MAKERERE UNIVERSITY

OPTIMISING SPAWNING CONDITIONS AND GROWTH PERFORMANCE

OF LARVAE AND JUVENILES IN altianalis (BOULENGER, 1900)

BY

ARUHO CASSIUS

BSC (Botany and Zoology), PGDE (Biology), MSc. (Zoology), PDPPM

(Project management)

A THESIS SUBMITTED TO THE DIRECTORATE OF RESEARCH AND

GRADUATE TRAINING FOR THE AWARD OF THE DEGREE OF

DOCTOR OF PHILOSOPHY OF MAKERERE UNIVERSITY

NOVEMBER 2018

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DEDICATION

My work is dedicated to my dear parents, Mr. and Mrs. Rubanju William, My dear Wife Judith

Aruho and my children who have stood by me in all I have gone through. They have sacrificed all they could for my sake in terms of encouragement, love and financial contributions. They are my heroes, and I thank Almighty God for them and everything that I am.

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ACKNOWLEDGEMENT

I am deeply indebted to my supervisors; Prof. Rutaisire Justus and Prof. Fredrick Bugenyi for their

effort they dedicated to my PhD project from its inception to its completion. Together with my PhD

committee members including Dr. Gladys Bwanika Namuswe, Dr. John Barilwa and Dr. Owori, I am

humbled by their academic counsel and support that has culminated into this beautiful scientific piece

of work. I salute them and may the Almighty God reward you abundantly.

My honest thanks go to the government of and the World Bank through “The Agricultural

Technology and Agribusiness Advisory Services (ATAAS) project, implemented by National

Agricultural Research Organization (NARO) for their financial support I received that fulfilled my

dream of attaining a PhD from Makerere University. I am humbled by the financial support I received

from my colleague Dr. John Walakira.

I acknowledge the support I received from National Fisheries Resource Research Institute

(NaFIRRI), National Crop Resources Research Institute (NaCRRI) and the College of Veterinary

Medicine, Resources and Bio-security (COVABs) at Makerere University who provided me

with laboratory facilities for my research work. I am also humbled by Mr. Sekyewa for providing his

Ssenya fish farm to conduct my research.

Special thanks go to Dr. Eric Sande, Dr. Ronald Semyalo, Dr. Jackson Effitri, Dr. Godfrey Kawoya

Kubiriza all from the Department of Zoology, Entomology and Fisheries Sciences; Mr. Magid

Kiseka and Dr. Kato Drago of COVABS; Dr. Namanya Ephraim of NACRRI; Ms. Kimera Bridget,

Ms. Ganda Egulance, Ms. Emalyn Kamusiime, Mr. Ddungu Richard, Dr. Victo Namulawa, Dr. v

Mbabazi Dismas, Dr. Walakira, Dr. Mathew Mwanja, Dr. Rose Basiita and Mujib Mukambo all from

Kajjansi Aquaculture Research and Development Center (ARDC), and Dr. Dickson Turyareeba of

Makerere University Business School for their professional advice, guidance and support in this research.

Heartfelt thanks go to my parents, Mr and Mrs Rubanju, my brothers, my dear wife Kemigisha

Judith, my dear Sons Pedro, Leon, Eugene and My lovely daughter Mariella, for their encouragement, inspiration, motivation, care, love and financial support. Also notwithstanding the fact that my children missed me a lot when I was doing my PhD, I salute my children. I sincerely thank Jackline Oidu my sister in-law for supporting my family during the period when family resources were constrained by my study.

Finally, I thank all people in their different capacities for every bit they have contributed to my study.

May the good Lord bless you and reward you abundantly.

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LIST OF PUBLISHED PAPERS

1. Aruho, C., Ddungu, R., Nkalubo, C., Ondhoro, C.C., Bugenyi, F., & Rutaisire, J. (2018). Optimizing selection of sexually mature Barbus altianalis for induced spawning: determination of size at sexual maturity of populations from Lake Edward and Upper Victoria in Uganda. Fisheries and aquatic sciences, 2018, 21: 34. https://doi.org/10.1186/s41240- 018-0110-3

2. Aruho, C., Mwanja, M. T., Bugenyi, F., & Rutaisire, J. (2017). Effectiveness of African catfish pituitary extracts, dagin and water flow for optimising eggproduction, fertilisation and hatchability in artificial spawning of Barbus altianalis. Uganda Journal of Agricultural Sciences, 17 (2), 183 - 195

3. Aruho, C., Namulawa, V., Kato, C. D, Kisekka, M., Bugenyi, F., & Rutaisire, J. (2017). Histo-morphological description of the digestive system of the Rippon Barbel Barbus altianals (Boulenger 1900): A potential species for culture. Uganda Journal of Agricultural Sciences, 17 (2), 197 – 217.

4. Aruho, C., Walakira, J. K., & Rutaisire, J. (2018). An overview of domestication potential of Barbus altianalis (Boulenger, 1900) in Uganda. Aquaculture reports, 11, 31-37. https://doi.org/10.1016/j.aqrep.2018.05.001

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TABLE OF CONTENTS

DECLARATION ...... Error! Bookmark not defined.

DEDICATION ...... iv

ACKNOWLEDGEMENT ...... v

LIST OF PUBLISHED PAPERS ...... vii

TABLE OF CONTENTS ...... viii

LIST OF FIGURES ...... xvi

ABBREVIATIONS (ACRONYMS) ...... xix

ABSTRACT ...... xx

CHAPTER ONE ...... 1 1.0 General Introduction ...... 1 1.1 Background ...... 1 1.2 Statement of the problem ...... 3 1.3 General objective of the study ...... 3 1.4 Specific objectives of the study ...... 3 1.5 Research questions ...... 4 1.6 Significance of the study ...... 5 1.7 Justification of the study ...... 5 1.8 Conceptual framework for increased seed production through increased survival of embryos, larvae and juveniles 7 1.9 Thesis organisation ...... 8 1.9.1 Chapter One ...... 8 1.9.2 Chapter Two (literature review-an overview of domestication potential of B. altianalis) ...... 8 1.9.3 Chapter Three, Four and Five (covered objective 1 of the study) ...... 8 1.9.4 Chapter Six and Seven (covered objective 2 of the study) ...... 9 1.9.5 Chapter Eight (covering objective 3 of the study) ...... 9 1.9.6 Chapter Nine ...... 9

CHAPTER TWO ...... 12

An overview of domestication potential of Barbus altianalis (Boulenger, 1900) in Uganda ...... 12 2.0 Abstract ...... 12 2.1 Introduction ...... 13 viii

2.1 Introduction ...... 13 2.2 Contribution of cyprinid fish to fish culture ...... 15 2.3 Distribution and ecology of Barbus altianalis ...... 16 2.4 Trends and socio-economic prospects of Barbus altianalis fishery ...... 21 2.5 Progress in Culture of Barbus altianalis and other indigenous cyprinids ...... 23 2.5.1 Artificial spawning and factors for optimal egg incubation and larval growth ...... 24 2.5.2 Feeding in Barbus altianalis ...... 25 2.6 Conclusions ...... 26 2.7 Acknowledgment ...... 27 2.8 References ...... 28

CHAPTER THREE ...... 42

Optimising selection of sexually mature Barbus altianalis for induced spawning; Determination of size at sexual maturity of populations from Lake Edward and Upper Victoria Nile in Uganda...... 42 3.0 Abstract ...... 42 3.1 Introduction ...... 43 3.2 Materials and Methods ...... 44 3.2.1 Collection of wild fish samples for determination of size at Maturity ...... 44 3.2.2 Monitoring maturity level of second generation fish using farm records at Ssenya fish farm, Lwengo District...... 45 3.3 Statistical analysis ...... 45 3.4 Results ...... 47 3.4.1 Sex ratios ...... 47 3.4.2 Macroscopic and microscopic description of female classification stages ...... 47 3.4.3 Macroscopic and microscopic description of male classification stages ...... 51 3.4.4 Size at sexual maturity, L50 from Lake Edward and River Nile ...... 54 3.4.5 Lengths-weight relationships ...... 56 3.4.6 Size frequency distribution (distribution of number of sampled fish in class size) ...... 57 3.4.7 Monitoring maturity level of second generation fish using farm records at Ssenya fish farm, Lwengo District 59 3.5 Discussion ...... 60 3.6 Conclusions ...... 64 3.7 Acknowledgment ...... 64 3.8 References ...... 65

CHAPTER FOUR ...... 69

Effectiveness of African catfish pituitary extracts, Dagin and water flow for optimising egg production, fertilisation and hatchability in artificial spawning of Barbus altianalis...... 69 ix

4.0 Abstract ...... 69 4.1 Introduction ...... 70 4.2 Method and Materials ...... 72 4.2.1 Preparation and Feeding of Broodstock ...... 72 4.2.2 Preparation of Catfish Pituitary Extract and Dagin ...... 72 4.2.3 Experiment I; Separation and treatment of broodstocks with hormones ...... 72 4.2.4 Stripping and incubation of eggs ...... 73 4.2.5 Experiment II ...... 74 4.3 Measuring parameters ...... 74 4.4 Data analysis ...... 75 4.5 Results ...... 75 4.6 Discussion ...... 77 4.7 Conclusion ...... 81 4.8 Acknowledgment ...... 81 4.9 References ...... 82

CHAPTER FIVE ...... 87

Optimal factors for egg hatchability and larvae development of Barbus altianalis under captivity ...... 87 5.0 Abstract ...... 87 5.1 Introduction ...... 88 5.2. Methods and Materials ...... 90 5.2.1 Optimal factors for egg hatchability ...... 90 5.2.1.1 Experiment I: Effect of Temperature and Aeration on Egg Hatching ...... 90 5.2.1.2 Experiment II; the Effect of Hatching Facility on Egg Hatching ...... 91 5.2.1.3 Experiment III: Effect of Light on Egg Hatchability ...... 93 5.2.1.4 Experiment IV: Hatching Water Level or Water Depth ...... 93 5.2.2 Optimal water temperature for larval survival and growth ...... 93 5.2.2.1 Experiment V: Effect of temperature on larval survival and growth ...... 93 5.2.3 Measured growth parameters ...... 94 5.2.4 Statistical analysis ...... 95 5.3 Results ...... 95 5.3.1 Optimal factors for egg hatchability ...... 95 5.3.1.1 Experiment I: Effect of Temperature and Aeration on Egg Hatching ...... 95 5.3.1.2 Effect of Temperature on Embryonic Developmental Stages (Critical Stages of Embryonic Development) ...... 97 5.3.1.3 Experiment II: The Effect of Hatching Facility on Egg Hatchability ...... 99 5.3.1.4 Experiment III: Effect of Light on Hatchability ...... 100 5.3.1.5 Experiment IV: Hatching Level (or Water Depth) ...... 101 5.3.2 Optimal water temperature for larval survival and growth ...... 101 x

5.4 Discussion ...... 104 5.5 Conclusions ...... 108 5.6 Acknowledgment ...... 108 5.7 References ...... 110

CHAPTER SIX ...... 115

Histo-morphological description of the digestive system of the Ripon Barbel Barbus altianalis (Boulenger 1900); a potential species for culture ...... 115 6.0 Abstract ...... 115 6.1 Introduction ...... 116 6.2 Materials and methods ...... 117 6.3 Results ...... 118 6.3.1 Gross morphology ...... 118 6.3.2 Histological description ...... 122 6.4 Discussion ...... 132 6.5 Conclusion ...... 136 6.6 Acknowledgement ...... 136 6.7 References ...... 137

CHAPTER SEVEN ...... 142

Morphology and functional ontogeny of the digestive tract of Barbus altianalis larvae ...... 142 7.0 Abstract ...... 142 7.1 Introduction ...... 143 7.2 Materials and methods ...... 144 7.2.1 Fish larvae, sampling and external morphological observations ...... 144 7.2.2 Histological and histochemical procedure ...... 145 7.2.3 Data analysis ...... 146 7.3 Results ...... 147 7.3.1 Larval growth and development ...... 147 7.3.2 The digestive tube ...... 148 7.3.3 The buccopharyngeal cavity and the oesophagus ...... 150 7.3.4 The intestine ...... 155 7.3.5 The liver and the pancreas ...... 159 7.4 Discussion ...... 161 7.5 Conclusion ...... 164 7.6 Acknowledgment ...... 165 xi

7.7 References ...... 166

CHAPTER EIGHT ...... 172

Growth and survival of Barbus altianalis larvae and juveniles in captivity ...... 172 8.0 Abstract ...... 172 8.1 Introduction ...... 173 8.2. Materials and methods ...... 175 8.2.1 Sampling and data collection ...... 175 8.2.1.1 Experimental study 1a; growth and survival of larvae on weaning diets and diet combinations ...... 175 8.2.1.2 Experimental study 1b; ontogenetic enzyme activity and regulation in larvae fed with best diet combination (MD)...... 176 8.2.1.2.1 Preparation of the enzyme extract ...... 176 8.2.1.2.2 Amylase activity ...... 177 8.2.1.2.3 Protease activity ...... 177 8.2.1.2.4 Lipase activity ...... 177 8.2.1.2.5 Total protein ...... 178 8.2.1.3 Experimental study 2: Consequential effects of larvae diets in growth of Juveniles ...... 178 8.2.1.4 Experimental study 3. Green water and larvae culture ...... 178 8.2.1.5 -8.2.1.7 Nursing of larvea and juveniles in outdoor facilities ...... 179 8.2.1.6 Experimental study 5; growth of larvae raised from the first generation (F1) broodstock...... 180 8.2.1.7 Experimental study 6; Juvenile growth in tanks ...... 181 8.2.1.8 Experimental study 7. Larvae rearing and parasitic infestation as an extraneous variable during experimentation ...... 181 8.2.1.8.1 Salt treatment tests for Trichodina, Apiosoma, and Epistylis ...... 182 8.2.1.8.2 Ichthyophthirius multifiliis (white spot disease) ...... 182 8.2.2 Preparation of live feeds and decapsulation of Artemia cysts ...... 183 8.2.2.1 Green water ...... 183 8.2.2.2 Moina micrura enriched with green water ...... 183 8.2.2.4 Decapsulating Artemia ...... 184 8.2.4 Data analysis ...... 185 8.3. Results ...... 188 8.3.1 Effect of live and microdiets on growth performance of larvae and juveniles (experimental study 1a &2) ... 188 8.3.2 Ontogeny of enzyme activity and larval development for the best diet MD (6 DAH to 45 DAH; experimental study 1b) ...... 194 8.3.3 Green water experiment ...... 196 8.3.4 Nursing larvae in outdoor green water concrete tanks ...... 197 8.3.5 Determining Growth rates of F2 generation larvae raised from the first generation (F1) broodstock at a local hatchery ...... 198 8.3.6 Nursing Juveniles in tanks up to 6640.03 ± 1972.02mg ...... 199 8.3.7 Larval rearing and parasite infestations ...... 201 8.3.7.1 Treatment of protozoans (ciliated) parasites with salt concentration ...... 201 8.3.7.2 Treatment of Ich (white spot disease) ...... 202

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8.4 Discussion ...... 207 8.5 Conclusion ...... 218 8.6 Acknowledgement ...... 219 8.7 References ...... 220

CHAPTER NINE ...... 232 9.0 General discussion ...... 232 9.1 Conclusions and recommendations ...... 234 9.2 Areas for further study ...... 235

APPENDIX ...... 239

Study Map ……………………………………………………………………………………………………………………………………………………………….239

Puplications ……………………………………………………………………………………………………………………………………………………………..240

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LIST OF TABLES Table 3. 1: Classification of maturity stages for female B. altianalis ...... 49

Table 3. 2: Measurements of B. altianalis oocytes for fish obtained from Lake Edward ...... 50

Table 3. 3: Measurements of B. altianalis oocytes for fish obtained from Victoria. Nile ...... 50

Table 3. 4: Classification of maturity stages for males B. altianalis ...... 52

Table 3. 5: Spermatogenic cell measurements in B. altianalis ...... 54

Table 3. 6: Sexual maturity and Length-weight relationships for B. altianalis from Lake Edward and Upper Victoria Nile ...... 57

Table 3. 7: Annual Female and male percentage ages of ripe individuals between 2012-2014; Stocked in January 2012...... 59

Table 4. 1: Calculated costs per kilogram of B. altianalis injected with African catfish Pituitary Extract and Dagin ...... 73

Table 4. 2 : Measured parameters of second generation B. altianalis females induced under ...... 76

Table 5. 1: Mean hatchability, dissolved oxygen and degree days for each temperature treatment. Means are shown as, Mean ± SD (Standard deviations) ...... 96

Table 5. 2: Mean hatchability, dissolved oxygen and degree days for each temperature treatment. Means are shown as, Mean ± SD (Standard Deviations) ...... 96

Table 5. 3: Effect of temperature on embryonic stages during incubation within each temperature treatment...... 99

Table 5. 4: Cross tabulation of survival (hatched) and mortality of B. altianalis embryos in hatching facilities ...... 100

Table 5. 5: Light effect on Hatchability in B. altianalis ...... 100

Table 5. 6: Egg hatchability with the level of water depth ...... 101

Table 5. 7: Average Larval weight (mg) for B. altianalis larvae at each temperature and each sampling. Values are presented as Means ± Standard Error ...... 102

Table 5. 8: Growth parameters of B. altianalis larvae at four treatment temperatures during the culture period. Values are presented as Means ± Standard Error ...... 103

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Table 6. 1: Variation of mean RGI values with fish weight and total length in class sizes...... 122

Table 7. 1: The mean values of epithelia folds, goblet cells and taste buds at various periods DAH) of development ...... 157

Table 8. 1: Fatty acid and amino acid profiles of microdiet (dry feed) DF ...... 186

Table 8. 2: Feeding schedule (estimates) for different diets at satiation during experimental period...... 187

Table 8. 3: Mean values (±Standard Error SE) of larval body weight fed different diets at p<0.05...... 188

Table 8. 4: F-tests for all the diets at each sampling at p < 0.05 ...... 189

Table 8. 5: Growth parameters of B. altianalis fed on different diets (mean ± SE) ...... 190

Table 8. 6: Contingency table showing proportions of mortalities and survival of larvae in each treatment at 48 DAH...... 192

Table 8. 7: Statistical significance between treatment mean weights at P = 0.05 from 62 DAH to 92 DAH. (Mean ± Standard Error SE) ...... 193

Table 8. 8: Mean values (±Standard Error SE) of growth parameters between green water and clear water treatments...... 196

Table 8. 9: Contingency table showing proportions of mortalities and survival of larvae in each treatment at 48 DAH...... 197

Table 8. 10: Contingency table showing proportions of mortalities and survival of larvae of B. altianalis in each replicate at 48 DAH...... 198

Table 8. 11: Mean values (± SE ) of growth parameters of juveniles raised in concrete nursing tanks at ARDC Kajjansi...... 199

Table 8. 12: Contingency table showing survival and mortalities of B. altianalis in Tanks ...... 200

Table 8. 13: Treatment regime for Ich with salt, potassium and formalin ...... 202

Table 8. 14: Growth parameters and features of infected larvae stocked in outdoor nursing concrete tanks. 203

Table 8. 15: Growth performance for various cyprinids species in captivity ...... 206

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LIST OF FIGURES

Figure 2.1: Map showing Lakes and rivers where B. altianalis is found……………...... 18 Figure 3.1: Cross section of female gonad showing various oocytes.………………...... 48 Figure 3.2: a cross section of testis showing spermatogenic cells at different stages. ……... 53 Figure 3.3: Maturity Ogives for male and female B. altianalis sampled from Lake Edward in 2015. ……………...... 55 Figure 3.4: Maturity ogives for male and female B. altianalis sampled from Victoria Nile in 2015. ……………………………………………………...... 56 Figure 3.5: Length-frequency distribution of B. altianalis from Lake Edward; Bars with

shaded textboxes in the middle indicate the class size with L50 ……………...... 58 Figure 3.6: Length-frequency distribution of B. altianalis from Victoria Nile; Bars with

shaded textboxes in the middle indicate the class size with L50 ……………...... 58 Figure 5.1: hatching facilities a) circular plastic tanks. b) Glass tank system. C) Plastic conical jars ……………...... 92 Figure 5.2: Interaction effect of aeration and temperature on hatchability in B. altianalis…. 97 Figure 5.3: a) Early cleavage (blastodisc), formation of blastomeres, after 30 minutes of fertilization. b) Morular formed after 3hrs of fertilization.. ……………...... 98 Figure 5.4: average larval weight variation with Days after Hatch (DAH) in B. altianalis ... 102 Figure 5.5: variation of final average weight and percentage survival with temperature in B. altianalis larvae……………...... 103 Figure 6.1: Gross anatomy of the digestive system of B. altianalis. In, intestine; Rlv, Right liver; Llv, left liver; Dlv, distal liver lobe;……...... 120 Figure 6.2: Gross anatomy of digestive tract of Barbus altianalis. Es, Esophagus; Ru, Rugae; Lv, liver; In, intestine; Fa, fat; Go, Genital opening; An, Anus; Gb, Gall bladder; As, Gas bladder; Sp, Spleen; Ha, Heart: Te, Testis. ……………...... 121 Figure 6.3: Sections through the lips of B. altianalis.. ……………...... 123 Figure 6.4: Sections through the oral and pharyngeal cavities. ……………...... 124 Figure 6.5: Sections through the eosophagus and Anterior (cranial) intestine of B. altianalis. ……………...... 127

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Figure 6.6: Sections through the intestine and the rectum. ……………...... 128 Figure 6.7: Sections through the liver of B. altianalis. ……………...... 130 Figure 6.8: Sections through the intestine and the spleen. a) Pancreatic tissue (Pc) around the intestine. H&E. a) Pancreatic acini (Pa) within the spleen (Sp), seen around the Vein (Ve). White pulp (white arrows) and Red pulp (black Arrows) are observed. MT. ……………...... 131 Figure 7.1: Variation of weight (mg) with Days after Hatch (DAH) in B. altianalis larvae. The micrographs show the intestinal coiling (single loop) in the upper abdomen at 30 DAH (H & E) and a double loop at 45 DAH (PAS, AB-PH 2.5). It, intestine; Sb, swim bladder. ………...... 147 Figure 7.2: Gut morphology of live B. altianalis larvae viewed under light Microscope. (a) larva at 1 DAH with a large yolk and an intestinal tube over the yolk. ………...... 149 Figure 7.3: a section through the head and trunk of 3 DAH B. altianalis Larvae……….. … 152 Figure 7.4: Longitudinal section of B. altianalis larvae at 10 DAH: (a) Feature of Larvae at 10 DAH, the yolk residues are still observed, anterior section is coiled, and pancreas & liver are enlarging. …………………………………….…...... 153 Figure 7.5: Histological sections of figure (4) of B. altianalis larvae at a higher magnification. (a) Differentiated pancreas and the liver at 7 DAH. ……………...... 154 Figure 7.6: Sections of digestive system of B. altianalis Larvae with PAS-AB (PH 2.5) stain showing mucus cells (goblet cells) at various ages. ……………...... 158 Figure 7.7: sections B. altianalis larvae showing the differentiation of the liver and the Pancreas at 3 DAH. ……………...... ……………...... 160 Figure 8.1: Growth performance of B. altianalis larvae fed with different diets during the experimental period. ……………...... ……………...... 189 Figure 8.2: Mean percentage survival of B. altianalis larvae at 48 DAH of feeding (42 DAF); and mean percentage mortalities by 24 DAH (18 DAF) and 45 DAH (39 DAF).. 191 Figure 8.3: Variation of average body weight (mg) of B. altianalis juveniles with days after Hatch (DAH) ……………...... ……………...... 193 Figure 8.4: variation of Amylase activity with Days after hatch (DAH) in B. altianalis larvae (n=3) ……………...... ……………...... 194

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Figure 8.5: Variation of protease activity with Days after hatch (DAH) in B. altianalis larvae (n=3). ……………...... ……………...... 195 Figure 8.6: Variation of lipase activity with Days after hatch (DAH) in B. altianalis larvae (n=3) ……………...... ……………...... 195 Figure 8.7: Variation of mean larval weight (mg) for green and clear water treatments with Days After Hatch (DAH). ……………...... 196 Figure 8.8: Average larvae weight variation with Days after hatch (DAH) ……………...... 197 Figure 8.9: Variation of average body weight of B. altianalis larvae with DAH …………... 198 Figure 8.10: Variation of average body weight of B. altianalis juveniles with DAH………. 200 Figure 8.11. Variation of salt concentrations with effective treatment period of B. altianalis larval bath and tolerance……………………………………………………… 201 Figure 8.12: graphical presentation of the relationship between survival and stocking size with respect to the DAH. ……………...... ……………...... 203 Figure 8.13: showing Ich parasite under the skin of B. altianalis Larvae as observed under the microscope. Mg x 40 ……………...... ……………...... 203 Figure 8.14: Scale formation. a) Scales forming round the neck and along the lateral region of B. altianalis larvae metamorphosising into Juveniles at 57 DAH (257mg, 3.0TL)….. 205

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ABBREVIATIONS (ACRONYMS)

NARO – National Agriculture Research Organisation NaFIRRI – National Resources Research Institute FAO – Food and Agricultural Organisation DFR – Department of Fisheries Resources (Uganda) MT – Million tones t – tonnes LIFDCs –Low Income Food-Deficit Countries WFC – World Fish Center DRC– Democratic Republic of Congo WHO – World Health Organisation TUFMAC – The Uganda Fish Marketing Cooperation factory UNDESP – United Nations Department of Economic and Social Affairs and Population Division. DAH – Day after Hatch ARDC– Aquaculture Research and Development Center GTHs – Gonado Trophin Hormones FSH – Follicle stimulating hormone COVABs – College of Veterinary Medicine, Animal Resources and Bio-security ATAAS – The Agricultural Technology and Agribusiness Advisory Services project SRAC– Southern Regional Aquaculture Center SE – standard error SD – Standard deviation ACPE – African catfish pituitary extracts

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ABSTRACT Barbus altianalis is an indigenous cyprinid that lives in lacustrine-riverine environments with limited distribution to Upper Victoria Nile and lakes Victoria, Edward, George and their associated rivers. The species is a delicacy in Uganda and surrounding regions and also vulnerable to overexploitation. B. altianalis is cultured by very few farmers and the biggest challenge has been lack of seed for its commercial propagation. This study was conducted to determine optimal spawning conditions, larval weaning, juvenile growth and survival with a view of producing mass quality seed for propagation. Two field and 17 lab/pond experiments were conducted to improve hatchability, growth and survival of larvae and juveniles. Results showed that fish for spawning is best picked at ≥ 30-34.9 cm and 35- 39.9 cm fork lengh for males and females, respectively. Fish treated with African catfish pituitary extracts (ACPE) performed slightly better than those treated with Dagin. However, the differences between females treated with ACPE and Dagin were not significant with respect to working fecundity (2314.40; 1207.37), fertilisation rates (80.27; 40.80%) and hatchability (42.20; 27.44%) at p > 0.0167 respectively. ACPE are equally effective and cheaper for inducing B. altianalis to spawn. Working fecundity and hatchability were significantly higher when only ripe running females were stripped after 4 hours (100 degree hours) of flashing water than those stripped after 10hrs (250 degree

hours) at 25⁰C. Optimal temperature for embryo hatchability was 24⁰C - 27⁰C. Hatchability was good and the same (p > 0.05) between re-circulating (84.3%) and glass tank systems (80.3%). High larval growth (158.61 ± 1.56; 195.03 ± 2.71mg) and survival (81.24 ± 1.55%; 78.96 ± 2.04%) were attained at 30⁰C and 27⁰C, respectively. The digestive tract of B. altianalis was simple and valveless and was on average 2.22 ± 0.37 times longer than its body length. It showed a strong ability to utilize and assimilate every diet but also showed preference of food items at early stages becoming all inclusive in its diet as it matures. Larval ontogeny of digestive structure and related enzyme activity confirmed that microdiets were acceptable at exogenous feeding (6-7 Day after hatch-DAH). But better growth and survival were obtained with a combination diet (Moina + microdiet). Hence, outdoor larvae nursing and microdiet manipulations for better growth and survival were successful≥ at 15 DAH. When prevalence of aquatic parasites was anticipated, outdoor nursing and or stocking were delayed until after or during larvae scalation process at 48 - 75 DAH. This study has now been able to determine the optimal spawning and growth performance conditions of larvae and juveniles in B. altianalis and it will now be possible to produce mass and quality seed for propagation. xx

CHAPTER ONE

1.0 General Introduction

1.1 Background

Barbus altianalis is a cyprinid belonging to the family of the Order . The family is the largest of all fish families constituting about 2400 fish species and is widely distributed in North America, Eurasia and Africa (FAO, 2010; Greenwood, 1966; Nelson, 2006; Snoeks, Kaningini, Masilya, Nyina-Wamwiza, & Guillard, 2012). In Uganda cyprinid species occur in all the major lakes and the river Nile system (Greenwood, 1966). Barbus altianalis closely resembles Barbus bynni of the same genus Barbus and they are thus both locally known as Kisinja. However, B. altianalis is also known as ‘Enjunguri’ around Lakes Edward and George. Both B. altianalis and B. bynni largely inhabit lacustrine-riverine systems and use upstream fresh running water as a conducive environment for spawning (de-Graaf, Nentwich, Osse & Sibbing, 2005; Rutaisire, Levavi‐Sivan, Aruho & Ondhoro, 2015; Skelton, Tweddle, & Jackson, 1991). B. bynni dominates the Albert Nile fishery where it contributes an estimated annual average of about 11% (700 metric tonnes) of the total catches of 5,800 metric tonnes (Mbabazi et al., 2012) while B. altianalis contributes about 18% of the total catches in the Upper Victoria Nile and less than 1% of the total catch (300,000t) from Lake Victoria (DFR report, 2012). Substantial commercial quantities of B. altianalis are reported from Lake Edward particularly at Katwe, Rwenshama and Kayanja landing sites where it contributes 10% of the annual average fish catch of 1294 tonnes (Kasese district fisheries department report, 2012). Rwenshama, Katwe and Kayanja areas are interconneced by various tributaries including rivers; Ishasha, Chiruruma, Nyamugashani, Rwindi, Rutshuru, Ntungwe, Nchera and Ruhibi. These rivers form a complex hydrological pattern that is a suitable habitat for the species’s spawning and growth.

There has been a gradual decline in catches of B. altianalis in Uganda due to increasing fishing pressure. For instance, B. altianalis catches reduced from 813 metric tonnes (mt) in 1967 to less than 1mt since 2000 (Balirwa et al., 2003). B. altianalis is highly priced and largely cherished by the population in central Uganda and Albertine region because of its aroma and taste when smoked. Catches of B. altianalis from Lake Edward constitute the largest individual mean size of 2.24kg and 1

fetches the highest price of 12,000 Uganda shillings per kilo (US$3.8) in Uganda and in the neighboring Democratic Republic of Congo, while other fishes from the same lake are much smaller (Kasese district fisheries department report, 2012). Additionally, the fish is relished by the riparian Congolese and Ugandan communities who believe it enhances men’s sexual ability and boosts women’s strength during pregnancy (Personal Communication with fishermen at Kayanja, 2013; 2017).

These facts are likely to lead to further reduction in stocks of B. altianalis in the lacustrine-riverine systems in Uganda It is therefore important that interventional strategies are quickly developed and strengthened to address the declining production from the lakes and rivers. In spite of the strict regulations in place geared towards fish conservation in Uganda, the decline in catches has not stopped. Currently the most viable alternative to increase production is to focus on the domestication. Complete domestication entails seed production and rearing thus starts with inducing spawning and feeding among other culture practices (Bilio, 2007). Although the culture of B. altianalis had been initiated by successfully studying the seasonal patterns and subsequent first induced spawning trials (Rutaisire et al., 2015), the developed spawning technology required optimisation and subsequently development of technologies for increased survival and feeding of larvae and juveniles in order to massively produce the seed for commercial farming. The works by Rutaisire et al. (2015) reported very low embryonic hatching rates, high mortality of larvae while very slow growth of both larvae and juveniles were later reported by farmers. Currently unavailability of seed hampers culture of the fish. There is paucity of information to support optimal induction of spawning activities, ontogeny of larval digestive system that later defines the larval weaning protocol and appropriate optimal feeds for larval and juveniles. This study investigated factors that should be optimised for spawning and larval and juvenile rearing.

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1.2 Statement of the problem

The potential for culture of indigenous cyprinids (carps) in Uganda including Barbus altianalis has not been fully exploited. B. altianalis was first induced to spawn successfully by Rutaisire et al., (2015). However further development of technologies for mass seed production and subsequent commercialization were required because poor egg hatchability and high larvae mortalities of more than 80% were reported by farmers. Rutaisire et al. (2015) also reported egg and yolk sac mortalities of more than 55 % and 11% respectively. Additionally, slow growth of larvae was reported by farmers. High embryo and larval mortalities are attributed to inappropriate water quality and nutritional conditions for spawning, egg incubation, larval nursing and juvenile growth performance (Kamler, 2008; Villamizar et al., 2011, Mwanja et al., 2015). These conditions were unknown for B. altianalis spawning, growth and survival. The underlying reasons for high embryo and larval mortalities were lack of knowledge of size at maturity to help identify the right broodstock size for spawning, appropriate hormone or dosage for induced spawning and improved egg quality, optimal temperature conditions for hatching and larval nursing, and larvae gut development structure to guide on appropriate nutrition requirements for growth and survival of larvae and juveniles. These factors were therefore determined and integrated into the spawning and larval weaning protocols and consequently they will facilitate mass production of B. altianalis seed or fingerlings adequate for commercial culture.

1.3 General objective of the study

To develop technologies for improving artificial spawning conditions, survival and growth of B. altianalis larvae and juveniles for mass seed production.

1.4 Specific objectives of the study

1. To determine optimal conditions for egg hatchability and larval survival in B. altianalis 2. To determine the earliest stage at which B. altianalis larvae begin to digest a microdiet 3. To determine the appropriate weaning diets for optimising growth and survival of larvae and juveniles in B. alitianalis under captivity

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1.5 Research questions

a) What are the optimal conditions for the hatchability and survival of Barbus altianalis embryos and larvae following induced spawning process? i) At what length is 50% of B. altianalis in a given size class sexually mature? ii) What is the best hormone for improved egg production, fertilisation and hatchability? iii) Does water flow alone have any significant role in induced spawning of B. altianalis? iv) What is the optimal temperature for the hatchability of B. altianalis embryos during the incubation and survival of hatched larvae? v) How does aeration or interaction between aeration and temperature affect hatchability? vi) Does light and water depth affect hatchability? vii) What type of hatching facility is suitable for hatchability B. altianalis b) At what age, size and stage are the hatched larvae of B. alitianalis able to appropriately to digest or assimilate artificial feed (microdiet)? i) What are key features that could be uniquely associated or identify the digestive structure in relation to feeding habits of B. altianalis? ii) When does B. altianalis begin exogenous feeding? iii) Is there a period of mixed feeding? iv) Can the digestive system of B. altianalis accept microdiet at exogenous feeding c) What are the appropriate weaning diets for optimal growth and survival of larvae and juveniles in B. alitianalis under captivity? i) What are the best diets for weaning larvae? ii) When do the larvae attain the digestive maturation competence level to efficiently be able to digest any microdiet with minimal mortalities? iii) What are the growth rates of larvae and juveniles in B. altianalis? iv) When do larvae become juveniles in B. altianalis and

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v) When is it suitable to transfer (or stock) larvae or juveniles from the hatchery to outdoor nursing facilities and or ponds?

1.6 Significance of the study

The study investigated and provided the optimal conditions required for artificial spawning. Knowledge of length-at-50%-sexual maturity was required for the identification of the appropriate size at maturity for breeding. The study identified the effective but also cheaper hormone and administration procedure to optimize egg hatchability rates. Suitable temperature and hatching facilities for egg (embryo) incubation and larval growth were identified. The optimal values obtained in this study greatly improved egg hatchability, larval growth and survival. Understanding the period and or age when larvae were able to competently digest microdiets helped to determine the stocking age of larvae and juveniles. The study identified suitable weaning diets that were recommended for improving the growth and survival of larvae and juveniles. Knowledge of these factors will guide farmers to reduce larval mortalities and improve growth rates for quality and sufficient seed for commercial farming of B. altianalis.

1.7 Justification of the study

Although considerable quantities of B. altianalis are reportedly caught in Lake Edward and the Upper Victoria Nile, reports indicate reduced catches from these habitats. B. altianalis is highly cherished by many communities in Uganda and in Eastern DRC because of its aroma, good taste and soft scales that are also relished. The species fetches lucrative prices with a kilogram costing 10,000 shillings (US$2.63) on average for traders in central and 12,000 shillings (US$3.16) compared to tilapia at between 7,000 to 8,000 shillings (US$1.84 to 2.11)(Traders, personal communication, July 10, 2016; Kasese district fisheries department report, 2012) in western Uganda as well as parts of eastern DRC and has potential to generate income to farmers and the country from local, regional and international markets once it is successfully commercialized. The culture of the related cyprinids in Asia has contributed over 72% of the 90 million metric tonnes produced in the continent and about 70% of the cultured species in the world are cyprinids (FAO, 2010; FAO, 2012; Lucas & Southgate, 2012). Hence successful domestication of indigenous cyprinids such as B. altianalis would largely boost fish 5

production in Uganda and in the region. Currently only the Nile tilapia (Oreochromis niloticus) and the African catfish (Clarias gariepinus) are cultured in Uganda. The country has not yet exploited the potential inherent in indigenous cyprinids (carps). This contrast with China where Chinese carps have been reared with other cultured fish species in polyculture systems to enhance utilization of applied feeds in the system for over 4000 years (Billard & Berni, 2004; Woynarovich, Moth-Poulsen & Peteri, 2010). B. altianalis is an omnivorous species (Balirwa, 1979) and could be of significance in such polyculture systems because it can feed on detritus material. In spite of the recent successful artificial spawning of this species, its seed is still unavailable for commercial propagation. This is attributed to lack of information on appropriate spawning conditions in the hatchery, high larvae mortality rates and lack of a weaning feed. The findings in this study provided the much needed information to culture the species under controlled optimal conditions. This is a step towards increased availability of its seed by the farmers in the East African region. Its commercial culture will also compliment effort to the management of the cyprinid stocks in the lakes and Rivers in Uganda by reducing fishing pressure on them since some of the demand will be met by production from the farms.

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1.8 Conceptual framework for increased seed production through increased survival of embryos, larvae and juveniles

Increased productivity of Barbus altianalis

Mass seed production

Increased larval growth & juvenile survival

Improved spawning technology Improved larval weaning technology

Improved embryo growth and Larval/Juvenile growth and survival (hatching) survival

Optimal factors for spawning Appropriate Larvae and hatching Diets Digestive structure

Light Depth Dissolved Temperature Oxygen Live diets – Artemia Algae Appropriate spawning hormone Moina Inert diets - Artemia eggs Dry processed Improved hatchability Increased Number Combination Moina & Dry of eggs produced per females Improved

fertilization rates

Appropriate minimum spawning size (L50) 7

1.9 Thesis organisation

The thesis establishes the optimal spawning conditions and growth performance of B. altianalis larvae and juveniles in captivity. It was a two-dimensional study with two key outputs namely, i) improved spawning technology and ii) improved larvae weaning technology thus ultimately led to increased seed production of B. altianalis. Objective one was designed to achieve the first output and objective two and three were made to achieve the second output. The thesis was organized in standalone chapters for paper/manuscript arrangement. For each of the result chapters, a title, introduction, materials and methods, results, discussion, and conclusion are provided.

1.9.1 Chapter One

The first chapter describes the abstract of the study, background to the study, the objectives, research questions, problem statement, study justification, study significance, the conceptual framework and the thesis organisation.

1.9.2 Chapter Two (literature review-an overview of domestication potential of B. altianalis)

The second chapter describes the literature review about domestication process of B. altianalis relating to studies and methods used to solve the critical gaps that were identified to be addressed.

1.9.3 Chapter Three, Four and Five (covered objective 1 of the study)

The third, fourth and fifth chapters were designed to answer the first objective of the study, ‘to determine optimal conditions for egg hatchability and larval survival in B. altianalis’. In the third chapter, the key question was what were the recommended sizes of females and males at sexual maturity (L50) that should be included in the spawning technology (and later a protocol) from Lake Edward and Upper River Nile? Chapter four describes how effectively the African catfish pituitary extracts were used to improve, the working fecundity (number of eggs), fertilization and hatchability of embryos. The fourth chapter examined the optimal temperatures and its interacting factors such as

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aeration that should be included in a spawning technology (or spawning protocal) for better hatchability of embryos (eggs) as well as growth and survival of larvae during the incubation process.

1.9.4 Chapter Six and Seven (covered objective 2 of the study)

The sixth and the seventh chapter described the digestive structure of both mature and ontogenetic larval development to determine the appropriate timing for exogenous feeding and acceptability of dry diets (micro-diets). It was imperative to first describe the nature and features associated with the digestive structure of mature before understanding the structure of the developing larvae because the digestive structure of the B. altianailis had not been described before. The description of the digestive structure of both juvenile and mature stages helped understand the feeding behaviours and consequently the feeding strategies vital for raising the B. altianalis in captivity. Chapter seven answered objective 2 of this study.

1.9.5 Chapter Eight (covering objective 3 of the study)

Having determined that the digestive structure of larvae was able to accept the dry feed (microdiet) at exogenous feeding, chapter eight examined the growth performance of larval and juveniles on a number of weaning diets that included both live and microdiets as well as their combinations. In order to answer the third objective of the study, chapter eight determined the best diet for weaning larvae and also laid a procedure for maximizing physiological larval growth and survival by establishing the enzyme activity evolution pattern in developing larvae. The enzyme pattern in developing larvae also helped identify the maturation competence level by which the larvae ware able to fully digest and assimilate a microdiet without compromising growth and survival. Under chapter eight, stocking of larvae or juveniles as well as further diet manipulations are suggested based on this maturation competence period.

1.9.6 Chapter Nine

This is the final chapter of the study, integrating the discussion of all the result chapters, the conclusions, recommendations and areas of further research arising from the study.

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1.10 References

Balirwa, J. B. (1979). A contribution to the study of the food of six cyprinid fishes in three areas of the Lake Victoria basin, , Hydrobiologia, 66, 65-72.

Balirwa, J. S., Chapman, C. A., Chapman, L. J., Cowx, I. G., Geheb, K., Kaufman, L., ….& Witte, F. (2003). Biodiversity and fishery sustainability in the Lake Victoria Basin: An unexpected marriage? Bioscience, 53,703-715.

Billard, R., & Berni, P. (2004). Trends in cyprinid polyculture. Cybium, 28 (3), 255-261.

Bilio, M. (2007) Controlled reproduction and domestication in aquaculture – the current state of the art, Part I. Aquaculture Europe, 32, 5–14. de-Graaf, D. M., Nentwich, E. D., Osse, J. W. M., & Sibbing, F. A. (2005). Lacustrine spawning: is this a new reproductive strategy among 'large' African cyprinid fishes? Journal of Fish Biology, 66, 1214-1236.

Department of Fisheries Resources (DFR). (2012). Annual Report 2010/2011 (pp. 1- 41). Entebbe, Uganda: Ministry of Agriculture Animal Industry and Fisheries (MAAIF), Retrieved from http://www.agriculture.go.ug/userfiles/DFR%20ANNUAL%20REPORT%202012.pdf,

Food and Agriculture Organization (FAO). (2010). The state of world fisheries and aquaculture. World review of fisheries and aquaculture (pp.1- 220). Rome, Italy: Author.

Food and Agriculture Organization (FAO). (2012). The state of world fisheries and aquaculture. World review of fisheries and aquaculture (pp.1-202). Rome, Italy: Author.

Greenwood, P. H. (1966). The fishes of Uganda (2nd ed.). Kampala, Uganda: Kampala Uganda Society

Kamler, E. (2008). Resource allocation in yolk-feeding fish. Reviews in Fish biology and Fisheries, 18(2), 143

Kasese District Fisheries Department Report (2012). Lake George and Edward fisheries statistics (District report, pp 10). Kasese District: Belgian Technical support department.

Lucas, J. S., & Southgate, P. C. (Eds.). (2012). Aquaculture: Farming aquatic and plants (2nd ed.) (pp. 629). John Wiley & Sons.

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Mbabazi, D., Taabu-Munyaho, A., Muhoozi, L., Nakiyende, H., Bassa, S., Muhumuza, …. & Balirwa, J. S. (2012). The past, present and projected scenarios in the Lake Albert and Albert Nile fisheries: Implications for sustainable management. Uganda Journal of Agricultural Sciences, 13 (2), 47-64.

Ondhoro, C. C., Masembe, C., Maes, G. E., Nkalubo, N. W., Walakira, J. K., Naluwairo, J & Efitre, J. (2016). Condition factor, length–weight relationship, and the fishery of Barbus altianalis (Boulenger 1900) in Lakes Victoria and Edward basins of Uganda. Environmental Biology of Fishes, 1-12100 (2), 99-110. doi:10.1007/s10641-016-0540-7.

Nelson, J. S. (2006). Fishes of the world (4th ed.) (pp, 601). Inc., New York: John Wiley & Sons.

Villamizar, N., Blanco-Vives, B., Migaud, H., Davie, A., Carboni, S., & Sanchez-Vazquez, F. J. (2011). Effects of light during early larval development of some aquacultured teleosts: a review. Aquaculture, 315(1), 86-94.

Rutaisire, J., Levavi-Sivan. B., Aruho, C., & Ondhoro, C. C. (2015). Gonadal recrudescence and induced spawning in Barbus altianalis. Aquaculture, Aquaculture, 46 (3), 669–678. Doi:10.1111/are.12213

Skelton, P. H., Tweddle, D., & Jackson, P. (1991). Cyprinids of Africa. In: I. J. Winfield & J. S. Nelson (Eds.), Cyprinid Fishes, Systematics, Biology and Exploitation (pp. 211–233). London, UK: Chapman & Hall.

Snoeks, J., Kaningini, B., Masilya, P., Nyina-Wamwiza, L., & Guillard, J. (2012). Fishes in Lake Kivu: diversity and fisheries. In Lake Kivu (pp. 127-152). Netherlands: Springer.

Woynarovich, A., Moth-Poulsen, T., & Peteri, A. (2010). Carp polyculture in Central and Eastern Europe, the Caucasus and Central Asia: a manual. FAO Fisheries and Aquaculture Technical Paper, 554 (pp.1- 73). Rome, Italy, FAO.

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CHAPTER TWO

An overview of domestication potential of Barbus altianalis (Boulenger, 1900) in Uganda

C. Aruho, J. K. Walakira & Rutaisire. J.

Aquaculture reports, 11 (2018) 31-37; doi.org/10.1016/j.aqrep.2018.05.0019-38pp

2.0 Abstract

Domestication of fish is a key strategy for diversification of farmed species to meet consumer’s choices and demands as well as conservation of the species for sustainable provision of nutritional benefits and incomes. Initial successful induced spawning of Barbus altianalis was achieved, but there is low adoption attributed to lack of sufficient quality seed. This paper reviews the ecological and social-economic trends, and potential prospects that justify the domestication of the indigenous high value species, and identify gaps that could be addressed to increase seed production for commercialization. Review findings show that due to overexploitation, there is a steady decline of B. altianalis in Ugandan water bodies, with no current record from Lake Victoria where catches had in the past been reported. B. altianalis shows ability to survive in interlacustrine-riverine environments although, the juveniles are largely confined in the river or stream water. Varying levels of adaptability and tolerance to environmental conditions including oxygen and temperature by different age groups occur. The species has a great potential for culture as an omnivorous species with high chances of adapting to varying feeding strategies. Knowledge gaps in size at maturity, appropriate inducing hormones, growth conditions, egg hatchability and larvae weaning were identified as key challenges associated with B. altianalis domestication. Understanding the underlying natural ecological dynamics of B. altianalis will guide further research in the areas mentioned to ensure advancement in domestication so as to meet the rising demand for B. altianalis. This will curtail its overexploitation in the wild and also improve the livelihoods of the communities in the region.

Key words: cyprinid, omnivorous, indigenous species, domestication, commercialization

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2.1 Introduction

The overall global demand for fish has significantly increased over the years and by 2014 the world registered a total fish production of 171 MT of which 80.3 MT were from farmed products (FAO, 2018). With a projected 9.7 billion world population at a consumption rate of 20.3 kg person-1 year-1, by 2050 a total production of 220 MT will be required to meet nutritional requirements. About 140 MT will come from aquaculture (Rice & Garcia, 2011; Merino et al., 2012; Waite et al., 2014; Bene et al., 2015). Much of the production from capture fisheries has stagnated with 28.8% of the fish stocks being exploited at biologically unsustainable levels (FAO, 2014; 2016). The only hope to meet the world’s fish demand is to exploit the potential of culturing fish in confined environments (FAO, 2016; 2018).

In Africa, the contribution of cultured fish was less than 2.5 % of the World’s total, with Egypt contributing 1.7%, Nigeria 0.4 and only 0.4% from sub-Saharan Africa excluding Nigeria (FAO, 2018). Estimated annual per capita fish supply in Africa (about 9.8 kg) is less than half the world‘s average; and from aquaculture (6kg) even less (FAO, 2016; WFC, 2005). This is less than that recommended by US dietary guidelines, Health Organization/FAO of 250mg-500mg of EPA/DHA (omega-3-fatty acids) per day especially for many fish species which have low omega-3 fatty acids and therefore one may need to take in more of the fish than what is stated (FAO/WHO, 2011; McGuire, 2011). For instance a minimum of 26.15g daily (9.55kg person−1 year−1) are recommended from Tilapia compared to 18.17g (6.84kg person−1 year−1) from the fatty acid rich salmon (Reames, 2012). According FAO (2016) statistics, by 2014 the sub-Saharan region contributed only 0.33% of world’s 73.8MT produced from aquaculture, yet GIS models have estimated that 37% of sub-Saharan region were potentially suitable for Aquaculture (Aguilar-Manjarrez & Nath, 1998; Brummett, Lazard & Moehl, 2008). This reflects an imbalance in the fish production, consumption and distribution in the world and increased production may not necessarily reflect the fair access to nutritional requirements of fish by the world populations especially from low income food-deficit countries (LIFDCs). This deficit represents a food security threat, and fish as an affordable protein source is increasingly becoming scarce and expensive. Given that the catches from capture fisheries are stagnating, aquaculture is seen as the only plausible option to meet the rising demand for fish in the sub-Saharan region. Efforts to encourage aquaculture in Africa are constrained by the low number

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of cultured species, and poor quality of fish seed and fish feeds (Aruho, Basiita, Kahwa, Bwanika & Rutaisire, 2013; Brummett et al., 2008; Rutaisire, Levavi‐Sivan, Aruho & Ondhoro, 2015).

Domestication of fish species entails adopting fish with wild characteristics into confined culture systems that are artificially managed. Fish with desirable biological, nutrition, social and economic characteristics are preferably selected from the wild and cultured for human consumption and or ornamental use (Bilio, 2007). The transformation of the wild fish into confined culture systems is a technical process that begins with understanding the environment of the fish and its reproductive biology before being artificially induced to spawn (Mylonas, Fostier & Zanuy, 2010; Rutaisire et al., 2015; Teletchea & Fontaine, 2014). Successful artificial spawning must be accompanied by technologies to make the fish available for propagation. The response to the growing demand of animal protein due to the increasing world population is enormous and is characterized by the great desire for domestication of high value fish species to bridge the demand gap.

Barbus altianalis (or altianalis) also known as the Ripon barbell is one of the potentially high-value fish species threatened with overexploitation (Ondhoro et al., 2016; Rutaisire et al., 2015). It is a large cyprinid that grows up to about 90 cm total length and 10 kg individual weight (Greenwood, 1966). The species is widely distributed in the Lake Victoria region and in many rivers and streams, except in Lake Albert. It is considered a delicacy among many communities in Uganda, Eastern Democratic Republic of Congo (DRC) and Eastern . The recent successful first induced spawning of wild B. altianalis (Rutaisire et al., 2015) opened an opportunity for its increased commercial production through domestication as well as for its conservation. However, like many other newly domesticated species, there are challenges of producing sufficient good quality fish seed (Mylonas et al., 2010; Naylor & Burke, 2005). Lack of sufficient quality fish seed has been attributed to high larval and embryonic mortalities as well as low-quality larval and juvenile feed. These are all linked to lack of knowledge about improved artificial breeding techniques, optimal conditions for egg hatchability and larval growth, feeding ecology, digestive structure of the species and better larval weaning strategies. This paper reviews a number of aspects related to the efforts directed toward B. altianalis commercialization. It highlights potential gaps to be addressed in future strategies for popularization of B. altianalis through domestication and its sustainable exploitation for nutritional benefits and economic development. 14

2.2 Contribution of cyprinid fish to fish culture

About 325 fish species (including hybrids) are cultured worldwide, the majority of them are from Asia (FAO, 2014; 2016; 2018), and with cyprinids and tilapias forming the bulk of the tonnage (Chiu et al., 2013; FAO, 2016; Statista, 2014). Over 70% of all the cultured species in India, Bangladesh, Iran, Pakistan, Thailand, China and Japan are cyprinids (FAO, 2012). Most of the cultured cyprinids have been reared for centuries in Asia. The domestication process has taken so many years of improving the culture of their indigenous species (Balon, 1995; Hulata, 1995). The culture of some of the Asian cyprinids is preferred because some may not require feeds and are thus polycultured with other non cyprinid species or together to maximize utilization of available feeds in the culture system (Billard & Berni, 2004; Dey et al., 2005; Reddy et al., 2002; Woynarovich, Moth-Poulsen & Peteri, 2010). For instance and big head carp are non-fed cyprinids that alone contributed 9.2 MT (18.58%) of the world’s total finfish culture of 49.86 MT in 2014 (FAO, 2016) and 8.8MT out of the 54.1MT produced in 2016 (FAO, 2018). Cyprinids have therefore significantly contributed to their economies and food security system in Asia. Some of these species were introduced into Africa, where in some countries they have become invasive and have interfered with other species (Britton et al., 2007; DeGrandchamp, Garvey & Colombo, 2008; Lorenzen, Beveridge & Mangel, 2012; Zambrano, Martinez-Meyer, Menezes & Peterson, 2006). To avoid these problems, promoting indigenous high-value species is considered a good strategy for production and conservation purposes (De Silva, Nguyen, Turchini, Amarasinghe & Abery, 2009; Funge-Smith & Phillips, 2001; Teletchea & Fontaine, 2014). The domestication of fish species however, must conform to the tenet of responsible aquaculture to ensure environmentally sound and sustainable culture, conservation of indigenous wild stocks as well as constant supply and replenishment of domesticated fish for sustained food security (Diana et al., 2013; Merino et al., 2012).

In sub-Saharan Africa much fewer species (< 27) are cultured and these are dominated by tilapias and the African catfish species (Brummett et al., 2008; Hecht, 2007; Machena & Moehl, 2001; Skelton, Tweddle, & Jackson, 1991). However, there are many indigenous African species of high value that can be cultured. Among them are the African cyprinids (carps). The Cyprinus carpio, an exotic species, is one of the most commonly cultured specie in Uganda and other parts of sub- Saharan region. The level of production of the common carp and other Chinese carps in Africa

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however, has remained very low (Hecht, 2007; Moreau & Costa‐Pierce, 1997). The local communities prefer culturing indigenous species including Nile Tilapia and Catfish. The potential for culture of indigenous cyprinids (carps) has not been fully exploited (Rutaisire et al., 2015) in Africa. In Uganda, efforts have focused on domestication of high-value species including the Nile perch, catfish, bagrids and cyprinids (Aruho et al., 2013; Basiita et al., 2011; Rutaisire & Booth, 2004; Rutaisire et al., 2015). The domestication of these species is expected to increase the diversity of farmed species, enhance production and productivity, as well as relieve pressure on wild stocks thus leading to genetic conservation of the species in the wild.

