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(This is a sample cover image for this issue. The actual cover is not yet available at this time.)
This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies areencouraged to visit:
Lipid composition and metabolism of European sea bass (Dicentrarchus labrax L.)fed diets containing wheat gluten and legume meals as substitutes for fish meal
M. Messina a,c,⁎, G. Piccolo b, F. Tulli a, C.M. Messina c, G. Cardinaletti a, E. Tibaldi a
a Department of Food Science, Aquaculture Section, Udine University, Udine, Italyb Department of Zootechnical and Food Inspection Science, Napoli University Federico II, Italyc Department of Experimental Biomedicine and Clinical Neuroscience, Palermo University, Italy
a b s t r a c ta r t i c l e i n f o
Article history:Received 11 September 2012Received in revised form 31 October 2012Accepted 1 November 2012Available online 13 November 2012
The role of dietary protein source on growth performance and lipid metabolism in European sea bass(Dicentrarchus labrax) was investigated, by the evaluation of fat content in different tissues, plasma metabo-lites, liver lipogenic activity and fatty acid composition of dorsal muscle tissue.Five isonitrogenous (490 g kg−1 crude protein dry-matter basis) and isolipidic (170 g kg−1 lipid dry-matterbasis) diets with graded levels of wheat gluten, soybean and pea meal in substitution of fish meal were eval-uated in European sea bass (initial body weight 23.9±0.1) for 96 days. There was a significant difference(pb0.05) in dorsal muscle and liver lipid contents. Plasma glucose and cholesterol levels decreased signifi-cantly (pb0.05) as diet included soybean and wheat gluten meal (pb0.05). The composition of fatty acidsin muscle triacylglycerols and phospholipids was affected by vegetable sources as n−6 PUFA percentagewas significantly increased and n−3 PUFA percentage was significantly decreased (pb0.05), without modi-fying EPA/DHA ratio, when wheat gluten and pea meal were included in the diet. The results confirm that thequality and quantity of vegetable protein source could affect lipid metabolism and lipid content in the dorsalmuscle of the European sea bass and suggest the possibility of manipulation of the diet for commercialpurpose.
In the last decade, the use of vegetable protein rich ingredients inpractical diets of carnivorous fish species, has deserved much atten-tion in order to wider the amount and diversity of plant ingredientsof aqua feeds. Their use in spite of fish meals is a major challengefor a sustainable aquaculture.
Despite a significant number of studies focused on the replacementof fishmeal by soybean or other protein rich plant ingredients in differ-ent carnivorous fish species (Carter and Hauler, 2000; Davis et al., 2005;Glencross et al., 2011; Gomes et al., 1995; Hardy, 2010; Kaushik et al.,1995; Nengas et al., 1996; Olli and Krogdahl, 1995; Robaina et al.,1997; Tusche et al., 2012; Yang et al., 2011) considerably less investiga-tions have been performed in the European sea bass and the availableinformation so far is limited to very few studies (Ballestrazzi et al.,1994; D'Agaro and Ravarotto, 1999; Dias et al., 1998, 2005; Kaushiket al., 2004; Robaina et al., 1999; Tibaldi et al., 1998, 1999, 2006; Tulliet al., 1999). In some cultivated fish species, the substitution of dietaryfish meal by certain protein-rich plant ingredients has been shown toinfluence quality traits like adiposity and fatty acid (FA) composition
of the edible tissue. Partial substitution of fish meal with soy proteinconcentrate or corn gluten meal up to 80% in feeds for the Europeansea bass was shown to affect liver and muscle adiposity (Dias et al.,2005). Total replacement of fish meal by a blend of corn gluten, wheatgluten, extruded peas and rapeseed meal in the diet for trout affectedtotal lipid content and fatty acid composition of fillet (de Francescoet al., 2004). Similarly, in gilthead sea bream replacing 75% of dietaryfish meal protein by the same blend of plant protein sources resultedin a different lipid distribution among tissue (de Francesco et al., 2007).
In general, vegetable protein sources entail a worsening of the n−3/n−6 PUFA ratio due to high level of n−6 PUFA in the lipid fractionof vegetable meals (de Francesco et al., 2004, 2007; Figueiredo-Silvaet al., 2010). The relationship among protein sources, adiposity, fatdistribution and muscle fatty acid composition has been scarcelystudied in fish and particularly in European sea bass where excess ad-iposity is considered a fault by the consumer.
