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Received: 8 February 2016 Accepted: 21 February 2017 DOI: 10.1111/anu.12567
ORIGINAL ARTICLE
Effects of increasing protein level on the performance, enzyme activity and body composition of the Brazilian sardine, Sardinella brasiliensis (Steindachner, 1879) F.C. Sterzelecki1 | J.K. Sugai2 | M. Baloi1 | G. Passini1 | C.V.A. de Carvalho2 | D.M. Fracalossi3 | V.R. Cerqueira1 1 Departamento de aquicultura, Laboratório de Piscicultura Marinha (LAPMAR), Centro de Ciências Agrárias, UFSC, Florianópolis, SC, Brasil 2
Departamento de Bioquímica, Laboratório de Enzimologia Aplicada, Centro de Ciências Biológicas, UFSC, Florianópolis, SC, Brasil 3
Departamento de aquicultura, Laboratório de Nutrição de Espécies Aquícolas (LABNUTRI), Centro de Ciências Agrárias, UFSC, Florianópolis, SC, Brasil Correspondence Fabio Carneiro Sterzelecki, Departamento de Aquicultura, Laboratório de Piscicultura Marinha, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina, Florianópolis, SC, Brasil. Email:
[email protected]
Abstract A six-week growth trial was performed to estimate the dietary protein requirements for maximum growth of juvenile Brazilian sardine (Sardinella brasiliensis) based on growth performance, feed utilization, body composition and digestive enzyme activity. Six isoenergetic diets were formulated to contain protein levels that increased from 250 to 500 g/kg. Each diet was randomly assigned to triplicate groups of 160 fish with mean initial body weight of 0.93 ± 0.13 g, which were fed four times a day to apparent satiation. Growth tended to increase with the increase in the dietary protein level up to 400 g/kg. Total protein intake was indirectly correlated to apparent protein utilization. No significant differences in whole-body composition were found between fish fed the different protein levels. Acid protease and neutral lipase activities did not show significant differences among the different protein dietary groups. Alkaline protease activity increased in fish fed up to 350 g/kg of protein and amylase activity in fish fed up to 400 g/kg. Using polynomial regression, 367 g/kg was estimated to be the optimum dietary protein requirement for maximum weight gain of juvenile Brazilian sardines. KEYWORDS
brazilian sardine, digestive enzymes, feed, protein requirement, Sardinella brasiliensis
1 | INTRODUCTION
conditions as well as overfishing (Jablonski, 2007). Production peaked in 1973 at 2,28,000 tons and has since declined continuously, reach-
The Brazilian sardine, Sardinella brasiliensis, is a schooling, pelagic fish
ing 51,781 tons in 2014 (FAO, 2016). In this context, aquaculture is
mainly distributed between Cabo de São Tomé, Rio de Janeiro State
one of the best options to meet demand and relieve pressure on the
(22°S) and Cabo de Santa Marta, Santa Catarina State (29°S) and is
over-fished stock (Naylor et al., 2009). Among the efforts to avoid a
one of the most important marine fishery resources in Brazil (Gigliotti,
population collapse, a scientific committee has recommended the pro-
Gherardi, Paes, Souza, & Katsuragawa, 2010; Perin & Vaz-dos-Santos,
duction in captivity of Brazilian sardine for use as live bait (Cergole &
2014). The Brazilian sardine is not only the foundation of an important
Dias-Neto, 2011).
canned sardine industry, but is also used extensively as live bait to
Brazilian sardine is a clupeid species with a short life cycle, high
capture skipjack tuna, Katsuwonus pelamis. Nevertheless, both markets
growth rate and fecundity (Perin & Vaz-dos-Santos, 2014). With diur-
are threatened by the declining populations of this species commonly
nal feeding behaviour, Brazilian sardine feeds on phyto and zooplank-
used as live bait (dos Santos & Rodrigues-Ribeiro, 2000).
ton at early and adult stages (Kurtz & Matsuura, 2001; Schneider &
Brazilian sardine production has decreased over the past 40 years
Schwingel, 1999). However, efforts to produce sardines in captivity
due to environmental changes related to atmospheric and oceanic
have only been initiated recently (Baloi, de Carvalho, Sterzelecki,
Aquaculture Nutrition. 2017;1–9.
wileyonlinelibrary.com/journal/anu © 2017 John Wiley & Sons Ltd | 1
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STERZELECKI et al.
