Aquaculture 301 (2010) 69–77

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Impact of diets with vegetable oils on the growth, histological structure of internalorgans, biochemical blood parameters, and proximate composition of pikeperchSander lucioperca (L.)

Agata Kowalska a,⁎, Zdzisław Zakęś a, Barbara Jankowska b, Andrzej Siwicki c

a Department of Aquaculture, Stanisław Sakowicz Inland Fisheries Institute, 10 - 719 Olsztyn, Oczapowskiego 10, Polandb Department of Meat Technology and Chemistry, Faculty of Food Science, University of Warmia and Mazury, 10 - 718 Olsztyn, Pl. Cieszyński 1, Polandc Department of Fish Pathology and Immunology, Stanisław Sakowicz Inland Fisheries Institute, 10 - 719 Olsztyn, Oczapowskiego 10, Poland

⁎ Corresponding author. Tel.: +48 89 524 01 71; fax:E-mail address: agatakow@infish.com.pl (A. Kowalsk

0044-8486/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.aquaculture.2010.01.028

a b s t r a c t

a r t i c l e i n f o

Article history:Received 17 November 2009Received in revised form 23 January 2010Accepted 25 January 2010

Keywords:Sander luciopercaFatty acidGrowthHistological structureBlood parameters

The aim of the study was to evaluate the impact feed supplemented with vegetable oils had on the growth,histological structure of the liver, intestines, and spleen, selected biochemical blood parameters, and proximatebody composition of juvenile pikeperch (initial body weight 33 g). The fish were fed isoenergetic (21.0 MJ kg−1

feed) and isoprotein feeds (450 g protein kg−1 feed) supplemented with fish and vegetable oils: linseed andpeanut— group LO/PO, fish and peanut oils— group FO/PO, fish and linseed oils— group FO/LO (oil supplementsfor each groupwere 40 and 100 g kg−1 feed, respectively). The fourth group of fishwas fed feedwith fish oil andsoy oil — group FO/SO, and the addition of these oils was 100 and 40 g kg−1 feed, respectively. The fish werereared for 70 days in three replicates for each feed treatment. The replacement of fish oil with vegetable oils(group LO/PO)didnot impactfishgrowth (PN0.05). Feedingfish peanut oil (groups LO/POand FO/PO) resulted ina significant increase in lipid retention coefficient and in aspartate and alanine aminotransferase activity(Pb0.05). Low hepatocyte vacuolization, increased supranuclear zone in intestinal enterocytes, and the highestceruloplasmin activity in the bloodwere noted in group LO/PO (Pb0.05). The amount of lipids in thefillets and/orwhole fish was significantly higher in groups LO/PO and FO/PO. A high level of vegetable oils in the feed (groupsLO/PO, FO/LO, FO/PO) was linked to increased viscerosomatic index (Pb0.05). The feed with a high content oflinseed oil (group FO/LO) caused a significant decrease in lipid content and an increase in protein in viscera. Thequantity of n-6 polyunsaturated fatty acids (PUFA) in group LO/POwas significantly the highest in thewholefish,viscera, fillet, and liver. The n3/n6 index in fish fillets ranged from 3.24 (group LO/PO) to 6.79 (group FO/SO)(Pb0.05). The quantity of eicosapentaenoic acid infish liver andfilletwas significantly thehighest in group FO/SO(Pb0.05). The contents of docosahexaenoic acid were similar in all groups (PN0.05).

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1. Introduction

Pikeperch (Sander lucioperca) is a valuable species in aquaculture,and recently attempts have been undertaken to improve rearingmethods (Fontaine et al., 2008; Zakęś, 2009). Pikeperch growth ratesare high, and they utilize formulated feeds recommended for salmonidseffectively when reared in recirculating systems (Molnár et al., 2006;Zakęś et al., 2003). Feeding pikeperch this type of feed increases the fatcontent of the whole body and fillet, in comparison with wild fish. Thefatty acid (FA) profile, however, does not change significantly(Jankowska et al., 2003). Consequently, the higher absolute content ofthe valuable eicosapentaenoic (EPA, C20:5 n-3) and docosahexaenoic(DHA, C22:6 n-3) fatty acids in themeat of cultivatedfishmight bemoreattractive to the consumer (Jankowska et al., 2003).

The primary lipid source in formulated feeds isfish oil, which is rich inhighly unsaturated fatty acids (HUFA), including EPA and DHA. Thedynamic development of global aquaculture has led to increasedproduction and consumption of formulated feeds. Vargas et al. (2008)report that the demand for fish oil among consumers (including feedmanufacturers) has reached its sustainability limits. Concurrently, theproduction of vegetable oils has been increasing consistently in recentyears, and is already a hundred times higher than that offish oil (Turchiniet al., 2009). Accordingly, vegetable oils are being used increasingly asmore available and sustainable sources of energy in fish feeds.

Vegetable oils such as soy, rapeseed, sunflower, and linseedhave beenused in pikerperch studies conducted to date (Zakęś et al., 2004; Schulzet al., 2005; Molnár et al., 2006; Kowalska et al., 2008). These oils arecharacterized by higher levels of linolenic (ALA, C18:3 n-3) and linoleic(LA, C:18:2 n-6) fatty acids than those in fish oil, and by a lack of EPA andDHA (NRC, 1993). Replacing fish oil with vegetable oils can cause adecrease in the valuable EPA and DHA in the tissues of fish, and,consequently, in the state of fish health (Watanabe et al., 1989; Tacon,

Table 1Proximate (g kg−1of wet weight) and selected fatty acid (FA) composition (g FA kg−1

of total FA) of the experimental diets.

