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Aquaculture 219 (2003) 279 – 290

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Transport of hatchery-reared and wild grouper


larvae, Epinephelus sp.
Chona B. Estudillo *, Marietta N. Duray
Southeast Asian Fisheries Development Center, Aquaculture Department (SEAFDEC/AQD),
5021 Tigbauan, Iloilo, Philippines
Received 5 February 2002; received in revised form 30 May 2002; accepted 2 August 2002

Abstract

Optimum packing conditions for the transport of hatchery-reared and wild grouper larvae were
investigated under simulated condition or actual air transport. Simulation of transport motion was
done through the use of an electric orbit shaker to identify the best packing conditions for the transport
of grouper larvae at various ages. Simulated transport was conducted in hatchery-reared grouper
larvae at day 35 (mean TL = 14.73 mm), 45 (mean TL = 15.23 mm) and 60 (mean TL = 28.16 mm) at
packing densities of 50, 100 and 200 larvae l 1 and at high (28 jC) or low (23 jC) temperatures.
Packing density of 50 larvae l 1 was best for 45- and 60-day-old larvae 8 h transport at low
temperature. However, packing density could be increased to a maximum of 100 larvae l 1 8 h
transport at 23 jC with mortality rates ranging from 2.3% to 5.3%. The increase in total NH3 level
was dependent on temperature, packing density and size of larvae. High packing density (100 – 200
larvae l 1) and temperature (28 jC) resulted in increased NH3 level and mortality rates during
transport. In addition, regardless of the temperature, NH3 levels were consistently higher for 60-day-
old larvae. Day-60 grouper larvae displayed strong resistance to handling/mechanical stress
compared to 35-day-old larvae probably because most are already fully metamorphosed at this stage.
Based on these results, a packing density of 50 larvae l 1, a temperature of 23 jC and larval age of
60 days were considered as the best transport conditions for hatchery-reared grouper larvae. When
these transport conditions were used in experiment 2, for 26-day-old hormone-metamorphosed, 60-
day-old naturally metamorphosed or 60-day-old pre-metamorphosing hatchery-reared grouper
larvae, a 100% survival rate was attained in all treatments. Seven days of hormone (T3) treatment did
not accelerate metamorphosis of wild-caught transparent grouper larvae (tinies) significantly.
Survival rates of hormone-treated transparent tinies (H-tinies), untreated black tinies (B-tinies) and
untreated transparent tinies (T-tinies) were also similar after 8 – 9 h air transport (experiment 3). The
results of the current study suggest that T3 treatment did not affect the performance of hatchery-
reared and wild-caught transparent tinies/larvae during transport. In addition, mass mortalities of

*
Corresponding author.
E-mail address: chonae@aqd.seafdec.org.ph (C.B. Estudillo).

0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 ( 0 2 ) 0 0 4 1 3 - 1
280 C.B. Estudillo, M.N. Duray / Aquaculture 219 (2003) 279–290

these transported tinies during the nursery phase were associated with nutritional aspect and the
sudden confinement of these undomesticated wild-caught grouper to small space rather than
transport or hormone treatment effects.
D 2003 Elsevier Science B.V. All rights reserved.

