Bol. Inst. Pesca, São Paulo, 44(vol. esp.): 74 - 84, 2017
Doi: 10.20950/1678-2305.2017.74.84
GROWTH PERFORMANCE AND CHEMICAL COMPOSITION OF PACUS (Piaractus
mesopotamicus) FED WITH VEGETABLE OIL SOURCES AND ALPHA-LIPOIC ACID*
Adja Cristina Lira de MEDEIROS
1
; Julio Guerra SEGURA
2
; Katia Rodrigues Batista de
OLIVEIRA
3
; Fábio Rosa SUSSEL
4
; Ricardo Henrique Franco de OLIVEIRA
1
; Elisabete Maria
Macedo VIEGAS
1
ABSTRACT
The objective of this study was to evaluate the effects of different oil sources and alpha-lipoic acid
levels on pacus performance, chemical composition of fillets and histological changes in the liver.
Were used 480 pacu juveniles (3.35 ± 0.78 g), distributed in 24 experimental units (n = 20). Six
treatments were delineated in a completely randomized design (CRD), in a 3x2 factorial scheme,
with three sources of oil (soybean, sesame and linseed) and two levels of alpha-lipoic acid (0 and
0.1 %), with four replications. Data were analyzed statistically by analysis of variance (ANOVA)
and Tukey’s test (5%). Diets containing linseed oil as the only lipid source provided better indices
of final body weight and individual weight gain in pacu juveniles. Crude protein content was
higher in fish fillets that fed diets without alpha-lipoic acid. Livers histology was not affected by
the experimental diets. In conclusion, linseed oil improved final body weight and individual
weight gain on pacus, and alpha-lipoic acid did not affect the performance of the animals, however
its absence promoted higher levels of crude protein in fish fillets.
Keywords: average weight; crude protein; individual weight gain; linseed; liver; sesame
DESEMPENHO PRODUTIVO E COMPOSIÇÃO QUÍMICA DE PACUS (Piaractus
mesopotamicus) ALIMENTADOS COM ÓLEOS VEGETAIS E ÁCIDO ALFA-LIPOICO
RESUMO
O objetivo do estudo foi avaliar os efeitos de óleos vegetais e veis de ácido alfa-lipoico sobre o
desempenho de pacus, composição química de filés e alterações histológicas no fígado. Foram
utilizados 480 juvenis de pacu (3,35 ± 0,78 g), distribuídos em 24 unidades experimentais (n = 20).
Foram testados seis tratamentos em delineamento inteiramente casualizado (DIC), no esquema
fatorial 3x2, sendo três fontes de óleo (soja, gergelim e linhaça) e dois níveis de ácido alfa-lipoico (0
e 0,1%), com quatro repetições. Os dados obtidos foram analisados estatisticamente por análise de
variância (ANOVA) e teste de Tukey (5%). As dietas contendo óleo de linhaça como única fonte
lipídica proporcionaram melhores índices de peso médio e ganho de peso individual nos juvenis de
pacu. O teor de proteína bruta foi maior nos filés dos peixes alimentados com as dietas sem ácido
alfa-lipoico. Os fígados de juvenis de pacu não foram afetados pelas dietas experimentais. Conclui-
se que o óleo de linhaça promoveu os melhores índices de peso médio e ganho de peso individual
em pacus e o ácido alfa-lipoico não afetou o desempenho dos animais, mas a sua ausência
promoveu maiores teores de proteína bruta nos filés dos pacus.
Palavras-chave: peso médio; proteína bruta; fígado; ganho de peso individual; linhaça; fígado;
gergelim
Original Article/Artigo Científico: Recebido em 15/11/2016 Aprovado em 18/08/2017
1
University of São Paulo (USP), College of Animal Science and Food Engineering (FZEA), Department of Animal Science,
Aquaculture Laboratory Av. Duque de Caxias Norte, 225 Campus Fernando Costa CEP: 13635-900 Pirassununga
SP Brazil. e-mails: adja.cristina@gmail.com; ricohfo@usp.br; emviegas@usp.br (corresponding author)
2
Central University of Ecuador, Aquaculture Laboratory. Jerónimo Leiton, s/n y Gato Sobral Quito Ecuador. e-mail:
jguerras@uce.edu.ec
3
Federal University of Lavras (UFLA). Av. Doutor Sylvio Menicucci, 1001 Kennedy CEP: 37200-000 Lavras MG
Brazil. e-mail: katiarbo@gmail.com
4
Agency of Agribusiness Technology of São Paulo (APTA). Av. Virgilio Baggio, 85 CEP: 13631-971 Cachoeira das
Emas SP Brazil. e-mail: fabiosussel@hotmail.com
* Financial support: National Council for the Improvement of Higher Education Personnel (CAPES); National Council for
Scientific and Technological Development (CNPq) (process 142482/2015-3).
