Pandian. 1990. Production of all-female triploid Oreochromis mossambicus. Aquaculture, 84:117-123. Velasco, R.R. 2003a. Reproductive biology of tilapias. p. 1-12. In: Training manual on broodstock management and sex reversal of tilapia using GIFT protocol. GIFT Foundation International Inc. (unpublished).
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Velasco, R.R. 2003b. Production of all male population. p. 25-40. In: Training manual on broodstock management and sex reversal of tilapia using GIFT protocol. GIFT Foundation International Inc. (unpublished). Wohlfarth, G.W., S. Rothbard, G. Hulata and D. Szweigman. 1990. Inheritance of red body coloration in Taiwanese tilapias and in O. mossambicus. Aquaculture, 84:219-234. WorldFish Center. 2004. GIFT technology manual: An aid to tilapia selective breeding. WorldFish Center, Penang, Malaysia, 56p. Worthington, E.B. and C.K. Ricardo. 1936. Scientific results of the Cambridge Expedition to the East African lakes, 1930-1. No. 15. THe fish of Lake Rudolf and Lake Baringo. J. Linn. Lond. (Zool) 39: 353-389.

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Index
Abdominal dropsy, 115 Absolute fecundity, 14 Aceto-carmine squash technique, 17, 78 Additive genetic gain, 22 Additive genetic variation, 21 Aerator, 97 Aeromonas punctata, 115 Allele, 18, 21, 48 All male monosex population, 42, 65, 83, 84 Androgenesis, 42 Aneuploid metaphase, 44 Anus, 12 Aquatic chicken, 3 Aquatic larvae, 11 Argulas sp., 119 Artificial feed, 11, 14, 33, 113 Artificial hatching system, 24 Artificial incubation, 20 Artificial incubation system, 24 Asian countries, 8 Asian Development Bank (ADB), 3 Atomic absorption spectrophotometry, 17 Automatic feed mixing machine, 76 Average genetic gain, 22 Blackchin tilapia, 1 Bamboo pole, 60, 76, 92, 103, 105 Base population, 21, 23 Biomass, 24, 54, 58-60, 70 Blotched type, 50 Blue tilapia, 1 Body colour inheritance, 45, 47 Body weight, 27, 29, 56 Brackish water, 7, 10, 98, 127
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Breeding behaviour, 9 Breeding candidate, 23 Breeding hapa, 19, 23, 60, 77 Breeding pond, 23, 69 Breeding population, 21, 30 Breeding tank, 56, 58-59 Breeding value, 27, 29 Brood stock, 19, 23, 40, 53 Brood stock management, 18 Brood stock replacement, 18-21 Cage, 27, 103, 107 Cattle dung, 86, 96 Caudal fin, 10 Chichlid, 1, 9-10, Chilodonella sp., 117 Chitralada strain, 6 Chlorampheniocol, 116 Chromosome, 31, 37 Chromosome karyotyping, 37, 42-43 Chromosome manipulation, 20, 31, 33 Circular tank, 108 Clonal line, 39 Closely related individual, 29 Coefficient of variation, 22 Colchicine solution, 42 Cold shock, 36 Combined selection, 22 Combined selection strategy, 23 Commercial tilapia farm, 95 Common carp, 103 Common farm environment, 27 Communal testing, 26-27 Control group, 7 Copepod, 11 Courtship behavior, 12

Farming of Tilapia

Cross breeding, 20, 45 Crude protein, 48, 58 Cryopreservation, 42 Cumulative weight gain, 7 DEGITA countries, 6 Demand feeder, 109 Dike construction, 95 Diploid, 3-32 Diploid metaphase, 44 Disease resistance, 21 Dissolve oxygen, 11 Dorsal fin, 10, 26 Earthen pond, 27, 53, 59, 77 Effective population size (Ne), 1921, 29-30 Egg, 13, 31, 40 Egg incubation system, 66 Egyptian red strain Endocrine hormone, 15 Endomitotic, 31 Ethyl alcohol, 72, 83 Existing strain, 6 Family selection, 22 Family selection strategy, 23 Fecundity, 14, 18, 21 Feeding intensity, 27, 96, 105 Feeding rate, 27, 56, 58-60, 87, 96, 105, 109, 112 Feeding trolley, 105 Fertilization, 13, 31, 34 Fertilized egg, 14, 20, 24, 36, 38, 70 Fine meshed hapa, 59 Fingerling, 20, 26-27, 56, 111 Finite population, 18 Fish meal, 54, 58 First cleavage, 36, 39 First feeding fry, 20, 24, 55, 71 First mitotic division, 32

