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Use of alternative feed ingredients and the effects on growth and flesh quality of Atlantic salmon (Salmo… Friesen, Erin 2008

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Use of alternative feed ingredients and the effects on growth and flesh quality of Atlantic salmon (Salmo salar) and sablefish (Anoplopoma fimbria).  by Erin Friesen B.Sc., Simon Fraser University, 2003  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Food Science)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) October 2008 © Erin Friesen, 2008  ii  Abstract Aquaculture feeds, traditionally composed mainly of fishmeal and fish oil, currently represent the largest cost to fish farmers. With aquaculture growing at an average of 8.8% per year and limited supply of fishmeal and fish oil, suitable alternatives must be found. In addition to increasing sustainability and lowering production costs, the use of plant and/or animal ingredients has the potential to lower flesh levels of persistent organic pollutants (POPs) such as polychlorinated biphenyls. Fish oil and to a lesser extent fishmeal, are considered to be the largest source POPs in farmed fish. Using alternative feed ingredients however, can compromise fish growth and the flesh quality of the final product. Lipid sources including flaxseed oil, canola oil, poultry fat and the protein sources canola protein concentrate, soy protein concentrate and poultry by-product meal were examined as alternatives to fish oil and fishmeal in one on-farm field study and one laboratory feeding trial with Atlantic salmon (Salmon salar) and two laboratory feeding trials conducted on sablefish (Anoplopoma fimbria), a relatively new marine aquaculture species. The nutritive value of the alternative ingredients was assessed on the basis of fish growth performance, proximate composition, fatty acid composition and apparent digestibility coefficients. Sensory attributes were evaluated in the sablefish studies while flesh POP levels were determined in both species. The use of alternative dietary lipids showed no negative effects on fish performance. However replacement of fishmeal with plant proteins in some cases, negatively affected fish growth. Flesh levels of persistent organic pollutants were significantly decreased (p<0.05) with the use of alternative dietary lipids, and flesh levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were also depressed. Activated carbon treated anchovy oil and finishing diets were examined in the Atlantic salmon laboratory feeding trial and were effective at lowering flesh POP levels while providing high levels of EPA and DHA. The use of alternative feed ingredients will soon be inevitable in aquaculture feeds. The current research shows alternative lipids and proteins can be incorporated successfully in sablefish and Atlantic salmon feeds with minimal effects on fish growth and quality.  iii  Table of Contents Abstract.......................................................................................................................................... ii Table of Contents ......................................................................................................................... iii List of Tables ................................................................................................................................ ix List of Figures............................................................................................................................. xiv List of Abbreviations ................................................................................................................ xvii Acknowledgments ....................................................................................................................... xx Co-Authorship Statement ......................................................................................................... xxi 1  Introduction....................................................................................................................... 1 1.1 RESEARCH AIMS ................................................................................................................... 3 1.2 LITERATURE REVIEW ........................................................................................................... 5 1.2.1 Aquaculture............................................................................................................... 5 1.2.1.1 Species ............................................................................................................. 5 1.2.2 Nutritional requirements ........................................................................................... 6 1.2.2.1 Energy .............................................................................................................. 7 1.2.2.1.1 Determination of apparent digestibility coefficients............................... 7 1.2.2.2 Proteins ............................................................................................................ 9 1.2.2.3 Lipids ............................................................................................................... 9 1.2.2.3.1 Elongation and desaturation of fatty acids............................................ 10 1.2.2.4 Micronutrients................................................................................................ 12 1.2.3 Feed ingredients ...................................................................................................... 12 1.2.3.1 Fishmeal supply ............................................................................................. 13 1.2.3.2 Fish oil supply................................................................................................ 13 1.2.3.3 Alternatives to fish oil and fishmeal .............................................................. 14 1.2.3.4 Costs............................................................................................................... 14 1.2.4 Vegetable and animal feed ingredients ................................................................... 15 1.2.4.1 Protein content and amino acid composition ................................................. 15 1.2.4.2 Fatty acid composition................................................................................... 16 1.2.4.3 Vitamins and minerals ................................................................................... 17 1.2.4.4 Carbohydrates ................................................................................................ 17 1.2.4.5 Fiber ............................................................................................................... 18 1.2.4.6 Anti-nutrient factors....................................................................................... 18 1.2.4.6.1 Trypsin inhibitors.................................................................................. 19 1.2.4.6.2 Phytic acid............................................................................................. 19 1.2.4.6.3 Hemagglutinins ..................................................................................... 19 1.2.4.6.4 Glucosinolates....................................................................................... 20 1.2.4.6.5 Saponins................................................................................................ 20 1.2.5 Effects of alternative dietary feed ingredients in fish feed ..................................... 20 1.2.5.1 Growth ........................................................................................................... 20 1.2.5.2 Flesh quality................................................................................................... 21 1.2.5.2.1 Fatty acids ............................................................................................. 21 1.2.5.2.2 Finishing diet ........................................................................................ 23 1.2.5.3 Aroma, flavour, texture and colour................................................................ 24 1.2.5.3.1 Sensory panel ........................................................................................ 24  iv 1.2.5.3.2 Colour ................................................................................................... 25 1.2.5.3.3 Texture .................................................................................................. 25 1.2.5.4 Effects of alternative lipids on sensory attributes .......................................... 26 1.2.5.5 Effects of alternative proteins on sensory attributes ...................................... 26 1.2.6 Persistent organic pollutants ................................................................................... 27 1.2.6.1 Polychlorinated dibenzodioxin/furans (PCDD/F).......................................... 27 1.2.6.2 Polychlorinated biphenyls (PCB) .................................................................. 28 1.2.6.3 Polybrominated diphenyl ethers (PBDE)....................................................... 28 1.2.6.4 Chemical structure ......................................................................................... 29 1.2.6.4.1 Congeners ............................................................................................. 29 1.2.6.5 Toxic effects................................................................................................... 29 1.2.6.5.1 Toxic equivalency factors (TEFs)......................................................... 30 1.2.6.5.2 Tolerable intakes................................................................................... 31 1.2.6.5.3 Human intake ........................................................................................ 31 1.2.7 Persistent organic pollutants in fish and feed ......................................................... 32 1.2.7.1 Bioconcentration, biomagnification, bioaccumulation .................................. 32 1.2.7.2 Levels of persistent organic pollutants in fish ............................................... 33 1.2.7.3 Organic contaminants in processed ingredients............................................. 34 1.2.8 Lowering persistent organic pollutants in farmed fish ........................................... 35 1.2.8.1 Use of alternative feed ingredients ................................................................ 35 1.2.8.2 Dietary protein to lipid ratio .......................................................................... 37 1.2.8.3 Contaminant-reduced fish oil and algae oil ................................................... 37 1.2.8.4 Handling and cooking techniques.................................................................. 38 1.2.9 Contaminants vs. fatty acids ................................................................................... 39 1.2.9.1 Acceptable persistent organic pollutant intake levels .................................... 39 1.2.9.2 Risks versus benefits...................................................................................... 40 1.2.9.3 Fatty acid and contaminant dynamics within the fish.................................... 41 1.3 FIGURES ............................................................................................................................. 43 1.4 REFERENCES ....................................................................................................................... 45 2 Evaluation of cold pressed flaxseed oil as an alternative dietary lipid source for juvenile sablefish ......................................................................................................................... 60 2.1 INTRODUCTION ................................................................................................................... 60 2.2 MATERIALS AND METHODS ................................................................................................ 62 2.2.1 Fish husbandry ........................................................................................................ 62 2.2.2 Experimental diets and feeding protocol ................................................................ 63 2.2.3 Fish handling........................................................................................................... 64 2.2.4 Sampling ................................................................................................................. 64 2.2.5 Chemical analysis ................................................................................................... 65 2.2.6 Data and statistical analyses.................................................................................... 66 2.3 RESULTS ............................................................................................................................. 67 2.3.1 Diet composition..................................................................................................... 67 2.3.2 Influence of dietary treatment on fish performance, diet digestibility and fish and fillet composition .............................................................................................................. 67 2.3.3 Influence of dietary treatment on terminal fillet fatty acid compositions............... 68 2.4 DISCUSSION ........................................................................................................................ 68 2.4.1. Influence of dietary treatment on fish performance................................................ 68  v 2.5 2.6 2.7 2.8  2.4.2 Influence of dietary treatment on fillet fatty acid compositions ............................. 71 CONCLUSIONS .................................................................................................................... 72 TABLES ............................................................................................................................... 73 FIGURES ............................................................................................................................. 79 REFERENCES ....................................................................................................................... 80  3 Evaluation of poultry fat and blends of poultry fat with cold pressed flaxseed oil as alternative dietary lipid sources for juvenile sablefish............................................................ 84 3.1 INTRODUCTION ................................................................................................................... 84 3.2 MATERIALS AND METHODS ................................................................................................ 85 3.2.1 Source and husbandry of experimental fish............................................................ 85 3.2.2 Experimental diets and feeding protocol ................................................................ 85 3.2.3 Fish handling........................................................................................................... 87 3.2.4 Sampling ................................................................................................................. 87 3.2.5 Chemical analysis ................................................................................................... 88 3.2.6 Energy and protein digestibility.............................................................................. 89 3.2.7 Data and statistical analyses.................................................................................... 89 3.3 RESULTS ............................................................................................................................. 90 3.3.1 Diet composition..................................................................................................... 90 3.3.2 Effect of dietary treatment on fish performance and whole body and fillet composition....................................................................................................................... 91 3.3.3 Influence of dietary treatment on terminal fillet fatty acid compositions............... 92 3.4 DISCUSSION ........................................................................................................................ 93 3.4.1 Sablefish growth ...................................................................................................... 93 3.4.2 Economic incentive of poultry fat............................................................................ 95 3.4.3 Fatty acid composition............................................................................................. 96 3.4.4 Proximate composition and digestibility ................................................................. 97 3.4.5 Changes in fatty acid composition and implications for human health ................... 98 3.5 CONCLUSIONS .................................................................................................................... 99 3.6 TABLES ............................................................................................................................. 101 3.7 FIGURES ........................................................................................................................... 107 3.8 REFERENCES ..................................................................................................................... 110 4 Effect of dietary lipid source on the sensory quality of juvenile sablefish (Anoplopoma fimbria)................................................................................................................ 113 4.1 INTRODUCTION ................................................................................................................. 113 4.2 MATERIALS AND METHODS .............................................................................................. 114 4.2.1 Feeding trial .......................................................................................................... 114 4.2.2 Dietary treatments................................................................................................. 115 4.2.3 Sample collection and storage .............................................................................. 115 4.2.4 Panel selection ...................................................................................................... 116 4.2.5 Training of Panel................................................................................................... 116 4.2.6 Sensory ballot........................................................................................................ 117 4.2.7 Sample preparation ............................................................................................... 118 4.2.8 Sample replications............................................................................................... 118 4.2.8.1 Trial 1............................................................................................................ 119  vi 4.2.8.2 Trial 2............................................................................................................ 119 4.2.9 Sensory sessions.................................................................................................... 119 4.2.10 Foreign flavour and aroma standards.................................................................. 120 4.2.11 Colour evaluation................................................................................................ 120 4.2.12 Statistical analysis............................................................................................... 121 4.3 RESULTS ........................................................................................................................... 121 4.3.1 Sensory analysis - Effects of dietary flaxseed oil ................................................. 121 4.3.2 Sensory analysis - Effects of dietary flaxseed oil and/or poultry fat .................... 123 4.3.3 Colour results ........................................................................................................ 125 4.4 DISCUSSION ...................................................................................................................... 125 4.4.1 Cooking method.................................................................................................... 125 4.4.2 Comparison between two sensory trials ................................................................ 125 4.4.3 High fishiness in 50AO:50FO treatment .............................................................. 126 4.4.4 Comparison to other sensory studies .................................................................... 127 4.5 CONCLUSIONS .................................................................................................................. 128 4.6 TABLES ............................................................................................................................. 129 4.7 FIGURES ............................................................................................................................ 136 4.8 REFERENCES ..................................................................................................................... 139 5 Replacement of supplemental dietary anchovy oil with flaxseed oil lowers flesh contaminant levels in cultured sablefish ................................................................................. 141 5.1 INTRODUCTION ................................................................................................................. 141 5.2 METHODS ......................................................................................................................... 143 5.2.1 Fish husbandry and dietary treatments ................................................................. 143 5.2.2 Sample collection.................................................................................................. 144 5.2.3 Proximate analysis and fatty acid composition..................................................... 144 5.2.4 Contaminant analysis ............................................................................................ 145 5.2.5 Statistical analysis................................................................................................. 145 5.3 RESULTS ........................................................................................................................... 146 5.3.1 Persistent organic pollutants in oils and diets ....................................................... 146 5.3.2 Persistent organic pollutants in sablefish muscles ................................................ 147 5.3.3 Accumulation efficiency....................................................................................... 148 5.4 DISCUSSION ....................................................................................................................... 149 5.4.1 Persistent organic pollutants in dietary treatments ............................................... 149 5.4.2 Reduction in flesh persistent organic pollutants ................................................... 150 5.4.3 Persistent organic pollutants in wild sablefish..................................................... 151 5.4.4 Changes in fatty acid composition........................................................................ 152 5.5 CONCLUSIONS .................................................................................................................. 153 5.8 FIGURES ........................................................................................................................... 156 5.7 REFERENCES ..................................................................................................................... 158 6 Use of alternative dietary lipid and protein sources in Atlantic salmon (Salmo salar): Effects on growth, diet digestibility, and flesh quality .......................................................... 162 6.1 INTRODUCTION ................................................................................................................. 162 6.2 MATERIALS AND METHODS .............................................................................................. 164  vii 6.2.1 Design of dietary treatments ................................................................................. 164 6.2.2 Mixing of diets...................................................................................................... 166 6.2.3 Pelleting ................................................................................................................ 166 6.2.4 Lipid sources and lipid coating of pellets ............................................................. 167 6.2.5 Digestibility diets .................................................................................................. 168 6.2.6 Experimental fish and husbandry procedures ....................................................... 168 6.2.7 Short feeding trial ................................................................................................. 170 6.2.8 Collection of samples............................................................................................ 170 6.2.9 Chemical analyses................................................................................................. 172 6.2.9.1 Proximate analysis ....................................................................................... 172 6.2.9.2 Fatty acid determinations............................................................................. 172 6.2.9.3 Digestibility.................................................................................................. 174 6.2.9.4 Contaminant analysis ................................................................................... 174 6.2.9.4.1 Sample preparation ............................................................................. 174 6.2.9.4.2 Chemicals............................................................................................ 175 6.2.9.4.3 Extraction and analysis ....................................................................... 175 6.2.10 Data calculation and statistical analyses ............................................................. 176 6.3 RESULTS ........................................................................................................................... 178 6.3.1 Ingredients.............................................................................................................. 178 6.3.1.1 Fatty acid compositions of lipid sources...................................................... 178 6.3.1.2 Contaminant concentrations in the protein and lipid sources ...................... 178 6.3.1.2.1 Polychlorinated biphenyls (PCB) ....................................................... 178 6.3.1.2.2 Polychlorinated dibenzo-dioxin/furans (PCDD/F) ............................. 179 6.3.1.2.3 Toxic equivalents (TEQ)..................................................................... 179 6.3.1.2.4 Polybrominated biphenyl ethers (PBDE)........................................... 179 6.3.1.3 Concentrations of total and digestible nutrients and energy........................ 179 6.3.1.4 Fatty acid compositions ............................................................................... 180 6.3.1.5 Contaminant concentrations......................................................................... 181 6.3.1.5.1 Polychlorinated biphenyls................................................................... 181 6.3.1.5.2 Polychlorinated dibenzo-dioxin/furans............................................... 181 6.3.1.5.3 Toxic equivalents ................................................................................ 181 6.3.1.5.4 Polybrominated biphenyl ethers ........................................................ 182 6.3.2 Growth performance and whole body and fillet proximate compositions............ 182 6.3.2.1 Phase 1 ......................................................................................................... 182 6.3.2.2 Phase 2 ......................................................................................................... 182 6.3.3 Short term feeding trial ......................................................................................... 184 6.3.4 Fatty acid composition of the fish fillets............................................................... 184 6.3.4.1 Phase 1 ......................................................................................................... 184 6.3.4.2 Phase 2 ......................................................................................................... 185 6.3.5 Flesh contaminant concentrations......................................................................... 186 6.3.5.1 Phase 1 ......................................................................................................... 186 6.3.5.1.1 PCBs, PCDDs, PCDFs........................................................................ 186 6.3.5.1.2 Toxic Equivalents ............................................................................... 187 6.3.5.1.3 PBDEs................................................................................................. 187 6.3.5.1.4 Bioaccumulation ................................................................................. 187 6.3.5.2 Phase 2 ......................................................................................................... 188 6.3.5.2.1 PCBs, PCDDs, PCDFs........................................................................ 188 6.3.5.2.2 Toxic equivalents ................................................................................ 188 6.3.5.2.3 PBDEs................................................................................................. 188  viii  6.4  6.5 6.6 6.7 6.8  6.3.6 Reduction and re-establishment of contaminants ................................................. 189 6.3.7 Retention efficiencies............................................................................................ 189 DISCUSSION ...................................................................................................................... 190 6.4.1 Influence of dietary treatment on fish performance.............................................. 190 6.4.1.1 Activated carbon treatment .......................................................................... 191 6.4.1.2 Alternative Protein sources .......................................................................... 192 6.4.2 Influence of dietary treatment on flesh fatty acid composition ............................ 194 6.4.3 Influence of finishing diets on flesh fatty acid composition................................. 195 6.4.4 Influence of dietary treatment on flesh contaminant levels .................................. 196 6.4.5 Deposition of contaminants in Atlantic salmon flesh ........................................... 201 6.4.6 Effect of the finishing diets on both fatty acids and flesh contaminant concentrations ................................................................................................................. 