2.3 Distribution and ecology of Barbus altianalis

Barbus altianalis is a lacustrine-riverline species that is widely found in Ugandan open water bodies, the rivers and streams that are interconnected within the Lake Victoria Basin (Figure 2.1) (Greenwood, 1966). Elsewhere in East Africa, B. altianalis is found in River Kagera, Lake Kivu and its effluent rivers (Snoeks, Kaningini, Masilya, Nyina-Wamwiza & Guillard, 2012) as well as in the rivers of Nyando, Nzoia, Yala and Sondu-Miriu on the Kenyan side of Lake Victoria (Chemoiwa et al., 2013). Along the River Nile the catches are reported only in the Upper Victoria Nile and the lake area where the river joins Lake Kyoga at Mbulamuti, Kamuli district but there are no reports of the species after Lake Kyoga (Greenwood 1966; Fishermen, personal communication, July 10, 2016). In South Western Uganda it is found in Lakes Edward and George which have a wide interconnectivity of rivers including, Ishasha, Chiruruma, Nyamugashani, Rwindi, Rutshuru, Ntungwe, Nchera and Ruhibi (De Vos & Thys van den Audenaerde, 1990; Muwanika, Nakamya, Rutaisire, Sivan & Masembe, 2012; Snoeks et al., 2012). These rivers drain from vast surrounding areas and are fed by other much smaller streams where this species is also reported. The rivers join together into Semliki River which moves northwards towards Lake Albert. However, there are no reports of this species in Lake Albert except for a closely related species B. bynni (Greenwood, 1966). The absence of this species in Lake Albert and the lower section of the Victoria Nile as well as the upper Nile after Lake Albert could be linked to both abiotic and biotic environmental factors. B. altianalis closely resembles B. bynni. Both species are morphologically similar except that the body depth of B. altianalis is equal or slightly longer than the length of the head. Comparatively the body depth of B.

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bynni is much deeper than the head. The head of B. altianalis is 3-4⅓ times its standard body length but the head of B. bynni is 2⅓-3⅓ its standard body length. The coloration of an adult B. altianalis ranges from golden yellow to green, while that of B. bynni ranges from silver to yellow-orange (Greenwood, 1966; Muwanika et al., 2012). The number of dorsal spines for B. altianalis are 3, dorsal soft rays are 9-11 and anal spines are 2-3, anal soft rays are 5-6 (De Vos & Thys van den Audenaerde, 1990). The dorsal spine for B. bynni is one, dorsal soft rays are 12-13 and the anal soft rays are 8 (Fishbase, 2017; Leveque, 2003; Muwanika et al., 2012). According to the competitive exclusion principle (Alley, 1982; Zaret & Rand, 1971), it would be expected that the two species occupy the same “niche” however; they may not co-exist due to competition for the same resources. Another reason could be that their response to physico-chemical water parameters is different. Despite the common origin of the species (Greenwood, 1981; Joyce et al., 2005), lakes in the Lake Victoria region can have different hydrological and limnological characteristics (Russell & Johnson, 2004) which could be why the two Barbus species are not found in the same water body. There is a need to better understanding of the ecology of both species in light of their close similarity. There is need for a better understanding of the ecology of both species in light of their close similarity. The observed differences in niche may point to the need for different conservation strategies or culture techniques. An investigation to ascertain the relationship between karyotypes of both species could be of great value if they possess superior characteristics that can be crossbred for hybrid performance under culture conditions.

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South Sudan

Figure 2.1: Map showing lakes and rivers where B. altianalis is distributed but excluding the lakes and rivers above the red line. The map was made using ArcGIS 10.2 soft ware.

The available genetic and phenotypic evidence does not confirm any cryptic process leading to evolution of different lineages for B. altianalis species in the Lake Victoria region (Nakamya, 2010; Muwanika et al., 2012). Worthington (1932) categorized B. altianalis by their location into subspecies that included B. altianalis altianalis Boulenger, 1900 (found in Lake Kivu and Rusizi River); B. altianalis eduardianus Boulenger, 1901 (from lakes Edward and George); and B. altianalis radcliffii Boulenger, 1903 (from lakes Victoria and Kyoga, and the Upper Victoria Nile). De Vos & Thys van den Audenaerde (1990) and Nakamya (2010) disagree with this classification, which was based on location and not on any species characteristics. Moreover, Banyankimbona, Vreven, Ntakimazi & Snoeks (2012) and Vreven et al., 2016 suggested classifying the species under the genus 18

Labeobarbus instead of Barbus. Before the development and conservation of broodstock for continuous replenishment of the cultured stocks, the existence of any strains, natural hybrids, subspecies or species across water bodies should be investigated. Until then, the species or “subspecies” are still regarded as one species under the genus Barbus.

Fish growth may vary due to varying environmental conditions across water bodies. For example, faster growth under culture conditions is reported for Oreochromis niloticus from Lake Victoria than from other lakes in Uganda (Mwanja et al., 2016). It is therefore necessary to compare growth rates of B. altianalis from various water bodies to ascertain its performance under culture conditions. However, in the early stages of domestication attention should be focused on its artificial reproduction (Bilio, 2007; Teletchea & Fontaine, 2014). The research by Rutaisire et al. (2015) was useful in inducing successful spawning in B. altianalis collected from the Upper Victoria Nile. Whereas the effect of seasonality on spawning can be expected to be the same within the same region for the same species, length at maturity and fecundity may vary across water bodies due to microclimatic differences and other factors. Such parameters must be defined specifically for each water body (Aruho et al., 2013). Some of the critical information, such as length at sexual maturity, was not provided in previous studies and is required to guide culturalists in collecting the appropriate size of fish for breeding.

Domestication of B. altianalis in culture systems requires determining appropriate conditions that will be suitable for spawning, egg incubation and larval growth. Barbus altianalis is found both in lacustrine and riverine environments. The adults are said to migrate upstream for breeding (Copley, 1958; Tomasson, Cambray & Jackson, 1985) and prefer breeding in clear gravel running water (Skelton et al., 1991). The juveniles are found moving in ‘schools’ in riverine waters (Greenwood, 1966; Witte & de-Winter, 1995). This may suggest that the eggs are laid in fresh slow running clean water with a constant oxygen supply during the breading season and that the juveniles require more oxygen for their energy requirements in the river environment. This also indicate that the incubation temperature required for the larvae and juveniles optimal growth and survival may be lower compared to that of the lacustrine environment since river water has a relatively lower temperature than the lake water (Wetzel, 2001). The oxygen demand should be met in subsequent culture systems by maintaining sufficient aeration during the incubation process. Most cyprinids, including Barbus 19

(Skelton et al., 1991), Labeobarbus (de-Graaf, Machiels, Wudneh & Sibbing, 2004) and victorianus (Rutaisire & Booth, 2004) are active in the running water during the breeding season. However, studies from Lake Tana on Labeobarbus by de-Graaf et al. (2004) have shown that some carps live and spawn in the lacustine environment. Most cyprinids may show a great propensity of divergence in relation to feeding behavior, breeding and habitat (Winfield & Nelson, 2012), and are capable of adapting to varying water environments. Whether B. altianalis could easily adapt to confined culture conditions and breed naturally in ponds or other culture systems requires further investigation. The association of Barbus species with clean water habitat (Skelton et al., 1991) may suggest a difference in habitat occupation strategy from the cultured common carp Cyprinus caprio. Contrary to common carp which survive well in a constantly turbid and silted environment (Basavaraju et al., 2002; Weber & Brown, 2009), B. altianalis may require a clear and un-disturbed water environment during its culture. Other factors that could influence spawning include the pH, the photoperiod (light) and water hardness (Bromage et al., 1993; Lam 1983; Lam & Munro, 1987; Harvey & Carolsfeld, 1993; Young et al., 1989). There is no information on how these particular aspects influence spawning of B. altianalis however, Harvey and Carolsfeld, (1993), and Lam, 1983 emphasized the need to manipulate these water quality parameters to obtain the optimum values because the response to environmental cues defers in species. These aspects therefore need to be assessed to determine their effect on spawning of B. altianalis.

Cyprinids have a diversity of feeding strategies and morphological adaptations depending on their habitat (Winfield & Nelson, 2012): some are carnivores, others are plankton feeders, suckers, insectivores, omnivores, plant feeders or detritivores (De Silva & Anderson, 1995; Rust, 2002). Even when they are omnivorous some may lean more toward carnivorous behavior and others plants (De Silva & Anderson, 1995). Field data suggests that B. altianalis has a generalist diet that include plants, fishes, , gastropod mollusks and (Corbet, 1961). Chironomids and ditritus material are added to the list (Balirwa, 1979). Witte & de Winter (1995) also included algae and weeds from Lake Victoria. The large spectrum of food items consumed by B. altianalis suggests that the fish feeds at all levels of the water column and utilizes most available food items in the environment, although this also depends on the habitat and the life stage of the fish. How the species will respond to feeding in a confined cultured environment needs to be investigated, especially when sinking or floating artificial diets will be used. 20

Morphology and histology of the gut system can provide additional information about the feeding behavior and adaptive strategy for B. altianalis. The digestive morphology of several species has been studied to relate structural functionality with adaptation to feeding habits (Cataldi, Cataudella, Monaco, Rossi & Tansion, 1987; Murray, Wright & Goff, 1996). Increased attention to such studies is primarily focused on the development of feeding technologies of candidate species for culture (Banan-Khojasteh, 2012). Structural variations probably confer differences in digestive capabilities. Different regions along the gut with varying specialized characteristics maximize different physiological processes to ensure uptake of nutrients (Buddington & Diamond, 1987; Dabrowisk & Celia, 2005). Better knowledge of digestive system of B. altianalis will provide more insights into its functionality and feeding behavior, which can form a basis for developing feeding strategies for this fish under culture.

2.4 Trends and socio-economic prospects of Barbus altianalis fishery

Fish were generally abundant in Ugandan water bodies at the turn of the 20th century. Declining stocks due to overfishing were first reported by Graham (1929), and by mid 20th century, Lakes Edward and George experienced drastic declines in all species that led to the collapse of the ‘The Uganda Fish Marketing Cooperation’-TUFMAC factory (Balirwa, 2007). From Lake Victoria, B. altianalis along with L. victorianus and other big catfishes constituted a substantial part of the catch in a fishery that was dominated by Oreochromis esculentus before the 1950s (Balirwa et al., 2003; Cadwalladr 1965; Ogutu-ohwayo, 1990). The introduction of the carnivorous Nile perch (Lates niloticus) in the late 1950s led to reduction of the stocks of haplochromines but also the other large fish species, including B. altianalis (Ogutu-ohwayo, 1990). Catches of B. altianalis from the Ugandan part of Lake Victoria declined from 859 tonnes in 1966 to less than 1 tonne in 2000 (Balirwa et al., 2003). Although contributing only 1.5% to the total catch reported in 1957 (LVFS, 1958), B. altianalis was and is still greatly cherished by many communities in Uganda for its cultural and taste attributes (FAO, 1991; ARDC Kajjansi, 2016). Currently it is difficult to find any B. altianailis species in fish catches from Lake Victoria (Fishermen, personal communication, August, 15, 2016; Ondhoro et al., 2016). The remaining few stocks are confined to Upper Victoria Nile, the river Kagera and other tributaries of Lake Victoria. The continued destruction and disturbances of

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their habitat due to unregulated human activities, including the construction of dams along the Nile River, are likely to further reduce the remaining stocks and can eventually lead to their extinction (Ondhoro et al., 2016). It is therefore vital to initiate strategies that not only involve precautionary principles of management but also domestication.

Catch statistic trends in other parts of the country are poorly documented just like it is for other fish species (Cowx, van der Knaap, Muhoozi & Othina, 2003; DFR, 2012). Apparently most catches are reported from Lake Edward (Kasese district fisheries department report, 2012; Ondhoro et al., 2016). The mean catch size of B. altianalis from Lake Edward is 2.24kg and fetches the highest price of 12,000 Uganda shillings per kilo (US$3.8) compared to other fish species that are much smaller on average (Traders, personal communication, July 10, 2016; Kasese district fisheries department report, 2012). Catches are seasonal and fishermen tend to catch them at night during the “dark nights” (no moon light) and during the rainy season when the moon is obscured by clouds (Fishermen, personal communication, July 10, 2016; Ondhoro et al., 2016). After this period, the fishers resort to catching other species. This seasonal fishing further escalates its demand and leads to higher prices. Records of B. altianalis catches from Upper Victoria Nile and many other inland streams are scanty. Informal estimates for fish sold at the Owen falls Dam fish market indicate that on average 70kg of B. altianalis are sold daily (Un published data from Jinja & Kayunga District fisheries Department, 2012). This translates into 2.5 t, worth of US$73,000. However, catches are low and fishing is sometimes interrupted by the electricity generation activities from Owen falls, Kiira and Bujjagali Dams along the River Nile (Ondhoro et al., 2016).

Barbus altianalis is not only cherished by communities in Uganda but also by populations in parts of Western DRC Congo and Eastern Kenya. In Uganda, the fish is a delicacy largely in the south west, central and eastern regions. In southwestern Uganda, demand is much larger and a large part of the catch is exported to DRC (unpublished data, ARDC Kajjansi, 2016). Its demand in the south western region is linked to cultural attachments across the Banyabutumbi, Bakonjo, Bamba and other tribes which form a fairly large and potential consuming market for B. altianalis along with other fish species (Nabwiiso, 2015; Odongkara, Kyangwa, Akumu, Wegoye & Kyangwa, 2005, Odongkara, Abila & Luomba, 2009). These communities believe that the fish has got nutritive value that increases milk to breast feeding mothers and offers incredible sexual libido to men (unpublished 22

report, ARDC Kajjansi, 2016). Ninety percent of respondents in central and eastern Uganda preferred the fish in smoked form while 90% of respondents from the western region (including DRC) preferred the fish fresh (unpublished ARDC report, 2016). The remaining 10% preferred the fish when it is salted. One of the key preparation advantages is that its scales are smooth and soft hence it’s not necessary to removed them when preparing the fish and can therefore be consumed directly. These scales may also contain useful nutrients for human consumption yet to be investigated.

2.5 Progress in Culture of Barbus altianalis and other indigenous cyprinids

There are several indigenous cyprinid species in Uganda and in the region that are potentially viable for commercial production. These include B. altianalis, B. byanii, Labeo horie, L. victorianus and Labeo coubie. Their domestication and development of technologies is a vital strategy to ensure their production and productivity through culture and conservation. A number of technologies for their domestication are under way. Artificial reproduction and feeding have been well established in L. victorianus (Owori, 2009; Rutaisire & Booth, 2004). This fish is now being popularized commercially in the country. The technology for artificial reproduction of B. altianalis has successfully been developed (Rutaisire et al., 2015) but production of seed for commercialization is yet to be realized. High embryonic and larval mortalities of up to 55% and 11%, respectively were recorded (Rutaisire et al., 2015). This could also be due to lack of feeding technologies, unfavorable environmental conditions for artificial spawning (or incomplete reproductive cycle) and diseases, challenges commonly associated with domestication process of new fish species (Bilio, 2007; Lorenzen et al., 2012; Migaud et al., 2013).

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2.5.1 Artificial spawning and factors for optimal egg incubation and larval growth

The supply of gamates all year round is made possible through induced spawning. This technique requires understanding of reproductive biology of the species to be able to manipulate and sustain continuous production of seed in captivity (Aruho et al., 2013; Lorenzen et al., 2012; Mylonas et al., 2010). Gravid individuals are initially collected from the wild and induced with hormones. There are various hormones that include both synthetic and natural hormones that are injected to induce spawning in brood fish. Manipulation of the reproductive systems to induce maturation and ovulation in fish species using inducing hormones (inducing agent) has widely been adopted in commercial aquaculture to facilitate continuous supply of sufficient seed required on regular basis by the farmers (Bromage, 1995; Crim, Evans & Vickery, 1983; Mylonas et al., 2010). Manipulation of reproductive system entails use of appropriate hormones that will facilitate egg ripeness. The response to the inducing agent by various cultured species is variable (Marte, 1989) and has been determined in various species to ensure final maturation and ovulation of good quality eggs prior to stripping (Mylonas, Hinshaw & Sullivan, 1992; Mylonas et al., 2010). The number of eggs produced (working fecundity), fertilization rates, hatchability of eggs (survival), quality of eggs, the hormonal dosages and the period of incubation at varying temperatures are a reflection or a measure of an effective hormonal agent (Mylonas et al., 2010). Thus the high embryonic and larval mortality observed during the induced spawning of B. altianalis (Rutaisire et. al., 2015) could partly be attributed to lack of knowledge of appropriate hormones and hence requiring urgent attention to improve the artificial spawning technique.

The refinement of artificial spawning technique is associated with the optimal conditions for incubating the eggs or embryos and growth of larvae. Temperature is one of the most critical and widely acceptable environmental factors that affect incubation of embryos and larvae development in fishes (Bjornsson, Steinarsson & Oddgeirsson, 2001; Haylor & Mollah, 1995; Kokurewicz, 1970; Kucharczyk, Luczynskim, Kujawa & Czerkies, 1997). Water temperature in the tropics is generally high with tolerant ranges by various species (Lowe-McConnell, 1987). But this may also depend on specific water body and the ability of the species to temperature tolerance (Wetzel, 2001). The optimal temperature variation in which species are able to breed may be species-specific (Delince, 1987; Herzig & Winkler, 1986; Laurel & Blood, 2011). Both high and low temperature could impair

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the physiological process during embryonic and larval development leading to high mortalities even in their zone of tolerance (Blaxter, 1991; El-Gamal, 2009; Hokanson, 1977). The egg and larvae incubation temperature must be simulated in the culture of the species to be able to facilitate successful hatching and survival of larvae. An observation has been made during artificial spawning in B. altianalis in which some critical stages of embryonic development seem to have been affected more than others even when the temperature was constantly maintained at the same temperature of incubation (observation by authors). Generally egg hatchability and larvae survival is not only affected by temperature alone but also a multiplicity of important environmental factors including dissolved oxygen, salinity, light regimes, ammonia levels and hatching facilities among others (Brooks et al., 1997; Downing & Litvak, 2001; Kamler, 2008; Villamizar et al., 2011). For instance, the dissolved oxygen fluctuation was reportedly lower between 3-4mgL-1 from Lake Edward and the Upper Victoria Nile (Ondhoro et al., 2016) compared to the minimum of 5gmL-1 that is generally suitable for culture of many species in confined culture systems (Boyd, 1998, Ondhoro et al., 2016). It is not clear how the oxygen requirements would affect the B. altianalis in captivity. Some species evolve to cope with changes in environmental factors under culture environment (Lorenzen et al., 2012) but these changes must be clearly understood because tolerance to such environmental factors differs for various species. It is imperative that these parameters are optimally established to ensure increased survival of the embryos and larvae in B. altianalis under captivity.

2.5.2 Feeding in Barbus altianalis

Feeds are the most important contributors to fish growth in culture systems constituting between 40% -70% of the total production costs in culture systems (Craig & Helfrich, 2002; Diana, Lin & Jaiyen, 1994; Erondu, Bekibela & Gbulubo, 2006; Naylor et al., 2000). The nutritional fish requirements may vary with the stage of development and the fish species as well (De Silva & Anderson, 1995). Inappropriate feeds will affect the growth and survival of the fish at every developmental stage. In young fish the influence of feed is much more critical and greatly influences their survival. The optimal feed and the period for weaning are critical factors for improving the larval survival and growth for mass seed production (Cahu & Zambonino, 2001; Mai et al., 2005). The introduction of a weaning feed will require knowledge of the ontogenetic digestive system. This will help reveal the right period when the fish can be able to assimilate the processed feed. The assessment of fish

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development has been studied in many cultured fishes to determine the period when the digestive system is ready to digest and assimilate the artificial feeds (Cahu & Zambonino, 2001; Sarasquete, Polo & Yufera, 1995; Segner et al., 1993; Zambonino-Ifante et al., 2008). The mortalities observed during the growth of B. altianalis larvae were suggestively linked to inappropriate feeds and timing (Rutaisire et al., 2015). The introduction of starter feeds, both in terms of feed type and time of introduction, is species-specific for most cyprinid larvae (Kujawa et al., 2010). In some fish species including Ide, Leuciscus idus a cyprinid, at first feeding the digestive system is well advanced with functional enzymes which may allow digestion of exogenous feed particles (Ostaszewska, Wegner, & Wegiel, 2003; Riubeiro et al., 1999). In other fish species including the Cyprinus carpio the digestive system may not be properly developed to digest introduced food particles at first feeding (Garcia et al., 2001; Sarasquete et al., 1995). The weaning diet of B. altianalis larvae that will provide optimum survival and growth under culture conditions still need to be clearly understood. It is equally imperative to establish the growth rates and survival of juveniles under culture system. The juveniles of B. altianalis are largely confined in riverine environment while the adult fish can freely move and survive in both riverine and lacustrine environments (Ondhoro et al., 2016). This may suggest varying strategies including feed response and preference for various age groups and may also adapt differently to available food in confined culture conditions. B. altianalis is said to be an omnivorous species (Balirwa, 1979; Corbet, 1961) hence its response to feeds in culture environment may be influenced by the presence of what is available as natural food and or other fish species especially in polyculture systems. For instance the silver carps, and the big head are good planktonic filters and in absence of artificial feed they will effectively utilize the plankton to grow and survive under well-fertilized ponds when cultured with non filter fish species (Winfield & Nelson, 2012).Therefore successful domestication of B. altianalis necessitates knowledge of its feeding dynamics in confined environments.

2.6 Conclusions

Adapting a new fish species into captive environment is a process largely characterized by a multiplicity of factors including appropriate water quality parameters for spawning, egg incubation and growth performance. These factors are specific to different cultured species. Evolution of domestication technologies for various high-value indigenous species is preferred and encouraged in

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many countries not only to provide alternative choices of local farmed fish for local consumers but also to preserve the integrity of the wild fish sources being overexploited. B. altianalis is one of the high-value cyprinid species that has been successfully induced to spawn but it is yet to be commercially propagated because of lack of seed and slow growth. The current review suggests that B. altianalis has a great potential for culture because being omnivorous there are high chances of adapting to varying feeding strategies especially when it is polycultured with other species. Solving the problem of high egg, larval and juvenile mortalities as well as slow growth requires understanding the underlying causes. These causes suggest knowledge gap in some reproductive aspects such as length at maturity, optimal hormone and or hormonal dosages, appropriate conditions for spawning, egg incubation and growth of larvae and juveniles. Knowledge of feeding dynamics in the captive environment and the digestive structure is a prerequisite to determining successful weaning and feeding regimes of B. altianalis. Although, artificial spawning was successful with wild collected specimens, complete domestication and sustainability of seed supply of B. altianalis must focus on completion and closing of the reproductive cycle of the species in captivity. Thus further research is required in the mentioned areas to ensure advancement in levels of domestication so as to meet the rising demand of B. altianalis and reduce its overexploitation in the wild and consequently improving the social economic lives of the communities in the region.

2.7 Acknowledgment

We thank the National Fisheries Resources Research Institute (NaFIRRI) for their financial support to facilitate collection of literature documents and information from fishermen at landing sites. We are grateful to Dr. Mathew Mwanja, Dr. Dismas Mbabazi, Dr. Bwanika, Prof. Fred Bugenyi and Dr. John Balirwa for their input and editing services to improve this work. We thank the NaFIRRI library attendants for providing the documents from which this review was made. We are also grateful to various individuals including fishers and some opinion leaders around the landing sites that provided some relevant information for this review.

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2.8 References

Aquaculture Research and Development Centre (ARDC) report. (2016). The market potential and assessment of B. altianalis in Uganda. Aquaculture Research and Development Centre, Kajjansi Kampala.

Aguilar-Manjarrez, J., & Nath, S. S. (1998). A strategic reassessment of fish farming potential in Africa. CIFA Technical Paper 32, Rome, Italy: FAO.

Alley, T. R. (1982). Competition theory, evolution, and the concept of an ecological niche. Acta Biotheoretica, 31(3), 165-179.

Aruho, C., Basiita, R. K., Kahwa, D., Bwanika, G., & Rutaisire, J. (2013). Reproductive biology of Bagrus docmak in the Victoria Nile, Uganda. African Journal of Aquatic Science, 38(3), 263- 271.

Balirwa, J. B. (1979). A contribution to the study of the food of six cyprinid fishes in three areas of the Lake: Victoria basin, East Africa, Hydrobiologia, 66, 65-72.

Balirwa, J. S. (2007). Ecological, environmental and socioeconomic aspects of the Lake Victoria's introduced Nile perch fishery in relation to the native fisheries and the species culture potential: lessons to learn. African Journal of Ecology, 45(2), 120-129.

Balirwa, J. S., Chapman, C. A., Chapman, L. J., Cowx, I. G., Geheb, K., Kaufman, L., ... & Witte, F. (2003). Biodiversity and fishery sustainability in the Lake Victoria basin: an unexpected marriage?. BioScience, 53(8), 703-716.

Balon, E. K. (1995). Origin and domestication of the wild carp, Cyprinus carpio: from Roman gourmets to the swimming flowers. Aquaculture, 129(1-4), 3-48.

Banan-Khojasteh, S. M. (2012). The morphology of the post-gastric alimentary canal in teleost fishes: a brief review. International Journal of Aquatic Science, 3(2), 71-88.

Banyankimbona, G., Vreven, E., Ntakimazi, G., & Snoeks, J. (2012). The riverine fishes of Burundi (East Central Africa): an annotated checklist. Ichthyological Exploration of Freshwaters, 23(3), 273.

28

Basavaraju, Y., Mair, G. C., Kumar, H. M., Kumar, S. P., Keshavappa, G. Y., & Penman, D. J. (2002). An evaluation of triploidy as a potential solution to the problem of precocious sexual maturation in common carp, Cyprinus carpio, in Karnataka, India. Aquaculture, 204(3), 407- 418.

Basiita, R. K., Aruho, C., Kahwa, D., Nyatia, E., Bugenyi, F. W., & Rutaisire, J. (2011). Differentiated gonochorism in Nile perch Lates niloticus from Lake Victoria, Uganda. African Journal of Aquatic Science, 36(1), 89-96.

Bene, C., Barange, M., Subasinghe, R., Pinstrup-Andersen, P., Merino, G., Hemre, G. I., & Williams, M. (2015). Feeding 9 billion by 2050–Putting fish back on the menu. Food Security, 7(2), 261-274. Retrieved from http://link.springer.com/article/10.1007/s12571-015-0427-z

Bilio, M. (2007) Controlled reproduction and domestication in aquaculture – the current state of the art, Part I. Aquaculture Europe, 32, 5–14.

Billard, R., & Berni, P. (2004). Trends in cyprinid polyculture. Cybium, 28(3), 255-261.

Bjornsson, B., Steinarsson, A., & Oddgeirsson, M. (2001). Optimal temperature for growth and feed conversion of immature cod (Gadus morhua L.). ICES Journal of marine Sciences, 58, 29– 38.

Blaxter, J. H. S. (1991). The effect of temperature on larval fishes. Netherlands Journal of Zoology, 42, 336-357.

Boyd, C. E. (1998).Water quality for pond aquaculture. Research and Development. Series No.43. International centre for aquaculture and aquatic environments. Alabama Agriculture Experiment Station, Auburn University, Auburn.

Britton, J. R., Boar, R. R., Grey, J., Foster, J., Lugonzo, J., & Harper, D. M. (2007). From introduction to fishery dominance: the initial impacts of the invasive carp Cyprinus carpio in Lake Naivasha, Kenya, 1999 to 2006. Journal of Fish Biology, 71, 239-257.

Bromage, N., Randall, C., Davies, B., Thrush, M., Duston, J., Carillo, M. and Zanuy, S. (1993). In B. Lahlou & P. Vitiell (Eds.), Photoperiodism and the control of reproduction and development in farmed fish. Aquaculture: Fundamental and Applied Research. (Volume Coastal and Estuarine Studies 43. pp. 81–102). Washington: American Geophysical Union.

29

Bromage, N. (1995). Broodstock management and seed quality—general considerations. In Roberts R. J. & N. Bromage (Eds), Broodstock Management and Egg and Larval Quality (pp. 1–24.). Oxford, Blackwell Science.

Bromage, N., Randall, C., Davies, B., Thrush, M., Duston, J., Carillo, M. and Zanuy, S. (1993). In B. Lahlou & P. Vitiell (Eds.), Photoperiodism and the control of reproduction and development in farmed fish. Aquaculture: Fundamental and Applied Research (Volume Coastal and Estuarine Studies 43. pp. 81–102). Washington: American Geophysical Union.

Brooks Jr, G. B. (1994). A simplified method for the controlled production and artificial incubation of Oreochromis eggs and fry. The Progressive Fish-Culturist, 56(1), 58-59.

Brooks, S., Tyler, C. R., & Sumpter, J. P. (1997). Egg quality in fish: what makes a good egg? Reviews in Fish Biology and Fisheries, 7(4), 387-416.

Brummett, R. E., Lazard, J., & Moehl, J. (2008). African aquaculture: realizing the potential. Food policy, 33(5), 371-385.

Buddington, R. K., Chen J. W., & Diamond, J., (1987). Genetic and phenotypic adaptation of intestinal nutrient transport to diet in fish. Journal of Physiology, 393, 261–281.

Cadwalladr, D. A. (1965). The decline in the Labeo victorianus Blgr.(Pisces: Cyprinidae) fishery of Lake Victoria and an associated deterioration in some indigenous fishing methods in the Nzoia River, Kenya. East African Agricultural and Forestry Journal, 30(3), 249-256.

Cahu, C., Zambonino Infante, J. (2001). Substitution of live food by formulated diets in marine fish larvae. Aquaculture, 200, 161–180.

Cataldi, E., Cataudella, S., Monaco, G., Rossi, A., & Tansion, L. (1987). A study of the histology and morphology of the digestive tract of the sea-bream Sparus auratus. Journal of Fish Biology, 30,135-45.

Chemoiwa, E. J., Abila, R., Macdonald, A., Lamb, J., Njenga, E., & Barasa, J. E. (2013). Genetic diversity and population structure of the endangered Ripon barbel, Barbus altianalis (Boulenger, 1900) in Lake Victoria catchment, Kenya based on mitochondrial DNA sequences. Journal of Applied Ichthyology, 29(6), 1225-1233. 30

Chiu, A., Li, L., Guo, S., Bai, J., Fedor, C., & Naylor, R. L. (2013). Feed and fishmeal use in the production of carp and tilapia in China. Aquaculture, 414, 127-134.

Copley, H. (1958). Common Freshwater fishes of East Africa (pp. 172). London: H. F & G. Witherby Ltd.

Corbet, P.S. (1961). The food of non-cichlid fishes in the Lake Victoria basin, with remarks on their evolution and adaptation to lacustrine conditions. Proceedings of the Zoological Society of London, 136: 1-101).

Cowx, I. G., van der Knaap, M., Muhoozi, L. I., & Othina, A. (2003). Improving fishery catch statistics for Lake Victoria. Aquatic Ecosystem Health & Management, 6(3), 299-310.

Craig, S., & Helfrich, L. A. (2002). Understanding Fish Nutrition, Feeds and Feeding (Techenical paper 420-456). Virginia, Department of Fisheries and Wild Life Sciences: Virginia Tech.

Crim, L. W., Evans, D. M., & Vickery, B. H. (1983). Manipulation of the seasonal reproductive cycle of the landlocked Atlantic salmon (Salmo salar) by LHRH analogues administered at various stages of gonadal development. Canadian Journal of Aquatic Sciences, 40, 61-67.

Dabrowisk, K., & Celia. M. P. (2005). Feeding Plasticity and Nutritional Physiology. In: W. S. Hoar, J. D. Randall & A. P. Farrell (Eds.), Fish Physiology: The Physiology of Tropical Fishes (21: 155-224). Elsevier, Academic press.

De Silva, S. S., & Anderson, T. A. (1995). Fish Nutrition in Aquaculture (pp. 319). London, Chapman and Hall.

De Silva, S. S. D., Nguyen, T. T., Turchini, G. M., Amarasinghe, U. S., & Abery, N. W. (2009). Alien species in aquaculture and biodiversity: a paradox in food production. Ambio: a journal of the human environment, 38(1), 24-28.

De Vos, L. & Thys van den Audenaerde, D .F. E. (1990). Petits Barbus (Pisces, Cyprinidae) du Rwanda. Rev. Hydrobio. Trop., 23: 141-159. de Graaf, M., Machiels, M. A., Wudneh, T., & Sibbing, F. A. (2004). Declining stocks of Lake Tana’s endemic Barbus species flock (Pisces, Cyprinidae): natural variation or human impact? Biological conservation, 116(2), 277-287.

31

DeGrandchamp, K. L., Garvey, J. E., & Colombo, R. E. (2008). Movement and habitat selection by invasive Asian carps in a large river. Transactions of the American Fisheries Society, 137(1), 45-56.

Delince, G. A. (1987). Seed production - an overview. In. G. A. Delince, D. Campbell, J. A. L. Janssen & M. N. Kutty (Eds.), African Regional Aquaculture Centre Port Harcourt, Nigeria, FAO. Retrieved October, 5th 2016 from ttp://www.fao.org/docrep/field/003/AC182E/AC182E01.htm#ch1. Department of Fisheries Resources (DFR). (2012). Annual Report 2010/2011 (pp. 1- 41). Entebbe, Uganda: Ministry of Agriculture Animal Industry and Fisheries (MAAIF), Retrieved from http://www.agriculture.go.ug/userfiles/DFR%20ANNUAL%20REPORT%202012.pdf

Dey, M. M., Paraguas, F. J., Bhatta, R., Alam, F., Weimin, M., Piumsombun, S., ... & Sang, N. V. (2005). In D. J. Penman, M. V. Gupta & M. M. Dey (Eds.), Carp production in Asia: past trends and present status in Asia. Carp genetic resources for aquaculture in Asia (pp. 6-15). Penang, Malaysia.

Diana, J. S., Egna, H. S., Chopin, T., Peterson, M. S., Cao, L., Pomeroy, R., & Cabello, F. (2013). Responsible aquaculture in 2050: valuing local conditions and human innovations will be key to success. BioScience, 63(4), 255-262.

Diana, J. S., Lin, C. K., & K. Jaiyen. (1994). Supplemental feeding of Tilapia in fertilized ponds. Journal of World Aquaculture Society, 25, 497-506.

Downing, G., & Litvak, M. K. (2001). The effect of light intensity and spectrum on the incidence of first feeding by larval haddock. Journal of Fish Biology, 59(6), 1566-1578.

El-Gamal, E. (2009). Effect of Temperature on Hatching and Larval Development and Mucin Secretion in Common Carp, Cyprinus carpio (Linnaeus, 1758). Global Veterinaria, 3 (2), 80- 90.

Erondu, E. S., Bekibela, D., & Gbulubo, A.T. (2006). Optimum crude protein requirement of catfish, Chrysichthys nigrodigitatus. Journal of Fisheries International, 1(1-2), 40-43.

Fishbase, (2017). Labeobarbus bynni. Retrieved from http://www.fishbase.org/summary/11320\

32

Food and Agricultural Organisation (FAO). (2018). The State of World Fisheries and Aquaculture. Meeting the sustainable development Goals (pp.1-200). Rome, Italy: Author.

Food Agriculture Organisation (FAO). (2014). The state of world fisheries and aquaculture. Opportunities and challenges (pp. 1-223). Rome, Italy: Author.

Food and Agricultural Organisation (FAO). (2016). The State of World Fisheries and Aquaculture. Contributing to food security and nutrition for all (pp.1-200). Rome Italy, Author.

Food and Agriculture Organization (FAO). (2012). The state of world fisheries and aquaculture. World review of fisheries and aquaculture (pp. 1-202). Rome, Italy: Author.

Food and Agriculture Organization (FAO). (1991). Marketing and consumption of fish in Uganda. Retrieved from http://www.fao.org/docrep/006/AD146E/AD146E01.htm

Funge-Smith, S., Phillips, M. J., 2001. Aquaculture systems and species. In R. P. Subasinghe, P. Bueno, M .J. Phillips, C. Hough, S. E. McGladdery & J. R. Arthur (Eds.), Technical Proceedings of the Conference on Aquaculture in the Third Millennium, (pp. 129–135). February 20–25, 2000., Bangkok/ Rome, NACA/FAO.

Garcia, H. M. P., Lozano, M. T., Elbal, M., & Agulleiro, B. (2001). Development of the digestive tract of sea bass Dicentrarchus labrax (L.). Light and electron microscopic studies. Anatomy of Embryology, 204, 39-57.

Graham, M. (1929). The Victoria Nyanza, and its Fisheries. A Report on the Fishing Survey of Lake Victoria 1927–1928, and Appendices (pp. 1-255). London, Crown Agents for the Colonies.

Greenwood, P. H. (1966). The fishes of Uganda (2nd ed.). Kampala, Uganda: Kampala Uganda Society.

Greenwood, P. H. (1981). The haplochromine fishes of East African lakes. Collected papers on their , biology and evolution (pp. 839). Munich, Germany: Kraus International publications.

Harvey, B., & Carolsfeld. J. (1993). Induced breeding in tropical fish culture (pp.144). Ottawa, Canada: International Development Research Centre.

33

Haylor, G. S., & Mollah, M. F. A. (1995). Controlled hatchery production of African catfish, Clarius gariepinus: the influence of temperature on early development. Aquatic Living Resources, 8, 431-438.

Hecht, T. (2007). Review of feeds and fertilizers for sustainable aquaculture development in sub- Saharan Africa. In M. R. Hasan, T. Hecht, S. S. De Silva, & A. G. J. Tacon (Eds.), Study and analysis of feeds and fertilizers for sustainable aquaculture development (pp. 77-109). Rome, Italy: FAO.

Herzig, A., & Winkler, H. (1986). The influence of temperature on the embryonic development of three cyprinid fishes, Abramis brama, Chalcalburnus chalcoides mento and Vimba vimba. Journal of Fish Biology, 28(2), 171-181.

Hokanson, K. E. (1977). Temperature requirements of some percids and adaptations to the seasonal temperature cycle. Journal of the Fisheries Board of Canada, 34(10), 1524-1550.

Hulata, G. (1995). A review of genetic improvement of the common carp (Cyprinus carpio L.) and other cyprinids by crossbreeding, hybridization and selection. Aquaculture, 129(1-4), 143- 155.

Jinja fisheries department, (2012). Fish catch statistics data (raw data). Jinja District

Joyce, D. A., Lunt, D. H., Bills, R., Turner, G. F., Katongo, C., Duftner, N., ... & Seehausen, O. (2005). An extant cichlid fish radiation emerged in an extinct Pleistocene lake. Nature, 435(7038), 90-95.

Kamler, E. (2008). Resource allocation in yolk-feeding fish. Reviews in Fish Biology and Fisheries, 18(2), 143.

Kasese District Fisheries Department Report (2012). Lake George and Edward fisheries statistics (District report, pp 10). Kasese District: Belgian Technical support department.

Kokurewicz, R. (1970). The effect of the temperature on embryonic development of Tinka tinka (L) and Rutilus rutilus (L). Zoology Polish, 20 (3), 317-337.

Kucharczyk, D., Luczynskim, M., Kujawa, R., & Czerkies, P. (1997). Effect of temperature on embryonic and larval development of bream (Abramis brama L). Aquatic Science, 59, 214- 224. 34

Kujawa, R., Kucharczky, D., Mamcarz, A., Jamroze, M., Kwiat, M., Katarzyna, T., & Zarski, D. K. (2010). Impact of supplementing natural feed with dry diets on the growth and survival of larval asp Aspius aspius (L) and nase, Chondrostoma nasus (L). Archives of Polish Fisheries, 18, 13-23.

Lake Victoria Fisheries Service (LVFS). (1958). Annual Report (1957/1958). Jinja, Uganda). Author.

Lam, T. J. (1983). 2 Environmental Influences on Gonadal Activity in Fish. Fish physiology, 9, 65- 116.

Lam, T. J., & Munro, A. D. (1987). In R. D. Ideler, W. L. Crim, J. M. Walsh (Eds.), Environmental control of reproduction in teleosts: An overview. Reproductive Physiology of Fish (pp. 279- 288).

Laurel, B. J., & Blood, D. M. (2011). The effects of temperature on hatching and survival of northern rock sole larvae (Lepidopsetta polyxystra). Fishery Bulletin, 109 (3), 282-291.

Leveque, C. (2003). Cyprinidae. In D. Paugy, C. Leveque and G.G Teugels (eds.), the fresh and brackish water fishes of (Volume 1 p. 322-436). Paris, IRD Editions.

Lorenzen, K., Beveridge, M., & Mangel, M. (2012). Cultured fish: integrative biology and management of domestication and interactions with wild fish. Biological Reviews, 87(3), 639- 660.

Machena, C., & Moehl, J. (2001). African aquaculture: a regional summary with emphasis on Sub- Saharan Africa. In R. P. Subasinghe, P. Bueno, M. J. Phillips, C. Hough, S. E. McGladdery, J. E Arthur (Eds.), Aquaculture in the third millennium. Technical proceedings of the conference on aquaculture in the third millennium, (pp 341–355). 20–25 February 2000. Bangkok/Rome, NACA/FAO.

Mai, K.,Yu, H., Ma, H., Duan, Q., Gisbert, E., Zambonino-Infante, J. L., & Cahu, C. (2005). A histological study of the development of the digestive system of Pseudosciaena crocea larvae and juveniles. Journal of Fish Biology, 67 (4), 1094-1106.

Marte, C. L. (1989). Hormonal –induced spawning of cultured tropical fin fishes. Advances in Tropical Aquaculture, 9, 519-539.

35

Lowe-McConnell, R. H. (1987). Ecological studies in tropical fish communities (pp. 382). Cambridge, UK: Cambridge University Press.

McGuire, S. (2011). US Department of Agriculture and US Department of Health and Human Services, Dietary Guidelines for Americans, 2010. Washington, DC: US Government Printing Office, January 2011. Advances in Nutrition: An International Review Journal, 2(3), 293-294.

Merino, G., Barange, M., Blanchard, J. L., Harle, J., Holmes, R., Allen, I., & Jennings, S. (2012). Can marine fisheries and aquaculture meet fish demand from a growing human population in a changing climate? Global Environmental Change, 22(4), 795-806.

Migaud, H., Bell, G., Cabrita, E., McAndrew, B., Davie, A., Bobe, J., ... & Carrillo, M. (2013). Gamete quality and broodstock management in temperate fish. Reviews in Aquaculture, 5(s1), S194-S223.

Moreau, J., & Costa‐Pierce, B. (1997). Introduction and present status of exotic carp in Africa. Aquaculture Research, 28(9), 717-732.

Murray, H. M., Wright. G. M., & Goff. G. P. (1996). A comparative histological and histochemical study of the post-gastric alimentary canal from three species of pleuronectid, the Atlantic halibut, the yellowtail flounder and the winter flounder. Journal of Fish Biology, 48: 187- 468 206. Doi: 10.1111/j.1095-8649.1996.tb01112.x

Muwanika, V. B., Nakamya, M. F., Rutaisire, J., Sivan, B., & Masembe, C. (2012). Low genetic differentiation among morphologically distinct Labeobarbus species (Teleostei: Cyprinidae) in the Lake Victoria and Albertine basins, Uganda: insights from mitochondrial DNA. African Journal of Aquatic Science, 37(2), 143-153.

Mylonas, C. C., Fostier, A., & Zanuy, S. (2010). Broodstock management and hormonal manipulations of fish reproduction. General and Comparative Endocrinology, 165(3), 516- 534.

Mylonas, C.C., Hinshaw, J. M., & Sullivan, C. V. (1992). GnRHa-induced ovulation of brown trout (Salmo trutta) and its effects on egg quality. Aquaculture, 106, 379–392.

36

Mwanja, T.M., Kityo, G., Achieng, P., Kasozi, J.M., Sserwada., M.,& Namulawa, V. (2016). Growth performance evaluation of four wild strains and one current farmed strain of Nile tilapia in Uganda. International Journal of Fisheries and Aquatic Studies, 4(3), 594-598.

Nabwiiso, S. (2015, March 01). East African business week, Uganda fishers get tipped on markets. Retrieved from, Posted March 01st, 2015. Accessed on 20th February 2017 on http://www.busiweek.com/index1.php?Ctp=2&pI=2859&pLv=3&srI=68&spI=107

Nakamya, M. F. (2010). The population genetic structure and evolutionary relationships of two Barbus species (Pisces: Cyprinidae) in the Lake Victoria region (Msc dissertation, Makerere University) 86pp.

Naylor, R. L., Goldburg, R. J., Primavera, J. H., Kautsky, N., Beveridge, M. C., Clay, J., ... & Troell, M. (2000). Effect of aquaculture on world fish supplies. Nature, 405(6790), 1017-1024.

Naylor, R., & Burke, M. (2005). Aquaculture and ocean resources: raising tigers of the sea. Annual Review of Environment and Resources, 30, 185-218. doi: 10.1146/annurev.energy.30.081804.121034

Odongkara K, M. Kyangwa, J. Akumu, J. Wegoye & Kyangwa, I. (2005). Survey of the regional fish trade. LVEMP Socio-economic Research Report 7 (pp.38). Jinja, Uganda. National Fisheries Resources Research Institute, Jinja, Uganda.

Odongkara, O. K., Abila, R. O., & Luomba, J. (2009). The contribution of Lake Victoria fisheries to national economies. African Journal of Tropical Hydrobiology and Fisheries, 12(1).

Ogutu-Ohwayo, R. (1990). The decline of the native fishes of Lakes Victoria and Kyoga (East Africa) and the impact of the introduced species, especially the Nile perch, Lates niloticus and the Nile tilapia, Oreochromis niloticus. Environmental Biology of Fishes, 27, 81–96.

Ondhoro, C. C., Masembe, C., Maes, G. E., Nkalubo, N. W., Walakira, J. K., Naluwairo, J & Efitre, J. (2016). Condition factor, Length–Weight relationship, and the fishery of Barbus altianalis (Boulenger 1900) in Lakes Victoria and Edward basins of Uganda. Environmental Biology of Fishes, 1-12100 (2), 99-110. doi:10.1007/s10641-016-0540-7.

Ostaszewska, T., Wegner, A., & Wegiel, M. (2003). Development of the digestive tract of Ide, Leuciscus idus (L) during the larvae stage. Archives of Polish Fisheries, 11, 79-92.

37

Owori, W. A. (2009). The feeding ecology, ontogeny and larval feeding in Labao victorianus Boulenger 1901 (Pisces: Cyprinidae). Unpublished Doctoral thesis, Makerere University, Uganda. Retrieved from http://hdl.handle.net/10570/2635

Reames, E. (2012). Nutritional benefits of seafood. Southern Regional Aquaculture Center (Publication No. 7300) (pp.1-6). Retrieved from http://2kjj1d3odhc3296co7jhe511.wpengine.netdna-cdn.com/files/2013/09/SRAC- Publication-No.-7300-Nutritional-Benefits-of-Seafood.pdf

Reddy, P. V., Gjerde, B., Tripathi, S. D., Jana, R. K., Mahapatra, K. D., Gupta, S. D., ... & Rye, M. (2002). Growth and survival of six stocks of rohu (Labeo rohita, Hamilton) in mono and polyculture production systems. Aquaculture, 203(3), 239-250.

Rice, J. C., & Garcia, S. M. (2011). Fisheries, food security, climate change, and biodiversity: characteristics of the sector and perspective on emerging issues. ICES Journal of Marine Science, 68(6), 1343–1353.

Riubeiro. L., Zambonino-Infante, J. L., Cahu, C., & Dinis, M. T. (1999). Development of digestive enzyme in Larvae of Solea senegalensis, Kaup 1858. Aquaculture, 179, 465-473.

Russell, J. M., & Johnson, T. C. (2004). A high-resolution geochemical record from Lake Edward, Uganda Congo and the timing and causes of tropical African drought during the late Holocene article in Press Gene genealogies. Molecular Ecology, 9, 1657–1660.

Rust, M.B. (2002). Nutritional physiology (3rd ed.). In J. E. Halver & R.W. Hardy (Eds.), Fish Nutrition (pp. 367-452). London, Academic Press.

Rutaisire, J., & Booth, A. J. (2004). Induced ovulation, spawning, egg incubation, and hatching of the cyprinid fish Labeo victorianus in captivity. Journal of the World Aquaculture Society, 35(3), 383-391.

Rutaisire, J., Levavi‐Sivan, B., Aruho, C., & Ondhoro, C. C. (2015). Gonadal recrudescence and induced spawning in Barbus altianalis. Aquaculture Research, 46(3), 669-678. 1-10. DOI: 10.1111/are.12213

Sarasquete, M. C., Polo, A., & Yufera, M. (1995). Histology and histochemistry of the digestive system of larval gilt-head sea bream Sparus aurata L. Aquaculture, 130, 79-92.

38

Segner, H., Rosch, R., Verreth, J., & Witt, U. (1993). Larval nutritional physiology: studies with Clarias gariepinus, Coregonus lavaretus and Scophthalmus maximus. Journal of the World Aquaculture Society, 24(2), 121-134.

Skelton, P. H., Tweddle, D., & Jackson, P. (1991). Cyprinids of Africa. In I. J. Winfield & J. S. Nelson, (Eds.), Cyprinid Fishes, Systematics, Biology and Exploitation (pp. 211–239). London, UK: Chapman & Hall.

Snoeks, J., Kaningini, B., Masilya, P., Nyina-Wamwiza, L., & Guillard, J. (2012). Fishes in Lake Kivu: diversity and fisheries. In Lake Kivu (pp. 127-152). Springer Netherlands.

Statista (2014). Leading species in global aquaculture production in 2014. Hamburg, German. Statista portal. Retrieved from https://www.statista.com/statistics/240268/top-global- aquaculture-producing-countries-2010/

Teletchea, F., & Fontaine, P. (2014). Levels of domestication in fish: implications for the sustainable future of aquaculture. Fish and Fisheries, 15(2), 181-195.

Tomasson, T., Cambray, J. A. & Jackson, P. B. N. (1984). Reproductive biology of four large riverine fishes (Cyprinidae) in a man-made lake, Orange River, South Africa. Hydrobiologia, 112, 179–195. doi:10.1007/BF00008084

Vreven, E. J. W. M. N., Musschoot, T., Snoeks, J., & Schliewen, U. K. (2016). The African hexaploid Torini (Cypriniformes: Cyprinidae): review of a tumultuous history. Zoological Journal of the Linnean Society. Advance online publication. http://dx.doi.org/10.1111/zoj.12366

Villamizar, N., Blanco-Vives, B., Migaud, H., Davie, A., Carboni, S., & Sanchez-Vazquez, F. J. (2011). Effects of light during early larval development of some aquacultured teleosts: a review. Aquaculture, 315(1), 86-94.

Waite, R., Beveridge, M., Brummett, R., Castine, S., Chaiyawannakarn, N., Kaushik, S., & Phillips, M. (2014). “Improving Productivity and environmental Performance of Aquaculture.”Working Paper, Installment 5 of Creating a Sustainable Food Future (pp. 1- 58). Washington, DC: World Resources Institute. Retrieved from http://www.worldresourcesreport.org.

39

Weber, M. J., & Brown, M. L. (2009). Effects of common carp on aquatic ecosystems 80 years after “carp as a dominant”: ecological insights for fisheries management. Reviews in Fisheries Science, 17(4), 524-537.

Wetzel, R. G. (2001). Limnology: lake and river ecosystems (3rd ed.). Gulf Professional Publishing. 1006pp.

Winfield, I., & Nelson, J. S. (Eds.). (2012). Cyprinid fishes: systematics, biology and exploitation (Vol. 3) (pp.667). Springer Science & Business Media.

Witte, F., & de Winter, W. (1995). Biology of the major fish species of Lake Victoria. In F. Witte & W. L. T. Van Densen (Eds.), Fish Stocks and Fisheries of Lake Victoria A. Handbook for Field Observations (pp. 301–320). Cardigan, UK: Samara Publishing.

World Fish Center (WFC). (2005). Fish and food security in Africa (pp. 1-11). Penang, Malaysia. Author.

World Health Organization. (2010). Joint FAO/WHO Expert Consultation on the Risks and Benefits of Fish Consumption (No. 978). FAO Fisheries and Aquaculture Report.pp Food and Agriculture Organisation (FAO)/World Health Organisation (WHO). (2011). Report of the Joint FAO/WHO Expert Consultation on the Risks and Benefits of Fish consumption (pp 1- 50). Rome, Geneva: Author. Retrieved from http://www.fao.org/docrep/014/ba0136e/ba0136e00.pdf 50.

Worthington, E. B. (1932). A report on the fisheries of Uganda (pp 1-5). London, Crown Agent.

Woynarovich, A., Moth-Poulsen, T., & Peteri, A. (2010). Carp polyculture in Central and Eastern Europe, the Caucasus and Central Asia: a manual. FAO Fisheries and Aquaculture Technical Paper, 554 (pp. 1-73). Rome, Italy: FAO.

Zambonino-Infante, J., Gisbert, E., Sarasquete, C., Navarro, I., Gutierrez, J., & Cahu, C. L. (2008). In J. E.O. Cyrino, D. Bureau & B. G. Kapoor (Eds.), ontogeny and physiology of the digestive system of marine fish larvae. Feeding and Digestive Functions of Fish (pp. 277–344). Enfield, USA: Science Publishers Inc.

Leveque, C. (2003). Cyprinidae. In D. Paugy, C. Leveque & G.G Teugels (Eds.). the fresh and brackish water fishes of West Africa (Volume 1 p. 322-436). Paris: IRD Editions.

40

Zambrano, L., Martinez-Meyer, E., Menezes, N., & Peterson, A. T. (2006). Invasive potential of common carp (Cyprinus carpio) and Nile tilapia (Oreochromis niloticus) in American freshwater systems. Canadian Journal of Fisheries and Aquatic Sciences, 63(9), 1903-1910.

Zaret, T. M., & Rand, A. S. (1971). Competition in tropical stream fishes: support for the competitive exclusion principle. Ecology, 52(2), 336-342.

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CHAPTER THREE

Optimising selection of sexually mature Barbus altianalis for induced spawning; Determination of size at sexual maturity of populations from Lake Edward and Upper Victoria Nile in Uganda.

C. Aruho, R. Ddungu, W. Nkalubo, C. C. Ondhoro, F. Bugenyi, J. Rutaisire

Fisheries and aquatic sciences journal, https://doi.org/10.1186/s41240-018-0110-3

3.0 Abstract

Sexual maturity (L50), the length at which 50% of fish in a size class are mature is a key aspect of domestication of new fish species because it guides the procedure for identification of appropriate broodstock size for artificial spawning. In this study the L50 was determined for 1083 Barbus altianalis samples obtained from Lake Edward and the Upper Victoria Nile. Gonads of freshly killed samples were examined macroscopically and verified with standard histological procedures for the maturation stages that were used to determine L50. Oocytes and spermatogenic cell sizes were compared for fish obtained from both water bodies. Results indicated that there were no variations in macro gonad features observed for fish from Lake Edward and Upper Victoria Nile. Similarly, there were no significant differences in oocyte sizes (p > 0.05) between the two populations but significant differences in spermatogenic cell sizes were noted (p < 0.05) except for spermatozoa (p > 0.05). This however did not suggest peculiar differences between the two populations for staging the gonads.

Consequently no staging variations were suggested for both populations in determination of L50.

Sexual maturity was found in the same class size of fork length (FL) 20-24.9 cm and 35-39.9 cm for males and females from both water bodies, respectively. At this FL however, males were too small and for good selection of vigor broodstocks for spawning and conservation purposes, they are better picked from class size of 30-34.9 cm FL and above. These findings were crucial for integration of appropriate breeding size in spawning protocol by farmers and fisheries scientists conserving wild B. altianalis populations.

Key words: Sexual maturation, Staging, Oocytes sizes, Spermatozoa

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3.1 Introduction

The size at maturity is the size at which 50% of the fish in a given class size is sexually mature (Aruho, Basiita, Kahwa, Bwanika & Rutaisire, 2013; Booth, 1997). Length and weight are the most commonly used mean indices for estimating the size at which 50% of the fish become mature in a given class size (Lambert, Yaragina, Kraus, Marteinsdottir & Wright, 2003). The size at maturity in fish is determined based on gonadal maturation and developmental patterns in a given species (Lambert et al., 2003). Fish grow through egg-larval, pre-mature and mature ontogenetic phases which are genetically and environmentally controlled (Gunnarsson, Hjorleifsson, Thorarinsson & Marteinsdottir, 2006; Nickolskii, 1969). Transformation from immature to sexually mature phases in teleosts is a critical developing period when the fish enters the reproductive cycle (Brown-Peterson, Wyanski, Saborido-Rey, Macewicz, & Lowerre-Barbieri, 2011). Thus the transition from juvenile to sexual maturity in both females and males occurs when appropriate environmental and biological cues trigger the initiation and transformation for the first time of pre-vitellogenic oocytes and spermatogonia into vitellogenic oocytes and spermatozoa, respectively. The attainment of size at maturity in the same species may vary with changing environmental conditions over time and in the same or different geographical locations (Lambert et al., 2003). The size at maturity has been determined for many teleosts and is regarded as a remarkable indicator of reproductive potential that has been widely used in fisheries to control and regulate fish exploitation for sustainable conservation. Its use has apparently been extended into studies for domestication of fish species, guiding culturalists to identify sexually mature and ripe individuals for induced spawning (Aruho et al., 2013; Rutaisire & Booth, 2004). Identification of a suitable candidate species for induced spawning requires adequate knowledge of the size of the fish with high chances of oocytes that have reached ripening stage ready for ovulation (Basiita et al., 2010; Rutaisire et al., 2015).

