Aquaculture Research, 2014, 45, 566570

doi:10.1111/j.1365-2109.2012.03241.x

SHORT COMMUNICATION
Salinity lessens the impact of high stocking densities
and metabolic cost in white muscle and liver of Nile
tilapia fingerlings
Mohamed A M Hegazi, Zeinab I Attia & Mona M Hegazi
Department of Zoology, Faculty of Science, Tanta University, Tanta, Egypt
Correspondence: M A M Hegazi, Department of Zoology, Faculty of Science, Tanta University, Tanta, Egypt. E-mail: m_a_m_
hegazi@hotmail.com

Introduction
Growth performance of the fish is governed not
only by its genetic potential but also by its immediate environmental conditions. Considerable progress has been made towards increasing the
growth rate and food conversion efficiency of Nile
tilapia reared in intensive aquaculture (Bailey,
Rakocy, Martin & Shultz 2000; El-Sayed 2002;
Ouattara, Teugels, Douba & Philippart 2003;
Diana, Yi & Lin 2004; Ridha 2006; Muangkeow,
Ikejma, Powtongsook & Yi 2007; Nguyen,
Ponzoni, Abu-Bakar, Hamzah, Khaw & Yee 2010).
In fish intensive culture, stocking density (rearing density) is considered one of the important factors that govern fish growth. In tilapia,
experiments on the effect of stocking density have
been conducted on different sizes and using different culture systems (Bailey et al. 2000; El-Sayed
2002; Ouattara et al. 2003; Diana et al. 2004;
Ridha 2006). Results of these studies generally
demonstrated an inverse relationship between
stocking density and growth rate. Stress in
aquaculture such as high stocking rate activates
the brain-sympathetic-chromaffin cell axis and the
brainpituitaryinterrenal axis (Balm, Haenen &
Bonga 1995), leading to the swift discharge and
remarkable increase in the concentrations of catecholamines and cortisol in the blood (Mommsen,
Vijayan & Moon 1999). Cortisol is the principal
corticosteriod in teleosts and is known to increase
the activity of amino acid metabolizing enzymes:
aspartate aminotransferase (AST), alanine amino-

566

transferase (ALT) and glutamate dehydrogenase

(GDH) in different animal tissues (Mommsen et al.
1999).
Reports have described the efficacy of salts to
alleviate the effects of stress in fish during handling, crowding and transport (Carneiro & Urbinati
2001) and in post-stress recovery (Tsuzuki, Ogawa, Strussmann, Maita & Takashima 2001).
Therefore, the aim of the present study was to
evaluate the effect of two salinities (0 and 8
NaCl L 1) and three stocking densities (90, 150 or
300 fish m 3) on growth rate and the activities of
the key enzymes phosphofructokinase (PFK), AST,
ALT and GDH in white muscle and liver of Nile
tilapia juveniles.
Material and methods
The experiment
Juveniles of Nile tilapia, Oreochromis niloticus were
obtained from the fish hatching pond in Fowa city
(Kafer El-Sheikh Governorate). Fish of initial weight
15 2.4 g were distributed in 36 glass aquaria of
33 L capacity and stocked in two water salinities:
0 or 8 g L 1 and three stocking densities:
90 fish m 3 (D1) (3 fish aquarium 1), 150
fish m 3 (D2) (5 fish aquarium 1) or 300 fish m 3
(D3) (10 fish aquarium 1) giving six salinity/density combinations: 0/90, 0/150, 0/300, 8/90, 8/
150 and 8/300. Each aquarium was equipped with
an air pump for continuous air flow via a sponge
biological filter. The filter was washed and cleaned

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Salinity lessens high stocking densities M A M Hegazi et al.

