Effects of dietary oil source on growth and fillet fatty acid composition of Murray cod, Maccullochella peelii peelii

Aquaculture 253 (2006) 547 – 556 www.elsevier.com/locate/aqua-online

Effects of dietary oil source on growth and fillet fatty acid composition of Murray cod, Maccullochella peelii peelii David S. Francis, Giovanni M. Turchini, Paul L. Jones, Sena S. De Silva ⁎ School of Ecology and Environment, Deakin University, P.O. Box 423, Warrnambool, Victoria 3280, Australia Received 15 June 2005; received in revised form 15 August 2005; accepted 15 August 2005

Abstract The Murray cod, an Australian native freshwater fish, supports a relatively small but increasing aquaculture industry in Australia. Presently, there are no dedicated commercial diets available for Murray cod; instead, nutritionally sub-standard feeds formulated for other species are commonly used. The aim of the present investigation was to assess the suitability of two plant based lipid sources, canola oil (CO) and linseed oil (LO), as alternatives to fish oil for juvenile Murray cod. Five iso-nitrogenous, iso-calorific, iso-lipidic semi-purified experimental diets were formulated with 17% lipid originating from 100% cod liver oil (FO), 100% canola oil, 100% linseed oil and 1 : 1 blends of canola and cod liver oil (CFO) and 1 : 1 blends of linseed and cod liver oil (LFO). Each of the diets was fed to apparent satiation twice daily to triplicate groups of 50 Murray cod with initial mean weights of 6.45 ± 1.59 g for 84 days at 22 °C. Final mean weight, specific growth rate and daily feed consumption were significantly higher for the FO and LFO treatments compared to the LO treatment. Feed conversion and protein efficiency ratios were not significantly different amongst treatments. Experimental diets containing vegetable oil and vegetable oil blend(s) had significantly higher concentrations of n-6 fatty acids, predominantly in the form of linoleic acid (LA), while n-3 fatty acids were present in significantly higher concentrations in LO and LFO treatments. The fatty acid composition of Murray cod fillet was reflective of the dietary lipid source. Fillet of fish fed the FO was highest in EPA (20:5n-3), ArA (20:4n-6) and DHA (22:6n-3). Fish fed the CO diet had high concentrations of oleic acid (OlA) (192.2 ± 10.5 mg g lipid− 1), while the fillet of Murray cod fed the LO diet was high in αlinolenic acid (LnA) (107.1 ± 6.7 mg g lipid− 1). The present study suggests that fish oil can be replaced by up to 100% with canola oil and by up to 50% with linseed oil in Murray cod diets with no significant effect on growth. © 2005 Elsevier B.V. All rights reserved. Keywords: Murray cod; Canola oil; Linseed oil; Fatty acids; Fish oil replacement

1. Introduction Fish oil replacement is not a new concept, and has been given considerable attention in recent years in the light of the perceived situation of world fisheries and aquaculture and potential limitations on fish oil supplies ⁎ Corresponding author. Tel.: +61 3 55 633 527; fax: +61 3 55 633 462. E-mail address: [email protected] (S.S. De Silva). 0044-8486/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.08.008

for aquaculture feeds (Caballero et al., 2002; Bell et al., 2003; Glencross et al., 2003; Regost et al., 2003). The steady increase of aquaculture production over the last two decades has resulted in an increased utilisation of fish meal and fish oil—the two dominant ingredients used in the production of aquaculture feeds (Tacon, 1996; New, 1999; Chamberlain and Barlow, 2000; New and Wijkstrom, 2002). It has been forecasted that by 2010 fish oil use in aquaculture will consume around 0.96 million t or around 75% of the potential supplies

548

D.S. Francis et al. / Aquaculture 253 (2006) 547–556

(Barlow, 2000). The substitution of fish oil with alternative oil sources is therefore, imperative for the successful expansion of the industry. Various alternatives have been identified and investigated over the last few years as a means of reducing this dependency on fish oil (Dosanjh et al., 1998; Martino et al., 2002; Raso and Anderson, 2003; Turchini et al., 2003a,b). Vegetable oils however, seem to be the favoured alternative because of ready availability and relative stability in price (Izquierdo et al., 2003). Providing that the essential fatty acid (EFA) requirements are met, partial substitution of fish oil with vegetable oils appears possible, particularly for freshwater fish species (Izquierdo et al., 2003; Raso and Anderson, 2003; Tocher, 2003). Notably, there is presently a lack of data on the effect of alternative lipid sources on the growth and feed efficiency of warmwater freshwater carnivorous fish species (Martino et al., 2002). The Murray cod, an iconic Australian warm freshwater fish species, supports a small but steadily growing aquaculture industry (Ingram and De Silva, 2000). The basic nutritional requirements for this species are known and a practical diet supplementing fish meal with defatted soybean meal has been formulated for this species, but as yet has not been adopted by the industry (De Silva et al., 2004). However, there is very little known on fish oil replacement in Murray cod diets (Turchini et al., 2003a), and it is the authors' belief that Murray cod would make a suitable model for vegetable oil substitution for warmwater freshwater carnivorous fish species. The aim of the present study was to evaluate the effects of partial and total fish oil replacement with canola and linseed oils, two relatively freely available, low priced oils, on growth, feed efficiency and fillet fatty acid composition of juvenile Murray cod. Furthermore, this study chose to use a semi-purified diet in order to gauge the full effect of the added oil source and therefore to minimise the effect of lipids derived from other ingredients. 2. Materials and methods Seven hundred and fifty captive bred, six-month-old Murray cod, Maccullochella peelii peelii, of the 2002 year-class were purchased from Uarah fish hatchery (Grong-Grong, N.S.W., Australia) and used in this study. Prior to the commencement of the experiment, fish were transported to Deakin University facilities and acclimatised to the new environmental conditions on a commercial barramundi diet (Ridley Agriproducts, Qld, Australia) for a two week period within a recirculating system.

