Survival, growth and food conversion of cultured larvae of Pangasianodon

hypophthalmus, depending on feeding level, prey density and sh density

J. Slembrouck
a,
, E. Baras
b
, J. Subagja
c
, L.T. Hung
d
, M. Legendre
b
a
IRD, UR 175, c/o Loka Riset Budidaya Ikan Hias Air Tawar (LRBIHAT) Depok, Indonesia
b
IRD, UR 175, BP 5095, Rue J.F. Breton 361, F-34196 Montpellier cedex 05, France
c
Pusat Riset Perikanan Budidaya, 20 Jl. Raya Ragunan, Pasar Minggu, 12540 Jakarta, Indonesia
d
National University of Ho Chi Minh City, Faculty of Fisheries, Thu Duc, Ho Chi Minh City, Vietnam
a b s t r a c t a r t i c l e i n f o
Article history:
Received 2 December 2008
Received in revised form 23 April 2009
Accepted 24 April 2009
Keywords:
Food density
Catsh
Aquaculture
In young sh larvae feeding efciency is generally proportional to prey density, so feeding in excess is needed
to maximise growth and survival. Increasing sh density might contribute to improve food conversion, but it
can also impact negatively on sh growth or survival. Larvae of Pangasianodon hypophthalmus were raised
until 192 h after hatching (hah) in 30-L tanks in a recirculating system (light regime: 12L:12D, 29.61.2 C)
at three stocking densities (10, 30 and 90 sh L
1
), and fed every 3 h with Artemia nauplii at 1, 3 or 9 times a
reference feeding level (RFL; 50% increase per day), thereby producing ve different prey densities (from 10
to 810 RFL L
1
). Except for the highest prey density, survival (2060%) was dependent on feeding level,
whereas sh growth (12.517.6 mm TL at 192 h AH) was more inuenced by prey density than by feeding
level. Both variables were negatively affected by sh density, but to a much lesser extent than by food
availability. At all sh densities, the gross conversion efciency (GCE, 0.130.42) was highest at 1 RFL, and
decreased for higher feeding levels, but not between 1 and 3 RFL at 90 sh L
1
, which provided the best
compromise between survival, growth and GCE in this study. Temporal variations in the effects of food
availability and sh density are interpreted in respect to the developmental pattern of P. hypophthalmus.
2009 Elsevier B.V. All rights reserved.
1. Introduction
The denition of adequate feeding strategies in sh culture is
proportionally more crucial during the early life stages, when
suboptimal feeding can impact not only on growth but also on
survival. The larvae of most sh species have a narrow resource
spectrum and they generally require live feed which are expensive,
manpower demanding or complicated to produce. On the other hand,
live feed possess one major advantage in that they remain alive and
accessible for a longer time (especially for species that do not feed on
the bottom), in contrast to inert food particles. Hence, when a live
prey has been found adequate for a particular life stage, the feeding
strategy essentially consists of determining how much food should be
delivered to full the requirements of the larvae. This can be deduced
fromthe relationships between sh size and food intake for meal size,
and between sh size and gut transit rate for meal frequency.
Such theoretical calculations rely on the assumption that sh are
capable of nding and eating all food particles that are offered. This
might be a false premise in sh, because the sensorial equipments and
food searching behaviours are generally less elaborate in larvae than
in juveniles or adults. The relationships between food density and
food intake vary among species (e.g. Perca uviatilis, Wang and
Eckmann, 1994; Gadus morhua, Gotceitas et al., 1996; Puvanendran
and Brown, 1999; Paralichthys olivaceus, Dou et al., 2000; Rhombosolea
tapirina, Shaw et al., 2006), and among developmental stages (e.g.
Solea solea, Day et al., 1996; Sparus aurata, Parra and Yfera, 2000;
Sander lucioperca, Ljunggren, 2002). These relationships also depend
on prey types (e.g. rotifers versus cladocerans in Chirostoma riojai,
Morales-Ventura et al., 2004). However, with few exceptions (e.g.
Sebastes spp., Laurel et al., 2001), the consumption of live prey by sh
larvaedoes not becomeasymptotic unless foodis giveninexcess (Houde
and Schekter, 1980). Growth and survival follow similar asymptotic
curves (Werner and Blaxter, 1980), except when excessively high prey
densities alter the water quality (Puvanendran and Brown, 1999). Not
only dolowprey densities affect shgrowththroughtheir effect onfood
intake, but they can also impair growth by increasing the swimming
costs during foraging (Ruzicka and Gallager, 2006).
A possible way of maintaining high survival and fast growth without
compromising the conversion efciency would consist of increasing the
sh density while keeping the same feeding level (i.e. number of prey
per sh). This would increase the prey density (i.e. number of prey per
volume unit) and thus the probability of prey encounter. However, a
number of side effects are generally associated with higher sh
densities, especially among larvae that exhibit cannibalistic behaviours
Aquaculture 294 (2009) 5259
Corresponding author.
