The studiesevaluated practical diet formulations in which bothmarine ¢sh meal and ¢sh oil have been completely re-placed with alternative sources of nutrients Table 1.The basal diets Die
Trang 2Physiological responses of pink abalone Haliotis
combinations of temperature and salinity
Zarina Medina Romo1, Ana Denisse Re1, Fernando D|¤az1& Alfredo Mena2
Physiological responses of pink abalone Haliotis
corru-gata were determined under di¡erent temperature and
salinity conditions Oxygen consumption rate was not
a¡ected by temperature and salinity Ammonium
ex-cretion 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
in-dicative 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
con-trol the life and distribution of aquatic organisms;
both factors have direct e¡ects on the physiological
responses of the marine and estuarine organisms,
such as oxygen consumption (Brown & da Silva1979; Moore & Sander 1983; Saucedo, Ocampo, Mon-teforte & Bervera 2004; Soria, Merino & von Brand2007), nitrogen excretion products (Livingstone,Wid-dows & Fieth 1979; Stickle & Bayne 1982; Regnault1987; Saucedo et al 2004; Soria et al 2007), energybudget (Newell & Branch 1980; Bayne & Newell 1983;Bricelj & Shumway 1991; Beiras, Pe¤rez-Camacho &Albentosa 1993), endogenous substrate utilization(Barber & Blake 1985; Mayzaud & Conover 1988; Ro-sas, Cuzon, Gaxiola, Taboada, Arena & VanWorm-houdt 2002) and osmoregulation pattern (Shumway1977; Hildreth & Stickle 1980; Cheng, Yeh, Wang &Chen 2002)
One of the physiological responses that can be related with a change in the environmental para-meters is the oxygen consumption rate, because it isrelated to the metabolic work and energy £ow that or-ganisms must channel to homeostatic control me-chanisms (Salvato, Cuomo, Di Muro & Beltramini2001) In aquatic organisms, the measurement of oxy-gen consumption is a valid method to assess the e¡ect
cor-of environmental factors such as temperature, nity, exposure to pollutants, light intensity and dis-solved oxygen It allows the estimation of the energycosts associated with the physiological stress thatthese factors impose on organisms (Brown & Terwilli-ger 1999; Lemos, Phan & Alvarez 2001; Altinok &Grizzle 2003; Brougher, Douglass & Soares 2005).Previous studies have investigated the e¡ects of sali-nity and temperature on the oxygen consumption inabalone such as Haliotis discus hannai (Ino) exposed
sali-to di¡erent environmental facsali-tors (Sano & Maniwa1962) Uki and Kikuchi (1975) investigated the e¡ectAquaculture Research, 2010, 41, 953^960 doi:10.1111/j.1365-2109.2009.02377.x
r 2009 The Authors
Trang 3of temperature and weight on the oxygen
consump-tion of H discus hannai In young disc abalone
Nordo-tis discus discus (Reeve), Segawa (1995) determined in
a preliminary study, the e¡ect of temperature on the
oxygen consumption rate Paul and Paul (1998)
deter-mined the e¡ect of di¡erent temperatures on the
re-spiration rate in Haliotis kamtschatkana (Jonas)
Both osmotic and ionic regulation have been
stu-died 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
osmoconfor-mers in which the haemolymph is close to the
osmo-tic pressure of seawater, and due to response,
ammonium excretion increases with decreasing
sali-nity (Shumway 1977; Bricelj & Shumway 1991)
The role of ammonium in the osmoregulation
pro-cesses 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
regula-tion of Na1in the haemolymph (Mangum,
Silver-thorn, 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
in-tegration of the values of the oxygen consumption
and nitrogen excretion to determine which
meta-bolic substrate is being used for the organisms
(May-zaud & 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,
Gax-iola, LePriol, Pascual, Rossygnyol, Contreras,
SaŁn-chez & 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
aba-lone 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
tem-perature 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 ( 0.9 g) were obtained from the commercial
farm of B C Abalone Hatchery Ere¤ndira, Ensenada(Baja California, Me¤xico) The organisms were trans-ported in a 10 L-Styrofoam cage with seawater In thelaboratory, the time to acclimate was 2 weeks at 35%,
18 1 1C temperature, measured under farm tions in three reservoirs of 2000 L with constantaeration and a 60mm ¢ltered seawater £ow with anexchange rate of 100% daily Each reservoir was pro-vided with abalone refuges and was heavily shaded;refuges and shaded were used to minimize distur-bance from exterior movements
condi-The organisms were fed during the entire mental period with macroalgae Macrocystis pyriferaand Egregia menziesii Acclimation of 489 abalones
experi-to the experimental temperature 20 and 24 1C wascompleted in 21 days The temperature was increased
at a rate of 2 1C everyday to reach the programmed temperature Both temperatures wereachieved by titanium heaters of 1000 W controlled byMedusa devices (Sea Life Supply, Sand City, CA, USA).For experimental salinities 480 abalones were ac-climated to 35%, 32%, 29%, 26%, 23% and 20%,where the lowest concentration was obtained by dilu-tions of seawater (35%) with tap water The rate ofsalinity decrease was 3% per 5 days to reach the ex-perimental salinity, and once the experimental sali-nity had been achieved, the abalones remainedunder those conditions for 15 days, which representsu⁄cient time to attain a steady internal medium ofabalone according to Cheng et al (2002)
experimental-To avoid interference with post-prandial lism of food and faeces production, acclimated aba-lone was kept unfed for 24 h Oxygen consumptionand nitrogen excretion were measured with the or-ganisms maintained at temperatures of 20 and
metabo-24 1C and experimental salinities of 35%, 32%,29%, 26%, 23% and 20% by using a semi-closed re-spirometric system described by D|¤az, Re, GonzaŁlez,SaŁnchez, Leyva and Valenzuela (2007), consisting of
21 chambers of 1000 mL each Twenty abalones ofeach temperature and salinity were individually in-troduced into the respiratory chamber 24 h before in-itiating measurements, which were made between09:00 and 13:00 hours to avoid interference due tothe rhythm circadian response
Water £ow in the chambers remained open for 2 h;before closing, one water sample was taken to mea-sure the initial concentration of dissolved oxygenusing a YSI 52 oxymeter (Yellow Spring Instruments,Yellow Springs, OH, USA), equipped with a polaro-graphic sensor accuracy of 0.03 mg L 1, whichwas inside an acrylic hermetic chamber with a
Trang 410 mL capacity with adequate stirring Subsequently,
the chambers were kept closed for 1h, to avoid
reduc-tion in the dissolved oxygen of 25^30%, as this is a
stress factor (Stern, Borut & Cohen 1984) Before
re-establishing the water £ow, one water sample was
ta-ken to measure the ¢nal concentration of dissolved
oxygen The 21st chamber was used as a control to
measure oxygen consumption by the
microorgan-isms present in the water, and the necessary
correc-tions were made Two repeticorrec-tions were carried out for
each test The results of oxygen consumption are
given in mg O2kg 1h 1on a wet weight basis
To determine the ammonium production (NH41),
initial and ¢nal samples were obtained from the
re-spirometric chamber in the same manner as that
de-scribed 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
spectrophot-ometer El|¤ptica (Ely-2000 Instruments, Ensenada,
Me¤xico) at a wavelength of 640 nm Ammonium
pro-duction was calculated as the di¡erence between the
¢nal and the initial measure and it was expressed as
mg NH41h 1g 1wet weight (w.w.)
The O:N ratio was estimated using the oxygen
con-sumption 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
ex-perimental 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,10mL from 20
or-ganisms from each experimental condition, were
placed on a blotting paper disc in a Wescor 5520
va-pour pressure osmometer (Wescor Logan, UT, USA)
The osmolality of the internal as well as the external
medium was expressed as mmol kg 1
The data for oxygen consumption and ammonium
excretion of abalone exposed to di¡erent
experimen-tal conditions were plotted in parallel boxes (Tukey
1977).Within the boxes, 50% of the data were
distrib-uted around the median and the con¢dence
inter-vals; the other 50% remained distributed in each
bar The relationships between haemolymph
osmol-ality and medium osmolosmol-ality were determined using
a linear regression after satisfying the test for ability of ¢t to a linear model (SIGMA STATversion 3.1)
suit-A two-way analysis of variance (ANOVA) was used asprevious determination of the normality and homo-scedasticity of the data (SIGMA STAT), to determine thee¡ect of the temperature and salinity on oxygen con-sumption, ammonium excretion, atomic ratio O:Nand the osmolality of the haemolymph of pink aba-lone (Zar 1999)
ResultsThe rate of oxygen consumption of pink abalone main-tained at 20 1C and acclimated at di¡erent salinitieswas in the range of 0.58^0.79 mg O2h 1g 1w.w Inthe organism acclimated to 24 1C, oxygen consump-tion was in the range of 0.64^0.81mg O2h 1g 1w.w.(Fig 1) AnANOVAindicated that temperature and sali-nity did not have a signi¢cant e¡ect on the oxygen
Figure 1 Oxygen consumption rate of Haliotis corrugataacclimated to two temperatures at di¡erent salinities Thezone marked by circles represents the 95% con¢dence in-terval of the median; 50% of the data are distributed invertical lines
Aquaculture Research, 2010, 41, 953^960 Physiological responses of pink abalone Z M Romo et al.
r 2009 The Authors
Trang 5consumption 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 NH4h 1g 1w.w.) at the
salinity of 35% In abalone maintained at 24 1C, the
ammonium excretion rate increased when the
or-ganism 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 NH4h 1g 1w.w An ANOVA indicated that
there was a signi¢cant e¡ect of salinity on the
ammo-nium 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
aba-lones acclimated to 24 1C, the lowest values of the
O:N atomic ratio of 6.8 and 7.7 were obtained at nities of 20% and 23%; the highest value of 35.5 wasfound in the juveniles acclimated to 35% salinity(Fig 3) AnANOVAindicated that salinity had a signi¢-cant e¡ect (d.f 55,1, F 5 43.01,1.03, Po0.001) on theatomic ratio
sali-Osmolality of haemolymph in the abalone niles acclimated to 20 and 24 1C was related in a lin-ear way with respect to the external medium,yielding the equations:
juve-IM in 20C¼ 69:25 þ 0:949X r2¼ 0:996
IM in 24C¼ 14:82 þ 1:045X r2¼ 0:986where IM (internal medium) is the haemolymph os-molality and X is the external medium osmolality.For the abalones acclimated to 20 1C and exposed
to experimental salinities the haemolymph ity was in the range of 675^1002.7 mmol kg 1, hav-ing a slightly hyperiso-osmoconformer pattern ofosmoregulation (Fig 4) In the organism acclimated
osmolal-to 24 1C, haemolymph osmolality was in the range
Figure 2 Ammonium excretion of Haliotis corrugata
ac-climated to two temperatures at di¡erent salinities The
zone marked by circles represents the 95% con¢dence
in-terval of the median; 50% of the data are distributed in
vertical lines
Figure 3 Atomic ratio O:N (median con¢dence val) of Haliotis corrugata acclimated at two temperatures
inter-at di¡erent salinities
Trang 6of 691^1054 mmol kg 1, indicating that abalones
had a slightly hyperiso-osmoconformer pattern (Fig
4) AnANOVAindicated 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)
In our experiment, the oxygen consumption ratewas independent of salinity and temperatures, sug-gesting that abalone could adjust their metabolismafter an acclimation period to di¡erent experimentalconditions to which they were exposed In the aba-lone H discus hannai exposed to chlorinities higherthan 14%, Sano and Maniwa (1962) obtained thatthe rate of oxygen consumption was kept constant
In Argopecten purpuratus (Lamarck) exposed to acombination of two temperatures and three salinitiesfor a period of 45 days; Soria et al (2007) reported thatthe rate of oxygen consumption was maintained in-dependent In aquatic organisms that have been ac-climated to di¡erent salinities, Kinne (1967)described four types of metabolic responses Pinkabalone exposed to di¡erent salinities exhibited thetype I response, because the oxygen consumptionwas not modi¢ed signi¢cantly For other marine or-ganisms, it has been shown that salinity did not have
a pronounced e¡ect on the oxygen consumptionwhen 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 main-tain FAA pools that vary directly with external salinityand this allows the preservation of cellular volumes bychanging haemolymph osmolality (Tang, Liu, Yang &Xiang 2005) The ammonium excretion of pink aba-lone increased when salinity decreased from 35% to23%, and this response may be related to an increase
in catabolism of the amino acids to form intracellularosmolytes to regulate their osmotic equilibrium whenexposed to lower salinities Under hyperosmotic stress,pink abalone shows an enhancement in the concen-tration of intracellular £uid by shrinking the cell vo-lume from the environment absorbing ions andcatabolizing self-tissue protein All these metabolic ac-tivities lead to increased ammonium excretion InMeretrix meretrix (Linnaeus) (Tang et al 2005) and
A purpuratus exposed to decreasing and increasingsalinities (Navarro & GonzaŁlez 1998); Soria et al.(2007) reported an increase in the ammonium excre-tion rate, suggesting that it regulates the cellular vo-lume by breakdown of amino acids as intercellularregulators with a reduction in salinity
A high value of O:N is taken to represent a minance of lipid and/or carbohydrate degradationover protein degradation (Mayzaud & Conover 1988).Ikeda (1977) reported an O:N ratio of 24 when proteinand lipids were metabolized in equal quantities at thesame time; hence, an O:N ratioo24 indicates a pro-tein-dominant metabolism and a ratio424 indicates
predo-Figure 4 Relation between haemolymph osmolality of
Haliotis corrugata and medium osmolality when they were
exposed to two temperatures at di¡erent salinities
Aquaculture Research, 2010, 41, 953^960 Physiological responses of pink abalone Z M Romo et al.
