Long-term effects of high-energy, low-fishmeal feeds ...

Aquaculture 312 (2011) 109–116

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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Long-term effects of high-energy, low-fishmeal feeds on growth and flesh characteristics of Atlantic salmon (Salmo salar L.) Chris André Johnsen a,⁎, Ørjan Hagen a, Eldar Åsgard Bendiksen b a b

Faculty of Biosciences and Aquaculture, University of Nordland, N-8049 Bodø, Norway BioMar AS, Nordregt. 11. N-7484 Trondheim, Norway

a r t i c l e

i n f o

Article history: Received 21 September 2010 Received in revised form 3 December 2010 Accepted 8 December 2010 Available online 15 December 2010 Keywords: Atlantic salmon Reduced fishmeal inclusion Growth Feed utilisation Flesh quality Sensory evaluation

a b s t r a c t Long-term effects of feeding high-energy, low-fishmeal feeds on growth and flesh characteristics were investigated in ~ 1–4.5 kg Atlantic salmon (Salmo salar). Feeds were a high-fishmeal control (HFM; 20% fishmeal of total feed ingredients), medium-fishmeal (MFM; 15%), and low-fishmeal (LFM; 10%). Growth and feed utilisation were assessed regularly over a 9 month growth trial, and flesh characteristics (fillet fat, pigment concentration, visual colour) were monitored at ~ 1 kg weight intervals. These were expanded to include instrumental colour and texture analyses and sensory evaluation in harvest-size (ca. 4.5 kg) salmon. There were no significant differences between feed treatments with respect to growth, feed utilisation, mortality and flesh characteristics; confirmed by a lack of cluster formation in a multivariate principal component analysis (PCA). The most pronounced correlations were found between flesh pigment and a*-value, SalmoFan, hue and intensity, between muscle fat and pigment, and between fat and sensory attributes (odour, taste, flavour, texture and colour). This study demonstrates that dietary fishmeal levels can be substantially reduced from present commercial levels without compromising growth performance or flesh quality of harvest-size salmon. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Feed formulation for carnivorous fish species, such as Atlantic salmon (Salmo salar), has traditionally been based on fishmeals and fish oils derived from a range of small, marine fish species. Fishmeal has been the preferred protein source because of its high protein content, good amino acid profile, high nutrient digestibility, lack of antinutrients, and wide availability and relatively low price (Francis et al., 2001; Gatlin et al., 2007). According to information from feed manufacturers fishmeal comprises 20–50% of the ingredients in feeds for large salmon (Tacon and Metian, 2008). However, since 80% of the world fish stocks are reported as fully or over-exploited (FAO, 2009), further growth in aquaculture production will require increased use of alternative feed ingredients. Possible alternatives are vegetable proteins, by-products from fish and terrestrial animal processing industries, organisms from lower trophic levels and industrially produced bio-protein (bacteria, algae) (Gillund and Myhr, 2010). Successful replacement of marine protein sources with vegetable alternatives could potentially improve the sustainability, increase raw material predictability and access, and reduce the production costs in salmon production (Tacon et al., 2006; Gatlin et al., 2007; Tacon and Metian, 2008). The Atlantic salmon farming industry is regarded as a

net fish consumer (Tacon et al., 2006; Glencross et al., 2007; Tacon and Metian, 2008), but since the use of alternative vegetable protein sources is increasing the salmon farming industry could be moving towards net production of marine proteins (Torstensen et al., 2008; Crampton et al., 2010). The outcomes of studies on salmonids that have examined reduced dietary fishmeal inclusion on fish performance have been equivocal. For example, reduced feed intake and growth have been reported in some studies on Atlantic salmon and rainbow trout (Oncorhynchus mykiss) (Espe et al., 2006; Aksnes et al., 2008; Hevrøy et al., 2008; Torstensen et al., 2008; Pratoomyot et al., 2010), but a dietary inclusion of 5% fishmeal was sufficient for salmon to maintain good growth provided that dietary amino acids were balanced (Espe et al., 2007). The effect of high replacement of dietary fishmeal protein by vegetable proteins on flesh characteristics of harvest-size salmon has not received much attention. Therefore, a trial was undertaken to examine long-term effects on growth and flesh characteristics in salmon fed high-energy, low-fishmeal feeds. 2. Materials and methods 2.1. Experimental feeds