Dietsa

FO/SO LO/PO FO/PO FO/LO

ComponentsCrude protein 450 450 450 450Crude fat 184 183 187 183NFEb 266 267 263 267Crude ash 80 80 80 80Gross energy (MJ kg−1 feed) 20.7 20.8 20.9 20.8

Fatty acid compositionC14:0 46.9 16.0 30.2 36.1C16:0 165.2 112.6 143.5 134.2C18:1 cis 9 124.6 334.8 333.1 158.1C18:2 n-6 38.3 130.3 123.4 97.3C18:3 n-3 11.3 104.4 8.4 235.5C20:4 n-6 11.9 4.3 4.3 4.2C20:5 n-3 178.2 59.2 64.6 62.0C22:6 n-3 94.9 48.1 57.1 49.4Total saturatedc 257.1 187.2 226.0 213.4Total monoenesd 327.5 440.2 482.4 302.3Total polyenese 415.4 372.6 291.6 484.3Total n-3f 310.1 220.2 139.7 356.7Total n-6g 55.0 134.6 127.7 101.5Total n-9h 164.9 369.4 382.2 195.6n-3 HUFAi 298.8 115.8 131.3 121.2n3/n6 5.6 1.64 1.09 3.51

a FO/SO-feed with fish and soy oils (rich in C14:0, C16:0, C20:5 n-3, C22:6 n-3), LO/PO-feed with linseed and peanut oils (rich in C18:1 cis 9, C18:2 n-6, C18:3 n-3), FO/PO-feed with fish and peanut oils (rich in C18:1 cis 9, C18:2 n-6), FO/LO-feed with fish andlinseed oils (rich in C18:3 n-3).

b NFE—Nitrogen free extract, calculated as 1000— (protein+lipid+ash+fiber) g kg−1.c Total saturated—C14:0, C15:0, C16:0, C18:0, C20:0, C22:0.d Total monoenes—C14:1, C16:1, C17:1, C18:1 cis 9, C18:1 cis 11, C20:1 n-9, C21:1 n-7,

C22:1 n-11, C22:1 n-9.e Total polyenes—C16:4, C20:2, C21:5, C18:2 n-6, C20:3 n-6, C40:4 n-6, C22:5 n-6,

C18:3 n-3, C20:3 n-3, C20:4 n-3, C20:5 n-3, C22:5 n-3, C22:6 n-3.f Total n-3-C18:3 n-3, C20:3 n-3, C20:4 n-3, C20:5 n-3, C22:5 n-3, C22:6 n-3.g Total n-6-C18:2 n-6, C20:3 n-6, C20:4 n-6, C22:5 n-6.h Total n-9-C18:1 cis 9, C20:1 n-9, C22:1 n-9.i n-3 HUFA-C20:3 n-3, C20:4 n-3, C20:5 n-3, C22:5 n-3, C22:6 n-3.

70 A. Kowalska et al. / Aquaculture 301 (2010) 69–77

1992). This can also change the nutritional value of the fish, which issignificant to the consumer (Turchini et al., 2009). However, it has beendemonstrated that freshwater fish, including pikeperch, have an innatecapacity to synthesize EPA and DHA from precursors with 18 carbonatoms (Sargent et al., 2002; Jankowska et al., 2003),whichmeans that it ispossible to use some vegetable oils to feed this species. However,changing the lipid source in feeds can impact both lipid metabolism andthe state of fish health. When used as the primary lipid source in diets ofEuropean sea bass (Dicentrarchus labrax) and salmonids, soy, olive, andrapeseed oils resulted in histological changes in the liver and intestine(Olsen et al., 2000, 2003; Parpoura and Alexis, 2001; Caballero et al.,2002). Thismight be linked to excessive vegetable oils that are difficult toassimilate, a deficit of essential fatty acids (EFA), and/or the inhibition ofHUFA synthesis (Metailler et al., 1981;Watanabe et al., 1989;McClellandet al., 1995; Parpoura and Alexis, 2001). Studies have confirmed thatpathological changes in the alimentary tract increase the values ofbiochemical blood parameters such as aspartate (AST) and alanine (ALT)aminotransferase, alkaline phosphatase (ALP), ceruloplasmin (Cp), andbilirubin (Dunier et al., 1995; Lanari et al., 1999; Nivedita et al., 2002).Thus, the analysis of these indices could beused as amarker in diagnosingchanges in fish resulting from inappropriately composed diets withoutthe necessity of sacrificing the fish for analyses.

The aim of the study was to identify the impact of feedsupplemented with vegetable oils (peanut, linseed, and soy with orwithout the addition of fish oil) on the growth, histological structureof the liver, intestines, and spleen, biochemical blood parameters, andproximate body composition of juvenile pikeperch.

2. Materials and methods

2.1. Rearing animals

The juvenile pikeperch were obtained from artificial spawningperformed at the Inland Fisheries Institute in Olsztyn (Zakęś andSzczepkowski, 2004). Initially, the fish were fed mixed feed (Artemiasp.+formulated feed; first 3 weeks of rearing), followed by commercialtrout feed (Perla, Skretting, Italy), which contained 620 g protein kg−1

feed, 110 g fat kg−1 feed, 100 gashkg−1 feed, andhadadigestibleenergyvalue of 18.5 MJ kg−1 feed. After the pikeperch had reached an averagebodyweight of 33 g, thefishwere stocked into12 rearing tanks, eachwitha volume of 0.2 m3, that were part of one of three recirculating systems.Initially, the stocking density was 9.9–10.0 kg m−3 (60 individuals pertank). The water temperature and oxygen content, total ammonianitrogen (TAN=NH3–N+NH4

+–N), and water pH at the rearing tankoutflow were as follows: 22.3±0.1 °C, 5.7±0.4 mg O2 L−1, 0.18±0.09 mg TAN L−1, 7.75–7.81.Water flow in the tanks was 4 Lmin−1. Thephotoperiod appliedwas LD 24:0. Light intensitymeasured at the surfaceof the rearing tanks was 40–50 lx (Luchiari et al., 2006).

2.2. Feeding animals

The feed used to prepare the experimental diets was an Aller Safirbase feed (Aller-Aqua, Golub-Dobrzyń, Poland) and a mix of fish oil(FO) (Peter Möller, Möller's Tran, Oslo, Norway) and/or peanut oil(PO) (F.LLI Ruata S.p.A, Goccia d'Oro, Italy), linseed oil (LO) (S.P.R.P.Gal, Poznań, Poland), soy oil (SO) (ZPT Olvit, Gdańsk, Poland). Thebase feed contained 450 g protein kg−1 feed, 40 g fat kg−1 feed, and80 g ash kg−1 feed, with an energy value of 14.7 MJ kg−1 feed(granule size 3.0 mm). The dominant lipid source in the base feed wasfish meal, while the source of protein was from fish meal and soymeal. The base feed was supplemented with oils using a vacuumpump (AGA Labor, Lublin, Poland). Three treatments of fish were fedbase feed that had been supplemented with mixtures of two differentoils: LO and PO (group LO/PO), FO and PO (group FO/PO), FO and LO(group FO/LO) in quantities of 40 and 100 g kg−1 feed, respectively.The fourth group of fish was fed feed in which fish oil was the main

lipid source, and which had a chemical composition similar to that ofthe commercial feed used in the rearing of pikeperch. This feedcomprised the base feed supplementedwith FO and SO (group FO/SO)at quantities of 100 and 40 g kg−1 feed. The total lipid content of thefeeds ranged from 183 to 187 g kg−1 feed. The contents of protein, fat,and carbohydrates in the experimental feeds were all within thoserecommended for juvenile pikeperch (Nyina-Wamwiza et al., 2005).The proximate composition and fatty acid profile of the feeds testedare presented in Table 1.

