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Selenium levels in early weaning diets for gilthead seabream larvae
Reda Saleh a,b,⁎, Mónica B. Betancor a, Javier Roo a, Daniel Montero a, María J. Zamorano a, Marisol Izquierdo a
a Grupo de Investigación en Acuicultura (IUSA & ICCM), University of Las Palmas de Gran Canaria, Carretera de Taliarte, s/n, 35200 Telde, Gran Canaria, Spainb Oceanography Department, Faculty of Science, Alexandria University, 21515, MoharramBek, Alexandria, Egypt
Article history:Received 17 October 2013Received in revised form 13 February 2014Accepted 14 February 2014Available online 20 February 2014
Keywords:Seabream larvaeSeleniumOxidative stressSkeletal development
The inclusion of complementary antioxidative factors, such as selenium (Se), could counteract the high oxidationrisk in early weaning diets high in polyunsaturated fatty acids (PUFA). The present study investigated the effectsof graded levels of Se derived yeast with krill phospholipids (KPL) on skeletal development, survival, stress resis-tance, oxidative status and biochemical composition of seabream larvae. Seabream larvae were completelyweaned at 16 dph and fed five microdiets for 30 days with different levels of Se: 2SE, 4SE, 6SE, 8SE and 12SE(1.73, 3.91, 6.41, 8.47, 11.65 mg kg−1 dietary dry weight, respectively). Increases in Se up to 11.65 mg kg−1 di-etary dryweight significantly improved survival rate (54%) and stress resistance, but did not affect larval growth.Seabream larvae fed diets supplemented with 12SE (11.65 mg kg−1) showed a gradual increase in this mineralaccording to dietary Se levels, denoting the progressive absorption of this nutrient. The degree of larval lipidoxidation, as indicated by malondialdehyde (MDA) content and antioxidant enzyme (AOE) gene expression,was significantly lower in larvae fed 8SE and 12SE diets compared to those fed 2SE and 4SE diets. Furthermore,a reactive response as a result of Se inclusion was observed by the increase in osteocalcin, osteonectin, osteopon-tin, alkaline phosphatase and matrix gla protein gene expression in larval tissues, suggesting a well skeletaldevelopment. These results denoted the high efficiency of Se as an antioxidant factor and the importance ofthe inclusion of adequate levels (11.65 mg Se kg−1 diet) in early weaning diets.
The oxidation of important nutrients like lipids and proteins thathave critical biological and physiological functions leads to harmful al-terations in fish causing several pathological conditions (Sakai et al.,1989) including cellular damages (Halliwell and Gutteridge, 1996),muscle injuries (Betancor et al., 2012a,b), negative growth, low feed in-take and delayed development in several fish species (Tacon, 1991).Moreover, the oxidation of dietary lipids may also lead to an increasedincidence of deformities in marine fish larvae (Lewis-McCrea and Lall,2007). It is well known that dietary lipids constitute a major source ofenergy and essential fatty acids for fish and play amain role in larval de-velopment (Izquierdo and Koven, 2010; Rainuzzo et al., 1997). Amongthem, dietary phospholipid (PL) levels have been found to positively af-fect survival, growth and resistance to stress and reduce the occurrenceof morphological anomalies in several fish and crustaceans (Kanazawaet al., 1985; Geurden et al., 1998a,b; Cahu et al., 2003a; Liu et al.,2002; Izquierdo and Koven, 2010; Saleh et al., 2013a,b). They constitute
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important sources of essential fatty acids that have a crucial role inmaintaining the structure and function of cellular membranes (Tocher,2003). Besides, theymay act as emulsifiers in the gut and improve intes-tinal absorption of long chain fatty acids (Fontagné et al., 2000). More-over, they stimulate lipoprotein synthesis in intestinal enterocytes(Saleh et al., 2014; Geurden et al., 1998b; Liu et al., 2002) and play animportant role in the transport and assimilation of dietary lipids(Izquierdo et al., 2001). However, dietary PL may have high levels ofpolyunsaturated fatty acids that are very sensitive to peroxidationresulting in production of harmful peroxides