2
Passini, & Cerqueira, 2014) and a definition of nutritional requirements
are secreted (Bakke, Glover, & Krogdahl, 2010). In some species, the
is still lacking. As nutrition plays a key role in the aquaculture industry,
secretion of these enzymes responds positively to increasing levels of
influencing fish growth, health and water pollution, and represents up
dietary proteins, lipids and carbohydrates (Buddington & Krogdahl,
to half of production costs (NRC, 2011), it is essential to determine the
2004). A change in the enzyme profile and concentration can be used
dietary nutrient requirement for a commercial species.
to optimize feed formulation (Lundstedt, Melo, & Moraes, 2004).
Protein is the most expensive nutrient required in the diet and is
Therefore, this study aimed to determine the effects of increasing
essential for fish development (Watanabe, 2002). It plays a key role in
dietary crude protein levels on growth performance, feed efficiency,
many biological functions, including structural, enzymatic, transport,
protein utilization, whole-body composition and digestive enzymes of
immune and cell signalling (NRC, 2011). The dietary protein is bro-
the Brazilian sardine.
ken down into smaller molecules in the gastrointestinal tract, which secrets fluids, electrolytes and digestive enzymes that allow the free amino acids to be absorbed and used (Buddington & Krogdahl, 2004). A diet that contains protein in excess of fish requirements promotes ammonia excretion and a high use of energy for amino acid catabolism,
2 | MATERIALS AND METHODS 2.1 | Experimental diets
which can reduce growth rate (Velazco-Vargas et al., 2014). On the
Six isoenergetic diets were formulated containing from 250 to 500 g/
other hand, low protein dietary content negatively affects fish growth
Kg of protein (Table 1), based on the nutritional requirements of two
and health. In both situations, production costs increase (Carvalho,
other omnivorous species, milk fish Chanos chanos (Borlongan &
Bianchini, Tesser, & Sampaio, 2010; Coutinho et al., 2016; Lall &
Coloso, 1993; Borlongan & Satoh, 2001; NRC, 2011) and Nile tilapia
Tibbetts, 2009).
Oreochromis niloticus (NRC, 2011).
The gastrointestinal tract is the first organ that processes feed-
Fish meal (salmon by-product meal), casein and gelatin were
stuffs and is where digestive enzymes such as acid proteases in the
used as protein sources; sunflower and cod liver oil, lipids; and dex-
stomach and alkaline proteases, lipases and amylase in the intestine
trin, carbohydrate source. To meet phosphorus requirements, dibasic
Ingredients (g/kg)
250
300
350
400
450
500
Fish meala
140.0
160.0
166.2
177.6
190.0
210.0
Fish oilb
87.0
82.5
82.5
71.5
67.5
50.0
Sunflower oil
13.0
12.5
12.5
8.5
7.5
7.4
Premixc
10.0
10.0
10.0
10.0
10.0
10.0
Dextrind
447.5
409.2
363.8
340.0
300.0
270.0
Gelatind
30.0
39.0
45.0
52.5
60.0
70.0
127.5
156.8
200.0
237.5
274.3
302.4
120.0
110.0
100.0
97.4
89.7
79.1
25.0
20.0
20.0
5.0
1.0
1.0
Casein
d
Cellulosed Dibasic calcium phosphate Diet Composition (g/kg) Dry matter
121.9
119.9
113.0
123.0
113.1
101.2
Crude protein
246.7
299.7
345.1
385.8
442.4
486.3
Crude lipid
119.8
119.6
113.4
114.4
113.8
101.2
20.6
26.1
21.1
17.2
23.7
22.3
Acid detergent fibre Ash
a
53.3
52.0
54.1
41.4
36.7
40.3
Gross energy (MJ/kg)e
21,21
21.05
20.88
20.79
20.62
20.19
Protein/Energy (g/MJ)
11.6
14.2
16.5
18.7
21.4
24.0
