Aquaculture Nutrition

doi: 10.1111/j.1365-2095.2009.00747.x 2011 17; 258–266

..............................................................................................

1 2 2
1 2
Department of Aquaculture, National Kaohsiung Marine University, Kaohsiung, Taiwan; Department of Aquaculture,
National Taiwan Ocean University, Keelung, Taiwan

Various synthetic and natural carotenoids (CD) have been

This study aimed to find out whether dietary carotenoid
used as dietary supplement to improve pigmentation of
(CD) supplement could influence the resistance of characins
aquaculture animals (Storebakken & No 1992; Gouveia et al.
(Hyphessobrycon eques Steindachner) to ammonia stress.
2003; Chien & Shiau 2005; Kalinowski et al. 2005) for higher
Two types of CD and its combination [astaxanthin (AX),
market value. On the other hand, dietary CD were also found
b-carotene (BC), 1 : 1 combination of AX and BC (MX)] at
to increase cultured animalsÕ resistance to various stressors.
three concentrations (10, 20 and 40 mg kg)1) were used
Dietary astaxanthin (AX), a naturally occurring CD pigment,
resulting in nine pigmented diets. No differences in growth
supplementation in penaeid postlarvae not only increased
and survival of the fish among treatments were found after
body AX, but also enhanced the resistance to hypoxia (Chien
8-week rearing. Experimental and control fish were then
et al. 1999), salinity (Darachai et al. 1998; Merchie et al. 1998;
exposed to 15 mg total ammonia nitrogen L)1 (stress group)
Chien et al. 2003), temperature (Chien et al. 2003), ammonia
and 0.15 mg total ammonia nitrogen L)1 (normal group) for
(Pan et al. 2003a) and pathological (Pan et al. 2003b) stres-
72 h, and their blood was withdrawn. No mortality resulted
sors. The reason for the development of resistance to stress
under such TAN concentrations. Serum total antioxidant
from CD can be attributed to their antioxidant properties
status (TAS), serum antioxidant enzymes [superoxide
(Torrissen & Christiansen 1995; Shimidzu et al. 1996). This is
dismutase (SOD), glutathione peroxidases (GPx)] and serum
to inactivate the free radicals produced from normal cellular
transaminases [alanine aminotransferase (ALT), aspartate
activity and various stresses so that the oxidative damage is
aminotransferase (AST)] were chosen as indices of fish anti-
eliminated (Halliwell & Gutteridge 1989; Chew 1995).
oxidant capacity or stress resistance. SOD, GPx and AST
Various types of dietary CD can affect deposition and
were affected by the interactions of dietary CD and ammonia
conversion of body CD in fish, their pigmentation (Torrissen
stress. The activities of TAS, SOD, GPx and AST increased
et al. 1989; Pan & Chien 2009) and possibly their antioxidant
under the stress. Dietary CD reduced serum SOD, GPx, ALT
capacity and stress resistance. b-carotene (BC) is recognized
and AST activities. In conclusion, dietary CD increased the
as a lipid antioxidant, i.e. a free radical trap and quencher of
resistance of characins to ammonia stress.
singlet oxygen (Bohm et al. 1997). AX contains a long con-
jugated double bond system with relatively unstable electron
KEY WORDS: ammonia stress, antioxidant capacity, asta-
orbitals; it may scavenge oxygen radicals in cells (Stanier
xanthin, b-carotene, Hyphessobrycon eques, superoxide
et al. 1971). The antioxidant activity of AX was found to be
dismutase
approximately 10· stronger than BC (Shimidzu et al. 1996).
Besides type of dietary CD, various stresses can also result in
Received 27 August 2009, accepted 23 October 2009
different responses in antioxidant capacity. It was shown that
Correspondence: Y.-H. Chien, Department of Aquaculture, National Tai-
the activities of hemolymph antioxidation enzymes of juvenile
wan Ocean University, Keelung 202, Taiwan. E-mail: yhchien@mail.
ntou.edu.tw tiger prawn varied not only with dietary AX types but also

..............................................................................................

 2010 Blackwell Publishing Ltd

 with the stressors including temperature and salinity drop
(Chien et al. 2003) and ammonia exposure (Pan et al. 2003b).
Ammonia is the most common toxicant in culture (Colt &
Armstrong 1981) and live-transportation systems (Taylor &
Solomon 1979). It comes from excretion of aquatic animals as
the end product of protein metabolism (Walsh & Wright 1995)
and mineralization of organic nitrogen in faeces, uneaten feed
and other organic matters (Avnimelech & Ritvo 2003). Am-
monia can be toxic at low concentrations in the aquatic envi-
ronment. Unionized ammonia concentration of 0.5 mg L)1
can be harmful to finfish and crustaceans (Van Rijn et al. 1990;
Tomasso 1994; Wasielesky et al. 1994). Short-term exposure
of fish and crustacean to high concentrations of ammonia
causes increased gill ventilation, hyperexcitability, loss of
equilibrium, convulsions and then death (Thurston et al. 1981;
Maltby 1995). Such stress also resulted in changes in haemol-
ymph total antioxidant status (TAS), superoxide dismutase
(SOD), aspartate aminotransferase (AST) and alanine amino-
transferase (ALT) in prawn (Pan et al. 2003a) and in AST and
ALT activity in fish (Jeney et al. 1992; Vedel et al. 1998).
Characins (Hyphessobrycon eques) is one of the most
important cultured and exported ornamental fishes in Tai-
wan. Routine feeding of CD-supplemented diets to this fish
in grow-out farms or aquaria is a common practice. This
study was undertaken to find out whether synthetic AX and/
or BC at various dietary concentrations would influence the
antioxidant capacity of characins, and concomitantly, their
resistance to high ammonia stress.
Control diet was composed of white fishmeal 500 g kg)1,
wheat flour 150 g kg)1, dextrin 270 g kg)1, fish oil 30 g kg)1,
vitamin mix 20 g kg)1 and mineral mix 30 g kg)1. Diets sup-
plemented with CD have the same composition as the control
diet (except for dextrin which was adjusted depending on CD
levels used) but supplemented with either synthetic AX (8%
AX) or BC (10%BC) or both. Water was added to the ingre-
dients to form a dough, which was then extruded through a 2-
mm-diameter die press. The extruded feed was air dried in the
dark to prevent the degradation of CD. The feed was then
crushed, sieved to attain particle size of 0.9–1.2 mm and stored
at )20 C to avoid oxidation of the CD. There were nine CD
diets composed of 3 · 3 factorial combinations of CD type
[AX, BC and 1 : 1 mixture of AX and BC (MX)] and CD
concentrations (10, 20, and 40 mg kg)1). Proximate analyses
of these diets are listed in Table 1.
Experimental fish were bought from an ornamental fish farm.
During acclimatization in the laboratory in a 0.5-ton tank, fish
were fed control diet for 2 weeks to equalize their body CD
content. Fish were then transferred to 30 aquaria
(44 cm · 33 cm · 21.5 cm) to receive their respective treat-
ments (three replicates per treatment) at stocking density of 30
fish aquarium)1. Fish size was 0.41 ± 0.09 g. Culture water
was passed through a 1-lm filter and sterilized by ultraviolet
light to eliminate microalgae, a possible source of CD. More-
over, all aquaria were covered with black screen to discourage
algal growth for the same precaution. Fish were fed twice daily
at 0800 and 1500 at 5% body weight. Dissolved oxygen (DO)
was maintained at 6–7 mg L)1 by constant aeration, temper-
ature at 26–28 C, pH of 7.5–8 and NH3 of 0.1–0.2 mg L)1.
Faeces and uneaten feeds were siphoned out daily, and one-
third of the water was exchanged. The fish were reared for
8 weeks. No mortality was observed throughout the experi-
ment. Final overall average fish size was 0.89 ± 0.18 g.

