Effects of Different Diet Regimes On Development of Gill and Rostrum Spines of Pacific White Shrimp

Aquacultura Indonesiana (2013) 14 (XX): XX-XX
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Hak cipta oleh Masyarakat Akuakultur Indonesia 2013
Effects of different diet regimes on development of Gill and Rostrum spines of
Pacific white shrimp Litopenaeus vannamei
Romi

Novriadi
Aquaculture Hall
Directorate General of Aquaculture, Ministry of Marine Affairs and Fisheries
Jl. Raya Barelang, Jembatan III, PO BOX 60 subdistrict of Sekupang of Batam city of Riau Islands
Email : Romi_bbl@yahoo.co.id
Telphone: (0778) 7027623 7027624, Faksimile: (0778) 3582557
A b s t r a c t
Romi Novriadi. XXXX. The effect of partial replacement of Artemia nauplii with artificial diets were
evaluated by analyzing the gill and rostrum spines development as well as the quality of white shrimp
(Litopenaeus vannamei, Boone) post larvae (PL). The treatments were: (1) live food control Artemia nauplii, (2)
65% replacement of Artemia with artificial diet, and (3) 85% replacement of Artemia with artificial diet. All
treatments were also compared to the L. vannamei PL generated from commercial hatcheries. Instar II Artemia
nauplii were cultured as a starter live food and Frippak microencapsulated feeds were provided as the artificial
diet to the L. vannamei. A significantly better quality, gill and rostrum spines development was achieved by post
larvae (from PL1 to PL 5) fed with live Artemia or the 65% replacement of Artemia in comparison to 85%
replacement of Artemia. Interestingly, even had the lowest quality, PL fed with 85% replacement of Artemia
nauplii still recorded a superior quality compared to commercial hatchery products at the same PL stages,
suggesting that the nutrition standard performed at the commercial hatchery in the sampling area does not
support the optimal development of gill and rostrum spines as well as the quality of L. vannamei at the post
larva stages. Additionally, the findings are important in aquaculture where the appropriate concentrations of
artificial diet are also able to demonstrate a better growth, osmoregulation and detoxification performance in L.
vannamei post larvae and this may contribute to the efficiencies towards a reduction of Artemia nauplii cost.
Keywords : Litopenaeus vannamei, Diet regime, Gill, Rostrum spines, Larva index quality
Introduction
Pacific white shrimp (Litopenaeus
vannamei, Boone), is an economically important
species that is widely cultured not only in their
native regions at western pacific coast of Latin
America but also in the expansive Asia (Liao and
Chien, 2011). However, the success story of L.
vannamei farming has always been crippled by
the lack of high-quality post larvae. One of the
major factors hampering the quality of hatchery-
reared post larvae is nutrition, especially during
the critical periode where there is an extensive
morphogenesis in the digestive system occurs at
the first 10 days of post larvae (Lovett and Felder,
1989). The behavioural changes from herbivorous
(filter feeders) to carnivorous (hunters), which
occur due to the life cycle development of
shrimps require appropriate nutrition and correct
prey size (Lavens and Sorgeloos, 1996).
Freshly hatched Artemia nauplii are the
most widely used as live feed for early stage
crustacean larvae due to its high nutritional
quality and ease of use (Sorgeloos et al., 1986).
The suitability of Artemia nauplii as live feed in
crustacean larviculture is also supported by the
presence of 20:5(n-3) fatty acid
(eicosapentaenoic acid or EPA) (Leger et al.,
1986). Artemia nauplii is usually given in live
condition to the larvae to give an optimum
feeding regime especially on the mysis and early
postlarval stages (Mc Fey and Fox, 1983). In
addition, several forms of Artemia have also been
used in most penaeid hatcheries to induce an
optimal growth: i.e. heat killed nauplii, frozen
and blended Artemia (Wilkenfeld et al., 1984;
Wouters and Van Horenbeeck, 2003; Juarez et
al., 2010).
Good post larval quality is a top priority
in penaeid hatchery. Several factors influence the
growth and quality performance of L. vannamei
post larva. One of the factors is the development
of gill that play a key role in respiration,
osmoregulation and detoxification. In their real
life, gills are in direct contact with the external
environment; therefore, their development are
crucial to enhance shrimp tolerance to external
biotic and abiotic factors (Clavero-salas et al.,
2007). Other than gill, the development of
rostrum spines also play an important role,
particularly as an indicator for larval stage
identification.
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In penaeids hatchery, the increase of the
operational cost has been considered as a major
contsraint in the use of live nauplii.
Consequently, novel approaches to substitute the
use of Artemia are needed. Recent research have
include the use of rotifers Brachionus plicatilis
(Naessens et al., 1995), nematodes Panagrellus
redivivus (Biedenbach et al., 1989) and extending
the use of algal food for the late mysis and post
larval stages as a potential alternatives for
substitute live feed Artemia (Gopalakrishnan,
1976). Moreover, micro-particulate and micro-
encapsulated formulated diets can also be used to
reduce the amount of Artemia cysts (Galgani and
Aquacop, 1988; Wouters et al., 2009). Many
scientists have studied the effect of the
supplement diets on survival, growth or total
length of penaeid shrimps (Biedenbach et al.,
1989; Hirata et al., 1985; Cobo, 2013; Brito et al.,
2001 and Naessens et al., 1995). Moreover, Brito
et al. (2001) have stated that partial substitution
(50%) of Artemia by artificial diet and the use of
algae beyond the first post-larval stage have a
benefit impact to the growth and nutritional state
of L. vannamei.
In present study, the use of Artemia
nauplii as a live food and their combination with
Frippak
microencapsulated diet was evaluated in

