Aquaculture 298 (2010) 267–274

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Aquaculture
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Effect of dietary protein level, initial body weight, and their interaction on the
growth, feed utilization, and physiological alterations of Nile tilapia,
Oreochromis niloticus (L.)
Mohsen Abdel-Tawwab a,⁎, Mohammad H. Ahmad b, Yassir A.E. Khattab b, Adel M.E. Shalaby c
a
Department of Fish Biology and Ecology, Central Laboratory for Aquaculture Research, Abbassa, Abo-Hammad, Sharqia 44662, Egypt
b
Department of Fish Nutrition, Central Laboratory for Aquaculture Research, Abbassa, Abo-Hammad, Sharqia 44662, Egypt
c
Department of Fish Physiology, Central Laboratory for Aquaculture Research, Abbassa, Abo-Hammad, Sharqia 44662, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: A 10-week feeding trial was conducted to assess the interaction between dietary protein level and fish
Received 11 August 2009 weight on the growth, feed utilization, and physiological alterations of Nile tilapia, Oreochromis niloticus (L.).
Received in revised form 26 October 2009 Fish were categorized into three weights; 0.4–0.5 g (fry), 17–22 g (fingerling), and 37–43 g (advanced
Accepted 27 October 2009
juvenile). Diets containing 25, 35, or 45% crude protein (CP) were fed by triplicate to each fish weight. Fish
growth, feed utilization, and protein turn-over were significantly affected by dietary protein level and fish
Keywords:
Nile tilapia
weight, meanwhile their interaction significantly affected specific growth rate and protein efficiency ratio
Dietary protein (PER) only. Unionized ammonia was significantly affected by dietary protein level, fish weight, and their
Fish weight interaction. Moreover, protein and lipid contents in whole-body of fish were significantly affected by dietary
Feed utilization protein level and fish weight, while their interaction significantly affected total lipids content only. Ash
Body composition content significantly differed with fish weight only. The optimum feed conversion ratio (FCR) was obtained
Physiological status with fry tilapia fed the 45%-CP diet; whereas, the poorest FCR was observed for advanced juveniles fed the
25%-CP diet. The lowest PER and protein productive value (PPV) values were obtained with the 45%-CP diet
fed to advanced juveniles; whereas, the highest values were obtained with the 25%-CP diet fed to fry. The
highest protein growth rate (PGR) was obtained with fry tilapia fed the 45%-CP diet, while the lowest one
was obtained with advanced juvenile fed the 25%-CP diet. Hematological variables were significantly affected
by protein level, fish weight, and their interaction except for serum lipids which was not significantly
affected by the interaction. Activities of aspartate amninotransferase (AST) and alanine aminotransferase
(ALT) in serum, liver, and muscles were significantly affected by dietary protein level and fish weight. The
interaction significantly affected enzyme activities except for serum AST, which was not significant. The
optimum growth of fry tilapia was obtained at 45% CP, while fingerling and advanced juvenile showed
optimum growth performance with the 35%-CP diet. Excess protein in fingerling and advanced juvenile
might be deaminated and used as energy source resulting in increased blood glucose, protein, and lipids as
well as increased unionized ammonia in the environment.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction
The feeding of prepared diets is a principal factor in aquaculture to
increase growth and production of reared fish (Thankur et al., 2004; Liti
et al., 2005; Abdel-Tawwab et al., 2007). Dietary protein is an important
aspect in achieving efficient fish production and its needs should
accommodate fish requirements due to age/weight. Protein is the most
expensive ingredient in prepared feeds and thus it should be carefully
formulated to meet the needs of the cultured organism. Understanding
⁎ Corresponding author. Tel.: +20 55 2319821/+20 120570607; fax: +20 55 3400498.
E-mail address: mohsentawwab@yahoo.com (M. Abdel-Tawwab).
the fish's protein requirement during the growth period is fundamental
in fish culture management leading to maximized feed conversion
efficiency, cost savings, and reduced nutrient loading into the aquatic
ecosystem (Abdel-Tawwab and Ahmad, 2009).
The physiological status of intensively farmed fish is an integral
part of evaluating their health status. Diet composition, metabolic
adaptations, and variations in fish activity are the main factors
responsible for seasonal changes in physiological variables (Cnaani
et al., 2004; Řehulka et al., 2004). Physiological alterations might be
indicative of unsuitable environmental conditions or the presence of
stressing factors such as toxic chemicals, excess organic compounds,
and even usual procedures in aquaculture (Barton and Iwama, 1991;
Wendelaar Bonga, 1997; Barcellos et al., 2004).