The fish were divided into four feeding treatment groups, each inthree replicates (n=3), and were reared for 10 weeks (70 days) andfed continually (19 hd−1) with automatic band feeders (4305 FIAP,Fishtechnic GmbH, Germany). The daily feed ration was determinedat weekly intervals and ranged from 1.2% stock biomass (first 3 weeksof rearing) to 0.8% stock biomass (last 3 weeks of rearing).

2.3. Experimental procedures

2.3.1. CalculationsOn the first and last days of the experiment the fish were weighed

(W±0.01 g) and measured (total length, TL±0.1 cm), and sampleswere collected to determine the proximate composition of the bodies.The data collected served for calculating the following parameters:

Specific growth rate (SGR, % d−1)=100×[(ln Wf− lnWi)×T−1];Daily growth rate (DGR, g d−1)=(Wf−Wi)×T−1;Condition factor (CF)=100×(W×TL−3);

71A. Kowalska et al. / Aquaculture 301 (2010) 69–77

Coefficient of variation for final bodyweight (CV, %)=100×(SD×W−1);Feed conversion ratio (FCR)=TFI×(FB− IB)−1;Protein efficiency ratio (PER)=(FB− IB)×TFP−1;Apparent net protein retention (ANPR, %)=100×[(Wf×FBP)−(Wi× IBP)×TFP−1] (Ellis and Reigh, 1991);Apparent lipid retention (ALR, %)=100×[(Wf×FBL)−(Wi×IBL)×TFL−1] (Sadowski et al., 1998);Apparent energy retention (ANER, %)=100×[(Wf×FEB)−(Wi×IEB)×FE−1] (Ellis and Reigh, 1991);

where: Wf and Wi are the final and initial body weights (g), T—time ofrearing (days), FB and IB are the final and initial absoluteweights (gfish),TFI—total feed intake (g), FBP—final body protein content (%), IBP—initialbody protein content (%), TFP—total feed protein intake (g), FBL—finalbody lipid content (%), IBL—initial body lipid content (%), TFL—total feedlipid intake (g), FEB—final body energy value (kJ g−1), IEB—initial bodyenergy value (kJ g−1), FE—feed energy value (kJ) (protein energy value24 kJ g−1, lipids 39 kJ g−1, carbohydrates 17 kJ g−1 (Jobling, 1994)).

At the beginning and the end of the experiment, 15 randomlychosen individuals were taken from each replicate (tank) and theirviscera (±0.01 g) and livers (±0.001 g) were weighed to determine,among other parameters, the values of the viscerosomatic (VSI, %) andhepatosomatic (HSI, %) indices:

HSI=100×(LW×W−1);VSI=100×(VW×W−1);

where LW—liver weight (g), VW—viscera weight (g).

2.3.2. Histological analysisHistological analyses were performed on a sample of seven

individuals from each group (for the entire treatment) on the first andlast days of the experiment. The livers, the anterior section of theintestines, and the spleenswere collected from thefish. The tissueswerefixed in Bouin's solution, dehydrated in ethanol, cleared in xylene,embedded in paraffin blocks, cut into 5-μm slices with a rotatingmicro-tome (Leica, Bensheim, Germany), and then stained with H&E. A NikonE600 (Japan) light microscope was used for the histological observa-tions, and MultiScanBase v. 8.08 (Computer Scanning System Ltd.,Warsaw, Poland) and the NIS-Elements F2.30 v. 2.21 (Nikon, Japan)programs were used for structural measurements and analyses.

The following were analyzed in the tissues collected from eachindividual: the size of the hepatocytes and their nuclei, the size of theenterocytes, their nuclei, and the supranuclear zone (±0.01 μm)(Ostaszewska et al., 2005). The histological measurements included 50cells and their nuclei in each of the tissue samples analyzed from eachindividual. Hepatocyte vacuolization was determined on a five-degreescale ranging from 0 to 4 that expresses the degree to which the lipidvacuole fills the cell cytoplasm (0—no lipid vacuolization , 1—low, 2—moderate, 3—high, 4—maximum; Figueiredo-Silva et al., 2005). Thedegree of vacuolization was determined on cells within a field with asurface area of 2500 μm2 (50×50 μm). Twenty such fields of this sizewere analyzed in each individual, and each hepatocyte within this fieldwas assigned a rating on this five-degree scale.

2.3.3. Biochemical analysis of bloodOn the final day of the experiment, blood samples were drawn from

the caudal veins of 10 individuals from each dietary treatment (for theentire treatment). The samples were centrifuged for 10 min at 5000 g.The aspartate AST (IU) and alanine ALT (IU) aminotransferase andalkaline phosphatase ALP (IU) activities were determined in super-natants isolated with the kinetic method (IFCC, PTH Hydrex, Warsaw,Poland). The bilirubin (mmol L−1) and ceruloplasmin Cp (IU) wereassayed with spectrophotometry (Rice et al., 1986). Colorimetric-basedbiochemical assays were performed on the blood plasmawith a EPOL 2

(Poll-Ltd., Warsaw, Poland) spectrophotometer (Siwicki et al., 2003;Svobodova et al., 1986).