Keywords: Epinephelus; Packing density; Temperature; Transport

1. Introduction

Metamorphosing grouper larvae ( < 2 cm TL, total length), which are referred to as
transparent ‘‘tinies’’ by fry gatherers, are quite abundant in some coastal waters of Panay,
Palawan, Negros Occidental, Davao and Ormoc, Leyte, Philippines (Castaños, 1999)
during the months of July –May. Tinies are very sensitive to handling stress (Chao and
Lim, 1991; Duray et al., 1997) and mass mortalities were often encountered by fry
gatherers and fish farmers during transport, temporary storage until disposal and in nursery
rearing (Juario et al., 1993). The high mortalities encountered during the first 3 weeks of
nursery rearing of these tinies were attributed to poor larval condition caused by
inadequate transport technology. Handling/mechanical stress leading to increased meta-
bolic rate of the fish, low oxygen capacity and rapid accumulation of toxic metabolites
such as ammonia and carbon dioxide in a closed system are important contributing factors
to fish mortalities during transport (reviewed by Berka, 1986; Ayson et al., 1990; Gou et
al., 1995; Kaiser and Vine, 1998).
A cost-effective packing technology that would require less water volume could be
obtained by increasing the packing densities. However, the limited oxygen supply together
with the accumulation of metabolites in a small and confined environment deteriorates the
water quality resulting to low survival rate of fish fry during transport. Packing densities
could be increased by maintaining water quality and fish control techniques such as lowering
the water temperature or application of anaesthetics (Teo et al., 1989; Ayson et al., 1990;
Kaiser and Vine, 1998). Temperature is an important environmental factor in the transport of
live fish. Cool water generally holds more oxygen compared to warm water and fish
consume less oxygen and excrete less toxic metabolites at low temperature due to reduce
metabolism (reviewed by Berka, 1986). Transport techniques have already been docu-
mented for various species (see review by Berka, 1986; Carmichael and Tomasso, 1988;
Chow et al., 1994; Kaiser and Vine, 1998). However, there is no documented report on this
aspect for hatchery-reared or wild caught grouper (Epinephelus) larvae.
Thyroid hormones, such as thyroxine (T4) and triiodothyronine (T3) accelerate meta-
morphosis of flounder (Inui and Miwa, 1985) and hatchery-reared grouper larvae (de Jesus et
al., 1998). Thyroid hormones also improve survival of other teleost larval species (reviewed
by Lam, 1994). Similar studies have not been done in wild-caught grouper larvae. The use of
hormone in accelerating metamorphosis of transparent ‘‘tinies’’ and the development of
effective transport technology may reduce mortality and give better profits to the fry
gatherers as well as to the fish farmers. This study aims to identify the optimum packing
density/temperature for the transport of hatchery-reared grouper larvae at different ages and
of wild-caught transparent grouper tinies to develop a transport technique for grouper larvae.
C.B. Estudillo, M.N. Duray / Aquaculture 219 (2003) 279–290 281

2. Methodology

2.1. Larval rearing of grouper larvae in the hatchery

Newly hatched grouper larvae were reared for 3 weeks in 5000 l concrete tank at
approximately 15 larvae l 1 following the method of Duray et al. (1997). After 3 weeks
(mean TL = 10.24 mm), the density was decreased to approximately 1 larva l 1 by splitting
the grouper population into two 5000 l concrete tanks. In one tank, grouper larvae were
treated with triiodo-L-thyronine (T3) for 6 days at a dose of 0.01 mg l 1 (de Jesus et al.,
1998). Grouper larvae reared in another 5000 l concrete tank without hormone treatment
(untreated) were used in the simulated transport, Experiment 1.

2.2. Experiment 1. Simulated transport of hatchery-reared grouper larvae

Simulated transport of hormonally untreated grouper larvae at various ages (35-, 45- and
60-day old) at packing densities of 50, 100, 200 and 400 larvae l 1 and at high (29 F 1.0 jC)
and low (23 F 1.0 jC) temperatures, was conducted to determine the age at which
metamorphosing larvae that can be transported with minimal mortalities and to identify
the best conditions for transport. Grouper larvae were starved for 12 h prior to transport.
They were then packed in double layered plastic bags filled with 8 l seawater and inflated
with medical oxygen (ratio H2O:O2 = 1:3), placed inside a styrofoam box and shaken with an
orbit shaker (Labline Instrument Incorporated, Model 3521) for 8 h. A set of small packs of
ice (5 g of ice 1 l 1 of the total volume of the water of the transport bag) were placed in each
styrofoam box to maintain the temperature of 23 F 1.0 jC during transport. All treatments
were replicated three times. Water samples were taken before and after transport for the
measurement of total ammonia (Phenate method, APHA, 1989) and nitrite (Colorimetric
method, APHA, 1989). Dissolved oxygen was determined after transport with YSI 51B DO
meter (Yellow Spring Instrument, Yellow Spring, OH, USA).

