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Improvement of larval rearing technique forHumpback grouper, Cromileptes altivelis

Ketut Sugama1, Suko Ismi2, Shogo. Kawahara3 and Mike Rimmer4

1: Gondol Research Institute for Mariculture. Present Address: Central Research Institute for Aquaculture, Indonesia, Jl.KS Tubun Petamburan VI Jakarta, 3. Japan International Cooperation Agency, 4. Queensland Department of Primary

Industries.

Broodstock Care

The techniques for the capture,transportation, handling, sexing,sampling and acclimation of humpbackgrouper broodstock have beenadequately developed. Prophylaxisusing freshwater, antibiotics andquarantine is necessary beforeintroducing broodstock into thematuration tank. A detailed descriptionof prophylaxis and quarantinetechniques is available in work bySugama et.al.(2001). Recent advanceshave led to improved handling toreduce stress coupled with improvedbroodstock nutrition and good waterquality and could increase overall eggquality. This leads to higher firstfeeding success rate and subsequenthigher survival throughout the earlydevelopmental stages.

Maturation and spawning

Following quarantine and acclimation,broodstock fish are stocked in amaturation tank. The tank system is aflow-trough, achieving 200-300% waterexchange daily. Generally, 30 fish arestocked in 100-ton tank with sex ratioof two females and one male. Fish arefed with mixed fresh or frozen trash fish(avoid using only Sardinella sp.) andsquid mixed with 1% vitamin mix at 2-3% of body weight per day.

Six to eight months after stocking intank, the fish spawn naturally incaptivity. The spawning usually occursevery month 7-10 days before and afterthe new moon phase and spawningtakes place from midnight to earlymorning.

Introduction

Breeding of humpback grouper,Cromileptes altivelis has beenunderway in Gondol Research Institutefor Mariculture Indonesia since 1995.Techniques for mass seed productionof this fish were successful in late 1998under research collaboration with JICAon the Multi-species Hatchery Projectand the ACIAR Improved HatcheryTechnology for Groupers Project andtransferred to private hatcheriesincluding backyard hatcheries in 1999.The techniques, which are based onnatural spawning in captivity and theintensive rearing of larvae in tanks, aredescribed in detail by Sugama et al.(2001). About one million juveniles (4-5cm TL) were produced in 2000 and morethan three million juveniles in 2001. Eggproduction is no longer a constraintsince private hatcheries routinelyproduce billions of eggs fromdomesticated broodstock. In theresearch result carried in ResearchInstitute, Sugama et.al. (2001) reportedthe highest known survival of 53.9 % in10-ton tank on day 50 for this species.However, in recent times survival has

been highly variable (low and irregular)due to various factors, chiefly due toinfection with Viral Nervous Necrosis(VNN) infection (Koesharyaniet.al.2001)

This paper summarize the larvalrearing techniques that has beenpracticing in grouper hatcheries withinIndonesia.

Broodstock management,maturation and spawning

Broodstock

At present, all of existing humpbackgrouper hatcheries are using wildcaught fish as broodstock. Thisspecies is a protogynous hermaprodite,meaning that it is first sexually matureas a female and later on changes to bemale. The smallest mature female is 1 kgbody weight and only amongbroodstock more than 2.5 kg can malessometimes be found. In some cases,females do not change the sex even iftheir body weight is more than 3kg.

Broodstock Tank

The recommended volume ofbroodstock tanks can range from aminimum of 20 to a maximum of 200tons. However, considering biologicaland management factors, the idealvolume of tank is 50-100 tons with 2.0-2.5 deep. The ideal shape of maturationtank is circular, with a flat bottom and5-10 % gradient toward the centraldrain. The broodstock tank is equippedwith a water inlet and outlet (over-flow)pipes and egg collection tank with afine net (400mm) that is connected withthe outlet pipe, and an aeration system.

July-September 2003 (Vol. VIII No. 3) 35

Egg selection

Collected eggs are transferred into atransparent polycarbonate tank filledwith filtered seawater, debris mixed withthe eggs is removed using a 1.0 mmmesh net. In the tank, the eggsseparate into three groups namely,floating, suspended and sinking eggs.