0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2009.10.027
268 M. Abdel-Tawwab et al. / Aquaculture 298 (2010) 267–274

Nile tilapia, Oreochromis niloticus (L.) accepts artificial feeding
from hatching and typically shows high survival rates and fast growth
(El-Sayed, 2006). The effect of dietary protein on the intermediary
metabolism of this species, however, remains scarcely known.
Assessing the nutritional demands and the effect of dietary protein
on the metabolism of this species at different weights is of particular
interest. Therefore, this study was carried out to assess the effect of
dietary protein level, initial body weight, and their interaction on
growth, feed utilization, carcass composition, and physiological
alterations of Nile tilapia.
paste extruder. The diets were dried in a forced-air drier at room
temperature for 24 h and stored in plastic bags at −2 °C for further
use. Each of the three diets was fed to fish in the three different size
categories (fry, fingerling, and advanced juvenile of Nile tilapia) and
three aquaria were assigned to each treatment. Fish were fed to
satiation twice daily at 9:00 and 14:00 h for 6 days a week over
10 weeks. The amount of consumed feed for each aquarium was
subsequently calculated as a summation of given diets during the
experimental period. Fish in each aquarium were fortnightly group-
weighed and dead fish were removed and recorded daily.

2. Materials and methods 2.3. Water quality measurements

2.1. Fish culture technique
The experiment design was factorial, including three weight classes
and three dietary protein levels, by triplicate. Healthy Nile tilapia, O.
niloticus (L.) of different weights were obtained from Abbassa fish
hatchery and nursery ponds, Central Laboratory for Aquaculture
Research, Abbassa, Abo-Hammad, Sharqia, Egypt. Fish were acclimated
in indoor tanks for 2 weeks by feeding a commercial diet containing 20%
crude protein (CP). After that they were categorized according their
weights into fry (0.4–0.5 g), fingerling (17–22 g), and advanced juvenile
(37–43 g). Fish of each weight class were distributed into 100-L glass
aquaria (75 × 60 × 50 cm) at a rate of 5 g/L. Each aquarium was supplied
with compressed air via air-stones from air pumps. Well-aerated water
was provided from a storage fiberglass tank. The temperature was
adjusted at 27 ± 1 °C by using thermostatically controlled heaters. Half
of the aquarium's water with fish excreta was siphoned every day and
replaced by an equal volume of well-aerated storage water.
2.2. Diet preparation and feeding regime
Three experimental diets were formulated to contain 25, 35, or
45% crude protein (CP) (Table 1). The ingredients of each diet were
blended together for 40 min to make a paste which was separately
passed through a grinder, and cold-pelleted (1-mm diameter) in a
Table 1
Ingredients and chemical composition of the experimental diets (on dry matter basis).
Ingredients (g/100 g) Dietary protein levels
25% 35%
Water samples were collected fortnightly at 15 cm depth from each
aquarium. Dissolved oxygen was measured in situ with an oxygen meter
(YSI model 58, Yellow Spring Instrument Co., Yellow Springs, OH, USA),
unionized ammonia using DREL/2 HACH kits (HACH Co., Loveland, CO,
USA), and pH with a pH meter (Digital Mini-pH Meter, model 55, Fisher
Scientific, Denver, CO, USA). In all treatments, dissolved oxygen
concentrations ranged from 6.9 to 7.2 mg/L and pH ranged from 7.8 to
8.1. All the water quality parameters were within the acceptable ranges
for fish growth (Boyd, 1984).
2.4. Proximate chemical analysis of diets and fish
The tested diets and whole-fish body from each treatment were
analyzed according to the standard methods of AOAC (1990) for
moisture, protein, fat, and ash. Moisture content was estimated by
drying the samples to constant weight at 85 °C in a drying oven (GCA,
model 18EM, Precision Scientific group, Chicago, Illinois, USA) and
nitrogen content using a microKjeldahl apparatus (Labconco, Labconco
corporation, Kansas, Missouri, USA). Crude protein was estimated by
multiplying nitrogen content by 6.25. Lipid content was determined by
ether extraction in a multi-unit Soxhlet extraction apparatus (Lab-Line
Instruments, Inc., Melrose Park, Illinois, USA) for 16 h. Ash was
determined by combusting dry samples in a muffle furnace (Thermo-
lyne Corporation, Dubuque, Iowa, USA) at 550 °C for 6 h.
2.5. Fish performance
Growth performance was determined and feed utilization was
calculated as following:
45%

