Physiological effects of natural olive oil antioxidantsutilization in rainbow trout (Onchorynchus mykiss)feeding

Benedetto Sicuro Æ Paola Badino Æ Franco Dapra Æ Francesco Gai ÆMarco Galloni Æ Rosangela Odore Æ Giovanni Battista Palmegiano ÆElisabetta Macchi

Received: 30 June 2008 / Accepted: 18 March 2009 / Published online: 12 April 2009� Springer Science+Business Media B.V. 2009

Abstract Olive mill vegetation water (VW) is an olive oil by-product rich in polyphenols

has powerful antioxidant effects. In light of the interest on the research of novel natural

antioxidants to use in fish feed, the aim of this research was to use VW as a potential

substitute for artificial antioxidants in rainbow trout diet as well as checking its effects on

the blood chemistry and digestive organ physiology of the fish. The experimental plan was

monofactorial, balanced (4 9 3) and the experimental factor considered was the fish diet.

Diets were isoproteic (CP 40%) and isoenergetic (18 MJ kg-1 DM) with two inclusion

levels of VW (1 and 5%: VW1 and VW5) tested against a control diet. A feeding trial was

performed on quadruplicate groups of 200 fish (mean body weight: 44.2 g) fed experi-

mental diets for 94 days. At the end of the trial, the growth performance traits were

determined and sampling of blood and different tissues (brain, ovary, stomach, liver, and

intestine) were carried out for haematology, endocrinology, histology, and digestive

enzyme analysis. The main results of the present experimentation are that VW inclusion in

rainbow trout feed slightly affects the productive traits and blood chemistry, while the

histological structure of digestive organs and digestive enzyme physiology were not

affected.

Keywords Antioxidants � Feed additives � Olive mill wastewater � Phytoestrogens �Rainbow trout

B. Sicuro (&)Department of Animal Production, Ecology and Epidemiolgy, Faculty of Veterinary Medicineof Torino, via L. da Vinci 44, Grugliasco, TO, Italye-mail: benedetto.sicuro@unito.it

P. Badino � R. OdoreDepartment of Animal Pathology, Faculty of Veterinary Medicine of Torino, via L. da Vinci 44,Grugliasco, TO, Italy

F. Dapra � F. Gai � G. B. PalmegianoISPA – National Research Council, via L. da Vinci 44, Grugliasco, TO, Italy

M. Galloni � E. MacchiDepartment of Veterinary Morphophysiology, Faculty of Veterinary Medicine of Torino, via L. daVinci 44, Grugliasco, TO, Italy

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Aquacult Int (2010) 18:415–431DOI 10.1007/s10499-009-9254-6

AbbreviationsBW Body weight

DEA Digestive enzyme activities

ER Estrogen receptor

EQ Ethoxyquin

VW Olive mill wastewater

RBC Red blood cells

SGR Specific growth rate

SBP Steroid-binding proteins

TPA Total alkaline protease activity

TP Total proteins

Introduction

In modern aquaculture, the principal food additives used to scavenge lipid peroxide rad-

icals in fish feeds and feed ingredients are synthetic antioxidants. Among these, butylated

hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and ethoxyquin (EQ) are the

most utilized compounds. On the other hand, these molecules (and in particular EQ) have

been investigated for carcinogenicity due to their potential for toxicological and adverse

health effects in both fish and fish consumers through ‘‘carry-over’’ processes (Little 1990;

He and Ackman 2000; Berdikova Bohne et al. 2006). In order to avoid these toxic effects,

several natural compounds have been investigated to find valid alternatives as partial

substitute of synthetic antioxidants molecules. Natural antioxidants are a wide class of

compounds coming mainly from spices, herbs and recently by agriculture by-product

(Moure et al. 2001). Several of these antioxidants are utilized in animal nutrition for feed

conservation (Sigurgisladottir et al. 1994), for improving animal health (Sutton et al. 2006)

for organic animal production and for improving the quality of final product (Hamre et al.

2004).

Among these, polyphenols contained in olive mill waste water (VW) have been studied

for their antioxidant properties (De Lucia et al. 2006; Di Benedetto et al. 2006). VW is an

olive oil by-product rich in polyphenols obtained by mechanical compression of olives

during oil extraction. Recent studies on human diet demonstrated that virgin olive oil with

a higher content of polyphenolic compounds shows protective effects in inflammation

models in rats (Papadopoulos and Boskou 1991; Martınez-Domınguez et al. 2001), and

these effects have been largely attributed to the antioxidant effects of olive oil bio-phenols

such as hydroxitirosol (Yang et al. 2007). At the same time, it is well known that some

vegetal polyphenols have hormonal activity, which sometimes gives rise to significant

endocrine effects in experimental animals. These effects could be linked to the presence of

non-steroid plant constituents, i.e., phytoestrogens, which elicit estrogen-like effects in one

or more target tissues in animals (Mazur and Adlercreutz 2000). Phytoestrogens belong to

various chemical families (isoflavones, coumestanes, lignans, and stylbens) and their

chemical structure presents similarities with estradiol, that has a critical impact on

reproductive and sexual functions, but also affects other organs. Phytoestrogens have been

found to affect the endocrine system of farmed fish (Latonnelle et al. 2002) and they are

largely present in fish feed ingredients (Pellissero and Sumpter 1992).

