Aquaculture 440 (2015) 6068

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Aquaculture
journal homepage: www.elsevier.com/locate/aqua-online

Growth performance and quality traits of European sea bass (D. labrax)
fed diets including increasing levels of freeze-dried Isochrysis sp. (T-ISO)
biomass as a source of protein and n-3 long chain PUFA in partial
substitution of sh derivatives
E. Tibaldi a,, G. Chini Zittelli b, G. Parisi c, M. Bruno a, G. Giorgi c, F. Tulli a, S. Venturini b, M.R. Tredici c, B.M. Poli c
a
b
c

Department of Food Science, University of Udine, Udine, Italy

CNR-Institute for the Study of Ecosystems, Florence, Italy
Department of Agri-food Production and Environmental Sciences, University of Florence, Florence, Italy

a r t i c l e

i n f o

Article history:
Received 29 September 2014
Received in revised form 3 January 2015
Accepted 3 February 2015
Available online 11 February 2015
Keywords:
Dicentrarchus labrax
Feeds
Flesh quality
Isochrysis sp. (T-ISO)
Nutrient digestibility

a b s t r a c t
The aim of this study was to evaluate nutrient digestibility, growth performance, biometry, dressing out parameters,
llet muscle proximate and fatty acid composition of European sea bass (Dicentrarchus labrax L.) of nearly
marketable size, fed increasing levels of a freeze-dried biomass of Isochrysis sp. (clone T-ISO) as a partial
substitute for protein and lipid from sh derivatives, in diets where the level of n-3 long chain (LC) PUFA were
reduced through a partial replacement of sh oil for crude palm oil. Since diets including the test ingredients
could possibly result in changes of certain quality attributes, sh were also subjected to overall sensory
evaluation by a trained panel of assessors according to a triangle test.
Three diets were formulated to be grossly iso-nitrogenous (N, 7.5% dry matter). A preparation in which sh meal/
trimmings and sh oil were the major protein and lipid sources was used as a positive control diet. Two test
complete diets were obtained from the control preparation so that approximately 10 and 20% crude protein
from a high quality sh meal and 18 and 36% lipids from sh oil were supplied by the dried microalgae biomass.
To stress the role of Isochrysis sp. as a source of n-3(LC)PUFA, the diets including increasing levels of the
microalgae biomass were made grossly iso-caloriclipidic by replacing a part of sh oil with palm oil which is
known to be virtually free from n-3 PUFA. Each diet was fed to apparent satiety to quadruplicate groups of sea
bass (142 g) over 121 days. The results have shown that replacing up to 20% crude protein from sh meal with
a dried Isochrysis biomass and up to 36% sh lipid for those supplied by the microalgae in a diet with reduced
level of sh oil, did not adversely affect feed intake or growth performance relative to controls even if the highest
substitution rate resulted in a decline (P b 0.05) in lipid and energy apparent digestibility coefcients and in a
slightly reduced n-3(LC)PUFA content of the edible muscle tissue (P b 0.05). No major diet-dependent changes
were observed in biometry traits and slaughter yield. At the actual dietary inclusion levels, the presence of
dried microalgae led to increased greenish pigmentation of the skin, but did not permit discrimination of the
cooked llets by sensory analysis using the triangle test.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Similar to what has occurred with other cultured marine sh
species, also in case of the European sea bass (D. labrax), a carnivorous
teleost of great economic relevance for the Mediterranean aquaculture,
the search for dietary alternatives to sh meals and oils has been mostly
directed towards the use of terrestrial plant protein-rich ingredients
and vegetable oils (Kaushik et al., 2004; Messina et al., 2013;

Corresponding author. Tel.: +39 0432494781; fax: +39 0432558130.

E-mail address: emilio.tibaldi@uniud.it (E. Tibaldi).

http://dx.doi.org/10.1016/j.aquaculture.2015.02.002
0044-8486/ 2015 Elsevier B.V. All rights reserved.

Mourente and Bell, 2006; Tibaldi et al., 2006) and little attention has
until recently been paid to explore the dietary potential of novel feed
sources such as macro- and microalgae biomass (Tulli et al., 2012;
Valente et al., 2006).
Microalgae in particular, besides their already established applications in aquaculture hatcheries, have attracted increasing attention as
animal feed supplements since they are a natural source of pigments,
antioxidants and other bioactive compounds which give them functional properties in addition to their basic nutritional value (Spolaore et al.,
2006). More recently, dry microalgae biomass have also been proposed
as potential raw materials in partial substitution for sh protein and
lipid sources in sh feeds, even if high costs and uncertain availability

