Effects of additive iron on growth, tissue distribution,haematology and immunology of gilthead sea bream,Sparus aurata

George Rigos • Alexandros Samartzis • Morgane Henry •

Eleni Fountoulaki • Efthimia Cotou • John Sweetman •

Simon Davies • Ioannis Nengas

Received: 28 April 2009 / Accepted: 13 January 2010 / Published online: 3 February 2010� Springer Science+Business Media B.V. 2010

Abstract The comparative effects of iron-supplemented levels on growth, tissue distri-

bution, haematology and immunology of gilthead sea bream, Sparus aurata (2 g) were

investigated, using four organic (50, 100, 200, 300 mg ORG/kg diet) and one inorganic

iron source (200 INOR mg/kg diet). Fish were treated for 12 weeks with the experimental

diets and maintained at a water temperature of 19–22�C. Growth (final weight and specific

growth rate), tissue distribution (spleen, liver and muscle), haematological parameters (red

blood cells, haematocrit, haemoglobin and mean corpuscular haemoglobin concentration)

and non-specific immune indexes (respiratory burst activity and antibacterial activity of

serum) were analysed. No significant differences were found in growth and iron tissue

distribution among the tested groups. Red blood cell counting was statistically higher in

fish given 50 ORG, 100 ORG, 200 ORG and 200 INOR feeds. However, haematocrit,

haemoglobin and mean corpuscular haemoglobin concentration were not significantly

affected by increasing dietary iron. Fish receiving the 100 ORG diet had the best per-

formance with respect to the respiratory burst activity and significantly higher values for

antibacterial activity of serum were obtained in fish fed with the 300 ORG diet. The present

findings provided no clear evidence of the optimum iron concentration. However, there

was adequate indication that iron supplementation enhanced the performance of gilthead

sea bream, mainly from a haematological and immunological point of view.

Keywords Gilthead sea bream � Growth performance � Sparus aurata �Mineral concentration � Antibacterial activity � Haematological parameters �Chemiluminescence

G. Rigos (&) � A. Samartzis � M. Henry � E. Fountoulaki � E. Cotou � I. NengasLaboratory of Fish Nutrition and Pathology, Institute of Aquaculture, Hellenic Centre for MarineResearch, Aghios Kosmas, 16777 Ellinikon, Attiki, Greecee-mail: grigos@ath.hcmr.gr

A. Samartzis � S. DaviesSchool of Biological Sciences, Plymouth University, Plymouth, Devon PL48AA, UK

J. SweetmanAlltech Aqua, Samoli, Livadi, 28200 Lixouri, Cephalonia, Greece

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Aquacult Int (2010) 18:1093–1104DOI 10.1007/s10499-010-9326-7

Introduction

Iron is one of the essential minerals in living organisms involved in several biological

processes such as oxygen transport, cellular respiration and lipid oxidation reactions (Lee

et al. 1981). As in higher vertebrates, the mechanisms involved in the absorption and

regulation of iron in fish are poorly understood (Lall 2002).

Limited iron passage is possible across the gills from the fresh water environment and

thus diet is the major source for iron especially in marine farmed fish (Bury and Grosell

2003). The difference in water-derived capacity for iron absorption between freshwater and

seawater is due to the adaptive physiology and thus variations in the lumen chemistry of

fish inhabiting the two environments (Bury and Grosell 2003). In order to fight dehydra-

tion, marine fish compensate the high accumulation of calcium and magnesium from the

continuous consumption of water by secreting large quantities of bicarbonate resulting in

the formation of alkaline precipitates, which are hostile for the transport of ferrous iron (the

absorvable form) in the intestine (Cooper et al. 2006). Conversely, freshwater fish in their

hypo-osmotic environment aim to reduce water intake and posses low bicarbonate levels

and mildly acidic environment in their gut (Wilson 1999).

The key factors influencing dietary iron absorption in fish include nutrient intake,

chemical form, proportion of organic and inorganic dietary components, digestibility of the

diet, interactions with other dietary components, animal age and health status of the digestive

tract (Andersen et al. 1996; Lall 2002). It is generally accepted that the organic forms of iron

can be absorbed better compared to the inorganic sources (Watanabe et al. 1997).