Considering both the interest in the investigation of novel natural antioxidants to be

used in fish feed (Sutton et al. 2006) as well as the possible negative effects of some of

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these molecules on the fish physiology, there are two main aims for this research. The first

aim is verifying the possible use of dietary VW antioxidants in the rainbow trout feeding

and the second is checking their possible physiological and histological effects on the

blood chemistry and digestive organs of the fish.

Materials and methods

Experimental plan, growth trial and fish feed

VW is characterized by about 14% of dry matter and it is chemically characterized for the

presence of polyphenols (Table 1) that could be considered interesting as potential natural

antioxidant. The experimental plan was at randomized blocks, monofactorial and balanced

with four replicates per level (4 9 3). The experimental factor considered was fish diet,

considering two levels of VW inclusion (1 and 5%) and a control diet. A preliminary fish

feedstuff palatability trial was carried out to evaluate the maximum level of VW tolerated

by rainbow trout. The growth trial was conducted at the Experimental Station of

Department of Animal Husbandry of the University of Turin. A total of 2,400 rainbow

trout weighed individually and having a mean initial body weight of 44.2 ± 0.1 g were

randomly allotted to 12 tanks of 1,500 l (1.0 mW, 0.5 mH, 3.0 ml; 200 fish per tank), with

a water flow rate of 6 l min-1. The growth trial started on March 15th 2007 and ended on

June 27th 2007. During the first 2 weeks of the acclimatization period, the fish were

progressively fed with experimental diets. The fish were fed by hand (feeding ratio 1.5% of

BW) 6 days per week, twice per day. The feeding trial lasted for 94 days. Fish were bulk-

weighed fortnightly in order to adjust the feeding rate and individually at the end of the

experiment.

Two different diets were formulated with increasing levels of olive mill wastewater

(VW1 and VW5, respectively), these diets were tested against a control diet without olive

oil by-product (Control). Feeds were prepared in a private feed manufacturer (Monge,

Torre S, Giorgio-Cuneo). Diets were analyzed by proximate composition according to

standard methods (AOAC 1990) shows that all diets were isonitrogeonous (CP 40%) and

isoenergetic (18 MJ kg-1 DM; Table 2).

Haematology and endocrinology

Blood samples were collected from all the randomly selected fish (n = 5), within 20 min

(range 10–20 min) from capture and after anaesthesia in 3-aminobenzoic acid ethyl ester

Table 1 Olive mill vegetationwater composition (mean ± SD,n = 3)

Compound

Sugars (g/l) 3.4 ± 0.9

Proteins (g/l) 3.2 ± 1.3

Lipids (g/l) 5.6 ± 1.6

Polyphenols (g/l) 4.99 ± 0.02

Ortho-diphenols (g/l) 0.46 ± 0.01

Total suspended solids (g/l) 44.26 ± 6.66

Natural organic material (g/l) 142.2 ± 21.3

pH 4.32 ± 0.39

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solution (100 mg l-1) by caudal vein puncture. Fresh blood was placed in heparinized

tubes for haematology determination. The rest of the blood was left to clot at 4�C for 2 h,

the clot removed after centrifugation, and the serum aliquoted and deep-frozen (-80�C)

for biochemistry and hormonal determination. Red blood cell (RBC) count was manually

performed after dilution, 9200, in modified Dacie’s fluid as previously reported (Blaxhall

and Daisley 1973) by using a Burker counting chamber. Values were expressed as 106 ll.

Haematocrit (Hct) values were determined in duplicate by using a microhaematocrit

capillary tubes, a michrohaematocrit centrifuge (1,525g for 5 min) and a microhaematocrit

reader. The values were expressed as the percentage of erythrocytes. The haemoglobin

(Hb) concentration (g 100 ml-1) was estimated spectrophotometrically (540 nm wave-

length) using the cyanomethaemoglobin method with Drabkin’s reagent (Blaxhall and

Daisley 1973).

For estradiol determination, three blood samples per treatment were collected, allowed

to clot, and then centrifuged to obtain sera, which were stored at -80�C until analysis.

