Aquaculture research, tập 41, số 7, 2010

Aquaculture Research, 2010, 41, 953^960

doi:10.1111/j.1365-2109.2009.02377.x

Physiological responses of pink abalone Haliotis

corrugata (Gray, 1828) exposed to different
combinations of temperature and salinity
Zarina Medina Romo1, Ana Denisse Re1, Fernando D|¤ az1 & Alfredo Mena2
1

Laboratorio de Eco¢siolog|¤ a de Organismos AcuaŁticos, Departamento de Biotecnolog|¤ a Marina, Centro de Investigacio¤n
Cient|¤ ¢ca y de Educacio¤n Superior de Ensenada (CICESE), Ensenada Baja California, Me¤xico
2

Departamento de Acuicultura-Biotecnolog|¤ a, Facultad de Ciencias Marinas (FACIMAR), Universidad de Colima, Manzanillo

Colima, Me¤xico
Correspondence: F D|¤ az, Departamento de Biotecnolog|¤ a Marina (CICESE), PO Box 430222, San Diego, CA 92143-0222, USA.
E-mail:

Abstract
Physiological responses of pink abalone Haliotis corrugata were determined under di¡erent temperature and
salinity conditions. Oxygen consumption rate was not
a¡ected by temperature and salinity. Ammonium excretion of pink abalone was inversely related to salinity.
The O:N ratio indicated that abalone maintained in
lower salinities had an interval of 4.9^7.7, which is indicative of a protein-dominated metabolism, whereas
the O:N in 35% was 28.8^35.5 for both temperatures,
suggesting that carbohydrates were used as energy
substrate. Haemolymph osmolality of abalone exposed

to 20 and 24 1C was slightly hyperiso-osmoconformic
in salinity ranges of 20^35%. The results of this study
suggested that for optimized culture, pink abalone
should be cultivated at 24 1C at a salinity of 35%.

Keywords: oxygen consumption rate, ammonium
excretion, atomic ratio O:N, osmoregulation, Haliotis
corrugata
Introduction
Haliotis corrugata (Gray 1828) is one of the species of
economic importance and is actually cultivated in
Baja California; it was observed that the growth of
cultivated abalone is a¡ected by many factors such
as temperature, salinity, dissolved oxygen, nitrogen
subproducts, pH, density, food and water quality
(Hahn 1989;Valde¤s-Urriolagoitia 2000).
Temperature and salinity are two factors that control the life and distribution of aquatic organisms;
both factors have direct e¡ects on the physiological
responses of the marine and estuarine organisms,

r 2009 The Authors
Journal Compilation r 2009 Blackwell Publishing Ltd

such as oxygen consumption (Brown & da Silva
1979; Moore & Sander 1983; Saucedo, Ocampo, Monteforte & Bervera 2004; Soria, Merino & von Brand
2007), nitrogen excretion products (Livingstone,Widdows & Fieth 1979; Stickle & Bayne 1982; Regnault
1987; Saucedo et al. 2004; Soria et al. 2007), energy
budget (Newell & Branch 1980; Bayne & Newell 1983;
Bricelj & Shumway 1991; Beiras, Pe¤rez-Camacho &
Albentosa 1993), endogenous substrate utilization

(Barber & Blake 1985; Mayzaud & Conover 1988; Rosas, Cuzon, Gaxiola, Taboada, Arena & VanWormhoudt 2002) and osmoregulation pattern (Shumway
1977; Hildreth & Stickle 1980; Cheng, Yeh, Wang &
Chen 2002).
One of the physiological responses that can be correlated with a change in the environmental parameters is the oxygen consumption rate, because it is
related to the metabolic work and energy £ow that organisms must channel to homeostatic control mechanisms (Salvato, Cuomo, Di Muro & Beltramini
2001). In aquatic organisms, the measurement of oxygen consumption is a valid method to assess the e¡ect
of environmental factors such as temperature, salinity, exposure to pollutants, light intensity and dissolved oxygen. It allows the estimation of the energy
costs associated with the physiological stress that
these factors impose on organisms (Brown & Terwilliger 1999; Lemos, Phan & Alvarez 2001; Altinok &
Grizzle 2003; Brougher, Douglass & Soares 2005).
Previous studies have investigated the e¡ects of salinity and temperature on the oxygen consumption in
abalone such as Haliotis discus hannai (Ino) exposed
to di¡erent environmental factors (Sano & Maniwa
1962). Uki and Kikuchi (1975) investigated the e¡ect

953


Physiological responses of pink abalone Z M Romo et al.

of temperature and weight on the oxygen consumption of H. discus hannai. In young disc abalone Nordotis discus discus (Reeve), Segawa (1995) determined in
a preliminary study, the e¡ect of temperature on the
oxygen consumption rate. Paul and Paul (1998) determined the e¡ect of di¡erent temperatures on the respiration rate in Haliotis kamtschatkana (Jonas).
Both osmotic and ionic regulation have been studied in a number of marine molluscs (Burton 1983).
Changes in salinity may disturb the osmotic balance
of marine molluscs. However, nothing is known on
the osmotic and ionic regulation of the Haliotis genus
(Cheng et al. 2002). Bivalve molluscs are osmoconformers in which the haemolymph is close to the osmotic pressure of seawater, and due to response,
ammonium excretion increases with decreasing salinity (Shumway 1977; Bricelj & Shumway 1991).
The role of ammonium in the osmoregulation processes has been studied in di¡erent organisms in two

aspects: as a constituent of free amino acids (FAA) for
intracellular osmotic regulation (Bishop, Gosselink &
Stone 1980) and as an exchange ion for the regulation of Na1 in the haemolymph (Mangum, Silverthorn, Harris, Towle & Krall 1976; Pressley, Graves &
Krall 1981; Re, D|¤ az & Go¤mez-Jime¤nez 2004).
The atomic ratio (O:N) is an index that uses the integration of the values of the oxygen consumption
and nitrogen excretion to determine which metabolic substrate is being used for the organisms (Mayzaud & Conover1988). This atomic ratio has also been
used as a stress indicator due to the changes in the
environment to which the organisms are exposed
(Ikeda 1977; Cli¡ord & Brick 1979; Rosas, Cuzon, Gaxiola, LePriol, Pascual, Rossygnyol, Contreras, SaŁnchez & VanWormhoudt 2001). Finally, the O:N ratio is
an important index that is used in both ecological
and aquacultural settings, and so linked studies of
oxygen consumption and nitrogen excretion in abalone would provide useful information on this index.
The main goal of this study was to determine the
e¡ect of di¡erent combinations of salinity and temperature on di¡erent physiological responses in H.
corrugata. Because of the commercial importance of
this species, the information reported in this paper
will be useful for managing these parameters under
controlled conditions.

Material and methods
About 720 juveniles with an average wet weight of
2.8 (Æ 0.9 g) were obtained from the commercial

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Aquaculture Research, 2010, 41, 953^960

farm of B. C. Abalone Hatchery Ere¤ndira, Ensenada
(Baja California, Me¤xico). The organisms were transported in a10 L-Styrofoam cage with seawater. In the
laboratory, the time to acclimate was 2 weeks at 35%,

18 Æ 1 1C temperature, measured under farm conditions in three reservoirs of 2000 L with constant
aeration and a 60 mm ¢ltered seawater £ow with an
exchange rate of 100% daily. Each reservoir was provided with abalone refuges and was heavily shaded;
refuges and shaded were used to minimize disturbance from exterior movements.
The organisms were fed during the entire experimental period with macroalgae Macrocystis pyrifera
and Egregia menziesii. Acclimation of 489 abalones
to the experimental temperature 20 and 24 1C was
completed in 21days. The temperature was increased
at a rate of 2 1C everyday to reach the experimentalprogrammed temperature. Both temperatures were
achieved by titanium heaters of 1000 W controlled by
Medusa devices (Sea Life Supply, Sand City, CA, USA).
For experimental salinities 480 abalones were acclimated to 35%, 32%, 29%, 26%, 23% and 20%,
where the lowest concentration was obtained by dilutions of seawater (35%) with tap water. The rate of
salinity decrease was 3% per 5 days to reach the experimental salinity, and once the experimental salinity had been achieved, the abalones remained
under those conditions for 15 days, which represent
su⁄cient time to attain a steady internal medium of
abalone according to Cheng et al. (2002).
To avoid interference with post-prandial metabolism of food and faeces production, acclimated abalone was kept unfed for 24 h. Oxygen consumption
and nitrogen excretion were measured with the organisms maintained at temperatures of 20 and
24 1C and experimental salinities of 35%, 32%,
29%, 26%, 23% and 20% by using a semi-closed respirometric system described by D|¤ az, Re, GonzaŁlez,
SaŁnchez, Leyva and Valenzuela (2007), consisting of
21 chambers of 1000 mL each. Twenty abalones of
each temperature and salinity were individually introduced into the respiratory chamber 24 h before initiating measurements, which were made between
09:00 and 13:00 hours to avoid interference due to
the rhythm circadian response.
Water £ow in the chambers remained open for 2 h;
before closing, one water sample was taken to measure the initial concentration of dissolved oxygen
using aYSI 52 oxymeter (Yellow Spring Instruments,
Yellow Springs, OH, USA), equipped with a polarographic sensor accuracy of Æ 0.03 mg L À 1, which

was inside an acrylic hermetic chamber with a

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Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 953^960


Aquaculture Research, 2010, 41, 953^960

10 mL capacity with adequate stirring. Subsequently,
the chambers were kept closed for 1h, to avoid reduction in the dissolved oxygen of 25^30%, as this is a
stress factor (Stern, Borut & Cohen 1984). Before reestablishing the water £ow, one water sample was taken to measure the ¢nal concentration of dissolved
oxygen. The 21st chamber was used as a control to
measure oxygen consumption by the microorganisms present in the water, and the necessary corrections were made. Two repetitions were carried out for
each test. The results of oxygen consumption are
given in mg O2 kg À 1 h À 1 on a wet weight basis.
To determine the ammonium production (NH1
4 ),
initial and ¢nal samples were obtained from the respirometric chamber in the same manner as that described for oxygen consumption; the di¡erence was
that the water samples were of10 mL and the method
for quanti¢cation was indophenol blue (Rodier 1998).
The samples were analysed using the spectrophotometer El|¤ ptica (Ely-2000 Instruments, Ensenada,
Me¤xico) at a wavelength of 640 nm. Ammonium production was calculated as the di¡erence between the
¢nal and the initial measure and it was expressed as
À1 À1
mg NH1
g wet weight (w.w.).
4 h
The O:N ratio was estimated using the oxygen consumption and the ammonium excretion values of the
abalones, both were obtained using the respirometric
system from all di¡erent experimental combinations.

