Microalgae and organic minerals enhance lipid retention efficiency and fillet quality in Atlantic salmon (Salmo salar L.)

Aquaculture 451 (2016) 47–57

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Microalgae and organic minerals enhance lipid retention efficiency and fillet quality in Atlantic salmon (Salmo salar L.) K. Kousoulaki a,⁎, T. Mørkøre a, I. Nengas c, R.K. Berge b, J. Sweetman c a b c

Nofima AS, Feed Technology Centre, Kjerreidviken 16, N-5141 Fyllingsdalen, Norway Department of Medicine, University of Bergen, Norway Alltech Inc., Sarney, Dunboyne, Co. Meath, Ireland

a r t i c l e

i n f o

Article history: Received 27 May 2015 Received in revised form 21 August 2015 Accepted 22 August 2015 Available online 31 August 2015 Keywords: Microalgae A. salmon Lipid retention efficiency Fillet quality Organic minerals

a b s t r a c t Pure spray dried DHA rich microalgae biomass (Schizochytrium sp.) was used to replace fish oil as a source of long chain n-3 polyunsaturated fatty acids (n-3 LC-PUFA) in medium (150 g kg−1; MF) and low fish (100 g kg−1; LF) meal diets for Atlantic salmon post smolt supplemented with either inorganic or organic minerals (OM: Zn, Cu, Mn, Fe and Se). The diets were balanced for total saturated fatty acids (SFA), sum DHA + EPA and n-3/n-6 ratio and had similar protein and energy digestibility and high pellet technical quality. Lipid digestibility was above 90% in all diets, nevertheless 2.3% lower in the diet containing 50 g kg−1 microalgae, compared to the control, mainly due to the lower digestibility of SFA in the microalgae rich diets. The experimental fish grew from 0.4 kg to 1.1 kg with no significant differences in growth rate (TGC 3.7–3.8) or feed conversion ratio (FCR 0.7) among the dietary treatments. The retention efficiency of EPA + DHA in salmon body was significantly improved in the fish fed diets containing increasing levels of microalgae, and thus lower EPA/DHA ratios. Moreover, liver lipid levels were decreased and dress-out percentage increased by increasing microalgae dietary supplementation level. The fillet levels of SFA and DHA + EPA were similar for all treatments. Improved fillet quality in terms of lower gaping scores was observed with increasing dietary inclusion level of Schizochytrium sp. and even more pronounced for salmon fed organic minerals. No significant effects on fish blood plasma chemistry and whole body mineral content were observed. Statement of relevance: Aquaculture is in urgent need of adequate volumes of new LCn-3PUFA sources, alternative to fish oil. The current work documents the feasibility of using practically relevant levels of heterotrophic microalgae as LCn-3PUFA source in the diet of Atlantic salmon in terms of extruded feed production feasibility, general fish performance, fish fillet product quality (nutritional and technical), nutrient retention efficiency and blood chemistry. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Microalgae are recognized as prominent sustainable sources of long chain n-3 polyunsaturated fatty acid (n-3 LC-PUFA) rich oils as food grade fisheries providing fish oil and fish meal have already reached their limit of sustainability (Christensen et al., 2003; Heal and Schlenker 2009; Hilborn et al., 2003; Pauly et al., 2002; Pike and Barlow, 2003). Phototrophic microalgae production is currently associated with high production costs, high technological development requirement, and limited production capacity (Jiang et al., 2004; Norsker et al., 2011). Nevertheless, efficient fermentation production technology of heterotrophic microalgae (HM) such as Schizochytrium sp. and other Thraustochytrids has been evolved during the past 3 decades (e.g. Barclay, 1994a; Bowles et al., 1999) resulting in a successful industrial sector that provides n-3 LC-PUFA rich ingredients in large scale for ⁎ Corresponding author. E-mail address: katerina.kousoulaki@nofima.no (K. Kousoulaki).

http://dx.doi.org/10.1016/j.aquaculture.2015.08.027 0044-8486/© 2015 Elsevier B.V. All rights reserved.

both food and feed products (e.g. Barclay, 1994b; Barclay and Zeller, 1996; Barclay et al., 2005). Schizochytrium sp. biomass typically contains high lipid levels (55–75% in dry matter), up to 49% of total lipids of docosahexaenoic acid (DHA) (Nakahara et al., 1996; Ren et al., 2010) and appears to be a good source of DHA for farmed fish species, such as seabream Sparus aurata (Ganuza et al., 2008) and Atlantic salmon, even at 100% replacement of the supplemental dietary fish oil (Carter et al., 2003; Kousoulaki et al., 2015; Miller et al., 2007). Heterotrophic microalgae can thus support further sustainable aquaculture growth maintaining good fish health and welfare standards and high consumer product quality in terms of n-3 LC-PUFA levels (Jones et al., 2014). In addition to DHA increase, substitution of dietary fish oil with Schizochytrium sp. oil introduces significant changes of other fatty acids (FA), including a significant reduction in eicosapentaenoic acid (EPA) and arachidonic acid (ARA). In formulating fish feeds with a given level of total n-3 LC-PUFA from Schizochytrium sp. and total lipids, even more space for inexpensive supplementary plant oils will be created, such as rapeseed oil, which may have as a consequence to further

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increase the total dietary n-6 LC-PUFA, than already practiced today. Changes in the composition of the dietary FA are expected to affect physiological processes of the fish, such as nutrient digestibility, lipogenesis, lipid deposition, storage and transport by lipoproteins, and FA uptake and metabolism in tissues (Tocher, 2003; Tocher, et al., 2003). However, no clear physiological effects were found analyzing by microarray 15,000 genes in liver samples of Atlantic salmon fed up to 150 g kg−1 microalgae, 152 g kg−1 rapeseed oil and 0 g kg−1 supplemental fish oil in the diet (Kousoulaki et al., 2015). Lipid metabolism, besides regulation by dietary FA, is also dependent on organic minerals (Lewis et al., 2013) that facilitate catalytic activity of LC-PUFA desaturases (Holloway and Wakil, 1970; Oshino et al., 1966), elongases (Nagi et al., 1989; Prasad et al., 1984) and enzymes in peroxisomal β-oxidation (Osumi and Hashimoto, 1979; Poirier et al., 2006). Sufficient intake of available minerals is therefore important to maintain good lipid metabolism and provide protection against lipid oxidation and the resulting oxidation products. Fish meal, as do the natural diets of salmon, contain minerals in organic form, while in most commercial aquatic feeds where fish meal levels are low, inorganic mineral supplements are used to cover the requirement of the fish and counteract the mineral binding effect of phytate present in the dietary plant raw materials (Denstadli et al., 2006; Persson et al., 1998). In the current trial we studied the performance of Atlantic salmon post smolts fed practical diets with medium and low levels of fish meal and commercially relevant levels of whole microalgae Schizochytrium sp. biomass as replacement of fish oil at relatively low levels of total dietary EPA + DHA. Unlike previous studies (e.g. Carter et al., 2003; Kousoulaki et al., 2015), in the current study we balanced the experimental diets for total EPA + DHA, n-3/n-6 ratio and total SFA in order to identify specific effects of the microalgae components on lipid and energy metabolism and general fish condition. Besides production performance and fillet quality, in the current study we studied dietary Schizochytrium sp. effects on extruded pellet's technical quality, and A. salmon blood chemistry and mineral content using either organic or inorganic mineral supplements, at medium or low levels of dietary fishmeal. 2. Materials and methods 2.1. Experimental diets and chemical analyses in feeds and schizochytrium sp. biomass Five commercially relevant Atlantic salmon diets were formulated (Table 2) based on the trends on average raw material levels and nutrient values used in salmon feeds by the three major salmon feed producers in Norway in 2012 and 2013 (Ytrestøyl et al., 2014). The experimental diets were formulated to contain 48.5 g kg− 1 EPA + DHA (in dietary oil) and were further balanced for crude protein, crude lipid, digestible energy, total saturated fatty acids (SFA), and n-3/ n-6 fatty acid ratio using different oil blends and plant protein mixes. Diet 1 (MFM_0_Sc) was the control diet containing medium fish meal level (MFM: 150 g kg− 1), 70 g kg−1 supplemental fish oil, and no microalgae. Diets 2 (MFM_25Sc) and diet 3 (MFM_50Sc) contained equal fish meal levels as the control but part of fish oil and plant raw materials were replaced by 25 or 50 g kg− 1 pure spray dried Schizochytrium sp. biomass (Sc) (Alltech Inc., USA), respectively. Diet 4 (LFM_50Sc) contained lower fishmeal level (LFM: 100 g kg−1) than diets 1–3 and 50 g kg−1 Sc. Diet 5 (LFM_50Sc_OM) contained 100 g kg−1 fish meal, 50 g kg−1 Sc, and was supplemented with a mineral mix (OM) containing organic Zn, Cu, Se, Mn and Fe (Alltech Inc., USA) instead of the respective standard inorganic minerals in the inorganic mineral mix (IM)used in diets 1–4 The experimental diets were produced at the Feed Technology Center of Nofima in Titlestad, Norway in the same production series using a Wenger TX-52 corotating twin-screw extruder with 150 kg h−1 capacity. The used settings of the extruder were “normal” i.e. the production can be up scaled