Table 1 Proximate analysis of the experimental diets

Carotenoid type None (C0) Astaxanthin (AX) b-carotene (BC) 1/2 AX + 1/2 BC (MX)

Carotenoid concentration(mg kg)1) 0 10 20 40 10 20 40 10 20 40

Diet notation C0 AX-10 AX-20 AX-40 BC-10 BC-20 BC-40 MX-10 MX-20 MX-40

Proximate analysis
Crude protein (g kg)1) 303.5 307.6 304.4 308.1 303.8 304.4 304.5 305.6 301.2 305.8
Crude fat (g kg)1) 54 56.8 54.4 55.4 55.8 57.4 58.0 52.3 50.4 54.7
Ash (g kg)1) 94.8 92.8 93.5 92.6 93.4 93.5 92.7 93.4 92.5 93.6
Moisture (g kg)1) 132.8 134.7 132.0 134.3 135.0 134.7 134.0 134.7 133.0 134.7
NFE + CF1 (g kg)1) 409.3 398.5 397.5 402.5 399.5 405.1 401.4 403.0 404.7 403.5
Astaxanthin (mg kg)1) 16 108.6 225.5 416.7 16 16 16 48 12.7 217.4
b-carotene(mg kg)1) 22 22 22 22 120.4 234.2 422.2 63 10.8 20.8
1
Nitrogen-free extracts and crude fibre.
..............................................................................................

Aquaculture Nutrition 17; 258–266  2010 Blackwell Publishing Ltd


A stock solution of ammonium chloride (NH4Cl; Merck) was
prepared using distilled water to a final concentration of
10 000 mg L)1 ammonia-N. Experimental test solution of
15 mg L)1 ammonia-N was then prepared from this stock
solution. The test was conducted using a static renewal
procedure (Hubert 1980).
Six fish were collected at random from each aquarium for
high ammonia stress test. They were first acclimatized for 24 h
in a 2-L bucket with flow-through fresh water (100 mL min)1).
Temperature was 25 C and DO saturated at 6.5 mg L)1.
They were then divided into two groups and put into two
2000-mL beakers containing 1600 mL of the test solution and
a 0-mg ammonia-N L)1 control. Aeration was provided
continuously during the trial. Each test solution was renewed
every 24 h (Buikema et al. 1982). The fish were fed twice a day at
6.5% body weight. After 72 h, blood samples were collected
through the brachial artery of all experimental and control fish
for analysis of serum antioxidants.
Immediately after withdrawing blood, samples were prepared
by mixing 200 lL isotonic NaCl solution containing
0.94 mmol L)1 EDTA with 50 lL blood. The samples were
chilled if not immediately used for the determination of
blood protein and antioxidants: TAS, SOD, AST, ALT and
GPx (glutathione peroxidases).
Blood protein was determined using a protein assay kit
(No.500-0006; Bio-rad Laboratories, Richmond, CA., USA)
with BAS (bovine serum albumin, 66 Kda; Sigma, St. Louis,
MO, USA) as standard. The method used was based on
Bradford (1976) using 200 lL of serum sample.
The different antioxidant parameters were analysed using
Randox Laboratories kits (Crumlin Co., Atrim, UK) by
spectrophotometry (U-2000; Hitachi Ltd., Tokyo, Japan).
The volumes of serum samples used were 20 lL for TAS and
GPx, 25 lL for SOD and 100 lL for AST and ALT. Activi-
ties were expressed in international enzyme unit (U L)1).
Two-way ANOVA was performed to find out the main effects
of dietary CD supplementation and high ammonia stress on
AX, TAS, SOD, GPx, ALT and AST, and the main effects of
CD type and concentration on AX and those antioxidation
enzyme activities. DuncanÕs multiple range test was used to
compare various levels within each main effect. Correlation
analyses were applied between AX and each of the antioxi-
dation enzyme activities under normal and high ammonia-
stressed environment. The significant level applied to all
analysis was set at 5%.
No differences were found in growth and survival of the fish
among treatments. No mortality resulted at high TAN con-
centration for 72 h.