L. vannamei post larvae. We examined variations
in gill and rostrum spines development as well as
the quality of L. vannamei at the first five days of
post larval development as a response to the
different diet regimes included Artemia nauplii as
live food control, 65% and 85% substitution of
Artemia with Frippak. We wished to determine if
the variations in quality, especially in gill and
rostrum spines development that have an
important role to enhance the salinity tolerance of
L. vannamei, is related to any combination of
food. Additionally, the result may eventually
contribute towards a reduction of Artemia cost to
support the sustainability of shrimp farming
production.
Material and methods
Experimental animal
Larvae of Pacific white shrimp (L.
vannamei, Boone) was used as an experimental
animal and obtained from a commercial hatchery
in Chonburi Thailand. They were reared until
Protozoea II in the concrete tank using a feeding
schedule proposed by SCRD Thailand. Even
though the L.vannamei nauplii at this stage still
deplete internal reserves and do not feed on
algae, as much as 20 cells/mL of Chaetoceros
calcitrans was added to guarantee the feed
supply once the animals reached the protozoea
stage. Feeds was provided 6 times per day and
the number of Chaetoceros calcitrans was
counted by using Haemocytometer.
Experimental design
At stage protozoea III, larvae were
transferred to spherical tank containing 175 L
saline water (30 mg/L). The spherical tank were
placed in hatchery containing water maintained
at 291
0
C using thermostatic heater. Each tank
were stocked with batches of 175 nauplii of L.
vannamei. The water quality parameters, the
photoperiod, temperature and the feeding regime
were adjusted. Water was exchanged at a rate of
aproximately 50% per day after removing waste
and uneaten feed by siphoning. Several water
quality parameters, i.e., NH
4
-N, NO
2
-N, and
NO
3
-N were measured every two days. Gentle
aeration was applied in all rearing tank to
maintain the dissolved oxygen concentration
above 5 mg/L. The photoperiod was set at 12 h
light at an intensity of 900-1000lx with
fluorescent lamps at the water surface. Newly
hatched Artemia (SepArt, New generation High 5
Artemia, INVE aquaculture) were used as live
food and different feeding regimes was applied
from Protozoea III until PL
5
(Five days after
metamorphosis) according to Table 1. The
Artemia dosage was calculated based on the
feeding level and the number of shrimp larvae in
the tank and split over two feedings period at
07.00 h and 16.00 h.

Table 1. Type and amount of feed provided for each successive stage of larval development of
Litopenaeus vannamei
Stage Live food (control) 65% substitution 85% substitution
Nauplii 5 C.calcitrans 20
cells/L
C.calcitrans 20 cells/l C.calcitrans 20 cells/l
Protozoea
1-2
C.calcitrans 50
cells/L
C.calcitrans 50 cells/l C.calcitrans 50 cells/l
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Protozoea
3 Mysis 3
C.calcitrans 50
cells/L
Artemia nauplii 2-
10/mL
C.calcitrans 50 cells/l
65% substitution of Artemia
nauplii with Frippak
C.calcitrans 50 cells/l
PL1 PL5 Artemia nauplii 5-
10/mL