⁎ Corresponding author. Tel.: +47 755 17546; fax: +47 755 17349. E-mail address: [email protected] (C.A. Johnsen). 0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2010.12.012

Three different experimental feeds; high-fishmeal diet (HFM; 20% of the total feed ingredients), medium-fishmeal diet (MFM; 15% of the

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C.A. Johnsen et al. / Aquaculture 312 (2011) 109–116

total feed ingredients) and low-fishmeal diet (LFM; 10% of the total feed ingredients) were produced by BioMar AS Myre, Norway. Feed formulations and analysed feed compositions are given in Table 1. The HFM feed represents a fishmeal inclusion at the lower end of inclusion presently used in commercial feeds for large salmon (Tacon and Metian, 2008). Gross energy (20.5–21.0 MJ kg−1), calculated from caloric values of 39.5, 23.6 and 17.2 kJ g−1 for fat, protein and carbohydrates respectively (Blaxter, 1989), was adjusted to body size in accordance with commercial practise, and levels were kept constant between feed treatments. Across formulations fishmeals were gradually replaced with vegetable protein sources to maintain dietary protein levels, and all feeds were formulated to meet the amino acid requirements of salmon by supplementation with b2% crystalline amino acids (L-lysine, DL-methionine and L-threonine). Pigments (~40 mg kg−1), vitamins and minerals were added according to BioMar commercial standards (Table 1). Each feed was produced as extruded 9 and 12 mm pellets in 6 batches of 100– 120 tonnes each, and the feeds were stored dark in sealed 600 kg bags at ambient temperature until use. Feed dry matter contents were determined by drying at 105 °C for 24 h, crude protein contents were estimated by Kjeldahl analyses (nitrogen × 6.25, Kjeltec Autoanalyser, Tecator, Sweden), crude fat was estimated on acid hydrolysed samples (3 M HCl) using the Soxhlet method with petroleum ether extraction, and ash was determined by combustion at 550 °C for 16 h. Dietary astaxanthine content was measured in stored feed samples after termination of the trial using HPLC technique (Schierle and Härdi, 1994). All feed analyses were performed at AnalyCen AS, Moss, Norway, using standard methods. 2.2. Feeding trial and biomass estimation The feed trial was carried out from 27th October 2007 to 28th July 2008 at Gildeskål Research Station AS (GIFAS), Norway (67°N); 275 feeding days, including a six-week prelude phase of acclimatisation (27.10.07 to 04.12.07), during which all salmon were fed the HFM feed. Nine circular 60 m sea cages were used, i.e. three for each feed

treatment, each containing approximately 18,300 Atlantic salmon (initial weight 1216 ± 35 g; mean ± SD). The fish were a SalmoBreed strain produced as underyearling smolt (0+), and transferred to GIFAS in January 2007. The salmon were exposed to natural light until 20th December, and continuous light was then provided until 15th May, followed by natural light until end of the experiment (Fig. 1). Ambient seawater temperature registered at 3 m depth ranged from 2.6 °C in March to 11.9 °C in July 2008 (Fig. 1). Three cages of fish were fed each of the HFM, MFM and LFM feeds using automatic feeders (Betten T1A, Betten Maskinstasjon AS, Vågland, Norway; volume 1000 l). One daily meal was provided when water temperature was low (b5 °C), and 2–3 meals were given each day at higher temperatures, with feed provision being registered for each cage. Any dead fish were removed every second day; low mortality (0.75 ± 0.12%; mean ± SD, n = 9 cages) was registered during the experiment, with no significant differences between groups (Table 2). Fish weights and cage biomasses were estimated at regular intervals (~ each month) using the Storvik biomass estimation system (Storvik Aqua AS, Sunndalsøra, Norway); the rectangular registration frame (17.5 cm deep, 85 cm high, and 67 cm wide) was submerged at 3–12 m, and 443–1490 fish were measured in each cage over 1–2 days. The biomass estimation for each cage was used for calculations of growth and feed:gain ratio as [feed provided (kg)] × [final biomass (kg) − initial biomass (kg) + dead fish (kg)]−1. 2.3. Sampling procedure for flesh characteristics Fish samples were taken at intervals (when fish were ~1.2, 2.2, 3.2 and 4.2 kg live weight) for the evaluation of flesh characteristics. Salmon were netted at random near the surface during the morning meal. Fish that deviated substantially from cage mean weight (outliers), as indicated by the Storvik measurements, were avoided during sampling. The fish were immediately killed with a sharp blow to the head, gills cut, bled in ice water for 30 min, and transported on ice to University of Nordland where body weight (WtR; precision ±1 g), gutted body weight (WtG; ±1 g) and fork length (LF; ±0.1 cm) were recorded.

