SEABASS BREADING

1. INTRODUCTION
The seabass (Lates calcarifer Bloch) is one of the species with a high potential for cultivation. Apart from the culinary characteristics that endear it to the consumer, the fish is fastgrowing and euryhaline. The latter fact is seen as a valuable attribute for a species enabling its adoption for pond and cage culture under marine, brackish, and freshwater environments. The propagation of seabass in captivity has two major objectives. The first is the production of fingerlings that are further reared in captivity to marketable size. The second objective consists of purely biological purposes such as the study of the morphology, ecology and ethology of larval fish, taxonomy, population dynamics analysis, and genetics. This manual deals mainly with the first objective aiming to describe the technologies that have been developed and successfully tested at the National Seafarming Development Centre, Hanura, Teluk Betung Lampung, from 1987 1989. The breakthrough in induced spawning of the seabass in captivity through hormone manipulation made at the Seafarming Development Centre in April 1987 has a positive impact on the long term development of Indonesian seabass culture industry. It not only solves a constraint in seed supply, but also reduces impact on wild stock juveniles. The processes involving in seabass propagation as practiced at the Centre are presented in Fig. 1. The terminology used in this manual for the larvae at different stages of development are : Hatchlings : This stage comprises yolk-sac individuals. It starts from Days 04. Larvae : Refers to individuals after absorption of the yolk sac to the fry stage. It starts from Days 521. Fry : This stage begins when the individual has completed metamorphosis, i.e., when the fish takes on the appearance of sub-adult. It starts from Days 2140; and Juveniles : This stage includes larger fingerlings after Day-41.

Fig. 1. General scheme of seabass culture as practiced in Indonesia.

2. TAXONOMY AND NOMENCLATURE

Seabass was first described by Bloch (1790) from specimens received from Dutch merchants returning from Indonesia. His type specimen was named Holocentrus calcarifer for the similarity between the preopercular spines and thorns. The genus Lates (Cuvier and Valenciennes) was erected somewhat later in 1982 to encompass other species including the closely related Nile perch (L. niloticus Linnaeus).

Dunstan (1959) noted that there were differences in body proportions both within and between localities. His late work (Dunstan 1962) also identified morphological differences between the Papuan and Australian fish and those of previous descriptions. Variation in coloration between juvenile and adult fish and between fish in salt and fresh water was also reported by Dunstan (1962) and Reynolds (1978). Notwithstanding the variability reported to that time, Greenwood (1976) proposed only a single species of Lates throughout the IndoPacific. A full taxonomic description of Lates calcarifer (Bloch) can be found in FAO, 1974 (Appendix 1).

3. BROODSTOCK DEVELOPMENT
Success of fish breeding depends primarily on the availability of mature brooders of high quality which produce high survival and fast growing fish. Normally it takes at least 34 years for a hatchery to have enough brood fish for the operation. The brood stock can be obtained either from the stock raising from the juvenile stage in ponds, netcages and/or caught from the wild.

3.1. Farm raised brood fish

The broodstock can be reared either from hatchery produced fry or from fry collected from the natural ground. The reared brood fish would be ready for spawning activities at the end of their second year when male brood fish reach 50 cm, total length (TL) and 2.5 kg in weight and female weigh about 3.5 kg. 3.1.1. Development of brood fish in netcages (Fig. 2) The healthy and fast growing fish fry with size 1.5 to 2.0 cm TL (total length) are selected, and kept in netcages measuring 2.02. 01.0 m with a net mesh size of 2.0 mm at the initial stocking density of 1 000 juveniles/m3. As the fry grows bigger, the stocking density per unit area is reduced and uses larger netcages with bigger mesh size net as shown in Table 1. Table 1. Netcage specification and stocking density in relation to fish size.
Size of fish (cm TL) 1.5 2.0 2.0 10.0 10.0 15.0 15.0 20.0 >25 size of cage (m) 2.02.01.0 2.02.02.0 2.02.02.0 5.05.02.0 5.05.02.0 Mesh size (mm) 2.0 (nylon hapa) 8.0 (nylon hapa) 10.0 (polyethylene) 12.5 (polyethylene) 25.0 (polyethylene) Stocking density (pcs/m3) 1000 250 125 60 <10 kg/m3

Fig. 2. Floating netcages used for broodstock development One month later, 50% of the healthy and fast growing fry are reselected for further rearing in a bigger netcage of 5.05.02.0 m. After second year, the fry are reselected again and only half of the healthy and fast growing fry are kept as the potential brood fish. The holding netcages size as practiced at the Center measuring about 5.05.02.0 m. The mesh size varies from 48 cm. Stocking density is 57 kg of fish per ton of water. To ensure good water exchange, the holding netcage is replaced monthly with a clean net. Trash fish is given once or twice daily. The size of the food depends on the size of fish. Fingerlings smaller than 10 cm TL are fed minced trash fish at rate of 20% body weight daily while those of 1015 cm TL are fed on chopped pieces of around l cm length at 15% body weight daily. Fish larger than 15 cm TL are fed on larger chopped pieces of around 2.5 cm at 10% body weight daily. At the size of 1 kg, the fish are fed at 5% body weight daily. Mature fish of 3 years or more are fed at 23% body weight daily. The feeding rate is further reduced to 1% body weight during the spawning season. After the third year, the fish weighing between 3.5 to 4 kg in weight can be used for the gonadal development 3.1.2. Holding brood fish in broodstock pond. Concrete tanks or earth ponds built on land are also good for brood fish development. Although these types of cultured facilities provide easier control of environmental factors, but they are much more expensive in comparison with netcages. The size of the broodstock holding tanks as practiced in Southeast Asia region are ranging from 75150 t (5.010.01.5 m and 10.010.01.5 m). Stocking density is 1 kg of fish/t of water.

Water quality in the tank should be maintained as good as natural seawater. Suggested water quality of the concrete holding tanks is given in Table 2.

3.2. Wild brood fish

Brood fish captured from the natural ground during spawning season can obtained by gillnet of 610 cm mesh. The net is set in direction perpendicular to the current. The set gill net is checked for captured seabass regularly. This is to ensure that the captured fish are not left struggling for long period. The captured seabass are stocked immediately in the holding tank on board the fishing vessel. Aeration is provided either from an air blower or oxygen from a cylinder. As all fish caught by this method usually suffer to some extent from body damage, they are treated directly in the holding tank with antibiotics or approximate chemical bath, eg. 5ppm acriflavine for 23 hrs. The fish should be transferred to broodstock ponds or netcages immediately after arrival in the hatchery. In general, at least 6 months are needed for the fish to recover from stress and injury and to condition to the confined environment in the broodstock ponds or netcages before they can be used for spawning. Table 2. Suitable ranges of water quality of the broodstock tank.
Parameters Temperature Salinity Alkalinity (as CaCO3) Acidity (pH) Dissolved oxygen Phosphate Unionized ammonia (NH3) Ionized ammonie (NH3+) Aluminium sulphate (Alum) Turbidity : suspended solid size > 1 um suspended solid size < 1 um BOD (5 day) Nitrite (NO2) Nitrate (NO3) Chlorine (CL2) Suitable range of values 28 32 C 29 32 ppt 80 120 mg/1 6.8 8.0 >6 mg/l preferably 100% saturation 10 100 mg/l < 0.5 mg/l preferably not detectable < 1.5 mg/l < 80 mg/l 2 10 mg/l 23 mg/l maximum 3 mg/l <1 mg/l preferably not detectable < 150 mg/l < 0.8 mg/l

4. BROODSTOCK MAINTENANCE
Nutrition has profound effect on gonadal growth and fecundity of brood fish. Although precise information on the nutritional requirements for gonadal maturation in seabass broodstock is not available at present, it has been recognized that quality and quantity of the diet significantly

affect broodstock reproductive success, spawning, hatching ability of eggs and survival rate of offspring. At the National Seafarming Development Centre, Lampung, it has been observed that broodstock fed with poor nutrition diet resulted in poor hatching ability of eggs and survival rate of larvae and fry. The condition of the brood fish was improved after good quality of fresh marine fish was given and supplemented with vitamin E once a week at 30 mg/kg of fish.

5. SEX DETERMINATION
Sex of seabass can only be determined accurately in mature fish, even though the the fish has some dimorphic characters to enable sex determination. For seabass of the same age, males are generally smaller and with a more slender body and narrower body depth than the female. During the spawning season, the milt can be observed at the genital opening if slight pressure is applied on the abdomen of the mature male. The female can be recognized from the big soft round belly (Fig. 3) with the red-pink papilla extruding out at the urogenital aperture. If the female has a fully ripe egg, the egg will be visible when the abdomen is pressed by hand (Fig.4).

Fig. 3. Mature seabass brooder

6. STAGE OF MATURITY
In order to avoid using immature spawners, the potential female can be selected on the basis of egg size. The stages of maturity for male and female seabass are given in Table 3. Spawning takes place between stage V (fully ripe), and stage VI (spent).

Fig. 4 Checking the readiness of broodfish by pressing on abdomen of the fish Table 3. Stage of gonodal development in seabass, Lates calcarifer.
Stage I Virgin II Maturing virgin and recovering spent. III Developed gonad IV Female Glassy, rounded and the body cavity in length Gonadal condition male Colorless thin strap lying along the blood vessel. One half body cavity in length. Whitish and has assumed a definite gonadal appearance. The same length as stage I.

Definite gonadal appearance. The same length as stage I Yellowish and easily detectable as female. Ovary about of body cavity. Fills half the body Eggs can be

Whitish with gonadal appearance. Fills half the body cavity. Whitish.

Developing V Fully ripe

distinguished separately. Eggs are separate and fill the entire body cavity Milt fills the body cavity and can be expelled without difficulty White and sticky. Testes thin although not as flaccid as female. Some spawner may have the testes remain and fill to one half of body cavity. Testes are small and thin. They are sharp viewed from the edge.

VI Spent

Ovary flaccid. May have some eggs remaining. Ovaries reddish and small. Easily confused with stage II. Identification under microscope may be necessary.