and, consequently, affecttheir biological and physiological functions. In previous studies, thiobar-bituric acid reactive substance (TBARS) contents and antioxidant en-zyme gene expression were raised in seabream larvae fed increasinglevels of dietary PL rich in n-3 highly unsaturated fatty acids (HUFA)or linoleic acid (Saleh et al., 2014). Polyunsaturated fatty acids includingthose with five and six ethylenic bonds, such as eicosapentaenoic acid(EPA) and docosahexaenoic acid (DHA), are very susceptible to oxida-tion due to their long chain lengths and the greater number of unsatu-rated carbon–carbon bonds (Betancor et al., 2012c). The mechanism oflipid oxidation begins with auto-oxidation involving the direct reactionof lipids with molecular oxygen to form hydroperoxides, followed bysecondary oxidation reactions yielding diperoxides, which are detri-mental toxic compounds. Besides, dietary lipid oxidation is increasedby factors such as the presence of lipoxidase, hematin, peroxides, light
a Alltech, Lexington, KY.b Rieber and Son, Bergen, Norway.c Qrill, high phospholipids, Aker BioMarine, Fjordalléen, Norway.d Merck KGaA, Darmstadt, Germany.
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(involve in the photolysis of peroxides), high temperature, trace metalnotably iron, copper, cobalt, and zinc (Dabrowski and Guderley, 2002;Sutton et al., 2006). The high dietary lipids may yield products of sec-ondary oxidation of lipids that contribute to off flavour and includetoxic compounds frequently associated with rancidity reducing theirnutritional value (Halliwell and Chirico, 1993). Besides, the larvalphase is one of themost critical stages in fish life cycle, among other rea-sons, for the high requirement of n-3 HUFA, as essential fatty acids fornormal growth and development, that leads to a high oxidative stressrisk in relation to the elevated larval metabolic rate (Evjemo et al.,2003). The lipid oxidation in marine fish larvae could be at least partlyresponsible for the higher disease incidence and subsequent larval mor-talities (Tocher et al., 2002).
The oxidative stress in fish is an aspect of aerobic life that results ofan imbalance between the production of reactive oxygen species(ROS) and antioxidant defence factors in living organisms (Nishida,2011). ROS at low concentrations may be beneficial or even indispens-able in processes such as defence against microorganisms, contributingto phagocytic bactericidal activity. However, the elevated levels of freeradicals that are produced by endogenous cellular sources duringnormal cell metabolism and endogenous ROS that are produced bymitochondrial respiration can cause oxidation of proteins and lipids, al-terations in gene expression, and changes in cell redox status giving riseto oxidative stress (Livingstone, 2003; Rando, 2002). In fish there aredifferent effective antioxidant enzymes (AOEs) capable of inhibitingthe lipid-peroxidation catalytic cycle by catalysing the decompositionof hydrogen peroxide into less reactive gaseous oxygen and watermolecules, including catalase (CAT), superoxide dismutase (SOD) andglutathione peroxidase (GPX). Dietary nutrients also constitute antiox-idant factors thatmust be added in feed diets such as selenium, vitaminsE and C or carotenoids (Betancor et al., 2011, 2012a,c,d; Díaz et al., 2010;Hamre et al., 2010;Montero et al., 2001). Selenium is an antioxidant es-sential tracemineral in the nutrition of marine organisms partly obtain-ed from the surroundingwater (Lall and Bishop, 1977) but mostly fromthe diet (Halver, 2002). Selenium, as an essential micronutrient, playsan important role in antioxidant defences being a cofactor for antioxi-dant enzyme GPX (Felton et al., 1996), which reduces hydroperoxidesby catalysing the oxidation reaction of glutathione (Arteel and Sies,2001). Selenium has been shown to prevent dietary hepatic necrosisand exudative diathesis (Schwarz and Foltz, 1957), its deficiencybeing characterized by cardiomyopathy and muscular weakness (Chenet al., 1980). Despite the fact of being a required micronutrient, Semay be toxic at concentrations only slightly greater than the nutritionalrequirements (Hilton et al., 1980).
