Scientific Article
ISSN 1678-2305 online version
BOLETIM DO INSTITUTO DE PESCA
SIGNOR et al. Bol. Inst. Pesca 2018, 44(3): e331. DOI: 10.20950/1678-2305.2018.331 1/8
VITAMIN A SUPPLEMENTED DIET FOR PACU FINGERLINGS
ABSTRACT
Vitamins are present in small amounts in food and are essential for the organic balance of fish.
Liposoluble vitamins are stored in the liver and may lead to hypo or hypervitaminosis which in
both cases can generate complications to animal homeostasis. This study aimed to evaluate the
effects of vitamin A supplementation in the diet of pacu fingerlings (Piaractus mesopotamicus).
A total of 240 fingerlings with the initial average weight of 17.55 ± 3.22 g were randomly distributed in
20 500 L-circular tanks. Diets were supplemented with retinol acetate (1,000,000 IU of vitamin A g
-1
)
to contain 0, 3,000, 6,000, 9,000, and 12,000 IU of vitamin A kg
-1
of diet. The fish were fed ad
libitum. Productive performance, carcass yield, visceral fat, hepatosomatic ratio, carcass chemical
composition, hematology blood parameters, and liver histology were evaluated. The quadratic
effect was observed on final weight and apparent feed conversion with optimum levels at 6,583 and
5,555 IU of vitamin A kg
-1
of feed, respectively. There was no influence of vitamin A supplementation
on survival, carcass chemical composition, hematology, and liver histology. The minimum
supplementation of 5,555 IU of vitamin A kg
-1
of ration is indicated to obtain enhanced results in
weight gain and apparent feed conversion in pacu fingerlings.
Key words: blood parameters; histology; liposoluble vitamin; native fish; pisciculture.
VITAMINA A NA DIETA DE ALEVINOS DE PACU
RESUMO
As vitaminas estão presentes em pequenas quantidades nos alimentos e são essenciais para o
equilíbrio orgânico dos peixes. As vitaminas lipossolúveis são armazenadas no fígado, podem
apresentar sinais clínicos de hipovitaminose ou hipervitaminose, e que em ambos os casos
podem trazer complicações a homeostase animal. O objetivo do presente trabalho foi avaliar
a suplementação de vitamina A em dietas para alevinos de pacu (Piaractus mesopotamicus).
Foram utilizados 240 alevinos com peso inicial médio de 17,55 ± 3,22, distribuídos
aleatoriamente em 20 caixas circulares de 500 L. As dietas foram suplementadas com acetato
de retinol (1.000,000 IU of vitamin A g
-1
), de forma a conter 0, 3.000, 6.000, 9.000 e 12.000 UI
de vitamina A/kg de dieta. Foi observado efeito quadrático sobre os parâmetros de ganho de
peso e conversão alimentar aparente com níveis ótimos de 6.583 e 5.555 UI de vitamina A kg
-1
de
ração, respectivamente. Não foram observadas influencias da suplementação de vitamina A sobre
sobrevivência, composição da carcaça, hematologia e histologia do fígado do pacu. Para obtenção
de melhores resultados de ganho de peso e conversão alimentar indica-se a suplementação
mínima de 5,555 UI de vitamina A kg
-1
de ração para o pacu.
Palavras-chave: parâmetros sanguíneos; histologia; vitamina lipossolúvel; peixes nativos;
piscicultura.
INTRODUCTION
Vitamins are organic compounds that are classified as liposoluble or hydrosoluble
and function as catalysts or metabolism regulators. Liposoluble vitamins, including
vitamin A, are absorbed through the small intestine along with lipids from the diet.
Thus, conditions that are favorable for the absorption of fats provide an increased
absorption of liposoluble vitamins (NRC, 2011).
Vitamin A participates in numerous functions in organisms and is involved in the
synthesis of some glycoproteins and glycosaminoglycans, which act as steroid hormones
in growth regulation and cell differentiation (HALVER, 2002; FURUITA et al., 2003).