r 2009 The Authors
Trang 7a 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
ex-posed to lower salinities had values of 4.9^7.7,
imply-ing that the organisms used protein catabolism as a
primary catabolic substrate, due to the stress caused
by exposure to lower salinities At intermediate
sali-nities (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
tempera-tures 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^carbohy-drate 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
corruga-ta that the preferred and optimal of growth
tempera-ture 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
ventri-culosus-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
aba-lone 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-osmocon-formed regulation pattern; the di¡erences in the
osmoregulation pattern due to the process of
trans-ference to the experimental salinities in H
diversico-lor supertexta were drastic, and these conditions were
maintained for 9 days For pink abalone, the
physio-logical changes were gradual and once the mental conditions were reached, the abalones weremaintained for 15 days In Argopecten ventriculosus-circularis, Signoret-Brailovsky et al (1996) observed
experi-an osmoconformer regulation pattern In C gigas posed to gradual and sudden changes in salinities,anosmoregulation osmoconformator pattern was re-ported (Hosoi et al 2003) The osmoconformer mar-ine organisms, including abalones, adapt to salinitychanges using intracellular isosmotic regulation, inwhich intracellular FAA contribute predominantly
ex-to intracellular osmolality and ex-to cell volume tion (Shumway 1977; Signoret-Brailovsky et al 1996;Cheng et al 2002; Hosoi et al 2003) The slope regres-sion line calculated from the relationship betweenhaemolymph osmolality and medium osmolalitywas parallel to the isosmotic line, with values from0.949 to 1.045 similar to those reported by Cheng
regula-et al (2002) for H diversicolor supertexta exposed todi¡erent salinity levels
Acknowledgements
We thank Jose M Dominguez and Francisco JavierPonce from the Drawing Department of CICESE forpreparing the ¢gures We are also grateful for theEnglish language editing of the manuscript provided
by Dr A Leyva
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Trang 10Engle-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
4 South Carolina Department of Natural Resources, Charleston, SC, USA
Correspondence: Tzachi M Samocha, AgriLife Research Mariculture Laboratory, AgriLife Research & Extension Center, 4301 Waldron Rd., Corpus Christi,TX 78418, USA E-mail: t-samocha@tamu.edu
Abstract
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
doco-sahexaenoic 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
com-bination of plant oil with/without DHA and ArA
sup-plements 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-supple-ment-free diet In both trials, the weights were
re-duced 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
fer-mented products is a viable option to replace marine
¢sh oil for L vannamei
Keywords: DHA, ArA, practical diets, Paci¢c
white shrimp, Litopenaeus vannamei
IntroductionMarine ¢sh meals and ¢sh oils are excellent sources
of high-quality essential amino acids, lipids, mins, minerals and attractants in aquaculture diets(Tacon & Akiyama 1997) However, the unstableprices associated with £uctuations in the supply ofthese marine ingredients and the sustainability ofthese practices are of prime concern (Chamberlain1993; Tacon & Akiyama 1997; Naylor, Goldberg, Pri-mavera, Kautsky, Beveridge, Clay, Folk, Lubchenco,Mooney & Troell 2000) Hence, replacement of thesemarine ingredients with cost-e¡ective alternativesources of proteins and lipids in aquaculture feeds is
vita-a high-priority tvita-ask for feed mills vita-and vita-aquvita-aculturists(Tacon & Akiyama 1997) Previous studies (Lim 1996;Davis & Arnold 2000; Samocha, Davis, Saoud & De-Bault 2004; Menoyo, Lopez-Bote, Obach & Bautista2005) showed that either animal or plant sourcescan be used as suitable substitutes for ¢sh meal and
¢sh oil in a small-scale tank system In their e¡orts toreplace ¢sh meal, researchers used plant proteinsources such as soybean meal (Sudaryono, Hoxey,Kailis & Evans 1995; Hertrampf & Piedad-Pascual2000; Olvera-Novoa & Olivera-Castillo 2000), sol-vent-extracted cotton seed meal (Lim 1996), lupinmeals (Sudaryono et al 1995), legumes, leaf meals(Eusebio & Coloso 1998; Li, Robinson & Hardy 2000)and papaya or camote leaf meal (Pena£orida 1995) infeed formulations for aquatic animals, with varyingAquaculture Research, 2010, 41, 961^967 doi:10.1111/j.1365-2109.2009.02378.x
r 2009 The Authors
Trang 11degrees of success Several studies have
demon-strated that with suitable adjustments, animal
by-product 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;
Samo-cha 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
with-out 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
tele-osts (Harel, Koven, Lein, Bar, Behrens, Stubble¢eld,
Zohar & Place 2002) In a recent study, Patnaik,
Sa-mocha, 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
Mor-tierella sp obtained by a proprietary commercial
fer-mentation process Although we have demonstrated
the ability to make these substitutions, the need to
in-clude a highly unsaturated fatty acids (HUFA)
supple-ment has not been established especially under
conditions when some natural productivity is
pre-sent 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-tem operated with no water exchange The studiesevaluated practical diet formulations in which bothmarine ¢sh meal and ¢sh oil have been completely re-placed 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 whichshowed good growth and survival of this species inprevious studies (Davis & Arnold 2000; Samocha et al.2004) Test-diets were formulated to meet all knownnutritional requirements for this species and had 35%crude protein (CP) and 8% lipid levels The HUFA wereprovided either from ¢sh oil or from a meal made fromspray-dried cells of Schizochytrium sp and Mortierella
sp (DHA GOLDsand AquaGrows-ARA, Advanced Nutrition, Columbia, MD, USA), collectively referred to
Bio-as a HUFA-rich source, which are the products of prietary fermentation processes A commercial diet(35% CP,8% lipid; Rangen, Buhl, ID, USA) was included
pro-in each trial as a reference diet
The ¢rst trial was conducted over a 12-week periodusing hand-sorted juvenile (6.07 0.3 g) shrimp.Diet 1 (‘HUFA-rich’ diet) was formulated with theHUFA from the spray-dried cells Diet 2 (‘HUFA-de¢-cient’diet) was formulated with no HUFA supplemen-tation to determine the e¡ect of this de¢ciency onshrimp performance A product of co-extruded soy-bean and poultry by-product meal served as the pro-tein source in Diet 1 and Diet 2 (ProfoundTM,American Dehydrated Foods,Verona, MO, USA.).The second trial was conducted over a 14-weekperiod using hand-sorted juveniles (0.95 0.04 g).For this series of diets, the poultry by-product meal,rather than the ProfoundTM, served as the primaryprotein source Diet 4 (‘HUFA-rich’ diet) was formu-lated with the same source of HUFA as that of Diet 1.Diet 3 was prepared with the same ingredients asthose used for Diet 4 but the HUFAwas provided frommenhaden ¢sh oil (‘menhaden-rich’ diet) Diet 5(‘HUFA-de¢cient’diet) was prepared with the same in-gredients as those used for the formulation of Diet 3and Diet 4 but without HUFA supplementation Todetermine whether dietary HUFA supplementation
is only required during the juvenile phase, a sixthtreatment 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 thestudy It is important to note that the performances
of the diets were compared within the same trialand not between the trials
The test diets were prepared in the feed laboratory
of Auburn University, Auburn, AL, USA, using
Trang 12stan-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
treat-ments were randomly assigned and the study was
run as a double-blind experiment
Both trials were conducted in an outdoor tank
sys-tem at the AgriLife Research Mariculture Laboratory
theTexas AgriLife Research and Extension Center,
Cor-pus Christi,TX, USA Each treatment was randomly
as-signed to ¢ve-replicate high-density polyethylene
circular tanks positioned under a shade with roo¢ng
made of clear and opaque panels Each tank had aworking volume of 650 L and a bottom area of0.85 m2 Tanks were covered with a net to preventshrimp from escaping Aeration was provided by twoair stones per tank (7^10 L min 1stone 1) that werefed by a common regenerative air blower Natural sea-water was used after initial chlorination, de-chlorina-tion 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 2 (40 shrimp m 3) and 36 shrimp m 2 (48shrimp m 3) for Trial 1 and Trial 2 respectively Uponreaching the 5 g size in Trial 2, ¢ve shrimp from eachtank were removed for sub-sampling The study wasresumed, after the removal of these shrimp, assuming
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
Profound TM 39.00 39.00
Poultry by-product mealw 16.00 16.00 16.00 Soybean mealz 30.20 30.20 40.50 40.50 40.50 Corn gluten, organic‰ 5.00 5.00 5.00 Schizochytrium meal (DHA)z 0.50 0.50
Co-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).
zDHA GOLD s
(Schizochytrium sp algae meal) and AquaGrows-ARA (Mortierella sp.) (Advanced BioNutrition, Columbia, MD, USA) kUnited States Biochemical (Cleveland, OH, USA).
Sigma (St Louis, MO, USA).
wwOmega Protein (Reedville, VA, USA).
zzAs g100 g 1premix: cobalt chloride 0.004, cupric sulphate pentahyrate 0.250, ferrous sulphate 4.0, magnesium sulphate drate 28.398, manganous sulphate monohydrate 0.650, potassium iodide 0.067, sodium selenite 0.010, zinc sulphate heptahydrate 13.193,
heptahy-¢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.
zzStay C s
( 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.
Aquaculture Research, 2010, 41, 961^967 Alternative HUFA source for Litopenaeus vannamei T M Samocha et al.
