"Land and Food Systems, Faculty of"@en . "DSpace"@en . "UBCV"@en . "Schlicht, Alberto"@en . "2009-11-17T00:00:00"@en . "2003"@en . "Master of Science - MSc"@en . "University of British Columbia"@en . "Salmon farming is a flourishing aquaculture industry throughout the world and\r\nrelies on intensive husbandry practices and well balanced diets to succeed. As in\r\nagriculture, genetics, health, husbandry, and nutrition are the main pillars of a\r\nsuccessful and sustainable industry. Astaxanthin, a xanthophyll pigment with\r\nantioxidant properties, is incorporated into farmed salmon diets to provide the desirable\r\nred colour in flesh. Dietary antioxidant supplements (i.e. vitamins, flavonoids) have been\r\nshown to fight free radicals normally formed during aerobic metabolism and generated\r\nin excess during some physiological and pathological processes (i.e. exercise, disease).\r\nThis research was conducted to investigate the role of dietary supplementation,\r\nwith grape seed extract and astaxanthin, on pigmentation, growth and the immune\r\nresponse, of pre-smolt and post-smolt chinook salmon spp maintained in freshwater\r\n(FW) and saltwater (SW). Four experimental diets were formulated that contained\r\nuniform amounts of astaxanthin (60 ppm) and one of two concentrations (low and high)\r\nof grape seed extract (Kikkoman Proanthocyanidins, KPA\u00AE). The control diet (no\r\nastaxanthin, no KPA\u00AE), astaxanthin diet (astaxanthin, no KPA\u00AE), low KPA\u00AE diet\r\n(astaxanthin, 100 ppm KPA\u00AE), and high KPA\u00AE diet (astaxanthin, 1000 ppm KPA\u00AE) were\r\nfed for 32 days to pre-smolt chinook salmon in FW tanks, and 155 days to post-smolt\r\nchinook salmon in SW growout seacages.\r\nPre-smolts fed fortified diets with the antioxidants KPA\u00AE and astaxanthin, showed\r\na significantly (p=0.043) larger weight gain than fish groups fed the control diet after 32\r\ndays. There were no significant weight differences among the groups ingesting the\r\nantioxidant-fortified diets. The specific growth rates (SGR) and feed conversion ratios\r\n(FCR) were not significantly different between groups fed the four experimental diets in\r\nFW. Conversely, muscle astaxanthin concentrations were significantly higher (p=0.019)\r\nin groups fed the astaxanthin-containing diets compared to baseline values and fish fed\r\nthe control diet. There were no significant differences either in the concentration of\r\nastaxanthin in fish muscle or on the apparent astaxanthin retention coefficients (AARC)\r\nin salmon fed either the astaxanthin, low or high KPA\u00AE diets.\r\nAfter a feeding period of 155 days in SW, post-smolts fed the high KPA\u00AE diet had\r\na significantly higher deposition of astaxanthin in muscle (p=0.036), higher visual colour score in fillets as measured by the Roche Salmofan\u00AE (p=0.029), and greater wet weight\r\ngain (p<0.001) than those fed the other three diets.\r\nFollowing a disease challenge with Vibrio anguillarum, pre-smolts fed the high\r\nKPA\u00AE diet had a significantly lower (p=0.038) cumulative mortality among diet\r\ntreatments, as well as a significantly greater (p=0.025) number of circulating leucocytes\r\n(p=0.025), neutrophils (p=0.018), and monocytes (p=0.034). Furthermore, the lysozyme\r\nactivity both in plasma and head kidney, and the neutrophil respiratory burst activity\r\nwere significantly greater in fish fed the high KPA\u00AE diet, (p=0.029, p=0.037, and\r\np=0.027, respectively).\r\nIt is concluded that the addition of antioxidants (proanthocyanidins and\r\nastaxanthin) to a chinook salmon diet significantly enhanced the humoral and cellular\r\nnon-specific immune response factors and was related to a lower cumulative mortality\r\nafter the disease challenge. Furthermore, the deposition efficiency of the carotenoid\r\nastaxanthin, and growth-related variables in farmed chinook salmon were also\r\nsignificantly affected by the combination of dietary antioxidants."@en . "https://circle.library.ubc.ca/rest/handle/2429/15076?expand=metadata"@en . "6398882 bytes"@en . "application/pdf"@en . "ENHANCEMENT OF THE NON-SPECIFIC IMMUNE RESPONSE, PIGMENTATION AND GROWTH OF FARMED CHINOOK SALMON (ONCORHYNCHUS TSHA WYTSCHA) FED A COMBINATION OF DIETARY FLAVONOIDS (GRAPE SEED EXTRACT, KPA\u00C2\u00AE) AND ASTAXANTHIN by Alberto Schlicht. MV Medico Veterinario, Universidad Austral de Chile (Valdivia, Chile), 1997 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE D E G R E E OF MASTER OF SCIENCE in THE FACULTY OF G R A D U A T E STUDIES (Animal Science) We accept this thesis as conforming Aits'the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 2003 \u00C2\u00A9 Alberto Schlicht, 2003 U B C Rare Books and Special Collections - Thesis Authorisation Form Page 1 of 1 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada http://www.library.ubc.ca/spcoll/thesauth.html 11/17/03 ABSTRACT Salmon farming is a flourishing aquaculture industry throughout the world and relies on intensive husbandry practices and well balanced diets to succeed. As in agriculture, genetics, health, husbandry, and nutrition are the main pillars of a successful and sustainable industry. Astaxanthin, a xanthophyll pigment with antioxidant properties, is incorporated into farmed salmon diets to provide the desirable red colour in flesh. Dietary antioxidant supplements (i.e. vitamins, flavonoids) have been shown to fight free radicals normally formed during aerobic metabolism and generated in excess during some physiological and pathological processes (i.e. exercise, disease). This research was conducted to investigate the role of dietary supplementation, with grape seed extract and astaxanthin, on pigmentation, growth and the immune response, of pre-smolt and post-smolt chinook salmon spp maintained in freshwater (FW) and saltwater (SW). Four experimental diets were formulated that contained uniform amounts of astaxanthin (60 ppm) and one of two concentrations (low and high) of grape seed extract (Kikkoman Proanthocyanidins, KPA\u00C2\u00AE). The control diet (no astaxanthin, no KPA\u00C2\u00AE), astaxanthin diet (astaxanthin, no KPA\u00C2\u00AE), low KPA\u00C2\u00AE diet (astaxanthin, 100 ppm KPA\u00C2\u00AE), and high KPA\u00C2\u00AE diet (astaxanthin, 1000 ppm KPA\u00C2\u00AE) were fed for 32 days to pre-smolt chinook salmon in FW tanks, and 155 days to post-smolt chinook salmon in SW growout seacages. Pre-smolts fed fortified diets with the antioxidants KPA\u00C2\u00AE and astaxanthin, showed a significantly (p=0.043) larger weight gain than fish groups fed the control diet after 32 days. There were no significant weight differences among the groups ingesting the antioxidant-fortified diets. The specific growth rates (SGR) and feed conversion ratios (FCR) were not significantly different between groups fed the four experimental diets in FW. Conversely, muscle astaxanthin concentrations were significantly higher (p=0.019) in groups fed the astaxanthin-containing diets compared to baseline values and fish fed the control diet. There were no significant differences either in the concentration of astaxanthin in fish muscle or on the apparent astaxanthin retention coefficients (AARC) in salmon fed either the astaxanthin, low or high KPA\u00C2\u00AE diets. After a feeding period of 155 days in SW, post-smolts fed the high KPA\u00C2\u00AE diet had a significantly higher deposition of astaxanthin in muscle (p=0.036), higher visual colour Ill score in fillets as measured by the Roche Salmofan\u00C2\u00AE (p=0.029), and greater wet weight gain (p<0.001) than those fed the other three diets. Following a disease challenge with Vibrio anguillarum, pre-smolts fed the high KPA\u00C2\u00AE diet had a significantly lower (p=0.038) cumulative mortality among diet treatments, as well as a significantly greater (p=0.025) number of circulating leucocytes (p=0.025), neutrophils (p=0.018), and monocytes (p=0.034). Furthermore, the lysozyme activity both in plasma and head kidney, and the neutrophil respiratory burst activity were significantly greater in fish fed the high KPA\u00C2\u00AE diet, (p=0.029, p=0.037, and p=0.027, respectively). It is concluded that the addition of antioxidants (proanthocyanidins and astaxanthin) to a chinook salmon diet significantly enhanced the humoral and cellular non-specific immune response factors and was related to a lower cumulative mortality after the disease challenge. Furthermore, the deposition efficiency of the carotenoid astaxanthin, and growth-related variables in farmed chinook salmon were also significantly affected by the combination of dietary antioxidants. iv TABLE OF CONTENTS Page Abstract ii Table of Contents iv List of Tables vi List of Figures vii List of Abbreviations ix Acknowledgments xi Dedication xii 1. G E N E R A L INTRODUCTION 1 2. G E N E R A L H Y P O T H E S E S 6 2.1 Specific hypotheses 6 3. G E N E R A L MATERIALS AND METHODS 7 3.1 FISH 7 3.2 EXPERIMENTAL DESIGN 7 3.3.1 F E E D PREPARATION 11 3.4 FEED ANALYSIS 15 3.4.1 Feed KPA\u00C2\u00AE analysis 15 3.4.2 Astaxanthin extraction from feed and muscle 15 3.4.3 H P L C analysis 16 3.5 PIGMENTATION A S S E S S M E N T 17 3.5.1 Muscle astaxanthin analysis 17 3.5.2 Apparent astaxanthin retention coefficient (AARC) 17 3.5.3 Roche Salmofan\u00C2\u00AE fillet scores 18 3.6 G R O W T H A S S E S S M E N T 18 3.6.1 Wet weight gain 18 3.6.2 Specific growth rate (SGR) 18 3.6.3 Feed conversion ratio (FCR) 19 3.6.4 Fulton's condition factor (CF) 19 3.7 DISEASE C H A L L E N G E 19 3.7.1 Bacterial injection protocol 19 3.7.2 Bacterial isolation and identification from moribund fish 20 4. CHAPTER ONE Assessment of flesh pigmentation and growth of pre-smolt farmed chinook salmon fed one of two concentrations of a natural flavonoid antioxidant and astaxanthin in diet. 4.1 INTRODUCTION 22 4.2 MATERIAL AND METHODS 23 4.2.1 PIGMENTATION A S S E S S M E N T 23 4.2.2 G R O W T H A S S E S S M E N T 23 4.3 STATISTICAL ANALYSIS 23 4.4 R E S U L T S 24 4.5 D ISCUSSON 33 V 5. CHAPTER TWO Assessment of flesh pigmentation and growth of post-smolt farmed chinook salmon fed one of two concentrations of a natural flavonoid antioxidant and astaxanthin in diet. 5.1 INTRODUCTION 38 5.2 MATERIAL AND METHODS 39 5.2.1 PIGMENTATION ASSESSMENT 39 5.2.2 GROWTH ASSESSMENT 39 5.3 STATISTICAL ANALYSIS 39 5.4 RESULTS 40 5.5. DISCUSSON -. 48 6. CHAPTER THREE Non-specific immune responses of pre-smolt chinook salmon fed one of two concentrations of a natural flavonoid antioxidant and astaxanthin in diet, to an intraperitoneal injection of Vibrio anguillarum 6.1 INTRODUCTION 55 6.2 MATERIAL AND METHODS 56 6.2.1 DISEASE CHALLENGE PROTOCOL 56 6.2.2 CLINICAL HAEMATOLOGY 56 6.2.2.1 haematocrit 56 6.2.2.2 haemoglobin 56 6.2.2.3 total red blood cell count (RBC) 57 6.2.2.4 mean corpuscular volume (MCV) 57 6.2.2.5 mean corpuscular haemoglobin (MCH) 57 6.2.2.6 mean corpuscular haemoglobin concentration (MCHC) 57 6.2.2.7 total white blood cell count (WBC) 57 6.2.2.8 differential blood cell count 57 6.2.3 NON-SPECIFIC IMMUNE RESPONSE ANALYSES 58 6.2.3.1 neutrophil respiratory burst activity, NBT assay 58 6.2.3.2 lysozyme activity in plasma and head kidney 58 6.2.3.3 liver SP70 59 6.3 STATISTICAL ANALYSIS.. 60 6.4 RESULTS 60 6.5 DISCUSSION 75 7. GENERAL DISCUSSION 79 8. CONCLUSION 86 9. REFERENCES 89 10. APPENDIX 1. Interaction between antioxidant systems 101 11. APPENDIX 2. Role of dietary antioxidants in the defense against oxidative damage in biomembranes 102 12. APPENDIX 3. Freshwater temperature record (experiments A and B) 104 13. APPENDIX 4. Saltwater temperature record (experiment C) 105 VI Table 3.2.1 Table 3.3.1 Table 3.3.2 Table 4.1 Table 5.1 Table 6.1 Table 6.2 LIST OF TABLES Experimental protocol layout for the disease challenge (3.2.1-A) and sham injection (3.2.1-B) groups 10 Experimental diet groups supplemented with pigment (astaxanthin, 0 and 60 ppm) and grape seed extract (KPA\u00C2\u00AE, 100 ppm and 1000 ppm), respectively (3.3.1-A); and final concentration of astaxanthin in diets (3.3.1-B) 13 Formulation of the basal diet and vitamin and mineral premix composition 14 Apparent Astaxanthin Retention Coefficient (AARC) in muscle of pre-smolt chinook salmon (O. tshawytscha) fed control diet (A\u00C2\u00B0KPA\u00C2\u00B0), astaxanthin diet (A 6 0KPA\u00C2\u00B0), High KPA\u00C2\u00AE diet ( A 6 0 K P A 1 0 0 0 ) and low KPA\u00C2\u00AE diet ( A 6 0 K P A 1 0 0 ) . Four replicate groups were fed each of the test diets for 32 days at 3.5% body weight (b.w.). Different letters indicate significant differences (p<0.05) between diet groups 31 Apparent Astaxanthin Retention Coefficients (AARC) in muscle of post-smolt chinook salmon (O. tshawytscha) fed four experimental diets containing no antioxidant supplementation (control diet), astaxanthin (astaxanthin diet), astaxanthin and two concentrations of grape seed extract (high and low KPA\u00C2\u00AE diets, respectively). Different letters indicate significant differences (p<0.05) between diet groups 46 Primary haematology indices in pre-smolt chinook salmon (O. tshawytscha) fed diets containing no supplemental astaxanthin and KPA\u00C2\u00AE (control diet), astaxanthin (astaxanthin diet), or astaxanthin and one of two concentrations of grape seed extract (high KPA\u00C2\u00AE and low K P A diet, respectively) following a challenge with Vibrio anguillarum and sterile peptone saline (sham) injection. Letters indicate significant statistical differences (p<0.05) between the means for diet treatment groups 63 Leucocyte differential counts in pre-smolt chinook (O. tshawytscha) salmon fed diets containing no supplemental astaxanthin and KPA\u00C2\u00AE (control diet), astaxanthin (astaxanthin diet), or astaxanthin and one of two concentrations of grape seed extract (high KPA\u00C2\u00AE and low KPA\u00C2\u00AE diet, respectively) for six days after a challenge with Vibrio anguillarum and. Values for Sham-injected controls are also shown \u00E2\u0080\u00A2 68 vii LIST OF FIGURES Figure 4.1 Net weight increase in pre-smolt chinook salmon (O. tshawytscha) fed four experimental diets containing various levels of antioxidant supplementation (A\u00C2\u00B0KPA\u00C2\u00B0, A 6 0 KPA\u00C2\u00B0, A 6 0 K P A 1 0 0 0 , A 6 0 K P A 1 0 0 ) after a 32-day feeding period \u00E2\u0080\u00A2 26 Figure 4.2 Mean (\u00C2\u00B1 SD) apparent FCR (4.2-A) and S G R (4.2-B) in pre-smolt chinook salmon (O. tshawytscha) fed four experimental diets after a 32-day feeding period 27 Figure 4.3 Mean (\u00C2\u00B1 SD) Fulton's condition factor in pre-smolt chinook salmon (O. tshawytscha) during a 32-day feeding period fed four experimental diets containing various levels of antioxidant supplementation (A\u00C2\u00B0KPA\u00C2\u00B0, A 6 0 KPA\u00C2\u00B0, A 6 0 K P A 1 0 0 0 , A 6 0 K P A 1 0 0 ) 28 Figure 4.4 Mean (\u00C2\u00B1 SD) astaxanthin concentration in flesh of chinook salmon (O. tshawytscha) in freshwater after a 32-day feeding period with four experimental diets supplemented with astaxanthin and two levels of KPA\u00C2\u00AE 29 Figure 4.5 Correlation between total accumulated dietary astaxanthin (DAX) and flesh astaxanthin concentration in muscle (4.5-A), and cumulative apparent astaxanthin retention coefficient (4.5-B) after a 32-day feeding period of four experimental diets supplemented with astaxanthin and two levels of grape seed extract (KPA\u00C2\u00AE) in chinook salmon (O. tshawytscha) in freshwater tanks 30 Figure 5.1 Weight gain in post-smolt chinook salmon (O. tshawytscha) fed four experimental diets containing various levels of antioxidant (astaxanthin and KPA\u00C2\u00AE) supplementation (A\u00C2\u00B0KPA\u00C2\u00B0, A 6 0 KPA\u00C2\u00B0, A 6 0 K P A 1 0 0 0 , A 6 0 K P A 1 0 0 ) after a 155-day feeding period in seacages under commercial farming conditions. Different letters indicate significant differences (p<0.05) between diet groups 42 Figure 5.2 Mean (\u00C2\u00B1 SD) values for apparent F C R (5.2-A) and S G R (5.2-B) of post-smolt chinook salmon (O. tshawytscha) fed diets supplemented with astaxanthin alone or together with one of two levels of grape seed extract, or no astaxanthin and grape seed extract (A 6 0KPA\u00C2\u00B0, A 6 \u00C2\u00B0KPA 1 0 0 0 , A 6 0 K P A 1 0 0 and A\u00C2\u00B0KPA\u00C2\u00B0) after a 155-day feeding period in seacages under commercial farming conditions. Common letters indicate significant differences (p<0.05) between diet groups 43 Figure 5.3 Temporal changes in mean (\u00C2\u00B1 SD) condition factors for post-smolt chinook salmon (O. tshawytscha) fed diets supplemented with astaxanthin alone or together with one of two levels of grape seed extract, or no astaxanthin and grape seed extract (A 6 0KPA\u00C2\u00B0, A 6 0 K P A 1 0 0 0 , A 6 0 K P A 1 0 0 and A\u00C2\u00B0KPA\u00C2\u00B0) after a 155-day feeding period in seacages under commercial farming conditions. Common letters indicate significant differences (p<0.05) between diet groups 44 viii Figure 5.4 Muscle astaxanthin concentration of post-smolt chinook salmon (O. tshawytscha) assessed by H P L C (5.4-A) and visual estimation of Roche salmofan\u00C2\u00AE scores for fillet (mean \u00C2\u00B1 SD) (5.4-B) after a 155-day feeding period in seacages. Groups were fed diets supplemented with astaxanthin alone or together with one of two levels of grape seed extract, or no astaxanthin and grape seed extract (A 6 0KPA\u00C2\u00B0, A 6 0 K P A 1 0 0 0 , A 6 0 K P A 1 0 0 and A\u00C2\u00B0KPA\u00C2\u00B0) in seacages under commercial farming conditions. Different letters indicate significant differences p<0.05) between diet groups 45 Figure 5.5 Correlation between Roche Salmofan\u00C2\u00AE scores (RSS) and concentrations of astaxanthin in chinook salmon muscle (HPLC) following a 155-day feeding period in seacages (n=28, r2=0.668, p=0.027) 47 Figure 6.1 Cumulative percent mortality in pre-smolt chinook salmon (O. tshawytscha) fed four experimental diets containing various levels of antioxidant supplementation (A\u00C2\u00B0KPA\u00C2\u00B0, A 6 0 KPA\u00C2\u00B0, A 6 0 K P A 1 0 0 0 , A 6 0 K P A 1 0 0 ) following an intraperitoneal injection with Vibrio anguillarum (0.1 mL, 10 9 viable cells/mL) and sham injection (0.1 mL, sterile peptone saline) 61 Figure 6.2 Mean (\u00C2\u00B1 SD) secondary haematology indices in pre-smolt chinook salmon (O. tshawytscha) fed four experimental diets containing different supplemental levels of KPA\u00C2\u00AE and astaxanthin (A\u00C2\u00B0KPA\u00C2\u00B0, A 6 0 KPA\u00C2\u00B0, A 6 6 K P A 1 0 0 0 , A 6 0 K P A 1 0 0 ) following a disease challenge with Vibrio anguillarum (0.1 mL, 10 9 cfu/mL) and sterile peptone saline injection (0.1 mL) 64 Figure 6.3 Total white blood cell counts (WBC) in pre-smolt chinook salmon (O. tshawytscha) fed four diets containing different concentrations of astaxanthin and KPA\u00C2\u00AE for 6 days following an experimental challenge with Vibrio anguillarum (0.1 ml, 10 9 cfu/mL) and pooled mean of sham injection groups (sterile peptone saline, 0.1 mL). Different letters indicate significant differences (p<0.05) between diet groups, and symbol (((>) means a statistical difference (p<0.05) between bacterial injected and sham- injected groups 67 Figure 6.4 Number of glass-adherent, NBT-positive neutrophils from pre-smolt chinook salmon (O. tshawytscha) fed four experimental diets that contained various levels of antioxidant supplementation (A\u00C2\u00B0KPA\u00C2\u00B0, A 6 0 KPA\u00C2\u00B0, A 6 0 K P A 1 0 0 0 , A 6 0 K P A 1 0 0 ) after intraperitoneal injection of Vibrio anguillarum (OA mL, 10 9 cfu/mL) 71 Figure 6.5 Mean (\u00C2\u00B1 SD) lysozyme activity in plasma (6.5-A) and head kidney (6.5-B) of pre-smolt chinook salmon (O. tshawytscha) fed four experimental diets that contained various levels of antioxidant supplementation (A\u00C2\u00B0KPA\u00C2\u00B0, A 6 0 KPA\u00C2\u00B0, A 6 0 K P A 1 0 0 0 , A 6 0 K P A 1 0 0 ) for 6 days after disease challenge with Vibrio anguillarum (0.1 mL, 10 9 cfu/mL). The responses of sham-injected control (sterile peptone saline, 0.1 mL) are also shown 72 Figure 6.6 Correlation between lysozyme activity (HEWL equivalent) in plasma (6.6-A) and anterior kidney (6.6-B), and the number of circulating neutrophils in blood from pre-smolt chinook salmon (O. tshawytscha) fed each of four experimental diets that varied in astaxanthin and KPA\u00C2\u00AE supplementation levels following an experimental challenge injection of Vibrio anguillarum Figure 6.7 Hepatic SP70 response in pre-smolt chinook salmon (O. tshawytscha) fed four experimental diets that contained various levels of antioxidant supplementation (A\u00C2\u00B0KPA\u00C2\u00B0, A 6 0 KPA\u00C2\u00B0, A 6 0 K P A 1 0 0 0 , A 6 0 K P A 1 0 0 ) following a bacterial challenge (Vibrio anguillarum). The responses of sham-injected control (sterile peptone saline, 0.1 mL) are also shown LIST OF ABBREVIATIONS apparent astaxanthin retention coefficient control diet, astaxanthin (0 ppm), KPA\u00C2\u00AE (0 ppm) high K P A diet, astaxanthin (60 ppm), KPA\u00C2\u00AE (1000 ppm) low K P A diet, astaxanthin (60 ppm), KPA\u00C2\u00AE (100 ppm) astaxanthin diet, astaxanthin (60 ppm), KPA\u00C2\u00AE (0 ppm) body weight catalase condition factor colony forming units chemoluminiscence Department of Fisheries and Oceans Food and Agriculture Organization feed conversion ratio free radical freshwater glutathione peroxidase hour haemoglobin haematocrit high density lipoprotein hydrogen peroxide high performance liquid chromatography intraperitoneal Kikkoman proanthocyanidins (grape seed extract) low density lipoprotein mean corpuscular haemoglobin mean corpuscular haemoglobin content mean corpuscular volume myeloperoxidase 3-aminobenzoic acid ethyl ester methanesulfonate (tricaine) sodium bicarbonate nitroblue tetrazolium solution (0.2% in 0.85% saline) NO nitric oxide *o2 superoxide anion radical 1o2 singlet oxygen 'OH hydroxyl radical OD optical density 0. tshawytscha Oncorhynchus tshawytscha PA proanthocyanidins ppm parts per million PUFA polyunsaturated fatty acids RBC red blood cells, erythrocytes RCC Roche Colour Card ROS reactive oxygen specie rpm revolutions per minute RSS Roche salmofan\u00C2\u00AE score SD standard deviation SGR specific growth rate SOD superoxide dismutase SP stress protein SW saltwater TBARS thiobarbituric acid reactive substances TGC thermal growth coefficient TSA trypticase soy agar Vang Vibrio anguillarum VHDL very high density lipoprotein VLDL very low density lipoprotein vs. versus WBC white blood cells Wt weight Wtj initial weight Wtf final weight WVL West Vancouver Laboratory YIAL Yellow Island Aquaculture Ltd \u00C2\u00AE registered ACKNOWLEDGMENTS I am pleased to acknowledge Dr. G.K Iwama for his guidance and encouragement throughout the process of this thesis. I will not forget our first meeting and your support during the process of application to the graduate program at UBC. I would like to thank the financial support and in kind donation of KPA\u00C2\u00AE made by Kikkoman Corporation (Noda city, Chiba, Japan), also an operating grant from N S E R C to Dr. G.K. Iwama. In addition, Yellow Island Aquaculture Ltd. (YIAL) on Quadra Island, BC provided the chinook salmon and research facilities. My deepest appreciation to Dr. D. Higgs for your support with the diet formulation, advice with the experimental design and insightful discussion of the results. I would also like to thank the members of my supervisory Dr. S. Samuels and Dr. B. Skura, for your commitment to my research. Also I would like to acknowledge Dr. T. Koga from Kikkoman Corp for his technical assistance and analysis of KPA\u00C2\u00AE in food. The collaboration of the personnel and technicians at the West Vancouver Laboratory, DFO, especially Mahmoud Rowshandeli for your help making the food, and Nahid Rowshandeli and Joanna Wieruszceski for your help with the HPLC analysis of astaxanthin. The technical assistance of Ms. G. Cho of YIAL is very much appreciated. I am most grateful of the Iwama Laboratory students: Paige, Anne, Rosalind, Nil, Luis, Kazumi, Carlos and Dominic. All your collaboration, encouragement, support, wisdom and critical review made this thesis find an end. Your bright minds lead you with excellence! XIII DEDICATION The completion of this thesis is dedicated to my families both South and North of the Equator line. I will always have one foot on each hemisphere. To Michelle and Oliver, your love and encouragement lightens my pace and comforts my spirit. Ad Astra Per Aspera. Je suis Prest. 