2.3. Experiment 2. Simulated transport of hormone-treated versus untreated hatchery-


reared grouper larvae

The survival rates during 8 h simulated transport of hatchery-reared hormone-meta-


morphosed (3-week old + 5 days hormone treatment, mean TL = 18.11 mm) was compared
with that of naturally metamorphosed (60-day old, mean TL = 28.23 mm) and metamor-
phosing grouper larvae (60-day old, mean TL = 19.7 mm), whose bodies are still trans-
parent and not fully covered with black stripes and dorsal spines not fully resorbed (Tay et
al., 1994). Larvae were packed in triplicates at the best packing density (50 larvae l 1)
and temperature (23 F 1.0 jC) obtained in Experiment 1.

2.4. Experiment 3. Transport of wild-caught grouper larvae

Wild-caught transparent (mean TL = 21.35 mm) and black tinies (mean TL = 25.41 mm)
were obtained from local supplier (Lanang, Davao City, Philippines). They were stocked
separately at 2 larvae l 1 in 300 l glass tanks filled with 250 l seawater. Wild caught
282 C.B. Estudillo, M.N. Duray / Aquaculture 219 (2003) 279–290

transparent tinies are those with bodies not fully covered by black stripes and are still
transparent, while black tinies are those with bodies fully covered with black stripes and
had taken on the appearance of the adult fish (Tay et al., 1994). Tinies were fed with wild
copepods (ad libitum) or Artemia metanauplii (36 –48 h old) throughout the experimental
period of 7 days. Each tank was provided with adequate aeration and rearing water was
changed daily at a rate of 20 –30%. Transparent tinies were either treated daily with 0.01
mg l 1 T3 following de Jesus et al. (1998) or reared in ambient seawater without hormone
treatment. The percentage of metamorphosed transparent tinies in the hormone-treated
group (H-tinies) was recorded daily and compared to those untreated transparent tinies (T-
tinies). Treatments were replicated twice. Black tinies (B-tinies) were not treated with
hormone but reared similarly with transparent tinies for later use in the transport
experiment together with H-tinies and T-tinies.
After a week of treatment, 1-day starved hormone treated transparent tinies (H-
tinies), black tinies (B-tinies), and transparent tinies (T-tinies) were packed in oxygen
inflated plastic bags at a density of 50 larvae l 1 at 23 F 1.0 jC and transported in
triplicates following the method described in experiment 1. They were transported for
a total 8– 9 h from the experimental site (Davao City, Philippines) by air to Iloilo,
Philippines and by land from Iloilo City to the Marine Fish Hatchery of SEAFDEC/
AQD, Tigbauan, Iloilo. Survival rates were determined upon arrival at the Fish
Hatchery. The tinies were then stocked (separated by treatments) in nine 200 l 1
fiberglass tanks at 1 larva l 1 and reared further for 3 weeks. Mortality rates were
recorded daily for 3 weeks to compare the performance of H-tinies, B-tinies and the
T-tinies after transport (nursery rearing phase). Adult Artemia were fed ad libitum
during the first week of nursery rearing. By the second week, they were slowly
weaned to trash fish/mysids (Acetes sp.). Water was changed at a rate of 50% every
other day during the first week and 60 –80% during the second and third week of
nursery rearing.

2.5. Statistical analysis

A completely randomized design was used in all experiments. Treatment means were
compared using analysis of variance (ANOVA) followed by Duncan’s Multiple Range
Test (DMRT) when significant at P < 0.05. All percentage data were normalized by arcsin
or square root transformation prior to statistical analysis (SAS, Statistical Analysis System
software 1998).

3. Results

3.1. Simulated transport of hatchery-reared grouper larvae

After 8 h of simulated transport, 35-day-old metamorphosing grouper larvae (mean


TL = 14.5 mm) showed lowest survival rates (mean = 29.7%) at packing density of 400
larvae l 1 in both temperatures (data not shown). Based on this result, the packing density
of 400 larvae l 1 was not used during the succeeding runs.
C.B. Estudillo, M.N. Duray / Aquaculture 219 (2003) 279–290 283