E. Tibaldi et al. / Aquaculture 440 (2015) 6068

compared to commodity feedstuffs, currently set a limit to their large

use in commercial aquafeeds (Chini Zittelli et al., 2013; Shields and
Lupatsch, 2012).
In general microalgae biomass contain medium to high crude
protein levels, often higher than 45% and ranging between 28 and 71%
DM, depending on the species and culture/harvesting conditions, with
an amino acid prole which compares favourably with that of other
food proteins (Becker, 2007). To date there have been very few investigations where palatability and digestibility of diets including dried
microalgae biomass have been estimated in monogastric animals
(Becker, 2007; Skrede et al., 2011) and even more so in sh, where
the information on the nutritive value of microalgae dried biomass is
very limited (Burr et al., 2011). Despite this, previous studies have
shown dried biomass of Spirulina spp., Chlorella spp., Scenedesmus
spp., Nanofrustulum spp. and Tetraselmis suecica, to be valuable supplementary protein sources or partial substitutes for sh meal protein in
the diet of various omnivorous and carnivorous sh species at the
juvenile stage (Badwy et al., 2008; Belay et al., 1996; Kiron et al.,
2012; Nandeesha et al., 2001; Olvera-Novoa et al., 1998; Palmegiano
et al., 2005; Vizcano et al., 2014; Walker and Berlinsky, 2011). In the
European sea bass, the dried biomass of T. suecica was recently shown
to be able to replace up to 20% sh meal protein without hampering
the growth performance of the sh (Tulli et al., 2012).
Certain marine microalgae can directly produce n-3 long chain
(LC) polyunsaturated fatty acids (PUFAs) and in this respect their
inclusion in the diet could possibly spare the use of sh lipid. Dry
preparations of microalgae particularly rich in docosahexaenoic acid
(DHA) such as Schizochytrium spp. and Crypthecodinium chonii, have
proven successful as partial replacers for sh oil in starter diets of
certain marine sh species (Atalah et al., 2007; Carter et al., 2003;
Miller et al., 2007).
In this contest, the dry biomass of Isochrysis sp. deserves particular
attention as it combines medium-high level and quality of protein
with high lipid and DHA contents (Ben-Amotz et al., 1987; Brown
et al., 1993; Snchez et al., 2000), which makes it a potential candidate
ingredient for highly sustainable diets, where besides sh meal, even
substantial levels of sh oil have to be replaced by alternative sources.
This would be particularly the case of diets for the European sea bass,
which is notably incapable of signicant biosynthesis of n-3(LC)PUFA
(Mourente et al., 2005) and where a substantial replacement of sh
oil by vegetable oils led to a marked reduction of the n-3(LC)PUFA
content of the esh (Montero et al., 2005; Mourente and Bell, 2006).
To date, despite a favourable nutrient composition and encouraging
results observed in gilthead sea bream juveniles (Palmegiano et al.,
2009), the use of dried Isochrysis biomass as a raw material in sh
diets has been poorly investigated in marine sh species. In addition,
there is limited information on the effects of including substantial
levels of dried marine microalgae biomass in the diet of growing
marine sh species where, apart from growth response feed efciency
and the effects on the nutritional quality of the edible portion, the role
of microalgae in affecting certain sensory attributes of the sh such as
the colour of skin and esh (Belay et al., 1996; Tulli et al., 2012; Walker
and Berlinsky, 2011) as well as the overall sensory characteristics of
the cooked llet deserve attention from the point of view of consumer
acceptance.
The aim of this study was to evaluate nutrient digestibility, growth
performance, biometry, dressing out parameters, llet muscle proximate and fatty acid composition of European sea bass (D. labrax L.) of
nearly marketable size, fed increasing levels of freeze-dried biomass
of Isochrysis sp. (clone T-ISO) as a partial substitute for protein and
lipid from sh derivatives, in diets where the level of n-3(LC)PUFA
were reduced through a partial replacement of sh oil for crude
palm oil. Since diets including the test ingredients could possibly
result in changes of certain quality attributes, sh were also subjected
to overall sensory evaluation by a trained panel of assessors according to a triangle test.

61

2. Materials and methods

2.1. Test ingredients and diets
The freeze-dried biomass of Isochrysis sp. F&M-M36 (T-ISO) was
produced by CNR-Institute for the Study of Ecosystems, Florence,
Italy, at the commercial plant of Microalghe Camporosso Srl. (Imperia,
Italy). The plant consists of four independent modules each including
a number of Green Wall Panel photobioreactors of the rst generation
(GWP-I) 1-m high, 12.5-m long, 4-cm thick and 500 L in culture
volume, placed vertically in parallel rows (Chini Zittelli et al., 2013;
Tredici and Rodol, 2004). Isochrysis sp. F&M-M36 was cultivated outdoors during summer using articial seawater (Adriatic Sea Aquarium
& Equipment, Rimini, Italy) at 30 g L1 salinity, ltered through 80, 10
and 1 m melt-blown polypropylene cartridges (Everblue, Parma,
Italy) and added with f medium as a source of nutrients (Guillard
and Ryther, 1962). Culture overheating was prevented by automatically activating heat exchangers when the temperature exceeded
28 C. pH was maintained at values of about 7.87.9 by injecting
pure CO2 into the culture through gas diffusers placed along the reactors. Algal biomass was harvested by centrifugation. About 1000 L of
the culture were continuously fed to a centrifuge separator (Westfalia
mod. KA6, Germany) that yielded a paste with an average moisture
content of 80% (mostly intracellular water) which was recovered
from the collection chambers in 1 kg pats, then frozen, freeze-dried
and vacuum-packed.
The chemical composition and the fatty acid prole of the freezedried Isochrysis biomass are shown in Tables 1 and 3, respectively.

Table 1
Chemical composition of Isochrysis sp. F&M-M36 freeze-dried biomass (data expressed on
dry matter basis).
Proximate composition (%)
Crude protein
Total lipid
Ash
Phosphorous (g/kg)
-Carotene (mg/kg)

45.4
27.3
9.7
0.3
761.7

Essential AA (%)
Arginine
Histidine
Isoleucine
Leucine
Lysine
Methionine + cysteine
Phenylalanine + tyrosine
Threonine
Tryptophan
Valine

2.52
0.91
1.76
3.92
2.46
1.41
3.75
2.38
0.56
2.37

Non-essential AA (%)
Alanine
Aspartic acid
Glutamic acid
Glycine
Proline
Serine
EAA:NEAA ratio

3.17
4.19
4.58
2.64
2.36
2.17
1.15

Fatty acid composition (%)

Total SFA
Total MUFA
Total n-6 PUFA
Total n-3 PUFA
20:5n-3
22:6n-3
n-3/n-6

5.66
3.89
1.98
5.52
0.19
1.81
2.8

62

E. Tibaldi et al. / Aquaculture 440 (2015) 6068

Three diets were formulated to be grossly iso-nitrogenous (N, 7.5%

DM) and iso-lipidic (total lipid, 17.5% DM). Crude protein and lipid
levels were chosen based on the gures suggested as optimal for the
European sea bass by Peres and Oliva-Teles (1999a,b).

Table 2
Ingredient composition, proximate analysis, gross energy content, essential amino acid
composition and PUFA content of the test diets.
Diets
Control

IPO1

IPO2

300
250
0
120
80
100
100
0
10
10
15
15

250
250
70
120
80
85
70
25
10
10
15
15

200
250
140
120
80
70
40
50
10
10
15
15

Ingredient composition (g/kg)

Chile prime sh meal
Fish trimmingsa
Freeze-dried Isochrysis sp.
Wheat glutenb
Soybean meal (46% CP)
Wheat meal
Fish oil
Crude palm oil
Mineral mixc
Vitamin mixd
Celite
Na lignosulphonate
Proximate composition (% as fed)
Moisture
Crude protein
Total lipid
Ash
Acid insoluble ash
ND bre
AD bre
-Carotene (mg/kg)
Gross energy (MJ/kg)