thoroughly every day. Temperature was maintained at 26 0.5C using high precision-thermostat-controlled heater. Fish were acclimated for
four weeks at 26 0.5C for complete temperature
acclimation (Hegazi & Hasanein 2010). Dissolved
oxygen level in the aquaria was maintained at 6.87.1 mg L 1. Salinity (8 g L 1) was attained by
exposing individuals to progressively increasing
salinity (2 g L 1 per 2 day) until all treatments
reached the target salinity. The daily ration was
3% of fish weight and the fish were fed commercially available tilapia diet pellets recommended by
the Egyptian Ministry of Agriculture & Land Reclamation of (E-MALR) (25.2% protein, lipids 5% and
5.7% fibres with total energy, 10487934 J kg 1)
once daily at 10:00 a.m. for 42 days. Fifty per cent
of the water aquarium was daily replaced by water,
which has the corresponding salinity for each
group and previously heated to 26C.
Tissue sampling and enzyme assays
At the end of the experiment, fish were killed by a
rapid blow on the head and weighed. A small piece
of white muscle and liver was carefully excised on
ice, avoiding squeezing the tissue, washed in icecold isotonic NaCl saline and blotted dry and
weighed. The cytosolic and mitochondrial enzyme
fractions were prepared according to the procedure
described by Moon and Ouellet (1979) as modified
by Hegazi, Attia, Hegazi and Hasanein (2010). The
buffer B1 contained 20 mM HEPES, 1 mM EDTA,
30 mM b-mercaptoethanol, 225 mM mannitol,
50 mM sucrose pH 7.6. While buffer B2 contained
20 mM HEPES, pH 7.6, 1 mM EDTA, 1% triton X100 and 30 mM b-mercaptoethanol. Buffer B1 was
used for the extraction of the cytosolic enzyme fraction, while the buffer B2 was used for the extraction of the mitochondrial enzyme fraction.
Tissue was homogenized (1:2 w:v) in cold buffer
B1 (4C) using an all-glass manual homogenizer
immersed in ice. About 200 lL of the resulting
homogenate was delivered to a centrifuge tube
and diluted using the B1 buffer to bring the dilution to 1:10 w:v. To another centrifuge tube, an
equal volume from the resulting homogenate was
also delivered and diluted using the B2 buffer to
bring the dilution to 1:10 w:v. The contents of
each tube were mixed thoroughly. The homogenate of B1 buffer was centrifuged using a cooling
centrifuge at 12 000 g for 15 min at 4C. The
yielded high-speed supernatant of B1 was decanted
2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 566570

and used immediately to examine PFK and the

cytosolic fraction of ALT and AST. The homogenate of B2 buffer was frozenthawed three times
for the complete disruption of mitochondria
(Salach 1978). The homogenate was then centrifuged using a cooling centrifuge at 6000 g for
15 min at 4C. The B2 fraction contained the
enzymes GDH, total c- and m-ALT and AST. The
supernatants were carefully collected, avoiding
contamination with the upper lipid layer at the
top of the tubes. The mitochondrial ALT and AST
activities represent the difference between the
enzyme activities in the two fractions. To test the
mitochondrial-residue contaminations in the cytosolic fraction of B1, the activity of GDH was
assayed in the cytosolic fraction.
The reaction rate for each enzyme assayed was
linear with time and protein content. Enzymes
were assayed by following the rate of NADH oxidation. NADH oxidation was followed spectrophotometrically at 25 in assays modified after
Bergmeyer, Bergmeyer and Grassl (1984) at
340 nm (Ex = 6.22 cm2 lM 1) using an UV/vis
Spectrophotometer (JENWAY 6505, UK). Assay
conditions were selected to give maximal activity
for enzymes, and suitable controls were run to
substrate non-specific activity in the supernatant.
All enzyme measurements were performed in
duplicate. Assays were carried out in 80 mM imidazole-HCl buffer pH 7.4 in a final volume of 1ml.
All enzyme activities were performed based on the
methods used by Mommsen et al. (1999):
1 Phosphofructokinase (PFK, EC 2. 7. 1. 11): 6 mM
fructose 6-phosphate, 0.15 mM NADH, 2mM
ATP, 100 mM KC1, 10 mM MgCI2 and excess
triose phosphate isomerase (TPI, EC 5.3.1.1),
glycerol 3-phosphate dehydrogenase (G3PDH EC,
1.1.1.8) and aldolase (Ald, EC 4.1.2.7).
2 Aspartate aminotransferase (AST, EC 2.6. 1.1):
40 mM aspartate, 7 mM a-ketoglutarate
0.15 mM NADH, 0.025 mM pyridoxal phosphate and excess malate dehydrogenase (MDH,
EC 1.1.1.37).
3 Alanine aminotransferase (ALT, EC 2.6.1.2):
200 mM alanine, 10.5 mM a-ketoglutarate
0.15 mM NADH, 0.025 mM pyridoxal phosphate and excess lactate dehydrogenase (LDH,
EC 1.1.1.27).
4 Glutamate dehydrogenase (c-GDH, EC 1.4.1.2):
10 mM a-ketoglutarate, 150 mM ammonium
acetate 1mM ADP and 0.15 mM NADH.