Table 1 Feed formulation and analysed composition of the experimental diets; adjusted to body size in accordance with commercial practise. Ingredients and fishmeal qualities were always kept constant across diets, with exception of fishmeal levels; high-fishmeal (HFM; 20% fishmeal of the total feed ingredients), medium-fishmeal (MFM; 15%) and low-fishmeal diet (LFM; 10%). Weight range

High fishmeal

Medium fishmeal

Low fishmeal

1.7–2.6kga

2.6–3.5 kg

3.5–4.6 kg

1.7–2.6 kg

2.6–3.5 kg

3.5–4.6 kg

1.7–2.6 kg

2.6–3.5 kg

3.5–4.6 kg

Ingredients, % Fishmealsb Soya protein concentrate Sunflower expeller Wheat gluten Peas Corn gluten Fish oilc Rapeseed oild Calcium phosphate Fat leakage stopper Vitamins and minerals Pigments and antioxidants Crystalline amino acids

21.4 10.6 12.1 3.8 18.9 3.3 10.9 17.5 0.89 0.30 0.31 0.06 0.35

19.3 9.7 14.6 4.9 18.3

19.3 8.5 17.5 2.0 18.3

14.7 16.9 12.4 3.4 18.1

14.7 17.2 12.6 2.0 18.1

9.8 25.4 8.7 2.0 18.1

14.0 19.6 0.42 0.79 0.27 0.07 0.02

14.6 18.3 0.75 0.54 0.27 0.07 0.17

17.0 16.7 0.68 0.78 0.26 0.07 0.08

10.1 23.1 6.2 5.6 18.9 3.3 10.9 18.2 1.45 0.30 0.31 0.06 1.39

9.8 25.9 7.4 3.4 18.1

15.2 17.2 0.59 0.44 0.29 0.07 0.17

16.3 19.0 7.8 3.8 18.9 3.3 10.9 17.8 1.16 0.20 0.31 0.06 0.42

16.8 16.3 1.04 0.68 0.27 0.07 0.19

18.0 16.0 0.94 0.78 0.26 0.07 0.14

Analysed composition, % Protein Fat Water Ash Astaxanthine, mg kg−1

35.0 32.4 5.0 6.0 31.3

33.1 34.2 4.8 5.9 40.5

31.9 33.3 4.4 6.2 41.0

34.9 33.1 4.4 5.5 33.0

33.7 33.9 4.6 5.6 35.5

31.8 33.3 4.9 5.6 41.0

36.0 31.0 4.4 5.5 33.7

33.5 32.5 5.6 5.6 36.0

32.8 33.4 4.8 5.7 41.0

a

Feed provided to all fish during the prelude feeding period (1.2–1.7 kg). North Atlantic LT fishmeal (71% protein) produced from herring (Clupea harengus) and South American Super Prime (68% protein) and Standard (67% protein) fishmeals produced from Anchoveta (Engraulis ringens). c Blend of North Atlantic and South American fish oils produced from herring and Anchoveta, respectively. d Double-low crude rapeseed oil derived from Brassica sp. seeds. b


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column chromatography (column; 1 g BioSil A, eluent; 15 ml 20% acetone in hexane). After evaporation, 10 ml pure hexane was added. The extract was transferred to a glass cuvette and the optical density (E) was measured at 472 nm (Shimadzu, UV 1700 spectrophotometer). Hexane was used as the blank, and the extinction coefficient (E1%,1 cm) was 1910. Pigment concentration (mg kg−1 wet weight of tissue) was estimated as [1,000,000 × E × 0.103] × [E1%,1 cm × 0.1 × (40 − 2.17 × (fat yield (g))]−1 (AquaLab AS, Bergen, Norway). 2.5. Texture and gaping

Fig. 1. Ambient seawater temperature, photoperiod and additional artificial light (20.12.2007–15.05.2008) during the experiment.

