SUSTAINABLE ALTERNATIVES TO FISH MEAL AND FISH OIL IN FISH NUTRITION: EFFECTS ON GROWTH, TISSUE FATTY ACID COMPOSITION AND LIPID METABOLISM THESIS SUBMITTED TO THE UNIVERSITY OF STIRLING FOR THE DEGREE OF DOCTOR OF PHILOSOPHY by VASILEIOS KARALAZOS Ptychio, M.Sc. INSTITUTE OF AQUACULTURE, UNIVERSITY OF STIRLING, STIRLING, SCOTLAND APRIL 2007 To my family ii Declaration I hereby declare that this thesis has been composed entirely by myself and has not been submitted for any other degree. Except where specifically acknowledged the work described in this thesis is the result of my own investigations. Vasileios Karalazos iii Abstract Traditionally, fish meal (FM) and fish oil (FO) have been used extensively in aquafeeds, mainly due to their excellent nutritional properties. However, various reasons dictate the use of sustainable alternatives and the reduction of the dependence on these commodities in fish feeds. Hence, the aim of the present thesis was to investigate the effects of the replacement of FM and FO with two vegetable oils (VO) and an oilseed meal on the growth performance, feed utilization, nutrient and fatty acids (FA) digestibility and tissue FA composition and metabolism in three commercially important European fish species. Specifically, in Experiment I crude palm oil (PO) was used to replace FO in diets for rainbow trout. In Experiments II and III FO was replaced with rapeseed oil (RO) in diets for Atlantic salmon at various dietary protein/lipid levels aiming also at further reductions of FM by using low protein (high lipid) diet formulations. In Experiments II and III the fish were reared at low and high water temperatures, respectively, in order to elucidate, also, the potential effects of temperature. Lastly, the effects of the replacement of FM with full fat soya meal (FFS) in Atlantic cod were investigated in Experiment IV. The results of the present thesis showed no negative effects on growth performance and feed utilization in rainbow trout when FO was replaced with PO. The dietary inclusion of RO improved the growth of Atlantic salmon, possibly, due to changes in the nutrient and FA digestibilities and FA catabolism while, the growth and feed utilization were unaffected by the dietary protein/lipid level. However, the growth of Atlantic cod was affected negatively by the replacement of FM with FFS. The proximate composition of the fish whole body was in most cases unaffected by dietary treatments. The changes in dietary formulations affected the dietary FA compositions and resulted in significant changes in the fish tissue FA compositions. It was clearly shown that the fish tissue total lipid FA composition reflects the FA composition of the diet, although specific FA were selectively utilized or retained in the tissues by the fish. These may have serious implications not only for fish metabolism and growth but also for the quality of the final product, especially in terms of possible reductions of n-3 HUFA. iv Acknowledgements I would like firstly to express my deep gratitude to my supervisor Dr. Gordon Bell for his continued guidance, advice, encouragement and support throughout this project. I would like also to thank my secondary supervisor Dr. Kim Jauncey for his assistance. I am thankful to the great people of the Nutrition Group of the Institute of Aquaculture, namely Dr. Douglas Tocher, Dr. Jim Henderson, James Dick, Fiona Strachan, Liz MacKinlay, Irene Younger, Allan Porter and Dr. Mike Bell and my colleagues Dr. Ioannis Karapanagiotidis, Dr. Jorge Fonseca Madrigal and Sean Kennedy for their help, advice and support, but above all for making me feel that I have a second “family” in Scotland. My studies, towards this PhD, were funded by a PhD Scholarship from the Greek Scholarship Foundation to whom I am indebted. I am also grateful to BioMar AS for generously supporting this project and special thanks should be given to Dr. Eldar Bendiksen, Dr. Patrick Campbell and Dr. Trine Galloway, from BioMar in Norway and Scotland, for their assistance. Moreover, I am thankful to all the partners of the “FFS in Cod” project, which was funded by DEFRA, and especially to Dr. Jim Treasurer. I would like also to thank the Fisheries Association of the British Isles for offering additional resources for conference attendance. Many thanks to the staff at the Gildeskål Research Station (GIFAS) and Fjord Research Station AS in Norway and the Scottish Association for Marine Science (SAMS) in Scotland for their excellent work in running the feeding trials of the present project and also to the colleagues and staff of the Institute of Aquaculture for their support during this work. I must sincerely thank my parents and my brother for their words of encouragement and fathomless support during my period of studies, with special thanks to my father for his valuable scientific advice. Last, but not least, I would like to thank all my friends for their support and encouragement, and of course all the wonderful people I met in Scotland for making my life here such a unique experience. v Abbreviations and Acronyms AA Amino Acid(s) ADC Apparent Digestibility Coefficient ANF antinutritional factors ANOVA Analysis of Variance ARA arachidonic acid (20:4n-6) conc. concentrated CSI Cardiosomatic Index DE Digestible Energy DHA docosahexaenoic acid (22:6n-3) DM dry matter DP Digestible Protein EAA Essential Amino Acid(s) EPA eicosapentaenoic acid (20:5n-3) FA fatty acid(s) FAME fatty acid methyl esters FCR Feed Conversion Ratio FFS full fat soy bean meal FM fish meal FO fish oil HSI Hepatosomatic Index HUFA Highly Unsaturated Fatty Acid(s) K Condition Factor NQC Norwegian quality cuts PCB Poly-chlorinated biphenyls PO crude palm oil POP Persistent Organic Pollutants PPV Protein Productive Value prot. protein PUFA Poly-Unsaturated Fatty Acid(s) RO rapeseed oil SBM soy bean meal SGR Specific Growth Rate TGC Thermal Growth Coefficient VO vegetable oil(s) VSI Viscerosomatic Index vi Table of Contents Declaration......................................................................................................................iii Abstract............................................................................................................................iv Acknowledgements...........................................................................................................v Abbreviations and Acronyms..........................................................................................vi Table of Contents............................................................................................................vii List of