The physiological rates were determined for both
components and transformed to atom-gram in order
to calculate the O:N index (Mayzaud & Conover1988).
This index was used to estimate the ratio of proteins,
lipids and carbohydrates that were used as energy
substrates for the organisms under the di¡erent experimental conditions.
An individual sample of haemolymph was obtained
from the membrane between muscle and mantle of
the shell of abalones using a hypodermic needle, in a
manner similar to that described by Cheng et al.
(2002). The samples of haemolymph,10 mL from 20 organisms from each experimental condition, were
placed on a blotting paper disc in a Wescor 5520 vapour pressure osmometer (Wescor Logan, UT, USA).
The osmolality of the internal as well as the external
medium was expressed as mmol kg À 1.
The data for oxygen consumption and ammonium
excretion of abalone exposed to di¡erent experimental conditions were plotted in parallel boxes (Tukey
1977).Within the boxes, 50% of the data were distributed around the median and the con¢dence intervals; the other 50% remained distributed in each
bar. The relationships between haemolymph osmol-

Physiological responses of pink abalone Z M Romo et al.

ality and medium osmolality were determined using
a linear regression after satisfying the test for suitability of ¢t to a linear model (SIGMA STAT version 3.1).
A two-way analysis of variance (ANOVA) was used as
previous determination of the normality and homoscedasticity of the data (SIGMA STAT), to determine the
e¡ect of the temperature and salinity on oxygen consumption, ammonium excretion, atomic ratio O:N
and the osmolality of the haemolymph of pink abalone (Zar 1999).

Results
The rate of oxygen consumption of pink abalone maintained at 20 1C and acclimated at di¡erent salinities

was in the range of 0.58^0.79 mg O2 h À 1 g À 1 w.w. In
the organism acclimated to 24 1C, oxygen consumption was in the range of 0.64^0.81mg O2 h À 1 g À 1 w.w.
(Fig. 1). An ANOVA indicated that temperature and salinity did not have a signi¢cant e¡ect on the oxygen

Figure 1 Oxygen consumption rate of Haliotis corrugata
acclimated to two temperatures at di¡erent salinities. The
zone marked by circles represents the 95% con¢dence interval of the median; 50% of the data are distributed in
vertical lines.

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955


Physiological responses of pink abalone Z M Romo et al.

Figure 2 Ammonium excretion of Haliotis corrugata acclimated to two temperatures at di¡erent salinities. The
zone marked by circles represents the 95% con¢dence interval of the median; 50% of the data are distributed in
vertical lines.

consumption rate of the pink abalone (d.f. 55, 1,
F 5 2.15,1.12, P 5 0.34).
The ammonium excretion of the pink abalone at
20 1C increased at salinities of 20% and 23% and
reduced when it was increased, yielding the lowest
excretion value (0.25 mg NH4 h À 1 g À 1 w.w.) at the
salinity of 35%. In abalone maintained at 24 1C, the
ammonium excretion rate increased when the organism was exposed to 20% and 23% salinities
(Fig. 2). Ammonium excretion decreased as salinity

increased until the rate was in the range of 0.30^
0.35 NH4 h À 1 g À 1 w.w. An ANOVA indicated that
there was a signi¢cant e¡ect of salinity on the ammonium excretion rate of H. corrugata (d.f. 55, 1,
F 516.39, 12.72, Po0.05).
The index of O:N values estimated for the juveniles
of H. corrugata acclimated to 20 1C was in the range of
23.9^28.8 in the organisms acclimated at salinities of
29^35%. The lowest values of the O:N index found in
the acclimated juveniles exposed to 20% and 23%
salinities were 4.9 and 6.2 respectively (Fig.3). In abalones acclimated to 24 1C, the lowest values of the

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Aquaculture Research, 2010, 41, 953^960

Figure 3 Atomic ratio O:N (median Æ con¢dence interval) of Haliotis corrugata acclimated at two temperatures
at di¡erent salinities.

O:N atomic ratio of 6.8 and 7.7 were obtained at salinities of 20% and 23%; the highest value of 35.5 was
found in the juveniles acclimated to 35% salinity
(Fig. 3). An ANOVA indicated that salinity had a signi¢cant e¡ect (d.f. 55,1, F 5 43.01,1.03, Po0.001) on the
atomic ratio.
Osmolality of haemolymph in the abalone juveniles acclimated to 20 and 24 1C was related in a linear way with respect to the external medium,
yielding the equations:
IM in 20 C ¼ 69:25 þ 0:949X r2 ¼ 0:996
IM in 24 C ¼ À14:82 þ 1:045X r2 ¼ 0:986
where IM (internal medium) is the haemolymph osmolality and X is the external medium osmolality.
For the abalones acclimated to 20 1C and exposed
to experimental salinities the haemolymph osmolality was in the range of 675^1002.7 mmol kg À 1, having a slightly hyperiso-osmoconformer pattern of
osmoregulation (Fig. 4). In the organism acclimated

to 24 1C, haemolymph osmolality was in the range

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Aquaculture Research, 2010, 41, 953^960

Figure 4 Relation between haemolymph osmolality of
Haliotis corrugata and medium osmolality when they were
exposed to two temperatures at di¡erent salinities.

of 691^1054 mmol kg À 1, indicating that abalones
had a slightly hyperiso-osmoconformer pattern (Fig.
4). An ANOVA indicated that temperature and salinity
had a signi¢cant e¡ect (d.f. 55, 1, F 5168.3, 17.85,
Po0.001) on the haemolymph concentration of pink
abalone; the interaction between temperature and
salinity did not have a signi¢cant e¡ect (P40.05).
Discussion
Oxygen consumption and ammonium excretion
rates in marine invertebrates are a¡ected by body
size, diurnal rhythm, feeding and environmental
parameters such as temperature and salinity to
which organisms are exposed (Crear & Forteath
2000; Salvato et al. 2001; Ahmed, Segawa, Yokota &
Watanabe 2008).

Physiological responses of pink abalone Z M Romo et al.

In our experiment, the oxygen consumption rate
was independent of salinity and temperatures, suggesting that abalone could adjust their metabolism
after an acclimation period to di¡erent experimental
conditions to which they were exposed. In the abalone H. discus hannai exposed to chlorinities higher
than 14%, Sano and Maniwa (1962) obtained that
the rate of oxygen consumption was kept constant.
In Argopecten purpuratus (Lamarck) exposed to a
combination of two temperatures and three salinities
for a period of 45 days; Soria et al. (2007) reported that
the rate of oxygen consumption was maintained independent. In aquatic organisms that have been acclimated to di¡erent salinities, Kinne (1967)
described four types of metabolic responses. Pink
abalone exposed to di¡erent salinities exhibited the
type I response, because the oxygen consumption
was not modi¢ed signi¢cantly. For other marine organisms, it has been shown that salinity did not have
a pronounced e¡ect on the oxygen consumption
when the experimental organisms were acclimated
to salinities and these are not extreme (Bishop et al.
1980; Gaudy & Sloan 1981; Salvato et al. 2001).
Many osmoconforming marine invertebrates maintain FAA pools that vary directly with external salinity
and this allows the preservation of cellular volumes by
changing haemolymph osmolality (Tang, Liu, Yang &
Xiang 2005). The ammonium excretion of pink abalone increased when salinity decreased from 35% to
23%, and this response may be related to an increase
in catabolism of the amino acids to form intracellular
osmolytes to regulate their osmotic equilibrium when
exposed to lower salinities. Under hyperosmotic stress,
pink abalone shows an enhancement in the concentration of intracellular £uid by shrinking the cell volume from the environment absorbing ions and
catabolizing self-tissue protein. All these metabolic activities lead to increased ammonium excretion. In
Meretrix meretrix (Linnaeus) (Tang et al. 2005) and
A. purpuratus exposed to decreasing and increasing

salinities (Navarro & GonzaŁlez 1998); Soria et al.
(2007) reported an increase in the ammonium excretion rate, suggesting that it regulates the cellular volume by breakdown of amino acids as intercellular
regulators with a reduction in salinity.
A high value of O:N is taken to represent a predominance of lipid and/or carbohydrate degradation
over protein degradation (Mayzaud & Conover 1988).
Ikeda (1977) reported an O:N ratio of 24 when protein
and lipids were metabolized in equal quantities at the
same time; hence, an O:N ratio o24 indicates a protein-dominant metabolism and a ratio 424 indicates

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Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 953^960

957


Physiological responses of pink abalone Z M Romo et al.

a lipid-dominant metabolism. Factors such as season,
temperature and salinity may in£uence the value of
O:N (Ikeda 1977; Mayzaud & Conover 1988). In the
present work, the O:N ratio found for abalone exposed to lower salinities had values of 4.9^7.7, implying that the organisms used protein catabolism as a
primary catabolic substrate, due to the stress caused
by exposure to lower salinities. At intermediate salinities (23% and 26%), the organisms yielded an O:N
of 16^24, indicative of protein and lipid catabolism in
equal levels for organisms exposed to both temperatures. In abalone acclimated to 24 1C and exposed to
high salinities, the O:N ratios were 35.5, indicating
that under these conditions, lipid and carbohydrate
were the metabolic substrates used by abalones. In
Argopecten irradians concentricus (Say), Barber and
Blake (1985) reported the same trend. We observed a

shift from protein to protein^lipid and lipid^carbohydrate as the source of energy metabolism associated
with an increase in experimental salinities and a
temperature. In abalones exposed to high salinities
and a temperature of 24 1C, the organisms found in
environment optimum due to D|¤ az, Re, Medina, Re,
Valdez and Valenzuela (2006) reported for H. corrugata that the preferred and optimal of growth temperature were 25 and 24.5 1C for abalones maintained in
35%; under salinity condition, pink abalone used
lipid^carbohydrates as the metabolic substrate. This
indicates that these combinations of salinity and
temperature do not produce stress in the organism,
and therefore, we recommend these conditions for
adequate maintenance of H. corrugata under culture
conditions.
Osmotic regulation in mollusks has been reported
for some species of bivalves such as Argopecten ventriculosus-circularis (Sowerby II) (Signoret-Brailovsky,
Maeda-Martinez, Reynoso-Granados, Soto-Galera,
Monsalvo-Spencer & Valle-Meza 1996), Crassostrea
gigas (Thunberg) (Hosoi, Kubota,Toyohara & Hayashi
2003) and the abalone H. diversicolor supertexta
(Lischke) (Cheng et al. 2002). These papers indicated
that the haemolymph osmolality varies directly with
medium osmolality. In the present study, pink abalone maintained an internal medium that was
slightly hyperiso-osmoconformed, in contrast to the
¢nding of Cheng et al. (2002) for Haliotis diversicolor
supertexta, which had a slightly hypoiso-osmoconformed regulation pattern; the di¡erences in the
osmoregulation pattern due to the process of transference to the experimental salinities in H. diversicolor supertexta were drastic, and these conditions were
maintained for 9 days. For pink abalone, the physio-

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Aquaculture Research, 2010, 41, 953^960

logical changes were gradual and once the experimental conditions were reached, the abalones were
maintained for 15 days. In Argopecten ventriculosuscircularis, Signoret-Brailovsky et al. (1996) observed
an osmoconformer regulation pattern. In C. gigas exposed to gradual and sudden changes in salinities,an
osmoregulation osmoconformator pattern was reported (Hosoi et al. 2003). The osmoconformer marine organisms, including abalones, adapt to salinity
changes using intracellular isosmotic regulation, in
which intracellular FAA contribute predominantly
to intracellular osmolality and to cell volume regulation (Shumway 1977; Signoret-Brailovsky et al. 1996;
Cheng et al. 2002; Hosoi et al. 2003). The slope regression line calculated from the relationship between
haemolymph osmolality and medium osmolality
was parallel to the isosmotic line, with values from
0.949 to 1.045 similar to those reported by Cheng
et al. (2002) for H. diversicolor supertexta exposed to
di¡erent salinity levels.