5.6
47.9
17.2
15.5
1.5
3.6
2.2
10.0
20.3

5.3
47.7
17.8
15.2
1.6
3.3
2.0
69.0
20.5

5.6
47.1
18.2
14.8
1.6
3.7
1.7
130.0
20.6

2.5
0.9
1.2
2.9
2.3
1.9
2.9
1.8
0.3
1.8

2.7
0.9
1.6
3.2
2.6
1.8
3.1
2.0
0.3
2.0

2.6
0.9
1.6
3.0
2.4
1.7
3.0
2.1
0.3
1.9

2.7
3.5
7.7
3.8
3.5
2.2
0.80

2.8
3.7
8.0
3.8
3.6
2.2
0.84

2.7
3.5
7.3
3.5
3.5
2.1
0.86

3.10
6.15
2.13
4.54
1.09
2.24

3.69
5.71
2.14
3.93
0.83
1.85

4.00
4.97
2.00
3.15
0.55
1.39

Essential AA (% as fed)
Arginine
Histidine
Isoleucine
Leucine
Lysine
Methionine + cysteine
Phenylalanine + tyrosine
Threonine
tryptophan
valine
Non-essential AA (% as fed)
Alanine
Aspartic acid
Glutamic acid
Glycine
Proline
Serine
EAA:NEAA ratio
PUFA content (g/100 g)
Total SFA
Total MUFA
Total n-6 PUFA
Total n-3 PUFA
20:5n-3
22:6n-3

a
Vereinigte Fischmehlwerke Cuxhaven GmbH & Co. KG, Cuxhaven, Germany (moisture,
6.9%; crude protein, 64.3% DM; crude lipid, 9.1% DM; ash, 23.8% DM).
b
Roquette, Lestrem, France.
c
Composition (% mix): HPO42H2O, 78.9; NaCl, 17.65; MgO, 2.725; FeCO3, 0.335; KI,
0.005; ZnSO4H2O, 0.197; MnSO4H2O, 0.094; CuSO45H2O, 0.027; Na Selenite, 0.067.
d
Composition (% mix): Thiamine HCl, 0.16; Riboavin, 0.39; Piridoxine HCl, 0.21;
Cyanocobalamine, 0.21; Niacin, 2.12; Calcium pantotenate, 0.63 Folic Acid, 0.10; Biotin
Vit H, 1.05; Choline Clorure, 83.99; Myoinositol, 3.15; Stay C DSM, 4.51; a-tocoferol Vit
E, 3.15; Menadione Vit K3, 0.24; Vit A (2500 IU/kg diet), 0.03; Vit D3 (2400 IU/kg diet),
0.05.

Table 3
Fatty acid prole of the dried microalgae biomass, palm oil and test diets (% total fatty acid
methyl esters)(a).

12:0
14:0
16:0
18:0
20:0
Total SFA

Freeze-dried
Isochrysis sp.

Palm oil

2.1
10.5
6.8
1.1
1.6
32.3
6.3
9.4
2.1
0.5
2.1
0.5

Diets
Control

IPO1

IPO2

0.2
1.6
43.9
5.0
0.4
51.1

tr.
4.3
11.7
2.1
0.4
19.2

tr.
4.6
16.0
2.3
0.4
23.6

tr.
4.3
20.4
2.6
0.4
28.1

0.1
39.2

0.1

39.4

4.3
17.1
2.1
5.3
0.7
0.7
7.8
38.1

3.4
19.8
1.9
4.2
0.8
0.4
6.1
36.5

2.6
22.1
1.7
3.0
0.4
0.4
4.3
34.9

9.7

11.7

0.4

13.2

12.2

0.4
0.4
13.7

12.8
0.4
0.4
0.4
14.0

2.9
2.1
0.7
6.8
1.4
13.9
28.1

3.0
2.7
0.8
5.3
1.1
11.8
25.1

3.4
3.4
0.4
3.8
0.9
9.8
22.1

16:1n-7 + 9
18:1n-9
18:1n-7
20:1n-9
20:1n-11
22:1n-9
22:1n-11
Total MUFA

22.6

18:2n-6
18:3n-6
20:4n-6
22:5n-6
Total n-6 PUFA

5.8
1.6
0.5
2.1
11.5

18:3n-3
18:4n-3
20:4n-3
20:5n-3
22:5n-3
22:6n-3
Total n-3 PUFA

7.4
11.0
0.3
1.1
1.0
10.5
32.0

9.7
0.1

0.1

(a)

The fatty acids 10:0, 11:0, 12:0, 13:0, 14:1n-5, 15:0, 16:2n-4, 16:3n-4, 17:1, 16:4n-1,
18:2n-4, 18:3n-4, 18:4n-1, 20:1n-7, 20:3n-6, 20:3n-3, 20:4n-3, 21:5n-3, 22:4n-6 were
considered in their respective composite fractions but are not shown in the Table.

The ingredient composition, proximate analysis, amino acid and

fatty acid content and proles of the test diets are shown in Tables 2
and 3. A preparation in which sh meal/trimmings and sh oil were
the major protein and lipid sources was used as a positive control treatment. The other two complete feeds, denoted as IPO1 and IPO2, were
obtained from the control preparation through partial substitution of
sh meal and sh oil with increasing levels of the freeze-dried
microalgae biomass. Such changes resulted in approximately 10 and
20% crude protein from a high quality sh meal and 18 and 36% crude
lipid from sh oil to be replaced with the same nutrients supplied by
the dried microalgae biomass. To stress the role of Isochrysis spp. as a
source of n-3(LC)PUFA the diets including increasing levels of the
microalgae biomass were made grossly isocaloric isolipidic by replacing
a part of sh oil with palm oil which is known to be virtually free from
n-3 PUFA (Ng and Gibon, 2011). As shown in Table 2, even in the highly substituted diet (IPO2) the level of n-3(LC)PUFAs was kept well
above the minimum dietary requirement of the European sea bass
(e.g. 7 g/kg dry diet; Skalli and Robin, 2004).
If not otherwise specied in Table 2, dietary raw materials and
additives were obtained through local providers. The diets were
manufactured in the laboratories of the Department of Food Science
(DIAL), University of Udine (Italy). All ingredients were ground through
a 0.5 mm sieve before nal mixing and dry pelleting through a 4.5 mm
dye. The diets were stored at 3 C until used.
2.2. Growth trial
One hundred and sixty-eight half-sib sea bass were selected
according to size from a resident stock and randomly divided into 12
groups each consisting of 14 specimens. The sh groups were stocked
in 250-L breglass tanks being part of an indoor partially-recirculating
water system (total volume, 9 m3; daily water renewal, 5%; articial