1 1. GENERAL INTRODUCTION Aquaculture is the rearing of aquatic species under controlled or semi-controlled conditions. The salmon and trout farming have developed explosively in the last 20 years (FAO, 2000). In 1981, the industry produced 17,000 tonnes worldwide (3% of world's total salmon production); in 2002 the industry supplied 1,445,000 tonnes (69% of world's production) (Anonymous, 2003). Farmed salmon is the most important agricultural commodity in British Columbia (BC), generating revenues well over US$310 million in 2000 (Egan, 2001). Farmed salmon production in the province of B C is based on three species: Atlantic salmon (Salmo salar) 81%, chinook salmon (Oncorhynchus tshawytscha) 16% and coho salmon (Oncorhynchus kisutch) 3% (Egan, 2001). As a highly intensive method of animal production, farmed salmon husbandry relies strongly on adequate feeding practices, both in feed quality and quantity; therefore, proper nutrition plays a critical role in maintaining normal growth and health of cultured fish. Infectious diseases are the major cause of economic loss in commercial aquaculture (Loyell 1996). Current problems with disease outbreaks in aquaculture include the limited number of government-approved antibiotics, improper administration, antibiotic resistance, and accumulation of drugs in the environment. Therefore the aquaculture industry needs to focus on prevention of diseases rather than on treatment. The influence that dietary factors may have on a disease outbreak in cultured fish has been recognized for many years (Blazer, 1992). The effects of supplementing diets with vitamins (vitamin A, C, E), micronutrients (minerals), macronutrients (protein, carbohydrate and lipids), and immunostimulants (lipopolysaccharides, probiotics, p-glucans) have been the focus of investigation. Enhancing the immune system through supplementation with dietary antioxidants is a common practice in human nutrition, and is gaining increased popularity in companion animal diets (Devlin et al. 2000). Such practice is almost non-existent in intensive animal farming. The effects of adding a potent natural antioxidant extracted from grape seeds (Kikkoman proanthocyanidins, KPA\u00C2\u00AE) in the diet of farmed salmon could positively affect the immune system as well as their pigmentation and growth. Therefore, these studies were designed to address the potential beneficial effects of feeding farmed salmon diets fortified with one of two concentrations of KPA\u00C2\u00AE and a uniform concentration of astaxanthin on deposition of the carotenoid astaxanthin in flesh, salmon growth and the fish immune response after a bacterial challenge. 2 The fish immune system: The immune system works in a coordinated manner fighting foreign biotic and abiotic insults. The defense mechanisms can be differentiated into non-specific (innate) immunity and specific (acquired) immunity. Both use humoral (i.e. lysozyme, complement, C-reactive Protein (CRP), interferon, transferrin, lectin, lysines, agglutins, precipitins, and opsonins) and cellular (neutrophils, eosinophils, monocytes and macrophages) mechanisms to provide protection against foreign substances and pathogens. The pathogenesis of disease include the generation of reactive oxygen species (ROS), during inflammation and the \"oxygen-killing dependant mechanisms\" (i.e. respiratory burst activity). A major innate defense mechanism consists of phagocytosis by monocytes, macrophages, neutrophils and eosinophils (Secombes, 1996). Once a pathogen is internalized in the body, phagocytic cells recognize and engulf antigenic particles, including bacteria, and damaged host cells in a three-step process involving attachment, phagocytosis, and digestion. This allows lysozomal enzymes to directly degrade the ingested particles. The respiratory burst activity produces toxic oxygen-, and nitrogen-free radicals, such as superoxide anion f O2\"), hydrogen peroxide (H2O2), reactive singlet oxygen ( 1 0 2 ) , and nitric oxide (NO), respectively (reviewed by Secombes and Fletcher, 1992). These components are toxic to bacteria and protozoan parasites and occur within the cytoplasm of the phagocyte (phagosome). However, they may also be released extracellularly (Lamas and Ellis, 1994). The toxicity of these substances will not only affects microorganisms but will also affect the stability of lipid membranes of surrounding cells. Farmed salmon are not only affected by the action of pathogens, but also by a number of chemical (i.e. pharmacological treatments) and environmental (i.e. pollutants, net antifouling, hyperoxic conditions) insults that expose fish to the actions of deleterious R O S . Free radicals are also produced under normal healthy conditions as part of the respiratory metabolism of aerobic organisms. These molecules are highly reactive and unstable, and when a biomolecule (lipid, nucleic acid, protein) reacts with a free radical, a new radical is created (chain reaction). Antioxidants work by suppressing ROS formation, inhibiting chain reactions, breaking chain propagation, and repairing and de novo synthesizing antioxidant enzymes. 3 The fish carotenoid deposition: Carotenoids are a group of naturally occurring pigments that are responsible for the red, orange and yellow colour in the skin, flesh, shell and exoskeleton of aquatic animals. The most abundant natural pigments in the flesh of salmonids (Salmo spp., Oncorhynchus spp., and Salvelinus spp.) are the xanthophylls astaxanthin (3,3' -dyhydroxy-4, 4'- diketo-p-carotene) and canthaxanthin (4-4'-diketo-p-carotene). Salmonids are incapable of synthesizing carotenoids de novo (Weedon, 1971; Simpson, 1982; Storebakken and No, 1992) or converting one carotenoid pigment into another (Torrissen et al. 1989). Thus, flesh pigmentation of farmed salmon can only be achieved by supplementing the feed of the fish with natural and/or synthetic astaxanthin or canthaxanthin. Deposition of carotenoids in salmonid flesh occurs as a result of several processes: absorption of pigments in the digestive tract, transport of pigment in the blood, retention in the muscle and metabolism of carotenoids. These processes depend on several factors such as fish age, diet, genetic and husbandry conditions. Among dietary factors, lipid and antioxidant levels in feed are known to play important roles. Achieving a deep red flesh colour is critical for maximizing the price of salmon in the marketplace and is therefore a crucial aspect of salmon husbandry. Well-pigmented flesh is regarded second after freshness as the most important flesh quality criteria of farmed salmon (Torrissen, 1995; Schiedt, 1998). In addition to their role as biological pigment, carotenoids possess strong antioxidant properties as shown by Burton (1989) and later confirmed in vivo in rainbow trout by Nakano et al. (1995). The antioxidant network: Antioxidants break the chain reaction caused by free radicals on biomolecules. Moreover, they scavenge and neutralize reactive oxygen metabolites before they can do significant damage to cells, tissues and organs. The antioxidant defense system includes enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), free radical scavengers (vitamin C, vitamin E, carotenoids, flavonoids), and metal chelators. These molecules work together in a highly organized and cooperative manner to regulate the equilibrium between the production of oxygen radicals and the counteracting defense mechanisms of antioxidant scavenging systems 4 and repair enzymes. The components of the antioxidant system exert their actions either on the lipid (lipophilic) or the aqueous (hydrophilic) phase of the cell membranes and there have been recognized synergistic interactions and regenerations between antioxidants of both phases (i.e. Vitamin E-Vitamin C, carotenoids-Vitamin E, flavonoids-Vitamin E) (Aruoma, 1994; Halliwell, 1996) (Appendix 1 and 2-A). Flavonoids are polyphenolic antioxidants naturally present in fruits, vegetables, nuts, seeds, flowers and barks. Proanthocyanidins (PA) are a group of flavonoids present in high concentration in the seeds of grapes. Proanthocyanidins have significantly more potent antioxidant activity than vitamin C, E or p-carotene (Buettner, 1993; Bagchi et al. 1997; Bagchi et al. 1998) singly, or in combination (Bagchi et al. 2000). In addition, PA exerts a wide range of biological effects including antibacterial, antiviral, anti-inflammatory, anti-allergic and vasodilatory actions (Buening et al. 1981; Afanas'ev et al. 1989; Kolodziej et al. 1995). Proanthocyanidin regulates the activity of enzymes participating in inflammation, including cyclooxygenase and lipooxygenase (Kolodziej et al. 1995), and have been shown to prevent lipid peroxidation and oxidation of low-density lipoproteins (LDL) (Frankel et al. 1993). The Kikkoman Corporation (Noda city, Chiba, Japan) has extracted natural flavonoid PA from grape seeds (KPA\u00C2\u00AE), which have been experimentally tested as a photo-oxidative protector in salmon fillets. The colour of salmon fillets can fade with time due to the action of light and oxygen. Soaking salmon fillets in a KPA\u00C2\u00AE-saline solution has been shown to preserve the fillet colour compared to a saline control solution (Kikkoman Corporation, personal communication). Rationale. Farmed salmon are raised in confined high-density production systems and they are fed balanced diets that contain high levels of protein and lipid together with supplemental micronutrients and carotenoids. They are exposed to a variety of deleterious stimuli that promote the production of ROS. In this study it was postulated that supplementation of salmon diets with antioxidants (i.e. grape seed extract and astaxanthin) would either correct a deficiency of or restore the balance between pro-oxidants and antioxidants, and consequently enhance body functions, namely immune responses, growth and pigmentation. The effects of supplementing fish feed with KPA\u00C2\u00AE have not been described yet. The supplementation of KPA\u00C2\u00AE in salmon feed might have positive impact on carotenoid pigment (astaxanthin) deposition efficiency in their flesh. In addition, the cell-mediated and humoral non-specific immune responses of salmon could be enhanced due to the fortification of their feed with a combination of both antioxidants. This is the first study that has used a combination of astaxanthin and a natural flavonoid supplement in salmon diet in an attempt to enhance the immune responses of the fish against a bacterial challenge, while concurrently improving their flesh pigmentation and growth under practical salmon farming conditions. 6 2. GENERAL HYPOTHESIS H0: Fortification of a salmon diet with a combination of grape seed extract (KPA\u00C2\u00AE) and astaxanthin has no effect on the immune response, pigmentation and growth of farmed chinook salmon (O. tshawytscha). Hy. Fortification of a salmon diet with a combination of grape seed extract (KPA\u00C2\u00AE) and astaxanthin has an effect on the immune response, pigmentation and growth of farmed chinook salmon (O. tshawytscha). 2.1 Specific hypotheses Experiments A and C: Assessment of flesh pigmentation and growth of pre-smolt and post-smolt farmed chinook salmon fed diets with one of two concentrations of a natural flavonoid antioxidant and a single astaxanthin level or no supplemental antioxidants. 1. H0: KPA\u00C2\u00AE-supplemented diet does not increase astaxanthin retention in muscle of pre-smolt and post-smolt chinook salmon (O. tshawytscha). Hi : KPA\u00C2\u00AE-supplemented diet does increase astaxanthin retention in muscle of pre-smolt and post-smolt chinook salmon (O. tshawytscha). 2. H0: KPA\u00C2\u00AE-supplemented diet does not improve growth performance of pre-smolt and post-smolt chinook salmon (O. tshawytscha). H<|-. KPA\u00C2\u00AE-supplemented diet does improve growth performance of pre-smolt and post-smolt chinook salmon (O. tshawytschaj. Experiment B: Non-specific immune response of pre-smolt chinook salmon fed diets with one of two concentrations of a natural flavonoid antioxidant and a single astaxanthin level or no supplemental antioxidants after a disease challenge 1. H0: Dietary KPA\u00C2\u00AE does not enhance immune response in pre-smolt chinook salmon after disease challenge with Vibrio anguillarum by i.p injection. H-i: Dietary KPA\u00C2\u00AE does enhance immune response in pre-smolt chinook salmon after disease challenge with Vibrio anguillarum by i.p injection. 2. H0: Dietary KPA\u00C2\u00AE does not increase disease resistance in pre-smolt chinook salmon after disease challenge with an i.p injection of Vibrio anguillarum. Hi -. Dietary KPA\u00C2\u00AE does increase disease resistance in pre-smolt chinook salmon after disease challenge with an i.p injection of Vibrio anguillarum. 7 3. GENERAL MATERIALS AND METHODS 3.1 FISH Half-siblings of chinook salmon (O. tshawytscha) were hatchery reared in freshwater tanks at Yellow Island Aquaculture Ltd (YIAL, Quadra Island, BC, Canada), and then transferred to growout seacages at the same site. 3.2 EXPERIMENTAL DESIGN 3.2.1 Experiment A. The experiment was carried out as a completely random design with four replicate groups of four diet treatments. In May 2000, 6400 chinook salmon alevins (mean weight 1.35 \u00C2\u00B1 0.3 g) were randomly distributed into 16 circular plastic tanks (200 L capacity each), each one containing 400 hundred fish (stocking density 1 kg/m3). Feeding trials started in June 2000 (mean fish weight 2.95 \u00C2\u00B1 0.2 g). The fish groups were hand-fed three times daily, at a feeding rate between 3.0% to 3.5% of body weight (b.w.) according to Lovell (1998b). Prior to the start of the experiment, fish were sampled for initial weight, length and astaxanthin concentration in muscle (10 fish/tank). During the experiment, non-lethal samples offish were weighed and measured (fork length) on a weekly basis over 32 days. At the end of the feeding trial, muscle samples (n=12/diet group) were analyzed by HPLC to determine the amount of astaxanthin in tissue. Feed amounts were carefully weighed and recorded; uneaten feed was weighed and recorded daily. Collected data served to estimate weight gain, Fulton's condition factor (CF), feed conversion ratio (FCR), specific growth rate (SGR), and apparent astaxanthin retention coefficient (AARC) in FW hatchery. The feeders did not know the diet that they were feeding to each tank which reduced any hand-feeding bias to specific groups. All groups were fed 3 times a day with three feeding opportunities each time. This protocol was adopted so that all fish ingested their daily feed ration with a minimum of feed wastage. The amount of feed was initially estimated by the projected weight given by the thermal growth coefficient (TGC) (Iwama and Tautz, 1981) and was corrected according to the observed weight gain offish after every weekly weight sampling. Fish weight projections were estimated using the UBC Aquaculture Production Analysis Computer Program, developed by Dr. G. Iwama and Dr. L. Fidler. The software was run using actual water temperature for the trial period and the recommended growth coefficient (Gc) for the month/temperature/species under 8 investigation (Iwama, 1996). Changes in feed size were made over a period of time, with small pellet sizes gradually replaced by larger ones according to the size increased of the fish. Experimental conditions: artificial photoperiod regime during the experiment was set at 16L.8D. Well water was supplied to all tanks and flow and quality during the experiment were as follows: water flow 10 L/min; temperature 9.10 \u00C2\u00B1 0.25\u00C2\u00B0C (Appendix 3); dissolved oxygen 8.0 \u00C2\u00B1 1.0 mg/L; oxygen saturation level 70 \u00C2\u00B1 15%. Temperature was recorded by an electronic probe every 4 h, and dissolved oxygen and oxygen saturation were recorded daily. Water flow was checked on a weekly basis. 3.2.2 Experiment B. After 28 days of feeding, disease challenge and sham injection groups were established. The numbers of fish were reduced from 400 fish/tank to 2 sets of 40 fish/tank (tank replicates 1 and 2), and 2 sets of 80 fish/tank (tank replicates 3 and 4) and these were fed for another 4 days, completing the 32-day feeding period. Four additional tanks, each containing a pool of 200 fish from each diet treatment, were prepared as sham injection control groups (Table 3.2.1-A and 3.2.1-B). Once fish were injected either with a bacterial suspension (disease challenge) or sterile peptone saline (sham injection), daily mortality was recorded and tissue samples were undertaken for a period of 10 days. Replicate tanks 1 and 2 served as mortality controls and replicate tanks 3 and 4 provided blood and tissue samples throughout the challenge experiment. Six fish (n=6/tank) from each diet treatment were sampled every day for haematology, immunocompetence and tissue analyses, as numbers permitted. Whole blood was analysed for haematocrit, haemoglobin, total erythrocyte and leucocyte counts, and differential leucocyte counts. Neutrophil respiratory burst activity was also estimated from fresh blood. Plasma was frozen on dry ice, and stored at -80\u00C2\u00B0C for later analyses of lysozyme activity. Liver and head kidney were rapidly dissected, frozen on dry ice, and stored at -80\u00C2\u00B0C for later analyses of stress protein 70 Kd (SP70) and lysozyme activity, respectively. Fish injected with peptone saline were sampled daily (n=3/diet group) for all the variables mentioned above. 9 3.2.3 Experiment C. Chinook salmon smolts from experiment A were transferred from the hatchery of YIAL to on-site growout seacages in August 2000. Four thousand and four hundred smolts (mean weight 7.57 \u00C2\u00B1 0.3 g) were distributed into four 5 x 5 x 10 m adjacent seacages (1100 fish/pen) suspended in a floating wooden cage. Fish groups were fed manually to apparent satiation every day (estimated between 2-2.5% b.w). As in freshwater, the feeding regime was blind-designed to reduce the hand-feeding bias to specific groups. The amount of feed each group received was estimated according to the biomass in each seacage and was increased weekly based on weight gain prediction. The feeding regime was adjusted monthly based upon the actual weight gain of each group after every weight sampling. The feeding period lasted for 155 days in saltwater. Water temperature fluctuated from 11.6 \u00C2\u00B1 0.8\u00C2\u00B0C in August to 8.6 + 0.2\u00C2\u00B0C in January (Appendix 4). Dissolved oxygen was 8.0 \u00C2\u00B1 1.0 mg/L, and oxygen saturation level was 70 \u00C2\u00B1 15%. Temperature was monitored by an electronic probe every 4 h, and dissolved oxygen and oxygen saturation were recorded daily. Mortalities were collected by a scuba diver every 7 to 14 days, counted and disposed of in the YIAL disposal pit. Fish weights and fork lengths were recorded before transferring smolts to seacages, and every month thereafter, for 155 days (n=25 fish/diet group). Astaxanthin concentrations as determined by HPLC at the end of the freshwater period were considered as the baseline for muscle concentration of pigment for the saltwater phase. Flesh samples (n=14 fish/group) for astaxanthin analyses were also taken in the middle (October) and at the end (January) of the experiment. Data regarding fish weight and length, fed intake and muscle pigmentation were used to estimate C F , FCR, S G R , and A A R C . Quantification of Roche Salmofan\u00C2\u00AE scores (n=14 fish/group) was only possible in the final sampling time (January), when the sizes offish fillets were large enough to avoid background interference. 10 Table 3.2.1 Experimental protocol layout for the disease challenge (3.2.1-A) and sham injection (3.2.1-B) groups A. Bacterial suspension injection groups (Vibrio anguillarum, 0.1 mL, 10 9 cfu/mL). Experimental Diet Groups A\u00C2\u00B0KPA\u00C2\u00B0 A6oK p Aiooo A 6o K p A ioo A60KPA\u00C2\u00B0 Replicate 1 40 fish 40 fish 40 fish 40 fish Replicate 2 40 fish 40 fish 40 fish 40 fish Replicate 3 80 fish 80 fish 80 fish 80 fish Replicate 4 80 fish 80 fish 80 fish 80 fish B. Sham injection groups (Sterile peptone saline, 0.1 mL) Experimental Diet Groups A\u00C2\u00B0KPA\u00C2\u00B0 A6oK p Aiooo A60KpA100 A60KPA\u00C2\u00B0 200 fish 200 fish 200 fish 200 fish 11 3.3 DIET PREPARATION Diet treatment groups were based on the supplemental concentrations of grape seed extract (KPA\u00C2\u00AE) and astaxanthin (Carophyll Pink\u00C2\u00AE) (Table 3.3.1). Carophyll Pink\u00C2\u00AE and KPA\u00C2\u00AE were weighed on an air-dry basis, and added to the fish feed as active ingredient. An experimental chinook salmon diet was formulated by Dr. Dave Higgs and manually prepared by A. Schlicht and M. Rowshandeli at the West Vancouver Laboratory; DFO (West Vancouver, BC, Canada). All diets were formulated to contain equivalent concentrations of protein, lipid and energy based upon ingredient proximate analyses. The composition of the basal diet formulation, as well as the vitamin and mineral premix are described in Table 3.3.2. Briefly, Austral fishmeal was ground in a hammer mill (Fitzpatrick Co., Fitz-Mill\u00C2\u00AE, JT model, IL, USA) to standardize particles size. Vitamins were individually weighed and thoroughly blended by hand in a mortar and then mixed altogether in raw starch in an electric mixer for 30 min. Minerals, astaxanthin and KPA\u00C2\u00AE were also weighed individually and ground manually in a mortar and mixed in a-cellulose for 30 min in a mixer (Blakeslee, model B-12, Chicago, IL, USA). All ingredients of the basal formulation were weighed on an air-dry basis and thoroughly mixed (Hobart mixer, M 802, OH, USA) for 30 min and then in a commercial paddle blender (Marion mixer, Iowa, USA) for another 45-60 min. Diets were steam-pelleted (maximum temperature 80\u00C2\u00B0C for less than 5 sec.) in order of increasing pigment and antioxidant concentration, i.e. control diet, astaxanthin diet, low KPA\u00C2\u00AE diet, and high KPA\u00C2\u00AE diet, respectively using a laboratory pellet mill (model CL-type 2, California Pellet Mill Co., San Francisco, CA, USA) and following pellet size recommendation given by Lovell (1998b). Astaxanthin (Carophyll Pink\u00C2\u00AE 2%, Hoffman-La Roche, Basel, Switzerland) was incorporated at the same proportion on an air-dry basis in all diets, except the control diet. The concentration of astaxanthin among the three diets containing astaxanthin was not significantly different (p=0.117) as measured by HPLC in duplicate samples (Table 3.3.1-B). Grape seed extract (KPA\u00C2\u00AE) containing 42.7 mg/g (as active ingredient) of the polyphenolic antioxidant P A (oligomeric flavan-3-ol) was incorporated in a high (1000 ppm) and low (100 ppm) concentration in diets designated as high and low KPA\u00C2\u00AE, respectively. 