202 6.4.7 Risks versus benefits of salmon consumption ...................................................... 204 CONCLUSION .................................................................................................................... 205 TABLES ............................................................................................................................. 207 FIGURES ........................................................................................................................... 224 REFERENCES ..................................................................................................................... 229  7 Production of farmed salmon low in contaminants and high in omega-3 (n-3) fatty acids: A field study conducted on British Columbia Atlantic salmon farms. ..................... 236 7.1 INTRODUCTION ................................................................................................................. 236 7.2 MATERIALS AND METHODS .............................................................................................. 237 7.2.1 Sample collection and analysis ............................................................................. 237 7.3 RESULTS ........................................................................................................................... 238 7.3.1 Flesh concentrations of PCBs and PCDD/Fs........................................................ 238 7.3.2 Flesh fatty acid composition ................................................................................. 239 7.4 DISCUSSION ...................................................................................................................... 240 7.5 CONCLUSION .................................................................................................................... 241 7.5 FIGURES ........................................................................................................................... 243 7.6 REFERENCES ..................................................................................................................... 246 8  Conclusions.................................................................................................................... 248 8.1 REFERENCES ..................................................................................................................... 253  Appendix 1................................................................................................................................. 254 Appendix 2................................................................................................................................. 256  ix  List of Tables Table 2-1 Ingredient and mean (+/- 1 SD; n = 3) proximate composition and gross energy content of each of the dry test diets fed to juvenile sablefish for 105 days. The diets differed only with respect to the percentages of anchovy oil (AO) and cold pressed-flaxseed oil (FO) comprising the supplemental lipid. ....................................................................................... 73 Table 2-2 Mean fatty acid contents (g 100g-1 total fatty acids) in anchovy oil (AO) and cold pressed flaxseed oil (FO) and the sablefish test diets (n=3 per dietary treatment, ± 1SD). Different postscripts within a row denote significant differences among the means (p<0.05). ............................................................................................................................................... 74 Table 2-3 Mean (+/- 1 SD) initial body weight (IBW, g), final body weight (FBW, g), weight gain (WG, g), specific growth rate (SGR, g g bw-1 day-1) dry feed intake (DFI, g fish-1day-1), feed efficiency ratio (FER, g g-1), protein efficiency ratio (PER, g g-1 protein intake), percent protein deposited (PPD, %), survival (S, %), fillet weight (g), liver weight (g), hepatosomatic index (HSI, %), and condition factor (K) of juvenile sablefish after being fed experimental diets for 105 days. ........................................................................................... 75 Table 2-4 Mean (± 1SD; n = 3) percent apparent digestibility coefficients (%) for crude protein (ADCp), organic matter (ADCorm) and energy (ADCen) in the test diets fed to juvenile sablefish. The supplemental lipid portion of the diets contained either 100% anchovy oil (AO) or different percentages of AO with cold pressed flaxseed oil (FO)........................... 76 Table 2-5 Initial (n = 6 analyzed in duplicate) and final (n = 3, average of 5 fish analyzed in duplicate) mean concentrations (% of wet weight ± 1SD) of proximate constituents in the fillet and whole body of juvenile sablefish fed the test diets for 105 days. The supplemental lipid in the test diets stemmed from either 100% anchovy oil (AO) or different percentages of AO with cold pressed flaxseed oil (FO). .......................................................................... 77 Table 2-6 Terminal mean (± 1SD) fatty acid contents (g 100g-1 total fatty acids) in the fillets of juvenile sablefish fed diets with either 100% anchovy oil (AO) or different percentages of AO and cold pressed flaxseed oil (FO) as the supplemental lipid. ....................................... 78 Table 3-1 Ingredient compositions, mean concentrations +/- 1 SD of proximate constituents and gross energy, levels of digestible protein (DP) and energy (DE) and ratios of DP to DE in the test diets......................................................................................................................... 101 Table 3-2 Mean fatty acid contents (g 100g-1 total fatty acids) of anchovy oil (AO), flaxseed oil (FO), and poultry fat (PF) and of the test diets (+/- 1 SD) where the supplemental lipid source was either 100% AO or 25% AO with different percentages of FO and PF........... 103 Table 3-3 Mean (+/- 1 SD) initial body weight (IBW, g), final body weight (FBW, g), weight gain (WG, g), specific growth rate (SGR, g g bw-1 day-1), daily dry feed intake (DFI, g fish-1 day-1), feed efficiency ratio (FER, g g-1), protein efficiency ratio (PER, g g-1), percent protein deposited (PPD, %), survival (S, %), fillet weight (FW, g), liver weight (LW, g), hepatosomatic index (HSI, %), and condition factor (K) of juvenile sablefish fed dry diets differing only in the percentages of anchovy oil (AO), cold pressed flaxseed oil (FO) and  x poultry fat (PF) comprising the supplemental lipid for 105 days. Refer to Materials and Methods for additional information. ................................................................................... 105 Table 3-4 Terminal mean (+/- 1 SD ) fatty acid contents (g 100g-1 total fatty acids) in the fillets of sablefish fed diets for 105 days where the supplemental lipid source was either 100% anchovy oil (AO) or 25% AO with different percentages of flaxseed oil (FO) and poultry fat (PF) or 75% PF. .................................................................................................................. 106 Table 4-1 Standards used to pre-screen the sensory panel. Prospective panelists were asked to identify the basic taste and rank the three samples in increasing concentration................. 129 Table 4-2 Standards used to train the sensory panel and for creating extreme values for the attribute scale bars............................................................................................................... 130 Table 4-3 Analysis of variance of raw sensory scores for sablefish fed diets in which flaxseed oil comprised either 0, 25, 50, or 75% of the supplemental lipid. *, **, ***, significant at p<0.05, p<0.01, p<0.001, respectively................................................................................ 131 Table 4-4 Mean, raw sensory scores of sablefish fed diets in which flaxseed oil (FO) comprised either 0, 25, 50, or 75% of the supplemental lipid by replacement of anchovy oil (AO). Different superscripts within the same row indicate significant differences (p<0.05) ....... 131 Table 4-5 Correlation matrix among sensory analysis terms for the first feeding trial. *, **, ***, significant at p<0.05, p<0.01, p<0.001, respectively.......................................................... 132 Table 4-6 Analysis of variance of raw sensory scores for sablefish fed diets with various blends of flaxseed oil and/or poultry fat. *, **, ***, significant at p<0.05, p<0.01, p<0.001, respectively ......................................................................................................................... 132 Table 4-7 Mean, raw sensory scores of sablefish fed the control treatment (100AO) or diets in which 75% of the supplemental anchovy oil (AO) was replaced with various blends of poultry fat (PF) and flaxseed oil (FO). Different postscripts within the same row indicate significant differences (p<0.05) .......................................................................................... 133 Table 4-8 Correlation matrix among sensory analysis terms for the second feeding trial. *, **, ***, significant at p<0.05, p<0.01, p<0.001, respectively.................................................. 134 Table 4-9 Minolta colour scores for L (lightness), a (red/green) and b (blue/yellow) for sablefish from feeding trial 1 (a) and feeding trial 2 ( b). Different postscripts within the same column indicate significant differences (p<0.05) ............................................................................ 135 Table 5-1 Composition of diets and levels of contaminants in the experimental feeds. ........... 154 Table 5-2 Contaminant levels in sablefish muscle prior to the feeding trial (day 0) and 105 days after being fed one of 4 experimental diets with varying levels of supplemental anchovy oil (AO) and flaxseed oil (FO). Different postscripts within the same row indicate significant differences (p<0.05) ............................................................................................................ 154 Table 5-3 Accumulation efficiencies (AE) of organohalogens. Percentages of ingested contaminants estimated to be deposited in the sablefish whole body (WB) and fillet. Standard deviations are shown in brackets. ........................................................................ 155  xi -1  Table 5-4 Selected fatty acid contents (mg 100g serving size) and total omega-3 to omega-6 fatty acid ratios in the flesh of sablefish fed the test diets. Values represent means and different letters within a row denote significant differences among means (p<0.05). ....... 155 Table 6-1 Ingredient and proximate compositions and levels and ratios of digestible protein and energy in the phase 1 test diets. The diets were supplemented with either 100% anchovy oil (AO) or various blends of this lipid source with poultry fat (PF), flaxseed oil (FO), or canola oil (CO). The latter lipid source was also used in combination with either canola protein concentrate (CPC) or soy protein concentrate (SPC) in diets 75CO50CPC and 75CO50SPC, respectively. These latter diets also contained some carbon treated-anchovy oil (CT-AO) to replace the residual lipid that was removed when CPC or SPC were substituted for half of the anchovy meal protein in diet 100AO. The IND diet was a reference diet that was formulated to be similar to the least-cost formulation being used by one of the major feed companies in British Columbia. Different superscripts denote significant differences among the means (p<0.05).............................................................................................................. 208 Table 6-2 Supplemental mineral concentrations in the diets fed to Atlantic salmon in phase 1 of the study (mg kg-1 dry diet)................................................................................................. 209 Table 6-3 Ingredient compositions of the diets used in phase 2 of the study. During phase 2, the source of supplemental lipid was either conventional anchovy oil (AO) or carbon-treated AO (diets 75CO50CPC and 75CO50SPC only). The dietary treatments fed to the fish in phase 1 are indicated (refer to Table 6.1 for additional information). ................................ 210 Table 6-4 Fatty acid compositions of the supplemental dietary lipid sources (expressed as % of total identified fatty acids). The sources of supplemental lipid included: conventional anchovy oil (AO), carbon treated-anchovy oil (CT-AO), cold pressed flaxseed oil (FO), crude super-degummed canola oil (CO), and poultry fat (PF). The totals for saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), n-3 highly unsaturated fatty acids (n-3 HUFA), n-3 and n-6 fatty acids, and the ratios of n-3 to n-6 fatty acids are indicated. ............................................................................................... 211 Table 6-5 Concentrations (pg g-1) of polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polybrominated diphenylethers (PBDEs) and toxic equivalent values (pg TEQ g-1) for PCBs, PCDFs and PCDDs in the test sources of supplemental dietary lipid i.e., anchovy oil (AO), carbon treated-anchovy oil (CT-AO), flaxseed oil (FO), canola oil (CO), poultry fat (PF) and the main test dietary protein sources viz., anchovy meal (AM), poultry-by-product meal (PBM), canola protein concentrate (CPC) and soy protein concentrate (SPC). .............................. 212 Table 6-6 Fatty acid compositions of the diets used in phase 1 of the study (mean ± SD; expressed as % of total identified fatty acids). Different superscripts within a row denote significant differences among the means (p<0.05). For more information on dietary treatments and fatty acids refer to Tables 6.1 and 6.4. Results are the average of 6 batches of feed top coated with the different lipids sources on 6 different occasions (n=6). .............. 213 Table 6-7 Fatty acid compositions of the diets used in phase 2 of the study. Diets were pelleted in two batches (mean ± SD) expressed as percentage of total identifiable fatty acids. Different superscripts within a row denote significant differences among the means (p<0.05). Refer to Tables 6.3 and 6.4 for additional information....................................... 214  xii -1  Table 6-8 Concentrations (pg g ) of polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polybrominated diphenylethers (PBDEs) and toxic equivalent values for PCBs, PCDFs and PCDDs in the diets used in phases 1 and 2 of the study. Refer to Tables 6.1 and 6.3 for additional information.......................................................................................................................... 215 Table 6-9 Mean values (± SD; n = 3) for final body weight (FBW; g), weight gain (WG; g ), specific growth rate (SGR; g g bw-1 day-1), condition factor (K), daily dry feed intake (DFI; g fish-1day-1), feed efficiency ratio(FER; g g-1), protein efficiency ratio (PER; g g-1), percent protein deposited or retained (%PD; %), percent lipid deposited (%LD; %), percent survival (%S; %), percent fillet yield (%FY; %), hepatosomatic index (HSI; %), and gonadosomatic index (GSI; %) of Atlantic salmon post-smolts at the end of phase 1 (day 168) and phase 2 (day 252) of the study. Different superscripts (a-c) within a row denote significant differences among the means (p<0.05). Significant differences (p<0.05) within a treatment between phase 1 and phase 2 are indicated by superscripts ‘y’ and ‘z’.............................. 216 Table 6-10 Overall mean concentrations ± SD of proximate constituents (n = 3) in the whole bodies and fillets of post-smolt Atlantic salmon at the end of phase 1 (day 168) and phase 2 (day 252). The values are averages across all dietary treatments given in phase 1 or 2 since, in each case, there was no significant effect of dietary treatment. Significant differences (p<0.05) between phase 1 and phase 2 proximate consituents are indicated by ‘y’ and ‘z’ superscripts. ........................................................................................................................ 218 Table 6-11 Mean values (± SD ; n = 3) for daily dry feed intake (DFI; g fish-1 day-1), initial body weight (IBW; g), final body weight (FBW; g), weight gain (WG; g), feed efficiency ratio(FER; g/g), and specific growth rate (SGR; g g bw-1 day-1) of pre-smolt Atlantic salmon fed diets in which the source of supplemental lipid was either 100% conventional anchovy oil (AO) or 30% AO and 70% carbon treated-anchovy oil (CT-AO). The fish were fed their respective diets for 49 days. Different superscripts within a column denote significant differences among the means (p<0.05). .............................................................................. 219 Table 6-12 Mean concentrations of fatty acids (± SD ; n = 3), expressed as percentages of the total identified fatty acids in post-smolt Atlantic salmon fillets on day 0 and at the end of phase 1 (day 168) in relation to dietary treatment. Different superscripts within a row denote significant differences among the means (p<0.05). Refer to Table 6.1 for additional information.......................................................................................................................... 220 Table 6-13 Mean concentrations of fatty acids (± SD ; n = 3), expressed as percentages of the total identified fatty acids in Atlantic salmon fillets at the end of phase 2 (day 252) in relation to the dietary treatment. During phase 2, the source of supplemental dietary lipid was either conventional anchovy oil (AO) or carbon treated-anchovy oil (CT-AO; used in diets 75CO50CPC and 75CO50SPC only). Different superscripts (a-e) within a row denote significant differences among the means (p<0.05). Within each dietary treatment, a superscript ‘y’ indicates the concentration the fatty acid was significantly higher in phase 1 fish, while ‘z’ indicates the fish fed the finishing diet (phase 2) had significantly higher levels of the fatty acid. Refer to Table 6.3 for additional information on dietary treatments. ............................................................................................................................................. 221 Table 6-14 Concentrations (pg g-1) of polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polybrominated  xiii -1  diphenylethers (PBDEs) and toxic equivalent values (pg TEQ g ) in the Atlantic salmon fillets on day 0 and at the end of phase 1 (day 168) and phase 2 (day 252) in relation to the dietary treatment given to the fish in phase 1. Different superscripts (a-c) within a row denote significant differences among the means (p<0.05). Within each dietary treatment, a superscript ‘y’ indicates the concentration the contaminant was significantly higher in phase 1 fish, while ‘z’ indicates the fish fed the finishing diet (phase 2) had significantly higher levels of the contaminant. ................................................................................................... 222 Table 6-15 Retention efficiencies (% of consumed lipid, fatty acids or organohalogens deposited in the fillet) ± SD for total lipid, individual fatty acids and various organic contaminants in the flesh of Atlantic salmon at the end of phase 1 (day 168) and phase 2 (day 252). Significant differences (p<0.05) between retention efficiency between phase 1 and phase 2 are indicated by superscripts ‘y’ and ‘z’. ............................................................................ 223  xiv  List of Figures Figure 1-1 Global wild sablefish harvest (metric tonnes) with most of the fish harvested from Alaskan waters. ..................................................................................................................... 43 Figure 1-2 Desaturation and elongation of omega-9 (n-9), omega-6 (n-6) and omega-3 (n3) fatty acid series and the enzymes involved in the different steps. Adapted from Sargent et al., (2002). ................................................................................................................................... 43 Figure 1-3 Oil prices from January 2001 to October 2006 for Peruvian anchovy oil (AO), Chilean AO, refined, bleached and deodorized canola oil (CO), crude-degummed soy oil and poultry fat. Data obtained from www.thejacobsen.com................................................ 44 Figure 1-4 Chemical structure of polychlorinated dibenzo-dioxin (a), polychlorinated dibenzofuran (b), polychlorinated biphenyl (c), and polybrominated diphenyl ether....................... 44 Figure 2-1 Relationship between the concentration of either 14:0, 16:0, 16:1n-7, 18:1n-9, 18:2n6, 18:3n-3, 20:5n-3 or 22:6n-3 in the diet and the respective subsequent terminal concentration in the sablefish fillet. A diagonal line in the centre of each graph represents equality between the concentration of the fatty acid of interest in the fillet and the dietary treatment. .............................................................................................................................. 79 Figure 3-1 Changes in water temperature, fish weight, specific growth rate, dry feed intake, feed efficiency ratio and protein efficiency ratio at 5-week intervals during the 15-week feeding trial. Graphs for the performance parameters are averages of all four dietary treatments since there were no significant differences between treatments for any of the parameters at each sample time................................................................................................................. 107 Figure 3-2 Relationship between fish weight and muscle or whole body lipid content (%). Each sample represents the average for the fish on day 105. The data for the individual dietary treatments were pooled (n = 5 per tank for a total of 60 fish) since there was no effect of dietary treatment on terminal fish weights or whole body or fillet (muscle) lipid concentrations. .................................................................................................................... 108 Figure 3-3 Relationships between selected fatty acid concentrations in the feed and their respective terminal concentrations in the sablefish fillet. Diagonal lines in the centre of each graph represent equality between the concentrations of the fatty acids in the fillet and dietary treatments................................................................................................................ 109 Figure 4-1 Location of where colour measurements were made on sablefish fillets. A vertical cut was made below the dorsal fin. The colour was then measured in the cross section of the fillet using a Minolta both towards the tail and towards the head. ..................................... 136 Figure 4-2 Principal component analysis (PCA) of the mean intensities of sensory attributes characterizing the flesh of sablefish (represented by black squares) fed diets in which either 0, 25, 50, or 75% of the supplemental lipid was comprised of flaxseed oil (FO) by replacement of anchovy oil (AO). For example, if a fish sample fed diet ‘x’ was located in the bottom left corner of the PCA, the sensory panel would have found that fish fed diet ‘x’ had high ocean aroma and sweet taste. ............................................................................... 137  xv Figure 4-3 Principal component analysis of the mean intensities of sensory attributes characterizing the flesh of sablefish (represented by black squares) fed diets in which 75% of the supplemental lipid originated from anchovy oil (AO), poultry fat (PF), or blends of PF with flaxseed oil (FO).................................................................................................... 138 Figure 5-1 Toxic equivalencies in diets (solid bars) and fish muscle (striped) on a wet weight (Figure A) and lipid-corrected basis (Figure B). Means +/- standard deviations; different letters denote significant differences between means (p<0.05). ......................................... 156 Figure 5-2 Contribution of individual PCB congeners to total TEQ pg g-1 wet weight in fish feed (Figure A) and sablefish muscle (Figure B). ...................................................................... 157 Figure 6-1 Anchovy oil was decontaminated in 30 kg batches. The oil was heated in the jacketed glass reactor pictured above. The vessel was equipped with a propeller stirrer, and it was mixed with 5g of Norit SA4 PAH activated carbon (Amersfoort, The Netherlands) kg-1 at 300 rpm for 30 min at 90˚C. ............................................................................................... 224 Figure 6-2 After mixing the oil and activated carbon for 30 min, the oil was cooled to 50˚C and then filtered twice using the equipment pictured above. Celite Hyflor Super-Cel filter aid (Denver, USA) was used to facilitate filtering.................................................................... 224 Figure 6-3 Fatty acid content of fillets (g/ 100g serving) and the percent increases (+) or decreases (-) in flesh levels of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), sum of omega-6 (n-6) fatty acids1 or total of omega-3 (n-3) fatty acids2 relative to the control treatment (100AO). An asterisk is located beside the percentage value when treatments were significantly different from the 100AO control (p<0.05). In phase 1 (168 days), fish were fed diets with alternative dietary lipids. In phase 2 (84 days) the supplemental lipid source was 100% AO or 100% CT-AO. .............................................. 225 Figure 6-4 Principal component analysis of the fatty acid composition of the fish fed the different dietary treatments in phase 1 (squares) and phase 2 (triangles) following the 12week finishing diet. The fatty acid vectors are indicated by diamonds. For details on dietary treatments see Figure 6-3. ................................................................................................... 226 Figure 6-5 Lipid-corrected concentrations for polychlorinated biphenyls (PCBs; ng g-1) (Figure 6-5A), polybrominated diphenyl ethers (PBDEs; ng g-1) (Figure 6-5 B) and toxic equivalency (TEQ; pg g-1) (Figure 6-5C) in the phase 1 diets and in the fillets in relation to dietary treatment. For details on dietary treatments see Figure 6-3.................................... 227 Figure 6-6 Percent increases (+) or decreases (-) in total polychlorinated biphenyls (PCBs; pg g-1), total PCB toxic equivalency (PCB TEQ; pg g-1) and total polybrominated diphenyl ethers (PBDEs; pg g-1) in salmon fillets relative to the control treatment, 100AO, after both phase 1 (day 168; left-black bars) and phase 2 (day 252; right-grey bars). An asterisk is located beside the percentage value when treatments were significantly different from the control (p<0.05). For details on dietary treatments see Figure 6-3..................................... 228 Figure 7-1 Toxic equivalent values (TEQ values) for PCBs (striped bars) and polychlorinated dibenzodioxins and polychlorinated dibenzofurans (PCDD/Fs; grey bars) in the skinned fillets from British Columbia farmed salmon fed traditional diets (2003) versus those fed diets with alternative lipids (2005) in relation to the TEQ values noted for wild Pacific  xvi salmon (2003 and 2005 data combined; Chk= Chinook, Soc= Sockeye). One feed supplier produced diets where canola oil comprised 35% of the lipid content (35%CO) whereas the other produced diets where poultry fat comprised 50% of the lipid content (50%PF). Values are means ± 1 SD and FL is average fork length. ............................................................... 243 Figure 7-2 Flesh PCB concentrations in relation to the size (fork length) of Atlantic salmon and source of feed (farmed salmon only) and year of sampling. Flesh PCB levels in market-size salmon sampled in 2005 were dramatically lower than those found for salmon sampled in 2003. Feeds with the higher replacement level of fish oil (50%PF) resulted in lower flesh levels of PCBs at all fish sizes relative to those with the lower replacement level (35%CO). ............................................................................................................................................. 244 Figure 7-3 Total concentrations for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in 100 g servings of BC farmed salmon fed traditional feeds based on marine fish oil in 2003, or new diet formulations based more extensively on alternate lipid sources in 2005 either from canola oil (35% of dietary lipid; 35%CO) or poultry fat (50% of dietary lipid; 50% PF) and for wild Pacific salmon (2003 and 2005 data combined; Chk = Chinook, Soc = Sockeye). Values are means ± 1 SD and FL is average fork length. .................................. 