M MASTER’S DEGREE IN MARINE SCIENCES - MARINE RESOURCES MARINE - SCIENCES MARINE IN DEGREE MASTER’S ECOLOGY AND BIOLOGY MARINE IN SPECIALIZATION Characterization of brown trout fitness fitness trout brown of Characterization along the maturation and gonadal cycle reproductive Diana Santiago dos Santos 2018 Diana Santiago dos Santos. Characterization of brown trout M.ICBAS 2018 fitness and gonadal maturation along the reproductive cycle Characterization of brown trout fitness and gonadal maturation along the reproductive cycle Diana Santiago dos Santos INSTITUTE OF BIOMEDICAL SCIENCES ABEL SALAZAR MASTER’S DEGREE IN MARINE SCIENCES – MARINE RESOURCES SPECIALIZATION IN MARINE BIOLOGY AND ECOLOGY Characterization of brown trout fitness and gonadal maturation along the reproductive cycle. Diana Santiago dos Santos M 2018 Diana Santiago dos Santos Characterization of brown trout fitness and gonadal maturation along the reproductive cycle Dissertation application to the master’s degree in Marine Sciences - Marine Resources, submitted to the Institute of Biomedical Sciences Abel Salazar, University of Porto Supervisor – Tânia Vieira Madureira Category – Auxiliary Researcher and Invited Auxiliary Professor Affiliation – Interdisciplinary Centre of Marine and Environmental Research and Institute of Biomedical Sciences Abel Salazar, University of Porto Co-supervisor – Eduardo Jorge Sousa da Rocha Category – Full Professor Affiliation – Institute of Biomedical Sciences Abel Salazar, University of Porto Acknowledgements I would like to thank everyone who, with words or gestures, helped me through this incredible experience. The empathy and friendship I felt were overwhelming and kept me going through the toughest times. To Professor Doctor Tânia Madureira, my supervisor, I cannot thank enough for all the encouragement and dedication. I feel eternally grateful for your guidance and for continuously trying to help me improve myself. To Professor Doctor Eduardo Rocha, my co-supervisor, I’m very thankful for your outstanding leadership and integrity, you are an inspiration to me. Thank you so much for the enthusiasm and for the time dedicated to my dissertation. Thank you so much, Célia Lopes and Fernanda Malhão, you have no idea how much your endless help meant to me. I am very thankful for all the kindness and generosity you have shown me over the past year, it has been an invaluable gift. To all the staff and students of the laboratory of Histology and Embryology at ICBAS, I thank you for kindly receiving me and providing me with a supportive environment. To all my friends, for entertaining me in the most stressful moments. I thank you for all the care and affection you’ve given me, you have made the good times better and the hard times a lot easier. To Telmo Silva, I want to thank you for all the wonderful ways you make me smile, for all the love and for always trying to understand me. It has been a very challenging year and knowing that I have you by my side made all the difference. To my family, I thank you for the unconditional love and affection. I also thank you for trusting me in my decisions and always supporting me. To my sister, I know I can always count on you in the most difficult times and for that, I’ll always be grateful. Thanks for looking out for me, sis. To my mother, I will never stop being grateful to you for how amazing you have been and still are as a parent to me. You are the best doctor, the best teacher and the best listener I could ever ask for. You have helped me in every away during this transition. To my parents, my eternal gratitude, for all the sacrifices, the difficulties and the efforts to get me where I am today. I will forever hold you close to my heart. iii This investigation was supported by ICBAS, the FCT – Foundation for Science and Technology, via the Strategic Funding project UID/Multi/04423/2013, the European Regional Development Fund (ERDF) via COMPETE (INNOVMAR, ECOSERVICES NORTE-01-0145-FEDER-000035) and ICNF. A special thanks for the opportunity given to me through this funding responsible entities. iv Table of Contents Acknowledgements ................................................................................................ iii Resumo ................................................................................................................. vii Abstract .................................................................................................................. ix List of Abbreviations ............................................................................................... xi List of Figures ........................................................................................................ xv List of Tables ........................................................................................................ xix I. INTRODUCTION ............................................................................................. 1 1.1. Reproduction and Gonad Types in Teleost Fish .............................................3 1.1.1. Reproductive strategies ...........................................................................................3 1.1.2. Description of gonad structure .................................................................................4 1.1.3. Spermatogenesis ....................................................................................................7 1.1.4. Oogenesis............................................................................................................. 10 1.2. Endocrinology of Teleost Reproduction ........................................................12 1.2.1. Primary mediators and secondary hormones ......................................................... 12 1.3. Reproductive Seasonal Patterns in Salmonids .............................................14 1.4. The Brown Trout ..............................................................................................15 1.5. Background and Aims of the Study ...............................................................17 II. MATERIALS AND METHODS ....................................................................... 19 2.1. Study Animals ..................................................................................................21 2.2. Physico-Chemical Parameters of Water .........................................................21 2.3. Sampling Procedures ......................................................................................21 2.4. Biochemical Analyses of Blood, Plasma and Serum ....................................22 2.5. Hormone Plasmatic Levels .............................................................................23 2.6. Histological Processes of the Gonads ...........................................................23 2.6.1. Paraffin procedures ............................................................................................... 23 2.6.2. Methacrylate procedures ....................................................................................... 24 2.7. Grading of Gonadal Maturation ......................................................................24 2.8. Immunohistochemistry of the Gonads ...........................................................24 v 2.9. Data Analysis and Statistics ...........................................................................25 III. RESULTS ................................................................................................... 27 3.1. Physico-Chemical Parameters of Water .........................................................29 3.2. Biometric Measurements ................................................................................29 3.3. Biochemical Analyses .....................................................................................32 3.4. Quantification of Plasma Steroid Hormones .................................................34 3.5. Histological Criteria Established for the Classification of Adult Brown Trout Gonadal Maturation Stages .......................................................................................35 3.6. Grading Stages of Gonadal Maturation in Adult Brown Trout ......................39 3.7. Immunolocalization of CYP17-I, CYP19 and 17β-HSD in Adult Brown Trout Gonads ........................................................................................................................40 3.7.1. Testis .................................................................................................................... 40 3.7.2. Ovaries ................................................................................................................. 44 IV. DISCUSSION ............................................................................................. 49 V. CONCLUSION ............................................................................................ 59 VI. REFERENCES ........................................................................................... 63 VII. APPENDIXES............................................................................................. 85 Appendix I – Histological processing protocols ......................................................87 Appendix II – Hematoxylin-eosin staining (H&E) protocol for paraffin sections ...88 Appendix III – Technovit 7100 adapted protocol ......................................................89 Appendix IV – Sirius red staining protocol for methacrylate sections (Kiernan)...90 Appendix V – Reticulin staining protocol for methacrylate sections