Paula Alexandra Morgado Canada Improving growth potential in Senegalese sole (Solea senegalensis) through dietary protein: An integrated approach using muscle cellularity, tracer studies and gene expression Tese de Candidatura ao grau de Doutor em Ciência Animal, Especialidade Nutrição, submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Orientador – Professora Doutora Luisa Maria Pinheiro Valente Categoria – Professora associada Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto Coorientador – Doutor Luís Eugénio Castanheira da Conceição Categoria – Director de projectos Afiliação – Sparos, Lda. Coorientador – Doutora Sofia Alexandra Dias Engrola Categoria – Investigadora auxiliar Afiliação – Centro de Ciências do Mar do Algarve This Thesis includes two scientific papers published in international journals originating from part of the results obtained in the experimental work referenced to as: Canada, P., Engrola, S., Richard, N., Lopes, A.F., Pinto, W., Valente, L.M.P., Conceição, L.E.C., 2016. Dietary indispensable amino acids profile affects protein utilization and growth of Senegalese sole larvae. Fish physiology and biochemistry. 42, 1493-1508. DOI: 10.1007/s10695-016-0235-1 Canada, P. Engrola, S., Mira, S., Teodósio, R., Fernandes, J.M.O., Sousa, V., Barriga- Negra, L., Conceição, L.E.C., Valente, L.M.P. 2016 The supplementation of a microdiet with crystalline indispensable amino-acids affects muscle growth and the expression pattern of related genes in Senegalese sole (Solea senegalensis) larvae. Aquaculture 458:158-169 DOI:10.1016/j.aquaculture.2016.03.010 Table of contents Acknowledgements i Summary iii Resumo vi List of abbreviations x Chapter 1 - General introduction 2 1.1 General aspects of Senegalese sole biology and production 2 1.2 Larval growth potential, the role of protein synthesis 4 1.3 Feeding fish larvae 7 1.3.1 Live feeds vs. inert feeds 7 1.3.2 Formulating Protein for fish larvae 8 1.3.2.1 Protein quality and indispensable amino acids requirements 8 1.3.2.2 Protein complexity and digestibility 11 1.4 Improving the utilization of dietary protein: tracers studies and the regulation of the digestive capacity 14 1.5 Somatic growth potential and muscle growth: muscle cellularity and the regulation of myogenesis 15 1.6 Dietary protein and Epigenetics 19 1.7 Objectives 21 1.8 References 23 Chapter 2 - Dietary indispensable amino acids profile affects protein utilisation and growth of Senegalese sole larvae 41 2.1 Introduction: 42 2.2 Material and Methods 45 2.2.1. Husbandry and experimental set-up 45 2.2.2 Feeding protocol 45 2.2.3 Feed manufacturing and quality analysis 46 2.2.4 Sampling and experimental design 49 2.2.4.1 Growth and larvae performance 49 2.2.4.2 Protein metabolism trials 50 2.2.5 Data analysis 51 2.3 Results 52 2.3.1 Diets 52 2.3.2 Growth 53 2.3.3 Protein metabolism 55 2.4 Discussion 57 2.5 Acknowledgements 61 2.6 References 63 Chapter 3 - The supplementation of a microdiet with crystalline indispensable amino-acids affects muscle growth and the expression pattern of related genes in Senegalese sole (Solea senegalensis) larvae 69 3.1 Introduction 71 3.2 Material and Methods 73 3.2.1 Experimental diets 73 3.2.2 Husbandry and experimental set-up 76 3.2.3 Larvae performance 77 3.2.4 Protein metabolism trials 78 3.2.5 Fast-twitch muscle cellularity 79 3.2.6 Gene expression 80 3.2.6.1 RNA extraction and cDNA synthesis 80 3.2.6.2 Quantitave real-time PCR (qPCR) 80 3.2.7 Data analysis 82 3.3 Results 82 3.3.1 Diets 82 3.3.2 Protein metabolism 83 3.3.3 Larval performance 84 3.3.4 Dietary effect on fast-twitch skeletal muscle growth 85 3.3.5 Expression of growth-related genes and DNA methyltransferases 87 Chapter 4 - Dietary protein complexity modulates growth, protein utilisation and the expression of protein digestion-related genes in Senegalese sole larvae 103 4.1 Introduction: 105 4.2. Material and Methods 107 4.2.1. Husbandry and experimental set-up 107 4.2.2. Experimental diets: manufacturing and quality analysis 109 4.2.3. Sampling and experimental design 111 4.2.3.1 Growth and larvae performance 111 4.2.3.2 Protein metabolism trials 112 4.2.3.3 Gene expression 113 4.2.3.3.1 RNA extraction and cDNA synthesis 113 4.2.3.3.2 Quantitave real-time PCR (qPCR) 113 4.2.4 Data analysis 114 4.3 Results 116 4.3.1 Larval performance 116 4.3.2 Protein metabolism 117 4.3.3 Expression of protein digestion-related genes 120 4.4. Discussion 121 4.5. Conclusion 128 4.6. Acknowledgements 128 4.7. References 129 Chapter 5 - Dietary protein complexity affects growth and the expression pattern of muscle growth related genes in Senegalese sole (Solea senegalensis) larvae 137 5.1 Introduction: 138 5.2. Material and