Nutritional Condition and Quality Assessment of the American Lobster, Homarus Americanus, at Different Stages of the Moult Cycle
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UNIVERSITY OF PRINCE EDWARD ISLAND Nutritional condition and quality assessment of the American lobster, Homarus americanus, at different stages of the moult cycle A Thesis Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of Master of Science In the Department of Pathology and Microbiology Faculty of Veterinary Medicine University of Prince Edward Island Michael A. Ciaramella Charlottetown, P. E. I. August 2011 © 2011 M. Ciaramella i Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington OttawaONK1A0N4 OttawaONK1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-82259-3 Our file Notre reference ISBN: 978-0-494-82259-3 NOTICE: AVIS: The author has granted a non L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. 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Copying or publication or any other use of the thesis for financial gain without approval by the University of Prince Edward Island and the author's written permission is prohibited. Requests for permission to copy or to make any other use of material in this thesis in whole or in part should be addressed to: Chair of the Department of Pathology and Microbiology Faculty of Veterinary Medicine University of Prince Edward Island Charlottetown, PE Canada CIA 4P3 ii SIGNATURE PAGE Gt'Q^OvJ REMOVED ACKNOWLEDGEMENTS I would like to thank my supervisors Dr. Andrea Battison and Dr. Barbara Homey for the help and support they have provided me with over the past two years. I would additionally like to thank the members of my supervisory committee Dr. Fred Kibenge, Dr. Jean Lavallee and Dr. Dave Speare for their guidance in conducting my MSc research and the thesis writing. An additional thanks to all those who have helped along the way including everyone at the Lobster Science Centre and Atlantic Veterinary College for being there to support me through my program. I gratefully acknowledge the Atlantic Veterinary College Lobster Science Centre, Innovation PEI, the Department of Pathology and Microbiology, and NSERC the financial support received to conduct my MSc research and present it around the world. Finally I would like to thank all the friends I have made along the way, my fellow graduate students at the University, and my family for all their support! v Table of Contents 1. General Introduction 1 1.1. The American lobster, Homarus americanus 1 1.1.1. General Background 1 1.1.2. Growth 2 1.1.3. Quality 4 1.1.4. Economic Importance 5 1.2. Moult Staging 6 1.2.1. Drach Staging 6 1.2.1. Pleopod Staging 8 1.3. Energy Reserves 8 1.4. Hemolymph Biochemistry 12 1.4.1. Circulating Metabolites 12 1.4.2. Hemolymph Osmolality 14 1.5. Research Objectives 15 1.6. References 17 2. Practical Optimisation of the Dataset 23 2.1. Abstract 23 2.2. Introduction 23 2.3. Materials and Methods 26 2.3.1. Population Monitoring for Optimisation of Sample Collection 26 2.3.2. Collection of Lobster Samples 26 2.3.3. Laboratory Processing of Lobsters Collected 30 2.3.4. Statistical Analyses of sampling data 31 2.4. Results 31 2.4.1. Population Monitoring for Optimisation of Sample Collection 31 2.4.2. Collection of Lobster Samples 34 2.4.3. Laboratory Processing of Lobsters Collected 35 2.5. Discussion 36 2.6. References 40 VI 3. Evaluation of Fluctuations in Tissue Water Content of the American Lobster Homarus americanus during the Moult Cycle 43 3.1. Abstract 43 3.2. Introduction 43 3.3. Materials and Methods 45 3.3.1. Homogenisation of Lobster Tissues 45 3.3.2. Lyophilisation of Lobster Tissues 46 3.3.3. Calculation of Percent Water in Tissue