2007 Abstract The concentrations of anti-oxidant enzymes such as superoxide dismutase SOD, cat-alase CAT and selenium-dependent glutathione peroxidase SeGPx, and low molecular weight fre
Trang 1Anti-oxidant status in embryonic, post-hatch and larval
stages of Asian seabass (Lates calcarifer)
N KalaimaniÆ N Chakravarthy Æ R Shanmugham Æ A R Thirunavukkarasu Æ
S V AlavandiÆ T C Santiago
Received: 11 October 2006 / Accepted: 2 July 2007 / Published online: 7 August 2007
Ó Springer Science+Business Media B.V 2007
Abstract The concentrations of anti-oxidant
enzymes such as superoxide dismutase (SOD),
cat-alase (CAT) and selenium-dependent glutathione
peroxidase (SeGPx), and low molecular weight
free-radical scavengers such as reduced glutathione
(GSH) and ascorbic acid (vitamin C) were evaluated
during the period from gastrulation (GS) to 25 days
post-hatch (dph) in the larvae of Asian Seabass, Lates
calcarifer Oxidative damage due to lipid
peroxida-tion (LPO) was also assessed, by evaluaperoxida-tion of the
formation of malondialdehyde (MDA) All the three
anti-oxidant enzymes, SOD, CAT and GPx, showed
high activities during gastrulation, suggesting an
increased metabolic rate during the period of
embry-onic development Though the SOD activity
apparently decreased progressively during 3–20 dph
of larval development, the difference was not
signif-icant CAT showed high activity during gastrulation
and remained constant up to 3 dph, suggesting an
increased need to metabolise hydrogen peroxide
(H2O2) and organic peroxides In contrast, SeGPx
activity increased progressively from 5 dph to 25 dph
during larval development, indicating an increased
need to detoxify lipid peroxides This is evident from
the observation of increased lipid peroxidation from
10 dph to 25 dph during larval development GSH levels were low at gastrulation, indicating increased metabolic rate and formation of lipid radicals during this period, corresponding to the decrease in the level
of ascorbic acid, which is consumed for regeneration
of GSH
Keywords Anti-oxidant enzymes Ascorbic acid Eggs Embryos Gastrulation Larvae Lates calcarifer Malondialdehyde Reactive oxygen species
Introduction
Approximately 0.1% of all oxygen entering the mitochondrial electron transport chain is released as reactive oxygen species (ROS), which can cause damage to lipids, proteins, and DNA (Fridovich
2004) ROS and other pro-oxidants are continually detoxified and removed in cells by anti-oxidant defence systems, which include anti-oxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase, glutathione reductase (GR) and glutathione-S-transferase (GST), and by non- enzymatic defences such as ubiquitous tripep-tide glutathione, vitamins E, C and A, carotenes, and ubiquinol (Wilhelm Filho1996)
Knowledge of anti-oxidant enzyme expression in early embryonic stages would be extremely important
N Kalaimani (&) N Chakravarthy
R Shanmugham A R Thirunavukkarasu
S V Alavandi T C Santiago
Central Institute of Brackishwater Aquaculture, 75,
Santhome High Road, R.A Puram, Chennai, Tamil Nadu
600028, India
e-mail: nkalaimani77@yahoo.com
DOI 10.1007/s10695-007-9155-4
Trang 2in understanding the origin and formation of
protec-tive mechanisms during the life history of organisms
Rudneva (1999) has studied the anti-oxidant enzymes
and low molecular weight scavengers in eggs and
larvae of various Black Sea species (molluscs,
crustacea and fish, including elasmobranchs and
teleosts) The levels of anti-oxidant enzymes during
larval development of Macrobrachium malcolmsonii,
Macrobrachium rosenbergii, and Dentex dentex have
been studied (Arun and Subramanian1998; Dandapat
et al 2003; Mourente et al 1999) Developmental
aspects of detoxifying enzymes in the fish Salmo
iridaeus has been reported by Aceto et al (1994)
Anti-oxidant enzyme activities in embryonic and
early larval stages of turbot and sprat (Sprattus