Serum estradiol concentrations were measured in triplicate by the use of a

Table 2 Ingredient (%), proximate composition (% DM) and phenol content (g/kg) of the experimentaldiets

Ingredients (%) Control VW1 VW5

Herring fish meal 47.0 47.0 47.0

Wheat meal 20.4 20.4 20.4

Soybean extraction meal 18.6 18.6 18.6

Fish oil 10.0 10.0 10.0

Mineral mixturea 1.5 1.5 1.5

Vitaminmixtureb 1.5 1.5 1.5

Liver-protector integrator 1.0 1.0 1.0

Vegetation waterc 0 1 5

Proximate composition (% DM)

Moisture 10.0 9.2 10.8

Crude protein 39.8 39.3 39.8

Ether extract 11.8 11.0 11.6

Ash 8.3 7.5 8.1

Crude fiber 2.0 2.4 1.7

Gross energy (Mj kg-1 DM) 18.4 18.0 18.0

Phenol content

Polyphenols (g/kg) 0.67 ± 0.02 0.92 ± 0.03 1.01 ± 0.05

Ortho-diphenols (g/kg) 0.37 ± 0.03 0.07 ± 0.04 1.00 ± 0.05

Antioxidant activity (DPPH %) 3.1 5.8 6.2

a Mineral mixture (g or mg/kg diet): bicalcium phosphate 500 g, calcium carbonate 215 g, sodium salt 40 g,potassium chloride 90 g, magnesium chloride 124 g, magnesium carbonate 124 g, iron sulphate 20 g, zincsulphate 4 g, copper sulphate 3 g, potassium iodide 4 mg, cobalt sulphate 20 mg, manganese sulphate 3 g,sodium fluoride 1 g, (GrandaZootecnica, Cuneo, Italy)b Vitamin mixture (IU or mg/kg diet): DL-a tocopherol acetate, 60 IU; sodium menadione bisulphate, 5 mg;retinyl acetate, 15,000 IU; DL-cholecalciferol, 3,000 IU; thiamin, 15 mg; riboflavin, 30 mg; pyridoxine,15 mg; B12, 0.05 mg; nicotinic acid, 175 mg; folic acid, 500 mg; inositol, 1,000 mg; biotin, 2.5 mg;calcium panthotenate, 50 mg; choline chloride, 2,000 mg (GrandaZootecnica, Cuneo, Italy)c Vegetation water is a liquid by-product of olive oil extraction, its inclusion did not alter the percentage ofother ingredient in fish feed

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radioimmunoassay kit (DSL-4800, IBL Hamburg, Germany). Cytosol fraction was pre-

pared (three samples per tissue per treatment) as described previously (Re et al. 1993), but

introducing some minor modifications. Briefly, frozen tissue samples were homogenized in

ice-cold buffer (50 mM Tris–HCl, 1 mM EDTA, 12 mM thioglycerol, 10 mM sodium

molybdate, 10% glycerol, pH 7.4). The homogenates were then filtered through a double

layer of cheese-cloth and centrifuged at 3,000g for 20 min at 4�C. The supernatants were

centrifuged at 105,000g for 45 min at 4�C and the resulting supernatants (cytosolic frac-

tions) were used for the measurement of estrogen receptors (ER) at a concentration of 5 mg

protein per ml. Protein concentrations were measured according to the method described

by Lowry et al. (1951).

Haematochemical analysis was performed using commercially available kits for vet-

erinary use (Biochemical Systems International s.r.l.—Arezzo-Italy) with an automated

biochemical analyser (VETSCREEN, Biochemical Systems International s.r.l.—Arezzo-

Italy) for the following variables: glucose (Glu), urea, total proteins (TP).

The ER binding assays were performed as described by Re et al. (1993) introducing

some minor modifications. Briefly, aliquots of cytosol (200 ll) were incubated overnight in

triplicate at 4�C with 50 ll of increasing concentrations of [3H]estradiol (0.1–2.8 nm). To

estimate non-specific binding, a parallel set of tubes containing a 250-fold molar excess of

non-labelled diethylstilbestrol as an estrogen competitor was prepared. After absorption of

the free hormone on dextran-coated charcoal and precipitation, 200 ll of the supernatant

were added to 4 ml of scintillation liquid (Pico Fluor, Canberra Packard, Groningen, The

Netherlands) and radioactivity was measured for 2 min using a b-counter (Tri-Carb 1600

TR, Canberra Packard) at an efficiency of 60%. The equilibrium dissociation constants

(Kd) and the maximum number of binding sites (Bmax), expressed as nanomolar values and

femtomoles of specifically bound hormones per milligram of cytosol protein, respectively,

were calculated by use of computer-assisted Scatchard (1949).