Although induced spawning was successfully achieved for B. atlianalis (Rutaisire et al., 2015), the appropriate size at sexual maturity was never determined. The selection of females and males for induced spawning was made by visual inspection of eggs in a size range between 2-4 kg. Rutaisire et al. (2015) recommended a semi-natural method as one of the propagation methods in which the brood fish could be collected, induced and left to spawn naturally in ponds. However the collection of wild species for acclimatization, conditioning and subsequent domestication requires known length at

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sexual maturity. Knowledge of size at sexual maturity is an important reproductive aspect that was crucial in defining and integrating the right size of brood fish in artificial spawning protocol of B. altianalis. Thus this study focused on determination of appropriate size (length and weight) at maturity of B. altianalis from two populations of Lake Edward and Upper Victoria Nile to guide in selection of right fish size for induction during artificial spawning.

3.2 Materials and Methods

3.2.1 Collection of wild fish samples for determination of size at Maturity

Barbus altianalis samples were collected from Lake Edward (S00.08835, E029.76159; S00.13530, E029.86539) and Upper Victoria Nile (33o05’E, 0o35’N; 33o05’E, 0o45’N) between February and April 2015. Samples from Upper Victoria Nile were collected using both hooks and nets. The hooks were of size 9-7 (14 -13 mm hook gape size, respectively) mounted on long lines in a fleet of 100- 500 m in length and baited with tilapia fingerlings. The fishing nets were used for catching fish in both Upper Victoria Nile and Lake Edward. A fleet of nets each of ply 36, 26m in width and 90m deep (for Lake Edward) and 25m in width, 10m deep (for Upper Victoria River) with mesh size ranging from 0.5 to 10 inches were joined together. However, the nets in Lake Edward were not fixed but left to drift in the fishing ground (this is the commonest method used by fishermen for catching B. altianalis in Lake Edward). The nets were set for 12h at night and for 3 consecutive days. However, the experimental nets could not catch enough specimens and were therefore supplemented by purchasing all the landed fish on the sampling day. In total, 220 fish were captured with experimental nets from Lake Edward but 303 were purchased. While 200 fish were caught with experimental nets in Upper Victoria Nile but 360 fish were purchase at the landing sites. The Total weight (g) was recorded to the nearest 0.1 g and Standard Length (SL), Fork Length (FL) and Total Length (TL) were measured using a calibrated fish measuring board to the nearest 0.1 millimeter. Fish was dissected to obtain the gonads that were classified into maturity stages based on modifications from Brown-Peterson (2011) staging of gonads (Table 1 and Table 4). To validate the stages, small portions of gonads for each fish were immediately preserved in Bouin’s solution for further histological analysis. The preserved samples were taken to the College of Veterinary Medicine, Animal Resources and Bio-security (COVAB) histology laboratory at Makerere University. They were processed based on standard histological procedures by Bancroft and Gamble (2002). Tissue 44

subsections of 5μm of the preserved gonads were dehydrated in different alcohol concentrations in an automatic tissue processor, embedded in wax, further sectioned with a microtome, mounted, rehydrated and stained using Gill’s haematoxylin and eosin (H&E) and/or Masson’s trichome (MT). The sections were then examined using a light microscope (model Leica DM 500, Made by Microsystems Switzerland Ltd) for identification of oocyte and spermatogenesis stages. The diameter of oocytes and spermatogenic cells were measured by randomly sub sampling at least 5 oocytes for each type in each egg (in17 - 25 ovaries from each lake) and at least 30 spermatogenic cells for each type in each testis (in12 -17 testis from each lake) under a light microscope with a calibrated a stage micrometer in the eyepiece lenses. For the oocytes, three planes of oocytes and their nuclei diameters were measured and an average size was recorded. The number of nucleoli was counted for each nucleus and recorded.

3.2.2 Monitoring maturity level of second generation fish using farm records at Ssenya fish farm, Lwengo District.

The maturation level of the second generation of farmed B. altianalis (from Upper Victoria Nile) was estimated from the records kept by the farm over a period of two and half years between 2012 and 2014. The larvae were bred from the wild parents and raised in ponds at Ssenya fish farm in Lwengo district (N0022194, E32.679772). The fish were raised in well-fertilized ponds supplemented by feeding using 35% pelleted extruded feed (UGACHICK) and were regularly monitored for growth by recording the weights and lengths. The size at which the fish began to breed was crudely and directly estimated by physically pressing the abdomen at every sampling to release either the milt from males or the eggs in females.

3.3 Statistical analysis

The differences in sex ratios of fish from each natural water body were estimated by use of a chi- squire method to determine if the ratios were significantly different from the hypothetical 1:1 female to male ratio. Differences in oocyte and spermatogenic cell sizes between fish from Lake Edward and Upper Victoria Nile were determined by student’s t-test statistic using SPSS statistical software (IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp, 2013). The fish were sampled 45

as experimental units from Lake Edward and River Nile. The length at which 50% (L50) of individuals in a class size were sexually mature, was estimated from the ratio of the coefficients of a binary logistic regression of length and maturity level (mature; immature individuals). These coefficients (α and β) of the binary regression were estimated by Excel-solver statistical programme in Excel micro software (Windows 2010). The length L50 = Alpha (α)/Beta (β), where α and β are the coefficients of two parameter non linear model (Booth, 1997) obtained by stabilizing the coefficients and fitting the logistic ogive curve using Excel-solver.

The two-parameter logistic ogive was described by the non-linear equation: (α-βL) = PL = 1 ⁄ {1+exp } Where PL = the predicted proportion of mature fish at length of the fish L, α and β were coefficients of the parameter model. All individuals in developing phase (stage II) and above (stage II, IV and V) were taken as mature individuals (Brown-Peterson et al.,2011) A regression between length and weight was run using Excel Microsoft to predict the corresponding weight at L50 for standard lengths SL, fork length FL and total length TL (cm). Comparisons of coefficient b of power equations obtained were based on Froese (2006). To obtain the size frequency distributions (number of sampled fish in each size class) for each size class for the collected data from each lake, individual sizes of fish were grouped in intervals of 5 units using a pivot table in Microsoft Excel. A graph of individual sizes was plotted against the class intervals.

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3.4 Results

3.4.1 Sex ratios

A total of 523 specimens from Lake Edward were collected. There were 314 females while the males were 209, constituting a female to male ratio of 1:0.64 which was statistically different from the hypothetical 1:1(X2 = 18.00, df =1, p < 0.001). Comparatively, there were 560 fish samples from Upper Victoria Nile. Females were 360 and males were 200 constituting a ratio of 1:0.56. This ratio was significantly deviating from the hypothetical 1:1 female to male ratio (X2 = 45.71, df =1, P < 0.001).

3.4.2 Macroscopic and microscopic description of female classification stages

Macroscopically the female gonads in stage I were thread like and transparent (Table 3.1). Stage I was dominated by the oogonia and primary growth oocytes which included chromatin nucleolar oocytes and the perinuclear oocytes (Figure 3.1a). In stage II, the primary oocytes developed into cortical alveolar oocytes that later transformed into primary yolk vesicles and secondary yolk vesicles (Figure 3.1b). Macroscopically, gonads in stage II began to show minute speckles which later became large and began to fill up the gonad. The gonad became pale yellow as the primary and secondary yolk vesicles began to fill up the gonads (Table 3.1). In stage III, the secondary oocytes were transformed into tertiary oocytes and mature oocytes. Microscopically all types of oocytes at this stage were present in the monthly samples indicating batch characteristic development (Figure 3.1c). Macroscopically the gonads swell in size and they became yellowish in colour with off-white and yellow egg batches (Table 3.1-stage III). Stage IV had some Post Ovulatory follicles (POFs), atretic oocytes and sometimes very few remaining oocytes (Figure 3.1d). The gonad became regressed in size and had relatively bloody looking vessels (Table 3.1-stage IV). In stage V, only the oogonia and other primary growth oocytes were proliferating. This stage was distinguished from the virgin females by the presence of melanomacrophage centers, some increased bloody vessels, old POFs and atresia of most of vitellogenic stages. The gonad was further reduced but looked dark reddish and not transparent (Table 3.1-stage V).

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a b OO CAO Vtg2 Vtg1

CN Vtg1 Vtg1

PNO CAO PG

Vtg2

PNO

50 μm 788 μm

c d

Vtg2 Vtg3 ATO

PG Vtg1 PG CA POF

MO

MO

560 μm 300 μm

Figure 3.1: Histological section of female gonad. a) Stage I gonad with Ogonia (OO), chromatin nucleolar oocytes (CNO) and perinucleolar oocytes (PNO). The three types constitute primary growth oocytes (PG). b) Stage II gonads with cortical alveolar oocytes (CA), primary vitellogenic oocytes (Vtg1) and secondary vitellogenic oocytes (Vtg2). c) Stage III constituted all other oocyte types with tertiary vitellogenic oocytes (Vtg3) and mature oocytes (MO). d) Stage V with primary growth oocytes (PG), old post ovulatory follicles (POFs) and degenerating atretic oocytes (ATO). (H & E).

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Table 3. 1: Classification of maturity stages for female B. altianalis

Stage I (immature): Strand like reddish gonads Stage II (Developing): Minute off white (dominated by oogonia and Primary growth speckles (cortical alveolar oocytes) are oocytes) observed within the ovary. Few Vtg1 and Vtg2 are also observed

Stage III (spawning capable): Early stage; clear Stage III (spawning capable): spawning stage; gonads with batches of off white and pale clear gonads with batches of off white and pale yellow eggs. This stage is dominated by Vtg1 yellow eggs parked in the ovary. The observed and Vtg2 oocytes. But still enlarging in size oocytes are largely dominated by Vtg3 and mature oocytes with some POFs. But all oocytes types are present qualifying it as a batch spawner.

Stage IV (regressing): Flabby gonads with Stage V (regenerating) : Same as II but sligtly observed residual eggs. Residues are bigger. Old POFs, melanomicrophage centers, degenerating atretic oocytes OO and PG oocytes are observed.

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There were no significant differences in oocyte sizes for each oocyte type between fish from Lake Edward and those from River Nile (p > 0.5) (Tables 3.2 and 3.3).

Table 3. 2: Measurements of B. altianalis oocytes for fish obtained from Lake Edward

Lake Edward N Oocyte type Average cell size Nucleus size Estimated Ratio Total o (μm) recorded for (μm) recorded Number of between number of diameter for diameter nucleoli for oocyte and oocytes each egg nuclei sizes Measured 1 Primary growth oocytes Oogonia 14.14 ± 2.97 9.21 ± 1.76 - 0.65 ± 0.83 194 Chromatin 51.10 ± 12.11 22.14±5.10 3.89±2.867 0.44 ± 0.10 159 nucleolar oocytes Perinucleolar 162 ± 52.98 72.76 ± 27.13 17.66 ± 5.39 0.42 ± 0.13 259 oocytes 2 Cortical 325.30 ± 45.34 122.75 ± 29.11 34.04 ± 13.16 0.38 ± 0.08 100 alveolar 3 Vtg1 oocytes 530.64±91.83 125.47 ± 37.35 46.97 ± 28.28 0.24 ± 08 80 4 Vtg2 oocytes 724.12 ± 120.56 166.81±51.00 38.61 ± 14.25 0.22 ± 0.06 92 5 Vtg3 oocytes 1046.21 ± 397.41 155.26±68.46 38.6 ± 13.35 0.14 ± 0.04 111 6 Mature oocytes 1510.67 ± 182 196.34 ± 42.70 26.14 ± 13.40 0.143 ±0.04 85

Table 3. 3: Measurements of B. altianalis oocytes for fish obtained from Victoria. Nile

River Nile N Oocyte type Average cell size Nucleus size Estimated Ratio Total o (μm) recorded for (μm) recorded Number of between number of diameter for diameter nucleoli for oocyte and oocytes each egg nuclei sizes Measured 1 Primary growth oocyte Oogonia 13.95 ± 2.97 9.40 ± 1.82 - 0.64 ± 0.10 168 Chromatin 48.85 ± 10.47 21.23 ± 4.36 1.81±1.17 0.44±0.10 175 nucleolar Perinucleolar 162 ± 64.82 67.40 ± 36.90 16.84 ± 7.94 0.32 ± 0.13 364 oocytes 2 Cortical alveolar 330.63 ± 62.75 121.93 ± 28.23 31.40 ± 9.43 0.37 ± 0.76 134 3 Vtg1 oocytes 502.88 ± 114.97 124.04 ± 0.34 28.82 ± 9.23 0.27 ± 0.07 102 4 Vtg2 oocytes 767.40 ± 124.08 154.02 ± 46.16 29.67 ± 12.06 0.21 ± 0.07 96 5 Vtg3 oocytes 1036.60 ± 286.93 160.56 ± 0.042 21.90 ± 9.37 0.15 ± 0.044 82 6 Mature oocytes 1540.05 ± 184.68 204.31 ± 76.34 22.32 ± 15.30 0.135 ± 0.04 80 Data are shown as Mean ± SD

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3.4.3 Macroscopic and microscopic description of male classification stages

In stage I only the primary spermatogonia were present. The testis was tiny, strand like and difficult to differentiate from that of the female. In stage II, spermatogonia developed into primary spermatocytes and secondary spermatocytes (Figure 3.2a). In stage II however, few of the secondary spermatocytes had transformed into spermatids and into spermatozoa. The gonads looked off-white with serrations or appearance of lobulations (Table 3.4). In late stages for stage II a number of spermatocytes transforming into spermatids and spermatozoa increased. Stage III was characterized by the presence of all spermatogenic cells dominated by the spermatozoa (Figure 3.2b). The gonads were bigger than other stages with off-white coloration (Table 3.4). In stage IV, the number of spermatozoa reduced tremendously living large empty spaces, though there was still presence of all other spermatogenic cells. The gonads were bloody looking. Stage V, was characterized by the presence of many spermatogonia with very few residual spermatozoa.

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Table 3. 4: Classification of maturity stages for males B. altianalis

Stages Description Stage I: immature Strand like Y-shaped gonad and transparent. This stage is dominated by spermatogonia only

Stage II: Developing Easily distinguishable as males and off-white colored gonads. The majority of the cells are spermatogonia, and spermatocytes but occasionally few groups of spermatids and very few spermatozoa are observed

Stage III: spawning capable Large gonads, off-white coloration; dominated by lobules filled with spermatozoa

Stage IV: regressed Reduced in size with bloody perches. Other portions still look off-white. Microscopically they are large empty spaces with residual spermatozoa. However other portions may still have some viable spermatozoa. Stage V: regenerating Further reduced in size with pale reddish colouration. This stage largely contains parked spermatogonia with reduced spaces containing very few residual spermatozoa.

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a St

Sz Sc1

Sc1

Sg1 Sg2

10 μm

b St

Sg1 SC Nu Sg2

Sz

Sg2

Sc2 Sc1

10 μm

Figure 3. 1: a cross section of testis showing spermatogenic cells at different stages. a) Stage II

testis dominated by primary spematocytes (Sc1) but fewer spermatids and spermatozoans (Sz) are observed. b) Stage III testis showing all the types of spermatogenic cells that include primary spermagonia (Sg1) with a large nucleus (Nu), secondary permatogonia (Sg2), primary spermatocytes (Sc1), secondary spermatocytes (Sc2) and spermatids (St). The stage is dominated by spermatozoa (Sz). The sertoli cell (SC) is also observed. (H & E)

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Significant differences in sizes were observed in spermatogonia, spermatocytes and spermatids between the individuals from Lake Edward and those from Upper Victoria Nile (Table 3.5). However, no significant differences were observed with spermatozoa between the two populations (P > 0.05). All the spermatogenic cells of males from the Upper Victoria Nile were slightly bigger than those from Lake Edward.

Table 3. 5: Spermatogenic cell measurements in B. altianalis

Cell type Lake Edward Victoria Nile Total T-Test (μm) (μm) Average a a Spermatozoa 2.09 ± 0.23 2.12 ± 0.23 2.10 ± 0.23 t403 = -1.08, p>0.289 a b Spermatids 2.31 ± 0.33 2.78 ± 0.60 2.49 ± 0.51 t211.57 = -8.914, p<0.001 a b Secondary spermatocyte 3.14 ± 0.45 4.13 ± 0.93 3.57± 0.86 t293.07 = -14.66, p<0.001 a b Primary spermatocyte 4.76±0.62 5.47±0.82 5.00±0.78 t237.74 = -9.21, p<0.001 a b Secondary Cell size 7.90 ± 1.55 8.63±2.35 8.28±2.032 t328 = -3.495, p<0.001 a b spermatogonia nucleus 6.12±1.12 7.09±1.65 6.61±1.50 t371 = -6.720, p<0.001 a b Primary Cell size 12.44±1.53 13.97±1.71 13.26±26 t393.08 = -9.55, p<0.001 a b spermatogonia nucleus 8.48 ± 1.17 10.40±1.78 9.51±1.82 t371.73 = -12.72, p<0.001

Different subscripts a, b across rows indicate significant differences; Data are shown as mean ± SD

3.4.4 Size at sexual maturity, L50 from Lake Edward and River Nile

Females and males from Lake Edward reached their sexual maturity at an earlier stage (smaller size)

than those from Upper Victoria Nile. Length at maturity L50 for females from Lake Edward and the Upper Victoria Nile were attained at 35.4 cm FL (Figure 3.3) and 36.9 cm FL (Figure 3. 4)

respectively and were all found in the same class size of 35-39.9 cm FL. Similarly the L50 for males from Lake Edward and Upper Victoria Nile were attained in the same class size of 20-24.9 cm FL. Males in Lake Edward attained their maturity at 21.1 cm FL (Figure 3.3) while those from River Nile

attained their L50 at 22.9 cm FL (Figure 3.4). Generally males from both water bodies attained their

L50 earlier (smaller size) than the females. The L50 at standard length SL and total length TL are also provided in Table 3.6. The smallest mature female caught from Lake Edward was 22.6 cm FL while the smallest mature male was 18.2 cm FL. The biggest female caught from Lake Edward was 89.5 cm FL (100 TL) while the biggest male was 73.2 cm FL (82.2 cm TL). All males above 37.9 cm FL were mature and all females above 47.7 cm FL were mature. Smallest mature female from Upper

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Victoria Nile was 24.6 cm FL while the biggest fish caught was 77.0 cm FL (83.0 cm TL). The smallest male caught was 14.5 cm FL while the biggest male caught was 66.2 cm FL (70 cm TL). All females above 52.7 cm FL were mature and all males above 37.6 cm FL were mature.

Figure 3. 2: Maturity ogives for male and female B. altianalis sampled from Lake

Edward in 2015. Ogive is fitted to estimate Length at maturity L50 in females and males represented by dotted lines.

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Figure 3. 3: Maturity ogives for male and female B. altianalis sampled from Upper Victoria Nile in 2015. Ogive is fitted to estimate Length at maturity L50 in females and males represented by dotted lines.

3.4.5 Lengths-weight relationships

There was a strong and positive correlation between length and weight (r≥ 0.90) for both individuals from Lake Edward and Upper Victoria Nile (Table 3.6). The weights corresponding to L50 (SL, FL & TL) were estimated by the power equations at each length (Table 3.6). The power equation value of “b” was ≥ 3.100 for both females and males from Lake Edward and Upper Victoria Nile which was significantly higher than the value of b = 3, as determined by (Froese, 2006) (Table 3.6).

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Table 3. 6: Sexual maturity and Length-weight relationships for B. altianalis from Lake Edward and Upper Victoria Nile

Lake Edward Upper River Nile Parameters at L50 Females Males Females Males Standard length SL Class size 30-34.9 15-19.9 30-34.9 15-19.9 SL (cm) 31.9 18.0 33.1 19.1 W(g) = aSLb W= 0.014SL3.116 W= 0.020SL3.130 W= 0.013SL3.153 W= 0.013SL3.150 W(g) 627.4 127.2 716.8 141.4 r 0.923 0.908 0.940 0.931 r2 0.978 0.972 0.991 0.984 Fork Length (FL) Class size (FL) 35-39.9 20-24.9 35-39.9 20-24.9 FL (cm) 35.4 21.1 36.9 22.9 W (g)= aFLb W= 0.01FL3.129 W= 0.010FL3.128 W= 0.008FL3.164 W= 0.0103.126 W(g) 702.8 135.8 726.4 178.7 r 0.925 0.915 0.939 0.930 r2 0.976 0.979 0.910 0.984 Total length TL Class size (TL) 40-45.9 20-24.9 40-44.9 20-24.9 TL 41.6 23.5 40.0 24.2 W(g)= aTLb W= 0.006x3.160 W= 0.006TL3.149 W= 0.004TL3.281 W= 0.004TL3.251 W (g) 784 124.6 726.4 126.10 r 0.926 0.911 0.933 0.921 r2 0.973 0.965 0.989 0.980

3.4.6 Size frequency distribution (distribution of number of sampled fish in class size)

Size frequency distributions of females and males in Lake Edward followed the same pattern, with the highest number of fish recorded in 25-29.9 cm FL class size for both sexes and gradually declining in other class sizes (Figure 3.5). The variations shown for males and females from Upper Victoria Nile did not follow a same pattern as those from Lake Edward (Figure 3.6). The males were more frequent in class size of 30-34.9 cm to 45-49.9 cm FL and there after they declined. The highest peak for females was obtained in a class size of 55-59.9 cm FL. The males from both water bodies were conspicuously absent or very few in upper class sizes above 60-64.5 cm FL compared to females. For the females from Upper Victoria Nile, high frequencies were noted in upper class sizes while males almost disappeared (Figure 3.6).

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Figure 3.5 : Length-frequency distribution of B. altianalis from Lake Edward; Bars with shaded textboxes in the middle indicate the class size with L50

Figure 3.6: Length-frequency distribution of B. altianalis from Victoria Nile; Bars

with shaded textboxes in the middle indicate the class size with L50

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3.4.7 Monitoring maturity level of second generation fish using farm records at Ssenya fish farm, Lwengo District

Farm records indicated that of 28 fish sampled 3% males were found to be ripe running in the first 6 months. By the end of first year (12 months), 62 % of the males in the subsample were ripe running. By the 22nd month after hatch all the males produced milt (Table 3.7). According to the records kept by the farm, 22% of the females were gravid by 22nd month after hatch and by the 27th month after hatch 72% of fish identifiable as females were mature, suggesting that 50% maturity level was attained between the 2nd and the 3rd year of growth. The 50% maturity level for males is estimated to have been reached between the first and the second year, earlier than the values for the females.

Table 3. 7: Annual Female and male percentage ages of ripe individuals between 2012-2014; Stocked in January 2012.

Year Sampl age Nu Number Females Number % Males ing (mo mbe and % of of ripe month nths r of ripe males ) sam females (ripe/total pled (ripe/total males) fish females) No. % Mean Wt FL (cm) No. % Wt (gs) FL (cm) (g) 1 Jun. 6 28 1/11 27 28 ± 2.2 13.9±2 (2012) 1 Sept. 9 32 4/15 36 61 ± 2.0 16.1 ± 0.8 (2012) 1 Dec. 12 30 7/12 63 120 ± 30 20.0 ± 2.1 (2012) 2 Oct. 22 27 3/15 20 365 ± 70 28.9 ± 2.4 12/12 100 348 ± 50 28.8 ± 2.9 (2013) 3 Feb. 27 25 10/14 72 680± 105 35.6 ± 9.0 11/11 100 492 ± 102 30.9 ± 3.3 (2014) 3 Jun. 31 42 23/25 93 800± 130 36.6 ± 5.6 (2014)

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3.5 Discussion

The availability of quality and sufficient seed is the cornerstone for the successful commercialization of domesticated fish species (George, Olaoye, Akande & Oghobase, 2010; Mair, 2002). The seed is a reflection of a healthy and quality broodstock that will eventually determine the availability of quality seed for commercial production of the species. In a number of cultured species, the brood fish is an integral process of a well-planned seed production system (Bondad-Reantaso, 2007). One of the key characteristics of the spawning technology is the appropriate size and the source as well as availability of both broodstock females and males. The wild sources are still the most significant sources of the broodstocks for newly domesticated fish and grading up of genetically depressed captive stocks. In this study sex distribution from Lake Edward and Upper Victoria Nile were the same, thus the sex ratios of females to males were similar in both water bodies. The ratios were skewed toward females. Skewed sex ratios could suggest some migratory activity related to breeding seasons (Mahmood, Ayub & Siddiqui, 2011), a shorter life span for males, a natural intrinsic characteristic for biased sexes or environmental variations (Baroiller, D'Cotta & Saillant, 2009; Vandeputte, Quillet & Chatain, 2012). In spite of the fact that samples were peaked in a dry month of February and wet months of March and April, a biased sex ratio was observed. Therefore it seems that sex ratio bias toward females is genetically predisposed. Nevertheless, the occurrence of males and females at the same sampling points suggested that it is much easier for the farmer to collect both males and females for breeding (Aruho et al., 2013).

This study observed that the general developmental and maturation process in B. altianalis was similar to that described in other cyprinid species (Booth & Weyl, 2000; Lone & Hussain, 2009; Maack & Segner, 2003; Smith & Walker, 2004). However, special specific processes including the colour and size of gonads, oocytes and spermatogenic cells, the location of the nucleus and number of nuclei may vary for each species and were inevitably described to ensure a clear guided description for identification of classification staging as suggested by Brown-Peterson et al. (2011). Although size variations were only noted with spermatogonia, spermatocytes and spermatids among the two populations the spermatozoa sizes emerging from the cysts during spermiation process were the same, attributing the variations to differences in cytoplasmic volume. As observed with spermatozoa there were no differences in oocyte sizes between the two populations from both water bodies.

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However, studies by Nakamya (2010) and Muwanika, Nakamya, Rutaisire, Sivan, and Masembe (2012) suggested that morphological and genetical differences occurred between the two population clusters but did not identify them as different morphs or species and recommended further investigations because the sample size was small and required robust genetic methods. Based on the current results, it seems the two populations remain the same with less divergent strategies or rather still ‘primitive’ in its evolutionary process. Slight differences observed in cytoplasic volume between the two populations, could be linked to localized environmental conditions for each water body. Localized changes in the water environment, preferential food requirements or fishing pressure may account for the observed differences (Lam, 1983). Genetic studies are required to help ascertain if the differences between these groups exist. However, since no significant differences in oocytes and spermatozoa were observed between the two populations, the same classification (staging) was maintained for B. altianalis from both water bodies for description of length at maturity. This is important for guiding famers and other fisheries scientists in staging B. altianalis gonads.

Results from this study showed that the size at maturity L50 for B. altianalis males from Upper Victoria Nile and from Lake Edward were obtained in the same class size as was for females. This finding further suggests that in spite of two water bodies being separated or having no close connection the species has not separated into other morphs or forms. Despite the fact that they were in the same class size, the L50 values were not exactly of the same length. In species such as Oreochromis niloticus (Duponchelle & Panfili, 1998), Peritalbagrus fulvidraco (Cao, 2009) and

Hippoglossoides platessoides (Morgan & Colbourne, 1999), the variations observed in L50 in their population cohorts in different sampling areas were attributed to fishing pressure and environmental differences (phenotypic plasticity) other than genetic differences. Therefore the minor differences in

L50 observed in B. altianalis were probably linked to environmental differences other than genetic variations. It seems B. altianalis still retains very primitive traits with longer evolutional characteristics and thus fish from Lake Edward and Upper Victoria Nile could easily be crossbred to obtain the best traits possible for culture. The study also revealed that males matured much earlier than females. Early maturation of males could ensure that all eggs produced by females are fertilized during spawning. The selection of an appropriate broodfish for spawning is largely linked to knowledge of maturation size and takes into cognizance of conservation of the wild stocks to avoid over exploitation. For 61

instance size frequency distribution noted in this study suggests that the 50% of the sampled population (L50) for B. altianalis males from Lake Edward and Upper Victoria Nile obtained in a class size of 20 - 24.9 cm FL (135.8g-178.7g) was smaller than the minimum legally recommended sizes of 30-34.9 cm FL (about 500g). The recommended fishing nets for catching this size is≥ 4.5 inches mesh size (amendment Fish Act, 1951). This implied that any size below 30-34.9 cm FL is not legally acceptable for harvesting as broodfish. Secondly, in spite of the fact that the noted L50 could appropriately be collected for spawning, the weight is rather small for handling in order to obtain milt and also not appropriate for selection of good breeds. Hence this study suggests that males for breeding fish could appropriately be picked from class size of≥ 30 - 34.9cm FL (417g - 675g) for males and ≥ 35 - 39.9 cm FL (700 - 726.4g) for females.

The study showed that males and females from Lake Edward were more frequent in a class size of 25-29.9 cm FL than in other class sizes suggesting an improved enforcement effort of fishing regulations for the right net sizes on the lake. The reduction thereafter implied fishing effort or activity. The pattern of females and males in Upper Victoria Nile did not suggest a relationship with fishing activity. The frequency of females was notably high in the class sizes of 15 - 19.9 to 25 - 29.9 cm FL and from 40 - 44.9 to 60-64.9 cm FL. While the males frequencies were high between 30 - 34.9 and 45 - 49.9 cm FL. This implied that there was little fishing pressure. There is a reported regular interruption of power generating activities at Kira and Bujagali power Dams that bars fishers from fishing daily and some portions are restricted and inaccessible by fishermen (personal communication with fishers; David Odong, head of fishermen at Kiira landing site). This could further explain why the L50 values for fish from Upper Victoria Nile were slightly higher than those from Lake Edward for both females and males. Hence sustained fishing pressure of B. altinalis is more likely to influence evolutionary changes in L50 for Lake Edward individuals first before those from Upper Victoria Nile. Sustained selective fishing pressure has been suggested to cause rapid changes in evolution of maturation schedules in many fish species (Audzijonyte, Kuparinen &

Fulton, 2013; Hunter, Speirs & Heath, 2015; Law, 2000). The frequency distribution and the L50 observed in B. altianalis from this study showed that very few or no males were caught above 65cm FL. In some fish species shorter male life spans were associated with aggressive behavior of males making them vulnerable to enemies during feeding or mating (King, Furtbauer, Mamuneas, James & Manica, 2013; Reichard, Polacik, Blazek & Vrtilek, 2014). However, further investigations are 62

necessary to ascertain if there is any behavior that makes B. altianalis susceptible to predators or fishing.

The coefficient “b” of the power curves obtained for the weight–lengths relationship of the sampled population suggested that natural growth rates were similar for both populations from Lake Edward and Upper Victoria Nile because all the “b” values were in the same range of 3.126-3.164 for both males and females. This was also reported by Ondhoro et al., (2016) for the same species. These values were significantly higher than b = 3 (Froese, 2006) implying that the fish girth increased much faster than the length (Froese, 2006; Paraskevi & Konstantinos, 2011). Therefore B. altianalis from Lake Edward and Upper Victoria Nile were accurately grouped under species with positively allometric growth. The weight-lengths power equation had a strong r2 of > than 97 percent hence the weights at each L50 were and can be accurately estimated using the power equations obtained in this study.

The farm records for the cultured B. altianalis (Table 3.7) also indicated that males matured at smaller sizes than females as in the wild populations. In spite of the fact that L50 was not determined for the farm record samples because of the very small numbers, the fewer number of fish from the records show that both females and males matured at smaller sizes compared to their counterparts in the wild. This is common with many cultured species and it is a strategy by the fish to ensure earlier production of offsprings under confined and or unpredictable environmental conditions (Brummett, 1995; Longalong, Eknath & Bentsen, 1999). It’s imperative though to use bigger males (≥ 30-34.9cm FL) as earlier suggested, than the indicated sizes for easier selection of good broodstocks (also had milt less than 1ml). However, a thorough experimental study is recommended for determination of appropriate values of L50 in ponds with appropriate feed.

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3.6 Conclusions

Evidence obtained from sex ratios, L50, power equations and germ cell sizes in this study indicated that B. altianalis males and females from Lake Edward were similar to those from Upper Victoria Nile hence there are no clear evolutionary lines detected for this species and the species has remained more or less primitive. The species could therefore be crossbred where applicable for the best cultured traits. The length at which 50% individuals were mature was 20-24.9 cm FL for males and 35-39.9 cm FL for females. But because of the need to strict adherence to fishing regulations, conservation of the wild resources as well as the need to have selection of good broodfish the study recommends selecting individuals from 30-34.9 cm FL for males and 35-39.9 cm FL for females to be included in the spawning protocol. These individuals could be picked from the same areas for breeding and during the periods when breeding fish are prominent (Rutaisire et al., 2015). Because the famed fish attained smaller sizes at maturity than their wild counterparts, this may point to the fact that more growth study experiments with appropriate conditions and diet could be required to improve the growth performance of B. altianalis.

3.7 Acknowledgment

We acknowledge Mr. Waiswa and Mandela from Katwe landing site on Lake Edward and Mr. Odong of Jinja town who organized and guided us in collection of samples on the Lake Edward and River Nile. We thank Mr. Magidu Kiseka of College of Veterinary Medicine, Animal Resources and Bio- security for having organized for us the laboratory and equipment we used in histological processing of samples. We also thank the Ssenya Fish Farm for providing us with the pond data they had recorded on growth of B. altianalis.

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3.8 References

Aruho, C., Basiita, R. K., Kahwa, D., Bwanika, G., & Rutaisire, J. (2013). Reproductive biology of Bagrus docmak in the Victoria Nile, Uganda. African Journal of Aquatic Science, 38(3), 263- 271.

Audzijonyte, A., Kuparinen, A., & Fulton, E. A. (2013). How fast is fisheries‐induced evolution? Quantitative analysis of modelling and empirical studies. Evolutionary Applications, 6(4), 585-595. https://doi.org/10.1111/eva.12044

Bancroft, J. D., & Gamble, M. (2002). Theory and practice of histological techniques, 5th edn. pp 85- 107. Edinburgh, London: Churchill Livingston Publishers.

Baroiller, J. F., D'Cotta H., & Saillant, E. (2009). Environmental effects on fish sex determination and differentiation. Sexual Development, 3,118–135. https://doi.org/10.1159/000223077

Basiita, R. K., Aruho, C., Kahwa, D., Nyatia, E., Bugenyi, F. W., & Rutaisire, J. (2011). Differentiated gonochorism in Nile perch Lates niloticus from Lake Victoria, Uganda. African Journal of Aquatic Science, 36(1), 89-96. http://dx.doi.org/10.2989/16085914.2011.559694

Bondad-Reantaso, M. G. (Eds.). (2007). Assessment of freshwater fish seed resources for sustainable aquaculture. Fisheries Technical paper no. 501(pp.628). Rome, Italy: FAO.

Booth, A. J. (1997). On the life history of the lesser gurnard (Scorpaeniformes: Triglidae) inhabiting the Agulhas Bank, South Africa. Journal of Fish Biology, 51, 1155-1173. https://doi.org/10.1111/j.1095-8649.1997.tb01133.x

Booth, A. J., & Weyl, O. L. (2000). Histological validation of gonadal macroscopic staging criteria for Labeo cylindricus (Pisces: Cyprinidae). African Zoology, 35(2), 223-231.

Brown-Peterson, N. J., Wyanski, D. M., Saborido-Rey, F., Macewicz, B. J., & Lowerre-Barbieri, S. K. (2011). A standardized terminology for describing reproductive development in fishes. Marine and Coastal Fisheries, 3(1), 52-70.

Brummett, R. E. (1995). Environmental regulation of sexual maturation and reproduction in tilapia. Reviews in Fisheries Science, 3(3), 231-248. https://doi.org/10.1080/10641269509388573

65

Cao, L., Song, B., Zha, J., Yang, C., Gong, X., Li, J., Wang, W. (2009). Age composition, growth, and reproductive biology of yellow catfish (Peltobagrus fulvidraco, Bagridae) in Ce Lake of Hubei Province, Central China. Environmental Biology of Fishes, 86, 75–88. https://doi.org/10.1007/s10641-008-9342-x

Duponchelle, F., & Panfili, J. (1998). Variations in age and size at maturity of female Nile tilapia, Oreochromis niloticus, populations from man-made lakes of Cote d'Ivoire. Environmental Biology of Fishes, 52(4), 453-465. https://doi.org/10.1023

Froese, R. (2006). Cube law, condition factor and weight–length relationships: history, meta‐analysis and recommendations. Journal of Applied Ichthyology, 22(4), 241-253. https://doi.org/10.1111/j.1439-0426.2006.00805.x

George, F. O. A., Olaoye, O. J., Akande, O. P., & Oghobase, R. R. (2010). Determinants of aquaculture fish seed production and development in Ogun State, Nigeria. Journal of Sustainable Development in Africa, 12(8), 22-34.

Gunnarsson, A., Hjorleifsson, E., Thorarinsson, K., & Marteinsdottir, G. (2006). Growth, maturity and fecundity of Wolffish Anarhichas Lupus L. in Icelandic waters. Journal of fish biology, 68(4), 1158-1176. https://doi.org/10.1111/j.0022-1112.2006.00990.x

Hunter, A., Speirs, D. C., & Heath, M. R. (2015). Fishery-induced changes to age and length dependent maturation schedules of three demersal fish species in the Firth of Clyde. Fisheries Research, 170, 14-23. https://doi.org/10.1016/j.fishres.2015.05.004

King, A. J., Fürtbauer, I., Mamuneas, D., James, C., & Manica, A. (2013). Sex-differences and temporal consistency in stickleback fish boldness. PLoS One, 8(12), e81116.

Lam, T. J. (1983). 2 Environmental Influences on Gonadal Activity in Fish. Fish physiology, 9, 65- 116.

Lambert, Y., Yaragina, N. A., Kraus, G., Marteinsdottir, G., & Wright, P. J. (2003). Using environmental and biological indices as proxies for egg and larval production of marine fish. Journal of Northwest Atlantic Fisheries Science, 33(115), 159.

Law, R. (2000). Fishing, selection, and phenotypic evolution. ICES Journal of Marine Science: Journal du Conseil, 57(3), 659-668. https://doi.org/10.1006/jmsc.2000.0731

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Lone, K. P., & Hussain, A. (2009). Seasonal and Age related variations in the ovaries of Labeo rohita (Hamilton, 1822): A Detailed Gross and Histological Study of Gametogenesis, Maturation and Fecundity. Pakistan Journal of Zoololgy, 41(3), 217-239.

Longalong, F. M., Eknath, A. E., & Bentsen, H. B. (1999). Response to bi-directional selection for frequency of early maturing females in Nile tilapia (Oreochromis niloticus). Aquaculture, 178(1), 13-25. https://doi.org/10.1016/S0044-8486(99)00132-5

Maack, G., & Segner, H. (2003). Morphological development of the gonads in zebrafish. Journal of Fish Biology, 62(4), 895-906. https://doi.org/10.1046/j.1095-8649.2003.00074.x

Mahmood, K., Ayub, Z., & Siddiqui, G. (2011). Sex-ratio, maturation and spawning of the Indian ilisha, ilisha melastoma (clupeiformes: pristigasteridae) in coastal waters of Pakistan (northern Arabian Sea). Indian Journal of Marine Sciences, 40(4), 516.

Mair, G. (2002). Genes and fish: supply of good quality fish seed for sustainable aquaculture. Aquaculture Asia, 7(2), 25-27.

Morgan, M. J., & Colbourne, E. B. (1999). Variation in maturity-at-age and size in three populations of American plaice. ICES Journal of Marine Science: Journal du Conseil, 56(5), 673-688. https://doi.org/10.1006/jmsc.1999.0487

Muwanika, V. B., Nakamya, M. F., Rutaisire, J., Sivan, B., & Masembe, C. (2012). Low genetic differentiation among morphologically distinct Labeobarbus species (Teleostei: Cyprinidae) in the Lake Victoria and Albertine basins, Uganda: insights from mitochondrial DNA. African Journal of Aquatic Science, 37(2), 143-153. https://doi.org/10.2989/16085914.2012.668850

Nakamya, M. F. (2010). The population genetic structure and evolutionary relationships of two Barbus species (Pisces: Cyprinidae) in the Lake Victoria region (Msc dissertation, Makerere University).86pp.

Nickolskii, G. V. (1969). Theory of fish population dynamics: As the biological background for rational exploitation and management of fishery resources (pp. 323). Edinburgh: Oliver and Boyd.

Ondhoro, C. C., Masembe, C., Maes, G. E., Nkalubo, N. W., Walakira, J. K., Naluwairo, J., ... & Efitre, J. (2016). Condition factor, Length–Weight relationship, and the fishery of Barbus

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altianalis (Boulenger 1900) in Lakes Victoria and Edward basins of Uganda. Environmental Biology of Fishes, 1-12. DOI 10.1007/s10641-016-0540-7

Paraskevi, K. K., & Konstantinos, I. S. (2011). Morphometrics and Allometry in Fishes. Retrieved from http://cdn.intechweb.org/pdfs/30107.pdf

Reichard, M., Polacik, M., Blazek, R., & Vrtilek, M. (2014). Female bias in the adult sex ratio of African annual fishes: interspecific differences, seasonal trends and environmental predictors. Evolutionary Ecology, 28(6), 1105-1120. https://doi.org/10.1007/s10682-014-9732-9

Rutaisire, J., & Booth, A. J. (2004). Induced ovulation, spawning, egg incubation, and hatching of the cyprinid fish Labeo victorianus in captivity. Journal of the World Aquaculture Society, 35(3), 383-391. https://doi.org/10.1111/are.12213.

Rutaisire, J., Levavi-Sivan. B., Aruho, C & Ondhoro, C. C. (2015). Gonadal recrudescence and induced spawning in Barbus altianalis. Aquaculture Research, 46 (3), 669-678. https://doi.org/10.1111/are.12213

Smith, B. B., & Walker, K. F. (2004). Spawning dynamics of common carp in the River Murray, South Australia, shown by macroscopic and histological staging of gonads. Journal of Fish Biology, 64(2), 336-354. https://doi.org/10.1111/j.0022-1112.2004.00293.x

Vandeputte, M., Quillet, E., & Chatain, B. (2012). Are sex ratios in wild European sea bass (Dicentrarchus labrax) populations biased? Aquatic Living Resources, 25(1), 77-81.

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CHAPTER FOUR Effectiveness of African catfish pituitary extracts, Dagin and water flow for optimising egg production, fertilisation and hatchability in artificial spawning of Barbus altianalis.

C. Aruho, M. T. Mwanja, F. Bugenyi and J. Rutaisire.

Uganda Journal of Agricultural Sciences, 2016, 17 (2): 183 – 195. doi.org/10.4314/ujas.v17i2.5

4.0 Abstract

Manipulation of reproductive systems to induce oocyte maturation and ovulation in fish species using Fish inducing hormones are widely used in commercial aquaculture to facilitate continuous supply of sufficient seed required on regular basis by the farmers. The objective of this study was to optimise production of viable eggs for improved hatchability during artificial spawning in Barbus altianalis. Two experiments were conducted, namely (i) experiment I evaluated the efficiency of using catfish pituitary extracts in spawning of second generation broodstocks compared to that of Dagin and water flow. (ii) experiment II examined ripe running females facilitated to spawn by running water only. Fish treated with pituitary extracts performed better than those treated with Dagin and those in control tanks. However, the differences between females treated with catfish pituitary extracts and Dagin with respect to working fecundity (2314.40; 1207.37), fertilisation rates (80.27; 40.80%) and hatchability (42.20; 27.44%) at p > 0.0167 respectively, were not significant. Differences between fish treated with catfish pituitary extracts and those in control tanks were significant with respect to fecundity (U = 20.5, p < 0.001), fertilization rates (U = 24.5, p < 0.001), and hatchability (U = 24.5, p < 0.001). No significant differences in all tested parameters were observed between females treated with Dagin and the control (p > 0.0167). In experiment II, hatchability and working fecundity were significantly higher when fish were stripped after 4 hours (100 degree hours hr at 25⁰C) of running water than those stripped after 10 hrs (250 degree hours) but fertilization rates were not different (p > 0.05). The findings indicate that catfish pituitary extracts are equally effective as Dagin in inducing B. altianalis to spawn. However, the observations made on ripe running females in both experiments suggest that they should not be induced with any hormone. Hence, the cost of spawning could further be reduced using running water, especially in wet seasons when the majority are ripe.

Key words: Spawning, hatchability, eggs, hormone 69

4.1 Introduction

Manipulation of reproductive systems to induce oocyte maturation and ovulation in fish species using inducing hormones has widely been adopted in commercial aquaculture to facilitate continuous supply of sufficient seed required on regular basis by fish farmers (Bromage, 1998; Zohar and Mylonas, 2001). The regular availability of this seed is essential for closing the life cycle of fish in captivity (Funge-Smith & Phillips, 2001; Stickney, 2005). Fish species have an inherent mechanism that relates to the gonadal structure and activity with appropriate environmental stimuli that triggers spawning (Munro, 1990; Rutaisire et al., 2015). Under appropriate environmental stimuli relayed to the brain through the neuron system, sustained levels of releasing hormone (GnRH) from the hypothalamus, triggers the release of luteinising hormone (LH) and follicle stimulating hormones (FSH), that control oocyte maturation and ovulation (Levavi-Sivan B., Bogerd, Mananos, Gomez & Lareyre; Peter and Yu, 1997). Under culture conditions, the extent to which the inducing hormone effectively initiates oocyte maturation and ovulation depends on the levels of the GnRH hormone released by the brain in response to the stimuli. The nature and the source of the inducing agents influence and permit timely maturation and ovulation of viable eggs for incubation in a given species. The response to the inducing agent is species specific and may vary as a result of dosages (Marte, 1989).

Although induced spawning was successful in B. altianalis (Rutaisire et al., 2015), the high mortality of eggs and yolk sac larval stage observed (about 55 and 11%, respectively) could have been partly associated with the levels and timing of inducing hormone required to achieve optimal egg ripeness or quality. Both synthetic GnRH analogs and pituitary extracts (PE) have been used for induced spawning in the Chinese and Indian carps; but with analogs, higher success was registered when they are administered with anti-dopamine agents (Afzal, 2008; Levavi-Sivan et al., 2010). However, at over US$ 100 for each vial used for only 10 kg, synthetic analogues are expensive, hence are not readily available to fish farmers in East Africa. The pituitary extracts become an alternative cheaper source and readily available option to the farmers than the synthetic hormones. In the initial artificial spawning experiments, ovulation in wild B. altianalis broodstock was successfully facilitated by water flow (Rutaisire et al., 2015). The effectiveness of water flow alone to induce spawning could be linked to the maturation level of gravid individuals, whereby water flow facilitated increased

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hormonal release levels by encouraging courtship and excitement. However, still low egg production and hatchability was recorded (Rutaisire et al., 2015). Thus, the high embryonic mortality was probably linked to inadequate hormonal levels among other factors.

One of the most widely used natural hormones in inducing fish is the carp pituitary extract (CPE), which has been used with many different fish species (Drori, Ofir, Levavi-Sivan & Yaron, 1994; Rottman, Shireman & Chapman, 1991; Yaron et al., 2009; Zohar and Mylonas, 2001; Zohar et al., 2010). The pituitary extracts contain two important gonadotropins (GTHs), Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) that consist of identical weakly associated non- covalent dimer’s of α subunits joined to the more specialized β units of amino acids (Weltzien, Norberg & Swanson, P2003). Increased levels of these hormones are associated with spawning activities in fish (Nagahama, 1994; Yaron et al., 2009). Although the effect of CPE is nonspecies specific across farmed carps (Chaudhuri, 1976), the response of the fish to CPE by increasing gonadotropin levels could vary in different species (Miah, Mamun, Khan & Rahman, 2008; Mahfuj, Hossein & Sarower, 2012). The CPE has registered success only with split dose administration (priming and resolving) in almost all the carps (Mahfuj et al., 2012; Indira, Damodaran & Priyadarshini, 2013). The CPE has never been used to induce spawning in B. altianalis. However, because of the scarcity of farmed carps in East Africa, alternative sources from other fish species like the African catfish Clarius gariepinus has been largely used to induce spawning in the same species (de Graaf & Janssen, 1996; Mwanja, Rutaisire, Ondhoro, Ddungu, Aruho, 2015) and could be used as aheterologous gland for induced spawning in B. altianalis. The objective of this study was to evaluate the effect of African catfish pituitary extract on induced spawning and ovulation of B. altianalis compared to that of Dagin ([D-Arg6, Pro9-NEt])-sGnRH; 10µg kg-1 + metoclopramide 20 mg kg-1), a GnRH synthetic analogue with an anti-dopamine drug (Drori et al., 1994), and use of running water alone with a view of determining its effectiveness in optimising egg production and larvae hatchability since it might be a cost effective option and readily available technique to farmers.

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4.2 Method and Materials

4.2.1 Preparation and Feeding of Broodstock

A total of 79 fish of the first generation of parents that were originally obtained from the wild, ranging from 282 to 900 g were fed on commercial fish feed (30% CP) for two months, in fertilised ponds of 300M2 (approx one bag of 100 kg cow dung in 300 M2 pond). The fish were fed 3 times daily, and the feed was applied at 5% body weight for the first one week and 3% body weight for the rest of the period (based on the farm’s broodstock management protocol for catfish breeding). The broodstock feeding and spawning were done during the months of November and December 2015, at Ssenya Fish Farm, a commercial fish firm located in the Lwengo District (N0022194, E32.679772) in Uganda. In the first month, 48 females were fed separately from 31 males. However, about 15 females were found to have less than 300 eggs, and none of the males produced milt. They were then fed together in the second month before most of them became ripe for spawning.

4.2.2 Preparation of Catfish Pituitary Extract and Dagin

The catfish pituitary extract was prepared from the pituitary glands from 31 catfish of average 700 ± 50 g. Thirty one pituitary glands weighing on average 0.04 g ± 0.008, were crushed in a mortar and mixed with 3 ml of 5% saline solution for each pituitary, as used by the same farm on catfish induction as well as some other farms (Mwanja et al., 2015). The solution contained a total of 1.33 g pituitary glands in 93 ml of solution. Each ml contained 0.014 g pituitary extract and Dagin was administered according to the manufacturer protocol for common carp (10 μg kg-1 + 20 mg kg-1 sGnRHa- [D-Arg6, Pro9-NEt]-sGnRH and metoclopramide, respectively). A standard prescribed dosage for carps, of 20 kg per 10 ml in a vial was used in this experiment.

4.2.3 Experiment I; Separation and treatment of broodstocks with hormones

Forty five females were randomly separated into groups of 5, and placed in 8 m3 (4 x 2 x 1 metres) concrete tanks making a total of 9 tanks. Three males were randomly placed in each of the nine tanks containing females and the fish were left to rest for 4 hrs from 15-19.00 hr (100 degree hours). Water flow was allowed through the tank at a rate of 1 litre per 20 second. The temperature was kept at 25

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⁰C. After 4 hrs, the fish in each tank was treated with catfish pituitary extract or Dagin. The fish in rest of the tanks were used as control and were not treated with any hormone. Each treatment had three replicates. Females that were treated with catfish pituitary extract were injected with the first dosage of 2 ml of the pituitary extract for approximately 1 kg of B. altianalis broodstock (0.028 g kg- 1). Those treated with Dagin, were injected with 0.5 ml of Dagin hormone per kg of B. altianalis. The cost of the two homones used is compared in the Table 4.1 below. Hormones were administered intramuscularly. After 5 hrs (cumulative; 225 degree hours) between 19:30-00:30 hr, the fish were given the resolving dosage of 3 ml for those treated with catfish pituitary extract ,and 3 ml for those treated with Dagin. At the time of the second injection, the males received 2 ml for those in the tanks treated with catfish pituitary extract and those that were treated with Dagin.

Table 4. 1: Calculated costs per kilogram of B. altianalis injected with African catfish Pituitary Extract and Dagin Hormone Number Number Total Total Weight Unit cost Cost per of of glands cost of weight of 1 ml per ml injection catfish obtained fish per of (shs) for per kg used vial pituitary (shs) (US$) extract (g) African 31 31 43.4US$ 1.33 0.014 0.46US$ 0.91US$ (2 catfish (21.7kg) ml) pituitary extract Dagin - - 100US$ - - 5US$ 10US$ (10mls/20kg) (0.5mls)

4.2.4 Stripping and incubation of eggs

After 6 hours of latency at 25 ± 0.9 ⁰C, the fish was removed from the tanks and stripped. During stripping, about 5-10 eggs from each female were examined for development using acetic acid solution, to ascertain the position of the nucleus (or to find out whether the eggs were ripe). The eggs from each fish were placed on 60 cm x 30 cm trays made of 1 mm mesh size. The eggs were incubated at a temperature of 26.5 ± 1⁰C in small concrete tanks of 100 L. Dissolved oxygen was 6 ±

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1.2 mg L-1and pH range of 7-8. The eggs were incubated until hatching that started at 47.00 hr (51.9 degree hours), and ended after 72.00 hr (79.5 degree hours) in all the trays.

4.2.5 Experiment II

The second experiment was conducted 3 months later (April, 2016) using the same specimens. In this experiment, only females that had ripe running eggs (released by slightly pressing on the belly) were used. The females and males were housed and fed together for one month, following the procedure described in Experiment I above. After seining and identifying the ripe broodstock, 3 females were randomly placed in each of the 6 (4 m x 2 m x 1 m) concrete tanks. Water was allowed to flow through at a rate of one 0.05 litres per second. Nine females from three tanks were stripped after 4 hrs, and the others were stripped after 10 hrs from the time they were distributed into the tanks. The water temperature was maintained at 25 ⁰C. The eggs were incubated at 26.5 ± 1 ⁰C in small concrete tanks of 100 L.

4.3 Measuring parameters

Fertilisation was determined after 30 minutes of incubation, by sampling the eggs to identify early cleavage stage observed under the light microscope. Eggs without observable cleavage were considered unfertilised. Fertilization rate was calculated using the equation.

Working fecundity (number of stripped eggs) was obtained by counting all the eggs for the fish that had fewer eggs; however, for fish with many eggs, a sample was taken and weighed and the number of eggs in the sample was counted. An extrapolation of the sampled eggs was made with the number of eggs in a total weight to obtain the total number of eggs for each female.

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The hatchability was obtained by counting the number of hatched larvae of the total eggs incubated.

4.4 Data analysis

Differences in the mean values among hormonal treatments were analysed using Kruskal-Wallis in SPSS statistical software (IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp, 2013) at 95% confidence level. Kruskal-Wallis was used because the mean values did not conform to the homogeneity rule of variances for ANOVA. Significant differences between treatment means were determined by Mann-Whitney U test. The p value was adjusted for 3pair-wise comparisons (p = 0.05/3=0.0167) made from catfish pituitary extracts, Dagin and water (as control). Data were presented as means and their ranges. For the fish stripped after 4 hr and those after 10hr data was analyzed using student t-test statistic in SPSS. Data were shown as means ± SEM (Standard Error Mean).

4.5 Results

During broodstock feeding, males that were initially raised separately from the females did not exude milt on pressing the abdomen, but on bringing them together in the second month, all males except 3 were found with some milt (varying from 1-5 ml). On the other hand, 26 females out of 45 released some eggs either by pressing on the belly and or by use of a catheter. Twenty two males responded to the hormone by producing clear running milt, which was more than 5 ml. However, 6 males from the control group (with water current) had less than 2 ml of the milt on slight pressure on the abdomen; hence, they were not considered to be suitable for fertilising the ripe females. When the catfish pituitary extract was administered, all females ovulated or produced some eggs when stripped. However, two females, one from a replicate treated with Dagin and another from the replicate in a control tank, did not produce eggs when pressed at the belly.

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Ovulation rate (all those that released eggs) for catfish treated pituitary extract was 100% (15 fish), 93.3% (14 fish) for Dagin treated fish and 93.3% (14 fish) for control water treatments (Table 4.2). However, not all those that produced eggs had the eggs fertilised and or hatched. Fertilisation rates ranged from 0 to 100%. Differences in fertilisation rates were observed among the hormonal treatments (Kruska-Wallis X2 = 15.13, df = 2, p < 0.001). Mean fertilisation rate for females treated with catfish pituitary was 80.27%, and was not significantly different from 40.8% for those treated with Dagin (p > 0.0.0167) but was significantly different from 12.60% for those in control tanks (U = 24.5, p < 0.001). There was no significant difference in fertilization rates between females treated with Dagin and those in control experiment (p > 0.0167).