2.1. Experimental diets Five iso-nitrogenous, iso-calorific, iso-lipidic semipurified experimental diets were formulated with 17% lipid originating from 100% fish oil (FO), 100% canola oil (CO), 100% linseed oil (LO) and 1 : 1 blends of canola and cod liver oil (CFO) and 1 : 1 blends of linseed and cod liver oil (LFO) (Table 1). The nutritional content of the diets were based on the results of previous findings (Gunasekera et al., 2000; Abery et al., 2002; De Silva et al., 2002). Diets were prepared and stored as reported previously (Abery et al., 2002; De Silva et al., 2002). 2.2. Husbandry This study was conducted indoors in a thermostatically controlled room. Fish were housed in a 15 tank recirculating system of 160 l capacity with an in-line oxygen generator and a physical and biological treatment plant (flow rate of 6 l min− 1). The fibreglass, circular rearing tanks had conical bottoms and were maintained at 100 l on a 12-h light : 12-h dark cycle. The experiment was conducted at 22.1 ± 0.8 °C, water quality parameters were measured every second day using Aquamerck test kits (Merck, Darmstadt, Germany) with a mean pH of 7.0 and levels of ammonia and nitrate below 0.1 mg l− 1. 2.3. Experimental design At the commencement of the experiment a sample of 15 fish was taken and euthanised in excess anaesthetic (Benzocaine 0.5 mg l− 1) for analysis. The growth experiment consisted of 750 individually weighed (to the nearest 0.1 g) and measured (to the nearest mm) juvenile Murray cod (6.45 ± 1.59 g) that were randomly distributed into 15 160-l fibreglass tanks (50 fish per tank) and randomly assigned to one of the 5 experimental treatments (3 replicate tanks for each treatment). Fish were fed twice daily at approximately 08.00 and 15.00 h to apparent satiation for a period of 84 days. At the termination of the experiment a sample of 24 fish (8 per tank) was randomly selected and euthanased for analyses. Every seventh day, faecal samples were collected during the course of the experiment. Prior to collection, tanks were siphoned to remove uneaten feed from the pm feed to avoid possible contamination. The following morning, faeces were siphoned from each tank, freeze-dried and stored in the freezer − 20 °C at until analysed.

D.S. Francis et al. / Aquaculture 253 (2006) 547–556 Table 1 Ingredient and proximate composition of the experimental diets (mg g− 1 dry diet) Dietary treatments a

b

Casein Gelatin b Dextrin b Fish meal c Defatted soybean meal c Wheat flour d Canola oil d Linseed oil e Fish oil f Mineral premix g Vitamin premix h Cr2O3 i

FO

CO

LO

CFO

LFO

320 80 100 100 105 53 – – 170 40 30 2

320 80 100 100 105 53 170 – – 40 30 2

320 80 100 100 105 53 – 170 – 40 30 2

320 80 100 100 105 53 85 – 85 40 30 2

320 80 100 100 105 53 – 85 85 40 30 2

Proximate composition mg g− 1 Moisture 94.3 94.7 98.1 94.9 97.5 Crude protein 467.2 471.6 474.0 465.3 484.5 Crude lipid 174.3 180.2 176.6 177.6 183.5 Ash 45.6 45.8 45.1 45.8 45.1 NFE j 218.7 207.7 206.2 216.3 189.4 Energy kJ/g k 21.7 21.8 21.7 21.8 21.9 a Diet abbreviations—FO: 100% fish oil; CO: 100% canola oil; LO: 100% linseed oil; CFO: 50% canola oil and 50% fish oil; LFO: 50% linseed oil and 50% fish oil. b Sigma-Aldrich, Inc., St. Louis, MO, USA. c Ridley Agriproducts, Queensland, Australia. d Bi-Lo Pty. Ltd., Tooronga, Victoria, Australia. e Nature First, Cheltenham, Victoria, Australia. f Sceney Chemicals Pty. Ltd., Sunshine, Victoria, Australia. g Contains(as g/kg): Ca (C6H10O6)·5H2O, 348.49; Ca (H2PO4)2·H2O, 136.0; FeSO4·7H2O, 5.0; MgSO4·7H2O, 132.0; K2HPO4, 240.0; NaH2PO4·H2O, 88.0; NaCl, 45.0; AlCl3·6H2O, 0.15; KI, 0.15; CuSO4·5H2O, 0.5; MnSO4·H2O, 0.7; CoCl2·6H2O, 1.0; ZnSO4·7H20, 3.0; Na2SeO3, 0.011 (Sigma-Aldrich, Inc., St. Louis, MO, USA; BDH Laboratory Supplies, Poole, England; Ajax Chemicals, Auburn, N.S. W, Australia). h Contains (as g/kg): ascorbic acid, 50.0; DL-calcium pantothenate, 5.0; choline bitartrate, 100.0; inositol, 5.0; menadione, 2.0; niacin, 5.0; pyridoxine·HCl, 1.0; riboflavin, 3.0; thiamine mononitrate, 0.5; DL-αtocopheryl acetate (250 IU/g), 8.0; vitamin A acetate (20 000 IU/g), 5.0; biotin, 0.05; cholecalciferol (1 μg = 40 IU), 0.002; folic acid, 0.18; vitamin B12, 0.002; cellulose, 815.13 (Sigma-Aldrich, Inc., St. Louis, MO, USA; BDH Laboratory Supplies, Poole, England; Ajax Chemicals, Auburn, N.S.W, Australia). i BDH Laboratory Supplies, Poole, England. j NFE: nitrogen free extract—calculated by difference. k Calculated on the basis of 23.6, 39.5 and 17.2 kJ g− 1 of protein, fat and carbohydrate, respectively.

2.4. Chemical analysis The 8 sampled fish from each of the replicates were randomly split into two groups for carcass proximate analysis (4 fish) and muscle fatty acid analysis (4 fish). Fish allocated for fillet analysis were