Histology

At the end of the trial, three fish for each tank, 12 trout for each diet, 6 h after the last meal

were randomly selected and anaesthesized in 3-aminobenzoic acid ethyl ester solution

(100 mg l-1) then killed with a blow to the head. Samples of the liver, pyloric caeca,

middle and distal intestine were carefully dissected and fixed in 4% formalin solution PBS

buffered (pH 7.2) cooled at 4�C. Fixed samples were dehydrated, following normal his-

tological procedures, in increasing ethanol solutions, methyl benzoate and embedded in

paraffin, and then the samples were sectioned into 5–7 lm thickness.

The intestinal tissue sections were evaluated following the criteria reported in the paper

dealing with soybean meal-induced enteritis in Atlantic salmon (Baeverfjord and Krogdahl

1996). The evaluated parameters were: the widening and shortening of the intestinal folds,

the extent of enterocytes supranuclear vacuolization, the villi lamina propria widening

status, lymphocytes infiltration in the lamina propria, and submucosa by hematoxylin-eosin

stain (HE).

The histological evaluations of liver specimens take into account more parameters than

the intestinal samples. The observed parameters were: the lipid vacuoles, the presence of

glycogenosis, and the presence of ceroid substances. The HE was used to evaluate the lipid

vacuolizations in the hepatocytes and blood vessels congestion. The PAS and PAS-diastase

stains were useful to evaluate glycogenosis and glycogen granulation content. The Sudan

black stain in paraffin embedded sections was used to highlight the ceroid substances.

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Tissue sampling for digestive enzyme assays

At the end of the experiment, the fish were killed 6 h after the last meal and two fish each

tank (eight for treatment) were sampled and the repletion of all gut was checked. This time

was chosen after previous investigations in order to obtain an adequate gastric empting and

intestinal fulfill status. The stomach and the intestine (without pyloric caeca) were sepa-

rated and all visible fat was removed. Both tissues were opened longitudinally, rinsed with

an ice-cold physiological solution, gently blotted dry, and immediately frozen in liquid

nitrogen and stored at -80�C before analysis. The tissues were homogenized using a

polytron (Kinematica, Luzern, Switzerland) in a cold Tris–HCl 50 mM (pH 7.0) buffer

with a 1:10 (w/v) ratio for the intestine and 1:5 (w/v) for the stomach. The homogenate was

then centrifuged at 4�C at 7,500g for 10 min. The supernatant containing the crude extracts

was picked-up and stored at -20�C before analysis as proposed by Santigosa et al. (2008).

All enzyme analysis were carried out in a period of 1 week. Results are reported as enzyme

units released per gram of wet tissue (units g-1tissue), in the case of alkaline total proteases

and pepsin while as enzyme units released per ml of homogenate (units ml-1homogenate)

for amylase and lipase. One enzyme unit was defined as the amount of enzyme that

catalyzed the release of 1 lg of product min-1.

Pepsin [E.C. 3.4.23.1]

Stomach pepsin activity was determined spectrophotometrically by hydrolysis of bovine

hemoglobin according to Worthington (1993), using a 2% (w/v) solution of bovine

hemoglobin dissolved in HCl 0.06 N as substrate. One unit of enzyme activity was defined

as 1 lg of tyrosine released per minute.

Total alkaline protease activity

The effect of different pH incubations on the intestinal alkaline proteolytic activities of the

crude enzyme extract was determined on the bases of the casein hydrolysis assay by Kunitz

(1947). The pH values of different buffers were previously optimized for alkaline prote-

ases: a Tris–HCl 0.1 M (pH 7.0–8.0–9.0) buffer was chosen. The enzyme–substrate

mixture consisted of 0.75 ml 2% (w/v) casein in water, 0.75 ml selected buffer, and 0.1 ml

crude enzyme extract incubated in a water bath for 1 h at 37�C. A total of 2.25 ml of

trichloroacetic acid (TCA), 5% (w/v) was then added to the reaction mixture to stop the

reaction. This mixture was then allowed to stand for 0.30 h at 4�C before centrifuging at

3,500 rpm for 15 min. Absorbance of the supernatant was recorded at 280 nm to measure

the amount of tyrosine produced. The blank used for this assay was prepared by incubating

a mixture of the casein, water and buffer for 1 h at 37�C, followed by the addition of a

crude extract and TCA. One unit of enzyme activity was defined as 1 lg of tyrosine

released per minute.

Lipase [E.C. 3.1.1.3]

Intestinal lipase activity was determined spectrophotometrically by hydrolysis of q-nitro-

phenyl myristate according to Iijima et al. (1998) with a modified method of Albro et al.

