Effect of dietary lipid source on the growth, tissue ...

Aquaculture 255 (2006) 210 – 222 www.elsevier.com/locate/aqua-online

Effect of dietary lipid source on the growth, tissue composition and hematological parameters of largemouth bass (Micropterus salmoides) Bobban Subhadra a , Rebecca Lochmann a,⁎, Steven Rawles b , Ruguang Chen a a

School of Agriculture, Fisheries and Human Sciences, University of Arkansas at Pine Bluff, AR 71601, United States b Harry K. Dupree Stuttgart National Aquaculture Research Center, Stuttgart, AR, United States Received 28 June 2005; received in revised form 4 October 2005; accepted 30 November 2005

Abstract We conducted a 12-week feeding trial with LMB fed practical diets differing primarily in lipid source. We reduced the fish oil content of commercially solvent-extracted menhaden fish meal to 0.5–1.2%, and used it as the primary protein source in all test diets. Diets were supplemented with 10% lipid as canola (CAN), chicken (CHK), menhaden fish oil (MFO) or CHK + MFO (50/ 50%). An additional diet contained non-extracted fish meal and 10% CHK (NEF + CHK), and a commercial trout diet (SC) was also included. Growth, survival, feed utilization, body composition and health parameters were measured to assess diet effects. Weight gain, survival, feed intake, feed conversion ratio, and protein efficiency ratio of LMB fed the test diets did not differ. Weight gain of LMB fed the SC diet was higher than that of LMB fed the test diets. Muscle lipid of fish fed the diet with 10% MFO was higher than that of fish fed diets with an equal mixture of CHK + MFO or NEF + CHK, and muscle lipid of LMB fed the SC diet was higher than that of fish fed the test diets. The n-3 to n-6 fatty acid ratio of the test diets was lower than expected because the poultry meal contained 16% lipid (high in 18:2n-6). The ratio of n-3 to n-6 fatty acids was higher in muscle and liver of fish fed the SC diet than those of fish fed the test diets. Docosahexaenoic acid (22:6n-3) and 20:4n-6 were conserved in fish fed diets without HUFA, but 20:5n-3 declined sharply from initial values. The index of atherogenicity (IA) and the index of thrombogenicity (IT) of LMB fed the test or SC diets were below 1.0 (considered healthful). Lymphocytes were higher in fish fed diets with N 4% n-3 fatty acids, including the CAN diet without HUFA. There were no differences in the Hk, Hb, MCHC, or serum lysozyme of LMB fed the test diets, but lysozyme was lower in LMB fed the SC diet than those fed the CHK + MFO diet. Fish fed diets with 10% MFO or CAN had higher alternative complement activity than fish fed diets with CHK. Tissue concentrations of HUFA (especially 20:4n-6 and 22:6n-6) were not depleted in 12 weeks, possibly due to conservation of initial HUFA, synthesis of HUFA from 18:3n-3 and 18:2n-6, or both. Therefore, non-fish oils might reduce diet cost with few deleterious effects for at least a quarter of the production cycle of LMB. © 2005 Elsevier B.V. All rights reserved. Keywords: Largemouth bass; Lipid sources; Chicken oil; Menhaden oil; Canola oil; Hematological parameters

1. Introduction ⁎ Corresponding author. Tel.: +1 870 575 8124; fax: +1 870 575 4639. E-mail address: [email protected] (R. Lochmann). 0044-8486/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.11.043

Largemouth bass (LMB) are economically important as sport fish as well as food fish for ethnic markets in the United States. Interest in commercial culture of LMB is

B. Subhadra et al. / Aquaculture 255 (2006) 210–222

being spurred by increasing demand and high market prices (averaging $10/kg wholesale and $18/kg retail sale). Most studies that have addressed LMB nutrition and feeding have focused on general requirements, but more specific information is needed to produce costeffective practical diet formulations for commercial production of LMB. Snow et al. conducted a series of studies on the culture of feed-trained LMB utilizing the Oregon Moist Pellet (Snow, 1968; Snow and Maxwell, 1970). Growth and body composition of juvenile LMB in response to dietary protein and energy (Brecka et al., 1996; Portz et al., 2001) and carbohydrate (Goodwin et al., 2002) have also been investigated. Coyle et al. (2000) determined the response of LMB to dietary supplementation of lysine, methionine and highly unsaturated fatty acids (HUFA). Bright et al. (2005) determined that 7–16% lipid (cod liver oil) diets with 30% fish meal resulted in good growth and desirable body composition of LMB. However, the effect of dietary lipid sources has not been fully addressed in LMB, particularly under circumstances where the influence of fish meal in the diet is not a confounding factor. Salmonid diets containing at least 40% protein and high levels of fish meal (50–70%) and oil (20–30%) are typically fed to cultured LMB (Anderson et al., 1981; Tidwell et al., 1996). There is increasing pressure to find alternatives to fish meal and oil because global fisheries are approaching optimal sustainable yield and demand for marine-based proteins and oils is intense among competing industries (Tidwell and Allan, 2002). Concern over pollutants such as dioxin in fish oils is also fueling efforts to reduce marine products in fish diets (Sargent et al., 2002). However, the benefits of long chain n-3 HUFA (i.e., fatty acids with 20 or more carbons and four or more double bonds) to human health and development are well documented (Simopoulus, 1999; Connor, 2000). Cultured fish, including LMB, that consume diets containing fishery products acquire elevated tissue concentrations of n-3 HUFA , which is appealing to health-conscious consumers. Some of the n-6 HUFA such as arachidonic acid (AA, 20:4 n-6) also have human health benefits, but western diets are normally rich in n-6 lipid sources (Kelley et al., 1997; Pablo et al., 2002). Dietary fatty acids can also significantly influence immune parameters in fish. Hematocrit, lysozyme and alternative complement activity indicate general health and humoral immune responses in fish (Obach et al., 1993; Tort et al., 1998; Ortune et al., 2000; Chen et al., 2003). Producing carnivorous fish with high levels of n-3 HUFA using diets with little or no marine products is a challenge. The carnivorous LMB might require HUFA

211

in the diet, similar to hybrid striped bass (Webster and Lovell, 1990), and walleye (Kolkovski et al., 2000). However, rainbow trout, a freshwater carnivore, can elongate and desaturate α-linolenic acid (18:3n-3) to n-3 HUFA and might not require them in the diet (Castell et al., 1972; Takeuchi and Watanabe, 1977). The ability of fishes to convert 18:3n-3 and linoleic acid (18:2n-6) into functionally active HUFA can be modulated by diet. If LMB can elongate 18:2n-6 and 18:3n-3 to their respective HUFAs, then a variety of nonfishery-based lipids might be effective dietary lipid sources. Use of non-fish lipids may enhance the sustainability of LMB culture because many lipid sources are less expensive than marine fish oils, and vegetable oils in particular are perceived as more “eco-friendly”. In this study we investigated growth and immune function of LMB in response to different dietary lipids. We minimized the endogenous oil content of menhaden fish meal by exhaustive solvent extraction and then used the extracted fish meal as the primary protein source in practical diets supplemented with 10% lipid as canola (CAN), chicken (CHK), menhaden fish oil (MFO) or CHO + CHK (50/50%). Growth, survival, feed intake, feed conversion ratio, protein efficiency ratio, body composition, fatty acid composition of liver and muscle, hematological parameters and lysozyme and alternative complement activity of LMB were measured to assess diet effects in a 12-week feeding trial. 2. Material and methods 2.1. Experimental diets Four isonitrogenous, isolipidic diets (Table 1) were formulated to contain either 10% canola oil (CAN), chicken oil (CHO), or menhaden fish oil (MFO), or 5% MFO and 5% CHO (CHO + MFO). These lipids were chosen because of their differences in content of n-3 and n-6 fatty acids that may be essential for LMB. The principle fatty acid composition of the diets is shown in Table 2. Chicken oil, containing mainly 18:2n-6, is less costly and more available than fish oils in states such as Arkansas with a significant poultry industry. Fish oil is commonly used in commercial diets fed to LMB, and is the only lipid source in this study containing both n-3 and n-6 HUFA. Canola oil was also selected because it is a vegetable oil with significant amounts of 18:3n-3, as well as 18:2n-6. Select™ Menhaden fish meal (Omega Protein, etc.) was used as the primary protein source in the test diets because of its prevalent use in diets fed to cultured LMB. However, menhaden fish meal contains approximately

212


Table 1 Formulation (g kg− 1) and proximate composition (percent of weight of dry feed) of diets with different lipid sources fed to largemouth bass for 12 weeks a Ingredient

CAN CHK CHK + MFO MFO NEF + CHK

Fish meal Poultry meal Blood meal Wheat CMC Vitamin premix b Mineral premix c Canola oil Chicken oil Menhaden fish oil Analyzed composition d Crude protein Total lipid Ash

300 200 120 230 20 20 10 100 0 0

47.8 13.8 12.5

300 200 120 230 20 20 10 0 100 0

47.8 13.7 12.3

300 200 120 230 20 20 10 0 50 50

47.6 13.7 12.4

300 200 120 230 20 20 10 0 0 100

47.7 13.9 12.5

300 200 120 230 20 20 10 0 100 0

46.8 15.4 11.8

a Diet abbreviations are as follows: CAN = canola oil; CHK = chicken oil; CHK + MFO = chicken oil + Menhaden fish oil (50:50), MFO = Menhaden fish oil; NEF + CHK = non-extracted fish meal + chicken oil. b Vitamin premix contains (g kg− 1 of premix): ascorbic acid, 50.0; D-calcium pantothenate, 5.0; choline chloride, 100.0; inositol, 5.0; menadione, 2.0; niacin, 5.0; pyridoxine–HCl, 1.0; riboflavin, 3.0; thiamin–HCl, 0.5; DL alpha tochopheryl acetate (250 IU g− 1), 8.0; vitamin A acetate (20,000 IU g− 1), 5.0; vitamin micro-mix, 10.0; cellulose, 805.5.Vitamin micro-mix contains (g kg− 1 of micro-mix): biotin, 0.5; cholecalciferol (1 μg = 40 IU), 0.02; folic acid, 1.8; vitamin B12, 0.02; cellulose, 97.66; cellulose, 805. c Mineral premix contains (g kg−1 of premix): calcium phosphate (monobasic) monohydrate, 136.0; calcium lactate pentahydrate, 348.49; ferrous sulfate heptahydrate, 5.0; magnesium sulfate heptahydrate, 132.0; potassium phosphate (dibasic), 240; sodium phosphate (monobasic) monohydrate, 88.0; sodium chloride, 45.0; aluminium chloride hexahydrate, 0.15; potassium iodide, 0.15; cupric sulfate pentahydrate, 0.50; manganese sulfate monohydrate, 0.70; cobalt chloride hexahydrate, 1.0; zinc sulfate heptahydrate, 3.0; sodium selenite, 0.011. d Ingredient composition of the trout diet (Silver Cup™, Nelson and Sons Inc.) used as a qualitative control was unknown. The analyzed composition was (g 100 g− 1): protein, 43.5; lipid, 19.9; and ash, 9.9.