VII Resting

(Modified from Broadhead, 1953)

7. SELECTION OF MATURE BROODER

The assessment of the maturity of the female fish can be made from eggs sampled by catherization from the mid-portion of the ovary, using a polyethylene cannula. The sampling process is made by inserting a polyethylene cannula 1.2 mm in diameter into the oviduct of the fish for a distance of 67 cm through the genital opening (Fig. 5). The eggs are placed in the test tube containing 1% formalin in 0.6% saline (sodium chloride) solution for subsequent checking of egg size and appearance. Egg diameters are measured on a glass slide using an ocular micrometer under a microscope. The diameter of egg is measured by everaging the long and short diameters. Females bearing uniform, spherical and nonadhesive eggs with a mean diameter 0.4 0.5 mm or more are selected for the inducement of gonadal maturation. Males are selected when white and creamy milt oozes out from the genital opening upon gentle stripping of the belly. Males yielding watery and curdled milt are not suitable for use in spawning.

Fig. 5 Checking maturity of eggs using a polyethylene cannula

8. INDUCED GONADAL MATURATION

In Lampung area, induced spawning by hormone manipulation can be started from NovemberJuly. Synthetic hormones, Human Chorionic Gonadotropin (HCG), Puberogen and Pregnyl and pituitary gland (PG) of common carp are commonly used. Normally 50 IU of HCG plus 0.51 dose of PG is the appropriate dose for the first injection to induce ovulation. The second dose 100200 IU of HCG plus 1.52.0 doses of PG is applied after 12 hours. Overdoses of HCG should be avoided, since it may result in fattening of the fish, causing regression of the female's ovaries. Male (stage IV-V), milt formation can also be stimulated by injection of 0.5 dose of PG plus 2550 IU HCG. At the Seafarming Development Centre at Hanura, Teluk Betung Lampung, brood fish, both male and female weighing about 25 kg injecting first dose of 50 RU Puberogen + 250 IU HCG at 08.00 hr and following a second dose of 100 RU Puberogen + 500 IU HCG at 08 00 hr of the following day, spawned between 23 00 04 00 hr after the second injection.

9. PROCESS OF HORMONE INJECTION

To immobilize the brood fish during hormones injection process, a tranguilizer such as Tricane (MS - 222) or Ether at doses ranging between 1 : 12 5001:25 000 is recommended Other fish anesthetics available in the market and suitable for general handling of fish are quinaldine, tertiary amulalcohol, choralhydrate and Phenoxethol. At the Centre, Ethylene glycol was used at dose between 1:8 0001 :10 000. The brood fish will be immobilized within 35 mins. Injection of hormones is given intramuscular on area between the lateral line, above the base of the pectoral, and dorsal fin (Fig.6) using a 5 ml disposal syringe and a gauge No.21 22 needle. All instruments used for the operation should be clean to avoid bacterial infection

10. SPAWNING IN CAPTIVITY

Seabass can be induced to spawn in captivity either by hormone-inducing or by natural spawning. Four weeks before spawning season, the brood fish is transferred into the spawning tank at a rate of 1 kg of fish/t of water with a sex ratio of 1:1 Due to handling during the transfer, the fish may refrain from eating one to two days. To maintain good water quality in the spawning tank, periodic draining and refilling of water are necessary. Normally 80100 percent of the total volume of water is changed every day. Salinity of water is maintained at 30 ppt. However, to ensure that the spawning tanks have the desirable water quality, the installation of a jet spray head and aeration system in the tank are preferred.

Fig. 6. Intramuscular injection of hormone

10.1 Natural spawning

If the water quality and environment in the spawning tank is suitable with proper feeding, the female fish gradually appears with swollen abdomen, swimming awkwardly. Approximately 12 weeks before spawning, the female fish separates from the school and reduces feeding activity while the male fish continues their normal activity and active. Natural spawning in controlled tank takes place at the same time as natural spawning ground. It starts from the beginning of November to the end of July. Spawning activity occurs between 19.00 and 23.00 hr on the first to the 8th day of the full moon or new moon. As the female approaches full maturity, there is an increase in spawning play activity. The ripe male and female swim together often turning laterally when swimming and then spawn.

10.2 Induced spawning

If the female fish is not in a fully ripe stage, a certain dose of hormones are required for inducing the spawning. The first injection of 250 IU HCG/kg (body weight of female fish) and 50 RU Puberogen/kg (body weight of male fish) is practiced at the Seafarming Development Centre. The second injection of 500 IU HCG/kg female fish and 100 RU Puberogen/kg male fish is followed after 24 hrs. The fish showed a good response with satisfactory result.

10.3 Artificial fertilization

For broodstock newly caught from the natural ground during the spawning season, the eggs and milt can be stripped and fertilized artificially. In this case, the readiness of spawner has to be checked before stripping. The healthy ripe running eggs have a diameter of 0.8 mm, contained oil globule of 0.2 mm in diameter, round with smooth surface, transparent, have light yellow color, loosely separate, float in water at 2830 ppt salinity, and there is no yolk vesicle. The stage of ripe running eggs can be checked also by dropping a sample of eggs into a clear glass of water. The ripe running eggs show individually scattering whereas the unripe running eggs tend to group together and sink down to the bottom of the glass. Strip eggs and milt into a dry clean tray. Stir it thoroughly with a feather. Add filtered fresh seawater salinity of about 2830 ppt to cover all the eggs. Stir continuously for 13 mins, then wash it with seawater 34 times through a strain to remove mucus and foreign substances which could cause subsequent bacterial infection. The eggs are placed in incubation tank for further hatching.

10.4 Environmental manipulation

One to two months before spawning season, the brood fish everage size of about 4 kg are transferred to spawning tanks. The stocking density is about one fish per 45 m3. The initial salinity in the spawning tank should be the same as the salinity in the netcages or ponds where the brood fish are kept before transferring. After the fish get used to the tank environment, normally it takes about 23 days, the salinity of water in spawning tank is reduced to 2025 ppt. Keep the broodfish in the 20 25 ppt salinity for 7 days then gradually increase it to 3032 ppt through daily water changing About 6070% of water are changed daily until the salinity reached 3032 ppt. Changing in salinity is a natural stimulation during its migration from feeding ground in the brackishwater to spawning ground in the sea environments. On the day during full moon or new moon period, the water is reduced to about 30 cm depth at noon and exposed to the sun light for 34 hrs to increase the water temperature to 3032 degree Celsius. New seawater is added to manipulate rising tide conditions and decrease the water temperature to 2728 degree Celsius as found in normal spawning environment. If the fish is in the right stage and good condition, it will spawn in the same night or the next after at 19 0022 00 hr. If the fish does not respond or responds but with abnormal eggs, the injection of hormones is required. Besides spawning in tanks on shore, the seabass can also be spawned at sea in floating netcages. For spawning in the floating netcages, the small mesh size of net is installed the night of the second dose of the inducement of the spawning to prevent the spawned eggs from being washed away by the tidal current. The brooders are not fed so long as they are in the spawning cage to prevent deterioration of water quality inside the netcages.

11. FACTORS AFFECTING SPAWNING

Major factors affecting gonadal maturation and spawning of seabass in captivity are food, water quality, salinity, stress, size of broodstock, age of brood fish and lunar cycle.

11.1 Food.
Quality and quantity of food given to brood fish affect the gonadal maturation. Normally fresh marine fishes such as sardine, anchovy and yellow strip caranx are used as a main food for brood fish at the rate of 5% of body weight. One to two months before spawning season, the feed is reduced to 1% of body weight and fed once a day. At this period vitamin E should be given at a dose of 3050 mg/kg of fish as a supplementary feed to increase tocopherol in the female brood fish.

11.2 Water quality

Keeping the brood fish in floating netcages have advantage of having natural good seawater. For broodstock tanks on shore, the water quality have to be maintained as good as in nature. Normally 5080% of water are changed daily to keep the water quality to the acceptable level (Table 2.) Poor water quality will result in a negative effect on gonadal maturation and spawning.

11.3 Salinity
Salinity of the water in broodstock tanks prior to spawning season can be maintained within the range of 2025 ppt, but it should be increased to the range of 2832 ppt during spawning season.

11.4 Stress
Disturbances to brood fish during spawning season should be avoided. Excessive noise and vibration due to vehicles near-by are also stressing the brood fish having a negative effect on spawning.

11.5 Size of brood fish

Male and female brood fish should have a uniform size. Sex ratio between male and female is usually 1:1. If the male are much smaller than the female, the number of male brood fish should be increased.

11.6 Age of brood fish

Mature females of 37 years (body weight 3.512kg) and males of 35 years (body weight 3.0 7.5 kg) are good for inducement of the spawning.

11.7 Lunar cycle

Spawning activity of seabass in captivity is found closely related to the lunar cycle. In Indonesia water natural spawning activity of seabass occurs between 19 00 and 23 00 hr during high tide

at 1st8th day after new moon or full moon. Usually it spawns at night both during the new moon and full moon phases. However, the fish will yield more eggs and better quality at full moon than at new moon phase.

12. EGG COLLECTION

Two methods may be used for collecting seabass eggs from a spawning tank.

12.1 Seine net

A small seine net mesh size about 200 u can be used to collect the egg from the spawning tank in the morning after spawning (Fig.7).

12.1 Water overflow method

Collecting the eggs from spawning tanks by seine net is necessary to stop the aeration to allow the eggs afloat in upper layer of the water column. This period might reduce the dissolved oxygen in water to a level that could stress the spawners especially in high stocking density broodstock tanks. More over turbulence caused by fish movement in the tank tends to redistribute the eggs in the water column resulting in uncompleted collection of eggs. To avoid the problems, the eggs can be transferred into the egg collector through the continuous flow of water. Seawater should be flowed after the fish spawned. The overflowing water carries the eggs into the egg collector made of a fine netting (200 u) of 1020 1 capacity (Fig. 8).

Fig. 7 Collecting seabass eggs using a small seine net.

13. CARE OF FERTILIZED EGG AND HATCHING

The eggs are placed in plastic buckets. The unfertilized eggs sinking to the bottom are discarded. The fertilized eggs are washed through 1.52.0 mm mesh screen in order to remove debris or foreign materials that may attach to the eggs. The number of fertilized eggs is estimated and treated with 5 ppm of acriflavine solution or in ovadine at dilution of 1 ml/ 100 ml water for 1 min for disinfection purposes. Since survival of eggs and disinfectant are most effective at neutrality, an adjustment of disinfectant solution to pH 7.0 is necessary. Rinse the eggs in seawater two to three times before placing in the incubation tanks.