245  xvii  List of Abbreviations AA ADC AE AhR ALA AM ANOVA AO Arnt BC bw CAER CFIA CO CPC cr CT-AO D DE DFI DFO DHA DP DWB EPA EU F FAME FAO FBW FER FO GC GC-HRMS GSI HSI HUFA IBW IND  Arachidonic acid (20:4n-6) Apparent digestibility coefficients Accumulation efficiencies Aryl hydrocarbon receptor α-linolenic acid (18:3n-3) Anchovy meal Analysis of variance Anchovy oil Ah receptor nuclear translocator protein British Columbia Body weight Centre for Aquaculture and Environmental Research Canadian Food Inspection Agency Canola oil Canola protein concentrate Chromic oxide Carbon treated-anchovy oil Diet Digestible energy Dry feed intake Department of Fisheries and Oceans Docosahexaenoic acid Digestible protein Dry weight basis Eicosapentaenoic acid European Union Feces Fatty acid methyl ester Food and Agriculture Organization of the United Nations Final body weight Feed efficiency ratio Flaxseed oil Gas chromatography Gas chromatography/high-resolution mass spectrometry Gonadosomatic index Hepatosomatic index Highly unsaturated fatty acid Initial body weight Industry diet  xviii  IOS K  Institute of Ocean Sciences Condition factor  Kow LA MFO MT MUFA n-3 n-6 ND  Octanol-water partition coefficient Linoleic acid (18:2n6) Marine fish oil Metric tonne Monounsaturated fatty acids Omega 3 fatty acid Omega 6 fatty acid Non-detectable  ng NRC NSP OA om p PBDE PBM PC PCA PCB PCDD PCDF PER PF  Nanogram (10-9 g) National Research Council Non-starch polysaccharide Oleic acid (18:1n-9) Organic mater Protein Polybrominated diphenyl ether Poultry by-product meal Principal component Principal component analysis Polychlorinated biphenyl Polychlorinated dibenzodioxin Polychlorinated dibenzofuran Protein efficiency ratio Poultry fat  pg POP PPD PUFA QA/QC RPC SA SD SFA SGR SPC TAG TBARs TDI  Picogram (10-12 g) Persistent organic pollutant Percent protein deposition Polyunsaturated fatty acid Quality assurance/quality control Rapeseed protein concentrate Stearic acid (18:0) Standard deviation Saturated fatty acid Specific growth rate Soy protein concentrate Triacylglycerols Thiobarbituric acid reactive substances Tolerable daily intake  xix  TEF TEQ TI TPA UB US EPA USA WG WHO WHOSUM-TEQ  Toxic equivalency factor Toxic equivalency Trypsin inhibitor Texture profile analysis Upper bound (Concentrations = limit of detection) United States Environmental Protection Agency United States Weight gain World Health Organization Sum of TEQs for dioxin like-PCBs, PCDDs and PCDFs  %S 2,3,7,8-TCDD  Percent survival 2,3,7,8-tetrachlorodibenzo-p-dioxin  xx  Acknowledgments This couldn’t have been possible without the love and support of my parents Mark and Katherine. Thank you to my brothers Bryan and Kevin for the help and for turning down the volume when required. Good friends like Stephanie, Lauren, Tahira and Jessica (x2) are hard to find. The laughs, talks, coffees, drinks and trips kept me grounded and will be remembered. Kevin, thanks for the phone calls, support and help cleaning and feeding tanks, you truly were there for me during the years. Supervisors like Dave Higgs and Brent Skura are one of a kind. Dave your knowledge, encouragement and positive attitude were well appreciated on the numerous rainy days. The lessons learnt will be used for years to come. Brent thank you for your patience and support, the help with the scholarship applications, the last minute urgent help with directed readings and the numerous delicious Christmas dinners. I would also like to thanks Michael Ikonomou and his staff at IOS for inviting me into his lab and provided me with assistance and Corey for the sample collection and preparation. A warm thanks to my scientific committee: Dave Kitts, ChenHuei Huang and Zhaoming Xu. Your comments and time spent reading the thesis and committee reports are very much appreciated. Lab work couldn’t have been completed without the help of the following students: Nassim, Jenny, Rob, Dave, Anna, Jedi, Erin and Nelly. Thanks for working extra hours when required and for keeping a smile on your faces through the unusual tasks. Without the extra hands provided by Nahid, Shannon, Susie, Amelia and Erica, sampling days would have taken forever. Mahmoud, your help, kindness and road trips to pick up samples were greatly appreciated over the years. Janice, what would I have done without you? Thank you for answering my questions and helping me solve my problems. You were not only a great teacher for myself but shared your knowledge with many of the other students who passed through the lab. Jill, you were the big sister I never had but wish I had. Thank you for all your words of wisdom. This research couldn’t have been possible without funding from the following sources: Department of Fisheries and Oceans, the University of British Columbia, AquaNet, ACRDP, Skretting Canada, Ewos Ltd., Taplow Feeds, MCN Bioproducts Inc. and Microtec. A final thanks to my new friends at Skretting who have been patient with the long awaited end to this journey.  xxi  Co-Authorship Statement Chapter 2: I was responsible for the experimental design, data collection, all statistical analysis and writing of the manuscript. Dave Higgs, Brent Skura, Michael Ikonomou, and Shannon Balfry contributed to the experimental design, the choice of statistical tests and presentation of the manuscript. Although not presented in the manuscript, Shannon Balfry performed and analyzed all fish health results. Chapter 3: I was responsible for the experimental design, data collection, all statistical analysis and writing of the manuscript. Dave Higgs, Brent Skura, Michael Ikonomou, and Shannon Balfry contributed to the experimental design, the choice of statistical tests and presentation of the manuscript. Although not presented in the manuscript, Shannon Balfry conducted and reported all fish health results. Chapter 4: I was responsible for the experimental design, data collection, all statistical analysis and writing of the manuscript. Nelly Lim assisted with data collection and Dave Higgs, Brent Skura and Nelly Lim contributed to the experimental design, the choice of statistical tests and presentation of the manuscript. Chapter 5: I was responsible for the experimental design, data collection, all statistical analysis and writing of the manuscript. Dave Higgs, Brent Skura and Michael Ikonomou contributed to the experimental design, the choice of statistical tests and presentation of the manuscript. Cory Dubetz assisted with data collection and presentation of the manuscript. Greg Deacon and Jason Mann contributed to the experimental design. Chapter 6: I was responsible for the experimental design, data collection, all statistical analysis and writing of the manuscript. Dave Higgs, Brent Skura, Micahel Ikonomou and Shannon Balfry contributed to the experimental design, the choice of statistical tests and presentation of the manuscript. Cory Dubetz and Janice Oaks assisted with data collection. Åge Otherhals decontaminated the anchovy oil, while Greg Deacon, Jason Mann and Ann Gannam contributed to the experimental design. Although not presented in the thesis, Shannon Balfry conducted and reported all fish health results. Also not reported in the thesis was the fish cardiovascular performance work conducted under the supervision of Tony Farrell. Chapter 7: I as well as, Michael Ikonomou and Keng Pee Ang were responsible for the experimental design. Whole fish were sent to the Institute of Ocean Sciences by employees at the farm sites and were analyzed by Cory Dubetz. Statistical analysis and writing of the manuscript was performed by myself. Michael Ikonomou and Dave Higgs contributed to presentation of the manuscript.  1  1 Introduction Today, over 49% of the fish consumed is produced by aquaculture. This industry has been growing at an average rate of 8.8 % per year since 1950 to keep pace with the global demand for fish products (FAO, 2006). Fish feed is currently the largest input cost and can account for up to 75% of production costs. Traditionally, fish feed has been mainly composed of fishmeal and fish oil. With no increases in fishmeal and fish oil supply due to the wild global capture fisheries being at their maximum annual yield, aquaculture production costs will continue to rise unless suitable alternatives are found. In recent years, there has been interest in diversifying the aquaculture industry to include high-value marine finfish species. These species have a higher requirement for certain nutrients in fish-based ingredients and growth of this sector of the industry will increase the need for new sources of feed ingredients. Vegetable oils, animal fats, plant protein meals as well as concentrates and isolates, and animal by-product protein meals have all been examined as alternatives to marine fish oil and fishmeal in aquafeeds. Apart from the plant protein concentrates and isolates, these ingredients can often be obtained at prices lower than those for fishmeal and fish oil. In 2006 alone, the price of fishmeal doubled from about $700 per metric tonne (MT) to over $1400 per MT (www.thejacobsen.com). Meanwhile, soy prices remained relatively stable at a price of $200300 MT (www.thejacobsen.com). Price has not been the only factor driving the aquaculture industry to use alternative feed ingredients. It has been predicted that within the next 5-10 years, the requirement for fish oil by the aquaculture industry alone will exceed the annual global supply (Tacon, 2005). For continued growth of the aquaculture industry, the use of alternative feed ingredients is inevitable. However, the use of alternative feed ingredients may negatively impact fish growth, decrease fish survival and reduce the quality of the market-size product. Fish have specific dietary requirements for protein and amino acids, lipids and fatty acids, energy, vitamins, and minerals. Replacement of fishmeal with vegetable or animal protein sources could result in decreased fish growth, health and survival if amino acid and mineral supplements are not used. Further, replacement of fish oil with vegetable oils and animal fats, especially in the case of marine species, can also lead to decreases in their growth and survival if their essential fatty acid requirements are not met. The alternative feed ingredients may also lead to decreased fish growth  2 and survival if the diets are not palatable. A notable example of this is chinook salmon which show aversion to feed that contains soy protein products (Twibell and Wilson, 2004). Plants through evolution have developed defense mechanisms against predators, drought and winter freeze. These defense compounds when present in fish feed through the incorporation of plant protein products often exert antinutritional effects that can negatively impact fish growth through their adverse effects on feed intake and/or feed and protein utilization. However, processing protocols that involve alcohol or aqueous extraction and the appropriate application of heat may reduce or inactivate most of the preceding deleterious components that are present within plant protein products. Changes in the ingredient composition of fish feed may also influence the taste, aroma or texture of the final fish product. The stability and shelf life of fish products may be influenced by changes in feed ingredients. From a human health perspective, use of alternative dietary lipids will cause changes in the fatty acid composition of edible muscle. Fish oils are rich in omega-3 highly unsaturated fatty acids that have numerous health benefits, especially the prevention of cardiovascular disease. Vegetable oils and animal fats by contrast do not contain omega-3 highly unsaturated fatty acids. These terrestrial lipid sources contain higher levels of omega-6 fatty acids in the form of linoleic acid. Also, the plant oils often contain significant concentrations of other C18 fatty acids such as linolenic acid, the parent acid of the omega-3 family of fatty acids, and oleic acid. Further, the animal lipid sources may also be high in oleic acid and may contain higher levels of saturated fatty acids than are present in fish oil. Thus, the use of alternative plant and animal lipid sources may result in fish products that have fewer potential human health benefits than those which originate from fish fed diets based on fishmeal and fish oil. On the other hand, the use of alternative feed ingredients can lead to the production of ‘safer’ fish products. In 2004, a highly publicized study was published in the prestigious journal Science (Hites et al., 2004). This study examined the levels of lipophilic organic contaminants in the flesh of wild Pacific salmon, and farmed Atlantic salmon sampled from different regions of the world. Higher levels of persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) were measured in the flesh of farmed Atlantic salmon. The foregoing compounds have been demonstrated to be carcinogens in animals, but studies involving humans have produced less convincing evidence in this regard. In the study published in Science, the higher levels of organic  3 contaminants found in the flesh of farmed Atlantic salmon were due to the high genetic propensity of this species to deposit lipid in its flesh, consumption of high fat diets and extensive use of fishmeal and oil in aquafeeds. The wild Pacific salmon species by contrast do not deposit as much lipid in their flesh even when the lipid content of their natural prey is high. Hence, the flesh of farmed Atlantic salmon contained more lipid than noted in wild salmon and, as indicated above, because the POPs are lipid soluble, the farmed salmon also contained higher levels of POPs. POPs are highly lipophilic and most of the aforementioned contaminants are absorbed in the gut (from the diet). Relatively low amounts of these compounds are obtained via the gills from the water in which the fish reside. Since farmed fish are held in confined and relatively well controlled environments, aquaculture, through dietary modification, has the potential to lower the levels of POPs in fish at rates much faster than the current elimination of POPs in wild fish. Fish oil and to a lesser extent, fishmeal are the largest sources of POPs in aquafeeds. Vegetable oils and animal fats (in grain eating animals) often contain lower levels of POPs because these lipid sources originate from a lower trophic level in the food chain. Even though the current levels of POPs in farmed-raised salmon products are not a threat to human health since they are at least 50-70 times below the level of concern established by Health Canada and the US Food and Drug Administration, the use of terrestrial plant and animal ingredients in aquafeeds has the potential to lower flesh levels of POPs in farmed salmon and marine finfish to levels within the range found for wild counterparts. This, in turn, will at least improve the perceived safety of these products by consumers.  1.1 RESEARCH AIMS The primary research objective of this thesis was to examine the use of alternative feed ingredients in two commercially important finfish species in British Columbia (BC); Atlantic salmon (Salmo salar) and sablefish (Anoplopoma fimbria). Production of farmed Atlantic salmon currently accounts for over 80% of total salmon production in BC which presently is in excess of sixty thousand tonnes. Sablefish, on the other hand, is only farmed in small quantities at a couple of farms in BC, although many salmon farms hold licences that would allow them to farm sablefish should they desire.  4 Specific aims of the research were to examine the effect of the alternative dietary feed ingredients on: (1) fish growth and survival, (2) apparent energy, protein and organic matter digestibility, (3) flesh fatty acid compositions, (4) sensory attributes such as taste, texture, and aroma and, (5) flesh POP concentrations. The ingredients examined included: cold-pressed flaxseed oil, crude super de-gummed canola oil, poultry fat, contaminant-reduced anchovy oil (processed with activated carbon), poultry by-product meal, canola protein concentrate and soy protein concentrate. The ingredients were evaluated in three feeding trials and one on-farm field study. The efficacy of flaxseed oil or poultry fat alone and various blends of these lipid sources as supplemental dietary lipid sources was studied in two 15-week laboratory feeding trials that were conducted on sablefish whereas all of the preceding alternative lipid and protein sources were evaluated in a 36-week laboratory feeding trial that was conducted on post-smolt Atlantic salmon in seawater. In addition, two of the alternative lipid sources, namely, poultry fat and canola oil, were assessed in a field study conducted on farmed BC Atlantic salmon that involved examining the fish at different stages of their life history in the marine environment. The overall goal of the study is to gain a better understanding how diets composed of alternative feed ingredients can be used to lowering flesh levels of POPs in cultured salmon and sablefish. The present findings can also be extrapolated to other aquatic finfish species that have high prosperity to deposit lipid in their flesh. Moreover, the research provides important insights into how to maintain and restore flesh concentrations of the highly unsaturated fatty acids of the omega-3 family of fatty acids that are known to be important for human health after the fish have been fed diets based upon the alternative terrestrial lipid and protein sources. In the Atlantic salmon study, for instance, finishing diets composed of either conventional or contaminantreduced fish oil were examined for their effectiveness to restore the preceding beneficial fatty acids in the flesh of Atlantic while monitoring the re-introduction of POPs. Ideally, it was hoped that this research would minimize flesh concentrations of POPs and concurrently maximize levels of the beneficial omega-3 fatty acids so that the market product would have the greatest potential human health benefits as well as acceptance by the consumer.  5 1.2 LITERATURE REVIEW 1.2.1 Aquaculture Fish are a highly nutritious food source since they are rich in protein, healthy fatty acids, minerals such as calcium, iron, selenium, and zinc, and vitamins A, B3, B6, B12, D and E (Sidhu, 2003). In most areas of the world, resource availability rather than income predicts fish consumption. People in Asian nations are one exception, since they tend to eat higher levels of fish as their economy develops (York and Gossard, 2004). With the population of Asian countries expected to grow by 75% over the next 40 years (United Nations, 2005) and countries such as China, Japan and Malaysia consuming 26, 66, and 56 kg of fish person-1 year-1, respectively (York and Gossard, 2004), it is clear that there will be increased demands for fish products will occur. The wild fisheries will not be able to supply the fish required to meet the increased demands. The wild fisheries reached their peak growth in the middle of the 20th century. Due to over fishing (and possibly other human interactions such as forestry and global warming), the growth of the wild fisheries has ceased and has been in decline since the 1990’s (Wilkinson, 2006). In fact, humans have decreased the biomass of large predatory fish such as tuna, marlin and flatfishes by 90% of pre-industrial levels (Myers and Worm, 2003).  Aquaculture is defined as the farming of aquatic organisms and these include fish, molluscs, crustaceans, and aquatic plants. It requires some form of intervention such as feeding or protection from predators and implies ownership of the stock being cultivated (FAO, 1989 ). To keep up with global demand for fish products, world aquaculture has grown at an average annual rate of 8.8% since 1950 and currently provides about 49% of the fish consumed (FAO, 2006).  1.2.1.1 Species There has been a considerable increase in the diversity and number of species being farmed. In 1950, a total of 72 species were cultured and currently there are over 336 species (FAO, 2006). In Canada, salmon currently represent about 90% of total aquaculture finfish production, 80% of which is Atlantic salmon (Salmo salar) (Statistics Canada, 2005). As the aquaculture industry continues to grow, more highly-valued species, particularly marine finfish species are raised  6 (FAO, 2006 ). The sablefish (Anoplopoma fimbria) is one of the highest valued marine finfish in demand for smoking due to its white flaky flesh and high oil content. The majority of sablefish is exported to Japan. In North America, the demand for sablefish is increasing as a delicacy item (Clarke et al., 1999; McFarlane, 1989). The wild sablefish fishery is currently highly regulated, but over fishing in the 1970’s has led to a decline in current wild catches (Figure 1-1). Atlantic salmon have been farmed since the 1960’s and over the years, selective breeding has favoured increased growth rate, decreased early maturity (jacks) and enhanced disease resistance (Kallio-Nyberg and Koljonen, 1997). At about the same time, in the 1960’s, sablefish were first examined for their potential for mariculture. Early studies indicated that sablefish have one of the fastest growth rates as juveniles, can withstand high rearing densities and were well suited for netpen aquaculture (Kennedy, 1972 ; Gores and Prentice, 1984; Shenker and Olla, 1986). However, unlike the Atlantic salmon that spawns in freshwater, the incubation of sablefish eggs in high salinity seawater proved to be challenging and the growth of the sablefish aquaculture industry was limited by the inability to produce juveniles (Alderdice et al., 1988). The first juveniles were successfully produced from eggs in 1998 (Clarke et al., 1999). Now, other areas of research, such as establishment of their nutritional requirements and optimization of their cultural conditions for grow-out, need to be undertaken.  1.2.2 Nutritional requirements In the aquaculture environment, pelleted fish feeds provide the fish with all of their required nutrients and energy. Minimal nutrient and energy input from the surrounding environment requires that the diets be formulated to meet all of the known nutritional requirements of the fish. The nutritional requirements have been determined for energy, protein and amino acids, lipids and fatty acids, minerals, and vitamins for many fish species (NRC, 1993). When nutritional requirements are not known for a specific species, the previously determined nutritional requirements for related fish species can be used as an aid in diet formulation (Lovell, 1991). However, there still may be specific differences among the species particularly in their essential fatty acid requirements and their ability to cope with different sources and levels of dietary carbohydrates (Lovell, 1991) and different protein sources. Examination of the nutrient composition of the natural prey of the species and/or chemical analysis of the fish species  7 sampled from the wild can help when formulating diets for species where there is little or no nutritional requirement data available (Higgs et al., 1995a).  1.2.2.1 Energy All organisms require energy for survival, maintenance and growth. Energy itself is not defined as a nutrient, but is released during the metabolism of carbohydrates, lipids and amino acids (NRC, 1993). Since fish are poikilotherms, they generally have lower energy requirements than terrestrial animals because they do not need to maintain a constant internal temperature. Fish also inhabit aqueous environments and can excrete nitrogen waste products as ammonia, rather than using energy to produce less toxic urea or uric acid. However, being in water can cause large fluctuations in energy requirements since rising water temperatures increase their standard metabolism and hence, energy requirements (Higgs et al., 1995a). Energy requirements can also change in relation to life history stage (Einen and Roem, 1997). Some feed components resist digestion and this can result in different feed ingredients having different bioavailabilities of their energy-yielding nutrients viz., protein, lipid and carbohydrate (organic matter constituents). Digestible energy is defined as the difference between the intake energy (gross energy) and the energy lost in the feces and is therefore the energy that is bioavailable to the fish (NRC, 1993). Digestible rather than gross energy and nutrient content should be used in the formulation of fish diets (Cho and Kaushik, 1990). Often when the energy requirements of fish are reported, it is useful to state them on a digestible protein to digestible energy basis because these two components need to be kept in balance for optimal fish growth. When diets are high in protein relative to energy, the protein will be used for maintenance before growth, while if there is an excess of energy and suboptimal protein in the diet, feed intake may decrease and not provide enough protein for growth (NRC, 1993). Fish have a higher requirement for dietary protein than terrestrial animals because they have lower energy requirements (Lovell, 1991).  1.2.2.1.1 DETERMINATION OF APPARENT DIGESTIBILITY COEFFICIENTS  Digestible energy and protein values can be derived through direct or indirect determinations. The direct method is time consuming and involves measuring all of the feed consumed by the  8 fish and all of the resulting waste products. It has been attempted using a modified metabolism chamber (Smith, 1971), but due to the challenges associated with the fish being in an aqueous environment (Bureau et al., 2002) and the stress responses of fish during this procedure (Hunn, 1982), indirect methods are often preferred. Indirect methods use a non-digestible marker in all of the test diets often at a concentration of 0.5%. An important characteristic of the marker is that it must not be absorbed by the animal. Chromic oxide is most commonly used while other markers such as yttrium oxide and titanium oxide have been examined. The apparent digestibility coefficients for nutrients and energy can then be determined by finding the difference between the marker concentration in the feed and the feces and the concentrations of the dietary nutrient of interest in the feed and feces (Fenton and Fenton 1979). The method of fecal collection can strongly influence digestibility results (Cho et al., 1982; Weatherup and McCracken, 1998; Hajen et al., 1993). Fecal collection methods have included: anal aspiration, surgical removal (intestinal dissection), stripping, settling columns, and continuous filtration of the feces from the tank effluent water (Cho et al., 1982; Higgs et al., 1995a). Each method has positive and negative features. For example, intestinal dissection of the feces requires that the fish be sacrificed. Also, the stripping of feces often results in under estimation of especially the digestibility coefficients for protein due to contamination of the fecal samples with mucus and intestinal cells and, the settling column technique for fecal collection can lead to overestimation of the digestibility coefficients for nutrients due to some leaching of nutrients from the feces (Hajen et al., 1993). The determined digestibility values are, as indicated above, often referred to as apparent digestibility values or coefficients as opposed to true digestibility coefficients. This is because there will inevitably be cells, proteins and secretions originating endogenously from the fish that are contained within the fecal samples (Bureau et al., 2002). These compounds increase the estimated protein and energy contents of the feces, and thus underestimate the true digestibility of protein and energy in the ingredients. However, when feed intake is high, the difference between the estimates for ‘true’ and apparent digestibility coefficients is negligible (Bureau et al., 2002).  9 1.2.2.2 Proteins Depending upon the fish species, protein can comprise >90% of the fish whole body on a dry weight basis. Proteins, composed of amino acids, are required for tissue repair and tissue growth and the amino acids can be catabolised for energy (NRC, 1993). Ten amino acids are essential for all fish species and these have been labelled as essential or indispensable amino acids because fish are unable to produce or synthesize them in sufficient quantities. These 10 essential amino acids are: arginine, histidine, isoleucene, leucine, lysine, methionine, phenylalanine, threonine, thryptophan and valine (NRC, 1993). While the other amino acids are not essential, their dietary inclusion will improve fish growth since the fish will not have to assemble them from the nitrogen provided by the essential amino acids. Also important in formulation of feeds is ensuring that amino acids are present in their optimum ratio relative to each other (Green et al., 2002).  1.2.2.3 Lipids Lipids are compounds that are relatively insoluble in water but are soluble in organic solvents such as chloroform and hexane. Lipids liberate approximately 9.45 kcal (39.5 kJ) of gross energy per gram and are the best sources of energy in terms of amount g-1 compared with carbohydrates (4.15 kcal or 17.4 kJ g-1) and proteins (5.65 kcal or 23.6 kJ g-1) (Maynard and Loosli, 1969). Lipids are the preferred source of metabolic energy in fish for growth, reproduction, and swimming (Tocher, 2003). Lipids have four main functions in the body. These include the provision of energy and essential fatty acids and their roles as structural components and in regulatory functions. Elevation of the dietary lipid content can spare protein (amino acids) for growth and thereby de-emphasize the use of amino acids to meet part of the energy needs of the fish. However, too much dietary lipid can result in excessive lipid deposition and in an imbalance in the digestible protein to energy ratio (NRC, 1993) which, if too low, can have negative effects on the growth performance of fish. When the lipid requirements of fish are discussed, it is useful to consider these together with the specific types of dietary fatty acids that are required. Oils and fats are primarily composed of triacylglycerols (TAGs) and these contain three fatty acids that are esterified to a glycerol molecule. The fatty acids that are affixed to the glycerol backbone differ in the number of carbons, the number of double bonds, and in the position of the double bonds. More specifically,  10 fatty acid nomenclature distinguishes between fatty acids on the basis of their carbon chain length, the degree of their unsaturation (number of double bonds) and the position of the first double bond that in one system considers the number of carbon atoms counting from the methyl end of the molecule. For example, the fatty acid 18:2n-6 (linoleic acid, LA), has 18 carbons, 2 double bonds, and the first double bond is located 6 carbon atoms from the methyl end of the molecule, with the second double bond occurring at the ninth carbon atom from the methyl end.  1.2.2.3.1 ELONGATION AND DESATURATION OF FATTY ACIDS  All vertebrates, including fish cannot form α-linolenic acid (18:3n-3, ALA) from oleic acid (18:1n-9, OA) or form LA from ALA due to the lack of ∆12 and ∆15 desaturases (Tocher, 2003). Thus, LA and ALA are considered to be essential fatty acids and must be provided in the diet of the fish. Rainbow trout fed diets with ALA (and no LA) had better growth and health than those fed diets with LA (and no ALA) and it was suggested that ALA must be included at >1% of the diet (Castell et al., 1972). Fish do, however, contain ∆9 desaturase and can produce OA from 18:0 (stearic acid) but it is thought that very little conversion occurs due to high availability of OA in the natural diet. Fish have the enzymes required to desaturate (addition of double bonds) and elongate (addition of carbons) LA to arachidonic acid (20:4n-6, AA) and ALA to the nutritionally important n-3 highly unsaturated fatty acids (n-3 HUFAs) i.e. eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA). This process occurs in the microsomal fraction of the liver through the actions of ∆6 and ∆5 fatty acid desaturases and fatty acid elongases as shown in Figure 1-2. The affinity of the enzymes, especially the desaturases, is higher for the n-3 series than for the n-6 series and this explains why fish often have higher levels of n-3 HUFAs than n-6 HUFAs (Tocher, 2003). The degree or rate at which the desaturation and elongation processes occur is species dependent and also may depend upon the life history stage of the fish as described below. Freshwater fish are generally capable of desaturating and elongating LA and ALA. However, marine fish and diadromous fish (during the salt water phase) have a nutritional requirement for EPA, DHA, and AA because they have suboptimal or no ability to desaturate and elongate LA and ALA (Mourente and Dick, 2002; Rodriguez et al., 2002; Bell et al., 2006). It is suspected  11 that an evolutionary down regulation of these enzymes occurred in the wild since their natural prey are high in EPA and DHA. Marine environments are rich sources of EPA and DHA, which are passed on from diatoms and flagellates at the base of the food chain to zooplankton and finally to fish (Tocher, 2003). By contrast, freshwater fish consume terrestrial and aquatic plants or prey items that have larger amounts of 18:3n-3 and 18:2n-6 (Higgs and Dong, 2000). In marine and diadromous fish, the enzymes required for desaturation and elongation are active in the hepatocytes and in the pyloric caecal enterocytes, but not at rates significant enough to cause changes in the fatty acid profiles of the fish. While all enzymes in the pathway have low reaction rates in saltwater fish, the limiting enzyme differs according to species. In sea bream, the ∆6 desaturase and C18-C20 elongase had the lowest activities (Mourente and Dick, 2005), whereas in Atlantic salmon these enzymes had the highest activities (Bell et al., 2001). Contradictory results in another study showed that the ∆6 desaturase and C18-C20 elongase had the highest activity in seabream (Mourente et al., 2002). Atlantic cod have very slow rates of desaturation and elongation, with elongation from C18 to C20 being the limiting step (Bell et al., 2006). Turbot have been noted to have greater elongation than desaturation rates (Rodriguez et al., 2002). Desaturation and elongation rates typically increase in salt water fish when they are fed diets with high levels of vegetable oil (Tocher et al., 2000; Bell et al., 2001; Rodriguez et al., 2002; Bell et al., 2006), but the rates of the foregoing enzymes are still much lower than in rainbow trout (Tocher et al., 2004). Very little change in the fatty acid profile occurs because β-oxidation rates for ALA and EPA are >100 fold higher than the desaturation and elongation rates (Mourente et al., 2002 ). If desaturation and elongation rates were to increase, there would likely be higher levels of 18:4n-3, 20:4n-3, EPA and DHA, with less of an apparent increase in n-6 polyunsaturated fatty acids (PUFAs) since the enzymes show a preference for n-3 fatty acids, (Sargent et al., 2002). As summarized by Sargent et al. (2002), freshwater fish and marine fish have different essential fatty acid requirements. In general, 1% of the diet must be composed of each of ALA and LA. In marine fish, due to their inability to desaturate and elongate LNA and LA, on average 1-2.5% of the diet must be composed of n-3 HUFAs (Sargent et al., 2002) or 10-20% of the dietary lipid content must be supplied as n-3 HUFAs (Higgs and Dong, 2000) to meet their essential fatty acid  12 needs. In some marine species there also appears to be a need for an optimal dietary ratio of EPA to DHA (Higgs and Dong, 2000).  