Table 4. 2 : Measured parameters of second generation B. altianalis females induced under Captivity Parameters Pituitary extracts Dagin Control Ovulation rate (released eggs on 100% (15) 93.3% (14♀) 93.3% (14♀) pressing belly) working fecund (no. eggs per fish) 2314.40b (3604) 1207.37ba (3020) 416.47a (3020) Mean fertilisation (%) 80.27b (100) 40.80ba (100) 12.60a (100) Mean hatchability (%) 42.20b (100) 27.44ba (91) 9.58a (62) Values in parenthesis designate ranges and different superscripts across rows show significant differences

There were differences in mean hatchability among treatments (Kruskal-Wallis X2 = 9.16, df = 2, p < 0.001). Hatchability mean value for females treated with catfish pituitary was 42.20% and was not significantly different from the mean of 27.44% for females treated with the Dagin (p < 0.0167). It was, however, significantly higher than 9.58 for females that had not received hormonal treatment (U = 46.0, p < 0.004). The difference between hatchability means for Dagin treatments and the control treatments mean (no hormone) were not significant (p > 0.0167). The ranges were highly variable in each treatments (Table 4.2).

There were differences in working fecundity (number of eggs produced per fish) among all the treatments (Kruskal-Wallis X2 = 14.64, df = 2, p < 0.01). The mean working fecundity was 2314.40 for females treated with catfish pituitary, 1207.73 for females treated with Dagin, and 416.47 for

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those in control treatment. Fecundity was not significant between females treated with catfish pituitary extract and those treated with Dagin (p > 0.0167). Similarly, significant differences were observed between the females treated with catfish pituitary extract and the control treatment (U = 20.5, p < 0.001). No significant differences were observed between females treated with Dagin and the control treatment (p > 0.0167).

In experiment II, the mean fertilisation rates for females stripped after 4 hrs (86.67 ± 4.04%) was comparatively higher than that for females stripped after 10 hrs (81.77 ± 5.03%); but were not significantly different from each other (p > 0.05). However, significant difference in mean hatchability was observed between the females stripped after 4 hrs (100 degree hours) and those that were stripped after 10 hrs (250 degree hours) (t16 = 3.91, p < 0.001). Hatchability was 72.67 ± 5.63% for the females stripped after 4hrs and 37.17 ± 7.12% for those stripped after 10 hr. Working fecundity was not significantly different between females stripped after 4hrs and those striped after 10hrs (p > 0.05). Average number of eggs per females after 4hrs was 2308.89 ± 301.554 and that of the females stripped after 10hr was 1926.33 ± 252.335.

4.6 Discussion

Fish species are largely seasonal breeders with spawning only occurring when the environmental cues are suitable to ensure survival of their offspring (Abidin, 1986; Aruho et al., 2013; Lam, 1983; Lowe- McConnell, 1979; Poulsen & Valbo-Jorgensen, 2000; Rutaisire et al., 2013). Spawning in the tropics is largely triggered by rains which increase flooding, favorable temperature and availability of food for the larvae to be hatched (Bruton, 1979; Munro, 1990). Gonadal maturation and egg ripening is a product of hormonal mediation with the environmental cues (Munro, 1990). In domestication of fish in captivity seasonal breeding is not a factor of massive seed strategy for commercialization of fish species; hence a constant year round production must be facilitated. This is possible by artificially manipulating the hormones. Results from this study indicated that, the fertilization rates, the working fecundity and hatchability rates were notably high when catfish pituitary extract was used compared to Dagin in a priming dosage. Pituitary extracts have been effective in inducing spawning in many migratory fishes (Zaniboni-Filho & Weingartner, 2007) including Brycon orbignyanus (Felizardo, Murgas, Drumond & Silva, 2010), Astyanax bimaculatus (Felizardo et al., 2012) and also in carps

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(Drori et al., 1994; Harvey & Carolsfeld, 1994; Yaron, Bogomolnaya & Levavi, 1984). But advancement in production of hormonal analogues, suggests that the development and introduction of analogues with anti-dopamine agents has become much easier and easily facilitate spawning with a single dosage in some fish (Drori et al., 1994; Yaron et al., 2009). In spite of the fact that there was no significant differences in fecundity, fertilization, and hatchability between fish treated with catfish pituitary and Dagin, the performance with Dagin in this study was generally not as good as that for pituitary extracts (Table 4. 2). This was probably because the hormone was insufficient or required more degree hours to have a positive effect. The dosage used was a standard prescription for carps as recorded on vials (Kibbutz Gan Shmuel Fish Breeding Centre, Israel; Drori et al., 1994), but it is probable that by the time of stripping it could not facilitate adequate ripening of eggs compared to the dosages used for catfish pituitary extracts. The effective dosages of hormones to facilitate ripening of mature females may vary with fish species (Marte, 1989). Hence, the effective levels of Dagin for this species could further be investigated. Nevertheless, it was apparently noted that a dosage of 0.014 g ml-1 made in 5% saline solution and provided in two consecutive dosages separated by 6 hrs (250 degree hours including the 4 hrs of resting) was effective in facilitating ovulation in B. altianalis. Hence, the use of catfish extracts is preferable given its low cost and availability in the region.

The very high variation of ranges in fertilization and hatchability rates observed during induced spawning (Table 4.2) may be reflective of the varying levels of egg maturation in B. altianalis. Barbus altianalis is a batch spawner with eggs developing in batches, one batch after the other (Rutaisire et al., 2015). In the event of induced spawning, the egg batches do not ripen at the same time. Whereas some eggs will be recruited to replace ovulated ones, if the ovulated eggs are held in the ovary for long, they will become overripen and at the time of striping they may not be viable. A similar situation was reported in other batch spawners such as Scophthalmus maximus L (McEvoy, 1984), in Gadus morhua (Kjesbu, 1989) and Hippoglossus hippoglossus (Bromage et al., 1994). This is typical of batch spawners and poses a challenge in induced spawning because the deterioration of egg quality is much faster, especially with spawners in tropics (Bromage et al., 1994). In females where fertilisation was very low or even zero, the produced eggs had nuclei in the middle of the eggs (observed when acetic acid was applied to ascertain the position of the nucleus). This situation was,

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especially observed in fish which had very few eggs, indicating that by the time of stripping the eggs had not matured to the level of ovulation. This subsequently affected the hatchability. However, there were instances where in some females the fertilisation rates were high, yet the hatchability was very low. This was attributed to over-ripening (Bromage et al., 1994; Kjesbu, 1989; Kjorsvik, Mangor- Jensen & Holmefjord, 1990; Thorsen, Trippel & Lambert, 2003).

The use of acetic acid to clear the eggs for nucleus observation revealed that no nucleus was observed and the eggs disintegrated easily when touched. This confirmed that the eggs became overripe. Over ripening was largely observed in females that produced eggs upon application of very slight pressure on the abdomen prior to induced spawning. This suggested that such fish did not require prior hormonal application and in the subsequent spawning experiment (experiment II), those fish were not induced with hormones. One of the biggest challenges in induced spawning of B. altianalis is that the fish does not show clear signs of breeding characteristics or bulging belly as observed in other cultured cyprinids, such as the Chinese and Indian carps (Jhingran & Pullin, 1985). Hence, the only option is to use a catheter and or pressing the belly to identify mature females.

In experiment II, when the females with some ripe running eggs were subjected to varying incubation latency period, the best fertilisation and hatchability rates were produced with those stripped after 4 hrs (100 degree hours), without inducing them to spawn. The egg viability reduced when the eggs were stripped at 10 hrs (250 degree hours). This also indicated deterioration of the egg quality, i.e. the eggs were slowly becoming overripe. At 100 degree hours, the eggs nuclei were observed to be clearly at the animal pole, hence the majority of the batch eggs were viable. Various experimental reports established a minimum period by which the broodfish can retain viable ovulated eggs in their ovary. In common carp Cyprinus carpio and Hypophthalmichthys nobilis, eggs were found to remain viable after ovulation for between 50-80 minutes and 30-45 minutes in Ctenopharyngodon idella (Rottman et al., 1991). In species such as Rainbow trout, Oncorhynchus mykiss, eggs remain viable much longer (approximately 7 days) after ovulation but this period can be shorter at between 4-6 hrs in Atlantic halibut Hippoglossus hippoglossus (Bromage et al., 1994). The delay to strip and fertilise the ovulated eggs will trigger overripening. It was clear in experimental study II, that by the time the running eggs in B. altianalis were observed at the beginning of the experiment they needed a shorter period of time to be stripped and fertilized before they became overripe. However, this study did not 79

ascertain the actual period of egg viability after ovulation and further investigation is required to confirm the actual egg viability period. Nevertheless, this was a cheap strategy of breeding B. altianalis because no hormones were used in experiment II, but is limited to seasonality. It was observed that the majority of fish with such running eggs when pressed on the belly were largely present during the rainy season. Most probably, this seasonal phenomenon observed with seasonal spawners is not only influenced by the presence of food availability alone (Bye, 1984; Bromage et al., 1994; Lam, 1983; Lowe-McConnell, 1997), but is imprinted and was still carried in the second generation of B. altianalis held in captivity. This phenomenon was also reported in some cultured species such as Atlantic cod Gadus morhua L. (Ottera et al., 2012). This became a challenge to all year round production concept of seed in captivity. However, farmers still have an opportunity to breed this fish with reduced costs during the rainy seasons (breeding season), but can as well use catfish pituitary extract to induce spawning whenever the fingerlings are required.

The successful induced spawning followed a process of bringing together males and females and feeding them in the same pond. This suggests a courtship behavior which facilitated maturation of gonads. The presence of males around females has been found to facilitate gonadal maturation and development in a number of cultured fish including the Indian carps (Nandeesha et al., 1990), the Chinese carps (Jhingran and Pullin, 1985), the African catfish Clarius gariepnus (Waal, 1974) and the Channel catfish Ictalurus punctatus (Tucker & Robinson, 1990). In Chinese carps however, such as Ctenopharyngodon idella, Hypophthalmichthys molitrix and Aristichthys nobilis, induced spawning may be successful without courtship or pairing the males and females (Woynarovich & Horvath, 1980). In Cyprinus carpio, courtship was observed when a priming dosage of pituitary extract was provided, hence facilitating ovulation (Drori et al., 1994). However, no young ones were physically observed in the ponds indicating that the pond is not a suitable area for B. altianalis spawning.

This study has confirmed that the males with running milt are readily available whenever they are needed and this is never a constraint in induced spawning of B. altianalis. Their hormonal dosaging is irrelevant provided they are brought together with female prior to spawning. The technique of raising the fish together as observed in this study was found to be a pre-requisite to spawning B. altianalis and must be included in a spawning protocol for use by prospective farmers or hatchery producers. 80

4.7 Conclusion

This study has ascertained that the use of catfish pituitary extracts (0.014 g ml-1 made in 5% saline solution) administered in two consecutive dosages (priming and resolving) is an effective and a cheaper option in inducing B. altianalis to spawn. However, more costs are reduced when ripe running females are only facilitated to release eggs without any hormonal application for a latency period of 100 degree hours using only flashing (running) water, especially during the rainy season. Prior to spawning, it is inevitable to bring the fish together in order to facilitate maturation and ripening of both eggs and sperms. The study has also registered successful completion of the breeding cycle in captivity, a good strategy to guarantee continued production of seed and selection for better broodstocks. It is recommended that an investigation should be conducted to ascertain the actual period of egg viability after ovulation. Despite the high cost of using Dagin, it may still be necessary to determine the appropriate dosage of Dagin for induced spawning in B. altianalis because the prescribed standard for carps used in this study did not perform well.

4.8 Acknowledgment

We acknowledge financial support from the Agriculture Technology Development and Advisory Services project (ATAAS), funded by Government of Uganda through a facility by the World Bank managed locally by the National Agriculture Research Organization (NARO). We are grateful to Ssenya fish Farm for allowing us to conduct the study on their farm in Lwengo district. We thank Kimera Bridget the project technician for her effort in feeding and participating in sampling activities at the Farm.

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4.9 References

Afzal, M. R, A., Akhtar, N., Ahmed, I. F., Khan M., & Qayyum, M. (2008). Growth performance of Aristichthys nobilis (Richardson) in monoculture system with and without supplementary feeding. Pakistan Veterinary Journal, 28(2), 57-62.

Aruho, C., Basiita, R. K., Kahwa, D., Bwanika, G., & Rutaisire, J. (2013). Reproductive biology of Bagrus docmak in the Victoria Nile, Uganda. African Journal of Aquatic Science, 38(3), 263- 271.

Bromage, N. (1998). Broodstock management and the optimisation of seed supplies. Aquaculture Science, 46(3), 395-401.

Bromage, N., Bruce, M., Basavaraja, N., Rana, K., Shields, R., Young, C., ... & Gamble, J. (1994). Egg quality determinants in finfish the role of overripening with special reference to the timing of stripping in the Atlantic halibut Hippoglossus hippoglossus. Journal of the World Aquaculture Society, 25(1), 13-21.

Bruton, M. N. (1979). The breeding biology and early development of Clarias gariepinus (Pisces: Clariidae) in Lake Sibaya, South Africa, with a review of breeding in species of the subgenus Clarias (Clarias). The Transactions of the Zoological Society of London, 35(1), 1-45.

Bye, V. J. (1984). The role of environmental factors in the timing of reproductive cycles. Fish reproduction: strategies and tactics, 187-205.

Chaudhuri, H. (1976). Use of hormones in induced spawning carps. Journal of the Fisheries Research Board of Canada, 33, 940-7. de Graaf,G., & Janssen H. (1996). Artificial reproduction and pond rearing of the African catfish Clarius gariepinus in subsahara Africa a hand book: FAO fisheries technical paper no. 362 (pp. 1-73). Rome, Italy: FAO.

Drori, S., Ofir, M., Levavi-Sivan, B., & Yaron, Z. (1994). Spawning induction in common carp (Cyprinus carpio) using pituitary extract or GnRH superactive analogue combined with metoclopramide: analysis of hormone profile, progress of oocyte maturation and dependence on temperature. Aquaculture, 119(4), 393-407.

82

Harvey, B., & Carolsfeld, J. (1993). Induced Breeding in Tropical Fish Culture. IDRC, Ottawa. 145 pp.

Felizardo, V. D. O., Murgas, L. D. S., Drumond, M. M., & Silva, J. D. A. (2010). Insemination dose used in the artificial fertilization of piracanjuba ovocyte (Brycon orbignyanus). Revista Ceres, 57(5), 648-652.

Felizardo, V. O., Murgas, L. D. S., Andrade, E. S., Lopez, P. A., Freitas, R. T. F., & Ferreira, M. R. (2012). Effect of timing of hormonal induction on reproductive activity in lambari (h). Theriogenology, 77(8), 1570-1574.

Funge-Smith, S., & Phillips, M. J. (2001). Aquaculture systems and species. In R. P. Subasinghe, P. Bueno, M. J. Phillips, C. Hough, S. E. McGladdery, J. R. Arthur (Eds), Technical Proceedings of the Conference on Aquaculture in the Third Millennium (pp 129-135). Bangkok / Rome: NACA/ FAO.

Indira, T., Damodaran. R., & Priyadarshini, R. (2013). Comparative Study of Synthetic Hormones Ovaprim and Carp Pituitary Extract Used in Induced Breeding in Indian Major Carp. Supplement to Advanced Bio Technology, 12(07), 49-52.

Jhingran, V. G. & Pullin, R. S. V. (1985). A Hatchery Manual for the common, Chinese and Indian Major Carps. Asian Development Bank and International Center for Living Aquatic Resources Management (ICLARM) Studies and Reviews, 11, 1 – 191.

Kjesbu, O. S. (1989). The spawning activity of cod, Gadus morhua L. Journal of Fish Biology, 34(2), 195-206.

Kjorsvik, E., Mangor-Jensen, A., & Holmefjord, I. (1990). Egg quality in fishes. Advances in Marine biology, 26, 71-113.

Lam, T. J. (1983). Environmental Influences on Gonadal Activity in Fish. Fish physiology, 9, 65-116.

Levavi-Sivan B., Bogerd, J., Mananos, E. L., Gomez, A., & Lareyre, J. J. (2010). Perspectives on fish gonadotropins and their receptors. General and Comparative Endocrinology, 165, 412-437.

Lowe-McConnell, R. H. (1979). Ecological aspects of seasonality in fishes of tropical waters. In Symposia of the Zoological Society of London (Vol. 44, pp. 219-241).

83

Mahfuj, M. S., Hossein, M. A., & Sarower, M. G. (2012). Effect of different feeds on larvae development and survival of ornamental Coi carp, Cyprinus carpio larvae in laboratory condition. Journal of Bangladesh Agricultural University, 10 (1): 179-183.

Marte, C. L. (1989). Hormonal –induced spawning of cultured tropical fin fishes. Advances in tropical aquaculture, 9, 519-539.

Mwanja, M., Rutaisire, J, Ondhoro, C., Ddungu, R., Aruho, C. (2015). Current fish hatchery practices in Uganda: the potential for future investment. International Journal of Fisheries and Aquatic Studies, 2(4), 224-232.

McEvoy, L. A. (1984). Ovulatory rhythms and over-ripening of eggs in cultivated turbot, Scophthalmus maximus L. Journal of Fish Biology, 24(4), 437-448.

Miah, M. I., Mamun, A. A., Khan. M. M. R., & Rahman. M. M. (2008). Dose optimization with pituitary gland hormone for induced breeding of Bata fish (Labeo bata). Bangladesh Journal of Animal Sciences, 37 (1): 70 – 77.

Munro, A. D. (1990). General introduction. In: A. D. Munro, A. P. Scott & T. J. Lam (Eds.), Reproductive seasonality in teleosts: environmental influences, (pp. 1-11). Boca Raton, Florida, USA: CRC Press.

Nagahama, (1994). Endocrine regulation of gametogenesis in fish. International Journal of Developmental Biology, 38(2), 217-29.

Nandeesha, M. C., Rao, K. G., Jayanna, R., Parker, N. C., Varghese, T. J., Keshavanath, P., & Shetty, H. P. (1990). Induced spawning of Indian major carps through single application of Ovaprim- C. In R. Hirano & I. Hanvu (Eds.), The Second Asian Fisheries Forum (pp. 581-585). Asian Fisheries Society, Manila, Philippines

Ottera, H., Agnalt, A. L., Thorsen, A., Kjesbu, O. S., Dahle, G., & Jorstad, K. (2012). Is spawning time of marine fish imprinted in the genes? A two-generation experiment on local Atlantic cod (Gadus morhua L.) populations from different geographical regions. ICES Journal of Marine Science: Journal du Conseil, fss135.

Peter, R. E & Yu, K. L. (1997). Neuralendocrine regulation of ovulation in fishes. Basic and applied aspects. Revisions in Fish Biology and Fisheries, 7, 173-197.

84

Poulsen., A. F. & Valbo-Jorgensen, J. (2000). Fish migrations and spawning habits in the Mekong mainstream - A survey using local knowledge. Assessment of Mekong Fisheries: Fish Migrations and Spawning and the Impact of Water Management Project (AMFC) Vientiane, Lao P. D. R. February 2000. 132 pp

Rottman, R. W., Shireman, J. V., & Chapman, F. A. (1991). Techniques for taking and fertilizing the spawn of fish, Publication No. 426. Stoneville, Mississippi: Southern Regional Aquaculture Center (SRAC).

Rutaisire, J., Levavi-Sivan. B., Aruho, C & Ondhoro, C. C. (2013). Gonadal recrudescence and induced spawning in Barbus altianalis. Aquaculture Research, 46(3): 669-678. DOI: 10.1111/are.12213

Stickney R. R. (2005). Aquaculture: An introductory text, CABI Publ, Cambridge, USA p. 256

Thorsen, A., Trippel, E. A., & Lambert, Y. (2003). Experimental methods to monitor the production and quality of eggs of captive marine fish. Journal of Northwest Atlantic Fishery Science, 33, 55-70.

Tucker, C. C., & Robinson, E. H. (1990). Channel catfish farming handbook. Springer Science & Business Media.

Waal, B. C. W. (1974). Observations on the breeding habits of Clarias gariepinus (Burchell). Journal of Fish Biology, 6(1), 23-27.

Weltzien, F. A., Norberg, B., & Swanson, P. (2003). Isolation and characterization of FSH and LH from pituitary glands of Atlantic halibut (Hippoglossus hippoglossus L.). General and Comparative Endocrinology, 131 (2): 97–10.

Woynarovich, E., & Horva, L., (1980). The artificial propagation of warm-water finfishes – a manual for extension, FAO Fisheries Techenical Paper 201(pp.1- 183 p). Rome, Itally: FAO.

Yaron, Z., Bogomolnaya, A., & Levavi, B. (1984). A calibrated carp pituitary extract as a spawning- inducing agent. Research on Aquaculture, 8, 151-168.

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Yaron Z., Bogomoinaya, A., Drori, S., Biton, I., Aizen, J., Kulikovsky, Z. and Levavi-Sivan, B. (2009). Spawning induction in the carp: past experience and future prospects-a review. Israel Journal of Aquaculture. Bamidgeh 61(1): 5–26.

Zaniboni-Filho, E., & Weingartner, M. (2007). Induced breeding in migratory fishes. Revista brasileira De reproducao Animal Belo Horizante, 31(3), 367-373.

Zohar, Y., & Mylonas, C. C. (2001). Endocrine manipulations of spawning in cultured fish: from hormones to genes. Aquaculture, 197(1), 99-136.

Zohar, Y., Munoz-Cueto, J. A, Elizur, A., Kah, O. (2010). Neuroendocrinology of reproduction in teleost fish. General and Comparative Endocrinology, 165, 438–455.

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CHAPTER FIVE Optimal factors for egg hatchability and larvae development of Barbus altianalis under captivity

C. Aruho, R. K. Basiita, B. Kimera, F. Bugenyi and J. Rutaisire.

5.0 Abstract

One of the most critical environmental factors in early development of fish is temperature, and the interaction of temperature with other factors to maximize physiological functioning processes for optimal larvae development and growth. Five experiments were conducted to identify the optimal temperature, light, water depth and hatching facility suitable for hatchability of embryos and larval growth of B. altianalis. In experiment I, eggs were incubated at 24⁰C, 27⁰C and 30⁰C with and without aeration. This was replicated after eight months in a second trial. In experiment II, egg batches were incubated in conical jars, re-circulating and glass tank systems at 27⁰C with regulated aeration. In experiment III, eggs were incubated at lux (lx) values of 54.20 ± 5.45 and 10701.80 ± 2224.29 during the day and all were kept in the dark at night. In experiment IV, eggs were spread on trays and some of these trays placed at the bottom of glass tank (60cm depth) and others at the surface. In experiment V larvae were subjected to 24⁰C, 27⁰C, 30⁰C and 31⁰C at day six after hatch (DAH) when external feeding began. Results indicated that optimal embryo hatchability ranged from

92.925±2.01 to 94.10 ± 2.92% (with no aeration) and from 89.325±1.04 to 91.77 ± 0.81% at 27⁰C (with aeration) in both trials. Better hatchability of embryos was obtained when re-circulating (84.3%) and glass tank systems (80.3%) were used (p > 0.05) and reduced significantly (p < 0.05) when conical jars were used (37.2%). Significant differences were noted in larvae growth (p < 0.05).

Optimal larvae growth and high survival was attained at 30⁰C and 27⁰C when the larvae were 195.03 ± 47.62mg (78.96 ± 2.04%) and 158.61 ± 33.43mg (81.24 ± 1.55%) respectively. No significant differences were observed in egg hatchability at high or low light intensity, and for water depth (p > 0.05). These results will guide culturalists to produce sufficient and quality seed to promote commercialization of the newly domesticated B. altianalis

Key words: incubation, eggs, larvae, hatching facility, temperature, light

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5.1 Introduction

Barbus altianalis, locally known as Kisinjja is a freshwater species inhabiting an inter-lacustrine- riverine system of Lake Edward and the Upper Victoria Nile in Uganda (Greenwood, 1966). B. altianalis is one of the candidate aquaculture species that was successfully induced to spawn artificially in Uganda (Rutaisire et al., 2015). Efforts to domesticate B. altianalis were made in response to the high fish demand and declining numbers in the wild largely due to increased fishing pressure. B. altianalis is a vital source of animal protein and income for communities in Central and Western regions of Uganda as well as the neighboring areas of Democratic Republic of Congo (DRC). Inspite of the successful artificial spawning, the species has not been fully commercialized given that trials and development of seed production technologies for quality seed in sufficient quantities is still in its early stages. One of the challenges being encountered in experimental trials conducted at the Aquaculture Research and Development Centre (ARDC), Kajjansi and on some commercial farms, is the high mortalities of B. altianalis embryos that occur during egg incubation and larval development (Rutaisire et al. 2015; unpublished data ARDC-Kajjansi). Although there are many environmental factors that could affect ontogenetic processes of fish species, temperature is one of the most critical and widely acceptable environmental factor that affects incubation of embryos and larvae development in fishes (Bjornsson, Steinarsson & Oddgeirsson, 2001; Haylor & Mollah, 1995; Kokurewicz, 1970; Kucharczyk, Luczynski, Kujawa & Czerkies, (1997); Rombough, 1997). Through varying rates of development, temperature can influence the size of the organisms at which ontogenetic development occurs (Green & Fisher, 2004; Haylor & Mollah, 1995). Incubation temperature will also affect the ability of larvae to swim in search for food and escape from predators (Johnston, Vieira & Temple, 2001).

The effect of temperature on incubation of eggs and larvae development is species specific (Herzig & Winkler, 1986; Laurel & Blood, 2011). Temperature has been found to reduce or delay incubation period in various species. Both high and low temperatures could impair physiological process during embryonic and larval development leading to high mortalities even within their zone of tolerance (Blaxter, 1991; El-Gamal, 2009; Hokanson, 1977). For instance Keckeis (2000) observed three periods of elevated mortalities in a rheophilic cyprinid Chondrostoma nasus that included, early mortality (shortly after fertilization), hatching mortality and starvation mortality (when yolk reserves

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are depleted in absence of external feeds). In B. altianalis some critical stages of embryonic development seem to have been affected more than others even when the temperature was maintained at 27⁰C (observation by authors). However, these stages have not been identified. In three cyprinid species of genus Leuciscus, varying thermal modifications made during incubation, directly and differently influenced the incubation period, hatching rates, hatching period, yolk sac re-absorption, the final size, survival and developmental rates of embryos and early larvae ontogenetic stages (Kupren, Mamcarrz & Kucharczyk, 2011). In Solea senegalensis it was observed that the egg incubating temperature had a significant effect on the occurrence of abnormalities in larvae (Dionisio et al., 2012). In the common carp, Cyprinus capio optimal temperature ranges have been found to play an important role in improving hatchability, growth, food intake and defense mechanism (El- Gamal, 2009). Thus in fish culture it is inevitable to determine the appropriate temperature regimes to optimize larvae rearing protocols and reduce the cost of production (Dionisio et al., 2012; Kupren et al., 2011; Wolnicki et al., 2004). Some species could however tolerate wider temperature variations as observed in bream Abramis brama (Kucharczyk et al., 1997; Moran et al., 2010) and the cultured African catfish Clarias gariepinus (Haylor & Mollah, 1995). Generally it has also been noted that some cyprinids larvae show a higher preference of temperature than embryos (Wolnicki & Appelbaum, 1993). However this is yet to be investigated in B. altianalis. Other factors that could affect hatchability in some fish species include light and hatching method (Brooks, 1994; Brooks et al., 1997; Watson & Chapman, 2002). In addition, mechanical agitation through aeration may also negatively affect eggs of some species during incubation (Jensen & Alderdice, 1989; Watson & Chapman, 2002).

Fish species in the wild will only spawn when environmental conditions are favorable for the survival of offspring (Bromage, 1998). The favorable range of water temperature for embryonic development is strongly linked to the reproduction temperature of a given species (Kupren et al., 2011). Therefore in captivity, the natural conditions are simulated to those in the wild in order to ensure successful spawning and maximum survival of offspring. Appropriate optimum conditions ought to be determined to ensure that good quality and sufficient quantities of seed are produced and accessed by farmers for commercial production given that aquaculture success requires all year round production of seed for farms (Bromage, 1998). Hence, the present study identified the critical embryonic stages

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affected by temperature, light, aeration agitation and hatching facility in order to identify optimal ranges and suitable facilities for improved hatchability of B. altianalis embryos, growth and survival of larvae.

5.2. Methods and Materials

5.2.1 Optimal factors for egg hatchability

5.2.1.1 Experiment I: Effect of Temperature and Aeration on Egg Hatching

Ripe brood fish were collected from the wild and transported to the Aquaculture Research and Development Centre (ARDC) at Kajjansi, where they were acclimatized before spawning. Females were induced, stripped and fertilized according to the procedure stipulated by Rutaisire et al., (2015). To ensure control of individual female variations, eggs from one female were incubated. Eggs ranging from 150-300 were randomly placed on floating wooden trays in each of the 50 litre glass tanks and incubated at different temperatures and subjected to either aeration (O2) or no aeration (O1). Six treatments were constituted 1) O1T1 (aeration at 24o C); 2) O2T1 (no aeration at 24 o C); 3) O1T2 (with aeration at 27o C), 4) O2T2 (with no aeration at 27o C); O1T3 (with aeration at 30o C) and O2T3 (with no aeration at 30o C). Each of these treatments had 4 replicates. For all aerated treatments, the air flow was measured and maintained at an average of 640.67 ± 119.3 Pa (partial pressure) and at an air velocity of 38.34 ± 1.0 ms-1 (metres per second), using an airflow meter (PCE- PFM 2; manufacture by PCE instruments UK). Temperature was maintained by thermostatic heating rods (Sera Aquarium heater thermostat; sera D 52518, Heinsberg) with a fluctuation degree of 1 unit (T±1oC). The dissolved oxygen (DO) levels were monitored using a DO meter (Yellow Spring Instrument YSI, model 556). During the egg incubation, embryonic development was monitored by sampling eggs in the additional 5th replicate for each treatment. The eggs were sampled in the first minute of fertilization and at intervals of 10 minutes until after 30 minutes. Eggs were then sub- sampled every 2 hours until 15 h when sampling was now done every 6 h until they hatched. Sampled embryos were examined under the microscope (model Leica DM 500, Made by Microsystems Switzerland Ltd) to identify the embryonic stages. The number of dead eggs/embryos at each stage was recorded and the survival rates determined. The hatching period by which 99 percent of the embryos hatched was recorded for each treatment. This experiment was repeated in a second trial

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(trial 2) after six months for validation following the same procedure. The first experiment is referenced as (trial 1) in this study.

5.2.1.2 Experiment II; the Effect of Hatching Facility on Egg Hatching

Eggs were incubated in three differently designed hatching facilities; circular plastic tanks (re- circulatory tank system), glass tanks and hatching conical shaped jars (Figure 5.1). In the circular plastic tank system, water was aerated in another tank before it was continuously pumped into the hatching tanks (200 litres) holding trays with eggs. Water dripped from pipes close to the sides of the circular plastic tanks. Water was replaced with fresh water to a third of the water in tanks once in 24 h. In glass tanks system little aeration (500.5 ± 82.3 Pa; 26 ± 5 MS-1 flow rate) was continuously provided with air stones placed in the corner of the tank and covered with small trays to avoid any agitation from the bubbling. Water in glass tanks was replenished with fresh water to a third of the water in glass tanks once every 24 h. In the hatching plastic conical jars aerated water was allowed to continuously flow upwards into the jar at a flow rate 0.0083 Ls -1 in each jar. The water was re- pumped back into the common tank which was aerated. Water was replaced once in 24 h. After arrangement of the hatching facilities, eggs from one female were randomly placed on the trays and in each hatching facility. About 10-15 eggs per litre of water in each container were incubated.

Treatments were made in quadruplicates. The temperature was maintained at 27⁰C ± 1 by thermostatic heating rods and the dissolved oxygen levels DO were maintained at an average range of 8.2 ± 1.3mgL-1 in all facilities.

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a b

c

Figure 5.1: hatching facilities a) circular plastic tanks. b) Glass tank system. C) Plastic conical jars

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5.2.1.3 Experiment III: Effect of Light on Egg Hatchability

Eggs were randomly distributed in six glass tanks of 50 litres each. Three tanks were wrapped in black polyethene material (1000 microns) and covered ¾ of the open side with a soft board (piece of wood) to reduce the amount of light and to allow exchange of air (practiced by some hatchery producers of Clarius gariepinus larvae). Three other tanks were left in open light. All the tanks received 12 h of darkness and during the day the tanks covered with polythene bags were treated to 54.20 ± 5.45 lx, compared to those that received a light of 10701.80 ± 2224.29lx. The light was measured by a digital lux metre (model Ms-1300 manufactured by voltcraft). Dissolved oxygen DO was kept in an average range of 8.2 ± 0.9mgL-1. Temperature was maintained at 27 ± 1oC using thermostatic heating rods. The number of dead eggs was counted to determine the hatchability rates.

5.2.1.4 Experiment IV: Hatching Water Level or Water Depth

In this experiment, eggs were placed on hatching trays and held at the bottom of the glass tank of 50 litres each and 60 cm depth and in the other tanks the eggs were placed on hatching trays and left to float, about 1cm from the surface (as this is a common practice in hatcheries for cultured species in the region). The two treatments each had three replicates. Temperature was maintained at 27 ± 1⁰C with thermostatic heating rods and dissolved oxygen was maintained by constant aeration (498 ± 73.3 Pa; 24 ± 3 ms-1 flow rate) at 7.78 ± 1.2mgL-1. The time taken for embryos to hatch in each tank was recorded and the dead embryos were counted to determine hatchability.

5.2.2 Optimal water temperature for larval survival and growth

5.2.2.1 Experiment V: Effect of temperature on larval survival and growth

To determine the optimal water temperature for the growth of B. altianalis larvae, hatched larvae (3 ±

1g) were randomly distributed into 50litre glass tanks at temperatures of 24, 27, 30, 31⁰C with four replicates each. The larvae were fed on a combination diet of Moina and dry feed (57% crude protein) until 40 days after hatch (DAH) when they started developing scales. Feeding was done to satiation at 8.00 h, 12.00 h and 17.00 h daily. Aeration was provided by an aeration pump and dissolved oxygen was maintained at 7.0 ± 1.2 mgL-1 and pH of between 7 and 8. Tank bottoms were 93

cleaned daily at 7.00 h and 16.00 h before feeding to avoid accumulation of solid wastes and ammonia. Also water exchange was done to replace half of the water in the tank after cleaning. Any dead larvae were removed and counted to determine the survival rates for each treatment. The lengths and weights were recorded at each sampling that was conducted every 14 days. Total Lengths (TL) was measured to the nearest 0.1mm using a calibrated ruler and the wet weight (g) of each larva was recorded to the nearest 0.001g. Water was cleaned from larvae by a tissue paper before weighing them.

5.2.3 Measured growth parameters

The hatchability percentage, weight gain percentage, specific growth rates (SGR) and survival were calculated using the following equations:-

Number of live embryos after hatch i) Hatchability (%) = ------x 100 Total number of eggs incubated

Final Weight FW-Initial Weight IW ii) Weight gain W % =) ------x 100 Initial Weight IW

In FW- In IW iii) Specific growth rates SGR % per day (daily increment) = ------x 100 Number of culture days Final Number FN iv) Survival (%) = ------x100 Total number TN of stocked fish

Weight-Length relationship =Weight W =aLb where a and b are coefficients, L is the total length (cm), and W is the wet weight in g.

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5.2.4 Statistical analysis

Differences among mean values of treatments were analyzed by a two way Analysis of Variances (ANOVA). Statistical significance between treatments means was determined using Duncan’s test. Relationships between weights and total lengths of larvae and juveniles were calculated by linear regression analysis. Chi-square statistical test (X2) was used to compare the larval survival and mortality frequencies using cross-tabulation (contingency table) analysis technique. Mortalities of embryos between stages at each temperature treatment was analysed by Kruskal Wallis and statistical significant differences between stages were determined by Mann-Whitney U-test. The p value was adjusted from p = 0.05 to p =0.05/10 = 0.005 (where 10 is the total number of combinations of embryo stages at each temperature treatment. Statistical analyses were all performed using IBM SPSS Statistics program for Windows, Version 22.0. (Armonk, NY: IBM Corp, 2013) at 95% confidence level.

5.3 Results

5.3.1 Optimal factors for egg hatchability

5.3.1.1 Experiment I: Effect of Temperature and Aeration on Egg Hatching

Hatchability was generally high and ranged from 84.72 ± 2.92% to 94.10 ± 2.92 % across all temperature treatments in the first experiment (Trial 1) and from 83.150±0.87 to 92.925 ± 2.01 in the second experiment (Trial 2). Hatchability was significantly affected by the temperature variations in both trials (Trial 1: F2, 18 = 7.187, p < 0.005; Trial 2: F2, 18 = 18.416, p < 0.001). The aeration alone did not affect hatchability in both trials (p > 0.05). However, the interaction between temperature and aeration significantly affected hatchability (Trial 1; F2, 18 = 7.219, p = 0.005; Trial 2: F2, 18 = 18.416, p < 0.001). In both trials hatchability reduced with increasing temperature in treatments where aeration was not provided (Figure 5.2). When aeration was provided hatchability was optimum at 27⁰C but reduced thereafter (Figure 5.2). In both trials the highest hatching percentages were achieved at 24⁰C without any aeration (O1T1) and at 27⁰C in aerated (O2T2) treatments but treatment O2T2 had the same performance as in O2T2 and O2T1 in both trials (Table 5.1 and Table 5.2). The least hatchability of 83.150 ± 0.87 in trial 1 and 84.72 ± 2.92% in trial 2 were recorded when the 95

temperature was increased to 30⁰C at a DO of 4.500 ± 0.51 and 4.75 ± 0.24 mgL-1 respectively. Hatchability variations however, were noted in other treatments in trial 1 and 2, i.e. the experimental hatchability results in trial 1 were not exactly the same when the experiment was repeated after six months (Tables 5.1 & 5.2). Significant difference in egg hatchability between embryos in aerated tanks at 24⁰C (O2T1) and those not aerated (O1T1) was observed when the differences in DO were insignificant (Table 5.1 & 5.2). The fastest period for completion of hatchability (at 99 %) was achieved after 63.00 ± 1.77⁰ days at 30oC, while the longest period for hatching was 85.75⁰ days at

24⁰C. The consistent observation made in both experiments (trials) was that where aeration was provided at each temperature there was delayed completion for hatching compared to where the aeration was not provided at the same temperature (Table 5.1 & Table 5. 2).

Table 5. 1: Mean hatchability, dissolved oxygen and degree days for each temperature treatment. Means are shown as, Mean ± SD (Standard deviations)

Treatment Temp. oC Hatchability % DOmgL-1 Maximum mean Degree days Hatching time (h) for 99% hatchability O1T1 24 94.10 ± 2.92a 7.73 ± 0.40a 82.75 ± 0.96 82.75 ± 0.96 O2T1 24 87.10 ± 3.04bc 8.20 ± 0.22a 88.75 ± 0.96 85.75 ± 0.96 O1T2 27 88.87 ± 2.10abc 6.73 ± 0.33b 65.00 ± 0.82 73.13 ± 0.92 O2T2 27 91.77 ± 0.81ab 8.45 ±0.68ca 68.75 ± 0.58 77.06 ±0.65 O1T3 30 84.72 ± 2.92c 4.75 ± 0.24d 51.00 ± 0.82 63.75±1.02 O2T3 30 86.72 ± 3.78c 5.52 ± 0.44e 57.00 ± 0.82 71.25±1.02

Table 5. 2: Mean hatchability, dissolved oxygen and degree days for each temperature treatment. Means are shown as, Mean ± SD (Standard Deviations)

Treatment Temp. oC Hatchability % DOmgL-1 Maximum mean Degree days Hatching time (h) for 99% hatchability O1T1 24 92.925±2.01a 7.725±0.49ab 82.50±0.58 82.50±0.58 O2T1 24 85.825±1.67bc 7.900±0.27a 85.50±1.00 85.50±1.00 O1T2 27 86.725±1.87bc 6.850±0.25b 64.25±1.50 72.28±1.69 O2T2 27 89.325±1.04ab 8.300±0.47a 67.75±1.26 76.23±1.42 O1T3 30 83.150±0.87c 4.500±0.51c 51.00±1.41 63.00±1.77 O2T3 30 85.250±2.50c 5.475±0.31d 55.75±1.70 69.67±2.14

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Figure 5.2: Interaction effect of aeration and temperature on hatchability in B. altianalis

5.3.1.2 Effect of Temperature on Embryonic Developmental Stages (Critical Stages of Embryonic Development)

The percentage mortalities for each egg developmental stage was determined for nine identified embryonic stages during incubation (Figure 5. 3). Temperature did not have a significant effect on survival of embryonic stages (p > 0.005 adjusted for Mann-Whitney U-test). The highest egg mortality however, was observed at late embryo stage for each temperature treatment. The extent of differences in mortalities at this stage within each temperature treatment varied (Table 5. 3). The least affected embryonic developmental stage across all temperature treatments was the gastrulation stage. No egg mortality was observed in all treatments at early cleavage (0-3 h.) and morular (3-8 h.) stages of development.

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a b c

d e f

g h i

Figure 5. 3. a) Early cleavage (blastodisc), formation of blastomeres, after 30 minutes of fertilization. b) Morular formed after 3 h. of fertilization. C) Early blastula formation with a carp like structure and a germ ring (6-8 h.). d) Early gastrulation with a germ ring (9-14 h). e) Late gastrulation (15-20 h.). f) Early embryo (15-20 h)-neural tube formation begins. g) Tail bud (17-25 h.). h) Tail free and twitching stage (21-30 h.). i) Late embryo stage (25 h >) Heart beat; palpation and lens formation were observed in this stage. Mgx40

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Table 5. 3: Effect of temperature on embryonic stages during incubation within each temperature treatment.

Mortality (%) at each treatment Temperature O1T1 O2T1 O1T2 O2T2 O1T3 O2T3 treatments (24oC) (27oC) (27oC) (27oC) (30oC) (30oC) Period Embryonic (h) Stages Early 0-3 cleavage 0 0 0 0 0 0 3-8 morular 0 0 0 0 0 0 9-20 Gastrulation 1.71(5.7) 0.33(1.10) 0.48(1.10) 0.55(1.10)a 0 0 early 15-20 embryo 0.13(0.50) 0.83(0.80) 0.98(1.2) 1.40(40) 0.85 (1.2) 0.75(0.9) 17-25 tail bud 0.30(1.2) 2.73(2.10) 0.73(1.4) 0.98(0.90) 4.05(5.43) 2.20(2.8) 21-30 tail free 1.20(1.8) 2.25(3.5) 1.4(0.30) 1.68(2.00) 2.70(1.30) 1.83(1.40) 25> Late embryo 3.00(4) 7.88(5.2) 6.58(5.4) 5.05(3.00) 8.00(4.1) 10.60(5.8) 2 Kruskal-Wallis X - X2=8.31, X2=15.52, X2=13.157, X2=12.23, X2=17.897, X2=16.90 test, df=4 ,p=0.81 ,p=0.002 ,p=0.011 ,p=0.016 ,p=0.001 ,p=0.002 Values shown in Parenthesis designate ranges

5.3.1.3 Experiment II: The Effect of Hatching Facility on Egg Hatchability

Overall the hatching facilities significantly affected hatchability (F (2, 9) = 292.901, p <0.001). Although the hatchability in circular tank system (84.30 ± 3.25%) was better than that for the glass tanks (80.3 ± 2.19%), the differences in egg hatchability from these two hatching facilities were not significantly different (p > 0.05). The lowest egg hatchability was obtained in conical shaped jars (37.20 ± 3.61%) (Table 5.4).

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Table 5. 4: Cross tabulation of survival (hatched) and mortality of B. altianalis embryos in hatching facilities

Survival Mortality (hatched (dead Hatching facility larvae) Embryos) Total Conical flask shaped –up-flowing water Number 343 579 922 % 37.20 62.80 100.00 Circular tanks –re-circulating water system Number 8430 1570 10000 % 84.30 15.70 100.00 Glass tanks non re-circulating system Number 1627 398 2025 % 80.30 19.70 100.00 Total number of survival for all the eggs in all the hatching facilities Number 10400 2547 12947 Percentage survival (hatchability) /mortality for all hatching facilities % 80.30 19.70 100.00

5.3.1.4 Experiment III: Effect of Light on Hatchability

No statistical differences were observed in hatchability between the eggs or embryos that were exposed to light (10701.80 ± 2224.29lx.) and those that were exposed to very low light (54.20 ± 5.45 lx) much of the hatching period (X2= 1.4264, df = 1, p > 0. 05) (Table 5.5). Hatchability was 72. 20 ± 3.91% (Mean ± Standard Error Mean) in treatments exposed to much light and 68.40% ± 4.09% (Mean ±Standard Error Mean) for treatments exposed to very low light.

Table 5. 5: Light effect on Hatchability in B. altianalis

Treatment Survival Mortality Total (Hatched Larvae) (Dead embryos) Light Number 353 136 489 % 72.20 27.80 100.00 No light Number 256 118 374 % 68.40% 31.60% 100.00 Total number Number 609 254 863 survival/mortalities from all the treatments Percentage survival % 70.60% 29.40% 100.00 (hatchability) /mortalities from both treatments 100

5.3.1.5 Experiment IV: Hatching Level (or Water Depth)

There was no significant difference in egg hatchability due to the depth at which the egg hatching trays were placed (x2 = 0.29, df = 1, p > 0.05) (Table 5.6). However the hatchability time for the eggs placed at the bottom was shorter at 72.28 ± 1.06⁰days compared to 77.63 ± 2.05⁰ days for eggs that were placed on the tray at the top of the glass tank.

Table 5. 6: Egg hatchability with the level of water depth

Survival Mortality Treatment (hatched larvae) (Dead Embryos) Total Bottom Number 755 185 940 % 80.3 19.7 100.0% Top Number 688 172 860 % 80.0 20.0 100.0% Total number of survival for Number 1443 357 1800 all the eggs in all the hatching facilities Percentage survival % 80.2 19.8 100.0% (hatchability) & mortality for all hatching facilities

5.3.2 Optimal water temperature for larval survival and growth

Final average larval weight was significantly different among temperature treatments (F3-1295 = 88.706, p < 0.0001). Significant differences in average larval weight were also obtained at each sampling during the growth period (Table 5.7). The least average weight was obtained at 24⁰C and the highest at 30⁰C and was maintained thoughout the experimental period (Table 5.7, Figure 5.4).

Survival rates were notably high at 27⁰C (81.24 ± 0.78%) and 30⁰C (78.96 ± 1.02%) with no significant difference among these two temperature treatments (p > 0.05). Mean larval weight increased with survival as temperature was increased from 24⁰C to 30⁰C and reduced slightly with reduced survival at 31oC (Figure 5.5). Significant differences were observed at different larval treatment temperatures between final specific growth rates (F3-11 = 90.453, p < 0.0001), average 101

larval weight gain (F3-11 = 88.71, p < 0.0001) and the larval survival (F3-11 = 54.04, p < 0.0001) (Table 5.8).

Table 5. 7: Average Larval weight (mg) for B. altianalis larvae at each temperature and each sampling. Values are presented as Means ± Standard Error

Days After Weight (mg) at Weight (mg) at Weight (mg) at Weight (mg) at F- test Hatch 24 oC 31 oC 27oC 30 oC P < 0.0001 6 3.00 ± 1 3.00 ± 1 3.00 ± 1 3.00 ± 1 14 20.35 ± 0.33a 22.16 ± 0.34b 23.15 ± 0.27c 25.34 ± 0.37d F3-832 = 34.520 28 55.33 ±1.23a 57.17 ± 0.95a 58.08 ± 0.73a 67.94 ± 0.95b F3-832 = 37.230 42 101.10 ± 2.74a 114.04 ± 1.96b 115.87 ± 1.52b 133.35 ± 1.85c F3-891 = 41.250 a b c d 56 138.13 ± 2.83 167.31 ± 2.14 158.61 ± 1.56 195.03 ± 2.71 F3-1295 = 88.706 Values with different superscripts a cross the rows are significantly different

Figure 5.4: average larval weight variation with Days after Hatch (DAH) in B. altianalis

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Figure 5.5: variation of final average weight (Means ± Standard Error) and percentage survival within each temperature in B. altianalis larvae

Table 5. 8: Growth parameters of B. altianalis larvae at four treatment temperatures during the culture period. Values are presented as Means ± Standard Error

Growth T24 C T31 C T27 C T30 C parameters ⁰ ⁰ ⁰ ⁰ Specific 3.29 ± 0.02a 3.47 ± 0.01b 3.43 ± 0.01c 3.60 ± 0.01d growth rate (SGR)% Weight gain 4504.46 ± 94.42a 5476.97 ± 41.40b 5187.00 ± 52.03c 6400.97 ± 40.22d WG % Larval survival 69.13 ± 1.64a 63.50 ± 0.90b 81.24 ± 0.78c 78.96 ± 1.02c % Values with different superscripts a cross the rows are significantly different

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5.4 Discussion

Environmental factors are critical parameters that need to be clearly determined for egg hatchability and larval development in culture systems as part of any fish spawning protocol. In the present study, efforts focused on ensuring that the fish’s natural breeding environment was simulated under culture conditions or even improved to maximize survival of offspring following previous recommendations by Bromage (1998 and 2001). Key factors that facilitate egg hatchability and larvae survival under culture systems including temperature, light regimes, dissolved oxygen profiles, salinity and hatching facilities among others have been well studied in other species (Alami-Durante et al., 2000; Blaxter, 1991; Brooks et al., 1997Dabrowski, 1984; Dahlberg, 1979; Dimichele & Taylor, 1980; Downing & Litvak, 2001; Jhingran & Pullin, 1985; Kamler, 2008; Villamizar et al., 2011). Although there were differences in hatchability among temperature treatments in the present study, the hatchability was generally high (ranging from 83.15 0± 0.87 to 94.10 ± 2.92) in all the various temperature treatments and in both trials. This high hatchability probably suggests that, the temperature treatment range used in the present study was suitable for B. altianalis. Noteworthy though was the interaction between temperature and aeration that significantly affected hatchability. When aeration was provided hatchability increased from a temperature of 24⁰C to 27⁰C and reduced thereafter. The highest hatchability for non aerated treatments was achieved at 24⁰C but it was reducing when temperature was increased. Aeration of eggs in most hatcheries is provided by bubbling air through water to ensure sufficient dissolved oxygen (DO) for hatchability. However, in this study aeration had some negative effect on the hatchability of B. altianalis eggs. At lower temperature of 24⁰C, aeration significantly affected the egg hatchability but at higher temperatures aeration did not significantly affect hatchability. It was also noted that across all the temperature treatments egg hatchability was delayed by a range of 4-6 h in aerated tanks compared to tanks that were not aerated. It can be deduced that since in all the treatments DO was sufficient for hatchability it seems that aeration caused egg agitation or mechanical shocks resulting in prolonged hatching period in B. altianalis and this effect was much pronounced at 24⁰C leading to increased mortalities. In salmonids mechanical chock increased mortalities in embryos at eye formation stage (Bromage et al., 1988). In fact in this study this embryonic stage (late embryo) was the more affected than other stages during incubation.

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In addition any delay in hatching increases the risk of infections resulting from the dead eggs (Khodabandeh & Abtahi, 2006).

The optimal temperature peaks recommended for inclusion in the spawning process were 24⁰C when no aeration was provided and at 27⁰C when aeration was provided. Therefore these two temperatures were considered optimal for the best physiological performance for hatching B. altianalis eggs. A similar study in Cyprinus carpio recorded optimal temperature for egg hatchability at 27⁰C but the hatchability range was between 24°C and 30 °C (El-Gamal, 2009). In an optimal range of 21-26°C was provided by Shireman and Smith (1983) although recent findings have recorded the optimum temperature at 32⁰C (Korwin-Kossakowski, 2008). The viable range for hatchability of

Labeo rohita eggs was 26⁰C-33⁰C and the optimal temperature was at 31°C (Das et al., 2006). In cultured Clarius gariepinus the viable hatchability range was much wider from 20-35°C but the optimal temperature was 30 ⁰C (Haylor & Mollah, 1995). These variations indicated that optimal temperatures for hatchability are species specific even within the same family but also could adjust to prevailing or changing regional or global temperature conditions as was noted in Labeo rohita (Das et al., 2006; Ponnuraj, Murugesan, Sukumaran, 2002). Determining optimal temperature for egg development has also been shown to subsequently influence positive growth of larvae in common carp and grass carp (Korwin-Kossakowski, 2008).

This study noted that although no significant differences occurred among the embryonic stages (at p = 0.0167), the late embryo stage was affected at each temperature treatment more than other stages. This is a critical stage because embryos are developing body myotomes, heartbeat, completion of brain and notochord development as well as lens formation (George & Chapman, 2015) and it takes the longest period during incubation. However, across all treatments the lowest mortalities were recorded at 24⁰C (without aeration) and at 27⁰C (with aeration and no aeration), reflecting the final hatchability values obtained in this study. Generally because this stage was more affected than other stages of development at each treatment temperature it can be inferred that temperature had more or less the same or a uniform effect across all temperature treatment but the extent of its effect in each

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temperature treatment varied. This suggested that temperature modifications during incubation may not be necessary for B. altianalis as was observed in cyprinid species of Leuciscus genus (Kupren et al., 2011). It is imperative for hatchery managers to ensure that temperature is strictly controlled or regulated at the late embryonic stages in order to maximize egg survival during egg incubation period. The results further suggest that since early embryonic stages were not affected by high temperature it is possible to begin with a high temperature (30⁰C) so as to accelerate embryo development to gastrulation stage but gradually reduce the temperature to 27⁰C or 24⁰C to limit the damage to the late embryonic stage.

Given that the delayed hatching was mainly due to direct air bubbling of the water that led to mechanical agitation of eggs, hatching facilities were designed to reduce or avoid egg agitation. Results indicated that the circular plastic tanks (re-circulating) used in the experiment and the glass tank system were suitable for hatching B. altianalis eggs compared to the conical jar systems. Various systems have been developed to suit hatchability of eggs in other species depending on the nature of eggs and their tolerance (Glenn & Tiersch, 1997; Watson & Chapman, 2002; Woynarovich & Horvath, 1980). In B. altianalis, the eggs are not sticky or adhesive, they do not absorb water and they sink to the bottom of the tank (Rutaisire et al., 2015). In such species where eggs are non adhesive the conical jar system is one of the best options (Watson & Chapman, 2002). This was not possible with B. altianalis as high mortalities were obtained with the jar hatching system. The poor performance of the conical jar incubator observed in the present study confirmed that not only mechanical agitation of B. altianalis eggs delayed hatchability, but also reduced hatchability levels and was therefore not suitable for hatching B. altianalis eggs. It seems B. altianalis lays its eggs and they sink to the bottom in a calm environment and will hatch without any disturbance.

Evidence from the present study revealed that water level or depth did not affect hatchability but delayed hatching by 5.5 h. In pacific herring hatching depth of 0-11 meters did not significantly affect egg hatching but beyond this depth hatchability was affected (Taylor, 1971). It could be postulated that depth exerts pressure on the eggs and they hatch much faster. This is subject to further investigation. Often eggs of some cultured species in hatcheries in the region are accustomed to the trays and let to float in the hatchery tank (Mwanja et al., 2015). These findings indicated that it is 106

useful to spread the eggs on the tray and place them at the bottom of the hatching tank to facilitate quick hatching. It was also observed that light intensity used in this experiment did not have any significant effect on the hatchability of B. altianalis eggs. Light intensities have influence on hatchability of embryos of many fish species (Politis, Butts & Tomkiewicz, 2014; Villamizar, Vera, Foulkes, & Sanchez-Vezquez, 2014) but in some species it may not have significant effect (Iglesias et al., 1995). Generally hatching of embryos in many hatcheries for the cultured species in Uganda and in the region does not occur under strict light regims but hatcheries tend to provide dark or very dim light during the hatching. The light range values stated in this study did not suggest any significant effect of light on hatchability and therefore the farmers need not to be worried with hatching eggs in presence of light as it is postulated in some species (Politis et al., 2014; Villamizar et al., 2014).