10–12% fish oil as purchased, which might provide sufficient HUFA to meet the essential fatty acid (EFA) requirements of LMB and obviate the effects of the lipids of interest (e.g., Wonnacott et al., 2004). Therefore, we exhaustively extracted the fish meal as previously described (Chen et al., 2003) to reduce the endogenous lipid to trace amounts (0.5–1.2%) before inclusion in the test diets (#1–4). An additional diet containing unextracted fish meal and 10% chicken oil (NEF + CHK) was included as a control for any possible effects of solvent extraction or lipid reduction. A commercial diet (SC) (Silver cup™, Nelson and Sons, Murray, UT) used currently by LMB producers was included for comparison. The ingredient composition of

the trout diet was unknown but the analyzed proximate composition was (g 100 g− 1): protein, 43.5; lipid, 19.9; and ash, 9.9. As expected, the test diets contained similar amounts of protein and lipid except for the NEF + CHK and SC diets, which contained slightly higher (1.4% and 5%, respectively) amounts of lipid (Table 1). 2.2. Culture system and experimental design Feed-trained juvenile LMB were obtained from a local farmer (Dunn's fish farm, Monroe, AR) for the feeding trial. Upon arrival fish were stocked in 110-l tanks and acclimated to laboratory conditions for 1 week. Fish were fed the commercial diet that was fed in the subsequent feeding trial. Five pretrial fish were frozen at − 70 °C for subsequent analysis of proximate and fatty acid composition. Ten fish initially averaging 5 g ± 0.05 g in mass were selected and stocked into each of four 110-l aquaria, which in turn were randomly assigned to each treatment. Aquariums were configured in a recirculation system Table 2 Selected fatty acid composition (percent of total fatty acids by weight) of the experimental diets a, b Fatty acids

CAN

CHK

CHK + MFO

MFO

NEF + CHK

Silver Cup™

16:0 18:0 Saturates c 16:1 18:1 d MUFA e 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:6n-3 n-6 f n-3 g n-3 HUFA h n-3/n-6 ratio

11.8 4.0 16.3 2.7 55.4 60.2 20.7 4.2 0 0 0 20.7 4.2 0.0 0.2

21.5 6.0 28.5 6.8 43.3 51.5 18.5 0.8 0.9 0.7 0.3 19.4 1.8 1.0 0.1

22.4 5.8 31.7 8.4 35.2 45.0 15.9 1.0 1.3 3.7 1.8 17.1 6.6 5.5 0.4

21.7 5.4 33.8 10.0 24.9 37.1 11.8 1.3 1.9 8.3 4.2 14.4 14.9 13.6 1.0

23.4 6.0 31.7 8.6 37.2 47.5 16.5 0.9 1.1 2.2 1.4 17.5 4.5 3.6 0.3

22.6 4.5 38.2 13.0 16.0 33.5 6.1 1.3 2.0 13.8 5.3 9.0 21.6 20.3 2.4

a

Diet abbreviations are as follows: CAN = canola oil; CHK = chicken oil; CHK + MFO = chicken oil + Menhaden fish oil (50:50); MFO = Menhaden fish oil; NEF + CHK = non-extracted fish meal + chicken oil; Silver Cup™ = commercial diet. b Fatty acids present at ≤0.1 g 100 g− 1 are not presented. c Saturates included 14:0, 16:0, 18:0 and 20:0. d Total n-9 and n-7 isomers. e Monounsaturates included 14:1, 16:1, 18:1 and 20:1. f Total n-6 fatty acids included 18:2n-6, 20:3n-6 and 20:4n-6. g Total n-3 fatty acids included 18:3n-3, 20:5n-3 and 22:6n-3. h Total n-3 HUFA included 20:5n-3 and 22:6n-3. The only n-6 HUFA detected was 20:4n-6.


that contained dechlorinated municipal water with a hardness of 40–50 mg l− 1 as calcium carbonate. Water quality was maintained by continuous aeration and a flow rate of 1 l min− 1 per aquarium. Dissolved oxygen and ammonia levels were maintained at acceptable levels for LMB (Tidwell et al., 2000) and water temperature was maintained at 25 ± 2 °C (mean ± S.D.). A diurnal light/dark cycle of 12:12 h was maintained during the feeding trial. Fish were fed to apparent satiation twice daily (08:30 and 16:30 h) for 12 weeks. During the trial, fish in each tank were counted and weighed every 2 weeks and mortalities were recorded daily. 2.3. Proximate composition and fatty acid analysis Muscle samples were analyzed for protein (Kjeldahl), dry matter and ash content according to standard methods (AOAC, 1995). Protein efficiency ratio (PER) = wet weight gain (dry weight of protein intake)− 1. Total lipids were extracted and quantified from muscle and liver (Folch et al., 1957) and then saponified and methylated for fatty acid quantification as previously described (Morrison and Smith, 1964). Fatty acid methyl esters (FAME) were analyzed using a flame ionization gas chromatograph (Varian, Model CP3800, Walnut Creek, CA) equipped with a capillary column (100 m 0.25 mm; Varian CP Select for Fame #CP7420) with helium as the carrier. Injector port and detector temperatures were maintained at 250 °C and 315 °C, respectively. The FAME samples were automatically injected on column in 1 μl of hexane using an autosampler (Varian Chrompack, Model 8200, Walnut Creek, CA). Flow rates were programmed to remain constant throughout the run (hydrogen: 30 ml min− 1; air: 300 ml min− 1; makeup gas: 31.7 ml min− 1). Column oven temperature was initially 100 °C for 10 min, increased to 165 °C at 15 °C min− 1 and held for 3 min, then increased to 250 °C at 1.5 °C min− 1 and held for 10 min. Total run time was 60 min per sample. Sample FAMEs were identified and quantified by comparing the retention times and areas of the peaks to those of serially diluted mixtures of reference standards (GLC-96, GLC-473B, Nu-Chek Prep, Elysian, MN). Tridecanoate methyl ester (13:0) served as the internal standard. The results of the individual fatty acids were expressed as g 100 g− 1 of total identified FAMEs. 2.4. Index of atherogenicity (IA) and index of thrombogenicity (IT) The indices of atherogenicity and thrombogenicity (Ulbricht and Southgate, 1991) were calculated using

213

the fatty acid composition of the muscle to determine the potential health impact on human consumers. The following equations were used: (1) Index of atherogenicity (IA) = [(12:0) + (4 × 14:0) + (16:0)] × [(PUFA n-6 and n-3) + MUFA)− 1 (2) Index of thrombogenicity (IT) = [(14:0) + (16:0) + (18:0)] × [(0.5 × MUFA) + (0.5 × n-6) + (3 × n-3) + (n-3 × n-6 − 1 )] − 1 where PUFA = polyunsaturated fatty acids including 18:2n-6, 18:3n-3, 20:4n-6 and 22:6n-3; and MUFA = monounsaturated fatty acids including all isomers of 14:1, 16:1, 18:1 and 20:1. Concentrations of the fatty acids were expressed as g 100 g− 1 to perform the calculations. 2.5. Blood sampling, hematology and differential cell counts At the conclusion of the trial, LMB that had not been fed for 24 h were anesthetized with tricaine methanesulfonate at 30 mg l− 1 (Sigma, St. Louis, MO). The caudal peduncles of 4 fish per aquarium were severed with a scalpel and blood samples were collected from the caudal vein of individual fish with a heparinized microhematocrit capillary tube. After centrifugation (3500×g for 10 min) of each blood sample, analysis of hematocrit (Hk), hemoglobin (Hb) (Hb cyanide method, Houston, 1990), and differential blood cell counts (Wright-Giemsa method) were performed. Mean corpuscular hemoglobin content (MCHC) was calculated according to the formula: MCHC = Hb concentration (g dl− 1 ) × Hk− 1 . Blood smears were stained using Hema 3 Stain (Biochemical Sciences, St. Louis, MO), and the differentiated leucocyte types (lymphocytes, thrombocytes and granulocytes) were counted and calculated as the number of cells of a specific type per 100 total leucocytes counted. The fish plasma was analyzed for alternative complement and lysozyme (Sections 2.6 and 2.7). Exsanguinated fish were stored at − 70 °C until proximate and fatty acid composition of muscle and liver were analyzed. 2.6. Alternative complement assay The hemolytic activity driven by the alternative complement pathway was measured using washed rabbit red blood cells as target cells in the presence of EGTA and Mg2+, as described by Tort et al. (1996). Twenty-five μl of serum from each fish was used for the analysis.