There are very few studies denoting the importance of Se supple-mentation in fish diets and, despite its particular importance inmicrodiets for marine fish larvae, none of them has aimed to determinethe larval requirements for this micronutrient by feeding at least 5different dietary levels. Thus, the objective of the present study was toinvestigate the effect of graded levels of yeast derived Se in earlyweaning microdiets rich in KPL on gilthead seabream (Sparus aurata)larva performance, resistance to stress, biochemical composition andexpression of genes related to oxidative stress and bone metabolism.
2. Material and methods
2.1. Fish
Gilthead seabream larvae were obtained from natural spawningsfrom Instituto Canario de Ciencias Marinas (Grupo de Investigaciónen Acuicultura (GIA), Las Palmas de Gran Canaria, Spain). Larvae(5.1 mm total length, 100 μg dry body weight), previously fed rotifers(Brachinous plicatilis) enriched with DHA Protein Selco® (INVE,Dendermond, Belgium) until 15 dph, were randomly distributed in15 experimental tanks at a density of 2100 larvae tank−1. All tanks(200 L fibreglass cylinder tanks with conical bottom and painted
with light grey colour) were supplied with filtered seawater (37 ppmsalinity) at an increasing rate of 0.4–1.0 L min−1 to assure good waterquality during the entire trial. Water entered from the tank bottomand exited from the top to ensure water renewal and to maintain highwater quality,whichwas tested daily andno deteriorationwas observed.Water was continuously aerated (125mLmin−1) attaining 6.3± 1 ppmdissolved O2. Average water temperature and pH along the trial were19.8 ± 1.5 °C and 7.89, respectively. Photoperiod was kept at 12 hlight: 12 h dark, by fluorescent daylights and the light intensity waskept at 1700 lx (digital Lux Tester YF-1065, Powertech Rentals,WesternAustralia, Australia).
2.2. Diets
Five experimental microdiets (pellet size 250–500 μm)were formu-lated containing a PL rich krill oil (Qrill, high PL, Aker BioMarine,Fjordalléen, Norway) and five levels of yeast derived Se (Sel-Plex®2000, 2000 mg Se kg−1, Alltech, Lexington, KY) as source of organicSe. Thus, analysed Se content of the diets was 1.73, 3.91, 6.41, 8.47 and11.65 mg kg−1 for diets 2SE, 4SE, 6SE, 8SE and 12 SE, respectively.Diet formulation and proximate analysis are showed in Table 1 andtheir fatty acids in Table 2. The microdiets were prepared by mixingsquid powder and water-soluble components, then the lipids and fat-soluble vitamins and, finally, the gelatine were dissolved in warmwater. The paste was compressed pelleted (Severin, Suderm,Germany) and dried in an oven at 38 °C for 24 h (Ako, Barcelona,Spain). Pellets were grounded (Braun, Kronberg, Germany) and sieved(Filtra, Barcelona, Spain) to obtain a particle size between 250 to500 μm. Diets were prepared and analysed for proximate and fattyacid composition at GIA laboratories.
The larvaewere completelyweaned at 16 dph and fed one of the dietstested in triplicate. Diets were manually supplied fourteen times per day45 min each from 9:00 to 19:00 for 29 days. Daily feed (pellet size b
250 μm) supply was maintained at 1.5 and 2.5 g per tank during thefirst and second week of feeding. The amount of feed added daily wasgradually increased to 4–5 g per tank with increasing in pellet size to250–500 μm, where an overlap using a mixture of both pellet sizes wasconducted during the third and fourth week of feeding. Larvae wereobserved under the binocular microscope to determine feed acceptance.