2.3.4. Biochemical analysis of proximate and fatty acid compositionThe proximate composition of the fish was analyzed on the first

(one sample) and final (samples from each tank) days of theexperiment. The proximate composition was determined as follows:whole fish body-5 individuals; skinless fillets-6 individuals; viscera-15 individuals; liver-20 individuals. Whole fish and body parts of fishfrom each tank were combined and analyzed together (n=3, for eachfeeding treatment). These samples were used to determine thefollowing: water content, total protein, crude lipid, crude ash (in wetweight); the content of various fatty acids (g kg−1 of total fatty acid(tFA)). The proximate composition and fatty acid content were alsodetermined in the experimental feeds. The protein content wasdetermined with the Kjeldahl method. Lipids were determined withthe Soxhlet method, and ash by combusting the sample at 550–600 °C(AOAC, 1975). The qualitative and quantitative analyses of fatty acidswere performed according to the methods by Folch et al. (1957). Amixture of chloroform, dehydrated methanol, sulfuric acid(100:100:1) was used for acid methylation (Peisker, 1964). Chro-matographic separation was done with an Agilent Technologies6890N gas chromatograph, with a flame-ionizing detector (FID) anda 30-m capillary columnwith an internal diameter of 0.32 mm. Liquidphase Supelcowax 10, film thickness 0.25 μm. Separation conditions:carrier gas helium; flow rate 1 mL min−1. Temperatures — detector250 °C, injector 250 °C, columns 180 °C. The detector signal wasregistered with a Philips device on a scale of 1 mV and at a tape speedof 10 mmmin−1. The fatty acids were identified using standards fromSupelco (Bellefonte, PA, USA).

2.4. Statistical analysis

The results of all the measurements and calculations weresubjected to statistical analysis with the GraphPad Prism program(Soft. Inc., Avenida de la Playa la Jolla, CA, USA). The means werecompared with single factor analysis of variance (ANOVA). Whenstatistically significant differences were confirmed among dietarytreatments (P≤0.05), further statistical analysis was performed withTukey's test (for the analysis of rearing indices, cell size structure,hematological indices, proximate composition of fish). The Kruskal–Wallis test was applied to analyze the degree of vacuolization. Allvalues expressed as percentages were transformed with arcsin beforestatistical processing.

3. Results

3.1. Effect of the diet on rearing parameters

The experimental feeds fed to the juvenile pikeperch for 70 daysdid not have a significant impact on growth rates, FCR and CF (PN0.05;Table 2).

Feeding the pikeperch with the FO/SO feed caused a significantincrease in CF value compared to that of fish raised on the LO/PO feed.The values of the VSI in groups LO/PO and FO/LO were statistically thehighest (Pb0.05; Table 2). The fish fed the feed supplemented withpeanut oil (groups LO/PO and FO/PO) had significantly higher lipidretention in their bodies (Pb0.05; Table 3).

3.2. Effect of diet on liver, intestinal tract, and spleen histology

No significant differences were noted in the size of the hepatocytesor enterocytes of fish fed the experimental feeds (PN0.05; Table 4,Fig. 1), while a lower degree of hepatocyte vacuolization and anincrease in the supranuclear zone of the enterocytes was noted in thegroup of fish fed feed supplemented exclusively with vegetable oils

Table 2Rearing parameters of pikeperch fed experimental diets for 70 days (mean±SD; n=3and n=15 for VSI and HSI).

Dietary treatmentsa

FO/SO LO/PO FO/PO FO/LO

Initial body weight(g)

33.21±0.17 33.21±0.27 32.83±0.29 32.97±0.43

Final body weight(g)

70.35±5.07 74.73±1.24 72.92±0.88 72.04±0.63

Daily growth rateDGR (g d−1)

0.42±0.06 0.47±0.01 0.46±0.01 0.44±0.01

Specific growthrate SGR (% d−1)

1.05±0.10 1.08±0.12 1.13±0.02 1.13±0.04

Initial coefficient ofbody weightvariation CVi (%)

28.26±5.85 28.37±1.86 26.36±0.01 27.64±1.86

Final coefficient ofbody weightvariation CVf (%)

60.48b±7.99 39.81a±8.45 47.20ab±2.92 56.43ab±7.73

Initial conditionfactor CFi

0.84±0.02 0.83±0.01 0.83±0.01 0.83±0.01

Final conditionfactor CFf

0.92±0.05 0.91±0.03 0.92±0.02 0.93±0.02

Viscerosomaticindex VSI (%)

9.82a±2.43 11.23b±2.25 10.86ab±2.33 11.29b±2.50

Hepatosomaticindex HSI (%)

1.18±0.33 1.13±0.35 1.29±0.47 1.26±0.30

Absence of superscript letters indicates no significant differences among treatments(PN0.05).aFO/SO-group fed feed with fish and soy oils, LO/PO-group fed feed with linseed andpeanut oils, FO/PO-group fed feed with fish and peanut oils, FO/LO-group fed feed withfish and linseed oils.

Table 4Comparison of hepatocyte morfometric characteristics of pikeperch fed experimentaldiets (see Table 1) (mean±SD; n=7).

Morfometric data Beginning ofexperiment

Dietary treatmentsa

FO/SO LO/PO FO/PO FO/LO

Size of hepatocyte(μm)

14.1±1.6 13.2±1.5 11.9±1.2 12.5±0.9 12.8±1.3

Size of nuclei (μm) 3.8±0.2 3.8±0.6 3.9±0.3 3.9±0.3 4.2±0.5Vacuolization degree 3.6±0.3 3.2c±0.2 1.9a±0.7 3.0bc±0.3 2.3ab±0.3

Data on the same line with different superscript letters are significantly different(Pb0.05).aFO/SO-group fed feed with fish and soy oils, LO/PO-group fed feed with linseed andpeanut oils, FO/PO-group fed feed with fish and peanut oils, FO/LO-group fed feed withfish and linseed oils.

72 A. Kowalska et al. / Aquaculture 301 (2010) 69–77

(group LO/PO; Pb0.05; Table 4, Figs. 1–3). Simultaneously, the spleensof fish from this treatment exhibited a greater accumulation ofbrownish-black pigments (Fig. 4).

3.3. Effect of the diet on biochemical blood parameters

The fish fed feed supplemented exclusively with vegetable oilsexhibited significantly higher blood ceruloplasmin activity (group LO/PO) (Pb0.05; Table 5). Supplementing feed with PO (groups LO/POand FO/PO) resulted in significantly increased AST and ALT activity(Pb0.05; Table 5). The blood of pikeperch from group FO/PO had highbilirubin contents (Pb0.05; Table 5).

Table 3Nutrient utilization indexes by pikeperch fed experimental diets for 70 days (mean±SD;n=3).