r 2009 The Authors
Trang 13that 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
esti-mate 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 and 1.2 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
ni-trite-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
ni-trogen (TAN), nitrite nini-trogen (NO2-N), reactive
phos-phorus (RP) and 5-day biochemical oxygen demand
(cBOD5)
At the conclusion of each trial, shrimp were
group-weighed 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 measuresANOVA Di¡erences
among treatments in TAN, NO2-N, RP and cBOD5on
the day of termination were analysed using one-way
ANOVA The same statistical test was used to
deter-mine di¡erences between treatment means
(Po0.05) in the ¢nal mean weight, survival and FCR
values Statistical analyses were performed only on
the test diets; the data for the reference diet were vided for informational purposes The Student^New-man^Keuls (SNK) test was used as a tool to identifythe di¡erence between treatment means Square roottransformation of per cent survival data was alsoevaluated but did not a¡ect the interpretation of theresults; hence, it is not presented Statistical analyseswere conducted usingSPSS(V 13 for Windows, SPSS,Chicago, IL, USA)
pro-Results and discussion
No statistically signi¢cant di¡erences were found tween treatments for both trials in the daily or theweekly water quality indicators (Tables 2 and 3).These daily values represent acceptable ranges re-ported for good growth and survival of penaeidshrimp and are typical for this system It is interest-ing to note that although there were no signi¢cantdi¡erences in the weekly water quality indicators be-tween treatments, on Day 56 of the study, one of thetanks experienced a short exposure (for about aweek) to a TAN level of 1.94 mg L 1and an NO2-N le-vel as high as 7.3 mg L 1 The fact that there was nosigni¢cant di¡erence in the mean shrimp ¢nalweight within treatment tanks, along with the highsurvival in this tank (89%), suggests that these levelshad no adverse e¡ect on the shrimp in this study.Shrimp survival rates in both trials were high andtypical for this research system Statistical analyses
be-of the data from Trial l indicated signi¢cant ences 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 commercialreference diet, which was lower than the 97.5^100% observed for the test diets A signi¢cant reduc-tion was observed in the ¢nal weights betweenshrimp reared on the ‘HUFA-rich’diet (Diet 1) and the
di¡er-Table 2 Summary of the daily water quality indicators (Mean SD 1
and range) from the growth trials conducted in outdoor tanks with Litopenaeus vannamei
(6.0–9.3) (5.8–7.5) (25.7–29.1) (26.6–31.3) (7.3–8.3) (7.5–8.6) (18–26)
Trang 14‘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
sig-ni¢cant di¡erences in survival between shrimp fed
the di¡erent diets in Trial 2 Signi¢cant di¡erenceswere observed between treatments in terms of the ¢-nal shrimp weights and FCR There were no di¡er-ences 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 ofshrimp fed the ¢sh oil diet were signi¢cantly higherthan those fed the two diets without the HUFA sup-plements (Diet 5 and Diet 6) Shrimp reared on the
‘HUFA-rich’ (Diet 4) were not signi¢cantly di¡erentbut numerically larger than those reared on theseHUFA-de¢cient diets (Diet 5 and Diet 6) Shrimpmaintained on the ‘menhaden-rich’ diet (Diet 3)showed a better FCR than those maintained on theHUFA-de¢cient diet (Diet 5)
In both growth trials, the ¢nal weight, survivaland FCR values of shrimp receiving diets with HUFAsupplements were similar to those observed for thecommercial diets Furthermore, there were no indica-tions of feed rejection, with all diets readily con-sumed These results demonstrate that marineingredients can be removed from practical diets forshrimp reared in outdoor tanks with no water ex-change in the presence of natural productivity Thereduced growth of shrimp reared on diets withoutHUFA supplements would indicate that the supple-mentation of HUFAs is a critical component of the re-placement strategy While complete replacement of
¢sh meal has been successful in the production diets
Table 3 Summary (mean SD) of the mean weekly
water quality parameters (mg L 1 ) recorded during the
growth trials conducted in outdoor tanks with Litopenaeus
zShrimp were fed Diet 4 up to the 5 g size Beyond this size
shrimp were switched to Diet 5.
TAN, total ammonium nitrogen.
Table 4 Final weights, survival rates, feed conversion ratio (FCR) for Litopenaeus vannamei juveniles reared in outdoor tanks and o¡ered the test diets
Diet 5 (w/o HUFA) 14.6 b 92.3 1.53 a
‘Diet 6’ (Diet 41Diet 5)‰ 14.0 b 98.1 1.44 ab
Based on Student^Newman^Keuls mean separation, treatment means with the same superscript letters are not signi¢cantly di¡erent.
Data represent the mean of ¢ve replicates Shrimp had a mean initial weight of 6.07 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 0.04 g.
‰Shrimp were fed Diet 4 to 5 g size and Diet 5 until the harvest.
Aquaculture Research, 2010, 41, 961^967 Alternative HUFA source for Litopenaeus vannamei T M Samocha et al.
r 2009 The Authors
Trang 15of 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
stu-dies 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
Pro-foundTMas 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
com-plete 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
commercial fermentation product (DHA GOLDsand
AquaGrows-ARA as HUFA source) without
impair-ing shrimp performance Furthermore, Browdy,
Sea-born, Atwood, Davis, Bullis, Samocha, Wirth and
Le¥er (2006), in an outdoor pond study at the
Wad-dell Mariculture Center, Blu¡ton, SC, USA, showed
no signi¢cant di¡erences in the production
para-meters between shrimp that were fed an
all-plant-based 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
ma-jor source of essential fatty acids such as
eicosapen-taenoic 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
lim-ited by the potential problems associated with
insuf-¢cient levels of essential fatty acids (Gonzalez-Felix,
Gatlin III, Lawrence & Perez-Velazquez 2002)
Doco-sahexaenoic 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)
results, we were able to completely replace the ine oil ingredient using a combination of plant oilssupplemented with fermentation products rich inHUFA as a source for the essential fatty acids
mar-As demonstrated in a previous research and
con-¢rmed by these trials, both ProfoundTMand the try by-product meal can serve as primary sources forprotein and essential amino acids in practical diets ofthe Paci¢c white shrimp The complete replacement
poul-of ¢sh meal and ¢sh oil using non-marine ingredientscan be accomplished using plant oils supplementedwith fermentation products as the HUFA source Theuse of a heterotrophically produced non-marineHUFA-rich product as the lipid source in feed is a re-cent concept in practical diet formulations for shrimpand is still in a preliminary stage of research Addi-tional research on the e¡ect of these ingredients ondi¡erent life stages of L vannamei under various en-vironmental conditions would be bene¢cial to ¢sh oiland ¢sh meal replacement e¡orts And, ¢nally, morestudies are needed to determine the economic viabi-lity of the large-scale use of these components inshrimp feed formulations
AcknowledgmentsThis research was supported by funding from Ad-vanced BioNutrition, Columbia, MD, USA Theauthors would like to thank the students and the em-ployees of AgriLife Research Mariculture Laboratory,Corpus Christi, TX, USA for their help Mention of atrademark or a proprietary product does not consti-tute an endorsement of the products
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r 2009 The Authors
Trang 17Initial 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
2 US Department of Agriculture, Agriculture Research Service, National Warmwater Aquaculture Center, Stoneville, MS, USA
Correspondence: C C Mischke, National Warmwater Aquaculture Center, Mississippi State University, 127 Experiment Station Road,
PO Box 197, Stoneville, MS 38776, USA E-mail: cmischke@drec.msstate.edu
Abstract
Using di¡erent sources of nitrogen as fertilizers in
nur-sery ponds may a¡ect water quality and plankton
re-sponses 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
ni-trate (34% N) and urea (45% N) in190-L microcosms at
equimolar nitrogen application rates Sodium
nitrite-fertilized microcosms had higher nitrite and nitrate
le-vels 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
cya-nobacteria were detected among treatments; desirable
zooplankton for cat¢sh culture was increased in
urea-fertilized microcosms Based on these results, any form
of nitrogen used for pond fertilization should perform
similarly without causing substantial water quality
de-terioration 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
ferti-lizer have an equal cost per unit of nitrogen, we
recom-mend 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
is to increase dissolved inorganic nutrient tions in the pond water The increased nutrients arethen incorporated into biomass (algal and zooplankton)and ultimately incorporated into ¢sh biomass However,complicated interactions (climate, water and bottomsoil characteristics and pond morphology) can a¡ecthow ponds respond to nutrient additions (Knud-Han-sen 1998) Additionally, management practices asso-ciated with di¡erent species (e.g., feeding and stockingrates) may a¡ect fertilization responses
concentra-The production of channel cat¢sh Ictalurus tus fry and ¢ngerlings is unique and utilizes di¡erenttechniques compared with other types of aquacul-ture Cat¢sh fry are stocked at relatively high densi-ties (40 000^120 000 ha 1) into newly ¢lled earthenponds (¢lled with well water 3^4 weeks before stock-ing) Prepared diets are o¡ered to the fry immediatelyafter stocking Zooplankton populations are impor-tant in cat¢sh fry culture during the ¢rst 3^4 weeks,but diminish in importance as fry grow and seek theprepared diets (Mischke, Wise & Lane 2003) For cat-
puncta-¢sh fry culture, it does not appear to be necessary tohave long, sustained populations of zooplankton afterthe initial weeks post stocking Therefore, the primarygoal of fertilizing cat¢sh fry ponds is to establish aphytoplankton bloom as quickly as possible to shademacrophytes and produce large stocks of copepodsand cladocerans for fry consumption during the ¢rst3^4 weeks after stocking (Mischke et al 2003; Mis-chke & Zimba 2004)
When high-nitrogen fertilizers are applied ratherthan high-phosphorus fertilizers, the phytoplanktonpopulation is shifted to desirable algal groups, thusproviding a quick algal bloom and adequate foragefor cladocerans without the use of organic fertilizers(Mischke & Zimba 2004)
Trang 18Primary 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
re-sponses 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
tertio-lecta grew 10^30% faster on ammonia versus nitrate
Lourenco, Barbarino, Mancin-Filho, Schinke and
Ai-dar (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
Me-socosms were placed in the pond on 19 May 2008 at
an equal depth, providing 190 L of water in each
con-tainer Mesocosms were slowly lowered into position
to allow water to ¢ll the mesocosms without
disturb-ing the pond sediments Treatments were randomly
assigned to each mesocosm; there were ¢ve
replica-tions of each treatment
All mesocosms were fertilized on an equal
nitro-gen basis based on the nitronitro-gen fertilization
recom-mendations of Mischke and Zimba (2004), with the
¢rst application providing 2.2 mg N L 1, followed by
two applications per week of 1.1mg N L 1 for 3
weeks The di¡erent nitrogen treatments used were:
calcium nitrate (12% N), sodium nitrite (20% N),
am-monium chloride (26% N), amam-monium nitrate (34%
N) and urea (45% N) All fertilizers were granular
and dissolved in water before application to the
meso-cosms
Water samples were collected from each
meso-cosm 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
labora-tory and immediately analysed for pH, soluble
reac-tive phosphorus (ascorbic acid method), total
ammonia^nitrogen (Nesslerization),
nitrite^nitro-gen (diazotization) and nitrate^nitronitrite^nitro-gen (cadmium
reduction, followed by diazotization) using the ods outlined by HACH (1999)
meth-Pigment analysis was used to assess ton community composition using the HPLC metho-dology (Zimba, Dionigi & Millie 1999) Knownvolumes of mesocosm water were ¢ltered (GF/C
phytoplank-¢lters, Whatman, Maidestone, UK) under reducedpressure in the dark Filters were immediately frozenand held at 80 1C until analysis Filters were ex-tracted in 100% acetone for 24 h, clari¢ed by syringe
¢ltration before ampulation and HPLC analysis ments (carotenoids and chlorophylls) were quanti¢edusing an HP1100 equipped with diode array and
Pig-£uorescence detectors (Agilent Technologies, PaloAlto, CA, USA) Identi¢cation of speci¢c divisions ofalgae is possible using taxon-speci¢c pigment bio-markers (Zimba, Tucker, Mischke & Grimm 2002)
A pigment library was used to identify samples; known samples were quanti¢ed by linear regression
un-of known commercial standards
Separate tube samples of 3.8 L were collected forzooplankton using the same schedule and methods
as those described for water quality sampling Thecollected sample was then concentrated by pouringthrough a 63-mm mesh net A 63-mm mesh net wasused to ensure capture of smaller rotifers Sampleswere preserved in bu¡ered formalin solution beforecounting by light microscopy (Geiger & Turner1990) All organisms in 1ml subsamples from eachmesocosm were counted using a Sedgwick^Raftercounting cell as described by Geiger and Turner(1990) and zooplankton were identi¢ed using thetaxonomic keys of Thorp and Covich (1991)
The experimental design was completely mized, with repeated measures taken on replicatemesocosms Data were analysed using the MIXEDprocedure inSASVersion 8.02 software (SAS Institute,Cary, NC, USA) (Littell, Milliken, Stroup & Wol¢nger1996) The covariance structure, autoregressive oforder 1, was used in the repeated measure model.Mean comparisons were made using an LSD testwith a signi¢cance level of Po0.05
rando-ResultsDissolved soluble reactive phosphorus, ammonia ni-trogen and pH were not a¡ected by nitrogen source.There was a signi¢cant interaction among the maine¡ects of date by treatment with nitrate (Fig 1) andnitrite (Fig 2) Sodium nitrate-fertilized ponds hadhigher concentrations of both nitrate and nitrite rela-Aquaculture Research, 2010, 41, 968^972 Fertilizer nitrogen types and water quality C C Mischke & P V Zimba
r 2009 The Authors
Trang 19tive to the other treatments during the ¢rst week of
sampling, but returned to similar levels for the
re-mainder of the study
Green algae, diatoms and cyanobacteria were
pre-sent in all mesocosms; however, analysis of
chloro-phyll a, chlorochloro-phyll 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
ef-fects of date by treatment (Fig 3) Mesocosms treated
with calcium nitrate tended to show a more rapid crease in the desirable zooplankton concentrations atthe beginning of sampling, and urea-fertilized meso-cosms showed an increase in desirable zooplanktonconcentrations at the end of sampling
in-DiscussionThe choice of nitrogen type to use as a pond fertilizerwould depend on the e¡ectiveness of the fertilizer toincrease desirable zooplankton and phytoplanktonconcentrations in the pond, minimal deleterious ef-fects on water quality (e.g., changes in ammonia and
Figure 1 Dissolved nitrate concentration (mg N L 1) in mesocosms treated with various nitrogen sources from 20 May