Whereas optimal temperature for egg hatchability was recorded at 24⁰C and 27⁰C in this study results of the final average larval weight, specific growth rates and survival indicated that optimal growth was achieved at a higher temperature of 30⁰C. This was similar to studies conducted in some cyprinids such as Koi carp Cyprinus carpio L (Wolnicki & Appelbaum, 1993), common bream Abramis brama L. (Kucharczyk et al.,, 1997) and common carp Cyprinus carpio (El-Gamal, 2009), in which temperature demand for optimal physiological performance in larvae was notably higher than that for their embryonic development. However, for embryo incubation temperature and larvae growth temperature in Crucian carp Carassius carassius (Laurila, Piironen & Holopainen, 1987) was the same. Different species have specific optimal temperature ranges that promote better physiological and metabolic performance interactions with other factors to influence optimal growth of larvae (Blaxter, 1991; Jobling, 1981). The study also noted that although significant differences occurred in average weight between larvae raised at 27⁰C and those at 30⁰C, survival was not significantly different suggesting that larval growth was equally good at 27⁰C for B. altianalis. At 14

DAH larvae weight at 24⁰C performed slightly better than those at 27⁰C. This could suggest that larvae’s physiological processes at that age were significantly affected by the high temperatures in this species. Consequently in subsequent samplings larvae began to adjust physiologically and were able to efficiently utilize the feeds and grew faster than larvae kept at 24⁰C. In fact by the end of the

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experiment at 56 DAH, larvae grew bigger at 31⁰C than those at 27⁰C. The noted increase in larvae weight from 24⁰C to 30⁰C and thereafter its decline in weight imply that unfavorable temperature will inhibit appropriate developmental and growth rates of larvae physiological processes (Kamler, 2008;; Kujawa, Furgała-Selezniow, Mamcarz, Lach & Kucharczyk, 2015; Laurila et al., 1987). Generally mortalities were higher before 20 DAH and significantly reduced in all treatments by 32 DAH. This could suggest that the larvae had developed strategic efficient gut structures to be able to appropriately utilize the feed at a given temperature. Under appropriate environmental conditions digestive system of fish larvae develops gradually and reaches an age where the digestive system is mature enough to properly digest and efficiently utilize the micro diets and this is also described as a period when larvae mortalities reduce drastically (Kolkovski, 2001; Zambonino-Infante et al., 2008) the reduced mortalities were equally observed at 31⁰C suggesting an adaptation mechanism for the species (Korwin-Kossakowski, 2008).

5.5 Conclusions

This study found out that over 80% of embryos successfully hatched within the temperature range of

24⁰C to 30⁰C but optimum hatchability was achieved at 24⁰C (without aeration) and 27⁰C (with aeration) in both trials. The present study established that direct bubbling of air led to mechanical agitation that disrupted embryo development and largely affected especially late embryo stage and consequently the ‘re-circulating plastic tank’ and glass systems were found to be effective in reducing this problem. Larvae of B. altianalis achieved optimum development and growth when temperature was increased from 27⁰C up to 30⁰C. This temperature range should be considered for integration into the spawning protocol for this newly domesticated species, Barbus altianalis.

5.6 Acknowledgment

We appreciate the financial support from the Government of Uganda through the World Bank funded ATAAS project. We thank the National Agricultural Research Organization (NARO) and Makerere

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University who provided research facilities at the Aquaculture Development Centre (ARDC) and College of Natural sciences (CONAS) respectively.

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5.7 References

Alami-Durante, H. E., Bergot, P., Rouel, M., & Goldspink, G. (2000). Effects of environmental temperature on the development of the myotomal white muscle in larval carp (Cyprinus carpio L.). Journal of Experimental Biology, 203(24): 3675.

Bjornsson, B., Steinarsson, A., & Oddgeirsson, M. (2001). Optimal temperature for growth and feed conversion of immature cod (Gadus morhua L.). ICES Journal of marine Sciences, 58, 29– 38

Blaxter, J. H. S. (1991). The effect of temperature on larval fishes. Netherlands Journal of Zoology, 42, 336-357.

Bromage, N., Porter, M., & Randall, C. (2001). The environmental regulation of maturation in farmed finfish, with special reference to the role of photoperiod and melatonin. Aquaculture, 197, 63–98.

Bromage, N. (1998). Broodstock management and the optimisation of seed supplies. Aquaculture Science, 46(3), 395-401

Brooks, G. B. (1994). A simplified method for the controlled production and artificial incubation of Oreochromis eggs and fry. The Progressive Fish-Culturist, 56(1), 58-59.

Brooks, S., Tyler, C. R., & Sumpter, J. P. (1997). Egg quality in fish: what makes a good egg?. Reviews in Fish Biology and Fisheries, 7(4), 387-416.

Dabrowski, K. (1984). The feeding of fish larvae: present «state of the art» and perspectives. Reproduction Nutrition Development, 24(6), 807-833.

Dahlberg, M. D. (1979). A review of survival rates of fish eggs and larvae in relation to impact assessments. Marine Fisheries Review, 41(3), 1-12.

Das, T., Pal, A. K., Chakraborty, S. K., Manush, S. M., Dalvi, R. S., Sarma, K., & Mukherjee, S. C. (2006). Thermal dependence of embryonic development and hatching rate in Labeo rohita (Hamilton, 1822). Aquaculture, 255(1), 536-541.

Dimichele, L., & Taylor, M. H. (1980). The environmental control of hatching in Fundulus heteroclitus. Journal of Experimental Zoology Part A: Ecological Genetics and Physiology, 214(2), 181-187.

110

Dionisio, G., Campos, C., Valente, L. M. P., Conceicao, L. E. C., Cancela M. L & Gavaia, P. J. (2012). Effect of egg incubation temperature on the occurrence of skeletal deformities in Solea senegalensis. Journal of Applied Ichthyology, 28 (3), 471–476.

Downing, G., & Litvak, M. K. (2001). The effect of light intensity and spectrum on the incidence of first feeding by larval haddock. Journal of Fish Biology, 59(6), 1566-1578.

El-Gamal, E. (2009). Effect of Temperature on Hatching and Larval Development and Mucin Secretion in Common Carp, Cyprinus carpio L. Global veterinaria 3 (2), 80-90.

George, A. E., & Chapman, D. C. (2015). Embryonic and larval development and early behavior in grass carp, Ctenopharyngodon idella: implications for recruitment in rivers. PloS one, 10(3), e0119023.

Glenn, D. W., & Tiersch, T. R. (1997). An alternative egg-incubation jar. The Progressive Fish- culturist, 59(3), 253-255.

Green, S. B., & Fisher, R. (2004). Temperature influences swimming speed, growth and larval duration in coral reef fish larvae. Journal of Experimental Marine Biology and Ecology, 299, 115– 132.

Greenwood, PH. 1966. The fishes of Uganda 2nd ed. The Uganda Society Kampala, pp 1-6.

Haylor, G. S., & Mollah, M. F. A. (1995). Controlled hatchery production of African catfish, Clarius gariepinus: the influence of temperature on early development on early development. Aquatic living resources, 8, 431-438.

Herzig, A., & Winkler, H. (1986). The influence of temperature on the embryonic development of three Cyprinid fishes, Abramis brama, Chalcalburnus chalcoides mento and Vimba vimba. Journal of Fish Biology, 28(2), 171-181.

Hokanson, K. E. (1977). Temperature requirements of some percids and adaptations to the seasonal temperature cycle. Journal of the Fisheries Board of Canada, 34(10), 1524-1550.

Iglesias, J., Rodriguez-Ojea, G., & Peleteiro, J. B. (1995). Effect of light and temperature on the development of turbot eggs (Scophthalmus maximus L.). In ICES Marine Science Symposia (Vol. 201, pp. 40-44). Copenhagen, Denmark: International Council for the Exploration of the Sea. 111

Jhingran, V. G. & Pullin, R. S. V. (1985). A Hatchery Manual for the common, Chinese and Indian Major Carps. Asian Development Bank and International Center for Living Aquatic Resources Management (ICLARM) Studies and Reviews, 11: 1 – 191

Jobling, M. (1981). Temperature tolerance and the final preferendum—rapid methods for the assessment of optimum growth temperatures. Journal of Fish Biology, 19(4), 439-455.

Johnston, I. A., Vieira, V. L. A., & Temple, G. K. (2001). Functional consequences and population differences in the developmental plasticity of muscle to temperature in Atlantic herring Clupea harengus. Marine Ecology Progress Series, 213, 285-300.

Jensen, J. O. T., & Alderdice, D. F. (1989). Comparison of mechanical shock sensitivity of eggs of five Pacific salmon (Oncorhynchus) species and steelhead trout (Salmo gairdneri). Aquaculture, 78(2), 163-181.

Kamler, E. (2008). Resource allocation in yolk-feeding fish. Reviews in Fish biology and Fisheries, 18(2), 143

Keckeis, H., Bauer-Nemeschkal, E., Menshutkin, V. V., Nemeschkal, H. L & Kamler, E. (2000) Effects of female attributes and egg properties on offspring viability in a rheophilic cyprinid, Chondrostoma nasus. Canadian Journal of Fisheries and Aquatic Science. 57,789–796.

Khodabandeh, S., & Abtahi, B. (2006). Effects of sodium chloride, formalin and iodine on the hatching success of common carp, Cyprinus carpio, eggs. Journal of Applied Ichthyology, 22(1), 54-56.

Kokurewicz, R. (1970). The effect of the temperature on embryonic development of Tinka tinka (L) and Rutilus rutilus (L). Zoology Polish, 20 (3), 317-337.

Kolkovski, S., (2001). Digestive enzymes in fish larvae and juveniles-implications and applications to formulated diets. Aquaculture, 2000, 181–201.

Korwin-Kossakowski, M. (2008). The influence of temperature during the embryonic period on larval growth and development in carp, Cyprinus carpio L., and grass carp, Ctenopharyngodon idella (Val.): theoretical and practical aspects. Archives of Polish fisheries, 16(3), 231-314.

112

Kucharczyk, D., Luczynski, M., Kujawa, R., & Czerkies, P. (1997). Effect of temperature on embryonic and larval development of bream (Abramis brama L.). Aquatic Sciences, 59(3), 214-224.

Kujawa, R., Furgała-Selezniow, G., Mamcarz, A., Lach, M., & Kucharczyk, D. (2015). Influence of temperature on the growth and survivability of sichel larvae reared under controlled conditions. Ichthyological Research, 62(2), 163-170.

Kupren, K., Mamcarrz, A., & Kucharczyk, D. (2011). Effect of variable and constant thermal conditions on embryonic and early larvae development of fish from the genus Leuciscus (Cyprinidae, teleostei). Czech Journal of Animal science, 56 (2): 70-80.

Laurel, B. J., & Blood D. M (2011). The effects of temperature on hatching and survival of northern rock sole larvae (Lepidopsetta polyxystra). Fishery Bulletin 109 (3) 282-291.

Laurila, S., Piironen, J., & Holopainen, I. J. (1987, January). Notes on egg development and larval and juvenile growth of crucian carp (Carassius carassius (L.). In Annales Zoologici Fennici (pp. 315-321). Finnish Academy of Sciences, Societas Scientiarum Fennica, Societas pro Fauna et Flora Fennica and Societas Biologica Fennica Vanamo.

Moran, R., Harvey, I., Moss, B., Feuchtmayr, H., Hatton, K., Heyes, T., & Atkinson, D. (2010). Influence of simulated climate change and eutrophication on three‐spined stickleback populations: a large scale mesocosm experiment. Freshwater Biology, 55(2), 315-325.

Politis, S. N., Butts, I. A., & Tomkiewicz, J. (2014). Light impacts embryonic and early larval development of the European eel, Anguilla anguilla. Journal of Experimental Marine Biology and Ecology, 461, 407-415.

Mwanja, M., Rutaisire, J, Ondhoro, C, Ddungu, R., & Aruho, C. (2015). Current fish hatchery practises in Uganda: The potential for future investment. International Journal of Fisheries and Aquatic Studies, 2(4): 224-232.

Ponnuraj, M., Murugesan, A. G., Sukumaran, N. (2002). In B. Venkataramani, N. Sukumaran (Eds.), Effect of temperature on incubation time, fertilization rate and survival of the spawn of Rohu, Labeo rohita (pp. 430–433)., Mumbai, India: thermal Ecology, DAE, BRNS.

113

Rombough, P.J., (1997). The effects of temperature on embryonic and larval development. In C. M. Wood, D. G. McDonald (Eds.), Global Warming. Implications for Freshwater and Marine Fish (pp. 177– 223). Cambridge: Cambridge Univ. Press

Rutaisire, J., Levavi-Sivan. B., Aruho, C., & Ondhoro, C. C. (2015). Gonadal recrudescence and induced spawning in Barbus altianalis. Aquaculture, 46 (3), 669–678. Doi:10.1111/are.12213

Taylor, F. H. C. (1971). Variation in hatching success in Pacific herring (Clupea pallasii) eggs with water depth, temperature, salinity and egg mass thickness. Reunions du Conseil International pour l'Exploration de la Mer, 160, 34-41.

Villamizar, N., Blanco-Vives, B., Migaud, H., Davie, A., Carboni, S., & Sanchez-Vazquez, F. J. (2011). Effects of light during early larval development of some aquacultured teleosts: a review. Aquaculture, 315(1), 86-94.

Villamizar, N., Vera, L. M., Foulkes, N. S., & Sanchez-Vezquez, F. J. (2014). Effect of lighting conditions on zebrafish growth and development. Zebrafish, 11(2), 173-181.

Watson, C. A., & Chapman, F. A. (2002). Artificial Incubation of Fish Eggs. Fact Sheet FA-32, Institute of Food and Agricultural Science, University of Florida Extension.

Wolnicki, J., Kaminski, R., Korwin-Kossakowski, M., Kuzsnierz, J., & Myszkowski, L. (2004). The influence of water temperature on laboratory reared minnow Eupallasella perenurus (Pallas) larvae and Juvenile. Archives of Polish fisheries, 12, 61-69.

Wolnicki, J., & Appelbaum, S. (1993). Optimal growth temperature for koi carp (Cyprinus carpio L.) larvae and juveniles reared under controlled conditions. Polish Archives of Hydrobiology, 40, 223–230.

Woynarovich, E., & Horvath, L., (1980). The artificial propagation of warm-water finfishes – a manual for extension (FAO Fish. Tech. Pap. 201; 183 p). Rome, Italy: FAO.

Zambonino-Infante, J., Gisbert, E., Sarasquete, C., Navarro, I., Gutierrez, J., & Cahu, C. L. (2008). In J. E. O. Cyrino, D. Bureau & B. G. Kapoor (Eds.), Ontogeny and physiology of the digestive system of marine fish larvae. Feeding and Digestive Functions of Fish (pp. 277–344). Enfield, USA: Science Publishers Inc.

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CHAPTER SIX

Histo-morphological description of the digestive system of the Ripon Barbel Barbus altianalis (Boulenger 1900); a potential species for culture

C. Aruho, V. Namulawa, C. D. Kato, M. Kisekka, J. Rutaisire and F. Bugenyi

Uganda Journal of Agricultural Sciences, 2016, 17 (2): 197 – 217 http://dx.doi.org/10.4314/ujas.v17i2.6

6.0 Abstract

Morphology of the digestive system can help define the feeding adaptation habits of a given fish species in a given environment. In a study to describe the nature and functionality of the digestive system of Barbus altianalis, samples of B. altianalis were taken from River Nile. Their Lengths and weights were measured and the gut structure preserved. The structure of the digestive tract of the Ripon Barbel, B. altianalis, was described using morphological observations and standard histological procedures. The digestive tube of Barbus altianalis is stomachless and valveless, progressively and uniformly reducing in size from the proximal to distal end. The digestive tract is on average 2.22 ± 0.37 times longer than its body length. The mouth is terminal and protrusible and the pharyngeal palatal organ is well developed. The last gill arch is modified into pharyngeal teeth and the eosophagus is short and muscular. Histological sections revealed the presence of taste buds from the lips to the cranial eosophagus and these regions of the digestive tract are lined by a stratified squamous epithelium. The intestines are lined by simple or pseudo-stratified columnar epithelial layer which is highly folded. Goblet cells containing both acidic and neutral mucins are present throughout the entire digestive tract and are more numerous in the pharynx and the proximal part of the intestine. Lobes of pancreatic acini are discrete and scattered among liver cells, around the intestine and few are seen in the spleen surrounding blood vessels. Thus, the liver could most accurately be termed a hepatopancreas structure.

Key words: Goblet cells, hepatopancreas, intestine, mucins

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6.1 Introduction

Morphology of the digestive system is a key aspect in fishes that defines the feeding adaptation habits of a given fish species in a given environment. Different fishes have different feeding habits and are thus broadly classified into carnivores, detritus feeders, herbivores and omnivores depending on what they eat (De Silva & Anderson, 1995; Rust, 2002). For this reason different fishes have varying modifications of their digestive tracts for the purpose of maximising nutrient uptake for survival and growth. The digestive morphology of several species has been studied to relate structural functionality with adaptation to feeding habits (Cataldi, Cataudella, Monaco, Rossi & Tansion, 1987; Murray, Wright & Goff, 1996). Increased attention to such studies has primarily focused on the development of feeding technologies of candidate species for culture (Banan-Khojasteh, 2012).

In sub-Saharan Africa there is growing need to diversify and promote the culture of indigenous high- value fish species to increase fish production (Namulawa, Kato, Nyatia, Britz & Rutaisire, 2011; Kato et al., 2014; Rutaisire, Levavi-Sivan, Aruho & Ondhoro, 2015). Efforts were successful in artificially inducing one of the high-value indigenous cyprinid B. altianalis to spawn (Rutaisire et al., 2015). However, concern arose due to the slow growth rate of the species that could partly be attributed to a knowledge gap of its feeding behavior. Cyprinids are largely cultured in Eurasia and contributed more than 70% of the total fish production (FAO, 2014). A number of cyprinids in Africa are potentially high value species that are being overexploited and can only be salvaged through culture. Successful domestication and subsequent commercial culture of B. altianalis, will increase fish production and revenues for commercial farms in the region (Rutaisire et al., 2015). The cyprinids, commonly referred to as carps are regarded as stomachless fish though with a diversity of structural and functional modifications of the digestive system (De Silva & Anderson, 1995). Although the gastrointestinal tract in all vertebrates possesses stereotypical structural similarities, variation can occur between species (Domeneghini, Arrighi, Radaelli, Bosi, & Mascarello, 1999; Buddington & Kuzmina, 2000) and hence great diversity in functionality (Banan-Khojasteh, 2012). Structural variations probably confer differences in digestive capabilities. Different regions along the gut with varying specialised characteristics maximise different physiological processes to ensure uptake of nutrients (Buddington & Diamond, 1987; Dabrowisk & Celia, 2005). The purpose of this study was to describe the digestive system of B. altianalis to provide insights into its functionality

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and feeding behavior, which will form a basis for developing feeding strategies for this fish under aquaculture conditions.

6.2 Materials and methods

Two hundred ninety four B. altianalis specimens of body weight ranging from 58.3g to 8,300g were collected from River Nile at two close landing sites (00⁰27´N; 33⁰11´E and 00⁰35´N; 33⁰04´ E) along and close to the source of the River Nile from July 2014 to January 2015. The specimens were collected using a long line of about 100 hooks of gap size 9-4, baited with Tilapia fingerlings across a 10 m section of the River. The fish were killed by anaesthetising them with an overdose of AQUI-S solution (1ml in 10liters). They were weighed to the nearest 0.1g using a digital weighing scale and their length was measured to the nearest mm by a calibrated ruler. All the fish were immediately dissected to expose the digestive system through an incision made from the anus to the mouth. The extent of the mouth gap, the length of eosophagus and intestine, as well as the weight of the liver were recorded. The mouth gap was measured using a vernier caliper. Using this tool, 20 fish covering all size groups (58.3-8300g) had the intestines and buccal pharyngeal sections removed and were immediately fixed in Bouin`s solution for more than 48 hours before they were histologically processed. The remaining fish were immediately observed macroscopically to record and describe the gut features from the mouth to the anus. Photographs of the morphology of the digestive tract were taken using a digital NV3, 4.2 V, 7. 2 megapixels Samsung camera. From the fixed specimens, smaller sections were obtained from the oral cavity, the eosophagus, the intestine, the spleen and the liver. Tissue processing was accomplished following standard histological methods (Bancroft & Gamble, 2002). The processed sections were stained using Gill’s haematoxylin and eosin (H & E) and/or Masson’s trichome (MT). Periodic Acid Schiff (PAS) and Alcian Blue (AB; pH 2.5 and pH 1.0) stains were used on some tissues to identify the type of glycoconjugates in goblet cells along the digestive tract. The stained sections were examined using a Leica Micro system microscope (Switzerland LTD, model PN: DM 500) at different magnifications. Photomicrographs were taken using a 10 mega pixels mounted canon digital Powershot camera (A640, China). The elationship between the total length TL and the intestinal length IL and the mouth gap GL, the liver weight LW and body weight BW was determined by correlation analysis using SPSS statistical version 20

(Armonk, NY: IBM Corp.). The relative gut index (RGI) was calculated by dividing IL by TL. To

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determine if there were differences in RGI among class sizes, ANOVA was performed and significant differences was estimated by Duncan’s test at p > 0.05 confidence level, using SPSS. Mean values were presented as mean ±SD (standard deviation).

6.3 Results

6.3.1 Gross morphology

Macroscopic observations indicated that the digestive system of B. altinalis constituted a simple stomachless tube that could only be categorised into the head gut (mouth, pharyngeal and eosophagus) and the hind gut (intestine). The intestines were positioned ventrally to the gas bladder, which was immediately ventral to the vertebrae column. On both sides of the digestive system the bivalent gonads stretched from the upper peritoneal cavity near the septum alongside the digestive tract to the anal opening (Figure 6.1a). The terminal mouth was protrusible (Figure 6.1b, c). Its gap depth (from the upper to the lower lip) measured 0.9 cm in a small fish of 13.5 cm to 7.5cm in a big fish of 84.3cm standard length SL. The mouth gap positively and strongly correlated with the TL (slope = 6.46; r2 = 0.90, p < 0.001). The mouth had a rostral cap on the upper side joined to the upper lip by a thin muscle layer that allowed flexibility in moving the mouth (Figure 6.1c). Associated with the mouth were two pairs of short barbels, one on the rostral cap and the other at the end of the upper lip. There were four pairs of gill arches on either side of the buccal cavity. The oral cavity had no teeth but the fifth gill arch was modified into pharyngeal teeth (8-11 in number) on either side (Figure 6.1d). The roof of the oral cavity had ridges running parallel to each other towards the palate (Figure 6.1e). The chewing pad was an ovoid-shaped structure located at the posterior end of the palatal organ (Figure 6.1e). At the caudal edge of the chewing pad toward the eosophagus was a palatal protrusion pointing to the ventral and perpendicular to the highly folded pharyngeal-funnel like pouch that narrowed toward the eosophagus (Figure 6.1e).

The eosophagus was a short tough uniform tubular structure continuous with the intestines and connected to the pharynx cranially (Figure 6.2a). The eosophagus was positioned dorsal to the heart and transverse septum separating the heart from the peritoneum. It constituted 0.65 ± 0.19% relative to the total intestinal length. The inner lining of the eosophagus had longitudinal strand-like rugae (Figure 6.2b), while the outer surface was made of circular muscles that appeared as concentric rings. 118

The outer surface gave the eosophagus a characteristic off-white colour that easily differentiated it from the much lighter and transparent intestine. In addition, a circular ring-like strand where the longitudinal rugae terminated caudally was observed to mark the separation of the oesophagus and the intestine (Figure 6.2b). At the cranial end of the eosophagus was a pneumatic duct that joined the eosophagus to the gas bladder midway the constriction separating the two halves of the gas bladder.

The small intestine was a coiled structure with no clear macroscopic demarcations along its length. The intestine was coiled several times forming a circular pattern located in the upper half of the peritoneal cavity beneath the gas bladder in a small fish but as the fish grew bigger the intestinal coiling spanned the entire cavity up to the anus. The intestinal length was on average 2.22 ± 0.37 times longer than the total body length TL. The TL was strongly correlated with IL (slope = 0.03; r2 =0.80.5, p < 0.001). A statistical significance of RGI is observed between the individuals in lower class sizes of 20-29 cm and 30-40 cm TL; and the rest of other classes (F4,290 = 8.014, p<0.001; Table 6.I). Generally the small intestine decreased uniformly from cranial (proximal) intestine to distal intestine. There was no evidence identifying any distinct portions and any valves along the intestine. The inner surface of the small intestine was thrown into mesh-like folds spanning the entire length (Figure 6.2b). In females and males with developing gonads in a reproductive cycle, large quantities of fat were visible surrounding and coiling alongside the intestine (Figure 6.2c). The anus was clearly separated from the genital opening (Figure 6.2d).

The liver was an elaborate brown coloured structure with two clear large lobes. The liver formed 0.73 ± 0.23 % of the total body weight BW (g) of the fish and showed a strong linear correlation with BW (Slope = 111.03; r2 =0.89, p < 0.001). The right lobe was longer and bigger than the left lobe and stretched from the anterior side near the heart and eosophagus down to the point just before the anus (Figure 6.1a, 6.2e). The liver lobes were tightly joined to the intestines and the gonads by thin muscle-like strands and visible blood vessels. The gall bladder between the liver and the intestines (Figure 6.2e) possessed bile duct connecting cranially at about 1/3rd length of the main liver lobe.

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Figure 6. 1: Gross anatomy of the digestive system of B. altianalis. In, intestine; Rlv, Right liver; Llv, left liver; Dlv, distal liver lobe; Gn, gonad; Rc, Rostral carp; Ul, Upper lip; Ll,

lower lip; RM, Rostral membrane; Bb, Barbel; Mf, Mouth floor; Gl, gills; Te, Pharyngeal

teeth; Pc, Pharyngeal cavity; Mu, Upper mouth cavity; Po, Palatal organ; Pp, Palatal

protrusion; Cp, chewing pad.

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Figure 6.2: Gross anatomy of digestive tract of Barbus altianalis. Es, Esophagus; Ru, Rugae; Lv, liver; In, intestine; Fa, fat; Go, Genital opening; An, Anus; Gb, Gall bladder; As, Gas bladder; Sp, Spleen; Ha, Heart: Te, Testis.

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Table 6. 1: Variation of mean RGI values (± SD) with fish weight and total length in class sizes. Class size TL (cm) Fish weight (g) Mean RGI N (number) 21-30.9 196.5±64.9 1.98a±0.35 45 31-40.9 428.66±132.19 2.13b±0.36 50 41-50.9 1270.6±379.6 2.28c±0.34 68 51-50.9 2190.8±543.2 2.29c±0.4 85 >60 4551.1±1480.5 2.31c±0.30 47 Mean values with different superscripts indicate significant differences

6.3.2 Histological description

The lip was lined by a stratified squamous mucosa epithelium. Beneath, was a sub-mucosa layer made of collagen fibres. Underlying the sub-mucosa, fewer collagen fibres were interspaced with adipose cells (Figure 6.3a). The collagens fibres made invaginations into the base of the mucosa epithelium culminating into papillae-like fusiform structure that terminally formed the taste buds (Figure 6.3a, 6.3b). The taste buds were interspersed with mucosa epithelial cells along the lips. These taste buds were also present on the barbels. The arrangement of the layers in the oral cavity was similar to that of the lips; however, the layer underlying the sub-mucosa comparatively possessed fewer adipocytes. The adipocytes in this region were interspersed among striated muscle bundles (Figure 6.4a, 6.4b). Taste buds were also interspersed in the epithelium of the oral cavity. Sparse mucus and club cells were visible within the mucosa epithelium (Figure 6.4a, 6.4c). The pharynx had similar layers as those seen in the oral cavity but with a highly folded mucosa having numerous mucous goblet cells (Figure 6.4d). Furthermore, there was evidence of sparsely scattered taste buds within the epithelium lining the pharynx, including that of the palatal organ. The layer beneath the mucosa epithelium in the pharyngeal region retained characteristic mixture of dense striated muscles interspersed with connective tissue as observed in the oral cavity.

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Figure 6. 2: Sections through the lips of B. altianalis. (a) Three layers of the lips are

observed, mucosa epithelium (Ep), sub-mucosa (Sm) and adipose tissue (Ad). Muscles (Mu), mucosa epithelial old cells (EPP) peeling off. (b) Magnified taste bud showing the taste pore (Tp), the basal cells (Bc) and taste receptors (Tr); Squamus (Sc). H & E.

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Figure 6. 3: Sections through the oral and pharyngeal cavities. a) layers of the oral cavity,

mucosa epithelium (Ep); the broad connective tissue of the sub mucosa sub-mucosa (Sm), Adipose tissue (Ad) with the striated muscle bundles. MT. b) Magnified part of the oral cavity showing striated fibres (St). MT. c) epithelium is enlarged to show the taste bud (Tb) in the oral cavity, mucus cells (Mc) and the club cells (Cb). MT. e) highly folded epithelium (Ep) of the pharyngeal tissue with numerous goblet cells (Gb). H & E

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The eosophagus had four tunics, the mucosa, sub-mucosa, muscularis and the serosa (Figure 6.5a). The mucosa constituted the epithelium lining the lumen and the lamina propria. The eosophagus was lined with a stratified squamous epithelium at the cranial side that gradually turned pseudo stratified columnar in the middle and became columnar at the distal end. The stratified squamous epithelium at the cranial side of the eosophagus was largely dominated by goblet cells but the goblet cells were comparatively fewer among the columnar cells at the distal end (Figure 6.5a, 6.5b, 6.5d, 6.5e). Taste buds were still evident at the cranial region of the oesophagus but were absent in the rest of the oesophagus (Figure 6.5b). The sub-musosa was generally thin beneath the lamina propria and was composed of loose connective tissue of collagen fibers interspersed with small circular bundles of striated muscle. The sub-mucosa lay over a thick wide muscularis. The muscularis appeared as a single layer. However, in cross-sectioned tissues it constituted interdigitation of both circular and longitudinal striated muscle bundles (Figure 6.5a, 6.5c) occasioned by an arrangement of some connective tissue fibres. The adjacent muscularis of the cranial intestine was not a single layer as in the oesophagus but had longitudinal and circular subdivisions (Figure 6.5d, 6.5f). At the distal end of oesophagus adjoining the intestine where morphologically a ring was observed, the muscularis was much thicker compared to the rest of the oesophagus. Beneath the muscularis was a thin layer called serosa.

The histological organisation of intestinal tract layers remained relatively the same from the oesophagus to the anus, with some differing structural details. Epithelial folds were comparatively deeper in the intestine and reduced toward the distal intestine, with the folds becoming narrower and smaller but relatively deeper only around the rectum and the anus. Although the extent of the epithelial folds differed along the entire length of the intestine, the epithelium comprised of largely columnar cells with regularly dispersed goblet cells and enterocytes. A brush border that constituted microvilli was observed at the luminal surface of the enterocytes and numerous eosinophils (leucocytes) spanned the entire lamina propria of the intestine (Figure 6.6a). The goblet cells constituted both neutral and acidic glycoconjugates (mucins) along the digestive tract. These mucins occurred together within the same goblet cell, a reason why sections stained with (AB pH 2.5)-PAS combination largely showed dark purple coloration for the goblet cells indicating double staining (Figure 6.6b). However, staining with AB (pH 1.0 and 0.5) was strongly observed with a deep blue colouration and were numerous within the pharyngeal cavity and moderately stained in oesophagus. 125

This indicated the presence of sulphated glycoconjugates in this region. These goblets were very few and weakly stained blue along other regions of the digestive system.

Unlike the oesophagus, the intestinal sub-mucosa was devoid of the striated muscle bundles and had a wavy arrangement of the loose connective tissue along the intestinal length (Figure 6.6c). Similarly, whereas muscularis in the oesophagus was not subdivided, the intestinal muscularis had two clear tunics, the inner circular layer and the outer longitudinal muscle layer. The inner circular layer was thicker than the outer longitudinal layer throughout the entire intestinal length and the two layers were separated by a thin layer consisting of largely nerve plexus (Figure 6.6c). The muscularis of the first portion of anterior intestine (about 1cm) adjacent to the oesophagus retained some circular and longitudinal striated muscle fibres found in the oesophagus. The rectal muscularis lacked muscle striations but beneath it the collagen tissue and blocks of striated muscles were observed (Figure 6.6d). The serosa beneath the muscularis remained very thin from the proximal to distal intestine. However, its clarity disappeared close to the rectum and the anus.

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Figure 6. 4: Sections through the eosophagus and anterior (cranial) intestine of B. altianalis. a) eosophagus tunics. The mucosa epithelium (Ep) with lamina propria (Lp), sub-mucosa(Sm) with circular striated muscle bundles (Stc), muscularis (Mu) and serosa (Se). b) Magnified portion of mucosa epithelium showing stratified goblets (Gb) among epithelial cells and the taste bud (Tb); Blood capillary (Bc). c) Magnified potion of Mu showing the striated muscle fibres (St). d) Sections cut at the transition junction between oesophagus (Oe) and the Anterior Intestine (Ai) H & E. Both show columnar arrangement of epithelial and goblet cells in magnified section (e). Blood capillary (Bc) are also observed in (e). f) Magnification of muscularis layer in cranial intestine showing longitudinal muscle (Ls) and circular muscle (Cm). H & E. 127

Figure 6. 5: Sections through the intestine and the rectum. a) Mucosa epithelium showing columnar cells with elongated nucleus (Cu), interspersed with goblet mucus cells (Gb). Brash border microvilli (Bb), basement membrane (Bm), oesinophilis and the erythrocytes (Es). H&E. b) goblet mucus cells (Gb) stained positive with PAS and AB PH 2.5. The figure shows two superimposed colors in each globule indicating presence of neutral mucins (Gbn) staining purple and Acidic mucins (Gba) staining blue. (c) Broad sub-mucosa (Sm) with

wavy loose connective collagen fibres stained blue. Nerve plexus (Np) are between the inner circular layer (Cm) and the outer longitudinal layer (Lm). d) Rectal circular muscle (Cm)

adjoined to a layer of striated muscle (St) and connective tissues (Ct). Single columnar epithelium is observed with numerous globules (Gb; in set).

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The liver hepatocytes were arranged in anastomosing cords towards the central vein. The hepatic cords were interspaced by numerous sinusoids oriented toward the direction of central veins The liver hepatocytes were arranged in anastomising cords towards the central vein. The hepatic cords were interspersed by numerous sinusoids oriented toward the direction of the central veins (Figure 6.7a.). The arrangement depicted a roughly hexagonal shape constituting a lobule with unclear demarcation. Within the liver parenchyma were tracts of hepatic venules, the bile ducts and the hepatic arterioles (Figure 6.7b, 6.7c). Pancreatic acini constituted triangular, polygonal, pyramidal or rhomboid shaped acinar with a basal located eosinophilic nucleus. The inactive zymogen granules were observed packed within the acinar (Figure 6.7d). The pancreatic tissue appeared as oval or longitudinal mass within the liver parenchyma and formed a hepato-pancreas (Figure 6.7a, 6.7d). The pancreatic acini were formed around the veins. In a number of sections the pancreatic acini were commonly observed in contact with the intestines in mesentery along its entire length (Figure 6.8a). Pancreatic tissue was also observed in the spleen around the veins (Figure 6.8b).

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Figure 6. 6: Sections through the liver of B. altianalis. a) Anastomosing pattern of hepatocyte

plates (Hp). The arrangement orients toward the central veins (Ve) and is reflected by the

narrow empty spaces, the sinusoids (Ss). The biliary-arteriolar tracts (Bat) are commonly

observed in the liver parenchyma. Pancreas (Pc) is present in the liver. b) Magnified section

of liver (7a) showing biliary-arteriolar tract constituting the bile duct (Bd) and the hepatic

arteriole (Ha). c) A liver section with a portal triad consisting of bile duct (Bd), hepatic

arteriole (Ha) and portal venule (Pv). d) A magnified section of the liver (6a) showing

pancreatic acinar (Pa), exocrine zymogen granules (Zm), endocrine pancreas (Ed) and 130 Acinar nucleus (Nu). H & E.

Figure 6. 7: Sections through the intestine and the spleen. a) pancreatic tissue (Pc) around the intestine. H&E. b) Pancreatic acini (Pa) within the spleen (Sp), seen around the Vein (Ve). White pulp (white arrows) and Red pulp (black arrows) are observed. MT.

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6.4 Discussion

Morphological and histological observations confirmed that B. altianalis species has a general simple stomachless digestive structure characteristic of that seen in most cyprinids (Banan-Khojasteh, 2012; Delashoub, Pousty & Hofer, 1991); though with some structural details unique to the species. The protrusible and flexible mouth suggests that this fish shows greater flexibility in its feeding strategy along the water column, including the ability to feed on the detritus material and other bottom dwellers. The fact that the bottom molluscs, crustaceans and small fish formed part of the fish’s diet (Per. Obs.), in addition to plankton and detritus (Balirwa, 1978) confirmed its feeding flexibility. The Relative Gut Index RGI (2.22 ± 0.37) coupled with the dietary composition, absence of stomach and the nature and pattern of the mucins present in the goblet cells classified B. altianalis as an omnivorous fish. This feeding behavior is also observed in other similar cultured omnivorous cyprinid, Cyprinus carpio whose RGI is estimated to be between 1.8 -2.0 (De Silva & Anderson, 1995). The RGI of most omnivorous fish species ranges between 0.8-5 reported by Rust, 2002. Takeuchi, Satoh and Kiron (2002) suggested that the noted RGI in common carp was effective in utilising and deriving sufficient energy for growth from both carbohydrate and lipid or protein food sources. However, in other Barbus species such as Barbus sharpeyi and Barbus grypzts, the RGI was recorded between 2·79 - 3·18 and 2·00 - 2·76 respectively (Al-Hamed, 1965). These are plant feeders but have an RGI close to that of B. altianalis. This could possibly illustrate phylogenetic diversion from the ancestors, meant to exploit diverse feeding environments. Current studies are using evolutionary history and aphylogenetic approach to interpreting gut morphometrics such as gut index in order to classify closely related species of diverging feeding behavior (German & Horn, 2006; Wagner, Peter, Kalmia, Danielle & Ellinor, 2009). Such an approach could be useful to establish feeding relationships of B. altianalis with other species of the same genus. The significant variation in RGI observed in the lower class sizes could suggests a transient feeding mode in this species, preferring a live feed dominated deit in its juvenile stages. It is envisaged that this behavior could suggest investigating possibility of fish nutritional requirements at varying stages of development.

The presence of numerous taste buds interspersed among the mucosa epithelial cells on the barbels, the lips, the oral-pharyngeal cavities and the oesophagus, illustrated their strong gustatory role in this region, suggesting the fish’s ability to scan the environment for preferred food items. The taste buds

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on the barbels suggested a hunting behavior or search for its food items as observed by Fox (1999) on a number of barbeled fish species. The barbels formed part of extra oral gustatory system that enhances the role of incitants and suppressants in fish, used to search and grasp the preferred food items before they are passed on to the oral-pharyngeal system that enhances stimulation for food palatability (Mearns, Ellingsen, Doving & Helmer, 1987). The presence of this system in B. altianalis equally suggests a selective behavior for preferred items thus necessitating addition of stimulants to artificial feeds to enhance stimulation and palatability for food items, a common practice for some cultured species (Kasumyan & Doving, 2003). The palatal organ at the roof of the buccal cavity was well developed as represented in some cultured carps such us Cyprinus capio and is associated with the mechanical separation of food items from inorganic debris during feeding (Osse, Sibbing, Boogaart & Van den, 1997; Sibbing, 1982). The study by Callan and Sanderson (2003) illustrated an important additional role of chemosensory function of the palatal organ that is initiated by the presence of taste buds to aid formation of wavy protrusions across the organ for expelling inorganic particles and retaining small food particles as well. The absence of the tongue at the floor of the mouth cavity illustrated inability of the fish to mastication of the food items, a common challenge in most fishes (Namulawa et al., 2011). The tongue is replaced by taste buds for gustatory role as observed in Cyprinus carpio (Curry, 1939; Farag, Wally, Daghash & Ibrahim, 2014; Kasumyan & Morsi, 1996) as well as the Catla catla and Barbus stigma species (Kooper, 1957). However, the presence of pharyngeal teeth and the palatal chewing pad in pharyngeal cavity made effective machinery for grinding the food items. This was further strengthened by histological revelation of deep mucosa folds with numerous epithelial goblet cells in the pharyngeal cavity that offered lubricating role and facilitating smooth movement for the crashed food items. The posterior palatal protrusion on the chewing pad was perpendicular to the eosophagus opening and probably acted as a stopper by preventing the backflow of food items into pharyngeal cavity.

The noted tough short oesophagus with its numerous epithelial goblet mucus cells seems to facilitate a quick swift movement of the crushed feed material into the intestine. The increased number of goblet mucus cells in the buccal pharyngeal cavity and the oesophagus in fish species is generally an adaptation that compensates the absence of salivary glands found in other vertebrates to facilitate food lubrication and movement (Abd-El-Hafez, Mokhtar, Abou-Elhamd & Hassan, 2013; Albrecht, Ferreira & Caramaschi, 2001; Cataldi, Crosetti, Conte, Ovidio & Cataudella, 1988). The striated 133

muscle fibres and rugae observed in the oesophagus offered flexibility in expanding, contracting and containment of pressure exerted on it by the food items and allowed the movement of the food material into the intestine. The presence of striated muscles has been reported to perform similar functions in some species (Albrecht et al., 2001; Dai, Shu & Fang, 2007; Namulawa et al., 2011). The oesophagus was comparatively smaller in size than the adjacent proximal intestinal tube, suggesting an additional role of effectively preventing the backflow of food items. The presence of taste buds in the cranial part of the oesophagus has been also reported in some species such as sturgeon (Domeneghini et al., 1999), and in Grass carp Ctenopharyngodon idella (Abd-El-Hafez et al., 2013). Their presence indicated that unpalatable food items could still be expelled.

The effectiveness of digestibility of food along the digestive tube of B. altianalis was enhanced and facilitated by the presence of both neutral and acidic mucins within the goblets cells. The acidic mucins were dominated by the sialytedgly coconjugates that stained positive with AB pH 2.5. Both neutral and sialyted mucins are responsible for the less viscous mucal characteristic nature that largely facilitates lubrication and swift movement of food materials within the gut system (Fiertak & Kilarski, 2002), and are important in protection of the mucosa epithelia against harmful degrading pathogenic effects (Namulawa, Kato, Nyatia, Kiseka & Rutaisire, 2014). The sialomucins have been found to protect the mucosa epithelia against the glycosidases degrading activity in fish species (Carrasson, Grau, Dopazo & Crespo, 2006.) and also facilitate the conducive pH environment for appropriate enzymatic activity within the intestine (Traving & Shauer, 1998). The increased presence of sulphated mucins in the pharyngeal cavity than other regions of the gut illustrated their importance in the feeding process in this region. Tibbetts (1997) suggested that the sulphomucins are highly viscous and could help trap small particles. This observation seems to support the gustatory role and mechanical filtration process of food particles by the well developed palatal organ that aids retention of useful particles and the removal of unwanted materials from the buccal pharyngeal cavity. The entrapment of small food particles by mucus and their aggregation into boluses within the pharynx was also reported in the common carp Cyprinus carpio (Sibbing & Uribe, 1985). Mucins in cyprinids have been found to vary from one species to another and from one intestinal section to another and could be influenced by the environment or feed formulations as well (Fiertak & Kilarski, 2002). This could further illustrate the flexibility in feed formulations for the newly domesticated B. altianalis provided they are continuously improved to optimize the growth of fish. However, since the uptake 134

capacity of nutrients by the intestine could be correlated with the natural diets in fish species (Buddington, Chen & Diamond, 1987), it is imperative that the feed formulation simulates that of the natural environment as close as possible.

Increased presence of enterocytes with epical brush border surface was noted in the proximal and the middle intestine where the mucosa epithelium forms deeper folds. Very few are observed in the rectal region. This indicated that these regions could largely represent the main site of digestion where the intestine effectively maximises the absorption of fine digested food material over a wide surface area before being expelled. The presence of enterocytes is typical of many fish species (Cyrino, Bureau & Kapoor, 2008) but their distribution along the gut varies among different species and they are a function of the mucosal epithelial folds ensuring maximum absorption of nutrients (Cao, Wang & Song, 2011; Murray et al., 1996). Numerous eosinophils in the lamina propria throughout the entire length of the intestine were crucial in containing pathogenic invasions from the intestinal lumen (Powell, Briand, Wright & Burka, 1993).

The lobular structural arrangement of hepatocytes is similar to that of some fishes (Buddington & Kuzmina, 2000) but its outline is not well delineated with either connective tissue or the portal tracts as observed in higher vertebrates (Wheater, Burkitt & Daniels, 1979) and some fish species such as the Nile perch Lates niloticus (Namulawa et al., 2011). The presence of the numerous tracts in the liver ensures effective transportation of nutrients and exchange of metabolites through blood circulation around the hepatocytes plates. It was observed that in B. altianalis, the liver had a strong interdigitation of its tissue with the pancreas tissue making it a hepatopancreas. This arrangement has been observed in some cyprinids such as the common carp Cyprinus carpio (Takashima & Hibiya, 1995) and Crucian carp Carassius auratus (Yang et al., 2009) as well as in some non-cyprinid species (Brusle & Anadon, 1996; Namulawa et al., 2011; Nejedli & TlakGajger, 2013). Furthermore, the presence of pancreatic acini spanning the entire length around the intestine within the mesentery elaborates the critical role of exocrine and endocrine secretions during digestion. The interconnectivity of numerous blood vessels within and between the liver with other visceral organs is consistent with its many functions of largely ensuring the synthesis and distribution of the required body nutrients as well as breakdown and removal of metabolites (Brusle & Anadon, 1996). In fact the liver and mesenteric pancreatic tissue will ensure utilization and conversion of the large amounts of 135

fat deposited around the intestines into energy during breeding as observed on some mature spawning fish in this study.

6.5 Conclusion

This study revealed the digestive structure of B. altianalis as a simple tube with less differentiated regions and that the general structure is consistent with that of other cyprinids. However, the location, the distribution and the glycoconjugates of goblet mucus cells together with the variation pattern of the intestinal epithelial folds along the digestive tract may suggest some varying functional and physiological strategies unique to its feeding behavior. The flexibility in feeding along the water column and on various food items makes this fish a potential candidate for polyculture. The study suggests that in spite of the omnivorous behavior, the described attributes form a basis for the continuous development or improvement of the available artificial feeds suitable for optimal growths rates. Such feeds could put into consideration a diet premised on age or size requirements under culture conditions.

6.6 Acknowledgement

We appreciate the financial support from the World Bank funded project, the Agricultural Technology Advisory and Agribusiness Services (ATAAS). We thank the National Fisheries and Resources Research Institute (NaFIRRI), the Makerere University Colleges of CONAS and COVAB that provided other research facilities to make this work a success.

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6.7 References

Abd-El-Hafez, E. A, Mokhtar, D. M, Abou-Elhamd, A. S., & Hassan, A. H. S. (2013). Comparative histomorphological studies on oesophagus of catfish and grass carp. Journal of Histology, 1- 10. Doi:10.1155/2013/858674.

Albrecht, M. P., Ferreira, M. F. N., & Caramaschi, E. P. (2001). Anatomical features and histology of the digestive tract of two related neotropical omnivorous fishes (Characiformes; Anostomidae). Journal of Fish Biology, 58(2), 419–430. Doi:10.1006/jfbi.2000.1462.

Al-Hamed, M.I., (1965). On the morphology of the alimentary tract of three cyprinid fishes of Iraq. Bulletin of the Iraq Natural History Museum (University of Baghdad), 3, 1- 25.

Banan-Khojasteh, S.M.(2012). The morphology of the post-gastric alimentary canal in teleost fishes: a brief review. International Journal of Aquatic Science, 3(2), 71-88.

Bancroft, J. D., & Gamble, M. (2002). Theory and practice of histological techniques, 5th ed. (pp.85- 107)., Edinburgh, London: Churchill Livingston Publishers

Balirwa, J. B., (1979). A contribution to the study of the food of six cyprinid fishes in three areas of the Lake: Victoria basin, East Africa, Hydrobiologia, 66, 65-72.

Brusle, J., & Anadon. G. G. (1996). The structure and function of the liver. In. J. S. Datta-Munshi, and H. Dutta, (Eds.), Fish Morphology: Horizon of New Research (pp.79-94). Fish Brookfield VT, CRC Press,

Buddington, R. K., Chen J. W., & Diamond, J. (1987). Genetic and phenotypic adaptation of intestinal nutrient transport to diet in fish. Journal of Physiology, 393, 261–281.

Buddington, R. K.,&Kuzmina, V. (2000). The Digestive System. In G. K.Ostrander, (eds.), Handbook of Experimental Animals. The Fish. 3(11): 173-178.

Buddington, R. K., & Diamond, J. M. (1987). Pyloric caeca of fish, a "new" absorptive organ. American Journal of Physiology, 252, G65-G76.

Callan, W. T., & Sanderson, S. L. (2003). Feeding mechanisms in carp: cross flow filtration, palatal protrusions, and flow reversals. Journal of Experimental Biology, 206, 883-892. Doi: 10.1242/jeb.00195.

137

Cao, X. J., Wang, W. M., & Song, F. (2011). Anatomical and Histological Characteristics of the Intestine of the Topmouth Culter (Culteralburnus). Anatomia Histologia Embryologia, 40, 292–298.

Carrasson, M., Grau, A., Dopazo, L. R., & Crespo, S. (2006). A histological, histochemical and ultrastructural study of the digestive tract of Dentexdentex (Pisces, Sparidae). Histology and Histopathology, 21, 579-593.

Cataldi, E., Crosetti, D., Conte, G.,Ovidio, D. D., & Cataudella, S. (1988). Morphological changes in the esophageal epithelium during adaptation to salinities in Oreochromis mossambicus, O. niloticus and their hybrid. Journal of Fish Biology, 32 (2), 191–196.

Cataldi, E., Cataudella, S., Monaco, G., Rossi, A., & Tansion, L. (1987). A study of the histology and morphology of the digestive tract of the sea-bream Sparus auratus. Journal of Fish Biology, 30, 135-45. DOI: 10.1111/j.1095-8649.1987.tb05740.x

Curry, E. (1939). The histology of the digestive tube of the carp (Cyprinus carpio communis). Journal of Morphology, lxv: 53-78.

Cyrino, J. E. P., Bureau, D., & Kapoor, B.G. (2008). Feeding and Digestive Function of fishes (pp. 575) Enfield: Science Publishers.

Dabrowisk, K., & Celia., M. P. (2005). Feeding Plasticity and Nutritional Physiology. In: W. S. Hoar, J. D. Randall & A. P. Farrell (Eds.), Fish Physiology: The Physiology of Tropical Fishes (21, 155-224). Elsevier, Academic press.

Dai, X., Shu. M., & Fang, W. (2007). Histological and ultra structural study of the digestive tract of rice field eel, Monopterusalbus. Journal of Applied Ichthyology, 23, 177-183. Doi: 10.1111/j.1439-0426.2006.00830.x

De Silva, S. S., & Anderson, T. A. (1995). Fish Nutrition in Aquaculture (pp. 319). Landon: Chapman and Hall,

Delashoub, M., Pousty, I., & Banan-Khojasteh, S. M. (2010). Histology of Bighead Carp

(Hypophthalmichthys nobilis) Intestine. Global Veterinaria, 5 (6): 302-306.

138

Domeneghini, C., S., Arrighi, Radaelli, G., Bosi, G., & Mascarello, F., (1999). Morphological and histochemical peculiarities of the gut in the white sturgeon, Acipencer transmontanus. European. Journal of Histochemistry, 43:135- 145.

Fiertak, A., & Kilarski, W. M. (2002). Glycoconjugates of the intestinal goblet cells of four cyprinids. Cellular Molecular Life Sciences, 59, 1724-1733. Doi 1420-682x/02/101724-10

Farag, F. M. M., Wally, Y. R., Daghash, S. M., & Ibrahim, A. M. (2014). Some gross morphological studies on the internal anatomy of the scaled common carp fish Cyprinus carpio) in Egypt. Journal of Veterinary Anatomy, 7 (1), 15 – 29.

Food and Agriculture Organisation (FAO). (2014). The state of world fisheries and aquaculture. World review of fisheries and aquaculture (pp. 1-202). Rome, Itally: FAO.

Fox, H. (1999). Barbels and barbel-like tentacular structures in sub-mammalian vertebrates: A review. Hydrobiologia, 403: 153-193.

German, D. P., Horn, M. H. (2006). Gut length and mass in herbivorous and carnivorous prickleback fishes (Teleostei: Stichaeidae): ontogenetic, dietary, and phylogenetic effects. Marine Biology, 148, 1123-1134.

Hofer, R. (1991). Digestion in Cyprinid Fishes. In I. J. Winfield & J. S, Nelson (Eds.), Systematics, Biology and Exploitation (pp. 413–425). London, UK: Chapman and Hall,

Kasumyan, A. O., & Doving, K. B. (2003). Taste preferences in fishes, Fish and Fisheries, 4, 289- 347.

Kasumyan, A. O., & Morsi, A. M. H. (1996). Taste Sensitivity of Common Carp Cyprinus Carpio to Free Amino Acids and Classical Taste Substances. Journal of Ichthyology, 36 (5), 391–403.

Kato, C. D., Nyatia. E., Matovu. E., Uni, Z., Kedar. O., Hakim, Y., Levavi-Sivan, B., & Rutaisire, J. (2014). Developmental changes in intestinal brush border enzyme activity in wild, juvenile Nile perch Lates niloticus (Linnaeus, 1758). International Journal of Fisheries and Aquaculture, 6 (6): 71-79. DOI: 10.5897/IJFA2013.0396.

Kooper, B. G. (1957). The study of the tongue of fishes. Japanese Journal of Ichthyology, XII, 82- g85.

139

Mearns, K. J., Ellingsen, O. F., Doving, K. B., & Helmer, S. (1987). Feeding behaviour in adult rainbow trout and Atlantic Salmon Parr, elicited by chemical fractions and mixtures of compounds identified in shrimp extract. Aquaculture, 64, 47 63.

Murray, H. M., Wright. G. M., & Goff. G. P. (1996). A comparative histological and histochemical study of the post-gastric alimentary canal from three species of pleuronectid, the Atlantic halibut, the yellowtail flounder and the winter flounder. Journal of Fish Biology, 48, 187- 206. Doi: 10.1111/j.1095-8649.1996.tb01112.x

Namulawa, V. T., Kato, C. D., Nyatia. E., Britz, P., & Rutaisire, J. (2011). Histomorphology description of the digestive system of Nile perch (L. niloticus). International Journal of Morphology, 29 (3), 723-732.

Namulawa, V. T., Kato, C. D., Nyatia, E., Kiseka, M., & Rutaisire, J. (2014). Histochemistry and PH Characterization of the Gastrointestinal Tract of Nile Perch Lates niloticus. World Journal of Fish and Marine Sciences, 6 (2), 162-168.

Nejedli, S., & Tlakgajger, I. (2013). Hepatopancreas in some sea fish from different species and the structure of the liver in teleost fish, common pandora, Pagellus erythinus(Linnaeus, 1758) and whiting, Merlangius merlangu seuxinus (Nordmann, 1840). Veterinary Archives, 83, 441-452.

Osse, J. W. M., Sibbing, F. A., Boogaart, J. G., & Van den., M. (1997). Intra-oral food manipulation of carp and other cyprinids: adaptations and limitations. Acta Physiologica Scandinavica, Supplementum, 161(638), 47-57.

Powell, M. D., Briand, H. A., Wright. G. M., & Burka, J. F. (1993). Rainbow trout (Oncorhynchus mykiss) intestinal eosinophilic granular cell (EGC) response to Aeromonas salmonicida and Vibrio anguillarum extracellular products. Fish and Shellfish Immunology, 3, 279-289. Doi: 10.1006/fsim.1993.1027

Rust, M. B. (2002). Nutritional physiology (3rd ed.). In, J. E. Halver & R.W. Hardy, (Eds.), Fish Nutrition (pp. 367-452). London, UK: Academic Press.

Rutaisire, J., Levavi-Sivan. B., Aruho, C., & Ondhoro, C. C. (2015). Gonadal recrudescence and induced spawning in Barbus altianalis. Aquaculture, 46 (3), 669–678. Doi:10.1111/are.12213

140

Sibbing, F. A. (1982). Pharyngeal mastication and food transport in the carp (Cyprinus carpio L.): A cineradiographic and electromyographic study. Journal of Morphology, 172, 223–258. Doi: 10.1002/jmor.1051720208

Sibbing, F. A., & Uribe, R. (1985). Regional specialisation in the oral-pharyngeal wall and food processing in carp (Cyprinus carpio L.). Netherlands Journal of Zoology, 35, 377–422.

Takashima, F., & Hibiya, T. (1995). An atlas of fish histology. Normal and pathological features, 2nd ed. (pp.195). Tokyo: Kodansha Ltd.