549

filleted (denuded of skin and bone) on both sides. The right fillet was stored at − 80 °C until analysed for fatty acids while the left hand fillet was stored at − 20 °C until used for fillet proximate analysis. Fish allocated for carcass analysis were dried (at 80 °C to constant weight), ground, homogenised and divided into three subsamples and used for proximate composition analysis. Proximate analysis was conducted using standard procedures (AOAC, 1990), percentage moisture (outlined above), protein (Kjeldahl nitrogen; N × 6.25) in an automated Kjeltech (Model 2300, Tecator, Sweden), total lipid by chloroform/methanol extraction (2 : 1 v/v) (Folch et al., 1957) as modified by Ways and Hanahan (1964) and ash by incineration in a muffle furnace (Model WIT, C & L Tetlow, Australia) at 550 °C for 18 h. Fatty acid analysis was performed in triplicate on three subsamples of each of the added dietary oils, three subsamples of the experimental diets and three pooled fillet samples from each of the replicates. After extraction, fatty acids were esterified into methyl esters using the acid catalysed methylation method (Christie, 2003), and followed the methods previously used in the laboratory (Turchini et al., 2003b; De Silva et al., 2004). Briefly, 250 μl of ethyl 13:0 (5 mg ml− 1) (Sigma-Aldrich, Inc., St. Louis, MO, USA) was added to monitor the extent of transesterification, and 800 μl of 23:0 (2.5 mg ml− 1) as an internal standard (Sigma-Aldrich, Inc., St. Louis, MO, USA). Fatty acid methyl esters were isolated and identified using a Shimadzu GC 17A (Shimadzu, Chiyoda-ku, Tokyo, Japan) equipped with an Omegawax 250 capillary column (30 m × 0.25 mm internal diameter, 25 μm film thickness, Supelco, Bellefonte, PA, USA), a flame ionisation detector (FID), a Shimadzu AOC—20i auto injector, and a split injection system (split ratio 50 : 1). The temperature program was 150 to 180 °C at 3 °C min− 1, then from 180 to 250 °C at 2.5 °C min− 1 and held at 250 °C for 10 min. The carrier gas was helium at 1.0 ml min− 1, at a constant flow. Each of the fatty acids was identified relative to known external standards. The resulting peak areas were then corrected by theoretical relative FID response factors (Ackman, 2002) and quantified relative to the internal standard. Feed and freeze-dried faecal samples were analysed for protein (Kjeldahl nitrogen; N × 6.25) and chromic oxide (Cr2O3) according to the method of Furukawa and Tsukahara (1966). Estimates of dry matter digestibility (%ADM), protein digestibility (%PD) and lipid digestibility (%LD) were calculated using standard formulae (Maynard and Loosli, 1972; Cho and Slinger, 1979).

550

D.S. Francis et al. / Aquaculture 253 (2006) 547–556

2.5. Statistical analysis All data were analysed by one-way Analysis of Variance (ANOVA) at a significance level of 0.05% using statistical packages SPSS v11.2 (SPSS Inc., Chicago, IL, USA). Assumptions of normality and homogeneity of variance were checked with box plots and residual plots. Where significant differences were detected, data was subjected to a Student–Newman– Keuls post hoc test. 3. Results 3.1. Diet composition The proximate compositions of the diets were similar across all treatments (Table 1). The fish oil diet (FO) contained the highest level of saturated fatty acids (226.3 mg g lipid− 1, 29.1%) predominantly in the form

of palmitic acid (16:0) and myristic acid (14:0) which accounted for 138.4 (17.8%) and 44.1 (5.7%) mg g lipid− 1, respectively (Table 2). Monounsaturated fatty acid concentrations were highest in the canola oil diet (CO) (594.8 mg g lipid− 1, 61.9%), represented mainly as oleic acid (18:1n-9, OlA) (541.2 mg g lipid− 1, 56.3%). The linseed oil diet (LO) was richest in polyunsaturated fatty acids (686.1 mg g lipid− 1, 72.8%) with α-linolenic acid (18:3n-3, LnA) (521.8 mg g lipid− 1, 55.3%) and linoleic acid (18:2n-6, LA) (145.9 mg g lipid− 1, 15.5%) as the principal fatty acids. The highest levels of eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) were in the FO diet, with 74.1 (9.5%) and 107.9 mg g lipid− 1 (13.9%), respectively. Fatty acids of the n-3 series were observed in highest concentration in the LO diet (538.7 mg g lipid− 1, 57.1%), whereas n-6 fatty acids were higher in the CO diet (188.2 mg g lipid− 1, 19.6%). The n-3 / n-6 ratio of

Table 2 Fatty acid composition of the oils and experimental diets, expressed in mg g lipid− 1 Dietary treatments a

Dietary oils

14:0 16:0 16:1n-7 18:0 18:1n-9 18:1n-7 18:2n-6 18:3n-6 18:3n-3 18:4n-3 20:1n-9 20:2n-6 20:3n-6 20:4n-6 20:3n-3 20:4n-3 20:5n-3 22:1 b 22:5n-3 22:6n-3 SFA MUFA PUFA HUFA n-3 HUFA n-6 HUFA n-3 / n-6 n-3 n-6 n-3 / n-6

Fish

Canola

Linseed

FO

CO

LO

CFO

LFO

49.4 147.9 67.8 36.9 163.4 31.4 36.7 – 7.6 12.0 16.7 2.7 1.6 6.6 – 10.9 75.0 8.6 34.5 108.5 243.4 291.0 297.7 228.9 9.9 23.1 248.4 49.3 5.0

– 43.4 2.6 22.2 557.7 32.2 184.2 – 74.5 – 10.2 – – – – – – 1.5 – – 75.9 604.1 259.8 – – – 75.7 184.2 0.4

– 60.9 – 23.5 136.4 6.4 162.4 – 644.2 – 0.4 – – – – – – – – – 84.4 146.1 806.7 – – – 644.2 162.4 4.0

44.1 138.4 61.4 33.6 153.1 15.4 39.5 1.0 9.1 11.4 16.8 2.6 1.6 6.4 0.6 10.2 74.1 2.8 33.6 107.9 226.3 253.4 298.4 226.4 8.4 26.8 246.9 51.5 4.8

3.7 49.4 4.9 21.5 541.2 32.6 187.6 – 75.6 1.4 5.3 – – 0.5 – – 6.4 3.2 1.4 8.5 85.0 594.8 281.4 16.3 0.5 29.6 93.3 188.2 0.5

3.0 56.7 3.1 27.8 148.3 8.0 145.9 0.5 521.8 2.1 2.1 – – 1.1 – – 5.3 0.9 1.2 8.3 91.3 165.6 686.1 14.7 1.1 13.5 538.7 147.4 3.7

22.5 91.7 31.9 27.4 356.8 32.1 116.3 0.5 42.1 6.0 13.5 1.4 – 3.3 – 4.9 38.0 2.2 16.9 55.7 152.2 439.2 285.1 115.5 3.3 35.5 163.6 121.5 1.3

21.7 92.9 29.6 28.2 137.1 17.3 86.4 – 258.3 6.7 9.1 1.7 1.3 3.8 1.1 5.3 36.7 1.6 16.0 53.3 149.1 198.0 470.6 112.3 5.2 21.8 377.3 93.3 4.0

– not detected. a See Table 1 for diet abbreviations. b 22:1 represents the sum of 22:1n-9 and 22:1n-11.