Acknowledgements
We thank Jose M Dominguez and Francisco Javier
Ponce from the Drawing Department of CICESE for
preparing the ¢gures. We are also grateful for the
English language editing of the manuscript provided
by Dr A. Leyva.

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Aquaculture Research, 2010, 41, 961^967

doi:10.1111/j.1365-2109.2009.02378.x

Use of commercial fermentation products as a
highly unsaturated fatty acid source in
practical diets for the Pacific white shrimp

Litopenaeus vannamei
Tzachi M Samocha1, Susmita Patnaik1, Donald A Davis2, Robert A Bullis3 & Craig L Browdy4
1

AgriLife Research Mariculture Laboratory, AgriLife Research & Extension Center, Corpus Christi,TX, USA

2

Department of Fisheries and Allied Aquacultures, Auburn University, Auburn, AL, USA

3

Advanced BioNutrition, Columbia, MD, USA
South Carolina Department of Natural Resources, Charleston, SC, USA

4

Correspondence: Tzachi M Samocha, AgriLife Research Mariculture Laboratory, AgriLife Research & Extension Center, 4301 Waldron
Rd., Corpus Christi,TX 78418, USA. E-mail:

Abstract

Introduction

Removal or reduction of marine ingredients (MI)
from feed formulations is critical to the sustainability
of the aquaculture industry. By removing MI, diets
may become limiting in several nutrients including
highly unsaturated fatty acids (HUFA) such as docosahexaenoic acid (DHA) and arachidonic acid (ArA).
To reduce reliance on MI in shrimp diets, two trials
were conducted with Litopenaeus vannamei juveniles
to determine the feasibility of using fermentation
meals rich in DHA and ArA as the primary source
for HUFA. A practical diet with no MI was formulated

with/without DHA and ArA supplements and fed in
the ¢rst trial. A diet with menhaden ¢sh oil or a combination of plant oil with/without DHA and ArA supplements was used in the second trial. To determine
whether HUFA is only needed in the early growth
stages, we also fed one group a HUFA-supplemented
diet to 5 g and then switched them to a HUFA-supplement-free diet. In both trials, the weights were reduced when HUFA supplements were not provided
either throughout the trial or from 5 g to harvest
(o16 g). These results suggest that supplementation
of plant oils with DHA- and ArA-rich oils from fermented products is a viable option to replace marine
¢sh oil for L. vannamei.

Marine ¢sh meals and ¢sh oils are excellent sources
of high-quality essential amino acids, lipids, vitamins, minerals and attractants in aquaculture diets
(Tacon & Akiyama 1997). However, the unstable
prices associated with £uctuations in the supply of
these marine ingredients and the sustainability of
these practices are of prime concern (Chamberlain
1993; Tacon & Akiyama 1997; Naylor, Goldberg, Primavera, Kautsky, Beveridge, Clay, Folk, Lubchenco,
Mooney & Troell 2000). Hence, replacement of these
marine ingredients with cost-e¡ective alternative
sources of proteins and lipids in aquaculture feeds is
a high-priority task for feed mills and aquaculturists
(Tacon & Akiyama 1997). Previous studies (Lim 1996;
Davis & Arnold 2000; Samocha, Davis, Saoud & DeBault 2004; Menoyo, Lopez-Bote, Obach & Bautista
2005) showed that either animal or plant sources
can be used as suitable substitutes for ¢sh meal and
¢sh oil in a small-scale tank system. In their e¡orts to
replace ¢sh meal, researchers used plant protein
sources such as soybean meal (Sudaryono, Hoxey,
Kailis & Evans 1995; Hertrampf & Piedad-Pascual
2000; Olvera-Novoa & Olivera-Castillo 2000), solvent-extracted cotton seed meal (Lim 1996), lupin

meals (Sudaryono et al. 1995), legumes, leaf meals
(Eusebio & Coloso 1998; Li, Robinson & Hardy 2000)
and papaya or camote leaf meal (Pena£orida 1995) in
feed formulations for aquatic animals, with varying

Keywords: DHA, ArA, practical diets, Paci¢c
white shrimp, Litopenaeus vannamei

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961


Alternative HUFA source for Litopenaeus vannamei T M Samocha et al.

degrees of success. Several studies have demonstrated that with suitable adjustments, animal byproduct meals could be used successfully as ¢sh meal
replacements to meet the nutrient and attractability
requirements for the target species (Davis & Arnold
2000; Forster, Dominy, Obaldo & Tacon 2003; Samocha et al. 2004). Other researchers showed that a
combination of animal by-product meal and/or plant
protein sources provided promising results as ¢sh
meal substitutes without a¡ecting the physical
and nutritional quality of the feeds (Wu, Rosati,
Sessa & Brown 1995; Viola, Mokady, Rappaport &
Arieli 1982; Viola, Arieli & Zohar 1988; Tidwell,
Webster, Yancey & D’Abramo 1993; Sudaryono et al.
1995; Webster,Yancey & Tidwell 1995; Davis & Arnold
2000; Samocha et al. 2004).
Considerable research has also been carried out on

¢sh oil replacement strategies in aquaculture diets.
Plant protein and vegetable oil use in aquafeeds without marine ¢sh meal or ¢sh oil is often limited by the
potential problems associated with insu⁄cient levels
of essential amino and fatty acids, anti-nutritional
factors and poor palatability (Francis, Makkar &
Becker 2001). Heterotrophically grown algae and
products obtained by fermentation processes have
been reported to be a good source of nutrients and
essential fatty acids for larval live food enrichment
and for formulated broodstock diets of marine teleosts (Harel, Koven, Lein, Bar, Behrens, Stubble¢eld,
Zohar & Place 2002). In a recent study, Patnaik, Samocha, Davis, Bullis and Browdy (2006) showed that
¢sh meal and ¢sh oil can be successfully replaced in
the diets for Litopenaeus vannamei using co-extruded
soybean and a poultry by-product meal (ProfoundTM)
and spray-dried cells of Schizochytrium sp. and Mortierella sp. obtained by a proprietary commercial fermentation process. Although we have demonstrated
the ability to make these substitutions, the need to include a highly unsaturated fatty acids (HUFA) supplement has not been established especially under
conditions when some natural productivity is present. Consequently, the objective of the current study
was to evaluate potential replacement of marine oil
sources in diets for L. vannamei using a mixture of
plant oils enriched with HUFA produced through
the fermentation process.

Materials and methods
Two growth trials were conducted with juvenile Paci¢c white shrimp, L. vannamei, in an outdoor tank sys-

962

Aquaculture Research, 2010, 41, 961^967

tem operated with no water exchange. The studies

evaluated practical diet formulations in which both
marine ¢sh meal and ¢sh oil have been completely replaced with alternative sources of nutrients (Table 1).
The basal diets (Diet 1 and Diet 4, for Trial 1 and Trial
2, respectively) were based on diet formulations which
showed good growth and survival of this species in
previous studies (Davis & Arnold 2000; Samocha et al.
2004). Test-diets were formulated to meet all known
nutritional requirements for this species and had 35%
crude protein (CP) and 8% lipid levels. The HUFA were
provided either from ¢sh oil or from a meal made from
spray-dried cells of Schizochytrium sp. and Mortierella
s
s
sp. (DHA GOLD and AquaGrow -ARA, Advanced BioNutrition, Columbia, MD, USA), collectively referred to
as a HUFA-rich source, which are the products of proprietary fermentation processes. A commercial diet
(35% CP,8% lipid; Rangen, Buhl, ID, USA) was included
in each trial as a reference diet.
The ¢rst trial was conducted over a12-week period
using hand-sorted juvenile (6.07 Æ 0.3 g) shrimp.
Diet 1 (‘HUFA-rich’ diet) was formulated with the
HUFA from the spray-dried cells. Diet 2 (‘HUFA-de¢cient’diet) was formulated with no HUFA supplementation to determine the e¡ect of this de¢ciency on
shrimp performance. A product of co-extruded soybean and poultry by-product meal served as the protein source in Diet 1 and Diet 2 (ProfoundTM,
American Dehydrated Foods,Verona, MO, USA.).
The second trial was conducted over a 14-week
period using hand-sorted juveniles (0.95 Æ 0.04 g).
For this series of diets, the poultry by-product meal,
rather than the ProfoundTM, served as the primary
protein source. Diet 4 (‘HUFA-rich’ diet) was formulated with the same source of HUFA as that of Diet 1.
Diet 3 was prepared with the same ingredients as
those used for Diet 4 but the HUFAwas provided from

menhaden ¢sh oil (‘menhaden-rich’ diet). Diet 5
(‘HUFA-de¢cient’diet) was prepared with the same ingredients as those used for the formulation of Diet 3
and Diet 4 but without HUFA supplementation. To
determine whether dietary HUFA supplementation
is only required during the juvenile phase, a sixth
treatment was included and will be referred to as
‘Diet 6’, in which the shrimp were fed a ‘HUFA-rich’
diet (Diet 4) up to a size of 5 g and the ‘HUFA-de¢cient’ diet (Diet 5) from this size until the end of the
study. It is important to note that the performances
of the diets were compared within the same trial
and not between the trials.
The test diets were prepared in the feed laboratory
of Auburn University, Auburn, AL, USA, using stan-

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Aquaculture Research, 2010, 41, 961^967

Alternative HUFA source for Litopenaeus vannamei T M Samocha et al.