E. Tibaldi et al. / Aquaculture 440 (2015) 6068

day-length, 12 h; light intensity at tank surface, 200 lx) provided with

thermostatic control of water temperature, mechanical sand-lter,
biological lter and an UV lamp apparatus. The system ensured nearly
constant and optimal water quality to sea bass (temperature,
22.8 0.90 C; salinity, 25 1; dissolved oxygen, 7.0 0.29 mg/L;
pH, 8.2 0.20; total ammonia nitrogen, b0.25 mg/L; nitritenitrogen,
b0.10 mg/L).
After stocking, sh were fed the control diet and adapted over
2 weeks to the experimental conditions. At the end of this preliminary
period, two sh per tank were randomly sampled, sacriced using an
overdose of anaesthetic (Finquel, Argent Laboratories, RedmontVI,
USA) then pooled, minced, freeze-dried and ground to be analysed for
initial whole body composition.
At the beginning of the growth trial there were 12 sh in each tank
(initial average body weight 142.0 0.5 g). Fish were weighed in
bulk and tank-groups assigned in quadruplicate to the three diets
according to a completely random design.
Sea bass were offered the test diets 6 days a week over 121 days. Fish
were fed two daily meals (8:00 and 16:30) to visual satiety (i.e. until the
rst feed item was refused). The actual feed intake per group was
recorded daily. Fish were group-weighed every 4 weeks, after a 24-h
fast and under moderate anaesthesia (Finquel, Argent Laboratories,
RedmontVI, USA, 2050 mg/L).
The handling procedures and sampling methods involving sh used
in the trial followed the guidelines of the European Union directive
2010/63/EU on the protection of animals used for scientic purposes.
Feed intake, specic growth rate (SGR), feed conversion ratio (FCR),
and protein efciency ratio (PER) were measured/calculated per
group over 121 days. At the end of the experiment, all sh of each
tank were killed by a percussive blow to the head and kept immersed
in an ice-slurry bath before being subjected to biometry, skin colour
measurements or processed for further analysis.
Of these, 4 sh per group were sampled at random then minced,
pooled and used as nal samples for subsequent whole body N content
analysis and to calculate gross protein retention (GPR).
After killing, thirty sh per dietary treatment were immediately subjected to individual skin colour measurements. Of these, 15 specimens
were then subjected to hardness and biometry measurements prior to
be dissected in order to evaluate major organ weights and slaughter
yield calculated on individual specimens. The 15 left llets obtained so
far were subjected to colour analysis then they were skinned and the
muscle mass obtained was minced and kept frozen ( 60 C) to be
subsequently analyzed for proximate and fatty acid composition.

63

2.4. Skin and muscle colour measurements

Individual skin and llet muscle colour measurements were
performed by a CM 2600d Chroma Meter (Minolta, UK). Skin colour
readings were carried out at three dorsal (post-cranial, medial, caudal)
and two ventral (post-cranial, medial) locations while llet muscle
colour measurements were taken at three locations of each left llet
(dorsal, ventral, caudal).
Data were expressed using the L* a* b* system, representing
lightness, red/greeness and yellow/blueness, respectively (CIE, 1976).
In addition, the values of Chroma = (a*2 + b*2)1/2, as a measure of
colour saturation and Hue = arctan(b*/a*) were calculated.
2.5. Fish hardness measurements
Individual whole sh hardness measurements were performed at
two different locations along the body from dorsal to caudal with the
Zwick Analog Shore Hardness instrument. This handheld instrument is
only able to measure the shore A hardness which is the relative hardness of elastic materials. A reading of 0 means a complete penetration
of the sample. Conversely a reading of 100 corresponds to no penetration. A test load of 8 N was used to calibrate the instrument.
2.6. Sensory evaluation
For sensory evaluation a triangle test (ISO, 1983) was organized. In
the triangle test the assessors are asked to determine overall sensory
difference among three samples (of which two are samples of the
same group and one from a different group) offered at the same time
in the same session, and to indicate the odd sample. This method, is a
procedure for determining whether a perceptible sensory difference
for all the attributes of the samples or similarity between samples exists
(Meilgaard et al., 1991). The test was carried out using 45 llets per dietary treatment (15 right llets obtained from sh previously analysed
plus 30 llets obtained after dissecting the remaining sh). To this end,
the epaxial (dorsal) portion of each llet with skin was divided into
three portions, wrapped in special microwave oven paper and cooked
in a microwave oven (Moulinex Optiquick Compact, Group SEB Italia
S.p.a., Assago, Milano, Italy) at 500 W for 70 s. The test was carried out
in two sessions (two replicates per session) where a trained panel
consisting of 9 (1st session) or 10 (2nd session) judges evaluated the
llets in air-conditioned individual boxes designed for sensory analysis
(ISO, 1988). In the rst session, the cooked portion of sh fed diets C vs.
IPO1 and in the second session those of sh given diets C vs. IPO2, were
compared.

2.3. Feed digestibility

2.7. Analytical methods
The apparent digestibility of the test diets was measured at the same
time as the growth trial described above and adopting the indirect
method. Acid-insoluble ash (AIA) was used as an external indigestible
marker (Celite, Prolabo, France, i.e. puried silica powder from diatoms, containing N95% w/w of acid-insoluble ash) added in equal
amounts (15 g/kg) to each diet before nal mixing and dry pelleting
(Table 2). The digestibility measurements were carried out using a
tank system developed by the University of Guelph (Guelph CYAQ-2;
Cho, 1992) consisting of three-tank units each tted with a common
drain pipe connected to a settling column for collecting faecal material.
Each 60-L tank was stocked with 10 sea bass (average body weight
107 12 g; 3.13.2 kg biomass per unit). The entire tank system
apparatus was connected to the partially-recirculating water system
described above. Fish were left to adapt to the culture conditions over
30 days before starting faecal collection. The digestibility measurements
of each diet were carried out in triplicate units using the procedures described by Tibaldi et al. (2006). The apparent digestibility coefcients
(ADC) of DM, crude protein, crude lipid and gross energy of the diets
were computed according to Maynard and Loosly (Cho, 1992).