D.S. Francis et al. / Aquaculture 253 (2006) 547–556

the five experimental diets varied according to the lipid source(s) used in each of the diets and ranged gradually from 0.5 (CO) to 4.8 (FO). Levels of n-3 and n-6 highly unsaturated fatty acids (HUFA) (fatty acids with ≥ 3 ethylenic bonds with 20 or more carbon chains) were found in highest concentrations in the FO diet with 226.4 (29.1%) and 8.4 mg g lipid− 1 (1.1%), respectively. 3.2. Growth The overall mortality was low and did not appear to be related to the dietary treatment. The mean final weights of juvenile Murray cod reared on FO (30.0 ± 1.11) and LFO (28.9 ± 1.82) diets (Table 3) were significantly (P < 0.05) higher than those fish fed the LO diet (23.3 ± 0.52), while no other difference in final weight was observed between other treatments. Similar trends were evident for other growth parameters (%gain, %SGR, PGR and TGC). %SGR was highest in fish fed the FO and LFO treatments

551

(1.84 ± 0.05% and 1.79 ± 0.06% day− 1, respectively) and lowest for fish fed the LO diet (1.53 ± 0.04% day− 1). There were no significant differences between treatments for FCR or ration%. The lowest FCR however (0.71 ± 0.01), was observed for the FO treatment while ration% ranged from 1.19 ± 0.03% to 1.24 ± 0.12%. PER and %NPU in Murray cod juveniles ranged from 2.42 ± 0.10 (LO) to 2.67 ± 0.04 (FO), and 38.60 ± 1.50% (LO) to 41.80 ± 0.49% (FO), respectively and did not differ significantly between the dietary treatments. No significant differences were observed also for the biometric parameters such as the dress-out percentage (DP%), condition factor (K) and hepatosomatic index (HSI%). The deposition of fat for fish in this study (FDR and VFI%) revealed some significant differences between the dietary treatments. Fish fed LO diet had a significantly lower (P < 0.05) FDR (2.39 ± 0.04) than fish fed the FO (2.64 ± 0.05), CO (2.61 ± 0.04) and LFO (2.63 ± 0.06) diets, while the VFI % for fish fed the FO (1.05 ± 0.06%), LO (0.97 ± 0.08%)

Table 3 Mean (±SE) of growth, feed utilisation and other body parameters of Murray cod reared on the experimental diets Dietary treatments 1

Initial weight (g) Final weight (g) Gain% 2 SGR 3 (% day− 1) TGC 4 Ration% 5 FCR 6 PER 7 PGR 8 %NPU 9 FDR 10 DP% 11 K 12 HSI% 13 VFI% 14

FO

CO

LO

CFO

LFO

6.4 ± 0.05 30.0 ± 1.11b 371.4 ± 21.0b 1.84 ± 0.05b 4.27 ± 0.21b 1.23 ± 0.02 0.71 ± 0.01 2.67 ± 0.04 2.03 ± 0.05b 41.8 ± 0.49 2.64 ± 0.05b 90.3 ± 0.11 1.10 ± 0.04 1.82 ± 0.12 1.05 ± 0.06b

6.5 ± 0.07 26.9 ± 0.84ab 315.7 ± 12.9ab 1.69 ± 0.04ab 3.68 ± 0.15ab 1.23 ± 0.07 0.76 ± 0.05 2.54 ± 0.17 1.88 ± 0.04ab 39.9 ± 2.61 2.61 ± 0.04b 90.8 ± 0.59 1.12 ± 0.01 1.76 ± 0.09 0.74 ± 0.03a

6.4 ± 0.06 23.3 ± 0.52a 262.2 ± 11.4a 1.53 ± 0.04a 3.04 ± 0.10a 1.19 ± 0.03 0.80 ± 0.03 2.42 ± 0.10 1.73 ± 0.04a 38.6 ± 1.50 2.39 ± 0.04a 90.1 ± 0.41 1.08 ± 0.01 1.78 ± 0.15 0.97 ± 0.08b

6.5 ± 0.10 26.1 ± 0.78ab 304.4 ± 18.2ab 1.66 ± 0.05ab 3.54 ± 0.16ab 1.19 ± 0.03 0.75 ± 0.04 2.56 ± 0.13 1.86 ± 0.05ab 40.7 ± 1.98 2.46 ± 0.05ab 91.1 ± 0.21 1.05 ± 0.01 1.66 ± 0.09 0.67 ± 0.04a

6.4 ± 0.09 28.9 ± 1.82b 351.1 ± 22.7b 1.79 ± 0.06b 4.06 ± 0.31b 1.24 ± 0.12 0.76 ± 0.09 2.58 ± 0.30 1.95 ± 0.06b 39.0 ± 4.50 2.63 ± 0.06b 91.4 ± 0.14 1.08 ± 0.01 1.72 ± 0.10 1.03 ± 0.11b

Values in the same row with the same superscripts are not significantly different (P > 0.05). 1 See Table 1 for diet abbreviations. 2 Gain%: = (final weight − initial weight) × (initial weight)− 1 × 100. 3 Specific growth rate: SGR (%day− 1) = [Ln(final weight) − Ln(initial weight)] × (number of days)− 1 × 100. 4 Thermal growth coefficient: TGC = [(final weight)1 / 3 − (initial weight)1 / 3 / temperature degree days] × 1000. 5 Ration% = (dry food fed per day) / [(final weight + initial weight) / 2]. 6 Feed conversion ratio: FCR = (dry feed fed) × (wet weight gain)− 1. 7 Protein efficiency ratio: PER = (final weight − initial weight) × (mass of protein fed)− 1. 8 Protein growth ratio: PGR = [Ln(final protein) − Ln(initial protein)] × (number of days)− 1. 9 Net protein utilisation: %NPU = (final body protein − initial body protein) × (protein intake)− 1 × 100. 10 Fat deposition rate: FDR = [Ln(final lipid) − Ln(initial lipid)] × (number of days)− 1. 11 Dress percentage: DP% = (gutted fish weight) × (total fish weight)− 1 × 100. 12 Condition factor: K = 100 × (final weight (g) × (fork length (cm)− 3. 13 Hepatosomatic index: HSI% = (weight of liver) × (total fish weight)− 1 × 100. 14 Visceral fat index: VFI% = (visceral fat weight) × (total fish weight)− 1 × 100.