sprattus) larvae have also been studied (Peters and
Livingstone1996; Peters et al 2001) The activities
of the anti-oxidative enzymes in the cephalopods
Sepia officinalis and Lolliguncula brevis have been
reported by Zielinski and Portner (2000) The
meta-morphosis period in Senegal sole species is especially
critical, with high utilisation of energy reserves, due
to an elevated metabolism, which consumes more
exogenous oxygen to meet the metabolic demands
(Fernandez-Diaz et al 2001; Yufera et al.1999)
Studies have been made on anti-oxidant status in
other animals Starrs et al (2001) examined the
activities of catalase, SOD and GPx in the developing
lungs of two oviparous vertebrate species, the
chicken (Gallus gallus), and an agamid lizard
(Pog-ona vitticeps), and in a metamorphosing vertebrate,
the anuran Limnodynastes terraereginae They
con-cluded that the anti-oxidant enzymes are
differentially regulated in different species and
appear to have evolved different levels of
depen-dency on environmental variables, and the late
developmental increase in anti-oxidant enzymes
activity seen in mammals is not as pronounced in
oviparous and metamorphosing vertebrates The
activity of the basic anti-oxidant enzymes superoxide
dismutases, catalase and glutathione peroxidase in
liver has already developed at early stages of
embryogenesis and is considerably enlarged in the
end embryogenesis of goose (Danchenko and Kalytka
2002) ‘‘Aging’’ was associated with increases in the
activity of red cell SOD and GPx, and significant
correlations amongst red cell GR, GPx and SOD
activities were found in old but not in younger adult
Japanese quails (Godin et al.2001)
Sea bass, Lates calcarifer, is a euryhaline hardy fish suitable for farming in marine, freshwater and brackishwater ecosystems It is a fast-growing fish, suitable for culture in both ponds and cages Tech-nology for seed production of seabass under captivity has been developed by the Central Institute of Brackishwater Aquaculture (CIBA), Chennai, India
It is also widely distributed in the Indo-Pacific region
It has been selected because of the potential for culture in this region The oxidation process and interaction and status of anti-oxidants will throw light
on the metabolic status of the early stages of Asian seabass, Lates calcarifer, larvae, which will have bearing on the health of the fish In the present work, the process of changes in anti-oxidant levels at various stages of development and growth were studied
Materials and methods
Larval rearing
Lates calcarifer larvae were obtained by induced spawning from captive brood stock at the facilities of CIBA, Chennai, India The different stages selected were fertilised eggs, gastrulation stage, 3 days after hatching (3 dph), 5, 10, 15, 20 and 25 dph Larvae were reared in 4–5-tonne capacity circular fibreglass reinforced plastic (FRP) tanks in a salinity of
30 ± 1 ppt Fertilised eggs of Lates calcarifer col-lected from the spawning tanks were incubated in the incubation tanks The hatched out larvae were transferred to the rearing tanks From the 3rd day, larvae were fed with rotifers, Brachionus plicatilis,
up to the 9th day, with rotifer density maintained around 20–50 individuals per millilitre From the 10th day to the 15th day, larvae were co-fed with rotifers and Artemia nauplii From the 16th day, feeding was done with Artemia nauplii, alone, up to the 25th day The Artemia nauplii density was maintained at 2,000 to 6,000 individuals per litre Initial larval density ranged from 40 to 50 individuals per litre Depending upon age and size, the larval density was reduced to 20–25 individuals per litre at
10 dph, and later, after 15 dph, the density was maintained around 10–15 individuals per litre For the experiment, larvae and eggs were collected from fish spawned through induced maturation at night To
