Scientific Article
ISSN 1678-2305 online version
BOLETIM DO INSTITUTO DE PESCA
Gomes et al. Bol. Inst. Pesca 2019, 45(2): e.465. DOI: 10.20950/1678-2305.2019.45.2.465 1/10
SOURCES OF LIPIDS IN DIETS FOR SILVER CATFISH
(Rhamdia quelen) JUVENILES
ABSTRACT
The purpose of this study was to evaluate different lipid sources in diets for silver catfish juveniles
regarding aspects of productive performance, centesimal composition, hepatic histology, biochemical
responses, and oxidative stress. A total of 300 juveniles with an initial mean weight of 18.45 ± 1.22g
were distributed in 20 net-pens of 1m
3
, arranged in a 200 m
2
masonry tank, for a period of 90 days.
The experimental diets were isoprotein (29.05% digestible protein) and isoenergetic (3,250 kcal.kg-1),
containing different sources of oils (soybean, sunflower, fish, canola and olive) at a concentration of
3.0%. The animals were distributed in a completely randomized design, with five treatments and
four replicates. The data were submitted to ANOVA, and the means, when significant, were compared
by the Tukey test at 5% significance level. Visceral fat index was higher for fish fed diets containing
sunflower oil (P<0.05). No differences (P>0.05) were observed for the variables of productive
performance, centesimal composition, and blood biochemistry. A difference (P<0.05) was observed
in hepatic histology, where the treatment with soybean oil had a higher number of hepatocytes.
The animals fed with fish oil had a higher TBARS lipid peroxidation and a higher GST enzyme activity
(P<0.05). Therefore, the different lipid sources can be used to feed this species without any damage to
productive performance. It is not advisable to include sunflower oil, as it provides greater deposition
of visceral fat.
Key words: fatty acids; aquaculture; fish nutrition; Rhamdia quelen.
FONTES DE LIPÍDIOS EM DIETAS PARA JUVENIS DE JUNDIÁ
(Rhamdia quelen)
RESUMO
O objetivo do presente estudo foi avaliar diferentes fontes lipídicas em dietas para juvenis de jundiá
sobre o aspecto do desempenho produtivo, composição centesimal, histologia hepática, respostas
bioquímicas e estresse oxidativo. Foram utilizados 300 juvenis com peso médio inicial de 18,45 ± 1,22g,
distribuídos em 20 tanques-rede de 1m
3
, dispostos em um tanque de alvenaria de 200 m
2
, por um
período de 90 dias. As dietas experimentais foram isoproteicas (29,05% de proteína digestível) e
isoenergéticas (3.250 kcal.kg
-1
), contendo diferentes fontes de óleos em sua composição: soja, girassol,
peixe, canola e oliva na concentração de 3,0%. Os animais foram distribuídos em um delineamento
inteiramente aleatório, com cinco tratamentos e quatro repetições. Os dados foram submetidos a

de gordura visceral foi maior para os peixes alimentados com dietas contendo óleo de girassol (P<0,05).
Não foram observadas diferenças (P>0,05) para as variáveis de desempenho produtivo, composição
centesimal e bioquímica sanguínea. Foi observado diferença (P<0,05) na histologia hepática, onde o
tratamento com óleo de soja apresentou maior número de hepatócitos. Os animais alimentados com
óleo de peixe apresentaram uma maior peroxidação lipídica TBARS e maior atividade da enzima GST
(P<0,05). Portanto, as diferentes fontes lipídicas podem ser utilizadas na alimentação desta espécie
sem que ocorram prejuízos sobre o desempenho produtivo. Não se recomenda a inclusão de óleo de
girassol, devido ao mesmo proporcionar uma maior deposição de gordura visceral.
Palavras-chave: ácidos graxos; aquicultura; nutrição de peixes; Rhamdia quelen.