According to HALVER (2002), the vitamin requirements in fish vary with age, size,
Arcangelo Augusto SIGNOR
1
Flavia Renata Potrich SIGNOR
2
Wilson Rogério BOSCOLO
2
Altevir SIGNOR
2
Aldi FEIDEN
2
Lauro VARGAS
3
1
Instituto Federal do Paraná - IFPR, Departamento de
Aquicultura, Av. Araucária, 780, CEP 85860-000, Foz do
Iguaçu, PR, Brasil. E-mail: arcangelo.signor@ifpr.edu.
br (corresponding author).
2
Universidade Estadual do Oeste do Paraná, Campus
Toledo, Rua da Faculdade, 645, CEP 85903-000,
Toledo, PR, Brasil.
3
Universidade Estadual de Maringá – UEM, Av. Colombo,
5790, CEP 87020-900, Maringá, PR, Brasil.
Received: December 15, 2017
Approved: March 08, 2018
VITAMIN A SUPPLEMENTED…
SIGNOR et al. Bol. Inst. Pesca 2018, 44(3): e331. DOI: 10.20950/1678-2305.2018.331 2/8
environmental factors, maturation, and gonadal relationships with
nutrients. However, nutrients can be required in up to 10 times
more under conditions of diseases, stress, and social interactions
than under normal culture conditions (TOGUYENI et al., 1997).
SIGNOR et al. (2013) used retinyl acetate Microvit
TM
A Supra 1000 as
the source of vitamin A (0; 3,000; 6,000; 9,000 and 12,000 UI of
vitamin A for kg diet) to supplement diets for pacu (66 grams of
initial weight) and showed no influence on productive performance
in fish cultivated in cages. However, the authors evidenced the
need for more research aimed at establishing the requirement of
vitamin A in this species.
Because the liver is the storage site of vitamin A, it is considered
the main organ related to the metabolism and homeostasis of
this vitamin. Its absorption begins in the intestine followed by
assimilation in enterocytes linked to crude protein. Retinyl esters
are incorporated in the chylomicron and secreted into the
bloodstream. Chylomicrons are incorporated through endocytosis
into hepatocytes in the liver where retinyl esters are stored,
subsequently hydrolyzed, and released as free retinyl; the retinyl
carrier protein transfers it to hepatic stellate cells where it is
re-esterified into retinyl esters and stored in cytoplasmic lipid
droplets (FERNANDEZ and GISBERT, 2011).
The ingestion of vitamin A is important in fish because it acts
on differentiation and cellular proliferation, vision, reproduction,
embryonic development, immune response (HALVER, 2002),
skeletal deformation (LALL and LEWIS-MCCREA, 2007;
FERNANDEZ et al., 2009), and growth (WESTON et al., 2003).
Its deficiency can cause skin bleeding, vision problems, changes in
the hepatosomatic ratio, and fish mortality (SALEH et al., 1995;
HAYASHIDA et al., 2004; MOREN et al., 2004). However, the
excess of vitamin A in the diet reduces the productive performance
and survival, causes the liver to become yellowish, and reduces
hemoglobin and hematocrit levels (HILTON, 1983).
Several studies demonstrate the effects of vitamin A on various
metabolic aspects in fish, either in reproduction and larval stages
or in growth. However, SIGNOR et al. (2013) is the only study
reporting that vitamin A does not influence growth performance,
chemical composition, blood parameters, and total lipid content in
the liver of juvenile pacus (P. mesopotamicus) grown in cages and
weighing more than 66 grams. Therefore, the authors indicated
that further research was needed to evaluate the influence of
vitamin A on growth performance in pacu. On the other hand,
BUENO et al. (2008) reported that the environment used by the
researchers in that study (SIGNOR et al., 2013) was oligotrophic,
which may have compromised the study results. Knowledge
about the effects of vitamin A on fish growth is essential for de
designing of diet formulations that meet the nutritional requirements
of fish (NRC, 2011). An excess, as well as a lack of vitamin A,
may impair growth and survival and cause skeletal formation in
fish (ORNSRUD et al., 2002; HU et al., 2006; PEIL et al., 2007;
FERNANDEZ et al., 2008; FERNANDEZ et al., 2009).