E-mail address: Jacques.Slembrouck@ird.fr (J. Slembrouck).
0044-8486/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2009.04.038
Contents lists available at ScienceDirect
Aquaculture
j our nal homepage: www. el sevi er. com/ l ocat e/ aqua- onl i ne
at an early developmental stage. Indeed, early cannibalism does not
require a size differential and is generally proportional to the probability
of encounteringsiblings, andthus toshdensity (for catshes, seeHecht
andAppelbaum, 1988; Baras, 1999; Baras andd'Almeida, 2001). Not only
does early cannibalism impact directly on survival, but it also increases
size heterogeneity, which favours the transition to complete cannibal-
ism, and further impacts on survival in a cascading pattern that can
hardly be mitigated among sh that are too small and delicate to be
sorted mechanically (syntheses in Hecht and Pienaar, 1993; Baras and
Jobling, 2002).
In brief, these considerations can be summarised by one practical
questioning: should we feed the sh or the tank? which in turn,
requires examining the interactions between sh density, feeding
level and prey density (per volume unit), and their consequences on
sh survival, growth, size heterogeneity and food conversion. The
present study was designed to investigate this issue in larvae of the
catsh Pangasianodon hypophthalmus (Sauvage, 1878) (Siluriformes,
Pangasiidae; formerly Pangasius sutchi or Pangasius hypophthalmus).
Pangasiid production in aquaculture in 2007 exceeded 1 million tons
per year (Lazard et al., 2009). It relies essentially on the culture of P.
hypophthalmus, which originates from the Mekong and Tchao Praya
river basins, but has been introduced throughout South-East Asia.
Larvae of this species reputedly exhibit an intense cannibalistic
behaviour at an early age (about 36 h after hatching; Subagja et al.,
1999), so the issues of sh density and feeding strategy are crucial to
dene appropriate rearing strategies in respect to local production
constraints in terms of sh survival, growth and food conversion.
2. Methods
2.1. Larval rearing
The sh used in this experiment were obtained from the articial
reproduction of 35 year old broodsh raised in ponds at the RCA
(Research Centre for Aquaculture) research station in Sukamandi,
Indonesia (for detailed information on hormonally induced reproduc-
tion, egg fertilisation and incubation, see Legendre et al., 2000). Twelve
hours after hatching (hereafter hah), sh were counted and transferred
in an indoor recirculating system. Fish were stocked in 30-L tanks with
little aeration after preliminary experiments demonstrated that strong
aeration resulted in greater mortality (Hung, 1999). The water owwas
0.25L min
1
until shwere 72hah, and0.5L min
1
thereafter. The light
regime was 12L:12D, with abrupt transitions between light and dark
phases, and light intensities of 50100 Lx, and b0.01 Lx, respectively.
Water temperature was monitored continuously throughout the
experiment. Water quality (pH, nitrite and ammonia concentrations)
was examined at daily intervals in the system. Oxygen concentrations
were measured daily in each tank. During the experiment, a 10 mg L
1
oxytetracycline concentration was maintained in the recirculating
system, following the conclusions of Subagja et al. (1999) that an early
application of disinfectant or antibiotic strongly reduced the mortality
among young larvae of P. hypophthalmus.
Food delivery started at 36 hah. From this moment on, newly
hatched brine shrimp (Artemia) nauplii were delivered every 3 h
(eight times per day). Brine shrimp nauplii were produced twice a day
(20-h incubation of cysts at 29 C in brackish water [25 g NaCl L
1
])
and maintained in brackish water in a refrigerator (46 C) during the
12 h following hatching. The concentration of brine shrimp nauplii
was determined by volumetric counts (mean of three replicates) in 1-
mL samples that were collected with a pipette. At each meal, an
identical volume of brackish water was collected, brine shrimp nauplii
were rinsed and placed in fresh water from the recirculating system a
few minutes before distribution to the experimental tanks, in order to
minimise the amount of salt that was transferred to the rearing
environment. At the time of food delivery, water ow in the tanks was
stopped for 30 min in order to maintain the prey density as
homogeneous as possible throughout the tank (i.e. otherwise brine
shrimp nauplii tend to drift and concentrate near the exit of the tank).
This 30-min period was adequate for the sh to feed maximally, by
reference to observations of food intake in larvae allowed to feed over
15 and 30 min (E. Baras, unpublished data).