The freeze dried microalgae biomass, test diets, faeces, whole body
and llet muscle tissue were subjected to the moisture and crude
protein analyses (AOAC, 1998);
The dried microalgae biomass and test diets were also analysed for
carotene, ash, total phosphorous and bre fractions (NDF and ADF)
content (AOAC, 1998).
The acid-insoluble ash (AIA) content of the test diets and faeces
was determined according to the method CEE-EU (G.U. European
Community n. L.155/21, 12.7.71) and their gross energy content was
measured by an adiabatic bomb calorimeter (IKA C7000, Werke
GmbH and Co., Staufen, Germany);
The amino acid analyses of Isochrysis sp. biomass and test diets were
performed using a HPLC system provided with a LC 200 Perkin Elmer
pump tted with an ISS-100 auto sampler (20 L loop) and a uorimetric detector (Perkin Elmer, Norwalk, CT, USA), EX 250 nm and EM
395 nm. Separation was achieved by using one AccQ.Tag Aminoacid
Analysis column (Waters Corporation, Milford, MA, USA) and one
Waters pre-column lter. The column was thermostated at 31 C and

64

E. Tibaldi et al. / Aquaculture 440 (2015) 6068

the ow rate was 0.8 mL/min (Liu et al., 1995). Mobile phase A consisted
of acetatephosphate aqueous buffer, and mobile phase B was acetonitrile 100%. Acid hydrolysis with HCl 6 M at 115120 C for 2224 h was
used for all amino acids except cysteine (Cys) and methionine (Met), for
which performic acid oxidation followed by acid hydrolysis was used
and tryptophan that was determined after lithium hydroxide (4 M) hydrolysis. After borate buffer addition, ltered hydrolysated samples
were derivatized at 55 C for 10 min with 20 L of AccQ.Fluor reagent
(6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) before injection
in the HPLC system (Bosch et al., 2006).
The total lipid fraction of the freeze-dried microalgae biomass, test
diets, faeces and llet muscle tissue, was extracted with chloroformmethanol (2:1 v:v) mixture (Folch et al., 1957). The fatty acid methyl
esters were obtained according to Morrison and Smith (1964), and
their quantitative composition was determined by Varian gas chromatograph 430-GC under the condition previously described (Tulli
et al., 2012). The fatty acid methyl esters were identied by comparison
to reference standards (Supelco, Bellefonte, PA, USA) and an internal
standard (C23:0) was used to obtain their quantication.
2.8. Statistical analysis of data
All response variables to dietary treatments were analysed by
ANOVA (single factor diet xed model) according to the GLM procedures of the SPSS/PC package for Windows release 16.0.0 (SPSS Inc.,
Chicago, IL, USA). When appropriate, dietary means were subjected to
Duncan's multiple comparison test, for P b 0.05.
The results obtained in each session of the triangular test were
analysed by comparing the number of correct assignments with the
number you would expect to obtain by chance alone using the statistical
table of Roessler et al. (1948) for P b 0.05.
3. Results
As shown in Table 4, the apparent digestibility coefcients of crude
protein and dry matter were not affected by dietary treatments
(P N 0.05), whereas ADCs of gross energy and crude lipid were signicantly reduced in the highly substituted diet compared to the control
one. Despite the above mentioned differences in ADC values, all diets
resulted in similar digestible protein to energy ratios.
The feed intake, growth performance, feed and protein conversion
efciency of sea bass fed the test diets over 121 days are shown in
Table 5. Compared to the control treatment a slight increase in feed
intake (+ 4/5%) was observed in sh fed the substituted diets with a
signicantly higher value only in case of those fed diet IPO2. The
observed differences in feed intake did not result in parallel changes
of the growth performance as all diets gave rise to very similar average

Table 4
Apparent digestibility coefcients (ADCi %) of dry matter, major nutrients, gross energy
and digestible protein and energy contents of the test diets (values are means of triplicate
measurements).

Diets

Initial weight (g)

Final weight (g)
Feed intake (g/sh/day)
SGR1
FCR2
PER3
GPR4

Control

IPO1

IPO2

78.4
93.2
92.4a
88.7a
446.9
18.0
24.8

76.7
93.4
91.7a
86.8ab
445.9
17.8
25.0

75.3
92.6
87.6b
85.0b
435.9
17.5
24.9

2.20
1.39
1.73
1.56

Row means with different superscript letters are signicantly different, a, b: P b 0.05.
1
ADC(%) = {[(% nutrient in the diet / % marker in the diet) (% nutrient in faeces / %
marker in faeces)] / (% nutrient in the diet / % marker in diet)} 100.
2
s.e.m. = standard error of the mean.

Control

IPO1

IPO2

s.e.m.

142.0
285.4
1.93 b
0.58
1.68
1.25
27.6

141.9
287.7
2.01ab
0.58
1.69
1.24
27.0

142.1
286.3
2.03 a
0.58
1.76
1.21
27.3

0.46
11.15
0.048
0.032
0.093
0.064
1.73

Row means with different superscript letters are signicantly different, a, b: P b 0.05.
1
SGR: 100 {(ln nal body weight ln initial body weight) / days}.
2
FCR: feed intake / weight gain.
3
PER: weight gain / crude protein intake.
4
GPR: 100 [(nal body protein content initial body protein content) / crude
protein intake].

individual nal weight or SGR (P N 0.05) and not signicantly different

FCR, PER and GPR values.
As shown in Table 6, no diet-induced effects were observed in major
biometry traits, slaughter yield, somatic indices or hardness (shore).
Similarly, the dietary treatments did not affect moisture, crude protein
and total lipid contents of sh esh (Table 7). On the other hand, the
fatty acid prole of the lipid fraction of llet muscle was substantially affected by the dietary treatment (Table 7). Compared to controls, the
proportion of saturated fatty acids (particularly 16:0) increased in a
stepwise fashion (P b 0.05) in llets of sh fed diets IPO1 and IPO2, according to increasing levels of dietary sh lipid replacement. There was
a parallel increase in the proportion of 18:1n-9 (P b 0.05), but no major
diet-dependent changes were observed in the incidence of total monounsaturated fatty acids (P N 0.05), linoleate or total n-6 PUFA (P N 0.05)
despite a marginal but signicantly lower proportion of 20:4n-6 in llets of sh fed both substituted diets. On the contrary, replacing increasing level of dietary sh oil with alternate lipid sources resulted in
slightly higher linolenate (P b 0.05) but in gradually declining values
of all n-3(LC)PUFAs (P b 0.05), except DHA which lower incidence in llets of sh fed diets IPO1 and IPO2 was statistically not signicant when
compared to controls. A similar tendency was evident also when the
quantitative fatty acid composition was considered (data not shown)
as well as in the quantitative n-3/n-6 PUFA ratio (1.7 vs. 1.4, P b 0.05).
As shown in Table 8, feeding the test diets over 121 days resulted in
some changes in the instrumental colour parameters of sh skin and llet muscle. Irrespective of the topographic position along the body or

Table 6
Total lenght, dressing out yield, somatic indices and hardness of European sea bass fed the
test diets over 121 days. Values are means of 15 individual measurements per dietary
treatment.
Diets

s.e.m.2

Diets

ADCi (%) :
Dry matter
Crude protein
Crude lipid
Gross energy
Digestible proteinDP (g/kg)
Digestible energyDE (MJ/kg)
DP/DE (g/MJ)

Table 5
Feed intake, growth performance, feed conversion ratio and gross protein retention efciency of European sea bass fed the test diets over 121 days. Values are means of quadruplicate groups per dietary treatment.