Table 2 Average body astaxanthin

Diet Environment
content and activities of serum antioxi-
Carotenoid Ammonia dation enzymes of control and caroten-
Control supplemented1 Normal stressed2 Interaction oid-fed characins, Hyphessobrycon
3 b a x x callistus exposed to normal and ammo-
AX 5.47 (1.25) 18.14 (2.74) 16.97 (2.27) 15.27 (2.05) ns
TAS4 1.15a (0.03) 1.19a (0.05) 1.16y (0.04) 1.22x (0.05) ns nia-stressed environment
SOD5 4.65a (4.78) 1.86b (1.90) 0.48y (0.08) 3.80x (2.44) *
GPx6 69.40a (30.28) 31.44b (20.30) 18.74y (8.73) 51.73x (22.97) *
ALT7 8.55a (1.07) 4.10b (1.84) 4.04x (2.21) 5.05x (2.18) ns
AST8 19.93a (13.04) 8.76b (5.81) 6.45y (1.79) 13.30x (9.21) ns

Under the same factor, values with different superscript are significantly different (P £ 0.05).
Probability of significance for * is 0.01 < P £ 0.05.
1
Diets were supplemented with astaxanthin and/or b-carotene at various concentration.
2
Fish were stressed by 15 mg L)1 TAN for 72 h.
3
Body astaxanthin content (mg kg)1).
4
Total antioxidative status (mmol L)1serum).
5
Superoxide dismutase (U mg)1 protein).
6
Glutathione peroxidase (U mg)1 protein).
7
Alanine transaminase (U mg)1 protein).
8
Aspartate transaminase (U mg)1 protein).
..............................................................................................

Aquaculture Nutrition 17; 258–266  2010 Blackwell Publishing Ltd

 Table 3 After high ammonia stress, the average body astaxanthin content and activities of serum antioxidation enzymes of characins,
Hyphessobrycon callistus fed diets supplemented with various types and concentrations of carotenoid

Carotenoid type1 (T) Carotenoid concentration2 (C)

Control AX MX BC 10 20 40 T·C
3 a a b y y x
AX 5.22 (1.12) 19.10 (2.96) 20.24 (3.62) 15.08 (1.17) 16.01 (1.93) 17.79 (2.49) 20.61 (3.96) ns
TAS4 1.17 (0.02) 1.21a (0.04) 1.25a (0.04) 1.21a (0.07) 1.23x (0.05) 1.21x (0.06) 1.23x (0.05) ns
SOD5 8.71 (1.65) 1.77b (0.10) 2.68b (0.56) 4.32a (1.41) 4.30x (1.37) 2.50y (1.94) 1.99y (1.49) ns
GPx6 95.30 (7.92) 49.53a (12.15) 40.68a (1.70) 50.45a (26.62) 60.75x (15.94) 48.22xy (19.12) 31.70y (4.49) ns
ALT7 9.35 (0.78) 4.51a (1.17) 4.63a (2.30) 4.58a (2.32) 6.10x (1.14) 4.11xy (1.53) 3.52y (1.28) *
AST8 31.15 (2.33) 11.18a (7.11) 9.92a (7.96) 12.86a (7.77) 20.03x (3.90) 9.41y (3.30) 4.52z (1.58) *

Under the same factor, values without a common superscript are significantly different (P £ 0.05).
Probability of significance for * is 0.01 < P £ 0.05.
1
AX-astaxanthin, BC-b-carotene, MX- mix of half AX and BC.
2
Total concentration of the supplement carotenoid (mg kg)1).
3
Body astaxanthin content (mg kg)1).
4
Total antioxidative Status (mmol L)1serum).
5
Superoxide dismutase (U mg)1 protein).
6
Glutathione peroxidase (U mg)1 protein).
7
Alanine transaminase (U mg)1 protein).
8
Aspartate transaminase (U mg)1 protein).

Table 2 presents the body AX content and activities of Table 4 The correlation matrix among body astaxanthin content
serum antioxidation enzymes of control and CD-fed fish and activities of five serum antioxidation enzymes under (a) normal
exposed to normal and ammonia-stressed environment. The environment and (b) high ammonia-stressed environment. The cor-
CD-fed fish had three times higher body AX content and relation between two parameters is shown by correlation coefficient
(r) value and significant level
lower SOD, GPx, ALT and AST activities than the control
fish. There were no differences in TAS between two groups of (a) SOD1 GPx2 ALT3 AST4 AX5
fish. Ammonia stress did not affect fishÕs body AX content TAS6 ns ns ns ns 0.4935*
but resulted in higher activities of all serum antioxidation SOD 0.5925** ns 0.6102** )0.6738**
GPx 0.7015** 0.5861** )0.8257***
enzymes except ALT. There was significant interaction effect
ALT 0.7908*** )0.6145**
of dietary carotenoid supplement and ammonia stress on AST )0.6326**
fishÕs SOD and GPx.
(b) SOD GPx ALT AST AX
Table 3 presents the body AX content and activities of
serum antioxidation enzymes in fish-fed diets supplemented TAS )0.4931* ns ns ns ns
SOD 0.7168** 0.6650** 0.8192*** )0.8010***
with various types and concentrations of CD after high GPx 0.7773*** 0.8476*** )0.7362**
ammonia stress. Dietary CD type only affected body AX ALT 0.8322*** )0.6494**
content and SOD. BC-fed fish had lower body AX content AST )0.8116***