The requirements of iron have been determined for several species. The minimum

dietary iron requirement has been reported to be between 60 and 100 mg/kg in Atlantic

salmon, Salmo salar (Andersen et al. 1996), 30 mg/kg in channel catfish, Ictaluruspunctatus (Gatlin and Wilson 1986), 170 mg/kg in Japanese eel, Anguilla japonica (Nose

and Arai 1979) and 150 mg/kg in red sea bream, Pagrus major (Sakamoto and Yone

1976). Deficiency of iron induces haematological suppression, reduced growth and poor

feed conversion in farmed fish (Tacon 1992). In particular, experimentally induced lack of

iron may cause microcytic anaemia or low haemoglobin levels in salmonids (Kawatsu

1972; Andersen et al. 1996), channel catfish (Gatlin and Wilson 1986), Japanese eel (Nose

and Arai 1979), yellowtail, Seriola quinqueradiata (Ikeda et al. 1973) and red sea bream.

On the other hand, excessive amounts of dietary iron may not always be beneficial to

farmed fish (Salte et al. 1994; Andersen et al. 1997) and can be connected with increased

bacterial load (Fouz et al. 1994).

Determining the optimum iron feed concentration is therefore a necessary task for the

performance of any farmed fish of interest. Such data is however unavailable for Medi-

terranean mariculture species. Moreover, gilthead sea bream, Sparus aurata, the most

commercialised farmed fish species in this industry, suffers regularly from anaemia and

substantial seasonal mortalities due to infestations of haematophagus monogenean gill

parasites (Microcotyle spp.) (Athanassopoulou et al. 2005). Preliminary in situ results

showed a significant reduction of mortalities with the incorporation of iron supplementa-

tion in the diet (200 mg/kg diet) due to a possible improvement of fish blood parameters

(Nengas, personal communication). Consequently, there is an increasing interest in using

iron dietary supplementation in order to improve disease resistance in gilthead sea bream,

Sparus aurata.

The aim of this study was therefore to investigate the effect of additive iron on gilthead

sea bream, Sparus aurata performances and determine the type (organic vs. inorganic) and

optimum level of iron supplementation in commercial diets in relation to growth,

1094 Aquacult Int (2010) 18:1093–1104

123

haematology, tissue distribution and immunology of this species. This information is to be

used in future experiments involving possibly challenge by natural infections of haema-

tophagus monogenean gill parasites.

Materials and methods

All experiments were carried out in the facilities of the Laboratory of Fish Nutrition &

Pathology of the Institute of Aquaculture in the Hellenic Centre for Marine Research in

Athens (HCMR).

Fish

Two thousand gilthead sea bream, Sparus aurata of an initial average weight of about

1.8 ± 0.1 g and length 2.8 ± 0.2 cm (n = 50) were transported from a local farm (Evia,

Central Greece) at a biomass of 2 kg/m3 with continuous supply of oxygen. Fish were

acclimatised for 1 month before the start of the experiment and were fed with the control

diet used in the experiment (Tables 1, 2). Triplicate groups of 50 fish for each of the six

treatments were weighed and randomly distributed in 18 fibre glass tanks (100 l) receiving

seawater of 38% salinity and maintained at a water temperature of 19–22�C.

Diets and sampling

Six practical diets were prepared in the facilities of HCMR. Diets were supplemented with

four levels of organic iron source (Bioplex, Alltech Inco) at 50, 100, 200 and 300 mg/kg

diet inclusion level and one level of inorganic iron source (Ferrous sulphate, DCM) at

200 mg/kg diet inclusion level (50 ORG, 100 ORG, 200 ORG, 300 ORG and 200 INOR,

respectively). A control diet was also used where no iron was added in the premix. The

composition and iron analysis of the experimental diets are given in Tables 1 and 2,

respectively. Triplicate groups of fish were fed with each one of the diets for 12 weeks at a

constant ratio close to satiation. At the end of the experiment, the total population of fish in

each tank was weighted and growth parameters were calculated. Six fish from each

replicate tank (n = 18) were anesthetised and were used for haematological evaluation