552

D.S. Francis et al. / Aquaculture 253 (2006) 547–556

Table 4 Mean percent (± SE) apparent dry matter (%ADM), protein (%PD) and lipid (%LD) of the test diets containing different oils as the lipid source Dietary treatments 1

Digestibility (%)

Dry matter (%ADM) 2 Protein (%PD) 3 Lipid (%LD) 4

FO

CO

LO

CFO

LFO

89.1 ± 0.36 97.6 ± 0.08 94.1 ± 0.19a

88.5 ± 0.21 97.7 ± 0.04 93.6 ± 0.11a

89.1 ± 0.23 97.9 ± 0.04 96.4 ± 0.08c

89.6 ± 0.39 97.8 ± 0.08 95.4 ± 0.17b

89.0 ± 0.49 97.9 ± 0.09 95.7 ± 0.23b

Values in the same row with the same superscripts are not significantly different (P > 0.05). 1 See Table 1 for diet abbreviations. 2 %ADCdm = 100 − [100 (Cr2O3 in diet) ÷ (Cr2O3 in faeces)]. 3 %ADCpd = 100 − [100 (Cr2O3 in diet) ÷ (Cr2O3 in faeces)] × [(%protein in faeces) ÷ (%protein in feed)]. 4 %ADCld = 100 − [100 (Cr2O3 in diet) ÷ (Cr2O3 in faeces)] × [(%lipid in faeces) ÷ (%lipid in feed)].

and LFO (1.03 ± 0.11%) diets were significantly higher than fish fed the CO (0.74 ± 0.03%) and CFO (0.67 ± 0.04%) diets.

significantly lower %LD than the other three experimental diets, while fish fed the LO diet had a significantly higher %LD than all other dietary treatments.

3.3. Digestibility 3.4. Carcass, fillet and liver proximate compositions The mean apparent dry matter digestibility (% ADM), protein digestibility (%PD) and lipid digestibility (%LD) for each of the experimental diets are presented in Table 4. The %ADM and %PD values of the diets were reasonably high (88.5 ± 0.2% to 89.6 ± 0.4% and 97.6 ± 0.1% to 97.9 ± 0.1%, respectively), with no statistically significant differences evident amongst the dietary treatments. Significant differences (P < 0.05) were however apparent amongst the dietary treatments for %LD (Table 4). FO and CO diets had

There were no significant differences observed between dietary treatments for moisture, protein, total lipid and/or ash content in juvenile Murray cod fed the experimental diets (Table 5). Total lipid content was low in the fillet and represented only 1.1%. The lipid content was highest in the carcass, varying from 49.4 ± 0.15 (FO) to 54.8 ± 0.2 mg g− 1 (CO), while the lipid content of the liver ranged from 18.0 ± 0.15 to 30.3 ± 0.24 mg g− 1.

Table 5 Carcass, fillet and liver proximate compositions in mg g− 1 wet weight (mean ± SE) of Murray cod reared on different diets Dietary treatments a INITIAL b

FO

CO

LO

CFO

LFO

Carcass Moisture Protein Lipid Ash

800.9 ± 2.22 128.0 ± 1.26 25.4 ± 0.57 36.7 ± 0.28

760.1 ± 1.26 150.3 ± 2.99 49.4 ± 1.54 34.0 ± 0.92

757.1 ± 1.83 150.0 ± 1.53 54.8 ± 2.44 33.5 ± 0.46

763.1 ± 2.33 151.0 ± 0.56 52.1 ± 1.26 33.1 ± 0.64

763.3 ± 0.82 151.1 ± 2.93 49.6 ± 1.41 33.5 ± 0.32

758.3 ± 0.69 145.9 ± 3.30 51.2 ± 3.78 32.0 ± 0.35

Fillet Moisture Protein Lipid Ash

835.0 ± 2.58 148.9 ± 0.14 10.9 ± 0.37 7.2 ± 0.07

788.3 ± 0.36 189.0 ± 1.29 10.7 ± 0.35 11.4 ± 0.07

790.4 ± 0.51 188.1 ± 0.24 9.7 ± 0.50 11.4 ± 0.13

787.9 ± 0.81 191.3 ± 1.19 12.1 ± 1.45 11.6 ± 0.07

790.0 ± 0.34 188.1 ± 0.55 11.5 ± 0.18 11.2 ± 0.20

785.9 ± 0.21 190.4 ± 0.56 11.2 ± 0.56 11.4 ± 0.06

Liver Moisture Protein Lipid Ash

795.5 ± 1.52 110.0 ± 2.19 75.6 ± 0.87 1.7 ± 0.03

727.4 ± 3.36 122.5 ± 8.77 18.0 ± 2.26 3.0 ± 0.38

701.4 ± 6.50 120.7 ± 1.42 30.3 ± 3.22 3.4 ± 0.65

701.1 ± 4.69 126.7 ± 1.56 27.7 ± 3.90 3.1 ± 0.39

718.4 ± 13.69 121.8 ± 7.65 26.0 ± 5.47 3.1 ± 0.41

729.7 ± 3.43 135.6 ± 1.98 19.4 ± 1.28 3.0 ± 0.29

a b

See Table 1 for diet abbreviations. Statistics not performed on the initial sample.

D.S. Francis et al. / Aquaculture 253 (2006) 547–556

553

Table 6 Muscle fatty acid composition of juvenile Murray cod reared on the different diets in mg g lipid− 1 Dietary treatments 1

14:0 16:0 16:1n-7 18:0 18:1n-9 18:1n-7 18:2n-6 18:3n-6 18:3n-3 18:4n-3 20:1n-9 20:2n-6 20:3n-6 20:4n-6 20:3n-3 20:4n-3 20:5n-3 22:1 3 22:5n-3 22:6n-3 SFA MUFA PUFA HUFA n-3 HUFA n-6 HUFA n-3 / n-6 n-3 n-6 n-3 / n-6

INITIAL 2

FO

CO

LO

CFO

LFO

9.8 ± 0.94 77.6 ± 1.93 14.6 ± 1.08 40.3 ± 1.49 39.9 ± 3.94 16.1 ± 0.64 18.4 ± 1.61 – 23.1 ± 2.32 6.6 ± 0.27 8.4 ± 0.65 3.2 ± 0.50 3.1 ± 0.42 23.2 ± 0.49 5.0 ± 1.60 6.9 ± 0.24 37.7 ± 0.72 5.0 ± 0.67 22.0 ± 0.81 126.8 ± 2.92 143.0 ± 3.44 86.3 ± 5.63 278.2 ± 7.16 198.4 ± 4.22 28.6 ± 0.63 6.9 ± 1.90 228.0 ± 4.50 50.2 ± 2.98 4.5 ± 0.08

13.1 ± 0.51b 99.3 ± 0.41b 12.4 ± 3.14b 33.5 ± 0.87a 112.3 ± 3.37b 20.2 ± 0.49cd 31.7 ± 1.09a – 8.5 ± 0.51a 4.0 ± 0.40ab 15.0 ± 0.75d 0.5 ± 0.52a 0.6 ± 0.62a 8.4 ± 0.33b – 4.4 ± 0.05a 25.4 ± 0.27c 9.5 ± 0.31b 19.7 ± 0.37 118.8 ± 1.46d 145.8 ± 1.38c 170.0 ± 6.62c 222.0 ± 3.21a 168.3 ± 1.52d 9.0 ± 0.30b 18.8 ± 0.66a 180.8 ± 1.85c 41.2 ± 1.57a 4.4 ± 0.14d