Only floating eggs are recommendedfor further use in larval rearing.Floating eggs are soaked in 20 ppmiodine for 10 minutes or washed withUV treated sea water for 30 minutes toprevent a possible contamination ofbacteria or other micro organism thatmay cause disease. In watertemperatures of 28-29oC, the eggs

hatch after 18-20 hours of incubation.Eggs from broodstock fed with freshand mixed trash fish four times andsquid three times a week with 1 %vitamin mix result in good qualitylarvae. The newly hatched larvaeincubated without feeding can surviveuntil seven days after hatching, whileeggs from broodstock fed with sardineonly mixed with 1% vitamin mix hadcompletely died five days afterhatching. I recommend using goodquality eggs for seed production ofthis fish (Fig 1).

Larval rearing

Larval Rearing Tank

A particular feature of grouperhatcheries in Indonesia is use of theindoor method. The recommended sizefor larval rearing tanks is 10 ton with 1-1.2 metre depth. Both circular andrectangular tanks with flat bottoms canbe used for larval raring. The tankshould be painted with a light blue oryellow colour. For backyard hatcheries,the larval rearing tank should be roofedto avoid direct sunlight and rainwater.In order to minimize water temperaturefluctuation, it is recommended to coverthe tank with a transparent plasticsheet. The sheet is partially openedduring the day and closed at night. Thelarval rearing tank should be filled withsand filtered sea water on the day ofegg inoculation.

Feeding and Water Management

The eggs are generally added directlyto the larval rearing tank with a densityof 5-10 eggs/litre. Occasionally theseare placed in hatching tanks and thenthe newly hatched larvae aretransferred to the rearing tank, thisprocess enables larval density to beestimated more accurately. The larvalrearing is undertaken using green watertechniques. The algal density(Nannochlosopsis) used for greenwater culture ranges from 300,000 to500,000 cell/ml. Variables such as algaldensity are measured only in researchhatcheries, commercial hatcheries andbackyard hatcheries just add algal cellsuntil the desired shade of green isreached. Two-day-old larvae are fedsmall rotifer (SS-strain, size 120-140m)usually for three days at density of 5-7

A closer view of the fry

Early Cromileptes fry produced at Gondol

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individuals/ml and followed by S-strainrotifer (180-200m) at density of 8-10individuals/ml until day 20-24. Rotifersare enriched using a commercial fattyacid booster (DHA protein Selco) or byusing concentrated Nannochloropsisbefore supply to the larval rearing tank.Commercial compound feed is used asan artificial diet introduced from day 17onward and enriched Artemia issupplied from day 20 onward at densityof 0.5 individuals/ml. A detailed feedingscheme and water management ispresented in Table 1.

Survival rate

In 1999-2000 during five trials, thesurvival rate ranged from 2.65 to 5.13 %with total production of 22,000juveniles (Sugama et.al 1999). At thattime most mortality of the larvaeoccurred during the initial 2-5 daysafter hatching. Larval mortality wasmainly due to poor quality of the newlyhatched larvae. The newly hatchedlarvae were very weak, hence, easilytrapped at the water surface by watertension. As well, the trapped larvaewould be stressed and produce mucusthat would accelerate the trapping ofother larvae. In the 2000 trials,improvement of broodstock feedproduced better quality of eggs andlarvae. Furthermore, spreading squidoil on the surface of larval rearing water

seemed to reduce such mortality.Beginning on day 10-11, larvae have

an elongated dorsal and pelvic finspines, which often entangle larvae,especially when they swim to acommon place in the tank wall near thewater surface, probably in response tothe light. Here they aggregated andclump together, which may be

accelerated by the mucus andeventually die. Consequently, a highmortality (20-30 %) frequently occurredbetween day 10-25. To prevent totalaggregation, a fluorescent tube lamp(40 watt) was hung above the larvalrearing tank with minimum lightintensity of 800 lux. The light intensitywas adjusted as evenly as possible onthe water surface. The colour of larvalrearing water was maintained greenthrough inoculation ofNaanochloropsis at a density of 300-500 x 103 cell/ml. This might reducelarval aggregation.