Samples 46 3.3.4. Statistical Analysis 47 3.4. Results 47 3.4.1. Lyophilisation Study 47 3.4.2. Analysis of Tissue Water Content Data 48 3.4.3. Statistical Analysis 49 3.5. Discussion 49 3.6. References 53 4. Fluctuations in Glycogen Stores of the Hepatopancreas and Muscle Tissues of the American Lobster Homarus americanus during the Moult Cycle 55 4.1. Abstract 55 4.2. Introduction 56 4.3. Materials and Methods 61 4.3.1. Optimisation of Tissue and Oyster Glycogen Standard Digestion 61 4.3.2. Evaluation of the Glucose Oxidase Method for Glucose Determination in Lobster Tissues 62 4.3.3. Evaluation of the Hexokinase Method for Glucose Determination in Lobster Tissues 63 4.3.4. Calculation of Oyster Glycogen Equivalents 63 4.3.5. Validation of Glucose Determination Using the Hexokinase Assay 64 4.3.6. Assessment of Background Glucose in Lobster Tissues 65 4.3.7. Relative Distribution of Tissue Glycogen at Different Stages of the Moult Cycle 66 4.3.8. Statistical Analysis of Glycogen Data 67 4.4. Results 67 4.4.1. Optimisation of Tissue and Standard Glycogen Digestion 67 VII 4.4.2. Evaluation of the Glucose Oxidase Method for Glucose Determination in Lobster Tissues 68 4.4.3. Evaluation of the Hexokinase Method for Glucose Determination in Lobster Tissues 69 4.4.4. Validation of Glucose Determination Using the Hexokinase Assay 70 4.4.5. Assessment of Background Glucose in Lobster Tissues 73 4.4.6. Relative Distribution of Tissue Glycogen at Different Stages of the Moult Cycle 74 4.5. Discussion 83 4.6. References 97 5. Fluctuations in Stored Lipid of the Hepatopancreas and Muscle Tissues of the American Lobster Homarus americanus during the Moult Cycle 102 5.1. Abstract 102 5.2. Introduction 102 5.3. Materials and Methods 105 5.3.1. Comparison of Different Extraction Methods for the Determination of Lipid Concentrations in Lobster Tissues 105 5.3.2. Precision of the Folch Centrifugation Method for Lipid Extraction 107 5.3.3. Long Term Stability of Hepatopancreas Tissue Samples Stored at -80°C 107 5.3.4. Assessment of Lipid Concentrations in Lobster Tissues Representing Different Moult Categories 108 5.3.5. Statistical Analysis of Lipid Data 108 5.4. Results 109 5.4.1. Comparison of Different Extraction Methods for the Determination of Lipid Concentrations in Lobster Tissues 109 5.4.2. Precision of the Folch Centrifugation Method for Lipid Extraction 110 5.4.3. Long Term Stability of Hepatopancreas Tissue Samples Stored at -80°C Ill 5.4.4. Assessment of Lipid Concentrations in Lobster Tissues Representing Different Moult Categories Ill 5.5. Discussion 119 5.6. References 127 6. Biochemical Analysis of Hemolymph Metabolites during the Moult Cycle in the American Lobster, Homarus americanus 130 6.1. Abstract 130 VIII 6.2. Introduction 131 6.3. Materials and Methods 136 6.3.1. Biochemical Analysis of Hemolymph Metabolites and Osmolality During the Moult 136 6.3.2. Statistical Analysis of Hemolymph Biochemistry and Osmolality Data 136 6.4. Results 137 6.4.1. Evaluation of Hemolymph Biochemical Parameters for the Primary 2009 Moult Cohort 137 6.4.2. Evaluation of Hemolymph Biochemical Parameters for the 2009 and 2010 Moult Cohorts 137 6.4.3. Evaluation of Year Effects on Hemolymph Biochemical Parameters 143 6.4.4. Correlations between the Hemolymph Biochemical Parameters 144 6.5. Discussion 145 6.6. References 152 7. Correlations among the Changes in Physiology and Biochemistry of the Tissues and Hemolymph in the American Lobster, Homarus americanus, during the Moult Cycle 156 7.1. Introduction 156 7.2. Materials and Methods 157 7.3. Results 158 7.3.1. Relative Concentrations of Water, Lipid and Glycogen in the Hepatopancreas across the Five Moult Categories 158 7.3.2.