(1985). Each assay (0.5 ml) contained 0.53 mM q-nitrophenyl myristate, 0.25 mM 2-

methoxyethanol, 5 mM sodium cholate, and 0.25 M Tris–HCl (pH 9.0). One unit of

enzyme activity was defined as 1 lg of q-nitrophenol released per minute.

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Amylase [EC 3.2.1.1/2]

Intestinal amylase activity was determined according to Bernfeld (1955) using soluble

starch as the substrate and maltose as standard disaccharides. One unit of enzyme activity

was defined as 1 lg of maltose released per minute.

Statistical analysis

Fish diet was the experimental factor tested. In the beginning of the growth trial, homo-

geneity of variance for individual fish weight was tested with Bartlett test in order to assess

similar intra group variability. All data were analyzed by one-way ANOVA using R

software (R version 2.5.0, 2007-04-23), except estradiol and estrogen receptor data that

was elaborated with non parametric ANOVA (Kruskall-Wallis test), in consideration of the

low number of replicates in these experimental parameters. R is open-source software and

may be freely redistributed. (www.r-project.org). After the ANOVA, differences among

means were determined by the Tukey test multiple comparisons of means, using the

significant level of P \ 0.05.

Results

Productive results and antioxidant inclusion

Considering the VW composition (Table 1), lipids are the more significant fraction and

oleic acid is the dominant fatty acid which slightly influenced the fish feed fatty acid

composition (B. Sicuro et al. unpubl. data). The antioxidant effect of VW (DPPH) in the

fish feed is provided in Table 2 and shows that the antioxidant activity increases with the

VW level inclusion, as indicated by the DPPH% quenched. These levels of inclusion have

been planned in order to prevent antinutritional effect of VW. Fish final mortality was 3%

in all the considered tanks. Considering the zootechnical performance traits (Table 3), the

control diet showed the best result with a mean biomass gain of 80.4 g. In comparison to

this diet, the other two diets highlighted statistical differences, with a mean biomass gain of

72.2 and 74.0 g, respectively, for the VW1 and VW5 diets. The same trend and statistical

differences appeared for the SGR, PER, and NPU values. No statistical differences were

observed on FCR values.

Table 3 Zootechnical performance parameters

Diets SGR FCR PER NPU Biomass gain

Control 1.10 ± 0.01a 1.40 ± 0.24NS 0.88 ± 0.10a 0.39 ± 0.01a 80.4 ± 1.3a

VW1 1.06 ± 0.02b 1.40 ± 0.04NS 0.96 ± 0.01b 0.36 ± 0.01b 72.2 ± 2.5b

VW5 1.08 ± 0.03b 1.40 ± 0.05NS 0.95 ± 0.10b 0.37 ± 0.01b 74.0 ± 3.3b

In the columns, different letters mean statistical difference at P \ 0.05

Biomass gain (BG) (g) = final total weight - initial total weight; Specific growth rate SGR (%) = (ln finalweight - ln initial weight) 9 100/feeding days; Feed conversion rate (FCR) = total feed supplied (g ofDM)/BG (g); Protein efficiency ratio (PER) = BG (g)/total protein fed (g); Net protein utilization(NPU) = utilized protein(%)/protein gain (%)

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Haematological and endocrinology results

The experimental treatments did not affect the haematological parameters considered,

except for the haematocrit (Hct), which shows a significant increase in the fish fed with

VW diets (Table 4). Considering other haematological parameters, in erythrocyte count

(RBC) and glucose level (Glu), an increase is evident in the VW groups, whereas the

haemoglobin level (Hb) decreased. Neither total proteins nor urea values showed a clear

trend among the experimental groups.

As far as endocrinology is concerned (Tables 5, 6), estradiol concentration was only

detectable in the group of fish fed with higher VW inclusion (i.e., VW5, Table 5). On the

other hand, the estrogen receptor (ER) concentrations were not influenced by VW inclusion

in fish feed (Table 6), even if an increase of ER concentration is evident in the ovary and in

the liver.

Histological results

Concerning the intestinal tract sections, no inflammatory processes of the digestive organs

were recorded among the fish groups. The histological patterns of the sampled livers are

summarized in the Table 7, the lipids vacuolizations were rare in all groups of trout

(Fig. 1). The presence of glycogenosis was higher in the fish of group VW1 and the

positivity was verified by the PAS-diastase stain (Fig. 2). Fish fed with VW1 diet also

Table 4 Haematology and serum biochemistry (n = 5)

RBC (106/ll) Hb (g/dl) TP (g/dl) Glu (mg/dl) Urea (mg/dl) Hct (%)

Control 4.5 ± 1.5NS 14.3 ± 1.2NS 5.0 ± 0.2NS 64.6 ± 12.2NS 3.0 ± 2.1NS 41.0 ± 0.7a