1.2.2.4 Micronutrients Vitamins and minerals are essential in fish feeds. However, these nutrients are only required in small quantities. Vitamins are organic substances that are essential for growth, health, reproduction and maintenance. There are two groups of vitamins: fat soluble which includes vitamins A, D, E and K, and water soluble e.g., biotin, choline, folic acid, riboflavin, thiamine and vitamin C. Vitamins are important since they facilitate the absorption of minerals, protect cells, and act as co-factors that are required for the metabolism of macro-nutrients. Also, vitamins are necessary for cell respiration (Webster and Lim, 2002). Unlike the situation in terrestrial animals, aquatic species easily absorb minerals from their surrounding aquatic environment. Minerals are important for the fish skeletal structure, electron transfer, regulation of acid-base equilibrium, osmoregulation and are important reaction cofactors (NRC, 1993). The aqueous environment can provide most minerals, but the phosphates and sulfates are often limiting and need to be provided in the diet (NRC, 1993). Of the 90 naturally occurring elements, 29 are essential in animals (Lall, 2002). Mineral deficiencies often occur not because of lack of adequate amounts in the diet, but due to their reduced bioavailability especially when the diets are strongly based upon plant protein sources that contribute substantial amounts of fibre and phytic acid (Roberts, 2002; Richardson et al., 1985; Gatlin, 2000). Mineral deficiencies can lead to problems such as: anemia, poor growth, skin and fin lesions, bilateral lenticular cataract development, erosion of fins, dwarfism (short body), anorexia, dark colouration, bone mineralization (softening), and deformities of the head, vertebrae and ribs (NRC, 1993). When diets are supplemented with minerals, their uptake from the water must be considered because toxicity can also result from excessive dietary concentrations of minerals such as copper, iron, selenium and calcium (Roberts, 2002). 1.2.3 Feed ingredients Fish feeds are typically made of by-products of the food processing industry coupled with fish commodities such as meals and oils derived from the processing of pelagic fish species e.g., anchovies, sardines, herring, capelin, and the Atlantic menhaden (Hardy and Barrows, 2002).  13 Fishmeal and fish oil can comprise as much as 60% and 30%, respectively of the grower diet for salmon and some marine finfish species. These ingredients are commonly utilised because they mimic the natural diet of the fish in the wild. Fishmeal is rich in highly digestible protein that has an optimal amino acid composition. Fish oils and the lipid portion of the fishmeals contain high levels of n-3 HUFAs. Fish feeds currently account for 35%-70% of the cost of salmon farming (Tacon, 2005). With increasing demands for products rich in n-3 HUFAs due to progressive growth of the aquaculture industry and little or no growth in the annual global wild capture fishery and consequently in fishmeal and oil supplies, the costs of these latter commodities are expected to continue to rise in price (Hardy et al., 2001).  1.2.3.1 Fishmeal supply Peru and Chile account for about one-third of the global fishmeal production and these nations provide two thirds of the fishmeal on the global market (Hardy, 2000). The annual worldwide production of fishmeal has remained at about 5.5-6.5 million metric tonnes (MT) over the past decade, and future prospects for growth of this supply remain small. The highest recorded annual production has been 7.5 million MT, with lowest production in El Niño years at around 4.5 and 5 million MT (Hardy, 2000). In 2003, it was estimated that the aquaculture industry utilized 2.9 million MT (Tacon, 2005) and in 2010 the fishmeal needs for aquaculture alone have been predicted to be 4.086 million MT, which will account for about 63% of the total fishmeal supply. Considering that the aquaculture industry currently uses about 46% of the total fishmeal supply, with other industries such as the production of cattle, pigs and poultry using the remainder, there will be shortages in fishmeal supply in the coming years (Tacon, 2005).  1.2.3.2 Fish oil supply The yield of fish oil from industrial fisheries is expected to remain stagnant at about 1.4 million MT (Sargent and Tacon, 1999). In 2003, it was estimated that the aquaculture industry alone was utilising 0.8 million MT of fish oil or 86.6% of the annual global fish oil supply (Tacon, 2005). With continual growth of the aquaculture industry, demand for fish oil from aquaculture alone will soon exceed supply. An extreme estimate suggests that the worldwide demand for fish oil in 2015 will be 145% of the total annual fish oil supply (New and WijkstrÖm, 2002). Climatic effects such as El Niño events off coastal South America could exacerbate this problem.  14 1.2.3.3 Alternatives to fish oil and fishmeal Globally, grains and legumes are grown on 12-15% of the Earths arable surface (Graham and Vance, 2003). Palm oil, soybean oil and rapeseed/canola oil which are currently the world’s largest sources of edible oil, accounted for 37.3, 35.1 and 17.3 million MT of oil, respectively in 2006 (FAO, 2007 ). The quantities of the foregoing oils therefore greatly exceed the estimated 1.4 million MT of fish oil available per year. Worldwide animal offal production in 2006 was 11.0 million MT (FAO, 2007). Since there are few available uses for animal waste products, these ingredients could be used to make up for fishmeal and fish oil shortfalls, while increasing overall sustainability of aquaculture feeds.  1.2.3.4 Costs In 2006, standard South American fishmeals were priced from $1000-1100 USD MT-1, while prime fish meals were about $1200 USD MT-1. On the contrary, vegetable protein sources such as soy meal, canola meal and corn gluten meal could be purchased for $172 USD MT-1, $108 USD MT-1and $312 USD MT-1, respectively (www.thejacobsen.com). As described later, although these ingredients are more economical, inclusion in the diets is limited due to the presence of anti-nutritional factors. Furthermore, unlike the standard and prime fishmeals that contain 65-66% and >67% protein, the vegetable meals contain lower levels of protein, ranging from 38% in soy and canola meals to 60% in corn gluten meal. The protein in these meals can be concentrated to produce plant protein concentrates and isolates, however, prices of these commodities can often be more expensive than fishmeals. Animal protein sources are an inexpensive alternative to fishmeal and can contain similar or higher levels of protein. In 2006, poultry by-product meal (65% protein) could be obtained for $450 USD MT-1, less than half the price of standard and prime fishmeals. Other animal by-products higher in protein, namely porcine blood meal (>85% protein) and feather meal (>80% protein), were sold for $470 USD MT-1 and $260 USD MT-1, respectively. Vegetable oil and fish oil prices vary with crude petroleum oil prices. Crude vegetable oils can often be obtained at prices slightly below fish oil, but when vegetable oils are bleached, deodorized and/or de-gummed, the additional processing steps can often result in finished products with higher prices than fish oil (Figure 1-3). A notable exception is in El Niño years when supply of fish oil is low, which consequently results in higher prices for fish oil. A more  15 recent phenomenon is the demand for fish oil from the pharmaceutical industry. In 2008, fish oil prices tripled from $800 USD MT-1 to over $2200 USD MT-1 (www.thejacobsen.com). Currently the most economical choice for the aquaculture industry is animal fats. Due to lower demand for these commodities, animal fats can be obtained at prices almost half those of fish oils.  1.2.4 Vegetable and animal feed ingredients The efficacy of alternative lipid and protein sources from terrestrial origin as partial or total replacements for fish oil and fishmeal in the diets of aquatic finfish species has been investigated for well over 30 years. Some of the early studies included the examination of beef tallow in carp and rainbow trout diets (Takeuchi et al., 1978), soybean meal and soy protein concentrate in rainbow trout diets (Cho et al., 1976), poultry by-product meal alone or blended with feather meal as well as soybean meal and canola meal in the diets of juvenile coho salmon (Higgs et al., 1979), animal lard in rainbow trout diets (Yu et al., 1977) and plant proteins in the diet of European plaice (Cowey et al., 1971). Lipid sources that are more commonly investigated in the present day include: canola/rapeseed oil, linseed/flaxseed oil, soybean oil and palm oil. Protein sources of recent interest have included: soybean meals/concentrates/isolates, canola/rapeseed meals/concentrates and isolates, cereal grain products such as wheat or corn gluten meal and animal by-product meals. The aquaculture industry has embraced the concept of using alternative feed ingredients, especially in Canada, where fishmeal and fish oil now only comprise 20-25% and 12-20% of the feed for salmon (mainly Atlantic salmon). This contrasts to the UK, where fishmeal levels are 35-40% and mean fish oil levels are 25-30% due to public and industry concerns that the use of alternative feed ingredients will result in decreased flesh quality (Tacon, 2005).  1.2.4.1 Protein content and amino acid composition Many protein meals vary in their crude protein content. Fishmeal, on average, contains 60-70% crude protein, while other protein sources such as corn, wheat, canola meal (prepress solventextracted) and soybean meal (solvent-extracted without hulls) contain 9, 13, 38 or 49% protein, respectively. Poultry by-product has a protein content of >60% and is more similar to some sources of fishmeal in protein content, whereas spray-dried blood meal contains 89% protein (NRC, 1993).  16 Higher protein contents in vegetable ingredients such as canola and soybean can be obtained by removing the soluble carbohydrate fraction (Berk, 2002). The soluble carbohydrates can be separated from the protein by centrifugation to yield concentrates by exposing the pretreated oilseeds or meals to acids, water, enzymes and alcohols. Isolates have even higher protein content than concentrates due to additional processing steps that remove insoluble components such as carbohydrates and antinutritional factors. These protein sources are typically composed of 90% protein on a dry weight basis, but they are high-valued commodities that are rarely used in aquafeeds. The vegetable and animal protein sources also vary in amino acid composition. Fishmeal is considered to be ideal in this regard because its amino acid profile closely mimics the known essential amino acid requirements of fish. By contrast, the amino acid concentrations in plant and animal by-product meals can be low in lysine and/or the sulphur amino acids methionine and cysteine (NRC, 1993). In addition to meeting the amino acid requirements of a fish species, interactions between amino acids must be considered in fish fed diets that contain high levels of some feed ingredients. For example, blood meal is high in histidine, leucine and valine and low in isoleucine. Deficiencies in isoleucine can be amplified when using excess dietary concentrations of blood meal due to an antagonistic relationship that is known to exist between isoleucine and leucine (Tacon, 1984).  1.2.4.2 Fatty acid composition Vegetable oils and animal fats differ from marine fish oils with respect to their compositions of fatty acids affixed to the triacylglycerol molecules. Vegetable oils typically are high in polyunsaturated fatty acids (PUFAs), but do not contain EPA and DHA. Vegetable oils often contain higher levels of omega-6 fatty acids than marine fish oils in the form of LA. Vegetable oil plant crops have historically been bred selectively to contain higher levels of n-6 fatty acids (LA) at the expense of n-3 fatty acids (mainly in the form of ALA) as a means of increasing the oxidative stability of the oils. Animal fats, on the other hand, may be low (poultry fat) or high (beef tallow) in saturated fatty acids and are good sources of monounsaturated fatty acids (OA) and very poor sources of EPA and DHA (the n-3 HUFAs may be undetectable in some sources and present in minimal concentrations in others). Further, some animal lipid sources may contain  17 moderate concentrations of LA (e.g., poultry fat). Vegetable oils and animal fats can also contain different concentrations of carotenoids and vitamin E.  1.2.4.3 Vitamins and minerals Approximately two-thirds of the total phosphorous in oilseed protein meals is present as phytic acid (Gatlin et al., 2007). As will be described in greater detail later, carnivorous fish species like salmon lack the enzyme phytase in their intestinal tract and consequently most of the phosphorous, and some of the calcium, magnesium and zinc are not available for uptake (Dong et al., 2000). Protein meals differ very little in their vitamin composition, but one notable exception is vitamin B12 which is present in fish and animal meals but is virtually absent in vegetable meals (NRC 1993). Alternatively, higher variation exists in mineral composition between protein sources. Plant protein meals, in general, are lower in calcium, phosphorous, chlorine, sodium and zinc and higher in potassium than fishmeals (NRC 1993). The mineral composition of poultry by-product meal is quite similar to that of fishmeal.  1.2.4.4 Carbohydrates Plant protein sources contain carbohydrates and, as previously described, the carbohydrate content can be reduced through processing techniques. However, processing adds additional costs, often making the protein concentrates and especially the isolates more expensive than fishmeal. Removing the carbohydrates from vegetable meals can be beneficial because fish typically have no dietary requirement for carbohydrates (Vangen and Hemre, 2003). Marine fish, in particular, are glucose intolerant, and consequently are unable to rapidly deal with glucose loads (Moon, 2001). Excessive dietary carbohydrate levels have been associated with impaired fish health due partially to the accumulation of glycogen in the liver. High dietary levels of carbohydrates in salmonids can also cause hyperglycemia, reduced fish growth, decreased feed efficiency ratio, lowered energy, protein, lipid and carbohydrate digestibility and increased fish mortality (NRC, 1993; Gatlin, 2000). The carbohydrate intolerance referred to above is not the result of insulin deficiency, since fish tend to have insulin levels that are in the 0.2-5 mM range; values higher than in most mammals (Moon, 2001). Fish have slower rates and lower magnitudes of insulin secretion compared to humans (Stone, 2003). It has been suggested that β-cells in fish do not sense glucose possibly  18 leading to insulin exocytosis (Hemre et al., 2002). Another possibility is that fish have low levels of insulin receptors. For example, rainbow trout tissues have been shown to have from 3-10% of the insulin receptors per microgram of protein compared to the white or red skeletal muscle of rats (NRC, 1993). The recommended dietary inclusion level for carbohydrates is up to 7-20% for carnivorous fish (Vangen and Hemre, 2003). The dietary addition of small quantities of carbohydrates can be beneficial because the energy derived from the catabolism of the carbohydrates can spare amino acids from entering the catabolic energy-providing processes and thereby direct the amino acids into protein synthesis (Stone, 2003). Additionally, starch polysaccharides are good binding agents for the production of hard and durable feed pellets. 1.2.4.5 Fiber Non-starch polysaccharides (NSP) may comprise a large proportion of plant carbohydrates. NSPs, which include cellulose, hemi-cellulose, β-glucans, pectins and gums, are primarily composed of monomers such as glucose, mannose, arabinose and xylose linked by β -glycosidic bonds (Stone, 2003). In plants, NSPs form structural networks, bind water and minerals, exchange cations and absorb organic compounds like sterols and acids (Krogdahl et al., 2005). In fish, NSPs do not supply nutrients but instead may act as anti-nutrients by reducing the utilization of other nutrients. Also, NSPs may increase gut transit time, interfere with enzyme activity and inhibit the uptake of protein and lipid (Stone, 2003; Dong et al., 2000). Cellulase and enzymes that hydrolyze the β-glycoside bonds of non-starch polysaccharides (β-glucanases, βgalactanases and β-xylanases) have not been reported in carnivorous fish (Stone, 2003). The lack of these enzymes limits the type of carbohydrates that fish can digest to only starch polysaccharides. 1.2.4.6 Anti-nutrient factors Plants, as a mechanism of defense, have developed compounds that are commonly known as antinutritional factors. These compounds are toxic, inhibit digestion, or decrease the bioavailability of other nutrients in the protein source (Dong et al., 2000). Soybean meal, for example, contains trypsin inhibitors, hemagglutinins, saponins, phytic acid, phytoestrogens and antivitamins. Rapeseed/canola meal contains glucosinolates, phytic acid and phenolic compounds such as tannins as the main antinutritional factors (Dong et al., 2000). Selective breeding has lowered the glucosinolates levels in Canadian canola meals. Some antinutrient factors such as trypsin inhibitors, phytic acid and hemagglutinins are heat sensitive and can be  19 partially deactivated during the production of protein concentrates or the extrusion pelleting of feeds. Several of the most important antinutrient factors are considered in more detail below.  1.2.4.6.1 TRYPSIN INHIBITORS  Trypsin and chymotrypsin are the two major enzymes of the pancreas. They are serine proteinases and can bind serine during protein digestion (Liu, 1997). Trypsin inhibitors (TIs) are the most common type of antinutrients in soybeans and are large globular proteins (Hendericks, 2002). There are two TIs present in soy, namely, the Kunitz trypsin inhibitor (typical TI) and the Bowman Birk inhibitor. TI’s adversely affect the growth of fish by reducing protein digestibility since they bind with the digestive enzyme trypsin or less commonly chymotrypsin (Dong et al. 2000). 1.2.4.6.2 PHYTIC ACID  Phytic acid (hexaphosphate of myo-inositol, IP6) is a natural plant compound that acts as the primary phosphorous reserve in plants. Phytic acid is utilized during seed germination to support plant growth, and the end products of its hydrolysis are used for cell wall formation (Oatway et al. 2001). However, this form of phosphorous is unavailable to monogastric animals because they lack the enzyme phytase (Dong et al., 2000). Phytic acid is strongly negatively charged (Oatway et al., 2001) and consequently this compound has the capacity to bind tightly to divalent cations such as calcium, magnesium, and zinc. Phytate can also bind to proteins preventing their digestion by enzymes (Sugiura et al., 2001). 1.2.4.6.3 HEMAGGLUTININS  Hemagglutinins or lectins are a group of proteins that are capable of binding to free sugars, or residues of polysaccharides, glycoproteins or glycolipids (including those on cell membranes) (Hendericks, 2002). Their ability to bind to sugars gives these proteins the property of being able to cause agglomeration of erythrocytes (Dong et al., 2000) and other types of cells and this can cause pancreas enlargement, reduction of blood insulin levels, inhibition of the disaccharidase and proteases in the intestine, degenerative changes in the liver and kidneys and interference with the absorption of nonheme iron and lipid from the diet (Liu, 1997). However, the extent to which hemmaglutinins affect fish is questionable because these compounds can be inactivated by stomach acid (Dong et al., 2000) and pepsin (Hendericks, 2002) and are very heat sensitive.  20 1.2.4.6.4 GLUCOSINOLATES  Glucosinolates are derivatives of amino acids, and contain sulphur. Glucosinolates themselves are not harmful compounds but can be hydrolyzed by intestinal microorganisms or by the enzyme myrosinase to yield products such as thiocyanate ions and goitrin, as well as isothiocyanates and nitriles, both precursors of thiocyanate (Dong et al., 2000). Iodide uptake by the thyroid is inhibited by thiocyanate (Dong et al., 2000) and goitrin inhibits the synthesis of thyroid hormones (Hendericks 2002), and thus supplemental dietary iodide in this situation is ineffective. The result of excessive dietary levels of glucosinolates in fish can include depressed growth and feed (protein) utilization as well as hypertrophy and hyperplasia of the thyroid and decreased plasma thyroid hormone titres (Higgs et al., 1995b; Dong et al., 2000). 1.2.4.6.5 SAPONINS  Saponins are a large group of structurally related compounds that contain a steroid or triterpenoid aglycone (sapongenin) linked to one or more oligosaccharide moieties (Messina, 1997). Saponins can decrease growth of chinook salmon, rainbow trout and catfish (Bureau et al., 1998; Twibell and Wilson, 2004). The undesirable tastes of saponins (Twibell and Wilson, 2004) and the notable intestinal damage that has been associated with their dietary consumption (Bureau et al., 1998) have been suggested to be the causes for decreased fish growth.  1.2.5 Effects of alternative dietary feed ingredients in fish feed 1.2.5.1 Growth The use of some dietary protein sources and to a lesser extent, lipid sources have caused negative effects on fish growth. Fish growth can be measured in terms of whole body weight gain, final body weight and percentage increase in body weight gain per day, also known as specific growth rate. Other parameters that are commonly assessed as part of following the growth performance of fish fed different diets include: feed intake, feed efficiency ratio(weight gain relative to feed intake) and protein efficiency ratio (protein gain relative to protein intake). The measurement of especially feed efficiency ratio is very important for fish farmers because when feeds are utilized less efficiently per unit weight gain, more feed will be required and this, in turn, will increase production costs.  21 In most cases, supplemental marine fish oils can be replaced by vegetable oils and/or animal fats provided that the essential fatty acid requirements of the aquatic finfish species have been met. In species such as red hybrid tilapia, rainbow trout, Atlantic salmon and sunshine bass, replacement of 100% of the supplemental lipid with vegetable oils did not result in any growth depression (Bell et al., 2004; Wonnacott et al., 2004; Fonseca-Madrigal et al., 2005; Richard et al., 2006; Bahurmiz and Ng, 2007). Moreover, in some cases, replacement of fish oil with vegetable oil has resulted in increased fish growth (Ng et al., 2004; Torstensen et al., 2005). On the other hand, replacement of 60% or 80% of supplemental marine fish oil in some marine fish studies have caused significant reductions in fish growth (Bell et al., 1999; Regost et al., 2003; Izquierdo et al., 2005; Montero et al., 2005). Replacement of fishmeal with vegetable or animal by-product protein products requires careful formulation of the diets to ensure that the mineral and essential amino acid requirements of the finfish species are met. In some species such as the African catfish (Goda et al., 2007), gibel trout (Yang et al., 2006) and gilthead seabream (Kissil and Lupatsch, 2004) complete replacement of fishmeal has been accomplished using poultry by-product meal, soy protein concentrate, wheat gluten or a blend of wheat gluten with corn gluten and soy protein concentrate. These results contrast with those from many other studies that indicate that fishmeal can only be partially replaced (often at levels <50%) without having any effects on fish growth (Teskeredzic et al., 1995; Kissil et al., 1997; Bransden et al., 2001; Thiessen et al., 2004; Rahnema et al., 2005; Deng et al., 2006; Yigit et al., 2006). The decreased growth of fish ingesting diets based extensively upon various plant protein sources likely results from one or more factors that have been described previously, i.e., diet palatability issues, decreased protein digestibility, amino acid deficiencies and/or imbalances, and mineral deficiencies as a consequence of the inherent composition of the protein and/or the presence of one or more antinutritional factors in the alternative protein product.  1.2.5.2 Flesh quality 1.2.5.2.1 FATTY ACIDS  The fatty acid composition of fish lipids reflect the fatty acid composition of the dietary lipids that are consumed (Dosanjh et al., 1998; Rosenlund et al., 2001; Bell et al., 2003a; Bell et al.,  22 2003b; Bell et al., 2004; Torstensen et al., 2004; Menoyo et al., 2005; Higgs et al., 2006; Kennedy et al., 2007; Martinez-Llorens et al., 2007; de Souza et al., 2007). Fish fed diets with higher replacements of marine fish oil had reduced flesh levels of EPA and DHA due to the lack of these fatty acids in vegetable oils and animal fats. Furthermore, as mentioned previously, because vegetable oils are generally rich in LA and animal fats may contain higher or similar levels of saturated fatty acids to marine fish oil (Ackman, 1990), fish fed alternative dietary lipids will have higher flesh levels of these fatty acids. Since the types and amounts of some fatty acids consumed by humans can influence health, care must be taken when altering the lipid composition of fish feeds to ensure that the potential human health benefits from the consumption of the market-size product are not compromised. One of the first indications that diet could influence the risk of coronary heart disease was observed during the Second World War. In Europe, a significant decrease in heart disease occurred during the war and this corresponded with a decrease in the availability of foods such as meats, butter, cheeses and eggs, which are all components of what is known as the western diet (Allport, 2006). Specifically, the excessive dietary intake of saturated fatty acids was correlated with an increased risk for coronary heart disease (summarized by Hu and Willett, 2002). The consumption of saturated fatty acids results in higher levels of total serum cholesterol and higher levels of low density lipoproteins (Mensink and Katan 1992, Hegsted et al., 1993, Clark and Holt, 1997). Stearic acid is one exception as it does not increase serum cholesterol and is considered neutral (Katan et al., 1994). LA is an essential fatty acid and it has been shown to reduce plasma cholesterol and low density lipoproteins, both factors that lower the risk for coronary heart disease (Mensink et al., 2003). But an optimum dietary ratio of n-6 to n-3 fatty acids of 6:1 (Wijendran and Hayes, 2004) is recommended especially when the dietary intake of EPA and DHA is low. Currently, the North American diet contains excessive LA from a variety of food sources in relation to ALA (Simopoulos, 2004). Similar to fish, humans have the ability to desaturate and elongate both n-3 and n-6 fatty acids. But consumption of high levels of n-6 fatty acids may cause them to compete with the n-3 fatty acids for the desaturases and elongases. As it is, humans have limited ability to convert ALA to EPA and DHA. Females have higher rates of conversion than males, since females can convert  23 up to 9% of consumed ALA to DHA (21% to EPA), while males can only convert up to 4% of ALA to DHA (8% to EPA) (Burdge et al., 2002; Pawlosky et al., 2003a). Conversion of docosaspentaenoic acid (DPA; 22:5n-3) is the limiting step in the elongation process (Pawlosky et al., 2003b). High intakes of EPA and DHA may further limit the conversion of ALA to EPA and DHA due to down regulation of the ∆6-desaturase or competition for the enzyme (Emken et al., 1999; Pawlosky et al., 2003b). EPA and DHA are important fatty acids for cardiovascular health, neural and ocular development, cognitive function and prevention of various inflammatory conditions and types of cancer (Shahidi and Miraliakbari, 2004; Mozaffarian and Rimm, 2006; Narayan, et al., 2006). Indeed, EPA and DHA are effective in lowering plasma triacylglycerides (Sanders and Hochland, 1983; Schectman et al., 1989; Harris, 1997). Also, EPA and possibly DHA are precursors of physiologically important compounds called eicosanoids. These short lived autocrines are involved in inflammation and immunity. Two enzymes are involved in the production of eicosanoids. Cyclooxygenases produce cyclic oxygenated compounds which include prostaglandins and thromboxanes, while lipoxygenases produce linear oxygenated leukotrienes. Eicosanoids derived from AA are generally pro-inflammatory in nature, whereas the EPA-derived eicosanoids are anti-inflammatory and can counteract the effects of those stemming from AA (Tapiero et al., 2002). Eicosanoid production is influenced by the cellular membrane ratio of AA:EPA and a suboptimal ratio may be damaging to the health of both fish and humans (Tocher, 2003).  