Total length (cm)

Carcass yield1 (%)
Fillets yield2 (%)
Viscerosomatic index3 (%)
Hepatosomatic index4 (%)
Mesenteric fat index5 (%)
Dorsal hardness (N)
Caudal hardness (N)
1
2
3
4
5

s.e.m.

Control

IPO1

IPO2

29.5
92.6
45.3
7.4
1.7
3.1
12.4
11.9

29.8
92.1
46.0
7.9
1.6
3.6
12.9
12.6

29.8
92.3
46.3
7.7
1.6
3.4
11.3
11.9

Carcass yield (%): (gutted body weight / body weight) 100.

Fillet yield (%): (llets with skin weight / body weight) 100.
Viscerosomatic index (%): (viscera weight / body weight) 100.
Hepatosomatic index (%): (liver weight / body weight) 100.
Mesenteric fat index (%): (mesenteric fat weight / body weight) 100.

0.18
0.15
0.34
0.82
0.05
0.12
0.29
0.26

E. Tibaldi et al. / Aquaculture 440 (2015) 6068

65

Table 7
Fillet muscle moisture, crude protein, total lipid contents (% of wet weight) and fatty acid
prole (% total fatty acid methyl esters) of European sea bass fed the test diets over 121

Table 8
Colour parameters (L*, a*, b*, C*, H*) in the skin ventral and dorsal regions and in the llet
muscle dorsal, caudal and ventral positions of European sea bass fed the test diets over 121

days. Values are means of 15 individual analysis per dietary treatment.

days.

Diets
Control
Moisture (%)
Protein (%)
Total lipids (%)

69.9
19.9
7.7

Fatty acids (% total fatty acids)1

14:0
3.1a
16:0
14.8c
18:0
3.1
Total SFA
21.7c

s.e.m.
IPO1

Diets

IPO2

69.1
20.1
8.0

Control

69.3
19.3
8.2
a

0.39
0.19
0.47

3.1
16.7b
3.1
23.6b

2.7
17.9a
3.2
24.5a

0.09
0.13
0.04
0.16

16:1n-7
18:1n-9
18:1n-7
20:1n-9
22:1n-11
Total MUFA

4.2a
21.7c
2.3a
4.1a
3.4a
35.4

3.4b
23.9b
2.1ab
3.7a
3.5a
35.1

3.1b
25.4a
1.7b
3.1b
2.6b
34.5

0.04
0.14
0.14
0.12
0.15
0.46

18:2n-6
20:2n-6
20:4n-6
Total n-6 PUFA

14.2
0.7b
0.5a
16.0

14.9
0.7b
0.4b
16.7

14.9
0.9a
0.4b
17.2

0.43
0.02
0.01
0.43

18:3n-3
18:4n-3
20:5n-3
22:5n-3
22:6n-3
Total n-3 PUFA

2.2b
0.2
5.4a
1.3a
8.7
25.1a

2.6a
0.2
3.8b
1.1b
7.7
23.7ab

2.7a
0.3
3.4b
0.9c
7.4
22.8b

0.03
0.01
0.04
0.02
0.15
0.54

Row means with different superscript letters are signicantly different, a, b: P b 0.05.
1
The fatty acids 12:0, 13:0, 14:1n-5, 15:0, 16:2n-4, 16:3n-4, 17:1, 16:4n-1, 18:2n-4,
18:3n-6, 18:3n-4, 18:4n-1, 20:1n-7, 20:3n-6, 20:3n-3, 20:4n-3, 21:5n-3, 22:4n-6 and
22:5n-6 were considered in the composite fraction but were not reported in the Table.

llet, the lightness (L*) of both skin and muscle tissue was not affected
(P N 0.05) by dietary treatments. Relative to controls, there was a slight
but signicant increase in the greenness (a*) in either dorsal or ventral
positions of the skin in sh fed the diet including the highest level of
dried microalgae (IPO2). This was coupled with increased Hue values
(P b 0.05) and slightly different colour saturation (Chroma). In case of
llet muscle, both diets IPO1 and IPO2, when compared to controls,
resulted in increasing values of the parameters a* (dorsal and caudal
position, P b 0.05), b* (dorsal and ventral position, P b 0.05), and Chroma
(ventral region, P b 0.05) and in decreasing values of Hue (P b 0.05) regardless of the sampling position.
A triangle test was carried out to evaluate possible overall sensory
differences among cooked llet portions of sh subjected to the different dietary treatments. In both sessions of the triangle test there
were no replicates where the least signicant threshold (P b 0.05)
of 6 right assignments out of 9 (session 1) or 7 out of 10 (session
2) had been reached, making the overall sensory attributes of cooked
llets from sh fed the different diets, indistinguishable by the
assessors.
4. Discussion
In the present investigation, complete feeds including the freezedried microalgae biomass, were highly palatable and this seems to be
in contrast with what it has been reported in some previous investigations. In cod juveniles, a diet containing 140 g/kg of a mixture of dried
marine microalgae biomass including a substantial proportion of
Isochrysis sp., resulted in depressed feed intake and growth (Walker
and Berlinsky, 2011) which was rst ascribed to palatability problems.
Reduced feed consumption was likely a major cause of impaired growth
in goldsh fry fed a diet containing 5% by weight of a dried biomass of I.
galbana to replace 25% of sh protein (Coutinho et al., 2006). On the