Salmon by-product meal. Pesquera Pacific Star (Chile). Crude protein 728.0 g/kg of dry matter; crude fat 123.1 g/kg. b Delaware Ltda (Porto Alegre, RS, Brazil). c Nutron Alimentos (Toledo, PR, Brazil) mg/kg diet—manganese, 130; zinc, 700; iron, 500; copper, 70; cobalto, 1; iodum, 3; selenium, 3; retinol, 50 000 (IU/kg diet); cholecalciferol 20 000, (IU/kg diet); α- tocopherol, 500; menadione, 25; thiamine, 125; riboflavin, 125; pyridoxine, 125; cyanocobalamin, 0.15; ascorbyl monophosphate, 1 750; folic acid, 25; pantothenic acid, 250; niacin, 500; biotin, 4; choline, 10,000; methionine, 6 500; inositol, 250; ethoxyquin, 750. d Rhoster Ltda. (São Paulo, SP, Brazil). e Metabolizable energy was calculated from standard values, in which 1 kg of carbohydrate (N-free extract), protein and lipid yields 16.7, 16.7 and 37,6 MJ, respectively (Carvalho et al., 2010).
T A B L E 1 Ingredient composition and proximate analysis of experimental diets (dry matter)
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STERZELECKI et al.
calcium phosphate was added. The experimental diets were produced at the Laboratório de nutrição de organismos aquáticos (LABNUTRI), Universidade Federal de Santa Catarina (UFSC), Florianópolis, SC,
2.4 | Homogenization and enzymatic assay The fish were individually dissected and their whole gut was ex-
Brazil. All the ingredients were mixed, pelletized (1.5 mm), extruded
tracted. Samples were homogenized in cooled distilled water (1:8,
(Inbramaq, MX-40, SP, Brazil) without addition of temperature and
w/v) using tissue-tearor (Biospec Products, Inc., Bartlesville, OK, USA)
pressure and dried at 50°C for 24 hours. The experimental diets were
for 3 minutes and centrifuged at 28 230 g for 15 minutes at 4°C. The
then ground and passed through a 0.6-mm sieve, placed in plastic bags
supernatant was used to determine enzyme activities and soluble pro-
and stored in a freezer at −20°C until used.
tein assays. All enzymatic assays were incubated at 25°C and absorbance was
2.2 | Experimental procedures
read with a microplate reader (Spectramax, Plus-384, Molecular devices, Sunnyvale, CA), by transferring an aliquot of 300 μl of the hydro-
All individuals were handled according to the guidelines approved by
lysis products to the well, except for the lipase activity, whose reaction
the University Animal Ethics Committee (PP00861/CEUA/PROPESQ/
was carried out directly in the microplate wells.
UFSC/2013). Adult Brazilian sardines were hormonally induced
Analysis of total alkaline protease activity was performed according
to spawn, and larvae were reared in a laboratory using rotifers and
to García-Carreño, del Toro, and Ezquerra (1997) by azocasein (Sigma
Artemia as feed, following previously developed protocol (Baloi et al.,
Chemical Co, St Louis, Missouri, USA) hydrolysis. The control (blank) was
2014). After weaning (34 days posthatch), 2880 juveniles were trans-
assayed by adding 200 g/kg trichloroacetic acid to the reaction system
ferred to 18 circular plastic tanks (with 400 L capacity) with 160 fish
before substrate addition. The total alkaline protease was expressed as
per tank in a flowthrough mode (1.0 L min1). During the first week, the
specific activity, the difference in the absorbance at 366 nm between
fish were acclimated and fed ad libitum with INVE NRD aquaculture,
sample and blank (Δ absorbance 366 nm. min−1 ml min−1 mg protein−1). Acid protease activity was determined using the Anson (1938)
Belgium (550 g/kg protein, 130 g/kg lipid and 4843 kcal/kg energy). Before feeding with the experimental diets, food was withheld from
method, with modifications as described by Vega-Orellana, Fracalossi,
the fish for 18 hours and thirty individuals in each tank were weighed
and Sugai (2006). The substrate bovine haemoglobin (Sigma, St. Louis,