2.2. Experimental design
Larvae of P. hypophthalmus aged 24 hah were stocked at three
densities (10, 30 and90 shL
1
). Three feeding levels (1, 3 and9 times
a reference feeding level, hereafter RFL) were tested for each sh
density, with two replications per treatment (i.e. sh densityfeeding
level). In the rest of the manuscript, each treatment is designated as x:
y, where x is the sh density (sh L
1
) and y is the number of RFL per
meal (Fig. 1). In addition to the nine aforementioned treatments, a
tenth treatment was evaluated, with larvae raised at 10 sh L
1
and
using a very high feeding level (81 RFL, thereby producing the same
prey density as in treatment 90:9). This aimed to test whether growth
was maximal with 9 RFL. No counterpart with very high feeding level
was evaluated for higher sh densities, essentially to minimise the risk
of degradation of water quality. Groups were randomly assigned to the
20 tanks of the water recirculation system.
As a result of the experimental design, the prey density (number of
brine shrimp nauplii per mL) was identical among treatments 10:3
and 30:1, among treatments 10:9, 30:3 and 90:1, among treatments
30:9 and 90:3, and among treatments 90:9 and 10:81 (see Fig. 1). The
design thus enabled testing within a single experiment the effects of
sh density, feeding level (nauplii per sh) and their interactions, as
well as those of prey density (nauplii per volume unit).
The RFL was based on feeding schedules that were determined
after preliminary culture trials in Viet Nam (Slembrouck, 1997), and
stood as Log (RFL)=0.377+0.176 A, where RFL is the number of brine
shrimp nauplii delivered per meal per sh stocked, and A is the age of
the sh (days after hatching, hereafter dah). In practice, the RFLs on 2
8 dah were 4, 6, 8, 12, 18, 27 and 41 nauplii per sh per meal,
respectively (i.e. a 50% increase per day).
2.3. Measurements and calculations
At 48, 96, 144 and 192 h after hatching, 10 sh were sampled in
each tank 30 min after the distribution of brine shrimp nauplii. They
Fig. 1. Experimental design for testing the combined effects of sh density, feeding level
(prey per sh) and prey density (prey per volume) on the survival, food intake, growth
and food conversion in larvae of P. hypophthalmus. Identical symbols connected by plain
lines highlight identical food densities (10810 RFL L
1
), obtained with different
combinations of sh densities and feeding levels (i.e. N times the reference feeding
level, RFL). An additional treatment (not shown on the gure) was also evaluated with
sh raised at 10 sh L
1
offered 81 RFL.
53 J. Slembrouck et al. / Aquaculture 294 (2009) 5259
were anaesthetised with 2-phenoxy-ethanol (0.4 mL L
1
) and their
total body length (TL, mm) was measured under the stereomicroscope
with a micrometer (accuracy ranging from 0.05 to 0.1 mm, depending
on sh size and magnication). The anaesthetised sh were killed
with a lethal solution of 2-phenoxy-ethanol (over 2 mL L
1
) and
preserved in 5% formalin. Thereafter their guts were dissected under
the stereomicroscope and food intake was determined by counting
the number of brine shrimp nauplii in the foregut (then stomach
when differentiated).
To evaluate growth, body length was examined preferentially to
body mass, since the sh were fed every 3 h and rarely had an empty
gut, so growth estimates from body mass measurements would
have been biased to variable extents depending on food intake and
thus presumably on treatment. Fish wet body mass (WM) was back-
calculated from the sh's total length (TL) with a mass-to-size model
for larvae of P. hypophthalmus with empty guts (E. Baras, unpublished
data), i.e. Log WM=176.0[882.8 Log(TL)] +[1737.3 log(TL)
2
]
[1684.4 Log(TL)
3
] +[808.9 Log(TL)
4
])[154.1 Log(TL)
5
]. The model is
unusually complex for a mass-to-size relationship in sh, but it
accurately depicts the various stages of allometric growth that occur
throughout the larval stages of P. hypophthalmus (here from 0.9 to
80 mg WM, R
2
N0.99, df =350).
At 192 hah, all tanks were emptied and survivors were counted.
During the experiment, the tanks were siphoned twice a day to
remove uneaten food and dead sh. However, larvae of P. hypophthal-
mus are small (about 0.9 mg at the start of exogenous feeding, at
36 hah), and they undergo a rapid decay in warmwater. Hence, many
dead sh probably passed unnoticed and the mortality dynamics
could not be traced accurately during this experiment. The knowledge
of the mortality dynamics is unimportant in respect to prey density
(nauplii per volume unit), whereas it matters for determining the
actual number of prey available per survivor, and its consequences on
food intake, growth and size heterogeneity. In absence of additional
information, daily mortality (M, % day
1
) was deemed almost
constant throughout the experiment, and was estimated as M=[Ln
(N
i
)Ln (N
f
)] /6.5, where N
i
and N
f
are the initial and nal numbers
of sh in a tank, and 6.5 is the duration of the experiment (days). The
number of survivors at a particular age in each tank was estimated
from these.