Dietary treatmentsa

FO/SO LO/PO FO/PO FO/LO

Feed conversionratio FCR

0.98±0.14 0.89±0.02 0.88±0.02 0.91±0.02

Protein efficiencyratio PER

2.29±0.30 2.48±0.02 2.52±0.05 2.43±0.04

Apparent proteinretention ANPR(%)

43.67±4.46 45.22±1.42 42.39±0.99 43.91 ± 4.23

Apparent lipidretention ALR(%)

48.75a±5.82 69.93b±2.50 67.83b±6.22 41.22a±6.61

Apparent energyretention ANER(%)

41.74a±4.79 49.93b±1.87 48.54ab±0.99 43.91a±4.23

Data on the same line with different superscript letters are significantly different(Pb0.05).aFO/SO-group fed feed with fish and soy oils, LO/PO-group fed feed with linseed andpeanut oils, FO/PO-group fed feed with fish and peanut oils, FO/LO-group fed feed withfish and linseed oils.

3.4. Effect of the diet on the proximate composition of whole body,viscera, fillet, and liver

Supplementing feeds with PO, which has a high oleic acid content(C18:1 n-9), significantly increased the content of lipids in whole fish(group LO/PO) and in fillets (group FO/PO) (Pb0.05; Tables 1, 6 and 7).When LO,whichhas a fatty acid profile dominated byALA,was themainlipid source in the feed (group FO/LO) the quantity of protein increasedsignificantly while that of fat decreased in pikeperch viscera (Pb0.05;Tables 1 and 8).

In comparison to group FO/SO, the large quantity of vegetable oils (LOand PO) in the pikeperch diets caused a significant decrease in thecontents of SFA in groups LO/PO, FO/PO, FO/LO in the whole fish and infillets, viscera, and livers (Pb0.05; Tables 1 and 6–9). Statisticallysignificant differences among the four feeding treatments were noted inthe content of monounsaturated fatty acids (MUFA) and n-3 and n-6PUFA (Pb0.05; Table 6). The n3/n6 index in fish fillets was the lowest ingroup LO/PO, and the highest in group FO/SO (3.24 vs. 6.79; Pb0.05;Table 7). Dominant among the unsaturated fatty acids analyzed in thefillets was DHA (groups FO/SO and FO/LO) and oleic acid (groups FO/POand LO/PO). TheDHA content in the fishfillets from the groups comparedwas similar (PN0.05; Table 7). The viscera of fish fed feed supplementedwith peanut oil also had the highest content of oleic acid and total MUFA.The amount ofMUFA in groups FO/SO, LO/PO, and FO/PO in the viscera offish was 383.3, 454.5, and 496.0 g kg−1 tFA, respectively (Pb0.05;Table 8), which was higher than the amount of SFA and PUFA. Only ingroup FO/LO was the content of MUFA in the viscera lower than that of

Fig. 1. Morphometry of enterocytes in pikeperch fed experimental diets. Mean values(±SD). Groups with different superscript letters are significantly different (Pb0.05)FO/SO-group fed feed with fish and soy oils, LO/PO-group fed feed with linseed andpeanut oils, FO/PO-group fed feed with fish and peanut oils, FO/LO-group fed feed withfish and linseed oils.

Fig. 2. Histological picture of the liver of pikeperch fed feed supplemented with peanutand linseed oils (group LO/PO) (a), fish and linseed oils (group FO/LO) (b); arrowsindicates small lipid droplets and lipid vacuoles of the hepatocytes.

Fig. 3. Interior intestinal epithelium of fish fed feed supplemented with peanut andlinseed oils (group LO/PO) (a), fish and peanut oils (group FO/PO) (b); arrows indicatelipid vacuoles of the enterocytes.

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PUFA (375.5 vs. 414.0 g kg−1 tFA), whichwas a reflection of the high ALAcontent of feeds supplemented with linseed oil (Tables 1 and 8).Supplementing feeds with vegetable oils exclusively (group LO/PO)significantly lowered the content ofmyristic (C14:0) andpalmitic (C16:0)acids in the fish livers. Vegetable oil supplementation caused an increasein the share of acids with 18 carbon atoms, such as LA (in groups LO/POand FO/PO) andALA (in group FO/LO) and lowered the contents of EPA inthefish livers (Tables 1 and 9). The highest relation of LA to ALA (LA/ALA)acids in group FO/POwas linked to the substantial decrease in the contentof EPA (Pb0.05; Table 9). Feeding pikeperch feeds supplemented withvegetable oils, which have a lowered content of HUFA, did not impact thequantity of DHA in the fish livers (PN0.05; Tables 1 and 9).

4. Discussion

The high content of vegetable oils (i.e., 140 g kg−1 feed) in thepikeperch diet of group LO/PO did not lower the growth rate of thefish incomparison to that of individuals fed feed inwhich the share of vegetableoilwas lowerat40 g kg−1 feed (groupFO/SO)or100 g kg−1 feed(groupsFO/PO and FO/LO). It also had no impact on FCR. Molnár et al. (2006) didnot note that an increased amount of LO (127 and 178 g kg−1 feed) usedas the lipid source in pikeperch feeds (W=64 g; rearing period of42 days) had an impact on the values of the SGR, DGR, or FCR indices incomparison with those of fish fed feed with similar lipid contents of fishorigin. Similarly, no differences were noted in the growth rates or the

value of the FCR during the rearing of Atlantic salmon (Salmo salar)(W=220 g; rearing period of 84 days) between the fish fed feedsupplemented with FO and SO or LO and SO in different proportions(100:0, 25:75, 50:50, 75:25; Menoyo et al., 2007). However, when thefeed was supplemented with PO, the values of ANER and ALR increasedsignificantly in pikeperch. This was probably because of the high contentof oleic acid in the feed becausemainlyMUFA are the energy reserves andare stored in the fish viscera (Lanari et al., 1999; Xu et al., 2001). This isalso why in groups LO/PO and FO/PO the value of VSI was higher than ingroup FO/SO. Itwas also confirmed that in group FO/LO thehighVSI valuemight indicate that a high quantity of ALA (n-3 PUFA) in the diet, alongwith the low content of MUFA, might lead to disturbances in themetabolic processes of the pikeperch. The dominant acids in group FO/SOwere the long-chained EPA and DHA from the n-3 PUFA family, while thequantity of MUFA was low, and in this group the value of the VSI wassignificantly the lowest. The assimilability of fat in the digestive processdepends on the length and the degree of saturation of the fatty acid(Bracco, 1994), which was also confirmed by the present study.