(red blood cell count, haematocrit and haemoglobin concentration), tissue concentration

Table 1 Composition (%) of the experimental diets

Control 50 ORG 100 ORG 200 ORG 300 ORG 200 INOR

Fish meal 35 35 35 35 35 35

Soya bean meal 20 20 20 20 20 20

Wheat meal 15 15 15 15 15 15

Wheat gluten 15 15 15 15 15 15

Fish oil 13 13 13 13 13 13

Premix 0.3 0.3 0.3 0.3 0.3 0.3

Cellulose 1.7 1.7 1.6 1.5 1.4 1.6

Bioplex iron 0.050 0.100 0.200 0.300

Ferrous iron 0.200

Total 100 100 100 100 100 100

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analysis (muscle, liver and spleen) and measurement of the immune status of the fish

(whole blood respiratory burst activity and serum antibacterial activity).

Blood sampling

Approximately 1 ml of blood was drawn by a caudal puncture using 23G needles (Mi-

crolance 23G 11/4 0.6 9 30, Becton Dickinson, Saragosa, Spain). Heparin-treated blood

was used for the measure of the respiratory burst activity and for the determination of the

haematological parameters, whereas antibacterial activity was assessed in serum (unhep-

arinised blood was left to coagulate overnight at 4�C, centrifuged twice at 9,000g for

10 min and serum was kept at -80�C until assayed).

Feed and tissue analysis

Feed and tissues (muscle, liver and pooled spleen) were freeze-dried with a CHRIST

GAMMA 1-20 lyophilisator. Spleen samples were pooled due to small quantities collected.

One gram of dried tissue was digested with 10 ml of HNO3 in a microwave-device (CEM

MDS 2100). The drying factor varied from 22 to 25% of the fresh weight. Following

digestion, the sample was diluted with distilled water to 20 ml. A Varian AA 20 Plus flame

Atomic Absorption Spectrophotometer was used for the determination of the metal con-

centrations. The accuracy and precision of the analytical methodology was tested with the

reference material NRCC-Dorm-2 of homogenised dogfish muscle.

Growth

Fish growth was evaluated by calculating the final weight of fish and specific growth rate

(SGR).

SGR ð%=dayÞ ¼ ½lnðW1Þ � lnðW0Þ�total days

� 100:

Haematology

Red blood cell count

Red blood cell count (RBC) was based on Blaxhall (1972). Blood was diluted (1:500) in

Dacie’s solution, and red blood cells were counted in 5 squares of a neubauer chamber.

Cell concentration per ml of blood was deduced.

Haemoglobin concentration

The haemoglobin concentration (Hb) was determined by a miniaturised method using the

Drabkin’s reagent (Drabkin and Austin 1935). In brief, 2 ll of blood was added to 500 ll

Table 2 Measurement of iron concentration (mg/kg) in the experimental diets following preparation

Control 50 ORG 100 ORG 200 ORG 300 ORG 200 INOR

Iron concentration 22.12 71.34 110.54 217.69 308.98 220.24

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of Drabkin’s reagent (Sigma) and 200 ll was transferred to duplicate wells of a transparent

96-flat bottomed well microplate (Greiner). The haemoglobin concentration of the blood

sample was calculated from a standard curve prepared from bovine haemoglobin (Fluka).

The absorbance was determined at 540 nm by a GeniosPro luminometer (TECAN, Aus-

tria). Results were expressed as g/dl blood.

Haematocrit

The haematocrit (Hct) was measured in 18 fish per treatment using duplicate heparinised

75-ll capillary tubes per fish. Capillary tubes were centrifuged at 13,000g for 10 min and

read against a percentage scale. Results were expressed as percentage haematocrit.

Mean corpuscular haemoglobin concentration

Mean corpuscular haemoglobin concentration (MCHC) expressed in g/dl blood was

deduced from the haemoglobin and haematocrit results (MHCH = Hb 9 100/Hct).