Using the information on growth and survival, sh production was
calculated and compared to the food offered in order to estimate the
gross conversion efciency (GCE=production/food offered). Dry
mass (DM) rather than wet mass (WM) ratios were used for
calculating the GCE, because the water content varies substantially
during the ontogeny of P. hypophthalmus, so comparisons between
sh of different WMs would have been biased. A standard DM:WM
ratio of 16.5% was used for brine shrimp nauplii (Sorgeloos et al.,
1986). The DM:WM ratios for sh larvae were calculated from
independent data sets produced during experiments where larvae
were fed maximally with brine shrimp nauplii (E. Baras, unpublished
data). At the start of exogenous feeding (sh of 0.9 mg WM), the
DM:WM ratio is about 11%. Thereafter, the ontogenetic changes in
the proportion of dry matter during the larval stages of well-fed
P. hypophthalmus can be described by the following model: DM:WM
(%) =9.048 [0.187] +3.689 [0.269] Log WM (R
2
=0.969, F=187.70,
Pb0.0001 both for the intercept and slope) (where DM and WM are
the dry and wet body mass of sh, and the values between brackets
are the standard errors of coefcients).
2.4. Statistics
The heterogeneity of food intake or sh size was expressed as the
coefcient of variation, CV=SD mean
1
, where SD is the standard
deviation of the sample. GCE, food intake, survival and growth rates
were compared between treatments by 2-way analysis of variance
(ANOVA), using sh density and feeding level as independent factors.
The 10:81 treatment could not be included into these analyses (since
the 81 RFL feeding level was not tested for all densities), so additional
post hoc tests (Scheffe F-tests) were used for pairwise comparisons
between all 10 treatments. These analyses also permitted to test for
differences between treatments with identical prey densities but
different combinations of sh density and feeding level (e.g. 10:9
versus 30:3 and 90:1). Stepwise multiple-regression analyses were
used to test for the preponderant inuence of variables (feeding level,
prey density, sh density) on the variables under study (survival,
growth, food conversion). Null hypotheses were rejected at Pb0.05.
3. Results
Temperature varied between 28.4 and 30.9 C during the ex-
periment. Water pH was 8.48.5 throughout, whereas nitrite and
ammonium increased slightly in the course of the experiment (from
0.005 to 0.012 mg NO
2

L
1
, and from 0.01 to 0.02 mg NH
4
+
L
1
). At
the very end of the experiment, however, the ammonium concentra-
tion in the system soared to 0.22 mg NH
4
+
L
1
, but no mass mortality
was observed by then. The oxygen level remained stable (around
7.6 mg O
2
L
1
) throughout the experiment in the tanks with the
lowest prey density (10 RFL L
1
, treatment 10:1). In all other
treatments, the oxygen level declined progressively, and proportion-
ally to the prey density (mean levels of 7.5, 7.0, 6.3 and 5.5 mg L
1
, at
prey densities of 30, 90, 270 and 810 RFL L
1
). The lowest oxygen level
(4.4 mg O
2
L
1
) was observed at 192 hah in a tank of the 90:9
treatment but no mass mortality was observed on this day.
3.1. Survival
Survival at 192 h PH ranged from 20 to 60.5% (Fig. 2). It was
signicantly dependent on feeding level (P=0.0003) and indepen-
dent from sh density (P=0.1080). At 10 and 30 sh L
1
, survival of
sh fed 1 to 9 RFL was proportional to the feeding level. By contrast, at
90 sh L
1
, survival was slightly lower among sh that were offered
9 RFL than among those receiving 3 RFL (33.5 versus 39%).
A stepwise multiple regression analysis revealed that survival at
192 hah (S
192h
, % initial; Log form) was more closely dependent on the
feeding level (FL, nauplii sh
1
meal
1
, Log form, partial R
2
=0.718)
than on prey density (PD, nauplii mL
1
, Log form, partial R
2
=0.190),
Fig. 2. Survival of larvae of P. hypophthalmus at 192 h after hatching (hah), as a function
of sh density and feeding level (see Fig. 1). Bars and whiskers are the means and
differences between two replicates. Bars with the same shade correspond to treatments
with identical prey density (nauplii per volume). Statistics (2-way ANOVA, excluding
the 10:81 groups): sh density: P=0.1080, feeding level: P=0.0003, interaction:
P=0.0663. Bars that share at least one superscript in common do not differ at Pb0.05
(Scheffe F-tests between all 10 treatments).
54 J. Slembrouck et al. / Aquaculture 294 (2009) 5259
and that sh density (FD, sh L
1
) impacted negatively on the
survival of sh that were offered the same feeding level, i.e.