Trang 3determine the total length (TL), and the status of
metamorphic development, we took samples of 20–
30 larvae periodically and examined them under a
light microscope Two replicates were performed
Biochemical analysis
Aliquots of larvae Lates calcarifer, weighing
approx-imately 1 g, were homogenised in
phosphate-buffered saline (PBS) solution with a pH of 7.6,
using a Potter homogeniser kept in an ice-water bath
After centrifugation (13,000 g, 5°C, 15 min), the
supernatant was used immediately for determination
of enzyme activities All assays were carried out in
duplicate at 25°C
Superoxide dismutase (Superoxide: Superoxide
oxidoreductase, EC 1.15.1.1) was assayed in the
homogenate by the method of Marklund and
Markl-und (1974) The enzyme activity was expressed as
units per milligramme of protein in tissues The
reaction was initiated by the addition of 0.5 ml
pyrogallol reagent, and the change in optical density
was measured at 480 nm for 3 min Fifty percent
inhibition of pyrogallol by the enzyme was taken as
one enzyme unit Catalase (Hydrogen peroxide:
Hydrogen-peroxide oxidoreductase, EC 1.11.1.6)
activity was assayed by the method of Sinha
(1972) Absorbance was read at 570 nm Catalase
activity was expressed as micromoles of H2O2
consumed per minute per milligramme of protein
Glutathione peroxidase (Glutathione:
Hydrogen-per-oxide oxidoreductase, EC 1.11.1.9) was assayed by
the method of Rotruck et al (1973), with some
modifications The inclusion of sodium azide in the
incubation medium inhibits catalase activity
Absor-bance was measured at 420 nm GPx activity was
expressed as microgrammes of glutathione utilised
per minute per milligramme of protein Protein
content of tissue samples was estimated by the
method of Lowry et al (1951) The blue complex
formed was measured at 640 nm after 15 min against
the blank
Tissue lipid peroxidation (LPO) was measured by
the method of Devasagayam (1986)
Malondialde-hyde (MDA), an end product of LPO, reacts with
thiobarbituric acid (TBA) to form a pink chromogen
(allegedly an MDA adduct) and is measured by its
absorbance at 532 nm Ascorbic acid was estimated
by the method of Omaye et al (1979) Ascorbic acid values were expressed as microgrammes per milli-gramme of protein The disappearance of reduced glutathione (GSH) was measured by its reaction with 5,50-dithio-bis (2-nitro benzoic acid) (DTNB) GSH
in homogenate was measured according to the method of Beutler and Kelley (1963), using Ellman’s reagent This method is based on the development of
a yellow complex upon the addition of 5,50-dithio bis (2-nitro benzoic acid) to compounds containing sulphydryl groups
Statistical analysis
Results are presented as mean ± SD (n = 4) Differ-ences between mean values of enzyme activities, levels of GSH, vitamin C and lipid peroxidation in different stages of development, starting from ferti-lised eggs till 25 dph, were analysed by one-way analysis of variance (ANOVA) followed by testing for multiple range comparisons between means (Duncan’s) The differences between means were reported as statistically significant when P < 0.05
Results
Fertilised eggs float on the surface of water imme-diately after spawning Gastrulation occurs 6 h after fertilisation Eighteen hours after spawning, the eggs hatch out (day 0) During the period up to 3 dph, absorption of the yolk takes place The size of the eggs after fertilisation and at gastrulation was observed to be 0.84 ± 0.3 mm and 0.87 ± 0.05 mm, respectively First feeding stage of larvae was studied
at 3 dph, when the mouth of the larvae opened, and the larvae were fed with the rotifer Brachionus plicatilis (strain S-1, O.F Muller, size range 120–