Floating raft, 105 Floy tag, 26 Formalin, 117 Formulated feed, 27, 53, 56, 59, 98, 105, 109, 112 Founder stock, 18 Freshwater, 10, 127 Freon, 31 Fry, 20, 26, 53, 111 Full-sib family, 26 Galilee tilapia, 1 Gametogenesis, 32 Genetically Improved Farmed Tilapia (GIFT), 2 Gene transfer, 20 Genome, 31, 37 Generic names, 9 Genetic gain, 21-22 Genetic improvement, 22-23, 29 Genetic stock deterioration, 18 Genetic selection, 22 Genetic variability, 18, 21-22 Genetic variation, 19, 21,23, 30 Genital papilla, 12, 34 Giemsa stain, 43 GIFT strain, 3-7, 22, 33, 103 Gill-raker, 10 Glacial acetic acid, 43 Glass aquaria, 48 Gonad development, 15 Growth performance, 22 Grow-out period, 27 Gynogenesis Half-sib, 29 Hapa, 20, 24 Haploid, 31 Haploid metaphase, 44 Hatching, 14 Hatching stage, 14, 37
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Farming of Tilapia

Heat shock, 36, 40-41 Heritability, 21-22 Heterogametic, 31 Heterozygous, 45-47 Heterogeneous, 21

Histological section, 15 Holding tank, 19 Homogametic, 31, 64 Homozygosity, 18 Homozygous, 45-48 Hormone mixed feed, 77
Hybridization, 20, 64 Hybrid strain, 6 Hydracarine, 11 Hydrolic pump, 36 Inbreeding, 18, 23, 30 Inbreeding depression, 18, 21, 29 Incubation system, 20, 34 Individual selection, 22-23 Intensity of selection, 21 Intensive culture, 56, 103, 1o3 In vitro, 34 Israel strain, 3 Larnaea sp., 120 Larvae, 20 Lateral line, 10, 27 Liming, 94 Lipophosphoprotein-calcium, 15 Livelihood, 103 Local strain, 6 Longfin tilapia, 1 Malachite green, 117 Malathion, 120 Manual stripping, 34 Marker chromosome, 44 Masculinization, 64, 78 Mass selection, 22, 29
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Mass selection strategy, 22 Maternal effect, 23 Maternal mouth brooder, 10, 14 Mating, 12 Meiotic gynogenesis, 38 Melanophores, 50 Metaphase chromosome, 44 Methanol-acetic acid, 43 Methylene blue, 117 Methyl testosterone-17, 63, 72 Microscope, 34, 78 Micro-pipette, 34 Milt, 13 Mixed sex, 89 Modified Cortlands solution, 40 Monosex population, 63-64 Monosex tilapia seed production system, 66 Morula stage, 14, 37 Mouth brooding, 9, 13 Mozambique tilapia, 1, 2 Mustard oilcake, 55, 58, 60 Myxobacterial infection, 115 Myxobolus sp., 119 Natural breeding, 12 Nile tilapia, 1-3, 6, 9-12, 14-15, 31, 47, 70, 92 Nitrous oxide, 31 Nest building, 9 None selected control group, 7 Non-genetic effect, 23 Nursery hapa, 20, 26, 77, 83 Nursery tank, 58 Oestradiol-17, 15, 63, 83 Oestrogen, 17 Oestrogenic control, 15 Omnivorous, 11 On-farm, 6 On-station, 6

Farming of Tilapia

Oocyte, 15, 78 Oocyte maturation, 17 Oogonia, 15 Oreochromis andersonii, 1 Oreochromis aureus, 1, 10, 37, 107, 110, 112 Oreochromis machrochir, 1 Oreochromis mossambicus, 1-3, 10, 41, 45, 47, 64, 112 Oreochromis niloticus, 1- 4, 7, 9-11, 14, 17, 31, 33, 37, 40-41, 45-46, 53, 65, 107, 110, 112 Outbred stock, 21 Ovary, 15 Oviduct, 12 Ovulation, 13 Oxytetracycline, 116 Paddle wheel, 97 Parallel pond, 95 Parasite, 113 Parental care, 14 Passive Integrated Transponder (PIT) tag, 27 Paternal mouth brooder, 10 Peak maturation, 15 Pectoral fin, 34 Pedigree, 21 Perennial pond, 90 Perspex, 33 pH, 10 Phenotype, 45 Phosphate buffer, 43 Photoperiod, 33 Physiological condition, 113 Phytoplankton, 11 Pigmentation stage, 14, 37 Plastic tray, 24, 71 Ploidy manipulation, 31 Polyculture, 90 Polyetheline, 69, 91

Polyploidy, 32 Pond construction, 93 Pond fertilization, 86-87 Poor genetic material, 18