INTRODUCTION
Brazil has an enormous water availability, which makes it a promising country in the
production of aquatic organisms. Over the past few years, the country has presented
significant growth in continental fish farming (FAO, 2018), and the cultivation of
Ricácio Luan Marques Gomes
1
Evandro Bilha Moro
1
Bruno dos Santos Sosa
1
Danielle Zanerato Damasceno
2
Mariana Lins Rodrigues
3
Fábio Bittencourt
3
1
Universidade Estadual Paulista “Júlio de Mesquita
Filho” – UNESP, Centro de Aquicultura, Via de Acesso
Professor Paulo Donato Castellane, s/n, Vila Industrial,
CEP 14884-900, Jaboticabal, SP, Brasil. E-mail:
ricacioluan@gmail.com (corresponding author).
2
Raguife Rações, Estrada Municipal SFS 14, s/n, Zona
Rural, CEP 15775-000, Santa Fé do Sul, SP, Brasil.
3
Universidade Estadual do Oeste do Paraná – UNIOESTE,
Rua da Faculdade, 645, Jardim La Salle, CEP 85903-000,
Toledo, PR, Brasil.
Received: November 15, 2018
Approved: February 18, 2019
SOURCES OF LIPIDS IN DIETS…
Gomes et al. Bol. Inst. Pesca 2019, 45(2): e.465. DOI: 10.20950/1678-2305.2019.45.2.465 2/10
native species increased considerably (IBGE, 2016). However,
there is still a lack for studies focused on management techniques
and production of these species in captivity, in order to boost the
productive chain of freshwater fish in Brazil and worldwide.
In the southern region of the country, Rhamdia quelen, popularly
known as Silver catfish, stands out due to its characteristics of rapid
growth in low temperature environments, resistance to handling,
easy adaptation to environmental variables and intensive cultivation,
easy reproduction, omnivorous eating habits with good use of the
most varied foods, in addition to the good acceptance of its meat
by the consumer market (Carneiro et al., 2003; Fracalossi et al.,
2004; Parra et al., 2008; Signor et al., 2013; Feiden et al., 2010;
Diemer et al., 2012).
Nutrition is a limiting factor for fish growth. The development
of highly balanced diets to meet the nutritional requirements of
animals in order to maximize their productive performance is
paramount. In fish feeds, protein is the most expensive nutrient
in diet formulation (Wilson, 2002). Additionally, protein is used
by aquatic organisms (in particular freshwater fish) as sources
of energy (Sargent et al., 2002). Nevertheless, the inclusion of
other nutrients in diets to serve as energy sources is interesting
at the nutritional and functional level, in order to contribute to
the reduction of the use of protein as energy in order to intend it
exclusively for animal growth (Shiau, 2002; Martino et al., 2002).
In this case, lipids may be an alternative, as this nutrient provides
twice as much energy as proteins and carbohydrates through the
oxidation process (Lehninger et al., 2011).
Lipids are organic molecules used as sources of energy and
essential fatty acids in animal nutrition (Bertechini, 2013).
They also act as carriers of fat-soluble vitamins, in addition to
performing important functions in biochemical and hormonal
processes, maintaining the permeability and flexibility of cell
membranes (Haliloǧlu et al., 2004; Leonard et al., 2004).
There are a variety of lipid sources providing essential fatty acids
of both animal and vegetable origin used as ingredients for fish
diets. Fish oil is a lipid source of animal origin, whose chemical
composition consists of approximately of 0.6-10.5% linoleic acid
(C18:2 n-6); of 0.4-2.5% linolenic acid (C18: 3 n-3); of 0.1-1.6%
arachidonic acid (C20: 4 n-6), 0.4-17.4% eicosapentaenoic acid
(C20: 5 n-3); and 1.3-23.5% docosahexaenoic acid (C22: 6 n-3)
(NRC, 2011).