2008 to 10 June 2008 Symbols represent means SE
Figure 2 Dissolved nitrite concentration (mg N L 1) in mesocosms treated with various nitrogen sources from 20 May
2008 to 10 June 2008 Symbols represent means SE
Trang 20nitrite 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 orb-carotene However, urea-fertilized microcosms
showed increased desirable zooplankton
concentra-tions at the end of the study Generally, cat¢sh
nur-sery ponds are ¢lled and fertilized for about 3 weeks
before fry are stocked Therefore, urea may have an
advantage over the other nitrogen fertilizers,
provid-ing higher desirable zooplankton concentrations at
the time of stocking
Although we expected ammonia concentrations to
be higher in microcosms treated with
ammonia-based fertilizers (ammonium chloride, ammonium
nitrate and urea), nitrite levels to be higher in
trite-fertilized microcosms (sodium nitrite) and
ni-trate to be higher in nini-trate-fertilized microcosms
(ammonium nitrate and calcium nitrate), this
ex-pected outcome did not occur The only di¡erences
in dissolved inorganic nitrogen concentrations
among treatments occurred in the sodium
nitrite-fertilized microcosms; this fertilizer resulted in
in-creased nitrite and nitrate levels for the ¢rst week of
the study, but then returned to concentrations
simi-lar to other fertilizer treatments This suggests that
algal utilization or absorption to sediments reduced
nitrogen concentrations more rapidly than
pre-viously observed in pond-scale fertilization (Mischke
& Zimba 2004)
Ammonia nitrogen is the preferred source of gen for phytoplankton according to Tepe and Boyd(2001); however, other evidence suggests that nitro-gen utilization is species speci¢c (Syrett1981; Louren-
nitro-co et al 2002) In the current study, there were nopeaks in dissolved ammonia concentrations whenmicrocosms were fertilized with ammonia-based fer-tilizers We assume that because nitrogen is limiting
in these ponds (Mischke & Zimba 2004), there was pid uptake of dissolved nitrogen by the phytoplank-ton Although water quality was similar by the end
ra-of the study, using a nitrite fertilizer did cause nitritelevels to increase slightly during the ¢rst week.Therefore, nitrite fertilizers may be less desirable foruse in nursery ponds relative to the other nitrogensources
Ammonium fertilizers are cheaper than nitratefertilizers per unit of nitrogen (Boyd & Tucker 1998).Urea and ammonium nitrate are generally similar incost per unit of nitrogen; however, ammonium ni-trate can be more di⁄cult to obtain and may requireextensive record keeping because of its potential use
in explosives
Based on the results of this study, any form of trogen used for pond fertilization should perform si-milarly in the short term without causing substantialwater quality deterioration Typical cat¢sh nurseryponds are only fertilized for a short time until fry arestocked (after 2^3 weeks of fertilization) or fry arereadily feeding at the pond surface (usually 3^4
ni-050100
Figure 3 Desirable zooplankton [i.e., adult copepods, cladocerans and ostracods (number L 1)] in mesocosms treatedwith various nitrogen sources from 20 May 2008 to 10 June 2008 Symbols represent means SE
Aquaculture Research, 2010, 41, 968^972 Fertilizer nitrogen types and water quality C C Mischke & P V Zimba
r 2009 The Authors
Trang 21weeks after stocking) Additional studies would be
needed to determine the longer-term e¡ects of these
fertilizers for situations requiring longer-term
fertili-zation practices Ammonium nitrate and urea
con-tain a higher percentage of nitrogen than other
nitrogen fertilizers, and so a smaller amount of
ferti-lizer would have to be added to the ponds Urea is
usually readily available, and may increase the
desir-able 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
pur-chased 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
de-sirable 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
ex-clusion of others that may be available
References
Boyd C.E & Tucker C.S (1998) Pond Aquaculture Water
Quality Management Kluwer Academic Publishers,
Bos-ton, MA, USA,700pp.
Geiger J.G & Turner C.J (1990) Pond fertilization and
zoo-plankton management techniques for production of
¢n-gerling striped bass and hybrid striped bass In: Culture
and Propagation of Striped Bass and its Hybrids (ed by
R.M Harrell, J.H Kerby & R.V Minton), pp 79^98
Ameri-can Fisheries Society, Bethesda, MD, USA.
Graves K.G & Morrow J.C (1998) Tube sampler for
zooplank-ton Progressive Fish-Culturist 50, 182^183.
HACH 1999 DR/4000 Spectrophotometer Procedure Manual HACH Chemical, Loveland, CO, USA.
Knud-Hansen C.F (1998) Pond fertilization: ecological proach and practical applications Pond Dynamics/Aquacul- ture Collaborative Research Support Program, Oregon State University, Corvallis, OR, USA, 125pp.
ap-Liang Y., Beardall J & Heraud P (2006) E¡ects of nitrogen source and UV radiation on the growth, chlorophyll £uores- cence and fatty acid composition of Phaeodactylum tricornu- tum and Chaetoceros muelleri (Bacillariophyceae) Journal of Photochemistry and Photobiology B: Biology 82, 161^172 Littell R.C., Milliken G.A., Stroup W.W & Wol¢nger R.D (1996) SAS System for Mixed Models SAS Institute, Cary,
NC, USA, 633pp.
Lourenco S.O., Barbarino E., Mancin-Filho J., Schinke J.K & Aidar E (2002) E¡ects of di¡erent nitrogen sources on the growth and biochemical pro¢le of 10 marine microalgae
in batch culture: an evaluation for aquaculture gia 41, 158^168.
Phycolo-Mischke C.C & Zimba P.V (2004) Plankton community responses in earthen channel cat¢sh nursery ponds under various fertilization regimes Aquaculture 233, 219^235.
Mischke C.C.,Wise D.J & Lane R.L (2003) Zooplankton size and taxonomic selectivity of channel cat¢sh fry North American Journal of Aquaculture 65, 141^146.
Paasche E (1971) E¡ect of ammonia and nitrate on growth, photosynthesis and ribosediphosphate carboxylase con- tent of Dunaliella tertiolecta Physiologia Plantarum 24, 294^299.
Syrett P.J (1981) Nitrogen metabolism of microalgae In: Physiological Bases of Phytoplankton Ecology (ed by T Platt), pp 182^210 National Research Council Canada, Ottowa, ON, Canada.
TepeY & Boyd C.E (2001) A sodium-nitrate-based, luble, granular fertilizer for sport ¢sh ponds North Amer- ican Journal of Aquaculture 63, 328^332.
water-so-Thorp J.H & Covich A.P (1991) Ecology and Classi¢cation of North American Freshwater Invertebrates Academic Press, San Diego, CA, USA, 911pp.
Zimba P.V., Dionigi C.P & Millie D.F (1999) Evaluating the lationship between photopigment synthesis and 2- methylisoborneol accumulation in cyanobacteria Journal
re-of Phycology 35, 1422^1429.
Zimba P.V., Tucker C.S., Mischke C.C & Grimm C.C (2002) Short-term e¡ect of diuron on cat¢sh pond ecology North American Journal of Aquaculture 64, 16^23.
Trang 22The antioxidant capacity response to hypoxia stress
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: yhchien@mail.ntou.edu.tw
Abstract
This study aimed to determine whether dietary
caro-tenoid (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 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 1
Normoxia was DO kept in saturation
Hypoxia led to an increase in the total antioxidant
status (TAS), superoxide dismutase (SOD),
glu-tathione peroxidases (GPx) and aspartate
amino-transferase (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
diet-ary 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
un-der hypoxia stress, because GPx, ALT and AST were
lower in AX-fed ¢sh Except TAS, the other four
en-zyme activities showed decreasing trends with
in-creasing dietary CD concentrations
Keywords: antioxidant capacity, astaxanthin,
b-carotene, Hyphessobrycon callistus, hypoxia,
superoxide dismutase
IntroductionTransport of live aquatic animals, especially duringtheir larval and fry stages, is a common procedure inaquaculture practice In ornamental ¢sh trade,transport of juvenile or adult ¢sh is characterized by
a long duration and small packaging.Various kinds ofacute and chronic stress, such as £uctuation in watertemperature, dissolved oxygen (DO), ammonia, pHand sometimes salinity, caused by handling, packa-ging, shipping, releasing and stocking (Taylor & Solo-mon 1979), are inevitable and result in unpredictablemortality and loss
When an organism is subjected to stress, a suddenshortage of oxygen causes abnormal oxidative reac-tions in the aerobic metabolic pathways, resulting inthe 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 areimportant parts of cellular constituents, includingmembranes, enzymes and DNA Radical damagecan be signi¢cant because it can proceed as a chainreaction
Ornamental ¢sh pigment is one of the most tant quality criteria dictating their market value.Var-ious synthetic [b-carotene (BC), canthaxanthin,zeaxanthin and astaxanthin (AX)] and natural(yeast, bacteria, algae, higher plants and crustaceanmeal) carotenoids (CD) have been used as dietarysupplements to enhance pigmentation of ¢sh andAquaculture Research, 2010, 41, 973^981 doi:10.1111/j.1365-2109.2009.02380.x
impor-r 2009 The Authors
Trang 23crustaceans (Shahidi, Metusalach & Brown 1998;
Ka-linowski, Robaina, Fernandez-Palacios, Schuchardt &
Izquierdo 2005) Dietary AX supplementation of
pe-naeid postlarvae increased not only bodyAX but also
enhanced the resistance to hypoxia (Chien, Chen,
Pan & Kurmaly 1999), salinity (Darachai,
Piyatiratiti-vorakul, Kittakoop, Nitithamyong & Menasveta 1998;
Merchie, Kontara, Lavens, Robles, Kurmaly &
Sorge-loos 1998; Chien, Pan & Hunter 2003), thermal
(Chien et al 2003), ammonia (Pan, Chien & Hunter
2003a) and pathological stressors (Pan Chien &
Hun-ter 2003b) Among the functions of AX in
aquacul-ture as proposed by Torrissen and Christiansen
(1995) and Shimidzu, Goto and Miki (1996),
antioxi-dant properties may be closely associated with stress
resistance The reason for the development of
resis-tance to stress from CDs can be attributed to the
anti-oxidant 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
ra-dicals in cells (Stanier, Kunizawa & Cohen-Bazire
1971) The antioxidant activity of AX was found to be
approximately 10 stronger than BC (Shimidzu
et al 1996)
In recent years, some enzymes involved in
protec-tion against active oxygen species, which are caused
by abnormal oxidative reactions at stress (Ranby &
Rabek 1978), have often been used to measure the
re-sponse to stress Total antioxidant status (TAS) is an
overall indicator of antioxidant defence against free
radical Superoxide dismutase (SOD), a cytosolic
en-zyme that is speci¢c for scavenging superoxide
radi-cals, is involved in protective mechanisms within
tissue injury following oxidative process and
phago-cytosis Oxidative stress was successfully assayed by
TAS and SOD (Seymen, Seven, Civelek, Yigit, Hatemi
& Burcak 1999; O’Brien, Slaughter, Swain,
Birming-ham, Greenhill, Elcock & Bugelski 2000)
Glu-tathione peroxidase (GPx) is involved in the reaction
of removal of H2O2and 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,
which are involved in protection against active gen species, are considered to be potential tools foridenti¢cation of stress caused by environmental fac-tors (Roche & Boge 1996) Aspartate aminotransfer-ase (AST) (or glutamate oxalate transaminase) andalanine aminotransferase (ALT) (or glutamate pyru-vate transaminase) are usually used as general indi-cators of the functioning of vertebrate liver High ASTand ALT generally, but not de¢nitively, indicate theweakening or damage of normal liver function Ala-nine aminotransferase and AST were often used asmarkers of hepatocellular injury (Seymen et al 1999;O’Brien et al 2000; Suzumura, Hashimura, Kubota,Ohmiza & Suzuki 2000)
oxy-Characins, Hyphessobrycon callistus (Boulenger), isone of the most important cultured and exported or-namental ¢sh in Taiwan This study aimed to com-pare the antioxidant capacity in Characins fed dietssupplemented with synthetic AX and/or BC at var-ious dietary concentrations when subjected to hypox-
ia stress during transportation
Materials and methodsDiet preparation
The control diet was composed of white ¢shmeal50%, wheat £our 15%, dextrin 27%, ¢sh oil 3%, vita-min mix 2% and mineral mix 3% Diets supplemen-ted with CD had the same composition as the controldiet (except for dextrin, which was adjusted depend-ing on the CD levels used) but supplemented witheither 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 diepress The extruded feed was air dried in the dark toprevent the degradation of CD The feed was thencrushed, sieved to attain a particle size of 0.9^1.2 mm and stored at 20 1C to avoid oxidation ofthe CD There were nine CD diets composed of 3 3factorial combinations of CD type (AX, BC and a 1:1mixture of AX and BC) and CD concentrations (10,
20 and 40 mg kg 1) Proximate analyses of thesediets are listed in Table 1
Fish rearing, feeding and samplingExperimental ¢sh were bought from an ornamental
¢sh farm During acclimatization in the laboratory
in a 0.5 tonne tank, ¢sh were fed the controldiet for 2 weeks to equalize their body CD
Trang 24contents Fish were then transferred to 30 aquaria
(44 cm 33 cm 21.5 cm) to receive their
respec-tive treatments (three replicates per treatment) at a
stocking density of 30 ¢sh/aquarium Fish size was
0.41 0.09 g Culture water was passed through a
1mm ¢lter and sterilized by ultraviolet light to
elimi-nate microalgae, a possible source of CD Moreover,
all aquaria were covered with a black screen to
dis-courage 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 1by constant aeration, a temperature of 26^
28 1C, pH of 7.5^8 and NH3of 0.1^0.2 mg L 1 Faeces
and uneaten feeds were siphoned out daily and
one-third of the water was exchanged The ¢sh were
reared for 8 weeks No mortality occurred
through-out the experiment The ¢nal overall average ¢sh size
was 0.89 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 0.4 1C and DO in saturation at 6.5 0.6
mg L 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 1
Each tle contained 2 g of zeolite, which is commonly used
bot-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
mon-itored After around 2.5 h when DO declined to
1.0 mg L 1and was maintained for 10 min, the
¢sh were taken out and blood samples were collectedthrough the branchial artery for the analysis of ser-
um antioxidants In the control group, the bottle wasnot capped and full aeration was provided
Analysis of antioxidant parameters and bloodprotein
Immediately after withdrawing the blood, sampleswere prepared by mixing 200mL isotonic NaCl solu-tion containing 0.94 mmol L 1 EDTA with 50mLblood The samples were chilled if not immediatelyused for determination of antioxidants: TAS, SOD,AST, ALT and GPx and blood protein
The di¡erent antioxidant parameters were lysed using Randox Laboratories kits (Crumlin,County Atrim, UK) by spectrophotometry (U-2000;Hitachi, Ibarake County, Japan) The volumes of ser-
ana-um samples used were 20mL for TAS and GPx, 25 mLfor SOD and 100mL for AST and ALT Activities wereexpressed in international enzyme unit (U L 1).Blood protein was determined using a protein as-say kit (No 500-0006, Bio-rad Laboratories, Rich-mond, 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
200mL of serum sample
Statistical analysisTwo two-wayANOVAs were performed: the ¢rst to de-termine the main e¡ects of dietary CD supplementa-tion and hypoxia stress on TAS, SOD, GPx, ALT andAST, and the second to determine the main e¡ects of
CD type and concentration on those antioxidant
ca-Table 1 Proximate analysis of the control and carotenoid diets
Nitrogen-free extracts and crude ¢bre.