Immunology

Antibacterial activity of serum

The antibacterial activity of serum was assessed against a strain of Escherichia colitransformed with a luciferase gene (Nikoskelainen et al. 2002). Fish sera were diluted with

Phosphate Buffer Saline (PBS) and 50 mM Mg2? Ca2? (MgCa, pH 7.4) and increasing

amount (0–100 ll) of the diluted serum were added to 12 wells of a white 96-wells flat-

bottomed microplate (Nunc) giving final serum concentration ranging from 0 to 40 ll/ml

for each fish. Volume was adjusted in each well to 100 ll with PBS and MgCa so that

equal amount of MgCa was present in all wells. Bacteria (E. coli K12pEGFLPLucTet;

kindly provided by S. Verho University of Turku, Finland) were grown overnight to log-

phase at 37�C in LB-broth containing 10 lg tetracycline/ml. Bacterial concentration was

then adjusted to OD450nm of 0.1 and 50 ll were added to the sera. A 3-h incubation at 23�C

allowed bacterial killing, then 100 ll of D-luciferin (Synchem, Germany) at 0.5 mM (in

0.1 M citrate buffer, pH 5.0) were added to each well. The emitted luminescence (RLU)

measured by GeniosPro luminometer (Tecan, Austria) was proportional to the number of

live bacteria. Curves representing RLU as a function of serum concentration (ll/ml)

starting by a plateau and rapidly decreasing to a final plateau were plotted using the Origin

software. From these curves, IC50 (concentration of serum necessary for 50% of bacterial

killing) was calculated, and bactericidal activity of serum in Units/ml was deduced (1000/

IC50).

Chemiluminescence

Induction of the respiratory burst (RB) activity in blood leucocytes was measured directly

from heparinised blood following the method described by Nikoskelainen et al. (2005)

with some modifications. Briefly, 275 ll of diluted blood (1:200) in gHBSS-hep-luminol

(Hanks’ Balanced Salt Solution containing 0.1% gelatin, 5 U/ml of heparin, 100 I.U./ml of

penicillin/streptomycin, 5 lg/ml of oxytetracycline, 0.19 mM luminol, pH 7.4) was dis-

pensed in triplicate wells of a white flat-bottomed 96-well microplate. The background

Aquacult Int (2010) 18:1093–1104 1097

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luminescence was allowed to stabilise for 10 min. The reaction started with the addition of

25 ll of zymosan at 5 mg/ml in each well, and luminescence was recorded for 2 h every

3 min at 23�C. The peak chemiluminescence was determined and expressed in relative

luminescent units (RLU).

Statistical analysis

Data from each measured parameter was checked for homogeneity of variances (Levene)

and normality (Kolmogorov–Smirnov) prior to statistical analysis. The triplicate tanks of

each diet showed no significant difference (One-Way ANOVA) and could be pooled for

each parameter analysed. Statistical analysis between dietary groups was performed by

One-Way ANOVA followed by a Tukey’s multiple range comparison test using STAT-

GRAPHICS Plus. A student t-test was used to compare the effect of organic and inorganic

source of iron for 200 ORG and 200 INOR. The level of significance for all tests was set at

P \ 0.05.

Results

Iron feed and tissue concentration

The analysis of the iron concentrations in the tested diets revealed values of expected

magnitude, since the measured levels were slightly above the supplemented concentrations

with concentration in the control non-supplemented diet of 22.12 mg/kg (Table 2). Iron

concentrations in the analyzed tissues are shown in Table 3. The iron levels in the three

tested tissue of fish fed with the 200 ORG diet displayed the highest values with however

no statistical difference in muscle (P = 0.507) and liver (P = 0.369) among the experi-

mental groups.

Growth

The results concerning the growth performance of tested fish are given in Table 4. Growth

was not affected by iron supplementation, since no statistical differences were observed in

final weight (P = 0.423) and SGR (P = 0.511) between the experimental groups.

