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Transport of hatchery-reared and wild grouper larvae, Epinephelus sp.
Aquaculture 219 (2003) 279 – 290 www.elsevier.com/locate/aqua-online
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-dayold 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, 60day-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 hatcheryreared and wild-caught transparent tinies/larvae during transport. In addition, mass mortalities of
*
Corresponding author. E-mail address: [email protected] (C.B. Estudillo).
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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 (Castan˜os, 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 metabolic 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 documented 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 metamorphosis 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.
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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 hatcheryreared grouper larvae The survival rates during 8 h simulated transport of hatchery-reared hormone-metamorphosed (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 metamorphosing grouper larvae (60-day old, mean TL = 19.7 mm), whose bodies are still transparent 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
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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 (Ttinies). 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 (Htinies), 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.
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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 60day-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.
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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 45and 60-day-old larvae. These indicate increased oxygen consumption of larger larvae at high packing densities and temperature. Total ammonia (NH3) increased significantly 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).
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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
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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 treatments). 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.
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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 45and 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 temperature (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 metamorphosis. It was suspected that the transparent tinies (T-tinies) used in the present study
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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
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>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. Castan˜os, M., 1999. SEAFDEC/AQD’s R&D on grouper. In: Castan˜os, 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.
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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.
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-dayold 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, 60day-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 hatcheryreared and wild-caught transparent tinies/larvae during transport. In addition, mass mortalities of
*
Corresponding author. E-mail address: [email protected] (C.B. Estudillo).
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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 (Castan˜os, 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 metabolic 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 documented 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 metamorphosis 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.
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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 hatcheryreared grouper larvae The survival rates during 8 h simulated transport of hatchery-reared hormone-metamorphosed (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 metamorphosing grouper larvae (60-day old, mean TL = 19.7 mm), whose bodies are still transparent 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
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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 (Ttinies). 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 (Htinies), 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.
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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 60day-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.
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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 45and 60-day-old larvae. These indicate increased oxygen consumption of larger larvae at high packing densities and temperature. Total ammonia (NH3) increased significantly 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).
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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
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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 treatments). 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.
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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 45and 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 temperature (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 metamorphosis. It was suspected that the transparent tinies (T-tinies) used in the present study
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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
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>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. Castan˜os, M., 1999. SEAFDEC/AQD’s R&D on grouper. In: Castan˜os, 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.
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