In our experiments, an open clear water system
was used with a daily water exchange of 50 %.
During water exchange, the remaining (heat
killed) Artemia and waste from the previous day
were removed by siphoning. This operation was
carried out with great care to avoid loss of larvae.
Feeding was done after water exchange. Three
different treatments with four replicates each
were tested (Table 1), namely: life food as control
(Artemia nauplii only), 65 % substitution of
Artemia nauplii with Frippak and 85 %
substitution of Artemia nauplii with Frippak,
respectively. The substitution was adjusted based
on the daily growth (length, wet weight and dry
weight) and the estimation number of L.
vannamei in the experiment tank. The
experiments was terminated after five days of
feeding at post larvae stage (PL-5) and quality,
length, gill and rostrum development were
determined.
Determination of gill and rostrum spines
development
Gill and rostrum spines development in
each treatment was observed under light
microscope equipped with a Canon EOS digital
camera connected to a PC. The images were
compared with gill and rostrum development at
the same post larvae stages from commercial
hatcheries in Chonburi-Thailand. The rearing
condition and feeding regime in commercial
hatchery was also evaluated by direct interview to
obtain a relation to the gill and rostrum spines
development.
Post larva quality
Post larva (PL) quality was checked at
every stage of PL by analyzing the osmotic stress,
dry weight, wet weight, and average length
among the treatments. Size of the PL was
measured according to Kitani (1986) from the
base of the antennal flagellum to the telson, and
the mean of total length and coefficient of
variation were calculated for each treatment and
PL stages of sampling. To evaluate the PL to
osmotic stress, a simple stress test was performed
according to Samocha et al. (1998) with some
modification. Sample for each treatment was left
for 60 minutes at different range salinity, 1, 2 and
3 , respectively and then the number of
survivors PL was counted after the specified
time. Dry weight analysis were performed by
drying 1 gram of PL in the oven for 24 hours at
60
0
C and measured using analytical balance.
Wet weight obtained by measuring 1 gram and
dividing by the number of existing PL. The post
larvae quality from the commercial hatchery was
also evaluated as a comparison to the post larvae
quality generated from the experiment.
Statistics
The results are presented as mean values
followed by the standard deviation and the
percentage data were arcsine transformed for
statistical comparisons to satisfy normal
distribution and homoscedasticity requirements.
Data were then subjected to one way analysis of
variances followed by Tukeys multiple
comparison range using the statistical software
SPSS version 21.0 to determine significant
differences among treatments. All significance
level of the statistical analysis was set at P<0.05.
Results
Gill and rostrum spines development
Microscopic analysis showed that the
development of gill and rostrum spines varies for
each treatment . At PL 1 stages, larvae fed only
live food (control) and fed Frippak 65 % have 3
dorsal rostral spines. Another growth indications
of spine was also observed between the first
spines (at the edge) and the second spines.
Larvae fed frippak 85 % treatment have only 3
dorsal rostral spines without any indication of
further spines growth. At PL 2 stages, larvae fed
only live food and fed frippak 65 % have 4 dorsal
rostral spines each, while the larvae fed frippak
85 % still have 3 dorsal rostral spines with new
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indication of spines growth. At PL 3 stages,
larvae fed live food and fed frippak 65% have 4
dorsal spines each with the addition of setae and
the second spines. However, at larvae fed frippak
85 % there are variations in the number of spines
(3 and 4 dorsal spines) with or without setae. At
PL 4 stages , larvae fed live food and fed frippak
65 % have 4 dorsal spines each with 2-3 setae at
the second dorsal spines, while the l;arvae fed
frippak 85 % also have 4 dorsal spines but only
with 1-2 setae at the second dorsal spines. Finally
at PL 5, larvae fed live food have 5 dorsal rostral
spines, while larvae fed frippak 85 % have 4
dorsal rostral spines. The difference in rostrum
spines became more and more pronounced
towards the end of the experiment (Fig.2).