Soybean oil is a lipid source of vegetable origin, consisting of
51% n-6 linoleic acid and 6.8% n-3 linolenic acid. Sunflower
oil has 65.7% of n-6 linoleic acid in its chemical composition,
however, it does not contain n-3 linolenic acid. Canola oil contains
20.2% n-6 linoleic acid and 12% n-3 linolenic acid. Olive oil has,
in its chemical composition, 7.9% linoleic acid in the n-6 series
and 0.6% linolenic acid in the n-3 series (NRC, 2011).
The adequate supply of animal and vegetable oil supplementation
can enhance growth without impairing the physiological responses
of the animals. This study aimed to evaluate the effects of
supplementation of different lipid sources on the productive
performance, centesimal composition, hepatic histology, biochemical
parameters and enzymatic activity in silver catfish juveniles.
MATERIAL AND METHODS
Place and period
The experiment was conducted at the Environmental Aquaculture
Research Institute (InPAA), Western Paraná State University
(Unioeste), Toledo, State of Paraná, Brazil. The experimental
period was 90 days (October to January 2016).
This study was approved by the Committee on Ethics in the
Use of Animals (CEUA) of the same institution under protocol
Nº. 08/17.
Animals and experimental design
A total of 300 silver catfish (Rhamdia quelen) juveniles with
an initial mean weight of 18.45 ± 1.22 g were distributed in
20 net-pens with 1 m
3
in volume, arranged in a 200 m
2
masonry
tank. The experimental design was completely randomized
with five treatments and four replicates, totaling 15 fish per
experimental unit.
Water quality
The physical-chemical variables were monitored during the
experimental period, with weekly measurements of dissolved
oxygen (6.0 ± 0.8 mg.L
-1
), pH (6.7 ± 0.2) and electrical conductivity
(36.1 ± 1.5 μS.cm
-1
), with the aid of a multiparameter probe
(YSI, Pro Plus, Yellow Springs-Ohio, United States). The water
temperature (23.6 ± 2.1 °C) was measured daily in the morning
and afternoon by means of a digital thermometer. The parameters
of water quality remained in the ideal range for the productive
performance of the species (Baldisserotto and Radünz Neto, 2004).
Experimental diets and food management
Five isoenergetic (3250 kcal ED/kg feed) and isoprotein (29.09%
PD) diets were prepared. The diets contained different oil sources
in their composition (soybean, sunflower, fish, canola, and olive)
in the concentration of 3.0% (Table 1). The ingredients were
ground in a hammer mill with a 0.6 mm mesh sieve, mixed and
extruded (Ex-Micro
extruder, Ex-Micro, Ribeirão Preto – São
Paulo, Brazil). Simultaneously, the feeds were subjected to forced
ventilation drying (55 °C) during the 24-hour period. The feeds
then received the necessary oils manually by spraying and were
mixed and stored in a freezer during the experimental period.
The fish were fed four times a day (8:30 am, 11:00 am, 2:00 pm,
and 5:30 pm) to apparent satiation.
Data collection and productive performance
At the end of the experiment, the fish were fasted for 24 hours
to empty the gastrointestinal tract and subsequently desensitized
with a solution containing 100 mg.L
-1
benzocaine for individual
measurements of weight (g) and length (cm) and blood tests.
Three fish from each experimental unit were euthanized in
benzocaine, at a dose of 250 mg.L
-1
(Gomes et al., 2001), and
then stored on ice for removal of visceral fat and liver.
SOURCES OF LIPIDS IN DIETS…
Gomes et al. Bol. Inst. Pesca 2019, 45(2): e.465. DOI: 10.20950/1678-2305.2019.45.2.465 3/10
The productive performance data evaluated were weight
gain (g) (final body weight – initial body weight); survival (%)
[(final number of fish / initial number of fish)] * 100; apparent
feed conversion (diet consumed / weight gain); specific growth
rate (% day
-1
) [(ln (final weight) – ln (initial weight)) / days of
experiment] * 100; protein efficiency ratio [weight gain (g) / crude
protein consumption in dry matter (g)]; hepatosomatic index (%)
[liver weight (g) * 100 / final weight (g)]; and visceral fat (%)
[visceral fat weight (g) * 100 / final weight (g).