to a feed factory (extruder settings considered: screw configuration (D), die opening (4.5 mm), knife speed (1494–1981 rpm), SME (7.7–9.5 kW), feed rate (150 kg/h) and amount of steam (0 kg/h) and water (0.15–0.41 kg/min) added to the process). Following production of the five experimental diets their pellet technical quality characteristics were tested, as described below: Doris Durability Index (DDI) was measured on oil coated pellets in an AkvaMarina DORIS Feed Tester (Aquasmart ASA, Bryne, Norway) by putting a pre-sieved sample of 350 g pellets into the inlet of the DORIS Feed Tester, conveyed by a screw onto a rotating paddle, and collected in an accumulation box at the end. The sample was then carefully sieved on three sieves (5.60, 3.35 and 2.80 mm) to determine the amount of whole pellet (N5.60 mm), breakage (3.35–5.60 mm), and dust (b2.80 mm). The DDI is given as the percentage of pellets in each category. Each diet was analyzed in triplicate. Pellet water stability was determined by stirring the feed samples in a water bath for 120 min, then sieved, weighed, dried and weighed again (Bæverfjord et al., 2006 slightly modified). Pellet hardness was measured by a texture analyzer (TA-HDi®, Stable Micro Systems Ltd., Surrey, UK) which consists of a load arm, equipped with a cylindrical flat-ended aluminum probe (70 mm diameter). The pellets were broken individually between the probe and the bottom plate, and the major break of the pellet (the peak force) was measured and presented in Newton (N). Measurements were conducted for 20 pellets from each of the feed samples and reported as the average. Schizochytrium sp. biomass and feeds were analyzed for proximate composition: Crude protein: (Kjeldahl method N × 6.25; ISO 59831997), moisture (ISO 6496-1999), ash (ISO 5984-2002) and lipid (Bligh and Dyer, 1959). Preparation of fatty acid methyl esters (FAME) for the determination of fatty acid profile in raw materials and feeds was realized according to the AOCS Official Method Ce 1b-89 using a trace GC gas chromatograph (Thermo Fisher Scientific) with flame ionization detector (GC–FID), equipped with a 60 m × 0.25 mm BPX-70 cyan propyl column with 0.25 μm film thickness (SGE, Ringwood, Victoria, Australia). Helium 4.6 was used as mobile phase under the pressure of 2.60 bar. The injector temperature was 250 °C and the detector temperature was 260 °C. The oven was programmed as follows: 60 °C for 4 min, 30 °C min− 1 to 164 °C, and then 1.0 °C min−1 to 213 °C, and 100 °C min−1 to 250 °C where the temperature was held for 10 min. The sample solution (3.0 μl) was injected split-less and the split was opened after 2 min. The FAMEs were identified by comparing the elution pattern and relative retention time with the reference FAME mixture (GLC-793, Nu-Chek Prep Inc., Elysian, MN, USA). Chromatographic peak areas were corrected by empirical response factors calculated from the areas of the GLC-793 mixture. FA composition were calculated by using 23:0 FAME as internal standard and reported on a sample basis as g/100 g fatty acid methyl esters. Dietary gross energy was determined in a Parr adiabatic bomb calorimeter. For total amino acid profile determination, samples were hydrolyzed in 6 M HCl for 22 h at 110 °C and analyzed by HPLC using a fluorescence technique for detection (Cohen & Michaud, 1993). The total levels of tryptophan and cysteine were analyzed in the Schizochytrium sp. biomass but not in the diets. Free amino acids, taurine and anserine in Schizochytrium sp. were analyzed similarly, as described by Bidlingmeyer et al. (1987). Total and soluble phosphorus in Schizochytrium sp. was determined by a spectrophotometric method (ISO 6491-1998). The water soluble fraction of the Schizochytrium sp. was extracted with boiling water, the extract was then filtered using paper filter and the crude protein content in the water phase was determined by the Kjeldahl method. Size distribution of peptides in Schizochytrium sp. was analyzed by HPLC size exclusion chromatography using a TSK G2000 column and spectrophotometric detection at 220 nm (Aksnes and Asbjørnsen, 2003). The samples were solubilized in water containing 3 g kg−1 sodium dodecyl sulfate, centrifuged for 10 min at 10,000 rpm, decanted and filtered before applied to the column. Bovine serum albumin, pepsin, carbonic anhydrase, lysozyme, cytochrome C, blue dextran,

K. Kousoulaki et al. / Aquaculture 451 (2016) 47–57

ovalbumin, aprotinin, polymyxin, leucylglycylglycine and glycylglycine were used to define the standard curve to relate molecular weight of peptides to elution time. All analyses were performed in duplicate. If differences between parallels exceeded standardized values, new duplicate analyses were carried out according to accredited procedures. 2.2. Fish feeding trial Each one of the experimental feeds was given to triplicate population of unfed (24 h) Atlantic salmon post smolt. The start mean body weight of the experimental fish was 400 g. The feeding trial commenced in November 2013, and lasted for 12 weeks. The original stocking density in the experimental tanks (volume 1.3 m3) was ca. 10 kg m− 3. The fish were fed continuously 120% of the ad libidum levels using automatic feeders until they were bulk weighed and sampled for different tissues in the end of the trial. Uneaten feed was collected and weighed daily for the estimation of total daily feed intake of the fish populations. The mean water temperature during the trial was 8.8 °C, the water flow was continuous (30 l min−1, 180% h−1), and water salinity ranged between 32 and 33 ppt. The water system was flow-through using UV treated and filtrated sea water from 40 m depth. At trial end 35 fish from each tank were stripped and their feces separated from urine and collected in a pre-weighed box per tank. The feces of each tank were mixed with ethoxyquin and frozen immediately at −20 °C prior until further analyses.

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experimental fish was analyzed in the blood samples which were taken from 5 anesthetized fed fish (that contained feed in stomach and intestine) in the end of the experiment. Plasma lipids were measured enzymatically on a Hitachi 917 system (Roche Diagnostics GmbH, Mannheim, Germany) using the triacylglycerol (GPO-PAP) and cholesterol kit (CHOD-PAP) from Roche Diagnostics, the free fatty acid (FFA) kit from DiaSys Diagnostic Systems GmbH (Holzheim, Germany), and the phospholipid kit from bioMerieux SA (Marcy l'Etoile, France). The plasma glucose (gluco-quant -glucose/HK) level was also determined enzymatically on the Hitachi 917 system. 2.5. Statistics Data were tested for normality using a Kolomogorov–Smirnov test and homogeneity of variance using Levene's test and, where necessary, transformed via arcsine function. Regression, one-, twoway ANOVA, uni- and multi-variate analyses of data was performed using IBM SPSS statistics 21 for Windows. When significant differences among groups were identified, multiple comparisons among means were made using the Tukey post hoc test. Differences were considered significant at the level of P b 0.05 and tendencies identified at P = 0.05–0.1. 3. Results 3.1. Schizochytrium sp. biomass (Table 1)

2.3. Fish fillet and whole body composition and fillet technical quality Fish fillet and whole body nutritional quality was evaluated in terms of fatty acid and mineral composition (ICP-MS) (minerals were analyzed only in whole bodies). The technical and sensory quality of salmon fillets was evaluated in terms of liquid loss, color, muscle pH, gaping and firmness as following: The cutlet between the posterior end of the dorsal fin and the gut (Norwegian quality cut, NQC) were removed from 5 fish from each tank at start and the end of the trial, pooled, homogenized and analyzed for fatty acid composition. Lipid extraction was realized according to Bligh and Dyer (1959) and the fatty acid composition according to AOCS Ce 1b-89 as described in Section 2.1 above. Liquid retention was analyzed as the amount of liquid loss during thawing overnight at 4 °C of frozen muscle at −20 °C for one week. Fillet color was evaluated visually using DSM SalmonFan™ (score 21–34). Instrumental, colorimetric measurements were performed using a Minolta Chroma Meter CR-200 (Minolta, Osaka, Japan). Measurements were made on pooled homogenized muscle per net-pen. Means of six repeated analyses were recorded as lightness (L-value), redness (a-value) and yellowness (b-value). Muscle pH measurements were performed using a pH meter, 330i SET (Wissenschaftlich-Technische-Werkstätten GmbH, Weilheim, Germany), connected to a BlueLine 21 electrode (Schott Instruments Electrode, SI Analytics GmbH, Mainz, Germany) and TFK 325 temperature compensator (Wissenschaftlich-Technische-Werkstätten GmbH, Weilheim, Germany). Fillet gaping was analyzed as the strength of the myofiber-myocommata detachments by pressing a finger on the center of a frozen/thawed 2.5 cm thick cutlet (NQC). Occurrence of gaping was recorded when the muscle segments separated upon the applied force of 300 g for 2 s and the results are given as % cutlets with gaping. Instrumental analyses of fish fillet firmness were performed parallel to the muscle fibers using a Texture analyzer, TA-XT2 (Stable Micro system Ltd., Surrey, UK) equipped with a flat-ended cylindrical probe (12.5 mm diameter, type p/0.5) and a 30 kg load cell. Firmness predicted using this method correlates well with sensory assessment of firmness of raw and smoked salmon fillets (Mørkøre and Einen 2003). 2.4. Blood chemistry Glucose and lipid class composition (cholesterol-HDL & LDL, free fatty acids, triglycerides, phospholipids) in the blood plasma of the