VW1 5.6 ± 1.9NS 13.7 ± 2.6NS 5.1 ± 0.4NS 74.4 ± 17.7NS 5.4 ± 2.9NS 45.6 ± 2.9b

VW5 6.4 ± 3.6NS 13.6 ± 2.0NS 4.7 ± 0.5 NS 70.1 ± 4.1NS 2.4 ± 1.7NS 45.0 ± 3.2b

In the columns, different letters mean statistical difference at P \ 0.05

Table 5 Estradiol concentration(pc/ml, n = 3)

ND, not detected

Concentration

Control ND

VW1 ND

VW5 2.02 ± 0.22

Table 6 Estrogen receptor con-centrations (fmol mg-1, n = 3)

Tissue Diets

Control VW5

Brain 53.3 ± 12.1NS 46.0 ± 7NS

Ovary 42.7 ± 6.5NS 50.0 ± 7.8NS

Liver 49.3 ± 17.5NS 56.5 ± 7.8NS

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showed a low number of livers that were poor or negative to glycogen granules. All livers

did not display presence of ceroid substances in the hepatocytes (Fig. 3).

Digestive enzyme results

The digestive enzyme activities (DEA) values are reported in Table 8.

Table 7 Histological evaluations of the trout livers (n = 12, livers sampled for each treatments)

Diets Histomorphologya Glycogenosisb

l-v h-v neg pos

Control 2 – 6 2

VW1 1 – 3 5

VW5 1 – 5 2

a Number of livers with low (l-v) or high (h-v) presence of lipids vacuolizationsb Number of samples with high (pos) or poor (neg) content of glycogen granulations

Fig. 1 Examples of liversections of fish fed experimentaldiets stained withhematoxylineosin stain:photograph a shows a normalliver. Photograph b showshepatocytes with moderate lipidvacuolizations (indicated byblack arrowheads)

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None of the determined enzymes showed statistical differences independently from

the experimental treatment. Although the results did not appear significant, a higher

protease activity was detected in the stomach tissues than in the intestinal tract. Pepsin

showed a slight increasing trend at the increasing VW inclusion level, while intestinal

total alkaline protease activity (TPA) values remain almost constant in all the examined

groups.

A similar trend was observed for the intestinal lipase and amylase DEA. In terms of

absolute values, higher values were detected for lipase than for amylase. Lipase showed an

irregular trend at the increasing VW inclusion level with the maximum value reached in

the VW1, while amylase values remain almost constant, (about 0.24), in all the examined

groups. The swinging trend revealed for the lipase activities could probably be due to the

high variability among the sampled subjects.

Fig. 2 Examples of liver sections of fish fed with experimental diets Pas (photo a and c) and PAS-Diastase(photo b and d) stained in order to evaluate the sample glycogen granulations

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Discussion

Zootechnical parameters

The fish feeds could be considered equivalent from the point of view of composition, the

only relevant difference is on the antioxidant activity. An ameliorative effect of VW

inclusion is clear on fish feeds. The low mortality recorded in this research suggests that

farmed fish were in optimal experimental conditions. The growth trial results showed that

the VW inclusion levels of 1 and 5% induced a growth reduction of about 10 and 7.5%,

respectively, and was confirmed by the SGR values. This effect could be explained by the

reduced protein utilization of fish fed with VW that is mirrored in the PER and NPU

values. VW polyphenols probably interacted with protein absorption-and/or digestibility

processes, but specific analysis is necessary to validate this hypothesis.

From an applicative point of view, for future utilization of these natural antioxidants,

the registered differences in fish growth could be reduced using different olive oil by-

products purified from antinutritional polyphenols. Moreover, antioxidant analyses on fish

fillet fed with VW diets showed an interesting antioxidant carry—over effect and a con-

sequent improvement of final product quality (Sicuro et al. 2007). In the future, this effect

could partially compensate farmers from the lower growth of fish producing a healthier and

Fig. 3 Example of a liversection stained with Sudan blackB stain, no ceroid substancegranulations are visible in thehepatocytes. Black arrowheadsindicate melanomacrophagecenter rich in melanin that appearblack

Table 8 Digestive enzyme activities: U/g tissue (total alkaline protease and pepsin), U/ml homogenate(amylase and lipase)

TPAa Pepsin Amylase Lipase

Control 0.24 ± 0.13NS 0.94 ± 0.36NS 0.25 ± 0.09NS 4.65 ± 2.14NS

VW1 0.25 ± 0.07NS 1.20 ± 0.27NS 0.24 ± 0.06NS 6.05 ± 2.99NS

VW5 0.19 ± 0.08NS 1.22 ± 0.35NS 0.24 ± 0.06NS 5.01 ± 2.95NS

a These data are the mean values determined at different pHs (pH = 7–8–9)

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more environmentally sustainable product than those obtained utilizing synthetic

antioxidants.