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Aquaculture Research, 2014, 45, 566570

ties (1519 psu) in the upper temperature range

(>18C) (Imsland, Foss, Gunnarsson, Berntssen,
FitzGerald, Bonga, Ham, Nvdal, Sigurd & Stefansson 2001). In European sea bass, Dicentrarchus labrax growth rate was affected by salinity (Rubio,
Sanchez-Vazquez & Madrid 2005). One of the physiological functions clearly influenced by water salinity in fish is growth (Buf & Payan 2001;
Engstrom-Ost, Lehtiniemi, Jonasdottir & Viitasalo
2005). In tilapia, the positive effect on growth was
reported by many authors where optimal growth
was between 5 and 18 g L (Watanabe, French &
Ernt 1989; Suresh & Lin 1992; Jonassen, Pittman &
Imsland 1997; Buf & Payan 2001; Chowdhury,
Yi, Lin & El-Haroun 2006).
The activity of the enzyme PFK in white muscle
and liver was significantly increased in 0/150, 0/
300 and 8/300 treatments (Table 2 and 3). This
might indicate high energy expenditure. The
increase in PFK activity in liver may indicate one
of two things or both: first, liver may rely on glucose as a source of energy at these conditions; the
second is the production of higher quantities of a
ketoglutarate, which are needed for biosynthetic
processes especially for the nitrogenous waste
products capture.
Similarly, the increase in the activity of mitochondrial AST, ALT and GDH in white muscle and
liver of fish in 0/150 and 0/300 and 8/300 treatments( (Table 2 and 3) may indicate an increased
rate of protein catabolism. Barcellos, Nicolaiewsky,
Souza and Lulhier (1999) reported high levels of
cortisol in high stocked Nile tilapia weighing
15 5 g (about 142 fish m 3).
In conclusion, the enzymes activity AST, ALT,
GDH and PFK increased in Nile tilapia reared in
freshwater at high stocking density (150 and

Statistical analyses
Results are presented as meansstandard deviation
(XSD). The statistical evaluation of all data was
performed using two-way analysis of variance (ANOVA) followed by Dunnetts test (D2 and D3 vs. D1,
the respective controls of each trial). P values
 0.05 were regarded as statistically significant.
Results
There was a significant interaction (P  0.05)
between the effect of salinity and stocking density
as indicated by two-way ANOVA. At both salinities,
final mean weight and daily growth rates
decreased as the density increased. However, fish
in the 8/150 treatment had better growth performance than at 0/150 treatment (Table 1).
A significant increase in enzymes activity was
observed in 0/150 and 0/300 fish m 3 and in 8/
300 fish m 3 treatments (Tables 2 and 3). Water
salinity 8 g NaCl improved the fish tolerances to
the high stocking level 8/150 (150 fish m 3) with
a notable effect on enzyme activity occurred only
in 8/300 (300 fish m 3). There was significant
(P  0.05) increase in the activity of the cytosolic
and mitochondrial AST, ALT of white muscle and
liver of fish at stocking densities 0/150, 0/300
and 8/300 were significantly higher than the
other groups.
Discussion
Salinity of 8 g L 1 (8 psu) used in the current study
improved fish growth significantly as indicated by
8/150 treatment. Better growth occurred if turbot,
Scophthalmus maximus reared at intermediate salini-