Thereafter the gutted fish were stored on ice in polystyrene boxes for four days to ensure rigor resolution and samples for flesh analyses were then taken from three locations (Fig. 2). The initial sample (15th October 2007; ~1.2 kg) comprised 20 fish which were weighed, length measured, and fillet fat, total pigment concentration and visual colour were evaluated. The same variables were analysed for fish in the second (19th February) and third (26th May) samplings, when 30 fish per treatment (10 per cage) were sampled. At the final sampling (11th July), 30 similar sized fish from each of the HFM (4131 ± 365 g; mean ± SD), MFM (4218 ± 397 g) and the LFM (4218 ± 341 g) groups were taken for analysis of fillet fat, pigment concentration, visual colour, fillet gaping, texture, and sensorial properties. Flesh colour and texture were monitored at University of Nordland, samples for fillet fat, visual colour and pigment concentration were stored at −40 °C for 14 days prior to analysis at AquaLab AS (Bergen, Norway), and samples for sensorial evaluation were stored at −40 °C for 1.5 months prior to analysis at Nofima Mat (Ås, Norway).

2.4. Fillet fat and colour Fillet fat was determined gravimetrically in the Norwegian Quality Cut (NQC) (Fig. 2) using duplicated 10 g homogenised post rigor white muscle samples extracted in ethyl acetate (NS-9402E, 1994). Flesh colour was evaluated using the Roche SalmoFan™ (Hoffmann LaRoche, Basel, Switzerland) under standardised conditions in a light cabinet (Ra N 90, colour temperature N 5000 K) according to Norwegian standards (NS-9402E, 1994). The SalmoFan evaluation was carried out on the same fillet sections used for analysis of fillet fat (Fig. 2). In addition, colour was analysed in duplicate on post rigor fillets from the left Scottish Quality Cut (SQC) (Fig. 2) with a Minolta Chromameter 300 (Minolta Camera Co. Ltd., Osaka, Japan) in the tristimulus L*a*b* measuring mode (NS-9402E, 1994). Pigment concentrations were determined on the same fillet cuts as used for the analysis of fillet fat (Fig. 2). Duplicate samples (10 g) were extracted in ethyl acetate (NS-9402E, 1994) and purified using Table 2 Growth performance, mortality and feed utilisation in the high (20%), medium (15%) and low (10%) fishmeal groups during the experiment. Results are means ± SD (n = 3); there were no significant differences between treatments for any parameter (ANOVA test).