Tables..................................................................................................................xii List of Figures.................................................................................................................xv Chapter 1. General Introduction...................................................................................1 1.1 Lipids in fish nutrition...................................................................................2 1.1.1 General..................................................................................................2 1.1.2 Lipids and fatty acids – a brief overview..............................................2 1.1.3 Biosynthesis of fatty acids....................................................................4 1.1.4 β-oxidation of fatty acids and energy production.................................6 1.2 Overview of aquaculture in Europe and the world........................................7 1.2.1 General..................................................................................................7 1.2.2 Atlantic salmon.....................................................................................7 1.2.3 Rainbow trout.......................................................................................9 1.2.4 Atlantic cod.........................................................................................11 1.2.5 Current and future status of aquaculture.............................................13 1.3 Fish meal and fish oil in fish feeds..............................................................14 1.3.1 General................................................................................................14 1.3.2 Raw material sources and production.................................................14 1.3.3 Nutritional properties..........................................................................15 1.3.4 Organic contaminants and human nutrition and health issues............16 1.3.5 Current and future production, consumption and demand.................16 1.3.6 Replacement of fish meal and fish oil................................................22 1.4 Sustainable alternatives to fish meal and fish oil.........................................22 1.4.1 Substitution with plant alternatives.....................................................22 1.4.2 Challenges and limitations..................................................................23 1.4.2.1 Growth performance...............................................................23 1.4.2.2 Quality of the final product and implications for human nutrition..................................................................................24 vii 1.4.2.3 Antinutritional factors.............................................................25 1.4.3 Palm oil in rainbow trout diets............................................................25 1.4.4 Rapeseed oil in Atlantic salmon feeds................................................26 1.4.5 Soybean meal in Atlantic cod diets.....................................................28 1.4.6 Energy dense / High lipid diets...........................................................29 1.5 General objectives........................................................................................31 Chapter 2. General Materials and Methods...............................................................32 2.1 Materials......................................................................................................33 2.2 Experimental diets.......................................................................................33 2.3 Experimental animals and husbandry..........................................................34 2.4 Sampling procedures....................................................................................34 2.4.1 Feed sampling.....................................................................................34 2.4.2 Fish sampling......................................................................................34 2.5 Proximate analysis of diets, tissues and faeces............................................36 2.5.1 Moisture..............................................................................................36 2.5.2 Freeze drying......................................................................................36 2.5.3 Crude protein......................................................................................37 2.5.4 Acid hydrolysis...................................................................................38 2.5.5 Crude fat.............................................................................................38 2.5.6 Ash......................................................................................................39 2.5.7 Gross energy.......................................................................................39 2.5.8 Yttrium oxide (Y O )..........................................................................40 2 3 2.6 Lipid extraction and analysis.......................................................................40 2.6.1 Total lipid extraction of diets, tissues and faeces...............................40 2.6.2 Preparation and purification of fatty acid methyl esters.....................41 2.6.3 Separation and identification of fatty acid methyl esters....................42 2.7 β-oxidation capacity.....................................................................................43 2.8 Calculations..................................................................................................44 2.9 Statistics.......................................................................................................46 Chapter 3. Effects of dietary crude palm oil on rainbow trout (Oncorhynchus mykiss).........................................................................................................48 3.1 Introduction..................................................................................................49 3.2 Materials and methods.................................................................................51 3.2.1 Fish and facilities................................................................................51 viii 3.2.2 Experimental feeds and feeding..........................................................51 3.2.3 Sampling procedure............................................................................52 3.2.4 Proximate analysis..............................................................................52 3.2.5 Lipid extraction and fatty acid analyses.............................................53 3.2.6 Calculations and statistical