Figure 1. Stage wise gill development of PL1 to PL5 of L.vannamei for each treatment

PL 1
PL 2
PL 3
PL 4
PL 5
Live food (control) 65 % frippak
85 % frippak
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Figure 2. Stage wise rostrum spines development of PL1 to PL5 of L.vannamei for each treatment

The treatments with only Artemia nauplii (live
food) and with Frippak 65 % at PL 2 indicate the
presence of 2-3 branched at their gill, while with
Frippak 85 % remain without branched. At PL 3,
treatment with only live food and with Frippak 65
% majority of shrimps have 5 branches in their
gill, while with Frippak 85 % varies from 2-5
branches. At PL 4, treatment with only live food
and with Frippak 65 % indicated 7-9 branches,
while with Frippak 85 % varied from 5-7
branches. And finally at PL 5, treatment with only
live food and with Frippak 65 % showed 10-11
branches in each of the gill, while the branches in
Frippak 85 % varied between 3-9.
Post larva quality analysis
1. Average length and dry weight
Larvae fed with Artemia nauplii were
significantly longer (P < 0.05) in average length
at PL 5 (0.87 0.011 cm) followed by 65 %
frippak substitution treatment (0.79 0.018 cm),
and this group was significantly different with
PL 2
PL 3
PL 4
PL 5
PL 1
Live food (control)
65 % Frippak
85 % Frippak
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treatment of 85% frippak substitution of Artemia
nauplii resulted in the shortest average length (
0.680.017 cm) at the end of experiment (PL5)
(Figure 3). Post larvae of L.vannamei fed with
Artemia nauplii (Live food) were significantly (P
< 0.05) heavier in terms of dry weight (0.218
0.03 mg) than those other treatments. Larvae fed
with 65 % frippak substitution were significantly
(P < 0.05) heavier in dry weight (0,191 0.01
cm) than fed 85 % frippak substitution (0,175
0.02 cm). Overall, the dry weight variation trends
were observed from PL3 stages.

Figure 3. Average length of L. vannamei from PL1 to PL 5, fed with Artemia naulii (LFC = Live
Food), 65% S and 85% S (S= substitution of Artemia nauplii with Frippak INVE Commercial feed),
respectively. Values are averageSE, n=4. Different letters denote significant differences (P<0.05).
Different types of superscript denote different comparison.

Figure 4. Average dry weight of L. vannamei post larvae from PL1 to PL 5 fed with Artemia naulii
(LFC = Live Food), 65% S and 85% S (S= substitution of Artemia nauplii with Frippak INVE
Commercial feed), respectively. Values are averageSD, n=4. Different letters denote significant
differences (P<0.05). Different types of superscript denote different comparison.
2. Salinity stress analysis
Post larvae of L.vannamei are maintained
in saline water (30 ) at 300,5
0
C before
salinity shock. All the treatments (live food 65%
frippak substitution and 85% frippak substitution)
were exposed to three low salinity solution (1 ,
2 and 3 ) at ambient temperature and
survival determined after 1 h. Twenty post larvae
were used per trial at every post larvae stages
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Figure 5. Mortality (%) of PL 1 to PL 5 L.vannamei post larvae fed Artemia nauplii with or without
commercial feed. In the experiment, LFC : Live food (only fed with Artemia nauplii), 65 % S : 65 %
substitution of Artemia nauplii with frippak, and 85 % S : 85 % substitution of Artemia nauplii with
frippak. All the treatments exposed to 1 solution. Values are averageSD, n=3, Different letters
denote significant differences (P<0.05).
The results presented in Figure 5 showed
that a significant low percentage mortality
(P<0.05) was observed in L. vannamei post
larvae fed with Artemia nauplii exposed to 1
salinity solution in comparison to 85% partial
substitution of Artemia with frippak at every
stage of post larvae development. However, no
significant difference in mortality was observed
between post larvae of L. vannamei fed with
Artemia in comparison to post larvae that receive
65% partial replacement of Artemia.
Similar trend was also noted at 2 and
3 treatments solution (Fig.6 and Fig.7). The
treatments with both live food and with 65 %
frippak substitution resulted in the lowest
mortality percentage in comparison to 85%
partial replacement of Artemia nauplii (P<0.05)

Figure 6. Mortality (%) of PL1 to PL5 L.vannamei post larvae fed Artemia nauplii with or without
commercial feed. in experiment, LFC : Live food (only fed with Artemia nauplii), 65% S : 65%
substitution of Artemia nauplii with commercial feeds, and 85% S : 85% substitution of Artemia
nauplii with commercial feeds. All the treatments exposed to 2 solution. Values are averageSD,
n=3, Different letters denote significant differences (P<0.05).