Fish meal 15.6 20.3 31.0 Specific growth rate (SGR; %/day) = 100(lnW2 − lnW1) / T; where
Soybean meal 20.0 40.0 50.0 W1 and W2 are the initial and final weight, respectively, and T is
Wheat bran 5.0 5.0 5.0 the number of days in the feeding period;
Ground corn 52.63 28.42 9.44 Feed conversion ratio (FCR) = feed intake (g)/weight gain (g);
Fish oil + corn oil (1:1) 2.0 2.0 2.0
Vitamins and minerals premixa 1.5 1.5 1.5
Protein efficiency ratio (PER) = weight gain (g)/protein intake (g);
Ascorbic acid 0.06 0.06 0.06 Protein productive value (PPV; %) = 100 × (protein gain (g) / protein
Starch 2.21 1.72 0.0 intake (g));
Carboxymethyl cellulose 1.0 1.0 1.0 Protein growth rate (PGR; %/day) =100(Ln final protein content− Ln
Total 100 100 100
initial protein content) /days of feeding;
Chemical analysis (%)b Hepatosomatic index (HIS; %) = 100 × [liver weight (g) / body
Dry matter 92.48 ± 0.7 92.69 ± 0.6 93.09 ± 0.6 weight (g)].
Crude protein 25.32 ± 0.24 35.41 ± 0.33 45.56 ± 0.46
Crude fat 5.87 ± 0.15 5.67 ± 0.25 5.99 ± 0.20 2.6. Physiological measurements
Ash 5.51 ± 0.23 6.31 ± 0.36 7.31 ± 0.37
Fiber 6.68 ± 0.15 5.50 ± 0.12 5.76 ± 0.13
NFEc 56.62 47.11 35.38 At the end of the feeding trial, fish were not fed during the 24 h
GE (Kcal/g)d 439.14 446.85 458.92 immediately prior to blood sampling. Fish were anaesthetized with
a
Vitamin and minerals premix: each 2.5 kg contain vitamin A 12 MIU; D3 2 MIU, E buffered tricaine methane sulfonate (20 mg/L) and blood was collected
10 g; K 2 g; B1 1 g; B2 4 g; B6 1.5 g; B12 10 mg; pantothenic acid 10 g; nicotinic acid 20 g; with a hypodermic syringe from the caudal vein. The extracted blood
folic acid 1 g; biotin 50 mg; choline chloride 500 mg; copper 10 g; iodine 1 g; iron 30 g; was divided in two sets of Eppendorf tubes. One set contained 500 U
manganese 55 g; zinc 55 g and selenium 0.1 g. sodium heparinate/mL, used as an anticoagulant, for hematology
b
Means of five replicates.
c
NFE (nitrogen free extract) = 100 − (protein% + lipid% + ash% + fiber%).
(hemoglobin, haematocrit and red blood cell counting). The second
d
GE (gross energy): calculated after NRC (1993) as 5.64, 9.44, and 4.11 kcal/g for set, without anticoagulant, was left to clot at 4 °C and centrifuged at
protein, lipid, and NFE, respectively. 5000 rpm for 5 min at room temperature. The collected serum was
 M. Abdel-Tawwab et al. / Aquaculture 298 (2010) 267–274 269

stored at −20 °C for further assays. Red blood cells (RBCs) were counted
under the light microscope using a Neubauer haemocytometer after
blood dilution with phosphate-buffered saline (pH 7.2). Hemoglobin
(Hb) level was determined colorimetrically by measuring the formation
of cyanomethaemoglobin according to Van Kampen and Zijlstra (1961).
Haematocrit values (Ht) were immediately determined after sampling
by placing fresh blood in glass capillary tubes and centrifuging for 5 min
in a microhematocrit centrifuge. Glucose was determined colorimetri-
cally according to Trinder (1969). Total protein and total lipid contents
in serum were determined colorimetrically according to Henry (1964)
and Joseph et al. (1972), respectively. Activities of aspartate amino-
transferase (AST) and alanine aminotransferase (ALT) in serum, liver,
and muscle were determined colorimetrically according to Reitman and
Frankel (1957).

2.7. Statistical analysis

Data were analyzed using a two-way ANOVA with protein levels

and fish weights as factors. Statistical significance was set at the 5%
probability level and means were separated using Duncan's new
multiple range test. The software SPSS, version 12 (SPSS, Richmond,
USA) was used as described by Dytham (1999).

3. Results

3.1. Growth performance

Fish growth was significantly affected by protein level and initial

weight, while their interaction significantly affected specific growth Fig. 1. Changes in live body weight (g) of Nile tilapia with different initial body weights
rate (SGR) only (P < 0.05; Table 2). The highest growth of fry tilapia and fed different protein levels for 10 weeks in glass aquaria.

was obtained at 45% CP, while fingerling and advanced juvenile

showed optimum growth performance at 35% CP (Fig. 1). The poorest there was no significant effect due to their interaction (P < 0.05;
fish growth was obtained with the 25%-CP diet irrespective of fish Table 2). FI increased and FCR decreased significantly with increasing
weight. Feed intake (FI) and feed conversion ratio (FCR) were protein level up to 35% CP (P < 0.05), although FI and FCR of fingerling
significantly affected by protein level and initial body weight and were not significantly different (P > 0.05). The optimum FCR was

Table 2
Final weight, specific growth rate (SGR), feed intake, feed conversion ratio (FCR), hepatosomatic index (HSI), and survival of Nile tilapia as affected by dietary protein levels and different
initial body weights.