Potassium permanganate, 120

Poultry manure, 86, 90, 96 Precocious maturation, 18, 63 Pressure shock, 36, 40 Primary spermatocyte, 15 Primary oocyte, 15 Progeny testing, 84 Prophylactic treatment, 99 Purebred red strain, 49-50 Raceway, 102, 107-108, 110-112 Radiometer, 40 Radioimmunoassay, 17 Receptor-mediated endocytotic process, 15 Recirculation system, 24, 37, 56, 111 Redbelly tilapia, 2 Red tilapia strain, 45, 47 Reduced growth rate, 18 Reference stock, 20-21 Retailing market, 122 Rice bran, 54-55, 58 Rotenone, 54 Rural market, 122 Salinity, 11, 98 Sarotherodon galilaeus, 1, 10, 107 Sarotherodon melanotheron, 2, 10 Sashimi, 124 Seasonal pond, 86 Secondary sexual characteristic, 12, 15 Secondary oocyte, 15 Secondary spermatocyte, 15 Second meiotic division, 32 Second polar body, 32, 38, 40 Seed production, 18
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Farming of Tilapia

Selective breeding, 7, 20, 23 Selection intensity, 29 Semen, 17 Semi-intensive culture, 11, 87, 91, 97 Serum calcium concentration, 17 Sex chromosome, 44 Sex determination, 31 Sex ratio, 17, 30, 54, 58, 70 Sex reversal, 20, 63, 78 Sex steroid hormone, 15, 17 Sexual dimorphism, 11 Sexual maturation, 15, 17 Sexual pheromones, 13 Sib cross, 49 Silver barb, 22 Site selection, 93-94 Small-scale farmer, 59 Spawning, 12 Sperm, 15, 17, 34, 40 Spermatogonia, 15, 78 Spermatozoa, 15 Spindle apparatus, 31 Standard length, 27 Standard population size, 19 Standard reference stock, 19 Steroid hormone, 32 Sodium Chloride, 118 Solid waste, 109 Stocking density, 27, 55, 77, 92, 97, 98-99, 105 Stock solution, 72 Streptomycin, 116 Substrate spawner, 9 Super Strain of GIFT, 8, 22, 70 Supplementary feed, 54, 60 Tagging, 26 Tank, 27, 102, 107 Taxonomic classification, 9 Terrestrial insect, 11 Tetraploidy, 32, 34, 37
148

Thai red strain, 46-47 Three spotted tilapia, 1 Tilapia zillii, 2, 9, 107 Tilapia farming, 8, 126-128 Tilapia hatchery, 66 Transitory hapa, 19 Trichodina sp., 118 Triploidy, 32, 34, 37 Triploid metaphase, 44 True breeder, 48, 70 Undifferentiated gonadal tissue, 65 Unfertilized egg, 12 Urethra, 12, 34 Urogenital opening, 12 Urogenital papilla, 15, 64 UV sterilization, 37

Vitellogenesis, 15 Vitellogenin, 15, 17

Water bath, 34 Water quality, 96, 106 Water tempertaure, 10 Wild type, 48, 50 World market, 124 Yolk, 15 Yolk sac resorption stage, 14 YY male, 20, 78, 84 Zooplankton, 11 Zygote, 39

Farming of Tilapia

About the author

Dr. M.G. Hussain was born in Mymensingh, Bangladesh on February 1954. He earned his Ph.D. in Aquaculture Genetics from the University of Stirling, Scotland, UK. He has been internationally reputed as tilapia geneticist due to his pioneer works on suppression of mitotic cleavage and production of genetic clones in Nile tilapia and genetic inheritance study leading to gene mapping in red tilapia strains. As the Team Leader of Fish Breeding and Genetic Research Group of Bangladesh Fisheries Research Institute (BFRI), he is presently involved with some international and national fish genetic research programs. The two most recent outstanding contributions of Dr. Hussain are: the development of a genetically improved strain of silver barb (Barbods gonionotus) and further improvement of GIFT strain through several generations of genetic selection. In recognition of such contributions, he has been conferred the prestigious National Fish Fortnight 2003 Gold Medal Award. He is instrumental in developing tilapia farming in Bangladesh particularly tilapia hatchery and monosex seed production system designing, operation and promotion of tilapia aquaculture. Dr. Hussain has published more than 100 peer reviewed journal papers and co-edited several proceedings of seminars and workshops. He has also co-authored several books; the recent one is entitled Genetic improvement and conservation of carp species in Bangladesh. He has served as Scientific Officer under Department of Fisheries, Bangladesh (1978-1981); UNV Fisheries/Aquaculture Specialist under UNDP, Geneva posted in Syria (1981-1986); Principal Scientific Officer and Chief Scientific Officer under BFRI (1986-2000); Director, Research and Planning under BFRI (2001-2004). Dr. Hussain is currently the Director, Admin. and Finance of BFRI.

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