1= water inlet, 0= water outlet

S= screen,

N= egg - collecting net, and

Fig. 8. Diagram of a spawning tank with egg-collecting system. The eggs are incubated in a circular tank. The stocking density recommended is around 60100 eggs/1. Hatching rates of seabass eggs vary with salinity (Table 4). The salinity ranging between 2830 ppt is recommended for hatching of the egg. During the hatching process, aeration should be given gently to allow the water to ciruculate and prevent the eggs from settle on the bottom. Unfertilized eggs are siphoned out by stopping the aeration so that fertilized eggs will float to the upper layer of the water column. After the 10th hour, 50% of the water in the incubator tank are changed by siphoning out the lower layer and replaced it with a new seawater of the same salinity (2830 ppt). Table 4. Effects of salinity on hatching rate of seabass eggs.
Salinity (ppt) 0 5 10 15 20 25 30 35 Rate of hatching (%) 00.0 02.9 58.5 75.0 82.4 83.4 80.8 46.9

Source : Tattanon and Maneewongsa, 1982a.

14. EMBRYONIC DEVELOPMENT

The fertilized eggs hatched out within 1718 hrs at a water temperature of 2628 degree Celsius. The stages of development of the seabass egg recorded by Maneewongsa and Tattanon (1982) is given in Table 5, Fig. 9. The newly hatched larvae (size about 1.5 mm) contain a big yolk sac with an oil drop at the anterior end. When it does not swim the fish has its head in an upward position. While moving, the body of the fish will be at a 4590 degree angle with the horizontal. The body is slender and compressed. The pigment is scattered in spots. The eye, digestive system and intestines can be seen clearly. The mouth parts appear when the fish is three days old. The yolk sac is completely absorbed at Day 4 (Table 6, Fig. 10). Table. 5 Embryonic development of seabass eggs at 27 C
Stage of development Fertilized egg 1 - cell 2 - cell 4 - cell 8 - cell 32 - cell 64 - cell 128 - cell Multi - cell Blastula Gastrula Morula Early embryo with eye vesicle Heart function, free movement of tail Hatching Hour Minutes 00 00 35 00 40 00 45 01 00 02 15 02 45 02 55 03 15 05 30 06 30 08 30 11 20 15 30 17 30

Source : Maneewongsa and Tattanon 1982. Table 6. Rate of absorption of the yolk
Diameter of Yolk (mm) 0.88 0.35 0.28 0.15 0.01 Days 0 1 2 3 4 Source : Maneewongsa and Tattanon, 1982

0.00

Fig. 9 Embryonic development of seabass (Source : Maneewongsa and Tattanon, 1982)

15. LARVAL DEVELOPMENT

Newly hatched larvae float in the upper layer of the water column. Chromatophores are observed behind the eyes, in the body, and on the oil globule. The larvae completed its larval stage within 3 weeks (Fig. 10).

Fig. 10. Larval development of seabass (Source : Konsutarak and Watanabe, 1984) Day 1 (TL 2.20 0.08 mm) Large part of the yolk sac absorbed. Mouth still closed. Anus can be observed. Eyes unpigmented. Pectoral fin appeared as bud. The larvae distributed uniformly in the rearing tank. Day 2 (TL 1.52 0.06 mm)

Yolk sac almost disappeared. Mouth opened. The larvae gather near aeration or in the direction of the light. Oil globule is still apparent (Konsutarak and Watanabe, 1984). Day 3 (TL 2.61 0.008 mm) Air bladder appeared. Yolk sac absorbed, but the oil globule is still observed. Day 4 (TL 2.78 0.15 mm) Mouth opened with developed upper and lower jaws. Nostrils appeared on snout. Pectoral fin developed in round shape as finfold. Digestive tract extended relatively in thickness. Melanopores presented on dorsal and ventral profiles, body mid-line and abdomen. Melanophores scattered on mid-brain and lower jaw. Oil globule disappeared. Day 5 (TL 3.08 0.09 mm) Teeth appeared on the upper jaw Day 6 (TL 3.10 0.13 mm) Under part of caudal end whitish Day 7 (TL 3.44 0.09 mm) Rudiments of dorsal and anal fins appeared. Serrated spine appeared at the pre-operculum. Melanophores from snout to tail pronounce, making the larvae black in color (Konsutarak and Watanabe, 1984). Day 8 (TL 3.58 0.13 mm) Teeth appeared in the lower jaw Day 9 (TL 3.49 0.26 mm) Caudal end of the notochord bent. Soft ray part of the caudal fin developed. Day 10 (TL 3.81 0.27 mm). Three serrated spines developed on the posterior margin of the pre-operculum. Head slightly rounded, and the body depth extended with developing dorsal and anal fin base. Tip of notochord flexed to develop the caudal fin. Segmented soft rays appeared. Melanophores pronounced from snout to tail and to the abdomen. Day 11 (TL 3.87 0.24 mm) Posterior margins of the dorsal and anal fins were deeply cut. Larval membrance in front of the anal fin small. The number of serrated spines at the posterior margin of the pre-operculum increased from three to four.

Day 12 (TL 4.41 0.09 mm) Segmented soft rays appeared in the dorsal fin. Day 13 (TL 4.58 0.17 mm) The number of serrated spines on the posterior margin of the preoperculum increased to four. Larval membrane in front of anal fin disappeared. Day 14 (TL 4.75 0.32 mm) Dorsal and anal fins separated from the caudal fin, and the rudiment of pelvic fin appeared. The chordal well developed and the vertebrae could be counted (11+14). Melanophore spreaded to the whole abdomen, dorsal and anal fins. A white band distinguished from the center of the dorsal fin to the anal fin with the naked eye. Day 15 (TL 5.41 0.50 mm) Spines and soft rays of dorsal and anal fins well developed. One-two serrated spines on the upper part of the posterior margin of the operculum developed. Day 16 (TL 6.56 0.56 mm) Each fin completely separated. Number of spines and soft rays of the dorsal and anal fins constant, 19 and 11 respectively. Serrated spines of the anterior margin of the pre-operculum disappeared. Day 18 (5.5 0.40 mm) Nostrils well developed. Maxillary reached to the center of eye. Single opercular spine presented on the upper part of operculum. Body depth increased relatively. The spines and soft rays of the dorsal, anal, and caudal fins well developed. Pectoral fin partially developed while the pelvic fin appeared as bud. Melanophores scattered on head and most of body and the posterior part. The body has two vertical bands and divided by mid-point of body. There is no melanophore on caudal peduncle. Day 21 (TL 8.91 1.19 mm) Numbers of spines and soft rays of the dorsal and anal fins constant. Scales appeared in the mid-lateral surface above the anal fin. The body color changes from black to brown (Konsutarak and Watanbe, 1984).

16. CARE OF HATCHLINGS

After hatching, undeveloped embryos and other dirts have to be siphoned out. This can be done by stopping aeration for a few minute to allow the sediments settle on the bottom. The sediments can be siphoned out as required. Hatching rate of seabass larvae also reflects the survival rate of the larvae in subsequent period. It has been observed from culture trials at the Centre as well as from hatcheries in Thailand that the batch with low hatching rate will have also poor survival

rate. For economical point of view only the batch with hatching rate above 50 percent will be kept for further development.

17. REARING OF LARVAE

The larval rearing can be divided into two phases. The primary rearing phase extends from hatching to a larval size of 46 mm TL or 1014 days after hatching. This rearing phase in mainly in indoor tanks. The secondary rearing phase covers the period up to 610 mm TL size or 1521 days after hatching. This phase is in outdoor tanks.

17.1 Indoor phase

During the first two weeks in indoor phase, the density of the larvae is about 4060 larvae/1. Water exchange is about 1020 % perday. Salinity is maintained at 2830 ppt. Two systems of larval rearing technique have been developed and tested. Each system has its own advantage and disadvantage depending on management and skill of technician. 17.1.1. Green water system Algae (both Chlorella and Tetraselmis) and rotifer are given to the larvae after attained two-three days old. Rotifer is given daily at the rate 23 pcs/ml on Day 2, 35 pcs/ml from Days 310, and 510 pcs/ml from Days 1114. The algae does not only serve as a food for the seabass larvae, but also serves as a food for rotifer and helping to improve the water quality. The algae is capable of converting the excretory products toxic to the fish larvae (un-ionized ammonia), which produced by the larvae, and the rotifer, and degrading of other decomposing feeds to less harmful nitrites. Every day before exchanging the water, the rotifer remaining in the rearing tanks is counted in order to adjust the amount of rotifer given daily. To ensure that the availability of food is adequate for the fry during 24 hrs period, the amount of rotifer remaining in the tank at the following day should not less than 5 pcs/ml. The amount of rotifer consumed by seabass fry relating to the size of fry is shown in Fig. 11. For the fry size about 4 mm TL consumes about 1 500 rotifers a day. The algae and rotifer are given until the fry reached 15 days old. Feeding schedule of seabass larvae and fry from Days 140 is given in Fig. 12.

Fig. 11. Number of rotifers consumed by a seabass fry per day corresponding to size. (Source : Tongrawd and Suteemechanikune, 1983). Artemia is given when the larvae reached the 810 days old or attained the size about 4 mm TL until 20 days old. The density of brine shrimp nauplii is given 11.5 pcs/1 first. The density is gradually increased to 45 pcs/ml when the fry are 15 days old, and 67 pcs/ml for the 20 days old fry. The amount of brine shrimp nauplii daily given should be adjusted according to the number of nauplii and larvae remaining in the rearing tank in the following day. The amount of nauplii eaten by seabass larvae increased linearly with the age of the fish (Fig. 13). For the 15 days old seabass larvae, it consumes as many as 300 nauplii per day.

Fig. 12. Feeding schedule and water management for seabass larvae and fry in nursery during the first 40 days period. The disadvantage of the green water system is the rapidly blooming of the phytopplankton in the larval rearing tanks. It may cause high mortality to larvae if the water cannot be changed in time. Rate of sedimentation on the bottom of the tanks is also high when compared with the clear water system. This increases work in cleaning the nursery tank and depresses the larvae during the cleaning process.