12 After pelleting, the diets were sprayed with a mix of fish and vegetable oil (anchovy oil: canola oil 5:2,122 g/kg of pellets) and stored in dark bags at room temperature until use. Fish feed was prepared no longer than 8 weeks from one batch to another. 13 Table 3.3.1 Experimental diet groups supplemented with pigment (astaxanthin, 0 and 60 ppm) and grape seed extract (KPA\u00C2\u00AE, 100 ppm or 1000 ppm), respectively (3.3.1-A); and final concentrations of astaxanthin in the test diets (3.3.1-B). A. Experimental diet treatments Experimental Diet Grape Seed Concentration (KPA\u00C2\u00AE, ppm) Astaxanthin Concentration (Carophyll Pink\u00C2\u00AE, ppm) Control Diet (A\u00C2\u00B0KPA\u00C2\u00B0) 0 0 High KPA Diet (A 6 0 KPA 1 0 0 0 ) 1000 57 Low KPA Diet (A 6 0 KPA 1 0 0 ) 100 57 Astaxanthin Diet (A60KPA\u00C2\u00B0) 0 57 B. Mean (\u00C2\u00B1 SD) HPLC concentrations of astaxanthin in the test diet (ug/g) fed to chinook salmon (Oncorhynchus tshawytscha); pooled data for different batches. Experimental Diet Diet A\u00C2\u00B0KPA\u00C2\u00B0 A6oK p Aiooo A60KpA100 A60KPA\u00C2\u00B0 Astaxanthin Concentration 0.21 \u00C2\u00B10 .02 b 60.49 + 1.51a 56.20 \u00C2\u00B1 2 . 4 3 a ' 57.57 \u00C2\u00B1 1.51a 14 Table 3.3.2 Formulation of the basal diet and vitamin and mineral premix composition. Ingredients g/kg as fed Austral fish meal 536.29 Blood flour; spray-dried 51.26 Squid meal 71.89 Wheat gluten meal 50.90 Pregelatinized wheat starch 52.38 Raw wheat starch 21.77 Vitamin premix1 ' 18.93 Mineral premix 2 ' 18.93 Anchovy oil; stabilized^' 84.88 Canola oil 37.86 Soybean lecithin 9.47 Choline chloride (60%) 4.73 Vitamin C (monophosphate, 42%) 0.95 Permapell\u00C2\u00AE 9.47 Finstimm\u00C2\u00AE 9.47 DL-methionine 1.89 Carophyll pink and/or a-cellulose 9.47 KPA\u00C2\u00AE and/or a-cellulose 9.47 1 / . Vitamin premix composition (amount/kg dry diet): D-calcium pantothenate, 168.40 mg; pyridoxine HCI, 49.30 mg; riboflavin, 60 mg; folic acid, 15 mg; thiamine mononitrate, 56 mg; biotin, 1.50 mg; Vitamin B12 (cyanocobalamin), 0.09 mg; menadione (as vitamin K), 18 mg; vitamin E (a-tocopherol), 50 IU; vitamin D 3 , 2400 IU; vitamin A, 5,000 IU; inositol, 400 mg; niacin, 300 mg; BHT, 22 mg; raw starch (carrier). 2 /. Mineral premix composition (mg/g dry diet): Mn (as MnSO 4 *H 2 0) , 75 mg; Zn (as ZnS0 4 *7 H 20), 60 mg; Co (as CoCI 2*6 H 20), 3 mg; Cu (as C u S 0 4 * 5 H 20), 7 mg; Fe (as F e S 0 4 * 7 H 20), 100 mg; I (as KI0 3 ) , 5 mg; I (as Kl), 5 mg; F (as NaF), 5 mg; Na (as NaCI), 1,500 mg; Se (as N a 2 S e 0 3 ) , 0.20 mg; Mg (as M g S 0 4 * 7 H 20), 400 mg; K (as K 2 S 0 4 ) , 850 mg; K (as K 2 C 0 3 ) , 850 mg; a-cellulose, (carrier). 3 / . Stabilized with 500 ppm Santoquin 15 3.4 FEED ANALYSIS 3.4.1 Feed KPA\u00C2\u00AE analysis Random samples (250 g) of each diet were vacuum packed in oxygen-impermeable bags and frozen at -40\u00C2\u00B0C until further analysis. Feed samples (20 g) were diluted in 400 mL of methanol. The mixture was extracted with methanol at 65\u00C2\u00B0C for 2 h by gently stirring, and then filtered through cellulose powder. The filtered mixture was then evaporated and the residue was dissolved in 50 mL of methanol prior to measuring the content of PA in the extract by the Porter method. The Porter method measures the procyanidin oligomers/polymers as the absorbance of a 1% w/v solution of methanol soluble sample material at 550 nm. Results are reported as Porter Value Units (PVU) rather than as percent, which correlates to the procyanidin content. Monomers are not included in the measurement and the degree of polymerization is not differentiated. Absorbance generally increases with average phenolic content. On a molar basis, hexamers will have higher PVUs than dimers. Therefore, if a grape seed extract is comprised of primarily larger polymers, it will have a higher PVU than an extract comprised of the same molar concentration of dimers. In our experience, grape seed extracts have a range from 25 to 325 P V U . The analysis of proanthocyanidins in feed were performed in the laboratory of Kikkoman Corporation in Japan (Noda City, Chiba, Japan), and unfortunately this laboratory failed to report the content of KPA\u00C2\u00AE in the final diet. 3.4.2 Astaxanthin extraction from feed and muscle Feed samples were pre-dissolved in warm water bath (<50\u00C2\u00B0C) while muscle samples were extracted directly. Samples of 10 g of feed were ground in a coffee grinder (Braun, type 4014) and astaxanthin was extracted using the Bligh and Dyer (1959) method for lipid extraction. One-gram (\u00C2\u00B1 0.005 g) samples of ground feed were suspended in 10 mL of deionised water in a thick-glass test tube. The suspension was warmed up in a water bath (Precision Scientific Co., Winchester, VA, USA) at 80\u00C2\u00B0C for 30 min with occasional shaking, allowing the release of astaxanthin from its gelatin matrix and cornstarch coating. The feed suspension was then extracted three times in a separatory funnel using a 2:2:1.8, chloroform: methanol: deionised water mixture. The chloroform layer from each subsequent extraction was pooled in a round-bottom flask 16 for rotatory evaporation under vacuum on a water bath (50\u00C2\u00B0C). Following evaporation, the oil-film coat containing carotenoids was dissolved in 2 mL of n-hexane (HPLC grade) and re-evaporated, and re-dissolved in one mL of n-hexane (HPLC grade). An aliquot of the solvent containing pigment was put in a 1.0 mL glass H P L C vial (Wheaton, USA) and immediately analyzed by HPLC. Duplicate samples of feed from each diet batch were analyzed for astaxanthin concentration. Muscle samples were weighed (1.0 \u00C2\u00B1 0.005 g), and then each one was placed directly in a separatory funnel and astaxanthin was extracted following the same protocol as described for feed astaxanthin analysis. 3.4.3 HPLC analysis Astaxanthin was quantitatively analyzed by H P L C according to Kiessling et al. (1995). Hexane solution (150 pL) (see 3.4.2.1) was injected into a Waters HPLC-module 1, equipped with a stainless steel silica column (pporasil 125 ,10 (inn, 3.9 mm X 30 cm). The system was equipped with a 600E Powerline\u00E2\u0084\u00A2 Controller pump, a 715 Ultra WISP\u00E2\u0084\u00A2 autoinjector, and a Waters 486 Tunable Asorbance Detector (Table 1.3). The mobile phase was hexane:acetone (83:17) with a flow rate of 1 mL/min. Authentic astaxanthin standards were prepared from crystalline all-E astaxanthin (Hoffman-La Roche, Basel, Switzerland). The absorbance was measured with spectrophotometer at 472 nm, and the concentration of astaxanthin was calculated according to the formula used by Schierle and Hardi (1992): Astaxanthin (mg/L) = Absorption * 10,000/21001 A standard curve was generated, and HPLC peaks detected at 472 nm were integrated and quantified using Waters Millennium\u00C2\u00AE software. The HPLC retention times for the pigment isomers (XZ, all-E, 9Z and 13Z) were estimated, and the amounts of the different isomers were calculated and corrected for their differences in extinction coefficients. In regard to the latter, the quantities of the three Z astaxanthin isomers were multiplied by 1.6, 1.2, and 1.1 (ratio of the isomer extinction coefficient for the 13Z, 9Z and XZ isomers relative to the all-E isomer coefficient, 2100) to account for their lower specific absorption. Total astaxanthin concentration in each sample was reported as the sum of all four isomers. 1 2100 = E (1%/1 cm) = standard absorption of a 1% astaxanthin solution (w/v) in a 1 cm cuvette at 470 nm in n-hexane 17 3.5 PIGMENTATION ASSESSMENT The deposition of astaxanthin in the muscle of chinook salmon was assessed for both the FW and SW phases of the study. Baseline values for astaxanthin concentration in muscle of salmon grown in freshwater were determined before the start of the feeding trial in June 2000. Astaxanthin deposition efficiency in pre-smolt chinook salmon was calculated after 32 days in freshwater. In this regard, a random sample of 40 alevins (10 fish/dietary treatment, mean weight 2.90 \u00C2\u00B1 0.21 g) were killed by a lethal dose of anaesthetic (MS-222\u00C2\u00AE), skinned and filleted. Salmon fillets were pooled and ground altogether and ten samples of 1 g were analyzed by H P L C to establish the baseline concentration of astaxanthin in muscle. After 32 days of feeding, astaxanthin concentration in flesh was assessed on individual fillets for each dietary treatment group (mean weight 5.31 \u00C2\u00B1 0.36 g). H P L C results reported at the end of experiment A were considered as the initial flesh concentration of astaxanthin in smolts that were transferred to SW (beginning of experiment B). A second muscle sample (n=7/diet group) was taken after 70 days, and the final sample (n=7/diet group) was taken at the end of the experiment, after 155 feeding days. All samples for astaxanthin quantification were immediately vacuum-sealed in oxygen-impermeable bags after filleting, wrapped in tin foil, frozen on dry ice, and then stored at -80\u00C2\u00B0C until H P L C analysis. 3.5.1 Muscle astaxanthin analysis Chinook salmon were killed by a lethal dose of anaesthetic, skinned and filleted. Fillets were wrapped in tin foil, rapidly frozen on dry ice, vacuum-packed in oxygen impermeable bags and stored at -80\u00C2\u00B0C until further analysis. Fillets were first thawed and then ground by hand in a mortar and pestle in order to achieve an even consistency and a homogeneous sample of flesh. A 1.0 g (\u00C2\u00B1 0.005 g) sample of fillet homogenate was extracted directly following the procedures outlined in sections 3.4.2 and 3.4.3. 3.5.2 Apparent astaxanthin retention coefficient (AARC) A A R C in chinook salmon fillets was calculated according to the formula used by Nickell and Bromage (1998): AARC: (( Axf - Ax,) (Wtf - Wti)) / DAX * 100 18 Where Axf is the final mean flesh astaxanthin concentration (ucj/g), AXJ is the initial flesh astaxanthin concentration (u.g/g), Wtf is mean final fish weight, Wtj is initial mean fish weight, and DAX means the total amount of dietary astaxanthin over the experimental period (u.g/g). 3.5.3 Roche Salmofan\u00C2\u00AE fillets scores (RSS) Immediately after filleting, the opposite halves of the salmon fillets sampled for flesh astaxanthin quantification were each placed in a one-side-open white box illuminated with two fluorescent light tubes (Spectralite\u00C2\u00AE F40T12 fluorescent lamp, Philips Lighting, USA). Two judges scored independently the colour of fillets using the Roche Salmofan\u00C2\u00AE (scale 20-34, Appendix 5). Scores were assessed following the cut recommended by the Norwegian general standardizing body as stated by Sigurgisladottir et al. (1997). Briefly, each R S S reading was taken just under the dorsal fin at an equidistant point between the dorsal ridge and the pelvic fins. The colours of the individual fillets were compared with the Salmofan and a score was assigned according to the matching colour of the Salmofan tiles (20-34). Fillets of the control diet group, as well as some of the smaller fillets from fish fed astaxanthin-fortified diet groups showed flesh colour below the minimum score of 20 (ranging from the palest green to the palest peach), and they were assigned a score of 20. 3.6 GROWTH ASSESSMENT 3.6.1 Wet weight gain Random samples offish were taken from all diet treatment groups. Fish were anaesthetized (MS-222\u00C2\u00AE and N a H C 0 3 , 50 mg/L and 100 mg/L, respectively) and weights and lengths were recorded individually (n=20). This permitted us to calculate the mean weight increase per dietary group. The fork length of each fish was determined by measuring from the tip of the snout to the fork of the tail (fork length). 3.6.2 Specific growth rate (SGR) S G R (percent of increase in weight/day) values were calculated according to the formula: SGR = (Ln Wtf - Ln Wti)/t *100 19 Where, Wtf is mean final weight, Wtj is initial mean weight, and t is time (in days) between samplings. 3.6.3 Feed conversion rate (FCR) FCR values were estimated according to the formula: FCR = amount of fed food (kg)/fish biomass increase (kg) Where, feed was the weight of feed fed to the fish groups population and biomass increase was the difference between the initial and final mean weights of the fish in each group. 3.6.4 Fulton's condition factor (CF) Condition factor was estimated according to the formula: CF = Wt/Length3 *100 Where, Wt is the mean weight of the fish population (g), and length is the mean fork length (cm) of the fish population. 3.7 DISEASE CHALLENGE IN FRESHWATER A virulent strain of Vibrio anguillarum (Vang) that was originally isolated from an adult chum salmon (Oncorhynchus keta) which exhibited signs of vibriosis was used in this experiment (isolate 98055, Pacific Biological Station, DFO, Nanaimo, B.C). The isolates were grown on tripticase soy agar (TSA) plates supplemented with 1.5% NaCI. Bacteria in their log phase (24 h at 20\u00C2\u00B0C) were transferred into sterile peptone-saline (PS, 0.2% peptone and 1.5% saline, pH 7.2) and vortexed to suspend the bacteria thoroughly in the solution. The concentration of Vang used to inoculate the fish was estimated from absorbance measurements made by spectrophotometer at 540 nm (one OD unit at 540 nm was estimated to contain 10 9 cfu/mL). The suspension was diluted to reach a final concentration of 1x10 9 cfu/mL, as determined by drop-plate counts. 3.7.1 Bacteria injection protocol A volume of 0.1 mL of a bacterial suspension of Vang (10 9 cfu/mL) was injected into the peritoneal (i.p) cavity offish. Sham fish groups were inoculated with 0.1 mL i.p of sterile peptone saline, following the same procedure as that described for the disease challenge groups. All injection procedures were carried out in fish anaesthetized in a 20 buffered solution of MS-222\u00C2\u00AE and N a H C 0 3 (50 mg/L and 100 mg/L, respectively) and the bacterial suspension and sham solutions were maintained on ice during the duration of the inoculation procedure. 3.7.2 Bacterial re-isolation and identification from moribund fish Vang was re-isolated and identified from moribund fish from the challenge groups. Samples from liver and head kidney were grown on trypticase soy agar (TSA) (Difco Laboratories, Oakville, ON, Canada) and 0.85% NaCI for 20-24 h. Well isolated suspicious colonies (smooth, opaque, creamy colour, medium size) were picked, smeared onto a microscope slide and Gram stained. Small Gram-negative bacilli (often curved) were considered Vibrio anguillarum suspicious. Biochemical confirmation of the bacteria was performed using API\u00C2\u00AE 20-E kit (BioMerieux, Missouri, MO, USA) (Appendix 6). A single well-isolated colony was removed from the agar plate and tested for an oxidase positive reaction. Another similar single colony was placed in 0.85% NaCI and emulsified to obtain a homogeneous suspension. Thereafter, this suspension was used to fill all 20 chambers of an API\u00C2\u00AE 20-E strip. Colonies exhibiting oxidase (+), gelatinase (+), oxidation and fermentation of glucose (sorbitol, sucrose, arabinose) (+), and Voges Proskauer (+) were identified as Vang positives. 4. CHAPTER ONE Assessment of flesh pigmentation and growth of pre-smolt farmed chinook salmon fed one of two concentrations of a natural flavonoid antioxidant and astaxanthin in the diet 22 4.1 INTRODUCTION The effects of feeding juvenile chinook salmon diets containing a combination of natural dietary antioxidants, other than antioxidant vitamins (i.e. vitamin A, vitamin C, vitamin E), and astaxanthin on flesh pigmentation and growth variables have not been described previously. Kikkoman proanthocyanidins are natural flavonoid antioxidants that have been shown to exert beneficial effects both in vitro and in vivo. Amongst other attributes, PA have been shown to enhance the growth and viability of gastric mucosal cells in humans and macrophages in rats (Ye et al. 1999). KPA\u00C2\u00AE has also been shown to provide photo-oxidative protection of flesh colour in salmon fillets soaked in a KPA\u00C2\u00AE-saline solution (Kikkoman Corporation, personal communication). Astaxanthin is a naturally occurring carotenoid pigment that provides salmonids with their characteristic red-pink colour to various tissues. Salmonids must obtain carotenoids from a dietary source. They are deposited into the muscle, skin, and gonads according to the physiological state of the fish (Torrissen, 1985). Carotenoids are an essential nutritional supplement in the diets of farmed salmonids during their early development (Christiansen et al. 1994). A minimum of 5 ppm of astaxanthin is required in the diet to ensure fish survival, optimal development and growth in Atlantic salmon swim-up fry (Salmo salar) during first-feeding. There are conflicting results in the literature regarding the ability of small salmonids to deposit carotenoid pigment in their flesh. Some reports indicate either no presence of astaxanthin in muscle or very little deposition of pigment. For instance, Christiansen era/. (1995b) reported negligible deposition of astaxanthin in the muscle of Atlantic salmon fry (weight ~4 g). Alternatively, Thomas (1999) and Wieruszewski (2000) reported some deposition of astaxanthin in the muscle of small chinook and Atlantic salmon (<100 g), respectively. The deposition of pigment in the flesh of young salmonids is not completely understood. Absorption, metabolism, and deposition of carotenoids in salmon depend on several endogenous and exogenous factors. In addition to the role of astaxanthin in pigmentation, astaxanthin has been shown to have an in vivo antioxidant activity in rainbow trout (Nakano et al. 1995). It may be speculated that feeding a combination of KPA\u00C2\u00AE and astaxanthin might increase the overall redox status in salmon and thereby exert a \"sparing effect\" on the availability of astaxanthin as a flesh pigmentation agent. 23 In this study, I compared the efficiency of astaxanthin deposition in the muscle of pre-smolt juvenile chinook salmon (initial mean weight 2.95 g) when the fish had been fed one of four experimental diets that contained either a uniform amount of astaxanthin and one of two levels of grape seed PA (KPA\u00C2\u00AE) or no supplemented astaxanthin or KPA\u00C2\u00AE for 32 days. In addition, the growth performance, as well as their condition factor and food conversions were assessed during the feeding trial. 4.2 MATERIAL AND METHODS 4.2.1 PIGMENTATION ASSESSMENT Muscle astaxanthin concentration, AARC, RSS The concentration of astaxanthin in flesh was measured before the start and after 32 days of feeding the experimental diets. Collected data regarding feed and muscle astaxanthin concentration, and total consumed feed by fish served to estimate A A R C . Pigmentation analysis and formulas were outlined in section 3.4 of the General Materials and Methods. Pigmentation analysis using the Roche Salmofan\u00C2\u00AE was not estimated at this time due to the small size of the fish. 4.2.2 GROWTH ASSESSMENT Wet weight gain, length, CF, FCR, and SGR Random samples of 20 fish were taken from all diet treatment groups. The salmon were anaesthetized (MS-222\u00C2\u00AE and N a H C 0 3 , 50 mg/L and 100 mg/L, respectively), then individually weighed and measured for fork length. Collected data was used to calculate all growth variables (CF, F C R , S G R ) according to the formulae described of section 3.6 in the General Materials and Methods section. 4.3 STATISTICAL ANALYSES Each diet was fed to four replicate groups and the results for all the parameters have been expressed as the mean \u00C2\u00B1 one standard deviation (SD). Quantitative data were analysed by A N O V A (p<0.05), and the significant differences among means were detected using Student-Newman-Keul's or Dunn's test where appropriate. All statistical analyses were performed by SigmaStat\u00E2\u0084\u00A2 software (SSPS Inc., Chicago, IL, USA). 24 4.4 RESULTS Mortality Mortality reached 3.5% during the freshwater phase of the experiment. There were no statistically significant differences in fish mortality between the diet treatment groups (p=0.872). Fish Growth and feed conversion The initial mean weights of the groups were not significantly different, averaged 2.95 \u00C2\u00B1 0.21 g (Fig 4.1). On day 32, fish fed the diet astaxanthin alone (astaxanthin diet) or together with a high or low dose of KPA\u00C2\u00AE had mean weights of 5.35 \u00C2\u00B1 1.34 g, 5.43 \u00C2\u00B1 1.18 g, and 5.66 \u00C2\u00B1 1.33 g, respectively. And these values were statistically equivalent and higher than fish fed the diet without any supplement KPA\u00C2\u00AE and astaxanthin (mean weight (4.75 \u00C2\u00B1 0.86 g). Specific growth rate values for the groups were uninfluenced by the diet treatment and varied between 2.16%/day for the control diet and 2.40%/day for chinook ingesting the diet with the high dose of KPA\u00C2\u00AE together with astaxanthin (Fig. 4.2-B). Likewise, the feed to gain ratios were also not significantly influenced by diet treatment and ranged from 1.36 \u00C2\u00B1 0.28 (low KPA\u00C2\u00AE diet) to 1.90 \u00C2\u00B1 0.54 (high KPA\u00C2\u00AE diet). Fulton's condition factor was recorded weekly throughout the feeding trial for all groups, but values foe the parameter were generally not affected by diet treatment, except for fish fed diet with high K P A which had a significantly higher C F (p=0.047) than noted for all the other fish groups on day 21 (Fig. 4.3). Pigmentation assessment Muscle astaxanthin Pre-smolt chinook salmon fed the control diet exhibited a statistically significant (p=0.019) lower concentration of astaxanthin in muscle after 32-day feeding period, compared with treatment groups fed diets containing 60 ppm astaxanthin (Figure 4.4). Moreover, fish fed the control diet had similar flesh astaxanthin concentration compared with the fish on day 0 (0.0290 pg/g vs. 0.0285 \xg/g, respectively). Chinook fed the astaxanthin, low KPA\u00C2\u00AE and high KPA\u00C2\u00AE diet had an overall three-fold increase in flesh 25 astaxanthin concentration after 32 days (mean values ranged from 0.098 Lig/g to 0.104 ng/g)-. Correlation between dietary astaxanthin (DAX), apparent astaxanthin retention coefficient (AARC) and HPLC astaxanthin concentration in muscle (Figure 4.5-A and 4.5-B) A significant positive correlation was detected both between DAX (|ag/g) and the concentration of astaxanthin in muscle (Figure 4.5-A, 0.996, p=0.029, n=4); and DAX and A A R C (Figure 4.5-B, r*= 0.994, p=0.004, n=4). Apparent astaxanthin retention coefficient, AARC Fish groups fed the high KPA\u00C2\u00AE diet for 32 days showed a higher, but not significant (p=0.709) apparent astaxanthin retention coefficient (0.069%) than fish groups fed low KPA\u00C2\u00AE (0.068%), and astaxanthin diet (0.060%), respectively (Table 4.1). 