Regardless of the larval age, survival rate was highest (97.7 – 100%) when packed at
50 larvae l 1 and transported at low temperature 23 jC (Fig. 1). In addition, at a
density of 50 larvae l 1, all 45- and 60-day-old grouper larvae survived when
transported at 23 jC. In general, survival rates were higher (93 – 100%) for day 60
compared with younger larvae (day 35, 69.3– 97.7%) at 23 jC in all packing densities.
Considerably high survival rate (97.7%) was attained at 100 larvae l 1 for 45- and 60-
day-old larvae transported at 23 jC. At high temperature (28 jC), survival rates in all
ages ranges from 94.7% to 97.7% at 50 larvae l 1. Survival decreased still further with
increasing packing densities.
In all ages and in both temperatures, dissolved O2 dropped significantly with
increasing packing densities (Fig. 2). Dissolved O2 levels were generally low ranging

Fig. 1. Mean survival rates of 35-, 45- and 60-day-old hatchery-reared grouper larvae under simulated transport at
different packing densities and temperatures. Vertical lines are F SD, bars with different superscripts denote
significant differences at P < 0.05 ANOVA.
284 C.B. Estudillo, M.N. Duray / Aquaculture 219 (2003) 279–290

Fig. 2. Mean dissolved O2 levels after simulated transport of 35-, 45- and 60-day-old hatchery-reared grouper
larvae at different packing densities and temperatures. Vertical lines are F SD, bars with different superscripts
denote significant differences at P < 0.05 ANOVA.

from 1.9 to 10.9 mg l 1 when larvae were transported at 28 jC, and were and near
critical levels (range 1.9 –3.0 mg l 1) at packing density of 200 larvae l 1 for 45-
and 60-day-old larvae. These indicate increased oxygen consumption of larger larvae
at high packing densities and temperature. Total ammonia (NH3) increased signifi-
cantly to highest level of 9.8 –19.9 mg l 1 for 60-day-old larvae transported at a
packing density of 200 larvae l 1 at both temperatures ( P < 0.05) (Fig. 3). Generally,
values were highest at 28 jC for the largest larvae (60-day old). The over all levels of
nitrite in all treatments ranges from 0.02 to 0.30 mg l 1, which are below critical
range for fish survival (Mohapatra and Rengaparajan, 1995), and did not vary
significantly between packing densities and temperatures (data not shown).
C.B. Estudillo, M.N. Duray / Aquaculture 219 (2003) 279–290 285

Fig. 3. Mean total NH3 levels after simulated transport of 35-, 45- and 60-day old hatchery-reared grouper larvae
at different packing densities and temperatures. Vertical lines are F SD, bars with different superscripts denote
significant differences at P < 0.05 ANOVA.

3.2. Experiment 2. Simulated transport of hormone treated grouper larvae

Survival of hatchery-reared hormone-metamorphosed, naturally metamorphosed and


metamorphosing hatchery-reared grouper larvae at a packing density of 50 larvae l 1
and at 23 jC was 100% in all treatments (data not shown).

3.3. Experiment 3. Transport of wild-caught grouper tinies

The effect of thyroid hormone on the metamorphosis of wild-caught transparent


tinies were not consistent and as effective as with the hatchery-reared pre-metamorphic
286 C.B. Estudillo, M.N. Duray / Aquaculture 219 (2003) 279–290

grouper larvae (data not shown). Although the rate of metamorphosis was slightly
faster in hormone treated transparent tinies (H-tinies), it was not significantly
accelerated compared with the transparent group (T-tinies). Survival rates of T-tinies,
H-tinies and B-tinies transported from the experimental site to the Fish Hatchery of
SEAFDEC/AQD did not vary significantly between treatments (100% in all treat-
ments). During the nursery rearing, survival rates were significantly highest (100%) for
H-tinies and the T-tinies on the first week of rearing (Fig. 4, P < 0.05). By the second
and third week of rearing, T-tinies showed better survival (80%) compared to those in
the two other treatments. Mortalities started to gradually set in on the second week of
rearing and mass mortalities occurred on the third week of nursery rearing in all
treatments.