FIG. 13. Number of brine shrimp nauplii consumed per day by a seabass fry of 1117 days old. (Source : Pechamanee et al, 1984). 17.1.2. Clear water system On Day-2, after the mouth is fully developed, the rotifer size about 100 are given at a density of 23/ml. The size of rotifer can be increased to 150 at Day-3, and 200 at Day-4. After Day5, the rotifer size bigger than 200 are given. The amount of rotifer given is also vary with the size of the larvae. It is maintained at 35/ml from Days 310, and 510/ml from Days 1114. After Day- 11, the larvae should attain size of about 4.5 mm TL, and are ready to accept brine shrimp nauplii. This technique even though has to grade the rotifer to the size eatable by the seabass larvae, but it requires less work in nursery tank management and gives more consistent result when compared with the green water system.

17.2 Outdoor phase

After 14 days in indoor tanks, the larvae are much stronger and are more adaptable to outdoor condition. The larvae are graded and transferred to outdoor larval rearing tanks until Day-21. At this stage the density of the larvae is reduced to 2040 larvae/1. Salinity is reduced to 2526 ppt. Water exchange is around 50% perday. The feed is switched to brine shrimp nauplii. To reduce the production costs, Daphnia or Moina are also used as a supplementary feed, eight times a day.

After Day- 16, Acetes, pre-adult Artemia, or compound feed are given, 8 times a day. Types and relative amounts of food given at various stages shown in Table 7 are only given as a guideline. The actual amount of food required per day should be adjusted by observing the eating and swimming behavior of the fry. If the fry are swimming fast around the tank, it means that more food is required. Alternatively, if the food remains, the amount of food given should be reduced. To minimize the deterioration of rearing water and increase the efficiency of feeding rate, a dropping feeding technique (Fig. 14) is adopted with a good result.

18. FRY REARING

After 21 days old, the fry are graded and transferred to the rearing tanks. Stocking density is around 1020 fry/1. Salinity is reduced to 2025 ppt. Water exchange is around 80% per day. Minced fish is given as the main food. At first, the fish might not accept the minced fish but it gradually accepts it, once it is accustomed to it. Amount of fish flesh given is about 1015 per cent of the body weight. For convenience the calculation is based on the everage weight of the 25 days old fry, 0.15 gm/fry. The amount of feed given should be adjusted according to fish conditions and water quality. Sub-adult and adult brine shrimp are also fed to the fry during Days 2130. After Day 30, the fry should be transferred into the nursery ponds and/or in floating net cages. Table 7 Types and relative amounts of food given at different stages of seabass larvae and fry.
Age (days) 37 8 15 16 20 21 30 31 40 >41 Chlorella etraselmis (cells/ml) 10000 5 10 5 10 Rotifer Artemia (pcs/ml) (pcs/ml) (pcs/ml) 57 6 10 12 45 67 23 35 0 0 1 2 3 30 35 25 30 15 20 15 (pcs/ml) (% body wt) Artificial plankton Acetes pre-dult Artemia Minced fish compound feed

18.1. Nursing in pond

In the nursery pond, a density of 2550 fry/m2 are recommended. To keep the fry healthy and fast growth, the daily water exchange rate should be about 30 per cent. The food given in this period can be either trash fish or compound feed or both.

Fig. 14. Dropping feeding technique

18.2 Nursing in netcage

Nursing of seabass fry in netcage has been successful since conducive environmental conditions. The size of cages suitable for the operation are vary from 2.02. 01.0 m to 5.02. 01.0 m. The mesh size of the net used for nursery netcages is 1.0 mm. Seabass fry size betwen 1.0 and 2.5 cm TL are stocked at the rate of 80100 fry per m2 . After 45 days in nursery

ponds or netcages, the fry attain weight of about 10 gm (body weight) which are ready for the grow out phase.

19. NURSERY MANAGEMENT

Rearing the fry in confinement is subjected to adverse effects of over-crowding and ecological problems inherent in the culture system. As environmental parameters fluctuate and other factors extend its adaptive response, the fry attempts to maintain or reestablish its normal physiological balance. If this process is within the adaptive range the chance of survival of the fry is high. The behavior and size distribution of the larvae and fry in the tank reflect the management and culture techniques that have been employed for the particular batch. i. Swimming behavior If the fry actively swim with head slightly downwards, and aggregate at the level near the bottom of the tank or at the certain level in the water column due to light activating, it suggested that the fry are in good condition and healthy. Healthy fry also prefer staying at a certain distance from the aeration spot, and move actively. ii. Feeding behavior The healthy fry shows fast swimming behavior around the tank seeking for food and accepts food vigorously. The fry swim slow and relax on the water surface after having enough food. iii. Size distribution If the stock is properly managed, the fry are uniform in size. Under confinement conditions, the uneven growth of the larvae and fry promotes competition among the individuals for food, space and other essentials of survival. The resulting additive effects of stresses on the smaller and weaker fry, are witnessed by the dark to black color, making them more susceptible to being preyed upon and contracting disease. Uneven growth can cause a significant nursery mortality. The uneven growth could be due to the cannibalistic behavior of the species, dietary and environmental factors involved. However each batch of fry, there are the normal and the subnormal fry in terms of growth and other biological characteristics, which cannot be detected until later. iv. Pigmentation The healthy fry have bright pigment and lively look. Head, body and tail are well developed. The survival rates of seabass larvae and fry are determined by temperature, salinity, light intensity, stocking density, feed and feeding, water quality, grading and cannibalism.

19.1. Temperature
Temperature affects the growth and survival of the seabass fry. Within the temperature ranges between 2632 degree Celsius survival rate of the fry increased as the temperature increased (Table 8, Fig. 15). From rearing trials, poor survival rate was observed if the temperature fluctuated according to the ambient temperature (between 2632 degree Celsius) during the 24hr cycle. Good survival rate was obtained, when the temperature was kept consistent either at 28 degree Celsius or above. On contrary slow growth and poor survival rate were observed when the larvae or fry were kept at 26 degree Celsius or below. Table 8. Survival rate of seabass fry from Days 2134 rearing in 20 ppt at different temperatures.
No. 1 2 3 Total Average Initial number of fry 200 200 200 600 200 Control 36 36 36 26 C 47 54 52 153 51 28 C 30 C 68 114 57 146 71 135 196 395 65 132 32 C 185 182 145 512 171

19.2 Salinity
Salinity affects survival rate of seabass larvae and fry. The results of the hatchery trials suggested that for good survival rate, the salinity of the larval tanks should be maintained at 28 30 ppt during the first tow weeks. After 2 week, the salinity can be reduced to 2528 ppt, and maintained at 25 ppt after the 3rd week (Table 9). Table 9. Survival rate of seabass larvae and fry from days 130 at different salinities.
Age (days) 17 8 14 15 23 24 30 Salinity (ppt) 28 30 28 30 25 28 25 Survival rate (%) 90 80 75 85

19.3 Aeration
Aeration should be provided in as many spots as possible to allow even distribution of air in the tank. Keep the flow rate at minimal in order to facilitate even distribution of the fry in the culture tank.

Fig. 15. Survival rate of seabass larvae kept in 20 ppt at different temperature.

19.4. Light intensity

Light intensity enhances the blooms of micro-organisms in the culture tanks. If the blooms occur too rapid and the water cannot be changed in time, the high mortality of the larvae might incur. Covering the tank is not only keeping the light intensity minimal and evenly distributed in the tank during the day time, but also makes the water temperature less fluctuation during the 24 hrs period.

19.5. Stocking density

Stocking density of the larvae and fry in the nursery tank also varies according to the age of fry (Table 10). During the first two weeks a stocking density should be about 60 80 larvae/1. It should be reduced to 2040 larvae/1 in 2nd week, and 1020/1 in the 3rd week. After 21 days, the density of 110/1 is preferable. The stocking density used in hatchery trials at the Nationel Seafarming Development Centre, Hanura, Teluk Betung, Lampung, with a good survival rate are given in Table 10. Table 10. Stocking density and survival rate of seabass larvae and fry at different ages.
Age (days) 1 14 15 20 21 28 29 35 36 42 Total lengt (mm) 1.5 5 58 8 10 10 13 13 30 Stocking density (per m3) 60 000 80 000 20 000 40 000 10 000 20 000 5 000 10 000 1 000 5 000 Survival rate % 70 80 60 80 70 80 80 90 80 90

High stocking density is another common cause of high nursery mortality especially in the absence of stock management measures. Chan (1982) observed that in the absence of stock management initial stocking density of fry size ranging between 2.0 and 2.2 cm TL at 375 fry/m3 would be reduced of 200 fry or by 47 percent after 25 days of culture. The remaining stock is much more even in size, comprising roughly 2% of 6.7 cm TL. 88% of 4.5 cm TL and 10% of 2.53.0 cm TL. Of this remaining stock, the lowest size group similarly exhibits a contrasting color pattern signifying a state of stress while the highest size group is also silvery except when disturbed.

19.6 Feed and feediing

During the first 21 days period, the larval fish are fed with live food. Quality and quantity of feed given to the larvae and fry during this period are significantly affect on tis growth and survival rate. It has been clearly shown in rearing trials that seabass fry fed with rotifer and Artemia containing high level of the highly unsaturated fattay acid (w3-HUFA) gave higher survival rate than those fed with the low w3-HUFA diet (Chantarasri et al., 1989). The result suggested that larvae and fry of Lates calcalifer are similar to seabass, Dicentrarchus, (Franicevic et al., 1986) and seabream, Sparus auratus) (Lisac, et al., 1986) that require diet containing high level of 20:5w3 and 22:6w3. The quantitative requirement is approximately 1.80% in dry weight of the diet for good growth, high efficiency and free from chronic essential fatty acid (EFA) deficiency symptom. The quantity of feed given must kept under control. Water from rearing tanks should be taken seven or eight times a day to determine the concentration of the feed to assure the sufficient amount and continous supply of food to the fish larvae. After Day-21, minced fish is given. It takes about 23 days to wean the seabass fry to the minced fish. After getting use to the feed, it accepts the minced fish nicely.