The pure heterotrophic Schizochytrium sp. microalgae spray dried biomass (984 g kg−1 dry matter) used in the present study contained 132 g kg−1 crude protein, of which as much as 394 g kg−1 was water soluble, 2.1 g kg−1 total phosphorus, of which nearly all was soluble, and 614 g kg−1 crude lipid, of which 920 g kg−1 were TAG, 26 g kg−1 free fatty acids and 10 g kg−1 phospholipids. The water soluble protein of the Schizochytrium sp. biomass was composed mainly of small and very small peptides and free amino acids and other nitrogenous compounds such as creatinine, 4-amino-butanoic acid, carnosine and anserine. The content of Schizochytrium sp. protein in lysine and methionine was higher than other commonly used plant raw materials, such as bean and wheat protein concentrates, but not as high as commonly found in microbial products and marine meals (e.g. yeast extracts, fish meal, krill meal) (NRC, 2011). Palmitate (16:0) accounted about half (528 g kg−1) and DHA for 285 g kg−1 of the crude lipids in the dried algae biomass. 3.2. Pellet technical quality (Table 3) At 25 g kg−1 Schizochytrium sp. inclusion level pellet durability was improved compared to the control. However at 50 g kg−1 Schizochytrium sp. inclusion level, in all 3 diets produced, pellet durability was reduced resulting in higher levels of pellet breakage and dust formation based on the Doris test. No significant differences were measured in terms of pellet water stability or hardness among the different experimental diets. 3.3. Apparent nutrient digestibility coefficient (ADC) of dietary macronutrients, energy (Table 4) and fatty acids (Table 5) Dietary protein ADC was significantly higher in the LFM_50Sc_OM treatment when compared to the control and the MFM_50Sc treatments, whereas dietary energy ADC was significantly higher in the MFM_25Sc treatment only compared to the LFM_50Sc treatment. Reduced lipid digestibility was observed in the Schizochytrium sp. supplemented diets except for treatment MFM_25Sc compared to the control. Both control and the Schizochytrium sp. biomass supplemented diets had similar DHA and EPA ADC of 98% and 99%, respectively. The ADC of 14:0, 16:0 and 18:0 was significantly reduced in the

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Table 1 Schizochytrium sp. spray dried biomass chemical composition.

Table 1 (continued)

Chemical composition (as is)

Analyzed level

Crude protein (%) Moisture (%) Ash (%) fat (Bligh & Dyer) (%) Total phosphorus (%) Soluble phosphorus (%) Water soluble protein (% protein) Carbohydrates (%) (calculated) Raw starch (%) Iodine value (in Bl&D extract)

13.2 1.6 4.4 61.4 0.21 0.20 39.4 19.4 1.20 164

Fatty acid and lipid classes composition 14:0 (% oil) 15:0 (% oil) 16:0 (% oil) 17:0 (% oil) 18:0 (% oil) 20:0 (% oil) 22:0 (% oil) 14:1 n-3 (% in oil) 16:1 n-5 (% oil) 16:1 n-7 (% oil) 16:1 n-7 (% oil) 18:1 (n-11) + (n-9) + (n-7) + (n-5) (% oil) 20:1 (n-11) + (n-9) + (n-7) (% oil) 22:1 (n-11) + (n-9) + (n-7) (% oil) 24:1 n-9 (% oil) 16:2 n-4 (% oil) 16:3 n-4 (% oil) 18:2 n-6 (% oil) 18:3 n-6 (% oil) 20:2 n-6 (% oil) 20:3 n-6 (% oil) 20:4 n-6 (% oil) 22:4 n-6 (% oil) 22:5 n-6 (% oil) 18:3 n-3 (% oil) 18:4 n-3 (% oil) 20:3 n-3 (% oil) 20:4 n-3 (% oil) 20:5 n-3 (% oil) 21:5 n-3 (% oil) 22:5 n-3 (% oil) 22:6 n-3 (% oil) Saturated fatty acids (% oil) Monounsaturated fatty acids (% oil) Total n-6 PUFA (% oil) Total n-3 PUFA (% oil) Total PUFA (% oil) Total fatty acids (% oil) Triacylglycerol (% B&D extract) Diacylglycerol (% B&D extract) Monoacylglycerol (% B&D extract) Free fatty acids (% B&D extract) Sterols (% B&D extract) Sterol esters (% B&D extract) Phosphatidylethanolamine (% B&D extract) Phosphatidylinositol (% B&D extract) Phosphatidylserine (% B&D extract) Phosphatidylcholine (% B&D extract) Lyso-phosphatidylcholine (% B&D extract) Total polar lipids (% B&D extract) Total neutral lipids (% B&D extract) Total lipids (% B&D extract)

4.1 2.0 52.8 0.7 1.5 0.2 0.1 0.1 b0.1 0.1 b0.1 0.1 b0.1 0.4 0.2 b0.1 b0.1 0.1 b0.1 b0.1 0.1 0.3 b0.1 6.9 b0.1 b0.1 0.1 0.1 0.3 b0.1 b0.1 28.5 61.4 0.8 7.4 29 36.4 98.6 92 b0.5 b1 2.6 1.2 b0.5 b0.5 b1 b1 b1 b0.5 1 95.5 96.5

Total amino acid composition (g kg−1 protein) Asparaginic acid Glutaminic acid Hydroxyproline Serine Glycine Histidine Arginine Threonine Alanine

70 99 b8 44 38 17 42 32 44

Chemical composition (as is)

Analyzed level

Proline Tyrosine Valine Methionine Isoleucine Leucine Phenylalanine Lysine Tryptophane Cystine Total amino acids

26 23 42 17 34 54 33 63 13 18 709

Free amino acid and other nitrogenous compound composition (% of sample) Creatinine 0.095 Asparaginic acid 0.017 Glutaminic acid 0.056 Hydroxyproline 0.001 Serine 0.071 Asparagine 0.049 Glycine 0.034 Glutamine 0.11 3-Amino-propionic acid b0.001 Taurine 0.018 Histidine 0.013 4-Amino-butanoic acid 0.059 Citrulline b0.001 Threonine 0.047 Alanine 0.084 Carnosine 0.016 Arginine 0.067 Proline 0.041 Anserine b0.001 Tyrosine 0.079 Valine 0.097 Methionine 0.047 Cystine b0.001 Isoleucine 0.074 Leucine 0.157 Phenylalanine 0.088 Tryptophane 0.027 Ornithine 0.002 Lysine 0.054 Total free amino acids 1.403 Soluble peptide molecular weight distribution (% of water soluble peptides) MW-peptide N20,000 (Da) MW-peptide 20,000–15,000 (Da) MW-peptide 15,000–10,000 (Da) MW-peptide 10,000–8000 (Da) MW-peptide 8000–6000 (Da) MW-peptide 6000–4000 (Da) MW-peptide 4000–2000 (Da) MW-peptide 2000–1000 (Da) MW-peptide 1000–500 (Da) MW-peptide 500–200 (Da)

0.4 b0.1 0.1 0.3 1 2.8 5.9 7.9 10.5 15.5 55.7

Schizochytrium sp. supplemented diets compared to the control. The low fish meal diet supplemented with inorganic minerals (LFM_50Sc) had lower 18:0 ADC compared to its medium fish meal control diet (MFM_50Sc). Substitution of the inorganic minerals with organic minerals in diet LFM_50Sc_OM led to small but significantly improved ADC of the total dietary fatty acids (FA) and total PUFA, and more specifically of n-6 PUFA, 22:1, 14:0 and 18:0 compared with the LFM_50Sc. 3.4. Fish performance, biometrics and liver lipid (Table 6) Fish growth rates were high and similar for all treatments (TGC of 3.7–3.8) and FCR was low (0.7 in dry matter) and did not differ among the dietary treatments. There was a tendency (P b 0.1) for lower feed intake and lower FCR in the treatments with low fish meal inclusion level. A significant positive correlation was observed

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Table 2 Formulation and chemical composition (g kg−1) of experimental diets. Diet name Fish meal Super Prime Schizochytrium sp.1 Organic minerals mix1 Inorganic mineral mix2 Herring oil Palm oil Camelina oil Rapeseed oil Horse beans Wheat Corn gluten Wheat gluten Soy protein concentrate Soya lecithin vitamin mix2 NaH2PO4 Betafin Inositol Carop. Pink (10%) Lys (99%, 19.41% Cl) Methionine 99% Threonine 98.5% Yttrium oxide Sum

MFM_0_Sc 150

5.2 66.5 24.8 167.3 100 46 35 124.7 210 5 20 24.5 5 0.3 0.5 11.7 2.3 0.7 0.5 1000

MFM_25Sc

MFM_50Sc

LFM_50Sc

150 25

150 50

100 50

5.2 38.4 14.4 7 183.9 100 44.8 35 135 190.8 5 20 24.5 5 0.3 0.5 11.7 2.3 0.7 0.5 1000

5.2 10.1 4.1 14 200.5 100 44 35 145.7 170.9 5 20 24.5 5 0.3 0.5 11.7 2.3 0.7 0.5 1000