Start weight (g) End weight (g) Mortality (%) g feed/fish Feed:gain

HFM

MFM

LFM

1182 ± 44 4659 ± 329 0.88 ± 0.22 3852 ± 394 1.11 ± 0.09

1252 ± 28 4625 ± 319 0.70 ± 0.12 3879 ± 268 1.13 ± 0.07

1214 ± 32 4592 ± 55 0.69 ± 0.01 3853 ± 202 1.14 ± 0.06

Flesh texture was determined on post rigor muscle samples according to Johnston et al. (2004), using a TA-XT2 PLUS texture analyser with Texture Expert Exceed 2.52 software (Stable Micro Systems, Surrey, England). The instrument was equipped with a 50 kg load cell, a knife edge steel blade (60°, not sharpened), and a slotted blade insert working as a measuring platform, and test speed was set at 1 mm s−1. All measurements were performed in duplicate for each fish using standardised blocks (4 cm × 4 cm × 2 cm) of white muscle from the epaxial myotomes in the left SQC (Fig. 2.). The area under the curve during shearing was calculated as the total work done (mJ) for cutting through the muscle block, transverse to the fibre direction. Gaping in the left fillet was scored subjectively using a nine point scale; 0 (no gaping), 0.5 (1–3 small breaks [b0.5 cm] in the myoseptum), 1.0 (4–6 small breaks), 1.5 (7–9 small breaks or 1–3 medium breaks [0.5–1.5 cm]), 2.0 (N10 small breaks or 4–6 medium breaks), 2.5 (7–9 medium breaks or 1–2 large breaks [N1.5 cm]), 3.0 (N10 medium breaks or 3–5 large breaks), 3.5 (N6 large breaks), and 4.0 (extreme gaping = myotomes falling apart). 2.6. Sensory evaluation Sensory evaluation of 15 post rigor AQC and NQC samples per treatment (Fig. 2.) was conducted at Nofima Mat (Ås, Norway). A trained panel of eight judges performed quantitative descriptive analysis (QDA) to profile each salmon in terms of 20 attributes relating to taste (sour, salty, metallic and bitter), odour (sea, vegetable, feed, metallic and sour), flavour (sea, vegetable, feed and rancid), colour (intensity, tone/hue, whiteness) and texture (coarseness, firmness, fatness and juiciness) (ISO-6564, 1985). The intensity of each sensory attribute was scored on a scale from 1 to 9 (1 = ‘low intensity’ and 9 = ‘high intensity’). Salmon in the HFM group was considered as the control, and the HFM feed was used as a reference for feed odour and flavour during panel testing. For analysis, samples were defrosted in a cold-room at 4 °C, 8 duplicate 1.5 cm thick samples were put in coded diffusion-tight plastic bags and vacuum sealed, and samples were then heated in a steam boiler at 80 °C for 11 min, and served to the judges in steel containers on a heated plate at 65 °C. The samples were served in a randomised order, but individual judges tasted samples from the same muscle sections across treatments. 2.7. Statistical analyses Data are presented as means ± SD (n = number of samples). Statistica v9.1 (StatSoft, Inc., Tulsa, USA) was used for data testing by analysis of variance (ANOVA), analysis of covariance (ANCOVA) and regression analysis. Unscrambler v.9.2 (Camo Process AS, Oslo, Norway) was used for principal component analysis (PCA). The effects of feed treatment on growth and feed utilisation were analysed by one-way ANOVA. The effects of feed treatments on flesh characteristics were analysed by ANCOVA and PCA. In the ANCOVA model, flesh characteristics were dependent variables, feed treatment the categorical variable and gutted fish weight the covariate. In the PCA model, full crossvalidation was used as the validation method and all dependent variables were weighted with 1/SD. Relationships between test variables were also analysed by regression analysis. Assumptions of statistical

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Fig. 2. Sample sites for the different flesh quality analyses done in the final sampling. Anterior Quality Cut (AQC), Scottish Quality Cut (SQC), and the Norwegian Quality Cut (NQC).

methods were tested prior to analysis, and in case of non-homogeneous (Bartlett's test) or non-normality (Shapiro Wilk's W test + visual evaluation of frequency histograms), data were subjected to log transformation or evaluated by a non-parametric Mann–Whitney U test. Significance was accepted at P-value of b0.05. 3. Results 3.1. Growth performance Similar amounts of feed were provided to all treatment groups (Table 2), and fish weights increased 4-fold from 1216 ± 35 g to 4625 ± 234 g (overall cage mean± SD, n = 9 cages), for an overall mean feed: gain ratio of 1.12 (±0.07) (Table 2). No significant differences in growth (Fig. 3A) or feed utilisation (Fig. 3B) were observed between treatments at any time during the experiment (Fig. 3; Table 2). This was confirmed in a PCA analysis that showed no grouping of samples; weight was most important in PC1, and feed:gain ratio most important in PC2.

significantly correlated to the hue (r 2 = 0.44; P b 0.001, n = 45) and colour intensity (r 2 = 0.65; P b 0.001, n = 45) assessments made on the cooked fish by members of the taste panel. Flesh texture (186.0 ± 31.3 mJ, overall mean± SD, n = 90) was not significantly affected by feed treatment (Table 3), and the incidence of gaping was very low across feed treatments (Table 3). PCA revealed no clustering, and the degree of overlapping in the score plot underscores the lack of significant differences between fish from different treatments with regard to flesh characteristics (Fig. 5); 59% of the variation in the PCA model is explained by PC1 (44%) and PC2 (15%), with pigment, SalmoFan, a* and b* as the most important variables in PC1, and L* as most important in PC2.