The adiposity in carnivorous fish species is related to dietaryenergy intake (Cowey, 1993), but there is also evidence that dietaryprotein source per se influences in fish, like in other animal models,the regulation of lipid metabolism affecting the gene expression andactivity of lipogenic enzymes (Barroso et al., 1994; Dias et al., 1998,2005; Iritani et al., 1986, 1996; Lupianez et al., 1989; Tulli et al.,1999; Walton, 1985) and in vitro adipocites lipolysis (Albalat et al.,2005).
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Due to the complexity of the mechanisms by which the dietaryprotein sources per se, without any manipulation of the dietary oilsource, could affect lipid metabolism in fish and because of the varietyof alternate protein sources, it is necessary to specifically focus on theconsequences of their inclusion. For this reason, the aim of the presentstudy was to investigate the effects of replacing dietary fish meal withgraded levels of wheat gluten, an ingredient which has been poorlystudied in fish species different from salmonids, alone or in associa-tions with legume seed derivatives such as pea or soybean meal ongrowth and lipid composition and metabolism in European sea bass(Dicentrarchus labrax) through the analysis of adiposity in tissues,plasma metabolites, selected lipogenic liver enzyme activities andfatty acid composition of muscle tissue.
2. Materials and methods
2.1. Test diets
Five experimental diets, whose composition is shown in Table 1,were formulated to be grossly iso-nitrogenous (7.9±0.13% DM),isolipidic (ether extract, 17.0±0.15% DM) and isocaloric (gross energy22.5 MJ kg−1 DM). Crude protein, starch and lipid levels were chosenbased on the figures suggested as optimal for this species by Peresand Oliva-Teles (1999, 2002). A positive control (diet F) contained680 g kg−1 Chile Super Prime fish meal as the sole protein source.The experimental diets WG50 and WG70 were respectively obtained
from preparation F by substituting 50 and 70% fish meal (proteinbasis) with wheat gluten. The test diets coined as WGpea and WGsbmwere obtained by replacing 70% fish meal (protein basis) in F dietwith a blend of wheat gluten and extruded pea meal or wheat glutenand dehulled, toasted, solvent-extracted soybean meal. The diets thatincluded vegetable proteins were proportionally supplemented withthe most limiting essential L-amino acids, to meet as close as possiblethe dietary ideal protein profile of the European sea bass (Tibaldi etal., 1996) (Table 1). The fatty acid profile (% of FAMES) of the experi-mental diets is shown in Table 2. The diets were manufactured at thefacilities of the Department of Food Science, Udine University. All ingre-dients were ground through a 0.5 mm sieve before final mixing, drypelleting through a 3.5 mm dye. The feeds were stored at 4 °C untilused. Dry matter, crude protein, crude fiber and the ash level of thetest diets were analyzed according to AOAC (1995) (Table 1).
The gross energy content was determined by an adiabatic bombcalorimeter (IKA C7000, Werke GmbH and Co., Germany). Starchlevel was analyzed according to a polarimetric method (AOAC, 1995).
2.2. Fish and experimental conditions
Five hundred and twenty five European sea bass juveniles(initial body weight 23.9±0.1 g), obtained from a commercial hatchery(Panittica Pugliese, Brindisi, Italy) were randomly divided among15 groups each consisting of 35 specimens and stocked in 250-l fiberglasstanks of an indoor partially-recirculating water system (total volume,10 m3, daily water volume renewal rate, 5%, artificial day length=12 h,light intensity, 400 lx) providedwith thermostatic control and regulationof water temperature, mechanical sand-filter, biological filter and UVlamp apparatus. The system ensured nearly constant and optimalwater quality to fishes (temperature 23.3±0.5 °C, salinity, 30.0±2‰,dissolved oxygen, 6.4±1.5 mg l−1, pH 7.2±0.5, total ammonialevelb0.15 mg l−1, nitrite levelb0.05 mg l−1). After stocking, fish werefed a commercial diet and adapted over 2 weeks to the experimentalconditions. At the end of this period the 15 groups were assigned to theexperimental diets according to a random design with triplicate groups(tanks) per treatment. Fish were hand-fed the experimental diets over96 days in two daily meals (9:00 am and 4:00 pm) until the first feeditem was refused. During this period the actual feed intake per groupwas recorded daily. Fish were group-weighed every 3 weeks, after a24-h fast and under moderate anesthesia (20–50 mg l−1 of 3-amino
Table 1Dietary formulation (g kg−1), proximate composition (% DM) and gross energycontent of the experimental diets.