LogS
192 h
= 1:517 0:084 + 0:352 0:051 Log FL 0:116 0:053 Log FD;
where variables are presented in order of entrance in the model, and
values between brackets are the standard errors of coefcients
(F=26.186, R
2
=0.789, df =17, P of b0.0001, b0.0001, and 0.0469
for the intercept, FL and FD, respectively).
3.2. Growth and size heterogeneity
The average size of sh at 192 hah varied between 12.5 and
17.6 mm TL, in the 10:1 and 90:9 groups, respectively (Fig. 3). Fish
fromthe 10:81 group did not attain signicantly larger sizes (17.7 mm
TL on average). The coefcients of variation of sh size at 192 hah
remained low in all groups (from 4.52 to 7.93%), and were not
inuenced signicantly by sh density (2-way ANOVA: P=0.0972),
by feeding level (P=0.9612) or by the interaction between the two
factors (P=0.4786). By contrast, at all sh densities and on all
sampling dates, the feeding level had a signicant positive effect on
sh size (Pb0.0001; Fig. 3). Fish density also had a signicant impact
on growth (P from b0.0001 to 0.0027), but it acted in contrasting
ways, depending on sh age and size. During the early feeding stages
(from 36 to 48 hah), sh density impacted negatively on sh growth.
Thereafter, growth progressively became faster among the sh
stocked at medium or high density, at least among those that were
offered 3 or 9 RFL.
A stepwise multiple regression analysis was conducted for
analysing the variations of sh growth (G, mg WM day
1
) against
the wet body mass of sh (WM, mg), sh density (FD, sh L
1
),
feeding level (FL, nauplii sh
1
meal
1
) and prey density (PD,
nauplii mL
1
). The analysis was restricted to the data collected until
144 hah for the following reason. From 144 to 192 hah, the growth of
sh fed 1 or 3 RFL was slower than on the previous days (Fig. 3), which
is most unusual in sh. By contrast, no such decrease was observed in
the groups fed 9 or 81 RFL. It was strongly suspected that this situation
originated from a lower quality and survival of brine shrimp on these
two days, which affected the sh fed the lowest feeding levels more
than those fed in large excess. Since the experimental conditions were
seemingly not fully respected, the data fromthe last feeding days were
not taken into account in the growth model, which stood as:
Log G = 0:372 0:108 + 0:691 0:061 Log WM + 0:221 0:050 Log PD
0:259 0:067 Log FD;
where variables are presented in order of entrance in the model, and
values between brackets are the standard errors of coefcients
(F=139.13, R
2
=0.948, df =53, P of 0.0022, b0.0001, 0.0002 and
0.0008 for the intercept, WM, PD and FD, respectively). The inuence
of the feeding level was insignicant after the effect of prey density
was taken into account by the model.
3.3. Gut content
The gut contents of larvae at 48, 96 and 144 hah were signicantly
dependent on the feeding level (Pb0.0001) but independent fromsh
density (P of 0.1491, 0.0634 and 0.1766) and from the interaction
between the two variables (P of 0.1235, 0.3420 and 0.0680). At all
sampling dates, the gut contents of larvae that received 1 RFL were
close to those predicted by the model that was used for calculating the
reference feeding level (Fig. 4). By contrast, larvae that were offered
larger amounts of food ate more than predicted. At 48 hah, the gut
contents of sh fed 3 to 81 RFL were about three times higher than
predicted by the model. Differences between predicted and observed
food intakes at 3, 9 and 81 RFL persisted in older sh, because sh
grewfaster in this experiment than in the feeding trials that were used
to elaborate the feeding schedule. At almost all sh densities, the
coefcient of variation of food intake was reduced when the feeding
level was increased (Fig. 4).
Fig. 3. Size (total body length, TL) of larvae of P. hypophthalmus as a function of sh age (hours after hatching, hah), sh density and feeding level. Bars and whiskers are the mean and
standard errors of 20 sh (two replications per treatment, 10 sh per sample). Bars and whiskers are the means and standard errors of 20 sh for each treatment (10 sh per sample
two replicate groups per treatment). Bars with the same shade correspond to treatments with identical prey density (nauplii per volume). Statistics (2-way ANOVA, excluding the
10:81 group): 48 hah: sh density: Pb0.0001, feeding level: Pb0.0001, interaction: P=0.4497; 96 hah: sh density: P=0.0015, feeding level: Pb0.0001, interaction: P=0.3826;
144 hah: sh density: P=0.0027, feeding level: Pb0.0001, interaction: P=0.0018; 192 hah: sh density: P=0.0013, feeding level: Pb0.0001, interaction: P=0.0012. In each graph,
bars that share at least one superscript in common do not differ at Pb0.05 (Scheffe F-tests between all 10 treatments).