2.3. Survival determination
Before the end of the experiment an activity test by thermal shockwas conducted allocating 25 larvae tank−1 in another aerated tank
Table 2Fatty acid (% dry weight) composition in total lipids of diets containing five seleniumlevels.
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supplied with seawater at a temperature of 24 °C and determiningtheir survival after 24 h. Final survival was calculated by individuallycounting all the living larvae at the end of the experiment.
2.4. Biochemical analysis
In addition, at the end of the trial and after 12 h of starvation, all re-maining larvae in each tank were washed with distilled water, sampledand kept at −80 °C for biochemical composition, TBARS and seleniumanalysis.
Moisture (A.O.A.C., 1995), protein (A.O.A.C., 1995) and crude lipid(Folch et al., 1957) contents of diets were analysed. Fatty acid methylesters for larvae and diets were obtained by transmethylation of crudelipids as described by Christie (1982). Fatty acid methyl esters wereseparated by GLC (GC-14A, Shimadzu, Tokyo, Japan) following theconditions described in Izquierdo et al. (1990) and identified bycomparison to previously characterized standards and GLC–MS.
Total seleniumconcentrationwasmeasured in total larvae and diets.Samples were acidified in a microwave digestor (MarsXpress, CEM,Kamp-Lintfort, Germany)with 5mLof 69% pure nitric acid, then pouredafter digestion into a 10 mL volumetric flask and made up to volumewith distilled water. A total of 0.4 mL of this solution was then addedto a 10 mL sample tube, 10 μL of the internal standard (Ga and Sc,10 ppm) was included and 0.3 mL of methanol added. The tubes were
Table 3Sequences of forward and reverse primers (5′–3′) for real-time quantitative-PCR of s
a RUNX2 (Runt-related transcription factor 2).b BMP4 (Bone morphogenetic protein 4).
made up to volume with distilled water and total selenium was mea-sured by collision/reaction ICP-MS (Thermo Scientific, Cheshire, UK)using argon and hydrogen as carrier gas.
The measurement of thiobarbituric acid-reactive substances(TBARS) in triplicate samples was performed using a method adaptedfrom that used by Burk et al. (1980). Approximately 20–30mg of larvaltissue per sample was homogenized in 1.5 mL of 20% trichloroaceticacid (w/v) containing 0.05 mL of 1% BHT in methanol. To this, 2.95 mLof freshly prepared 50 mM thiobarbituric acid solution was addedbefore mixing and heating for 10 min at 100 °C. After cooling proteinprecipitates were removed by centrifugation (Sigma 4K15, Osterodeam Harz, Germany) at 2000 ×g and the supernatant read in a spectro-photometer (Evolution 300, Thermo Scientific, Cheshire, UK) at 532 nm.The absorbance was recorded against a blank at the same wavelength.The concentration of TBA-malondialdehyde (MDA) expressed as μmolMDA per g of tissue was calculated using the extinction coefficient0.156 μM−1 cm−1.
2.5. Gene expression analysis
Fresh larva samples (100mgper tank) formolecular biology analysiswere well washed and conserved into 500 mL RNA Later (SIGMA). Thelarvae were stored overnight in RNA Later at 4 °C and after that RNALater was removed before storing samples at −80 °C.