Kalogeropoulos et al. (1992), Piedecausa et al. (2007), and Rennertet al. (2005) reported that the quantity of SFA and PUFA impacts themetabolism of lipids in the liver. In the current experiment supplement-ing feed with vegetable oils with low contents of C14:0 and C16:0 acidsand of EPA and DHA caused a significant decrease in the degree ofhepatocyte vacuolization, an increase in the supranuclear zone of theenterocytes, and the accumulation of lipid droplets in the enterocytes

Fig. 4. Histology of pikeperch spleen fed a commercial diet supplemented with linseedand peanut oils (group LO/PO) (a), fish and peanut oils (group FO/PO) (b); arrowindicates brownish-black pigments.

Table 6Proximate (g kg−1 of wet weight) and main fatty acid (FA) composition (g FA kg−1

total FA) of whole body from pikeperch fed diets with different lipid sources (seeTable 1) (mean±SD; n=3).

Dietary treatmentsa

FO/SO LO/PO FO/PO FO/LO

ComponentsCrude protein 180.0±5.1 174.8±0.9 169.9±4.6 174.9±5.1Crude fat 93.1b±1.9 107.1c±1.3 107.4c±6.4 83.1a±5.6Crude ash 36.6±0.5 34.3±0.6 36.6±1.4 36.5±1.0

Fatty acid compositionC14:0 64.4b±5.7 38.3a±5.2 49.5ab±5.3 49.5ab±8.5C16:0 177.9b±5.0 147.7a±7.8 159.6ab±3.0 155.5ab±10.8C18:1 cis 9 173.6a±5.5 314.0c±6.6 330.0c±7.2 193.2b±3.7C18:2 n-6 48.5a±2.4 107.3d±1.4 92.9c±2.6 83.2b±2.4C18:3 n-3 15.3b±4.4 69.2c±1.6 9.1a±1.0 136.9d±5.7C20:4 n-6 9.5b±0.6 5.1a±0.3 5.1a±0.1 5.8a±0.5C20:5 n-3 122.3b±8.0 63.7a±3.4 66.8a±3.5 71.2a±6.2C22:6 n-3 88.5b±4.3 60.7a±5.0 69.8a±3.3 71.4a±5.5Total saturated 266.0b±1.6 208.0a±2.9 229.6a±8.3 227.2a±17.4Total monoenes 384.8b±3.0 446.0c±4.4 482.6d±4.8 359.1a±4.1Total polyenes 349.2b±0.8 346.0b±1.5 287.8a±5.1 413.7c±21.2Total n-3 248.9c±9.7 207.3b±0.1 160.0a±2.9 295.1d±17.9Total n-6 62.4a±2.2 114.6d±1.2 100.8c±2.7 91.6b±2.4Total n-9 201.3a±5.8 340.4b±4.6 364.0b±6.8 214.3a±17.1n-3 HUFA 233.6b±13.3 138.1a±9.2 150.9a±2.5 138.1a±9.1n3/n6 3.99c±0.21 1.81a±0.07 1.59a±0.05 3.22b±0.13

Different superscript letters on the same line denotes significant differences (Pb0.05).aFO/SO-group fed feed with fish and soy oils, LO/PO-group fed feed with linseed andpeanut oils, FO/PO-group fed feed with fish and peanut oils, FO/LO-group fed feed withfish and linseed oils; for explanation of fatty acid groups see Table 1.

Table 7Proximate (g kg−1 of wet weight) and main fatty acid (FA) composition (g FA kg−1

total FA) of fillet from pikeperch fed diets with different lipid sources (see Table 1)(mean±SD; n=3).

Dietary treatmentsa

FO/SO LO/PO FO/PO FO/LO

Components

74 A. Kowalska et al. / Aquaculture 301 (2010) 69–77

combined with localized sloughing of the intestinal epithelium (groupLO/PO). Changes such as these can occur when the transport ofhydrolyzed lipid products to the circulatory system is suppressed, andthe lipids are stored in the liver (Ostaszewska et al., 2005). Caballero et al.(2002) and Olsen et al. (2000) confirmed that the accumulation of lipid

Table 5Biochemical parameters of blood of pikeperch fed experimental diets (see Table 1)(mean±SD; n=10).

Dietary treatmentsa

FO/SO LO/PO FO/PO FO/LO

AspartateaminotransferaseAST (IU)

9.77a±0.36 11.39b±0.50 11.21b±0.46 9.99a±0.47

AlanineaminotransferaseALT (IU)

5.01a±0.14 6.15b±0.25 7.20c±0.26 5.07a±0.22

Alcaline phosphataseALP (IU)

2.68±0.17 2.50±0.21 2.64±0.21 2.69±0.18

Bilirubin(mmol L−1)

4.94a±0.17 5.20a±0.23 5.71b±0.41 4.95a±0.25

Ceruloplazmin (IU) 28.36a±0.65 31.18b±0.64 28.53a±0.87 27.96a±0.45

Absence of superscript letters indicates no significant differences among treatments(PN0.05).a FO/SO-group fed feed with fish and soy oils, LO/PO-group fed feed with linseed andpeanut oils, FO/PO-group fed feed with fish and peanut oils, FO/LO-group fed feed withfish and linseed oils.

droplets in the enterocytes of salmonids fed feed with vegetable oilsresulted from the insufficient quantity of phospholipids required for thesynthesis of lipoprotein. The low share of myristic and palmitic acids inthe feed of group LO/PO and the associated suppression of phospholipidsynthesis (Rennert et al., 2005) could have influenced low dietary fatassimilability in pikeperch. In the current experiment, when the quantity

Crude protein 201.3b±0.4 201.5b±1.0 195.4ab±4.2 194.7a±2.1Crude fat 7.2a±1.7 7.0a±0.9 11.3b±1.3 6.8a±1.2Crude ash 12.1±0.2 12.2±0.3 12.2±0.2 12.5±0.5