Centesimal composition analysis
The centesimal composition of the fish was determined by
the methodology proposed by AOAC (2005), being: humidity
(pre-drying in forced air ventilation oven at 55 °C for 72 hours,
followed by drying at 105 °C for 8 hours); proteins (Kjeldhal
method; Modle MA-036, Piracicaba - São Paulo, Brazil); ethereal
extract (Soxhlet extractor with petroleum ether as solvent; Modle
TE-0,44, Piracicaba - São Paulo, Brazil); and mineral matter
Table 1. Percentage and centesimal composition of the experimental feeds containing different oils, for silver catfish (Rhamdia
quelen) juveniles.
Ingredients (%)
Lipid sources
Soybean oil Sunflower oil Fish oil Canola oil Olive oil
Fish flour 33.30 33.30 33.30 33.30 33.30
Corn grain 18.89 18.89 18.89 18.89 18.89
Rice flour 12.65 12.65 12.65 12.65 12.65
Poultry offal flour 12.55 12.55 12.55 12.55 12.55
Soybean meal 45% 10.00 10.00 10.00 10.00 10.00
Wheat meal 8.00 8.00 8.00 8.00 8.00
Soybean oil 3.00 - - - -
Sunflower oil - 3.00 - - -
Fish oil (tilapia) - - 3.00 - -
Canola oil - - - 3.00 -
Olive oil - - - - 3.00
Premix
1
1.00 1.00 1.00 1.00 1.00
Table salt 0.30 0.30 0.30 0.30 0.30
Choline chloride 0.10 0.10 0.10 0.10 0.10
Antifungal 0.30 0.30 0.30 0.30 0.10
Vitamin C 0.10 0.10 0.10 0.10 0.10
Antioxidant (BHT) 0.02 0.02 0.02 0.02 0.02
Total 100 100 100 100 100
Nutrients (%)
Digestible protein 29.09 29.09 29.09 29.09 29.09
Linoleic acid 2.97 2.97 2.97 2.97 2.97
Starch 25.00 25.00 25.00 25.00 25.00
Total arginine 2.67 2.67 2.67 2.67 2.67
Calcium 3.21 3.21 3.21 3.21 3.21
Digestible energy (kcal) 3250 3250 3250 3250 3250
Crude fiber 1.68 1.68 1.68 1.68 1.68
Total phosphorus 1.45 1.45 1.45 1.45 1.45
Fat 9.36 9.36 9.36 9.36 9.36
Total lysine 2.01 2.01 2.01 2.01 2.01
Total methionine 0.71 0.71 0.71 0.71 0.71
Chemical composition (natural matter %)
Gross energy (Kcal) 4540.00 4480.00 4500.00 4480.00 4480.00
Crude protein 38.43 39.06 39.08 38.28 39.47
Fat 8.60 8.03 8.68 8.79 8.84
Mineral matter 3.84 4.05 3.64 4.23 4.05
1
Guarantee levels per kilogram of product: vit. A = 500,000 IU; vit. D3 = 250,000 IU; 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; calcium pantothenate = 4,000 mg; vit. C = 10,000 mg; biotin = 10 mg; Inositol = 1,000; nicotinamide = 7,000; choline =
10,000 mg; Cobalt = 10 mg; Copper = 1,000 mg; Iron = 5,000 mg; Iodine = 200 mg; Manganese = 1,500 mg; Selenium = 30 mg; Zinc = 9,000 mg.
SOURCES OF LIPIDS IN DIETS…
Gomes et al. Bol. Inst. Pesca 2019, 45(2): e.465. DOI: 10.20950/1678-2305.2019.45.2.465 4/10
(calcination of samples at 550 °C for 6 hours, Modle 2000B,
Belo Horizonte - Minas Gerais, Brazil).