Fig. 4. Survival rates of wild-caught hormone-treated transparent tinies (H-tinies), black tinies (B-tinies), and
transparent tinies (T-tinies) after 3 weeks rearing in the hatchery. Vertical lines are F SD, bars with different
superscripts denote significant differences at P < 0.05 ANOVA.
C.B. Estudillo, M.N. Duray / Aquaculture 219 (2003) 279–290 287

4. Discussion

The present study was conducted in order to determine the optimum condition for the
transport of hatchery-reared and wild-caught grouper larvae. The results showed that 45-
and 60-day-old hatchery-reared grouper larvae can be transported successfully at a packing
density of 50 larvae l 1 (100% survival) at 23 jC. Day-35 grouper larvae were more
sensitive to handling stress than with 45- and 60-day-old larvae. Fin resorption occurred at
this stage and when the fish were handled, they exhibited ‘‘shock syndrome’’ characterized
by erratic swimming behavior followed by sudden death. This behavior has been reported
by various authors for grouper larvae (Chao and Lim, 1991; Duray et al., 1997). On the
other hand, 60-day-old grouper larvae displayed a strong resistance to handling stress. At
this age, fin resorption is completed and most larvae were morphologically similar to adult
fish (e.g. black stripes). Optimum transport density of 45- and 60-day-old grouper larvae
in the current study was at 100 larvae l 1 with mortality rates ranging from 2.3% to 5.3%
when transported at 23 jC. The transport density of fish vary between species, fish size/
age and is affected by the water quality (see review by Berka, 1986; Ayson et al., 1990;
Kaiser and Vine, 1998). In guppy, Poecilia reticulata (body weight range 15.2– 16.5 g),
survival rate was 100% when transported at 20 guppies 400 ml 1 at 25 jC (Teo et al.,
1989). However, these authors anaesthetized (0.11 g l 1 2-phenoxyethanol) the fish
during transport to decrease their metabolism, therefore maintaining the water quality.
Without chemical treatment (control) 20% mortality was observed. At ambient temper-
ature (28 jC), 100% survival rate was attained for Siganus guttatus (47 days old;
2.1 F 0.11 cm mean TL) transported for 8 h at 100 fish l 1 (Ayson et al., 1990). At 28
jC, increasing the packing density of S. guttatus to 200, 300 and 400 larvae l 1 resulted
to a significant reduction in survival rates (48 –18%) after 8 h simulated transport.
Dissolved O2 depletion and increased total ammonia levels were the two most
important factors that may have caused fish death at higher packing densities and
temperature in the current study. Kaiser and Vine (1998) suggested minimum dissolved
oxygen level of 4 mg l 1 for the transport of goldfish Carassius auratus. In this study,
dissolved oxygen reached a critical range of 1.9– 3.0 mg l 1 when 45- and 60-day-old
hatchery-reared grouper larvae packed at 200 l 1 were transported at 28 jC. At this
condition, grouper larvae died with their mouth and gills open suggesting hypoxic
condition and increased ammonia levels in the water. This was attributed to increased
metabolic rate resulting from increased physical activity under crowded conditions and the
limited supply of dissolved oxygen at high temperature. Oxygen deficiency and physical
injuries caused by frequent physical contact between individual fish due to crowding
contributed to the high mortalities of young rabbitfish S. guttatus at higher packing
densities (200 –400 larvae l 1) transported at 28 jC (Ayson et al., 1990). Results of the
current study showed increased survival rates of grouper larvae at low temperature even at
high packing densities. Lowering the water temperature during transport reduces fish
activity and stress as well as decreases metabolic rate of the fish and favors the dissolution
of oxygen in the water, reduces the toxicity of ammonia and carbon dioxide and inhibits
population growth of microorganisms during fish transport (Gou et al., 1995).
Hormone treatment (T3) of transparent tinies did not significantly accelerate meta-
morphosis. It was suspected that the transparent tinies (T-tinies) used in the present study
288 C.B. Estudillo, M.N. Duray / Aquaculture 219 (2003) 279–290