Experimental diets
F WG50 WG70 WGpea WGsbm
Ingredients (g kg−1)Chile super prime fish meal 680 340 195 195 195Wheat gluten meala – 292 410 325 164Solvent-extracted soybean mealb – – – – 383Extruded pea mealc – – – 280 –
f Carboximethylcellulose, Laboratori Dottori Piccioni.g Celite®, from Prolabo, France.h From Sigma chemicals, Milan, Italy.i Gross energy (MJ kg−1 DM).
Table 2Fatty acid profile (% of FAMEs) of the experimental diets.
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benzoic acid ethyl ester,MS222, Sigma-Aldrich,Milan, Italy). The trial hasbeen carried out in accordance with the EU Directive 2010/63/EU.
At the end of the trial, specific growth rate (SGR=100×[(ln FBW− ln IBW)/days]), feed conversion ratio (FCR=feed intake/biomass gain), dry matter intake (DMI=feed intake/average bodyweight/day), protein gain (PG=[final whole body protein con-tent− initial whole body protein content]/average body weight/day) and energy gain (EG=[final whole body energy content− initialwhole body energy content]/average body weight/day) were calcu-lated per tank.
2.3. Tissue sampling and analysis
At the endof the growth trial 4fish per tankwere randomly sampledfrom each group 6 h after the morning meal. Individual blood sampleswere withdrawn from caudal vessels using heparinized syringesunder moderate anesthesia. Plasma was obtained after centrifugationat 2000 ×g for 20 min at 4 °C and stored at−20 °C until analyzed. Be-sides, 3 fish per tankwere sacrificed by lethal anesthetic dose after 24 hfasting period, pooled and stored at−20 °C for subsequent whole bodycomposition analysis. At the same time, other 3 fish per tank weresacrificed by a sharp blow to the head to carry out liver enzymatic andlipid analysis. Liver, portion of muscle, and mesenteric fat were quicklycollected, weighed, frozen in liquid nitrogen and stored at −80 °C forsubsequent individual analysis. Total lipid content was determinedaccording to Folch et al. (1957) in whole body pooled samples and inindividual samples of muscle, liver and mesenteric fat.
The separation of phospholipids (PL) and triacylglycerols (TAG) inmuscle was performed by solid phase partitioning chromatographyaccording to Juaneda and Rocqueline (1985).
The fatty acid profile of the diets and of individual muscle PL andTAG has been determined. Briefly, a 5% solution of HCl in methanolwas used to prepare fatty acid methyl esters (FAMEs) according toChristie (2003). FAMEs were separated and quantified by gas chroma-tography (TRACE GC2000, Fisher Scientific SAS, F67403 Illkirch Cedex,France) using a 30 m×0.32 mm i.d. Omegawax™ Capillary GC Column(Supelco Bellefonte, PA, USA) and a flame ionization detection at250 °C. Carrier gas (H2) flow rate was 1 ml min−1 and samples (1 μl)were directly injected in a split ratio of 1:30. Oven temperature wasprogrammed from 180 °C for 6 min, then at 3 °C/min till 225 °C andheld for 10 min. The chromatograms were acquired and processedusing ChromQuest integration software (ThermoQuest Italia, Rodano(MI), Italy) and individual methyl esters were identified by comparisonto known standards (Ackman, 1980). Data on individual fatty acidcomposition are expressed as percent of total identified fatty acids.
Plasma levels of total cholesterol (chol), HDLchol, TAG and glucose(Glu) were determined using commercial kits validated for Europeansea bass (Eurokit, Gorizia, Italy).
The hepatic activities of glucose-6-phosphate deydrogenase(G6PD, EC 1.1.1.49), malic enzyme (ME, EC 1.1.1.40) and ATP citratelyase (ACL, EC 4.1.3.8) were determined according to Dias et al.(1998). Briefly, frozen liver samples were homogenized in eightvolumes of ice-cold buffer (0.02 M Tris–HCl, 0.25 M sucrose, 2 mMEDTA, 0.1 M sodium fluoride, 0.5 mM phenyl methyl sulfonylfluoride, 10 mM β-mercaptoethanol, pH 7.4) and centrifuged at30,000 ×g, at 4 °C for 20 min. The enzymatic activity was assayed onsupernatant: G6PD according to Bautista et al. (1988), ME accordingto Ochoa (1955) and ACL according to Srere (1962). The hepatic solubleproteins on each sample were measured according to Bradford (1976).Enzyme activity (IU, International Unit), was defined as μmol of sub-strate converted to product per minute, at assay temperature. Specificactivity was expressed as enzyme activity mg−1 of hepatic solubleprotein per minute or per gram of liver tissue (wet weight) per minute.To take into account possible variations in the liver to bodyweight ratio,activities were also calculated per 100 g fish.