Growth performance and chemical composition of pacus 75
Bol. Inst. Pesca, São Paulo, 44(vol. esp.): 74 - 84, 2017
INTRODUCTION
The addition of lipids, from vegetable or
animal sources, in fish diets is common. In
general, vegetable oils, especially soybean oils, are
used, and the quality of these products is assessed
primarily in relation to nutritional quality
(DUNFORD, 2004). Besides playing an important
rule in food quality, lipids are extremely
important for living organisms in general (PORTZ
and FURUYA, 2013), as they participate in the
formation and maintenance of various tissues,
organs and structures, as well as being sources of
essential fatty acids (SARGENT et al., 2003). In
addition, the amount and/or quality of the lipids
in the diets may influence the structure of the
hepatocytes of the fish livers (CABALLERO et al.,
1999) and even the hepatosomatic index of the
animals (CHEN et al., 2013).
For fish, lipids are the main source of energy,
releasing more available energy than proteins
and carbohydrates (GLENCROSS, 2009). There
are several factors that may influence the
optimum level of lipids needed in fish diets, such
as the species and environmental conditions to
which they are adapted (fresh water or salt water,
water temperature), food habits (carnivorous or
omnivore/herbivore), development phase (larvae,
juveniles, adults or breeding), and the amount of
proteins and carbohydrates present in the diet
due to the interactions between lipid,
carbohydrate and protein metabolism (GARCIA
et al., 2013), and the lipid source to be used
(ZHOU et al., 2014).
The vegetable oils that are used in fish feed
are soybean oil and linseed oil. Soybean oil, which
is rich in linoleic acid (MCCLEMENTS and
DECKER, 2010), can be used as a source of n-6
fatty acid, and linseed oil, which is rich in
linolenic acid (MCCLEMENTS and DECKER,
2010), can be used as source of n-3 fatty acid
(REGOST et al., 2003). Sesame oil is known to
exhibit high oxidative stability due to the
antioxidant properties of bioactive compounds
present in its composition, such as sesamin and
sesamol (KOCHHAR, 2002). High concentrations
(5.8 g kg
-1
) of sesamin in salmon diets are capable
of affecting the energy metabolism of fish,
especially the metabolism of liver and white
muscle (WAGNER et al., 2014).
Alpha-lipoic acid (ALA) is also a bioactive
compound, found in green leaves (spinach,
broccoli), potato, barley, wheat germ, red meat,
rice and derivatives, among others (PORTELA
et al., 2014), and presents neuroprotective and
anti-inflammatory properties (HOLMQUIST et al.,
2007).
Among native species, pacu (Piaractus
mesopotamicus) has stood out in recent years as a
species of great economic interest in Brazil. As
well as its excellent characteristic of adapting to
the climatic conditions, it has omnivorous food
habit, with strong tendency to herbivorous, as it is
able to feed on fruits, crustaceans, organic debris,
small fishes and molluscs (URBINATI et al., 2005).
In pacu juveniles fed with diets supplemented
with ALA (1,000 mg kg
-1
of feed) and/or ascorbate
(500 mg AA kg
-1
of feed), eicosapentaenoic acid
(EPA, 20:5n-3) levels increased significantly in the
muscles, but not significantly in the brain,
suggesting that the brains are influenced very
little by supplementation of this substance in the
diet, a fact that can be explained by a possible
defensive reaction of these animals to
environmental changes (TRATTNER et al., 2007).
In view of the importance of the use of
vegetable oil sources and bioactive components,
such as alpha-lipoic acid, in fish nutrition, this
study aimed to evaluate the effects of adding of
the three sources of vegetable oils (soybean,
sesame and linseed) and the two concentrations of
alpha-lipoic acid on the performance and
chemical composition of fillets and histological
changes of the liver of pacus.
MATERIAL AND METHODS
The experiment was approved by the
Research Ethics Committee of the Faculty of
Animal Science and Food Engineering (N°
5451281015). The experimental period lasted for
90 days, using 480 pacu juveniles (3.35 ± 0.78 g)
from a commercial fish farm.