1.2.5.2.2 FINISHING DIET  The use of a finishing diet based extensively on marine fish oil has been examined as a means to re-instate the levels of EPA and DHA in the flesh lipids of fish that have been previously fed with diets based on alternative lipids. Using this method, fish are fed the less expensive diet containing the alternative lipids for most of their grow-out period and then at some point prior to harvesting the fish, the finishing diet is fed. This approach was first examined in the 1980’s in chinook salmon (Dosanjh et al., 1988) and since this first study it has been tested on a variety of species including the Atlantic salmon (Bell et al., 2003a; Bell et al., 2003b; Bell et al., 2005; Torstensen et al., 2005). There has been some controversy with respect to how quickly EPA and DHA levels in the flesh are restored. The ‘washout theory’ is based on the theory that the fatty acids that are deposited when the fish are consuming the alternative lipid-based diet can be  24 turned over and replaced by those that are present within the marine fish oil when the finishing diet is used. But in reality, there is very little turnover of fatty acids. This process has been more accurately termed the ‘dilution theory’ since the finishing diet results in an increase in EPA and DHA simply because of temporal deposition of these fatty acids and therefore there is dilution in the concentrations of the fatty acids that had been deposited previously as more lipid is stored in the flesh (Jobling, 2003; Robin et al., 2003). In most cases, depending upon the length of time that the fish are fed the finishing diet, EPA and DHA levels can be restored to almost the same levels as are observed in the control fish fed the marine fish oil diet for the whole duration of the grow-out period (Bell et al., 2005; Torstensen et al., 2005).  1.2.5.3 Aroma, flavour, texture and colour Fish nutrition has a direct influence on the flesh quality of the market product such as, its colour, appearance, smell, taste, texture, nutritional quality and shelf life (Lie, 2001). Instrumental techniques can be used to measure colour, texture and aroma compounds. However, human subjects are often the best means for evaluating the overall quality of foods.  1.2.5.3.1 SENSORY PANEL  The training of the sensory panel is a crucial step when conducting sensory analysis because humans can easily be influenced by psychological factors. For example, sample order or differences in sample size or colour may affect how the panelists perceive the sample (Poste et al., 1991). Sensory panels can be conducted to simply determine if there are differences between samples (discriminative tests) or can be used to characterize the differences between samples (descriptive tests). A widely used discriminative test is the triangle test, where panelists receive three coded samples, two of which are identical and then they are asked to identify the odd sample. Descriptive tests require the use of a scale bar to measure the perceived intensity of a certain sensory characteristic, for instance, bitterness. The scale bar can be a structured line with pre-defined categories or numbers. However, the use of a structured scale bar presents some challenges because panelists tend to avoid using the extreme ends of the scales. Furthermore the distance between two descriptors such as ‘very sweet’ and ‘extremely sweet’ may not be the same as the distance between the descriptors ‘low sweetness’ and ‘not sweet’ (Poste et al. 1991). The use of an unstructured scale allows panelists to record intensities at any point along the scale and this approach eliminates the challenges associated with a structured scale. When using the  25 unstructured scales, the two anchor points at either end must clearly be defined and labelled. Quantitative descriptive analysis is a commonly used descriptive sensory analysis method. It requires the development of a sensory language, uses an unstructured scale and allows for analysis of variance (ANOVA) between attributes. Also, the correlation between attributes can be determined (Poste et al. 1991).  1.2.5.3.2 COLOUR  Colour can be measured both by the human eye and instrumentally. The Roche SalmofanTM is often used on farms and in industry to assess the red/pink flesh colouration of salmon. The technique is quick and practical, but cannot be applied to white-fleshed fish such as the sablefish. Furthermore, due to differences in colour perception/colour processing between individuals, instrumental techniques often result in more consistent colour results (Nickell and Springate, 2001). Carotenoids can be measured using techniques such as high performance liquid chromatography and colour can be measured using spectrophotometers and colourimeters. Colour is often reported in CIELAB uniform colour space, which is based on opponent colour theory (Hunter, 1948).. For example, you will never see a reddish-green, nor a yellowish-blue object. Using this method of reporting colour, samples are given L,a,b values where L is the degree of lightness (White = 100, black = 0), while a is the degree oaf greenness (-a) or redness (+a) and b is the degree of blueness (-b) or yellowness (+b) (Hunter, 1948).  1.2.5.3.3 TEXTURE  The texture of fish is defined by its dryness, chewiness and juiciness. The amount and distribution of lipid can affect these characteristics (Lie, 2001). Texture is an important quality attribute, especially when the product looks and smells fine. Texture can be assessed instrumentally with compression tests, shearing tests or puncture tests. The texture profile analysis method (Bourne, 1978) is a commonly utilized technique that measures fracturability, hardness, cohesiveness, adhesiveness, springiness, gumminess and chewiness of food samples. The machine measures these seven parameters using a flat-faced cylinder that is compressed down onto the food sample twice to mimic the chewing action of a jaw. Meanwhile the forcetime curve is measured during the process. However, measuring the texture of fish instrumentally has proven to be challenging (Hyldig and Nielsen 2001; Barroso et al., 1998). Instrumental methods often produce inconsistent results due to slippage of the muscle myotomes  26 during compression (Borderias et al., 1983). Furthermore, fish are most often consumed cooked, so their muscle texture should ideally be measured on cooked fish. Cooking increases the flakiness of the texture and further increase inconsistencies between replicate samples (Barroso et al., 1998). The analysis of flesh texture by sensory panel is often the best choice when fish samples are under evaluation.  1.2.5.4 Effects of alternative lipids on sensory attributes Mixed results have been obtained with respect to the effect of alternative dietary lipids on the sensory attributes of fish. Feeding trials with Atlantic salmon (Hardy et al., 1987; Rørå et al,. 2005; Koshio et al., 1994) and brook charr (Guillou et al., 1995) found no effects on the taste or aroma of the fish when dietary fish oil was replaced with canola or soy oil. Similarly, rainbow trout (Liu et al., 2004) and brown trout (Turchini et al. 2003) fed diets with poultry fat were not different from those fed diets with marine fish oil. By contrast, studies assessing alternative lipids often find that the use of vegetable oils or animal fats in the diet result in fish fillets with a less fishy flavour or aroma. The inclusion of canola oil or soybean oil in the diets of Atlantic salmon (Thomassen and Rosjo, 1989), brown trout (Turchini et al,. 2003), red seabream (Glencross et al., 2003) and turbot (Regost et al., 2003) were noted to significantly affect their flavour and/or aroma. Similarly, when turbot were fed a diet in which 100% of the supplemental lipid originated from flaxseed oil, the fish fed flaxseed oil had ventral fillets with a more intense odor and more fatty fishy odor than fish fed the diet based on marine fish oil (Regost et al., 2003). Also, tench (freshwater fish) fed diets containing flaxseed oil were noted to have less off-flavour than those fed a diet with 100% soy oil (Turchini et al,. 2007).  1.2.5.5 Effects of alternative proteins on sensory attributes Most of the studies that have assessed the effects of alternative dietary protein sources on the sensory attributes of fish have involved soybean meal. Soy can leave a grassy, beany, sour, bitter, or astringent off-flavour in the fillet of a fish due to the action of lipoxygenases or presence of isoflavones (Refstie et al. 2001). A study using rainbow trout found that fish fed a diet with soy protein concentrate were least stale, while those fed diets with soybean meal and soy flour were staler than the fish fed the fishmeal-based diet (Adelizi et al., 1998). In another study, fish  27 receiving the diet with soy had higher levels of rancidity, and lower levels of freshwater flavour and sweetness than those fed the diet with fishmeal (Kaushik et al.,1995). Further, rainbow trout fed a diet containing a blend of plant protein sources, i.e., corn gluten, wheat gluten, extruded peas, and rapeseed meal were harder, less sweet and had a lower odour intensity than those fed a diet with fishmeal (de Francesco et al., 2004). Other studies that have examined the effects of alternative dietary proteins have revealed no effect on sensory attributes. For example, Atlantic salmon fed a diet with full fat soybean meal were not significantly different with respect to their intensity of total flavour, flavour freshness, bitterness, sourness, rancidity or off-flavour attributes from those fed a fishmeal-based diet (Bjerkeng et al. 1997). Additionally, rainbow trout fed diets containing several types of plant protein sources did not differ in their sensory attributes when they were examined fresh and at various times during a 2-week refrigeration storage trial (Ozogul et al., 2006).  1.2.6 Persistent organic pollutants Alternative feed ingredients have the potential to lower flesh levels of persistent organic pollutants (POPs) such as polychlorinated dibenzodioxins (PCDDs) polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs; flame retardants). These compounds are highly lipophilic and have a widespread global distribution. They are ubiquitous in the environment due to their long half-life, lipophilic nature and ability to transfer through the food web. POPs are present in almost every organism on earth and make their way into the atmosphere through the burning of waste, into soil and landfills by rainfall, and into water by contaminated industrial and sewage effluents (Smith and Gangolli, 2002). Because POPs have low water solubility and are lipophilic, they are absorbed and retained in the fatty tissues of fish and invertebrates.  1.2.6.1 Polychlorinated dibenzodioxin/furans (PCDD/F) Dioxins and furans are formed unintentionally as by-products of manufacturing processes or incineration processes. Industrial processes that result in the formation of these chemicals include: manufacturing of PCBs and pesticides, steel mills, metal processing plants and the pulp  28 and paper industry through bleaching of wood pulp with chlorine (Wright and Welbourne, 2002). These toxic compounds are also produced when materials composed of chlorine are burned.  1.2.6.2 Polychlorinated biphenyls (PCB) Unlike dioxins and furans, PCBs were intentionally made. The compounds were used in industrial and commercial applications due to their non-flammability, chemical stability, high boiling point and electrical insulating properties. PCBs were first synthesized in the late 19th century and by 1980 total worldwide production of PCBs was estimated to be in excess of 1 million MT (Smith and Gangolli, 2002). Over 90% of PCBs were produced in the United States under the trade name Arochlor (Wright and Welbourn , 2002). The mounting evidence that PCBs were accumulating in the environment and evidence of their carcinogenicity in animals led many governments to prohibit the manufacturing of these compounds. In 2000, diplomats from 122 countries finalized a treaty eliminating the deliberate production of PCBs. Production in the United States was halted in 1977, but levels in the environment have persisted due to their long half-lives and the continued use of equipment containing PCBs (Wright and Welbourn, 2002). 1.2.6.3 Polybrominated diphenyl ethers (PBDE) PBDEs quickly became the most widely used flame retardant after the ban of PCBs. PBDEs are used in the manufacturing of resins and polymers, used in building materials such as thermal insulation as well as in electronics such as cables, TVs and computers. Also, the compounds are used in household fabrics including carpets and furniture upholstery (Mikula and Svobodova, 2006). PBDEs function as flame retardants by releasing bromine atoms when exposed to heat. The bromine atoms neutralize the energy-rich radicals which prevents them from combusting (Mikula and Svobodova, 2006). Until recently, 70,000 MT of PBDEs were produced every year, with over half of these compounds used in the United States and Canada (Stapleton, 2006). Three commercial groups/types of PBDEs have been manufactured and each differs in the number of bromine molecules. Penta-mixtures contain PBDEs with 4 or 5 bromines, octamixtures contain mostly 7 or 8 bromines, and deca-mixtures are composed of about 97% DecaBDE, with very small quantities of compounds with 8 or 9 bromines. Penta and octa mixtures have been banned in the European Union since 2004. In 2005, the sole manufacturer of PBDEs in the United States voluntarily stopped production of these penta and octa mixtures.  29 1.2.6.4 Chemical structure The aforementioned chemicals are all structurally related (Figure 1- 4). In general, they are all composed of two 6-carbon rings and are halogenated by up to 8 or 10 chlorine or bromine atoms. PCDDs are composed of two benzyl rings that can be chlorinated with up to 8 chlorine atoms, and the two rings are connected by two oxygen bridges. Similarly, PCDFs can be halogenated with 8 chlorine atoms, but the two benzene rings are only connected with one oxygen bridge and a carbon-carbon bond in the place of the other oxygen bridge. PCBs are composed of a biphenyl (two benzene rings connected by a C-C bond) and can have up to 10 chlorine atoms. Finally, PBDEs are composed of two phenyl rings connected by an ether bond and are halogenated with up to 10 bromine atoms.  1.2.6.4.1 CONGENERS  Since these POPs can be halogenated with a number of chlorine or bromine atoms, numerous chemical structures are possible and each configuration is referred to as a congener. The number of congeners varies according to chemical structure. PCDDs have 75 possible congeners and contain the lowest amount of congeners due to the mirror image nature of the compounds. PCDFs have 135 possible congeners, while PCBs and PBDEs each contain 10 possible binding locations for atoms and have 209 possible congeners each.  1.2.6.5 Toxic effects Some POP congeners induce toxic responses including wasting syndrome, reproductive and developmental toxicity, neurotoxicity and hepatotoxic effects including porphyria. Moreover, POPs can cause immune suppression, modulation of endocrine responses, and chloracne and initiate drug-metabolizing enzymes that can both induce and suppress genes (Safe, 1998). Tetrachlorodibenzo-p-dioxin with chlorine atoms substituted at positions 2,3,7 and 8 is the most potent congener of all of the compounds (Van den Berg et al., 1998). This chemical is thought to act as a ligand and binds to an aromatic (aryl) hydrocarbon receptor (AhR) found in the cytosol of cells (Safe, 1998, Mandal, 2005, Larsen, 2006). The AhR and dioxin molecule form a complex with another protein (the Ah receptor nuclear translocator proteins; Arnt) and the complex is translocated to the nucleus. Here, it activates gene transcription by binding to DNA  30 (Mandal, 2005). This can lead to induction of the CYP1A family of enzymes which form more reactive metabolites that exhibit carcinogenicity (Shimada and Fujii-Kuriyama, 2004). In humans, the AhR can be detected in a wide variety of tissues including the placenta, liver and lung as well as primary and established human-derived cell lines (Van den Berg et al, 1998). The ability of a specific congener to induce toxic effects depends on the structure of the compound. The congeners that are more structurally related to 2,3,7,8-tetrachlorodibenzo-pdioxin (2,3,7,8-TCDD) will have a greater receptor-binding affinity to the AhR. For example PCDD and PCDFs with chlorine atoms substituted into position 2,3,7 and 8 are the most toxic due to their highly coplanar (flat) structure. Additional chlorine atom substitutions decrease toxicity by changing the structure of the compound. PCB molecules have the greatest toxicity when there are no chlorine atoms in the ortho position (carbons 2, 2’, 6 or 6’). Mono-ortho PCBs (chlorination of one of the ortho carbons) are still considered to be toxic, but have reduced ability to bind to the AhR due to stearic interactions that reduce the coplanar nature of the compound (Safe, 1998). PCBs with two chlorine atoms in the ortho position do not bind to the AhR (Safe, 1998). Binding affinities of PBDEs to the AhR is not related to the planarity of the molecule (Chen et al., 2001).  1.2.6.5.1 TOXIC EQUIVALENCY FACTORS (TEF)  In order to compare the toxicity of the different congeners, the concept of toxic equivalency factors (TEFs) was developed so that the toxicity of each congener could be compared and then the sum of the toxicities of the individual congeners could be used to estimate the total toxicity of the sample otherwise known as the toxic equivalency (TEQ). Since 2,3,7,8-TCDD is the most toxic congener, it was assigned a TEF of 1.0. All other congeners were subsequently assigned TEF based upon their toxicity relative to 2,3,7,8-TCDD. The compound 2,3,4,7,8-PentaCDF for example, is suspected to be half as toxic as 2,3,7,8-TCDD so it has been assigned a TEF of 0.5 (Van den Berg et al, 1998). Since the toxicity of these congeners is assumed to be additive, the total toxicity of a sample or TEQ can be determined as follows: TEQ = ∑ [PCDDi *TEFi] + ∑ [PCDFi * TEFi] + ∑ [PCBi*TEFi]  31 If a compound is to be assigned a TEF value, it must show structural relationship to 2,3,7,8TCDD, bind to the AhR, cause AhR-mediated biochemical and toxic responses and be persistent and accumulate in the food chain (Van den Berg, 2000). TEF values are discussed and assigned at expert meetings held by the World Health organisation (WHO). TEFs were derived using a database that includes results from studies that have compared the relative potencies of different congeners. TEF values were first assigned in 1993 (Ahlborg et al., 1994), and were re-evaluated in 1997 (Van den berg et al., 1998). The most recent revaluation of TEF values was conducted in June 2005 (Van den Berg et al., 2006). The TEFs are based upon scientific judgment where more weight is given to data obtained from longer-term feeding experiments or studies with endpoints such as cancer and developmental and reproductive toxicity (Safe, 1998). More weight is also given to in vivo toxicity data than in vitro or biochemical data (Van den Berg et al., 1998). Furthermore, ideal studies are those that examine different concentrations so that dose response curves can be developed for both test congeners and for 2,3,7,8-TCDD (Van den Berg et al., 2006).  1.2.6.5.2 TOLERABLE INTAKES  The concept of tolerable daily intake (TDI) was designed as a method to evaluate human health risks associated with contaminants and the values are obtained in a similar manner as TEF values. A meeting was held in the Netherlands in 1990 and a TDI of 10 pg TEQ kg-1 body weight (bw) was established. In 1998, these values were re-examined and a new TDI was set to 1-4 pg TEQ kg-1 bw (Van den Berg, 2000). Since 2,3,7,8-TCDD and related compounds have long half lives and humans may have periodic exposure to higher levels of contaminants, a tolerable weekly intake (TWI) of 14 pg kg-1 bw and a monthly intake of 70 pg kg-1 bw was also established. In Canada, the TDI has yet to be updated and remains at 10 pg TEQ kg-1 bw per day (Health Canada, 1996). However, Canada generally follows TDI’s established by the WHO/FAO Joint Expert Committee on Food Additives and Contaminants (JECFA, 2001). In 2001, JECFA established a tolerable monthly intake of 70 pg TEQ kg-1 bw.  1.2.6.5.3 HUMAN INTAKE  In most cases the global intake of dioxin-like contaminants is below the TDI’s that have been established by the various health organisations. In Asian communities such as China and Japan, where fish consumption is high, total daily intake has been estimated at 1.0-1.7 WHO-TEQ kg–1  32 -1  bw ( Jiang et al., 2007; Mato et al., 2007). Additionally, intake of 1.5 pg WHO-TEQ kg bw has been determined for residents of Finland, another country with high fish consumption (Kiviranta et al., 2004). In the United States (USA), mean intake in 2001-2002 was estimated to be 0.2-0.3 pg-TEQ kg-1 bw day-1 (Charnley and Doull, 2005). The lower levels of contaminant intake are likely the result of lower fish consumption in the USA. In Japan and Finland, fish and shellfish were estimated to contribute 45-90% of the total TEQ consumed (Mato et al., 2007; Kiviranta et al., 2004). Alternatively, in the USA, fish were estimated to account for 5.8% of the total TEQ intake (Charnley and Doull, 2005). Meat products, followed by dairy, fruits and vegetables, and poultry contributed more to the total daily intake than fish products in the USA (Charnley and Doull, 2005). Daily consumption of PBDEs via food in European countries ranges from 0.6 ng kg-1 bw in Finland and Belgium (Kiviranta et al., 2004; Voorspoels et al., 2007) to 1.2-1.306 ng kg-1 bw in Spain and the UK (Bocio et al., 2003; Harrad et al., 2004). In the USA, daily consumption of PBDEs has been estimated to be 0.9 ng kg-1 bw and this value is in the same range as levels consumed in Europe, but PBDE body burdens in Americans are higher than those for European residents (Schecter et al., 2006). In the USA consumption of meat followed by dairy products and fish contribute most highly to the daily PBDE intakes (Schecter et al., 2006). However, household dust concentrations, meat consumption and dairy consumption are significantly correlated to PBDE body burdens in the United States, while fish consumption is not (Wu et al., 2007). 1.2.7 Persistent organic pollutants in fish and feed 1.2.7.1 Bioconcentration, biomagnification, bioaccumulation POPs that are present on land, in the atmosphere and in the water migrate into organisms and may be transported from one organism to the next through the food chain. In fish, the absorption of chemicals can occur through the gills or the gut, but due to the highly lipophilic nature of PCDD/Fs, PCBs and PBDEs, most are absorbed through the gut. The terms used to describe the accumulation of contaminants through the food chain include bioconcentrate, bioaccumulate, and biomagnify. Bioconcentration occurs when contaminants are absorbed from the water directly and the organism accumulates levels of contaminants that are  33 higher than those in the water source. This term does not include contaminants that are absorbed through the food chain. Typically, only the more water-soluble compounds can bioconcentrate because they must be dissolved in water and taken up by the fish’s gills. The octanol-water partition coefficient (Kow) is often used to predict the environmental fate of contaminants. Kow is the ratio of the concentration of a chemical in octanol and in water at equilibrium and at a specified temperature. Chemicals with a Kow >7 are predominantly found in the octanol layer, are highly lipophilic and tend not to bioconcentrate through aqueous media. Chemicals with Kow <3 are generally absorbed by the gills (Heath, 1995). Chemicals with Kow ranging from 3-6 can be absorbed via the gills and the gut (Qiao et al., 2000). Bioaccumulation is said to occur when contaminants are absorbed through the gut and the gills and are present in the organism at a concentration higher than in the water. As indicated above, chemicals with large Kow values are absorbed mostly through the gut. POPs tend to biomagnify when metabolism is the rate-limiting step for the elimination of POPs from the body (Van den Berg, 2000). Biomagnification occurs when the POP concentration in the organism expressed on a lipid-corrected basis exceeds the lipid-corrected concentration in the food consumed. Bioconcentration, bioaccumulation and biomagnification of contaminants can be challenging processes to measure in growing organisms since the elaboration of new tissue can dilute their concentrations, and thus reduce the observed estimates for bioaccumulation or biomagnification (Herendeen and Hill, 2004). Accumulation of PCBs, PCDD, PCDF and PBDEs are favoured through the gut based upon their respective Kow values. Mono and di-PCBs have Kow values of 4.5-5.0 and as the degree of their chlorination increases, so do the corresponding Kow values which can rise to >8.0 in Octa-PCB (Zhou et al., 2005). Similarly, Kow values increase from 5 in low chlorinated and low brominated PCDD/Fs and PBDEs, to >8.0 in Octa-PCCD/F or DecaPBDE (SaÇan et al., 2005; Braekevelt et al., 2003).  1.2.7.2 Levels of persistent organic pollutants in fish Fish tend to have higher concentrations of POPs on a wet weight basis than other food groups such as meats, dairy, and fruits and vegetables (Darnerud et al., 2006; Kiviranta et al., 2004; Schecter et al., 2004). Higher levels of POPs are detected in fish because of bioaccumulation, especially in fish at higher trophic levels in the food chain. Fish also tend to have higher POP  34 concentrations than other organisms simply because they have higher fat contents and more extensive lipid deposition than terrestrial animals. Several recent studies have reported higher flesh levels of POPs or organohalogens in farmed Atlantic salmon from Europe, North America and Chile than in wild Pacific salmon from Canada and Alaska (Easton et al., 2002; Hites et al., 2004; Shaw et al., 2006; Ikonomou et al., 2007). Following the publication of the first two of these reports, negative media reports increased public fear regarding the safety of farmed fish. In fact, importation of farmed fish to the United States decreased by 20% in the first quarter of 2004 following the publication in the paper by Hites and co-workers in the prestigious journal Science (Knapp et al., 2007). The earlier studies ignored several important facts. On a lipid-corrected basis, farmed Atlantic salmon flesh had similar levels of POPs to those determined for the wild Pacific salmon (Ikonomou et al., 2007). In the wild, Atlantic salmon are generally known to have higher lipid contents than those in Pacific salmon species and this also is true in the flesh of farmed Atlantic salmon. Farmed Atlantic salmon have a higher genetic propensity to deposit lipid in their flesh. The majority of the studies (Easton et al., 2002; Hites et al., 2004; Shaw et al., 2006) on this theme have failed to acknowledge that wild Pacific salmon are among the cleanest (least contaminated) salmon in the world and contain much lower levels of POPs in their flesh than wild salmon caught in the Great Lakes (Jackson et al., 2001; Giesy et al., 1999) or in Europe (Isosaari et al., 2006). Furthermore, it should be emphasized that the levels of POPs observed in both the wild and farmed salmon in the aforementioned studies were well below those in Norwegian flounder, shrimp, eel and herring (Knutzen et al., 2003) and lower than in wild fish from Spain and Korea (Moon and Ok, 2006; Bocio et al., 2007). In the study published in Science (Hites et al., 2004), the lowest levels of contaminants were measured in the flesh of farmed Atlantic salmon produced in North and South America. European fish oils on average have POP levels that are 8 times higher than those in Pacific fish oils i.e., 24 versus 3 pg g-1 WHOSUM-TEQ (SCAN, 2000). Also, salmon feeds produced in Canada and Chile contain lower levels of fishmeal and fish oil than those of European origin (Tacon, 2005).  