300 lm) The length of the larvae on 3 dph was 2.4 ± 0.2 mm The fifth day after hatching was included in our evaluation so that we could observe changes in anti-oxidant level corresponding to the rotifer in the diet During this period, the dorsal and anal fins start appearing and the serration appears in the pre-operculum The length of the larvae on 5 dph increased to 2.8 ± 0.5 mm Anti-oxidant status at the 10th day of larval development (length 3.0 ± 0.2 mm) was observed when Artemia nauplii
Trang 4(Commercial strain OSI PRO 80) were fed in addition
to rotifer Morphologically, on the 15th day of larval
development, the dorsal and anal fins are separated
from the caudal fin and the pelvic fin appears A
white band from the centre of the dorsal fin to the
anal fin is visible The anti-oxidant status after the
inclusion of Artemia nauplii in the diet of seabass
larvae at 15 dph was evaluated The length of larvae
was observed to be 4.5 ± 0.5 mm, 8.0 ± 0.5 mm and
10 ± 0.5 mm on 15, 20 and 25 dph, respectively On
20 dph the number of spines and soft rays of the
dorsal and anal fins become constant Scales appear
in the mid-lateral surface above the anal fin The
body colour changes from black to pale brown
Feeding was continued with Artemia nauplii The
20th and 25th days of larval development
represent-ing the metamorphosis period were also included in
our study The metamorphosis period in fish is
especially critical, with high utilisation of energy
reserves due to elevated metabolism, which
con-sumes more exogenous oxygen to meet the metabolic
demands (Fernandez-Diaz et al 2001; Yufera et al
1999)
The activities of the anti-oxidant enzymes SOD,
catalase, selenium (Se)-glutathione peroxidase, level
of lipid peroxidation and the amounts of glutathione
and vitamin C during different development stages of
seabass larvae, including fertilised eggs and
gastru-lation, are presented in Table1
The activities of the enzymes SOD and catalase
are presented in Figs.1 and2, respectively SOD
activity apparently found to be maximum at
gastrulation (GS) was observed to decline gradually, and the decrease was not significant The activity of catalase was the highest in the gastrulation stage The minimum values were observed for SOD during
20 dph and for catalase at 15 dph The decrease was significant in catalase, and the activities increased gradually on 20 dph and 25 dph The increase in the activity of catalase was significant at 25 dph com-pared to 15 dph
GPx activity (Fig.3) was observed to be high during gastrulation and minimal at 5 dph GPx activity was found to increase gradually after 5 dph
of larval development This increase in GPx activity directly correlates with the increase in lipid peroxi-dation observed in this period of larval development Selenium-dependent glutathione peroxidase (SeGPx) activity in different stages varied significantly, except
on 5 dph and 10 dph The rate of lipid peroxidation (Fig.4) was very high at gastrulation and decreased
to a minimum at 5 dph The significant differences in GPx activities and lipid peroxidation during various stages are presented in Table 1
At gastrulation, GSH level (Fig.5) was observed
to be low A smaller increase in GSH level than that found in fertilised eggs (FEs) was noted after gastrulation up to 3 dph After 3 dph, the level of GSH was found to decline gradually But again, GSH showed a small increase at 25 dph Ascorbic acid concentration (Fig.6) was very low at gastrulation, and there was a drastic increase in vitamin C at
3 dph, when the larvae were fed with rotifers After that the level of ascorbic acid was found to decrease
Table 1 Status of anti-oxidant enzymes and anti-oxidants and
lipid peroxidation in different developmental stages of Lates
calcarifer Results are mean ± SD (n = 4) Values within each
column bearing different superscript letters are significantly different at P < 0.05 (SeGPx selenium-dependent glutathione peroxidase, FE fertilised egg, GS gastrulation)
Different
stages of
development
SOD (units/mg
protein)
Catalase (lmol H2O2 consumed/min per mg protein