Local
The trial was conducted at the Paulista
Agribusiness Technology Agency (APTA,
Pirassununga-SP). The analyses of the chemical
composition of the diets and muscles and the
76 MEDEIROS et al.
Bol. Inst. Pesca, São Paulo, 44(vol. esp.): 74 - 84, 2017
histology of the livers were carried out at the
Faculty of Animal Science and Food Engineering,
University of São Paulo (FZEA/USP).
Experimental design
Six treatments were distributed in a
completely randomized design (CRD), in a 3x2
factorial scheme, with three sources of vegetable
oils (soybean, sesame and linseed) and two levels
of alpha-lipoic acid (0 and 0.1%), with four
replicates. Each treatment corresponded to an
experimental diet and was calculated in relation
to the total percentage of the diet, as described
below: T1 (control diet) 5% soybean oil (SBO);
T2 2.5% sesame oil (SO) + 2.5% linseed oil (LO);
T3 5% LO; T4 5% SBO + 0.1% alpha-lipoic acid
(ALA); T5 2.5% SO + 2.5% LO + 0.1% ALA; T6
5% LO + 0.1% ALA.
Experimental diets
The analysis of the chemical composition of
the ingredients and experimental diets were
performed in triplicate, according to the AOAC
(2000) methodology: dry matter was obtained
after drying the samples in an oven at 105 ºC until
constant weight; crude protein was obtained by
the micro-Kjeldahl method, after acid digestion
(total nitrogen x 6.25); ether extract was
determined by extraction by heating the
petroleum ether in a Soxhlet apparatus for six
hours; mineral content was obtained by
incineration in a muffle furnace at 550 °C for four
hours; and gross energy was determined in a
calorimetric pump.
After previous analysis of the chemical
composition of the ingredients, six isoproteic (22%
crude protein - CP) and isoenergetic (4,200
kcal.kg
1
of GE; gross energy) diets were
formulated as presented in Table 1.
In order to prepare the experimental diets, the
ingredients were ground, weighed, homogenized
and submitted to the extrusion process (2 mm
granulometry) using an extruder with a
processing capacity of 30 kg hour
-1
(NX 30
Imbramaq, Ribeirão Preto-SP, Brazil) at Fisheries
Laboratory of ESALQ USP (Piracicaba-SP). After
extrusion, soybean, linseed and sesame oils, as
well as alpha-lipoic acid, were added in the
proportions of each treatment and homogenated.
After drying in a forced ventilation oven (55 °C
for 24 h), the diets were conditioned in identified
plastic containers and stored in a cold room (4 °C)
until use.
Fish and experimental conditions
Prior to the beginning of the experiment, fish
were acclimatized to experimental conditions for
seven days and fed with a commercial diet (Pirá
Alevinos 1.7 mm, Guabi Nutrição Animal,
Campinas-SP, Brazil) with 40% crude protein,
which had been ground in a meat grinder, and the
granules retained in a 1 millimeter mesh were
selected. After the acclimatization period, fish
were individually weighed (3.35 ± 0.78 g) and
randomly distributed in 24 tanks with a capacity
of 50 L, in a recirculating system with thermostatic
heaters and a biological filter. Each tank
corresponded to an experimental unit.
The parameters of water quality evaluated
were temperature (T), dissolved oxygen (DO),
hydrogenation potential (pH), nitrite (NO
2
-
) and
toxic ammonia (NH
3
). Temperature was measured
with a digital thermometer (Incoterm, Porto
Alegre-RS, Brazil) with minimum and maximum
daily temperature records. The other water
parameters were evaluated weekly throughout
the experimental period. Dissolved oxygen was
measured with a digital oximeter (YSI Pro2,
Yellow Springs Instruments, Ohio, USA), and pH,
nitrite and toxic ammonia were measured daily
using commercial kits (Labcon Test). The animals
were fed ad libitum until apparent satiation twice a
day (8:00 a.m. and 5:00 p.m.) for 90 days.
Two individual biometrics were performed,
one before starting the administration of the
experimental diets (Day 0) and another at the end
of the experimental period (Day 90), in order to
measure the parameters of growth performance of
the pacus. In the first biometry, the fish were
anesthetized with eugenol (20 mg L
-1
). In the
second biometry, the fish were immersed in a
lethal dose (250 mg L
-1
) of benzocaine (BRASIL,
2013). Six fish per experimental unit were filleted
and the fillets were frozen separately in
polyethylene packages and stored in a freezer
(-20 °C) for further analysis.