2.4. Statistical analysis
Data were subjected to one-way ANOVA and, if necessary, meanswere compared using the Duncan's test (significant level 95%, a, b,and c or 99%, A, B, and C).
Overall data on tissue FA composition were analyzedchemometrically by multivariate principal components analysis(PCA), with the score plots being used as a pattern recognition tool.Similarities between the FA profiles of tissue for each lipid fractionfrom the dietary treatments were studied using the D coefficient ofdistance (McIntire et al., 1969). All analyses were completed usingthe SPSS package (SPSS Inc., Chicago, IL).
3. Results
3.1. Growth performance
As shown in Table 3, the final body weight (values range amongdiets 77.5–80.5 g fish−1) and SGR (1.26–1.31%BW day−1) were notsignificantly affected by the dietary treatment, while a significantincrease in FCR was observed in fish fed diets WGsbm and WGpeacompared to fish fed diet F and WG50 and WG70 mainly due toincreased voluntary feed intake. Whole body protein (16.6±0.18%fresh weight) and energy contents (10.31±0.32 kJ/g fresh weight),did not differ among dietary treatments as well as whole body proteinand energy gains (p>0.05).
3.2. Tissue lipid content
Total lipid content of whole body and of different tissues of fish fedthe experimental diets are shown in Table 4. Lipid content in wholebody and mesenteric fat (g 100 g fish−1) did not change in responseto dietary treatments. Fish fed diets including legume seed deriva-tives resulted in reduced fat content in liver relative to those giventhe control and wheat gluten-based diets (pb0.05).
The decrease in fat liver is reflected on hepatosomatic index ofWGsbm fed fish, which had the lowest value, 2.90 vs. 2.36 (pb0.05)(Table 4).
Relative to the controls, fish fed WGsbm diet showed the lowestlipid content in muscle tissue (2.5 vs. 4.0 g 100 g tissue−1, pb0.05)while higher values were observed in fish fed diets with increasinglevels of wheat gluten (pb0.05). In general, levels of PL and TAGlipid in muscle reflect those of total lipid content in the same tissue,and PL content was always higher and cholesterol always lower infish fed vegetable protein based diets relative to controls (pb0.05)(Table 4).
Table 3Feed intake, growth performance, feed conversion ratio, protein and energy gain in Eu-ropean sea bass fed the experimental diets over 96 days.
Row means with the same superscript letters are not significantly different (pb0.05).a Initial body weight (g).b Final body weight (g).c Specific growth rate [(% BW) day−1].d Voluntary feed intake (g kg ABW−1 day−1).e Feed conversion ratio.f Protein gain (g kg ABW−1 day−1).g Energy gain (kJ kg ABW−1 day−1).
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The fatty acid profiles of muscle TAG and PL are shown in Tables 5and 6. Saturated and monounsaturated fatty acid profiles in muscletissue were little or not affected by dietary treatments. On thecontrary, major changes occurred in the n−6 PUFA profile, whichshowed increased incidence in the muscle TAG lipid fraction and toa lesser extent in the PL fraction of fish fed vegetable protein baseddiets relative to controls, mostly mirroring the higher percentage inα-linoleic acid in the replaced diets. On the other hand, percentageof n−3 PUFA in the muscle TAG fraction were significantly lower infish fed the diets containing the highest amount of vegetable proteinsrelative to controls, reflecting the lower percentage of EPA and DHAin diets including vegetable protein sources (Table 2). This effectwas less marked in the polar lipid fraction where not a clear trendbetween controls and vegetable protein fed fish was observed in then−3 PUFA, EPA and DHA incidences and in the EPA/DHA ratio.
The relationship between dietary intake and dorsal muscle con-tent of single fatty acid or fatty acid categories was also studied by aregression approach. A significant correlation (pb0.05) was foundfor EPA (r=0.83), DHA (r=0.52) and n−6 PUFA (r=0.49) only in
the TAG fraction, while scarce of absent correlation was observedfor other individual fatty acid or other fatty acid classes.