The aim of the present study was to evaluate the supplementation
of vitamin A in diets for pacu fingerlings (Piaractus mesopotamicus)
in regards to productive performance responses, carcass chemical
composition, and hematological and liver histological parameters.
METHODS
The experiment was performed for 102 days in the Aquaculture
Laboratory at the State University of Western Paraná in Toledo-PR.
This methodology was approved by the Committee on Ethical
Conduct for the Use of Animals in Experimentation from the
State University of Maringá under protocol number 066/2009.
Preparation and analysis of diets
Prepared mineral and vitamin supplements containing
0, 3,000, 6,000, 9,000, and 12,000 IU of vitamin A per kilogram
of diet, through the use of retinyl acetate (Microvit™ A Supra
1000) in the concentration of 1,000,000 IU of vitamin A g
-1
were
used in the experiments. The vitamin was the last ingredient added
to the diets (Table 1). The ration was formulated according to the
nutritional requirements of 28% crude protein (PB) and 3,000 kcal
of digestible energy (ED) kg
-1
of ration.
The ingredients (corn, soybean, and wheat bran, and poultry viscera
and fish meal) were weighed, blended, and ground in a hammer
mill until reaching 0.8 mm in diameter. Micronutrients, mineral
and vitamin supplements, antifungal, and oil were subsequently
added and mixed. The diets were subjected to extrusion producing
pellets with 2 mm that were dried in a forced convection oven at
Table 1. Percentage and chemical composition estimated in the
basal ration for pacu fingerlings, Piaractus mesopotamicus.
Ingredients %
Corn grain 41.36
Soybean meal 26.77
Poultry viscera meal 14.65
Fish meal 11.44
Wheat bran 3.00
Soybean oil 1.65
Mineral and vitamin supplement
(1)
0.50
Sodium chloride 0.30
DL-methionine 0.21
Antifungal (calcium propionate) 0.10
Antioxidant (BHT) 0.02
Total (g) 100
Chemical composition
(2)
%
Dry matter 91.00
Protein 28.10
Fat 7.00
Mineral matter 6.40
(1)
Assurance levels per kilogram of product: Vit. D
3
, 400,000 IU; Vit. E, 30,000 IU;
Vit. K3, 2,000 mg; Vit. B1, 4,000 mg; Vit. B2, 4,000 mg; Vit. B6, 2,000 mg;
Vit. B12, 8 mg; Folic acid, 1,000 mg; Ca Pantothenate 1,000 mg; Vit. C, 60,000 mg;
Biotin, 200 mg; Inositol, 20,000; Niacin, 20,000; Choline, 100,000 mg; Co, 140 mg;
Cu, 2,000 mg; Fe, 16,000 mg; I, 200 mg; Mn, 10,000 mg; Se, 80 mg; and Zn,
16,000 mg.
(2)
Analysis performed in the Laboratory of Food Quality/Unioeste.
VITAMIN A SUPPLEMENTED…
SIGNOR et al. Bol. Inst. Pesca 2018, 44(3): e331. DOI: 10.20950/1678-2305.2018.331 3/8
55 °C, packaged, labeled, and stored in a refrigerator until use.
The fish were fed daily to satiety at 8 and 11 am, and 2 and 5 pm.
Experimental design
A total of 240 fingerlings were used with an average weight
of 17.55 ± 3.22 g and 9.29 ± 0.69 cm in total length. They were
randomly distributed in five treatments with four replicates; each
tank contained 12 fish was considered one experimental unity.
Twenty circular 500 L-fiberglass tanks with a conical bottom
and constant oxygenation produced by a central air blower were
used; these tanks contained a water recirculation system with
central and low water outlets. The water circulated through a
mechanical and biological filter, and a heating system. Feeding
was done ad libutum at 8 an 11 am, 2 and 5 pm.