19.7 Water quality

Water quality in nursery tanks have to be maintained as good as in the nature or at least as good as suggested in Table 2. The nursery tank should be cleaned as often as necessary. The rate of water replacement in nursery tanks depends on feed and feeding at different ages. During the period of rotifer feeding 1020% of water is changed every day. It is increased to 50% during the Artemia feeding period, and almost complete change during minced fish feeding period. Water exchange is siphoned through strainer box (Fig. 16). Dirts and wastes settled on the tank bottom are siphoned out.

SB : Strainer box. SI : Siphon, PP : 1 inch PVC pipe, FH : Flexible hose,

Fig. 16 Strainer box to facilitate water exchange.

Fig. 17. Grading of seabass fry.

19.8 Grading
First grading starts after the fry attained 12 days old using a net mesh size of 1.5 mm (Fig. 17) A small fish pass through the net but retained the larger ones. The second grading is needed after the fry attained 15 days old or whenever the difference in size is distinguished to separated the fast-growing fry from slogrowing ones. In order to reduce unnecessary injury and stress to the fish fry, and to speed up the operation, the second grading is done by using a series of fish graders with different pore sizes. The pore size of graders to retain the different sizes of fish is given in Table 11. Fish are placed in the fish grader and floated in the newly prepared larval tank. Table 11. Pore size of grader and retained size of fish.
Pore size (mm) 2 6 10 15 20 Minimum retained size (mm) 34 10 25 50 75

The smaller size fish pass through the pore to the new tank. The remainings are transferred into another tank. This procedure is able to sort the fish into different size groups as required and faster. Each grading may cause at least 5 percent mortality to the stock due to stress or injury. The rate will alleviate, if technicians lack of experience in handling the fry during the grading process. After grading, the larvae and fry are treated with 5 ppm acriflavine solution for disinfection purposes.

19.9 Cannibalism
Seabass is highly cannibalistic especially in early stages when the individuals tend to congregate at high density in a certain place. This offers an opportunity for the stronger fry to take their prey. Upon its first success to prey on its kind, a seabass fry will exhibit increasing vigour and urge to feed mainly through its cannibalistic behavior. Chan (1982) observed that while the bigger individual readily takes the smaller fry, it is also not uncommon for one to take another of the same size. The cannibalistic behavior is most pronounced at dawn and dusk when light intensity is low and also when flow rate of culture medium is minimal. The cannibalistic behavior of the species is also pronounced when the fry congregate in high densities, fighting for food during feeding time. The cannibalistic rate increases with increasing stocking density, clarity of water, and the number of feeding sessions per day. It also increases with decreasing percentage satiation per feeding, the number of feeding sessions per day, and the intensity of light.

20. GROWTH
After 30 days, the fish fry attaining a size of about 12 mm TL (Table 12, Fig. 18), the fry can be either transferred into nursery ponds, netcages or sold to farmers for furhter growth.

21. HARVESTING
Harvesting of the fry can be done either using a small seine net with 2 mm mesh size after the water of the rearing tank is lower to 1015 cm depth for large scale operation or collecting at the outlet for small scale operation.

22. SURVIVAL RATE

Under normal conditions, average hatching rate of fertilized eggs is around 80% Survival rate of larvae at day-15 and fry at Day-30 are expected at 7080%, and 3050% respectively. Table 12. Normal growth of seabass fry in 30 days.
Age (days) Fertilized egg (dia) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 21 30 Total length (mm) 0.87 1.60 0.04 2.20 0.08 2.52 0.06 2.61 0.08 2.78 0.15 3.08 0.09 3.10 0.13 3.44 0.09 3.58 0.13 3.49 0.26 3.81 0.27 3.87 0.24 4.41 0.29 4.58 0.17 4.75 0.32 5.41 0.50 6.56 0.56 8.91 1.19 12.05

(Source : Konsutarak and Watanabe, 1984)

23. DISEASE CONTROL

Disease infection is another major cause of nursery mortality. The term disease here generally refers to viruses, bacteria, fungi, protozoans and other harmful pathogens. Disease control in the hatchery will not be efficient, if any of the three factors; namely diagnosis, preventive measure of treatment, is omitted. The recommendations to control diseases in the seabass hatchery are listed below:

23.1 Diagnosis
Correct diagnosis, as well as an understanding of the life cycle and ecology of the pathogen, is essential in order to identify a proper control programme. The diseases infected seabass fry show symptoms of loss of appetite, loss of scales, change in body color from gray to black and occurrence of white spot on the body (for white spot syndrome).

Fig. 18. Growth of seabass fry during the first 30 days.

23.2 Preventive measures

To control the disease, the following preventive measures should be applied : i. ii. iii. iv. v. vi. vii. viii. Maintenance of water quality up to standard required in a seabass nursery. Reduction of environment stress such as low DO, build-up of waste products, etc., as much as possible. Development of resistant disease stocks. Vaccine development for immunological protection. Environmental manipulation (e.g growing of larvae in salinities above or below that at which pathogen will survive). Regulations to prevent transfer of pathogens from one host population to another. Chemical prophylaxis. Hatchery sanitation and disinfection.

23.3 Treatment
Chemical control should be considered as a last resort in disease control. Before treatment, it is better to have base line information on water quality such as pH and temperature of the culture media. When treating the fish, few fish should be treated to see how they react before treating the entire group. Only those drugs and chemicals which have been cleared by an authorized agency should be used on food fish. Treatments for specific groups of pathogens are suggested in Table 13 and Table 14.

24. LIVE FOOD

Live food used for nursing seabas from newly hatched larvae (after the yolk sac is absorbed) to 30 days old are Tetraselmis, rotifer, marine yeast, brine shrimp and Moina. The culture techniques being used successfully at the Seafarming Development Centre are follows :

24.1 Tetraselmis
Nutrients required for mass production of the diatom are give in Tables 15, 16, 17 and 18. To prevent the diatom settling on the bottom of the culture tank, the culture media should be continuously stirred. To maintain the pH level of the culture medium within 78, an excess of CO2 should be provided.

24.2 Chlorella
The cultivation of marine green algae is usually done in an outdoor concrete tank with a capacity of 550 t with seawater of 11.5 m depth. Chlorella cultivation is based on the following procedures : i. ii. iii. iv. supply of carbon dioxide and inorganic fertilizer in the proper amounts, adequate supply of lighting, holding the temperature at the proper levels, stirring of the water to keep an even density of chemical components and to prevent settling of the algae.

Pure culture of the algae at density of 10 million cells/ml from 1-gallon bottles are inoculated into a sufficiently aerated 1-ton tank filled up to one third of its capacity with fresh sea water. The chemical fertilizers usually used are ammonium sulphate, calcium phosphate, urea. The concentration of each of these elements (Table 19) is adjusted according to the nature of seawater supplied to the tank, which in turn varies by localities and other factors. The range of optimum temperature for algal growth is 2425 degree Celsius. It often disappears in water with temperature more than 30 degree Celsius. As the Chlorella cells increase in number, water is gradually added and appropriate amounts of fertilizers are applied. The amount of Chlorella produced although varying according to several factors, has a range of 24 million cells/ml within a 7-day period (Fig. 19). After 5 days a portion of the stock can be harvested. An equal volume of fresh seawater is added to the remaining stock and appropriate amounts of fertilizers are also supplied. A stock of Chlorella can last for a considerable period if there is good water management and contamination is minimized. Table 13. Treatment of diseases commonly found in seabas fry and fingerlings.
Size 3 8 days (0.5 cm) 10 20 days (0.51.5 cm) Disease Gas-buble syndrome Black body syndrome White faeces syndrome 2.58.0 cm White spot Cause Unknown Unknown Unkown Cryptocaryon irritans Ichthyopthirius multifilis Popeye Vibriosis 1 Formalin 200 ppm, 3060 mins, depending on the fish's endurance (Chong and Chao, 1986) 2. Formalin 100 ppm + acriflavine 10 ppm for 1 har (Chong and Chao, 1986) 1. Oxytetracycline 0.5 gm/kg feed for 7 days. 2. Sulphonamides or potentiated sulphonamides, 0.5 gm active in gradients per kg feed, 7 days. 3. Chloramphenicol 0.42 gm/kg feed for 4 days. 4. Bath in Nitrofurazone 15 ppm for at least 4 hrs, 5. Bath in Sulphonamides 50 ppm active ingredients for at least 4 hrs (Chong & Chao, 1986). Ampicillin 50 100 ppm, 57 days (Yongprapat, 1988) 1. Acriflavine 3 ppm, 3 days or bath NaCl 35%, 3 days or Tetracycline 25 mg/kg fish *(Yongprapat, 1988) 2. Nitrofurazone 15 ppm, 4 hrs, or Sulphonamide 50 ppm, 2 hrs, or Neomycin sulphate 50 ppm, 2 hrs, or Treatment Formalin 25 30 ppm 24 hrs. (Yongprapat, 1988) Formalin 100 200 ppm 1520 min or Tetracycline 25 ppm 24 hrs. (Yongprapat, 1988).

4.55.0 cm 7.5 cm

Kidney Colunaris

Vibrio sp syndrome Flixibacter columnaris Aeromonas hyrophilia A. puctata

finrot, tailrot

Myxobacter 10.017.5 Lymphocystis Virus

Chloramphenicol 50 pm, 2 hrs, or Acriflavine 100 ppm, 1 min, or 100% freshwater for 1 hr (Chong & Chao, 1986). unknown

Table 14 Treatment of diseases in young and adult seabass

Product name 1. Chloramphericol (Chloromycetin) 2. Furazolidone (nf-180-Furoxone) 3. Nitrofurozons 4. Oxytetracycline (Terramycine) 5. Sulfamethazine Suggested uses Bacteria, protozoa, and virus Bacteria Antimicrobial Dose and treatment Food : 50 100 mg/kg of fish per day for five day. Food : 2575 mg/kg of fish for 14 consecutive days Food : 7.5 gm/kg of fish daily for 2 weeks. Food : 1.8 mg/gm of fish approximately 3% of body weight/day for 8 days. Food : 100 mg/kg of fish per day for 10 15 days. Food : 120 mg sulfaguanidine + 250 mg sulfamerazine/kg of fish per day for 3 days followed by 80 mg sulfaguanidine plus 120 mg sulfamerazine per kg of fish for other 7 days. Food : 18 gm/kg of fish per day for 14 days. Food : 200 mg/kg of fish per day for 710 days. Dissolve in culture medium at concentration of 1 ppm.