Molecular biology analysis of genes related to antioxidant enzymesand bone formation and mineralization was carried out. Total RNAfrom larva samples (average weight per sample 60 mg) was extractedusing the RNeasyMini Kit (Qiagen). Total body tissuewas homogenisedusing the TissueLyzer-II (Qiagen, Hilden, Germany)with QIAzol lysis re-agent (Qiagen). Samples were centrifuged with chloroform for phaseseparation (12,000×g, 15min, 4 °C). The upper aqueous phase contain-ing RNAwasmixedwith 75% ethanol and transferred into a RNeasy spincolumn where total RNA bonded to a membrane and contaminantswere washed away by RW1 and RPE buffers (Qiagen). Purified RNAwas eluted with 30 μL of RNase-free water. The quality and quantity ofRNA were analysed using the NanoDrop 1000 Spectrophotometer(Thermo Scientific, Wilmington, DE, USA). Synthesis of cDNA was con-ducted using the iScript cDNA Synthesis Kit (Bio-Rad) according tomanufacturer's instructions in an iCycler thermal cycler (Bio-Rad,Hercules, CA, USA). Primer efficiency was tested with serial dilutionsof a cDNA pool (1, 1:5, 1:10, 1:15,1:20 and 1:25). The product size of
Fig. 1. Larvae survival rate (%) fed the diets 2SE, 4SE, 6SE, 8SE or 12SE (A) at the end of theexperimental trial (44 dph) or (B) 24 h after thermal shock of 24 °C at 44 dph. Values(mean ± standard deviation) with the same letters are not significantly different(p N 0.05).
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the real-time q PCR amplification was checked by electrophoresis anal-yses using a PB322 cut with HAEIII as a standard. Real-time quantitativePCR was performed in an iQ5 Multicolor Real-Time PCR detection sys-tem (Bio-Rad, Hercules, CA, USA) using β-actin as the house-keepinggene in a final volume of 20 μL per reaction well, and 100 ng of totalRNA reverse transcribed to complementary cDNA. Each gene samplewas analysed once per gene. Detailed information on primer sequencesand accession numbers is presented in Table 3.
3. Statistical analysis
All data were tested for normality and homogeneity of varianceswith Levene's test, not requiring any transformation and were treatedusing one-way ANOVA. Means compared by Duncan's test (p b 0.05)using a SPSS software (SPSS for Windows 11.5; SPSS Inc., Chicago, IL,USA).
Table 4Fatty acid (% total identified fatty acids) composition in total lipids of larvae (44 dph) fed diets
Values (mean ± standard deviation) with the same letters in the same row are not significant
4. Results
All the experimental diets were well accepted by gilthead seabreamlarvae according to the microscopic observations. Generally, survivalwas very high for this type of studies and it was significantly correlatedto the dietary Se contents in Fig. 1. Thus, average survival of larvae fed12SE diet (54% survival rate) was significantly higher to 2SE, 4SE and6SE treatments, but was not significantly different to 8SE treatment.Generally, there was a very good survival of the thermal stress andonly survival in larvae fed 12SE diet was significantly different thanthat of the other larvae. The resistance to thermal shock stress wascorrelated to the dietary Se contents in Fig. 1.
The larval growth after thirty days of feeding in terms of total lengthand bodyweight was very good regardless the experimental diets used.
The fatty acid composition of the experimental diets was very simi-lar andwas not affected by the inclusion of different Se levels in Table 2.However, the fatty acid composition of the larvae was significantly af-fected by the dietary Se inclusion in Table 4. Thus, the larvae fed 8SE,and particularly, 12SE diets, were higher in n-3 and n-3 HUFA, mainlydue to higher DHA, than 2SE, 4SE and 6SE larvae. Moreover, thelarval DHA contents were correlated to the Se contents in larval tissues(y = 0.0053x + 24.248, R2 = 0.86269). No significant difference wasfound in n-6, n-9, EPA and ARA contents among the different larvae.
Biochemical composition of the larvae reflected the dietary content.Se content in seabream larvae was correlated to the dietary Se levels.Thus larvae fed diet of 12SE showed the significantly highest contentof this mineral, being 6, 3 and 2 times higher than in the larvae fed2SE, 4SE and 6SE respectively, denoting the progressive absorption ofthis nutrient in Fig. 2.
The degree of larval lipid oxidation in Fig. 2, as indicated by larvalmalondialdehyde (MDA) content, was significantly lower in larvae fed8SE and 12SE diets (152 and 119 nmol g−1 larvae respectively) com-paredwith those of 2SE and 4SE diets (216 and 220 nmol g−1 larvae re-spectively) and was negatively correlated with the dietary Se contents.Besides, theMDA contents in the larvae were also negatively correlatedto the larval Se contents (y = −0.2041x + 257.94, R2 =0.94804).