6.6 ± 1.91a 85.5 ± 1.69a 7.0 ± 0.53b 35.0 ± 2.15a 192.2 ± 10.50d 22.9 ± 1.92d 83.1 ± 3.27d 11.1 ± 3.06b 20.1 ± 3.26a 4.6 ± 0.30b 6.5 ± 0.44b 3.9 ± 0.73b 11.7 ± 0.71c 4.4 ± 0.46a 6.3 ± 3.32 6.8 ± 0.57b 10.7 ± 1.98a – 16.5 ± 1.91 63.3 ± 0.87a 127.2 ± 3.61ab 228.6 ± 11.65d 242.6 ± 9.01ab 103.6 ± 6.63a 16.1 ± 1.09c 6.4 ± 0.15a 128.3 ± 3.52a 114.3 ± 6.43c 1.1 ± 0.05a

3.8 ± 1.43a 87.5 ± 1.03a 1.9 ± 0.98a 46.0 ± 0.65b 86.8 ± 3.94a 9.2 ± 0.21a 62.4 ± 1.99c 1.9 ± 1.14a 107.1 ± 6.68c 13.2 ± 0.46d 1.7 ± 1.73a 0.7 ± 0.75a – 4.0 ± 0.84a 7.8 ± 0.35 12.9 ± 0.30c 9.9 ± 0.13a – 16.5 ± 0.30 65.2 ± 2.42a 137.3 ± 2.07bc 99.7 ± 6.41a 301.6 ± 11.41c 112.3 ± 2.35ab 4.0 ± 0.84a 31.1 ± 7.79b 232.6 ± 6.97e 69.1 ± 4.55b 3.4 ± 0.12c

7.8 ± 0.05a 78.6 ± 2.46a 10.2 ± 0.31b 31.2 ± 0.86a 140.5 ± 0.71c 18.7 ± 0.25c 51.9 ± 0.55b 2.9 ± 0.06a 15.8 ± 0.04a 3.0 ± 0.06a 10.5 ± 0.17c 2.4 ± 0.20ab 3.5 ± 0.12b 5.9 ± 0.20a 3.6 ± 1.60 3.9 ± 0.11a 16.4 ± 0.24b 3.5 ± 1.00a 15.6 ± 0.44 98.0 ± 2.27c 119.2 ± 3.67a 184.2 ± 1.46c 223.1 ± 2.16a 137.6 ± 1.61c 9.4 ± 0.32b 14.6 ± 0.39a 156.5 ± 1.60b 66.7 ± 0.89b 2.3 ± 0.03b

7.5 ± 0.79a 83.0 ± 4.05a 12.0 ± 0.45b 35.5 ± 2.90a 99.6 ± 1.19ab 13.5 ± 0.36b 46.9 ± 1.16b 2.3 ± 0.15a 70.7 ± 1.10b 7.7 ± 0.13c 9.7 ± 0.14c 1.3 ± 0.31a 1.6 ± 0.24a 5.3 ± 0.60a 6.6 ± 1.14 7.1 ± 0.20b 15.7 ± 0.98b 4.8 ± 0.70a 15.7 ± 1.05 83.4 ± 7.59b 127.9 ± 6.50ab 141.0 ± 1.99b 264.8 ± 9.99b 128.5 ± 9.02bc 7.6 ± 0.62b 17.0 ± 0.52a 206.8 ± 8.57d 58.0 ± 1.47b 3.6 ± 0.07c

Values in the same row with the same superscripts are not significantly different (P > 0.05). – not detected. 1 See Table 1 for diet abbreviations. 2 Statistics not performed on initial sample. 3 22:1 represents the sum of 22:1n-9 and 22:1n-11.

3.5. Fillet fatty acid composition Individual fatty acids found in the highest concentration across the chief fatty acid classes (SFA, MUFA and PUFA) were palmitic acid, OlA, LnA and DHA, respectively (Table 6). The level of SFA was observed in higher (P < 0.05) concentrations for fish fed the FO diets compared to fish fed CO, CFO and LFO diets. Levels of MUFA ranged from 99.7 ± 6.4 (LO; 18.5%) to 228.6 ± 11.6 (CO; 38.2%) mg g lipid− 1 and were observed to be significantly higher in fish fed the CO diet. The fillet of fish fed with the CO diet were particularly rich with OlA (192.2 ± 10.5; 32.1%) while fish fed on the LO diet had high concentrations of LnA (107.1 ± 6.7; 19.9%). EPA, arachidonic acid (20:4n-6, ArA) and DHA levels were found in higher concentrations in the

fillet than in the diets. The highest observed level of each of the aforementioned fatty acids was found in fish fed the FO diet (P < 0.05). However, DHA was found in high concentrations across all of the dietary treatments, ranging from 63.3 ± 0.9 (CO; 10.6%) to 118.8 ± 1.5 (FO; 22.1%) mg g lipid− 1. Levels of n-3 and n-6 fatty acids were higher in the fillet than the diet for each of the treatments, with n-3 / n-6 ratios ranging from 0.5 to 4.8 in the diet and 1.1 ± 0.05 to 4.4 ± 0.14 in the fillet. The highest HUFA n-3 concentrations (P < 0.05) were found in fish fed the FO diet (168.3 ± 1.52 mg g lipid− 1); while the lowest amount was observed in fish fed the CO diet (103.6 ± 6.63). HUFA n-6 was statistically higher for fish fed the CO diet, predominantly in the form of 20:3n-6 and ranged from 4.0 ± 0.84 (LO) to 16.1 ± 1.09 (CO) mg g lipid− 1.