Fig. 1A and B shows the factor score plot of the principal compo-nents analysis for the fatty acid profile of polar and neutral lipids indorsal muscle of fish fed the experimental diets studied throughPCA. The first two components accounted for 56% of the totalvariation when PL were considered (Fig. 1A). Twenty-nine percentwas explained by component 1 and 27% by component 2 whichseparated the control diet and the substituted diets. In TAG compo-nents 1 and 2 accounted for 76% of the total variation and contributedrespectively for 47% and 29% (Fig. 1B). In order to find the influence oforiginal variables on the PC axes and consequently the influence oforiginal variables on the formed clusters, data (not shown) of theloading plot suggest that the first component, PC1, is associatedmain-ly with the myristic (14:0), palmitic (16:0) and stearic (18:0) acids,more abundant in the F diet, while the second component, PC2, rep-resents mainly the oleic and linolenic (18:3n3) acid.
3.3. Activity of lipogenic enzymes
The activities of the liver lipogenic enzymes in European sea bassfed the experimental diets, are presented in Table 7.
G6PD activity, measured as IU 100 g fish−1, resulted in a narrowrange of values with a maximum ratio of 1.62 between WG70 andWGsbm diets, and it was 2–20 fold higher than the analogous valuesof ACL and ME. A significant lower activity of G6PD was found only infish fed diet WGsbm in comparison with the control diet and dietWG70 (54.60 vs. 87.13 IU/100 g−1
fish; pb0.05). The hepatic activityof ME (IU 100 g fish−1) varied within a broader range of figures witha maximum ratio of 3.4 between WG70 and WGpea diet. Replacing50% and 70% of fish meal protein with wheat gluten meal resultedin a significant increase of the liver activity of ME compared to theother diets (6.8 vs. 2.58 IU 100 g fish−1; pb0.05) which were similarto the control. A similar trend was observed for ACL activity thatresulted to be significantly increased when wheat gluten meal wasused to substitute 50 or 70% protein from fish meal, with a maximumratio of 4.3 between WG70 and F, while the presence of legumesmaintained the values similar to those of the F diet. Besides, a
Table 4HSI, total lipid content in the whole body, mesenteric fat, liver, dorsal muscle and TAG,PL and Chol concentration in dorsal muscle of fish fed the experimental diets over96 days.
Row means with the same superscript letters are not significantly different (a,b,c=pb0.05; A,B,C=pb0.001).
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significant positive correlation was observed between each enzymeactivity, G6PD (r=0.474, pb0.05), ME (r=0.646, pb0.01), ACL (r=0.721, pb0.01) and dietary starch content.
3.4. Plasma metabolites
Plasma concentrations of TAG, Chol, HDLChol and Glu are presentedin Fig. 2. PlasmaTAG levelwas little affected by the dietary protein source,while plasma Chol level was significantly reduced in fish fed diet
containingwheat gluten associatedwith soybeanmeal. The latter dietarytreatment also significantly reduced plasma HDLChol (112.27 vs.149.45 mg dl−1, pb0.05) and Glu (82.69 mg dl−1 vs. 97.92 mg dl−1,pb0.05) related to the control diet F.
When the plasma glucose levels were regressed against thedietary starch content a positive relationship was found (r=0.437,pb0.01).
4. Discussion
The results of the present study show that replacing up to 70% fishmeal protein by wheat gluten in diets supplemented with the mostlimiting amino acids did not adversely affect feed intake, growth,feed and nutrient conversion efficiency in European sea bass. Otherstudies carried out with different carnivorous fish species have alsoshown that wheat gluten can replace significant proportions of die-tary fish meal without hampering growth performance and feed con-version efficiency (Davies et al., 1997; Helland and Grisdale-Helland,2006; Pfeffer et al., 1992; Robaina et al., 1999; Rodehutscord et al.,1994; Schumacher et al., 1997; Storebakken et al., 2000), providedthat diets high in wheat gluten are supplemented with lysine, thefirst limiting amino acid in wheat gluten. The need for a dietary addi-tion of arginine, which is the second limiting amino acid in wheat glu-ten, still appears controversial based on some investigations carriedout in salmonids (Davies et al., 1997; Fournier et al., 2003; Pfefferet al., 1992) and in the European sea bass (Tulli et al., 2007) fedWG-containing preparations. In this experiment combining wheatgluten with pea meal resulted in nearly optimal growth responseand reduced the need of a huge dietary supplementation with essen-tial amino acids. This is not surprising because pea meal has beenshown to have a high nutritive value in diets for the European seabass (Gouveia and Davies, 1998; Gouveia and Davies, 2004) as wellas in other carnivorous species (Collins et al., 2012; de Francescoet al., 2007). The diet containing wheat gluten and soybean mealresulted in the worst FCR mostly due to increased feed consumptionwhich is consistent with a compensatory mechanism given thequite high inclusion level of dehulled oil-extracted toasted soybeanmeal (383 g kg−1, Table 1) and the lower protein and energy digest-ibility values of this ingredient in sea bass if compared with wheatgluten and pea meal (Tulli et al., 2004).