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wet weight Dry weight average of
( gr) ( gr ) length (cm) 1 g/L 2 g/L 3 g/L Total T (C) sal ( g/L)
Prom-Phong Hatchery Penaeus Vannamei Zoea : C. Calcitrans + arti fi ci al di et+ heat ki l l ed Artemia PL 6 0,0013624 0,000326 0,71032 5 5 0 10 Brand "whi te bear" 50% 25 30
Mysi s : C. Cal ci trans + arti fi ci al di et+ heat ki l l ed Artemi a
PL 1-12 : Daphnia + Artemia + arti fi ci al di et
Pichitpon Farm Penaeus Vannamei Zoea : C. Calcitrans + arti fi ci al di et+ heat ki l l ed Artemia PL 8 0,00223214 0,00047 0,91085 1 0 0 1 Brand "Gal axy" 60% 27 25 (rai ny day)
PL 1-12 : Artemia + arti fi ci al di et+ Bl ack fl akes
Lab-Inter2 Farm Penaeus Vannamei Naupl i i : C. Calcitrans PL 7 0,00174216 0,0004546 0,8326 0 0 0 0 Brand " Swan GSL" 50-60 % 30 30
Zoea : C. Calcitrans + arti fi ci al di et+ heat ki l l ed Artemia
PL 1-12 : Artemia + arti fi ci al di et+ Bl ack fl akes
55 Farm Penaeus Vannamei Naupl i i : C. Calcitrans PL 6 0,00110865 0,000276 0,7116 1 0 0 1 Brand "Eagl e" Artemi a 50% 30 30
Zoea : C. Calcitrans + arti fi ci al di et+ heat ki l l ed Artemia
PL 1-12 : Artemia + Daphnia + arti fi ci al di et+ Bl ack fl akes
Phanh Tib Farm Penaeus Vannamei Naupl i i : C. Calcitrans PL 12 0,00649351 0,0015633 0,9951 2 0 0 2 Brand " Aqua " AQP 50% 30 30
Zoea : C. Calcitrans + Spirulina + heat ki l l ed Artemia
PL 1-12 : Artemia +arti fi ci al di et
Chaleam Farm 1 Penaeus Vannamei Thi s hatchery i s onl y as a transi t step at PL 8-12 PL 8 0,0037594 0,00053867 0,9817 1 0 0 1 Brand "Gal axy" 50% 29 20
Artemi a onl y appl i ed when packagi ng to avoi d the stress -
condi ti on i n shri mp
Chanchira Farm 2 Penaeus Monodon Mysi s to PL 1 : heat ki l l ed artemi a (Instar-1) PL 2 0,0015008 0,00021067 0,704 20 20 20 60 Brand M.T.I > 80 % 30 30
Chanchira Farm 1 Penaeus Monodon Mysi s to PL 1 : heat ki l l ed artemi a (Instar-1) PL 3 0,0013298 0,00019933 0,7548 20 20 20 60 Brand M.T.I > 80 % 30 30
Chanchira Farm Penaeus Vannamei Mysi s to PL 1 : heat ki l l ed artemi a (Instar-1) PL 3 0,00065934 0,000096 0,4914 5 2 2 9 Brand M.T.I 60% 30 30
PL 1-12 : Artemia +arti fi ci al di et
Pasit Farm 1 Penaeus Monodon Naupl i i : C. calcitrans PL 8 0,0117375 0,0002967 0,70165 20 20 20 60 Brand " Premi um" > 80 % 30 30
Mysi s to Zoea 1 : heat ki l l ed Artemia (i nstar 1) Red and green package
Zoea 2 to PL 4 : Artemia
PL 5 onwards : Daphnia
Pasit Farm Penaeus Vannamei Naupl i i : C. calcitrans PL 3 0,00105568 0,000099 0,5973 5 3 2 10 Brand " Premi um" 60-70 % 30 30
Mysi s to Zoea 1 : heat ki l l ed Artemia (i nstar 1) Red and green package
Zoea 2 to PL 4 : Artemia
PL 5 onwards : Daphnia
Ma win Farm Penaeus Vannamei Zoea I-III : Spi rul i na powder + Chaetoceros PL 10 0,00325733 0,00043313 0,8103 2 0 0 2 Brand " M.C.C " 10-50% 30 30
zoea II they appl i ed the heat ki l l ed artemi a
PL 1 onwards : Artemia + arti fi ci al di et
someti mes mi x wi th Vi tami n
Observation Parameter
SR
water quality (at sampling) Hatchery Farm Feeding scheme Type of shrimp mortality with osmotic stress (1 H)
PL stages
Type of Artemia