Aquaculture Research, 2010, 41, 973^981 Antioxidant capacity response to hypoxia of characins C-H Pan et al.
r 2009 The Authors
Trang 25pacity 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
le-vel applied to all analysis was set to 5%
Results
Hypoxia stress increased the SOD, GPx and AST
ac-tivities 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 on TAS 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
ef-fects on SOD, GPX and AST, but not on TAS and ALT
Dietary CD type a¡ected all enzyme activities
ex-cept 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
ef-fect on TAS of the ¢sh exposed to hypoxia stress
Ex-cept TAS, the other four enzyme activities showed a
decreasing trend with increasing dietary CD
concen-trations The GPx of ¢sh fed with 40 mg kg 1CD was
the lowest among the various concentrations tested
The SOD, ALT and AST of ¢sh fed with 40 mg kg 1
CD were lower than those fed with 10 mg kg 1CD,
but not di¡erent from those fed with 20 mg kg 1CD
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
para-meters Superoxide dismutase, GPx, ALT and AST
had positive correlations among themselves (Table 4)
Discussion
Hypoxia stress
Numerous signi¢cant studies and reviews have been
carried out on the physiological and biochemical
re-sponses 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 1
(equivalent to
2 mL O2L 1or 91.4 mM) (Diaz & Rosenberg 1995)
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 1 serum)
Diet
Control Carotenoid Mean
Environmentw Normoxia x 1.14 a (0.03) y 1.17 a (0.04) y 1.16 (0.04) Hypoxia x 1.12 b (0.01) x 1.31 a (0.11) x 1.29 (0.12) Mean 1.13 a (0.02) 1.24 a (0.11)
SOD (U mg 1protein)
Diet
Control Carotenoid Mean
Environmentw Normoxia y 0.59 a (0.10) y 0.47 b (0.07) y 0.48 (0.08) Hypoxia x 1.35 a (0.46) x 0.60 b (0.07) x 0.67 (0.26) Mean 0.97 a (0.51) 0.53 b (0.10)
GPx (U mg 1protein)
Diet
Control Carotenoid Mean
Environmentw Normoxia y 43.50a(1.98) y 15.99b(2.21) y 18.74 (8.73) Hypoxia x 77.15a(1.91) x 55.02a(16.91) x 57.23 (17.40) Mean 60.33 a (19.49) 35.50 b (23.09)
ALT (U mg 1 protein)
Diet
Control Carotenoid Mean
Environmentw Normoxia x 7.75 a (0.49) x 3.62 b (1.91) x 4.04 (2.21) Hypoxia x 8.35 a (0.35) x 3.74 b (1.52) x 4.21 (2.02) Mean 8.05 a (0.49) 3.68 b (1.70)
AST (U mg 1protein)
Diet
Control Carotenoid Mean
Environmentw Normoxia y 8.70 a (0.85) y 6.20 b (1.70) y 6.45 (1.79) Hypoxia x 35.00 a (0.07) x 21.46 a (10.58) x 22.81 (10.83) Mean 21.83 a (15.16) 13.83 a (10.75)
In Duncan’s multiple range test, means in the same row with di¡erent superscripts or in the same column with di¡erent sub- scripts are signi¢cantly di¡erent (P 0.05).
Control: no carotenoid supplemented in diet; Carotenoid: tenoid (astaxanthin, carotene or mix of the two) supplemented at
caro-10, 20 or 40 mg kg 1
in diet.
wNormoxia: dissolved oxygen (DO) remained at 6.5 mg L 1
; poxia: DO declined from 6.5 to o1.0 mg L 1
hy-in 2.5 h and mained at o1.0 mg L 1
re-for 10 min.
TAS, total antioxidative status; SOD, superoxide dismutase; GPx, glutathione peroxidase; ALT, alanine transaminase; AST, aspar- tate transaminase.
Trang 26In our study, ¢sh was exposed to low DO
( 1.0 mg L 1
) for around 10 min, which could be
well justi¢ed as under hypoxia stress Low oxygen
availability may result in oxidative stress, which is
characterized by excessive ROS (Sies 1991; Storey
1996) In this study, when the ¢sh was exposed to
hy-poxia stress, abnormally high levels of oxygen
radi-cals could have been potentially generated as shown
by an increase in SOD (129%), GPx (77%) and AST
(302%) compared with the normoxia condition in
¢sh fed with control diets (Table 2) The increase inSOD and GPx activity under hypoxia stress is inagreement with few studies on this aspect Hypoxiaappeared to trigger SOD activity in the gill and mus-cle tissue of an estuarine ¢sh exposed to 10% DO sa-turation for 12 h (Cooper, Clough, Farwell & West2002) Following exposure to hypoxia,Vig and Nemc-sok (1989) found increases in SOD activity in the gill,liver and brain of common carp (Cyprinus carpio L.).Hypoxia exposure (25% of normal oxygen level) for
5 h also stimulated an increase in GPx in the brain
of common carp by 1.3-fold (Lushchak, Lushchak,Mota & Hermes-Lima 2001)
TASHypoxia stress also increased TAS of the ¢sh fed the
CD diet but had no e¡ect on TAS of the ¢sh fed thecontrol diet (Table 2) The antioxidant capacity thatTAS expresses includes enzymatic and non-enzy-matic antioxidant activities The higher the TAS va-lue, the higher the antioxidant capacity In thisstudy, the higher TAS value in stressed ¢sh than that
in normal ¢sh might be attributed to the enzymaticand/or non-enzymatic antioxidant activity induced
by hypoxia stress In the study by Chien et al (2003),TAS of tiger prawn juvenile was not in£uenced byeither osmotic stress (32% salinity decline) or ther-mal stress (22 1C temperature decline) but by the in-teraction of both In this study, all types and
Table 3 After hypoxia stress,the average and standard deviation (in parentheses) activities of serum antioxidation zymes of Characins (Hyphessobrycon eques Steindachner) fed diets supplemented with various type and concentration of car- otenoid
(0.06) (0.08) (0.08) (0.07) (0.06) (0.04) GPx (U mg 1 protein) 40.08 b 60.47 a 64.5 a 67.4 x 56.17 y 41.48 z 0.48
(14.79) (13.05) (13.13) (15.06) (8.83) (16.20) ALT (U mg 1 protein) 2.58 b 4.23 a 4.42 a 4.72 x 3.80 xy 2.72 y 0.92
(0.62) (1.41) (1.75) (1.51) (1.01) (1.47) AST (U mg 1 protein) 13.30 b 23.83 a 27.25 a 30.13 x 19.05 y 15.20 y 0.80
(8.10) (9.20) (10.17) (5.90) (10.26) (9.81)
In Duncan’s multiple range test, means without a common superscript are signi¢cantly di¡erent (P 0.05).
Dissolved oxygen declined from 6.5 to o1.0 mg L 1
in 2.5 h and remained at o1.0 mg L 1
for 10 min.
wInteraction e¡ects of two main e¡ects ^ carotenoid type and carotenoid concentration and the probability to be signi¢cant.
AX, astaxanthin; BC, b-carotene; MX, mix of half AX and BC; TAS, total antioxidative status; SOD, superoxide dismutase; GPx, tathione peroxidase; ALT, alanine transaminase; AST, aspartate transaminase.
glu-Table 4 Correlation coe⁄cient and signi¢cant levelsw of a
correlation matrix (n 5 30) between antioxidant parameters
of Characins (Hyphessobrycon eques Steindachner) fed diets
supplemented with combinations of various types and
con-centrations of carotenoid for 8 weeks.
TAS, total antioxidant status; SOD, superoxide dismutase; GPx,
glutathione peroxidase; ALT, alanine transaminase; AST,
aspar-tate transaminase.