Variables Protein level (%) Final weight (g)a SGR (%/day) Feed intake (g feed/g fish) FCR HSI (%) Survival (%)
b
Individual treatment means
Fish size
Fry 25 5.1 3.289 c 8.3 1.81 3.29 96.7
35 7.7 3.900 b 11.8 1.65 2.47 100
45 10.3 4.287 a 14.5 1.49 1.79 100
Fingerling 25 41.1 1.007 e 46.2 2.22 2.01 100
35 45.2 1.143 d 47.9 1.92 1.91 100
45 44.3 1.107 d 47.3 1.98 1.69 100
Advanced juvenile 25 58.6 0.524 g 50.2 2.79 2.30 100
35 64.7 0.672 f 55.7 2.29 1.82 100
45 62.9 0.635 f 55.5 2.45 1.57 100
Pooled SE 0.421 0.022 0.409 0.020 0.202 0.258

Means of main effectsc

Fish size
Fry 7.7 r 3.825 11.5 q 1.65 q 2.52 p 98.9
Fingerling 43.5 q 1.086 47.1 p 2.04 p 1.87 q 100
Advanced juvenile 62.1 p 0.61 53.8 p 2.51 p 1.90 q 100
25 34.9 y 1.607 34.9 y 2.27 x 2.53 98.9
35 39.2 x 1.905 38.5 x 1.95 y 2.07 100
45 39.2 x 2.010 39.1 x 1.97 y 1.68 100

ANOVA: P values
Protein level 0.001 0.0001 0.001 0.0001 0.211 0.099
Fish size 0.0001 0.0001 0.0001 0.0001 0.034 0.099
Protein level × fish size 0.445 0.0001 0.426 0.062 0.181 0.068
a
Initial weights for fry, fingerlings, and advanced juvenile were 0.51 ± 0.006, 20.33 ± 0.577, and 40.43 ± 1.528, respectively.
b
Treatments means represent the average values of three aquaria per treatment. Duncan multiple range test was conducted for individual means only if there was a significant
interaction (ANOVA: P < 0.05). Means followed by the same letter are not significantly different.
c
Main effect means followed by the same letter are not significantly different at P < 0.05 by Duncan multiple range test; p, q, and r for fish size and x, y, and z for protein level.
270 M. Abdel-Tawwab et al. / Aquaculture 298 (2010) 267–274

obtained when fry tilapia were fed the 45%-CP diet (1.49); whereas,
the poorest one was obtained when advanced juvenile were fed the
25%-CP diet (2.79).
The hepatosomatic index (HSI) was significantly affected by fish
weight only (P < 0.05; Table 2) where it decreased significantly with
increasing fish weight. The highest HSI was obtained by fry tilapia fed
the 25%-CP diet; whereas, the lowest one was obtained by advanced
juveniles fed the 45%-CP diet. No significant differences in fish survival
were observed among fish groups and it was almost 100% except for
that of fish fry fed the 25%-CP protein diet (96.7%; Table 2).
3.2. Protein utilization
Protein utilization parameters, i.e., PER, PPV, and PGR were signifi-
cantly affected by protein level and fish weight (P < 0.05; Table 3). The
interaction of both factors significantly affected PER (P > 0.05). The
dietary protein level inversely affected PER and PPV, while it positively
affected PGR at different fish weights. The lowest PER and PPV were
obtained with the 45%-CP diet for advanced juvenile (0.99 and 19.70%,
respectively); whereas, the highest ones were obtained with the 25%-CP
diet for fry tilapia (2.35 and 35.95%, respectively). PGR increased
significantly with increasing fish weights; it was affected by dietary
protein level for fingerling and advanced juvenile. The highest PGR was
obtained with fry tilapia fed the 45%-CP diet (4.32%/day), while the
lowest one was obtained with advanced juvenile fed the 25%-CP diet
(0.69%/day). Unionized ammonia (UA) was significantly affected by
protein level, fish weight, and their interaction (P < 0.05; Table 3). UA
excreted in rearing water increased significantly with increasing protein
level and fish weights. However, the highest UA value was obtained
when advanced juvenile were fed the 45%-CP diet (1.622 mg/L);
whereas, the lowest one was obtained with fry tilapia fed the 25%-CP
diet (1.148 mg/L).
3.3. Body composition
No significant changes in moisture content were observed at the
different treatments (Table 4). Protein and lipid contents in whole-body
fish were significantly affected by dietary protein level and fish weight,
while their interaction significantly affected lipids content only (P < 0.05;
Table 4). Ash content significantly differed due to fish weight (P< 0.05).
The highest protein content in whole body was obtained with the 45%-CP
diet in all weight classes. The body lipid content decreased with increased
dietary protein level within each fish weight; whereas, the highest lipid
content was recorded for the 25%-CP diet at all fish weights. The lowest
lipid content was obtained in fish fed the 35- and 45%-CP diets for
fingerling and advanced juvenile. Ash content in whole body was
unaffected by dietary protein levels at all fish weights, and the lowest
contents were observed in fry (P< 0.05).
3.4. Physiological alterations
RBCs, Hb, and Ht values were significantly affected by protein level,
fish weight, and their interaction (P < 0.05; Table 5). Likewise, serum
glucose, protein, and lipids were significantly affected by protein level
and fish weight (P < 0.05; Table 5), while plasma lipids were not affected
by their interaction. All physiological variables significantly increased
with increasing dietary protein level in all fish weights. In some cases,
these variables did not exhibit significant differences between fish fed
the 35- or 45%-CP diets. AST and ALT activities in serum, liver, and
muscles were significantly affected by dietary protein level and fish
weight (P < 0.05; Table 6). The interaction significantly affected all