214


2.7. Lysozyme analysis

3. Results

Fifty μl of fish plasma was added into each well in a 96-well plate. One hundred and fifty μl of suspended Micrococcus lysodeikticus (0.4 mg ml− 1 in phosphate buffer) was mixed with the fish plasma in each well, and the reduction in the absorbance reading at 450 nm was taken every 10 s for 5 min using the basic kinetic protocol of the microplate reader with SoftMax Pro 4.3 (Molecular Devices, Sunnyvale, CA). One unit of lysozyme activity was defined as a reduction in absorbance of 0.001 min− 1 (1 mOD min− 1).

3.1. Growth, survival and feed utilization

2.8. Statistical analysis Each of the dietary treatments was assigned to four aquaria in a completely randomized design. Weight gain, survival, feed intake, FCR, PER, hematocrit, hemoglobin content, IA, IT, lysozyme, and complement data from the test diets were analyzed by one way Analysis of Variance (ANOVA) with a StatView program. When significant differences among treatments were found (P b 0.05), treatment means were compared using Fishers least significance difference test. Data from fish fed the commercial diet (SC) was not included in the ANOVA because the diet composition was unknown. Instead, differences between test diets and the control diet were separated using contrast statements in the Tukey–Kramer procedure for pairwise comparisons (Tukey, 1953; Kramer, 1956).

Survival was 100% in all treatments. Weight gain of LMB fed the test diets ranged from 26.7 to 29.0 g and did not differ among treatments (Table 3). Feed intake, feed conversion ratio and protein efficiency ratio also did not differ among dietary treatments (Table 3). Weight gain of LMB fed the SC diet (30.9 g) was higher than that of fish fed the test diets, but FCR and PER were similar to those of fish fed the test diets (Table 3). 3.2. Proximate composition and fatty acid analysis Moisture, protein and ash contents of muscle did not differ among dietary treatments (Table 3). Muscle lipid content of fish fed the diet supplemented with 10% MFO was higher than that of fish fed diets with CHK and lipid derived from menhaden (CHK + MFO or NEF + CHK). Crude protein, moisture and ash in the muscle of fish fed the commercial diet were similar to that of fish fed the test diets, but muscle lipid was approximately twice as high in fish fed the commercial diet as in fish fed the test diets (Table 3). Muscle fatty acid composition was similar to that of the diet. Total saturated fatty acids were highest in muscle of fish fed the diets with MFO and the diet with

Table 3 Performance and muscle composition (percent of fresh weight) of largemouth bass fed diets with different lipid sources for 12 weeks 1, 2 Parameter

CAN

CHK

CHK + MFO

MFO

NEF + CHK

Silver Cup™

Weight gain (g) Feed intake 3 FCR 4 PER 5 Moisture 6 Crude protein 6 Total lipid 6 Ash 6

26.7 ± 1.4* 33.2 ± 1.6 1.33 ± 0.03 1.59 ± 0.05 77.4 ± 0.1 16.5 ± 0.6 1.6 ± 0.1ab* 1.9 ± 0.1

28.0 ± 0.3* 37.1 ± 1.5 1.37 ± 0.02 1.59 ± 0.07 77.5 ± 0.5 16.9 ± 0.5 1.6 ± 0.2ab* 1.8 ± 0.1

28.0 ± 0.9* 37.6 ± 0.8 1.36 ± 0.001 1.68 ± 0.13 77.5 ± 0.2 16.6 ± 0.6 1.4 ± 0.1b* 1.7 ± 0.1

29.0 ± 1.2* 36.5 ± 1.5 1.32 ± 0.03 1.59 ± 0.04 78.4 ± 0.9 16.5 ± 0.6 2.1 ± 0.2a* 1.7 ± 0.1

27.2 ± 1.1* 35.4 ± 15 1.28 ± 0.06 1.55 ± 0.10 77.3 ± 0.2 17.0 ± 0.1 1.5 ± 0.1b* 1.8 ± 0.1

30.9 ± 1.0 37.4 ± 0.9 1.26 ± 0.02 1.86 ± 0.04 76.8 ± 0.6 16.3 ± 0.6 3.4 ± 0.3 1.6 ± 0.1

1 Diet abbreviations are as follows: CAN = canola oil; CHK = chicken oil; CHK + MFO = chicken oil + menhaden fish oil (50:50); MFO = Menhaden fish oil; NEF + CHK = non-extracted fish meal + chicken oil; Silver Cup™ = commercial diet. 2 Values are means ± S.E.M. of four replicate tanks per diet (N = 10 fish per tank). A one-way ANOVA was used to determine differences among treatment means of the test diets (P b 0.05). When significant differences were found, treatment means were compared using Fishers least significance difference test. The composition of the commercial diet was unknown so differences between test diets and the commercial diet (SC) were separated using contrast statements in the Tukey–Kramer procedure for pair-wise comparisons (Tukey, 1953; Kramer, 1956). An asterisk indicates that the mean is different (P b 0.05) from that of the SC diet. 3 Feed intake = average weight of feed consumed (g, as fed) per fish. 4 Feed conversion ratio (FCR) = dry feed weight (wet weight gain)− 1. 5 Protein efficiency ratio (PER) = wet weight gain (dry weight of protein intake)− 1. 6 Samples from each replicate were analyzed in duplicate.


NEF, and lowest in fish fed the diet with CAN (Table 4). Total MUFA in muscle were highest in fish fed the diets with CAN or 10% CHK and lowest in those fed diets with MFO (Table 4). Concentrations of MUFA were intermediate for fish fed the other diets. Total n-6 fatty acids of muscle were highest in fish fed the CAN and 10% CHK diets, lowest in fish fed the 10% MFO diet, and intermediate in fish fed the other diets (Table 4). Total n-3 fatty acids of muscle were highest in fish fed the diets with MFO or NEF + CHK, and lowest in fish fed the diets with CAN, 10% CHK or CHK + MFO. Total n-3 HUFA followed the same pattern as total n-3 fatty acids except that the CHK + MFO diet had concentrations of n-3 HUFA similar to diets with 10% CHK or NEF + CHK. The only n-6 HUFA detected in muscle was 20:4n-6, and there were no differences in concentrations of this fatty acid among fish fed different diets. The ratio of n-3 to n-6 fatty acids was highest in muscle of fish fed the diets with MFO, and lowest in fish fed the diets with CAN or 10% CHK (Table 4).

215

Total saturates in muscle of fish fed the commercial diet were higher than those of fish fed the diets with 10% CAN or CHK and similar to those of fish fed the other diets (Table 4). The MUFA were similar in fish fed the SC diet and those fed the diets with MFO, and MUFA were lower in fish fed the SC diet than in fish fed the other test diets (Table 4). Total n-6 fatty acids were similar in fish fed the SC and 10% MFO diets, and lower in fish fed the SC diet than in fish fed the other test diets. Total n-3 fatty acids and the n-3 to n-6 fatty acid ratio were higher in fish fed the SC diet than in those fed the test diets (Table 4). The concentration of total saturated fatty acids in the liver of LMB were highest in fish fed the diets with 10% MFO or CHK + MFO, lowest in fish fed the diet with CAN, and intermediate in fish fed the other diets. The MUFA concentration in the liver of LMB followed an inverse pattern from the saturates—fish fed the CAN diet had the highest concentration, fish fed the diets with CHK + MFO or 10% MFO had the lowest, and fish fed

Table 4 Fatty acid composition of muscle (percentage of total fatty acids by weight) of largemouth bass fed diets with different lipid sources for 12 weeks 1, 2 Fatty acids 3 16:0 18:0 Saturates 4 16:1 18:1 5 MUFA 6 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:6n-3 Σn-6 7 Σn-3 8 Σn-3 HUFA 9 n-3/n-6 ratio IA 10 IT 11 1

Initial fish 20.8 3.2 35.1 11.7 18.3 33.2 4.8 0.9 2.2 5.9 8.6 8.9 16.7 14.5 1.9 0.46 0.31

CAN

CHK b

15.5 ± 0.5 4.5 ± 0.4 23.3 ± 2.4c* 3.5 ± 0.7c* 42.1 ± 2.3a* 48.2 ± 1.3a* 17.8 ± 0.6a* 2.5 ± 0.0a* 1.8 ± 0.6 1.1 ± 0.3c* 4.4 ± 1.4bc 0.2 ± 1.1a* 8.1 ± 1.6cd* 5.9 ± 1.4bc* 0.4 ± 0.1cd* 0.29 ± 0.06d* 0.41 ± 0.08ab

CHK + MFO a

20.5 ± 0.5 5.2 ± 0.2* 27.2 ± 0.5bc* 6.6 ± 0.4b* 39.3 ± 0.9a* 46.7 ± 1.0a* 17.4 ± 0.3a* 0.6 ± 0.3b 2.0 ± 0.2 1.1 ± 0.2c* 3.9 ± 0.6c* 20.5 ± 0.5a* 5.7 ± 0.7d* 5.4 ± 0.8c* 0.3 ± 0.0d* 0.35 ± 0.01ad* 0.53 ± 0.03a*

a

20.1 ± 1.2 5.7 ± 0.1* 29.7 ± 1.2ab 6.5 ± 0.3b* 32.3 ± 0.7b* 40.0 ± 0.7bc 11.5 ± 2.9bc* 0.7 ± 0.0b 2.6 ± 0.0 2.8 ± 0.2ab* 9.4 ± 1.1a 13.6 ± 0.6c* 8.8 ± 0.9cd* 10.2 ± 0.8ab 0.8 ± 0.1ab* 0.38 ± 0.03cd 0.38 ± 0.04b