Aquaculture Research, 2010, 41, 973^981 Antioxidant capacity response to hypoxia of characins C-H Pan et al.
r 2009 The Authors
Trang 27concentrations of dietary CD used had no e¡ects on
TAS in this ¢sh This could be attributed to the
insen-sitive response of this ¢sh towards TAS
SOD
When the ¢sh was exposed to hypoxia stress,
abnor-mally high levels of oxygen radicals could have been
potentially generated as shown by a 40% increase in
SOD under stress than under a normal condition
in ¢sh fed with control or CD-supplemented diets
(Table 2) Under hypoxia stress, SOD of CD-fed ¢sh
was 56% lower than that of control ¢sh (Table 2)
The decrease in SOD with increasing dietary CD
con-centrations (Table 3) indicated that dietary CD
e¡ec-tively reduced SOD in ¢sh Because SOD is a
superoxide reductase that can protect the tissue from
damage by the oxidative process and phagocytosis,
the lower the SOD value the higher the protection of
the cells Without hypoxia stress, SOD of CD-fed ¢sh
was already 20% lower than that of control ¢sh
(Table 2) It is speculated that after feeding with the
CD diet, the increase in body CD can result in more
oxygen reserves within the cells (Ghidalia 1985;
Latscha 1990; Oshima, Ojima, Sokamoto, Ishiguro &
Terao 1993) or can inhibit the singlet oxygen reaction
that is induced by stress (Di Mascio, Murphy & Sies
1991) Consequently, the need to produce SOD to
sca-venge superoxide radicals was reduced Under
hypox-ia stress, although SOD of CD-fed ¢sh was lower than
that of control ¢sh, no di¡erences in SOD were found
among the ¢sh fed with various CD (Table 3),
indicat-ing that the protection against superoxide radicals
was similar among CD types
GPx
A signi¢cant correlation between GPx and SOD
(Table 4) could indicate that the removal of H2O2by
GPx was related to the reaction of superoxide radicals
by SOD Lushchak et al (2001) found that SOD
and GPx activity all increased under hypoxia stress,
showing a synergistic relationship between SOD and
GPx Despite such a correlation, there was still some
discrepancy between GPx and SOD in the oxidative
reaction The response pattern of GPx to hypoxia
stress was similar to that of SOD but stronger: 40%
higher in stressed ¢sh than in normal ¢sh for SOD
compared with 205% for GPx (Table 2) When under
hypoxia stress, dietary CD reduced SOD production
but had no e¡ects on GPx, as no di¡erence in GPx
was found between CD-fed ¢sh and control ¢sh Alsolike SOD, GPx decreased with increasing dietary CDconcentration (Table 3) However, di¡erent from SOD,which responded equally to various dietary CD types,GPx was a¡ected by CD type Astaxanthin-fed ¢shhad the lowest GPx, being 50.87% and 60.93% lowerthan GPx of BC-fed ¢sh and MX-fed ¢sh respectively.This indicated that AX could be most e¡ective in re-moving H2O2induced by hypoxia stress Di Mascio
et al (1991) found that AX had twice the ability as BCand 80 times that of vitamin E in inhibiting the gen-eration of singlet oxygen Terao (1989) pointed outthat AX was 50% stronger in eliminating free radi-cals than BC In inhibiting the formation of lipidsuperoxide, AX was also more e¡ective than BC(Lim, Nagao, Tera, Tanaka, Suzuki & Takama 1992).The peroxyl-trapping activity of AX was greater thanthat of BC (Jorgensen & Skibsted 1993) Both AX and
BC inhibited the production of lipid peroxides, AXbeing about 2-fold more e¡ective than BC The e⁄-cient antioxidant activity of AX is suggested to bedue to the unique structure of the terminal ring moi-ety (Goto, Kogure, Abe, Kimata, Kitahama,Yamashita
& Terada 2001)
ALT and ASTALT and AST were highly correlated (Table 4) as bothare indices for the diagnosis of liver function (Ozaki1978; Yamamoto 1981) and damage (Oda 1990), buttheir reaction may not be consistent in all aspects
In this study, hypoxia stress had no e¡ect on ALT tivity but increased ASTactivity by 254% Dietary CDreduced ALT activity but had no e¡ect on AST How-ever, both ALT and AST showed the same decreasingtrend when the dietary CD concentration increased.Our results are in accordance with the studies ofNakano, Tosa and Takeuchi (1995) and Nakano,Kanmuri, Sato and Takeuchi (1999), in which dietary
ac-AX signi¢cantly decreased the levels of lipid ides in the liver and reduced the ALTof rainbow trout
perox-A recent study showed that CD treatment cantly altered the total lipid pro¢le and hepatic mu-copolysaccharide contents of the livers of rainbowtrout (Page, Russell & Davies 2005) Both ALT andAST in AX-fed ¢sh were lower than BC-fed and MX-fed ¢sh Overall, AX-fed ¢sh had 63.95^104.89% high-
signi¢-er capability in reducing ALT and AST than BC-fedand MX-fed ¢sh This clearly shows that AX can pro-tect liver damage caused by hypoxia stress betterthan BC and MX
Trang 28In this study, ¢sh fed the CD supplement had lower
SOD, GPx and ALT activities than control ¢sh,
show-ing that dietary CD could increase the antioxidant
capacity and protection of the liver However, the
ef-fects of various dietary CD on the activity of these
en-zymes also varied In general, dietary AX was more
e¡ective in terms of antioxidant capacity than BC
alone and MX when under hypoxia stress This is
evi-denced by lower GPx, ALT and AST in AX-fed ¢sh
than in BC-fed and MX-fed ¢sh As for the e¡ects of
dietary CD concentration, although SOD, ALT and
AST in ¢sh fed with 40 mg kg 1dietary CD were
not di¡erent from those fed with 20 mg kg 1, GPx in
¢sh fed with 40 mg kg 1dietary CD was still lower
than GPx in ¢sh fed with 20 mg kg 1 The
decreas-ing trend of all these four activities with the increase
in the CD concentration, as shown in this study,
indi-cates that the most e¡ective CD supplementation for
maximal protection through antioxidant capability
against hypoxia stress is at least 40 mg kg 1AX
Acknowledgment
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
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reac-r 2009 The Authors
Trang 31Effect of replacing soybean meal with canola meal
on growth, feed utilization and haematological indices
Qi-Cun Zhou & Yi-Rong Yue
Laboratory of Aquatic Economic Animal Nutrition and Feed, College of Fisheries, Guangdong Ocean University, Zhanjiang
524025, China
Correspondence: Q-C Zhou, Laboratory of Aquatic Economic Animal Nutrition and Feed, College of Fisheries, Guangdong Ocean versity, Zhanjiang 524025, China E-mail: zhouqc@gdou.edu.cn or qicunzhou@tom.com
Uni-Abstract
An 8-week feeding trial was conducted to evaluate
the e¡ects of replacing soybean meal (SBM) with
ca-nola meal (CM) on growth, feed utilization, body
composition and haematological indices of juvenile
hybrid tilapia (Oreochromis niloticus Oreochromis
aureus) Six isonitrogenous diets containing graded
levels of CM (0, 95, 190, 285, 380 and 634 g kg 1of
diet corresponding to 0%, 15%, 30%, 45%, 60%
and 100%, respectively, of protein from SBM) to
re-place SBM on an equal protein basis were fed to
tripli-cate groups of juvenile ¢sh (initial weight 5 6.3 g)
The results indicated that up to 30% of SBM could
be replaced by CM without causing a signi¢cant
reduction in growth performance Fish fed with diets
in which CM replaced over 45% of SBM had a
cantly lower protein e⁄ciency ratio and a
signi¢-cantly higher feed conversion ratio than ¢sh fed
with other diets The apparent digestibility
coe⁄-cients (ADC) of dry matter, protein and phosphorus
were lowest for ¢sh fed the CM100 diet Signi¢cant
di¡erences in haemoglobin, haematocrit and white
blood cell concentration were found in ¢sh fed diets
with di¡erent CM levels It is concluded that up to
19.02% CM can be used to replace 30% of SBM in
diets for juvenile hybrid tilapia without
compromis-ing growth, feed conversion and protein utilization
Keywords: hybrid tilapia, canola meal, soybean
meal, growth, haematology
IntroductionTilapia are widely cultured in many tropical and sub-tropical regions of the world and constitute the thirdlargest group of farmed ¢n¢sh (after carps and sal-monids), with more than 22 species being culturedworldwide (El-Sayed 1999) In the past, the tilapiawas mainly consumed in Africa and Asia, but in re-cent years, it has been touted as the‘new white ¢sh’toreplace the depleted ocean stocks of cod and hake(Costa-Pierce 1997), such that the demand for tilapiahas become worldwide As the international trade oftilapia products developed, tilapia aquaculture inChina expanded rapidly, especially in the southernprovinces of China To sustain such expanded tilapiatrade development, a matching increase in ¢sh feedproduction is imperative Because of the high price
of ¢sh meal, it is used sparingly in commercial feedsfor tilapia in China; whereas, soybean meal (SBM) iswidely used as a main protein source in tilapia feed.Soybean meal is the leading oilseed crop producedglobally and was projected to exceed 200 mmt al-ready in 2004^2005 (Gatlin III, Barrows, Brown,Dabrowski, Gaylord, Hardy, Herman, Hu, Krogdahl,Nelson, Overturf, Rust, Sealey, Skonberg & Souza2007) Soybean meal is considered to be one of themost nutritious of all protein feedstu¡s and is cur-rently the most commonly used plant protein source
in ¢sh feeds (Lovell 1988; El-Sayed 1999) Because ofits high protein content, high digestibility, relativelywell-balanced amino acid pro¢le, reasonable price
Trang 32and steady supply, SBM is widely used as a
cost-e¡ec-tive feed ingredient for many aquaculture animals
(Storebakken, Refstie & Ruyter 2000) Moreover,
when SBM inclusion in aquafeed exceeds a certain
level, impaired utilization occurs due to reduced feed
intake, growth and development of intestinal
enteri-tis; these conditions may be related to antinutrients
such as lipoxygenase, lectins, urease, trypsin
inhibi-tors, oestrogenic compounds and phytic acid as well
as possibly by an immunological-based food
intoler-ance that is essentially a food allergy (Gatlin III et al
2007) However, other plant protein sources
gener-ally cost less than SBM, such as canola meal (CM)
and cottonseed meal; thus, replacing SBM with less
expensive plant protein sources would be bene¢cial
in reducing feed costs (Barros, Lim & Klesius 2002)
Canola meal as a potential substitute for ¢sh meal
has been the object of numerous studies with
rain-bow trout (Yurkowski, Bailey, Evans, Tabachek, &
Burton Ayles 1978; Hilton & Slinger 1986; Mccurdy &
March 1992; Gomes, Corraze & Kaushik 1993),
Chi-nook salmon (Higgs, McBride, Markert, Dosanjh,
Plotniko¡ & Clarke 1982), tilapia (Sarotherodon
mos-sambicus) (Jackson, Capper & Matty 1982; Davies,
McConnell & Bateson 1990), channel cat¢sh
(Web-ster, Tiu, Tidwell & Grizzle 1997) and shrimp
(Litope-naeus vannamei) (Lim, Beames, Eales, Prendergast,
Mcleese, Shearer & Higgs 1997) However, in relation
to supply, the quality of canola/rapeseed protein that
is potentially available for inclusion in aquatic animal
diets surpasses the global amount of ¢sh meal
pro-tein that is produced each year (Higgs, Dosanjh,
Beames, Prendergast, Mwachireya & Deacon 1996)
In general, the nutritional value of CM to ¢sh was
re-ported to vary according to the amount of residual oil
content of the meal, the levels of glucosinolates in the
meal and the need to ensure that these glucosinolates
do not impair circulating thyroid hormone levels
and/or feed intake (Burel, Boujard, Kaushik, Boeuf,
Mol, Van der Geyten, Darras, Kuhn, Pradet-Balade,
Querat, Quinsac, Krouti & Ribaillier 2001) The
incor-poration of CM containing high levels of
glucosino-lates to animal feed may lead to reduced feed intake,
enlarged thyroid, reduced plasma thyroid hormone
levels and occasionally organ (liver and kidney)
ab-normalities and even mortality (VanEtten & Tookey
1983) Limited information is available on the
poten-tial adverse e¡ects of CM on ¢sh
Therefore, this study was undertaken to (1)
evalu-ate growth performance, feed and protein utilization
and digestibility of nutrients of hybrid tilapia diets in
which SBM was incrementally replaced with CM and
(2) investigate the e¡ect of replacing SBM with CM onthe body composition and haematology of juvenilehybrid tilapia