Growth performance
To evaluate the efficiency of the experimental
diets, some growth performance parameters were
Growth performance and chemical composition of pacus 77
Bol. Inst. Pesca, São Paulo, 44(vol. esp.): 74 - 84, 2017
determined: final body weight (FBW), individual
weight gain (IWG), feed intake (FI), feed conversion
ratio (FCR), protein efficiency ratio (PER), specific
growth rate (SGR) and survival rate (SR)
according to the formulas described below (NRC,
2013): FBW = total biomass/number of animals;
IWG = final weight initial weight; FI = total feed
intake per experimental unit; FCR = FI/IWG; PER
= [FBW/(FI x crude protein of diet)] x 100; SGR =
[(log final weight log initial weight)/time (days)]
x 100; SR = [(number of alive animals number of
dead animals)/number of total animals] x 100.
Table 1. Ingredients (%) and chemical compositions (dry basis; %) of experimental diets for pacu juveniles.
T1 (control diet) 5% soybean oil (SBO); T2 2.5% sesame oil (SO) + 2.5% linseed oil (LO); T3 5% LO; T4
5% SBO + 0.1% alpha-lipoic acid (ALA); T5 2.5% SO + 2.5% LO + 0.1% ALA; T6 5% LO + 0.1% ALA.
Ingredient (%)
T1
T2
T3
T4
T6
Soybean oil
5.00
0.00
0.00
5.00
0.00
Sesame oil
0.00
2.50
0.00
0.00
0.00
Linseed oil
0.00
2.50
5.00
0.00
5.00
Alpha-lipoic acid
0.00
0.00
0.00
0.10
0.10
Corn gluten meal
0.18
0.18
0.18
0.18
0.18
Soybean meal
24.65
24.65
24.65
24.65
24.65
Wheat meal
25.30
25.30
25.30
25.30
25.30
Broken rice
11.44
11.44
11.44
11.44
11.44
Ground corn
25.75
25.75
25.75
25.75
25.75
Dicalcium phosphate
1.50
1.50
1.50
1.50
1.50
Premixed vitamins and minerals
1
1.00
1.00
1.00
1.00
1.00
BHT
0.05
0.05
0.05
0.05
0.05
Corn starch
4.21
4.21
4.21
4.21
4.21
Lysine
0.82
0.82
0.82
0.82
0.82
Sand (inert)
0.10
0.10
0.10
0.00
0.00
Chemical composition
DM (%)
90.38
90.81
90.43
90.86
91.14
CP (%)
22.23
22.28
22.15
22.21
22.20
EE (%)
5.07
5.02
5.02
5.06
5.02
MM (%)
4.29
4.28
4.32
4.31
4.30
GE (kcal kg
-1
)
4,202
4,201
4,205
4,204
4,202
1
Composition of premixed vitamins and minerals: vit. A 500,000 UI; vit. D3 250,000 UI; vit. E 5,000 mg; vit. K3
500 mg; vit. B1 1,500 mg; vit. B2 1,500 mg; vit. B6 1,500 mg; vit. B12 4,000 mg; folic acid 500 mg; pantothenate
Ca 4,000 mg; vit. C 10,000 mg; biotin 10 mg; Inositol 1,000; nicotinamide 7,000; choline 10,000 mg; Co 10 mg;
Cu 1,000 mg; Fe 5,000 mg; I 200 mg; Mn 1,500 mg; Se 30 mg; Zn 9,000 mg
3
. BHT: Butylated hydroxytoluene;
DM: dry matter; CP: crude protein; EE: ether extract; MM: mineral matter; GE: gross energy.
Chemical composition of pacu fillets
For each experimental unit, three fish were
randomly collected, filleted, stored in polyethylene
packages in a freezer (-20 °C) and kept frozen
until the analysis. Analyses of the chemical
composition of fish fillets were carried out in
triplicate according to the AOAC (2000)
methodology: moisture, crude protein, ether
extract and mineral matter, as previously
described.
Liver histology
At the end of the experimental period, livers
from three fish per experimental unit were
collected and stored frozen at -20 °C for further
analysis. After thawing, the samples were fixed in
Bouin for 24 hours and then stored in 70% alcohol.