1.2.7.3 Organic contaminants in processed ingredients In most cases, vegetable oils and animal fats are less contaminated than marine fish oils (Eljarrat et al., 2002; Jacobs et al., 2004; Drew et al., 2007) and vegetable protein sources are less  35 contaminated than fishmeal (Drew et al., 2007). However, accidental contamination of these ingredients can occur. In 1995, farmed Mississippi catfish fed diets with soy products had high levels of PCDD/Fs (Cooper et al.1995; Fiedler et al. 1997). Subsequently, it was determined that the ball clay anti-caking agent used in the filtration stage was contaminated. After investigating possible sources of contamination, the source of the PCDD/Fs in the clay was suggested to be a result of contact with lake and/or river sediments contaminated with sewage sludge from southeastern USA (Rappe et al. 1998). The importance of careful selection of feed ingredients can be highlighted further by the recent findings of melamine contamination of fish feed and pet food in North America. In this case, it appears that nitrogen-rich melamine was added by a Chinese company to ‘wheat meal’ as a means of increasing its apparent protein content and was presented for sale as wheat gluten. The elevation of the non-protein nitrogen content resulted in a fallaciously high estimate for the protein content in the product (The Lancet, 2007) since this was determined as % nitrogen x 6.25 (nitrogen generally comprises about 16% of the protein content in feed ingredients). While contamination events are quite rare, the European Union was the first organisation to set guidelines for the maximum permissible levels of POPs in feed ingredients and finished feeds (European Union, 2006). Within this same guideline, action threshold levels were also established. When plant and animal ingredients are above the action levels, the source of contamination must be identified and action must be taken to remove the contamination source. In the case of fishmeal and fish oil, where POPs are expected, no investigation into the source of the contaminants needs to be done when levels are above action levels. However, information such as sampling time, location, and fish species should be recorded. Aquafeed companies also are highly involved in the monitoring of feed ingredients, and those ingredients known to contain POPs, such as fish oils, are routinely monitored for their concentrations of POPs. 1.2.8 Lowering persistent organic pollutants in farmed fish 1.2.8.1 Use of alternative feed ingredients To date, three published studies have examined the use of alternative feed ingredients and the potential for these to reduce contaminant levels in the fish flesh. Two of these studies examined vegetable oil blends as replacements for marine fish oil in Atlantic salmon diets (Bell et al., 2005; Berntssen et al., 2005), while a third study examined the use of a vegetable oil blend and  36 partial replacement of fishmeal with canola protein concentrate in rainbow trout diets (Drew et al., 2007). Bell et al., (2005) using Atlantic salmon, examined diets in which 100% of the supplemental herring/capelin oil was replaced with a 1:1 blend of linseed oil and rapeseed oil. The two oils sources were examined at two dietary lipid inclusions i.e., high lipid (>30% lipid) and low lipid (<20% lipid). The fish were fed the diets formulated with the vegetable oils or fish oil for 115 weeks and then all groups regardless of treatment were fed the high lipid content fish oil diet for another 24 weeks during the finishing period. In the initial 115-weeks, the flesh of fish fed the high lipid content fish oil diet had the highest total TEQ for dioxins and dioxin-like PCBs (2.01 pg g-1), while the flesh of fish fed the low lipid content marine fish oil had the lowest total TEQ (0.68 pg g-1 which was 66% less than detected in control fish). After the finishing diet period, the fish fed the high lipid content fish oil diet for the whole duration of the feeding trial once again had the highest total TEQ (1.61 pg g-1), but fish previously fed the high lipid content vegetable oil diet had the lowest total TEQ (0.86 pg g-1) which was still 47% lower than noted for the control fish. After the initial 115-week feeding period, the n-3 HUFAS were lowered by 72% in fish fed the high lipid content vegetable oil diet relative to those fed the high lipid content fish oil diet. After the finishing diet period, the n-3 HUFAs in the fish that had previously been given the high lipid content vegetable oil diet were restored to 83% of the value observed in the flesh of the preceding control fish. In another feeding trial on Atlantic salmon, Berntssen et al. (2005) in Norway, examined whole fish and flesh levels of PCDD/Fs and dioxin-like PCBs after 22 months of feeding either a diet with 100% of the supplemental lipid as a mixture of rapeseed oil (55%), palm oil (30%) and linseed oil (15%) or a diet in which 100% of the supplemental lipid originated from capelin oil. The fish attained a final bw of ~2kg. At the end of the feeding trial, fish ingesting the vegetable oil diet had muscle TEQ concentrations that were 90% lower than those observed in control fish (~2 pg g-1 versus 0.2 pg g-1), while the amounts of n-3 HUFAs (EPA, DHA and 22:5n-3) in the flesh were only 71% lower than the control fish. Due to the frequent analyses of contaminants in the study, it was possible to determine the factors that influenced the changes in the fish contaminant levels. Fish feed had the greatest influence on the body burdens for contaminants, but decreased fish growth rates and decreased feed efficiencies also increased contaminant levels  37 in the Atlantic salmon. Lipid content, and deposition efficiency of the consumed lipids did not influence contaminant levels. In a 20-week feeding trial, Drew et al. (2007) examined fillet concentrations of PCDD/Fs and dioxin-like PCBs in rainbow trout which had been fed diets where 100% of the supplemental lipid was furnished by menhaden oil or by a 65:35 blend of canola oil and linseed oil. The study also examined the effects of replacing either 0%, 50%, 75%, or 100% of the fishmeal by canola protein concentrate in the diets containing the vegetable oil blend only. Contaminant concentrations in the flesh decreased by ~90% in fish fed the diet with 100% vegetable oil and no fishmeal, while those in fish fed the 100% vegetable oil with 100% fish meal were ~80% lower in total TEQ relative to the levels seen in the fishmeal/fish oil control fish. Corresponding decreases in fillet percentages of EPA and DHA in the two aforementioned treatment groups were not as great as those mentioned for the contaminants since EPA and DHA were respectively only 80% and 67% lower than in the control fish.  1.2.8.2 Dietary protein to lipid ratio As mentioned above, Bell et al. (2004) showed that altering the dietary protein to lipid ratio can lower flesh contaminant levels. Currently high energy feeds, or high lipid feeds, are being used to promote higher growth rates since lipid contains more energy and can be obtained at lower prices. Lowering dietary lipid levels would lower flesh contaminant levels, but fish growth would be compromised unless there were subsequent parallel increases in dietary protein levels. With fishmeal costing $400-600 MT-1 more then fish oil and this strategy would result in substantially higher production costs.  1.2.8.3 Contaminant-reduced fish oil and algae oil Several methods can be employed to reduce the concentrations of POPs in marine fish oils. For example, activated carbon, with a highly porous surface, adsorbs PCBs, PCDD/Fs and PBDEs that have a co-planar conformation (Oterhals et al., 2007). Activated carbon has been shown to lower the concentrations of PCCD/Fs in fish oils by 97-100%, non-ortho PCBs by 70-90% and mono-orthos by only 10-25% (Maes et al., 2005; Oterhals et al., 2007). The efficiency of the process increases with the length of time that the oil is exposed to the activated carbon and if the oil is heated (Oterhals et al., 2007). These factors, however, can increase lipid peroxidation  38 and thus decrease the quality of the oil. Furthermore, processing marine fish oils with activated carbon will increase production costs through capital costs such as the purchasing of filtering equipment (to remove the spent activated carbon) and equipment to heat and mix the oil and the activated carbon which currently costs $7.30 (USD) kg-1 (Norit personal communication). Typically, the activated carbon is used at a concentration of 0.5%, and this adds $0.04 for each kilogram of oil used. Furthermore, additional costs are required to dispose of the spent and contaminated activated carbon. Other options include the use of genetically modified vegetable oils. With current technology, it is possible to obtain vegetable oils with 3% EPA (Napier and Sayanova, 2005). However, public concern with respect to the safety and acceptability of genetically modified foods will likely limit the use of these ingredients. Another option is to use transgenic farmed fish that have the ability to over-express the desaturation and elongation enzymes so that higher flesh levels of EPA and DHA are obtained (Alimuddin et al., 2005; Alimuddin et al., 2007). Also, oils produced from single cells are an upcoming and promising source of n-3 HUFAs (Harel et al., 2002), but these oils are costly, produced in small quantities, and contain high levels of DHA but very little EPA.  1.2.8.4 Handling and cooking techniques The consumption of different regions of the fish could result in lower contaminant intake. For instance, lipid content, PCB content, and PBDE content decreases from the head to the tail of Atlantic salmon (Bayen et al., 2005). Therefore the ingestion of portions of fish from the tail region would lower POP intake. However, this would also decrease the intake of lipid and of EPA and DHA since lipid level decreases from head to tail. By removing the belly fat of fish, it may be possible to decrease POPs while retaining more n-3 HUFAs. In a study that examined the distribution of fatty acids in different parts of Atlantic salmon, n-3 HUFAs were at higher percentage in white muscle than in the belly flap region (Nanton et al., 2007). Moreover, another study on Atlantic salmon found that the belly flap region contained higher levels of PCBs both on a wet weight basis and on a lipid-corrected basis (Persson et al., 2007). Hence, the findings of these studies collectively suggest that the removal of the belly flap could reduce PCBs and raise the relative proportion of n-3 HUFAs, but this idea should be confirmed in a future study that concurrently measures both parameters.  39 The skin of fish contains high levels of lipids (Bayen et al., 2005) and removal of the skin can lower lipid content by up to 90% in low fat species such as chum and coho salmon (Ikonomou, et al., 2007). In addition the removal of skin can lower PCB concentrations by 23-70% in chinook salmon (Zabik et al., 1995) and by 10% in trout (Zabik et al., 1996). Cooking can also lower flesh levels of POPs. Broiling, charbroiling, and salt boiling decreased PCB levels in Great Lake trout by 15%, while hot smoking lowered PCBs by 40% (Zabik et al., 1996). Smoking has also been found to be most effective at lowering PCBs in North Atlantic bluefish (Salama et al., 1998). In this regard, smoking removed 55% of the PCBs while microwaving, charbroiling (skin-off) and charbroiling (skin-on) removed 48%, 29% and 18% of the PCBs, respectively. Smoking was suspected to have more pronounced effects on the removal of POPs because of the longer cook times and higher temperatures involved (Zabik et al., 1996), but this process also results in higher levels of polycyclic aromatic hydrocarbons due to the burning of wood chips (Larsson, 1982). Cooking processes lower POPs through loss of lipid. Indeed, the loss of POPs has been directly correlated to the loss of lipids in a 1:1 ratio (Bayen et al., 2005). No selective retention of n-3 HUFAs occurs during cooking (Al-Saghir et al., 2004). Consequently, the loss of POPs will coincide with an equal loss in EPA and DHA 1.2.9 Contaminants vs. fatty acids 1.2.9.1 Acceptable persistent organic pollutant intake levels Several groups of scientists have evaluated the risks and benefits associated with the consumption of fish products. In most cases, scientists have concluded that the benefits from n-3 HUFAs far outweigh the risks from PCBs and PCDD/Fs. As described previously, the WHO uses the tolerable daily intake to assess contaminant risk and this has been defined as the amount of TEQ from food that a person could consume per day over their whole lifespan that would not cause harm. The TDI was set to 1-4 pg TEQ kg-1 bw day-1 and was determined based on results from accidental human exposure, occupational exposure, background exposure, activation of the AhR, toxicokinetics and laboratory studies involving both non-carcinogenic and carcinogenic effects (Van Leeuwen et al., 2000). In a similar manner, the United States Environmental Protection Agency (US EPA) has developed exposure limits but these express the risk as the dose required to cause 1 extra case of cancer per 100,000 people. The method is considered to be  40 highly conservative as cancer risk was made using the assumption that a linear relationship exists between cancer and exposure and also uses upper confidence interval estimates (US EPA, 2000). If one compares the two risk-based consumption methods, first using the US EPA guidelines, a 70kg person should eat no more then 0.5kg of fish per month when the concentration of POPs in the fish is 0.2-0.3 pg g-1 WHO-TEQ. On the other hand, if one uses the WHO monthly limit of 70 pg kg-1 bw, the same person could consume 16.3-24.5 kg of fish if one assumes no other intake of contaminants. The large difference in the amount of fish that one can consume according to the two risk models is due to the numerous uncertainties involved in determining risk. For example, very little data exist regarding the effects of POPs in humans. Hence, uncertainty factors must be used to extrapolate animal data to humans. The use of different uncertainty factors in the two models has resulted in one model, namely, that based on the US EPA, being much more conservative than the other.  1.2.9.2 Risks versus benefits Even when the more conservative US EPA risk guidelines are used, most scientists conclude there are more benefits from consumption of fish than risks, but careful selection of the type of fish may be required. Evaluation of data from an Italian prevention trial, that involved subjects with coronary artery disease, revealed that for every case of cancer caused by fish consumption, an additional 92 Italians would avoid death from coronary heart disease (Valagussa,1999; Rembold, 2004). Further, in a more recent comprehensive meta-analysis conducted at the Harvard Medical Centre, it was concluded that consumption of farmed or wild Atlantic salmon (1 serving per week to provide 250mg EPA+DHA day-1) could result in 7125 fewer coronary heart deaths per 100,000 people over a 70-year period. Meanwhile the lifetime risk of cancer death was estimated to be only 6 for those who consumed farmed salmon or 2 for those ingesting wild salmon (Mozaffarian and Rimm, 2006). In another risk analysis, the number of lives saved per 100 000 people was estimated to be nearly 300 times greater than the number of excess deaths from cancer if farmed salmon were consumed at levels great enough to give 1g of EPA and DHA per day (Foran et al., 2005). Additionally, epidemiological data collected in Norway did not show any increased risk of cancer in self-reported high consumers of farmed salmon versus those with low farmed salmon  41 intake (Lund et al., 2004). After weighing the health benefits of fish consumption versus the risks, health organizations such as Health Canada maintain that farmed salmon do not pose a risk and are a healthy food choice for part of a balanced diet (Health Canada, 2004). Additionally, following a request from the European Parliament, a scientific panel of experts examined the risks and benefits of fish consumption and concluded that with respect to safety there is no difference between wild and farmed fish (EFSA, 2005). 1.2.9.3 Fatty acid and contaminant dynamics within the fish At the physiological level, no studies have concurrently examined the digestion, absorption, transportation, deposition, metabolism, and excretion of both POPs and fatty acids. Consumed lipids are emulsified by bile salts and intestinal lipases degrade the triacylglycerols into free fatty acids and monoglycerides. The fatty acid breakdown products then diffuse across a thin aqueous layer into the intestinal mucosa where they are converted back to TAGs and transported in chylomicrons via the lymphatic and blood stream to tissues (Nelson and Cox, 2005). The exact mechanisms of POP uptake are unknown. As reviewed by Kelly et al. (2004), some researchers believe that POPs are transported with lipids, while others suggest that the POPs are separated from the lipids in the aqueous layer prior to uptake into the intestinal mucosa. POPs absorbed by this mechanism, would be transported by passive diffusion. Supporting this model, contaminants with a Kow > 7 diffuse less efficiently across the aqueous layer due to highly lipophilic nature of these compounds (McLachlan, 1993; Gobas et al., 1988; Drouillard and Norstrom, 2000; Moser and MacLauchlan, 2001). Other POP absorption models are based on the fugacity theory. Fugacity is a measure of the potential of the chemical to leave the present medium. It is related to both the molar concentration of the substance as well as its solubility in that phase. When the chemical fugacities of two compartments differ, the chemical will flow from high fugacity to low fugacity. The fugacity model and not micelle transportation is supported by the findings that when fish are fed diets with higher levels of lipids, the contaminants are absorbed less efficiently (Gobas et al, 1993). Frequently, contaminants are present at concentrations that are higher in the organism than in the food source, i.e., they have been biomagnified. To explain the transport against possible fugacity barriers, it has been suggested that during the digestion process, lipids may be absorbed prior to the contaminants thereby increasing the fugacity in the gut contents. This, in  42 turn, would cause the contaminants to diffuse into the intestinal wall at a later time (Kelly et al., 2004). Another possibility is that the fugacity capacity of the intestinal cells increases during periods of food digestion (Schlummer et al., 1998). Regardless of the exact fugacity mechanism, if this is indeed the means for controlling POP uptake, it may be possible to lower contaminant uptake in fish by formulating diets using a non-digestible lipid such as olestra. In rats, the use of a non-absorbable oil decreased the fugacity in the intestine causing more contaminants to stay in the gut rather than favoring absorption (Volpenhein et al., 1980). Mobilization of deposited fatty acids generally only occurs in periods of starvation or during sexual maturation and gonad development (Higgs and Dong, 2000). Elimination of contaminants, on the other hand, is driven by chemical half-lives. For the lower chlorinated PCBs, half-lives can be less than 100 days, while higher chlorinated PCBs remain for longer periods of time (Niimi, 1996). In Atlantic salmon, PCDDs and PCDFs are eliminated at rates higher then PCBs (Berntssen et al., 2007). By researching the exact mechanisms of deposition, transport and elimination of both contaminants and fatty acids, it may be possible to design formulated diets that will maximize the deposition of key fatty acids in the fish while simultaneously minimizing the uptake of POPs.  43 1.3 FIGURES 80000  MT  60000 40000 20000 0 1950 1955 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 Year  (www.globefish.org.) Figure 1-1 Global wild sablefish harvest (metric tonnes) with most of the fish harvested from Alaskan waters.  n-9 Series  n-6 Series  n-3 Series  18:1n-9  18:2n-6  18:3n-3  18:2n-9  18:3n-6  18:4n-3  20:2n-9  20:2n-6  20:4n-3  ∆ 6 Desaturase  Elongase  ∆ 5 Desaturase 20:5n-3 Elongase 22:5n-3  22:6n-3  Elongase  B -oxidation 24:5n-3  24:6n-3 Desaturase  Figure 1-2 Desaturation and elongation of omega-9 (n-9), omega-6 (n-6) and omega-3 (n3) fatty acid series and the enzymes involved in the different steps. Adapted from Sargent et al., (2002).  44  Figure 1-3 Oil prices from January 2001 to October 2006 for Peruvian anchovy oil (AO), Chilean AO, refined, bleached and deodorized canola oil (CO), crude-degummed soy oil and poultry fat. Data obtained from www.thejacobsen.com.  (A) Polychlorinated dibenzo dioxin (PCDD)  (C) Polychlorinated biphenyl (PCB)  (B) Polychlorinated dibenzo furan (PCDF)  (D) Polybrominated diphenyl ether (PBDE)  Figure 1-4 Chemical structure of polychlorinated dibenzo-dioxin (a), polychlorinated dibenzofuran (b), polychlorinated biphenyl (c), and polybrominated diphenyl ether.  45 1.4 REFERENCES Ackman, R.G., 1990. Canola fatty acids-an ideal mixture for health, nutrition, and food use. In: Shahidi, F., (Ed.), Canola and Rapeseed, Production, Chemistry, Nutrition and Processing Technology. Van Nostrand Reinhold, New York, pp. 81-98. Adelizi, P. D., Rosati, R. R., Warner, K., Wu, Y. V., Muench, T. R., White, M. R., and Brown, P. B., 1998. Evaluation of fish-meal free diets for rainbow trout, Oncorhynchus mykiss. Aquacult. Nutr. 4, 255-262. Ahlborg, U.G., Becking, G.C., Birnbaum, L.S., Brouwer, A., Derks, H., Feeley, M., Golor, G., Hanberg, A., Larsen, J.C., Liem, A.K.D., Safe, S.H., Schlatter, C., Waern, F., Younes, M., and Yrjanheikki, E. 1994. Toxic equivalency factors for dioxin-like PCBs. Chemosphere 28, 1049-1067. Alderdice, D.F., Jensen, J.O.T., and Velsen, F.P.J., 1988. Preliminary trials on incubation of sablefish eggs (Anoplopoma Fimbria). Aquaculture 69, 271-290. Alimuddin, Yoshizaki, G., Kiron, V., Satoh, S., and Takeuchi, T., 2005. Enhancement of EPA and DHA biosynthesis by over-expression of masu salmon delta 6-desaturase-like gene in zebrafish. Transgenic Res. 14, 159-165. Alimuddin, Yoshizaki, G., Kiron, V., Satoh, S., and Takeuchi, T., 2007. Expression of masu salmon delta 5-desaturase-like gene elevated EPA and DHA biosynthesis in zebrafish. Mar. Biotechnol. 9, 92-100. Allport, S. 2006. The queen of fats: Why omega-3s were removed from the western diet and what we can do to replace them. University of California Press. Berkley, California, pp. 1-222. Al-Saghir, S., Thurner, K., Wagner, K.H., Frisch, G., Luf, W., Razzazi-Fazeli, E., and Elmadfa, I., 2004. Effects of different cooking procedures on lipid quality and cholesterol oxidation of farmed salmon fish (Salmo salar). J. Agr. Food Chem. 52, 5290-5296. Bahurmiz, O.M., and Ng, W.K., 2007. Effects of dietary palm oil source on growth, tissue fatty acid composition and nutrient digestibility of red hybrid tilapia, Oreochromis sp., raised from stocking to marketable size. Aquaculture 262, 382-392. Barroso, M., Careche, M., and Borderias, A.J., 1998. Quality of frozen fish using rheological techniques. Trends Food Sci. Technol. 9, 223-229. Bayen, S., Barlow, P., Lee, H.K., and Obbard, J.P., 2005. Effect of cooking on the loss of persistent organic pollutants from salmon. J. Toxicol. Environ. Health 68A, 253-265. 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Effect of dietary echium oil on growth, fatty acid composition and metabolism, gill prostaglandin production and macrophage activity in Atlantic cod (Gadus morhua L.). Aquacult. Res. 37, 606-617.  46 Bell, J.G., Tocher, D.R., Farndale, B.M., McVicar, A.H., and Sargent, J.R., 1999. Effects of essential fatty acid-deficient diets on growth, mortality, tissue histopathology and fatty acid compositions in juvenile turbot (Scophthalmus maximus). Fish Physiol. Biochem. 20, 263-277. Bell, J.G., Tocher, D.R., Henderson, R.J., Dick, J.R., and Crampton, V.O., 2003b. Altered fatty acid compositions in Atlantic salmon (Salmo salar) fed diets containing linseed and rapeseed oils can be partially restored by a subsequent fish oil finishing diet. J. Nutr. 133, 2793-2801. Bell, J.G., McEvoy, J., Tocher, D.R., McGhee, F., Campbell, P.J., and Sargent, J.R., 2001. Replacement of fish oil with rapeseed oil in diets of Atlantic salmon (Salmo salar) affects tissue lipid compositions and hepatocyte fatty acid metabolism. J. Nutr. 131, 1535-1543. Berk, Z., 2002. FAO Agricultural services bulletin: Technology of production of edible flours and protein products from soybeans. Rome. pp. 1-178. Berntssen, M.H.G., Lundebye, A.K., and Torstensen, B.E., 2005. Reducing the levels of dioxins and dioxin-like PCBs in farmed Atlantic salmon by substitution of fish oil with vegetable oil in the feed. Aquacult. Nutr. 11, 219-231. Berntssen, M.H.G., Giskegjerde, T.A., Rosenlund, G., Torstensen, B.E., and Lundebye, A.K., 2007. Predicting world health organization toxic equivalency factor dioxin and dioxinlike polychlorinated biphenyl levels in farmed Atlantic salmon (Salmo salar) based on known levels in feed. Environ. Toxicol. Chem 26, 13-23. Bjerkeng, B., Refstie, S., Fjalestad, K. T., Storebakken, T., Roedbotten, M., and Roem, A. J., 1997. 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Struc. 755, 137-145.  60  2 Evaluation of cold pressed flaxseed oil as an alternative dietary lipid source for juvenile sablefish* 2.1 INTRODUCTION In recent years, there has been interest in diversifying finfish culture in various regions of the world to include economically valuable marine finfish species. In Canada, farmed salmon represent about 90% of total finfish production (Statistics Canada, 2005) and the farming of marine finfish species could expand traditional markets and contribute to economic renewal in coastal communities. The sablefish (Anoplopoma fimbria), a marine species found in the Pacific Ocean, has a range extending from Mexico to Alaska and from the West coast of North America to Japan. Juvenile sablefish inhabit surface waters, but as they approach weights of 1 kg they seek water depths of up to 1500m. Sablefish, more commonly known as blackcod, is a highly valued finfish due to its high oil content, and white flaky flesh. Japan is currently the world’s largest importer of sablefish but recently there have been increased demands for sablefish in other Asian markets e.g., Korea and Hong Kong, and in North America as a delicacy item (Sonu, 2000). Research aimed at assessing the potential of sablefish as a species for intensive culture began in the late 1960’s. Kennedy (1972) for example, indicated that sablefish can withstand high rearing densities and low dissolved oxygen concentrations. Juvenile sablefish have one of the fastest recorded growth rates of all teleost species (Shenker and Olla, 1986; Sogard and Olla, 2001) and grow well in marine net pens (Gores and Prentice, 1984). While early research indicated great potential for sablefish aquaculture, the industry was limited mainly by the inability to produce marine fish larvae. With successful rearing of sablefish from eggs now possible (Clarke et al., 1999), there is now a need to develop more cost effective diet formulations than those currently used in sablefish farming. Feed represents the single largest operational expense in finfish aquaculture and can account for 35-70% of the costs of fish production. Sablefish grow well on Atlantic salmon (Salmo salar) feeds that are based extensively on fishmeal and fish oil (Sogard and Olla, 2001; Minkoff and *  A version of this chapter has been submitted for publication. Friesen, E.N., Balfry, S.K., Skura, B.J., Ikonomou M.G., Higgs, D.A. 2008. Evaluation of cold pressed flaxseed oil as an alternative dietary lipid source for juvenile sablefish (Anoplopoma fimbria).  61 Clarke, 2003). Fish oil is a rich source of omega-3 (n-3) highly unsaturated fatty acids (n-3 HUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are known to be essential fatty acids for the growth and health of marine finfish species (Kanazawa et al., 1979; Higgs and Dong, 2000). However, the global supply of fish oil is finite and rising demands for this commodity is escalating not only the prices of fish oil, but also the costs of formulated diets (Higgs, 1997). In 2003, it was estimated that the aquaculture industry alone was utilising 86.6% of the annual global fish oil supply (Tacon, 2005). Moreover, with aquaculture growing at a rate of 8.9% per year since 1970 and the global capture fisheries remaining static since 1990 (FAO, 2004), demand for fish oil may exceed supply unless suitable alternatives of plant and/or animal origin are found. Vegetable oils offer a good alternative to fish oil due to their higher availability and lower prices. However, these oils are rich in C18 polyunsaturated fatty acids and lack the n-3 HUFAs that are characteristically high in fish oil. As mentioned above, marine fish have a nutritional requirement for n-3 HUFA’s and have little or no ability to desaturate and elongate the parent acid of the n-3 family of fatty acids viz., linolenic acid (ALA; 18:3n-3) to EPA (Mourente and Dick, 2002; Rodriguez et al., 2002; Bell et al., 2006). Thus the extent to which marine fish oil can be replaced by vegetable oil in the diet of a marine finfish species must be carefully be examined to avoid negative impacts on fish growth and health. The use of alternative lipids may also impact human health since the fatty acid composition of the edible flesh reflects that of the dietary lipid source. From a human health perspective, EPA and DHA are important fatty acids for cardiovascular health, neural and ocular development, cognitive function and prevention of various inflammatory conditions and types of cancer (Connor, 2000; Shahidi and Miraliakbari, 2004; Mozaffarian and Rimm, 2006; Narayan, et al., 2006). Therefore, when fish oil is replaced by vegetable oil, care should also be taken to ensure that the health benefits associated with the consumption of fish are not compromised. One way that this can be accomplished is to choose a vegetable oil that has a favourable fatty acid profile for human health. Flaxseed oil is one such example and is a richer source of 18:3n-3 and poorer source of linoleic acid (LA; 18:2n-6; parent acid of the n-6 family of fatty acids) than either soybean oil or canola oil (Ackman, 1990), two important sources of 18:3n-3 in the North American human diet. Presently, the North American diet contains an excessive amount of omega-6 fatty acids relative to omega-3 fatty acids (Simopoulos, 2004). Although adequate  62 ingestion of the n-3 HUFAs is most important for reducing cardiovascular disease risk factors (Mozaffarian and Rimm, 2006), some benefits in this regard may also be derived from adequate intake of 18:3n-3 (Djoussé et al., 2001; Zhao et al., 2004). The efficacy of flaxseed oil (also referred to as linseed oil) as a dietary lipid source for marine finfish species has been investigated in gilthead seabream (Izquierdo et al., 2003; Izquierdo et al., 2005), European sea bass (Izquierdo et al., 2003; Montero et al., 2005) and turbot (Bell et al., 1994; Bell et al., 1999; Regost et al., 2003). The results from these studies indicate that fish oil can be replaced partially by flaxseed oil. However, high substitution levels of flaxseed oil for fish oil in diets for the preceding species have resulted in reductions in their growth performance possibly due to suboptimal or imbalanced levels of n-3 HUFAs (Bell et al., 1999 ; Regost et al., 2003 ; Izquierdo et al., 2005; Montero et al., 2005). At present, there is no knowledge about the n-3 HUFA requirements for sablefish and it is assumed, because of excellent growth responses of juvenile sablefish fed salmon diets that are based upon fishmeal and oil, that the fatty acid composition of fish oil meets the needs of this species for essential fatty acids. However, it is unknown to what extent the fish oil concentration in diets for sablefish can be reduced through the use of a vegetable oil such as flaxseed oil before there are adverse consequences on the growth performance of the fish. Hence, the objective in this study was to determine whether up to 75% of the supplemental anchovy oil could be replaced by less expensive cold-pressed flaxseed oil in a premium quality diet for juvenile sablefish without compromising their growth, feed efficiency ratio and proximate composition. Additionally, the fish were examined from a flesh quality perspective with respect to the effects of the dietary treatments on the terminal fatty acid compositions of the muscle lipids. 2.2 MATERIALS AND METHODS 2.2.1 Fish husbandry A total of 325 unvaccinated juvenile sablefish (size range, 60-150g) were purchased from Cluxewe Enterprises (Cedar, British Columbia). Subsequently, the fish were transported to the Department of Fisheries and Oceans/University of British Columbia, Centre for Aquaculture and Environmental Research (CAER), West Vancouver, British Columbia, Canada. Upon arrival, the fish were separated into three 1100L fibreglass tanks according to fish weight to minimise size  63 disparity and prevent cannibalism (as seen by Sogard and Olla, 2000). For 7 weeks, the fish were acclimated to the experimental conditions and during this time the fish were fed a commercial Atlantic salmon feed (Ewos Canada Ltd., Surrey, BC, 2mm pellets). On November 24th, 23 sablefish were randomly distributed into each of 12 indoor 1100L fibreglass tanks (range in mean initial weight, 153.4 - 155.6 g). The fish in each tank were subjected to a natural photoperiod (daylight fluorescent lights) and were provided with running (11-15 L min-1), filtered, and oxygenated sea water. During the experiment (November- March) the temperature, dissolved oxygen concentration and salinity of the sea water were measured daily at 1200 hr and these parameters ranged from 7.7 - 11.1 ºC, 7.5 - 10.4 mg L-1 and 28 - 31 ‰, respectively. 