The general pattern of antioxidant enzyme gene expression ofseabream larvae was a decreased expression with increasing dietarySe levels in Fig. 3. Thus, larvae fed 12SE diets showed significantlylower CAT gene expression than larvae fed 2SE, 4SE and 6SE diets,being CAT gene expression in larvae negatively correlated to the Se con-tent in the larvae (y = −0.0136x + 10.83, R2 = 0.90955). Larvae fed12SE diet also showed significantly lower GPX gene expression thanlarvae fed 2SE and lower SOD gene expression than 2SE and 4SE treat-ments. There was no significant difference between larvae fed 4SE,6SE and 8SE diets in all antioxidant enzyme gene expression.
Fig. 2. Seabream larva selenium contents (A) and lipid peroxidation products (thiobarbi-turic acid-reactive substances (TBARS)) (B) after eating any of the five experimentaldiets for 30 days. Values (mean ± standard deviation) with the same letters are notsignificantly different (p N 0.05).
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The osteological genes that have role in bone-building, formation ofcartilage and bone remodelling and mineralization were expressed inslightly higher levels in larvae fed higher dietary SE contents (8SE and12SE). The osteocalcin, osteonectin, osteopontin, alkaline phosphataseand matrix gla protein genes showed a significantly higher expressionin larvae fed 8SE and 12SE diets than larvae fed 2SE, 4SE and 6SEdiets, while there were no significant differences among the last treat-ments in Fig. 4. A significant correlation was found between ALP geneexpression and larval Se contents (y = 0.0179x + 6.7818, R2 =0.97113) or larval n-3 HUFA contents (y = 1.7433x − 57.472, R2 =0.9068). There were no significant differences among all treatments inRUNX2 (Runt-related transcription factor 2) gene expression in Fig. 4.The larvae fed 8SE diet have higher BMP4 gene expression than the alltreatments, while there was no significant difference between the 4SE,6SE and 12SE treatments in Fig. 4.
Fig. 3. Catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX)gene expression levels measured by real-time PCR in Sparus aurata larvae when fed thediets 2SE, 4SE, 6SE, 8SE and 12SE with increasing levels of selenium for 30 days. Values(mean ± standard deviation) with the same letters are not significantly different(p N 0.05).
5. Discussion
The aim of the present study was to evaluate the effect of gradedlevels of Se derived yeast in diets containing KPL rich in n-3HUFA on lar-val production performance, osteological gene expression, oxidativestatus and biochemical composition of seabream larvae. Although theincreased Se levels did not affect larval growth, the highest dietary Secontent (11.65 mg kg−1 diet) promoted larval survival rate and resis-tance to stress. This positive effect in larval survival or stress resistancecould be related in one hand to the n-3 HUFA increase on larval tissuescaused by a higher protection of these fatty acids against oxidation bythe increased Se content in the larvae. Indeed, n-3 HUFA are known toplay a crucial role in larval survival (Izquierdo and Koven, 2010). Onthe other hand, the higher survival and stress resistance could be alsorelated to the reduction in oxidation products, which can be severelyharmful for fish causing several pathological damages and larvalmortal-ities (Betancor et al., 2011; Halliwell and Gutteridge, 1996). For in-stance, the inclusion of Se in diets for European seabass proved to beefficient in controlling the damage caused by the reactive oxygen
species, reducing the incidence of muscle injury to almost half as com-pared with the diet without Se supplementation (Betancor et al.,2012c).