Fatty acid compositionC14:0 37.7b±4.4 23.7a±3.8 24.3a±4.5 25.5a±1.7C16:0 206.1±10.0 192.8±7.1 185.0±20.6 179.3±4.0C18:1 cis 9 132.3a±1.6 222.3c±2.8 230.3c±3.4 159.2b±3.8C18:2 n-6 32.4a±1.5 77.3d±0.3 67.1c±1.6 62.9b±0.8C18:3 n-3 14.0b±5.2 47.6c±2.1 6.0a±0.1 107.6d±3.0C20:4 n-6 15.4b±0.8 10.5a±0.3 11.3a±0.7 10.5a±0.4C20:5 n-3 130.7b±6.5 73.7a±2.5 80.8a±7.7 82.9a±3.4C22:6 n-3 184.8±12.3 160.9±6.4 188.1±23.2 161.7±3.1Total saturated 289.4b±14.6 267.1a±10.2 260.0a±27.7 248.5a±6.0Total monoenes 278.9a±4.4 328.5b±3.0 352.8c±3.0 286.6a±6.1Total polyenes 431.7ab±17.5 404.4a±11.6 387.2a±30.7 464.9b±1.9Total n-3 353.6b±17.6 297.7a±11.1 290.3a±33.1 369.2b±3.4Total n-6 52.1a±1.5 92.0d±0.7 80.9c±2.4 76.3b±1.3Total n-9 154.6b±3.2 144.1a±3.1 261.3d±2.7 188.0c±5.0n-3 HUFA 339.6b±17.8 250.1a±9.0 284.4a±33.1 261.6a±6.3n3/n6 6.79c±0.32 3.24a±0.09 3.59a±0.52 4.84b±0.12

Different superscript letters on the same line denotes significant differences (Pb0.05).aFO/SO-group fed feed with fish and soy oils, LO/PO-group fed feed with linseed andpeanut oils, FO/PO-group fed feed with fish and peanut oils, FO/LO-group fed feed withfish and linseed oils; for explanation of fatty acid groups see Table 1.

Table 8Proximate (g kg−1 of wet weight) and main fatty acid (FA) composition (g FA kg−1

total FA) of viscera from pikeperch fed diets with different lipid sources (see Table 1)(mean±SD; n=3).

Dietary treatmentsa

FO/SO LO/PO FO/PO FO/LO

ComponentsCrude protein 25.9a±6.5 14.8a±3.2 14.8a±3.2 202.5b±15.5Crude fat 802.9b±24.5 818.6b±0.9 814.2b±7.2 655.8a±16.5Crude ash 5.9±0.8 1.9±0.3 2.1±0.1 1.9±0.1

Fatty acid compositionC14:0 83.1b±4.8 47.0a±9.6 51.6a±4.3 46.4a±7.5C16:0 179.1b±3.9 146.2a±7.4 151.2a±1.9 148.3a±6.8C18:1 cis 9 170.2a±9.4 322.9c±14.2 339.7c±8.3 206.0b±6.0C18:2 n-6 47.3a±2.9 110.3d±6.4 98.9c±2.5 87.2b±2.8C18:3 n-3 11.7a±0.6 69.8b±4.7 9.4a±0.3 155.8c±6.3C20:4 n-6 8.4b±0.3 4.2a±0.1 4.6ab±0.3 3.4a±2.0C20:5 n-3 122.1c±1.2 61.6a±1.4 69.6b±3.6 66.0ab±2.3C22:6 n-3 76.6c±0.9 48.4a±2.4 58.1b±2.3 55.2b±0.7Total saturated 280.7b±2.4 211.5a±17.3 220.0a±6.6 210.5a±14.6Total monoenes 383.3a±4.3 454.5b±7.4 496.0c±6.0 375.5a±0.3Total polyenes 336.0b±4.7 334.0b±11.4 284.0a±5.9 414.0c±14.9Total n-3 233.1c±2.3 193.2b±6.0 151.4a±6.1 291.8d±9.5Total n-6 60.0a±3.4 117.2c±6.5 106.3bc±2.5 93.9b±5.5Total n-9 180.1a±7.9 348.2c±14.2 373.6c±8.1 237.6b±7.4n-3 HUFA 221.5c±2.1 123.4a±2.9 142.0b±5.8 136.1b±3.2n3/n6 3.89c±0.20 1.65a±0.08 1.42a±0.08 3.11b±0.08

Different superscript letters on the same line denotes significant differences (Pb0.05).aFO/SO-group fed feed with fish and soy oils, LO/PO-group fed feed with linseed andpeanut oils, FO/PO-group fed feed with fish and peanut oils, FO/LO-group fed feed withfish and linseed oils; for explanation of fatty acids groups see Table 1.

75A. Kowalska et al. / Aquaculture 301 (2010) 69–77

of C16:0 in the feed was about 13% tFA, then no significant changes werenoted in the histological structure of the intestines or liver of thepikeperch. Similarlywith salmon, a content of palmitic acid above10% tFAin the feed did not affect the digestibility of dietary fat (Menoyo et al.,

Table 9Proximate (g kg−1 of wet weight) and main fatty acid (FA) composition (g FA kg−1

total FA) of livers from pikeperch fed diets with different lipid sources (see Table 1)(mean±SD; n=3).

Dietary treatmentsa

FO/SO LO/PO FO/PO FO/LO

ComponentsCrude protein 216.8±19.3 175.4±0.7 198.8±13.3 204.5±35.1Crude fat 139.9±13.4 165.3±1.8 155.7±18.3 151.6±6.9Crude ash 8.9±0.2 08.1±0.3 7.4±0.5 8.5±0.6

Fatty acid compositionC14:0 57.4b±2.0 25.7a±1.3 38.5ab±8.8 38.4ab±5.6C16:0 192.6b±9.5 154.0a±7.4 168.7ab±2.2 169.6ab±3.2C18:1 cis 9 156.1a±4.1 301.6b±4.0 331.7b±14.3 198.0a±16.9C18:2 n-6 44.5a±1.7 103.8d±7.4 84.7c±1.1 68.9b±3.2C18:3 n-3 8.8a±0.4 36.9ab±26.9 6.8a±0.2 94.7b±23.0C20:4 n-6 7.7b±0.8 5.0ab±0.2 4.4a±0.5 5.1ab±0.8C20:5 n-3 101.1b±0.4 58.4ab±2.2 45.1a±5.6 63.3ab±20.2C22:6 n-3 141.3±20.3 105.7±6.3 94.1±5.6 120.4±29.4Total saturated 270.7b±9.2 202.6a±7.4 229.0a±9.0 228.0a±9.8Total monoenes 360.5a±12.5 441.9b±3.4 499.4c±2.3 366.9a±17.5Total polyenes 368.8bc±21.7 355.5b±0.8 271.6a±11.4 405.2c±27.3Total n-3 284.0b±26.2 223.0ab±17.2 162.3a±12.2 303.2b±30.2Total n-6 56.5a±2.3 111.9c±7.0 92.1b±0.4 77.8b±2.2Total n-9 176.2a±13.7 324.4b±3.3 360.0b±10.3 223.7a±16.9n-3 HUFA 275.1b±26.6 186.0a±9.7 155.4a±12.0 208.5a±53.2n3/n6 5.03c±1.93 1.99a±0.28 1.76a±0.86 3.90b±1.79LA/ALA 5.06b±0.06 3.93b±3.06 12.51c±0.56 0.75a±0.15

Different superscript letters on the same line denotes significant differences (Pb0.05).aFO/SO-group fed feed with fish and soy oils, LO/PO-group fed feed with linseed andpeanut oils, FO/PO-group fed feed with fish and peanut oils, FO/LO-group fed feed withfish and linseed oils; LA—linoleic acid, C18:2 n-6, ALA-linolenic acid C 18:3 n-3; forexplanation of fatty acid groups see Table 1.