and measured (0.93 ± 0.13 g; total length 5.01 ± 0.20 cm). A batch of
USA) hydrolysis was determined using the tyrosine (Merck S.A.,
100 g (± 100 individuals) of fish was anaesthetized with 50 mg/L of
Darmstadt, Germany) standard curve read at 280 nm. A blank with
benzocaine (Reagen Ltda), sacrificed with cold water (2°C) and sub-
enzyme extract, added after the addition of TCA, was prepared to cor-
mitted to whole-body composition analysis. Each diet was randomly
rect endogenous hydrolysis. The specific activity of acid protease was
assigned to triplicate tanks and the fish were fed by hand four times a
expressed as μmol of TCA-soluble tyrosine min−1 ml−1 mg−1 protein. Amylase activity was estimated using soluble starch as a substrate
day to apparent visual satiation. The trial lasted 6 weeks using a natural photoperiod (14-hour light:
as described by Aguilar-Quaresma and Sugai (2005). The released re-
10-hour dark cycle), water temperature at 20.65 ± 0.37°C, dissolved
ducing sugar was assayed using dinitrosalicylic acid as a reagent as
oxygen at 6.34 ± 0.33 mg/L, salinity at 35 ppt, pH at 8.27 ± 0.42 and
described by Miller (1959) and using maltose (E. Merck, Darmstadt,
ammonia-N (total ammonia) and nitrite less than 1 mg/L.
Germany) as the standard read at 540 nm. To quantify endogenous
At the end of the feeding trial, food was withheld from the fish
reducing sugars in the homogenates, and in the starch solution (sub-
for 18 hours before sampling. Total body length, weight and mortality
strate), assay mixtures of enzyme and substrate blanks were also used.
of fish in each tank were noted to calculate weight gain (WG), specific growth rate (SGR), feed efficiency (FE), apparent protein utiliza-
Specific activity of amylase was expressed as μmol of maltose min
−1
ml −1 mg−1 protein.
tion (APU) and survival. To determine whole-body composition and
Neutral lipase activity measurements were taken according to
the hepatosomatic (HSI) and visceral fat index (VFI), 40 fish from each
Sæle, Nordgreen, Olsvik, and Hamre (2010). The activity of the enzyme
tank were anaesthetized with benzocaine (50 mg/L) and sacrificed
was assayed by reading every minute for 30 minutes at 404 nm the
with cold water (2°C). Two other fish per tank were sacrificed and im-
product of substrate 4-nitrophenyl myristate (Sigma-Aldrich, St. Louis,
mediately frozen in liquid nitrogen and stored at −20°C to determine
MO) hydrolysis. The specific activity of neutral lipase was expressed as
digestive enzyme activity.
μmol of 4-nitrophenyl myristate hydrolysed min −1 ml −1 mg−1 protein. The soluble protein of crude enzyme extracts was evaluated using
2.3 | Proximate composition The diets and whole-body compositions were analysed at the LABNUTRI.
the principle of protein-dye method by Bradford (1976) and determined by the bovine serum albumin (Sigma Chemical Co, St. Louis, USA) standard curve read at 595 nm.
All analyses followed standard procedures of the Association of Official Analytical Chemists (AOAC, 1999). Dry matter was obtained by drying in an oven at 105°C until constant weight; ash, by incineration in a muf-
2.5 | Statistical analyses
fle furnace at 450°C for 4 hours; lipid content, by the ether extraction
The data were first subjected to homoscedasticity and homogene-
method; and fibre, by acid digestion; and crude protein was determined
ity. Body composition, HSI, VFI, enzymatic activities were analysed
by Kjeldahl method, multiplying nitrogen by 6.25.
by one-way ANOVA followed by a Tukey test (α=0.05). Second-order
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STERZELECKI et al.