Figure 7. Mortality (%) of PL1 to PL5 L.vannamei post larvae fed Artemia nauplii with or without
commercial feed. in experiment, LFC : Live food (only fed with Artemia nauplii), 65% S : 65%
substitution of Artemia nauplii with commercial feeds, and 85% S : 85% substitution of Artemia
nauplii with commercial feeds. All the treatments exposed to 3 solution. Values are averageSD,
n=3, Different letters denote significant differences (P<0.05).
Comparison of post larva quality with
commercial hatcheries.
Twelve shrimp hatcheries was visited in
the frame of sampling campaign to obtain a
comparative data between post larva quality
generated from trial work with post larva quality
from commercial hatchery. Sampling was carried
out using local equipment and sample were kept
on plastic filled with oxygen during
transportation.

Table 2. Summary of sampling campaign in 12 commercial hatcheries
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There were two commercial hatcheries
that could be compared due to the availability of
PL stage based on the observed post larva stage,
namely: Chanchira Farm (PL 3 of L.vannamei)
and Pasit Farm (PL 3 of L.vannamei). Based on
the post larva quality and microscopic analysis,
PL from commercial hatcheries appeared to grow
slightly lower in terms of dry weight and average
length even when compared with PL fed Frippak
85% substitution. Percentage mortality in 1
solution also slightly higher compared with
larvae fed with live food and with 65% frippak
substitution treatments, but equal to the mortality
number of 85% frippak substitution treatment.
Similar trend was also seen in the 2 and 3
solution treatments, no replicate measurements
are available for commercial hatcheries sample to
enable statistical evaluation. Gill and rostrum
development was also slower for both
commercial hatcheries sample.