Table 1 Diet formulation (% as is basis) for practical diets designed to contain 35% protein and 8% lipid using various ¢sh
meal and ¢sh oil replacement strategies
Trial 1

ProfoundTMÃ
Poultry by-product mealw
Soybean mealz
Corn gluten, organic‰

Schizochytrium meal (DHA)z
s
AquaGrow -ARAz
Soy oilk
Flax oil (linseed oil)ÃÃ
Menhaden fish oilww
Wheat starchk
Whole wheatk
Trace Mineral premixzz
Vitamin premix‰‰
Stay C 250 mg/kgzz
CaP-diebasick
Lecethin (soy refined)k
Lecethin, organic crudekk
Cholesterolk

Trial 2

Diet 1

Diet 2

Diet 3

Diet 4

Diet 5

HUFA

w/o HUFA

MFO

HUFA

w/o HUFA

39.00

39.00

30.20

30.20

16.00
40.50
5.00

16.00
40.50
5.00

0.50
0.13
1.53
1.23

1.30

1.80

16.00
40.50
5.00
0.50
0.13
3.15
1.60

2.34
20.00
0.50
2.00
0.07
2.00

1.63
21.00
0.50
2.00
0.07
2.00

5.02
1.61
26.00
0.50
2.00
0.07

2.60
0.50

1.25
26.00
0.50
2.00
0.07
2.60
0.50

1.64
26.00
0.50
2.00
0.07
2.60
0.50

0.50

0.50
0.20

0.20

0.20

3.20
1.79

ÃCo-extruded soybean and poultry by-product meal (American Dehydrated Foods, Verona, MO, USA.).

wGri⁄n Industries (Cold Springs, KY, USA).
zDehulled solvent-extracted soybean meal, Southern States, Cooperative, Richmond, VA, USA.
‰Grain Processing, (Muscatine, IA, USA).
s
s
zDHA GOLD (Schizochytrium sp. algae meal) and AquaGrow -ARA (Mortierella sp.) (Advanced BioNutrition, Columbia, MD, USA).
kUnited States Biochemical (Cleveland, OH, USA).
ÃÃSigma (St. Louis, MO, USA).
wwOmega Protein (Reedville, VA, USA).
zzAs g100 g À 1 premix: cobalt chloride 0.004, cupric sulphate pentahyrate 0.250, ferrous sulphate 4.0, magnesium sulphate heptahydrate 28.398, manganous sulphate monohydrate 0.650, potassium iodide 0.067, sodium selenite 0.010, zinc sulphate heptahydrate 13.193,
¢ller 53.428.
‰‰g kg-1 premix: thiamine HCl 0.5, ribo£avin 3.0, pyrodoxine HCl 1.0, DL Ca-Pantothenate 5.0, nicotinic acid 5.0.
s
zzStay C (L -ascorbyl-2-polyphosphate 35% Active C) (Roche Vitamins, Parsippany, NJ, USA).
kkOrganic lecithin, Clarkson Grain (Cerro Gordo, IL, USA).
HUFA, highly unsaturated fatty acid; DHA, docosahexaenoic acid; ArA, arachidonic acid.

dard practices. Dry ingredients and oil were mixed in
a food mixer (Hobart, Troy, OH, USA) for 15 min. Hot
water was then blended into the mixture to attain a
consistency appropriate for pelleting. Each diet was
pressure pelleted using a meat grinder and a 2 mm
die. After pelleting, diets were dried to a moisture
content of 8^10% and stored at 4 1C. Dietary treatments were randomly assigned and the study was
run as a double-blind experiment.
Both trials were conducted in an outdoor tank system at the AgriLife Research Mariculture Laboratory
theTexas AgriLife Research and Extension Center, Corpus Christi,TX, USA. Each treatment was randomly assigned to ¢ve-replicate high-density polyethylene

circular tanks positioned under a shade with roo¢ng

made of clear and opaque panels. Each tank had a
working volume of 650 L and a bottom area of
0.85 m2. Tanks were covered with a net to prevent
shrimp from escaping. Aeration was provided by two
air stones per tank (7^10 L min À 1 stone À 1) that were
fed by a common regenerative air blower. Natural seawater was used after initial chlorination, de-chlorination by aeration and salinity was adjusted to
30 ppt. Tanks were stocked with 26 and 31 shrimp
to provide an initial stocking density of 31shri
mp m À 2 (40 shrimp m À 3) and 36 shrimp m À 2 (48
shrimp m À 3) for Trial 1 and Trial 2 respectively. Upon
reaching the 5 g size in Trial 2, ¢ve shrimp from each
tank were removed for sub-sampling. The study was
resumed, after the removal of these shrimp, assuming

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Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 961^967

963


Alternative HUFA source for Litopenaeus vannamei T M Samocha et al.

that each tank had 26 shrimp. However, the actual
number of shrimp in each tank after the culling was
not determined to minimize shrimp stress. One tank
in each treatment was provided with a feed tray, which
covered about 45% of the tank’s bottom area, to estimate feed consumption, and was considered to be the
treatment indicator tank. Five shrimp from each of the

indicator tanks were collected weekly to estimate
growth (group weights) and to adjust rations. Weekly
rations were calculated assuming 100% survival, feed
conversion ratio (FCR) of 1:1.5 and predicted weekly
growth that varied between 1.0 and1.2 g. Daily rations
were divided into four equal portions, which were fed
at 08:30, 11:30, 14:30 and 16:30, hours 7 days a week.
Both studies were conducted with no water exchange.
To o¡set evaporative losses and to prevent an increase
in salinity, chlorinated municipal freshwater was
added to each tank when needed. Physicochemical
parameters including pH, temperature, salinity and
dissolved oxygen were measured twice daily in all the
tanks. Total ammonium-nitrogen (NH3-NH4) and nitrite-nitrogen (NO2-N) were measured in every tank
once a week. On the day of termination, water samples
from each tank were analysed for total ammonium nitrogen (TAN), nitrite nitrogen (NO2-N), reactive phosphorus (RP) and 5-day biochemical oxygen demand
(cBOD5).
At the conclusion of each trial, shrimp were groupweighed and counted to provide the mean ¢nal
weight and survival for each tank. Feed conversion
ratio values were calculated based on feed inputs
and the biomass gain for each tank. Di¡erences in
the weekly and daily water quality indicators were
analysed using repeated measures ANOVA. Di¡erences
among treatments in TAN, NO2-N, RP and cBOD5 on
the day of termination were analysed using one-way
ANOVA. The same statistical test was used to determine di¡erences between treatment means
(Po0.05) in the ¢nal mean weight, survival and FCR
values. Statistical analyses were performed only on

Aquaculture Research, 2010, 41, 961^967

the test diets; the data for the reference diet were provided for informational purposes. The Student^Newman^Keuls (SNK) test was used as a tool to identify
the di¡erence between treatment means. Square root
transformation of per cent survival data was also
evaluated but did not a¡ect the interpretation of the
results; hence, it is not presented. Statistical analyses
were conducted using SPSS (V. 13 for Windows, SPSS,
Chicago, IL, USA).

Results and discussion
No statistically signi¢cant di¡erences were found between treatments for both trials in the daily or the
weekly water quality indicators (Tables 2 and 3).
These daily values represent acceptable ranges reported for good growth and survival of penaeid
shrimp and are typical for this system. It is interesting to note that although there were no signi¢cant
di¡erences in the weekly water quality indicators between treatments, on Day 56 of the study, one of the
tanks experienced a short exposure (for about a
week) to a TAN level of 1.94 mg L À 1 and an NO2-N level as high as 7.3 mg L À 1. The fact that there was no
signi¢cant di¡erence in the mean shrimp ¢nal
weight within treatment tanks, along with the high
survival in this tank (89%), suggests that these levels
had no adverse e¡ect on the shrimp in this study.
Shrimp survival rates in both trials were high and
typical for this research system. Statistical analyses
of the data from Trial l indicated signi¢cant di¡erences in shrimp ¢nal mean weights, with no signi¢cant di¡erences in shrimp survival and FCR values
(Table 4). The lowest survival value (93.5%)
was found for shrimp maintained on the commercial
reference diet, which was lower than the 97.5^
100% observed for the test diets. A signi¢cant reduction was observed in the ¢nal weights between
shrimp reared on the ‘HUFA-rich’diet (Diet 1) and the

Table 2 Summary of the daily water quality indicators (Mean Æ SD1 and range) from the growth trials conducted in outdoor
tanks with Litopenaeus vannamei

Trial 1
Trial 2

964

Dissolved oxygen
(mg L À 1)

Temperature ( 1C)

a.m.

p.m.

a.m.

p.m.

a.m.

p.m.

Salinity (g L À 1)

6.5 Æ 0.4
(5.6–7.6)
7.0 Æ 0.5

(6.0–9.3)

6.5 Æ 0.5
(5.1–7.8)
6.8 Æ 0.4
(5.8–7.5)

27.2 Æ 0.9
(24.3–28.6)
27.4 Æ 0.8
(25.7–29.1)

28.7 Æ 1.2
(24.7–30.52)
28.9 Æ 0.9
(26.6–31.3)

7.7 Æ 0.3
(6.4–8.4)
7.8 Æ 0.3
(7.3–8.3)

7.9 Æ 0.2
(7.4–8.2)
8.1 Æ 0.2
(7.5–8.6)

32 Æ 1
(27–36)
22 Æ 1

(18–26)

pH

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Aquaculture Research, 2010, 41, 961^967

Alternative HUFA source for Litopenaeus vannamei T M Samocha et al.

‘HUFA-de¢cient’diet, indicating a possible de¢ciency
of HUFAs.
The second growth trial was initiated with smaller
shrimp (0.6 vs. 6 g shrimp), allowing for more tissue
replacement and the evaluation of phased feeding of
the HUFA diets. As in the ¢rst trial, there were no signi¢cant di¡erences in survival between shrimp fed
Table 3 Summary (mean Æ SDÃ) of the mean weekly
water quality parameters (mg L À 1) recorded during the
growth trials conducted in outdoor tanks with Litopenaeus
vannamei

Trial 1
Diet 1
Diet 2
Reference
P value
Trial 2
Diet 3

Diet 4
Diet 5
Diet 6z
Reference
P value

TAN (mg L À 1)

NO2-Nw (mg L À 1)

0.11 Æ 0.19
0.16 Æ 0.20
0.13 Æ 0.21
0.18

1.19 Æ 1.57
1.19 Æ 0.54
0.49 Æ 0.7
0.07

0.13
0.10
0.12
0.11
0.13
0.93

Æ
Æ
Æ

Æ
Æ

0.43
0.34
0.37
0.38
0.38

0.67
0.34
0.87
0.50
0.47
0.42

Æ
Æ
Æ
Æ
Æ

1.08
0.75
1.41
0.79
0.87

ÃStandard deviation.
wNitrite-nitrogen.

zShrimp were fed Diet 4 up to the 5 g size. Beyond this size
shrimp were switched to Diet 5.
TAN, total ammonium nitrogen.

the di¡erent diets in Trial 2. Signi¢cant di¡erences
were observed between treatments in terms of the ¢nal shrimp weights and FCR. There were no di¡erences in the ¢nal weights between the ‘HUFA-rich’
diet (Diet 4), the ‘menhaden-rich’diet (Diet 3), as well
as the commercial reference diet. The ¢nal weights of
shrimp fed the ¢sh oil diet were signi¢cantly higher
than those fed the two diets without the HUFA supplements (Diet 5 and Diet 6). Shrimp reared on the
‘HUFA-rich’ (Diet 4) were not signi¢cantly di¡erent
but numerically larger than those reared on these
HUFA-de¢cient diets (Diet 5 and Diet 6). Shrimp
maintained on the ‘menhaden-rich’ diet (Diet 3)
showed a better FCR than those maintained on the
HUFA-de¢cient diet (Diet 5).
In both growth trials, the ¢nal weight, survival
and FCR values of shrimp receiving diets with HUFA
supplements were similar to those observed for the
commercial diets. Furthermore, there were no indications of feed rejection, with all diets readily consumed. These results demonstrate that marine
ingredients can be removed from practical diets for
shrimp reared in outdoor tanks with no water exchange in the presence of natural productivity. The
reduced growth of shrimp reared on diets without
HUFA supplements would indicate that the supplementation of HUFAs is a critical component of the replacement strategy. While complete replacement of
¢sh meal has been successful in the production diets

