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Effect of graded levels of protein in diet on growth, proximate and elemental composition of fingerling Grass carp (Ctenopharyngodon idellus) from Southern Punjab, Pakistan Candidate’s Name: MUHAMMAD KHALID Supervisor: Prof. Dr. MUHAMMAD NAEEM Institute of Pure & Applied Biology, Bahauddin Zakariya University, Multan. THESIS SUBMITTED TO BAHAUDDIN ZAKARIYA UNIVERSITY, MULTAN. For the Degree of Doctor of Philosophy In the Subject of ZOOLOGY Institute of Pure and Applied Biology (Zoology Division), BAHAUDDIN ZAKARIYA UNIVERSITY, MULTAN. STATEMENT AND DECLARATION The work submitted in this thesis under title, “Effect of graded levels of protein in diet on growth, proximate and elemental composition of fingerling Grass carp(Ctenopharyngodon idellus) from Southern Punjab, Pakistan” is fulfillment of the requirements for the degree of Doctor of Philosophy. I declare that this work is the result of my own investigations and has not already been accepted in substance for any degree, nor is it currently being submitted for any other degree. All author works referred to in this thesis have been fully acknowledged. Candidate______________________ Date___________________________ I certify that above statement is correct Supervisor: ________________________________________ Prof. Dr. Muhammad Naeem External Examiner:__________________________________ Director:___________________________________________ Prof. Dr. Muhammad Naeem “It Is He Who Has Made The Sea Subject, That Ye May Eat Thereof Flesh That Is Fresh And Tender. And That Ye May Extract There From Ornaments To Wear.” (26: 14) – THE HOLY QURAN I Dedicate This Research Work to My Parents & My Respected Teachers Prof. DR. MUHAMMAD NAEEM who always provides me dexterous guidance in my academic and research field & PROF. DR. ABDUS SALAM who provides care and a great deal of support in every walk of my life (i) ACKNOWLEDGEMENTS I bow my head before Allah, the Almighty, the most merciful, the most beneficent who bestowed upon me the ability to undertake and complete this work; and the Holy Prophet Hazrat Muhammad (Peace Be Upon Him) who inspired me for the truth. I would like to thank the enviable administration of Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan, who provided me the research opportunity on the investigated topic. I deem it an utmost pleasure to be able to express the heartiest gratitude and deep sense of devotion to my worthy supervisor, Prof. Dr. Muhammad Naeem, Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, who always inspired and guided me in the real sense and spirit of a teacher. By dint of his constant help, keen interest and kind advice the completion of this thesis became possible. I feel great pleasure in expressing heartfelt gratitude to Prof. Dr. Muhammad Naeem, Director, Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, for his encouragement, co-operation and providing all research facilities. I am also greatly thankful to Mr. Abir Ishtiaq (Ph.D. Scholar) who remained very helpful to me and provided every possible assistance related to my research work. I wish to extend my sincere thanks to Fiaz Ali, Shoaib Khan, Prof. M. Azam Sheikh, Danyl Naeem, Vijay lal, Abubaker, Uzair , Talha Habib for providing composing facilities & moral support. I am also thankful to Dr. Mohsin, Dr. Qaswar Ali Shah, and Prof. Malik Ashraf for their moral support. In the end, I acknowledge my mother, and wife for their love, prayers, whole-hearted support and encouragement, from the core of my heart. Nothing can replace their positions. May they live long to see all my future progression and dreams come true. Muhammad Khalid (ii) ACRONYMS, ABBREVIATIONS AND SYMBOLS a Intercept b Regression Coefficient cm Centimeter Conc. Concentration g Gram HNO3 Nitric Acid K Condition factor L3 Cube of Length LLR Length Length Relationship LWR Length Weight Relationship mg Milligram n Number of samples n.s Non Significant (P > 0.05) P Probability P < 0.001 Highly Significant P < 0.01 Significant P < 0.05 Least Significant ppm Parts Per Million r Correlation Coefficient S.D. Standard Deviation S.E. Standard Error µg Micrograms W Wet Body Weight % Percentage < Less than > Greater than TL Total length FL Fork length SL Standard length ED Eye diameter HL Head length (iii) HW Head width BD Body depth BG Body girth PelFL Pelvic fin length DFL Dorsal fin length AFL Anal fin length CFL Caudal fin length CFW Caudal fin width Ca Calcium Cd Cadmium Cr Chromium Co Cobalt Cu Copper Fe Iron K Potassium Mn Manganese Ni Nickel Na Sodium Pb Lead Zn Zinc (iv) TABLE OF CONTENTS Chapter Description Page number number ACKNOWLEDGEMENTS i ACRONYMS, ABBREVIATIONS AND SYMBOLS ii TABLE OF CONTENTS iv LIST OF TABLES vii LIST OF