MFO

NEF + CHK a

20.8 ± 1.3 5.1 ± 0.5 32.1 ± 2.1a 8.3 ± 0.5a* 26.0 ± 1.3c* 36.0 ± 1.8c 9.7 ± 0.5c 0.9 ± 0.0b 2.2 ± 0.2 3.8 ± 0.3a* 7.8 ± 0.9ab 10.1 ± 0.1d 14.4 ± 1.5ab* 13.4 ± 1.4a 1.1 ± 0.1b* 0.54 ± 0.02b 0.43 ± 0.02ab

a

20.9 ± 0.4 5.7 ± 0.7* 29.3 ± 0.4ab 6.9 ± 0.8ab* 34.5 ± 0.9b* 41.8 ± 1.9b* 14.9 ± 0.5ab* 0.5 ± 0.2b 2.5 ± 0.5 2.4 ± 0.7b* 8.0 ± 2.0ab 18.1 ± 0.6b* 11.5 ± 2.2ac* 11.0 ± 2.4a 0.6 ± 0.1ac* 0.40 ± .02ac 0.44 ± 0.04ab

Silver Cup™ 19.3 ± 1.7 3.5 ± 0.2 34.9 ± 2.0 12.0 ± 1.1 20.6 ± 0.7 34.1 ± 1.2 5.7 ± 0.2 1.1 ± 0.0 2.4 ± 0.2 8.2 ± 0.7 10.6 ± 2.0 11.1 ± 0.4 24.2 ± 3.6 23.1 ± 3.6 2.2 ± 0.2 0.68 ± 0.13 0.32 ± 0.06

Diet abbreviations are as follows: CAN = canola oil; CHK = chicken oil; CHK + MFO = chicken oil + Menhaden fish oil (50:50); MFO = Menhaden fish oil; NEF + CHK = non-extracted fish meal + chicken oil; Silver Cup™ = commercial diet. 2 Values are means ± S.E.M. of 3–4 replicate tanks per diet (N = 10 fish per tank). A one-way ANOVA was used to determine differences among treatment means (P b 0.05). When significant differences were found, treatment means were compared using Fishers least significance difference test. The composition of the commercial diet was unknown so differences between test diets and the commercial diet (SC) were separated using contrast statements in the Tukey–Kramer procedure for pair-wise comparisons (Tukey, 1953; Kramer, 1956). An asterisk indicates that the mean is different (P b 0.05) from that of the SC diet. 3 Fatty acids present at ≤0.1percentage of total fatty acids by weight are not included. 4 Saturates included 14:0, 16:0, 18:0 and 20:0. 5 Total n-9 and n-7 isomers. 6 Monounsaturates included 14:1, 16:1, 18:1 and 20:1. 7 Total n-6 fatty acids included 18:2n-6, 20:3n-6 and 20:4n-6. 8 Total n-3 fatty acids included 18:3n-3, 20:5n-3 and 22:6n-3. 9 Total n-3 HUFA included 20:5n-3 and 22:6n-3. The only n-6 HUFA detected was 20:4n-6. 10 Index of atherogenicity. 11 Index of thrombogenicity.

216


other diets had intermediate concentrations (Table 5). Total n-6 fatty acids in liver were similar in fish fed diets with CAN, 10% CHK or NEF + CHK, and lower in fish fed other diets (Table 5). Total n-3 fatty acids in liver were highest in fish fed diets with MFO and lowest in fish fed diets with CAN or CHK (Table 5). The pattern of n-3 HUFA in liver was similar to that of the total n-3 fatty acids (Table 5). The only n-6 HUFA found in liver was 20:4n-6 and concentrations of this fatty acid did not differ among diets. The ratio of n-3 to n-6 fatty acids in liver was highest in fish fed the diet with 10% MFO, lowest in fish fed the diets with CAN or 10% CHK, and intermediate in fish fed the other diets (Table 5). Total saturates in liver of fish fed the SC diet were lower than those of fish fed the diets with CHK + MFO or 10% MFO and similar to those of fish fed the other diets (Table 5). Total MUFA in liver of fish fed the SC diet were higher than those of fish fed the diets with CHK + MFO or 10% MFO and similar to those of fish fed the other diets (Table 5). Total n-6 fatty acids in the liver of LMB fed the SC diet were lower than those of

fish fed the test diets. Total n-3 fatty acids in the liver of LMB fed the SC diet were higher than those of fish fed the diets with 10% CAN or CHK. Total n-3 HUFA in liver were similar among fish fed the SC and the test diets (Table 5). The ratio of n-3 to n-6 fatty acids in liver of LMB fed the SC diet was higher than that of fish fed the test diets (Table 5). 3.3. Index of atherogenicity (IA) and index of thrombogenicity (IT) The IA was highest in muscle of fish fed the diet with 10% MFO, lowest in fish fed the CAN diet, and intermediate for fish fed the other diets (Table 4). The IT was similar in muscle of fish fed the diets with 10% CHK, 10% MFO or NEF + CHK. The IT in muscle of fish fed the CAN diet was similar to that of fish fed the diet with CHK + MFO and lower than that of fish fed the other diets (Table 4). The IA of the muscle of fish fed the SC diet was higher than that of fish fed the diets with 10% CAN or CHK (Table 4). The IT of the muscle of fish fed the

Table 5 Fatty acid composition of liver (percentage of total fatty acids by weight) of largemouth bass fed diets with different lipid sources for 12 weeks 1, 2 Fatty acids 3

Initial fish

CAN

CHK

CHK + MFO

MFO

NEF + CHK

Silver Cup™

16:0 18:0 Saturates 4 16:1 18:1 5 MUFA 6 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:6n-3 Σn-6 8 Σn-3 9 Σn-3 HUFA 10 n-3/n-6 ratio

20.7 2.4 27.9 17.9 28.8 48.7 3.6 0.8 1.7 2.9 5.1 5.3 11.2 8.0 2.1

14.7 ± 0.6c 5.3 ± 0.1b 22.4 ± 0.6d 4.4 ± 0.4b* 45.5 ± 0.6a* 52.0 ± 0.9a 15.1 ± 0.7a* 1.6 ± 0.2a 2.8 ± 0.2 0.5 ± 0.2bc* 4.7 ± 0.6d 18.9 ± 0.6a* 6.7 ± 0.6c* 7.2 ± 1.6b 0.04 ± 0.0d*

20.9 ± 2.1ab* 8.4 ± 1.4a* 30.6 ± 3.4c 6.9 ± 0.5a* 37.4 ± 2.8b* 46.1 ± 2.9a 14.4 ± 0.7ad* NDc, 6 4.5 ± 0.9* NDc*,6 6.9 ± 1.5cd 20.0 ± 1.8a* 6.9 ± 1.5c* 8.7 ± 1.3b 0.3 ± 0.0d*

22.8 ± 1.0ab* 9.2 ± 0.6a* 34.9 ± 0.8ab* 7.0 ± 0.3a* 28.4 ± 1.2cd* 36.6 ± 1.6bc* 10.8 ± 0.9bc* NDc, 7 4.0 ± 0.0* 1.8 ± 0.1b* 11.8 ± 1.2ab 14.8 ± 0.9bc* 14.5 ± 2.2ab 14.2 ± 1.9a 1.0 ± 0.1b*

23.3 ± 0.8a* 7.1 ± 0.6ab* 36.6 ± 1.6b* 8.2 ± 0.6a* 26.8 ± 1.9c* 36.1 ± 2.4c* 9.1 ± 0.4c* 0.7 ± 0.0b 3.7 ± 0.4 3.8 ± 0.3a 7.8 ± 0.9d 13.0 ± 0.5c* 15.7 ± 0.8a 15.1 ± 1.0a 1.2 ± 0.1a*

19.2 ± 1.0b* 6.4 ± 1.2ab* 27.6 ± 1.6cd 7.3 ± 0.7a* 35.3 ± 3.2bd* 44.3 ± 3.8ac 12.5 ± 0.9bd* 0.3 ± 0.2cd 3.6 ± 0.7* 1.2 ± 0.1b* 10.0 ± 1.6bc 16.8 ± 1.2ab* 12.0 ± 1.2b 10.8 ± 1.9ab 0.7 ± 0.1c*

17.2 ± 0.7 2.9 ± 0.3 28.5 ± 0.8 17.0 ± 0.3 33.7 ± 0.6 51.9 ± 0.8 4.6 ± 0.1 0.7 ± 0.0 2.0 ± .0.01 3.1 ± 0.1 7.7 ± 0.6 8.0 ± 0.2 13.7 ± 0.7 8.8 ± 1.4 1.8 ± 0.1

1 Diet abbreviations are as follows: CAN = canola oil; CHK = chicken oil; CHK + MFO = chicken oil + Menhaden fish oil (50:50); MFO = Menhaden fish oil; NEF + CHK = non-extracted fish meal + chicken oil; Silver Cup™ = commercial diet. 2 Values are means ± S.E.M. of 3-4 replicate tanks per diet (N = 10 fish per tank) except for diet 3, where N = 2 due to sample loss. A one-way ANOVA was used to determine differences among treatment means (P b 0.05). When significant differences were found, treatment means were compared using Fishers least significance difference test. The composition of the commercial diet was unknown so differences between test diets and the commercial diet (SC) were separated using contrast statements in the Tukey–Kramer procedure for pair-wise comparisons (Tukey, 1953; Kramer, 1956). An asterisk indicates that the mean is different (P b 0.05) from that of the SC diet. 3 Fatty acids present at ≤0.1 percentage of total fatty acids by weight are not included. 4 Saturates included 14:0, 16:0, 18:0 and 20:0. 5 Total n-9 and n-7 isomers. 6 Monounsaturated fatty acids (MUFA) included 14:1, 16:1, 18:1 and 20:1. 7 ND = not detected. 8 Total n-6 fatty acids included 18:2n-6, 20:3n-6 and 20:4n-6. 9 Total n-3 fatty acids included 18:3n-3, 20:5n-3 and 22:6n-3. 10 Total n-3 HUFA included 20:5n-3 and 22:6n-3. The only n-6 HUFA detected was 20:4n-6.


commercial diet was lower than that of fish fed the diets with 10% CAN or CHK (Table 4). 3.4. Hematology, differential count, complement and lysozyme There were no differences in the Hk, Hb, or MCHC among fish fed different diets (Table 6). The percentage of lymphocytes was highest in fish fed diets with CAN, CHK + MFO or 10% MFO, and lowest in fish fed the diet with 10% CHK (Table 6). The percentage of thrombocytes was higher in fish fed the diet with NEF + CHK than in those fed with CHK + MFO and there were no other differences among diets. The percentage of granulocytes was similar among fish fed different diets. Fish fed diets containing 10% MFO or CAN had higher alternative complement activity than fish fed any of the diets containing CHK (Table 6). There were no differences in serum lysozyme activity among fish fed different diets (Table 6). The alternative complement activity, Hk, Hb, MCHC, and the percentage of thrombocytes and granulocytes in LMB fed the SC diet were similar to those of fish fed the test diets (Table 6). Lysozyme of LMB fed the SC diet was lower than that of fish fed the diet with CHK + MFO (Table 6). The percentage of lymphocytes was higher in LMB fed the SC diet than in those fed the diet with 10% CHK (Table 6).