Takeuchi, T., Satoh, S., & Kiron, V. (2002). Common carp, Cyprinus carpio. In C.D.Webster and C. E. Lim (Eds.), Nutrient Requirements and Feeding of Finfish for Aquaculture (pp. 245-261). CABI Publishing. Oxon.

Tibbetts, I. R. (1997). The distribution and function of mucus cells and their secretions in the alimentary tract of Arramphus sclerolepis krefftii. Journal of Fish Biology, 50, 809–820. Doi: 10.1111/j.1095-8649.1997.tb01974.x

Traving, C., & Shauer, R. (1998). Structure, function and metabolism of sialic acids. Cellular Molecular Life Sciences, 54, 1330–1349.

Wagner, C. E., Peter, B. M., Kalmia, S. B., Danielle, M. G., & Ellinor, (2009). Diet predicts intestine length in Lake Tanganyika’s cichlid fishes. Functional Ecology, 23, 1122–1131. Doi:10.1111/j.1365-2435.2009.01589.x

Wheater. P. R., Burkitt, H. G., & Daniels, V. G. (1979). Functional histology; a text and colour atlas (2nd ed.) (pp. 348). London: Church Hill Livingstone.

Yang, F., Su, W. J., Lu, B. J., Wu, T., Sun, L. C., Hara, K., & Cao, M. J. (2009). Purification and chemo characterization of chymotripsins from the hepatopancreas of Crucian carp (Carassius auratus). Food chemistry, 116 (4), 860–866.

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CHAPTER SEVEN

Morphology and functional ontogeny of the digestive tract of Barbus altianalis larvae

C. Aruho, J. Walakira; F. Bugenyi, J. Rutaisire, B. J. Reading

7.0 Abstract

The ontogenetic development of digestive structures in Ripon barbel (Barbus altianalis) larvae was investigated using standard histological and histochemical procedures from hatching up to 60 days after hatch (DAH). At hatching, the digestive tube, the mouth and the anus were closed. Complete separation of the whole gut was observed on 5 DAH while the opening of mouth and the anus were observed on 3-4 DAH. Exogenous feeding started at 5-6 DAH, but complete yolk exhaustion occurred on 7-8 DAH, indicating a period of mixed feeding. This period coincided with the beginning of the constriction of the swim bladder that facilitated active swimming from the bottom in search for food. The mucosal epithelial folds were first noted on 3 DAH in the anterior intestine, but by 6 DAH they became profound and included some goblet cells (mucus cells). At 7 DAH the mucus cells had started secreting both neutral and acid glycoconjugates. Intestinal coiling first occurred at 28-30 DAH making a single loop and the double loop occurred at 45-50 DAH. Each coiling was proceeded by larval weight increase. By 7 DAH the bucco-pharyngeal cavity was lined by a layer of squamous epithelial cells including scattered goblet cells and taste buds that became numerous by 15 DAH. At hatching, the liver and the pancreas were undifferentiated, but on 3 DAH the hepatocytes and zymogen granules of the pancreas became apparently clear. By 7 DAH both organs enlarged, making extensions into the posterior. The first coiling of intestines at 28-30 DAH coincided with the beginning of external dressing of the scales. This is a period when B. altianalis started transforming into a Juvenile. By 7-8 DAH the digestive structure showed all the necessary digestive features characteristic of a juvenile fish that could enable the larvae to digest any compound diet.

Key words: Ripon Barbel, digestive tube, mucins, exogenous feeding

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7.1 Introduction

The Nile Ripon Barbel, Barbus altianalis is a newly domesticated cyprinid whose larval survival rates are still very low, which hinders its massive seed production potential for commercial aquaculture. It is a commercial species of high value that is cherished in the region and it is distributed in Lakes Victoria, Edward and George including all the rivers associated with these lakes (Chemoiwa et al., 2013; Snoeks, Kaningini, Masilya, Nyina-Wamwiza, & Guillard, 2012). One of several aspects in fish culture is mass seed production and it entails reducing larval mortalities and increasing larval growth rates. But this requires knowledge of morphological and functional structure of the digestive system of the developing larvae. Understanding larval ontogenetic development is crucial for larval fish rearing in any economically important aquaculture species (Chen, Qin, Kumar, Hutchinson & Clarke, 2006), in part because introducing a correct feed type at a particular larval developmental stage is a key challenge in intensive fish culture (Kolkovski, 2001; Navarro & Serasquete, 1998; Ostaszewska, Wegner & Wegiel, 2003). The assessment of larval development has been studied in many cultured fishes to determine the period when the digestive system is ready to breakdown and assimilate the artificial feeds (Cahu & Zambonino, 2001; Sarasquete, Polo & Yufera, 1995; Segner, Roch, Verreth, & Witt, 1993; Zambonino-Ifante et al., 2008). The developmental stage of the digestive structure determines the timing and defines the protocol for larval weaning. How soon the larvae will feed and get weaned to a microdiet will depend on the functional development of the digestive system. In some fish species such as ide, Leuciscus idus, another cyprinid, the digestive system is well advanced with functional enzymes which may allow digestion of microdiets at the time of first exogenous feeding (Ribeiro, Sarasquete, Dinis, 1999a; Ostaszewska et al., 2003). In other fish species including common carp, Cyprinus carpio the digestive system is not properly developed to digest introduced feed particles at first feeding (Sarasquete et al., 1995; Hamlin, Herbing & Kling, 2000; Garcia-Hernandez, Lozano, Elbal & Agulleiro, 2001). First feeding, a period when exogenous feeding begins is considered the most critical period as delays or early feeding may affect larval growth and survival (Kamler, 1992; Mai et al., 2005). In spite of the fact that most fishes may develop through the same basic phases, the rate and the complexity of development of the digestive tract may defer as the larvae matures, indicating varying nutritional requirements at different stages and periods in different fish species (Chen et al., 2006; De Silva De Silva, Anderson & Sargent, 1995; Jian, 2008; Zambonino-Ifante et al., 2008).

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In B. altianalis it was morphologically observed that the yolk was only visible within about 2-3 days after hatching (DAH) and feeding started on 3 DAH (Rutaisire, Levavi‐Sivan, Aruho & Ondhoro, 2015). The newly hatched larvae were fed on Raanan feed (56 Crude Protein CP %, made by Raan Feed Company from Akko City in Israel). Despite the feeding there were high mortalities from the larvae and slow growth. Although there are other factors that might contribute to larvae mortality it is indispensable that the ontogenetic development in B. altianalis is studied and understood. Knowledge of ontogeny of the digestive structure will contribute to the understanding of larval physiology and determine the appropriate period of first or mixed feeding, acceptability of compound diets as well as the nutritional requirements at each developmental stage for effective development of the weaning technology of B. altianalis in hatcheries. The aim of this study was to describe the ontogenetic development of the digestive system from hatching until metamorphosis with the view of understanding the organization and functionality of the system, to provide the basis for developing nutritional requirements, and to identify the period for introducing compound feed to the larvae.

7.2 Materials and methods

7.2.1 Fish larvae, sampling and external morphological observations

Barbus altianalis broodstock with ripe eggs were obtained from the Victoria Nile at Jinja (E 33.5, N00.35) and transferred to the Aquaculture Research and Development Center (ARDC) Kajjansi (E32.679; S00.36.246) where they were facilitated to spawn using water flow (Chapter 4). Eggs were incubated in three hatching tanks of 50 litres at 27 ⁰C and hatched after 68.00 ± 0.88 hrs (77 degree days). Immediately after hatching they were transferred and randomly assigned into three glass tanks for better management and growth. The hatching day was considered as Day 0. The larvae started feeding at 5-6 DAH on decapsulated Artemia until 30 DAH. A commercial dry feed (57% Crude Protein CP) was gradually introduced for four days until the amount of Artemia were reduced to half the original quantity. Feeding with both Artemia and the dry feed continued for 15 more days until 45 DAH when the decapsulated Artemia was gradually and completely removed in a transitional period of four days (protocol followed by the Ssenyafish farm in Masaka-Lwengo). The larvae were kept on dry (microdiet) feed alone until 60 DAH. Feeding was done to satiation. To avoid accumulation of ammonia in tanks, tanks were cleaned at 7.00 h and 17.00 h daily with replacement of two-thirds of

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the water in the tanks after every cleaning. A sample of 10 larvae was randomly drawn from each of the three tanks. The weight and length of this sample were measure. They were anaesthetized with AQUI-S (manufactured by AQUI-S Newzland LTD), weighed and preserved in Bouins solution for more than 45 hrs before being processed following standard histological procedures by Bancroft & Gamble (2002). The samples were preserved daily from day 0 for the first 10 days, then every two days up 20 DAH, every three days up 30 DAH, and every 5 days from 30 DAH up to 60 DAH. However, in order to estimate the Specific Growth Rate (SGR) accurately with adequate sampling, about 40 larvae were sampled from each tank, weighed after every 15 days before being returned into the tanks. All the larvae were weighed to the nearest 0.001 g and their lengths recorded to the nearest 1.0 mm. In every sub-sampling, another 3 larvae were taken from each of the 3 tanks, anaesthetized with AQUI-S and were examined under the light microscope (Model Leica DM 500, Made by Microsystems Switzerland Ltd) to observe any changes in the gut development that would be correlated with the preserved samples. Because the young larvae were very transparent, the internal gut structures were easily observed under the light microscope until 20 DAH, when the gut became opaque.

7.2.2 Histological and histochemical procedure

Small larval tissues of 5-7 µm size were processed following standard histological methods by Bancroft & Gamble (2002). They were stained using Gill’s haematoxylin and eosin (H & E). To determine the presence of neutral and acidic glycoconjugates (mucins), some sections were stained with periodic acid-Schiff/PAS and Alcian blue/AB (pH 1, and 2.5) and were processed following the procedure by Pearse (1985). Both longitudinal and cross tissue sections were examined using a Carl Zeiss light microscope (model Leica DM 500) at different magnifications. Photomicrographs were taken using a canon digital Powershot A640, 10 mega pixels Camera mounted onto the microscope to aid with description of the digestive tube of the fish.

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7.2.3 Data analysis

The larval growth was expressed as average increase in weight versus the culture period and specific growth rates (SGR) as a percentage per daily increment (Hopkins, 1992; Chen et al., 2006). SGR= Ln Fw – Ln Iw/ΔT*100. Where Ln = the natural log; Iw = initial weight; Fw = final weight; ΔT = number of culture days. Relationship between weight and length as well as mean weight were analysed using IBM SPSS (Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp, 2013). Data are shown as means ± SD (Standard Deviation).

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7.3 Results

7.3.1 Larval growth and development

At hatch average total weight W of B. altianalis Larvae was 2.8 ± 0.63 g while the total length TL was 7.9 ± 0.32 mm. The mean weight W increased exponentially over the experimental period (Figure 7.1). The mean weight W was strongly and positively correlated to the total length TL and their relationship was of a power nature (r2 = 0.97, y = 4.508x3.321). The specific growth Rate SGR was 3.02%.

Vn It

It It Sb

It It

Figure 7.1: Variation of weight (mg) with Days after Hatch (DAH) in B. altianalis larvae.

The micrographs show the intestinal coiling (single loop) in the upper abdomen at 30 DAH (H & E) and a double loop at 45 DAH (PAS, AB-PH 2.5). Vn: vein, It, intestine;

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At hatch the body of the larvae was transparent with a large oval shaped yolk sac which reduced gradually until it flattened at 7-8 DAH (W 4.5 ± 0.98 mg; TL 1.1 ± 0.02 cm) (Figure 7.2a, 7.2b, 7.2c). The fins were very transparent and the tail was not forked. The larvae were confined and congregated together at the bottom of the hatching tank most of the time until 5-6 DAH (W 4.3 ± 1.26 mg; TL 1.05 ± 0.05 cm) when they began swimming slowly upwards in search for food. At hatch the eyes were brown and gradually become more pigmented, turning dark by 5 DAH when the larvae began to search for food. Active swimming at 7 DAH (W 4.4 ± 0.71 mg; TL 1.11 ± 0.02 cm) and onwards was characterized by a constriction at the anterior side of the swim bladder that separated the swim bladder into two portions (Figure 7.2d) and coincided with division of the tail forming a ‘fork’. Under the light microscope, hexagonal transparent cells (the hepatocytes) of live larvae were observed at 2 DAH and grew from just beneath the cranial position of the swim bladder and increased in size, number and length, growing towards the posterior region (Figure 7.2b). The digestive tract at hatch was a small short and closed tube, but by 7 DAH the epithelial invaginations forming folds could be observed. These intestinal folds grew much deeper as the animal increased in size and also formed visible intestinal loops under the microscope (Figure 7.2f, 7.2g, 7.2h). At 28-30 DAH (W 36 ± 9.0 mg to 40.2 ± 12.77 mg; TL 1.80 ± 0.11 cm to 1.95 ± 0.16 cm) the larvae began developing scales and this processes continued even after the experiment was terminated at 60 DAH (W 193.85 ± 35.24 mg; TL 2.90 ± 0.24 cm).

7.3.2 The digestive tube

At hatching, the larvae had a large acidophilic yolk volume (H & E stain) with a thin cyncytium separating it from the primordial digestive tract (Figure 7.2; Figure 7.3). The digestive tract was not communicating with the exterior as both the mouth and the anus were closed. The digestive tube was separated into a Buccal-pharyngeal cavity, the oesophagus, the intestine and the associated digestive organs (the liver and the pancreas).

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a Dt b C B

Yk E L Sb Op E M

c d Yk C Dt Dt An n At Sb Sb E Op L Yk

e Cn If f

Sb

At Sb At If An Cy At It g h

At

If Lv

Lv Sc

Ic Lv

Ic 29μm

Figure 7.2: Gut morphology of live B. altianalis larvae viewed under light Microscope. (a) larva at 1 DAH with a large yolk and an intestinal tube over the yolk. (b) Larva at 5 DAH with

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reduced yolk, open mouth, liver cells and a constriction which begins as an outgrowth (c) abdominal section of larva of figure (b) at 5 DAH, with a digestive tube containing a single Artemia cyst. (d) Larva at 7 DAH; there is a constriction on the swim bladder separating it into two. The liver below the swim bladder enlarges along and around the gut length toward the posterior. (e) Larvae at 10 DAH; intestine with folds also defined by the increased number of Artemia cysts (inset are folds). (f) At 15 DAH; numerous and enlarged intestinal folds (also shown inset). (g) At 22 DAH; a bend in the intestine; intestine filled with Artemia cysts. (h) At 30 DAH; large liver around the intestine, intestine loop is observed. At, Artemia; An, anus; Bb, back bone; Dt, Digestive tube; Yk, yolk; E, eye; M, mouth; Op, operculum; Lv, liver; Sb, swim bladder; Cn, constriction of the swim bladder; Cy, Cyncytial layer; Sc, scaleration; Ic, intestinal coiling; It, intestine; If, intestinal folds.

7.3.3 The buccopharyngeal cavity and the oesophagus

At hatching the mouth was closed and it opened between 3-4 DAH (W 4.15 ± 2.5 mg; TL 9.6 ± 0.47 mm). The buccal-pharyngeal cavity was clearly and largely lined by a single layer of squamous epithelial cells. Intermittent with epithelia cells were taste buds, goblet (mucus) cells and club cells that were clearly noticed by 2-3 DAH (4.37 ± 1.9 mg; 9.1 ± 0.7 mm). Between 6-7 DAH (W 4.29 ± 1.19 mg; TL 10.9 ± 0.038 mm), the buccopharynx had epithelium made of stratified squamous and cuboidal shaped cells. The mucosa epithelium formed short pharyngeal folds and had several taste buds and numerous scattered goblet cells along the buccopharynx (Table 7.1; Figure 7.4b, 7.5c, 7.5d, 7.5e). These goblet cells were PAS-AB (pH 2.5) positive giving a characteristic weak blue–purplish colouration. But at 12 DAH the colour became moderate and after 15 DAH (W 12.87 ± 2.09 mg; TL 13.8 ± 0.89 mm) it was intense with numerous mucus cells (Figure 7.7). However, the goblet cells tested negative with AB (pH 1) throughout the experimental period. By 15 DAH both the goblet cells and taste buds increased in number and size (Table 7. 1) as the larvae grew. The taste buds became elongated and the epithelium grew in thickness (Table 7.1) constituting largely pseudo-stratified squamous cells. The mucosa epithelial pharyngeal folds became prominent and by 45 DAH (W 102.98 ± 21.60 mg; TL 23.4 ± 1.4 mm) they become much deeper with numerous goblet cells (Figure 7.6). The pharyngeal cavity at this point was characterized by a clear palatal organ with several layers of cells. On 3 DAH the oesophagus was observed connecting the pharynx to the intestines. It was

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lined by a pseudo-stratified columnar mucosa epithelium. The epithelium had few goblet cells, and beneath it was a thin layer of sub-mucosa surrounded by a thick layer of a mixture of connective tissue with smooth and striated muscles. By 7 DAH the folds were conspicuously present with increasing goblet cells. At 11 DAH (W 6.0 ± 1.15 mg; TL 12.1 ± 0.6 mm) the folds were elongated further with numerous oil globules.

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Figure 7.3: A section through the head and trunk of 3 DAH B. altianalis larva. (a) A small intestinal tube placed dorsal to the yolk. (b) Buccal and mouth cavities. (c) Magnified section of (a) showing intestine. (d) Magnified section of (a) showing a portion of liver. (e) Magnified section of (b) showing epithelium around the outer lip. Oe, oesophagus; Ep, epithelium; Bc, buccal cavity; Bv, blood vessels; Cc, club cells; Ic, intestinal cavity; Et, enterocytes; Mu, mucus cells; Gl, gills; Ph, pharynx; Yks, yolk sac; Br, Brain; Epl, epithelium around the outer lip.

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Figure 7.4: Longitudinal section of B. altianalis larva at 7 DAH: (a) Feature of larvae at 10 DAH, the yolk residues are still observed, anterior section is coiled, and pancreas and liver are enlarging. (b) Features of a middle section of (a) shown at a higher magnification. Itw, intestinal wall; Ita, anterior intestine; Pa, pancreas; Eps, eye position.

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Figure 7.5: Histological sections of figure (7. 4) of B. altianalis larva at a higher magnification. (a) Differentiated pancreas and liver at 7 DAH. (b) Mucosa epithelium of intestines showing columnar cells. (c) Buccocavity lined with taste buds and mucus cells. (d) Epithelium organized into pharyngeal folds. (e) Taste buds interspaced with mucus cells in the epithelium of the pharynx. Phf, pharyngeal folds; He, heart.

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7.3.4 The intestine

At hatching much of the digestive tract length was an intestine that dorsal to the yolk mass and was closed with no clear lumen observed. The intestine was lined by a mucosa epithelium consisting of a single lining of squamous and cuboidal shaped cells. The epithelium had no intestinal folds. On 1 DAH (W 4.0 ± 2.28 g; TL 0.81 ± 0.064 cm) the gut epithelial layers separated at some points forming a lumen beginning with the anterior side, just below the swim bladder. Subsequently on 2-3 DAH thin ‘villi’ (folds) were seen in the anterior (cranial) intestine. Mucosa epithelial cells at 3 DAH elongated and became columnar shaped in a single layer (Figure 7.3c). By 5 DAH the whole intestinal length had opened with a visible lumen. The anus and the mouth were opened between 3-4 DAH. The opening of the mouth and anus coincided with the reduction in the yolk size and the beginning of the constriction of the swim bladder at 5 DAH. However, commencement of feeding on decapsulated Artemia cysts began with about 25% of the larvae at 5 DAH whose guts contained about 2-5 Artemia cysts (Figure 7.2c). Feeding by the majority of the larvae (75%) occurred at 6 DAH. At exogenous feeding (6-7 DAH) the intestine was a short tube wider at the anterior with prominent folds and narrower at the posterior and its length or shape was clearly marked by the presence of decapsulated Artemia cysts (Figure 7.2d; Figure 7.4).

Histological sections further revealed that little yolk was still observed by 7 DAH in some of the larvae (Figure 7.4) and complete yolk exhaustion occurred by 8 DAH (W 4.44 ± 0.9 g; TL 11.1 ± 0.2 mm) in few larvae. The depletion of the yolk coincided with complete division of swim bladder by a constriction (Figure 7.2d; 7.4), making the animal more capable to of active swimming in search for food. On 7 DAH, the mucosa epithelial folds were well patterned and prominent in the proximal region (cranial) of the intestine, but became much more pronounced and grew toward the distal end by 10 DAH (W 5.25 ± 1.25 g; TL 11.6 ± 0.5 mm). The epithelium folds constituted clear pseudo or single columnar cells of variable lengths with large basal nuclei (Figure 7.5b). The columnar cells were lined by an epical eosinophilic brush border (H & E stain). In live larvae observed under the microscope the whole tract still looked simple and was defined by the presence of Artemia cysts (Figure 7.2c, 7.2f). The folds were associated with few scattered goblets cells on 6-7 DAH, but increased in number and together with the goblet cells they became numerous by 10 DAH. The intestinal goblets stained very faintly (weakly) blue with PAS and AB (pH 2.5) at 10 DAH. No

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colored globules were observed with AB (pH 1) along the intestine until 45 DAH when few faintly blue goblet cells were noticed (Figure 7.6). The brush border along the epithelium was intensely purplish-blue with PAS and AB (pH 2.5). At 10 DAH the oesophagus had mucosa epithelial folds with numerous goblets compared to the intestine. By 15 DAH well patterned folds were observed (Figure 7.2f) and continued to increase in number and length through 22 DAH (Figure 7.2g).

Between 28 and 30 DAH the first gut coiling was observed. The coiling processes began with a bend observed at 22 DAH (Figure 7.2g) and became pronounced with a clear bend by 30 DAH (Figure 7.2 h; 7.1). By 45-55 DAH three sections of the gut were histologically observed in the same slide indicating that the gut had coiled twice (Figure 7.1). The gut mucosal epithelial folds became more elongated (Table 1) and were well patterned. After each coiling an exponential growth was observed (Figure 7.1). The gut coiling coincided with external appearance of glittering colors indicating the beginning of the scale formation process. The scales began forming on the lower part of the lateral line which was now visible and by 60 DAH the scales were clear observed above the lateral line and had covered more than 60 percent of the body. At this point the larvae had metamorphosed into juvenile fish. The coiling of the digestive tube and the presence of scales characterized the fish as a juvenile fish. The mean values of epithelia, folds, goblet cells and stain tests at various stages of development are provided in the Table 7.1.

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Table 7. 1: The mean values (±SD) of epithelia folds, goblet cells and taste buds at various periods DAH) of development

Day After 2-3 7-8 14-15 28-30 50-60 Hatch Buccopharynx 12 ± 2 20 ± 2.1 - 25.62±10.98; 41.48±5.2; n=10 48 ± 4 (n=10) layer (µm) n=9 125±10 n=6 Nature of Simple Simple- Stratified Pseudo- Pseudo- Epithelium in squamous stratified squamous squamous squamous buccopharynx stratified stratified Size of taste 15.86±1.2; 20.5±4.5; 26.8±2.44 39.04±9.76; 45.8±8.3 buds ( μm) n=8 n=12 n=11 n=11 n= 12

Goblet cells in 9±2.3; 10.8 ± 2.9; 11.03±1.9 12.30±2.0 13.8 ± 2.9; buccocavity n=5 n=15 n=10 n=6 n=15 (μm) Intestinal Single tube Single tube Single tube Double Double loops (number) goblet cells in 5.6±1.1 7.8±2.1 9.92±1.2 10.30±2.03 13.8±1.22 intestines n=10 n=5 n=7 n=8 n=10 (µm)

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Figure 7.6: Sections of digestive system of B. altianalis larva with PAS-AB (pH 2.5) stain showing mucus cells (goblet cells) at various ages. (a) At 7 DAH faint blue coloured mucus cells are observed. (b) Mucus cells at 15 DAH in intestines. (c) Mucus cells are of various stains (intense) at 45 DAH. (d) Intense blue stain mucus cells at 55 DAH. Only blue coloured stains are observed in this region. Sm, submucosa; Mcp, purplish stained mucus cells; Mcb, blue stained mucus cells; Msc, Muscularis.

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7.3.5 The liver and the pancreas

At hatching the liver and the pancreas were undifferentiated. The liver cells (hepatocytes) were conspicuously observed on 3 DAH as ‘penta’ or ‘hexagonal’ shaped transparent cells from live larvae under the light microscope as well as in the histologically processed samples (Figure 7.2b, 7.2e, 7.2h; 7.3d & 7.4). They began as very few cells growing from around the oesophageal region just below the cranial side of the swim bladder and by 6-7 DAH they had increased in size and number extending toward the posterior region around the gut. By 15 DAH the liver had extended dorsally past the swim bladder and on 45 DAH the liver was very large and had grown up to the posterior end of the gut toward the anus. Histologically, between 0-2 DAH the liver and pancreatic tissue could not easily be differentiated because the cells looked alike. However, on 3 DAH the pancreas was clearly differentiated from the liver by the puplish-redish zymogen granules that tested positive with PAS- AB stain (Figure 7.7). From the time the pancreatic cells were observed they were separated from the liver and by 45 DAH some pancreatic cells were now seen to be embedded within the liver around the blood vessels forming a hepatopancreas (also see Chapter 6).

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Figure 7.7: sections B. altianalis larvae showing the differentiation of the liver and the pancreas at 3 DAH. (a) The liver and the pancreas lay over the yolk mass. (b) Magnified section of (a) showing the pancreas with pancreatic acinar (plates) represented by the * (stars). The zymogens are shown by the arrows. Lu, lumen of the vein; V, vein.

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7.4 Discussion

Digestion of a compound diet at the start of exogenous feeding by larvae of most cultured species is a bottleneck to mass seed production (Hamre et al., 2013; Kolkovski, 2001; Ostaszewska et al., 2003; Trevino et al., 2011). This is because the digestive capabilities at that particular period of exogenous feeding for some of the species may not be mature or ready to effectively digest and or absorb required nutrients by the larvae from micro-processed (compound) diets (Garcia et al., 2001; Zambonino-Ifante et al., 2008). Larval requirements are species specific, age dependent and linked to the development and maturation level of digestive tract and its associated organs (Herrera, Hachero- Cruzado, Naranjo, & Mancera, 2010; Hoar & Randall, 1988; Kumar, Sharma & Chakrabarti, 2000; Ruan, Yang & Wang, 2012; Zhang et al., 2016). This study noted that the B. altianalis hatched with a big yolk reserve which took 7-8 days to get depleted contrary to the 3 DAH indicated by Rutaisire et al. (2015). Confirmation was made by histological examination which was not done in an earlier study. Active feeding started at 5-6 DAH and this implied that there was a period of mixed feeding ranging from 5-7 or up to 8 DAH. In a related cyprinid, Labeo victorianus that was recently domesticated a mixed feeding period of 2 days from 3 DAH to 5 DAH when yolk was depleted, was observed (Owori, 2009). Variations at first feeding by other cultured cyprinids occur (George & Chapman, 2013; Zhang et al., 2016) and the amount or size of yolk determined how long the mixed feeding period would take. The longer the larvae take to utilize the yolk reserves, the bigger they will grow before they search for their own food sources (Helfman, Collette, Facey & Bowen, 2009; Rutaisire et al., 2015). This is an advantage in farmed species as the larvae will grow bigger with a mature digestive system that will quickly and easily accept dry feed at first feeding. The mixed feeding period gives the larva an advantage to gradually adapt to searching for food when the yolk is completely exhausted (Lucas & Southgate, 2012). This study noted that despite the fact that the mouth opened at 3-4 DAH no Artemia cysts were seen in the gut until 5 DAH implying that feeding immediately in B. altianalis after opening of the mouth and the anus could disrupt developmental process of the gut and cause mortalities. This was also reported in bay snook Petenia splendida (Trevino et al., 2011). The period when exogenous feeding begins is considered the most critical period as delays or early feeding may affect larval growth and survival (Kamler, 1992; Mendoza et al., 2003; Mai et al., 2005). However, this period could be influenced by temperature since the rate at which the yolk is depleted will be influenced by temperature. Accelerated temperatures will drain the

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yolk faster than reduced temperature (George & Chapman, 2013; Heming, 1982; Herrera et al., 2010). This may render difficulty for farmer to determine the actual period when first feeding begins. In this study the beginning of exogenous feeding was visually and morphologically identified when the yolk reduced almost to the level of head or the lower jaw and this was the same period when the larvae began to lift up from the bottom of the tank with definite constriction of the swim bladder that eventually develop a second portion that also inflates.

Evidence from morphological, histological and histo-chemical examination revealed that in B. altianalis, all the necessary features for digestion were ready by 6-7 DAH. The taste buds, mucus cells, the intestinal folds as well as the intestinal goblet cells were present by 6-7 DAH. The presence of neutral and acidic glycoconjugates (mucins) was detected by 7 DAH, implying that they were active in the digestion process. The neutral and sialo-mucins are characteristic key components of fluid secretions of low viscosity (Fiertak & Kilarski, 2002) and their presence in the buccopharyngeal cavity largely facilitated lubrication of food items and protection against mechanical abrasion of the mucosa epithelium in fish (see Chapter 6; Fiertak & Kilarski, 2002). Sylated glycoconjugates (sialo- mucins) can prevent glycosidase degrading activity against the gut epithelium so that the larval gut is protected from infections (Carrasson et al., 2006; Namulawa, Kato, Nyatia, Kiseka & Rutaisire, 2014). However, the highly dense sulphonated mucins were negative with AB (pH 1) stain, hence they were absent in this region at this stage. In mature B. altianalis, they are present in the buccopharyngeal cavity and their presence suggests a role to facilitate trapping of small particles and filtration of plankton and other desirable microorganisms or feed particles in larger juvenile or adult fish (Aruho et al., 2017). Together with the well developed palatal organ, the sulphomucins are effective in filtering planktons for ingestion by the fish (see also Chapter 6; Sibbing & Uribe, 1985). Lack of sulpho-mucins in the pharyngeal cavity indicates that the filtration process, especially for micro particles including algae, is poorly developed in larvae, after all the palatal organ was still immature and the mechanical filtration process was therefore less efficient at this stage. This could indicate that the larvae are limited in their mechanical digestive capability, but were able to easily ingest relatively larger sized particles than the algae for instance. It is presumed that the range of 19- 24 µm in size of Artemia or fine larval microdiet as recorded in this study can be easily captured and ingested compared to the algae or other smaller particles (<19 µm). The sulphomucins began to appear in the intestine section by 50 DAH, the period when the fish are metamorphosing into 162

juveniles implying that they had matured and the mode of feeding could have improved. Differences in mucins between age groups (excluding larvae) have also been found in cultured Cyprinus carpio and have been attributed to the increasing need for their role as the fish grows and becomes more diverse in feeding strategies (Neuhaus et al., 2007).

Both neutral and acid mucins were also present along the intestine by 7 DAH, but the acid mucins (sialo-mucins) were prominently observed staining faintly blue, becoming moderately blue by 14 DAH and intense at 30 DAH, compared to the neutral mucins that stained weakly (or faintly) throughout the experimental period. Neutral mucins are very intense in larvae with mature stomachs where they neutralize the acidity of the HCl acid produced during the digestion of proteins (Namulawa et al., 2014). In stomachless fish, in addition to facilitating quick movement of bolus through the intestine, they are important in preventing the produced enzymes from self-digestion (Fiertak & Kilarski, 2002; Namulawa et al., 2014). It can be postulated that the increasing level of the intensity of mucins as the larvae grew was commensurate with increasing number and size of taste buds and goblet cells, lengthening of the intestinal folds as well as the associated liver and pancreas; coupled with the enzyme activity resulting in improved digestive capability. Differences in mucin amount in common carp between age groups (excluding larvae) were linked to increased intestinal folds (Neuhaus et al., 2007). The strength of the acid mucins (stain colour strength) was an indicator of improved functionality of mucins, which were vital in facilitating transportation of ions across the epithelium, inducing immunity or defense of the gut and stabilizing enzymes (Gomez, Sunyer & Salinas, 2013; Fiertak & Kilarski, 2002; Neuhaus et al., 2007; Shephard, 1994). The identification of these mucins and the level of gut development at 7 DAH therefore suggested that the larvae of B. altianalis were possibly able to digest a compound diet at first exogenous feeding. In spite of the fact that some species may directly be weaned to dry (compound) diets at the exogenous feeding, in other species it is possible that more mortalities may still occur even when they can be started on dry feed (Akbary, Imanpoor, Sudagar & Makhdomi, 2010; Kolkovski, 2001; Policar et al., 2011; Ramesh et al., 2014; Wang et al., 2005). Therefore this implies that the effectiveness and efficiency of the larval gut system to digest the feed could further be tested with various diets including micro-diets to indentify and confirm the weaning diet or combination of diets that will provide appropriate nutrients for optimal growth and survival of larvae.

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The intestinal coiling followed a relatively sharp rise in larval growth after 28-30 and 45-55 DAH. Variations in the timing of the intestinal coiling in some farmed cyprinids occurred during ontogenetic development of the digestive tract (Ruan et al., 2012). The gut coiling increased the surface area over which digestion and absorption occurred thereby facilitating rapid growth and development of the larvae (Falk-Petersen, 2005; Ronnestad et al., 2013; Ruan et al., 2012; Trevino et al., 2011). The initial gut coiling at 28-30 DAH coincided with initiation of scalation and continued even after the experiment was terminated. The scaleration process is critical as part of the skin, in protecting the fish from diseases and parasitic infections, facilitating swiftness during swimming as well as aiding the fish to adapt to various aquatic environments, thereby increasing chances of survival (Creaser, 1926; Long, Hale, Mchenry & Westneat, 1996; Sherman, Yaraghi, Kisailus & Meyers, 2016). This also implied that the fish had started the transition process to the juvenile stage, a stage when the larvae resembled an adult (Jones, Martin & Hardy, 1978; Kendall & Moser, 1984).

7.5 Conclusion

The digestive structure of B. altianalis was presumed to be ready to accept the micro-processed diets at first exogenous feeding between 6-7 DAH, because all the required gut structures for digestion were present by this time. The digestive structure and its associated organs together with the histo- chemical characteristic pattern of mucins gradually became prominent as the larvae grew, conferring to the larvae a system for efficient digestion. By 28-30 DAH, the digestive tube had coiled and formed a single loop and at 45-55 DAH it formed a double loop. The intestinal coiling indicated increasing efficiency of the gut system for utilisation of consumed diets and as a result there was a noted increase in the larval growth. Understanding the ontogenetic development process of the larval digestive system is an important step that will guide hatchery operators to harmonize the larval nutrition requirements with larvae digestive capability in development of the weaning protocol. Hence further study is inevitable to evaluate the effectiveness of the digestive system with various feeds including the dry feed from the first exogenous feeding period with the view of maximising growth and minimising mortalities.

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7.6 Acknowledgment

We thank the World Bank funded project, the Agricultural Technology Advisory and Agribusiness Services (ATAAS) for the financial support. We thank the National Fisheries and Resources Research Institute (NaFIRRI), the Makerere University Colleges of CONAS and COVAB that provided other research facilities to make this work a success.

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7.7 References

Akbary, P., Imanpoor, M., Sudagar, M., & Makhdomi, N. M. (2010). Comparison between live food and artificial diet on survival rate, growth and body chemical composition of Oncorhynchus mykiss larvae. Iranian Journal of Fisheries Science, 9 (1), 19-32.‎

Aruho, C., Namulawa, V., Kato, C. D., Kisekka. M., Rutaisire, J., & Bugenyi, F. (2016). Histo- morphological description of the digestive system of the Rippon Barbel Barbus altianalis

(Boulenger 1900): A potential species for culture. Uganda Journal of Agricultural Sciences, 17 (2): 197 – 217.

Bancroft, J. D., & Gamble, M. (2002). Theory and practice of histological techniques (5th edn. pp 85-107). Edinburgh, London: Churchill Livingston Publishers.

Cahu, C. & Zambonino Infante, J. (2001). Substitution of live food by formulated diets in marine fish larvae. Aquaculture, 200, 161–180.

Carrasson, M., Grau, A., Dopazo, L. R., & Crespo, S., (2006). A histological, histochemical and ultrastructural study of the digestive tract of Dentex dentex (Pisces, Sparidae). Histology and Histopathology, 21, 579-593.

Chemoiwa, E. J., Abila, R., Macdonald, A., Lamb, J., Njenga, E., & Barasa, J. E. (2013). Genetic diversity and population structure of the endangered ripon barbel, Barbus altianalis (Boulenger, 1900) in Lake Victoria catchment, Kenya based on mitochondrial DNA sequences. Journal of Applied Ichthyology, 29(6), 1225-1233.

Chen, B. N., Qin, J. G., Kumar, M. S., Hutchinson, W., & Clarke, S. (2006). Ontogenetic development of the digestive system in yellowtail kingfish Seriola lalandi larvae. Aquaculture, 256(1), 489-501.

Creaser, C. W. (1926). The structure and growth of the scales of fishes in relation to the

interpretation of their life-history, with special reference to the sunfish Eupomotis gibbosus ( Miscellaneous Publication No. 17). University of Michigan Museum of Zoology, Ann Arbor.

De Silva, S. D., Anderson, T. A., & Sargent, J. R. (1995). Fish nutrition in aquaculture. Reviews in Fish Biology and Fisheries, 5(4), 472-473.

166

Falk-Petersen, I. B. (2005). Comparative organ differentiation during early life stages of marine fish. Fish & Shellfish Immunology, 19(5), 397-412.

Fiertak, A. & Kilarski, W. M., (2002). Glycoconjugates of the intestinal goblet cells of four cyprinids. Cellular Molecular Life Sciences, 59, 1724-1733. Doi 1420-682x/02/101724-10

Garcia-Hernandez, M., Lozano, M. T., Elbal, M. T., & Agulleiro, B. (2001). Development of the digestive tract of sea bass (Dicentrarchus labrax L). Light and electron microscopic studies. Anatomy and embryology, 204(1), 39-57.

George, A. E., & Chapman, D. C. (2013). Aspects of embryonic and larval development in bighead carp Hypophthalmichthys nobilis and silver carp Hypophthalmichthys molitrix. PloS one, 8(8), e73829.

Gomez, D., Sunyer, J. O., & Salinas, I. (2013). The mucosal immune system of fish: the evolution of tolerating commensals while fighting pathogens. Fish & Shellfish Immunology, 35(6), 1729- 1739.

Hamlin, H. J., Herbing, I. H., & Kling, L. J. (2000). Histological and morphological evaluations of the digestive tract and associated organs of haddock throughout post‐hatching ontogeny. Journal of Fish Biology, 57(3), 716-732.

Hamre, K., Yufera, M., Ronnestad, I., Boglione, C., Conceiçao, L. E., & Izquierdo, M. (2013). Fish larval nutrition and feed formulation: knowledge gaps and bottlenecks for advances in larval rearing. Reviews in Aquaculture, 5(s1), S26-S58.

Helfman, G., Collette, B. B., Facey, D. E., & Bowen, B. W. (2009). The diversity of fishes: biology, evolution, and ecology. John Wiley & Sons.

Heming, T. A. (1982). Effects of temperature on utilization of yolk by chinook salmon (Oncorhynchus tshawytscha) eggs and alevins. Canadian Journal of Fisheries and Aquatic Sciences, 39(1), 184-190.

Herrera, M., Hachero-Cruzado, I., Naranjo, A., & Mancera, J. M. (2010). Organogenesis and histological development of the wedge sole Dicologoglossa cuneata M. larva with special reference to the digestive system. Reviews in Fish Biology and Fisheries, 20(4), 489-497.

167

Hoar, W. S., & Randall, D. J. (1988). The Physiology of developing fish. Part A: Eggs and Larvae. Fish Physiology, 11, 253-346.

Hopkins, K. D. (1992). Reporting fish growth: a review of the basics. Journal of the World Aquaculture Society, 23(3), 173-179.

Jian, G. Q. (2008). Larval fish Nutrition and rearing technologies: In H. Stephen S (Ed.), State of the Art and future. Aquaculture Research trends, 3, 113-148.

Jones, P. W., Martin, F. D., & Hardy, J. J. D. (1978). Development of fishes of the Mid-Atlantic Bight: An atlas of egg, larval and juvenile stages. Bio-logical Services Program, U.S. Fish and Wildlife Service, 1, 1-366.

Kamler. E. (1992). Endogenous feeding period. In Chapman & Hall (Eds.), Early life history of fish. An energetic approach. Fish and fisheries series (IV: 107- 196). London. Springer Netherlands.

Kendall, A. W., Ahlstrom, E. H., & Moser, H. G. (1984). Early life history stages of fishes and their characters. In: H. G. Moser, W. J. Richard & D. M. Cohen (Eds.), Ontogeny and systematic of fishes. American society of Ichthyologist and Herpertologists, special publication 1: 11-22.

Kolkovski, S. (2001). Digestive enzymes in fish larvae and juveniles-implications and applications to formulated diets. Aquaculture, 2000, 181–201

Kumar, S., Sharma, J. G., & Chakrabarti, R. (2000). Quantitative estimation of proteolytic enzyme and ultrastructural study of anterior part of intestine of Indian major carp (Catla catla) larvae during ontogenesis. Current Science-Bangalore, 79(7), 1007-1010.

Long, J., Hale, M., Mchenry, M., & Westneat, M. (1996). Functions of fish skin: flexural stiffness and steady swimming of longnose gar, Lepisosteus osseus. Journal of Experimental Biology, 199(10), 2139-2151.

Lucas, J. S., & Southgate, P. C. (2012). Aquaculture: Farming aquatic animals and plants (pp.629). John Wiley & Sons.

Mai, K., Yu, H., Ma, H., Duan, Q., Gisbert, E., Zambonino-Infante, J. L., & Cahu, C. L. (2005). A histological study on the development of the digestive system of Pseudosciaena crocea larvae and juveniles. Journal of Fish Biology, 67(4), 1094-1106. 168

Namulawa, V. T., Kato, C. D., Nyatia, E., Kiseka, M., & Rutaisire, J. (2014). Histochemistry and PH Characterization of the Gastrointestinal Tract of Nile Perch Lates niloticus. World Journal of Fish and Marine Sciences, 6 (2), 162-168.

Navarro, N., & Sarasquete, C. (1998). Use of freeze-dried microalgae for rearing gilthead seabream, Sparus aurata, larvae: I. Growth, histology and water quality. Aquaculture, 167(3), 179-193.

Neuhaus, H., Van der Marel, M., Caspari, N., Meyer, W., Enss, M. L., & Steinhagen, D. (2007). Biochemical and histochemical study on the intestinal mucosa of the common carp Cyprinus carpio L., with special consideration of mucin glycoproteins. Journal of Fish Biology, 70(5), 1523-1534.

Ostaszewska, T., Wegner, A., & Wegiel, M. (2003). Development of the digestive tract of Ide, leuciscus idus (L) during the larvae stage. Archives of Polish Fisheries, 11, 79-92.

Owori, W. A. (2009). The feeding ecology, ontogeny and larval feeding in Labao Victorianus Boulenger 1901 (Pisces: Cyprinidae). Unpublished Doctoral thesis, Makerere University, Uganda. Retrieved from http://hdl.handle.net/10570/2635

Pearse, A. G. E. (1985): Histochemistry-Theoretical and applied. Vol. II. Edinburgh (pp. 441-1055). London. Churchill Livingstone Inc.

Policar, T., Podhorec, P., Stejskal, V., Kozak P., Svinger, V., & Alavi, S. M. H. (2011). Growth and survival rates, puberty and fecundity in captive common barbel (Barbus barbus L.) under controlled condition. Czech Journal of Animal Science, 56 (10), 433–442.

Ramesh, R., Dube, K., A. K., Prakash, C., Tiwari, V. K., Rangacharyulu, P. V., & Venkateshwarlu, G. (2014). Growth and survival of pengba, Osteobrama belangeri (Val.) larvae in response to co-feeding with live feed and microparticulate diet. Ecology Environment & Conservation, 20(4), 1715-1721.

Ribeiro, L., C. Sarasquete, & Dinis, M.T. (1999a). Histological and histochemical development of the digestive system of Solea senegalensis (Kaup 1858) larvae. Aquaculture, 171, 293-308.

Ronnestad, I., Yufera, M., Ueberschar, B., Ribeiro, L., Sæle, O., & Boglione, C. (2013). Feeding behaviour and digestive physiology in larval fish: current knowledge, and gaps and bottlenecks in research. Reviews in Aquaculture, 5, s59–s98.

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Ruan, G. L., Yang, D. Q., & Wang, W. M. (2012). Ontogeny of the digestive tracts in grass carp (Ctenopharyngodon idella), yellowcheck carp (Elopichthys bambusa) and topmouth culter (Culter alburnus). Acta Hydrobiol Sin, 36, 1164-1169.

Rutaisire, J., Levavi‐Sivan, B., Aruho, C., & Ondhoro, C. C. (2015). Gonadal recrudescence and induced spawning in Barbus altianalis. Aquaculture Research, 46(3), 669-678.

Sarasquete, M. C., Polo, A., & Yufera, M. (1995). Histology and histochemistry of the digestive system of larval gilhead sea bream Sparus aurata L. Aquaculture, 130, 79-92.

Segner, H., Rosch, R., Verreth, J., & Witt, U. (1993). Larval nutritional physiology: studies with Clarias gariepinus, Coregonus lavaretus and Scophthalmus maximus. Journal of the World Aquaculture Society, 24(2), 121-134.

Shephard, K. L. (1994). Functions for fish mucus. Reviews in Fish Biology and Fisheries, 4(4), 401- 429.

Sherman, V. R., Yaraghi, N. A., Kisailus, D., & Meyers, M. A. (2016). Microstructural and geometric influences in the protective scales of Atractosteus spatula. Journal of the Royal Society Interface, 13(125), 20160595.

Sibbing, F. A., & Uribe, R. (1985). Regional specialisation in the oral-pharyngeal wall and food processing in carp (Cyprinus carpio L.). Netherlands Journal of Zoology, 35, 377–422

Snoeks, J., Kaningini, B., Masilya, P., Nyina-Wamwiza, L., & Guillard, J. (2012). Fishes in Lake Kivu: diversity and fisheries. In Lake Kivu (pp. 127-152). Netherlands: Springer.

Trevino, L., Alvarez‐González, C. A., Perales‐García, N., Arévalo‐Galán, L., Uscanga‐Martínez, A., Marquez‐Couturier, G., ... & Gisbert, E. (2011). A histological study of the organogenesis of the digestive system in bay snook Petenia splendida Gunther, 1862 from hatching to the juvenile stage. Journal of Applied Ichthyology, 27(1), 73-82.

Wang, C., Xie, S., Zheng, K., Zhu, X., Lei, W., Yang, Y., & Liu, J. (2005). Effects of live food and formulated diets on survival, growth and protein content of first‐feeding larvae of Plelteobagrus fulvidraco. Journal of Applied Ichthyology, 21(3), 210-214.

Zambonino-Infante, J., Gisbert, E., Sarasquete, C., Navarro, I., Gutierrez, J., & Cahu, C. L. (2008). Ontogeny and physiology of the digestive system of marine fish larvae. In J. E. O. Cyrino, 170

Bureau, D., & Kapoor, B.G. (Eds.), Feeding and Digestive Functions of Fish (pp. 277–344). Enfield, USA: Science Publishers Inc

Zhang, J., Yang, R., Yang, X., Fan, Q., Wei, K., & Wang, W. (2016). Ontogeny of the digestive tract in mud loach Misgurnus anguillicaudatus larvae. Aquaculture Research, 47(4), 1180-1190.

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CHAPTER EIGHT

Growth and survival of Barbus altianalis larvae and juveniles in captivity

C. Aruho, O. Wadunde, E. Nuwamanya, F. Bugenyi, R. J. Borski, J. Rutaisire

8.0 Abstract

In fish culture massive production of quality seed is vital for commercial fish culture and requires prior knowledge of appropriate larval diets and their utilization. This study evaluated the effect of different diets on growth and survival of the Barbus altianalis larvae and juveniles, identified the age when digestive system is mature enough to competently assimilate microdiets without compromising growth performance and suggested appropriate periods for stocking based on the maturation age and prevalence of aquatic parasites. Eight experiments were conducted at different periods to evaluate the effect of live and microdiets on growth of B. altianalis larvae and juveniles. Larvae were fed exclusively on live prey (Moina and or Artemia nauplii), microdiet (57% Crude Protein), decapsulated Artemia cysts and in combination (Moina + microdiet). The effect on growth was further evaluated in subsequent juvenile trials by co-feeding. Green water effect on larval growth was also evaluated in a separate experiment. Results indicated that each diet affected larval growth significantly different (p < 0.05) with the combination diet (152.05 ± 2.51mg) and decapsulated Artemia cysts (141.14 ± 2.43mg) performing better than microdiet, Moina and Artemia nauplii in that order. Similar trend was observed in subsequent juvenile trial. No direct effect was noted on larval weight, specific growth rate and survival between tanks where green water was added and those where it was not (p > 0.05). The ontogenetic pattern of amylase, lipase and protease activity identified larvae maturation age at 14-21 DAH (3rd Week; 14.93 ± 0.36mg) with the combination diet. Hence stocking and further microdiet manipulations could suitably be done after this date. In the event of anticipation of parasitic infestations for outdoor facilities, stocking can be delayed until after larvae metaphormosize into juvenile stage by 75 DAH because larvae survival increased with squamation. The B. altianalis larvae were also confirmed to utilize the microdiet efficiently from exogenous stage but co-feeding produced the best growth parameters.

Key words: Larval diets; weaning; enzyme activity

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8.1 Introduction

To promote hatchery rearing of a particular species it is imperative to identify effective diets for the development and growth of larval fish (Cahu & Zambonino-Infante 2001; Herath & Atapaththu, 2013; Peteri, Nandi & Chowdhury, 1992). A quality diet delivered at the appropriate period for weaning are critical factors for larval survival and growth (Bisht, Anand, Bhadula, Pal 2013; Mai et al. 2005). The sustainable production of profitable quality seed also entails reducing production costs by gradually orientating larvae to cheaper diets while maximizing their growth and survival (Herath & Atapaththu, 2013; Mokolensang, Yamasaki & Onoue, 2003; Bondad-Reantaso, 2007). This is dependent on the availability of suitable diets that are readily consumed and efficiently digested to support rapid growth (Mokolensang et al., 2003, Giri, Sahoo, Sahu 2002). Large quantities of larvae cannot be sustained on live cultures alone as they are expensive to prepare and require considerable time to maintain (Conceicao, Yufera, Makridis, Morais, Dinis 2010; Kolkovski, 2001; Liu et al., 2012). They may also, at times, carry harmful disease agents (Lahnsteiner, Kletzl & Weismann, 2009). Processed microdiets (dry feeds) are preferred because they are cheaper and easier to apply for weaning larvae (Chatain, 1997). A good larval weaning strategy would enable the introduction of microdiets at a particular time and stage when fish can easily digest and utilize the feeds efficiently. This process varies among species as the gradual acceptance of food and digestive capability depend on developmental progression of the gastrointestinal tract and its capacity to digest and absorb nutrients (Cahu & Zambonino-Infante, 2001; Kujawa et al., 2010). An improper weaning diet could impair or delay the development of the digestive system causing chronic stress leading to physiological malfunctions or even death (Cahu & Zambonino-Infante, 1994). In cyprinids such as rheophilic species (Kwiatkowski et al., 2008 & Wolnicki, 2005), the Rudd, Scardinius erythrophthalmus (Wolnicki, Sikorska & Karminiski, 2009), Aspius aspius (L.) and Chondrostoma nasus (Kujawa et al., 2010), Cuspian Katum Rutilus frisii kutum (Falahatkar, Mohammadi & Noveirian, 2011) and common carp Cyprinus carpio (Mahfuj, Hossein & Sarower, 2012), larval growth and survival rates were strongly and differently affected by both the type of diet given and the larvae age when the feed was introduced.

The performance of weaning diets can be evaluated through growth and survival parameters and also by determining ontogenetic patterns in enzyme activity to identify the earliest age of digestive 173

competence for larvae provided a given diet (Zambonino-Infante et al., 2008). Cost-effective weaning strategies are focused on partial or total replacement of live diets by micro particulate diets (Murray, Perez-Casanova, Gallant, Johnson & Douglas, 2004; Liu et al., 2012). While some species can successfully be started on processed microdiets exclusively others are weaned on a combination of live diets and microdiets (Kolkovski, 2001). Co-feeding of live prey and microdiets may enhance enzyme activity to aid digestion of the microdiet and facilitate maximum uptake of nutrients since some of the larvae lack sufficient endogenous enzymes (Dabrowski, 1984). It is imperative to assess species larvae nutritional requirements and their comparative response to various diets, to ascertain and identify appropriate weaning strategies. Most of the commonly cultured live prey for fish larvae include rotifers, Artemia nauplii, copepods and cladocerans that are maintained on algal cultures (Chlorella sp) for their nutrient enrichment (Das, Mandal, Bhagabati, Akhtar, Singh, 2012). However, some studies report a direct role of algae (green water) in growth and survival as part of the larvae weaning strategy (Nicolas, Robic & Ansquer, 1989; Reitan, Rainuzzo, Oie & Olsen, 1997; Sanaye, Dhaker, Tibile & Mhatre, 2014). It has also been reported that the omnivorous adult B. altianalis has a well developed pharyngeal pad for straining algae and other important smaller particles (Aruho et al., 2017). It is unknown whether green water has any direct effect on B. altianalis larvae development. The Artemia spp (including its eggs/cysts) are said to be a good diet that can produce quality larvae for some cultured fish species but the high costs and importation encumbrances are prohibitive for most farmers in developing nations. Alternative, cheaper options could be a better strategy (Dhert, Divanach, Kentouri & Sorgeloos, 1998; Olurin & Oluwo, 2010).

Previous attempts to produce B. altianalis were successful using induced spawning techniques but reports suggest this species exhibits slow growth and relatively low larval survival (Rutaisire, Levavi‐Sivan, Aruho & Ondhoro, 2015; personal observation). Hence, there is little seed available for propagation of B. altianalis. Low survival rates may be attributed, in part, to a lack of knowledge on larval rearing. Recent investigations in unpublished report by Aruho (2017; also see chapter 7) on ontogenetic development of the digestive system using histology revealed that B. altianalis larvae may have the capacity to digest a microdiet by 7 days post-hatch. However, whether larvae and juveniles effectively utilize such diets or can improve growth and survival of larvae is unclear. This study evaluated survival and growth of B. altianalis larvae raised on live prey, a microdiet, decapsulated Artemia cysts and green water. The study evaluated changes in amylase, protease and 174

lipase enzymatic activity over the course of larval development to establish the earliest period the digestive system matures and can utilize different diets. Based on the growth performance and digestive maturation competence level of larvae, this study determined the best growth rates, survival and appropriate period for stocking or nursing larvae in outdoor rearing facilities. The findings were of great significance in developing a weaning strategy for production of B. altianalis fingerlings under culture conditions.

8.2. Materials and methods

8.2.1 Sampling and data collection

8.2.1.1 Experimental study 1a; growth and survival of larvae on weaning diets and diet combinations

The B. altianalis hatchlings for the experiment were obtained by inducing one ripe female using a running water technique described previously (Rutaisire et al., 2015). Eggs were fertilized and incubated at 27oC in a basin where they hatched after 77 days. The larvae were kept in the hatching basins at 27 ± 1⁰C and the ammonia level (monitored by LaMotte Fresh water aquaculture kit Code 6665-02-CC) was kept below 0.1 ppm by flow through of water until the yolk gradually resorbed at 6 days after hatch (DAH). Fifty-five larvae (3.0 ± 1.0 mg) were randomly allocated to 15 glass aquaria (55 L) capacity each. Five diet treatments, each in triplicate, were randomly assigned to the 15 glass tanks. The treatments were as follows; Moina alone (MO), a combination of Moina + microdiet (MD), hatched Artemia nauplii (HA), decapsulated Artemia cysts (DA), and microdiet or dry feed (DF) alone. Diet DF was a commercial feed of 57% crude protein (CP). Nutritional composition of the DF, including fatty acid and the amino acid profiles was analyzed at the nutritional laboratory of the University of Ghent, Belgium and Nitric lab Company in the Netherlands respectively (Table 8.1). Temperature in all experimental tanks was maintained at 27 ± 1⁰C using thermostatic heating rods (Sera Aquarium heater thermostat; sera D 52518, Heinsberg Germany). Ammonia levels were maintained below 0.1 ppm by cleaning tanks and exchanging water twice daily at 7:00h and 17.00h. Dissolved oxygen (DO) was maintained at 7.0 ± 2.7 mg/l throughout the experimental period. The hatched larvae were fed to satiation, 3 times a day at 8.00h, 12.00h and 18.00h (Table 8.2). For the combination diet, larvae were fed Moina + microdiet (MD) at the same time during each feeding

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time. The experiment was conducted for a period of 48 days (48 DAH). About 45 larvae were sampled from each tank throughout the experiment. The wet weight of each larva was recorded to the nearest 0.001 g using an electronic micro weighting scale (Model DJ V320A; 320 g/0.001 g, PAN SCALE Hardware) and the length was recorded to the nearest 0.1 cm using a calibrated ruler.