5.2 17.2 2.0 13 194.3 100 44 35 169 191.5 10 20 26.0 5 0.3 0.5 12 3 1.5 0.5 1000

LFM_50Sc_OM 100 50 6.86 17.2 2.0 13 194.3 100 42.34 35 169 191.5 10 20 26.0 5 0.3 0.5 12 3 1.5 0.5 1000

Dietary fatty acid composition (% in B&D extract) 14:0 16:0 18:0 20:0 22:0 16:1 n-7 18:1 (n-9) + (n-7) + (n-5) 20:1 (n-9) + (n-7) 22:1 (n-11) + (n-9) + (n-7) 24:1 n-9 16:2 n-4 18:2 n-6 20:2 n-6 18:3 n-3 18:4 n-3 20:4 n-3 20:5 n-3 21:5 n-3 22:5 n-3 22:6 n-3 Saturated fatty acids Monounsaturated fatty acids Total n-6 PUFA Total n-3 PUFA Total PUFA Total fatty acids EPA (% feed) DHA (% feed) EPA + DHA (% feed) DHA/EPA ω3/ω6

2 10.9 1.9 0.3 0.2 1.6 43.7 4.2 4.9 0.2 0.1 15.8 0.1 6 0.8 0.1 2.1 0.1 0.1 2.8 15.3 54.6 15.9 12 28 97.9 0.6 0.8 1.4 1.3 0.8

1.4 11.2 1.8 0.4 0.2 1.1 45.6 3.1 2.8 0.2 0.1 17.5 0.1 7.3 0.5 0.1 1.4 b0.1 0.1 3.3 15 52.8 17.6 12.7 30.4 98.2 0.4 0.9 1.35 2.4 0.7

1 12.2 1.7 0.4 0.2 0.6 46.5 2.3 1.4 0.1 b0.1 18.7 0.1 8.3 0.2 0.1 0.7 b0.1 b0.1 4 15.5 50.9 18.8 13.3 32.1 98.5 0.2 1.2 1.4 5.7 0.7

1.1 12.6 1.6 0.4 0.2 0.6 43.1 2.3 1.4 0.1 b0.1 18.9 0.1 7.8 0.2 0.1 0.8 b0.1 b0.1 4.3 15.9 47.5 19 13.2 32.2 95.6 0.2 1.2 1.4 5.4 0.7

1.1 12.3 1.6 0.4 0.2 0.6 43.3 2.3 1.4 0.1 b0.1 18.9 0.1 7.9 0.2 b0.1 0.7 b0.1 b0.1 4.2 15.6 47.7 19 13 32 95.3 0.2 1.2 1.4 6.0 0.7

Chemical composition in dry matter (DM) Crude protein (%) Ash (%) Fat (Bligh & Dyer) (%) Crude energy (Mj/kg)

44.6 6.6 31.7 25.5

44.9 6.7 31.4 25.4

44.3 6.8 31.1 25.4

45.1 6.5 29.3 25.1

44.7 6.4 30.9 25.4

1 Alltech Inc., Kentucky, USA. The mineral mix contains the following products (mineral level in the product): a) Bioplex Zn (15%); Bioplex Cu (10%); Sel-Plex 2300 (0.29% Se); Bioplex Mn (15%); Bioplex Fe (15%), as well as inorganic Mg and K at similar levels to those present in the inorganic mineral mix. 2 Supplied by Norsk Mineralnæring, Norway and mixed at Nofima AS providing in the final diet 3000 IU Vit D3,160 mg kg−1 Vit E, 20 mg kg−1 Vit K3, 200 mg kg−1 Vit C, 20 mg kg−1 Vit B1, 30 mg kg−1 Vit B2, 25 mg kg−1 Vit B6, 0.05 mg kg−1 Vit B12, 60 mg kg−1 Vit B5, 10 mg kg−1 folic acid, 200 mg kg−1 Vit B3, 1 mg kg−1 Vit B7, 92.31 mg kg−1 MnSO4 + H20, 3125 mg kg−1 MgHPO4 + 3H2O, 182.4 mg kg−1 FeSO4 + H20, 352.9 mg kg−1 ZnSO4 + H20, 23.62 mg kg−1 CuSO4 + H20, 1413 mg kg−1 K2CO3, and 6.67 mg kg−1 Se.

between feed intake and TGC (n = 15; R2 = 0.732; P b 0.01) and also between feed intake and FCR (n = 15; R2 = 0.685; P b 0.05). The CF and HSI showed no significant difference among the dietary

treatments, but salmon fed 50 gkg − 1 microalgae had significantly higher dress-out percentage and the lipid levels in the liver tended to be lower (P b 0.1).

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K. Kousoulaki et al. / Aquaculture 451 (2016) 47–57

Table 3 Experimental diet pellet technical quality (the values are given as mean ± standard deviation; hardness: n = 20, durability; water stability: n = 3).

Durability Doris (DDI)

Whole pellet (%) Broken pellet (%) Dust (%)

Water stability (%) Hardness (N)

MFM_0_Sc

MFM_25Sc

MFM_50Sc

LFM_50Sc

LFM_50Sc_OM

ANOVA (P⁎)

92.5 ± 0.5c 6.7 ± 0.3b 0.8 ± 0.1b 86.5 ± 2.1 68.5 ± 9.4

96.1 ± 0.2d 3.4 ± 0.2a 0.5 ± 0.01a 88.3 ± 1.2 63.0 ± 11.2

82.3 ± 0.6a 16.5 ± 06d 1.1 ± 0.1c 86.2 ± 3.4 67.1 ± 8.4

89.7 ± 1.2b 9.0 ± 1.1c 1.3 ± 0.1d 85.4 ± 5.1 61.2 ± 10.1

88.4 ± 0.3b 10.0 ± 0.2c 1.6 ± 0.1e 88.5 ± 2.7 64.3 ± 12.2

b0.001 b0.001 b0.001 ns⁎⁎ ns

⁎ Values not sharing common superscript letters are significantly different (P ≤ 0.05) as determined by ANOVA followed by Tukey post hoc test. ⁎⁎ Non-significant.

3.5. NQC fillet (Table 7) and whole body (Table 8) fatty acid composition and total fatty acid retention efficiency (Table 9)

3.6. Whole body mineral composition No statistically significant differences were found in the whole body mineral composition of the fish fed the diets with varying levels of fish meal, microalgae and either organic or inorganic mineral supplementation. The analyzed minerals and respective levels (in mg kg−1) were: Ca (980–1044), Mg (804–866), Zn (91–128), Fe (10.9–14.6), Cu (4.51–5.85), Mn (3.23–3.88), As (0.74–0.98), Co (0.17–0.26), Cd (0.007–0.014).

The NQC fillet lipids generally mirrored the dietary composition. Thus, the control fish had higher levels of 14:0, 16:1, 20:1, 22:1, 20:4n-3, 20:5n-3 (EPA) and 22:5n-3 (DPA) in their fillet and lower levels of 20:2n-6, 20:4n-6 (AA; arachidonic acid) and the essential fatty acids 18:3n-3 (ALA; a-linolenic acid) and 22:6n-3 (DHA) compared to the Schizochytrium sp. supplemented diets (with some exceptions regarding the LFM_50Sc treatment which had in general lower levels of fillet lipids). LA, the precursor for the production of arachidonic acid, was found in higher level in the fillet of the fish fed the Schizochytrium sp. supplemented diets, was higher but not significantly in the fillet of the fish fed the Schizochytrium sp. supplemented diets. Alpha-linolenic acid was higher in the Schizochytrium sp. supplemented diets, according to the dietary composition. The NQC fillet saturated fatty acids were lower in the fish fed the LFM_50Sc diet compared to the control fish. The fillet of the fish fed the organic mineral supplemented diet (LFM_50Sc_OM) contained higher levels of DHA and monounsaturated FA (18:1 and total) compared to that of the fish fed the diet LFM_50Sc supplemented with inorganic minerals. Reduction of fish meal in the diet resulted in a reduction in NQC lipid level, significant only when compared to the control fish (MFM_0Sc). The fillet of the fish fed diet LFM_50Sc contained lower levels of total monounsaturated FA (significantly also for 18:1 and 20:1) compared to that of the fish fed the MFM_50Sc diet. Unlike in the fillet, there was no difference in the whole body lipid levels between the medium fish meal and low fish meal diet fed fish, or between the fish that were fed with diets supplemented with either IM or OM. The individual fatty acid profile of the whole body had similar trends among the different treatments as described for the NQC fillet above. The retention efficiency of 18:0 and monounsaturated fatty acids (significantly for 16:1 and 20:1) was higher in the fish fed the groups fed the diets supplemented with Schizochytrium sp. biomass, which contained lower levels of those fatty acids compared to the control. The same was observed for 20:2n-6, 18:4n-3, EPA (significantly at MFM and 50 g kg− 1 Schizochytrium sp. supplementation). Based on total amounts of lipids fed and those incorporated in the fish body, EPA + DHA retention efficiency was significantly higher (P b 0.05) in the Schizochytrium sp. supplemented diets compared to the control.