4
polynomial regression was applied to weight gain to determine the
activities decreased to levels similar to those found at low dietary pro-
protein requirement (α = 0.05). Pearson correlation coefficients (r)
tein (Figure 2).
among digestive enzymes, feed intake and growth were calculated.
4 | DISCUSSION
3 | RESULTS
Deficiencies or excesses of each major dietary component may have All of the treatments had a high survival rate, above 98%, without
profound impacts on disease development and survival of fish (Lall
significant differences. In relation to growth parameters, the final
& Tibbetts, 2009). Nevertheless, after the six-week experiment, the
weight was higher at the intermediate protein concentration, from
juvenile Brazilian sardines showed no significant difference in survival,
300 to 450 g/kg of crude protein and specific growth rate from 300
indicating an adaptation to different levels of protein intake.
to 400 g/kg. Although not significant, a clear increase in the hepato-
The optimum protein utilization is not only related to its concentra-
somatic index with an increase in the dietary protein level was no-
tion in the diet, but also to the availability of energy (Watanabe, 1982).
ticed. The visceral fat index displayed no changes. Concerning feeding
A diet deficient in energy in relation to protein, or high protein-to-
parameters, although there was no significant difference in feed in-
energy ratio (P/E), will mean that protein is used for energy to satisfy
take and feed efficiency, protein intake was higher as dietary protein
maintenance before growth (Lovell, 1993) and is usually associated to
increased and reached 8.81 g for the fish fed the 500 g/kg crude
a decreasing nitrogen retention (NRC, 2011). This occurs because the
protein diet in contrast to 4.65 g for the fish fed 250 g/kg. On the
excess amino acid is not stored or excreted, it is converted into in-
other hand, the higher the feed protein concentration, the lower the
termediate metabolic fuel sources, as fatty acids and carbohydrates,
apparent protein utilization (Table 2). The crude protein requirement
and the nitrogen excreted mainly as ammonia (Evans, Piermarini, &
for juvenile Brazilian sardine was estimated to be 367.7 g/kg, accord-
Choe, 2005). Although fish are known to utilize protein preferentially
ing to the polynomial analyses of weight gain (Figure 1). Whole-body
to lipids or carbohydrates as fuel (Mohanta, Mohanty, Jena, & Sahu,
moisture, ash, lipid and protein were not affected by the experimental
2008a), some economy can be made if other dietary energy-yielding
diets (Table 3).
nutrients are present in adequate amounts to reduce amino acid ca-
Acid protease and neutral lipase activity showed no significant dif-
tabolism, an effect commonly referred to as “protein sparing” (NRC
ference among the treatments. In contrast, alkaline protease activity
2011). Therefore, the decreasing protein-to-energy ratio is associated
increased when the diet contained up to 350 g/kg crude protein and
with increasing nitrogen retention of fishes (Green & Hardy, 2008);
amylase activity increased when fish were fed up to 400 g/kg crude
however, if dietary protein or amino acids are provided below require-
protein. When protein content exceeded 400 g/kg, both enzyme
ments, growth and feed utilization are compromised (Coutinho et al.,
T A B L E 2 Growth parameters of juvenile Brazilian sardine fed different crude protein levels during 6 weeks1 Dietary Protein (g/kg) Variables
250
Survival (%)
98.75
100
350
400
450
500
99.79
99.38
98.75
99.58
Initial weigh (g)
0.94 ± 0.21
0.86 ± 0.23
0.90 ± 0.31
0.82 ± 0.33
1.06 ± 0.23
0.99 ± 0.29
Final weight (g)
2.56 ± 0.17c
2.64 ± 0.16abc
2.86 ± 0.23ab
2.88 ± 0.20a
2.75 ± 0.06abc
2.62 ± 0.15bc
Specific growth rate (%)
2.37 ± 0.03b
2.67 ± 0.15ab
2.78 ± 0.17ab
3.03 ± 0.37a
2.28 ± 0.37b
2.35 ± 0.35b
Hepatosomatic index (%)
0.47 ± 0.09
0.49 ± 0.12
0.50 ± 0.09
0.55 ± 0.07
0.59 ± 0.18
0.70 ± 0.19
Visceral fat index (%) Feed intake (g) Total protein intake (g) Feed efficiency (%) Apparent protein utilization (%) 1
300
2.20 ± 0.71
2.45 ± 1.14
1.77 ± 0.34
2.24 ± 0.54
2.14 ± 0.14
1.57 ± 0.43
18.84 ± 2.05
19.74 ± 0.75
22.77 ± 0.86
20.79 ± 2.15
18.25 ± 1.92
18.13 ± 2.66
4.65 ± 0.51c 34.72 ± 1.48 21.88 ± 0.84
a
5.92 ± 0.22bc 36.49 ± 2.26 ab
18.88 ± 0.55
7.85 ± 0.30ab 34.98 ± 1.82
8.02 ± 0.83a 40.29 ± 3.18
bc
16.16 ± 1.06
b
16.53 ± 2.03
8.07 ± 0.85a 37.82 ± 4.37 bc
14.81 ± 1.16
8.81 ± 1.30a 36.92 ± 5.71 11.88 ± 2.90c
All parameters were analysed by ANOVA and Tukey post hoc test. Results are shown as mean ± standard deviation (n = 3). Significant differences at the same row are shown by different letters (p .05).