Table 3 Tissue iron concentrations (lg/g) of gilthead sea bream fed with iron-supplemented diets for12 weeks

Diet Control 50 ORG 100 ORG 200 ORG 300 ORG 200 INOR

Muscle 10.9 ± 0.75a* 11.1 ± 0.16a 9.9 ± 0.7a 11.8 ± 3.5a 11.3 ± 0.7a 9.6 ± 0.3a

Liver 110.5 ± 12.7a 131.4 ± 21.4a 134.3 ± 15.4a 140 ± 21.1a 138.1 ± 23.1a 128.8 ± 5.7a

Spleen(pooled)

593.6 679.6 726.52 764.6 761.8 696.1

ANOVA: P = 0.507 for muscle, P = 0.369 for liver

* Values are the means (n = 18) of three replicate tanks expressed with the standard deviation betweentanks. Means sharing a common letter in a row are not significantly different at P \ 0.05

1098 Aquacult Int (2010) 18:1093–1104

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Haematological analysis

The haematological analysis showed significantly higher levels (P = 0.003) of RBC in fish

fed with the 50, 100, 200 ORG and 200 INOR diets compared to the fish receiving the

control feed (Fig. 1), whereas Hct (P = 0.105; Fig. 2), Hb (P = 0.642; Fig. 3) and MHCH

(P = 0.321; Fig. 4) measurements showed no significant differences between the experi-

mental groups. A tendency for increased Hb and MCHC was observed in fish fed with 200

ORG. Student t-test revealed no significant differences in Htc (P = 0.435), in Hb

(P = 0.687) nor in MHCH (P = 0.101) between groups given the 200 ORG and 200

INOR diets.

Immunological parameters

Respiratory burst activity showed no significant increase in response to the inclusion of

dietary iron (P = 0.404; Fig. 5). A tendency for increased levels was however present in

fish fed with 100 ORG and 200 ORG, but large standard deviations between fish masked

any significant differences. Antibacterial activity of serum was significantly higher

(P = 0.004; Fig. 6) in fish given the highest dosage of organic iron (300 ORG) compared

Table 4 Growth performance factors of gilthead sea bream fed with iron-supplemented diets for 12 weeks

Diet Control 50 ORG 100 ORG 200 ORG 300 ORG 200 INOR

Initial weight 1.8 ± 0.1a* 1.7 ± 0.2a 1.8 ± 0.1a 1.8 ± 0.3a 1.7 ± 0.1a 1.8 ± 0.1a

Final weight 12.2 ± 0.4a 12.8 ± 0.3a 12.4 ± 0.4a 12.7 ± 0.5a 12.6 ± 1.0a 12.2 ± 1.5a

SGR 2.6 ± 0.1a 2.7 ± 0.1a 2.7 ± 0.1a 2.6 ± 0.1a 2.7 ± 0.1a 2.6 ± 0.1a

ANOVA: P = 0.423 for final weight, P = 0.511 for SGR

* Values are the means (n = 18) of three replicate tanks expressed with the standard deviation betweentanks. Means sharing a common letter in a row are not significantly different at P \ 0.05

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

Control 50 ORG 100 ORG 200 ORG 300 ORG 200 INOR

Fish Diet

RB

C/m

l

a

c

b

c

abc

c

1. ANOVA: P=0.003 2.tukey multiple range test

Fig. 1 Effect of dietary iron on red blood cell count in gilthead sea bream fed with supplemented diets for12 weeks. Values are the means (n = 18) of three replicate tanks expressed with the standard deviationbetween tanks. Means sharing a common letter are not significantly different at P \ 0.05

Aquacult Int (2010) 18:1093–1104 1099

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to 100 ORG and 200 INOR. Student t-test revealed no significant differences in respiratory

burst activity (P = 0.400) nor in antibacterial activity of serum (P = 0.326) between

groups given the 200 ORG and 200 INOR diets.

Fig. 2 Effect of dietary iron onhaematocrit count in gilthead seabream fed with supplementeddiets for 12 weeks. Values are themeans (n = 18) of three replicatetanks expressed with the standarddeviation between tanks. Meanssharing a common letter are notsignificantly different at P \ 0.05

Fig. 3 Effect of dietary iron onhaemoglobin count in giltheadsea bream fed with supplementeddiets for 12 weeks. Values are themeans (n = 18) of three replicatetanks expressed with the standarddeviation between tanks. Meanssharing a common letter are notsignificantly different at P \ 0.05