and higher SOD than MX- and AX-fed fish. After high Probabilities of significance are ns, P ‡ 0.05; *, 0.01 < P £ 0.05;
ammonia stress, body AX content of the fish fed 40 mg kg)1 **, 0.0001 < P £ 0.01 and ***, P £ 0.0001.
1
Superoxide dismutase (U mg)1 protein).
CD was higher than those fed 20 and 10 mg kg)1 CD 2
Glutathione peroxidase (U mg)1 protein).
(referred hereafter as 40-, 20-, and 10-fish). Decreasing trends 3
Alanine transaminase (U mg)1 protein).
in SOD, GPx, ALT and AST activities with increasing die- 4
Aspartate transaminase (U mg)1 protein).
tary CD concentration were also observed. The descending
5
Body astaxanthin content (mg kg)1).
6
Total antioxidative status (mmol L)1serum).
orders were 10-fish > (20-fish = 40-fish) for SOD, 10-
fish ‡ 20-fish ‡ 40-fish for GPx, 10-fish ‡ 20-fish ‡ 40-fish
for ALT and 10-fish > 20-fish > 40-fish for AST. Dietary under normal environment (Table 4). Among those
CD type and concentration had interaction effects on ALT correlations, body AX content had negative correlations with
and AST, but not on body AX content, TAS, SOD and GPx. SOD, GPx, ALT and AST. TAS positively correlated with
Under either normal or high ammonia-stressed environ- body AX content under normal environment and nega-
ment, close correlations among body AX content, SOD, GPx, tively correlated with SOD under high ammonia-stressed
ALT and AST were observed except between SOD and ALT environment.
..............................................................................................