Table 4 Final weights, survival rates, feed conversion ratio (FCR) for Litopenaeus vannamei juveniles reared in outdoor tanks
and o¡ered the test diets

Trial 1Ã

Diet 1 (HUFA)
Diet 2 (w/o HUFA)
PSEw
P value
Reference diet
Trial 2z
Diet 3 (MFO)
Diet 4 (HUFA)
Diet 5 (w/o HUFA)
‘Diet 6’ (Diet 41Diet 5)‰
PSE
P value
Reference diet

Mean final weight (g)

Survival (%)

FCR

17.4a
16.4b
0.385
0.0206
18.0

97.5
100
2.28
0.5161

93.5

1.20
1.23
0.041
0.5844
1.16

16.4a
15.7ab
14.6b
14.0b
0.478
0.0143
16.7

98.1
97.7
92.3
98.1
2.119
0.1637
91.0

1.28b
1.34ab
1.53a
1.44ab
0.061
0.0465

1.36

Based on Student^Newman^Keuls mean separation, treatment means with the same superscript letters are not signi¢cantly di¡erent.
ÃData represent the mean of ¢ve replicates. Shrimp had a mean initial weight of 6.07 Æ 0.3 g.
wPooled standard error.
zData represent the mean of ¢ve replicates. Two tanks being o¡ered Diet 3 and Reference diet, have been excluded from the data set due
to discrepancy from human error. The shrimp had a mean initial weight of 0.95 Æ 0.04 g.
‰Shrimp were fed Diet 4 to 5 g size and Diet 5 until the harvest.

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965


Alternative HUFA source for Litopenaeus vannamei T M Samocha et al.

of various ¢sh such as cat¢sh and tilapia (Webster &
Lim 2002) and crustaceans such as Macrobrachium
rosenbergii (Tidwell et al. 1993), the replacement of
marine protein and oil ingredients in practical diets
for L. vannamei is still under development. Earlier studies have reported partial substitution of ¢sh meal
using a solvent-extracted soybean meal (Lim &
Dominy 1990) and a solvent-extracted cotton seed
meal in a diet formulation for L. vannamei (Lim
1996). Similarly, studies with L. vannamei using ProfoundTM as a partial substitute for ¢sh meal have
shown encouraging results: Davis and Arnold
(2000) demonstrated that 80% of the ¢sh meal in a
diet could be substituted by this product without an
apparent negative e¡ect on shrimp survival or

growth. Samocha et al. (2004) also reported a complete replacement of the ¢sh meal by ProfoundTM,
with no apparent palatability problems or negative
impact on shrimp performance. In a more recent
study, Patnaik et al. (2006) showed that both ¢sh
meal and ¢sh oil can be completely replaced by a
s
commercial fermentation product (DHA GOLD and
s
AquaGrow -ARA as HUFA source) without impairing shrimp performance. Furthermore, Browdy, Seaborn, Atwood, Davis, Bullis, Samocha, Wirth and
Le¥er (2006), in an outdoor pond study at the Waddell Mariculture Center, Blu¡ton, SC, USA, showed
no signi¢cant di¡erences in the production parameters between shrimp that were fed an all-plantbased diet with a HUFA supplement and those fed a
conventional ¢sh meal-based diet.
The importance of ¢sh oil in the aquaculture diets
has already been well documented. Fish oil is the major source of essential fatty acids such as eicosapentaenoic acid, The DHA and arachidonic acid (ArA).
Researchers have attempted to substitute ¢sh oil with
various types of vegetable oils. The use of vegetable
oil in feeds without marine oil sources is often limited by the potential problems associated with insuf¢cient levels of essential fatty acids (Gonzalez-Felix,
Gatlin III, Lawrence & Perez-Velazquez 2002). Docosahexaenoic acid- and ArA-rich products created by
fermentation process have been used successfully in
the past to enrich live larval food or in maturation
diets of many aquatic species (Barclay & Zeller 1996).
The oil from the Schizochytrium sp. has as high as
50% DHA (Barclay & Zeller 1996) and can serve as a
potential candidate for replacement of conventional
sources for marine HUFA. In the present study, we
have demonstrated a successful 100% replacement
of ¢sh meal by poultry meal in a practical shrimp
diet. Furthermore, similar to Patnaik et al.’s (2006)

966

Aquaculture Research, 2010, 41, 961^967

results, we were able to completely replace the marine oil ingredient using a combination of plant oils
supplemented with fermentation products rich in
HUFA as a source for the essential fatty acids.
As demonstrated in a previous research and con¢rmed by these trials, both ProfoundTM and the poultry by-product meal can serve as primary sources for
protein and essential amino acids in practical diets of
the Paci¢c white shrimp. The complete replacement
of ¢sh meal and ¢sh oil using non-marine ingredients
can be accomplished using plant oils supplemented
with fermentation products as the HUFA source. The
use of a heterotrophically produced non-marine
HUFA-rich product as the lipid source in feed is a recent concept in practical diet formulations for shrimp
and is still in a preliminary stage of research. Additional research on the e¡ect of these ingredients on
di¡erent life stages of L. vannamei under various environmental conditions would be bene¢cial to ¢sh oil
and ¢sh meal replacement e¡orts. And, ¢nally, more
studies are needed to determine the economic viability of the large-scale use of these components in
shrimp feed formulations.

Acknowledgments
This research was supported by funding from Advanced BioNutrition, Columbia, MD, USA. The
authors would like to thank the students and the employees of AgriLife Research Mariculture Laboratory,
Corpus Christi, TX, USA for their help. Mention of a
trademark or a proprietary product does not constitute an endorsement of the products.

References
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and n-6 fatty acids in rotifers and Artemia nauplii by feeding spray dried Schizochytrium sp. Journal of the World
Aquaculture Society 27, 314^322.

Browdy C.L., Seaborn S., Atwood H., Davis D.A., Bullis R.A.,
Samocha T.M., Wirth E. & Le¥er J.W. (2006) Comparison
of pond production e⁄ciency, fatty acid pro¢les, and
contaminants in Litopenaues vannamei fed organic plantbased and ¢sh meal-based diets. Journal of the World
Aquaculture Society 37, 437^451.
Chamberlain G.W. (1993) Aquaculture trends and feed projections.World Aquaculture 24, 19^29.
Davis D.A. & Arnold C.R. (2000) Replacement of ¢sh meal in
practical diets for the Paci¢c white shrimp Litopenaeus
vannamei. Aquaculture 185, 291^298.

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Eusebio P. & Coloso R.M. (1998) Evaluation of leguminous
seed meals and leaf meals as plant protein sources in diets
for juvenile Penaeus indicus. Israeli Journal of Aquaculture ^
Bamidgeh 50, 47^54.
Forster I.P., DominyW., Obaldo L. & Tacon A.G.J. (2003) Rendered meat and bone meals as ingredients of diets for
shrimp Litopenaeus vannamei (Boone, 1931). Aquaculture
219, 655^670.
Francis G., Makkar H.P.S. & Becker K. (2001) Antinutritional
factors present in plant derived alternate ¢sh feed ingredients and their e¡ects in ¢sh. Aquaculture 199, 197^227.
Gonzalez-Felix M.L., Gatlin D.M. III, Lawrence A.L. & PerezVelazquez M. (2002) E¡ect of various dietary lipid levels on
quantitative essential fatty acid requirements of juvenile
Paci¢c white shrimp Litopenaeus vannamei. Journal of

World Aquaculture Society 33, 330^340.
Harel M., KovenW., Lein I., BarY., Behrens P., Stubble¢eld J.,
ZoharY. & Place A.R. (2002) Advanced DHA, EPA and ArA
enrichment materials for marine aquaculture using single cell heterotrophs. Aquaculture 213, 347^362.
Hertrampf J.W. & Piedad-Pascual F. (2000) Handbook on Ingredients forAquaculture Feeds. Kluwer Academic Publishers, Dordrecht, the Netherlands.
Li M.H., Robinson E.H. & Hardy R.W. (2000) Protein sources for
feed. In: Encyclopedia of Aquaculture (ed. by R.R. Stickney),
pp. 688^695. John Wiley and Sons, NewYork, NY, USA.
Lim C. (1996) Substitution of cottonseed meal for marine animal protein in diets for Penaeus vannamei. Journal of the
World Aquaculture Society 27, 402^409.
Lim C. & Dominy W. (1990) Evaluation of soybean meal as a
replacement for marine animal protein in diet for shrimp
Penaeus vannamei. Aquaculture 87, 53^64.
Menoyo D., Lopez-Bote C.J., Obach A. & Bautista J.M. (2005)
E¡ect of dietary ¢sh oil substitution with linseed oil on the
performance, tissue fatty acid pro¢le, metabolism, and
oxidative stability of Atlantic salmon. Journal of Animal
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Naylor R.L., Goldberg R.J., Primavera J.H., Kautsky N., Beveridge M.C., Clay J., Folk C., Lubchenco J., Mooney H. &
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Olvera-Novoa M.A. & Olivera-Castillo L. (2000) Potencialidad del uso de las leguminosas como fuente proteica en
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Patnaik S., Samocha T.M., Davis D.A., Bullis R.A. & Browdy
C.L. (2006) The use of algal meals as highly unsaturated
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Samocha T.M., Davis D.A., Saoud I.P. & DeBault K.
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poultry by-product meal in practical diets for the Paci¢c
white shrimp, Litopenaeus vannamei. Aquaculture 231,
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and complete replacement of ¢shmeal by soybean meal
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Aquaculture Research, 2010, 41, 968^972

doi:10.1111/j.1365-2109.2009.02379.x

Initial influence of fertilizer nitrogen types on
water quality
Charles C Mischke1 & Paul V Zimba2
1

National Warmwater Aquaculture Center, Mississippi State University, Stoneville, MS, USA
US Department of Agriculture, Agriculture Research Service, National Warmwater Aquaculture Center, Stoneville, MS, USA

2

Correspondence: C C Mischke, National Warmwater Aquaculture Center, Mississippi State University, 127 Experiment Station Road,
PO Box 197, Stoneville, MS 38776, USA. E-mail:

Abstract
Using di¡erent sources of nitrogen as fertilizers in nursery ponds may a¡ect water quality and plankton responses. We evaluated water quality variables and
plankton population responses when using di¡erent
nitrogen sources for cat¢sh nursery pond fertilization.