26 9.0 8.5 8.0 7.5 I 7 . 0 H ^ 6.0 g 5.0 -I \u00C2\u00A3 4.5 \u00E2\u0080\u00A25 4.0 5 3.5 .<\u00C2\u00A3 3.0 LL 2.5 2.0 1.5 1.0 Control Diet V \" High KPA Diet \u00E2\u0080\u00A2H\u00E2\u0080\u0094 Low KPA Diet Astaxanthin Diet D a y 0 D a y 7 D a y 1 4 D a y 2 1 D a y 2 8 D a y 3 2 Sampling Date Figure 4.1 Wet weight increase in pre-smolt chinook salmon (O. tshawytscha) fed four experimental diets containing various levels of antioxidant supplementation (A\u00C2\u00B0KPA\u00C2\u00B0, A 6 &KPA\u00C2\u00B0, A 6 0 K P A 1 0 0 0 , A 6 0 K P A 1 0 0 ) after a 32-day feeding period. Letters indicate a statistical d ifference between d ietary g roups (n=20/diet treatment, four replicates/diet, p=0.043). 27 Dietary Group mmm Control Diet V//////A High K P A Diet Low K P A Diet Astaxanthin Diet Figure 4.2 Mean (\u00C2\u00B1 SD) apparent values for F C R (4.2-A) and S G R (4.2-B) in pre-smolt chinook salmon (O. tshawytscha) fed four experimental diets after a 32-day feeding period. Different letters indicate significant differences (n=20/diet treatment, four replicates/diet, p<0.05) between diet groups. 28 n o U) o (0 LL c o c o o Hp Day 0 Day 7 Day 14 Day 21 Day 28 Day 32 Sampling Date Control Diet P%3?a High KPA Diet Low KPA Diet Astaxanthin Diet Figure 4.3 Mean (\u00C2\u00B1 SD) Fulton's condition factor for pre-smolt chinook salmon (O. tshawytscha) during a 32-day feeding period fed four experimental diets containing various levels of antioxidant supplementation (A\u00C2\u00B0KPA\u00C2\u00B0, A 6 0 KPA\u00C2\u00B0, A 6 0 K P A 1 0 0 0 , A 6 0 K P A 1 0 0 ) . Solid bars represent dietary group mean, error bars indicate SD of diet group. Different letters indicate significant differences (n=20/diet treatment, four replicates/diet, p<0.05) between the diet groups. 29 0.20 Dietary Group t^m Control Diet mm High KPA Diet Low KPA Diet Astaxanthin Diet Flesh HPLC Baseline (previous experiment) Figure 4.4 Mean (\u00C2\u00B1 SD) astaxanthin concentration in flesh of pre-smolt chinook salmon (0. tshawytscha) after a 32-day feeding period with four experimental diets supplemented with astaxanthin and two levels of KPA\u00C2\u00AE. Solid bars represent dietary group mean, error bars indicate \u00C2\u00B1 1 SD. Different letters indicate significant differences (n=20/diet treatment, four replicates/diet, p<0.05) between diet groups. Horizontal hatched bars (far right) represent the astaxanthin concentration in the fish flesh prior to the start of the experiment. 30 0 . 1 2 - , 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 D i e t a r y A s t a x a n t h i n I n g e s t e d ( D A X , f i g / g ) c ro x 2 < 0! Q . Q. < C 0) o i t Q) O O c o c 0.05) affected by diet treatment during this part of the study (Fig. 5.2-A and 5.2-B). There were however, trends for improvements in fish growth rates and feed utilization when they were fed the high and low KPA\u00C2\u00AE diet and the astaxanthin diet. The observed FCR after 155-day feeding period was 1.27 \u00C2\u00B1 0.58, 1.35 \u00C2\u00B1 0.47, 1.40 \u00C2\u00B1 0.52 and 1.54 \u00C2\u00B1 0.66, for diet high and low KPA\u00C2\u00AE, astaxanthin only and control diet, respectively. The observed S G R was 1.58 \u00C2\u00B1 0.72, 1.44 + 0.56, 1.43 \u00C2\u00B1 0.57 and 1.34 \u00C2\u00B1 0.65, for the same diet treatment. Diet treatment had no significant effect on the Fulton's condition factors of the fish during seawater phase of the study (Fig 5.3) regardless of the sampling time. Pigmentation assessment Muscle astaxanthin (Figure 5.4-A) Post-smolt chinook salmon cultured in seacages for 155 days and fed diets either a combination of astaxanthin and KPA\u00C2\u00AE or astaxanthin-alone had significantly higher deposition of astaxanthin in their muscle than those fed a control diet without supplemental astaxanthin (p<0.001). Fish fed the high KPA\u00C2\u00AE diet had significantly more astaxanthin in their muscle (0.864 p.g/g, p=0.036) than those fed the low KPA\u00C2\u00AE (0.630 u,g/g), although the difference was not significant when compared with those fed the astaxanthin diet (0.754 u,g/g, p=0.543) (Figure 5.4-A). Visual Roche Salmofan values (Figure 5.4 b) Attempts to visually score the colour of the fillets using the Roche Salmofan\u00C2\u00AE (Hoffman-La Roche, Basel, Switzerland) were unsuccessful at all sampling times in freshwater and saltwater except at the last weight sampling in January 2001, when all fish had reached the proper size to enable visual assessment of differences in the colour if the fillets. Post-smolts fed the high KPA\u00C2\u00AE diet had a significantly higher fillet score (24.3 \u00C2\u00B1 1.8, p=0.029) than fish fed the diet with astaxanthin only (23.0 \u00C2\u00B1 0.8) or low KPA\u00C2\u00AE (22.0 \u00C2\u00B1 1.7) or the control diet (20.0 \u00C2\u00B1 0.0). There were no statistical differences between the fillet scores offish fed the low KPA\u00C2\u00AE and astaxanthin diets (p=0.489). Fish fed the astaxanthin-containing diets had significantly higher scores than those fed the diet without astaxanthin (p<0.001). Apparent astaxanthin retention coefficient, AARC (Table 5.1) Fish fed the high KPA\u00C2\u00AE diet for 155 days in SW showed a higher apparent astaxanthin retention coefficient (1.28%) than groups fed the astaxanthin-only diet (1.09%) and low KPA\u00C2\u00AE diet (0.90%), respectively (Table 5.1). Correlation between Roche Salmofan scores and astaxanthin concentrations in muscle (Figure 5.5) A statistically significant positive correlation (1^=0.668, p=0.027) was detected between the visual salmofan scores in salmon fillets and the muscle concentrations of astaxanthin quantified by HPLC. 42 100 90-<5 80 CO 3 7 0 o J2 60 o c 40 O) 0 \u00C2\u00A7 30 \u00C2\u00A3 20 10 50 -Control Diet V High KPA Diet \u00E2\u0080\u0094 L o w KPA Diet O Astaxanthin Diet s \u00E2\u0080\u00A2A w /-/$-*\u00E2\u0080\u00A2 * / s f a ab 0 August September October NovemlDeilDecember January Sampling Date Figure 5.1 Weight gain in post-smolt chinook salmon (O. tshawytscha) fed four experimental diets containing various levels of antioxidant (astaxanthin and KPA\u00C2\u00AE) supplementation (A\u00C2\u00B0KPA\u00C2\u00B0, A K P A 0 , A 6 0 K P A 1 0 0 0 , A 6 0 K P A 1 0 0 ) after a 155-day feeding period in seacages under commercial farming conditions. Different letters indicate significant differences (n=25/diet treatment, single diet groups, p<0.05) between diet groups. 43 3.2 -n Dietary Group 3.2 - , Dietary Group \u00E2\u0080\u00A2 M H Con t ro l Diet V//////A High K P A Diet P M M Low K P A Diet mm\u00C2\u00AEt As taxan th in Diet Figure 5.2 Mean (\u00C2\u00B1 SD) values for apparent FCR (5.2-A) and S G R (5.2-B) of post-smolt chinook salmon (O. tshawytscha) fed diets supplemented with astaxanthin alone or together with one of two levels of grape seed extract, or no astaxanthin and grape seed extract (A 6 0KPA\u00C2\u00B0, A 6 0 K P A 1 0 0 0 , A 6 0 K P A 1 0 0 and A\u00C2\u00B0KPA\u00C2\u00B0) after a 155-day feeding period in seacages under commercial farming conditions. Common letters indicate significant differences (n=25/diet treatment, single diet groups, p<0.05) between diet groups. 44 September October November December Date of Sampling \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 Control Diet 777777), High KPA Diet ^ 3 Low KPA Diet Astaxanthin Det January Figure 5.3 Temporal changes in mean (\u00C2\u00B1 SD) condition factors for post-smolt chinook salmon (0. tshawytscha) fed diets supplemented with astaxanthin alone or together with one of two levels of grape seed extract, or no astaxanthin and grape seed extract (A 6 0KPA\u00C2\u00B0, A 6 0 K P A 1 0 0 0 , A 6 0 K P A 1 0 0 and A\u00C2\u00B0KPA\u00C2\u00B0) after a 155-day feeding period in seacages under commercial farming conditions. Common letters indicate significant differences (n=25/diet treatment, single diet groups, p<0.05) between diet groups. 45 1.20 - i August October January Sampling Date 34 - . 32 -January Sampling Date Figure 5.4 Muscle astaxanthin concentration of post-smolt chinook salmon (O. tshawytscha) assessed by HPLC (5.4-A) and visual estimation of Roche salmofan\u00C2\u00AE scores for fillet (mean \u00C2\u00B1 SD) (5.4-B) after a 155-day feeding period in seacages. Groups were fed diets supplemented with astaxanthin alone or together with one of two levels of grape seed extract, or no astaxanthin and grape seed extract (A 6 0KPA\u00C2\u00B0, A 6 0 K P A 1 0 0 0 , A 6 0 K P A 1 0 0 and A\u00C2\u00B0KPA\u00C2\u00B0) in seacages under commercial farming conditions. Different letters indicate significant differences (n=25/diet treatment, single diet groups, p<0.05) between diet groups. 46 Table 5.1 Apparent Astaxanthin Retention Coefficients (AARC) in muscle of post-smolt chinook salmon (O. tshawytscha) fed four experimental diets containing no antioxidant supplementation (control diet), astaxanthin (astaxanthin diet), astaxanthin and two concentrations of grape seed extract (high and low KPA\u00C2\u00AE diets, respectively). Single diet groups were raised in seacages for 155 days and fed between 2-2.5% b.w/day. Different letters indicate significant differences (n=25/diet treatment, single diet groups, p<0.05) between diet groups, (n.a. not analyzed) Feeding Period: September 2000 - January 2001 Wi Wf Axd Feed DAx Axi Axf AARC Diet Group (g) (g) (ng/g) (kg) (mg) (ng/g) (ng/g) (%) 7.3a 58.2 c 0.21 D 0.028\u00C2\u00B0 0.047 c A\u00C2\u00B0KPA\u00C2\u00B0 (1.7) (14.1) (0.01) 62.656 13.1 (0.013) (0.018) n.a 7.4 a 82.6 a 60.4 a 0.104 a 0.864a A 6 o K p A i o o o (1.6) (15.5) (1.51) 73.825 4466.4 (0.033) (0.159) 1.28a 8.0 a 75.8 a D 56.20 a 0.101 a 0.632\" A 6 0 K P A 1 0 0 (1.7) (8.7) (2.43) 69.690 3916.6 (0.027) (0.164) 0.90 b 7.6 a 69.4\u00C2\u00B0 57.57 a 0.098 a 0.754 a D A60KPA\u00C2\u00B0 (1.3) (10.9) (1.51) 64.635 3723.0 (0.030) (0.194) 1.09\u00C2\u00B0 47 Figure 5.5 Correlation between Roche Salmofan\u00C2\u00AE scores (RSS) and concentrations of astaxanthin in chinook salmon muscle (HPLC) following a 155-day feeding period in seacages (n=28, ^=0.668, p=0.027). 48 5.5 DISCUSSION Growth, in simple terms, results from the elaboration of new tissue and is the principal aim offish farming. Pre-smolt chinook salmon (<6 g) fed diets fortified with a combination of grape seed PA and astaxanthin for a short period of time in FW did not result in significant growth differences compared with fish fed diets containing astaxanthin alone, although a significant low weight gain was observed in fish fed diets containing no dietary astaxanthin and KPA\u00C2\u00AE. The mean initial weights of post-smolts transferred to growout seacages were not significantly different among diet treatment groups (average 7.57 \u00C2\u00B1 2.26 g, p=0.389). The observed final mean weights of chinook salmon reared for 155 days in seacages, however, showed important diet differences. In this regard, the final mean weight in relation to diet treatment was as follows: high KPA\u00C2\u00AE diet (82 g) > low KPA\u00C2\u00AE diet (74 g) > astaxanthin diet (69 g) > control diet (58 g). Fish groups fed the high KPA\u00C2\u00AE, low KPA\u00C2\u00AE and astaxanthin only diet exhibited percent gain in weight of 42%, 30% and 18%, respectively, compared to that offish fed the control diet. As discussed in chapter 1, feeding level directly affects fish growth and feed conversion efficiency. The overestimated feeding ratio of 3.5% b.w a day in experiment A (fresh water trial) allowed pre-smolt chinook salmon to achieve maximum growth but adversely affected values for feed utilization (FCR). The daily feeding rate used for the post-smolt chinook salmon was established at 2.0-2.5% b.w a day. The Ewos (Surrey, BC, Canada) feeding chart recommendations for chinook salmon cultured in saltwater at water temperatures ranging from 8\u00C2\u00B0C to 12\u00C2\u00B0C varies between 1.46% to 1.85% b.w./day. Although this recommendation is based on higher energy diets than the employed in my research, hence the results support that the feeding rate chosen for the experiment in saltwater was more realistic for local salmon farming practice than that used in the experiment in freshwater. The specific growth rates obtained for post-smolt chinook salmon in growout seacages ranged from 1.27% to 1.58%/day. Fish fed the high KPA\u00C2\u00AE diet had the best growth rate (1.58%/day), followed by those fed the low KPA\u00C2\u00AE and astaxanthin-only diet (both 1.40%/day). These results agree with those reported previously by Austreng et al. (1987) for both Atlantic salmon and rainbow trout (-1.60%) reared at comparable water temperatures (8\u00C2\u00B0C to 12\u00C2\u00B0C) and also under practical salmon farming conditions. My 49 results show better growth rates of chinook salmon than the observed by March and McMillan (1996), who reported a S G R value of 0.67%/day in chinook salmon fed for 25 weeks. Kreiberg (1991) found a growth rate of 0.70% per day for chinook salmon fed a commercial grower diet to satiation for 6 months during their first year in seawater (initial weight 70 g). The S G R values of this latter study agree with those previously observed by Kreiberg et al. (1989). The predicted growth and estimated final weight in fish reared in seacages using the thermal growth coefficient (TGC) formula was achieved only by the fish group fed the high KPA\u00C2\u00AE diet. The other diet groups showed less growth than the predicted weights and the percent differences were -11% in the low KPA\u00C2\u00AE, -17% in the astaxanthin-only, and -30% in the control diet groups. Best fed to gain ratios or utilization of feed for growth was attained by fish fed diets supplemented with the combination of astaxanthin and either high or low KPA\u00C2\u00AE, 1.27 and 1.35, respectively. The fish group fed the astaxanthin-only diet showed a F C R value of 1.40, and those fed the control diet had a F C R of 1.54. Feed conversion ratios obtained in experiment C are within acceptable feed efficiency standards for cultured salmon in seawater at this life history stage. Thomas (1999) noted that the feed efficiency of chinook salmon ranging in weight from 40 g to 120 g was 1.1 after they were fed an experimental diet and held in SW. The initial Fulton's condition factors in the present study varied from 1.10-1.15, and these values fall within the C F range reported for recently transferred post-smolt salmonids. This low weight to length ratio is a consequence of a transient reduction in appetite after transfer of smolts from FW to SW. (Usher et al. 1991; Jorgensen and Jobling, 1994). Afterwards, C F values showed an increasing trend towards the end of the feeding trial. The values ranged from 1.35-1.40. Such C F values have previously been reported for chinook salmon cultured in saltwater (Mazur, 1986). Furthermore, the observed pattern of increasing condition factors in the fish group fed the high KPA\u00C2\u00AE diet suggests that well fed fish have higher condition factors than fish of the same length fed a less optimal diet (Piper et al. 1982). Salmon diets contain large amounts of lipids and these are mainly of marine origin. Marine fish oils contain 20% to 25% polyunsaturated fatty acids (PUFA). PUFA of the n-3 family serve as structural building blocks in cell membranes, precursors of bioactive eicosanoids compounds, and as reservoirs of available energy. 50 Polyunsaturated fatty acids present in fish diets, biomembranes and plasma lipids are more susceptible to lipid peroxidation. The presence of dietary antioxidants has shown to reduce lipid peroxidation than less unsaturated fatty acids. Proanthocyanidins contained in red wine have also been shown to significantly reduce lipid peroxidation of LDL in humans (Frankel etal. 1993). Moreover, rainbow trout fed astaxanthin-containing diets had reduced lipid peroxidation in vivo (Nakano etal. 1999). The salmon feed used in this experiment was stored at room temperature in a shed during late summer, fall and early winter. Heat and oxygen are environmental factors that are known to initiate, or to lead to intermediate metabolite participants in the oxidation of lipids and other oxidation-sensitive compounds. Autoxidation of unsaturated fatty acids produces a large number of free radicals and peroxide compounds, which are active pro-oxidants. These compounds may react with other diet ingredients (i.e. vitamins) and reduce their nutritional value, or after ingestion, react with oxidation-sensitive phospholipids in the cellular and subcellular membranes and cause oxidative damage. Roberts and Bullock (1989) have described that the ingestion of oxidised fish oils may reduce growth rate, provoke anaemia, nutritional muscular dystrophy, and ceroidosis in the liver offish. The quality of the dietary lipid or extent of lipid peroxidation was not followed in this study but it is conceivable that a low level of lipid oxidation occurred during the experimental period especially in diets not supplemented with KPA\u00C2\u00AE. Grape seed PA have been shown to enhance the growth and viability of normal human gastric mucosal cells and murine macrophage J774A.1 cells (Ye et al. 1999). Cell health, communication, membrane fluidity, cell receptors, enzymes, and RNA and protein production might be protected by the actions of PA and their potential synergistic interaction with other antioxidants in the body. This overall cellular integrity might be reflected in greater ability for use of dietary protein, lipids and vitamins for new tissue growth. The dietary antioxidant combination effects were not evident after the short rearing time in freshwater tanks, but when they were fed for longer time in saltwater seacages, they were able to exhibit important differences in weight gain, growth rate. They appeared to be related to the dietary concentration of KPA\u00C2\u00AE as described above. The distinctive red-pink colour of salmon flesh, produced by the retention of astaxanthin in the muscle, provides an immediate indication of product quality and is, therefore, an essential aspect offish farming, product marketing and commercial feed 51 production. As fish cannot synthesize these pigments, they must be provided in the diet. A deep and even red colour in fish muscle is more than a cosmetic effect and the consumer associates this characteristic with a high quality, healthy product (Schiedt, 1998). Chinook salmon fry (< 7 g) housed in FW and fed astaxanthin-containing diets for 32 days had very limited capacity for carotenoid pigment deposition in their flesh. Diets fortified with astaxanthin (60 ppm), and astaxanthin with one of two levels of KPA\u00C2\u00AE (100 and 1000 ppm) resulted in no significant deposition of astaxanthin in, salmon muscle, although a higher level of astaxanthin was observed in both of the KPA\u00C2\u00AE-astaxanthin fed groups (p=0.543). Post-smolt chinook salmon were transferred to growout seacages and fed the same experimental diets for 155 days. At the conclusion of this feeding trial, astaxanthin concentrations in the muscle of chinook salmon fed the diets supplemented with astaxanthin with or without KPA\u00C2\u00AE showed a six to eight-fold increase over initial values. Initial astaxanthin concentrations in the muscle of the fish consuming the astaxanthin-supplemented diets showed no significant differences, ranged from 0.098 to 0.104 ux|/g, whereas the mean value offish fed the astaxanthin free diet was 0.028 ug/g. Fish fed the high KPA\u00C2\u00AE diet attained the highest deposition of astaxanthin in muscle (0.863 u,g/g), followed by those fed the astaxanthin diet (0.751 u,g/g), and the low KPA\u00C2\u00AE diet (0.632 p.g/g). Muscle astaxanthin deposition in fish fed the control diet remained negligible (0.047 ug/g). As mentioned previously, salmon have to reach a certain size to deposit carotenoids efficiently in the actomyosin complex of their muscle. Torrissen (1985) and March etal. (1990) have positively correlated astaxanthin deposition in muscle of salmonids with an increase in body weight. Chinook salmon that were held in seacages and had an average final weight of less than 85 g showed little deposition of astaxanthin in their muscle (<1.0 u.g/g) and estimated apparent astaxanthin retention coefficients were low (<1.3%). Several experiments have reported carotenoid concentrations in muscle of salmonids ranging between 1.0 uxj/g and up to 3.0 u,g/g in salmonids weighing less than 200 g (Christiansen and Wallace 1988; Bjerkeng et al. 1992; Smith etal. 1992; Hatlen etal. 1995; Thomas, 1999; Wieruszewski, 2000). Christiansen etal. (1995b) reported a two-fold increase of astaxanthin deposition from 1.2 ug/g to 2.7 (ig/g 52 in Atlantic salmon parr muscle (initial to final weight, 16 g to 58 g, respectively) fed a diet fortified with 60 ppm astaxanthin for 10.5 months. However, these authors failed to mention the total dietary amount of astaxanthin ingested by the fish. In contrast, Spinelli and Mahnken, (1978) found no pigment deposition in coho salmon and Storebakken et al. (1987) also reported no pigment deposition in Atlantic salmon weighing less than 80 g and fed astaxanthin-containing diets (30, 50 and 90 ppm). These authors found that muscle pigment carotenoid deposition only began when fish reached a body weight over 100 g. The reason for the meagre deposition of astaxanthin in the flesh of young salmonids is still unknown. The linear model hypothesized for carotenoid deposition by Torrissen etal. (1989) might explain the low final astaxanthin concentration in muscle of salmon that size. They propose that there is a linear increase in carotenoid concentration as the fish increases in weight, assuming there is also an