Liver histology
For hepatic histological assessment, the livers of three animals
per experimental unit were collected to determine the number of
hepatocytes, totaling 12 fish per treatment. These samples were
fixed in an Alfac solution for 24 hours and then transferred to flasks
containing 70° alcohol. The material was dehydrated by passages in
increasing series of alcohols, diaphanized in xylol and embedded in
paraffin, to obtain serial cross sections (7μm in thickness) with the
aid of a rotating microtome (Thermo Scientific – Microm HM 340E).
To determine the number of hepatocytes per area (counting area:
2,000μm
2
), the slides were stained with Hematoxylin and Eosin
(HE), as described by Bancroft and Stevens (1982).
The photodocumentation (image capture) was obtained by
means of an optical microscope (P1 Olympus BX 50 – Manila,
Philippines) coupled to a camera (Olympus PMC 35 B – Berlin,
Germany), using a 40x, in 8 images per cut (24 images per animal),
totaling 192 images per treatment. Morphological alterations
were evaluated qualitatively through a lesion index calculated
according to Bernet et al. (1999).
The changes were classified into three factors of importance (w):
(1) reasonable injury; (2) moderate injury; and (3) irreversible injury,
which leads to partial or total organ loss. Each histopathological
change was evaluated by using scores (α) that varied from 0 to 6,
depending on the degree of alteration, being (0) without change;
(2) low occurrence; (4) moderate occurrence; (6) serious injury.
To determine the lesions, we used the Bernet index = Σ factor of
importance (w) x score (α). A table was developed for the present
study (Table 2), with the main histopathological lesions found in
the study. These measurements were performed with the aid of an
image analysis system (cellSens Standard 1.15
software program).
Blood tests
For the evaluation of the blood parameters, an aliquot of blood
was collected in three fish from each experimental unit through the
caudal puncture, with the aid of a heparinized syringe. Subsequently,
biochemical analyses of total protein (g.dL
-1
), total cholesterol
(mg.dL
-1
), glucose (mg.dL
-1
) and triglycerides (mg.dL
-1
) were
performed. To obtain the plasma, the samples were centrifuged
at 2,500 rpm for five minutes. Commercial kits (Gold Analisa
)
were used for the analysis, the reading being processed in a
spectrophotometer, according to the manufacturers instructions.
Oxidative stress
At the end of the experimental period, analyses were
performed on thiobarbituric acid reactive substances (TBARS),
Glutathione-S-transferase (GST), and catalase (CAT). Two fish
from each experimental unit, packed in ice, were used. A liver
sample was then removed and stored in liquid nitrogen.
The liver samples were homogenized in 9 mL of 0.3M sodium
phosphate buffer (140mM KCl, pH 7.4). They were then centrifuged
at 10,000 x g for 10 minutes at 4 °C. The liver supernatant was
used to perform the TBARS, GST and CAT analyses.
For the determination of TBARS, the methodology described
by Buege and Aust (1978) was used, and the results were
expressed in n mol MDA/mg protein. The catalase activity
(CAT) was determined according to Nelson and Kiesow (1972)
at a wavelength of 240 nm, and the results were expressed as
µmol/mg protein/min. In turn, the Glutathione S-transferase
(GST) activity was measured at 412 nm in a spectrophotometer
according to the Ellman method (1959) using 20% Trichloroacetic
Acid (20% TCA = 20g TCA in 100 mL distilled water) in the
proportion of 0.2 g of tissue per 1 mL of TCA. The reading was
performed in a spectrophotometer at a wavelength of 412 nm,
and the activity was expressed in μmol.mg
-1
protein.
Statistical analysis
The data were submitted to the analysis of normality and
homoscedasticity and, considering these assumptions, were
submitted to analysis of variance (ANOVA). When they presented
significant differences, a comparison test was performed by the
Tukey means, at 5% significance level. The Bernet index was
submitted to non-parametric Kruskal-Wallis statistics. The analyses
were performed by Statistic 7.1 software
.