Antimicrobial

Antimicrobial

6. Sulfaguanidine

Antimicrobial

7. Sulfamerazine 8. Sulfasoxazole 9. Copper sulphate

Antimicrobial Antimicrobial Protozoa

24.3. Rotifer
The nutritional value of rotifer is greatly affected by its diet. Food for rotifer, Brachionus plicatilis, are Chlorella, Tetraselmis, yeast, bacteria, and protozoans in which fatty acids have been assimilated. Propagation of rotifer for hatchery can be done using any of three methods : thinning method practiced in large tank, in a canvas cage, and the repeated stocking method in a small tank. Only the first method is described here because it is the one most common used and practiced at the Centre. Table 15. Ingredients of Conway medium.
Nutrients 1. Nutrient enrichment solution for Chlorella, Tetraselmis. FeCl3 6H2O MnCl2.4H2O H3BO EDA (Sodium salt) NaH2 PO4. 2H2O NaNO3 Trace metal solution Distilled water to One ml of enrichment solution is added to each liter of seawater. 2. Trace metal solution ZnCl2 CoCl2. 6H O (NH4) 6Mo 7O24. 4H2O CuSO4. 5H2O Distilled water to 3. Vitamin stock solution Vitamin B 12 Thiamin HCL Distilled water 10 ml of vitamin stock solution is added to 100 liters of seawater. 10.00 mg 200.00 mg 200.00 ml mg 2.10 gm 2.00 gm 0.90 gm 2.00 gm 100.00 ml 2.60 gm 0.72 gm 67.20 gm 90.00 gm 40.00 gm 200.00 gm 2.00 ml 2.00 l Concentration

Feeding rotifer with Chlorella combined with marine yeast may be done as following : Starting with the culture of Chlorella, Tetraselmis and marine yeast until the density increases to 5, 3, and 1 million cells/ml respectively. On the 4th day a starter of rotifer eith a density of 1020 cells/ml is inoculated; the rotifer will increase its density to 70100 cells/ ml after the 10th day, and reach its peak on the 12th day, 1001 000 cells/ml (Fig.13). To maintain the culture of the rotifer at a considerable level, the Chlorella, Tetraselmis and marine yeast are added after the 11th day. Rotifer can be partially harvested after the 10th day. Harvesting is carried out by draining the culture through a nylon net (60 u) leaving one third of the original volume to serve as a starter for the next batch. Before use, the rotifer should be enriched with cod liver oil (100ml/ton) and raw egg yolk (1 gm/ton) to increase its nutritional value.

24.4 Marine yeast

The stock of marine yeast can be obtained from local drainage of the hatchery. Nutrients required for the marine yeast culture are 15 gm of brown sugar, 3 gm of ammonium sulphate, 1 gm of potassium phosphate dissolved in one liter of water. One mg of Hydrochloric acid is added to bring pH of the solution down to 4. Within few days, the yeast can be transferred to 101 bottle. Adding the same the nutrients but without hydrochloric acid in strongly aerated seawater. The yeast then is further transferred into 100150 liter tank. After the marine yeast increases its density to 1 million cells/ml, it can be ready for feeding rotifer. Table 16. Nutrients for 1-liter stock culture of Tetraselmis.
Nutrients Sodium nitrate Any one of the following : Monobasic sodium phosphate Tribasic sodium phosphate Calcium phosphate Ferric chloride EDTA Thiamin HCl (B1) Biotin Vitamin B12 CuSO4. 5H2O ZnSO4. 7H2O NaMoO4. 2H2O MnCl2. 4H2O CoCl2. 6H2O Concentration 84.0 mg/l 10.0 mg/l 27.6 mg/l 11.2 mg/l 2.9 mg/l 10.0 mg/l 0.2 ug/l 1.0 ug/l 1.0 ug/l 0.02 mg/l 0.04 mg/l 0.02 mg/l 0.13 mg/l 3.6 mg/l

Table 17. Nutrients for 3-liter culture of Tetraselmis.

Nutrients Urea 46 K 2 HPO4 Concentration (mg/l) 100.00 10.00

Fecl2 Agrimin EDTA Vitamin B1 Vitamin B12

2.00 11.00 2.00 0.005 0.005

Table 18 Nutrients for 200 - liter and 1-ton culture of Tetraselmis

Nutrients KNO3 Na2HPO4. 12H2O CaHCO3 FeCl3 Concentration (gm/l) 100 50 25 5

Table 19 Nutrients for 1-ton culture of Chlorella.

Nutrients Ammonium sulphate Calsium phosphate Urea Concentration (gm/t) 50 200 10 50 5 25

Fig. 19 Rotifer culture showing a blooming peak of rotifer after inoculation.

Table 20. Nutrients for marine yeast culture.

Nutrients Brown sugar Ammonium sulphate Potassium phosphate Hydrochloric acid Concentration 15 gm/l 3 gm/l 1 gm/l 1 mg/l

Table 21. Content of W3-HUFA of the rotifer fed with different diets.
Kinds of rotifer Rotifer fed on baker's yeast only Rotifer fed on baker's yeast enriched with 8% fish liver oil Rotifer fed on baker's yeast enriched with 15% pollock liver oil Rotifer fed with baker's yeast enriched with 15% cuttle fish liver oil Rotifer cultured with Chlorella Rotifer fed on baker's yeast and secondary fed on Chlorella 3 hrs prior to administration to the fish larvae Rotifer fed on baker's yeast and secondary cultured on Chlorella 12 hrs prior to administration to the fish larvae w3-HUFA (%) 5.9 7.0 10.7 13.6 30.0 7.2 20.1

(Source : Foscarini, 1988)

24.5 Brine shrimp

Brine shrimp. Artemia salina, is the best live food for seabass larvae aged between 8 and 20 days. At present many brands and varities of brine shrimp cysts are on markets. Each brand contains different nutrition values, survival rates as well as prices. If the required amount of brine shrimp is 5 nauplii/ml. For feeding the seabass larvae in a 2-t tank, the amount of brine shrimp nauplii require can be calculated as follows :
Feeding density Hatching rate (Argentemia grade 1) 2t water Nauplii required (2 000 0005) The brine shrimp cyst required (10 000 000/280 000). 5 280 000 2 000 000 10 000 000 36 nauplii/ml nauplii/gm ml nauplii gm/2t water

24.5.1 Decapsulation In order to improve hatching rate as well as eliminating potential diseases that might be carried with the brine shrimp cysts, the cysts should be decapsulated first before hatching. The decapsulation can be done as follows:

Place required amount of brine shrimp cysts in hatching jar (conical fiberglass container). Add 1 200 ml seawater per 100 gm of a brine shrimp cysts. Aerate for 1 hr. Add 1 000 ml NaOCL or CaO per 100 gm of brine shrimp cysts. Stir thoroughly. Then add 25 gm of bleaching powder per 100 gm of brine shrimps cysts. Continue stirring. During the process of decapsulation, the temperature might gradually increase, if necessary using ice to keep the temperature below 40 degree Celsius. After 58 mins, the temperature should become steady. At this stage the cyst should change color from dark brown to white or orange. Clean the cysts in a fine meshed strainer and rinse with freshwater or seawater until the chlorine odor is removed. The cysts can be fed directly to the fish larvae and fry or put in incubation for further nauplii hatching. The cyst can be stored in concentrated brine (salinity of 300 ppt) or in refined salt (30 gm NaCl/100 gm of Artemia cyst) until needed. Neutralize the chlorine may be required by applying a 0.05 gm of sodium thiosulphate (Na2S203.5h2O) per 100 gm Artemia cysts. Add 100 ml of water and stir for 25 mins The decapsulated cysts will float. 24.5.2 Hatching To obtain the high hatching rate of the brine shrimp nauplii, the following hatching procedures are recommended : The cyst is hatched in conical hatching tank (Fig. 20) at density of 2 gm/l. For practical reasons, natural seawater is used as hatching medium. However it has been demonstrated that at lower salinities (e.g 5 ppt), the hatching rate increases and the nauplii have a higher energy content. Water temperature is maintained at 30 degree Celsius and pH is between 8 and 9. Continuous aeration is provided. The dissolved oxygen is maintanined close to saturation level. If necessary Na 2CO3 (1 ml of 0.5 M solution/1 medium) or CaO (65 mg/l) may be added to increase the buffer capacity. The cysts will be hatched out within 2024hrs. All cysts are kept in suspension. Accumulation of cysts on the bottom tank creates anaerobic zones which interrupts the cyst metabolism. To assure a maximum hatching rate, the culture is exposed to a continuous illumination of about 1 000 lux. This light intensity is attained when the hatching container is placed at about 20 cm from a florescent light tube of 60 W. Harvest of brine shrimp nauplii can be done by stopping aeration to allow egg shells afloat. Cover the lid to allow the nauplii to concentrate at the bottom. Drain the nauplii into a strainer and rinse with seawater to remove any remaining empty egg shells. Refill the hatching container with new seawater for further hatching of the remaining cysts. The brine shrimp cysts used at the Centre is Argetemia brand grade 1. It contained 62% protein, 22.0% 1 carbohydrate, 6.1% ash on dry weight basis. Its fatty acid profile is 5.6%, 20:5 w3HUFA.

24.6 Moina
The freshwater Moina can be used to substitute brine shrimp for seabass aged between 15 and 30 days old. It can be produced in large quantities at low cost. The stock is also easily obtained

from an aquarium shop or from local drainage. The Moina can be produced in large quantity in outdoor tank. The following procedures are recommended for mass production of Moina in 25-t outdoor concrete tank. Clean and disinfect the culture tank as necessary. Bring the water in at 25 cm depth. Add 15 l of dried chicken manure and 5 l of rice bran. Stir the tank thoroughly. On the 4th day about 500 ml of Moina stock is added. During this period, all mosquito eggs laid along the surface of water must be removed to minimize the competition in food between mosquito larvae and young Moina. Water level of the tank is gradually increase at the rate of 10 cm per day. The amount of water added can be adjusted according to the growth of the micro-organisms and Moina in the tank. The bloom of the Moina will reach a peak at the 7th day or three days after adding the stock. A partial harvest can be made. The culture will last for three days. After the 10th day the population of the Moina will decline, and the new culture is required. Since the Moina have a low level of w3-HUFA in its nutrition, the organism should be enriched with enrichment diets to increase its nutritional value before feeding to the seabass fry.