3.2. Flesh characteristics Percentage fat (Fig. 4A) and pigment concentrations (Fig. 4B) in the muscle increased gradually during the 9 months feeding trial from 12.3 ± 1.5% (overall mean ± SD) to 17.3 ± 1.5%, and from 4.30 ± 0.68 mg kg−1 to 7.31 ± 0.75 mg kg−1, respectively. There were no significant differences between treatments in either percentage fat or pigment concentrations (Fig. 4A and B), and fat and pigment concentrations were correlated positively (r2 = 0.38; P b 0.001, n = 290) across treatments and sampling times. At the final sampling, percentage fat content was negatively correlated to the sensory attributes flesh feed odour (r2 = 0.13; P = 0.015, n = 45), flavour (r2 = 0.10; P = 0.037, n = 45), bitter taste (r2 = 0.09; P = 0.042, n = 45) and hardness (r2 = 0.15; P = 0.009, n = 45), and positively correlated to the attributes flesh juiciness (r2 = 0.14; P = 0.013, n = 45) and intensity of sourness odour (r 2 = 0.14; P b 0.001, n = 45). As such, percentage fat content was identified as a flesh characteristic that had the largest impact on the sensory profile of the cooked salmon muscle. Colour score indicators (Table 3) and pigment concentrations (Fig. 4B) both tended to increase during the trial, but there were no significant differences between feed treatments with regard to the colour attributes L*a*b*-values and SalmoFan score in the harvest-size fish (Table 3). Muscle pigment concentrations were positively correlated to the Minolta a*-values (r 2 = 0.68; P = 0.012) and the SalmoFan scores (r 2 = 0.82; P b 0.001). The pigment concentrations were also

Fig. 3. Weight development (A) and feed utilisation (B) during the experiment based on a biomass estimation system.


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from feed treatments (Fig. 7). 40% of the variation in the PCA model is explained by PC1 (26%) and PC2 (14%); with sea odour and flavour, sour odour and taste, rancid and feed flavour as the most important variables in PC1, and whiteness, juiciness, colour tone and intensity as most important in PC2 (Fig. 7). Other variables had only minor effects on the overall variation in the PCA model. 4. Discussion 4.1. Growth performance

Fig. 4. Development in fillet fat (A) and total pigment concentration (B) in salmon muscle analysed in the Norwegian Quality Cut (NQC) during the experiment. The HFM, MFM and the LFM group contained 20%, 15% and 10% fishmeal of the total feed ingredients respectively. Significant difference between diets is indicated by an asterisk determined by ANOVA (P b 0.05).

Lower fishmeal inclusion (10–15%) in feeds for salmon did not have any significant impact on growth performance in the present study (Table 2), which is in agreement with other investigations (Espe et al., 2007; Torstensen et al., 2008; Crampton et al., 2010). In contrast, an adverse effect on growth of salmon provided low-fishmeal feeds was shown by others (Espe et al., 2006; Hevrøy et al., 2008) in combination with low fish oil inclusion (Torstensen et al., 2008; Pratoomyot et al., 2010). Impaired growth was mainly a consequence of reduced feed intake (e.g. Pratoomyot et al., 2010), but antinutrients (Francis et al., 2001), less hydroxyproline (Aksnes et al., 2008), reduced starch digestibility (Torstensen et al., 2008) and impaired protein utilisation (Aksnes et al., 2008; Hevrøy et al., 2008) may also contribute. Feeds with good palatability and quality of fishmeal replacers (e.g. low in antinutritional factors and sufficient amino acids) are crucial parameters for maintaining a high growth rate in salmon fed lower fishmeal diets (Espe et al., 2007). Growth was maintained in the present study and it is therefore reasonable to assume the nutritional and energetical requirement of the fish were sufficiently met. It is previously suggested that salmon need an adaptation period before being fully able to accept a plant based diet due to changed palatability (Torstensen et al., 2008; Pratoomyot et al., 2010). However, the frequent biomass estimations conducted

The taste panel did not detect any significant differences between fish from different treatments (Fig. 6). The sensory profile was characterised by high (N4) juiciness, colour tone and intensity, and whiteness, medium (~4) sea odour and flavour, metallic odour and taste, sour odour, bitter taste, coarseness and fatness, and by low (b4) intensity of feed odour and flavour, vegetable oil odour and flavour, rancid flavour, salty taste and firmness (Fig. 6). PCA revealed no clustering of sensory data which underscores the lack of influence

Table 3 Biometry and flesh quality data in Atlantic salmon at start (n = 20) and after 270 days of feeding feed with high (20%), intermediate (15%), and low (10%) fishmeal inclusion (n = 90). Round weight (WtR), gutted weight (WtG), fork length (LF), and condition factor (K-factor = 100 × [(round body mass (g)) × (fork length (cm))3]). Data are given as means ± SD. There were no significant differences between treatments for any parameter (ANCOVA test with gutted weight as covariate). Initial sampling