3.7. Fillet technical quality (Table 10) Fish fillet liquid losses and texture were not different in salmon fed the Schizochytrium sp. supplemented diets compared to the control, but fillet redness was lower (instrumental analyses). No significant color differences were however detected by visual assessment using the SalmoFan scoring (sensory assessment). There was relatively high amount of fish fillets with gaping in the medium fish meal treatments, but no significant effect of Schizochytrium sp. supplementation was observed between the MF_0_Sc, MFM_25Sc and MFM_50Sc. Salmon fed low fish meal diets had significantly higher gaping frequency than salmon fed medium fish meal diets (13% units increase), but gaping frequency was significantly lowest for salmon fed the low fish meal diet supplemented with organic minerals instead of inorganic minerals. 3.8. Blood chemistry No statistically significant differences were found in the A. salmon plasma glucose or different lipid class levels of this trial and the analyzed levels were similar to those of the control group in the study by Kortner et al. (2014). 4. Discussion All dietary treatments demonstrated high performance confirming that Schizochytrium sp. is a feed ingredient that can be incorporated in Atlantic salmon diets without compromising growth or feed conversion ratio as previously shown with dietary Thraustochytrids in Carter et al. (2003) as well as the same microalgae strain as the one we used in our present study in Kousoulaki et al. (2015). The dietary treatments had significant effects on the development of tissues and energy deposition. In particular Schizochytrium sp., showed stimulated muscle growth (1.1%-unit higher D%) rather than visceral fat deposition at

Table 4 Dietary macronutrient and energy apparent digestibility coefficient (ADC1) in A. salmon. MFM_0_Sc ADCProtein ADCLipids ADCEnergy

a

88.2 ± 0.2 93.9 ± 0.2c 79.0 ± 0.0ab

MFM_25Sc ab

89.0 ± 0.6 93.2 ± 0.2bc 80.3 ± 1.0b

MFM_50Sc a

88.4 ± 0.3 91.6 ± 0.2a 78.8 ± 0.3ab

LFM_50Sc

LFM_50Sc_OM ab

89.4 ± 0.7 91.1 ± 0.7a 77.9 ± 1.0a

b

89.9 ± 0.3 92.1 ± 0.6ab 79.2 ± 0.4ab

ANOVA (P⁎) b0.01 b0.001 b0.05

⁎ Values not sharing common superscript letters are significantly different (P ≤ 0.05) as determined by ANOVA followed by Tukey post hoc test. 1 ADC of nutrients and energy in the test diets was calculated from the following formula: ADC = 100 − 100 × Yd × Nf / Nd/Yf, were d is diet, f is feces, Y yttrium content and N nutrient content.

K. Kousoulaki et al. / Aquaculture 451 (2016) 47–57

53

Table 5 Dietary fatty acid (FA) apparent digestibility coefficient.

14:0 16:0 18:0 20:0 22:0 16:1 n-7 18:1 (n-9) + (n-7) + (n-5) 20:1 (n-9) + (n-7) 22:1 (n-11) + (n-9) + (n-7) 24:1 n-9 18:2 n-6 20:2 n-6 18:3 n-3 20:5 n-3 22:6 n-3 Total saturated FA Total monounsaturated FA Total PUFA (n-6) FA Total PUFA (n-3) FA Total PUFA FA Total FA

MFM_0_Sc

MFM_25Sc

MFM_50Sc

LFM_50Sc

LFM_50Sc_OM

ANOVA (P⁎)

96.5 ± 0.3 d 91.6 ± 0.7c 94.5 ± 0.2c 95.9 ± 0.1a 94.9 ± 1.8 99.0 ± 0.2 98.9 ± 0.1a 97.7 ± 0.2a 97.0 ± 0.2a 93.9 ± 0.2bc 98.7 ± 0.1 93.9 ± 0.2 99.5 ± 0.0 99.4 ± 0.0 98.2 ± 0.3 92.8 ± 0.5c 98.6 ± 0.1a 98.6 ± 0.1ab 99.3 ± 0.0c 98.9 ± 0.0ab 97.8 ± 0.1d

90.8 ± 0.3 c 85.2 ± 0.6b 93.9 ± 0.2c 96.6 ± 0.1b 93.2 ± 0.2 98.8 ± 0.6 99.1 ± 0.1ab 98.5 ± 0.2b 98.0 ± 0.1c 95.5 ± 1.7c 98.8 ± 0.1 93.2 ± 0.2 99.6 ± 0.0 99.0 ± 0.0 97.7 ± 0.4 87.2 ± 0.5b 99.0 ± 0.1bc 98.8 ± 0.1b 99.1 ± 0.1ab 98.9 ± 0.1ab 97.2 ± 0.1c

76.5 ± 0.6a 76.3 ± 0.6a 92.6 ± 0.2b 95.8 ± 0.1a 93.0 ± 2.4 98.6 ± 0.0 99.0 ± 0.1a 98.3 ± 0.2ab 97.6 ± 0.1bc 91.6 ± 0.2ab 98.7 ± 0.1 97.2 ± 4.9 99.4 ± 0.1 98.8 ± 0.0 97.8 ± 0.2 78.8 ± 0.5a 98.9 ± 0.1b 98.7 ± 0.1ab 98.9 ± 0.1a 98.8 ± 0.1ab 95.7 ± 0.2ab

77.4 ± 1.2a 75.5 ± 1.5a 91.7 ± 0.6a 95.5 ± 0.3a 91.1 ± 0.7 98.5 ± 0.1 99.2 ± 0.1ab 98.6 ± 0.3b 97.5 ± 0.2b 91.1 ± 0.7a 98.6 ± 0.1 97.1 ± 5.0 99.5 ± 0.1 98.9 ± 0.1 97.9 ± 0.3 77.9 ± 1.4a 99.1 ± 0.1bc 98.6 ± 0.1a 99.0 ± 0.1ab 98.8 ± 0.1a 95.4 ± 0.3a

79.7 ± 0.8b 77.4 ± 0.9a 92.8 ± 0.1b 96.0 ± 0.3ab 94.8 ± 1.8 98.7 ± 0.1 99.3 ± 0.0b 98.9 ± 0.1b 97.9 ± 0.3c 92.1 ± 0.6ab 98.8 ± 0.0 100 ± 0.0 99.6 ± 0.0 99.3 ± 0.6 98.3 ± 0.1 79.8 ± 0.8a 99.2 ± 0.0c 98.8 ± 0.0b 99.2 ± 0.1bc 99.0 ± 0.0b 96.0 ± 0.1b

b0.001 b0.001 b0.001 b0.01 b0.1 ns b0.01 b0.01 b0.001 b0.01 b0.1 ns⁎⁎ ns ns b0.1 b0.001 b0.001 b0.05 b0.01 b0.05 b0.001

⁎ Values not sharing common superscript letters are significantly different (P ≤ 0.05) as determined by ANOVA followed by Tukey post hoc test. ⁎⁎ Non-significant.

50 g kg− 1inclusion level. These results are in line with our previous study Kousoulaki et al. (2015) revealing that Schizochytrium sp. increased the carcass yield, thus increasing the relative amount of fillets that are the best paid parts of salmon. Fat content in the whole body was not affected by dietary treatment, but the results on lipid retention efficiency indicate that Schizochytrium sp. supplementation combined with higher levels of rapeseed oil and camelina oil resulted in more efficient retention of n-3 LC-PUFA. The energy deposition in fillets showed significant variation between the dietary groups, with lower fat content of the LFM_50Sc compared with the MFM_0_Sc, and indications of increased in vivo fillet glycogen deposition in salmon fed 50 g kg−1 Schizochytrium sp. (significantly lower pH). The high ADC of DHA and MUFA (≥98%) indicate that decreased

ADC of SFA originating from Schizochytrium sp. in the present and a previous study (Kousoulaki et al., 2015) is related to additional factors than incomplete cell wall disruption. For example the positioning of the SFA in the microalgae triglycerides can be a contributing factor, as previously suggested by Bracco (1994). Supplementation of organic Zn, Cu, Se, Mn and Fe did not affect the retention of these minerals in the whole body, but the organic minerals improved FA digestibility and slightly altered the fillet FA composition, including 13% increased DHA concentration. Fe (Stangl and Kirchgeßner, 1998) and Zn (Eder and Kirchgessner, 1994a,b) are among minerals that are important in stimulating the bioconversion of ALA into EPA and DHA. Our results indicate that organic minerals may be more efficient than inorganic minerals.

Table 6 Growth, feeding performance, biometrics and liver composition of A. salmon fed diets with different levels of fish meal (FM) and microalgae (Schizochytrium sp.). Values are mean ± standard variation (n = 3 tanks per treatment). Dietary FM/Sc level (g/kg)

MFM_0_Sc

MFM_25Sc

MFM_50Sc

150/0

150/25

150/50

Start fish number/tank Mortality (%) Initial weight (g) Final weight (g) Total feed intake (kg) Fl12 (% mbw/day) FCR2 TGC3 (⁎1000) PER4 CF5 D%6 HSI7 Liver lipid % Liver Cu mg/kg

35 0.0 ± 0.0 398 ± 2 1074 ± 39 16.8 ± 0.9 0.84 ± 0.02 0.72 ± 0.00 3.82 ± 0.16 3.14 ± 0.01 1.49 ± 0.03 87.4 ± 0.4a 1.56 ± 0.13 11.4 ± 2.8 83.67 ± 9.07

35 0.0 ± 0.0 403 ± 6 1053 ± 35 16.3 ± 1.3 0.82 ± 0.04 0.72 ± 0.02 3.69 ± 0.10 3.13 ± 0.09 1.45 ± 0.03 87.7 ± 0.3ab 1.64 ± 0.06 10.9 ± 0.6 85.33 ± 6.81