We compared calcium nitrate (12% N), sodium nitrite
(20% N), ammonium chloride (26% N), ammonium nitrate (34% N) and urea (45% N) in190-L microcosms at
equimolar nitrogen application rates. Sodium nitritefertilized microcosms had higher nitrite and nitrate levels during the ¢rst week; no other di¡erences in the
water quality were detected among fertilizer types
(P40.05). No di¡erences in green algae, diatoms or cyanobacteria were detected among treatments; desirable
zooplankton for cat¢sh culture was increased in ureafertilized microcosms. Based on these results, any form
of nitrogen used for pond fertilization should perform
similarly without causing substantial water quality deterioration. Ammonium nitrate and urea contain a
higher percentage of nitrogen, requiring less volume
to achieve dosing levels. If both urea and ammonium
nitrate are available, we recommend using the one with
the least cost per unit of nitrogen. If both types of fertilizer have an equal cost per unit of nitrogen, we recommend using urea because of the potential advantage of
increasing desirable zooplankton concentrations.

Keywords: nitrogen fertilizer, channel cat¢sh fry,
plankton, water quality

Introduction
Fertilizing nursery ponds is common practice among all
cultured species of ¢sh. The primary aim of fertilization

968

is to increase dissolved inorganic nutrient concentrations in the pond water. The increased nutrients are
then incorporated into biomass (algal and zooplankton)
and ultimately incorporated into ¢sh biomass. However,
complicated interactions (climate, water and bottom
soil characteristics and pond morphology) can a¡ect
how ponds respond to nutrient additions (Knud-Hansen 1998). Additionally, management practices associated with di¡erent species (e.g., feeding and stocking
rates) may a¡ect fertilization responses.

The production of channel cat¢sh Ictalurus punctatus fry and ¢ngerlings is unique and utilizes di¡erent
techniques compared with other types of aquaculture. Cat¢sh fry are stocked at relatively high densities (40 000^120 000 ha À 1) into newly ¢lled earthen
ponds (¢lled with well water 3^4 weeks before stocking). Prepared diets are o¡ered to the fry immediately
after stocking. Zooplankton populations are important in cat¢sh fry culture during the ¢rst 3^4 weeks,
but diminish in importance as fry grow and seek the
prepared diets (Mischke, Wise & Lane 2003). For cat¢sh fry culture, it does not appear to be necessary to
have long, sustained populations of zooplankton after
the initial weeks post stocking. Therefore, the primary
goal of fertilizing cat¢sh fry ponds is to establish a
phytoplankton bloom as quickly as possible to shade
macrophytes and produce large stocks of copepods
and cladocerans for fry consumption during the ¢rst
3^4 weeks after stocking (Mischke et al. 2003; Mischke & Zimba 2004).
When high-nitrogen fertilizers are applied rather
than high-phosphorus fertilizers, the phytoplankton
population is shifted to desirable algal groups, thus
providing a quick algal bloom and adequate forage
for cladocerans without the use of organic fertilizers
(Mischke & Zimba 2004).

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Aquaculture Research, 2010, 41, 968^972

Fertilizer nitrogen types and water quality C C Mischke & P V Zimba

Primary nitrogen sources in pond fertilizers can be
from urea, ammonium salts, nitrite or nitrate. Using

di¡erent sources of nitrogen as fertilizers in nursery
ponds may a¡ect the water quality and plankton responses. Liang, Beardall and Heraud (2006) reported
that nitrogen source had no e¡ect on two species of
marine diatoms grown in photobioreactors. Paasche
(1971) reported that the green algae Dunaliella tertiolecta grew 10^30% faster on ammonia versus nitrate.
Lourenco, Barbarino, Mancin-Filho, Schinke and Aidar (2002) assessed the growth of 10 algal species on
nitrogen and concluded that algal responses were
not uniform within the same taxonomic group
(among diatoms and green algae). The purpose of this
study was to evaluate water quality variables and
plankton population responses when using di¡erent
nitrogen sources for nursery pond fertilization.

Materials and methods
Twenty-¢ve bottomless mesocosms (200-L capacity)
were placed in a newly ¢lled 0.4-ha nursery pond
(depth 0.75 m) and held in place with steel posts. Mesocosms were placed in the pond on 19 May 2008 at
an equal depth, providing 190 L of water in each container. Mesocosms were slowly lowered into position
to allow water to ¢ll the mesocosms without disturbing the pond sediments. Treatments were randomly
assigned to each mesocosm; there were ¢ve replications of each treatment.
All mesocosms were fertilized on an equal nitrogen basis based on the nitrogen fertilization recommendations of Mischke and Zimba (2004), with the
¢rst application providing 2.2 mg N L À 1, followed by
two applications per week of 1.1mg N L À 1 for 3
weeks. The di¡erent nitrogen treatments used were:
calcium nitrate (12% N), sodium nitrite (20% N), ammonium chloride (26% N), ammonium nitrate (34%
N) and urea (45% N). All fertilizers were granular
and dissolved in water before application to the mesocosms.
Water samples were collected from each mesocosm with a tube sampler (modi¢ed from Graves &
Morrow 1998), which is designed to sample the entire
water column. The samples were taken 24 h after a

fertilization treatment between 07:00 and 08:00
hours.Water samples were transported to the laboratory and immediately analysed for pH, soluble reactive phosphorus (ascorbic acid method), total
ammonia^nitrogen (Nesslerization), nitrite^nitrogen (diazotization) and nitrate^nitrogen (cadmium

reduction, followed by diazotization) using the methods outlined by HACH (1999).
Pigment analysis was used to assess phytoplankton community composition using the HPLC methodology (Zimba, Dionigi & Millie 1999). Known
volumes of mesocosm water were ¢ltered (GF/C
¢lters, Whatman, Maidestone, UK) under reduced
pressure in the dark. Filters were immediately frozen
and held at À 80 1C until analysis. Filters were extracted in 100% acetone for 24 h, clari¢ed by syringe
¢ltration before ampulation and HPLC analysis. Pigments (carotenoids and chlorophylls) were quanti¢ed
using an HP1100 equipped with diode array and
£uorescence detectors (Agilent Technologies, Palo
Alto, CA, USA). Identi¢cation of speci¢c divisions of
algae is possible using taxon-speci¢c pigment biomarkers (Zimba, Tucker, Mischke & Grimm 2002).
A pigment library was used to identify samples; unknown samples were quanti¢ed by linear regression
of known commercial standards.
Separate tube samples of 3.8 L were collected for
zooplankton using the same schedule and methods
as those described for water quality sampling. The
collected sample was then concentrated by pouring
through a 63-mm mesh net. A 63-mm mesh net was
used to ensure capture of smaller rotifers. Samples
were preserved in bu¡ered formalin solution before
counting by light microscopy (Geiger & Turner
1990). All organisms in 1ml subsamples from each
mesocosm were counted using a Sedgwick^Rafter
counting cell as described by Geiger and Turner
(1990) and zooplankton were identi¢ed using the
taxonomic keys of Thorp and Covich (1991).

The experimental design was completely randomized, with repeated measures taken on replicate
mesocosms. Data were analysed using the MIXED
procedure in SAS Version 8.02 software (SAS Institute,
Cary, NC, USA) (Littell, Milliken, Stroup & Wol¢nger
1996). The covariance structure, autoregressive of
order 1, was used in the repeated measure model.
Mean comparisons were made using an LSD test
with a signi¢cance level of Po0.05.

Results
Dissolved soluble reactive phosphorus, ammonia nitrogen and pH were not a¡ected by nitrogen source.
There was a signi¢cant interaction among the main
e¡ects of date by treatment with nitrate (Fig. 1) and
nitrite (Fig. 2). Sodium nitrate-fertilized ponds had
higher concentrations of both nitrate and nitrite rela-

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969


Fertilizer nitrogen types and water quality C C Mischke & P V Zimba

Aquaculture Research, 2010, 41, 968^972

3.00

Ammonium chloride
Ammonium nitrate

2.50
Nitrate (mg N L-1)

Calcium nitrate
2.00

Sodium nitrite
Urea

1.50
1.00
0.50
0.00
5/20/08

5/23/08

5/27/08

6/3/08

6/10/08

Figure 1 Dissolved nitrate concentration (mg N L À 1) in mesocosms treated with various nitrogen sources from 20 May
2008 to 10 June 2008. Symbols represent means Æ SE.

1.2

Ammonium chloride

Ammonium nitrate

1.0
Nitrite (mg N L-1)

Calcium nitrate
0.8

Sodium nitrite
Urea

0.6
0.4
0.2
0.0
5/20/08

5/23/08

5/27/08

6/ 3/08

6/10/08

Figure 2 Dissolved nitrite concentration (mg N L À 1) in mesocosms treated with various nitrogen sources from 20 May
2008 to 10 June 2008. Symbols represent means Æ SE.

tive to the other treatments during the ¢rst week of
sampling, but returned to similar levels for the remainder of the study.

Green algae, diatoms and cyanobacteria were present in all mesocosms; however, analysis of chlorophyll a, chlorophyll b and b-carotene, zeaxanthin
and fucoxanthin showed no signi¢cant di¡erences
among the various nitrogen sources used. Individual
zooplankton groups were not signi¢cantly di¡erent
among treatments; however, desirable zooplankton
for cat¢sh fry culture (i.e., the sum of adult copepods,
cladocerans and ostracods) (Mischke et al. 2003) did
show a signi¢cant interaction among the main effects of date by treatment (Fig. 3). Mesocosms treated

970

with calcium nitrate tended to show a more rapid increase in the desirable zooplankton concentrations at
the beginning of sampling, and urea-fertilized mesocosms showed an increase in desirable zooplankton
concentrations at the end of sampling.

Discussion
The choice of nitrogen type to use as a pond fertilizer
would depend on the e¡ectiveness of the fertilizer to
increase desirable zooplankton and phytoplankton
concentrations in the pond, minimal deleterious effects on water quality (e.g., changes in ammonia and

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Aquaculture Research, 2010, 41, 968^972

Fertilizer nitrogen types and water quality C C Mischke & P V Zimba

Ammonium chloride

400

Desirable Zooplankton (Number L-1)

Ammonium nitrate
350

Calcium nitrate

300

Sodium nitrite

250

Urea

200
150
100
50
0
5/20/08

5/23/08

5/27/08

6/3/08

6/10/08

Figure 3 Desirable zooplankton [i.e., adult copepods, cladocerans and ostracods (number L À 1)] in mesocosms treated
with various nitrogen sources from 20 May 2008 to 10 June 2008. Symbols represent means Æ SE.

nitrite concentrations), cost per unit nitrogen and
local availability. At the nitrogen fertilization rate
and the time frame used in this study, it appears that
the nitrogen source does not in£uence chlorophyll a,
b or b-carotene. However, urea-fertilized microcosms
showed increased desirable zooplankton concentrations at the end of the study. Generally, cat¢sh nursery ponds are ¢lled and fertilized for about 3 weeks
before fry are stocked. Therefore, urea may have an
advantage over the other nitrogen fertilizers, providing higher desirable zooplankton concentrations at
the time of stocking.
Although we expected ammonia concentrations to
be higher in microcosms treated with ammoniabased fertilizers (ammonium chloride, ammonium
nitrate and urea), nitrite levels to be higher in nitrite-fertilized microcosms (sodium nitrite) and nitrate to be higher in nitrate-fertilized microcosms
(ammonium nitrate and calcium nitrate), this expected outcome did not occur. The only di¡erences
in dissolved inorganic nitrogen concentrations
among treatments occurred in the sodium nitritefertilized microcosms; this fertilizer resulted in increased nitrite and nitrate levels for the ¢rst week of
the study, but then returned to concentrations similar to other fertilizer treatments. This suggests that
algal utilization or absorption to sediments reduced
nitrogen concentrations more rapidly than previously observed in pond-scale fertilization (Mischke
& Zimba 2004).