2.2.2 Experimental diets and feeding protocol Four highly palatable diets were formulated to contain 45% crude protein and 20% lipid. Chromic oxide (0.5% final concentration) was added to the diets as an indigestible marker. The composition of the basal diet used for the study was similar to that used by Clarke et al. (unpublished data) in a previous nutrition study on juvenile sablefish. The basal diet was prepared at the beginning of the study, and the diets were steam pelleted on three occasions over the course of the study to ensure appropriately sized pellets (4-6 mm) for the growing fish. All diets were identical in composition except for the source of supplemental lipid (134.4g kg-1 diet) . Since the basal ingredients such as fishmeal and squid meal contained some lipid, the supplemental lipid provided 67.7% of the total dietary lipid content which was 198g kg-1 diet (Table 2.1). The control diet was supplemented with 100% South American anchovy oil (diet 1, 100AO) whereas the three experimental diets were supplemented with either 75% AO and 25% cold pressed flaxseed oil (diet 2, 75AO:25FO), an equal mixture of both lipid sources (diet 3, 50AO:50FO) or 25%AO and 75% FO (diet 4, 25AO:75FO). The flaxseed oil was produced at CAER by cold pressing (Gusta 1 HP Model 11 laboratory-scale cold press equipped with a 7 mm die; Gusta Cold Press, St. Andrews, Manitoba, Canada) whole brown flaxseeds furnished by InfraReady Products Ltd., Saskatoon, Saskatchewan. Thereafter, the oil was stabilized with 500 ppm of ethoxyquin (final concentration) and stored in 4L brown bottles under nitrogen at 15ºC. Each of the aforementioned sources of supplemental lipid was sprayed onto batches of the basal diet using an electrically operated sprayer and a cement mixer and then each of the diets was stored at 4°C in an air-tight container. Between diets, the cement  64 mixer was scrubbed with hot water and soap and rinsed until clean. The mixer was then rubbed down with acetone (Anachemia, HPLC grade, 99.9%) and left to dry for over 20 minutes prior to coating the next diet. Each of the four diets was fed to triplicate groups of fish using a randomized complete block design. Fish were fed their prescribed diet by hand twice daily to satiation (beginning at 08:00 and 13:00). The fish were fed two tanks at a time for approximately 20-30 minutes and then left for 20 min to ensure that the fish had time to consume pellets that had fallen to the bottom of the tank. At this point, all uneaten pellets were siphoned off the bottom of the tank into buckets with bottom mesh, and counted. Accurate estimates of the daily ration consumed by each group were subsequently derived by deducting the weight of the uneaten feed (number of pellets x mean pellet air-dry weight) from the total daily feed dispensed in each case. 2.2.3 Fish handling Following 18 hours of starvation, individual fish in each group were weighed and measured (fork length) on day 0 and every 5 weeks thereafter using a dual anaesthetic treatment. Clove oil (0.5 ppm; Hill Tech Canada Inc.) was used to sedate the fish in their rearing tanks, immediately prior to their removal for sampling. The fish were then fully anaesthetized using 150 ppm tricainemethanesulfonate (MS 222; Syndel Laboratories Ltd., Vancouver, BC). To protect the fish from scale loss, a water conditioner (Vidalife, Syndel International Inc.) was used on all surfaces that came into contact with the fish and this was added to the anaesthetic bath (50 ppm). Excess moisture was removed from each fish using an absorbent cloth before weighing. Following weight and length measurements, each fish was placed into an aerated recovery bath, and the entire group was then returned to its respective experimental tank. 2.2.4 Sampling On day 0 of the feeding trial, 12 fish sampled prior to grouping were killed using a lethal dose of MS 222 (>1000 ppm) for determination of whole body proximate analysis (n = 6) and fillet proximate composition and fatty acid analysis (n = 6, right fillets, skinned). On day 105, fish from each tank were selected randomly and killed with a swift blow to the head for measurement of whole body proximate analysis (n = 5), fillet proximate analysis and fatty acid composition (n = 5, right fillets, skinned). Fish samples as well as diet samples were placed into 20.3 cm by 25.4  65 -2  cm gold deli bags (oxygen transmission, 0.7 cc m in 24 hrs at 23°C dry; West Coast FoodPak Systems) that were vacuum-sealed and immediately stored at -20ºC until analysis. Faeces samples were also collected by dissection on day 105. Fecal samples collected from individual fish were pooled by tank to obtain 3 faecal samples per dietary treatment. The samples were frozen at –20°C and freeze-dried. Prior to analysis the samples were homogenized by grinding them using a mortar and pestle. 2.2.5 Chemical analysis Fish samples were thawed overnight at 4ºC. Partially digested feed and faeces were removed from the intestinal tract of each fish. The fish was subsequently cut into small pieces and homogenized in a blender (Braun Type 3210-325) before analysis. Samples from all the experimental diets were finely ground using a coffee grinder before analysis. Whole body, skinned fillets, and feed were analysed in duplicate for proximate analysis according to the procedures of Higgs et al. (2006). Percent nitrogen was multiplied by 6.25 to obtain percentage crude protein. A portion of the lipid/chloroform layer resulting from lipid extraction of each sample prepared according to Bligh and Dyer (1959) was collected and stored at -80ºC in a 10ml glass vial for subsequent determination of fatty acid composition. Fatty acid methyl esters (FAMEs) were obtained from concentrated lipid samples using basecatalysed transesterification (Christie, 1973) and stored in 2ml gas chromatography (GC) vials (Varian) prior to GC analysis. Separation and analysis of FAMES were conducted using a Varian model 3400 GC equipped with a flame ionization detector and CP-Sil 88 fused silica column (Varian). The GC injector and detector temperatures were set at 250ºC and helium was used as the carrier gas at a rate of 1.0 ml min-1. The oven was initially set at a temperature of 60ºC which was raised to 160ºC at a rate of 15ºC min-1. FAMEs were then eluted as the oven increased in temperature at a rate of 4ºC min-1 to 220 ºC. The column was held at this final temperature for 15 minutes for a total run time of 38 minutes per sample. Individual FAME peaks were identified using external standards (FAME mix 37, and other individual standards; Supelco Inc.) and concentrations were calculated as a percentage of the sum of the total identifiable fatty acids. To assess the extent of dietary lipid oxidation, thiobarbituric acid reactive substances (TBARs; secondary products of lipid peroxidation), were measured in all 4 test diets following the  66 completion of the feeding trial according the methods of Tarladgis et al. (1960) as modified by Sutton et al. (2006). Levels of chromic oxide in the diets and faeces were measured according to Fenton and Fenton (1979) and the moisture, protein and ash contents of the faeces were analysed according to Higgs et al. (2006) in duplicate. The gross energy contents of the diets and faecal samples were determined using adiabatic bomb calorimetry (IKA Calorimeter System C5000 duo control, IKA-WERKE, Staufen, Germany). 2.2.6 Data and statistical analyses The effect of dietary treatment on the fish growth performance was assessed by the following: (1) Wet weight gain (WG) (g) = final mean wet weight (FBW) (g) – initial mean wet weight (IW) (g) (2) Specific growth rate (SGR) (g g-1 bw-1 day-1) = [(ln FBW (g) – ln IBW (g))/time (days)] x 100 (3) Dry feed intake (DFI) (g fish-1 day-1) = mean daily dry feed intake/fish over 105 days (4) Feed efficiency ratio (FER) (g g-1) = WG (g)/DFITOT (g fish-1) where DFITOT is the total dry feed intake/fish consumed over 105 days (5) Protein efficiency ratio (PER) (g g-1) = WG (g)/protein intake (g) (6) Percent protein deposited (PPD) (%) = protein gain (g) x 100/protein intake (g) (7) Survival (%) = (number of fish in each group remaining on day 105/initial number of fish) x 100 (8) Hepatosomatic index (HSI) = liver weight (g) x 100/fish weight (g) (9) Condition factor (K) = fish weight (g) x 100/fork length (cm)3 (10) Apparent digestibility coefficients for dietary protein (ADCp), energy (ADCen) and organic matter (ADCorm) (%) = [1-(F/D x Dcr/Fcr)] x 100 were calculated where F = % nutrient (p or orm) or energy content (MJ g-1) of faeces, D = % nutrient (p or orm) or energy content (MJ g-1) of diet, Dcr = % chromic oxide in diet and Fcr = % chromic oxide in faeces (Cho et al., 1985). Statistical analysis was conducted using the mean results for each tank using data from individual fish. The results for each of the preceding parameters were analyzed by randomized  67 block ANOVA using JMP (version 5 release 5.0.1.2). Percentage data (e.g., proximate components, individual fatty acids) were arcsine square root-transformed to achieve normalized distribution of the data and homogeneity of variance before statistical analysis. Tukey’s test with P = 0.05 was used to detect significant differences among means where appropriate. 2.3 RESULTS 2.3.1 Diet composition All test diets had almost identical concentrations of proximate constituents and gross energy content (Table 2.1). However, the dietary fatty acid compositions reflected the differences in the supplemental lipid composition (Table 2.2). Flaxseed oil had higher concentrations of 18:1n-9 (oleic acid), 18:2n-6, 18:3n-3 and lower concentrations of 14:0, 16:0, total saturated fatty acids, and 16:1n-7 relative to anchovy oil. Flaxseed oil also had an absence of 18:4n-3, 20:5n-3, 22:5n3, 22:6n-3 and 20:4n-6. Thus dietary concentrations of 18:1n-9, 18:2n-6, 18:3n-3 and totals for n-3, n-6 and polyunsaturated fatty acids increased in dietary treatments with higher dietary flaxseed oil concentration. The opposite was true for the other fatty acids mentioned above, as flaxseed oil content increased in the diets, the concentrations of 14:0, 16:0, 16:1n-7, 20:4n-6, 20:5n-3, 22:5n-3, 22:6n-3, and the total for n-3 HUFAs decreased (Table 2.2). Although the dietary levels of polyunsaturated fatty acids increased with the use of flaxseed oil, all diets at the end of the study had minimal levels of lipid peroxidation since TBAR values were less than 10 µmoles kg-1 in every case. 2.3.2 Influence of dietary treatment on fish performance, diet digestibility and fish and fillet composition Dietary treatments did not significantly affect values for WG, SGR, K, DFI, FER, PER, and PPD during the 15-week study (Table 2.3). On average, sablefish fed all 4 dietary treatments generally exhibited a 2.7 fold increase in average weight during the study and there were no cases of fish mortality in any of the treatments. Further, terminal fillet and liver weights, and values for HSI (Table 2.3) as well as values for ADCp, ADCorm and ADCen (Table 2.4) and terminal concentrations of protein, lipid, moisture, and ash in the whole bodies and fillets of sablefish (Table 2.5) were not affected by dietary treatment. Mean whole body concentrations of moisture  68 and protein were observed to be lower and lipid higher than respective values observed for the fillets of the sablefish (Table 2.5). 2.3.3 Influence of dietary treatment on terminal fillet fatty acid compositions The terminal fatty acid compositions of the fillets (Table 2.6) generally reflected the fatty acid profiles of the dietary treatments. Examination of the eight fatty acids that were present at the highest concentrations in the sablefish muscle concentrations (Figure 2-1), revealed a strong positive correlation in relation to their respective dietary fatty acid composition in every case. The Pearson R2 values except for 18:1n-9, were greater than 0.98. The slopes for each of the fatty acids shown in Figure 2-1 were less than one. This indicates reduced retention efficiency of each fatty acid in the fillet as the dietary concentration (intake) increased. The range (difference between the highest and lowest concentration) for mean overall concentrations of saturated fatty acids in the fillets of the sablefish across treatments (20.8%27.2%) was less than that observed for the diets (20.6%-33.4%). This was also true for 18:1n-9 where the range was 26.6%-29.0% in the fillets versus 9.5%-16.9% in the diets. No evidence of bioconversion of 18:3n-3 to 20:5n-3 was observed for sablefish in this study since the fillet concentrations of 18:4n-3 across treatments were lower than respective values observed for the diets. Also, the other metabolic derivatives of 18:3n-3 were each inversely related to the dietary flaxseed oil concentration. A similar percent decline was observed for flesh concentrations of n-3 HUFAs when fish fed diets with higher flaxseed oil concentrations. By contrast, the mean overall flesh concentrations of n-3 fatty acids and polyunsaturated fatty acids in the sablefish bore a direct relationship to the dietary concentration of flaxseed oil mostly because of progressive increases in 18:3n-3 as more anchovy oil was replaced by flaxseed oil in the supplemental dietary lipid. Interestingly, the ratios of n-3 to n-6 fatty acids were not significantly influenced by dietary treatment and ranged from 3.12 to 3.24. 2.4 DISCUSSION 2.4.1. Influence of dietary treatment on fish performance The results of this study demonstrate that the growth performance (i.e., growth, feed intake, feed and protein utilization), survival, condition factor (weight/length relationship) and yield of edible  69 tissue of juvenile sablefish were not compromised by any of the dietary concentrations of flaxseed oil that were substituted for AO in this study. Although very few studies have been conducted on sablefish of similar size to those used in the present study, these results agree closely with those of Clarke et al. (1999). Both studies showed that sablefish grew from a weight of 150g to 420 g in about 100 days. Juvenile sablefish of weights less then 10g can have specific growth rates that exceed 10% of body weight day-1 (Sogard and Olla, 2001). These high growth rates appear to be short lived, as the fish used in this study had a maximum growth rate of 1.2 % body weight day-1. While no other studies have examined the use of alternative lipids in diets for juvenile sablefish, vegetable oils have been incorporated successfully into the feeds of other marine species such as red seabream (Glencross et al., 2003), gilthead seabream (Izquierdo et al., 2005), European sea bass (Mourente et al., 2005), and Atlantic cod (Bell et al., 2006). Depending upon the lipid contributed by the dietary protein sources (e.g., fish meal), complete replacement of the supplemental fish oil with vegetable oil has not been successful since marine fish species have essential dietary requirements for n-3 HUFAs (Kanazawa et al., 1979; Higgs and Dong, 2000). Substitution of vegetable oil for either 80% or 100% of the supplemental fish oil in diets for gilthead seabream (Izquierdo et al. 2005) and sea bass (Yildiz and Sener, 2004) respectively, depressed both the growth and feed efficiency ratio of each species. In the present study on sablefish, the dietary n-3 HUFA concentrations ranged from about 2.7% (Diet 25AO:75FO) to 5.7% (diet 100AO) of the dry weight or varied between 14% and 29% of the dietary lipid concentration without any differences in fish performance. However, these n-3 HUFA estimates are likely to be overestimated since fatty acids were not measured quantitatively. The n-3 HUFAs were determined as a percentage of total fatty acids and assumed 100% of the lipid was present as fatty acids. Marine fish generally have n-3 HUFA essential fatty acid requirements at around 1% of the dry diet (reviewed by Sargent et al., 2002), but in some species such as the yellow tail flounder, n-3 HUFA requirements can be as high as 2.5% of the dry diet (Whalen et al. 1999). While the aim of the current study was not to determine the essential fatty acid requirements of sablefish, the results suggest that the preceding range in dietary levels of n-3 HUFAs was adequate for growth of sablefish. But until more work and long-term feeding trials are conducted to define the essential fatty acid needs of sablefish, this conclusion must remain tentative. For instance, other  70 studies on the essential fatty acid needs of marine finfish species indicate there may be optimal dietary levels (and ratios) of 20:5n-3 and 22:6n-3, with some requirement for 20:4n-6 (reviewed by Higgs and Dong, 2000). The estimated apparent digestibility coefficients for protein, energy and organic matter in the test diets were not influenced by dietary treatment. However, the values for each of the foregoing digestibility coefficients were relatively low. This likely occurred because of the faeces collection procedure used. Dissection of faeces can underestimate digestibility coefficients especially for protein due to contamination of samples with intestinal fluids and mucus (Hajen et al., 1993). This undoubtedly occurred in this study but intestinal dissection had to be employed for faecal collection because the sablefish had soft bellies that prevented collection of faeces by stripping. Conducting a separate digestibility trial using the modified “Guelph system” of faecal collection as described by Hajen et al. (1993) would have been ideal, however, it was not possible to obtain a sufficient number of juvenile sablefish. In the study by Clarke et al. (unpublished data) and in similar size sablefish, much higher estimates of digestibility were obtained for protein, organic matter, and energy using diets of similar composition to those employed in this study. Dietary treatment did not influence whole body or fillet proximate compositions. Since there were no differences in dietary protein and energy contents or ratios of digestible protein to lipid or in feed intake among the groups given the different dietary treatments, these results are not surprising (Higgs et al., 1995). Fillet lipid concentrations were similar to those in the test diets (~19%). Sablefish are known for their high lipid contents and can commonly be known as butterfish. In the wild, lipid values of 18.7% (Nakayama et al., 1978) and 15.1% (Stansby, 1976) have been measured. The present results did not suggest a propensity of sablefish to deposit dietary lipid into the liver since liver weights expressed as a percentage of body weight remained less than 3.2% across all dietary treatments. Minkoff and Clarke (2003) also indicated that juvenile sablefish are able to utilize dietary lipid up to 22% of dry matter without enlargement of the liver. The lack of a dietary effect on liver size also suggests that the sablefish were not limiting in essential fatty acids since increases in liver lipid deposition can be associated with essential fatty acid deficiency in marine fish (Izquierdo et al., 2005; Mourenete et al., 2005). However, once again  71 this does not signify that an optimal balance between 20:5n-3, 22:6n-3 and 20:4n-6 was achieved for maximum growth of juvenile sablefish. In relation to possible economic benefits for sablefish culture related to these findings, at the time of this study in 2006, the price of one metric tonne (MT) of fish oil was $800 US. Alternatively, cold pressed flaxseed oil could be produced for US$ 600 MT-1. Realization of the latter price could be achieved by efficiently (80% lipid extraction; Zheng et al., 2005) cold pressing whole brown premium grade flaxseeds (contain 42.1 % oil and 8.3% moisture; Canadian grain Commission, 2006) that cost between US$ 200 and 236 MT-1; prices observed for brown flaxseed in 2006/2007 (Agriculture and Agri-Food Canada, 2006). 2.4.2 Influence of dietary treatment on fillet fatty acid compositions The fatty acid compositions of the sablefish fillets generally mirrored their respective dietary fatty acid compositions. These results are consistent with those of other studies on marine fish that have substituted flaxseed oil for fish oil (Izquierdo et al. 2005; Montero et al. 2005). Some fatty acids, especially the saturated fatty acids, 18:2n-6, and 18:3n-3 may have been preferentially utilized by the sablefish as sources of non-protein energy since reduced ranges for the preceding fatty acids were noted in the fillets across treatments relative to respective values in the test diets. The elevated concentration of 18:1n-9 in the flesh lipids versus dietary lipids of all groups may reflect the high retention of this exogenous fatty acid in the triglyceride fraction of the sablefish muscle over time. Since the sablefish inhabit deep waters as adults, they may have higher rates of ∆9 desaturase which converts stearic acid to oleic acid. Oleic acid has a markedly lower melting point than stearic acid and may regulate the viscosity of cell membranes in these deep cold waters (Tocher, 2003). Progressively lower levels of EPA and DHA were measured in the flesh of sablefish when they were fed diets that contained increased amounts of flaxseed oil by replacement of supplemental anchovy oil. This partially occurred because flaxseed oil does not contain n-3 HUFAs. Additionally, marine fish have insufficient elongase and desaturase activity and consequently do not produce significant amounts of either n-3 HUFAs or arachidonic acid from C18 fatty acids (Rodriguez et al. 2002; Mourente and Dick, 2002; Bell et al., 2006). In this study on juvenile sablefish there was no indication of bioconversion of dietary 18:3n-3 to n-3 HUFAS in the fillet  72 lipids. Adequate consumption of EPA and DHA by humans has numerous health benefits such as prevention of cardiovascular disease, and reduction in the likelihood of some cancers and inflammatory conditions as well as promotion of neural and ocular development and cognitive function (see Introduction). To restore flesh levels of EPA and DHA in sablefish that are fed diets based extensively on alternate lipid sources of plant origin during their commercial culture, it may be desirable to employ a finishing diet period where the fish are fed a 100% fish oil-based diet before their harvest (Bell et al. 2003). 2.5 CONCLUSIONS Flaxseed oil was a cost effective alternative to anchovy oil in sablefish feed under the conditions of this study. Seventy five percent of the supplemental dietary anchovy oil or half of the dietary lipid content (mostly fish oil) was replaced without any negative effects on fish growth, feed intake, feed efficiency ratio, protein utilization, condition factor, survival or the apparent digestibility coefficients for dietary protein, organic matter and energy. The fillet fatty acid compositions were similar to those of the dietary lipid compositions. Although the muscle EPA and DHA concentrations in the sablefish decreased as the dietary flaxseed oil concentration was increased, there were attendant elevations in the concentrations of linolenic acid and total polyunsaturated fatty acids without marked elevations in the linoleic acid concentration that may prove in subsequent studies to have positive benefits for human health.  73 2.6 TABLES Table 2-1 Ingredient and mean (+/- 1 SD; n = 3) proximate composition and gross energy content of each of the dry test diets fed to juvenile sablefish for 105 days. The diets differed only with respect to the percentages of anchovy oil (AO) and cold pressedflaxseed oil (FO) comprising the supplemental lipid. Ingredient (g kg-1 dry weight) LT-anchovy meal Blood flour; spray-dried Squid meal Krill meal Wheat gluten meal Wheat starch; pregelatinized Vitamin supplement a Mineral supplement b Anchovy oilc Flaxseed oil; cold pressed d Soybean lecithin Choline chloride (60%) Vitamin C monophosphate (42%) α-cellulose Permapell (lignin sulfonate binder) DL-methionine Chromic oxide Proximate constituents (g kg-1 dry weight) Dry matter Protein Lipid Ash Gross energy (MJ kg-1 dry matter) a  100AO 389.7 39.1 57.0 77.7 56.8 100.0  Diet 75AO:25FO 50AO:50FO 389.7 389.7 39.1 39.1 57.0 57.0 77.7 77.7 56.8 56.8 100.0 100.0  25AO:75FO 389.7 39.1 57.0 77.7 56.8 100.0  20.0 30.0 134.4 0.0 10.0 5.0 3.6  20.0 30.0 100.8 33.6 10.0 5.0 3.6  20.0 30.0 67.2 67.2 10.0 5.0 3.6  20.0 30.0 33.6 100.8 10.0 5.0 3.6  59.8 10.0  59.8 10.0  59.8 10.0  59.8 10.0  2.0 5.0  2.0 5.0  2.0 5.0  2.0 5.0  927 ± 1.8 460 ± 8.1 196 ± 4.7 95.8 ± 0.5 23.1  927 ± 2.3 460 ± 3.8 204 ± 5.1 95.9 ± 0.5 23.4  929 ± 2.2 460 ± 5.4 197 ± 6.3 95.9 ± 0.6 23.2  929 ± 2.7 460 ± 4.9 197 ± 5.2 96.0 ± 0.5 23.1  -1  Supplemental levels of vitamins (amounts kg dry diet) were: vitamin A (asVitamin A acetate), 5000 IU; vitamin D3, 2400 IU; vitamin E, 300 IU; inositol, 400.0 mg; niacin, 300.0 mg; pantothenate (as D-calcium pantothenate), 165.0 mg; riboflavin, 60.0 mg; pyridoxine (as pyridoxine HCl), 40.0 mg; thiamine (as thiamine mononitrate), 50.0 mg; menadione (as MSBC), 18.0 mg; folic acid, 15.0 mg; biotin, 1.5 mg; vitamin B12, 0.09 mg; BHT, 22mg. -1 b Supplemental levels of minerals (mg kg dry diet) were: potassium (as K2SO4 and K2CO3, 1:1), 2110; magnesium (as MgSO4·7H2O), 250; zinc (as ZnSO4·7H2O), 100; iron (as FeSO4·7H2O), 100; manganese (as MnSO4·H2O), 75; iodine (as KIO3 and KI, 1:1), 10.0; copper (as CuSO4· 5H2O), 5.0; fluorine (as NaF), 5.0; cobalt (as CoCl2·6H2O), 3.0; selenium (as Na2SeO3), 0.1. c Anchovy oil stabilized with 200–250 ppm BHA d Flaxseed oil stabilized with 500 ppm ethoxyquin.  74 Table 2-2 Mean fatty acid contents (g 100g-1 total fatty acids) in anchovy oil (AO) and cold pressed flaxseed oil (FO) and the sablefish test diets (n=3 per dietary treatment, ± 1SD). Different postscripts within a row denote significant differences among the means (p<0.05). Fatty acid 12:0 14:0 15:0 16:0 17:0 18:0 20:0 Σ Saturated 16:1n-7 18:1n-9 18:1n-7 20:1n-9 22:1 24:1n-9 Σ mono-unsaturated 18:2n-6 18:3n-6 18:3n-3 18:4n-3 20:2n-6 20:3n-6 20:4n-6 20:4n-3 20:5n-3 22:4n-6 22:5n-3 22:6n-3 Σ n-3 Σ n-6 n-3/n-6 Σ polyunsaturated Σ n-3 HUFAS  Supplemental lipid sources FO AO 0.17 9.27 0.65 20.4 0.98 3.65 0.31 35.5 10.3 9.11 2.72 1.04 1.75 0.38 25.3 1.45 0.20 1.07 3.08 0.25 0.25 0.86 0.97 16.7 0.53 2.43 11.4 35.7 3.54 10.1 39.2 28.2  0.00 0.06 0.02 5.27 0.00 3.27 0.21 8.84 0.37 22.2 0.00 0.00 0.00 0.00 22.6 13.6 0.00 55.0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 55.0 13.6 4.06 68.6 0.00  Diet 100AO  75AO:25FO a  0.15 ± 0.02 6.99 ± 0.63 a 0.57 ± 0.03 a 20.6 ± 0.31 a 0.90 ± 0.02 a 3.93 ± 0.12 0.25 ± 0.07 a 33.4 ± 0.43 a 7.72 ± 0.74 a 9.48 ± 0.30 c 2.69 ± 0.13 1.80 ± 0.40 a 1.43 ± 0.04 a 0.16 ± 0.01 23.3 ± 0.90 6.53 ± 1.42 c 0.15 ± 0.03 1.08 ± 0.06 c 2.50 ± 0.29 a 0.17 ± 0.03 a 0.14 ± 0.01 a 0.62 ± 0.04 a 0.74 ± 0.07 a 14.0 ± 0.70 a 0.52 ± 0.02 a 1.89 ± 0.12 a 15.0 ± 1.18 a 35.2 ± 0.05 d 8.13 ± 1.36 c 4.33 ± 0.79 43.3 ± 1.29 d 29.0 ± 1.18 a  b  0.10 ± 0.01 5.43 ± 0.11 b 0.46 ± 0.02 b 17.8 ± 1.28 b 0.77 ± 0.08 b 4.06 ± 0.55 0.19 ± 0.12 ab 28.8 ± 1.16 b 5.98 ± 0.14 b 12.2 ± 1.35 b 2.07 ± 0.58 0.75 ± 0.09 b 1 .07 ± 0.08 b 0.20 ± 0.09 22.3 ± 0.41 8.41 ± 0.27 bc 0.12 ± 0.11 11.8 ± 4.20 b 2.04 ± 0.04 ab 0.09 ± 0.03 b 0.10 ± 0.01 ab 0.38 ± 0.10 b 0.51 ± 0.09 b 10.9 ± 0.81 b 0.44 ± 0.09 ab 1.36 ± 0.18 b 12.8 ± 2.33 ab 39.4 ± 0.70 c 9.54 ± 0.35 bc 4.13 ± 0.22 48.9 ± 0.80 c 23.7 ± 2.10 a  50AO:50FO b  0.09 ± 0.01 4.51 ± 0.08 c 0.38 ± 0.01 c 15.0 ± 0.49 c 0.64 ± 0.02 c 3.66 ± 0.04 0.15 ± 0.03 ab 24.5 ± 0.58 c 4.96 ± 0.06 c 14.6 ± 0.42 ab 1.13 ± 0.05 0.54 ± 0.02 bc 0.87 ± 0.01 bc 0.11 ± 0.01 22.2 ± 0.41 9.55 ± 0.44 ab 0.03 ± 0.04 21.5 ± 1.14 a 1.85 ± 0.26 b 0.07 ± 0.00 bc 0.09 ± 0.00 ab 0.42 ± 0.04 b 0.46 ± 0.02 b 8.44 ± 0.15 c 0.31 ± 0.04 bc 1.21 ± 0.03 b 9.34 ± 0.52 bc 42.8 ± 0.69 b 10.5 ± 0.41 ab 4.09 ± 0.20 53.3 ± 0.69 b 17.8 ± 0.64 b  25AO:75FO 0.06 ± 0.00 c 2.95 ± 0.04 d 0.28 ± 0.02 d 13.0 ± 1.29 c 0.50 ± 0.03 d 3.79 ± 0.28 0.06 ± 0.02 b 20.6 ± 1.21 d 3.13 ± 0.09 d 16.9 ± 1.46 a 0.81 ± 0.70 0.25 ± 0.13 c 0.60 ± 0.11 c 0.14 ± 0.06 21.8 ± 0.62 11.5 ± 0.60 a 0.02 ± 0.03 29.5 ± 3.62 a 1.19 ± 0.08 c 0.03 ± 0.01 c 0.07 ± 0.03 b 0.27 ± 0.03 b 0.29 ± 0.01 c 5.82 ± 0.44 d 0.24 ± 0.06 c 0.79 ± 0.06 c 7.87 ± 1.87 c 45.5 ± 1.36 a 12.1 ± 0.74 a 3.75 ± 0.33 57.6 ± 0.68 a 13.7 ± 2.31 b  74  75 Table 2-3 Mean (+/- 1 SD) initial body weight (IBW, g), final body weight (FBW, g), weight gain (WG, g), specific growth rate (SGR, g g-1 bw-1 day-1) dry feed intake (DFI, g fish-1 day-1), feed efficiency ratio (FER, g g-1), protein efficiency ratio (PER, g g-1 protein intake), percent protein deposited (PPD, %), survival (S, %), fillet weight (g), liver weight (g), hepatosomatic index (HSI, %), and condition factor (K) of juvenile sablefish after being fed experimental diets for 105 days. a Performance parameters IBW FBW WG SGR DFI FER PER PPD S (%) Right fillet weight Liver weight HSI K a  Diet 100AO 154.5 ± 0.97 424.8 ± 6.72 270.3 ± 6.83 0.96 ± 0.02 3.33 ± 0.06 0.77 ± 0.03 1.68 ± 0.06 21.3 ± 0.70 100.0 79.4 ± 3.51 13.8 ± 0.25 3.19 ± 0.12 2.02 ± 0.06  75AO:25FO 154.4 ± 0.57 423.5 ± 27.0 269.1 ± 26.9 0.96 ± 0.06 3.45 ± 0.42 0.74 ± 0.02 1.62 ± 0.04 20.0 ± 0.41 100.0 75.1 ± 12.1 12.9 ± 1.77 3.17 ± 0.07 2.00 ± 0.07  50AO:50FO 154.1 ± 0.56 420.9 ± 11.6 266.9 ± 11.1 0.96 ± 0.02 3.27 ± 0.09 0.78 ± 0.02 1.69 ± 0.05 20.9 ± 0.68 100.0 83.2 ± 6.01 13.8 ± 0.38 3.17 ± 0.04 2.02 ± 0.08  25AO:75FO 154.5 ± 0.64 430.4 ± 15.0 275.9 ± 15.1 0.98 ± 0.03 3.40 ± 0.09 0.77 ± 0.03 1.68 ± 0.06 20.5 ± 0.78 100.0 80.8 ± 9.46 13.6 ± 1.63 3.14 ± 0.16 2.07 ± 0.05  The data for each parameter (n = 3) were analyzed by randomized block ANOVA. No significant differences were found (p>0.05) due to dietary treatment among any of the experimental groups for any of the performance parameters.  76 Table 2-4 Mean (± 1SD; n = 3) percent apparent digestibility coefficients (%) for crude protein (ADCp), organic matter (ADCorm) and energy (ADCen) in the test diets fed to juvenile sablefish. The supplemental lipid portion of the diets contained either 100% anchovy oil (AO) or different percentages of AO with cold pressed flaxseed oil (FO).a  Parameter ADCp ADCorm ADCen a  Diet 100AO 81.1 ± 2.36 70.1 ± 2.43 79.2 ± 1.80  75AO:25FO 78.8 ± 5.21 67.7 ± 6.45 77.8 ± 5.15  50AO:50FO 78.4 ± 1.93 67.9 ± 1.45 77.9 ± 1.24  25AO:75FO 77.2 ± 2.01 68.3 ± 1.04 77.4 ± 1.06  The percentages for each of the digestibility coefficients were arcsine square root-transformed and then analyzed by randomized block ANOVA. No significant diet or block effects were found (p>0.05) for any of the digestibility coefficients.  77 Table 2-5 Initial (n = 6 analyzed in duplicate) and final (n = 3, average of 5 fish analyzed in duplicate) mean concentrations (% of wet weight ± 1SD) of proximate constituents in the fillet and whole body of juvenile sablefish fed the test diets for 105 days. The supplemental lipid in the test diets stemmed from either 100% anchovy oil (AO) or different percentages of AO with cold pressed flaxseed oil (FO). a  Source Fillet  Day 0 105 105 105 105  Whole body  a  0 105 105 105 105  Diet 100AO 75AO:25FO 50AO:50FO 25AO:75FO  Moisture 63.3  Proximate constituent Ash Protein 1.20 14.0  Lipid 20.7  66.1 ± 0.74 66.0 ± 1.77 65.1 ± 0.53 65.7 ± 0.85  1.31 ± 0.02 1.31 ± 0.03 1.28 ± 0.04 1.30 ± 0.02  14.5 ± 0.36 14.7 ± 0.33 14.9 ± 0.15 14.9 ± 0.21  18.5 ± 0.46 18.3 ± 2.12 19.7 ± 0.94 18.8 ± 1.05  66.3  2.04  12.9  18.0  1.87 ± 0.08  12.7 ± 0.08  21.2 ± 0.94  1.84 ± 0.08 1.90 ± 0.02 1.87 ± 0.04  12.6 ± 0.08 12.6 ± 0.05 12.4 ± 0.15  21.5 ± 0.94 22.5 ± 0.36 21.5 ± 1.62  100AO 63.8 ± 1.06 75AO:25FO 63.5 ± 1.06 50AO:50FO 63.4 ± 0.55 25AO:75FO 64.5 ± 1.15  The terminal percentages for each proximate constituent were arcsine square root-transformed before randomized block ANOVA. No significant diet or block effects were found (p>0.05).  