were of the same age as the black tinies (B-tinies), but had delayed development or that the
black tinies could be the ‘‘shooters’’ in the batch. In the hatchery, thyroid hormones (T3)
was successfully used to accelerate metamorphosis of 3-week-old grouper larvae (mean
TL range: 14 –16.5 mm) (de Jesus et al., 1998). The levels of T3 of 20-day-old grouper
larvae is low (1.9 F 0.2 ng g 1) and reaches its peak on day 40 (4.0 ng g 1) (de Jesus et
al., 1998). Therefore, addition of thyroid hormone to 3-week-old grouper larvae is needed
to accelerate methamorphosis. For the transparent wild tinies (mean TL range: 22.5– 29.0
mm), thyroid hormone levels could have been high so that any addition of thyroid
hormone have no effect and may even be harmful to the fish health. The effect of thyroid
hormones is bi-phasic, it may accelerate growth/metamorphosis at a proper dose but may
be lethal in excess (de Jesus et al., 1998).
Survival after transport of wild-caught hormone-treated transparent tinies (H-tinies),
untreated black tinies (B-tinies) and transparent tinies (T-tinies) was similar (100%). High
mortality rates of wild tinies in the nursery phase were attributed to poor larval condition
due to stress from inadequate transport technique (Juario et al., 1993). To address this
reported problem, transported H-tinies, B-tinies and T-tinies were reared further for 3
weeks in the hatchery. High survival rate during the first week of rearing was obtained
when they were fed live food (adult Artemia), however, when weaned to trash fish during
the second and third week, mortalities set in. Gut examination showed no trace of food
implying starvation as the cause of fish mortalities. Similar observation had been
experienced by the fish farmers (Schlageter, pers. comm.) wherein mass mortalities are
often encountered after 3 weeks of nursery rearing in net cages. Apparently, mass
mortalities in the nursery phase is associated with feeding behavior (weaning from live
food to trash fish) and the sudden confinement to small spaces (cages or ponds). In
Taiwan, fish farmers preferred hatchery-reared rather than the wild grouper fry due to the
undomesticated behavior of the wild fry that often results to uneven growth and poor
resistance to stress (Liao, 1998). Hatchery-reared grouper larvae, on the other hand, are
more resistant to handling stress and are already acclimatized to small spaces which make
them more easier to culture in net cages.

5. Summary and recommendation

The results of the present study suggest that the best packing density for transport of
grouper larvae/tinies is 50 larvae l 1 and that packing density could be increased to a
maximum of 100 larvae l 1 when transported at 23 jC. The limiting factor in increasing
the packing density of 45- and 60-day-old hatchery-reared grouper larvae is the
deterioration of water quality. At high temperature (28 jC), increased metabolism occurred
leading to increased oxygen consumption and excretion of toxic metabolites. At present,
50 fish 8.0 l 1 seawater (28 – 30 jC) is the standard packing density used in the
SEAFDEC/AQD Marine Finfish hatchery. This packing density is far too low compared
to the suggested packing density (50 larvae l 1) in the present study. However, grouper
fry normally transported from the hatchery are two-fold larger (1.5 –2.5 in. TL), older
(75 – 90 days old) and transport time was longer (f24 h) compared to the present study. It
is recommended that further studies be conducted for long-term transport (48 h) using
C.B. Estudillo, M.N. Duray / Aquaculture 219 (2003) 279–290 289

>60-day-old grouper fry at 23 jC. Anaesthetic and chemicals (MS222, Phenoxy-ethanol,


methylene blue) have been used by several authors (Chow et al., 1994; Gou et al., 1995)
for fish transport but has not been covered by this study and is also worth pursuing in the
future. Result of a preliminary run showed that temperature less than 22 jC produced very
low survival rates ( < 50%), therefore it is suggested that the ideal temperature for transport
of grouper fry is between 23 and 25 jC. Furthermore, the use of T3 in wild-caught
transparent tinies is impractical but may be profitable in the hatchery-reared grouper
larvae. The abrupt change from live food to trash fish and the sudden confinement of
undomesticated wild-caught grouper fry could be some of the critical factors for their
survival in the nursery rearing phase.

Acknowledgements

The authors thank Dr. E. de Jesus for her critical review of the manuscript. Mr. and Mrs.
Mitchell Schlageter for allowing us to use their facilities for the transport experiment of
wild grouper tinies. We are also grateful for the help extended by L. Alpasan, B. Eullaran,
J. Damaso, E. Bolivar and the SEAFDEC Marine Fish Hatchery staff.