RESULTS
The silver catfish fed a diet containing animal and vegetable
oils presented similar results (P>0.05) for weight gain, survival,
apparent feed conversion, specific growth rate, protein efficiency
ratio and hepatosomatic index. Meanwhile, the visceral fat index
was higher (P<0.05) in fish fed diets containing sunflower oil
(Table 3).
The centesimal composition of the fish did not differ (P>0.05)
between diets containing animal and vegetable oils (Table 4).
Regarding hepatic histology (Table 5), the amount of liver cells
was higher for the treatment containing soybean oil (P <0.05).
No significant difference was found between the treatments for
the lesion index in the liver (Figure 1). In the present study, the
Table 2. Changes found in the liver of silver catfish (Rhamdia
quelen) juveniles fed diets containing animal and vegetable oils.
Bernet index
Changes observed in
the liver
Factor of
importance
Hepatocyte
IFL 2
MM 1
N 3
CS 1
LLC 2
VC 1
IFL = Leukocyte inflammation; MM = Melanomacrophagous; N = Necrosis;
CS = Congestion of sinusoids; LLC = Loss of cell limit; VC = Cytoplasmic
vacuolization.
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Table 3. Productive performance of silver catfish (Rhamdia quelen) juveniles fed diets containing animal and vegetable oils.
Variables*
Oils
Soybean Sunflower Fish Canola Olive CV P
WG (g) 104.83 106.12 100.84 106.62 102.21 9.13 NS
SO (%) 98.33 100 93.33 95 95 3.36 NS
AFC 1.38 1.37 1.53 1.42 1.4 11.19 NS
SPR (%/day) 2.28 2.28 2.03 2.2 2.16 4.37 NS
PER 2.07 2.08 1.97 2.14 2 11.81 NS
HSR (%) 2.21 2.07 2.44 2.2 2.28 16.54 NS
VSFI (%) 2.24a 3.26b 2.38a 2.35a 2.42a 18.19 0,01
WG = Weight Gain (g); SO = Survival (%); AFC = Apparent Food Conversion; SPR = Specific Growth Rate (% / day); PER = Protein Efficiency Rate; HSR = Hepatosomatic
Rate (%); VSFI = Visceral Fat Index (%). *Values followed by distinct letters on the same line differ statistically by the Tukey test (P<0.05). CV = Coefficient of variation.
Table 4. Centesimal composition of silver catfish (Rhamdia quelen) juveniles fed diets containing animal and vegetable oil.
Variables*
Oils
Soybean Sunflower Fish Canola Olive CV P
HU (%) 71.32 70.65 70.93 72.76 71. 06 2.28 NS
CP (%) 15.8 16.03 15.72 18.17 16.33 8.41 NS
EE (%) 10.3 10.03 10.07 9.45 9.64 10.39 NS
MM (%) 3.22 3.48 3.34 2.93 3.57 9.39 NS
HU = Humidity (%); CP = Crude Protein (%); EE = Ethereal Extract (%); MM = Mineral Matter (%); CV = Coefficient of variation; *Not significant (P>0.05). *Not
significant (NS). Probability of significance (P).
Table 5. Hepatic histology of silver catfish (Rhamdia quelen) juveniles fed diets containing animal and vegetable oil.
Variables*
Oils
Soybean Sunflower Fish Canola Olive CV P
HE** 172b 115.91a 146.08ab 131.69a 123.70a 17.02 0.02
HE = Hepatocytes; CV = Coefficient of variation; **Counting area: 2,000 µm
2
; *Values followed by distinct letters on the same line differ statistically by the Tukey test
(P<0.05). *Not significant (NS). Probability of significance (P).
Figure 1. Index of histophatological lesions of silver catfish (Rhamdia quelen) juvenilies fed diets containing animal and vegetable
oil. Values followed by similar letters no differ statistically by the Tukey test (P>0.05).