Final sampling HFM

MFM

LFM

Biometry WtR (g) WtG (g) LF (cm) K-factor

1234 ± 138 1094 ± 123 45.7 ± 1.7 1.29 ± 0.07

4131 ± 365 3616 ± 322 68.9 ± 2.1 1.26 ± 0.07

4218 ± 397 3669 ± 348 69.1 ± 1.8 1.27 ± 0.08

4218 ± 341 3686 ± 278 69.3 ± 1.8 1.27 ± 0.08

Flesh quality L*-value a*-value b*-value SalmoFan Texture (mJ) Gaping

41.04 ± 0.90 4.65 ± 0.99 13.70 ± 1.36 25.1 ± 0.9 – –

43.97 ± 1.97 6.80 ± 1.03 16.22 ± 1.71 26.8 ± 0.9 185.2 ± 28.9 0.03 ± 0.18

43.06 ± 1.84 6.69 ± 0.97 15.76 ± 1.41 27.0 ± 0.8 179.1 ± 30.4 0.00 ± 0.00

42.83 ± 1.50 6.64 ± 0.87 15.78 ± 0.99 27.0 ± 0.8 193.7 ± 33.7 0.00 ± 0.00

Fig. 5. PCA score plot and correlation loadings plot for the HFM (20% fishmeal of the total feed ingredients), MFM (15%) and the LFM (10%) group at the final sampling point for flesh quality characteristics.

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Fig. 6. Sensory profile of cooked flesh (80 °C, 11 min) evaluated by a trained taste panel at Nofima Mat (Ås, Norway). The HFM, MFM and LFM dietary group were formulated with 20%, 15% and 10% fishmeal respectively. Significant difference between diets is indicated by an asterisk determined by ANOVA (P b 0.05).

throughout the experiment indicated that growth of salmon was unaffected also in the first months provided low-fishmeal feeds. No significant differences in feed:gain ratio were detected between feed treatments in the present study (Table 2), in agreement with previous studies performed on Atlantic salmon (Espe et al., 2006; Torstensen et al., 2008). Similar results have also been achieved in rainbow trout provided feed with a blend of plant proteins (Valente et al., 1999) or a single soy protein concentrate (Kaushik et al., 1995) as fishmeal

replacers. In contrary adverse effects of reduced fishmeal inclusions were reported from Atlantic salmon (Sveier et al., 2001; Opstvedt et al., 2003; Aksnes et al., 2008), rainbow trout (de Francesco et al., 2004; Aksnes et al., 2008) and Atlantic cod (Gadus morhua) (Hansen et al., 2007). It is likely that differences in quality and quantity of fishmeal replacers, fishmeal qualities, fish size, environmental conditions, and duration of test period may explain discrepancies in growth performance between various fishmeal substitution studies. 4.2. Flesh characteristics

Fig. 7. PCA score plot and correlation loadings plot for the HFM (20% fishmeal of the total feed ingredients), MFM (15%) and the LFM (10%) group at the final sampling point for sensory attributes of cooked flesh, evaluated by a trained taste panel.

Reduction to 10% fishmeal inclusion in feed for ~1–4.5 kg salmon had no significant impact on flesh taste, odour, flavour, colour and texture characteristics compared to salmon provided feeds containing 15 and 20% prior to harvest (Figs. 6 and 7). The present results are in agreement with a previous study showing that Atlantic salmon provided a feed with 10% reduced fishmeal inclusion had no effect on flesh characteristics (Bjerkeng et al., 1997). Bjerkeng et al. (1997) used single full-fat soybean meal as main fishmeal replacer at lower replacement level under small-scale conditions with smaller fish (2.6 kg at harvest). Fishmeal substitution has also been tested on juvenile rainbow trout using soy flour (50%), soy protein concentrate (100%) or a blend of vitamin-free casein and soy flour (1:1, 100%) as fishmeal replacers. These authors found significant impact on flesh physical and sensory quality of the fish (Kaushik et al., 1995). In a 24 weeks feeding trial with rainbow trout morphometric traits, fat deposits, fillet chemical composition and organoleptic characteristics of rainbow trout were influenced (de Francesco et al., 2004). Fish fed plant proteins were firmer, and had less sweetness and lower odour intensity than the higher fishmeal group (de Francesco et al., 2004). In contrast, feed with soya did not influence flesh quality in rainbow trout, but the L*-value was significantly higher (~6%) when 40% of the fishmeal was replaced although the consumer's acceptability remained unaffected (D'Souza et al., 2006). Higher levels of soybean meal also resulted in a significantly slower lipid oxidation in the muscle (D'Souza et al., 2006), which might have a positive effect on shelf life and especially during long-term freeze storage. Fish weight (Torrissen and Naevdal, 1988; Shearer, 1994) and growth rate (Mørkøre and Rørvik, 2001) are known to affect flesh characteristics in salmon, but did not contribute in the present study since no differences were detected between feed treatments (Table 2). Different fishmeal replacers, substitution levels, quality of ingredients,