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STERZELECKI et al.
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F I G U R E 2 Activity of acid protease, alkaline protease, amylase and lipase in the gastrointestinal tract of juvenile Brazilian sardine fed different crude protein levels during 6 weeks. Data are showed as mean ± SEM (n = 6), and U represents μM of specific subtract formed per min and units represents the Δ absorbance 366 nm. min−1. Means with different letters are significantly different two-banded sea bream, (357 g/kg, Ozório et al., 2009) and blue gourami Trichogaster trichopterus (350 g/kgMohanta et al., 2013). Dose–response on growth parameter is broadly used to estimate
and pintado, Pseudoplatystoma corruscans (Lundstedt et al., 2004). In this study, total proteolytic acid activity in Brazilian sardine 18 hours after feeding was unresponsive to the protein content of the diet.
the nutrient requirements of determined species. However, the profile
In contrast, total alkaline protease activity responded positively
of digestive enzyme activity could be used to improve meal formula-
to dietary protein in Brazilian sardine. In some species, pancreatic en-
tion (Debnath et al., 2007). Changes in diet composition can modulate
zymes such as proteases, amylases and lipases increase with increase
enzymatic activities and nutrient absorption capacity, to improve feed
in dietary protein, lipids and carbohydrates (Buddington & Krogdahl,
use and assure growth performance (García-Meilán et al., 2013). The activity of digestive enzymes can be influenced by many
2004; García-Meilán et al., 2013). However, after inclusion of 400 g/ kg, alkaline protease activity and growth decreased in Brazilian sar-
factors, such as the quality of the diet, feeding management, stage
dine. When the optimal dietary level of protein is surpassed, enzyme
of life and the sampling time after feeding (García-Meilán, Ordóñez-
activity responsible for its breakdown begins to decrease (Debnath
Grande, Valentín, et al., 2016). Pepsin activity, for example, is linked
et al., 2007). Although no correlation was found (r = 0.67, p = .15) to
to gastric pH that becomes acidic after feeding and, depending on
weight gain, the increased activity at 350–400 g/kg was quite similar
sampling time, could yield different results (García-Meilán, Ordóñez-
to the best growth (367.7 g/kg) defined by polynomial regression. This
Grande, Machahua, et al., 2016). This may be why we found contrast-
association was also found in studies on rohu Labeo rohita (300 g/kg,
ing results in acid protease activity in fish fed different levels of dietary
Debnath et al., 2007), pintado (400 g/kg, Lundstedt et al., 2004) and
protein. While pepsin-like activity responded positively in tambaqui,
cachara cat fish (492 g/kg, Cornélio et al., 2014). Nevertheless, pro-
Colossoma macropomum, and cachara catfish, Pseudoplatystoma retic-
tease activity is not always associated with the best fish growth, as
ulatum (Cornélio et al., 2014; de Almeida, Avilez, Honorato, Hori, &
observed in mullet (Carvalho et al., 2010).