increase in retention efficiency. This theory contemplates a 1% carotenoid retention in fish weighing 0.2 to 1 kg, reaching a maximum of 8.8% for fish weighing 4 to 5 kg (Torrissen et al. 1989). Also, juvenile salmonids show a preferential deposition of carotenoids in skin rather than in flesh (Torrisen et al. 1989). Finally, circulating levels of the main carotenoid-carrying lipoproteins (HDL and VDHL) are present in low quantities in juvenile salmonids (Choubert etal. 1992, 1994) compared to circulatory levels of these lipoproteins in adult salmonids. The fortification of astaxanthin-containing diets with an antioxidant, such as KPA\u00C2\u00AE, that has demonstrated strong FR scavenging properties, might have improved the deposition of astaxanthin in muscle by reducing the use of astaxanthin as an antioxidant (\"sparing effect\"), hence there may have been more astaxanthin available for deposition in the actomyosin complex. Nakano et al. (1999) showed the dual pigment-antioxidant properties of the carotenoid astaxanthin in rainbow trout. Other possibilities include an increased functionality of astaxanthin receptors in the muscle cell membrane, or a reduction in lipid peroxidation of astaxanthin-carrying lipoproteins due to the presence of KPA\u00C2\u00AE. The reduced efficiency of deposition of astaxanthin in salmon fed the experimental diets in this research might have been due to loss of astaxanthin during diet storage due to heat, oxygen decomposition and some lipid peroxidation in the food. Unfortunately astaxanthin concentrations in the feed at the end of the experiment were 53 not determined. Christiansen et al. (1995a) reported loss of astaxanthin from 59.6 ppm to 47.8 ppm (20%) in fish diets kept for 10.5 months at -20\u00C2\u00B0C. The supply of freshly made fish feed in my research did not exceed more than 8 weeks between different feed batches. Thus the loss of astaxanthin during diet storage was probably minimal. Regardless of the cause of the low astaxanthin deposition, the supplementation of high concentration of grape seed PA in the diet significantly increased both the efficiency of pigment deposition in chinook salmon muscle and the total final concentration level of astaxanthin in the flesh after 155-day feeding period. Assessing flesh colour in salmonids at slaughter by using visual means is an industry standard. Roche Salmofan\u00C2\u00AE scores observed for fillets from fish fed the high KPA\u00C2\u00AE diet were significantly greater than those noted from the other 3 diets. Although there were no statistical difference between the low concentration of KPA\u00C2\u00AE diet and astaxanthin-only diet, the Salmofan\u00C2\u00AE score were significantly greater than those for fish fed the control diet. The visual assessment of the flesh colour obtained using the Roche Salmofan\u00C2\u00AE agreed with the muscle astaxanthin concentration results obtained by chemical analysis (HPLC) of flesh. The use of R S S in chinook salmon (weights ranging between 50 to 80 g) fillets proved to be a helpful and practical tool to estimate and compare the colour of salmon flesh. Visual pigmentation scores assessed by R C C have been reported to correlate well with HPLC concentrations of astaxanthin in the flesh of Atlantic salmon (Christiansen et al. 1995c), rainbow trout (Smith et al. 1992), and chinook salmon (Thomas, 1999). In the present study, a good correlation was found between Salmofan\u00C2\u00AE and astaxanthin concentrations in muscle ((^=0.668) as measured chemically by HPLC. This result supports the high coefficient regression found by Smith et al. 1992 (1^=0.997), and Thomas, 1999 (r^O.911). 6. CHAPTER THREE Non-specific immune responses of pre-smolt chinook salmon fed of two concentrations of a natural flavonoid antioxidant and astaxanthin in the diet, to an intraperitoneal injection of Vibrio anguillarum 55 6.1 INTRODUCTION The bacterial fish disease vibriosis is caused by Vibrio anguillarum. Vibriosis is one of the most economically important diseases of marine cultured and feral fish (Toranzo et al. 1997). Vibrio anguillarum is a ubiquitous, facultative pathogen of aquatic organisms living in marine and brackish environment worldwide (Groff and LaPatra, 2001) The immune system in fish is comprised of precise defensive mechanisms to prevent and control disease caused pathogens entering, spreading and multiplying in the body. The immune system is composed of cells and molecules and when the body reacts to an infection, many powerful antimicrobial processes are brought into play (i.e. respiratory burst). These defense mechanisms result in the production of vast amounts of free radicals. Dietary supplementation of antioxidants has been postulated to enhance the immune responses of animals by protecting cell membranes from the oxidative damage caused by free radicals, modulating the activity of molecules and enzymes that participate in the immune response (Halliwell et al. 1994), and working synergistically with one to another to exert a \"sparing effect\". Fortification of diets with large amounts of antioxidant vitamins (i.e. vitamin E, C, (3-carotene) has received considerable attention in fish (Sealey and Gatlin, 1999). Nakano etal. (1995, 1999) demonstrated the in vivo antioxidant activity of astaxanthin since lipid peroxidation was reduced in rainbow trout tissues when the diet was supplemented with this pigment source. Grape seed extract has been shown to exert great cellular protection by scavenging free radicals, reducing lipid peroxidation, and modulating the activity of inflammatory enzymes such as cyclooxygenase and lipooxygenase (Hollman and Katan, 1998). There has been no work documenting the use of antioxidants other than antioxidant vitamins, micronutrients and astaxanthin in fish diets within the current feeding practice of intensive salmon farming. This study assessed the effects of feeding a combination of a natural flavonoid antioxidant (grape seed extract, KPA\u00C2\u00AE) and astaxanthin in salmon feed on the non-specific immune responses of pre smolt chinook salmon after a disease challenge by an i.p injection with a bacterial suspension (Vibrio anguillarum). 56 6.2 MATERIALS AND METHODS 6.2.1 EXPERIMENTAL DESIGN, GENERAL HUSBANDRY AND DISEASE CHALLENGE PROTOCOL Fish used in this experiment and challenge protocol were described in sub-section 3.1, 3.2.2, and 3.7 in the General Materials and Methods section. After bacterial suspension and sterile peptone saline (0.1 mL i.p.) were inoculated, samples (n=6 fish/tank) were obtained daily for blood and tissue analysis. The water temperature was 9.0 \u00C2\u00B1 0.5\u00C2\u00B0C throughout the sampling period. 6.2.2 CLINICAL HAEMATOLOGY Primary haematology indices (haemoglobin, haematocrit, and total red blood cell) and their derived indices (mean corpuscular volume, MCV; mean corpuscular haemoglobin, M C H ; and mean corpuscular haemoglobin concentration, MCHC)were determined according to the protocols outlined by Houston (1990) and Klontz (1994). Fish from each treatment group were rapidly sacrificed in a lethal buffered solution of anaesthetic (500 mg/L MS-222\u00C2\u00AE + 500 mg/L N a H C 0 3 ) . Blood was collected in a microhaematocrit capillary tube after severing the caudal peduncle with a scalpel, a few millimetres posterior to the adipose fin. Primary haematology indices 6.2.2.1 Haemoglobin (Hb) . Haemoglobin was analysed as cyanmethemoglobin according to the procedure described by Houston (1990) using Drabkin's reagent (Kit 525, Sigma, ON, Canada) and samples were read in a microplate spectrophotometer SpectraMAX 340pc (Molecular Devices, Sunnyvale, CA, USA) at 540 nm. The concentration of haemoglobin in the blood sample is directly proportional to the absorption read in the spectrophotometer. 6.2.2.2 Haematocrit (Hct) Fish haematocrit was analysed according to Klontz (1994). Blood was directly sampled into a 75 mm heparinized microhaematocrit capillary tube (Fisherbrand; Fisher, Inc., Pittsburgh, PA, USA), sealed and centrifuged in a haematocrit centrifuge (IEC Micro-MB Microcentrifuge, Philadelphia, PA) at 11,500 rpm for 5 min. 57 6.2.2.3 Total red blood cell count (RBC) The total numbers of circulating erythrocytes (RBC) were estimated using a haemocytometer according to the technique described by Houston (1990). The cell concentration for each sample was calculated according to Klontz (1994): RBCs/mL = number of erythrocytes counted x 10 (counts for depth of chamber, 0.1 mm) x 5 (corrects the chamber area of 0.2 mm 2) x 200 (dilution factor). Secondary haematological indices 6.2.2.4 Mean Corpuscular Volume (MCV) Expresses the mean volume of an individual erythrocyte. MCV= (Hct (%) x 10) / R B C (reported in nm 3 , Houston 1990). 6.2.2.5 Mean Corpuscular Haemoglobin (MCH) Expresses the mean amount of haemoglobin contained within an erythrocyte. MCH= (Hb x 10) / R B C (reported in uo/cell, Houston 1990). 6.2.2.6 Mean Corpuscular Haemoglobin Concentration (MCHC) Expresses the relative amount of haemoglobin per unit volume of cell. MCHC= (Hb x 100) / Hct (reported as g/100 mL, Houston 1990). 6.2.2.7 Total leucocytes count (WBC) Total circulating leucocytes were estimated according to Klontz (1994). The number of total leucocytes per mL was estimated as: WBC/mL = number of total leucocytes counted x 10 (counts for depth of chamber, 0.1 mm) x 200 (dilution factor) / 4 (corrects for the chamber area of 4 mm 2). 6.2.2.8 Differential blood cell count Blood smears were stained in a modified Wright-Giemsa stain (Diff-Quick\u00C2\u00AE, Dade diagnostics Inc, Aguada, PR, USA) according to the manufacturer's directions. Smears were air-dried before staining the slides using Diff-Quick, rinsed with deionized water, and air-dried before being examined under immersion oil microscopy. Cells that touched the outer boundaries of the microscope field were not included in the count. A thousand erythrocytes were counted per slide in ten randomly selected microscopic fields (min. 50 58 RBCs; max. 200 RBCs) . In each field, the number of R B C s was tallied and leucocytes (lymphocytes, neutrophils, thrombocytes and monocytes) were differentiated and counted (Houston, 1990). 6.2.3 NON-SPECIFIC IMMUNE RESPONSE ANALYSES The non-specific immune responses were assessed by estimating the cellular and humoral immune responses in fish elicited by the bacterial pathogen Vang. Respiratory burst activity was measured in neutrophils, and lysozyme activity was assessed in plasma and head kidney tissue. In addition, the cellular stress response was estimated in hepatic tissue by measuring the production of stress proteins 70 kd (SP70) (Forsyth etal. 1997). 6.2.3.1 Neutrophil respiratory burst activity (NBT assay) The detection of superoxide anion (*02) as a result of respiratory burst activity in neutrophils was estimated using the technique described by Anderson (1992a). Briefly, a drop of blood was placed directly onto a glass microslide with 4 reaction wells (Steinmetz, Surrey, BC , Canada). The slides were incubated for 30-60 min and the excess of cells was washed away with PBS (pH 7.4). A drop of nitro blue tetrazolium (NBT) solution (Sigma cat N\u00C2\u00B0 N-5514, ON, Canada) was placed onto the cells and covered with a glass coverslip. After incubating the slides for 30 min in a moist chamber, they were observed under a microscope (1000X). The total number of positive neutrophils (polymorphonuclear leucocytes with a purple-blue stain halo) were tallied and compared with negative neutrophils between the different diet treatment groups. 6.2.3.2 Lysozyme activity in plasma and head kidney Plasma was removed from the 75 mm microhaematocrit tubes after reading haematocrit values of every blood sample. Subsequently the plasma was placed in a 0.5 mL microcentrifuge tube (cat N\u00C2\u00B0 05-407-16, ON, Canada) and immediately frozen on dry ice and stored at -80\u00C2\u00B0C until further analysis. The head kidney was dissected out and frozen on dry ice in a 1.5 mL microcentrifuge tubes, then stored at -80\u00C2\u00B0C. Plasma and head kidney samples were thawed on ice. One mL of P B S was added into the tubes containing anterior kidney tissue and the suspension was sonicated in a Vibra-Cell\u00E2\u0084\u00A2 (Sonics and Materials, Danbury, CT, USA) for 30 s. The lysates were centrifuged 59 (MSE, Microcentaur, London, England) at 13,000 rpm for 3 min at room temperature and the clear aliquot used for lysozyme assay. Lysozyme activity in plasma and head kidney was measured by microplate assay adaptation of Litwack's method (1955) as applied by Muona and Soivio (1992) and Maule et al. (1996). The decrease in optical density (OD) readings at 450 nm over 20 min of incubation at 25\u00C2\u00B0C was expressed as u.g/mL equivalent of hen egg white lysozyme activity (HEWL, Sigma, cat N\u00C2\u00B0 L-6876, ON, Canada), which was used as the standard. 6.2.3.3 Liver SP70 Hepatic tissue was sonicated in lysis buffer kept on ice (contained 100 mM Tris-HCI, 0.1% sodium dodecyl sulphate (SDS), 1 mM ethylenediamine tetra acetic acid (EDTA), and protease inhibitors (1 uM Pepstatin, 1 u.M a-Toluenesulfonyl fluoride (PMSF), 1 uJVI Leupeptin, and 0.01 u.M Aprotinin). The homogenate was centrifuged in a microcentrifuge (MSE, Microcentaur, London, England) at 16,500 g for 2 min at room temperature. Protein concentration in the lysate was estimated in duplicate aliquots of homogenate using the Bicinchoninic acid assay (BCA, Sigma Diagnostics protein kit) and using bovine serum albumin (BSA) as a reference. Ten ui. of supernatant was added to an equal volume of SDS-sample dilution buffer (Laemmli, 1970) and boiled for 3 min and then frozen at -80\u00C2\u00B0C until further SP70 analysis. Stress proteins 70 were separated by S D S - P A G E according to Laemmli (1970). Equal amounts of protein (10 u.g) and control sample were loaded onto separate gel lanes (4% stacking and 12.5% resolving gel) on a Mini-Protean II electrophoresis cell (Bio-Rad Laboratories, Hercules, CA, USA). The molecular mass of each protein band in the electrophoretic gel was estimated using pre-stained molecular weight markers (Gibco-BRL, Burlington, ON, Canada). Protein samples from the control diet group were used to normalize the data. After electrophoretic separation, strips of gel containing proteins in the 70-kda range were cut out and transferred onto nitrocellulose membranes (0.2 um pore size, Bio-Rad, Hercules, CA, USA) for western immunoblotting according to Forsyth et al. (1997). Nitrocellulose membranes were incubated for 1 h in a polyclonal rabbit antibody raised against rainbow trout (RTG-2) SP70. Following the incubation in primary 60 antibody, membranes were incubated in an alkaline phosphatase conjugated goat anti-rabbit IgG (Gibco-BRL,Burlington, ON, Canada) secondary antibody for another 1 h. Both antibodies were diluted with 2% w/v skim milk powder in TTBS (20 mM Tris, 500 mM NaCI, 0.05% v/v Tween-20, pH 7.5). The intensity of the SP70 bands was determined using a ScanJet II p (Hewlett Packard) and SigmaGel software (Jandel Scientific, CA, USA). Stress Protein 70 levels were expressed in relative units to a known positive control sample that was run simultaneously on every gel. 6.3 STATISTICAL ANALYSIS Experiments were performed using replicates groups. Quantitative data were analysed by an analysis of variance (one-way ANOVA, a=0.05) using Student-Newman-Keuls's and Dunn's tests where appropriate. Data expressed as percentages (cumulative mortality and haematocrit) were arcsine square root transformed prior to performing A N O V A analysis. Correlation between circulatory neutrophils and lysozyme activity in plasma and head kidney were determined using the Pearson's correlation test. Where significant differences were detected, all pair-wise multiple comparison tests were used to identify significantly different treatment means (p<0.05). All statistical analyses were performed using SigmaStat software (SSPS Inc., Chicago, IL, USA). Results were expressed as the mean \u00C2\u00B1 one SD. 6.4 RESULTS Mortality (Figure 6.1) Fish fed the high KPA\u00C2\u00AE diet had significantly lower (p=0.038) cumulative mortality (88.7%) compared with those ingesting the other 3 treatment diets (astaxanthin diet, 97.5%; control diet, 98.7%; low KPA\u00C2\u00AE diet, 100%) following a disease challenge with Vang injection (Figure 6.1). There were no immediate mortalities at the end of the bacteria injection procedure in any group. Mortality started within 24 h and continued up to day 10 in all diet groups. Mortality in the sham-injected groups ranged from 0.5-1.5% (up to 3 fish/tank) and was not significantly affected by diet treatment (p=0.677). There were no mortalities after 36 h following the injection with sterile peptone saline and no mortalities were attributed to Vibrio. There was a significantly lower mortality (p<0.001) in sham-injected groups compared to the bacterial injected groups. 61 100 90 J / 60 0 \u00E2\u0080\u0094 80 | 70 . ' < V Q . r / .... CD J ^ 50 \ /\u00E2\u0080\u00A2\u00E2\u0080\u00A2/ |y Control diet \u00C2\u00A7 40-j '\u00E2\u0080\u00A2/\u00E2\u0080\u00A2 V High KPA diet \u00E2\u0080\u00A2= <^ J/ - 3 - Low KPA diet 3 30- I 0 Astaxanthin diet i J 1 Sham injection groups O 20 J 3 (pooled mean) 10 0 1 2 3 4 5 6 7 8 9 10 11 Days Post Challenge Figure 6.1 Cumulative percent mortality in pre-smolt chinook salmon (O. tshawytscha) fed four experimental diets containing various levels of antioxidant supplementation (A\u00C2\u00B0KPA\u00C2\u00B0, A K P A 0 , A 6 0 K P A 1 0 0 0 , A 6 0 K P A 1 0 0 ) following an intraperitoneal injection with Vibrio anguillarum (0.1 mL, 10 9 viable cells/mL) and sham injection (0.1 mL, sterile peptone saline). Different letters indicate a significant difference (n=6/diet treatment, two replicates tanks/diet treatment, p=0.038) between dietary groups and symbol (\u00C2\u00A7) represents a significant disease difference (p<0.001) between bacteria injected groups and sham injected groups. 62 Clinical haematology Primary indices Hct, Hb, RBC (Table 6.1) There were no significant diet differences in circulating red blood cells (RBC) at any sample time throughout the disease challenge (p=0.392 to p=0.926). A significant erythrocytopenia (p<0.001 to p=0.045) was observed from day 3 throughout day 6 between bacteria-injected and sham injected groups. Haematocrit values began fluctuating at day 2 after Vang injection and no consistent significant differences were observed between fish groups in relation to dietary treatment. There was a trend towards a lower Hct in fish fed the astaxanthin diet during the disease challenge (p=0.076 to p=0.095). There were no significant diet differences in Hb values between groups (p=0.221 to p=0.249). However, on day 3 of the challenge, those fish fed the control and low KPA\u00C2\u00AE diet had significantly higher Hb values (p=0.025) compared to those fed the high KPA\u00C2\u00AE and astaxanthin diets. Secondary indices MCV, MCH, MCHC (Figure 6.2) Erythrocyte size (MCV) was uninfluenced by diet treatment (p=0.589 to p=0.975) and disease treatment (p=0.094 to p=0.533) following the bacteria and sham injections. There were significant differences in M C H after bacterial inoculation. The amount of Hb carried in erythrocytes was significantly (p=0.025) lower in fish fed the high KPA\u00C2\u00AE diet from day 1 to day 3, and in fish fed the astaxanthin diet on day 3 (p=0.033) relative to the other groups after injection with Vang. The amount of haemoglobin per 100 mL (MCHC) was not significantly different between sham and bacteria inoculation groups (p=0.190). There was a significant decrease in M C H C in fish fed the high KPA\u00C2\u00AE diet during the first 3 days of the challenge period (p=0.014 to p=0.028) compared to those fed the low and astaxanthin-only diet. Table 6.1 Primary haematology indices in pre-smolt chinook salmon (O. tshawytscha) fed diets containing no supplemental astaxanthin and KPA\u00C2\u00AE (control diet), astaxanthin (astaxanthin diet), or astaxanthin and one of two concentrations of grape seed extract (high KPA\u00C2\u00AE and low KPA\u00C2\u00AE diet, respectively) following a challenge with Vibrio anguillarum and sterile peptone saline (sham) injection. Letters indicate significant statistical differences (n=6/diet treatment, two replicates tanks/diet treatment p<0.05) between the means for diet treatment groups. Days post Vibrio anguillarum injection Haematology 1ary Index Dietary Group Prior to challenge Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Control Diet n.a 1.25\u00C2\u00B10.14a 1.20\u00C2\u00B10.29a 1.07\u00C2\u00B10.31 b 0.89 \u00C2\u00B1 0.27\u00C2\u00B0 0.77 \u00C2\u00B10.12\u00C2\u00B0 n.a Red Blood Cell High KPA Diet n.a 1.30\u00C2\u00B10.12a 1.24\u00C2\u00B10.17a 0.88 + 0.39\u00C2\u00B0 0.98 \u00C2\u00B10.28\u00C2\u00B0 0.96 \u00C2\u00B10.31\u00C2\u00B0 n.a Count Low KPA Diet n.a 1.31 +0.26a 1.26\u00C2\u00B10.25a 1.05 \u00C2\u00B10.29\u00C2\u00B0 0.98 \u00C2\u00B1 0.24\u00C2\u00B0 1.00 \u00C2\u00B10.32\u00C2\u00B0 0.78\u00C2\u00B10.20 b (x 106 cell/mL) Astaxant. Diet n.a 1.18\u00C2\u00B10.56a 1.00\u00C2\u00B10.24a 0.97 + 0.25\u00C2\u00B0 0.97 \u00C2\u00B1 0.26\u00C2\u00B0 0.85 \u00C2\u00B1 0.26\u00C2\u00B0 0.85 \u00C2\u00B1 0.34b Sham Injection n.a 1.46\u00C2\u00B10.16a 1.39 \u00C2\u00B1 0.17a 1.37 + 0.19a 1.40\u00C2\u00B10.23a 1.35\u00C2\u00B10.20a 1.36\u00C2\u00B10.40a Control Diet 48.9 + 3.3 46.5 \u00C2\u00B1 7.6a 42.8\u00C2\u00B15.8 a 36.6 \u00C2\u00B1 5.8DC 32.6 \u00C2\u00B1 7.5C 30.6 \u00C2\u00B1 1.2C n.a Haematocrit (%) High KPA Diet Low KPA Diet 50.4 \u00C2\u00B13.1 50.8 + 2.6 45.2 \u00C2\u00B1 3.9a 46.7 \u00C2\u00B1 7.8 a 44.2 \u00C2\u00B14.0 a 43.6\u00C2\u00B16.3 a 30.3 \u00C2\u00B18.1\u00C2\u00B0 38.7 + 5.4\u00C2\u00B0 36.5 \u00C2\u00B1 4.0 b c 38.9 \u00C2\u00B14.3\u00C2\u00B0 37.8\u00C2\u00B13.2 b 39.1 \u00C2\u00B15.8 b n.a 28.5\u00C2\u00B14.0 b Astaxant. Diet 48.7 + 1.7 36.8\u00C2\u00B18.3 a 34.2 \u00C2\u00B1 5.5\u00C2\u00B0 36.5 \u00C2\u00B1 7.5\u00C2\u00B0\u00C2\u00B0 38.7 \u00C2\u00B13.4\u00C2\u00B0 34.7\u00C2\u00B15.1 b c 31.2\u00C2\u00B19.0b Sham Injection n.a 45.4 \u00C2\u00B1 4.0 a 45.7 \u00C2\u00B1 3.8a 46.4 \u00C2\u00B1 3.6a 46.1 \u00C2\u00B12.7 a 46.3 \u00C2\u00B1 2.3a 45.8 \u00C2\u00B1 3.2a Control Diet 6.8 \u00C2\u00B1 1.8 7.5 \u00C2\u00B12.5\" 7.6 \u00C2\u00B12.2\u00C2\u00B0 9.0 \u00C2\u00B12.0\u00C2\u00B0 5.9 \u00C2\u00B1 2.1 \" 5.5 \u00C2\u00B1 1.0 a u n.a Haemoglobin (g/dL) High KPA Diet Low KPA Diet 5.7\u00C2\u00B10.7 6.6 \u00C2\u00B1 1.4 6.7 \u00C2\u00B1 1.6a 9.4 +2.0a 6.2 \u00C2\u00B1 0.6a 8.9\u00C2\u00B13.1 a 5.1 \u00C2\u00B12.0\u00C2\u00B0 8.4 \u00C2\u00B1 1.0a 6.2\u00C2\u00B12.3 a 5.4 \u00C2\u00B1 1.7a 6.0 \u00C2\u00B1 1.8a b 6.5\u00C2\u00B12.9 a b n.a 5.6 \u00C2\u00B1 1.2b Astaxant. Diet 6.2 \u00C2\u00B1 1.2 8.3 + 2.5a 6.5 \u00C2\u00B1 1.4a 6.5 \u00C2\u00B12.4\u00C2\u00B0 6.3 \u00C2\u00B1 1.7a 4.8\u00C2\u00B12.1 b 4.1 \u00C2\u00B1 2 . 