s.e.m
IPO1

IPO2

Skin
Dorsal1
L*
a*
b*
Chroma
Hue

57.61
1.68a
13.59a
13.70a
83.1b

58.83
0.66b
11.59b
11.62b
93.5a

3.59
0.31
0.66
0.67
4.25

86.16
0.67a
8.53b
8.59b
95.8b

85.46
0.68a
9.90a
9.96a
95.4b

86.92
1.60b
7.62b
7.80b
102.7a

4.91
0.29
0.69
0.66
22.96

Dorsal1
L*
a*
b*
Chroma
Hue

27.57
5.57b
3.64b
6.73
147.1a

28.25
3.87a
6.43a
7.84
123.6b

29.29
3.56a
5.76a
7.20
127.1b

0.60
0.23
0.37
0.26
2.7

Caudal1
L*
a*
b*
Chroma
Hue

30.87
4.86b
5.35
7.55
136.2a

29.35
3.28a
6.95
8.44
107.7b

30.26
3.17a
6.74
7.66
118.2b

0.75
0.78
0.45
0.29
3.5

Ventral1
L*
a*
b*
Chroma
Hue

36.90
2.13
4.57b
5.23b
117.5a

38.04
0.72
9.02a
10.08a
96.74b

40.99
0.04
8.88a
8.95a
90.69b

1.95
0.89
0.51
0.45
2.5

Ventral1
L*
a*
b*
Chroma
Hue

58.90
1.59a
12.50ab
12.61ab
82.9b

Fillet

Row means with different superscript letters are signicantly different, a, b: P b 0.05.
1
Values for the different location in the same region were pooled because not signicantly different.

other hand, Palmegiano et al. (2009) did not note reduced palatability
in gilthead seabream juveniles fed a diet including 70% dry weight of
Isochrysis sp.(T-ISO) dry biomass, which indeed resulted in improved
feed intake, growth and feed conversion efciency when compared to
controls given a commercial diet. However, in this latter experiment
higher feed consumption could have been the result of a compensatory
response of sea bream to reduced energy and protein densities in the
microalgae containing diet relative to the control one. Reduced feed
consumption and growth performance have also been reported in tilapias fed diets including more than 200 g/kg of dry biomass of different
microalgae species such as Chlorella, Scenedesmus and Spirulina spp.,
whereas at lower or even at higher dietary inclusion levels, the same
microalgae biomass resulted in higher or same feed intake and similar
or improved growth response relative to controls (Badwy et al., 2008;
Olvera-Novoa et al., 1998; Vizcano et al., 2014). In earlier studies summarized by Belay et al. (1996), the inclusion of dried biomass of Spirulina sp. up to 10% in the dry diet of various marine carnivorous sh
species, gave rise to similar growth response and feed efciency relative
to that of sh fed sh meal-based diets. In other recent investigations,
the use of a dried biomass of the marine microalgae Nanofrustulum sp.,
at inclusion levels up to 174 and 323 g/kg, in the diet of juvenile Atlantic
salmon and common carp respectively, resulted in the same feed consumption and growth performance when compared to that of sh fed
the control diets largely based on sh proteins and oils (Kiron et al.,
2012). Similarly, in the European sea bass, Tulli et al. (2012) noted

66

E. Tibaldi et al. / Aquaculture 440 (2015) 6068

equal feed intake, growth and feed conversion ratio in sh given a control feed or diets including up to 160 g/kg of a dried biomass of T. suecica
to replace 20% sh meal protein of the control preparation.
Hence, based on the available literature there is no ready explanation for the different results to diets including different species and
levels of microalgae biomass. It seems however that responses in
terms of feed consumption and growth parameters depend to some
extent on the sh species and sh size and varies with the level of inclusion, the nutritional value and possibly the palatability of the microalgae
biomass which are known to be widely affected by species as well as the
cultivating, harvesting, processing and drying conditions (Brown et al.,
1997; Reitan et al., 1994; Snchez et al., 2000).
A slight increase in feed intake with preparations containing
the dried microalgae biomass and CPO in the present study, seems
consistent with a compensatory response to a parallel slight dilution
of available nutrient/energy density in the same diets. In fact, replacing
increasing levels of sh meal and oil with dried microalgae and CPO
resulted in a tendency towards reduced apparent digestibility of lipid
and gross energy. Given that all diets resulted in similar crude protein
apparent ADC values, it seems that at the levels of inclusion of the
present study, protein digestibility of the dried Isochrysis sp. biomass
compared favourably with that of the control diet largely based on
highly digestible protein sources such as sh meals and wheat gluten.
Unfortunately, the simple layout of the experiment does not help establishing to what extent the observed depression of lipid and gross energy
ADC values is attributable to the inclusion of microalgae biomass in the
diet. Comparisons with other studies are difcult since there have been
very few investigations where the digestibility of dried microalgae
biomass have been estimated in monogastric animals (Becker, 2007;
Skrede et al., 2011) and even more so in sh, where the available
information on this subject is particularly scarce. In the Arctic char and
Atlantic salmon, Burr et al. (2011) found the apparent digestibility of
crude protein and gross energy of a sun-dried Spirulina sp. biomass at
30% dietary inclusion level, to range between 82 and 85% in both species, comparing favourably with those of commonly used plant protein
derivatives such as canola protein or soy protein concentrate. In a recent
study with cod and Atlantic salmon quoted by Reitan et al. (2013), a
substitution up to 12% by weight, of dietary sh meal by a freezedried biomass of Pheodactylum tricornutum resulted in similarly high
apparent nitrogen (9092%) and energy (8588%) digestibilities in
both sh species irrespective of the inclusion level of the marine
microalgae biomass. On the other hand, in the European sea bass
(Tulli et al., 2012), a preparation including 16% by weight of a freezedried T. suecica biomass to replace sh trimmings of a control diet, resulted in still high but signicantly lower ADC values of crude protein,
lipid and organic matter. To our knowledge, the only study reporting
estimates of the apparent digestibility of diets including a freeze-dried
I. galbana biomass in a comparison with those of other marine
microalgae such as Nannochloropsis oceanica and P. tricornutum was
carried out by Skrede et al. (2011) in mink as a carnivorous model for
salmon. In that study, different to the present one, crude protein
digestibility of diets including I. galbana and N. oceanica in graded levels
(from 0 up to 24% dry weight) to replace same proportions of sh meal
in a basal diet, was substantially reduced when just 6% of microalgae
biomass was included in the diet whereas lipid apparent digestibility
was adversely affected only at the highest level of substitution. Reduced
nutrient digestibility was supposedly ascribed to the resistence of the
thick and rigid cell wall of the two algal species to disruption by digestive processes and to the possible presence of lipase inhibitor activity
in marine microalgae as quoted by Bitou et al. (1999). Being less marked
than those observed in the study on mink, the declining lipid apparent
digestibility values in response to increasing dietary levels of dried
Isochrysis sp. biomass in this study could only partly be explained by
the same mechanisms given that the cell wall of Isochrysis sp. is
particularly thin. However, depressed fat and gross energy apparent
digestibilities observed here with the substituted diets could have also