Table 3
Changes in protein efficiency ratio (PER), protein productive value (PPV), protein Table 4
growth rate (PGR), and unionized ammonia (UA) of Nile tilapia fed different levels of Proximate chemical analysis (%; on fresh-weight basis) of whole body of Nile tilapia fed
dietary protein at different initial body weights. different levels of dietary protein at different initial body weights.

Variables Protein PER PPV (%) PGR UA Variable Protein Moisture Crude Total Ash
level (%) (%/day) (mg/L) level (%) protein lipids

Individual treatment meansa Individual treatment meansa

Fish size Fish size
Fry 25 2.35 a 35.95 3.98 1.148 f Fry 25 71.6 15.4 9.7 a 3.3
35 1.85 b 28.97 3.94 1.194 ef 35 72.0 15.6 8.9 b 3.5
45 1.58 c 25.18 4.32 1.234 e 45 72.5 16.0 7.8 c 3.7
Fingerling 25 1.92 b 26.80 1.00 1.212 e Fingerling 25 75.2 14.0 5.8 d 5.0
35 1.58 c 24.17 1.21 1.316 d 35 74.9 14.7 5.1 e 5.3
45 1.19 d 19.73 1.24 1.438 b 45 74.2 15.4 5.1 e 5.3
Advanced juvenile 25 1.53 c 27.72 0.69 1.372 c Advanced juvenile 25 73.1 14.6 6.5 e 5.8
35 1.33 d 21.92 0.81 1.482 b 35 74.7 14.3 5.3 de 5.7
45 0.99 e 19.70 0.89 1.622 a 45 73.5 15.5 5.1 e 5.9
Pooled SE 0.016 0.254 0.024 0.007 Pooled SE 0.709 0.145 0.065 0.047

Means of main effectsb Means of main effectsb

Fish size Fish size
Fry 1.93 30.03 p 4.08 p 1.192 Fry 72.0 15.7p 8.8 3.5q
Fingerling 1.56 23.57 q 1.15 q 1.322 Fingerling 74.8 14.7q 5.3 5.2 p
Advanced juvenile 1.28 23.11 q 0.80 r 1.492 Advanced juvenile 73.8 14.8q 5.6 5.8 p
25 1.93 30.16 x 1.89 y 1.244 25 73.3 14.7y 7.3 4.7
35 1.59 25.02 y 1.99 y 1.331 35 73.9 14.9y 6.4 4.8
45 1.25 21.54 z 2.15 x 1.431 45 73.4 15.6x 6.0 5.0

ANOVA: P values ANOVA: P values

Protein level 0.0001 0.0001 0.001 0.0001 Protein level 0.933 0.026 0.0001 0.099
Fish size 0.0001 0.0001 0.0001 0.0001 Fish size 0.294 0.033 0.0001 0.001
Protein level × fish size 0.048 0.063 0.179 0.009 Protein level × fish size 0.981 0.731 0.032 0.629
a a
Treatment means represent the average values of three aquaria per treatment. Treatment means represent the average values of three aquaria per treatment.
Duncan multiple range test was conducted for individual means only if there was a Duncan multiple range test was conducted for individual means only if there was a
significant interaction (ANOVA: P < 0.05). Means followed by the same letter are not significant interaction (ANOVA: P < 0.05). Means followed by the same letter are not
significantly different. significantly different.
b b
Main effect means followed by the same letter are not significantly different at P < 0.05 Main effect means followed by the same letter are not significantly different at P < 0.05
by Duncan multiple range test; p, q, and r for fish size and x, y, and z for protein level. by Duncan multiple range test; p, q, and r for fish size and x, y, and z for protein level.
 M. Abdel-Tawwab et al. / Aquaculture 298 (2010) 267–274 271

Table 5
Changes in red blood corpuscles (RBCs), haemoglobin (Hb), haematocrit (Ht), serum glucose, protein, and lipids in Nile tilapia fed different levels of dietary protein at different initial
weights.