SeGpx GSH utilised/min per mg protein
LPO (nmol of malondialdehyde released/mg protein)
GSH (lg/mg protein)
Vit C (lg/mg protein)
FE 43.93 ± 5.04a 16.63 ± 2.80a 50.25 ± 2.09a 34.56 ± 2.91a 95.25 ± 2.49a 27.72 ± 2.88a
GS 46.81 ± 5.9a 20.19 ± 3.56b 56.07 ± 2.05b 64.29 ± 3.02b 25.36 ± 3.13b 8.12 ± 1.46b
3 dph 43.77 ± 5.3a 18.53 ± 1.90ab 31.07 ± 4.20c 5.18 ± 1.49c 102.72 ± 6.25c 69.21 ± 2.39c
5 dph 43.38 ± 5.45a 6.59 ± 1.02c 17.02 ± 1.34d 4.10 ± 1.06c 85.41 ± 6.24d 36.11 ± 3.37d
10 dph 43.33 ± 5.23 a 1.71 ± 1.00 de 16.98 ± 1.67 d 11.49 ± 1.36 d 66.33 ± 4.67 e 36.06 ± 3.78 d
15 dph 43.27 ± 5.01 a 1.24 ± 0.49 d 28.99 ± 1.82 c 11.02 ± 1.14 d 57.17 ± 2.37 f 36.03 ± 2.63 d
20 dph 43.17 ± 4.83a 3.47 ± 0.41de 35.06 ± 1.84e 17.19 ± 1.52e 20.23 ± 2.58b 12.09 ± 2.94be
25 dph 43.44 ± 4.91a 4.30 ± 0.65ce 41.57 ± 1.24f 18.09 ± 2.38e 80.26 ± 3.48d 13.40 ± 1.96e
Trang 5gradually From 20 dph to 25 dph the vitamin C
content remained constant The significant
differ-ences are presented in Table1
Discussion
Measurable amounts of SOD, catalase (CAT) and
SeGPx were present in the fertilised eggs of Lates
calcarifer, suggesting that the eggs are well protected
against peroxidation, despite high levels of
polyunsaturated fatty acids (PUFAs) present in the eggs, as observed in Dentex dentex (Mourente et al
1999) SOD and CAT are responsible for the inactivation of superoxide and hydrogen peroxide, respectively SOD, by converting superoxide to
0
10
20
30
40
50
60
Stage
SOD
FE - Fertilised eggs
GS - 6hrs after spawning
Fig 1 Superoxide dismutase activity in different larval stages
of Lates calcarifer
0
5
10
15
20
25
FE
Stage
Catalase
FE - Fertilised eggs
GS - 6hrs after spawning
GS 3dph 5dph 10dph 15dph 20dph 25dph
Fig 2 Catalase activity during different larval stages of Lates
calcarifer
0
10
20
30
40
50
60
70
FE GS 3dph 5dph 10dph 15dph 20dph 25dph
Stage
Glutathione Peroxidase
FE - Fertilised eggs
GS - 6hrs after spawning
Fig 3 Glutathione peroxidase activity in different larval
stages of Lates calcarifer
0 10 20 30 40 50 60 70 80
FE
Stage
LPO
FE - Fertilised eggs
GS - 6hrs after spawning
GS 3dph 5dph 10dph 15dph 20dph 25dph
Fig 4 Lipid peroxidation during different larval stages of Lates calcarifer
0 20 40 60 80 100 120
Stage
Glutathione
FE - Fertilised eggs
GS - 6hrs after spawning
FE GS 3dph 5dph 10dph 15dph 20dph 25dph
Fig 5 Glutathione (reduced) levels in different larval stages
of Lates calcarifer
0 10 20 30 40 50 60 70 80
Stage
Vitamin C
FE - Fertilised eggs
GS - 6hrs after spawning
FE GS 3dph 5dph 10dph 15dph 20dph 25dph
Fig 6 Vitamin C levels in different larval stages of Lates calcarifer
Trang 6hydrogen peroxide, provides substrate for CAT
(Miller et al 1993) Contradicting results for SOD
activity during early development of fish have been
reported The activity of SOD decreased throughout
larval development from egg in S maximus (Peters
and Livingstone 1996) and in Dentex dentex
(Mou-rente et al 1999), and an increase in activity was
reported in other species (Aceto et al.1994; Rudneva
1999) In our study the levels of SOD were not
significantly different with larval development
According to Starrs et al (2001), the response of
this enzyme to both natural and experimental oxygen
tension is difficult to interpret In the lung of the
bearded dragon, SOD activity continued to decrease
throughout late development While no change in
SOD activity occurred between days 14 and 18 in the
embryonic chicken lung, there was a significant
decrease between days 18 and 19
Catalase activity was high at 3 dph, when seabass
larvae started feeding on rotifers, and this phase is
considered as a highly energy demanding phase in the
larval life-cycle, with a high requirement for oxygen
(Sole et al 2004) Most of the fish species showed
strong enzymatic changes in anti-oxidant defences
when their larvae changed from endogenous feeding
to exogenous feeding (Mourentex et al.1999; Peters