Gradual larval mortality was usuallyobserved after day 25, which wassuspected to be due to nutritionaldeficiency. To prevent this problem,early weaning of larvae onto artificialdiets that have sufficient nutritionalvalue is recommended. In 2000-2001trials, artificial diets were introduce atday 15 prior to feeding Artemia, andthis minimized the demand of Artemiaas food. The remaining Artemia in thelarval rearing water should not be keptfor more than one day. With thisfeeding management, mortality couldbe reduced and resulted in absence oflordosis. Based on our observationcannibalism was not the main factor ofmortality in humpback grouper.

Fingerlings being graded for sale to local nurseries and farms

Figure 1. Survival of larvae without feeding from eggs that spawned by differentbroodstock

20

30

40

50

60

70

80

90

100

110

120

1 2 3 4 5 6 7 8

Days after hatching

Surv

ival

rate

(%)

Good eggsNot good eggs

July-September 2003 (Vol. VIII No. 3) 37

The success of larval rearingdepends on the control of VirusNervous Necrosis (VNN). Once VNNbroke out during larval rearing, most ifnot all larvae died within 2-3 days(Koesharyani et.al.2001). No treatmentmethod is presently available. To avoidVNN infection, use only VNN-freebroodstock by checking sperm andoocyte by PCR. In 2001-2002, an effortwas made to reduce larval stress bydecreasing the stocking density,improve nutritional quality of live feed(rotifer and Artemia) by enrichment andearly weaning of artificial diet. In 2001-2002 data have shown that inGovernment hatcheries the survival atday 50 ranged from 23.4-53.9% in fourcommercial hatcheries ranged from 3.1-51.4 % and in 15 farmers backyardhatcheries ranged from 7.0-35.01 %.

Production

In 2001-2002, more than one millionjuveniles have been produced byhatcheries within Indonesia. Atpresent, two Government, sevencommercial and more than one hundredfarmer backyard hatcheries are activelyproducing juveniles.

References

Sugama Ketut, Trijoko, Wardoyo, J. H. Hutapea and S.Kumagai, 1999. Natural spawning and larval rearingof Cromileptes altivelis. Report the APEC-NACACooperative Grouper Aquaculture Workshop, 7-9April Hat-Yai, Thailand. 91-98 pp.

Sugama, Ketut, Trijoko, B. Slamet, S. Ismi, E. Setiadi andS. Kawahara. 2001. Manual for the seed productionof Humback grouper Cromileptes altivelis, GRIM-JICA, Agency for Marine and Fisheries Research ofIndonesia 37 p.

Koesharyani, I, D. Roza, K. Mahardika, F. Johnny, Zafranand K. Yuasa. 2001. Manual for fish diseasesdiagnosis II. 2001. GRIM-JICA, Agency for Marineand Fisheries Research of Indonesia 48 p.

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Feeding scheme Day 2-25 Nannochloropsis (3-5 105 cell/ml Control at 8:00 AM and 5 PM Day 3-5 SS-strain rotifer (5-8 ind/ml) Control at 8:00 AM and 5 PM Day 5-25 S-strain rotifer (8-10 ind/ml) Control at 8:00 AM and 5 PM Day 15-31 Artificial diet (200-400µ) 1.5 g. each, four times daily Day 20- 45 Artemia (0.5-1.0 ind./ml) Supply at 5 PM Day 28-39 Artificial diet (400-600µ) 2-5 g each, five times daily Day 36 onward Artificial diet (600-800µ) 5-10 g each, 7- 12 times daily Water management Day 2-5 Spread squid oil in water surface 1 ml each, 10 AM and 15 PM Day 11-17 10 % water exchange Bottom siphoning at 9 AM Day 18-30 20 % water exchange Bottom siphoning at 9 AM Day 30-35 50 % water exchange Bottom siphoning at 9 AM Day 35 onward Running Water Exchanged rate 100 %/day

Table 1. Feeding scheme and water management in larval rearing of humpback grouper Cromileptes altivelis