The effects of the dietary protein source on adiposity have beenstudied with controversial results in the fish species investigated todate. In the present trial, feeding European sea bass diets varying inprotein source over 96 days didn't affect whole body and mesentericfat content. Similar to our results, no effects on whole body were ob-served in a number of carnivorous fish species fed different vegetableprotein-rich ingredients (Brown et al., 1997; Carter and Hauler, 2000;de Francesco et al., 2007; Dias et al., 2005; Hansen et al., 2007;Oliva-Teles et al., 1998; Tibaldi et al., 2006; Yang et al., 2011). Itshould be noted however that in the present study as well as inmost of the experiments quoted above, fish were fed to apparent sa-tiety isolipidic and nearly isocaloric diets and this could have maskeda possible influence of the diet composition on body fat content. Onthe contrary, in red drum a decrease in whole body adiposity wasreported when fish meal was substituted by graded amounts of soy-bean meal (Reigh and Ellis, 1992), and Weeks et al. (2010) obtaineda negative dose-dependent response of body lipid content to dietarysoybean level from 0 to 30% in Atlantic salmon. In the same speciesOlli et al. (1994) and Olli and Krogdahl (1995) also reported a de-creasing effect of dehulled solvent-extracted soybean meal on carcassand viscera lipid content. On the other hand Kaushik et al. (2004)observed increased whole body fat content in European sea bass feddiets containing graded levels of wheat gluten but already includinga blend of vegetable protein sources such as corn gluten, soybeanand rapeseed meals.
A
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Fig. 1. Factor score plot of the principal components analysis for the fatty acid profile ofpolar (A) and neutral (B) lipids in dorsal muscle in fish fed fish meal (solid rhombus),WG50 (circles), WG70 (triangles), WGpea (crosses), and WGsbm (solid squares) over96 days. Data are represented as single value (n=3).
Table 7Activity of the lipogenic enzymes of liver in fish fed the experimental diets over96 days (n=9).
Enzyme Experimental diets
F WG50 WG70 WGpea WGsbm SEM
G6PDIU/100 g fish 85.35a 71.85ab 88.91a 71.99ab 54.60b 19.05IU/g of liver 27.33 23.39 25.78 27.09 25.2 5.87IU/mg protein 0.621a 0.507ab 0.508ab 0.41b 0.371b 0.051
MEIU/100 g fish 3.21b 6.43 a 7.24a 2.15b 2.39b 2.26IU/g of liver 1.4ab 1.91 a 2.07a 0.89b 1.18b 0.64mIU/mg protein 30.34ab 36.33a 41.04a 17.08bc 14.28c 14.04
ACLIU/100 g fish 9.59c 25.23b 41.32a 17.42bc 12.14c 8.97IU/g of liver 3.03c 6.61b 12.25a 7.3b 5.75b 2.05mIU/mg protein 74.0b 118.16b 229.27a 92.74b 69.87b 50.02
Row means with the same superscript letters are not significantly different (pb0.05).
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Despite no absolute changes in overall adiposity and mesentericfat, in the present experiment a different lipid distribution betweendorsal muscle and liver was observed. Wheat gluten meal, even atthe highest dietary inclusion, did not affect liver lipid content whilethe dietary inclusion of both legume seed derivatives resulted in a re-duction of liver lipid content. On the contrary, dorsal muscle lipidcontent increased at the highest level of fish meal protein substitution(WG70 and WGpea) except in case of the diet including substantiallevel of soybean meal which resulted in a reduction in fat concentra-tion. These different partitioning could depend on the dietary pro-tein sources as it was shown also by Dias et al. (2005), whichfound the lipid content of dorsal muscle in European sea bass fed asoybean protein concentrate-based diet to be similar to that of fishfed fish meal, but significantly lower than that of fish fed a corngluten-based diet.