8.2.1.2 Experimental study 1b; ontogenetic enzyme activity and regulation in larvae fed with best diet combination (MD).

The ontogeny and regulation of enzyme activity of amylase, protease and lipase enzymes were determined to explain the growth changes during larval development for the best performing combination diet (MD) during experimental study 1a. Enzyme activity helped identify the maturation digestive competent period for break down and utilization of starch, proteins and lipids by larvae. This experiment determined the period of larval digestive maturation level (or efficiency) for which the larvae was competent enough or easily able to digest and assimilate a microdiet with minimal mortalities and improved weight. In this experiment (1b), an additional 10 aquaria identical to the 15 in experiment 1a were treated with the combined Moina and microdiet (MD). The aquaria were randomly stocked with 100 larvae each. Rearing conditions and feeding regimes were maintained similar to experiment 1a until 45 DAH. Larvae were randomly sampled in equal proportion from the 10 tanks before the morning feeding, anaesthetized with an overdose of clove oil and weighed to achieve 5 g (constituting between 50 to 150 individuals depending on the age). Larvae were immediately put in 20 ml plastic bottles and preserved in nitrogen filled tank (-80 C). Samples were collected every 2 days from 1 DAH up to 8 DAH, then every 3 days up to 35 DAH,⁰ and there after every 5 days up to 45 DAH. Regular sampling to measure the weights and lengths for determination of the specific growth rates was synchronized with other replicate tanks in experiment 1a. The samples were transferred to the bioscience lab at the National Crop Research Institute at Namulonge (NaCRI) for analysis of enzymatic profiles.

8.2.1.2.1 Preparation of the enzyme extract

In the laboratory, fish samples earlier stored at -80⁰C were placed in 50ml falcon tubes containing 15 ml of phosphate buffered saline (PBS) to stabilize the reaction mixture and prevent possible 176

hydrolytic activities (Dawson, Elliott & Jones 1986). The samples were homogenized using an ultrasonic homogenizer (model 150 V/T-Biologics Inc) at -20⁰C. The homogenate was then centrifuged at 10,000 rpm for 15 minutes. The supernatant was decanted and stored at -20⁰C for later enzyme activities analyses.

8.2.1.2.2 Amylase activity Enzyme activity was determined following a method modified from Kimura and Robyt (1995). Starch solution was prepared by weighing 0.2g of commercial potato starch (98% purity). Ten milliliters of 0.2M NaOH was added to the starch solution, which was vortexed and incubated in a warm water bath at 80⁰C for 30 minutes and then allowed to cool to room temperature. Acetic acid (0.4 ml of 0.1M solution) was added to a test tube containing 0.2mls of starch solution and vortexed. Distilled water (10 ml) was added followed by 0.2mls of iodine solution. The starch-iodine solution (0.2 ml) was transferred to a 5ml cuvette and placed into the spectrophotometer (Biochrom WPA Biowave II/UV/visible). Larval enzyme extract (0.1 ml) was added and the. change in absorbance was recorded every 30 seconds for 12 minutes at a wavelength of 620nm, when the blue black coloration disappeared. One unit of enzyme was expressed as milligrams (mg) of glucose released per 30 seconds. Specific activity of amylase was presented as a unit of glucose per mg of protein.

8.2.1.2.3 Protease activity The protease activity was determined by a method modified from a method by Sigma Aldrich (1999). About 2ml of fish extract was incubated with 2ml of casein protein (commercial) solution at 250C. The amount of tyrosine released was estimated spectrophotometrically at a wavelength of 595nm wave length. The absorbance of the solution was taken every 30 seconds, until the absorbance values leveled. A unit of enzyme was defined as mg of tyrosine produced per 30 seconds. Protease specific activity was expressed as enzyme activity per total mg of protein

8.2.1.2.4 Lipase activity Lipase activity was determined following a method modified from Sugihara et al., (1991). A mixture of 1 ml olive oil, 4.5 ml of 50 mM acetic acid, 3.5 ml of 0.1 M calcium chloride (CaCl2) and 1 ml extract was incubated for 30 min at 30ºC with continuous stirring at 500 rpm. The enzyme reaction 177

was stopped by adding 20 ml of ethanol. Lipase (EC 3.1.1.3) activity was assayed by titrating fatty acids extracted from olive oil with KOH. The enzyme activities were calculated in accordance with the amount of KOH read from the burette. One unit of lipase activity was determined as the amount of enzyme that releases 1 ml of free fatty acids.

8.2.1.2.5 Total protein Protein was essayed based on Bradford, (1976) method. A standard curve was made using bovine serum albumin (BSA) of 0.1-0.6mg/ml. 240µl of Bradford reagent was mixed to 10 µl of homogenate; 200 µl from each aliquot was placed in spectrophotometer and absorbance values were read off at 595nm.

8.2.1.3 Experimental study 2: Consequential effects of larvae diets in growth of Juveniles

To determine whether there was a compensatory growth on any of the feeds used as a result of introducing a better feed combination, the four treatments MO, HA, DA and DF (in experiment 1a) received either Moina or microdiet (co-feeding). MD treatment was maintained on the same diet as a control or a reference because it was the best diet in the experimental study 1a. The experiment was conducted from 48 DAH to the 92 DAH. At 48 DAH, the diet MD was gradually used to replace the original diets in experiment 1a (Table 8.2) for 5 days until when the original feeds were completely replaced. Sampling was done after every 10 days of culture to record the weights and the lengths. During the whole experimental period for experiments 1a, b & 2 (from 6 DAH to 92 DAH) about 5 fish were observed of their squamation process every sampling to identify the period of larvae transition into juvenile stage. Observations were made using light microscope.

8.2.1.4 Experimental study 3. Green water and larvae culture

In another set of experiment to determine whether green water had any direct effect on growth of B. altianalis larvae, 75 larvae of average weight 3.0 ± 1.0mg (6 DAH) each were randomly distributed into 6 glass tanks of 55 litres. Three tanks were given green cells (Green water cultures- mixed microalgae-dominated by chlorella spp) estimated at 6.3x106 cells per litre. The rest of the tanks were used as control. Larvae in all the tanks were fed with Moina + microdiet (DM) following the same

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procedure as used in experiment 1a. Temperature was kept at 27 ± 1 C by thermostatic heaters. Green water was added daily in tanks after cleaning them of feaces or uneaten⁰ feed at 8.00h and 16.00h and before feeding the larvae. The tanks were kept in a greenhouse and received 12 h of day light and 12 h of darkness. The weights and the lengths of the larvae were recorded at every sampling done after 14 days for a period of 42 days (6 DAH to 48 DAH).

8.2.1.5 -8.2.1.7 Nursing of larvea and juveniles in outdoor facilities

The three experiments (4, 5 & 6) indicated were designed and conducted in triplicates (only one treatment) to evaluate the growth rates (specific growth rates SGR) for B. altianalis larvae bred from the wild and those of F1-generation bred from the ponds.

8.2.1.5 Experimental study 4: Growth performance of larvae co-fed microdiet from outdoor concrete tanks

After a series of indoor growths experiments to determine the best diet, an outdoor experiment to evaluate growth rates (Average body weight WB, SGR, W and CF) for mass fingerling production of B. altianalis larvae nursed in a semi natural controlled environment was conducted at ARDC- Kajjansi (N 13'19.182". E 32 32'4.9092"). The larvae used in this experiment were bred from the 2 broodstocks⁰ obtained from the⁰ wild. Three concrete tanks (100m ) were thoroughly cleaned and disinfected with sodium hypochlorite (NaOCl). They were fertilized with application of NPK (nitrogen, phosphorous & potassium) at a rate of 80g/1000litres to encourage growth of both phytoplankton and zooplankton. When the tanks were green, 3000 larvae (14.0g ± 1.82mg; 15 DAH) raised indoor on MD diet were randomly and equally distributed into the three replicate tanks. Water inlet and the outlet were tightly screened with a 250µm mesh to avoid entrance of unwanted natural predators and loss of zooplanktons. The tanks were covered with mosquito nets to prevent birds and other insects from falling into the tanks. Feed supplementation was done 3 times a day by feeding the larvae with DF diet (57% CP) at 8.00h, 12.00h and 17.00h. Sampling to record the wet weight was conducted only twice on the 45 DAH (after one month) and on 75 DAH (second month) when the larvae were observed to have metarmophosised into the Juveniles. Two hundred larvae were weighed at each sampling. Larvae were counted to ascertain the mortalities. The water quality (ammonia and dissolved oxygen) was maintained by flashing out the water at slow flow rate of 10 seconds per litre 179

for one hour at 1.00hr, 5.00h, 12.00h and 7.00hr. Average ammonia was 0.13 ± 0.06 ppm and dissolved oxygen was 4.6 ± 1.2mg/l. Ammonia was monitored using LaMotte Fresh water aquaculture kit (Code 6665-02-CC-) while the dissolved oxygen was monitored by OxyGuad Handy Polaris 2 Portabe DO Metre.

8.2.1.6 Experimental study 5; growth of larvae raised from the first generation (F1) broodstock.

This experiment was conducted at the farmer’s fish farm in Masaka-Lwengo district (N0022194, E32.679772) to determine growth rates of F2 generation in captivity. Two F1 generation females were induced with a catfish pituitary extract to produce eggs, which were fertilized with F1 generation males, incubated and hatched (following the procedure described in Chapter 4). At 6 DAH, they were retained where they were hatched in 1.06 m3 concrete tank and fed with green water containing a mixture of young Moina and rotifers (prepared based on farmer’s protocol for catfish production). The green water was added to the tank twice a day at 8.00 h and at 16.00 h. The green water was supplemented with a micro particulate diet (DF diet) that was fed to the larvae 3 times a day at 8.00hrs, 12.00 h and 17.00 h. Feeding was done to satiation (adbilitum). Temperature was maintained at 27.0 ± 1⁰C by warming water in a tank using a solar energy and mixing it with cold water in another tank before being allowed into the nursing tank. The water was brought into the tank at a rate of 1 litre per minute. However, during the day there was no warming as the Farmer’s indoor hatchery had been constructed to absorb and maintain the temperature. Warming was done at night when the temperature dropped to 25⁰C. Ammonia was kept below 0.1ppm by ensuring continuous flow-through of fresh warm water brought into the nursing tank at night and siphoning out the remaining feed at the bottom of the tank before feeding at 8.00 h and 17.00 h. Dissolved oxygen DO was recorded at 6.3 ± 1.2 and the pH was 7.5 ± 0.8. The water inlet and the outlet pipes of the nursing tanks were screened by a 50μm mesh to prevent entry of unwanted microorganisms such as copepods or other aquatic organisms that could eat the larvae. The outlet prevented escape of Moina and rotifers as well as the larvae. At 25 DAH when the larvae were 10 ± 2g (1.3cm TL), about 300 larvae were randomly stocked in each of the three happas of 2*2*1m (4m3). The happas were placed in a 200m2 green water pond that was fertilized one week before stocking with 2.5 kilograms per 100m2 of soya powder to encourage rapid growth of micro organisms (soya is used at the farm initially to

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fertilize the nursing ponds for catfish larvae). The happas were fed with DF 57% CP microdiet thrice a day at 8.00 h, 12h and 17.00 h. Their weights and lengths were recorded at each sampling that was conducted every after 14 days up to 48 DAH. The number of dead larvae were removed and counted throughout the experimental period.

8.2.1.7 Experimental study 6; Juvenile growth in tanks

The experiment was done to determine the specific growth rates of juveniles grown in captivity.

Two hundred B. altianalis juveniles (170 ± 40 mg; 2.84cm at 60 DAH) were stocked in each of the three 100m3 outdoor concrete tanks at ARDC Kajjansi. The tanks were fertilized with chicken manure to support zooplankton growth for the fish. They were also fed microdiet (DF, 57% CP) for one month and later given 1.5mm pellets of the same composition up to the 180 DAH when the experiment was terminated. Feeding was done three times a day at 8.00 h, 1200 h and 17.00 h. The tanks received the same water source from the reservoir tank. To ensure appropriate conditions for growth of larvae, pH, DO and temperature were monitored daily. A continuous flow of water at a rate of 9 seconds L-1 was allowed through the tanks for four hours from 7.00 hrs to 11.00hrs to avoid accumulation of ammonium in tanks and prevent low dissolved oxygen levels. The juveniles were raised up to an average of 6640.03±1972.02mg (8.74±1.09.0cm TL), big enough for further stocking into larger ponds or cages. Sampling was done after every 14 days to record the weights and lengths. The number of surviving larvae was counted after every sampling.

8.2.1.8 Experimental study 7. Larvae rearing and parasitic infestation as an extraneous variable during experimentation

The initial experiment was prepared for determining growth rates of B. altianalis larvae fed on various weaning diets but was terminated due to infestation of parasites that eventually killed most of the larvae. The experimental arrangement had been set up as that indicated in experimental study 1a at the Aquaculture Research and Development Center (ARDC-Kajjansi). In this experiment stream water was pumped to the laboratory where all the indoor larvae experiments were conducted. Throughout the experimental period from 27 DAH when the parasites were first noticed to the termination of the experiment at 94 DAH, a number of larval mortalities were observed to be 181

associated with several parasites. The parasites that were associated with poor larval growth or mortalities were identified and short treatment experiments were conducted. The stream water was abandoned for the rest of the indoor experiments and only the ground water was instead used.

8.2.1.8.1 Salt treatment tests for Trichodina, Apiosoma, and Epistylis Four salt concentrations of 0.9% (normal saline), 1%, 1.2% and 1.5% were prepared from commercial salt (NaCl). The concentrations were made by weighing 0.9g, 1g, 1.2g and 1.5 g in 100 ml of water respectively. About 20 litres of each concentration (experimental treatment) was made and placed in 40 litre oval plastic basins. Each concentration was made in 3 replicates. The presence of parasites was detected at about 27 DAH when the larvae weighed 40.5 ± 10.8mg. Then about 15 larvae with parasites, identified with the aid of microscope were randomly placed in the salt bath for up to 120 minutes (2 h). Five larvae were sampled every 5 minutes to check if the parasites were still attached to the larval skin or fin. The behavioral activity of the larvae was followed to determine and record the period when the larvae would begin to show signs of tress due to salt treatment. This is the time the larvae would be removed.

8.2.1.8.2 Ichthyophthirius multifiliis (white spot disease) This parasite was first identified when an entire tank of larvae at 27 DAH (40.5 ± 10.8mg; 20 mm) was killed. Varying concentrations of salt (1%, 1.2% and 1.5%), formalin (1ml/3L, 1ml/2L and 1ml/L) and potassium permanganate (0.01g/L, 0.02g/L, 0.04g/L and 0.05g/L) were prepared. About 15 larvae with Ich parasites were randomly put in each 20 litre prepared solutions in plastic basins. Larvae were sampled to observe the state of parasites at an interval of 10 minutes until 60 minutes (1h) when the experiment was terminated. The reduced inactive behavior of the larvae was monitored to establish the tolerance of larvae to the concentrations. Subsequently immediately after the tests, the larvae were later transferred to outdoor 100m2 concrete tanks based on the period when the parasite would attack each tank. At 27 DAH, about 110 larvae (40.5 ± 8.0mg) were transferred into the outdoor concrete tank (Tank 1). At 47 DAH, 96 Ich infected larvae (123.2 ± 10 mg) were transferred to another outdoor concrete tank (Tank 2). The last batch of 120 infected larvae was transferred to 100m2 concrete tank (Tank 3) when the larvae had reached 168.2 ± 38.6 mg at 64 DAH. The tanks were provided with constant flow-through of stream water and the larvae were fed with a microdiet

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(DF 57% Crude Protein). The tanks were sampled after one month from the period when the last tank was stocked, to establish whether there were some larvae surviving and if they were some surviving they would be examined to establish if they still had Ich the parasites. Scale formation (scalation) on the larvae was also observed under the light microscope (model Leica DM 500, Made by Microsystems Switzerland Ltd) to establish if their development had a positive impact on reducing parasite infestations.

8.2.2 Preparation of live feeds and decapsulation of Artemia cysts

8.2.2.1 Green water

Two fibre glass tanks of 1000 litres were filled with water and 0.08gL-1 of NPK fertilizer added to the tanks. Microalgae (green water) were concentrated from the ponds to make approximately 9x106 cells per litre using 50µm planktonic net that prevented unwanted zooplanktons to pass through. About 10 litres were used to inoculate each of the tanks and they became green within 5 days.

8.2.2.2 Moina micrura enriched with green water

Three large fibre tanks of 1000 liters were filled with water and inoculated with green water following the procedure indicated above (8.2.2.1). When the tanks became green they were inoculated with Moina micrura that were concentrated from the ponds with 150 µm net. However as the Moina multiplied it consumed the green algae and thus necessitated continuous addition of green water daily to maintain and support the growth of Moina. The density of the Moina increased from the 5th day until the 14th day when they reached their peak and when their density was 100 Moina per ml and were harvested to feed the larvae. The culture was maintained by addition of green water daily for more two weeks until the cycle was terminated to avoid loss of nutrients along the culture generations. The tanks were synchronized to ensure continued provision of Moina for feeding the larvae under experiment.

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8.2.2.3 Hatching Artemia nauplii

Artemia nauplii were hatched following a method modified from Sorgeloos (1980). The Artemia fransciscana strain were hatched into nauplii and used to feed the fish larvae. A concentration of 35 % salt solution (commercial salt) was prepared and placed in inverted two litre plastic empty bottles. A pipette was connected to an aeration tube and placed into the bottle to allow sufficient aeration from the bottom of the bottle. About 50 grams of the Artemia cysts were added to each of the bottle and a bright light was kept on top of the bottles to attract the hatching nauplii. The solution was kept at a temperature of 26 ± 1⁰C using thermostatic heaters (Sera Aquarium heater thermostat; sera D 52518, Heinsberg). About 95% hatchability was achieved after 24 h of incubation. The nauplii were harvested after 10 h and used to feed the fish larvae. Only 12 hrs old Artemia nauplii were used to feed the fish larvae in order to avoid loss of nutrients. The production was synchronized to ensure availability of the live Artemia to fish larvae in the experiment.

8.2.2.4 Decapsulating Artemia

Preparation of decapsulated cysts followed a method modified from Spotte (1992). A table spoon full (50g) of shrimp cysts were put in a conical shaped 3 litre plastic bottle. A litre of water was added and aeration provided to keep the cysts in suspension for 2 hours at 25⁰C. One litre of sodium hypochlorite (NaOCl; a commercial Jik) added to the mixture to facilitate decapsulation. Decapsulation of cysts occurred after 5-15 minutes and the brown colour of the cysts turned orange. A litre of acetic acid (commercial vinegar) was added to neutralize the NaOCl in the mixture. The decapsulated Artemia cysts were rinsed with tap water until the smell of sodium hypochlorite had completely disappeared. The cysts were kept in a plastic bottle under -20oC.

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8.2.3 Growth parameters

The weight gain percentage, specific growth rates (SGR), survival and condition factors were calculated using the following equations:-

Final Weight FW-Initial Weight IW) (i) Weight gain W % = ------x100. Initial Weight IW

In FW- InIW (ii) Specific growth rates SGR % per day = ------x100. (Daily increment) Number of culture days n

Final Number FN (iii) Survival (%) = ------x100. Total number TN of stocked fish

Weight W (vi) Fulton’s condition factor K (Froese, 2006) = ------x 100. Length L3

(v) Weight-Length relationship =Weight W =aLb where a and b are coefficients, L is the total length (cm), and W is the wet weight in g.

8.2.4 Data analysis

An interactive effect between sampling period and the treatment means (weight) due to larval diets was analysed using repeated measures, Analysis of Variances (ANOVA). Statistical significance between treatment means at each sampling level (period) was determined using Duncan’s test in oneway ANOVA. Relationships between weights and total lengths of larvae and juveniles were calculated by linear regression analysis. Chi-square statistical test (X2) was used to compare the larval survival and mortality frequencies using cross-tabulation (contingency table) analysis technique. Student’s t-test was used to determine differences in effective treatment time between salt concentrations for parasitic treatment. T-test was also used to analyze the weight differences between green water fed larva and non green water fed larvae. Statistical analyses were all performed using IBM SPSS Statistics for Windows (Version 22.0. Armonk, NY: IBM Corp, 2013) for windows at 95% confidence level.

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Table 8. 1: Fatty acid and amino acid profiles of microdiet (dry feed) DF

FAME: procentual composition & quantitation (mg/g dry weight) Protein profiles mg/g mg/g Fatty acids Area% Fatty acid Area% DW DW Amino Acids %/100g 14:0 6.4 9.4 I.S. Alanine 3.32 14:1(n-5) 0.2 0.3 21:0 Arginine 3.12 15:0 0.6 0.9 20:3(n-6) 0.1 0.1 Aspartic acid 4.91 15:1(n-5) 0.2 0.2 20:4(n-6) 1.1 1.6 Cystine 0.65 16:0 18.9 27.6 20:3(n-3) 0.1 0.1 Glutamic acid 9.85 16:1(n-7) 7.0 10.2 20:4(n-3) 0.7 1.0 Glycine 3.47 17:0 0.7 1.1 22:0 0.1 0.1 Histidine 1.18 17:1(n-7) 0.1 0.2 20:5(n-3) 15.5 22.6 Isoleucine 2.28 18:0 4.2 6.2 22:1(n-9) 0.5 0.8 Leucine 4.21 18:1(n-9) 9.5 13.9 22:1(n-7) 0.2 0.3 Lysine 3.93 18:1(n-7) 3.1 4.5 23:0 Methionine 1.51

18:2(n-6)-t 0.1 0.1 21:5(n-3) 0.6 0.9 Phenylalanine 2.3 18:2(n-6)-c 3.6 5.3 23:1(n-9) Proline 3.26 19:0 0.2 0.2 22:4(n-6) Serine 2.57 18:3(n-6) 22:3(n-3) Threonine 2.36 19:1(n-9) 0.2 0.3 22:5(n-6) 0.3 0.4 Tyrosine 1.59 18:3(n-3) 0.8 1.2 22:4(n-3) Valine 2.38 18:4(n-3) 2.2 3.3 24:0 Tryptophan 0.58 20:0 22:5(n-3) 2.1 3.1 20:1(n-9) 0.8 1.2 24:1(n-9) 20:1(n-7) 0.3 0.4 22:6(n-3) 11.5 16.8 Proximate analysis Sum (n-3) >or= Crude 30.4 44.5 20:3(n-3) Protein 57% Sum (n-6) >or= 5.1 7.4 18:2(n-6)-t Crude Fiber 11% Crude fat 15%

g wet 0.0579 0.0531 Ash 11% % DW 91.645 9.4196 Phosphorous 18% Total mg FAME/g DW 146.2 CuSO4 8mg/kg Total lipid % VitaminA on DW ui/kg 70000

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Table 8. 2: Feeding schedule (estimates) for different diets at satiation during experimental period.

Experiment 1a; (live prey estimates per individual) Days Week Moina Artemia Microdiet) Moina + dry feed Decaps. of (MO) nauplii DF (g) (MD) Artemia culture (HA) Moina Dry feed (g) per tank 5-7 1 30-60 115-400 0.4 20-30 0.2 20-30 Rotifers Rotifers 8-14 1 136-300 115-400 0.8 70-100 0.4 20-30 15-21 2 200-400 200-600 0.9 100-200 0.5 30-60 22-29 3 300-500 400-600 1 150-250 0.6 40-80 30-37 4 500-600 500-800 1 250-400 0.6 70-90 30-45 5 700-1000 600-1200 1 400-600 0.6 90-100 46-48 6 1000-1500 >1200 1 600-700 0.7 >100 Experiment 2; (Moina +Microdiet) proportions provided during transition period Treatments Days of MO HA DF MD DA culture- +Microdiet +Moina +Moina +Moina & +Moina transition &Microdie microdiet & microdiet (50%+50%) (50%+50%) (50%+50%) 48 +20% +20% +20% 100% +20% 49 +40% +40% +40% 100% +40% 50 +60% +60% +60% 100% +60% 51 +80% +80% +80% 100% +80% 52 +100% +100% +100% 100% +100% Note that: the number of Moina provided was constant across all the treatments during transition Up 92 DAH >2000 Moina per individual + Microdiet (ranging from 1.2g to -1.5g for each tank)

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8.3. Results

8.3.1 Effect of live and microdiets on growth performance of larvae and juveniles (experimental study 1a &2)

The interaction between treatments (diets) and sampling period was significant throughout the culture period (p > 0.001). Meaning that at each sampling, larval weight was significantly higher than the previous sampling for each particular diet. That is, there was significant weight increase due to the diet effect after every sampling for each diet. At first sampling, 13 DAH (7 DAF), larvae fed diet MD attained significantly higher average body weight BW than the larvae on all other diets (F4, 10 = 146.19, p < 0.001) followed by larvae fed diets DA and DF diets. There was no statistical significance in average body weight BW between larvae fed diets DA and DF (p > 0.05). The larvae fed diets MO and HA attained the least average BW and there was no statistical difference between them (p > 0.05). In all the subsequent samplings that were conducted at 20, 27, 34, 41 and 48 DAH, average larval weights BW for all diets were significantly different from each other (Table 8.3, and Table 8.4). Larvae fed MD diet maintained a significantly better growth performance than all other diets followed by DA, DF, MO and HA in that order. Only a little change in BW was noted with larvae fed diets MO and HA diets in the first week while a slight drop was observed in the larvae fed diet HA in the last week (Figure 8.1). In spite of the significant differences in larval BW between diets MD and DA, their means were closer and bigger than the rest of the treatments. Significant variations in the final specific growth rates SGR (F4, 10 = 45.66, p < 0.001), weight gain W (F4, 10 =

58.85, p < 0.001) and condition factor K (F4, 10 = 60.25, p <0.001) were observed for all diets with a better growth performance recorded for diet MD followed by DA, DF, MO and HA (Table 8.5).

Table 8. 3: Mean values (±Standard Error SE) of larval body weight fed different diets at p<0.05.

DAH DAF MO (mg) MD (mg) HA (mg) DA (mg) DF (mg) 13 7 3.30±0.10 a 14.93±0.36 c 3.21± 0.08a 12.87±0.21 b 13.31±0.25 b 20 14 8.63±0.30a 31.05±0.58e 14.80±0.35b 24.16±0.32d 20.99+0.42c 28 21 19.17±0.53a 63.64±1.08e 24.81±0.53b 40.30±1.13d 32.52±1.00c 34 28 37.24±0.75a 87.59±1.45e 42.07±0.66b 70.04±1.72d 55.61±1.65c 41 35 44.66±0.93a 110.65±1.70e 55.07±0.86b 102.98±1.97d 65.40±1.73c 48 42 73.67±1.48b 152.05±2.51e 60.35±0.83a 141.14±2.43d 87.30±2.63c Mean values with different superscripts show significant differences.

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Table 8. 4: F-tests for all the diets at each sampling at p < 0.05

DAH DAF F Level of Degree of freedom No. of fish significance (treatments, subjects) N 13 7 146.19 <0.001 4, 10 678 20 14 65.97 <0.001 4, 10 678 28 21 62.72 <0.001 4, 10 678 34 28 18.52 <0.001 4, 10 678 42 35 75.80 <0.001 4, 10 678 48 42 58.45 <0.005 4, 10 678

Figure 8.1: Growth performance of B. altianalis larvae fed with different diets during the experimental period.

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Table 8. 5: Growth parameters of B. altianalis fed on different diets (mean ± SE)

Growth MO MD HA DA DF Parameters Experiment 1a; 48 DAH (42 DAF) Initial Weight (mg) 3.00±1.00 3.00±1.00 3.00±1.00 3.00±1.00 3.00±1.00 Final weight (mg) 73.67±1.48b 152.05±2.51e 60.35±0.83a 141.14±2.43a 87.30±2.63c Weight gain W % 2355.80±49.33b 4968.40±83.74e 1912.26±27.50c 4604.69±81.00d 2809.88±87.59c SGR% 3.28±0.02b 4.06±0.01e 3.10±0.01a 3.96±0.02d 3.41±0.0.04c Final Condition factor K 0.70±0.01b 0.88±0.01e 0.64±0.01a 0.85±0.01d 0.72±0.01c Survival% 93.3b 93.0b 92.9b 92.9b 82.9a Experiment 2; 92 DAH (86 DAF)

Initial Weight (mg) 73.67±1.48b 152.05±2.51e 60.35±0.83a 141.14±2.43a 87.30±2.63c Final weight (mg) 355.47±6.08a 500.20±11.80b 361.01±5.85a 510.13±11.93b 354.69±12.60a Weight gain W % 382.514±8.26d 228.85±7.76a 498.19±9.69e 261.43±8.45b 306.29±14.43c SGR% 1.53±0.02d 1.14±0.02a 1.75±0.02c 1.23±0.02b 1.30±0.04c Final Condition Factor K 0.89±0.02a 0.87±0.01a 0.85±0.02a 0.85±0.01a 0.89±0.01a Weight length y = 0.002x4.022; y = 0.004x3.503; y = 0.002x3.974; y = 0.004x3.465; y = 0.003x3.660; relation r2=0.969 r2=0.959 r2=0.963 r2=0.979 r2=0.968 (power curves) Different superscripts across the rows indicate significant differences.

At the time of termination of experimental study 1a, larval percentage survival was generally high in all the treatments. There were no significance differences in larval survival among MO, MD, HA, and DA diets (p > 0.05) but DF was significantly different from all other diets (X2= 21.173, df = 4, p < 0.0001). However, mortalities were comparatively higher in larvae that were fed MO MD, DF and DA diets by the 24 DAH (18 DAF) than mortalities for larvae fed on the same diets by 45 DAH (35 DAF) (Figure 8.2). Larvae mortalities remained relatively constant for those fed on DF diet throughout the experimental period (Figure 8.2). The proportions of larval mortalities to survival for the whole experimental period are shown in a contingency table (Table 8.6). 190

Figure 8.2: Mean percentage survival of B. altianalis larvae at 48 DAH of feeding (42 DAF); and mean percentage mortalities by 24 DAH (18 DAF) and 48 DAH (39 DAF). Bars are shown as Mean±Standard deviation

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Table 8. 6: Contingency table showing proportions of mortalities and survival of larvae in each treatment at 48 DAH.

Diet Mortality Survival Total MO Number 13 152 165 % 7.9 92.1 100.0 MD Number 15 150 165 % 9.1 90.9 100.0 HA Number 15 150 165 % 9.1 90.9 100.0 DA Number 15 150 165 % 9.1 90.9 100.0 DF Number 37 128 165 % 22.4 77.6 100.0 Total Number 95 730 825 % 11.5 88.5 100.0

In the experimental study 2 where all the treatments in experiment 1a received a combination of Moina + microdiet (MD), the interaction between treatment (diets) and sampling period was significant throughout the culture period (p > 0.001). No significant differences in final average BW were noted between larvae in treatments MD and DA (p > 0.05) throughout the experimental period. Similarly no significant differences in average BW was noted among the larvae in treatments MO, DF and HA (p > 0.05). However, larvae in MD and DA treatments maintained a significantly higher final average body weight than larvae in MO, DF, and HA (Table 8.7; Figure 8.3). Significant differences among all treatments were observed in larval weight gain W (F4, 10 = 28.98, p < 0.001) and specific growth rates SGR (F4, 10 = 32.36, p < 0.001). No significant differences in condition factor were observed among treatments (p > 0.05). In spite of the higher average body weight BW of larvae in DM and DA treatments, the weight gain W and SGR were much higher for larvae in treatments MO, HA and DF, signifying a previously and comparatively much deeper growth depression and therefore a higher compensatory performance than larvae in MD and DA treatments (Table 8.8). There was no death recorded during the transition period of introducing new diet MD (co-feeding) between 48 DAH and 52 DAH and until when the experiment was terminated at 92 DAH. The power curve equations relating the standard weight and the total length showed a very

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strong relationship r2 of greater than 95% for each diet and all showed positive allometric growth in all the treatments (Table 8.5). The power curve equations for diet DM and DA; and those for MO and HA were generally similar showing a similar growth trajectory for the diets.

Table 8. 7: Statistical significance between treatment mean weights at P = 0.05 from 62 DAH to 92 DAH. (Mean ± Standard Error SE)

DAH DAF MO (mg) MD (mg) HA(mg) DA (mg) DF (mg) 62 56 170.00±3.39a 263.36±4.30b 167.82±2.36a 279.04±5.13b 179.71±5.63a 72 66 251.18±3.77a 355.33±6.44b 237.81±3.46a 373.11±6.97b 236.9±6.41a 82 76 325.17±3.28a 427.30±8.22b 315.90±4.21a 443.22±9.69b 305.96±12.60a 92 86 355.47±6.08a 500.20±11.80b 361.01±5.85a 510.13±11.93b 354.69±12.60a

Treatments with the same superscripts along the rows are statistically not significant and treatments with different scripts are significantly difference.

Figure 8.3: Variation of average body weight (mg) of B. altianalis juveniles with days after Hatch (DAH) 193

8.3.2 Ontogeny of enzyme activity and larval development for the best diet MD (6 DAH to 45 DAH; experimental study 1b)

Growth performance curves amoung all the 13 replicates for MD means were similar (Figure 8.1). Very little or no amylase activity was detected before and until the mouth opening. The amylase activity surfaced on the 6 DAH (8 ± 2 U/mg) when feeding started and increased sharply from that day reaching the peak at 14 DAH (23 ± 4 U/mg) and then reduced to 17 DAH (11 ± 2 U/mg) and gradually continued to reduce up to 45 DAH (Figure 8.4). The alkaline proteases was detected at the beginning of exogenous feeding, increased sharply to 17 DAH (100 ± 9 U/mg) and dropped low to 20 DAH (40 ± 4 U/mg) and gradually until the end of the experiment period (Figure 8.5). Lipase activity increased to the highest peak at 17 DAH (11 ± 0.5 U/ml) then slightly reduced to 20 DAH (8 ± 0.9 U/ml) and rose again to 28 DAH (9.5 ± 0.3 U/ml) before it gradually declined at 40 DAH (3 ± 0.30 U/ml) (Figure 8.6).

Figure 8.4: Variation of amylase activity with Days after hatch (DAH) in B. altianalis larvae (n=3)

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Figure 8.5: Variation of protease activity with Days after hatch (DAH) in B. altianalis larvae (n=3).

Figure 8.6: Variation of lipase activity with Days after hatch (DAH) in B. altianalis larvae (n=3)

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8.3.3 Green water experiment

A significant difference in larval growth was observed between the treatment that was given algae

(green water) and that of the clear water at 20 DAH (t374 = -5.67, p < 0.0001). Conversely, no significant differences that were observed in larval weight between the treatments in all the subsequent samplings at 34 and 48 DAH (p > 0.05) (Table 8.8; Figure 8.7). No significant differences in SGR and W were observed between the two treatments (p > 0.005) (Table 8.8). Percentage survival and mortalities were not significantly different between green water and clear water treatments (p > 0.05) (Table 8. 9).

Table 8. 8: Mean values (±Standard Error SE) of growth parameters between green water and clear water treatments.

DAF DAH Green water (mg) Clear water (mg) 14 20 35.33±0.57a 39.95±0.58b 28 34 89.84±2.00a 92.22±1.00a 42 48 147.19±2.98a 148.59±2.90a Final weight (mg) 147.19±2.98a 148.59±2.98a Specific growth rates SGR% 3.989793±0.02a 3.994740±0.02a

Weight gain W % 4806.4030±99.32a 4852.9411±96.36a

Figure 8.7: Variation of mean larval weight (mg) for green and clear water treatments with Days after Hatch (DAH).

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Table 8. 9: Contingency table showing proportions of mortalities and survival of larvae in each treatment at 48 DAH.

Treatment Mortality survival Total Green water Number 66 234 300

% 22.0 78.0 100.0 Clear water Number 58 242 300 % 19.3 80.7 100.0 Total Number 124 476 600 % 20.7 79.3 100.0

8.3.4 Nursing larvae in outdoor green water concrete tanks

Final average weight BW was 1112 ± 42.70mg (± standard Error SE) at 75 DAH after two months (Figure 8.8). Specific growth rates were 4.159 ± 0.04% (± SE) between 15 DAH and 45 DAH and 2.12 ± 0.03% between 45 DAH and 75 DAH. The SGR for the whole period was 3.149 ± 0.02%. The weight gain W between 15 and 45 DAH was 1691.19 ± 27.37% and W was 343 ± 17.02% between 45 DAH and 75 DAH. But for the whole period from 15 DAH to 75 DAH, W was 7843.32 ± 169.44%. Survival was 89.23% at 45 DAH and 95.3% between 45 DAH and 75 DAH. Water temperature was 24 ± 1.2⁰C, dissolved oxygen was recorded at 6.3 ± 1.5mgL-1 (± standard deviation) and the Ammonia was ≤ 0.01ppm.

Figure 8.8: Variation of average body weight of B. altianalis larvae with DAH

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8.3.5 Determining Growth rates of F2 generation larvae raised from the first generation (F1) broodstock at a local hatchery

Average body weight increased gradually with Days after Hatch (Figure 8.9). Survival was over 80% in each happa (not that there are no treatments) (Table 8.10). The specific growth rate SGR was 3.039 ± 0.23 (Mean ± SE) and the weight gain W was 6893.84 ± 207.81 (Mean ± SE) between 21 DAH and 85 DAH.

Table 8. 10: Contingency table showing proportions of mortalities and survival of larvae of B. altianalis in each replicate at 48 DAH.

Replicates Mortality Survival Total Happa 1 Number 39 261 300 % 13.0 87.0 100.0 Happa 2 Number 42 258 300 % 14.0 86.0 100.0

Happa 3 Number 45 255 300 % 15.0 85.0 100.0 Total Number 126 774 900 % 14.0 86.0 100.0

Figure 8.9: Mean (±SE) larvae weight variation with Days after hatch (DAH) 198

8.3.6 Nursing Juveniles in tanks up to 6640.03 ± 1972.02mg

There was a gradual weight change over time (DAH) as the juveniles grew (Figure 8.10, Table 8.11). However, in the last sampling at 180 DAH mean values of juveniles in Tank 2 and Tank 3 were comparatively better than the mean of juveniles in Tank1 (Table 8.11). There were consistently increased ammonia levels (0.20 ± 0.03 ppm) and low dissolved oxygen levels (2.6 ± 1.2mgL-1) in tank 1 (replicate) at 180 DAH. The ammonia levels in all replicates at all samplings other than that of tank 1 at 180 DAH remained below 0.01ppm and at DO of 3.5 ± 1.6 mgL-1. The weight-length relationship throughout the experimental period was best predicted by a power function, y = 0.007x3.139 where r² = 0.988 indicating a positive allometric growth. The daily specific growth increment (SGR) of 2.403 ± 0.01 was comparatively higher between 60 DAH and 90 DAH, than that of rest of the experimental period from 90 DAH to 180 DAH recorded at 0.98 ± 0.080. There was no significant difference in percentage larval survival among the replicates (p > 0.05) (Table 8.12).

Table 8. 11: Mean values (± SE ) of growth parameters of juveniles raised in concrete nursing tanks at ARDC Kajjansi.

Growth Replicate 1 (tank Replicate 2(tank 2) Replicate (tank 3) parameters 1) Final average 6054.9±223.19 7023.7 ±217.67 6925.5± 248.59 weight (mg) Average 1.27±0.01 1.33±0.01 1.33±0.01 Specific growth rates % at 60 DAH-180 DAH Final Weight 3461.69±131.29 4031.58±128.04 3973.81±146.23 gain %

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Figure 8.10: Variation of average body weight of B. altianalis juveniles with DAH

Table 8. 12: Contingency table showing survival and mortalities of B. altianalis in Tanks

Replicate (tank) Mortality Survival Total 1 Number 58 142 200 % 29 71 100 2 Number 55 145 200 % 27.5 72.5 100 3 number 61 139 200 % 30.5 69.5 33.3 Total Number 174 426 600 % 29.0 71.0 100.0%

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8.3.7 Larval rearing and parasite infestations

The identified ciliated parasites included; the Trichodina, Apiosoma, and Epistylis. These were successful treated with a salt concentration

8.3.7.1 Treatment of protozoans (ciliated) parasites with salt concentration

Significant effect of salt concentrations on the parasites with respect to treatment period (minutes) was observed between concentrations of 1% and 1.2% (t2=39.38, p < 0.001), 1% and 1.5% (t2=22.00, p < 0.002) but was not significant at adjusted p = 0.0167 (0.05/3) of between 1.2% and 1.5% -1 (t2=6.42, p < 0.023). The shortest treatment effect of salt bath was recorded with 1.5gL for a mean period of 6.67 ± 1.53 minutes with larval tolerance period of 51.7 minutes. It was followed by 1.25g L-1 salt bath for a mean period of 16 ± 1minutes with a larval tolerance of 61.7 minutes. The longest effective salt bath was 1% for a mean period of 50.6 ± 2.08 minutes with a larval tolerance of >120 minutes (Figure 8.11). Salt did not have any treatment effect at 9g L-1 (normal saline).

Figure 8.11: Variation of salt concentrations with effective treatment period of B. altianalis larval bath and tolerance 201

8.3.7.2 Treatment of Ich (white spot disease)

Treatment was only successful with Ich parasites that were attached on the fins and on the skin but was not successful with those under the skin and inside the gills (Table 8.13). Under the microscope most of the live ich (Figure 8.12.) were still observed active under the gills and the skin.

Table 8. 13: Treatment regime for Ich with salt, potassium and formalin

Chemical Salt Potassium Formalin (40%) Time 10g/L 12.5g/L 15g/L 0.01g/L 0.02/L 0.04g/L 0.05g/L 1ml/3 1ml/2 1ml/Litre (minutes) Litres Litre 10 - - -+ ------+ 30 -+ -+ -+ ------+ -+ 40-60 -+ -+ -+ - -+ -+ -+ -+ -+ -+ Period of >60 60 50 >60 >60 >60 60 >60 60 40 larval tolerance (minutes) (-) represents a negative effect of concentration of chemical on the parasites (no dead parasites) (-+) represents both negative and positive effects on the parasites; (-) negative indicates no effect on parasites inside the skin or the gills while (+) positive indicates the parasites appended on the fins and on skin were all killed. (-+) also indicates that all other parasites other than the Ich were killed.

At the first sampling day (94 DAH), it was noted that some of the Ich infected larvae that were transferred into the concrete tanks had survived and had no parasites. However, the number of the larvae that survived varied with the stocking size (Table 8.13; Figure 8.13) and the relationship was positively exponential (r2=0.988, y = 4.365e13.83x). The stocking size reflected the level of larval development with reference to the nature of scale formation (Table 8.14). At 94 DAH all the larvae had scales covering the head, trunk, dorsal, ventral accept for the peduncle and the small portion of the lower abdomen (Figure 8.14). The mean weight of surviving larvae significantly varied only

between tanks 1 and the rest of the tanks (F1, 74=7.567, p < 0.001). There were no significant growth differences between tanks 2 and 3 (p = 0.05) and the specific growth rates were notably the same in all the tanks (Table 8.14).

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Figure 8.12: Graphical presentation of the relationship between survival and stocking size with respect to the DAH.

Table 8. 14: Growth parameters and features of infected larvae stocked in outdoor nursing concrete tanks.

Tank stocking Stocki Scale Number Surv Larval Final Period to Specific age ng size formation at stocked ival survival weight at sampling growth (DAH) stocking N0. (%) 94 DAH (days) rates (%) 1 27 0.038 No scales 110 8 7.27 0.851a±0. 67 1.21 21 2 47 0.123 Only 96 24 25.25 0.667b±0. 47 1.16 abdominal 19 scales & along the lateral line 3 64 0.168 Abdominal & 120 52 43.33 0.561b±0. 30 1.31 dorsal scales 20 present

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Figure 8.13: showing Ich parasite under the skin of B. altianalis Larvae as observed under the microscope. Mg x 40

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Figure 8.14: Scale formation. a) Scales forming around the neck and along the lateral region of B. altianalis larvae which is metamorphosising into Juveniles at 57 DAH (257mg, 3.0TL). b) The animal has transited into juvenile and the whole body is almost covered by scales at 75 DAH (427mg, 3.9TL). The juvenile clearly resembles an adult fish. c) Magnified section of the skin of young fish showing formed scales. The scales gave a characteristic glittering colouration of an adult B. altianalis Mg X 100.

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Table 8. 15: Growth performance for various cyprinids species in captivity

Species DAH Final Growth Initial Feeds Reference rates (mg) wt Carassius auratus 11 8.70±1.10 Dry and live Lubzens, feed Tandler & Minkoff, (1989) Cyprinus carpio 28 325 Dry feed Charlon & Bergot (1984) Cyprinus carpio 21 189 (112-336) 1.0 Dry feed with Alami- Premix & Durante, 31 755(265-1385) vitamins Charlon, Escaffre, & Bergot, (1991) Ctenophrangodon 16 101.6±27 1.3±0.4 Live feed JF Prinsloo, idella HJ Schoonbee 20 157.9±46.9 1.3±0.4 Live feed (1986) 20 26.5g±26.5 Dry feed Hypophthalmichthys 15 103.0±19.8 1.0 Live feed molitrix 15 27.5 ±20.0 1.0 Dry feed Barbus altianalis 20 31.05+6.76 3.0±1 Live feed Current study &Dry feed 20 14.80+4.11 3.0±1 Live feed 20 20.99+4.87 3.0±1 Dry feed 34 87.59+16.81 3.0±1 Live feed & dry feed 48 152.05±29.19 3.0±1 Live feed & dry feed 180 6640.3±1972 3.0±1 Live feed & dry feed Barbus altianalis 45 250.7667±38.64131 14.2±1.8 zooplankton + Current study dry feed Outdoor, In 75 1112.065±0.238 14.2±1.8 zooplankton + green water dry fee tanks Osteobrama 45 4.56±0.12 <0.15 Live feed & Ramesh et al., Belangerii dry feed (2014) 45 3.82±0.16 <0.15 Live feed Moina 45 3.03±0.03 Dry feed Barbus barbus L 44 200±30 10±2 Dry feed Policar et al 358 5400±500 10±2 Dry feed .,(2011) 723 29200±5900 10±2 Dry feed 206

8.4 Discussion

Larval growth and survival in captivity are generally influenced by a multiplicity of factors ranging from nutritional quality, maturation and digestive capability of larvae, management and control of key physico-chemical water parameters, hygiene in the hatchery (Braum, 1967; Delince, Campbell, Janssen & Kutty, 1987; Mwanja, Rutaisire, Ondhoro, Ddungu & Aruho, 2015) and often genetic quality of the species (Lorenzen, Beveridge & Mangel, 2012). In this study larval growth and survival were found to be significantly affected by the diets and the age at which the diets were offered. Results from experimental study 1a showed that in B. altianalis the specific growth rates (SGR), weight gain (W), condition factor (CF) and final average body weight (BW) for larvae co-fed Moina + microdiet (MD) performed better than fish on all other diets. This diet was followed by larvae fed on decapsulated Artemia DA, the microdiet alone DF, the Moina MO and the hatched Artemia (HA) respectively. These results agree with those obtained from some other cultured species including, Aristichthys nobilis (Fermine & Recometa 1988), Clarias batrachus (Giri et al., 2002), Migurnus anguillicaudatus (Wang, Hu, Cao, Yang & Wang, 2008), M. anguiillicaudatus (Wang et al., 2009), Oncorhynchus mykiss (Akbary, Imanpoor, Sudagar & Makhdomi, 2010) and Osteobrama belangeri (Ramesh et al., 2014). These authors reported that larvae fed a combination diet resulted in better growth and survival than those fed a microdiet or live food alone. The current results also concurred with the same studies in A. nobilis and O. mykisss in which larvae fed microdiet performed better than those fed live feed alone. However, the microdiet given to the O. belangeri larvae performed poorly with high larvae mortilities than Moina diet (Ramesh et al., 2014). With a better final BW for larvae fed diet DF (83.3 ± 30.53mg) than MO (77.67 ± 17.19mg) and HA (60.35±9.65mg), this study confirmed that B. altianalis larvae could be directly weaned to a microdiet though this was not the best diet since significant mortalities were recorded compared to other diets. Segner, Rosch, Verreth, & Witt, (1993) suggested that the ability by the given species to start with a microdiet was linked to the size of the larvae. The larvae hatched with a big size will more likely accept the microdiet because the digestive structure is probably at an advanced stage to be able to digest the diets. But for small larvae they are vulnerable and will have very high mortalities. B. altianalis has big egg (2.97 ± 0.1 mm in diameter) and is hatched with a big yolk (at 8 ± 1mm Tl) thus such size is possibly linked to the early development of the digestive structure. Histological evidence (unpuplished data; Chapter 7) confirmed that by the 7 DAH B. altianalis larvae

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possessed a relatively advanced digestive structure indicating a possibility of the species being started on a microdiet. However, variations in nutrient composition for different microdiets will also influence how fast the digestive capability of the larvae develops to digest and assimilate the microdiet for better larval growth (Cahu & Zambonino–Infanty, 2001; Cara, Moyano, Cardenas, Fernandez‐Diaz & Yufera, 2003). The analysis on DF diet used in this study showed that this commercial diet was well formulated with minimum nutritive requirements of all the essential amino acids and total HUFAs (20: n-3) of 44.5mg/g dry weight for better larvae growth and survival. Hence larvae performance was consequently limited by other factors such as sufficient endogenous enzymes for digestion. Variations in CP percentage and other dietary constituents of microdiets as those reported in some of the species mentioned above (Akbary et al., 2010; Giri et al., 2002; Ramesh et al., 2014; Wang et al., 2005; Wang, et al., 2009) may influence growth and survival of larvae differently. This therefore suggests that a microdiet should continuously be improved for a higher growth performance of B. altianalis larvae.

The good performance of diet MD in B. altianalis was attributed to a contribution of nutrients from Moina and microdiet. This was because Moina alone and the microdiet alone could not independently perform better than their combination. Live feeds are said to contribute enzymes either for self digestion or facilitate breaking down of the microdiets (Dabrowski, 1984; Kolkovski, Tandler, Kissil & Gertler, 1993). However, some authors suggest that the enzyme contribution from the live feed is very small and that co-factors carried by live prey stimulate larval pancreatic secretions that facilitate maturation of the digestive process to efficiently digest and assimilate the microdiets (Cahu & Zambonino-Infante, 2001; Kurokawa, Shiraishi, Suzuki, 1998; Koven, Kolkovski, Hadas, Gamsiz & Tandler, 2001; Liu et al., 2012). Additionally, visual and chemical stimulation by the live prey facilitate capture and ingestion of microdiet for quick growth (Canavate & Fernandez, 1999). The results of the current study are in agreement with the later two assertions since even the larvae fed exclusively on microdiet performed better than those fed on Moina and or Artemia nauplii. Hence the contribution of enzyme from Moina could not have directly broken down the microdiet for it to be assimilated. Also during the experiment it was clearly observed that in glass tanks (aquaria), larvae fed with a combination diet were very active than those in tanks fed with other diets, supporting the visual and chemo stimulation activity attributed to the live preys during the feeding process. Nevertheless, co-feeding maximized nutritional benefits of both live and microdiet influencing 208

growth and survival of B. altianalis. The findings recommend inclusion of a co-feeding strategy in larval rearing protocol.

The larval ontogenetic enzyme pattern indicating an early increase in activity, followed by a sharp decline for larvae fed combination diet, is a characteristic of larvae maturation and development manifested in many other species at different stages (Zambonino-Infante et al., 2008). All enzymes were detected at the time of exogenous feeding and their initial increase was attributed to the development and size increase of the pancreas as was observed in several cultured cyprinids (Chakrabarti, Rathore & Kumar, 2006; Chakrabarti & Rathore, 2010; Farhoudi, Nazari & Ch, 2013). Early increase in amylase and then its sharp decline is said to be genetically programmed in larvae development (Cahu & Zambonino-Infante, 2001; Peres, Cahu, Zambonino-Infante, Le Gall & Quazuguel, 1996). The decline of other pancreatic enzymes as the larvae grew did not reflect enzyme reduction but suggests a normal increase in protein tissue as the fish gain weight (Cara et al., 2003; Zambonino-Infante et al., 2008). The evolution of enzyme activity pattern in developing larvae relates the digestive maturation with food assimilation mechanisms, and this helps to indentify the period when the larvae are competent enough to effectively digest and assimilate the microdiet (Gisbert, Gimenez, Fernandez, Kotzamanis & Estevez, 2009; Zambonino-Infante et al., 2008). Thus at a time when the pancreatic enzyme activity began to decline, the larvae developed an efficient digestive mechanism that was attributed to gradual increase of enterocyte enzymatic activity as a result of increased gut microvilli structural development (Gisbert et al., 2009; Kolkovski, 2001). This observation was affirmed by increased clarity and size of brash border or microvilli of larval intestinal folds by the 15 DAH (unpuplished data; Chapter 7). The attainment of digestive maturation level in common carp larvae was able to facilitate complete diet replacements in a related study (Farhoudi et al., 2013). The variation patterns exhibited by pancreatic enzymes in B. altianalis larvae indicated that the assimilation mechanisms by the digestive structure are also age dependent in this species. It can therefore be inferred that the earliest B. altianalis larvae fed with the best diet MD can reach maturation stage with minimal mortalities and better BW was at 15 DAH, 20 DAH and 17 DAH for amylase, protease and lipase respectively. By the third week of maturation after hatch, the B. altianalis larvae were easily able to adapt to dietary changes i.e. digest and assimilate complex diets with inevitable manipulations or complete replacement of live diets with microdiets. Diet manipulation and or complete replacement of live preys by microdiets is an inevitable strategy in 209

mass seed culture in order to lower the production costs and increase growth performance (Conceicao, Yufera, Makridis, Morais, Dinis, 2010; Kolkovski, 2001).

The gradual declined of enzyme activity noted in this study in larvae fed MD diet coincided with the reduction of larvae mortality. Larvae mortality was generally higher before 24 DAH and significantly declined further than other diets by the end of the experiment period at 48 DAH confirming the possibility of gradual maturity and stabilization of the digestive capability of the larvae. In spite of the fact that the growth parameters in larvae fed exclusively with microdiets DF were better than in those fed with Moina and Artemia nauplii, this study observed that the survival was significantly lower in those fed on microdiet than other diets. Most studies attribute the poor performance of the microdiets to limitations in production of enzymes to facilitate digestion of the microdiet which in turn is related to rudimentary structure of the digestive system during early stages of larvae development (Day, Howell, & Jones, 1997; Kolkovski, 2001; Ramesh et al., 2014; Segner et al., 1993). However, the survival reported in such studies has been notably too low compared to the 77% obtained for B. altianalis in the current study (experiment 1a). Besides, the SGR and Final BW of larvae fed dry diet was higher than that of the live diets. It can be suggested that whereas there are sufficient nutrients for larval uptake from the DF diet; there could still be an insufficient mechanism for self released enzymes to appropriately breakdown and utilized the processed microdiets. The final BW variation of larvae fed diet DF was high and most of the dead larvae or the weak ones were largely small individuals. It is hypothesized that the feed could have stressed the larvae’s maturing digestive system rendering them unable to adjust and produce sufficient enzyme to digest the microdiet compared to larvae which were bigger. The stress factor is justified by the fact that the larvae fed with MO and HA diets had comparatively fewer dead larvae and yet their growth was lower than that of the microdiet DF. Since the enzyme activity in DF was not investigated, and basing on the enzyme activity pattern observed in larvae fed diet MD, it can be stated that the microdiet may have stressed the digestive system and delayed or retarded some larvae to reach a maturation age. Thus the role of stress effect by diets could further be investigated in this species.