217

4. Discussion Growth of largemouth bass fed diets containing lipids with n-3 to n-6 fatty acid ratios ranging from 0.1 to 1.0 were very similar, suggesting that a variety of lipid sources may be used in practical diets for this species. Fish fed the commercial diet with a higher n-3 to n-6 ratio (2.4) gained more weight, but the magnitude of the differences in weight gain of LMB fed the test and commercial diets was not large, and nutrients other than fatty acids might have contributed to the differences. For instance, higher lipid in the SC diet might have spared protein for additional growth. Feed utilization (intake, conversion and protein efficiency ratio) were similar in fish fed the test and commercial diets. The higher lipid content of the commercial diet and possibly larger fish size explained the higher muscle lipid of fish fed the SC diet. Other indices of muscle composition were not differentially affected by diet. Although the menhaden fish meal in the test diets was exhaustively extracted prior to diet formulation to minimize the contribution of endogenous lipid, the poultry meal was not. The poultry meal contained 16% lipid, predominantly from n-6 fatty acids, and the resulting ratios of n-3 to n-6 fatty acids were lower than expected for the diets supplemented with CAN and MFO. Diets containing rendered fats such as tallow, lard or chicken oil supported growth of rainbow trout, carp (Takeuchi et al., 1978), channel catfish (Fracalossi and

Table 6 Hematological parameters, differential cell counts 1, alternative complement activity (ACH50), and lysozyme activity of largemouth bass fed diets with different lipid sources for 12 weeks 2, 3 Parameters

CAN −1

ACH50 (units dl ) Lysozyme Hematocrit (Hk; %) Hemoglobin (Hb; g dl− 1) MCHC 4 Lymphocytes Thrombocytes 5 Granulocytes 6

CHK a

56.8 ± 3.7 76.5 ± 2.4 51.6 ± 0.9 3.8 ± 0.1 7.2 ± 0.2 54.7 ± 1.2a 31.8 ± 2.0ab 13.6 ± 0.9

CHK + MFO b

46.6 ± 3.1 77.5 ± 2.2 52.4 ± 1.0 3.8 ± 0.1 7.3 ± 0.1 45.6 ± 0.9c* 37.2 ± 4.7ab 13.9 ± 0.9

b

45.1 ± 3.1 82.1 ± 3.7* 51.2 ± 1.2 3.7 ± 0.1 7.3 ± 0.2 53.2 ± 0.8a 29.2 ± 3.7b 14.2 ± 0.8

MFO

NEF + CHK a

62.3 ± 0.8 74.5 ± 4.8 53.9 ± 0.9 3.9 ± 0.1 7.5 ± 0.2 53.7 ± 1.0a 31.2 ± 1.3ab 15.1 ± 0.9

b

47.3 ± 3.8 76.7 ± 3.2 52.0 ± 0.9 3.6 ± 0.1 6.9 ± 0.3 49.1 ± 0.7b 37.9 ± 1.2a 13.0 ± 0.9

Silver Cup™ 56.1 ± 3.1 63.8 ± 4.4 54.0 ± 1.1 3.9 ± 0.1 7.3 ± 0.2 51.3 ± 1.3 33.7 ± 1.2 15.0 ± 0.9

1 Differential cell types including lymphocytes, thrombocytes and granulocytes were calculated as a percentage of the total number of leucocytes counted (100 per slide). Unidentified or fragile cells were excluded. Three slides from three individual fishes per replicate (N = 36) were used for the counts. 2 Diet abbreviations are as follows: CAN = canola oil; CHK = chicken oil; CHK + MFO = chicken oil + Menhaden fish oil (50:50); MFO = Menhaden fish oil; NEF + CHK = non-extracted fish meal + chicken oil; Silver Cup™ = commercial diet. 3 Values are means ± S.E.M. of four replicate tanks per diet (N = 10 fish per tank). A one-way ANOVA was used to determine differences among treatment means (P b 0.05). When significant differences were found, treatment means were compared using Fishers least significance difference test. The composition of the commercial diet was unknown so differences between test diets and the commercial diet (SC) were separated using contrast statements in the Tukey–Kramer procedure for pair-wise comparisons (Tukey, 1953; Kramer, 1956). An asterisk indicates that the mean is different (P b 0.05) from that of the SC diet. 4 Mean corpuscular hemoglobin concentration (MCHC) = hemoglobin content (g dl− 1) × 100 hematocrit− 1. 5 Includes both spiked and spindle shaped cells. 6 Includes neutrophils, monocytes, basophils and eosinophils.

218


Lovell, 1994; Fracalossi et al., 1995), red sea bream (Glencross et al., 2003), and European eel (Luzzana et al., 2003) as well as diets containing lipid only from marine fish. Similar growth of fish fed diets with total or partial replacement of fish oil with chicken oil or canola oil were observed in coho salmon (Dosanjh et al., 1984), chinook salmon (Dosanjh et al., 1988), rainbow trout (Greene and Selivonchick, 1990; Liu et al., 2004), brown trout (Turchini et al., 2003), brook charr (Guillou et al., 1995), and Atlantic salmon (Rosenlund et al., 2001). The comparative efficacy of different lipid sources for fish is affected by the initial body concentration of essential fatty acids, the total diet composition, the length of the feeding trial, and the overall growth obtained during the trial. The growth of LMB in this trial was high (440–500%). However, the initial tissue concentrations of n-3 HUFA also were high because fish initially were trained to accept a trout diet (SC) and were maintained on this diet exclusively prior to use in this study. Tissue concentrations of EFA in fish fed the test diets may not have been sufficiently depleted to reveal differential effects of the dietary lipid sources during the 12-week trial, because no growth reduction or other signs of EFA deficiency were observed. The fatty acid composition of the liver and muscle of LMB were similar to those of the diets, as expected (e.g., Stickney and Andrews, 1972; Xu et al., 1993; Legendre et al., 1995). The n-3 to n-6 ratios of LMB muscle reflected the pattern of ratios in the diets. However, the concentrations of n-3 and n-6 HUFA in muscle never reached zero even in fish fed the CAN diet devoid of HUFA. This pattern indicates selective retention of the HUFA, as in other fish (Bell et al., 1995a, 2001, 2002; Caballero et al., 2003; Ng et al., 2003; Regost et al., 2003; Mourente et al., 2005). However, fish fed diets with low concentrations of n-3 HUFA (CAN and diets with CHK) had low concentrations of n-3 HUFA in muscle relative to the initial fish, indicating that LMB have little ability to synthesize n-3 HUFA from 18:3n-3. The concentration of 20:4n-6 was very similar in muscle of initial fish and that of fish fed the commercial or test diets for 12 weeks (1.8–2.5%). Arachidonic acid is probably maintained at a physiologically optimum level in tissues due to its importance in eicosanoid formation (Greene and Selivonchick, 1990; Bell et al., 1995b; Fountoulaki et al., 2003). Interestingly, the concentration of 20:4n-6 was higher in livers of all fish after 12 weeks compared to the initial fish and to final muscle concentrations, despite low (≤2.0%) concentrations in the diets. Docosahexaenoic acid is the main component of the phosphoglycerols in fish biomembranes (Henderson and

Tocher, 1987) and is critical for normal development of the brain and retina (Mourente et al., 1991; Bell et al., 1995c). Eicosapentaenoic acid is more likely to be catabolized for energy and less likely to be retained in biomembranes than DHA (Madsen et al., 1999). Eicosapentaenoic acid was greatly reduced in muscle of LMB fed the test diets relative to initial values, and in liver also with the exception of fish fed the 10% MFO diet. In rainbow trout 20:5n-3 was catabolized for energy in the muscle but retained in the liver (Torstensen et al., 2004). In LMB, the muscle concentrations of 22:6n-3 were only maintained near or above initial values in fish fed diets with non-extracted fish meal and/ or oil (including the commercial diet). By contrast, liver concentrations of 22:6n-3 were equal to or higher than initial values regardless of diet, which is consistent with selective retention of EFA. The differences in retention of 22:6n-3 between tissues may be due to the central role of the liver in lipid and fatty acid metabolism (Greene, 1990). Dietary fatty acids regulate the expression of genes involved in lipid and energy metabolism. The n-3 HUFA, 18:2n-3, and 18:3n-3 suppress the induction of hepatic lipogenic genes and HUFA biosynthesis depends on the relative dietary concentrations of substrates (18:3n-3 and 18:2n-6) and end products. The formation of 22:6n-3 from both 18:3n-3 and 20:5n-3 in hepatocytes of rainbow trout is stimulated by omitting 22:6n-3 from the diet (Buzzi et al., 1996, 1997). Replacing fish oil with vegetable oils in the diets of Atlantic salmon resulted in increased activity of fatty acyl desaturation and elongation pathways in isolated hepatocytes (Tocher et al., 1997, 2002; Bell et al., 2001, 2002). The activity of desaturases and elongases in the HUFA biosynthetic pathway increased with increases in dietary vegetable oils in salmon parr (Bell et al., 1997; Tocher et al., 2000) and other freshwater salmonids (Tocher et al., 2001a,b). Transcription and enzyme activity were stimulated in salmon fed diets rich in 18:2n-6 and 18:3n-3 compared to fish fed diets rich in n-3 HUFAs (Zheng et al., 2004). The diets with 10% CAN or CHK had relatively high concentrations of 18-carbon precursors for HUFA synthesis and low concentrations of HUFA, which should favor HUFA synthesis. However, after 12 weeks LMB fed diets with 10% CAN had a lower concentration of n-3 HUFA in the liver than initial fish and those fed the other diets with at least 1% n-3 HUFA. Concentrations of n-3 HUFA were lower in muscle than in liver, but they were proportional to the concentrations in the diet and they declined relative to initial values in fish fed the test diets. Early studies with