35 0.0 ± 0.0 401 ± 6 1076 ± 26 17.2 ± 0.8 0.83 ± 0.03 0.73 ± 0.04 3.80 ± 0.06 3.10 ± 0.15 1.46 ± 0.06 88.5 ± 0.4b 1.56 ± 0.15 9.8 ± 1.6 85.33 ± 2.52

LFM_50Sc_OM

ANOVA (P⁎)

100/50

100/50

Treatment

FM

Sc

35 0.0 ± 0.0 399 ± 2 1054 ± 37 15.9 ± 1.2 0.77 ± 0.04 0.69 ± 0.02 3.73 ± 0.14 3.23 ± 0.09 1.47 ± 0.08 88.5 ± 0.6b 1.45 ± 0.10 7.6 ± 0.3 89.00 ± 2.00

35 0.0 ± 0.0 399 ± 6 1061 ± 32 16.3 ± 1.0 0.79 ± 0.03 0.71 ± 0.01 3.75 ± 0.08 3.25 ± 0.02 1.47 ± 0.03 88.0 ± 0.5ab 1.65 ± 0.09 10.2 ± 2.8 88.00 ± 8.19

ns⁎⁎ ns ns ns ns ns ns ns b0.05 ns ns ns

ns ns b0.1 b0.1 ns ns ns ns ns ns ns

ns ns ns ns ns ns ns b0.05 ns b0.1 ns

LFM_50Sc

⁎ Values not sharing common superscript letters are significantly different (P ≤ 0.05) as determined by ANOVA followed by Tukey post hoc test. ⁎⁎ Non-significant. 1 Mean total feed consumption per tank (in dry matter). Feed moisture was MFM_0_Sc = 87 g kg−1; MFM_25Sc = 87 g kg−1; MFM_5_Sc = 64 g kg−1; LFM_5_Sc = 55 g kg−1; LFM_5_Sc_BP = 64 g kg−1. 2 FCR; feed conversion ratio is feed consumed/biomass increase. 3 1/3 TGC; thermal growth coefficient is (w1/3 2 − w1 ) × 1000 / ∑(t × feeding days), where ∑(t × feeding days) is the sum of water temperatures (°C) for every feeding day in the experiment (Cho, 1992). 4 PER; protein efficiency ratio is fish weight gain/protein consumption. 5 CF; condition factor is bw (g) × fish fork length-3 (cm) × 1000. 6 D%; dress out percentage is gutted fish weight/bw × 100. 7 HIS; hepatosomatic index is liver weight / bw × 100.

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K. Kousoulaki et al. / Aquaculture 451 (2016) 47–57

Table 7 Fillet fatty acid composition (mg fatty acid per 100 g serve (i.e. NQC fillet) of Atlantic salmon fed diets with different levels of fish meal (FM) and microalgae (Schizochytrium sp.). Values are mean ± standard variation (n = 3 tanks per treatment).

Fat Bligh & Dyer 14:0 16:0 18:0 20:0 16:1 n-7 18:1 (n-9) + (n-7) + (n-5) 20:1 (n-9) + (n-7) 22:1 (n-11) + (n-9) + (n-7) 24:1 n-9 16:2 n-4 18:2 n-6 (LA) 18:3 n-6 20:2 n-6 (AA) 20:3 n-6 20:4 n-6 18:3 n-3 (ALA) 18:4 n-3 20:3 n-3 20:4 n-3 20:5 n-3 (EPA) 22:5 n-3 (DPA) 22:6 n-3 (DHA) Sum saturated fatty acids Sum monoene fatty acids Sum PUFA (n-6) fatty acids Sum PUFA (n-3) fatty acids Sum PUFA fatty acids

Start

MFM_0_Sc

MFM_25Sc

MFM_50Sc

LFM_50Sc

LFM_50Sc_OM

ANOVA (P⁎)

4950 ± 210 190 ± 140 620 ± 350 120 ± 70 10 ± 70 160 ± 70 1200 ± 710 250 ± 70 270 ± 70 20 ± 0 10 ± 0 320 ± 210 0±0 20 ± 0 0 10 ± 0 110 ± 0 50 ± 70 0±0 30 ± 140 190 ± 140 70 ± 70 570 ± 420 940 ± 640 1890 ± 780 0360 ± 210 1040 ± 570 1420 ± 710

12,470 ± 230b 260 ± 0c 1400 ± 260 300 ± 120 20 ± 0ab 250 ± 60c 4670 ± 420b 590 ± 60c 490 ± 0c 20 ± 0 10 ± 0b 1430 ± 60 20 ± 0 110 ± 0a 50 ± 0 10 ± 0a 410 ± 0a 90 ± 60b 20 ± 0 80 ± 60b 230 ± 60c 100 ± 60b 680 ± 150ab 2000 ± 380b 6010 ± 450b 1630 ± 60 1620 ± 400 3270 ± 400

11,770 ± 1100ab 210 ± 60b 1350 ± 350 290 ± 100 20 ± 0a 0.200 ± 60b 4550 ± 1080ab 500 ± 60b 340 ± 120b 20 ± 0 10 ± 0b 1450 ± 250 30 ± 60 120 ± 60ab 50 ± 60 20 ± 60ab 460 ± 100ab 90 ± 60ab 30 ± 60 70 ± 60ab 180 ± 60b 70 ± 60ab 720 ± 360ab 1890 ± 470ab 5600 ± 1270b 1680 ± 420 1620 ± 510 3310 ± 900

11,770 ± 510ab 180 ± 60ab 1360 ± 310 290 ± 100 40 ± 0b 160 ± 100ab 4630 ± 1560b 450 ± 100b 240 ± 150a 20 ± 0 10 ± 0ab 1530 ± 670 30 ± 60 140 ± 0ab 60 ± 60 20 ± 0b 500 ± 210b 80 ± 100ab 40 ± 0 60 ± 100a 160 ± 100ab 60 ± 60a 740 ± 200ab 1880 ± 460ab 5510 ± 1770b 1770 ± 740 1650 ± 510 3440 ± 1240

10,130 ± 450a 150 ± 100a 1170 ± 120 250 ± 60 20 ± 60ab 150 ± 120a 3860 ± 350a 370 ± 100a 210 ± 120a 20 ± 0 0a 1360 ± 100 30 ± 60 120 ± 60ab 50 ± 60 20 ± 0b 430 ± 60ab 70 ± 0a 30 ± 0 50 ± 0a 140 ± 60a 50 ± 60a 670 ± 150a 1610 ± 210a 4600 ± 640a 1570 ± 120 1450 ± 170 3020 ± 150

12,030 ± 1060ab 180 ± 100ab 1380 ± 400 300 ± 150 30 ± 60ab 170 ± 150ab 4620 ± 1450b 440 ± 0120ab 240 ± 150a 20 ± 0 0a 1600 ± 500 20 ± 0 140 ± 0b 60 ± 60 20 ± 0b 500 ± 200b 80 ± 60ab 40 ± 0 50 ± 60a 160 ± 100ab 60 ± 100a 760 ± 380b 1900 ± 700ab 5500 ± 1860b 1850 ± 500 1650 ± 900 3500 ± 1330

b0.05 b0.001 b0.1 b0.1 b0.05 b0.001 b0.05 b0.001 b0.001 ns⁎⁎ b0.01 b0.1 ns b0.05 ns b0.05 b0.01 b0.05 ns b0.01 b0.001 b0.01 b0.05 b0.05 b0.01 0.05 0.1 b0.1

⁎ Values not sharing common superscript letters are significantly different (P ≤ 0.05) as determined by ANOVA followed by Tukey post hoc test (the start values are not included in the statistics) Data in bold are not included in the statistical analysis. ⁎⁎ Non-significant.

Fillet gaping is caused by rupture of the connective tissue so that slits or holes appear in the fillet (Love, 1970). Salmon fed the low fish meal diet had the highest gaping incidence of 40%, but addition of organic

minerals to the same diet resulted in a six fold gaping reduction (6.7%), and the lowest gaping incidence among all dietary treatments. The significant effect of dietary organic minerals on salmon fillet

Table 8 Whole body fatty acid (FA) composition (g FA in 100 g homogenized fish body mass) in Atlantic salmon fed diets with different levels of fish meal (FM) and microalgae (Schizochytrium sp.). Values are mean ± standard variation (n = 3 tanks per treatment).