Table 1 Combined effect of stocking density and salinity on growth performance of Nile tilapia at 26 C (mean SD,
n = 10)
Salinity

0/90

0/150

0/300

FBM (g)
IBM (g)
DGR g day

15.1 2.4
26.3 2.8
0.26 0.3

15.2 2.40
21.1 3.10a
0.16 0.02 a

15.1 2.40
18.6 3.00
0.08 0.01

a
a

8/90

8/150

8/300

15.3 2.4
26.5 2.8
0.27 0.30

15.1 2.4
24.9 2.60
0.23 0.04

15.2 2.40
19.1 2.60
0.09 0.01

b
b

FBM, Final body mass; IBM, Initial body mass; DGR, daily growth rate. 0/90, 90 fish stocked in 0 salinity; 0/150, 150 fish m 3
stocked in 0 salinity; 0/300, 300 fish m 3 stocked in 0 salinity; 8/90, 90 fish m 3 stocked in 8 g NaCl L 1 salinity; 0/150,
150 fish m 3 stocked in 8 g NaCl L 1 salinity; 0/300, 300 fish m 3 stocked in 8 g NaCl L 1 salinity.
Values in rows of 0 salinity with superscript letter a and rows of 8 g NaCl L 1 with superscript letter b are significantly different at
P  0.05.

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2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 566570

Salinity lessens high stocking densities M A M Hegazi et al.

Aquaculture Research, 2014, 45, 566570

Table 2 Combined effect of stocking density and salinity on enzymes activity in white muscle of Nile tilapia at 26 C
(mean sd, n = 10)
Salinity

0/90

PFK
GDH
c-AST
m-AST
c-ALT
m-ALT

5.6
5.7
13.3
12.2
14.2
15.8

0/150

0.60
0.60
1.4
1.5
1.6
1.2

7.1
9.9
19.2
17.5
19.2
20.8

0/300
0.70 a
1.00 a
2.0 a
1.9 a
1.8 a
2.4 a

7.9
11.7
24.3
29.9
23.6
23.1

8/90
0.90 a
1.7 a
2.6 a
2.8 a
2.1 a
2.3 a

5.4
5.9
14.1
12.1
13.7
15.3

8/150

0.70
0.70
1.4
1.2
1.4
1.6

5.1
6.2
14.1
13.1
13.6
16.3

8/300

0.70
0.80
2.0
1.5
1.2
1.2

7.2
8.7
21.3
24.6
21.6
22.1

0.90 b
1.00 b
2.6 b
2.8 b
3.0 b
3.0 b

0/90, 90 fish stocked in 0 salinity; 0/150, 150 fish m 3 stocked in 0 salinity; 0/300, 300 fish m 3 stocked in 0 salinity; 8/90,
90 fish m 3 stocked in 8 g NaCl L 1 salinity; 0/150, 150 fish m 3 stocked in 8 g NaCl L 1 salinity; 0/300, 300 fish m 3 stocked
in 8 g NaCl L 1 salinity.
Values in rows of 0 salinity with superscript letter a and rows of 8 g NaCl L 1 with superscript letter b are significantly different at
P  0.05.

Table 3 Combined effect of stocking density and salinity on enzymes activity in liver of Nile tilapia at 26 C (mean
SD, n = 10)
Salinity