rainbow trout indicated that 18:3n-3 could meet the dietary EFA requirement (Castell et al., 1972; Takeuchi and Watanabe, 1977), but the HUFA concentrations in rainbow trout decline markedly with prolonged feeding of diets devoid of HUFA (Bell and Dick, 2004). The n-3 HUFA concentration of the muscle of LMB fed the test diets without n-3 HUFA was lower than that of fish fed test diets with n-3 HUFA, even though performance was similar. The consumption of n-3 fatty acids and fish oils (high in n-3 HUFA) have numerous beneficial effects on human health (e.g., Connor, 2000; Kris-Etherton et al., 2002; Covington, 2004). Cultured fish fed diets rich in marine fish meal and oil are good sources of n-3 HUFA but diets with reduced levels of marine products are frequently cheaper, contain fewer polychlorinated biphenyls (PCBs) and dioxins (Bell et al., 2005; Berntssen et al., 2005), and are less prone to peroxidation (Baker and Davis, 1997; Refsgaard et al., 1998). Saturated fatty acids, MUFA and unsaturated fatty acids other than n-3 HUFA also affect human health in positive or negative ways, depending on the relative amounts in the diet (Simpson et al., 1991; Conner et al., 1996). The IA and IT take the interactions among different fatty acids into account, allowing an integrated assessment of dietary lipid on human coronary health (Ulbricht and Southgate, 1991). Higher values of IT and IA (N1.0) are detrimental to human health (Bobe et al., 2004). There were statistically significant differences in the IT and IA of muscle from LMB fed diets with different lipids, but the values did not exceed 0.54 and 0.68 for fish fed the test and commercial diets, respectively. Therefore, the differences in potential health impact of fillets from LMB fed diets with CAN, CHK, MFO or combinations of these appear to be minor. Similar results were obtained in brown trout fed a diet with fish oil compared to fish fed diets with canola or poultry lipids (Turchini et al., 2003). Dietary fatty acids also have direct or indirect effects on the immune system (Pablo et al., 2002), either by stimulating the production of cytokines (Endreas et al., 1989; Yaqoob and Calder, 1995) or by affecting lymphocyte proliferation (Endreas et al., 1989; Meydani et al., 1991; Secombes et al., 1994; Yaqoob et al., 1994). Leukotriene B4 (LTB4), produced from 20:4n-6, is the main agent of lymphocyte proliferation. Leukotriene B5 (LTB5), derived from 20:5n-3, has a similar but less potent physiological effect (Secombes et al., 1994). Differential cell counts in LMB revealed that the percentage of lymphocytes was highest in LMB fed diets with 10% CAN, CHK + MFO, 10% MFO, or the commercial diet and lowest in LMB fed the diet with

219

10% CHK. Fish fed diets with at least 4% n-3 fatty acids, including the CAN diet without HUFA, appeared to have a health advantage over those fed the 10% CHK diet with about 2% n-3 fatty acids because higher lymphocyte counts indicate greater immunocompetence (Clark and Lane, 1991; Pablo et al., 2002). The n-3 fatty acids have been associated with both immunosuppressive and immunostimulatory effects in fish (Lall, 2000), and the specific mechanism for the differential effect of dietary lipids on lymphocytes in LMB is unclear. The hematocrit and lysozyme of LMB fed the test diets were similar, but LMB fed the diet with CHK + MFO had higher lysozyme activity than fish fed the commercial diet. Hematocrit and lysozyme activity in gilthead sea bream fed diets with fish oil partially replaced with soybean oil, rapeseed oil, linseed oil or a mixture of these also were similar to those of fish fed a diet with only fish oil for 101 days (Montero et al., 2003). However, prolonged feeding (201 days) of the diets caused a reduction in humoral immune parameters, possibly due to altered production of complement and cortisol. Increasing dietary lipid (a mixture of fish and corn oils, 50:50 (by weight)) enhanced alternative complement and lysozyme activity in grouper (Lin and Shiau, 2003), indicating an immunostimulatory effect. Mourente et al. (2005) replaced 60% of the fish oil in diets for European seabass with rapeseed oil, linseed oil and or olive oil, and observed a reduction in circulating leucocytes and respiratory burst activity characteristic of immune suppression. The complement activity of LMB fed diets with CAN or MFO was significantly higher than that of fish fed any of the diets supplemented with CHK. Because the CAN diet was devoid of HUFA it appears that dietary HUFA were not necessary to maximize complement activity in LMB during the 12-week trial. Tissue concentrations of EFA may have been sufficient to maintain normal complement activity for 12 weeks. In addition, the reduction in complement activity of fish fed the diets with CHK might have resulted from competitive inhibition between the n-3 and n-6 fatty acids in the diet, resulting in altered eicosanoid production (Lall, 2000). However, the effect was not clearly associated with individual dietary fatty acids or with dietary ratios of n-3 to n-6 fatty acids. Alternative complement activity was reduced in gilthead sea bream fed diets deficient in n-3 HUFA (Montero et al., 1998), but replacement of fish oil with beef fat or poultry fat in diets of rainbow trout did not affect the non-specific immune response (Bureau et al., 1997). In summary, there was no conclusive evidence from this study that LMB can elongate and desaturate 18:2n-6

220


and 18:3n-3 to their respective HUFA enough to obviate the need for a dietary source for an extended period of culture. However, diets without HUFA can be used in place of traditional salmonid diets for juvenile LMB for several months without growth reduction or obvious deleterious effects. In addition, diets low in n-3 HUFA still resulted in LMB fillets with positive human-health benefits, as indicated by the IA and IT. Acknowledgments We thank Dunn's Fish Farm (Monroe, AR) for donating the largemouth bass, Omega Protein, Inc. (Hammond, LA) for donating the fish meal and oil, and ARKAT, Inc., Dumas, AR, for donating the poultry meal and oil for this project. Rebecca Jacobs assisted with fatty acid analysis. Harold Phillips, Felicia Bearden and Troy Davis assisted with proximate analysis of diets and tissues. Funding for the study was provided partly by the State of Arkansas. References Anderson, R.J., Kienholz, E.W., Flickinger, S.A., 1981. Protein requirement of smallmouth and largemouth bass. J. Nutr. 111, 1085–1097. AOAC (Association of Official Analytical Chemists), 1995. Official Methods of Analysis, 16th ed. AOAC, Arlington, VA. 1832 pp. Baker, R.T.M., Davis, S.J., 1997. Muscle and hepatic fatty acid profiles and alpha-tocopherol status of African catfish (Clarius garipinus) given diets varying in oxidative state and vitamin E inclusion level. Anim. Sci. 64, 187–195. Bell, M.V., Dick, J.R., 2004. Changes in capacity to synthesize 22:6n3 during early development in rainbow trout (Oncorhynchus mykiss). Aquaculture 235, 393–409. Bell, J.G., Tocher, D.R., MacDonald, F.M., Sargent, J.R., 1995a. Effects of borage oil (enriched in γ -linoleic acid, 18:3 (n-6) or marine oils (enriched in eicosapentanoic acid, 20:5n-3) on growth, mortalities, liver histopathology and lipid composition of juvenile turbot (Scophthalmus maximus). Fish Physiol. Biochem. 14, 373–383. Bell, J.G., Castell, J.D., Tocher, D.R., MacDonald, F.M., Sargent, J.R., 1995b. Effects of different arachidonic acid: docosahexanoic acid ratios on phospholipid fatty acid compositions and prostaglandin production in juvenile turbot (Scophthalmus maximus). Fish Physiol. Biochem. 14, 139–151. Bell, M.V., Batty, R.S., Dick, J.R., Fretwell, K., Navarro, J.C., Sargent, J.R., 1995c. Dietary deficiency of docosahexanoic acid impairs vision at low light intensities in juvenile herring (Clupea harengus L.). Lipids 30, 373–376. Bell, J.G., Tocher, D.R., Farnada, B.M., McKinney, R.W., Sargent, J.R., 1997. The effect of dietary lipids on polyunsaturated fatty acid metabolism in Atlantic salmon (Salmo salar) under parr– smolt transformation. Lipids 32, 525. Bell, J.G., McEvoy, J., Tocher, D.R., McGhee, F., Campbell, P.J., Sargent, J.R., 2001. Replacement of fish oil with rape seed oil in diets of Atlantic salmon (Salmo salar) affects tissue lipid