Material and methodsSix isonitrogeous (containing about 320 g kg 1crude protein) and isoenergetic experimental dietswere formulated and a proximate analysis of the diets
is mentioned in Table 1 The experimental diets wereformulated to produce diets in which 0 (CM0), 15(CM15), 30 (CM30), 45 (CM45), 60 (CM60) and 100%(CM100) of protein from SBM were replaced with thatfrom CM The diets were supplemented with lysine to
a level similar to that in a CM0 diet and to satisfy therequirement for juvenile hybrid tilapia (Yue & Zhou2008) Fish oil and soybean oil (w/w 51:1) wereadded to keep the lipid and energy constant in alltreatments All of the dry ingredients were thor-oughly mixed until homogeneous in a Hobart-typemixer, and then lipid and water were added and thor-oughly mixed Two-millimetre-diameter pellets werewet-extruded, airdried to about 10.0% moisture andsealed in vacuum-packed bags and stored frozen( 20 1C) until feeding
Juvenile hybrid tilapias (Oreochromis niloticusOreochromis aureus) were obtained from a local tila-pia breeding farm Before the start of the feeding trial,
¢sh were acclimated to the experimental conditionsand fed a commercial diet (CP 5 32%, lipid 5 5.6%)for 2 weeks At the beginning of the feeding trial, ¢sh(initial weight 6.3 g) were weighed, and sorted into
18, 500 L cylindrical ¢breglass tanks, with 20 ¢sh ineach tank Three replicate groups of ¢sh were ran-domly assigned to each diet They were provided with
a continuous £ow of water (2 L min 1) with ous aeration to maintain the dissolved oxygen level atsaturation Fish were fed to apparent satiation threetimes daily during the feeding trial The amount offeed consumed by the ¢sh in each tank was recordeddaily, and the rations were adjusted according to thefeed consumed the previous day Tanks were cleanedweekly, and the feeding trial lasted for 8 weeks.Waterquality parameters were monitored daily between09:00 and 15:00 hours During the feeding trial, tem-peratures ranged from 28 to 30 1C, pH from 7.4 to 7.6,ammonia nitrogen was o0.05 mg L 1, nitrite waso0.04 mg L 1, nitrate was o1.0 mg L 1 and dis-solved oxygen was not less than 6.0 mg L 1.Triplicate groups of ¢sh were fed the experimentaldiets to visual satiety at 18:00 hours daily Two hoursAquaculture Research, 2010, 41, 982^990 E¡ect of replacing SBM with CM Q-C Zhou & Y-R Yue
continu-r 2009 The Authors
Trang 33after feeding, the uneaten feed and faecal residues
were removed Faeces were then allowed to settle
over-night, and faecal samples were collected at 08:00
hours each morning before the next feeding Faeces
collected from the settling columns were immediately
¢ltered with ¢lter paper or maintained for 60 min at
4 1C and stored at 18 1C for chemical analyses Daily
faecal samples from each tank were pooled over the
course of the experiment until su⁄cient sample was
available for chemical analyses (Yue & Zhou 2008)
At the termination of the 8-week feeding trial, ¢sh
in each tank were individually weighed and sampled
for tissue analysis 24 h after the last feeding Twenty
¢sh at the start were sampled and stored frozen
( 18 1C) for the analysis of whole-body composition
Three ¢sh from each tank were used for whole-body
composition analysis, and the livers and viscera of
¢ve ¢sh per tank were weighed for the calculation of
hepatosomatic index (HSI) and viscerosomatic index
(VSI) Dorsal muscles of ¢sh were sampled, sealed in
plastic bags and stored frozen ( 18 1C) until analysis
for the muscle nutrient composition Blood samples
were drawn from the caudal vein of ¢ve ¢sh from
each tank; these samples were considered to be
repli-cates and were used to determine blood tics, according to the method described by Barros
characteris-et al (2002)
Crude protein, crude lipid, moisture and ash indiets and whole-body samples were determined fol-lowing standard methods (AOAC 1995) Moisturewas determined by oven-drying at 105 1C until a con-stant weight was achieved Crude protein (N 6.25)was determined using the Kjeldahl method after aciddigestion with an Auto Kjeldahl System (1030-Auto-analyzer,Tecator, Hoganos, Sweden) Crude lipid wasdetermined using the ether-extraction method with
a Soxtec System HT (Soxtec System HT6, Tecator).Ash content was determined after placing the sam-ples in a mu¥e furnace at 550 1C for 24 h Chromicoxide content in diets and faeces were determined
by acid digestion with nitric acid and perchloric acid,according to the method described by Zhou,Tan, Maiand Liu (2004)
The following variables were calculated:
Weight gainðWG; %Þ
¼ 100 Final body weight initial body weight
Initial body weight
Table 1 Formulation and proximate analysis of the experimental diets (g kg 1dry matter)
Ingredient (%)
Experimental diets
Fish meal 60.0 60.0 60.0 60.0 60.0 60.0 Soybean meal 560.0 476.0 392.0 308.0 224.0 0.0 Canola meal 0.00 95.1 190.2 285.3 380.4 634.3 Wheat middlings 251.3 251.3 251.3 251.3 251.3 251.3 Fish oil/soy oil (1:1) 20.0 20.0 20.0 20.0 20.0 20.0 Dicalcium phosphate 15.0 15.0 15.0 15.0 15.0 15.0 Vitamin mixture 2.0 2.0 2.0 2.0 2.0 2.0 Choline chloride 0.5 0.5 0.5 0.5 0.5 0.5 Mineral mixture 5.0 5.0 5.0 5.0 5.0 5.0 Chromium oxide 5.0 5.0 5.0 5.0 5.0 5.0
Cellulose 81.2 69.0 56.9 44.8 32.6 0.0 Proximate composition (g kg 1 )
Dry matter 910.2 906.5 903.7 912.1 907.4 908.6 Crude protein 321.1 318.7 320.2 322.5 325.7 321.4 Crude lipid 32.2 33.3 32.5 34.0 32.2 31.4
Fibre 119.3 113.4 107.5 101.7 95.8 79.1 Nitrogen-free extractw 465.2 470.1 472.9 472.5 475.6 491.1 Phosphorus 9.6 10.0 10.7 11.2 11.8 12.9 Gross energyz (MJ kg 1 ) 12.05 12.09 12.15 12.30 12.35 12.39
Mineral mixture and vitamin mixture according to Yue and Zhou (2008).
wNitrogen-free extract, calculated as 100 (protein1lipid1ash1¢bre).
zGross energy was calculated using the equivalents 18.81, 35.57 and 14.59 MJ kg 1 g for protein, lipid and digestible carbohydrate spectively.
Trang 34¼ 100 Final amount of fish
Initial amount of fish
Specific growth rateðSGRÞ
¼ 100 ½lnðfinal weightÞ lnðinitial weightÞ
timeFeed conversion ratioðFCRÞ
¼ feed consumed ðg; dry weightÞ=weight gain ðgÞ
Protein efficiency ratioðPERÞ
¼ weight gain ðgÞ=protein intake ðgÞ
Condition factorðCFÞ ¼100 ðbody weight; gÞ=
ðbody length; cmÞ3Hepatosomatic indexðHSIÞ
¼ 100 ðliver weight; g=whole body weight; gÞ
Viscerosomatic indexðVSIÞ
¼ 100 ðviscera weight; gÞ=
ðwhole body weight; gÞ
Results were expressed as mean sd All data
were subjected to one-way analysis of variance
When signi¢cant di¡erences occurred, the group
means were further compared with Duncan’s
multi-ple-range tests All statistical analyses were
per-formed using theSPSS11.5 (SPSS, IL, USA)
Results
The data for apparent digestibility coe⁄cients (ADCs)
of dry matter, protein, lipid and phosphorus are
pre-sented in Table 2 The ADCs of dry matter, protein and
phosphorus were signi¢cantly a¡ected by the
repla-cement level of SBM with CM The ADC of dry matter
for the diets in which SBM was replaced with CM
from 0% to 30% was signi¢cantly higher than the
other diets, and signi¢cantly decreased with the
re-placement level from 45% to 100% The ADCs of
pro-tein for the CM100 diet were signi¢cantly lower thanthe other diets, with no signi¢cant di¡erences found
in CM replacement level from 0% to 60% The ADCs
of lipid did not di¡er among diets Finally, the ADCs ofphosphorus for the CM100 diet were lower than theother diets, while no signi¢cant di¡erences werefound in those diets in which CM level increased from0% to 60%
The growth, feed utilization e⁄ciency, HSI,VSI and
CF data are shown in Table 3 High survival was served in all treatment and survival did not di¡er sig-ni¢cantly among treatments Weight gain (WG),speci¢c growth rate (SGR), protein e⁄ciency ratio(PER) and feed conversion ratio (FCR) were signi¢-cantly a¡ected by the replacement level, with WGand SGR signi¢cantly decreasing as the level of repla-cement of SBM with CM was over 45% Protein e⁄-ciency ratio of ¢sh fed the CM60 and CM100 dietswas signi¢cantly lower that those of ¢sh fed the otherdiets Feed conversion ratio of ¢sh fed the CM30 dietwas signi¢cantly lower than those of ¢sh fed theCM45, CM60 and CM100 diets These results indi-cated that up to 30% of SBM protein can be replacedwith CM without causing a signi¢cant reduction ingrowth and feed utilization
ob-No signi¢cant di¡erences among treatments wereobserved for the condition factor (CF) of juvenile hy-brid tilapia However, the HSI and VIS were signi¢-cantly a¡ected by the replacement level of SBM withCM; the HSI signi¢cantly increased as the replace-ment level increased, and the HSI and VIS of ¢sh fedthe CM100 diet was signi¢cantly higher than those of
¢sh fed the CM0 diet
Whole-body composition data are presented in ble 4 No signi¢cant di¡erences among treatmentswere detected for the ash content in wholebody sam-ples, but moisture, protein and lipid content in whole-body were signi¢cantly a¡ected by dietary CM level.The haematological data are presented in Table 5.Red blood cell was signi¢cantly a¡ected by the diet-
Ta-Table 2 Apparent digestibility coe⁄cients (ADC) of dry matter, protein, lipid and phosphorus of juvenile hybrid tilapia fed the experimental diets
Aquaculture Research, 2010, 41, 982^990 E¡ect of replacing SBM with CM Q-C Zhou & Y-R Yue
r 2009 The Authors
Trang 35ary CM supplemental level.White blood cell and
hae-matocrit values of ¢sh fed the CM45 diet were
signi¢-cantly higher than those of ¢sh fed the control diet
Haemoglobin of ¢sh fed the control and CM100 diets
were signi¢cantly lower than those of ¢sh fed the
CM30, CM45 and CM60 diets
Discussion
The present study indicates that CM can be
incorpo-rated in the diets of hybrid tilapia up to a level of
19.02%, which can replace 30% protein of SBM, out signi¢cant negative e¡ects on growth perfor-mance and feed utilization The WG, SGR and PER ofjuvenile hybrid tilapia fed diets in which the replace-ment level of SBM with CM was over 30% were signif-icantly lower than those in the other dietarytreatments Our results agree with the previous study
with-of Davies et al (1990), who reported that only15% CMcould e¡ectively replace FM/SBM in tilapia (Oreochro-mis mossambicus) diets Similar results were also ob-tained in rainbow trout (Yurkowski et al 1978; Hardy
& Sullivan 1983; Hilton & Slinger 1986; Leatherland,
Table 3 Growth performance and feed utlization of juvenile hybrid tilapia fed the experimental diets
Item
Experimental diets
IBM (g) 6.26 0.11 6.22 0.10 6.31 0.16 6.22 0.08 6.29 0.20 6.27 0.16 FBW (g) 65.11 3.99a 62.66 2.30ab 63.08 1.24ab 59.21 0.97bc 57.14 2.39c 49.57 2.19d
WG (%) 939.35 67.94a 906.89 22.69ab 900.14 45.55ab 852.63 25.89bc 809.91 57.72c 691.06 14.85d SGR (%) 3.99 0.03ab 4.00 0.11ab 4.11 0.08a 3.99 0.01ab 3.88 0.05b 3.63 0.08c Survival (%) 90.00 5.00 93.33 7.64 100.00 0.00 98.33 2.89 96.67 5.77 96.67 5.77 FCR 1.27 0.03d 1.34 0.03cd 1.35 0.05cd 1.37 0.01c 1.45 0.01b 1.70 0.02a PER 2.47 0.06a 2.33 0.09a 2.32 0.04a 2.26 0.02ab 2.12 0.01b 1.83 0.02c HSI 1.42 0.21b 1.54 0.16ab 1.56 0.03ab 1.69 0.26ab 1.69 0.08ab 1.73 0.10a VSI 7.98 0.55b 7.76 0.84b 8.11 0.28ab 8.15 0.25ab 8.36 0.26ab 8.93 0.41a
CF 3.79 0.16 3.84 0.08 3.83 0.20 4.05 0.08 3.80 0.12 3.82 0.14 Values are mean SEM Values in the same row with di¡erent alphabets are signi¢cantly di¡erent (Po0.05).
WG, weight gain; SGR, speci¢c growth rate; FCR, feed conversion ratio; PER, protein e⁄ciency ratio; HSI, hepatosomatic index; VSI, viscerosomatic index; CF, condition factor.
Table 4 Wholebody composition of juvenile hybrid tilapia fed the experimental diets
Composition (g kg 1wet weight)
Experimental diets
Moisture 732.8 5.6ab 738.6 6.7a 737.1 6.7a 735.1 7.2ab 723.9 7.4b 728.6 3.4ab Ash 40.3 1.5 40.3 1.8 41.1 1.4 42.8 1.5 43.9 1.3 41.9 1.9 Protein 165.8 8 1ab 168.2 2.8b 165.9 2.6ab 161.9 8.4ab 160.9 4.7a 158.5 8.8a Lipid 52.2 2.7a 50.3 1.6a 53.2 2.4ab 51.5 2.9a 60.8 3.9b 59.7 2.8b Values are mean SEM Values in the same row with di¡erent alphabets are signi¢cantly di¡erent (Po0.05).