Ammonia nitrogen is the preferred source of nitrogen for phytoplankton according to Tepe and Boyd
(2001); however, other evidence suggests that nitrogen utilization is species speci¢c (Syrett1981; Lourenco et al. 2002). In the current study, there were no
peaks in dissolved ammonia concentrations when
microcosms were fertilized with ammonia-based fertilizers. We assume that because nitrogen is limiting
in these ponds (Mischke & Zimba 2004), there was rapid uptake of dissolved nitrogen by the phytoplankton. Although water quality was similar by the end

of the study, using a nitrite fertilizer did cause nitrite
levels to increase slightly during the ¢rst week.
Therefore, nitrite fertilizers may be less desirable for
use in nursery ponds relative to the other nitrogen
sources.
Ammonium fertilizers are cheaper than nitrate
fertilizers per unit of nitrogen (Boyd & Tucker 1998).
Urea and ammonium nitrate are generally similar in
cost per unit of nitrogen; however, ammonium nitrate can be more di⁄cult to obtain and may require
extensive record keeping because of its potential use
in explosives.
Based on the results of this study, any form of nitrogen used for pond fertilization should perform similarly in the short term without causing substantial
water quality deterioration. Typical cat¢sh nursery
ponds are only fertilized for a short time until fry are
stocked (after 2^3 weeks of fertilization) or fry are
readily feeding at the pond surface (usually 3^4

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Fertilizer nitrogen types and water quality C C Mischke & P V Zimba

weeks after stocking). Additional studies would be
needed to determine the longer-term e¡ects of these
fertilizers for situations requiring longer-term fertilization practices. Ammonium nitrate and urea contain a higher percentage of nitrogen than other
nitrogen fertilizers, and so a smaller amount of fertilizer would have to be added to the ponds. Urea is
usually readily available, and may increase the desirable zooplankton concentrations for cat¢sh culture.

If both urea and ammonium nitrate are available,
we would recommend using the one with the least
cost per unit of nitrogen. In 2009, urea could be purchased from a local dealer (Greenville, MS, USA) for
US$17.50/50 lb bag (78b/lb N), and ammonium
nitrate could be purchased for US$14.75/50 lb bag
(87b/lb N). If both types of fertilizers have an equal
cost per unit of nitrogen, we recommend using urea
because of the potential advantage of increasing desirable zooplankton concentrations.

Acknowledgment
This is article J-11584 of Mississippi State University,
Mississippi Agricultural and Forestry Experiment
Station. Mention of a trade name, proprietary product
or speci¢c equipment does not constitute a guarantee
or a warranty by the US Department of Agriculture
and does not imply approval of the product to the exclusion of others that may be available.

References
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Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 968^972


Aquaculture Research, 2010, 41, 973^981

doi:10.1111/j.1365-2109.2009.02380.x

The antioxidant capacity response to hypoxia stress
during transportation of characins (Hyphessobrycon

callistus Boulenger) fed diets supplemented with
carotenoids
Chih-Hung Pan1,Yew-Hu Chien2 & Yi-Juan Wang2
1

Department of Aquaculture, National Kaohsiung Marine University,Taiwan, Republic of China

2

Department of Aquaculture, National Taiwan Ocean University,Taiwan, Republic of China

Correspondence: Y-H Chien, Department of Aquaculture, National Taiwan Ocean University, Keelung 202, Taiwan, Republic of China.

E-mail:

Abstract

Introduction

This study aimed to determine whether dietary carotenoid (CD) supplements could a¡ect the antioxidant
capacity of characins Hyphessobrycon callistus upon
hypoxia stress at live transportation. Two types of
CD [astaxanthin (AX), b-carotene (BC)] and their 1:1
combination (MX) at three concentrations (10, 20
and 40 mg kg À 1) were supplemented, resulting in
nine CD diets. After 8 weeks’ rearing, the resulting
¢sh were divided into two subgroups and exposed to
hypoxia or normoxia. Hypoxia involved a gradual
decrease in dissolved oxygen (DO) from 6.5 to
o1.0 mg L À 1. Normoxia was DO kept in saturation.
Hypoxia led to an increase in the total antioxidant
status (TAS), superoxide dismutase (SOD), glutathione peroxidases (GPx) and aspartate aminotransferase (AST) activity of blood serum in ¢sh, but
had no e¡ect on alanine aminotransferase (ALT).
Under hypoxia, ¢sh fed CD diets had lower SOD, GPx
and ALT activity than control ¢sh, showing that dietary CD could increase the antioxidant capacity and
protection of the liver. Dietary AX was more e¡ective
for antioxidant capacity than BC and MX when under hypoxia stress, because GPx, ALT and AST were
lower in AX-fed ¢sh. Except TAS, the other four enzyme activities showed decreasing trends with increasing dietary CD concentrations.

Transport of live aquatic animals, especially during
their larval and fry stages, is a common procedure in
aquaculture practice. In ornamental ¢sh trade,
transport of juvenile or adult ¢sh is characterized by

a long duration and small packaging.Various kinds of
acute and chronic stress, such as £uctuation in water
temperature, dissolved oxygen (DO), ammonia, pH
and sometimes salinity, caused by handling, packaging, shipping, releasing and stocking (Taylor & Solomon 1979), are inevitable and result in unpredictable
mortality and loss.
When an organism is subjected to stress, a sudden
shortage of oxygen causes abnormal oxidative reactions in the aerobic metabolic pathways, resulting in
the formation of excessive amounts of singlet oxygen
(Ranby & Rabek1978) and the subsequent generation
of radicals or reactive oxygen species (ROS).
Reactive oxygen species can impair lipids, proteins,
carbohydrates and nucleotides (Yu 1994), which are
important parts of cellular constituents, including
membranes, enzymes and DNA. Radical damage
can be signi¢cant because it can proceed as a chain
reaction.
Ornamental ¢sh pigment is one of the most important quality criteria dictating their market value.Various synthetic [b-carotene (BC), canthaxanthin,
zeaxanthin and astaxanthin (AX)] and natural
(yeast, bacteria, algae, higher plants and crustacean
meal) carotenoids (CD) have been used as dietary
supplements to enhance pigmentation of ¢sh and

Keywords: antioxidant capacity, astaxanthin,
b-carotene, Hyphessobrycon callistus, hypoxia,
superoxide dismutase

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Journal Compilation r 2009 Blackwell Publishing Ltd

973

Antioxidant capacity response to hypoxia of characins C-H Pan et al.

crustaceans (Shahidi, Metusalach & Brown 1998; Kalinowski, Robaina, Fernandez-Palacios, Schuchardt &
Izquierdo 2005). Dietary AX supplementation of penaeid postlarvae increased not only bodyAX but also
enhanced the resistance to hypoxia (Chien, Chen,
Pan & Kurmaly 1999), salinity (Darachai, Piyatiratitivorakul, Kittakoop, Nitithamyong & Menasveta 1998;
Merchie, Kontara, Lavens, Robles, Kurmaly & Sorgeloos 1998; Chien, Pan & Hunter 2003), thermal
(Chien et al. 2003), ammonia (Pan, Chien & Hunter
2003a) and pathological stressors (Pan Chien & Hunter 2003b). Among the functions of AX in aquaculture as proposed by Torrissen and Christiansen
(1995) and Shimidzu, Goto and Miki (1996), antioxidant properties may be closely associated with stress
resistance. The reason for the development of resistance to stress from CDs can be attributed to the antioxidant role that CDs play to inactivate the free
radicals produced from normal cellular activity and
various stressors so that the oxidative damage is
eliminated (Halliwell & Gutteridge 1989; Chew 1995).
Pigmentation e⁄ciency of BC and AX can vary with
the animal species (Meyers & Chen 1982), and so can
their antioxidant capacity. b-carotene is recognized
as a lipid antioxidant, i.e. a free radical trap and
quencher of singlet oxygen (Bohm, Edge, Land,
McGarvey & Truscott 1997). Astaxanthin contains a
long conjugated double-bond system with relatively
unstable electron orbitals; it may scavenge oxygen radicals in cells (Stanier, Kunizawa & Cohen-Bazire
1971). The antioxidant activity of AX was found to be
approximately 10 Â stronger than BC (Shimidzu
et al. 1996).
In recent years, some enzymes involved in protection against active oxygen species, which are caused
by abnormal oxidative reactions at stress (Ranby &
Rabek 1978), have often been used to measure the response to stress. Total antioxidant status (TAS) is an

overall indicator of antioxidant defence against free
radical. Superoxide dismutase (SOD), a cytosolic enzyme that is speci¢c for scavenging superoxide radicals, is involved in protective mechanisms within
tissue injury following oxidative process and phagocytosis. Oxidative stress was successfully assayed by
TAS and SOD (Seymen, Seven, Civelek, Yigit, Hatemi
& Burcak 1999; O’Brien, Slaughter, Swain, Birmingham, Greenhill, Elcock & Bugelski 2000). Glutathione peroxidase (GPx) is involved in the reaction
of removal of H2O2 and is recognized as one of the
most important antioxidant defences against oxygen
toxicity in organisms (Kappus & Sies 1981; Cohen &
Doherty 1987). Superoxide dismutase and GPx,

974

Aquaculture Research, 2010, 41, 973^981

which are involved in protection against active oxygen species, are considered to be potential tools for
identi¢cation of stress caused by environmental factors (Roche & Boge 1996). Aspartate aminotransferase (AST) (or glutamate oxalate transaminase) and
alanine aminotransferase (ALT) (or glutamate pyruvate transaminase) are usually used as general indicators of the functioning of vertebrate liver. High AST
and ALT generally, but not de¢nitively, indicate the
weakening or damage of normal liver function. Alanine aminotransferase and AST were often used as
markers of hepatocellular injury (Seymen et al. 1999;
O’Brien et al. 2000; Suzumura, Hashimura, Kubota,
Ohmiza & Suzuki 2000).
Characins, Hyphessobrycon callistus (Boulenger), is
one of the most important cultured and exported ornamental ¢sh in Taiwan. This study aimed to compare the antioxidant capacity in Characins fed diets
supplemented with synthetic AX and/or BC at various dietary concentrations when subjected to hypoxia stress during transportation.