0/90

PFK
GDH
c-AST
m-AST
c-ALT
m-ALT

1.3
40.1
37.3
25.2
35.2
22.4

0/150

0.20
5.1
2.2
2.3
3.2
2.7

2.0
82.7
54.3
36.3
48.8
25.2

0/300
0.20 a
8.9 a
4.1 a
31 a
4.4 a
2.3 a

2.1
95.2
63.5
42.7
55.3
36.2

8/90
0.22 a
8.9 a
7.5 a
5.3 a
4.2 a
4.7 a

1.2
38.9
35.9
25.8
34.6
20.1

8/150

0.14
4.6
3.1
2.9
2.9
2.2

1.3
40.3
36.7
24.5
33.9
26.4

8/300
0.2
5.1
3.3
2.9
3.3
2.7

1.7
65.2
52.5
34.7
46.3
31.1

0.20 b
8.9 b
4.5 b
5.3 b
4.2 b
3.7 b

0/90, 90 fish stocked in 0 salinity; 0/150, 150 fish m 3 stocked in 0 salinity; 0/300, 300 fish m 3 stocked in 0 salinity; 8/90,
90 fish m 3 stocked in 8 g NaCl L 1 salinity; 0/150, 150 fish m 3 stocked in 8 g NaCl L 1 salinity; 0/300, 300 fish m 3 stocked
in 8 g NaCl L 1 salinity.
Values in rows of 0 salinity with superscript letter a and rows of 8 g NaCl L 1 with superscript letter b are significantly different at
P  0.05.

300 fish m 3). This could be considered a tool

that can maintain higher source of energy for
maintenance at the present conditions. The of 8 g
NaCl L salinity mitigated the adverse effect of high
stocking density on growth and enzymes activity
up to 150 fish m 3, and did not show any
increased metabolic expenditure.
References
Bailey D.S., Rakocy J.E., Martin J.M. & Shultz R.C.
(2000) Intensive production of tilapia fingerlings in
recirculating system. In: Proceeding of the Fifth International Symposium on Tilapia in Aquaculture, Rio de
Janeiro, Brazil (ed. by K. Fitzsimmons, & J.C. Filho),
pp 328333. Panorama da Aquicultura Magazine, Rio
de Janeiro, Brazil.
Balm P.H.M., Haenen H.E.M.G. & Bonga S.E.W. (1995)
Regulation of interrenal function in fresh water and
seawater adapted tilapia (Oreochromis mossambicus).
Fish Physiology and Biochemistry 14, 3747.

2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 566570

Barcellos L.J.G., Nicolaiewsky S., de Souza S.M.G. &

Lulhier F. (1999) Plasmatic levels of cortisol in the
response to acute stress in Nile tilapia, Oreochromis
niloticus (L.), previously exposed to chronic stress.
Aquaculture Research 30, 437444.
Bergmeyer H.U., Bergmeyer J. & Grassl M. (1984) Methods of Enzymatic Analysis, Vol 2 (2nd English edition,
translated from the third German edition)Verlag Chemie International, Deerfield Beach, Florida.
Buf G. & Payan P. (2001) How should salinity influence fish growth? Comparative Biochemistry and Physiology 130C, 411423.
Carneiro P.C.F. & Urbinati E.C. (2001) Salt as a stress
response mitigator of matrinxa, Bryconcephalus
(Gunther), during transport. Aquaculture Research 32,
297304.
Chowdhury M.A.K., Yi Y., Lin C.K. & El-Haroun E.R.
(2006) Effect of salinity on carrying capacity of adult
Nile tilapia Oreochromis niloticus L. in recirculating systems. Aquaculture Research 37, 16271635.
Diana J.S., Yi Y. & Lin C.K. (2004) Stocking densities
and fertilization regimes for Nile tilapia (Oreochromis

569

Salinity lessens high stocking densities M A M Hegazi et al.

