Title Nile crocodile (Crocodylus niloticus) urine as sample for biochemical and hormonal analyses LASYA CHRISTINA BEKKER Thesis submitted to the Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, in fulfilment of the requirements for the degree Doctor of Philosophy (PhD) SUPERVISOR: PROF Jan G Myburgh (PhD) CO-SUPERVISOR: PROF A Duncan Cromarty (PhD) 2017 © University of Pretoria QUOTE God sleeps in the minerals, awakens in plants, walks in animals, and thinks in man. Arthur Young (1741-1820) i © University of Pretoria TABLE OF CONTENTS QUOTE ...............................................................................................................................i TABLE OF CONTENTS ..................................................................................................... ii ACKNOWLEDGEMENTS .................................................................................................. vi DEDICATION .................................................................................................................. viii DECLARATION ................................................................................................................. ix FORMAT OF THIS THESIS ...............................................................................................x LIST OF ABBREVIATIONS ............................................................................................... xi LIST OF FIGURES .......................................................................................................... xiii LIST OF TABLES ............................................................................................................ xvi LIST OF EQUATIONS ................................................................................................... xviii LIST OF APPENDICES ................................................................................................... xix ABSTRACT ...................................................................................................................... xx CHAPTER 1: INTRODUCTION ......................................................................................... 1 CHAPTER 2: AIM AND OBJECTIVES ............................................................................ 14 2.1. Aim ....................................................................................................................... 14 2.2. Objectives ............................................................................................................. 14 CHAPTER 3: LITERATURE REVIEW ............................................................................. 15 3.1. Urine sample analysis for diagnostic tools ................................................................ 15 3.1.1. Introduction .......................................................................................................... 15 3.1.2. Laboratory analysis of urine .................................................................................. 16 3.1.3. Specific laboratory analyses ................................................................................. 19 3.1.4. Routine testing of urine samples in veterinary medicine ....................................... 20 3.2. Urine from crocodilians ............................................................................................ 21 3.3. Crocodilian blood parameters ................................................................................... 24 CHAPTER 4: JUSTIFICATION ........................................................................................ 26 ii © University of Pretoria CHAPTER 5: Nile crocodile (Crocodylus niloticus) urine and plasma biochemistry investigation .................................................................................................................... 28 5.1. Introduction .............................................................................................................. 28 5.2. Materials and methods ............................................................................................. 33 5.2.1. Collection of samples ........................................................................................... 33 5.2.2. Laboratory examination of the urine samples ....................................................... 34 5.2.3. Laboratory examination of the plasma samples .................................................... 35 5.2.4. Statistical assessment .......................................................................................... 35 5.3. Results ..................................................................................................................... 37 5.3.1. Urine .................................................................................................................... 37 5.3.2. Plasma .................................................................................................................. 38 5.3.3. Relation of the urine- to the plasma biochemical parameters ................................ 38 5.4. Discussion ................................................................................................................ 41 5.5. Conclusion ............................................................................................................... 47 5.6. Acknowledgements .................................................................................................. 47 5.7. Proposed publication from this chapter ..................................................................... 47 CHAPTER 6: Clinical validation of the analysis of steroids and steroid metabolites in Nile crocodile urine .......................................................................................................... 48 6.1. Introduction .............................................................................................................. 48 6.2. Materials and methods ............................................................................................. 52 6.2.1. Subjects ............................................................................................................... 52 6.2.2. Materials ............................................................................................................... 52 6.2.3. Instrumentation ..................................................................................................... 54 6.2.4. Preparation procedures ........................................................................................ 54 6.2.5. Extraction Procedure ............................................................................................ 55 6.2.6. Chromatographic conditions ................................................................................. 57 6.2.7. Validation of method ............................................................................................. 57 6.2.8. Calibration of analytes .......................................................................................... 57 iii © University of Pretoria 6.2.9. Quality control ...................................................................................................... 58 6.2.10. Quantitation procedures ..................................................................................... 59 6.3. Results ..................................................................................................................... 60 6.3.1. Calibration results ................................................................................................. 64 6.3.2. Quality Control Results ......................................................................................... 65 6.4. Discussion ................................................................................................................ 65 6.5. Conclusion ............................................................................................................... 66 CHAPTER 7: Steroid profiling of crocodilian urine ........................................................... 68 7.1. Introduction ........................................................................................................... 68 7.2. Materials and methods ......................................................................................... 70 7.2.1. Sample collection and subjects ...................................................................... 70 7.2.2. Sample preparation ....................................................................................... 72 7.2.3. Instrumentation .............................................................................................. 74 7.3. Results ................................................................................................................. 75 7.3.1. Human control sample ................................................................................... 75 7.3.2. Subject 1: A reproductively active female Nile crocodile ................................ 76 7.3.3. Subject 2: Pooled sample of adult Nile crocodile urine ................................... 77 7.3.4. Subject 3: Female Nile crocodile; Izintaba Crocodile Farm ............................ 79 7.3.5. Subject 4: Juvenile male Nile crocodile; Kruger National Park ....................... 80 7.3.6. Subject 5: Sub-adult male Nile crocodile; Loskop Dam .................................. 81 7.3.7. Subject 6: Sub-adult male Nile crocodile; Flag Boshielo Dam ........................ 82 7.3.8. Subject 7: Female spectacled