Variables Protein level (%) RBCs (× 106/μL) Hb (g/L) Ht (%) Glucose (mg/L) Protein (g/L) Lipids (g/L)

Individual treatment meansa

Fish size
Fry 25 1.262 d 75.4 c 21.8 f 648.9 c 25.0 e 15.0
35 1.298 d 84.4 c 23.8 e 1191.7 b 28.2 d 15.4
45 1.866 bc 119.4 a 26.8 d 1282.0 b 29.3 cd 16.2
Fingerling 25 1.856 bc 83.7 c 29.0 c 641.2 c 30.3 cd 15.8
35 1.972 bc 100.6 b 31.0 ab 653.0 c 34.9 bc 16.8
45 2.186 ab 102.2 b 32.2 a 1274.4 b 37.7 ab 19.6
Advanced juvenile 25 1.728 c 79.6 c 29.6 bc 714.9 c 33.3 bc 16.8
35 2.166 ab 99.8 b 31.6 a 1425.5 a 41.0 a 19.7
45 2.412 a 109.1 b 32.2 a 1442.0 a 43.1 a 20.5
Pooled SE 0.041 1.274 0.206 13.361 0.709 0.174

Means of main effectsb

Fish size
Fry 1.475 93.1 24.1 1040.9 27.5 15.5 r
Fingerling 2.005 95.5 30.7 856.2 34.3 17.4 q
Advanced juvenile 2.102 96.2 31.1 1194.1 39.1 19.0 p
25 1.6153 79.6 26.8 668.3 29.5 15.9 y
35 1.812 94.9 28.8 1090.1 34.7 17.3 xy
45 2.155 110.2 30.4 1332.8 36.7 18.8 x

ANOVA: P values
Protein level 0.011 0.0001 0.0001 0.0001 0.038 0.0001
Fish size 0.001 0.015 0.0001 0.0001 0.0001 0.0001
Protein level × fish size 0.001 0.001 0.046 0.0001 0.015 0.596
a
Treatment means represent the average values of three aquaria per treatment. Duncan multiple range test was conducted for individual means only if there was a significant
interaction (ANOVA: P < 0.05). Means followed by the same letter are not significantly different.
b
Main effect means followed by the same letter are not significantly different at P < 0.05 by Duncan multiple range test; p, q, and r for fish size and x, y, and z for protein level.

enzyme activities except serum AST. In all fish weights, enzyme
activities increased significantly with the increase of dietary protein
level. The highest activities were observed in advanced juvenile fed the
45%-CP diet; whereas, the lowest values were obtained for fry fed 25%-
CP diet.
4. Discussion
The present study shows that the dietary protein level markedly
affects the growth, feed utilization, and physiological status of Nile
tilapia in all weight classes. The optimum dietary protein required for

Table 6
Changes in AST and ALT activities in serum (IU/L), liver (IU/g fresh weight), and muscles (IU/g fresh weight) of Nile tilapia fed different levels of dietary protein at different initial
weights.

Variables Protein level (%) Serum AST Liver AST Muscle AST Serum ALT Liver ALT Muscle ALT

Individual treatment meansa

Fish size
Fry 25 13.5 225.7 d 460.8 d 14.7 c 235.0 f 337.3 d
35 14.3 258.5 c 612.5 c 18.5 b 292.5 de 427.1 bc
45 17.4 301.3 b 764.0 b 20.3 ab 436.3 c 476.0 ab
Fingerling 25 14.1 229.4 d 458.0 d 15.1 c 255.1 ef 340.3 d
35 17.3 264.8 c 647.3 c 19.1 b 363.7 cd 411.2 bc
45 19.5 303.4 b 817.2 a 20.1 ab 528.9 b 530.0 a
Advanced juvenile 25 15.5 261.3 c 590.0 c 19.0 b 367.5 cd 373.0 d
35 17.3 312.8 b 782.0 ab 20.7 ab 535.5 b 417.3 bc
45 21.6 414.5 a 819.8 a 22.9 a 675.3 a 548.1 a
Pooled SE 0.223 6.613 7.785 0.371 9.223 7.541

Means of main effectsb

Fish size
Fry 15.1 q 261.8 612.4 17.8 321.3 413.5
Fingerling 17.0 p 265.9 640.8 18.1 382.6 427.2
Advanced juvenile 18.1 p 329.5 730.6 20.9 526.1 446.1
25 14.4 z 238.8 502.9 16.3 285.9 350.2
35 16.3 y 278.7 680.6 19.4 397.2 418.5
45 19.5 x 339.7 800.3 21.1 546.8 518.0