Aquaculture Nutrition 17; 258–266  2010 Blackwell Publishing Ltd


The mechanisms of toxicity and lethal concentrations of
ammonia to various fishes and crustaceans are relatively well
documented (Tomasso 1994), and physiological responses of
environmental ammonia in fish is also extensively reviewed
(Randall & Tsui 2002). However, information on antioxidant
enzymatic responses of fish to environmental ammonia is
quite limited. Ammonia assumes two chemical forms in
aqueous solution, the unionized (NH3) and the ionized form
(NH4+) (Butler 1964). Only the NH3 is toxic (Hampson
1976), as it has high lipid solubility and is able to diffuse
easily across cell membranes in the direction favoured by its
pressure gradient (Fromm & Gillete 1968; Emerson et al.
1975). When NH3 concentration in water increases, the rate
of diffusion from the blood decreases. The consequences are
adverse effects on membrane stability and enzyme-catalysed
reaction, an elevation of haemolymph pH, reduction in the
transport of oxygen, increase in oxygen consumption by
tissues (Colt & Armstrong 1981; Chen et al. 1991) and
sometimes gill damage (Schreckenbach & Spangenberg 1978;
Tomasso 1994), which may eventually lead to death.
As ammonia toxicity ensues, like hypoxia or anoxia stress,
its low oxygen availability may result in oxidative stress
(Storey 1996). This is characterized by excessive production
of reactive oxygen species (Sies 1991) and increased antioxi-
dant defence (Lushchak et al. 2001). In the present study,
when fish were exposed to ammonia stress, there would have
been potential generation of abnormally high levels of oxy-
gen radicals and induction of radical scavenging enzymes
such as SOD, acting on superoxide (O2·)), and GPx on H2O2
and lipid hydroperoxides (Halliwell & Gutteridge 1984). This
has resulted in higher SOD and GPx and lower TAS in
ammonia-stressed group. Chronic high ammonia level can
also impact fishÕs antioxidant enzyme response. Liu et al.
(2008) reported that hepatic SOD activity of Chinese
longsnout catfish (Leiocassis longirostris) increased signifi-
cantly with long-term exposure (32 day) to different NH3
concentrations (0.004, 0.037 and 0.292 mg L)1). This is
regardless whether the fish are fed ascorbic acid (AA)-sup-
plemented diet or the control diet.
In freshwater teleosts, ALT and AST play an important
role in ammonia detoxification (DÕApollonia & Anderson
1980). The physiologically toxic NH3 level is neutralized in
the organism by increase in AST and ALT activities (Nem-
csok et al. 1982). The responses of AST and ALT to
ammonia stresses in fish can be similar but are not necessarily
parallel to each other. In rainbow trout (Oncorhynchus
mykiss), an increase in AST activity was observed after 4 day
exposure to high ammonia concentration, but plasma ALT
activity did not change (Vedel et al. 1998). Similar results
were obtained in our study where ammonia stress resulted in
106% increase in AST, but had no effect on ALT. On the
other hand, an increase in AST and ALT activities was
observed in carp Cyprinus carpio (Jeney et al. 1992) and
black seabream Acanthopagrus schlegeli (Kwon & Chang
1996) after exposure to high levels of ammonia.
As a result of the oxidative stress, fish like many other ver-
tebrates try to reduce the damage through antioxidant
defence system. Besides radical scavenging and associated
enzymes, the defence system includes many other antioxi-
dants such as carotenes, vitamins E, C and A, glutathione
(GSH) and ubiquinol Q10 (Wilhelm 1996). In this study,
after high ammonia stress, lower SOD, GPx, ALT and AST
activities in CD-fed fish were observed, which could be
attributed to the development of antioxidant defence system
induced by higher body AX content. Because AX contains a
long conjugated double bond system with relatively unstable
electron orbital, it may scavenge oxygen radicals in cells
(Stanier et al. 1971) and therefore reduce cellular damage.
Dietary CD effectively reduce SOD in fish. As SOD is a
superoxide reductase, which can protect the tissue from the
damage by oxidative process and phagocytosis, lower SOD
value may indicate higher cell protection in a certain period
of time. It is speculated that after feeding with CD diet, the
increase in body CD can result in more oxygen reserves
within the cells (Ghidalia 1985; Latscha 1990; Oshima et al.
1993) or can inhibit singlet oxygen reaction, which is induced
by stress (Di Mascio et al. 1991). Consequently, the need to
produce SOD to scavenge superoxide radicals is lessened.
The antioxidant defence systems, which have been devel-
oped through dietary antioxidants, can respond to oxidative
stresses differently depending on internal factors (e.g.
enzymes, tissues, and fish species) and external factors (die-
tary antioxidants, concentrations, and oxidative stresses).
AA which was proven to be beneficial in reducing oxidative
damage to tissues (Dabrowski 2001; Khassaf et al. 2003) was
found to play an important role in rainbow trout O. mykiss
in resisting hypoxia stress (Dabrowski et al. 2004). However,
in Chinese longsnout catfish L. longirostris, dietary AA did
not show any effect on antioxidant activities such as
hepatic SOD (Liu et al. 2008). In other studies, vitamin E
..............................................................................................
Aquaculture Nutrition 17; 258–266  2010 Blackwell Publishing Ltd
 (tocopherol acetate) supplement in diet, in the presence of
oxidative stress by oxidized oil, increased SOD but reduced
GPx in seabream (Sparus aurata), had no effect on SOD and
GPx in turbot (Scophthalmus maximus), and increased
SOD and had no effect on GPx in halibut (Hippoglossus
hippoglossus) (Tocher et al. 2002).
AX has higher antioxidant activity than BC (Miki 1991;
Naguib 2000; Goto et al. 2001). Such higher antioxidant
activity includes eliminating free radicals (Terao 1989),
inhibiting the generation of singlet oxygen (Di Mascio et al.
1991), trapping peroxyl (Jorgensen & Skibsted 1993), and
inhibiting the formation of lipid superoxide (Lim et al. 1992;
Goto et al. 2001). In this study, however, when fish was under
ammonia stress, dietary CD type did not reflect the superiority
of AX over BC on all antioxidant activities except SOD, where
BC-fed fish showed lower activity than AX- and MX-fed fish.
This is because of the conversion of dietary BC into body AX