Haematology and endocrinology

The impact of nutritional state of the fish was determined by haematochemical parameters

and plasma concentrations of proteins, glucose, and urea, all good indicators of general

state of health in fish and the values are in the physiological range for Salmonid (Rehulka

and Minarik 2003; Hemre and Sandnes 2008). Looking at the slight increase of erythrocyte

count, our results are opposite as expected and VW seems to exert a positive effect on

erythropoiesis. It must be considered that in mammals oestrogens are erythropoiesis

inhibitors and evidence exists that similar effects also occur in fish (Valenzuela et al.

2007). This promoting effect can be explained by the estradiol level increase in the higher

level group of VW. Haematocrit values are a useful indicator for physiological stress in fish

(Kiron et al. 2004; Hemre and Sandnes 2008). It can also be an indicator of oxidative stress

since red blood cells (RBC) are one of the principal production sites of free radicals and

some of them can induce peroxidation of unsaturated fatty acids in phospholipids, thus

altering their quantity. Changes in haematocrit and haemoglobin are explained as a strategy

to increase oxygen-carrying capacity of blood or a consequence of increased swimming

activity during periods of high energy demand (Caruso et al. 2005). In this case, the

haematocrit increase could be explained as a nutritional stress indicator caused by poly-

phenol antinutritional effect. Also, glucose level increase is considered a stress response

(Caruso et al. 2005; Sala-Rabanal et al. 2003) and in this case, there is a variability in the

VW1 group that could mask the statistical differences. In any case, an increased level of

haematic glucose is evident in the VW groups. Studies examining the glucose levels in fish

nutrition showed that it could be an important marker to elucidate the mechanisms

underlying stress response (Alves-Martins et al. 2007). Our results indicate an increase in

glucose, which is classically related to stress conditions, even if the measured levels were

within the physiological range for Salmonids (Hemre and Sandnes 2008). The present

results also indicated that the presence of polyphenols in fish feed did not pose a challenge

to the erythrocytes, even if an increase is visible in fish fed with VW inclusion. Further

haemoglobin did not vary with dietary polyphenols content, indicating that the diets were

not deleterious enough to the fish. The measured values for haemoglobin and erythrocytes

seems to be a little bit higher than reference (Valenzuela et al. 2007), even if there are no

differences between the experimental treatments.

Changes in the concentration of serum protein have been used as indicators of stress

response in fish (Caruso et al. 2005; Sala-Rabanal et al. 2003); Cho et al. 2007 for instance,

showed a total protein (TP) increase in flounder fed on green tea polyphenols, while in this

research TP is on the physiological range of the species.

A primary concern associated with estrogenic compound bioaccumulation in fish is

interference with reproduction or development (Donahoe and Curtis 1996; Kiparissis et al.

2003) and phytoestrogens have been reported to have many biological activities in fer-

tilization, immunostimulation, anabolism, and balancing hormones (Lee et al. 2005; Zierau

et al. 2005; Leusch et al. 2006). Considering that estrogens typically induce biological

responses through interaction with the ER, either as agonist or antagonist, in this research

ER have been investigated in control and VW5 group of fish in order to give a preliminary

view of possible endocrinological effects of olive oil VW inclusion in rainbow trout feed.

However, it must be considered that receptor-binding assays are an initial step in the

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mechanism of action of steroid hormones and it does not necessarily imply activation or

inhibition of the receptor-mediated cascade.

A variety of organic chemicals have been documented to activate the ER and conse-

quently induce estrogenic effects in different animals, in particular xenoestrogens have

been studied in rainbow trout (Sabo-Attwood et al. 2007; Tollefsen 2007). Our results

show that a diet with olive oil polyphenols elicits the activation of ER in ovary and liver

(Table 6) and could consequently induce estrogenic effects in fish as documented in a

previous study on the hepatic ER from Atlantic salmon (Salmo salar) and rainbow trout

(Oncorhynchus mykiss) activated by environmental xenoestrogens (Tollefsen et al. 2002).

The biological activity of phytoestrogens is usually studied on the level of ER binding.

Although their structure is not related to vertebrate steroids, they are able to produce

estrogen-like effects. Evaluation of these results allows us to conclude that the tested levels

of VW inclusion had no adverse effects on the haematology of rainbow trout. The sta-

tistically significant differences found in the haematological profile do not show any

negative effects on the state of the health of the fish.