ANOVA: P values
Protein level 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001
Fish size 0.0001 0.0001 0.0001 0.004 0.0001 0.028
Protein level × fish size 0.096 0.043 0.006 0.046 0.039 0.002
a
Treatment means represent the average values of three aquaria per treatment. Duncan multiple range test was conducted for individual means only if there was a significant
interaction (ANOVA: P < 0.05). Means followed by the same letter are not significantly different.
b
Main effect means followed by the same letter are not significantly different at P < 0.05 by Duncan multiple range test; p, q, and r for fish size and x, y, and z for protein level.
272 M. Abdel-Tawwab et al. / Aquaculture 298 (2010) 267–274
Nile tilapia is weight dependant; fry tilapia (~0.5 g) required the 45%-
CP diet for optimal growth; whereas, fingerling (~ 20 g) and advanced
juvenile (~40 g) performed optimally with the 35%-CP diet. El-Sayed
and Teshima (1991) found similar results in terms of the protein
requirement of Nile tilapia; with values ranging between 20% and 56%
CP. The studies of Balarin and Haller (1982) showed that fry of tilapia
required a diet ranging between 35 and 50% CP, and 5–25 g fish
between 25 and 35% CP. Tacon (1987) reported dietary protein levels
varying from 42% for fry to 35% for growing adults of omnivorous fish
species. Our results show a more accurate description of dietary CP
needs for different life history changes, as we tested the possible
variations within these ranges (25–45%). Variations in dietary CP
needs related to fish weights may be attributed to different protein
needs at different life history stages. El-Sayed and Teshima (1991)
found that dietary protein requirements decreased with increasing
fish weight and age. Both in fingerling and advanced juvenile of Nile
tilapia, excess protein could not be utilized efficiently and might have
been used for energy.
Khattab et al. (2000) studied the optimum dietary protein level for
three Nile tilapia strains of the same weight class (12 g) collected from
different locations in Egypt and found that optimum dietary protein
level ranged from 27 to 37%. Such variations in optimum dietary
protein requirements for tilapia growth might be due to the variation
in stocking density, hygiene, and/or environmental conditions in their
natural environments. In cultured fish, such as those used in this
study, the environmental variability was controlled and the effect of
protein in the diet was not confounded by other factors.
Feed utilization was significantly affected by protein level and fish
weight but not their interaction. FCR increased with fish weight and
coincided with previously published ranges for Nile tilapia (Siddiqui
et al., 1988; Al-Hafedh, 1999; Abdelghany, 2000; Khattab et al., 2000).
The hepatosomatic index was inversely affected by dietary CP in all
fish weights except fingerlings due to the increased fish weight at higher
CP, the liver to body weight ratio decreased. Likewise, Gallagher (1999)
found that HSI was significantly higher in sunshine bass fed lower
protein diets.
The carcass proximate analysis of all weight classes was signifi-
cantly influenced by dietary protein level and fish weight; the ash
content was only significantly affected by fish weight. Gallagher
(1999), however, did not find significant differences in moisture,
protein, lipid, and ash in whole-body of sunshine bass fed different
protein levels. Nile tilapia fed the 25%-CP diet had lower content of
protein and higher lipid content than fish fed the 35%- or 45%-CP diet,
for all weight classes. Due to the high feed intake, nutrient utilization,
and the high nutrient digestibility, the deposited nutrients increased.
Changes in protein and lipid contents in fish body could be linked with
changes in their synthesis, deposition rate in muscle and/or different
growth rates (Smith, 1981; Fauconneau, 1984; Soivio et al., 1989;
Abdel-Tawwab et al., 2006). Similar results were obtained by Wee and
Tuan (1988), Al-Hafedh (1999), and Khattab et al. (2000).
All different measures of protein metabolism, including PER, PPV,
and PGR, and ammonia production were affected by the treatments.
PER, PPV, and PGR were significantly affected by protein level and fish
weight. Protein utilization decreased with increasing dietary protein
levels and with larger fish. These results may occur because the major
part of weight gain was related to the deposition of protein, and protein
accretion is a balance between protein anabolism and catabolism.
Gastric emptying rate or solubility of the protein has been shown to
affect the utilization of dietary protein (de la Higuera et al., 1998; Espe
et al., 1999). In our study, fingerling and advanced juvenile of Nile tilapia
did not use the excess protein over 35% suggesting that some dietary
protein might be deaminated and produced ammonia. The increase
of nitrogenous excretion is a consequence of using amino acids as
energetic compounds (Hidalgo and Alliot, 1988; Kim et al., 1991). A
direct relationship between protein intake and ammonia excretion has
been found in fish (Li and Lowell, 1992; Chakraborty and Chakraborty,
1998). The increased protein breakdown in fish, resulting in increased
plasma ammonia concentrations, was observed in Bidyanus bidyanus,
Dicentrarchus labrax (Yang et al., 2002; Peres and Oliva-Teles, 2001),
Anguilla australis australis (Engin and Carter, 2001) and Rhamdia quelen