Fat Bligh & Dyer 14:0 16:0 18:0 20:0 16:1 n-7 18:1 (n-9) + (n-7) + (n-5) 20:1 (n-9) + (n-7) 22:1 (n-11) + (n-9) + (n-7) 24:1 n-9 16:2 n-4 18:2 n-6 (LA) 18:3 n-6 20:2 n-6 20:3 n-6 20:4 n-6 (AA) 18:3 n-3 (ALA) 18:4 n-3 20:3 n-3 20:4 n-3 20:5 n-3 (EPA) 21:5 n-3 22:5 n-3 (DPA) 22:6 n-3 (DHA) sum saturated fatty acids sum monoene fatty acids sum PUFA (n-6) fatty acids sum PUFA (n-3) fatty acids sum PUFA fatty acids

Start

MFM_0_Sc

MFM_25Sc

MFM_50Sc

LFM_50Sc

LFM_50Sc_OM

ANOVA (P⁎)

10.80 ± 0.71 0.43 ± 0.04 1.33 ± 0.12 0.27 ± 0.03 0.02 ± 0.00 0.38 ± 0.03 2.72 ± 0.26 0.56 ± 0.04 0.59 ± 0.04 0.05 ± 0.01 0.03 ± 0.00 0.73 ± 0.06 0.01 ± 0.00 0.05 ± 0.01 0.01 ± 0.00 0.03 ± 0.00 0.25 ± 0.02 0.11 ± 0.01 0.01 ± 0.00 0.07 ± 0.00 0.43 ± 0.02 0.02 ± 0.00 0.17 ± 0.01 1.09 ± 0.11 2.06 ± 0.19 4.31 ± 0.38 0.83 ± 0.07 2.14 ± 0.16 3.03 ± 0.24

15.40 ± 0.17 0.35 ± 0.01c 1.76 ± 0.05 0.40 ± 0.01 0.03 ± 0.00 0.32 ± 0.02c 5.76 ± 0.12 0.76 ± 0.00c 0.64 ± 0.02c 0.05 ± 0.00 0.02 ± 0.00 1.73 ± 0.05a 0.03 ± 0.01 0.14 ± 0.00a 0.06 ± 0.01a 0.03 ± 0.01 0.49 ± 0.01a 0.12 ± 0.01 0.03 ± 0.00 0.10 ± 0.01b 0.31 ± 0.02b 0.02 ± 0.00b 0.12 ± 0.00c 0.89 ± 0.04a 2.56 ± 0.06 7.53 ± 0.13 1.98 ± 0.07a 2.08 ± 0.06 4.07 ± 0.08a

16.07 ± 0.70 0.31 ± 0.03b 1.83 ± 0.15 0.40 ± 0.04 0.03 ± 0.00 0.28 ± 0.02b 6.20 ± 0.50 0.70 ± 0.06bc 0.49 ± 0.05b 0.04 ± 0.01 0.02 ± 0.00 1.97 ± 0.14ab 0.03 ± 0.01 0.17 ± 0.02ab 0.06 ± 0.01a 0.03 ± 0.00 0.60 ± 0.05b 0.11 ± 0.01 0.04 ± 0.01 0.10 ± 0.01b 0.26 ± 0.02a 0.01 ± 0.01ab 0.11 ± 0.01bc 0.96 ± 0.05b 2.58 ± 0.21 7.71 ± 0.63 2.25 ± 0.17ab 2.20 ± 0.15 4.46 ± 0.33ab

16.37 ± 0.70 0.28 ± 0.01ab 1.88 ± 0.12 0.43 ± 0.03 0.04 ± 0.01 0.26 ± 0.01ab 6.43 ± 0.41 0.64 ± 0.03ab 0.37 ± 0.02a 0.04 ± 0.01 0.01 ± 0.01 2.05 ± 0.12b 0.03 ± 0.00 0.18 ± 0.01b 0.08 ± 0.00b 0.03 ± 0.00 0.67 ± 0.05b 0.11 ± 0.00 0.05 ± 0.00 0.10 ± 0.00ab 0.25 ± 0.01a 0.00 ± 0.00a 0.10 ± 0.01ab 1.03 ± 0.03c 2.65 ± 0.17 7.74 ± 0.47 2.38 ± 0.14b 2.31 ± 0.10 4.70 ± 0.23b

15.47 ± 0.70 0.25 ± 0.01a 1.75 ± 0.08 0.40 ± 0.03 0.03 ± 0.00 0.24 ± 0.01a 5.82 ± 0.35 0.57 ± 0.02a 0.35 ± 0.01a 0.03 ± 0.00 0.01 ± 0.01 2.00 ± 0.13ab 0.03 ± 0.00 0.17 ± 0.01b 0.06 ± 0.00a 0.03 ± 0.00 0.62 ± 0.04b 0.10 ± 0.01 0.05 ± 0.00 0.07 ± 0.01a 0.23 ± 0.00a 0.00 ± 0.00a 0.09 ± 0.01a 0.99 ± 0.03c 2.44 ± 0.09 7.01 ± 0.37 2.29 ± 0.14ab 2.15 ± 0.07 4.45 ± 0.21ab

15.37 ± 0.35 0.26 ± 0.01a 1.79 ± 0.03 0.39 ± 0.01 0.04 ± 0.01 0.25 ± 0.01ab 5.81 ± 0.06 0.59 ± 0.01a 0.37 ± 0.01a 0.04 ± 0.01 0.02 ± 0.00 1.97 ± 0.01ab 0.04 ± 0.01 0.16 ± 0.01ab 0.07 ± 0.01ab 0.04 ± 0.01 0.61 ± 0.01b 0.11 ± 0.00 0.04 ± 0.01 0.09 ± 0.00ab 0.24 ± 0.01a 0.00 ± 0.00a 0.09 ± 0.00a 0.99 ± 0.02c 2.49 ± 0.06 7.05 ± 0.09 2.28 ± 0.03ab 2.16 ± 0.02 4.46 ± 0.03ab

ns⁎⁎ b0.001 ns ns ns b0.001 ns b0.001 b0.001 ns ns b0.05 0.05 b0.05 b0.01 ns b0.01 ns b0.1 b0.05 b0.01 b0.01 b0.01 b0.01 ns ns b0.05 0.100 b0.05

⁎ Values not sharing common superscript letters are significantly different (P ≤ 0.05) as determined by ANOVA followed by Tukey post hoc test. ⁎⁎ Non-significant.

K. Kousoulaki et al. / Aquaculture 451 (2016) 47–57

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Table 9 Fatty acid (FA) retention efficiency (g FA retained in body mass of 100 g fatty acids eaten) in Atlantic salmon fed diets with different levels of fish meal (FM) and microalgae (Schizochytrium sp.). Values are mean ± standard variation (n = 3 tanks per treatment). ANOVA (P⁎) Mean

MFM_0_Sc

MFM_25Sc

MFM_50Sc

LFM_50Sc

LFM_50Sc_OM

diet

Sc

FM

minerals

14:0 16:0 18:0 20:0 22:0 16:1 n-7 18:1 (n-9) + (n-7) + (n-5) 20:1 (n-9) + (n-7) 22:1 (n-11) + (n-9) + (n-7) 24:1 n-9 18:2 n-6 (LA) 20:2 n-6 18:3 n-3 (ALA) 18:4 n-3 20:4 n-3 20:5 n-3 (EPA) 22:6 n-3 (DHA) EPA + DHA Total saturated FA Total monoene FA Total PUFA (n-6) FA Total PUFA (n-3) FA Total PUFA FA Total FA

67 ± 3 82 ± 3 111 ± 5a 53 ± 0 40 ± 0 79 ± 6a 76 ± 2 93 ± 2a 60 ± 3 99 ± 1 65 ± 2 845 ± 8a 47 ± 0 68 ± 8a 537 ± 60 50 ± 5a 122 ± 8b 91 ± 7 82 ± 3 76 ± 2a 73 ± 3 75 ± 3 74 ± 2 76 ± 2

73 ± 11 85 ± 7 117 ± 10a 43 ± 2 43 ± 2 86 ± 12a 81 ± 5 113 ± 10b 67 ± 9 86 ± 32 69 ± 3 1059 ± 75b 50 ± 3 94 ± 12a 538 ± 77 51 ± 10a 119 ± 8b 99 ± 8 86 ± 7 82 ± 5ab 79 ± 4 78 ± 6 78 ± 5 82 ± 5

84 ± 9 80 ± 7 134 ± 11ab 62 ± 16 43 ± 3 139 ± 14b 82 ± 6 131 ± 9b 75 ± 13 139 ± 52 67 ± 5 1139 ± 61b 49 ± 4 261 ± 19b 507 ± 33 93 ± 16b 110 ± 6ab 107 ± 7 85 ± 7 85 ± 6ab 77 ± 5 80 ± 6 78 ± 5 83 ± 6

66 ± 4 78 ± 4 149 ± 19b 45 ± 3 45 ± 3 121 ± 14b 88 ± 6 124 ± 7b 71 ± 4 99 ± 12 72 ± 5 1203 ± 85b 54 ± 4 245 ± 17b 363 ± 84 64 ± 7ab 106 ± 4ab 100 ± 5 83 ± 4 90 ± 5b 83 ± 5 80 ± 4 81 ± 5 86 ± 5

64 ± 7 77 ± 1 134 ± 3ab 51 ± 15 41 ± 1 124 ± 7b 81 ± 1 120 ± 1b 76 ± 4 167 ± 70 66 ± 2 1066 ± 52b 48 ± 2 245 ± 7b

b0.05 ns⁎⁎ b0.05 ns b0.1 b0.001 b0.1 b0.001 ns ns ns b0.000 b0.1 b0.001 b0.05 b0.01 b0.05 b0.1 ns b0.05 ns ns ns ns

b0.1 ns b0.1 0.1 ns b0.001 ns b0.001 ns ns ns b0.001 ns b0.001 ns b0.01 ns b0.05 ns 0.1 ns ns ns ns

b0.05 ns ns b0.1 ns b0.1 b0.1 ns ns ns b0.1 ns b0.05 ns b0.05 b0.05 ns ns ns ns ns ns ns ns

ns ns ns ns b0.05 ns b0.1 ns ns b0.1 b0.05 b0.05 b0.05 ns ns ns ns ns ns b0.1 b0.1 ns ns ns