for storage (Wang et al. 2006), which was further supported
by this study. Although BC-fed fish had lower body AX
content than AX- and MX-fed fish, it was sufficient enough to
enhance an antioxidant defence system against ammonia
stress, which is equivalent to that of AX- and MX-fed fish. As
for the dietary CD concentration effect, although 40-fish had
higher body AX content than 20-fish, no further improvement
on the antioxidant defence capacity was observed.
ALT and AST in fish have been used as indices for the
diagnosis of liver function (Yamamoto 1981) and damage
(Oda 1990). A study by Nakano et al. (1995) demonstrated for
the first time that a dietary AX supplement had an effect on
liver function and increased defence potential against oxida-
tive stress of rainbow trout. Further study by Nakano et al.
(1999) indicated that dietary red yeast, Phaffia rhodozyma,
which is rich in AX, had a reducing effect on oxidized oil-
induced oxidative stress in rainbow trout. In their study, the
fishÕs serum ALT and AST increased significantly when fed
oxidized oil. The supply of red yeast considerably lowered
ALT and AST activities. In our study, results also showed
that the increase in body AX content through dietary CD
supplementation reduced both ALT and AST under ammonia
stress.
The significant correlation between GPx and SOD could
indicate that the removal of H2O2 by GPx was related to the
reaction of superoxide radicals by SOD. Lushchak et al.
(2001) observed an increase in SOD and GPx activities in
goldfish Carassius auratus under hypoxia stress, showing
synergistic relationship between SOD and GPx. However,
..............................................................................................
Aquaculture Nutrition 17; 258–266  2010 Blackwell Publishing Ltd
the responses of different antioxidant enzymes in various
tissues to an oxidative stress may not be all the same. In the
study of Marcon & Filho (1999) on tambaqui, Colossoma
macropomum, a freshwater migratory teleost showing good
tolerance to chemical–physical environment fluctuation, they
found that antioxidant defences located in different tissues
and sub-cellular compartments act in harmony. There were
good correlations between liver SOD and red cell SOD,
between red cell GSH and liver SOD, between red cell GSH
and red cell SOD and between plasma thiobarbituric acid-
reactive substances and plasma SOD. Meyer et al. (2003)
reported that a short-term (up to 8 day) exposure of killifish
(Fundulus heteroclitus) to contaminated sediments led to
upregulated antioxidant defences, including MnSOD protein
levels and GPx. They considered these responses were pro-
tective against the acute toxicity of sediments, and thus
represented part of the physiological process of acclimation.
Monteiro et al. (2006) reported that exposure of characid
fish, Brycon cephalus, to 2 mg L)1 of methyl parathion, an
organophosphate insecticide, for 96 h resulted in a significant
induction of SOD activity, catalase and glutathione
S-transferase in liver, white muscle and gills. However, the
GPx activity decreased significantly in white muscle and gills,
whereas no change was observed in the liver. Hypoxia stress
appeared to trigger SOD activity in gill and muscle tissues of
Leiostomus xanthurus, but had no significant effect in catalase
activity (Cooper et al. 2002).
TAS had poor correlations with antioxidant enzyme
activity and body AX content. TAS is an indicator of the
status of overall antioxidant defence against reactive oxygen
species and intermediates. SOD is specific in catalyzing the
dismutation of O2) into H2O2. While an organism is first
subjected to stress, SOD should be able to respond immedi-
ately to the production of superoxide anion. During ammo-
nia stress, TAS might not respond as sensitive as SOD and
did not have a complete inverse (r = )0.4931) relationship
with SOD.
ALT and AST were highly correlated, where both show
similar decreasing trend with increasing dietary CD concen-
trations. On the other hand, ammonia stress had no effect on
ALT activity but increased AST activity by 106%. Our results
opposed the findings of Nakano et al. (1995, 1999) on rainbow
trout where dietary AX significantly decreased the levels of
lipid peroxides in the liver and ALT activity, but not AST.
When characins were exposed to 15 mg TAN L)1 for 72 h,
regardless if they had been fed CD-supplemented diets for
8 weeks or not, activities of TAS, SOD, GPx and AST
increased, indicating positive responses against oxidative
stress. The enhanced body AX content through dietary CD
supplement helped to develop antioxidant defences and liver
protection during ammonia stress. Dietary CD types (AX,
BC and MX) did not affect TAS, GPx, ALT and AST,
because most of the dietary BC consumed was converted into
body AX and made AXÕs superiority over BC in antioxidant
defence much less pronounced. As there was no difference in
body AX content, and all antioxidant parameters measured
between MX- and AX-fed fish and no difference in TAS,
SOD, GPx and ALT between 20- and 40-fish, dietary sup-
plement of 10 mg kg)1 each of MC is sufficient to develop
antioxidant defences for ammonia stress.
This work was supported by the National Science Council
Project No. NSC 92-2318-B019-002 partially by Center for
Marine Bioscience and Biotechnology, National Taiwan
Ocean University.
Avnimelech, Y. & Ritvo, G. (2003) Shrimp and fishpond soils: pro-
cesses and management. Aquaculture, 220, 549–567.
Bohm, F., Edge, R., Land, E.J., McGarvey, D.J. & Truscott, T.G.
(1997) Carotenoids enhance vitamin E antioxidant efficiency.
J. Am. Chem. Soc., 119, 621–622.
Bradford, M.M. (1976) A rapid and sensitive method for the quan-
titation of microgram quantities of protein using the principle of
protein-dye binding. Anal. Biochem., 72, 248–254.
Buikema, A.L. Jr, Niedertehner, R.R. & Cairns, J. Jr (1982) Biolog-
ical monitoring, Part IV. Toxicity testing. Water Res., 16, 239–262.
Butler, J.N. (1964) Ionic Equilibrium. Addison-Wiley Publishing,
Reading, MA, USA.
Chen, J.C., Nan, F.H. & Kuo, C.M. (1991) Oxygen consumption and
ammonia-N excretion of prawns (Penaeus chinensis) exposed to
ambient ammonia. Arch. Environ. Contam. Toxicol., 21, 377–382.
Chew, B.P. (1995) Antioxidant vitamins affect food animal immunity
and health. J. Nutr., 125, 1804S–1808S.
