MUSCLE GROWTH AND FLESH QUALITY OF FARMED ATLANTIC HALIBUT (HIPPOGLOSSUS HIPPOGLOSSUS) IN RELATION TO SEASON OF HARVEST Ørjan Hagen A Thesis Submitted for the Degree of PhD at the University of St. Andrews 2008 Full metadata for this item is available in the St Andrews Digital Research Repository at: https://research-repository.st-andrews.ac.uk/ Please use this identifier to cite or link to this item: http://hdl.handle.net/10023/642 This item is protected by original copyright Muscle growth and flesh quality of farmed Atlantic halibut (Hippoglossus hippoglossus) in relation to season of harvest. Ørjan Hagen A thesis submitted for the degree of Doctor of Philosophy University of St Andrews St Andrews, July 2008 ii This thesis is dedicated to my family. I could not have managed without their loving support. Thank you so much! iii Acknowledgements Taking a PhD abroad at a highly respected University across the world has been a milestone of my life. I am very proud to have been a PhD student at the University of St Andrews and very grateful to the people helping me reaching my goals. Looking back at the beginning (October 2003) these last few years have been an educational expedition, full of challenges and memories. I have met people from Australia and Taiwan in the east to Canada in the west, wonderful people that I hope to have collaboration with in the future. There are so many to thank, and I would like to start with my supervisor and the head of the “Fish Muscle Research Group” (FMRG), Prof. Ian A. Johnston. I am forever grateful to have been a member of the FMRG, and without Ian’s skills my path would without doubt have been much more troublesome. I would also like to thank Prof. Christel Solberg, at the Faculty of Biosciences and Aquaculture (FBA) at Bodø University College where I have spent most of my research time, for always encouraging me, trusting my decisions and giving me free hands when setting up the new labs. I am very grateful to the people in the FMRG for giving me such a warm welcome when I started my PhD, providing a good and steady working environment and making me feel like home. I would especially like to mention Dr Vera Vieira, Dr Ralph Bickerdike, Dr Jorge Fernandes, Mr Hung-Tai Lee, Dr Matthew MacKenzie and Ms. Marguerite Abercromby. The faces in the FMRG have changed through the years, but the spirit within the group has not. Dr Ralph Bickerdike was the only person in the lab working with seafood quality at the time I started, and without his support regarding the collagen/cross-link method, things would have been more difficult. I am hoping that I someday can be of any assistance to you. iv Dr Xuejun Lee was very helpful and most valuable when the enzyme assays were established at FBA and the frequent e-mail correspondences during the winter of 2006 solved most of the problems. I am forever grateful. Without the help of Dr Neil Bower, Dr Lara Meichke and especially Dr Jorge Fernandes, the molecular biology part of my PhD sure would have been most difficult. You all introduced me to the world of molecular biology, a valuable and most interesting lesson. I am very grateful. Dr Charles Paxton is gratefully acknowledged for his help in solving some of the statistical obstacles encountered during these last few years. I would like to thank FBA and the people working there for providing a good and stable working environment. I am looking forward to future collaborations. I am very grateful to Mørkvedbukta research station, Aga Marin AS and to the people working there for taking care of and providing the fish needed for my experiments. The Research Council of Norway is gratefully acknowledged for the funding of this project and Bodø University College for providing the salary and the laboratory facilities needed during my PhD period. My parents Irene and Geir Hagen’s strong belief, support and cheerful words have been a blessing to me, making these last few years less difficult. My dearest Ellen Sirnes, chemist and girlfriend, what should I have done without you? I do not even know where to begin. Your chemistry (both work related and private) has proven to be an unbeatable combination. Proof reading, practical assistance in the lab, encouraging words and with a lovely smile always in store (just to mention a few), has made this journey a lot more joyful. You truly are an angle! If I have forgot to mention anyone I am truly sorry, you know who you are, so thank you! v Table of contents Page Acknowledgements iii Table of contents v List of figures xi List of tables xiv Publications xv Declarations xvi List of abbreviations xvii Thesis abstract 1 Chapter 1 General introduction 3 1.1 Biology of the Atlantic halibut 4 1.2 Myotomal and connective tissue architecture 7 1.2.1 