Table 5 Concentration of haematological characteristics of juvenile hybrid tilapia fed the experimental diets
Trang 36Hilton & Slinger 1987; Gomes et al 1993) However,
Jackson et al (1982) indicated that none of the diets
(25%, 50% and 75% FM protein replacement with
ra-peseed meal) were obviously limited even when 75%
FM protein was replaced by rapeseed meal; the
de-crease in growth rate at this level may have been due
to the toxic e¡ects of glucosinolates As a matter of
fact, due to cost, ¢sh meal is not used or less used in
tilapia diets in China (Yue & Zhou 2008) Moreover,
Webster et al (1997) reported that channel cat¢sh
can be fed diets containing up to 36% CM without
ad-verse e¡ects on growth or body composition
Suppression of feed intake may be one of the main
reasons for reduced growth performance of aquatic
animals as the dietary concentration of CM protein
products increase (Lim & Klesius 1997) Some of the
antinutritional factors in CM such as sinapine, are
known to impart a bitter taste to animals (McCurdy
& March 1992) Hilton & Slinger (1986) reported that
the reduced growth of rainbow trout fed CM-based
diets was due to a reduced diet intake In the present
study, when SBM protein replacement level with CM
was over 30%, feed utilization decreased
signi¢-cantly, perhaps because of poor palatability, which
may lead to a lower feed intake These ¢ndings
sug-gest that hybrid tilapia cannot be cultured
success-fully using a diet containing CM as the single
protein source Other studies have reported that
ra-peseed and canola protein concentrates could not
to-tally replace ¢sh meal in diets for rainbow trout
unless a feed attractant was added (Higgs, Dosanjh,
Prendergast, Beames, Hardy, Riley & Deacon 1995)
Davies et al (1990) reported that PER and net
pro-tein utilization (NPU) in tilapia were not di¡erent in
¢sh fed a diet with 30% CM compared with ¢sh fed a
control diet, although growth was reduced Similar
results were found for channel cat¢sh (Webster et al
1997) These results may indicate that reduced
growth was due to an imbalance in the amino acid
composition of CM, or reduced protein digestibility
In the present study, PER and protein digestibility
were similar among ¢sh fed the control diet and those
diets that contain between 15% and 45% CM,
reveal-ing that the protein value of diets with up to 45% CM
were similar to a control diet
The depression of growth performance could be
at-tributed to several factors, among which the presence
of the following antinutrients is likely important:
(1) The glucosinolate metabolites, such as
isothio-cyanates, vinyloxazolidinethiones, have a
goitro-genic activity in ¢sh (Yurkowski et al.1978; Hardy
& Sullivan1983; Leatherland et al.1987; Hossain &
Jauncey 1988; Teskeredzic, Higgs, Dosanjih,McBride, Hardy, Beames, Jones, Simell, Vaara &Bridges 1995; Burel, Boujard, Esca¡re, Kaushik,Boeuf, Mol, Geyten & Kuhn 2000) Furthermore,
a deleterious e¡ect of isolated isothiocyanates onthe digestive utilization of nutrients, togetherwith thyroid disturbances, have been reported incarp (Cyprinus carpio) (Hossain & Jauncey 1988)
In the present study, with the increase of dietary
CM, growth performance and feed utilization ciency decreased signi¢cantly The reason may bedue to the dietary glucosinolates concentrationincreasing It is well known that glucosinolatesare compounds found in canola or rapeseed meal,and their hydrolysis products are antithyroid sub-stances High glucosinolates in diet will inhibitthe organic binding of iodine, and may cause phy-siological e¡ects in ¢sh ((Gatlin III et al 2007).(2) Phytate can reduce the bioavailability of miner-als, reduce the protein digestibility by the forma-tion of phytic acid^protein complexes anddamage the pyloric caecum by depressing the ab-sorption of nutrients (Francis, Makkar & Becker2001) It has been reported that phytic acid canimpair the growth of rainbow trout (Spinelli,Houle & Wekell 1983) and common carp (Hossain
e⁄-& Jauncey 1993)
(3) Tannins and other phenolic constituents wereknown to in£uence amino acid availability, diges-tible and metabolizable energy content (Yapar &Clandinin 1972; Kozlowska, Sabir, Sosulski & Cox-worth 1975; Seth, Cladinin & Hardin 1975; Sosuls-
ki 1979) Richter, Siddhuraju and Becker (2003)also reported that total phenolic substances re-duced the protein digestibility and amino acidavailability through phenolics^protein and/orphenolics^protein^enzyme complexes
(4) Fibre is another constituent in CM that can press mineral bioavailability as well as decreasetransit time of intestinal contents (increases fae-cal nitrogen and lipid excretion as re£ected bylowered protein and energy digestibility) (Rein-hold, Ismail-Beigi & Faradji 1975) Hilton, Atkin-son and Slinger (1983) also reported a similarreduction in growth performance of rainbowtrout when fed with a high-¢bre diet A furtherpossible reason for low growth at high CM inclu-sion levels might be an inferior amino acid bal-ance (Leslie & Summers 1975)
de-The proximate analysis data of this study indicatedthat the ash content in whole body, was not a¡ected
by the dietary CM levels; however, there were Aquaculture Research, 2010, 41, 982^990 E¡ect of replacing SBM with CM Q-C Zhou & Y-R Yue
signi¢-r 2009 The Authors
Trang 37cant di¡erences in wholebody protein, lipid and
moisture content Similar results were also found in
channel cat¢sh (Webster et al 1997), rainbow trout
(Teskeredzic et al 1995) and tilapia (Davies et al
1990) Teskeredzic et al (1995) reported that these
phenomena might be explained by the di¡erences
be-tween groups in size, feed intake (available energy)
and proportions of total available dietary energy
ori-ginating from protein and lipid were not of su⁄cient
magnitude to a¡ect body composition Biological
in-dices, such as VSI and HSI, were signi¢cantly higher
in ¢sh fed the CM100 diet than the control diet
Simi-lar results were reported for hybrid tilapia (Yue &
Zhou 2008) and cobia (Lunger, Craig & McLean
2006), and this phenomenon can be attributed to
their low growth rates
In the present study, the ADCs of dry matter,
pro-tein and phosphorus were signi¢cantly a¡ected by
the dietary CM supplemented level, while the ADCs
of lipid were not a¡ected by the dietary CM
supple-mentation Similar results were also found in
rain-bow trout (Gomes et al 1993) This might be
explained by the fact that ¢sh have e⁄cient lipid
di-gestibility (Sullivan & Reigh 1995; Allan, Parkinson,
Booth, Stone, Rowland, Frances & Warner-Smith
2000) The results indicated that tilapia digested lipid
e⁄ciently, regardless of the ingredient source In the
present study, higher ADC values of phosphorus were
observed In contrast, Burel et al (2000) reported that
the ADC values of phosphorus ranged from 33% to
15%, when the content of phosphorus was about
1.5% in diets, while the dietary phosphorus ranged
from 0.96% to 1.29% The high ADC values of
phos-phorus might be due to low dietary phosphos-phorus Zhou
et al (2004) reported that the lower the dietary
con-centration of phosphorus, the better is its digestibility
No studies have been conducted to evaluate the
ef-fect of dietary CM on ¢sh haematological values.White
blood cell, haemoglobin and haematocrit increased as
dietary CM levels increased from 0% to 28.53%, and
then decreased when the dietary CM increased from
28.53% to 63.43% Red blood cell was not signi¢cantly
a¡ected by the dietary CM supplementation But, the
values of RBC, HCT and HGB are within the range
re-ported for normal, healthy juvenile channel cat¢sh
(Lim & Klesius 1997; Barros et al 2002; Peres, Lim &
Klesius 2003) Furthermore, studies will be conducted
to obtain some data between the relationship of
hae-matological indices and ¢sh health
In conclusion, this study has shown that diets in
which 30% of SBM was replaced with 19.02% CM
and supplemented with lysine yielded the best
growth and feed utilization But it appears that tion of 38.04% CM causes reduced growth in juvenilehybrid tilapia
addi-Acknowledgments
We would like to thank Dr Gatlin III who kindlyreviewed the draft manuscript This study wasfunded by a research project of the GuangdongOcean University
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Trang 40di-Stress mitigating and immunomodulatory effect of
fingerlings
Mohammad Shahbaz Akhtar1, Asim Kumar Pal1, Narottam Prasad Sahu1, Ciji Alexander1, SanjayKumar Gupta1, Arup Kumar Choudhary1, Ashish Kumar Jha1& Mysore Govindrajan Rajan2
1 Division of Fish Nutrition, Biochemistry and Physiology, Central Institute of Fisheries Education, Mumbai, India
2 Laboratory Nuclear Medicine Section, Bhabha Atomic Research Centre,TMH annex, Mumbai, India
Correspondence: Md Shahbaz Akhtar, FNB&P Division, Central Institute of Fisheries Education, Fisheries University Road, Versova, Mumbai 400061, India E-mail: mdshahbazakhtar@gmail.com
Abstract
A 60-day experiment was carried out to delineate
stress mitigating and immunomodulatory role of
dietary pyridoxine (PN) in Labeo rohita ¢ngerlings
exposed to endosulfan Two hundred and seventy
¢ngerlings were randomly distributed into six
treat-ments in triplicates Five iso-caloric and
iso-nitrogen-ous puri¢ed diets were prepared with graded levels
of pyridoxine Six treatment groups were T0
(10 mg PN1without endosulfan),T1(0 mg
PN1endo-sulfan), T2 (10 mg PN1endosulfan), T3 (50 mg PN1
endosulfan), T4 (100 mg PN1endosulfan) and T5
(200 mg PN1endosulfan) The role of pyridoxine on
stress mitigation and immunomodulation was
as-sessed by biochemical and haemato-immunological
parameters like aspartate aminotransaminase,
ala-nine aminotransaminase, lactate dehydrogenase,
malate dehydrogenase, superoxide dismutase and
catalase were signi¢cantly (Po0.05) lower while
acetylcholinesterase was signi¢cantly (Po0.05)
higher in pyridoxine-fed groups Erythrocytes count,
haemoglobin content and total serum protein,
albu-min, globulin, nitroblue tetrazolium and lysozyme
activity were signi¢cantly (Po0.05) higher while
cortisol and blood glucose were decreased
signi¢-cantly (Po0.05) in pyridoxine-fed groups
Percen-tage survival after challenge with Aeromonas
hydrophila was highest in T0group The results
ob-tained in present study indicate that dietary
pyridox-ine supplementation at 100 mg PN kg 1diet reduces
the endosulfan-induced stress and triggers immune
response in L rohita ¢ngerlings
Keywords: stress, pyridoxine, cortisol, lysozyme,AChE, Labeo rohita
IntroductionAquaculture is the fastest growing agricultural sec-tor with a growth rate of about 8.8% in the world(FAO 2007) To sustain this growth rate and to accom-plish the huge gap between demand and supply, there
is a need to augment the aquaculture production.The indiscriminate use of antibiotics, syntheticgrowth promoters and pesticides has become a regu-lar practice in aquaculture industry This in turnleads to ecological imbalance and stressful condi-tions to the aquatic animals especially in ¢sh (Elia,Waller & Norton 2002) Kilgore and Mingyuli (1975)emphasized that concentration of pesticide residues
is more in aquatic ecosystem than the terrestrial system The stressful condition in ¢sh is not due to asingle stressor but in fact, it is a cumulative and sy-nergistic e¡ect of many stressors like temperature,heavy metals, crowding, pesticides, low dissolvedoxygen, etc Among these stressors, pesticides are ofprime concern as far as aquaculture industry is con-cern Pesticide like endosulfan is an organochlorinelipophilic insecticide with immunosuppressive ef-fects that alters the immune response of ¢sh Endo-sulfan in water is incorporated in ¢sh by ingestion
eco-or direct contact with skin, scales eco-or mucus, and thenenters the food chain It is a well-known blocker ofneuronal GABA-gated chlorine channels (KamijimaAquaculture Research, 2010, 41, 991^1002 doi:10.1111/j.1365-2109.2009.02383.x
r 2009 The Authors