Materials and methods
Diet preparation
The control diet was composed of white ¢shmeal
50%, wheat £our 15%, dextrin 27%, ¢sh oil 3%, vitamin mix 2% and mineral mix 3%. Diets supplemented with CD had the same composition as the control

diet (except for dextrin, which was adjusted depending on the CD levels used) but supplemented with
either synthetic AX (8%AX) or BC (10%BC) or both.
Water was added to the ingredients to form a dough,
which was 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 a particle size of 0.9^
1.2 mm and stored at À 20 1C to avoid oxidation of
the CD. There were nine CD diets composed of 3 Â 3
factorial combinations of CD type (AX, BC and a 1:1
mixture of AX and BC) and CD concentrations (10,
20 and 40 mg kg À 1). Proximate analyses of these
diets are listed in Table 1.

Fish rearing, feeding and sampling
Experimental ¢sh were bought from an ornamental
¢sh farm. During acclimatization in the laboratory
in a 0.5 tonne tank, ¢sh were fed the control
diet for 2 weeks to equalize their body CD

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Aquaculture Research, 2010, 41, 973^981

Antioxidant capacity response to hypoxia of characins C-H Pan et al.

Table 1 Proximate analysis of the control and carotenoid diets

Control diet
Carotenoid type
Carotenoid concentration (mg kg À 1)
Proximate analysis

None
0

Crude protein (%)
Crude fat (%)
Ash (%)
Moisture (%)
NFE1CFÃ (%)
Astaxanthin (mg kg À 1)
b-carotene (mg kg À 1)

30.35
5.4
9.48
13.28
40.93
1.6
2.2

Carotenoid diet
b-carotene (BC)

Astaxanthin (AX)

1/2 AX11/2 BC (MX)

10

20

40

10

20

40

10

20

40

30.76
5.68
9.28
13.47
39.85
10.86
2.2

30.44
5.44
9.35

13.20
39.75
22.55
2.2

30.81
5.54
9.26
13.43
40.25
41.67
2.2

30.38
5.58
9.34
13.50
39.95
1.6
12.04

30.44
5.74
9.35
13.47
40.51
1.6
23.42

30.45

5.80
9.27
13.40
40.14
1.6
42.22

30.56
5.23
9.34
13.47
40.30
4.8
6.3

30.12
5.04
9.25
13.30
40.47
12.7
10.8

30.58
5.47
9.36
13.47
40.35
21.74
20.8

ÃNitrogen-free extracts and crude ¢bre.

contents. Fish were then transferred to 30 aquaria
(44 cm  33 cm  21.5 cm) to receive their respective treatments (three replicates per treatment) at a
stocking density of 30 ¢sh/aquarium. Fish size was
0.41 Æ 0.09 g. Culture water was passed through a
1 mm ¢lter and sterilized by ultraviolet light to eliminate microalgae, a possible source of CD. Moreover,
all aquaria were covered with a black screen to discourage algal growth for the same precaution. Fish
were fed twice daily at 08:00 and 15:00 hours at 5%
body weight. Dissolved oxygen was maintained at 6^
7 mg L À 1 by constant aeration, a temperature of 26^
28 1C, pH of 7.5^8 and NH3 of 0.1^0.2 mg L À 1. Faeces
and uneaten feeds were siphoned out daily and onethird of the water was exchanged. The ¢sh were
reared for 8 weeks. No mortality occurred throughout the experiment. The ¢nal overall average ¢sh size
was 0.89 Æ 0.18 g. Six ¢sh were then randomly
sampled from each aquarium for a hypoxia stress
test.

Hypoxia stress test
The six ¢sh were ¢rst acclimatized for 24 h in a
2 L bucket, where the temperature was maintained
at 25.3 Æ 0.4 1C and DO in saturation at 6.5 Æ 0.6
mg L À 1. They were then divided into two groups
and placed in two 250 mL bottles in which the water
was pre-saturated with DO ! 6.6 mg L À 1. Each bottle contained 2 g of zeolite, which is commonly used
for controlling ammonia during live transport
(Amend, Croy, Goven, Johnson & McCarthy 1982;
Teo, Chen & Lee 1989; Cole,Tamaru, Bailey, Brown &
Ako 1999; Singh, Vartak, Balange & Ghughuskar

2004). In the stress treatment, the bottle was tightly
capped to block the oxygen supply and DO was monitored. After around 2.5 h when DO declined to

1.0 mg L À 1 and was maintained for 10 min, the
¢sh were taken out and blood samples were collected
through the branchial artery for the analysis of serum antioxidants. In the control group, the bottle was
not capped and full aeration was provided.

Analysis of antioxidant parameters and blood
protein
Immediately after withdrawing the blood, samples
were prepared by mixing 200 mL isotonic NaCl solution containing 0.94 mmol L À 1 EDTA with 50 mL
blood. The samples were chilled if not immediately
used for determination of antioxidants: TAS, SOD,
AST, ALT and GPx and blood protein.
The di¡erent antioxidant parameters were analysed using Randox Laboratories kits (Crumlin,
County Atrim, UK) by spectrophotometry (U-2000;
Hitachi, Ibarake County, Japan). The volumes of serum samples used were 20 mL for TAS and GPx, 25 mL
for SOD and 100 mL for AST and ALT. Activities were
expressed in international enzyme unit (U L À 1).
Blood protein was determined using a protein assay kit (No. 500-0006, Bio-rad Laboratories, Richmond, CA, USA) with bovine serum albumin
(66 KDa, Sigma, St Louis, MO, USA) as the standard.
The method used was based on Bradford (1976) using
200 mL of serum sample.

Statistical analysis
Two two-way ANOVAs were performed: the ¢rst to determine the main e¡ects of dietary CD supplementation and hypoxia stress on TAS, SOD, GPx, ALT and
AST, and the second to determine the main e¡ects of
CD type and concentration on those antioxidant ca-

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Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 973^981

975


Antioxidant capacity response to hypoxia of characins C-H Pan et al.

pacity parameters. Duncan’s multiple range test was
then used to compare various levels within each
main e¡ect. Correlation analyses were conducted
among all antioxidant parameters. The signi¢cant level applied to all analysis was set to 5%.

Table 2 The average and standard deviation (in parenthesis)
of activities of serum antioxidation enzymes of control diet fed
and carotenoid diet fed Characins (Hyphessobrycon eques
Steindachner) exposed to normoxia and hypoxia environment
TAS (mmol L À 1
serum)

Results
Hypoxia stress increased the SOD, GPx and AST activities of all ¢sh, but had no e¡ect on ALT. Hypoxia
stress also increased TAS of the ¢sh fed the CD diet
but had no e¡ect onTAS of the ¢sh fed the control diet
(Table 2). Dietary CD reduced SOD and ALT activities
whether the ¢sh was under hypoxia stress or not. The
interaction of hypoxia stress and dietary CD had effects on SOD, GPX and AST, but not on TAS and ALT.
Dietary CD type a¡ected all enzyme activities except TAS and SOD of the ¢sh exposed to hypoxia
stress. Astaxanthin-fed ¢sh had lower GPx, ALT and
AST than BC-fed and MX-fed ¢sh. No di¡erences

were found in GPx, ALT and AST between BC-fed
and MX-fed ¢sh. Dietary CD concentration had no effect on TAS of the ¢sh exposed to hypoxia stress. Except TAS, the other four enzyme activities showed a
decreasing trend with increasing dietary CD concentrations. The GPx of ¢sh fed with 40 mg kg À 1 CD was
the lowest among the various concentrations tested.
The SOD, ALT and AST of ¢sh fed with 40 mg kg À 1
CD were lower than those fed with 10 mg kg À 1 CD,
but not di¡erent from those fed with 20 mg kg À 1 CD.
The interaction of CD type and concentration had an
e¡ect only on ALT and AST (Table 3).
Except for a negative correlation with SOD, TAS
had no correlations with the other antioxidant parameters. Superoxide dismutase, GPx, ALT and AST
had positive correlations among themselves (Table 4).

Hypoxia stress
Numerous signi¢cant studies and reviews have been
carried out on the physiological and biochemical responses of aquatic animals to hypoxia, especially on
¢sh (e.g. Holton & Randall 1967; Dunn & Hochachka
1986; Ip, Chew & Low 1991; Val, Lessard & Randall
1995; Hochachka 1997; Wu 2002). However, none of
these studies reported hypoxia e¡ects on antioxidant
enzyme activity. Hypoxia is de¢ned as a condition
where the DO level is o2.8 mg L À 1 (equivalent to
2 mL O2 L À 1 or 91.4 mM) (Diaz & Rosenberg 1995).

976

Environmentw
Normoxia
Hypoxia
Mean

SOD (U mg À 1
protein)
Environmentw
Normoxia
Hypoxia
Mean

DietÃ
Control

Carotenoid

Mean

a
x1.14
b
x1.12
a

a
y1.17
a
x1.31
a

y1.16

(0.03)

(0.01)
1.13 (0.02)

Environmentw
Normoxia
Hypoxia
Mean

ALT (U mg À 1
protein)
Environmentw
Normoxia
Hypoxia
Mean

(0.04)
(0.11)
1.24 (0.11)

x1.29

(0.04)
(0.12)

DietÃ
Control

y0.59

a

x1.35

a

(0.10)
(0.46)
0.97a (0.51)

DietÃ
GPx (U mg À 1
protein)
Control

a
y43.50
a
x77.15
a

(1.98)
(1.91)
60.33 (19.49)

Carotenoid

Mean

b
y0.47

b
x0.60
b

y0.48

(0.07)
(0.07)
0.53 (0.10)

x0.67

Carotenoid

Mean

b
y15.99
a
x55.02
b

y18.74

(2.21)
(16.91)
35.50 (23.09)

x57.23

(0.08)
(0.26)

(8.73)
(17.40)

DietÃ
Control

Carotenoid

Mean

a
x7.75
a
x8.35
a

b
x3.62
b
x3.74
b

x4.04

(0.49)
(0.35)
8.05 (0.49)

DietÃ
AST (U mg À 1
protein)
Control
Environmentw
Normoxia
Hypoxia
Mean

Discussion

Aquaculture Research, 2010, 41, 973^981

a
y8.70
a

(0.85)
x35.00 (0.07)
21.83a (15.16)

(1.91)
(1.52)
3.68 (1.70)

Carotenoid

b
y6.20

a

(1.70)
(10.58)
13.83 (10.75)

x21.46
a

x4.21

(2.21)
(2.02)

Mean

y6.45
x22.81

(1.79)
(10.83)

In Duncan’s multiple range test, means in the same row with
di¡erent superscripts or in the same column with di¡erent subscripts are signi¢cantly di¡erent (P
0.05).
ÃControl: no carotenoid supplemented in diet; Carotenoid: carotenoid (astaxanthin, carotene or mix of the two) supplemented at
10, 20 or 40 mg kg À 1 in diet.
wNormoxia: dissolved oxygen (DO) remained at 6.5 mg L À 1; hypoxia: DO declined from 6.5 to o1.0 mg L À 1 in 2.5 h and remained at o1.0 mg L À 1 for 10 min.
TAS, total antioxidative status; SOD, superoxide dismutase; GPx,
glutathione peroxidase; ALT, alanine transaminase; AST, aspartate transaminase.

r 2009 The Authors
Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 973^981