niloticus) production in ponds with supplemental feeding. In: Proceedings of the Sixth International Symposium
on Tilapia in Aquaculture, Manila, Philippines (ed. by
R. Bolivar, G. Mair, & K. Fitzsimmons), pp 487499.
BFAR, Philippines.
El-Sayed A.M. (2002) Effect of stocking density and feeding levels on growth and feed efficiency of Nile tilapia
(Oreochromis niloticus L.) fry. Aquaculture Research 33,
621626.
Engstrom-Ost J., Lehtiniemi M., Jonasdottir S.H. &
Viitasalo M. (2005) Growth of pike larvae (Esox lucius)
under different conditions of food quality and salinity.
Ecology of Freshwater Fish 14, 385393.
Hegazi M.M. & Hasanein S.S. (2010) Effects of chronic
exposure to ammonia concentrations on brain monoamines and ATPases of Nile tilapia (Oreochromis niloticus). Comparative Biochemistry and Physiology 151C,
420425.
Hegazi M.M., Attia Z.I., Hegazi M.A.M. & Hasanein S.S.
(2010) Metabolic consequences of chronic sublethal
ammonia exposure at cellular and subcellular levels in
Nile tilapia brain. Aquaculture 299, 149156.
Imsland A.K., Foss A., Gunnarsson S., Berntssen M.H.G.,
FitzGerald R., Bonga S.W., Ham E.v., Nvdal G.,
Sigurd O. & Stefansson S.O. (2001) The interaction of
temperature and salinity on growth and food conversion in juvenile turbot (Scophthalmus maximus). Aquaculture 198, 353367.
Jonassen T.M., Pittman K. & Imsland A.K. (1997) Seawater acclimation of tilapia, Oreochromis spilurus spilurus
Gunter, fry and fingerlings. Aquaculture Research 28,
205214.
Mommsen T.P., Vijayan M.M. & Moon T.W. (1999) Cortisol in teleosts: dynamics, mechanisms of action, and
metabolic regulation. Reviews in Fish Biology and Fisheries 9, 211268.
Moon T.W. & Ouellet G. (1979) The oxidation of tricarboxylic acid cycle intermediates, with particular reference to isocitrate, by intact mitochondria isolated from
the liver of the American eel, Anguilla rostrata L.
Archives of Biochemistry and Biophysics 195, 438452.
Muangkeow B., Ikejma K., Powtongsook S. & Yi Y.
(2007) Effects of white shrimp, Litopenaeus vannamei
(Boone), and Nile tilapia, Oreochromis niloticus L.,

570

Aquaculture Research, 2014, 45, 566570

stocking density on growth, nutrient conversion rate

and economic return in integrated closed recirculation
system. Aquaculture 269, 363376.
Nguyen N.H., Ponzoni R.W., Abu-Bakar K.R., Hamzah A.,
Khaw H.L. & Yee H.Y. (2010) Correlated response in fillet weight and yield to selection for increased harvest
weight in genetically improved farmed tilapia (GIFT
strain), Oreochromis niloticus. Aquaculture 305, 15.
Ouattara N., Teugels G., Douba V. & Philippart J. (2003)
Aquaculture potential of the black-chinned tilapia,
Sarotherodon melanotheron (Chiclidae): comparative
study of the effect of stocking density on growth performance of landlocked and natural populations under
cage culture conditions in Lake Ayame (Cote dIvoire).
Aquaculture Research 32, 12231229.
Ridha M.T. (2006) Comparative study of growth performance of three strains of Nile tilapia, Oreochromis niloticus, L. at two stocking densities. Aquaculture
Research 37, 172179.
Rubio V.C., Sanchez-Vazquez F.J. & Madrid J.A. (2005)
Effects of salinity on food intake and macronutrient
selection in European sea bass. Physiology & Behavior
85, 333339.
Salach J.I. (1978) Preparation of monoamine oxidase
from beef liver mitochondria. Methods in Enzymology
153, 494501.
Suresh A.V. & Lin C.K. (1992) Tilapia culture in saline
waters. Aquaculture 106, 201226.
Tsuzuki M.Y., Ogawa K., Strussmann C.A., Maita M. &
Takashima F. (2001) Physiological responses during
stress and subsequent recovery at different salinities in
adult pejerrey. Odontesthes bonariensis, Aquaculture
200, 349362.
Watanabe W.O., French K.E. & Ernt D.H. (1989) Salinity
during early development influences growth and survival of Florida red tilapia in brackish and seawater.
Journal of the World Aquaculture Society 20, 134142.

Keywords: Nile tilapia, stocking density, salinity, phosphofructokinase, glutamate dehydrogenase, cytosolic- and mitochondrial aspartate- and
alanine- aminotransferases

2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 566570