2007). Additionally, an increased degree of hepatocyte vacuolization inpikeperchwas linked to higher total contents of EPA and DHA in the feed(from 10.73% tFA in group LO/PO to 27.31% tFA in group FO/SO). ThehigherCpactivity ingroupLO/POcouldhave resulted fromthe lowdegreeof vacuolization in liver cells and consequently from disturbances in theregulation of its excretion (Cousin, 1985). This could have caused thegreater number ofmelanin granules in the spleens offish from this group.It has been confirmed that these are linked to fish immunologicalresponse (Chacrabarti and Rao, 2009, unpub. observ.), that they are areaction of the fish to some nutritional components (Evans et al., 2005),and that they can also stem from a lower tolerance of feeds rich invegetable lipids.

It is possible that the low contents of n-3 PUFA in the feed ofgroups LO/PO and FO/PO might have caused an increase in theactivities of Cp, AST, and ALT. Increased activities of aminotransferasesin marine fish fed feed with vegetable oils are caused by deficits ofPUFA (Lanari et al., 1999; Sanchez-Muros et al., 2003; Roncarati et al.,2006). Further, low levels of EPA and DHA in diets (5.09 and 8.75%tFA) caused pathological changes in the livers of pikeperch (Kowalskaet al., 2008). Anomalies in hepatocyte structure were also observed insea bass fed feeds poor in EPA and DHA (6.90% tFA), but this effect wasleveled by increasing the contents of EPA and DHA in the feed to10.50% tFA (Parpoura and Alexis, 2001). Thus, feeding fish feedssupplemented with vegetable oils might negatively impact the stateof their health (Bell et al., 1991; Thompson et al., 1996). The studiescited above suggest that when vegetable oils replace fish oils in fishdiets it is important to ensure that the contents of myristic andpalmitic acids and EPA and DHA are sufficient since deficits of thesecan disturb dietary fat metabolism and lead to poor fish health.

Supplementing diets with vegetable oils resulted in lowered SFA inall of the pikeperch body parts analyzed. This dependency wasconfirmed in other studies of this species (Schulz et al., 2005) and instudies of rainbow trout (Oncorhynchus mykiss) (Caballero et al.,2002). Moreover, the addition of only vegetable oils to base feed(group LO/PO) resulted in the lowest content of palmitic acid in thefish. Adding fish oil to the feeds (groups FO/LO and FO/PO) leveledthese inter-group differences. The study suggests that using vegetableoils (100 g kg−1 feed) in pikeperch feed combined with fish oil(40 g kg−1) is key for the proper synthesis of phospholipids and themetabolism of dietary fats.

The principal site of fatty acid metabolism is the liver, and thequantity of HUFA in fish bodies can influence the bioconversioncapacity (Tocher et al., 2000; Sargent et al., 2002;Menoyo et al., 2007).The addition of vegetable oils to pikeperch feed resulted in anincreased share of LA (groups LO/PO and FO/PO), ALA (group FO/LO),and a decrease in the quantity of EPA in fish livers. Similarly, highlevels of C:18 fatty acids were noted in the tissues of rainbow trout,Atlantic salmon, and brown trout (Salmo trutta morpha fario), andbrook trout (Salvelinus fontinalis) when these species were fed feedswith vegetable oils (Greene and Salivonchick, 1990; Arzel et al., 1994;Boggio et al., 1985). This could have been the result of suppressedeffectiveness in the bioconversion process of fatty acids provided bythe diet (Jordal et al., 2007). In the current experiment, supplement-ing the feed with linseed oil increased the feed content of ALA nearlytenfold, but this did not result in higher contents of EPA or DHA in thefish bodies. Schulz et al. (2005) reported that potential capacity for thesynthesis of n-3 HUFA is not fully exploited in pikeperch whenvegetable oils are used to supplement feed. Exclusive vegetable oilsupplementation can result in the low assimilability of fatty acids,which was confirmed by Olsen et al. (2003) and by the histologicalresults from group LO/PO in the current study.

One important aspect of replacing fish oil with vegetable oil is toensure that the high nutritional values of the fishmeat (i.e., high levelsof EPA and DHA) are maintained, while simultaneously ensuring thatthe content of LA is limited (Sargent and Tacon, 1999). The contentof DHA in skinned pikeperch fillets from each of the four feeding

76 A. Kowalska et al. / Aquaculture 301 (2010) 69–77

treatments was similar. This resulted from the high capacity of fish toaccumulate long-chain fatty acids in fillets (Tocher et al., 2000).Significant inter-group differences were also confirmed, however, inthe contents of LA and ALA. This is also why the amount of LA and ALA,and not that of DHA or EPA, had the greatest impact on the value of then3/n6 index. It should be underscored, though, that DHA wasdominant among PUFA. It cannot be ruled out that the directabsorption of fatty acids into the fish meat could also have impactedthe fatty acid profile of pikeperch fillets (Henderson, 1996).

In summation, it can be concluded that vegetable oils can beapplied to pikeperch diets, but that diets for juvenile pikeperch mustbe composedwith the appropriate quantities of myristic, palmitic, andoleic acids and ALA, EPA, and DHA. In the current experiment, thefeeds supplemented with vegetable oils (100 g kg−1 feed) and fish oil(40 g kg−1 feed) were more beneficial for pikeperch than were feedssupplemented exclusively with vegetable oils (140 gkg−1 feed). Acertain addition of fish oil to pikeperch diets with vegetable oils mightprove to be essential. Determining theminimal, essential quantities offish oil in formulated feeds for pikeperch that are supplemented withvegetable oils is a subject for further study.

Acknowledgments

The authors would like to thank feed manufacturer Aller-Aqua ofGolub-Dobrzyń for their donation of the base feed used in the study.

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