Indeed, in the present study, increased dietary Se levels resulted inhigher Se contents in larval tissues and an improved protection againstperoxidation denoted by the decreasingMDA concentrations and show-ing the essential role of Se as an antioxidant factor that reduces hydro-peroxides (Arteel and Sies, 2001; Betancor et al., 2012c). Moreover,the increase in Se tissue contents was negatively correlated to the CATgene expression andwas also related to lower SOD and GPX, suggestingaswell a reduced peroxidation risk and a better oxidative stress status inlarvae fed the higher dietary Se levels. These results are in agreementwith those found in Manchurian trout (Brachymystax lenok) and seabass (Dicentrarchus labrax) larvae, where feeding high dietary lipid
Fig. 4. Bonemorphogenetic protein 4 (BMP4; A), runt-related transcription factor 2 (RUNX2, B), alkaline phosphatase (ALP, C), osteocalcin (D), osteopontin (E), osteonectin (F) andmatrixGla protein (G) gene expression levelsmeasured by real-time PCR in seabream larvaewhen fed the diets 2SE, 4SE, 6SE, 8SE and 12SEwith increasing levels of selenium for 30 days. Values(mean ± standard deviation) with the same letters are not significantly different (p N 0.05).
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levels increased MDA production and induced an antioxidant responsenoticeable by an increase in the activity of antioxidant enzyme genes(Betancor et al., 2012a,c,d; Zhang et al., 2009). These results agree wellwith other studies that demonstrated that Se supplementation has aprotective effect against oxidative stress caused by high dietary DHAin sea bass or methyl parathion in Brycon cephalus, as denoted by thedecrease in CAT and SOD activity (Monteiro et al., 2009) or gene expres-sion (Betancor et al., 2012a). Thus, the inclusion of antioxidative factors,such as Se, could counteract the high oxidation risk in early weaningdiets high in PUFA that are more susceptible to oxidation not only inthe inert diets but also in larval tissues.
The impact of the inclusion of Se on microdiets with high dietary PLon the expression of genes considered as biomarkers of bone develop-ment and mineralization is still limited. Antioxidants may interactwith cellular receptors and transcriptional factors that may furtherlead to changes inmRNA and protein levels or directly interact with en-zymes through protein–protein-binding properties (Olsvik et al., 2011).Elevated intracellular ROS production may ultimately damage DNA,proteins and lipids (Halliwell andGutteridge, 1999). Also, ROS can affectthe transcription of many genes, acting either via various transcriptionfactors or directly as a result of oxidative damage (Di Giulio andMeyer, 2008). As observed in our study the higher dietary Se levelsled to the promoted expression of bone formation and mineralizationgenes, where the BMP4, alkaline phosphatase, osteocalcin, osteonectin,osteopontin, and matrix gla protein genes, all important for skeletaldevelopment, bone formation and mineralization, were up-regulated
by the dietary inclusion at 8.47 and 11.65 μg Se mg−1 diet. This up-regulation of several bone metabolism related genes could be due tothe higher larval tissue content in n-3 HUFA, and particularly DHA, inagreement with the higher expression of these genes when DHArose in gilthead seabream larval tissues (Saleh et al., 2014). In other ver-tebrates, these genes, particularly BMP-4, are up-regulated by dietary n-3 HUFA exerting a beneficial effect (Kruger et al., 2010). Among the im-portant mechanisms of PUFA action are their effects on gene transcrip-tion that can be mediated by fatty acid binding to the peroxisomeproliferator activator receptor (PPAR) transcription factors that haveimportant effects on bone physiology (Kruger et al., 2010). The presentstudy demonstrated improved osteological gene expressionwith higherlarval DHA content, where DHA enhances expression of key transcrip-tion factors such as specific transcription factors such as RUNX2 (alsoknown as core binding factor-1, Cbf1) and osterix that enhances differ-entiation of pre-osteoblasts into mature osteoblasts that elevate bonemass (Kruger et al., 2010). The present study had shown a coincidencebetween the lower larval MDA content and lower AOE gene expres-sion and the higher larval n-3 HUFA content in the larvae fed higherdietary Se levels that resulted in improving the regulation of the genesinvolved in skeletal development and mineralization of BMP4,osteocalcin, osteonectin, osteopontin, alkaline phosphatase and matrixgla protein genes.