Fig. 20 Artemia hatching tank

25. ENRICHMENT OF LIVE FOOD

Besides increasing the level of w3-HUFA in the Artemia, and rotifer by feeding the organisms with baker's yeast, cod and pollock liver oil as shown in Table 21, the Artemia, and rotifer can also be enriched with enrichment diets which are now commercially available in markets. Among of those are TOPAL, SELCO, SUPER SELCO, SELCO BASE etc. Details and method of enrichment of the selected enrichment diets are given in Appendix 3.

26. INERT FOOD

Inert food can also be given as a supplementary feed to reduced the operation costs or to substitute the live food organisms if there are a shortage during the operation.

26.1 Micro-encapsulated egg particles

Micro-encapsulated egg particles size between 150200 u is given after Day-4 at density of 35 pcs/ml, three times a day at 8 00, 10 00 and 12 00 hr. After feeding with the micro-encapsulated egg, 80% of water is changed. Method of preparation of the micro encapsulated egg given in Appendix 2.

26.2 Artificial plankton

This type of inert food is now available on markets under different trade names such as Nippai artificial plankton BP, AS and artificial rotifer. It is convenience and can be used to substitute the rotifer with the similar results. The particle size suitable for seabass Day-3 larvae is 50150 u. The rate given is 510 particles ml water. Feeding interval is 68 times/day. After feeding 20% of water is changed.

26.3 Compound feed

Compound feed with crude protein higher than 40 percent can be used as a feed for seabass fry after Day-20 to replace minced fish and Artemia. The compound feed available in markets are salmon starter, trout starter, and shrimp starter. At the Seafarming Development Centre, the shrimp starter of President feed was used to feed the seabass fry after Day-20 to reduce the operation costs such as the costs of Artemia, fish flesh, and to reduce the labor in preparation of feed and changing the water. The fry accept the feed nicely after 12 days weaning period. The compound feed is given 3 times a day at satiation. The excess feed is siphoned out after feeding. After feeding about 50% of the water is changed. The result showed that the fry fed with compound feed has a better growth and survival rate when compared with those fed with minced fish.

27. PACKING AND TRANSPORT

The seabass fry can be transported in airtight carriers with oxygen. The basic needs are plastic polyethylene size 4060 cm, with 0.11 mm thick, insulating boxes, rubber bands and pure oxygen. The main causes for high mortality in transportation of live fish are : i. ii. iii. mistakes made before transport (fed fish, fish density too high). too high concentrations of poisonous ammonia at the end of transhipment (a symptom of poor preparation e.g unbalance between density and transporting time or feeding before transport). improper handling of fish after arrival (too quick transfer into new water, wrong treatment of diseases).

27.1 Preparing fish for transport

A few days before transport, fish are kept in clean water in separate tanks. The fish should not be fed for several days, depending on size. The last feed for larvae should be 12 to 24 hrs before transhipment while for fish up to 3 gm body weight it is 48 hrs. Larger fish should not be fed for three days. Weak or diseased fish have to be removed. The fry are graded according to size. Before packing, the fry is conditioned for 24 hr in plastic baskets floating in a concrete tank (Fig. 21). This procedure is necessary to allow the fish to empty their stomach, and as a part of sanitary control to prevent transporting any disease that might be carried with the fry. The fry is treated with 10 ppm acriflavine solution for 30 mins or 0.5 ppm of copper sulphate solution for 510 mins.

27.2 Packing fish

The plastic bags should be filled with three parts oxygen to one part seawater. After filling with water and fish, the plastic bags are inflated with oxygen. The oxygen level is measured by collapsing the bag to water level then placing hand down 10 inches from top and filling with oxygen until there is a feel of resistance. The top of the bag is then bent and tied with six or eight rubber bands (Fig.22) A crushed ice and sawdust can also be used to control the water temperature in plastic bags during the transporting period. For maintaining the water temperature between 1923 degrees Celsius the ratio of crushed ice and sawdust (by weight) is 1:2 for 45 hrs transporting time and 1:1 for 1216 hrs transporting period.

Fig. 21. Conditioning of seabass fry before packing

Fig. 22. Sequence of packing fish in plastic bag. Dry ice can be also used as an effective refrigerant to maintain the temperature in the plastic bag. However, the ratio of dry ice and sawdust to maintain the water temperature between 19 23 degree celsius at the required transporting time is subjected for further studies Density of fry per bag depends on age and size of fry, duration of transport, temperature control system, nature of container, and climate. If the temperature in the plastic bag is maintained between 19 and 23 degrees Celsius, about 500 fry size of 23 cm can be packed per bag with good survival rate after the 16 hr transporting period (Table 22). Table 22. Survival rate of seabass fry packed in 4060 cm plastic bags at different age, size and density.
Age (days) 7 15 20 22 30 60 Size(TL) (cm) 0.20.3 0.5 1.01.5 2.03.0 fry/bag water temperature(C) 10 000 5 000 1 000 5 000 19 23 " " " Duration (hrs) 16 16 16 16 Survival (%) 90 90 90 90

(Source : Tatanon and Maneewongsa, 1982b).

27.3 Preparing for the arrival of the fish

Before arrival, tanks containing clean seawater should be prepared. The water should be aerated and of appropriate temperature, If possible, a separate tank should be made available for each bag of fish.

27.4 Acclimatization of the fish

The main reason for mortalities after arrival in hasty transfer from transport water into new water. By the time of arrival, fish have become acclimatized to conditions in the bag namely high concentrations of carbon dioxide and ammonia, and pH between 56. These concentrations may be reduced gradually by a simple method. First open the bags and leave them in the boxes or in baskets. Then pour new water from the tanks into the bags, until the water volume is three or four times the initial quantity. This procedure should last about half an hour. The transport water must not be aerated, as this would drive out carbon dioxide, increase the pH and turn harmless ionized ammonia into poisonous unionized ammonia. A simple device to allow the gradual changing of water is shown in Fig.23. After the fish acclimatized to the new water, the fish may be transferred into the tanks. The tanks should be covered to avoid stressing the fish and preventing them from jumping out the tank, especially at the corners. The fish should be fed the day after arrival.

27.5 Treatment of diseases

All fish should be prophylactically treated against ectoparasites (0.6 mg Malachite Green per 10 liters of water) the day after arrival. Heavy bacterial and fungal infections can be treated with Tetracycline (2 gm per 100 l) and Chloramphenicol (1 gm per 100 l).

28. SPERM PRRSERVATION TECHNIQUE

One of the most crucial problems of spawning seabass is that the eggs and sperm are not available at the same time. Sometimes viable eggs are available but sperm is not, or vice versa. The problem could be reduced if the milt are collected first and preserved at low temperature. Seabass sperm can be kept for five days in the refrigerator at a temperature of 48 degree Celsius. The sperm is more active if suspended in 10% of dimethyl suphoxide before chilled storage (Withler and Lim, 1982).

Fig. 23. A device for gradual changing of water in a transport bag (After Frose, 1986). The long term preservation technique can be done by storing the fish sperm in liquid nitrogen at a temperature of-196 degree Celsius. To prevent cell breakdown due to extremely low temperature, a drop of dimethyl sulphoxide can be mixed with the sperms. By this method the sperm can be kept for about 2 years; and approximately 98 percent survival of sperm is obtained (Bhinyoing, 1980). The following method of freezing fish spermatozoa is modified from Harvey (1983) which have been tested successfully in Tilapia, goldfish, seabass, and grouper.

28.1. Preparation of diluent

Add 1 ml methanol to 9 ml 0.9% Na Cl in tap water. Add 1.5 gm dry powdered milk and mix well. Make up fresh daily; keep powdered milk frozen until use.

28.2 Mixing and freezing milt

Add 1 part milt to 5 parts diluent in a plastic vial. Mix well and bury in dry ice for 10 mins., then transfer to liquid nitrogen. Freeze no more than 0.5 ml (total volume) in one container. Milt frozen in liquid nitrogen will keep indefinitely.

28.3 Thawing and fertilization

Remove from nitrogen and agitate in a 40 degree Celsius water for 30 second or until ice just melts. Dilute 10:1 with seawater and add immediately to eggs.

29. ELEMENTS IN THE SUCCESS OF SEABASS HATCHERY OPERATORS

Seabass hatchery operation is a new kind of business. All techniques described in the manual can be considered as pioneering and need to be applied with caution. Although there have been rapid advances in developing appropriate seabass hatchery techniques since 1970, the techniques are still far from perfect. As with any art from, mastering the art of producing fish fry requires inherent ability, training and hard work. The required inherent ability, although hard to define, is indeed real. Some are better at rearing fish larvae and fry than others. The skill of those who are able to make decisions on day-to-day operation is probably the most critical factor in success or failure of the hatchery. The art of producing fish fry cannot be learned in any depth by reading, by discussion or by observing, but learning to handle fish fry under good supervision of experts would broaden the skill. Training could consist, in whole or in part, of gaining experience by trial and error. The hard work must be intelligently directly towards improving skill and know-know by whatever means. As in any business, success or failure will depend largely on the manager's ability to manage well.

30. REFERENCES
Broadhead, G.C. 1953 Investigations of black mullet, Mugil cephalus L In North-west Florida. State Board of Conservation Technical Series No. 7, Marine Laboratory, University of Miami, Fla., 34p. Bhinyoying, S. 1980 Sperm preservation technique, hormone bank, and fish seed production in Thailand. Asean Meeting of Technical Experts on Aquaculture, 34 Jan 1980 National Inland Fisheries Institute Bangkok, Thailand, Asean 80/Aquaculture 1/XI, 5 p. Chan, W.L. 1982 Management of the nursery of seabass fry. In : Report of training course on seabass spawning and larval rearing. SCS/GEN/82/39. South China Sea Fisheries Development and Coordinating Programme, Manila, Philippines p.3437. Chantarasri, 1989 Hanung Santosa, Hardoto and Sumbodo Kresno Yuwono. Induced spawning and larval rearing of seabass, Lates calcarifer in captivity. INS/ 81/008/Technical Paper No.8, 13pp. Dunstan, D.J. 1959 The barramundi in Queensland waters. Technical Paper Division of Fisheries and Oceanography CSIRO Australia, No.5, 22p. 1962 The barramundi in New Guinea waters. Papua New Guinea Agriculture Journal 15: 2331. FAO. FAO 1974 Species identification sheets for fishery purposes. Eastern Indian Ocean (Fishing area 57) and Western Central Pacific (Fishing area 71) Vol 1 Foscarini, 1988 Roberto. Intensive farming procedure for red sea bream (Pagrus major) in Japan. Aquaculture 72:191246.