(Melo et al., 2006). Excess of ammonia is promptly excreted through the
gills (VanWaarde et al., 1983). Therefore, the increase in unionized
ammonia in the present study reflects an increased protein catabolism.
Webb and Gatlin (2003) found similar results for red drum; when it was
fed high-protein diet (45% CP); it excreted significantly more ammonia
than those fed on low-protein diet (35% CP).
Changes in the metabolic profile are proxies of fish performance
and ability to cope with different dietary conditions (Bidinotto and
Moraes, 2000; Moraes and Bidinotto, 2004; Lundstedt et al., 2004;
Melo et al., 2006). Changes in RBCs, Hb, and Ht were significantly
affected by dietary protein, fish weight, and their interaction. The
increase in RBC count may have occurred because of its release from
the storage pool in the spleen (Vijayan and Leatherland, 1989;
Pulsford et al., 1994). Thus, it seems that spleen activity is different for
different fish weight and that it is affected by dietary protein level. In
addition, fish blood contains heterogeneous populations of erythro-
cytes where immature cells are generally smaller and contain less Hb
than older and mature ones (Härdig and Høglund, 1983). These
authors suggested that Hb synthesis occurs in the erythrocytes after
their release into circulation. The knowledge herein gained on
biochemical features may provide the basis for better understanding
the handling and rearing of Nile tilapia.
In serum, glucose, protein, and lipid tended to increase with
increased diet protein content. Similar results were observed in On-
corhynchus mykiss (Lone et al., 1982), European eel (Suárez et al.,
1995), and R. quelen (Melo et al., 2006). These increments may be
because any excess of amino acids could be converted into carbohy-
drates or, in smaller amounts, to fat (Driedzic and Hochachka, 1978).
In the present study, the surplus of amino acids in Nile tilapia was
reflected in the increased amino acid concentration in the tissues due
to the increased protein levels. This can be associated with increased
absorption of amino acids from protein digestion (Yamamoto et al.,
1999). Serum protein depends on many factors such as digestion
efficiency, fish weight, composition of the diet, and temperature (Grove
et al., 1981; Darcy, 1984). Although there was an increase in serum
protein, the increased levels of ALT and AST suggests protein catabolism
at high protein levels in the diet. The amino acids surplus from protein-
rich diets cannot be directly stored in fish and they might be deaminated
and converted into energetic compounds (Ballantyne, 2001; Stone et al.,
2003). In this study, the rise of plasma protein with dietary protein could
likely be due to the enhancement of digested protein (Lundstedt et al.,
2002). Increased glucose in serum suggests gluconeogenesis as a
consequence to increased dietary protein level. Serum lipids slightly
increased due to the increase in protein level and it may be because the
muscle is a pivotal compartment directly linked to amino acid turnover.
This involves protein synthesis or breakdown of those molecules as
energetic substrates.
The expression of key enzymes of intermediary metabolism is
modulated by nutritional status in fish (Metón et al., 1999, 2003). The
levels of amino acid-metabolizing enzymes and nitrogen excretion are
reliable indicators of dietary protein availability. Metabolism of amino
acids involves deamination and transamination reactions. The activities
of transaminases and deaminases are useful to evaluate the feeding
status in some fish (Alexis and Papaparaskeva-Papoutsoglou, 1986;
Moyano et al., 1991; Melo et al., 2006). The rise of ALT and AST activities
observed in Nile tilapia fed the 45%-CP diets may reflect the use of excess
hydrocarbons from amino acids to supply energetic demands. High
protein/carbohydrate ratios in the feeding of Sparus aurata increased
ALT and AST activity in the liver (Metón et al., 1999). Similar responses
were observed in O. mykiss for ALT (Sánchez-Muros et al., 1998) and in
R. quelen for AST and ALT (Melo et al., 2006). The rise in the hepatic
activity of protein-metabolizing enzymes when fish were fed the 45%-
 M. Abdel-Tawwab et al. / Aquaculture 298 (2010) 267–274
CP diet may denote use of excess dietary amino acids for growth as well
as substrate for gluconeogenesis, particularly for AST and ALT activities.
Physiological alterations were significantly affected by diet
composition and weights of Nile tilapia and could be used as a
metabolic tool for assessing the proper concentration of dietary
protein in the feeding of Nile tilapia. Moreover, the proper ratio of
protein/carbohydrate in the diet is fundamental to establish the
optimal nitrogen content in the diet, to increase fish gain efficiency
while preventing nitrogen waste and environmental damage. The
ratio of protein/energy in the Nile tilapia diet seems to be specific and
further studies concerning types and sources of carbohydrates and
lipids to replace protein as energy sources are necessary.
Acknowledgements
The authors would like to thank Mamdouh A.A. Mousa, Depart-
ment of Fish Biology and Ecology, Central Laboratory for Aquaculture
Research, Abbassa, Abo-Hammad, Sharqia, Egypt, for his great help in
doing the physiological assays and for his valuable comments and
advises during the writing of this manuscript.
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