Water quality
The water electrical conductivity (µS cm
-1
), dissolved oxygen
(mg L
-1
), and pH were monitored weekly while temperature (
o
C)
was monitored twice daily, in the morning (8 am) and afternoon
(5 pm). Water samples were collected in dark polyethylene bottles
and evaluated in the Laboratory of Food Quality Control at the
State University of Western Paraná, in Toledo-PR for turbidity,
ammonia, orthophosphate, and available phosphate. Turbidity
was measured using HANNA instruments (Hanna Instruments
);
these samples were subsequently filtered using a vacuum pump
for the analyses of ammonia, orthophosphate, and dissolved
phosphate. The ammonia content was analyzed according to the
methodology described by STRICKLAND and PARSONS (1972),
and orthophosphate and dissolved phosphate were analyzed
according to the methodology described by MACKERETH et al.
(1978). The average values of water quality parameters were
27.14 ± 0.54 °C; 6.89 ± 0.39 pH; 6.84 ± 0.97 mg L
-1
dissolved oxygen;
91.0 ± 4.92 µS cm
-1
electrical conductivity; 0.77 ± 0.13 turbidity;
0.04 ± 0.01 mg L
-1
ammonia; 0.83 ± 0.01 mg L
-1
orthophosphate;
and 0.80 ± 0.01 mg L
-1
dissolved phosphate.
Productive performance
The fish were submitted to fasting for 12 h at the end of the
experimental period and euthanized with benzocaine at the
concentration of 250 mg L
-1
of water (GOMES et al., 2001) for
the analyses of productive performance, yield, carcass chemical
composition, and liver histology.
All fish were weighed and counted for the calculation of
final weight, weight gain (final weight-initial weight), daily
weight gain (weight gain/days of cultivation), final length,
condition factor ((final weight/final length
3
)*100), apparent
feed conversion (ration intake/weight gain), and survival
((final number of animals/initial number of animals)*100).
After being weighted, the animals were identified, immersed
in ice, and sent to the Laboratory of Fish Technology at the State
University of Western Paraná, in Toledo-PR. These fish were
subsequently eviscerated for the removal of visceral fat and liver for the
calculation of body yield (weight without viscera*100)/total weight),
the hepatosomatic ratio (liver weight*100)/total weight), and
visceral fat (visceral fat weight*100)/total weight).
Carcass chemical composition
Six eviscerated fish per replicate were frozen for later analysis
on carcass chemical composition. Whole fish were stored at -18 °C
for the analyses of dry matter, chemical composition, crude
protein, lipid, and mineral matter. Dry weight was calculated
after dehydration in a forced convection oven at 55 °C. The crude
protein analysis was performed by the Kjeldahl method. Lipids were
assessed using the Soxhlet method, and the mineral matter was
obtained using a muffle furnace at 550 ºC.
Hematology parameters
Eight specimens were collected in each experimental unit for
blood evaluation; the fish were anesthetized with benzocaine
(75 mg L
-1
of water) (GOMES et al., 2001) and analyzed in the
Hematology Laboratory of the State University of Western Paraná,
in Toledo-PR. One milliliter of blood was collected from each fish
through caudal puncture using a disposable heparinized syringe.
These blood samples were used for erythrocyte counts and hemoglobin
and hematocrit analysis according to the methodology described by
COLLIER (1944) and GOLDENFARB et al. (1971), respectively.
The hematimetric indexes were calculated based on the average of
corpuscular hemoglobin (hemoglobin*10/erythrocytes), average of
corpuscular hemoglobin concentration (hemoglobin*100/hematocrit),
and corpuscular volume (hematocrit*10/erythrocytes).
Liver histology
Two livers were collected per replicate (8 per treatment), weighted,
and macroscopically evaluated in the Laboratory of Animal
Histotechniques from the Department of Animal Morphology
at the State University of Maringá for the histological analyses.