55 J. Slembrouck et al. / Aquaculture 294 (2009) 5259
A model of maximal food intake against sh size was constructed
from the data on gut contents (Fig. 5). Based on this model, it became
obvious that sh offered 1 RFL were underfed at the start of the
experiment. The question whether this feeding level remained
insufcient during the subsequent days cannot be answered directly,
because it requires knowledge on the dynamics of growth and
mortality. For each treatment and each rearing day, the number of
survivors was calculated on the basis of a constant mortality rate (see
methods), sh size was modelled from growth curves constructed
from samples at 2-day intervals, then sh body mass was back-
calculated, using the model described in the methods. Finally, sh
number and body mass were equated to determine how many nauplii
would have been needed to feed the survivors maximally on each day,
and this value was compared to the number of nauplii that was
actually distributed on this particular day. The results of this analysis
(Fig. 6) clearly indicate that, at all sh densities, the sh that were
offered 1 RFL were underfed throughout, except for the last feeding
day, and even those given 3 RFL were slightly underfed from 60 to
108 hah.
Fig. 6. Comparison between the amounts of food that were actually distributed and
those that would have been needed to feed maximally the survivors (nauplii available:
needed). Calculations are based on sh survival, size and maximum food intake (Figs. 2,
3 and 5, see text for details). All points below the bold horizontal line correspond to
situations where sh were fed less than maximally. The 10:81 treatment is not
illustrated here.
Fig. 5. Ontogenetic variation of food intake in larvae of P. hypophthalmus, based on the
gut contents of sh sampled in all treatments at 48, 96 and 144 hah. A model of food
intake against sh size was constructed from the sh with the highest gut contents
relative to their size (circled symbols), which were deemed to have replenished, and
stands as:
FI
max
= 74:01 3:39 + 14:51 0:34 TL
where FImax is the maximum food intake (nauplii sh
1
meal
1
), TL is the total body
length of sh (mm), and values between brackets are the standard errors of coefcients
(F = 1840.62, R
2
= 0.986, df = 27, P b 0.0001 both for intercept and slope).
Fig. 4. Gut content of larvae of P. hypophthalmus, depending on age, sh density and feeding level. The lower right graph illustrates the coefcients of variation (CV, %) of gut content
between treatments. Bars and whiskers are the means and standard errors (10 sh per sample two replicate groups per treatment). Bars with the same shade correspond to
treatments with identical prey density (nauplii per volume). The dotted horizontal lines in the rst three graphs indicate the reference feeding level on these days (nauplii per meal
per sh stocked). Statistics for gut content (2-way ANOVA, excluding the 10:81 group): 48 hah: sh density: P=0.1491, feeding level: Pb0.0001, interaction: P=0.1235; 96 hah: sh
density: P=0.0634, feeding level: Pb0.0001, interaction: P=0.3420; 144 hah: density: P=0.1766, feeding level: Pb0.0001, interaction: P=0.0680. Bars that share at least one
superscript in common do not differ at Pb0.05 (Scheffe F-tests between all 10 treatments).
56 J. Slembrouck et al. / Aquaculture 294 (2009) 5259
3.4. Apparent food conversion efciency
The apparent gross conversion efciency (GCE, DM:DM) until
144 hah was signicantly dependent on feeding level (P=0.0006),
but was not inuenced by the sh density (P=0.9301) or by the
interaction between the two factors (P=0.2583; Fig. 7). At all sh
densities, the GCE was highest (0.35) at 1 RFL, and decreased
signicantlyat higher levels (except between1 and3 RFL at 90 shL
1
).
4. Discussion
4.1. Main outcomes of the study
This study is among the very rst to analyse the interactions
between feeding level, prey density and sh density in sh larvae. It
provided evidence that sh density and prey density, rather than
feeding level, govern the growth of P. hypophthalmus larvae, whereas
their survival is inuenced to a greater extent by the feeding level than
by prey density, and little affected by sh density. The results also
provide information on how the rearing and feeding strategies can be
dened in respect to production constraints, in terms of survival,
growth or food conversion. In particular, the study permitted us to
model the relationships between food intake and sh size during the
larval stages of P. hypophthalmus, thereby giving the opportunity of
adjusting feeding charts to sh size, which can be rapidly obtained
from sh samples. The maximal meal size is low during the early
feeding stages (12% WM at 5.5 mm TL and 0.72 mg), but increases
rapidlyas it amounts to22 and26%WM, respectivelyat 6.0and6.5mm
TL (corresponding WM of 1.2 and 1.6 mg). From 7 mm TL onwards,
meal size starts decreasing in a curvilinear way, and it amounts to
about 10% WM at 15 mm TL (WM of 25 mg). Based on stomach
residence time (b34 h at 29 C in 6.57.0 mm TL sh; E. Baras and Y.