In experimental study 2 where all the larvae in all treatments were introduced to a combination of Moina + microdiet, no larvae mortality was recorded throughout the growth period. The absence of 210

mortalities coincided with a sharp rise in larval weight at the start of the experiment and was attributed to the gut coiling reported in a previous study on the same species at 35 DAH (Unpublished data; Chapter 7). The intestinal coiling provided an impetus for rapid growth after coiling because it meant that there was an increased intestinal surface area over which the diets were digested and assimilated (Gisbert et al. 2009; Ronnestad et al., 2013; Ruan et al., 2012; Trevino et al., 2011). This change also marked the beginning of scale formation characterizing a transition process into juvenile stage. A compensatory growth was also noted in all growth parameters including the final body weight, weight gain, condition factor and specific growth rates in larvae that were formally fed diet MO, HA, DA, and DF (in experiment 1a). Compensatory growth was observed in all treatments but the juveniles in treatment DA (initially fed decapsulated Artemia at larval stage) grew comparatively faster to the same rate with those in control treatment MD than other treatments. In experiment 1a, larvae fed decapsulated Artemia had exhibited a close growth performance to those fed diet MD and therefore reached a digestive maturation level, much earlier than larvae fed diets MO, HA and DF before all the treatments were introduced to co-feeding. The decapsulated Artemia have good and well composed nutrients with sufficient energy for larvae growth (Leger, Bengtson, Simpson & Sorgeloos, 1986) and hence the introduction of co-feeding (Moina + microdiet) provided a quick impulse for further (or better) growth. The current study concurred with larval studies by Kaiser, Endemann and Paulet (2003) and Paulet (2003) in a related cyprinid Carassius auratus where decapsulated Artemia performed better than Artemia nauplii. In some species such as Solea solea, decapsulated Artemia cysts retarded the larval growth but performed well with Artemia nauplii (Leger et al., 1986). Decapsulated Artemia cysts resulted in low growth and survival of early fry of Paracheirodon innesi (Sanaye et al., 2014). In Hippoglossus hippoglossus decapsulated Artemia cysts showed a consequential negative effect on pigmentation in larvae (Naess, Germain-Henry & Naas, 1995). However, in other farmed species including Cyprinus carpio (Vanhaecke, Vrieze, Tackaert & Sorgeloos, 1990), Leuciscus cephalus L. (Shiri Harzevili, De Charleroy, Auwerx, Vught & Van Slycken, 2003) and Clarias gariepinus (Garcia-Ortega, Verreth & Segner, 2000; Olurin & Oluwo, 2010) the decapsulated Artemia cysts provided better larval growth. From this study it can be stated that the decapsulated Artemia cysts provides a better alternative diet for growth of B. altianalis larvae to reach a digestive maturation level earlier than Artemia nauplii. The decapsulated Artemia cysts contain more energy than other forms of Artemia nauplii (Bengtson, Leger, Sorgeloos, 1991; Leger, Bengtson, Sorgeloos, Simpson & Beck, 1987; Vanhaecke, Lavens & Sorgeloos, 1983) and 211

therefore B. altianalis larvae easily consolidated this energy in decapsulated form. The decapsulated Artemia cysts are more readily acceptable by the B. altianalis larvae as an initial starter diet for this species and there is no need of hatching them. The challenge with Artemia diets is the high cost; and even though it can serve as an alternative better source of B. altianalis larval feed, it might not be a better option for local producers in mass seed production.

In experimental study 2, growth performance of larvae in MO, HA and DF treatments was the same. Despite the fact that the final BW was lower in MO, HA and DF treatments than in MD and DA, the weight gain and specific growth rates were significantly higher than those of MD and DA treatments. This indicated a relatively deeper growth depression in these treatments and that the larvae growth had earlier (in experiment 1a) been limited by lack of nutrients especially for larvae that were initially fed on live diets (MO and HA) and inadequate enzymes factors to sufficiently trigger digestion and assimilation of the microdiet DF. Therefore when larvae in all treatments were introduced to co- feeding they maximized available nutrients of the combination diets and grew comparatively faster than the larvae in treatments MD and DA. This showed a level of compensatory growth in these treatments. This is typical of fish when they have been deprived of food for a while (Jobling, 1994). However, at no point during growth period did the larvae in MO, HA and DF treatments attain a final average BW as that for the larvae in MD and DA treatments. That is the larvae in MD and DA treatments still maintained a significantly higher average larvae BW than larvae in MO, HA and DF treatments. At this point larvae in all the treatments had reached their normal physiological potential under the conditions in the aquaria and therefore the nutritional composition of diets was no longer a limiting factor but perhaps other factors. First, larvae growth could have been limited by the delay of the digestive maturation mechanism shown in experimental study 1a, subsequently affecting larval performance in experimental study 2. Comparatively larvae fed the combination diet (MD) attained maturation level by the third week of after hatch (in experiment 1a). Its performance was expected to be maintained because the larvae were able to maximize and efficiently utilize nutrients for faster growth by its mature digestive system than other larvae in other treatments whose level of maturation could have been initially delayed or been affected by the nature of their diets. Secondary, in some instances the level of growth depression or deprivation in larvae will influence whether a species will reach full compensatory growth or will not at all and this is also species specific (Ali, Nicieza, Wootton, 2003). This result had a significant implication in growth of B. altianalis larvae because 212

initial diet provided to the larvae will consequently influence growth and survival in juvenile stages. High larval growth may facilitate size advantages in accessing limited resources and/or evading and escaping predators (Leggett & Deblois, 1994; Pechenik & Cerulli, 1991). In tropic marine fishes high larval growth rates were associated with survival during metamorphosis to juvenile stages (McCormick & Hoey, 2004). With B. altianalis it is inevitable to maintain an initial feeding protocol of co-feeding and or using decapsulated Artemia cysts to ensure early larvae maturation. These results also suggested that early introduction of a microdiet before 48 DAH to Moina-fed larvae could shorten the period of growth depression thus increasing growth rates during a compensation period. However, this will necessitate further investigations. The third limitation factor was attributed to the environmental stress in which the larvae and or juveniles had been kept i.e. the indoor confined tanks. Larvae reached upper physiological growth potential for all diets in all aquaria and it was possible that there was limitation attributed to stress. Manipulation of environment can largely influence compensatory growth (Ali, Nicieza & Wootton, 2003) including the stocking densities as was observed in Oreochromis niloticus (Basiao, Doyle & Arago, 1996). Hence transferring the larvae in another environment such as the outdoor nursing tanks or ponds could further influence growth positively. In fact in another experiment (experimental study 4) when two weeks old larvae were transferred to outdoor concrete tanks for further nursing, larvae reached a final BW of 1112 ± 42.7mg by 75 DAH which was 3 times bigger than the larvae (373.11 ± 6.97mg) raised on best diet in indoor aquaria within the same period. B. altianalis larvae from indoor systems for outdoor nursing could therefore be best done after two weeks (15-17 DAH) as demonstrated by the ontogenetic enzyme pattern.

In the green water experiment (experimental study 3), the green water concentrates did not directly influence growth of larvae. Many studies have shown that green water has a significant growth effect on larvae (Reitan et al., 1997; Bengtson, Lydon & Ainley, 1999; Sanaye et al., 2014). However the effect and or the mechanisms by which the green algae are utilized in improving the larvae survival and growth vary. The green algae may increase the nutritional value of the prey (Silva, 1999), improve digestibility and assimilation efficiency by the larvae (Lazo, Dinis, Holt, Faulk & Arnold, 2000; Makridis, Libeiro, Rocha & Dinis, 2010), or improve better prey contrast through improved light penetration (Naas, Huse, & Iglesias, 1996; Van der Meeren, Mangor-Jensen & Pickova, 2007). The results of this study do not suggest a direct role of green algae. Since the Moina had been 213

enhanced with the green water (micro-algae) before it was fed to larvae in the tanks, and yet no significant differences in growth parameters were observed between larvae fed with green water and clear water, it can be deduced that micro-algae did not have a direct influence on B. altianalis larvae during the experimental period but only enhanced the nutritional value of Moina. In fact in a related study by Fermine & Recometa (1988) on bighead carp, Aristichthys nobilis it was noted that larvae fed on algae alone did not survive after 5 days of feeding and that enhanced growth and survival was observed when larvae were fed on green water + Moina + a dry feed combination. The workers in the same study also showed that larvae fed on combination diet of green water with Moina + dry feed was not significantly different from those fed with Moina + dry feed. These results concurred with studies emphasizing the nutritional enhancement of the prey as indicated by Silva (1999). The role of green water cannot be underestimated and since it is cheaper to prepare, farmers need to carefully prepare and include it in the rearing protocol as the key source of feed for zooplankton proliferation. It is also important to note that the role of microalgae in a more stable semi natural system such as well fertilized bigger nursing ponds or tanks (outdoor) could even offer a characteristic and complex environment that supports robust growth and survival than smaller confined indoor tanks tanks. For instance studies on some larvae of marine species such as Atlantic halibut Hippoglossus hippoglosuss (Skjermo & Vadstain, 1993) and herring Clupea harengus (Hansen, Strom & Olafsen, 1992) indicated that the addition of green algae to rearing tanks (>1000m3) influenced microbial balance mechanism in rearing water and with in the gut system of the larvae and consequently affected the growth and survival of larvae in these species. In grass carp Ctenopharyngodon idella and in Aristichthys nobilis outdoor facilities provided better growth than indoor facilities (Opuszynski, Shireman, Aldridge & Rottmann, 1985). This result is similar to what was obtained in experimental study 4 when 2 weeks old larvae were nursed in outdoor facility; and the mechanisms for better growth may have been largely facilitated by complex microalgae interactional role. B. altianalis is an omnivorous species and shows a strong structural composition for filtering and concentrating micro- organisms before ingesting them (Unpuplished data; Chapter 6). Hence the results from green water experimental study 3 may also indicate that there is a gradual dietary shift from predominantly zooplankton diets to a more diversified range of diets thus the juveniles could probably be suited to directly utilize the algae than the larvae. Moreover in Sparus aurata L. and Solea senegalensis the interaction between light, different algae species, the type of live prey offered and the age of the larvae greatly influenced the prey selection differently, subsequently affecting growth and survival of 214

larvae (Rocha, Ribeiro, Costa & Dinis, 2008). Further investigations are recommended to identify the age when B. altianalis juveniles are able to directly use and incorporate algae diets into their digestive systems and also the possibility of the microbial role as a result of green water technique that influences the survival of larvae or juveniles among other factors. The raised mortalities together with the low larval weight noted before the 20 DAH in green water fed larvae were likely due to inability by the larvae to properly visualize the feed or prey. Most fish larvae are largely visual feeders (OBrien, 1979). In Atlantic cod Gadus morhua (Puvanendran & Brown, 1998) and Clupea harengus (Batty, 1987) light intensity influenced larval growth differently. It seems that in B. altianalis larvae the acuity for prey contrast improved with age and and as a result by 35 DAH growth had significantly improved.

The stocking of larvae and juveniles in subsequent experiments (experiment 4, 5 & 6) conducted for outdoor nursing was based on growth of larvae in previous experiments (1 a, b &2). The weight gain and specific growth rates were higher for both the second generation larvae stocked at the farm (experimental study 5) and the first generation larvae raised at the research station (experimental study 5 & 6) compared to the larvae that were raised indoors (experimental study 2). Thus the indoor facilities (glass tanks or aquaria) were a limiting factor for further growth of larvae and it was imperative the larvae were transferred to nursing facilities earlier to allow them grow faster. In fact in experimental study 5 where the larvae were transferred to larger concrete tanks for further nursing after two weeks, the larvae grew faster and attained a final BW of 1112 ± 42.70mg compared to 373.11± 6.97mg larvae that had earlier been raised indoor in glass tanks by the 75 DAH. This was the highest growth noted in all the experiments.

In spite of the fact that the experiments for the two generations were conducted under different conditions (different places) the specific growth rates were generally within the same range of 2.12% to 2.54% for the same period between 60 DAH and 90 DAH. Often it is expected that the second or subsequent generations raised in captive environment grow faster than the wild or first generation larvae bred from the wild broodstock (Lorenzen, 2000) largely because of adaptability to feeding and water environment under captivity. Farmed species have a high growth performance than the wild species (Pauly et a1., 1988). The evidence from this study apparently does not suggest a big variation

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in the specific growth rates however, what was generally observed was that survival was significantly higher in larvae bred from the brood fish that had been held in captivity for a long time (experiment 4 & 5) than those that were bred from broodfish obtained from the wild (experimental study 6). This could indicate the beginning of a gradual adaptability process of the species to captive environment (Frankham, 2008). A new species in captivity will undergo a complex of phenotypic, behavior and genetic interactions over successive generations before better growth rates are realized (Lorenzen, 2000b; Lorenzen et al., 2012). Although growth rates improved far better than those reported by NaFIRRI, 2014; Rutaisire et al.,15), both the first and second generation larvae showed relatively slow growth rates compared to other cultured cyprinid (Table 8.15). However, much slower growth rates have also been observed in other cyprinids such as Osteobrama Belangeri (Ramesh et al., 2014) and Barbus barbus L (Policar et al ., 2010) which are also being domesticated. These results suggest that it could perhaps take several generations of larvae to adapt to faster pond growths. In spite of the influence of both physical and biological factors in the environment, the size or maximum potential growth rates and how long the species lives could be controlled by genetic factors (Beverton, 1987; Gilbert & Bolker, 2003). No aging studies for B. altianalis have been conducted to ascertain the age of fish and their corresponding size in the wild for further comparison with the cultured fish. Continued investigations of the feeding strategies commensurate to larvae and juvenile growths are required and it is also probable that at a certain level of development there could be faster stages that could quickly gain weight during development. The observed slight decline of larvae body weight in one of the tanks (in experimental study 6) was attributed to consistently high ammonia levels due to the accumulation of rotten leaves of a nearby tree falling into the tanks.

In all the experiments conducted the fish exhibited a positive allometric growth that was tending toward the exponential b = 3 (isometric growth) as calculated for the weight-length relationships by Froese (2006). The b values calculated for all the larvae in treatments MD, DA, DF, MO, HA and from 6 DAH to 93 DAH (experimental study 1a and 2) were significantly higher than b = 3 (Froese, 2006) and ranged from 3.4 - 4.022 beginning with the feed that produced larvae of the least average weight to the highest respectively. This meant that the larvae grew in depth or width than their length (Froese, 2006). This was possible given that the animal is hatched when it is about 8 mm in length much thinner than the body weight and will therefore require putting on more weight as it grows. This study is clearly in agreement with exponential values of b obtained for the cultured Cyprinus 216

carpio that showed gradual reduction from 4.48 to 3.12 (Osse, 1990), tending toward isometric growth (b=3) in which the juveniles exhibited equal growth between weight and length. This pattern was attributed to initial but sequential development of inevitable body structures such as the head or brain and the tail for swimming which are critical for early survival of hatched larvae (Osse & Boogaart, 1995). This shows that the larvae ultimate cost was focused on the development of the head and tail before the development of trunk. However, on the one hand it is also feasible that since the B. altianalis larvae hatched when it is about 8mm in length (Chapter 7; Rutaisire et al., 2013) much thinner than the body weight; they will therefore require putting on more weight than length as it grows. The variation of b values from the diets used in this study reflected the importance of the feeds to the larvae in attaining better growth. Moreover investigating allometry for individual larvae or juvenile body parts could further reveal the stage in relation to the importance of body structures and their key nutritional requirement (Osse & Boogaart, 1995).

Results from experimental treatment of parasitic infestations of B. altianalis larvae and juveniles revealed that the type and nature of emerging aquatic parasites severely affected and influenced the larval development and the age when the larvae were stocked in outdoor rearing ponds or tanks for further growth. During larvae indoor rearing, parasites associated with the B. altianalis mortalities were identified and they included Trichodina, Apiosoma, Epistylis and Ichthyophthirius multifiliis. These ciliated epibionts appended to the host by anchoring or boring into the skin, gills, eyes and fins of the larvae causing exhaustion, stress, and reduced the ability of the animal to feed leading to reduced growth, secondary infections and in some instances death (Klinger & Floyd, 2002; Nigrelli, Pokorny & Ruggieri, 1976). The treatment of these parasites is variable for fish species and age (Floyd, 2002). In this study effective concentrations of salt, potassium permanganate and formalin were determined for effective treatment of Trichodina, Apiosoma and Epistylis parasites (Table 8.13). Since the salt is cheaper and effective, concentrations of salt ranging from 1%, 1.2% and 1.5% for the period of up to 16, 50 and > 120 minutes respectively were highly recommended to be effective in treatment of B. altianalis larvae in captivity. The most difficult parasites to treat were the Ichthyophthirius multifiliis. The presence of this parasite in Uganda was first reported by Paperna (1972) as a protozoan which heavily infested Barbus amphigramma and Lebistes reticulatus at ARDC Kajjansi. In this study the parasites affected the larvae rendering them weak and unable to feed and killed the B. altianalis larvae massively in less than 72 hrs. A high concentration of salt and 217

formalin was only able to effectively kill the Ich parasites appended on the skin surface and fins of the infected larvae but failed to eliminate those that had penetrated inside the larval’s body (Table 8.13). The Ich parasites influenced the stocking size of fish in ponds and tanks. The study revealed that survival of the infected larvae that were gradually removed from indoor tanks increased with scalation and age. The infected larvae that were stocked at the age of 64 DAH had a higher survival percentage than those stocked at 47 DAH and 28 DAH respectively. At 64 DAH, the larvae had almost the whole body covered with abdominal and dorsal scales while those stocked at 47 DAH had only abdominal scales. The larvae stocked at 28 DAH had no scales hence they had the highest mortalities though final body weight was higher implying that the surviving fish grew faster in abundance of live prey and reduced stress. Paperna (1972) reported that Cyprinus carpio larvae from the same area (ARDC-Kajjansi) had mild parasitic infections between 35 and 120mm which spontaneously disappeared in a month. This further implied that the resilience of B. altianalis larvae to withstand environmental epibiotic parasites including Ich was related to increased formation of body scales, although other factors such as the quality of water and feeds may not be ignored. The results suggest that the farmers must first ascertain the possibility of presence of Ich parasite in their rearing system before stocking the B. altianalis larvae and or can be encouraged to keep them indoors until when the larvae have metarmophosized into juveniles (after two months) before they are stocked.

8.5 Conclusion

Several studies define the larvae transition age to juvenile stages as the period of metamorphosis until the larvae acquire all adult features (Jones et al., 1978; Kendall & Moser, 1984). Based on the evidence adduced from this study of scale formation process, growth parameters and ontogenetic control of enzymatic activity coupled with structural ontogenetic histological development of the digestive system as well as the prevalence of water parasites, it can be concluded that the larvae of B. altianalis transformed into juveniles between the age of 48 (150mg) and 75 DAH (427mg). But these were also influenced by the type of diet provided. The study affirmed that the larvae of B. altianalis are vulnerable in outdoor nursing environments especially before two weeks after hatch but the chance of their survival was increased with the transformation of the larvae into juveniles. Thus stocking B. altianalis juveniles in bigger culture systems is preferable during or after larvae have

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transformed into juveniles (after two months). With better controlled environment of free parasites and good water quality rich in live feed, outdoor nursing of larvae offers faster growth rates and survivals of B. altianalis with supplementation of a well nutrient balanced microdiet. This is a feasible and cheaper option for its production in large quantities.

8.6 Acknowledgement

We appreciate the financial support from the World Bank funded project, the Agricultural Technology Advisory and Agribusiness Services (ATAAS), Uganda/AFRICA- P109224. We thank the National Fisheries and Resources Research Institute (NaFIRRI), the Makerere University Colleges of CONAS and National Crop Research Institutes (NaCRI) that provided other research facilities to make this work a success. We thank Ms Bridget Kimera for her support role in lab and outdoor facilities at ARDC during experimentation.

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8.7 References

Akbary, P., Imanpoor, M., Sudagar, M., & Makhdomi, N. M. (2010). Comparison between live food and artificial diet on survival rate, growth and body chemical composition of Oncorhynchus mykiss larvae. Iranian journal of fisheries science, 9(1), 19-32.‎

Alami-Durante, H., Charlon, N., Escaffre, A. M., & Bergot, P. (1991). Supplementation of artificial diets for common carp (Cyprinus carpio L.) larvae. Aquaculture, 93(2), 167-175. doi:10.1016/0044-8486(91)90215-S

Ali, M., Nicieza, A., & Wootton, R. J. (2003). Compensatory growth in fishes: a response to growth depression. Fish and fisheries, 4(2), 147-190.

Aruho, C., Namulawa,V., Kato, C.D., Kisekka. M., Rutaisire, J., Bugenyi. F., (2016, 2017). Histo- morphological description of the digestive system of the Rippon Barbel Barbus altianalis

(Boulenger 1900): A potential species for culture Uganda Journal of Agricultural Sciences, 2016, 17 (2), 197 – 217.

Basiao, Z. U., Doyle, R. W., & Arago, A. L. (1996). A statistical power analysis of the ‘internal reference’technique for comparing growth and growth depensation of tilapia strains. Journal of fish biology, 49(2), 277-286.

Batty, R. S. (1987). Effect of light intensity on activity and food-searching of larval herring, Clupea harengus: a laboratory study. Marine Biology, 94(3), 323-327.

Bengtson, D. A., Leger, P., & Sorgeloos, P. (1991). Use of Artemia as a food source for aquaculture. Artemia biology, 11, 255-285.

Bengtson, D. A., Lydon, L., & Ainley, J. D. (1999). Green-water rearing and delayed weaning improve growth and survival of summer flounder. North American Journal of Aquaculture, 61(3), 239-242.

Beverton, R. J. H. (1987). Longevity in ¢sh: some ecological and evolutionary perspectives. In A. D. Woodhead, M. Witten & K. Thompson (eds.), Ageing processes in animals (pp. 161-186).

Bisht, A., Anand, S., Bhadula, S., Pal, D. (2013).“Fish seed production and hatchery management: A Review”. New York Science Journal, 6, 42-48.

220

Bondad-Reantaso, M. G. (2007). Assessment of freshwater fish seed resources for sustainable aquaculture. FAO Fisheries Technical Paper. No. 501(pp. 1-628). Rome: FAO.

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry, 72(1-2), 248- 254.

Braum, E. (1967). The survival of fish larvae with reference to their feeding behavior and the food supply. In S. D. Gerking (ed.), The biological basis of freshwater fish production (pp. 113- 131). John Wiley and Sons, N.Y.

Cahu, C. L., & Infante, J. Z. (1994). Early weaning of sea bass (Dicentrarchus labrax) larvae with a compound diet: effect on digestive enzymes. Comparative Biochemistry and Physiology Part A: Physiology, 109(2), 213-222.

Cahu, C., & Zambonino-Infante, J. (2001). Substitution of live food by formulated diets in marine fish larvae. Aquaculture, 200, 161–180.

Canavate, J. P., & Fernandez-Dıaz, C. (1999). Influence of co-feeding larvae with live and inert diets on weaning the sole Solea senegalensis onto commercial dry feeds. Aquaculture, 174(3), 255- 263.

Cara, J. B., Moyano, F. J., Cardenas, S., Fernandez‐Diaz, C., & Yufera, M. (2003). Assessment of digestive enzyme activities during larval development of white bream. Journal of Fish Biology, 63(1), 48-58.

Chakrabarti, R., & Rathore, R. M. (2010). Ontogenic changes in the digestive enzyme patterns and characterization of proteases in Indian major carp Cirrhinus mrigala. Aquaculture Nutrition, 16(6), 569-581.

Chakrabarti, R., Rathore, R. M., & Kumar, S. (2006). Study of digestive enzyme activities and partial characterization of digestive proteases in a freshwater teleost, Labeo rohita, during early ontogeny. Aquaculture Nutrition, 12(1), 35-43.

Chatain, B. (1997). Development and achievements of marine fish-rearing technology in France over the last 15 years. Hydrobiologia, 358: 7-11.

221

Conceicao, L. E. C., Yufera, M., Makridis, P., Morais, S., Dinis, M.T. (2010). Live feeds for early stages of fish rearing. Aquaculture Research, 41, 613–640.

Dabrowski, K. (1984). The feeding of fish larvae: present state of the art and perspectives. Reproduction Nutrition Development, 24, 807–833.

Das, P., Mandal, S. C., Bhagabati, S. K., Akhtar, M. S., Singh, S. K. (2012). Important live food organism and their role in aquaculture. Frontiers in Aquaculture, 5, 69–86.

Dawson, R. M., Elliott, D. C., Elliott W. H., and Jones, K. M. (1986). Data for Biochemical Research. 3rd ed. New York: Clarendon Press.

Day, O. J., Howell, B. R., & Jones, D. A. (1997). The effect of dietary hydrolysed fish protein concentrate on the survival and growth of juvenile Dover sole, Solea solea (L.), during and after weaning. Aquaculture Research, 28(12), 911-921.

Delince, G. A., Campbell, D., Janssen, J. A. L., & Kutty, M., N. (1987). Seed production (pp. 115 - 125.) Port Harcourt, Nigeria. FAO, African Regional Aquaculture Centre.

Dhert, P., Divanach, P., Kentouri, M., & Sorgeloos, P. (1998). Rearing techniques for difficult marine fish larvae. World Aquaculture Society, 29, 48-55.

Falahatkar, B., Mohammadi, H., & Noveirian, H. (2011). Effects of different starter diets on growth indices of Caspian Kutum, Rutilus frisii kutum larvae. Iranian Journal of Fisheries Sciences, 11(1), 28-36.

Farhoudi, A., Nazari, R. M., & Ch, M. (2013). Changes of digestive enzymes activity in common carp (Cyprinus carpio) during larval ontogeny. Iranian Journal of Fisheries Sciences, 12(2), 320-334.

Fermin, A. C., & Recometa, R. D. (1988). Larval rearing of bighead carp, Aristichthys nobilis Richardson,using different types of feed and their combinations. Aquaculture Research, 19(3), 283-290. DOI: 10.1111/j.1365-2109.1988.tb00431.x

Frankham, R. (2008). Genetic adaptation to captivity in species conservation programs. Molecular Ecology, 17(1), 325-333.

222

Froese, R. (2006). Cube law, condition factor and weight–length relationships: history, meta‐analysis and recommendations. Journal of Applied Ichthyology, 22(4), 241-253.

Garcia-Ortega, A., Verreth, J., & Segner, H. (2000). Post-prandial protease activity in the digestive tract of African catfish Clarias gariepinus larvae fed decapsulated cysts of Artemia. Fish Physiology and Biochemistry, 22(3), 237-244.

Gilbert, S. F., & Bolker, J. A., (2003). Ecological developmental biology: preface to the symposium. Evolution & Development, 5, 3-8.

Giri, S. S., Sahoo, S. K., & Sahu, B. B. (2002). Larval survival and growth in Wallago attu (Bloch and Schneider): effects of light, photoperiod and feeding regimes. Aquaculture 213, 151– 161

Gisbert, E., Gimenez, G., Fernandez, I., Kotzamanis, Y., & Estevez, A. (2009). Development of digestive enzymes in common dentex Dentex dentex during early ontogeny. Aquaculture, 287(3), 381-387.

Hansen, G. H., Strom, E., & Olafsen, J. A. (1992). Effect of different holding regimens on the intestinal microflora of herring (Clupea harengus) larvae. Applied and Environmental Microbiology, 58(2), 461-470.

Herath, S. S., & Atapaththu, K. S. S. (2013). Sudden weaning of angel fish Pterophyllum scalare (Lichtenstein) (Pisces; Cichlidae) larvae from brine shrimp (Artemia sp) nauplii to formulated larval feed. Springer Plus, 2,102. http://doi.org/10.1186/2193-1801-2-102

Jobling, M. (1994). Fish Bioenergetics (Pp 121–145). London: Chapman & Hall.

Jones, P. W., Martin F. D., & Hardy, J. D. (1978). Development of fishes of the Mid- Atlantic Bight: An atlas of egg, larval and juvenile stages (1: pp366). United States Department of the Interior.

Kaiser, H., Endemann, F., & Paulet, T. G. (2003). A comparison of artificial and natural foods and their combinations in the rearing of goldfish, Carassius auratus (L.). Aquaculture Research, 34(11), 943-950.

Kendall, Jr. A.W., Ahlstrom, E. H. & Moser, H. G. (1984). "Early life history stages of fishes and their characters". American society of Ichthyologist and Herpertologists, special publication 1: 11-22. 223

Kimura, A., & Robyt, J. F. (1995). Reaction of enzymes with starch granules: kinetics and products of the reaction with glucoamylase. Carbohydrate Research, 277(1), 87-107.

Klinger, R., & Floyd, R. F. (2002). Introduction to freshwater fish parasites. Document CIR716. Institute of Food and Agricultural Science, University of Florida New York: Plenum Press.

Kolkovski, S. (2001). Digestive enzymes in fish larvae and juveniles-implications and applications to formulated diets. Aquaculture, 2000, 181–201.

Kolkovski, S., Tandler, A., Kissil, G. W., & Gertler, A. (1993). The effect of dietary exogenous digestive enzymes on ingestion, assimilation, growth and survival of gilthead seabream (Sparus aurata, Sparidae, Linnaeus) larvae. Fish Physiology and Biochemistry, 12(3), 203- 209.

Koven, W., Kolkovski, S., Hadas, E., Gamsiz, K., & Tandler, A. (2001). Advances in the development of microdiets for gilthead seabream, Sparus aurata: a review. Aquaculture, 194(1), 107-121.

Kujawa, R., Kucharczyk, D., Mamcarz, A., Jamroz, M., Kwiatkowski, M., Targonska, K., & Żarski, D. (2010). Impact of supplementing natural feed with dry diets on the growth and survival of larval asp, Aspius aspius (L), and nase, Chondrostoma nasus (L). Archives of Polish Fisheries, 18, 13–23. doi: 10.2478/v10086-010-0002-3

Kurokawa, T., Shiraishi, M., & Suzuki, T. (1998). Quantification of exogenous protease derived from zooplankton in the intestine of Japanese sardine (Sardinops melanotictus) larvae. Aquaculture, 161(1), 491-499.

Kwiatkowski, M., Zarski, D., Kucharczyk D., Kupren K., Jamroz M., Targoñska K., Krejszeff S., Hakuc-Blazowska, Kujawa, R., & Mamcarz, A. (2008). Influence of feeding chosen rheophilic cyprinid larvae using natural and compound diet. Archives of Polish Fisheries, 16 (4), 383-396.

Lahnsteiner, F., Kletzl. M., & Weismann, T. (2009). The risk of parasite transfer to juvenile fishes by live copepod food with the example Triaenophorus crassus and Triaenophorus nodulosus. Aquaculture, 295:120–125.

224

Lazo, J. P., Dinis, M. T., Holt, G. J., Faulk, C., & Arnold, C. R. (2000). Co-feeding microparticulate diets with algae: toward eliminating the need of zooplankton at first feeding in larval red drum (Sciaenops ocellatus). Aquaculture, 188(3), 339-351.

Leger, P., Bengtson, D. A., Simpson, K. L., & Sorgeloos, P. (1986). The use and nutritional value of Artemia as a food source. Oceanogr. Marine. Biol. Ann. Rev, 24, 521-623.

Leger, P., Bengtson, D. A., Sorgeloos, P., Simpson, K. L., & Beck, A. D. (1987). The nutritional value of Artemia: a review. Artemia Research and its Applications, 3, 357-372.

Leggett, W. C., & Deblois, E. (1994). Recruitment in marine fishes: is it regulated by starvation and predation in the egg and larval stages? Netherlands Journal of Sea Research, 32(2), 119-134.

Liu, B., Zhu, X., Lei, W., Yang, Y., Han, D., Jin, J., & Xie, S. (2012). Effects of different weaning strategies on survival and growth in Chinese longsnout catfish (Leiocassis longirostris Günther) larvae. Aquaculture, 364, 13-18.

Lorenzen, K. (2000). Population dynamics and management. In Tilapias: biology and exploitation (pp. 163-225). Netherlands: Springer

Lorenzen, K., Beveridge, M., & Mangel, M. (2012). Cultured fish: integrative biology and management of domestication and interactions with wild fish. Biological Reviews, 87(3), 639- 660.

Lubzens, E., Tandler, A., & Minkoff, G. (1989). Rotifers as food in aquaculture. Hydrobiologia, 186(1), 387-400.

Mahfuj, M. S., Hossein, M. A., & Sarower, M. G. (2012). Effect of different feeds on larvae development and survival of ornamental Coi carp, Cyprinus carpio larvae in laboratory condition. Journal of Bangladesh Agricultural University, 10 (1), 179-183.

Mai, K., Yu, H., Ma, H., Duan, Q., Gisbert, E., Zambonino-Infante, J. L., & Cahu, C. (2005). A histological study of the development of the digestive system of Pseudosciaena crocea larvae and juveniles. Journal of Fish Biology, 67 (4), 1094-1106.

Makridis, P., Libeiro L., Rocha R., Dinis, M.T. (2010). The influence of microalgae supernatant, and bacteria isolated from microalgae cultures, on microbiology, and digestive capacity of larval

225

gilthead seabream, Sparus aurata, and senegalese sole, Solea senegalensis. The journal of world aquaculture society, 44 (5), 757, DOI: 10.1111/j.1749-7345.2010.00420.x

McCormick, M. I., & Hoey, A. S. (2004). Larval growth history determines juvenile growth and survival in a tropical marine fish. Oikos, 106(2), 225-242.

Mokolensang, J. F., Yamasaki, S., & Onoue, Y. (2003). Utilization of Shochu distillery by-products for culturing the common carp Cyprinus carpio L. On Line Journal of Biolological Science, 3(5), 502-507.

Murray, H. M., Perez-Casanova, J. C., Gallant, J. W., Johnson, S. C., & Douglas, S. E. (2004). Trypsinogen expression during the development of the exocrine pancreas in winter flounder (Pleuronectes americanus). Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 138(1), 53-59.

Mwanja, M., Rutaisire, J, Ondhoro, C, Ddungu, R., & Aruho, C. (2015). Current fish hatchery practises in Uganda: The potential for future investment. International Journal of Fisheries and Aquatic Studies, 2(4), 224-232.

Naas, K., Huse, I., & Iglesias, J. (1996). Illumination in first feeding tanks for marine fish larvae. Aquacultural Engineering, 15(4), 291-300.

Naess, T., Germain-Henry, M., & Naas, K. E. (1995). First feeding of Atlantic halibut (Hippoglossus hippoglossus) using different combinations of Artemia and wild zooplankton. Aquaculture, 130(2), 235-250.

Nicolas, J. L., Robic, E., & Ansquer, D. (1989). Bacterial flora associated with a trophic chain consisting of microalgae, rotifers and turbot larvae: influence of bacteria on larval survival. Aquaculture, 83(3), 237-248.

Charlon, N., & Bergot, P. (1984). Rearing system for feeding fish larvae on dry diets. Trial with carp (Cyprinus carpio L.) larvae. Aquaculture, 41(1), 1-9.

Nigrelli R. F., Pokorny K. S., & Ruggieri G. D. (1976). Notes on Ichthyophthirius multifiliis, a ciliate parasitic on freshwater fishes, with some remarks on possible physiological races and species. Transactions of the American Microscopical Society, 95: 607–613.

226

OBrien, W. J. (1979). The predator-prey interaction of planktivorous fish and zooplankton: recent research with planktivorous fish and their zooplankton prey shows the evolutionary thrust and parry of the predator-prey relationship. American Scientist, 67(5), 572-581.

Olurin, K. B., & Oluwo, A. B. (2010). Growth and survival of African catfish (Clarias gariepinus) larvae fed decapsulated Artemia, live Daphnia, or commercial starter diet. Israel Journal of Aquaculture, 62(1), 50-55

Opuszynski, K., Shireman, J. V., Aldridge, F. J., & Rottmann, R. (1985). Intensive culture of grass carp and hybrid grass carp larvae. Journal of fish biology, 26(5), 563-573.

Osse, J. W. M. (1990). Form changes in fish larvae in relation to changing demands of function. Neth. J. Zool. 40(1-2): 362-385.

Osse, J. W. M., & Van den Boogaart, J. G. M. (1995). Fish larvae, development, allometric growth, and the aquatic environment. In ICES Marine Science Symposia (Vol. 201, No. 0, pp. 21-34). Copenhagen, Denmark: International Council for the Exploration of the Sea, 1991.

Sorgeloos, P. (1980). “The use of the brine shrimp Artemia in aquaculture”. In G. Persoone, P. Sorgeloos, O. Roels & E. Jaspers (Eds.) The brine shrimp Artemia: ecology, culturing, and use in aquaculture (pp. 25-46). Proceedings of the international symposium on the brine shrimp Artemia salina, Wetteren, Belgium, Universal Press.

Paperna, I. (1972) Infection by Ichthyophthirius multifiliis of fish in Uganda. Progressive Fish Culturalist 34: 162–164.

Paulet, T. G. (2003). The effect of diet type and feeding rate on growth, morphological development and behaviour of larval and juvenile goldfish (Carassius auratus)(L.) (Doctoral dissertation, M. Sc. Thesis, Rhodes University).

Pauly, D., Moreau, J., & Prein, M. (1988). A comparison of overall growth performance of tilapia in open waters and aquaculture. In The second international symposium on tilapia in aquaculture (Vol. 15, pp. 469-479). ICLARM Conference Proceedings.

Pechenik, J. A., & Cerulli, T. R. (1991). Influence of delayed metamorphosis on survival, growth, and reproduction of the marine polychaete Capitella sp. I. Journal of Experimental Marine Biology and Ecology, 151(1), 17-27.

227

Peres, A., Cahu, C. L., Zambonino-Infante, J., Le Gall, M. M., & Quazuguel, P. (1996). Amylase and trypsin responses to intake of dietary carbohydrate and protein depend on the developmental stage in sea bass (Dicentrarchus labrax) larvae. Fish physiology and biochemistry, 15(3), 237-242.

Peteri, A., Nandi, S., Chowdhury, S. N. (1992). Manual on Seed Production of Carps. Institutional Strengthening in the Fisheries Sector, Bangladesh, Project reports (not in a Series) -No.24, (pp 61). http://www.fao.org/docrep/field/003/AC376E/AC376E00.htm

Policar, T., Podhorec, P., Stejskal, V., Hamackova, J., Alavi, S. M. H. (2010). Fertilization and hatching rates and larval performance in captive common barbel (Barbus barbus L.) throughout the spawning season. Journal of Applied Ichthyol, 26, 812–815

Policar T., Podhorec, P., Stejskal, V., Kozak P., Svinger, V., Alavi, S. M. H. (2011). Growth and survival rates, puberty and fecundity in captive common barbel (Barbus barbus L.) under controlled condition. Czech Journal of Animal Science, 56 (10), 433–442.

Prinsloo, J. F & Schoonbee, H. J. (1986). Comparison of early larval growth rates of Chinese grass carps Ctenophrangodon idella and Hypophthalmichthys molitrix Chinese silver carp using live feed and artificial feed. Water 12 (4), 229-234.

Puvanendran, V., & Brown, J. A. (1998). Effect of light intensity on the foraging and growth of Atlantic cod larvae: interpopulation difference? Marine Ecology Progress Series, 167, 207- 214.

Rocha, R. J., Ribeiro, L., Costa, R., & M. T. Dinis. (2008). “Does the presence of microalgae influence fish larvae prey capture?” Aquaculture Research, 39, 362-369.

Ramesh, R., Dube, K., Reddy, A. K., Prakash, C., Tiwari, V. K., Rangacharyulu, P. V., & Venkateshwarlu, G. (2014). Growth and survival of pengba, Osteobrama belangeri (Val.) larvae in response to co-feeding with live feed and microparticulate diet. Ecology environment & conservation, 20 (4), 1715-1721.

Reitan, K. I., Rainuzzo, J. R., Oie, G., & Olsen, Y. (1997). A review of the nutritional effects of algae in marine fish larvae. Aquaculture, 155(1), 207-221.

228

Ronnestad, I., Yufera, M., Ueberschar, B., Ribeiro, L., Sæle, O., & Boglione, C. (2013). Feeding behaviour and digestive physiology in larval fish: current knowledge, and gaps and bottlenecks in research. Reviews in Aquaculture, 5, s59–s98.

Ruan, G. L., Yang, D. Q., & Wang, W. M. (2012). Ontogeny of the digestive tracts in grass carp (Ctenopharyngodon idella), yellowcheck carp (Elopichthys bambusa) and topmouth culter (Culter alburnus). Acta Hydrobiol Sin, 36, 1164-1169.

Rutaisire, J., Levavi‐Sivan, B., Aruho, C., & Ondhoro, C. C. (2015). Gonadal recrudescence and induced spawning in Barbus altianalis. Aquaculture Research, 46(3), 669-678.

Sanaye, S. V., Dhaker, H. S., Tibile, R. M., & Mhatre, V. D. (2014). Effect of Green Water and Mixed Zooplankton on Growth and Survival in Neon Tetra, Paracheirodon innesi (Myers, 1936) during Larval and Early Fry Rearing. World Academy of Science, Engineering and Technology, International Journal of Biological, Biomolecular, Agricultural, Food and Biotechnological Engineering, 8(2), 159-163.

Segner, H., Rosch, R., Verreth, J., & Witt, U. (1993). Larval nutritional physiology: studies with Clarias gariepinus, Coregonus lavaretus and Scophthalmus maximus. Journal of the World Aquaculture Society, 24 (2), 121-134.

Shiri Harzevili, A., De Charleroy, D., Auwerx, J., Vught, I., & Van Slycken, J. (2003). Larval rearing of chub, Leuciscus cephalus (L.), using decapsulated Artemia as direct food. Journal of applied ichthyology, 19(2), 123-125.

Silva, A. (1999). Effect of the microalga Isochrysis galbana on the early larval culture of Paralichthys adspersus. Ciencias Marinas, 25(2), 267-276.

Sigma Aldrich (1999). Enzymatic assay of protease activity using casein as a substrate. VELIPA-07

Skjermo, J., & Vadstein, O. (1993). The effect of microalgae on skin and gut bacterial flora of halibut larvae. Fish Farming Technology, 1, 61-67.

Spotte, S. (1992). “Decapsulation of Brine Shrimp cysts”. In: Spotte, S. (Ed.), Captive Sea water Fishes Science and Technology (pp. 411-413). New York/ China/ Toronto/Singapore: A Wing-interscience Publication, John Wiley and sons, Inc.

229

Sugihara, A., Tani, T., & Tominaga, Y. (1991). Purification and characterization of a novel thermostable lipase from Bacillus sp. Journal of Biochemistry, 109(2): 211-216.

Trevino, L., Alvarez‐Gonzalez, C. A., Perales‐Garcia, N., Arevalo‐Galan, L., Uscanga‐Martinez, A., Marquez‐Couturier, G., ... & Gisbert, E. (2011). A histological study of the organogenesis of the digestive system in bay snook Petenia splendida Gunther, 1862 from hatching to the juvenile stage. Journal of Applied Ichthyology, 27(1), 73-82.

Van der Meeren, T., Mangor-Jensen, A., & Pickova, J. (2007). The effect of green water and light intensity on survival, growth and lipid composition in Atlantic cod (Gadus morhua) during intensive larval rearing. Aquaculture, 265(1), 206-217.

Vanhaecke, P., Lavens, P., & Sorgeloos, P. (1983). International study on Artemia: XVII. Energy consumption in cysts and early larval stages of various geographical strains of Artemia. Annales Societe Royale Zoologique de Belgique, 113, 155-164.

Vanhaecke, P., Vrieze, L. D., Tackaert, W., & Sorgeloos, P. (1990). The use of decapsulated cysts of the brine shrimp Artemia as direct food for carp Cyprinus carpio L. larvae. Journal of the World Aquaculture Society, 21(4), 257-262.

Wang, C., Xie, S., Zheng, K., Zhu, X., Lei, W., Yang, Y., & Liu, J. (2005). Effects of live food and formulated diets on survival, growth and protein content of first‐feeding larvae of Plelteobagrus fulvidraco. Journal of Applied Ichthyology, 21(3), 210-214.

Wang, Y., Hu, M., Cao, L., Yang, Y., & Wang, W. (2008). Effects of daphnia (Moina micrura) plus chlorella (Chlorella pyrenoidosa) or microparticle diets on growth and survival of larval loach (Misgurnus anguillicaudatus). Aquaculture International, 16(4), 361-368.

Wang, Y., Hu, M., Wang, W., & Cao, L. (2009). Effects on growth and survival of loach (Misgurnus anguillicaudatus) larvae when co‐fed on live and microparticle diets. Aquaculture Research, 40(4), 385-394.

Wolnicki, J. (2005). Intensive rearing of early stages of cyprinid fish under controlled conditions Archives of Polish Fisheries, 13 (1), 5-87.

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Wolnicki, J., Sikorska, J., & Karminiski, R. (2009). Response of larval and juvenile Rudd Scardinius erythrophthalmus (L) to different diets under controlled conditions. Czech Journal of Animal Science, 54 (7), 331-337

Zambonino-Infante, J., Gisbert, E., Sarasquete, C., Navarro, I., Gutierrez, J., & Cahu, C. L. (2008). In J. E.O. Cyrino, D. Bureau & B. G. Kapoor (Eds.), ontogeny and physiology of the digestive system of marine fish larvae. Feeding and Digestive Functions of Fish (pp. 277–344). Enfield, USA: Science Publishers Inc.

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CHAPTER NINE

9.0 General discussion

This study was two dimensional, refining the artificial spawning conditions for improved egg fertilization rates, hatchability and larvae growth; and also developing optimal feeding techniques or procedure for larvae and juvenile growth and survival of Barbus altianalis. The optimal factors for spawning, growth and survival of larvae and juveniles of cultured fish are critically investigated with a focus of defining standard protocols for mass seed rearing (Migaud et al., 2013; Mylonas, Fostier & Zanuy, 2010; Bilio, 2007; Teletchea & Fontaine, 2014; Yong-Sulem, Brummett & Tchoumboue, 2008). In this study the size at sexual maturity, appropriate hormonal profiles for optimal fertilization rates and hatchability, suitable temperature regimes and hatching facility were determined and resulted in improved egg and larvae survival ultimately leading to increased seed or fingerling production of Barbus altianalis. These factors are an integral process of a spawning protocol for any successful domestication of fish species for commercial production (Bilio, 2007; Migaud et al., 2013). Successful seed production is not only determined by optimal spawning conditions but is equally influenced by the health state of the brood stock with reference to diet provided (Bromage, 1998; Izquierdo, Fernandez-Palacios, & Tacon, 2001; Migaud et al., 2013; Mylonas et al., 2010). Although this study successfully closed spawning cycle with first generation of broodfish in captivity, most of the experiments conducted in this study relied on wild broodstock sources. It is therefore imperative to continuously focus more investigations into development of broodstocks fed with good nutritive feed source to ultimately improve the quality and quantity of the eggs produced per female. Good brood stocks ensure all year round production of quality eggs in mass seed production cycle (Bromage, 1998).

Another key dimension of this study was understanding and development of desirable weaning diets for better growth and higher survival of larvae and juveniles of B. altianalis. The ability of the larvae to quickly adapt to efficient digestion and utilization of microdiets is of great importance to larviculture because this will reduce the cost of seed production in hatcheries (Zambonino-Ifante et al., 2008). Knowledge of the digestive structure and its ontogenetic development in B. altianalis from this study facilitated the understanding of physiological mechanisms and feeding behavior in this

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species. The evolution of the enzyme profiles and the digestive structure identified an appropriate stage in which digestibility of the microdiet promoted better growth without compromising the survival of larvae. Consequently, the digestive larval maturation competence period in B. altianalis determined for the best combination diet of Moina + dry MD was 15 DAH (In the 3rd week of development). This is the period when further manipulation of diets is possible without compromising the growth and survival of the larvae (Kolkovski, 2001; Rangsin, Areechon, & Yoonpundh, 2012; Zambonino-Ifante, & Cahu, 2007). Further studies to improve diets that are prominently nutritious and promote faster growth are inevitable in larviculture and are better done after this period. Growth performance and survival of larvae significantly improved when stocking was done 15 DAH in outdoor nursing tanks. This study however noted that stocking of larvae in outdoor facilities was also influenced by prevalence of aquatic parasites. In anticipation of such circumstances stocking was delayed until or during the period when the larvae transformed into the Juveniles, between 48 and 75 DAH.

Although growth parameters of B. altianalis larvae and juveniles obtained in this study were improved with the best combination diet (Moina + microdiet), the growth rates were comparatively lower than those of most of the cultured species such as the common carp, tilapia or catfish. Growth rates are influenced by a multiplicity of aquatic environmental factors including biotic, abiotic and the organism’s genetic factors (Lorenzen et al., 2012). However, provision of appropriate feeds or feeding regimes for fish growth is one of the key factors that define faster growth rates in captive environment (Jobling, 2016). This study affirmed the nature of the feeding behavior of B. altianalis as an omnivorous species, but its digestive structure revealed and suggested that as the fish grows there is continued diversification of diets at particular developmental stages. For instance the role of algae in survival and growth of larvae and juveniles or the stage at which its impact is critical was unclear. The implication of sequential food requirements at each stage of development is that the species develop capacity to acquire and maximize required nutrients from diversity of feeds at each developmental stage (Dabrowski, 1986; De Silva & Anderson, 1995). This indicates that in captivity and in absence of the diversification of feed sources there is a need to understand the dynamics and feeding mechanisms for requirements nutrients at each stage and formulate optimal feeds for rapid growth at each stage. The implications of monoculture and polyculture feed dynamics need to be investigated in order to optimize the growth of B. altianalis for successful commercial production. 233

9.1 Conclusions and recommendations

Barbus altianalis is an indigenous cyprinid of high value because it’s cherished by local communities in the region and has therefore potential to generate income and provide nutritional needs. Previous studies successfully established technologies for its induced spawning but in the current study artificial spawning conditions, larval and juvenile growth rates have been improved. Larvae survival was improved from less than 11% to over 80%. Males in 30-34.9 cm and females from 35-39.9 cm size class and above from both Lake Edward and Upper River Nile were determined as appropriate to be picked from the wild for acclimatization and subsequent spawning. During the induced spawning process in this study, the African catfish pituitary extracts of 0.014g per ml in 5% saline solution were effective in improving fertilization rates and hatchability of larvae when administered in two priming dosages separated by 250 degree hours to. However, when it is a breeding season and the females are ripe running, hormonal application is not required and spawning is only facilitated by water flashing with stripping being done after 100 degree hours. Males and females become ripe when they are put together in the same pond and fed for not less than a month prior to striping. Over

80% of the embryos hatched within a temperature range of 24⁰C to 30⁰C within which the experiment was conducted but high hatchability values were obtained at 24⁰C and at 27⁰C. Hatchery operators are cautioned to avoid direct aeration that causes egg agitation because it was found out that aeration caused mortalities at lower temperature of 24⁰C and delayed hatchability at other temperatures than

24⁰C. The best designed hatching facilities to be used by farmers to reduce mechanical agitation of B. altianalis eggs and subsequwntly the embryo mortalities during incubation process were The re- circulating and aquaria tank systems (as designed in this study). Improved average larvae weight and survival were obtained at an optimal temperature range of 27-30⁰C which is recommended for incubating B. altianalis larvae.

The digestive structure of B. altianalis was able to accept cheaper micro-processed diets at exogenous feeding (6-7 DAH) but better growth rates and reduced mortalities were achieved when the microdiet was provided in combination with live diets (Moina). Ontogenetic digestive structure and

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development of enzymatic profiles suggested that farmers can get better growth rates and high survivals if larvae are nursed in well set outdoor facilities after 15 DAH (during and after third week of hatch) because by this period the larvae had developed digestive maturation competence to efficiently utilize microdiets. In the likely hood that farmers anticipate poor water quality with reference to prevalence of parasites, larvae transfer should be delayed until when they are transformed into juveniles between 48-75 DAH (150-427mg). Feeding is paramount in any successful domestication of any high value species. Therefore nutritional requirements and the species interaction mechanisms in polyculture systems are recommended for further investigations.

9.2 Areas for further study Arising from the literature review was the fact that B. altianalis and B. bynni are species that closely resemble each other but do not occupy the same water bodies. There is a possibility that these two species could crossbreed and also may have the same method of inducing them to spawning. More studies are required to establish whether they could breed together and if possible the superior traits could be bred for faster growth. In addition, because of their close resemblance it is imperative to find out whether B. bynni can be induced to spawn the same way as B. altianalis.

Little data for determining length at sexual maturity (L50) in captivity was provided by the famer (from Ssenya farm records) and this data was not sufficient to be analyzed by the two parameter logistic ogive models, hence more data will be required before true values are established in captive environment. However, the B. altianalis growth values could as well be influenced by the type of feed provided which in turn could influence the L50 in captivity.

Although the performance of African catfish pituitary extracts in this study was comparatively better than that of Dagin (an artificial hormone), this study does not confirm that Dagin was not very effective in inducing the fish to spawn. There is a need to study the actual dosages by varying the amount of hormone that could particularly make Dagin a more effective inducing hormone for B. altianalis. Of kin interest though is looking at purification processes of catfish pituitary extracts and their effectiveness (viability) in different dosages at different stages of catfish development so that they can be availed to famers in preserved form ready for use.

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The literature review and relative gut index (RGI) of B. altianalis obtained from this study suggested a preference for food items at juvenile and adult stages of development. The focus here is to continue developing feeds suitable for these stages in captivity. B. altianalis was identified to be an omnivorous species, whose mouthparts are flexible and enable the species to feed at any level in the water column. This confers upon the species ability to be polycultured with other farmed fish. Further studies are encouraged to ascertain the growth performance of B. altianalis with various cultured species.

The best larval feed combination was the live feed and the dry combination diet (Moina + microdiet). In this study, diet manipulations were only possible during and after the third week of larvae hatch. Investigations to improve growth and survival should continue through conducting diet experiments that include formulation of appropriate feeds for B. altianalis larvae and juveniles during and or after the third week of hatch.

The current study focused on improving growth and survival of larvae and juveniles with a view of providing quality seed for commercial up-scaling of B. altianalis. The next stage is to up-scale production of B. altianalis by conducting on farm trials, formulating feeds and comparing growth performance of grow-out table sizes in different regions of the country as well as marketing the farmed B. altianalis.

The study looked at specific growth rates of larvae bred from the wild and the second generation larvae bred from the pond. These studies were conducted in the tanks and ponds independently i.e. the experiments did not deliberately target comparing them in the same culture systems. Further studies should compare the growth rates of larvae bred from the wild parents and those bred from the first generation parents raised in captivity.

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9.3 References

Yong-Sulem, S., Brummett, R. E., & Tchoumboue, J. (2008). Hatchability of African catfish Clarias gariepinus eggs in hapas and in basins: a diagnostic study of frequent inhibition by rainfall and water stagnation. Sommaire/Inhoud/Sumario, 26(1), 39-42

Bilio, M. (2007) Controlled reproduction and domestication in aquaculture – the current state of the art, Part I. Aquaculture Europe, 32, 5–14.

Mylonas, C. C., Fostier, A., & Zanuy, S. (2010). Broodstock management and hormonal manipulations of fish reproduction. General and Comparative Endocrinology, 165(3), 516- 534.

Izquierdo, M. S., Fernandez-Palacios, H., & Tacon, A. G. J. (2001). Effect of broodstock nutrition on reproductive performance of fish. Aquaculture, 197(1), 25-42.

Bromage, N. (1998). Brood stock management and the optimisation of seed supplies. Aquaculture Science, 46(3), 395-401.

Teletchea, F., & Fontaine, P. (2014). Levels of domestication in fish: implications for the sustainable future of aquaculture. Fish and fisheries, 15(2), 181-195.

Migaud, H., Bell, G., Cabrita, E., McAndrew, B., Davie, A., Bobe, J., ... & Carrillo, M. (2013). Gamete quality and broodstock management in temperate fish. Reviews in Aquaculture, 5(s1), S194-S223.

Rangsin, W., Areechon, N., & Yoonpundh, R. (2012). Digestive enzyme activities during larval development of striped catfish, Pangasianodon hypophthalmus (Sauvage, 1878). Kasetsart Journal of Natural Science, 46, 217-228.

Kolkovski, S., (2001). Digestive enzymes in fish larvae and juveniles-implications and applications to formulated diets. Aquaculture, 2000, 181–201.

Lorenzen, K., Beveridge, M., & Mangel, M. (2012). Cultured fish: integrative biology and management of domestication and interactions with wild fish. Biological Reviews, 87(3), 639- 660

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Jobling, M. (2016). Fish nutrition research: past, present and future. Aquaculture International, 24(3), 767-786.

De Silva, S. S. & Anderson, T. A. (1995). Fish Nutrition in Aquaculture (pp.319). Landon, Chapman and Hall,

Dabrowski, K. R. (1986). Ontogenetical aspects of nutritional requirements in fish. Comparative Biochemistry and Physiology Part A: Physiology, 85(4), 639-655.

Zambonino-Ifante, J., & Cahu, C. L. (2007). Dietary modulation of some digestive enzymes and metabolic processes in developing marine fish: applications to diet formulation. Aquaculture, 268(1), 98-105.

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APPENDIX

The studay map

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