Chien, Y.H. & Shiau, W.C. (2005) The effects of dietary supple-
mentation of algae and synthetic astaxanthin on body astaxanthin,
survival, growth and low dissolved oxygen stress resistance of
kuruma prawn, Marsupenaeus japonicus Bate. J. Exp. Mar. Biol.
Ecol., 318, 201–211.
Chien, Y.H., Chen, I.M., Pan, C.H. & Kurmaly, K. (1999) Oxygen
depletion stress on mortality and lethal course of juvenile tiger
prawn Penaeus monodon fed high level of dietary astaxanthin.
J. Fish. Soc. Taiwan, 26, 85–93.
Chien, Y.H., Pan, C.H. & Hunter, B. (2003) The resistance to
physical stresses by Penaeus monodon juvenile fed diets supple-
mented with astaxanthin. Aquaculture, 216, 177–191.
Colt, J.E. & Armstrong, D.A. (1981) Nitrogen toxicity to crusta-
ceans, fish, and mollusks. In: Proceedings of the Bio-Engineering
Symposium for Fish Culture. Fish Culture Section, American Fish-
eries Society. Northeast Society of Conservation Engineers (Allen,
L.J. & Kinney, E.C. eds), pp. 34–47. Bethesda, MD.
Cooper, R.U., Clough, L.M., Farwell, M.A. & West, T.L. (2002)
Hypoxia-induced metabolic and antioxidant enzymatic activities in
the estuarine fish Leiostomus xanthurus. J. Exp. Mar. Biol. Ecol.,
279, 1–20.
DÕApollonia, S. & Anderson, P.D. (1980) Optimal assay conditions
for serum and liver glutamate oxalacetate transaminase, glutamate
pyruvate transaminase, and sorbitol dehyddrogenase from the
rainbow trout. Sulmo guirdneri. Can. J. Fish. Aquat. Sci., 37, 163–
169.
Dabrowski, K. (2001) History, present and future of ascorbic acid
research in aquatic organisms. In: Ascorbic Acid in Aquatic
Organisms Status and Perspectives (Dabrowski, K. ed), pp. 255–
277. CRC Press, Boca Raton, FL, USA.
Dabrowski, K., Lee, K.J., Guz, L., Verlhac, V. & Gabaudan, J.
(2004) Effects of dietary ascorbic acid on oxygen stress (hypoxia or
hyperoxia), growth and tissue vitamin concentrations in juvenile
rainbow trout (Oncorhynchus mykiss). Aquaculture, 233, 383–392.
Darachai, J., Piyatiratitivorakul, S., Kittakoop, P., Nitithamyong, C.
& Menasveta, P. (1998) Effects of Astaxanthin on larval growth
and survival of the giant tiger prawn, Penaeus monodon. In:
Advances in Shrimp Biotechnology (Flegel, T.W. ed), pp. 117–121.
National Center for Genetic Engineering and Biotechnology,
Bangkok.
Di Mascio, P., Murphy, M.E. & Sies, H. (1991) Antioxidant defense
systems: the role of carotenoids, tocopherols and thiols. Am. J.
Clin. Nutr., 53, 194S–200S.
Emerson, K., Russo, R.C., Lund, R.E. & Thurston, R.V. (1975)
Aqueous ammonia equilibrium calculations: effect of pH and
temperature. J. Fish. Res. Board Can., 32, 2379–2388.
Fromm, P.O. & Gillete, J.R. (1968) Effect of ambient ammonia on
blood ammonia and nitrogen excretion of rainbow trout (Salmo
gairdneri). Comp. Biochem. Physiol., 26, 887–896.
Ghidalia, W. (1985) Structural and biological aspects of pigments.
In: The Biology of Crustacea, Vol. 9 (Bliss, D.E. & Mantel, L.H.
eds), p. 301. Academic Press, New York.
Goto, S., Kogure, K., Abe, K., Kimata, Y., Kitahama, K.,
Yamashita, E. & Terada, H. (2001) Efficient radical trapping at the
surface and inside the phospholipids membrane is responsible for
highly potent antiperoxidative activity of the carotenoid astaxan-
thin. Biochim. Biophys. Acta, 1512, 251–258.
Gouveia, L., Rema, P., Pereira, O. & Empis, J. (2003) Colouring
ornamental fish (Cyprinus carpio and Carassius auratus) with
microalgal biomass. Aquac. Nutr., 9, 123–129.
Halliwell, B. & Gutteridge, J.M.C. (1984) Oxygen toxicity, oxygen
radicals, transition metals and disease. Biochem. J., 219, 1–14.
Halliwell, B. & Gutteridge, J.M.C. (1989) Free Radicals in Biology
and Medicine, 2nd edn. Clarendon Press, Oxford.
Hampson, B.L. (1976) Ammonia concentration in rainbow trout
rearing experiment in a closed freshwater-seawater system. Aqua-
culture, 9, 61–70.
Hubert, J.J. (Ed.) (1980) Bioassay. Kendall Hunt Publishing,
Toronto, Ontario, Canada. 164 pp.
Jeney, G., Nemcsok, J., Jeney, Z.S. & Olah, J. (1992) Acute effect of
sublethal ammonia concentrations on common carp (Cyprinus
carpio L.). 2. Effect of ammonia on blood plasma transaminases
GOT, GPT, G1DH enzyme activity, and ATP value. Aquaculture,
104, 149–156.
Jorgensen, K. & Skibsted, L.H. (1993) Carotenoid scavenging of
radicals. Z. Lebensm. Unters. Forsch., 196, 423–429.
Kalinowski, C.T., Robaina, L.E., Fernandez-Palacios, H., Schuc-
hardt, D. & Izquierdo, M.S. (2005) Effect of different carotenoid
..............................................................................................
Aquaculture Nutrition 17; 258–266  2010 Blackwell Publishing Ltd
 sources and their dietary levels on red porgy (Pagrus pagrus)
growth and skin colour. Aquaculture, 244, 223–231.
Khassaf, M., McSrdle, A., Dsamu, C., Vasilaki, A., McArdle, F.,
Griffiths, R.D., Brodie, D.A. & Jackson, M.J. (2003) Effect of
vitamin C supplements on antioxidant defence and stress proteins
in human lymphocytes and skeletal muscle. J. Physiol., 549, 645–
652.
Kwon, J.Y. & Chang, Y.J. (1996) Effect of ammonia concentration
on histological and physiological status in black seabream
Acanthopagurs schlegeli. J. Korean Fish. Soc., 29, 828–836.
Latscha, T. (1990) Carotenoids in Animal Nutrition: Their Nature and
Significance in Animal Feeds. Roche Publication No. 2175, Animal
Nutrition and Health. F. Hoffmann-La Roche, Basel, Switzerland.
Lim, B.P., Nagao, A., Tera, J., Tanaka, K., Suzuki, T. & Takama, K.
(1992) Antioxidant activity of xanthophylls on peroxyl radical –
mediated phospholipid peroxidation. Biochim. Biophys. Acta,
1126, 178–184.
Liu, H., Xie, S., Zhu, X., Lei, W., Han, D. & Yang, Y. (2008)
Effects of dietary ascorbic acid supplementation on the growth
performance, immune and stress response in juvenile Leiocassis
longirostris Günther exposed to ammonia. Aquac. Res., 39,
1628–1638.
Lushchak, V.I., Lushchak, L.P., Mota, A.A. & Hermes-Lima, M.
(2001) Oxidative stress and antioxidant defenses in goldfish Car-
assius auratus during anoxia and reoxygenation. Am. J. Physiol.
Regul. Integr. Comp. Physiol., 280, 100–107.
Maltby, L. (1995) Sensitivity of crustaceans Gammarus pulex (L.) and
Astllus aquaticus (L.) to short-term exposure to hypoxia and
unionized ammonia: observations and possible mechanism. Water
Res., 29, 781–787.
Marcon, J.L. & Filho, D.W. (1999) Antioxidant processes of the wild
tambaqui, Colossoma macropomum (Osteichthyes, Serrasalmidae)
from the Amazon. Comp. Biochem. Physiol., 123C, 257–263.