The blocks were made with paraffin and
histological sections were made using a microtome,
with a thickness of 5 μm. In order to evaluate
the morphology of the structures under light
78 MEDEIROS et al.
Bol. Inst. Pesca, São Paulo, 44(vol. esp.): 74 - 84, 2017
microscopy and to measure the area of the
epithelial mucosa, histological sections were
dewaxed, rehydrated according to routine
histological methods and submitted to staining
with hematoxylin and eosin (BANCROFT and
GAMBLE, 2008). The slides were photographed
using a light microscope (Carl Zeiss, Germany)
coupled to a digital camera and analyzed with the
aid of Axio Vision 4.1 software.
Statistical analysis
The data were analyzed using Statistical
Analysis System (SAS, 2011), version 9.3, by
analysis of variance ANOVA and significant
differences were verified by Tukey’s Test with a
significance of 5%. Means within each factor (oil
sources and ALA levels) were compared by
ANOVA and Tukey’s Test (5%). In the analyses of
variance, SAS procedure PROC GLM was used.
All assumptions of the statistical model were
verified using SAS/LAB of SAS (R). When the
assumption of homogeneity of variance was
rejected, SAS procedure PROC GLIMMIX was
used by associating different variances with each
treatment. When the assumptions were satisfied,
SAS procedure PROC GLM was used.
RESULTS
Growth performance
Growth performance data are presented in
Table 2, and there were no differences (P>0.05)
among the treatments evaluated.
Although no differences were observed
between the treatments for growth performance
(P>0.05), there was a difference between FBW and
IWG within each factor (oil sources and ALA
levels) (Table 3).
Table 2. Parameters of growth performance (mean ± standard deviation) of pacu juveniles fed different
experimental diets after 90 days. T1 (control diet) 5% soybean oil (SBO); T2 2.5% sesame oil (SO) + 2.5%
linseed oil (LO); T3 5% LO; T4 5% SBO + 0.1% alpha-lipoic acid (ALA); T5 2.5% SO + 2.5% LO + 0.1%
ALA; T6 5% LO + 0.1% ALA.
Variable
T1
T2
T3
T4
T5
T6
FBW (g)
50.12 ± 7.09
52.63 ± 3.80
56.84 ± 7.62
48.08 ± 6.60
53.15 ± 6.02
60.65 ± 8.91
IWG (g)
46.85 ± 7.04
49.11 ± 3.67
53.49 ± 7.75
44.72 ± 6.51
49.89 ± 5.92
57.33 ± 8.92
FI (g)
66.84 ± 9.91
71.06 ± 8.08
74.62 ± 8.98
65.78 ± 9.06
68.92 ± 7.55
75.22 ± 9.20
FCR
1.43 ± 0.10
1.45 ± 0.11
1.40 ± 0.08
1.47 ± 0.07
1.38 ± 0.08
1.32 ± 0.05
PER (%)
3.16 ± 0.22
3.12 ± 0.23
3.23 ± 0.19
3.06 ± 0.14
3.24 ± 0.18
3.42 ± 0.13
SGR (%.day
-1
)
1.32 ± 0.07
1.31 ± 0.03
1.37 ± 0.07
1.36 ± 0.13
1.35 ± 0.04
1.40 ± 0.07
SR (%)
88.75 ± 13.15
87.50 ± 11.90
90.00 ± 10.80
88.75 ± 6.29
97.50 ± 2.89
86.25 ± 15.48
FBW: final body weight; IWG: individual weight gain; FI: feed intake; FCR: feed conversion ratio; PER: protein efficiency
ratio; SGR: specific growth rate; SR: survival rate.
Table 3. Mean values of final body weight (FBW) and individual weight gain (IWG) of pacu juveniles fed
different experimental diets, within each factor (oil sources and ALA levels).
Variable
Oil Sources
P-value
Levels of ALA
P-value
SBO
SO+LO
LO
0%
0.1%
FBW (g)
49.10
b
52.89
ab
58.74
a
0.04
*
53.20
53.96
0.79
ns
IWG (g)
45.78
b
49.50
ab
55.41
a
0.04
*
49.81
50.65
0.77
ns
FBW: final body weight; IWG: individual weight gain; SBO: soybean oil; SO+LO: sesame oil + linseed
oil; LO: linseed oil; ALA: alpha-lipoic acid;
ns
non-significant (P>0.05);
*
significant (P<0.05). Means of
the same factor followed by different letters in the same row differ from each other (P<0.05).