the use of a single plant source vs. mixture of several, in addition to fish size, species, environmental conditions and duration of experiments are factors that might influence results compared to the previous studies. The present results may be of particular high value for the salmon farming industry since the results were obtained from commercial conditions both in terms of feed used, feeding and rearing. To back up traditional variance (statistical) analysis, a multivariate principal component analysis (PCA) was performed to identify trends in the data. PCA analysis is suited for evaluation of large data sets and for detecting variables responsible for cluster formations in the score plot (Torstensen et al., 2005; Veiseth-Kent et al., 2010). In the present study, all sample points overlapped in the PCA score plot (Figs. 5 and 7) and no cluster formation or other trends were found. This supports the results of the ANCOVA analysis and it is concluded that reduced fishmeal inclusion in feeds had no negative impact on physical flesh characteristics of raw salmon muscle compared to fish fed the higher fishmeal feeds. Colour visualisation (e.g. SalmoFan) was high (~27) and all fish met the requirement of the Norwegian industry standard (NBS10-01, 1999). Espe et al. (2004) reported that fat content in salmon should be b18% to meet the requirements of a French standard (NF-V45-065, 1997), and a pigment concentration at 7 mg kg−1 or higher will in most cases meet the consumers' demand for a high quality product (Ytrestøyl, 2006). Fat content and pigment concentration in salmon muscle were analysed at regular intervals throughout the experimental period. No differences were found between feed treatments at any time (Fig. 4) which underscores the assumption that lowfishmeal feeds did not have any significant impact on fat and pigment concentration. Flesh pigment concentration and visual colour (e.g. Minolta and SalmoFan) were significantly correlated. In addition, the colour of raw flesh (e.g. pigment, Minolta and SalmoFan) were positively correlated to the colour attributes hue and colour intensity in cooked muscle, as assessed by the trained taste panel. Fillet fat was identified as the physical flesh characteristic having the largest impact on the organoleptic properties of cooked salmon muscle in the present study which is in agreement with others (Robb et al., 2002). In rainbow trout juiciness was related to fat content and water-holding capacity (Johansson et al., 1991). Feed and feeding strategies affecting fillet fat content are therefore likely to affect the consumers' perception of the edible end product. Fish muscle collagen and proteins are known to denaturate during heat treatment (Ofstad et al., 1996; Kong et al., 2007) forming new extracellular structures of coagulated proteins and gelatine (Ofstad et al., 1996) explaining the lack of relationship between raw and cooked muscle texture. However, raw and cooked muscle texture were unaffected by fishmeal substitution, and indicates that flesh characteristics analysed by instrumental (e.g. raw flesh) or sensory methods (e.g. cooked muscle) were suited for detecting treatment effects in the present experiment. Instrumental analyses are often cheaper and faster, and with a higher throughput compared to sensory methods, but results might be more valuable for consumers when analyses are performed on samples comparable to commercial products (i.e. cooked products) and by recognizable methods (i.e. sensory evaluation). As such the sensory data from present trial were particularly valuable for validation of results obtained from the instrumental analyses, and confirmed that flesh characteristics were identical across treatment groups also after heat treatment. In conclusion, the present study demonstrates that the fishmeal level in high-energy feeds for farmed Atlantic salmon can be substantially reduced without compromising growth, feed utilisation, mortality, flesh characteristics or sensory attributes of the edible product. Acknowledgements This experiment was funded by BioMar AS with support from Norwegian Research Council (project no: 180007). We would like to

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thank Gildeskål Research Station AS, and in particular Roy Arne Eilertsen for fish husbandry, and the technical staff working in the Seafood Quality Group at Faculty of Biosciences and Aquaculture at University of Nordland for skilful assistance during sampling. We also thank Prof. Malcolm Jobling for critical comments on earlier versions of the manuscript, and for help with English and structural organization of the paper.

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