Merchie, G., Kontara, E., Lavens, P., Robles, R., Kurmaly, K. &
Sorgeloos, P. (1998) Effect of vitamin C and astaxanthin on stress
and disease resistance of postlarval tiger shrimp Penaeus monodon
(Fabricius). Aquac. Res., 29, 579–585.
Meyer, J.N., Smith, J.D., Winston, G.W. & Di Giulio, R.T. (2003)
Antioxidant defenses in killifish (Fundulus heteroclitus) exposed to
contaminated sediments and model prooxidants: short-term and
heritable responses. Aquat. Toxicol., 65, 377–395.
Miki, W. (1991) Biological functions and activities of animal carot-
enoids. Pure Appl. Chem., 63, 141–146.
Monteiro, D.A., de Almeida, J.A., Rantin, F.T. & Lúcia Kalinin,
A.L. (2006) Oxidative stress biomarkers in the freshwater characid
fish, Brycon cephalus, exposed to organophosphorus insecticide
Folisuper 600 (methyl parathion). Comp. Biochem. Physiol., 143C,
141–149.
Naguib, Y.M.A. (2000) Antioxidant activities of astaxanthin and
related carotenoids. J. Agric. Food. Chem., 48, 1150–1154.
Nakano, T., Tosa, M. & Takeuchi, M. (1995) Improvement of bio-
chemical features in fish health by red yeast and synthetic asta-
xanthin. J. Agric. Food. Chem., 43, 1570–1573.
Nakano, T., Kanmuri, T., Sato, M. & Takeuchi, M. (1999) Effect of
astaxanthin rich red yeast (Phaffia rhodozyma) on oxidative stress
in rainbow trout. Biochim. Biophys. Acta, 1426, 119–125.
Nemcsok, J., Gyore, K., Olah, J. & Boros, L. (1982) Effects of NH3
on blood glucose level, GOT, GPT, LDH enzyme activity and
respiration of three fish species. Aquacult. Hung., 3, 63–68.
Oda, T. (Ed.) (1990) The Biology of Liver. University of Tokyo Press,
Tokyo. 140 pp. (In Japanese).
Oshima, S., Ojima, F., Sokamoto, H., Ishiguro, Y. & Terao, J. (1993)
Inhibitory effect of b-carotene and astaxanthin on photosynthe-
..............................................................................................
Aquaculture Nutrition 17; 258–266  2010 Blackwell Publishing Ltd
sized oxidation of phospholipid bilayers. J. Nutr. Sci. Vitaminol.,
39, 607–615.
Pan, C.H. & Chien, Y.H. (2009) Effects of dietary supplementation
of alga Haematococus pluvialis (Flotow), synthetic astaxanthin
and b-carotene on survival, growth, and pigment distribution of
red devil, Cichlasoma citrinellum (Gunther). Aquac. Res., 40, 871–
879.
Pan, C.H., Chien, Y.H. & Hunter, B. (2003a) The resistance to
ammonia stress of Penaeus monodon Fabricius juvenile fed diets
supplemented with astaxanthin. J. Exp. Mar. Biol. Ecol., 297, 107–
118.
Pan, C.H., Chien, Y.H. & Hunter, B. (2003b) Alterations of anti-
oxidant capacity and hepatopancreatic enzymes in Penaeus mon-
odon (Fabricius) juveniles fed diets supplemented with astaxanthin
and exposed to Vibrio damsela challenge. J. Fish. Soc. Taiwan, 30,
279–290.
Randall, D.J. & Tsui, T.K.N. (2002) Ammonia toxicity in fish. Mar.
Pollut. Bull., 45, 17–23.
Schreckenbach, K. & Spangenberg, R. (1978) pH-wert-abhangige
Ammoniakvergiftung bei Fischen und Moglichkeiten ihrer Beein-
flussung. Z. Binnenfisch. D.D.R., 25, 299–314.
Shimidzu, N., Goto, M. & Miki, W. (1996) Carotenoids as
singlet oxygen quenchers in marine organisms. Fish. Sci., 62,
134–137.
Sies, H. (1991) Oxidative stress: from basic research to clinical
application. Am. J. Med., 91, 31S–38S.
Stanier, R.Y., Kunizawa, M.M. & Cohen-Bazire, G. (1971) Purifi-
cation and property of unicellular blue-green algae (Order Chro-
ococales). Bacteriol. Rev., 35, 120–171.
Storebakken, T. & No, H.K. (1992) Pigmentation of rainbow trout.
Aquaculture, 100, 209–229.
Storey, K.B. (1996) Oxidative stress: animal adaptations in nature.
Brasil. J. Med. Biol. Res., 29, 1715–1733.
Taylor, A.L. & Solomon, D.J. (1979) Critical factors in transport of
living freshwater fish. I. General considerations and atmospheric
gases. Aquac. Res., 10, 27–32.
Terao, J. (1989) Antioxidant activity of beta-carotene-related carot-
enoids in solution. Lipids, 24, 659–661.
Thurston, R.V., Russo, R.C. & Vinogaradov, G.A. (1981) Ammonia
toxicity to fishes: effect of pH on the toxicity of the unionized
ammonia species. Environ. Sci. Technol., 15, 837–840.
Tocher, D.R., Mourente, G., Van Der Eecken, A., Evjemo, J.O.,
Diaz, E., Bell, J.G., Geurden, I., Lavens, P. & Olsen, Y. (2002)
Effects of dietary vitamin E on antioxidant defence mechanisms of
juvenile turbot (Scophthalmus maximus L.), halibut (Hippoglossus
hippoglossus L.) and sea bream (Sparus aurata L.). Aquac. Nutr., 8,
195–207.
Tomasso, J.R. (1994) Toxicity of nitrogenous wastes to aquaculture
animals. Rev. Fish. Sci., 2, 291–314.
Torrissen, O.J. & Christiansen, R. (1995) Requirements for carote-
noids in fish diets. J. Appl. Ichthyol., 11, 225–230.
Torrissen, O.J., Hardy, R.W. & Shearer, K.D. (1989) Pigmentation
of salmonids-carotenoid deposition and metabolism. Crit. Rev.
Aquat. Sci., 1, 209–225.
Van Rijn, J., Shilo, M., Bejerano, T. & Nizan, S. (1990) The effect of
inorganic nitrogen on microorganisms and fish in fishponds. In:
Research in Modern Aquaculture (Sarig, S. & Rosenthal, H. eds),
pp. 3–27. Proceedings of the 3rd Status Seminar. Special Publi-
cation of European Aquaculture Society, vol. 11. EAS, Oostende,
Belgium.
Vedel, N.E., Korsgaard, B. & Jensen, F.B. (1998) Isolated and
combined exposure to ammonia and nitrite in rainbow trout
(Oncorhynchus mykiss): effects electrolyte status, blood respiratory
 properties- and brain glutamine/glutamate concentration. Aquat.
Toxicol., 41, 325–342.
Walsh, P.J. & Wright, P.A. (1995) Nitrogen Metabolism and Excre-
tion. CRC Press, Florida, USA.
Wang, W.N., Wang, A.L. & Wang, Y. (2006) Effect of supple-
mental L-ascorbyl-2-polyphosphate in enriched live food on
the antioxidant defense system of Penaeus vannamei of differ-
ent sizes exposed to ammonia-N. Aquac. Nutr., 12, 348–
352.
Wasielesky, W. Jr, Marchiori, M.A. & Santos, M.H. (1994) Effect of
ammonium on the growth of the pink-shrimp Penaeus paulensis,
Perez Farfante, 1967 (DECAPODA: PENAEIDAE) post larvae.
Nauplius, 2, 99–105.
Wilhelm, D. (1996) Fish antioxidant defenses. A comparative
approach. Braz. J. Med. Biol. Res., 29, 1735–1742.
Yamamoto, Y. (1981) Determination of toxicity by biochemical
method. In: Fishes as Laboratory Animals (Egami, N. ed) Chapter
4, pp. 63–80. Soft Science, Tokyo.
..............................................................................................
Aquaculture Nutrition 17; 258–266  2010 Blackwell Publishing Ltd