been a consequence of a parallel increase in the dietary level of saturated fatty acids due to the inclusion of a SFA-rich oil such as CPO. This
seems to be supported by the models proposed by Hua and Bureau
(2009) which found the apparent digestibility of dietary lipid in a
variety of sh species, to be inversely related to the proportion of SFA
in total dietary fatty acids. Reduced fat and SFA apparent digestibilities
were noted in salmonids in response to graded levels of dietary SFArich oil like crude palm oil with a more marked declining tendency
when sh were kept at low water temperatures (Ng et al., 2003). Significantly lower fat and SFA digestibility has been reported also in red
hybrid tilapia kept at 29 C when dietary sh oil was totally substituted
by palm oils (Bahurmiz and Ng, 2007). In the abovementioned investigations the drop in apparent lipid and SFA ADC values has been attributed to the high melting point and to increased resistence to digestion of
the dietary triacylglycerols rich in SFA.
As consistently noted in cultured sh species even in the present
study, changes in dietary fatty acid composition were mirrored in the
fatty acid prole and composition of the edible muscle tissue of the
European sea bass. A stepwise increase in the incidence of 16:0, total
SFA and 18:1n-9 in muscle were expected due to the relative abundance
of the same fatty acids in the corresponding diets which in turn mostly
reected the increasing contribution of a SFA-rich oil lipid source such
as palm oil to total dietary lipid. Similar changes in the same fatty acid
prole of the total lipid fraction in sh muscle tissue have been observed
in other studies when increasing levels of palm oils replaced marine
lipids in the diet of a vast range of sh species including salmonids
and marine carnivorous teleosts (Ng and Gibon, 2011). A decline in
the proportion and content of n-3(LC)PUFA in llets of sh fed diets
IPO1 and IPO2 was also expected due to a concurrent shortage of the
same fatty acids in the corresponding diets. However, such a decline,
although statistically signicant, appeared less marked in magnitude
than in the corresponding diets indicating that n-3(LC)PUFAs were
selectively deposited and retained in the esh as extensively observed
in cultivated sh species. In particular tissue DHA was reduced only by
23% and EPA by less than 35% when sh were fed the diet where up to
36% lipid from sh derivatives were replaced by lipids supplied by
Isochrysis compared to control groups, whereas in the same diet these
n-3(LC)PUFAs were only 58% of the concentrations in the control
preparation. This seems to support the idea that the DHA supplied by
the microalgae biomass was actually bioavailable and effectively deposited in the edible portion of sh. Given the recognised nutritional and
healthy roles of n-3(LC)PUFAs and n-3/n-6 ratio to humans, any changes towards the use of alternatives to marine lipids in the diet of cultured
sh species should not be at the expense of the nutritional value of the
nal product. In this regard the present study showed a moderate
reduction of EPA, DHA and n-3/n-6 ratio in the esh of sea bass fed
the substituted diets. This could have some impact in terms of human
weekly recommended n-3(LC)PUFAs intake (ISSFAL, 2009) and claims
for further studies to be conducted in order to optimize the dietary
inclusion of dried Isochrysis biomass as a partial alternative to marine
sh lipids.
Besides the nutritional properties, if a novel dietary treatment could
affect certain quality attributes of the whole sh or esh, the sensory
characteristics also deserve attention from the point of view of consumer acceptance. In this study the addition of microalgae imparted a green
colour to the diets, due to chlorophyll and other pigments, as it was also
observed in other investigations (Walker and Berlinsky, 2011). It is well
known that the presence of various pigments can result in enhanced
pigmentation of skin or esh or both, in sh (Belay et al., 1996; Tulli
et al., 2012; Walker and Berlinsky, 2011). This was also evident in the
present study where increased greenish skin pigmentation was associated to a slightly enhanced yellowish esh in sh fed the diets containing the dried microalgae. To what extent the observed changes in
European sea bass pigmentation patterns could impact on consumer
appeal is presently unpredictable. However, based on the sensory
evaluation by triangle test, the trained panellists were not able to

E. Tibaldi et al. / Aquaculture 440 (2015) 6068

discriminate among cooked llets obtained from sh fed the different

diets.
In conclusion, the results of the present study have shown that
replacing up to 20% crude protein from sh meal by dried Isochrysis
sp. biomass and nearly 36% sh lipid by lipid provided by microalgae
in the diet of adult European sea bass, did not adversely affect feed intake and growth performance even if it resulted in a slight decline of
lipid and energy digestibility and in a moderate reduction of n-3(LC)
PUFA content in the edible muscle tissue. No major diet-dependent
changes were observed in biometry traits and slaughter yield. At the
actual dietary inclusion levels, the presence of dried microalgae led to
increased greenish pigmentation of the skin, but did not allow discrimination of the cooked llet based on sensory evaluation.
To date, based on very limited available data, the use of microalgae in
aquafeeds is not economically feasible (Vizcano et al., 2014). Feed
markets require large quantities of biomass produced at low cost (less
than 1 kg1) (Draaisma et al., 2013) whereas microalgae production
costs are currently higher than 45 kg1, although recent analyses
foresee a decrease to 12 kg1 (Norsker et al., 2011).
From this perspective the results of the present investigation suggest
that further studies are needed to nd optimal dietary combinations of
marine microalgae biomass and cheap alternative lipid sources which
could make feed costs more affordable, while reducing reliance on sh
meal and sh oil and preserve the nutritional value of cultured marine
sh species.

Acknowledgements
The authors wish to thank B. Piani and G.P. Martincig of the Dept. of
Food Science of the University of Udine and A. Bonelli, D. Benvenuti, A.
Pezzati of the Dept. of Agri-food Production and Environmental Sciences
of the University of Florence for technical assistance during sampling
and laboratory analyses.
This study was supported by Scientic Research Projects funded by
the Universities of Florence and Udine, grant number EX60%-2010.

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