Betancor et al. (2012c) found increased CAT, SOD and GPX expres-sion in sea bass larvae fed a high-DHA and Se-free diet but similarly, ex-posure to high-DHA diets caused a significant increase in CAT and GPX
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in the larvae fed the Se-supplementeddiets, that canbe interpretedby thehigher requirements of sea bass larvae of Se (more than 6.27 μg mg−1
diet) to be more effective. This is similar to the present study, wherethe increase of Se levels up to 6.41 μg mg−1 diet did not affect signifi-cantly larval performance, MDA content and AOE gene expression, butthe increase up to 8.47 μg mg−1 diet and 11.65 μg mg−1 diet resultedin better larval survival and resistance to stress, and also resulted inlower MDA content and AOE expression levels. It can be concludedthat, supranutritional levels of Se (more than 8.47 μgmg−1 diet) are re-quired formarine fish larvae to reduce significantly the larvalMDA con-tent and consequently AOE gene expression. Also, another studyshowed that supranutritional levels of Se are required to reduce the in-cidence of human and animal diseases (Brown and Arthur, 2001).
Finally, the Se levels used did not seem to be excessive, as the larvaefed the highest Se dietary contents did not show reduced growth or anyother detrimental effects. Excessive levels of dietary Se have been asso-ciated with reduced growth. Chronic Se toxicity for rainbow trout juve-niles (1.3 g) occurred at feed levels of around 13mg Se kg−1 supplied assodiumselenite that resulted in reduced growth (Hilton et al., 1980) butRider et al. (2010) demonstrated that therewere no toxic effects at 8mgSe kg−1 supplied mainly as Se via selenoyeast for rainbow trout juve-niles. While in cod larvae, reduction in growth occurred at 7–17 dphwhen fed rotifers with 3 mg Se kg−1 DW that may indicate excessivedietary Se at this larval age (Penglase et al., 2010). In agreement withour previous results (Betancor et al., 2012c), the Se organic sourceused derived from yeast could had a lower toxicity than inorganic Se(Hilton et al., 1980; Rider et al., 2010), since Se toxicity is highly depen-dent on its speciation (Tinggi, 2003).
Hamre et al. (2010) found that Se content in rotifers are consid-erably low (0.08–0.09 mg kg−1 DW) than the fish requirements(0.5–0.3 mg kg−1 DW; NRC, 1993), where for both Atlantic cod andEpinephelus malabaricus juveniles, the Se requirements are 0.25 and0.7 mg Se kg−1 DW, respectively. Increase in the level of Se in rotifersenhanced the mRNA expression of GPX in cod larvae (Penglase et al.,2010), suggesting that extra supplementation is needed to protect lar-vae against lipid oxidation and the oxidative derivatives, which can beabundant in feeds enriched with n-3 LC-PUFA.
In the present study, the improved larval survival and stress resis-tance by increased organic Se dietary levels could be related in onehand to the reduction of toxic free radicals and in another hand to theimproved utilization of dietary lipids. The low levels of MDA and AOEcontent observed in the larvae fed 11.65 μg Se mg−1 diet demonstratean adaptive response in attempting to neutralize the generated ROS.Moreover, a reactive responsewas observedby the increase in BMP4, al-kaline phosphatase, osteocalcin, osteonectin osteopontin, and matrixgla protein gene expression in larval tissues, suggesting a well skeletaldevelopment. Inclusion of 11.65−1 mg Se kg−1 diet inmicrodiets is rec-ommended to enhance the larval antioxidant capacity and regulation ofthe genes involved in skeletal development.
Acknowledgements
This study was partially supported by a grant from the SpanishAgency of International Cooperation and Development (AECID) toReda Saleh Mohamed Ibrahim. The present study was partly fundedby the Spanish Ministry of Sciences and Education (AGL2009-14661).This work has been partly funded under the EU Seventh FrameworkProgram by the ARRAINA project No 288925: Advanced Research Initia-tives for Nutrition and Aquaculture.
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