79 ± 15ab 101 ± 2a 98 ± 3 81 ± 2 83 ± 1ab 76 ± 2 76 ± 0 76 ± 1 80 ± 0

⁎ Values not sharing common superscript letters are significantly different (P ≤ 0.05) as determined by ANOVA followed by Tukey post hoc test. ⁎⁎ Non-significant.

technical quality, i.e. the connective tissue strength, is interesting to the processing industry as fillet gaping is a major cause to quality downgrading in the secondary processing industry (Michie, 2001). Recent studies have shown that glutamate (a functional amino acid) has a positive effect on fillet firmness of salmon (Larsson et al. 2012) and cod fillets (Ingebrigtsen et al., 2014), but the present study is the first published documentation showing improved fillet integrity of salmon fed diets supplemented with organic minerals. A positive correlation between copper concentration in the muscle and firmness of salmon is reported (Mørkøre and Austreng, 2004), and dietary vitamin E supplementation has shown improved firmness, collagen stability and sensory freshness (Mørkøre et al. 2012). A synergism between dietary selenium and vitamin E is known (Lall, 2002), and usually the organic forms of Se (Mahan et al., 1999; Mahmoud and Edens, 2003) and other micro minerals (Creech et al., 2004) have higher bioavailability than inorganic forms. Clearly gaping is a complex defect as we also found an overall reverse correlation between fillet total PUFA level and gaping (R2 = 0.729; P b 0.05). Hence, in addition to explore the importance of each Zn, Cu, Mn, Fe and Se for gaping, the impact of dietary lipid profile should also be considered in future studies. Besides the biological functionality, feed pellets with added microalgae raw materials must have high physical quality to withstand

impacts during transport, storage and feeding. In our study, pellet durability improved at 25 g kg−1 dietary Schizochytrium sp. supplementation and deteriorated at 50 g kg−1 dietary inclusion level. However, no correlation between fish performance and pellet technical quality was found, and the deterioration in pellet durability was not combined with reduced water stability and hardness, as described before (Aas et al., 2014). This may be due to the fact that all experimental diets had superior pellet technical quality in terms of water stability, hardness and durability when compared to those tested by Aas et al. (2014) where some negative effects in ADC of dietary nutrients in rainbow trout were observed at reduced pellet technical quality. Thus, the variation in pellet durability among the experimental diets did not contribute to variation in fish performance and feed quality of the current study, similarly to the study with Atlantic salmon by Oehme et al. (2014). The reason behind the observed variation in pellet technical quality is difficult to explain, as more than one raw materials varied between the experimental diets. Some microalgae species have previously shown to have good binding properties whereas others cause competitive interactions and thermodynamic incompatibility effects with other proteins, e.g. pea protein (Batista et al., 2007). The physicochemical and rheological properties of the microalgal biomass will vary related to species and processing methods and conditions as seen for other plant and

Table 10 NQC fillet technical quality in Atlantic salmon fed diets with different levels of fish meal (FM) and microalgae (Schizochytrium sp.) (n = 3 tanks per treatment).

Liquid loss, % SalmoFan score Lightness, L value Redness, a value Yellowness, b value Gaping1 (%) Firmness2 (N) Muscle pH

MFM_0_Sc

MFM_25Sc

MFM_50Sc

LFM_50Sc

LFM_50Sc_OM

ANOVA (P⁎)

2.1 ± 0.5 22.0 ± 0.3 79.4 ± 0.3 8.9 ± 0.3b 20.2 ± 0.2 33.3 ± 2.5bc 1.57 ± 0.08 6.07 ± 0.01b

3.0 ± 0.2 21.2 ± 0.2 79.7 ± 0.4 8.0 ± 0.3a 19.8 ± 0.6 26.7 ± 2.5b 1.61 ± 0.10 6.03 ± 0.02ab

2.5 ± 0.5 21.3 ± 0.3 79.9 ± 0.3 7.9 ± 0.2a 19.6 ± 0.4 26.7 ± 2.5b 1.61 ± 0.08 6.03 ± 0.01a

2.8 ± 0.3 21.9 ± 0.3 79.1 ± 0.2 7.5 ± 0.3a 19.5 ± 0.3 40 ± 4.4c 1.55 ± 0.06 6.01 ± 0.00a

2.5 ± 0.4 21.5 ± 0.3 80.0 ± 0.5 7.9 ± 0.4a 19.3 ± 0.6 6.7 ± 2.5a 1.44 ± 0.10 6.02 ± 0.01a

ns⁎⁎ ns ns b0.05 ns b0.001 ns b0.05

⁎ Values not sharing common superscript letters are significantly different (P ≤ 0.05) as determined by ANOVA followed by Tukey post hoc test. ⁎⁎ Non-significant. 1 Percentage of fish with gaping. 2 Breaking strength.

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marine raw materials, and this variation may impact the extrusion process and pellet physical quality (Hill et al., 2010a,b; Samuelsen et al., 2014). New basic knowledge of the technical properties of microalgal biomass is necessary in order to secure uniform and high extruded pellet quality when high dietary levels of microalgae are used in order to avoid negative economic and environmental effects due to pellet breakage, product losses and discharge of nutrients to the aqueous environment (Aas et al., 2014). 5. Conclusions Spray dried microalgae biomass (Schizochytrium sp.) included up to 5% in extruded feeds for salmon can successfully replace fish oil as source of n-3 LC-PUFA without compromising fish growth rate and FCR, dietary protein and energy digestibility and flesh quality. Dietary Schizochytrium sp. improved the retention efficiency of EPA + DHA and monounsaturated fatty acids, improved the technical quality of salmon fillets in terms of gaping. Dietary organic minerals included in low fish meal diets for Atlantic salmon significantly improved fillet quality technically, in terms of lower gaping incidence, and nutritionally, in terms of increased DHA and monounsaturated fatty acid levels. Acknowledgments The experimental works undertaken through the Alltech – Nofima Strategic Research Alliance were realized by the supply of products and full financial support from Alltech Inc. under the scope of the Alltech-Nofima Strategic Research Alliance (SRA). The authors further acknowledge the significant contributions of Nofima's employees at The Feed Technology Center for the production of the experimental feeds, at the Research station at Sunndalsøra for a well-run fish feeding trial and at BioLab for the realization of chemical analyses in fish, feeds and raw materials. References Aas, T.S., Terjesen, B.F., Sigholt, T., Hillestad, M., Holm, J., Refstie, S., Baeverfjord, G., Rørvik, K.-A., Sørensen, M., Oehme, M., Åsgård, T., 2014. Nutritional responses in rainbow trout (Oncorhynchus mykiss) fed diets with different physical qualities at stable or variable environmental conditions. Aquac. Nutr. 17, 657–670. Aksnes, A., Asbjørnsen, B., 2003. Method for the definition of peptide size in fish hydrolysate, ensilage and gelatine. Fiskeriforskning Report K-289 (in Norwegian). Bæverfjord, G., Reftsie, S., Krogedal, P., Åsgård, T., 2006. Low feed pellet water stability and fluctuating water salinity cause separation and accumulation of dietary oil in the stomach of rainbow trout (Oncorhynchus mykiss). Aquaculture 261, 1335–1345. Barclay, W., Zeller, S., 1996. Nutritional enhancement of n-3 and n-6 fatty acids in rotifers and Artemia nauplii by feeding spray-dried Schizochytrium sp. J. World Aquacult. Soc. 27, 314–322. Barclay, W.R., 1994a. Process for growing Thraustochytrium and Schizochytrium using nonchloride salts to produce a micro-floral biomass having omega-3 highly unsaturated fatty acids. US Patent 5,340,742. Barclay, W.R., 1994b. Food product having high concentrations of omega-3 highly unsaturated fatty acids. US Patent 5,340,594. Barclay, W.R., Weaver, C., Metz, J., 2005. Development of a docosahexaenoic acid production technology using Schizochytrium: a historical perspective. In: Cohen, Z., Ratledge, C. (Eds.), Single Cell oil. AOCS, Champaign, Illinois. Batista, A.P., Gouveia, L., Nunes, M.C., Franco, M.F., Raymundo, A., 2007. Microalgae Biomass as a Novel Functional Ingredient in Mixed Gel Systems. 14th Gums and Stabilisers for the Food Industry Conference, North East Wales Institute, Wrexham, June. Bidlingmeyer, B.A., Cohen, S.A., Tarvin, T.L., Frost, B., 1987. A new, rapid, high sensitive analysis of amino-acids in food type samples. J. Assoc. Off. Anal. Chem. 70, 241–247. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Phys. 37, 911–917. Bowles, R.D., Hunt, A.E., Bremer, G.B., Duchars, M.G., Eaton, R.A., 1999. Long-chain n-3 polyunsaturated fatty acid production by members of the marine protistan group the thraustochytrids: screening of isolates and optimisation of docosahexaenoic acid production. J. Biotechnol. 70, 193–202. Bracco, U., 1994. Effect of triglyceride structure on fat absorption. Am. J. Clin. Nutr. 60, 1002–1009. Carter, C.G., Bransden, M.P., Lewis, T.E., Nichols, P.D., 2003. Potential of thraustochytrids to partially replace fish oil in Atlantic salmon feeds. Mar. Biotechnol. 5, 480–492. Cho, C.Y., 1992. Feeding systems for rainbow trout and other salmonids with reference to current estimates of energy and protein requirements. Aquaculture 100, 107–123.

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