Myotomal architecture 7 1.2.1.1 Myotendious junctions (MJ) in teleosts 12 1.2.2 Connective tissue architecture 12 1.3 Muscle fibre types 21 1.4 Muscle growth in teleosts 27 1.5 Response of muscle to the environment 34 1.5.1 Temperature 34 1.5.2 Photoperiod 37 1.5.3 Food 38 1.6 IGF-system in fish, a general perspective 40 1.7 Atlantic halibut farming 43 vi 1.8 General parameters influencing flesh quality in fish 47 1.8.1 Chemical composition 48 1.8.2 Colour 48 1.8.3 Water holding capacity 50 1.8.4 Texture 53 1.8.5 Proteolytic enzymes 55 1.8.5.1 Lysosomal cathepsins 56 1.8.5.2 Matrix metalloproteases 58 1.10 Project aims 60 Chapter 2 Sexual dimorphism of muscle growth in Atlantic halibut (Hippoglossus hippoglossus) results in a lower final fibre number in males. 61 2.1 Abstract 62 2.2 Introduction 63 2.3 Material and methods 65 2.3.1 Fish 65 2.3.2 Sample preparation 66 2.3.3 Histochemistry and Immunohistochemistry 66 2.3.4 Morphometric studies 68 2.3.5 Statistics 69 2.4 Results 71 2.4.1 Muscle fibre differentiation 71 2.4.2 Muscle fibre size and number 71 vii 2.5 Discussion 81 Chapter 3 Sexual dimorphism of fast muscle fibre recruitment in farmed Atlantic halibut (Hippoglossuss hippoglossus). 84 3.1. Abstract 85 3.2 Introduction 86 3.3 Material and methods 88 3.3.1 Fish husbandry 88 3.3.2 Morphometric studies 89 3.3.3 General chemistry 90 3.3.4 Statistics 92 3.4. Results 93 3.4.1 Growth characteristics 93 3.4.2 Condition-factor, GSI and general chemistry 95 3.4.3 Muscle growth pattern 98 3.4.3.1 Total cross section area of fast muscle 98 3.4.3.2 Change in muscle fibre number 98 3.4.3.3 Muscle fibre diameter and distribution 103 3.5 Discussion 107 Chapter 4 Biochemical and structural factors contributing to seasonal variation in the texture of farmed Atlantic halibut (Hippoglossus hippoglossus) flesh. 111 4.1 Abstract 112 viii 4.2. Introduction 113 4.3. Material and methods 115 4.3.1 Fish husbandry 115 4.3.2 Instrumental texture measurement 115 4.3.3 Sample preparation 117 4.3.4 Collagen and crosslink assay 117 4.3.5 Statistics 122 4.4 Results 123 4.4.1 Validation of the texture over the fillet (pilot study) 123 4.4.2 Seasonal variation in texture and pH 125 4.4.3 Effect of season on muscle collagen and pyridinoline cross-link concentration 125 4.4.4 Impact of pH, fibre density, water, collagen and pyridinoline cross-link on texture 127 4.5 Discussion 133 Chapter 5 The influence of cathepsins and collagenase on protein and water holding capacity in muscle of commercially farmed Atlantic halibut (Hippoglossus hippoglossus). 137 5.1 Abstract 138 5.2 Introduction 139 5.3 Material and methods 142 5.3.1 Fish farming 142 5.3.2 Sample preparation 142 ix 5.3.3 Water holding capacity 142 5.3.4 Enzyme kinetics 143 5.3.4.1 Cathepsin B, B + L and H activity assay 143 5.3.4.2 Cathepsin D activity assay 144 5.3.4.3 Collagenase activity assay 145 5.3.5 Statistics 148 5.4 Results 150 5.4.1 Validation of pH optimum for cathepsin B, B + L and H 150 5.4.2 Seasonal variation and influence of enzymes on protein and liquid loss. 150 5.5 Discussion 162 Chapter 6 Muscle expression of growth-related genes during fasting and refeeding in juvenile Atlantic halibut, Hippoglossus hippoglossus. 165 6.1 Abstract 166 6.2 Introduction 167 6.3 Material and method 170 6.3.1 Fish maintenance and sample preparation 170 6.3.2 RNA extraction and cDNA synthesis 171 6.3.3 Cloning of IGF-I, IGF-II, ctsb and ctsd 171 6.3.4 Design of real-time PCR primers 172 6.3.5 Quantitative real-time PCR analysis 175 6.3.6 Statistical analysis 177 6.4 Results 178 x 6.4.1 Cloning and sequencing of IGF-I, IGF-II, ctsb and ctsd 178 6.4.2 Fasting-refeeding experiment 184 6.4.3 Gene expression 186 6.5 Discussion 190 Chapter 7 General discussion 195 7.1 Muscle growth 196 7.2 Flesh quality 201 7.3 Future perspectives 205 Chapter 8 References 208 xi List of figures Chapter 1 Page Figure 1.1 Evolutionary tree 6 Figure 1.2 Macro and micro myotomal structures 11 Figure 1.3 Collagen compartments in Atlantic halibut muscle 15 Figure 1.4 Collagen synthesis and structure 20 Figure 1.5 Muscle fibre types 22 Figure 1.6 Variation in slow muscle between fish species 23 Figure 1.7 Variation in slow muscle along the body axis 23 Figure 1.8 Stratified and mosaic hyperplasia 32 Figure 1.9 Myogenesis 33 Figure 1.10 The IGF system 42 Figure 1.11 Commercial halibut farming 46 Figure 1.12 Water binding to proteins 52 Chapter 2 Figure 2.1 Sampling site and fibre types 72 Figure 2.2 Fast fibre distribution and the number of sampled blocks 73 Figure 2.3 Relationship between body size and sampled blocks 75 Figure 2.4 Probability density function 76 Figure 2.5 Comparison of fast fibre distribution of equal sized males and females 78 Figure 2.6 Gompertz curves 80 Chapter 3 Figure 3.1.