Fig. 4 Effect of dietary iron on mean corpuscular haemoglobin concentration in gilthead sea bream fedwith supplemented diets for 12 weeks. Values are the means (n = 18) of three replicate tanks expressedwith the standard deviation between tanks. Means sharing a common letter are not significantly different atP \ 0.05

1100 Aquacult Int (2010) 18:1093–1104

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Discussion

The present study represents the first investigation on the effects of iron supplementation in

Mediterranean farmed fish species. No mortalities were recorded in any of the treated

groups during experimentation. Iron addition either as organic (up to 300 mg/kg diet) or

inorganic (200 mg/kg) form was not found to improve final weight (P = 0.423) and SGR

(P = 0.511) of gilthead sea bream, Sparus aurata indicating that growth cannot be used as

a criterion for evaluating iron supplementation in this species. In accordance with this

finding, Atlantic salmon (Andersen et al. 1996, 1997, 1998) and red sea bream (Sakamoto

and Yone 1976) showed no significant weight gain when receiving additional iron in their

feeds, whereas channel catfish showed a growth retardation when given a purified diet

without iron supplementation (Gatlin and Wilson 1986). These data suggested that iron

present in the dietary fish meal provides the fish with iron levels sufficient for adequate

growth of Sparus aurata.

Fig. 5 Effect of dietary iron on respiratory burst activity in blood of gilthead sea bream fed withsupplemented diets for 12 weeks. Values are the means (n = 18) of three replicate tanks expressed with thestandard deviation between tanks. Means sharing a common letter are not significantly different at P \ 0.05

Fig. 6 Effect of dietary iron on antibacterial activity in serum of gilthead sea bream fed with supplementeddiets for 12 weeks. Values are the means (n = 18) of three replicate tanks expressed with the standarddeviation between tanks. Means sharing a common letter are not significantly different at P \ 0.05

Aquacult Int (2010) 18:1093–1104 1101

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Although no significant differences were revealed in iron tissue concentrations among

the experimental groups (P = 0.507 and P = 0.369 for muscle and liver, respectively), an

increasing trend was apparent with the increase of dietary organic iron (up to 200 mg/kg

diet) with, however, lower levels measured in fish receiving the highest organic iron

concentration (300 mg/kg diet). It was previously demonstrated that iron absorption

capability and thus tissue levels in Atlantic salmon are not proportional to the dietary levels

(Andersen et al. 1996). In particular, the latter study revealed that a stabilisation in iron

storage occurred when high dietary iron was supplemented (100–400 mg/kg). This was in

accordance with the previous findings in the same species studied by Rasmussen (1994),

where similar amounts of stored iron were measured despite wide differences in dietary

iron intake (210–307 mg/kg). These results support a previous hypothesis that there is a

regulation mechanism for iron metabolism in fish that is not thoroughly known yet

(Andersen et al. 1996). Even if not significantly, organic iron sources in the present work

showed higher availability compared to the inorganic iron form as seen in all three tissues

tested confirming the fact that organic iron is more available than inorganic sources in fish.

Haematological analyses revealed clear differences among the treated groups only in

RBC counts (P = 0.003) where iron levels from both organic and inorganic sources up to

200 mg/kg feed displayed a positive effect on this parameter. This probably indicates that

the iron content of the control feed (22 mg/kg) may cover some of the haematological

requirements of healthy gilthead sea bream, Sparus aurata, which however may not be

adequate during infestations of haematophagus monogeneans. Andersen et al. (1996) found

that RBC count in Atlantic salmon was not significantly affected by the supplemental iron

levels of 10–400 mg/kg (given as iron sulphate) for 12 weeks, but a tendency for increased

values was observed. Other blood parameters such as Hb and Hct were actually affected by

iron addition in the latter study, whereas Andersen et al. (1998) reported that the hae-

matological parameters of the same fish species were not influenced by iron treatment for

20 weeks (iron sulfate; 400 mg/kg). Iron supplementation in gilthead sea bream, Sparusaurata did not significantly affect Hb concentrations (P = 0.642) but its concentration in

fish fed with the 200 ORG feed increased almost 10% in relation to the control diet.