Moreau, unpublished data), the daily food intake can be 6 to 8 times
higher than meal size, thus 155205% WM. These estimates concur
with those obtained during independent experiments where the food
intake of cannibals was estimated from the number of siblings
consumed during 24 h (i.e. up to 203% WM in a cannibal of 5 mg
WM; E. Baras, unpublished data). They also support the empirical
recommendation of Hardjamulia et al. (1981) that larvae should be fed
250% WM per day during the rst feeding days.
4.2. Effects on sh growth
The model between food intake and sh size emphasised that sh
offered 1 RFL were underfed. This statement remained unchanged
after survival rates were taken into account in the analysis, except for
the last feeding day (Fig. 6). Similarly, it turned out that from 60 to
108 hah sh offered 3 RFL were not fed in excess but just maximally.
However, these ndings do not compromise the relevance of the
experimental design, because the highest feeding level that was
evaluated at all sh densities (9 RFL) was enough to produce maximal
or near-maximal growth in this species (comparison with the 10:81
treatment; Fig. 3). The growth model that was constructed with a
stepwise multiple-regression analysis provided evidence that prey
density (per volume unit) prevailed over prey availability (per sh),
and that sh growth at a particular prey density was inversely
proportional to the sh density.
The effect of prey density on the growth of P. hypophthalmus larvae
can be accounted as follows. Young larvae of this species swim
throughout the water column with their mouth wide open (Hard-
jamulia et al., 1981; Subagja et al., 1999) and possess little ability for
manoeuvring, since their pectoral ns are not developed at the start of
exogenous feeding (E. Baras, J. Slembrouck and M. Legendre,
unpublished data). With such behavioural and morphological traits,
the probability of encountering and capturing food items is directly
proportional to prey density, to a greater extent than in larvae of other
sh species with greater manoeuvring abilities. Whatever the intrinsic
foraging efciency of a larva of P. hypophthalmus, it is presumably
reduced in presence of conspecics, which either remove prey from
the trajectory of a particular forager or interact with its movements.
Hence, it presumably takes longer for a particular sh to replenish in
the presence of numerous conspecics, This interpretation is
supported by the observation that the food intake of 48-h old larvae
at a particular prey density was negatively correlated with sh
density, even when food was in excess (Fig. 3). The longer the foraging
time, the higher the swimming costs (Ruzicka and Gallager, 2006),
and the slower the growth.
There was no adverse impact of high prey densities on food intake
or sh growth, in contrast to the situation in redsh Sebastes spp.
(Laurel et al., 2001). Redsh larvae offered high prey densities
frequently abandoned a prey capture sequence and re-orientated
towards other prey, thereby resulting in slower growth. The absence
of such confusion effect in P. hypophthalmus is consistent with their
very simple foraging behaviour, which merely relies on constant
swimming and little orientation towards prey.
4.3. Effects on food conversion efciency
At all sh densities, the lowest gross conversion efciency (GCE,
DM:DM) was observed when sh were offered 9 RFL (or 81 RFL at 10
sh L
1
): these feeding levels largely exceeded the maximal needs
of sh (cf. Figs. 6 and 7), so food was inevitably wasted. The highest
GCE at all sh densities (about 0.350.40) was obtained with the
lowest feeding level. However, this nding is not an encouragement
to feeding submaximally the larvae of this species, especially in view
of the negative effects of underfeeding on sh growth and survival.
Furthermore, the GCE was almost equally high with other treatments
that produced signicantly faster growth and higher survival
than among groups fed 1 RFL (e.g. 90:3 treatment). The best GCE
values during this study were just below those (0.45 DM:DM) ob-
tained in experiments where the growth of cannibalistic larvae
of P. hypopthalmus was compared to their actual food intake (E.
Baras, unpublished data). This comparison suggests that, in spite
of the apparent simplicity of their foraging behaviour, larvae of
P. hypophthalmus are capable of nding and capturing food quite
efciently, even when prey are scarce or just abundant enough to
full their needs (Fig. 6).
Fig. 7. Comparison between the gross food conversion efciency (GCE, DM:DM) of
larvae until 144 hah, depending on sh density and feeding level. Bars and whiskers are
the means and differences between two replicates. Bars with the same shade
correspond to treatments with identical prey density (nauplii per water volume).
Statistics (2-way ANOVA, excluding the 10:81 group): sh density: P=0.9301, feeding
level: P=0.0006, interaction: P=0.2583 Bars that share at least one superscript in
common do not differ at Pb0.05 (Scheffe F-tests between all 10 treatments).
57 J. Slembrouck et al. / Aquaculture 294 (2009) 5259
4.4. Effects on survival
The effects of feeding level and sh density on sh survival were
less straightforward than those on sh growth or food conversion.