554

D.S. Francis et al. / Aquaculture 253 (2006) 547–556

4. Discussion The results of the present study suggest that canola and linseed oils can be used to replace fish oils by up to 50% with minimal adverse effects on juvenile Murray cod growth, as reported for other fish species (Martino et al., 2002; Regost et al., 2003; Turchini et al., 2003b). This was evident by the weight gain and feed conversion of fish fed CO, CFO and LFO diets which ranged from 304.4 ± 18.2% to 351.1 ± 22.7% and 0.75 ± 0.04 to 0.76 ± 0.09, respectively, with no significant differences from fish fed the control diet and generally comparable to values reported previously for this species (Gunasekera et al., 2000; Abery et al., 2002; De Silva et al., 2002, 2004; Turchini et al., 2003a; Abery and De Silva, 2005). The 100% substitution of fish oil with linseed oil did however result in reduced weight gain (262.2 ± 11.4%) and can therefore be deemed unsuitable for complete fish oil substitution for this fish species. The apparent dry matter digestibility (%ADM), protein digestibility (%PD) and lipid digestibility (%LD) values for juvenile Murray cod were similar for other species fed semi-purified diets (Gunasekera et al., 2002). The present results indicate that the %PD in juvenile Murray cod was not influenced by the oil source, as reported for the Australian shortfin eel and Atlantic salmon (Gunasekera et al., 2002; Bendiksen et al., 2003). Values of %LD were high and differed between dietary treatments, with the lowest being observed for the CO diet (93.6 ± 0.11%) and the highest for the LO diet (96.4 ± 0.08%). Similar differences in the lipid digestibility of fish fed alternative oils have been reported previously for rainbow trout, as reported by Caballero et al. (2002) who reported a preference for diets rich in polyunsaturated fatty acids over those with higher concentrations of saturated and monounsaturated fatty acids. Unfortunately, due to a lack of faecal material, faecal fatty acid analysis was not performed and the digestion of individual fatty acids remains unknown and ultimately requires additional investigation. Considering the formulated diets were isonitrogenous, iso-calorific and iso-lipidic; varying only in lipid source, the differences in growth of juvenile Murray cod signify that the fatty acid profile of the test diets may have an influence on growth for this species. This is further supported by the relative uniformity of the proximate composition of the fish amongst the dietary treatments. In agreement with previous studies of similar nature (Caballero et al., 2002; Martino et al., 2002; Glencross et al., 2003; Turchini et al., 2003b), considerable

differences were evident in the fatty acid composition of juvenile Murray cod fed different lipid sources. There was a prominent increase in levels of LA for treatments where fish were fed either of the vegetable oils or the vegetable oil blends. Similarly, there was an increase in LnA in treatments where fish were fed linseed oil and linseed oil blends. As reported previously for this species (Turchini et al., 2003a), and for many other fish species (Guillou et al., 1995; Bell et al., 2003; Turchini et al., 2003b; Torstensen et al., 2004), high correlations for individual fatty acids as well as MUFA and PUFA were observed between the diets and fillets of juvenile Murray cod. There was, however, no correlation between the amount of SFA in the diet and SFA in the fillet, in conformity with the findings of Turchini et al. (2003a,b) who postulated that SFA are not used efficiently by Murray cod as an energy source and are subsequently deposited at an optimal level in preference to the other chief fatty acid classes. It is well known that freshwater fish have a dietary requirement for n-3 and n-6 fatty acids, predominantly in the form of LA and LnA (Kanazawa et al., 1979, 1980; Guillou et al., 1995; Martino et al., 2002; Izquierdo et al., 2003; Tocher, 2003). In comparison to marine fish species, freshwater fish are also generally better equipped to desaturate and elongate these base fatty acids to higher homologs (Guillou et al., 1995; Tocher, 2003). Despite the LO diet having the highest %LD of all the experimental diets, this study observed LnA in lower concentrations in the muscle than in the diets, it is therefore suspected that a high degree of metabolism of this fatty acid for β-oxidation and/or desaturation and elongation is taking place in juvenile Murray cod. This is further bolstered by the presence of n-3 desaturation and elongation enzyme products in the form of 18:4n-3, 20:3n-3 and 20:4n-3 in fish fed LO and LFO diets. These fatty acids were either not detected or were found in much lower concentrations in the diets and the initial fish samples. Likewise, fish fed the CO and CFO diets contained n-6 desaturation and elongation intermediates (18:3n-6 and 20:3n-6) and indicate an elongation and desaturation of LA via Δ6 desaturase. However, the further desaturation of 20:3n-6 to ArA and 20:4n-3 to EPA and ultimately DHA was shrouded by high concentrations of these fatty acids within the fillet of initial fish samples. 5. Conclusion In juvenile Murray cod, the substitution of fish oil up to a level of 50% with linseed oil and up to 100%

D.S. Francis et al. / Aquaculture 253 (2006) 547–556

with canola oil has been shown possible in this study without negative effects on growth and feed efficiency. However, the reflection of the dietary oil source on the muscle fatty acid composition of the juvenile Murray cod could be a potential drawback for vegetable oil substitution from a human nutritional point of view, given the decreases in levels of EPA and DHA in fish fed the vegetable oil diets. Further investigation into the benefits of other vegetable oils or indeed a blend of various vegetable oils is required in order to reduce usage of traditionally used fish oils, while simultaneously avoiding a reduction in the human health protective properties found within fish flesh. Acknowledgements This project was funded by the Victorian State Government, Science and Technology Innovation Initiative (STI). The authors are in debt to Bob Collins and staff of the STI for technical assistance throughout the project. References Abery, N.W., De Silva, S.S., 2005. Performance of Murray cod, Maccullochella peelii peelii (Mitchell) in response to different feeding schedules. Aquac. Res. 36, 472–478. Abery, N.W., Gunasekera, R.M., De Silva, S.S., 2002. Growth and nutrient utilization of Murray cod Maccullochella peelii peelii (Mitchell) fingerlings fed diets with varying levels of soybean meal and blood meal. Aquac. Res. 33, 279–289. Ackman, R.G., 2002. The gas chromatograph in practical analyses of common and uncommon fatty acids for the 21st century. Anal. Chim. Acta 465, 175–192. AOAC, 1990. In: Helrich, K. (Ed.), Official Methods of Analysis of the Association of Official Analytical Chemists. Association of Official Analytical Chemists, Arlington, VA, USA. Barlow, S., 2000. Fishmeal and fish oil: sustainable feed ingredients for aquafeeds. Advocate 4, 85–88. Bell, J.G., McGhee, F., Campbell, P.J., Sargent, J.R., 2003. Rapeseed oil as an alternative to marine fish oil in diets of post-smolt Atlantic salmon (Salmo salar): changes in flesh fatty acid composition and effectiveness of subsequent fish oil “wash out”. Aquaculture 218, 515–528. Bendiksen, E.A., Berg, O.K., Jobling, M., Arnesen, A.M., Masøval, K., 2003. Digestibility, growth and nutrient utilisation of Atlantic salmon parr (Salmo salar L.) in relation to temperature, feed fat content and oil source. Aquaculture 224, 283–299. Caballero, M.J., Obach, A., Rosenlund, G., Montero, D., Gisvold, M., Izquierdo, M.S., 2002. Impact of different dietary lipid sources on growth, lipid digestibility, tissue fatty acid composition and histology of rainbow trout, Oncorhynchus mykiss. Aquaculture 214, 253–271. Chamberlain, G.W., Barlow, S.M., 2000. A balanced assessment of aquaculture. Advocate 3, 7.