and Livingstone 1996; Rudneva1999) In our study
significant increase was noted in CAT activity from
15 dph to 25 dph During this period of
metamor-phosis the Artemia nauplii feed concentration was
increased The result as of our study was similar to
that of Fernandez-Diaz et al (2006) The activity
profile of anti-oxidant enzymes during larval
devel-opment under an Artemia diet demonstrated a
stage-specific compensatory mechanism to neutralise
per-oxides (Dandapat et al.2003) Fernandez-Diaz et al
(2006), studied the variation in larval development
and stress defences in Solea senegalensis larvae fed
on live and microencapsulated diets and observed
that CAT, SOD, GPx and lipid peroxidation levels in
the sole larvae showed diet and age dependence in
their responses Larvae fed with an inert diet showed
similar biomarker activities and were different from
larvae fed with Artemia (P < 0.05)
At gastrulation, GPx level was high, which may be
due to high metabolic activity resulting in increased
H2O2 accumulation From embryo to hatching, the
level of GPx gradually decreased and reached its
minimum at 5 dph During this period, activities of
other anti-oxidant enzymes (SOD, CAT) were ele-vated, and after 5 dph the levels were stable and there was a gradual increase in GPx up to 25 dph These observations indicate a progressive need to remove
H2O2 and lipid peroxides from the cells This could
be due to increase in LPO from 5 dph to 25 dph The increase in GPx is apparently not due to production of
H2O2from dismutation of O2, since larval SOD and CAT activity declined from the 5th day onwards The increase in catalytic activity of CAT and GPx after the initiation of feeding with Artemia nauplii, corre-sponding with the increase in its density, may have been a response to strong metabolic changes, such as metamorphosis, occurring during this time (Mourente
et al 1999; Sole et al 2004) The increase in lipid peroxidation with larval age with higher GPx activity has been observed in Artemia fed sole larvae from hatching to 28 dph (Sole et al 2004) Our observa-tion in Lates calcarifer is also in agreement with earlier finding
Lipid peroxidation was the highest at gastrulation and gradually declined up to 5 dph A similar result was obtained in Dentex larvae (Mourente et al.1999) However, LPO increased gradually from 10 dph to
25 dph in seabass larvae During metamorphosis at
20 dph and 25 dph, an increase in LPO was observed that may have been due to altered metabolic rate Under an Artemia diet, co-evolution of lipid perox-idation and GPx is also expected, since the role of GPx is to eliminate not only hydrogen peroxide but also lipid peroxides (Fernandez-Diaz et al.2006) At the stage of metamorphosis only, an enhanced level
of LPO occurred During this period the exogenous feed is changed, both in quality and quantity, which,
in turn, leads to altered metabolic rate resulting in oxidation of PUFAs
Vitamin C level is high at 3 dph of development However, the levels of vitamin C on 5, 10 and 15 dph were not significantly different From 15 dph vita-min C level distinctly decreased During early larval development, vitamin C level counterbalances the oxidative damage and lipid peroxidation, but, when the concentration of feed was increased, the scav-enging rate of vitamin C decreased because of the high level of LPO The decrease in vitamin C indirectly suggests a high rate of lipid peroxidation Vitamin E reacts with lipid peroxy and alkoxy radicals and donates its labile hydrogen, generating vitamin E in its radical form Vitamin E radical is
Trang 7reduced back to vitamin E by vitamin C (McCay
1985) Thus, the gradual depletion of vitamin C level
relates to the gradual increase in vitamin E, which, in
turn, counterbalances the lipid radicals Vitamin E
level could be strictly correlated to a metabolic
acceleration or to high rates of ROS generation, as