References

American Public Health Association, 1989. In: Clesceri, L.S., Greenberge, A.E., Trussell, R.R.(Eds.), Standard
Methods for Examination of Wastewater, 17th ed. Academic Press, New York. 1193 pp.
Ayson, F.G., Parazo, M.M., Reyes Jr., D.M., 1990. Survival of young rabbitfish (Siganus guttatus Bloch) under
simulated transport conditions. J. Appl. Ichthyol. 6, 161 – 166.
Berka, R., 1986. The transport of live fish: a review. EIFAC Tech. Pap. 48 (52 pp.).
Carmichael, G.J., Tomasso, J.R., 1988. Survey of fish transportation equipment and techniques. Prog. Fish-Cult.
50, 155 – 159.
Castaños, M., 1999. SEAFDEC/AQD’s R&D on grouper. In: Castaños, M. (Ed.), SEAFDEC Asian Aquaculture
Vol. XXI No. 1. Aquafarm News, Southeast Asian Fisheries Development Center (SEAFDEC), Tigbauan,
Iloilo, Philippines, p. 21.
Chao, T.M., Lim, L.C., 1991. Recent developments in the breeding of grouper (Epinephelus spp.) in Singapore.
Singap. J. Prim. Ind. 19, 78 – 93.
Chow, P.S., Chen, T.W., Teo, L.H., 1994. Physiological responses of the common clownfish, Amphiprion ocellaris
(Cuvier), to factors related to packaging and long-distance transport by air. Aquaculture 127, 347 – 361.
de Jesus, E.G., Toledo, J.D., Simpas, S.M., 1998. Thyroid hormones promote early metamorphosis in grouper
(Epinephelus coioides) larvae. Gen. Comp. Endocrinol. 112, 10 – 16.
Duray, M.N., Estudillo, C.B., Alpasan, L.G., 1997. Larval rearing of the grouper Epinephelus suillus under
laboratory condition. Aquaculture 150, 63 – 76.
Gou, F.C., Teo, L.H., Chen, T.W., 1995. Effects of anaesthetics on the water parameters in a simulated transport
experiment of platyfish, Xiphophorus maculatus gunther. Aquacult. Res. 26, 265 – 271.
Inui, Y., Miwa, S., 1985. Thyroid hormone induces metamorphosis of flounder larvae. Gen. Comp. Endocrinol.
60, 450 – 454.
Juario, J.V., Silapan, J.R., Silapan Jr., L.L., 1993. The commercial production of green grouper fingerlings,
Epinephelus suillus, from wild-caught fry and industry experience. In: Marte, C.L., Quinitio, G.F., Emata,
A.C. (Eds.), Proceedings of the Seminar – Workshop on Breeding and Seed Production of Cultured Finfishes
of the Philippines, 4 – 5 May 1993. SEAFDEC/AQD, Tigbauan, Iloilo, Philippines, pp. 132 – 139.
Kaiser, H., Vine, N., 1998. The effect of 2-phenoxyethanol and transport packing density on the post-transport
survival rate and metabolic activity in the goldfish, Carassius auratus. Aquar. Sci. Conserv. 2, 1 – 7.
290 C.B. Estudillo, M.N. Duray / Aquaculture 219 (2003) 279–290

Lam, T.J., 1994. Hormones and egg/larval quality in fish. J. World Aquacult. Soc. 25, 2 – 12.
Liao, I.C., 1998. A review of the nursery and grow-out techniques of high-value marine finfishes in Taiwan.
Tungkang Marine Laboratory, Taiwan Fisheries Research Institute, Tungkang, Pintung, Taiwan. Fish. Res.,
pp. 121 – 137.
Mohapatra, B.C., Rengaparajan, K., 1995. A manual in bio-assays in the laboratory and their techniques. CMFRI
Spec. Publ. 64, 1 – 75.
Tay, H.C., Goh, J., Yong, A.N., Lim, H.S., Chao, T.M., Chou, R., Lam, T.J., 1994. Effect of thyroid hormone in
metamorphosis in greasy grouper, Epinephelus tauvina. Singap. J. Prim. Ind. 22, 35 – 38.
Teo, L.H., Chen, T.W., Lee, B.H., 1989. Packaging of guppy, Poecilia reticulata, for air transport in a closed
system. Aquaculture 78, 321 – 332.