555

Cho, C.Y., Slinger, S.J., 1979. Apparent digestibility measurements in feedstuffs for rainbow trout. In: Halver, J.E., Tiews, K. (Eds.), Finfish Nutrition and Fish Feed Technology Proceeding of a World Symposium. Heenemann, Berlin, pp. 239–247. Christie, W.W., 2003. Lipid Analysis. Isolation, Separation, Identification and Structural Analysis of Lipids, 3rd ed. The Oily Press, PJ Barnes and Associates Bridgewater, United Kingdom, 416 pp. De Silva, S., Gunasekera, R., Collins, R., Ingram, B., 2002. Performance of juvenile Murray cod, Maccullochella peelii peelii (Mitchell), fed with diets of different protein to energy ratio. Aquac. Nutr. 8, 79–85. De Silva, S.S., Gunasekera, R.M., Ingram, B.A., 2004. Performance of intensively farmed Murray cod Maccullochella peelii peelii (Mitchell) fed newly formulated vs. currently used commercial diets, and a comparison of fillet composition of farmed and wild fish. Aquac. Res. 35, 1039–1052. Dosanjh, B.S., Higgs, D.A., McKenzie, D.J., Randall, D.K., Eales, J.G., Rowshandeli, N., Rowshandeli, M., Deacon, G., 1998. Influence of dietary blends of menhaden oil and canola oil on growth, muscle lipid composition, and thyroidal status of Atlantic salmon (Salmo salar) in sea water. Fish Physiol. Biochem. 19, 123–134. Folch, J.M., Lees, M., Sloane-Stanley, G.H., 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497–509. Furukawa, A., Tsukahara, H., 1966. On the acid digestion method for the determination of chromic oxide as an indicator substance in the study of digestibility in fish. Bull. Jpn. Soc. Sci. Fish. 32, 502–506. Glencross, B., Hawkins, W., Curnow, J., 2003. Evaluation of canola oils as alternative lipid resources in diets for juvenile red seabream, Pagrus auratus. Aquac. Nutr. 9, 305–315. Guillou, A., Soucy, P., Khalil, M., Adambounou, L., 1995. Effects of dietary vegetable and marine lipid on growth, muscle fatty acid composition and organoleptic quality of flesh of brook charr (Salvelinus fontinalis). Aquaculture 136, 351–362. Gunasekera, R.M., De Silva, S.S., Collins, R.A., Gooley, G., Ingram, B.A., 2000. Effect of dietary protein level on growth and food utilization in juvenile Murray cod Maccullochella peelii peelii (Mitchell). Aquac. Res. 31, 181–187. Gunasekera, R.M., Leelarasamee, K., De Silva, S.S., 2002. Lipid and fatty acid digestibility of three oil types in the Australian shortfin eel, Anguilla australis. Aquaculture 203, 335–347. Ingram, B., De Silva, S., 2000. Overview of the Murray cod aquaculture project. In: Ingram, B.A. (Ed.), Murray cod aquaculture: a potential industry for the new millennium. Proceedings of a workshop held 18th January 2000, Eildon, Victoria. Department of Natural Resources and Environment Marine and Freshwater Resources Institute, pp. 42–43. Izquierdo, M.S., Obach, A., Arantzamendi, L., Montero, D., Robaina, L., Roselund, G., 2003. Dietary lipid sources for seabream and seabass: growth performance, tissue composition and flesh quality. Aquac. Nutr. 9, 397–407. Kanazawa, A., Teshima, S.-I., Ono, K., 1979. Relationship between essential fatty acid requirements of aquatic animals and the capacity for bioconversion of linolenic acid to highly unsaturated fatty acids. Comp. Biochem. Physiol 63B, 295–298. Kanazawa, A., Teshima, S.I., Sakamoto, M., Awal, M., 1980. Requirements of Tilapia zillii for essential fatty acids. Bull. Jpn. Soc. Sci. Fish. 46, 1353–1356. Martino, R.C., Cyrino, J.E.P., Portz, L., Trugo, L.C., 2002. Performance and fatty acid composition of surubim (Pseudoplatystoma coruscans) fed diets with animal and plant lipids. Aquaculture 209, 233–246.

556

D.S. Francis et al. / Aquaculture 253 (2006) 547–556

Maynard, L.A., Loosli, J.K., 1972. Animal Nutrition, 6th ed. McGrawHill, New York. New, M.B., 1999. Global aquaculture: current trends and challenges for the 21st century. World Aquac. 30, 8–13. New, M.B., Wijkstrom, U.N., 2002. Use of fishmeal and fish oil in aquafeeds: further thoughts on the fishmeal trap. FAO Fisheries Circular No. 975 FIPP/C975. Raso, S., Anderson, T.A., 2003. Effect of dietary fish oil replacement on growth and carcass proximate composition of juvenile barramundi (Lates calcarifer). Aquac. Res. 34, 813–819. Regost, C., Arzel, J., Robin, J., Rosenlund, G., Kaushik, S.J., 2003. Total replacement of fish oil by soybean or linseed oil with a return to fish oil in turbot (Psetta maxima): 1. Growth performance, flesh fatty acid profile, and lipid metabolism. Aquaculture 217, 465–482. Tacon, A.G.J., 1996. Feeding tomorrow's fish. World Aquac. 27, 20–32. Tocher, D.R., 2003. Metabolism and functions of lipids and fatty acids in teleost fish. Rev. Fish. Sci. 11, 107–184.

Torstensen, L., Frøyland, 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 and lipoprotein composition and lipogenic enzyme activities. Aquac. Nutr. 10, 175–192. Turchini, G.M., Gunasekera, R.M., De Silva, S.S., 2003a. Effect of crude oil extracts from trout offal as a replacement for fish oil in the diets of the Australian native fish Murray cod Maccullochella peelii peelii. Aquac. Res. 34, 697–708. Turchini, G.M., Mentasti, T., Frøyland, L., Orban, E., Caprino, F., Moretti, V.M., Valfre, F., 2003b. Effects of alternative dietary lipid sources on performance, tissue chemical composition, mitochondrial fatty acid oxidation capabilities and sensory characteristics in brown trout (Salmo trutta L.). Aquaculture 225, 251–267. Ways, P., Hanahan, D.J., 1964. Characterization and quantification of red cell lipids in normal man. J. Lipid Res. 5, 318–328.