Survival increased with increasing feeding levels, except for the
highest prey densities (90:9 and 10:81 treatments). Brine shrimp
nauplii release nitrogenous metabolites, the accumulation of which in
recirculating systems can impact sh survival, especially at densities
above 8 nauplii mL
1
(Houde, 1975; Lger et al., 1986; Puvanendran
and Brown, 1999). The prey density culminated at 33 nauplii mL
1
on
the last feeding day of this study. Freshly hatched brine shrimp nauplii
survive no more than a few hours in fresh water at 29 C, and their
decay alters the water quality. However, it is unlikely that in this study
P. hypophthalmus larvae died from a decline in the chemical quality of
the water. First, the total ammonia nitrogen (TAN) in the recirculation
system remained low throughout the experiment, except for the very
last day. However no high mortality ensued. Second, recent experi-
ments indicated that larvae and juveniles of this species had a rather
high tolerance toTAN(DL
50
in 5-g sh is about 35 mg TANL
1
at 28 C
and pH 8.2; D. Caruso and E. Baras, unpublished data 2009). Finally,
survival rates of 85% have been obtained for larvae of P. hypophthalmus
raised from 8 to 28 dah in a recirculating system where nitrite and
ammonia concentrations varied between 0.2 to 7.5 mg L
1
, and 0.5 to
1.0 mg L
1
, respectively (J. Slembrouck, unpublished data). Alter-
natively, it is likely that the large amounts of uneaten food at high prey
density facilitate the proliferation of bacteria. Bacterial growth puts
larvae at risk, especially those that grow long oral teeth and are prone
to injure each other, as is the case of P. hypophthalmus. Yet, antibiotics
were applied throughout the experiment (10 mg L
1
oxytetracyclin).
This concentration gives satisfactory results with standard feeding
schedules (Subagja et al., 1999), but it might be insufcient for a rapid
bacterial growth with high loads of wasted food.
The low survival of P. hypophthalmus at low feeding levels cannot
be blamed on sh dying out of hunger, since the P
50
mortality of larvae
deprived of exogenous food lies at 89 dah at 28 C (E. Baras,
unpublished data). Cannibalism is generally higher when food is
rationed (syntheses in Hecht and Pienaar, 1993; Baras and Jobling,
2002). However, cannibalism generally causes the elimination of the
smallest sh, while the largest sh gain a rapid growth advantage and
become jumpers. If cannibalism had been the main source of
mortality in this study, the survivors in underfed groups would have
been expected to grow as fast as, or faster than in other groups, while
the opposite trend was observed. Other factors can be invoked, in
relation to the morphology of P. hypophthalmus larvae. At the onset of
exogenous feeding, these larvae exhibit a large gape (18% TL), and long
oral teeth (0.10 mm). They swim continuously throughout the water
column but in absence of pectoral ns, they are probably incapable of
ne swimming manoeuvres. Encounters result in deadly clashes,
because the larvae cannot get rid of the siblings they grasped and
rapidly suffocate. This situation is most transient, as the mouth and
teeth exhibit a strongly negative allometric growth. The faster the sh
grow through this interval, the shorter the risk period and the lower
the mortality. Furthermore, sh probably need to forage over longer
periods if food is scarce, thereby increasing the risks of deadly
encounters with siblings during each meal. Altogether, these inter-
pretations might account for why survival was about three times
lower among the groups that were underfed than when food
availability was higher.
4.5. Conclusions
The study highlights how the production settings in nurseries of P.
hypophthalmus could be adjusted if the premiumis growth, survival or
food conversion. These priorities can vary substantially between
producers, depending on local constraints in terms of food supply, size
and type of rearing system (ponds versus recirculating water
systems), number of broodsh relative to the production of weaned
larvae or juveniles, water renewal rate or water quality. If growth,
survival and food conversion are equally important, the best
compromise until 192 hah is a density of 90 sh per litre, with slight
overfeeding (3 RFL). It is possible that higher performance be obtained
with intermediate combinations of feeding levels and sh densities, or
with feeding schedules that better track the ontogenetic variations in
sh behaviour and morphology. For example, it might be worth
testing very high feeding levels during the rst 24 or 36 h of
exogenous feeding, in order to maximise early survival and growth,
then decreasing the feeding level. This would have little negative
impact on the food conversion, because young larvae of P.
hypophthalmus consume few nauplii per capita.
Acknowledgements
The authors acknowledge Kamlawi and Wawan (Sukamandi
research station) for their technical assistance, and Rmi Dugu
(IRD, UR 175, Montpellier) for the constructive criticisms on a draft of
this manuscript. This work was part of a project on the biodiversity
and culture of Southeast Asian catshes supported by the European
Union (contract IC18-CT96-0043). Mrs. Dominique Caseau contrib-
uted to improve the English style of the manuscript.
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