Liver samples were fixed in Bouin’s solution (750 mL picric acid,
250 mL formaldehyde, and 50 mL glacial acetic acid) for 24 h, and
transferred and stored in 70° GL alcohol solution. These samples
were subsequently dehydrated through an ascending alcohol series,
diaphanized in xylene, and embedded in paraffin for the production
of 6.0 µm thick semi-serial cross-sectional slices using a rotary
microtome (Leica RM 2145). These slices were mounted on slides
that were stained with hematoxylin-eosin (HE) for the evaluation
and description of liver morphology and tissue integrity under a
microscope (400X). Fifty images/slides/fish were captured for
the quantification of hepatocytes using a high-resolution digital
camera (Pro-series
Cybertecnics Average) coupled to an Olympus
Bx 41
microscope and image analysis system (Image-Pro Plus
4.5.1-Media Cybernetics Inc.), totaling 400 images/treatment.
The total area in the microscopic field (90,570.13 mm
2
) subtracted
from the area occupied by the centrilobular vein (9,630.75 mm
2
),
corresponding to 80,939.25 mm
2
of the counting useful area, was
considered as the standard measurement area.
VITAMIN A SUPPLEMENTED…
SIGNOR et al. Bol. Inst. Pesca 2018, 44(3): e331. DOI: 10.20950/1678-2305.2018.331 4/8
Statistical analyzes
The data were submitted to the test of homogeneity and
normality of Cramer-Von Mises. The ANOVA regression analysis,
applied at the 5% significance level, was used to determine the
requirements of pacu fingerlings according to the NRC (2011)
using the statistical program SAS (SAS, 2004).
RESULTS
Productive performance
The data demonstrates that the vitamin A supplementation did
not influence (P > 0.05) the parameters of survival, final length,
condition factor, weight eviscerated fish, and visceral fat (Table 2).
However, weight gain and apparent feed conversion in pacus were
influenced by the diet supplementation with vitamin A.
The quadratic effect was observed on daily weight gain
(y = -6E
-07
x
2
+ 0.0079x + 78.627, R
2
= 0.96) and apparent feed
conversion (y = 9E
-09
x
2
- 0.0001x + 1.6086, R
2
= 0.84) through
the regression analyses; the derivation of the equation showed
optimal levels at 6,583 and 5,555 IU of vitamin A per kilogram
of diet, respectively (Figure 1).
Carcass chemical composition
In this study, the levels of vitamin A supplemented in the diets
for pacu did not show effects on crude protein, lipids, and carcass
mineral matter (Table 3).
Hematology parameters
No differences were observed in hematological parameters such
as hematocrit, erythrocytes, hemoglobin, corpuscular volume
average, corpuscular hemoglobin average, and corpuscular
hemoglobin concentration average between different levels of
vitamin A supplementation in the diet (P > 0.05) (Table 4).
Liver histology
The vitamin A supplementation in the diet for pacu fingerlings
showed no effects on the hepatosomatic index and number of
hepatocytes (P > 0.05) (Table 5) in this study.
Table 2. Productive performance of pacu fingerlings fed with different levels of vitamin A.
Parameters
Vitamin A (IU kg
-1
)
Effect
0 3,000 6,000 9,000 12,000
Initial weight (g) 17.45 ± 1.4 17.86 ± 1.9 17.72 ± 1.8 17.86 ± 1.3 17.67 ± 1.0 NS
Survival (%) 95.83 ± 0.3 97.22 ± 4.2 97.20 ± 4.0 100.00 97.90 ± 4.2 NS
Final length (cm) 16.09 ± 0.2 17.38 ± 0.6 15.57 ± 0.7 17.46 ± 0.5 16.83 ± 0.6 NS
Final condition factor 2.56 ± 0.1 2.24 ± 0.1 2.14 ± 0.1 2.10 ± 0.1 2.15 ± 0.1 NS
Weight eviscerated fish (%) 84.06 ± 1.9 83.89 ± 3.2 85.06 ± 1.8 85.76 ± 0.6 85.76 ± 0.4 NS
Visceral fat (%) 1.27 ± 0.3 1.27 ± 0.1 1.53 ± 0.2 1.46 ± 0.2 1.75 ± 0.3 NS
NS = not significant (P > 0.05).
Figure 1. Effect of different levels of vitamin A supplementation in the diet for pacu fingerlings on weight gain and apparent feed
conversion.