1b Sham Injection n.a 8.4\u00C2\u00B12.1 a 6.9\u00C2\u00B10.9 a 8.6\u00C2\u00B12.4 a 7.2 \u00C2\u00B1 1.0a 8.3 \u00C2\u00B1 1.5a 7.8 \u00C2\u00B1 1.6\" 64 700 -600 - a a a a a a a a Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Days After Bacterial Injection Control Diet VZm High KPA Diet Low KPA Diet Astaxanthin Diet \u00E2\u0080\u0094O\u00E2\u0080\u0094 Sham Injection (pooled mean) Figure 6.2 Mean (\u00C2\u00B1 SD) secondary haematology indices in pre-smolt chinook salmon (O. tshawytscha) fed four experimental diets containing different supplemental levels of KPA\u00C2\u00AE and astaxanthin (A\u00C2\u00B0KPA\u00C2\u00B0, A 6 0 KPA\u00C2\u00B0, A 6 0 K P A 1 0 0 0 , A 6 0 K P A 1 0 0 ) following a disease challenge with Vibrio anguillarum (0.1 mL, 10 9 cfu/mL) and sterile peptone saline injection (0.1 mL). Different letters indicate significant dietary difference (n=6/diet treatment, two replicates tanks/diet treatment p<0.05) between diet groups. Symbol (()>) indicates a statistical difference (p<0.05) among sham injection and challenge injection groups. 65 Total White Cell Count (WBC) (Figure 6.3) There were a statistically significant (p<0.025) diet and disease treatment effect on W B C throughout the 4 day period after Vang and sham injection. Fish fed the high KPA\u00C2\u00AE diet had significantly higher (p=0.003 to p=0.025) circulating numbers of W B C during day 3 to 5 of the disease challenge compared with ingesting the other the other 3 diets. There were no differences between fish receiving the control diet, low KPA\u00C2\u00AE diet and astaxanthin diet between days 1 to 4 (p=0.104 to p=0.416), but on day 5, fish fed the control diet had a significantly lower W B C number (p=0.021) than those fed the other 3 diets. Differential Leucocyte counts (Table 6.2) Neutrophil cell counts Fish fed the high KPA\u00C2\u00AE diet had significantly higher numbers of circulating neutrophils in blood (p=0.018) on days 2, 3 and 4 after injection with Vang than noted for the other groups (Table 6.2). There were no significant differences among the fish groups fed the other 3 diets at any of the sampling times (p=0.155). Significantly higher numbers of circulating neutrophils (p=0.003) were observed in bacteria challenged groups compared to fish injected groups during the sampling period. Lymphocyte cell counts Fish fed the high KPA\u00C2\u00AE diet on days 2, 3, 4 and 5 of the disease challenge period exhibited significantly greater numbers of circulating lymphocytes than the other groups. Circulating lymphocyte numbers in all infected fish were significantly higher (p=0.002) than found for sham-injected fish (Table 6.2). Thrombocyte cell counts A marked thrombocytopenia was evident when comparing bacteria-injected groups to sham-injected groups; the reduction of thrombocytes in blood was statistically significant (p=0.040) for the former groups. No significant diet differences were observed in numbers of circulating thrombocytes during the challenge sampling (p=0.104), but on day 1, fish fed the high KPA\u00C2\u00AE diet, and on day 2 offish fed the astaxanthin diet had significantly greater numbers of circulating cells (p=0.036 and p=0.046, respectively) than observed in other groups (Table 6.2). 66 Monocyte cell counts Numbers of circulating monocytes were significantly affected by both the diet and disease treatments. Fish fed the high KPA\u00C2\u00AE diet had significantly (p=0.021) greater numbers of monocytes in blood compared to those fed with the other 3 diets. Moreover, fish injected with sterile peptone saline had significantly fewer (p=0.044) circulating monocytes than fish injected with Vang (Table 6.2). 67 36 Day1 Day 2 Day 3 Day4 Day 5 Day 6 Days After Bacterial Injection ContolDet Hgn KPA Diet Lew KPA Diet Asta>onfrihDet Shairinj9dion(pooiclrnean) Figure 6.3 Total white blood cell counts (WBC) in pre-smolt chinook salmon (O. tshawytscha) fed four diets containing different concentrations of astaxanthin and KPA\u00C2\u00AE for 6 days following an experimental challenge with Vibrio anguillarum (0.1 ml, 10 9 cfu/mL) and pooled mean of sham injection groups (sterile peptone saline, 0.1 mL). Different letters indicate significant differences (n=6/diet treatment, two replicates tanks/diet treatment, p<0.05) between diet groups, and symbol () indicates a statistical difference (p<0.05) between sham-injected and bacteria-injected fish. 73 P 3 0) c 3 o o T3 O O m c O 3 0) z O) c 3 u O o m c 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Head Kidney Lysozyme Activity (HWEL units/mL) 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 B \u00E2\u0080\u00A2 Control Diet V High KPA Diet \u00E2\u0080\u00A2 Low KPA Diet O Astaxanthin Diet 0.5 1.0 1.5 . 2.0 2.5 3.0 3.5 Plasma Lysozyme Activity (HEWL units/g) 4.0 Figure 6.6 Correlation between lysozyme activity (HEWL equivalent) in plasma (6.6-A) and anterior kidney (6.6-B), and the number of circulating neutrophils in blood from pre-smolt chinook salmon (O. tshawytscha) fed each of four experimental diets that varied in astaxanthin and KPA\u00C2\u00AE supplementation levels following an experimental challenge injection of Vibrio anguillarum. 74 55.0 n Day1 Day 2 Day 3 Day 4 Day 5 Day 6 Days After Bacterial Injection Control Diet m% High KPA Diet Low KPA Diet HHia Astaxanthin Diet \u00E2\u0080\u0094o\u00E2\u0080\u0094 Sham Injection (pooled mean) Figure 6.7 Hepatic SP70 response in pre-smolt chinook salmon (O. tshawytscha) fed four experimental diets that contained various levels of antioxidant supplementation (A\u00C2\u00B0KPA\u00C2\u00B0, A 6 0 KPA\u00C2\u00B0, A 6 0 K P A 1 0 0 0 , A 6 0 K P A 1 0 0 ) following a bacterial challenge (Vibrio anguillarum). The responses of sham-injected control (sterile peptone saline, 0.1 mL) are also shown. Different letters indicate significant differences (n=6/diet treatment, two replicates tanks/diet treatment, p<0.05) between diet groups. Symbol (<(>) indicates a statistical difference (p<0.05) between sham-injected and bacteria-injected fish. 75 6.5 DISCUSSION The nutritional status of an organism may affect its immunocompetence. Furthermore, it is accepted that both nutritional deficiencies arid excesses compromise the immune responses and disease resistance of animals (Chandra, 1993). Good nutritional status prior to an outbreak of infectious disease will increase disease resistance and reduce mortality during a period when feed intake is limited (Waagb0, 1994). The use of immunostimulants can enhance protection against disease, by enhancing non-specific defense mechanisms (Anderson, 1992b). Natural antioxidants, including carotenoids and vitamins A, C and E, are among the most important nutrients that influence the immune system (Chew, 1996). Antioxidants are known to have synergistic interactions in fish (Hilton, 1982). Grape seed PA have been shown to have potent antioxidant capabilities singly and in combination with vitamins, and might interact synergistically with carotenoids and other antioxidants in fish (Bagchi et al. 2002). The effects of feeding farmed salmon with diets containing supplemental astaxanthin and concurrently fortified with proanthocyanidins flavonoids on their immunocompetence has not been tested previously. Vibriosis is a serious bacterial disease that is responsible for severe losses in many aquatic species (Santos et al. 1996). Vibriosis is characterized by a haemorrhagic septicaemia. The early external clinical signs of this disease in salmonids include anorexia, discoloration of the skin, and inactivity. Internally, there are haemorrhages in the liver and kidney, and the gut becomes distended and filled with a clear-yellow fluid. As the disease progresses, the surface haemorrhages may become ulcerative, and the vent may be red and swollen. The high concentration of bacterial suspension injected into the fish in this study was highly virulent and resulted in severe mortality. Fish mortalities started within 24 h after the injection challenge. Vibrio anguillarum was re-isolated from liver and head kidney of moribund fish, and identified based on Gram-stained smears, colony morphology on T S A + 1.5% NaCI plates, and biochemical reactions using an API-20 E\u00C2\u00AE kit (BioMerieux, Missouri, MO, USA). The severity of a disease outbreak in fish is modulated by a triage of pathogen-host-environment factors. It is known that the course of an infectious disease challenge is more severe when is caused by inoculation of fish with the pathogen rather than by 76 immersion of the fish in a bacterial suspension or cohabitation of uninfected fish with an infected fish (Balfry, 1997). The pathogenesis of vibriosis following intraperitoneal injection of bacteria was particularly severe in this study due to: 1) an immunologically naive host (chinook salmon in FW) to the marine pathogen, 2) inoculation of a large concentration of bacteria, and 3) bypassing the primary immune barriers such as physical barriers (scales, integument, epithelial cilia) and humoral non-specific agents present in mucus (i.e. lysozyme, C-reactive protein, complement, etc). Early fish mortalities did not exhibited any external or internal signs of infection. This likely was due to acute toxaemia because the overwhelming amounts of endotoxins and exotoxins present in Vang as a result of the very large number of bacteria injected to the fish. Peracute cases of vibriosis without evident pathological signs of disease have been reported in young fish by Anderson and Conroy (1970), and Home et al. (1977). Furthermore, the fish used in this experiment were in the process of smoltification as noted by the loss of parr marks and silvering of the body. Smoltification is a physiological stage of anadromous salmonids that is characterized by physical, behavioural, and physiological changes that include increasing levels of circulating Cortisol in the blood (Barton era/. 1985; Maule etal. 1987) and depression of immunocompetence variables (Maule et a/. 1989). Hence, this might contributed to the observed findings. In the latter course of infection, internal examination offish from all diet groups showed clinical signs of Vibrio infection such as severe haemorrhaging and inflammation in the liver, spleen and kidney. These findings are consistent with the lesions that have been observed in chum, chinook and coho salmon infected with Vang as reported by Ransom et al. (1984), who found extensive vascular and haemorrhagic damage to different tissues during a vibriosis outbreak. The primary haematology index parameters haematocrit (Hct), haemoglobin (Hb), and red blood cells number (RBC) test the oxygen-carrying capability of erythrocytes in the body. The values of R B C , Hct and Hb in this experiment fluctuated in relation to the diet treatment. Therefore no consistent diet differences were observed for these haematology variables. Ackerman and Iwama (2001) also reported fluctuating and insignificant haematology results in juvenile rainbow trout subjected to the same bacterial strain used in my disease challenge. Despite the lack of diet effect of treatment on the haematological variables, there was a significant effect of Vibriosis on 77 haematology and anaemia was observed in the fish 3 days post bacterial injection in all dietary groups compared with the haematology variables observed in the sham-injected groups. In agreement with these results, Cardwell and Smith (1971) reported significantly decreased values for all primary and secondary haematology variables during a natural outbreak of vibriosis in chinook salmon. The progressive deterioration in RBC, Hct, and Hb values suggests either damage to the erythropoietic system or accelerated destruction of red blood cells due to the action of haemolysins in Vang as described by Lamas et al. (1994). Decreased values during the course of the infection could have also been due to to renal dysfunction, caused by destruction of renal excretory tissue of the posterior kidney due to the effect of Vang. Moreover, kidney damage will affect the osmoregulatory processes resulting in haemodilution in salmon housed in freshwater. Finally, decreased haematological values are inherently related to a decrease in plasma protein values that affect the osmotic contribution of plasma proteins to the fluid balance and homeostasis. Secondary haematological indices are calculated from R B C , Hct and Hb values. They provide clinical insights regarding blood characteristics, and they identify pathological effects of a variety of conditions on the number and size of circulating erythrocytes, and the amount of haemoglobin contained within them. The observed normocytic (same erythrocyte size) and normochromic (normal amount of haemoglobin) anaemia is clinically related to haemorrhage and haemolysis of erythrocytes, total loss of blood from the circulatory system and haemodilution. Vibrio anguillarum possesses potent extracellular products such as haemolysins and proteases (Lamas et al. 1994), which may be responsible for such haemorrhagic anaemia. There were significant differences in the differential leucocyte and monocyte counts throughout the infection period due to treatment. In this regard, the numbers of circulating neutrophils and monocytes in blood from fish fed the high KPA\u00C2\u00AE diet were significantly greater than in the other 3 treatment groups. The increase in the number of circulating neutrophils and monocytes was associated with greater plasma lysozyme activity since the neutrophils synthesize and secrete the enzyme. The larger number of circulating neutrophils were also coincident with the significantly higher number of neutrophil-NBT positive cells measured by the nitro blue tetrazolium assay in the fish fed the high KPA\u00C2\u00AE diet. The neutrophil cell counts in the blood were reduced in chinook salmon fed the control diet, which was not supplemented with astaxanthin and KPA\u00C2\u00AE. In 78 addition, lysozyme activity in the plasma was also reduced in the fish fed the control diet. KPA\u00C2\u00AE-mediated antioxidant effects may have enhanced both the chemotactic and chemokinetic properties (increased production of cytokines) of neutrophils. Besides providing protection to the phagocytic membranes of neutrophils, KPA\u00C2\u00AE may have increased the persistency and responsiveness of the reacting immune cells in the oxidative environment (oxygen-dependent killing) during the respiratory burst. Finally, grape seed antioxidant properties may have exerted important scavenging effects on the ROS produced by phagocytes to kill invading microbes, thus preventing damage the body's own cells and tissues and thereby reducing some immunopathological effects associated with infection and inflammation. It should be mentioned that astaxanthin-fed fish had a significantly lower neutrophil-NBT positive reaction on days 1 and 2 after bacterial injection, which coincided with the highest cumulative mortality during that period. There was also a marked thrombocytopenia observed during the second day post challenge, although there was no statistical difference between treatment groups. Haemorrhagic anaemia is characterized by a decrease in the number of circulating thrombocytes, due to their active role in clotting processes. The inflammatory response elicits a strong stress protein induction (reviewed by Jacquier-Sarlin et al. 1994). Increases in SP70 levels have been reported to occur in head kidney and liver during bacterial infections with Renibacterium salmoninarum (Forsyth et al. 1997) and Vibrio anguillarum (Ackerman and Iwama, 2001). In this experiment, there was an increase of hepatic SP70 throughout the course of the infection challenge, and values reached their highest levels on day 3 and day 4 after bacterial injection, although there was not a significant effect of diet treatment during the challenge period. 79 7. GENERAL DISCUSSION The current depletion of wild fish stocks throughout the world in recent years has occurred concomitantly with an explosive increase in demand for fish by health conscious consumers and a rise in world population. This has created a need that can no longer be met by the existing wild fisheries and therefore commercial aquaculture production has increased to meet this growing demand for aquatic protein and lipid. Historically, the wild salmon catch has supplied the world market demand for salmon almost completely; however, in the last 2 decades salmonid farming (salmon and trout) has grown to such an extent that it has surpassed the gross tonnage supplied by salmon fisheries to the international market (FAO, 2000). Proper nutrition plays a critical role in maintaining normal growth and health of cultured fish. Intensive fish farming demands not only precisely formulated and cost-efficient diets, but also diets that can mitigate the deleterious effects of diseases and improve pigmentation deposition efficiency. Dietary antioxidants can impart health benefits. The fortification of commercial diets with antioxidants other than vitamins is a novel nutritional practice in animals, despite their long history of health benefits in human nutrition. There have been several studies on the effects of supplementing fish diets with very high doses of the antioxidant vitamins C and E, singly and in combination, on their immune system, and growth (Hamre etal. 1997; Mulero et al. 1998; Montero etal. 1999; Ortuno etal. 1999; Ortuno etal. 2000; Cuesta etal. 2001; Sealey and Gatlin. 2002). However, there are no reports on the benefits of the supplementing fish diets with antioxidants other than vitamins and carotenoids. Therefore the potential benefits of this nutritional strategy remain unknown. Proanthocyanidins are naturally occurring flavonoids in grape seeds, and PA extracts are readily available in the market (i.e. KPA\u00C2\u00AE). Grape seed PA exhibit a broad spectrum of pharmacological, therapeutic and chemoprotective properties, while concurrently enhancing the growth and viability of normal cells (Reviewed by Bagchi et al. 2002; Joshi et al. 2000). Also, they are significantly more potent antioxidants than vitamin E, C and (3-carotene (Buettner, 1993, Bagchi etal. 1998). Proanthocyanidins have highly protective effects against lipoperoxidative damage, which depend on the hydrogen-donating capacity of a hydroxy! group in their structure (Saija et al. 1995), as well as their incorporation rate 80 into cells, orientation in biomembranes (Thomas etal. 1992), and strong affinity in chelating iron (Afanas'ev etal. 1989). It ws not the intend of this research to unravel the mechanism of action of orally ingested proanthocyanidins and astaxanthin in chinook salmon, but rather to describe for the first time the possible beneficial effects of the preceding on chinook salmon immunocompetence, pigmentation and growth under practical farming conditions in British Columbia, Canada. Diets fortified with flavonoid PA and astaxanthin improved growth and diet utilization variables, as well as astaxanthin deposition and retention coefficient in salmon muscle after 155-day feeding period in SW, but not after a short feeding period of 32 days in FW. In addition, the immune system and some of the non-specific immune factors were significantly enhanced by diets containing a combination of KPA\u00C2\u00AE and astaxanthin in FW. As a result, the total cumulative mortality observed in pre-smolt chinook salmon fed the high KPA\u00C2\u00AE diet after a disease challenge was significantly lower than observed for the other 3 dietary groups. Observed pre-smolt chinook salmon growth in FW surpassed projections by T G C for the 32-day trial. Likely was due to the very high feeding rate (3.5% b.w./day) that was selected fro the trial. The observed weight gain in FW exceeded the projected estimation by T G C by +5% (control diet group) and +13% (astaxanthin diet group). On one hand, this high feeding rate allowed fish to reach their maximum growth rate, but it also negatively depressed values noted for feed utilization. In contrast to the situation observed in FW, the final weight gain observed for chinook salmon in SW did not reach the projected weight estimated by T G C . Despite the fact that the growth and diet utilization variables (SGR and FCR) that were observed among groups were within farmed salmon standards, the projected weight gain estimated by T G C was not reached by fish fed the low KPA\u00C2\u00AE, astaxanthin, or control diets. Fish fed the control, astaxanthin-only, and low KPA\u00C2\u00AE diets grew less well, i.e. -417o, -21%, and -13%, respectively compared to their respective projected weights by T G C . Only the fish group fed the high KPA\u00C2\u00AE diet attained the projected weight (no difference between projected and actual final wet weight). Indeed, the fish fed the diet with the high concentration of KPA\u00C2\u00AE together with astaxanthin had significantly greater weight gain, but not values for S G R and FCR, related to those fed the other diets after 155 days in SW. In addition, this 81 group had significantly higher astaxanthin deposition in their muscle compared to the fish ingesting the other diets. The relatively low carotenoid pigment deposition and weight gain in fish groups fed diets other than high KPA\u00C2\u00AE diet after 155 days in SW may have been due to some oxidation of the PUFA-rich marine oil contained in the salmon diets during storage. Environmental conditions during storage (oxygen, heat) may have affected the nutritional composition of lipids, vitamins and the levels of astaxanthin in the diets. Carotenoid pigment breakdown in diets has been reported even under temperature-controlled storage (-20\u00C2\u00B0C) after 10.5 months (Christiansen, 1995a). Thus, one may expect some degree of nutrient deterioration under room temperature storage. This was probably not extensive due to the frequent preparation of the diets. . The fortification of diets with grape seed PA may have protected the diets against oxidative deterioration. Proanthocyanidins have been shown to possess strong antioxidant properties (Ariga et al. 1988; Ricardo da Silva et al. 1991) and prevent lipid peroxidation. Also KPA\u00C2\u00AE has exhibited protective photo-oxidative properties in reducing astaxanthin breakdown in salmon fillets exposed to refrigerated conditions for 5 days (Kikkoman personal communication). Therefore, it is conceivable that an increased level of grape seed P A with astaxanthin may have protected the highly susceptible PUFA-rich oils and astaxanthin in the feed. Moreover, the increased astaxanthin concentration in muscle as well as the apparent pigment retention coefficient achieved by feeding the high KPA\u00C2\u00AE diet over 155 days may be related to the antioxidant protection of PA. Pigmentation involves not only the occurrence of synthetic carotenoids in the feed, but also other factors like absorption, transport in blood and deposition. The impact that KPA\u00C2\u00AE might have exerted on the