Similarly, Hct (P = 0.105) and MHCH (P = 0.321) were not significantly affected by iron

supplementation in the treated groups and this finding was possibly related to the fact that

the actual iron concentration was up to 22 mg/kg higher than the supplementation level in

the experimental diets due to the supply of iron through the fishmeal present in all diets

including the control. The decrease in RBC count, Hb concentration and MHCH in fish fed

with 300 ORG compared to fish fed with 200 ORG, although not significant, suggested that

supplementation doses above 200 mg/kg should be avoided as they may reduce the ben-

eficial effect of iron supplementation on the fish haematological parameters.

Berger (1996) reported that either a deficiency or an excess of iron could compromise

the immune system. In the present study, no immunosuppression was observed compared

to control diet even at the highest dietary iron concentration tested (300 ORG). Immu-

nostimulation was even observed in the current study after 12 weeks feeding of gilthead

sea bream, Sparus aurata in the antibacterial activity of serum of fish fed with 300 ORG

compared to fish fed with 100 ORG or 200 INOR (P = 0.004). Also, a tendency for

increased respiratory burst activity was observed in fish fed with 100 and 200 ORG

compared to other diets (P = 0.405). Given the fact that the actual iron content of control

diet was 22 mg/kg, this treatment appeared to induce no iron-deficiency. The reduction of

respiratory burst activity at dietary iron levels of 300 ORG compared to 100 ORG,

although not significant, suggested that higher iron levels may reduce the extent of

immunostimulation. However, the antibacterial activity of serum was still high at the

1102 Aquacult Int (2010) 18:1093–1104

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highest concentrations tested (200 and 300 ORG). In Atlantic salmon, the serum total

antibody, specific haemolytic complement activity and spontaneous haemolytic activity

and serum, head–kidney and spleen lysozyme activities were not significantly influenced

by dietary iron administration for 8 and 20 weeks (Andersen et al. 1998). In channel

catfish, iron-deficiency had no effect on the specific antibody response (Sealey et al. 1997)

but decreased macrophage chemotaxis (Lim and Klesius 1997; Sealey et al. 1997) and

accelerated the onset of mortality in channel catfish exposed to Edwardsiella ictaluri (Lim

and Klesius 1997; Lim et al. 2000). Cumulative mortality results were either not influenced

by dietary levels of iron (Lim and Klesius 1997; Lim et al. 2000) or increased in iron-

deficient fish (Sealey et al. 1997). In Atlantic salmon, a significant association between

high concentrations of serum iron and mortality of Atlantic salmon infected with Vibrioanguillarum was reported (Sherman 1992). Ravndal et al. (1994) indicated that a delicate

balance exists between the need of iron for host defence mechanisms and the need for iron

to sustain microbial growth. The results of the respiratory burst activity in the present study

suggested that a dose of 100–200 mg/kg gives satisfactory stimulation and higher doses

may reduce the extent of the immunostimulation, although the antibacterial activity of

serum was still positively influenced by 300 mg/kg.

In conclusion, the present findings provided no clear evidence respecting the optimum

iron concentration. However, there was indication that iron supplementation enhanced the

performance of gilthead sea bream, Sparus aurata, mainly from a haematological and

immunological point of view. Possibly a longer than 12 weeks experimentation with

additive dietary iron could have induced more apparent effects. The superiority of organic

vs inorganic iron sources can be only suggested as higher values of 200 ORG (in muscle,

liver and spleen iron concentrations, final fish weight and respiratory burst activity) were not

significantly different from values obtained with 200 INOR. It can be preliminary proposed

that an iron concentration between 100–200 mg/kg diet should be the recommended

additive level in feeding schedules of gilthead sea bream, Sparus aurata. Considering the

cost of the ingredient, the 100 mg/kg additive iron concentration should be considered on a

yearly basis for the dietary schedules of this species, while the 200 mg/kg concentration

should be used as a preventive measure when attacks of haematophagus monogeneans are

expected and when the water oxygen levels are low and the haematological status of the fish

might become highly compromised. Further experiments will investigate iron dietary

supplementation in association with hypoxia and/or parasitical natural challenges.

Acknowledgment This work was funded by Alltech, Department of Aquaculture Nutrition and Health.

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