Franicevic, 1986 V.D. Lisac, J. Buble, Ph. Leger, and P. sorgeloos. International study on Artemia XLII. The effect on the nutrition qauality of Artemia on the growth and survival of seabass (Dicentrarchus labrax L.) larvae in a commercial hatchery. In : Proceedings of the Conference on Production in marine hatcheries. Rovinj-Zadar (Yufoslavia) 1028 Feb., 1986, 10pp. Frose, Rainer. 1986 How to transport live fish in plastic bags. Infofish Marketing Digest 4: 3536. Greenwood, P.H. 1976 A review of the family Centropomidae (Pisces Perciformes). Bulletin of the British Museum of Natural History (Zoology) 29 : 181. Harvey, B. 1983 Cryopreservation of Sarotherodon mossambicus spermatozoa. Aquaculture matozoa. Aquaculture 32:313320. Konsutarak, P. And T. Watanabe. 1984 Notes on the development of larval and juvenile stages of seabass, Lates calcarifer. Report of Thailand and Japan Joint Costal Aquaculture Research Project No. 1 : 3645. Lisac, D.,V. Franicevic, Z. Vejmlka, J. Buble, Ph. Leger and P. Sorgeloos. 1986 International study on Artemia. XLIII. The effect of live food fatty acid content on growth and survival of sea bream (Sparus aurata) larvae. Paper presented at the conference Ichtyphotology in Aquaculture October 2124, 1986, Inter-University Center, Dubrovnik, 10 pp. Maneewongsa, S. and T.Tattanon. 1982 Nature of eggs, larvae and of the seabass. In : Report of training course on seabass spawning and larval rearing. SCS/GEN/82/39. South China Sea Fisheries Development and Coordinating Programme, Manila, Philippines p. 2224. Pechmanee, T., 1984 P. Ugkayanon and S. Maneewong. Growth comparison of 1118 days old seabass larvae, Lates calcarifer, fed with birne shrimp nauplii, Artemia salina, and with rotifer, Brachionus plicatilis. Report of Thailand and Japan Joint Coastal Aquaculture Research Project. No 1 : 134139. Reynolds, L.F. The population dynamics of barramundi Lates Calcarifer (Pisces : Centropomidae) in Papua New Guinea. MSc Thesis, University of Papua New Guinea, Port Moresby. Tattanon, T. and S. Maneewongsa. 1982a Larval rearing seabass. In : Report of training course on seabass spawning and larval rearing. SCS/GEN/82/39, South China Sea Fisheries Development and Coordinating Programme, Manila, Philippines p. 2930. Tattanon, T. and S. Maneewongsa. 1982b Distribution and transport of seabass. In : Report of training course on seabass spawning and larval rearing. SCS/GEN/82/39. South China Sea Fisheries Development and Coordinating Programme, Manila, Philippines p. 33. Tongrawd, S. and N. Suteemeechaikune. 1983 Feeding rate of seabass larvae fed on larvae rotifer. Contribution No.8 Satul Brackishwater Fisheries Station (in Thai) Withler, F.C and L.C. Lim 1982 Preliminary observations of chilled and deep frozen storage of grouper (Epinephelus tauvina) sperm. Aquaculture 27 38992.

Appendix 1. Taxonomic classification

Phylum Chordata Subphylum Vertebrata Class Pisces Subclass Teleostomi Order Perciformes Family Centropomidae Genus Lates Species Lates calcarifer ( Bloch) Taxonomic description Body elongate, compressed, with a deep caudal peduncle. Head pointed, with concave dorsal profile become convex in front of dorsal fin. Mouth large slightly oblique, Upper jaw reaching to behind eye; teeth veliform, no canines present. Lower edge of preoperculum with a strong spine; operculum with a small spine and with a serrated flap above origin of later line. Dorsal fin with 7 9 spines and 1011 soft rays; a very deep notch almost dividing spiny from soft part of fin; pectoral fin short and rounded, several short, strong serration above its base; dorsal and anal fins both have scaly sheaths; anal fin rounded. Scales large, ctenoid (rough to touch). Color : two phases, either olive brown above with silver sides and belly (usually juveniles) or green/blue above and silver below. No spots or bars present on fins or body. (Source : FAO, 1974)

Appendix 2. Production of prepared inert food for seabass larvae and fry.
As mentioned in the text, many different inert food are used in rearing seabass larvae and fry. Some inert food can be prepared and used local available materials. This appendix describes method of preparation of two types of the inert food that currently use at the Centre. These tow feeds can be prepared as follows. 1. Micro-encapsulated egg particles Crack an egg into heat resistent container. Soak 20 gm of soya been in water for 24 hrs. Wash it two-three times and blend it with a blender. Filter the soya milk through strainer of 150200 u. Add 10 gm of soybean milk and 10 gm of fish meal per egg. Add a drop of cod liver oil or vitamins as required. Homogenize egg and soyabean and milk with a mechanical blender. Steam it for about 15 mins. A fine opalescent suspension is obtained. Particles of the required size for food can be obtained by passing through sieve of appropriate size. Wash thoroughly until the wash water is clear. Feed directly to larvae. Unused food can be stored in a tight container in refrigerator for 23 days. Micro-encapsulated egg particles with 44% crude protein
Ingredient Quantity

Egg Soy bean Fish meal Non-fat dry milk powder Cod liver oil Vitamin C Antibiotic

1 pc 20 gm 10 gm 10 gm 1 drop 1 tablet 1 capsule

2. Minced fish Skipjack tuna, caranx, bonito, and mackerel are the fish of choice for preparing this feed. Fillet the fish, discarding head, bones, and viscera. Grind and liquidize the flesh in a blender. Force the flesh through the stainless steel sieves. The mesh sizes should be chosen to produce particles of size relevant to the age of the seabass fry. The fish flesh may be used directly or formed into bals of known weight for storage. It may be kept in the refrigerator for 23 days.

Appendix 3. Instruction for use of selected enrichment diets.

ENRICHMENT OF Artemia WITH SELCO OR SUPER SELCO

Weight the required amount of SELCO or SUPER SELCO and add small volume of water. Mix vigorously (e.g with kitchen blender). Prepare Artemia that want to be enriched, separating the newly hatched Artemia from cyst shell and rinse with the seawater, Add the prepared emulsion to the Artemia tank. Concentration of the enrichment medium is 0.3 gm SELCO or SUPER SELCO per 300 000 Artemia nauplii/1 water, Apply strong aeration to maintain the level of the dissolved oxygen in enriching Artemia tank above 4 ppm throughout the enrichment period, Another 0.3 gm SELCO or SUPER SELCO was add again after 12 hrs of the first period of the enrichment. After 24 hrs the Artemia can be harvested and rinse thoroughly with seawater before feeding it to the fry.

ENRICHMENT OF ROTIFER WITH SUPER SELCO

Concentration recommended is 0.1 gm of SELCO or SUPER SELCO per 10 million rotifer/1 water. Add the enrichment medium to the rotifer tank 3 hars prior to feed it to the larvae, Maintaining the level of the dissolved oxygen in the culture tank above 4 ppm. Rinse the enriched rotifer with seawater before use.

Appendix 4. ABBREVIATIONS
< > Less than. Greater than

 hr 1 mm cm m m2 m3 ml min mg gm kg ppm ppt DO2 TL HUFA HCG IU RU PG

Micron (sometimes written as um). Hour. Litre. Millimeter. Centimeter. Meter. Square meter. Cubic meter Milliliter. Minute. Milligramme Gramme Kilogramme Parts per million. It is equivalent to 1ml/m3 1 gm/t, lug/g, 1mg/l Parts per thousan (also expressed as o/oo). Dissolved oxygen. Total length. Highly unsaturated fatty acid. Human chorionic gonadotropin. International unit Rabbit unit and Rat unit. Pituitary gland.

Appendix 5 CONVERSIONS
1 um = 0.001 mm 1 um = 0.0394 in = 0.001 m 1 cm = 0.394 in = 10 mm = 0.01 m 1 m = 3.28 ft = 1.094 yd 1 in = 25.38 mm = 2.54 cm 1 ft = 0.305 m = 12 in 1 yd = 0.915 m = 3 ft WEIGHT: 1 gm = 0.0353 oz

LENGTH:

1 kg = 1 000 g = 2.205 lb 50 kg = 110.25 lb 1 000 kg = 1 t 1 t = 0.9842 UK t = 1.102 US t 1 oz = 28.349 g 1 lb = 16 oz = 453.59 g 1 cwta = 112 lb = 50.80 kg 1 US cwt= 100 lb = 45.36 kg 1 ta = 20 cwta = 2 240 lb 1 US t = 20 cwt = 2 000 lb 1 UK t = 1.016 t = 1.12 US t 1 liter = 1 000 ml = 0.220 gallona = 0.264 US gallon 1 m3 = 35.315 ft3 = 1.308 yd3 1 m3 = 1 000 liters = 219.97 gallonsa = 264.16 US gallon 1 ft3 = 0.02832 m3 = 6.229 gallonsa = 28.316 liters VOLUME: 1 gallona = 4.546 liters = 1.2009 US gallon 1 US gallon = 3.785 liters = 0.833 gallona 1 MGDa = 694.44 GPMa = 3.157 m3 / minute = 3 157 liters/minute 1 m2 = 10.764 ft2 = 1.196 yd2 1 ha = 10 000 m2 = 2.471 acres 1 km2 = 100 ha = 0.386 mi2 AREA: 1 ft2 = 0.0929 m2 1 yd2 = 0.836 m2 1 acre = 0.405 ha 1 mi2 = 640 acres = 2.59 km2

TEMPERATURE:

PRESSURE:
a

1 psi = 70.307 gm/cm2

British or Imperial units