deposition of astaxanthin in salmon muscle could have been due to several factors such as: 1) the reduction of astaxanthin oxidation in feed, 2) the reduction of PUFA-rich marine oil oxidation contained in salmon diets, thus reducing the breakdown of vitamins, triglycerides, phospholipids, etc, 3) the synergistic interaction with the antioxidant's network (vitamin A, C, E, glutathione, etc), thereby increasing astaxanthin's bioavailability, 4) the reduction of astaxanthin's use as a free radical scavenger in the gut of salmon, thus increasing availability and efficiency of absorption of astaxanthin from the digestive tract, and 5) reduced oxidation of transporting lipoprotein fractions (HDL and VHDL), thus increasing the quantity of xanthophyll carried 82 in the blood. Finally, KPA\u00C2\u00AE might have enhanced the non-specific carotenoid receptors in the actomyosin complex in salmon muscle due to a general improvement of the phospholipid fraction contained in the muscle cell membrane. Movileane et al. (2000) demonstrated a positive interaction between flavonoids affecting biomembranes and planar lipid bilayers. The effect of grape seed PA on the immune system of pre-smolts was investigated by conducting a disease challenge test. Pre-smolts fed the high KPA\u00C2\u00AE diet for 32 days had significantly enhanced non-specific immune response during the disease challenge. The total number of circulating white blood cells, circulating neutrophils and monocytes, number of NBT-positive neutrophils, and plasma and head kidney lysozyme activities were significantly higher in fish fed the high KPA\u00C2\u00AE diet, when compared to the responses noted for the other diet groups. Pathological processes such as infection, inflammation and tissue injury generate a pro-oxidant environment with the release of oxidative cellular components. The immune system is particularly sensitive to oxidative stress, primarily because the coordination of an effective immune response relies heavily on cell-to-cell communication. One of the events during the inflammatory response is the movement of phagocytic cells from the blood or surrounding tissue to the site of microbial invasion attracted by chemotactic factors (Secombes and Fletcher, 1992). Phagocytic cells described in fish are mononuclear cells (tissue macrophages and circulating monocytes) and polymorphonuclear granulocytes (particularly neutrophils)(Secombes, 1996). Phagocytes attack the pathogen either through digestive enzymes (oxygen independent killing mechanism) or via the oxygen dependent mechanism. During oxygen dependent killing, oxygen is converted into a number of microbicidal R O S in a process termed the respiratory burst. The stimulation of the cell membrane activates the enzyme complex NADPH oxidase that is capable of the univalent reduction of molecular oxygen into *0 2 \" (Kang et al. 1994) and it has been demonstrated in fish by Secombes (1996). The nitro blue tetrazolium assay (NBT) reacts by reducing NBT by *0 2 \" , producing a blue precipitate called formazan. The neutrophils that had a blue halo were counted as NBT positive cells, and these indicate the cells that were participating actively in the oxygen-dependent killing mechanism. The NADPH is generated from the pentose phosphate shunt (Kang et al. 1994). Part of the 'O2 produced is dismutased to H2O2 either spontaneously or catalyzed by 83 SOD. Also 1 0 2 i s produced during spontaneous dismutation and this is detectable by chemiluminescence (CL) measurement following the respiratory burst. Excess H2O2 production can be regulated by catalase. The extremely harmful OH* is produced when H 2 0 2 reacts with *0 2 \" . Further, there has been evidence for the existence of the myeloperoxidase (MPO) H 202-halide system in fish granulocytes (not present in macrophages). M P O catalyzes the oxidation of halide ions by H 2 0 2 to form hypohalites and chloramines with enhanced microbicidal actions (Secombes, 1996). Finally, since the production of R O S increases during certain immune responses and during inflammation, and since oxidant enzymes are involved in the regulation of the respiratory burst, cellular antioxidants can be regarded as part of the non-specific immune system (Kang et al. 1994) illustrating the closer inter-relationship between the two protective systems. Secombes (1996) pointed out the important participation of eicosanoids such as prostaglandins, leucotrienes and thromboxanes produced by leucocytes in the enhancement and development of the inflammatory response in fish. Eicosanoids are lipid mediators derived from arachidonic acid and have been shown to possess a number of non-specific and specific immune functions such as augmenting phagocytosis and increasing chemotaxis for neutrophils. KPA\u00C2\u00AE might have helped to control the excessive production of free radicals during the respiratory burst activity of neutrophils and therefore prevented the peroxidation of such eicosanoids. In addition, lipid peroxidation promotes the formation of peroxides that compromise the cellular membrane structure and function, thereby disrupting signal transduction through which cells communicate. As a result, a number of changes may indicate the possible loss in cell function, such as reduction in receptor binding (Nunez and Glass, 1982; Riley and Carlson, 1988), loss in membrane transport (Yuli et al. 1981) and alterations in enzyme activity (Kim and Yeoun, 1983; Riley and Carlson, 1987). In addition to the reported individual effects of PA (reviewed by Bagchi et al. 2002), dietary antioxidants and antioxidant vitamins have been shown to act synergistically exhibiting a \"sparing effect\" against the damage caused by oxidative stress. Vitamin E, in particular a-tocopherol, is regarded as the primary lipid-soluble antioxidant that operates synergistically with Vitamin C (ascorbic acid, which is a water-soluble antioxidant) to protect lipids against peroxidative damage (Burton et al. 1985; Sato etal. 1990; Buettner, 1993; Kamal-Eldin and Appelqvist, 1996). This interaction 84 has been demonstrated in phospholipid model membranes, microsomes, human platelets, and LDL against lipid peroxidation in vitro (Niki, 1987; Wefers and Sies, 1988; Chan et al. 1991; and Kagan et al. 1992). This synergistic interaction has also been demonstrated in juvenile Atlantic salmon by Hamre et al. (1997) by measuring vitamin concentrations in the liver. Shiau and Hsu (2002) measured and quantified plasma and liver vitamin concentrations, as well as hepatic T B A R S in juvenile tilapia. A combination of vitamins C and E has been shown to positively affect growth but not the immune system in juvenile hybrid striped bass (Sealey and Gatlin, 2002). In contrast, Hamre et al. (1997) showed that Atlantic salmon fed a combination of vitamins C and E had an improved immune system but growth was not affected. Additionally, several recent studies have suggested that carotenoids, including p-carotene, astaxanthin and canthaxanthin possess potent antioxidant properties in cell membranes and operate synergistically with Vitamin E (Palozza and Krinsky, 1992a and 1992b; Nishigaki et al. 1994). The vitamin E and flavonoid regeneration in membranes has been discussed by Terao and Piskula (1998). Synergistic a-tocopherol and quercetin (also a natural flavonoid) regeneration has been shown in micellar solutions by Mukai etal. (1996); and the synergistic and regenerative activity between vitamin E and flavonoids have been shown by Pedrielli and Skibsted (2002). In spite of the efforts made by the chemical analysis laboratory of Kikkoman Corporation in Japan in retrieving and quantifying the amount of PA in fish feed, the procedure was unsuccessful. This situation has not been unique, Yamakoshi et al. (1999) also failed to recover and quantify the amount of PA from blood in rabbits fed PA-containing diet, although they were able to recover and quantify PA when the grape seed extract was singly force-fed to rats. Biological properties of PA depend on their bioavailability. Proanthocyanidins from red wine are highly available due to their high rate and extent of intestinal absorption (Tapiero et al. 2002). It has also been shown by Jimenez-Ramsey er al. (1994) that PA solubles in water and ethanol, such as KPA\u00C2\u00AE, are highly absorbed from the intestinal tract and extensively distributed in all tissues and plasma. Furthermore, Bravo (1998) demonstrated that plasma PA can be maintained following a regular intake of sufficient quantities of fresh fruits or supplementation with bioavailable PA's . Based on the evidence from the studies mentioned above, one might expect the bioavailability of PA in chinook salmon fed diets fortified with KPA\u00C2\u00AE to be high. 85 Finally, and supported by the literature cited above, it is conceivable that the combination of KPA\u00C2\u00AE and other antioxidant vitamins and minerals in sufficient quantities in a balanced diet may have acted synergistically and exerted positive and beneficial effects on the immunological, pigmentation and growth variables in chinook salmon under the conditions of these studies. 86 8. CONCLUSION Free radicals are normally produced under physiological conditions as part of the oxygen metabolism in aerobic organisms. Their levels increase significantly in specific situations (i.e. inflammation, as part of its regular defense functions). Farmed salmon are susceptible to environmental, chemical and pathological insults that will increase the production of these compounds. They possess different mechanisms to cope with the increasing production of free radicals (i.e. antioxidant enzymes, FR scavengers, metal chelators). The balance between oxidants and antioxidants is crucial for the integrity and functionality of cell membrane, signalling transduction and gene expression. In addition to the endogenous enzymatic antioxidant defense, consumption of dietary antioxidants appears to be of great importance because of their protective antioxidant effects. Feeding dietary antioxidants other than antioxidant vitamins is a well-established practice in the human diet. By contrast, this is a novel practice in small animal diets (i.e. dogs and cats), and it is non-existent in livestock and aquaculture (i.e. salmon farming). This is the first study that has assessed the effects of feeding farmed salmon diets supplemented with grape seed flavonoids (proanthocyanidins) in combination with astaxanthin with respect to their growth, flesh pigmentation and immune response. The effects of feeding a combination of the foregoing dietary antioxidants resulted in significant positive effects on immunocompetence after a short feeding period in FW (32 days). The circulating number of leucocytes (neutrophils and monocytes), oxidative burst activity, and the activity of lysozyme both in plasma and anterior kidney were significantly higher in fish fed the high KPA\u00C2\u00AE diet after a disease challenge with Vibrio anguillarum. The increase of these non-specific immune factors may have contributed to the significantly lower cumulative mortality seen in this diet group. Muscle pigmentation and growth of pre-smolts (less than 7 g) were not significantly affected by feeding the antioxidant-fortified diets over the short trial in FW. However, when the feeding period was extended to 155 days in smolts transferred to saltwater (mean initial weight 7 g to final weight 80 g), significant increases were observed for growth (weight gain) and pigmentation (concentration of astaxanthin in muscle, A A R C , RSS) variables in fish fed the high KPA\u00C2\u00AE diet. As farmed salmon production has increased every year in the world, the supply of salmon has at times exceeded demand and the market prices have declined. The 87 reduction in salmon prices has forced fish researchers to investigate new methods for improving general efficiency while reducing costs of production. The muscle retention of carotenoids represents only 1-5% of the ingested pigments (Choubert and Luquet, 1983; Torrissen et al. 1990). The price of extruded salmon feed supplemented with 40 to 80 ppm of astaxanthin comprises as much as 15-20% of the total feed costs, or about 6-8% of the total production cost (Torrissen, 1995). Therefore, reducing fish mortalities, and effective feed and pigment management are vitally important to the economics of intensive salmon aquaculture. The results of this research indicate that the supplementation of 1000 ppm of KPA\u00C2\u00AE in combination with 60 ppm of astaxanthin in a high quality salmon diet resulted in significant differences in valuable farmed salmon production traits, such as weight gain, flesh pigmentation and fish mortality. In order to realize the magnitude of the results obtained in this research we may extrapolate them into a full-scale farming situation. Seacages in SW are 15x15x15 m (a volume of 3,375 m 3) and transferred chinook salmon smolts are held at stocking densities of 10-12 kg/m 3 (total biomass between 33,750 to 40,500 kg). Vibriosis caused by an immersion challenge in a Vang suspension has resulted in mortalities that have varied between 30 and 60% in coho salmon (Balfry, 1997). If one projects the results observed in fish fed the high KPA\u00C2\u00AE diet in the disease challenge to a commercial situation (40,500 kg biomass/seacage), the use of the high KPA\u00C2\u00AE diet would prevent the loss of 1,200 to 2,400 kg of biomass/seacage considering outbreak mortalities ranging from 30-60%. If the results observed in the SW experiment are considered, the attainment of an increase in S G R from 1.43%/day (astaxanthin diet) to 1.58%/day (high KPA\u00C2\u00AE and astaxanthin diet) would increase the biomass between 50 to 60 kg/day/seacage, and after 155 growing days it will be between 7,850 to 9,420 kg/seacage. In addition, increasing the fish biomass by 1,000 kg would require 1,400 kg of food at a F C R of 1.40 (as seen in fish fed the astaxanthin diet) or 1,270 kg with a F C R of 1.27 (high KPA\u00C2\u00AE diet), which represents a 10% saving in food. Also, feeding salmon with a diet fortified with a high concentration of KPA\u00C2\u00AE increased significantly the retention coefficient of astaxanthin from 0.90% to 1.28%. Finally, the use of salmon diets fortified with a high concentration of KPA\u00C2\u00AE would enable farmed salmon to reach a 88 market size faster than those fed diets without KPA\u00C2\u00AE supplementation and with higher concentrations of astaxanthin in their flesh. While the findings in this study are very promising, it is important to conduct additional research to confirm the present findings at other stages of the life history and with extensive replication of each dietary treatment. Additionally, the relative efficiency of K P A as an antioxidant versus vitamins C and E should be considered in the future studies. 89 9. REFERENCES Ackerman P.A and G.K. Iwama. 2001. Physiological and cellular stress responses of juvenile rainbow trout to Vibriosis. Journal of Aquatic Animal Health, 13: 173-180. Afanas'ev, I.B., Dorozhko, A.I., Brodskii, A.V., Kostyuk, V.A. and A.I., Potapovitch.1989. Chelating and free radical scavenging mechanisms of inhibitory action of rutin and quercetin in lipid peroxidation. Biochemical Pharmacology, 38(11): 1763-1769. Anderson J.I.W and D.A Conroy. 1970. Vibrio disease in marine fish. In: a symposium on diseases offish and shellfish (Ed. By S.F. Snieszko), pp: 266-272. American Fisheries Society, special publication N\u00C2\u00B0 5, Washington, DC, USA. Anderson, D. 1992a. In Vitro immunization offish spleen sections and NBT, phagocytic, P F C and antibody assays for monitoring the immune response. In: Techniques in Fish Immunology, 1992. J . S. Stolen, T. C. Fletcher, D. P. Anderson, S. L Kaatari and A. F Rowley (Editors). S O S Publications, 43 DeNormandie Ave., Fair Haven, NJ , USA. pp: 79-88. Anderson, D.P. 1992b. Immunostimulants, adjuvants, and vaccines carriers in fish: applications to aquaculture. Annu Rev Fish Dis, 2: 281-307. Anonymous, 2003. Chilean Salmon Farmers Association. Statistics. http://www.salmonchile.cl/estadisticas/tabla3_2.htm Ariga, T., Koshiyama, I., and D. Fukushima. 1988. Antioxidants properties of Procyanidins B-1 and B-2 from azuki beans in aqueous systems. Agricultural and Biological Chemistry, 52: 2717-2722. Aruoma, O.I. 1994. Nutrition and health aspects of free radicals and antioxidants. Food and Chemistry Toxicology (7): 671-683. Austreng, E., Strorebakken, T., and T. Aasgaard. 1987. Growth rate estimates for cultured Atlantic salmon and rainbow trout. Aquaculture, 60: 157-160. Bagchi, D., Garg, A., Krohn, R.L., Bagchi, M., Tram, M X , and S .J . Stohs. 1997. Oxygen free radical scavenging abilities of vitamins C and E, and a grape seed proanthocyanidin extract in vivo. Research Communications in Molecular Pathology and Pharmacology, 95(2): 179-189. Bagchi, D., Garg, A., Krohn, R.L., Bagchi, M., Bagchi, J.L., Balmoori, J . , and S.J . Stohs. 1998. Protective effects of grape seed Proanthocyanidins and selected antioxidants against TPA-lnduced hepatic and Brain lipid peroxidation and DNA fragmentation, and peritoneal macrophage activation in mice. General Pharmacology, 30 (5): 771-776. Bagchi, D., Bagchi, M., Stohs, S .J , Das, D.K., Ray, S.D., Kuszynski, C.A., Joshi, S .S. and H.G. Preuss 2000. Free radicals and grape seed proanthocyanidins extract: importance in human health and disease prevention. Toxicology, 148(2-3): 187-197. 90 Bagchi, D., Bagchi, M., Stohs, S .J , Ray, S.D., Sen, C.K., and H.G. Preuss. 2002. Cellular protection with proanthocyanidins derived from grape seeds. Annals of the New York Academy of Sciences, 957: 260-270. Balfry, S.K. 1997. The non-specific immune system and innate disease resistance in different strains of teleost fish. Doctoral Dissertation. University of British Columbia, Vancouver. Barton, B.A., Schreck, C.B. , Ewing, R.D., Hemmingsen, A.R., and R. Patino. 1985. Changes in plasma Cortisol during stress and smoltification in coho salmon, Oncorhynchus kisutch. General and Comparative Endocrinology, 59: 468-471. Bird, J .N. , and G.P. Savage. 1989. The absorption of astaxanthin by chinook salmon (Oncorhynchus tshawytscha). Proceedings of the Nutrition Society of New Zealand, 14: 174-177. Bjerkeng, B, Storebakken, T., and S. Liaaen-Jensen. 1992. Pigmentation of rainbow trout from start feeding to sexual maturation. Aquaculture, 108: 333-346. Blazer, V .S . 1992. Nutrition and disease resistance in fish. Annual Review of Fish Diseases, 2: 309-323. Bligh, E.G, and J . Dyer. 1959. A rapid method of total lipid extraction and purification. Canadian Journal of Medical Sciences, 37(8): 911-917. Bravo, L. 1998. Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutrition Reviews, 56: 317-333. Buening, M.K., Chang, R.L., Huang, M.T., Forther, J .G. , Wood, A.W., and A .H. Conney. 1981. Activation and inhibition of benzo(a)pyrene and aflatoxin B1 metabolism in human liver microsomes by naturally occurring flavonoids. Cancer Reserch, 41(1): 67-72. Buettner, G.R. 1993. The pecking order of free radicals and antioxidants: lipid peroxidation, a-tocopherol, and ascorbate. Archives of Biochemistry and Biophysics, 300: 535-543. Burton, G.W. 1989. Antioxidant action of carotenoids. Journal of Nutrition, 119: 109-111. Cardwell R.D. and L.S. Smith. 1971 .Hematological manifestations of vibriosis upon juvenile chinook salmon. The Progressive Fish-Culturist, 33 (4): 232-235 Chan, J.C.K.; Mason, J . ; Rowshandeli, M.; Rowshandeli, N.; and D.A. Higgs. 2002. Effects of feeding diets containing various dietary protein and lipid ratios on the growth performance and pigmentation of post-juvenile coho salmon Oncorhynchus kisutch reared in sea water. Aquaculture Research, 33:1137-1156. Chan, A .C. , Tran, K., Raynor, T., Ganz, P.R., and C.K. Chow. 1991. Regeneration of vitamin E in human platelets. Journal of Biological Chemistry, 266: 17290-17295. 91 Chandra, R.K. 1993. Nutrition and the immune system. Proceedings of the Nutrition Society, 52: 77-84. Chapman, J . , Goldstein, S., Mills, G.L., and C. Leger. 1978. Distribution and characterisation of the serum lipoproteins and their apoproteins in the rainbow trout Salmo gairdneri. Biochemistry, 17: 4455-4464. Chew, B.P. 1996. Importance of antioxidant vitamins in immunity and health in animals. Animal Feed Science Technology, 59: 103-114. Cho C.Y. 1992. Feeding systems for rainbow trout and other salmonids with reference to current estimates of energy and protein requirements. Aquaculture, 100: 107-123. Choubert, G., and P. Luquet. 1983. Utilization of shrimp meal for rainbow trout (Salmo gairdneri Rich.) pigmentation, influence of fat content of the diet. Aquaculture, 32: 19-26. Choubert, G, Milicua, J .C. , Gomez-Martinez, R., Sance, S., Petit, H., Negre-Sadargues, G. Castillo, R., and J .P Trilles. 1992. Distribution of canthaxanthin in immature rainbow trout Oncorhynchus mykiss serum. Comparative Biochemistry and Physiology, 103A: 403-405. Choubert, G, Milicua, J .C. , and R. Gomez. 1994. The transport of astaxanthin in immature rainbow trout Oncorhynchus mykiss serum. Comparative Biochemistry and Physiology, 108A (2/3): 245-248. Christiansen, J .S . , and J .C . Wallace. 1988. Deposition of canthaxanthin and muscle lipid in two size groups of Artie charr (Salvelinus alpinus). Aquaculture, 69(1-2): 69-78. Christiansen, R., Lie, 0 . , and O.J.Torrisen. 1994. Effect of astaxanthin and vitamin A on growth and survival during first feeding of Atlantic salmon, Salmo salarL. Aquaculture and Fisheries Management, 25: 903-914. Christiansen, R., Glette, J . , Lie, 0 . , Torrisen, O.J. , and R. Waagbo. 1995a. Antioxidant status and immunity in Atlantic salmon, Salmo salarL., fed semi-purified diets with and without astaxanthin supplementation. Journal of Fish Diseases, 18: 317-328. Christiansen, R., Lie, O., and O.J.Torrisen. 1995b. Growth and survival of Atlantic salmon, Salmo salarL., fed different dietary levels of astaxanthin. Aquaculture Nutrition, 1:189-198. Christiansen, R., Struksnaes, G. , Estermann, R., and O.J . Torrisen. 1995c. Assessment of flesh colour in Atlantic salmon, Salmo salarL. Aquaculture Research, 26 (5): 311-321. Clarke, W.C. , Saunders, R.L. and S.D. McCormick. 1996. Smolt production. In: Principles of Salmonid Culture. Developments in Aquaculture and Fisheries Science, Vol. 29. W. Pennell and B.A. Barton, Eds. Elsevier Science. Netherlands, pp: 517-555. 92 Cuesta, A., Esteban, M.A., Ortuno, J . , and J . Meseguer. 2001. Vitamin E increases natural cytotoxic activity in seabream (Sparus aurata L.) Fish and Shellfish Immunology, 11(4): 293-302. Devlin, P., Koelsch, S., Heaton P.R., Charlton, C .J . , O'Reilly, J.D., Smith, B.H.E., and E.J. Harper. 