Figure 7. Rostrum spines and gill of L.vannamei at PL 3 in Pasit Farm (upper row : A and B) and
rostrum spines and gill of L.vannamei at PL 3 (lower row : C and D) images in Chanchira farm
Discussion
In this study, we investigated the effect of
different diet regimes on the pacific white shrimp
L.vannamei post larvae performance from PL1 to
PL5 and on the rostrum spines and gill
development. During their life cycle, Shrimp will
be more herbivorous at zoea stages and therefore
require microalgae such as diatoms Chaetoceros
spp., Thalassiosira spp. and Skeletonema spp
(Cook and Murphy, 1966; Emmerson, 1980;
Martins et al., 2006; Soares et al., 2006), whereas
at the mysis and postlarva stage, penaeid shrimp
becomes more carnivorous and require
zooplankton (Lavens and Sorgeloos, 1996).).
It is evident that the administration of
live Artemia nauplii from Protozoea III to Post
larvae 5 resulted in better gill and rostrum spines
development (Figure 1 and 2). Moreover, These
results showed no significant differences with
65% partial replacement of Artemia in respect to
the gill and rostrum spines development along
with the quality of post larvae. In contrast, 85%
replacement of Artemia nauplii resulting in the
lowest quality in comparison to other treatments.
In term of gill development, as an euryhaline
tropical shrimp, L. vannamei highly depend on
gill, mainly for regulation of their
osmoregulatory capacity and tolerance of salinity
fluctuations in the water (Wickins and Lee.
2002). Salinity is one of the most important
abiotic factors for marine and estuarine
organisms, which affect their growth and
survival (Saoud et al., 2003; Buranajitpirom et
al., 2010; Pqueux, 1995; Fielder et al., 2001;
Atwood et al., 2003 and Kumlu et al., 1999 ).
Moreover, mass mortalities in grow out ponds of
penaeid shrimp is often related to the
A B
C D
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fluctuations of salinity levels and less observed
in smaller sized organisms because they are better
osmoregulators (Vargas-Albores and Ochoa,
1992).
Our results also indicate that the different
diets directly influenced the rostrum spines
development (Fig. 2). Although there is no clear
relation, for post larvae it is likely that protein
quality in diet have an important role to induce
the development of rostrum spines in L. vannamei
and this development are related to the quality
and exact stage of post larvae.
Another important feature arising from
this trial is the impact of different feeding regime
to the growth and survival of L. vannamei post
larvae when tested with osmotic stress factor.
Importantly, we found that control and 65%
substitution of Artemia nauplii, which had a better
development of gill and rostrum spines had
essentially the same survival levels (Fig. 5) when
exposed to 1, 2 and 3 solution from 30 for 1
hour. Their survival was significantly higher in
comparison to 85% substitution (P < 0.05).
Dietary manipulations have important roles to
improve the osmoregulatory abilities of culture L.
vannamei subjected to osmotically stressful
conditions which in turn is expected to increase
the quality and productivity of aquaculture
production (Romano and Zeng, 2012). This
mainly due to the osmoregulation process require
energy that are sourced from protein (Setiarto et
al., 2004, Rosas et al., 1999, and Silvia et al.,
2004) and/or lipids (Palacios et al., 2004;
Luvizotto-Santos et al., 2003; Lemos et al.,
2001 and Sang and Fotedar. 2004). Therefore it
seems reasonable to assume that providing an
easy digestible live food and readily available
energy supply with optimal substitution
concentration could effectively improve the
survival of L. vannamei in extreme change of
environment salinity.
In addition to the importance of diet,
every stage of L. vannamei PL responded
similarly to several feed and microencapsulated
diet (Cousin et al., 1993). If there is a difference
in digestibility, potential exist appear between
larvae and postlarvae. Figure 3 showed that from
PL 1 to PL4, there is no significant difference (P
< 0.05) in average length between live food and
65% substitution treatment. However, both of the
treatments (life food and 65% frippak
substitution) have a significant differences (P <
0.05) compared to 85 % frippak substitution.
Furthermore, at the end of observation (PL 5),
treatment live food had a larger average length
(P < 0.05) than 65% frippak substitution. From
PL 1 to PL 5, treatment of 85% frippak
substitution resulted in poor growth in terms of
average length. The trials emphasized that native
protein seems to be better hydrolyzed than the
processed one (Zwilling et al., 1981). This is in
agreement with studies of Cuzona et al (2004),
who reported that more digestible nutrients
absorbed promotes more growth to the cultured
aquatic animals. The provision of feed by using
Artemia nauplii also appears to provide a better
growth, as the post larvae stage had a
significantly heavier (P<0.05) dry weight
compared to 65% and 85% substitution of
Artemia nauplii. This difference is very evident
from PL 3 to PL 5 and 65% substitution still have
a better performance than the 85% substitution.
In comparison of our PL stages with
those from several commercial hatcheries, the
quality of L. vannamei post larvae was not as
expected. The gill and rostrum spines
development in both hatcheries: Pasit and
Chanchira farm was slower in terms of gill
lamellae branch and rostrum spines. Microscopic
analysis show that the PL 3 stages from Pasit and
Chanchira farm is nearly equal to PL 3 of 85 %
substitution and PL 2 of life food treatment,
respectively. In general, the quality performance
of PL from both hatcheries were still lower than
PL fed with life food and with 65% substitution.
We note that these conditions were caused by
inadequate nutrition in their Artemia cyst due to
the diversity of cyst products in the market and
improper Artemia hatching procedure and
feeding regime.
In the present study, appropriate
concentrations of Frippak can also provide a
better growth and performance of L. vannamei
post larvae as well as gill and rostrum spines
development in comparison to post larvae that
fed with Artemia nauplii. The results showed that
an appropriate concentrations of
microencapsulated diet in the diet will also make
the feed more digestible and provide adequate
nutrition to induce gill and rostrum development
resulting in beneficial effects on L. vannamei
post larvae performance.
Conclusion
Artemia nauplii had good dietary and
promote the optimal development of gill and
rostrum spines for Pacific white shrimp
(Litopenaeus vannamei, Boone) at post larvae
stages. The constant dietary availability on
ISSN 0216-0749

microencapsulated diet in Frippak show no
significant differences in gill and rostrum spines
development compared to Artemia nauplii live
food only when 65% of Artemia was substituted
with this diet, while the differences become
significant when the live food substitution
increase up to 85%. In this work, we found that
the development of gill increase the salinity
tolerance of L. vannamei PL mainly due to the
higher survival percentage when exposed to lower
salinity solution.
In addition, from the sampling campaign
activity, the quality of post larvae generated from
this study was better than the post larvae coming
from commercial hatchery. Even when we
compared to 85% Artemia substitution that have a
lower quality of post larvae. The apparent
capacity of Artemia nauplii to promote better
quality of post larvae production offers an
interesting reality that the substitution of Artemia
with microencapsulated diet in early development
of L. vannamei remains a challenge.
Acknowledgements
The author acknowledges the grant
support by De Vlaamse Interuniversitaire Raad
(VLIR) Belgium for the Internship program and
technical assistance by Patipon Srianek.
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Effects of Different Diet Regimes On Development of Gill and Rostrum Spines of Pacific White Shrimp

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