Phytoestrogens effects have been investigated in fish (Ng et al. 2006); Ko et al. (1999)

reported that in yellow perch, genistein induced vitellogenin synthesis by the liver, and had

a growth- promoting effect similar to that of estradiol-17b. Genistein, daidzein, and soy-

bean-based diets also increased plasma vitellogenin concentrations in male and female

rainbow trout, Siberian sturgeon Acipenser baeri (Pelissero et al. 1991a, 1991b), and

juvenile striped bass Morone saxatilis (Pollack and Ottinger 2003). As regard as phyto-

estrogen metabolism, it is known that the steroid-like compounds are collected by hepatic

circulation and in these vessels they can bind steroid-binding proteins (SBP) and in

rainbow trout it has been demonstrated that certain phytoestrogens are able to bind to SBP.

This fact can explain the increase of the oestrogen receptor concentrations in the ovary and

mainly in the liver (Table 6).

Histology

Over the past decade, researchers began to evaluate histological modification during fish-

feeding trials and the addition of this investigation is useful in understanding if some

ingredients in the diets can display detrimental effects on the fish gut. A high concentration

of soybean meal induces intestinal histology modifications (Baeverfjord and Krogdahl

1996; Van den Ingh et al. 1996; Krogdahl et al. 2003; Romarheim et al. 2006), even

displayed by high content of blends of different plant protein and starch sources (Sanden

et al. 2005; Aslaksen et al. 2007). According to Escaffre et al. (2007), in this work the VW

inclusion did not exceed 5%, therefore a dietary low inclusion level is comparable to

protein or starch sources in the diets of the above-cited articles. The VW did not induce any

evident modification of the intestinal and liver histology at this inclusion level. Some plant

compounds can display detrimental effects also at low inclusion level in the diets, as has

been reported for red kidney bean lectins (Dapra et al. 2005). It is possible to suppose that

the 1% of VW slightly improved the liver glycogen PAS reactivity compared to the control

and VW5 diets, however, to validate this possible effect, further study focused on this

aspect is necessary.

Digestive enzymes

Although the digestive process in fish is not studied as much as in mammals, the enzyme

profile seems to be a good physiological indicator of feed utilization. In particular, when an

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anti-nutritional factor effect is suspected, DEA analysis is a useful method that gives clear

information for the utilization of a new formulated fish feed by the fish (Refstie et al. 2006;

Correa et al. 2007).

Total alkaline protease activity (TPA) values obtained in this study are comparable to

those reported by Correa et al. (2007) for trypsin activity in the proximal intestine of

tambaqui (Colossoma macropomum) fed with different dietary cornstarch levels. Similar

values of trypsin activity are also shown by Robaina et al. (1995) in gilthead seabream fed

with control and 20% soybean meal inclusion levels. Moreover, it is interesting to note that

TAP did not change significantly in rainbow trout fed with VW-added diets, contrary to

those reported in seabream, tilapia, and African sole that were inhibited by vegetable meals

(Moyano Lopez et al. 1999).

In this study, rainbow trout showed similar lipase values to those reported by Lundstedt

et al. (2004) in the anterior intestine of Brazilian Siluriformes fish fed with a dietary crude

protein and a cornstarch content ranging from 20 to 40% and from 36 to 13%, respectively.

Considering the examined DEA results, is possible to suppose that VW inclusion on

rainbow trout fish diets did not negatively affect the fish digestive processes, contrary to

that reported for the rohu (Labeo rohita) fingerlings digestive enzymes fed with tannins

(Maitra and Ray 2003).

Conclusions

The inclusion of vegetal feedstuffs, even at low levels, could have negative effects on fish.

It is for this reason that an investigation into fish physiology and digestive tract histology is

of primary importance as a preliminary step for fish-feed experimentation. In this case, the

VW inclusion in rainbow trout feed partially affected the productive traits and the values of

haematological biochemical parameters whereas it did not affect the histological structure

of intestine and liver and the digestive enzyme physiology. The VW polyphenols show a

promoting effect on ER and on estradiol level, which could be considered carefully for

future VW inclusion in fish feeds. Moreover, other results on similar experimentation on

rainbow trout (unpublished data) show that VW inclusion in feed partially improve the

antioxidant parameters of fish fillets. These results confirm the possible agriculture by-

products utilization as a source of natural antioxidants in fish nutrition. This fact could

contribute to the valorization of olive oil VW, which is the main olive oil by-product in all

Mediterranean countries.

Acknowledgments The authors thank Dr. Davide Sburlati (CNR- ISPA) and Mrs. Cristina Vignolini(Department of Veterinary Morphophysiology) for their excellent technical assistance during laboratoryanalysis. The authors are also indebted to Prof. Sebastiano Vilella (University of Lecce) who investigatedthe olive mill vegetation water composition.

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