2000. Effect of antioxidant supplementation on the immune response in weaned puppies. Proceedings 18th American College of Veterinary Internal Medicine forum, Seattle, WA, pp: 738. Egan, D. 2001. Salmon Farming Overview 2000. Presentation for the BC Salmon Farmers Association Annual General Meeting. Campbell river, BC . July 5 t h , 2001. FAO. 2000. Fisheries Department, Fisheries Information, Data and Statistics Unit. FishStatPlus: Universal Software for Fishery. Statistical time series, version 2.3. Aquaculture Production Quantities Database 1970-2000. Rome. Italy. Frankel, E.N., Kanner, J . , German, J.B., Parks, E., and J .E Kinsella. 1993. Inhibition of oxidation in human Low-density proteins by phenolic substances in red wine. The Lancet, 341 (8843): 454-457. Fremont, L., and D. Marion. 1982. A comparison of lipoprotein profiles in male trout Salmo gairdneri before maturity and during spermiation. Comparative Biochemistry and Physiology, 73 (4): 849-855. Forsyth, R.B., Candido, E.P.M. , Babich, S.L., and Iwama, G.K. 1997. Stress protein expression in coho salmon with bacterial kidney disease. Journal of Aquatic Animal Health, 9: 18-25. Groff, J .M. and S.E. , LaPatra, 2001. An overview of economically important diseases of salmonids. In: Nutrition and Fish Health. C. Lim and C D Webster, Eds. Food Products Press, Binghamton, NY. pp: 11-78 Groot, C , and L. Margolis. 1995. Pacific Salmon Life Histories. U B C Press, Vancouver, BC, Canada, pp: 102-152. Halver, J .E . 1989. Fish Growth In: Fish Nutrition. J .E. Halver (Ed). Academic Press, San Diego, CA. pp: 31-109. Halliwell, B., Evans, P.J . , Kaur, H., and O.I. Aruoma. 1994. Free radicals, tissue injury, and human disease: a potential for therapeutic use of antioxidants? In: Organ Metabolism and Nutrition: Ideas for Further Critical Care. Kinney, J .M. , and H.N. Tucker, Eds. Raven Press Ltd, New York, pp: 425-445. Halliwell, B. 1996. Oxidative stress, nutrition and health. Experimental strategies for optimization of nutritional antioxidant intake in humans. Free Radical Research, 25(1): 57-74. 93 Hamre, K., Waagbo, R., Berge, R.K. and O. Lie. 1997. Vitamins C and E interact in juvenile Atlantic salmon (Salmo salar, L.) Free Radical Biology and Medicine, 22(1-2): 137-149. Hatlen, B., Aas, G.H. , Joergensen, E.H., Storebakken, T., and U.C. Goswami. 1995. Pigmentation of 1, 2 and 3 year old Arctic charr {Salvelinus alpinus) fed two different dietary astaxanthin concentrations. Aquaculture, 138(1-4): 303-312. Hilton, J.W. 1982. The interaction of vitamins, minerals and diet composition in the diet of fish. Aquaculture, 79: 233-244. Hoar, W.S. 1988. The physiology of smolting salmonids. In: Fish Physiology, Vol. Xlb. Hoar, W.S. & D.J. Randall Eds. Academic Press, N.Y., pp: 275-434. Hollman, P.C.H. , and M.B. Katan.1998. Absorption, metabolism, and bioavailability of flavonoids. In: Rice-Evans, C A . and L. Packer (Eds). Flavonoids in Health and Disease. Marcel Dekker Inc. New York, NY, USA. pp: 483-522. Home M.R., Richards, R.H., Roberts, R.J and P.C. Smith. 1977. Peracute vibriosis in juvenile turbot, Scophthalmus maximus. Journal of Fish Biology, 11: 355-361. Houston, A .H. 1990. Blood and Circulation. In: Methods for Fish Biology. Schreck, C.B. , Moyle, P.B. (Eds). American Fisheries Society. Bethesda, Maryland, pp: 273-334. Iwama, G.K. & A.F.Tautz. 1981. A simple growth model for salmonids in hatcheries. Canadian Journal of Fisheries and Aquatic Sciences, 38(6): 649-656. Iwama, G.K. 1996. Growth of salmonids. . In: Principles of Salmonid Culture. Developments in Aquaculture and Fisheries Science, Vol. 29. W. Pennell and B.A. Barton, Eds. Elsevier Science. Netherlands, pp: 467-515. Jacquier-Sarlin MR, Fuller K, Dinh-Xuan AT, Richard MJ , Polla BS. . 1994. Protective effects of hsp70 in inflammation. Experientia 30;50(11-12):1031-8. Jimenez-Ramsey, L.M., Rogler, J .C. , Housley, T.L., Butler, L.G., and R.G. Elkin. 1994. Absorption and distribution of 1 4C-labeled condensed tannins and related sorghum phenolics in chickens. Journal of Agriculture and Food Chemistry, 42: 963-967. Jobling, M. 1994. Fish bioenergetics. New York. Chapman & Hall. Jorgensen, E.H. and M. Jobling. 1994. Feeding and growth of exercised and unexercised juvenile Atlantic salmon (Salmo salar) in freshwater, and performance after transfer to seawater. Aquaculture International, 2:154-164. Joshi, S .S. , Kuszinski, C.A., Bagchi, M., and D. Bagchi. 2000. Chemopreventive effects of grape seed proanthocyanidin extract on Chang liver cells. Toxicology, 155(1-3):83-90. 94 Kagan, V .E . , Serrbonova, E.A., Forte, T., Scita, G. and L. Packer. 1992. Recycling of vitamin E in human low-density lipoproteins. Journal of Lipid Research, 33: 385-397. Kamal-Eldin, A., and L.A. Appelqvist. 1996. The chemistry and antioxidant properties of tocopherols and tocotrienols. Lipids, 31: 671-701. Kang, C.R., Sweetser, S., Boylan, L.M., and J .E. Spallholz. 1994. Oxygen toxicity, biological defence systems and immunity- a historical perspective. Journal of Nutritional Immunology, 3: 51-84. Kiessling, A, Dosanjh, B, Higgs, D, Deacon, G, Rowshandeli, N. 1995. Dorsal aorta cannulation: a method to monitor changes in blood levels of astaxanthin in voluntarily feeding Atlantic salmon, Salmo salarL. Aquaculture Nutrition, 1: 43-50. Kim, I., and D.S. Yeoun. 1983. Effect of prostaglandin F2\u00C2\u00AB and Na + -K + -ATPase activity in luteal membranes. Biology of Reproduction, 29: 48-55. Klontz, G.W. 1994. Fish Hematology. In: Techniques in Fish Immunology 3, 1994. J .S. Stolen, T.C. Fletcher, A.F Rowley, J.T Zelikoff, S.L Kaatari, and S.A Smith (Editors). SOS Publications, 43 DeNormandie Ave., Fair Haven, NJ , USA. pp: 121-132. Kolodziej, H., Haberland, C , Woerdenbarg, H.J., and A.W.T. Konings. 1995. Moderate cytotoxicity of proanthocyanidin to human tumour cells lines. Phytotherapy Research, 9(6): 410-415. Kreiberg, H., Withler, R.E., and W.C. Clarke. 1989. Early seawater growth of chinook salmon strains at rearing sites in British Columbia. World Aquaculture, 20(2): 54-55. Kreiberg, H. 1991. Effect of ration level and water temperature on growth and conversion efficiency in chinook salmon in seawater. World Aquaculture, 22 (1): 84-85. Laemmli, U.K. 1970. Cleavage of structural proteins during assembly of the head bacteriophage T4. Nature, 227: 680-685. Lamas, J . , Santos, Y., Bruno, D., and A.E . Toranzo. 1994. A comparison of pathological changes caused by Vibrio anguillarum and its extracellular products in rainbow trout. Fish Pathology, 29(2): 79-89. Lamas, J . , and A .E . Ellis. 1994. Atlantic salmon (Salmon salar) neutrophil responses to Aeromonas salmonicida. Fish and Shellfish Immunology, 4 (3): 201-219. Litwack, G. 1955. Photometric determination of lysozyme activity. Proceedings for the Society for Experimental Biology and Medicine, 89: 401-403. Lovell, T. 1996. Feed deprivation increase resistance of channel catfish to bacterial infection. Aquaculture Magazine, 22(6): 65-67. Lovell, T. 1998a. Digestion and Metabolism. In: Nutrition and Feeding of Fish, 2 n d Ed. T. Lovell, editor. Kluwer Academic Publisher. Norwell, Massachusetts, USA. pp: 71-93. 95 Lovell, T. 1998b. Feeding salmon and trout. In: Nutrition and Feeding of Fish, 2 n d Ed. T. Lovell, editor. Kluwer Academic Publisher. Norwell, Massachusetts, USA. pp: 175-197. March, B.E., Hajen, W.E. , Deacon, G., MacMillam, C., and M.G. Walsh. 1990. Intestinal absorption of astaxanthin, plasma astaxanthin concentration, body weight, and metabolic rate as determinants of flesh pigmentation in salmonid fish. Aquaculture, 90: 313-322. March, B.E., and C. Macmillan. 1996. Muscle pigmentation and plasma concentrations of astaxanthin in rainbow trout, chinook salmon, and Atlantic salmon in response to different dietary levels of astaxanthin. Progressive Fish-Culturist, 58 (3): 178-186. Maule, A . G , C.B . Schreck and S.L. Kaatari. 1987. Changes in the immune system of coho salmon, Oncorhynchus kisutch, during the parr-to-smolt transformation and after implantation of Cortisol. Canadian Journal of Fisheries and Aquatic Sciences, 44: 161-166. Maule, A .G , Tripp, R.A., Kaatari, S.L. and C.B. Schreck. 1989. Stress alters immune functions and disease resistance in chinook salmon (Oncorhynchus tshawytscha). Journal of Endocrinology, 120: 135-142. Maule, A .G , Schrock, R., Slater C , Fitzpatrick, M. S., and C.B. Schreck. 1996. Immune and endocrine responses of adult chinook salmon during freshwater immigration and sexual maturation. Fish and Shellfish Immunology, 6: 221-233. Mazur, C F . 1986. Growth, incidence of bacterial kidney disease and immunological function of salmonids reared in captivity. M.Sc. Dissertation. University of British Columbia, Vancouver, BC , Canada. Montero, D., Marrero, M., Izquierdo, M.S., Robaina, L., Vergara, J .M. , and L. Tort. 1999. Effect of vitamin E and C dietary supplementation on some immune parameters of gilthead seabream (Sparus aurata) juveniles subjected to crowding stress. Aquaculture, 171(3-4): 269-278. Muona, M., and A. Soivio. 1992. Changes in lysozyme and blood leukocyte levels of hatchery-reared Atlantic salmon (Salmo salar) and sea trout (Salmo trutta L.) during parr-smolt transformation. Aquaculture, 106: 75-87. Movileane.L., Neagoe, I., and M.L. Flonta. 2000. Interaction of the antioxidant quercetin with planar lipid bilayers. International Journal of Pharmaceutics, 205: 135-146. Mukai, K., Oka, W., Egawa, Y., Nagaoka, D.,and J . Terao. 1996. A kinetic study of the free-radical scavenging action of flavonoids in aqueos triton x-100 micellar solution. Proceedings of International symposium on natural antioxidants. In: Molecular mechanisms and health effects. Packer, L., Traber, M.G., and W. Xin (Eds). A O C S Press, Champaign, IL. pp: 557-568. 96 Mulero, V., Esteban, M.A., and J . Meseguer. 1998. Effects of in vitro addition of exogenous vitamins C and E on gilthead seabream (Sparus aurata L.) phagocytes. Veterinary Immunology and Immunopathology, 66(2): 185-199. Nakano, T., Tosa, M., and M., Takeuchi. 1995. Improvement of biochemical features in fish health by red yeast and synthetic astaxanthin. Journal of Agriculture and Food Chemistry, 43: 1570-1573. Nakano, T., Kanmuri, M., Sato, M., and M., Takeuchi. 1999. Effect of astaxanthin rich red yeast (Phaffia rhodozyma) on oxidative stress in rainbow trout. Biochimica et Biophysica Acta, 1426(1): 119-125. Nickell, D.C and N.R Bromage. 1998. The effect of timing and duration of feeding astaxanthin on the development and variation of fillet colour and efficiency of pigmentation in rainbow trout (Oncorhynchus mykiss). Aquaculture, 169: 233-246. Niki, E. 1987. Antioxidants in relation to lipid peroxidation. Chemistry and Physics of Lipids, 44: 227-253. Nishigaki, I., Dmitrovskii, A.A., Miki, w., and K. Yaki. 1994. Suppresive effect of astaxanthin on lipid peroxidation induced in rats. Journal of Clinical Biochemistry and Nutrition, 16: 161-166. Nordgarden, U., Hemre, I., and T. Hansen. 2002. Growth and body composition of Atlantic salmon (Salmo salarL.) parr and smolt fed diets varying in protein and lipid contents. Aquaculture, 207: 65-78. Nunez, M.T. and J . Glass. 1982. Reconstitution of the transferrin receptor in lipid vesicles. Effect of cholesterol on the binding of transferrin. Biochemistry, 21: 4139-4143. Ortuno, J . , Esteban, M.A., and J . Meseguer. 1999. Effect of high dietary intake of vitamin C on non-specific immune response of gilthead seabream (Sparus aurata L.) Fish and Shellfish Immunology, 9(5): 429-443. Ortuno, J . , Esteban, M.A., and J . Meseguer. 2000. High dietary intake of alpha -tocopherol acetate enhances the non-specific immune response of gilthead seabream (Sparus aurata L.) Fish and Shellfish Immunology, 10(4): 293-307. Palozza, P., and N.I. Krinsky. 1992a. (3-carotene and a-tocopherol are synergistic antioxidants. Archives of Biochemistry and Biophysics, 297: 184-187. Palozza, P., and N.I. Krinsky. 1992b. Astaxanthin and canthaxanthin are potent antioxidants in a membrane model. Archives of Biochemistry and Biophysics, 297: 291-295. Pedrielli, P. and L.H. Skibsted. 2002. Antioxidant synergy and regeneration effect of Quercetin, (-)-Epicatechin, and (+)-Catechin on a-Tocopherol in homogeneous solutions of peroxidating Methyl Linoleate. Journal of Agricultural and Food Chemistry, 50: 7138-7144. 97 Pennell, W., and W.E . Mclean. 1996. Early rearing. In: Principles of Salmonid Culture. Developments in Aquaculture and Fisheries Science, Vol . 29. W. Pennell and B.A. Barton, Eds. Elsevier Science. Netherlands, pp: 365-467. Piper, R.G., McElwain, I.B. Orme, L.E., McCraren, J .P. , Fowler, L.G., and J.R Leonard. 1982. Fish Hatchery Management. U.S. Fish and Wildlife Services, Washington, DC, 517 p. Ransom, D.P, Lannan, C.N, Rohovec, J . S and J . L Fryer. 1984. Comparison of histopathology caused by Vibrio anguillarum and Vibrio ordalii in three species of pacific salmon. Journal of Fish Diseases, 7: 107-115. Ricardo da Silva, J .M. , Darmin, N., Fernandez, Y., and S. Mitjavila. 1991. Oxygen free radical scavenger capacity in aqueous models of different proanthocyanidins from grape seeds. Journal of Agriculture Food and Chemistry, 39: 1549-1552. Riley, J .C .M. and J .C. Carlson. 1987. Involvement of phospholipase A activity in the plasma membrane of the rat corpus luteum during luteolysis. Endocrinology, 121: 776-781. Riley, J .C .M. and J .C . Carlson.1988. Impairment of gonadotropin binding occurs during membrane rigid if ication in plasma membrane samples prepared from rat corpora lutea. Canadian Journal of Physiology and Pharmacology, 66: 76-79. Roberts, R.J. and A .M. Bullock. 1989. Nutritional Pathology. In: Fish Nutrition. J .E. Halver (Ed). Academic Press, San Diego, CA. pp: 423-437. Saija, A., Scalese, M., Lanza, M., Marzullo, D., Bonina, F., and F. Castelli. 1995. Flavonoids as antioxidant agents: importance of their interaction with biomembranes. Free Radical Biology & Medicine, 19(4): 481-486. Santos, Y., Pazos, F., and A .E . Toranzo. 1996. Biochemical and serological analysis of Vibrio anguillarum related organisms. Diseases of Aquatic Organisms, 26(1): 67-73. Sato, K, Niki, E., and H. Shimasaki. 1990. Free radical-mediated chain oxidation of low density lipoprotein and its synergistic inhibition by vitamin E and vitamin C. Archives of Biochemistry and Biophysics, 279: 402-405. Schierle, J . and W. Hardi. 1992. Revised supplement: Determination of stabilized astaxanthin in CAROPHYLL\u00C2\u00AE Pink, premixes and fish feeds. In: Analytical Methods for Vitamins and Carotenoids in Feeds. Hoffman, P., Keller, H.E. Schierle, J . , and W. Schuep. Roche, Switzerland. Schiedt, K. 1998. Absorption and metabolism of carotenoids in birds, fish and crustaceans. In: Carotenoids. Volume 3, Biosynthesis and Metabolism. Ed. Britton, S. Liaaen-Jensen & H. Pfander. Birkhauser Verlag, Basel, Boston and Berlin, pp: 285-358. Sealey, W.M. and D.M. Gatlin. 1999. Overview of nutritional strategies affecting health of marine fish. Journal of applied aquaculture, 9(2): 11-26 98 Sealey, W.M., and D.M. Gatlin. 2002. Dietary vitamin C and vitamin E interact to influence growth and tissue composition of juvenile hybrid striped bass ((Morone chrysops (female) x M. saxatilis (male)) but have limited effects on immune responses. Journal of Nutrition, 132(4): 748-55. Secombes, C .J . , and T.C. Fletcher. 1992. The role of phagocytes in the protective mechanism offish. Annual Review of Fish Diseases, 2: 51-57. Secombes, C .J . 1996. The non-specific immune system: cellular defences. In: The fish immune system. Iwama, G., and T. Nakanishi (Eds). Academic Press Limited, London, UK, pp: 63-103. Shiau, S.Y and C.Y. Hsu. 2002. Vitamin E sparing effect by dietary vitamin C in juvenile hybrid tilapia, Oreochromis niloticus x O. aureus. Aquaculture, 210: 335-342. Smith, B.E., Hardy. R., and O.J. Torrissen. 1992. Synthetic astaxanthin deposition in pan-size coho salmon (Oncorhynchus kisutch). Aquaculture, 104: 105-119. Sigurgisladottir, S., Torrissen, O.J. , Lie, 0 . , Thomassen, M., and H. Hafsteinsson. 1997. Salmon quality: methods to determine the quality parameters. Reviews in Fisheries Science, 5(3): 223-252. Simpson, K.L. 1982. Carotenoid pigments in seafoods. In: R.E Martin et al. (Editors), Chemistry and Biochemistry of Marine Food Products. Avi Publishing Company, Wesport, C N . pp. 115-136. Smith, B.E., Hardy. R.W., and O.J. Torrisen. 1992. Synthetic astaxanthin deposition in pan-size coho salmon (O. kisutch). Aquaculture, 104: 105-119. Spinelli, J . , and C. Mahnken. 1978. Carotenoid deposition in pen-reared salmonids fed diets containing oil extracts of red crab (Pleuroncodes planipes). Aquaculture, 13: 213-223. Steffens.W. 1989. Principles of metabolism. In: Principles of Fish Nutrition. Stephens, W. Ellis Horwood Ltd, Chichester, England, pp: 11-40. Storebakken, T., Foss, P., Schiedt, K., Austreng, E., Liaasen-Jensen, S., and Mans, U. 1987. Carotenoids in diet for Salmonids. IV. Pigmentation of Atlantic salmon with astaxanthin, astaxanthin dipalmitate and canthaxanthin. Aquaculture, 65: 279-292. Storebakken, T., and H.K, No. 1992. Pigmentation of rainbow trout. Aquaculture, 100: 209-229. Tapiero, H., Tew, K.D., Nguyen Ba, G., and G. Mathe. 2002. Polyphenols: do they play a role in the prevention of human pathologies? Biomedicine and Pharmacotherapy, 56: 200-207. 99 Terao, J . , and M.K. Piskula, 1998. Flavonoids as inhibitors of lipid peroxidation in membranes. In: Rice-Evans, C A . and L. Packer (Eds). Flavonoids in Health and Disease. Marcel Dekker Inc. New York, NY, USA. pp277-293. Thomas, C.E. , McLean, L.R., Parker, R.A., and D.F. Ohlweiler. 1992. Ascorbate and phenolic antioxidant interactions in prevention of liposomal oxidation. Lipids, 27: 543-550. Thomas, A. 1999. Astaxanthin in juvenile farmed chinook salmon (Oncorhynchus tshawytscha): effective dietary levels for flesh pigmentation and influence on fatty acids profile during cold temperature storage of fillets. M.Sc. Dissertation. University of British Columbia, Vancouver, BC , Canada. Toranzo.A.E., Santos, Y., J.L Barja. 1997. Immunizations with bacterial antigens: Vibrio infections. Developments in Biological Standardization, 90: 93-105. Torrisen, O.J. 1985. Pigmentation in Salmonids: factors affecting carotenoid deposition in rainbow trout (Salmo gairdneri). Aquaculture, 46: 133-142. Torrisen, O.J. , Hardy, R.W., Shearer, K. D. 1989. Pigmentation of Salmonids-Carotenoid Deposition and Metabolism. C R C Critical Reviews in Aquatic Sciences, 1(2): 209-225 Torrisen, O.J. , Hardy, R.W., Shearer, K. D, Scott, T.M, and F.E. Stone. 1990. Effects of dietary canthaxanthin level and lipid level on apparent digestibility coefficients for canthaxanthin in rainbow trout. Aquaculture, 88 351-362. Torrisen, O.J. 1995. Strategies for salmonid pigmentation. Journal of Applie Ichthyology, 276-281. Usher, M.L., Talbot, C , and F.B. Eddy. 1991. Effects of transfer to seawater on growth and feeding in Atlantic salmon smolts (Salmo salar). Aquaculture, 94: 309-326. Waagbo, R. 1994. The impact of nutritional factors on the immune system in Atlantic salmon, Salmo salar L.: a review. Aquaculture and Fisheries Management, 25: 175-197. Weedon, B.C.L. 1971. Occurrence. In: Carotenoids. O. Isler (Editor). Birkhauser Verlag, Basel, pp: 29-59. Wefers, H. and H. Sies. 1988. the protection of Ascorbate anf glutathione against microsomal lipid peroxidation is dependent on vitamin E. Eur. Biochem. 174: 353-357. Wieruszewski, J .B. 2000. Astaxanthin bioavailability, retention efficiency and kinetics in Atlantic salmon (Salmo salar) as influenced by pigment concentration and method of administration (kinetics only). M.Sc. Dissertation. Simon Fraser University, Burnaby, BC , Canada. 99 p. 100 Yamakoshi, J . , Kataoka, S., Koga, T., and T. Ariga. 1999. Proanthocyanidin-rich extract from grape seeds attenuates the development of aortic atherosclerosis in cholesterol-fed rabbits. Atherosclerosis, 142:139-149. Ye, X., Krohn, R.L., Liu, W., Joshi, S.S. , Kuszynski, C.A., McGinn, T.R., Bagchi, M., Preuss, H.G., Stohs, S .J . , and D. Bagchi. 1999. The cytotoxic effects of a novel IH636 grape seed proanthocyanidin extract on cultured human cancer cells. Molecular and Cellular Biochemistry, 196(1-2): 99-108. Yuli, I., Wilbrandt, W., and M. Shinitsky. 1981. Glucose transport through cell membranes of modified lipid fluidity. Biochemistry, 20: 4250-4256. Appendix 1 Interactions between antioxidant systems. ROOH ROH ROO*; R a-Tocopheryl-a-Tocotrienoxyl-radical ^ Vitamin E Cycle Lipid/Water Interface cx-Tocopherol a-Tocotrienol Vitamin C Cycle AscH* Antioxidants GSSG Lipoate NADPH + H + PUFA \ 0 2 \" and other FLO' NADH + H + F L O - + H + DHA Flavonoid Cycling ^ V NADPH + H + Thiol 1 Cycle I Glutathione Reductase (GR) GSH Dihydrolipoate Enzymes: Thiol transferase GSH-dependent dehydroascorbate Protein disulfide isomerase Thioredoxin (TRX) reductase FLO\" Pro-oxidants UVA, UVB, Ozone Flavonoid Species: FLO\" reduced (dissociated) FLO*\" radical (semiquinone) FLO=0 oxidized fauinone} Ascorbate Species: AscH\" reduced (dissociated) AscH*\" radical DHA oxidized Interactions between antioxidant systems, the antioxidant network: Lipophilic and hydrophilic antioxidants do not stand-alone but interact with each other in multiple ways. Vitamin E scavenges radicals in the lipid phase (vitamin E cycle) and can be regenerated by vitamin C, which in turn can be regenerated by different substrates (vitamin E cycle). One of the main substrates is G S H , which can regenerate vitamin C enzymatically. Other Thiols fulfil similar functions. Thiols finally can be regenerated using metabolic energy (Thiol cycle). Thus antioxidants form a highly organized network. Appendix 2 A. Role of dietary antioxidants in the defense against oxidative damage in biomembranes. VITAMIN C: scavenging of water-soluble radicals Regeneration of vitamin E FLAVONOIDS: scavenging of water-soluble radicals Chelation of metal ions WATER-PHASE VITAMIN E: scavenging of chain-propagating radicals l l l\u00C2\u00BBMliJft CAROTENOIDS: quenching of singlet oxygen LIPID-PHASE B. The overall mechanism of lipid oxidation consists of three phases (Newer, 1996) 1. Initiation: RH + 0 2 => R* + *OH 2. Propagation: R* + 0 2 => ' + ROO* ROO* + RH => R* + ROOH R O O H => RO* + HO* 3. Termination: R* + R* => RR R' + R O O ' ^ R O O R R O O # + ROO* => ROOR + 0 2 Appendix 3 Freshwater temperature record (experiments one and three) 11.0-1 10.5 A 10.0 A CO CD CD _ -*\u00E2\u0080\u00941 CO CD 8.5 -CL E CD H 8.0 -7.5 7.0 1 1 1 1 1 1 1 1 week 1 week 2 week 3 week 4 week 5 week 6 week 7 week 8 Record Date (Week 1-4= June 2000; Week 5-8= July 2000) Appendix 4 Saltwater temperature record (experiment C) 14.0 i 13.5 -13.0 -"@en . "Thesis/Dissertation"@en . "2004-05"@en . "10.14288/1.0099755"@en . "eng"@en . "Animal Science"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Enhancement of the non-specific immune response, pigmentation and growth of farmed Chinook salmon (Oncorhynchus tshawytscha) fed a combination of dietary flavonoids (grape seed extract, KPA\u00AE) and astaxanthin"@en . "Text"@en . "http://hdl.handle.net/2429/15076"@en .