There is evidence from different animal models, including fish, thatthe effect of varying the dietary protein source on fat content coulddepend, to some extent, on changes in the activity of liver lipogenicenzymes. In rats fed different protein sources the activities of G6PD,ME, ACoAC and FAS were reduced when the animals were fed glutenor soybean protein in comparison to those fed casein or fish protein(Iritani et al., 1986). In a second experiment with rats, the differencesin enzyme activity between soybean and casein were mirrored bysame changes inmRNA concentration suggesting that lipogenic enzymeinduction is regulated at a transcriptional level (Iritani et al., 1996). Adepressing effect on all lipogenic hepatic enzymes was also observedby Dias et al. (2005) in European sea bass fed diets containing soy pro-tein concentrate but not in fish fed corn gluten based diet where onlythe activity of G6PD, ME and ACL were depressed when compared tothose of controls fed a fish meal-based diet.
Differences in the amino acid composition (EAA balance/imbalance;ketogenic vs. glucogenic amino acid ratio) of diets including vegetablevs. animal proteins have been quoted as a major factor in the mecha-nisms modulating de novo fatty acid synthesis (Dias et al., 2005;Herzberg, 1991; Iritani et al., 1986), even if apart from this, other dietarynon-protein components of the plant protein sources could also be in-volved inmodulating lipid tissue balance not only through the lipogenicbut also stimulating the lipolytic pathway (Iritani et al., 1996).
In the present study, however, the role of the amino acid compo-sition of the different protein sources on fat synthesis in liver remainsspeculative given that we did not measure the activities of majorlipogenic enzymes such as FAS and ACoAC while in case of G6PD,ME and ACL activities, the effects of dietary protein source and starchlevel were confounded. In fact, a significant positive correlation wasfound between dietary starch content, glycemia and the activities ofsuch lipogenic enzymes as already reported by Meton et al. (1999)in sea bream and by Alvarez et al. (2000) in isolated trout liver cellcultures. In general this is also in agreement with the well known ef-fect of digestible carbohydrates intake on de novo fatty acid synthesis(Herzberg, 1991). These three NADPH citoplasmatic producing en-zymes supply the reducing power to de novo fatty acid synthesis.G6PD provides information on the reducing equivalents originatedfrom glucose metabolism, ME has a role in supplying reducing equiv-alents for the fatty acid synthetase activity and in supplying piruvate,which returns to the citric acid cycle (Iniesta et al., 1985), and ACLprovides acetyl groups to acetyl CoA carboxylase to support de novolipogenesis. In the present trial, G6PD activity resulted to be 2–20fold higher than those of ME and ACL, which is similar to the observa-tion of Bautista et al. (1988) in the same species, demonstrating thatNADPH production via pentose phosphate pathway in European seabass is a major pathway to supply reducing power for fatty acidsynthesis. Similarly very high activity of G6PD relative to that of MEhas been found in Atlantic salmon (Lin et al., 1977), channel catfish(Likimani and Wilson, 1982), rainbow trout (Barroso et al., 1994)and European sea bass (Dias et al., 1998).
Plasma glucose levels observed in the present trial were similar tothose reported in European sea bass fed diets containing almost adouble amount of starch (30%) (Enes et al., 2010), thus confirmingthe different availability of starch according to different vegetablefeedstuffs (reviewed by Enes et al., 2011). The significant positive re-lationship between dietary starch level and plasma glucose concen-tration observed in the present trial confirms that European seabass is able to efficiently digest and absorb glucose from gelatinizedwheat starch and pea meal and likely to utilize dietary amino acidsto maintain the homeostasis of glucose. Optimal availability of peameal starch was also reported by Gouveia and Davies (2004) in the
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Fig. 2. Concentration of plasma metabolites in fish fed the experimental diets over 96 days (n=4). Columns with the same letters are not significantly different (pb0.05).
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same fish species fed diets including 40% of pea meal providing, likein the present experiment, the bulk of the dietary starch.