Methods 141 5.2.1. Experimental diets 141 5.2.2. Husbandry and experimental set-up 144 5.2.3. Somatic growth and survival 145 5.2.4. Fast-twitch muscle cellularity 145 5.2.5 Gene expression 146 5.2.5.1 RNA extraction and cDNA synthesis 146 5.2.5.2 Quantitave real-time PCR (qPCR) 146 5.2.6 Data analysis 147 5.3 Results 149 5.3.1 Larval performance 149 5.3.2 Dietary effect on white skeletal muscle growth 150 5.3.3 Expression of growth-related genes and DNA methyltransferases 153 5.4 Discussion 155 5.4.1 Effect of dietary protein complexity on larval performance 155 5.4.2 Effect of dietary protein complexity on the regulation of muscle growth 156 5.4.3 Epigenetic effect 159 5.5 Conclusion 159 5.6 Acknowledgements 160 5.7 References 161 6. General discussion 168 6.1 Feed formulating strategies affect protein utilization and somatic growth 168 6.2 Formulation strategies affect early muscle growth and somatic growth potential 173 6.3 Formulation strategies may induce epigenetic effects 175 6.4. Conclusions and recommendations 177 6.5 References 179 Acknowledgements Firstly, I acknowledge Fundação para a Ciência e a Tecnologia (FCT) for the financial support through the PhD grant SFRH/BD/82149/2011 and through the projects EPISOLE (PTDC/MAR/110547/2009) and CCMAR/Multi/04326/2013 (Portugal). I acknowledge POAlgarve 21, QREN and European Union, for additional financial support, through the project MICALA — I&DT Co-Promoção No. 13380 (supported by POAlgarve 21, QREN and European Union). I would like to deeply acknowledge the invaluable contribution of all the people that contributed for the successful completion of this Thesis. First of all, I am mostly grateful for having the chance to work with my supervisors Professora Luisa Valente, Doutor Luís Conceição and Doutora Sofia Engrola. Firstly I should thank Professora Luisa Valente for giving me this opportunity. Professora Luisa was the one who first challenged me to work in this project and apply to a PhD grant, almost in a leap of faith, regarding my background. I thank all the knowledge she shared with me, all the support and motivation from the first to the last day, for always being so helpful in pretty much anything. I want to sincerely thank Doutor Luís Conceição for all his guidance and great support. Luís Conceição has an immense scientific knowledge, which demanded me for studying and learning a lot. In every discussion, I always had the chance to, not only learn a lot, but also think at higher level. Luís was crucial in setting up all the workplan, from the experimental design to the analysis of the results. Last but not the least, I want to acknowledge Doutora Sofia Engrola for setting up the experimental design, based on all the knowledge and experience that she has on larval rearing and nutrition. I want to thank Sofia for providing me the conditions to run the trials, for hosting me in Aquagroup for 4 years, for all her technical support and helping me analyzing my results on a daily basis. Most of all, I want to thank Sofia for her support and good friendship. I want to particularly thank Professor Jorge Fernandes. His contribution from the experimental design, supervision of molecular analysis to the analysis of the results and manuscript revision was invaluable. Furthermore, I am extremely thankful for his unreserved support, help and kindness. I want to thank my friends and colleagues from Aquagroup/CCMar, for their invaluable help. In particular, I want to thank Sara Ferreira, for performing the HPLC analysis, for being my favourite trouble shooter, always ready to help, for all the stimulating conversations that we i had. I also want to thank Ana Filipa Lopes and Carmen Barrios, for helping me perform the growth trials – we learnt how to make little soles together. More than to anyone, I want to thank Rita Teodósio, most of all for her support and friendship for all this time. As a research fellowship holder within the EPISOLE project her help was invaluable in pretty much everything. Particularly I want to thank her for introducing me to molecular biology techniques, together with Doutora Sara Mira, to whom I also thank for hosting me in the EDGE lab/CCMar. I want to thank Nadège Richard for her friendship, technical and moral support, and for helping me analyse this PhD achievements at the necessary distance and in a positive way. I want to thank SPAROS Lda, in particular the collaboration of Wilson Pinto and André Santos. At the colleagues from Lanuce/CIIMAR, I specially thank Vera Sousa for performing the diets proximal analysis, performing the histological cuts and helping me with the histological analysis, but most of all, for always being so helpful and friendly.