compositions and hepatocyte fatty acid metabolism. J. Nutr. 131, 1535–1543. Bell, J.G., Henderson, R.J., Tocher, D.R., McGhee, F., Dick, J.R., Porter, A., Smullen, R., Sargent, J.R., 2002. Substituting fish oil with crude palm oil in the diet of Atlantic salmon (Salmo salar) affects tissue fatty acid compositions and hepatic fatty acid metabolism. J. Nutr. 132, 222–230. Bell, J.G., McGhee, F., Tocher, J.R., 2005. Dioxin and dioxin-like polychlorinated biophenyls (PCBs) in Scottish farmed salmon (Salmo salar): effects of replacement of dietary marine fish oil with vegetable oils. Aquaculture 243, 305–314. Berntssen, M.G., Lundbye, A., Torstensen, B.E., 2005. Reducing levels of dioxins and dioxin-like PCB's in farmed Atlantic salmon by substituting fish oil with vegetable oil in the feed. Aquac. Nutr. 11, 219–231. Bobe, G., Zimmerman, S., Hammond, E.G., Freeman, G., Lindberg, G.L., Beitz, D.C., 2004. Texture of butters made from milks differing in indices of atherogenicity. Iowa State University Animal Industry Report 2004. A.S. Leaflet R1902. Brecka, B.J., Wahl, D.H., Hooe, M.I., 1996. Growth, survival and body composition of large mouth bass fed various commercial diets and protein concentrations. Prog. Fish-Cult. 58, 104–110. Bright, L.A., Coyle, S.D., Tidwell, J.H., 2005. Effect of dietary lipid level and protein energy ratio on growth and body composition of largemouth bass Micropterus salmoides. J. World Aquac. Soc. 36, 129–134. Bureau, D.P., Harris, A.M., Cho, C.Y., 1997. The effect of dietary lipid and long-chain n-3 PUFA levels on growth, energy utilization, carcass quality and immune function of rainbow trout (Oncorhynchus mykiss). Final Report of OMAFRA/University of Guelph Project Number 14250/95-42-12, May 1997, University of Guelph, Ontario, Canada. Buzzi, M.R., Henderson, R.J., Sargent, J.R., 1996. The desaturation and elongation of linolenic acid and eicosapentanoic acid by hepatocytes and liver microsomes from rainbow trout (Onchorhynchus mykiss) fed diets containing fish oil or olive oil. Biochim. Biophys. Acta 1299, 235–244. Buzzi, M.R., Henderson, R.J., Sargent, J.R., 1997. Biosynthesis of docosahexanoic acid in trout hepatocytes proceeds via 24 carbon intermediates. Comp. Biochem. Physiol. 116, 263–267. Caballero, M.J., Izquierdo, M.S., Kjorscik, E., Montero, D., Socorro, J., Fernandez, A.J., Rosenlund, G., 2003. Morphological aspects of intestinal cells from gilthead seabream (Sparus aurata) fed diets containing different lipid sources. Aquaculture 225, 325–340. Castell, J.D., Lee, D.J., Sinhuber, R.O., 1972. Essential fatty acids in the diet of rainbow trout (Salmo gairdineri): lipid metabolism and fatty acid composition. J. Nutr. 102, 93–100. Chen, R., Lochmann, R.T., Goodwin, A., Praveen, K., Dabrowski, K., Lee, K.J., 2003. Alternative complement activity and heat stress in golden shiners (Notemigonus crysoleucas) are increased by dietary vitamin C levels in excess of requirements for prevention of deficiency signs. J. Nutr. 133, 2281–2286. Clark, E.A., Lane, P.J.L., 1991. Regulation of human B cell activation and adhesion. Annu. Rev. Immunol. 9, 97–127. Conner, W.E., Lin, D.S., Colvis, C., 1996. Differential mobilization of fatty acids from adipose tissue. J. Lipid Res. 37, 290–298. Connor, W.E., 2000. Importance of n-3 fatty acids in health and disease. Am. J. Clin. Nutr. 71, 171S–175S. Covington, M.B., 2004. Omega-3 fatty acids. Am. Fam. Phys. 70, 133–140. Coyle, S.D., Tidwell, J.H., Webster, C.D., 2000. Response of large mouth Bass Micropterus salmoides supplementation of lysine,

B. Subhadra et al. / Aquaculture 255 (2006) 210–222 methionine and highly unsaturated fatty acids. J. World Aquac. Soc. 31, 89–95. Dosanjh, B.S., Higgs, D.A., Plotnikoff, M.D., Markert, J.R., Buckley, J.T., 1984. Efficacy of canola oil, pork lard and marine oil singly and in combination as supplemental dietary lipid sources for juvenile coho salmon (Oncorhynchus kisutch). Aquaculture 36, 333–345. Dosanjh, B.S., Higgs, D.A., Plotnikoff, M.D., McBride, J.R., Markert, J.R., Buckley, J.T., 1988. Preliminary evaluation of canola oil, pork lard and marine oil singly and in combination as supplemental dietary lipid sources for juvenile chinook salmon (Oncorhynchus tshawytscha). Aquaculture 68, 325–345. Endreas, S., Ghorbani, R., Kelley, V.E., Georgilis, K., Lonemann, G., van der Meer, J.M.V., Cannon, J.G., Rogers, T.S., Klempner, M.S., Weber, P.C., Schaeffer, E.J., Wolff, M.S., Dinarello, C.A., 1989. The effect of dietary supplementation with n-3 polyunsaturated fatty acids on the synthesis of interleukin-1 and tumor necrosis factor by mononuclear cell. N. Engl. J. Med. 320, 265–271. Folch, J., Lees, M., Sloane-Stannly, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226, 497–509. Fountoulaki, E., Alexis, M.N., Nengas, I., Venou, B., 2003. Effects of arachidonic acid (20:4n-6), on growth, body composition, and tissue fatty acid profile of gilthead bream fingerlings (Sparus aurata L.). Aquaculture 225, 309–323. Fracalossi, D.M., Lovell, R.T., 1994. Dietary lipid sources influence responses of channel catfish (Ictalurus punctatus) to challenge with the pathogen Edwardsiella ictaluri. Aquaculture 119, 287–298. Fracalossi, D.M., Craig-Schmidt, M.C., Lovell, R.T., 1995. Effect of dietary lipid sources on production of leukotriene B by head kidney of channel catfish held at different water temperatures. J. Aquat. Anim. Health 6, 242–250. Glencross, B.D., Hawkins, W.E., Curnow, J.G., 2003. Restoration of fatty acid composition of red seabream (Pagrus auratus) using fish oil finishing diets after a grow-out on plant based diets. Aquac. Nutr. 9, 409–418. Goodwin, A., Lochmann, R.T., Tieman, D.M., 2002. Massive hepatic necrosis and nodular regeneration in largemouth bass fed diets high in carbohydrate. J. World Aquac. Soc. 33, 466–477. Greene, D.H., 1990. Lipid metabolism in fish. In: Stansby, M.E. (Ed.), Fish Oils in Nutrition. Van Nostrand Reinhold, New York, NY, pp. 226–246. Greene, D.H.S., Selivonchick, D.P., 1990. Effects of dietary vegetable, animal and marine lipids on muscle lipid and hematology of rainbow trout (Oncorhynchus mykiss). Aquaculture 89, 165–182. Guillou, A., Soucy, P., Khailil, M., Adambounou, L., 1995. Effects of dietary vegetable and marine lipid on the growth and organoleptic quality of flesh of brook charr (Salvelinus fontinalis). Aquaculture 136, 351–362. Henderson, R.J., Tocher, D.R., 1987. The lipid composition and biochemistry of freshwater fish. Prog. Lipid Res. 26, 281–347. Houston, A.H., 1990. Blood and circulation. In: Schreck, C.G., Moyle, P.B. (Eds.), Methods for Fish Biology. American Fisheries Society, Bethesda, MD, pp. 273–334. Kelley, D.S., Taylor, P.C., Nelson, G.J., Schmidt, P.C., Mackey, B.E., Kyle, D., 1997. Effects of dietary arachidonic acid on human immune response. Lipids 32, 449–456. Kolkovski, S., Czesny, S., Yackey, C., Moreau, R., Cihla, F., Mahan, D., Dabrowski, K., 2000. The effect of vitamin C and E in (n-3) highly unsaturated fatty acids-enriched Artemia nauplii on growth,

221

survival and stress resistance of fresh water walleye Stizostedion vitreum larvae. Aquac. Nutr. 6, 199–206. Kramer, C.Y., 1956. Extension of multiple range tests to group with means of unequal number of replications. Biometrics 12, 307–310. Kris-Etherton, P.M., Harris, W.S., Appel, L.J., 2002. Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Circulation 106, 2747–2757. Lall, S.P., 2000. Nutrition and health of fish. In: Cruz-Suarez, L.E., Ricque-Marie, D., Tapia-Salazar, M., Olvera-Novoa, M.A., Civera-Cerecedo, R. (Eds.), Avances en Nutricion Acuicola V. Memorias del V Simposium Internacional de Nutricion Acuicola. 19–22 Noviembre, 2000, Merida, Yucatan, Mexico. Legendre, M., Kerdchuen, N., Corraze, G., Bergot, P., 1995. Larval rearing of an African catfish , Heterobranchus longifilus: effect of dietary lipids on growth, survival and fatty acid composition of fry. Aquat. Living Resour. 8, 355–363. Lin, Y.H., Shiau, S.Y., 2003. Dietary lipid requirement of grouper, Epinephelus malabaricus, and effects on immune responses. Aquaculture 225, 243–250. Liu, K.M., Barrows, F.T., Hardy, R.W., Dong, F.M., 2004. Body composition, growth performance, and product quality of rainbow trout (Onchorhynchus mykiss) fed diets containing poultry fat, soybean/corn lecithin or menhaden oil. Aquaculture 238, 309–328. Luzzana, U., Scolari, M., Campo Dall'Orto, B., Caprino, F., Turchini, G., Orban, E., Sinesio, F., Valfrè, F., 2003. Growth and product quality of European eel (Anguilla anguilla) as affected by dietary protein and lipid sources. J. Appl. Ichthyol. 19, 74–78. Madsen, L., Rustan, A.C., Vaagenes, H., Berge, K., Dyroy, E., Berge, R.K., 1999. Eicosapentanoic and docosahexanoic acid affect mitochondrial and peroxisomal fatty acid oxidation in relation to substrate preference. Lipids 34, 951–963. Meydani, S.N., Endreas, S., Woods, M.M., Goldin, B.R., Soo, C., Morrill-Labrode, A., Dinerello, C.A., Gorbach, S.L., 1991. Oral (n-3) fatty acid supplementation suppresses cytokine production and lymphocyte proliferation: comparison between young and older women. J. Nutr. 17, 138–146. Montero, D., Tort, L., Izquierdo, L., Robaina, L., Veragara, J.M., 1998. Depletion of serum alternative complement pathway activity in gilthead sea bream caused by α-tocopherol and n-3 HUFA dietary deficiencies. Fish Physiol. Biochem. 18, 399–407. Montero, D., Kalinowski, T., Obach, A., Robiana, L., Tort, L., Caballero, M.J., Izquierdo, M.S., 2003. Vegetable lipid sources for gilthead sea bream: effects on fish health. Aquaculture 225, 353–370. Morrison, W.R., Smith, L.M., 1964. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoridemethanol. J. Lipid Res. 53, 600–608. Mourente, G., Tocher, D.R., Sargent, J.R., 1991. Specific accumulation of docosahexanoic acid (22:6(n-3)) in brain lipids during development of juvenile turbot Scophthamus maximus L. Lipids 26, 871–877. Mourente, G., Good, J.E., Bell, J.G., 2005. Partial substitution of fish oil with rapeseed, linseed and olive oil for European sea bass (Dicentrarchus labrax L.): effects on flesh fatty acid composition, plasma prostaglandin E2 and F2, immune functions and effectiveness of fish oil finishing diet. Aquac. Nutr. 11, 25–40. Ng, W.K., Lim, P.K., Boey, P.L., 2003. Dietary lipid and palm oil source affects growth, fatty acid composition and muscle αtocopherol concentration in African catfish, Claris gariepinus. Aquaculture 215, 229–243.