analysis....................................................53 3.3 Results..........................................................................................................53 3.3.1 Diet proximate and fatty acid composition.........................................53 3.3.2 Growth performance...........................................................................54 3.3.3 Whole body proximate composition...................................................55 3.3.4 Tissue fatty acid compositions............................................................55 3.3.4.1 Muscle fatty acid composition................................................55 3.3.4.2 Liver fatty acid composition...................................................56 3.3.4.3 Gill fatty acid composition.....................................................57 3.3.4.4 Heart fatty acid composition...................................................58 3.3.4.5 Kidney fatty acid composition................................................58 3.4 Discussion....................................................................................................69 3.4.1 Growth performance...........................................................................69 3.4.2 Body composition...............................................................................70 3.4.3 Tissue fatty acid composition.............................................................71 3.5 Conclusions..................................................................................................74 Chapter 4. Dietary protein / lipid ratio and rapeseed oil interactions in Atlantic salmon – I.....................................................................................75 4.1 Introduction..................................................................................................76 4.2 Materials and methods.................................................................................78 4.2.1 Fish and culture conditions.................................................................78 4.2.2 Experimental diets..............................................................................78 4.2.3 Sampling procedure............................................................................79 4.2.4 Proximate analysis..............................................................................80 4.2.5 Lipid extraction and fatty acid analyses.............................................80 4.2.6 Calculations and statistical analysis....................................................81 4.3 Results..........................................................................................................81 4.3.1 Growth performance and feed efficiency...........................................81 4.3.2 Proximate composition of whole body...............................................82 4.3.3 Fatty acid composition of diets and tissues........................................82 4.4 Discussion....................................................................................................90 4.4.1 Growth performance...........................................................................90 ix 4.4.2 Whole body proximate composition...................................................91 4.4.3 Tissue fatty acid composition.............................................................93 4.5 Conclusions..................................................................................................95 Chapter 5. Dietary protein / lipid ratio and rapeseed oil interactions in Atlantic salmon – II...................................................................................96 5.1 Introduction..................................................................................................97 5.2 Materials and methods.................................................................................99 5.2.1 Fish and culture conditions.................................................................99 5.2.2 Experimental diets and feeding.........................................................100 5.2.3 Sampling procedure..........................................................................101 5.2.4 Proximate analysis............................................................................102 5.2.5 Lipid extraction and fatty acid analyses...........................................102 5.2.6 Peroxisomal β-oxidation capacity.....................................................102 5.2.7 Calculations and statistical analysis..................................................103 5.3 Results........................................................................................................103 5.3.1 Diet proximate and fatty acid composition.......................................103 5.3.2 Growth..............................................................................................104 5.3.3 Apparent nutrient digestibility..........................................................105 5.3.4 FA apparent digestibility..................................................................106 5.3.5 Whole body proximate composition.................................................108 5.3.6 Tissue fatty acid compositions..........................................................109 5.3.7 Peroxisomal β-oxidation capacity.....................................................110 5.4 Discussion..................................................................................................125 5.4.1 Growth performance.........................................................................125 5.4.2 Nutrient and fatty acid digestibility..................................................127 5.4.3 Whole body proximate composition.................................................130 5.4.4 Tissue fatty acid composition...........................................................131 5.4.5 Peroxisomal β-oxidation capacity.....................................................133 5.5 Conclusions................................................................................................135 Chapter 6. Effects of dietary full fat soy bean meal inclusion on Atlantic cod (Gadus morhua)........................................................................................136 6.1 Introduction................................................................................................137 6.2 Materials and methods...............................................................................140 6.2.1 Fish and culture conditions...............................................................140 6.2.2 Experimental diets and feeding.........................................................140 x