78 Table 2-6 Terminal mean (± 1SD) fatty acid contents (g 100g-1 total fatty acids) in the fillets of juvenile sablefish fed diets with either 100% anchovy oil (AO) or different percentages of AO and cold pressed flaxseed oil (FO) as the supplemental lipid. a Fatty acid 12:0 14:0 15:0 16:0 17:0 18:0 20:0 Σ Saturated 16:1n-7 18:1n-9 18:1n-7 20:1n-9 22:1 24:1n-9 Σ monounsaturated 18:2n-6 18:3n-6 18:3n-3 18:4n-3 20:2n-6 20:3n-6 20:4n-6 20:4n-3 20:5n-3 22:4n-6 22:5n-3 22:6n-3 Σ n-3 Σ n-6 n-3/n-6 Σ polyunsaturated Σ n-3 HUFAS a  Day 0 0.09 4.04 0.33 16.9 0.61 3.91 0.18 26.0 7.31 31.3 2.79 1.61 0.82 0.02 43.8  100AO 0.09 ± 0.01 a 4.96 ± 0.68 a 0.40 ± 0.05 a 17.6 ± 1.13 a 0.35 ± 0.01 a 3.60 ± 0.08 0.21 ± 0.03 a 27.2 ± 1.63 a 8.07 ± 0.80 a 26.8 ± 1.80 4.06 ± 0.40 a 1.02 ± 0.06 0.55 ± 0.01 0.38 ± 0.04 a 40.8 ± 0.63 a  Diet 75AO:25FO 0.08 ± 0.01 ab 4.31 ± 0.47 ab 0.35 ± 0.03 ab 16.2 ± 0.80 ab 0.29 ± 0.04 ab 3.62 ± 0.10 0.20 ± 0.02 ab 25.1 ± 1.24 ab 6.91 ± 0.42 b 26.6 ± 0.77 3.83 ± 0.57 a 0.97 ± 0.06 0.57 ± 0.05 0.37 ± 0.04 a 39.3 ± 0.19 ab  8.45 1.67 0.21 1.24 0.40 0.20 0.62 0.85 7.44 0.32 1.89 6.85 18.5 11.7 1.58 30.2  5.27 ± 0.18 d 0.27 ± 0.05 1.36 ± 0.07 d 1.69 ± 0.04 a 0.28 ± 0.03 0.23 ± 0.09 1.05 ± 0.09 a 0.90 ± 0.06 a 9.01 ± 0.31 a 0.46 ± 0.03 2.14 ± 0.11 a 9.33 ± 0.52 a 24.4 ± 1.04 c 7.56 ± 0.38 d 3.23 ± 0.04 32.0 ± 1.41 c  6.36 ± 0.40 c 0.26 ± 0.03 6.52 ± 0.47 c 1.45 ± 0.01 b 0.26 ± 0.06 0.18 ± 0.07 0.97 ± 0.08 a 0.82 ± 0.05 ab 7.95 ± 0.37 b 0.37 ± 0.02 1.92 ± 0.15 ab 8.53 ± 0.63 a 27.2 ± 1.52 bc 8.40 ± 0.35 c 3.24 ± 0.29 35.6 ± 1.29 bc  7.37 ± 0.06 b 0.25 ± 0.07 12.1 ± 0.36 b 1.21 ± 0.08 c 0.23 ± 0.02 0.19 ± 0.11 0.75 ± 0.06 b 0.71 ± 0.08 b 6.27 ± 0.26 c 0.31 ± 0.09 1.72 ± 0.25 b 6.89 ± 0.87 b 28.9 ± 1.75 ab 9.10 ± 0.40 b 3.18 ± 0.07 38.0 ± 2.13 ab  8.48 ± 0.08 a 0.23 ± 0.07 17.6 ± 0.55 a 0.93 ± 0.05 d 0.20 ± 0.01 0.16 ± 0.06 0.60 ± 0.10 c 0.58 ± 0.06 c 4.74 ± 0.25 c 0.30 ± 0.12 1.27 ± 0.16 c 6.03 ± 0.75 b 31.2 ± 1.77 a 9.97 ± 0.23 a 3.12 ± 0.11 41.1 ± 1.99 a  14.3  18.3 ± 0.79 a  16.5 ± 0.99 a  13.2 ± 1.12 b  10.8 ± 0.99 c  50AO:50FO 0.06 ± 0.00 b 3.54 ± 0.33 bc 0.30 ± 0.03 bc 15.1 ± 0.86 bc 0.27 ± 0.02 bc 3.64 ± 0.11 0.17 ± 0.02 ab 23.0 ± 1.18 bc 5.79 ± 0.34 c 27.8 ± 0.94 3.42 ± 0.57 ab 0.99 ± 0.03 0.55 ± 0.09 0.31 ± 0.05 b 38.9 ± 1.32 ab  25AO:75FO 0.05 ± 0.00 c 2.71 ± 0.48 c 0.24 ± 0.03 c 13.7 ± 1.13 c 0.23 ± 0.00 c 3.70 ± 0.06 0.17 ± 0.01 b 20.8 ± 1.60 c 4.61 ± 0.52 d 29.0 ± 0.77 2.76 ± 0.73 b 0.93 ± 0.03 0.61 ± 0.03 0.26 ± 0.02 c 38.1 ± 0.39 b  The percentage data for each fatty acid were arcsine square root- transformed and then analyzed by randomized block ANOVA. Different postscripts within a row denote differences among the means (p<0.05). Day 0 fillet fatty acid content is the average of 6 individual fish, while fillet fatty acid content after 105 days is the average of 3 tanks (n=3) per dietary treatment based on the analysis of 5 individual fish per tank.  79 2.7 FIGURES  Fillet (g fatty acid/100g total fatty acids)  14:0 7  16:0  y = 0.516x + 1.045 R2 = 0.980  6  20  5  18  4  16  3  14  2  12 2  3  4  5  6  7  y = 0.433x + 7.334 R2 = 0.997  10  5 3 12  14  16  18  20  22  3  5  20  4  y = 0.534x + 0.237 R2 = 0.992  12 10  0 10  12  9 9  0  10  20  9  14  20:5n-3  10  6  7  y = 0.350x + 20.979 R2 = 0.854  14  30  15 y = 0.485x + 1.748 R2 = 0.979  6  7  4  5 8  10  29  11 9  6  24  y = 0.417x + 2.451 R2 = 0.994  13  8  4  19 22:6n-3  14  8  8  y = 0.694x + 2.113 R2 = 0.985  18:3n-3  y = 0.635x + 0.675 R2 = 0.997  6  24  7  30  4  29  19  18:2n-6 12  18:1n-9  16:1n-7 9  22  12  14  5  7  9  11  13  15  Diet (g fatty acid/100g total fatty acids)  Figure 2-1 Relationship between the concentration of either 14:0, 16:0, 16:1n-7, 18:1n-9, 18:2n-6, 18:3n-3, 20:5n-3 or 22:6n-3 in the diet and the respective subsequent terminal concentration in the sablefish fillet. 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Influence of partial substitution of dietary fish oil by vegetable oils on the metabolism of [1-C-14] 18 : 3n-3 in isolated hepatocytes of European sea bass (Dicentrarchus labrax L.). Fish. Physiol. Biochem. 26, 297-308. Mourente, G., Good, J.E., and Bell, J.G., 2005. Partial substitution of fish oil with rapeseed, linseed and olive oils in diets for European sea bass (Dicentrarchus labrax L.): effects on flesh fatty acid composition, plasma prostaglandins E2 and F2α, immune function and effectiveness of a fish oil finishing diet. Aquacult. Nutr. 11, 25-40. Mozaffarian, D., and Rimm, E.B., 2006. Fish intake, contaminants and human health, evaluating the risks and the benefits. JAMA 296, 1885-1899.  82 Nakayama, Y., Kawai, N., Mori. T., Matsuoka. S., and Akehashi H., 1978. Studies on the muscle lipids of deep-sea fishes (I) Investigation about the lipids and their unsaponifiable matters. (in Japanese). Shokuhin Eiseigaku Zasshi. 19, 68-72. Narayan, B., Miyashita, K., and Hosakawa, M., 2006. Physiological effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) – A review. Food Rev. Int., 291- 307. Regost, C., Arzel, J., Robin, J., Rosenlund, G., and Kaushik, S.J., 2003. Total replacement of fish oil by soybean or linseed oil with a return to fish oil in turbot (Psetta maxima) - 1. Growth performance, flesh fatty acid profile, and lipid metabolism. Aquaculture 217, 465-482. Rodriquez, C., Perez, J.A., and Henderson, R.J., 2002. The esterification and modification of n-3 and n-6 polyunsaturated fatty acids by hepatocytes and liver microsomes of turbot (Scophtalmus maximus). Comp. Biochem. Physiol. 132B, 559-570. Sargent, J.R., Tocher, D.R., Bell, J.G. 2002. The lipids. In: Halver, J.E., Hardy, R.W. (Eds). Fish Nutrition. Academic Press, New York, pp. 181-257 Shahidi, F., and Miraliakbari, H., 2004. Omega-3 (n-3) fatty acids in health and disease: Part ICardiovascular disease and cancer. J. Med. Food 7, 387-401. Shenker, J.M., and Olla, B.L., 1986. Laboratory feeding and growth of juvenile sablefish, Anoplopoma fimbria. Can. J. Fish. Aquat. Sci. 43, 930-937. Simopoulos, A.P., 2004. Omega-6/omega-3 essential fatty acid ratio and chronic diseases. Food Rev. Int. 20, 77-90. Sogard, S.M., and Olla, B.L., 2000. Effects of group membership and size distribution within a group on growth rates of juvenile sablefish Anoplopoma fimbria. Environ. Biol. Fishes. 59, 199-209. Sogard, S.M., and Olla, B.L., 2001. Growth and behavioural responses to elevated temperatures by juvenile sablefish Anoplopoma fimbria and the interactive role of food availability. Mar. Ecol.-Prog. Ser. 217, 121-134. Sonu, S.C., 2000. Japanese supply and market for sablefish. NOAA Technical Memorandum, NOAA-TM-NMFS-SWR-037. 61 pp. Stansby, M.E., 1976. Chemical characteristics of fish caught in the northeast Pacific Ocean. Mar. Fish. Rev. 38 (9), 1-11. Statistics Canada. 2005. Aquaculture statistics 2004.Catalogue no. 23-222-XIE. 46pp. Sutton, J., Balfry, S., Higgs, D., Huang, C.-H., and Skura, B., 2006. Impact of iron-catalyzed dietary lipid peroxidation on growth performance, general health and flesh proximate and fatty acid composition of Atlantic salmon (Salmo salar L.) reared in seawater. Aquaculture 257, 534-557. Tacon, A.G.J., 2005. State of information on salmon aquaculture feed and the environment. Report prepared for the WWF US Initiated Salmon Aquaculture Dialogue. 80p. Available at: www.worldwildlife.org/cci/dialogues/salmon.cfm Tarladgis, B.G., Watts, B.M., and Younathan, M.T., 1960. A distillation method for the quantitative determination of malondialdehyde in rancid foods, J. Am. Chem. Oil Soc. 37, 44–48. Tocher, D.R., 2003. Metabolism and functions of lipids and fatty acids in teleost fish. Rev. Fish. Sci. 11, 107-184. Whalen, K.S., Brown, J.A., Parrish, C.C., Lall, S.P and Goddard, J.S. 1999. Effect of dietary n-3 HUFA on growth and body composition of juvenile yellowtail flounder. (Pleuronectes ferrugineus). Bull. Aquacult. Assoc. Can. 98, 21-22. Yildiz, M., and Sener, E., 2004. The effect of dietary oils of vegetable origin on the performance, body composition and fatty acid profiles of sea bass (Dicentrarchus labrax L. 1758) Juveniles. Turk. J. Vet. Anim. Sci. 28, 553-562.  83 Zhao, G., Etherton, T.D., Martin, K.R., West, S.G., Gillies, P.J., and Kris-Etherton, P.M., 2004. Dietary -linolenic acid reduces inflammatory and lipid cardiovascular risk factors in hypercholesterolemic men and women. J. Nutr. 134, 2991-2997. Zheng, Y.L., Dennis P. Wiesenborn, D.P., Tostenson, K., and Kangas, N., 2005. Energy analysis in the screw pressing of whole and dehulled flaxseed. J. Food Eng. 66, 193-202.  84  3 Evaluation of poultry fat and blends of poultry fat with cold pressed flaxseed oil as alternative dietary lipid sources for juvenile sablefish* 3.1 INTRODUCTION As discussed previously in Chapter 2, sablefish (Anoplopoma fimbria) show great potential for marine aquaculture, but apart from their dietary needs for protein and energy very little is known about their nutritional requirements. In the previous study (Chapter 2), it was determined that 75% of the supplemental marine fish oil (MFO) in a marine protein and MFO-based dry diet for juvenile sablefish could be replaced with cold-pressed flaxseed oil without adversely influencing their growth performance, survival and whole body and flesh proximate composition. It was also determined in this study that cold pressed flaxseed oil could be an economical replacement for MFO. However, this depended upon whether or not the flaxseeds were cold pressed as part of the feed mill operation. Existing cold pressed flaxseed oil stemming from cold pressing operations is presently used as a high value source of essential fatty acids (especially linolenic acid, ALA, and to a much lesser extent linoleic acid, LA) in the human diet. In this study, we examined the potential for using an inexpensive rendered animal lipid source namely, poultry fat, alone or together with cold pressed flaxseed oil as economical replacements for 75% of the supplemental MFO in a premium quality diet for sablefish. Poultry fat, unlike cold pressed flaxseed oil, offers great potential to improve the cost effectiveness of sablefish culture since the cost of poultry fat is far below that of MFO (Higgs et al., 2006). Poultry fat has successfully been used in feeds for trout (Greene and Selivonchick, 1990; Turchini et al., 2003; Liu et al., 2004) and Atlantic salmon (Rosenlund et al., 2001; Higgs et al., 2006) and like flaxseed oil, this animal lipid source can be included in the diets of the preceding species without compromising their growth performance and health provided that their essential fatty acids needs are concurrently met. However, flaxseed oil and poultry fat contain respectively either no eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) or low concentrations of these fatty acids. Hence, the replacement of MFO with  *  A version of this chapter has been submitted for publication. Friesen, E.N., Balfry, S.K., Skura, B.J., Ikonomou M.G., Higgs, D.A. 2008. Evaluation of poultry fat and blends of poultry fat with cold pressed flaxseed oil as alternative dietary lipid sources for juvenile sablefish (Anoplopoma fimbria).  85 flaxseed oil, poultry fat or blends of these lipid sources will reduce flesh levels of the aforementioned fatty acids which are known to have important human health benefits. The present study comprehensively assessed the nutritive value and cost effectiveness of using poultry fat or blends of poultry fat with cold pressed flaxseed oil as replacements for 75% of the supplemental anchovy oil in a premium quality diet for juvenile sablefish. Fish performance in this 105-day study was assessed by following the effects of dietary treatment on sablefish growth, feed intake, feed and protein utilization, percent survival, dietary protein, energy and organic matter digestibility, whole body and muscle proximate composition, liver weight in relation to body weight, fillet fatty acid composition and production costs. 3.2 MATERIALS AND METHODS 3.2.1 Source and husbandry of experimental fish In December 2003, 600 juvenile sablefish (~300g) were obtained from the Department of Fisheries and Oceans, Pacific Biological Station in Nanaimo, British Columbia (BC). Subsequently, the fish were acclimated to outdoor 4000L fibreglass tanks at the Department of Fisheries and Oceans/University of British Columbia, Centre for Aquaculture and Environmental research (CAER; 49º 15’N, 123º 10’W) and until the start of the study the sablefish were fed Ewos Vita 5 mm unpigmented salmon feed. On April 27, 2004, the fish (mean initial wt., 422 g.) were distributed randomly into 12-4000L outdoor fibreglass tanks so that each contained 30 fish. The tanks were supplied with running (50-60L min-1), filtered, and oxygenated seawater. Additionally, each tank was fitted with landscape fabric on its cover and on the sides of the cover to prevent direct entry of sunlight into the tank, since sablefish at this stage of their life history exhibited clear avoidance of bright light. Over the course of the 15-week experiment (April August) the water temperature, dissolved oxygen content and salinity of the seawater were measured daily at noon and these parameters varied between 9.8-14.0 ºC, 6.5 –10.6 ppm and 2831 g L-1, respectively. 3.2.2 Experimental diets and feeding protocol The experimental feeds were formulated and manufactured at CAER and were identical in ingredient composition except for the supplemental lipid sources (Table 3.1). The latter were  86 chosen on the basis of the results from the previous feeding trial (Chapter 2) which, once again, showed that 75% of the supplemental anchovy oil (AO) in a premium quality diet for sablefish could be replaced by cold-pressed flaxseed oil (FO) without having any negative effects on their growth and other aspects of their performance. Poultry fat (PF) alone or mixed with FO were selected as major sources of supplemental dietary lipid for sablefish in this study because in BC this lipid is much lower in cost than either FO or MFO (Personal communication: West Coast Reduction Ltd., Vancouver, BC). Thus, as more PF was included in the supplemental lipid at the expense of MFO and/or FO, the diets became progressively less expensive. The percentage composition of each of the supplemental lipid sources always contained 25% South American AO (source; Skretting Canada, Ltd., Vancouver, BC) in an attempt to ensure that the dietary needs of the sablefish for essential fatty acids were met from this source as well as from the residual lipids furnished by the fishmeal, squid meal, and krill meal. The supplemental lipid treatments examined in this study were as follows: diet 1, 100% AO (100AO; control diet); diet 2, 25% AO, 50% FO, 25% PF (25AO:50FO:25PF); diet 3, 25% AO, 25% FO, 50% PF (25AO:25FO:50PF); diet 4, 25% AO, 75% PF (25AO:75PF). FO was prepared at CAER using whole brown flaxseeds (InfraReady Products Ltd., Saskatoon, Saskatchewan) that were cold pressed using a Gusta 1HP Model 11 Cold Press (7mm die, 7000Hz; St. Andrews, Manitoba, Canada). Subsequently, the cold pressed oil was stabilized with ethoxyquin (500 ppm) and then stored in 4L brown bottles under a nitrogen atmosphere at 15 ºC. In addition, it should be mentioned that AO and PF were stabilized with 200-250 ppm BHA and 250 ppm of BHA/BHT, respectively and PF was held at 4ºC. Chromic oxide (5g kg-1) was included in each diet as an indigestible indicator during the last week of the study. All diets were mixed and steam pelleted (6mm die) 7 times during the course of the study using the procedures of Higgs et al. (1979). Each of the aforementioned sources of supplemental lipid was sprayed onto batches of the basal diet using an electrically operated sprayer and a cement mixer and then each of the diets was stored at 4°C in an air-tight container. In between oiling each diet, the cement mixer was scrubbed with hot water and soap and rinsed until clean. The mixer was then rubbed down with acetone (Anachemia, HPLC grade, 99.9%) and left to dry for over 20 minutes prior to coating the next diet.  87 The four test diets were assigned to the different groups of fish using a randomized complete block design (n=3 groups per dietary treatment). Subsequently, the fish were fed their prescribed diet by hand twice daily (0800 and 1300) to apparent satiation. Pellets were left on the bottom of the tanks for 30 minutes following each feeding time and then the uneaten pellets were siphoned into buckets with bottom mesh to be counted. Accurate feed intakes were recorded daily for each group by deducting the weight of waste feed (number of uneaten pellets x air-dry mean weight of the pellets for a given dietary treatment) from the amount of feed dispensed each day. During the final week of the feeding trial, the fish were fed the diets containing the chromic oxide to determine their respective apparent digestibility coefficients for protein, energy and organic matter. 3.2.3 Fish handling The fish were individually weighed (to nearest 0.1 g) and measured (to nearest 0.1 cm) on days 0, 35, 70 and 105 following 18 hours of starvation. Before weighing and measuring, all fish in each tank were sedated using 0.5 ppm clove oil (Hilltech Canada Inc., Vankleek Hill, Ontario, Canada). Thereafter the fish were removed from their respective tank and were fully anaesthetized using 150 ppm tricainemethanesulfonate (MS 222; Syndel Laboratories Ltd., Vancouver, BC). To protect the fish from scale loss, a water conditioner (Vidalife, Syndel International Inc., Vancouver, BC) was used on all surfaces that came into contact with the fish during sampling. Vidalife (50 ppm) was also added to the anaesthetic bath. Further, fish were blotted dry with an absorbent towel before the sablefish were weighed and measured (fork length). Following the weighing and measuring procedure, the fish were allowed to recover before returning the fish to their respective experimental tank. 3.2.4 Sampling On day 0 of the feeding trial, 12 fish common to all groups were killed using a lethal dose (>1000 ppm) of MS 222 for subsequent determinations of concentrations of whole body (n=6 fish) and fillet proximate constituents and fillet fatty acid compositions (n=6 fish, skinned right fillets). On day 105, fish from each tank were selected randomly and then killed by a swift blow to the head for determinations of concentrations of whole body (n=5 fish) and fillet proximate constituents and fillet fatty acid compositions as well as various haematological and innate immunological parameters (n=5 fish; skinned right fillets used for the proximate and fatty acid  88 analyses; health results will be reported elsewhere). Fish samples, as well as diet samples, were placed into 20.3 cm by 25.4 cm gold deli bags (oxygen transmission, 0.7 cc m-2 in 24 hrs at 23°C dry; West Coast FoodPak Systems, Vancouver, BC), that were vacuum-sealed and then stored immediately at -20ºC pending analysis. Faecal samples were collected by intestinal dissection (Hajen et al., 1993) of 15 fish from each tank on day 105. The faecal samples from the individual fish in each tank were pooled to obtain 1 sample per tank and 3 samples per dietary treatment. 3.2.5 Chemical analysis Feed samples were ground in a coffee grinder and fish samples were thawed overnight at 4ºC and then homogenized in a blender (Braun Type 3210-325 blender) before proximate analysis. Feed and undigested material were removed from the digestive tract of each whole fish before homogenization. Samples were analyzed in duplicate according to Higgs et al. (2006). Briefly, 2g samples were weighed into porcelain crucibles and heated at 100ºC for 16 hours in a drying oven and at 600ºC for 2 hours in a muffle furnace to obtain moisture and ash content respectively. Crude protein was measured using a micro Kjeldahl method (Technicon industrial method No. 334-74W/B, revised March, 1977; Technicon Industrial Systems, Tarrytown, NY, USA) and percent nitrogen was multiplied by 6.25 to obtain the percentage of protein. Crude lipid content was obtained using the chloroform/methanol procedure of Bligh and Dyer (1959). In the case of each fillet, a portion of the lipid/chloroform layer was collected and stored at -80ºC in a 10ml glass vial for determination of fatty acid composition. To ensure the stability of dietary lipids, thiobarbituric acid reactive substances (TBARs), which are secondary products of lipid peroxidation, were measured in all 4 test diets following the completion of the feeding trial according the methods of Tarladgis et al. (1960) as modified by Sutton et al. (2006). The stored lipid and chloroform samples originating from fillet analyses were concentrated by nitrogen evaporation to obtain 0.06 g of lipid. Subsequently, the samples were base-catalysed transesterified using benzene and sodium methoxide (Christie, 1973). Then the fatty acid methyl esters (FAMEs) were stored in 2ml gas chromatography (GC) vials (Varian) at -80ºC prior to gas chromatography (GC) analysis. Separation and analysis of FAMEs was conducted using a Varian model 3400 GC equipped with a flame ionization detector and CP-Sil 88 fused silica column (Varian). The GC injector and detector temperature were set at 250ºC and helium was used as the carrier gas. The column was initially set at a temperature of 60ºC and this was raised  89 -1  to 160ºC at a rate of 15ºC min ; FAMEs were then eluted as the column increased in temperature at a rate of 4ºC min-1 to 220 ºC. The column was held at this final temperature for 15 minutes for a total run time of 38 minutes per sample. Individual FAME peaks were identified using external standards (FAME mix 37, and other individual standards; Supelco Inc., Bellfonte, USA) and concentrations were calculated as a percentage of the sum of the total identifiable fatty acids. 3.2.6 Energy and protein digestibility The faecal samples that been collected by intestinal dissection were frozen at –20 °C and then freeze-dried. Samples were homogenized before analysis by grinding them using a mortar and pestle. Levels of chromic oxide were measured according to Fenton and Fenton (1979) and the moisture, protein, and ash contents were analysed in duplicate as described previously (Higgs et al. 2006). The gross energy contents of the faeces and test diets were measured by adiabatic bomb calorimetry (IKA Calorimeter System C5000 duo control, IKA-WERKE, Staufen, Germany). 3.2.7 Data and statistical analyses The effect of dietary treatment on the growth performance of the fish was assessed by the following: (1) Wet weight gain (WG) (g) = final mean wet weight (FBW) (g) – initial mean wet weight (IBW) (g) (2) Specific growth rate (SGR) (g g-1 bw-1 day-1) = [(ln FBW (g) – ln IBW (g))/time (days)] x 100 (3) Dry feed intake (DFI) (g fish-1 day-1) = mean daily dry feed intake/fish over 105 days (4) Feed efficiency ratio(FER) (g g-1) = WG (g)/DFITOT (g fish-1) where DFITOT is the total dry feed intake/fish consumed over 105 days (5) Protein efficiency ratio (PER) (g g-1) = WG (g)/protein intake (g) (6) Percent protein deposited (PPD) (%) = protein gain (g) x 100/protein intake (g) (7) Survival (%) = (number of fish in each group remaining on day 105/initial number of fish) x 100 (8) Hepatosomatic index (HSI) = liver weight (g) x 100/fish weight (g)  90 3  (9) Condition factor (K) = fish weight (g) x 100/fork length (cm)  (10) Apparent digestibility coefficients were calculated for dietary protein and energy (ADCen) (%) = [1-(F/D x Dcr/Fcr)] x 100 where F = % nutrient (p) or energy content (MJ g-1) of faeces, D = % nutrient (p) or energy content (MJ g-1) of diet, Dcr = % chromic oxide in diet and Fcr = % chromic oxide in faeces (Cho et al., 1985). The results for each of the preceding parameters were analyzed by randomized block ANOVA using JMP (version 5 release 5.0.1.2). Percentage data (e.g., proximate components and individual fatty acids) were arcsine square root-transformed to achieve a normal distribution of the data and homogeneity of variance before statistical analysis. Tukey’s test with P = 0.05 was used to detect significant differences among means where appropriate. 3.3 RESULTS 3.3.1 Diet composition All of the test diets contained similar concentrations of moisture, ash, protein, lipid and gross energy (Table 3.1). Moreover, the estimated dietary concentrations of digestible protein and energy as well as the ratios of digestible protein to energy were similar in all diets since the apparent digestibility coefficients for protein (ranged from 86.8%-88.9%) and energy (varied between 83.6% and 86.4%) showed little variation between diets (Table 3.1). Likewise, the apparent digestibility coefficients for organic matter in the diets varied little (ranged from 76.4% -80.6%). The dietary fatty acid compositions were influenced strongly by the fatty acid compositions of each of the supplemental lipid sources i.e., AO, FO, and PF and their respective concentrations in the supplemental lipid (Table 3.2). For instance, saturated fatty acids in the supplemental lipid sources followed the sequence AO > PF > FO and in the test diets, the observed sequence was 100AO > 25AO:75PF > 25AO:25FO:50PF > 25AO:50FO:25PF. The monounsaturated fatty acids in the supplemental lipid sources were dominated by 18:1n-9 (OA) and this fatty acid followed the sequence PF > FO > AO. Thus the diets that were richest in PF, 25AO 75PF followed by 25AO:25FO:50PF had the highest levels of OA and totals for monounsaturated fatty acid whereas diet 100AO had the lowest. The n-3 fatty acids were dominated by ALA in FO (54%) and n-3 HUFA in AO (25.4%) with EPA > DHA. By contrast PF contained negligible  91 concentrations of n-3 HUFAs and while the level of ALA was low (1.39%) it was nevertheless higher than in AO (1.09%). The sequence noted for ALA in the test diets viz., 25AO:50FO:25PF > 25AO:25FO:50PF > 25AO:75PF > 100AO, reflected the preceding trends noted for this fatty acid in the dietary lipids. Likewise, the three test diets that contained 25%AO in the supplemental lipid had significantly reduced concentrations of EPA, DHA and n-3 HUFAs relative to their respective levels in diet 100AO. The aforementioned differences in dietary fatty acid composition did not result in differences in their susceptibility to lipid oxidation. All four test diets had TBAR values that were less than 7 µmoles kg-1. 3.3.2 Effect of dietary treatment on fish performance and whole body and fillet composition Replacement of 75% of AO in the supplemental dietary lipid with PF or blends of PF with FO did not affect values for FBW, WG, SGR, DFI, FER, PER or PPD, percent survival, fillet or liver weights, HSI or K (Table 3.3). But several of these parameters decreased with time when there were attendant increases in fish size and water temperature. The mean weights of the sablefish rose throughout the study along with water temperature which reached a maximum of 14°C (Figure 3-1). However, the foregoing trends were accompanied by progressive declines in values for SGR that were noted for fish across all treatments. The reductions in SGR values were observed to mainly result from significant declines in values for FER and PER during weeks 5 to 10 and in values for DFI during weeks 10 to 15 (Figure 3-1). While the sablefish used in this study were not pit-tagged to permit their individual identification, it is probable that some of the fish in all groups did not grow during the last 5 weeks of the study and some appeared to even lose weight. Some evidence for this viewpoint was obtained by ranking the fish in each tank in order of their descending weights and then comparing the weights of the fish at the different sampling intervals. For example, in one group fed 100AO, the 3 smallest fish at the end of week 10 weighed 368.5, 495.1 and 516.9g while at week 15, the three smallest fish in this group weighed, 393.3, 487.1 and 500.4g. Dietary treatment did not significantly influence the terminal concentrations of whole body or fillet proximate constituents in the sablefish. The values observed for whole body protein and ash ranged respectively from 11.9-12.6% and 1.82-1.98% and these ranges were respectively lower and higher than for the fillets i.e., 14.1-15.8% and 1.39-1.52%. The ranges observed for  92 concentrations of lipid in the whole bodies versus the fillets were similar viz., 20.6-24.0% and 20.1-22.8%, respectively and this was also true for moisture contents i.e., 61.5-63.8% and 60.662.2%. The fillet (muscle) and whole body concentrations of lipid in the sablefish were directly related to their weight (Figure 3-2). 3.3.3 Influence of dietary treatment on terminal fillet fatty acid compositions The terminal fatty acid compositions in the fillets of the sablefish (Table 3.4) were strongly influenced by the fatty acid compositions of their respective dietary lipids. For example, an examination of the eight fatty acids that were most prevalent in the sablefish flesh, showed a strong positive correlation (Pearson R2 values ≥ 0.96) with the corresponding dietary fatty acid concentration (Figure 3-3). The slopes for each of the fatty acids shown in Figure 3-3. Two of the fatty acids, myristic (14:0) and palmitic (16:0), had slope values of 0.293 and 0.216, respectively indicating that large increases in the dietary concentrations of these two fatty acids resulted in only small increases in their respective concentrations in the muscle. The range found for the concentrations of total saturated fatty acids in the fillets of the fish varied narrowly (23.225.5%) compared to that noted for the dietary lipids (22.2-32.3%). Similar to the trends described above for the saturated fatty acid concentrations in the flesh versus the dietary lipids, the range noted for the flesh concentrations of total monounsaturated fatty acids (45.8-51.6%) was narrower than that observed for the dietary lipids (24.6-39.2%). However, in contrast to the saturated fatty acids, the totals for the monounsaturated fatty acids in the flesh lipids of the fish were also higher than their respective values observed in the dietary lipids due to extensive deposition or retention of especially OA in the flesh lipids (Figure 3-3) and to a lesser extent 16:1n-7 (Figure 3-3). The greatest retention of OA relative to the concentrations of this fatty acid observed in the dietary lipids was noted in the flesh of fish fed the diets with the lowest monounsaturated fatty acid contents because of either no addition of PF to the supplemental lipid (diet 100AO) or a low level of inclusion of this lipid source (diet 25AO:50FO:25PF). Nevertheless, the flesh concentrations of OA and total monounsaturated fatty acids were still directly related to the dietary con