Document 1
Document 1
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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.
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.
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.
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).
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.
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
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.
11.3 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.
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.
S= screen,
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 : 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. 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).
rate. For economical point of view only the batch with hatching rate above 50 percent will be kept for further development.
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.
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.
ponds or netcages, the fry attain weight of about 10 gm (body weight) which are ready for the grow out phase.
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.
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.
19.8 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.
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).
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.
24.1 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
finrot, tailrot
Chloramphenicol 50 pm, 2 hrs, or Acriflavine 100 ppm, 1 min, or 100% freshwater for 1 hr (Chong & Chao, 1986). unknown
Antimicrobial
Antimicrobial
6. Sulfaguanidine
Antimicrobial
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.
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
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.
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.
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
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.
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.
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 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
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.
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.
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
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