Moraes, 2006), the activity level seems to be weakly influenced by
Omnivorous fish have lower protein requirements than carnivorous
dietary protein in larval species (Zambonino Infante & Cahu, 2007) or
fish because of their higher capacity to utilize carbohydrates as energy
even unresponsive as found in studies on tambaqui (Kohla et al., 1992)
sources (Polakof, Panserat, Soengas, & Moon, 2012). In a number of
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STERZELECKI et al.
fish species, amylase activity correlates positively with dietary carbo-
Federal de Santa Catarina (LAPMAR, LABCAI, LABNUTRI and
hydrate levels and feeding intensity (Krogdahl, Hemre, & Mommsen,
Laboratório de Enzimologia Aplicada) for their technical support and
2005). Amylase is an enzyme that has a central role in polysaccha-
Hilton Hiroshi Oshima (Cargill Animal Nutrition) for providing the min-
ride hydrolysis, a common nutrient present in plankton (Krogdahl
eral and vitamin premix.
et al., 2005) that composes the Brazilian sardine diet in nature. In this study, amylase showed increased activity between 350 and 400 g/kg of protein concentration, which corresponds to 363.8–340 g/kg of dextrin, respectively. The gradual replacement of protein in the sardines’ diet was conducted mainly by adding dextrin, a highly digestible carbohydrate. Therefore, we can suppose that the Brazilian sardine is capable of digesting high levels of carbohydrates, up to 363.8 g/kg. After that point, dextrin impaired the induction of amylase, possibly, due to intolerance to higher levels, as was observed in pacu Piaractus mesopotamicus (Honorato et al., 2015) and pintado (Lundstedt et al., 2004). High starch levels can also provoke absorption of amylase onto starch molecules and inhibits the enzyme activity (García-Meilán et al., 2013). In general, omnivorous and herbivorous species can tolerate high levels of carbohydrates in their diets without detrimental effects (Polakof et al., 2012). For example, no negative influence on growth performance was observed on the Nile tilapia (Azaza et al., 2015) and Lebranche mullet (Zamora-Sillero, Ramos, Romano, Monserrat, & Tesser, 2013) when they were fed up to 322–463.9 g/kg of carbohydrates, respectively. The Grass carp Ctenopharyngodon idella even showed improved growth at 314 g/kg cornstarch inclusion (Gao et al., 2010). The most important digestive lipase in teleost fish is bile salt- dependent neutral lipase (Sæle et al., 2010). In general, lipase activity responds to dietary lipids, as in tambaqui Colossoma macropomum (de Almeida et al., 2006) and in Silver barb, Puntius gonionotus (Mohanta et al., 2008b). The neutral lipase activity in Brazilian sardine showed no significant difference among the treatments, probably due to the constant lipid content in the experimental diets. Indian carp also showed no significant difference in lipase activity when fingerlings were given diets with different protein concentrations and similar lipid content (Debnath et al., 2007). In conclusion, based on growth parameters (weight gain and SGR), juvenile Brazilian sardines showed a protein requirement of 367.7 g/ kg for crude protein. Despite the fact that feed efficiency was not significantly different, increasing the P/E ratio was associated with decreasing protein retention. The results on survival, whole-body composition, HSI and VGI show that Brazilian sardine is capable of dealing with high-protein diets without pronounced effects over the range of experimental diets. Alkaline protease and amylase activity were influenced by the experimental diets. The higher activities can be associated with higher digestibility, feed utilization and growth and, therefore, are considered good tools for feed formulation.
ACKNOWLE DG E MEN TS We thank CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for its financial support to this project and for the doctoral scholarship for the first author (AUX-PE 879/2010 Ciências do Mar). We would also like to thank the laboratories of the Universidade
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How to cite this article: Sterzelecki FC, Sugai JK, Baloi M, et al. Effects of increasing protein level on the performance, enzyme activity and body composition of the Brazilian sardine, Sardinella brasiliensis (Steindachner, 1879). Aquacult Nutr. 2017;00:1–9. https://doi.org/10.1111/anu.12567