In this trial, feeding diets differing in source and level of protein-richvegetable ingredients had a slight effect on plasma triacylglycerolswhile resulted in lower cholesterol level in tissues. The inclusion ofsoybean meal in the diet caused a significant reduction of plasmacholesterol while dorsal muscle cholesterol was significantly reducedin fish fed all the plant protein based diets. The causes that determinethe well known cholesterol-lowering effect of soybean derivativesin tissues of different animal models, are complex (Carrol andKurowska, 1995; Cho et al., 2007; Potter, 1995) and at least partiallyattributable to the lack of cholesterol in vegetable ingredients. Thishas been assumed as the main reason to explain similar effects in fishspecies like rainbow trout (Kaushik et al., 1995; Romarheim et al.,2006) and European sea bass (Dias et al., 1998, 2005; Tibaldi and Tulli,2002; Tulli et al., 1999). It is not clear yet whether or not the cholesterollowering effect of soybean meal depends on its different protein com-position, or in the presence of isoflavones (Setchell and Cassidy,1999). In fact, a recent study refers to the direct effect of soy isoflavoneson the expression of FAT/CD36 and muscle VLDL receptors that partici-pates in plasma fatty acid transport andmuscle uptake in rainbow trout(Kolditz et al., 2010).
Interestingly the other legume seed derivatives tested here (e.g. peameal), in combination with wheat gluten, did not affect neither plasmametabolites nor hepatic lipogenic enzyme activities, while dorsal mus-cle cholesterol content was significantly reduced although lessmarked-ly than in the case of fish fed the diet containing the higher level ofwheat gluten meal alone. A cholesterol decreasing effect of pea mealhas been reported even in mammals by Rigamonti et al. (2010).
The causes by which dietary vegetable proteins affect cholesterolmetabolism remain to be better elucidated even if some of the mech-anisms proposed for mammals could be applied to fish as well. Morestudies are needed in this direction to better understand the contra-dictory results on muscle cholesterol levels observed so far whenfeeding diet similar in ingredient composition to different fish specieslike rainbow trout and gilthead sea bream (de Francesco et al., 2004,2007).
It is well known that the fatty acid composition is considered aquality parameter of seafood products. Wild and reared marine fishare a food of choice mainly due to their high supply of polyunsaturatedfatty acid, such as long chain (LC) n−3 PUFA. In the present experi-ment, replacing substantial amounts of fish meal by vegetable proteinsources led to some significant changes in fatty acid composition ofTAG and PL in the dorsalmuscle of European sea bass. Fish fed the high-ly substituted diets (70% replacement) similarly resulted in reducedn−3 LC PUFA incidence and in significant increased incidence of n−6LC PUFA in both TAG and PL mainly due to the high content ofα-linoleic acid in the vegetable ingredients, according to the wellestablished relationship between dietary fatty acid composition andflesh fatty acid composition (Wood et al., 2008). Similar results havebeen reported by Izquierdo et al. (2003) and de Francesco et al.(2007) in sea bass and sea bream fed diets high in vegetable proteinsources. It is worth to note that such an effect was more pronouncedin the TAG fraction that confirms to be more dependent from dietcomposition, than was in the PL. This factor should be taken into ac-count in tailoring flesh fatty acid composition of fish with high musclefat stores. The substitution of protein from fish meal with high levelsof plant protein did not reduce the levels of EPA and DHA in the PL,with a selective accumulation of DHA in this fraction, and with minorchanges in the DHA level of the TAG.
Apart from changes in single fatty acid, PCA of the TAG fatty acidpattern allowed the distinction of muscle samples according to thediet. Those originating from fish given the fish meal-based diet wereclearly spaced out from those of specimens fed the WG-based prepa-ration, while those of fish subjected to the diet containing soybeanlied in an intermediate position.
5. Conclusion
The results of the present study indicate that major changes indietary protein source can affect lipid metabolism in European seabass without affecting the growth performance.
The substitution of fish meal by substantial amounts of wheatgluten alone or in combination with pea or soybean meal resultedin different patterns of hepatic lipogenesis and a modified fat distri-bution among tissues. Due to concomitant changes in starch level ofthe diets, the effects of replacing varying levels of fish meal by vege-table proteins on lipid metabolism and tissue composition, weresomewhat confounded because of different digestible carbohydrateintake in sea bass fed the different experimental diets.
It is interesting to note that a diet including wheat gluten andsoybean meal could markedly reduce muscle adiposity. However, alldiets including higher proportions of wheat gluten alone or in combi-nation with legume seed derivatives led to an increased incidence ofn−6 PUFA and reduced levels of n−3 PUFA and in particular DHAin fish muscle tissue relative to controls. These last findings couldhave consequences on the nutritional value of the edible portion ofEuropean sea bass.
Acknowledgments
This research was supported byMIPAAF Italy, research code 5-C-23.Data were partially presented at ASPA 17th Congress, 2007, by the firstauthor.
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