222


Obach, A., Quental, C., Laurencin, F.B., 1993. Effects of alphatocopherol and dietary oxidized fish oil on the immune response of sea bass Dicentrarchus labrax. Dis. Aquat. Org. 15, 175–185. Ortune, J., Esteben, M.A., Meseguer, J., 2000. High dietary intake of alpha tocopherol acetate enhances the non-specific immune response of gilthead seabream (Sparus aurate). Fish Shellfish Immunol. 10, 293–307. Pablo, M.A., Puertollano, M.A., Cienfuegos, G.V., 2002. Biological and clinical significance of lipids as modulators of immune functions. Clin. Diagn. Lab. Immunol. 9, 945–950. Portz, L., Cyrino, J.E.P., Martino, R.C., 2001. Growth and body composition of juvenile largemouth bass (Micropterus salmoides) in response to dietary protein and energy levels. Aquac. Nutr. 7, 247–251. Refsgaard, H.H.F., Brockhoff, P.B., Jensen, B., 1998. Biological variation of lipid constituents and distribution of tocopherols and astaxanthin in farmed Atlantic salmon (Salmo salar). J. Agric. Food Chem. 46, 808–812. Regost, C., Arzel, J., Robin, J., Rosenlund, G., Kaushik, S.J., 2003. Total replacement of fish oil by soybean oil or oil with return to fish oil in turbot (Psetta maxima): 1. Growth performance, flesh fatty acid profile and lipid metabolism. Aquaculture 217, 465–482. Rosenlund, G., Obach, A., Sandberg, M.G., Standal, H., Tviet, K., 2001. Effect of alternative lipid sources on long term growth performance and quality of Atlantic salmon (Salmo salar). Aquac. Res. 32 (Suppl. 1), 323–328. Sargent, J.R., Henderson, R.J., Tocher, D.R., 2002. The lipids, In: Halver, J.E., Hardy, R.W. (Eds.), Fish Nutrition, 3rd edition. Academic Press, San Diego, pp. 181–257. Secombes, C.J., Clements, K., Ashton, I., Rowley, A.F., 1994. The effect of eicosanoids on rainbow trout, Oncorhynchus mykiss, leucocyte proliferation. Vet. Immunol. Immunopathol. 42, 367–378. Simopoulus, A.P., 1999. Essential fatty acids in health and chronic disease. Am. J. Nutr. 70, 560S–569S. Simpson, J.L., Mills, J.L., Rhoads, G.G., 1991. Vitamins, folic acid and neural tube defects: comments on the investigations in the United States. Prenat. Diagn. 11, 641–648. Snow, J.R., 1968. Production of six to eight inch largemouth bass for special purposes. Prog. Fish-Cult. 30, 144–152. Snow, J.R., Maxwell, J.I., 1970. Oregon moist pellet as a production ration for largemouth bass. Prog. Fish-Cult. 32, 101–102. Stickney, R.R., Andrews, J.W., 1972. Effects of dietary lipids on growth, food conversion, lipid and fatty acid composition of channel catfish. J. Nutr. 102, 249–258. Takeuchi, T., Watanabe, T., 1977. Dietary levels of methyllaurate and essential fatty acid requirement of rainbow trout. Bull. Jpn. Soc. Sci. Fish. 43, 893–898. Takeuchi, T., Watanabe, T., Ogino, C., 1978. Use of hydrogenated fish oil and beef tallow as a dietary energy source for carp and rainbow trout. Bull. Jpn. Soc. Sci. Fish. 44, 875–881. Tidwell, J.H., Allan, G.L., 2002. Fish as food: aquaculture contribution. World Aquac. 33, 44–48. Tidwell, J.H., Webster, C.D., Coyle, S.D., 1996. Effects of dietary protein level on second year growth and water quality for large mouth bass (Micropterus salmoides) raised in ponds. Aquaculture 145, 213–223. Tidwell, J.H., Coyle, S.D., Woods, T.A., 2000. Species profile: Largemouth bass. SRAC Publication, vol. 722. Southern Regional Aquaculture Center. Tocher, D.R., Bell, J.G., Dick, J.R., Sargent, J.R., 1997. Fatty acid desaturation in isolated hepatocytes from Atlantic salmon (Salmo salar): stimulation by dietary borage oil containing gammalinolenic acid. Lipids 32, 1237–1247.

Tocher, D.R., Bell, J.G., Henderson, J.R., McGhee, F., Mitchell, D., Morris, P.C., 2000. The effect of dietary linseed and rapeseed oils on polyunsaturated fatty acid metabolism in Atlantic salmon (Salmo salar) undergoing parr–smolt transformation. Fish Physiol. Biochem. 23, 59–73. Tocher, D.R., Bell, J.G., MacGlaughlin, P., McGhee, F., Dick, J.R., 2001a. Hepatocyte fatty acid desaturation and polyunsaturated fatty acid composition of liver in salmonids: effects of dietary vegetable oil. Comp. Biochem. Physiol. 130, 257–270. Tocher, D.R., Agabe, M., Hastings, N., Bell, J.G., Dick, J.R., Teale, A. J., 2001b. Nutritional regulation of hepatocyte fatty acid desaturation and polyunsaturated fatty acid composition in zebrafish (Danio rerio) and tilapia (Oreochromis nilotica). Fish Physiol. Biochem. 24, 309–320. Tocher, D.R., Fonseca-Madrigalm, J., Bell, J.G., Dick, J.R., Henderson, R.J., Sargent, J.R., 2002. Effects of diets containing linseed oil on fatty acid desaturation and oxidation in hepatocytes and intestinal enterocytes in Atlantic salmon (Salmo salar). Fish Physiol. Biochem. 26, 157–170. Torstensen, B.E., Froyland, L., Lie, O., 2004. Replacing dietary fish oil with increasing levels of rapeseed oil and olive oil-effects on Atlantic salmon (Salmo salar L) tissue composition and lipoprotein lipid composition and lipogenic enzyme activities. Aquac. Nutr. 10, 175–192. Tort, L., Gomez, E., .Montero, D., Sunyer, J.O., 1996. Serum hemolytic and agglutinating activity as indicators of fish immunocompetence: their suitability in stress and dietary studies. Aquac. Int. 4, 31–41. Tort, L., Sunyer, J.O., Gomez, E., Molinero, A., 1998. Crowding stress induced changes in serum haemolytic and agglutination activity in gilthead sea bream, Sparus aurata. Vet. Immunol. Immunopathol. 51, 179–188. Tukey, J. W., 1953. The problem of multiple comparisons. Unpublished manuscript. In The Collected Works of John W. Tukey VIII. Multiple Comparisons: 1948–1983, 1–300. Chapman and Hall, New York. Turchini, G.M., Mentasti, T., Froyland, L., Orban, E., Caprino, F., Moretti, V.M., Valfre, F., 2003. Effects of alternative lipid sources on performance, tissue chemical composition, and mitochondrial fatty acid oxidation capabilities and sensory characteristics in brown trout (Salmo trutta L). Aquaculture 225, 251–267. Ulbricht, T.L.V., Southgate, D.A.T., 1991. Coronary heart disease: seven dietary factors. Lancet 338, 985–992. Webster, C.D., Lovell, R.T., 1990. Response of striped bass larvae fed brine shrimp from different sources containing different fatty acid compositions. Aquaculture 90, 49–61. Wonnacott, E.J., Lane, R.L., Kohler, C.C., 2004. Influence of dietary replacement of menhaden oil with canola oil on fatty acid composition of sunshine bass. N. Am. J. Aquac. 66, 243–250. Xu, R., Hung, S.S.O., German, J.B., 1993. White sturgeon tissue fatty acid composition is affected by dietary lipids. J. Nutr. 123, 1685–1692. Yaqoob, P., Calder, P.C., 1995. The effects of dietary lipid manipulation on the production of murine T-cell derived cytokines. Cytokine 7, 548–247. Yaqoob, P., Newsholme, E.A., Calder, P.C., 1994. Inhibition of natural killer cell activity by dietary lipids. Immunol. Lett. 41, 241–247. Zheng, X., Tocher, D.R., Dickson, C.A., Bell, J.G., Teale, A.J., 2004. Effects of diets containing vegetable oil on expression of genes involved in highly unsaturated fatty acid biosynthesis in liver of Atlantic salmon (Salmo salar). Aquaculture 236, 467–483.