FIGURES xii ABSTRACT xx GENERAL INTRODUCTION 1 INTRODUCTION Grass carp (Ctenopharyngodon idella) 6 AIMS AND OBJECTIVES 11 Chapter GROWTH 1 1.1 INTRODUCTION 12 1.2 MATERIALS AND METHODS 17 1.2.1 Study Sites And Experimental Design 17 1.2.2 Preparation of Diet 17 1.2.3 Fish Growth Studies 17 1.2.4 Physico-Chemical Parameters 18 1.3 RESULTS 19 1.4 DISCUSSION 21 Chapter 2 EXTERNAL MORPHOMETRY 2.1 INTRODUCTION 30 2.2 MATERIALS AND METHODS 34 2.3 RESULTS 37 2.3.1 Length- weight relationship (LWR) 37 2.3.2 Relationships of Condition Factor with Total Length & 37 Wet Weight (v) 2.3.3 Length length relationship (LLR) 38 2.3.4 2 Studied morphometric variables versus wet body weight 38 2.4 DISCUSSION 39 Chapter 3 PROXIMATE COMPOSITION 3.1 INTRODUCTION 59 3.2 MATERIALS AND METHODS 65 3.2.1 Wet body weight, total length measurement 65 3.2.2 Assessment of water 65 3.3.3 Ash content assessment 66 3.2.4 Estimation of fat contents 66 3.2.5 Protein mass estimation 67 3.2.6 Carbohydrates estimation 67 3.2.7 Condition factor calculation 68 3.2.8 Assessment of multiple regression 68 3.2.9 Statistical analysis 68 3.3 RESULTS 69 3.3.1 Relationship of % water constituent other body 69 constituents 3.3.2 Relationship of Fish wet body weight versus body 69 constituents 3.3.3 Relationship of fish total length versus other body 70 constituents 3.3.4 Relationship of condition factor (k) with other body 70 constituents. 3.3.5 Analysis of multiple regression 70 3.4 DISCUSSION 111 3.4.1 Impact of water constituent on other body constituents 111 3.4.2 Relationship of body size versus body constituents 113 (vi) 3.4.3 Influence of condition factor (k) on body composition 113 Chapter 4 ELEMENTAL COMPOSITION 4.1 INTRODUCTION 115 4.1.1 Magnesium (Mg) 119 4.1.2 Copper (Cu) 119 4.1.3 Zinc (Zn) 119 4.1.4 Manganese (Mn) 120 4.1.5 Cadmium (Cd) 120 4.1.6 Calcium (Ca) 120 4.1.7 Lead (Pb) 121 4.1.6 Nickel (Ni) 121 4.1.9 Chromium (Cr) 121 4.1.10 Iron (Fe) 121 4.1.11 Potassium (K) 122 4.1.12 Cobalt (Co) 122 4.2 MATERIALS AND METHODS 123 4.3 RESULTS 124 4.3.1 Conjunction of concentration of elements with wet body 124 weight 4.3.2 Association between various concentration of elements with 124 body length 4.3.3 Relationship between elemental concentration and 124 condition factor 4.3.4 Interelemental regression 125 4.3.5 Multiple regression analysis 125 4.4 DISCUSSION 127 Chapter 5 REFERENCES 161 (vii) LIST OF TABLES Table Title Page no. no. 1.1 Growth performance, survival, production and feed 24 utilization by grass carp reared to three different feeds for 90 days in 3 ponds (one acre each) at Mushtaq fish farm Muzaffar Garh, Punjab, Pakistan under polyculture system 1.2 Various parameters of water quality of experimental aquaria 25 1.3 Growth performance, survival, production and feed 25 utilization by Oreochromis niloticus. 1.4 Growth performance, survival, production and feed 25 utilization byLabeo rohita 1.5 Growth performance, survival, production and feed 26 utilization by Oreochromis niloticus 1.6 Growth performance, survival, production and feed 26 utilization byOreochromis niloticus 1.7 Growth performance, survival, production and feed 26 utilization by Ompok pabda 1.8 Growth performance, survival, production and feed 27 utilization by Cyprinus carpio 1.9 Growth performance, survival, production and feed 27 utilization by Clarias gariepinus burchell 1.10 Growth performance, survival, production and feed 27 utilization of (Clarias gariepinus burchell). 2.1 Mean values and ranges of weight and various external morphology of 42 grass carp (n = 72) 2.2 Descriptive statistics and regression parameters of total length (TL, cm) 43 with different morphometrics of.grass carp 2.3 Descriptive statistics and regression parameters of total 44 length (TL, cm) with different morphometrics for grass carp 2.4 Descrptive statistics and regression parameters of body weight ( 45 (gm) with different morphometrics of female grass carp (viii) 2.5 Descrptive statistics and regression parameters of body 46 weight (gm) with different morphometrics of female grass carp 2.6 Mean values and ranges of grass carp 47 2.7 Length-weight relationships for grass carp 48 2.8 Length-weight relationship for different fishes from 49 different localities Appendix Mean and range of different components of grass carp (n = 71 3.1 72) Appendix Comparison of relationship between % of body constituent 72 3.2 and % content of water of grass carp (n=72) Appendix Comparison of relationship between body weight ( gm), 73 3.3 total length (cm) and constituents (%) of body (wet, dry weight) of grass carp (n = 72) Appendix Comparison of relation between wet body weight ( gm), 74 3.4 body ingredients (wet weight, gm) of grass carp (n = 72 ) Appendix Comparison of relation between total length