demonstrated in animal systems (Chamiec et al
1996; Jialal et al 2001) Thus, the increase and
decrease in vitamin C level has an inverse relation
with vitamin E During marine animal
embryogene-sis, the activities of most of the anti-oxidant enzymes
examined tend to increase, especially in eggs and
hatching larvae, while the contents of low molecular
weight anti-oxidants decrease (Rudneva 1999) The
eggs of Lates were found to contain anti-oxidant
enzymes, and, during embryogenesis, the levels of
these enzymes tended to increase, while low
molec-ular weight anti-oxidants, such as vitamin C and
GSH, were found to decrease These results are
similar to those of Rudneva (1999), who suggested
that, in the early stages of embryogenic development
of marine organisms, the low molecular weight
anti-oxidants play an important protective role against
oxygen damage During embryogenesis, the
concen-tration of low molecular weight scavengers
decreased, with an enhancement of anti-oxidant
enzyme activities, possibly as a result of gene
expression
GSH is a water-soluble anti-oxidant, found in the
cytosol and mitochondria (Halliwell and Gutteridge
1989) Glutathione, a tripeptidethiol consisting of
glycine, cysteine and glutamic acid moieties, exists in
high concentration virtually in all types of living
cells It has been demonstrated that glutathione plays
an important role in scavenging free radicals and
active oxygen species (Comporti 1987; McLennan
et al 1991; Ogino et al 1989) It occurs mainly by
detoxifying hydrogen peroxide and lipid peroxides
through reactions catalysed by glutathione
peroxi-dase In our study an inverse relationship with
peroxide decomposition of membrane
poly-unsatu-rated fatty acids and GSH was clearly established,
which is in agreement with the earlier finding of Shen
et al (1990) At gastrulation, GSH level was at its
minimum, which might be due to an increased
metabolic rate and increased formation of lipid
radicals When the levels of free radicals are high,
more reduced glutathione (GSH) is converted to its
oxidised form [glutathione disulphide (GSSG)], and,
hence, there is a reduction in the level of GSH (Oup
et al 1996) GSH level also shows an inverse relationship with LPO decomposition The levels of GSH and vitamin C displayed a decreasing trend from 5 dph to 20 dph of larval development How-ever, from 20 dph to 25 dph, the level of vitamin C remained constant, and GSH showed a significant increase, which may have been due to a small increase in the activities of CAT and GPx during this period Story (1996) suggested that NADPH-depen-dent glutathione reductase (GR) replenishes the GSH substrate for GPx and glutathione-S-transferase from GSSG This trend was observed during 20 dph to
25 dph, which may be an indication of the activity of secondary enzymes in Lates calcarifer larvae during this period
Our study indicates that oxidant stress is high during the embryonic and early larval stages, which may be attributed to metamorphosis and the initiation
of external feeding, possibly linked to altered respi-ration rates at these stages These results are in line with those of the earlier studies confirming that the initiation of external feeding and metamorphosis are amongst the critical and most stressful periods for fish larvae The results of this study should pave the way for further research on the effects of the enrichment
of live feed with PUFAs and other anti-oxidants for the management of stress during larval rearing of Lates calcarifer
Acknowledgements The authors are grateful to A.G Ponniah, Director, CIBA, for critically going through the manuscript and for his valuable suggestions The help rendered
by R Subburaj, Technical Officer, R Thiagarajan, Technical Assistant and V Anuradha, Senior Research Fellow, is gratefully acknowledged.
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